DEPARTMENT OF MICROBIOLOGYTHE OXFORD COLLEGE OF SCIENCE

(Accredited ‘A’ grade by NAAC, Accredited by IAO)

Final Report of the Work Done on the Minor Research Project

On

“EXPLORATION AND EXPLOITATION OF NOVEL STRAINS FOR THE PRODUCTION OF L-GLUTAMINASE- AN ANTI-TUMOR AGENT”

Submitted to:

University Grant CommissionSouth Western Regional Office

Prasanna Kumar BlockPalace Road

Bangalore-560 001

Principal InvestigatorDr. Pramod.T. M.Sc. Ph.D

Associate Professor,Department of Microbiology,The Oxford College of Science

2015-17

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CERTIFICATE

This is to certify that the research work entitled “Exploration and Exploitation of Novel strains for the production of L-Glutaminase-An antitumor Agent” has been carried out at the Department of Microbiology, “The Oxford College of Science” during the period 2015-17 awarded by the University Grants Commission, South Western Regional Office, Bangalore via sanction order No. 1399-MRP/14-15/KABA 108/UGC-SWRO/2014-15, dated 10th December 2014.

Principal

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Serial Number

Title

Page Number

1 Introduction 1-4

2 Review of Literature 5-15

3 Materials And Methods 16-22

4 Results and Discussion23-43

5 Summary 44

7 References 45-51

CONTENTS

ACKNOWLEDGEMENT3

At the outset I would like to express my gratitude to the University Grants Commission, South Western Regional Office, Bangalore for funding to carry out the research project.

I would like to thank The Chairman, of The Oxford College of Science, for providing the necessary infrastructure facilities to carry out this Project.

I thank Dr. Kavyashree, Principal, The Oxford College of Science, Bangalore for her support and encouragement.

I am also greatly thankful to Dr. Bharathi.S., The Head, Department of Microbiology for her suggestions, support and encouragement.

I would also like to thank the Purchase Committee, The Oxford College of Science for their timely help during the project tenure.

My thanks to all my colleagues in the Department of Microbiology, The Oxford College of Science, for their help and co-operation during the project tenure, without which this project would not have been successfully completed.

I thank our Lab Assistant, Mr. Ramana Reddy for his help during this work.

I would lastly thank my students for their help and cooperation during the research work.

I would like to take this opportunity to express my gratitude to all the people who have directly or indirectly helped me in this project period.

Dr. Pramod.T

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INTRODUCTION

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1.0 INTRODUCTION

Cancer, a disease which has enormously increased over the last few decades, attacking ten of

millions of people all over the world irrespective of gender, age or color. The reason for its high

incidence rates may be both internal (genetic, mutations, hormonal, poor immune conditions etc)

and external or environmental factors (food habits, industrialization, over population etc).

Cancer is actually a general term that describes a large group of related diseases. Cancer may

affect people at all ages, even fetuses, but risk for the more common varieties tends to increase

with age. Cancer causes about 13% of all deaths (WHO 2006). Every case of cancer is unique,

with its own set of genetic changes and growth properties. Some cancers grow quickly while

others can take years to become dangerous to the patient. These many differences between cases

of cancer, even of the same organ (i.e. different cases of breast cancer), is one of the main

reasons that treatment is so difficult. But we must at the same time be thankful that the treatment

of the different types of cancer has also dramatically improved and hundreds of thousands lives

have been saved by the correct treatment.

In 2000, cancer accounted for over 7 million deaths (135 of total mortality) and there were

more than 10 million new cancer cases worldwide in 2000. More than 60% of cancer deaths and

approximately half of new cases occurred in developing regions. Therapeutic enzymes are

gaining importance mainly because of their broad variety of specific uses such as oncolytics,

thrombolytics or anticoagulants and as replacements for metabolic deficiencies (Prakasham et

al., 2006). Industries based on biotechnology are harnessing the metabolic activities of

microbes, plants and animal cells to produce a wide variety of diverse compounds which are

used by industries such as chemical, food, pharmaceutical and health-care. The application of

enzyme technologies to pharmaceutical research, development and manufacturing is a growing

field and is the subject of interest. The concept of therapeutic enzymes has been around for at

least 40 years.

One of the prime candidates in the treatment of debilitating human cancers includes a family

of significant enzymes referred to as L-glutaminases. L-glutaminase (L-glutamine

amidohydrolase E.C 3.5.1.2) is the enzyme that catalyzes the deamidation of L-glutamine to L-

glutamic acid and ammonia (Balagurunathan, R et al., 2010), (Roberts, J et al., 1970).This is an

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essential enzyme for the synthesis of various nitrogenous metabolic intermediates. Tumor cells

have an absolute requirement for glutamine as a growth substrate. Glutamine is required as a

precursor for both DNA synthesis and protein synthesis and may also be used as a respiratory

substrate.

As the demand of L-glutaminase is increasing due of its vast applications in various

industrial sectors therefore, it is necessary to produce L-glutaminase to reach its demand in

various fields. There are several factors like search for new isolate with high production

capacity, media designing and optimization for maximum production or genetic manipulation to

achieve a hyperproducing microorganism etc. Interest on amidohydrolases started with the

discovery of their antitumor properties (Broome, 1961), (El-Asmar and Greenberg, 1966)

Santana et al., 1968;Roberts et al., 1970) and since then, a lot of efforts have gone into extensive

studies on microbial L-glutaminases with the intention of developing them as antitumor agents.

Glutamine Synthetase

L-glutamate + NH3 + ATP L—glutamine+ ADP + Pi

Glutaminases

L—glutamine + H20 L-glutamic acid + NH3

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The research work on L-glutaminase enzyme was initially started in the year 1956. The

importance of L-glutaminase enzyme was accidently found when Alexander B. Gutman and

Tsai-Fan (1963) were working on the measurement of the total uric acid-N15 enrichment and

the N15 abundance of each of the four uric acid enrichment and the N15 abundance of each of

the four uric acid nitrogen’s, found that an abnormality of glutamine metabolism in primary gout

is implied. It is suggested that the abnormality in glutamine metabolism may have significance

for the pathogenesis of primary gout. Thus, from then the study on this enzyme was focused.

1.1 AIM OF THE PRESENT STUDY

The aim of the present study is to evaluate the potential of Bacillus subtilis subsp.inaquosorum

for L-glutaminase production using coconut cake extract under submerged fermentation.

Groundnut cake is the residue obtained after the extraction of oil and it is high in protein and

used as supplement in feed and food. The groundnut cake is usually fried in oil and is used as a

delicious snacks or food supplement. The decorticated cake and meal are safe feeds for all

classes of livestock because of their good protein balance. For mature ruminants there are no

restrictions on the use of groundnut cake. The high fiber content of undecorticated cake makes it

a useful corrective for cattle feeding on grass that is low in fiber. Groundnut oil cake is a cheap

and locally available substrate. This is the first report on the utilization of groundnut oil cake as

a substrate/medium for the production of L-glutaminase is scanty. The present work suggests

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that this process can be exploited as an alternative to the other processes, as this is cost effective

and economically viable.

1.2 OBJECTIVES OF THE STUDY

The present study was undertaken with the following objectives:

1. Isolation and screening of novel strains for the production of L-glutaminase.

2. Screening of strains for L-glutaminase production.

3. Screening of agro-residues as substrates for the production of L-glutaminase.

4. Optimization of process parameters like initial moisture content, pH, incubation

temperature and inoculum size for L-glutaminase production under submerged

fermentation.

5. Process economization by supplementation of carbon and nitrogen sources.

6. Partial purification and characterization of L-glutaminase.

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REVIEW OF LITERATURE

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2.1. ENZYMES

Most of the specific biochemical reaction that takes place within cell for its growth repair and

development involves the catalyst called enzyme. Enzymes are proteins and highly specific on

their actions on substrates and serves as biocatalyst. These organic proteinaceous catalysts exist

throughout the nature and produced by living things only, hence the name biocatalyst. In a

biochemical reaction enzymes act on substrates and speed up reaction by lowering the activation

energy, the whole process is called as catalysis.

2.2. BIOPROCESS OF ENZYME PRODUCTION

Enzymes occur in every living cell, hence in microorganisms. The presence of a particular or

specific enzyme in microorganisms may vary with the type of microbes (Bacteria, Fungi, Yeast

and Actinomycetes), their genus, species and strain, niche and the available nutrients. The first

projection of employing microbes in industry for the production of enzymes goes back to the

year 1914 wherein Jakichi Thakamine (1914) has elucidated that, particular enzymes generated

by the genus Aspergillacea during its growth have significant application in fermentation

industries. Further he reported about few applications of the enzymes produced by Aspergillus

oryzae in food and beverage industries, but he was mainly concerned with fungal enzymes. In

the year 1917, exploitation of bacteria for enzyme production was pioneered in France (Boidin

and Effront, 1917) and in next two decades, the technological progresses in this particular field

has been so great that, so many microbial cultivated enzymes have replaced by animal or plant

enzymes (Underkofler et al., 1958). In continuation to this, until 1980 there were several

advanced and feasible methods that were developed and employed for the production of

microbial enzymes. The microbial genera, Bacillus and Aspergillus were most reliable for the

production of enzymes at industrial scale during that period. In that era, microbial fermentation

methods such as solid state fermentation and submerged fermentation were considerably

developed along with strain improvement techniques to enable the good yield of enzymes. Apart

from this, enzyme recovery and purification techniques were improved and became very useful

in enzyme producing industries (Lambert and Meers, 1983). By the time, attempts were done for

the production of quite a lot of enzymes of industrial, environmental and pharmaceutical

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importance from various microbial sources employing different fermentation methods and

cultivation conditions (Jen-Kuo Yang et al., 2000; Rajeev Kumar and Chandrasekaran,

2003;Vidyalaxmi et al., 2009; Saleem Basha et al., 2009; Soniyamby et al., 2011).

