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Purohit, Megha K., 2011, “Cloning, Over-expression and Characterization of Alkaline Proteases from Cultivatable and Non-cultivable Halophilic and / or
Haloalkaliphilic Bacteria Isolated from Saline Habitats of Coastal Gujarat”, thesis PhD, Saurashtra University
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CLONING, OVER-EXPRESSION AND CHARACTERIZATION
OF ALKALINE PROTEASES FROM CULTIVABLE AND NON-
CULTIVABLE HALOPHILIC AND/OR HALOALKALIPHILIC
BACTERIA ISOLATED FROM SALINE HABITATS OF
COASTAL GUJARAT
A THESIS SUBMITTED FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
IN
BIOTECHNOLOGY
MEGHA K. PUROHIT
REGISTRATION NO: 3617 DATE: FEBRUARY 28, 2007
SUPERVISOR
Dr. S. P. SINGH
PROFESSOR & HEAD
DEPARTMENT OF BIOSCIENCES
SAURASHTRA UNIVERSITY
RAJKOT – 360 005, GUJARAT, INDIA
AUGUST, 2011
SAURASHTRA UNIVERSITY DEPARTMENT OF BIOSCIENCES RAJKOT – 360 005, Gujarat, India Phone: Office + 91 281 2586419, Fax : + 91 281 2586419 E-mail [email protected]
Satya P. Singh, Ph.D. Ref: SU/Bio/
Professor & Head Date:
CERTIFICATE I take pleasure in forwarding the thesis entitled “CLONING, OVER-
EXPRESSION AND CHARACTERIZATION OF ALKALINE PROTEASES FROM
CULTIVABLE AND NON-CULTIVABLE HALOPHILIC AND/OR
HALOALKALIPHILIC BACTERIA ISOLATED FROM SALINE HABITATS OF
COASTAL GUJARAT” of Ms. Megha K. Purohit for the acceptance of
the degree of Doctor of Philosophy in Biotechnology.
Thesis presented here embodies records of original results and
investigations carried out by her.
Date :
Place : Rajkot
Forwarded through:
Prof. S. P. Singh Prof. S. P. Singh Supervisor Professor & Head Department of Biosciences Department of Biosciences Saurashtra University Saurashtra University Rajkot- 360 005, (INDIA) Rajkot- 360 005, (INDIA)
ACKNOWLEDGEMENT
Man is a social animal and all his achievements are the cooperative
actions of many well wishers. It is a matter of immense pleasure
and proud privilege to me to express my heartful gratitude to all
those personality who have been helping me in diversified ways.
This Doctoral research work was my humble effort to give some
contribution in the field of Biotechnology which could not have been
possible without inspiring guidance of my mentor, Prof. S. P. Singh,
Head, Department of Biosciences, Saurashtra University, Rajkot,
Gujarat, India. I am thankful for his valuable guidance, creative
ideas, thoughtful discussions and untiring supervision assisted to
shape up my research to the existing element. He gently but firmly
led to me along the difficult path of rectitude, his guidance denotes
to the high spots of excellence. I am and shall ever remain very
grateful for his warm affection that he has showered upon me at
all the time. He was always more than a Guide; a make-feel-home
friend who make the rough road easy walking.
I express my gratitude to Prof. V. C. Soni, Prof. A. N. Pandey,
Prof. V. S. Thaker, Dr. R. Kundu, Dr. S.V. Chanda, Dr. B.R.M. Vyas,
Dr. N. Panchal, Ms. V. M. Trivedi and Ms. J. Patel for their valuable
instruction and suggestion pertaining to my work.
I am thankful to Dr. Sanjay Kapoor, University of Delhi-South
Campus, New Delhi, India for the exposure and training related to
my research work. I also acknowledge valuable discussions with
Prof. S. K. Khare, Indian Institute of Technology, New Delhi, India.
I am thankful to Council of Scientific and Industrial Research, New
Delhi, India (CSIR, New Delhi) for awarding me Senior Research
Fellowship (SRF).
I thank all the non teaching staff members of our Department,
Dr. S. Bhatt, Mr. R. Purohit, Mr. P. C. Pandya, Mr. Joshi,
Mr. R. Solanki and others for the support they provided during my
work.
I am thankful to Kailash bhai, Chandresh bhai, Nitesh bhai, Raju
Bhai, Vaghela bhai and others for extending their help for all their
jobs
I express my sincere gratitude to my colleagues; Mr. Chirantan
Raval, Mr. Sandeep Pandey, Mr. Himanshu Bhimani, Ms. Sangeeta
Gohel, Ms. Viral Akabari, Mr. Vikram Rawal, Mr. Bhavtosh Kikani,
Mr. Rushit Shukla, Ms. Sejal Patel, Ms. Rupal Pandya, Ms. Mansi
Khokhra and Ms. Prinsa Siddhpura for their humble support.
I cherish the love, affection rendered by my friends and all M.Sc.
Biotechnology and Microbiology students since 2006 for their
constant support. I would always bare a fruits of being a part of
Biotechnology programme at Department of Biosciences.
From the deep within, I acknowledge my family. No word would
suffice to express my gratitude to my dearest parents for their
cherished devotation, inspiration, care and blessing. I thank my
husband, Paresh who always encouraged me during the time of
disappointment with his love and support. The words of thanks are
not enough to express my feelings for his support. Thanks to
Mummy2, Papa2, Uncle, Aunty, Badal, Girish, Chandan, Urja, Mira
and all my cousins and family members for their affection and
moral support, without which the work would not have been
completed. I assure them to be worthy of whatever they have done
for me. I humbly bow down to almighty God for making me
capable of carrying out this research tasks.
Megha Purohit
CONTENTS
Chapter 1 General Introduction 1-4
Chapter 2 Review of Literature 5-50
Chapter 3 Isolation and Diversity of Haloalkaliphilic Bacteria 51-101
3.1 Introduction 51
3.2 Materials and Methods 53
3.3 Results and Discussion 59
Chapter 4 Purification and Characterization of Alkaline
Proteases 102-136
4.1 Introduction 102
4.2 Materials and Methods 104
4.3 Results and Discussion 108
Chapter 5 Recombinant Proteases-
Cloning, Over-expression and Characterization 137-172
5.1 Introduction 137
5.2 Materials and Methods 139
5.3 Results and Discussion 149
Chapter 6 Metagenomic study-
Metagenome Isolation and Capturing Functional
Attributes
173-208
Section-I: Metagenomics studies for bacterial
diversity and its amenability for further functional
attributes (Alkaline Proteases)
173-194
6.1.1 Introduction 174
6.2.2 Materials and Methods 176
6.2.3 Results and Discussion 182
Section-II: Capturing of alkaline proteases from
saline soil metagenome: A culture independent
approach
195-208
6.2.1 Introduction 196
6.2.2 Materials and Methods 197
6.2.3 Results and Discussion 198
Chapter 7 Comparative analysis of native haloalkaline,
recombinant and metagenomic alkaline proteases as
a function of their tolerance against organic solvents
209-229
7.1 Introduction 209
7.2 Materials and Methods 211
7.3 Results and Discussion 213
Chapter 8 Remarks 230-236
Chapter 9 Summary 237-242
Conclusion 243-244
Bibliography 245-279
Appendices 280-290
I. Publications 280
II. Paper Presented 285
III. Scholarships/Awards 289
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CHAPTER
GENERAL
INTRODUCTION
1
1
GENERAL INTRODUCTION
Extremophiles, able to live in unusual habitats, can potentially serve in a verity of
industrial applications (Horikoshi, 2008). As a result of adaptation to extreme
environments, extremophiles have evolved unique properties, which can provide
significant commercial opportunities. The groups of bacteria that can grow under
alkaline conditions in the presence of salt are referred as haloalkaliphiles. The dual
extremity of halophiles and alkaliphiles make them interesting models for fundamental
research and exploration of biotechnological potential (Dodia et al., 2008a and b; Joshi
et al., 2008; Bominadhan et al., 2009; Purohit and Singh, 2011). Haloalkaliphilic
bacteria have largely been studied from the concentrated hyper saline environments;
Soda Lake, Solar Saltern, Salt brines, Carbonate springs and Dead Sea. The
exploration of the natural saline and alkaline environments beyond the above
boundaries is just the beginning (Patel et al., 2006; Thumar and Singh, 2007a and b;
Dodia et al., 2008a and b; Joshi et al., 2008; Thumar and Singh, 2009; Purohit and
Singh, 2011).
The enzymes from extremophilic organisms, particularly halophilic and
haloalkaliphilic bacteria and archaebacteria are relatively less explored. Among the
enzymes, proteases are among the most important groups of industrial enzymes and
account for about 60% of the total worldwide commercial enzymes (Horikoshi, 2008),
two-third of them being obtained from microbial sources (Carvalho et al., 2008;
Carolina et al., 2009). Several microbes have been investigated for these enzymes and
over the years, Bacillus species have emerged as the key producers of extracellular
proteases having potential applications in detergent, food, pharmaceutical, leather and
chemical industries (Boominadhan et al., 2009; Carvalho et al., 2009; Joshi et al.,
2008; Raj et al., 2010; Purohit and Singh, 2011). Most of the studies on
haloalkaliphilic bacteria; however, have focused on diversity and phylogenetic
analysis of the organisms and only limited information is available on their enzymatic
and other biotechnological potential. With the advancement in molecular tools, it
would be possible to expand the horizons of biocatalysis (Purohit and Singh, 2009;
Siddhpura et al., 2010; Purohit and Singh, 2011; Horikoshi et al., 2011a and b; Karan
et al., 2011a and b; Sudhir et al., 2011).
2
Maintenance of stability and activity in high salt is a challenge for halophilic and
haloalkaliphilic proteins (Dodia et al., 2008; Wang et al., 2009). The enzymes from
extremely halophilic archaea and bacteria require high concentrations of salt for
activity and stability and inactivated in Escherichia coli unless refolded in the
presence of salts under in-vitro conditions. Recombinant DNA technology in
conjunction with other molecular techniques is being used to improve and evolve
enzymes leading to new opportunities for biocatalysts (Zhang et al., 2009;
Ni et al., 2009; Singh et al., 2010a). Therefore, cloning of the potential genes coding
for different enzymes would be an attractive preposition to begin with. The
developments related to cloning and expression of genes and solubilization of
expressed proteins from halophilic and other extremophilic organisms in heterologous
hosts will facilitate enzyme-driven catalysis (Machida et al., 1998; Kim et al., 1998;
Machida et al., 2000; Singh et al., 2002; Singh et al., 2009; Kumar et al., 2011).
Since 95-99% of microorganisms in majority of the habitats are not cultivable,
significantly limited information is available about their genomes, genes and encoded
enzymatic activities. Metagenomics is the large-scale study of the DNA of naturally
existing microbial communities rather than laboratory-cultivable organisms. It’s an
emerging approach to explore diversity and harness microbial potential based on the
analysis of the total genomic DNA of microbial communities in their natural
environments, followed by cloning and expression of the genes into a cultivable host
organism.
In the direction of metagenomics studies, isolation and analysis of environmental
DNA are the key steps, which would allow to mine microbial diversity and help
understanding the dynamics, properties and functions of these organisms (Desai and
Madamwar, 2007; Purohit and Singh, 2009; Siddhpura et al., 2010). For extreme
habitats, the metagenomics is least explored and understood. The advances in
metagenomics would revolutionize the investigations in microbial ecology and
biotechnology, leading to exploration of uncultured microbial population and
discovery of new enzymes for various applications (Risenfield, 2000). Diversity of
the organisms from a given habitat would certainly play significant role in commercial
successes, particularly in applications for which bulk enzyme or product quantities
have to be produced. The phylogenetic complexity of the environments can range over
3
orders of magnitude. Therefore, the potential for applications of haloalkaliphilic
organisms in biotechnology seems endless.
India has enormous and unique coastline of the two oceans, a couple of gulfs and a
bay. Among the various parts, Gujarat (Western India) accounts for 1600 km long
coast, with industrial activities of mega projects, representing the remarkable diversity
within the natural microbial flora. Of this, the Saurashtra region under Kathiawar
peninsula occupies a total stretch of 865 km. Existence of halotolerant,
haloalkalitolerant and haloalkaliphilic bacteria clearly indicated the wide spread
distribution of such organisms in natural saline environment (Joshi et al., 2008).
Although, the field of microbial biotechnology is diverse, only limited attention has
been paid to haloalkaliphiles particularly from moderately saline habitats. In
realization of these facts, the present work in this thesis aims at the investigation of
microbes from saline habitats of the Coastal Gujarat. The work was majorly focused
on the microbial diversity, phylogeny, secretion of proteases and enzymatic
characteristics. Further, cloning, over-expression and characterization of recombinant
enzymes was undertaken. Metagenomics to find novel sequences of alkaline proteases
from the saline habitat has also been attempted. Comparative studies of native,
recombinant and metagenomic derived alkaline proteases for solvent tolerance would
be significant for non-aqueous enzymology.
4
OBJECTIVES Isolation of Halophilic/ Haloalkaliphilic bacteria from saline habitats
Detection, screening and optimization of extracellular enzymes among the
Haloalkaliphilic bacteria obtained from saline environment
Purification and characterization of alkaline proteases from potential strains with
respect to enzyme catalysis and stability
Cloning and sequencing of alkaline protease genes from potential strains of
Haloalkaliphilic bacteria and characterization of recombinant enzymes
To explore the diversity and novel alkaline protease genes from the uncultured
organisms by sequence and function based approaches
To elucidate structure and function relationship of alkaline proteases through
bioinformatics tools
Molecular Phylogeny & Capturing Function
16S rRNA Amplification
DGGE Analysis
Diversity BasedMorphology,
Gram Reaction
Enzyme secretion,
Biochemical
Studies on Growth & Enzyme
Secretion ,
Partial Purification &
Purification
Characterization
Haloalkaliphilic bacteria(Isolation,
Enrichment)
Sample collection from Okha Madhi site
Cloning &
Sequencing of
alkaline Protease
gene(two isolates)
Expression
Purification and
characterization of
enzyme
Isolation, Alkaline Protease Purification, Cloning and expression of enzyme
Metagenomics of saline Habitats :Phylogeny, Retrieval of genes, Cloning
of Enzyme
Metagenomics of saline Habitats :
Phylogeny & Retrieval of Novel genes for
biocatalysts
Isolation of Total Metagenomic DNA: Chemical &
Mechanical Method
Capturing Function
Alkaline protease gene
amplification
Sequence analysis of gene
Cloning of protease gene
Schematic representation of overall plan of work
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CHAPTER
REVIEW
OF
LITERATURE
2
5
REVIEW OF LITERATURE
Besides the extreme halophiles, the moderate halophiles are also important group of
microorganisms adapted to live in hyper saline habitats and constitute a
heterogeneous group, which includes a great variety of bacteria (Manikandan et al.,
2009; Ramesh et al., 2009). Moderately halophilic bacteria have the capabilities for
exciting and promising applications and hence, could be among the most potential
candidates, compared with other extremophiles.
During the past decades, the studies on ecology, physiology, and taxonomy of
halophilic organisms revealed an impressive diversity. Till now haloalkaliphiles were
studied extensively for the microbiological classification and phylogeny; only limited
attempts have been made to explore molecular basis of adaptation, enzymatic
potential and their other biotechnological implications. The diversity of the halophilic,
haloalkaliphilic and alkaliphilic microbes has been studied from the hyper saline and
hyper alkaline environment.
Microbial species represent the largest species diversity on the Earth and their
composition is of complex and dynamic nature (Torsvik and Ovreas, 2002). They
provide an infinite source of gene sequences, encoding functional components of
numerous catabolic pathways and regulation systems, of which many remain to be
discovered. At present, only a minor fraction of the microorganisms on Earth have
been explored (Burg, 2003). The development of new strategies for isolation, of
uncultured microbes; particularly extremophiles, is a challenging issue for the
scientists. It is of great value to make available the unexplored world of organisms,
besides that, their investigation is likely to generate enough knowledge of many
basics questions of biology towards harsh conditions.
Now, it has become clear that apart from soil and surface water, microbes colonized
almost every noxious environment on the Earth, considered to be extreme
(Horikoshi 2011a and b; Thomas and Dieckmann, 2002). Such extremophiles have
specialized skill, rarely seen in nature (Eichler, 2001). Some of them, such as,
thermoacidophiles, thermoalkaliphiles, thermohalophiles and haloalkaliphiles could
sustain in more than one extremity and are known as poly-extremophiles. These
groups of bacteria are mainly habituated from the fresh water to the (hyper) saline and
alkaline environments, Dead Sea, saltern crystallizer ponds and other places saturated
6
with respect to sodium chloride. So far, large numbers of hyper saline environments
have been studied for these bacteria from the ecology and diversity point of view. On
the basis of the physical parameters and chemical compositions, hyper saline
environments are mainly classified in to two; thalassohaline (arising from sea water
and contain sodium chloride as the predominant salt) and athalassohaline (largely
derived from the solution of evaporative deposits and contain different ion ratios
(Grant, 1993; Madern et al., 2004).
2.1 Hyper saline and alkaline environments
Hyper saline environments can be expected to have a relatively simple ecosystem
structure. The diversity of saline and hyper saline habitats with respect to their
properties reflected in the great diversification of microbial communities adapted to
prevailing conditions (Oren, 2002). Salt Lakes and other ecosystems with salt
concentrations at or approaching saturation are, therefore, convenient model systems
for studies in microbial ecology. As a result of natural and man-made global changes,
hyper saline environments are increasing.
Hyper saline waters are defined as having salt concentrations greater than that of sea
water (3.5%, w/v) (Grant et al., 1998). Several halophilic biotopes have been
identified, including saline lakes; evaporate lagoon sediments and coastal salterns.
Saline soils and the salt-excreting surfaces of animals are among the less explored
habitats, but almost all hyper saline biotopes are thought to harbor significant
populations of microorganisms (Grant et al., 1998).
2.1.1 Athalassohaline Environments
Athalassohaline environments are those, in which the ionic composition differs
greatly from that of sea water and in which the salts are of non-marine proportion.
The concentration of sea water leads to precipitation of NaCl, leaving a high
concentration of potassium and magnesium salts. This point marks the upper limit of
resistance of all biological forms (Das Sarma and Arora, 2001). Dead Sea, some
alkaline Soda Lakes, carbonate springs, salterns brines and alkaline soil are the
examples of thalassohaline environments.
2.1.2 Soda Lake
Soda Lakes, which represent stable and extremely productive aquatic ecosystems,
exhibit ambient pH values around 10 or higher. Most of the alkaline Soda Lakes in
7
Africa, India, China and elsewhere with pH values of 11 and higher and salt
concentrations exceeding 300 g/l are teeming with life (Oren, 2002). Soda Lakes are
widely distributed; however, as a result of their inaccessibility, few such Lakes have
been explored from the microbial diversity and ecological point of view.
2.1.3 The Dead Sea
The Dead Sea presents unique challenges to the halophilic microorganisms inhabiting
it because of its peculiar ionic composition. The concentration of divalent cations
(presently about 1.9M Mg2+ and 0.4M Ca2+) is dominant over monovalent cations
(1.6M Na+ and 0.14M K+) and the pH is relatively low (about 6.0). Even such a
hostile environment periodically supports dense microbial blooms (Oren, 1988). The
Dead Sea in the Middle East is the largest hyper saline environment studied in great
detail (DasSarma and Arora, 2001) (Table 2.1).
2.1.4 Carbonate springs
Carbonates rich springs and alkaline soils provide organic matter for diverse groups of
heterotrophs, primarily alkaliphilic Bacillus spp. and several species of
Cynobacterium are also normally abundant in such habitats. Decomposition of the
protein and hydrolysis of urea leads to microbial ammonification at particular place
resulting in high concentration of ammonia that raises pH of the habitat and
encourages the growth of alkaliphiles (Horikoshi, 2011a and b).
2.1.5 Thalassohaline environment
Many hyper saline environments originated by evaporation of sea water are known as
thalassohaline environments. Their salt composition is similar to that of sea water:
sodium and chloride are the dominating ions, and the pH is near neutral to slightly
alkaline. When evaporation proceeds, some changes occur in the ionic composition
due to the precipitation of gypsum (CaSO4·2H2O) and other minerals after their
solubility has been exceeded (Oren et al., 2005).
2.1.6 Salt Lakes and alkaline environments
There are two kinds of naturally occurring stable alkaline environments in the world;
high Ca2+ (ground waters bearing high calcium hydroxide) and low Ca2+
environments (Soda Lakes and Soda deserts are dominated by sodium carbonate)
(Grant, 1991 and 1992). Besides, natural hyper saline natural lakes, numerous
8
artificial solar lakes have been constructed for producing sea salts (Satyanarayana et
al., 2005).
2.1.7 Solar salterns
Multi-pond solar salterns present a gradient of salinities, from sea water salinity to
halite saturation. The salt concentration in each pond is kept relatively constant and
microbial community densities are generally high. Although, salterns are superficially
similar all over the world, they differ with respect to nutrient status and retention time
of the water, depending on climatic conditions (Javor, 1983). NaCl saturated brines
such as; saltern crystallizer ponds often display a bright red color due to the large
numbers of pigmented microorganisms (Oren, 2002).
2.1.8 Sea water
When 1 cubic foot of sea water evaporates it yields about 2.2 pounds of salt but 1
cubic foot of fresh water from Lake Michigan contains only one-hundredth (0.01) of a
pound of salt, thus, sea water is 220 times saltier than the fresh lake water. The
salinity of ocean water varies as affected by many factors; melting of ice, inflow of
river water, evaporation, rain, snowfall, wind, wave motion, and ocean currents that
cause horizontal and vertical mixing of the saltwater. The saltiness of sea is due in
part to the high water temperature, causing a high rate of evaporation. Sodium and
chloride constitute 85% of the dissolved solids in sea water and account for the
Ion (g/l) Sea
water Dead Sea
Great Salt
Lake
Na+ 10.8 39.2 105.4
K+ 0.4 7.3 6.7
Mg+ 1.3 40.7 11.1
Ca2+ 0.4 16.9 0.3
Cl- 19.6 212.4 181.4
Br- 0.1 5.1 0.2
SO42- 2.7 0.5 27
HCO3-/CO3
2- 0.1 0.2 0.7
Total salinity 35.2 322.6 333.6
pH 8.2 5.9-6.3 7.7
Table 2.1: Characteristics of (hyper) saline
environments (Grant,1992)
9
characteristic salty taste. Certain constituents in sea water, such as calcium,
magnesium, bicarbonate and silica are partly taken out of solution by biological
organisms, chemical precipitation, or physical-chemical reactions. Thus, the sea
contains many moderately halophilic or at least extremely halotolerant bacteria.
2.1.9 Saline and alkaline soils
The soil habitat is inherently inhomogeneous and wide range of salinities might be
present in any saline soil (Grant, 1991). Saline soils appear to yield mostly
halotolerant rather than halophilic microorganisms, presumably reflecting adaptation
to periodic episodes of relatively high dilution (Ruiz-Garcia et al., 2005a and b).
However, isolation of novel halophilic Actinopolyspora and Nocardiopsis species
from salty soils in Death Valley (Calif.), Alicante, and Iraq (Al-Tai and Ruan, 1994)
suggests that a wealth of interesting unknown halophilic microorganisms may be
present in such saline soils.
2.1.10 Other saline habitats
Extensive microbiological studies have been carried out in the Antarctic, especially
the cold saline lakes in the Vestfold Hills (East Antarctica) region and the saline soils
of the Dry Valleys. The best studied is Organic Lake, a meromictic lake with a
maximum depth of 7.5m. The lake is stratified, with salt concentrations increasing
from 0.8 to 21%. Many strains of moderate halophiles, belonging to genera including
Halomonas, Flavobacterium and Cytophaga, were isolated from the lake (Dobson et
al., 1991; Franzmann et al., 1987). Many moderately halophilic bacteria have been
isolated from salted fish, meat, and other food products (Onishi et al., 1980;
Vilhelmsson et al, 1996). Besides, moderately halophilic bacteria may be found in
some unusual environments, such as on desert plants (Simon et al., 1994) and desert
animals (Deutch, 1994).
2.2 Halophiles, Alkaliphiles and Haloalkaliphiles: Diversity and
Molecular Phylogeny The diversity of an ecosystem is dependent on the physical characteristics of the
environment, the diversity of species present, and the interactions that the species
have with each other and with the environment. Environmental disturbance on a
variety of temporal and spatial scales can affect the species richness and,
consequently, the diversity of an ecosystem.
10
The traditional and classical methods of the classification are not sufficient to
generate the evolutionary relationship between different groups. Ribosomal RNA is an
ancient molecule, functionally constant, universally distributed and moderately well
conserved across broad phylogenetic distances (Madigan et al., 1997). Moreover,
there is no evidence of lateral gene transfer of rRNA genes between different species
and therefore rRNA genes can bring true information regarding evolutionary
relationships (Pace, 1997). 16S rRNA studies have shown to support a different but
equally diverse population of halophilic, alkaliphilic and haloalkaliphilic bacteria and
archaea (Jones et al., 1994).
To describe halophilic bacteria, several classifications or categories have been
proposed according to their behavior towards salt, (Kushner, 1978; Vreeland, 1987;
Ramos-Cormenzana, 1989). The most widely used is that of Kushner. According to
him, one can distinguish between slight halophiles (many marine organisms; sea
water contains about 3-5% (w/v) NaCl), moderate halophiles (optimal growth at 3–
15% (w/v) salt), extreme halophiles (optimal growth at 25% (w/v) NaCl;
Halobacteria and Halococci), and borderline extreme halophiles (requirement of at
least 12% (w/v) salt). This diversity of halophilic and highly halotolerant
microorganisms is expressed both at the phylogenetic level; halophiles are found in all
three domains of life: Archaea, Bacteria, and Eucarya and at the physiological level,
most modes of energy generation known in non-halophiles are also used by halophilic
counterparts. Still, most microbiologists do not realize the true extent of the diversity
of halophilic and halotolerant microorganisms in nature (Oren, 2002a; Guranthon et
al., 2010).
2.2.1 Halophilic bacteria and archea
Bacterial halophiles are abundant in environments such as salt lakes, saline soils, and
salted food products (Oren, 1999). Further, they are also found in both; the aerobic
branches (Bacillus and related organisms) and anaerobic branches. There is even an
order, the Halanaerobiales, consisting of two families (the Halanaerobiaceae and the
Halobacteroidaceae) that consist solely of halophilic anaerobic microorganisms
(Oren, 2002b). DasSarma and Arora (2001) and Oren (2002c) have extensively
reviewed the diversity and molecular ecology of the halophilic and extremely
halophilic bacteria and archaea in different groups of microbes. The 250 million year
old halotolerant bacteria, Bacillus sphaericus, was isolated from spore, extracted from
11
a salt crystal buried at more than 1,500 feet underground in Carlsbad (Vreeland et al.,
1987).
2.2.2 Alkaliphiles
Alkaliphiles consist of two main physiological groups of microorganisms; alkaliphiles
and haloalkaliphiles. Alkaliphiles require an alkaline pH of 9 or more for their growth
and have an optimal growth pH of around 10, whereas haloalkaliphiles require both an
alkaline pH (>pH 9) and high salinity (up to 33% (w/v) NaCl) (Horikoshi, 2011).
Horikoshi have spent the major part of his research career to investigate the
physiology, ecology, taxonomy, enzymology, molecular biology and genetics of the
alkaliphiles.
2.3 Haloalkaliphilic bacteria Haloalkaliphiles possess special adaptation mechanisms for survival in highly saline
and alkaline pH. These properties make them interesting not only for fundamental
research but also for industrial application (Margesin and Schinner, 2001). So far,
moderately haloalkaliphilic bacteria have been isolated from the different saline and
alkaline environments (Ventosa et al., 1998; Xu et al., 2001; Xin et al., 2001; Zhang
et al., 2002; Doronina et al., 2003a; 2003b; Hoover et al., 2003; Patel et al., 2005a;
Dodia, 2005; Patel et al., 2006a; Dodia et al., 2008a and b; Nowlan et al., 2006).
2.4 Adaptation strategies Life is based on organic chemistry and the mechanisms of such chemistry must be
allowed to function for life to continue. Extremophiles adopt two distinct approaches
to living within extreme environments; they adapt to function within the physical and
chemical bounds of their environment or they maintain mesophilic conditions
intracellularly, guarding against the external pressures. Among them, halophiles are
an interesting class of extremophilic organisms that have adapted to harsh, hyper
saline environments.
The organisms living in extreme conditions possess special adaptation strategies that
make them interesting not only for fundamental research but also towards exploration
of their applications, Horikoshi (2008). These organisms may hold secret for the
origin of life, apart from that it will unfold many basic questions about the stability of
12
the macromolecules under extreme conditions. Therefore, their studies would provide
important clues for adaptation under salinity.
To cope up with the high and often changing salinity of their environment, the aerobic
halophilic bacteria, similar to all other microorganisms, need to balance their
cytoplasm with the osmotic pressure exerted by the external medium (Oren, 2008;
Oren, 2010). Osmotic balance can be achieved by the accumulation of salts, organic
molecules or similar mechanism. Alternatively, the cell is able to control water
movement in and out and maintain a hypo-osmotic state of their intracellular space.
The extremely halophilic archaea and bacteria adapt various strategies, viz. molar
concentrations of chloride is pumped into the cells by co-transport with sodium ions
and/or using the light-driven primary chloride pump halorhodopsin (Oren, 2010).
Distribution of charged amino acids could also serve as one of the major approach.
2.4.1 Chloride Pumps
A very high requirement for chloride was demonstrated in two groups of bacteria;
anaerobic Halanaerobiales and the aerobic extremely halophilic Salinibacter rubber,
that accumulate inorganic salts intracellularly rather than using organic osmotic
solutes. Thus, it becomes clear that chloride has specific functions in halo-adaptation
in different groups of halophilic microorganisms (Müller and Oren, 2003).
2.4.2 Osmoregulation in bacteria
Osmoregulation is a fundamental phenomenon developed by bacteria, fungi, plants
and animals to overcome osmotic stress. The most widely distributed strategy of
response to hyperosmotic stress is the accumulation of compatible solutes, which
protects the cells and allows growth. Adaptation of bacteria to high solute
concentrations involves intracellular accumulation of organic compounds called
osmolytes. Osmolytes are often referred to as compatible solutes because they can be
accumulated to high intracellular concentrations without adversely affecting cellular
processes it can be either taken up from the environment or synthesized de novo, and
they act by counterbalancing external osmotic strength, thus preventing water loss
from the cell and plasmolysis. Since the water permeability of the cytoplasmic
membrane is high, imposed imbalances between turgor pressure and the osmolality
gradient across the bacterial cell wall are short in duration. Bacteria respond to
osmotic upshifts in three overlapping phases: dehydration (loss of some cell water);
13
adjustment of cytoplasmic solvent composition and rehydration and cellular
remodeling.
2.4.3 Compatible solutes
The accumulation of organic solutes is a prerequisite for osmotic adjustment of all
organisms. Archaea synthesize unusual solutes such as β-amino acids, N -acetyl-β-
lysine, mannosylglycerate and di-myo-inositol phosphate. Among all of them, uptake
of solutes such as glycine betaine is preferred over de novo synthesis. Most
interestingly, some solutes are not only produced in response to salt but also to
temperature stress.
Glycine Betaine
The ability of the organism to survive both high salt concentrations and low
temperatures is attributed mainly to the accumulation of the compatible solute glycine
betaine. One of the most effective compatible solutes widely used by bacteria is
glycine betaine, the N-trimethyl derivative of glycine, which can be accumulated
intracellularly at high concentration through either synthesis or uptake or both.
Bacillus subtilis has been shown to possess three transport systems for glycine
betaine: the secondary uptake system opuD and two binding-protein-dependent
transport systems, opuA and opuC (proU).
Distribution of amino acids
The cell wall halophilic archaea Halobacterium has a high proportion of the acidic
amino acids; aspartate and glutamate as sodium salts. Interestingly, this sodium
binding is essential to maintain the cell wall and dilution of the medium leads to
repulsion between the free carboxylate groups leading to cell wall disintegration and
cell lysis.
Molecular aspects of salinity
Marine microbes are known to play an essential role in the global cycling of nitrogen,
carbon, oxygen, phosphorous, iron, sulfur and trace elements (Nada et al., 2011).
Salinity tolerance comes from genes that limit the rate of salt uptake from the soil or
water and the transport of salt throughout the plant, adjust the ionic and osmotic
balance of cells in roots and shoots and regulate leaf development and the onset of
senescence (Munns and Tester, 2008). However, very little progress has been made in
this regard so far, as the gene expression pattern and analysis has been difficult. Most
14
of the sequenced culturable microorganisms from the deep-sea are Alteromonadales
from the Gammaproteobacteria. Unique properties of sequenced deep-sea microbes
are that they all have a high ratio of rRNA operon copies per genome size, and that
their intergenic regions are larger than average (Lauro and Bartlett, 2008). These
properties are characteristic of bacteria with an opportunistic lifestyle and a high
degree of gene regulation to respond rapidly to environmental changes when
searching for food.
Study of the molecular basis of osmoadaptation and its regulation in archaea is still in
its infancy, but genomics and functional genome analyses combined with classical
biochemistry shed light on the processes that confer osmoadaptation in archaea.
Furthermore, they showed that betS is constitutively expressed, whereas BetS
activity depends on posttranslational activation by high osmolarity and is most likely
the emergency system transporting betaines for immediate osmotic protection. Many
microorganisms possess two or more glycine betaine transport systems. Salmonella
typhimurium, for example, possesses two genetically distinct pathways, a constitutive
low affinity system (ProP) and an osmotically induced high-affinity system (ProU),
while B. subtilis has three glycine betaine transport systems, OpuD, OpuA, and OpuC.
2.4.4 pH
Internal pH maintenance in alkaliphilic bacteria is achieved by both; active (sodium
ion channels) and passive regulation (through cytoplasmic pools of polyamines and
low membrane permeability). The pools of cytoplasmic polyamines are rich in amino
acids with positively charged side groups (lysine, arginine and histidin).
The cell wall play a key role in protecting the cell from alkaline environments, hence
in addition to peptidoglycan, alkaliphilic Bacillus sp. contains certain acidic polymers,
such as galacturonic acid, gluconic acid, glutamic acid, aspartic acid, and phosphoric
acid (Aono et al., 1999; Kitada et al., 2000). Some alkaliphilic bacteria, however,
have developed sodium ion channels that actively drive the entry of protons across the
membrane through H+/Na+ antiporters, thus decreasing the overall pH of the
cytoplasm. Besides controlling protons, Na+ dependent pH homeostasis requires
reentry of Na+ into the cell. Na+ coupled solute symporter and Na+-driven flagella
rotation ensure a net sodium balance.
15
2.4.5 Light
Halophilic archaea live in shallow evaporation pond encounter with very high
temperature and ultraviolet light. They have developed a special retinal pigments
called carotenoid. These pigments provide protective barrier to the ultraviolet light.
These pigments not only found in halophilic archaea (Lakatos et al., 2002) but also in
haloalkaliphiles (Bivin and Stoeckenius, 1986).
2.5 Industrial and Biotechnological Relevance of Halophiles,
Alkaliphiles and Haloalkaliphiles
Besides their important role in ecology of hyper saline environments, these groups of
prokaryotes have received considerable interest because of their potential for use in
various biotechnological and industrial applications, such as biomedical and chemical
sciences, food, leather, laundry detergent and pharmaceutical industries (Rothschild
and Mancinelli, 2001). Moreover, some archaeal metabolites, such as some proteins,
extracellular enzymes, osmotically active substances (compatible solutes), exo-
polysaccharides and special lipids have potential industrial applications (Schiraldi,
2002). They appear to be a very good source of various biomolecules and can open
the dimensions for the development of novel value based products because of unique
properties, which can withstand at harsh environment.
2.5.1 Compatible solutes
Halophilic bacteria produce compatible solutes that maintain a positive water balance
in the cell and are compatible with the cellular metabolism. These low molecular
weight substances are excellent stabilizers for whole cells and biomolecules (Galinski,
1993; Da Costa et al., 1998; Welsh, 2000; Santos and Da Costa, 2002; Vargas et al.,
2004). The stabilizing effect of mannosylglycerate (MG) and diglycerol phosphate is
higher for several enzymes subjected to heating or freeze-drying to that of other
stabilizers (Lamosa et al., 2000). Ectoine and derivatives have been patented as
moisturizers in cosmetics (Montitsche et al., 2000), although the most promising
application may be as stabilizers in the polymerase chain reaction (Sauer and
Galinski, 1998).
2.5.2 Antimicrobial substances
The biological diversity of the marine environment, in particular, offers enormous
scope for the discovery of novel natural products, several of which are potential
16
targets for biomedical developments (Austin, 1989; Fenical, 1997). Extremophiles
have been recognized as valuable sources of novel bioproducts and this may well
include antimicrobials (Horikoshi, 1993; Kokare et al., 2004; Fiedler et al., 2005).
Halocins are bacteriocin-like proteins or peptides produced by many species of
Halobacteriaceae, Haloferax mediterranii and Haloferax gibbonsii, which act against
haloarchaea and haloalkaliphilic rods which suggests that these different archaeal
kingdoms may share a common archaeal-specific target (Platas et al., 2002; Yun et
al., 2003).
2.5.3 Bacteriorhodopsin
Certain extremely halophilic and haloalkaliphilic bacteria contain membrane bound
retinal pigments called Bacteriorhodopsin (BR) and halorhodopsin (HR) (Lanyi,
1993). The applications comprise holography, special light modulators, artificial
retina, neural network optical computing and volumetric and associative memories.
Recently, cloning and functional expression of archaerhodopsin gene from
Halorubrum xinjiangense was successfully achieved in E. coli, where the purple
membrane was fabricated into films and photoelectric responses depending on the
light-on and light-off stimuli were observed (Feng et al., 2006).
2.5.4 Biosurfactants
Biosurfactants enhance the remediation of oil-contaminated soil and water and have
potential for pollution treatment in marine environments and coastal region (Banat et
al., 2000). Biosurfactants from these extremophiles are also used for in-situ
microbially enhanced oil recovery (MEOR) but the production cost is limiting factor
for exploitation of these biosurfactants.
2.5.5 Exopolysaccharides
Halophilic exopolysaccharide (EPS) producers could be interesting source for MEOR,
where polymers with appropriate properties act as emulsifiers and mobility
controllers. The exopolymer polyg-D-glutamic acid (PGA) can be used as a
biodegradable thickener, humectant, sustained release material, or drug carrier in the
food or pharmaceutical industry (Kunioka, 1997). Hezayen et al., (2000) reported the
first description of a PGA-producing extremely halophilic archaeon related to the
genus Natrialba.
17
2.5.6 Liposomes
Liposomes are used in medicines and cosmetics for the transport of compounds to
specific target sites in the body. Liposomes prepared from polar archaeal glycerolipids
are of special interest because of their adjuvant activities in mammals (Sprott et al.,
2003).
2.5.7 Food Biotechnology
Halotolerant microorganisms play important role in various fermentation processes,
occurring in the presence of salt and producing various compounds that give
characteristic taste, flavor and aroma to the resulting products. In the production of
pickles (fermented cucumbers), brine strength is increased gradually from 5-15.9%
(w/v) NaCl. Certain species of halophiles; Halobacterium salinarum, Halococcus sp.,
Bacillus sp., Pseudomonads and Coryneform bacteria are used in the production of an
Asian (Thai) fish sauce, in which fish is fermented in concentrated brine (Thongthai
and Suntinanalert 1991; 2001). Canthaxanthin is used in cosmetics to decrease the
necessary exposure time in sunlight to acquire a tan and to intensify the tan as the
compound attaches to the subcutaneous layer of fat (Margesin and Schinner, 2001).
2.5.8 Biological waste treatment
Degradation of aromatic compound
Relatively few reports have addressed for the degradation of aromatic compounds
under highly saline conditions by halophilic and haloalkaliphilic bacteria. The ability
of halophiles/halotolerants to oxidize hydrocarbons in the presence of salt is useful for
the biological treatment of saline ecosystems, which are contaminated with petroleum
products (Margesin and Schinner, 2001). Several studies have demonstrated bacterial
degradation of aromatic compounds in saline conditions (Piedad Diaz, et al., 2000;
Mellado and Ventosa, 2003; Peyton et al., 2004). However, the ecological studies
concerning the ability of these microorganisms to degrade different aromatic
compounds are still in their infancy.
Development of a bioprocess
It is quite expensive and difficult to dispose the briny alkaline waste, produced after
resin regeneration from the process of ion exchange. The biological removal of nitrate
and subsequent reuse of these brines can potentially provide a cost-saving alternative
to disposing of this waste product.
18
Bioplastics
Polyhydroxyalkanoates (PHA) is intracellularly accumulated bacterial storage
compounds. Due to the unique characteristic of polyhydroxybutyrate (PHB), such as
biodegradable thermopolyester that can be produced from renewable resources, and
has properties similar to those of petroleum derived plastics (Lee, 1996; Steinb¨uchel
and F¨uchtenbush, 1998). Production of PHB was very recently reported by a
moderate halophile, Halomonas boliviensis LC-1, isolated from Bolivian highlands
(Quillaguam´an et al., 2006).
2.6 Approaches for the study of haloalkaliphilic bacteria
2.6.1 Classical Approaches
The three major techniques for identification of bacteria are biochemical tests, fatty
acid profiling, and DNA sequencing. Each technique has its strong points and
weaknesses. Biochemical test-based identification systems are familiar to most
microbiologists and require little training to operate. Systems range from strip cards
for specific groups of bacteria to large plate arrays that may be automatically scanned
for changes due to pH shifts or redox reactions. The strength of identification in
enteric is generally quite good and the ease of use and cost per sample for
identification is considerably less than for other molecular approaches.
2.6.2 Molecular approaches
DNA sequencing
DNA-based technology for the identification of bacteria typically uses only the 16S
rRNA gene as the basis for identification. This technique has the advantage of being
able to identify difficult-to-cultivate strains, and is growth and operator independent.
As the 16S rRNA gene is highly conserved at the species level, speciation is
commonly quite good, but as a result, subspecies and strain level differences are not
shown.
16S rRNA sequencing
Molecular approaches based on 16S ribosomal RNA (rRNA) sequence analysis allow
direct investigation of the community structure, diversity, and phylogeny of
microorganisms in almost any environment, while quantification of the individual
types of microorganisms or entire microbial communities may be addressed by
nucleic acid hybridization techniques (Maidak et al., 1996).
19
To identify bacteria in sample material, ribosomal sequences are analyzed by
transcribing ribosomal RNA into cDNA, which can then be cloned. Alternatively,
extracted DNA can be used as a template to amplify ribosomal gene fragments with
primers for universal sequences by PCR (Polymerase Chain Reaction). The PCR
amplified fragments can be cloned as well. The result of both strategies is a clone
library, containing ribosomal sequences as inserts. By sequencing individual inserts
and comparing the obtained sequences with sequences present in databases, it is
possible to identify the phylogenetic position of the corresponding bacteria without
their cultivation. An alternative to this approach is the Denaturing Gradient Gel
Electrophoresis (DGGE) of PCR-amplified gene fragments coding for rRNA (Muyzer
et al., 1993). This technique allows the separation of partial 16S rDNA amplified
fragments of identical length but different sequence due to their different melting
behaviour in a gel system containing a gradient of denaturants. As a result, a band
pattern is obtained, which reflects the complexity of the microbial community.
FAME (Fatty acid microbial identification)
The FAME (Sherlock System) identifies microorganisms based on gas
chromatographic (GC) analysis of extracted microbial fatty acid methyl esters
(FAMEs). Microbial fatty acid profiles are unique from one species to another, and
this has allowed for the creation of very large microbial libraries. There is a large
library with over 1,500 bacterial species, along with 200 species of yeast. A
combination of features makes the system attractive for use in all fields of
microbiology. These features include, but are not limited to: accurate identifications,
large environmental libraries, the ability to perform presumptive “strain tracking” (for
finding the source of a contaminant), high throughput, and a low cost per sample for
consumables.
2.7 Extremozymes The focus on industrial enzymes that can withstand harsh conditions has greatly
increased over the past decade. Considerable efforts have been made to study
extracellular salt-tolerant enzymes of the moderately halophilic and haloalkaliphilic
bacteria, towards developing a new era in biotechnological processes. These enzymes
include hydrolases (proteases, nucleases, lipases, phosphatases) and many polymer-
degrading enzymes (amylases, cellulases and chitinases), viewed as important
20
candidates for various industries such as food, detergent, chemical, pharmaceutical,
paper and pulp or waste-treatment, (Patel et al., 2006; Thumar and Singh, 2007;
Arikan, 2008; Carvalho et al., 2008; Dodia et al., 2008a and 2008b; Ghorbel et al.,
2008; Joshi et al.,2008; Boominidhan et al., 2009; Ramesh, 2009; Sorror, 2009;
Sorror et al., 2009; Raj et al., 2010). Extremozymes have gained considerable
attention in the various industrial communities and several products based on
particularly proteases have been launched successfully in the market in past few
years.
While the biotechnological applications of enzymes from extremophiles offer great
horizons, it‟s still long way to go to capture the opportunities. Nevertheless, in view
of the great potential of biocatalysis, it is quite likely that new concepts will be
developed resulting in the application of enzymes from extremophiles. Bacteria
secrete variety of enzymes, many of them being commercially significant. Beside, the
patterns of enzyme secretion and characteristics may also suggest on the population
heterogeneity in a particular extreme habitat. Enzymes from extreme microbes have
great potential for biocatalysis and biotransformation, due to their stability under
number of extreme conditions.
Several microbes have been investigated for their ability to secrete these enzymes and
over the years, Bacillus species have emerged as the key producers of extracellular
proteases having potential applications in detergent, food, pharmaceutical, leather and
chemical industries (Patel et al., 2005; Patel et al., 2006a; Patel et al.,2006b ; Dodia et
al., 2008a and 2008b ; Sorror et al., 2009; Singh et al., 2009). During recent years,
there has been increasing emphasis on the search and development of enzymes with
capabilities to function and maintain stability under multitude of extreme conditions.
The results indicated that different proteolytic bacteria release different amounts or
activities of proteases (Dodia et al., 2006; 2008a; 2008b; Joshi et al., 2008; Purohit
and Singh, 2009; Siddhpura et al., 2010; Purohit and Singh, 2011). The proteolytic
bacterial communities may play a major role in determining the population dynamics
in context with the available nutrition.
This is mainly due to the discovery of novel enzymes from extremophilic
microorganisms. However, the ability to withstand the rigorous environments is not
21
sufficient for commercial success. In additions, number of other factors must also be
considered and investigated.
Both the discovery of new extremophilic species and the determination of genome
sequences provide a route to new enzymes, with the possibility that these will lead to
novel applications. Of equal importance, protein engineering and directed evolution
provide approaches to improve enzyme stability and modify specificity in ways that
may not exist in the natural world (Takahashi et al., 2010; Sato et al., 2010).
2.7.1 Proteases
Proteases are degradative enzymes that catalyze the hydrolysis of the proteins.
Proteases have occupied an important position with respect to their applications in
both physiological and commercial context. They have been studied in great detail not
only because they play important role in cellular metabolic processes but also for their
pivotal role in industrial community. The quantity of proteases produced on a
commercial scale worldwide is greater than any other enzymatic group of
biotechnological relevance (Horikoshi et al., 2008; Horikoshi et al., 2010).
Since proteases are physiologically necessary for all living organisms, they are
ubiquitously being found in a wide diversity of sources such as plants, animals and
microorganisms. The inability of the plants and animals proteases to meet current
demands had led to an increase interest in microbial proteases. Microbial proteases
account for 40% of the total worldwide enzymes sales (Horikoshi, 2008) and around
two third share of the commercial protease production in the world (Horikoshi, 2010).
Many scientists have reviewed microbial proteases discussing their different aspects.
The major studies are the selection of the microbes and fermentation of proteases, as
well sources of microbial proteases and their possible functional role in nature.
Different types of proteases and their commercial applications (Dodia et al., 2008a
and b, Joshi et al., 2008; Thumar et al., 2008; Manikandan et al., 2009, Toyokawa et
al., 2010) and the role of molecular biology in protease research (Ni et al., 2009;
Zhang et al., 2008a and b). The bioindustrial viewpoints of microbial alkaline
proteases from production to downstream processing, characterization and
commercial applications have also been reviewed in some recent publications. In
many cases, these enzymes retain their catalytic activity not only at elevated
22
temperatures but also in the presence of detergents or other denaturing agents (Dodia
et al., 2008; Rasch et al., 2010; Manabe et al., 2010; Vijayanand et al., 2010).
2.7.2 Microbial proteases
Classification of Proteases
According to the nomenclature committee of the international union of biochemistry
and molecular biology, proteases are classified in subgroup 4 of group 3 (hydrolases)
(IUBMB, 1992). Depending on the site of action, proteases are mainly subdivided in
to two major groups, i.e., exopeptidases (cleave the peptide bond proximal to the
amino or carboxy termini of the substrate) and endopeptidases (cleave the peptide
bonds distant from the termini of the substrate).
Based on the functional group present at the active site, proteases are further classified
into four prominent groups, i.e., serine proteases, aspartic proteases, cysteine
proteases, and metalloproteases (Hartley, 1960). Table 2.2 describes the classification
of the protease.
Metalloproteases
Metalloproteases are the most diverse of the catalytic types of proteases (Manni et al.,
2008; Sorror et al., 2009). They are characterized by the requirement of a divalent
metal ion for their activity. Entomopathogenic bacterium Photorhabdus Sp. Strain
EK1, purification and characterization was carried by Sorror et al., 2009. Based on
the specificity of their action, metalloproteases can be divided into four groups, (i)
neutral, (ii) alkaline, (iii) Myxobacter I and (iv) Myxobacter II.
Aspartic proteases
Aspartic acid proteases, commonly known as acidic proteases, are the endopeptidases
that depend on aspartic acid residues for their catalytic activity. Most aspartic
proteases show maximal activity at pH; 3 to 4 and have isoelectric points in the range
of pH 3 - 4.5.
23
Proteases EC number
Aminopeptidases 3.4.11
Dipeptidyl peptidase 3.4.14
Tripeptidyl peptidase 3.4.14
Carboxypeptidase 3.4.16–3.4.18
Serine type protease 3.4.16
Metalloprotease 3.4.17
Cysteine type protease 3.4.18
Peptidyl dipeptidase 3.4.15
Proteases EC number
Dipeptidases 3.4.13
Omega peptidases 3.4.19
Endopeptidases 3.4.21–3.4.3
Serine protease 3.4.21
Cysteine protease 3.4.22
Aspartic protease 3.4.23
Metalloprotease 3.4.24
Endopeptidases 3.4.99
Table 2.2: Classification and EC number of proteases (Hartley, 1960)
Cysteine/ Thiol proteases
The activity of all cysteine proteases depends on a catalytic site consisting of cysteine
and histidine. Generally, cysteine proteases are active only in the presence of reducing
agents such cysteine. Based on their side chain specificity, they are broadly divided
into four groups: (i) papain-like (ii) trypsin-like with preference for cleavage at the
arginine residue (iii) specific to glutamic acid (iv) others. Papain is the best-known
cysteine protease.
Serine proteases
Serine proteases are characterized by the presence of a serine group in their active
site. They are numerous and widespread among viruses, bacteria, and eukaryotes,
suggesting that they are vital to these organisms. Serine proteases are subdivided in to
four class; chymotrypsin (SA), subtilisin (SB), carboxypeptidase C (SC), and
Escherichia D-Ala–D-Ala peptidase A (SE).
Serine proteases are recognized by their irreversible inhibition by 3, 4-
dichloroisocoumarin (3,4-DCI), L-3-carboxytrans 2,3-epoxypropyl-leucylamido (4-
guanidine) butane (E.64), DFP, phenylmethylsulfonyl fluoride (PMSF) and tosyl-L-
lysine chloromethyl ketone (TLCK). Their molecular masses range between 18-35
kDa.
Serine alkaline proteases
The alkaline serine proteases are the most important group of enzymes exploited
commercially. It is produced by several bacteria, molds, yeasts, and fungi. They are
24
inhibited by DFP or a potato protease inhibitor but not by tosyl-L-phenylalanine
chloromethyl ketone (TPCK) or TLCK. They hydrolyze a peptide bond, which has
tyrosine, phenylalanine, or leucine at the carboxyl side of the splitting bond. Their
molecular masses are in the range of 15 - 30 kDa.
Subtilisin
The microbial proteases are generally secreted extracellularly for the purpose of
scavenging nutrients and are specific for aromatic or hydrophobic residues such as
tyrosine, phenylalanine and leucine. They are highly sensitive towards PMSF. There
are two major classes of subtilisin as highlighted below.
Subtilisin Carlsberg
Gutenlberg and Ottesen (1952) discovered enzyme capable of converting ovalbumin
to plakalbumin. This enzyme was known as subtilisin carlsberg. The source of this
enzyme was B. pumilis and B. licheniformnis. Subtilisin Carlsberg is widely used in
detergents. Enzyme has a wide pH range 5.0-11.0 for stability. They are most active at
pH 10.0 with molecular weight between 15-39 kDa (Gupta et al., 2005). The
Carlsberg enzyme has broader substrate specificity and does not depend on Ca2+ for
its stability.
Subtilisin Novo or bacterial protease Nagase (BPN’)
This alkaline serine protease was first purified and crystallized by Hagihara, (1958). It
is usually present as a side activity in commercial preparation of Bacillus α-amylases.
25
2.8 Purification of the alkaline protease
As described above, proteases are among the most commercially exploited enzyme. A
number of alkaline proteases have been purified and characterized. However, to reach
the current industrial demand for the enzymes with specific features, further sources
need to be explored. Generally, crude preparations of alkaline proteases are widely
used in industries such as detergent; however, purification of the proteases is
important for the better understanding of the structural and functional relationship
(Purohit and Singh, 2011). Besides, many applications would require enzyme in
homogeneity.
2.8.1 Enzyme concentration
After separating the culture from the biomass by filtration or centrifugation, the
culture supernatant is concentrated by means of ultra-filtration (Kang et al., 1999;
Smacchi et al., 1999), salting out by solid ammonium sulfate (Thumar and Singh,
2006; Reza et al., 2008; Purohit and Singh, 2011). Besides, salt precipitation solvent
extraction methods using acetone (Kumar et al., 1999; Thangam et al., 2002) and
ethanol are also effective. In order to purify the enzymes many chromatographic
techniques can be used in different combinations.
2.8.2 Affinity Chromatography
Reports on the purification of alkaline proteases by different affinity chromatographic
methods showed that an affinity adsorbent hydroxyapatite can be used to separate and
purify the proteases from a Bacillus sp. (Dodia et al., 2008a and b; Gupta et al.,
2005). However, the cost of enzyme supports and the labile nature of some affinity
legends limit the used of this technique at large scale.
2.8.3 Ion Exchange Chromatography
Alkaline proteases are generally positively charged and thus could not bind to anion
exchangers (Fujiwara et al., 1993; Kumar, 1999). However, cation exchangers can be
a rational choice and the bound molecules are eluted from the column by an
increasing salt or using pH gradient (Joshi et al., 2008). Positively charged proteins
(cationic proteins) can be separated on negatively charged carboxymethyl-cellulose
(CM-cellulose) columns. The adsorbed protein molecule is eluted by a gradient
change in the pH or ionic strength of the eluting buffer or solution.
26
2.8.4 Hydrophobic interaction chromatography
This approach exploits the variability of external hydrophobic amino acid residues on
different proteins. These hydrophobic interactions are strengthened by high salt
concentrations and higher temperatures, and are weakened by the presence of
detergents or miscible organic solvents. Hydrophobic interactions are much more
variable in behavior than ion exchangers and, thus, resolution is generally poor than
ion exchange. The most commonly used hydrophobic adsorbents are octyl- (C8 -) and
phenyl-substituted matrices.
2.8.5 Affinity precipitation
Affinity precipitation is a function of a soluble macromolecule (ligand polymer and
macroligand) that has two functions: (1) it contains an affinity ligand (polyvalent
macromolecule), and (2) it can be precipitated in many ways, i.e., by change in pH,
temperature or ionic strength. After elution of proteins the polymer can be recycled.
2.8.6 Gel filtration
In addition to the above chromatographic techniques, gel filtration is used for rapid
separation of macromolecules based on size. Recently, many new agarose based and
more rigid and cross-linked gels, such as Sephacryl, Superose, Superdex and
Toyopearl are also being used for purification purposes. They are generally used
either in the early-to-middle stage of purification or in the final stages of purification
(Joshi et al., 2008). An extreme halophilic bacterium Chromohalobacter sp. strain
TVSP101 protease was purified using this chromatography to 180 fold with 22%
yield (Vidyasagar et al., 2009). Major disadvantages of this method are the lower
capacity for loading proteins and that the desired protein gets too diluted.
2.8.7 High-Pressure Liquid Chromatography
The resolving power of all of the column techniques can be improved substantially
through high-pressure liquid chromatography (HPLC) giving high resolution as well
as rapid separation. Halophilic archaebacterium strain 172 P1 was purified by HPLC
technique (Seno, 2009).
2.8.8 Aqueous two-phase systems
This technique has been applied for purification of alkaline proteases using mixtures
of polyethylene glycol (PEG) and dextran or PEG and salts such as H3PO4, MgSO4
(Sharma et al., 2007; Sinha et al., 1996; Hotha et al., 1997).
27
2.9 Properties of Alkaline Proteases
2.9.1 Influence of temperature and thermostability on protease activity and
stability
Thermostable enzymes are of special interest for industrial applications due to their
stability under typical operation conditions; such as high temperatures and wide pH
range. The thermophilic proteases catalyze the reaction and maintain the stability at
higher temperatures. In addition, higher temperatures can accelerate the reaction rates,
increase the solubility of non-gaseous reactants and products and decrease the
incidence of microbial contamination by mesophilic organisms. Many thermophiles,
such as Bacillus stearothermophilus, Thermus aquaticus, Bacillus licheniformis,
Bacillus pumilus and Thermoanaerobacter yonseiensis, produce a variety of
thermostable extracellular proteases (Carvalho et al., 2008; Ueda et al., 2008; Wang
et al., 2008; Zhang et al., 2008a and b; Toyokawa et al., 2008). It has been known that
enzymes from thermophilic bacteria are unusually thermostable, while possessing
other properties identical with enzymes found in mesophilic bacteria (Battestein and
Macedo, 2007).
According to some reports, salt enhanced the thermostability of alkaline proteases.
Similarly, Ca2+ and Polyethylene glycol also plays a very important role in enhancing
the temperature stability of the enzymes (Ghorbel et al., 2007; Dodia et al., 2008a and
b, Manni et al., 2008). The sequencing, structure, and mutagenesis information
accumulated during the last 20 years have confirmed that hydrophobicity (Luke 2007;
Vielle et al., 2008; Berezovsky and Shakhnovich, 2008). Enzyme producing
industries use cloning and expression as one of the approaches to obtain high quantity
of desired proteins (Guo et al., 2008; Ni et al., 2009). Protein engineering could be
considered as one of the important approaches to obtain improved biocatalysts. As an
alternate to such modern but expensive and time consuming techniques, exploration
of microbial resources from extreme environments can provide much needed
biocatalytic platform (Reza et al., 2009).
While there are number of thermostable proteases reported from thermophilic
organisms, similar citations from non-thermophilic organisms are quite rare (Ramesh
et al., 2009). Search for thermostable enzymes from other groups of extremophiles
28
would be quite attractive in providing the biocatalysts with the abilities to function
under multitudes of non-conventional conditions.
2.9.2 Salt adaptation of halophilic and haloalkaliphilic proteins
The enzymes from halophiles and haloalkaliphiles can not only withstand higher
NaCl concentrations but actually its fundamental requirement for the function and
stability of those enzymes. Stability and activity are strongly depend on protein
dynamics, which is itself solvent environment dependent. Stability is needed to ensure
the appropriate geometry for ligand binding, as well as to avoid denaturation, while
flexibility is necessary to allow catalysis at a metabolically appropriate rate.
The understanding of the processes of protein folding, stability and solubility is of
fundamental importance in basic molecular biology, and also in the development of
methods in protein engineering. High salt concentration affects the conformational
stability of proteins and in general the salt conditions that favor the solubility
destabilize the folded form. Halophilic proteins have evolved specific mechanisms
that allow them to be stable and soluble at high salt concentrations (Madern et al.,
2000).
Negative charges on the halophilic proteins bind significant amounts of hydrated ions,
thus reducing their surface hydrophobicity and decreasing the tendency to aggregate
at high salt concentration. Halophilic proteins are distinguished from their non-
halophilic homologous proteins by exhibiting remarkable instability in solutions with
low salt concentrations and by maintaining soluble and active conformations in high
concentrations of salt, for example, up to 5 M NaCl (Madern et al., 2000). The
requirement of high salt concentration for the stabilization of halophilic enzymes, on
the other hand, is due to a low affinity binding of the salt to specific sites on the
surface of the folded polypeptide, thus stabilizing the active conformation of the
protein. It has been proposed that salts exert charge screening, reducing electrostatic
repulsion and enhancing hydrophobic interaction, favoring a compact folded structure
of halophilic proteins (Karan et al., 2011)
2.9.3 Effect of salt on protease activity and stability
As discussed above, proteins from halophilic and haloalkaliphilic organisms require
salt (NaCl/KCl) for their activity and stability. However, the requirement of salt was
highly varied among them. Most of the halophilic proteins active and stable up to
29
4M, optimum being at 1-2M (Gimenez et al., 2000; Thumar and Singh, 2007; Dodia
et al., 2008a and b; Joshi et al., 2008) and inactivated and denatured at concentrations
below 1M NaCl or lost their activity in the absence of salt. In general for
haloalkaliphiles; salt strongly increases enzyme activity, solubility, stability and
thermal stability. Similar kind of trend has been also reported by many other proteins
of halophilic origins. Alkaline protease from the novel haloalkaliphilic Bacillus sp.
were active up to 0.2-0.5M NaCl and the decreased with the further increase of NaCl
(Gupta et al., 2005; Patel et al., 2006b Dodia et al., 2008a; Joshi et al., 2008; Purohit
and Singh, 2011).
2.9.4 Effect of pH
Protease activity is highly pH dependent and they are generally active in the range of
pH 8-10 (Sanchez-porro et al., 2003; Hiraga et al., 2005; Gupta et al., 2005; Patel et
al., 2006b; Dodia et al., 2006, Purohit and Singh, 2011). However, there are many
examples where optimum pH was higher; pH 11, with broad range of activity from
pH 8-11(Purohit and Singh, 2011). However, the pH optimum was quite low pH 7.5
for the two novel halotolerant extracellular proteases from Bacillus subtilis strain FP-
133 (Setyorini et al., 2006) and pH 8 for alkaline protease from novel haloalkaliphilic
Bacillus sp. (Patel et al., 2006b).
2.9.5 Metal ion requirement
Alkaline proteases require divalent cations, viz. Ca2+, Mg2+ and Mn2+ or a
combination of these cations for maximum activity. It is believed that these cations
protect the enzyme against thermal denaturation and play a vital role in maintaining
the active conformation of the enzyme at high temperatures (Manni et al., 2008).
