OSUAGWU, UCHECHUKWU O. - University of Nigeria...Osuagwu, Uchechukwu O., a postgraduate student with...
Transcript of OSUAGWU, UCHECHUKWU O. - University of Nigeria...Osuagwu, Uchechukwu O., a postgraduate student with...
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EXTRACTION, PARTIAL PURIFICATION AND
CHARACTERIZATION OF CELLULASE FROM Aspergillus
fumigatus AND Aspergillus flavus IN SUBMERGED
FERMENTATION SYSTEM USING BREADFRUIT HULLS
Digitally Signed by: Content manager’s Name
DN : CN = Webmaster’s name
O = University of Nigeria, Nsukka
OU = Innovation Centre
Agboeze Irene E.
OSUAGWU, UCHECHUKWU O.
(PG/MSc/12/61930)
BIOLOGICAL SCIENCES
BIOCHEMISTRY
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TITLE PAGE
EXTRACTION, PARTIAL PURIFICATION AND CHARACTERIZATION OF
CELLULASE FROM Aspergillus fumigatus AND Aspergillus flavus IN SUBMERGED
FERMENTATION SYSTEM USING BREADFRUIT HULLS AS CARBON SOURCE
A DISSERTATION SUBMITTED IN PARTIAL FULFILLMENT OF THE
REQUIREMENT FOR THE AWARD OF THE DEGREE OF MASTER OF SCIENCE
(M.Sc) IN ENZYMOLOGY AND PROTEIN CHEMISTRY, UNIVERSITY OF
NIGERIA, NSUKKA
BY
OSUAGWU, UCHECHUKWU O.
(PG/MSc/12/61930)
DEPARTMENT OF BIOCHEMISTRY
UNIVERSITY OF NIGERIA
NSUKKA
DECEMBER, 2014
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CERTIFICATION
Osuagwu, Uchechukwu O., a postgraduate student with registration number
PG/M.Sc/12/61930 in the Department of Biochemistry has satisfactorily completed the
requirements for the research work for the degree of Master of Science (M.Sc.) in
Biochemistry. The work embodied in this report is original and has not been submitted in part
or full for any other diploma or degree of this or any other university.
PROF F. C. CHILAKA DR S. O. O. EZE
(Supervisor) (Supervisor)
______________________________
PROF OFC NWODO
(Head of Department)
EXTERNAL EXAMINER
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DEDICATION
This project is dedicated to my family members- Mr and Mrs Osuagwu, Nonye Osuagwu,
Uloma Ofole and Obioma Osuagwu.
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ACKNOWLEDGEMENT
I wish to express my gratitude to my supervisors- Prof. F. C. Chilaka and Dr. S. O. O. Eze for
their guidance and support during the course of this work. I have benefitted immensely under
their tutelage both at undergraduate and postgraduate levels.
I am also thankful to the Department of Biochemistry for providing me with the necessary
equipment which were used to carry out this work. I am equally indebted to the Lecturers in
the Department who at some point indicated interest in the work and encouraged me to carry
on. They include: Prof. OFC Nwodo, Prof. L. U. S. Ezeanyika, Prof. P. N. Uzoegwu, Prof.
I.N.E. Onwurah, Prof. O.U. Njoku, Dr. V. N. Ogugua, Prof. E.O. Alumanah, Prof. H. A.
Onwubiko, Dr. O.C. Enechi, Dr. C. A. Anosike, Dr. C. S. Ubani, Dr. V. E. O. Ozougwu, Mr.
P. A. C. Egbuna, Mr. O. Ikwuagwu, Dr. O. U. Njoku and Mr. C. C. Okonkwo. I am
especially grateful to Dr. P. E. Joshua for his guidance and input to the success of this project
work. I also appreciate the external examiner for taking out his time to examine this project
I am grateful to my friends and colleagues who contributed in one way or the other to make
this a successful venture- Cliff Victor, Onos Iruoghene, Christopher Ugwu, Angela
Igboanugo, Onyedika Aruma and many other members of my class. I also thank Arinze Linus
for the necessary support and encouragement throughout the duration of work.
Finally, I owe immense gratitude to my parents- Mr & Mrs C. O. Osuagwu, and to my
siblings- Nonye, Uloma and Obioma for their patience and kindness during the duration of
the work. God bless you all.
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ABSTRACT
Cellulase is a complex enzyme system commercially produced by filamentous fungi under
solid state or submerged cultivation. It has wide applications in textile, food and beverage
industry for effective saccharification process. In this study, Treculia africana (breadfruit)
hulls were used to induce cellulase production from Aspergillus flavus and Aspergillus
fumigatus grown under submerged fermentation conditions. Crude cellulase was harvested
after 5 days of growth with activities of 2.97 and 3.87 U/ml for enzymes produced by A.
flavus and A. fumigatus respectively. The protein concentrations for the crude enzymes were
found to be 4.03 and 4.17 mg/ml for A. flavus and A. fumigatus respectively. The enzymes
were then subjected to a two step purification process of ammonium sulphate precipitation
and gel filtration. Gel elution fractions were assayed for total cellulase activity. Two
prominent peaks indicating isoforms were observed. The isoforms were designated A and B
for enzymes of A. flavus and C and D for enzymes of A. fumigatus. These 4 fractions were
characterised separately. The pH optima of enzymes of A. flavus were 6.5 and 7.0
corresponding to the isoforms A and B with activities 3.31 and 3.53 U/ml respectively.
Enzymes from A. fumigatus had optimum pH of 5.0 for both isoforms C and D with
corresponding activities of 3.07 and 3.42 U/ml. The temperature optimum of enzymes of A.
flavus was 50⁰C with peak activities of 2.72 and 2.58 U/ml while enzymes of A. fumigatus
had maximum activities of 3.01 and 3.14 U/ml at a temperature of 55⁰C for both isoforms.
The Michaelis-menten constants Km, were 59.02, 47.67, 27.82 and 32 mg for isoforms A, B,
C and D respectively. Also, the maximum velocity, Vmax were 142.9, 166.7, 128.21 and
90.91 µmol/min for the isoforms A, B, C and D respectively. Using cellobiose as substrate,
Vmax values were 588.2, 476.2, 833.3 and 666.67 µmol/min for isoforms A, B, C and D
respectively. Km values of 7.7, 3.3, 11.1 and 9.1 mM were obtained for isoforms A, B, C and
D respectively.
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TABLE OF CONTENT
Title Page - - - - - - - - - - i
Certification- - - - - - - - - - ii
Dedication - - - - - - - - - - iii
Acknowledgement- - - - - - - - - - iv
Abstract- - - - - - - - - - - v
Table of Content- - - - - - - - - - vi
List of Figures - - - - - - - - - - vii
List of Tables - - - - - - - - - viii
CHAPTER ONE: INTRODUCTION AND LITERATURE REVIEW
1.1 Cellulose - - - - - - - - - - 2
1.1.1 Structure of cellulose - - - - - - - - 2
1.1.1.1 Chemical structure - - - - - - - - 2
1.1.1.2 Crystalline structure - - - - - - - - 4
1.1.2 Cellulose biosynthesis - - - - - - - - 6
1.1.3 Sources of cellulose - - - - - - - - 6
1.2 Breadfruit (Treculia africana)- - - - - - - 7
1.3 Cellulose hydrolysis - - - - - - - - 8
.1.3.1 Acid hydrolysis - - - - - - - - - 8
1.3.2 Enzyme hydrolysis - - - - - - - - 8
1.4 Cellulases - - - - - - - - - - 9
1.4.1 Cellulase classification - - - - - - - 10
1.4.2 Types of cellulases - - - - - - - - 11
1.4.3 Endoglucanase (EC 3.2.1.4) - - - - - - - 11
1.4.4 Exoglucanase (EC 3.2.1.91) - - - - - - - 11
1.4.5 β-glucosidase (EC 3.2.1.21) - - - - - - - 11
1.5 Mechanism of action of cellulases - - - - - - 12
1.6 Molecular biology of cellulase - - - - - - - 13
1.7 Cellulase production - - - - - - - - 15
1.7.1 Cellulase producing microoganisms - - - - - - 15
1.7.1.2 Mesophilic microorganisms - - - - - - - 19
1.7.1.3 Thermophilic microorganisms - - - - - - 22
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1.7.2 Aspergillus spp - - - - - - - - - 24
1.8 Fermentation methods - - - - - - - - 24
1.8.1 Solid-State fermentation (SSF) - - - - - - - 24
1.8.2 Submerged fermentation (SmF)/ Liquid fermentation (LF) - - - 24
1.9 Factors affecting cellulase enzyme production- - - - - - 25
1.9.1 Chemical Factors - - - - - - - - 25
1.9.1.1 Effect of carbon sources - - - - - - - 25
1.9.1.2 Effect of nitrogen sources - - - - - - - 27
1.9.1.3 Phosphorus sources - - - - - - - - 27
1.9.2 Physical factors - - - - - - - - - 27
1.9.2.1 pH - - - - - - - - - - 27
1.9.2.2 Temperature - - - - - - - - - 27
1.10 Applications of cellulases - - - - - - - 28
1.10.1 Cellulases in brewing and wine biotechnology - - - - 28
1.10.1.1 Beer brewing process - - - - - - - 28
1.10.1.2 Wine production - - - - - - - 29
1.10.2 Cellulases in pulp and paper biotechnology - - - - - 29
1.10.2.1 Biomechanical pulping - - - - - - - 30
1.10.2.2 Biodeinking - - - - - - - - - 30
1.10.3 Cellulases in textile and laundry biotechnology - - - - 31
1.10.3.1 Biostoning and biopolishing - - - - - - - 31
1.10.3.2 Laundry - - - - - - - - - 32
1.11 Aim of study and objectives - - - - - - - 33
1.11.1 Aim of study - - - - - - - - - 33
1.11.2 Specific objectives of the study - - - - - - 33
CHAPTER TWO: MATERIALS AND METHODS
2.1 Materials - - - - - - - - - 34
2.1.1 Reagents - - - - - - - - - - 34
2.1.2 Apparatus - - - - - - - - - 34
2.2 Method - - - - - - - - - - 35
2.2.1 Collection of breadfruit - - - - - - - - 35
2.2.2 Collection of microorganism - - - - - - - 35
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2.2.3 Preparation of ground breadfruit hulls - - - - - - 35
2.2.4 Storage of pure fungal isolates - - - - - - - 36
2.2.5 The Fermentation broth - - - - - - - - 36
2.2.6 Inoculation of the broth - - - - - - - - 36
2.2.7 Harvesting of the fermented broth - - - - - - 37
2.2.8 Mass production of enzyme - - - - - - - 37
2.2.9 Procedure for protein determination - - - - - - 37
2.2.10 Cellulase assay - - - - - - - - 38
2.2.10.1 Glucosidase (Cellobiase) assay - - - - - - 38
2.2.10.2 Endoglucanase (CMCase) assay - - - - - - 38
2.2.10.3 Total cellulase (Filterpaperase) assay - - - - - 39
2.2.11 Partial purification of protein - - - - - - - 39
2.2.11.1 Determination of percentage ammonium
sulphate saturation suitable for cellulase precipitation - - - 39
2.2.11.2 Ammonium sulphate precipitation of cellulase - - - - 40
2.2.11.3 Gel filtration - - - - - - - - - 40
2.2.12 Studies on partially purified enzyme - - - - - - 40
2.2.12.1 Enzyme progress curve - - - - - - - 40
2.2.12.2 Effect of pH change on total cellulase activity - - - - 40
2.2.12.3 Effect of temperature change on total cellulase activity - - - 41
2.2.12.4 Determination of kinetic parameters - - - - - - 41
2.2.12.5 Further studies with partially purified enzyme - - - - 41
CHAPTER THREE: RESULTS
3.1 Incubation period (pilot studies) - - - - - - - 42
3.2 Studies on crude enzyme - - - - - - - - 43
3.2.1. Protein concentration of crude enzyme- - - - - - 43
3.2.2 Cellulase activity of crude enzyme - - - - - - 44
3.3 Ammonium sulphate precipitation profile of cellulases- - - - - 45
3.4 Gel filtration (elution profile of cellulase enzymes) - - - - 46
3.5 Summary of purification steps - - - - - - - 48
3.6 Changes in protein concentration of partially purified enzymes- - - 50
3.6.1 Changes in protein concentration of enzymes from A. flavus. - - - 50
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3.6.2. Changes in protein concentration of enzymes from A. fumigates - - 51
3.7 Changes in total cellulase activity of partially purified enzymes - - - 52
3.7.1 Changes in total cellulase activity of
partially purified enzymes from A. flavus - - - - - 52
3.7.2 Changes in total cellulase Activity of
partially purified enzymes from A. fumigatus -- - - - - 53
3.8 Changes in specific activities of partially purified enzymes - - - 54
3.8.1 Changes in specific activities of partially purified enzymes from A. flavus - 54
3.8.2 Specific activities of partially purified enzymes from A. Fumigatus - - 55
3.9 Enzyme characterization - - - - - - - - 56
3.9.1 Enzyme progress curve - - - - - - - - 56
3.9.1.1 Enzyme progress curve of partially purified enzymes from A. flavus - 56
3.9.1.2 Enzyme progress curve of
partially purified enzymes from A. fumigatus - - - - 57
3.9.2 Effect of pH change on cellulase activity - - - - - 58
3.9.2.1 Effect of pH change on cellulase produced by A. flavus - - - 58
3.9.2.2 Effect of pH change on cellulase produced by A. fumigatus - - - 59
3.9.3 Effect of temperature change on cellulase activity - - - 60
3.9.3.1 Effect of temperature on cellulase produced by A. flavus - - - - 60
3.9.3.2 Effect of temperature on cellulase produced by A. fumigatus - - - 61
3.9.4 Determination of kinetic parameters (using filter paper as substrate) - - 62
3.9.4.1 Determination of kinetic parameters for enzymes of A. flavus - - - 62
3.9.4.2 Determination of kinetic parameters for enzyme of A. fumigates - - 64
3.9.5 Determination of kinetic parameters (using cellobiose as substrate) - - 66
3.9.5.1 Determination of kinetic parameters for enzymes of A. flavus - - 66
3.9.5.2 Determination of kinetic parameters for enzymes of A. fumigatus - - 68
CHAPTER FOUR: DISCUSSION
4.1 Discussion - - - - - - - - - 71
4.2 Conclusion - - - - - - - - - 76
4.3 Suggestions for further studies - - - - - - - 76
REFERENCES - - - - - - - - - 77
APPENDICES - - - - - - - - - 90
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LIST OF FIGURES
Fig. 1: Cellulose structure - - - - - - - - 4
Fig. 2: Crystalline forms of Cellulose I- - - - - - - 5
Fig. 3: Cellulose biosynthesis- - - - - - - - 7
Fig. 4: Breadfruit seed hulls - - - - - - - - 8
Fig. 5: Mechanism of cellulolysis - - - - - - - 13
Fig. 6: 3D structure of cellulase E4 from T. fusca- - - - - - 14
Fig. 7: A. flavus - - - - - - - - - 23
Fig. 8: A. fumigatus - - - - - - - - - 23
Fig. 9: Effect of incubation period on cellulase production - - - - 42
Fig. 10: Protein concentration of crude enzyme produced by the microoganisms- - 43
Fig. 11: Total cellulase, glucanase and cellobiase activities of crude enzyme - 44
Fig. 12: Ammonium sulphate precipitation profiles for celluluases
isolated from A. flavus and A. fumigatus.- - - - - - 45
Fig. 13a: Gel elution profile of proteins produced by A. flavus.- - - - 46
Fig. 13b: Gel elution profile of proteins produced by A. fumigatus - - - 47
Fig. 14: Changes in protein concentration after
partial purification of cellulases from A. flavus - - - - 50
Fig. 15: Changes in protein concentration after
partial purification of cellulases from A. fumigatus - - - - 51
Fig. 16: Changes in total cellulase activity after
partial purification of enzymes from A. flavus - - - - 52
Fig. 17: Changes in total cellulase activity
after partial purification of enzymes from A. fumigatus- - - - 53
Fig. 18: Changes in specific activity after partial
purification of cellulases from A. flavus.- - - - - - 54
Fig. 19: Changes in specific activity after partial
purification of cellulases from A. fumigatus- - - - - - 55
Fig. 20: Progress curve of cellulases isolated from A. flavus - - - - 56
Fig. 21: Progress curve of cellulases isolated from A. fumigatus - - - 57
Fig. 22: Effect of pH on cellulases produced by A. flavus- - - - - 58
Fig. 23: Effect of pH on cellulases produced by A. fumigatus- - - - 59
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Fig. 24: Effect of temperature on cellulases produced by A. flavus - - - 60
Fig. 25: Effect of temperature on cellulases produced by A. fumigatus - - 61
Fig. 26: Lineweaver-Burke plot of cellulase A
from A. flavus using filter paper as substrate- - - - - 62
Fig. 27: Lineweaver-Burke plot of cellulase B
from A. flavus using filter paper as substrate-- - - - - 63
Fig. 28: Lineweaver-Burke plot of cellulase C
from A. fumigatus using filter paper as substrate- - - - - 64
Fig. 29: Lineweaver-Burke plot of cellulase D
from A. fumigatus using filter paper as substrate- - - - - 65
Fig. 30: Lineweaver-Burke plot of cellulase A
from A. flavus using cellobiose as substrate- - - - - - 66
Fig. 31: Lineweaver-Burke plot of cellulase B
from A. flavus cellobiose as substrate - - - - - - 67
Fig. 32: Lineweaver-Burke plot of cellulase C
from A. fumigatus cellobiose as substrate - - - - - 68
Fig. 33: Lineweaver-Burke plot of cellulase D
from A. fumigatus cellobiose as substrate - - - - - 69
LIST OF TABLES
Table 1: Lignocellulose composition of several agricultural wastes - - - 7
Table 2: Some mesophilic cellulolytic Bacteria- - - - - - 15
Table 3: Some mesophylic cellulolytic fungi - - - - - - 18
Table 4: Some (hyper) thermophilic cellulolytic Bacteria and Archaea - - 20
Table 5: Some (hyper) thermophilic cellulolytic Fungi- - - - - 21
Table 6: Summary of purification steps of cellulase from A. flavus- - - - 48
Table 7: Summary of purification steps of cellulase from A. fumigatus - - 49
Table 8: Cellulase characterization table- - - - - - - 70
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CHAPTER ONE
INTRODUCTION
Enzymes are the catalysts of biological processes. Like any other catalyst, an enzyme brings
the reaction catalyzed to its equilibrium position more quickly than would occur otherwise.
