“PRODUCTION, PURIFICATION AND CHARACTERIZATION OF

CELLULASES FROM WILD AND MUTANT STRAINS OF

ASPERGILLUS SPECIES”

By

SOFIA QAISAR

Research Supervisor

DR. SHAH ALI UL QADER

Associate Professor

Dr. A. Q. Khan Institute of Biotechnology and

Genetic Engineering (KIBGE),

University of Karachi

Pakistan

2014

THESIS SUBMITTED FOR

THE DEGREE OF DOCTORATE IN

BIOTECHNOLOGY

BY

SOFIA QAISAR

Acknowledgments

Abstract

Section 1

Introduction

Section 2

Plan of Work

Section 3

Materials and Methods

Section 4

Results and Discussion

Section 5

References & Publications

ACKNOWLEDEMENTS

In writing this thesis, I feel that I have plunged into a course of surveying a new field and

treading an unbeaten roadway. Allah Tala was always there behind my hard work and

effort.

I owe my deepest recognition to the Director of the A.Q. Khan Institute of

Biotechnology and Genetic Engineering, Prof. Dr. Abid Azhar and former Director

General, Dr. Irtifaq Ali. They have made available their benevolence in a number of ways

in facilitating my thesis.

I am extremely grateful to Prof. Dr. Shah Ali Ul Qader for the sustained support which he

has provided over the years. He was the one who provided much-needed criticism as well

as encouragement. . This thesis would not have been possible without endeavors of

Khatoon always gave me courage in time of difficulty. I offer my regard to Hanif ur

Rehman, Muhammad Asif Nawaz, Rashida Rehmat Zohra, Maria Ghani, Asma Ansari,

Zainab Bibi, Hafsa Sattar, Sidra Pervaiz, Rizma Khan and all researchers in Industrial

Biotechnology Wing C.

It is with immense gratitude that I acknowledge the support and help of my family. The

light that made my journey possible came from Muhammad Qaisar Hayat Khan and my

mother Mrs. Effat Qaisar Khan and my husband Mr. Hamayoon Khan Jadoon whom

unbounded learning and round the clock efforts enriched my work. Without their adroit

guidance, the present work would not have seen the light of the day.

DEDICATION

THE THESIS IS DEDICATED TO MY BELOVED PARENTS

AND HUSBAND WHOM LOVE, PRAYERS AND INTENSE

SUPPORT HAS MADE THIS POSSIBLE

CONTENTS

S. No. DESCRIPTION Pg No.

i ACKNOWLEDGEMENT i

ii Dedication ii

iii List of Tables iii

iv List of Figures iv

v ABBREVIATIONS vii

vi ABSTRACT ix

vii ABSTRACT (Urdu) x

SECTION

1

INTRODUCTION

1.1 Cellulose and Cellulase 1

1.2 Organism responsible for cellulase production 2

1.3 Cellulase system 3

1.4 Classification of cellulase based upon cellulolysis 4

1.5 Cellulose Hydrolysis Mechanisms 4

1.6 Synergism of cellulase components 6

1.7 Cellulase structure 8

1.8 Cellulase configurations 9

1.8.1 Pocket configuration 10

1.8.2 Cleft configuration 11

1.8.3 Tunnel configuration 11

1.9 Cellulase activity assay 12

1.10 Endoglucanases 12

1.11 Exoglucanases 13

1.12 β-D-glucosidases 13

1.13 Screening strategies of cellulases producing organism 14

1.14 Production of cellulase 16

1.15 Kinetics of cellulase 16

1.16 Applications of cellulases 16

1.16.1 Food Industry 16

1.16.1.1 Processing of pulp/Juices 16

1.16.1.2 Total soluble solids (TSS) 16

1.16.1.3 pH of juice 17

1.16.1.4 Titratable acidity of juice 17

1.16.1.5 Clarity 17

1.16.1.6 Flavor 17

1.16.1.7 Sweetness 17

1.16.1.8 Overall Acceptability 18

1.16.2 Baking Industry 18

1.16.2.1 CMCase in bread making 18

1.16.2. 2 Crust color 18

1.16.2.3 Symmetry of form 18

1.16.2.4 Character of crust 18

1.16.2.5 Aroma 19

1.16.2. 6 Taste 19

1.16.2.7 Texture 19

1.16.3 Other uses 20

1.16.4 Brewing Industry 20

1.16.4.1 Beer brewing 20

1.16.4.2 Wine brewing 21

1.16.5 Feed Industry 21

1.16.5.1 Toxicity 21

1.16. 5.2 Future concerns 22

1.16.6 Textile Industry 22

1.17.6.1 Bio-stoning 22

1.16.6.2 Fabric biopolishing 23

1.16.6.3 Defibrillation 23

1.16.6.4 De starching 23

1.16.7 Detergent Industry 23

1.16.7.1 Color brightness 23

1.16.7.2 Washing efficiency 24

1.16.7.3 New trends in laundry detergents 24

1.16.8 Edible oil Industry 24

1.16.9 Extraction of pigments 25

1.16.10 Vitamins and proteins 25

1.16.11 Protoplast research 25

1.16.12 Disinfection 25

1.16.13 Pulp and paper industry 26

1.16.13.1 Bio Mechanical pulping 26

1.16.13.2 Fiber modification 26

1.16.13.3 Bleaching of pulp 26

1.16.13.4 De inking 26

1.16.13.5 Drainage in paper mill 27

1.16.13.6 Fiber studies 27

SECTION

2

PLAN OF WORK

2.1 Isolation and identification of fungi from different sources 28

2.2 Mutation Induction 28

2.3 Optimization of growth and culture conditions for cellulase

production

28

2.4 Enzyme Kinetics 29

2.5 Purification of Cellulases 29

2.6 Electrophoresis and Zymography of purified cellulases 29

SECTION MATERIAL AND METHODS

3

A GENERAL SECTION 30

3.1 Chemicals 31

3.2 Instruments 31

3.3 Buffers used 31

3.3.1 Phosphate Buffer 32

3.3.2 Citrate Phosphate Buffer 32

3.3.3 Tris-HCl Buffer 32

3.4 Reagent 33

3.4.1 Ditrosalicyclic acid reagent (DNS) 33

3.5 Solution 33

B EXPERIMENTAL SECTION

3.6 Samples Collection 33

3.7 Isolation and purification of cellulolytic fungi 34

3.8 Processing of Samples 34

3.9 Identification of Fungi 35

3.10 Screening for potential Cellulolytic Fungus 35

3.10.1 Qualitative Method 35

3.10.2 Quantitative Method 36

3.11 Selection of culturing medium 37

3.12 Selection of fermentation mode 38

3.13 Optimization of fermentation Conditions for Cellulase

Production

39

3.13.1 Effect of temperature on cellulase production 39

3.13.2 Effect of initial pH of medium on cellulase production 40

3.13.3 Effect of incubation time on cellulase production 40

3.13.4 Effect of different CMC concentration on cellulase production 40

3.13.5 Effect of peptone on cellulase production 40

3.13.6 Effect of Tween 80 on cellulase production 41

3.13.7 Effect of calcium chloride (CaCl2) on cellulase production 41

3.13.8 Effect of sodium nitrate (NaNO3) on cellulase production 41

3.13.9 Effect of potassium dihydrogen phosphate on cellulase

production

41

3.14 Optimization of kinetic parameters for cellulase production 41

3.14.1 pH maxima 42

3.14.2 Temperature maxima 42

3.14.3 Selection of buffer for enzyme activity 42

3.14.4 Ion strength of buffer 42

3.14.5 Storage stability 42

3.15 Mutation induction 43

3.15.1 Selection of strain after mutation 43

3.15.2 Stability of mutant strain 43

3.16 Purification of cellulase enzyme 43

3.16.1 Crude enzyme extraction 43

3.16.2 Partial Purification of CMCase 44

3.17 Gel permeation chromatography 44

3.18 Genome characterizations 44

3.19 Electrophoresis SDS PAGE 44

3.20 Zymography of CMCase 45

3.20.1 Reagents 45

3.20.2 Methodology 45

3.21 N-terminal Protein Sequencing 45

C ANALYTICAL SECTION

3.22 CMCase Assay 46

3.22.1 Principle 46

3.22.2 Procedure 46

3.23 Glucose standard curve 47

3.24 Estimation Of Protein 47

SECTION RESULTS AND DISCUSSION

4

4.1 Sample collection and Handling 49

4.2 Identification 49

4.3 Phylogenetic tree 51

4.4 Fermentation mode for the CMCase production 53

4.5 Effect of fermentation conditions for CMCase production 54

4.5.1 Time course for CMCase production 54

4.5.2 Effect of substrate concentration on CMCase production 56

4.5.3 Effect of temperature on CMCase production 58

4.5.4 Effect of pH on CMCase production 60

4.5.5 Effect of peptone concentration on CMCase production 62

4.5.6 Effect of Tween 80 concentration on CMCase production 64

4.5.7 Effect of NaNO₃ concentration on CMCase production 66

4.5.8 Effect of CaCl₂ concentration on CMCase production 68

4.5.9 Optimized medium for native Aspergillus versicolor KIBGE-

IB37

70

4.6 Partial Purification of CMCase using ammonium sulphate 71

4.7 Gel permeation chromatography of KIBGE-IB37 72

4.8 Catalytic Properties of CMCase from KIBGE-IB37 73

4.8.1 Kinetic Parameters (Km and Vmax) 73

4.8.2 Effect of pH on CMCase activity 74

4.8.3 Effect of Temperature on CMCase activity 75

4.8.4 Effect of different buffers on CMCase activity 76

4.8.5 Effect of ionic strength of buffer on CMCase activity 77

4.8.6 Storage stability of CMCase 78

4.9 SDS-PAGE and Zymography of the partially purified CMCase

from KIBGE-IB37

79

4.10 Mutation Induction 80

4.11 Optimization of fermentation conditions for maximum CMCase 81

production from mutant KIBGE-IB37MT

4.11.1 Effect of pH on CMCase production 82

4.11.2 Effect of Incubation Period on CMCase Production 83

4.11.3 Effect of Substrate Concentration on CMCase Production 84

4.11.4 Effect of Peptone Concentration on CMCase Production 85

4.11.5 Effect of Tween 80 Concentration on CMCase production 86

4.12 Partial Purification of KIBGE-IB37MT 87

4.13 Gel permeation chromatography of KIBGE-IB37MT 88

4.14 Catalytic properties of CMCase from KIBGE-IB37MT 89

4.14.1 Kinetic parameters (Km and Vmax) 89

4.14.2 Effect of pH on CMCase Activity 90

4.14.3 Effect of Temperature on CMCase Activity 91

4.14.4 Effect of different buffers on CMCase activity 92

4.14.5 Effect of ionic strength of buffer on CMCase activity 93

4.14.6 Storage stability of CMCase 94

4.15 SDS-PAGE and Zymography of the partially purified CMCase

from KIBGE-IB37MT

95

4.16 N-terminal Protein Sequence Analysis of CMCase form Mutant

KIBGE-IB37MT

96

4.17 Conclusion 96

SECTION 5

A) REFERENCES 98

B) PUBLICATIONS 115

LIST OF TABLES

TABLE DESCRIPTION PAGE#

Table 1 Cellulase families 9

Table 2 Contents of potato dextrose agar medium 32

Table 3 Vogel's medium composition 35

Table 4 CMC specific medium composition 35

Table 5 Colony characteristics of different Aspergillus strains on the

zepack’s agar medium

46

Table 6 Colony characteristics of different Aspergillus strains on the

Malt agar medium

46

Table 7 Colony characteristics of different Aspergillus strains on the

CMC agar medium

46

Table 8 Highlighting the CMC medium constituents 60

Table 9 Partial purification of CMCase from KIBGE-IB37 using

gradient (NH4)2SO4 precipitation.

61

Table 10 Partial purification of CMCase from KIBGE-IB37MT using

gradient(NH4)2SO4 precipitation

77

LIST OF FIGURES

FIGURES DESCRIPTION Page#

Figure 1 Representation of different cellulose sources and their structure 1

Figure 2 Describing the cellulose chemical structure 5

Figure 3 Explain the arrangement of cellulose chains forming sheets 5

Figure 4 Cellulose fibrils attack by endoglucanase and exoglucanase 6

Figure 5 Model representing a fungal cellulose hydrolysis mechanism 8

Figure 6 Fungal cellulose binding module 10

Figure 7 Cellulase configurations 11

a) Pocket configuration A. awamori

b) Cleft T. fusca

c) Tunnel T. reesei

Figure 8 Standard curve for the estimation of reducing sugars by DNS

method

43

Figure 9 Standard curve for the estimation of total proteins by Lowry’s

method using BSA

44

Figure 10 a) Aspergillusversicolor KIBGE-IB37 colony

b) Aspergillusversicolor KIBGE-IB37 sporangium

45

Figure 11 Nucleotide sequence of 18S rDNA from KIBGE-IB37 48

Figure 12 Phylogentic clustering showing the relation of KIBGEIB-37

with other species

48

Figure 13

Shows unit production from selected strains of Aspergillus at

different mode of fermentation

49

Figure 14 Effect of different time interval on CMCase production 51

Figure 15 Effect of CMC concentrations on CMCase production by

KIBGE-IB37

52

Figure 16 Effect of temperatures on CMCase production by KIBGE-IB37 53

Figure 17 Effect of pH on CMCase production by KIBGE-IB37 55

Figure 18 Effect of peptone on CMCase production by KIBGE-IB37 56

Figure 19 Effect of Tween 80 on CMCase production by KIBGE-IB37 57

Figure 20 Effect of NaNO3% on CMCase production by KIBGE-IB37 58

Figure 21 Effect of CaCl2 concentration on CMCase production by

KIBGE-IB37

59

Figure 22 Chromatogram showing purification of cellulase from KIBGE

IB-37

62

Figure 23 Michaelis Menten and Lineweaver-Burk plot of CMCase by

KIBGE-IB37

63

Figure 24 Effect of pH on enzyme activity by KIBGE-IB37 64

Figure 25 Effect of temperature on enzyme activity by KIBGE-IB37 65

Figure 26 Effect of different buffers on enzyme activity by KIBGE-IB37 66

Figure 27 Effect of ionic strength of citrate buffer by KIBGE-IB37 67

Figure 28 Storage stability at different temperatures by KIBGE-IB37 68

Figure 29 SDS PAGE Profile of CMCase from KIBGE-IB37 70

Figure 30 a) KIBGE-IB37MT colony

b) KIBGE-IB37MT Sporangium morphology

71

Figure 31 Effect of pH on the CMCase production by KIBGE-IB37MT 72

Figure 32 Effect of time on CMCase production by KIBGE IB37MT 73

Figure 33 Effect of CMC concentration on CMCase production from

KIBGE-IB37MT

74

Figure 34 Effect of peptone concentration on CMCase production

by KIBGE-IB37MT

75

Figure 35 Effect of Tween 80 on CMCase production by KIBGE-

IB37MT

76

Figure 36 Elution pattern of CMCase produced by KIBGE-IB37MT. 78

Figure 37

Michaelis Menten and Lineweaver-Burk plot of CMCase

by KIBGE-IB37MT.

