REVIEW OF LITERATURES

REVIEW OF LITERATURES

During the course of human history fungi have been exploited in many

ways. Even before the microorganisms were recognized they were used in

making of beer, wine, bread, cheese, milk products and even for simple eating

(edible mushrooms). The Babylonians used the yeast S.cerevisiae in the brewing

of beer in 6000 BC (Pommerville, 2004). In the eastern world rice was used

instead of malt or mashed grapes for ethanol production and Aspergillus oryzae

was used for hydrolysis of rice starch (Purohit, 2001 )._Mould was later on utilized

for enzyme production Penicillium camemberti and P. roqueforti were used for

secretion of different enzymes (Hamlyn, 1997).

Enzymes are natures answer to many industrial and environmental

challenges. Microbial enzyme plays a key role in microorganism's metabolic

activities and ability to survive under various environmental conditions (Germano

et.al. (1998). As a biocatalyst enzymes replace harsh chemicals in number of

industrial process (Satyanarayana, 2006). This capability has launched an

initiative in scientific community dedicated to enzyme discovery and

development. Because of enzymes very specific catalytic properties only small

quantities of them are required to perform the desired conversions and product

yields are often higher than those obtained with chemical based routes. Derived

from renewable sources, they are fully biodegradable Nester et. a/. (2005).

The study of enzymes is a subject, which has a special interest because

enzymes are of supreme importance in biology. In 1833 Payen and Persoz made

first clear recognition of an enzyme and found that an alcohol precipitate of malt

extract contained a thermolabile substance, which converted starch into sugars.

They named them diastase (Voet, 2006). In eighteenth century many workers

used the name 'ferment' for enzymes (Prescott, 1996). During the second half of

the nineteenth century Liebig postulated that process of fermentation was due to

the action of chemical substances but according to Pasteur the process of

fermentation was inseparable from living cells (Adria and Demain, 2003). The

name 'unorganized ferment' and 'organized ferment' were also used for extracted

9

enzyme. In order to avoid these unsatisfactory names, Kuhne in 1878 introduced

the name 'enzyme' (Stryer, 2008).

Towards the end of the nineteenth century increasing knowledge in the

structural and organic chemistry of biomolecules made it possible to study the

specificity of enzymes. In 1897 Buchner succeeded in obtaining the fermentation

system from yeast in a cell free extract. Emil Fisher gave the idea of enzyme

specificity Pelczar et.al. (2002). The serious purification of enzymes began after

1920. Willstatter and his colleagues carried out purification of enzymes between

1922 and 1928 (Laskin and Lechevalier, 1984 ). The next important development

was the preparation of enzymes in crystalline form. The first enzyme to be

crystallized was urease by Sumner in 1926. This work was soon followed by

classical isolation of crystalline proteolytic enzymes by Northrop and his

colleagues (Dixon et.a/.1979).

Studies on penicillin from Penicillium were carried out by McKee et.al.

(1944). Lowry et.al. (1951) discovered a new technique for protein measurement

with folin phenol reagent. Glucose was estimated by Miller (1959). Identification

of fungal species was reported by Barnett ( 1969) and Ellis ( 1949). Methods for

estimation of proteolytic activity in an organism is defined by Nakagawa (1970)

and also quantitatively measured by Yasunobu and McConn (1970).

Identification of fungi by using new classification system was suggested by

Margulis (1971). The effect of glucose and manganese on adenosine 3', 5'

monophosphate levels during growth and differentiation of Aspergillus nidulans

was studied by Zonneveld (1976). Comparison of a-amylase activity from

different assay method was analysed by Yoo et.al. (1987). Fungi have various

applications in biotechnology (Wainwright, 1990 and Wainwright, 1995). Process

for protein enrichment was analysed by Nigam and singh (1994). Optimization of

culture conditions for Aspergillus niger on addition of nitrogen sources was

studied by Pandey et.al. (1994). Safer procedure for routine staining of vesicular­

arbuscular mycorrhizal fungi was discovered by Grace and Stridbley (1995).

Isolation of microorganism from soil by serial dilution method was reported by

Tate (1995). Heterologous protein production in Aspergillus was studied by

10

Hombergh ven den et.al. (1997). Screening of fungi for enzyme producing ability

was performed by Dehran and Davies ( 1999). Applications of industrial enzyme

were reported by Ole Kirk et.a/. (2002). Protein quantitation and its biochemistry

were studied by Walsh and Walsh (2002). Effects of process parameters on

heterologous protein production in Aspergillus niger fermentation was studied by

Wang et.al. (2003). Sallam et.al. (2003) measured role of some fermentation

parameters on cyclosporine production by Aspergillus terreus. Molecular and

cellular biology of filamentous fungi was reported by Talbot (2005). Production of

enzymes from fungi was also reported by Nutan et.al. (2003). Physiology and

biochemistry of Aspergillus was studied by Ward et.al. (2006). Shimizu et.a/.

(2009) studied the proteomics of Aspergillus nidu/ans.

In past century number of purified enzymes isolated was very small,

whereas now the pure and crystalline enzymes exceeds more than fifteen

hundred but still the requirement for purified enzymes are high. Thermostable

protease had find major application in detergent industry as they have high

temperature and pH tolerance and are able to withstand harsh conditions that

occur during washing. Hashimoto et.al. (1972) found a thermostable acid

protease produced by Penicillium duponti K1014, was a true thermophilic fungus

isolated from compost. They further characterized the acidic protease as thermo

tolerant and acid sensitive. Gill and Modi (1981) suggested extracellular protease

activity in Aspergillus nidulans. Many strains of the thermophilic fungus

Thermomyces lanuginosus were screened for their ability to produce proteases

by Chuan Li et.a/. (1997). According to their finding selected strain Thermomyces

lanuginosus P134, showed high enzymatic activity for proteases in submerged

culture. They found two protease isoenzymes by SDS-PAGE which was serine

protease. A thermostable alkaline protease was purified and characterized by

Kaur et.al. (1998) from a Bacillus polymyxa which was useful in laundry

industries. A study on thermostable alkaline protease from alkalophilic Bacillus

sp. /S-3 was performed by Purva et.al. (1998). Thermostable alkaline proteases

from Bacillus sp. have attracted the attention of many researchers due to their

use m detergents (Sumandeep et.al. 1999). Merheb et.al. (2007) partially

II

characterized a protease from a thermophilic fungus Thermoascus aurantiacus,

and found its hydrolytic activity on bovine casein.