An enzyme cannot bring about a reaction with an unfavourable change in free energy unless
that reaction can be coupled to one whose free energy change is more favourable (Nelson and
Cox, 2000). The activities of enzymes have been recognized for thousands of years.
However, only recently have the properties of enzymes been understood properly (Wolfgang,
2007). Indeed, research on enzymes has now entered a new phase with the fusion of ideas
from protein chemistry, molecular biophysics, and molecular biology which have given rise
to applications in fields ranging from agriculture to industry (Wolfgang, 2007).
The enzyme industry as we know it today is the result of a rapid development seen primarily
over the past four decades and thanks to the evolution of modern biotechnology (Ole et al.,
2002). Enzymes found in nature have been used since ancient times in the production of food
products, such as cheese, sourdough, beer, wine and vinegar, and in the manufacture of
commodities such as leather, indigo and linen (Ole et al., 2002). All of these processes relied
on either enzymes produced by spontaneously growing microorganisms or enzymes present
in added preparations such as calves’ rumen or papaya fruit. The development of
fermentation processes during the later part of the last century, aimed specifically at the
production of enzymes by use of selected production strains, made it possible to manufacture
enzymes as purified, well-characterized preparations even on a large scale (Wolfgang, 2007)
Microbial cellulases have shown their potential application in various industries including
pulp and paper, textile, laundry, biofuel production, food and feed industry, brewing and
agriculture. Due to the complexity of the enzyme system and immense industrial potential,
cellulases have been a potential candidate for research by both academic and industrial
research groups (Shang, 2013). The growing concerns about depletion of crude oil and the
emissions of greenhouse gases have motivated the production of bioethanol from
lignocellulose, especially through enzymatic hydrolysis of lignocellulosic materials (Bayer et
al., 2004; Himmel et al., 1999)
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1.1 Cellulose
Cellulose is a linear polymer of β-D-glucose units linked through 1,4-β-linkages with a
degree of polymerization ranging from 2,000 to 25,000 (Kuhad et al., 1997). Cellulose chains
form numerous intra- and intermolecular hydrogen bonds, which account for the formation of
rigid, insoluble, crystalline microfibrils (Golan, 2011). Natural cellulose compounds are
structurally heterogeneous and have both amorphous and highly ordered crystalline regions
(Morana et al., 2011). The degree of crystallinity depends on the source of the cellulose and
the highly crystalline regions are more resistant to enzymatic hydrolysis (Morana et al.,
2011). Cellulosic materials are particularly attractive because of their relatively low cost and
abundant supply. As the most abundant polysaccharide in nature, cellulose decomposition
plays not only a key role in the carbon cycle of nature, but also provides a great potential for
a number of applications, most notably biofuel and chemical production (Lynd et al., 2012).
The central technological impediment to more widespread utilization of this important
resource is the general absence of low-cost technology for overcoming the recalcitrance of
cellulosic biomass.
1.1.1 Structure of Cellulose
1.1.1.1 Chemical Structure
Payen first used the term cellulose for this plant constituent which is the most widespread
organic compound on Earth (Payen 1938; Guo et al., 2008]). The total amount of this
polysaccharide on our planet has been estimated at 7 × 1011 tons (Coughlan, 1985) and
constitutes the most abundant and renewable polymer resource available today. Cellulose is
an insoluble crystalline substrate, flavourless, odourless, hydrophilic, insoluble in water and
in most organic solvents, chiral, and with a wide chemical variability (Coughlan, 1985). It is
a structural component of the cell wall of green plants accounting for almost 33% of the total
biomass. It is also biosynthesized in other living systems such as Bacteria and Algae.
Cellulose produced by plants usually exists within a matrix of other polymers primarily
hemicellulose, lignin, pectin and other substances, forming the so-called lignocellulosic
biomass, while microbial cellulose is quite pure, has a higher water content, and consists of
long chains (Jagtap and Rao, 2005) . It is a carbohydrate polymer with formula (C6H10O5)n ,
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consisting of a linear chain of several hundred to over ten thousand 1,4-β-D-glucose units
linked through acetal functions between the equatorial -OH group of C4 and the C1 carbon
atom (Jagtap and Rao, 2005). The high stability of this conformation leads to a decreased
flexibility of the polymer, so this is usually described as a real tape.
There are two different types of intra- and one interchain hydrogen bonds in the structure, and
it has been considered that the intrachain hydrogen bonds determine the single-
chainconformation and the stiffness of cellulose, while the interchain hydrogen bond is
responsible for the sheetlike nature of cellulose (Watanabe and Tokuda, 2001; Klemm et al.,
2002; Klemm et al., 2005). The chains are arranged parallel to each other and form
elementary fibrils that have a diameter between 1.5 and 3.5 nm (microfibrils), the length of
the microfibrils is about of several hundred nm (Watanabe and Tokuda, 2001; Klemm et al.,
2002; Klemm et al., 2005) .
16
Fig. 1: Structure of Cellulose (Matheus et al., 2013)
1.1.1.2 Crystalline structure
The high degree of hydrogen bonds within and between cellulose chains can form a 3-D
lattice-like structure, while amorphous cellulose lacks this high degree of hydrogen bonds
and the structure is less ordered (Morana et al., 2011). The physical and chemical properties
of cellulose are defined by intermolecular interactions, cross-linking reactions, polymer
lengths, and distribution of functional groups on the repeating units and along the polymer
chains (Morana et al., 2011).
Initially, crystalline structure of native cellulose (cellulose I) has been studied by X-ray
diffraction and has been defined as monoclinic unit cells with two cellulose chains with a
twofold screw axis in a parallel orientation forming slight crystalline microfibrils (Gardner
and Blackwell, 2004; Klemm et al., 2005). Moreover, there are other types of crystal
17
structures: cellulose II, III, and IV (Gardner and Blackwell, 2004); the cellulose I, result the
less stable thermodynamically, while the cellulose II is the most stable structure (Klemm et
al., 2005). The cellulose I can turn into other forms using different treatments; for example,
by mercerization, using aqueous sodium hydroxide or dissolution followed by precipitation
and regeneration (formation of fiber and film) (O'Sullivan, 1997; Nishiyama et al., 2002).
However, additional information on the structure of noncrystalline random cellulose chain
segments are needed because it is very important for the accessibility and reactivity of the
polymer and the characteristics of cellulose fibers (Paakkari et al., 1989).
Fig. 2: Crystalline forms of Cellulose I (Matheus et al., 2013)
1.1.2 Biosynthesis of Cellulose
Cellulose is synthesized by a variety of living organisms, including plants, algae, bacteria,
and animals. It is the major component of plant cell walls with secondary cell walls having a
much higher content. The biosynthesis of cellulose essentially proceeds by the
polymerization of glucose residues using an activated substrate UDP-glucose (Saxena et al.,
200).
In plants, cellulose is synthesized on the plasma membrane by the enzyme cellulose synthase
that is present in the membrane. In the bacterium Acetobacter xylinum, the enzyme cellulose
synthase is present on the cytoplasmic membrane, and the cellulose is obtained
18
extracellularly. However, in other organisms, cellulose is found to be synthesized in other
regions of the cell. In the alga Pleurochrysis, cellulose scales are formed in the Golgi
apparatus and then deposited on the cell surface (Saxena et al, 2000).
The biosynthesis of Cellulose proceeds in at least two stages – polymerization and
crystallization. The first stage is catalyzed by the enzyme cellulose synthase, and the second
stage is dependent on the organization of the cellulose synthases possibly with other proteins
such that the glucan chains are assembled in a crystalline form (Saxena et al, 2000).
Fig. 3: Biosynthesis of Cellulose (Peter, 2008)
1.1.3 Sources of Cellulose
The plant cell wall is the major source of cellulose. Cellulose therefore abounds in
agricultural wastes of plant origin.
19
Table 1: Lignocellulose composition of several agricultural wastes
Lignocellulosic materials Cellulose (%) Hemicellulose(%) Lignin (%)
Hardwood 40-55 24-40 18-25
Softwood 45-50 25-35 25-35
Nut shell 25-30 25-30 30-40
Chestnut shell 27.4 10 44.6
Grape stalk 38 15 33
Corn stover 36.7 13.33 33
Wheat straw 30 50 15
Rice straw 32.1 24 18
Brewer‘s spent grain 16.8 28.4 27.8
Paper 85-99 0 0-15
Leaves 15-20 80-85 0
Cotton seeds hairs 80-95 5-20 0________
(Jorgensen et al., 2007)
1.2 Breadfruit (Treculia africana)
Treculia africana is a multipurpose tree species commonly known as African breadfruit. It
belongs to the family Moraceae and it grows in the forest zone, particularly the coastal
swamp zone (Agbogidi and Onomeregbor, 2008). African breadfruit is a traditionally
important edible fruit tree in Nigeria (Okafor, 1985) whose importance is due to the potential
use of its seeds, leaves, timber, roots and bark. It is increasingly becoming commercially
important in Southern Nigeria. Baiyeri and Mbah (2006) described African Breadfruit as an
important natural resource which contributes significantly to the income and dietary intake of
the poor. The seeds are used for cooking and are highly nutritious as pointed out by various
authors including; Okafor and Okolo (1974), Okafor (1990) and Onyekwelu and Fayose
(2007). However an important by-product of processing breadfruit seed is the seed hull (seed
coat or seed shell) and this may pose a risk to health as well as environment ( Atuanya et al.,
2012). These hulls are particularly rich in cellulose that could be harnessed for cellulase
production using microbes (Sonde and Odomelam, 2012)
20
Fig. 4: Breadfruit seed hulls (Atuanya et al., 2012)
1.3 Hydrolysis of Cellulose
Cellulose and can be hydrolyzed to sugars and microbially fermented into various products
such as ethanol or chemically converted into other products (Wyman, 1999). The primary
challenge is that the glucose in cellulose is joined by beta bonds in a crystalline structure that
is far more difficult to depolymerize than the alpha bonds in amorphous starch (Wyman,
1999). There are 2 broad based approaches to cellulose hydrolysis. These are acid based
hydrolysis and enzymatic hydrolysis.
1.3.1 Acid hydrolysis
When heated to high temperatures with dilute sulfuric acid, long cellulose chains break down
to shorter groups of molecules that release glucose that can degrade to hydroxymethyl
furfural (McParland et al., 1982). Generally, most cellulose is crystalline, and harsh
conditions (high temperatures, high acid concentrations) are needed to liberate glucose from
these tightly associated chains. Furthermore, yields increase with temperature and acid
concentration, reaching about 70% at 260 C (McParland et al., 1982). However, pyrolysis
21
and other side reactions become very important above about 220⁰C, and the amount of tars
and other difficult to handle by-products increases as the temperature is raised above these
levels (Brennan et al., 1986). In addition, controlling reaction times for maximum glucose
yields of only about 6 seconds at about 250ºC with 1% sulphuric acid presents severe
commercial challenges (Brennan et al., 1986).
1.3.2 Enzyme hydrolysis
Enzymatic hydrolysis has a potential to overcome many of the drawbacks of acid hydrolysis.
The conversion is carried out under mild conditions, thus greatly reducing the cost of
hydrolysis equipment. Sugars decomposition is avoided, thus eliminating this cause for loss
in yield (Hinz et al., 2009). Costly neutralization and purification equipment is unnecessary,
and disposal of waste streams from acid neutralization are eliminated. Balancing these
potential savings, extensive pre-treatment to breakdown to lignin and increase cellulase
accessibility is required to achieve good yields. The cost of high activity cellulolytic enzymes
is at very present very high (Wyman, 1999). Mutations and selection methods have been used
to develop and isolate Fusarium and Trichoderma strains of high cellulolytic activity
(Wyman, 1999).. In all, enzyme hydrolysis is a better alternative to industrial acid hydrolysis,
given the relatively low cost of enzyme hydrolysis and also higher yields of products
obtained via enzyme hydrolysis.
1.4 Cellulases
Cellulase refers to a class of enzymes produced chiefly by fungi, bacteria, and protozoans that
catalyze the cellulolysis (or hydrolysis) of cellulose (Golan, 2011). Cellulases catalyze the
hydrolysis of 1,4-β-D-glucosidic linkages in cellulose, and play a significant role in nature by
recycling this polysaccharide, which is the main component of the plant cell wall. Cellulases
work in synergy with other hydrolytic enzymes in order to obtain the full degradation of the
polysaccharide to soluble sugars, namely cellobiose and glucose, which are then assimilated
by the cell (Morana et al, 2011).
22
The enormous potential that cellulases have in biotechnology is the driving force for
continuous basic and applied research on these biocatalysts from fungi and bacteria.
Cellulases are found in many fields, such as animal feeding, brewing and wine, food, textile
and laundry, pulp and paper products. The growing interest in the conversion of
lignocellulosic biomass into fermentable sugars has generated an additional interest for
cellulases and their related enzymes (Morana et al, 2011).
1.4.1 Classification of Cellulase
Enzymes are designated according to their substrate specificity, based on the guidelines of the
international union of biochemistry and molecular biology (IUBMB). The cellulases are
grouped with many of the hemicellulases and other polysaccharidases as o-glycoside
hydrolases (EC 3.2.1.x) (Morana et al., 2011). Since the substrate specificity classification is
sometime little informative, because the complete range of substrates is only rarely
determined for individual enzymes, an alternative classification of glycoside hydrolases (GH)
into families based on amino acid sequence similarity has been suggested (Henrissat, 1991;
Henrissat and Bairoch, 1993; Henrissat and Bairoch, 1996). In addition, Henrissat et al.
(1998) have proposed a new type of nomenclature for glycoside hydrolases in which the first
three letters designate the preferred substrate, the number indicates the glycoside hydrolase
family, and the following capital letter indicates the order in which the enzymes were first
reported. For example, the enzymes CBHI, CBHII, and EGI of trichoderma reesei are
designated cel7a (CBHI), cel6a (CBHII), and cel6b (EGI).
Due to the great increase of identified glycoside hydrolases, Coutinho and Henrissat have
created an integrated database which is continuously updated (http://www.cazy.org/)
(Coutinho and Henrissat, 1999). At the 13 july 2010 update, glycoside hydrolases were
grouped into 118 families. In addition, 876 glycoside hydrolases have not yet assigned to a
family (glycoside hydrolase family ―non-classifiedǁ) because some of them display weak
similarity to established GH families, but they are too distant to allow a reliable assignment.
Cellulases are found in several different GH families (5, 6, 7, 8, 9, 12, 44, 45, 48, 51, 61, and
74), suggesting convergent evolution of different folds resulting in the same substrate
specificity (Morana et al., 2011). Gh family 9 contains cellulases from bacteria (aerobic and
anaerobic), fungi, plants and animals (protozoa and termites). Other families only group
23
hydrolases from a specific origin, as GH family 7 which contains only fungal hydrolases and
gh family 8 which contains only bacterial hydrolases. Cellulases from the same
microorganism can also be found in different families (e.g. The Clostridium thermocellum
cellulosome contains endoglucanases and exoglucanases from families 5, 8, 9, 44, and 48)
(Morana et al., 2011).
1.4.2 Types of cellulases
Cellulases, responsible for the hydrolytic cleavage of cellulose, are composed of a complex
mixture of enzymes with different specificities to hydrolyse glycosidic bonds. Cellulases can
be grouped into three major classes viz. Endoglucanase, exoglucanase and β-glucosidase.
1.4.2.1 Endoglucanase (EC 3.2.1.4)
Endoglucanases, often called carboxy methyl cellulases (CMCase), are proposed to initiate
random attack at multiple internal sites in the amorphous regions of the cellulose fiber to
open up sites for subsequent attack of cellobiohydrolases (Sunil et al., 2011). This action
results in a rapid decrease of the polymer length and in a gradual increase of reducing sugars
concentration (Morana et al., 2011)
1.4.2.2 Exoglucanase (EC 3.2.1.91)
Exoglucanase, better known as cellobiohydrolase, is the major component of the microbial
cellulase system accounting for 40-70% of the total cellulase proteins and can hydrolyse
highly crystalline cellulose. It removes mono-and dimers from the end of the glucose chain
(Sunil et al., 2011).
1.4.2.3 β-glucosidase (EC 3.2.1.21)
Β-glucosidase also known as cellobiase hydrolyses glucose dimers (cellobiose) and in some
cases cello-oligosaccharides to release glucose units (Sunil et al., 2011). These enzymatic
components act sequentially in a synergistic system to facilitate the breakdown of cellulose
and the subsequent biological conversion to β-glucose.