Bioprocess technology has provided an unlimited and pure source of enzymes as an

alternative to the harsh chemicals traditionally used in industry for accelerating chemical

reactions. The enzyme market is a significant market estimated at € 3.4 billion with an annual

growth of about 6.5 to 10% (pharmaceutical enzymes excluded). Products, such as amylase

(Maltogenase) and lipase (Lipolase) were first biotech enzymes marketed in the 80's. And while

enzymes are present everywhere and comes in thousands of different forms, it is still only about

25 of them, which have actually been industrialized and commercialized. Some of them are

amylase, lipase, cellulase, glycomylase, glucose isomerase, chymosin, lactase, pullulanase,

xylanase, protease and so on (http://www.lifescience-online.com/article.htm).

2.3. METHODS OF ENZYME PRODUCTION

Enzymes being biocatalysts produced by plants, animals and microorganisms, but the microbial

populations were the most exploited and being exploited sources of the enzymes, especially of

commercial importance. For thousands of years natural enzymes produced by microorganisms

have been used to make products such as cheese, bread and alcoholic products like wine and

beer. Microbes are still the most common sources of industrial enzymes, this is because, it is

easy to cultivate microbes in controlled environment at laboratory, their rapid growth and

reproducibility, the well advanced methods and techniques for enzyme recovery and purification

from microorganisms. There are several industrial fermentation processes and conditions, where

microbes are used to produce enzymes which have well defined uses and application in various

fields. These microbial fermentation methods have been broadly classified into two types, firstly

Solid State Fermentation (SSF) and secondly Submerged Fermentation (SmF). The adaptation

and suitability of specific fermentation method i.e., SSF or SmF may vary with type of

microorganism, its nature, growth conditions, media provided and the desired end product.

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2.3.1. Solid State Fermentation (SSF)

Solid State Fermentation is a traditional and ancient microbial fermentation process wherein

microbes are cultivated under controlled conditions in the absence of free water. It can also be

described as a fermentation process involving solid substrates in absence or near absence of free

water. However, the substrate must possess enough moisture to support growth and metabolism

of microorganisms (Pandey, 1992). The existence and practice of SSF goes back to 2,600 BC

where the Egyptians were applying this technique for bread making (Toca-Herrera et al., 2007).

Over the centuries this technique was updated with respect to its practice and applications in

several fields of industrial microbiology. Since a few decades, SSF has emerged as potential

technology for the production of several microbial metabolites including enzymes (Susana and

Angeles, 2006). Despite a few controllable obstacles in cultivation conditions such as build up of

temperature, O2, pH, substrates and moisture gradients, SSF appears to possess several

biotechnological advantages towards the production of microbial metabolites at laboratory scale

(Holker et al., 2004). SSF has been considered as an expensive and ecofriendly method (Bashir

et al., 2011, Amena 2010) and has proven to be the best bioprocess method for the production of

several enzymes of industrial importance from a range of diverse microbes.

In addition to this, SSF has considerable application in the production of antibiotics, single cell

proteins, unsaturated fatty acids, organic acids, biopesticides, biofuels and aroma products

(Bhargav et al., 2008). Despite, SSF has significant application in agro-food industry for the

bioconversion of agro based byproducts into valuable animal feeds and in environmental control

as biodegradation tool (Prabhakar et al., 2005).

2.3.2. Submerged Fermentation (SmF)

Submerged Fermentation is the cultivation of microorganisms in liquid media, wherein the

microbe is submerged in aqueous solution containing all the nutrients needed for its growth and

development. This involves carefully growing of selected microorganisms in closed vessels

containing defined cultivation media with enough concentration of oxygen. As the microbes

break down the available/provided nutrients in culture media, they release desirable metabolites

such as fatty acids, organic acids, vitamins, amino acids including vast kind of enzymes. In the 13

present scenario, due to the developments of large scale ideal fermentors with several facilities

and advances in fermentation technologies, the production of microbial enzymes accounts for

significant proportion of the biotechnology industries total output. Submerged fermentation is

used for pickling vegetables, producing yoghurt, brewing beer and producing wine and soy sauce

(Chisti, 1999). Prior to five decades, submerged fermentation was emerged as important

alternative to solid state fermentation for industrial operations (Hiroshi and Tsunada, 1965).

Although SSF is a predominant method being used for the production of majority of the

microbial metabolites worldwide, submerged fermentation has its unique importance for the

production of most commercial products such as alcoholic beverages and antibiotics like

penicillin. At a time when many microbial metabolites and biomolecules are being produced by

employing SSF, several researchers have emphasized the importance and application of SmF in

the field of industrial microbiology. The studies of Anita et al. (2009) suggest that, submerged

fermentation reduces the cost of cellulase production by Aspergillus heteromorphus. (Garcia et

al., 2002).

2.4. L-GLUTAMINASE

L-glutaminase (EC 3.5.1.2) is a glutamine aminohydrolase which catalysis the hydrolytic

deamidation of L-glutamine to produce L-glutamic acid by releasing ammonia. The catalytic

action of L-glutaminase has vital role in nitrogen metabolism in both eukaryotes and

prokaryotes. Recently this enzyme attracted a significance attention owing to its diverse

application in several fields of pharmaceutical and food industry as anti tumor agent (Roberts et

al., 1970; Pal and Maity, 1992, 1994), anti retroviral agent (Roberts and McGrigor, 1991) and as

an aroma and flavor enhancing agent (Nakadai and Nasuno, 1989, Chou and Hwan, 1994). L-

glutaminase inhibits the proliferation and as well as kills leukemic cells by depriving them from

L-glutamine supply (Roberts et al., 1979). The treatment of leukemic cells with L-glutaminase

may represent a clear example of a therapy based on specific nutritional differences between

certain leukemic and normal cells. Even though L-asparaginase, the first enzyme to be

extensively studied as an anti tumor agent in human, it is highly effective in the treatment of

acute lymphoblastic leukemia but this enzyme has little or no activity against on other neoplasm

cells in human body. The enzyme is active against much broader range of cancers than the

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asparaginase. The discovery of antitumor properties of microbial glutaminase and asparaginase

enzymes was the key pace which shaped a great enthusiasm in researchers towards developing

them as potent antitumor agents. The recombinant glutaminase produced by Pseudomonas sp.

got patent in the year 1993 and the claims reveals that, it is useful in treating breast, lung and

colon cancer and also in the treatment of HIV infected cells (Roberts et al., 1994).

2.5 SOURCES OF L-GLUTAMINASE

The research work on L-glutaminase enzyme was initially started in the year 1956. The

importance of L-glutaminase enzyme was accidently found when (Gutman 1963) was working

on the measurement of the total uric acid-N15 enrichment and the N15 abundance of each of the

four uric acid nitrogen’s, found that an abnormality of glutamine metabolism in primary gout is

implied. It is suggested that the abnormality in glutamine metabolism may have significance for

the pathogenesis of primary gout. Thus, from then the study on this enzyme was focused. In the

following year, the effect of a number of sulphonamide derivatives was studied on the synthesis

of urea and on the activity of the enzyme L-glutaminase in vitro in liver slices and in isolated

mitochondria. Later El-Asmar and Greenberg (1966) detected that L-glutaminase of

Pseudomonas stutzeri arrest the initial growth of several murine carcinomas but have little effect

on the survival time of animals. Roberts et al. (1970) found that L-glutaminase of a Gram

negative rod-shaped bacterium suppressed Ehrlich ascites carcinoma (one type of breast

carcinoma). Imada et al. (1973) extensively studied the distribution of L-glutaminase in various

microorganisms. The authors tested 464 bacterial strains, 1326 yeast strains and 4185 fungal

strains for L-glutaminase activity. They observed that Bacillus sp showed L-glutaminase activity.

Streptomyces and Nocardia genera contain only intracellular amidases. In fungal strains

Fusarium sp. and Pencillium sp. are capable to produce the asparaginase, whereas Moniliaceae

sp. produced L-glutaminase only without asparaginase activity. The Ascomycetous fungi

reported to be producing either L-glutaminase or D -glutaminase based on substrate. Among

I326 yeasts, L-glutaminase or D-glutaminase occurred frequently in certain serological groups of

yeasts. Several strains of Cryptococcus and Rhodotorula possess L-glutaminase. L-Glutaminase

alone was formed in many strains of Candida scottii and Cryptococcus albidus, both of which

are related to Basidiomycetes. Japanese, while making traditional food, soya sauce, add some

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yeast and fungus mold to increase the taste of sauce. They isolated the two strains of L-

glutaminase producing Aspergillus sojae from soya mash (Mahesh Divtar 2015). Based on the

therapeutic and food industry use of L-glutaminase many researchers worked on the various

aspects of L-glutaminase. Still a lot of research work is been carried out world-wide.