Aspergillus, Rhizopus, Mucor, and Actinomucor have long been used in

oriental food fermentations. Solid state fermentation for the production of

industrial enzymes was performed by Pandey ( 1991 ). Malathi and Chakraborty

(1991) examined the production of alkaline protease by a new Aspergillus flavus

isolates under solid-substrate fermentation conditions and also found the use of

alkaline protease as a depilation agent. Bockelmann (1995) suggested proteases

from Bacterium. Production of proteolytic enzymes by

solid state fermentation was performed by Mitra et.al. (1996). Keratinolysis by

poultry farm soil fungi was studied by Kaul and Sumbali (1997). In recent years

several fungi and bacterial strains have been immobilized with different type of

supporting materials for production of enzymes. Production of alkaline protease

by immobilized Aspergillus mycelia was carried out by Nehra et.a/. (1998).

Sindhu and Shashidhar (1998) found protease producing fungi, which was

qualitatively enumerated, from marine and estuarine environments of Kerala

according to their studies species of Aspergillus, Rhizopus, Penicillium,

Saccharomyces, Mucor and Neurospora were predominant for protease

production. Growth and autolysis of two strains of the entomopathogenic

Deuteromycete fungus Metarhizium anisopliae var. anisop/iae were studied by

Braga et.al. (1999) in the medium containing casein or glucose. Parameters such

as economic coefficient and degree of autolysis were determined for each strain

and also the protease production during growth and autolysis of submerged

Metarhizium anisopliae cultures. The extracellular serine proteases are important

for hydrolysis of proteins in cell free environment and enable the cell to absorb

and utilize hydrolytic products. Alkaline protease from Aspergillus oryzae apply

as a silver recovery agent from used X-ray film was evaluated by Warin and

Tanticharoen (1999). Van Kuyk et.a/. (2000) carried out analysis of two

Aspergillus nidulans genes encoding extracellular proteases. A gene, isp-b,

encoding an intracellular serine protease from Bacillus sp WRD-2 was cloned

and characterized by Sun-Young An et. a/. (2004 ). Proteases are used in laundry

12

detergents for over 50 years to facilitate release of proteinaceous material in

stains (blood and milk) and account for approx 25% of total worldwide sales of

enzymes. Kumar and Bhalla (2004) had isolated new protease as a laundry

additive from Bacillus sp. APR-4. Using a phylogenomic approach with 10 fungi

of very different virulence and habitat Hu and Leger (2004) determine proteases

in Ascomycetes. Sandhya et.al. (2005) observed neutral protease activity in A.

oryzae.

Proteases play a specific catalytic role in hydrolysis of proteins and are

also used in industries like leather, food, photography and pharmaceuticals.

Studies on process and nutritional parameters for the production of extracellular

alkaline protease from Thermoactinomyces thalpophi/us PEE14 was carried out

by Ellaiah et.al. (2005). Dutta et.al. (2005) studied the kinetics of a low molecular

weight protease from newly isolated Pseudomonas sp. using artificial neural

network. Most of the fungi are characterized by their ability to produce enzymes

to the external environment. Many species of genus Mucor have important

biotechnological potential, which are also responsible for production of industrial

enzymes. Maria et.al. (2005) evaluated the ability of protease production in

twelve species of genus Mucor. Depilation or dehairing of hides and skins, in

leather industry, was traditionally done by chemical method but now a day it is

done by using enzyme protease. Mitra and Chakrabartty (2005) extracted an

extracellular protease with depilation activity from Streptomyces nogulator.

Srinivasan et.al. (2005) also reported high alkaline protease activity by a

saprophytic strain of Conodiobolus. Bajra et.al. (2005) suggested the existence

of Ca2·-dependent protease II in crude extracts of Neurospora crassa and

Uromyces appendiculatus demonstrated by immunoblotting using specific

antibodies. Immunofluorescence of the enzyme was predominantly localized in

the apical regions of germlings and growing hyphae which suggested a functional

role for the enzyme in hyphal growth.

According to Banik and Prakash (2006) an alkalophilic bacterium, Bacillus

cereus produced an extracellular alkaline protease, which was found to be active

at high temperature and pH range, suitable for commercial laundry detergents.

Serine proteases secreted by nematode and insect pathogenic fungi are bio­

control agents, which have commercial potential for developing into effective bio­

pesticides. A thorough understanding of the structural and functional features of

these proteases was carried out by Liu et. a/. (2007) who had characterized the

structural features of cuticle-degrading proteases from fungi by molecular

modeling. Charles et.al. (2008) reported protease in Aspergillus nidulans.

Alpha amylase is starch-hydrolyzing enzyme, which has many

applications in various fields. A study on Intracellular uptake and a-amylase,

lactate dehydrogenase releasing actions of divalent cations ionophore A23187 in

dissociated pancreatic acinar cells was performed by Chandler and Williams

(1977). Ramesh and Lonsane (1990) dealt about the importance of moisture

content of the medium in alpha-amylase production by Bacillus /icheniformis M27

in a solid-state fermentation system. They observed large reduction in the

production of alpha-amylase by Bacillus licheniformis M27 in standardized wheat

bran medium under solid-state fermentation when the moisture content of the

medium is high. Yang and Wang (1999) detected amylase and protease activity

in Streptomyces rimosus by submerged and solid state cultivations. A study on

catabolic repression in solid state fermentation for biosynthesis of fungal

amylases was performed by Nandakumar et.al. (2002). Ramachandran et.al.