24
1.5 Mechanism of action of cellulases
Cellulolytic enzymes hydrolyze the 1,4-β-glycosidic bonds in cellulose, but they differ in
their specificities based on the macroscopic features of the substrate. They are progressive
enzymes when they interact with a single polysaccharide strand continuously, and non-
progressive types when they interact once and then, the polypeptidic chain disengages to
attack another polysaccharide strand (Morana et al., 2011) the enzymatic hydrolysis of
cellulose requires a carbohydrate binding module (CBM) that binds and arranges the catalytic
components on the surface of the substrate. Cellulases from fungi have a two-domain
structure with one catalytic domain, and one cellulose binding domain, that are connected by
a flexible linker. However, there are also cellulases that lack cellulose binding domain
(Morana et al., 2011)
The following are three types of reaction catalyzed by cellulases:
1) breakage of the non-covalent interactions present in the crystalline structure of
cellulose (endo-cellulase)
2) hydrolysis of the individual cellulose fibers to break it down into smaller sugars
(exo-cellulase)
3) hydrolysis of disaccharides and tetrasaccharides into glucose (beta-glucosidase).
25
Fig. 5: Mechanism of cellulolysis (Zhang et al., 2006).
1.6 Molecular biology of cellulase
Here, we shall briefly consider cellulase e4 from Thermomonospora fusca. T. Fusca is a
filamentous thermophilic soil bacterium and an important species degrading cellulose and
hemicelluloses in plant residues (Lykidis et al., 2007). Cellulase produced by T. Fusca is
unusual in that it has characteristics of both exo- and endo- cellulases (Sakon et al., 1997).
26
Fig. 6: The 3D structure of cellulase E4 from T. fusca ( Sakon et al., 1997)
The complete genome sequence shows that T. fusca has a single circular chromosome of
3,642,249 bp predicted to encode 3,117 proteins and 65 rna species with a coding density of
85% (Lykidis et al., 2007). Genome analysis reveals the existence of 29 putative glycoside
hydrolases in addition to the previously identified cellulases and xylanases. T. fusca has been
the source organism for isolating and studying multiple secreted cellulases and other
carbohydrate degrading enzymes (Hu and Wilson, 1988). Using classical biochemical
methods, six different cellulases have been identified. Four endocellulase genes (Hu and
Wilson, 1988) and two exocellulase (Irwin et al., 2000). In addition an intracellular β-
glucosidase and extracellular xyloglucanase have been cloned and characterized (Irwin et al.,
2000).
27
1.7 Production of Cellulase
Cellulases are well established in different industrial areas, and are currently the third largest
industrial enzyme worldwide, by dollar volume, mainly because of their use in cotton
processing and paper recycling, as detergent industry enzymes, and in juice extraction and
animal feeding additives as well (Nascimento and Coehlo, 2011). Here, we discuss cellulase
producing organisms, methods of fermentation and factors affecting enzyme production.
1.7.1 Cellulase Producing Microganisms
Microganisms involved in cellulase production will be grouped into two broad groups-
mesophilic microorganisms and thermophilic microorganisms.
1.7.1.1 Mesophilic microorganisms
Microorganisms growing best at moderate temperatures (between 10ºC and 45°C) are named
mesophiles. They represent the majority of microbial species on Earth, and their habitats
include the soil, the human body, the animals, etc. There are many mesophilic Bacteria and
Fungi that play a significant role in the carbon cycle on Earth, and there is increasing interest
in the enzymes from these microorganisms, since they have a key function in the conversion
of plant biomass into useful products (Morana et al., 2011).
28
Table 2. Some mesophilic cellulolytic Bacteria
___________________________________________________________________________ Microorganism Gram reaction Growth (°C) Growth conditions
Temperature
___________________________________________________________________________ Acetivibrio cellulolyticus - 37 Anaerobic
Bacillus megaterium + 30 Aerobic
Bacillus pumilus + 30 Aerobic
Bacteroides cellulosolvens - 35 Anaerobic
Butyrivibrio fibrisolvens + 37 Anaerobic
Cellulomonas fimi + 30 Aerobic
Cellulomonas fermentans + 30 Aerobic
Cellulomonas flavigena + 30 Aerobic
Cellulomonas gelida + 30 Aerobic
Cellulomonas iranes + 28 Aerobic
Cellulomonas persica + 28 Aerobic
Cellulomonas uda + 30 Aerobic
Cellvibrio mixtus - 20 Aerobic
Clostridium acetobutylicum + 37 Anaerobic
Clostridium cellulolyticum + 35-37 Anaerobic
Clostridium cellulofermentans - 40 Anaerobic
Clostridium cellulovorans - 37 Anaerobic
Clostridium herbivorans + 37 Anaerobic
Clostridium hungatei - 30 Anaerobic
Clostridium josui - 45 Anaerobic
Clostridium papyrosolvens - 25 Anaerobic
___________________________________________________________________________
(Morana et al., 2011)
Identification, purification and characterization of cellulases are continuously increasing and
always in progress, with incessant research and isolation of new microorganisms able to
produce novel cellulolytic activities. As an example, a bacterial strain, TR7-06(T), showing
high sequence similarity (98.5 %) to Cellulomonas uda DSM 20107(T), was isolated from
29
compost at a cattle farm near Daejeon, Republic of Korea. The isolated type strain of a novel
Cellulomonas species, named Cellulomonas composti sp. nov., possesses endoglucanase and
β-glucosidase activities (Kang et al., 2007). A microorganism capable of hydrolyzing rice
hull, one of the major cellulosic waste materials in Korea, was isolated from soil and
identified as Bacillus amyloliquefaciens DL-3 (Lee et al., 2008). Based on the characteristics
of this novel strain of Bacillus, Lee et al., (2008) aimed to develop an economical process for
production of cellulases by using cellulosic waste as inexpensive and widely distributed
carbon source. The new isolate produced an extracellular cellulase with an estimated
molecular mass of about 54.0 kDa. The deduced amino acid sequence of the cellulase from B.
amyloliquefaciens DL-3 showed high identity to cellulases from other Bacillus species, a
modular structure containing a catalytic domain of the GH family 5, and a cellulose-binding
module type 3 (CBM3). The purified enzyme was optimally active at 50°C and pH 8.0, and
showed broad thermal and pH stability ranging from 40 to 80°C and from 4.0 to 9.0,
respectively (Lee et al., 2008)
30
Table 3. Some mesophylic cellulolytic fungi
___________________________________________________________________________ Microorganism Growth Growth (°C) Growth conditions
Temperature
__________________________________________________________________________
Acremonium cellulolyticus 24 Aerobic
Anaeromyces mucronatus 37 Anaerobic
Aspergillus glaucus 30 Aerobic
Aspergillus niger 30 Aerobic
Aspergillus terreus 35 Aerobic
Caecomyces communis 37 Anaerobic
Ceratocystis paradoxa 20 Aerobic
Chrysosporium lucknowense 25-43 Aerobic
Cyllamyces aberensis 37 Anaerobic
Fusarium solani 25 Aerobic
Neocallimastix frontalis 37 Anaerobic
Neocallimastix patriciarum 37 Anaerobic
Orpinomyces sp. 37 Anaerobic
Penicillium funiculosum 24 Aerobic
Penicillium pinophilum 24 Aerobic
Phanerochaete chrysosporium 35 Aerobic
Piptoporus betulinus 25 Aerobic
Piromyces sp. 39 Anaerobic
Piromyces equi 39 Anaerobic
Pycnoporus cinnabarinus 24 Aerobic
Rhizopus oryzae 30 Aerobic
___________________________________________________________________________
(Morana et al., 2011)
Fungal cellulases are well-studied enzymes used in various industrial processes (Bhat, 2000).
A variety of aerobic and anaerobic Fungi are producers of cellulose-degrading enzymes. The
aerobic Fungi play a major role in the degradation of plant materials and are found on the
decomposing wood and plants, in the soil, and on the agricultural residues. The cellulase
31
systems of the aerobic Fungi Trichoderma reesei, T. koningii, Penicillium pinophilum,
Phanerochaete chrysosporium, Fusarium solani, Talaromyces emersonii, and Rhizopus
oryzae are well characterized (Bhat and Bhat, 1997). Much of the knowledge on enzymatic
depolymerization of cellulosic material has come from Trichoderma cellulase system. In
particular, the cellulase system of T. reesei (initially called T. viride) has been the focus of
research for 50 years (Reese and Mandels, 1971). A lot of work on cellulases has been
directed toward this fungus since it produces readily, and in large quantities, a complete set of
extracellular cellulases, and consequently, it has a high commercial value (Claeyssens et al.,
1998; Miettinen-Oinonen and Suominen 2002). In fact, T. reesei is capable of secreting more
than 30 g/L of protein into the extracellular medium (Conesa et al., 2001). It has been
reported that T. reesei possesses two CBH (cellobiohydrolase) genes, cbh1-2, and eight EG
(endoglucanase) genes, egl1-8, and that CBH I–II and EG I–VI are secreted proteins
(Foreman et al., 2003).
1.7.1.2 Thermophilic Microorganisms
The thermophilic microorganisms represent a unique group growing at temperatures that may
exceed 100°C. More precisely, thermophilic microorganisms thrive at temperatures from 65
to 85°C, and hyperthermophiles grow at temperatures of above 85°C (Morana et al., 2011).
Hyperthermophiles are microorganisms within the Archaea domain although some bacteria
are able to tolerate temperatures around 100°C. An extraordinary heat-tolerant
hyperthermophile is Methanopyrus kandleri, discovered on the wall of a black smoker from
the Gulf of California at a depth of 2000 m, at temperatures of 84-110°C. It can survive and
reproduce at 122°C (Takai et al., 2008). Thermophilic and hyperthermophilic
microorganisms have received considerable attention as sources of thermostable cellulolytic
enzymes, as the properties of these biocatalysts make them interesting candidates for
industrial applications. Running biotechnological processes at elevated temperatures has
many advantages. High temperature has a significant influence on the solubility of the
substrates (especially if viscous or polymers) and on the reaction rate. Moreover, problems of
microbial contamination can be avoided when a reaction is performed at elevated temperature
(Takai et al., 2008).
32
Table 4: Some (hyper) thermophilic cellulolytic Bacteria and Archaea
___________________________________________________________________________ Microorganism Gram reaction Growth Growth condition
Temperature (°C)
___________________________________________________________________________
Acidothermus cellulolyticus + 55 Aerobic
Alicyclobacillus acidocaldarius + 60 Aerobic
Anaerocellum thermophilum + 75 Anaerobic
Aquifex aeolicus - 85-95 Aerobic
Caldibacillus cellulovorans + 68 Aerobic
Caldicellulosiruptor saccharolyticus - 70 Anaerobic
Clostridium stercorarium + 65 Anaerobic
Clostridium thermocellum + 60 Anaerobic
Dictyoglomus thermophilus - 73 Anaerobic
Dictyoglomus turgidus - 72 Anaerobic
Pyrococcus abyssi - 96 Anaerobic
Pyrococcus furiosus - 98 Anaerobic
Pyrococcus horikoshii - 98 Anaerobic
(Morana et al., 2011)
Thermostable cellulases are of great biotechnological interest (Hongpattarakere, 2002). A
number of cellulolytic thermophilic Bacteria have been isolated, and many cellulose
degrading enzymes have been identified, characterized, cloned and expressed (Bergquist et
al., 1999). Conversely, screening of hyperthermophilic Bacteria for cellulose-degrading
enzymes has revealed that the presence of such enzymes is rather rare in this group. In
addition, among the Archaea, only the genus Pyrococcus and Sulfolobus have been found to
process thermoactive cellulases. Few aerobic thermophilic microorganisms have been
described to produce cellulases in comparison with the anaerobic ones. Acidothermus
cellulolyticus, isolated from 55-60°C acidic water and mud samples collected in Yellowstone
National Park, produces at least three thermostable endoglucanases (Mohagheghi, 1986). One
of them, E1 belonging to GH family 5, was crystallized, while properties and application of
33
the other enzymes are protected by patents (Sakon et al., 1996). The aerobic thermophilic
bacterium Rhodothermus marinus, isolated from a submarine hot spring at Reykjanes, NW
Iceland (Alfredsson et al., 1988), produces one higly thermostable cellulase (Cel12A) which
retains 50% activity after 3.5 h at 100°C (Hreggvidsson et al., 1996).
Table 5: Some (hyper) thermophilic cellulolytic Fungi
___________________________________________________________________________ Microorganism Growth Temperature (°C) Growth conditions
___________________________________________________________________________ Chaetomium thermophilum 45-55 Aerobic
Humicola grisea 45 Aerobic
Humicola insolens 40-50 Aerobic
Melanocarpus albomyces 45-55 Aerobic
(Morana et al., 2011)
Among the thermophilic Fungi, only a few number is described to be cellulase-producer The
thermophilic filamentous fungus Humicola sp. has been known to produce several cellulases,
and some of the genes have been cloned, sequenced and expressed (Takashima et al., 1996).
The cellulase system of the thermophilic fungus Humicola insolens possesses a battery of
enzymes that allows the efficient utilization of cellulose. This system, homologous to that of
T. reesei, contains five endoglucanases: EGI (Cel7B), EGII (Cel5), EGIII (Cel12), EGV
(Cel45A), and EGVI (Cel6B) in addition to two cellobiohydrolases: CBHI (Cel7A), and
CBHII (Cel6A) (Schulein, 1997).
The thermophilic fungus Chaetomium thermophile var. dissitum, was able to produce in the
culture medium all the enzymes involved in cellulose breakdown, namely endoglucanase
(41.0 kDa), exoglucanase (67.0 kDa) and β-glucosidase (Eriksen and Goksoyr, 1977). Lu et
al. (2002) reported that C. thermophile secreted in the culture medium a glycosylated
endocellulase with an apparent molecular weight of 67.8 kDa, as determined by SDS-PAGE.
The enzyme was optimally active at pH 4.0-4.5 and 60°C, and it retained 30% activity after
60 min at 70°C.
34
Melanocarpus albomyces, a rare true thermophilic Ascomycete capable of growing copiously
at 50°C, has been documented to produce high levels of endoglucanases under optimized
culture conditions (Jatinder et al., 2006). The endoglucanases from this fungus have been
recognized as potentially important in denim washing. In fact, the supernatant from M.
albomyces worked well in biostoning, with low backstaining. Three cellulases were identified
and purified to homogeneity, and two of them were endoglucanases with apparent molecular
masses of 20.0 kDa (Cel45A) and 50.0 kDa (Cel7A) (Miettinen-Oinonen et al., 2004).
The thermophilic fungus Thermoascus aurantiacus produces high levels of cellulase
components when grown on lignocellulosic carbon sources such as corncob and cereal straw
(Khandke et al., 1989). As these enzyme components are remarkably stable over a wide
range of pH and temperatures, they appear to have great commercial potential. A major
extracellular endoglucanase, with a molecular mass of 34.0 kDa, was purified and
characterized (Parry et al., 2002). It was optimally active at 70-80°C and pHs 4.0-4.4, and it
was stable at pH 5.2 and up to 60°C for 48 h. At 70°C and pH 5.2 the enzyme retained 40%
of the original activity after 48 h (Parry et al., 2002). The cellulase exhibited the highest
activity toward CMC; barley β-glucan and lichenan were also hydrolyzed, but the enzyme
was inactive on laminarin, confirming that it was an endoglucanase and was specific toward
β-1,4 linked polysaccharides (Parry et al., 2002).
1.7.2 Aspergillus spp
A large number of fungi have been reported in municipal solid waste. The Aspergillus sp.
was predominantly high among the other fungal species (Gautam et al., 2010). Most of the
work on fungal Cellulase is centered on the saccharification of cellulose by Aspergillus
(Milala et al., 2005) although Cellulase production on different carbon sources by Aspergillus
and other fungi have been reported (Ruijter and Visser, 1997).
Aspergillus is a genus consisting of several hundred mold species found in various climates
worldwide (Bennet, 2010). Aspergillus is a potential producer of cellulases (Mohammed et
al., 2005). The organisms are widespread in nature and are typically found in soil and
decaying organic matter, such as compost heaps, where they play an essential role in carbon
35
and nitrogen recycling (Bennet, 2010). The two species of aspergillus used for this work are
A. fumigatus and A. flavus.
Fig. 7: A. flavus (Winiati, 2013)
Fig. 8: A. fumigatus (Mirhendi, 2000)
36
1.8 Fermentation Methods
Fermentation is the technique of biological conversion of complex substrates into simple
compounds by various microorganisms such as bacteria and fungi. In the course of this
metabolic breakdown, they also release several additional compounds apart from the usual
products of fermentation, such as carbon dioxide and alcohol (Subramaniyam and Vimala,
2012). These additional compounds are called secondary metabolites. Secondary metabolites
range from several antibiotics to peptides, enzymes and growth factors (Machado et al.,
2004). Two broad methods will be considered for the production of cellulase enzyme. These
are solid state fermentation (SSF) and submerged fermentation (SmF) processes.
1.8.1 Solid-State Fermentation (SSF)
SSF utilizes solid substrates, like bran, bagasse, and paper pulp. The main advantage of using
these substrates is that nutrient-rich waste materials can be easily recycled as substrates
(Subramaniyam and Vimala, 2012). In this fermentation technique, the substrates are utilized
very slowly and steadily, so the same substrate can be used for long fermentation periods
(Subramaniyam and Vimala, 2012). Hence, this technique supports controlled release of
nutrients. SSF is best suited for fermentation techniques involving fungi and microorganisms
that require less moisture content. However, it cannot be used in fermentation processes
involving organisms that require high water activity (aw), such as bacteria. (Babu and
Satyanarayana, 1996).
