Report

INTRODUCTION

Enzymes are proteins that catalyze chemical reactions (Grisham and Reginald, 1999). In

enzymatic reactions, the molecules at the beginning of the process, called substrates, are

converted into different molecules, called products. Almost all chemical reactions in a biological

cell need enzymes in order to occur at rates sufficient for life. Since enzymes are selective for

their substrates and speed up only a few reactions from among many possibilities, the set of

enzymes made in a cell determines which metabolic pathways occur in that cell.

Like all catalysts, enzymes work by lowering the activation energy (Ea‡) for a reaction, thus

dramatically increasing the rate of the reaction. As a result, products are formed faster and

reactions reach their equilibrium state more rapidly. Most enzyme reaction rates are millions of

times faster than those of comparable un-catalyzed reactions. As with all catalysts, enzymes are

not consumed by the reactions they catalyze, nor do they alter the equilibrium of these reactions.

However, enzymes do differ from most other catalysts in that they are highly specific for their

substrates. Enzymes are known to catalyze about 4,000 biochemical reactions (Bairoch, 2000).

A few RNA molecules called ribozymes also catalyze reactions, with an important example of

being some parts of the ribosome (Lilley, 2005) and Cech, 2000). Synthetic molecules

called artificial enzymes also display enzyme-like catalysis (Groves, 1997).

Enzyme activity can be affected by other molecules. Inhibitors are molecules that decrease

enzyme activity; activators are molecules that increase activity. Many drugs and poisons are

enzyme inhibitors. Activity is also affected by temperature, chemical environment (e.g., pH), and

the concentration of substrate. Some enzymes are used commercially, for example, in the

synthesis of antibiotics. In addition, some household products use enzymes to speed up

biochemical reactions (e.g., enzymes in biological washing powders break down protein

or fat stains on clothes; enzymes in meat tenderizers break down proteins into smaller molecules,

making the meat easier to chew).

Cellulase refers to a suite of enzymes produced chiefly by fungi, bacteria, and protozoans

that catalyze cellulolysis (i.e. the hydrolysis of cellulose). However, there are also cellulases

produced by a few other types of organisms, such as some termites and the microbial intestinal

symbionts of other termites (Watanabe, 1998). Several different kinds of cellulases are known,

which differ structurally and mechanistically.

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Reaction: Hydrolysis of 1, 4-beta-D-glycosidic linkages in cellulose, lichenin and cereal beta-D-

glucans.

Other names for 'endoglucanases' are: endo-1, 4-beta-glucanase, carboxymethyl cellulase

(CMCase), endo-1, 4-beta-D-glucanase, beta-1, 4-glucanase, beta-1, 4-endoglucan hydrolase,

and celludextrinase. The other types of cellulases are called exocellulase. The expression

'avicelase' refers almost exclusively to exo-cellulase activity as avicel is a highly micro-

crystalline substrate. Cellulase action is considered to be synergistic as all three classes of

cellulase can yield much more sugar than the addition of all three separately. Beta-glucosidase

can also be considered as yet another group of cellulases.

Five general types of cellulases based on the type of reaction catalyzed:

Endocellulase (EC 3.2.1.4) randomly cleaves internal bonds at amorphous sites that create

new chain ends.

Exocellulase (EC 3.2.1.91) cleaves two to four units from the ends of the exposed chains

produced by Endocellulase, resulting in the tetrasaccharides or disaccharides, such

ascellobiose. There are two main types of exocellulase [or cellobiohydrolases (CBH)] -

CBHI works processively from the reducing end, and CBHII works processively from the

non-reducing end of cellulose.

Cellobiase (EC 3.2.1.21) or beta-glucosidase hydrolyses the exocellulase product into

individual monosaccharides.

Oxidative cellulases depolymerize cellulose by radical reactions, as for instance cellobiose

dehydrogenase (acceptor).

Cellulose phosphorylases depolymerize cellulose using phosphates instead of water.

In the most familiar case of Cellulase activity, the enzyme complex breaks down cellulose to

beta-glucose. This type of cellulase is produced mainly by symbiotic bacteria in

the ruminating chambers of herbivores. Aside from ruminants, most animals (including humans)

do not produce cellulase in their bodies and can only partially break down cellulose through

fermentation, limiting their ability to use energy in fibrous plant material. Enzymes that

hydrolyze hemicellulose are usually referred to as hemicellulase and are usually classified under

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cellulase in general. Enzymes that cleave lignin are occasionally classified as cellulase, but this is

usually considered erroneous.

Applications

Pulp and Paper Industry

Mixtures of cellulases (endoglucanases I and II) and hemicellulases have been used for

biomodification of fiber properties with the aim of improving drainage and beatability in the

paper mills before or after beating of pulp (Dienes et al., 2004). Cellulases alone, or used in

combination with xylanases, are beneficial for deinking of different types of paper wastes. Most

applications proposed so far use cellulases and hemicellulases for the release of ink from the

fiber surface by partial hydrolysis of carbohydrate molecules (Kuhad et al., 2010).

Textile Industry

Cellulases are the most successful enzymes used in textile wet processing, especially finishing of

cellulose-based textiles (Hebeish and Ibrahim, 2007). Endoglucanases activity-rich cellulase is

also proved better for biofinishing. Most cotton or cotton-blended garments, during repeated

washing, tend to become fluffy and dull, which is mainly due to the presence of partially

detached microfibrils on the surface of garments. The use of cellulases can remove these

microfibrils and restore a smooth surface and original color to the garments. While the bio-

polishing is usually carried out during the wet processing stages, which include desizing,

scouring, bleaching, dyeing, and finishing. The acidic cellulases improve softness and water

absorbance property of fibers, strongly reduce the tendency for pill formation, and provide a

cleaner surface structure with less fuzz.

Bio-Ethanol Industry

Enzymatic saccharification of lignocellulosic materials such as Sugarcane Bagasse, corncob, rice

straw, Prosopis juliflora, Lantana camara, switch grass, saw dust, and forest residues by

cellulases for biofuels production is perhaps the most popular application currently being

investigated (Sukumaran et al., 2005). Bioconversion of lignocellulosic materials into useful

and higher value products normally requires multistep processes (Wyman et al., 2005). To

reduce the enzyme cost in the production of fuel ethanol from lignocellulosic biomass, two

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aspects are widely addressed: optimization of the cellulase production and development of a

more efficient cellulase-based catalysis system.

Wine and Brewing Industry

Microbial glucanase and related polysaccharides play important roles in fermentation processes

to produce alcoholic beverages including beers and wines (Sukumaran et al., 2005). These

enzymes can improve both quality and yields of the fermented products. Glucanases are added

either during mashing or primary fermentation to hydrolyze glucan, reduce the viscosity of wort,

and improve the filterability. Beer brewing is based on the action of enzymes activated during

malting and fermentation. Malting of barley depends on seed germination, which initiates the

biosynthesis and activation of α- and β-amylases, carboxypeptidase, and β-glucanase that

hydrolyze the seed reserves (Bamforth., 2009). In wine production, enzymes such as pectinases,

glucanase, and hemicellulases play an important role by improving color extraction, skin

maceration, must clarification, filtration, and finally the wine quality and stability.

Food Processing Industry

Cellulases have a wide range of potential applications in food biotechnology as well. The

production of fruit and vegetable juices requires improved methods for extraction, clarification,

and stabilization. Cellulases also have an important application as a part of macerating enzymes

complex (cellulases, xylanases, and pectinases) used for extraction and clarification of fruit and

vegetable juices to increase the yield of juices (Minussi et al., 2002).

Animal and Feed Industry

Applications of cellulases and hemicellulases in the feed industry have received considerable

attention because of their potential to improve feed value and performance of animals (Dhiman

et al., 2002). Β-Glucanases and xylanases have been used in the feed of monogastric animals to

hydrolyze nonstarch polysaccharides such as β-glucans and arabinoxylans. Cellulases, used as

feed additives alone or with proteases, can significantly improve the quality of pork meat.

Glucanases and xylanases reduce viscosity of high fibre rye- and barley-based feeds in poultry

and pig. These enzymes can also cause weight gain in chickens and piglets by improving

digestion and absorption of feed materials (Bhat, 2002).

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Waste Management

The wastes generated from forests, agricultural fields, and agro industries contain a large amount

of unutilized or underutilized cellulose, causing environmental pollution .Nowadays, these so-

called wastes are judiciously utilized to produce valuable products such as enzymes, sugars,

biofuels, chemicals, cheap energy sources for fermentation, improved animal feeds, and human

nutrients (Kuhad et al., 2010).

Objectives of the project

• Isolation of Bacteria from normal soil.

• Morphological studies of mixed cultures.

• Purification of obtained mixed culture.

• Screening of purified culture for cellulase production.

• Study of growth parameters of culture showing maximum activity during screening.

• Identification of culture showing maximum growth.

• Optimization of the media.

• Production of the Cellulase producing Bacteria

• Extraction of Enzyme.

• Purification of Enzyme.

• Enzyme Assay.

• Characterization of Enzyme.

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REVIEW OF LITREATURE

Lee and Blackburn, 1975, isolated strain M7, a thermophilic, anaerobic, terminally sporing

bacterium (0.6 by 4.0µm) from manure. It degraded filter paper in 1 to 2 days at 60°C in a

minimal cellulose medium but was stimulated by yeast extract. It fermented a wide variety of

sugars but produced cellulase only in cellulose or carboxymethylcellulose media. Cellulase

synthesis not only was probably repressed by 0.4% glucose and 0.3% cellobiose, but also

cellulase activity appeared to be inhibited by these sugars at these concentrations. Both C,

cellulase (degrades native cellulose) and Cx cellulase (13-1, 4-glucanase) activities in strain M7

cultures were assayed by measuring the liberation of reducing sugars with di-nitrosalicylic acid.

Both activities had optima at pH 6.5 and 67°C. One milliliter of a 48-h culture of strain M7

hydrolyzed 0.044-meq of glucose per min from cotton fibers. The cellulase(s) from strain M7

was extracellular, produced during exponential growth, but was not free in the growth medium

until approximately 30% of the cellulose was hydrolyzed. Glucose and cellobiose were the major

soluble products liberated from cellulose by the cellulase. ZnCl2 precipitation appeared initially

to be a good method for the concentration of cellulase activity, but subsequent purification was

not successful. Iso-electric focusing indicated the presence of four C; cellulases (pI 4.5, 6.3, 6.8,

and 8.7). The rapid production and high activity of cellulases from this organism strongly

support the basic premise that increased hydrolysis of native cellulose is possible at elevated

temperature.