(2004) carried out solid-state fermentation (SSF) using coconut oil cake (COC)

as substrate for the production of amylase using a fungal culture of Aspergillus

oryzae, optimization of process was also done by changing carbon sources,

temperature of fermenter, different nitrogen sources. They also found the

relationship of amylase production with biomass of fungi. A study on solid state

fermentation (SSF) or solid substrate fermentation for growth of microorganisms

in a moist solid substrate with air as continuous phase was carried out by

Rahardjo et.al. (2005) and also described presence of aerial mycelia of A. oryzae

resulted in a strong increase in fungal biomass and a-amylase production. Patel

et.al. (2005) worked on partial purification and biochemical characterization of an

alpha amylase produced by a mesophilic mould, Aspergillus oryzae by solid-state

fermentation using spent brewing grains as substrate. Traditionally amylase has

14

been produced by submerged fermentation. In recent years, however solid-state

fermentation process has been increasingly utilized for production of enzyme.

Kunamneni et.al. (2005) worked on amylase production in solid-state

fermentation by the thermophilic fungus Thermomyces lanuginosus.

Sivaramakrishnan et.al. (2007) studied the production of amylase from

Aspergillus oryzae employing solid-state fermentation.

Barnett and Fergus (1 971) investigated the relation of extracellular

amylase production with time in some thermophilic and mesophilic Humicola

species. They also found the relationship between starch concentration and

amylase production. Many thermophilic fungi are very well known for amylase

production. Adams and Deploey (1976) screened and selected amylase

producing fungi which were thermophilic also. According to them Mucor meihei

and M. pusil/us was amylase producing thermophilic fungi. Basaveswara et.al.

( 1981) carried out purification and characterization of a thermostable

glucoamylase from the thermophilic fungus Thermomyces lanuginosus. Jensen

et.al. (1987) reported an extracellular amylase by a thermophilic fungi

Thermomyces lanuginosus in shake flask cultures with different carbon sources

in the growth medium. According to them the greatest yield of amylolytic activity

was found with dextrans as carbon source. Bunni el.a/. (1989) studied

production, isolation and partial characterization of an amylase system produced

by Talaromyces emersonii CBS 814.70. Thermostable a-amylases have

applications in a variety of industrial processes and enzymes from a substantial

number of thermophilic bacteria and fungi have been screened and characterized

to varying degrees. Haasum et.al. (1991) discussed the production of glycogenic

amylase from the thermophilic fungus Thermomyces lanuginosus in shake flasks

and laboratory fermenters in the synthetic medium in which nitrogen source was

replaced by some other source and also found out the stability of enzyme at

different pH as well as in absence of substrate. Different factors affecting growth

and amylase production by fungi inhabiting poultry feeds was examined by

Mahmud (1993). Ward et.al. (1995) stated about thermotolerant, ethanol­

producing yeast strain, Kluyveromyces marxianus IMB3 which was shown to

15

produce ethanol at 45°C on starch-containing media supplemented with a crude

amylase preparation derived from the thermophilic, filamentous fungus

Talaromyces emersonii CBS 814.70. Uguru et.al. (1997) used yam peel as a

carbon source to produce extracellular amylase in shake flask cultures from a

thermophilic strain of Aspergillus niger and also optimized the culture conditions

for better amylase production. Arnesen et.al. (1998) studied the thermophilic

fungus Thermomyces lanuginosus. Protein production was also recognized in

those fungi. Rubinder et.al. (2000) performed a detailed studied on a

recombinant strain produced by intraspecific protoplast fusion of thermophilic

fungus Thermomyces lanuginosus strains which were amylase hyper-producing

fungi characterized by 2-deoxy-D-glucose resistant markers. Petrova et.al.

(2000) examined the production and characterization of extracellular a-amylases

from the thermophilic fungus Thermomyces lanuginosus (in both wild and mutant

strains). Optimization of culture conditions for thermostable amylase by Bacillus

sp. was done by Carlos and Meire (2000). Studies on a thermostable a-amylase

from the thermophilic fungus Scytalidium thermophilum was carried out by

Aquino et.al. (2003). Fossi el.a/. (2005) measured the production and partial

characterization of a thermostable amylase from ascomycetes yeast strain

isolated from starchy soils.

Kundu and Das (1970) studied the production of amylase in liquid culture

by a strain of Aspergillus oryzae and also studied the effect of different media

and pH on the formation of amylase by Aspergillus oryzae El 212 For secretion

of amylase, some of the properties of the partially purified a-amylase were

different from a-amylases from other sources. The production of a-amylase (a-

1 ,4 glucan-4-glucan hydrolase; E.C. 3.2.1.1.) by a strain of Bacillus /icheniformis

has been studied by Meers ( 1972) in batch and continuous cultures by addition

of glucose as repressor and starch as inducer. Hankin and Anagnostakis (1975)

used solid media on which production of different enzymes like amylase,

protease was detected by different pathogenic and saprophytic fungi. Oso (1979)

studied the mycelial growth and amylase production by Talaromyces emarsonii,

in stationary liquid starch- yeast-extract after autolysis. Wilson and lngledew

16

(1982) performed isolation and characterization of amylolytic enzyme from

Schwanniomyces al/uvius. Federici (1982) showed that Aureobasidium pullulans

was a promising source of lytic enzymes by screening ninety eight strains of

Aureobasidium and tested many extracellular enzymes like amylase. Production

and characterization of amylase from Calvatia gigantean was perfomed by Kekos

and Macris (1983). Kindle (1983) characterized enzymes and studied the

genetics of a-amylase production in Bacillus subtilis and recombinant DNA

approaches to increasing a-amylase production. A bacterial strain, Bacillus

licheniformis was isolated by Bajpai and Bajpai (1987), which produced high

temperature alkaline alpha amylases. They had also optimized culture conditions

and studied high temperature resistance amylase that can tolerate 100°C

temperature. Kekos et.al. (1987) investigated a number of nutritional factors

affecting a-amylase production by the edible fungus Calvatia gigantean,

cultivated in a wheat bran liquid medium. Mountfort and Asher (1988) analysed

the production of a-amylase by the rumina! anaerobic fungus Neocallimastix

frontalis. Cloning, characterization, and expression of two a-amylase genes from

Aspergillus niger var. awamori (Aspergillus awamori) was evaluated by Korman

et.al. (1990). Laderman et.al. (1993) reported a a-amylase from Pyrococcus

furiosus, a hyperthermophilic archaebacterium. Sogaard et.al. (1993) did the

characterization of post-translational modifications of barley a-amylase produced