79

Figure 38 Effect of pH on enzyme activity by KIBGE-IB37MT 80

Figure 39 Effect of temperature on enzyme activity by KIBGE-IB37MT 81

Figure 40 Effect of different Buffers on enzyme activity by KIBGE-

IB37MT

82

Figure 41 Effect of ionic strength of citrate buffer by KIBGE-IB37MT 83

Figure 42 Storage stability at different temperatures by KIBGE-IB37MT 84

Figure 43 SDS PAGE profile of CMCase from KIBGE-IB37MT 85

ABBREVATIONS

(NH4)2SO4 Ammonium Sulfate

µl Micro liter

A Aspergillus

APNEDP Atmospheric pressure non equilibrium discharge plasma

BC Bacterial celluloses

BCA 2, 2’-bicinchroninate

BSA Bovine serum albumin

CaCl2 Calcium chloride

CBH Cellobiohydrolase

CFF Cell free filtrate

CMC Carboxy methyl cellulose

CMCase Carboxy methyl cellulase

CrI Cellulose crystallinity index

DNS Dinitrosalicylic acid

DP Degree of polymerization

DS Degree of substitution

EDTA Ethylene diamine tetraacetic acid

FPA Filter paper assay

g Gram

h Hour

IUPAC International Union of Pure and Applied Chemistry

KIBGE-IB37

KIBGE-IB37MT

Native strain of Aspergillus versicolor

Mutant strain of Aspergillus versicolor

Km Michaelis constant

M Molar

M.W molecular weight

mg Milligram

MgSO4 Magnesium sulfate

min Minutes

ml Milliliter

mm Millimeter

mM Millimolar

NaCl Sodium chloride

nm Nanometer

O.D Optical density

oC Degree centigrade

PDA Potato Dextrose Agar

r.p.m. Revolution per minute

Rs Selection ratio

SDS-PAGE Sodium dodecyl sulfate-Polyacrylamide gel electrophoresis

SmF Submerged fermentation

sp. Specie

SSF Solid state fermentation

TNP-CMC Trinitrophenyl-carboxymethyl cellulose

Tris-HCl Tris-hydrochloric acid

UV Ultra violet radiations/rays

v/v Volume by volume

Vmax Maximum velocity

w/v Weight by volume

ABSTRACT

Microbes are ubiquitous tiny organisms/biomachines which have enormous potential to

produce many kind of industrially important products (enzymes, metabolites, phenolics,

antibiotics etc) when feeding upon specific medium. This study is about the exploration

of native aspergillus members for the production of cellulase enzyme and to get enhanced

production through mutation.

All the isolates assessed for the production ability of CMCase (carboxymethyl cellulase)

and taxonomically identified as A.niveus, A. fumigatus, A.versicolor, A. niger, A. wentti,

A. terreus, A. nidulance, A.flavus. Optimized medium for selected Aspergillus versicolor

(Gen Bank Accession KF905652) was 0.5% CMC, 4 pH, 0.075% peptone, 0.1% tween

80, 1.5% NaNO3, 0.05% CaCl2 at 120 hrs and 30°C. CMCase purified by 40%

ammonium sulfate followed by gel permeation chromatography with 11 folds of

purification and molecular weight was 59 kDaltons. Km and Vmax determined as 1.134

mg/ml and 1435 U/ml/minute. CMCase maximum reactivity attained by 50mM Citrate

Phosphate buffer, 0.5% CMC, pH 4 and 30°C for 30minutes.Storage stability at -18°C,

4°C and 25°C retained 69%, 44% and 12% activities at 25th day respectively.

KIBGE-IB37MT was a mutant of Aspergillus versicolor (KIBGE-IB37) the optimum

medium was 0.5% CMC, pH 4, 0.1% peptone, 0.1% Tween 80, 120 hrs and 30°C.

CMCase purified up to 12.26 folds with 59 kDaltons molecular weight. CMCase maxima

obtained by 25mM Citrate Phosphate buffer at 30°C for 30 minutes while Km and Vmax

were 1.13 mg/ml and 1435 U/ml/minute respectively. Shelf life at -18°C, 4°C and 25°C

was estimated as the retention of 70, 63 and 49 % activities respectively.

1.1 CELLULOSE & CELLULASE

Cellulose is most important resource from plants and biologically decomposable by

specific microbes. During photosynthesis cellulose production is a major primary product

of terrestrial environment and such a bulk quantity coined this compound as most

abundant renewable bioresource which is naturally produced in the biosphere (Figure 1)

(Zhang and Lynd, 2004). The fate of cellulose compound is changing from carbon flow

from fixed carbon sinks to atmospheric CO2 due to the biodegradation of cellulose

molecule by various microorganisms which are capable to produce cellulases and

cellulosomes.

Figure 1: Representation of different cellulose sources and their structure.

Enzymatic digestion/breakdown of such diverse lignocelluloses produces various

products under the favorable environmental conditions which are very cheap and cost

effective method. Cost effectiveness and decomposition of lignocelluloses in different

important sugars are very important factors involve in the performance of cellulose

biorefineries. The role of microorganism is very important for the treatment of waste

(Schloss et al, 2005). Engineering for the production of cellulase conventionally based

upon enhancement of cellulase related specific activities of microorganism.

Application of cellulase enzyme in different industries is now making much more

attention due to the diversity of their uses. High production cost of cellulase is the main

problem causing limitation of cellulase formation on industrial scale. Therefore at this

stage use of low cost carbon source and effectiveness of fermenters are being critical

point of consideration. Absolute hydrolysis of the cellulose using a combination of

complex enzyme system is also an important factor. Fungal cellulases contain multi-

enzymes which act in a naturally complex way and hydrolyze entire cellulose.

Recently cellulase enzymes were produced from Aspergillus genera and used in textile

industry as cotton softening agent, finishing in denim cloths and stone effects. In

detergent market they were used for the color care, cloth soother, fast cleaning and for

anti deposition step and in food industry for mashing, clearing agent in juicesand

liquefying agent in toffees and peeling of fruits and nuts.

1.2 ORGANISM RESPONSIBLE FOR CELLULASE PRODUCTION

The variety of organisms digests cellulose to gain energy for their metabolic processes

like some protozoan (symbiotically present in the gut of other organisms), bacteria

(aerobic and anaerobic origin) and fungi ranging through primitive to the advanced

genera (chytridomycetes, homobasidiomycetes and basidiomycetes). Extensively studied

organisms are Trichoderma, Aspergillus, Cladosporium, and Chetomium etc.

1.3 CELLULASE SYSTEM

Mainly two kind of system identified,

1) Bacterial complex system (cellulosome dependant transport mechanism for intracellular

hydrolysis of large oligosaccharides)

2) Fungal simpler system. (Extracellular hydrolysis)

Bacterial system of cellulase is complex because the enzymes are fixed upon cell wall of

the organism to partially digest the substrate and to facilitate the uptake of stable

intermediate complexes where remaining intracellular hydrolysis takes place. It was

reported that cellulosome clutch enzyme and responsible to locate substrate and to

improve diffusion of hydrolysis products into the cell body at the expense of fewer ATP

(Bayer et al, 1998).

Fungi have simple kind of cellulase system as the hydrolysis do not form intermediate

compounds of higher molecular weight and hydrolysis products simply diffused through

hyphae. This property of fungal cellulose system enhances the efficiency of hydrolysis

hence this property make aerobic cellulolytic fungus ideal for a variety of industrial

usage.

1.4 CLASSIFICATION OF CELLULASE BASED UPON

CELLULOLYSIS

A complete cellulase system basically comprises of three parts which are:

1) Endoglucanase (EC 3.2.1.4)

2) Exoglucanase (EC 3.2.1.91)

3) β-glucosidase (EC 3.2.1.21)

As above classification is assigned with respect to the specific function during hydrolysis

mechanism so it is important to briefly overlook to the cellulose hydrolysis mechanism.

1.5 CELLULOSE HYDROLYSIS MECHANISMS

Cellulose is the polymeric condensate of linear chained D-anhydroglucopyranose which

are connected through the β-1,4-glycosidic linkages along with the degree polymerization

where as the anhydro cellobiose is the repeating unit of cellulose chain (Figure 2). Stable

molecular fibers of high tensile strength with low accessibility finished through the

pairing of adjoining cellulose chains and ultimately into the straight sheets of cellulose

(Figure 3) with the help of hydrogen bonds and van der Waal’s forces (Demain et al,

2005). Cellulose molecule is highly stable in nature and stability could be assessed by its

5-8 million years half life (Wolfenden and Snider, 2001).

Mechanism of the cellulose enzymatic digestion is the result of synergistic actions of

endoglucanase (EC 3.2.1.4), exoglucanase or cellobiohydrolase (EC 3.2.1.91) and β-

glucosidase (EC 3.2.1.21). Endoglucanases hydrolyzes β-1, 4-glucosidic bonds of

cellulosic chains randomly resulting in formation of new chain which releases soluble

glucose or cellobiose; and β-glycosidase hydrolyze cellobiose into glucose (Figure 4).

Primary hydrolysis by endoglucanase and exoglucanase attacks on surface of the solid

substrate and liberates soluble sugars with a degree of polymerization (DP) up to 6 into

the liquid phase.

Figure 2: Describing the cellulose chemical structure.

Figure 3: Explain the arrangement of cellulose chains forming sheets.

(http://www.generalbiomass.com)

glucose or cellobiose and β-glycosidase hydrolyze cellobiose into glucose (figure 4).

Primary hydrolysis by endoglucanase and exoglucanase attacks on surface of the solid

substrate and liberates soluble sugars with a degree of polymerization (DP) up to 6 into

the liquid phase.

Figure 4: Cellulose fibril attack by endoglucanase and exoglucanase.

Cellulose digestion process bring about two types of changes; change the chain end

number of cellulose long chain and consumption by exoglucanases (Zhang and Lynd,

2005) and second is about to enhance cellulose accessibility resulting from substrate

consumption and cellulose fragmentation (Wang et al, 2003). Present literature depicts an

enzymatic cellulose hydrolysis in a distinct rather an articulating manner to congregate a

snapshot of the phenomenon which illustrate basic concepts (Figure 5).

1.6 SYNERGISM OF CELLULASE COMPONENTS

Components of cellulase always work in synergistic manner as individual components

are unable to hydrolyze cellulose effectively. Synergistic mechanism improves the total

efficiency of hydrolysis in comparison to its components efficiencies. The synergistic

action enables substrate attack at different sides by the components which form new sites

for the components to work on.

Synergy is of two types

endo-exo synergy

exo-exo synergy

In endo-exo synergy the endocellulase component cut substrate to form new chains with

reducing and non-reducing ends for the progressive attack of cellobiohydrolases. Such

synergy reported for the Trichoderma. reesei, in which four major cellulase follow the

pattern as Cel7B and Cel5A work in synergy with Cel6A, Cel7A (Nidetzky et al, 1994).

On the other hand, exo-exo synergy is attributed as inherent ability of cellobiohydrolases

which is difficult to explain because of the chain end specificities and directionalities

(Boisset et al. 2001). Cel6A from Humicola insolens is an example of exo-exo synergy.

Figure 5: Model representing a fungal cellulose hydrolysis mechanism (Lynd et al., 2002).

1.7 CELLULASE STRUCTURE

Cellulase structure varies on the basis of their origin and evolutionary relationship among

different cellulase enzyme explored their functional inferences. Three dimensional

structure of cellulase enzyme subjected to a constant evolutionary pressure by nature

which brings changes in its protein interactions but not in its primary sequence (Mornon,

2003). Some time sequences of cellulase are diverged enough that their similarity with

standard sequence harder to find but their structure and functions remains the same

(Gilkes et al, 1991). So three dimensional structure of enzyme usually preferred over

primary sequence information. Cellulases are of three types according to its structural and

mechanism pattern but have similarities in catalytic site, similar overall protein folding

patterns and reaction mechanism (Sandgren et al, 2005). The database CAZy put

cellulases into fourteen families on the basis of catalytic action and protein fold

(http://www.cazy.org), shown in table below.

Table 1: Cellulase families (http://www.cazy.org)

Family Mechanism Type EC No.