Singh and Kumar, 1998, isolated Bacillus brevis from the soil. It has been found to secrete

cellulase extracellularly whose production increased almost five times on addition of galactose in

the culture medium. Production of cellulase has been found optimal at pH 5.5, 37°C and 175 rpm

speed using environmental orbital shaker. The cellulase has been purified using ultra filtration

and Sephadex G-200 column chromatography. The native molecular weight of the enzyme is

found to be 33,000 + 2000 using Sephadex G-200 gel filtration chromatography. The subunit

molecular weight (33,000 + 2,000) indicates monomeric nature of the enzyme. The enzyme

showed Michaelis Menten kinetics exhibiting Km -1.7 + 0.1 mg/ml for CMC. The enzyme

activity got inhibited by heavy metals viz. Hg2+ and Ag2+.

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Kurup et al., 2005, reported that most of the freshwater systems in tropical countries are infested

with one kind of aquatic weed or the other causing serious environmental problems. All efforts to

control the growth and spread of these weeds have failed miserably and hence the concept of

eradication through utilization is being adopted by many researchers. Solid state fermentation,

the culturing of microorganisms on moist solid substrates in the absence or near absence of free

water, has generated great deal of interest among researchers because of its various advantages

over the submerged fermentation technique. Cellulase enzyme is used extensively in various

industries, especially in textile, food and in the bioconversion of lignocellulosic wastes to

alcohol. The extensive use of cellulase in industries depends on the cost of the enzyme and hence

considerable research is being carried out to isolate better microbial strains and also to develop

new fermentation processes with the aim to reduce the product cost. The objective of the present

study is to determine whether water hyacinth, one of the commonly found aquatic weeds, can be

used as a substrate for cellulase production, by three native bacterial isolates named WHB 3,

WHB 4 and SMB 3, under the process of solid state fermentation. Results indicate that all the

three isolates produced cellulase enzyme by using water hyacinth as the solid support. Under

optimized conditions of moisture, pH, temperature, incubation time and inoculum concentration,

the enzyme yield increased from 16.8 to 94.8 units for SMB 3, from 25.2 to 110.4 units for WHB

3 and from 18.0 to 127.2 units for WHB 4. The addition of nitrogen and carbon sources resulted

in a significant increase in cellulase yield and WHB 3 produced the maximum amount of 216

units followed by SMB 3 and WHB 4.

Bakare et al.,2005, purified cellulases from the wild-type (WT) and two improved mutants

(catabolite repression resistant mutant 4 and 24, abbreviated CRRmt 4 and CRRmt 24,

respectively) of Pseudomonas fluorescens to apparent homogeneity by ammonium sulphate

precipitation, ion exchange chromatography on DEAE Sephadex A-50 and gel filtration on

Sephadex G-100. Purification fold of about 5 was obtained for the WT and CRRmt24 while

purification fold of about 7 was achieved for CRRmt4 by ammonium sulphate precipitation. Ion

exchange chromatography gave purification fold of about 24, 22 and 25 for WT, CRRmt 4 and

CRRmt 24, respectively. Gel filtration chromatography step yielded a homogeneous preparation

with a specific activity of 6.8, 5.9 and 6.9 units/mg protein for the WT, CRRmt4and CRRmt24

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respectively. The purified cellulase gave a single protein band on polyacrylamide gel

electrophoresis. The molecular weights of the three cellulases were estimated to be 36, 26 and 36

kDa for the wild-type, CRRmt4 and CRRmt24, respectively. Km values of 3.6, 3.1, and 5.3

mg/ml were obtained for the wild-type, CRRmt4 and CRRmt24, respectively. The optimum pH

value for the purified cellulases was 6.5 – 7.0 and the enzymes were optimally active at

temperature of 35°C. The activities of the purified cellulases were stimulated by low

concentrations (10-30 mM) of Na+ and Mg 2+ while EDTA was found to inhibit enzyme activity

at all concentrations.

Keshk et al., 2006, produced bacteria friendly cellulose from beet molasses using

Gluconacetobacter xylinus ATCC 10245. The yield of the bacterial cellulose (BC) produced

from beet molasses was higher than that using glucose as a sole carbon source. The structure of

BC produced in presence of beet molasses was studied using IR spectroscopy and X-ray

diffractometry. IR spectra show the relative absorbance of CO- C ether linkage (at 1120 cm-1) in

BC using glucose has a relatively lower value than that from molasses. This indicates that BC

produced from glucose has a relatively higher degree of polymerization. From X-ray pattern, no

remarkable differences in crystallinity index of cellulose between the two media were recorded.

Sheble Ibrahim and El-diwany, 2007, isolated thermophilic cellulases producing bacteria from

an Egyptian hot spring by enrichment of the water and soil samples with cellulose for 3 weeks at

70 °C. Three isolates termed EHP1, EHP2 and EHP3 had been isolated. The phylogenetic

analysis of these strains using its 16S rDNA sequence data showed that strain EFP1 had highest

homology (98.5%) with Anoxybacillus flavithermus, EFP2 showed 98.5 % similarity with

Geobacillus thermodenitrificans and EHP3 showed 99.0 % similarities with Geobacillus

stearothermophilus. Maximal cellulases production by Anoxybacillus flavithermus EHP2 was

detected at the end of the stationary phase (36 h). The crude cellulase had activity toward avicell,

CMC, cellobiose, and xylan, but there was no detectable activity on p-nitrophenyl-â-d-

glucopyranoside. The rate of CMC degradation was higher than any other substrates used in this

study. The optimum temperature and pH for the crude enzyme activity was 75 °C and 7.5,

respectively.

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Chawla et al., 2009, reported that bacterial cellulose, an exopolysaccharide produced by some

bacteria, had unique structural and mechanical properties and is highly pure as compared to plant

cellulose. This article presents a critical review of the available information on the bacterial

cellulose with special emphasis on its fermentative production and applications. Information on

the biosynthetic pathway of bacterial cellulose, enzymes and precursors involved in bacterial

cellulose synthesis has been specified. Characteristics of bacterial cellulose with respect to its

structure and physicochemical properties are discussed. Current and potential applications of

bacterial cellulose in food, pharmaceutical and other industries are also presented.

Boonmee, 2009, investigated screening of cellulolytic microorganisms for degrading rice straw.

There are twenty nine bacterial isolates and 30 fungal isolates were selected for further study on

their cellulolytic activity. All isolates were assayed for exoglucanase, endoglucanase and β-

glucosidase specific activities, by which following substrates were used: filter paper (Whatman

No.1), carboxymethyl cellulose and cellobiose, respectively. Specific activities of those enzymes

were determined by measuring reducing sugar released from substrates. From the results, the

following isolates showed the highest specific activity of each category of cellulases; FR14 for

Filter paper cellulase (FPase) (0.032 unit/ mg protein), FR4 for CMCase (0.5 unit/ mg protein)

and FC1 for cellobiose (0.6 unit/ mg protein). Two isolates showed nearly equal activity of

CMCase and Cellobiase. The isolate FR3 had 0.22 unit/ mg protein CMCase specific activity and

0.23 unit/ mg protein Cellobiase specific activity, while the isolate FR18 showed CMCase

specific activity with 0.25 unit/ mg protein and Cellobiase specific activity with 0.30 unit/ mg

protein.

Kumar et al., 2009, evaluated newly isolated strains of Bacillus sp. FME 1 and FME 2 for the

cellulolytic enzymes production during submerged fermentation (SmF) of different substrates

including rice husk, Whatman filter paper and cellulose powder CF 11. Extracellular enzyme

assays for CMCase, FPase and β-glucosidase were examined up to 8 days of submerged

fermentation. Among the three substrates, rice husk was the most suitable substrate for higher

production of cellulolytic enzymes. Maximum titers of 100, 45, and 3.5 U/mL in respect of

CMCase, FPase and β-glucosidase in Bacillus sp. FME 2 were recovered as against 45, 12, and

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0.39 U/mL in Bacillus sp. FME 1 respectively, at their respective peak time intervals. Bacillus

sp. FME 2 was found to produce higher cellulolytic enzyme activities than Bacillus sp. FME 1.

Krairitthichai and Thongwai, 2009, had isolated one hundred and twenty-five isolates of

bacteria from soils, decomposing logs and composts collected from the Northern part of

Thailand. The bacteria isolated were grown on carboxymethylcellulose (CMC) agar at 45, 50 or

55 ºC for 24 hours prior to examine their cellulase production by using a Congo red test. It was

found that sixty-two isolates showed positive results with clear zone around the cultures. All

isolates were evaluated their cellulase activity by growing in CMC broth. It was found that

isolate CM120-1 displayed the highest enzyme activity of 22.84 U/ml and specific activity of

0.15 U/mg proteins. The optimal conditions for cellulase production were at 45 ºC, pH 7 and 96

hours of incubation. According to the morphological and biochemical studies, the isolate

CM120-1 was primarily identified as the genus Bacillus.

Yin et al., 2010, isolated cellulase-producing bacterium from soil and identified

as Cellulomonas sp. YJ5. Maximal cellulase activity was obtained after 48 h of incubation at 30

°C in a medium containing 1.0% carboxymethyl cellulose (CMC), 1.0% algae powder, 1.0%

peptone, 0.24% (NH4)2SO4, 0.20% K2HPO4, and 0.03% MgSO4·7H2O. The cellulase was

purified after Sephacryl S-100 chromatography twice with a recovery of 27.9% and purification

fold of 17.5. It was, with N-terminal amino acids of AGTKTPVAK, stable at pH 7.5−10.5 and

20−50 °C with optimal pH and temperature of 7.0 and 60 °C, respectively. Cu 2+, Fe2+, Hg2+, Cr3+,

and SDS highly inhibited, but cysteine and β-mercaptoethanol activated its activity. Substrate

specificity indicated it to be an endo-β-1, 4-glucanase.

Maki et al., 2011, reported that, there is wide variety of bacteria in the environment which

permits screening for more efficient cellulases to help overcome current challenges in biofuels

production. The study focused on the isolation of efficient cellulase producing bacteria found in

organic fertilizers and paper mill sludges which can be considered for use in large scale

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Biorefining. Pure isolate cultures were screened for cellulase activity. Six isolates: S1, S2, S3,

S4, E2, and E4, produced halos greater in diameter than the positive control (Cellulomonas

xylanilytica), suggesting high cellulase activities. A portion of the 16S rDNA genes of cellulase

positive isolates were amplified and sequenced, then BLAST was performed to determine the

genera. Phylogenetic analysis revealed genera belonging to two major Phyla of Gram positive

bacteria: Firmicutes and Actinobacteria. All isolates were tested for the visible degradation of

filter paper; only isolates E2 and E4 (Paenibacillus species) were observed to completely break

down filter paper within 72 and 96 h incubation, respectively ,under limited oxygen condition.