in yeast by electrospray mass spectrometry. Okolo et.al. (1995) isolated

Aspergillus niger from rotting cassava which produced raw starch degrading

amylase on cassava, maize, sorghum and soluble potato-derived starch as the

sole carbon source without prior gelatinization. Covalent immobilization of a­

amylase onto pHEMA microspheres and its application to fixed bed reactor was

carried out by Arica et.al. (1995). Continuous cultivations of an a-amylase

producing strain of Aspergillus oryzae were carried out by Carlsen et.al. (1996)

using a chemically defined medium with glucose as the growth-limiting

component. Woloshuk (1997) investigated about the aflatoxin biosynthesis

induced by compounds in filtrates (EF) obtained from cultures consisting of

ground maize kernels colonized by Aspergillus flavus. Amylase activity was

17

detected in the EF. Analysis of the enzyme by isoelectric focusing

electrophoresis indicated the PI of amylase. Gouka et.al. (1997) studied

filamentous fungi for the production of homologous and heterologous proteins

but, compared to homologous proteins, the levels of production of heterologous

proteins were usually low; a detailed analysis of the levels of production of

several proteins and glucoamylase fusion proteins in defined recombinant

Aspergillus awamori strains was also carried out by them.

Rajanikanth and Ravi ( 1998) had partially purified amylase produced from

Bacillus sp. In recent years, several amylolytic yeast species especially of the

genera Saccharomycopsis and Schwanniomyces have been characterized in

view of their potential utilization for the conversion of starch biomass into ethanol.

An amylolytic yeast Saccharomycopsis fibuligera was exploited by Gogoi et.al.

(1998) for their extracellular a-amylase production. Three amylase-producing

strains of Aspergillus oryzae used for recombinant protein production were

studied by Spohr et.al. (1998) in fed-batch and continuous cultures. They also

compared the three strains with respect to morphology of the fungus and alpha

amylase production during submerged growth. A novel amylase was isolated by

Mohapatra et.a/. (1998) from the Mucor sp. associated with the marine sponge

Spirastrella sp., grown at 30'C, optimum pH, temperature and half lives was also

calculated followed by estimation of activation, deactivation energies. Reddy and

Reddy (1998) studied amylase production from three isolates of Myrothecium

roridum and found the variability in the production of amylase in three isolates.

Goto et.al. (1998) isolated a strain of Aspergi/lus fumigatus from soil; strain was

able to produce amylase in media containing a-methyl-o-glucoside (aMG), a

synthetic analogue of maltose, as the only carbon source. aMG was a more

effective inducer of alpha amylase than starch and maltose. Soni et.al. (1998)

analyzed catabolic repression of f3-glucosidase and amylase in Chaetomium

erracticum in the presence of glucose and glycerol, which accompanied an

increase in protease production. Aspergillus oryzae has efficient system for

secretion of protein and it is extensively used to produce amylases. Singh et.al.

(1998) considered the importance of protoplast in genetic and strain

IX

improvement and various physiochemical factors affecting protoplast isolation in

Thermomyces lanuginosus, which was a amylase producing fungi. Aspergillus

oryzae is extensively used to produce several industrial enzymes including

amylases. Ray and Chakraverty ( 1998) evaluated many characters of

extracellular [beta]-amylase from Syncephalastrum racemosum.

Chen et.al. (1999) observed the inhibition of fungal growth in potato

dextrose broth medium by the 14-kDa-corn trypsins inhibitor (TI) protein. Further

investigation found that Tl inhibited fungal production of extracellular a-amylase

from Aspergillus flavus. Several types of enzymes are involved in degradation of

starch mainly a-amylase, [3- amylase and glucoamylase and among these a­

amylase plays important role. a-amylase are common in Aspergillus sp. and

Rhizopus sp. and are often used as sources of industrial amylases. Petersen

et.al. (1999) studied the transcriptional activators for amylase genes in

Aspergillus. Moreira ( 1999) considered a strain of Aspergillus tamarii isolated by

soil during a screening programme for xylanases, the purpose of that work was

to investigate the ability of Aspergillus tamarii to produce amylase. In that

investigation he also studied the effect of pH and temperature in the enzyme

activity. Agger et.al. (2000) investigated the growth and enzymes formation from

Aspergillus oryzae during submerged cultivations with the help of morphologically

structured model using fluorescent probes. The model was able to produce

accurate simulations of steady state and transient conditions in chemostats, of

batch cultivations, and even the formation of a single hypha! element from a

spore. A selection and characterization of a high a-amylase producing variant in

glucose limited continuous cultures of Aspergillus oryzae performed by

Zangirolami et.al. (2000). Sago starch degrading enzyme production from

Acremonium sp. endophytic fungus was isolated by Marlida et.a/. (2000).

Fusarium moniliforme and Aspergillus flavus have been reported to

produce fumonisins, mycotoxins. Figueira and Hirooka (2000) study mycelial

growth and amylase production by these mycotoxigenic strains and optimized the

culture conditions for the proper growth and development of fungi. Socking et.al.

