PRODUCTION AND CHARACTERIZATION OF

CELLULASE FROM Bacillus circulans ISOLATED FROM

DECAYED WOOD

ARUWAJOYE GABRIEL SANJO

B.Tech. (Hons.) (Ogbomoso).
(SCP10/11/H/1028)

A THESIS SUBMITTED TO THE DEPARTMENT OF

BIOCHEMISTRY, FACULTY OF SCIENCE, OBAFEMI
AWOLOWO UNIVERSITY, ILE-IFE, IN PARTIAL
FUFILMENT OF THE AWARD OF A MASTER OF SCIENCE
DEGREE IN BIOCHEMISTRY

2014

APPROVAL
This research project was supervised by me and approved in accordance with the
partial fufilment for the award of Master of Science (M.Sc.) Degree in Biochemistry,
Obafemi Awolowo University, Ile-Ife, Nigeria.

_______________________

___________________

Date

Dr. F.K. Agboola

Supervisor

ii

DEDICATION

This work is dedicated to

Almighty God

The source of knowledge and strength, without whom I am nothing.

iii

ACKNOWLEDGEMENTS
Firstly, I thank my God for the grace, strength, wisdom and enthusiasm given to me to
complete this project.
My profound gratitude goes my supervisor, Dr. F. K. Agboola, for his advice, interest
and encouragement throughout the period this project. More than a supervisor, you have been
to me a paradigm of resilience and perseverance, without which I would not have been able
to complete this project.
My appreciation also goes to the former Head of Department, Prof. O. O. Oyedapo. I
am also grateful to Dr. (Mrs). A. Kuku, Dr. O. Osoniyi, Dr. E. M. Obuotor, Dr. O. O.
Babalola, Dr. R. E. Okonji, Dr. (Mrs). B. A. Akinpelu, Dr. O. O. Odekanyin, Dr (Mrs). K. F.
Akinwumi, Miss O. O. Ogunruku and the non-academic staff of the Department. In
particular, I wish to appreciate the efforts of Mr. Olumide Omoboye of the Department of
Microbiology towards the success of this work. I pray that God will reward your efforts,
contributions and encouragement.
I am grateful to all the postgraduate students of the department especially Mr. Kunle
Bamigbade, Mr. Dare Agunbiade, Mrs. Yetunde Ayinuola, Mrs. Blessing Gold, Mr. Tope
Atere and others who gave up their time at one point or the other to make this study a
success. I will never forget Daramola Ifeoluwa and Odeyinde Ebunoluwa for their moral
support, concern and contributions. Also, I must say that I have enjoyed the companionship
of my laboratory colleagues, Tolu, Tosin, Joba and Labake. I look forward to seeing you at
the top.
My sincere appreciation also goes to my family especially my parents, Mr. Gabriel T.
Aruwajoye and Mrs. Mercy O. Aruwajoye for your spiritual, moral and financial support
throughout the period of this study. By the grace of God you will live long to reap the fruits
and dividends of your labour. Posterity will never forget you. Thank you so much. I also

iv

appreciate my brother Peter Aruwajoye and my siblings, Blessing, Gifty and Samson for their
expression of love and concern throughout the period of this programme.
Finally, I appreciate my classmates (Philomena, Eugene, Bimpe, Roland, Tunde,
Funke, Mukail, Kabir, Wole and Idowu). I have really enjoyed your companionship and
friendship during the period of this project. God bless you all.

Gabriel S. Aruwajoye
January, 2014.

TABLE OF CONTENTS
Title

Approval

ii

Dedication

iii

Acknowledgements

iv

Table of Contents

vi

List of Figures

xi

List of Tables

xiii

List of Abbreviations

xiv

Abstract

xv

CHAPTER ONE
1.0

INTRODUCTION

1.1

Cellulose Containing Materials

1.2

Cellulases

1.3

Microbial Sources of Cellulase

1.4

Statement of Research Problem

1.5

Objectives of the Research

1.6

Expected Contribution to Knowledge

CHAPTER TWO
2.0

LITERATURE REVIEW

2.1

Cellulose Accessibility Prior to Hydrolysis

2.2

Reactions Catalyzed by Cellulase

vi

2.3

Process of Cellulose Breakdown

2.4

Substrates of Cellulase

11

2.5

Assay Methods of Cellulase

13

2.6

Purification of Cellulases

15

2.7

Properties of Cellulase

18

2.7.1 Molecular Weight

18

2.7.2 Kinetics of Cellulose Hydrolysis

18

2.8

Inhibition of Cellulase

20

2.9

Structural properties of Cellulases

20

2.9.1 Active Sites of Cellulase

22

2.9.2 Cellulose Binding Domains

23

2.9.3 Catalytic Domains

24

2.9.4 Multiple Combination of Domains

24

2.10

27

Industrial Application of Cellulase

2.10.1 Textile Industry

30

2.10.2 Laundry and Detergents

30

2.10.3 Food and Animal Feed

30

2.9.4 Pulp and Paper Industry

31

2.9.5 Biofuel

31

CHAPTER THREE
3.0

MATERIALS AND METHODS

32

3.1

Materials

33
vii

3.2

Methods

33

3.2.1 Preparation of Reagents

34

3.2.2 Sample Collection and Isolation of Bacteria

34

3.2.3 Characterization and Identification of Isolates

34

3.2.3.1 Macroscopic Morphology

34

3.2.3.2 Gram Staining

34

3.2.3.3 Spore Staining

35

3.3

Biochemical Tests

35

3.4

Cellulase Assay

37

3.4.1 Glucose Standard Preparation

38

3.4.2 Determination of Protein Concentration

38

3.4.3 Protein Standard Preparation

39

3.5

39

Production of Cellulase

3.5.1 Inoculation of Basal Medium

39

3.5.2 Growth and Enzyme Production

39

3.6

40

Optimization of Cellulase Production Medium

3.6.1 Optimum pH for Cellulase Production

40

3.6.2 Optimum Temperature for Cellulase Production

40

3.6.3 Effect of Percentage Substrate Concentration on Enzyme Production

40

3.6.4 Effect of Some Nitrogen Sources on Enzyme Production

40

3.6.5 Effect of Some Carbon Sources on Cellulase Production

41

3.7

41

Enzyme Purification

3.7.1 Activation and Acetylation of Dialysis Bag

viii

41

3.7.2 Dialysis

41

3.7.3 Purification by Ion-Exchange Chromatography on CM-Sepharose CL-6B

41

3.7.4 Determination of Subunit Molecular Weight

41

3.7.5 Determination of Kinetic Parameters

42

3.7.6 Effect of Temperature on Activity of B. circulans Cellulase

42

3.7.7 Heat Stability of Cellulase from B. circulans

42

3.7.8 Effect of pH on Activity of B. circulans Cellulase

43

3.7.9 Effect of pH on Stability of B.circulans Cellulase

43

CHAPTER FOUR
4.0

RESULTS

44

4.1

Isolation of Cellulolytic Bacteria

44

4.2

Growth of Bacillus circulans and Changes in pH

44

4.3

Optimization of Cellulase Production

44

4.4

Enzyme Purification

53

4.4.1 Dialysis

53

4.4.2 Purification of the Enzyme using Ion-Exchange Chromatography on CMSepharose CL-6B

53

4.5

Molecular Weight Determination

53

4.6

Determination of Kinetic Parameters

53

4.7

Effect of pH on Cellulase Activity

53

4.8

Effect of pH on Stability of Cellulase Activity

60

4.9

Effect of Temperature on Cellulase Activity

60

4.10

Effect of Temperature on the Stability of Cellulase

60

ix

CHAPTER FIVE
5.0

DISCUSSION

64

REFERENCES

70

APPENDIX

88

List of Figures
Figure

Title

Page

1.1

The Structure of Cellulose and Related Compounds

2.1

The Mechanism of Cellulose Hydrolysis

10

2.2

The Two Different Mechanisms for the Enzymatic Hydrolysis of Glycosidic

Bonds in Cellulase I

12

2.3

Two Domain Architecture of Many Cellulases

21

2.4

The 3D Fold of the Catalytic Domain of Trichoderma ressei Cellobiohydrolase II 25

2.5

Ribbon Representation of the 3D Structure of Clostridium thermocellum

Endoglucanases D

2.6

26

Multiple Binary Combinations of Catalytic Domain Families and Cellulose

Binding Domain Families in Cellulases

28

2.7

The Additional Complexity of Cellulases

29

4.1

Screening for Cellulose Hydrolysis

45

4.2

Growth of Bacillus circulans Isolated from Decayed Wood

47

4.3

Profile of the Effect of pH on Cellulase Production by Bacillus circulans

48

4.4

Effect of Temperature on the Production of Cellulase by Bacillus circulans

49

4.5

Effect of Percentage Substrate Concentration on Enzyme Production and Cell

Growth

50

4.6

Effect of Nitrogen Sources on Enzyme Production

51

4.7

Effect of Carbon Sources on Cellulase Production by Bacillus circulans

52

4.8

Elution profile of Crude Cellulase obtained from Bacillus circulans on

CM-Sepharose CL-6B Ion-Exchange Column

xi

54

4.9

Photograph of SDS-PAGE of the Purified Cellulase from Bacillus circulans

56

4.10

Calibration Curve for Subunit Molecular Weight Determination on SDS-PAGE

57

4.11

Lineweaver-Burk Plot for the Determination of Kinetic Parameters of Purified

Cellulase Obtained from Bacillus circulans

58

4.12

Effect of pH on Purified Cellulase of Bacillus circulans

59

4.13

Effect of pH on Stability of Cellulase activity Obtained from Bacillus circulans

61

4.14

Effect of Temperature on the Activity of Cellulase Obtained from Bacillus

circulans

4.15

62

Effect of Temperature on the Stability of Cellulase Obtained from

Bacillus circulans

63

xii

Table
4.1

4.2

List of Tables

Page

Biochemical and Morphological Characteristics of Cellulolytic Bacterium Isolated

from Decayed Wood

46

Summary of the Purification of Cellulase Obtained from Bacillus circulans

55

xiii

List of Abbreviations
BSA Bovine Serum Albumin
CBD Cellulose Binding Domain
CBH Cellobiohydrolase
CMC Carboxymethylcellulose
CMCA Carboxymethylcellulose Agar
DEAE Diethylaminoethyl
DNSA Dinitrosalicylic Acid
EDTA Ethylene Diamine Tetraacetic Acid
EG Endoglucanase
FP Filter Paper
FPU Filter Paper Unit
GHF9 Glucosyl Hydrolase Family 9
HEC Hydroxylethyl Cellulose
HPLC High Performance Liquid Chromatography
MCC Microcrystalline Cellulose
MeUMB Methyl Umbelliferyl
MUG Methyl Umbelliferyl -D-glucoside
NMR Nuclear Magnetic Resonance
SDS-PAGE Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis
Rf Relative Mobility

xiv

ABSTRACT
Cellulolytic organisms were isolated from decayed wood and the best bacteria
candidate was optimized and used to produce cellulase that was characterized. This was with
a view to identifying cellulolytic bacteria needed for producing cellulase for industrial uses.
Decayed wood sample was collected from a site at Road 2 Area, Obafemi Awolowo
University, Ile-Ife. The sample was cultured on Nutrient Agar in order to isolate cellulolytic
bacteria. Bacteria isolated were screened for cellulolytic activity using serial dilution and
pour-plate method after which they were characterized. The bacterial isolate with the highest
carboxymethylcellulose (CMC) hydrolytic capacity was picked for further studies. The
growth curve, nitrogen sources, percentage substrate concentration, carbon sources and pH
under which cellulase can be produced maximally by the isolate were also determined.
Cellulase activity was determined by measuring the amount of reducing ends released from
CMC using Dinitrosalicylic acid method. The cellulase was purified using a combination of
dialysis and CM-Sepharose CL-6B ion-exchange chromatography. The molecular weight of
the enzyme was determined using Sodium Dodecyl Sulphate-Polyacrylamide Gel
Electrophoresis (SDS-PAGE). The kinetic parameters, effects of pH and temperature on the
purified cellulase were determined.
Fourteen bacteria were isolated from the sample. Seven of them were found to be
cellulolytic. The isolate with the highest cellulolytic potential was characterized and
tentatively identified as Bacillus circulans. The stationary phase of the organism was found to
be the thirtieth hour, while the nitrogen source, percentage CMC substrate concentration,
carbon source and pH under which the enzyme could be maximally produced was found to be
Tryptic soy broth, 0.15 % CMC, maltose and 6.5 respectively. In a typical purification
procedure, the specific activity of the purified cellulase was determined to be 29.13 g of
glucose/ml/min/mg protein with a purification fold of 4.37. The molecular weight of the

xv

purified cellulase was determined to be 43 KDa. The apparent Km value for the hydrolysis of
CMC was determined to be 1.061 1.17 mg/ml and its Vmax was determined to be 13.75
1.51 g of glucose produced/ml/min. The optimum pH and temperature were determined to
be 9.0 and 50oC respectively.
The study concluded that Bacillus circulans was capable of producing alkalophilic
and moderately thermostable cellulase which could be of industrial and biotechnological
applications.

xvi

CHAPTER ONE
1.0

INTRODUCTION

1.1

Cellulose Containing Materials

Cellulose microfibrils are insoluble structures typically composed of approximately

36 hydrogen-bonded chains containing 500 to 14,000 -1,4-linked glucose molecules.