Activity of an alkaline serine protease from Bacillus subtilis increased in the presence
of Ca2+, Mg2+ and Mn2+ (Bayoudh et al., 2000; Adinarayana et al., 2003; Gessesse et
al., 2003).
2.9.6 Effect of surfactant and detergents
As discussed, the alkaline proteases are most commercial viable enzyme and widely
applied for the detergent industries, due to its unique properties. More and more
alkaline proteases were explored from varied categories of microbes and characterized
from this point of view. Some alkaline proteases were stable with sodium dodecyl
sulphate (SDS) and sodium linear alkyl benzene sulphonate (Joshi et al., 2008). The
30
alkaline protease from Bacillus clausii was highly stable with 5% SDS and 10% H2O2
(Joo et al., 2003). The extracellular alkaline proteases from the haloalkaliphilic
Bacillus sp. were stable in the presence of SDS, Triton X-100 and Tween-80 (Dodia,
2005; Patel et al., 2006b; Dodia et al., 2008b; Joshi et al., 2008).
2.9.7 Protein folding
To have biologically active protein, it must fold into proper secondary and tertiary
structures. These structures are held together by chemical interactions between the
side chains of the amino acids, including; hydrogen bonds, hydrophobic interactions,
and, at times, covalent bonds. Regardless of its function, a protein must be properly
folded to carry out its biological role. Genes from extremophiles are being cloned in
mesophilic bacteria to generate the protein in large amount. Fast and high-level
expression of heterologous proteins in bacterial hosts often results in the accumulation
of almost pure aggregates, inclusion bodies of the target protein. Hence, renaturation
of the over expressed but wrongly folded proteins have gained considerable attention.
While denaturation behaviors of alkaline proteases have been studied by few
scientists (Kamatari et al., 2003; Dodia et al., 2008b).The process of protein folding is
quite significant during cloning and over-expression, the over-expressed protein need
to be identical and correctly folded. High level expression of recombinant protein
produced in E. coli often forms aggregate, in insoluble fraction (Machida et al., 1998;
Fu et al., 2003; Singh et al., 2009; Yan et al., 2009; Purohit et al., 2008; Siddhpura et
al., 2010). In order to address the problem of inclusion bodies formation during over-
expression various in-vitro and in-vivo strategies have been attempted (Yan et al.,
2009). The association of molecular chaperone is required for the stability and
function of over-expressed protein (Singh et al., 2009).
Alkaline protease from haloalkaliphilic Bacillus sp. was sensitive to urea denaturation
and denatured within 30 min (Patel et al., 2006b). However, this finding was in
contrast with some of our own studies with other strains of haloalkaliphilic bacteria,
where the extracellular proteases were highly resistant to urea denaturation and the
resistance nature was salt dependent (Dodia, 2005; Dodia et al., 2008; Purohit and
Singh, 2011).
During the last several years, various in-vitro methods have been developed to obtain
successful renaturation of the proteins. Among these approaches, gentle removal of
31
denaturant by modified dialysis (Maeda et al., 1995), a resin bound dialysis, rapid
dilution method and folding in immobilized state by FPLC (Singh et al., 2002) have
led to attractive options for protein folding. Another useful strategy to improve the
refolding yield of proteins is to use small molecular weight additives; low
concentrations of denaturants, polyethylene glycol, polyols and sugars in the refolding
buffer (Baynes et al., 2005). The renaturation of urea-denatured alkaline protease
from haloalkaliphilic Bacillus sp., in-vitro conditions was significantly enhanced at
lower protein concentrations (Patel et al., 2006b). In contrast, the renaturation of
another alkaline protease from haloalkaliphilic Bacillus sp., was not achieved by
conventional dialysis and at even at lower protein concentrations (Dodia, 2005).
2.10 Applications of alkaline protease As reviewed above, many alkaline proteases have stability in wide range of pH and
many of them are thermophilic and halophilic in nature. These properties make them
attractive candidates in enzyme market and account for major share of the enzyme
market (Horikoshi, 2008). Major applications of alkaline protease are listed below.
2.10.1 Detergent industries
Alkaline proteases have long been of interest to the detergent industry for their ability
to aid in the removal of proteinaceous stains and to deliver unique benefits that cannot
otherwise be obtained with conventional detergent technologies. Microbial alkaline
protease especially from the Bacillus sp. dominated commercial applications with a
significant share of the detergent market (Horikoshi, 2008). The evaluation of
detergent proteases is mainly dependent upon parameters such as the pH and ionic
strength of the detergent solution, the washing temperature and pH, mechanical
handling, level of soiling and the type of textile (Gupta et al., 2002a). Reports have
been published on the compatibility of alkaline protease with detergent (Joshi et al.,
2008; Raj et al., 2010).
2.10.2 Photographic industries
Alkaline proteases find potential application in the bioprocessing of used X-ray films
for silver recovery. Used X-ray film contains approximately 1.5-2.0 % (by weight)
silver in its gelatin layers (Kumar and Takagi, 1999). The enzymatic hydrolysis of the
gelatin layers on the X-ray film allows the silver as well as the polyester film base, to
be recycled. Alkaline proteases can also be used for silver recovery.
32
2.10.3. Medical usage
Alkaline proteases are also used for developing products of medical importance.
Kudrya and Simonenko (1994) exploited the elastolytic activity of B. subtilis 316M
for the preparation of elastoterase, which was applied for the treatment of burns,
purulent wounds, carbuncles, furuncles and deep abscesses. Kim and his coworkers
(1998) have reported the use of alkaline protease from Bacillus sp. strain CK 11-4 as a
thrombolytic agent having fibrinolytic activity. Similiarly, for medical usage
Purification and characterization of a clostripain-like protease from a recombinant
Clostridium perfringens culture was studied by, Sado et al., (2010) and full-length
protease domain of murine MMP-9 was expressed in Drosophila S2 cells by Rasch et
al., (2010).
2.10.4. Food industries
Alkaline proteases have broad substrate specificity and can hydrolyze proteins from
plants, fishes, or animals to produce hydrolysates of well-defined peptide profile and
high nutritional value. The commercial alkaline protease Alcalase, was used in the
production of a less bitter hydrolysate (Adler-Nissen, 1986) and a debittered
enzymatic whey protein hydrolysate which play an important role in blood pressure
regulation, in infant food formulations and therapeutic dietary products (Neklyudov et
al., 2000).
2.10.5. Leather industry
The traditional methods of bating and dehairing of leather using sodium sulfide
treatment creates a lots of environmental pollution problem, which contributes to
100% of sulfide and over 80% of the suspended solids in tannery effluents (Malathi
and Chakraborty, 1991). Thus, the biotreament of leather using an enzymatic
approach is preferable as it offers several advantages, e.g. easy control, speed and
waste reduction, thus being ecofriendly (Boominadhan et al., 2009). Alkaline
proteases are eco-friendly enzymes that can be used as an alternate to chemical
processes of pre-tanning operations in tanning industry (Jaswal et al., 2009).
2.10.6. Enzymatic Cleansing of Contact Lenses
Several microbial enzymes from Bacillus sp., Streptomyces sp. and Aspergillus sp.
were reported for cleansing of tear films and debris of contact lens. With the view of
overcoming these drawbacks and to make the cleansing composition odorless and safe
33
i.e., not producing an allergic response or causing irritation to the eyes, bacterial
proteases are gaining importance. Several reports are available on production of
proteases from bacterial cultures and Bacillus sp. is the dominating organism (Tari et
al., 2007; Nilegaonkar et al., 2007). Therefore, it is essential to explore bacterial
protease based cleansing solutions for lens cleansing. Recently protease isolated from
Bacillus sp. 158 has potential application in contact lens cleansing (Pawar et al.,
2009).
2.10.7 Fish Sauce Fermentation
Fish sauce is a popular seasoning in Southeast Asia, as typified by nam pla in
Thailand, nuoc mam in Vietnam, and patis in the Philippines, and in Thailand, it is
produced by mixing fish, such as anchovies with salts and fermenting for 6 to 12
months at room temperature. The fermentation liquid is rich in fish soluble proteins,
peptides, and amino acids that are characterized by Umami taste (Curtis, 2009). They
are produced during proteolytic degradation by endogenous proteases in the muscles
or digestive tracts of fish, and various microorganisms exist in the fermentation broth
(Taira et al., 2007). Hence, microbial halotolerant proteases are considered to greatly
contribute to fish sauce fermentation in the food industry. Thus the halotolerant
proteinase from B. licheniformis RKK-04 is a key enzyme for fish sauce fermentation
isolated from a fermented Thai fish sauce broth showing capable to digest the myosin
heavy chain of fish protein completely can be used for fish sauce fermentation
(Toyokawa et al., 2010).
2.11 Cloning and Expression of Alkaline Proteases enzyme Among the enzymes from extremophilic organisms, relatively limited awareness
exists about enzymes from haloalkaliphilic bacteria. Extremozymes offer new
opportunities for biocatalysis and biotransformations as a result of their extreme
stability (Niehaus et al., 1999). From recent work, major approaches to extending the
range of applications of extremozymes have emerged. Both the discovery of new
extremophilic species and the determination of genome sequences provide a route to
new enzymes, with the possibility that these will lead to novel applications. Of equal
importance, molecular gene cloning and over-expression of protein, protein
engineering and directed evolution provide approaches to improve enzyme stability
and modify specificity in ways that may not exist in the natural world (Colquhouna,
34
2002). In the aspect of novel enzymology, the enzymes from extremophilic organisms
are relatively less explored (Horikoshi, 2011). All that is known/ explored about the
extremophilic enzymes is its character to work at relative high/ elevated temperatures.
The well known examples are the Taq polymerases from various thermophilic
organisms. In the past few decades, biocatalysts have been successfully exploited for
the synthesis of complex drug intermediates, specialty chemicals and even commodity
chemicals in the pharmaceutical, chemical and food industries. Recent advances in
recombinant DNA technologies, high-throughput technologies, genomics and
proteomics have fuelled the development of new catalysts and biocatalytic processes.
In particular, gene cloning and directed evolution have emerged as powerful tools for
biocatalyst engineering in order to develop enzymes with novel properties, even
without requiring knowledge of the enzyme structure and catalytic mechanisms.
The approach of directed evolution has been reviewed several times by a number of
researchers. Also very important is the cloning of these important genes which in turn
code for extremophilic enzymes and novel proteins (Matsuo et al., 2001; Yan et al.,
2009). Cloning of potential proteins, gradually leads to cloned gene is it‟s over
expressed form in stable host or suitable mesophilic host which produces desired
protein in bulk quantities.
2.12 Expression Systems
2.12.1 Bacteriophage CE6
Expression can be induced from a host strain without a source of T7 RNA polymerase
by infection with Bacteriophage CE6. CE6 is a lambda recombinant that carries the
cloned polymerase gene under control of the phage pL and pI promoters, the cI857
thermolabile repressor, and the Sam7 lysis mutations (pET Manual, Novagen,USA).
When CE6 infects an appropriate host, the newly made T7 RNA polymerase
transcribes target DNA so actively that normal phage development cannot proceed
with the transcription. Although this method is less convenient than induction of DE3
lysogens, it can be used if target gene products are too toxic to be maintained any
other way. As, in a host system, there is no presence of T7 RNA polymerase before
infection, so it should be possible to express any target DNA that can be cloned under
control of a T7 promoter.
35
2.12.2 Induction of λDE3 Lysogens with IPTG
After a target plasmid is established in a λDE3 lysogen, expression of the target DNA
is induced by the addition of IPTG to a growing culture. IPTG induction results in
uniform, concentration-dependent entry into all cells in the population. A range of
IPTG concentrations from 25 μM to 4 mM is required for target protein activity and
solubility.
2.13 Recombinant Protein Purification The methods chosen for protein purification depend on variable factors, including the
properties of the protein of interest, its location and form within the cell, the vector,
host strain background, and the intended application for the expressed protein. Culture
conditions can also have a dramatic effect on solubility and localization of a given
target protein. Many approaches can be used to purify target proteins expressed with
the pET System. One advantage of the system is that in many cases the target protein
accumulates to such high levels that it constitutes a high percentage of the total cell
protein. Therefore, it is relatively straightforward to isolate the protein in two or three
chromatographic steps by conventional methods (ion exchange, gel filtration, etc.).
Before purification or activity measurements of an expressed target protein,
preliminary analysis of expression levels, cellular localization, and solubility of the
target protein should be performed. The target protein may be found in any or all of
the following fractions: soluble or insoluble cytoplasmic fractions, periplasm, or
medium. Depending on the intended application, preferential localization to inclusion
bodies, medium, or the periplasmic space can be advantageous for rapid purification
by relatively simple procedures.
2.14 Solubilization and Refolding Proteins A variety of methods have been published describing refolding of insoluble proteins
(Kurucz et al., 1995; Burgess, 1996; Frankel et al., 1996; Rudolph et al., 1996;
Mukhopadhyay, 2006; Machida et al.,2002; Singh et al., 2002; Vincentelli et al.,
2004; Willis et al., 2005). Most protocols describe the isolation of insoluble inclusion
bodies by centrifugation followed by solubilization under denaturing conditions. The
protein is then dialyzed or diluted into a non-denaturing buffer where refolding
occurs. Because every protein possesses unique folding properties, the optimal
refolding protocol for any given protein must be empirically determined (Dodia et al.,
36
2008a and b). Optimal refolding conditions can be rapidly determined on a small scale
by a matrix approach, in which variables, such as protein concentration, reducing
agent, redox treatment, divalent cations, etc., are tested. Once the optimal
concentrations are found, they can be applied to a larger scale solubilization and
refolding of the target protein.
The protein refolding requires CAPS buffer at alkaline pH in combination with
N-lauroylsarcosine to achieve solubility of the inclusion bodies, followed by dialysis
in the presence of DTT to promote refolding. Depending on the target protein,
expression conditions, and intended application, proteins solubilized from washed
inclusion bodies may be >90% homogeneous and may not require further purification.
In current procedures, purification under fully denaturing conditions (before
refolding) is possible using fusion proteins and immobilized metal.
2.15 Purifying Target Proteins Fusion proteins solubilized from inclusion bodies using 6M urea can be purified under
partially denaturing conditions by dilution to 2M/ 1M urea prior to chromatography
on the appropriate resin. Refolded fusion proteins can be affinity purified under native
conditions using appropriate affinity tags (e.g., GST•Tag and T7•Tag).
2.16 Examples of Successful Cloning Approaches of Several Enzymes With reference to halophilic proteins particularly, maintenance of stability and
activity in high salt is major challenge (Ueda et al., 2008; Wang et al., 2008a and b).
Most typical halophilic enzymes from extremely halophilic archaea and bacteria
require high concentrations of salt for their activity and stability and are inactivated in
Escherichia coli unless refolded in the presence of salts under in-vitro conditions.
Recombinant DNA technology in conjunction with other molecular techniques is
being used to improve and evolve enzymes and opening new opportunities for the
construction of genetically modified microbial strains with the selected biocatalysis
(Caralino et al., 2008). Many newer preparations, such as Durazym, Maxapem and
Purafect, have been produced, using techniques of site-directed mutagenesis and/or
random mutagenesis. Directed evolution has also paved the way to a great variety of
subtilisin variants with better specificities and stability. Knowledge of full nucleotide
sequences of the enzyme genes has facilitated the deduction of the primary structure
of the encoded enzymes and in many cases, identification of various functional
37
regions. These sequences also serve as the basis for phylogenetic analysis of proteins
and assist in predicting the secondary structure of proteins, leading to the
understanding of structure and function relationship of the enzymes.
Several examples are available in literature where successful cloning and expression
has been analyzed, the gene encoding a ferredoxin of nucleoside diphosphate kinase
from a moderately halophilic eubacterium was cloned and protein was over expressed
in E. coli (Matsuo et al., 2001).
Some alkaline protease-encoding bacterial genes have been cloned and expressed in
new hosts, the two major organisms for cloning and over-expression being E. coli and
B. subtilis. The gene of a highly thermostable alkaline protease from an alkaliphilic
bacillus was cloned by PCR and nucleotide sequence was determined. Similarly,
around 1242 base pair DNA fragment from Bacillus halodurans isolated from
alkaline sediments coding for a potential protease was cloned and sequenced (Zang et
al., 2008a and b).
Earlier, to study gene expression in halophilic archaea, a reporter system was
analyzed by β–glycosidase enzyme. The developments related to cloning and
expression of the genes from halophilic organisms in heterologous hosts will certainly
boost the number of enzyme-driven transformations in chemical, food,
pharmaceutical and other industrial applications (Singh et al., 2009).
The gene encoding the protease Nep from haloalkaliphilic archaeon Natrialba
magadii was cloned and sequenced. The nep gene was expressed in Escherichia coli
and Haloferax volcanii resulting in production of active Nep protease. The nep-
encoded polypeptide had a molecular mass of 56.4 kDa, a pI of 3.77 and included a
121-amino acid propeptide not present in the mature Nep. The primary sequence of
Nep was closely related to serine proteases of the subtilisin family from archaea and
bacteria (50–85% similarity). The Hfx. volcanii synthesized protease was active in
high salt, high pH and high DMSO (Rosana et al., 2008). A homology search of the
N-terminal amino acid sequence of the purified PseA protease revealed an exact
match to a P. aeruginosa PST-01 protease gene, las B (Gupta et al., 2008).
38
2.17 Metagenomics Aspects Microbiology has experienced a transformation during the last 25 years that has
altered microbiologists‟ view of microorganisms and how to study them. The
realization that most microorganisms cannot be grown readily in pure culture forced
microbiologists to question their belief that the microbial world had been conquered.
We were forced to replace this belief with an acknowledgment of the extent of our
ignorance about the range of metabolic and organism diversity. In principle, any study
that addresses all the individuals of community as a single genomic pool can be seen
as an exercise in metagenomics.
Metagenomics allows to insight into specific physiological and ecological functions,
metabolic variability of an environment. The vast majority of the biosphere‟s genetic
and metabolic diversity is currently locked up within the world‟s microbial
communities, containing a staggering number of yet uncharacterized microbial. It has
become well accepted that the diversity of microorganisms represented in culture
collections is highly skewed toward those taxa that are amenable to growing under
laboratory conditions, making our discovery of microbial genes through cultivation-
dependent conventional genome sequencing equally skewed. In the late 1980s, the
direct analysis of rRNA gene sequences had also shown that the vast majority of
microorganisms present in the environment had not been captured by culture-
dependent methods. Even with the recent success of novel and high throughput
culturing strategies, we are still unable to mimic most microbial environments
sufficiently to induce growth of many environmentally relevant microbes.
Among the methods designed to gain access to the physiology and genetics of
uncultured organisms, metagenomics, the genomic analysis of population of
microorganisms, has emerged as a powerful centerpiece.
Direct isolation of genomic DNA from an environment circumvents culturing the
organisms under study, and cloning of it into a cultured organism captures it for study
and preservation. Advances have derived from sequence-based and functional
analysis in samples from water and soil and associated with eukaryotic hosts. In a
nutshell, metagenomics allows isolation of large portions of genomes which provide
access to genes for protein-coding for biochemical pathways.
39
2.18 Extraction of Metagenome (Total DNA) Metagenomic studies begin with the extraction of total DNA from a particular
environment (Purohit and Singh, 2008). Metagenomics, the analysis of DNA isolated
from environmental samples, has proved particularly useful for the knowledge of
uncultured bacteria. In the core of the metagenomic approaches, establishing better
DNA extraction techniques is of prime significance. Detecting the rare members of a
microbial community is a challenge (Voget et al., 2003). However, it is equally
important to know about a small number of microbes that play a critical role in the
community. Improved DNA extraction techniques could help ensure that a
metagenomic library adequately represents the entire community‟s genome and has
little or no contamination. Extending the analyses beyond the DNA sequence to study
the proteins and metabolites (the products of cellular processes) generated by a
community is critical for understanding how the microbial community operates and
interacts within the habitat.
Among the key factors responsible for the success of metagenomics, the isolation of
quality environmental DNA in appreciable amount from a given habitat holds
significance (Raes, 2007). The isolation of total DNA appears to be of prime
importance and a bottleneck step in metagenomic studies, as the extracted DNA
should be of high quality to pursue molecular biological applications (Voget et al.,
2003; Desai and Madamwar, 2007; Gilbert, 2010). Standardization of total DNA
extraction technique is desirable as the composition of different habitats varies with
respect to their matrix, organic and inorganic compounds and biotic factors (William,
1998). Improved DNA extraction techniques should also ensure a metagenomic
library adequately representing the entire community‟s genome without inhibitory
substances (Santosa, 2001).
Mostly metagenomics projects currently focus on the microbes found in the sample
environment that have smaller amounts of DNA, such as bacteria and other microbes
which can live in extreme environments (Risenfeild et al., 2004; Sharma et al., 2007).
During the last 10 years, number of protocols for DNA extraction from environmental
sample have been reported (Kauffman et al., 2004; Handelsmann et al., 2004) and
commercial soil DNA extraction kits (Mo Bio, Maidegen, USA) are also available.
These kits and most of the published methods have improved the original direct DNA
40
extraction procedures mainly in terms of DNA yield and quality. The protocols for
isolating total DNA from environmental sample could be broadly classified as direct
and indirect methods. The variability in the outcome among the methods is viewed
with respect to the degree of shearing, purity and quantity of the extracted DNA
(Desai and Madamwar, 2007).
2.19 Approaches and Techniques The DNA is extracted from a sample followed by the construction of a genomic
library containing pieces of the genomes of all the microbes. This metagenome can
further be applicable by two approaches:
2.19.1 Sequence-Based Metagenomics
Entire information‟s of genetic sequences could be determined by sequence-based
approaches, which reflect DNA profile and addresses population heterogeneity in
particular (Tringe et al., 2005; Glockner et al., 2010). Phylogenetic and large-insert
metagenomic approaches, provide access to genetic information contained within
microbial populations only known to us in the form of specific phylogenetic marker
gene sequences (Rondon et al., 2000).
2.19.2 Function-Based Metagenomics
It explores and aims at the specific products from the microbes in a community. In
function-based metagenomics, researchers screen metagenomic libraries for various
functions, such as biocatalysts, vitamins or antibiotic production (Raes et al., 2007).
Through this approach, scientists can search and identify the functions that are largely
unknown. Similarly, recent technological advances enable to directly extract and
identify novel proteins and metabolites (the products of cellular processes) from a
microbial community (Jeffrey, 2010). Moreover, metagenomics analyses microbial
communities as systems that have functional properties beyond individual genes or
individual microbes (or even single-taxon populations). Metabolic cascades, for
example, can be distributed over different members of multi-taxa communities,
(Rajenderan and Gunashankar, 2008). Product or activity-driven metagenomic studies
are often approached with a more applied perspective in view, exploring useful
properties aencoded within the metagenome (Raes et al., 2007).
41
2.19.3 Techniques
A wide range of techniques has been employed to gain access to metagenomes,
among them shotgun analysis of community genomes is a rather simple exercise
(Wooley, 2010). Metagenomics, however, has more to offer than merely providing
lots of interesting DNA sequence data. It takes a non-traditional focus on the genomic
resources of a dynamic microbial community, rather than on individual strains of
microbes or individual genes and their functions. Community genomics perspectives
aim to explore how horizontal transfer allows otherwise distantly related organisms.
Therefore, metagenomic analyses of microbial communities focus on systems having
functional properties beyond the individual genes or individual microbes (Jeroen et
al., 2007). Through automated high-throughput methods, it‟s possible to recover and
sequence as many clones as necessary (Handelsman, 2004; Handelsman, 2005).
2.20 Metagenome Screening An enormous variety of different biocatalysts or other functional products can be
theoretically obtained using DNA extracted from a given environmental sample. An
example for the impressive diversity of metagenome-encoded enzymes was provided
by Diversa Corporation (San Diego). By fragmenting total DNA from an alkaline
marine sample, cloning it into an expression vector, and screening for esterase/lipase
activity in an easily cultivable host strain, 120 new enzymes were discovered, falling
into 21 protein families (Miller, 2008). During the past five years, cloning of genes
from the metagenome has become the most popular tool for cultivation-independent
enzyme discovery, leading to the recovery of a range of new biocatalysts by academic
and commercial institutions (Kennedy, 2008). While in almost all studies, E. coli was
used as expression host, there is an example of cloning in a broad host range vector
and expression in Streptomyces lividans (Tringe et al., 2003; Hugenholt, 2008).
Vector systems used for the cloning of environmental DNA range from small-insert
cloning vectors such as plasmids or phage vectors (up to 15 kb inserts) to bacterial
artificial chromosomes (BACs) that can harbor as much as 100kb fragments. While
BAC vectors are usually applied when activities are targeted on the expression of
large gene clusters (e.g. metabolite formation), small-insert libraries are usually
prepared for the screening of single genes or small operons (Gilbert, 2010).
42
However, smaller cloned fragments necessitate larger gene banks required for a
comprehensive and comparable coverage of the genetic information, which ultimately
leads to more laborious screening procedures. For example, one amylase-expressing
clone could be isolated per 450 clones screened using a BAC vector (Gilbert, 2010).
2.21. Molecular Tools Used In Metagenomics Molecular tools developed during the past 20 years by molecular biologists have
facilitated the extraction, cloning, screening and sequencing of genes and genomes.
Many of these approaches have also allowed microbial ecologists to access and study
the microbial diversity in its totality, regardless of our ability to culture organisms.
This has opened the doors of unexplored domains of non-cultivable microbes,
allowing unprecedented access to the world of natural products encoded by
community genomes (Gilbert, 2010; Glockner et al., 2010).
The advent of culture-independent techniques has transformed the field of
microbiology and microbial ecology in particular. PCR-based techniques allow the
classification of microorganisms based on particular genetic markers and the profiling
of complex microbial communities on the basis of sequence diversity (Bach, 2001).
The most commonly used marker for profiling bacterial communities is the 16S rRNA
gene. The size of this gene (1.5kb) is large enough for reliable phylogenetic
information. Hierarchal domain specific primers are designed, which can target
broadly or with high specificity. Different functional genes can also be used in order
to target specific groups of bacteria. Domain specific primers are designed on the
basis of conserved residues of sequences.
One technique that is now routinely used is denaturing gradient gel electrophoresis
(DGGE) and the analogous temperature gradient gel electrophoresis (TGGE)
(William, 1998). It is a genetic fingerprinting technique that is used to separate
individual sequences from a complex mixture. In principle, this means that DNA
fragments of the same length are separated on the basis of differing sequences, even
by a single base (Ercolini, 2004).
We have attempted to find 16S rRNA sequence/s of unculturables from saline soils of
Coastal Gujarat, India by the DGGE protocols perfected in our laboratory. Towards
this end, we aim to focus on identifying signature sequences of
43
halophiles/haloalkaliphiles; based on shotgun sequencing approaches and designing
specific primers for halophiles/haloalkaliphilies (Siddhpura et al., 2010).
The results indicated that different proteolytic bacteria release different amounts or
activities of proteases (Dodia et al., 2008a and b; Joshi et al., 2008; Purohit and
Singh, 2008; Siddhpura et al., 2010). The proteolytic bacterial communities may play
a major role in determining the population dynamics in context with the available
nutrition. In the overall scenario of the secretion of extracellular proteins by the
microbes in their surrounding, the recently published idea on the economic synthesis
of the proteins/enzymes by the microbes assumes significance (Smith and Chapman,
2010). According to this proposed theme supported by the analysis of the data, the
organisms spend minimum energy on the synthesis of extracellular proteins.
The widespread use of molecular techniques in studying microbial communities has
greatly enhanced our understanding of microbial diversity and function in the natural
environment and contributed to an explosion of novel commercially viable enzymes.
Technological advances in sequencing and cloning methodologies as well as
improvements in annotation and comparative sequence analysis, generate information
for microbial ecologists. e. g. Natural products isolated from sponges are an important
source of new biologically active compounds (Raes et al., 2007; Glockner, 2010).
Metagenome of marine microbial communities have been shown to contain genes and
gene clusters typical for the biosynthesis of biologically active natural products
(Mitchell et al., 2000; Kauffman et al., 2004; Keneddy et al., 2007; Keneddy et al.,
2008).
Combining metagenomic approaches with heterologous expression holds much
promise for the sustainable exploitation of the chemical diversity present in the
marine microbial community. A PCR-based method targeting a 59-base
recombination site highlighted on the diverse bacterial taxonomic groups and that
flanks gene cassettes are associated with integrons. The recovered gene cassettes
contained complete open reading frames, most of which did not show homology to
any database entry, and which potentially encode enzymes of biotechnological
interest (Glockner, 2010). PCR-based cloning methods are also being employed to
recover novel enzymes. In most cases, degenerate primers are used, hybridizing with
44
conserved regions that preferentially are located close to the extremities of the target
genes (Liles, 2008; Ni et al., 2009).
We relied on similar approach for our own studies, where degenerate primers were
designed for alkaline proteases by using bioinformatics tools. Designed sets of
primers were specifically based on halophilic/ haloalkaliphilc alkaline proteases
available from marine environment. We identified several such sequences and
successfully cloned, over-expressed and characterized them in E. coli host system, our
unpublished data (Singh et al., 2010a). The characteristic features of native and
recombinant enzymes were studied, interestingly, we noticed that recombinant clones
have maintained their nascent properties, specific activity of enzyme was found to be
around five times higher activity then purified native enzymes.
However, this approach does not sound equally well in capturing functional attributes
of sequences that share some sequence identity with already identified sequences
(Craig and Venter 2004; Craig et al., 2010). Besides, expression-based identification
of biocatalysts, large-scale shotgun sequencing projects and in silico identification of
enzyme-coding regions are currently carried out, for instance by The Monterey Bay
Coastal Ocean Microbial Observatory on marine picoplancton (Nakumura et al.,
2009).
2.22 Gleaning Information out of the data: Bioinformatics and data
analysis
Metagenomic approaches have the potential to generate enormously huge body of
sequences. However, the knowledge gleaned from such studies is not proportional to
the sequencing effort involved, and it depends on the bioinformatics interpretation of
the informations.
2.23 Metagenomics: Commercial successes in Biotechnology As the excitement about genetic access to the boundless realms of microbial diversity
slowly gives way to the reality of tapping into this diversity, the usual challenge of
heterologus gene expression needs to be addressed to turn metagenomic technologies
into commercial successes, particularly in applications for which bulk enzyme or
product quantities have to be produced at competitive prices.
45
Given that the majority of natural products are of microbial origin, and that the vast
majority of microbial genomes have yet to be explored, it follows that microbial
metagenomes contain a great economic potential. Due to their huge diversity and
history as sources of commercially valuable molecules with agricultural, chemical,
industrial, and pharmaceutical applications, soil environments have been the most
common subjects of metagenome interrogation in this way. Functional screening
methods potentially provide a means to discover new variants of functions of interest.
Metagenomics, together with in vitro evolution and high-throughput screening
technologies, provides industry with an unprecedented chance to bring biomolecules
into industrial application.
The goals of researchers venturing into the microbial metagenome vary from directed
product discovery to total community characterization, and the phylogenetic
complexity of the environments studied can range over orders of magnitude.
Metagenomics has redefined the concept of a genome, and accelerated the rate of
gene discovery. The potential for application of metagenomics to biotechnology
seems endless.
A high-throughput pipeline has been constructed to provide high-performance
computing to all researchers interested in using metagenomics (Morgan et al., 2010).
The pipeline produces automated functional assignments of sequences in the
metagenome by comparing both protein and nucleotide databases. Phylogenetic and
functional summaries of the metagenomes are generated, and tools for comparative
metagenomics are incorporated into the standard views.
2.24 Enzymes from solvent-tolerant microbes: A way towards non-
aqueous enzymology Biocatalysis under a water-restricted medium has undergone tremendous development
during the last decade and numerous reactions have been introduced and optimized
for synthetic applications (Carrea and Riva, 2000). By using enzymes in a solvent
medium, it is now becoming possible to synthesize novel compounds which are
difficult to synthesize conventionally and to obtain the biologically active enantiomer
for which the racemic solution is either very complex or difficult (Karadzic et al.,
2006; Klibanov, 2001). However, this necessitates efficient catalysis, and the stability
46
of the enzymes in organic solvents is a prerequisite. Enzymes, in general, get
denatured or give very low rates of reaction in solvent media because of the
unfolding, structural disfunctioning, and stripping of the essential water layer from the
enzyme molecule (Khare et al., 2000a, b; Klibanov, 2001; Vulfson et al., 2001).
The screening of solvent-stable enzymes in natural sources has come to be accepted
as a better and more promising approach than directed evolution, chemical
modification and protein engineering. Several solvent-tolerant microbial strains, some
of which produce solvent-stable enzymes, have been discovered. However, less
attention has been paid to their enzymes, which logically should be stable and
efficient catalysts for functioning in solvent media and which could be nature‟s very
own toolbox for solvent-stable enzymes for applications in non-aqueous systems
(Fang et al., 2006; Ogino and Ishikawa, 2001; Takeda et al., 2006).
2.24.1 Microbial adaptation to solvents
It is necessary to look into the cellular toxicity of solvents for an understanding of the
generic adaptation mechanisms in this class of microbes. Solvent accumulation leads
to specific permeabilization of the cell membranes, leading to the leakage of ATP,
potassium and other ions, RNA, phospholipids, and proteins (Heipieper et al., 1991;
Ramos et al., 1997; Woldringh, 1973). Further, the membrane fluidity is also affected
by organic solvents (Sikkema et al., 1994). Several studies have shown that a
correlation exists between the solvent toxicity and its hydrophobicity, i.e. the log Pow
value (the partition coefficient of a given solvent in an equimolar mixture of octanol
and water) (Inoue and Horikoshi, 1991). Solvents in a log P range of 1–4 are more
water soluble and partition well to the membrane. Thus these solvents are much more
toxic in comparison to lipophilic solvents (log P > 4), which do not reach a high
membrane concentration owing to their low water solubility (Sardessai and Bhosle,
2004). Solvent-tolerant bacteria circumvent the solvents‟ toxic effects by virtue of
various adaptations which have been excellently studied and reviewed (Heipieper et
al., 2007). Hence these are only briefly discussed here. Rigidification of the cell
membrane (a) a shift in the ratio of saturated to unsaturated fatty acids in cell
membrane (Mohammad et al., 2006). (b) Isomerization of the naturally synthesized
cis-isomer of an unsaturated fatty acid to the trans-isomer by an energy-independent,
periplasmic isomerase enzyme (Mohammad et al., 2006; Nielsen et al., 2005) (c)
Change in fatty acid composition; the phospholipids head group‟s composition also
47
alters during solvent adaptation (Nielsen et al., 2005). (d) Changes in the composition
of lipopolysaccharides (LPS), lipid–protein ratios, and outer membrane proteins
(Pinkart et al., 1996; Ramos et al., 1997). These adaptations change the fluidity of the
membrane and in this way suppress the effects of the solvents on membrane stability,
biotransformation and degradation of toxic organic solvents.
2.24.2 Solvent-efflux pumps
Several solvent-efflux pumps involved in solvent tolerance in various bacteria have
been described in the last few years and most of them belong to the RND (resistance/
nodulation/ cell division) family. Only a few efflux pumps for organic solvents,
namely tolC, mar, rob, soxS and acrAB have been identified in Pseudomonas sp.
(Kieboom et al., 1998; Li et al., 1998; Ramos et al., 1998) and E. coli (Asako et al.,
1997; Kobayashi et al., 2001). Increase in cell size of P. putida and Enterobacter sp.
adapt to toxic organic compounds by increasing their cell size. A bigger size reduces
the relative surface and consequently reduces the attachable surface for toxic organic
compounds. It is obvious that the functioning of solvent-efflux pumps is more
effective, if the overall membrane surface is reduced. This leads to a reduction in the
area that allows diffusion and partitioning of solvents into the membrane where they
are recognized and excluded by the efflux-pump proteins (Neumann et al., 2005).
2.24.3 Enzymes from solvent-tolerant microbes
Solvent-tolerant microbes have been less studied from the perspective of non-aqueous
enzymology. It is now becoming obvious that their enzymes display striking novel
properties and that they attain a higher level of catalytic activity of their enzymes in
organic solvents (Ogino and Ishikawa, 2001). Some of industrially important enzymes
such as lipases, proteases, and amylases from solvent-tolerant microbes (Doukyu et
al., 2003; 2007; Geok et al., 2003; Ghorbel et al., 2003; Gupta et al., 2005; Karadzic
et al., 2004). Surprisingly, halophiles have also been noticed to exhibit the properties
of solvent-tolerant enzymes. Examples are amylase from extremely halophilic
archaea, Haloarcula sp. strain S-1 (Fukushima et al., 2005), and protease from the
moderately halophilic bacterium Saliniovibrio sp. strain AF-2004 (Heidari et al.,
2007). The halotolerant actinomycetes derived alkaline proteases have also been
reported having growth, activity and stability in the presence of solvents (Thumar and
Singh, 2007a). This opens up the possibility of the availability of enzymes that have
combinations of tolerances of extreme conditions, such as to salts and solvents, which
48
could be beneficial for industrial processes involving the use of high salt
concentrations and hydrophobic organic solvents. The enzymes produced by the
solvent-tolerant microbes that have been studied are mainly those wherein the reverse
reactions are of industrial significance or the substrates are sparingly soluble in water
but soluble in solvents.
The general features observed in enzymes from such sources include (i) better
stability in hydrophobic solvents especially alkanes, (ii) monomeric proteins of
molecular weight ranging from 20 kDa to 80 kDa, (iii) hydrophobicsurfaces and the
marked presence of disulphide bonds, (iv) hydrophobic interaction chromatography
(HIC) or ion exchangers tend to work more selectively for their purification over
conventional protocols, (v) most of the reported solvent stable proteases belong to the
group of metalloproteases, and (vi) these have been mainly resourced from
pseudomonas and few from Bacillus sp. (Ogino et al., 1994 and1995; Geok et al.,
2003; Ghorbel et al., 2003; Gupta et al., 2005). It is interesting to note that their
characteristics vary even for enzymes produced from one strain of Pseudomonas to
another. The common attribute, however, is their stability in alkanes. In general, most
of the reported enzymes from solvent-stable strains are stable-to-long-chain aliphatic
hydrocarbons, benzene, toluene, and alcohols.
2.24.4 Solvent stable proteases
Proteases stable in organic solvents are desirable for effective peptide synthesis.
Protease catalyzed synthesis has several advantages over chemical catalysis, e.g.
regio- and stereo-selectivity, absence of racemization, lack of requirement of side
chain protection and milder non-hazardous reaction conditions (Gill et al., 1996;
Klibanov, 1986; Rahman et al., 2007). Several peptides, such as the analgesic
dipeptide kyotorphin (Tyr-ArgS) (Jönsson et al., 1996; Sareen et al., 2004a and b) and
aspartame (Eichhorn et al., 1997), have been synthesized in aqueous or nonaqueous
media using proteases. Subtilisin, thermolysin, and other proteolytic enzymes have
been used in the presence of organic solvents as catalysts for peptide synthesis (Isowa
and Ichikawa, 1998; Oka and Morihara, 1978; 1980; Pauchon et al., 1993). But the
rate of peptide synthesis is low in the presence of organic solvents because of
denaturation or inactivation of the enzymes (Ogino et al., 1999a; Vulfson et al.,
2001).
49
Recently it has also been shown that the amino acid residues located at the surface of
the protein molecule played an important role in exhibiting the organic solvent
tolerant nature of the protease (Gupta et al., 2007; Ogino et al., 2007). In general,
these protease producers have been isolated from soil except in a few cases from
fishing-industry wastewater (Ghorbel et al., 2003) and cutting oil used in industrial
metal-working processes (Karadzic et al., 2004). Most of the reported solvent-stable
proteases are mainly from Pseudomonas sp. In a few recent cases, Bacillus spp. have
also been found to be endowed with solvent-stable proteases (Ghorbel et al., 2003;
Sareen et al., 2004a and b). Both Pseudomonas and Bacillus proteases exhibit better
stability towards hydrophobic solvents, especially alkanes (Ogino et al., 1995; Gupta
and Khare, 2006a and b, Rahman et al., 2006 and 2007). However, in some cases,
stability towards alcohols has also been reported (Ghorbel et al., 2003; Karadzic et
al., 2004; Sana et al., 2006). These proteases are mainly being purified by using anion
exchange and/or hydrophobic-interaction chromatography.
2.24.5 Biotechnological applications of solvent-tolerant microbes and their
enzymes in bioremediation/ biotransformation
Apart from their enzymes, solvent tolerant bacteria can also be of vital importance in
bioremediation/ biotransformation. The microbial transformation of hydrocarbons,
soil remediation and waste-stream purification necessitates the survival and growth of
microbes in toxic effluent (Kieboom et al., 1998). The persistence of many solvents in
contaminated sites is indicative of the lack of natural systems that can efficiently
degrade these compounds. Numerous problems appear in the application of biological
systems to solvent treatment due to the toxic effects of organic solvents on the
bacteria (Mohammad et al., 2006). Organic solvent tolerant bacteria can serve as an
invaluable tool for such processes. The enzymes have been in use for bioconversions
in two-phase systems for a very long time. The exploitation of solvent-tolerant
bacteria for biotransformation in two phase fermentation systems has been recently
reviewed by Heipieper et al., 2007.
2.24.6 Conclusions and future perspectives of solvent tolerant bacteria and
enzymes
Biocatalysis in low water/solvent media has undergone tremendous development
during the last decade and numerous new reactions have been introduced for synthetic
applications. The stability and efficiency of enzymes in solvents, however, remains a
50
necessary prerequisite for such applications. The research efforts have been entwined
with the development of new strategies to obtain solvent-stable enzymes. In this
context, enzymes from solvent-tolerant microbes, with the requisite natural catabolic
potential, seem to have a major advantage. Solvent-tolerant bacteria also show great
promise for the future development of cost effective solvent bioconversion or
remediation processes. Their ability to tolerate and mineralize high concentrations of
toxic solvents widens the opportunities for biological treatments into areas
traditionally served by chemical and physical techniques, which do not involve a
pretreatment step for effluents so as to render them suitable for „normal‟ biological
conditions. These possibilities represent a future avenue of research for both
microbiologists and enzymologists.
CHAPTER Launch Internet Explorer Brow ser.lnk
CHAPTER
HALOALKALIPHILIC
BACTERIA
PHYLOGENY, DIVERSITY, ENZYMATIC
POTENTIAL
3
51
3.1 INTRODUCTION
Microbial diversity includes, the diversity of bacteria, protozoan, fungi, and
unicellular algae, constitutes the most extraordinary reservoir of life in the biosphere.
In a very particular term; diversity is composed of two elements: richness and
evenness, so that the highest diversity occurs in communities with many different
species present (richness) in relatively equal abundance (evenness) (Huston, 1994).
The richness and evenness of bacterial communities reflect selective pressure that
shape diversity within communities.
For much of the last century, microbiologists have been aware that we know the
nature and identity of only a tiny fraction of the inhabitants of the microscopic
landscape. While most people are very familiar with the diversity of life in the plant
and animal kingdoms, few actually realize the vast amounts of variability present in
the bacterial populations. Interestingly; microorganisms represent the richest
repertoire of molecular and chemical diversity in nature as they underlie basic
ecosystem processes. The current inventory of the world‟s biodiversity is very
incomplete and that of microorganisms is especially deficient. Scientists have
identified about 1.7 million living species on our planet. Studies indicate that the
5,000 identified species of prokaryotes represent only 1-10% of all bacterial species;
therefore we have only a small idea of our true microbial diversity (Bowen et al.,
2011).
In particular, extremophiles are organisms able to live in unusual habitats, can
potentially serve in a verity of industrial applications (Burg, 2003; Horikoshi, 2008;
Horikoshi, 2011). As a result of adaptation to extreme environments, extremophiles
have evolved unique properties, which can provide significant biotechnological and
commercial opportunities. Major categories of extremophiles include halophiles,
alkaliphiles, acidophiles, thermopiles and haloalkaliphiles. The groups of bacteria able
to grow under alkaline conditions in the presence of salt are referred as
haloalkaliphiles. They possess special adaptation mechanisms for their under salinity
and alkaline pH. These properties of dual extremity of halophiles and alkaliphiles
make them interesting from both, fundamental research and biotechnological points of
view (Dodia et al., 2008a and b; Purohit and Singh, 2011; Romano et al., 2011).
52
The microbial diversity has focused renewed emphasis and in this regard
extremophiles hold great significance among microbial world. Limited studies have
identified a huge diversity of extremophiles. Large number of haloalkaliphilic
bacterial strains depicted wide diversity, as reflected through microbiological
examinations, biochemical characteristics and molecular approaches (Dodia et al.,
2008; Joshi et al., 2008; Purohit and Singh, 2011; Siddhpura et al., 2010; Singh et al.,
2010 a and b).
53
3.2 MATERIALS AND METHODS
3.2.1 Sample Collection For the isolation of the halophilic and haloalkaliphilic bacteria, the soil samples were
collected from the different sites along the coast of Gujarat; particularly from saline
soil across the coastline and artificial salt pane of Okha Madhi. The samples were
collected in sterile plastic bags; the pH and temperature of all the samples were
measured manually at the time of the sample collection, and processed within four
days after the sample collection. From the total collected samples; two samples
(O.M.6.2 and O.M.6.5) were selected for further studies. All the collected samples
were stored at 4°C.
3.2.2 Physical and chemical analysis of the samples Before proceeding for the isolation, the samples were subjected to the physical and
chemical analysis, such as pH, temperature, conductivity, total dissolved solids
(TDS), turbidity, alkalinity, total hardness and Mg+2 concentration as per the method
BIS (Bureau of Indian Standards).
3.2.3 Enrichment and isolation For the isolation of the halophilic and haloalkaliphilic bacteria, 1.0 gm of the soil
sample was inoculated into the 100ml of the enrichment medium. The bacteria were
isolated by using enrichment culture techniques in Complex Medium Broth (CMB)
consisting, (g/liter): Glucose, 10; Peptone, 5; Yeast extract, 5; KH2PO4, 5; with
varying concentration of NaCl (10% and 30%, w/v) at different pH (8 and 10). The
pH of the medium was adjusted by adding separately autoclaved Na2CO3 (20%, w/v).
After inoculation, flasks were incubated on environmental shaker at 37°C with regular
monitoring on the turbidity of the enrichment media. After 48-72h of growth, a loop
full culture was streaked on the CMB agar (agar, 3%, w/v) plate and incubated at
37°C. After 48h of the incubation, on the basis of colony characteristics, various
isolated colonies were selected and pure cultures were obtained by subsequent
streaking on the CMB agar plate (Fig.3.2.1).
54
Fig. 3.2.1: A schematic representation of enrichment and isolation procedure
(Joshi, 2006).
3.2.4 Maintenance and preservation The pure cultures were preserved on the CMB agar media (10% w/v NaCl; and pH 8-
10) and stored at 4°C. After screening for the extracellular enzymes, the protease
producers were preserved on gelatin agar medium respectively. The cultures were
subsequently transferred on fresh CMB agar at 3 months intervals.
3.2.5 Characterization of the organisms
3.2.5.1 Colony characteristics
For the primary characterization, the pure culture of all the isolated bacteria were
streaked on the CMB agar plate with corresponding enrichment conditions of the
NaCl (10 and 30%, w/v) and pH (8 and 10) and their colony characteristics were
observed.
3.2.5.2 Cell morphology and Gram reaction
For the differentiation on the basis of the cell morphology and cell arrangement,
individual bacterium was studied for the Gram reaction, in activated culture in CMB
at the corresponding enrichment conditions.
55
3.2.5.3 Biochemical characterization
For further differentiation, the isolates were studied for biochemical and metabolic
activities. The biochemical tests included production of catalase, oxidase, H2S,
ammonia, indole, hydrolysis of urea, reduction of nitrate and litmus; fermentation of
the sugars such as glucose, fructose, sucrose, maltose, lactose and xylose. All the
biochemical media and their test reagents were prepared as mentioned by Cappuccino
and Sherman (Cappuccino and Sherman, 2004). Because of the halophilic nature of
the organisms, all the biochemical media were supplemented with 5% (w/v) NaCl.
The individual isolate was inoculated to the respective biochemical medium and
incubated at 37°C for 24-48h and results were subsequently observed.
3.2.6 16S rRNA amplification and nucleotide sequencing For potential strains; having enzymatic potential to secrete proteases; genomic DNA
was isolated from the pure culture of O.M.A18, O.M.E12, O.M.C28 and O.M.C14. The
~1.5 kb rDNA fragment was amplified through high-fidelity PCR polymerase by
using consensus primers. The PCR product was bi-directionally sequenced by using
the forward; 5‟-AGAGTTTGATCATGGCTCAG-3‟ and reverse primer;
5‟-TACGGTTACCTTGTTACGACTT-3‟.
3.2.7 Phylogenentic analysis of the 16 S rRNA sequences The sequence of a selection of published 16S rRNA genes were obtained in aligned
form from the Ribosomal Database Project (RDP) (http;//rdpwww.life.uiuc.edu,
Maidak et al., 1996) using the „subalign‟ service. The Rt3 sequence was added to this
alignment and manually aligned in accordance with RDP “align sequence” report,
using the alignment editor AE2 (Larsen Likelihood (ML). The phylogeny of the
aligned sequence was obtained using the RDP „suggest tree‟ service from fast DNAml
program (version 1.08).
3.2.8 FAME analysis (Fatty acid based microbial identification
software) The fatty acids are extracted from haloalkaliphiles by a procedure which consists of
saponification in dilute sodium hydroxide/methanol solution followed by
derivatization with dilute hydrochloric acid/methanol solution to give the respective
methyl esters (FAMEs). The FAMEs are then extracted from the aqueous phase by
56
the use of an organic solvent and the resulting extract is analyzed by GC. Fatty acids
were analyzed by Sherlock software which, automates all analytical operations and
uses a sophisticated pattern recognition algorithm to match the unknown FAME
profile to the stored library entries for identifications.
3.2.9 Detection of antibiotic resistance and sensitivity
For the detection of antibiotic resistance and sensitive nature of the isolates, Bauer-
Kirby test was performed by using the octadiscs (Hi Media Life science, India)
specific for the Gram negative and Gram positive bacteria. The isolates were tested
against different antibiotic octadiscs on the basis of its Gram‟s reactions. The
abbreviations of antibiotics used are: Ampicillin (A); Carbenicillin (Cb);
Cephotaxime (Ce); Cholarmphenicol (C); Co-Trimazine (Cm); Gentamicin (G);
Norfloxacin (Nx); Oxacillin (Ox); Cephaloridine (Cr); Kanamycin (K); Lincomycin
(L); Methicillin (M); Oleandomycin (Ol); Penicillin-G (P); Tobramycin (Tb);
Tetracycline (T); Co-Trimaxazole (Co); Cloxacillin (Cx); Cephradin (Cv);
Erythomycin (E); Cefuroxime (Cu); Ceprofloxacin (Cf); Colistin (Cl); Nitrofurantoin
(Nf); Steptomycin (S); Cephalexin (Cp); Nalidixic Acid (Na); Furazolidone (Fr);
Oxytetracyclline (O).
The melted CMB agar medium (10%, NaCl w/v; pH 9) was inoculated with 5%
inoculum and poured in sterile plate followed by the addition of antibiotics
impregnated octadisc onto the agar surface. The plates were incubated at 37°C for 24-
48h. Antibiotic sensitivity was detected by measuring zone of the clearance (zone of
inhibition) around the individual antibiotic disc while growth in the vicinity or
surrounding the disc indicates the resistance of particular isolate against that
antibiotic.
3.2.10 Screening for extracellular alkaline protease enzyme secretion Actively growing cultures of different isolates were prepared in the Complete
Medium Broth (CMB) at its optimum NaCl (0-25%); pH (8-10) and used as inoculum
(A540>1.0) for the primary screening of alkaline protease. The cultures were
inoculated in the form of regular spots on gelatin agar medium containing (g/liter);
Gelatin, 30; Peptone, 10; NaCl, 100; pH, 8-10 and Agar, 30. The pH of the medium
adjusted to 8-10 by adding separately autoclaved 20% Na2CO3 (w/v). The plates were
incubated for 48-72h at 37°C and Frazier's reagent (g/liter: HgCl2, 150g; concentrated
57
HCl, 200ml) was poured on plate for the gelatin liquefaction. The clear zone
surrounding the colony indicated the secretion of extracellular protease. The colony
diameter and zone of clearance was measured. The ratio was calculated to assess the
relative enzyme secretion as a function of colony size.
3.2.11. PCR Amplification of alkaline protease gene The DNA preparations described above were used as template to amplify region
coding alkaline protease. The four pair of primers used for amplification profile was
synthetic degenerate oligonucleotides based on the previously known sequence of
alkaline protease gene from Bacillus halodurans, Bacillus cerus, Oceanobacillus
iheyensis serine proteases and haloalkaliphilic Bacillus sp.
Primer designation Sequence
SPS-1F 5‟-gga tcc ttg aaa aac aaa atc att-3‟
SPS-1R 5‟-gtc gac tta aga agc ttt att taa c-3‟
SPS-3F 5‟-gga tcc ttg aaa aca aaa tca ttg-3‟
SPS-3R 5‟-gtc gac tta aga agc ttt att taa c-3‟
SPS-4F 5‟-gga tcc cta ctt gat gta ga-3‟
SPS-4R 5‟-gtc gac atg cat atc gga aaa c-3‟
SPS-5F 5‟-gga tcc gcc gcc gag gac gac-3‟
SPS-5R 5‟-gtc gac atg gga tat tat gac-3‟
SPS-6F 5‟-gga tcc gcc gcc gag gac gac-3‟
SPS-6R 5‟-gtc gac gga cca gac cgt cg-3‟
SPS-7F 5‟-cat atg ccg ccg agg agg ac-3‟
SPS-7R 5‟-gtc gac ggc ctt cgt gtg g-3‟
Table 3.2.1: Primer sequences used for amplification procedures
The other two primer pairs were designed on the basis of conserved sequences of
Haloalkaliphilic Bacillus species, by using multiple sequencing tool followed by
block generation using degenerate primer designing bioinformatics tool-CODEHOP
(Table 3.2.1). To 100 ng of DNA as the template, 25 pmol of each Forward and
oligonucleotides primer (Sigma Aldrich,life sciences), 25µl of 2X Red Mix Plus
(Merk, Life sciences) were added. Two negative controls; one without template and
another without primer, were also included in the PCR reactions to check validity of
the experiment. The amplified products were visualized on agarose gel as described
above, further purified as discussed below and stored at -20oC till further use.
58
1. Initial denaturation at 94°C for 2 mins.
2. Denaturation at 94°C for 1 min.
3. Gradient of annealing at 60°C with gradient of 8°C for 45 secs.
4. Extension at 72°C for 1.5 mins.
5. Repeat step 2 to 4 for 29 cycles
6. A final elongation was done at 72°C for 5 min
7. Hold at 4°C
59
3.3 RESULTS AND DISCUSSION
It has been always fascinating to study the microbial community especially
extremophiles in particular. The extreme environments are often more complex and to
maintain them under laboratory conditions is a difficult task. So, the development of
new strategies of isolation, particularly for the extremophiles, is a challenging issue
for the scientists. Furthermore, it is of great value to make available the unexplored
world of organisms, as our knowledge is restricted to less than 1-5% of the total
microbial population in nature. Up till now majority of the halophiles and
haloalkaliphiles have been isolated from athalassohaline environments (Demergasso
et al., 2004; Wang et al., 2007), where as thalassohaline environments have relatively
less explored (Munoz et al., 2001; Amoozegar et al., 2003; Guranthan et al., 2010). In
view of these facts, we isolated haloalkaliphilic and haloalkalitolerant microbes from
the thalassohaline environments.
3.3.1 Sites for sample collection Around 34 different haloalkaliphilic bacteria were isolated from the saline soil;
particularly artificial salt pane samples located near the coastal region of the Western
Gujarat (India). The sites of the isolation as depicted in map (Fig. 3.3.1). Several
samples were collected as described in Table. 3.3.1. Among them, site designated 6:
Okha Madhi; particularly, O.M.6.2 and O.M.6.5 were selected for isolation and
enrichment procedures. Selection of site was done on the basis of its physico-
chemical properties (Table 3.3.2). Both the sampling sites used for studies were
artificial salt pane; having heavy deposition of salt; pane was heavily saturated with
salt as well ring of different colors; pink, red and orange were seen around the pane,
which could be interesting with diversity view point. The sampling site was around
1.5- 2.0 km long; with several panes located in close proximity with each other. The
temperature was around 37°C at the time of sample collection. The salinity and pH of
the samples varied from 3.5-4% and 7.8-9, respectively, presence of different color;
indicates undissolved salts on the surface. The complete description of the sites,
samples, their physical parameters and isolates isolated from each sample along with
their enrichment condition is given in Table 3.3.2 and Table 3.3.3.
60
Fig. 3.3.1: Map of Gujarat displaying site of Isolation, Okha, Gujarat, India.
Table 3.3.1: Sample collection details (date of collection: 07-10-07)
Site of collection No. of sample Description
Okha-Madhi 6.2 Soil collected from red ring from the site,
crystalline soil
Okha-Madhi 6.5 Sticky and smooth mud soil; with high salt
concentration
Okha
Madhi
61
Table 3.3.2: Physical and Chemical Properties of Soil {*Tests were performed as per
the BIS (Bureau of Indian Standards) IS: 3025}
3.3.2 Physical and chemical analysis of the sample
The salinity and alkalinity of the collected soil samples were nearly equal but the
values of the turbidity, TDS, total hardness and Mg+2 concentrations varied
(Table 3.3.2).
3.3.3 Enrichment and isolation
In present study, we isolated 34 haloalkaliphilic and haloalkalitolerant bacteria from
saline salt pane along the coastal region of Gujarat. Existence of halotolerant,
haloalkalitolerant and haloalkaliphilic bacteria clearly indicating the wide spread
distribution of such organisms in moderate saline environment beyond the
conventionally described habitats of salt lakes, solar salt evaporation ponds and salt
deserts. Depending on their optimum growth at 10% (w/v) NaCl and pH 9, the
isolated haloalkaliphilic bacteria can be put under the class of moderate
haloalkaliphiles. Therefore, in the present thesis the isolates have been referred as
haloalkaliphilic instead of halophilic organism. Interestingly, number of isolates from
a given site decreased with increasing degree of extremity of salt and pH. From the
two soil sample, total 34 different haloalkaliphilic/ haloalkaliphilic bacteria were
isolated by using different enrichment conditions of NaCl and pH.
Tests Okha madhi
(O.M.6.2)
Okha madhi
(O.M.6.5)
pH 7.88 8.0
*Salinity(g/l) 35.9 35.6
*Turbidity(NTU) 0.6 0.3
*TDS (mg/l) 1310 1295
*Alkalinity (mg/l) 110 115
*Total hardness(mg/l) 630.22 650.28
*Magnesium(mg/l) 341.268 361.95
62
Organisms were preliminary distinguished on the basis of enrichment conditions and
colony characteristics. Out of 34 isolates, 16 were isolated at combination of 10%
NaCl, (w/v) and pH-8 while 18 organism were isolated at combination of 30% NaCl,
(w/v) and pH-10 (Table 3.3.3). Both the combinations were selected for enrichment
procedures to isolate both haloalkalitolerant/ haloalkaliphilic bacteria (Fig. 3.3.3).
Almost equal numbers of isolates were obtained with both the combinations. The
overall profile for the isolation with different enrichment combinations is given in
Table 3.3.3 A and B.
Designation Enrichment condition No. of isolates
O.M.6.2 O.M. 6.5
A pH-8, 10% NaCl 2 5
B pH-8, 30% NaCl -- 3
C pH-10,10% NaCl 4 8
D pH-10, 30% NaCl 8 --
------ Total isolates 14 16
Table 3.3.3A: Nomenclature of organism isolated from saline soil of coastal Gujarat.
We have noticed by changing growth conditions that there were few isolates growing
optimally at 0% NaCl. Along the same line isolates were able to grow at higher
concentration i.e. upto 3-4M NaCl concentration. Growth of organism upto 2M NaCl
was preliminary characteristic of all isolates. However, along the same line, similar
results were not observed with respect to alkaliphiles, organism was able to grow at
higher alkaline pH. Maximum amount of organisms were able to grow optimally at
pH-9. However the range of growth was quite broad from pH-8 to pH-11. O.M.E11
and O.M.E12 were able to grow optimally at pH-11; however there was not much
variation in growth from pH-9 or 10. Similiarly this isolates were able to grow upto
20-25% of NaCl, although, few isolates, of interesting features, were isolated from
extreme condition. The important point emerged indicated that both diversity and
63
number of the organisms decreased with the increasing level of extremities,
supporting the general view that ultra extreme environments support the growth of
true extremophiles only.
Table 3.3.3B: Site description and characteristics of O.M.6.2 and O.M.6.5 site
Fig. 3.3.2A: Distribution of bacteria on the basis of pH profile
Fig. 3.3.2B: Isolation and enrichment
on the basis of NaCl
Fig. 3.3.2C: Isolation and enrichment
on the basis of pH
Site
Designation
Site description Characteristics of samples
Physical appearance pH Salinity Temp
(˚C)
O.M.6.2 Soil with presence of salt crystal 10 High 27
O.M.6.5 Soil with presence of salt crystal and
pink pigmentation 10 High 27
64
Table 3.3.4: Total number of isolates from O.M.6.2 and O.M.6.5 site
Site No. of
isolates
Enrichment
conditions
O.M.6.5 pH NaCl (%)
1 O.M.A21 8 10
2 O.M.A22 8 10
3 O.M.A23 8 10
4 O.M.A24 8 10
5 O.M.A25 8 10
6 O.M.A27 8 10
7 O.M.A28 8 10
8 O.M. B28 8 30
9 O.M. B21 8 30
10 O.M. B22 8 30
11 O.M. B23 8 30
12 O.M.C21 10 10
13 O.M.C2 2 10 10
14 O.M.C23 10 10
15 O.M.C24 10 10
16 O.M.C25 10 10
17 O.M.C26 10 10
18 O.M.C28 10 10
Site No. of
isolates
Enrichment
conditions
O.M.6.2 pH NaCl (%)
1 O.M.A11 8 10
2 O.M.A14 8 10
3 O.M.A16 8 10
4 O.M.A17 8 10
5 O.M.A18 8 10
6 O.M.C11 10 10
7 O.M.C12 10 10
8 O.M.C13 10 10
9 O.M.C14 10 10
10 O.M.D116 10 30
11 O.M.D17 10 30
12 O.M.D18 10 30
13 O.M.D114 10 30
14 O.M.D115 10 30
15 O.M. E12 10 30
16 O.M. E11 10 30
65
3.3.4 Characterization of the organisms Although, the genetic data and molecular techniques are extensively being used for
the identification and phylogenetic relatedness of organisms belonging to prokaryotes
and archaebacteria during the last many years, the traditional classification methods
based on phenotypic, morphological and microbiological observation have its own
importance in studying.
3.3.4.1 Colony characterization
The isolated haloalkaliphilic bacteria were primarily diversified on the basis of their
cultural characteristics, as described in Table 3.3.5. Some common characters are
collectively displayed by the majority of isolates from same site as well as those from
different sites; such as, round and regular shape with opaque colony, smooth texture
and creamish white pigmentation (Fig.3.3.3). The overall impression for the
comparison of all the colony characteristics for the isolates of the different sites is
described in Fig. 3.3.3A.