Thus E2 and E4 were selected for the FP assay for quantification of total cellulase activities. It

was shown that 1% (w/v) CMC could induce total cellulase activities of 1652.2±61.5 and

1456.5±30.7 μM of glucose equivalents for E2 and E4, respectively. CMC could induce cellulase

activities 8 and 5.6X greater than FP, therefore CMC represented a good inducing substrate for

cellulase production. The genus Paenibacillus are known to contain some excellent cellulase

producing strains, E2 and E4 displayed superior cellulase activities and represent excellent

candidates for further cellulase analysis and characterization.

Deka et al., 2011, reported that, the cellulase activity of Bacillus subtilis AS3 was enhanced by

optimizing the medium composition by statistical methods. The enzyme activity with un-

optimized medium with carboxymethylcellulose (CMC) was 0.07U/mL and that was

significantly enhanced by CMC, peptone, and yeast extract using Placket-Burman design. The

combined effects of these nutrients on cellulase activity were studied using 22 full factorial

central composite designs. The optimal levels of medium components determined were CMC

(1.8%), peptone (0.8%), and yeast extract (0.479%). The maximum enzyme activity predicted by

the model was 0.49U/mL which was in good agreement with the experimental value 0.43U/mL

showing 6-fold increase as compared to un-optimized medium. The enzyme showed

multisubstrate specificity, showing significantly higher activity with lichenan and β-glucan and

lower activity with laminarin, hydroxyethylcellulose, and steam exploded bagasse. The

optimized medium with lichenan or β- glucan showed 2.5- or 2.8-fold higher activity,

respectively, at same concentration as of CMC.

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Otajevwo and Aluyi, 2011, collected a total of 115 samples made up of 42 (36.5%) rumen fluid,

36 (31.3%) cow dung and 37 (32.2%) soil samples with the aid of sterile swab sticks except for

rumen fluid samples which were collected by use of stomach tubes inserted into mouths of cows

and by suction, liquor was collected into pre-warmed thermo flasks under continuous flushing

with carbon-dioxide. Soil samples were collected into sterile universal containers. All samples

were obtained from abattoirs situated at three locations in Benin City, Nigeria. Samples were

investigated for cellulolytic bacteria by Filter Paper Yeast Mineral broth method. Cellulase

production was assayed by Carboxymethyl cellulose submerged broth culture while residual

sugar yield and other cellulolytic activities were determined by 3, 5 – Di-nitrosalicylic acid,

Filter Paper, Microcrystalline and Viscometric methods. Cellulolytic bacterial organisms isolated

from both soil and rumen fluids were Bacillus subtilis, Clostridium cellobioparum and

Clostridium thermocellum. Pseudomonas aeruginosa was isolated from both soil and cow dung

samples while Erwinia sp was obtained from both rumen fluid and cow dung samples. Bacillus

circulans and Serratia sp were obtained from soil samples only. Clostridium thermocellum and

Erwinia sp produced the highest and lowest cellulase yields respectively. All isolates at 40°C and

pH 6, recorded optimal sugar yields in culture broth of which Clostridium thermocellum

recorded the highest. Lowest yields were recorded at 30°C and pH 3 although there was

significant difference in individual yields (P < 0.05). Clostridium thermocellum recorded optimal

cellulolytic activities at 50°C and pH 6. All isolates attained optimal cellulolytic activities at 32.6

± 6.2°C and pH 6.29 ± 0.9 with other broth cultural conditions kept constant.

Shankar and Isaiarasu, 2011, tested, Bacillus pumilus EWBCM1 isolated from earthworm gut

(Eudrilus eugeniae) for its abilities to hydrolyze the structural polysaccharides. They studied the

effect of different production parameters such as pH, temperature, carbon source, nitrogen source

(Organic and Inorganic), NaCl concentration, surfactants, metal ions, and inoculum size and

incubation time on cellulase production by the isolated bacterial strain. The enzyme production

was assayed in submerged fermentation (SmF). Maximum cellulase activity was found at pH 6,

37°C, galactose, malt extract, ammonium molybdate, calcium chloride, 2.5% NaCl, Tween-20,

72 hrs, 2% inoculum. A higher titer of cellulase enzyme activity (0.5851±0.006 IU/ml) was

obtained in the optimized production medium.

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Gupta et al., 2011, isolated eight isolates of cellulose-degrading bacteria (CDB) from four

different invertebrates (termite, snail, caterpillar, and bookworm) by enriching the basal culture

medium with filter paper as substrate for cellulose degradation. To indicate the cellulase activity

of the organisms, diameter of clear zone around the colony and hydrolytic value on cellulose

Congo red agar media were measured. CDB 8 and CDB 10 exhibited the maximum zone of

clearance around the colony with diameter of 45 and 50mm and with the hydrolytic value of 9

and 9.8, respectively. The enzyme assays for two enzymes, filter paper cellulase (FPC), and

cellulase (endoglucanase), were examined by methods recommended by the International Union

of Pure and Applied Chemistry (IUPAC). The extracellular cellulase activities ranged from 0.012

to 0.196 IU/mL for FPC and 0.162 to 0.400 IU/mL for endoglucanase assay. All the cultures

were also further tested for their capacity to degrade filter paper by gravimetric method. The

maximum filter paper degradation percentage was estimated to be 65.7 for CDB 8. Selected

bacterial isolates CDB 2, 7, 8, and 10 were co-cultured with Saccharomyces cerevisiae for

simultaneous saccharification and fermentation. Ethanol production was positively tested after

five days of incubation with acidified potassium dichromate.

Sangkharak et al., 2012, reported that, high concentration of cellulase can be achieved by

mutagenesis and optimization of the media. Among 328 mutant strains of Cellulomonas sp.

TSU-03, the mutant M23, NTG mutant, gave the highest value of cellulase activity 2008 U/mg

protein) followed by mutant M17 (1884 U/mg protein) in CMC medium. The optimum medium

and environmental conditions for cellulase production consisted of 4% wastepaper, 1% NaNO3

under cultivation temperature at 35°C with initial pH and agitation speed at 6 and 100 rpm,

respectively. Cellulomonas sp. strain M23 produced the highest cellular growth (28.09 ± 2.28

g/L) and FPase, CMCase as well as, β-glucosidase activities at 325, 2420 and 152 U/mg protein,

respectively. Under optimal condition, the cellulase activity achieved from strain M23 is 1.28

and 1.30-fold higher than cellulase from mutant M17 and wild type, respectively. After being

subculture 12 times, the cellulase production of the mutant M23 was stable. The results

suggested that Cellulomonas sp. M23 had a good potential for production of cellulase by

fermentation using a cultivation medium containing wastepaper as the main substrate.

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Sheng et al., 2012, isolated the hindgut contents of Holotrichia parallela, 93 cellulolytic

bacterial isolates after enrichment in carboxymethyl cellulose medium. Among these isolates, a

novel bacterium, designated HP207, with the highest endoglucanase productivity was selected

for further study. This bacterium was identified as Pseudomonas sp. based on the results of the

16S ribosomal DNA analysis, morphological characteristics, and biochemical properties.

The production of the endoglucanase was optimized by varying various physical culture

conditions using a submerged fermentation method. Under the optimized fermentation

conditions, the maximum endoglucanase activity of 1.432 U mL(-1) in bacterial cultures was

obtained, higher than those of the most widely studied bacteria and fungi, which are the

attractive candidates for the commercial producer of cellulase. And the crude endoglucanase

enzyme was also highly thermo stable; approximately 55 % of the original activity was

maintained after pretreatment at 70 °C for 1 h. Thus, from the present study, the bacterium can

be added up to the database of cellulolytic bacteria.

Wilson, 2012, reported that cellulases are key enzymes used in many processes for producing

liquid fuels from biomass. Currently there are many efforts to reduce the cost of cellulases using

both structural approaches to improve the properties of individual cellulases and genomic

approaches to identify new cellulases as well as other proteins that increase the activity of

cellulases in degrading pretreated biomass materials. Fungal GH-61 proteins are important new

enzymes that increase the activity of current commercial cellulases leading to lower total protein

loading and thus lower cost. Recent work has greatly increased our knowledge of these novel

enzymes that appear to be oxido-reductases that target crystalline cellulose and increase its

accessibility to cellulases. They appear to carry out the C1 activity originally proposed by Dr

Reese. Cellobiose dehydrogenase appears to interact with GH-61 proteins in this function,

providing a role for this puzzling enzyme. Cellulase research is making considerable progress

and appears to be poised for even greater advances.

14

MATERIALS AND METHODS

COLLECTION OF SOIL SAMPLE

Soil sample were collected from the soil were leaves were decaying near railway line on

Daliganj Crossing, Lucknow. Soil was collected from 2.5 cm below the ground level in sterile

polybag and transferred to laboratory.

ISOLATION OF BACTERIA FROM SOIL SAMPLE

Requirements: Soil Sample , Test Tubes, Normal Saline, Weighing Balance, Petri plates, NA

Media , Spreader, Laminar Air Flow, Serially Culture, Micropipettes.

Principle:

Dilution allows the number of living bacteria to be determined in suspensions that contain even

very large numbers of bacteria. The number of bacteria obtained by dilution of a culture can

involve growth of the living bacteria on a solid growth source, the so-called dilution plating

technique. The objective of dilution plating is to have growth of the bacteria on the surface of the

medium in a form known as a colony. Theoretically each colony arises from a single bacterium.

Procedure:

30ml of normal saline (0.85% NaCl) was prepared.

5ml f he saline was transferred to the test tubes labeled as 10-1, 10-2, 10-3, 10-4, 10-5.

Now 0.5gm of the soil was dissolved in the saline test tube labeled as 10 and frothing is

done to mix well.

0.5ml of the sample was transferred to the test tube labeled as 10-1.

Similarly the sample was transferred to one test tube to another tube.

20µl of the inoculums from the serially diluted test tube was spreaded on the respective

solidified NAM plates.

Finally the plates were incubated at 37°C for 24 hours.