Roughly one-third of the total mass of many plants is cellulose. The long, inelastic,
microfibrils wrap around cells in spatially oriented overlapping layers that provide resistance
to osmotic pressures that are similar in magnitude to the air pressure in a car tire (Somerville
2004). Cellulose can take on at least four different forms as determined by X-ray
crystallography (Marchessault and Sundararajan, 1983). The predominant native form is
referred to as cellulose I. Microcrystalline cellulose (cellulose I) has been shown to be made
up of two different crystal phases. Early Nuclear Magnetic Resonance (NMR) and X-ray
diffraction studies of extracted cellulose indicated that there are two distinct types of
cellulose I called cellulose I and I (Brown, 1996). Cellulose I exists as a single unit and is
dominant in bacterial and algal cellulose whereas cellulose I has two chain units and is
dominant in higher plants. In both forms the cellulose chains are parallel, and successive
glucose residues are rotated 180o, forming a flat ribbon in which cellobiose is the repeating
unit. Native cellulose (Cellulose I) can be converted to other crystalline forms (II-IV) by
various treatments (Zhang and Lynd, 2004). The cellulose chains are held in crystalline
structure by hydrogen bonds and Van der Waal forces to form micro fibrils (Nishiyama et al.,
2002; 2003).
Lignocellulosics provide an exclusive renewable resource as it makes up the plant
biomass which are consequently the most abundant terrestrial organic molecules (Gilbert and
Hazlewood, 1993). Lignocellulosics are abundant carbohydrate which is continually
replenished by photosynthetic reduction of carbon dioxide by sunlight energy, thus, making

them the most promising feedstock for the production of energy, food and chemical
(Solomon et al., 1999; Ojumu et al., 2003). Lignocellulosic is made up of cellulose (40-60%
of dry mass), hemicelluloses (20-40%) and lignin (10-25%) (Hamelinck et al., 2005).
Hemicellulose and lignin traps the cellulose fibrillar architecture which often affects the
accessibility of enzyme to cellulose (Mansfield et al., 1999). The parallel cellulose chains are
tightly linked by hydrogen bonds which results in rigidity of plant cell wall and the
hydrophobic lignin acts as a physical barrier to microorganisms and water (Bidlack et al.,
1992). Hemicellulose is the linking material between cellulose and lignin.
Cellulosic materials present in cotton, paper and wood are composed of a range of
polymeric material which reflects their origins and the degree of processing. Cotton is
composed primarily of cellulose, whereas wood and paper contain a range of polymers.
Wood contains significant amount of both cellulose and hemicelluloses. (Deobald and
Crawford, 1997). Lignin is an aromatic polymer, which forms cross links with polysaccharide
components of wood providing mechanical strength and resistance. Shorter oligosaccharides
(cellobiose) and related compounds of cellulose are generated after action by cellulolytic
enzymes (Fig.1).
1.2

Cellulases
Cellulase refers to a suite of enzymes produced by fungi and bacteria that catalyze the

hydrolysis of cellulose. Cellulose has a compact structure and its hydrolysis require a
complex system of cellulolytic enzymes for its breakdown into glucose units. There are three
major groups of enzymes in cellulase system, endo--1,4-glucanase (EC 3.2.1.4,), exo--1,4glucanase (EC 3.2.1.21,) and -1,4-glucosidase (EC 3.2.1.91). Endoglucanases, which cleave
-glucosidic bonds at random in the middle of cellulose molecules; cellobiohydrolases, which
attack cellulose molecules stepwise from the non reducing end, liberating cellobiose subunits;
-glucosidases, which hydrolyze cellobiose and low molecular

Fig. 1.1: The structure of cellulose and related compounds. Cellulose is the polymer.
Cellobiose is the dissacharide resulting from cellulose breakdown and glucose is the
monomer result of cellobiose hydrolysis.

weight cellodextrins into glucose (Beguin, 1990). Cellulases can be found in some animals,
plants, protozoans and microorganisms but are obtained basically from fungal and bacterial
sources.
Most animals cannot use cellulose because they do not have cellulolytic enzymes in
their systems. Contrari-wise, termites can efficiently degrade cellulose with the aid of
intestinal microbiont (Breznak and Brune, 1994). Termites are among the most important
lignocelluloses digesting insects and possess a great variety of symbiotic microorganisms in
their hindguts, including bacteria, archaea and eukarya, i.e. protozoa and yeasts (Konig et al.,
2002). In the digestive tracts of lower termites, cellulose seems to be synergistically degraded
by flagellates, bacteria, and yeasts (Breznak and Brune, 1994) as well as by the termites own
cellulases (Watanabe et al., 1998). Both the termites and their symbiotic flagellates can
produce cellulases (Watanabe et al., 2002). Cellulases of termite origin belong to glycosyl
hydrolase family 9 (GHF9). Flagellated protozoa belonging to the Archaezoa are unique
symbionts in the phylogenetically lower termites. They are cellulolytic and produce acetate
from cellulose for the benefit of their hosts (Odelson and Breznak, 1985).
Apart from termites, several cellulase studies have been undertaken in molluscs,
including snails (Parnas, 1961), a sea slug (Anzai et al., 1984), a periwinkle (Elyakova et al.,
1968), and some bivalves (Xu et al., 2000), some reported possible endogenous enzyme
sources such as the hepatopancreas (Elyakova et al., 1968), gastric teeth (Anzai et al., 1984),
and crystalline styles {needlelike structures made of crystalline proteins forming a motor
organ in the stomach of bivalves (Brusca and Brusca, 1990)} (Xu et al., 2000). In the snail,
Helix pomatia, cellulase and chitinase activities were detected in sterile hepatopancreas
(Strasdine and Whitaker, 1963). In addition, both cellulase and chitinase activity in the
digestive juices (luminal fluid of the gut) increased in proportion to body weight and the total
protein content of the hepatopancreas, but not in relation to increases in digestive juice

bacterial counts when, for example, the snails emerge from hibernation and start eating. This
suggests that these enzymes are secreted endogenously (Strasdine and Whitaker, 1963).
Cellulases find extensive application on cellulosic materials, and about 10% of the
finished product is estimated to be performed by these enzymes to achieve various effects
(Oinonen, 2004). They also find application in animal feed, textile, brewing and wine-making
industries (Beguin and Aubert, 1994).
1.3

Microbial Sources of Cellulase

Cellulolytic microbes are primarily carbohydrate degraders and are generally unable

to use proteins or lipids as energy sources for growth (Lynd et al., 2002). Cellulolytic
microbes notably the bacteria cellulomonas and cytophaga and most fungi can utilize a
variety of other carbohydrates in addition to cellulose (Poulsen and Peterson, 1988). One of
the most extensively studied fungi used for the production of cellulase is Trichoderma reesei,
which converts native as well as derived cellulose to glucose (Sukumaran et al., 2005). Most
commonly studied cellulolytic organisms include: Fungal species-Trichoderma, Humicola,
Penicillium, Aspergillus; Bacteria-Bacilli, Pseudomonas, Cellulomonas; and Actinomycetes,
Actinomucor, and Streptomyces (Sukumaran et al., 2005).
1.4

Statement of Research Problem

Ethanol, which can be used as biofuel in place of petroleum can be made synthetically

by microbial conversion of cellulosic biomass through enzymatic hydrolysis by cellulase(s)

and subsequent fermentation. There is therefore the need for search for cellulase(s) with
suitable industrial uses and high catalytic activity, hence this study.
1.5

Objectives of the Research

The specific objectives are to
(a) screen, isolate and identify cellulolytic bacteria from decayed wood;

(b) determine optimum conditions for the production of cellulase from one of the
cellulolytic bacteria; and
(c) investigate the characteristics of the cellulase produced.
1.6

Expected Contribution to Knowledge

This study will provide useful information on one of the sources of bacteria for

cellulase production for biotechnological uses.

CHAPTER TWO
2.0

LITERATURE REVIEW

2.1

Cellulose Accessibility Prior to Hydrolysis.

Cellulose is the most abundant organic source of food, fuel and chemicals. However,

its usefulness is dependent upon its accessibility and subsequent hydrolysis to glucose.
Cellulose can be hydrolyzed into its glucose subunits by chemical methods and biological
methods by enzymatic hydrolysis. Enzymatic hydrolysis has benefits over chemical methods
(acid and alkaline hydrolysis) which usually involve extreme conditions of pH and high
temperatures which can cause glucose degradation into non-specific products and also give
rise to spent acid and alkali that are of environmental concerns (Chinedu et al., 2008).
Enzymatic processes are supposed to have roughly equal costs as that of chemical
methods today but are more environmentally friendly. Nevertheless, it has its own costs
especially in cellulosic ethanol production where high enzyme doses are needed. Specifically,
cellulase loadings of approximately fifteen filter paper unit (FPU) per gram cellulose is
typically used to achieve economically viable sugar yields from pretreated biomass which is
equivalent to approximately 30 g of enzyme per liter of ethanol made (Yang et al., 2011).
Therefore most studies focus on enzymatic hydrolysis (Lynd, 1996; Lee et al., 2008;
Mawazda et al., 2000; Adeleke et al., 2012). In other to increase the yield of hydrolysis, a
pre-treatment step is needed that softens biomass and breaks down cell structures to a large
extent. The bioconversion of cellulose to soluble sugars and glucose is catalyzed by a group
of enzymes called cellulases that are produced by microorganisms (Wood and GarciaCampayo, 1990). Fibre swelling and fragmentation of cellulose aggregations into short fibers
occurs during enzymatic hydrolysis of cellulose before any detectable amount of reducing
sugars is released (Coughlan, 1985; Teeri et al., 1992). This initial stage in enzymatic
saccharification of cellulose has been termed amorphogenesis (Arantes and Saddler, 2010).

The original mechanistic model for enzymatic degradation of cellulose postulated by

the pioneering work of Mandel and Reese introduced the C1 -Cx model (Reese et al., 1950;
Mandels and Reese, 1964). They hypothesized that an unknown component of the cellulase
system (C1, the so called swelling factor) opens up the cellulose matrix, allowing the now
more accessible substrate to be depolymerized by the truly hydrolytic enzymes (Cx) (Reese et
al., 1950; Mandels and Reese, 1964). Although hydrolytic enzymes that could account for the
suggested Cx action have been identified and characterized, so far the identification and
characterization of the C1 factor remains elusive (Arantes and Saddler, 2010).
Cellobiohydrolase I (CBH 1), along with a number of other proteins (expansins,
expansin-like proteins, swollenin), contains a polysaccharide binding surface. These proteins
have been suggested to be able to non-hydrolytically loosen or disrupt the packaging of the
cellulose fibril network. The cellulose-disrupting activity of such proteins has recently been
shown to interact synergistically with cellulase enzymes when they are used to hydrolyze
insoluble cellulose, apparently by increasing the accessibility of the cellulose to the enzymes
(Baker et al., 2000; Kim et al., 2009). Several factors that affect enzyme hydrolysis of the
substrate (cellulose) include: Lignin/hemicelluloses association; degree of cellulose
crystallinity and polymerization; and extent of surface area while from the enzymes we have
factors such as end-product inhibition; need for synergism; and irreversible enzyme
adsorption. These have been suggested as possible factors that account for the recalcitrance of
cellulose to enzyme hydrolysis (Arantes and Saddler, 2010).
Cellulolytic microorganisms play an important role in the biosphere by recycling
cellulose, the most abundant and renewable biopolymer on Earth (Li et al., 2009). Fungal
cellulases are produced in large amounts, which include all the components of a multienzyme system with different specificities and mode of action, that is, endoglucanases (or
CMCase), exoglucanases (cellobiohydrolase), and -glucosidases (or cellobiases), acting in

synergy for the complete hydrolysis of cellulose (Bhat and Bhat 1997; Henrisat et al., 1985;
Knowles et al., 1987).
2.2

Reactions Catalyzed by Cellulases

There are three types of reaction catalyzed by cellulases: 1. Breakage of the non

covalent interaction present in the amorphous structure of cellulose (endocellulase) 2.