(2000) developed highly branched mutants of two strains of Aspergillus oryzae

19

(IF04177), which produced alpha amylase and a transformant of IF04177, which

produced heterologous glucoamylase in addition to a-amylase; they were

generated by UV or nitrous acid mutagenesis. Teodoro and Martins (2000)

studied an a-amylase from a bacterial strain isolated from soil. Studies involved

cultivation of bacterial colonies in a mineral medium containing soluble starch as

sole carbon source. They also optimized and characterized the amylase of

bacterial origin. Culture medium for amylase production by toxigenic fungi was

studied by Luiz et.al. (2000). Molecular basis of glucoamylase overproduction by

mutagenesis industrial strain of Aspergillus niger was studied by MacKenzie

et.al. (2000). Agger et.al. (2001) studied the effect of biomass concentration on

the formation of a-amylase from Aspergillus oryzae, during submerged cultivation

with Aspergillus oryzae and recombinant Aspergillus nidulans strains. It was

suggested that the specific rate of a-amylase formation in chemostats decreased

significantly with increasing biomass and also reported the induction of amylase

required a substrate having a 1-4 glucoside bond including maltose, dextrins and

starch. Aspergillus ochraceus which produced amylase was reported by Nahas

and Waldemarin (2002) and they studied the control of amylase production by

Aspergillus ochraceus. A study on amylases of fungus Aspergillus flavipes

associated with Fucus evanescens performed by Frolova et.al. (2002). Isolation

and screening of fungi for the biosynthesis of alpha amylase was carried out by

Haq et.al. (2002). A study on glucoamylase production under solid state

fermentation by a newly isolated Aspergillus sp. was performed by Ellaiah et.al.

(2002). Properties and applications of starch-converting enzymes of the a­

amylase family were reported by Marc et.al. (2002).

Wanderley et.al. (2004) also performed biochemical characterization of

alpha- amylase from the yeast Cryptococcus flavus. Carlsen and Nielsen (2004)

studied the influence of carbon source on alpha amylase production by

Aspergillus oryzae they indicated that glucose acts as an inducer for amylase

production. Ravikumar et.al. (2004) studied the cleavage of the precursor

enzyme by autocatalysis secretion of multiple amylases by Aspergillus niger.

Vahidi et.al. (2005) stated that optimization of cultivation conditions was expected

20

to improve the enzyme production. They investigated the effect of different

cultivation conditions on growth and amylase production by the isolated Mucor

sp. using the variable size simplex algorithm analyses. The characterization of

extracellular microbial enzymes is important for understanding their role in the

pathogenesis of infectious diseases, as they play a major role in causing

cytotoxicity in mammalian cells as well as their application in biotechnology.

Serratia a gram-negative enteric bacterium highly pathogenic to humans was

isolated from soil and tested for its extracellular amylase, protease-producing

capability by Sharma and Tiwari (2005). Interaction of a-amylase produced by

Bacillus amyloliquefaciens with magnesium ions and its thermodynamic study

was performed by Saboury et.al. (2005). Noman et.al. (2005) found some

properties of alpha amylase from post harvest Pachyrhizus erosus L tuber.

Isolation and characterization of amylase from fermentated cassava waste was

perfomed by Oboh (2005). Mithu et.a/. (2005) purified and characterized a­

amylase from the culture filtrate of Bacillus amyloliquefaciens NCIM 2829.

Endosulfan, a chlorinated hydrocarbon insecticide of cyclodiene subgroup acts

as a contact poison in a wide variety of organisms. A thermostable maltose

tolerant amylase was isolated from Aspergillus tamari by Moreira et.al. (2005).

Identification and transcriptional regulation of starch modifying enzymes in

Aspergillus niger genome was examined by Yuan et.al. (2005). Nagarajan et.al.

(2006) carried out purification and characterization of a maltooligosaccharide

forming alpha amylase from a Bacillus subtilis KCC103. Hernandez et.al. (2006)

performed amylase production by Aspergillus niger in submerged cultivation on

two wastes from food industries. Tolan and Ensari (2006) investigated the effect

of endosulfan on growth a-amylase activity and plasmid amplification in Bacillus

subti/is system. Rodriguez (2006) reported the enzymatic hydrolysis of soluble

starch with amylase from Bacillus licheniformis at pH 7.5; the enzyme activity

was measured by iodometric method. They had also studied the kinetic

properties of amylase. Djekrif-Dakhmouche et.al. (2006) worked on the

production optimization of a-amylase (E.C.3.2.1.1.) from Aspergillus niger ATCC

16404, it was obtained with statistical experimental designs, using orange waste

21

powder as substrate. Kathiresan and Manivannan (2006) studied alpha amylase

production by Penicillium fellutanum isolated from mangrove rhizosphere soil.

Ezeji and Bahl (2006) characterized and purified amylase from Geobacillus

thermodenitrificans HR010. Qader et.al. (2006) analysed production and

extracellular activity of commercially important amylolytic enzyme by Bacillus sp.

AS-1. Ramesh et.al. (2006) studied purification and characterization of a

thermophilic a-amylase of Aspergillus niger van Tieghem. Characterization of a­

amylase immobilized on collagen membranes was performed by Strumeyer et.al.

(2006). Prakasham et.al. (2007) evaluated the influence of factors on acid

amylase production by isolated Aspergillus awamori. According to their study the

pH of the fermentation medium and substrate concentration regulates maximum

enzyme production process.

Alva et.al. (2007) isolated Aspergillus species from various seeds and

screened for their ability to produce amylase. Rao et.al. (2007) studied the

bioprocess strategies involved in the production of amylase from different

microbial sources. They also studied about glucoamylase, molecular biology of

amylases and application of commercially available enzymes. Optimization of

nutrients and cultivation conditions in glucoamylase production was studied by

Lewis and Sinkar (2007). The development of industrial biotechnology

processing has led to utilization of microbial enzymes in various applications.