5 Retaining Endoglucanase 3.2.1.4

6 Inverting Endoglucanase 3.2.1.4

6 Inverting Cellobiohydrolase 3.2.1.91

7 Retaining Endoglucanase 3.2.1.4

7 Retaining Cellobiohydrolase 3.2.1.91

8 Inverting Endoglucanase 3.2.1.4

9 Inverting Endoglucanase 3.2.1.4

10 Retaining Endoglucanase -

12 Retaining Endoglucanase 3.2.1.4

26 Retaining Endoglucanase 3.2.1.4

44 Inverting Endoglucanase 3.2.1.4

45 Inverting Endoglucanase 3.2.1.4

48 Inverting Cellobiohydrolase 3.2.1.91

51 Retaining Endoglucanase 3.2.1.4

74 Inverting Endoglucanase 3.2.1.4

1.8 CELLULASE CONFIGURATIONS

Universal structural design of a cellulase comprises of a catalytic domain (contained

active sites to carry out hydrolysis reaction) along linker (rich in proline/serine/threonine)

and cellulose binding module (for adsorption of crystalline cellulose) (Gilkes et al, 1991).

Generally, structure of fungal cellulose binding module is small in size in comparison to

the other carbohydrate binding modules. Universal attribute of a fungal cellulase

comprises of amino acid sequence (up to 40 amino acids chain length) and disulphide

bridges (2-3 to strengthen the structure) (Figure 6).

Three kinds of architect of cellulases depict different globular protein configurations

upon the basis of active sites are; a) Pocket b) Cleft c) Tunnel.

Figure 6: Fungal cellulose binding module. Green Arrows stand for disulphide bridges,

Green rope presenting Carbon chain and contain cellulose tangled into binding

face in red (Kraulis et al, 1989).

1.8.1 Pocket configuration

Topology helps the enzyme to recognize non reducing ends and thus attributed to the β-

glucosidase of cellulase consortium (Figure 7a). Usually attack the available chain ends

which are fewer in cellulose fibrils so low in efficiency and follow endo-exo synergy.

Starch granules are best substrate due to the excess of available chain ends at radial

granule surface.

1.8.2 Cleft configuration

Structure of cleft is open type and has affinity to bind the sugar units at random sites. In

cellulase consortium the endocellulase has an example of clef configuration (Figure 7b).

1.8.3 Tunnel configuration

This configuration is similar to cleft one but a long loop covers up the groove which

results in a tunnel formation. Catalytic sites lie in between the tunnel so polysaccharide

chain has to be threaded through. Cellobiohydrolases of cellulase consortium are example

(Figure 7c). Enzyme substrate binding may be result of the opening up loop or due

entrapment of loosen substrate.

Figure 7: Cellulase configurations a) pocket configuration Aspergillus awamori b) cleft.

Trichoderma fusca c) tunnel T. reesei. (Davies and Henrissat, 1995)

a

b

c

1.9 CELLULASE ACTIVITY ASSAY

Cellulase activity enhances by adjusting the different important kinetic parameters

involve in the enzyme substrate reaction. Ex-situation applications of enzyme need high

specificity, long shelf life and reusability of enzyme, cost and equipments. In situation

and ex-situation conditions are different as first one is about the production of enzyme

using particular medium at specific pH, temperature etc and second one is for working of

that specific enzyme in an industrial process to avoid any kind of hindrance/interactions.

Cellulase activity measure by the two ways one is the total cellulase activity and other is

by measuring individual enzyme (endoglucanases, exoglucanases, and β-glucosidases).

The hydrolase enzyme activity is measured in terms of initial hydrolysis or the detection

of end-point in standard assay conditions. There is a difference in the initial and final

cellulose hydrolysis measurement in terms of activity assays (Sheehan and Himmel,

1999). Therefore it is necessary to estimate the individual cellulase enzymes activity in

consistent manner.

1.10 ENDOGLUCANASES

Endoglucanases are known to cleave the β-1, 4-glucosidic linkages at random positions

and relative activities are generally calculated upon cellulose derivatives. Endoglucanases

perform cleavage at the intramolecular places and lower down the specific viscosity of

CMC (Zhang and Lynd, 2004). Endoglucanase activity was calculated on the basis of

decrease in substrate viscosity or by measuring reducing sugar found after enzyme

substrate reaction. It was reported by several scientist that BCA method is very reliable as

compared to DNS method for reducing sugar assay of endoglucanase (Carcia et al, 1993).

In modified BCA method enzyme substrate reaction was performed at75oC to restrict the

cleavage of β-glucosidic bonds which results in the reducing ends of cellodextrins

independent of the carbon chain lengths, hence proved to be a high sensitivity of the

procedure (Zhang and Lynd, 2005). CMC agar plates are also a easy approach for the

detection of endoglucanase activity using different dyes to stain the cellulose surface

(Jang et al, 2003 and Ten et al, 2004).

1.11 EXOGLUCANASES

Exoglucanases is enzyme use for its potential to produce cellulosic ethanol to reduce the

world’s energy requirement. Exoglucanases cleave cellulose molecules at the ends of

carbon chain to release glucose and cellobiose. Cellobiohydrolase (CBH) I and II both

cleave reducing and non-reducing cellulose (Zhang and Lynd, 2004). Insoluble cellulosic

substrates exoglucanase activity estimated by Avicel method as it contains the highest

ratio of FNR/Fa. Exoglucanases are purified by the use of chromatographic fractionation

technique and as the result a layer formed which characterized as enzymes with low

activity on soluble CMC but also have relatively high avicel activity.

1.12 β-D-GLUCOSIDASES

β-D-glucosidases hydrolyse all cellodextrins and soluble cellobiose to glucose in a

reaction mixture and the hydrolysis rates tend to fall down with increase in DPs. (Zhang

and Lynd, 2004). A wide range of simple but sensitive assay methods are available which

release colored or fluorescent products such as; 6-bromo-2-naphthyl-β-D-

glucopyranoside, 4-methylumbelliferyl-β-D-glucopyranoside, pnitrophenyl β-D-1,4-

glucopyranoside, β-naphthyl-β-D-glucopyranoside (Setlow et al, 2004). β-D-glucosidase

activity could also be calculated using cellobiose as a substrate (McCharthy et al, 2004).

1.13 SCREENING STRATEGIES OF CELLULASES PRODUCING

ORGANISM

Screening is a one of the basic step for the use of newly isolated strains and only purified

isolated could assure the reliability. Large numbers of wild and mutant samples screened

through this technique to discover a single required isolate/mutant. There are two basic

approaches are;

1) Random screening: picking of one species at random position. 2) Facilitated screening:

based upon distinct phenotype, chromospheres and halos formed. Screening was carried

out upon solid agar plates and characteristic zone confirmed the presence of cellulase

producing organism. Zone of identity is due to the production of specific products

released during the growth of inoculated species on agar gel. In some cases during the

growth of organisms, enzymes are responsible for the production of chromophores which

gives the coloration to a zone. It was also reported that in some enzyme assays the first

enzyme coupled with the second enzyme which give specific product hence easily be

observed likewise in the case of cytochrome P450 turns up into horseradish peroxides

(Joo et al, 1999; Delagrave et al, 2001). Endoglucanases activities forms a zone on solid

CMC agar plate and upon Congo red dye staining with successive washing enable to

detect specific qualitative activity. The larger zone usually related to hyper hydrolysis

activity of selected isolates/mutants.

1.14 PRODUCTION OF CELLULASE

Protein purification techniques are involve to get pure enzyme with high specific

activities usually related with increase in production cost thus it is necessary to find out

different strategies to come across this impediment. Use of low cost but abundantly

available resources to overcome this obstacle is recommended in this regard. Agricultural

countries have a vast range of agricultural waste to be spent as a potential substrate for

cellulase enzyme production and recycling of waste for proper management.

Aspergillus niger usually used for the enhanced cellulase production in submerged

fermentation with slight alterations in different ingredients such as nitrogen sources by

the ammonium dihydrogen phosphate/thiophosphate, ammonium sulphate and corn step

liquor (Gokhale et al, 1991).

Combination of different types of substrates such as cellulose with lactose, cellulose with

xylose, bagasse with lactose, bagasse with xylose, rice straw with lactose and rice straw

with xylose are also resulted in enhance enzyme production (Muthuvelayudham and

Viruthagiri, 2006).

1.15 KINETICS OF CELLULASE

CMCase enzyme reactivity improves through the kinetic studies of related parameters

like enzyme substrate reaction time, pH maxima, Michealis-Menten constant, thermal

stability, and use of organic solvents as buffering agents and shelf life etc.

1.16 APPLICATIONS OF CELLULASES

1.16.1 Food Industry

1.16.1.1 Processing of pulp/Juices

Fruits/vegetables are rich in minerals, vitamins, the specific taste and aroma thus they are

used to make value added food grade commercial products. The important steps in juice

industry are extraction, clarification and stabilization. Whereas, the use of suitable

enzymes in juice filtration and other related aspects gained attention and the food grade

microorganisms are thus needed (Uhlig, 1998).

1.16.1.2 Total soluble solids (TSS)

Cellulase along with pectinase enzyme in, different combinations, applied to the pulp of

vegetables/fruits to enhance the total dissolve solids (TSS) which make available by the

digestion of cellulose and pectin components thus, improving digestibility of the juices

(Saha, 2004).

1.16.1.3 pH of juice

CMCase activity have not been associated with the pH changes of the juices but when

applied along with pectinase tend to decrease in pH because of the pectin degradation

into galacturonic acid in processing of guava puree (El-Zoghbi et al, 1992).

1.16.1.4 Clarity

Clarity of juices is measured by the percent transmittance and commercially very

important factor in juice quality. Lee et al, (2006) made a trial of CMCase enzymatic

treatment to banana pulp which resulted in better clarity of banana juice.

1.16.1.5 Flavor

Flavor enhancement is associated with the availability of more polysaccharide contents

which retain the sweetness in flavor of the pulp/juices. Similar findings were reported by

the Riu-Aumatell et al, (2004).

1.16.1.6 Overall Acceptability

The overall acceptability improved by the enzyme application as digestibility, quality,

clarity, taste and aroma increased in pulp/juices (Grassin and Fauquembergue, 1996).

1.16.2 Baking Industry

1.16.2.1 CMCase in bread making

Bread making the focused characteristics are crust color, volume, texture, taste and aroma.

Many enzymes with endo-xylanases, hemicellulases, amylases and proteases in different

cocktails, have proven beneficent, though, an appropriate mixture could yield maximum in

baking process.

1.16.2.2 Crust color

An attractive appeal urges a purchaser at first sight; therefore, the use of enzyme CMCase

has proven effective for the crust beauty (Harada et al, 2000).

1.16.2.3 Symmetry of form

Symmetry of form significantly depends upon the CMCase activity as the cellulose fibers

loosen up which will improve the symmetry of the bread (Harada et al, 2000).

1.16.2.4 Character of crust

Crisp and softness of the baked bread is a quality measure and by the use of certain

proportions of enzyme cocktails the degree of softness and crisp could easily be achieved.

1.16.2.5 Aroma

Aroma is a basic qualitative characteristic of the baked products, especially, in fresh and

preserved items. Only aroma could make a product favorite or rejected (Matz, 1972).

1.16.2.6 Taste

The CMCase treatment enhances taste and flavor of backed items due to the fermentative

effect of cellulose compound. Haros et al, (2002) also described similar effects of

CMCase in process of wheat tempering.

1.16.2.7 Texture

Improved texture in baking is reported by Harada et al, (2000); but a negative correlation

exhibited by the baked item possibly due to the release of sugars. In fact the sensory

attributes of the loaves of bread along with good texture is of commercially important.

Haros et al, (2002) applied CMCase to wheat flour which increased volume of bread.

1.16.3 Other uses

Human grade cellulases are used as digestive aid to cure malabsorption. They help in

removal of toxins and cholesterol by adsorption in other words prevents them to go into

blood stream. Cellulase treatment is also helpful in food allergies, pain relieving in colon

related diseases, gastrointestinal infections etc.

Enzymatic treatment of cellulases or their components (in various proportions) are also

playing promising role in processing of beans, rice polishing, wood finishing and coffee

processing. Cereals contain non starch polysaccharides which increases viscosity and

decrease product quality therefore, cellulase not only solve these issues but also improve

the product digestibility and quality.

1.16.4 Brewing Industry

Brewing was the most primitive biotechnology. Brewing generally of two types and

named as beer, wine. Malting of barley is beer while the juice fermentation through yeast

is wine. Enzyme treatment is advanced methodology to improve taste and quality and

reduction in processing time (Galante et al, 1998).

1.16.4.1 Beer brewing

Barly seeds germinate by the natural phenomenon which contains many enzymes for

malting. Some time poor quality barley, cultivar type, seasonal differences or the

mishandling may lead to malting enzyme insufficiency, forms gel which produce toxins

and hinder filtration. For maceration of polymers the cellulases play an important role.

Galante et al, (1998) indicated the use of Aspergillus niger, Trichoderma reesei,

Penicillium emersonii and bacteria Bacillus subtilis for the better crushing, more

clarification and improved quality with high yield from this process.

1.16.4.2 Wine brewing

In wine making initially carried out through the bacteria but afterwords the use of other

exogenous enzyme treatment found better. The enzyme mixtures are of pectinases,

cellulases and hemicellulases and they may be from bacterial source or fungal in origin.

Gunata et al, 1990 discovered the cellulase ability in aroma improvement.

1.16.5 Feed Industry

Cellulases in feed utilized for the removal of anti nutritional factors, enhance nutrient

contents and for the digestive aid (Galante et al, 1998). Feed generally formulated

according to the need of specific animal and for the specific purpose of farming as for

broiler (farmed for meet) feed is different than those of the layers (farmed for eggs).