2.6. MICROBIAL PRODUCTION OF L-GLUTAMINASE

Microbial L-glutaminase was reported to produce in both solid state and submerged

fermentation. The suitability of the bioprocess may be varying with the microorganism which is

involved in the fermentation. There are many reports on SSF for L-glutaminase production

employing various microorganisms and different solid substrates. Few authors reported that SSF

with wheat bran was found to be a preferable substrate for enzyme production (Prema et al.,

2002; El-Sayed, 2009; Nathiya et al., 2011a; Nagamony et al., 2012). There are also reports in

which the glutaminase production was found to be impressive under SSF with the wheat bran,

rice bran, copra cake powder, groundnut cake powder and sesamum oil cake as solid substrates

by employing different microbes (Chanakya et al., 2010). Worth mentioning bacterial genera

producing L-glutaminase under SSF are Vibrio costicola ACMR 267 (Prabhu and

Chandrasekaran, 1997), Lactobacillus casei (Nagamony et al., 2012). Among fungi notable L-

glutaminase producers are Beauveria sp. BTMS-10 (Sabu et al., 2000a), Actinomucor elegans

(Han et al., 2003), Rhizopus oligosporus (Han et al., 2003), A. fumigatus (Nathiya et al., 2011b),

Trichoderma harzanium (Palaniswamy et al., 2011), A. flavus (Rashmi et al., 2012). Various

microbes were used for the production of the L-glutaminase in submerged fermentation.

Bacterial isolates like P. fluorescence (Chitanand and Shete, 2012), Pseudomonas aeruginosa

BGNAS-5 (Rashmi et al., 2012a), Pseudomonas sp. BTMS 51(Rajeevkumar and

Chandrasekaran, 2003) and Bacillus subtilis (Satish and Prakasham, 2010) were reported to

produce considerable titer of enzyme under submerged fermentation. Other than bacteria several

microbes like Cryptococcus nodaeinsis (Sato et al., 1999), Zygosaccharomyces rouxii (Iyer and

Singhal, 2008) Aspergillus sp. (Prashant Kumar et al., 2009), Streptomyces griseus

(Muthuvelayudham,2013) and Streptomyces avermitilis (Abdallah et al., 2013) reported produce

L-glutaminase under SmF.

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2.7 BIOLOGICAL ROLE OF L-GLUTAMINASE IN NORMAL CELLS AND TUMOR

CELLS

Cancer cells, especially acute lymphoblastic leukemia (ALL) cells cannot synthesize L-

glutamine and hence there is a demand for large amount of L-glutamine for its growth. The use

of amidases deprives the tumor cells from L-glutamine and causes selective death of L-glutamine

dependent tumor cells. L-glutaminase can bring about degradation of L-glutamine and thus can

be a possible source for enzyme therapy. Cancer cells require a continuous supply of reduced

nitrogen to produce nucleotides, non-essential amino acids and a high cellular redox activity.

Glutamine provides a major substrate for respiration as well as nitrogen for the production of

proteins, hexosamines, and macromolecules. Therefore, glutamine is one of key molecules in

cancer metabolism during cell proliferation (Unissa et al.,2014).

The concept of targeting glutamine metabolism in cancer, justified by the number of pathways

fed by this nutrient, has been strengthened by more recent studies demonstrating that its

metabolism is regulated by oncogenes. Glutamine can exert its effect by modulating redox

homeostasis, bioenergetics, nitrogen balance or other functions, including by being a precursor

of glutathione, the major non-enzymatic cellular antioxidant. Glutaminase (GA) is the first

enzyme that converts glutamine to glutamate, which is in turn converted to alpha-ketoglutarate

for further metabolism in the tricarboxylic acid cycle. Different GA isoforms in mammals are

encoded by two genes, Gls and Gls2. GA encoded by Gls gene (GLS) has been demonstrated to

be regulated by oncogenes and to support tumor cell growth. GA encoded by Gls2 gene (GLS2)

reduces cellular sensitivity to reactive oxygen species associated apoptosis possibly through

glutathione-dependent antioxidant defense, and therefore to behave more like a tumor

suppressor. Thus, modulation of GA function may be a new therapeutic target for cancer

treatment (Unissa et al.,2014).

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2.8.0 THERAPEUTIC APPLICATIONS OF L-GLUTAMINASE ENZYME

L-glutaminases according to the current studies is therapeutically suitable if they display high

enzyme activity at physiologic pH, i.e., between about pH 6.5 and 8.5. Therapeutically suitable

glutaminases should have a low KM, i.e., between 10−6 and 10−4 M. Additionally desirable

properties of glutaminase enzymes for therapeutic use include:

High stability at physiological pH.

Retains high activity and stability in animal and human sera and blood.

Cleared slowly from the circulation when injected into animals or humans. A plasma

half-life (t½) for glutaminase greater than six hours in mice and sixteen hours in humans

is desirable.

Not strongly inhibited by the products of the reaction it catalyzes or by other constituents

normally found in body fluids.

Does not require cofactors or prosthetic groups that can easily dissociate from the

enzyme.

Narrow substrate specificity.

Effective irreversibility of the enzymatic reaction under physiologic conditions.

Available from an organism that contains low levels of endotoxin.

Low immunogenicity. (Unissa et al.,2014)

2.8.1 L-Glutaminase and its development as a chemotherapeutic agent

A characteristic nature of High rate of glutamine consumption is seen in some types of

cancerous cells (Natiyal et al., 2012). Based on this character experimental therapies have been

developed to deprive L-glutamine to tumour cells. Tumour growth regulation can be achieved

by inhibition of both protein and nucleic acid biosynthesis in the cancerous cells due to the lack

of availability of any component of these macromolecules. Inhibition of the tumour cell uptake

of glutamine is one of the possible ways to stop the growth and this is the best accomplished by

the use of L-Glutaminase, which breaks down L-glutamine.

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2.8.2 Application of L-glutaminase in food industry

L-Glutamine is the most important amino acid in food manufacture, for a delicious taste (Nanda

Kumar, 2003, Fukushima & Motai 1990). The pleasant and palatable tastes of oriental fermented

foods like soy sauce, miso and sufu are considered to be related to the content of L-glutamic acid

accumulated in them (Hamada & Mashall 1988, Chou & Hawn 1994) due to the hydrolysis of a

protein component catalysed by proteolytic enzymes, including L-glutaminase, protease and

peptidases. Hydrolysis of glutamine by L-glutaminase may also contribute significantly to the

high content of L- glutamate in these products. Several attempts were made to improve the

quality of soy sauce and miso utilizing the action of microbial L-Glutaminases (Sugiama 1984,

Chou et al., 1993).

2.8.3 Analytical applications

L-Glutaminase applied as a biosensor to determine the L-glutamine levels was investigated by

Kikkoman Corporation, Japan, Sabu et al, Botre et al. Huang et al., Mulchandani and Bassi, and

hybridoma culture media by flow injection analysis. Analysis of L-glutamine and glutamate

levels of the body fluids is important in clinical diagnostics and health monitoring. L-

Glutaminases are used currently both in free enzyme or immobilized on membrane forms as

biosensors for monitoring glutamine and glutamate levels of fluids.

2.8.4 Commercial production of microbial L-glutaminase

Researchers at Kikkoman Corporation, Japan carried out some of the remarkable works on

production of the enzyme. The research group in the company used mutation and protoplast

fusion techniques for the improvement of L-Glutaminase production by the koji mould, A. sojae,

(Kikuchi & Sakaguchi 1973). The company developed a process which was patented, for the

submerged fermentation process for production and purification of thermostable L-glutaminase

by C. nodaensis. (Sato et al., 1999).

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2.9 DEVELOPMENT OF BIOPROCESS FOR L-GLUTAMINASE PRODUCTION

A bioprocess is a specific process and its operations make exploitation of microbial cells for the

production of several useful products such as fermented foods, alcoholic beverages, biofuels,

biopolymers, and proteins including enzymes. The use of microbes for transform of biological

materials into alcoholic beverages and fermented foods has its origins in antiquity. Since, then

bioprocesses have been developed for an enormous range of commercial products like organic

solvents, antibiotics, therapeutic enzymes and vaccines. Production of a specific biomolecule

from microbes involves controlled environment which includes method of fermentation,

cultivation media, cultivation conditions and nutritive value of fermentation media.

Development of bioprocess encompasses wide range of modification and their application in its

practices. The modification may include usage of chiefly available cost effective agro-based

byproducts as raw materials instead of defined production media, formulation of cultivation

media, evaluation of physical parameters optima for the production of desired end product of

microbial origin and genetic modification of microbes. The existing literature gives the enough

evidences on the development of bioprocesses for the production of various biomolecules

including enzymes of microbial origin.