One of the industrially important enzymes is amylase. Isolation of amylase

producers is generally performed on starch agar plates, which restrict starch as

sole carbon source, and agar is an expensive component this component can be

replaced by sago (sabu) plates which is cheaper (Binky et.al. 2007). Whereas

Van der kaaji et.al. (2007) performed phylogenetic and biochemical

characterization of a novel cluster of intracellular fungal alpha- amylase

enzymes. Purification, biochemical characterization and gene cloning of a new

extracellular thermotolerant and glucose tolerant maltooligosaccharide-forming a­

amylase from endophytic Ascomycetes Fusicoccum sp. BCC4124 was carried

out by Champreda et.al. (2007). Gouda and Elbahloul (2008) measured

statistical optimization and partial characterization of amylases produced by

halotolerant Penicillium sp. The effect of growth temperature on the production of

amylases by yeast Lipomyces kononenkoae was analysed by Estrela et.al.

(2008). Amylase activity of a starch degrading bacteria isolated from soil

receiving kitchen waste was estimated by Mishra and Behera (2008).

Many raw food materials are known to contain various types of inhibitors

which affect nutritional quality and out of these some of them are proteins which

decrease specific enzyme activity. A strain of fungi Cladosporium herbarum

extracellularly produced inhibitors specific for mammalian a-amylase. Saito

(1 982) had purified inhibitor of Cladosporium herbarum by freeze thawing, heat

treatment and column chromatography on DEAE-cellulose, Sephadex G-75

because they may be of value as novel therapeutic and dietetic agents. Column

studies for biosorption of dyes from aqueous solutions on immobilized

Aspergillus niger fungal biomass was performed by Fu and Viraraghavan (2003).

Varbanets et.al. (2004) specified the ability of some species to synthesized

compounds of germanium with bioligands to affect the biosynthesis and activity

of alpha amylase. Inhibitors of amylase have received considerable attention in

recent years. Biosynthesis of a-amylase of Bacillus licheniformis 234 was

inhibited by a number of synthesized compounds which are inhibitor of amylase.

Fossum and Whittaker (2007) find out amylase inhibitors in biological materials.

Purification of amylases is important step because contaminated enzyme

with other materials affects specific activity. Purified amylases works well in

important industrial process. Zakowski and Bruns (1 982) describe a column

chromatography method for measuring amylase activity by using mini column

DEAE-Sephadex system. Zakowski et.al. (1984) develop a method for

purification of alpha amylase. They develop a DEAE Sephadex ion exchange

chromatography and isoelectric focusing. Machaiah and Vakil (1984) had purified

and separated a-amylase by gel chromatography into two distinct entities a­

amylase I and a-amylase II. An extreme thermophilic fungus, Thermus sp. was

exploited by Shaw et.al. (1995) for their extracellular a- amylase. They further

purified amylase and its molecular weight was determined by SDS PAGE.

1' -·'

Laemmli (1970) developed method for separating proteins using SDS

PAGE electrophoresis. Ferraz et. a/. (2000) studied protein profiles of

Mycoplasma gal/isepticum on SDS-PAGE. Ibrahim et. a/. (2001) studied metal

binding protein of Pseudomonas diminuta using SDS PAGE. According to Lanoot

et. a/. (2002) whole-cell proteins can be used for grouping bacteria like

Streptomyces aurantiacus, Streptomyces cacaoi, Streptomyces caeruleus and

Streptomyces violaceus by SDS- PAGE.

The bioconversion of agro-waste based lignocellulosic material to energy

has gained much interest during the recent past. The enzymatic degradation of

waste cellulose by fungal enzymes has been suggested as a feasible alternative

for the conversion of lignocellulosic material. Mandels and Reese (1960) studied

induction of cellulase in fungi by cellbiose. Hulme and Stranks (1970) suggested

about induction and the regulation of cellulase production by fungi and

investigated that synthesis of cellulases from fungi was considered to be induced

by cellulose substrates, or more specifically by their water soluble short chain

depolymerization products such as cellobiose. Varadi (1972) studied the effect of

aromatic compounds on cellulase and xylanase production from fungi

Schizophyllum commune and Chaetomium globosum. Coutts and Smith (1976)

analyzed the factors influencing the production of cellulases by Sporotrichum

thermophile. Cellulase production and growth of a strain of Sporotrichum

thermophile were studied by using a mineral salts medium supplemented with

yeast extract and insoluble cellulose. The effects of cultural conditions, such as

pH, nitrogen source, substrate concentration, and temperature, were also

examined by them.

Sternberg (1976) was performed a detail study on cellulase complex by

optimizing the culture conditions and their repression in Trichoderma viride.

Further improvements of cellulase yields are being sought by continued

mutagenesis and increased nutrient levels in the growth medium. Hurst et.al.

(1977) reported cellulase activity in A. niger. Mountfort and Asher (1985)

discussed production and regulation of cellulase by two strains of the rumen

anaerobic fungus Neocallimastix frontalis. Grajek (1987) examined six

24

thermophilic fungi for their ability to produce cellulolytic enzymes in liquid (LF)

and solid-state fermentation (SSF). The best cellulase activities were also

determined in Thermoascus aurantiacus and Sporotrichum thermophile, thermal

and pH characteristics of cellulases were evaluated. Mordcawa et.al. (1985)

reported improvement of cellulase production in Trichoderma reesei. Saccobolus

saccoboloides a coprophilous fungus was grown in synthetic liquid media.