For O.M.6.2 site; the differentiation of isolates, was quite evident from the different
colony characteristic features (Fig. 3.3.4 A, B, C, D, E, F). On the basis of colony
size, among the isolates, around 60% of bacteria, size range between 1-3mm in colony
size while about 40% had large (4-6mm) colony size. However, for O.M.6.5 the
diversity profile was reversed where around 70% were in the size range of (4-6mm),
and only 30% of total isolates were of small size (Fig. 3.3.3B).
With reference to colony shape, for O.M.6.2; no diversity was noticed and all
organisms were totally of regular shape; while with respect to O.M.6.5 although
majority of them were noticed with regular shape, however 20% were found to be of
irregular shape (Fig. 3.3.3C).
With respect to elevation parameter only sheared numbers of isolates in O.M.6.2 and
no isolates in O.M.6.5 were noticed with flat elevation. With respect to elevation
parameter; in O.M.6.2 around 60% were with raised elevation and 40% were slightly
raised. Comparing the other site, exactly reverse side was observed. On studying these
parameters it is quite obvious analysis, that although the site of isolation was in close
proximity of each other; wide difference was seen with respect to diversity of
organisms (Fig. 3.3.3D).
66
For, colony texture parameter, although majority of organisms were found to be
smooth in nature for both the sites; however, ratio was quite different for both site. In
O.M.6.5 site around 20% were rough while only 3-4% was found to be of rough
texture in O.M.6.2 (Fig. 3.3.3E). There was not much diversity noticed with reference
to opacity parameter; and only 2-3% of bacteria were noticed of translucent and rest
all were opaque in nature and none of the total collected pool of bacteria has
transparent opacity (Fig. 3.3.3F).
Such phenotypic characters were useful for primary characterization, which could be
used to assess the initial level diversity among the isolates.
Fig 3.3.3 A
Fig 3.3.3B Fig 3.3.3C
67
Fig. 3.3.3: Distribution of isolates on the basis of colony characteristics: (A) Percent
distribution (B) Colony size (C) Colony shape (D) Colony Elevation (F) Opacity.
Fig 3.3.3E Fig 3.3.3D
Fig 3.3.3F
68
Fig. 3.3.4A: Distribution of organism
on the basis of different cell
morphology.
Fig. 3.3.4B: Distribution of organism
on the basis of different cell
arrangement.
Fig. 3.3.4C: Schematic distribution of
O.M.6.2 organism site on the basis of
cell morphology
Fig. 3.3.4D: Schematic distribution of
O.M.6.5 organism site on the basis of
cell morphology
69
Isolates Size (mm)
Shape Margin Elevation Opacity Texture Pigmentation
O.M.6.2 O.M.A1 1 5 Round Regular Slightly raised Opaque Smooth Creamish white
O.M.A1 4 4 Round Regular Raised Opaque Smooth Creamish white
O.M.A1 6 3 Round Regular Raised Opaque Smooth Creamish white
O.M.A1 7 3 Round Regular Slightly raised Opaque Smooth Creamish white
O.M.A1 8 4 Round Regular Raised Opaque Smooth Creamish white
O.M.C11 5 Round Regular Slightly raised Opaque Smooth Creamish white
O.M.C1 2 4 Round Regular Flat Opaque Smooth Creamish white
O.M.C1 3 3 Round Regular Flat Opaque Smooth Creamish white
O.M.C1 4 4 Round Regular Slightly raised Opaque Smooth Creamish white
O.M.D116 3 Round Regular Slightly raised Opaque Smooth Creamish white
O.M.D17 4 Round Regular Slightly raised Opaque Smooth Creamish white
O.M.D18 2 Round Regular Slightly raised Opaque Smooth Creamish white
O.M.D114 4 Round Regular Raised Opaque Smooth Creamish white
O.M.D115 3 Round Regular Raised Opaque Smooth Creamish white
O.M. E12 3 Round Regular Raised Opaque Smooth Brownish red
O.M. E11 4 Round Regular Raised Translucent Smooth Creamish white
O.M.6.5
O.M.A21 5 Round Regular Flat Opaque Smooth Creamish white
O.M.A22 3 Round Regular Flat Opaque Smooth Creamish white
O.M.A23 3 Round Regular Flat Opaque Smooth Brownish yellow
O.M.A24 3 Round Regular Raised Translucent Smooth Creamish yellow
O.M.A25 3 Round Irregular Flat Opaque Rough Creamish white
O.M.A27 4 Round Irregular Flat Opaque Rough Creamish white
O.M. A28 3 Irregular Irregular Flat Opaque Rough Creamish white
O.M. B28 3 Round Regular Flat Opaque Smooth Creamish white
O.M. B21 4 Round Regular Flat Opaque Rough Creamish white
O.M. B22 3 Round Regular Flat Opaque Smooth Creamish white
O.M.B23 4 Round Regular Raised Opaque Smooth Creamish yellow
O.M.C22 3 Round Regular Raised Opaque Smooth Creamish yellow
O.M.C23 5 Round Regular Raised Opaque Smooth Creamish yellow
O.M.C24 3 Round Regular Flat Opaque Smooth Red pigment
O.M.C25 3 Round Regular Flat Opaque Smooth Red pigment
O.M.C26 3 Round Regular Flat Opaque Smooth Creamish sticky
O.M.C28 4 Round Iregular Flat Translucent Smooth White
Table 3.3.5: Diversity of the organism on the basis of colony characteristics
70
Table 3.3.6: Characterization of organism on the basis of Gram‟s reaction
Similarly, looking at the gram reactions, Gram positive character was dominated over
the Gram negative for the same site. Gram variable characters were much less evident
among the isolates (Fig. 3.3.4). On the basis of Gram‟s reaction organism were
majorly; 75% were gram negative in nature and only 25% were gram positive. While,
Isolates Gram reaction Size and Shape Arrangement
O.M.6.2 O.M.A11 Negative Short thin rod Singly and in pair O.M.A14 Negative Very short thick rod Singly and in chain O.M.A16 Variable Long thin rod Singly O.M.A17 Negative Small thin rod Singly O.M.A18 Negative Medium thin rod Singly and in pair
O.M.C11 Negative Small thin rod Singly and in pair, most of in “V” shape
O.M.C1 2 Positive Short thin rod Singly and in pair O.M.C1 3 Positive Long thick rod Singly O.M.C1 4 Positive Short thick rod Singly and in clusters O.M.D116 Variable Short thick rod Singly and in pair O.M.D17 Positive Small cocci Singly and most of in pair O.M.D18 Positive Small cocci Singly O.M.D114 Positive Small cocci Singly O.M.D115 Positive Very small cocci Singly and in cluster O.M. E12 Positive Very small cocci Singly and in cluster O.M. E11 Positive Small cocci Singly
O.M.6.5 O.M.A21 Negative Short thick rod Singly and in pair O.M.A22 Positive Small thin rod Singly and some in pair also O.M.A23 Positive Short thick rod Singly
O.M.A24 Positive Very short thin rod, with middle spore
Singly and most of in pair and chains
O.M.A25 Variable Small thin rod Singly and in pair O.M.A27 Positive Short thick rod Singly O.M. A28 Positive Small cocci Singly and in clusters
O.M. B28 Positive Small oval shape cocci In tetrad only
O.M.A11 Negative Thick rod Singly and in pair O.M.A14 Positive Very Short thick rod Singly O.M.A16 Positive Small thin rod Singly O.M.A17 Positive Short thick rod Most of singly and some in pair O.M.A18 Positive Long thin rod Singly O.M.C11 Positive Small thick rod Singly and in pair O.M.C12 Positive Small cocci Singly O.M.C13 Positive Small cocci In pair and in clusters O.M.C14 Positive Very small cocci Singly and in tetrad
71
with respect to O.M.6.5; all the 18 isolates were negative and none of them were gram
positive. From, majority of the isolates only 10% were of gram variable character for
O.M.6.5 while none of such isolate was noticed in O.M.6.5 (Table 3.3.6).
According to literature, moderate halophiles with Gram negative characteristics have
been studied in great detailed while information relating to Gram positive is scarce
(Mormile et al., 1999). Our studies on O.M.6.2, however, highlighted the dominance
of the Gram positive organisms over the Gram negative ones. From the literature,
Gram negative haloalkaliphilic bacteria appear to be widely described (Xin et al.,
2001; Doronina et al., 2003a; Doronina et al., 2003b; Loiko et al., 2003;
Hoover et al., 2003; Banciu et al., 2004; Romano et al., 2002). We found similar
results for O.M.6.5 where we have found all the isolates were gram negative in nature.
Some of our isolates displayed Gram variable properties. With respect to cell shape,
coccid shape was widely observed among the bacteria enriched at higher NaCl
concentrations (15-20%); where as the rod shape was frequently distributed at lower
NaCl concentration (10%) for O.M.6.2, similar results were obtained by Joshi (2006)
from haloalkaliphiles isolated from sea water of coastal Gujarat. However, similar
observation was not observed for O.M.6.5.
With respect to cell arrangement; among all the isolates of the O.M.6.2, approx. 55%
of total isolates were singly and in pair and 35% were singly and 5% were singly and
in clusters and 5% were tetrad in nature, interestingly O.M.C11, displayed very unique
pattern of the cell arrangement in “V” shape (Fig. 3.3.4 A and B). Different diversity
profile was observed from both the sample. There were not much peculiar
characteristic features observed of bacteria isolated from this site. Although the sites
of isolation were quite nearby, there is much diversity noticed among the total isolates
from both the sites (Fig. 3.3.4 C and 3.3.4 D).
3.3.4.3 Biochemical characterization
In the present day of increasing emphasis on the molecular tools and chronometers,
the metabolic and physiological status of the organisms is still important to diversify
and differentiate organisms. The microorganisms have their own identifying
biochemical characteristics. These biochemical fingerprints are the properties
controlled by the cell‟s vital molecules and they are responsible for the bioenergetics,
biosynthesis and biodegradation.
72
With these objectives, the biochemical and metabolic activities of all the isolates
were studied for the further differentiation and characterization. The detail outline for
the biochemical reactions of all the isolates is depicted in Table 3.3.7, Fig. 3.3.5A and
3.3.5E. Among isolated haloalkaliphilic bacteria, approximately 70-75% of the
isolates were catalase and oxidase positive, although the extent of the production of
catalase varied among the isolates of the same site as well of the other sites (Fig.
3.3.5B). Isolates from O.M.6.5 site were more catalase positive than O.M.6.2 (Fig.
3.3.5C and 3.3.5D). Organisms were also able to utilize and generate diversified result
with respect to other biochemical parameters. Maximum organism were able to utilize
citrate with 62%, ammonia production was noticed to be 55%, while gelatin
utilization was 42%, around 33% of the total isolates were noticed producing H2S gas.
Nitrate reduction and casein hydrolysis was by 22% of the isolates. Indole production
was by 10% of the isolates. However extent of utilization was quiet variable among
the positive isolates (Fig. 3.3.5E, Table 3.3.7).
On analyzing, the profile of the individual sites for the different biochemical
activities, isolates of the O.M.6.2 site were more positive towards catalase with 100%,
while for O.M.6.5 it was around 60% (Fig. 3.3.5B). However, oxidase positive were
distributed equally. Citrate utilizers were more noticed in O.M.6.5, with 90%;
significant numbers i.e. 78% were noticed in the other studied site. Amylase
producers were around one-fifth of total isolates in O.M.6.2 and O.M.6.5 judged on
the basis of starch liquifications. Overall, 29% isolates were screened as H2S
producers, Significant difference were seen in ammonium production; 90% of the
isolates were positive in O.M. 6.5 while in O.M.6.2, its number were reduced to half.
While reverse observation was noticed in nitrate reduction, H2S gas production and
urea utilization, for O.M.6.5 was 62, 42 and 48%, while for O.M.6.5 it was only 10%
of isolates able to generate positive results for all the three above mentioned tests.
With respect to gelatin liquefaction, which was an important parameter for functional
attributes of protease enzyme, it was known that almost half of the isolates were
positive. However, for all the positive results, extent of positivity was a variable
feature.
With reference to TSI test, maximum alkaline reaction was observed in the slant and
butt of isolates of both the sites. However, if we observe the data within the specific
site O.M.6.2, slants were more alkali with 64%.
73
Table 3.3.7: Biochemical profile of haloalkaliphilic organism (Color Indications: Red
– Negative, Yellow – Partial Positive, Blue – Positive).
For the site O.M.6.5, both slants and butt were found to be of alkaline nature with 66 -
68% (Table 3.3.8, Fig. 3.3.6). Although, site of isolation of all isolates is within the
same coastline, the extent of catalase production varied significantly among the
isolates. Organisms from O.M.6.2 were found to be highly aerobic, as all the isolates
were catalase positive and 92% of them were oxidase positive. For, O.M.6.5, we can
say that they were moderate aerobic in nature, as around 40% of the studied isolates
were catalase test negative. The variation in O2 requirements reflects the differences
in bio-oxidative enzyme systems presents in the organisms. Only 10% of the total
isolates form both the sites were indole positive, which suggest lack of tryptophanase
in these organisms. Similarly, only 10% of organisms from site O.M.6.5 were able to
utilize urea.
These results were also supported by the literature where many moderately halophilic
and alkaliphilic bacteria did not produce indole or utilized urea (Mota et al., 1997;
Muntyan et al., 2002; Reddy et al., 2003; Romano et al., 2005; Dodia, 2005). The
results were further supported by Mizuki and his co-workers, who screened for the
urease activity among 71 extremely halophilic strains but only 4 were able to secret
the urease (Mizuki et al., 2004). Results of O.M.6.5 contradict to O.M.6.2 as we
found 42% of the total isolates which are able to generate positive reaction in urea.
Isolates Tests
Ammonia Production
Nitrate Reduction
Indole Production
H2S Prod
Urea Hydolyis
Gelatin Hydrolyis
Casein Hydolysis
Starch Hydro
Citrate Utilization Catalase Oxidase
O.M.A11 2 0 0 1 0 0 0 2 2 2 1
O.M.A1 4 1 2 0 1 0 2 1 1 2 1 1
O.M.A16 0 0 0 1 0 2 0 0 2 1 1
O.M.A1 7 1 0 1 1 2 0 0 0 1 2 2
O.M.A1 8 2 0 0 0 0 2 1 0 2 1 1
O.M.C1 1 1 0 2 1 2 2 0 1 0 1 0
O.M.C1 2 2 1 0 1 1 1 0 1 1 1 1
O.M.C1 3 2 1 0 0 0 1 0 0 2 1 1
O.M.C1 4 2 1 0 0 1 2 0 0 1 1 1
O.M.D1 16 0 1 0 1 2 0 0 0 2 1 1
O.M. E1 2 0 1 0 1 0 0 0 0 1 1 1
O.M. E21 0 1 0 1 0 2 1 0 1 1 1
O.M.A21 1 0 0 0 0 1 1 0 2 1 2
O.M.A23 1 0 0 1 0 0 0 0 1 0 0
O.M.A24 2 1 1 0 2 2 1 0 1 0 0
O.M. B28 2 0 0 0 0 1 0 0 1 1 1
O.M. B21 0 0 0 0 0 1 0 0 0 1 1
O.M. B23 1 0 0 0 0 2 0 0 1 1 1
O.M.C2 1 1 0 0 0 0 0 0 1 1 0 1
O.M.C2 2 1 0 0 0 0 1 0 0 2 0 1
O.M.C2 3 2 0 0 0 0 2 1 1 1 1 1
O.M.C2 4 2 0 0 0 0 2 1 0 0 2 1
O.M.C28 1 0 0 0 0 2 1 0 1 1 1
74
The isolates varied extensively with respect to H2S and ammonia production and
nitrate reduction, which clearly reflected the metabolic diversity among them. H2S
production was maximally produced by O.M.6.2 site with 42%, while for the other
site, it was only one-tenth of total isolates displaying positive results. In the site
O.M.6.5, organisms were more ammonia utilizers with 90%, while was only 10% in
the site O.M.6.2. However, as revealed in the literature, a number of haloalkaliphilic
bacteria possess the ability of nitrate reduction (Vreeland et al., 1980; Mormile et al.,
1999; Sorokin et al., 2003a, b). Results of H2S and ammonia production are of vital
importance as they can utilize sulfur-containing amino acids as a carbon source from
the protein-rich medium. This may also imply that the concerned habitats are rich in
proteinaceous substances occupying the nutritional dynamics where easily utilizable
carbohydrates are scares. The high-energy requirement of these organisms could also
be attributed to the energy required for the synthesis and transport of compatible
solutes to compensate the high osmotic pressure present in the surroundings. In
general much of the diversity was observed among the sites, as well characteristic
features of the organism with respect to its site of isolation, energy requirement and
nutrient parameters differ to an extent.
Fig. 3.3.5A: Overall profile of
biochemical test for total number of
positive isolate
Fig. 3.3.5B: Overall % scenario of
biochemical test
75
Fig. 3.3.5C: % of positive isolate for
O.M.6.2
Fig. 3.3.5D: % of positive isolate for
O.M.6.5
Fig. 3.3.5E: Overall representation of biochemical test
76
Fig. 3.3.6A
Fig. 3.3.6B
Fig. 3.3.6C
Fig.3.3.6: TSI Profile of isolates, where slant (■), Butt (■), Gas (■).
77
Table 3.3.8: Triple Sugar Ion test of haloalkaliphilic organisms
3.3.4.4 Sugar fermentation
Ability of the organisms to metabolize different sugars for the bioenergetics purpose
is one of the approaches to diversify the organisms. The extent of sugar utilization
highly varied among the isolates from the same site as well as those from different
site. None of the isolates were able to produce gas in Durham‟s tube (Table 3.3.9).
Similarly, none of them were able to ferment ribose sugar.
For O.M.6.2; around 25% of total isolates were able to utilize glucose and sucrose as
a carbon source. Utilization of maltose and manitol was by around 20% of the
isolates. Lactose was utilized by half of the total isolates utilizing glucose and
Isolates Slant Butt Gas
O.M.A11 Acid Acid Nil O.M.A1 4 Acid Acid Nil O.M.A16 Acid Acid Nil O.M.A17 Acid Acid Nil O.M.A18 Alkali Alkali Nil O.M.C11 Alkali Acid Nil O.M.C1 2 Alkali Acid Nil O.M.C1 3 Alkali Alkali Nil O.M.C1 4 Alkali Alkali Nil O.M.D116 Acid Acid Nil O.M. E12 Alkali Alkali Nil O.M. E21 Alkali Acid Nil O.M.A21 Alkali Alkali Nil O.M.A23 Acid Acid Nil O.M.A24 Acid Acid Nil O.M. B28 Alkali Alkali Nil O.M. B21 Acid Acid Nil O.M. B23 Acid Acid Nil O.M.C21 Alkali Alkali Nil O.M.C22 Alkali Alkali Nil O.M.C23 Alkali Alkali Nil O.M.C24 Alkali Alkali Nil O.M.C28 Alkali Alkali Nil
78
sucrose. Striking point emerged is that only O.M.E11 as a candidate was able to utilize
xylose sugar (Fig. 3.3.7A and 3.3.7B). An interesting point was noticed that each
isolates as an individual was able to consume only single sugar for their metabolic
activities and growth. In much simpler way, e.g. the organism displaying positive test
with glucose was displaying negative results with other six sugars tested. This was
noticed as a general trend for all the isolates, except O.M.A11, O.M.D18 and
O.M.D116. O.M.A11 was able to utilize maltose, lactose, mannitol and sucrose.
Among this, four positive sugars with maltose it showed partial positive result, while
with other three the utilization was profound (Fig. 3.3.7C and 3.3.7D).
For O.M.6.5; maximum amount of isolates were able to utilize maltose as an energy
source with total 55% of total isolates. 15% of isolates were burning glucose for
carbon and 10% of isolates used lactose. Only 5% of bacteria used partially mannitol
and sucrose. The results were quite contrasting in terms of intake of sugar with
respect to O.M.6.2. In this case, utilization of such disaccharides, compared to simple
carbon sources, suggested the adaptation of different metabolic pathways for their
energy generation. Spirochaeta americana sp. nov, a haloalkaliphilic, obligately
anaerobic, Gram negative spirochaete (Hoover et al., 2003) and alkaliphilic and
moderately halophilic strain Salinicoccus alkaliphilus sp. nov. (Zhang et al., 2002)
were able to utilized range of sugars such as D-glucose, fructose, maltose, lactose,
sucrose, starch and D-mannitol.
79
Isolates Sugar Utilization Glucose Ribose Maltose Lactose Xylose Manniitol Sucrose
O.M.6.2 O.M.A11
-- ++ -- ++ ++ O.M.A14 -- -- -- -- -- -- -- O.M.A16 + -- -- -- -- -- -- O.M.A17 -- -- -- -- -- -- ++ O.M.A18 -- -- -- -- -- -- ++ O.M.C11 ++ -- -- -- -- -- -- O.M.C12 -- -- -- + -- -- -- O.M.C13 -- -- -- -- -- + -- O.M.C14 -- -- -- -- -- -- -- O.M.D116 + -- + -- -- -- --
O.M.D17 -- -- -- -- -- -- -- O.M.D18 ++ -- ++ -- -- ++ ++ O.M.D114 -- -- -- -- -- -- --
O.M.D115 -- -- -- -- -- -- --
O.M. E12 -- -- -- -- -- -- --
O.M. E11 -- -- -- -- + -- --
O.M.6.5 O.M.A21 ++ -- -- -- -- -- -- O.M.A22 -- -- -- -- -- -- ++ O.M.A23 -- -- -- -- -- -- -- O.M.A24 -- -- -- -- -- ++ -- O.M.A25 -- -- ++ -- -- -- -- O.M.A27 -- -- ++ -- -- -- -- O.M. A28 -- -- ++ -- -- -- -- O.M. B28 -- -- + -- -- -- -- O.M. B21 -- -- ++ -- -- -- -- O.M. B22 -- -- ++ -- -- -- -- O.M.B23 -- -- ++ -- -- -- -- O.M.C22 ++ -- ++ -- -- -- -- O.M.C23 -- -- -- -- -- -- -- O.M.C24 -- -- -- -- -- -- -- O.M.C25 -- -- -- -- -- -- -- O.M.C26 -- -- -- -- -- -- -- O.M.C28 -- -- -- -- -- -- --
Table 3.3.9: Sugar utilization profile of isolates
80
Fig. 3.3.7A: Characterization of
isolates for sugar utilization for
O.M.6.2
Fig. 3.3.7B: % of isolates utilizing
sugar by O.M.6.2 site
Fig. 3.3.7C: Characterization of
isolates for sugar utilization for
O.M.6.5
Fig. 3.3.7D: % of isolate utilizing
sugar for O.M.6.5
Fig. 3.3.7D
81
3.3.5 Phylogenetic identiifciation
3.3.5.1 16S rRNA amplification, nucleotide sequencing and homology prediction
Potential isolate showing interesting results for bioctalytic studies; organism
designated as O.M.A18, O.M.E12, O.M.C14 from O.M.6.2 site and O.M.C28 from
O.M.6.5 site were identified on the basis of 16S rRNA gene homology. As described
in materials and method, the 1500bp rRNA gene was amplified by using forward and
reverses primers. Fig. 3.3.8, Table 3.3.10, 3.3.11 provides the aligned sequenced data
of 1501 bp. The sequence data were further analyzed for finding the closest homologs
for the microbe by comparing gene sequence with reference strains. Using consensus
primers, the ~1.5 kb 16S rDNA fragment was amplified using high-fidelity PCR
polymerase. The PCR product was bi-directionally sequenced using the forward,
reverse and an internal primer. Sequence data was aligned and analyzed for finding
the closest homologs for the microbe. The alignment view table (Table 3.3.10);
distance matrix (Table 3.3.11) and phylogentic position studied by Mega align
software (Fig. 3.3.8).
Fig.3.3.8A: Phylogenetic Tree made in MEGA 3.1 software using Neighbor Joining
method for O.M.A18
'AY647302' 'O. M A1 8'
'BA000028' 'AJ276808' 'EF512732' 'AY505535' 'AY505536' 'AY121439' 'EF660762' 'AB188089' 'DQ089679' 'EU311210'
0.002 0.012
0.004 0.001
0.019 0.013
0.004 0.003 0.007
0.002 0.010
0.002
0.020
0.009
0.007
0.002
0.002
82
Fig.3.3.8B Phylogenetic Tree made in MEGA 3.1 software using Neighbor Joining
method for O.M.E12
Fig.3.3.8C: Phylogenetic Tree made in MEGA 3.1 software using Neighbor Joining
method for O.M.C28
'EU090232' 'EU118360' 'EU118361' 'OM E12'
'AF406790' 'AB201795' 'AB201799' 'EU091310' 'EU135674' 'AY121430'
'DQ333301'
0.021 0.019
0.003 0.022
0.015 0.008
0.031
0.008
0.012 0.022
0.003
0.008
0.004
0.024
0.014 0.002
83
Table 3.3.10: Alignment view using combination of NCBI GenBank and RDP
database for O.M.A18 and O.M.E12
ID Alignment results Sequence description
OM A1 8 0.87 Studied sample (O.M. A18)
BA000028 0.96 Oceanobacillus iheyensis
AY647302 0.92 Oceanobacillus iheyensis strain MSU3110
DQ089679 0.84 Oceanobacillus oncorhynchi strain Iii13
AB188089 0.85 Oceanobacillus oncorhynchi strain: R-2
EF660762 0.82 Oceanobacillus cibarius strain A34
EU311210 0.80 Oceanobacillus oncorhynchi strain zyj2-1
AJ276808 0.86 Virgibacillus picturae strain LMG-19416
EF512732 0.86 Oceanobacillus picturae strain JL85
AY505535 0.86 Virgibacillus picturae strain GSP52
AY505536 0.86 Virgibacillus picturae strain GSP60
AY121439 0.88 Salibacillus sp.
Alignment View ID Alignment results Sequence description
OM E12 0.60 Studied sample (O.M. E12)
EU118361 0.66 Haloalkaliphilic bacterium AH-6
EU118360 0.65 Haloalkaliphilic bacterium S-20-9
EU090232 0.62 Bacillus pseudofirmus strain SJ2
AF406790 0.97 Bacillus pseudofirmus strain FTU
AB201795 0.97 Bacillus pseudofirmus
AB201799 0.99 Bacillus pseudofirmus
EU135674 0.84 Virgibacillus sp.
AY121430 0.89 Salibacillus sp.
DQ333301 0.91 Planococcus maritimus isolate LLQ
EU091310 0.85 Bacillus halodurans strain C7
84
Distance Matrix of O.M.A18
1 2 3 4 5 6 7 8 9 10 11
DQ089679 --- 0.990 0.972 0.961 0.990 0.960 0.970 0.960 0.960 0.960 0.982
EU311210 0.010 --- 0.968 0.952 0.988 0.951 0.968 0.951 0.954 0.951 0.973
AY647302 0.028 0.032 --- 0.955 0.974 0.955 0.991 0.955 0.958 0.955 0.967
AJ276808 0.039 0.048 0.045 --- 0.955 0.997 0.954 0.997 0.960 0.997 0.948
AB188089 0.010 0.012 0.026 0.045 --- 0.955 0.973 0.955 0.955 0.955 0.980
EF512732 0.040 0.049 0.045 0.003 0.045 --- 0.953 1 0.959 1 0.947
BA000028 0.030 0.032 0.009 0.046 0.027 0.047 --- 0.953 0.957 0.953 0.966
AY505535 0.040 0.049 0.045 0.003 0.045 0.000 0.047 --- 0.959 1 0.947
AY505536 0.040 0.049 0.045 0.003 0.045 0.000 0.047 0.000 --- 0.053 0.947
EF660762 0.018 0.027 0.033 0.052 0.020 0.053 0.034 0.053 0.049 --- 0.440
OM A1 8 0.040 0.042 0.014 0.055 0.036 0.055 0.017 0.055 0.051 0.055 ---
Distance Matrix of O.M.E12
1 2 3 4 5 6 7 8 9 10 11
EU090232 --- 0.938 0.939 0.926 0.938 0.927 0.940 0.911 0.932 0.938 0.922
AB201799 0.062 --- 0.911 0.956 1 0.965 0.916 0.948 0.989 1 0.897
EU118360 0.061 0.088 --- 0.909 0.911 0.910 0.965 0.903 0.909 0.911 0.948
EU135674 0.074 0.044 0.091 --- 0.956 0.978 0.915 0.946 0.948 0.956 0.900
AF406790 0.062 0.000 0.088 0.044 --- 0.965 0.916 0.948 0.989 1 0.897
AY121430 0.073 0.035 0.090 0.022 0.035 --- 0.914 0.953 0.958 0.965 0.894
EU118361 0.060 0.084 0.035 0.085 0.084 0.086 --- 0.905 0.913 0.916 0.975
DQ333301 0.089 0.052 0.097 0.054 0.052 0.047 0.095 --- 0.941 0.948 0.889
EU091310 0.068 0.011 0.091 0.052 0.011 0.042 0.087 0.059 --- 0.989 0.894
AB201795 0.062 0.000 0.088 0.044 0.000 0.035 0.084 0.052 0.011 --- 0.897
OM E12 0.077 0.103 0.052 0.100 0.103 0.106 0.025 0.111 0.106 0.103 ---
Table 3.3.11: Distance Matrix based on Nucleotide Sequence Homology (Using
Kimura-2 Parameter) for O.M.A18 and O.M.E12 (indicates nucleotide similarity
(above diagonal) and distance (below diagonal).
85
Aligned Sequence Data of O.M.A18: size (1447bp)
GCTGGCGGCGTGCCTAATCATGCAAGTCGAGCGCAGGAAGTTATCTGATCCTCTTTAGAGGTGACGATAAT
GGAATGAGCGGCGGACGGGTGAGTAACACGTAGGCAACCTGCCTGTAAGACTGGGATAACTCGTGGAAAC
GCGAGCTAATACCGGATAACACTTTTCATCTCCTGATGAGAAGTTGAAAGGCGGCTTTTGCTGTCACTTACA
GATGGGCCTGCGGCGCATTAGCTAGTTGGTAAGGTAATGGCTTACCAAGGCGACGATGCGTAGCCGACCTG
AGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCT
TCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAACTCTG
TTGTTAGGGAAGAACAAGTGCCAAAGTAACTGATGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTA
ACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGCGCTC
GCAGGCGGTTCTTTAAGTCTGATGTGAAATCTTACGGCTCAACCGTAAACGTGCATTGAGAAACTGGGGACT
TGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAG
TGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGAT
ACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAGGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGAAGTTA
ACGCATTAAGCACTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAAGAATTGACGGGGGCCCGC
ACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGAA
CACTCTAGAGATAGAGTTTTCCCTTTGGGGACAGAATGACAGGTGGTCCATGGTTGTTGTCAGCTTGTGTCG
TGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCTTTGATCTTAGTTGCCAGCATTCAGTTGGGCCCTCT
AAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGACGACGTCAAATCATCATGCCCCTTATGACCTGG
GCTACACACGTGCTACAATGGATGGAACAAAGGGAAGCGAACCCGCGAGGTCAAGCAAATCCCACAAAAC
CATTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGCCGGAATCGCTAGTAATCGCGGATCAGCA
TGCCGCGGTGAATACGTTTCCCGGGCCTTGTACACACCGCTAGTCACACCACGAGAGTTGGTATCACCCCGA
AGTCGGTGAGGTAACCTTTTGGAGC
Aligned Sequence Data of O.M.E12: size (1485 bp)
AGAGTTTGATCATGGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAACTCGAGCGAACCCGGGGT
GCTTGCACCCTGAGGGTTAGCGGCGGACGGGTGAGTAACACGTGGGGAACCTGCCTTGCTGTCTGGGATAA
CACCGGGAAACCGGTGCTAATACCGGATGTCCCCTTTCCGGCACCTGCCGGAGAGGGAAAAGGCGGCTTTG
AGCCGCCGCAGCAAGAGGGGCCCGCGGCGCATTAGTTAGTTGGCAGGGTAACGGCCTCCCAAGGCGACGA
TGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGACACACGGCCCAGACTCCTACGGGAGGCA
GCAGTAGGGAATCATCCGCAATGGACGAAAGTCTGACGGTGCAACGCCGCGTGAGTGATGAAGGTTTTCGG
ATCGTAAAGCTCTGTTGTGAGGGAAGAATAAGATGGGGAGGAAATGCCCGATCTGTGACGGTACCTCACCA
GAAAGCCCCGGCTAAGTACGTGACAACAGCCGCGGTTATACGTGGGGGGCAAGAGTTGACCGGAATTATTG
GGCGTAAAGGGCACGCAGCGTTCCGGCATGTCTGTTGTGAAAGGCCGTGGCTCAACCACGGAATGGCATTG
GAAACTGCCAGACTTGAGTACAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATG
TGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGTGCGAAAGCGTGGGGAGC
GAACGGGATTAGATACCCTGGTAGTCCACGCCGTAAACGTTGAGTGCTAGGTGTTAGGGGTTTCGATACCC
GTAGTGCCAAGCAACGATTAAGACTCCGCCTGGGAGACACCGCAGGTTGAAACTCAAAGGAATGACGGGG
CCGCACAAGCGGTGGAGCATGTGGTTTAATTCGACGCCACGCGAAGAACCTTACCCGGTCTTGACATCTTCT
GACCGCCTGGAAACAAGGTTTCCTTTCGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCG
TGAGATGTTGGGTTAAGTCCCGTAACGAGCGCAACCCTTGAATGTCGTTGCCAGCATTGAGTTGGGCACTTT
ACATTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGG
GCTACACACGTGCTACAATGGACGGTACAACGGGAAGCGAAACCGTGAGGTGGAGCGAATCCTGAAAAGC
CGTTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTGCATGAAGCTGGAATCGCTAGTAATCGCGGATCAGAA
TGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGCCAGCAACACCCGAA
GTCGGTGAGGCAACCGTTTGGAGCCAGCCGCCGAAGGTGGGGCCGGTGATTGGGGTGAAGTCGTA
C28 ) was AB188089 )
86
Aligned sequence data of O.M.C28 (1450bp) AAGTTCGTGGAACGAGCGGCGGACGGGTGAGTAACACGTAGGCAACCTGCCTGTAAGACTGGGATAACTCGCGGAAACGCG
AGCTAATACCGGATAACACTTTCTATCACCTGATGGAAAGTTGAAAGGCGGCTTTTGCTGTCACTTACAGATGGGCCTGCG
GCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGG
GACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGC
CGCGTGAGTGATGAAGGTTTTCGGATCGTAAAACTCTGTTGTCGGGGAAGAACAAGTATGATAGTAACTGATCGTACCTTG
ACGGTACCCGACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATT
ATTGGGCGTAAAGCGCTCGCAGGCGGTTCTTTAAGTCTGATGTGAAATCTTGCGGCTCAACCGCANACGTGCATTGGAAAC
TGGAGGACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTG
GCGAAGGCGACTCTCTGGTCTGTAACTGACGCTNAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTC
CACGCCGTANACGATGAGTGCTAGGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGAAGTTAACGCATTAAGCACTCCGCCTG
GGGAGTACGGCCGCAAGGCTGAAACTCAAAAGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAG
CAACGCGAAGAACCTTACCAGGTCTTGACATCCTTTGAGCCCTCTAGAGATAGAGCTTTCCCTTCGGGGACAAAGTGACAG
GTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTAATCTTAGTTGC
CAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGCGAAAGTGGGGATGACGTCAAATCATCATGCCCCT
TATGACCTGGGCTACACACGTGCTACAATGTAAGGAACAAAGGGAACCGAACCCGCGAGGTCCAGCAAATCCCATAAAACC
GTTCTCAGTTCGGATTGCAGGCTGCAACTCGCCTGCATGAAGCCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTG
AATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCACGAGAGTTCGTAACACCCGAAGTCGGTGAGGTAACCTTTTG
GAGCCAGCCGCCGAAGGTGGGACGAATGATTGGGGTGAAGTCGTAACAAGGTAGCCGTATCGGAAGGTGCGGC
Based on nucleotides homology and phylogenetic analysis the microbe
(Sample: OM E12) was detected to be Haloalkaliphilic bacterium (GenBank accession
number: EU118361). Nearest homolog genus-species was found to be Bacillus
pseudofirmus (accession no. EU090232). Information about other close homologs for
the microbe can be found from the alignment view table (Table 3.3.11) its distance
matrix is displayed in (Table 12). The relatedness is displayed in Fig.3.3.8.
Aligned sequence data of O.M.C28
Based on nucleotides homology and phylogenetic analysis the Microbe
(Sample: OM C28) was detected to be Oceanobacillus oncorhynchi (GenBank
accession number: EU118361). Nearest homolog genus-species was found to be
Oceanobacillus sp. (accession no. AY553089).
87
3.3.5.2 FAME Analysis
Microbial fatty acid profiles are unique from one species to another, and this has
allowed for the creation of very large microbial libraries.
O.M.A18 Datasheet Volume: DATA File: E089028.04A Samp Ctr: 3 ID Number: 1257
Type: Samp Bottle: 2 Method: RTSBA6
Created: 9/2/2008 7:35:13 PM
ECL Deviation: 0.003 Reference ECL Shift: 0.002
Number Reference Peaks: 11
Total Response: 202535 Totals Named: 200394
Percent Named: 98.94% Total Amount: 189750
RT Response Ar/Ht RFact ECL Peak Name Percent Comment
0.7352 2.22E+9 0.018 ---- 6.6505 SOLVENT PEAK ---- < min rt
1.6740 361 0.009 1.055 11.999
8
12:0 0.20 ECL deviates 0.000
2.1298 2527 0.009 0.989 13.628
5
14:0 iso 1.32 ECL deviates 0.000
2.2399 1038 0.009 0.976 13.999
9
14:0 0.54 ECL deviates 0.000
2.4376 86552 0.009 0.957 14.632
2
15:0 iso 43.82 ECL deviates 0.000
2.4665 28796 0.009 0.954 14.724
9
15:0 anteiso 14.54 ECL deviates 0.000
2.5525 837 0.011 0.947 14.999
9
15:0 ---- ECL deviates 0.000
2.6859 3528 0.010 0.937 15.415
6
16:1 w7c alcohol 1.75 ECL deviates 0.002
2.7557 11781 0.009 0.933 15.633
1
16:0 iso 5.82 ECL deviates 0.000
2.8037 1928 0.010 0.930 15.782
6
16:1 w11c 0.95 ECL deviates 0.001
2.8733 7890 0.009 0.926 15.999
3
16:0 3.87 ECL deviates -0.001
3.0067 1522 0.010 0.921 16.414
6
17:1 iso w10c 0.74 ECL deviates 0.001
3.0374 2491 0.010 0.920 16.510
2
Sum In Feature 4 1.21 ECL deviates -0.002
3.0778 18340 0.009 0.918 16.635
9
17:0 iso 8.91 ECL deviates -0.001
3.1091 30426 0.009 0.917 16.733
3
17:0 anteiso 14.77 ECL deviates 0.000
3.1952 350 0.009 0.916 17.001
3
17:0 0.17 ECL deviates 0.001
3.4479 364 0.010 0.915 17.794
0
18:1 w9c 0.18 ECL deviates 0.000
3.4605 555 0.009 0.915 17.833
5
Sum In Feature 8 0.27 ECL deviates -0.014
3.5136 951 0.010 0.916 17.999
8
18:0 0.46 ECL deviates 0.000
3.6360 430 0.011 0.919 18.393
2
18:0 10-methyl, TBSA 0.21 ECL deviates -0.002
3.6887 565 0.013 0.921 18.562
7
17:0 3OH 0.28 ECL deviates -0.002
3.7224 628 0.015 ---- 18.670
9
----
3.7349 909 0.014 ---- 18.711
0
----
4.0384 605 0.010 ---- 19.703
0
----
---- 2491 --- ---- ---- Summed Feature 4 1.21 17:1 iso I/anteiso B
---- 555 --- ---- ---- Summed Feature 8 0.27 18:1 w7c
88
min0.5 1 1.5 2 2.5 3 3.5 4
pA
15
20
25
30
35
FID1 A, (E08902.804\A0031257.D)
0.7
35
1.6
74 2
.13
0
2.2
40
2.4
38
2.4
67
2.5
53
2.6
86
2.7
56
2.8
04
2.8
73
3.0
07
3.0
37
3.0
78
3.1
09
3.1
95
3.4
48
3.4
61
3.5
14
3.6
36
3.6
89
3.7
22
3.7
35
4.0
38
O.M.E12 Datasheet
Volume: DATA File: E089028.04A Samp Ctr: 6 ID Number:
1260
Type: Samp Bottle: 5 Method: RTSBA6
Created: 9/2/2008 8:00:41 PM
RT Response Ar/Ht RFact ECL Peak Name Percent Comment 0.7344 2.224E+9 0.017 ---- 6.6441 SOLVENT
PEAK
---- < min rt 2.1297 4558 0.009 0.989 13.6282 14:0 iso 6.99 ECL deviates
0.000 2.2399 1620 0.009 0.976 14.0005 14:0 2.45 ECL deviates
0.000 2.4371 16776 0.008 0.957 14.6311 15:0 iso 24.89 ECL deviates -
0.001 2.4663 23538 0.009 0.954 14.7247 15:0 anteiso 34.83 ECL deviates
0.000 2.7556 5719 0.009 0.933 15.6334 16:0 iso 8.27 ECL deviates
0.000 2.8732 7255 0.009 0.926 16.0000 16:0 10.42 ECL deviates
0.000 3.0779 1754 0.009 0.918 16.6374 17:0 iso 2.50 ECL deviates
0.000 3.1086 5184 0.009 0.917 16.7330 17:0 anteiso 7.38 ECL deviates
0.000 3.4608 785 0.011 0.915 17.8365 Sum In Feature
8
1.11 ECL deviates -
0.011 3.5129 814 0.009 0.916 17.9998 18:0 1.16 ECL deviates
0.000 3.7341 628 0.012 ---- 18.7110 ---- 4.0380 626 0.010 ---- 19.7040 ---- 4.2713 1016 0.021 ---- 20.4712 ---- > max rt
---- 785 --- ---- ---- Summed
Feature 8
1.11 18:1 w7c
Library Sim Index
Entry Name
RTSBA6 6.00
0.280 Geobacillus-stearothermophilus-GC subgroup A (55C, Bacillus)
89
ECL Deviation: 0.004 Reference ECL Shift: 0.002
Total Response: 69258 Total Named: 68003
Percent Named: 98.19% Total Amount: 64479
Library Sim Index Entry Name
RTSBA6 6.00 0.687 Virgibacillus-pantothenticus (Bacillus)
min0.5 1 1.5 2 2.5 3 3.5 4
pA
15
17.5
20
22.5
25
27.5
30
32.5
FID1 A, (E08902.804\A0061260.D)
0.7
34
2.1
30
2.2
40
2.4
37
2.4
66
2.7
56 2.8
73
3.0
78
3.1
09
3.4
61
3.5
13
3.7
34
4.0
38
4.2
71
3.3.6 Antibiotic resistance and sensitivity
The haloalkaliphilic bacteria were highly diversified in terms of their antibiotic
sensitivity and resistance property. The detailed antibiotic profile for all the isolates is
depicted in Table 3.3.12. The strategy conceived for antibiotic profiling were gram-
positive organisms were checked for antibiotics related to gram positive nature and
gram negative were assessed for gram negative features. From, the total isolates from
gram reaction it was observed that all the isolates were gram positive in O.M.6.2 site
90
and in O.M.6.5 majority of isolates were gram positive; however few isolates were
gram negative. The profile of the sensitivity varied among the isolates from the same
site and those from the other site. Some of the antibiotics used were specific for both,
Gram positive and negative bacteria viz., Tetracyclin, Co-Trimaxazole, Ampicillin,
Gentamycin, Cephotaxime and Nalidixic Acid.
Table 3.3.12A: Antimicrobial properties of gram positive isolates for O.M.6.5
Table 3.3.12B: Antimicrobial properties of isolates for O.M.6.2 site
Ab+v
e
Mcg
Isolates with gram positive character from O.M.6.5
O.M
.A21
O.M
.A22
O.M
.A23
O.M
.A24
O.M
.A25
O.M
.A27
O.M
.A28
O.M
.B28
O.M
.B21
O.M
.B22
O.M
.B23
O.M
.C21
O.M
.C22
O.M
.C23
O.M
.C24
O.M
.C25
O. M
.C26
O.M
.C27
O.M
.C28
At 15 30 32 21 302 44 R 7 22 R3 30 22 20 R 22 29 33 36 18 10
Ak 30 27 R R 38 R R R 33 44 30 22 18 21 27 R 41 17 10 R
G 10 R 26 R 36 R R R 36 24 20 R 18 26 19 33 36 R 15 10
Cf 5 23 32 38 44 48 22 32 22 40 37 35 30 41 22 35 39 41 11 18
Cq 30 12 R 26 42 26 32 21 38 18 21 20 27 24 29 21 40 43 40 24
Ro 30 15 R 32 22 R 20 R 32 38 35 30 36 31 30 38 7 30 37 29
Ax 10 29 44 R 12 26 9 6 38 10 15 9 6 30 32 31 5 18 28 33
Ce 30 21 R R R R 10 R R R 5 R R 3 6 10 6 11 R 7
Cs 75 28 24 50 22 32 R 35 38 28 21 27 33 30 37 31 22 26 30 39
Cw 15 31 27 28 30 12 22 R 44 35 32 31 42 37 43 R 33 8 R 41
Sc 5 6 44 7 32 24 20 7 40 11 7 32 19 24 20 26 45 9 31 22
Cu 30 22 26 30 42 44 8 24 R 26 21 35 31 13 17 22 41 32 33 24
Ab+ve
mcg
Isolates with gram positive character from O.M.6.2
O.M.A1 1 O.M.A1 4 O.M.A1 6 O.M.A1 7 O.M.A1 8 O.M.C1 1
At 15 33 31 21 22 44 R
Ak 30 24 R R 30 R 26
G 10 41 20 R 35 R R
Cf 5 R 32 38 44 40 31
Cq 30 10 R 26 40 29 32
Ro 30 13 R 32 23 28 18
Ax 10 28 30 R 20 26 8
Ce 30 20 R R R R 10
Cs 75 27 23 36 27 30 R
Cw 15 30 28 29 25 16 27
Sc 5 6 41 28 34 20 21 Cu 30 21 28 33 40 42 11
91
Ab+ve mcg
Isolates with gram negative character from O.M.6.2
O.M
.C12
O.M
.C13
O.M
.C1 4
O.M
.D11
6
O.M
.D17
O.M
.D18
O.M
.D11
4
O.M
.D11
5
O.M
. E12
O.M
. E11
Ak 15 30 22 21 R 22 29 33 36 33 33
Lo 30 33 22 11 20 27 R 41 17 35 22
Cq 10 26 R 28 24 19 33 36 R 21 11
Sc 5 27 35 36 44 22 35 39 41 38 20
Nt 30 20 10 17 28 29 21 40 43 31 27
Ca 30 30 31 36 30 30 38 7 30 35 R
Ci 10 11 R 6 36 32 31 5 18 20 40
Cf 30 4 12 R 30 6 10 6 11 18 22
Ce 75 32 22 33 32 37 31 22 26 17 23
G 15 21 28 42 35 43 R 33 8 36 R
Cs 5 7 31 19 20 20 26 45 9 31 21
Table 3.3.12C: Antimicrobial properties of isolates for O.M.6.2 site
Fig.3.3.9A: % of isolates displaying
positive results in antimicrobial test for
O.M.6.5
Fig.3.3.9B: Distribution of organism
on the basis of antimicrobial test
92
Fig.3.3.9C: % of positive tests for
O.M.6.2 Gram positiive isolates
Fig.3.3.9D: Distribution of organism
on the basis of antimicrobial tests for
O.M.6.2
Fig.3.3.9E: % of positive tests for
O.M.6.5 Gram positive isolates
Fig.3.3.9F: Distribution of organism
on the basis of antimicrobial tests for
O.M.6.5
93
Overall, for O.M.6.5 site; the isolates were highly resistance towards Cu, Se, Cw, Ce,
Ro, Cf, while in response to Cs and Cq around 14% of isolates were sensitive,
however sensitivity towards Ax was more than 50% (Fig. 3.3.9.1). With reference, to
gram positive isolates of O.M.6.2; it was observed that all the isolates were having
85% sensitivity towards all the tested antibiotics except Ak and Ce (Fig.3.3.9.2). With
respect to gram negative isolates; there was not much diversity noticed and all the
isolates were found sensitive to the array of the antibiotics (Fig. 3.3.9.3). In
comparison to O.M.6.2, isolates of O.M.6.5 were found more sensitive (Table 3.3.12).
In general, the isolates were sensitive against antibiotics that affect purine
biosynthesis and DNA replication. Isolates showed higher degree of resistance against
antibiotics which affect the protein synthesis. However, the molecular and genetic
basis of the resistance remains to be unexplained and further investigations would be
required in this context. Nevertheless, antibiotic resistance genes, in general, are
plasmid born (Vargas and Nieto, 2004).
3.3.7 Screening isolates for functional attributes by detecting
proteolytic activity All the isolates earlier studied for other classical and molecular parameters to judge
their diversity profile; were also assessed for its proteolytic activity by subjecting
them to protein rich medium under sets of conditions as described in materials and
methods sections. Efficiency of proteolytic secretion was done at varying physico-
chemical parameters; pH, temperature and NaCl (Table 3.3.13).
Among the total isolates screened from both the isolates; it was found that isolates of
O.M.6.2 were able to secret protease in large amount as compared to other site. From
total isolates screened for protease secretion on gelatin agar plate; total 17 isolates
were found protease positive. Within them around 78% were from O.M.6.2 (Fig.
3.3.10.2). In O.M.6.2; it was observed that two isolates O.M.A16 and O.M.A18 were
able to produce profound amount of enzyme. All the isolates enriched at pH-10 with
NaCl 10% and pH-10 with NaCl-30% were protease positive along with O.M.E12.
94
Table 3.3.13: Screening for extracellular enzyme (proteases) in isolated cultures;
effect of physico-chemical factors (pH, temperature and NaCl) on enzyme secretion.
Screening was done on gelatin agar plate
3.3.7.1 Protease secretion with varying NaCl concentration
Efficiency of protease secretion and growth was judged by varying salt concentration
in the range of 0-25%. In detail; isolate O.M.A16 was able to grow in range of 0-15%;
while protease secretion was noticed in 0 and 5% NaCl. Isolate O.MA17 was unable
to secrete protease however, organism were able to grow upto 20% of NaCl
concentration. Similarly, O.M.A18 was able to secrete enzyme upto 10; however with
increase in NaCl, enzyme secretion was reduced and growth was enhanced. It is
clearly demonstrated there is not straightforward role between growth and enzyme
Isolates Protease activity
Range of pH
Optimum pH
Range of
NaCl (%
w/v)
Optimum NaCl
(% w/v)
Range of Temperature
(oC)
Optimum Temperature
(oC)
O.M.A11 -- --- --- --- --- --- --- O.M.A14 -- ---- ---- ---- ---- ---- ---- O.M.A16 ++ 8-10 9 5 5 37- 50 37 O.M.A17 -- --- -- -- -- -- -- O.M.A18 +++ 8-10 9 0-10 10 37- 50 50 O.M.C11 +++ 8-10 9 0-10 0 37- 50 37 O.M.C12 +++ 8-10 9 5-10 5 37- 50 37 O.M.C13 +++ 8-10 8 5-10 5 37- 50 37 O.M.C14 +++ 8-10 9 0-10 0 37- 50 37 O.M.D16 +++ 8-10 9 0-10 5 37 37 O.M.D17 +++ 8-10 9 0-10 5 37 37 O.M.D18 +++ 8-10 9 0-10 5 37 37 O.M.D13 +++ 8-10 9 0-10 5 37 37 O.M.D14 +++ 8-10 9 0-5 0 37 37 O.M.D15 +++ 8-10 9 0-5 5 37 37 O.M. E12 +++ 8-11 11 5-20 10 37 37 O.M. E11 -- -- -- -- -- -- -- O.M.A21 -- -- -- -- -- -- -- O.M.A22 -- -- -- -- -- -- -- O.M.A23 -- -- -- -- -- -- -- O.M.A24 -- -- -- -- -- -- -- O.M.A25 -- -- -- -- -- -- -- O.M.A27 -- -- -- -- -- -- -- O.M.A28 -- -- -- -- -- -- -- O.M.B28 -- -- -- -- -- -- -- O.M. B21 -- -- -- -- -- -- -- O.M. B22 -- -- -- -- -- -- -- O.M.B23 +++ 8-10 8 0-10 5 37-50 37 O.M.C22 +++ 8-10 9 0-5 0 37-50 50 O.M.C23 +++ 8-10 9 0-5 0 37-50 50 O.M.C24 +++ 8-10 9 0-10 10 37-50 37 O.M.C25 -- -- -- -- -- -- -- O.M.C26 -- -- -- -- -- -- -- O.M.C28 +++ 8-10 9 0-10 10 37-50 37
95
secretion among the isolates, in fact enzyme secretion is noticed only among the early
growth. Along the same line; halotolerant organisms would have adopted to some
alternate strategy to grow in protein rich medium without secreting protease into the
medium. Both O.M.B23 and O.M.B28 were able to grow and secrete protease in range
of 0-10% NaCl, O.M.B28 was secreting protease in 5-10% NaCl. As organism was
unable to secret enzyme at 0% NaCl concentration; it clearly demonstrates that
although organism were not able to secrete enzyme in higher range of concentration;
it was mandatory for enzyme secretion, this describes the haloalkaline nature of the
studied isolates.
Similar results were obtained for C series of organisms from same site. Within the D
series of the isolates; the unique feature which was observed that with increase in
NaCl concentration, the growth of organisms were increased 9-10 times in D113,
D114; D117; D118.; although the organisms were halotolerant in nature, the increase in
growth with increase in NaCl emerged as a noticeable feature (Fig 3.3.10).The
moderate haloalkaliphilic organism with unique features was O.M.E12, an organism
was able to grow and secrete enzyme in range of 5-20%, with optimum in range of
10-20% NaCl (Table 3.3.13).
3.3.7.2 Effect of pH on enzyme secretion
For all the protease producers; effect of pH was assessed on enzyme secretion profile.
The variable pH of enzyme; pH-8-11 was set by supplementing 20% Na2CO3. All the
organisms were able to secrete enzyme in alkaline environment. Organisms were able
to secret protease in a broader range from pH-8 to 10, for O.M.E12 it was up to 11 pH.
Optimum level of secretion was noticed at pH-9 for all isolates, except O.M.E12 and
O.M.B23 having optimum pH-11 and 8 respectively for enzyme secretion. There was
no variable pattern observed for enzyme secretion for both the sites, no striking
diversity was observed with respect to pH (Table 3.3.13).
3.3.7.3 Effect of Temperature on enzyme secretion
The effect of temperature was analyzed on the enzyme secretion profile; protease
enzymes from all the isolates were having optimum temperature 37°C; except
O.M.C23 and O.M.C24 isolates of O.M.6.5 having 50°C. All the isolates were able to
secrete enzyme in quite broad range from 37-50°C; however organisms of D series
from O.M.6.2 site were able to secrete enzyme only at 37°C (Table 3.3.13).
96
Fig. 3.3.10: Effect of NaCl on growth and enzyme secretion on different organism of
O.M.6.2 and O.M.6.5 site.
Col
ony
Dia
met
er (m
m)
Rat
io (m
/z)
97
Contd...
Col
ony
Dia
met
er (m
m)
Rat
io (m
/z)
98
3.3.8 Alkaline protease amplification All the sets of primers designed for alkaline proteases were used for amplification
(Table 3.3.14). The detail description of designed primer and its specificity is as
described in materials and methods. For, amplification of aprox. 700-bp to 1.2 kb
ORF of the protease gene, the genomic DNA of protease positive strains were
isolated. PCR reactions were carried out at three gradient of annealing temperatures
using Gradient Thermocycler (Eppendorf). By using different primers, PCR reactions
were carried, to ensure complete amplicon generation. For O.M.E12; amplicon size of
product obtained from SPS-6 was of aprox. 1kb.
The concentration of product varied with respect to gradient of temperature and
primer pair used for the amplification profile generation (Fig.3.3.14). Partial amplified
product was generated by SPS- 1, 4, 5. For O.M.A18; amplicon were generated by
using three different sets of primer; SPS-3, 4, 7. A quite satisfactory product size was
obtained, SPS-3 and SPS-4 generated 1 and 1.1 kb band, however SPS-7 generated
0.8 kb band. For O.M.A16, only partial amplified products were obtained of 0.3 kb
with SPS-1 and 0.5 kb with SPS-5 and SPS-7 (Fig 3.3.11). With reference to
O.M.C14, SPS-3 generated amplified band with product size of 1kb while SPS-1
generated only partial amplified band of product size 0.5 kb. For O.M.C13 a partial
amplified products were generated by SPS-5, 6 and 7. For O.M.C14; SPS-4 generated
intense amplified product of 1 kb while SPS-3 generated only partial product.
In general, numbers of amplicon, with product size ranging from 1.2-0.5 kb, were
visualized on agarose gel. While one of the reasons for the multiple bands from the
site could be due to the annealing of the primer at different sites within the template.
The different size of products was tapered with reference to primer pair combinations,
which is primarily due to specificity of primer sequence with template sequence at
variable positions. The product size was also dependent on Ta; and there was a
noticeable change in concentration of product with gradient of annealing temperature.
For assessment and quantification of PCR products; amplicons were resolved on an
agarose gel. Amplification of template was found with multiple primers.
99
Fig.3.3.11: Amplification profile for potential alkaline proteases producers.
3.3.9 Environmental Studies Sea water and soils are often contaminated with heavy metals or other compounds
from anthropogenic manufacturer of chemicals and oil industries Oren et al., 1992;
Margesin and Schinner, 2001). Thus, protecting the integrity of our biosphere
resources is one of the most essential environmental issues of 21st century. Usually,
biodegradation of commercial dyestuff is the friendliest method as it does not require
100
large amount of energy and does not generate toxic substances. Conventional
microbiological treatment employing normal flora do not function at alkaline pH and
high salt concentrations. To cope with this problem, we tried to explore the dye
degradation potential of haloalkaliphilic bacteria. Our initial studies on dye
decolorization indicated that some of our haloalkaliphilic bacterial strains could
decolorize the azo dyes within 6h of incubation. Among 3 isolates studied; O.M.E12,
O.M.A18 and O.M.C28 we found that O. M. C28, was potent candidate for the
decolorization of consortium of dyes. In secondary screening we emphasized
specially with various parameters as same as before we proceeded for other strain.
In addition to that each parameters where checked for influence of NaCl concentration
(0%, 5% and 10%) in dye decolorization. The extent of decolorization enhanced
under shaking condition at higher concentrations. Such properties would be useful in
biological waste treatment and bioremediation purpose. The progressive growth and
decolorization of acidred-4 dye by strain O.M.C28 under static and shaking condition
with respect to incubation time from 0-31 hr were noted for each salt concentration
(Table 3.3.15).
Incubation in static condition Incubation in shaking condition
Time in hr
Growth control
(660nm)
Growth (660nm)
Dye decolorization
( 527nm)
Percent Dye
decolorization
Growth control
(660nm)
Growth (660nm)
Dye decolorization
( 527nm)
Percent Dye decolorization
0 0.32 0.027 2.242 0.0068 1.256 0.32 2.242 0.0068
2 0.334 0.029 2.139 4.6006 1.346 0.334 2.139 4.6006
4 0.359 0.075 1.892 15.6168 1.569 0.359 1.892 15.6168
6 0.373 0.089 1.851 17.4454 1.694 0.373 1.851 17.4454
8 0.441 0.14 1.615 27.971 1.902 0.441 1.615 27.971
11 0.501 0.149 1.567 30.1118 1.91 0.501 1.567 30.1118
14 0.52 0.192 1.532 31.6728 1.976 0.52 1.532 31.6728
15 0.537 0.208 1.51 32.654 2.061 0.537 1.529 31.8066
18 0.596 0.246 1.25 44.25 2.279 0.596 1.51 32.654
20 0.601 0.281 1.215 45.811 2.322 0.601 1.297 42.1538
24 0.72 0.319 0.983 56.1582 2.393 0.803 1.25 44.25
27 0.803 0.419 0.743 66.8622 2.44 0.924 1.215 45.811
29 0.924 0.543 0.65 71.01 2.5 0.983 1.095 51.163
31 0.983 0.643 0.54 75.916 2.559 1.066 1.1 53.734
Table 3.3.15: Secondary screening of strain C-28 (5% NaCl concentration) to access the dye (Acidred-4) decolorizing potential in static and shaking flask conditions.
101
The study highlighted in this chapter, basically focuses on haloalkaliphilic bacterial
diversity and phylogeny, the study was planned to asses the microbial heterogeneity
among the haloalkaliphilic bacteria isolated from the unexplored habitat of Gujarat by
using certain conventional and traditional approaches. Such primary and basic
information‟s are really helpful to understand the biochemical, physiological and
genetic basis of these organisms. Besides, their investigation is likely to generate
enough knowledge towards many basics questions of biology. We have attempted to
explore some unique and novel properties of the organisms.
CHAPTER Launch Internet Explorer Brow ser.lnk
CHAPTER
PURIFICATION &
CHARACTERIZATION OF
ALKALINE ROTEASES
4
102
4.1 INTRODUCTION
A fraction of microbial world on earth has been commercially explored and more than
3000 different enzymes have been screened and characterized from mesophilic
organisms (Horikoshi et al., 2011). However, similar attempts on this enzyme from
extremophilic organisms are less attended (Horikoshi et al., 2008). In view of these
limitations, scientists have diverted their attention towards exploration of enzymes
that function under extreme conditions, in which their mesophilic counterparts could
not survive. A wide variety of biotechnological products such as bacteriorhodopsins,
halorhodopsins, compatible solutes, biopolymers, biosurfactants, exopolysaccharides,
polyhydroxyalkanoates, flavouring agents, extracellular enzymes (isomerases,
nucleases, amylases, cellulases, chitinases, proteases and lipases), anti-tumor drugs
and liposomes are of halophilic/haloalkaliphilic origin (Raj et al., 2010). In addition,
they play important roles in fermenting fish sauces, modifying food textures and
flavors, and in transforming and degrading waste and organic pollutants (Toyokawa,
2010). In particular, proteases constitute one of the most important groups of
industrial enzymes and account for about 60% of the total worldwide enzyme sales
(Horikoshi et al., 2008).
The biocatalysis from extremozymes contribute to filling the gap between chemical
and biological processes. Production of extremozymes can be improved and explored
from laboratory scale to large scale for the industrial application by increasing the
biomass production through optimization strategies (Dodia et al., 2008a and b; Joshi
et al., 2008; Ramesh et al., 2009; Manikandan et al., 2009a, b, c). Alternatively, the
gene encoding the biocatalyst can be cloned and expressed in a suitable mesophilic
host, to obtain large quantities of enzymes (Zhang et al., 2008a and b; Boominadhan
et al., 2009; Ni et al., 2009). Moreover, the cultivation is relatively easier and chances
of contamination during the large scale production would be highly minimized.