Hydrolysis of chain ends to break the polymer into smaller sugars (exocellulase) 3.
Hydrolysis of disaccharides and tetrasaccharides into glucose (beta-glucosidase) (Fig. 2.1).
In most familiar case of cellulase activity, the enzyme complex breaks down cellulose
to beta-glucose. Enzymes that hydrolyze hemicelluloses are usually referred to as
hemicellulase and are usually classified under cellulase in general. Enzymes that cleave
lignin are occasionally classified as cellulase, but this is usually considered erroneous.
2.3

Process of Cellulose Breakdown

To hydrolyse cellulose, cellulase must hydrolyze the glycosidic bonds connecting the

-D-glucosyl residues of cellulose. The two mechanisms by which enzymes hydrolyze

glycosidic bonds have been studied in great detail. The molecular catalysis usually involves
two amino acids of the protein performing one or two nucleophilic substitutions at the
anomeric centre (Koshland, 1953; Sinnot, 1990).
The two different mechanisms for the enzymatic hydrolysis of glycosidic bonds are
shown in Fig. 2.2. The main difference between the two mechanisms is that they lead to
retention or inversion of the anomeric configuration. Another difference is that the retaining
mechanism almost invariably leads to transglycosylation capability while the inverting
mechanism leads exclusively to hydrolysis (Sinnot, 1990). However, it is sure that only one
mechanism prevails for a given family of enzyme.

Fig. 2.1: The mechanism of cellulose hydrolysis. The final product of the reaction is as a
result of the synergistic action of the three enzymes.

10

2.4

Substrates of Cellulase
Although cellulolytic enzymes hydrolyze -1,4-glucosidic bonds of glucan polymers,

their specificities differ. The endo-glucanases attack randomly in the interior of the cellulose
structure not being very active against crystalline cellulose. The major structural features of
substrate that determine its susceptibility to enzyme degradation include: the degree of
swelling and crystallinity. The composition of enzyme e.g. amino acid sequence and
composition, isoelectric point, carbohydrate content and adsorbability on cellulose and
catalytic specificity contribute to the enzyme specificity. Also, variation in enzyme source
and method of extraction result in exhibition of different properties (Beguin and Aubert,
1994).
A wide variety of substrates are available for the studies of cellulase activities in
microorganisms. The potential substrates for the assay of cellulase activity include native
cellulose, regenerated cellulose and cellulose derivatives such as Carboxymethylcellulose (CMC) and hydroxylmethylcellulose (HEC). Out of all these materials,
Carboxymethylcellulose has been used extensively for the studies of cellulase synthesis in
microorganisms (Adeleke, 2010).
There are three major groups of insoluble cellulose used, namely: those slowly
hydrolyzed by cellulase which is derived from native fibre, for example, cotton and filter
paper; those that are often extremely hydrated but not degraded, for example, swollen
celluloses and cellophane and those that are extremely degraded and hydrated, for example,
cellodextrin (Adeleke, 2010). There are two types of soluble substrate in common use namely
cello-oligoglucosides (cellobiose and cellohexose) and soluble derivative of cellulose (CMC
and HEC) (Willis et al., 2010).

11

Fig. 2.2: The two different mechanisms for the enzymatic hydrolysis of glycosidic bonds in
cellulose. (I), mechanism leading to the overall retention of the anomeric configuration; (II),
mechanism leading to overall inversion of the anomeric configuration; A and B represent two
catalytic residues of the enzyme (Henrissat, 1994).

12

2.5

Assay Methods of Cellulase

Assay methods used are normally measured by loss in weight of insoluble substrate;

decrease in mechanical properties of fibres or films; increase in reducing end groups;

decrease of viscocity of cellulose derivatives; colorimetric determination of dissolved
decomposed products of cellulose and measurements of clearance zones in cellulose agar
(Waksman, 1991). In most cases, the degradation of cellulase substrates is measured using
modifications of the 3,5-dinitrosalicylic acid (DNSA) (Miller, 1959) or terazolium blue (Jue
and Lipke, 1985) assays, which detect reducing sugars generated during cellulose
degradation. Initially, the DNSA assay was found susceptible to interferences when using
native or pretreated lignocellulosic materials, which limited its use depending on the
substrates (Rivers et al., 1984). Optimized microplate assays based on the DNSA method to
quantify degradation of modified cellulose (Xiao et al., 2005), filter paper (FP) (Xiao et al.,
2004), or pretreated lignocelluloses material (King et al., 2009), have been developed to
overcome these limitations. Other techniques to quantify production of glucose in solution
include the alkaline copper (Somogyi, 1952) and the glucose oxidase/peroxidase (Dahlqvist,
1968) methods.
Methods based on high Perfomance liquid chromatography (HPLC) or thin layer
chromatography seperations to estimate produced sugars have also been described (Berlin et
al., 2006; Chundawat et al., 2008). To further characterize the activity of specific cellulolytic
enzymes, activity is measured by spectrophotometry using substrates containing pnitrophenol (Marana et al., 2004; Yapi et al., 2009) or methyl umbelliferyl (MU) groups
(Marana et al., 2001). Use of diverse cellulase substrates can help differentiate the digestion
of plant material (Tokuda et al., 2005), which can contribute to a more complete
characterization of cellulolytic process. Due to their low levels of chemical modification,
filter paper (FP), microcrystalline cellulose (MCC) and cotton, have been used as preferred

13

cellulase substrates to determine the existence of complete cellulolytic systems

(endoglucanase, cellobiohydrolase and -glucosidases) (Scrivener and Slaytor, 1994).
The main limitation in the use of this CBH substrates is their solubility, which limits
their use to in-solution assays with continuous mixing and complicates the removal of
particulates. A high throughput microplate assay for cellulolytic activity was developed using
MCC as substrate (Chundawat et al., 2008). An alternative approach is the detection by
absorbance or fluorescence of p-nitrophenol or methyl-umbelliferyl (MU) cleavage after
hydrolysis of glycosides such as p-nitrophenyl--D-cellobioside (pNPC) (Zhou et al., 2007),
or 4-methylumbelliferyl--cellobiopyranoside (MeUMB) (Jacobson and Schlein, 1997).
An alternative to insoluble cellulase substrates are modified celluloses, such as CMC
which are derived to improve water solubility. In CMC, the hydroxyl groups are methylated
resulting in high water solubility compared to crystalline or armophous cellulose. Due to its
ease of use and easy degradation by EG activity, CMC is the most documented cellulase
substrate used for solution assays or incorporation into agar or acrylamide gel matrices
(Willis et al., 2010). CMC has been used as substrate in agarose plates for qualitative
determination of EG activity (Zverlove et al., 2003); as early screening for cellulolytic
symbionts (Delalibera et al., 2005) and to screen modified (Zhang et al., 2009) or
heterologously produced cellulases (Wei et al., 2006). In this method, plates are incubated
with inoculums and activity revealed as clear zones when staining undigested CMC with
congo red dye. The diameter of this activity area has been utilized as a relative measurement
to compare activity of heterologously expressed enzymes (Zhang et al., 2009). In a similar
strategy, cellulolytic zymograms (a medium showing electrophoresis result) with CMC as
substrate can be used to detect proteins with cellulolytic activity after electrophoretic
separation of proteins (Schwarz et al., 1987). The advantage of this method over activity
assays in agar plates is that specific protein bands with cellulolytic activity can be visualized

14

and their molecular weight estimated. To prevent CMC degradation during electrophoresis,
proteins are only partially denatured and gels run at low temperature so that discrete bands,
rather than smears, can be detected (Li et al., 2008). These zymograms have also been used
to characterize cellulolytic activity of cellulases overproduced in heterologous systems
(Zhang et al., 2009). Due to the diverse range of specificities of -glucosidases, a variety of
substrates are used for detecting and quantifying this enzymatic activity.
Cleavage of -D-glucopyranosides (such as salicin, octyl -glucoside or helicin), or
disaccharides (such as cellobiose or amygdalin) by -glucosidases is generally quantified by
detection of the generated glucose using the glucose oxidase/peroxidase method (Yapi et al.,
2009). Alternatively, -glucosidase activity is also quantified by measuring the hydrolysis of
p-nitrophenol from glycoside derivatives (NP -glycosides) such as glucoside (NP -Glu)
(Yapi et al., 2009), or methyl-umbelliferyl (MU) fluorescence after hydrolysis of MU
glycosides such as 4-methyl-umbelliferyl -D-glucoside (MUG) (Marana et al., 2001).
Combined use of substrates allowed determination of enzymatic specificities in glucosidases (Ferreira et al., 1998). Protein bands displaying -glucosidase activity after
electrophoretic separation can be detected using MUG as substrate (Genta et al., 2006).
2.6

Purification of Cellulases
There is need to purify enzymes as they degenerate during storage even when stored

at temperature as low as 40C. Therefore, the purification strategy adopted must preserve
enzyme function and hence minimize denaturation and proteolysis (Wilson and Walker,
1996). Purification methods are based on separation due to differences in stability to heat;
purification by fractional precipitation; electrophoretic techniques; paper chromatography and
column chromatography (Yapi et al.,2009).
Purification procedures usually consist of multiple steps, including size exclusion,
anion exchange and/or hydrophobic chromatography (Yapi et al., 2009). Proteins have been

15

purified and characterized using different liquid chromatographic procedures (Genta et al.,
2003). Alternative purification methods have also been reported which include isoelectric
focusing and preparative electrophoresis (Willis et al., 2010). Once the protein is purified,
protein sequencing may facilitate primer design for polymerase chain reaction (PCR)
(Marana et al., 2001).
Bause et al. (1986) reported a three step purification method for purification and
characterization of glucosidase I, namely, DEAE-sephacel chromatography, affinity
chromatography on AH-sepharose 4B-linked N-5-carboxypentyl-1-deoxynojirimycin and
Con A-sepharose chromatography which yielded about 1900-fold from yeast microsomal
preparations. Trichoderma reesei secrets several different proteins into the culture liquid. To
separate these from each other, adsorption and ion-exchange chromatography were the
earliest methods used. Molecular sieving, chromatofocusing and affinity to different specific
sorbents have also been used. To obtain a pure enzyme, several successive steps are
necessary.
Purification procedures are, under continuous development. The development of new
sensitive detection methods also sets new demands on the degree of purification. Difficulties
in purification are caused by the close similarity in physicochemical properties of fungal
extracellular proteins as shown, for example, by combined polyacrylamide gel
electrophoresis immunodiffusion (Nunmi et al., 1983). The similar physicochemical
properties of contaminating proteins make the purification of endoglucanases difficult using
methods based on ionic properties and molecular size. (Sprey and Lampbert, 1983).
Endoglucanase preparations will always contain these contaminants, If rigorous methods are
not used. The composition of the culture medium affects the amount and quality of the noncellulolytic protein in the culture medium and thus the impurities which interact with the
cellulolytic enzymes (Enaria and Niku-Paavola, 1987).