There are reports on the development of bioprocess for the production of enzymes from

microbial sources. Wheat bran and neem bark solely and their combination in various ratios were

used as production media as the part of process development for laccase enzyme production by

Pleurotus ostreatus under SSF (Jayanti and Sangeeta, 2014). The research conducted by Anis

highlights on the bioprocess modifications that achieved and employed to develop bioprocess for

the production of phytase (Anis. Ph.D. Thesis, 2008). Some of the agro-industrial byproducts

were employed as inexpensive substrate for asparaginase enzyme production by Pseudomonas

plecoglossicida. Further, studies revealed that sugar cane industry effluent was found to be the

suitable production media at low economy (Ganeshan et al., 2015).

In case of the production of L-glutaminase there have been various modifications were reported

to be employed in fermentation practices aiming to develop a feasible bioprocess. In this regard

there are several reports on exploitation of agro-based byproducts as raw materials, optimization 20

of process variables, strain improvement strategy, whole cell immobilization and implementation

of response surface methodology for the enhanced production of L-glutaminase from vast kind

of microbes.

2.9.1. Exploitation of Agro based Byproducts

Since last two decades as the part of bioremediation, biodegradation and biological

detoxification, agro-industrial residues and crop residues have became the hot sources of raw

materials for the production value added products such as antibiotics, organic acids, biopesticides

and various enzymes of microbial origin (Pandey et al., 2000). In fast few years, agro-based

byproducts are being used as raw materials for the production of enzymes from microorganisms.

Wheat bran, coconut oil cake, sesamum oil cake and ground nut oil cake were evaluated for L-

glutaminase production by Zygosaccharomyces rouxii.

The consequences of the investigation uncover that; sesamum oil cake was the best raw material

(Prema et al., 2002). Husks of different grains, brans of various cereals and oil seed cakes were

utilized for the production L-glutaminase (Pandey, 2003). Another study suggests that, wheat

bran is the best substrate for induction of L-glutaminase by Trichoderma koningii (Ashraf, 2009;

Chanakya et al., 2010). The investigation outcome on the production of L-glutaminase by

Aspergillus fumigates reports that, paddy straw is the inexpensive substrate under solid state

fermentation (Nathiya et al., 2011). (Pramod et al. 2014) exploited various agro-based

byproducts such as green gram husk, bengal gram husk, cattle feed, wheat bran and ground nut

oil cake as substrate for L-glutaminase production by Pseudomonas stutzeri and they have

reported that, cattle feed was a the suitable substrate for enzyme production (Pramod et al.,

2014).

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MATERIALS AND METHODS

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3.0 CHEMICALS:

Chemicals were procured from SRL, and L-glutamine used in the study was procured from Hi-

Media Laboratories, Bombay, India.

3.1.0 SAMPLE COLLECTION AND ISOLATION OF STRAINS

Sea water and soil samples were collected from different locations in Kerala and Goa in sterile

bottles and polythene bags and were refrigerated till use. Sea water and soil samples were

subjected to serial dilution by taking 1ml of the water sample and 1g of soil and was subjected to

shaking on a gyratory shaker for 30 min. 1 ml of these samples were taken for serial dilutions (up

to 10-6) and 0.1 mL of aliquots were aseptically spread on Sea water agar medium and minimal

agar medium.

Composition (Gms/Litre):

Part A:-Yeast extract-5.000, Peptic digest of animal tissue-5.000, Beef extract-3.000, Agar-

15.000 Part B: - Sodium chloride-24.000, Potassium chloride- 0.700, Magnesium chloride.

6H2O- 5.300 Magnesium sulphate 7H2O-7.000, Calcium chloride-0.100, Final pH (at 25°C)

7.5±0.2 supplemented with cycloheximide (50μg/mL).

Composition (Gms/Litre): Potassium dihydrogen phosphate-3.0, Disodium hydrogen

phosphate 6.0, Sodium chloride-5.0, Ammonium chloride-2.0, Magnesium sulphate-0.1,

Glucose -8.0, Agar-15.0, Final pH - 7.0±0.2.

23

3.2.0 SCREENING OF STRAINS

0.1ml from the soil samples after serial dilution were inoculated onto minimal media

supplemented with L-glutamine and phenol red as indicator and incubated at 37°C for 24-48 hr

for rapid screening as per the method of (Gulati et al., 1997) (Plate 1). L-glutaminase producing

colonies were selected on the basis of formation of pink zone around the colonies on the

medium.

3.3.0 CONFIRMATION OF (L-GLUTAMINASE) GLUTAMIC ACID PRODUCTION BY

TLC:

The culture extract of the best strain obtained after initial screening were confirmed by TLC for

(L-glutaminase) glutamic acid production using standard Glutamic acid. The extract was loaded

on silica gel plates and subjected to TLC (Thin Layer Chromatography). The mobile phase used

was Acetic acid: n-butanol: water (1:4:5) ratio as per the modified method of (Shah et al.,

2010).

3.4 IDENTIFICATION OF STRAINS The identification of the strains showing maximum production of L-glutaminase was carried out

tentatively by Gram’s staining, catalase test and oxidase test. The strains were also subjected to

16s rRNA sequencing for identification.

3.5 SCREENING OF AGRO-INDUSTRIAL RESIDUES AS SUBSTRATES Various agro-industrial waste substrates/residues and their extracts were evaluated for the

production of L-glutaminase under submerged fermentation. A total of six different substrates

24

like Fried gram husk, green gram husk, groundnut cake, wheat bran, cattle feed and coconut oil

cake were utilized for enzyme production. All the substrates used in the study were procured

from the local market.

3.6 PREPARATION OF THE SUBSTRATES The extracts of the various agro-based residues were used as substrates for the production of L-

glutaminase under submerged fermentation employing bacterial isolates Bacillus subtilis subsp.

inaquosorum and Acinetobacter baumannii. The extracts were obtained by using ten grams of

each of the powdered substrates by mixing in 100 ml of distilled water taken separately in a 250

ml Erlenmeyer flask. The contents of the flasks were heated for about 10 min and cooled to

room temperature. It was filtered using cheese cloth (Fig 4). The extract thus obtained was used

for submerged fermentation studies. The pH of the medium was adjusted to 7.0 using (0.1N

HCl/NaOH) and autoclaved at 1210C for 15 min. The flasks were inoculated with one ml of the

bacterial isolates Bacillus subtilis subsp. inaquosorum and Acinetobacter baumannii

respectively. Shake-flask experiments were carried out with the flasks incubated at 130 rev/min

on a rotary shaker maintained at 37ᴼC. The L-glutaminase activity was determined after every 24

hr intervals for a period of 5 days.

As per the above fermentation methods used for L-glutaminase production Groundnut

oil cake and Coconut oil cake extract yielded maximum amount of L-glutaminase under

submerged fermentation. Hence the same substrates were employed for further studies.

3.7.0 SUBMERGED FERMENTATION FOR L-GLUTAMINASE PRODUCTION The medium prepared as above were inoculated with one ml of the bacterial isolate Bacillus

subtilis subsp. inaquosorum. Shake-flask experiments were made with flasks incubated at130

rev/min on a rotary shaker maintained at 37ᴼC (De Azeredo et al., 2005).

3.8.0 EXTRACTION OF CRUDE ENZYME To determine the enzyme activity, 2ml of the fermented broth was withdrawn aseptically from

the flasks at an interval of every 24 h. The broth was filtered using Whatman filter paper No.1

and then centrifuged at 10,000 rpm for 10 min. The supernatant thus obtained was used as crude

extract for L-glutaminase assay.

25

3.9.0 ASSAY OF L-GLUTAMINASE L-glutaminase activity was determined in culture filtrate by quantifying the ammonia formation

using Nessler’s reagent. Assay of enzyme was carried out as per Imada et al., (1973). 0.5 ml of

0.04M L-glutamine was taken in a test tube, to which 0.5 ml of 0.05 M buffer (Tris –HCl buffer

pH 8), 0.5 ml of crude enzyme extract and 0.5 ml of distilled water was added to make up the

volume to 2 ml and the reaction mixture was incubated for 30 min at 37ᴼC. After the incubation

period the reaction was stopped by adding 0.5 ml of 1.5 M trichloroacetic acid (TCA). 0.1 ml

was taken from the above reaction mixture and added to 3.7 ml of distilled water and to that 0.2

ml of Nessler’s reagent was added and incubated for 15-20 min at 20ᴼC. The O.D was measured

at 450 nm. The blank was prepared without adding enzyme. The enzyme activity was expressed

in international unit (IU).

3.10.0 INTERNATIONAL UNIT One IU of L-glutaminase is the amount of enzyme which liberates 1µmole of ammonia per ml

per min (µmole/ml/min).

3.11.0 OPTIMIZATION OF PROCESS PARAMETERS FOR L-GLUTAMINASE

PRODUCTION

Optimization of various process parameters required for maximum L-glutaminase production by

Bacillus subtilis subsp. inaquosorum was performed. The parameters studied for submerged

fermentation included initial pH (6.0-10.0) of the medium, temperature of incubation (25-450C),

inoculum size (1-5%) and subjected to constant agitation (130 rev/min). The procedure adopted

for optimization of various parameters influencing L-glutaminase production was to evaluate the

effect of independent parameters keeping others constant and to incorporate it at the optimized

level in the next experiment while optimizing another parameter.