Besides, many haloalkaliphiles also produce extracellular pigments, osmolytes and
compatible solutes that could be obtained as byproducts. Alkaline proteases from such
extremophiles can be subjected to harsh environments including elevated
temperatures, high pH and salt; non aqueous medium, surfactants, bleach chemicals
and chelating agents where applications of many other enzymes are limited because of
their low activity or stability (Gupta et al., 2008; Manikandan et al., 2009).
103
Bacteria display large variations in optimum growth temperatures, often reflected in
thermal stabilities of their extracellular enzymes. Over the years, Bacillus species
have emerged as prolific producers of extracellular proteases with a potential for wide
range of applications in detergent, food, pharmaceutical, leather and chemical
industries (Aguilar et al., 1998; Patel et al., 2005; Patel et al., 2006; Thumar and
Singh, 2007a and b; Carolina et al., 2008; Reza et al., 2008; Gupta et al., 2008; Dodia
et al., 2008a and b, Manikandan et al., 2009; Thumar and Singh, 2009;
Boominadhan et al., 2009; Tayokawa et al., 2010).
So far, only few alkaline proteases are purified and characterized from the halophilic
and haloalkaliphilic bacteria, which is primarily due to the difficulties associated with
the protein stability in the absence of salt (Dodia et al., 2008 a and b; Patel et al.,
2006). Characterization of such haloalkaliphilic enzymes would provide important
clue, for the adaptation strategies and stability of the biomolecules under which they
can sustain with more than one extremity of NaCl, pH and sometimes temperature
(Sorokin et al., 2008). The folding, stability and therefore, function of proteins are
critically dependent on the interactions between macromolecules and the solvent in
which they are placed (Gupta et al., 2008; Thumar and Singh, 2009). It is necessary
and interesting to investigate, whether the folding and functioning of recombinant
protein is identical to normal protein (Berzovancky and Shaknovich, 2008).
The present investigation focused on the exploration of purification and
characterization of enzymes of haloalkaliphilic bacteria from the unexplored salt
pane, Okha Madhi, Gujarat, India. As described in chapter-3, several isolates secreted
a novel serine protease active at wide range of alkaline pH, salt and moderate
temperatures. In the present chapter, we have also described a purification and
characterization of protease from a two potent haloalkaliphilic bacterial strains;
Haloalkaliphilic bacterium O.M.A18 and Oceanobacillus iheyensis O. M. E12.
104
4.2 MATERIALS AND METHODS
4.2.1 Production of alkaline protease in liquid culture The inoculum was prepared by adding a loop full of pure culture of protease
producers into 50ml sterile CMB medium (10% (w/v) NaCl; pH 9) and incubated at
37°C on environmental shaker (100rpm) for 24h. From the activated culture, 5% (v/v)
inoculum (A540; >1) was inoculated into 100ml Gelatin Broth (GB) containing (g/liter:
Gelatin, 10; Peptone, 5; Yeast extract, 5; KH2PO4, 5; NaCl, 100; pH 9). Culture
samples were withdrawn aseptically and the growth was measured at A540. Samples
were centrifuged at 10,000 rpm for 10min at 4°C and the cell free extract was used as
crude enzyme preparation to estimate protease activity.
4.2.2 Protease and Total Protein estimation
Alkaline protease activity was measured by Anson-Hagihara's method
(Hagihara,1958).The enzyme (0.5 ml) was added to 3.0 ml casein (0.6%, w/v in
20mM NaOH-Borax buffer, pH 10) and the reaction mixture was incubated at 37°C
for 20 min. The reaction was terminated by the addition of 3.2 ml of TCA mixture
(0.11M Trichloro acetic acid, 0.22M Sodium acetate, and 0.33M Acetic acid) and
incubated at room temperature for 20 min. The precipitates were removed by filtration
through Whatman-1 filter paper and absorbance of the filtrate was measured at 280nm.
One unit of alkaline protease activity was defined as the amount of enzyme liberating
1μg of tyrosine per minute under assay conditions. Enzyme activity was measured
using tyrosine (0-100μg) as standard. Protein was measured by using Bradford
method (Bradford, 1976) with bovine serum albumin as standard protein.
4.2.3 Partial purification of proteases The enzyme was partially purified by ammonium sulphate fractionation. The
organism O.M. A18 and O.M. E12 were grown as described above and after 72 hour
of growth, the cells were separated by centrifugation at 6,000 rpm, 4°C for 15 min.
The proteins in the culture supernatant were precipitated by ammonium sulphate
(80% saturation, w/v) and the precipitate was suspended in the minimum volume of
20mM Borax-NaOH buffer (pH-10). The enzyme activity and total protein were
measured as described earlier. After dialysis against the same buffer, the dialysate
were concentrated on sucrose bed and loaded on SDS- PAGE.
105
4.2.4 One-step purification by hydrophobic interaction
chromatography Purification was achieved by a single step purification method using hydrophobic
interaction chromatography. Hydrophobic interaction chromatography on a phenyl
sepharose 6 FF column (1 cm × 6.5 cm), equilibrated with 0.1 M sodium phosphate
buffer (pH 8.0) containing 1 M ammonium sulfate, was performed. The crude
protease preparation (20.0 ml in 1M ammonium sulfate) was loaded onto this column
and the bound enzyme was eluted by 0.1 M sodium phosphate buffer, pH 8.0
containing decreasing ammonium sulfate (1000mM, 500mM, 200mM and 100mM).
Fractions at a flow rate of 0.7 ml min−1 were collected by BIO-RAD fraction collector
(BIO-RAD, California, USA) and analyzed for protease activity. The protein content
was measured according to the method of Bradford (1976), using bovine serum
albumin (10µg/ml) as a standard (4 - 20µg/ml). The purity of the enzyme was judged
on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The
active fractions were pooled and used for further characterization.
4.2.5 SDS-Polyacryalamide gel electrophoresis Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was
carried out according to the method of Laemmli using 12% cross linked
polyacrylamide gel. To monitor crude, partially purified and purified fractions of
enzyme preparations, the respective samples were loaded onto the gel. The status of
purity and molecular weight was determined with reference to molecular weight
marker (Broad Range Marker, Merck Life sciences). The protein bands were
visualized on the gel by comassie blue staining.
4.2.6. Enzyme characterization
4.2.6.1 Effect of NaCl on protease activity and stability
For the influence of NaCl on enzyme activity, the reaction mixture was supplemented
with 0-3MNaCl. Assay was carried out at optimum temperature. To study the stability
of protease, the enzyme was incubated with NaCl in the range of 0-4M NaCl and the
aliquots were withdrawn at regular time intervals for monitoring residual activity. The
protease activity in the absence of additional NaCl was considered as a 100%.
106
4.2.6.2. Effect of pH on protease activity and stability
The effect of pH on protease activity was determined by preparing the substrate in
various buffers (20mM) at different pH. Reaction mixtures were incubated for 20mins
at optimum temperature. The different buffers used were: Citrate phosphate (pH 5-7);
Succinate (pH 5.5-6); Sodium Phosphate (pH 6-8); Tris-HCl (pH 8-9); NaOH-Borax
(pH 9.5-10); Glycine-NaOH (pH 9-11.5); Bicarbonate (pH 9.5-10.5) and KCl-NaOH
(pH 12-13). For the determination of pH stability, the pH of the enzyme was adjusted
to 5-13 with respective buffers (listed above). After incubation of 1, 2 and 24h, the
residual activities were estimated.
4.2.6.3 Effect of temperature on protease activity
The temperature profile for the protease activity was examined, by incubating the
assay reaction mixtures at different temperatures in the range of 37-90°C. The
protease activity was determined as mentioned in section (4.2.2).
4.2.6.4 Effect of NaCl on temperature profile of the protease
To investigate the effect of NaCl on temperature optimum, the substrate was prepared
with varying NaCl concentrations (0-3 M NaCl) and then the assays cocktail were
incubated for 20min at different temperatures; 37-90°C.
4.2.6.5 Thermostability of protease
The thermal stability of protease was studied by incubating the enzyme at different
temperatures (37-90°C). The aliquots were withdrawn at every 10 min intervals up to
120min and the reaction mixture was incubated at optimum temperature. Then, the
residual protease activities were measured.
4.2.6.6. Effect of NaCl on the thermostability of protease
In order to determine the influence of NaCl on thermostability, enzyme was incubated
at various temperatures (60-90°C) for 20min with varying concentrations of NaCl
(0.25-3M). The aliquots were withdrawn at definite time interval and the residual
activities were calculated.
4.2.6.7 Denaturation kinetics of proteases and commercial enzymes
The proteases were incubated with 2-8M urea. The aliquots were withdrawn at
definite time intervals and subjected to protease assay. In case of commercial
enzymes, the working stock of the enzyme was prepared at 1mg/ml. The % residual
107
activities were calculated by considering the activity of the urea -untreated enzyme as
100%
4.2.6.8 Effect of cations on protease activity
Various cations were assessed for their effect on enzyme activity by supplementing
the substrate with varying concentrations of divalent and monovalent cations under
standard assay conditions. The cations used were; NaCl (0-3M); CaCl2 and K2HPO4
(0-5M) and MgCl2 (0-1M).
4.2.6.9 Effect of various inhibitors on protease activity
To study the effect of inhibitors, the reaction mixtures were supplemented with
various protease inhibitors (0-10mM). After incubation at optimum temperature for
20 min, the residual enzyme activities were calculated. The inhibitors were; p-
Chloromercuribenzoic (p-CMB), dithiothritol (DTT), Phenyl Methane Salfonayl
Fluoride PMSF (PMSF), Phenethroline, Thiourea and EDTA (Ethylene Diamine
Tetra Acetate).
4.2.6.10 Effect of reducing and oxidizing agents on the protease activity
Hydrogen peroxide (oxidizing agent) and β-mercaptoethanol (reducing agent) were
supplemented at 0-50mM final concentrations in the reaction mixture. The assay was
carried out at the optimum temperature.
4.2.6.11 Effect of surfactants on protease activity
Different surfactants such as SDS, Tween-80 and Triton X-100 were incorporated into
the substrate at the final concentrations of 0-2% (w/v) and the protease assay was
performed as per standard protocol to estimate the protease activity.
4.2.7 Sequence prediction and analysis of the physicochemical properties of
proteases
Phylogenetic position and prediction of protein properties were carried out by
searching the National Center for Biotechnology Information NCBI; BLAST. The
amino acid sequences of proteases from O.M.A18 and O.M.E12 were determined by
Reverse translating the gene sequence on the basis of correct CDS frame (CLC
Workbench). Further, physicochemical properties of protein were analyzed by using
protein server database- Expasy proteomics tool.
108
4.3 RESULTS AND DISCUSSIONS
As described and discussed earlier in chapter 3, Haloalkaliphilic bacteria enriched at
higher NaCl concentrations (10-30%, w/v), and pH in range of 8-10; were screened as
a potent producers of alkaline proteases and produced significant amount of protease
in Gelatin Broth (GB) (Table 4.3.1).
As described earlier, besides their physiological importance, proteases constitute a
class of enzymes with great application potential (Horikoshi et al., 2008). To meet the
current industrial demand for the enzymes having specific features, there is constant
need to search for new sources. Generally, crude preparations of alkaline proteases are
widely used in many industries; however, purification of the proteases is important for
the better understanding of the structure and function relationship (Karan and Khare,
2010; Karan et al., 2011). Besides, many applications would require enzyme in
homogeneity. While several proteases from mesophilic microorganisms have been
purified and characterized and the corresponding genes are cloned; however, similar
studies from halophilic organisms are less frequently investigated (Carvalho et al.,
2008; Ni et al., 2009; Zhang et al., 2009).
Enzyme characterization from halophiles and haloalkaliphiles in general has been
restricted due to the coupled extremities of pH and salt (Madern et al., 2004) and thus,
the enzyme has been studied from only few haloalkaliphilic bacteria. Extracellular
alkaline proteases (Nep) from genus Natronobacterium magadi, a haloalkaliphilic
strain was partially purified and characterized by Zhang and his coworkers (2009).
Quite recently, two novel halotolerant extracellular metallo-proteases were purified
and characterized from a halotolerant Bacillus subtilis and Bacillus spp. (Manni et al.,
2009; Sudhir et al., 2011).
Earlier, our research group have published several reports of purifying and
characterizing enzymes of isolates of natural habitat, i.e sea water and saline soil from
coastal region of Gujarat (Patel et al., 2005; Patel et al., 2006a and b; Thumar et al.,
2007a and b; Dodia et al., 2008a and b; Joshi et al., 2008; Thumar et al., 2009). The
study reported in this chapter is an attempt to purify and characterize an alkaline
protease from two potent haloalkaliphilic strains; O.M.A18 and O.M.E12 from
109
artificial salt pane of Okha Madhi, Coastal Gujarat. A comparative study of enzymes
was drawn for its crude, partially purified and purified state preparations.
4.3.1 Purification of alkaline protease
4.3.1.1 Partial purification of alkaline proteases
O.M.A18 and O.M.E12 proteases were partially purified by ammonium sulfate
fractionation. For, 500ml of crude enzyme; specific activity of O.M.A18 enzyme after
partial purification was 13,380.60 with 12.41 fold purification and 18.63% yield. On
the other hand, the specific activity of O.M.E12 enzyme was 17049.6 with fold
purification and % yield of 16.465 and 16.33, respectively (Table 4.3.2A and 4.3.2B)
(Purohit and Singh, 2011).
4.3.1.2. Enzyme purification by hydrophobic interaction chromatography
Hydrophobic interaction chromatography has been a successful technique for the
purification of many alkaline proteases. The purification led to 23.75 fold purification
with a yield of 29.09% for O.M.E12 protease, while the corresponding values for
O.M.A18 were 17.33 and 14.77. The purification was achieved with specific activities
of 16945 and 24600 units for O.M.A18 and O.M.E12 proteases, respectively by single
step on phenyl sepharose 6 FF affinity column (Fig.4.3.1) (Table 4.3.2A and 4.3.2B).
SDS-PAGE revealed that the apparent molecular mass of the proteases were
estimated 35 and 29 kDa respectively by using Rf values of the reference proteins for
O.M.A18 and O.M.E12 respectively (Table 3.1) (Fig. 3.1) (Purohit and Singh, 2011).
Earlier, a single step purification technique with phenyl sepharose 6 FF column have
been successfully used to purify alkaline proteases from the haloalkaliphilic Bacillus
spp. in our laboratory (Gupta et al., 2005; Dodia, 2005; Dodia et al., 2005, Dodia et.
al., 2008a and b). The molecular mass of alkaline proteases from other
haloalkaliphilic and alkaliphilic Bacillus spp. were quite comparable with our values
(Gupta et al., 2005; Carolina et al., 2008). However, it was quite lower as compared
to some other halophilic and alkaline proteases, where the molecular weight ranged
from 60-130kDa (Lama et al., 2005).
110
Features O.M.A18 O.M.E12
1. Site and Isolation
Site of Isolation Okha, Gujarat, India Latitude 22.20 N, Longitude 70.05 E
Okha, Gujarat, India Latitude 22.20 N, Longitude 70.05 E
Phylogenetic
Identification Oceanobacillus iheyensis O.M.A18 –
(Gene bank Accession No. EU680961)
Haloalkaliphilic bacterium O.M.E12-
(Gene bank Accession No.- EU680960)
2. Growth and Protease production
pH 11( range 8-11) 11( range 8-11)
NaCl 15% (range 5-20%) 20% ( range 5-20%)
Temperature 37oC ( range 30 -50oC) 37oC ( range 30 -50oC)
3. Mol. weight 35kDa 29kDa
4. Enzyme Characteristics
4.1 Temperatures for
Enzyme catalysis Optimum
Temperatures Range Optimum
Temperatures Range
Crude 90oC (37 90oC) 50oC (37 -70oC)
Partially Purified 90oC (37 90oC) 50oC (37 -70oC)
Dialyzed 60oC (37 70oC) 50oC (37 -70oC)
Purified 50oC (37 50oC) 50oC (37 -50oC)
4.2 pH for Enzyme
catalysis Optimum pH Range Optimum pH Range
Crude 11 (9-11) 11 (9-11)
Partially Purified 11 (9-11) 11 (9-11)
Dialyzed 11 (9-11) 11 (9-11)
Purified 11 (9-11) 11 (9-11)
Table 4.3.1: Comparative profile on the Growth, Protease Secretion and enzymatic
characteristics of O.M.A18 and O.M.E12.
111
A
B
Table 4.3.2: Protease Purification from (A) O.M.A18 and (B) O.M.E12
Enzyme
Preparations
Activity
(U/ml)
Total
Units
Protein
(mg/ml)
Total
Protein
(mg)
Specific
activity
(U/mg)
Purifn
Fold
Yield
(%)
Crude 293.85 146925 0.3 150.25 978.68 1.0 100
Partially purified
fraction 2,943.74 44,156.1 0.220 3.3 13,380.6 12.41 18.63
One Step Purification
Phenyl
Sepharose 6FF 3101 21707 0.183 1.281 16945.35 17.33 14.77
Enzyme
Preparation
Activity
(U/ml)
Total
activity
(U)
Protein
(mg/ml)
Total
protein
(mg)
Specific
Activity
(U/mg)
Purifn
Fold
Yield
(%)
Crude 202.95 101475 0.196 98 1035.45 1.0 100
Partially
purified fraction 2,557.44 43476.48 0.150 2.55 17049.6 16.465 16.33
One Step Purification
Phenyl
Sepharose 6FF 5904.00 29520 0.240 1.20 24600.00 23.75 29.09
112
Fig.4.3.1: Protein Purifcation of haloalkaliphilic organism O.M.E12 and O.M.A18 by
phenyl Sepharose 6FF. SDS-PAGE analysis of protein purifcation: Lane 1: Crude
O.M.A18; Lane 2: Partially purified enzyme O.M.A18; Lane 3: Purified enzyme
O.M.A18; Lane 4: Protein molecular weight marker (3500-205kDa); Lane 5: Purified
enzyme O.M.E12; Lane 6: Partially purified enzyme O.M. E12; Lane 7: Crude
enzyme O.M.E12.
4.3.2 Effect of NaCl on protease activity
Effect of NaCl (25-3000mM) was investigated on the crude, partially purified and
purified alkaline protease activity. For O.M.E12; the activity of crude enzyme
decreased with the increasing concentrations of NaCl for the enzyme preparations.
Crude enzyme retained nearly almost 90% of its total activity up to 100mM NaCl;
however, at higher concentrations, the activity gradual declined, almost marginal
activity was retained at 2-3M (Fig. 4.3.2) (Table 4.3.1). Similar trends were also
observed for partially purified enzyme preparations. Where as, addition of NaCl to its
purified state enhanced the activity to 25-30%. The activity of partially purified
enzyme, however, gradually decreased and reached to 8% at 3000mM. Where as, the
activity of purified was gradually decline to its one-fifth of activity at 3000mM NaCl.
The trend of the reduction in activity was comparable for both the isolates. For, both
the isolates the effect of NaCl was quite distinct (Table 4.3.1) (Fig. 4.3.2)
(Purohit and Singh, 2011).
205kD
a
29kD
1 2 3 4 5 6 7
113
Fig. 4.3.2: Effect of NaCl on protease activity; Crude ( ), partially purified ( ) and
Purified ( )
4.3.3 Effect of NaCl on Enzyme Thermostability
Effect of NaCl on temperature profile was explored by incubating the reaction
mixtures supplemented with various concentrations of NaCl (0-3M) at different
temperatures; 37-80°C. An interesting trend emerged, when NaCl was added to the
reaction mixtures of both enzymes. The addition of NaCl led to the gradual shifting in
temperature optima towards higher temperatures with crude, partially purified and
purified proteases (Fig. 4.3.3). As the optimum range for protease catalysis were quiet
broad, influence of NaCl could not be observed at lower temperatures, an observation
also supported by our earlier report (Joshi et al., 2008). However, further increase in
A
ctiv
ity (U
/ml)
114
NaCl, resulted in a sharp decline in activity. It was also observed that NaCl enhanced
the activity coupled with the shift in temperature profile (Fig. 4.3.3).
As compared to crude O.M.A18 enzyme, the purified enzyme preparation had
enhanced catalysis at higher concentrations of salt. Thus, to retain its maximal activity
at higher temperatures, the enzyme required higher concentrations of NaCl. In
purified protease, the temperature optimum shifted from 60 to 70°C with 0.25M NaCl
and finally to 80°C with 2-3M NaCl (Fig. 4.3.3A). The requirement of NaCl increased
from 0.5 to 2M with the increasing temperatures, displaying a maximum activity at
2M for O.M.A18 at 80°C (Fig. 4.3.3B). The maximal activity enhanced by 6 folds
from 37 to 80°C with 2M NaCl and remained stable up to 3M NaCl (Fig. 4.3.3).
Features O.M.A1 8 O.M.E12
Thermostability of Enzyme
Time required to retain 50 % of the Residual Activity
Time required to retain 50 % of the Residual Activity
Crude 24 24
Partially Purified 48 48
Dialyzed 48 48
Purified 3 1
Chemical Denaturation of the Enzymes
Time (hours)to retain 100%
Residual Activity Temperature
Time(hours) to retain 100%
Residual Activity Temperature
Partially Purified 24 hrs 70oC 48 hrs 50oC
Dialyzed 24 hrs 80oC 24 hrs 60oC
Purified 30 min 90oC 1 hr 70oC
Table 4.3.3: Comparative profile on stability characteristics of O.M.A18 and
O.M.E12
115
Fig.4.3.3 A: Effect of NaCl on protease stability at different temperatures on alkaline
protease O.M.A18.
Rel
ativ
e ac
tivity
(%)
116
Fig. 4.3.3 B: Effect of NaCl on protease stability at different temperatures on alkaline
protease O.M.E12.
Rel
ativ
e ac
tivity
(%)
117
4.3.4 Effect of pH on protease activity
Effect of pH in the range of 8.0 to 11.5 was investigated for the purified enzyme
preparations. Enzyme was less active at lower moderate pH; around 8-8.5; the
optimum pH for enzyme secretion was 9 pH. However, there was not much variation
found in % relative activity with change in pH to 9.0 and 10.0; However drop down of
activity of around 15-20% observed in both the enzyme preparations (Fig 4.3.4). The
trend observed was quite similar for both the studied enzymes in its purified state. The
pH 9, 9.5 and 10; crucial for alkaline protease production was studied by incubating in
two different buffer systems, Glycine buffer (8.5-10) and Borax-NaoH buffer (9-11)
pH, although in our earlier studies we observed that change in buffers leads to drastic
variation in activity (Dodia, 2005; Joshi, 2006). However, similar observation was
noticed in our present studies. The trend displayed was quite similar in our both the
isolates, however loss of activity in O.M.E12 was quite significant, where with
increase in pH from 10, loss of activity was of 20% with increase in 0.5 pH
(Fig.4.3.4).
4.3.5 Effect of pH on protease stability
The pH stability of purified enzyme was investigated in the range of pH; 8-11.
Enzyme was found to be highly stable in crude and purified state. Stability of enzyme
was assessed at variable time interval; 0, 2 and 24 hour incubation. Comparing crude
and purified enzyme; it was observed that there is no noticable loss of activity in
purified enzyme (Fig.4.3.5). Although, there was significant diversity noticed with
reference to other characteristic properties of enzyme, no much change is noticed in
present studies.
118
% R
esid
ual a
ctiv
ity
%
Rel
ativ
e a
ctiv
ity
Fig: 4.3.4: Effect of pH on enzyme activity of alkaline proteases of O.M.A1 8
(Upper panel) and O.M.E12 (Lower panel)
Fig: 4.3.5: Effect of pH on enzyme stability of alkaline proteases of
O.M.A1 8(Upper panel) and O.M.E12 (Lower panel).
119
4.3.6 Effect of temperature on protease activity The effect of temperature on the activity of crude, partially purified, dialyzed and
purified enzyme preparations was evaluated at pH 10. The optimum temperature for
enzyme catalysis by O.M.A18 protease was over a wide range, 50-90oC; fact which
relates to rare enzymes from mesophilic groups. Moreover, we have not come across
with any protease having such a temperature profile from haloalkaliphilic organisms
(Fig. 4.3.6). However, this range of temperature profile was not displayed by dialyzed
and purified enzyme preparations. The dialyzed enzyme had optimum temperature at
60oC over a range of 37-70oC and for purified enzyme; it was 50oC with a relatively
narrow range of 37-50oC. On the other hand, O.M.E12 protease showed optimum
temperature around 50oC. The rate of catalysis increased significantly from 37 to
50oC, after which it declined leading to a total loss of activity at 90oC (Fig. 4.3.6).
Enzyme was able to maintain its activity in the range of 37-70oC, which turned
narrower with purified enzyme. The key point emerged; enzyme is highly
thermostable before purified state and decreased the thermostability when purified.
However, data holds novelty as very few reports are available where such a unique
characteristic are observed. Along, the same line, results are equally interesting from
diversity viewpoint due to its novel features; particularly with reference to moderate
haloalkaliphiles. To give insight into its characteristic features; comparative studies
on the growth patterns, enzyme production and enzymatic characteristics, as depicted
in Table 4.3.3, highlighted that although the organisms were isolated from the same
site, they had distinct properties. The foremost point which emerged from the study
was the optimum rate of catalysis over a broader range of elevated temperature with
significantly higher half-life of the enzyme.
4.3.7. Effect of NaCl on temperature profile for protease activity Effect of NaCl on temperature was further explored by incubating the reaction
mixtures supplemented with various concentrations of NaCl (0-3M) at different
temperatures (37-90°C). An interesting trend emerged, when NaCl was added to the
reaction mixtures. The addition of NaCl led to the gradual shifting in temperature
optima towards higher temperatures with crude, (Figure 4.3.7A), and other enzyme
preparations; partial purified and purified. Addition of 0.25M NaCl shifted the
temperature optimum from 50-60°C to 60-70°C.
120
Fig. 4.3.6: Effect of Temperature on Enzyme catalysis of crude, partially purified
and purified alkaline protease sample of strain O.M.A1 8(■) and O.M.E1 2(♦).
Act
ivity
(U/m
l)
121
The NaCl based temperature shift continued at higher and finally, it reached to 80°C
at 3M NaCl. Interestingly, addition of NaCl enhanced the activity coupled with shift
in temperature profile (Figure 4.3.7A). As the temperature increased from 37 to 70°C,
the requirement of NaCl gradually increased from 0.5 to 1M and then to 2M at 80°C
for the maximal activity. Moreover, the activity was enhanced by 4 folds with 2M
NaCl at 80°C. As mentioned above, to retain its maximal activity at higher
temperatures, the enzyme required higher concentrations of NaCl (Fig.4.3.7A).
However, the optimal activity sharply declined at 90°C even in the presence of 3M
NaCl. Similar trends were also observed for the partially purified and purified
enzymes. Characteristic features of O.M.E12 were as observed for O.M.A18, with
increase in salt concentration, the enzyme activity increased. As well synergistic
effect of NaCl and temperature was also noteworthy. However, as observed in our
earlier results, in present case, addition of NaCl up to 0.5M did not have any effect on
enzyme activity or temperature. With further supplement of NaCl, the effect of
temperature was very sharp, where optimum temperature at 0.5M was 50oC, which
shifted to 70-80oC at 2M concentration. However, further increase in NaCl, led to
drop down in enzyme activity and optimum temperature. The enzyme activity of
O.M.E12 was reduced to its one-third as compared to 2M NaCl in higher
concentration to 3M (Fig 4.3.7B).
4.3.8. Thermostability of protease The thermal stability of crude, partially purified and purified preparations of O.M.A18
enzyme were assessed for 48 hours at 50-90oC and pH 10. Similarly, thermal
denaturation of O.M.E12 enzyme was followed at 50, 60 and 80oC under similar
conditions. The O.M.A18 enzyme maintained its stability at temperatures; 60-90oC for
24 hours. However, on extending the time of incubation to 72 hour, less than 25% of
the original activity was retained (Fig. 4.3.8A.). Purified enzyme retained about 50%
of the activity after 3 hours of incubation at 60oC for OM.E12. A total loss in activity
of O.M.E12 enzyme was evident at all tested temperatures (50, 60, 80oC) when
incubated for 48 hours (Fig. 4.3.8B). The thermal denaturation patterns of O.M.E12
protease corresponds well with some of our earlier findings on haloalkaliphilic
enzymes, where at 50oC for 72 hours, there was a total loss in activity. Purified
O.M.E12 enzyme maintained around 25% of its activity for 3 hours at 90oC, while
O.M.A18 enzyme retained 30% of its activity at 80oC for 1hour. Thus, it was evident
122
that the alkaline proteases from two strains isolated from the same site displayed
distinct characteristics in terms of their catalysis and stability at high temperatures.
With particular reference to extracellular alkaline proteases, it’s evident from the
literature that the optimum temperatures for enzyme catalysis exceed those for growth
and enzyme production. It’s quite logical to suggest that the stability of proteases
could be due to their tertiary structures and genetic adaptability to carry out biological
functions at higher temperatures. The high activity at 90°C; probably may also be due
to the protection of the enzyme by substrate from heat inactivation under the assay
conditions. The temperature response of O.M.E12 protease, on the other hand,
resembled with the patterns of temperature profiles and thermal stability of other
proteases, as reported in literature (Dodia et al., 2008 a and b, Carolina et al., 2008,
Gupta et al., 2008). The comparative data on the temperature stability and
denaturation profile of O.M.E12 and O.M.A18 alkaline proteases suggested that
O.M.A18 enzyme was extremely resistant against thermal and urea denaturation
(Purohit and Singh, 2011). Chemical denaturation profile of both organisms indicated
that the action of denaturant was salt independent. However, no variation in the trend
was observed in partially purified enzyme indicating that the other proteins might not
be playing a role in enzyme protection. Some of the previous studies including our
own have earlier established that denaturation of certain enzymes from
haloalkaliphilic organisms was significantly affected by NaCl and presence of other
proteins (Dodia et al.,2005; Joshi, 2006; Joshi et al., 2008; Dodia et al., 2008 a and b).
4.3.9. Denaturation kinetics of proteases with urea The partially purified and purified proteases from O.M.A18 and O.M.E12 were
subjected to urea denaturation. For partially purified enzyme of O.M.A18 was
exceptionally resistant against urea denaturation and retained 30% activity at 90oC
after 48 hours with 8M urea, followed by nearly a total loss of activity on further
incubation to 72 hours (Fig.4.3.8A).
123
Fig.4.3.7: Effect of NaCl on temperature optima of alkaline proteases of O.M.A1
8(Upper panel) and O.M.E12 (Lower panel). Activity was measured at different
temperature; 37-90oC.
The O.M.E12 enzyme, however, was relatively less resistant to urea denaturation and
retained 50% activity after 24 hours in 8M urea, with a complete loss in activity at 72
hour (Fig. 4.3.9B). Percent residual activity was related to zero hour enzyme activity
as 100%. To assess the effect of NaCl on urea denaturation, the above studies were
conducted with the dialyzed enzyme preparations. The findings revealed that there
was no significant change in the denaturation profile of crude, partially purified and
dialyzed samples (Fig. 4.3.9 A and B). However, a change in the profile of purified
% R
elat
ive
act
ivity
124
enzyme was evident indicating enhanced sensitivity against denaturant, with a loss of
80% activity after 2-3 hours at 70oC for O.M.E12, leading to a total loss of activity at
80oC for the same period of incubation (Fig. 4.3.9) (Purohit and Singh, 2011).
4.3.10 Denaturation kinetics of commercial enzymes with urea Denaturation studies with urea were also extended to 2 commercial hydrolytic
enzymes; pepsin and papain to compare the resistant nature of our enzymes against
urea denaturation. Pepsin was relatively more resistant as compared to papain. It
retained 50% of its original activity after 3h of incubation with 4 M urea (Fig.4.3.9
C). However, at 8M urea, the enzyme lost all activities within 3h. Papain was much
more sensitive to urea as within 1h of incubation, the enzyme lost 60-70% of the
original activity with 8M urea and was completely denatured after 24h (Fig. 4.3.9D).
4.3.11 Effect of cations on crude protease activity
Effects of various cations with varying concentrations were investigated on the
activity of the crude and partially purified enzyme. Crude enzyme responded variably
towards effect of cations. Crude enzyme was stable in the presence of KCl upto
50mM, with increase in concentration there was marginal loss in activity; 20% loss in
activity was observed at 10mM and subsequently with further increase in cation
concentration, enzyme activity was reduced by 120%.
Similar, results were also observed for NaCl, where 92% of enzyme activity was
retained up till 250mM. Enzyme activity was increased in presence of K2HPO4; the
activity enhanced only marginal with increase in concentration upto 250mM while
35% of activity was enhanced at 500mM concentration. The activity reduced
significantly in presence of CaCl2, where 50% activity was lost at 500mM CaCl2 and
only sparse amount of activity was maintained thereafter. MgCl2 affected differently
where 60% activity was retained at 25mM and it remained almost static up to
250mM. The activity was enhanced to 120% at 500mM and then further reduced to
30% at 1000mM (Fig. 4.3.10).
C D
125
.
B
Fig. 4.3.8: Effect of NaCl on thermostability at different temperatures {50oC (♦); 60oC (■);
80oC (▲)} on alkaline protease O.M.A1 8(A) and O.M.E12 (B).
A
126
Fig. 4.3.9: Effect of urea denaturation on alkaline protease O.M.A1 8 (A) at different
temperatures; 70oC (♦); 80oC (■); 90oC (▲) and alkaline protease O.M.E12 (B) at
different temperatures; 50oC (♦); 60oC (■); 70oC (▲).
127
Fig. 4.3.9 C & D: Denaturation of Commercial enzymes; Pepsin( C) and papain (D)
with 4M urea (Left panel) and 8M urea (Right panel) at different temperatures 37°C
(▬) and 50°C (▬).
Comparatively, for O.M.E12; similar trend was observed as of O.M.A18 enzyme,
however, the activity of the enzyme was significantly enhanced in presence of
K2HPO4 to 142%. In addition, the activity was also enhanced by 100mM NaCl
(130%). The activity was not affected by KCl upto 50mM and around 15-20% loss of
activity was observed at further increased concentration.
4.3.12. Effect of cations on purified protease
Effect of cations was also assessed on purified proteases, where although enzyme
activity was found to be reduced significantly with reference to its crude state; in its
purified form activity was retained to 100%. Similiarly for MgCl2 and CaCl2 activity
was reduced to 95-60%.Comparing the studies with crude enzyme; enzyme was able
to maintain more activity in presence of inhibitors (Fig. 4.3.11). For purified enzyme
preparations; trends were distinctly similar for both the enzyme preparations; where
enzyme activities were marginally reduced with supplementing of MgCl2 and
K2HPO4, however there was no change of activities with other cations when
compared with control sets as described in 4.3.11.
% R
esid
ual a
ctiv
ity
128
Fig.4.3.10: Effect of various cations {CaCl2 (●), MgCl2 (♦), NaCl (▲), KCl (×)} on
crude protease enzyme.
% R
esid
ual a
ctiv
ity
129
Fig.4.3.11: Effect of various cations (CaCl2, MgCl2, K2HPO4, HgCl2, NaCl, KCl) on
protease activity (Upper panel: O.M.A18 and Lower panel: O.M.E12).
4.3.13. Inhibition studies on protease activity Effect of various inhibitors (0-10mM) was investigated on the purified enzyme. The
purified protease was completely inhibited by Phenyl methane salfonayl fluoride
(PMSF) (10mM), a specific inhibitor of serine proteases. However, 60 and 38% loss
of activities were noticed at 5 and 10mM of p-Chloromercuribenzoic (pCMB, an
inhibitor of thiol protease), respectively. However, the activity of purified enzyme
was enhanced in the presence of 1-10mM dithiothritol (DTT), another inhibitor of
thiol protease. The enhancement of activity by 5-10mM DTT was 50-60%. The
activity, however, was not affected by the EDTA and thiourea, which are
metallo-protease specific inhibitors (Fig. 4.3.12). The effects of both the studied
enzyme were quite similar and displayed similar trend. Both the alkaline proteases
were characterized as serine proteases, based on strong inhibition by PMSF.
% R
elat
ive
activ
ity
130
Fig. 4.3.12: Effect of inhibitors: Thiourea, p- henylmethanesulfonyl Fluoride (PMSF),
(EDTA), Dithiothritol (DTT on O.M.A18 in crude (♦), partial purified (■) and purified
state (▲).
Fig. 4.3.12: Effect of inhibitors: Thiourea, p-henyl methane sulfonyl Fluoride
(PMSF), (EDTA), Dithiothritol (DTT on O.M. A18 in crude, partially purified and
purified enzyme preprations at variable concentration where bar represents; 0 mM
(■), 1mM(■); 5mM(■) 10mM(■).
% R
esid
ual a
ctiv
ity
131
Majority of the alkaline proteases of halophilic and haloalkaliphilic origins were
classified as serine proteases (Gupta et al., 2005; Lama et al., 2005; Dodia, 2005;
Patel et al., 2006b; Joshi et al., 2008; Dodia et al., 2008; Manikandan et al., 2009).
However, some are also metalloproteases (Manni et al., 2008; Sorror et al., 2009).
Fig. 4.3.12: Effect of inhibitors: Thiourea, p- henylmethanesulfonyl Fluoride (PMSF),
(EDTA), Dithiothritol (DTT) on O.M. E12 in crude (♦), partial purified (■) and
purified state (▲).
4.3.14. Effect of reducing and oxidizing agents on the protease
activity and stability Effect of H2O2 and β-mercaptoethanol was investigated on the activity of purified
protease. The enzyme activity was reduced with the increasing concentrations of H2O2
and β-mercaptoethanol. About 20% activity was lost with the addition of 5mM β-
mercaptoethanol, beyond which, the activity was quite stable up to 25mM. With
further increase in concentration to 50mM around 50% loss of activity was observed.
For, O.M.E12, reduction of activity was noticed with increase in effectors
concentration. However, the rate of reduction was gradual process. With increase in
effectors concentration, activity was getting declined by 10%; with increase in
% R
esid
ual a
ctiv
ity
132
concentration from 25 to 50%, around half of the total percentage of activity was
retained. In case of β-mercaptoethanol, 100% activity was retained upto 10mM after
which it decreased to 45% at 50mM (Fig. 4.3.13).
Fig.4.3.13: Effect of oxidizing and reducing agent on purified alkaline proteases
O.M. A18 (Upper panel) and O.M.E12 enzyme (Lower panel) where H2O2 (■) and
β-mercaptoethanol (▲).
4.3.15. Effect of surfactants on protease activity and stability Effect of SDS, Tween-80 and Triton X-100 was investigated on the crude, partially
purified and purified protease preparation. In general, the enzyme was found to be
highly stable in the presence of SDS (Fig.4.3.14) and Tween-80 (Fig. 4.3.14); along
the same line, Tween-80 within limited concentrations also enhanced the protease
activity however; there was profound decline of activity when substrate was
supplemented with Triton X-100 (Fig.4.3.14).
For O.M.A18, crude enzyme retained 100% activity in the presence of 0.6 and 0.8%
of Tween 80. With further increase in concentration; only 50% of the total activity
was retained with total loss of activity at 2% of surfactant concentration. There was
no variable in trend observed at any of enzyme preparations for Tween 80.
The activity of enzyme was enhanced in magnitudes in presence of Trition X-100.
A bell shaped result was observed with respect to the said surfactant, 0.4% of Tween
80 was found optimum for all the studied enzyme preparations. The activity of
% R
esid
ual a
ctiv
ity
133
purified enzyme was enhanced 4 times to its original; similarly activity of other
enzyme preparations was also enhanced; however there was difference of two times in
partially purified and crude enzyme (Fig. 4.3.14).
Fig.4.3.14: Effect of surfactant on crude, partially purified and purified alkaline
proteases of O.M.A18; where SDS (■), Tween 80(■) and Triton X-100(■).
Enz
yme
activ
ity (U
/ml)
134
Fig. 4.3.14: Effect of surfactant (SDS, Tween 80 and Triton X-100) on crude (♦)
partially purified (■) and purified (▲) alkaline proteases of O.M.A18.
% R
es
idu
al a
cti
vit
y
% R
elat
ive
activ
ity
135
3.9 Amino acid sequence prediction and analysis of the enzyme
Protein similarity and phylogenetic analysis was carried out by using nBLASTp
(prediction of protein sequence by submitting nucleotide sequence as a query) to
identify the protein. Predicted N-terminal sequence for; O.M.A18 was
5’MNPGSAWRSPVVPFSSLGMSPAYG and that for O.M.E12 was
5’KLRVIIEFKEDAVEAGIQSTKQLMKK.
Features O.M.A18 enzyme O.M. E12 enzyme
Basis Information
N-terminal Sequence 5’MNPGSAWRSPVVPFSSLGMSPAYG 5’KLRVIIEFKEDAVEAGIQSTKQLMKK…
Original
Source(Sequence) Nucleotide Nucleotide
Homology (%) 100 100
Homologus protein (BLAST analysis)
Bacillus sp.KP43, complete CDS
gene-protease gene
Bacillus pumulius SAFA-032 -protease gene
NCBI Genbank ID: HM219179 HM219182
Physicochemical Properties
pI 5 5
instability index (II) 39.57 27.30
Stability Yes Yes
Aliphatic Index 65.60 42.94
Grand average of
hydropathicity
RAVY)
0.016 -0.747
Total numbers of
negatively charged
residues (Asp + Glu)
30 40
Total numbers of
positively charged
residues
30 40
Table 4.3.4: Sequence analysis of the recombinant alkaline proteases from O.M.A18 and
O.M.E12
136
A 100% homology of both the sequences was found with extracellular protease
sequence gene. Similarly, O.M.A18 showed complete similarity with Bacillus
sp.KP43. We have predicted physico-chemical properties of the native enzyme by
using our nucleotide sequences for the two alkaline proteases. By submitting query
sequence to EXpasy protein database, we found the characteristic features of the
sequences closely resembled to nascent protease enzyme. Estimated theoretical PI for
both isolates was about 5. The instability index (II) was computed as 27.30 and 39.57
for O.M.E12 and O.M.A18 proteases, respectively. On the basis of data, we can
predict that structure of enzyme is quite stable, a fact also reflected by our
experimental data on thermal stability and resistance against chemical denaturation.
Other properties, as aliphatic index and grand average of hydropathicity (GRAVY)
for O.M.E12 were 65.60 and 0.016, while those for O.M.A18 and were 42.94 and -
0.747, respectively. Total numbers of negatively and positively charged residues were
~30 and ~40, respectively for both proteases (Table 4.3.4).
The significance of the work relates to the fact that despite saline habitat in the study
possessed significant bacterial diversity, it remains unexplored in terms of its
characterization, biocatalytic potential, enzymatic characteristics and phylogenetic
status. Moreover, some of the novel features of the enzymes, such as stability over the
wide range of pH and salt, catalysis and thermostability of enzyme at higher
temperatures make them attractive candidates for future studies. The results are also
important from the diversity viewpoint. Although both strains are from the same site,
they display distinct features of growth, protease secretion and enzymatic
characteristics, highlighting their ecological significance.
CHAPTER Launch Internet Explorer Brow ser.lnk
CHAPTER
RECOMBINANT
PROTEASES
CLONING, OVER-EXPRESSION AND
CHARACTERIZATION
5
137
5.1 INTRODUCTION
Genes from the extremophiles are often cloned and over expressed in domestic host
systems to obtain large quantities of enzymes (Corolina et al., 2008; Ni et al., 2009).
It is necessary and interesting to investigate, whether the folding and functioning of
recombinant protein is identical to normal protein.
So far, only few alkaline proteases are purified and characterized from the halophilic
and haloalkaliphilic bacteria, which primarily may be due to the difficulties associated
with the protein stability in the absence of salt. Characterization of such
haloalkaliphilic enzymes would provide important clue for the adaptation strategies
and stability of the biomolecules under which they can sustain with more than one
extremities of NaCl, pH and sometimes temperature.
In this context, during recent years gene cloning from extremophiles into mesophilic
bacteria has focused considerable attention. However, as said earlier in chapter 4, only
few alkaline proteases from halophiles and haloalkaliphiles are purified and
characterized due to its instability in the absence of salt (Dodia et al., 2008a; Ni et al.,
2009). Most typical halophilic enzymes require high concentrations of salt for their
activity and stability and are inactivated in Escherichia coli unless refolded in the
presence of salts under in-vitro conditions.
Recombinant DNA technology in conjunction with other molecular techniques is
being used to improve, evolve enzymes and for opening new opportunities for the
construction of genetically modified microbial strains with the selected biocatalysis
(Battestein, 2007; Caralino et al., 2008). Knowledge of full nucleotide sequences of
enzymes has facilitated the deduction of the primary structure of the encoded enzymes
and, in many cases, identification of various functional regions. These sequences also
serve as the basis for phylogenetic analysis of proteins and assist in predicting the
secondary structure of proteins, leading to the understanding of structure and function
relationship of the enzymes (Purohit and Singh, 2009; Siddhpura et al., 2010).
Therefore, cloning of the potential genes coding for different enzymes would be an
attractive approve to begin with. Some alkaline protease-encoding bacterial genes
have been cloned and expressed in new hosts, the two major organisms for cloning
and over-expression being E. coli and B. subtilis (Fu et al., 2003; Wang et al., 2008).
138
Developments in molecular approaches to improve the cloning and expression of
genes leads to enhanced solubilization of the expressed proteins from halophilic and
other extremophilic organisms in heterologous hosts will certainly boost the number
of enzyme-driven transformations in chemical, food, pharmaceutical and other
industrial applications. This process will add to the prospect of enzyme-driven
catalysis (Kim et al., 1998; Machida et al., 1998; Machida et al., 2000; Singh et al.,
2002). In this chapter we have described our studies on alkaline protease gene from
haloalkaliphilic bacteria which has been cloned and expressed in E. coli as host. The
properties of the recombinant enzyme were then compared with the native one. The
major aspects of cloning and over-expression discussed in detail are as depicted in
Fig.5.1.1
Fig.5.1.1: The schematic representation of major aspects covered in Chapter-5
Expression of recombinant clones
Gelatin Agar Plate SDS-PAGE
Cloning of Alkaline Protease gene from potential candidates
Colony PCR Confirmation of the insert
Amplification of Alkaline Protease gene
Seven Sets of primers designed for amplification of alkaline protease genes.
139
5.2 MATERIALS AND METHODS
5.2.1 Bacterial strain and plasmids The bacterial strains used for cloning procedures and expression analysis were E. coli
TOP10 and BL21a+ (DE3) (Invitrogen, USA). Plasmid over-expression vector
pET21a+ (Invitrogen, USA) was used for expression analysis of serine alkaline
proteases.
5.2.2 Sample collections and growth conditions Sample collections, growth conditions and phylogenentic determination of O.M.A18
and O.M.E12 were as described in details in chapter-2.
5.2.3 DNA manipulations
Isolation and assessment of genomic DNA and plasmid DNA Genomic DNA of haloalkaliphilic bacterial strains O.M.E12 and O.M.A18 were
isolated by enzymatic method (Sambrook and Russell, 2001). Plasmids DNA were
retrieved by using SDS-Miniprep method (Sambrook and Russell, 2001) from
transformed clones of BL21 (DE3) harboring pET21a+. Purity and yield of DNA
preparations were judged and analyzed by spectrophotometric assessment/Nanodrop
and agarose gel electrophoresis. The 1,000-bp PCR-amplified products were gel
purified by using the QIAgen PCR purification kit (Qiagen, Germany) according to
the manufacturer’s instructions after resolving on a 0.8% agarose gel.
PCR primer designing
In order to amplify the complete ORF (1.0 kb) of the protease gene, six pairs of
primers were designed specifically for haloalkaliphilic extracellular alkaline protease.
As described in brief in chapter-3; among the six pairs, three pairs of primers used for
amplification profile SPS-1, 3 and 4 were synthetic degenerate oligonucleotides based
on the previously known sequence of extracellular alkaline protease gene from
Bacillus halodurans, Bacillus cerus and Oceanobacillus iheyensis serine proteases
respectively. UTR region of known sequences were used as a frame for primer
designing procedures. Primer pair, SPS-5 was designed on the basis of identification
of conserved residues among the already reported alkaline proteases sequences
belonging to haloalkaliphilic bacteria. The conserved pattern within the UTR’s was
identified using multiple sequence alignment tool-CLUSTAL W (Thompson et al.,
140
1994) (www.ebi.ac.uk/Tools/msa/clustalw2/). All the above described primer pair
combinations were designed manually, while two primer pairs were designed on the
basis of conserved sequences of Haloalkaliphilic Bacillus species, by generating block
using degenerate primer designing bioinformatics tool-CODEHOP. A set of forward
and reverse primers were designed on the basis of conserved sequences of 15
haloalkaliphilic Bacillus species alkaline proteases, by using multiple sequencing tool,
followed by block generation using degenerate primer designing bioinformatics tool-
CODEHOP (Timothy et al., 2003) (Fig.5.2.1). All the designed primers were
commercially synthesized (Sigma Aldrich, Life Sciences). The sequence and
description of all six pairs of primers are described in details in materials and methods
section of chapter-3. The primer set that yielded the specific amplified product was:
SPS-6 forward 5’-cat atg ccg ccg agg agg ac-3’(Tm 66oC) and SPS-6 reverses 5’-gtc
gac ggc ctt cgt gtg g-3’ (Tm 64oC).
CODEHOP Results Oligo Summary Not all overlapping primers are shown
CODEHOP Version 10/14/04.1 COPYRIGHT 1997-2004, Fred Hutchinson Cancer Research Center, Seattle, WA, USA Parameters: Amino acids PSSM calculated with odds ratios normalized to 100 and back-translated with Standard genetic code Maximum core degeneracy 128 Core strictness 0.00 Clamp strictness 1.00 Target clamp temperature 60.00 C DNA Concentration 50.00 nM Salt Concentration 50.00 mM Codon boundary 0 Most common codon 0 Suggested CODEHOPS: The degenerate region (core) is printed in lower case, the non-degenerate region (clamp) is printed in upper case.
Block x25568xblB Oligos
P E A A E E N K K D Y L I CCGCCGAGGAGGACAAGrarrantayyt -3' Core: degen=128 len=11 Clamp: score=66, len=17 temp= 63.0 GCCGCCGAGGAGGACrarrarranta -3' Core: degen=128 len=11 Clamp: score=63, len=15 temp= 61.6
Complement of Block x25568xblB
P E A A E E N K K D Y L I No suggested primers found.
Block x25568xblC
E F N D E V D I Q S E E E E Y D No suggested primers found.
Complement of Block x25568xblC
E F N D E V D I Q S E E E E Y D No suggested primers found.
Block x25568xblD Oligos
D V I H E F E T I P V I H A E L S P K E L K K L K K D P N I N Y I E E D A E V T CCAACATCAACTACATCGAGGAGraygynsargt -3' Core: degen=128 len=11 Clamp: score=72, len=23 temp= 60.9 AAGGACCCCAACATCAACTACAThgarrarrayg -3' Core: degen=96 len=11 Clamp: score=70, len=23 temp= 60.9
141
AAGGACCCCAACATCAACTACathgarrarra -3' Core: degen=48 len=11 Clamp: score=65, len=21 temp= 60.9 GAGGTGAAGAAGCTGAAGAAGgayccnamnrt -3' Core: degen=128 len=11 Clamp: score=69, len=21 temp= 61.4
Complement of Block x25568xblD Oligos
D V I H E F E T I P V I H A E L S P K E L K K L K K D P N I N Y I E E D A E V T ctrggntknyaGTTGATGTAGCTCCTCCTGC -5' Core: degen=128 len=11 Clamp: score=68, len=20 temp= 60.3 tadctyytyytGCGGCTCCACTGT -5' Core: degen=48 len=11 Clamp: score=71, len=13 temp= 52.7 *** CLAMP NEEDS EXTENSION adctyytyytrcGGCTCCACTGT -5' Core: degen=96 len=12 Clamp: score=69, len=11 temp= 42.3 *** CLAMP NEEDS EXTENSION ctyytyytrcrGCTCCACTGT -5' Core: degen=64 len=11 Clamp: score=69, len=10 temp= 22.6 *** CLAMP NEEDS EXTENSION
Block x25568xblE Oligos
M S Q T V P W G I S R V N T Q Q A H N R G I F G N G I K V A V L D T G I S Q H P D L N I Q G G A S F I P S E P GGTGGCCGTCCTGgayacnggnat -3' Core: degen=32 len=11 Clamp: score=73, len=13 temp= 60.1 CGTCAAGGTGGCCGTCCTngayacnggna -3' Core: degen=128 len=11 Clamp: score=74, len=18 temp= 64.6 GCGTCAAGGTGGCCGTCytngayacngg -3' Core: degen=64 len=11 Clamp: score=70, len=17 temp= 64.0 CGGCGTCAAGGTGGCCrtnytngayac -3' Core: degen=128 len=11 Clamp: score=72, len=16 temp= 65.0
Complement of Block x25568xblE Oligos
M S Q T V P W G I S R V N T Q Q A H N R G I F G N G I K V A V L D T G I S Q H P D L N I Q G G A S F I P S E P anctrtgnccntAGAGGTGCGTGGGGC -5' Core: degen=128 len=12 Clamp: score=62, len=15 temp= 61.7 ctrtgnccntaGAGGTGCGTGGGGC -5' Core: degen=32 len=11 Clamp: score=56, len=14 temp= 61.7
Block x25568xblF Oligos
S T H D N N G H G T H V A G T I A A L N N S I G V L G V A P S A E L Y A V K V L N R N G S G S Y S S I A Q G L GCCGAGCTGTACGCCgynaargtnyt -3' Core: degen=128 len=11 Clamp: score=73, len=15 temp= 62.8 CATCGGCGTGCTGggnrtngcncc -3' Core: degen=128 len=11 Clamp: score=64, len=13 temp= 62.2 ACCCAGGACGACAACggncayggnac -3' Core: degen=32 len=11 Clamp: score=71, len=15 temp= 60.5 TCCACCCAGGACGACAAyggncayggna -3' Core: degen=64 len=11 Clamp: score=72, len=17 temp= 59.5 *** CLAMP NEEDS EXTENSION TCCACCCAGGACGACaayggncaygg -3' Core: degen=16 len=11 Clamp: score=64, len=15 temp= 59.5 *** CLAMP NEEDS EXTENSION TCCACCCAGGACGAnaayggncayg -3' Core: degen=64 len=11 Clamp: score=69, len=14 temp= 52.3 *** CLAMP NEEDS EXTENSION TCCACCCAGgayrrnaaygg -3' Core: degen=64 len=11 Clamp: score=60, len=9 temp= 37.3 *** CLAMP NEEDS EXTENSION
Complement of Block x25568xblF Oligos
S T H D N N G H G T H V A G T I A A L N N S I G V L G V A P S A E L Y A V K V L N R N G S G S Y S S I A Q G L ctryynttrccGGTGCCGTGGGT -5' Core: degen=64 len=11 Clamp: score=77, len=12 temp= 62.5 ttrccngtrccGTGGGTGCACCGG -5' Core: degen=16 len=11 Clamp: score=74, len=13 temp= 63.5 trccngtrccntGGGTGCACCGGCC -5' Core: degen=64 len=12 Clamp: score=77, len=13 temp= 61.2 ccngtrccntgGGTGCACCGGCC -5' Core: degen=32 len=11 Clamp: score=73, len=12 temp= 61.2 ccnyancgnggGCTGCGGCTCGA -5' Core: degen=128 len=11 Clamp: score=56, len=12 temp= 60.7 crnttycanraCCTGTCCTTGCCGTAGC -5' Core: degen=128 len=11 Clamp: score=58, len=17 temp= 64.3
Block x25568xblG
E W A I N N N M H I I N M S L G S T S P S K T L E Q A V N R A N N A G V L L V G A S G N N G R Q S V N Y P A R No suggested primers found.
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Complement of Block x25568xblG Oligos
E W A I N N N M H I I N M S L G S T S P S K T L E Q A V N R A N N A G V L L V G A S G N N G R Q S V N Y P A R ctyacccgnnmGTTGTTGTTGTACGTGTAGCA -5' Core: degen=64 len=11 Clamp: score=74, len=21 temp= 60.5
Block x25568xblH Oligos
Y E N V M A V G A T D Q N N Q R A S F S Q Y G P G L E I V A P G V N V Q S T Y Q G N R Y V S L S G T S M A T P CGTGGCCCCCggngtnaaybt -3' Core: degen=96 len=11 Clamp: score=70, len=10 temp= 60.0 TCGAGATCGTGGCCccnggngtnaa -3' Core: degen=64 len=11 Clamp: score=66, len=14 temp= 61.8 GGGATCGAGATCGTGgcnccnggngt -3' Core: degen=64 len=11 Clamp: score=61, len=15 temp= 60.1
Complement of Block x25568xblH Oligos
Y E N V M A V G A T D Q N N Q R A S F S Q Y G P G L E I V A P G V N V Q S T Y Q G N R Y V S L S G T S M A T P cgnggnccncaCTTGCACTTCTGGTGGATGG -5' Core: degen=64 len=11 Clamp: score=68, len=20 temp= 60.8 ggnccncanttGCACTTCTGGTGGATGGGC -5' Core: degen=64 len=11 Clamp: score=63, len=19 temp= 63.5 ccncanttrvaCTTCTGGTGGATGGGCCC -5' Core: degen=96 len=11 Clamp: score=58, len=18 temp= 63.2
Block x25568xblI Oligos
H V A G V A A L V W S Q N P H W D N N Q I R Q H L K Q T A T Y L G N P N L Y G N G N V N A N R A T F GAACCCCCACTGGACCaayrwncanat -3' Core: degen=128 len=11 Clamp: score=62, len=16 temp= 61.8 CACGTGGCCggngyngcngc -3' Core: degen=128 len=11 Clamp: score=65, len=9 temp= 48.5 *** CLAMP NEEDS EXTENSION
Complement of Block x25568xblI Oligos
H V A G V A A L V W S Q N P H W D N N Q I R Q H L K Q T A T Y L G N P N L Y G N G N V N A N R A T F ccncrncgncgGGACCAGACCGTCG -5' Core: degen=128 len=11 Clamp: score=66, len=14 temp= 61.6 ttrywngtntaGGCCTTCGTGTAGTTCG -5' Core: degen=128 len=11 Clamp: score=59, len=17 temp= 61.2 Oligos Degenerate alphabet D P N I N Y I E E D A ctrggntknyaGTTGATGTAGCTCCTCCTGC oligo:5'-CGTCCTCCTCGATGTAGTTGaynktnggrtc-3' degen=128 temp=60.3 I E E D A E V T tadctyytyytGCGGCTCCACTGT oligo:5'-TGTCACCTCGGCGtyytyytcdat-3' degen=48 temp=52.7 Extend clamp E E D A E V T adctyytyytrcGGCTCCACTGT oligo:5'-TGTCACCTCGGcrtyytyytcda-3' degen=96 temp=42.3 Extend clamp E E D A E V T ctyytyytrcrGCTCCACTGT oligo:5'-TGTCACCTCGrcrtyytyytc-3' degen=64 temp=22.6 Extend clamp Block x25568xblE G I K V A V L D T oligo:5'-CGGCGTCAAGGTGGCCrtnytngayac-3' degen=128 temp=65.0 I K V A V L D T G oligo:5'-GCGTCAAGGTGGCCGTCytngayacngg-3' degen=64 temp=64.0 I K V A V L D T G I oligo:5'-CGTCAAGGTGGCCGTCCTngayacnggna-3' degen=128 temp=64.6 V A V L D T G I oligo:5'-GGTGGCCGTCCTGgayacnggnat-3' degen=32 temp=60.1 Complement of Block x25568xblE D T G I S Q H P D anctrtgnccntAGAGGTGCGTGGGGC oligo:5'-CGGGGTGCGTGGAGAtnccngtrtcna-3' degen=128 temp=61.7 D T G I S Q H P D ctrtgnccntaGAGGTGCGTGGGGC oligo:5'-CGGGGTGCGTGGAGatnccngtrtc-3' degen=32 temp=61.7 Block x25568xblF S T H D N N G oligo:5'-TCCACCCAGgayrrnaaygg-3' degen=64 temp=37.3 Extend clamp S T H D N N G H G oligo:5'-TCCACCCAGGACGAnaayggncayg-3' degen=64 temp=52.3 Extend clamp S T H D N N G H G oligo:5'-TCCACCCAGGACGACaayggncaygg-3' degen=16 temp=59.5 Extend clamp S T H D N N G H G T
143
oligo:5'-TCCACCCAGGACGACAAyggncayggna-3' degen=64 temp=59.5 Extend clamp T H D N N G H G T oligo:5'-ACCCAGGACGACAACggncayggnac-3' degen=32 temp=60.5 I G V L G V A P oligo:5'-CATCGGCGTGCTGggnrtngcncc-3' degen=128 temp=62.2 A E L Y A V K V L oligo:5'-GCCGAGCTGTACGCCgynaargtnyt-3' degen=128 temp=62.8 Complement of Block x25568xblF D N N G H G T H ctryynttrccGGTGCCGTGGGT oligo:5'-TGGGTGCCGTGGccrttnyyrtc-3' degen=64 temp=62.5 N G H G T H V A ttrccngtrccGTGGGTGCACCGG oligo:5'-GGCCACGTGGGTGccrtgnccrtt-3' degen=16 temp=63.5 G H G T H V A G trccngtrccntGGGTGCACCGGCC oligo:5'-CCGGCCACGTGGGtnccrtgnccrt-3' degen=64 temp=61.2 G H G T H V A G ccngtrccntgGGTGCACCGGCC oligo:5'-CCGGCCACGTGGgtnccrtgncc-3' degen=32 temp=61.2 G V A P S A E L ccnyancgnggGCTGCGGCTCGA oligo:5'-AGCTCGGCGTCGggngcnayncc-3' degen=128 temp=60.7 V K V L N R N G S G crnttycanraCCTGTCCTTGCCGTAGC oligo:5'-CGATGCCGTTCCTGTCCarnacyttnrc-3' degen=128 temp=64.3 Complement of Block x25568xblG E W A I N N N M H I I ctyacccgnnmGTTGTTGTTGTACGTGTAGCA oligo:5'-ACGATGTGCATGTTGTTGTTGmnngcccaytc-3' degen=64 temp=60.5 Block x25568xblH G L E I V A P G V oligo:5'-GGGATCGAGATCGTGgcnccnggngt-3' degen=64 temp=60.1 E I V A P G V N oligo:5'-TCGAGATCGTGGCCccnggngtnaa-3' degen=64 temp=61.8 V A P G V N V oligo:5'-CGTGGCCCCCggngtnaaybt-3' degen=96 temp=60.0 Complement of Block x25568xblH A P G V N V Q S T Y Q cgnggnccncaCTTGCACTTCTGGTGGATGG oligo:5'-GGTAGGTGGTCTTCACGTTCacnccnggngc-3' degen=64 temp=60.8 P G V N V Q S T Y Q ggnccncanttGCACTTCTGGTGGATGGGC oligo:5'-CGGGTAGGTGGTCTTCACGttnacnccngg-3' degen=64 temp=63.5 G V N V Q S T Y Q G ccncanttrvaCTTCTGGTGGATGGGCCC oligo:5'-CCCGGGTAGGTGGTCTTCavrttnacncc-3' degen=96 temp=63.2 Block x25568xblI H V A G V A A oligo:5'-CACGTGGCCggngyngcngc-3' degen=128 temp=48.5 Extend clamp N P H W D N N Q I oligo:5'-GAACCCCCACTGGACCaayrwncanat-3' degen=128 temp=61.8 Complement of Block x25568xblI G V A A L V W S Q ccncrncgncgGGACCAGACCGTCG oligo:5'-GCTGCCAGACCAGGgcngcnrcncc-3' degen=128 temp=61.6 N N Q I R Q H L K Q ttrywngtntaGGCCTTCGTGTAGTTCG oligo:5'-GCTTGATGTGCTTCCGGatntgnwyrtt-3' degen=128 temp=61.2
Fig. 5.2.1: Conserved primer designing of alkaline proteases by block generation
using online CODEHOP (Consensus Degenrate Oligonucleotide Primer) tool.