16

To facilitate the purification of separate cellulolytic enzymes, it is advantageous to

choose cultivation conditions where the amount of impurities and interacting proteins is
minimized, even if the production of cellulolytic enzymes is not maximal. The cellulolytic
enzyme preparations obtained from the different purification procedures show differences in
enzymatic properties. For example, pure cellobiohydrolases include a powerful hydrolyzing
activity of microcrystalline Avicel (Berghem et al., 1975), but also enzymes which hardly
attack even swollen amorphous cellulose (Gritzali and Brown, 1979). These differences may
depend on traces of other cellulolytic enzymes and on the interacting impurities. The
differences may also be due to modification and inactivation caused by rigorous purification
treatments and too many purification steps. Concerning cellobiohydrolase, it was reported
that alkaline pH and even the temperature used in assays caused disintegration of the enzyme
into inactive fragments (Nunmi et al., 1983). During the several successive purification steps
used, cellobiohydrolase is partly inactivated. Also, the history of starting material may affect
the properties of the enzymes obtained after purification. Freeze-dried commercial enzyme
preparations hardly correspond to the original form of enzymes secreted by the fungus.
Endoglucanases exist in the culture medium as a series of immunologically related
components.
During purification and storage, the endoglucanases are spontaneously modified: the
relative amounts of components have greater molecular size, more alkaline pH, and lower
specific activity is increased (Niku-Paavola et al., 1985). Evidence for proteolytic cleavage of
cellulolytic enzymes during storage of culture liquids has been presented. To obtain enzymes
in their original form, the freshness of the starting material is crucial. Also, the number of
purification steps should be kept at a minimum to avoid modification of the enzymes, with
these; the specific affinity methods are to be preferred (Enari and Niku-Paavola, 1987).

17

2.7

Properties of Cellulase

2.7.1 Molecular Weight

The molecular weight of cellulase varies from one organism to another. It can be as
low as 5,600 to 88,000 (Selby and Maitland, 1965; Ng and Zeikus, 1981). The molecular
weights of thee components E1, E3a and E4 from Trichoderma koningii were determined
using a column of sephadex G-100 to be 13,000, 48,000 and 31,000 respectively (Wood and
McCrae, 1977). Using sodium dodecylsulphate polyacrylamide gel electrophoresis it was
found that molecular weight of thee endoglucanases (II, III and IV) out of four from the same
fungus were 37,000, 52,000 and 49,000 respectively (Shoemaker and Brown, 1978). It has
been established that there is apparently diversity among the glucanohydrolases even from
the same organism (Berghem et al., 1975). Molecular weights from different sources have
also been determined. Using gel filtration on calibrated column of Bio gel P-60, the
molecular weights of three endoglucanases from Trichoderma aurantiacus were 78,000,
48,000 and 33,000. Ng and Zeicus (1981) reported that using amino acid composition
analysis the molecular weight of Clostridium thermocellum was 88,000 2000, Two
thermostable cellulases A CelA and CelB purified from Thermotoga neapolitana had
molecular weights of 29,000 and 30,000 respectively (Bok et al., 1998).
2.7.2 Kinetics of Cellulose Hydrolysis
Earlier studies of cellulose hydrolysis kinetics were restricted to crude cellulase
mixtures. Regardless of the cellulase used, it was often found that the hydrolysis rate
decreased far more rapidly than expected from the total degree of solubilisation. Hydrolysis
exhibited a typical biphasic pattern. The cause of this gradual drop in reaction rates is not
fully understood, but it has been postulated that both enzyme- and substrate-related properties
contributed to this effect (Mansfield et al., 1999). Conflicting observations have prevented
scientists from identifying a single factor as the sole cause of the gradual loss of efficiency

18

during the hydrolysis of cellulose. In general, the proposed explanations fall into three
distinct groups.
(a) Bulk cellulose substrate contains several regions that differ in susceptibility to
enzymatic attack. Consumption of the small amount of easily degradable parts in
cellulose causes the rate to fall.
(b) Strong inhibition of cellulases by the reaction product, cellobiose.
(c) Inactivation of the cellulase.
The simplest model for the hydrolysis kinetics is based on the assumption that
cellulose consists of many different regions, each with its own rate constant for enzymatic
hydrolysis, and that the hydrolysis proceeds as a first-order reaction with respect to the
concentration of each region (Sattler et al., 1989; Nidetzky and Steiner, 1993). Since
cellulose is known to consist of amorphous and crystalline regions, it has long been suggested
that the high initial rates of hydrolysis are due to the amorphous regions: As the reaction
proceeds and the amorphous regions become depleted, the overall rate slows to the value
corresponding to the hydrolysis of crystalline regions (Fan et al., 1980; Wald et al., 1984).
However, the above hyphothesis seems to especially relevant in the case of EG-s. They
exhibit a very low and abrubtly decreasing activity toward crystalline substrates, whereas the
activity toward amorphous substrates is far higher than that of CBH-s.
Kinetic parameters of some microbial cellulases have been reported. Bacillus strains
CH 43 and HR 68 have been reported to have a Km of 1.5 and 1.7 mg/ml respectively, the
two strains also had a Vmax of 0.93 and 1.70 mmol glucose min-1 mg protein-1 respectively
(Mawazda et al., 2000). Adeleke et al. (2012) has also reported cellulase Km value of 0.18
0.06 mg/ml of carboxymethylcellulase as well as Vmax of 37.94 2.98 units/mg of protein.

19

2.8

Inhibition of Cellulase
Apart from natural reaction, product inhibition of cellulase by cellobiose and glucose

has been reported (Bulcholz et al., 1983). It can also be completely inhibited by other salts in
their ionic forms such as mercury and slightly inhibited by manganese, silver, copper and
zinc (Bulcholz et al., 1983). Most chloride salts are formed either by direct union of chlorine
with a metal or by reaction of hydrochloric acid with metal, metal oxide or an inorganic base.
Most chloride salts such sodium chloride and ammonium chloride are readily soluble in water
but lead chloride is slightly soluble while mercury chloride is insoluble in water. Chlorides
had been reported to inhibit cellulase activities (Lakshmesha et al., 2005).
2.9

Structural Properties of Cellulases

Cellulases are comprised of independently folding, structurally and functionally

discrete units called domains or modules, making cellulases modular (Henrisaat et al., 1998).
A typical free cellulase is composed of a cellulose binding domain (CBD) at the C-terminal
joined by a short poly-linker region to the catalytic domain at the N-terminal (Fig. 2.3). There
are only two modes of action for the hydrolysis of cellulose by cellulases, either inversion or
retention of the configuration of the anomeric carbon. At least two amino acids with carboxyl
groups located within the active site catalyze the reaction by acid-base catalysis.

20

Fig. 2.3: Two-domain architecture of many cellulases (Knowles et al., 1987). The
catalytic domain is at the N-terminal while the cellulose-binding domain is at the C-terminal.

21

The commonly described mode of action for cellulases on polymers is either exo- or
endo-cleavage, and all cellulases target the specific cleavage of -1,4-glycosidic bonds
(Wood and McCrae, 1979). Using this classification system, cellobiohydrolases
(exoglucanases) were classified as exo-acting based on the assumption that they all cleave 1,4-glycosidic bonds from chain ends. As well, those enzymes truly exo-acting often have a
tunnel-shaped closed active site which retains a single glucan chain and prevents it from readhering to the cellulose crystal (Rouvinen et al., 1990; Divine et al., 1994; Divine et al.,
1998). While endoglucanases on the other hand, are often classified as endo-acting cellulases
because they are thought to cleave -1,4-glycosidic bonds internally only and appear to have
cleft-shaped open active sites. Endoglucanase are active on amorphous regions of cellulose
and thus their activity can be assayed using soluble cellulose substrates; i.e., the
carboxymethylcellulase assay (CMCase). However, there is now supporting evidence that
some cellulases display both modes of action, endo- and exo- (Davies and Henrissat, 1995).
Thus classification has changed; cellobiohydrolases (exoglucanases) are described as active
on the crystalline regions of cellulose; whereas, endoglucanases are typically active on the
more soluble amorphous region of the cellulose crystal.
2.9.1 Active Sites of Cellulase
Cellulases catalyze the random hydrolysis of the glycosidic bonds of cellulose and
related cellooligosaccharides. Through kinetic and chemical modification studies on
cellulases, some workers have tried to identify the amino acids present at the active sites.
Hurst et al. (1977a and 1977b) have reported the participation of carboxyl residues in the
active site of a cellulase from Aspergillus niger. Tryptophan and histidine are thought to be
involved in the active site of a cellulase of Penicillium notatum (Erickson and Petterson,
1968). Two highly homologous forms of endoglucanases (EG I, and EG II) produced by a
white rot fungus Schizophyllum commune have been characterized and differ only in their

22

amino acid terminal sequence (Paice et al., 1984). Ionizable carboxyl groups were essentially
required for EG I activity (Clarke and Yaguchi, 1985). Through sequence homology studies
Yaguchi et al. (1983) suggested that enzymes follow the same pathway as hen egg white
lysozyme. Two specific carboxyl groups of endoglucanases, Glu-33 and Asp-50 were
postulated to participate in the catalytic site of the enzyme. Glu-33 was assumed to act as
general acid catalyst while Asp-50 would be negatively charged and stabilize the incipient
carbonium ion of the substrate. Studies made on ultraviolet difference adsorption spectra of
the cellulase from white rot fungus S. commune, suggested the presence of tryptophan
residues in the binding region of this enzyme (Clarke and Yaguchi, 1986). One residue
appears to be involved in the binding of substrate, while second residue proposed to
constitute an integral part of the catalytically sound active centre (Clarke, 1987). Implication
of histidine at the active site of exoglucanases from Basidiomycetes species has also been
reported. (Jeffcoat and Kirkwood, 1987)
2.9.2 Cellulose Binding Domains
CBD is the most common accessory module of cellulases and there are 54 distinct
families (Henrissat et al., 1998). The major function of CBDs is to deliver its resident
catalytic domain to crystalline cellulose. Binding brings the catalytic domain into close
contact with the crystalline cellulose for efficient hydrolysis. Binding of the cellulase via
CBD is extremely stable, yet still allows the enzymes to diffuse laterally across the surface of
the substrate and in some cases CBD has also been shown to catalyze the disruption of non
covalent interactions between cellulose chains of crystalline cellulose. Some other CBDs are
preferential to binding noncrystalline cellulose (Din et al., 1991; Johnson et al., 1996; Jervis
et al., 1997). Cellulases have been classified based on their cellulose binding domains and
catalytic binding domains. Due to the discovery of the amino acid sequences of cellulose

23

binding domains of the enzyme, it was classified into five families (I-V) (Coutinho et al.,
1992).
2.9.3 Catalytic Domains
Careful comparison of the amino acid sequences of cellulase catalytic domains have
shown that these enzymes cannot be grouped to only two categories as was the case before
i.e. endoglucanases and cellobiohydrolases. They were grouped into six families A-F
(Henrissat et al., 1989). Since significant sequence similarities are a strong indication of
three-dimensional (3D) structure similarities, it was proposed that each family of cellulase
catalytic domains would be characterized by a conserved fold, whereas different folds were
expected in different families (Henrisaat et al.,1991). With steady increase in new cellulase
sequences, classification in six families were confirmed (Beguin, 1990) and new families
were added later: H (Gilkes et al., 1991), I (Henrissat, 1991), J and K (Henrissat and Bairoch,
1993). Besides the fact that they contain endoglucanases and sometimes cellobiohydrolases,
they sometimes also contain polysaccharides which are not cellulases. For instance, Family A
was shown to also contain a -mannase, an exo-1,3--glucanase and a cellodextrinase
(Henrissat, 1992; Henrissat and Bairoch, 1993). The validity of the classification of cellulases
based on amino acid sequence similarities can be ultimately verified by the determination and
comparison of the 3D structure of several members of each family (Fig. 2.4 on 3D structures
of catalytic domains). An indirect approach is the determination of the stereochemistry of the
catalytic reaction which should be conserved within each family (Fig. 2.5 on the catalytic
mechanism and the catalytic residues).
2.9.4 Multiple Combinations of Domains
The structural complexity of cellulolytic enzymes originating from their multipledomain architecture is further increased by the various combinations one can observe
between catalytic domains and cellulose-binding domains (Gilkes et al., 1991).