MORPHOLOGICAL STUDIES OF THE MIXED CULTURES

15

The following outline will be helpful for verbally communicating the appearance of observed

colonial growth.

Form – The form refers to the shape of the colony. These forms represent the most common

colony shapes which are likely to be encounter. The different forms are: Circular, Irregular,

Filamentous and Rhizoid.

Surface – Bacterial colonies are frequently shiny and smooth in appearance. Other surface

descriptions might be: veined, rough, wrinkled (or shriveled), glistening.

Texture – Several terms that may be appropriate for describing the texture or consistency of

bacterial growth are: dry, moist, mucoid, brittle, and Viscous.

Color – It is important to describe the color or pigment of the colony.

Elevation – This describes the “side view” of a colony. These are the most common. Flat,

Raised, Umbonate, convex, Pulvinate.

Margin- The margin or edge of a colony (or any growth) may be an important characteristic in

identifying organisms. They are: Entire, Undulate, Lobate, curled or filiform.

PURIFICATION/SUBCULTURING OF THE OBTAINED MIXED CULTURES

Requirements: Petri plates, NAM Media, Inoculation loop, Mixed Cultures, Sprit lamp.

Principle:

Pure culture, a laboratory culture containing a single species of organism. A pure culture is

usually derived from a mixed culture (one containing many species) by transferring a small

sample into new, sterile growth medium in such a manner as to disperse the individual cells

across the medium surface or by thinning the sample many fold before inoculating the new

medium. Both methods separate the individual cells so that, when they multiply, each will form a

discrete colony, which may then be used to inoculate more medium, with the assurance that only

one type of organism will be present. Isolation of a pure culture may be enhanced by providing a

mixed inoculum with a medium favoring the growth of one organism to the exclusion of others.

Procedure:

16

100ml of NAM was prepared and poured in sterile petri plates after autoclaving.

The inoculation was done by Discontinuous Quadrant Streaking by inoculation loop.

All the plates were incubated at 37°C for 24 hours.

SCREENING OF PURIFIED CULTURES FOR CELLULASE PRODUCTION

Requirements: Petri plates, Screening Media (MAM), Inoculation loop, 0.1%Congo red

solution, 1N NaCl (destaining) and Magnetic Stirrer.

Principle:

Screening is done to detect the potency of fungi for the production of Cellulases. Cellulase

producing bacteria is detected with the help of screening media in which Carbon is the limiting

factor and additional substrates is provided in order to see whether the bacteria is able to degrade

the substrate. The substrate here provided was 1%CMC. The isolated pure strains were screened

for the production of extracellular cellulase. This is visualized by the presence of zone of

hydrolysis on the plate after the treatment of Congo red solution.

Procedure:

100ml of screening media was prepared.

Media was poured in sterile petri plates after autoclaving.

Central streaking is done from the various pure cultures.

Petri plates were incubated at 37°C for 24 hours.

After incubation plates were flooded with 0.1 % Congo red Solution, for 15 mins.

Destaining was done by 1N NaCl for 10 mins thrice, for removing extra stain.

The Zone of hydrolysis was observed.

STUDY OF THE GROWTH PARAMETERS SHOWING MAXIMUM ACTIVITY

a) GROWTH KINETICS:

Requirements: Flask, NB, Test Tube containing the culture, Sprit Lamp, LAF,

micropipette , autoclaved tips , Magnetic Stirrer and Colorimeter.

17

Principle: Growth kinetics, i.e., the relationship between specific growth rate and the

concentration of a substrate, is one of the basic tools in microbiology. Growth kinetics

is applied to determine the time period at which the culture show optimum activity.

Growth of any microbe occurs in different distinct stages which are indicated by

growth curve. Growth curve shows four distinct phases.

(1) Lag Phase-This phase shows that the microbe adjusts to the provided

environment. There is no such growth seen in the culture. The specific growth

rate is zero (µmax=0).

(2) Log/Exponential Phase- This phase shows the maximum growth, characterized

by cell doubling. The actual rate of this growth (i.e. the slope of the line in the

figure) depends upon the growth conditions, which affect the frequency of cell

division events and the probability of both daughter cells surviving. Growth rate

is independent of nutrient concentration, as nutrients are in excess. If the microbe

is re-inoculated in the fresh media its Lag phase increases and will produce the

product in large amount. The specific growth rate is maximum (µnet=µmax).

(3) Stationary phase-The growth rate slows as a result of nutrient depletion and

accumulation of toxic products. This phase is reached as the bacteria begin to

exhaust the resources that are available to them. This phase is a constant value as

the rate of bacterial growth is equal to the rate of bacterial death. Cells undergo

internal restructuring to increase their chances of survival. The secondary

metabolites are form in this phase.

(4) Death Phase-With the exhaustion of nutrients (S≈0) and build-up of waste and

secondary metabolic products, the growth rate became equals the death rate.

There is no net growth in the organism population. Cells may have active

metabolism to produce secondary metabolites. Primary metabolites are growth-

related product, in this case cellulase enzyme. Secondary metabolites are non-

growth-related and have no such role in the production of the primary product, it’s

a by-product. The specific growth rate is again zero.

18

Stationary phase is the most important to study because of the production of

secondary products are being formed. These secondary products may be some

antibiotics or pigments.

Procedure:

130ml of NB was prepared and dispensed into two flasks, 100 ml in first and

30 ml in second, as a blank.

Both of them were autoclaved.

The first flask was cooled and inoculated with the culture (50µl).

The blank was stored into refrigerator.

The Optical density of the culture broth was taken up to five days from

inoculation a 620nm.

b) EFFECT OF TEMPERATURE:

Requirements: NAM, Four petri plates, Inoculation loop, Sprit lamp, Culture plate,

Magnetic Stirrer and LAF.

Principle: Effect of temperature was studied at different temperature to observe that at

which temperature shows the maximum growth of the cultures. The different

temperatures were at 20°C, 28°C, 37°C and 50°C.

Procedure:

100ml of the NAM was prepared for four petri plates.

The media and the plates were autoclaved.

The media was poured in different four petri plates and quadrant discontinuous

streaks were done.

The plates were kept at different temperatures like, at 20°C(fridge), 28°C(room

temperature),37°C(incubator) and 50°C(hot air oven) for 24 hours.

The plates were observed for the growth.

c) EFFECT OF pH :

19

Requirements: pH meter, four conical flasks, Culture plate, Inoculation loop, Sprit

lamp, Magnetic Stirrer and LAF.

Principle: The pH is the most important aspect for the growth of the culture. Slight

difference in the pH will result in no growth or maximum growth of the culture. There

are microbes which are very sensitive to the pH range. There are microbes which

grows in acidic medium (pH 2- 5), in alkaline medium (pH 9-14), and those grows at

Neutral pH, i.e. at pH 7.

Procedure:

125 ml of NB was prepared and dispensed in four conical flasks (30ml each)

and 5ml of the NB was kept as a blank.

The pH meter was calibrated and was set at different pH as, pH 5, 7, 9 and 11.

The media was then autoclaved.

After cooling them the culture was inoculated in to the four different conical

flasks and was kept in incubator shaker for 24 hours.

OD of the flasks was taken at 620nm.

IDENTIICATION OF THE BACTERIAL STRAIN

Gram Staining

Requirements: MJRM1209 culture plate, microscope, slide, Crystal violet, Iodine solution,

safranine, ethanol and inoculation loop.

Principle: It is a method of differentiating bacterial species into two large groups (Gram-

positive and Gram-negative).It is based on the chemical and physical properties of their cell

walls. Primarily, it detects peptidoglycan, which is present in a thick layer in Gram positive

bacteria. A Gram positive results in a purple/blue color while a Gram negative results in a

pink/red color. The Gram stain is almost always the first step in the identification of a bacterial

organism, and is the default stain performed by laboratories over a sample when no specific

culture is referred. While Gram staining is a valuable diagnostic tool in both clinical and research

settings, not all bacteria can be definitively classified by this technique, thus forming Gram-

variable and Gram-indeterminate groups as well.

20

Procedure:

MJRM1209 culture was taken in inoculation loop and a smear was made onto slide.

Smear was heat fixed.

Smear was flooded with crystal violet for 1 min.

Wash the extra stain with D/W.

Smear was again flooded with iodine solution for 1 min.

Again washed with D/W.

Smear was again flooded with 95% ethanol for 30secs.

Washed with D/W.

Smear was again flooded with counter stain safranine for 1min.

Washed with D/W.

The smear was again air dried and observes under microscope.

Endospore Test

Requirements: Slide, Malachite green, safranine, filter paper, water bath, MJRM1209 culture.

Principle: Due to the highly resistant nature of endospores, they are not easily penetrated by

stains. Thus, it is necessary to steam the stain into endospores. The Schaeffer Fulton method is

the most commonly used endospores staining technique, and it uses Malachite green as the

primary stain. Once the endospores have absorbed the stain, it is resistant to decolourisation,

but the vegetative cell is easily decolorized with water (leaving the vegetative cells colorless).

Finally, the vegetative cells are counterstained with safranine to aid in their visualization. When

viewed under a microscope, the endospores appear green, while the vegetative are red or pink.

Procedure:

A thin smear of culture MJRM1209 was made on glass slides.

The smear was heat fixed.

Put a filter paper over the smear. The slide was put on a water bath (at 100°C) and

flooded with malachite green drop by drop for 7 minutes.

21

Let the slide cool down.

The slide was washed with distilled water.

The smear was covered with safranine for 2 minutes and allows it to dry.

The slide was washed with distilled water.

The slide was allowed to air dry.

Then it was observe under microscope.

Catalase Test

Requirements: Slide, MJRM1209, Hydrogen Peroxide, Inoculation loop.

Principle: It is a common enzyme found in nearly all living organisms exposed to oxygen. It

catalyzes the decomposition of hydrogen peroxide to water and oxygen. The reaction of Catalase

in the decomposition of hydrogen peroxide is:

2 H2O2 → 2 H2O + O2

The presence of catalase in a microbial or tissue sample can be tested by adding a volume

of hydrogen peroxide and observing the reaction. The formation of bubbles, oxygen, indicates a

positive result. This easy assay, which can be seen with the naked eye, without the aid of

instruments, is possible because catalase has a very high specific activity, which produces a

detectable response.

Procedure:

A small amount of culture was taken onto the slide from the MJRM1209 culture.

Few Drops of Hydrogen Peroxide was put onto the culture.

Mannitol Fermentation Test.

Requirements: Phenol red, Mannitol, weighing balance, MJRM1209 culture, Shaker Incubator,

Test tubes.