144
Polymerase chain reaction for amplification of protease gene
Polymerase chain reaction was carried using Gradient Eppendorf Thermocycler.
To 100ng of DNA as the template, 25 pmol of each forward and reverse
oligonucleotides primer, 25µl of 2X red mix plus which contains all the reagents and
enzymes required for PCR reaction except primers and template DNA (Merk, Life
sciences, India) were added. Negative controls were included in the PCR reactions to
establish the validity of the experiment. Experiment was carried out under the thermal
cycling conditions as described in Materials and Method section of Chapter-3.
In general cycles were designed as: [95°Cx5 mins] x1, [95°Cx1 mins/50°C x 45s/and
72°C x1 min] x 30, [72°C x1 min] x 1.
5.2.4 Cloning of PCR Product (Digestion and ligation procedure) Approximately 200ng of plasmid DNA and 50ng of the insert DNA samples were
digested in 30µl reaction mixtures with BamHI for 4 hours under the conditions
specified by the manufacturer (Merk life science, India). The digested samples
(10-15µl) were resolved on 1.2% agarose gel along with broad range DNA marker
(Merk life sciences, India) to analyze the restriction patterns. Purification of RE
digested products were subsequently done by using PCR purification kit (Merk Life
sciences, India). For ligation procedures, 250ng of the purified fragment and 50ng
pET21a+ was added in a sterile eppendorf tubes, final reaction mixture was made
upto 25µl with sterile MilliQ grade D/W. Tubes were incubated overnight at 4°C as
per manufacturer’s instructions (Promega, Madison, Wisc., USA). This ligation
mixture was used to transform E. coli strain Top10 (Novagen) (Sambrook and Russell,
2001). The bacterial colonies containing recombinant plasmids were selected on LB
agar medium containing 0.5mM IPTG (isopropyl-D-thiogalactopyranoside) and 50
µg/ml ampicillin. The overall flow chart of cloning procedures is as described in
5.2.2.After sub-cloning the PCR product - pET21a+ plasmids were re-extracted from
Top10 for further confirmation of positive clones.
Sequence of the insert was confirmed by sequencing (Merk life science, India), and
found to be in frame to vector. Further, it was re-transformed in over-expression host;
BL21 (DE3) by using standard calcium transformation procedures (Sambrook and
Russell, 2001).
145
5.2.5 Cloning confirmation by restriction analysis Plasmids harbored by positive clones were re-digested in 30µl reaction mixtures with
NotI restriction enzyme and incubated at 16°C for 4 hours (Merk life science, India).
The digested samples (10–15µl) were resolved on 1.2% agarose gel along with broad
range DNA marker (Merk Life Science) to analyze the restriction patterns.
Fig.5.2.2: The major steps of cloning of alkaline protease genes
5.2.6 Expression Analysis
Effect of temperature and IPTG Induction on the growth and expression of
alkaline protease enzyme
Recombinant clones were screened on gelatin agar plate (pH-7) for enzyme secretion
at different IPTG concentrations (0.1-3mM) and growth temperatures (27 and 37oC).
Positive clones were grown in LB broth containing ampicillin (30µg/ml). At regular
interval of time, 2 ml of culture was withdrawn and centrifuged at 5000 rpm for 5 min
at 4°C. The growth was measured at 660nm. The cells were suspended in 1ml
potassium phosphate buffer (pH-8) and subjected to sonication at 30Hz for 30 seconds
•Amplicon and pET 21a+ was Digested by BamH1
•Amplicon and pET 21a+ was Digested by Sal1RE Digestion-
• Double digested amplicon and vector was ligated in ratio of 1:3
•Ligation carried by Quick Ligase, FermentasLigation
•Ligated vector transformed to Top 10(E.coli Host strain)
•5 Poisitive clones for O.M.A18 and 7 positive clones for O.M.E12 obtained on plate containing LB+Ampicillin
Transformation-I
•Colony PCR
•To check for release of vectorConfirmation of transformation
•Positive clone from both the strains vector transformed in BL21 Host strain(Expression strain)Transformation-II
146
in 6 cycles. Samples were cooled in ice for 30 seconds between each cycle. The
resulted supernatant after sonication was treated as soluble fraction. The pellet was
then treated with 8M urea for 30 min at 30oC, followed by centrifugation at 5000rpm
for 5 minutes at 10oC to obtain supernatant, which was treated as insoluble fraction.
The insoluble fractions were dialyzed against phosphate buffer (pH-8) to renature the
denatured enzyme for further analysis. Insoluble and soluble fractions in required
aliquot judged on the basis of total protein estimation were taken as an enzyme
sample for activity analysis and SDS-PAGE confirmation.
Enzyme Assay and Protein estimation
The alkaline protease activity and total protein estimation was measured by Anson-
Hagihara's and Bradford method as described in detail in Materials and Method
section of chapter-4.
SDS-polyacryalamide gel electrophoresis
Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was
carried out according to the method of Laemmli as described in detail in Materials and
method section of chapter-4. To visualize protease expression, soluble and insoluble
fractions (20μg) were loaded onto gel. The molecular weight of the enzyme was
determined using reference molecular weight marker (Middle range ruler, Merk life
science, India).
One-step purification of enzyme
Purification fractions having enzyme activities were pooled together on the basis of
elution profile. Sample was purified to homogeneity by one step affinity
chromatography using Nickel as a matrix column. Purification procedure was
performed as described by using gradient of Immidazole concentration from
(0-20mM) (Sambrook and Russell, 2001).
5.2.7 Enzyme characterization Effect of temperature and pH
The effect of pH and temperature was monitored by incubating the reaction mixture at
the set conditions. For the effect of temperature on catalysis, the activity was
determined by incubating the assay mixture at temperatures, 37, 50, 60 and 70°C.
Similarly, the effect of pH was assessed by performing the enzyme activity in the
range of pH 7-10.
147
Effect of urea on enzyme activity
The effects of chemical denaturing agent, urea (8M), on the enzyme preparations were
studied. The recombinant enzymes were incubated with urea at different
temperatures; 50, 60 and 70°C as similar to its native counterparts (Purohit and Singh,
2011. The enzyme mixtures were incubated at set conditions for 3 hours followed by
measurement of the residual activity to ascertain the loss of enzyme activity.
Effect of NaCl on enzyme stability
Effect of NaCl on temperature profile was explored by incubating the reaction
mixtures supplemented with various concentrations of NaCl (0-3M).
Enzyme Thermostability
The thermal stability of the enzyme was studied by incubating it at different
temperatures; 37, 50 and 60°C. The enzyme aliquots were withdrawn at regular
intervals up to 24 hours and the enzyme activities were measured at optimum
temperature.
5.2.8 DNA Sequencing and in-silico analysis Plasmids were retrieved from positive clones and sequenced from both ends, using
standard T7 promoter and terminator sequence which was on a flanking region of
insert by chromosome walking method, using custom based service of Merk Life
sciences, India. Sequence homologies to known nucleotide sequences in the GenBank
database were determined using the BLAST algorithm of the NCBI at the National
Library of Medicine. A phylogenetic tree for recombinant O.M.A18 and O.M.E12
were constructed of aligned sequences by the Neighbor-Joining method clustering
strategy in Mega 4.0(www.megasoftware.net) (Tamura et al., 2007). Other DNA
analyses, required for cloning confirmation i.e. restriction analysis and primer
prediction were carried out by CLC main workbench (Dainith, 2004). The amino acid
sequence for both of them were deduced using translate tool of Expasy
(http://expasy.org/tools/translate), exploiting correct ORF predicted by NCBI
(www.ncbi.nlm.nih.gov.in) (Purohit and Singh, 2011). The protein properties
prediction and hydropathy plots were performed by EXpasY (http://expasy.org/tools)
freely available protein server database (Kyte and Doolittle 2007).
148
5.2.9 Modeling of the 3D Structure Three-dimensional structures of both the serine proteases protein were modeled using
the online I-TASSER server for protein 3D structure prediction (Wu et al., 2007,
Zhang and Zeng, 2008, Zhang et al., 2008). The server also predicts other molecular
information’s, such as distribution of amino acids, active site moiety, primary and
secondary protein structure properties by Profile Profile Alignment (PPA) Threading
techniques. For the O.M.A18 and O.M.E12 proteases, 5 models were obtained.
5.2.10 Nucleotide sequence accession numbers The DNA sequence of the protease genes cloned and studied in present work were
submitted in the GenBank database under the accession number HM219179 for
O.M.A18 and HM219182 for O.M.E12 with its characteristic properties in native and
recombinant system.
149
5.3 RESULTS AND DISCUSSION
5.3.1 Amplification profile and cloning of the protease gene The amplification of the alkaline protease gene was carried out using the genomic
DNA of Oceanobacillus iheyensis O.M.A18 (gene bank accession number-
EU680961) and Haloalkaliphilic bacterium O.M.E12 (Gene bank accession no.
EU680960). Virtual PCR was carried out to analyze the product size of amplicons.
The predicted amplicons are as described in Fig. 5.3.1 A and B. Restriction analysis
of amplified PCR was judged by RE cutter. The results are as described in Fig.5.3.2.
PCR reaction was carried out at three gradient of annealing temperatures using
Gradient Thermocycler (Eppendorf).
The 1-kb coding region of the gene was PCR amplified by using primer pair designed
as described in materials and methods section. Amplicon size of product obtained
from SPS-6 was of aprox. 1kb for O.M.A18 and O.M.E12 at Ta of 60°C, however as
described earlier in Result and Discussion section of Chapter-3 in protease
amplification section, concentration of product varied with respect to gradient of
temperature and primer pair used for the amplification profile generation (Fig.5.3.3 A
and B). For, visual assessment and quantification of PCR products; both the
amplicons were resolved on an agarose gel.
Amplicon were further purified by using PCR purification kit (Merk life sciences,
India) to remove traces of enzymes and chemicals. After, successful restriction
digestion procedures, O.M.A18 and O.M.E12 digested amplicons were cloned into
suitable over-expression vector- pET21a+ individually; vector constructs were
transformed into over-expression host Escherichia coli BL21 (Fig.5.3.4; 5.3.5).
Selections of positive clones were done on the basis of ampicillin (30µg/ml) as a
marker trait (Fig.5.3.4).
Further, confirmation of positive clones harboring O.M.A18 protease and O.M.E12
protease were done on the basis of excision with BamHI and SalI restriction sites, all
the plasmids were individually digested with these enzymes (Fig. 5.3.6). Two excised
bands were visualized on agarose gel (0.8%) which confirmed the cloning procedures
(Fig.5.3.6). The complete analysis of nucleotide sequence was done by chromosome
150
walking method using T7 promoter and terminator sequence of pET21 a+ as a primer
sequence (Fig.5.3.7A and 5.3.7B).
Fig.5.3.1A: Virtual PCR of alkaline protease gene of Oceanobacillus iheyensis to
check amenability of designed PCR primer
151
Fig.5.3.1B: Virtual PCR of alkaline protease gene of Haloalkaliphilic Bacillus, to
check amenability of designed PCR primers.
152
Fig.5.3.2: Snapshot of virtual restriction analysis of conserved region of alkaline proteases
153
Fig.5.3.3: Amplification profile of alkaline proteases of O.M.A18 and O.M.E12.
0
0.2
0.4
0.6
0.8
1
1.2
SPS-1F&R
SPS-3F&R
SPS-4F&R
SPS-5F &R
SPS-6F &R
SPS7F&R
Size(kb) 1 1.1 0.8
0
0.2
0.4
0.6
0.8
1
1.2
Primer pair combination
Siz
e (k
b)
154
Fig.5.3.4A: PCR simplification profile of alkaline protease genes M: Middle range
marker, Merk Life Sciences, India (A) Amplification by using SPS7F and SPS7R
primer Lane 1: 62oC-O.M.A18; Lane 2: 62oC-O.M..E12 Lane 3: 60.1oC-O.M.A18;
Lane 4: 60.1oC-O.M.E12; Lane 5: 62.3oC-M.A18; Lane 6: 62.3oC-M.A18; Lane 7:
Positive control (B) Amplification by using SPS5F and SPS6R Lane 1: 62oC-
O.M.A18; Lane 2: 62oC-O.M..E12 Lane3: 60.1oC-O.M.A18; Lane 4: 60.1oC-
O.M.E12; Lane 5: 62.3oC-M.A18; Lane 6: 62.3oC-M.A18; Lane 7: Positive control
M 1 2 3 4 5 6 7
A
B
3kb
1kb
3kb
1kb
155
Fig.5.3.4: Expressed clones of alkaline proteases gene of O.M.A18 and O.ME12.
Fig. 5.3.5: Virtual Confirmation of ligation insert in pET 21a+
Fig.5.3.6: Confirmation of cloning by release of insert; where colony designated 1, 2,
3 are of O.M.A18 recombinant clone and colony number: 4, 5, 6 are of O.M.E12 clone
O.M.A18
156
Fig.5.3.7A: Nucleotide sequence analysis of O.M.A18 by chromosome walking
method. The presentation layout was redrawn by CLC main workbench.
157
Fig.5.3.7B: Nucleotide sequence analysis of O.M.E12 by chromosome walking
method. The presentation layout was redrawn by CLC main workbench.
158
5.3.2 Analysis of the nucleotide and protein sequence The plasmids isolated from clones were used for sequencing. As the size of the insert
was 1.0 kb, two internal primers, SPS 6 forward and SPS 6 reverse, were used to
obtain the partial sequence. The sequence contained 50% G + C base pairs. A 100%
homology of the sequence was found with Bacillus sp.KP43 protease. This ORF had a
codon bias towards C or G at position 3 (Purohit and Singh, 2011). Protein similarity
and phylogenetic analysis was carried out by using nBLASTp (prediction of protein
sequence by submitting nucleotide sequence as a query) to identify the protein.
Predicted N-terminal sequence for Oceanobacillus iheyensis O.M.A18 was
5’MNPGSAWRSPVVPFSSLGMSPAYG (Purohit and Singh, 2011) and for;
Haloalkaliphilic bacterium O.M.E12 was 5’KLRVIIEFKEDAVEAGIQSTKQLMKK.
On the basis of physico-chemical properties predicted by in-silico projection and our
experimental results on thermal stability and denaturation profile were compared and
analyzed (Purohit and Singh, 2011).
5.3.3 Protein solubilization Protein folding is a specific process that leads to functional molecules under in-vivo
conditions. There are various physico-chemical factors required to maintain the stable
structure. However, the aggregation of newly synthesized proteins emerges as a
process that competes with in-vivo folding (Kim et al., 1998; Machida et al., 1998;
Machida et al., 2000; Singh et al., 2009). The growth and production of foreign
proteins in the host cells is influenced by different factors, such as temperature, pH
and ionic strength. Aggregation of partially folded intermediates leads to the
production of insoluble inclusion bodies, which may be mainly due to unstable
folding intermediate of the target protein at higher temperature and/or during over-
expression of a gene. The optimum secretion of recombinant enzyme was at 27oC on
gelatin plate; however, level of expression at 37oC was also significant for both
recombinant O.M.A18 and O.M.E12. The growth, as expected, was higher at 37oC as
compared to 27oC.
159
Fig.5.3.8A: Effect of induction on growth and enzyme secretion of O.M.A18 (Upper
panel) and O.M.E12 (Lower panel): Effect of inducer (IPTG) on growth and enzyme
production: 1mM IPTG at 37oC; 1mM IPTG at 27oC; 3mM IPTG at 37oC; 3mM
IPTG at 27oC were analyzed.
As similar to temperature, with respect to effect of inducer, both the isolates were
reflecting same trends with different concentration of IPTG. Different concentrations
of isopropyl β-d-thiogalactopyranoside (IPTG); 1.0-3.0mM, were used as inducer to
induce the expression of the target protease gene in E. coli harboring recombinant
plasmids.
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
0
1
2
3
4
5
6
7
8
9
10
1 2 3 4
Colony number
colony diameter(mm) at 27oC and 1mM IPTG colony diameter(mm) at 37oC and 3mM IPTG
zone ratio(mm) at 27oC and 1mM IPTG zone ratio(mm) at 37oC and and 3mM IPTG
C
olon
y di
amet
er (m
m)
Zon
e ra
tio (m
m)
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
0
1
2
3
4
5
6
7
Effect of Induction
160
1 2 3 4 5 6 7 1 2 3 4 5 6 7
(A) (B)
Fig.5.3.9 A: Expression analysis of protein in soluble and insoluble fractions.
Soluble fractions prepared at different hours after IPTG induction (A) Expression
analysis was carried at 27oC: Lane 1: PCR control; Lane 2: Molecular weight marker
(Middle range, Merk life science); Lane 3: 24 hours sample; Lane 4: 6 hours sample;
Lane 5: 4 hours sample; Lane 6: 2 hours sample; Lane 7: Pre-induction sample
Insoluble fractions prepared at different hours after IPTG induction
(B) Expression analysis was carried at 27oC: Lane 1: Molecular weight marker
(Middle range, Merk life science); Lane 2: Pre-induction sample; Lane 3: 2 hours
sample; Lane 4: 4 hours sample; Lane 5: 4 hours sample; Lane 6: 6 hours sample;
Lane 7: 24 hours sample 1 2 3 4 5 6 7
Fig.5.3.9 B: Expression analysis of protein in soluble fraction. Lane 1: Middle
range Marker. Lane 2: Crude Soluble fraction E12, Lane 3: His tag elution Fraction
(50mM), Lane 4: His tag elution Fraction (100mM Fraction no: 11), Lane 5: His tag
elution Fraction (100mM Fraction no: 12), Lane 6: His tag elution Fraction (200mM
Fraction no: 13), Lane 7: Binding buffer Wash
30kDa
29kDa
29kDa
161
At 1.0mM IPTG induction, higher level of enzyme was produced as compared to
0.5mM, while the optimum enzyme production was evidently with 1mM
concentration (Fig.5.3.8 A and B). Level of induction, however, did not significantly
affect growth of the host cells (Fig.5.3.8 A and B).
Synergistic effect of temperature and IPTG induction on protein solubilization was
examined at; 27oC and 1mM IPTG and 37oC and 3mM IPTG. For both the isolates, it
was quite distinct that at 27oC and 1mM IPTG, higher level of protein expression was
evident for both the recombinant enzyme preparations. However, substantial enzyme
was expressed at other conditions of growth and induction. The SDS-PAGE patterns
and protease activity revealed that with increasing time after induction, there was
gradual increase in the target protein in soluble fraction (Fig. 5.3.9A). Similar profile
was apparent for insoluble fraction. Although activity was lower than soluble fraction,
it increased with increasing time after induction (Fig. 5.3.9B).
5.3.4 Protease activity assay and enzyme purification The expressed proteins were fractionated into soluble and insoluble fraction as
described in materials and method section. Fractions collected at different hours i.e. 0,
2, 4, 6, 24 were analyzed for both, soluble and insoluble components. Enzyme
samples of different hours were further analyzed on SDS PAGE (Fig.5.3.9). SDS
PAGE results were quite comparable with the patterns of protease assay. The apparent
molecular weight of the enzyme was estimated as 29 and 30 kDa for O.M.A18 and
O.M.E12, which was quite comparable to our bioinformatics, based prediction as well
our studies on native enzyme preparations of same isolates (Purohit and Singh, 2011).
Purification of protein
To facilitate purification, the recombinant alkaline proteases were expressed with a
His tag at its C-terminal in BL21. Purification was achieved at its homogeneity, which
was evident from SDS-PAGE as well activity of enzyme increased to 5.7 fold, with
specific activity of 3,052 for O.M.A18 while for O.M.E12 activity increased to 6.12
fold, with specific activity of 3076.92 and yield of 88.15% (Table 5.3.1).
162
Fig. 5.3.10: Recombinant alkaline proteases purification by nickel
chromatography:
SDS-PAGE profile of recombinant enzyme purified on Ni-Column of recombinant
O.M.A18( Left panel) and O.M.E12(Right panel) Left panel: Lane 1: Crude
Soluble fraction E12, Lane 2: His tag elution Fraction (50mM), Lane 3: His tag
elution Fraction (100mM Fraction no: 11), Lane 4: His tag elution Fraction (100mM
Fraction no: 12), Lane 5: His tag elution Fraction (200mM Fraction no: 13), Lane 6:
Binding buffer Wash, Lane 7: Middle range Marker. Right panel: Lane 1: Crude
Soluble fraction O.M.A18, Lane 2: His tag elution Fraction (50mM), Lane 3: His
tag elution Fraction (100mM Fraction no: 11), Lane 4: His tag elution Fraction
(100mM Fraction no: 12), Lane 5: His tag elution Fraction (200mM Fraction no: 13),
Lane 6: Binding buffer Wash, Lane 7: Middle range Marker
1 2 3 4 5 6 7 1 2 3 4 5 6 7
7
En
zym
e a
ctivity (
U/m
l)
163
Fig.5.3.11: Elution profile of recombinant purified O.M.E12(Upper panel) and
O.M.A18(Lower panel) alkaline protease: Fractions: 1-12, Binding ; 13-23, Buffer
wash; 24-25, 50mM Immidazole ( SRL,Sisco Laboratories,India), 26-27,100mM
immidazole; 28-29, 200mM immidazole, 30; Wash buffer
These results are quite encouraging and interesting in light of verity that, over-
expression of enzyme was achieved in one step and high level of enzyme was
obtained in simple bacterial system and profound amount of enzyme was also noticed
in soluble fractions (Fig.5.3. 10, Fig. 5.3.11 A and B, Table.5.3.1).
0
0.5
1
1.5
2
2.5
0
200
400
600
800
1000
1200
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29
Fraction number
OM.A18 enzyme
Enzyme activity (U/ml) A 280
0
0.5
1
1.5
2
2.5
0
200
400
600
800
1000
1200
O.M.E12 enzyme
Prot
ein
cont
ent (
A28
0)
Enz
yme
activ
ity (U
/ml)
164
Table 5.3.1: One step purification of enzyme sample by Nickel chromatography :
O.M.A18 (Upper panel) and O.M.E12 (Lower panel)
5.3.5 Characterization of enzyme
Effect of Temperature and pH
The pH profile of enzymes was quite broad, being able to grow and secrete protease at
pH, 7-10. However, the optimum pH for recombinant enzyme secretion and growth
was 8 (Fig. 5.3.11 Upper panel), while as discussed in detail in Chapter-4, native
enzyme in its natural host secreted enzyme at pH-8 to 11, with optimum at 11 (Purohit
and Singh, 2011). Effect of temperature was examined at 37 and 50oC. Both the
clones were able to grow and secrete enzyme efficiently at 37oC. Profound level of
enzyme secretion was also found at 50oC. The findings indicated that haloalkaline
attributes of the enzyme were maintained when produced in E.coli.
Enzyme
Preparations
Activity
(U/ml)
Total
activity
(U)
Protein
(mg/ml)
Total
protein
(mg)
Specific
Activity
(U/mg)
Purification
fold
Yield
(%)
Recombinant
fraction
80 640 0.212 0.696 535.11 - 100
Purified
enzyme
293 586 0.096 0.192 3052.00 5.7 91.5
Enzyme
Preparations
Activity
(U/ml)
Total
activity
(U)
Protein
(mg/ml)
Total
protein
(mg)
Specific
Activity
(U/mg)
Purification
fold
Yield
(%)
Recombinant
fraction
121 726 0.241 1.446 502.07 - 100
Purified
enzyme
320 640 0.104 0.208 3076.92 6.12 88.15
O.M.E12
O.M.A18
165
5.3.11 (Upper panel) Effect of pH (7, 8, 9, 10) on recombinant alkaline proteases.
(Lower panel) Effect of temperature (37oC, and 50oC) on recombinant alkaline
protease
Effect of NaCl on enzyme stability
Effect of NaCl on temperature profile was explored by incubating the reaction
mixtures supplemented with various concentrations of NaCl (0-3M). The recombinant
enzyme maintained its 100% activity with up to 1M NaCl; however with increasing
NaCl, the activity sharply declined (Fig. 5.3.12). In the moderate haloalkalophilic
organisms, the enzyme activity increases with increasing NaCl up to a threshold level
(Dodia et al., 2008a and b; Joshi et al., 2008). As compared to the purified native
O.M.A18 protease, the recombinant enzyme was relatively more sensitive towards
NaCl. O.M.E12 enzyme was able to maintain its total activity upto 1M NaCl,
however, with further supplement of salt, enzyme activity was reduced with total loss
of activity at 24 hours (Fig.5.3.12). It is well studied fact that in moderate
haloalklaiphiilc organism, with increase in NaCl concentration upto threshold there is
increase in activity (Dodia et al., 2008b, Joshi et al., 2008, Purohit and Singh, 2011).
As compared to purified haloalkaliphilic O.M.E12 enzyme, the recombinant enzyme
was found to be very sensitive in the presence of NaCl.
0
50
100
150
200
250
37 50
7 8 9 10
0
50
100
150
200
250
7 8 9 10
37 50
z
% R
esid
ual a
ctiv
ity
166
Fig.5.3.12: Stability of NaCl on recombinant enzyme O.M.A18 (Upper panel) and
O.M.E12 (Lower panel) Stability of NaCl; where (1M-■-), (2M-●-), (3M-♦-) was
checked on recombinant alkaline proteases after different hours of incubations
Thermostability of Enzyme
The thermal stability of the O.M.A18 recombinant alkaline protease was assessed for
36 hours at, 37, 50 and 60oC, and pH 8. The recombinant enzyme maintained its
stability at 37 and 50oC for 3 hours, with a complete loss of activity at 60oC after 3
hour for O.M.A18 while for O.M.E12 enzyme maintained 50% of its stability at 37, 50
temperatures up to 3 hours (Fig. 5.3.13). However, on extending the time of
incubation to 24hour, both the enzymes preparations were completely denatured at
lower temperatures (Fig. 5.3.13).
% R
esid
ual a
ctiv
ity
0
20
40
60
80
100
120
0 1 2 3 24
Time(h)
1M 2M 3M
0
20
40
60
80
100
120
0 1 2 48 72
167
Fig.5.3.13. Thermostability profile of recombinant enzyme O.M.A18
(Upper panel) and O.M.E12 (Lower panel): Thermostability of enzyme was
characterized after different hours (1, 2, 3, and 24) of incubation at (37oC▬); (50oC -
■-), (60oC -▲-).
On the whole, it is quite logical that the stability of the alkaline protease in its native
form at elevated temperature could be attributed due to the inherent haloalkaliphilic
cellular components, metabolites and other complex machinery.
Effect of chemical denaturant urea
Recombinant O.M.A18 protease was treated with urea at 50, 60 and 70oC. Enzyme
retained marginal activity at 60 and 70oC. At 50oC, 75% of the residual activity was
retained after 30 mins, which was further reduced to 50% after an hour of incubation.
A complete loss of activity was observed after 3 hours (Fig.5.3.14). When compared
with the urea sensitivity of native enzyme, the recombinant enzyme was more
sensitive (Purohit and Singh, 2011).
% R
esid
ual a
ctiv
ity
0
20
40
60
80
100
120
140
0
20
40
60
80
100
120
1 2 3 24 36Time(h)
37 50 60
168
Fig.5.3.14. Urea Denaturation profile: Effect of urea after different time intervals at
50oC (♦), 60oC (■) and 70oC (▲).
With increase in temperature to 60oC for O.M.E12, only half of its activity was
maintained, with further increase in activity only marginal activity was observed.
Enzyme was found to be highly sensitive to chemical denaturant urea, as after an hour
of incubation, only 10-20% of activity was observed at all the temperature range and
total loss of activity was observed after 3 hours (Fig.5.3.14). On comparison of the
results with its native counterparts, we can observe that native counterparts were more
resistant to harsh conditions (Purohit and Singh, 2011).
5.3.6 Hydropathy determination The hydropathy profile of the nucleotide sequence of O.M.A18 and O.M.E12 protease,
plotted according to the method of Kyte and Doolittle (Kyte and Doolittle, 2007) by
using pscale tool available at Expasy. The results showed increased presence of
0
20
40
60
80
100
120
0 30 60 180
Time(mins)
50 60 70
0
20
40
60
80
100
120
% R
esid
ual a
ctiv
ity
169
hydrophobic residues in both the sequence analysis (Fig. 5.3.15). For general
information, the results showed increased presence of hydrophobic residues, the peak
above +1 indicate residues are more hydrophobic in nature while its value below 0
indicates its hydrophilicity, the net charge of amino acids is observed to be +1,
indicating structure to be hydrophobic in nature (Fig. 5.3.15). There are several
reports where we found that the distribution of hydrophobic amino acids is one of the
mechanisms of halophilic organisms to thrive in extreme salt concentrations (Oren,
2008; Nada, 2010).
Figure 5.3.15: Hydropathy analysis for O.M.A18 (Left panel) and O.M.E12
(Right panel) protease according to Kyte and Doolittle.
On the plot, a positive peak indicates a probability that the corresponding polypeptide
fragment is hydrophobic (a negative peak indicates a probable hydrophilic segment).
5.3.7 3D structure prediction In order to correlate our work on this enzyme, in its native (Purohit and Singh, 2011)
and recombinant forms, three-dimensional structures of O.M.A18 protease were
modeled using the online I-TASSER 3D structure prediction (Fig.5.3.16). We
evaluated that the distribution of alpha helix and beta sheets were repeated after
approximately every 20 amino acids in O.M.A18. While there was no such uniform
confirmation observed in O.M.E12. We further predicted that enzyme contained serine
amino acids at its active site by I-TASSER tool, which was supported by our findings
on the inhibitor studies, where polymethyl sulfonyl chloride (PMSF) strongly
inhibited the enzyme activity. The stability of in-silico structure was predicted by
170
Ramachandran plot-PROCHECK expasy tool, plotting psi vs. phi value; where it was
analyzed that molecule was stable in its confirmation (Fig.5.3.17).
A
B
Fig. 5.3.16: 3D structure prediction by I-TASSER structure prediction tool for
O.M.A18 (A) and O.M.E12 (B)
171
Fig. 5.3.17: Ramachandran analysis of predicted 3D structure by PROCHECK for
O.M.A18 (Left panel) and O.M.E12 (Right Panel)
We were not able to analyze any special distribution pattern of helix and sheets in its
secondary structure confirmation. We further predicted that enzyme contained serine
amino acids at its active site by I-TASSER tool, which was supported by our findings
on the inhibitor studies, where polymethyl sulfonyl chloride (PMSF) strongly
inhibited the enzyme activity.
172
In brief, recombinant enzyme maintained features and attributes of native protein
O.M.A18 and O.M.E12. The characteristics of the native haloalkaliphilic serine
alkaline protease were by and large maintained by recombinant clones O.M.A18 and
O.M.E12; with respect to urea denaturation, thermal and salt stability.
Overall, the results in this chapter are highlighted on the cloning, over expression and
inexpensive method of purifying the over-expressed protein. The results hold novelty
in that the over expressed recombinant protein was obtained in active form. The fact
that only limited enzymes from halophiles and haloalkaliphiles have been cloned and
studied for heterologous expression further adds to the study. The results and trends
highlighted in this chapter are therefore of value addition to the recombinant
enzymology and significant from biotechnological stand point.
CHAPTER Launch Internet Explorer Brow ser.lnk
CHAPTER
METAGENOMIC
STUDIES
METAGENOME ISOLATION AND CAPTURING
FUNCTIONAL ATTRIBUTES
6
173
SECTION-I
METAGENOMICS STUDIES FOR
BACTERIAL DIVERSITY AND ITS
AMENABALITY FOR FURTHER
FUNCTIONAL ATTRIBUTES
(ALKALINE PROTEASES)
174
6.1.1 INTRODUCTION
Metagenomics is an emerging approach based on the extensive analysis of the DNA
of microbial communities in their natural environment. Metagenomics has been
developed over the last several years to assess the genomes of the non-cultivable
microbes towards better understanding of global microbial ecology and to trap vast
biotechnological potential of a given habitat. The basic strategies encompass sequence
and functional based approaches. Since it is widely accepted that the majority of the
microbes are not cultivable, the not-yet-cultivated microbes represent a shear
unlimited and intriguing resource for the development of novel genes, enzymes and
other compounds for applications in biotechnology.
Studies on metagenomes have revealed vast scope of biodiversity in a wide range of
environment, and new functional capacities of individual cells and communities,
including complex evolutionary relationships between them (Kennedy and Marchesi,
2007). Microorganisms offer huge potential for new biocatalysts for industrial and
commercial applications. Of late metagenomic based strategies have recently been
employed as powerful tools to isolate and identify enzymes with novel biocatalytic
activities from the unculturable component of microbial communities from various
terrestrial and aquatic environmental niches. Besides, attention on the diversity and
phylogeny of unculturable organisms is also being focused, as marine environment
has enormous microbial biodiversity, yet to be explored (Kennedy and Marchesi,
2007; Kennedy et al., 2008; Purohit and Singh, 2009; Siddhpura et al., 2010).
Several metagenomic mega projects such as Sargasso Sea, Acid-mine drainage,
Human-Microbial Gut are completed worldwide successfully. However, similar
efforts have not been paid in context with saline habitats. The initial results hold
significance in the light of the fact that although saline environments display
enormous microbial biodiversity, it remains largely unexplored. The application of
metagenomic strategies embraces great potential to study and exploit the enormous
microbial biodiversity present within the saline habitats.
One of the hurdles in the way of metagenomics is the extraction of total
environmental DNA (metagenome) from a given habitat. We have explored various
175
protocols, in terms of DNA purity, yield and humic acid content, for the isolation of
metagenome from various saline soils of Gujarat, to substantiate its applications for
further molecular biological work. Diversity based assessment has been elucidated on
the basis of 16S rRNA amplicons – DGGE analysis (Molecular Fingerprinting
Technique). Beside, the source would also provide a huge and comprehensive
platform for capturing novel gene sequences. As an extension of our on-going work
on haloalkaliphilic bacteria from the saline habitats of Coastal Gujarat, we have taken
alkaline proteases as model system for the assessment of genetic diversity among
these habitats by designing degenerate primers with the aid of bioinformatics tools.
Successful cloning and expression of alkaline proteases revealed unidentified gene/s
with interesting features.
176
6.1.2 MATERIALS AND METHODS
6.1.2.1 Environmental Soil Sampling and Storage Two soil samples, designated as O.M.6.2 and O.M.6.5 were collected in September
2007 from Coastal region of Okha Madhi (Latitude 22.20 N, Longitude 70.05 E)
Gujarat, India. They represent a typical saline soil with heavy deposition of salt. At
the site of collection, a block of soil was removed and transported to laboratory in
sterile plastic bags for storage at 4°C. Total DNA extraction and further analyses were
carried out from these samples within 15 days.
6.1.2.2 Direct DNA extraction methods
A. Soft Lysis method
DNA extraction using Lysis Buffer
Soil sample (1g) was suspended in 10 ml of extraction buffer and incubated at 37°C
for 10- 12 h with shaking at 150 rpm. The samples were re-extracted in 1ml of
extraction buffer (100mM Tris HCl (pH-8.2); 100 mM EDTA (pH-8); 1.5 M NaCl).
Supernatant were collected by low speed centrifugation (5000rpm) for 10min. A 4ml
of Lysis buffer (20% (w/v) SDS; Lysozyme 1mg/ml; ProtinaseK 1mg/ml; N-lauroyl
sarcosine 10mg/ml;1%(w/v) CTAB(Cetyltrimethyl ammonium bromide) was added
and incubated at 65°C for 2 hours with vigorous shaking at every 15 min. Samples
were centrifuged at10000 rpm for 10 min at 4°C. The upper aqueous phase was
extracted with equal volume of P: C: I (25:24:1) at 10000 rpm for 20 min at 4°C.
Again upper aqueous phase was extracted with equal volume of C: I (24:1) at 10000
rpm for 10 min at 4°C. DNA was precipitated by adding 1/10th volume of 7.5M
potassium acetate and DNA was subsequently precipitated by adding 2 times of
chilled ethanol. DNA precipitate was collected by centrifugation at 10000 rpm for
10min, air dried and suspended in 20-50 µl TE buffer.
B. Harsh methods
DNA extraction using bead beating method
Soil sample (1g) was suspended in 10 ml of extraction buffer and incubated at 37°C
for 10-12 h with shaking at 150 rpm. Re-extract the sample in 1ml of extraction
177
buffer. Supernatant were collected by low speed centrifugation (5000 rpm) for
10mins. Glass beads (5g) were added and the sample blended for 15min and
incubated at 65°C for 2 hours. Samples were then centrifuged at 10000 rpm for 10min
at 4°C. The upper aqueous phase was extracted with equal volume of P: C: I (25:24:1)
at 10000 rpm for 20min at 4°C. Upper aqueous phase was again extracted with equal
volume of C: I (24:1) at 10000 rpm for 10 min at 4°C. DNA was precipitated by
adding 1/10th volume of 7.5M potassium acetate and DNA was subsequently
precipitated by adding 2 times of chilled ethanol. DNA precipitate was collected by
centrifugation at 10000 rpm for 10min, air dried and suspended in 20-50 µl TE buffer.
DNA extraction using sonication treatment method
Soil sample (1g) was suspended in 10 ml of extraction buffer and incubated at 37°C
for 10-12h with shaking at150 rpm. The sample was re-extracted in 1ml of extraction
buffer and the supernatant was collected by low speed centrifugation (5000rpm) for
10min. The supernatant was sonicated using a high intensity ultrasonic processor
(Sartorious, India) with a standard 13mm horn solid probe for 3 pulses of 30 seconds
each in a chilled ice bath. The sample was cooled in ice and repeatedly sonicated (6
cycles of 30 seconds) followed by incubation at 65°C for 10mins. Samples were then
centrifuged at 10000rpm for 10min at 4°C. The upper aqueous phase was extracted
with equal volume of P: C: I (25:24:1) at 10000 rpm for 20 min at 4°C. The upper
aqueous phase was again extracted with equal volume of C: I (24:1) at 10000 rpm for
10 min at 4°C. DNA was treated by adding 1/10 volume of 7.5M potassium acetate
and subsequently precipitated by adding 2 volumes of chilled ethanol. DNA
precipitate was collected by centrifugation at 10000 rpm for 10min, air dried and
suspended in TE buffer.
DNA extraction by a combination of Bead beating and Sonication treatment
Soil sample (1g) was suspended in 10 ml of extraction buffer and incubated at 37°C
for 10-12 h with shaking at 150rpm. The sample was re-extracted in 1ml of extraction
buffer. Supernatant were collected by low speed centrifugation (5000rpm;10min) and
sample was blended with glass beads (1g) for 15min followed by sonication using a
high intensity ultrasonic processor (Sartorious) with a standard 13mm horn solid
probe for 3 pulses of 30 seconds each in a chilled ice bath. The sample was cooled in
ice and sonicated repeated (6 cycles of 30 seconds) and incubated at 65°C for 10 min.
178
Samples were centrifuged at 10000 rpm for 10min at 4°C. The upper aqueous phase
was extracted with equal volume of P: C: I (25:24:1) at 10000 rpm for 20 min at 4°C.
The upper aqueous phase was again extracted with equal volume of C: I (24:1) at
10000 rpm for 10 min at 4°C. DNA was treated with 1/10 volume of 7.5M Potassium
acetate and subsequently precipitated by adding 2 volumes of chilled ethanol. DNA
precipitate was collected by centrifugation (10000 rpm; 10min) and air dried before
suspending in 20-50 µl TE buffer.
C. DNA extraction by Combination of Soft and Harsh Method
DNA extraction using bead beating combined with Lysis buffer treatment
Soil sample (1g) was suspended in 10 ml of extraction buffer and incubated at 37°C
for 10-12 h with shaking at 150 rpm. Re-extract the sample in 1ml of extraction
buffer. Supernatant were collected by low speed centrifugation (5000rpm) for 10min.
Glass beads (5g) were added and the sample blended for 5min and 15min. A 4ml of
lysis buffer was added and incubated at 65°C for 2 h and shake vigorously at every 15
min. Samples were centrifuged at 10000rpm for 10min at 4°C. The upper aqueous
phase was extracted with equal volume of P: C: I (25:24:1) at 10000 rpm for 20min at
4°C. Again upper aqueous phase was extracted with equal volume of C: I (24:1) at
10000 rpm for 10 min at 4°C. The upper aqueous phase was again extracted with
equal volume of C: I (24:1) at 10000 rpm for 10 min at 4°C. DNA was treated with
1/10 volume of 7.5M potassium acetate and subsequently precipitated by adding 2
volumes of chilled ethanol. DNA precipitate was collected by centrifugation (10,000
rpm; 10min) and air dried before suspending in 20-50 µl TE buffer.
DNA extraction using sonication treatment combined with lysis buffer
Duplicate 1g of soil sample was suspended in 10 ml of Extraction buffer and
incubated at 37°C for 10-12 h with shaking at 150 rpm. Re-extract the sample in 1ml
of extraction buffer. Supernatant were collected by low speed centrifugation
(5000rpm) for 10min. The supernatants were sonicated using a high intensity
ultrasonic processor (sartorious) with a standard 13mm horn solid probe for 3 pulses
& 6 pulses of 30 seconds each in a chilled ice bath. A 4ml of lysis buffer was added
and incubated at 65°C for 2 hours and shake vigorously at every 15 min. Samples
were centrifuged at 10,000rpm for 10min at 4°C. The upper aqueous phase was
extracted with equal volume of P: C: I (25:24:1) at 10000 rpm for 20min at 4°C. The
179
upper aqueous phase was again extracted with equal volume of C: I (24:1) at 10,000
rpm for 10 min at 4°C. DNA was treated with 1/10 volume of 7.5M Potassium acetate
and subsequently precipitated by adding 2 volumes of chilled ethanol. DNA
precipitate was collected by centrifugation (10,000 rpm; 10min) and air dried before
suspending in 20-50 µl TE buffer.
DNA extraction using bead beating combined with lysis buffer containing 30%
PEG.
1g of soil sample was suspended in 10 ml of extraction buffer and incubated at 37°C
for 10-12 h with shaking at 150rpm. Re-extract the sample in 1ml of extraction buffer.
Particles (supernatant) were collected by low speed centrifugation (5000rpm) for
10min. Glass beads (5g) were added and the sample blended for 15min. A 4ml of
lysis buffer containing 30%PEG was added and incubated at 65°C for 2 hours and
shake vigorously at every 15 min. Samples were centrifuged at 10000rpm for 10min
at 4°C. The upper aqueous phase was extracted with equal volume of P: C: I (25:24:1)
at 10000 rpm for 20min at 4°C. The upper aqueous phase was again extracted with
equal volume of C: I (24:1) at 10,000 rpm for 10 min at 4°C. DNA was treated with
1/10 volume of 7.5M potassium acetate and subsequently precipitated by adding 2
volumes of chilled ethanol. DNA precipitates were collected by centrifugation
(10,000 rpm; 10min) and air dried before suspending in 20-50 µl TE buffer.
DNA extraction using small and big beads together with lysis buffer
Duplicate 1g of soil sample was suspended in 10 ml of extraction buffer and
incubated at 37°C for 10-12 h with shaking at 150rpm. Re-extract the sample in 1ml
of extraction buffer. Particles (supernatant) were collected by low speed
centrifugation (5000 rpm) for 10min. Small glass bead (0.5g) & big glass beads (0.5g)
were added and the sample blended for 5min &15min. A 4ml of lysis buffer was
added and incubated at 65°C for 2 hours and shake vigorously at every 15 min.
Samples were centrifuged at 10000 rpm for 10min at 4°C. The upper aqueous phase
was extracted with equal volume of P: C: I (25:24:1) at 10,000 rpm for 20 min at 4°C.
Again upper aqueous phase was extracted with equal volume of C: I (24:1) at 10,000
rpm for 10 min at 4°C. The upper aqueous phase was again extracted with equal
volume of C: I (24:1) at 10,000 rpm for 10 min at 4°C. DNA was treated with 1/10
volume of 7.5M potassium acetate and subsequently precipitated by adding 2 volumes
180
of chilled ethanol. DNA precipitate was collected by centrifugation (10,000 rpm;
10min) and air dried before suspending in 20-50 µl TE buffer.
6.1.2.3 Determination of purity and yield of DNA
Co- extracted humic acids are the major contaminant when DNA is extracted from
soil. These compounds absorbs at 230nm, DNA at 260nm and protein at 280nm. To
evaluate the purity of the extracted environmental DNA (eDNA), absorbance ratios at
260nm/230nm (DNA/humic acid) and 260nm/280nm (DNA/protein) were determined.
6.1.2.4 Gel Electrophoresis DNA extracts (10µl) from each method were mixed with 5µl loading buffer and
analyzed on 0.8% agarose gels using TAE as electrophoresis buffer. Gels were stained
with ethidium bromide and gel photographs were scanned and analyzed by syngene
Gene Genius Bio-imaging system. A DNA marker (DNA Ruler-Middle range, Merk
life sci, India) was included in each run.
6.1.2.5 PCR amplification of 16S rRNA gene The DNA preparations described above were used as a template to amplify a DNA
fragment encoding 16S rRNA gene. The reaction mixture preparation and
amplification protocol was as described in detail in Materials and Method section of
Chapter-3.
6.1.2.6 Denaturing Gradient Gel Electrophoresis Denaturing Gradient Gel Electrophoresis was performed according to Muyzer and
Smalla (1998). 50µl of 16S rRNA amplicon were subjected to increasingly higher
concentrations of urea and formamide which act as a chemical denaturant (20- 50%).
The amplicon migrated through polyacrylamide gel containing denaturants at constant
voltage; 200V for 1 hour followed by 30V for 10 hour. Gels were visualized and
analyzed by Syngene Gene Genius Bio-imaging system after staining with ethidium
bromide (5µg/ml). The extracted DNA was assessed by PCR amplification of 16S
rRNA region followed by DGGE analysis.
181
6.1.2.7 PCR primer designing and amplification of alkaline protease
gene/s Amplification of extracellular alkaline proteases directly from soil was carried out by
designing degenerate primers using CODEHOP method (Timothy et al., 2003). The
designing of primers is as described in detail in materials and method section of
chapter 5. The primer set that yielded the specific amplified product is as follows:
SPS-5 forward 5’-gga tcc gcc gcc gag gac gac-3’and reverse 5’-5’-gtc gac atg gga tat
tat gac-3’; SPS-6 forward 5’-gga tcc gcc gcc gag gac gac-3’and reverse 5’-gga tcc gcc
gcc gag gac gac-3’; SPS-7 forward 5’-cat atg ccg ccg agg agg ac-3’ and reverse 5’-gtc
gac ggc ctt cgt gtg g-3’.
The DNA preparations obtained in the present study were used as template to amplify
region/s coding alkaline protease. The reaction mixture preparation and amplification
protocol for protease gene amplification from soil DNA was as described in detail in
Materials and Method section of Chapter-5.
182
6.1.3 RESULTS AND DISCUSSION
6.1.3.1 Isolation of Total DNA (Metagenome/Environmental DNA) We have assessed and compared various methods for the extraction of total
environmental DNA from saline soil of coastal Gujarat and optimized them for the
quality, yield and PCR amplification ability. The applicability of various methods for
the extraction of total DNA using small quantity of soil sample was explored. Total
DNA was isolated from the two samples collected from Okha Madhi site by soft lysis,
bead beating, sonication and by combination of these methods. In comparison to these
methods, we also attempted the extraction of total DNA by Clean Gene Isolation kit
(Mo Bio laboratories) and also tried several other approaches.
DNA extraction from saline soil in the present study had three folds objectives; lysis
of representative microbes within the sample, obtaining high molecular weight intact
DNA and removal of inhibitors from the extracted DNA for subsequent molecular
manipulations. Therefore, various methods were examined for DNA extraction
towards fulfilling these objectives. We have developed an improved method for
isolating total metagenomic DNA from saline soil through which intact and unsheared
DNA, amenable for further molecular biology work was obtained.
6.1.3.2 Purity and Yield assessment on the basis of spectrophotometer
and agarose gel electrophoresis The extracted DNA was assessed for purity and yield on the basis of absorbance ratios
at 260/230nm (DNA/Humic acids) and 260nm/280nm (DNA/protein) (Table 6.1.3.1).
High ratio of 260/230nm indicated the purity of extracted DNA with respect to humic
acid contamination, whereas high 260/280nm ratio was an indicative of the purity with
respect to protein contamination. DNA samples were analyzed using 0.8% agarose gel
using λDNA/Hind III digest (Merk life science,India) as marker and smart ladder
(0.2-10kbp) (invitrogen). There was no noticeable variation in the quality and quantity
of DNA on the basis of agarose gel patterns (Fig. 6.1.3.1).The striking features of the
extraction methods highlights that sonication alone was not suitable for DNA
extraction from O.M.6.5, as humic acids content was not reduced and the purity and
concentration of the extracted DNA did not compare favorably with other methods.
183
1 2 3 4 5 6 7
Fig.6.1.3.1: Isolation of metagenomic DNA by various methods for saline soil
sample O.M.6.2 and O.M.6.5. Lane 1: Lamda DNA /HindIII Marker (Banglo
Genei), Lane 2: Soft Lysis, Lane 3: Bead Beating, Lane 4 : Bead beating+Lysis,
Lane 5 : Sonication+Lysis Lane 6 : Sonication Lane 7: Sonication+Bead Beating
1 2 3 4 5 6 7 8 9 10 11 12 13
Fig.6.1.3.2: Agarose gel electrophoresis of the Total Genomic DNA. Lane 1: DNA
ruler (Middle range marker, Merk life sciences, India), Lane 2: lysis buffer treatment
(sample-6.2), Lane-3: Lysis Buffer Treatment (sample-6.5), Lane 4: Bead Beating
only (sample-6.2); Lane 5: Bead Beating only (sample-6.5); Lane 6: Bead Beating
+ Lysis Buffer Treatment (sample-6.2), Lane 7: Bead Beating + Lysis Buffer
Treatment (sample-6.5); Lane 8: Bead Beating + sonication treatment (sample-6.2);
Lane 9: Bead Beating + sonication treatment (sample-6.5); Lane 10: lysis buffer +
sonication treatment (sample-6.2); Lane 11: lysis buffer + sonication treatment
(sample-6.5); Lane 12: sonication treatment (sample-6.2); Lane 13: sonication
treatment ( (sample-6.5) (Fig.6.1.3.2).
However, the method based on sonication yielded better results with another sample,
O.M.6.2 (Fig.6.1.3.2). The differential results by sonication may reflect on the fact
2.31kb
564bp
3kb
1kb
184
that the matrix of the concerned habitat might be causing a barrier against the
extraction and lysis of the cell. Spectrophotometric assessment revealed that there
were quite similarity in purity and yield of retrieved DNA from both sites.
Comparative analysis indicated that the soft lysis and bead beating method yielded
pure form of DNA from both the sites. Contradictorily, sonication method was not
suitable for both the samples as the yield of DNA was very low when compared to
other methods. Combinations of the above methods were quite encouraging as bead
beating combined with lysis buffer treatment yielded pure DNA in good quantity as
compared to bead beating and lysis buffer method independently, from both the site.
On the other hand, sonication in combination with lysis buffer treatment did not
emerge as a better alternate, as compared to their independent outcomes.
Bead beating emerged as equally effective method for the extractions of quality DNA
in appreciable quantity from both environmental samples. Standardization of method,
for 5 mins and 15 mins, on the basis of assessment was found that agitation for 15
mins gives optimum concentration, similarly size of bead also have a great influence
on yield of DNA. The major factor, responsible for choosing bead size and time is
type and characteristic of soil material. In present study, moderate size beads gave
better result as compared to its counterpart. On the other hand, combinations of
different mechanical methods lead to decreased concentration and purity of the
extracted DNA with excessive shearing.
Soft lysis method proved best for O.M.6.2 and O.M.6.5, as it yielded higher
concentration and eliminated humic acid to significant extent. Modification of soft
lysis method with PEG (30%), was also tried for better yield, however encouraging
results were not observed. The best quality of DNA was obtained by employing
combination of soft lysis with bead beating method, while its combination with
sonication was not as good in terms of DNA yield. The DNA preparations were
considered for molecular biology applications, as obtained environmental DNA
should not only satisfy the yield and purity criteria but it should also be amenable for
further work (Desai and Madamwar, 2007).
As, we evaluated DNA extraction methods to identify a procedure that results in high
molecular weight DNA that is relatively free from contaminants and maximizes
detectable diversity. Bead beating treatment, an easy to perform method, is based on
185
the ballistic disintegration of the cells, where the results depend upon the time of
agitation and bead size. The efficiency of cell disruption and consequently, the
damage to the DNA strands during sonication mainly depends on the energy input.
Even under optimized conditions, harsh treatment may result in shearing of high
molecular weight DNA, low yields and small fragment sizes. This method may have a
possibility of introducing a bias in microbial community analysis.
The enzymatic method relied on the proteinase K and lysozyme digestion of microbial
cells to release DNA, while the treatment of soil with surfactants and chelating agents
resulted into removal of inhibitors and prevented chemical flocculation with minimal
loss of DNA yield. PVPP treatment was employed for removing traces of humic acid
in DNA sample. Surprisingly, although literature reports that better quality of DNA is
resulted after treatment, noticeable difference was not seen in our sample. A
combination of mild bead beating and enzymatic lysis treatment emerged as the most
successful protocol for recovering higher yields and inhibitor free DNA from saline
soil sample. In order to obtain pure form of DNA in an easiest way, a modification of
soft lysis method with cesium chloride and Ethidium Bromide was also attempted, in
this case although the pure form of DNA was found, yield was quite less, which
certainly limited its further applications.
Although, based on the spectroscopic analysis, humic acid was detectable to varying
extent; the extracted DNA preparations were amenable for further molecular biology
work. This finding appears to be a favorable observation in comparison to some
reports in literature where humic acid strongly inhibited the DNA application in
molecular biology (Kauffmann et al., 2004; Santosa 2001; Desai and Madamwar,
2007). The quality of the extracted metagenome is of prime importance in
metagenomics, as the DNA should be suitable to proceed for molecular biological
applications such as molecular diversity and functional genomics (Rajendhran and
Gunasekaran, 2008).
6.1.3.3 PCR amplification of 16S rRNA gene The environmental DNA extracted by all the above mentioned methods was used as
template for PCR amplification. Amplification of the 16S rRNA gene (aproximately-
1.5kb) directly from undiluted DNA samples (Okha Madhi) indicated the high purity
of DNA (Fig. 6.1.3.3, 6.1.3.4, 6.1.3.5).
186
1 2 3 4 5 6 7 1 2 3 4 5 6 7
Fig. 6.1.3.3: 16S rRNA amplification of Total DNA isolated by various method by using eubacterial universal primer Left Panel: 16S rRNA PCR of environmental sample using isolation methods Bead beating + Sonication Method, Lane 1: 0.2-10Kb ladder, Lane 2: Site 6.2 (Ta=52.4), Lane 3: Site 6.2 (Ta=55.7), Lane 4 : Site 6.2(Ta=56.9), Lane 5 : Site 6.5(Ta=52.4), Lane 6 : Site 6.5 (Ta=55.7), Lane 7 : Site 6.5 (Ta=56.9). Right panel :( Bead Beating Method) Lane 1: 0.2-10Kb ladder, Lane 2: Site 6.2 (Ta=52.4), Lane 3: Site 6.2 (Ta=55.7), Lane 4 : Site 6.2(Ta=56.9), Lane 5 : Site 6.5(Ta=52.4), Lane 6 : Site 6.5 (Ta=55.7), Lane 7 : Site 6.5 (Ta=56.9) 1 2 3 4 5 6 7 8 9 10 11 12 13
Fig. 6.1.3.4: 16S rRNA Amplification from Total DNA of sample O.M.6.2 (Ta- 64oC) Lane 1, smart ladder 0.2-10 kbp ladder (invitrogen); Lane 2, Lysis treatment; Lane 3, Soft Lysis + Bead Beating; Lane 4, Soft Lysis +Sonication; Lane 5, Bead beating; Lane 6, Sonication; Lane 7, Sonication+ Bead Beating. 16S rRNA Amplification from Total DNA of sample O.M.6.5 (Ta- 64oC) Lane8, Lysis treatment; Lane 9, Soft Lysis +Bead Beating; Lane 10, Soft Lysis +Sonication; Lane 11, Bead beating; Lane 12, Sonication; Lane 13, Sonication+ Bead Beating
16S
rRNA
1
5
0
0
b
p
b
p
1500bp
1500bp
3000bp
3000bp
187
1 2 3 4 5 6 7 8 9 10 11 12 13
Fig.6.1.3.5: 16S rRNA Amplification from Total DNA of sample O.M.6.2
(Ta- 62.5oC) Lane 1, Broad range ruler (Merk life science, India); Lane 2, Lysis
treatment; Lane 3, Soft Lysis + Bead Beating; Lane 4, Soft Lysis +Sonication; Lane
5, Bead beating; Lane 6, Sonication; Lane 7, Sonication+ Bead Beating.
16S rRNA Amplification from Total DNA of sample O.M.6.5 (Ta- 62.5oC) Lane
8, Lysis treatment; Lane 9, Soft Lysis +Bead Beating; Lane 10, Soft Lysis
+Sonication; Lane 11, Bead beating; Lane 12, Sonication; Lane 13, Sonication+
Bead Beating; Lane 14: Positive control
Fig.6.1.3.6 16S rRNA Amplification from Total DNA of sample O.M.6.5
(Ta- 62.5oC) Lane 1, Broad range ruler (Merk life science,India); Lane 2, Lysis
treatment; Lane 3, Soft Lysis + Bead Beating; Lane 4, Soft Lysis +Sonication; Lane
5, Bead beating; Lane 6,Sonication; Lane 7, Sonication+ Bead Beating
16S rRNA Amplification from Total DNA of sample O.M.6.5 (Ta- 62.5oC)
Lane 8, Lysis treatment; Lane 9,Soft Lysis +Bead Beating; Lane 10, Soft Lysis
+Sonication; Lane 11,Bead beating; Lane 12,Sonication
1 2 3 4 5 6 7 8 9 10 11 12
1500bp 16S
rRNA
16S
rRNA
1500bp
188
Total DNA preparations extracted by chemical lysis and bead beating method from
the samples of both sites were used as template for PCR amplification of 16S rRNA
gene. Amplification was successfully carried out in all the gradient range of
temperatures selected for annealing by gradient PCR (Fig.6.1.3.3, 6.1.3.4, 6.1.3.5,
6.1.3.6). Intense amplified band of 1.5 kb from the saline soil sample O.M.6.3 and
O.M.6.5 was observed from both the sites. However, the intensity of amplicon varied
at different Ta used for profile generation (Fig. 6.1.3.6). In general, good amplification
was observed at all the tested temperature conditions.
6.1.3.4 Denaturing Gradient Gel Electrophoresis To gauge the utility of extracted DNA in molecular fingerprinting methods, especially
in microbial ecology studies, the amplified 16S rRNA DNA was subjected to
denaturing gradient gel electrophoresis. In DGGE at threshold concentrations of
denaturant, different sequences of DNA, presumably from different bacteria,
denatured resulting in a pattern of bands. As revealed in Fig 6.1.3.7, the DGGE band
patterns of 16S rRNA amplified from different DNA samples obtained by various
extraction protocols, were quite comparable. The observation was also reflected with
the extracted DNA from different sample sites. Therefore, differences in DGGE
banding pattern suggested that there was not much bias generated from DNA
extraction procedures (Fig.6.1.3.7). The diversity in the banding profile, however,
revealed population heterogeneity and differences in both samples. Marker DNA
(smart ladder, 10kbp) in denaturing gel did not generate bands according to standards
(Fig. 6.1.3.7).
The described methods could allow the use of large scale preparations providing
greater probability of detecting genes present in low abundance in the soil
environment. These methods would be applicable to more challenging and heavily
contaminated soils; therefore, microbial biodiversity assessment can now be more
readily assessed and useful sequences could be retrieved.
189
1 2 3 4 5 6 7
1 2 3 4 5 6 7
Fig.6.1.3.7 DGGE (Denaturing gradient gel electrophoresis) of 16S rRNA
amplicons
Upper panel, DGGE Analysis (Urea and Formamide as denaturant) of the PCR
amplified product from Total DNA of sample O.M.6.1.2. Lane 1, smart ladder 0.2-
10kbp ladder (invitrogen); Lane 2, Lysis treatment; Lane 3, Soft Lysis + Bead
Beating; Lane 4, Soft Lysis +Sonication; Lane 5, Bead Beating; Lane 6, Sonication;
Lane 7, Sonication+Bead Beating
Lower panel, DGGE Analysis (Urea and Formamide as denaturant) of the PCR
amplified product from Total DNA of sample O.M.6.5. Lane 1, Lysis treatment;
Lane 2, Soft Lysis + Bead Beating; Lane 3, Soft Lysis +Sonication; Lane 4,
Beadbeating; Lane 5, Sonication; Lane 6, Sonication+BeadBeating; Lane 7, smart
ladder 0.2-10kbp ladder (Invitrogen).
6.1.3.5 Alkaline protease gene amplification Total DNA extracted by chemical lysis method from both the sample of Okha Madhi
(O.M.6.2 and 6.5) were used as template for alkaline protease gene amplification.
Selection of template for amplification was based on the results of quantification and
190
purity. All the sets of primers designed for alkaline proteases were used for
amplification. Amplicons of varied size and concentration were obtained by using
different sets of primer. Intense amplified bands were obtained by using combination
of SPS-5, SPS-6 and SPS-7. Range of amplicons, were obtained of different size SPS-
5 gave 0.5kb product for O.M.6.2 and 0.7 kb for O.M.6.5, the size of the product is
quite less as compared to the size of alkaline protease judged from literature. SPS-5F
and SPS-6R gave 1kb product for O.M.6.2 and for O.M. 6.5 no products were
obtained (Fig.6.1.3.8). Similar, results were also obtained for SPS-5F and SPS-7R; on
the basis of this it could be judged that availability of alkaline proteases in the sample
O.M.6.5 would be less as compared to its counterpart soil sample. SPS-6 and
combination of SPS6F and 5R generated no product. SPS-7F and SPS-7R gave
product size of 1.2kb with O.M.6.5 while partial product of 0.5kb, 0.7kb, 1.1kb and
2.8kb was obtained with same primer combination (Fig. 6.1.3.9). However, SPS-6F
and SPS-7R gave 1kb product with O.M.6.2 (Fig.6.1.3.9). Different sizes of bands
were obtained after amplification procedure, which were quiet interesting for
generating profile of alkaline proteases (Fig.6.1.3.9).