1.8.2 Submerged Fermentation (SmF)/Liquid Fermentation (LF)
SmF utilizes free flowing liquid substrates, such as molasses and broths. The bioactive
compounds are secreted into the fermentation broth. The substrates are utilized quite rapidly;
Hence, they need to be constantly replaced/supplemented with nutrients (Subramaniyam and
Vimala, 2012). This fermentation technique is best suited for microorganisms such as bacteria
that require high moisture content. An additional advantage of this technique is that
purification of products is easier (Subramaniyam and Vimala, 2012). SmF is primarily used in
37
the production of secondary metabolites that need to be used in liquid form. Submerged
fermentation method for enzyme production is usually preferred since the enzyme will be
obtained in liquid form thus making for easy purification and characterization.
1.9 Factors Affecting Cellulase Enzyme Production
1.9.1 Chemical Factors
1.9.1.1 Effect of Carbon Sources
Since any cellulose biotechnological process is likely to base on crude enzymes, it is
important to increase their activities in the culture supernatants by selecting the best carbon
and nitrogen sources and optimizing their concentrations (Gomes et al., 2000). Cellulase
production is dependent on the nature of the carbon source used in the culture medium.
Various lignocellulose carbon sources have been tested for their ability to induce cellulase
production. Besides, the efficiency of enzyme production also depends on the bare chemical
composition of the raw material, accessibility of various components and their chemical and
physical associations. Wheat straw, rice straw and corn stover have been known as ideal
substrate for cellulose production (Panagiotou et al., 2003; Mishra and Nain, 2010). Several
investigations have indicated that cellulases are inducible enzymes, and different carbon
sources have been used to find their role in effecting the enzymatic levels. Cellobiose (2.95
mM) may act as an effective inducer of cellulases synthesis in Nectria catalinensis (Pardo
and Forchiassin, 1999). An increased rate of endoglucanase biosynthesis in Bacillus sp. was
reported in the presence of cellobiose or glucose (0.2%) in the culture medium (Paul and
Verma, 1990). Yeoh et al. (1986) had reported the inhibition of β-glucosidase activity at
higher concentrations of cellobiose to an extant of 80%; similarly, laminaribiose and glucose
also led to a 55–60% reduction in the enzymatic activity. Later, Shiang et al. (1991)
described a possible regulation mechanism of cellulose biosynthesis and proposed that sugar
alcohols, sugar analogues, xylose, glucose, sucrose, sorbose, cellobiose, methylglucoside etc.
at a particular concentration may induce a cellulose regulatory protein called cellulase
activator molecule (CAM). The level and yield of CAM get affected possibly due to substrate
concentration and some unknown factors imparted by moderators. Many different agro-
38
industrial wastes, synthetic or natural, have been examined as the carbon source for the
process. Among the cellulosic materials, sulfate pulp, printed papers, mixed waste paper,
wheat straw, paddy straw, sugarcane bagasse, jute stick, carboxymethylcellulose, corncobs,
groundnut shells, cotton, ball milled barley straw, delignified ball milled oat spelt xylan, larch
wood xylan, etc. have been used as the substrates
for cellulase production (Singh et al., 1991; Mishra and Nain, 2010). The observations
indicated that the production of cellulases increased with increase in substrate concentration
up to 12% during solid-state-fermentation using Aspergillus niger. Further increase in
substrate concentration decreased the production levels. This might have been due to
limitation of oxygen in the central biomass of the pellets, and exhaustion of nutrients other
than energy sources. Martins et al. (2008) and Steiner et al. (1993) also demonstrated that
carboxymethycellulose or cereal straw (1%, w/w) would be the best carbon source compared
to sawdust for CMCase and β-glucosidase production using Chaetomium globosum as the
producer organism. In contrast, 3% malt extract or water hyacinth was found optimum for
CMCase, FPase and β-glucosidase as observed with lactose as an additional carbon sources
(Mukhopadhyey and Nandi, 1999). However, the saccharification of alkali-treated bagasse at
higher substrate levels of 4% w/v was also reported (Singh et al., 1991). Interestingly, higher
concentrations (2.5–6.2% w/v) of carbon source were observed to be suitable for maximum
saccharification when cellobiose was supplemented in the medium containing delignified rice
straw, news print or other paper wastes as substrates ( Ju and Afolabi, 1999).
1.9.1.2 Effect of Nitrogen Sources
The effects of nitrogen sources on cellulase production are variable with respect to the fungi
used (Kachlishvili et al., 2006). Enzyme production is affected significantly under different
concentrations of nitrogen sources (Panagiotou et al., 2003). With different nitrogen sources,
enzyme activities are higher with organic nitrogen (Gao et al., 2008). Maximum cellulase
activity has been obtained with yeast extract (Gao et al., 2008), though other researchers
found that inorganic nitrogen sources produce an optimal result (Kalogeris et al., 2003). The
effect of different inorganic nitrogen sources such as ammonium sulfate, ammonium nitrate,
ammonium ferrous sulfate, ammonium chloride and sodium nitrate have been studied.
Among these, ammonium sulfate (0.5 g /L) led to maximum production of cellulases (Singh
et al., 1991). In contrast, Menon et al. (1994) observed a significant decrease in enzymatic
39
levels in the presence of ammonium salts as the nitrogen source. However, an increase in the
level of β-glucosidase was reported when corn steep liquor (0.8% v/v) was added into the
production medium. Corn steep liquor also resulted in 3-5 fold induction of endo- and
exoglucanase levels with synthetic cellulose, wheat straw and wheat bran as the substrates.
Enzyme production was sensitive to corn steep liquor (0.88 g/L), and production increased
significantly when mixed nitrogen sources (corn steep liquor and ammonium nitrate) were
supplied (Steiner et al., 1993). However, additional incorporation of nitrogen sources into
medium scale up the cost of the process (Sunil et al., 2011).
1.9.1.3 Phosphorus Sources
Phosphorus is an essential requirement for fungal growth and metabolism. It is an important
constituent of phospholipids involved in the formation of cell membranes. Besides its role in
linkage between the nucleotides forming the nucleic acid strands, it is involved in the
formation of numerous intermediates, enzymes and coenzymes essential in carbohydrate
metabolism, other oxidative reactions and intracellular processes (Singh et al., 1991).
Different phosphate sources such as potassium dihydrogen phosphate, tetra-sodium
pyrophosphate, sodium β-glycerophosphate and dipotassium hydrogen phosphate have been
evaluated for their effect on cellulases production (Garg and Neelkantan, 1982). It has been
widely known that potassium dihydrogen phosphate is the most favorable phosphorus source
for cellulase production (Sunil et al., 2011)
1.9.2 Physical Factors
1.9.2.1 pH
Different physical parameters influence the cellulose bioconversion, and pH is an important
factor affecting cellulase production (Pardo and Forchiassin, 1999). The effect of pH on
cellulase production has been analysed using Aspergillus niger, and found that pH 5.5 was
optimal for maximum cellulase production. On other side, the pH range of 5.5–6.5 was
optimal for β-glucosidase production from Penicillium rubrum (Menon et al., 1994). Eberhart
et al. (1977) has reported that production and release of cellulase from Neurospora crassa
depends on pH of the medium and maximum release occurs at pH 7.0, whereas the enzyme
40
remained accumulated in the cell at pH 7.5. Similarly, pH 7.0 is suitable for extracellular
production of cellulase from the Humicola fuscoatra (Rajendran et al., 1994). The adsorption
behavior of cellulases has been found to be affected by pH of the medium. Kim et al. (1988)
had reported maximum adsorption of cellulase from Aspergillus phoenicus at pH 4.8–5.5.
The pH range 4.6–5.0 has been found suitable for CMCase, filterpaperase (FPase) and β-
glucosidase production with Aspergillus ornatus and Trichoderma reesei (Mukhopadhyey
and Nandi, 1999).
1.9.2.2 Temperature
Temperature has a profound effect on lignocellulosic bioconversion. The temperature for
assaying cellulase activities is generally within 50–65 °C for a variety of microbial strains
(Menon et al., 1994; Steiner et al., 1993), whereas growth temperature of these microbial
strains was found to be in the 25–30 °C (Macris et al., 1989). Similarly Penicillium
purpurogenum, Pleurotus florida and Pleurotus cornucopiae show higher growth at 28 °C
but maximum cellulase activities at 50 °C (Steiner et al., 1993) and about 98, 59 and 76% of
the CMCase, FPase and β-glucosidase activities, respectively, retained after 48 h at 40 °C.
Researchers have shown that temperature influences the cellulose-cellulase adsorption
behaviour. A positive relationship between adsorption and saccharification of cellulosic
substrate was observed at temperature below 60 °C. The adsorption activities beyond 60 °C
decreased possibly because of the loss of enzyme configuration leading to denaturation of the
enzyme activity (Van-Wyk, 1997). Bronnenmeier and Staudenbauer (1988) reported that
extracellular as well as cell bound β-glulcosidase from Clostridium stercorarium required an
identical temperature of 65 °C for their activity. Further increase in temperature led to a sharp
decrease in the enzyme activity. Some of the thermophilic fungi having maximum growth at
or above 45–50 °C produce cellulase with wide temperature optima (50–78 °C) (Wojtczak et
al., 1987).
41
1.10 Applications of Cellulases
1.10.1 Cellulases in Brewing and Wine Biotechnology
The macerating enzymes, comprising cellulases, hemicellulases and pectinases, hydrolyze the
plant cell wall and, consequently, can be used in brewing and wine biotechnology to improve
the quality of finished products and avoid the use of chemicals. Enzyme preparations are used
in the brewing and distilling industries to decrease the viscosity of the mash and to improve
the overall efficiency of the process. In fact, cellulolytic and hemicellulolytic enzymes allow
the conversion of undigestible lignocellulosic biomass into fermentable sugars, with
consequent increase of alcohol yield.
1.10.1.1 Beer Brewing Process
Barley is the most common cereal used for the production of beer although wheat, corn, and
rice are also widely used. The main processes involved in beer production include milling to
reduce the size of the dry malt in order to increase the availability of the carbohydrates;
mashing where water is added to the malt; lautering where spent grains are removed from the
wort, boiling of the wort with flavouring hops, fermentation of the wort liquor, maturation,
conditioning, filtration and packaging of the final product. The high concentration of β-
glucan in the brewing process, resulting from unsuitable brewing process or low quality
barley, produces high viscosity of beer, formation of gelatinous precipitate, decrease of the
extract yield, and lower run-off of wort (Bamforth, 1994; Bhat, 2000; Guo et al., 2010). In
brewing process, cellulases are used during the mashing stage in order to hydrolyze excess β-
glucans and reduce the viscosity, thus improving the separation of the wort from the spent
grains. Oksanen et al. (1985) observed that the endoglucanase and the cellobiohydrolase from
the Trichoderma cellulase system produced a large reduction of the degree of polymerization
of the β-glucans, and wort viscosity. Moreover, the increased addition of enzymes used
resulted in improved filtering. A. niger, T. reesei, and P. funiculosum, which are generally
recognized as food grade microorganisms, are the major source of cellulases currently used in
the mashing step, as these enzymes provide technological benefit to beer manufacture
(Karboune et al., 2008).
42
1.10.1.2 Wine Production
Wine manufacture is a biotechnological process in which yeast cells and enzymes are
indispensable for ensuring a high quality product. The use of cellulases, hemicellulases and
pectinases during wine making, allows a better skin maceration, and superior color
extraction, particularly important in the production of red wine; in addition, it improves
clarification, filtration, and the overall quality and stability of the wine (Galante et al., 1998).
Pectinase preparations, used in wine making, were lately modified by addition of cellulases
and hemicellulases in small quantities to realize a more complete breakdown of the cells with
consequent fruit liquefaction in a moderately short time period (Plank and Zent, 1993). It has
also been demonstrated that the mixture of macerating enzymes worked better than pectinases
alone in grape processing (Haight and Gump, 1994).
1.10.2 Cellulases in Pulp and Paper Biotechnology
1.10.2.1 Biomechanical Pulping
Mechanical pulping process is electrical energy intensive and results in low paper strength.
Biomechanical pulping, defined as the enzymatic treatment of lignocellulosic materials
before the mechanical pulping step, has shown at least 30% savings in electrical energy
consumption, and significant improvements in paper strength properties. The potential of
enzymatic treatments has been assessed and the processes have proved successful (Gubitz et
al., 1998). Utilization of cellulases from fungal sources (T. reesei, Aspergillus sp.) (Buchert
et al., 1998; Suurnakki et al., 2000) saves 33% electrical energy and significantly improves
paper strength properties. A cellulase preparation produced by the ascomycete fungus
Chrysosporium lucknowense for using in the pulp and paper industry represents, at present,
an attractive alternative to the well-known cellulases from Fungi like Aspergillus sp. and T.
reesei for protein production on a commercial scale (Bukhtojarov et al., 2004; Hinz et al.,
2009).
43
1.10.2.2 Biodeinking
All over the world people give more attention to the environment and so, the recycle of waste
paper has to be considered also as a necessity for the protection of forest and economy. Paper
mill will gain profit from the utilization of recycled fiber, since it is profitable to decrease
pollution, cost, and investment. Conventional deinking technology with alkali is
characterized by a low efficiency on laser printed paper and is not considered
environmentally friendly. Consequently, researchers have concentrated their attention on new
deinking technologies (Moon and Nagarajan, 1998). The principle of enzymatic deinking is
based on the weakening of the connections between toner and fibers due to the enzyme attack
with separation of toner particles from fibers (Yingjuan et al., 2005; Shufang et al., 2005).
The enzymatic deinking allows us to avoid the use of alkali; moreover, using enzymes at
acidic pH it is possible to prevent the yellowing, modify the distribution of the ink particle
size, improve fiber brightness strength, pulp freeness and cleanliness, reduce fine particles
and reduce environmental pollution. Until 2000, the use of enzymes to perform biodeinking
was only investigated at the laboratory scale (Buchert et al., 1998; Bhat, 2000). Subsequently,
a mixture of cellulase, lipase, and amylase was employed in biodeinking process at industrial
level (Morbak and Zimmermann, 1998). The effect of combined deinking technology with
ultrasounds, UV irradiation and enzyme on laser printed paper was investigated. The results
confirmed that the dose of alkali can be reduced using biodeinking technology. Cellulases
from different microorganisms such as A. niger, T. reesei, Humicola insolens, Myceliophtora
fergusii, Chrysosporium lucknowense, Fusarium sp. were used for this purpose (Marques et
al., 2003).
1.10.3 Cellulases in Textile and Laundry Biotechnology
Since the early part of the last century, enzymes such as the cellulases have been used for a
wide range of applications in textile processing in replacement of the traditional methods.
44
1.10.3.1 Biostoning and Biopolishing
Jeans manufactured from denim are one of the world's most popular clothing items. In the
late 1970s and early 1980s, industrial laundries developed methods for producing faded jeans
by washing the garments with pumice stones, which partially removed the indigo dye
revealing the white interior of the yarn, which leads to the faded, worn and aged appearance.
This process was designated as ―stone-washing (Cavaco- Paulo, 1998). The use of 1-2 kg
stones per kg of jeans for 1 h during stone-washing met the market requirements, but caused
several problems including rapid consumption of washing machines, and unsafe working
conditions. As an alternative to the stone-washing, biostoning is by far the most economical
and environmental friendly way to treat denim. The cotton fabrics treated with the enzymes
loose the indigo, which later is easily removed by mechanical abrasion in the wash cycle
(Cavaco- Paulo, 1998; Yamada et al., 2005). The substitution of pumice stones by an
enzymatic treatment has many advantages: washing machines lower consumption and
elevated productivity, short treatment times and less intensive working conditions. Moreover,
it is possible to operate in a more safe environment because pumice powder is not produced,
and the process can be mechanized controlling, with the use of computer, the dosing devices
of liquid cellulase preparations (Bhat, 2000).
In the textile wet processing, the biopolishing is usually carried out with desizing, scouring,
bleaching, dyeing and finishing by utilization of cellulases. However, there are no clear
indications about the best cellulase mixture to use. (Miettinen-Oinonen and Suominen, 2002).
The use of these enzymes allow many improvements such as the removal of short fibers,
surface fuzziness smooth, polished appearance, more color uniformity and brigthness,
improved finishing, and fashionable effects. At last, due to increasing environmental
concerns and constraints being imposed on textile industry, cellulase treatment of cotton
fabrics is an environmentally friendly way of improving the property of the fabrics. In 2007,
Anish et al. (2006) isolated an endoglucanase from the alkalothermophilic bacterium
Thermomonospora sp. The enzyme which is used for denim biofinishing under alkaline
conditions, was effective in removing hairiness with negligible weight loss and imparting
softness to the fabric. Higher abrasive activity with lower back-staining was a preferred
property for denim biofinishing exhibited by the Thermomonospora endoglucanase.
45
1.10.3.2 Laundry
The most important reason to use enzymes in detergents is that they are biodegradable and a
very small quantity of these inexhaustible biocatalysts can replace very large quantity of
chemicals. Since detergents hold ionic and anionic surfactants, and bleaching agents
(oxidizing agents) that can partially or completely denature proteins, the enzymes for laundry
must be resistant to anionic surfactants and oxidizing agents. The accumulation of
microfibrils on the surface of the fabrics makes the fabrics look hairy and scatters incident
light, thereby lessening the brightness of the original colors. In detergent industry, cellulases
are used to remove microfibrils from the surface of cellulosic fabrics, enhancing color
brightness, hand feel and dirt removal from cotton garments that during repeated washings
can become fluffy and dull.
Other notable applications of cellulase are found in the treatment of wastes, production of
biofuels and also in the animal feed industry.