Extracellular ~-glucosidase, ~-1, 4 endoglucanase and ~-1, 4 exoglucanase

induction and repression by carbohydrates were investigated. Cellulase

production by this fungus is inducible and subject to a complex repression by

easily metabolized sugars. Acebal et.al. (1986) were able to enhance the

cellulase production from Trichoderma reesei OM 9414 on physically treated

wheat straw. Detection and quantitation of cellulase by congo red was carried out

by Carder (1986). Improvement of enzyme production in Aspergillus was studied

by Finkelstein (1987). Brown et.al. (1987) isolated and discussed the properties

of a mutant fungus Penicillium pinophi/um with enhanced cellulase and ~­

glucosidase production. Feldman and Lovett (1988) studied the isolation and

regulation of the cellulase enzymes from the thermophilic fungus Thermoascus

aurantiacus. Maivan and Shearer (1988) examined wood decay activity and

coupled cellulase production for freshwater lignicolous, Ascomycetes,

Deuteromycetes and an Oomycete. Wood decay ability was assessed by weight

changes in wood and bark blocks of ash and cottonwood colonized by test fungi.

Changes in wood components were also measured. Durand et.al. (1988)

investigated strategy based on plate screening tests designed for the selection of

mutant strains of the fungus Trichoderma reesei suitable for cellulase (EC

3.2.1 .4) production on an industrial scale. The selected mutant generations

successively isolated was able to fulfill all of the three criteria: ( 1) improved

productivity compared to the previous one, (2) high stability (3) ability to be

further improved. Bagga et.al. (1989) investigated that the cellulase complex of

Aspergillus nidulans was found to undergo catabolite repression in the presence

of glucose and glycerol accompanied by an increase in protease production,

which apparently caused inactivation of cellulolytic enzymes in vitro. Cheng et.a/.

25

(1990) isolated a pyr G mutant of Trichoderma viride, a very efficient cellulase

producer, from among 5-fluoroorotic acid-resistant mutants. The mutation was

complemented with the pyr 4 gene of Neurospora crassa and used as a selection

marker for the transformation ofT. viride. A plasmid vector, pDJB1-Taa, carrying

both the pyr 4 gene and a gene encoding Taka-amylase from Aspergillus

oryzae, was constructed and introduced into protoplasts of T. viride pyr G.

Esterbauer, et.al. (1991) reported the production of Trichoderma cellulase in

laboratory and pilot scale from a mutant Trichoderma reesei QM 9414, MCG 77,

MCG 80, RUT C 30, CL-847, VTT-D, and SVG. Persson et.al. (1991) suggested

that cellulolytic enzyme is easily produced and purified from Trichoderma reesei

mutant species, the enzyme yield can be increases by strain improvement,

substrate concentration and cultivation conditions. Optimization for cellulase

production by Aspergillus niger NCIM 1207 was performed by Gokhale et.al.

( 1991 ). Wayman and Chen ( 1992) investigated cellulase production by

Trichoderma reesei using whole-wheat flour as a carbon source. Garcia-Garrido

et.al. (1992) discussed the production of endoglucanase (EC 3 2 1.4) and

exoglucanase (EC 3 2.1 91) enzymes during penetration of the host and

development of the vesicular-arbuscular mycorrhizal (VAM) fungus, Glomus

mosseae, in roots of lettuce (Lactuca sativa) and onion (Allium cepa). Cellulase

activity was detected in VAM roots which was attributed to the fungus tested on

the basis of electrophoretic mobility. Malviya et.al. (1992) carried out synthesis

and regulation of extracellular keratinase in three fungi isolated from the grounds

of a gelatin factory, Jabalpur, India. A study on ethanol production in SSF

through saccharification was performed by Xin et.al. (1993) in which paper mill

waster fiber (WF) from the ammonium sulfite pulp process was used. The

process was improved by the supplementation of the cellulase from Penicillium

decumbens JU-A10 with the ~-glucosidase-rich cellulase from Aspergillus niger

L22. Steiner et.al. (1994) maintained the culture conditions for enhanced

cellulase production by a native strain of Penicillium purpurogenum.

Duenas et.al. (1995) studied Trichoderma reesi LM-UC4 and Aspergillus

phoemcis QM 329 culture in ammonia-treated bagasse with 80 % ( wlw) moisture

26

content through solid-substrate fermentation (SSF) in flask or pot fermenters, for

cellulase production. Significantly higher activities of all the enzymes of the

cellulase complex were studied. Keranen and Penttila (1995) investigated the

production of recombinant proteins in the filamentous fungus. They exploited the

potential of the filamentous fungus Trichoderma reesei for producing

heterologous proteins and also determine rate-limiting steps and ways of

improving the production, especially using antibody Fab fragments. Major

improvements were achieved by producing the foreign protein fused to the fungal

cellulase cellobiohydrolases. Pardo (1996) described the effect of different

nonionic surfactants (Tween 80, Tween 20, Triton X-100) and polyethylene glycol

(PEG 6000) on cellulolytic enzyme system production by Nectria catalinensis.

Gutierrez-Correa and Tengerdy (1997) investigated Trichoderma reesei

LM-UC4 and its mutant LM-UC4E1 and co-cultured with Aspergillus phoenicis

QM329 for cellulase production on bagasse by mixed culture solid substrate

fermentation and successfully analyzed a mutual synergism between the parent

Trichoderma strain and the Aspergillus which enhanced combined biomass

production and corresponding increase in cellulase, endoglucanase and

glucosidase activities. Umikalsom et.a/. (1997) worked on the feasibility of using

delignified oil palm empty-fruit-bunch (OPEFB) fibres as a substrate for cellulase

production by Chaetomium globosum strain 414 in shake-flask cultures

containing different types and concentrations of nitrogen source. Cellulose

degrading enzymes and their potential industrial applications was analysed by

Bhat and Bhat (1997). The cellulase mixture, partially purified by ammonium

sulphate precipitation, capability of hydrolyzing delignified OPEFB fibers was also

characteized. Correia et.al. (1998) established a modified method for direct

determination of cellulolytic activity using Avicel colored with Remazol dye in

Basidiomycetes. Romero et.al. (1999) reported the production of the cellulase

complex by the fungus Neurospora crassa on milled and sieved wheat straw.

The effects of straw concentration, temperature, and initial pH on the production

of the ~-glucosidase, exoglucanase, and endoglucanase activities, as well as

27

extracellular and mycelial protein were also observed. Potential application of

enzymes in waste treatment was reported by Karam and Nicell (1999).