1.16.5.1 Toxicity

Trials for the toxic effects upon experimental animals confirm the toxicity level of feed

additives (Coenen et al, 1995). The Trichoderma reesei (fungal cellulase) in 28 days

chicken trial had no adverse effects upon skin irritation, eye rashes, inhalation toxicity

and mutagenic effects (Coenen et al, 1995).

1.16.5.2 Future concerns

Nowadays, the focused point in enzymatic feed is to obtain such enzymes which could

stay in digestive tract for longer period of time under the safety guidelines (Ali et al,

1995). Another thought is to clone genes of such feed enzymes in to the gastrointestinal

tracts of animals so that there will be no need of enzyme dosage in feed for these

transgenic animals but the socioeconomic and ethnic considerations must be satisfied in

this regard (Hall et al, 1993).

1.16.6 Textile Industry

Textile industry is a big consumer of cellulase enzyme as many processes of this industry

utilize enzyme technology like, bio-polishing, stoning effect, fabric quality, modify

structure of fabric, de starching etc.

1.16.6.1 Bio-stoning

Denim cloths are stiff and have aged effect with repeated washing and nowadays

biostoning refer to a procedure in which cellulase enzyme applied at denim surface for

softness, appealing print patterns and smoother look (Uhlig, 1998). The cellulase

treatment not only improve the efficiency but also the labor and wear tear loss of

equipments as in traditional method two kg of stones per kg of denim was spin with

bleach for two hours of time interval (Galante et al,1998).

1.16.6.2 Fabric biopolishing

It is a wet process in textile manufacturing to remove fuzz of cloths enzymatically which

in turn appeared as a glossy look, fine texture, uniformly color to the cloths (Galante et

al, 1998).

1.16.6.3 De starching

Fabric sheets starched for piling and handling purpose and the excessive stiffness

washed out by the cellulase enzymes to give fabric glossy and smooth look (Uhlig, 1998).

1.16.7 Detergent Industry

Cellulases are very important in laundry detergent because of ease in cloth washing

process with good hand feel and no fabric damage due to excessive rubbing which

ultimately reduces the cost and process time (Godfrey, 1996)

1.16.7.1 Color brightness

Faded colors of cotton and cotton mix stuff are mainly due to repeated use and rubbing

and washing but addition of enzyme aid this problem no longer been harmful for cloths.

Therefore the time consumption and electricity cost reduced and color brightness prevails

longer (Godfrey, 1996).

1.16.7.2 Washing efficiency

The dirt particles trapped in microfibers of cellulose and cellulase not only helps in

removal of these entangle particles but also remove the dust/trapped particles. Stains of

different kind need just minutes to be washed out because of active enzymes.

1.16.7.3 New trends in laundry detergents

Many other enzymes like lipase, xylanase etc are incorporated in different combinations

keeping in view of the cleansing requirements. Alkaline enzymes of moderate

temperature are usually laundry favorite to minimize heating cost (Uhlig, 1998). To meet

the need of cold climatic conditions the enzyme capable of activity at low temperatures

are focused. Strong competition exist in Global laundry market is to formulate a variety

of detergents for specific conditions.

1.16.8 Edible oil Industry

Olive oil industry gained industrial attention worldwide because of health benefits and oil

quality. Previously, extraction of oil done through laborious work with lesser yield of oil

but with the introduction of enzyme based crushing solved these issues and shortens the

process duration. Galante et al, (1998) reported maximum yield by using specific

enzymes of different species like, cellulase and hemicellulase of Trichoderma, pectinases

of Aspergillus in a cocktail mixture. Advantages of ezyme based extraction are low

rancidity, richness in antioxidants, increased yield, and improved fractionation of mist.

1.16.9 Extraction of pigments

The use of cellulase enzyme increases pigment extraction by plant sources like peeling of

different fruits yields β-carotenes and food grade additives.

1.16.10 Vitamins and proteins

Clostridium thermocellum cellulosome an affinity column (CBD-based affinity tagging)

had been made for the purification of antibodies (Bayer et al, 1995).

1.16.11 Protoplast research

In protoplast research the cell wall posses a barier and for disintegration of cell wall a

large quantity of solvents are needed with laborious and time consuming protocols, so the

enzymatic digestion is preferred. Fungal cellulases/enzymes are mainly obtained from

Trichoderma sp., Geocladium sp., Chaetomium sp., Penicillium sp., Rhizopus nigricans,

Fusarium roseum (Harman and Kubicek, 1998). Trichoderma reesei and Aspergillus

niger used by Joutsjoki et al, (1993) for the heterologous proteins.

1.16.12 Disinfection

Cellulase consortium sprayed over the surfaces to digest microbial cell wall. These

disinfectants contain pectinases and chitinases to kill all type of pathogens with complex

cell wall composition (Benitez et al, 1998).

1.16.13 Pulp and paper industry

1.16.13.1 Bio Mechanical pulping

Biomechanical pulping is the reduction of wood shavings into the soft raw pulp and

carried out with the help of cellulase and xylanases up to establishment of hand-sheet

strength properties (Buchert et al, 1998).

1.16.13.2 Fiber modification

Plant fiber has unique specification and could be modified according to the industrial

demand. However the goal of fiber modification based upon the paper quality. Pere et al,

(1996), used Trichoderma enzymes for fiber modification and yield better drainage and

beatability in product.

1.16.13.3 Bleaching of pulp

In late eighties cellulases with xylanase was experimented for bleaching improving

efficiency. Enzymatic digestion of lignin and large cellulose fiber results in low dose of

chlorine for the bleaching stage (Mansfield et al, 1996).

1.16.13.4 De inking

De inking based on the principle that cellulase and hemicellulase erode the cell wall

surface so removal of ink became easy. Prasad et al, (1993) reported that the treatment

with pure alkaline cellulase improved brightness level of photocopied and laser printed

papers relative to pulping in water without enzymes. .

1.16.13.5 Drainage in paper mill

Turbidity of filtrates cases serious threats to paper mills whereas the enzyme treatment

hydrolyze the pitch, lignin and other molecules thus drainage efficiency finally improved

(Buchert et al, 1998).

1.16.13.6 Fiber studies

Enzymes are specific scissor which cuts undesirable structures from the compound so is

the case with fibers which exposed for further studies like stereoscopy and derivitization

etc (Buchert et al, 1998; Teleman et a1, 1995).

2.1 ISOLATION AND IDENTIFICATION OF FUNGI FROM

DIFFERENT SOURCES

Several fungal strains will be isolated from the environment

Taxonomical identification

Pure culture studies

Screening of Aspergillus spices for maximum cellulases production

Characterization by phenotypic features, qualitative and quantitative aspects

2.2 MUTATION INDUCTION

Potent strains capable of producing cellulase will be used for the strains improvement

will be mutant using UV radiation.

2.3 OPTIMIZATION OF GROWTH AND CULTURE

CONDITIONS FOR CELLULASE PRODUCTION

Fermentation conditions will be optimized for the newly isolated strain for maximum

cellulase production.

Medium selection

Optimization of time course

Optimization of substrate maxima

Optimization of temperature maxima

Optimization of pH maxima

2.4 ENZYME KINETICS

Optimization of enzyme kinetics will be performed using following parameters

Enzyme-substrate reaction time

Substrate maxima

Temperature maxima

pH maxima

Buffer selection

Ion strength of Buffer

Thermal stability of enzyme

Storage stability

2.5 PURIFICATION OF CELLULASE

Enzyme will be purified using different techniques such as salt precipitation, dialysis and

gel permeation chromatography for single band purification.

2.6 ELECTROPHORESIS AND ZYMOGRAPHY OF PURIFIED

CELLULASE

Electrophoresis and Zymography of purified cellulases will be performed for the

determination of homogeneity and molecular weight determination.

A: GENERAL SECTION

All the used chemicals were of analytical grade and purchased through the recognized

vendors. All glassware (Pyrex) were rinsed and socked in deionized water and also

sterilized by autoclaving (Astell, UK) before and after use.

3.1 CHEMICALS

CMC (Merck)

Peptone (Oxoid)

Yeast extracts (Merck)

Glucose (Merck)

KH2PO4 (Scharlau)

Tri sodium citrate (Merck)

NaCl (scharlau)

Tween 80 (Merck)

CaCl2 (Merck)

(NH4)2 SO4 (Scharlau)

(NH4)2 NO3 (Sigma)

Mg2SO4 (Merck)

Agar Agar (Oxide)

Acrylamide (Sigma)

3.2 INSTRUMENTS

1. Analytical balance (Sartorius, Germany)

2. Autoclave (Astell, UK)

3. pH meter (digital pH meter, PCSIR Laboratories Complex Karachi, Pakistan)

4. Spectrophotometer

Optizen 1412 Spectrophotometer, Korea

Microtech 3000, Germany

5. Incubator (Memmert, Binder) Germany

6. Magnetic Stirrer (Combimag-RCO) Germany

7. Electrophoresis unit (Thermo EC 120Mini vertical gel system) USA

8. High speed centrifuge (Sigma 3K 30)

3.3 BUFFERS USED

Following buffers were used during the study:

3.3.1 Phosphate Buffer (0.05M, pH 7.0)

Reagent A: Dihydrogen potassium phosphate (0.68 g) was dissolved in deionized

water and volume was made upto 100 ml in a volumetric flask.

Reagent B: Di-Potassium hydrogen phosphate (0.87 g) was dissolved in deionized

water and volume was made upto 100 ml in a volumetric flask.

In a beaker both the 0.05M solutions of reagent A and B were gradually mixed and the

pH was adjusted at 7.0.

3.3.2 Citrate Phosphate Buffer (0.05M, pH 7.0)

REAGENT A: Dissolve 0.96 gm citric acid (M.W. 192.1) in deionized water and

volume was made upto 100 ml in a volumetric flask.

REAGENT B: Disodium hydrogen phosphate (0.89 gm) was dissolved in

deionized water and the volume was made up to 100 ml.

In a beaker both the reagent A and B solutions were gradually mixed and the pH was

adjusted at 7.0.

3.3.3 Tris-HCl Buffer (0.05M, pH 7.0)

REAGENT A: Tris (Hydroxymethyl)-aminomethane (0.6 gm) was dissolved in

deionized water and the volume was made up to 100 ml.

REAGENT B: Concentrated hydrochloric acid 0.42 ml was added in 100 ml

volumetric flask and the volume was made up to 100 ml with deionized water.

In a beaker both the reagent A and B solutions were gradually mixed and pH was

maintained at 7.0.

3.4 REAGENT

Na-K tartarate (182 gm), Dinitrosalicylic acid (10 gm), Phenol (2.0 gm), sodium sulfite

(0.5 gm), sodium hydroxide (10 gm) dissolved in deionized water and volume was made

upto 1 litre with deionized water.

3.5 SOLUTIONS

1. 1% CMC: CMC (1.0 gm) was dissolved in 100 ml of distilled water.

2. 1% Congo red: Congo red (1.0 gm) was dissolved in 100 ml distilled water.

3. 1 M NaCl: Sodium chloride (5.95 gm) was dissolved in 100 ml of distilled water.

B. EXPERIMENTAL SECTION

3.6 SAMPLES COLLECTION

All the samples were collected from different soil of banana and sugarcane fields,

skimmed milk, partially decompose litter, rotting sugar cane bagasse and from putrefied

fruits and vegetables. Samples were collected in sterilized plastic bags and kept at 4°C.

All the samples were labeled with care to avoid errors in future processing of samples in

order to isolate cellulolytic fungi for further experimentation.

3.7 ISOLATION OF CELLULOLYTIC FUNGI

Isolation was performed on Potato Dextrose Agar plates (Table 2). Medium was

sterilized using an autoclave at 121oC at 15 lb2 for 15 minutes. Potato Dextrose Agar

(PDA) was then poured into sterilized Petri dishes under aseptic conditions.

Table 2: Contents of Potato Dextrose Agar medium (pH adjusted to 5.0)

Ingredients Concentration (g/dl)

Potato 125.0

Dextrose 30.0

Agar 4.2

3.8 PROCESSING OF SAMPLES

One gram of each sample was taken and added in 10.0 ml sterilized distilled water and

further diluted to make four dilutions from each stock tube. Inoculation was carried out

by spreading 0.2 ml of liquid on sterilized Potato Dextrose Agar (PDA) plates with the

help of a sterilized glass spreader and incubated at 30oC until complete growth of fungal

strains. Sub-culturing of different fungal colonies was performed to obtained pure culture

of an isolated fungus. These pure cultures were renewed after every 20 days.

3.9 IDENTIFICATION OF FUNGI

Important macroscopic features such as colony color, growth pattern, color imparted into

the Czapek’s solution gel and back color of the plate and malt extract agar plates were

used for specimens. Microscopic characteristics observed under a compound microscope

includes; shape of the conidia head, pattern of the arrangement of spores, spore shapes,

and shape of condidiophores to facilitate identification of an isolate.A universal key of

identification (Domsch et al, 1980; Kiffer and Morelet, 2000) was used to identify a

specimen upon the basis of routine cultural and morphological characteristics.

3.10 SCREENING OF POTENTIAL CELLULOLYTIC FUNGUS

For the screening of different strains of fungi capable of producing cellulolytic enzymes

two methods were used;

Qualitative Method

Quantitative Method

3.10.1 Qualitative Method

Specific culture medium was prepared by adding 1.0 % CMC in agar agar (2.0%) and

volume was made upto 100 ml with distilled water. This medium was autoclaved and

poured in Petri plates. Screening of twelve isolated specimens for cellulase production

was performed in accordance to the methodology of Bhat and Bhat, (1997). A spore

suspension was made by pouring 2.0 ml sterilized distilled water into a slant containing a

pure fungus culture, mixed by inverting many times. A hole was made in the centre of

agar plate gel and filled with spore suspension of each fungus specimen and plates were

incubated at 30oC for three (03) days. To establish a halo zone 5.0 ml of Congo red dye

(0.5%) was flooded in Petri plates for 30 min followed by washing with 1.0 N NaCl

solution to remove excess dye. The presence of a zone of clearance (a halo) around the

colony is actually an indication of cellulase production (Ten et al, 2004).