1.11 Aim and Objectives of study
1.11.1 Aim of study
This study is aimed at using microorganisms cultivated on agricultural waste to produce
cellulase enzyme with industrial potential.
1.11.2 Specific Objectives of the Study
This work is therefore designed to achieve the following specific objectives:
• Isolation of crude cellulase secreted by Aspergillus fungi.
• Determination of the protein content of the enzyme.
• To Assay for activity of the cellulase enzymes
• Partial purification of cellulase.
• Characterization of purified cellulase.
46
CHAPTER TWO
MATERIALS AND METHODS
2.1 Materials
2.1.1 Reagents
Chemicals/Regents Manufacturer
Bovine serum albumin (BSA) Sigma Chemical Company (USA)
Folin –Ciocalteau Sigma-Aldrich (USA
Ammonium sulphate British Drug House (BDH) Chem. Ltd (USA)
Tris HCL salt Merek specialist Private limited (Mumbai)
Sephadex G- 50 Sigma Chemicals Company Limited (USA)
All other chemicals used in this work were of analytical grade and were obtained from
reputable sources. Distilled water was used for all preparations of solutions and pH
measurements were made at room temperature using a pH meter.
2.1.2 Apparatus
Weighing balance: Ohaus Dial-O- Gram, Ohaus Cooperation, N.J. USA.
Water bath: Model DK.
Magnetic stirrer: AM-3250B Surgi Friend Medicals, England.
Milling machine: Thomas Willey Laboratory Mill Model 4, Anthor H
(Thomas Company, Philadelphia, USA)
Autoclave: UDAY BURDON’s Patient Autoclave, India.
Incubator: B and T Trimline incubator.
Centrifuge: Finland Nigeria 80-2B.
Oven: Gallenkamp, England.
pH meter: Ecosan pH meter, Singapore.
47
Microscope: WESO microscope.
Glass wares: Pyrex, England
Uv/visible spectrophotometer: Jenway 6405, England
Visible spectrophotometer: Labscience 721, England
2.2 Methods
2.2.1 Collection of Breadfruit hulls
Breadfruit hulls were obtained from breadfruit processing centres at the Ogige market in
Nsukka town, Enugu State of Nigeria.
2.2.2 Collection of Microorganism
Aspergillus fumigatus and Aspergillus flavus strains were obtained from the post-graduate
laboratory of the Department of Biochemistry, University of Nigeria, Nsukka.
2.2.3 Preparation of Ground Breadfruit hulls
The hulls were sun dried for seven days and then ground into powder with the help of a
milling machine.
2.2.4 Storage of Pure Fungal Isolates
Fungal isolates were obtained from the Department of Microbiology. The pure fungal isolates
were maintained on potato dextrose agar (PDA) slopes or slants as stock cultures. PDA media
were prepared according to the manufacture’s description. In the procedure, 3.9g of PDA
powder was weighed and added to a small volume of distilled water and made up to 100 ml.
The medium was autoclaved at 121oC (15 psi) for 15 min. It was allowed to cool to 45
oC and
then poured into Petri dishes and allowed to gel. The plates were then incubated in a B & T
Trimline incubator at 37oC for 24hr to check for sterility.
48
2.2.5 The Fermentation Broth
Submerged fermentation (SmF) technique was employed using a 250 ml Erlenmeyer flask
containing 100 ml of sterile cultivation medium optimized for cellulase with 0.1% NH4NO3,
0.1% NH4 H2PO4, 0.1% MgS04.7H2O and 1% breadfruit hulls. The flask was stoppered with
aluminium foil and autoclaved at 121oC (15 psi) for 15 min. The experiment was performed
in duplicate for both fungal species.
2.2.6 Inoculation of the Broth
From the PDA slants, fresh plates were prepared and inoculated. Three days old cultures were
used to inoculate the flasks. In every 50 ml of the broth, two discs of the respective fungal
isolates were added using a cork borer of diameter 10 mm and then plugged properly. The
culture was incubated for 7 days at room temperature (30oC). This experiment was also
performed for both species of Aspergillus.
2.2.7 Harvesting of the Fermented Broth
At each day of harvest, flasks were selected from the respective groups and mycelia biomass
separated by filtration. Each day, the filtrate was analyzed for cellulase activity till the 7th
day of fermentation.
2.2.8 Mass Production of Enzyme
After the 7 days pilot studies, the day of peak cellulase activity was chosen for mass
production of enzyme from the respective fungal isolates. Several 250 ml Erlenmeyer flasks
were used to produce 750 ml of the enzymes using the method described in sections 2.2.6 and
2.2.7. Harvesting was carried out on the respective peak days of enzyme activity.
49
2.2.9 Procedure for Protein Determination
Protein determination was done by Lowry’s method (1951). For protein standard curve, the
reaction mixture contained 0.0-1.0 ml of protein stock solution (2 mg/ml BSA) in test tubes
arranged in triplicates. The volume was made up to 1 ml with 0.05M sodium acetate buffer.
But for the test mixture, 0.1 ml of sample enzyme was mixed with 0.9ml of buffer. In either
case, 2ml of solution E was added and allowed to stand at room temperature for 10min. Then,
0.2ml of solution C (dilute Folin-Ciocalteau phenol reagent) was added with rapid mixing.
After standing for 30min, absorbance was read at 750nm using UV spectrophotometer.
Absorbance values were converted to protein concentration by extrapolation from the
standard curve.
2.2.10 Cellulase Assay
Cellulase activity was evaluated by assaying for the glycosidic activity of the enzyme. This
was achieved by measuring the release of reducing groups from filter paper, carboxymethyl
cellulose (CMC) and cellobiose using a modification of the 3, 5-dinitrosalicylic acid (DNS)
reagent assay method described by Miller (1959) as contained in Ghose (1997).
2.2.10.1 Cellobiase assay
The reaction mixture containing 0.2 ml 15 mM cellobiose in 0.05 M sodium acetate buffer of
pH 5.5 and 0.2 ml enzyme solution was incubated for 30 mins at 50 ºC. 1ml of DNS reagent
was added and the reaction was stopped by boiling the mixture in a boiling water bath for
10mins. The mixture volume was made up to 4 ml with 1 ml of Rochelle salt solution. The
reaction mixture was allowed to cool and then the absorbance read at 540 nm. One unit of
enzyme activity was defined as the amount of enzyme that catalyzed the release of one
micromole of glucose per minute
50
2.2.10.2 Endoglucanase (CMCase) assay
The reaction mixture containing 0.2 ml CMC (2%) in 0.05 M sodium acetate buffer pH 5.5
and 0.5 ml enzyme solution was incubated for 30 min at 50 ºC. Then, 1 ml of DNS reagent
was added and the reaction was stopped by boiling the mixture in a boiling water bath for 10
mins. The mixture was made up to 4 ml with 1 ml of Rochelle salt solution. The reaction
mixture was allowed to cool and then the absorbance read at 540 nm. One unit of enzyme
activity was defined as the amount of enzyme that catalyzes the release of one micromole of
glucose per minute
2.2.10.3 Total cellulase (Filterpaperase) assay
The reaction mixture containing 50 mg (1 cm x 6 cm) filter paper in 0.05 M sodium acetate
buffer of pH 5.5 and 0.5 ml enzyme solution was incubated for 1 hour at 50 ºC and 1 ml of
DNS reagent was added and the reaction was stopped by boiling the mixture in a boiling
water bath for 10 min. The volume was made up to 4 ml with Rochelle salt solution. The
reaction mixture was allowed to cool and then the absorbance read at 540 nm. One unit of
enzyme activity was defined as the amount of enzyme that catalyzed the release of one
micromole of glucose per minute.
2.2.11 Partial Purification of Protein
2.2.11.1 Determination of Percentage Ammonium Sulphate Saturation Suitable for
Cellulase Precipitation
Nine test tubes were used to form an ammonium sulphate precipitation profile. Cellulases
were precipitated with gentle stirring at 20-100% saturation of solid ammonium sulphate at
intervals of 10% in each test tube. The ammonium sulphate-crude enzyme solutions were
allowed to stand at cold temperature of 4oC for 30 hr till the supernatant could be gently
decanted off. The test tubes were centrifuged at 3500 rpm for 10 mins. Precipitates from the
individual percentage ammonium sulphate saturations were re-dissolved, respectively, in
equal volumes of 0.05 M acetate buffer pH 5.0. Cellulase activities of the precipitates were
51
assayed to determine the percentage ammonium sulphate saturation that precipitated enzyme
with maximum activity.
2.2.11.2 Ammonium Sulphate Precipitation of Cellulase
A known value, 750 ml of crude enzyme filtrate was used in the process. Also, 80% and 70%
ammonium sulphate saturation (for Aspergillus fumigatus and Aspergillus flavus,
respectively) were found suitable for mass precipitation of the enzymes from the fungal
isolates. Ammonium sulphate precipitation (at 80% and 70% saturation) was carried out by
dissolving gently 516 g and 436 g of the salt in the filtrates and stirring gently till the salt was
completely dissolved as seen in section 2.2.11.1. The precipitate was re-dissolved in 55 ml of
0.05 M acetate buffer pH 5.0 after centrifugation and then kept under cold condition for
further studies.
2.2.11.3 Gel filtration
The enzyme was introduced onto Sephadex G-25 packed column (2.6 × 61.50 cm) pre-
equilibrated with 0.05M Na-acetate buffer, pH 5.5. The protein was eluted with 0.05 M Na-
Acetate buffer, pH 5.5. The fractions with high cellulase activity were pooled together.
Cellulase activity from each of the eluted fraction were monitored at wave length of 540 nm
and protein absorbance read at 280 nm.
2.2.12 Studies on Partially Purified Enzyme
2.2.12.1 Enzyme Progress Curve
The enzyme was incubated with its substrate (filter paper) at 50 C and activity was assayed
at 5, 10 , 20, 30, 40, 50, 60, 70, 90 and 120 min respectively.
52
2.2.12.2 Effect of pH on Total Cellulase Activity
The optimum pH for enzyme activity was determined using 0.05 M sodium acetate buffer pH
4.0 - 5.5, phosphate buffer pH 6.0 - 7.5 and Tris-HCl buffer pH 8.0 – 9.0 at intervals of 0.5. A
known quantity, 0.2 ml of partially purified enzyme was dispersed into 0.8 ml of buffer of
different pHs into which 50 mg (1cm x 6cm) had been added to. Total Cellulase activity was
assayed as seen in section 2.2.10.3
2.2.12.3 Effect of Temperature on Total Cellulase Activity
The optimum temperature was determined by incubating the enzyme with 50mg filter paper
at 25-70oC, interval of 5
oC for 1 hour and at pH 5.5. The activity was then assayed using the
method described in 2.2.10.3
2.2.12.4 Determination of kinetic parameters
Kinetic parameters were determined using different concentrations of filter paper and
cellobiose. The Vmax and Km values of the enzymes were calculated using the Lineweaver-
Burke double reciprocal plot of 1/V against 1/[S].
53
CHAPTER 3
RESULTS
3.1 Incubation Period (Pilot Study)
Five (5) days of incubation produced the highest total cellulase activity at 2.97 and 3.87 U/ml
for enzymes produced by A. flavus and A. fumigatus respectively.
Fig. 9: Effect of incubation period on cellulase production
54
3.2 Studies on crude enzyme
3.2.1 Protein concentration of crude enzyme
Protein concentration of crude enzyme produced by A. flavus and A. fumigatus were found to
be 4.03 and 4.17 mg/ml respectively.
Fig. 10: Protein concentration of crude enzyme produced by the microoganisms
55
3.2.2 Cellulase activity of crude enzymes.
Cellulolytic activities of enzymes from the two fungi are shown below.
The total cellulase activity of 750 ml of crude enzyme produced by each microorganism was
found to be 3.04 and 4.16 U/ml for A. flavus and A. fumigatus respectively. Glucanase
activity was 2.86 and 4.84 U/ml for enzymes produced by A. flavus and A. fumigatus
respectively. Cellobiase activity was observed to be highest for the three enzyme assays. A.
flavus had an activity of 10.09 U/ml while A. fumigatus had an activity of 10.16 U/ml.
Fig. 11: Total cellulase, glucanase and cellobiase activities of crude enzyme
56
.3 Ammonium sulphate precipitation profile of cellulases
As in Fig. 12, 70% and 80% ammonium sulphate had highest cellulase activities at 3.55 and
3.3 U/ml for A. flavus and A. fumigatus respectively. Hence, 70% and 80% were chosen for
the precipitation of the enzymes from the two microorganisms.
Fig. 12: Ammonium sulphate precipitation profiles for celluluases isolated from A. flavus and
A. fumigatus.
57
3.4 Gel filtration (Elution profile of cellulase enzymes)
For cellulase isolated from A. flavus, two prominent peaks (A and B) were observed with
activities of 3.99 U/ml and 3.64 U/ml respectively as shown in Fig. 13a.
Fig. 13a: Gel elution profile of proteins produced by A. flavus.
58
For cellulases isolated from A. fumigatus, two prominent peaks C and D were also identified
with activities of 2.94 and 3.11 U/ml as show in Fig. 13b.
Fig. 13b: Gel elution profile of proteins produced by A. fumigatus
59
3.5 Summary of purification steps
Table 6: Summary of purification steps of cellulase from A. flavus
Purification step Volume
(ml)
Protein
conc.
(mg/ml)
Activity
(U/ml)
Spec.
Activity
(U/mg)
Total
Activity
(U)
Purification
fold
% Yield
Crude Enzyme
Filtrate
750 4.03 3.04 0.75 2280 1 100
% (NH4)2SO4
Precipitation
55 4.1 5.1 1.24 280.5 1.65 12.3
Gel filtrate A 20 2.75 3.99 1.43 59.8 1.91 2.62
Gel fitrate B 20 2.28 3.64 1.59 52.8 2.12 2.34
Table 6 shows the summary of the purification steps of cellulase from A. flavus. An initial
volume of 750 ml of crude enzyme extract yielded a protein concentration of 4.03 mg/ml
with an activity of 3.04 U/ml. and specific activity of 0.75 U/mg. Ammonium sulphate
precipitation re-dissolved in 55 ml of buffer yielded a protein concentration of 4.1 mg with
activity of 5.1 U/ml and specific activity of 1.24 U/mg. Gel filtrates of 20 ml each for the 2
isoforms of cellulase A and B yielded protein concentrations of 2.75 mg/ml and 2.28 mg/ml
for isoforms A and B respectively. The activities of the filtrates were 3.99 and 3.64 U/ml for
the corresponding isoforms. Specific activities were 1.43 and 1.59 U/ml respectively. The
overall percentage yield fell from 100% to 2.62 and 2.34% after gel filtration. Total activity
decreased from 2280 U to 59.8 and 52.8 U respectively after gel filtration.
60
Table 7: Summary of purification steps of cellulase from A. fumigatus
Purification step Volume
(ml)
Protein
conc.
(mg/ml)
Activity
(U/ml)
Spec.
Activity
(U/mg)
Total
Activity
(U)
Purification
fold
% Yield
Crude Enzyme
Filtrate
750 4.17 4.16 1.0 3120 1 100
% (NH4)2SO4
Precipitation
55 4.79 5.9 1.23 324.5 1.23 10.4
Gel filtrate A 20 2.16 2.94 1.36 58.8 1.36 1.88
Gel fitrate B 20 2.09 3.11 1.49 62.2 1.49 1.99
Table 7 shows the summary of the purification steps of cellulase from A. fumigatus. An initial
volume of 750 ml of crude enzyme extract yielded a protein concentration of 4.17 mg/ml
with an activity of 4.16 U/ml. and specific activity of 1.0 U/mg. Ammonium sulphate
precipitation re-dissolved in 55 ml of buffer yielded a protein concentration of 4.79 mg with
activity of 5.9 U/ml and specific activity of 1.23 U/mg. Gel filtrates of 20 ml each for the two
isoforms of cellulase A and B yielded protein concentrations of 2.16 mg/ml and 2.09 mg/ml
for isoforms A and B respectively. The activities of the filtrates were 2.94 and 3.11 U/ml for
the corresponding isoforms. Specific activities were 1.36 and 1.49 U/ml respectively. The
overall percentage yield fell from 100% to 1.88 and 1.99% after gel filtration. Total activity
reduced from 3120 U to 58.8 and 62.2 U respectively after gel filtration.
61
3.6 Changes in Protein Concentration of Partially Purified Enzymes
3.6.1 Changes in protein concentration of enzymes from a. flavus.
It was observed in Fig. 14 that the protein concentration increased from 4.03 to 4.1 mg/ml
after ammonium sulphate precipitation and decreased to 2.75 mg/ml and 2.28 mg/ml
(corresponding to isoforms A and B) after gel filtration.
Fig. 14: Changes in protein concentration after partial purification of cellulases from A. flavus
62
3.6.2 Changes in protein concentration of enzymes from A. fumigatus
As shown in Fig. 15, for A. fumigatus, the protein concentration increased from 4.17 to 4.79
mg/ml after ammonium sulphate precipitation and decreased to 2.16 and 2.09 mg/ml for
isoforms C and D respectively after gel filtration.
Fig. 15: Changes in protein concentration after partial purification of cellulases from A.
fumigatus
63
3.7 Changes in Total Cellulase Activity of Partially Purified Enzymes
3.7.1 Changes in total cellulase activity of partially purified enzymes from a. flavus
The activity of the enzyme increased 3.04 to 5.1 U/ml after ammonium sulphate precipitation
as shown in Fig 16. There was a reduction in activity after gel filtration to 3.99 and 3.64 U/ml
for the 2 isoforms A and B present.