Principle: Mannitol is an organic substance which has carbon chain and hydroxyl group.

Mannitol work as reducing agent which can be oxidized by microbial fermentation and so the –

OH group of Mannitol converted into –COOH group on oxidation. The fermentation broth

22

contains mannitol and a pH indicator (phenol red) which is red at a neutral pH 7 and turns yellow

at or below pH 6.8 due to the production of organic acids.

Procedure:

20 ml of the Mannitol fermentation broth was prepared.

Then it was dispensed into two test tubes, one is kept as blank, and autoclaved.

Add MJRM1204 culture to the test tubes and keep 1 test tube as control.

Incubate for 48 hours in incubator shaker at 37°C.

MR-VP Test

Requirements: M.R.V.P broth, 6 test tubes, reagents- methyl red, VP-I, VP-II and MJRM1209

culture.

Principle: This test is done for facultative aerobes. They sometimes yield organic acids (pH less

than 7.0) or acetoin (pH-7.0).The reagents used in this test are methyl red, VP-I (it contains α-

napthol) and VP-II (40% KOH). If some color change is seen in VP-I it means the bacteria is

producing acetoin and if color change occurs due to methyl red it means the bacteria is producing

organic acids.MR negative is always VP positive and vice versa.

Procedure:

20 ml of M.R.-V.P broth was prepared.

Dispense the media in two test tubes and autoclave them.

Now add MJRM1204 culture into test tubes and keep the other test tube as Blank.

Incubate for 48hours at 37°C in incubator shaker.

DNS ASSAY

Requirements: Test tubes, DNS reagent, Glucose (0.5mg/ml) solution, Crude enzyme, water

bath, colorimeter, cuvettes, Refrigerated centrifuge, LAF and Micropipettes.

Principle: Cellulase is an enzyme which converts polymeric cellulase to monomeric glucose.

Cellulose+ Cellulase Glucose

23

Thus, if in any solution Cellulose is converted into glucose that means that there is an

involvement of cellulase enzyme. To check the degradation of Cellulase or presence of Glucose

in solutions, DNS test is performed and activity of cellulase and amount of glucose liberated in

solution is determined. Since glucose is a reducing sugar and so reduces yellow colored DNS to

deep brown color ANSA (Amino nitro salicylic acid) which confirms the presence of glucose in

the solution after incubation at 100°C for 15mins. DNS is added to stop the reaction and to show

the color change.

DNS (yellow color) + Glucose ANSA (deep brown color)

After the reaction is complete O.D. was taken at 540nm and a standard graph was made. The

amount of glucose released from CMC, it can easily determine the enzyme activity.

Activity (U/ml/min) = (mg/ml glucose released)* 0.180/ml of enzyme used/incubation

time.

Procedure:

Preparation of Standard Graph

10ml of D/W and 0.5mg/ml glucose solution was prepared.

11 test tubes were taken and 0.1mg/ml,0.2mg/ml,0.3mg/ml so on was dispensed into 10

test tubes

The volume of each test tube was maintained up to 1ml by adding D/W.

The test tubes were incubated in incubator (37°C) for 15mins.

After 15mins 1ml of the DNS was added in every test tube to stop the reaction.

The test tubes were then again incubated for 15min at 100°C in water bath.

After incubation 5ml of D/W was added to dilute and then the O.D. was taken at 540nm.

Standard graph was plotted between concentration and Absorbance at 540nm.

Enzyme Assay

Two test tubes were taken, and 500µl of 1%CMC was taken in one test tube and labeled

as test sample.

24

The other test tube was labeled blank, and 0.5ml of D/W and 0.5ml of 1% CMC was

added to it.

The crude sample was taken after four days of incubation till there is decline in the

enzyme activity.

1ml of the sample was dispensed into eppendorf and centrifuged at 5000rpm for 5min at

4°C.

500µl of the Supernatant was collected and dispensed into the test tube labeled as test

sample.

Both the test tube was incubated in incubator (37°C) for 15mins.

After 15mins of incubation the DNS reagent (1ml) was added to both the test tube.

The test tubes were again kept in water bath for 15mins at 100°C.

After incubation 5ml of D/W was added to the test tubes and absorbance was read at

540nm.

The Graph was plotted against glucose concentration and absorbance at 540nm.

OPTIMIZATION OF PHISIO-CHEMICAL FACTORS FOR MAXIMUM YEILD OF

CELLULASES

PHYSICAL FACTORS

a) Incubation Time

Requirements: MJRM1209 culture, conical flask, Production Media, LAF, Inoculation loop, pH

meter, Magnetic Stirrer and shaker incubator.

Principle: Fermentation is a process of production of industrially important substance by help of

the microorganism under controlled physical and chemical condition. Micro-organism utilizes

the nutrients present in the medium for the survival and thus when it is supplied with CMC

supplemented production media, the micro organism is stressed to utilize CMC as a carbon

source and thus it has to secrete Cellulase enzyme for the degradation of the cellulose.

Procedure:

25

100ml of the production media supplemented with 1%CMC was prepared and other

components were also added.

The pH was maintained at 7.

Media was autoclaved.

1ml of 24 hours old grown culture MJRM1209 was inoculated into production media by

inoculation loop.

It was then incubated at 37°C for four days in incubator shaker and every day DNS assay

was performed.

b) Optimization of pH

Requirements: conical flaks, MJRM1209 culture, inoculation loop, pH meter, LAF,

Incubator shaker, Magnetic Stirrer and spirit lamp.

Principle: The pH is the most important aspect for the growth of the culture. Slight

difference in the pH will result in no growth or maximum growth of the culture. There

are microbes which are very sensitive to the pH range. There are microbes which grows

in acidic medium (pH 2- 5), in alkaline medium (pH 9-14), and those grows at Neutral

pH, i.e. at pH 7.

Procedure:

30ml in each flask media was prepared.

1%w/v of CMC was dissolved in each flask and yeast extract was added in

different conical flask, the other components were also added.

The pH was set as, pH5, 7, 9 and 11.

It was then autoclaved.

Loop full of inoculums from MJRM1209 culture was taken and inoculated in each

flask and kept in incubator shaker for 48 hours at 37°C.

DNS assay was done and the best pH was selected.

CHEMICAL FACTORS

c) Optimization of Nitrogen Source

26

Requirements: Four conical flaks, MJRM1209 culture, inoculation loop, Different Nitrogen

sources, LAF, Sprit lamp, Magnetic Stirrer and Incubator shaker.

Principle: The main aim for optimizing the nitrogen source is to show that which nitrogen

source like, Peptone, Ammonium chloride, Beef extract and Urea supplemented with CMC (1%)

has maximum effect on the growth of the micro organism.

Procedure:

30ml in each flask media was prepared.

1%w/v of CMC was dissolved in each flask and different nitrogen source was added in

different conical flask, the other components were also added and pH was maintained at

7.

The flasks were autoclaved.

Loop full of inoculums from MJRM1209 culture was taken and inoculated in each flask

and kept in incubator shaker for 48 hours at 37°C.

DNS assay was done and the best nitrogen source was selected.

d) Optimization of Substrate at Different Concentrations

Requirements: conical flaks, MJRM1209 culture, inoculation loop, pH meter, LAF,

Incubator shaker, Magnetic Stirrer and spirit lamp.

Principle: The different substrate concentration was used, to show that which showed the

maximum growth of the micro organism. For this CMC was used in different

concentration like, 0.1, 0.5, 1.0, 1.5 and 2.0%w/v concentration.

Procedure:

30ml in each flask media was prepared

Different concentration was dissolved in four different conical flasks.

The other component of the media was added and the pH was maintained at 7.

It was then autoclaved.

Loop full of inoculum from MJRM1209 culture was taken and inoculated in each

flask and kept in incubator shaker for 48 hours at 37°C.

DNS assay was done and the best substrate concentration was selected.

27

PRODUCTION OF CELLULASE BT MJRM1209 IN OPTIMIZED PRODUCTION

MEDIA BY SHAKE FLASK FERMENTATION

Requirements: 250ml conical flask, Optimized production media supplemented with CMC, pH

meter, MJRM12049 culture, inoculation loop, LAF, Spirit Lamp, Magnetic Stirrer and incubator

shaker.

Principle: After the optimization of the media, the Cellulase production was under taken for

high yield of enzyme from the micro organism.

Procedure:

CMC was dissolved in the flask and optimized media was used.

The pH was maintained and it was autoclaved.

The loop full of culture was inoculated in the flask.

It was then incubated at 37°C in incubator shaker for four days.

Then DNS assay of crude enzyme will be performed.

PREPARATION OF CELL FREE EXTRACT

CENTRIUGATION

Requirements: Refrigerated centrifuge, centrifuge tubes, micropipette, tips, beaker and ice pack.

Principle: In order to obtain crude enzyme the fermented broth was centrifuged so that all the

cell debris is collected in the form of pellet and the supernatant was collected in a beaker which

contains enzyme.

28

Procedure:

The fermented broth was dispensed into the centrifuge tubes.

The tubes were centrifuged at 5000rpm, for 5mins at 4°C.

The supernatant was collected and stored in a beaker.

The beaker was kept on ice pack to maintain the temperature at 4°C.

The process was repeated again, till all the fermented broth was not centrifuged.

PROTEIN ESTIMATION OF CRUDE ENZYME BY LOWRY’S METHOD

Requirements: Test tubes, BSA stock solution, Reagent A (1N NaOH+Na2CO3), B (Sodium

potassium tartrate+0.5% CuSO4), C (A+B) and D (Folin-Ciocalteau reagent+ D/W), D/W, Crude

enzyme, Micropipette, tips, Vortex mixer and Colorimeter.

Principle: The phenolic group of tyrosine and tryptophan residues (amino acid) in a protein will

produce a blue purple color complex, with maximum absorption in the region of 660 nm

wavelength, with Folin- Ciocalteau reagent which consists of sodium tungstate molybdate and

phosphate. Thus the intensity of color depends on the amount of these aromatic amino acids

present and will thus vary for different proteins.

Procedure:

Preparation of reagents

70ml of reagent A was made by mixing 0.25g of 1N NaOH and 1.4g of Na2CO3.

10ml of reagent B was made by mixing 0.1g of Sodium potassium tartrate in 0.05

gm of CuSO4.

Reagent C was made by mixing 70ml of Reagent A and 1.4ml of Reagent B.

29

Reagent D was prepared by mixing equal volume of FC reagent (3.5ml) and D/W

(3.5).