3.10.2 Quantitative Method

Six different types of isolates were selected for quantitative analysis. Seven days old

culture was used for inoculation and 2.0mm agar blocks of Potato Dextrose Agar gel

(containing growth) were taken as inoculums for the 100 ml Vogel’s medium in 250 ml

flask, at 30o C (Table 3).

For determination of cellulase activity, fermented broth was centrifuged at 4°C with

10000 r.p.m. for 30 minutes and supernatant was used to determine cellulase activity

through CMCase assay.

Both CMCase and β-glucosidase activities were checked; units of β-glucosidase enzymes

were very poor. And in some cases, units were not detected, therefore only CMCase

assay method was selected for further studies.

3.11 SELECTION OF CULTURING MEDIUM

Different media were tested in order to compare cellulase activities so that a better

medium could be sorted out. This media include;

Vogel’s medium

CMC medium (0.5% CMC in 100 ml water)

3.12 SELECTION OF FERMENTATION MODE

Submerged fermentation is the method of choice due to its high significance and ease in

handling.

Table 4: CMC specific medium composition (at pH 5.0)

Table 3: Vogel's medium composition (at pH 5.0)

Ingredients Concentration (g/dl)

CMC 1.00

Peptone 0.08

NaNO3 1.00

Tween 80 0.20

KH2PO4 0.20

CaCl2 0.05

Ingredients Concentration

(g/dl)

Tri-sodium citrate 0.25

Potassium di hydrogen phosphate 0.50

Ammonium nitrate 0.20

Ammonium sulphate 0.40

Magnesium sulphate 0.02

Peptone 0.10

Yeast extract 0.20

Glucose 2.00

3.13 FERMENTATION CONDITIONS FOR CELLULASE

PRODUCTION

Different parameters were optimized for maximum cellulase production with shaking

mode of fermentation;

Effect of temperature on cellulase production

Effect of initial pH of medium on cellulase production

Effect of incubation time on cellulase production

Effect of different CMC concentration on cellulase production

Effect of Peptone on cellulase production

Effect of Tween 80 on cellulase production

Effect of CaCl2 on cellulase production

Effect of NaNO3 on cellulase production

Effect of KH2PO4 on cellulase production

3.13.1 Effect of temperature on cellulase production

Different temperatures ranging from 20°C to 60 °C were used for the production of

cellulase (CMCase). Spores suspension (1.0 ml) was inoculated in 250 ml sterile medium

in 500ml Erlenmeyer conical flasks for five days and then enzyme activity was

performed.

3.13.2 Effect of medium pH on cellulase production

Maximum production of carboxymethyl cellulase (CMCase) at different pH was achieved

using same media composition having different pH ranging from 4.0 to 7.0. Media pH

was adjusted using HCl / NaOH before sterilization. After sterilization equal amount of

spore suspension was inoculated for five days at 30°C.

3.13.3 Effect of incubation time on cellulase production

Innocula (50 ml) of 48 hours was transferred in different flasks of 450 ml media and

incubated at 30°C for different time intervals ranging from 24-168 hours (7 days) and

after every 24 hours CMCase activity was performed. (Millati et al, 2002).

3.13.4 Effect of different CMC concentration on cellulase production

Maximum carboxymethyl cellulase (CMCase) production with different concentration of

CMC ranging from 0.25 to 1.5 % was optimized in separate 250 ml Erlenmeyer flasks

keeping the other constituent of medium constant.

3.13.5 Effect of peptone on cellulase production

Different concentrations of peptone ranging from 0.025 to 0.15 % (0.025%, 0.05%,

0.075%, 0.1%, 0.125%, and 0.15%) were added in separate flasks for maximum

production of CMCase before autoclaving. These media were incubated for five days at

30 °C for fungal growth.

3.13.6 Effect of Tween 80 on cellulase production

Different concentrations of Tween 80 (0.25 ml, 0.5 ml, 1.0 ml, 1.5 ml and 2.0 ml) were

added in each flask having 250 ml medium after sterilization. Flasks were incubated for

fungal growth for five days at 30 °C.

3.13.7 Effect of calcium chloride (CaCl2) on cellulase production

Different concentrations of CaCl₂ (0.025, 0.050, 0.075 and 0.1%) were added in

fermentation media separately before sterilization and these media were incubated for

five days at 30 °C.

3.13.8 Effect of sodium nitrate (NaNO3) on cellulase production

Different concentrations of sodium nitrate (0.25, 0.5, 1.0, 1.5 and 2.0%) were added in

fermentation media before sterilization and these media were incubated for five days at

30 °C.

3.13.9 Effect of potassium dihydrogen phosphate on cellulase production

Different concentrations of potassium dihydrogen phosphate (0.1, 0.2, 0.3, and 0.4 %)

were added in fermentation media separately before sterilization and these media were

incubated for five days at 30 °C.

3.14 OPTIMIZATION OF KINETIC PARAMETERS OF CELLULASE

Different kinetic parameters of CMCase was optimized which are discussed below

3.14.1 pH maxima

The pH ranged in between 3-6 with different buffers to determine the maximum CMCase

activity while keeping substrate (0.5%) and temperature (30°C) constant.

3.14.2 Temperature maxima

CMCase activity was performed at different temperatures ranging from 30 -60°C to

determine the temperature maxima.

3.14.3 Selection of Buffer for enzyme activity

Different buffers (Citrate Phosphate, Phosphate and Tris-HCl) were used to estimate the

best suited buffer for the CMCase activity while the rest of reaction parameters were kept

at constant.

3.14.4 Ion strength of Buffer

Selected buffer was used with varying strengths such as 25, 50 and 100 mM to estimate

the appropriate strength for the CMCase enzyme activity.

3.14.5 Storage stability

CMCase enzyme was kept at 4, 18 and 25 °C to estimate its storage stability which was

tested after five days interval up to one month.

3.15 MUTATION INDUCTION

Ultra Violet (UV) radiation was selected as a physical mutagenic agent to induce random

mutations in strain. UV lamp of 2.6 × 106 J/m2/s power was used for the purpose. Dried

spore powder form seven days old culture was exposed to UV light for the duration of 5,

10, 15, 20, 25, 30 minutes respectively and kept in dark for one day.

3.15.1 Selection of strain after mutation

Mutants were screened for CMCase producing ability by qualitative test as reported

earlier (Bhat and Bhat, 1997; Aharoni, 2005). Zone of clearance was the indication and

criteria in selection of enhanced enzyme production by mutants and bigger diameter of

the zone is related to the production of CMCase from mutant strains.

3.15. 2 Stability of mutant strain

Repeated sub culturing was done upon PDA slants to check the stability of mutant strain

and capability to produce enzyme at standard conditions after five days incubation.

Retention of almost same activity at each step was the stability assessment criteria (Kim

et al, 2000).

3.16 PURIFICATION OF CELLULASE ENZYME

3.16.1 Crude enzyme extraction

Flasks obtained after incubation was centrifuged at 10,000 r.p.m. for 15 min at 4°C.

Under aseptic conditions the supernatant was collected in 10 ml vials while pellet was

discarded.

3.16.2 Partial Purification of CMCase

CMCase were subjected to partial purification using ammonium sulphate by gradient

precipitation technique. Different concentrations of ammonium sulphate were prepared

and protein from cell free filtrate was precipitated at 4°C with constant stirring and kept

for 60 minutes for equilibration. All precipitated proteins were collected after

centrifugation (15000 r.p.m.) and dissolved in citrate phosphate buffer (pH 4.0) and

dialyzed with the same buffer.

3.17 GEL PERMEATION CHROMATOGRAPHY

Dialyzed pure sample was subjected to the size exclusion chromatography using Econo

pump EP-1 Bio-Rrad, USA system.The fractions were analyzed for the enzyme activity,

pooled and concentrated through 10 KDa filter membrane.

3.18 GENOME CHARACTERIZATIONS

DNA extraction procedure was followed using the method of Moller et al, (1992).

Universal fungal primers were used during PCR amplification process and purified PCR

product was sequenced for similarity index through the service of NCBI

(http:www.ncbi.nlm.nih.gov/BLAST). The N-terminal Protein Sequence, gene sequence

were determined (Gen Bank: Acc # KF905652.1) and Phylogenetic tree was constructed.

3.19 ELECTROPHORESIS (SDS PAGE)

SDS PAGE (Sodium dodecyl sulphate polyacrylamide gel electrophoresis) was

performed to determine the molecular mass of the purified CMCase with 10.0 % w/v gel

(Laemmli, 1970). Protein bands visualized after staining with Coomassie blue (R-250)

and excess of dye was removed using destining solution. Standard molecular weight

marker was also run parallel to sample (Bano et al, 2009).

3.20 ZYMOGRAPHY OF CMCase

3.20.1 Reagents

Buffer; Phosphate buffer (50mM.pH 7.5) with 0.5% (v/v) Triton-X-100.

Commassie brilliant blue R-250; (0.1g) dissolved in 40ml methanol, 10ml acetic acid in

100ml deionized water.

3.20.2 Methodology

Purified enzyme extract was used in accordance with the methodology as performed by

Bano et al., 2009. After the electrophoresis the gel immersed in buffer (0.5% ‹v/v›

Triton-X-100) for 15 minutes to remove SDS and afterwards incubated in substrate

solution for 20 minutes at 30 0C. Then gel was stained with the Commassie brilliant blue

R-250 solution by dipping it for 15 minutes. Band appeared showing the CMCase

activities in a pattern.

3.21 N-TERMINAL PROTEIN SEQUENCING

CMCase was blotted upon PVDF membrane and then provided to the Alta Biosciences

Limited (Birmingham, United Kingdom) for N-terminal analysis.

C: ANALYTICAL SECTION

3.22 CMCase ASSAY

3.22.1 Principle

Principle is based upon the capability of the cellulase enzyme to hydrolyze

carboxymethyl cellulose (CMC) in the form of reducing sugars. Estimation of sample is

use to determine an increase in the reducing sugar (glucose) by the help of 3, 5-

dinitrosalicylic acid.

Cellulase (CMCase) unit may be defined as “One unit (IU) of CMCase activity was

defined as the amount of enzyme required to release 1.0μ mole of glucose per minute at

standard conditions.” A linear glucose standard curve was used to interpret the values of

absorbance from a sample which was converted into the glucose.

3.22.2 Procedure

Partially purified enzyme (1.0 ml) and 1.0 ml substrate (1.0 % CMC prepared in 0.05 M

citrate buffer) were added in test tube. A control was also prepared by adding inactive

enzyme with substrate. Blank was also prepared by replacing reaction mixture with the

distilled water. All the test tubes were incubated at 30oC for 30 minutes. Reaction was

stopped by adding 1.0 ml DNS in test, control and blank and placed in boiling water bath

for 5.0 minutes. After boiling all tubes cooled at room temperature and 9.0 ml distilled

water was added. Absorbance was noted at 540 nm.

3.23 GLUCOSE STANDARD CURVE

The various concentrations of anhydrous glucose were prepared for standard curve and

DNS was added for color reaction. Boiling and cooling time remain same as in assay

procedure and absorbance was taken at 540 nm to prepare standard curve.

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

0 0.1 0.2 0.3 0.4 0.5

Ab

sorb

an

ce

Glucose Conc (mg/ml)

Figure 8: Glucose standard curve for the estimation of reducing sugars by DNS method

3.24 ESTIMATION OF TOTAL PROTEIN

Estimation of protein was done using method of Lowery et al, (1951). Alkaline reagent of

copper 5.0 ml was added in 0.5ml of fermentation broth, incubated for 15min at room

temperature. Afterwards the Follin’s reagent (0.5) ml was added in it and kept for 15

minutes in dark to develop a Bluish color and absorbance was noted at 650 nm. Many

known dilution of Bovine serum was prepared to be serving as reference.

Total Protein = mean absorbance x standard factor BSA

Figure 9: Standard curve for the estimation of total proteins by Lowry’s method

a b

4.1 SAMPLE COLLECTION AND HANDLING

All the samples were collected from the active sites and brought to the lab in sterile

plastic bags properly tagged. For research work the samples were kept at standard

conditions and fungi were isolated by microbiological techniques. Isolated pure species

after identification sub cultured on regular basis. The Aspergillus versicolor named

endorsed as KIBGE-IB37 (Figure 10) was selected to proceed for research work.

Figure 10: Aspergillus versicolor KIBGE-IB37 colony (a). Aspergillus versicolor (b).

KIBGE-IB37 sporangium

4.2 IDENTIFICATION

Morphological characterization studied in detailed and investigated for its identification

by the universal key of aspergillus genera (Carmichael et al, 1980; Domsch et al, 1980;

Kiffer and Morelet, 2000). Some phenotypic traits noted upon CMC, malt and czepak’s

agar plates which are summarized in tables below:

Table 5: Colony characteristics of different Aspergillus strains on Zepack’s agar medium

a b

a b

Medium pH was kept 5.5 before sterilization and incubated at 30°C

Table 6: Colony characteristics of different Aspergillus strains on Malt agar medium

Aspergillus

(A) Growth Spore Appearance Spore color

Gel color

(front)

Gel color

(back)

A.wentti slow/++ 4th day Radish brown Brown tinge Radish brown

A.versicolor slow/++ 5th day Dark green Orange brown Faded brown

A.flavus medium/++ 4rth day lime green Yellowish Cream brown

A.nidulance medium/++ 3rd day dark green Opaque yellow Brownish

Medium pH was kept 5.5 before sterilization and incubated at 30°C.