Amplification of varied size of products were obtained by SPS-7, as the primer were
designed by using degenerate primer designing tool-CODEHOP (Fig.6.1.3.9).
In this procedure, the chances of getting different types of proteases are quiet higher
as compared to primer designed on the basis of individual known sequences. As, a
whole CODEHOP primer under the optimized sets of protocol, would certainly allow
to capture proteases sequences present in the particular saline soil at a given habitat.
Along, with functional attributes this would also give us some of the information on
diversity of proteases, particularly alkaline proteases. Further, for O.M.6.2, nucleotide
sequence explored by chromosome walking method (Fig.6.1.3.10). On the basis of
nucleotide sequence and ORF prediction, amino acid sequence was predicted
(Fig.6.1.3.10). Alkaline protease sequence could be further explored to use as a
marker trait for identification, knowledge driven process of saline soil of Okha Madhi
(Gujarat, India).
While there is no doubt that multiple bands in Okha Madhi site could be due to the
annealing of primer at multiple sites within the template, as this question could be
better addressed by sequencing of the amplicons. To address, this ambiguity, PCR
191
products were run on low melting agarose (low EEO). Sample were gel eluted and
sequenced by using SPS-6F and SPS-6R primer by using chromosome walking
method. Complete sequence was determined and subjected to Mega 4.0 for
phylogenetic determination. Sequence was found most homologus to protease type of
family. All the characteristic features related to physico-chemical properties and
structure details were elucidated.
Along the same line, it was further analyzed and confirmed by sequencing of
dominant bands that multiple bands generated by a primer within a single reaction, is
not the result of binding of primer at several position in same gene. The amplification
profile was found to be reproducible from both the sites, a fact which could be
explained on the basis of equal distribution of organism producing alkaline protease
gene within a particular habitat and a good concentration of template DNA. Results of
16S rRNA PCR amplification and banding profiles visualized in DGGE provided the
evidence for expediency of the DNA extraction protocol in studies related to
molecular diversity (Ercolini, 2004). In view of the heterogeneity of the
environmental samples, it is quite obvious that the extraction procedures would have
to be case specific and hence need to be optimized for different soil samples (Santosa,
2001; Vereshchagin and Kostornova, 2008; Purohit and Singh, 2009; Siddhpura et al.,
2010). However, the methods described in the present study appear to have wide
applicability in investigating molecular diversity and exploring functional genes from
the total DNA.
In a nutshell, given that the majority of natural products are of microbial origin, and
that the vast majority of microbial genomes are yet to be explored, it’s quite logical
that microbial metagenomes harbour a great economic potential. Due to their huge but
largely unexplored diversity and history as sources of commercially valuable
molecules with agricultural, chemical, industrial and pharmaceutical applications,
marine environments would be among the most common habitats to explore from
metagenomics view point (Morrissey et al., 2010). Improved functional screening
methods would potentially provide a means to discover new variants of functions of
interest.
With the possibilities to access vast genetic resources in different ecosystems, the
unlimited realms of microbial diversity would slowly but steadily lead to new
192
knowledge and novel biotechnological avenues. However, the usual challenge of
heterologus gene expression needs to be addressed to turn metagenomic technologies
into commercial successes, particularly in applications where bulk enzyme or product
have to be produced at viable cost (Kennedy et al., 2007).
The goals of researchers venturing into the microbial metagenome vary from directed
product discovery to total community characterization and assessment of the
phylogenetic complexity of the environments. Metagenomics has redefined the
concept of a genome, and accelerated the rate of gene discovery. The potential for
application of metagenomics to biotechnology seems endless. Metagenomics, together
with in-vitro evolution and high-throughput screening technologies would provide
unprecedented opportunities to bring new generation of biomolecules into various
fields, besides adding to new knowledge in our understanding on biotic and abiotic
interactions in ecosystems.
Fig. 6.1.3.8: Functional attributes of alkaline proteases: Amplification profile of
alkaline proteases gene by different sets of combination of primer for O.M.6.2 and
O.M.6.5: Sets of primers: SPS5F and SPSR; SPS5F and 6R; SPS5F and 7R; SPS6F
and 6R; SPS6F and SPS5R; SPS6F and SPS7R; SPS7F and SPS7R; SPS7F and
SPS6R; SPS7F and SPS5R
Siz
e (k
b)
193
Fig 6.1.3.9: PCR amplification of alkaline proteases genes
(A) PCR amplification of alkaline protease gene by SPS7F and SPS7R Lane 1: 62oC-
O.M.6.2; Lane 2: 63.5oC-O.M.6.2; Lane 3:60.1oC-O.M.6.5; Lane 4:62.3oC-O.M.6.5;
Lane 5: Low range DNA Ruler (3000bp), (Merk Life Science).
(B) PCR amplification of alkaline protease gene by SPS5F and SPS6R Lane 1: Low
range DNA Ruler (3000bp) (Merk life science); Lane 2: 62oC -O.M.6.2; Lane 3:
63.2oC-O.M.6.2; Lane 4: 60.1oC- O.M.6.5; Lane 5: 62.3oC-O.M.6.5; Lane 6: 63.5oC-
O.M.6.5
(C) PCR amplification of alkaline protease gene by SPS6F and SPS6R Lane 1: 62oC-
O.M.6.2; Lane 2: 63.2oC-O.M.6.2; Lane 3: 60.1oC-O.M.6.5; Lane 4: 62.3oC-
O.M.6.5; Lane 5: Medium range DNA Ruler (5000bp)(Merk Life Science)
(D) PCR amplification of alkaline protease gene by SPS5F and SPS5R Lane 1: DNA
marker; Lane 2: 63.2oC-O.M.6.2; Lane 3: 60.1oC-O.M.6.5; Lane 4: 62.3oC-O.M.6.5;
Lane 5: 62oC-O.M.6.2
1 2 3 4 5 1 2 3 4 5 6
1 2 3 4 5 1 2 3 4 5
A
B
C
D
Protease gene
amplicons
Protease gene
amplicons
194
Fig.6.1.3.10: Partial nucleotide sequence analysis of O.M.6.2 alkaline proteases by
chromosome walking method and partial amino acid sequence prediction of O.M.6.2
alkaline proteases by reverse translate tool (ExPASY).
195
SECTION-II
CAPTURING OF ALKALINE
PROTEASES FROM SALINE SOIL
METAGENOME:
A CULTURE INDEPENDENT
APPROACH
196
6.2.1 INTRODUCTION
An enormous variety of different biocatalysts or other functional products can be
theoretically obtained using DNA extracted from a given environmental sample. By
fragmenting total DNA from an alkaline marine sample, cloning it into an expression
vector, and screening for protease/esterase/lipase activity in an easily cultivable host
strain, 120 new enzymes were discovered, falling into 21 protein families (Miller,
2000). As, smaller cloned fragments are created; further necessitate larger gene banks
required for a comprehensive and comparable coverage of the genetic informations
(Kennedy et al., 2008). During the past five years, cloning of genes from the
metagenome has become the most popular tool for cultivation-independent enzyme
discovery, leading to the recovery of a range of new biocatalysts by academic and
commercial institutions (Kennedy and Marchesi, 2007; Kennedy et al., 2008).
197
6.2.2 MATERIALS AND METHODS
6.2.2.1 Amplification, cloning procedures, expression analysis and
one-step purification of alkaline protease enzyme Amplification of metagenomics DNA was carried as described in section 6.1.2.2.1.8
of this chapter. Cloning procedures, expression analysis and one step procedures were
carried out as described in cloning and over-expression of haloalkaliphilic isolates in
chapter-5.
6.2.2.2 DNA Sequencing, In-silico analysis and 3D structure modeling Plasmids were re-retrieved from positive clones and sequenced from both ends, using
standard T7 promoter and terminator sequence which is on a flanking region of insert
by chromosome walking method (Merk Life sciences, India).The amino acid
sequence was deduced using CLC main workbench (Daintith, 2004). Sequence
homologies and three dimensional structures of serine protease were modeled using
the online I-TASSER server as described in detail in Chapter-5.
6.2.2.3 Nucleotide sequence accession number The DNA sequence of the protease gene cloned and studied in present work was
submitted in the GenBank database under the accession number HM219181 with its
characteristic properties in native and recombinant system.
198
6.2.3 RESULT AND DISCUSSION
6.2.3.1 Cloning confirmation Positive clones exhibiting resistance towards ampicillin (30µg/ml) were selected for
plasmid isolation. For cloning confirmation, protease gene from environmental
sample was sequenced by custom based service of Merk life science, India.
Phylogenetically, protease sequence was found homologus to protease sequence of
Bacillus sp. by the neighbor-joining method clustering strategy in Mega
4.0(www.megasoftware.net) (Tamura, 2007).
6.2.3.2 Functional analysis of recombinant clone PCR-based cloning methods are being employed to recover novel enzymes. In most
cases, degenerate primers are used, hybridizing with conserved regions that
preferentially are located close to the extremities of the target genes (Liles et al.,
2008; Ni et al., 2009). The characteristic features of native and recombinant enzymes
were studied; interestingly, we noticed that recombinant clones have maintained their
nascent properties with higher specific activity.
In detail, functional attributes of metagenomic clone was judged on gelatin agar plate.
The zone of clearance was quite comparable with our studies on other reported
haloalkaliphilic bacteria, halotolerant actinomycetes and other recombinant clones
(Thumar and Singh, 2007; Dodia et al., 2008a and b; Joshi et al., 2008; Thumar and
Singh, 2009; Singh et al., 2010a and b; Purohit and Singh, 2011). Similar results were
observed on recombinant clones studied for over-expression and characterization
from the same soil sample.
6.2.3.3 Protein solubilization
An enormous variety of different biocatalysts or other functional products can be
theoretically obtained using DNA extracted from a given environmental sample.
However, to check for functional clone; within a metagenome DNA, by designing
primer based approach is to search needle in a haystack (Handelsman, 2004; 2005;
2008). However, in our present studies it has been observed that, protein was able to
solubilize. Along the same line, it was quiet interesting that to get alkaline protease as
a signature sequence from a total DNA. Although, equally challenging is to get active
199
enzyme from a soil DNA. However, if draw a comparative picture of over-expression
profile and enzyme activity of recombinant clones of haloalkaliphiles and
metagenome derived clone; it is general trend seen that enzyme studied in current
studies is far more sensitive as compared to cultivable alkaline proteases.
6.2.3.4 Effect of temperature For, a soil derived recombinant clone, as compared to results of chapter-5, growth
temperature of 27°C was optimum for secretion of recombinant protein on gelatin
plate and SDS-PAGE; however, level of expression was also found satisfactory at
other parameters. However, obviously growth as expected was definitely higher at
37oC as compared to 27oC.
6.2.3.5 Effect of IPTG induction As described in chapter-5, levels of induction have a profound impact on expression
analysis as transcriptions of genes are controlled by T7 strong promoter in pET21a+
(Novagen, Madisen, USA). At 1mM IPTG induction, higher amount of enzyme was
produced as compared to 3mM which was evidently seen on SDS-PAGE. Level of
induction, however, did not have significant effect on growth of host cell.
6.2.3.6 Synergistic effect of IPTG induction and temperature Synergistic effect of temperature and IPTG was checked both at best combination i.e.
27oC and 1mM IPTG and 37oC and 3mM IPTG, to check the effect of best factors on
protein solubilization (Fig.6.2.3.1). According to literature study, it is generally
observed that at low level of temperature and induction subsequent level of enzyme is
over-expressed (Singh et al., 2009; Yan et al., 2009; Xu et al., 2009), similar results
were also observed in the present studies; i.e. 27oC and 1mM IPTG; high level of
protein was expressed (Fig. 6.2.3.1). However, in our studies subsequent amount of
enzyme was also expressed at other mentioned parameter. The SDS-PAGE profile
and determination of proteolytic activities patterns bear that there was no activity at
basal level; however after four hours of induction, there was gradual increase of the
target protein in soluble and insoluble fraction.
200
Fig.6.2.3.1: Synergistic effect of induction: Effect of inducer (IPTG) was checked
on growth of cells and enzyme production of O.M.A18 (Colony no:1 and 2) and O.M.
E12(Colony no: 3 and 4); where effect of 1mM IPTG at 37oC; 1mM IPTG at 27oC;
3mM IPTG at 37oC; 3mM IPTG at 27oC
6.2.3.7 Proteolytic activity assay A result of SDS PAGE was quite comparable with the proteolytic assay in terms of its
activity. No basal level activity was seen in uninduced sample, with increase in time
significant amount of activity was monitered after 6 hours of induction (Fig.6.2.3.3).
6.2.3.8 Purification of protein To facilitate purification, the recombinant alkaline proteases, which carries His-tag at
its C-terminal in pET 21a+ was exploited. Purification was achieved at its
homogeneity by one step chromatography; by using 50mM immidazole concentration.
Complete purification was evident from SDS-PAGE and specific activity of 6765.76
with fold purification of 6.41 and high yield (Table 6.2.3.1). Profound amount of
enzyme was noticed in soluble and insoluble fractions. Result holds significance as,
within one step of purification significant amount of enzyme is produced. Along this
line, characteristic features of alkaline proteases derived from haloalkaliphilies and
metagenome clone would be quite similar; evident from similar purification strategies
201
Fig. 6.2.3.2: Effect of IPTG and temperature on growth and enzyme
secretion:SDS PAGE of comparative effect of temperature and IPTG induction on
growth and secretion of alkaline protease enzyme.
Left panel Soluble fraction: Lane 1:Protein molecular weight marker (3500-
205000Da); Lane 2:27oC;1 mM (0 hr); Lane 3:27oC;1 mM (2 hr); Lane 4:27oC;1
mM (4hr); Lane 5:37oC;3 mM (6 hr); Lane 6:37oC; 3mM (24hr). Right panel
Insoluble fractions: Lane 1:Protein molecular weight marker (3500-205000Da);
Lane 2:27oC;1 mM (0 hr);Lane 3:27oC;1 mM (2 hr); Lane 4:27oC;1 mM (4 hr);
Lane 5:27oC;1 mM (4hr); Lane 6:37oC;3 mM (6 hr); Lane 7:37oC; 3mM (24hr).
exploited (Purohit and Singh, 2008). These results are quite encouraging and
interesting in light of verity that, purification was achieved in one step, and substantial
level of enzyme was obtained in simple bacterial system. The approximate calculated
size of protein is estimated to be around 30 KDa, which is quite comparable to our
bioinformatics based prediction and our study on the same protein secreted from
several haloalkaliphilic bacterium (Fig.6.2.3.2).
1 2 3 4 5 6 1 2 3 4 5 6 7
30 kDa 30kDa
202
Table 6.2.3.1: One step purification of enzyme O.M.6.2 by affinity chromatography
6.2.3.10 Characterization of enzyme
Effect of Temperature and pH
The pH profile for the isolate was quite broad and they were able to grow and secrete
protease at pH, 7-10 (Fig. 6.2.3.3A). Although, the optimum pH for enzyme secretion
was pH-7, there was only marginal difference in enzyme secretion from pH-7 and 9;
enzyme was not able to maintain its activity at 10. On the basis of our study on
moderate saline habitats from coastal region of Gujarat, from last 15 years, we have
not come across enzyme; which is not catalytically active at higher alkaline range;
infact we have several reports of alkaline protease active at pH-9, 10. Similar,
contradictory results were obtained in studying effect of temperature; the organisms
were able to secrete enzyme efficiently at 37oC, however sparse activity was seen at
50oC, while enzyme was completely denatured at 60oC(Fig.6.2.3.3B). These results
are equally intriguing, as we have several isolates isolated from same soil sample
O.M.6.2; which are active upto 50oC-90oC (i.e., Oceanobacillus iheyensis O.M.E12
(EU680960); Haloalkaliphilic bacterium O.M.A18 (EU680961). Infact, it was found
that an enzyme activity increases in magnitudes with increase in temperature to its
optimum. Similar trend is not noticed in our present studies.
Enzyme Preparations
Activity (U/ml)
Total activity
(U)
Protein (mg/ml)
Total protein
(mg)
Specific Activity
(U/mg)
Yield Purification fold
Recombinant fraction
216 1728 0.204 1.632 1054.94 100 -
Purified enzyme
751 1502 0.111 0.222 6765.76 86.92 6.41
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Fig. 6.2.3.2: Effect of pH (7, 8, 9, 10) and Temperature (35, 50, 60, 70) on
recombinant alkaline proteases.
Thermostability of enzyme
The thermal stability of the recombinant enzyme was assessed for 12 hours at
temperatures, 37, 50 and 60oC, and pH 8. Broad range of activity difference was
noticed with respect to elevated temperature. At, 37oC around 80% of activity was
retained while with increase in temperature to 60oC, more than 90% of the activity
was lost. With prolonged incubation upto 2 hours, only 30% of total activity was
maintained at 37oC and marginal activity at 50oC. While, total loss of activity was
observed in higher range of temperature, i.e.60oC (Fig. 6.2.3.4). Further, on extending
the time of incubation to 24 hour, enzyme was completely denatured at set
temperature range (Fig. 6.2.3.4). From our current study, it’s apparent from the results
that haloalkaliphilic nature of enzyme is maintained. Although, the resistant nature of
enzyme is comparably very low as compared to extracellular alkaline proteases from
haloalkaliphilic bacterial systems as reported in our literature (Patel et al., 2005 a and
b; Patel et al., 2006 a and b; Dodia et al., 2008a and b; Joshi et al., 2008; Thumar and
Singh, 2007; Thumar and Singh, 2009) and our studies on characterization of
recombinant proteases. On the basis of these studies; it is quite logical to suggest that
the stability of proteases could be due to their genetic adaptability to carry out their
biological activity at a higher temperature in haloalkaliphiles. But, the characteristic
feature of enzyme is quite different as compared to our own studies and also available
pH Temp(°C)
204
literature. However, more detailed information on enzyme confirmation and structure
could be explored by studying 3D structure and enzyme energetic of novel proteases.
Effect of NaCl on enzyme stability
Effect of NaCl on enzyme secretion was studied by incubating the reaction mixtures
supplemented with various concentrations of NaCl (0-3M) (Fig. 6.2.3.5). Enzyme was
found to be more sensitive in presence of salt, in all gradient of NaCl concentration
around 25-40% of activity was reduced within an hour of incubation. With increase in
time to 2 hours; at 4M concentration, complete activity was lost; however at 1, 2, and
3M concentration around 50% activity was retained which was completely lost at 12
hours of incubation. As discussed in our earlier results; these observations are also
quite more sensitive than our similar studies to other recombinant enzymes; this itself
provokes as a unique characteristic of this enzyme. In similar studies done with
haloalkaliphilic organism; it was a general trend observed; with increase in NaCl
concentration upto threshold there is increase in activity (Dodia et al., 2008a; Joshi et
al., 2008). However, similar results are not noticed in present studies.
Fig. 6.2.3.4: Thermostability profile of recombinant enzyme: Thermostability of
enzyme was characterized after different hours (1, 2, 3, and 24) of incubation at
(37oC▬); (50 oC -■-), (60oC -▲-).
% R
esid
ual a
ctiv
ity
Time (mins)
205
Fig. 6.2.3.5: Stability of NaCl : Stability of NaCl; where (1M-♦-); (2M -■-),
(3M-▲-) was checked on recombinant alkaline proteases after different hours of
incubation
Urea denaturation
Maximum amount of enzyme was able to maintain its active confirmation in the
presence of chemical denaturant urea in narrow temperature; 37oC. However, enzyme
was also able to maintain its marginal activity at 50oC for 30 mins, however total loss
of activity was observed at both the temperature with further increase in temperature.
(Fig.6.2.3.6). Percent residual activity was related to zero hour enzyme activity as
100%.
Fig. 6.2.3.6: Urea Denaturation profile: Effect of chemical denaturant urea was
checked on enzyme after different time interval (37oC (-♦-), 50oC (-■ -).
% R
esid
ual a
ctiv
ity
% R
esid
ual a
ctiv
ity
Time (mins)
Time (mins)
206
6.2.3.11 In-silico analysis of protease gene A salient feature of protein sequence was analyzed; by reverse translating the
nucleotide sequence using translate tool (ncbi.nlm.nih.gov.in). The instability index
(II) is computed to be 39.57. This classifies the protein as stable. Aliphatic index:
42.94. Grand average of hydropathicity (gravy): -0.747 theoretical
PI/MW:5.15/46683.44. Predicted N-terminal sequence for; O.M.6.2
clone"MRQSLKVMVLSTVALLFMANPAAASEEKKEYLIVVEPEEVSAQSVEES
YDVDVIHEFEEIPVIHAELTKKELKKLKKDPNVKEHPAGA.On the basis of data,
as similar to results of O.M.A18 and O.M.E12 described earlier in chapter-4 and 5, we
can predict that structure of enzyme is quite stable, a fact which is strongly, reflected
by our experimental data on thermal stability and resistance against chemical
denaturation.
6.2.3.12 Hydropathy and 3D structure determination The hydropathy profile of the nucleotide sequence of O.M.6.2 protease, showed
increased presence of hydrophobic residues (Fig. 6.2.3.7A). We further predicted that
enzyme contained serine amino acids at its active site by I-TASSER tool
(Fig.6.2.3.7B), which was supported by our findings on the inhibitor studies, where
polymethyl sulfonyl chloride (PMSF) strongly inhibited the enzyme activity. The
stability of in-silico structure was predicted by Ramachandran plot-PROCHECK
expasy tool, plotting psi vs. phi value; where it was analyzed that molecule was stable
in its confirmation (6.2.3.8). To judge the relatedness, known alkaline proteases
sequences were aligned with metagenomic O.M.6.2 alkaline proteases The
phylogenetic position in constructed tree is shown in Result and Discussion section of
chapter-7.Phylogentic analysis of recombinant metagenomics enzyme confirmed its
100% homology with extracellular protease sequence gene using Mega 4.0..
207
Fig.6.2.3.7A: Hydropathy analysis for O.M.6.2 protease according to Kyte and
Doolittle. On the plot, a positive peak indicates a probability that the corresponding
polypeptide fragment is hydrophobic (a negative peak indicates a probable
hydrophilic segment).
Fig.6.2.3.7B: 3D structure prediction of O.M.6.2 protease enzyme by I-TASSER
structure prediction tool
208
Fig.6.2.3.8: Ramachandran analysis of predicted 3D structure by PROCHECK
Overall, the results discussed in section II of chapter-6, are quite novel with view
point that enzyme studied is metagenomic in nature and characteristic feature
reflected are quite different than our earlier studied enzymes from same saline soil
sample. Further exploration of enzyme from soil; would address heterogeneity or
diversity of soil sample. Identification of such unique properties could also be served
as marker properties of proteases. Availability, of active recombinant protein;
possessing ability to work under moderate to harsh condition would be of significance
important from biotechnological standpoint. Enzyme could also be further explored
for its commercial applications and for studying structural and functional properties of
serine alkaline proteases.
CHAPTER Launch Internet Explorer Brow ser.lnk
CHAPTER
COMPARATIVE ANALYSIS OF
NATIVE, RECOMBINANT AND
METAGENOMIC ALKALINE
PROTEASES WITH RESPECT TO
THEIR TOLERANCE AGAINST
ORGANIC SOLVENTS
7
209
7.1 INTRODUCTION
We have discussed in detail regarding characteristics, applications and commercial
exploitation of proteases from the haloalkaliphilic bacteria in our previous chapters.
Among them, one of the characteristic feature of proteases is that they are among the
most valuable catalysts used in food, pharmaceutical and detergent industries as they
hydrolyze peptide bonds in aqueous environments, while synthesize peptide bonds
under microaqueous conditions (Ogino et al., 2001). In addition to proteolytic activity
of protease, its application in organic synthesis has generated significant interest
(Meos et al., 1993; Klibanov, 2001; Bordusa, 2002; Diego et al., 2007; Karan and
Khare, 2010).
Organic solutions are ideal for synthesis reactions since the solubility of polar
substrates increases in solutions supplemented with organic solvents and the stability
of the alkaline proteases in organic solvents would be an attractive feature of the
biocatalysis. Along with stability, it favors reversal of thermodynamics equilibrium
over hydrolysis and decreases microbial contamination. Among the major
applications of protease-catalyzed reaction is the synthesis of dipeptides, such as
kyotorphin (Tyr-Arg) precursors (Meos et al., 1993; Sareen et al., 2004 a, b).
In recent years, several new proteases that are able to maintain stability and activity in
organic solvents have been discovered (Patil et al., 2008; Reza et al., 2008 a and b;
Hamid et al., 2011). There are many approaches to capture the non-aqueous
biocatalytic potential; the most obvious being the microorganisms from extreme
environments or contaminated areas enriched with various organic solvents (Hamid et
al., 2011). The property of tolerating organic solvents makes these bacteria better
candidates for exploiting solvent-stable enzymes. However, alternatively, genetic
engineering and molecular biology could be considered as one of the approaches, to
transfer solvent resistant gene/s. Genetic engineering is instrumental in opening new
opportunities for the construction of genetically modified microbial strains with
selected enzymes properties.
The demand for potentially useful proteases with specific properties continues to
stimulate the search for new sources of organic-solvent tolerant proteases. With
210
diversity view point, most of the reported solvent tolerant strains belong to genera;
Pseudomonas, Bacillus and Arthrobacter. However, exploration of haloalkaliphilic
bacteria with such unique characteristic feature is in infancy. Therefore, studies on the
solvent tolerant nature of haloalkaliphilic bacteria from moderate saline habitats may
open new arena for the basic research in non-aqueous enzymology.
In this chapter, we selected several organic solvents on the basis of their Log pOW
and analyzed their effect on the native and recombinant proteases from O.M.A18 and
O.M.E12 strains. The studies were then compared with a metagenomically derived
alkaline protease from the saline habitat. Further, effect of physico-chemical
parameters such as temperature, pH and NaCl were analyzed.
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7.2 MATERIALS AND METHODS
7.2.1 Bacterial strains Haloalkaliphilic bacteria, Oceanobacillus iheyensis O.M.A18 (EU680961) and
Haloalkaliphilic bacterium O.M.E12 (EU680960) were isolated as earlier described in
detail in chapter 2 and Purohit and Singh (2011).
7.2.2 Recombinant clones Construction of recombinant clones of O.M.A18 and O.M.E12 is described in detail in
chapter-5 and construction of metagenomic clone O.M.6.2 is described in chapter-6
was used for solvent studies. Sequencing of clones was done as described in detail at
appropriate place in chapter-5 and 6.
7.2.3 Organic Solvents Glycerol, Xylene, Methanol, n-Hexane, Acetone and Chloroform, with log Pow values
as 1.07, 3, 0.82, 0.25, 0.2 and 1.9 respectively, were obtained from Merk chemicals
(India).
7.2.4 Effect of organic solvents on enzyme catalysis Protease activities were measured in a reaction mixture (Hagihara, 1958) with varied
concentrations of 10-30% (v/v) of above mentioned solvents. Controls for each set
were also carried out simultaneously.
7.2.5 Effect of organic solvents on enzyme stability The solvent stability was studied by incubating the enzymes in different solvents
(Methanol, Glycerol and Hexane) at 10% (v/v). The aliquotes of enzyme preparations
were withdrawn at regular intervals for 15 hours and the residual enzyme activities
were measured.
7.2.6 Effect of pH on enzyme catalysis Effect of pH on native and recombinant proteases were examined by carrying out enzyme
assay at different pH in the presence of (10% (v/v)) Hexane, using buffers systems
(20mM): phosphate (pH-7), Tris-HCl (pH 8), NaOH-Borax (pH 9) and Glycine - NaOH
(pH 10).
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7.2.7 Effect of NaCl on enzyme activity To assess the influence of NaCl and organic solvent, Hexane in conjunction, the
reaction mixtures were supplemented with 1-4M NaCl and protease assay was carried
out at 37°C with 10% (v/v) of the solvents.
7.2.8 Effect of Temperature on proteases catalysis The temperature profile for protease activity was examined in the presence of Hexane
by incubating the assay reaction mixtures at different temperatures, 30-60°C. The
proteases activity was determined as mentioned above.
7.2.9 Multiple sequence alignment and phylogenetic determination Multiple sequence alignment of three nucleotide sequences (recombinant O.M. A18,
recombinant O.M.E12, metagenomic clone) were constructed by using CLUSTALW
(www.ebi.ac.uk/Tools/msa/clustalw2/)(Thompson et al., 1994); data were further
interpreted by CLC workbench to analyze conserved residue and consensus pattern
(Dainith, 2004). A phylogenetic tree was constructed of aligned three enzymes studied in
present work. The phylogenetic relatedness of these enzymes was also established with
other proteases sequences of haloalkaliphilic organisms by the Neighbor-Joining method
clustering strategy in Mega 4.0 (www.megasoftware.net) (Tamura et al., 2007).
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7.3 RESULTS AND DISCUSSIONS
While there are many studies on the solvent resistant strains, the sensitivity of the
organisms and their enzymes from saline habitat are scarce (Reza et al., 2009; Karan
and Khare, 2011). There are several reports where effect of solvent is checked on
enzyme activity (Thumar and Singh, 2007a; Reza et al., 2008) Similiarly,
construction of recombinant clones and to study similar aspects are also studied (Reza
et al., 2009; Hamid et al., 2011). However, to the best of our knowledge, study on
comparison of catalytic efficiency of native and recombinant preparations is not found
in literature. In Chapter-7, we have studied varied enzyme preparations; native,
recombinant and metagenomic clone in a comparative manner.
7.3.1 Native, Recombinant and Metagenomic derived alkaline
enzymes Alkaline proteases were purified to its homogeneity from both the selected
haloalkaliphilic bacterial strains using Phenyl Sepharose 6FF described in detail in,
Purohit and Singh (2011) and Chapter 4. The construction of recombinant clones and
purification of recombinant enzymes is described in detail in chapter 5 and 6.
Sequence analysis of recombinant clones and the amino acid sequence prediction and
analysis of its physico chemical properties were carried out as described at
appropriate place in detail in chapter 5 and 6.
7.3.2 Catalysis of alkaline proteases in organic solvents The five enzyme preparations; two native, two recombinants and one metagenomic,
were studied with respect to their alkaline protease activities in the presence of
various organic solvents described in Materials and Methods.
As regard to the effect of ethanol on the catalysis, for O.M.A18 native enzyme, 73%
of the residual activity was retained at 5% ethanol, which on further increase in
ethanol to 30%, reduced to 16.33%. Compared to native, the recombinant enzyme was
relatively more sensitive, with 62 % loss of the residual activity at 5%. The activity
was substantially reduced at 10% solvent. Further, enzyme was completely denatured
at 30% ethanol (Fig.7.3.1).
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In general, the native O.M.E12 protease was more sensitive towards organic solvents
as compared to its O.M.A18 counterpart. The activity reduced to 52% of total activity
with 10% methanol, while only 11.56% of the residual activity was evident at 20%
solvent, followed by a total loss at 30%. The recombinant O.M.E12 enzyme was
relatively more sensitive than the native enzyme. For metagenome derived protease,
only 21.5% residual activity was observed at 5% methanol (Fig. 7.3.1).
Around half of the activity of O.M.A18 native enzyme was lost in the presence of
10% glycerol, with a complete loss at 30%. On the other hand, with reference to
O.M.E12 enzyme, 60% loss in enzyme activity at 10% and complete denaturation at
20% glycerol was evident. While, for recombinant O.M.A18 enzyme, only one-tenth
residual activity was apparent at 10% glycerol, with a total loss at 20%. The
metagenomically derived protease maintained 12% of the residual activity at 10%
glycerol.
The trends of enzyme responses in acetone and chloroform were quite similar for both
native enzymes. At 5% acetone and chloroform, 35-40% loss in activities was
observed for O.M.A18 and O.M.E12 native enzymes, leading to a total at 30%
solvents. For recombinant enzymes, trends were quite different with different
solvents. The O.M.A18 recombinant protease maintained 30% of the residual activity
at 5% of acetone and chloroform, while at 20% (v/v) solvents, nearly total loss of the
activity was evident. The O.M.E12 recombinant enzyme had 20-30% of the residual
activity at 5% acetone and chloroform, and with increase in solvent to 20%, the
activity reduced to 50%. The metagenomically derived enzyme was highly sensitive
to acetone and chloroform (Fig.7.3.1) (Table 7.3.1, Table 7.3.2). The solvent tolerance
of the enzymes was greater towards hexane and xylene for native O.M.A18 and
O.M.E12 proteases. These enzymes retained approximately 70-80% activity at 5%
hexane and xylene. However, the residual activities of both native enzymes reduced to
30-35% at 20% hexane. The recombinant enzymes, O.M.A18 and O.M.E12 had
similar trends. The maximum activity at 40% residual level was apparent with 20%
solvent (Fig.7.3.1), (Table 7.3.1, Table 7.3.2).
As described in review of literature, the log P values which are less than 4 are
considered extremely toxic, as required water molecules on the enzyme surface are
easily replaced with solvents (Ogino et al., 2001; Gupta et al., 2006b). In brief, the
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native proteases retained substantial catalysis at lower concentrations of both
hydrophobic and hydrophilic solvents. Interestingly, at increased solvent
concentrations, comparatively better activities were evident with hydrophobic
solvents. Such trends of enzymatic efficiency for native enzymes are frequently sited
in literature (Reza et al., 2008; Reza et al., 2009).
7.3.3 Effect of organic solvents on enzyme stability The stability of enzymes was determined with the solvents: methanol, glycerol and
hexane. The solvents were selected on the basis of trends displayed for enzyme
catalysis. The enzymes were incubated with 10% (v/v) solvent up to 15 hours to
monitor stability (Fig.7.3.2).
O.M.A18 native enzyme in methanol, at zero hours itself, it lost 43% residual activity
compared to its control. The time required for native O.M.A18 to reduce to 50% of its
initial activity was 3 hours (Table 7.3.3, Table 7.3.4). For, O.M.E12 native enzyme,
the enzyme activity was lost by 10-15% at every 3 hours of incubation and after 15
hours, 30% of the residual activity was recorded (Fig.7.3.2). Native O.M.E12 enzyme
was resistant against solvents as half life in methanol was 9 hours (Table 7.3.3, Table
7.3.4). In comparison, recombinant O.M.A18 enzyme in methanol had 70% of the
residual activity after 6 hours of incubation. For recombinant O.M.E12, trend was
quite similar to native enzyme (Table 7.3.3, Table 7.3.4).
In general, for hexane and xylene trends were quite similar to methanol. With increase
in incubation time, as the concentration of the solvents increased, the enzyme stability
significantly decreased. With respect to metagenomic clone, around 40% of the
activity was maintained between 0-9 hours of incubations. Overall, stability of n-
hexane was highest as compared to other two solvents (Fig. 7.3.2), (Table 7.3.3, Table
7.3.4).
In general, proteases are relatively more stable in n-hexane, a hydrophobic solvent
than hydrophilic solvents: methanol and glycerol. Almost similar stability trends for
proteases in the presence of various organic solvents have been reported by others
(Ogino et al., 1995; Gupta et al., 2006a).
216
Fig.7.3.1: Effect of solvents on enzyme catalysis of native O.M.A18 and O.ME12,
recombinant O.M.A18 and O.M.E12 and metagenomics sample O.M.6.2. Organic
solvents are; Glycerol, Xylene, n-Hexane, Methanol, Acetone, Chloroform.
217
`
Solvents Concentration(range) (%) for O.M.A18 native enzyme
Concentration(range)(%) for O.M.A18 recombinant enzyme
0 5 10 20 30 0 5 10 20 30 Methanol 100 73 65.6 24.25 16.33 100 38.4 16.2 8.2 0 Glycerol 100 69.1 53.2 20.16 0 100 31 11.96 0 0 Acetone 100 62.56 45.46 39.73 0 100 28.8 9.7 3.6 0 Chloroform 100 66.46 52.21 35.55 0 100 30.2 10.4 6.1 0 n-hexane 100 80.83 69.98 34.94 18.98 100 65.1 54.8 43.7 0 Xylene 100 78.46 57.31 30.19 20.94 100 60.7 46.5 24.1 0 Solvents Concentration(range) (%) for
O.M.E12 native enzyme Concentration(range)(%) for O.M.E12 recombinant enzyme
0 5 10 20 30 0 5 10 20 30 Methanol 100 67.3 52.86 12.45 0 100 28.55 11.56 0 0 Glycerol 100 73.45 39.73 0 0 100 17.55 14.46 0 0 Acetone 100 60.56 50.66 44 0 100 28.68 16.67 7.45 0 Chloroform 100 69.46 56.76 39.99 0 100 21.5 10.56 0 0 n-hexane 100 67.66 65.2 38.17 0 100 15.7 8.9 0 0 Xylene 100 80.54 59.75 54.7 29.91 100 15.9 10.38 0 0
Table 7.3.1: Comparative analysis of effect different solvents with range of solvent concentration on enzyme preparations
Table 7.3.2: Solvent concentrations (%) required to reduce enzyme catalysis to its half.
Solvents Concentration(range) (%) for recombinant O.M.6.2 enzyme
0 5 10 20 30 Methanol 100 21.5 10.56 0 0 Glycerol 100 28.55 11.56 0 0 Acetone 100 15.7 8.9 0 0 Chloroform 100 15.9 10.38 0 0 n-hexane 100 28.68 16.67 7.45 0 xylene 100 17.45 14.46 0 0
Solvent concentration (%)
O.M.A18 O.M.E12 O.M.6.2 metagenome
Native Recombinant Native Recombinant Glycerol 10 10 10 < 5 < 5 Xylene 10 10 10 < 5 < 5 Hexane 10 5 10 < 5 < 5 Methanol 10 5 10 < 5 < 5 Acetone 10 5 10 < 5 < 5 Chloroform 10 5 10 < 5 < 5
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Fig.7.3.2: Stability of enzyme in the presence of organic solvent at different time interval (hours)
219
Solvents O.M.A18 native enzyme O.M.A18 recombinant enzyme Time (hours)
0 3 6 9 12 15 0 3 6 9 12 15 Control 100 100 100 100 100 100 100 100 100 100 100 100
Methanol 57.3 50.3 43.9 34.8 30.5 16.9 74.5 70.6 67.8 40.7 31.6 28.6 Glycerol 54.3 49.6 39.8 33.6 28.4 12.4 68.6 50.9 49.8 36.7 29.6 16.1 n-Hexane 62.5 58.6 51.6 46.8 40.2 20.4 70.6 61.6 54.6 50.6 38.6 29.6
Solvents O.M.E12 native enzyme O.M.E12 recombinant enzyme
Time (hours) 0 3 6 9 12 15 0 3 6 9 12 15
Control 100 100 100 100 100 100 100 100 100 100 100 100 Methanol 83.5 72.6 67.8 49.7 38.6 29.6 83.5 72.6 67.8 49.7 38.6 29.6 Glycerol 64.6 56.9 44.8 36.7 29.6 16.1 64.6 56.9 44.8 36.7 29.6 16.1 n-Hexane 77.6 64.8 59.36 48.6 31.6 28.6 77.6 64.8 59.36 48.6 31.6 28.6
Table 7.3.3: Effect of organic solvents of different concentration on enzyme stability
Solvent concentration
(%) O.M.A18 O.M.E12 O.M.6.2
metagenome Native Recombinant Native Recombinant
Methanol 3 6 9 9 3 Glycerol 3 6 6 6 3 n-Hexane 6 6 9 9 3
Table 7.3.4: Stability of enzyme in the presence of organic solvent: Time required by
enzymes to reduce its activity to 50%.
7.3.4 Influence of pH on enzyme catalysis
Effect of pH was determined on all five recombinant enzyme preparations in the
presence of varying concentrations of Hexane. With increase in solvent concentration,
the enzyme activity was profoundly reduced at different pH. However, extent of the
activity loss varied with pH. The trends of enzyme activity at different concentrations
were assessed. pH profile were quite similar for both, native and recombinant
enzymes. Native and recombinant O.M.A18 enzymes had maximum activity at pH-8.
With increase in pH, residual activities marginally reduced. However, the difference
in activity profile was evidently revealed at 10% solvent concentration. While with
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higher solvent concentrations, the differential effect of pH was less evident at pH-8, 9
and 10. With 30% solvent, around 30-35% of the residual activity was maintained at
all pH (Fig.7.3.3).
With increase in solvent concentrations, there was gradual decrease in enzyme
activity. Even at lower pH, significant residual activity was observed. There was no
significant difference in the activity at pH-9 with 10 and 20% solvent for the native
enzyme.For alkaline protease clone generated from soil sample, most favorable pH
was 8. There the residual activity was significantly lost (Fig.7.3.3).
7.3.5 NaCl effect on enzyme catalysis In presence of NaCl there was not much difference in trend revealed at pH-7 and 9 on
catalysis in presence of n-hexane. With increasing solvent concentrations in the
presence of NaCl, there was reduction in residual activities. For O.M.E12 native and
recombinant enzymes, the residual activity was reduced to 50% at 1M NaCl. With,
further increase in salt concentration to 2M, there was marginal increase in activity
(Fig.7.4.4).For O.M.A18 enzymes, the observed trends were quite similar to O.M.E12,
where although there was significant loss of activity at 1M NaCl, with increase in salt
concentration to 2M, the percent residual activity was enhanced (Fig.7.4.4). With
reference to metagenomic clone, the reduction of activity was similar to above
discussed trends of native and recombinant clones; however increase in activity with
enhanced salt concentration to 2M was not noticed in metagenomic clone.
7.3.6 Effect of Temperature in enzyme catalysis Temperature profiles of all enzyme preparations were studied in the presence of n-
Hexane, where it was clearly indicated that optimum temperature was 37oC. For,
native and recombinant O.M.A18; with increased solvent concentrations, there was
loss in activity. With increase in solvent concentration to 30%, around half of the
residual activity was maintained compared to 10% of its residual activity. With
reference to pH profile, there was not much change in percent residual activity of
native, recombinant and metagenomic enzyme preparations. This clearly indicates the
efficacy of recombinant clones.For O.M.E12, both native and recombinant enzyme
displayed similar trends to O.M.A18 (Fig.7.3.5). The optimum temperature of
metagenomic clone was 37oC. The loss of activity was quite significant as compared
to earlier studied enzyme systems (Fig.7.3.5).
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7.3.7 Phylogenentic identification Nucleotide sequence of recombinant enzymes; O.M.A18, O.M.E12 and
metagenomically derived protease were identified. The consensus amino acid
sequence was deduced from the DNA sequence. On the basis of phylogenentic tree
constructed by neighbor joining (NJ) method, using Mega 4.0, it was revealed from
the inter node that O.MA18 was closely related to uncultured bacterium O.M.6.2.
O.M.E12 and both this described sequences are diverged from nodes of constructed
tree. To get more insight into phylogenentic relatedness, the sequences in the present
studies were aligned with known protease sequences available in the database. The
enzyme sequences were quite diverse in its sequence similarity; they were more
closely related to enzyme sequences of other halophilic organisms as compared to
enzyme studied in present case which were of the haloalkaliphilic organism isolated
from the same saline soil (Fig.7.3.6, Fig.7.3.7).
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Fig.7.3.3: Effect of pH on enzyme catalysis in presence of Hexane with varied solvent concentrations (0-30%).
223
Fig.7.3.4: Effect of NaCl on enzyme catalysis in presence of Hexane with varied solvent concentrations (0-30%).
224
Fig.7.3.5: Effect of Temperature on enzyme catalysis in presence of Hexane with varied solvent concentrations (0- 30%).
225
226
227
Fig.7.3.6: Snapshot of Multiple sequence alignment of alkaline proteases sequences
of O.M.A18, O.M.E12 and metagenomics sample O.M.6.2 by Clutal W and CLC
workbench.
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Fig.7.3.7: Phylogentic analysis of sequences by Neighbor joining method using
Mega 4.0. Upper Panel: Phylogenetic relatedness of O.M.A18, O.M.E12 Lower
Panel: Phylogenetic relatedness of several alkaline proteases
229
Although, there is much advancement in the field of molecular biology and
biochemistry, only sparse number of archeal and bacterial haloalkaliphilic proteases
have been reported as an organic solvent-stable protease (Diego et al., 2007). With
this above objective; the behavior of alkaline proteases in its native and recombinant
counterpart as well as functional attribute based metagenome alkaline protease were
studied in presence of solvent. To, the best of our knowledge, this would be among
the few reports dealing with comparative study of enzyme preparations, particularly
organic solvents.
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CHAPTER
CONCLUDING
REMARKS
8
230
CONCLUDING REMARKS
The overall study was planned in view of the realization that only fraction of
microbial population; particularly extremophiles, are cultivated and known to us.
Moreover, most of the ecological and physiological studies on haloalkaliphilic
bacteria in the past have largely focused on hyper saline environments; Solar saltern,
Dead Sea, Soda lake. Only limited studies are focused on the diversity of the extreme
organisms from moderate saline habitats. Therefore, exploration of more extreme
habitats would be of great significance. The research group in the Department of
Biosciences, Saurashtra University is working on these bacteria and has indicated
their wide occurrence in moderately natural saline environment of Coastal Gujarat. In
the present study, both conventional and metagenomic approaches to get insight into
the total diversity of microorganism and their biocatalytic potential has been
undertaken. The saline environment taken into consideration for the present
investigation has not been explored for microbial diversity and their biotechnological
potentials through both cultivable and non-cultivable approaches.
Haloalkaliphiles hold many interesting biological secrets, such as the biochemical
limits to macromolecular stability and the genetic information for synthesizing
macromolecules stable to more than one extremity. Despite the significance of
extremophiles, particularly those with dual extremities, such organisms have been
paid only limited attention towards the exploration of biotechnologically relevant
products and enzymatic potential.
In the light of above realization, a pool of 34 haloalkaliphilic bacteria was obtained
from salt pane using different enrichment conditions of NaCl and pH. Existence of
halotolerant, haloalkalitolerant and truly haloalkaliphilic bacteria clearly indicated the
widespread distribution of these bacteria in saline environments beyond the
conventionally explored habitats. Isolates were diversified on the basis of their
phenotypic characters, antibiotic sensitivity, physiological properties and protease
secretion. The studies of the saline environment established the relationship between
the extent of extremity and diversity.
The distribution of organisms were almost equal in both the combinations, pH-8 with
10% NaCl and pH-10 with 30% NaCl used for enrichment and isolation. The
231
community structure and the complexion of the population dynamics largely depends
upon the available energy sources in a given habitats.
Studies on biochemical and metabolic reactions of haloalkaliphilic bacteria revealed
the common occurrence and secretion of catalase and oxidase. Ability of the
organisms to metabolize different sugars for the bio-energetic purpose is one of the
approaches to diversify the organisms. The organisms utilized disaccharides,
compared to simple carbon sources, suggested that the adaptation of different
metabolic pathways for their energy generation.
The studies on haloalkaliphilic strains indicated that the concerned habitats may be
rich in proteinaceous substances occupying the nutritional dynamics where easily
utilizable carbohydrates are scares. Majority of the haloalkaliphilic bacteria (90% for
O.M.6.2) had ability of denitrification, indicating their possible applications in
wastewater treatment. Organisms producing H2S and ammonia, utilize sulfur-
containing amino acids as carbon source from the protein-rich medium. Further,
biotransformation and degradation of aromatic compounds in hypersaline
environments has become increasingly important. One of the isolate, O.M.C28 was
preliminary characterized for its biodegradation potential.
Antibiogram approach appeared to be quite useful and reflected useful trends on the
microbial diversity. The Gram reaction patterns closely matched with the trends
reflected in antibiogram, as most of the isolates were sensitive against Gram positive
antibiotics. The differential response towards antibiotics may reflect on the gene
expression and/or regulation, where expression of certain genes might be salt
dependent (Oren, 2010).
The present studies on the diversity of haloalkaliphilic bacteria, on the basis of the
phenotypic, physiological and biochemical characteristics assume significance in the
light of recent emphasis on the microbial ecology with a new dimension. However,
for judging the microbial diversity among these isolates, classical methods are not
sufficient. Phylogentic identification based on 16S rRNA analysis of key strains
producing alkaline proteases reflected diversity among them. In addition, fatty acid
methyl ester (FAME) profile and carbon utilization patterns were also explored to
diversify the organisms.
232
Despite ample opportunities, only few instances are reported for actual exploitation of
haloalkaliphilic bacteria. In additions to the above discussed aspects of microbial
diversity in the arena of reemphasized microbial ecology of the haloalkaliphilic
bacteria, the second phase of the study was the exploration of biotechnological
potentials and microbial diversity among these bacteria with respect to secretion and
properties of the extracellular alkaline proteases. They are quite important with
respect to their physiological significance and commercial applications.
The isolates displayed varying degree of diversity with respect to growth patterns and
enzyme secretion. While the salt and pH range for growth and enzyme secretion did
not vary extensively among the isolates, the variation in optimum pH and salt was
pronounced and the growth did not necessarily correspond to enzyme secretion. The
results, however, established the specificity and stability of alkaline proteases at
higher NaCl concentrations. Most of the protease producers were moderately
haloalkaliphilic in nature; however, few were truly haloalkaliphilic. Apparently, the
occurrence of enzymes could also be used as biochemical marker to judge the
microbial heterogeneity among moderately haloalkaliphilic bacteria. Besides, the dual
extremities of alkaline pH and salinity project them as promising candidates for
various biotechnological applications.
With the knowledge of basic properties and optimum production conditions, two
potential strains O.M.A18 and O.M.E12 were purified to homogeneity by single step
chromatography on phenyl sepharose 6FF and characterized. The enzymes from with
respect to salt, pH, temperature, chemical denaturation, surfactants, cations, oxidizing
and reducing agents followed by the comparisons of these fetures with the enzyme
preparations at different state of purity. The alkaline proteases from the two isolates
from the same site displayed distinct characteristics in terms of their catalysis and
stability at high temperatures. The novel features of the enzymes, such as stability
over the wide range of pH and salt, catalysis and thermostability of the enzyme at
higher temperatures make them attractive candidates for biotechnological stand point.
Some other properties of the enzymes, as emerged from the present studies, such as
stability in the presence of cations, surfactants, oxidizing and reducing agents would
make them attractive candidates for certain key applications including detergent
industries. Although both strains were from the same site, their features on growth,
233
protease secretion and enzymatic characteristics were distinct, reflecting their
ecological significance. Further, investigation of this phenomenon would be quite
interesting to understand the structural basis of the haloalkaliphilic proteins.
As emphasized earlier, alkaline proteases are quite important with respect to their
physiological significance and commercial applications. Further, the global advances
in molecular and computational biology, search for new sources of enzymes,
combinatorial methodologies and biochemical engineering of single and multi
component enzyme systems would certainly stimulate enzyme technology.
To identify gene/s for proteases from haloalkaliphiles, several sets of primers specific
for protease sequence were designed and used for the amplification of proteases.
Number of amplicons varying in concentration and size reflected the diversity of the
alkaline protease genes among the bacteria dwelling in saline habitats. Alkaline
protease genes cloned and expressed in E.coli were characterized and compared with
the native one. Characteristic features of the recombinant protease were similar with
those of the native enzyme. However, the recombinant enzyme became more
sensitivity as compared to its native system.
We confirmed hydrophobic tendency and predicted 3D structure of the proteins and
classified as serine protease. Confirmative stability of folded protein is of significant
importance as the stability of enzymes in-vitro conditions remains critical for the
scientists and hence prediction of protein structure and function is one of the major
limitations in the field of proteomics. Structure was stable in its confirmation and
configuration on the basis of Ramachandran plot analysis.
Metagenomics is an emerging approach based on the extensive analysis of the DNA
of microbial communities in their natural environment. It has been developed over the
last several years to assess the genomes of the non-culturable microbes towards better
understanding of global microbial ecology and to trap vast biotechnological potential
of a given habitat. With the advent and availability of newer applications of PCR,
applications of DNA sequences of the microbial communities from natural and
extreme habitats has become possible without culturing the organisms.
234
The application of metagenomic strategies embraces great potential to study and
exploit the enormous microbial biodiversity present within the saline habitats. With
these objectives, we have tried for sequence and functional approaches. One of the
hurdles in metagenomics is the extraction of total environmental DNA (metagenome)
from a given habitat. We explored and optimized various protocols and assessed in
terms of DNA purity, yield and humic acid content. Diversity based assessment was
based on the 16S rRNA amplicons-DGGE analysis (Molecular Fingerprinting
Technique). Further, we took alkaline proteases as model system for the assessment of
genetic diversity among these habitats by designing degenerate primers with the aid of
bioinformatics tools. Successful cloning and expression of alkaline proteases by
adopting similar strategy as of haloalkaliphiles revealed unidentified gene/s with
interesting features.
The ever emphasized aspects of exploring new habitats and metagenomics along with
the rapidly developed new approaches such as genomics, proteomics and
metabolomics will stimulate the developments of new industrial processes on the
basis of biocatalysts from these groups of bacteria. With the advancement in
metagenomics and their implications in our current research, it would be possible to
get insight into the biocatalysis for novel applications. Capturing the alkaline
proteases gene from metagenome DNA holds significance in the light of the fact that
although saline environment has enormous microbial biodiversity, it is relatively less
explored.
Comparative analysis of native haloalkaline, recombinant and metagenomic alkaline
proteases as a function of their tolerance against organic solvents was carried out. The
native enzymes were resistant towards organic solvents as compared to recombinant
counterparts. While, metagenomics derived enzyme had minimum resistance. The
novelty of this study lies in the fact that major studies so far on solvent tolerant
enzymes are from mesophilic enzymes and only rarely archeal and bacterial
haloalkaliphilic proteases are reported.
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FUTURE PERSPECTIVES
New tools and techniques for exploring microbial resources through culture
dependent conventional approaches and culture-independent metagenomic tools
provide great opportunities to bring biomolecules for varied applications. The
objectives, however, vary from directed product discovery to ecosystem analysis and
assessment of the population dynamics. Metagenomics has redefined the concept of a
genome and has added enormously to the gene discovery. Although, the field of
microbial biotechnology is quite diverse, only limited attention has been paid to
haloalkaliphiles particularly from moderately saline habitats. To explore such
habitats, we studied halolaklaiphilic bacteria with respect to diversity, distribution and
production of extracellular enzymes. However, the larger umbrella of genomics and
proteomics is yet to come up with respect to the microbial diversity of saline habitats.
Work on these microbes is the beginning to long journey and further studies would
provide information and insights into the adaptation to extremity as well potential
application avenues. The future studies would, therefore, focus on the diversity,
molecular phylogeny, population dynamics in totality, structural basis of protein
stability under multitude of extreme conditions, development of expression systems,
over-expression and understanding protein folding.
The work presented in this thesis could be further extended along the following lines:
The taxonomic status of other isolates should be pursued by using molecular
techniques to understand phylogenic relatedness and to get further insight into
the microbial diversity in saline soil of Gujarat coast.
Organisms were screened for biocatalytic potential of alkaline proteases.
However, other enzymes; lipase, amylases; catalase; chitinase should also be
explored. This would be of vital importance from biotechnological view point.
Purification and characterization of alkaline proteases from other strains would
be important for comparing the profile of the enzyme. Similarly, cloning,
sequencing, over-expression and characterization could be further expanded to
proteases from other potential candidates. Effect of molecular chaperones,
236
protein folding and renaturation and several biochemical features could be
explored.
The role of salt and other factors on global gene expression based on recent
tools of genomics and proteomics should be undertaken. Similarly, salt-plasmid
relationship would be an excellent perspective of these haloalkaliphilic bacteria
to explore.
Exploration of habitat by metagenomics was a beginning and should be
extended to other saline habitats.
Newer fingerprinting methodology could be further adopted for getting insight
into non-cultivable.
Genomic library creation would generate ample of opportunities for sequence
based analysis and functional attributes.
Developing footprints in these aspects would certainly generate novel information’s
from both, diversity and biotechnological view point.
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CHAPTER
SUMMARY
9
237
SUMMARY
ISOLATION AND DIVERSITY OF HALOALKALIPHILIC
BACTERIA
Thirty four different haloalkaliphilic bacteria were isolated from salt-enriched
soil collected from salt panes located in Okha (Latitude 22.20 N, Longitude
70.05 E), Gujarat, India. Isolates were initially diversified based on their
enrichment conditions followed by the studies on colony characteristics, gram
and metabolic reactions, cell morphology, antibiotic profiles, enzyme
secretion and protease amplification profile.
Based on 16S rRNA gene homology, three potential isolates, O.M.A18,
O.M.E12, O.M.C28 were related to their nearest homologus and the sequences
were deposited in NCBI as Oceanobacillus iheyensis O.M.A18,
Haloalkaliphilic bacterium O.M.E12 and Oceanobacillus oncorhynchi
O.M.C28. The two potential isolates, O.M.A18 and O.M.E12 were also
analyzed by FAME confirming their status as genus Bacillus.
Along with classical and molecular parameters, proteolytic activity was
considered as one of the parameter for judging the diversity of haloalkaliphilic
bacteria. Isolates secreted alkaline proteases in the broad range of NaCl (0-
20%, w/v) and pH (8-11) and were further diversified on the basis of their
optimum pH and salt. Alkaline protease genes were amplified by using sets of
primers. Diversity was evident in terms of product size and concentration of
amplicons. Size of the protease gene products varied form 0.5 to 1.2 kb.
The antibiogram profile was quite useful in generating the microbial diversity
profile among the isolates. Overall, isolates were more resistant against
Tobramycine and highly sensitive towards Oleandomycin, Cephaloridine,
Norfloxacin, Cephradin, Erythomycin, and Cefuroxime. The biochemical and
metabolic reactions revealed the production of catalase and oxidase by all
isolates. Isolates were diversified on the basis of sugar utilization and none
238
produced gas in Durham’s tube. Only few isolates from O.M.6.2 and O.M.6.5
utilized all the sugars.
PURIFICATION AND CHARACTERIZATION OF
ALKALINE PROTEASES
The enzyme characteristics reflected that although O.M.A18 and O.M.E12
strains were from the same site they reflected quite distinct traits.
The enzymes were purified to their homogeneity by single step on phenyl
sepharose 6 FF affinity column. The purification was achieved with specific
activities of 16945 and 24600 U/mg for O.M.A18 and O.M.E12 proteases. The
apparent molecular mass of the proteases were estimated 35 and 29 kDa for
O.M.A18 and O.M.E12, respectively. The effect of NaCl was assessed on
enzyme activity and thermostability. NaCl enhanced the enzyme activity of
purified enzyme, while activity declined in crude and partial purified stage of
the enzyme. For both; O.M.A18 and O.M.E12 strains, there was shift in
temperature profile with NaCl. Enzyme preparations maintained their
activities and stability in wide range of pH; 8-11, with maximum activity at
10.
One of the foremost points of the study was related to the effect of temperature
on enzyme catalysis. The O.M.A18 enzyme had broader range of temperature
for catalysis (37-90oC), while that for O.M.E12 was over a relatively narrower
range (37-70oC). The high activity at 90°C for O.M.A18, would be among the
few reports where mesophilic organisms catalyze reaction at elevated
temperature. This feature holds novelty from diversity view point as well;
since there may be only limited reports where such a unique and contrasting
characteristic features are observed from the same site of isolation.
The partially purified and purified proteases from O.M.A18 and O.M.E12 were
subjected to urea denaturation. Partially purified enzyme from O.M.A18 was
exceptionally resistant against urea denaturation at 90oC after 48 hours with
8M urea. The O.M.E12 enzyme, however, was relatively less resistant to urea.
The effect of NaCl was assessed on urea denaturation; the findings revealed
239
that there was no significant change in the denaturation profile of crude,
partially purified and dialyzed enzymes. Crude and partially purified enzymes
were quite stable in various commercial detergents and oxidizing and reducing
agents.
On the basis of phylogenetic prediction, the next most homologus sequences
for all the proteases sequences were found with the reported extracellular
protease sequences. The characteristic features; i.e. G+C content, PI, aliphatic
index, hydrophobicity resembled to known haloalkaline proteases.
The significance of the work relates to the fact that while alkaline proteases
are extensively studied, only few haloalkaliphilic bacteria have been explored
towards this end. Despite the fact that the saline habitat in the study possessed
significant bacterial diversity, it remains unexplored in terms of it
characterization, biocatalytic potential, enzymatic characteristics, enzyme
structure-function analysis and phylogenentic status.
Further, some of the novel features of the enzymes, such as stability over the
wide range of pH and salt, catalysis and thermostability of enzyme at higher
temperatures make them attractive candidates for future studies. The results
are also important from the diversity viewpoint as although, both the strains
are from the same site, they displayed distinct features of growth, protease
secretion and enzymatic characteristics, highlighting their ecological
significance.
CLONING, SEQUENCING, OVER EXPRESSION AND
CHARACTERIZATION OF SERINE ALKALINE
PROTEASES
Cloning of alkaline protease genes from the genomic DNA of O.M.A18 and
O.M.E12 was carried out in an over-expression vector, pET21a+ (Novagen).
Vector construct of O.M.A18 and O.M.E12 were transformed in Escherichia
coli strain BL21. The positive clones exhibiting resistance against ampicillin
(30µg/ml) were selected for plasmid isolation.
240
With respect to over-expression of protease genes from both isolates, the
optimum production of recombinant proteases was at 27oC and 1mM IPTG.
The molecular weights of both proteases were estimated ˜30 kDa, which were
quite comparable to the enzymes produced in native haloalkaliphilic
organisms.
Over-expressed proteases were purified to the homogeneity by single step
purification using nickel column exploiting His-tag property of pET21a+. High
level of purified enzymes obtained as evident from the specific activity and
yield.
Enzymes were characterized for physico-chemical properties, as for their
native counterparts. In general, the recombinant enzymes were more sensitive
towards NaCl, temperature and chemical denaturant compared to the native
O.M.A18 and O.M.E12 proteases.
Molecular characterization of recombinant enzymes; distribution of amino
acids, 3D structure analysis, protein secondary structure analysis and
hydropathy analysis were carried out. The presence of serine residues in active
site confirmed the serine nature of the enzyme. Further, stability of enzyme
structure was confirmed by in-silico analysis.
METAGENOMICS: ISOLATION OF ENVIRONMENTAL
DNA AND CAPTURING ALKALINE PROTEASES GENES
Several protocols were attempted and optimized for the isolation of
metagenomics DNA from O.M.6.2 and O.M.6.5 the saline sites used for the
isolation and enrichment of bacterial isolates. DNA extraction methods were
evaluated in terms of DNA purity, yield and humic acid content. Diversity
based analysis and amenability for molecular biology work was assessed by
16S rRNA amplicons. Followed by amplification profile, metagenomics
nature of amplicons was elucidated from DGGE-molecular fingerprinting
technique. Beside, the source also provided a huge and comprehensive
platform for capturing novel alkaline protease gene sequences. Successful
241
cloning and expression of alkaline protease from metagenomic derived
amplicons revealed interesting features.
Physico-chemical and molecular characterization of metagenomic protease
confirmed that some characteristic properties of un-cultivable clone were quite
similar to recombinant clones earlier discussed. However, the enzyme was
found to be more sensitive. These findings were further enriched by protein
structure and function predictions.
ORGANIC SOLVENTS TOLERANCE OF NATIVE,
RECOMBINANT AND METAGENOMIC DERIVED
PROTEASES
Native alkaline proteases exhibited resistance towards organic solvents as
compared to recombinant enzymes. While, metagenomics derived enzyme
displayed minimum resistance against solvents.