Fig. 16: Changes in total cellulase activity after partial purification of enzymes from A. flavus
64
3.7.2 Changes in Total Cellulase Activity of Partially Purified Enzymes from A.
fumigatus
Enzyme activity increased from 4.16 to 5.9 U/ml after ammonium sulphate precipitation as
depicted in Fig. 17. However, it reduced to 2.94 U/ml and 3.11 U/ml (for isoforms C and D
respectively) after gel filtration.
Fig. 17: Changes in total cellulase activity after partial purification of enzymes from A.
fumigatus
65
3.8 Changes in Specific Activities of Partially Purified Enzymes
3.8.1 Changes in specific activities of partially purified enzymes from A. flavus
Specific activity of crude enzyme was found to be 0.75 U/mg as observed in Fig 18. This
value increased to 1.24 U/mg after ammonium sulphate precipitation. It further increased to
1.43 U/mg and 1.59 U/mg (corresponding to the two isoforms) after gel filtration.
Fig. 18: Changes in specific activity after partial purification of cellulases from A. flavus.
66
3.8.2 Specific activities of partially purified enzymes from A. fumigatus
Fig. 19 shows specific activity of 1.23 U/mg for crude enzyme after ammonium sulphate
precipitation. This value increased to 1.36 U/mg and 1.49 U/mg corresponding to the two
isoforms present after gel filtration.
Fig. 19: Changes in specific activity after partial purification of cellulases from A. fumigatus
67
3.9 Enzyme Characterization
3.9.1 Enzyme Progress Curve
3.9.1.1 Enzyme progress curve of partially purified enzymes from A. flavus
The A isoform exhibited maximum activity after 60 min of incubation as shown in Fig. 20
The B isoform, however, had maximum activity after 50 min of incubation.
Fig. 20: Progress curve of cellulases isolated from A. flavus
68
3.9.1.2 Enzyme progress curve of partially purified enzymes from A. fumigatus
Fig. 21 shows the progress curve of partially purified enzyme. For cellulase isolated from A.
fumigatus, the C isoform had highest activity after 70 min of incubation while the D isoform
had highest activity after 60 minutes of incubation.
Fig. 21: Progress curve of cellulases isolated from A. fumigatus
69
3.9.2 Effect of pH Change on Cellulase Activity
3.9.2.1 Effect of pH change on cellulase produced by A. flavus
Fig. 22 shows that the enzymes had optimum activities of 3.31 and 3.53 U/ml corresponding
to the two isoforms A and B of the enzymes at pHs of 6.5 and 7.0 respectively. Further
increase in pH led to a decline in enzyme activity.
Fig. 22: Effect of pH on cellulases produced by A. flavus.
70
3.9.2.2 Effect of pH Change on Cellulase Produced by A. fumigatus
For cellulase isolated from A. fumigatus, the enzyme exhibited highest activities of 3.07 U/ml
and 3.42 U/ml corresponding to the isoforms C and D at a pH of 5 for both forms as shown in
Fig. 23
Fig. 23: Effect of pH on cellulases produced by A. fumigatus
71
3.9.3 Effect of Temperature Change on Cellulase Activity.
3.9.3.1 Effect of temperature on cellulase produced by A. flavus
For cellulase isolated from A. flavus as shown in Fig. 24, maximum activity was observed at
a temperature of 50⁰C for both isoforms of the enzyme when assayed at pHs of 6.5 and 7.0
Fig. 24: Effect of temperature on cellulases produced by A. flavus
72
3.9.3.2 Effect of temperature on cellulase produced by A. fumigatus
Cellulase produced by A. fumigatus had optimum activity at 55 ⁰C for both isoforms when
assayed at a pH of 5.5 for both isoforms as shown in Fig. 25.
Fig. 25: Effect of temperature on cellulases produced by A. fumigatus
73
3.9.4 Determination of Kinetic Parameters (Using Filter paper as Substrate)
Kinetics parameter Vmax and Km determined from lineweaver burk plots (using filter paper as
substrate)
3.9.4.1 Determination of Kinetic parameters for enzymes of A. flavus
Isoform A of cellulase isolated from A. flavus had Vmax and Km values of 142.9 µmole/min
and 59.02 mg respectively as shown in Fig. 26.
Fig. 26: Lineweaver-Burke plot of cellulase A from A. flavus using filter paper as substrate
74
Isoform B of cellulase isolated from A. flavus had Vmax and Km values of 166.7 umole/min
and 47.67 mg respectively as shown in Fig. 27
Fig. 27: Lineweaver-Burke plot of cellulase B from A. flavus using filter paper as substrate
75
3.9.4.2 Determination of Kinetic parameters for enzymes of A. fumigatus
Isoform C of cellulase isolated from A. fumigatus had Vmax and Km values of 128.21
µmole/min and 27.82 mg respectively as shown in Fig. 28
Fig. 28: Lineweaver-Burke plot of cellulase C from A. fumigatus using filter paper as
substrate
76
Isoform D of cellulase isolated from A. fumigatus had Vmax and Km values of 90.91
µmole/min and 32 mg respectively as shown in Fig. 29
Fig. 29: Lineweaver-Burke plot of cellulase D from A. fumigatus using filter as substrate
77
3.9.5 Determination of Kinetic Parameters (Using Cellobiose as Substrate)
Kinetics parameter Vmax and Km determined from lineweaver burk plots (using cellobiose as
substrate)
3.9.5.1 Determination of Kinetic parameters for enzymes of A. flavus
Isoform A of cellulase isolated from A. flavus had Vmax and Km values of 588.2 µmole/min
and 7.7 mM as shown in Fig. 30
Fig. 30: Lineweaver-Burke plot of cellulase A from A. flavus using cellobiose as substrate
78
Isoform B of cellulase isolated from A. flavus had Vmax and Km values of 476.2 µmole/min
and 3.33 mM as shown in Fig. 31
Fig. 31: Lineweaver-Burke plot of cellulase B from A. flavus using cellobiose as substrate
79
3.9.5.2 Determination of Kinetic parameters for enzymes of A. fumigatus
Isoform C of cellulase isolated from A. fumigatus had Vmax and Km values of 833.3
µmole/min and 11.1 mM as shown in Fig. 32
Fig. 32: Lineweaver-Burke plot of cellulase C from A. fumigatus using cellobiose as substrate
80
Isoform D of cellulase isolated from A. fumigatus had Vmax and Km values of 666.67
µmole/min and 9.1 mM as shown in Fig. 33
Fig. 33: Lineweaver-Burke plot of cellulase D from A. fumigatus using cellobiose as substrate
81
Table 8: Characterization of Cellulase
Properties A.flavus (Isoform
A)
A.flavus (Isoform
B)
A.fumigatus (Isoform
C)
A.fumigatus (Isoform
D)
pH 6.5 7.0 5.0 5.0
Temperature (◦C) 50 50 55 55
Vmax (µmole/min)
(Filter paper)
142.9 166.7 128.21 90.91
Km (mg)
(Filter paper)
59.02 47.67 27.82 32
Vmax (µmole/min)
(Cellobiose)
588.2 476.2 833.3 666.67
Km (mM)
(Cellobiose)
7.7 3.33 11.1 9.1
82
CHAPTER 4
DISCUSSION
This study deals with the production, partial purification and characterization of cellulolytic
enzymes produced by two species of the Aspergillus genus- flavus and fumigatus. Studies
were undertaken to optimize the production of extracellular cellulases by these organisms.
The enzymes were then isolated and purified and their characteristics were studied.
The microbes when cultivated in the presence of breadfruit hulls as carbon source secreted
cellulolytic enzymes which exhibited highest activity after 5 days of incubation. Both species
of Aspergillus used in this work were grown over a 7 day period. The growth of fungi on a
medium is usually affected by several factors which include: nutrients, temperature, light,
aeration, pH, and water activity (Raim, 1998). Fungi generally exhibit four basic growth
stages: the spore, which is the dormant phase, followed by the spore germination or lag
phase, the growth or hyphae phase and the spore formation phase (Carlile et al., 2001). It is
expected that as the fungi grow, they secrete more proteins into the containing medium until
growth approaches the lag phase (Raim, 1998). Other researchers have previously reported
different days for obtaining maximum activity during incubation. Nwobodo and Okochi
(2011) reported maximum activity after 3 days of incubation using Aspergillus niger grown
on sawdust. Charitha and Kumar (2012) also observed highest activity after 7 days of
incubation using A. niger grown with waste paper. However, Adekunle et al (2012) reported
maximum enzyme activity at 6 day of growth when A. niger was grown on rice as carbon
source. Recently, Das et al (2013) reported maximum activity after 3 days growth when A.
fumigatus ABK9 was cultivated under submerged fermentation condition. Incubation time of
a fermentation experiment has a direct relationship with the production of extracellular
enzymes; however, microbial growth and enzyme production plateaus as the growth
approaches the lag or stationary phase. (Raim, 1998). The variation in optimum activities can
be attributed to the different environmental conditions which affect the growth of fungi (Das
et al., 2013). The findings in this work suggest that in order to obtain a commercial quantity
of cellulases (in an industrial setting) from A. flavus and A. fumigatus, especially under
submerged conditions using breadfruit hulls as carbon source, the crude enzymes should be
harvested on the 5th
day of growth. However, fresh pilot studies should be carried out when
83
other cellulosic substrates are used as carbon source or when other cellulase producing
microorganisms are used instead of those used in this work
Ammonium sulphate was used to salt out the proteins. Ammonium sulphate precipitation is
one of the most commonly used methods for protein purification from a solution. The
principle behind ammonium sulphate precipitation is the altering of the solubility of proteins
in the presence of a salt (Green and Hughes, 1955). In solution, proteins form hydrogen
bonds with water molecules through their exposed polar and ionic groups (Mitchinson and
Pain, 1986). When high concentrations of small, highly charged ions such as ammonium
sulphate are added, these groups compete with the proteins to bind to the water molecules.
This removes the water molecules from the protein and decreases its solubility, resulting in
precipitation (Green and Hughes, 1955; Harriette and Charles, 1913). Critical factors that
affect the concentration at which a particular protein will precipitate include: the number and
position of polar groups, molecular weight of the protein, pH of the solution, and temperature
at which the precipitation is performed. Protein concentration and activity were higher for the
precipitated enzymes when compared with that of the crude form with the greater activity
being attributed to the increased concentration of proteins in solution. The proteins were
purified by precipitating with ammonium sulphate at 70% and 80% for A. flavus and A.
fumigatus respectively as shown in the result. Charitha and Kumar (2012) reported 80%
saturation for enzymes of A. niger fermented with paper and timber sawmill industrial waste.
Bakare et al., (2005) reported maximum precipitation of proteins at 90% saturation for
proteins from mutants of Pseudomonas fluorescens. Shanmu et al., (2012) reported 80%
saturation for cellulases produced by bacteria that were grown on cow dung Guo et al.,
(2013) obtained 60% saturation for cellulase from a commercial enzyme preparation. It is
important to note that in all these precipitation experiments including the one performed in
this work, the supernatant had almost no activity and was therefore discarded. From the
results obtained from literature and from this work, cellulases tend to precipitate out at
increasing concentrations of ammonium sulphate from 60% to 90%.
On further subjection to gel filtration, two prominent peaks were observed for both cellulases
produced by both fungi indicating two forms or isoforms of the enzymes as seen in the
results. Multiplicity of cellulases produced by microbes is a general phenomenon (Marsden
and Gray, 1986; Brown and Gritzali, 1984). Both microbes showed at least two forms of
84
these enzymes. Heterogeneity of cellulases may be attributed to one or more of the following:
posttranslational modification (glycosylation, proteolysis, phosphorylation, acetylation etc.),
multiple gene expression (Agelos and Panagiotis, 1991)
After the purification, it was observed that the specific activity of the enzymes increased after
each step as can be deduced from the result. For enzymes from A. flavus, the initial specific
activity was 0.75 U/mg. After ammonium sulphate precipitation, this value increased to 1.24
U/mg and further to 1.43 U/mg and 1.59 U/mg for isoforms A and B respectively after gel
filtration. For enzymes of A. fumigatus on the other hand, the initial specific activity was 1.0
U/mg. After ammonium sulphate precipitation, this value increased to 1.23U/mg and further
to 1.36 U/mg and 1.49 U/mg for isoforms C and D respectively after gel filtration. Specific
activity is a measure of enzyme purity. This value increases as an enzyme preparation
becomes purer, since the amount of protein (mg) is typically less, but the rate of reaction
stays the same or increases due to reduced interference or removal of inhibitors (Nelson and
Cox, 2000).
The enzyme was characterized and effects of incubation time, pH and temperature on enzyme
activity were observed. Kinetic parameters were also studied. Cellulase isolated from A.
flavus had highest activities after 60 min and 50 min (corresponding to forms A and B) of
incubation as shown in the results. Those of A. fumigatus were observed to have highest
activity after 70 minutes and 60 minutes for forms C and D respectively of incubation as
shown in the results. The longer the enzyme was incubated with its substrate, the greater the
amount of product formed up to a point when there is a decline with time. This was probably
due to the combined effects of substrate utilization and product accumulation (Duggleby,
1986).
The effect of pH on the total cellulase activity of both enzymes produced by the two species
of Aspergillus was studied. A. flavus exhibited highest activities at pHs of 6.5 and 7.0
corresponding to the isoforms A and B identified after gel filtration. Enzymes produced by A.
fumigatus on the other hand had maximum activity at a pH of 5.5 for both forms of the
enzymes. On further increase in pH, activity fell probably due to changes in total net charge
of the enzymes. This effect of pH on charge distribution on the ionizable groups interrupts the
tertiary structure of the enzyme and thus causes its denaturation. Saraswati et al. (2012)
85
reported a pH of 7.0 for cellulase produced by bacillus isolated from cow dung. Sunita and
Sumit (2012) also reported an optimum pH of 7 for cellulase produced by Trichoderma viride
using sawdust and coir waste as carbon sources. Mawadza et al (2000) reported highest
activity at a pH range of 5-7 for bacillus sp CH43 strain and a pH optimum 5-6.5 for Bacillus
sp HR68 strain with the enzyme having highest activity at 6.5. The results are in line with the
observed pHs of 5.5, 6.5 and 7.0 reported in this work using 2 species of the Aspergillus
genus. The results obtained indicate that optimum pH is around neutral and slightly acidic
pH. Thus, cellulase enzyme obtained from these organisms will best be utilized for industrial
applications at neutral or slightly acidic pH.
The temperature optima of the total cellulase activity for both isoforms A and B secreted by
A. flavus was 50 ºC while the highest total cellulase activity was obtained at 55 ºC for both
forms C and D isolated from A. fumigatus. Enzyme-catalyzed reactions tend to be slower at
temperatures below the optimum. They then tend to go faster with increasing temperature but
only until a temperature optimum is reached. Above the optimum temperature, the kinetic
energy of the enzyme increases to the extent that the weak intermolecular attractions that
maintain the shape of proteins are broken and the enzyme molecule is disrupted - the enzyme
becomes denatured. Changing the shape of the enzyme results in less efficient binding of the
substrate (reactants) resulting in a significant decrease in enzyme activity (Duggleby, 1986).
Mawadza et al (2000) had earlier reported maximum activity at 45 ºC for total cellulase
activity of the enzymes produced by fusarium and penicilium. A temperature of 60 ºC was
also observed in same work by Mawadza et al (2000) to give maximum activity for enzymes
produced by Aspergillus sp. In a study carried out by Immanuel et al. (2006) the enzyme
produced by A. niger had less activity at 20 ºC but on further increase to 50 ºC, maximum
activity was obtained while maximum enzyme activity was recorded at 50 ºC for A.
fumigatus. Rahna et al. (2012) reported maximum activity at 50 ºC for total cellulase activity
of enzymes produced by pseudomonas sp using salvinia as substrate. These temperature
optima are relatively in agreement with those observed in this study. The implication of these
findings is that cellulolytic enzymes would best be utilized at 50 ºC for converting cellulosic
biomass into desirable products.
The effect of substrate concentration on the total cellulase activity of the enzymes was
studied. Thereafter, the kinetic constants were determined. With fixed enzyme concentration,
86
an increase in substrate concentration resulted in increase in enzyme activity until a saturation
point was reached at which further increases in substrate concentration did not result in
activity. This could be due to the formation of unreactive complexes formed between the
enzyme and substrate. Also to be considered is the fact that when substrate molecules are in
high concentration around the enzyme, they may bind to other sites other than the enzyme
active site or alternatively crowd the active site. Total cellulase activity, using filter paper as
substate, had maximum velocity of 142.9 and 166.7 µmol/min for both isoforms A and B of
the enzyme secreted by A. flavus while Km values of 59.02 and 47.67 mg were observed for
the corresponding isoforms. On the other hand, for enzymes produced by A. fumigatus, they
showed maximum velocity of 128.21 and 90.91 µmol/min for the 2 isoforms C and D
respectively. The Km values were 27.82 and 32 mg respectively. Using cellobiose as
substrate, Vmax values were 588.2, 476.2, 833.3 and 666.67 µmol/min for isoforms A, B, C
and D respectively. Km values 7.7, 3.3, 11.1 and 9.1 mM were obtained for isoforms A, B, C
and D respectively. The Km values serve to indicate the substrate concentration required to
achieve half the maximum initial reaction velocity. Km therefore measures the relative
affinity an enzyme has for its substrate. A smaller Km denotes a greater affinity that an
enzyme has for its substrate. From the results obtained, enzymes of A. fumigatus have a
greater affinity for cellulose when compared with those of A. flavus and are thus better
applied for an industrial saccharifying process, the reason being that the enzyme will act at a
more or less constant rate, regardless of variations in concentrations of substrate.