Table 7: Colony characteristics of different Aspergillus strains on the CMC agar medium

Aspergillus

(A) Growth

Spore

Appearance Spore color

Gel color

(front)

Gel color

(back)

A. wentti medium/++ 6th day Brown Light brown Light brown

A.versicolor medium/++ 5th day Dull green Brownish Red brown

A.flavus slow/+ 7th day Yellow green Cream color Pale yellow

A.nidulance fast/+++ 6th day Orange green No color Yellow tinge

Medium pH was kept 5.5 before sterilization and incubated at 30°C.

Aspergillus (A) Growth Spore Appearance Spore color Gel color (front) Gel color (back)

A.wentti Fast/+++ 3rd ay Yellowish brown Opaque white No color

A.versicolor Slow/+ 6th day Grayish green Radish brown Brownish

A.flavus Medium/++ 4th day Yellow green No color Pale yellow

A.nidulance Fast/+++ 4th day Plane green Yellow Olive brown

4.3 PHYLOGENETIC TREE

The nucleotide sequence of KIBGE-IB37 (accession number KF905652) by the GenBank

database (Figure 11) was aligned with other sequences for the construction of

phylogenetic clustering of different fungal species (Figure 12).

1 tacctggttg attctgccat tagtcatatg cttgtctcaa agattaagcc atgcatgtct

61 aagtataagc aatctatact gtgaaactgc gaatggctca ttaaatcagt tatcgtttat

121 ttgatagtac cttactacat ggatacctgt ggtaattcta gagctaatac atgctaaaaa

181 ccccgacttc gggaggggtg tatttattag ataaaaaacc aatgcccctc ggggctcctt

241 ggtgaatcat aataacttaa cgaatcgcat ggccttgcgc cggcgatggt tcattcaaat

301 ttctgcccta tcaactttcg atggtaggat agtggcctac catggtggca acgggtaacg

361 gggaattagg gttcgattcc ggagagggag cctgagaaac ggctaccaca tccaaggaag

421 gcagcaggcg cgcaaattac ccaatcccga cacggggagg tagtgacaat aaatactgat

481 acggggctct tttgggtctc gtaattggaa tgagtacaat ctaaatccct taacgaggaa

541 caattggagg gcaagtctgg tgccagcagc cgcggtaatt ccagctccaa tagcgtatat

601 taaagttgtt gcagttaaaa agctcgtagt tgaaccttgg gtctggctgg ccggtccgcc

661 tcaccgcgag tactggtccg gctggacctt tccttctggg gaatcccatg gccttcactg

721 gctgtgggtg gaaccaggac ttttactgtg aaaaaattag agtgttcaaa gcaggccttt

781 gctcgaatac attagcatgg aataatagaa taggacgtgc ggttctattt tgttggtttc

841 taggaacgcc gtaatgatta atagggatag tcgggggcgt cagtattcag ctgtcagagg

901 tgaaattctt ggatttgctg aagactaact actgcgaaag cattcgccaa ggatgttttc

961 attaatcagg gaacgaaagt taggggatcg aagacgatca gataccgtcg tagtcttaac

1021 cataaactat gccgactagg gatcgggcgg cgtttctatg atgacccgct cggcacctta

1081 cgagaaatca aagtttttgg gttctggggg gagtatggtc gcaaggctga aacttaaaga

1141 aattgacgga agggcaccac aaggcgtgga gcctgcggct taatttgact caacacgggg

1201 aaactcacca ggtccagaca aaataaggat tgacagattg agagctcttt cttgatcttt

1261 tggatggtgg tgcatggccg ttcttagttg gcggagtgat ttgtctgctt aattgcgata

1321 acgaacgaga cctcggccct taaatagccc ggtccgcgtc cgcgggccgc tggcttctta

1381 gggggactat cggctcaagc cgatggaagt gcgcggcaat aacaggtctg tgatgccctt

1441 agatgttctg ggccgcacgc gcgctacact gacagggcca gcgagtacat caccttggcc

1501 gagaggcccg ggtaatcttg ttaaaccctg tcgtgctggg gatagagcat tgcaattatt

1561 gctcttcaac gaggaatgcc tagtaggcac gagtcatcag ctcgtgccga ttacgtccct

1621 gccctttgta cacaccgccc gtcgctacta ccgattgaat ggctcggtga ggccttcgga

1681 ctggcgcagg agggttggca acgacccccc cgcgccggaa agttggtcaa aaccggtcat

1741 ttagaggaag taaaagtcgt aaca

Figure 11: Nucleotide sequence of 18S rDNA from Aspergillus versicolor KIBGE-

IB37 (GenBank database)

Figure 12: Phylogenetic clustering showing the relation of Aspergillus versicolor

KIBGE-IB37 with other species.

4.4 FERMENTATION MODE FOR CMCASE PRODUCTION

Type of fermentation affects the production quality and also on the quantity of enzyme

and different strains use to devour upon different environmental conditions (Baker, et al,

2005). All the selected isolates were grown on same media to assess the most suitable

fermentation type in relation to the CMCase production (Figure 13).

0

100

200

300

400

500

A. versicolor A. wentti A. flavus A. nidulance

En

zy

me a

cti

vit

y

(U/m

l/m

in)

Fungal strains

Static Shaking

Figure 13: CMCase production from selected strains of Aspergillus at different mode of

fermentation.. (Means± S.E., n = 6).

4.5 EFFECT OF FERMENTATION CONDITIONS FOR CMCASE

PRODUCTION

Selected mode of fermentation further studied and explored for the production of

maximum CMCase and different physical and chemical parameters were optimized.

4.5.1 Time course for maximum CMCase production

Aspergillus versicolor KIBGE-IB37 was incubated on CMC containing medium for

maximum CMCase production at different time periods. It was found that the maximum

enzyme production was achieved after 120 hours and as the time exceeded from 120

hours, a declined in enzyme production was noted (Figure 14). It was also observed that

the increased in cell growth also increased the enzyme production. This increase in cell

growth started after 24 hours and reached to maximum upto 120 hours which is directly

related to enzyme production. Decreased in enzyme production after 120 hrs is might be

due to the repressions of different metabolites produced during fermentation. It was

reported earlier that the maximum CMCase production was found in 120 hours from

Aspergillus japonicus URM5620 (Herculano et al, 2011) and similarly cellulase

production was also reported in 72 hours from fungus isolated from a rain forest (Vega et

al, 2012). Different bacterial strains are also capable to produce CMCase and it was

reported that the Bacillus pumilus EB3 produced CMCase in 24 hrs (Ariffin et al, 2006).

Figure 14: Effect of different time interval on CMCase production. Symbols

(means ± S.E., n = 6) having similar letters are not significantly different

from each other (Bonferroni test, P < 0.05).

4.5.2 Effect of substrate concentration on CMCase production

CMC specific medium is known to induce the production of CMCase enzyme as reported

by many scientists (Acharya et al, 2008; Ahmed et al, 2009; Ramanathan et al, 2010).

Maximum CMCase production from Aspergillus versicolor KIBGE-IB37 was achieved

when carboxymethyl cellulose (0.5%) was used as substrate in fermentation medium. It

was also found that when concentration of CMC increased from 0.5% in fermentation

medium, CMCase production decreased drastically (Figure 15). This decreased in

CMCase production was in fact due to the substrate feedback inhibition which activated

due to the high concentration of substrate. It was reported that higher substrate

concentration resulted in poor fungal growth and low CMCase production (Liu and yang,

2007). It was also reported that the carbon sources induce production of CMCase but

amount of enzyme produced is variable. This is because of the influence of substrate

(carbon source) on the growth of organisms (Mandels and Reese, 1999; Lakshmikant and

Mathur, 1990).

Figure 15: Effect of CMC concentrations on CMCase production by Aspergillus

versicolor KIBGE-IB37. Symbols (means ± S.E., n = 6) having similar

letters are not significantly different from each other (Bonferroni test, P <

0.05).

4.5.3 Effect of temperature on CMCase production

Aspergillus versicolor KIBGE-IB37 was cultured at various temperatures by keeping

other variables constant. It was found that at 20ºC comparatively low amount of CMCase

(403 U/ml/min) was produced while at 30ºC the maximum CMCase production (874

U/ml/min) was achieved. After further increased in temperature up to 60ºC continuous

decreased in CMCase production was observed (Figure 16). It was reported that

maximum cellulase production from Aspergillus niger was achieved at 20ºC and as the

temperature increased up to 40ºC decreased in 48% of cellulase production was observed

(Sakthi et al, 2011). This decrease in enzyme production might be due to the low

availability of oxygen in the fermentation medium and at high temperatures the solubility

of oxygen gas decreases resulting in limited available quantity of dissolved oxygen in the

medium hence anaerobic condition prevail and thus making aerobic strain impossible to

grow which ultimately lead to low production of enzyme (Stewart and Parry, 1981; Rao

et al, 1983).

Figure 16: Effect of temperatures on CMCase production by Aspergillus versicolor

KIBGE-IB37. Symbols (means ± S.E., n = 6) having similar letters are not

significantly different from each other (Bonferroni test, P < 0.05).

4.5.4 Effect of pH on CMCase production

Effect of pH on enzyme production was performed from pH 3.0 to 7.0 and maximum

activity was obtained at pH 4 (acidic media) with activity of 820 U/ml/min. It was found

that 35 % increased in production was observed when pH was raised from 3.0 to 4.0,

while at pH 5.0 suddenly decreased and almost 53 % enzyme production was found

which continue to declined as the pH increased from 5.0 to 7.0 (Figure 17). It was

reported that maximum CMCase production was found when medium pH was kept at 4.0

( Prasetyo et al, 2011) and it was also reported that in case of cellulase production by the

Bacillus pumilus EWBCM1 isolated from earthworm mid-gut, maximum production was

recorded at pH 6.0 and minimum production was recorded at pH 3.0 (Shankar and

Isaiarasu, 2011). It was also reported that cellulases from Bacillus thuringiensis showed

maximum production under acidic conditions with optimum pH at 4.0 and presented

relatively wider pH adaptability, showing more than 20% of maximum production from

pH 3.0 to 7.0 (Lin et al, 2012).

Figure 17: Effect of pH on CMCase production by A. versicolor KIBGE-IB37.

Symbols (means± S.E., n = 6) having similar letters are not significantly

different from each other (Bonferroni test, P < 0.05)

4.5.5 Effect of peptone concentration on CMCase production

Nitrogen source is one of the important sources to increase the production of different

commercial enzymes (Das et al, 2008). Different CMCase production from Aspergillus

versicolor was found at different concentration of peptone in fermentation medium.

Various concentration of peptone was incorporated in fermentation medium and

maximum production of CMCase was found when 0.075 gm% peptone was incorporated

in the fermentation medium and as the peptone concentration increased more than 60%

decreased in CMCase production was noted and on further increase in peptone

concentration in medium the continuous decreased in CMCase production was observed

(Figure 18).

Figure 18: Effect of peptone on CMCase production by A. versicolor KIBGE-IB37.

Symbols (means ± S.E., n = 6) having similar letters are not significantly

different from each other (Bonferroni test, P < 0.05).

4.5.6 Effect of Tween 80 concentration on CMCase production

Tween 80 is a surfactant and responsible for equilibration of components of a medium

and indirectly cure coagulation of certain products/metabolites (Zhang and lynd 2004).

Many researchers reported that the surfactant cleans the surface of microorganism from

adsorbed compounds and the accumulation of product formed during fermentation

process which causes problem in the formation of desired product. Therefore removal of

component is very essential with the proper agitation/shaking. For this purpose Tween 80

is incorporated in various concentrations upto 0.5% and it was observed that maximum

CMCase production was achieved when 0.1% Tween 80 was added in medium (Figure

19). It was observed that addition of high concentrations of Tween 80 in fermentation

medium, decreased in production of CMCase. It was also reported by Percival et al,

(2006) and Atsushi and Eiichi, (1998) that surfactant used above optimum values

produces foaming in fermentation vessel which causes negative impact on product

formation (Woods et al, 2001).

Figure 19: Effect of Tween 80 on CMCase production by Aspergillus versicolor

KIBGE-IB37. Symbols (means ± S.E., n = 6) having similar letters are not

significantly different from each other (Bonferroni test, P < 0.05).

4.5.7 Effect of NaNO3 concentration on CMCase production

Biosynthesis of CMCase was affected by the type of nitrogen sources and its quantity in

the fermentation medium (Wang et al, 2003). Sodium nitrate was added in the

fermentation medium in different concentration for the production of CMCase from

Aspergillus versicolor KIBGE-IB37 and it was found that maximum CMCase production

was achieved when 1.5 % sodium nitrate was added. It was also noted that CMCase

production was also found without the addition of sodium nitrate in the medium but

addition of sodium nitrate in the medium increased CMCase production and it was

reported that NO3 yon induced the enzyme production ( Rajoka, 2004). The production of

CMCase increased from 50 units (medium having no NO3) to 900 units (Figure 20). It

was also noted that after reaching maximum at 1.5 % NaNO3, the CMCase production

declined on further addition. It was reported that high concentration of NaNO3 causes

increased in pH of the medium resulting a decreased in CMCase production (Lin and

Cornish, 2002). Muthuvelayudham and Viruthangiri (2006) reported hydrophobicity of

cell wall decreased due to increased concentration of NaNO3 in the medium which

ultimately decreases the CMCase production.