Similar trends were also reflected with reference to enzyme stability. The
results on organic solvents holds novelty as similar results exhibiting
comparative view on varied enzyme preparations are not studied extensively.
Catalysis in the presence of solvents was further studied in combination of
salt, pH and temperatures.
To get more insight into phylogenentic relatedness, the protease sequences of
O.M.A18, O.M.E12 and metagenome clone O.M.6.2 were aligned with known
protease sequences available in the database. The enzyme sequences were
quite diverse in its sequence similarity; they were more closely related to
enzyme sequences of other halophilic organisms as compared to enzymes
studied in the present study.
The present study highlighted the properties and capabilities of the
haloalkaliphilic bacteria and their enzymes. The properties of the O.M.A18
and O.M.E12 native enzymes and its recombinant counterparts displayed
unique features with its ability to function under extreme conditions. Besides,
the biotechnological potentials, extensive studies on diversity, physiology and
242
metabolic reactions would further be supportive to understand the
bioenergetics and adaptation strategies of these organisms as they are
subjected to different environmental stresses. Moreover, the wide occurrence
of the alkaline protease and their properties among cultivable isolates as well
by soil derived clone clearly indicated that they could be used as marker for
studying bacterial heterogeneity and population dynamics of unexplored
habitats. The result on catalytic efficiency in the presence of organic solvents
significantly addressed that they can be employed in non-aqueous
enzymology.
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CONCLUSIONS
243
CONCLUSIONS
Existence of the haloalkaliphilic bacteria in saline soil clearly indicated the
wide spread distribution of these extremophiles in saline habitats. Requirement
of NaCl (5-20%) and pH (8-11) for the optimum growth indicated the
moderate haloalkaliphilic nature of these organisms. Along with phenotypic
properties; metabolic reactions, antibiogram, and occurrence of alkaline
proteases clearly emerged as effective tools to judge the microbial
heterogeneity.
The wide occurrence of the alkaline protease and their properties among these
bacteria clearly indicated that they could be used as marker for studying
bacterial heterogeneity and population dynamics of unexplored habitats.
Alkaline proteases were purified to the homogeneity by single step
purification by affinity chromatography. Stability and catalysis of the
O.M.A18 and O.M.E12 proteases under three extremities of pH, salt and
temperature would attract unique biotechnological applications, besides
providing a unique model to study protein stability. The commercial
exploitation of enzymes explored on the basis of unique characteristic features
of enzymes reflected the novelty with particular reference to chemical
denaturant, oxidizing and reducing agent.
Molecular studies of alkaline proteases were carried out by gene amplification
profile, cloning, over expression and inexpensive method of purifying the
over-expressed protein followed by the enzymatic.
The recombinant enzymes produced in E.coli. by and large maintained the
characteristic features as reflected by the native enzymes produced in
haloalkaliphilic strains of the bacteria. The over expressed recombinant
proteins were obtained in active form. The characteristics of the
haloalkaliphililc serine alkaline protease were maintained with respect to
thermal and salt stability. The results, therefore, are a value addition to the
recombinant enzymology.
244
The fact that only limited enzymes from haloalkalophiles have been cloned
and studied for heterologous expression further adds to significance of the
study. Studies on recombinant enzymes and their characteristics were further
supported by in-silico structure and function analysis based on nucleotide and
protein sequences using bioinformatics tools and approaches.
Metagenomics has opened new areas for diversity based exploration unlocking
biotechnological potential for novel enzymes/genes. The designed and
optimized metagenomic DNA extraction protocols were simple, short and
facilitated rapid isolation of PCR amplifiable total genomic DNA from saline
soil. The methods yielded good quality of the DNA suitable for metagenomic
studies.
The findings are also significant as only few extreme environments,
particularly saline habitats, are explored for their metagenomic potential.
Further, to access to functional attributes of metagenome, recombinant clones
for alkaline proteases were constructed from the total DNA isolated from
saline habitats. The characteristic features of the recombinant enzymes were
studied with reference to its physico-chemical parameters and in-silico
molecular structure analysis.
On the basis of comparative analysis of solvent tolerance of native
haloalkaline, recombinant and metagenomic alkaline proteases, we observed
that native enzymes were quite resistant nature towards organic solvents as
compared to recombinant enzymes. While, metagenomics derived enzyme had
minimum resistance.
We observed wide diversity among the isolated haloalkaliphilic bacteria on the
basis of its microbiological and molecular traits. Single-step purification and
characterization of O.M.A18 and O.M.E12 proteases under multitude of
extremities promises its biotechnological significances. Cloning, sequencing,
over-expression and characterization of serine proteases from two potential
strains will enhance our knowledge on serine proteases. The studies on
metagenomics generated vital information, as only few extreme environments,
particularly saline habitats, are explored for their metagenomic potential.
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APPENDICES
APPENDIX I
I. PAPERS COMMUNICATED/ACCEPTED/PUBLISHED:
PUBLISHED
1. Purohit, M.K and Singh, S. P. 2008. Assessment of various methods for
extraction of Metagenomic DNA from saline habitats of Coastal Gujarat
(India) to explore Molecular Diversity. Letters in Applied Microbiology.
49(3): 338-344.
2. Singh, S.P., Purohit, M.K., Aoyagi, C., Kitaoka, M. and Hayashi K. 2009.
Effect of growth temperature, induction and molecular chaperones on the
solubilization of over-expressed Cellobiose Phosphorylase from Cellvibrio
gilvus under in-vivo conditions. Biotechnology Bioprocess Engineering.
15: 273-276.
3. Siddhpura, P. K., Vanparia, S., Purohit, M. K. and Singh, S. P. 2010.
Comparative studies on the extraction of metagenomic DNA from the saline
habitats of Coastal Gujarat and Sambhar Lake, Rajasthan (India) in prospect of
molecular diversity and search for novel biocatalysts. International Journal
of Biological Macromolecules. 47: 375-379.
4. Purohit, M. K. and Singh, S.P. 2011.Comparative analysis of highly
thermostable extracellular alkaline proteases produced by two Haloalkaliphilic
bacterial strains isolated from Coastal Gujarat (India). International Journal
of Biological Macromolecules. 49:103–112.
5. Surani, J.J., Akbari, V.G., Purohit, M.K. and Singh, S.P. 2011. PAHbase, a
freely available functional database of Polycyclic Aromatic Hydrocarbons
(pahs) degrading bacteria. Journal of Bioremediation & Biodegradation 2:1.
281
6. Ukani H., Purohit, M.K., Georrge, J.J., Paul, S. and Singh, S.P. HaloBase:
Development of Database System for Halophilic Bacteria and Archaea with
respect to Proteomics, Genomics & other Molecular Traits. Journal of
Scientific and Industrial Research. (In Press).
COMMUNICATED
1. Purohit, M. K. and Singh, S.P. Cloning, Over-expression and characterization
of haloalkaliphilic bacteria strain isolated from coastal region of Gujarat.
Process Biochemistry
IN-PROGRESS
1. Purohit, M. K. and Singh, S.P. Microbiological and molecular diversity of
haloalkaliphilic bacterial isolates of Coastal Gujarat (India).
2. Purohit, M. K., Pandey, S and Singh, S.P. Comparative analysis of native,
recombinant and metagenomic alkaline proteases with respect to their
tolerance against organic solvents.
3. Purohit, M. K. and Singh, S.P. Gene cloning, over-expression and
characterization of an alkaline protease from Haloalkaliphilic bacterium
O.M.E12
4. Purohit, M. K. and Singh, S.P. An alkaline protease from metagenome of
saline habitat: cloning, over-expression, characterization and functional
attributes
282
II. BOOK CHAPTERS
PUBLISHED/ACCEPTED/COMMUNICATED
PUBLISHED
1. Singh, S. P., Purohit, M. K., Thumar, J. T., Pandey, S., Rawal, C. M. and
Bhimani, H. G. 2008. “Biocatalytic Potential of Haloalkaliphilic Bacteria” In
“Biocatalysis Research Progress" NOVA Publisher, New York, USA.
2. Singh, S. P., Purohit, M. K., Bhimani H.G. and Rawal, C. M. 2007.
Metagenomic: a culture independent approach to study biotechnological
potential of unexplored/uncultivable microorganism In Vak Journal,
Saurashtra University.
3. Singh, S.P., Purohit, M.K., Raval, V.H., Pandey, S., Akbari V. G. and Rawal
C. M. 2010a. Capturing the potential of Haloalkaliphilic Bacteria from the
saline habitats through culture dependent and metagenomic approaches. In
Current Research Technology and Education Topics in Applied Microbiology
and Microbial Biotechnology. (Ed. A. Mendez-Vilas) Formatex Publications.
pp.81-87
4. Singh, S.P., Thumar J.T., Gohel S.D. and Purohit, M.K. 2010b. Molecular
diversity and enzymatic potential of salt-tolerant alkaliphilic actinomycetes. In
Current research technology and Education Topics in applied Microbiology
and Microbial Biotechnology. (Ed. A. Mendez-Vilas) Formatex Publications.
pp. 280-286.
283
ACCEPTED
1. Purohit, M. K., Singh, S. P. 2010a. Metagenomics of saline habitats with
respect to bacterial phylogeny and biocatalytic potential. Microbes in
environmental management and biotechnology. Springer publication.
2. Singh, S.P., Raval, V. H., Purohit, M. K, Thumar J.T., Gohel, S.D., Pandey,
S., Rawal C.M. and Akbari V.G. 2010. Haloalkaliphilic bacteria and
actinobacteria from the saline habitats: new opportunities for biocatalysis and
bioremediation. Microbes in environmental management and biotechnology.
Springer publication.
COMMUNICATED
1. Singh, S.P., Raval, V. H., Purohit, M. K. 2011. Strategies for the salt tolerance
in bacteria and archeae and its implications in developing crops for adverse
conditions. In Crop improvement under adverse conditions.
Springer publication.
284
III. SEQUENCES SUBMITTED IN NCBI
16S RIBOSOMAL RNA GENE
1. Singh S.P. and Purohit M.K, Rawal C.M. Haloalkaliphilic bacterium
O.M. E12 16S ribosomal RNA gene, partial sequence. GenBank: EU680960.
2. Singh S.P. and Purohit M.K, Bhimani H.G. Oceanobacillus iheyensis strain O.M.A18 16S ribosomal RNA gene, partial sequence. GenBank: EU680961.
3. Singh S.P. and Purohit M.K. Oceanobacillus oncorhynchi strain O.M.C28
16S ribosomal RNA gene, partial sequence GenBank: GQ162110.
4. Singh, S. P., Bhimani, H.G., Rawal, C. M. and Purohit, M. K. Micrococcus luteus isolate PAH-8 16S ribosomal RNA gene, partial sequence. GenBank: EU095950.
5. Singh, S. P., Rawal, C. M., Bhimani, H. G. and Purohit, M. K. Bacillus halodurans strain C7 16S ribosomal RNA gene, partial sequence. GenBank: EU091310.
ALKALINE PROTEASES GENE
6. Singh S.P., Purohit M.K. and Siddhpura P.K. An alkaline protease gene cloned from Haloalkaliphilic bacterium O.M.C14, isolated from Okha Madhi, Coastal Gujarat, India, into pET 21a+ plasmid vector. GenBank: HM219180.
7. Singh S.P., Purohit M.K. Alkaline Protease gene cloned from
Haloalkaliphilic bacterium Oceanobacillus iheynsis O.M.A18, isolated from Okha Madhi, Coastal Gujarat, India: GenBank: HM219179.
8. Singh S.P., Purohit M.K. An alkaline protease gene cloned from Haloalkaliphilic bacterium O.M.E12, isolated from Okha Madhi, Coastal Gujarat, India, into pET 21a+ plasmid vector. GenBank: HM219182.
9. Singh S.P., Purohit M.K. Alkaline Protease sequence retrieved from saline soil sample, Okha Madhi, Gujarat, India by metagenomic approaches. OkM.6.2: GenBank: HM219181.
285
APPENDIX II
I. PAPER/POSTER PRESENTATIONS
1. Purohit, M. K. and Singh, S.P “Cloning, Over-expression and
Characterization of Alkaline Protease from Non.-Cultivable and Cultivable
Organisms obtained from Halophilic/or Haloalkalophilic organism isolated
from Coastal region of Gujarat” in Recent trends in Biology at Saurashtra
University, Rajkot, 16-17 Sep-2010.
2. Purohit, M. K. and Singh, S.P “Culture dependent and metagenomic
approaches to unlock biotechnological potential and search for signature of
saline habitats of Coastal Gujarat” in Science Excellence at Gujarat
University, Ahmedabad (India), 10Jan-2010(First Rank).
3. Siddhpura P.K., Purohit, M.K., Singh S.P “Assessment of various methods
for the extraction of Metagenomic DNA from saline habitats in Coastal
Gujarat to judge its amenability for molecular biological applications” in
Science Excellence at Gujarat University, Ahmedabad (India), 10Jan-2010.
4. Singh S.P., Purohit, M.K., Pandey, S., Raval, V. and Raval, C “Alkaline
proteases among haloalkalipliic bacteria dwelling in saline habitats of coastal
Gujarat: distribution, biochemical properties and metagenomics” in National
Symposium on Recent trends in Cellular Research at Saurashtra University
Rajkot (India), 09 March, 2009.
5. Singh S.P., Purohit, M.K., Pandey, S., Raval, V. and Raval, C “Biocatalytic
Potential under Multitude of Extremities: Vast Opportunities for Industrial and
Environmental Applications” in National Conference on “ Microbial
Technology on Sustainable Environment” at Gujarat University Ahmedabad
(India), 02-03 March, 2009.
286
6. Purohit, M. K. and Singh, S.P “Exploration of Microbial Diversity and
Biotechnological potential by Metagenomic Approach” in Science Excellence
at Gujarat University, Ahmedabad(India), 10Jan-2009.
7. Siddhpura P.K., Purohit, M.K., Singh S.P “Extraction and assessment of
various protocol for isolation of Total Metagenomic DNA from saline
habitats of Coastal Gujarat (India) to confirm its amenability for further
metagenomic work” in Science Excellence at Gujarat University,
Ahmedabad, 10Jan-2009.
8. Savsani. K. A., Purohit, M.K., Singh S.P “Alkaline proteases of
Haloalkaliphilic bacteria from the saline habitats of Coastal Gujarat” in
Science Excellence at Gujarat University, Ahmedabad, 10Jan-2009
9. Gohel S., Dalsania T.L., Purohit, M.K., Singh S.P “Halo-tolerant and
alkaliphilic actinomycetes from the saline habitats of Coastal Gujarat:
Diversity based on morphological features, enzyme secretion, antibiotic
sensitivity and molecular parameters in Microbial Technology for Sustainable
Environment” at Gujarat University, Ahmedabad. 2-3 March, 2009.
10. Purohit M. K., and Singh, S.P “Accessing Non-Cultivable from saline
habitats of Coastal Gujarat by metagenomic approaches towards
understanding global ecology and exploration of microbial potential” in
Microbial Technology for Sustainable Environment at Gujarat University,
Ahmedabad. 2-3 March, 2009 (First Rank).
11. Singh S.P., Purohit, M.K., Pandey, S., Raval, V. and Raval, C “Diversity,
Molecular phylogeny and Biocatalytic Potential of Haloalkaliphilic bacteria
from Coastal Gujarat”Extremophile-2008, Cape Town, South Africa, 07-11
September 2008.
287
12. Purohit, M. K. and Singh, S.P “Assessment of various methods for extraction
of Metagenomic DNA from saline habitats of Gujarat (India) to explore
Molecular Diversity and Alkaline Protease genes” Extremophile-2008, Cape
Town, South Africa, 07-11 September 2008.
13. Singh S.P., Purohit, M.K., Pandey, S., Raval,V, Kikani, B., ,V.G., Akbari
“Haloalkaliphilic bacteria from coastal Gujarat: Diversity and Biocatalytic
potential under multitude of extremities” in National Conference on “ Recent
advances in Molecular Biology” at Nirma university of Science &
Technology, Ahmedabad (India), 26 March, 2008.
14. Purohit. M. K, Maniar E.V, Makani S, Joshi A.Y, Bhimani H.G, Kothari
R.K. and Singh. S.P. “Biodegradation of textile dyes by seven membered
Bacterial consortium and its plasmid profiling “at National level Conference
on “Bioresource and its Conservation” on 17th and 18th February, 2006 held at
Department of Biosciences, Saurashtra University, Rajkot.
II. WORKSHPS/SEMINARS ATTENDED
1. National Level Science Symposium on Global Prospective on Parma Patents
held at Department of Pharmaceutical Sciences, Saurashtra University Rajkot
in 2008.
2. National Level Symposium on Recent Advances in Molecular Biology &
Biotechnology RAMB-2008.
3. National Level Science Symposium on Current Trends in Science organized
by Christ College, Rajkot in 2008.
4. National Level Science Symposium on “Silencing the message”: mi and si
RNA organized by Christ College, Rajkot in 2008.
5. ICMR Sponsored One Day National Workshop on “Clinical Trials: Scope,
Challenges, Regulation & Methodology of working in India” organized by S.J.
Thakkar Pharmacy College, Rajkot in 2007.
288
6. Workshop on Nano-technology at Saurashtra University, Rajkot.Attended
National level Workshop on” Current Drug Patent Régime” on 5thMarch,
2006 held at S. J. Thakkar College, Rajkot.
7. One day National level Seminar on “50 Years of DNA Double Helix –
Retrospect and Prospects” held at M & N Virani Science College on 11th
October, 2004.
8. State level Science Symposium organized by Christ College Rajkot, India on
January 25, 2004.
9. International symposium on molecular medicine with special reference to the
molecular biology of infectious disease held at Baroda on 26th and 27th Dec.
2003.
10. State Level Competition on “Microobial Interaction” organized by Microbial
Study Circle, Nadiad 2004.
11. Conference on “Basic Aspects of Bioinformatics” held at Christ College
Rajkot, India from July 10-11, 2002.
289
APPENDIX III
I. TRAINING UNDERTAKEN ORGANIZATION Department of Plant Molecular Biology
Delhi University South Campus, Delhi
BRIEF DESCRIPTION Cloning of Alkaline Protease enzyme and its expression
analysis-as an extension of research activity part of Ph.D.
programme.
DURATION 2 months (1September-30 October 2008)
ORGANIZATION
BRIEF DESCRIPTION
DURATION
Indian Institute of Advanced Research(IIAR), Gandhinagar,
Gujarat
Hands on Training Programme in “Bioinformatics”
5 Days(February 2008)
ORGANIZATION
BRIEF DESCRIPTION
DURATION
DURATION
National Research Centre for Groundnut (NRCG), Junagadh,
Gujarat
Advanced Molecular Biological techniques-Molecular
Markers
45 Days(15 May-30 June 2005)
290
II.SCHOLARSHIPS/AWARDS
Awarded Senior Research Fellowship (SRF) by CSIR, New Delhi from March 2010.
Stood 1st in Science Excellence, Ahmedabad in January 2010 for Paper Presentation.
1st Prize in Paper presentation in Science Excellence, Gujarat University,
Ahmedabad. 10Jan-2009.
Stood 1st in Saurashtra University in 2006 for M.Sc Biotechnology.
1st in Christ College and 2nd in Saurashtra University during. T.Y. B.Sc. Biotechnology exam in 2004.
2nd in Christ College during. S.Y. B.Sc. Biotechnology exam in 2003.
1st Prize in Oral presentation in State Level competition organized by Christ College in 2004, Rajkot.
Stood 52nd in National Level Talent Search Examination-Senior Level held by Biotech Helpline Foundation, Jaipur.
ORIGINAL ARTICLE
Assessment of various methods for extraction ofmetagenomic DNA from saline habitats of coastal Gujarat(India) to explore molecular diversityM.K. Purohit and S.P. Singh
Department of Biosciences, Saurashtra University, Rajkot, Gujarat, India
Introduction
The impact of molecular studies on our knowledge of
microbial diversity cannot be overstated. As a conse-
quence, emphasis and focus on microbiology, from basic
ecological research into the organization of microbial
communities to bioprospecting for commercially relevant
enzymes and metabolic potential, have changed (Lakay
et al. 2007; Mitchell et al. 2008).
According to the current estimates, c. 99% of the micro-
organisms present in nature are not cultivable by standard
techniques. Therefore, the genetic information and bio-
technological potential of the majority of the organisms
would be untapped by conventional approaches (Green
and Keller 2006; Chernitsyna et al. 2008). Recovery, clon-
ing and expression of environmental DNA without necessi-
tating cultivation is a recent approach to exploit the
potential of microbial communities present in environ-
mental samples, as it has been of growing interest to both
microbial ecologists and biochemists looking for novel bio-
catalysts and metabolites (Risenfeld et al. 2004; Liles et al.
2008). Besides, metagenomic approaches also highlight on
the population heterogeneity and phylogenetic status of a
habitat in totality (Gabor et al. 2003; Galperin 2008; Mes
2008). Detecting the rare members of a microbial commu-
nity is a challenge; however, it is very important as they
play a critical ecological role (Yeates et al. 1998; Voget et al.
2003). The genetic information on all indigenous organ-
isms can be accessed theoretically, including the predomi-
nant fraction of micro-organism that is recalcitrant to
cultivation, by applying metagenomics approaches (Raes
et al. 2007). The biotechnological applications currently
targeting microbial metagenomic studies range from the
search for new antibiotics to environmentally sound biocat-
alysts (Mitchell et al. 2008).
Among the key factors responsible for the success of
metagenomics, the isolation of quality environmental
DNA in appreciable amount from a given habitat holds
Correspondence
Satya P. Singh, Department of Biosciences,
Saurashtra University, Rajkot 360 005,
Gujarat, India.
E-mail: [email protected],
2008 ⁄ 1825: received 23 October 2008,
revised and accepted 14 May 2009
doi:10.1111/j.1472-765X.2009.02663.x
Abstract
Aims: To develop total DNA extraction protocol from saline soil for further
metagenomic applications.
Methods and Results: The protocols combine the application of mechanical
(Beads and Sonicator) and Soft Lysis (SDS and enzymes) method for the isola-
tion of total DNA from saline soil of coastal Gujarat followed by its quantifica-
tion and purity assessment. The quality and purity of metagenomic DNA was
quite consistent and reliable, although it contained residual concentartions of
humic acid. The extracted DNA was used to successfully amplify 16S rRNA
region. The amplicons were suitable for further applications such as diversity-
based analysis by denaturing gradient gel electrophoresis (DGGE).
Conclusions: The methods appear to have wide applicability in investigating
molecular diversity and exploring functional genes from the total DNA.
Significance and Impact of the Study: The protocol is simple, short and facili-
tates rapid isolation of PCR amplifiable total genomic DNA from saline soil.
The method yielded good quality of the DNA suitable for metagenomic studies.
The results are also significant as only few extreme environments, particularly
saline habitats, are explored for their metagenomic potential.
Letters in Applied Microbiology ISSN 0266-8254
338 Journal compilation ª 2009 The Society for Applied Microbiology, Letters in Applied Microbiology 49 (2009) 338–344
ª 2009 The Authors
significance (Voget et al. 2003; Kennedy and Marchesi
2007; Sharma et al. 2007). During the last 10 years, many
protocols for the extraction of environmental DNA have
been published, and some of them are commercialized as
soil DNA extraction kits. The methods vary with respect to
shearing, purity and quantity of the isolated DNA.
However, the basic concept of cell lysis by enzymatic
(lysozyme) and hot detergent (SDS) treatment is still the
core of many DNA extraction methods (Rondon et al.
2000). Besides, some protocols also apply mechanical forces
generated by bead beating, freeze-thawing and sonication
methods to disrupt the rigid cell structure (Voget et al.
2003; Kennedy and Marchesi 2007; Sharma et al. 2007).
The extraction protocols are generally classified as
direct and indirect DNA extraction procedures. Direct
DNA isolation is based on cell lyses within the sample
matrix and subsequent separation of DNA from the
matrix and cell debris (Voget et al. 2003). While the indi-
rect approach involves the extraction of cells from the
environmental material prior to the lytic release of DNA
(Santosa 2001; Kauffmann et al. 2004), direct DNA
extraction protocol involves soft and harsh lysis methods.
Soft lysis method is based on the disruption of micro-
organism solely by enzymatic and chemical means,
whereas harsh lysis approach involves the mechanical cell
disruption by bead beating, sonication, freeze-thawing
and grinding. The indirect DNA extraction protocols
involved blending, cation-exchange method (Desai and
Madamwar 2007) or other new approaches, such as the
use of super paramagnetic silica–magnetite nanoparticles
for the isolation and purification of DNA from soil sam-
ples (Sebastianelli and Bruce 2008).
Because the composition of different habitats varies
with respect to their matrix, organic and inorganic com-
pounds and biotic factors, standardization of total DNA
extraction technique is desirable. Improved DNA extrac-
tion techniques could help to ensure a metagenomic
library that adequately represents the entire community’s
genome without inhibitory substances. Therefore, as an
extension of our ongoing research work on haloalkaliphil-
ic bacteria form coastal Gujarat (Gupta et al. 2005; Now-
lan et al. 2006; Thumar and Singh 2007; Dodia et al.
2008a,b). The present study aims at the optimization and
assessment of the extraction methods for total environ-
mental DNA from saline soil near salt pan of Okha
Madhi, Gujarat coast, India.
Materials and methods
Environmental soil sampling and storage
Two soil samples, designated as Ok.M.6Æ2 and Ok.M.6Æ5,
were collected in September 2007 from coastal region of
Okha Madhi (Latitude 22Æ20�N, Longitude 70Æ05�E),
Gujarat, India. They represent a typical saline soil with heavy
deposition of salt. At the site of collection, a block of soil was
removed and transported to laboratory in sterile plastic bags
for storage at 4�C. Total DNA extraction and further
analyses were carried out from these samples within 15 days.
DNA extraction methods
Soft lysis method
Soil samples (1 g) in duplicate were suspended in 10 ml
of extraction buffer (100 mmol l)1 Tris–HCl (pH-8Æ2);
100 mmol l)1 EDTA (pH-8); 1Æ5 mol l)1 NaCl) and incu-
bated at 37�C for 10–12 h with shaking at 150 rev min)1.
Samples were re-extracted in 1 ml of the same extraction
buffer. Supernatants were collected by low speed centrifu-
gation at 2097 g for 10 min. Four millilitres of lysis buf-
fer; 20% (w ⁄ v) SDS; lysozyme, 20 mg ml)1; protinase K,
10 mg ml)1; N-lauroyl sarcosine, 10 mg ml)1; CTAB
(cetyltrimethylammonium bromide), 1% (w ⁄ v) were
added to the supernatant followed by incubation at 65�C
for 2 h with vigorous shaking at every 15 min. Samples were
centrifuged at 8385 g for 10 min at 4�C. The upper aqueous
phase was extracted with equal volume of
phenol : chloroform : isoamylalcohol (P : C : I = 25 :
24:1) at 8385 g for 20 min at 4�C. The upper aqueous
phase was again extracted with equal volume of C : I
(24 : 1) at 8385 g for 10 min at 4�C. DNA was treated by
adding 1 ⁄ 10 volume of 7Æ5 mol l)1 potassium acetate and
subsequently precipitated by adding two volumes of
chilled absolute ethanol for 30 min at 4�C. DNA precipi-
tate were collected by centrifugation at 6708 g for 10 min,
air-dried and suspended in 20 ll sterile D ⁄ W.
Harsh methods
DNA extraction using bead beating. Soil samples (1 g) in
duplicate were suspended in 10 ml of extraction buffer
and incubated at 37�C for 10–12 h with shaking at
150 rev min)1. Samples were re-extracted in 1 ml of the
extraction buffer, and supernatants were collected by low
speed centrifugation at 2097 g for 10 min. Glass beads
(1 g) were added to the supernatant and blended for
15 min following incubation at 65�C for 2 h. Samples
were then centrifuged at 8385 g for 10 min at 4�C.
Extraction was then continued as per soft lysis method.
DNA extraction using sonication. The initial steps for sam-
ple treatment for extraction were essentially the same as
described earlier. The supernatant were then sonicated
using a High Intensity Ultrasonic processor (Sartorius,
Goettingen, Germany) with a standard 13-mm horn solid
M.K. Purohit and S.P. Singh Extraction of metagenomic DNA from saline habitat and its assessment for further molecular biology applications
ª 2009 The Authors
Journal compilation ª 2009 The Society for Applied Microbiology, Letters in Applied Microbiology 49 (2009) 338–344 339
probe for three pulses of 30 s each in a chilled ice bath. The
sample was cooled in ice and repeatedly sonicated (six
cycles of 30 s) followed by incubation at 65�C for 10 min.
Samples were then centrifuged at 8385 g for 10 min at 4�C.
Further extraction was continued as per soft lysis method.
DNA extraction by a combination of bead beating and
sonication treatment. The initial steps for sample treatment
for extraction were essentially the same as described earlier.
The supernatants were treated by glass bead followed by
sonication as per the earlier description of these methods.
Samples from sonication were centrifuged at 8385 g for
10 min at 4�C. The supernatants were extracted with equal
volume of P : C : I = 25 : 24 : 1 at 8385 g for 20 min at
4�C. Further extraction was continued as per soft lysis
method.
DNA extraction by combination of soft and harsh method
DNA extraction using bead beating combined with lysis
buffer. This method was a combination of bead beating
treatment followed by the extraction using lysis buffer. The
DNA was finally extracted by the method as described in
soft lysis approach.
DNA extraction using sonication treatment combined with
lysis buffer. This method was a combination of sonication
treatment followed by the extraction using lysis buffer. The
DNA was finally extracted by the method as described in
soft lysis approach.
DNA extraction by ultra clean soil DNA isolation kit. DNA
extraction from the soil sample was also attempted using a
kit provided by Mo Bio Laboratories Inc. according to the
protocol described (Mo Bio Laboratories, Carlsbad, CA).
Determination of purity and yield of DNA
Co-extracted humic acids are the major contaminant
when DNA is extracted from soil. Humic acids absorb at
230 nm, while DNA and protein at 260 and 280 nm
respectively. To evaluate the purity of the extracted DNA,
absorbance ratios at 260 ⁄ 230 nm (DNA ⁄ humic acid) and
260 ⁄ 280 nm (DNA ⁄ protein) were determined.
PCR amplification of 16S rRNA gene
The DNA preparations obtained by the methods described
were used as template to amplify DNA fragment encoding
16S rRNA gene. To 100 ng of DNA as template; 25 pmol of
each, forward (5¢-AGA GTT TGA TCC TGG CTC AG-3¢)and reverse (5¢-ACG GCT ACC TTG TTA CGA CTT-3¢)oligonucleotide primers (Invitrogen) followed by the addi-
tion of 25 ll of 2· Red Mix Plus (Banglo Genei, Bangalore,
India) was added. The final reaction mixture was adjusted
to 50 ll by adding miliQ grade water. The amplification
protocol was: Step 1, initial denaturation at 94�C for
1 min; Step 2, denaturation at 94�C for 30 s; Step 3,
annealing at 54�C for 30 s; Step 4, extension at 72�C for
2 min. Steps 2–4 were repeated for 29 cycles with a final
elongation at 72�C for 2 min. Two negative controls, one
without template and another without primer, were also
included in the PCR. The amplified products were stored at
)20�C till further use.
Gel electrophoresis
Amplicons of each reaction (10 ll) were analysed with
smart ladder (0Æ2–10 kbp; Invitrogen) as DNA marker on
0Æ8% agarose gels using TAE (pH-8) as electrophoresis
buffer. Gels were stained with ethidium bromide
(5 lg ml)1) and analysed by Syngene Gene Genius Bio-
imaging system (Syngene, Frederick, MD).
Denaturing gradient gel electrophoresis
Denaturing gradient gel electrophoresis was performed
according to Muyzer and Smalla (1998). Fifty microlitres
of 16S rRNA amplicon were subjected to increasingly
higher concentrations of urea and formamide that acted
as chemical denaturant (20–50%). The amplicons
migrated through polyacrylamide gel containing denatur-
ants at constant voltage; 200 V for 1 h followed by 30 V
for 10 h. Gels were visualized and analysed by Syngene
Gene Genius Bio-imaging system after staining with ethi-
dium bromide (5 lg ml)1).
Results
DNA extraction from saline soil in the present study had
threefold objectives; lysis of representative microbes within
the sample, obtaining high molecular weight intact DNA
and removal of inhibitors from the extracted DNA for
subsequent molecular manipulations. Therefore, various
methods were examined for DNA extraction towards ful-
filling these objectives. We have developed an improved
method for isolating total metagenomic DNA from saline
soil through which intact and unsheared DNA, amenable
for further molecular biology work was obtained. The
extracted DNA was assessed by PCR amplification of 16S
rRNA region followed by DGGE analysis.
Extraction of metagenomic DNA from saline habitat and its assessment for further molecular biology applications M.K. Purohit and S.P. Singh
340 Journal compilation ª 2009 The Society for Applied Microbiology, Letters in Applied Microbiology 49 (2009) 338–344
ª 2009 The Authors
Purity and yield assessment
Total DNA was isolated from the two samples collected
from Okha Madhi site by soft lysis, bead beating, sonica-
tion and in combination with these methods. In compari-
son with these methods, we also attempted the extraction
of total DNA by Clean Gene Isolation kit (Mo Bio Labo-
ratories). The extracted DNA was assessed for purity and
yield on the basis of absorbance ratios at 260 ⁄ 230 nm
(DNA ⁄ Humic acids) and 260 ⁄ 280 nm (DNA ⁄ protein;
Table 1). High ratio of 260 ⁄ 230 nm indicated the purity
of extracted DNA with respect to humic acid contamina-
tion, whereas high 260 ⁄ 280 nm ratio was an indicative of
the purity with respect to protein contamination. DNA
samples were analysed using 0Æ8% agarose gel with smart
ladder (0Æ2–10 kbp) as marker (Invitrogen; Fig. 1). There
was no noticeable variation in the quality and quantity of
DNA on the basis of agarose gel patterns. The striking
features of the extraction methods highlight that sonica-
tion alone was not suitable for DNA extraction from
Ok.M.6Æ5, as humic acid content was not reduced, and
the purity and concentration of the extracted DNA did
not compare favourably with other methods. However,
the method based on sonication yielded better results
with another sample, Ok.M.6Æ2. The differential results by
sonication may reflect on the fact that the matrix of the
concerned habitat might be causing a barrier against the
extraction and lysis of the cell.
Bead beating emerged as equally effective method for the
extractions of quality DNA in appreciable quantity from
both environmental samples. On the other hand, combina-
tions of different mechanical methods lead to decreased
concentration and purity of the extracted DNA with
excessive shearing. Soft lysis method proved best for Ok.M.
6.2 and Ok.M.6Æ5, as it yielded higher concentration and
eliminated humic acid to significant extent. The best
quality of DNA was obtained by employing combination of
soft lysis with bead beating method, while its combination
with sonication was not as good in terms of DNA yield.
The DNA preparations were considered for Molecular
Biology applications, as obtained environmental DNA
should not only satisfy the yield and purity criteria but it
should also be amenable to further work.
PCR amplification of 16S rRNA gene
The environmental DNA extracted by all the above-
mentioned methods were used as template for PCR
Table 1 Concentration and purity of total DNA isolated from soil sample Ok.M.6Æ2 and Ok.M.6Æ5 by using various isolation methods
Sr.No. Method
260 ⁄ 280 260 ⁄ 230 Concentration (lg ml)1)
Sample Sample
Ok.M.6Æ2 Ok.M.6Æ5Ok.M.6Æ2 Ok.M.6Æ5 Ok.M.6Æ2 Ok.M.6Æ5
1 Sonication 1Æ466 0Æ952 1Æ523 0Æ833 153Æ72 145Æ00
2 Beads beating 1Æ525 1Æ442 1Æ642 1Æ242 160Æ00 172Æ00
3 Sonication + bead beating 1Æ255 1Æ043 1Æ112 0Æ743 142Æ00 150Æ00
4 Soft lysis 1Æ726 1Æ570 1Æ732 1Æ672 212Æ00 225Æ00
5 Soft lysis + bead beating 1Æ755 1Æ780 1Æ712 1Æ550 205Æ00 197Æ00
6 Soft lysis + sonication 1Æ652 1Æ585 1Æ652 1Æ452 185Æ00 178Æ00
1 2 3 4 5 6 7
Figure 1 Isolation of total metagenomic DNA from sample 6Æ2 by
various methods. Lane 1: Lamda DNA ⁄ HindIII Marker (Banglo Genei);
Lane 2: soft lysis; Lane 3: bead beating; Lane 4: bead beating + lysis;
Lane 5: sonication + lysis; Lane 6: sonication; Lane 7: sonica-
tion + bead beating.
1 2 3 4 5 6 7
1500 bp
1 2 3 4 5 6 7
1500 bp
Figure 2 Left panel: 16S rRNA PCR of environmental sample using
isolation methods. Bead beating + sonication method, Lane 1:
0Æ2–10 Kb ladder; Lane 2: site 6Æ2 (Ta = 52Æ4); Lane 3: site 6Æ2
(Ta = 55Æ7); Lane 4: site 6Æ2 (Ta = 56Æ9); Lane 5: site 6Æ5 (Ta = 52Æ4);
Lane 6: site 6Æ5 (Ta = 55Æ7); Lane 7: site 6Æ5 (Ta = 56Æ9). Right panel:
(bead beating method) Lane 1: 0Æ2–10 Kb ladder; Lane 2: site 6Æ2
(Ta = 52Æ4); Lane 3: site 6Æ2 (Ta = 55Æ7); Lane 4: site 6Æ2 (Ta = 56Æ9);
lane 5: site 6Æ5 (Ta = 52Æ4); Lane 6: site 6Æ5 (Ta = 55Æ7); Lane 7: site
6Æ5 (Ta = 56Æ9).
M.K. Purohit and S.P. Singh Extraction of metagenomic DNA from saline habitat and its assessment for further molecular biology applications
ª 2009 The Authors
Journal compilation ª 2009 The Society for Applied Microbiology, Letters in Applied Microbiology 49 (2009) 338–344 341
amplification. Optimum annealing temperature as deter-
mined by gradient was 54�C. Amplification of the 16S
rRNA gene (c. 1Æ5 kb) directly from undiluted DNA
samples (Okha Madhi) indicated the high purity of DNA
(Figs 1 and 2).
Denaturing gradient gel electrophoresis
To gauge the utility of extracted DNA in molecular
fingerprinting methods, especially in microbial ecology
studies, the amplified 16S rRNA DNA was subjected to
denaturing gradient gel electrophoresis. In DGGE at
threshold concentrations of denaturant, different
sequences of DNA, presumably from different bacteria,
denatured resulting in a pattern of bands. As shown in
Fig. 3, the DGGE band patterns of 16S rRNA amplified
from different DNA samples obtained by various extrac-
tion protocols were quite comparable. The observation
was also reflected with the extracted DNA from different
sample sites. Therefore, differences in DGGE banding
pattern suggested that there was not much bias generated
from DNA extraction procedures. The diversity in the
banding profile, however, revealed population heterogene-
ity and differences in both samples. Marker DNA (smart
ladder, 10 kbp) in denaturing gel did not generate bands
according to the standards (Fig. 4).
Discussion
We evaluated DNA extraction methods to identify a pro-
cedure that results in high molecular weight DNA that is
relatively free from contaminants and maximizes detect-
able diversity. Bead beating treatment, an easy-to-perform
method, is based on the ballistic disintegration of the
cells, where the results depend upon the time of agitation
and bead size. The efficiency of cell disruption and conse-
quently the damage to the DNA strands during sonication
mainly depend on the energy input. Even under opti-
mized conditions, harsh treatment may result in shearing
of high molecular weight DNA, low yields and small frag-
ment sizes. This method may have a possibility of intro-
ducing a bias to microbial community analysis.
The enzymatic method relied on the proteinase K and
lysozyme digestion of microbial cells to release DNA, while
the treatment of soil with surfactants and chelating agents
resulted into the removal of inhibitors and prevented
chemical flocculation with minimal loss of DNA yield. A
combination of mild bead beating and enzymatic lysis
treatment emerged as the most successful protocol for
recovering higher yields and inhibitor-free DNA from
saline soil sample. Although, based on the spectroscopic
analysis, humic acid was detectable to varying extent; the
extracted DNA preparations were amenable for further
molecular biology work. This finding appears to be a
favourable observation in comparison with some reports in
literature where humic acid strongly inhibited the DNA
application in molecular biology (Santosa 2001; Kauffmann
et al. 2004; Desai and Madamwar 2007).
1 2 3 4 5 6 7 8 9 10 131211
16S rRNA Amplicon 1500 bp
Figure 3 16S rRNA amplification from total DNA of sample
Ok.M.6Æ2. Lane 1, smart ladder 0Æ2–10 kbp ladder (Invitrogen);
Lane 2, lysis treatment; Lane 3, soft lysis + bead beating; Lane
4, soft lysis + sonication; Lane 5, bead beating; Lane 6, sonication;
Lane 7, sonication + bead beating. 16S rRNA amplification from total
DNA of sample Ok.M.6Æ5. Lane 8, lysis treatment; Lane 9, soft lysis + -
bead beating; Lane 10, soft lysis + sonication; Lane 11, bead beating;
Lane 12, sonication; Lane 13, sonication + bead beating.
1 2 3 4 5 6 7 1 2 3 4 5 6 7
16S rRNA amplifiedproduct of Ok.M.6·2
and Ok.M.6·5
Figure 4 Left panel, DGGE analysis (urea and formamide as denaturant) of the PCR-amplified product from total DNA of sample Ok.M.6Æ2.
Lane 1, smart ladder 0Æ2–10 kbp ladder (Invitrogen); Lane 2, lysis treatment; Lane 3, soft lysis + bead beating; Lane 4, soft lysis + sonication;
Lane 5, bead beating; Lane 6, sonication; Lane 7, sonication + bead beating. Right panel, DGGE analysis (urea and formamide as denaturant) of
the PCR amplified product from total DNA of sample Ok.M.6Æ5. Lane 1, lysis treatment; Lane 2, soft lysis + bead beating; Lane 3, soft lysis + soni-
cation; Lane 4, bead beating; Lane 5, sonication; Lane 6, sonication + bead beating; Lane 7, smart ladder 0Æ2–10 kbp ladder (Invitrogen).
Extraction of metagenomic DNA from saline habitat and its assessment for further molecular biology applications M.K. Purohit and S.P. Singh
342 Journal compilation ª 2009 The Society for Applied Microbiology, Letters in Applied Microbiology 49 (2009) 338–344
ª 2009 The Authors
The quality of the extracted metagenome is of prime
importance in metagenomics, as the DNA should be
suitable to proceed for molecular biological applications
such as molecular diversity and functional genomics
(Rajendhran and Gunasekaran 2008). Results of 16S rRNA
PCR amplification and banding profiles visualized in
DGGE provided the evidence for expediency of the DNA
extraction protocol in studies related to molecular diversity
(Ercolini 2004; Laverick et al. 2004). In view of the hetero-
geneity of the environmental samples, it is quite obvious
that the extraction procedures would have to be case
specific and hence need to be optimized for different soil
samples (Zhou et al. 1996; Santosa 2001; Amorim et al.
2008; Sagova-Mareckova et al. 2008). However, the
methods described in the present study appear to have wide
applicability in investigating molecular diversity and
exploring functional genes from the total DNA.
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Extraction of metagenomic DNA from saline habitat and its assessment for further molecular biology applications M.K. Purohit and S.P. Singh
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ª 2009 The Authors
Biotechnology and Bioprocess Engineering 15: 273-276 (2010)
DOI 10.1007/s12257-009-0023-1
Effect of Growth Temperature, Induction, and Molecular
Chaperones on the Solubilization of Over-expressed Cellobiose Phosphorylase from Cellvibrio Gilvus under in vivo Conditions
S. P. Singh, M. K. Purohit, C. Aoyagi, M. Kitaoka, and K. Hayashi
Received: 4 February 2009 / Revised: 16 May 2009 / Accepted: 7 August 2009
© The Korean Society for Biotechnology and Bioengineering and Springer 2010
Abstract In vivo folding of many proteins can be
facilitated by growth temperature, extent of induction, and
molecular chaperones, which prevent over-expressed protein
from being trapped into insoluble inclusion bodies. In the
present report, we describe the role of molecular chaperones
and growth temperature on the solubilization of over-
expressed Cellobiose Phosphorylase (CBP) in Escherichia
coli. The growth of host at low temperature enhanced
enzyme in soluble fraction. Similarly, induction of target
gene at low level of IPTG also yielded higher enzyme in
soluble fraction. The synergistic effect of low temperature
and induction on the prevention of inclusion bodies was
also evident from our results. In addition, co-expression of
the target gene with two types of molecular chaperones
(GroESL and KODHsp) was also attempted. However,
none of these chaperones enhanced the solubilization under
in vivo conditions. Nevertheless, effective role of low growth
temperature coupled with low level of induction appeared
to be an attractive feature for producing recombinant protein.
Keywords: over-expression, solubilization, molecular
chaperone, cellobiose phosphorylase, Cellvibrio gilvus
1. Introduction
Cellobiose Phosphorylase is generally produced by a strain
because it is beneficial for a strain to hydrolyze cellulose
only to cellobiose and not to glucose, since glucose can
also be utilized by many other microorganisms, but cellobiose
can be utilized only by limited number of microorganisms
[1]. The accumulated cellulose is then digested by the
enzyme cellobiose phosphorylase into glucose and α-
glucose-1-phosphate in the cell [2].
The cloning and sequencing of the gene encoding
cellobiose phosphorylase, cyclodextran glycosyltransferase
(CGTase) was successfully achieved and the gene was
expressed in E. coli as a host, to yield active enzyme in
large quantity [2-4]. Use of heterologous gene expression
generally increases the space-time yield of active
phosphorylase by three orders of magnitude, compared to
production of the enzyme with the natural organism [5].
Cloning and sequencing of the gene leads enzymatic
process to produce amylose from cellobiose, as a useful
tool in converting β-1,4-linked-polysaccharide into α-1,4-
linked-polysaccharide [6].
The process of protein folding is quite significant during
cloning and over-expression, the over-expressed protein
need to be identical and correctly folded. High level
expression of recombinant protein produced in E. coli often
forms aggregate, in insoluble fraction [7-9]. In order to
address the problem of inclusion bodies formation during
over-expression, various in vitro and in vivo strategies have
been attempted [9-12]. The association of molecular
chaperone is required for the stability and function of over-
expressed protein [15]. Besides, other factors, such as
growth, temperature, concentration of inducer, and combi-
nation of both may also affect the folding of recombinant
proteins during over-expression. In the present report, we
described in vivo strategies including growth temperature,
level of induction, and co-expression with molecular
S. P. Singh*, M. K. PurohitDepartment of Biosciences, Saurashtra University, Rajkot 360 005, IndiaTel: +91-281-2586419; Fax: +91-281-2576802 E-mail: [email protected]
C. Aoyagi, M. Kitaoka, K. HayashiNational Food Research Institute, Tsukuba, Ibaraki-305, Japan
RESEARCH PAPER
274 Biotechnology and Bioprocess Engineering 15: 273-276 (2010)
chaperone to enhance solubilization of over-expressed
CBP gene.
2. Materials and Methods
2.1. Cloning of cellobiose phosphorylase gene
Cloning of cellobiose phosphorylase gene from Cellvibrio
gilvus genomic was carried out as reported in our earlier
report [2]. The genomic DNA was isolated and digested
with Sau3AI and followed by ultracentrifugation to obtain
20 kbp fractions. This fraction was then subjected to in
vitro packaging into Lamda Phages by using Gigapacks II
Gold Kit (Stratagene). The resulting Phage Library was
screened by using probe, which was designed by labeling
cellobiose phosphorylase gene (which codes for five of the
peptides derived from native cellobiose phosphorylase with
3’oligolabelling kit (Amersham) and further by digesting
with Sac and Pst I. DNA fragment, thus generated, was
cloned in PUC18 plasmid, which was previously digested
with Sac I and Pst I. The recombinant plasmids were
transformed into E. coli JM109 and the complete sequence
of entire cellobiose gene was determined.
2.2. Effect of temperature, IPTG induction, and molecular
chaperones on the growth and expression of cellobiose
phosphorylase gene
In order to determine the effect of molecular chaperone on
the CBP expression and solubilization, 3 µL each of the
plasmids; GroEL/ES (PkY206), KODHsp, and CBP (pET)
were co-transferred with 40 µL of host, E. coli BL21 cells.
To the transformed cells, 160 µL SOC medium was added
and incubated for 1 h at 37oC under shaking condition.
From this, 80 µL aliquot was spread on Luria Bertani
plates containing tetracycline (12.5 µg/mL) and kanamycin
(30 µg/mL). The selected clones were then grown in LB
broth containing kanamycin (30 µg/mL) and tetracycline
(12.5 µg/mL), using 10% of inoculum in 20 mL of Luria
Bertani medium supplemented with 0.1 mM IPTG and 1
mM IPTG as an inducer, and incubated at 30 and 37oC. At
regular interval of time, 1 mL of culture was withdrawn
and cells were centrifuged at 5,000 rpm for 5 min at 4oC
and growth was measured at 660 nm. The cell pellet was
mixed with 1 mL of sonication buffer and subjected to
sonication at 30 Hz for 30 secs in 6 cycles. Samples were
kept under chilled conditions for 30 secs between each
cycles and the resulted supernatant was treated as soluble
fraction. The pellet was treated with 8 M urea for 30 min at
30oC, followed by centrifugation at 5,000 rpm for 5 min at
10oC to obtain supernatant, which was treated as insoluble
fraction. CBP activity was measured at 505 nm as described
below.
2.3. Enzyme assay
Enzyme assay buffer (0.5 mL) containing 10 mM cellobiose,
10 mM phosphate buffer, 50 mM MOPS; pH 7.0 and
0.02% BSA was added to 1 µL of cellobiose phosphorylase
enzyme, and allowed to react for 15 min. The reaction was
terminated by using 0.5 mL of 40 mM KCN and the
absorbance was measured at 505 nm. The unit of enzyme
activity was defined as the amount of enzyme liberating 1
mM of glucose per minute under assay conditions. Enzyme
activity was measured using cellobiose as standard protein
and was measured by using Bradford method using bovine
serum albumin as standard protein.
2.4. SDS-polyacryalamide gel electrophoresis
Sodium dodecyl sulphate polyacrylamide gel electrophoresis
(SDS-PAGE) was carried out according to the method of
Laemmli using 12% cross-linked polyacrylamide gel. To
visualize CBP expression, both soluble and insoluble
fractions (20 µg) were loaded onto gel and molecular weight
was determined to be 90 kD by using reference molecular
weight marker (1 kD).The protein bands were visualized
on the gel by comassie blue staining.
3. Results and Discussion
Protein folding is an exquisitely optimized process that
leads to functional molecules under in vivo conditions,
despite physico-chemical factors that are quite challenging.
Not surprisingly, aggregation of newly synthesized proteins
emerges as a process that competes with in vivo folding. It
has also been observed that growth and production of foreign
proteins in the host cells is influenced by environmental
factors such as temperature. Especially in bacterial cells,
aggregation of partially folded intermediates manifests
itself in the production of insoluble inclusion bodies, which
may be mainly due to unstable folding intermediate of the
target protein at higher temperature and/or during over
expression of a gene.
3.1. Effect of temperature
With reference to CBP over-expression in the present
study, at a growth temperature of 23-30oC, 30~90% (Fig.
1) of the recombinant protein were in the soluble state.
Therefore, when the cells were grown and induced at 25oC,
higher activity was evident in soluble fraction as compared
to that obtained at 30 and 37oC, presumably due to rapid
accumulation of over-expressed protein at high temper-
atures (Fig. 1). Although, growth at temperatures below its
optimum level was substantially low, the fractions of the
functional over-expressed protein were enhanced (Fig. 2).
The solubilization of the over-expressed CBP was enhanced
Effect of Growth Temperature, Induction, and Molecular Chaperones on the Solubilization of Over-expressed Cellobiose… 275
nearly by a factor of two when cells were grown at 25oC
(Fig. 1).
3.2. Effect of IPTG induction
Different concentrations of isopropyl β-d-thiogalacto-
pyranoside (IPTG); 0.1-1.0 mM, were used as inducer to
induce the production of the enzyme cellobiose phosphor-
ylase by E. coli harboring plasmids for the target gene and
molecular chaperones. At 1.0 mM IPTG induction, the
enzyme activity in soluble fraction declined as compared to
induction with 0.1 mM IPTG, although its effect on growth
of host cells was not seen (Fig. 2).
3.3. Synergistic effect of IPTG induction and temper-
ature
At 37oC with 0.1 and 1.0 mM IPTG, although there was no
effect on the growth of cells, the enzyme activity decreased
significantly at 1.0 mM IPTG. The same trend of synergistic
effect was also observed at 25oC with 0.1 and 1.0 mM
IPTG. The SDS-PAGE patterns revealed that with increasing
time after induction, there was gradual increase of the
target protein in soluble fraction (Fig. 3).
3.4. Effect of molecular chaperones
Two different molecular chaperones; GroESL (pKY206)
and KODHsp (pACYC-cpk) were co-expressed with the
Fig. 1. Effect of temperature and IPTG induction on the expression
of cellobiose phosphorylase gene in E. coli (▲: 37oC, 0.1 mM; ---– ---37oC, 1 mM; ---■--- 30oC, 0.1 mM; ■: 30oC, 1 mM; −25oC,0.1 mM; and ●: 25oC, 1 mM).
Fig. 2. Effects of temperature and IPTG on growth of E. coli (▲:37oC, 0.1 mM; ---– --- 37oC, 1 mM; ---■--- 30oC, 0.1 mM; ■:30oC, 1 mM; −25oC, 0.1 mM; and ●: 25oC, 1 mM).
Fig. 3. SDS-PAGE profile of the expressed CBP in solublefraction at different hours after induction (Lane 1, Marker; Lane 2,1 h; Lane 3, 6 h; Lane 4, 9 h; Lane 5, 12 h; Lane 6, 24 h; and Lane7, Control (Pre-induction)).
Fig. 4. SDS-PAGE profile of the expressed CBP in response tomolecular chaperones. Lane 1: Marker; Lane 2: Soluble fraction,Control (0 h) with Chaperone; Lane 3: Soluble fraction, Control (0h) without Chaperone; Lane 4: Soluble fraction, 6 h withChaperone; Lane 5: Soluble fraction, 6 h without Chaperone; Lane6: Soluble fraction, 24 h with Chaperone; Lane 7: Insolublefraction, Control (0 h) without Chaperone; Lane 8: Insolublefraction Control (0 h) with Chaperone; Lane 9: Insoluble fraction,6 h without Chaperone; Lane 10: Insoluble fraction, 6 h withChaperone; Lane 11: Insoluble fraction, 24 h without Chaperone;and Lane 12: Insoluble fraction 24 h with Chaperone.
276 Biotechnology and Bioprocess Engineering 15: 273-276 (2010)
target CBP gene (pET). However, there was no effect of
these chaperones on the solubilization of over-expressed
CBP gene as reflected by SDS-PAGE patterns (Fig. 4).
In fact, on measuring the growth and enzyme activity in
the presence of molecular chaperone, it was observed that
although there was is no effect on growth, the enzyme
activity was marginally decreased (Data not shown).
Thus, molecular chaperone assisted protein folding that
had earlier yielded encouraging results [7-9], did not
improve the solubilization in the present case of CBP,
primarily due to the fact that the action of molecular
chaperones depend on the individual target protein.
On the other hand, the effect of growth temperature and
the level of induction, independently and in synergistic
manner, were pronounced on the solubilization of the
expressed enzyme. Expression of protein at a relatively
high temperature during cultivation has been earlier reported
to accelerate the formation of inclusion bodies [7,15]. This
trend might be explained on the ground that certain folding
intermediates of the target protein would be unstable at
higher temperatures under in vivo conditions and hence
the native conformation is not formed. In addition or
alternatively, the protein folding machinery might not be
able to keep pace with the high rate of protein synthesis.
In the event of failure of in vivo approaches for protein
solubilization, folding under in vitro conditions could be
attempted. Various methods of dialysis have proved to be
successful for renaturation of denatured proteins and towards
this end, a modified form of dialysis for the slow removal
of denaturant has been particularly attractive [12]. Dialysis,
a rapid dilution and a newly devised method of folding
immobilized proteins yields active enzyme [8,12]. Towards
this end, the application of an artificial chaperone appeared
as an effective tool in overcoming the folding problem of
over-expression of target proteins [14,15]. Overall, the
results described in the present report highlight on the
simple and inexpensive methods of getting over-expressed
recombinant enzymes in active form and hence it bears
significance from biotechnological stand point.
Acknowledgements SPS is grateful to Japan Research
Corporation (Previously, Japan Research & Development
Corporation), Government of Japan for STA Visiting
Scientist Fellowship, and JSPS Invitation Award. Research
collaboration with the National Food Research Institute,
Tsukuba Science City, Japan-305 is duly acknowledged.
References
1. Tanaka, K., K. Kawaguchi, T. Imada, T. Ooi, and M. Arai(1995) Purification and properties of cellobiose phosphorylasefrom Clostridium thermocellum. J. Ferment. Bioeng. 79: 212-216.
2. Liu, A., H. Tomita, H. Li, H. Miyaki, C. Aoyagi, S. Kaneko,and K. Hayashi (1998) Cloning, sequencing, and expression ofthe cellobiose phosphorylase gene of Cellvibrio gilvus. J.Ferment. Bioeng. 85: 511-513.
3. Ohdan, K., K. Fujii, M. Yanase, V. Takaha, and T. Kuriki (2007)Phosphorylase coupling as a tool to convert cellobiose intoamylose. J. Biotechnol. 127: 496-502.
4. Charoensakdi, R., S. Murakami, K. Aoki, V. Rimphanitchayakit,and T. Limpaseni (2006) Cloning and expression of cyclodextringlycosyltransferase Gene from Paenibacillus sp. T16 isolatedfrom hot spring soil in northern Thailand. J. Biochem. Mol. Bio.40: 333-340.
5. Nidetzky, B., R. Griessler, A. Schwarz, and B. Splechtna (2004)Cellobiose phosphorylase from Cellulomonas uda: gene cloningand expression in Escherichia coli, and application of therecombinant enzyme in a ‘glycosynthase-type’ reaction. J. Mol.Cata. B: Enzymatic 29: 241-248.
6. Ito, S., S. Hamada, K. Yamaguchi, S. Umene, H. Ito, H. Matsui,T. Ozawa, H. Taguchi, J. Watanabe, J. Wasaki, and S. Ito (2007)Cloning and sequencing of the cellobiose 2-epimerase genefrom an obligatory anaerobe, Ruminococcus albus. Biochem.Biophys. Res. Commun. 360: 640-645.
7. Machida, S., Y. Yu, S. P. Singh, J. D. Kim, K. Hayashi, and Y.Kawata (1998) Overproduction of β-glucosidase in active formby an Escherichia coli system coexpressing the chaperoninGroEL/ES. FEMS Microbiol. Lett. 159: 41-46.
8. Singh, S. P., J. D. Kim, S. Machida, and K. Hayashi (2002)Overexpression and protein folding of a chimeric β-glucosidaseconstructed from Agrobacterium tumefaciens and Cellvibriogilvus. Ind. J. Biochem. Biophys. 39: 235-239.
9. Kim, D., S. Singh, S. Machida, Y. Chika, Y. Kawata, and K.Hayashi (1998) Importance of five amino acid residues at C-terminal region for the folding and stability of β-Glucosidase ofCellvibrio gilvus. J. Ferment. Bioeng. 85: 433-435.
10. Machida, S., S. Ogawa, S. Xiaohua, T. Takaha, K. Fujii, and K.Hayashi (2000) Cycloamylose as an efficient artificial chaperonefor protein refolding. FEBS Lett. 486: 131-135.
11. Dodia, M. S., C. M. Rawal, H. G. Bhimani, R. H. Joshi, S. K.Khare, and S. P. Singh (2008) Purification and stabilitycharacteristics of an alkaline serine protease from a newlyisolated haloalkaliphilic bacterium sp. AH-6. J. Ind. Microbiol.Biotechnol. 35: 121-131.
12. Dodia, M. S., H. G. Bhimani, C. M. Rawal, R. H. Joshi, and S.P. Singh (2008) Salt dependent resistance against chemicaldenaturation of alkaline protease from a newly isolatedHaloalkaliphilic Bacillus sp. Bioresour. Technol. 99: 6223-6227.
13. Benech, R. O., X. Li, D. Patton, J. Powlowski, R. Storms, R.Bourbonnais, M. Paice, and A. Tsang (2007) Recombinantexpression, characterization, and pulp prebleaching property ofa Phanerochaete chrysosporium endo-β-1,4-mannanase. Enzy.Microb. Technol. 41: 740-747.
14. Maeda, Y., H. Koga, H. Yamada, T. Ueda, and T. Imoto (1995)Effective renaturation of reduced lysozyme by gentle removal ofurea. Protein Eng. 8: 201-205.
15. Maeda, Y., H. Koga, H. Yamada, T. Ueda, and T. Imoto (1996)Effect of additives on the renaturation of reduced lysozyme inthe presence of 4 M urea. Protein Eng. 9: 461-465.
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International Journal of Biological Macromolecules 49 (2011) 103–112
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules
journa l homepage: www.e lsev ier .com/ locate / i jb iomac
omparative analysis of enzymatic stability and amino acid sequences ofhermostable alkaline proteases from two haloalkaliphilic bacteria isolated fromoastal region of Gujarat, India
egha K. Purohit, Satya P. Singh ∗
epartment of Biosciences, Saurashtra University, Rajkot 360005, India
r t i c l e i n f o
rticle history:eceived 5 March 2011eceived in revised form 1 April 2011ccepted 4 April 2011vailable online 12 April 2011
eywords:
a b s t r a c t
Thermostable alkaline proteases from two haloalkaliphilic bacteria, Oceanobacillus iheyensis O.M.A18(EU680961) and Haloalkaliphilic bacterium O.M.E12 (EU680960) were studied for enzymatic propertiesand amino acid sequences in comparative manner. The bacteria were isolated from salt enriched soillocated in Okha, Coastal Gujarat, India. The unique aspect of the study was that alkaline protease fromHaloalkaliphilic bacterium O.M.A18 optimally catalyzed the reaction over a wide range of temperature,50–90 ◦C, with a half-life of 36 h at 90 ◦C. The molecular weights of O.M.A18 and O.M.E12 were 35 kDa
aloalkaliphilic bacterialkaline proteasesrotein purificationhermostability
and 25 kDa, respectively. The enzyme secretion was over the broader range of pH 8–11, with an optimumat 11. The alkaline proteases from the two haloalkaliphilic strains isolated from the same site reflectedquite different characteristics features. To the best of our knowledge, we have not come across with anysuch report on the thermal stability of alkaline proteases from haloalkaliphiles. Amino acid sequencesfor both enzymes were deduced from the nucleotide sequences of their corresponding genes followedby the analysis of physico-chemical properties of the enzymes.