24

Fig. 2.4: The 3D fold of the catalytic domain of Trichoderma ressei cellobiohydrolase II
as determined by X-ray crystallography (Rouvinen et al.,1990). Only the -carbons of the
polypeptide chain are shown. Two loops close the active site (AS) into a tunnel and are
shown by the arrows (Spezio et al., 1993).

25

Fig. 2.5: Ribbon representation of the 3D structure of Clostridium thermocellum

endoglucanases D (Juy et al., 1992). The active site cleft is shown by the arrow (Henrissat,
1994).

26

(a) some cellulases lack the cellulose binding domain, i.e. contain only a catalytic domain
(Fig. 2.6)
(b) cellulose-binding domains of one family can be linked to catalytic domains of different
families (Fig. 2.6)
(c) catalytic domains of one family can carry cellulose-binding domains of different families
(Fig. 2.6)
(d) cellulose-binding domains can be found apparently indifferently at the N-or the Cterminus of the catalytic domains (Fig. 2.7A and B)
(e) in some instances a cellulose binding domain can be linked to two catalytic domains
resulting in a bifunctional enzyme (Fig. 2.7C)
(f) cellulases sometimes carry additional discrete domains of a yet unknown function (Fig
2.7D)
2.10

Industrial application of Cellulase

Cellulose can provide very cheap raw material for the production of many

commercially important products such as paper, textile, cellophane, food, fuel and chemical.
The conversion of cellulose hitherto a waste to useful products can be achieved by cellulase
enzyme system from microorganisms. Commercial production of cellulases has been tried by
either solid or submerged culture including batch, fed batch and continuous flow processes.
Media used in cellulase fermentations contain cellulose in different degrees of purity or as
raw lignocellulosic substrates (Doppelbauer et al., 1987; Aiello et al., 1996; Reczey et al.,
1996).
Cellulose production on a commercial scale is induced by growing the organism on
solid cellulose or by culturing the organism in the presence of a disaccharide inducer such as
lactose. However, on an industrial scale, both methods of induction result in high costs. Since
the enzymes are inducible by cellulose, it is possible to use cellulose containing media for

27

Fig. 2.6: Multiple binary combinations of catalytic domain families and cellulosebinding domain families in cellulases. The first row schematically represents 5 out of the 10
catalytic domain families. The columns show the various combinations arising from the
various cellulose-binding domain families that may be attached to each catalytic domain
(Henrissat, 1994).

28

Fig. 2.7: The additional complexity of cellulases; resulting from two types of possible
attachment of the cellulose-binding domain onto the N-or C-terminus of the catalytic
domain. In A and B, cellulose binding domain is linked to the C-terminal and the N-terminal
respectively. In C; there is presence of two catalytic domains while in D; there is presence of
a number of discrete domains. (Henrissat, 1994).

29

production but the process is controlled by the dynamics of induction and repression. At low
concentrations of cellulose, glucose production may be too slow to meet the metabolic needs
of active cell growth and function. On the other hand, cellulase synthesis can be halted by
glucose repression when glucose generation is faster than consumption. Thus, expensive
process control schemes are required to provide slow substrate addition and monitoring of
glucose concentration (Ju and Afolabi, 1999).
2.10.1 Textile Industry
Cellulases have become the third largest group of enzymes used in industries. They
are used in the biostoning of denim garments for producing softness and the faded look of
denim garments replacing the use of pumice stones which were traditionally employed in the
industry (Belgith et al., 2001). They are used on the cellulose fiber to release the indigo dye
used for coloring the fabric producing the faded look of denim. H. insolens cellulase is most
commonly employed in the biostoning, though the use of acidic cellulase from Trichoderma
along with proteases is found to be equally good.
2.10.2 Laundry and Detergents
Cellulases are commonly used in detergents for cleaning textiles. Cellulase
preparations, mainly from species of Humicola (H. insolens and H. grisea var. thermoidea)
that are active under mild alkaline conditions and at elevated temperatures, are commonly
added in washing powders, and in detergents (Uhlig, 1998).
2.10.3 Food and Animal Feed
In food industry, cellulases are basically used in the extraction and clarification of
fruit and vegetable juices, production of fruit nectars and purees, and in the extraction of
olive oil (Galante et al., 1998). Glucanases are added to improve the malting of barley in beer
manufacturing, and in wine industry, better maceration and color extraction is achieved by

30

use of exogenous hemicellulases and glucanases. Cellulases are also used in carotenoid
extraction in the production of food coloring agents (Pajunen, 1986). Improvements in feed
digestibility and animal performance have also been reported with the use of cellulases in
feed processing (Lewis et al., 1996).
2.10.4 Pulp and Paper industry
In the pulp and paper industry, cellulases and hemicellulases have been employed for
biomechanical pulping for modification of the coarse mechanical pulp and hand sheet
strength properties, de-inking of recycled fibers and for improving drainage and runnability
of paper mills. They are also employed in the removing of inks, coating and toners from
paper, biocharacterisation of pulp fiber is another application where microbial cellulases are
empoloyed (Yang et al., 2004).
2.10.5 Biofuel
Perhaps the most important application currently being investigated actively is in the
utilization of lignocellulosic wastes for the production of biofuel. A potential application of
cellulase is the conversion of cellulosic materials to glucose and other fermentable sugars,
which in turn can be used as microbial substrates for the production of variety of
fermentation products like ethanol.
The strategy employed currently in bioethanol production from lignocellulosic
residues is a multi-step process involving pre-treatment of the residue to remove lignin and
hemicelluloses fraction, cellulose treatment at 500C to hydrolyze the cellulosic residue to
generate fermentable sugars, and finally use of a fermentative microorganism to produce
alcohol from the hydrolyzed cellulosic material (Sudha et al., 1997).

31

CHAPTER THREE
3.0

MATERIALS AND METHODS

3.1

Materials
Bovine serum albumin (BSA), D-glucose, ovalbumin, lysozyme, chymotrypsinogen

A, CMC, Tris HCl, Tris Base, malachite green and dipotassium hydrogen orthophosphate
were obtained from Sigma Chemical Company, Limited, St. Louis, MO., USA. Acrylamide
N,N-methylene bisacrylamide (TEMED), ammonium persulphate and sodium dodecyl
sulphate were products of Feinbiochemica, Heidelberg, Germany. Sodium hydroxide was
purchased from Merck Millipore International, Darmstadht, Germany while dinitrosalicylic
acid was purchased from Avishkar Scientific and Surgicals, Hyderabad, India. Disodium
hydrogen phosphate and sodium metabisulphite were purchased from Tianjin Kermel
Reagent Company Limited, Tianjin China. Crystal violet, safranin and peptone were obtained
from BDH Chemicals Limited, Poole, England. CM-Sepharose CL-6B was obtained from
Pharmacia Fine Chemicals, Upsalla, Sweden. All other reagents and chemicals were of
analytical grade. Glass distilled water was used for all preparation of solutions.
Apparatus used include inoculating loop, cotton wool, aluminum foil, paper tape, test
tubes and test tube racks. Illuminated cooled incubator and hot air oven from Gallenkamp,
Weiss United Kingdom (U.K.). Haier Thermocool refrigerator was purchased from Nigeria,
and Microfeild portable autoclave was purchased from Heatcon Composite Systems, Seattle,
Washington. Also, digital weighing balance was purchased from Mettler Toledo,
Switzerland. Microprocessor pH meter was purchased from Hanna Instruments, U.K. and
Spectrophotometer was purchased from Gulfex Medical and Scientific, England. Light
microscope also was purchased from Olympus Scientific Equipment Group, America.

32

3.2

Methods

3.2.1 Preparation of Reagents

(a)

Nutrient Agar
It was prepared by dissolving 2.8 g of Nutrient Agar in 100 ml of distilled water after

which it was autoclaved. The resulting solution was then poured into plates and allowed to
set. The organisms were introduced to the plates via streaking and then incubated to allow
them grow (subculturing).
(b)

Carboxymethylcellulose Agar (CMCA)

The composition includes the following components: yeast extract (0.2% w/v),

peptone (2% w/v), KH2PO4 (0.1%w/v), Mg2SO4.7H2O (0.5%w/v), NaCl (0.075%w/v),

carboxymethylcellulose (0.2% w/v) and nutrient agar (1.5% w/v).
(c)

Preparation of Bradford Reagent

100 mg of Coomasie Brilliant Blue G-250 was dissolved in 50 ml of 90% ethanol and

100 ml of 85% phosphoric acid (s.g. 1.75) was added. The mixture was made up to one litre
using distilled water.
(d)

Preparation of Dinitrosalicylic Acid

Its composition were 1% 3,5-dinitrosalicylic acid (DNSA), 0.2% phenol, 0.05%

sodium metabisulfite, 1% sodium hydroxide and 20% sodium tatarate all dissolved in
distilled water and made up to the desired quantity. It was then stored in a dark container at
4o C (Bailey et al., 1992)
(e)

Preparation of 1% Carboxymethylcellulose
1% carboxymethylcellulose was prepared by dissolving 1 g of carboxylmethyl-

cellulose in 100 ml of 0.05 M Tris buffer, pH 8.

33

3.2.2 Sample Collection and Isolation of Bacteria

Five gram of decayed wood samples collected from Road 2 Area of Obafemi
Awolowo University, Ile-Ife was weighed and mixed with 45 ml of sterile distilled water.
Subsequently, 1 ml of the suspension was added to 9 ml of sterile distilled water and 1 ml of
the resulting mixture was pipetted and added to another 9 ml of sterile distilled water. This
process was repeated until the fifth test tube. One millilitre of the diluents was mixed with
sterile nutrient agar inside Petri dishes. Plates were incubated at 37o C for 48 h and different
types of colonies were chosen based on their sizes and purified. Cellulose hydrolysis ability
of the isolated strains was tested on Carboxymethylcellulose agar (CMCA). Single streak of
the pure isolates were made on CMCA plates. After the incubation period, the plates were
stained with 0.1% congo red and destained with 1 M NaCl solution, a clear halozone around
the line of streak indicated a positive result. The isolates that produced halozones around the
line of streak were considered as cellulolytic and the isolate with the highest halozone based
on the Carboxymethylcellulose hydrolysis capacity (HC value) that is, ratio of the diameter of
clear zone and line of streak (Lu et al., 2005) was picked for cellulase production.
3.2.3 Characterization and Identification of Isolates.
Characterization of the bacterial isolates was carried out by observing their
macroscopic and microscopic morphology. The bacterial isolates obtained were classified
using Bergeys Manual of Determinative Bacteriology (Buchannan and Gibbons, 1985).
3.2.3.1 Macroscopic Morphology
The growth pattern of the isolates on the agar was studied for shape, surface,
appearance, elevation, edge and transparency of the colonies.
3.2.3.2 Gram Staining
A heat fixed smear of 18-24 h old culture of the organism was prepared on grease free
microscopic slide. The smear was stained with crystal violet for 20 seconds and then washed

34

off with distilled water. Grams iodine solution was then added for 60 seconds and washed
off with 95% ethanol, which was used to decolourize the smear until no violet colour was
noticed. Another round of washing off with distilled water was done and the smear was
counter stained with Safranin for 20 seconds. The slide was then rinsed off with distilled
water, air dried and observed under oil immersion objective lens of a microscope. Gram
positive bacteria appeared purple while Gram negative bacteria appeared pinkish. (Benson,
1990).
3.2.3.3 Spore Staining
The spore staining was carried out by heat fixing the thin smear of 5 days old culture
of the isolates on a grease free slide. Malachite green was added and steamed for 5-10
minutes, ensuring that the stain did not dry out nor boil off. The Malachite green was
carefully rinsed off with distilled water and counter stained with 0.25% Safranin solution for
15 seconds and washed with distilled water. The slide was blotted dry and examined under
the oil immersion objective lens of the microscope. Bacteria endospores stained green while
vegetative cells stained red (Harrigan and McCance, 1976).
3.3

Biochemical Tests
The biochemical tests performed were catalase test, hydrolysis of gelatin and starch.