Figure 20: Effect of NaNO3 on CMCase production by Aspergillus versicolor

KIBGE-IB37.Symbols (means ± S.E., n = 6) having similar letters are not

significantly different from each other (Bonferroni test, P < 0.05).

4.5.8 Effect of CaCl2 concentration on CMCase production

Fermentation rate boost up by the addition of calcium chloride (CaCl2) during

fermentation. Different concentrations of calcium chloride were added in fermentation

media and it was found that maximum CMCase production was achieved when 0.05%

calcium chloride was added in fermentation medium. It was also noted that in the absence

of calcium chloride very low CMCase production was found from Aspergillus versicolor

KIBGE-IB37. It was also found that a sharp declined in the production was noted after

reaching maxima (Figure 21). Bajpai (1999) also obtained analogous results with the use

of 0.02% CaCl2 in the fermentation medium for maximum enzyme production. Calcium

chloride is a typical ionic halide and in the liquid state splits to provide calcium and

chloride ions which have minimal effect on the pH of the medium (Asghar et al,. 2002).

a

b

c

d

a0

200

400

600

800

1000

1200

Control 0.025 0.05 0.075 0.1

En

zym

e a

cti

vit

y

(U/m

l/m

in)

Calcium chloride (gm%)

Figure 21: Effect of CaCl2 concentration on CMCase production by Aspergillus

versicolor KIBGE-IB37.Symbols (means ± S.E., n = 6) having similar

letters are not significantly different from each other (Bonferroni test, P <

0.05)

4.5.9 Optimized medium for native Aspergillus versicolor KIBGE-IB37

Optimized media has the basic role in a process designing and engineering (Asenjo et al,

1991). Ingredients minimal factors limit product formation during fermentation

(Ashokkumar et al, 2001). Certain basic factors when altered in dosage, effect product

formation as in pattern closed to stress environmental conditions (Chang and Holtzapple,

2000). All the parameters were optimized for the maximum CMCase production and final

composition of fermentation medium was achieved (Table 8)

Table 8. Highlighting the CMC medium constituents

Contents Reference

medium

Optimized

medium

CMC 1.000 0.500

Peptone 0.075 0.075

NaNO3 1.000 1.500

KH2PO₄ 0.200 0.200

CaCl2 0.050 0.050

pH 5.500 4.000

4.6 PARTIAL PURIFICATION OF CMCASE USING AMMONIUM

SULPHATE

The crude enzyme was partially purified through gradient precipitation by using various

concentration of ammonium sulphate (20%, 40% and 60%). It was observed that the

precipitation of CMCase in term of specific activity was increased by increasing the

ammonium sulphate concentration and maximum enzyme (9.55 folds) was purified in

40% ammonium sulphate saturation (Table 9), while no activity was detected with the

further increased in ammonium sulphate upto 60%, therefore it was not shown in Table 9.

Bano et al, (2009) also reported similar findings about precipitation of CMCase.

Table 9: Partial purification of CMCase from KIBGE-IB37 using gradient ammonium

sulphate precipitation

(NH4)2SO4 Enzyme activity

(U/ml/min)

Total proteins

(mg)

Specific activity

(U/mg)

Fold

Purification

CFF 608 4.26 142.7 1

20% 742 1.14 650.88 4.56

40% 886 0.65 1363.07 9.55

60% - - - -

4.7 GEL PERMEATION CHROMATOGRAPHY FOR CMCase

PURIFICATION

Cellulases from Aspergillus versicolor KIBGE-IB37 were purified through the CL-6B

(GE, Healthcare. 1.5 cm × 50.0 cm) sepharose column and chromatographic runs were

repeated to gain maximum protein yield. Fraction (2.0 ml) was loaded on the pre

equilibrated buffer column keeping flow rate 1.0 ml/min with1.0 ml fraction size.

CMCase activity was found in fraction 50 to 79 (Figure 21) which was pooled and stored

at -20ºC for further studies. Specific activity of purified CMCase from the Aspergillus

versicolor KIBGE-IB37 was 1637.42 U/mg which represent 11 fold of purification.

Figure 22: Chromatogram showing purification of cellulase from Aspergillus versicolor

KIBGE IB-37.

4.8 CATALYTIC PROPERTIES OF CMCASE FROM KIBGE-IB37

4.8.1 Kinetic Parameters (Km and Vmax)

The kinetic parameter of CMCase from native strain was calculated by performing the

experiment using different concentration of substrate ranging from 0.1% to 2.0%. The

respective Km and Vmax of CMCase from the KIBGE-IB37 were found to be 0.932 mg

ml-1and 186.0 U min-1(Fig 23). The Km value basically represent the affinity of

CMCase for cellulose degradation and as compared to previously reported

CMCasefrom Trichoderma reesei and Humicola insolens, the CMCase from

A. versicolor has higher affinity for cellulose degradation (Boisset et al., 2001; Busto

et al 1996).

Figure 23: Michaelis Menten and Lineweaver-Burk plot of CMCase by KIBGE-IB37.

4.8.2 Effect of pH on CMCase activity

The pH greatly affect the enzyme activity by bringing protonization and

deprotonization changes on the amino acids present on active site resulting in

deformity in enzyme (protein) structure or mal functioning (Carle et al, 2004;

Chandra et al, 2007). CMCase activity from A. versicolor KIBGE-IB37 was carried

out at different pH and it was found that CMCase activity was increased by increasing

the pH and maximum activity was observed at pH 4.0 (Figure 24). It was found that

further increased in pH beyond 4.0, decreased in enzyme activity and more than 67 %

and 89 % activity lost was found at pH 5.0 and 6.0 respectively. It can be concluded

from the results that the CMCase from the KIBGE-IB37 has optimum pH in acidic

range and it almost lost its complete activity near neutral pH. Similar findings about

the optimum pH of CMCase activity have been previously reported (Carbett, 1963).

Figure 24: Effect of pH on enzyme activity by KIBGE-IB37. Symbols (means ±

S.E.,n = 6) having similar letters are not significantly different from

each other (Bonferroni test, P < 0.05).

4.8.3 Effect of temperature on CMCase activity

Temperature is one of the important factors that bring changes in medium viscosity

and enzyme surface reactivity through conformational changes. It was reported that at

high temperature the enzyme became denatured or decreased its catalytic activity

(Chang and Holtzapple, 2000). The effect of temperature on CMCase activity was

analyzed by measuring the enzyme assay at various reaction temperatures ranging

from 20 °C to 60 °C. It was observed that the catalytic activity of CMCase reached its

maximum at 30 °C and gradually decreased after further increasing the temperature

from 30 °C to 50 °C and 76 % activity was lost at 60 °C (Figure 25). Coral et al,

(2002) reported that the CMCase from A. niger Z10 wild-type strain has optimum

temperature at 35°C for maximum activity. CMCase from Sporotrichum thermophilie

showed its maximum activity at 50°C (Coutts and Smith, 1975).

a

b

c

d

e

0

200

400

600

800

1000

20 30 40 50 60

CM

Ca

se a

cti

vit

y

(U/m

l/m

in)

Temperaure ( C)

Figure 25: Effect of temperature on enzyme activity by KIBGE-IB37. Symbols

(means ± S.E., n = 6) having similar letters are not significantly different

from each other (Bonferroni test, P < 0.05).

4.8.4 Effect of different buffers on CMCase activity

The effect of different buffers were tested on the CMCase activity from native strain

of A. versicolor KIBGE-IB37 and it was found that the CMCase performed maximum

cellulose degradation in the reaction medium of citrate phosphate buffer as compared

to potassium phosphate and Tris-HCl buffer (Figure 26). The buffers have weak acid

and conjugate base in its constituents to maintain the pH of reaction medium in

optimum range and provide good ionic conditions for enzymes to perform their

activity (Gokhale et al, 1991; Godfrey 1996). It was reported that citrate phosphate

buffer is also a suitable buffer for CMCase activity from native strain of Aspergillus

awamori (Enari et al, 1975).

Figure 26: Effect of different buffers on enzyme activity by KIBGE-IB37. Symbols

(means ± S.E., n = 6) having similar letters are not significantly different

from each other (Bonferroni test, P < 0.05).

4.8.5 Effect of ionic strength of buffer on CMCase activity

Ionic strength of the buffer also influences the enzyme activity (Iwashita, 2002;

Carmichael et al, 1980). The effect of ionic strength of citrate phosphate buffer on

CMCase activity was determined by performing the assay in the reaction medium of

citrate phosphate buffer having different ionic strength with constant pH. It was

observed that 50 mM of citrate phosphate buffer provide enough ionic strength of

reaction medium for binding of charged substrate to charged amino acids present at

active site of enzyme to perform their maximum hydrolytic activity (Figure 27). Some

native fungal isolates were also reported to have enhanced CMCase activity with

50 mM concentration of citrate phosphate buffer (Jahangeer et al, 2005).

Figure 27: Effect of ionic strength of citrate phosphate buffer on CMCase activity

(Means± S.E., n = 6). Symbols (means ± S.E., n = 6) having similar letters

are not significantly different from each other (Bonferroni test, P < 0.05).

4.8.6 Storage stability of CMCase at different temperature

Purified CMCase from Aspergillus versicolor was kept at different temperatures

(-18°C, 4°C and 25°C) for 30 days and after interval of every 5 days enzyme activity

of stored enzyme was performed.. It was observed that the activity of CMCase was

gradually decreased at these temperatures with reference to time and only 31% lost of

activity was found at -18C, 56 % lost at 4C after 30 days. Major activity lost of 88%

was found at 25C after 30 days (Figure 28). At low temperature the retention of

enzyme activity usually is far better as compared to those at elevated temperatures

Kitamoto (2000).

Figure 28: Storage stability at different temperatures by KIBGE-IB37. (Means±

S.E., n = 6).

4.9 SDS-PAGE AND ZYMOGRAPHY OF THE PARTIALLY PURIFIED

CMCASE FROM KIBGE-IB37

For the determination of molecular weight, the crude and partially purified CMCase

were subjected to SDS-PAGE and in-situ electrophoresis according to Bano et al.,

(2009). The SDS-PAGE gel showed several bands of proteins in crude enzyme

solution and whereas purified enzyme was obtained after gel permeation

chromatography showed only single band (Figure 29). The in-situ electrophoresis

revealed that the cellulolytic activity corresponded with the protein band of commassie

stain having the molecular weight of 59 kDa. The molecular weight of extracellular

CMCase from Aspergillus versicolor KIBGE-IB37was very closed to the molecular

weight of extracellular CMCase produced by the Bacillus sp isolated from a paddy

field having 58 kDa (Vijayaraghavan and Vincent, 2012). Ariffin et al, (2006) reported

30-65 kDa CMCase from Bacillus pumilus, whereas Giorgini, (1992) observed two

bands (60 and 70 kDa) of CMCase. A wild type strain Aspergillus niger Z10 was

found to produce CMCase having 83 kDa of molecular weight (Coral et al, 2002).

Figure 29: SDS PAGE Profile of CMCase from KIBGE-IB37. A = Molecular weight

marker, B = crude enzyme, C= purified CMCase, D = Zymography of

purified CMCase.

4.10 MUTATION INDUCTION

Various techniques have been applied to induce mutation into microorganisms for the

enhancing their production skill upto industrial level. UV (Ultra violet) radiation is one

of the oldest and reliable methods to induce random mutation in microorganism for the

generation of mutant strain capable to produce high production yield. A. versicolor

colonies were treated with Ultra violet UV radiations to cause mutation and nine

different mutants were picked. These mutants were screened qualitatively as well as

quantitatively for CMCase production and mutant KIBGE-IB37MT was selected on

the basis of high production of CMCase (Figure 30). The survival rate of a mutant is

usually time and dose dependant (Coutts and Smith, 1975) and 15 minutes of UV

exposure was observed to generate maximum number of mutants with high survival

rate as compared to others.

Figure 30. A. Versicolor KIBGE-IB37MT (a) sporangium, (b).colonial morphology a

a b

4.11 OPTIMIZATION OF FERMENTATION CONDITIONS FOR

MAXIMUM CMCase PRODUCTION FROM MUTANT KIBGE-

IB37MT

The physico-chemical parameters for maximum production of CMCase by mutant

strain of Aspergillus versicolor KIBGE-IB37MT were optimized similarly like native

strain using one variable at a time approach.

4.11.1 Effect of pH on CMCase production

The effect of pH on the production of CMCase from KIBGE-IB37MT was

investigated. It was observed that no change in the optimum pH was observed and

maximum production was achieved at pH 4.0 (Figure 31). Due to the mutation

CMCase production increased in comparison with wild strain of A. versicolor KIBGE-

IB37. Cherry and fidantsef, (2003) discovered more or less same conditions required

for the enhanced enzyme production by the mutated progeny in comparison to their

ancestors.

Figure 31: Effect of pH on the CMCase production by KIBGE-IB37MT. Symbols

(means ± S.E., n = 6) having similar letters are not significantly different

from each other (Bonferroni test, P < 0.05).

4.11.2 Effect of incubation period on CMCase production

Production of enzyme is a growth dependant factor and maximum production of

enzyme is usually occurred in growth phase. The production of CMCase from KIBGE-

IB37MT was analyzed with reference to incubation time. It was observed that KIBGE-

IB37MT started CMCase production after 24 hours and reached at maximum after 120

hours (Figure 32). But after 120 hours the CMCase production was gradually

decreased and more than 70% production was decreased after 168 hours. Thus after

mutation the optimal fermentation time for maximum CMCase production was

increased and as compared to native strain. It was observed that A. versicolor KIBGE-

IB37MT required one more day for maximum production of CMCase.

Figure 32: Effect of time on CMCase production by KIBGE IB37MT.Symbols (means

± S.E., n = 6) having similar letters are not significantly different from each

other (Bonferroni test, P < 0.05).