. Introduction
Bacteria secrete variety of enzymes, many of them being com-ercially significant. Beside, the patterns of enzyme secretion and
haracteristics may also reflect on the population heterogeneity inparticular extreme habitat. Proteases constitute one of the most
mportant groups of industrial enzymes and account for about 60%f the total worldwide enzyme sales [1]. Bacteria display large vari-tions in optimum growth temperatures, often reflected in thermaltabilities of their extracellular enzymes. Over the years, Bacilluspecies have emerged as prolific producers of extracellular pro-eases with a potential for wide range of applications, in detergent,ood, pharmaceutical, leather and chemical industries [2–15].
Thermostable enzymes are of special interest for industrialpplications due to their stability under typical operation con-itions; such as high temperatures and wide pH range. Thehermophilic proteases catalyze the reaction and maintain the sta-ility at higher temperatures. In addition, higher temperatures
an accelerate the reaction rates, increase the solubility of non-aseous reactants and products and decrease the incidence oficrobial contamination by mesophilic organisms. Many ther-∗ Corresponding author. Tel.: +91 281 2586419; fax: +91 281 2586419.E-mail addresses: [email protected], [email protected]
S.P. Singh).
141-8130/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.ijbiomac.2011.04.001
© 2011 Elsevier B.V. All rights reserved.
mophiles, such as Bacillus stearothermophilus, Thermus aquaticus,Bacillus licheniformis, Bacillus pumilus and Thermoanaerobacter yon-seiensis, produce a variety of thermostable extracellular proteases[9,16–20]. It has been known that enzymes from thermophilic bac-teria are unusually thermostable, while possessing other propertiesidentical with enzymes found in mesophilic bacteria [21]. Usu-ally, alkaline proteases used in detergents are from thermophiles,having optimum temperatures between 50 and 70 ◦C [22–24], andstability in the range of 37–70 ◦C [25].
An alkaline serine protease from Bacillus sp. was highly ther-mostable and retained 100% activity at 60–70 ◦C for 350–400 min[23]. According to some reports, salt enhanced the thermostabilityof alkaline proteases. Similarly, Ca2+ and polyethylene glycol alsoplays a very important role in enhancing the temperature stabilityof the enzymes [10–13,26].
The thermostability of enzymes is understood to be a character-istic due to structure of the protein itself [27–31]. The sequencing,structure, and mutagenesis information accumulated during thelast 20 years have confirmed that hydrophobicity [28], hydrogenbond, ion pairs and hydrophobic interactions [32], decrease in theuncharged polar residues and increase in charged polar residues inthe polypeptide chain of the thermophilic protein contributed sig-
nificantly in protein stability at higher temperatures and solvents[33–36]. Now a days, enzyme producing industries use cloning andexpression as one of the approaches to obtain high quantity of bio-catalysts [26,37,38]. Protein engineering could be considered as
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04 M.K. Purohit, S.P. Singh / International Journa
ne of the important approaches to obtain improved biocatalysts.s an alternate to such modern but expensive and time consum-
ng techniques, exploration of microbial resources from extremenvironments can provide much needed biocatalytic platform [39].
While there are number of thermostable proteases reportedrom thermophilic organisms, similar citations from non-hermophilic organisms are quite rare [40]. Search for thermostablenzymes from other groups of extremophiles would be quitettractive in providing the biocatalysts with the abilities to func-ion under multitudes of non-conventional conditions. Towardshis end, we studied proteases from two haloalkaliphilic bac-eria; Haloalkaliphilic bacterium O.M.E12 (Gene bank AccessionU680960) and Oceanobacillus iheyensis O.M.A18 (Gene bankccession EU680961) isolated from a saline habitat in Coastalujarat (India). The study reflected on some unique proper-
ies of the alkaline proteases and we understand that it woulde among the rare citations where, although the optimumrowth of the organism is 37 ◦C, an extremely high optimumemperature range for protease catalysis was evident. Despitehe site of isolation being the same, properties of the alkalineroteases from the two haloalkaliphilic bacteria were quiteistinct.
. Materials and methods
.1. Isolation of Haloalkaliphilic organism
Haloalkaliphilic bacteria, O.M.A18 and O.M.E12 were isolatedrom salt-enriched soil collected from salt panes located in OkhaLatitude 22.20N, Longitude 70.05 E), Gujarat, India. One gram ofoil sample was suspended in sterile distilled water and transferredo 50 ml of enrichment medium which contained (g/l): NaCl, 300;lucose, 100; yeast extract, 50; peptone, 50; KH2P04, 50 in a 250 mlf flask. The inoculated medium was mixed homogeneously and pHas adjusted to 8 and 10 by adding separately autoclaved Na2CO3
20%, w/v). Cultures were incubated at 37 ◦C under shake flask con-itions at 200 rpm for 72 h followed by spreading it on agar plates7,11]. The well isolated colonies were selected and re-streaked tobtain pure cultures. The isolates were preserved on agar slants andn glycerol medium.
.2. Characterization of the organisms
Actively growing cultures were prepared in complete mediumroth (CMB) (NaCl, 10%, pH 8–10) and used as inoculums forhe primary screening of alkaline proteases. The cultures werenoculated as regular spots on gelatin agar medium that con-ained (g/l): gelatin, 30; peptone, 10; NaCl, 100–300; pH, 8–10nd agar, 30. The pH of the medium was adjusted to 8–10 bydding separately autoclaved 20% Na2CO3 (w/v). The plates werehen incubated at 37 ◦C for 48–72 h. After growth, Frazier’s reagentg/l: HgCl2, 150 g, concentrated HCl, 200 ml) was poured into thelates to detect gelatin utilization, where appearance of a clearone surrounding the colony indicated the presence of extracel-ular protease [3,11]. The ratio of zone of clearance to the colonyiameter was calculated to assess the relative enzyme secretions a function of colony size. O.M.A18 and O.M.E12 showing max-mum ratio were selected for further studies. The two isolates
ere characterized on the basis of 16S rRNA gene sequencingnd morphological features. The genomic DNA was used as tem-late for 16S rRNA amplification using consensus universal primer.
he PCR product was sequenced by using pair of forward, reversend internal primers. Sequence data was aligned and analyzed forlosest homologous bacteria by using Mega 4.0, Neighbor Joiningethod.ological Macromolecules 49 (2011) 103–112
2.3. Effect of NaCl, pH and temperature on growth and alkalineproteases secretion
The effect of NaCl, pH and temperature on the growth and alka-line protease secretion was studied by inoculating actively growingcultures as a spot on the gelatin agar medium in triplicate. For theeffect of salt, the medium was supplemented with varying concen-trations of NaCl (0–25%, w/v) at pH 9. The pH of the gelatin agarmedium was adjusted in the range of pH 8–11, at NaCl, 10% (w/v),by adding 20% Na2CO3 (w/v) as described above. The plates wereincubated for 48–72 h at 37 ◦C [4–6,10].
To assess the effect of temperature, the growth medium (10%NaCl and pH 10) after inoculation were incubated at 30–60 ◦C for48–72 h. The growth and enzyme secretion was monitored for theeffect of NaCl, pH and temperature. The effect of above factors ongrowth and enzyme secretion was also carried out in liquid cul-tures. To follow the effect of pH on growth and protease production,5% (v/v) inoculum of the activated cultures were added into theGelatin Broth (10%, w/v, NaCl) at varying pH 8–11 and incubatedat 37 ◦C under shake flask conditions. Similarly, the effect of NaClwas assessed in GB (pH 9) medium at NaCl concentrations, 0–25%.The cultures were grown as described above. Culture samples werewithdrawn aseptically and the growth was measured at A600. Cul-ture aliquots were then centrifuged at 10,000 rpm for 10 min at 4 ◦Cand the cell free extracts were used as crude enzyme preparation.
2.4. Enzyme production
The inoculums was prepared by adding a loop full of pure cul-ture into 50 ml sterile CMB medium (NaCl, 15% for O.M.E12 and 20%for O.M.A18 (w/v) at pH 10) and incubated at 37 ◦C on environmen-tal shaker for 24 h. From the activated cultures, 5 ml was inoculatedinto 100 ml gelatin broth and grown as described above. Sampleswere withdrawn and processed for the growth and protease esti-mation.
2.5. Protease and total protein estimation
Alkaline protease activity was measured by Anson–Hagihara’smethod [41]. The enzyme (0.5 ml) was added to 3.0 ml casein (0.6%,w/v in 20 mM NaOH–Borax buffer, pH 11) and the reaction mixturewas incubated at 37 ◦C for 20 min. The reaction was terminatedby the addition of 3.2 ml of TCA mixture (0.11 M trichloro aceticacid, 0.22 M sodium acetate, and 0.33 M acetic acid) and incubatedat room temperature for 20 min. The precipitates were removedby filtration through Whatman-1 filter paper and absorbance ofthe filtrate was measured at 280 nm. One unit of alkaline proteaseactivity was defined as the amount of enzyme liberating 1 �g oftyrosine per minute under assay conditions. Enzyme activity wasmeasured using tyrosine (0–100 �g) as standard. Protein was mea-sured by using Bradford’s method with bovine serum albumin asstandard protein.
2.6. Partial purification of proteases
The enzyme was partially purified by ammonium sulfate frac-tionation. The organism O.M.A18 and O.M.E12 were grown asdescribed above and after 72 h of growth, the cells were separatedby centrifugation at 6000 rpm, 4 ◦C for 15 min. The proteins in theculture supernatant were precipitated by ammonium sulfate (80%saturation, w/v) and the precipitate was suspended in the mini-
mum volume of 20 mM Borax-NaOH buffer (pH 10). The enzymeactivity and total protein were measured as described earlier. Afterdialysis against the same buffer, the dialysate were concentratedon sucrose bed and loaded on SDS–PAGE [12].
M.K. Purohit, S.P. Singh / International Journal of Biological Macromolecules 49 (2011) 103–112 105
Table 1Comparative profile on the growth, protease secretion and enzymatic characteristics of O.M.A18 and O.M.E12.
Features O.M.A18 O.M.E12
Site and isolationSite of isolation Okha, Gujarat, India Latitude 22.20N,
Longitude 70.05EOkha, Gujarat, India Latitude 22.20N,Longitude 70.05E
Enrichment conditions pH 8, NaCl 30% and temperature 37 ◦C pH 10, NaCl 30% and temperature 37 ◦CPhylogenetic identification Oceanobacillus iheyensis O.M.A18 –16 S
rRNA gene Sequence (Gene bankAccession No. EU680961)
Haloalkaliphilic bacterium O.M.E12-16 SrRNA gene Sequence (Gene bankAccession No. EU680960)
Growth and protease productionpH 11 (range 8–11) 11 (range 8–11)NaCl 15% (range 5–20%) 20% (range 5–20%)Temperature 37 ◦C (range 30–50 ◦C) 37 ◦C (range 30–50 ◦C)Molecular weight 35 kDa 29 kDa
Enzyme characteristics Optimum temperatures Range Optimum temperatures Range
Temperatures for enzyme catalysisCrude 90 ◦C 37–90 ◦C 50 ◦C 37–70 ◦CPartially purified 90 ◦C 37–90 ◦C 50 ◦C 37–70 ◦CDialyzed 60 ◦C 37–70 ◦C 50 ◦C 37–70 ◦CPurified 50 ◦C 37–50 ◦C 50 ◦C 37–50 ◦C
Enzyme characteristics Optimum pH Range Optimum pH Range
pH for enzyme catalysisCrude 11 9–11 11 9–11
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Partially purified 11Dialyzed 11Purified 11
.7. One step purification by hydrophobic interactionhromatography
Purification was achieved by a single step purification methodsing hydrophobic interaction chromatography. Hydrophobic
nteraction chromatography on a phenyl sepharose 6 fast flow col-mn (1 cm × 6.5 cm), equilibrated with 0.1 M sodium phosphateuffer (pH 8.0) containing 1 M ammonium sulfate, was performed.he crude protease preparation (20.0 ml in 1 M ammonium sulfate)as loaded onto this column and the bound enzyme was eluted
y 0.1 M sodium phosphate buffer, pH 8.0 containing decreasingmmonium sulfate (1000 mM, 500 mM, 200 mM and 0.1 M). Frac-ions at a flow rate of 0.7 ml min−1 were collected by BIO-RADraction collector (BIO-RAD, California, USA) and analyzed for pro-ease activity. The active fractions were pooled and used for furtherharacterization [11].
.8. SDS–polyacryalamide gel electrophoresis
Sodium dodecyl sulfate polyacrylamide gel electrophoresisSDS–PAGE) was carried out according to the method of Laemmlising 12% cross-linked polyacrylamide gel. To monitor crude, par-ially purified and purified fractions of enzyme preparations, theespective samples were loaded onto the gel. The status of puritynd molecular weight was determined with reference to moleculareight marker (Broad Range Marker, Merck Life sciences). The pro-
ein bands were visualized on the gel by Commassie blue staining.
.9. Effect of temperature on enzyme activity and stability
The enzymes were characterized to assess the effect of tem-erature on its activity and stability. The temperature profile wasetermined by following the activity at temperatures; 37–90 ◦C.
he thermal stability was studied by incubating the enzymes atifferent temperatures; 50, 80 and 90 ◦C for O.M.A18 and 50, 60,0 ◦C for O.M.E12. The aliquots of crude, partially purified and puri-ed enzyme preparations were withdrawn at regular intervals for9–11 11 9–119–11 11 9–119–11 11 9–11
72 h and the residual enzyme activities were measured at optimumtemperatures [11].
2.10. Effect of pH on protease activity
The effect of pH on protease activity was determined by prepar-ing the substrate in various buffers (20 mM) of different pH (8,9, 10 and 11) and reaction mixtures were incubated at optimumtemperature. The different buffers used for enzyme preparationswere; Tris–HCl (pH 8–9); NaOH–Borax (pH 10); Glycine–NaOH (pH11).The pH stability of the enzymes was studied by incubating it atdifferent pH for O.M.A18 and O.M.E12. The aliquots of crude, par-tially purified and purified enzyme preparations were withdrawnat regular intervals for 72 h and the enzyme activities were mea-sured at optimum conditions [12].
2.11. Denaturation of the enzyme
The effects of chemical denaturing agent, urea (8 M), on the par-tially purified and purified protease were studied. The enzyme wasincubated with urea at different temperatures; 70, 80 and 90 ◦C forO.M.A18; 50, 60 and 70 ◦C for O.M.E12. The enzyme mixture wasincubated at set conditions for 72 h followed by measurement ofthe residual activity to ascertain the loss of enzyme activity [11].
2.12. Effect of NaCl on the thermostability of proteases
In order to determine the influence of NaCl on thermostability,enzyme was incubated at different temperatures (37, 50 and 60 ◦C)with varying concentrations of NaCl (0.25–3 M). The aliquots werewithdrawn at definite time interval and the residual activities werecalculated.
2.13. Sequence prediction and analysis of the physicochemical
properties of proteasesPhylogenetic position and prediction of protein properties werecarried out by searching the National Center for Biotechnology
106 M.K. Purohit, S.P. Singh / International Journal of Biological Macromolecules 49 (2011) 103–112
F vity (-1 ).
IflWa
3
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ned7eearhileamp
ig. 1. (Upper panel) Effect of NaCl (5%, 10%, 15% and 20%) on growth (-�-) and acti0, 11) on growth (�) and activity (�) of O.M.A18 (left side) and O.M.E12 (right side
nformation NCBI (BLAST). The amino acid sequences of proteasesrom O.M.A18 and O.M.E12 were determined by Reverse trans-ating the gene sequence on the basis of correct CDS frame (CLC
orkbench). Further, physicochemical properties of protein werenalyzed by using protein server database—Expasy proteomics tool.
. Results and discussion
.1. Bacterial isolation, identification and characterization
The present study describes the isolation and phylogeny ofew haloalkaliphilic bacterial strains and characteristics of theirxtracellular proteases. After enrichment and isolation proce-ure, 27 moderate haloalkaliphiles were screened. Among them,
organisms were found having potential to secrete proteasextracellularly. On the basis of high specific activity and differ-nt characteristic feature, all detailed study was done on O.M.A18nd O.M.E12. For its phylogenetic determination, based on 16SRNA gene homology, the organisms were related to their nearestomologus and the sequences deposited in NCBI as Oceanobacillus
heyensis O.M.A18 (Gene bank Accession EU680961) and Haloalka-iphilic bacterium O.M.E12 (Gene bank Accession EU680960). The
ffect of salt, pH and temperature were assessed on the growthnd protease secretion. Besides, aspects of enzyme stability underultitude of extreme conditions were also investigated. The tworoteases from two different strains isolated from the same habi-
�-) of O.M.A18 (left side) and O.M.E12 (right side). (Lower panel) Effect of pH (8, 9,
tat reflected distinct patterns of catalysis at elevated temperature,thermostability and chemical denaturation. Both, O.M.A18 andO.M.E12, colony and cell morphology displayed Gram positive char-acteristics (Table 1).
3.2. Effect of NaCl, pH and temperature on growth and alkalineprotease production
Maximum protease production for both isolates was in the rangeof 5–20% (w/v), NaCl. Therefore, although optimum range of NaClwas quite broader they were unable to grow at 0% NaCl and the opti-mum salt concentrations for growth and production were 15% and20% for O.M.A18 and O.M.E12, respectively (Fig. 1). The pH profilesfor both isolates were quite broad and they were able to grow andsecrete protease at pH 8–11. However, the optimum pH for growthand enzyme secretion was 11 for both organisms (Fig. 1). The pHrange was quite higher than some of our earlier reports [10–12]and those reported in literature [3,15], where we have found thatoptimum pH is in the range of pH 8–9 [10–12]. Effect of tempera-ture was examined at 37, 50 and 60 ◦C. The organisms were able togrow and secrete enzyme efficiently at 37 ◦C, limited growth with-out enzyme secretion was evident at 50 ◦C. However, the organisms
were unable to grow at 60 ◦C. The trends demonstrated the haloal-kaliphilic nature of both strains as they were unable to grow in theabsence of salt, but could grow and secrete proteases efficiently athigh salt and alkaline pH (Table 1).
M.K. Purohit, S.P. Singh / International Journal of Biological Macromolecules 49 (2011) 103–112 107
Table 2AProtease purification from O.M.A18.
Enzyme preparations Activity unit (ml) Total units Protein (mg/ml) Total protein (mg) Specific activity (U/mg) Fold purification Yield (%)
Crude 293.85 146,925 0.3 150.25 978.68 1.0 100Partially purified fraction 2943.74 44,156.10 0.220 3.3 13,380.60 12.41 18.63One step purificationPhenyl Sepharose 6FF 3101 21707 0.183 1.281 16945.35 17.33 14.77
Table 2BProtease purification from O.M.E12.
Enzyme preparation Activity (U/ml) Total activity (U) Protein (mg/ml) Total protein (mg) Specific activity (U/mg) Purification fold Yield (%)
Crude 202.95 101,475 0.196 98 1035.45 1.0 1002.55 17,049.6 16.465 16.33
1.20 24,600.00 23.75 29.09
3
as1hw(
3c
f[yvaOprt
3
fip
FpOOpe
Partially purified fraction 2557.44 43,476.48 0.150One step purificationPhenyl Sepharose 6FF 5904.00 29,520 0.240
.3. Partial purification of alkaline proteases
O.M.A18 and O.M.E12 proteases were partially purified bymmonium sulfate fractionation. For, 500 ml of crude enzyme;pecific activity of O.M.E12 enzyme after partial purification was7049.6 with 16.5-fold purification and 16.30% yield. On the otherand, the specific activity of O.M.A18 enzyme was 13,380, 60ith fold purification and % yield of 12.40 and 18.60, respectively
Table 2A and 2B, Fig. 2).
.4. Enzyme purification by hydrophobic interactionhromatography
Hydrophobic interaction chromatography has been a success-ul technique for the purification of many alkaline proteases11,24,25]. The purification led to 17.30-fold purification with aield of 14.80% for O.M.A18 protease, while the correspondingalues for O.M.E12 were 23.75 and 29.10. The purification waschieved with specific activities of 16,945 and 24,600 units for.M.A18 and O.M.E12 proteases, respectively by single step onhenyl sepharose 6 FF affinity column (Table 2A and 2B). SDS–PAGEevealed that the molecular weights of O.M.A18 and O.M.E12 pro-eases were 35 and 29 kDa, respectively (Fig. 2).
.5. Effect of temperature on enzyme activity
The effect of temperature on the activity of crude, partially puri-ed, dialyzed and purified enzyme preparations was evaluated atH 10. The optimum temperature for enzyme catalysis by O.M.A18
ig. 2. Protein purifcation of haloalkaliphilic organism O.M.E12 and O.M.A18 byhenyl Sepharose 6FF. SDS–PAGE analysis of protein purifcation: Lane 1: Crudek.M.A18; lane 2: partially purified enzyme Ok.M.A18; lane 3: purified enzymek.M.A18; lane 4: protein molecular weight marker (3500–205,000 Da); lane 5:urified enzyme Ok.M.E12; lane 6: partially purified enzyme O.M.E12; lane 7: crudenzyme O.M.E12.
Fig. 3. Effect of temperature on enzyme catalysis of crude, partially purified andpurified alkaline protease sample of strain O.M.A18 ( ) and O.M.E12 (-�-).
1 l of Bi
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08 M.K. Purohit, S.P. Singh / International Journa
rotease was over a wide range, 50–90 ◦C, and a fact which relateso rare enzymes from mesophilic groups. Moreover, we have notome across with any protease having such a temperature pro-le from haloalkaliphilic organisms (Fig. 3). However, this rangef temperature profile was not displayed by dialyzed and purifiednzyme preparations. The dialyzed enzyme had optimum tempera-ure at 60 ◦C over a range of 37–70 ◦C and for purified enzyme; it was0 ◦C with a relatively narrow range of 37–50 ◦C. On the other hand,.M.E12 protease showed optimum temperature around 50 ◦C. The
ate of catalysis increased significantly from 37 to 50 ◦C, after whicht declined leading to a total loss of activity at 90 ◦C (Fig. 3). Enzyme
as able to maintain its activity in the range of 37–70 ◦, whichurned narrower with purified enzyme. The key point emerged;nzyme is highly thermostable before purified state and decreasedhe thermostability when purified. However, data holds noveltys very few reports are available where such unique characteris-ics are observed [10–12,22–26,32]. Along, the same line, resultsre equally interesting from diversity viewpoint due to its noveleatures; particularly with reference to moderate haloalkaliphiles.o give insight into its characteristic features; comparative studiesn the growth patterns, enzyme production and enzymatic char-cteristics, as depicted in Table 1, highlighted that although therganisms were isolated from the same site, they had distinct prop-rties. The foremost point which emerged from the study was theptimum rate of catalysis over a broader range of elevated temper-ture with significantly higher half-life of the enzyme.
.6. Thermostability of enzymes
The thermal stability of crude, partially purified and purifiedreparations of O.M.A18 enzyme were assessed for 48 h at 50–90 ◦Cnd pH 10. Similarly, thermal denaturation of O.M.E12 enzyme wasollowed at 50, 60 and 80 ◦C under similar conditions. The O.M.A18nzyme maintained its stability at temperatures; 60–90 ◦C for 24 h.owever, on extending the time of incubation to 72 h, less than5% of the original activity was retained (Fig. 4A.). Purified enzymeetained about 50% of the activity after 3 h of incubation at 60 ◦Cor OM.E12 (Table 3). A total loss in activity of O.M.E12 enzyme wasvident at all tested temperatures (50 ◦C, 60 ◦C, and 80 ◦C) whenncubated for 48 h (Fig. 4B). The thermal denaturation patterns of.M.E12 protease corresponds well with some of our earlier find-
ngs on haloalkaliphilic enzymes, where at 50 ◦C for 72 h, thereas a total loss in activity. Purified O.M.E12 enzyme maintained
round 25% of its activity for 3 h at 90 ◦C, while O.M.A18 enzymeetained 30% of its activity at 80 ◦C for 1 h. Thus, it was evidenthat the alkaline proteases from two strains isolated from the sameite displayed distinct characteristics in terms of their catalysis andtability at high temperatures. With particular reference to extra-ellular alkaline proteases, it is evident from the literature thathe optimum temperatures for enzyme catalysis exceed those forrowth and enzyme production. It is quite logical to suggest thathe stability of proteases could be due to their tertiary structuresnd genetic adaptability to carry out biological functions at higheremperatures. The high activity at 90 ◦C, probably may also be dueo the protection of the enzyme by substrate from heat inactivationnder the assay conditions.
The temperature response of O.M.E12 protease, on the otherand, resembled with the patterns of temperature profiles andhermal stability of other proteases, as reported in literature8,12,24,42]. The comparative data on the temperature stabilitynd denaturation profile of O.M.E12 and O.M.A18 alkaline proteasesuggested that OMA18 enzyme was extremely resistant against
hermal and urea denaturation. Chemical denaturation profile ofoth organisms indicated that the action of denaturant was saltndependent. However, no variation in the trend was observed inartially purified enzyme indicating that the other proteins might
ological Macromolecules 49 (2011) 103–112
not be playing a role in enzyme protection. Some of the previousstudies including our own have earlier established that denat-uration of certain enzymes from haloalkaliphilic organisms wassignificantly affected by NaCl and presence of other proteins [12].
3.7. Denaturation kinetics
The partially purified and purified proteases from O.M.A18 andO.M.E12 were subjected to urea denaturation. For partially puri-fied enzyme from O.M.A18 was exceptionally resistant against ureadenaturation and retained 30% activity at 90 ◦C after 48 h with 8 Murea, followed by nearly a total loss of activity on further incuba-tion to 72 h (Fig. 5A). The O.M.E12 enzyme, however, was relativelyless resistant to urea denaturation and retained 50% activity after24 h in 8 M urea, with a complete loss in activity at 72 h (Fig. 5B).Percent residual activity was related to zero hour enzyme activ-ity as 100%. To assess the effect of NaCl on urea denaturation, theabove studies were conducted with the dialyzed enzyme prepara-tions. The findings revealed that there was no significant changein the denaturation profile of crude, partially purified and dialyzedsamples (Fig. 5A and B). However, a change in the profile of purifiedenzyme was evident indicating enhanced sensitivity against denat-urant, with a loss of 80% activity after 2–3 h at 70 ◦C for O.M.E12,leading to a total loss of activity at 80 ◦C for the same period ofincubation (Fig. 5, Table 3).
3.8. Effect of NaCl on enzyme thermostability
Effect of NaCl on temperature profile was explored by incubatingthe reaction mixtures supplemented with various concentrationsof NaCl (0–3 M) at different temperatures; 37–80 ◦C. An interest-ing trend emerged, when NaCl was added to the reaction mixturesof both enzymes. The addition of NaCl led to the gradual shiftingin temperature optima towards higher temperatures with crude,partially purified and purified proteases (Fig. 6). As the optimumrange for protease catalysis were quiet broad, influence of NaClcould not be observed at lower temperatures, an observation alsosupported by our earlier report [12]. However, further increase inNaCl, resulted in a sharp decline in activity. It was also observedthat NaCl enhanced the activity coupled with the shift in temper-ature profile (Fig. 6). As compared to crude O.M.A18 enzyme, thepurified enzyme preparation had enhanced catalysis at higher con-centrations of salt. Thus, to retain its maximal activity at highertemperatures, the enzyme required higher concentrations of NaCl.In purified protease, the temperature optimum shifted from 60to 70 ◦C with 0.25 M NaCl and finally to 80 ◦C with 2–3 M NaCl(Fig. 6A). The requirement of NaCl increased from 0.5 to 2 M withthe increasing temperatures, displaying a maximum activity at 2 Mfor O.M.A18 at 80 ◦C (Fig. 6B). The maximal activity enhanced by 6folds from 37 to 80 ◦C with 2 M NaCl and remained stable up to 3 MNaCl (Fig. 6).
3.9. Amino acid sequence prediction and analysis of the enzyme
Protein similarity and phylogenetic analysis was carried outby using nBLASTp (prediction of protein sequence by submittingnucleotide sequence as a query) to identify the protein. PredictedN-terminal sequence for; Oceanobacillus iheyensis O.M.A18 was5′MNPGSAWRSPVVPFSSLGMSPAYG and that for Haloalkaliphilicbacterium O.M.E12 was 5′KLRVIIEFKEDAVEAGIQSTKQLMKK. A 100%homology of both the sequences was found with extracellularprotease sequence gene. Similarly, O.M.A18 showed complete sim-
ilarity with Bacillus sp. KP43. We have predicted physico-chemicalproperties of the native enzyme by using our nucleotide sequencesfor the two alkaline proteases. By submitting query sequenceto EXpasy protein database, we found the characteristic features
M.K. Purohit, S.P. Singh / International Journal of Biological Macromolecules 49 (2011) 103–112 109
F me a
T samp
oEba
ig. 4. (A) Thermostability of crude, partially purified, dialyzed and purified enzy
hermostability of crude, partially purified, dialyzed and purified alkaline protease
f the sequences closely resembled to nascent protease enzyme.stimated theoretical PI for both isolates was about 5. The insta-ility index (II) was computed as 27.30 and 39.57 for O.M.E12nd O.M.A18 proteases, respectively. On the basis of data, we
lkaline protease sample of strain O.M.A18 (50 ◦C (�); 60 ◦C ( ); 80 ◦C ( ). (B)
le of strain O.M.E12 (60 ◦C (�); 80 ◦C ( )); 90 ◦C ( )).
can predict that structure of enzyme is quite stable, a fact alsoreflected by our experimental data on thermal stability and resis-tance against chemical denaturation. Other properties, as aliphaticindex and grand average of hydropathicity (GRAVY) for O.M.E12
110 M.K. Purohit, S.P. Singh / International Journal of Biological Macromolecules 49 (2011) 103–112
Table 3Comparative profile on stability characteristics of O.M.A18 and O.M.E12.
Features O.M.A1 8 O.M.E12
Time required to retain 50% of the residual activity Time required to retain 50% of the residual activity
Thermostability of enzymeCrude 24 24Partially purified 48 48Dialyzed 48 48Purified 3 1
Features O.M.A18 O.M.E12
Time to retain 100% residual activity Temperature Time (h) to retain 100% residual activity Temperature
Chemical denaturation of the enzymesPartially purified 24 h 70 ◦C 48 h 50 ◦CDialyzed 24 h 80 ◦C 24 h 60 ◦CPurified 30 min 90 ◦C 1 h 70 ◦C
Fig. 5. (A) Effect of urea denaturation on alkaline protease O.M.A18 at different temperatures (70 ◦C (�); 80 ◦C (�); 90 ◦C (�)). (B) Effect of urea denaturation on alkaline
protease O.M.E1 2 at different temperatures (70 ◦C (�); 80 ◦C ( ); 90 ◦C ( )).
Table 4Sequence analysis of the recombinant alkaline proteases from O.M.A18 and O.M.E12.
Features O.M.A18 enzyme O.M.E12 enzyme
Basis informationN-terminal sequence 5′MNPGSAWRSPVVPFSSLGMSPAYG 5′KLRVIIEFKEDAVEAGIQSTKQLMKK. . .Original source (Sequence) Nucleotide NucleotideHomology (%) 100 100Homologus protein (BLAST analysis) Bacillus sp.KP43, complete CDS gene-protease gene Bacillus pumulius SAFA-032 -protease geneNCBI Genbank ID: HM219179 HM219182Physicochemical propertiespI 5 5Instability index (II) 39.57 27.30Stability Yes YesAliphatic index 65.60 42.94Grand average of hydropathicity (GRAVY) 0.016 −0.747Total numbers of negatively charged residues (Asp + Glu) 30 40Total numbers of positively charged residues 30 40
M.K. Purohit, S.P. Singh / International Journal of Biological Macromolecules 49 (2011) 103–112 111
F 0 ◦C (
t .
w−c(
pttbapocmaape
A
(
[
[
[
ig. 6. (A) Effect of NaCl on thermostability at different temperatures (37 ◦C (�); 5
emperatures (37 ◦C (�); 50 ◦C ( ); 60 ◦C ( )); 80 ◦C on alkaline protease O.M.E12
ere 65.60 and 0.016, while those for O.M.A18 and were 42.94 and0.747, respectively. Total numbers of negatively and positively
harged residues were∼30 and∼40, respectively for both proteasesTable 4).
The significance of the work relates to the fact that while alkalineroteases are extensively studied, only few haloalkaliphilic bac-eria have been explored towards this end [3,6,7,10–12]. Despitehe fact that the saline habitat in the study possessed significantacterial diversity, it remains unexplored in terms of its char-cterization, biocatalytic potential, enzymatic characteristics andhylogenetic status [43,44]. Moreover, some of the novel featuresf the enzymes, such as stability over the wide range of pH and salt,atalysis and thermostability of enzyme at higher temperaturesake them attractive candidates for future studies. The results are
lso important from the diversity viewpoint. Although both strainsre from the same site, they display distinct features of growth,rotease secretion and enzymatic characteristics, highlighting theircological significance.
cknowledgements
Ms. Megha Purohit is a recipient of Senior Research FellowshipSRF) sponsored by Council of Scientific and Industrial Research,
[
[[
); 60 ◦C ( )); 80 ◦C on alkaline protease O.M.A18. (B) Effect of NaCl at different
New Delhi, India (CSIR, New Delhi). The work was sponsored bySaurashtra University, Rajkot.
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Comparative studies on the extraction of metagenomic DNA from the salinehabitats of Coastal Gujarat and Sambhar Lake, Rajasthan (India) in prospect ofmolecular diversity and search for novel biocatalysts
P.K. Siddhapura, S. Vanparia, M.K. Purohit, S.P. Singh ∗
Department of Biosciences, Saurashtra University, Rajkot 360 005, Gujarat, India
a r t i c l e i n f o
Article history:Received 30 April 2010Received in revised form 4 June 2010Accepted 10 June 2010Available online 23 June 2010
Keywords:Saline habitatsEnvironmental DNAMetagenomics
a b s t r a c t
Extraction of total DNA from a given habitat assumes significance in metagenomics, due to the require-ment of inhibitor free and high quality metagenome in good quantity for applications in molecularbiology. DNA extraction and its quality assessment for PCR applications from saline soils of Coastal Gujaratand Sambhar Soda Lake, Rajasthan in India is described in a comparative manner. The mechanical andsoft lysis methods were simple and efficient for rapid isolation of PCR amplifiable total genomic DNA.The results are significant as only few extreme environments, particularly saline habitats are exploredfor their metagenomic potential.
© 2010 Elsevier B.V. All rights reserved.
1. Introduction
Current estimates indicate that approximately 99% of themicroorganisms in nature cannot be cultivated by standard tech-niques. Isolation and further applications of total genomic DNA(metagenomic DNA) from soil microorganisms without cultivation[1,2], is a recent approach in molecular biology [3–5]. Marine envi-ronment has enormous microbial biodiversity that remains largelyunexplored. The metagenomic approaches have highlighted thepopulation heterogeneity and phylogenetic status of a habitat, inentirety [6,7]. This novel approach has been further aided by bioin-formatics based software for analyses and interpretations [8–10].
Among the key factors for the successful metagenomics, the iso-lation of quality environmental DNA in appreciable amount from agiven habitat holds significance [10,11]. While the isolation of totalDNA is highly significant, it remains as one of the bottlenecks inmetagenomic studies [12,13], as the extracted DNA should be ofhigh quality in good yield to pursue molecular biological applica-tions [14–16].
During the last 10 years, number of protocols for DNA extractionfrom environmental sample is reported [17–19]. Some commercialsoil DNA extraction kits are also available [20–21]. These kits andmost of the published methods have improved the original DirectDNA extraction procedures mainly in terms of DNA yield and ease
∗ Corresponding author. Tel.: +91 281 2586419; fax: +91 281 2586419.E-mail addresses: [email protected], [email protected]
(S.P. Singh).
of application [22–24]. These protocols are broadly classified asdirect and indirect methods. The variability among the methodsis viewed on account of the degree of shearing, purity and yield ofDNA [25].
Direct DNA isolation methods involve cell lysis within the sam-ple matrix followed by the separation of DNA from cell debris[4,20]. However, in indirect methods, cells are extracted from theenvironmental sample before lytic release of DNA [14]. DirectDNA extraction protocols involve soft and harsh lysis methods.Soft lysis is based on the disruption of microorganism by enzy-matic and chemical means. The enzymatic lysis treatment relies onenzymatic digestion of microbial cells to release DNA [22,23]. Thetreatment of soil with surfactants and chelating agents resulted intoinhibitors free high quality DNA in good quantity, where as harshlysis approaches involve the mechanical cell disruption by beadbeating, sonication, freeze-thawing and grinding. The indirect DNAextraction protocols are based on blending and cation-exchange[26].
Standardization of total DNA extraction technique is desirableas the composition of different habitats varies with respect totheir matrix, organic and inorganic compounds and biotic factors[27–29]. Improved DNA extraction techniques should also ensurea metagenomic library adequately representing the entire commu-nity’s genome without inhibitory substances [30–32].
As an extension of our on-going research on haloalkaliphilicbacteria form Coastal Gujarat [33–35], the present study aims atthe optimization, assessment and comparison of the extractionmethods for total environmental DNA from two different habitats;saline soil near salt pan of Okha Madhi, Gujarat Coast and Samb-
0141-8130/$ – see front matter © 2010 Elsevier B.V. All rights reserved.doi:10.1016/j.ijbiomac.2010.06.004
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har Soda Lake, Rajasthan, both in India. The protocols describedin the present report signify the extraction of DNA from differentsaline habitats without any prior treatment. The methods provedwide amenability of the extracted DNA for further molecular biol-ogy applications, such as capturing functional genes from the totalhabitat and assessment of diversity and phylogeny in entirety.
2. Materials and methods
2.1. Environmental soil sampling and storage
Two soil samples, designated as Ok.M.3.6 and Ok.M.3.7 werecollected from Coastal region of Okha Madhi (Latitude 22.20 N, Lon-gitude 70.05 E) Gujarat and SL1.1 from Sambhar Lake (Rajasthan),India. They represent a typical saline soil with heavy depositionof salt with pH; 9–11. From the site of collection, a block of soilwas removed and transported to laboratory in sterile plastic bagsfor storage at 4 ◦C. Total DNA extraction and further analyses werecarried out from these samples within 7 days.
2.2. Direct DNA extraction methods
2.2.1. Soft lysis methodDNA extraction using lysis bufferSoil samples (1 g) in duplicate were suspended in 10 ml of
extraction buffer (100 mM Tris–HCl (pH 8.2); 100 mM EDTA (pH8); 1.5 M NaCl) and incubated at 37 ◦C for 10–12 h under shak-ing at 150 rpm. Samples were re-extracted in 1 ml of extractionbuffer and supernatant were collected by low speed centrifu-gation (5000 rpm) for 10 min. A 4 ml of Lysis buffer (20%, w/v)SDS; Lysozyme 20 mg/ml; ProtinaseK10 mg/ml; N-lauroyl sarco-sine 10 mg/ml; 1% (w/v) CTAB (cetyltrimethylammonium bromide)was added and incubated at 65 ◦C for 2 h with vigorous shaking atevery 15 min. Samples were centrifuged at 10,000 rpm for 10 min at4 ◦C. The upper aqueous phase was extracted with equal volume ofP:C:I phenol:chloroform:isoamylalcohol (25:24:1) at 1000 rpm for20 min at 4 ◦C. Upper aqueous phase was again extracted with equalvolume of chloroform:isoamylalcohol (C:I) (24:1) at 10,000 rpm for10 min at 4 ◦C. DNA preparation was further treated by adding 1/10volume of 7.5 M potassium acetate and subsequently precipitatedby adding 2 times of chilled ethanol. DNA precipitates were col-lected by centrifugation at 10,000 rpm for 10 min, air dried andsuspended in 20–50 �l sterile D/W.
2.2.2. Harsh lysis method
A. DNA extraction using bead beating methodSoil samples (1 g) in duplicate were suspended in 10 ml of
extraction buffer and incubated at 37 ◦C for 10–12 h with shak-ing at 150 rpm. Samples were re-extracted in 1 ml of extractionbuffer and supernatants were collected by low speed centrifuga-tion (5000 rpm) for 10 min. To the supernatants, glass beads (1 g)were added and the sample blended for 15 min followed by incuba-tion at 65 ◦C for 2 h. Samples were then centrifuged at 10,000 rpmfor 10 min at 4 ◦C. Further steps of the extractions were then con-tinued as per soft lysis method.
B. DNA extraction by sonication treatmentSoil samples (1 g) in duplicate were suspended in 10 ml of
extraction buffer and incubated at 37 ◦C for 10–12 h with shakingat 150 rpm. Samples were re-extracted in 1 ml of extraction bufferand the supernatants were collected by low speed centrifugation(5000 rpm) for 10 min. The supernatants were sonicated using ahigh intensity ultrasonic processor (Sartorious) with a standard13 mm horn solid probe for 3 pulses of 30 s each in a chilled ice bath.The sample was cooled in ice and repeatedly sonicated (6 cycles of
30 s) followed by incubation at 65 ◦C for 10 min. Samples were thencentrifuged at 10,000 rpm for 10 min at 4 ◦C. Further extraction wascontinued as per soft lysis method described above.
2.2.3. DNA extraction by combination of soft and harsh method
A. DNA extraction using bead beating combined with lysisbuffer treatment
Soil samples (1 g) in duplicate were suspended in 10 ml ofextraction buffer and incubated at 37 ◦C for 10–12 h with shaking at150 rpm. Samples were re-extracted in 1 ml of extraction buffer andsupernatant were collected by low speed centrifugation (5000 rpm)for 10 min. Glass beads (1 g) were added and the sample blendedfor 15 min. A 4 ml of lysis buffer was added and incubated at 65 ◦Cfor 2 h with vigorous intermittent shaking at 15 min intervals. Sam-ples were centrifuged at 10,000 rpm for 10 min at 4 ◦C. Extractionwas then continued as per soft lysis method.
B. DNA extraction by a combination of bead beating with soni-cation treatment
Soil samples (1 g) in duplicate were suspended in 10 ml ofextraction buffer and incubated at 37 ◦C for 10–12 h with shak-ing at 150 rpm. Samples were re-extracted in 1 ml of extractionbuffer and supernatants were collected by low speed centrifugation(5000 rpm; 10 min). The preparations were then blended with glassbeads (1 g) for 15 min followed by sonication using a high intensityultrasonic processor (Sartorius) with a standard 13 mm horn solidprobe for 3 pulses of 30 s each in a chilled ice bath. The sample wascooled in ice and sonicated repeated (6 cycles of 30 s) followed byincubation at 65 ◦C for 10 min. The preparations were centrifugedat 10,000 rpm for 10 min at 4 ◦C. The upper aqueous phase wasextracted with equal volume of P:C:I (25:24:1) at 10,000 rpm for20 min at 4 ◦C. Further extraction was continued as per soft lysismethod.
C. DNA extraction using sonication treatment combined withlysis buffer
Soil samples (1 g) in duplicate were suspended in 10 ml ofextraction buffer and incubated at 37 ◦C for 10–12 h with shaking at150 rpm. Samples were re-extracted in 1 ml of extraction buffer andsupernatant were collected by low speed centrifugation (5000 rpm)for 10 min. The supernatants were sonicated using an ultrasonicprocessor with similar conditions as described in above methods.Extraction was further continued from lysis buffer treatment as persoft lysis method.
2.3. Determination of purity and yield of DNA
Co-extracted humic acids are the major contaminant when DNAis extracted from soil. These compounds absorbs at 230 nm, DNA at260 and protein at 280 nm. To evaluate the purity of the extractedenvironmental DNA (eDNA), absorbance ratios at 260 nm/230 nm(DNA/humic acid) and 260 nm/280 nm (DNA/protein) were deter-mined. A high A260 to A230 ratio of greater than 2 indicates purityof DNA.
2.4. Gel electrophoresis
DNA extracts (10 �l) from each method were mixed with 5 �lloading buffer and analyzed on 0.8% agarose gels using TAE aselectrophoresis buffer. Gels were stained with ethidium bromideand analyzed by syngene gene genius Bio-imaging system. A DNAmarker of middle range from Banglo Genei, India was included ineach run.
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2.5. PCR amplification of 16S rRNA gene
DNA preparations from the saline soil were used as templateto amplify the region encoding 16S rRNA gene. To 100 ng of DNAas the template, 25 pmol of each Forward (5′-aga gtt tga tcc tgg ctcag-3′) and Reverse (5′-acg gct acc ttg tta cga ctt-3′) oligonucleotidesprimer (Imperial biosciences, India) [20] and 25 �l of 2× Red MixPlus (Invitrogen) were added. The amplification was carried out ingradient PCR thermocycler (eppendorf) a protocol of 30 cycles. Thesteps were: (1) initial denaturation at 94 ◦C for 1 min; (2) denatura-tion at 94 ◦C for 30 s; (3) gradient of annealing at 50–58 ◦C for 30 s;(4) extension at 72 ◦C for 2 min; (5) steps 2–4 were repeated for 29cycles; (6) final elongation at 72 ◦C for 2 min; (7) hold at 4 ◦C.
2.6. PCR amplification of alkaline protease gene
The DNA preparations obtained in the present study were usedas template to amplify region/s coding alkaline protease. Num-ber of primer sets was designed based on conserved sequences ofhaloalkaliphilic Bacillus species, by using multiple sequencing toolsfollowed by block generation using degenerate primer designingbioinformatics tool-CODEHOP [36]. For this process, the conservedresidues were taken into accounts which were selected on the basisof blocks generated by primer designing tool, on subjecting themultiple sequence alignment file of several already known alkalineproteases sequences from Halophilic/haloalkaliphilic organism. To100 ng of DNA as template, 25 pmol of each forward (5′-cat atgccg ccg agg agg ac-3′) and reverse (5′-gtc gac ggc ctt cgt gtg g-3′)oligonucleotides primers and 25 �l of 2× Red Mix Plus (Invitrogen)were added. The amplification steps consisted: (1) initial denatu-ration at 94 ◦C for 5 min; (2) denaturation at 94 ◦C for 1 min; (3)gradient of annealing at 60 ◦C with gradient of 8 ◦C for 45 s; (4)extension at 72 ◦C for 1 min; (5) steps 2–4 were repeated for 29cycles; (6) a final elongation was at 72 ◦C for 5 min; (7) hold at 4 ◦C.
3. Results and discussion
We developed an improved method for isolating total metage-nomic DNA from saline soil to obtain intact unsheared DNA,amenable to further molecular biology applications. The quality ofthe extracted DNA was assessed by PCR amplification of 16S rRNAregion and alkaline protease genes.
3.1. Spectrophotometric assessment for purity and yield of theextracted DNA
Total environmental DNA was isolated from Okha Madhi(3.6 and 3.7) and Sambhar Lake (SL1.1) using various meth-ods and assessed for purity and yield by following absorbanceratios at 260 nm/230 nm (DNA/humic acids) and 260 nm/280 nm(DNA/protein). High ratios indicated purity of the extracted DNA,whereas lower ratios pointed towards the contamination by pro-teins (280 nm) and humic acids (230 nm) (Table 1).
Spectrophotometeric assessment revealed that there were dif-ferences in purity and yield of the extracted DNA from the two sites.Comparative analysis indicated that the soft lysis method yieldedpure DNA from both samples. While bead beating method wasquite suitable for Gujarat site, it did not yield good concentrationof DNA from Rajasthan Soda Lake. Sonication was not suitable foreither of the samples, as the yield was quite low when compared toother methods. Combinations of above methods were encouragingas bead beating combined with lysis buffer treatment yielded pureDNA in good quantity as compared to bead beating and lysis buffermethods applied independently. On the other hand, sonication incombination with lysis buffer treatment did not emerge as a betteralternate, as compared to their independent outcomes (Table 1).
3.2. Visualization of total DNA on agarose gel
Total soil DNA extracted by different methods were furtheranalyzed on 0.8% agarose gel. Quality of the DNA obtained fromdifferent method varied depending upon the methods used for itsisolation (Fig. 1).
Results on the concentration and quality of the extracted DNA,as assessed by agarose gel electrophoresis, were in agreement withthe spectrophotometric analysis. Low concentrations of DNA werevisualized in preparations from sonication method from both sites(Okha Madhi and Sambhar Lake). Bead beating coupled with thelysis buffer treatment and lysis buffer treatment on its own gener-ated intense bands, a finding which corresponded with our earlierresults [20]. The results also indicated that the DNA was contam-inated with RNA or other compounds to varying degree. Over all,the humic acid concentarations in Sambhar Lake DNA preparationsobtained from all the methods were much higher, as evident fromlow ratio of A260/A230, as compared to Okha Madhi DNA. The co-purification of humic materials in soil was primarily due to itssimilar size and charge to DNA. Humic contaminants also inter-fere in DNA quantification since they exhibit absorbance at both,230 and 260 nm [10,11] (Fig. 1A and B). Extracted DNA from allthe preparations were of high molecular weigh indicative from itsposition which is mainly due to its metagenomic nature.
3.3. 16S rRNA gene amplification
Total DNA preparations extracted by chemical lysis methodfrom the samples of both sites were used as template for PCR ampli-fication of 16S rRNA gene. Amplification was successfully carriedout in all the gradient range of temperatures selected for annealingby gradient PCR (Figs. 2 and 3). Intense amplified bands at 1.5 kbfrom the saline soil; Ok.M.6.3 and Ok.M.6.5 were observed. How-ever, the intensity of amplicon varied at different Ta used for profilegeneration. Significant amplification was evident at annealing tem-peratures, 52.4 ◦C for Ok.M.6.3 and 54.7 ◦C for Ok.M.6.5. While othertemperatures were also satisfactory, the product concentrationsvaried. SL1.1 generated product size of 1.5 kb at temperatures; 52.4and 56.9 ◦C (Figs. 2 and 3).
Table 1Spectrophotometric assessment for purity and yield of total DNA.
S.No. Method Okha Madhi (Ok.M.3.6) Okha Madhi (Ok.M.3.7) Sambhar Lake (SL1.1)
A260/A230 A260/A280 Concentration(�g/ml)
A260/A230 A260/A280 Concentration(�g/ml)
A260/A230 A260/A280 Concentration(�g/ml)
1 Lysis buffer treatment 1.72 1.51 605 1.63 1.425 125 0.502 1.132 2002 Bead beating 1.57 1.30 160 1.53 1.015 75 0.782 1.132 109.73 Sonication 1.21 1.12 60 1.11 1.052 72 0.956 1.131 55.04 Beadbeating + sonication 1.75 1.18 60.72 1.10 1.167 120 0.682 1.242 207.005 Bead beating + lysis buffer treatment 1.76 1.14 735 1.25 1.074 370 0.652 1.200 120.006 Sonication + lysis buffer treatment 1.82 1.12 58 1.08 1.052 70 0.672 1.243 130.00
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Fig. 1. Isolation of total metagenomic DNA from sample by various methods. (A) Saline soil of Okha Madhi, Gujarat, India. Lane M: DNA ruler (middle range, Merk Life Science,India), Lane 1: Lysis buffer treatment (Ok.M.3.6), Lane 2: lysis buffer treatment (Ok.M.3.6), Lane 3: bead beating only (Ok.M.3.6), Lane 4: bead beating (Ok.M.3.6), Lane 5: beadbeating + lysis buffer treatment (Ok.M.3.6), Lane 6: bead beating + lysis buffer treatment (sample-3.6), Lane 7: bead beating + sonication treatment (Ok.M.3.7), Lane 8: beadbeating + sonication treatment (Ok.M.3.7), Lane 9: lysis buffer + sonication treatment (Ok.M.3.7), Lane 10: lysis buffer + sonication treatment (Ok.M.3.7), Lane 11: sonicationtreatment (Ok.M.3.7), Lane 12: sonication treatment (Ok.M.3.7). (B) Sambhar Soda Lake, Rajasthan, India. Lane M: Lamda DNA/HindIII Marker (Merk Life Science, India), Lane1: lysis buffer treatment (SL1.1), Lane 2: bead beating only (sample-SL1.1), Lane 3: bead beating + lysis (SL1.1), Lane 4: sonication + lysis (SL1.1), Lane 5: sonication (SL1.1),lane 6: sonication + bead beating (SL1.1).
Fig. 2. 16S rRNA amplification profile from DNA extracted by Soft Lysis method from different samples (Ok.M.3.7, Ok.M.3.7 and SL1.1). (A) M: DNA ruler (middle range,Merk Life Science, India), Lane 1: lysis buffer treatment (Ok.M.3.6, Ta 52.4◦C), Lane 2: lysis buffer treatment (Ok.M.3.6, Ta 54.7 ◦C), Lane 3: lysis buffer treatment (Ok.M.3.6,Ta 56.9), Lane 4: lysis buffer treatment (Ok.M.3.7, Ta 52.4 ◦C), Lane 5: lysis buffer treatment (Ok.M.3.7, Ta 54.7 ◦C), Lane 6: lysis buffer treatment (Ok.M.3.6, Ta 56.9), Lane7: bead beating + sonication treatment (Ok.M.3.6), Lane 8: bead beating + sonication treatment (Ok.M.3.7), Lane 9: lysis buffer + sonication treatment (Ok.M.3.6), Lane 10:lysis buffer + sonication treatment (Ok.M.3.7), Lane 11: sonication treatment (Ok.M.3.6), Lane 12: sonication treatment (Ok.M.3.7). (B) M: DNA ruler (middle range, Merk LifeScience, India), Lane 1: lysis buffer treatment (SL1.1, Ta 52.4 ◦C), Lane 2: lysis buffer treatment (SL1.1, Ta 54.7 ◦C), Lane 3: lysis buffer treatment (SL1.1, Ta 56.9).
3.4. Alkaline protease gene amplification
Total DNA extracted by chemical lysis method from the sampleof Okha Madhi (Ok.M.3.6 and 3.7) and total DNA extracted by bead
Fig. 3. Graph depicting amplification profile of 16S rRNA by using soft lysis methodfrom Okha Madhi soil samples (Ok.M.3.6 andOk.M.3.7) and Sambhar Soda Lake(SL1.1).
beating plus lysis buffer treatment from the Sambhar Lake sample(SL1.1) were used as template for alkaline protease gene amplifi-cation. Selection of template for amplification was based on theyield and purity of the DNA preparations. Three amplicons, withproduct size ranging from 1.5 to 0.8 bp, were visualized on agarosegel from Ok.M.3.6 and 3.7. The amplification profile was quitesimilar with Okha Madhi sample, a fact which indicated towardsthe similar distribution of organism producing alkaline proteaseswithin a particular habitat. However, from Sambhar Lake salinesoil, with the same amplification protocol, a single band of 1.2 kbwas generated. While one of the reasons for the multiple bandsfrom the Okha Madhi site could be due to the annealing of theprimer at different sites within the template, the question wouldbe better addressed by sequencing of the corresponding amplicons(Figs. 4 and 5).
In brief, a combination of mild bead lysis and enzymaticlysis buffer treatment and lysis buffer treatment on its ownwere most successful approaches in recovering higher yields andinhibitor free DNA, compatible with PCR reactions, from samplesof Ok.M.3.6 and 3.7 sites of Okha Madhi. However, for Samb-har Lake sample (SL1.1), the bead beating in combination withlysis buffer treatment and lysis buffer treatment on its own, con-sistently led to the higher yields of the extracted DNA [17,19](Figs. 4 and 5).
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Fig. 4. Alkaline protease amplification profile for soil sample using soft lysis methodfrom Okha Madhi soil samples (Ok.M.3.6 andOk.M.3.7) and bead beating + lysisSambhar Soda Lake (SL. 1). M: DNA ruler (middle range, Merk Life Science, India),Lane 1: lysis buffer treatment (Ok.M.3.6, Primer pair, SPS-7), Lane 2: lysis buffertreatment (Ok.M.3.7, Primer pair, SPS-7), Lane 3: bead beating + lysis buffer treat-ment (SL1.1, Primer pair, SPS-7).
Fig. 5. Amplification profile of alkaline proteases by using soft lysis method fromOkha Madhi soil samples (Ok.M.3.6 and Ok.M.3.7, by using SPS-7 primer) and Samb-har Soda Lake (SL1.1, by using SPS-1 primer).
Isolated DNA was subjected to 16S rRNA and alkaline proteasegene amplification, which proved it suitability for sequence basedand functional metagenomic approaches confirming its amenabil-ity for molecular diversity and retrieval of gene/s of specificenzymes.
The methods explored and developed for the extraction oftotal DNA from saline soil in the present study are rather simpleand do not require any specific sample treatment. Therefore, theapproaches could also be applicable to other saline habitats withsimilar physico-chemical conditions.
The DNA extraction methods have resulted in rapid performanceof the molecular techniques, avoiding extensive purification steps.The described methods could allow the use of large-scale prepa-rations providing greater probability of detecting genes present inlow abundance in soil. These methods could be applicable to morecontaminated soils leading to assessment of microbial diversity andretrieval of useful sequences.
Acknowledgement
We acknowledge the financial assistance from the SaurashtraUniversity, Rajkot for carrying out the work.
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Volume 2 • Issue 1 • 1000116J Bioremed Biodegrad ISSN: 2155-6199 JBRBD, an open access journal
Research Article Open Access
Surani et al. J Bioremed Biodegrad 2011, 2:1http://dx.doi.org/10.4172/2155-6199.1000116
Research Article Open Access
Bioremediation & Biodegradation
Keywords: PAHs; Biodegradation; Database; PAHbase
Introduction Microbial population is a highly diverse and a ubiquitous group
among the living world. One of the novel features of the microbes relates to their versatility in utilizing a large numbers of natural and manmade compounds. This property proves highly valuable in bioremediation for the complete destruction and removal of pollutants [1]. Contamination of soils and sediments by Polycyclic Aromatic Hydrocarbons (PAHs) is widespread, which raises enormous environmental concerns. It has been observed that PAH degradation in soil is dominated by bacterial strains belonging to a very limited number of taxonomic groups such as Sphingomonads, Burkholderia, Pseudomonas, Bacillus, Micrococcus and Mycobacterium [2-9] Members of these genera are specialized in the degradation of aromatic chemicals [10,11]. As such, bioremediation may provide relatively low-cost and less intensified technology with high public acceptance.
Bioinformatics based analysis and prediction is playing a pivotal role in understanding and capturing the in-depth knowledge of biological molecules particularly with reference to proteomics and genomics. Although with this advancement, there have been only limited efforts on the collection of all relevant information for a specific field of interest. With this realization, present study focuses on the wide spread data and information related to the occurrence and potential of PAH degrading bacteria. The information and detailed account on these bacteria are quite limited and scattered in scientific journals. Therefore, details from the research papers were extracted, analyzed and presented in form of a precise informative database: PAHbase reflecting the diversity and functional analysis of PAHs degrading bacteria.
MethodologyPAHbase contains information regarding PAHs degrading
bacteria with respect to morphology, Gram reaction, degradation potential, metabolic pathways and genetic basis. Backhand database of PAHbase was created in MS Access and front end was done by PHP: Hypertext Preprocessor which provided the easy web access to database for data entry, retrieval and analysis. Data was collected and extracted from original research publications and public databases, i.e. NCBI, DDBJ and EMBL.
Data input
In PAHbase, we selected 45 PAHs degrading bacteria and integrated
detailed information comprising multiple fields of interest; such as name of the organism, habitat, site of isolation, Gram’s reaction, activity, country, extremophilic nature (Halophilic/Thermophilic/Mesophilic), taxonomy and phylogenetic relatedness with nearby species, preferred PAH source of utilization as carbon source, media used in laboratory to access its potential for respective PAH, physical, chemical and environmental conditions provided for degradation, degradation potential, enzymes involved in degradation, gene/s involved and gene location, metabolic pathway,16S ribosomal gene sequence and references.
Data retrieval
PAHbase is a freely accessed web database constructed using PHP on windows platform. “PAHbase” is the database which gives user friendly search criteria and also provides easy access, retrieval and manipulation with secure administrator and users.
Results and DiscussionPAHbase creation
The PAHbase database was constructed primarily in Access 2007 as back hand and exported to PHP as front hand with MySQL for the easy access and portability. Considerable efforts have added to the field of environmental biotechnology with reference to PAHs degrading bacteria. Over the years our group has focused on capturing diversity and degradation aspects of PAHs degrading bacteria and as an extension of our current research work, we constructed a web driven database system for PAHs degraders to focus on their different aspects. This data base would be of immense help for scientific fraternity conducting their research on PAHs degrading bacteria.
*Corresponding author: Satya P. Singh, Department of Biosciences, Saurashtra University, Rajkot, Gujarat, India-360 005, E-mail: [email protected], [email protected]
Received December 22, 2011; Accepted January 30, 2011; Published February 02, 2011
Citation: Surani JJ, Akbari VG, Purohit MK, Singh SP (2011) Pahbase, a Freely Available Functional Database of Polycyclic Aromatic Hydrocarbons (Pahs) Degrading Bacteria. J Bioremed Biodegrad 2:116. doi:10.4172/2155-6199.1000116
Copyright: © 2011 Surani JJ, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
AbstractPAHbase is a freely available functional database of Polycyclic Aromatic Hydrocarbons (PAHs) degrading
bacteria. The database consists of relevant information obtained from scientific literature and databases. The database provides a comprehensible representation of PAH degrading bacteria with reference to its occurrence, phylogeny, and stress adaptation, potential to withstand extreme conditions, biodegradative ability, metabolic pathways and genetic basis of the degradation. The narrow search and limit options of the constructed database provide comparable information from the relevant PAH degrading candidates. The user friendly approach of PHP front end facilitates to add sequences of reported entries leading to scientific information for the specific purpose. The functional PAH database available freely on internet under URL: www.pahbase.in.
Pahbase, a Freely Available Functional Database of Polycyclic Aromatic Hydrocarbons (Pahs) Degrading BacteriaJaimin J. Surani, Viral G. Akbari, Megha K. Purohit and Satya P. Singh*
Department of Biosciences, Saurashtra University, Rajkot, Gujarat, India-360 005
Citation: Surani JJ, Akbari VG, Purohit MK, Singh SP (2011) Pahbase, a Freely Available Functional Database of Polycyclic Aromatic Hydrocarbons (Pahs) Degrading Bacteria. J Bioremed Biodegrad 2:116. doi:10.4172/2155-6199.1000116
Volume 2 • Issue 1 • 1000116J Bioremed Biodegrad ISSN: 2155-6199 JBRBD, an open access journal
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‘PAHbase’ provides platform for the easy access and retrieval of data from the database (Figure 1). To the best of our knowledge, this would be the first report on the submission of specialized database for PAHs degrading organisms. The functionality to add and edit data to the database through a user friendly web based portal facilitates to update and maintain database system. All information required by the researchers for the study of PAHs degrading bacteria including basic microbiological features, ecological aspects, PAHs substrate specificity and molecular biological studies relevant to the source organisms were thrust area for database construction. Retrieval and analyzing all important parameters under a single system assures wide applicability of constructed PAHbase.Acknowledgement
We gratefully acknowledge the financial and other logistic support from Saurashtra University, Rajkot, India. VGA and MKP are the recipients of Meritorious Research Fellowship, UGC, New Delhi and Senior Research Fellowship from CSIR, New Delhi, respectively.
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Figure 1: Screen shot of PAHbase for Polycyclic Aromatic Hydrocarbons degrading bacteria database develop for the diversity, PAH degradation, substrate specificity, metabolic pathway and genetic base.
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