BSA sock solution of 10ml was made by adding 10mg of BSA in D/W and the

working solution was made by diluting in the ratio of 1:4, i.e. 1ml of BSA stock

solution and 4ml of water.

Preparation of the standard graph

11 test tubes were taken and working BSA (0.2mg/ml) was dispensed in all tubes starting

from 0µl to 100µl, 200µl up to 1000µl.

The volume of each test tube was maintained up to 1ml by adding D/W, in accordance to

the concentrations provided.

5ml of Reagent C was added to each test tube and was incubated at room temperature for

15mins.

0.5ml of Reagent D was added to each test tube and was incubated in dark for color

change for 30mins.

O.D. was taken at 660nm and standard graph was prepared.

Protein estimation of Crude Enzyme

Two test tubes were taken and 500µl of crude enzyme and 500µl of D/W were added in

one test tube and labeled as test sample.

The other test tube was labeled as blank, 0.5ml of D/W and 0.5ml of 1% CMC was

added.

5ml of Reagent C was added and incubated at room temperature for 10mins

After incubation 0.5ml of FC Reagent was added to both the test tube and was kept in

dark for 30mins.

O.D. was taken at 660nm and compared with the standard graph.

ENZYME PURIFICATION

Salt Precipitation

Requirements: Magnetic Stirrer, Beaker, Ice, Ammonium sulphate and Tris Buffer.

30

Principle:  At low concentrations, the presence of salt stabilizes the various charged groups on a

protein molecule, thus attracting protein into the solution and enhancing the solubility of protein.

This is commonly known as salting-in. However, as the salt concentration is increased, a point of

maximum protein solubility is usually reached. Further increase in the salt concentration implies

that there is less and less water available to solubilize protein. Finally, protein starts to precipitate

when there are not sufficient water molecules to interact with protein molecules. This

phenomenon of protein precipitation in the presence of excess salt is known as salting-out.

Procedure:

2ml of crude extract was kept for preservation.

Rest volume of the crude extract was taken in a small beaker and this beaker was kept in

big beaker full of ice.

The beaker was kept on the magnetic stirrer and ammonium sulphate was added pinch by

pinch until the first one completely dissolves. It is done till the ammonium sulphate

which was weighed is not finished.

When it is dissolved completely, it was put in the refrigerator for 24 hours.

Now it was taken in the centrifuge tube and centrifuged at 10000rpm for 10mins at 4°C.

Supernatant was removed and the pellet was collected.

10 ml of Tris buffer was added to the pellet obtain and was completely dissolved.

The crude enzyme as well as the pellet was put in the refrigerator.

Dialysis

Requirements: Tris Buffer, Dialysis bag, 0.1%SDS solution, Distilled Water and Crude extract.

Principle: Dialysis is one of the common operations in biochemistry to separate dissolved

molecules by passing through a semi-permeable membrane according to their molecular

dimensions. Semi-permeable membrane is containing pores of less than macromolecular

dimensions. These pores allow small molecules, such as those of solvents, salts, and small

metabolites, to diffuse across the membrane but block the passage of larger molecules.

31

Cellophane (cellulose acetate) is the most commonly used dialysis material although many other

substances such as nitrocellulose and collodion are similarity employed. So, dialysis is a method

in which an aqueous solution containing both macromolecules and very small molecules which

are placed in a dialysis bag which is in tern placed in a large container of a given buffer or

distilled water. Thus small solute molecules freely pass through the membrane, and after several

hours of stirring the equilibrium will reach (the concentration inside and outside the bag are the

same). Thus, at equilibrium the concentration of small molecules outside and inside the bag is

the same while the macromolecules remain inside the bag.

Procedure:

To start the process of dialysis, first dialysis bag need to be activated.

Dialysis bag activation is done by boiling the bag in D/W for 1mins twice and then

transferring it to 0.1%SDS solution and boiled twice for 1mins, then again it was boiled

in D/W for 1mins, and then finally transferred to the D/W.

Once the bag is activated, the crude enzyme was transferred to the bag and was sealed.

This bag was kept in 100mM Tris buffer for 90mins and was again changed and kept

overnight in fridge.

After keeping it for overnight, the buffer was again changed and kept for 90mins.

Protein Estimation and Enzyme Activity in Pure Enzyme by Lowry’s Method and DNS

Assay

Lowry’s Method

Procedure:

Two test tubes were taken and 500µl of crude enzyme and 500µl of D/W were added in

one test tube and labeled as test sample.

The other test tube was labeled as blank, 0.5ml of D/W and 0.5ml of 1% CMC was

added.

5ml of Reagent C was added and incubated at room temperature for 10mins

32

After incubation 0.5ml of FC Reagent was added to both the test tube and was kept in

dark for 30mins.

O.D. was taken at 660nm and compared with the standard graph.

DNS Assay

Procedure:

Two test tubes were taken, and 500µl of 1%CMC was taken in one test tube and labeled

as test sample.

The other test tube was labeled blank, and 0.5ml ml of D/W and 0.5ml of 1% CMC was

added to it.

The pure enzyme sample was taken for the enzyme activity.

Both the test tube was incubated in incubator (37°C) for 15mins.

After 15mins of incubation the DNS reagent (1ml) was added to both the test tube.

The test tubes were again kept in water bath for 15mins at 100°C.

After incubation 5ml of D/W was added to the test tubes and absorbance was read at

540nm.

The Graph was plotted against glucose concentration and absorbance at 540nm.

CHARACTERIZATION OF PURE ENZYME

a) Characterization at different Temperatures

Requirements: Test tubes, DNS reagent, Crude enzyme, water bath, colorimeter, cuvettes,

Incubator and Micropipettes.

Principle: The Enzyme activity was observed at different incubation temperatures, like 20°C,

28°C, 37°C and 50°C.

Procedure:

33

Five test tubes were taken, and 500µl of 1%CMC was taken in four test tubes and labeled

as 20°C, 28°C, 37°C and 50°C.

The other test tube was labeled blank, and 0.5ml of D/W and 0.5ml of 1% CMC was

added to it.

The pure enzyme sample was taken for the enzyme activity.

The test tubes were kept at different temperature like, 20°C (refrigerator), 28°C (Room

temperature), 37°C (Incubator) and 50°C (Hot air oven) for 15mins incubation.

After 15mins of incubation the DNS reagent (1ml) was added to all the test tube.

The test tubes were again kept in water bath for 15mins at 100°C.

After incubation 5ml of D/W was added to the test tubes and absorbance was read at

540nm.

Then graph was plotted for the same.

b) Characterization at Different pH

Requirements: Test tubes, DNS reagent, Crude enzyme, water bath, colorimeter, cuvettes,

Incubator and pH meter, Micropipettes.

Principle: The Enzyme activity was observed at different pH, so as to observe the enzyme

stability at different pH like, pH 5, 7, 9 and 11.

Procedure:

Five test tubes were taken, and 500µl of 1%CMC was taken in four test tubes and labeled

as pH5, 7, 9 and 11.

The other test tube was labeled blank, 0.5ml of D/W and 0.5ml of 1% CMC was added to

it.

The pure enzyme sample was taken for the enzyme activity.

All the test tube was incubated in incubator (37°C) for 15mins.

After 15mins of incubation the DNS reagent (1ml) was added to all the test tube.

The test tubes were again kept in water bath for 15mins at 100°C.

34

After incubation 5ml of D/W was added to the test tubes and absorbance was read at

540nm.

The Graph was plotted for the same.

c) Characterization Of Different Activators And Inhibitors

Requirements: Test tubes, DNS reagent, Crude enzyme, water bath, colorimeter, cuvettes,

Incubator, Micropipettes, Activators (CaCl2 and MgCl2) and Inhibitors (SDS and EDTA).

Principle: The enzyme activity was observed under different Activators and Inhibitors present in

the medium.

Procedure:

Five test tubes were taken, and 500µl of 1%CMC was taken in four test tubes and labeled

as CaCl2, MgCl2 (0.5mg/ml), SDS and EDTA (0.1mg/ml) were used.

The other test tube was labeled blank, 0.5ml of D/W and 0.5ml of 1% CMC was added to

it.

The pure enzyme sample was taken for the enzyme activity.

All the test tube was incubated in incubator (37°C) for 15mins.

After 15mins of incubation the DNS reagent (1ml) was added to all the test tube.

The test tubes were again kept in water bath for 15mins at 100°C.

After incubation 5ml of D/W was added to the test tubes and absorbance was read at

540nm.

The Graph was plotted for the same.

RESULTS

ISOLATION OF CELLULASE PRODUCING BACTEIA

35

Cellulase producing Bacterial strain were isolated by the help of serial dilution and Spread plate

method and mixed culture plates were obtained, isolates were differentiated based on the

differences in morphological characteristics and named as MJRM1206, MJRM1207,

MJRM1208, MJRM1209, MJRM1210, and MJRM1211, pictures of mixed culture plates can

be seen in Figure 1 and the morphological characteristics can be seen in a Table 1 below.

Figure 1: Mixed Culture Plate

Table 1: Colony Morphology of Different Bacterial Colony

Bacterial

Colony

Margin

type

Colony

Elevation

Colony

Surfac

e

Colony

Texture

Light

Transmission

Pigmentation

MJRM1206 Entire Flat Smooth Soft Opaque Yellowish

MJRM1207 Entire Flat Smooth Soft Opaque White

MJRM1208 Undulating Convex Smooth Soft Transparent Creamish

MJRM1209 Entire Convex Soft Sticky Opaque Creamish

MJRM1210 Irregular Bulge Rough Hard &

Sticky

Opaque White

MJRM1211 Circular Flat Smooth Sticky Transparent White

36

PURIFICATION OF OBTAINED MIXED CULTURES

Mixed cultures obtained after spreading were purified by the help of Discontinuous Quadrant

Streaking technique, and the result of the same can be seen in Figure 2 below.

MJRM1206 MJRM1209

MJRM1207 MJRM1210

MJRM1208

Figure 2: Pure Cultures

37

SCREENING OF PURE CULTURES FOR CELLULASE PRODUCTION BY CONGO

RED TEST

Purified plates were streaked on minimal agar media supplemented with 1% CMC, and

incubated for 72 hours. After incubation the screening plates were flooded with 0.1% Congo red

dye and de-stained with 1N NaCl solution, a clear zone of hydrolysis of CMC was obtained,

culture MJRM1204 showed largest zone of hydrolysis around its colony. Screening results were

ranked on the basis of maximum zone of hydrolysis. The results were ranked on the basis of zone

of hydrolysis as: intense +++, moderate ++, slight +, and no hydrolysis -. The same can be seen

in Figure 3 and Table 2 below.