Other tests include production of ammonia from peptone, methyl red-Vogues Proskauer
(MRVP) test, citrate utilization test, growth in 6.5% NaCl and arabinose test. Fresh culture
(18-24 h old) of the isolate was routinely used for all these tests and for each test, an
uninnoculated medium served as control.
Catalase Test
A drop of 3% hydrogen peroxide was put on a clean slide and a colony of the test
isolate was added. The occurrence of effervescence as a result of oxygen gas released

35

represents a positive reaction, while the absence of effervescence represents a negative

reaction.
Citrate Utilization Test
Sterile Citrate medium was inoculated with 18-24 h old broth culture of the test
bacterial isolate. The inoculated medium was incubated at 35o C for 4 days. A change in
colour of the medium from green to blue indicated positive reaction showing that the
organism was able to utilize citrate as a sole carbon source. Negative reaction was indicated
by no colour change meaning the organism was not able to utilize citrate as a sole carbon
source (Harrigan and McCance, 1976).
Production of Ammonia from Peptone
Sterile peptone water in test tube was inoculated with 0.1 ml of broth culture of the
organism. The inoculated tubes and sterile un-inoculated control tube which contained
peptone water were incubated at 35o C for 5 days. About 1 ml of Nesslers reagent was added
to each 1 ml of the peptone water culture and control peptone water in clean test tubes. The
presence of ammonia was indicated by the conversion of orange to brown colour. Sample
from the sterile control tube or from a tube in which no ammonia was produced turned pale
yellow or showed no colour change (Harrigan and McCance, 1976).
Hydrolysis of Gelatin
Sterile nutrient gelatin in test tubes were inoculated with 24 h old broth cultures of the
isolate and incubated for 7 days. The test tubes were then placed in the refrigerator at 4o C for
15 minutes. The test tubes in which gelatin had been hydrolyzed i.e. liquefied, indicated
positive results while those that solidified indicated negative result (Harrigan and McCance,
1976).

36

Hydrolysis of Starch
Sterile molten starch agar was poured into sterile petri-dish and allowed to set. The isolate
was streaked across the surface of the sterile starch agar plate. The plate was incubated at 35 o
C for 3-5 days. The starch agar culture plate was then flooded with some quantity of grams
iodine while a clear zone along the line of streak indicated starch hydrolysis (positive result).
Methyl Red-Vogues Proskaeur test (MRVP)
Sterile 5 ml of MRVP medium (Glucose phosphate broth) in a test tube was
inoculated with the isolate. The inoculated tube was incubated at 35o C for 5 days. At the end
of incubation, the content of the tube was aseptically divided into two portions and labeled M
and V respectively.
To tubes labeled M, 5 drops of methyl red solution was added and the colour
examined. Development of red colour indicated positive reaction i.e. acid produced while
yellow colour indicated negative reaction. To tubes labeled V, 0.5 ml of 6% naphthol solution
and 0.5 ml of 16% KOH were added to 1 ml and the tube was shaken. Development of a red
colouration usually within 5 minutes indicated a positive reaction (acetoin production).
Growth at 6.5% Sodium Chloride Concentration
Five milliliters (5 ml) of nutrient broth containing 6.5% NaCl respectively was
dispensed into test tubes and sterilized by autoclaving. The bacterial isolate was then
inoculated into the broths and incubated for 4 days at 35o C. Turbidity of the medium was
noted as positive reaction. Uninoculated tubes served as control (Harrigan and McCance,
1976).
3.4

Cellulase Assay
Cellulase activity was measured by the presence of reducing end groups released by

the action of the enzyme on the substrate using DNSA method (Miller, 1959). The culture
media after 48 h was centrifuged at 6,000 rpm for 30 min and the supernatant taken as crude

37

enzyme. The reducing ends released were determined by incubating 0.75 ml of 1% w/v CMC
(in 0.05 M Tris buffer pH 8.0) with 0.25 ml of crude enzyme and inactivated (boiled) crude
enzyme as blank (Bailey et al., 1992). The reaction mixture was then incubated at 400C for
20 min and the reaction terminated by addition of 1 ml of DNSA reagent. The reaction
mixture was heated at 100o C for 15 min in boiling water. The tubes were then cooled under
running water. The optical density (OD) was read at 540 nm. The amount of reducing sugars
produced was interpolated from the glucose standard curve.
One unit of cellulase activity was expressed as the amount of enzyme that liberated
reducing sugar equivalent to 1 g of glucose per minute under the assay condition. The
specific enzyme activity was expressed as the unit of enzyme activity per mg of protein.
3.4.1 Glucose Standard Preparation
Anhydrous glucose (0.001 g) was dissolved in 10 ml of distilled water to give 100 g
per ml of working solution. From the stock solution, 0, 0.2, 0.4, 0.6, 0.8 and 1 ml was
pipetted into test tubes and corresponding volume of distilled water was added to make up the
volume to 1 ml to give a final working concentration of 0, 20, 40, 60, 80 and 100 g of
glucose per ml respectively. One ml of different working concentration was assayed under
the same condition using the assay method described above. One millilitre of DNSA reagent
was added to it and the entire solution was boiled in water for 15 min. The tubes were cooled
under running water. The OD was read at 540 nm.
3.4.2 Determination of Protein Concentration
Protein concentration was determined using Bradford (1976) method with bovine
serum albumin as the standard protein. To 0.25 ml of enzyme, 1.35 ml of distilled water was
added and mixed; then, 0.4 ml of Bradford reagent was added to the reaction mixture. The
absorbance was read at 595nm and the amount of protein in the sample was determined by
interpolation from protein standard curve.

38

3.4.3 Protein Standard Preparation

Bovine serum albumin (BSA) (0.001 g) was dissolved in 10ml of distilled water to
give 100 g per ml of working solution. From the stock solution, 0, 0.2, 0.4, 0.6, 0.8 and 1 ml
was pipetted into test tubes and corresponding volume of distilled water was added to make
up each of the volume to 1.6 ml; then, 0.4 ml of Bradford reagent was added to the reaction
mixture. The absorbance was read at 595 nm. The absorbance at 595 nm was then plotted
against BSA concentration (g).
3.5

Production of Cellulase

3.5.1 Inoculation of the Basal Medium

The organism was cultured on modified liquid basal medium containing peptone (2%
w/v),

KH2PO4

(0.1%

w/v),

Mg2SO4.7H2O

(0.5%

w/v),

NaCl

(0.075%

w/v),

carboxymethylcellulose (0.2% w/v) and 0.1 M phosphate buffer (pH 7.0) (Kotchoni and
Shonukan, 2002). Aqueous suspension of pure bacterium isolate was made in sterile distilled
water and the medium containing 0.2% (w/v) CMC was inoculated with an aqueous
suspension of the organism from a 24 h old culture. The mixture was incubated at 37 o C for
48 h on a rotatory shaker at 140 rpm. This procedure was applied to the bacterial isolate with
the highest hydrolytic capacity value on the carboxymethylcellulose agar plates.
3.5.2 Growth and Enzyme Production
Aqueous suspension (1 ml) of 24 h old pure culture of the isolate was made in sterile
distilled water and used to inoculate 50 ml of sterile basal medium contained in a 250 ml
tightly closed Erlenmeyer flask and incubated for 24 h. This was in turn used to inoculate 150
ml of sterile basal medium contained in a 250 ml tightly closed Erlenmeyer flask. The culture
was incubated at 37o C for 48 h with moderate aeration in a water bath with shaker at 140
rpm. At two hours interval, Two milliliter samples were aseptically collected and the
turbidity of the culture was determined by measuring the increase in optical density at 680

39

nm with a spectrophotometer (Spectrumlab 25 model). The pH of the samples were also

determined.
3.6

Optimization of cellulase production medium

3.6.1 Optimum pH for Cellulase Production

The basal medium (50 ml each), with pH in the range 6.0 7.5 were inoculated with 1
ml of inoculums of DW-5 and incubated at 37 C for 48 h with agitation at 140 rpm. The
cells were thereafter removed by centrifugation and the activity of cellulase present in the
supernatant was quantified.
3.6.2 Optimum Temperature for Cellulase Production
The temperature optimal for cellulase production was determined by varying
incubation temperature of the culture from 30 40 C. Fifty millilitre of sterile production
medium was inoculated with One millilitre of inoculum of DW-5 and grown at the respective
temperature for 48 h with agitation at 140 rpm. The supernatant obtained was assayed for
cellulase activity.
3.6.3 Effect of Percentage Substrate Concentration on Enzyme Production
CMC in the percentage range of 0.1 0.25% was constituted in the production
medium in different Erlemeyer flasks and inoculated with one millilitre of inoculum of DW-5
The media was incubated at 37 C for 48 h and agitated at 140 rpm. The enzyme production
was determined as previously stated.
3.6.4 Effect of Some Nitrogen Sources on Enzyme Production
The production medium was composed with different nitrogen sources, which
included peptone, tryptic soy broth, gelatin, NH4Cl and NaNO3, at a constant quantity. The
pH of the medium previously adjusted to 6.5 was inoculated with 1 ml of the inoculum and
incubated for 48 h at 37 C with agitation at 140 rpm.

40

3.6.5 Effect of Some Carbon Sources on Cellulase Production

The effect of carbon sources was studied by replacing CMC with different sugars
(0.1%, w/v) sucrose, maltose and lactose while other media components were kept constant.
3.7

Enzyme Purification

3.7.1 Activation and Acetylating of Dialysis Bag.

The dialysis bag was first activated by boiling it in 1 mmol/L EDTA solution
containing 10 g/L sodium carbonate for 30 min. The bag was then filled and soaked with
distilled water for 24 h. The tube was then soaked in turns for 30 min each in 50% ethanol,
absolute ethanol, and diethylether respectively. It was then finally soaked in combined
mixture of benzene-acetic anhydride-pyridine reagent for 18 h (Whitaker et al.,1963). The
tube was then rinsed properly and kept in distilled water to be used for the dialysis process.
3.7.2

Dialysis
The cell free supernatant obtained from the culture medium was poured into treated

dialysis bag in batches and then dialyzed in a solution made up of 50 ml of glycerol and 50
ml of 0.1 M phosphate buffer pH 7.0. The dialysis was done for 8 h at room temperature.
3.7.3 Purification by Ion-Exchange Chromatography on CM-Sepharose CL-6B.
The dialysate obtained above was layered on a 1 cm x 10 cm column of CMSepharose CL-6B which had previously been equilibrated with 0.1 M phosphate buffer, pH
7.0. Fractions (2 ml) were collected at a flow rate of 12 ml/h and bound proteins were eluted
by stepwise elution with 0.5 and 1.0 M NaCl. Cellulase activity in the fractions and the
protein concentration were determined. Active fractions were pooled and dialyzed against
50% glycerol in the elution buffer.
3.7.4 Determination of Subunit Molecular Weight
The subunit molecular weight of purified cellulase was determined by sodium
dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) according to the method

41

of Weber and Osborn (1975) in 4% stacking gel and 15% separating gel. One hundred
microlitre (100 l) of the purified enzyme was mixed with 100 l 25mM Tris buffer
containing 4% SDS, 2M 2 mercaptoethanol, 40% v/v glycerol and 0.02% bromophenol
blue, pH 8.3. The mixture was boiled for 5 minutes and cooled prior to loading. An aliquot
(20 l) of the denatured enzyme and the low molecular weight calibration kit standard marker
proteins were loaded separately on slab gel. The proteins were allowed to stack at 100 V and
run at 150 V for 2 h in a Bio-Rad electrophoresis pack. The gel was fixed for an hour in a
fixing solution composed of 10 % (v/v) acetic acid and 40 % (v/v) methanol after which it
was stained for 4 h with a solution containing Coomasie brilliant blue R-250 (1.25 g), 227 ml
methanol, 46 ml glacial acetic acid and 227 ml of distilled water. The gel was, thereafter,
destained extensively using a solution of 5% methanol plus 7.5% glacial acetic acid in
distilled water.
3.7.5 Determination of Kinetic Parameters
The apparent kinetic parameters (Km and Vmax) were determined for the purified DW5 cellulase by incubating an aliquot of the enzyme with varied concentrations of CMC (6.5
11.5 mg/ml). The amount of reducing ends produced was estimated and the units of activity
were calculated.
3.7.6 Effect of temperature on activity of DW-5 cellulase
The effect of temperature on B. circulans cellulase was studied by incubating an
aliquot of the enzyme with the substrate at temperatures ranging from 30 700 C. The
enzyme activity was then assayed according the assay procedure earlier described.
3.7.7 Heat Stability of Cellulase from DW-5
This was carried out by assaying the enzyme at various temperatures ranging between
30 60 C for 60 min. The residual activities were expressed as a percentage of the activity

42

at the optimum temperature which was taken to be 100%. The percentage residual activity
was plotted against the various temperatures.
3.7.8 Effect of pH on Activity of DW-5 Cellulase
The effect of pH on B. circulans cellulase was performed in the pH range of 5.0 to
11.0. The optimum pH of the purified cellulase was determined by incubating the mixture
of the purified enzyme and the substrate in the presence of appropriate buffers; 50mM citrate
buffer (pH 4.0-6.0), 50 mM sodium phosphate (pH 7.0), 50 mM Tris-HCl (pH 8.0) and 50
mM glycine-NaOH (pH 9.0-11.0).
3.7.9 Effect of pH on Stability of DW-5 Cellulase
The purified enzyme was incubated at the various pH ranging from 4.0 to 11.0 for 60
min. The residual activity of each sample for hydrolysis of CMC was then assayed. The
residual activities were expressed as a percentage of the activity at the optimum pH which
was taken to be 100%. The percentage residual activity was plotted against the various pH.