3.11.1 EFFECT OF INITIAL pH ON L-GLUTAMINASE PRODUCTION

To determine the effect of initial pH on L-glutaminase production, a set of six conical flasks

each containing 100 ml of coconut oil cake extract was used. The pH range of the study was 6.0-

10.0 with an increment of 1. The desired pH level was adjusted using dilute (0.1N) NaOH/HCl.

Thus prepared flasks were autoclaved and inoculated with the culture suspension of Bacillus

26

subtilis subsp. inaquosorum and the flasks were then incubated at 370C in a shaker incubator

maintained at 130 rev/min. The enzyme activity was assessed every 24 hr for 5 days.

3.11.2 EFFECT OF INCUBATION TEMPERATURE ON L-GLUTAMINASE

PRODUCTION

A set of three conical flasks, each containing 100 ml of extract adjusted to pH 6.0 was

autoclaved and inoculated with 1% inoculum. The flasks were then incubated at different a

temperature ranging from 25ᴼC-45ᴼC, and the activity was assessed every 24 hr.

3.11.3 EFFECT OF INOCULUM SIZE ON L-GLUTAMINASE PRODUCTION

The conical flasks containing the media at pH 6.0 were inoculated separately with 1 ml of

inoculum and incubated at 37ᴼC. The culture inoculum was prepared at different levels 1-5%

with increments of 1% (Pushpa & Madhava Naidu 2010). Enzyme activity was assayed every

24 h for 5 days.

3.12.0 PROCESS ECONOMIZATION

3.12.1 EFFECT OF SUPPLEMENTATION OF NUTRIENTS ON THE YIELD OF L-GLUTAMINASE

The medium described above was taken as a basal medium and the effect of nutrients was

studied. In order to achieve higher yields of L-glutaminase, various nutrient sources like carbon,

nitrogen were supplemented to the coconut oil cake extract and their concentrations were

optimized.

3.12.2 EFFECT OF SUPPLEMENTATION OF CARBON SOURCES ON L-GLUTAMINASE PRODUCTION

A set of conical flasks containing 100 ml of coconut cake extract was supplemented separately

with different carbon sources like glucose, maltose, and lactose. The carbon sources were

evaluated at a concentration of 1% for each carbon source.

27

3.12.3 EFFECT OF SUPPLEMENTATION OF NITROGEN SOURCES FOR L-GLUTAMINASE PRODUCTION

Conical flasks with 100 ml of coconut cake extract supplemented with (1%) nitrogen sources

such as ammonium sulphate, yeast extract, ammonium nitrate and peptone.

3.13.0 ESTIMATION OF PROTEIN

Total protein content was estimated by using Lowry’s method. First of all standard curve of

Tyrosine was made. A stock solution 0f 100µg/ml of tyrosine was prepared by dissolving 10mg

tyrosine in 100ml of distilled water in a standard volumetric flask. Using this stock solution,

dilution ranging from 10-100 µg/ml was prepared. To 1 ml of each of the dilution, 5 ml of the

alkaline reagent ‘C’ was added 7 the test tube was incubated at 400C for 15min.To the incubated

tubes, 0.5ml of Folin’s reagent was added & tubes were further incubated at 400C for 15min for

the reaction to be completed. The OD was then taken at 640 nm. Using a blank solution, the

colorimeter was adjusted. A standard graph is plotted using the value of concentration on the X

axis and the OD values on the Y-axis to obtain a straight line. The OD of the protein sample was

plotted on the standard graph & extrapolated to get the concentration of protein.

3.14.0 PURIFICATION OF L-GLUTAMINASE

The purification was carried out using crude enzyme extract. The enzyme was purified by the

following steps at 0-4ᴼC.

3.14.1 PARTIAL PURIFICATION BYAMMONIUM SULPHATE PRECIPITATION

Finely powdered ammonium sulphate was added to 500ml of the crude extract. The L-

glutaminase activity was associated with the fraction precipitated at 80% saturation. The

precipitate was collected by centrifugation at 10,000 rpm for 15 min, dissolved in 50mM Tris-

HCl and dialysis was carried out using the same buffer.

3.14.2 DIALYSIS Selective particles such as ionic particles, amino acids, peptides removed through selective

semipermeable membrane, the process is dialysis. The dialysis membrane was activated by

boiling for 10 minutes and dead particles removed by adding 2% sodium bicarbonate and 28

boiling was continued for another 10 minutes. The water replaced and boiled for another 10

minutes. After the activation of the dialysis membrane the sample was poured, sealed tightly and

dialyzed overnight at 40°C. The dialysis bag with sample was placed in a magnetic stirrer for 2

hours and the water was replaced at every half an hour intervals. The sample collected was

breaking the dialysis membrane. The sample was stored for further enzyme assay and protein

estimation.

3.15.0 ENZYME CHARACTERIZATION

3.15.1 EFFECT OF pH ON L-GLUTAMINASE ACTIVITY The activity of L-glutaminase was evaluated at different pH values. Purified enzyme was

incubated with 0.04M buffer of pH 6-10, under assay conditions, with an increment of pH 1.

Buffers used were Potassium phosphate (pH 6.0-7.0), Tris-HCl (pH 8.0-9.0), glycine-NaOH (pH

10.0).

3.15.2 EFFECT OF TEMPERATURE ON L-GLUTAMINASE ACTIVITY

The optimum temperature determined for L-glutaminase activity by incubating the assay

mixtures at different temperature from 15, 25, 37, 60 and 100°C.

3.15.3 EFFECT OF NaCl CONCENTRATION ON L-GLUTAMINASE ACTIVITY The activity of L-glutaminase was evaluated at different sodium chloride concentration. The

activity was determined at NaCl concentration of range 1-5%, with an increment of 1%.

3.15.4 EFFECT OF METAL IONS ON L-GLUTAMINASE ACTIVITY

The effect of divalent cations on the enzyme activity was investigated. The metal ions studied

were Ca2+,Mg2+,Cu2+,Zn2+ and Fe2+.The purified enzyme was assayed in the presence of the

cation to be examined.

3.15.5 EFFECT OF VARIOUS CHEMICALS L-GLUTAMINASE ACTIVITY

The effect of various chemicals on the enzyme activity was investigated. The enzyme was

assayed in the presence of various chemicals like Sodium dodecyl sulphate (SDS) and Ethylene

Diamine tetra acetic acid (EDTA).

29

RESULTS AND DISCUSSION

4.0.1 ISOLATION OF BACTERIAL STRAINS

30

In the present study water samples from four different locations were used for the

isolation of strains and then screened for the L-glutaminase production.The isolation pattern of

the strains is presented in Table 4.1. In the present study 132 strains were isolated. Among them

44 bacterial strains were selected and named according to the source of isolation. Among the

different samples used for the isolation the water samples from Shimogga yielded 20, Kerala

yielded 29 and 65 respectively from sea water and soil, Goa yielded 10, Murdeshwara yielded 10 and

Mandavi yielded 18 strains from sea water respectively.

Sl.NO SOURCE OF LOCATION

TOTALISOLATES

NO.OF POSITIVE BACTERIAL STRAINS

1. SHIMOGGA (S) 20 11

KERALA (SW) 29 10KANNUR (S) 18 7KOZHIKODE (S) 10 3PATHANAMTHITTA (S) 17 5

2. GOA(SW) 10 02

3. MURUDESHWARA(SW) 10 03

4. MANDAVI(SW) 18 03

TOTAL 132 44

Table- 4.1 Isolation of Bacterial strains (SW) Sea water, (S) Soil

4.2 SCREENING OF STRAINS FOR L-GLUTAMINASE PRODUCTION BY RAPID

PLATE ASSAY METHOD

The results of the rapid plate assay method are presented in Plate 4.2. All these isolates

were subjected to rapid plate assay for the detection of L-glutaminase producing strains as per

Gulati et al., (1997). The results revealed that among the132 isolates,44 strains showed L-

glutaminase activity. The rapid plate assay also revealed distinct zone of hydrolysis. Among all

the L-glutaminase producers the strain Bacillus subtilis subsp.inaquosorum (2.19 cm) and

Acinetobacter baumannii showed the highest zone of hydrolysis i.e. (2.05 cm) at 48 h of

incubation. These strains Bacillus subtilis subsp. inaquosorum and Acinetobacter baumannii

showing maximum L-glutaminase production were identified by 16s rRNA sequencing.

31

As per the results Bacillus subtilis subsp. inaquosorum strain emerged as a potential strain for L-

glutaminase production, hence further studies were carried out with the strain Bacillus subtilis

subsp. inaquosorum.

Plate 4.2 Plate assay of L-Glutaminase

4.3 IDENTIFICATION OF STRAINS

The identification of the strain showing maximum production of L-glutaminase was

identified by 16s rRNA sequencing.

DNA extraction and PCR Amplification:

The genomic DNA was isolated from the given organism using genomic DNA extraction

Kit. Amplification of the 16s rRNA gene was performed using the universal primers.PCR

product was purified to remove unincorporated dNTPS and Primers before sequencing.

Sequencing:

Both strands of the rDNA region amplified by PCR were sequenced by automated DNA

sequencer -3037xl DNA analyzer from Applied Biosystems using BigDye® Terminator v3.1

cycle sequencing Kit (Applied Biosystems).Sequence data were aligned and dendrograms were

generated using Sequence analysis software version 5.2 from Applied biosystems. The sequences

obtained for upper and lower strands were manually aligned before performing the analysis.