Table 2: Ranking of different isolates on the basis of zone of hydrolysis

Figure 3: Screening Plates

38

S. No. Culture Ranking (zone of

hydrolysis)

1. MJRM1206 -

2. MJRM1207 +

3. MJRM1208 +

4. MJRM1209 +++

5. MJRM1210 ++

6. MJRM1211 ++

STUDY OF GROWTH PARAMETERS OF MJRM1209

a) Growth Kinetics:

Growth curve of the isolate was studies in order to have an idea of the time at which stationary

phase is reached as at this stage of growth curve stationary phase is reached. The study of growth

kinetics of MJRM1209 culture show different growth phases on different days. The different

phases are shown in Table 3 and Figure 4 below.

Table 3: Growth Kinetics of MJRM1209

S. No. Day Optical Density at

600nm

Activity

(U/ml/min)

1. 1 0.0 0.00

2. 2 0.34 0.008

3. 3 0.65 0.015

4. 4 0.75 0.018

5. 5 0.84 0.020

6. 6 0.80 0.019

7. 7 0.74 0.017

1 2 3 4 5 6 70

0.005

0.01

0.015

0.02

0.025

Growth Curve

No. of Days

Activ

ity (U

/ml/

min

)

Figure 4: Growth Curve

39

b) Effect of Temperature on Growth of MJRM1209

At different temperatures the growth of the MJRM1209 culture was seen and it was ranked as:

intense +++, moderate ++, slight +, and no growth -. It has been seen that temperature 37°C

showed maximum growth. The Figure 5a, b, c and d Table 4 shows the same.

Table 4: Effect of Temperature on MJRM1209 Culture

S. No. Incubation

Temperature

Remarks

a) 20°C -

b) 28°C ++

c) 37°C +++

d) 50°C -

A: 20°C b: 28°C c: 37°C d: 50°C

Figure 5: Effect of temperature on MJRM1209

40

c) Effect of pH on Growth of MJRM1209

At different pH the growth of the MJRM1209 culture was studied and it was observed that pH 7

showed the maximum growth. The O.D. was read at 600nm. The Table 5 and Figure 6 showed

the results.

Table 5: Effect of pH on MJRM12049 culture

S. No. pH O.D. at 600nm

1. 5 0.27

2. 7 0.41

3. 9 0.08

4. 11 0.00

Figure 6: Effect of pH on MJRM1209

IDENTIFICATION OF ISOALTE MJRM1209

The isolate MJRM1209 was identified as Bacillus subtilis based on Bergey’s manual (Aneja,

2003) by performing various staining and biochemical activities, Table 6 below shows results of

various staining and biochemical activities. Figure 7-8 below show the results of biochemical

tests.

41

pH5 pH7 pH9 pH110

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

Column2

Abso

rban

ce a

t 600

nm

Table 6: Morphological properties of MJRM1209

S. No. Staining/

Biochemical Tests

Results

1. Gram Staining +ve Rods in

chain

2. Endospore Staining +ve

3. Catalase +ve

4. Mannitol

Fermentation

+ve

5. Voges-Proskeur’s +ve

Figure 7: Mannitol Test Figure 8: MR-VP Test

42

DNS ASSAY

Standard Graph

A standard graph was prepared for different Glucose concentrations so that activity and amount

of Glucose concentration released can be found for the bacteria. Thus, firstly the standard graph

was prepared.

Table 7: Standard Graph for DNS assay

Volume of

Glucose

(5mg/ml)

Distilled

Water

Conc. of

Glucose

(mg/ml)

(x-axis)

DNS

(1ml)

Incu

bat

ion

At

(100

°C)

for

15m

ins

Distilled

water to

dilute

O.D.

at

540nm

(y-

axis)

Activity

(U/ml/min)

0.00(blank) 1.00 0.00 1ml 5ml 0.00 0.00

0.10 0.90 0.05 1ml 5ml 0.05 0.0012

0.20 0.80 0.01 1ml 5ml 0.08 0.0015

0.30 0.70 0.15 1ml 5ml 0.14 0.0034

0.40 0.60 0.20 1ml 5ml 0.19 0.0045

0.50 0.50 0.25 1ml 5ml 0.22 0.0053

0.60 0.40 0.30 1ml 5ml 0.25 0.0060

0.70 0.30 0.35 1ml 5ml 0.30 0.0072

0.80 0.20 0.40 1ml 5ml 0.32 0.0075

0.90 0.10 0.45 1ml 5ml 0.40 0.010

1.00 0.00 0.50 1ml 5ml 0.49 0.012

43

Figure 9: Standard Graph for DNS assay

OPTIMIZATION OF PHISIO-CHEMICAL FACTORS FOR MAXIMUM

PRODUCTION OF CELLULASES

PHYSICAL FACTORS

a) Incubation Time

The isolate was inoculated in the production media and incubated at 37 °C at 120 rpm in

shaker incubator. In order to have an idea of the best time after which maximum

cellulases are produced, 1ml of fermented broth was sampled out after every 24 hour of

inoculation and was subjected to DNS assay as explained earlier. The results are shown

in Table 8 and Figure 10 below.

Table 8: Effect of Incubation Time on Cellulase Production

S. No. Days O.D. at 540nm Activity

(U/ml/min)

1. 1 0.00 0.00

2. 2 0.35 0.007

3. 3 0.41 0.009

44

0 0.05 0.01 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.50

0.002

0.004

0.006

0.008

0.01

0.012

0.014

Standard Graph for DNS assay

Glucose concentration

Activ

ity (U

/ml/

min

)

4. 4 0.45 0.011

5. 5 0.36 0.008

6. 6 0.34 0.006

Figure 10: Incubation time

b) Optimization of pH

At different pH the bacterial cultures were incubated, to observe the maximum growth of

bacteria, this also shows that at which pH the enzyme produce by the bacteria is stable

and gave maximum results. Its DNS assay was performed after incubating it for 48 hours

at 37°C in incubator shaker. Thus, pH 7 showed the maximum growth of bacteria .The

Table 9 and Figure 11 below show the results.

Table 9: Optimization of pH for MJRM1209 culture

45

1 2 3 4 5 60

0.002

0.004

0.006

0.008

0.01

0.012

Activity(U/ml/min)

Incubation Time

Activ

ity (U

/ml/

min

)

Figure 11: Optimization of pH for MJRM1209 culture.

CHEMICAL FACTORS

c) Optimization of Nitrogen Source

After giving the incubation of 48hours at 37°C, its DNS assay was done to observe the

maximum growth of the bacterial culture at different Nitrogen sources. The best Nitrogen

46

blank pH5 pH7 pH9 pH110

0.001

0.002

0.003

0.004

0.005

0.006

0.007

Different pH

Activ

ity (

U/m

l/m

in)

S.NO. Different pH Optical

Density at

540nm

Activity

(U/ml/min)

1 Blank 0.00 0.00

2 pH 5 0.22 0.0052

3 pH 7 0.27 0.0063

4 pH 9 0.26 0.0062

5 pH 11 0.24 0.0057

source for the bacteria is Beef Extract as it favors maximum growth. Table 10 and

Figure 12 show the result below.

Table 10: Optimization of Nitrogen Source

Different

Nitrogen source

Optical

Density at

540nm

Activity

(U/ml/min)

Blank 0.00 0.00

Ammonium

Chloride

0.35 0.007

Peptone 0.32 0.008

Beef Extract 0.44 0.010

Urea 0.35 0.007

Figure 12: Optimization of Nitrogen Source.

d) Optimization of Substrate at different concentrations

At different substrate concentrations CMC was dissolved and the bacterial growth was

observed after 48 hours at 37°C. DNS assay was performed for the different nitrogen

sources and optical density was observed at 540nm, the result showed that CMC 1%w/v

47

Blank

Ammon

ium Ch

loride

Pepto

ne

Beef 

Extra

ctUr

ea0

0.002

0.004

0.006

0.008

0.01

0.012

Different Nitrogen Source

Activ

ity (U

/ml/

min

)

concentration was the best concentration for the growth of the bacteria. The Table 11 and

Figure 13 are shown below.

Table 11: Optimization of Substrate at different concentrations

S.NO. Different

substrate

concentrations

Optical

Density at

540nm

Activity

(U/ml/min)

1 0.1%w/v 0.15 0.0036

2 0.5%w/v 0.16 0.0038

3 1.0%w/v 0.41 0.0098

4 1.5%w/v 0.23 0.0055

5 2.0%w/v 0.27 0.0064

0.10% 0.50% 1.00% 1.50% 2.00%0

0.002

0.004

0.006

0.008

0.01

0.012

Substrate concentrations

Activ

ity (U

/ml/

min

)

Figure 13: Optimization of Substrate at different concentrations

DNS for the Enzyme

After preparing the standard graph, DNS assay for the test sample was performed and its

Enzyme activity and amount of Glucose released were calculated. The Optical density of

48

the reaction was 0.22nm and thus the liberated concentration of Glucose was 0.25mg/ml

which was calculated according to the standard graph. Activity was 0.006 U/ml/min.

Total activity of enzyme was 0.51 and specific activity was 0.075U/mg. Results of the

same are shown in Table 12 and Figure 14.

Table 12: Activity of the crude enzyme by DNS assay

Test

Tube

Vol. of

Glucose

(5mg/ml)

Vol. of

Crude

enzyme

Conc.

Of

Glucos

e in

mg/ml

Incubation

time at

37°c

Vol.

of

DNS

Incu

bat

ion

tim

e at

100

°C

For

15m

ins.

O.D.

at

540nm

Activity

U/ml/min

Blank 0.00 0.00 0.00 15mins 1ml 0.00 0.00

Crud

e

0.5ml 0.50 0.25 15mins 1ml 0.22 0.006

Figure 14: Graph for the DNS assay of crude enzyme

49

Calculation:

Amount of Glucose released=0.25mg/ml

Activity of Enzyme = 0.25*0.18/0.5/15 = 0.006U/ml/min.

Total activity was = volume of crude enzyme*activity of Enzyme = 85*0.006 = 0.51

Specific activity = total activity of enzyme/total protein of enzyme = 0.51/6.8 = 0.075U/mg.

PROTEIN ESTIMATION OF CRUDE ENZYME BY LOWRY’S METHOD

Table 13 and Figure 15 show the standard graph for the Lowry’s method.