4.2 CONCLUSION
The results indicate possibility of using agricultural waste such as breadfruit hulls to induce
cellulase production in saprophytic microbial fungi, thus converting waste into wealth in the
form of enzymes of industrial importance. Most of the work done on the genus Aspergillus
have been on A. niger which is still currently the fungi of choice to produce these enzymes.
This work however provides options that could be considered for production of cellulases
using raw waste materials that could be easily obtained from the environment.
87
4.3 SUGGESTIONS FOR FURTHER RESEARCH
Based on the findings in this work, the following suggestions are made
1. Studies on the other physiological properties of the cellulase enzymes such as thermal
stability and pH stability should be conducted to understand their effects on the
enzyme activity.
2. Further purification of the enzymes using ion exchange chromatography and gel
electrophoresis should be conducted and Sodium Dodecyl Sulphate Polyacrilamide
Gel Electrophoresis (SDS PAGE) used to find out the molecular weight of these
enzymes.
3. X-ray crystallography and NMR should be used to elucidate the active sites of these
enzymes as well as their tertiary structures.
88
REFERENCES
Adekunle, O. A., Olanike, O. I. and Olabisi, A. (2012). Production of amylase from Aspergillus
niger using a defined synthetic growth medium and also rice as growth source. E3
Journal of Medical Research, 1(7):91-94.
Agbogidi, O. M. and Onomeregbor, V. A. (2008). Morphological changes in the seedlings of
Treculia africana grown in crude oil impacted soils. In: Climate Change and
Sustainable Renewable Natural Resources Management (Ed) Popoola L. Proceeding of
32nd Annual Conference of the Forestry Association of Nigeria, held in Umuahia,
Abia-State, Nigeria. pp.170-182.
Agelos K. And Panagiotis K. (1991). Cellulase occurs in multiple forms in ripe avocado fruit
mesocarp. Plant Physiology, 98: 530-534.
Alfredsson, G. A., Kristjansson, J.K., Hjorleifsdottir, T. S. and Stetter, K. O. (1988).
Rhodothermus marinus, gen. nov., sp. nov., a thermophibic, halophilic bacterium from
submarine hot springs in Iceland. Journal of Genetics and Microbiology, 134: 299-306.
Anish, R., Rahman, M. S., Rao, M. (2006). Application of cellulases from an alkalothermophilic
Thermomonospora sp. in biopolishing of denims. Biotechnology Bioengineering, 96:
48-56.
Atuanya, C. U., Aigbodion, V. S. and Nwigbo, U. (2012) Characterization of breadfruit seed
hull ash for potential utilization in metal matrix composites for automotive application.
Peoples Journal of Science and Technology, 2(1): 2249- 5847.
Babu, K. R. and Satyanarayana, T. (1996) Production of bacterial enzymes by solid state
fermentation. Journal of Scientific and Industrial Research, 55: 464-467.
Baiyeri, K. P. and Mbah, B. N. (2006). Effect of soiless and soil-based nursery media on
seedling emergency, growth and response to water stress of african breadfruit (Treculia
africana Decne). African Journal of Biotechnology, 5(15): 1400-1405.
Bamforth, C. (1994). β-glucan and β-glucanases in malting and brewing: practical aspects.
Brewery Digest, 69: 12-16.
Bayer, E. A., Belaich, J. P., Shoham, Y. and Lamed, R. (2004). The cellulosomes: multienzyme
machines for degradation of plant cell wall polysaccharides . Annual Review of
Microbiology, 58: 521–554.
89
Bennet. J. W. (2010). An overview of the genus Aspergillus: Molecular Biology and Genomics.
Caister Academic Press. New York. pp. 23-35
Bergquist, P. L., Gibbs, M. D., Morris, D. D., Te‘o, V. S. J., Saul, D. J. and Morgan, H. W.
(1999) Molecular diversity of thermophilic cellulolytic and hemicellulolytic bacteria.
FEMS Microbiology and Ecology, 28: 99-110.
Bhat, M. K and Bhat, S. (1997). Cellulose degrading enzymes and their potential industrial
applications. Biotechnology Advances,15: 583-620.
Bhat, M. K. (2000). Cellulases and related enzymes in biotechnology. Biotechnology Advances,
18: 355-383.
Bhat, M. K. (2000). Cellulases and related enzymes in biotechnology. Biotechnology Advances,
18: 355-383.
Brennan, A. H., Hoagland, W. and Schell, D. J. (1986) High temperature acid-hydrolysis of
biomass using an engineering scale plug flow reactor: Results of low solids testing.
Biotechnology and Bioengineering Symposium, 17: 53- 66.
Bronnenmeier, K. and Staudenbauer, W. L. (1988). Purification and properties of an
extracellular β-glucosidase from the cellulolytic thermophilic Clostridium stercorarium.
Applied Microbiology and Biotechnology, 28: 380-386.
Brown, R. D. and Gritzali, M. (1984). Microbial enzymes and lignocelluloses utilization. Basic
life sciences, 28: 239-244.
Buchert, J., Oksanen, J., Pere, J., Siika-Aho, M., Suurnäkki, A. and Viikari, L. (1998)
Applications of Trichoderma reesei enzymes in the pulp and paper industry. In: Harman
G. E, Kubicek C. P (Eds.). Trichoderma and Gliocladium. London: Taylor & Francis.
pp 343-364.
Bukhtojarov, F. E., Ustinov, B. B., Salanovich, T. N., Antonov, A. I., Gusakov, A. V., Okunev,
O. N. and Sinitsyn, A. P. (2004). Cellulase complex of the fungus Chrysosporium
lucknowense: isolation and characterization of endoglucanases and cellobiohydrolases.
Biochemistry (Mosc), 69: 542-551.
Carlile, M. J., Watkinson, S. C. and Goodday, G. W. (2001). The Fungi. 2nd
Edn. Academic
Press. San Diego. Pp 23-41.
Cavaco-Paulo, A. (1998). Mechanism of cellulase action in textile processes. Carbohydrate
Polymers, 37: 273-277.
90
Charita D. M. and Kumar S. M. (2012). Production, opitimization and partial purification of
cellulase by aspergillus niger fermented with paper and timber saw mill industrial
wastes. Journal of Microbiology and Biotechnology Research, 2 (1): 120-128.
Claeyssens, M., Nerinckx, W. and Piens, K.(1998). Carbohydrases from Trichoderma reesei and
other microorganisms: structures, biochemistry, genetics and applications. Cambridge:
The Royal Society of Chemistry. pp 65-98.
Conesa, A., Punt, P. J., Luijk, N. and Hondel, C. A. (2001). The secretion pathway in
filamentous fungi: a biotechnological view. Fungal Genetics and Biology, 33: 155-171.
Coughlan, M. P. (1985). The properties of fungal and bacterial cellulases with comment on their
production and application. Biotechnology and Genetic Engineering Review, 3: 39-109.
Coutinho, P. M. and Henrissat, B. (1999) The modular structure of cellulases and other
carbohydrate-active enzymes: an integrated database approach. In: Ohmiya, K.,
Hayashi, K., Sakka, K., Kobayashi, Y., Karita, S., Kimura, T. (Eds). Genetics,
biochemistry and ecology of cellulose degradation, Uni Publishers Co., Tokyo, Japan.
pp. 15–23.
Das, A., Paul, T., Halder, S. and Maity, C. (2013). Study on the regulation of growth and
biosynthesis of cellulolytic enzymes from newly isolated fumigatus ABK9. Polish
Journal of Microbiology, 62(1): 31-43.
de Castro, A. M., de Albuquerque de Carvalho, M. L., Leite, S. G. and Pereira, N. (2010)
Cellulases from Penicillium funiculosum: production, properties and application to
cellulose hydrolysis. Journal of Industrial Microbiology and Biotechnology, 37: 151-
158.
Duggleby, R. G. (1986). Progress curves of reactions catalysed by unstable enzymes. A
theoretical approach. Journal of Theoretical Biology, 123(1): 67-80.
Eberhart, B. M., Beek, R. S. and Goolsby, K. M. (1977). Cellulase of Neurospora crassa.
Journal of Microbiology, 130: 181-186.
Eriksen, J. and Goksoyr, J. (1977). Cellulases from Chaetomium thermophile var. dissitum.
European Journal of Biochemistry, 45: 445-450.
Foreman, P. K., Brown, D., Dankmeyer, L., Dean, R., Diener, S., Dunn-Coleman, N. S.,
Goedegebuur, F., Houfek, T. D., England, G. J., Kelley, A. S., Meerman, H. J.,
Mitchell, T., Mitchinson, C., Olivares, H. A., Teunissen, P. J., Yao, J. and Ward, M.
(2003). Transcriptional regulation of biomass-degrading enzymes in the filamentous
fungus Trichoderma reesei. Journal of Biological Chemistry, 278: 31988-31997.
91
Galante, Y. M., De Conti, A. and Monteverdi, R. (1998). Application of Trichoderma enzymes
in textile industry. In: Harman, G. F, Kubicek, C. P. (Eds.). Trichoderma and
Gliocladium-Enzymes, biological control and commercial applications. Vol. 2. London:
Taylor and Francis. pp. 311–326.
Gao, J., Weng, H., Zhu, D., Yuan, M., Guan, F. and Xi, Y. (2008). Production and
characterization of cellulolytic enzymes from the thermoacidophilic fungal Aspergillus
terreus M11 under solid-state cultivation of corn stover. Bioresource Technology, 99:
7623- 7629.
Gardner, K. H. and Blackwell, J. (2004) The structure of native cellulose. Biopolymers. 13:
1975- 2001.
Garg, S. K. and Neelkantan, S. (1982). Effect of nutritional factors on cellulose enzyme and
microbial protein production by Aspergillus terrus and its evaluation. Biotechnology
and Bioengineering, 24: 109-125.
Gautam, S., Bundela, P., Pandey, A., Awasthi, M. and Sarsaiya, S. (2010). Effect of different
carbon sources on production of cellulases by Aspergillus niger. Journal of Applied
Sciences in Environmental Sanitation, 5(3): 295-300.
Ghose, T.K. (1997). Measuring of cellulase activities. Pure and Applied Chemistry, 59: 257-
268.
Green, A. A. and Hughes, W. L. (1955). Protein solubility on the basis of solubility in aqueous
solutions of salts and organic solvents. Methods in Enzymology. 1:67‐90.
Golan, A. E. (2011). Cellulases, types and actions, mechanism and uses. Nova Science
Publishers, Inc. New York. p.4
Gomes, I., Gomes, J., Gomes, D. J. and Steiner, W. (2000). Simultaneous production of high
activities of thermostable endoglucanase and betaglucosidase by the wild thermophilic
fungus Thermoascus aurantiacus. Applied Microbiology and Biotechnology, 53: 461-
468.
Gubitz, G. M., Mansfield, S. D., Bohm, D. and Saddler, J. N. (1998). Effect of endoglucanases
and hemicellulases in magnetic and flotation deinking of xerographic and laser-printed
papers. Jornal of Biotechnology, 65: 209-215.
Guo, R., Ding, M., Zhang, S., Xu, G. and Zhao, F. (2008). Molecular cloning and
characterization of two novel cellulase genes from the mollusk Ampullaria crossean.
Journal of Comparative Physiology, 178: 209-215.
92
Guowei, S., Hui, Y. and, He, C. (2013). Research on extraction and characterization of cellulase
from commercial enzyme preparation. Advanced Journal of Food Science and
Technology, 5(7): 839-842.
Haight, K. G and Gump, B. H. (1994). The use of macerating enzymes in grape juice
processing. American Journal Enology and Viticulture, 45: 113-116.
Harvey W. B., Stephen, D. and Daniel C. W. (Eds) (1983). Comprehensive Biotechnology 3:
862- 871.
Henrissat, B. A. (1991). Classification of glycosyl hydrolases based on amino acid sequence
similarities. Biochemical Journal, 280: 309-316.
Henrissat, B. and Bairoch, A. (1993). New families in the classification of glycosyl hydrolases
based on amino acid sequence similarities. Biochemical Journal, 293: 781-788.
Henrissat, B., Teeri, T. T., and Warren, R. A. (1998). A scheme for designating enzymes that
hydrolyse the polysaccharides in the cell walls of plants. FEBS Letters, 425: 352-354.
Henrissat, B; Bairoch, A. (1996). Updating the sequence-based classification of glycosyl
hydrolases. Biochemical Journal, 316: 695-696.
Himmel, M. E ,, Ruth, M. F. and Wyman, C. E. ( 1999). Cellulase for commodity products from
cellulosic biomass . Current Opinions in Biotechnology, 10: 358–364.
Hinz, S. W., Pouvreau, L., Joosten, R., Bartels, J., Jonathan, M. C., Wery, J. and Schols, H. A.
(2009). Hemicellulase production in Chrysosporium lucknowense C1. Journal of
Cereal Science, 50: 1- 6.
Harriette, C. and Charles, J. M. (1913). The precipitation of egg albumin by ammonium
sulphate: A contribution to the theory of salting out of proteins. Biochemical Journal.
7(4): 380-398
Hongpattarakere, T. (2002) Hyperthermostable cellulolytic and hemicellulolytic enzymes and
their biotechnological applications. Journal of Science and Technology, 24: 481-491.
Hu, Y. J. and Wilson, D. B. (1988 ) Cloning of Thermonospora fusca genes coding for beta-1,4-
endoglucanases E1, E2 and E5. Gene, 71: 331-337.
Immanuel, G., Bhagavath, P., Iyappa, P. and Esakkiraj, A. (2006). Production and partial
purification of cellulase by Aspergillus niger and Aspergillus fumigatus fermented in
coir waste and sawdust. The Internet Journal of Microbiology, 3(1): 221-229.
93
Irwin, D. C., Zhang, S. and Wilson, D. B. (2000 ). Cloning, expression and characterization of a
family 48 exocellulase , Cel48A from Thermobifida fusca. European Journal of
Biochemistry, 267: 4988-4997.
Jagtap, S. and Rao, M. (2005). Purification and properties of a low molecular weight 1,4-beta-d-
glucan glucohydrolase having one active site for carboxymethyl cellulose and xylan
from an alkalothermophilic Thermomonospora sp. Biochemical and Biophysical
Research Communication, 329: 111-116.
Jatinder, K., Chadha, B. S. and Saini, H. S. (2006). Optimization of medium components for
production of cellulases by Melanocarpus spp. MTCC 3922 under solid-state
fermentation. World Journal of Microbiology and Biotechnology, 22: 15-22.
Jorgensen, H., Kristensen, J. B and Felby, C. (2007). Enzymatic conversion of lignocellulose
into fermentable sugars: Challenges and opportunities. Biofuels Bioproducts and
Biorefining, 1: 119- 134.
Ju, L. K. and Afolabi, O. A. (1999). Waste papers hydrolysate as soluble inducing substrate for
cellulase production in continuous culture of Trichoderma reesei. Biotechnology
Progress, 15: 91-97.
Kachlishvili, E., Penninckx, M. J., Tsiklauri, N. and Elisashvili, V. (2006). Effect of nitrogen
source on lignocellulolytic enzyme production by white-rot basidiomycetes under solid
state cultivation. World Journal of Microbiology and Biotechnology, 22: 391-397.
Kalogeris, E., Christakopoulos, P., Katapodis, P., Alexiou, A., Vlachou, S., Kekos, D. and
Macris, B. J. (2003). Production and characterization of cellulolytic enzymes from the
thermophilic fungus Thermoascus aurantiacus under solid state cultivation of
agricultural wastes. Process Biochemistry, 38: 1099-1104.
Kang, M. S., Im, W. T., Jung, H. M., Kim, M. K., Goodfellow, M. and Kim, K. K. (2007).
Cellulomonas composti sp. nov., a cellulolytic bacterium isolated from cattle farm
compost. International Journal of Systematic Evolutionary Microbiology, 57: 1256-
1260.
Karboune, S., Geraert, P. A. and Kermasha, S. (2008). Characterization of selected cellulolytic
activities of multi-enzymatic complex system from Penicillium funiculosum. Journal of
Agriculture and Food Chemistry, 56: 903-909.
Khandke, K. M., Vithayathil, P. J. and Murthy, S. K. (1989). Purification of xylanase, beta-
glucosidase, endocellulase, and exocellulase from a thermophilic fungus, Thermoascus
aurantiacus. Archives of Biochemistry and Biophysics, 274: 491-500.
Kim, D. W., Yang, J. H. and Jeong, Y. K. (1988). Adsorption of cellulose from Trichoderma
viride on microcrystalline cellulose. Applied Microbiology and Biotechnology, 28: 148-
154.
94
Klemm, D., Heublein, B., Fink, H. P. and Bohn, A. (2005) Cellulose: Fascinating biopolymer
and sustainable raw material. Polymer Science, 44: 3358-3393.
Klemm, D., Schmauder, H. P. and Heinze, T. (2002) In: Biopolymers: Polysaccharides II;
Vandamme E., De Baets A., Steinbu¨chel A., (Eds)., Wiley-VCH, Weinheim. pp. 275-
319.
Kuhad, R. C., Singh, A. and Eriksson, K. E. (1997) Microorganisms and enzymes involved in
the degradation of plant fiber cell walls. Advances in Biochemical Engineering and
Biotechnology, 57:45-125.
Lee, Y. J., Kim, B. K., Lee, B. H., Jo, K. I and Lee, N. K. (2008). Purification and
characterization of cellulase produced by Bacillus amyoliquefaciens DL-3 utilizing rice
hull. Bioresources Technology, 99: 378-386.