Identification of the strain showing maximum production of L-glutaminase was identified

by 16 rRNA sequencing.

32

Aligned sequence:

>TOCS_907RC_Bacillus subtilis subsp. inaquosorum strain (1468bp)

ATGCAAGTCGAGCGGACAGATGGGAAGCTTGCTCCCTGATTGTTAGCGGCGGACGG

GTGAGTAAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCCG

GGGCTAATAACCGGATGCTTGTTTGAACCGCATGGTTCAAACATAAAAGGTGGCTTC

GGCCAACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTTGGTGAGGTAATGG

CTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGAC

TGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGA

CGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGC

TCTGTTGTTAGGGAAGAACAAGTACCGTTCGAATAGGGCGGTACCTTGACGGTACCT

AACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCA

AGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGAT

GTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGC

AGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGG

AACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGC

GTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGC

TAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGC

CTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAA

GCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGA

CATCCTCTGACAATCCTAGAGATAGGACTTCCCCTTCGGGGGCAGAGTGACAGGTGG

TGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCG

CAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTG

ACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGG

CTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCC

AATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGC

TGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTG

TACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAAC

CTTTTAGGAGCCAGCCGCCGAAGGTGGGACAGATGATTGGGGGTGAAGTCGTAACA

AGGG

33

>TOCS 7_907RC_ACINETOBACTER BAUMANNII (713bp)

CATGCAAGTCGAGCGGGGGAAGGTAGCTTGCTACTGGACCTAGCGGCGGACGGGTGAGTAATGCTTAGGAATCTGCCTATTAGTGGGGGACAACATCTCGAAAGGGATGCTAATACCGCATACGTCCTACGGGAGAAAGCAGGGGATCTTCGGACCTTGCGCTAATAGATGAGCCTAAGTCGGATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCGACGATCTGTAGCGGGTCTGAGAGGATGATCCGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGGGGAACCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGCCTTATGGTTGTAAAGCACTTTAAGCGAGGAGGAGGCTACTTTAGTTAATACCTAGGGATAGTGGACGTTACTCGCAGAATAAGCACCGGCTAACTCTGTGCCAGCAGCCGCGGTAATACAGAGGGTGCGAGCGTTAATCGGATTTACTGGGCGTAAAGCGTGCGTAGGCGGCTTATTAAGTCGGATGTGAAATCCCCGAGCTTAACTTGGGAATTGCATTCGATACTGGTGAGCTAGAGTATGGGAGAGGATGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGATGGCGAAGGCAGCCATCTGGCCTAATACTGACGCTGAGGTACGAAAGCA

Bioinformatics analysis:

Sequences were compared to the non-redundant NCBI database by using BLASTN, with the

default settings used to find the most similar sequence and were sorted by the E score. A

representative sequence of 10 most similar neighbours was aligned using CLUSTAL W2 for

multiple alignment with the default settings. The multiple-alignment file was then used to create

phylogram using MEGA5 software.

4.4 CONFIRMATION OF (L-GLUTAMINASE) GLUTAMIC ACID PRODUCTION BY

TLC

The results for the confirmation of glutamic acid were done by thin layer chromatography. To

confirm the zone of hydrolysis of the different isolates were indeed of L-glutaminase, they were

subjected to TLC. The TLC separation effectively separated the metabolite to confirm glutamic

acid. The extract of Bacillus subtilis subsp. inaquosorum showed similar Rf values (0.6) when

compared to the standard glutamic acid (0.62). In the present study the strain Bacillus subtilis

subsp. inaquosorum exhibited similar Rf value as that of standard glutamic acid, indicating that

34

the fermentation product was glutamic acid. (Table 4.4). The results obtained show close

proximity with the observations of (Shah et al., 2010)

Table-4.4. Rf values by thin layer chromatography

Sl. No Types Rf Values

1 Standard Glutamic acid -Control 0.62

2

Glutamic acid- hydrolyzed product

from L-glutamine from L-

glutamine

0.6

Lane-1 Std Lane-2 Lane-3 Fermented Fermented

Broth Broth duplicate

Plate-4.4 Confirmation of L-Glutaminase by TLC

35

4.5 SCREENING OF AGRO-INDUSTRIAL WASTE EXTRACTS FOR PRODUCTION

OF L-GLUTAMINASE UNDER SUBMERGED FERMENTATION

Taking into account the importance of L-glutaminase for its various applications, an

attempt was made to explore the use of newer and inexpensive agro-based substrates for L-

glutaminase production by using Bacillus subtilis subsp. inaquosorum. One of the problems

involved in industrializing a process is to design a cost-effective medium suitable for

fermentation. Mostly this is done by trial and error. No single production medium is the best or

an ideal one for all purposes. Hence a suitable production medium is designed/chosen based on

the nutritional characters of the raw materials available in the market. Production medium should

contain a source of carbon, nitrogen, growth factors and mineral salts. Moreover the agro-

industrial waste materials contain a large amount of potentially usable proteins and

carbohydrates. They are renewable, economic and not a part of human food, hence can be chosen

as best suitable raw material for media designing (Reddy et al., 2007).

In the present study screening of various agro-based substrates like Fried gram husk,

groundnut cake, wheat bran and cattle feed were carried out for the production of L-glutaminase.

Among the four substrates employed for the L-glutaminase production, groundnut cake was

found to be a potential substrate for the production of L-glutaminase. Hence, it was selected for

further detailed study and this appears to the first report with this substrate for the production of

L-glutaminase either under SSF or Smf.

The results on screening of various agro-based waste extracts for L-glutaminase

production under submerged fermentation by Bacillus subtilis subsp. inaquosorum are presented

in Fig 4.5. The results revealed that the maximum L-glutaminase activity of 18.34 IU was

observed in case of groundnut cake after 72 h of fermentation period and gradually decreased.

36

cattle feed groundnut cake Fried gram husk wheat bran

Figure 4.

4.6 BIOCHEMICAL ANALYSIS OF GROUNDNUT CAKE

Always it is essential to analyze the chemical components of the substrate before

a specific substrate is chosen for further fermentation studies because such analysis reveal the

suitability of the substrate for the required product synthesis. The biochemical composition of the

groundnut cake satisfies the condition of the fermentation process (Table 4.6)

It has good proportion of carbon, nitrogen and also has sufficient nutrients for growth of

the fermenting organisms as revealed by its biochemical composition. Further it does not have

any component toxic to the organism. Developing a concept for L- glutaminase production

process based on the utilization of ground nut cake extract under submerged fermentation is

economically attractive as it is a cheap and readily available raw material in agriculture-based

countries. Further, this appears to be the first study with this substrate for L-glutaminase

production under Smf employing Bacillus subtilis subsp. inaquosorum.

The results on the biochemical analysis of groundnut cake are presented in Table 4.6. The results

showed that the groundnut cake contains moisture (5.8%), total carbohydrates (1.81%), protein

(38.61%), fat content (47%), ash (3.8%), crude fiber (3.7%) et

Table 4.6 Composition of Groundnut cake37

Sl.No. Constituent Percentage%

1. Moisture 5.8%

2. Protein 38.61%

3. Total Carbohydrate 1.81%

4. Crude Fiber 3.7%

5. Ash 3.8%

6. Fat 47%

7. Na 42.0 mg/100g

8. K 705.1 mg/100g

9 Mg 3.98 mg/100g

10. Ca 2.28 mg/100g

11. Fe 6.97 mg/100g

12. Zn 3.20 mg/100g

13. P 10.35 mg/100 g

(Atasie et al., 2009)

4.7 OPTIMIZATION OF PROCESS PARAMETERS FOR L-GLUTAMINASE

PRODUCTION

4.7.1 EFFECT OF INITIAL pH ON L-GLUTAMINASE PRODUCTION

Initial pH of the substrate is one of the crucial factors for the successful L-

glutaminase fermentation. It is noticed herein and most of the industrial fermentations that

controlling pH of the medium at optimum levels is essential for achieving maximum product

formation (Shankarananand and Lonsane, 1994).

Most microbial extra cellular enzymes are produced at high levels at a growth pH that is

near to the optimal pH required for the maximal activity (Sabu et al., 2000). The initial pH of

the medium mainly depends on the carbon sources used during the fermentation. However, the

38

kinetics of pH variation depends highly on the microorganism (Raimbault, 1998). The response

of the organisms to variations in pH differs in strains of the same species.

The data on the effect of initial pH on L-glutaminase production by Bacillus subtilis

subsp. inaquosorum is presented in Fig. 4.7.2 The initial pH of the medium influenced L-

glutaminase production such that the pH optimum was obtained at pH 8.5, yielding maximum

enzyme activity of 18.43U/ml/min. The least enzyme production was recorded at pH 5.0 yielding

11.62 U/ml/min of L-glutaminase.

These variations in pH optimum for L-glutaminase production may be due to the strain of

the organism used, chemical composition of the medium, fermenting system and finally the

conditions under which the fermentation takes place.