43

CHAPTER FOUR
4.0

Results

4.1

Isolation of Cellulolytic Bacteria

Thirteen different bacteria were isolated from the decayed wood sample. Six of the

isolates showed a zone of clearance around the line of streak on Carboxymethylcellulose agar
(CMCA) plates (Fig. 4.1). All the isolates showing cellulolytic activity were characterized
(Table 4.1) while the isolate with the highest halozone was picked for further studies.
Based on the results of the biochemical and the physiological tests performed, the
bacterial isolate with the highest halozone was tentatively identified as Bacillus circulans
with the aid of Bergeys Manual of Determinative Bacteriology (Holt, 1994). (Table 4.1)
4.2

Growth of Bacillus circulans and changes in pH

The bacterium isolate with the highest carboxylmethyl hydrolysis capacity value was

considered for growth with respect to changes in pH. Exponential phase of growth was
observed till the twenty-eighth hour of incubation after which stationary phase was observed
(Fig. 4.2). There was continual increase in pH of the culture medium during the exponential
and the stationary phases of growth of the organism.
4.3

Optimization of Cellulase Production

The pH and temperature that gave the highest cellulase production were 6.5 and 370C
respectively (Figs. 4.3 and 4.4). At a higher pH, there was decrease in growth of the organism
and the enzyme activity The organism also had a lower growth and lower enzyme
productivity at a lower pH. Also at a lower and higher temperature, the enzyme activity
decreased. The optimum substrate concentration, nitrogen source, best carbon source were
0.15% Carboxymethylcellulose, Tryptic soy broth and maltose respectively (Figs. 4.5, 4.6
and 4.7).

44

Fig. 4.1: Screening for cellulose hydrolysis: Isolates 1, 3, 5, 6, 7 and 9 showed halozones.
Light yellow colour around the line of streak indicates the presence of halozone, this means
the isolate is cellulolytic.

45

Table 4.1: Biochemical characteristics of cellulolytic bacteria isolated from decayed wood
Isolate code

Gram

Spore

Catalase

Methyl

Vogues

Starch

stain

stain

test

red (MR)

Proskaeur

hydrolysis

6.5% NaCl

Citrate test

Gelatinase

Arabinose test

Probable organism

test

(VP)

DW-1

NA

NA

Bacillus azotoformans

DW-3

NA

NA

Bacillus larvae

DW-5

NA

NA

Bacillus circulans

DW-6

NA

NA

Bacillus circulans

DW-7

NA

NA

Streptococcus/enterococcus
sp

DW-8

NA

NA

NA

NA

NA

Bacillus circulans

DW9

NA

NA

Bacillus brevis

46

14

7,6

7,4

10
8

7,2
6
4

2
0

6,8
0

10

15

20

25

30

35

Time (hours)
Cell OD@680nm

pH

Fig. 4.2: Growth of Bacillus circulans isolated from decayed wood.

The OD of cell was read at 680 nm with corresponding pH of the aliquots determined using pH meter.

47

40

45

pH

OD@ 680nm (x10-1)

12

9,6

2,5
2,4
2,3
2,2

9,2

2,1
9

2
1,9

8,8

1,8
1,7

8,6

1,6
8,4

1,5
6

6,2

6,4

6,6

6,8

7,2

pH

Fig. 4.3: Profile of the effect of pH on cellulase production by Bacillus circulans

Optimum pH for cellulase production was found to be 6.5.

48

7,4

Activity (units/ml) X 10-1

OD@ 680 nm (x10-1)

9,4

cell OD @ 680nm
Activity (units/ml)

1,12

3,2

1,1

1,08

2,8

1,06

2,6

1,04

2,4

1,02

2,2

OD @ 680nm

Activity (units/ml)

3,4

Activity (units/ml)
cell OD @ 680 nm

0,98

30

32

34

36

38

40

42

Temperature 0C

Fig 4.4: Effect of temperature on the production of cellulase by Bacillus circulans. Production of cellulase was optimum at 37o C.

49

3,6

1,16

3,4

1,15

3,2

2,8

1,13

2,6

1,12

OD @680nm

Activity (units/ml)

1,14

Activity (units/ml)

cell OD @ 680 nm

2,4
1,11
2,2
1,1

2
1,8

1,09
0,1

0,12

0,14

0,16

0,18

0,2

0,22

0,24

0,26

% Substrate concentration

Fig. 4.5:

Effect of percentage substrate concentration on enzyme production and cell growth.

At 0.15% Carboxymethylcellulose concentration, the enzyme activity was found to be maximum, a change in this concentration brought about a
decrease or increase in cellulase production as well as the growth of the organism.

50

0,06
0,05

3
0,04

2,5
2

0,03

1,5

0,02

1
0,01

0,5

Protein conc. (mg/ml)

Enzyme activity (units/ml)

3,5

KEY
PEP: Peptone
TSB: Tryptic Soy Broth
GEL: Gelatin
AM CL: Ammonium
Chloride
SO NIT: Sodium Nitrate

ACTIVITY (units/ml)

0
PEP

TSB

GEL

AM CL

Protein(mg/ml)

SO NIT

Nitrogen sources

Fig. 4.6:

Effect of nitrogen sources on enzyme production. Tryptic soy broth showed the maximum activity.

51

Activity (units/ml) x 10-1

4
KEY
1: Sucrose
2: Maltose
3: Lactose
4: Carboxymethyl cellulose

3,5
3
2,5
2
1,5
1
0,5
0
1

Carbon sources

Fig. 4.7:

Effect of carbon sources on cellulase production by Bacillus circulans. Maltose proved to be the best carbon source.

52

4.4

Enzyme Purification

4.4.1 Dialysis
In other to concentrate the crude enzyme and reduce the supernatant volume,
ammonium sulfate precipitation was attempted but it led to total loss of enzyme activity,
therefore the process of dialysis against 50% glycerol was employed. The specific activity
was 5.61 while the percentage yield was 51.4%.
4.4.2 Purification by Ion Exchange Column Chromatography on CM Sepharose CL6B.
The enzyme did not bind to the column. A single peak (Fig. 4.8) of activity was
observed with a yield of 150.2% and the specific activity rose from 5.61 to 29.13 units/mg
protein. The purification fold of the cellulase was 4.37 (Table 4.2). The peak was pooled and
concentrated further by dialysis against 50% glycerol.
4.5

Molecular Weight Determination

The molecular weight was determined using SDS-PAGE (Fig. 4.9). The calibration

curve for the determination of the subunit molecular weight is shown in Fig. 4.10. Using slab
gel SDS-PAGE, the purified cellulase showed a single band and the molecular weight was
estimated to be 43 KDa.
4.6

Determination of Kinetic Parameters

Kinetic parameters of the purified cellulase of B. circulans were determined to be Km

of 1.061 1.17 mg/ml for CMC and its Vmax 13.75 1.51 g of glucose produced/ml/min.
The Lineweaver-Burk plot is shown in Fig. 4.11.
4.7

Effect of pH on Cellulase Activity

The effect of pH on the activity of cellulase is shown on Fig. 4.12. The pH of the reaction
mixture affected the activity of the purified cellulase. The optimum pH for the purified
cellulase was found to be 9.0. The cellulase activity increased with increase in pH up to 9.0

53

0,3

0,25

4
3

0,2
0,15

0,1

0,05

Protein conc. (mg/ml)

1M NaCl

0,35

0.5M
NaCl

Activity (g/ml)

Enzyme activity (g/ml)

Protein conc. (mg/ml)

0
0

10

15

20

25

30

35

Fraction number

Fig. 4.8: Elution profile of cellulase obtained from Bacillus circulans on CM-Sepharose CL 6B ion exchange column. This was carried
out at room temperature and a flow rate of 12ml/h. The enzyme did not bind to the resin, a single peak of activity was observed.

54

Table 4.2: Summary of the Purification of Cellulase Obtained from Bacillus circulans
Purification steps

Crude

Volume

Activity

Total

Protein

Total

Specific

(ml)

(units/ml)

units

(mg/ml)

protein (mg)

activity

Yield (%)

Purification fold

275

2.13

585.80

0.32

88

6.66

100

1.00

145

2.075

300.90

0.37

53.65

5.61

51.4

0.84

57

15.44

880.08

0.53

30.21

29.13

150.2

4.37

Dialysis against 50%

glycerol
CM-Sepharose CL-6B
followed by dialysis
against 50% glycerol

55

Fig 4.9: Photograph of SDS-PAGE of the purified cellulase from Bacillus circulans.
The pure enzyme was found to be 43 KDa after interpolation from the calibrate curve
showing the molecular weight of marker proteins.

56

Log of molecular weight

1,8

1,6

1,4

1,2

0,8
0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

Relative mobility (Rf)

Fig 4.10: Calibration curve for subunit molecular weight determination on SDS-PAGE.
The relative mobility (Rf ) was calculated by measuring the distance moved by each of the
band relative to that of the dye front. The Rf of the purified cellulase is indicated by the arrow

57

0,09
0,085

1/v (units/ml)-1

0,08
0,075
0,07
0,065
0,06
-0,15

-0,1

-0,05

0,05

1/[S]

0,1

0,15

0,2

(mg/ml)-1

Fig. 4.11: Lineweaver-Burk plot for the determination of kinetic parameters of purified
cellulase obtained from Bacillus circulans. A stock substrate concentration was prepared
out of which volumes of the substrate representing a range of concentration [S] was taken and
subjected to enzyme assay.

58

2,35

Enzyme activity (U/ml)

2,3
2,25
2,2
2,15
2,1
2,05
4

10

11

12

pH

Fig. 4.12: Effect of pH on purified cellulase of Bacillus circulans isolated from decayed
wood. The different buffers at the various pH was used to incubate the assay mixture.
Purified enzyme showed highest activity at pH 9.

59

except for pH 7.0 where there was decrease in activity. After the optimum pH there was
gradual decrease in activity up to pH 11
4.8

Effect of pH on Stability of Cellulase Activity

The activity of purified cellulase was relatively stable throughout the period of

incubation at the different pH values (Fig. 4.13). Only less than 20% of the residual activity
of the enzyme was lost both at a lesser pH and a higher pH than that of the optimum (100%).
4.9

Effect of Temperature on cellulase activity

The effect of temperature on purified cellulase is shown on Fig. 4.14. The activity of

purified cellulase was notably affected by the different temperatures of 30, 40, 50, 60, 70 0 C.
There was gradual increase in activity of the enzyme as the temperature was increase up to
500C after which there was decrease in activity of the enzyme.
4.10

Effect of Temperature on the stability of cellulase

There was relative stability in the enzyme activity at a lower temperature of 400C and

a higher temperature of 600C such that only less than 5% of the activity was lost (Fig. 4.15).
However, at a much lower temperature of 300C almost 50% of the enzyme activity was lost
in comparism with the optimum temperature

60

100

%Residual activity (U/ml)

90
80
70
60
50
40
30
20
10
0
4

10

11

12

pH

Fig 4.13: Effect of pH on stability of cellulase activity obtained from Bacillus circulans.
The enzyme was incubated for 60 min at various pH indicated before carrying out the assay.
The activity obtained for the optimum pH was taken as 100%.