Table 13: Lowry’s standard Graph

S.NO. BSA

Working

(mg/ml)

Distille

d

Water

Conc.

Of

BSA

Reagent

C

Incu

bat

ion

tim

e at

roo

m t

emp

erat

ure

.

For

10m

ins

Reagent

D

Incu

bat

ion

tim

e in

dar

k

For

30m

ins.

O.D. at

660nm

Activity

(U/ml/

min)

1 0.0 1.0 0.0 5ml 0.5ml 0.0 0.00

2 0.1 0.9 0.02 5ml 0.5ml 0.07 0.0016

3 0.2 0.8 0.04 5ml 0.5ml 0.10 0.0024

4 0.3 0.7 0.06 5ml 0.5ml 0.15 0.0036

5 0.4 0.6 0.08 5ml 0.5ml 0.23 0.0055

6 0.5 0.5 0.10 5ml 0.5ml 0.24 0.0057

7 0.6 0.4 0.12 5ml 0.5ml 0.29 0.0069

8 0.7 0.3 0.14 5ml 0.5ml 0.36 0.0086

9 0.8 0.2 0.16 5ml 0.5ml 0.44 0.0105

10 0.9 0.1 0.18 5ml 0.5ml 0.52 0.0125

11 1.0 0.0 0.20 5ml 0.5ml 0.56 0.0134

50

Figure 15: Standard Graph for Lowry’s method

Protein Estimation of Crude Enzyme

Concentration of Protein in the crude was determined by comparing the absorbance reading of

the test sample with the standard graph and it was found out to be 0.08mg/ml.

Table 14: Protein estimation by Lowry’s Method

51

0 0.020.040.060.08 0.1 0.120.140.160.18 0.20

0.002

0.004

0.006

0.008

0.01

0.012

0.014

0.016

Conc. of BSA

Activ

ity(U

/ml/

min

)

Test

tube

Vol. of

Crude

Enzym

e

Vol.

of

CMC

in ml

Conc.

Of

Protein

(mg/ml)

Vol. of

reagent

C in ml

Incu

bat

ion

tim

e at

roo

m

tem

per

atu

re

For

10m

ins.

Vol. of

Reagen

t D in

ml

Incu

bat

ion

tim

e in

dar

k

For

30m

ins.

O.D.

at

660nm

Total

Protein

(mg)

Blank 0.00 0.00 0.00 5ml 0.5ml 0.00 0.00

Crude

Enzyme

0.5 0.5 0.08 5ml 0.5ml 0.22 6.8

Figure 16: Graph for Crude Enzyme

Calculation:

Concentration of protein in crude enzyme was=0.08mg/ml

Total protein was= amount/ml * total volume of crude enzyme

52

0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.18 0.20

0.002

0.004

0.006

0.008

0.01

0.012

0.014

0.016

Activity (U/ml/min)

BSA Concentration

Activ

ity (U

/ml/

min

)

= 0.08*85= 6.8mg.

Protein Estimation and Enzyme Activity in Purified Enzyme by Lowry’s

Method and DNS Assay

Lowry’s Method

The enzyme after purification was 0.0432mg and its activity was 0.003U/ml/min with the

specific activity of 0.07U/mg.

Table 15: Protein Estimation by Lowry’s Method

Test

tube

Vol. of

Crude

Enzym

e

Vol.

of

CMC

in ml

Conc.

Of

Protein

(mg/ml)

Vol. of

reagent

C in ml

Incu

bat

ion

tim

e at

roo

m

tem

per

atu

re

For

10m

ins.

Vol. of

Reagen

t D in

ml

Incu

bat

ion

tim

e in

dar

k

For

30m

ins.

O.D.

at

660nm

Total

Protein

mg

Blank 0.00 0.00 0.00 5ml 0.5ml 0.00 0.00

Crude

Enzyme

0.5 0.5 0.12 5ml 0.5ml 0.28 0.0432

53

Figure 17: Graphical representation of Protein estimation by Lowry’s Method.

DNS Assay

Table 16: Activity of Pure Enzyme by DNS Assay

Test

Tube

Vol. of

Glucose

(5mg/

ml)

Vol. of

Crude

enzyme

Conc.

Of

Glucose

in

mg/ml

Vol.

of

DNS

Incu

bat

ion

tim

e at

100

°C

For

15m

ins.

O.D.

at

540nm

Activity

U/ml/min

Blank 0.00 0.00 0.00 1ml 0.00 0.00

Crude 0.5ml 0.50 0.20 1ml 0.20 0.003

54

Figure 18: Graphical representation of Activity of Pure Enzyme by DNS Assay

CHARACTERIZATION OF PURE ENZYME

Effect of Temperature on Enzyme Activity

During incubation for 15mins, the enzyme was kept at different temperatures to study their

characteristics features. The enzyme was kept at 20°C, 28°C, 37°C and 50°C for 15mins and it

was found that the Enzyme activity was low at 20°C and was high at 37°C. Table 17 and Figure

19 shows the result.

Table 17: Effect of Temperature on Enzyme Activity

S.NO. Temperature O.D. at 540nm Activity

(U/ml/min)

1 20°C 0.18 0.0043

2 28°C 0.22 0.0052

3 37°C 0.27 0.0064

4 50°C 0.25 0.0060

55

20°c 28°c 37°c 50°c0

0.001

0.002

0.003

0.004

0.005

0.006

0.007

Different Tempeatures

Activ

ity (U

/ml/

min

)

Figure 19: Graphical representation at different Temperatures

Effect of pH on Enzyme Activity

During incubation for 15mins, the enzyme was kept at different pH to study their characteristic

features. The Enzyme was kept at pH5, 7, 9 and 11 for studying the enzyme activity and it was

found that pH 5 showed the minimum activity while pH 7 showed the maximum activity. This

also shows that the enzyme is sensitive to pH change and loses its activity at alkaline or acidic

medium. Table 18 and Figure 20 showed the same.

Table 18: Effect of pH on Enzyme Activity

56

pH 5 pH 7 pH 9 pH 110

0.001

0.002

0.003

0.004

0.005

0.006

0.007

0.008

Different pH

Activ

ity (U

/ml/

min

)

20°c 28°c 37°c 50°c0

0.001

0.002

0.003

0.004

0.005

0.006

0.007

Different Tempeatures

Activ

ity (U

/ml/

min

)

S.NO. pH O.D. at 540nm Activity

(U/ml/min)

1 5 0.22 0.0052

2 7 0.28 0.0067

3 9 0.23 0.0055

4 11 0.24 0.0053

Figure 20: Graphical representation at different pH

Effect of Activators and Inhibitors on Enzyme Activity

During incubation for 15mins, the enzyme was kept at different activators and inhibitors to study

the effect on the protein activity. It was found that CaCl2 had a little effect on the protein activity

while MgCl2 showed the maximum protein activity. The inhibitors showed the decrease activity

in the protein. The Table 19 and Figure 21 and Figure 22 showed the same.

Table 19: Effect of Activators and Inhibitors on Enzyme Activity

S.NO. Activators O.D. at 540nm Activity

(U/ml/min)

1 CaCl2 0.23 0.0055

2 MgCl2 0.29 0.0071

Inhibitors O.D. at 540nm

1 EDTA 0.15 0.0045

2 SDS 0.12 0.0041

57

CaCl2 MgCl20

0.001

0.002

0.003

0.004

0.005

0.006

0.007

0.008

Activators

Activ

ity (U

/ml/

min

)

pH 5 pH 7 pH 9 pH 110

0.001

0.002

0.003

0.004

0.005

0.006

0.007

0.008

Different pH

Activ

ity (U

/ml/

min

)

Figure 21: Graphical representation of different Activators

Figure 22: Graphical representation of different Inhibitors

58

EDTA SDS0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

Inhibitors

Activ

ity (U

/ml/

min

)

CaCl2 MgCl20

0.001

0.002

0.003

0.004

0.005

0.006

0.007

0.008

Activators

Activ

ity (U

/ml/

min

)

DISCUSSION

Cellulase producing bacteria were isolated from soil and grown on Minimal Agar Medium

having limited glucose concentration and CMC as supplemented, which was earlier used by

Deka et al., 2011 and Gupta et al., 2011 and screening was done by Congo red Dye test as done

earlier by Krairitthichai and Thongwai, 2009.

The Bacterial culture was identified by performing different biochemical tests and stains on the

culture. The isolated culture was identified as Bacillus subtilis based on Bergey’s Manual

(Aneja, 2003).

Media was optimized on the basis of different temperature, pH, Nitrogen source, Incubation time

and substrate concentration on the isolated culture, and it was earlier done by Kurup et al, 2005;

Shankar and Isaiarasu, 2011.

Production of Cellulase by the help of isolated bacteria was done through shake flask

fermentation process by production media containing Beef extract, KH2PO4, MgSO4, NaNO3,

KCl and CMC. The temperature was kept at 37°C, pH was kept at 7 and substrate concentration

of 1%w/v for four days was used.

After incubation the cell free extract was obtained from fermented media by centrifugation at

5000rpm for 5mins at 4°c and supernatant was collected. The enzyme obtain was then purified

by salt precipitation and Dialysis Bakare et al., 2005.

59

Then it’s Protein Estimation and Enzyme activity was done by Lowry’s method (Lowry’s et al,

1951) and DNS Assay (Gail Lorenz miller, 1959). The pure enzyme activity (0.003 U/ml/min)

and specific activity (0.075U/mg) was observed after four days of incubation, activity

(0.006U/ml/min) and (0.0851IU/ml) have been reported by Shankar and Isaiarasu, 2011.

The activity of pure Cellulase was stimulated by low concentrations (1 mg/ml) of MgCl2 and was

inhibited by EDTA (1mg/ml).

CONCLUSION

Based on the present research it can be concluded that soil is the best source for isolation of

Cellulase producing bacteria. The purified Enzyme obtain can be widely use in many

applications at industrial level. The Enzyme obtain was 6.8mg with the specific activity of

0.075U/mg.

Future prospect for the present work include purification of enzyme in order to gain higher

specific activity of cellulase by the help of sophisticated purification procedures including

Salt/Solvent precipitation, Dialysis, Ion Exchange chromatography, Affinity chromatography,

and HPLC, also the purified enzyme can be characterized for the effect of temperature, pH,

activators, Inhibitors, Nitrogen and carbon sources. Molecular weight of purified enzyme can be

determined by SDS-PAGE.

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