Davidson et al (1976) reported L-glutminase production at pH 7.5. Hiremath Jyothi et al

(2011) have reported maximum L-glutaminase activity at pH 6.0. Prabhu and Chandrashekaran

(1999) reported L-glutaminase production to be maximum at pH 7.4. Sivakumar et al (2006)

reported L-glutminase production from esturine fish, Chanos chanos at pH 9.0.Keerthi et al

(1999) have reported maximum L-glutaminase activity by an alkalophilic bacillus at pH 9.0

isolated from marine sediment.

The effect of initial pH of the fermentation medium on the yield of L-glutaminase is depicted in

Fig. 4.7.1. There was a steady increase in the enzyme production as the initial pH of the medium

was raised from 5.0-9.0. The L-glutaminase yields were found to decrease thereafter. Maximum

enzyme activity of 18.43 U/ml/min was observed at pH 8.5 and the minimum 11.62 U/ml/min

was found at pH 5.0 at 72 h of fermentation.

Thus, the above study clearly indicates that the production of L-glutaminase mainly

depends on the strains employed during the fermentation. Chitanand and Shete (2012) have

reported the production of extracellular L-glutaminase production from Pseudomonas

fluorescens at pH 8.0 As such, our findings are in close agreement with the findings of Chitanand

and Shete (2012).

39

40

Fig - 4.7.1

4.7.2 EFFECT OF INCUBATION TEMPERATURE ON L-GLUTAMINASE

PRODUCTION

Temperature is an important parameter that governs the process of fermentation

as well as the recovery of desired product. Various temperature optima have been observed for

L-glutaminase production employing different microbial strains. Hence the influence of

temperature on L-glutaminase synthesis by Bacillus subtilis subsp. inaquosorum using

groundnut cake extract was utmost need. The L-glutaminase production pattern by Bacillus

subtilis subsp. inaquosorum at different temperatures is depicted in Fig. 4.7.2. A temperature of

350C was found to be optimum in the present study yielding an enzyme activity of 22.42 IU at 72

h of fermentation. The present study revealed that rise in incubation temperature from 25-400C

raised the enzyme titers significantly and temperatures beyond this were found to lower the

enzyme yields. Any temperature beyond the optimum range is found to have some adverse effect

on the metabolic activities of the microorganisms and it is also reported by various scientists that

the metabolic activities of the microbes become slow at lower or higher temperatures (Tunga et

al., 1999; Pandey et al., 2000).

Sivakumar et al (2006) studied the effect of temperature on growth and L-glutaminase

production by actinomycete strain from estuarine fish Chanos chanos, and the reported

temperature for L-glutaminase being 270C. Keerthi et al (1999) have reported maximum L-

glutaminase production from alkalophilic bacillus strain at a temperature of 270C. According to

Thadikamal Sathish and Prakasham (2012) have reported maximum temperature required for

the production of L-glutaminase from Bacillus subtilis RSP-GLU was found to be 370C.

Balagurunathan et al (2010) working on actinomycetes from marine sediments have reported

maximum L-glutaminase production at a temperature of 300C. Optimum L-glutaminase

production has been reported by Suresh Kumar et al (2013) by Streptomyces griseus strain at a

temperature of 330C. Nagwa et al (2012) have reported maximum L-glutaminase activity from

actinomycetes at a temperature of 300C.

41

Temperature of incubation has shown a considerable impact on L-glutaminase production by

Bacillus subtilis subsp. inaquosorum. The results of effect of initial temperature levels on the

enzyme yield are presented in Fig. 4.7.2. The data revealed that increasing the incubation

temperature from 250C-500C lead to an increase in L-glutaminase production. Temperatures

higher than 350C lowered the enzyme yield. The highest L- glutaminase activity (22.42

U/ml/min) was found at 350C at 72 h of incubation and the lowest (8.82 U/ml/min) was recorded

at 500C.

Chitanand and Shete (2012) have reported the production of extracellular L-glutaminase

production from Pseudomonas fluorescens at a temperature of 370C. Our results from the study,

are in close agreement with the results of Chitanand and Shete (2012) from Pseudomonas

fluorescens.

Figure - 4.7.2.

4.7.3 EFFECT OF INOCULUM SIZE ON L- GLUTAMINASE PRODUCTION

42

The effect of inoculum size on L-glutaminase production by Bacillus subtilis

subsp. inaquosorum in groundnut cake extract was studied because low density of spores leads to

insufficient biomass and end product synthesis as well as, permits the growth of undesirable

contaminants and too high densities of spores may cause a quick and too much biomass

production thereby leading to fast nutrient depletion and ultimately reduction in the end product

quality.

It is evident from Fig. 4.7.3 that inoculum size influenced the synthesis of L-glutaminase

by Bacillus subtilis subsp. inaquosorum under submerged fermentation. With the increase in

inoculum size there was a gradual increase in the production of enzyme and maximum L-

glutaminase was produced when an inoculum size of 5% was used, while the lowest yield of

enzyme was observed with an inoculum size of 6% at 72h of incubation. With a further increase

in the size of inoculum, there was a reduction in the enzyme activity.

An increase in inoculum generally improves the growth and growth-related activities of the

organism up to a certain level and with further increase in inoculum size there could be a

reduction in microbial activity due to nutrient limitations. Lower inoculum size results in a lesser

number of cells in the production medium. This requires a longer time to grow to optimum

number to utilize the substrate and form the desired product (Kashyap et al., 2002). The

inoculum size has some optimum value depending upon the microbial species and fermentation

processes (Tunga et al., 1999).

Thadikamal Sathish and Prakasham (2012) have reported maximum production of L-

glutaminase from Bacillus subtilis RSP-GLU with a inoculum size of 2%. Pushpa and Madhav

Rao (2010) have reported maximum protease production at an inoculums size of 3%.

The effect of inoculum size on L- glutaminase production by Bacillus subtilis subsp.

inaquosorum is shown in Fig. 4.7.3. An inoculum size of 5% was found to be optimum for the

production of L- glutaminase by Bacillus subtilis subsp. inaquosorum. The enzyme activity

observed at the inoculum size was 25.39U/ml/min at 72 h of fermentation. The lowest enzyme

yield 3.30U/ml/min was recorded with an inoculum size 6%.

43

Mario Khalil Habeeb et al., (2012) have reported maximum production of L-glutaminase

from Streptomyces avermitilis at a inoculums concentration of 5%. The results reported by

Khalil Habeeb et al (2012) are in accordance with our results.

Figure - 4.7.3.

4.8 PROCESS ECONOMIZATION

During the manufacture of any product, there is an always a trend towards economization

of the process, i.e., to reduce the cost and also time required for production of the product, so that

the process of fermentation that is aimed at obtaining a specific product. In such fermentation

processes, always importance was given to supplementation of nutrients and improvement of

methods. The production of desired product is often coupled with the stationary growth phase of

the microorganism and depends on N or P limitation together with excess carbon and energy

source (Holker et al., 2004).

4.8.1 EFFECT OF SUPPLEMENTATION OF CARBON SOURCES ON L-

GLUTAMINASE PRODUCTION

The main product of the fermentation process will often determine the choice of carbon

source, particularly if the product results from the direct dissimilation of it. Energy for growth

comes from either the oxidation of the medium components or from light. Most industrial 44

microbes utilize the commonest form of energy in the form of carbon sources such as

carbohydrates, lipids and proteins. It is a common practice to use carbohydrates as the carbon

source in microbial fermentation processes. The rate at which the carbon source is metabolized

often influences the formation of biomass or production of primary or secondary metabolites

(Stanbury et al., 1995).

Results on the effect of supplementation of submerged fermentation medium with

different carbon sources such as glucose, xylose, lactose and maltose on L-glutaminase

production are shown in Fig. 4.8.1. The carbon sources were supplemented in the range of 1% of

the medium. However, the enzyme activity was highest with 1% of Glucose i.e., 40.53U/ml/min.

The results indicated that this enzyme is directly correlated with the concentration of substrate

used during fermentation. Hence glucose at 1% concentration emerged as the best carbon source

in our studies.

Various reports are available on the supplementation of carbon sources to fermentation media for

L-glutaminase synthesis. Hiremath Jyothi et al (2011) reported maximum L-glutaminase

production from a synthetic medium supplemented with 1% glucose with Pseudomonas as the

fermenting organism. Mario Khalil Habeeb et al (2012) report maximum L-glutaminase

production from an actinomycete strains from Egypt with the supplementation of glucose at a

concentration of 1%. Chitanand and Shete (2012) have also reported L- glutaminase production

to be maximum when the medium was supplemented with 1% glucose.

Figure 4.8.1 shows the effect of supplementation of various carbon sources to groundnut

cake extract on L-glutaminase production by Bacillus subtilis subsp. inaquosorum. The data

revealed that the supplementation of all the carbon sources tested did not enhance the L-

glutaminase production. Among all the carbon sources employed in the present study, maltose

and glucose significantly increased the enzyme yields whereas lactose and xylose proved to be

detrimental for the production of L- glutaminase. Glucose (1%), xylose (1%), lactose (1%), and

maltose (1%), yielded a maximum of 40.53U/ml/min, 24.76 U/ml/min, 29.23U/ml/min and

37.23U/ml/min of L- glutaminase respectively at 76 h of fermentation. However, glucose was

found to be the best carbon source at a concentration of 1.0% yielding 40.53U/ml/min of L-

glutaminase at 72 h of fermentation.