4.11.3 Effect of substrate concentration on CMCase production

The effect of substrate concentration on the production of CMCase from KIBGE-

IB37MT was determined. The CMCase production was increased with the gradual

increase in substrate concentration (CMC) and maximum production was achieved

when 0.5 % CMC (substrate) was incorporated in the production medium (Figure

33). Further increased in CMC concentration in fermentation medium decreases the

enzyme production and 60% production was reduced when 1.5% CMC was

incorporated in the medium. The optimum substrate remained 0.5% for native and

mutant but CMCase production was increased in KIBGE-IB37MT.

a

b

a

c

0

300

600

900

1200

1500

1800

0.25 0.50 1.00 1.50

CM

Ca

se a

cti

vit

y

(U/m

l/m

in)

Ccrboxy methyl cellulose (%)

Figure 33: Effect of CMC concentration on CMCase production from KIBGE IB37MT .

Symbols (means ± S.E., n = 6) having similar letters are not significantly

different from each other (Bonferroni test, P < 0.05).

4.11.4 Effect of peptone concentration on CMCase production

Peptone is used as nitrogen source in fermentation medium and known to enhance the

product formation (Lin and Chen, 2006). The effect of peptone concentration on

CMCase production from KIBGE-IB37MT was analyzed by adding various

concentrations of peptone in production medium. It was observed that the cellulolytic

activity of fungal strain was increased by increasing the peptone concentration and

maximum CMCase production was achieved at 0.1 % (Figure 34). It was observed that

the mutation increased the demand of peptone and thus the mutant strain needs more

peptone for maximum CMCase production as compared to wild strain.

a

b

c

d

e

f

0

300

600

900

1200

1500

1800

0.025 0.050 0.075 0.100 0.125 0.150

CM

Ca

se a

cti

vit

y

(U/m

l/m

in)

Peptone (%)

Figure 34: Effect of peptone concentration on CMCase production by KIBGE-

IB37MT. Symbols (means ± S.E., n = 6) having similar letters are not

significantly different from each other (Bonferroni test, P < 0.05)

4.11.5 Effect of Tween 80 Concentration on CMCase production

The effect of Tween 80 on the production of CMCase from mutant strain KIBGE-

IB37MT was measured by incubating the fugal strain in various production medium

containing different concentration of Tween 80 ranging from 0.1% to 0.5% (Figure

35). It was found that maximum production of CMCase was achieved in medium

containing 0.1 % Tween 80 while in case of wild strain maximum CMCase production

was achieved at 0.2% Tween 80. It was found that as the concentration of Tween 80

increased the production declined and 78% decreased in production was observed

when 0.5% Tween 80 was incorporated in the medium.

Figure 35: Effect of Tween 80 on CMCase production by KIBGE-IB37MT. Symbols

(means ± S.E., n = 6) having similar letters are not significantly different

from each other (Bonferroni test, P < 0.05).

4.12 PURIFICATION OF CMCase FROM KIBGE-IB37MT

4.12.1 Partial purification of CMCase from KIBGE-IB37MT

CMCase enzyme from KIBGE-IB37MT was precipitated from the cell free filtrate

using ammonium sulphate ammonium sulphate (Bano et al, 2009). The partial

purification was performed using 40% ammonium sulphate which had 1768 U/ml/min

enzyme activity, proteins 2.5 mg/ml and 707.21 U/mg specific activity with 3.0 folds

purification (Table 10). The 60% ammonium sulphate concentration resulted in loss of

enzyme activity. Similar findings were found in case of A. versicolor KIBGE-IB37

strain where 40% ammonium sulphate showed maximum enzyme precipitation upto

884 units (U/ml/min) with specific activity of 1363.07 (U/mg) and yield 9.55 folds of

purification. Kim et al, (1994) studied CMCase from Trichoderma viride and

suggested deactivation or denaturation of CMCase active sites due to the extraction of

enzyme with concentrated buffers.

Table 10: Partial purification of CMCase from KIBGE-IB37MT using gradient

(NH4)2SO4 precipitation

Ammonium

sulphate (%)

Enzyme

activity

(U/ml/min)

Total Protein

(mg/ml)

Specific

activity (U/mg)

Fold

purification

CFF 1503 6.4 235,21 1.0

20 1663 3.8 437.63 1.9

40 1768 2.5 707.21 3.0

60 - - - -

4.12.2 Gel permeation chromatography of CMCase from KIBGE-IB37MT

Partial Purified enzyme from the KIBGE-IB37MT was subjected to gel permeation

chromatography to get purified CMCase. Similar conditions were followed as in case

of CMCase from native species but in this case enzyme activities were detected in the

fraction 58 to 79. These fractions were pooled and stored at -20 ºC. It was found that

CMCase from KIBGE-IB37MT had specific activity 2883.26 U/mg and folds of

purification were 12.26 (Figure 36).

Figure 36: Elution pattern of CMCase produced by KIBGE-IB37MT

1

4.13 CATALYTIC PROPERTIES OF CMCASE FROM KIBGE-

IB37MT

4.13.1 Kinetic parameters (Km and Vmax)

The Km and Vmax values of CMCase from KIBGE-IB37MT were also calculated by

Michaelis Menten and Lineweaver-Burk plot (Figure 37). It was observed that the value

of Km was 1.134 mg ml-1 and Vmax was 1435 U min-1.

Figure 37: Michaelis Menten and Lineweaver-Burk plot of CMCase by KIBGE-IB37MT

2

4.13.2 Effect of pH on CMCase activity

The effect of pH on the activity of CMCase from mutant was determined and it was

found that the optimum pH for maximum activity remained same i.e. at 4.0 with

reference to native strain. However mutation resulted in 100% increased in enzyme

activity as compared to its native form (Figure 38). Mäntylä et al, (1998) reported some

recombinant strains of Trichoderma reesei (as Industrial mutants) showed optimized pH

alike to their ancestral Trichoderma reesei but their efficiency was far better in

commercial applications. Gouka et al, (1996) also observed similar pH value for

recombinant Aspergillus awamori strains.

Figure 38: Effect of pH on enzyme activity by KIBGE-IB37MT. Symbols (means ±

S.E., n = 6) having similar letters are not significantly different from each

other (Bonferroni test, P < 0.05).

4.13.3 Effect of temperature on CMCase activity

3

CMCase from mutant strain of A. versicolor KIBGE-IB37MT showed similar

temperature for maximum activity i.e. 30 °C as that of its native form (Figure 39). Bakar

et al, (2005) used extracellular purified cellulase from two improved mutants of

Pseudomonas fluorescens to optimize the temperature and found 37 °C as optimum for

both of them which was ancestral (wild-type) optimal temperature.

Figure 39: Effect of temperature on enzyme activity by KIBGE-IB37MT. Symbols

(means ± S.E., n = 6) having similar letters are not significantly different from

each other (Bonferroni test, P < 0.05).

4.13.4 Effect of different buffers on CMCase activity

4

The effect of buffer on CMCase activity from KIBGE-IB37MT was similarly determined

like CMCase activity from wild strain by performing the assay in different buffers

including citrate phosphate, potassium phosphate and Tris-HCl of same pH. Among all

these three buffers, citrate phosphate buffer was found to be the best buffer CMCase

activity (Figure 40). The ions in the buffers influence the structure and physicochemical

properties of enzyme, and therefore, the suitable buffer increases the activity efficacy and

shelf life of enzymes (Ramos et al, 2005). McCarthy et al, (2004) also reported similar

findings about the role of specific buffer on 1, 4-β-D-glucan glucohydrolase activity from

both wild and hybrid strains but production capacities of the microbial strain was

increased after mutation.

0

300

600

900

1200

1500

1800

Citrate Phoshate

Buffer

Tris-HCl Buffer Potassium Phosphate

Buffer

CM

Case a

cti

vit

y

(U/m

l/m

in)

Buffers

Figure 40: Effect of different buffers on enzyme activity by KIBGE-IB37MT.

(Means± S.E., n = 6).

4.13.5 Effect of ionic strength of buffer on CMCase activity

5

The effect of ionic strength of buffer on the CMCase activity from KIBGE-IB37MT was

analyzed by measuring the reaction in various ionic strengths of buffer and it was found

that 25 mM strength is best for maximum enzyme activity of CMCase from mutant strain

(Figure 41). It was already mentioned in Figure 27 that for maximum CMCase activity

from native strain KIBGE-IB37 a buffer of 50mM is required. It was found that in case of

CMCase from mutant strain the increased in buffer ionic strength decreased the enzyme

activity.

Figure 41: Effect of ionic strength of citrate buffer by KIBGE-IB37MT. (Means± S.E., n

= 6)

4.13.6 Storage stability of CMCase

6

The storage stability of partially purified CMCase from KIBGE-IB37MT was determined

by keeping the enzyme solutions at various temperatures (-18, 4, 30 °C) for 30 days and

after every 5 days residual activity of CMCase was performed. The CMCase showed 70,

63 and 49 % residual activity at -18, 4 and 30 °C, respectively after 25 days of (Figure

42). It was also noted that CMCase from Aspergillus versicolor KIBGE-IB37MT is very

stable enzyme and can be stored for more than one month even at 30°C. Pernilla et al,

(2007) described that the enzymes can stored for longer period of time at low

temperatures.

Figure 42: Storage stability at different temperatures by KIBGE-IB37MT. (Means±

S.E., n = 6).

7

4.14 SDS-PAGE AND ZYMOGRAPHY OF THE PURIFIED CMCASE FROM

KIBGE-IB37MT

The molecular weight of CMCase from the KIBGE-IB37MT was determined by

performing SDS-PAGE and in-situ electrophoresis as described by Bano et al, (2009).

Molecular weight of mutant CMCase was found to be same as that of wild i.e. 59 kDa

(Figure 43). Ogawa (1990) reported cellulases with molecular weight with a wide range

of 38-58 kDa from Trichoderma viride. Qin et al, (2008) observed molecular weight of

54 kDa for the recombinant endoglucanase of Trichoderma reesei. Otten and Quax

(2005) found CMCase by mutant and native to have same catalytic activity and the

weight as well.

Figure 43: SDS PAGE profile of CMCase from KIBGE-IB37MT. A = marker, B =

crude enzyme, C= purified CMCase, D = Zymography of purified CMCase

4.15 N-TERMINAL PROTEIN SEQUENCE ANALYSIS OF CMCASE FORM

MUTANT KIBGE-IB37MT

8

After final purification step, purified CMCase was sent to Alta Biosciences, University of

Birmingham, UK for protein sequencing. Amino terminal of the protein was found to be

unblocked and initial ten amino acids were sequenced. The sequence “NH2- Val- Ala -

Ala - Ile - Gln - Thr - Val – Leu – Gly” obtained was cross matched with other

available CMCase sequences and found to have no similarity with them. Native form of

enzyme could not be N-terminally sequenced as the enzyme production from strain was

low and purified sample was insufficient.

4.16 CONCLUSIONS

A novel CMCase hyper producing strain was isolated from the samples.

9

Selection of stain among (A.versicolor, A. wentii, A. flavus and A. nidulance) was

performed after quantitative assay.

Taxonomy criteria were phenotypic and strains were stored at KIBGE lab where

Aspergillus versicolor renamed as KIBGE-IB37, deposited in Gene Bank

(Accession no # KF905652).

Shaking mode of fermentation proved better over static fermentation.

The optimized temperature was 30°C, CMC 0.5 % , pH 4, total time course of 120

hrs, Tween 80 0.1%, NaNO₃1.5%, peptone 0.075% and CaCl₂ 0.05% for

maximum CMCase production.

CMCase yield was purified upto 11 folds with molecular weight of 59 kDa.

Michaelis constants, Km 0.9322 mg/ml and Vmax for purified CMCase were

determined as 186.0 U/ml/min.

The CMCase exhibited maximum activity when it was incubated with 0.5%

substrate for the period of 30 min, pH 4 and citrate phosphate buffer (50mM).

The CMCase from wild strain Aspergillus versicolr when kept at -18 °C it

retained 69% activity as compared to the 44 % at 4°C and 12% at 25°C.

The native strain was mutated (tagged KIBGE-IB37MT) through ultra violet

radiations (UV) to get enhanced production of CMCase enzyme.

Peptone intake optimized at 0.1% in contrast to the ancestor stain which was at

0.075% while surfactant Tween 80 was 0.1% , 120 hrs and pH 4, 0.5% CMC as of

ancestor .

CMCase from KIBGE-IB37MT purified up to12.26 folds and molecular weight

remained 59kDa.

10

Michaelis constants, Km and Vmax for partially purified CMCase were

determined as 1.134 mg/ml and 1435 U/ml/min.

The CMCase from KIBGE-IB37MT exhibited maximum activity when it was

incubated with 0.5% substrate for the period of 30 min at pH 4.0.

Maximum CMCase activity from KIBGE-IB37MT optimized with citrate

phosphate buffer (25mM).

Storage stability at different temperatures of -18, 4, 25 and 30 °C displayed the

retention of respective activities 70, 63, 60 and 49 % at 25th day of storage from

CMCase by KIBGE-IB37MT.

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Publication

Carbohydrate Polymers

Volume 104, 15 April 2014, Pages 199–203

Enhanced production of cellulose degrading CMCase by newly

isolated strain of Aspergillus versicolor

Sofia Qaisar, Rashida Rahmat Zohra, Afsheen Aman, and Shah Ali Ul Qader

The Karachi Institute of Biotechnology and Genetic Engineering (KIBGE),

University of Karachi, Karachi 75270, Pakistan

Received 19 November 2013, Revised 21 December 2013, Accepted 3 January

2014, Available online 10 January 2014