61

Enzyme activity (U/ml)

7
6
5
4
3
2
1
0
25

35

45

55

Temperature

65

75

(0 C)

.
Fig. 4.14: Effect of Temperature on the activity of cellulase obtained from Bacillus
circulans. The enzyme was added to the reaction mixture and the reaction was carried out at
indicated temperatures. Purified enzyme showed highest activity at 50 C.

62

120

%Residual activity

100
80
60
40
20
0
30

35

40

45

50

Temperature

55

60

65

(0 C)

Fig. 4.15: Effect of Temperature on the stability of cellulase activity obtained from
Bacillus circulans. The enzyme was incubated for 60 min at indicated temperatures. Then
samples were measured under the same conditions for activity assay.

63

CHAPTER FIVE
5.0

DISCUSSION
Degradation of cellulosic materials is a complex process requiring participation by a

number of microbial enzymes. Habitats that contain these substrates are the best sources in
which to find these microorganisms. Decayed wood was the source used in this study. Wood
is a lignocellulosic material having 40-50% cellulose content. Many microorganisms
especially bacteria thrive successfully on fallen trees that have decayed over a period of time.
This is due to their ability to produce cellulose digesting enzymes which break down the
cellulose polymer into smaller units for easy utilization.
In this study, seven cellulolytic bacteria were isolated from decayed wood sample
obtained from Road 2 Area, Obafemi Awolowo University, Ile-Ife. Bacteria present an
attractive potential for the exploitation of cellulases due to their rapid growth rate, enzyme
complexity and extreme habitat variability (Maki et al., 2009). All the bacteria isolated were
characterized

and

identified

as

Bacillus

azotoformans,

Bacillus

larvae,

Streptococcus/enterococcus sp, Bacillus brevis and Bacillus circulans. Bacillus circulans

(DW-5) was found to have the highest potential for cellulase production as it showed the
highest halozone on CMCA plates. Enzyme productions by most microorganisms are growth
associated and usually is induced in the presence of its substrate in the culture medium.
Generally, enzyme production takes place during the log phase of microorganisms growth
and enzyme levels decrease towards the end of this phase or during the stationary phase. This
behavior may be due to catabolite repression by glucose and other readily metabolizable
monosaccharides released as the stationary phase continues or due to the rise in protease
levels in the environment or as a result of drastic change in pH levels in a bid for
microorganisms to conserve energy (Goyal et al., 2005). Bacillus circulans grew
logarithmically from the 2-30 h and then began to enter into stationary phase.

64

Media optimization is an important aspect to be considered in the development of

fermentation technology. Media requirement for optimum production of extracellular
cellulase by microorganisms may differ from one strain to another. Every microorganism has
its own peculiar physicochemical and nutritional requirements for cellulase production.
Bacillus circulans was able to produce cellulase using different substrates such as:
Lactose, Maltose, Carboxymethylcellulose and Sucrose. However, maltose gave the highest
cellulase production value in comparison to the other carbon sources including
carboxymethylcellulose, this is probably due to the fact that the compound is not a polymer
like carboxymethylcellulose and so could be metabolized faster. Also, it was found out that
the organism tested positive to starch hydrolysis during the process of characterization and it
is the same -1,4-glycosidic bonds that were present in both of them. Therefore, the ability of
the organism to produce cellulase in larger quantity using maltose as inducer as against
carboxymethylcellulose may suggest the possibility of the organism being more amylolytic
than cellulolytic. However, findings have revealed that molasses as well as cellulose
supported high cellulase productivity (Shabeb et al., 2010). Some investigators have shown
that agro-industrial residues such as rice bran, rice straw and sugar cane bagasse could be
used as substrates for cellulase production as in the case of Bacillus subtilis CBTK 106,
Bacillus subtilis BL62 and Bacillus pumillus (Heck et al., 2002 ; Poorna and Prema, 2007).
Also, a wide range of nitrogenous compounds, either organic or inorganic can affect
the productivity of cellulase (Shabeb et al., 2010). So, the effect of nitrogen sources on
cellulase production was studied using both organic and inorganic nitrogen sources such as
ammonium chloride, sodium nitrate, gelatin, tryptic soy broth and peptone. Maximum
cellulase production was observed using tryptic soy broth which is similar to the observation
of Ray et al. (2007) who reported that organic nitrogen sources were found to be more
suitable for optimizing cellulase production than inorganic sources. Other workers have also

65

found that maximum cellulase productivity was obtained by Bacillus pumilus BpCRI 6,
Pseudomonas fluorescens, Monascus purpureus and Streptomyces sp. BRC2 when tryptone
was added as an organic source (Bakare et al., 2005b; Chellapandi and Himanshu, 2008;
Daniel et al., 2008).
Effect of pH on cellulase production was also studied by culturing the organism at
various pH levels (6, 6.5, 7.0, 7.5). Enzyme activity was found to be high at pH 6.5 and this
was in accordance with the optimum pH for the production of cellulase by Bacillus sp. at pH
6.5 (Vijayarachavan and Vincent, 2012). Also, similar results were obtained with Bacillus
Licheniformis-1 (Dhillon et al., 1985) and Bacillus coagulans (Odeniyi et al., 2009).
The specific activity of cellulase in this study after it was produced in large quantity
was found to be 6.66 units/mg protein and this is higher than 0.421 and 0.093 units/mg
protein reported in Schinorhizobium fredii and the extreme thermophilic eubacterium
Rhodothermus marinus (Chen et al., 2004; Ayers et al., 1978). However, Lee et al. (2008)
reported a higher initial specific activity of 292.1 units/mg protein in Bacillus
amyoliquefaciens DL-3.
The harvested crude enzyme was concentrated by dialysis against 50% glycerol. This
is because an attempt to precipitate the protein using ammonium sulphate resulted in total
loss of enzyme activity. Pre-treated (acetylated) dialysis bag was used for the dialysis process
in other to prevent the cellophane bag from susceptibility to hydrolysis by the enzyme.
Purification was subsequently done using ion exchange chromatography on CM-Sepharose
CL-6B. The run on CM sepharose produced just one peak. This peak was pooled and
concentrated again by dialysis against 50% glycerol. This purification step followed by
dialysis against 50% glycerol yielded 150.2% of overall recovery with a specific activity of
29.13 units/mg protein. The high yield obtained may be due to the removal of enzyme

66

inhibitors during the purification process. The purification fold was 4.37 which is higher than
1.2 reported by Chen et al. (2004) for Sinorhizobium fredii.
The subunit molecular weight of purified cellulase as determined by SDS-PAGE was
43 KDa. This is in line with the report of Cole (1980) who reported values within the range of
20 to 60 KDa for cellulase from different cellulolytic organisms. Chen et al. (2004) reported
a higher molecular weight of 94 KDa for carboxymethylcellulase (CMCase) obtained from
Sinorhizobium fredii. Shoseyov and Doi (1990) reported a range of molecular weight of 17170 KDa for cellulase of different subunits obtained from Clostridium cellulovorans.
Molecular weight within the range of 20 KDa to 170 KDa has also been reported for
Clostridium cellulolyticum (Madarro et al., 1991).
The Km value of the purified cellulase was 1.061 1.17 mg/ml for CMC. The Km
value in this study is lower than that from thermophilic fungus M. thermophilia (3 mg/ml)
and the enzyme from T. aurantiacus. However, the Km is relatively higher than that of
Bacillus coagulans Co4 as reported by Adeleke et al. (2012). The Vmax value observed in this
study was 13.75 1.51 units/ml. The value is higher than 3.33 units/ml reported for
Pseudomonas fluorescens by Bakare et al. (2005a). At fixed enzyme concentration, an
increase in the concentration of the substrate will result in increase in enzyme activity until a
saturation point is reached beyond which enzyme activity decreases (Conn and Stumpf,
1976). Dixon and Webb (1971) suggested that under conditions of substrate saturation, the
substrate molecules tend to crowd on the enzyme and this could lead to the formation of
ineffective enzyme complexes.
The optimum temperature for the purified cellulase in this study was 50o C though the
enzyme was active at temperatures of 30 to 60o C, the enzyme was relatively inactive at 70o
C. The temperature optimum was similar to that of cellulase produced by Bacillus sp. and
that of Bacillus CH43 and HR68 (Vijayaraghavan and Vincent, 2012; Mawazda et al., 2000).

67

There was relative stability in the enzyme activity at a lower temperature of 40o C and a
higher temperature of 60o C such that only less than 5% of the activity was lost. However, at
a much lower temperature of 30o C almost 50% of the enzyme activity was lost in comparism
with the optimum temperature. Also, almost all the activity was lost at a much higher
temperature of 70o C. Usually, the initial rate of enzyme activity increases as temperature
rises and then decreases abruptly (Fructon and Simmonds, 1963). The initial increase in
activity has been ascribed to increase in kinetic energy which invariably increases collision
between reacting molecules (Murray et al., 1990). Sharp decline in the rate of reaction (as
observed in this study at 700C) could be associated with thermal inactivation of the enzyme
due to denaturation (Murray et al., 1990). At a critical temperature, an enzyme tends to lose
its compact three dimensional structure accompanied by a parallel loss of catalytic activity
(Murray et al., 1990). It has been suggested that the active sites of many enzymes may be the
region of highest instability in thermal denaturation of the enzyme since they are less readily
denatured in the presence of their substrates than in their absence. This phenomenon has been
attributed to the protective action of the substrate on the enzyme when it is bound to the
active centre (Dixon and Webb, 1971).
The purified cellulase had optimum activity at pH of 9.0 which is similar to what was
obtained from Bacillus sp. cellulase (Fukumori et al., 1985). However, many workers have
reported the optimum pH of cellulase to be around neutrality (7.0 - 7.5) as in the case of
cellulases produced by Sinorhizobium fredii, Bacillus amyoliquefaciens and Bacillus
cogulans Co4 (Chen et al., 2004; Lee et al., 2008; Adeleke et al., 2012). However, the
activity of purified cellulase was relatively stable throughout the period of incubation at the
different pH values. Only less than 20% of the residual activity of the enzyme was lost both
at a lesser pH and a higher pH than that of the optimum (100%). The enzyme displayed a
broad pH activity profile over a range of 5-11. Bacillus amyoliquefaciens cellulase also

68

showed activity in a pH range of 3.0-11.0 and retained more than 40% of the original purified
cellulase activity when maintained in a pH ranging from pH 4.0 to pH 9.0 (Lee et al., 2008).
The purified enzyme in this study can therefore be classified as an alkaline enzyme.
Enzymatic activities are usually sensitive to changes in pH values. This may be due to ionic
composition of the medium which may contribute to the stability of the enzyme.
In conclusion, this study has shown that there exists Bacillus species capable of
producing cellulase in decayed wood lignocellulosic wastes. The optimum pH and
temperature are essential characteristics of the cellulase purified from Bacillus circulans that
may make the enzyme suitable for industrial applications. Further biochemical, genetic and
biophysical works are required in order to engineer the enzyme for biotechnological and
industrial uses.

69

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APPENDIX
1
0,9
0,8

y = 7,9636x

OD @540nm

0,7
0,6
0,5
0,4
0,3
0,2
0,1
0
0

0,02

0,04

0,06

0,08

0,1

0,12

Glucose conc. (mg/ml)

Glucose standard curve: A stock concentration was prepared from which different
concentrations were calculated.

88

0,35
y = 0,0031x

Absorbance @ 595nm

0,3
0,25
0,2
0,15
0,1
0,05
0
0

20

40

60

80

100

BSA Conc. (g/ml)

Protein Standard curve: The same volume of Bradford reagent was added across the
various concentrations

89