45

Our results from the study are in agreement with the results of Mario Khalil Habeeb

(2012) and Chitanand and Shete (2012).

Figure 4.8

4.8.2 EFFECT OF SUPPLEMENTATION OF NITROGEN SOURCES ON L-

GLUTAMINASE PRODUCTION

Most of the industrial microorganisms can utilize inorganic or organic sources of

nitrogen. The requirement of nitrogen source mainly depends on the strain and the substrate

employed during fermentation (Pintado et al., 1993). Nitrogen can be a limiting factor in the

microbial production of enzymes (Sabu et al., 2000). It is clear that the organism shows slight

differences in the growth pattern in the presence of nitrogen sources than in the case of nitrogen-

free medium (Chandrashekhar Naik, 2002).

Organic nitrogen may be supplied as amino acids, protein and urea. In many instances

growth will be faster with a supply of organic nitrogen. Amino acids are more commonly added

as complex organic nitrogen sources which are non-homogenous, cheaper and more readily

available. The proteinaceous nitrogen compounds serving as sources of amino acids include beef

extract, yeast extract, tryptone, cornsteep liquor, soyabean meal etc (Stanbury et al., 1995). The

source of nitrogen is an important factor which plays a key role in the production of L-

46

glutaminase. Hence studies were conducted to investigate the influence of supplementation of

various inorganic and organic nitrogen sources to groundnut cake extract on L-glutaminase

production by Bacillus subtilis subsp. inaquosorum.

The nitrogen sources employed in the present study were peptone, casein, yeast extract,

ammonium sulphate (45.54U/ml/min) and ammonium nitrate at a concentration range of 1%.

The results of influence of supplementation of these nitrogen sources on enzyme production are

depicted in Fig. 4.8.2.

Suresh Kumar et al (2013) showed the positive effect of supplementation of yeast extract

(1%), in the production of L-glutaminase. The stimulatory effect of malt extract on L-

glutaminase synthesis has been demonstrated in estuarine fish by Sivakumar et al (2006). Mario

Khalil Habeeb et al., (2012) have reported the highest L-glutaminase production when peptone

(2%) was used as the sole source of nitrogen.

The present study revealed that the supplementation of ammonium salts to groundnut cake

extract enhanced the enzyme production by Bacillus subtilis subsp. inaquosorum. The

observations made in the present study are consistent with the findings of Gaffar (1976),

Albanese and Kafkewitz, (1978).A total of four different nitrogen sources like peptone, yeast

extract, ammonium sulphate and casein were individually supplemented to the groundnut cake

extract at a concentration range of 1%. Figure 4.8.2 depicts the effect of supplementation of these

nitrogen sources on L- glutaminase production by Bacillus subtilis subsp. inaquosorum. There

was a considerable increase in the enzyme production when the groundnut cake extract was

supplemented with peptone and ammonium sulphate. On the other hand, yeast extract and casein

proved to be inferior for L- glutaminase synthesis by Bacillus subtilis subsp. inaquosorum.

Among all the nitrogen sources employed in our study, ammonium sulphate at 1% was found to

be the best, yielding 45.54U/ml/min of enzyme at 72 of fermentation. Yeast extract (1%) peptone

(1%) and caesin yielded maximum activity of 26.76U/ml/min, 36.76U/ml/min and

21.58U/ml/min respectively. The present study revealed that among all the nitrogen sources

ammonium sulphate and peptone at 1% proved to be the most suitable for the production of L-

glutaminase by Bacillus subtilis subsp. inaquosorum.

47

Figure 4.8.2

4.9 PURIFICATION OF L-GLUTAMINASE

The purification of L-glutaminase was carried out by four steps as shown in Table 4.9,

with a final yield of 48.4%. The total protein decreased from 115 to 36 mg and the specific

activity increased from 45.54 to 49.33 IU/mg in the ammonium sulphate precipitation step.

These steps lead to a 1.5 fold purification of the enzyme.

4.9.1 EFFECT OF PH ON L-GLUTAMINASE ACTIVITY

The activity of L-Glutaminase was evaluated at different pH values. Purified enzyme

activity was found to be maximum at pH8 for L-glutaminase activity from Bacillus subtilis

subsp. inaquosorum. As such, our findings are in close agreement with the findings of Chitanand

and Shete (2012).

48

Figure 4.9.1

4.9.2 EFFECT OF TEMPERATURE ON L-GLUTAMINASE ACTIVITY

The optimum temperature determined for L-Glutaminase activity from Bacillus subtilis subsp.

inaquosorum by incubating the assay mixtures at different temperature ranging from 4-45°C and

optimum being 37°C. Our findings are in close agreement with the findings of Thadikamal

Sathish and Prakasham (2012)

49

Figure 4.9.2

4.9.3 EFFECT OF NaCl CONCENTRATION ON L-GLUTAMINASE ACTIVITY

The effect of NaCl concentration on L-Glutaminase activity is shown in the figure 4.15.3.

The activity of L-Glutaminase was evaluated at different sodium chloride concentrations.

Krishna Kumar et al., (2011) reported an increase in the activity of NaCl concentration of 3.5%.

The enzymes maximum activity was found at 6% NaCl with 13.98U/ml/min and minimum

activity of 2.15U/ml/min at 8% NaCl.

Figure 4.9.3

4.9.4 EFFECT OF METAL IONS

Different metal ions of 0.1% like magnesium chloride, potassium chloride, calcium chloride

and sodium chloride were used .It was found that MgCl2 gave maximum enzyme activity

(24.56U/ml/min) which was in agreement with the results obtained by Prabhu and

Chandrasekaran(1996) and KCl was found to give least enzyme activity(8.632U/ml/min).

50

Figure 4.9.4

51

SUMMARY

52

5.0.0 SUMMARY

L-glutaminase is an amidohydrolase which catalyses the hydrolytical deamination of

L- glutamine resulting in the production of L-glutamic acid and ammonia. L-Glutaminases have

been the choice of interest in the treatment of lymphoblastic leukemia. In recent years, L-

glutaminase has received significant attention owing to its potential applications in medicine as

an anticancer agent, as an efficient anti-retroviral agent and as a biosensor. In food industries it is

used as a flavor and aroma enhancing agent.

This study presents new and alternative bacterial strains for the production of glutaminase

enzyme which has great potential to be used in industrial and biotechnological processes. One

hundred and thirty two bacterial strains were isolated from various marine water and soil

sources. They were screened in both sea water agar and minimal agar incorporating L-glutamine

and phenol red as pH indicator for glutaminase production. Among them 44 strains were positive

for glutaminase activity by rapid plate assay, as evidenced by pink halos. Two best organisms

identified as Bacillus subtilis subsp. inaquosorum and Acinetobacter baumanii by 16s rRNA

sequencing. Amongst them the Gram positive bacterial isolate, Bacillus subtilis subsp.

inaquosorum, was further subjected to submerged fermentation, using different agro waste

residues.

Various agro waste substrates like Fried gram husk, groundnut cake, wheat bran and cattle feed

were used. Amongst them groundnut oil cake emerged as the best substrate, resulting in high

quantities of glutaminase enzyme (48.5 U/ml) with a zone of 2.2 cm when cultured at 37°C for 5

days. Then different parameters were optimized with optimum pH 8.5, optimum temperature

37oC, supplemented carbon source was found to be maltose, organic nitrogen source as peptone,

inorganic nitrogen source as ammonium sulphate and NaCl concentration around 6%. Crude

enzymes were partially purified using ammonium sulphate precipitation and dialysis. Enzyme

activity was characterized with activity at pH 8, temperature 37°C and Magnesium chloride was

used as activator and potassium chloride used as an inhibitor.

53

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Shah, A.J., Karadi, R.V. and Parekh P.P. (2010). Isolation, Optimization and production of L-asparaginase from coliform bacteria. Asian Journal of Biotechnology 2(3): 169-177.

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Suresh Kumar (2013) Medium optimization for production of L-Glutaminase (EC 3.5.1.2) by Streptomyces griseus under submerged fermentation.

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PAPERS PRESENTED IN THE CONFERENCES DURING PROJECT PERIOD:

1. Attended UGC sponsored One Day State Level Conference “Emerging Trends in Biosciences & Bio Industry”, Organized by the Dept. of Biotechnology, KLE College, Bangalore and presented a poster on 20th February, 2016

2. Attended National Level Technical Symposium “Bio-Chess” 16, Organized by the Dept. of Chemical Technology & Biotechnology, Siddaganga Institute of Technology, Tumkur and presented a paper on 12th March, 2016.

3. Attended DBT sponsored National Symposium “An Integrated Approach to Diagnosis and Therapy in Cancer” and presented a poster on 7th and 8th March 2017.

4. Amrutha Sivanandan, Arjun Sasidharan, Dakshita S. Setty, Sekar K.V., Bharathi. S, and Pramod. T., “Some Investigation on the Isolation and Screening of L-Glutaminase producing strains from various Agro residues by submerged fermentation” 2016, Proc. Of the Conf. on Emerging Trends on Biosciences and Bio Industry., 201-213.

PRINCIPAL INVESTIGATOR PRINCIPAL (Signature with

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