Domingues et.a/. (2000) examined the morphology of Trichoderma reesei

Rut C-30, during submerged cultivations in shake flask, and the influence of the

size of inoculums, composition of the fermentation medium on the morphology

and cellulase production. Effect of factors like Tween 80 on enzyme production

was also confirmed. Luiza (2000) suggested that lignocellulosic biomass

(especially agricultural wastes) is known to be an excellent carbon source for

microbial enzyme production. Cellulase production from lignocellulosic materials

under solid state fermentation (SSF) was studied. The effects of fermentation

conditions, such as moisture content, initial pH, temperature, and composition of

mixed substrate (wheat straw and wheat bran) on endoglucanase production by

Aspergillus niger 38 were also observed. Kim et.al. (2001) described cellulase

production by a solid-state culture system of Trichoderma reesei QM9414 and

Sporotrichum cellulophilum on wheat bran by using moisture controlled solid

culture equipment, the effect of water content of wheat bran on cell growth and

cellulase production, cellular biomass grown on solid substrate was also

investigated.

Levinskaite (200 1) investigated the effect of copper metal on growth,

reproduction and cellulase production from filamentous fungi. Lignocellulosic

biomass can be utilized to produce ethanol, a promising alternative energy

source for the limited crude oil. Genetical and physical mapping of two

centromere proximal regions of chromosomes IV in Aspergillus nidu/ans was

carried out by Aleksenko et.al. (2001 ). A study on processes involved in the

conversion: hydrolysis of cellulose in the lignocellulosic biomass to produce

reducing sugars, and fermentation of the sugars to ethanol was carried by Sun

and Cheng (2002), optimization of the cellulase enzymes and the enzyme

loading which improve the hydrolysis and ethanol production was also performed

by them. Seiboth et.al. (2002) studied lactose metabolism and cellulase

production in Hypocrea jecorina the gal 7 gene, encoding galactose-1-phosphate

uridylyltransferase, which was essential for growth on galactose but not for

28

cellulase induction. Expression profiling of pectinolytic genes from Aspergillus

niger was studied by (de Vries et.al., 2002 and de Varies, 2003). Wanjiru et.a/.

(2002) studied cell wall degrading enzymes produced by Fusarium graminearum

during infection of wheat heads.

The mushroom mycelium is able to grow on a wide spectrum of

lignocellulosic waste material, which can be attributed to its ability to secrete a

range of degradatory enzymes. Thygesen et.al. (2003) examined the production

of cellulose and hemicellulose-degrading enzymes by filamentous fungi

cultivated on wet-oxidised wheat straw. Fawole and Odunfa (2003) reported

some factors affected production of pectic enzymes by Aspergillus niger. Bailey

and Tahtiharju (2004) reported an efficient cellulase production by Trichoderma

reesei in continuous cultivation on lactose medium. Effect of cultural factors on

cellulase activity and protein production by Aspergillus terreus was performed by

Garg and Neelakantan (2004). Role of lignocellulosic enzymes during

basidiomata production by P/eurotus djamor var roseus was studied by

Periasamy and Natarajan (2004). Kaur and Satyanarayana (2004) studied

production of extracellular pectinolytic, cellulolytic enzymes by thermophilic

mould Sporotrichum thermophile in solid-state fermentation. Production of

cellulase on sugar cane bagasse by fungal mixed culture in solid substrate

fermentation was carried out by Tengerdy (2004). Cellulolytic enzymes of

Trichoderma lignorum produced on banana agro-waste and their culture medium

and conditions were optimized by Baig (2005). Chand et.al. (2005) isolated

cellulase producing fungi and studied increasing cellulase production using novel

mutations technique. This new method could be applied to obtain potent fungal

mutants for more enzymes production. Martens et.al. (2005) analysed pectinase

spectrum of Aspergillus niger.

Lignocellulosic substrate has recently gained considerable interest

because of their possible use in secondary fermentation process for the

production of food, fuel and chemicals. Aspergillus terreus is prodigious

cellulolytic fungi, Vyas et.al. (2005) isolated cellulases from Aspergillus terreus

AV49 pretreated on groundnut. The cost of raw material is the limiting factors in

29

developing an economic process for cellulase production. The economy of

cellulase production could be improved by the use of cheaper cellulosic substrate

for enzyme production. Singhania et.a/. (2006) attempted to use agro-industrial

residues and waste as raw material for the production of cellulase using cellulase

hyper producing fungus, Trichoderma reesei NRRL 11460, and the influence of

various parameters were evaluated to design a suitable SSF process for

cellulase production. Fukuda et.a/. (2006) improved the cellulolytic activity of a

yeast strain displaying endoglucanase II from Trichoderma reesei; a

combinatorial library of the cellulose-binding domain of EGII was constructed by

using cell surface engineering. Stoner et.al. (2006) demonstrated that surfactant

induced enfolding is a significant degradation pathway for detergent enzymes.

This study examines the kinetics of surfactant-induced unfolding for

endoglucanase Ill, a detergent cellulase, under conditions of varying pH,

temperature, ionic strength, surfactant type, and surfactant concentration.

Ximenes et.al. (2007) contributed in study of cellulolytic enzymes

production from fungi like Trichoderma reesei and Aspergillus niger and

according to them production of enzymes increases when they are cultured on

co-product from corn dry grind ethanol plants. Due to molecular arrangement

cellulose is tough and fibrous which is difficult to breakdown; cellulase was

identified as one key enzyme degrading cellulose. Rajagopal et.al. (2007)

isolated a cellulolytic bacterial species APS-8 from the soil of Vishakhapatnam

region and estimated as well as characterized the cellulase produced from

Streptobacillus sp. Production and characterization of cellulolytic enzymes from

the thermoacidophilic fungal Aspergillus terreus M11 was carried out by Gao

et.al. (2008).

30