Lowry, O.H., Rosebrough, N.J., Farr, A. l. and Randall. R.J .(1951). Protein measurements
with follin –phenol reagents. Journal of Biological Chemistry, 93:265-275.
Lu, M., Li, D. and Zhang, C. (2002). Purification and properties of an endocellulase from the
thermophilicfungus Chaetomium thermophile.Wei Sheng Wu Xue Bao, 42: 471-477.
Lykidis, A., Mavromtis. K., Ivanova, N., Anderson, I., Land, M., Dibatolo G., Martinez M. and
Lapidus, A. (2007). Genome sequence and analysis of the soil cellulolytic
actinomycete Themofida fusca. Journal of Bacteriology, 189(6): 2477-2489.
Lynd, L. R., Weimer, P. J., van Zyl, W. H. and Pretorius, I. S. (2012) Microbial cellulose
utilization: fundamentals and biotechnology. Microbiology and Molecular Biology
Review, 66: 506-577.
Machado, C. M., Oishi, B. O, Pandey, A. and Soccol, C.R. (2004) Kinetics of Gibberella
fujikori growth and gibberellic acid production by solid state fermentation in a packed-
bed column bioreactor. Biotechnology Progress, 20: 1449-1453.
Macris, B. J., Kekos, D. and Evangelidou, E. (1989). A simple and inexpensive method for
cellulose and β-glucosidase production by Neurospora crassa. Applied Microbiology
and Biotechnology, 31: 150-151
Marques, S., Pala, H., Alves, L., Amaral-Collaco, M. T., Gama, F. M. and Girio, F. M.(2003).
Characterisation and application of glycanases secreted by Aspergillus terreus CCMI
498 and Trichoderma viride CCMI 84 for enzymatic deinking of mixed office waste
papers. Journal of Biotechnology, 100: 209-219.
Marsden, W. L. and Gray, P. P. (1986). Enzymatic hydrolysis of cellulose in lignocellulosic
materials. Critical review of biotechnology, 3:235-246.
95
Martins, L.F., Kolling, D., Camassola, M., Dillon, A.J., Ramos, L.P. (2008). Comparison of
Penicillium echinulatum and Trichoderma reesei cellulases in relation to their activity
against various cellulosic substrates. Bioresource Technology, 99: 1417–1424.
Matheus P., Vinícios P. and Ademir J. Z. (2013). Structural characteristics and therm al
properties of native cellulose. Retrieved December 3, 2013 from the world wide web:
http://www.intechopen.com/books/cellulose-fundamental-aspects/structural-
characteristics-and-thermal-properties-of-native-cellulose
Mawadza, C., Rajni, H., Remigio, Z. and Mattiasson, B. (2000). Purification and
characterization of cellulases produced by two Bacillus strains. Journal of
Biotechnology. 83: 177–187.
McParland, J. J., Grethlein, H. E. and Converse, A. O. (1982). Kinetics of acid hydrolysis of
corn stover. Solar energy, 28: 55-73
Menon, K., Rao, K. K. and Pushalkar, S. (1994). Production of β-glucosidase by Penicillium
rubrum O stall. Indian Journal of Experimental Biology, 32: 706-709.
Miettinen-Oinonen, A. (2004). Trichoderma reesei strains for production of cellulases for the
textile industry. VTT Publications, Finland, Espoo. pp. 67- 79.
Miettinen-Oinonen, A. and Suominen, P. (2002). Enhanced production of Trichoderma reesei
endoglucanases and use of the new cellulase preparations in producing the stonewashed
effect on denim fabric. Applied Environmental Microbiology, 68: 3956-3964.
Miettinen-Oinonen, A., Londesborough, J., Joutsjoki, V., Lantto, R. and Vehmaanperä, J. (2004)
Three cellulases from Melanocarpus albomyces for textile treatment at neutral pH.
Enzyme Microbial Technology, 34: 332-341.
Milala, M. A., Shugaba, A., Gidado, A. C., Ena, D. and Wafar, J. (2005). Studies on the use of
agricultural wastes for cellulase enzyme production by Aspergillus niger. Journal of
Agriculture and Biological Science, 1(4): 325-328.
Mirhendi, H. (2000). Aspergillus fumigatus. Retrieved December 2, 2013 from the world wide
web: www.pfdb.net.html.species/s11.htm
Mishra, B. K. and Nain, L. (2010). Rice straw as a substrate for lignocellulolytic
enzymesproduction form Phanerochaete chrysosporium and cellulolytic bacteria.
Journal of Mycology and Plant Pathology, 40: 110-114
Mitchinson, C. and Pain, R.H. (1986). The effect of sulphate and urea on the stability and
reversible unfolding of β‐lactamase from Staphylococcus aureus. Journal of Molecular
Biology. 184:331‐342.
96
Moon, T. and Nagarajan, R. (1998). Deinking of xerographic and laser-printed paper using
block copolymers. Colloids and Surfaces A: Physiochemical Engineering Aspects, 132:
275-288.
Mohammad M. J., Ikram, U. H., Tehmin, S. K. and Zafar S. (2005). Cotton saccharifying
activity of cellulases produced by co-culture of Aspergillus Niger and Trichoderma
Viride. Research Journal of Agriculture and Biological Science. 1(3): 241-245.
Mohagheghi, A., Grohmann, K., Himmel, M., Leighton, L. and Updegraff, D. M. (1986).
Isolation and characterization of Acidothermus cellulolyticus gen. nov., sp. nov., a new
genus of thermophilic, acidophilic, cellulolytic bacteria. International Journal of
Systematic Bacteriology, 36: 435-443.
Morana, A., Maurelli, L., Ionata, E., La Cara F. and Rossi M. (2011) Cellulases from fungi and
bacteria and their biotechnological applications In Golan, A. E. (Ed.), Cellulases, types
and actions, mechanism and uses. (pp. 1-80) Nova Science Publishers, Inc. New York.
Morbak, A. L. and Zimmermann, W. (1998). Deinking of mixed office paper, old newspaper
and vegetable oil based ink printed paper using cellulase, xylanases and lipases.
Progress in Paper Recycling, 7: 14-27.
Mukhopadhyey, S. and Nandi, B. (1999). Optimization of cellulose production by Trichoderma
reesei ATTCC 26921 using a simplified medium on water hyacinth biomass. Journal of
Scientific and industrial Research, 58: 107-111.
Nascimento, R. P. and Coelho, R. R. (2011). Cellulases: From production to biotechnological
applications. In: Golan, A. E. (Ed.) Cellulases, types and actions, mechanism and uses.
Nova Science Publishers, Inc. New York.
Nelson, D. and Cox, M. (2000). Lehninger Principles of Biochemistry, 3rd
Edn.. Worth
Publishers, New York, p. 214.
Nishiyama, Y., Sugiyama, J., Chanzy, H. and Langan, P. (2002). Crystal structure and hydrogen
bonding system in cellulose Ir from synchrotron X-ray and neutron fiber diffraction.
Journal of American Chemical Society, 124: 9074–9082.
Nwobodo, C. S. and Okochi V. I. (2011). Cellulase production by wildtype Aspergillus niger,
Penicillium chrysogenum and Trichoderma harzanium using waste cellulosic materials.
Ife Journal of Sciences, 13(1): 57-62.
Okafor, J. C. (1985). Selection and improvement of indigenous tropical fruit trees: Problems and
prospects. Journal of Forest Reserve. 1(2): 87-95.
Okafor, J. C. and Okolo, H. C. (1974). Potentials and some indigenous fruit trees of Nigeria.
Paper Presented to the 5th Annual Conference of the Forestry Association of Nigeria,
Jos.
97
Okafor, J.C. (1990). Indigenous Trees of the Nigerian Rainforest. A Paper Presented in a
Symposium on the Potentials for Domestication and Rebuilding of Forest Resources.
Yaounde, Cameroon, pp.34-38.
Oksanen, J., Ahvenainen, J. and Home, S. (1985). Microbial cellulase for improving filtrability
of wort and beer. Proceedings of European Brewery and Chemistry Helsinki, 5: 419-
425.
Ole, K., Torben, V. B. and Claus, C. F. (2002). Industrial enzyme applications. Current
Opinions in Biotechnology, 13: 345-351.
Onyekwelu, J. C. and Fayose, O. J. (2007). Effect of storage methods on the germination and
proximate composition of Treculia africana seeds. Paper Presented at the Conference
on International Agricultural Research for Development. Tropentas, Germany,
O'Sullivan, A. C. (1997). Cellulose: the structure slowly unravels. Cellulose, 4: 173-207.
Paakkari, T., Serimaa, R. and Fink, H. P. (1989). Structure of amorphous cellulose. Acta
polimerica, 40: 731-734.
Panagiotou, G., Kekos, D., Macris, B. J. and Christakopoulos, P. (2003). Production of
cellulolytic and xylanolytic enzymes by Fusarium oxysporum grown on corn stover in
solid state fermentation. Industrial Crop Production, 18: 3745-3752.
Pardo, A. G. and Forchiassin, F. (1999). Influence of temperature and pH on cellulase activity
and stability in Nectria catalinensis. Revista Argentina de Microbiologia, 31: 31-35.
Parry, N. J., Beever, D. E., Owen, E., Nerinckx, W., Claeyssens, M. and Van Beeumen, J.
(2002). Biochemical characterization and mode of action of a thermostable
endoglucanase purified from Thermoascus aurantiacus. Archives of Biochemistry and
Biophysics, 404: 243-253.
Paul, J. and Verma, A. (1990). Influence of sugars on endoglucanase and β-xylanase of a
bacillus strain. Biotechnology Letters, 22: 61-64.
Payen, A. (1938). Mémoire sur la composition du tissu propre des plantes et du ligneux. C R
Hebdomadaires des Seances de L Academie des Sciences, 7: 1052–1056.
Peter, K. L. (2008). Cellulose biosynthesis. Retrieved December 15, 2013 from the World Wide
Web: http://www.public.iastate.edu/pkeeling/glucanbio_files/cellulosesynthesis.htm
Plank, P. F. and Zent, J. R. (1993). Use of enzymes in wine making and grape processing. In:
Gump, B. H., Pruett, D.J. (Eds). Beer and wine production analysis, characterisation,
and technological advances. Washington : American Chemical Society DC. pp. 191-
196.
98
Rahna, K. R., Sangeetha, G., Maringa, T. and Selby. A. (2012). Effective utilization of aquatic
weed Salvinia molesta as substrate for production of cellulase enzyme- Eradication
through utilization. International Journal of Environmental Science, 3(1): 497-501.
Raim, B. M. (1998). General microbiological aspects of solid substrate fermentation. Electronic
Journal of Biotechnology, 1:3-9.
Rajendran, A., Gunasekaran, P. & Lakshmanan, M. (1994). Cellulase activity of Humicola
fuscoatra. Indian Journal of Microbiology, 34: 289-295.
Reese, E. T and Mandels, M. (1971) Enzymatic degradation. In: Bikales, N. M. and Segal, L.
(Eds). Cellulose and cellulose derivatives. Wiley Interscience, New York. pp. 1079-
1094.
Ruijter, G .J. G. and Visser, J. (1997). Carbon repression in Aspergilli. FEMS Microbiology.
Letters., 151:103- 114.
Sakon, J., Irwin, D., Wilson, D.B., Karplus, P.A.(1997). Structure and mechanism of
endo/exocellulase E4 from Thermomonospora fusca. Natural Structural Biology. 4:
810-818.
Sarawasti B., Ravi, K., Mukosh, M.D. and Bala, D. (2012) Cellulase production by Bacillus
subtilis isolated from cow dung. Achives of Applied Science Research, 4(1): 269-279.
Saxena, I. M., Brown, R. M. and Dandeka, T. (2000). Structure-function characterization of
cellulose synthase: relationship to other glucosyltransferases. Phytochemistry, 57:1135-
1148.
Schülein, M. (1997). Enzymatic properties of cellulases from Humicola insolens. Journal of
Biotechnology, 57: 71-81.
Shang, T. Y. (2013). Bioprocessing technologies and biorefinery for sustainable production of
fuels, chemicals and polymers.1st
Edn. John Wiley and Sons Inc, New York, pp. 131-
146.
Shanmu, K., Saravana, P., and Joseph, S. (2012). Isolation, screening and partial purification of
cellulase producing bacteria. International Journal of Advanced Biotechnology and
Research, 3(1): 509-514.
Shiang, M., Linden, J. C., Mohagheghi, A., Grohmam, K. & Himmel, M. E. (1991).
Characterization of eng F, a gene for a non-cellulosomal Clostridium cellulovoras
endoglucanase. Gene, 182: 163-167.
99
Shufang, W., Shaojun, D. and Zhongzheng, L. (2005). Function of endocellulase in the
deinking process of mixed office waste paper. Chemistry and Industry of Forest
Products, 25: 87-90.
Singh, A., Abidi, A. B., Darmwal, N. S. and Agrawal, A. K. (1990). Saccharification of
cellulosic substrates by Aspergillus niger cellulase. World Journal of Microbiology and
Biotechnology, 6: 333-336.
Singh, A., Abidi, A. B., Darmwal, N. S. and Agrawal, A. K. (1991). Influence of nutritional
factors on cellulose production from natural cellulosic residues by Aspergillus niger
AS101. Agriculture and Biology Research, 7: 19-27.
Sonde, C. U. and Odoemelam, S. A. (2012). Sorption studies on the use of African Breadfruit
(Treculia africana) seed hull as adsorbent for the removal of Cu2+, Cd2+ and Pb2+
from aqueous solutions. American Journal of Physical Chemistry. 1(1): 11-21.
Steiner, J., Saccha, C. and Enzyaguirre, J. (1993). Culture condition for enhanced cellulose
production by a native strain of Penicillium purpurogenum. World Journal of
Microbiology and Biotechnology. 10: 280-284.
Subramaniyam, R. and Vimala, R. (2012). Solid state and submerged fermentation for the
production of bioactive substances: A comparative study. International journal of
science and nature, 3(3): 480-486
Sunil, K., Brijesh, K., Mishra, T. and Subramanian, P. (2011) Cellulases from fungi and bacteria
and their biotechnological applications. In: Golan, A. E. (Ed.) Cellulases, types and
actions, mechanism and uses. Nova Science Publishers, Inc. New York.
Sunita, A. and Sumit R. D. (2012). Cellulase production from from trichoderma viride and
trichoderma reseei using saw dust and coir waste as carbon source. International
Journal of Pharmacy and Life Sciences, 2(4): 22-24.
Suurnäkki, A., Tenkanen, M., Siika-aho, M., Niku-Paavola, M. L., Viikari, L. and Buchert, J.
(2000). Trichoderma reesei cellulases and their core domains in the hydrolysis and
modification of chemical pulp. Cellulose, 7: 189-209.
Takai, K, Nakamura, K, Toki, T., Tsunogai, U., Miyazaki, M. and Miyazaki, J. (2008). Cell
proliferation at 122°C and isotopically heavy CH4 production by a hyperthermophilic
methanogen under high-pressure cultivation. Proceedings of the National Academy of
Sciences USA, 105: 10949–10954
Takashima, S., Nakamura, A., Masaki, H. and Uozumi, T. (1996). Purification and
characterization of cellulases from Humicola grisea. Bioscience Biotechnology and
Biochemistry, 60: 77-82.
100
Tong, C. C., Cole, A. L and Shephard, M. G. (1980). Purification and properties of the
cellulases from thermophilic fungus Thermomoascus auranticus. Biochemistry journal
of Great Britain, 191: 83-94.
Tzi B. U and Randy C. F. (2011). Cellulase: types, actions, mechanisms and uses. In: Golan, A.
E. (Ed.) Cellulases, types and actions, mechanism and uses. Nova Science Publishers,
Inc. New York. pp.251-264.
Van-Wyk, J. P. H. (1997). Cellulose adsorption–desorption and cellulose saccharification during
enzymatic hydrolysis of cellulose material. Biotechnology Letters, 19: 775-778
Watanabe, H. and Tokuda, G. (2001). Animal cellulases. Cellular and Molecular Life Science.
58, 1167-1178.
Winiati, S. (2013) Prevention and reduction of mycotoxin by antagonistic microorganisms.
Retrieved December 2, 2013 from the world wide web: http://wpr.staff.ipb.ac.id/
Wojtczak, G., Breuil, C., Yamuda, J. and Saddler, J. N. (1987). A comparision of the
thermostability of cellulose from various thermophilic fungi. Applied Microbiology and
Biotechnology, 27: 82–87.
Wolgang, A. (2007). Enzymes in Industry. Wiley-VCH, Leiden. pp. 1-3
Wyman, C. E. (1999) Biomass ethanol: Technical progress, opportunities, and commercial
challenges. Annual Review of Energy and Environment, 24: 189- 193.
Yamada, M., Amano, Y., Horikawa, E., Nozaki, K. and Kanda, T. (2005). Mode of action of
cellulases on dyed cotton with a reactive dye. Biosciences Biotechnology and
Biochemistry, 69: 45-50.
Yeoh, H. H., Tan, T. K. and Koh, S. K. (1986). Kinetic properties of β-glucosidase from
Aspergillus ornatus. Applied Microbiology and Biotechnology, 25: 25-28.
Yingjuan, F., Menghua, Q. and Huiren, H. (2005). Effect of types and properties on the
deinkability of wastepaper. Trans Chin Pulp Paper, 20: 155-159.
Zhang, Y. H. P., Himmel, M. E. and Mielenz, J. R. (2006). Outlook for cellulase improvement:
Screening and selection strategies. Biotechnology Advances, 24:452–481.
101
APPENDICES
1.0 Preparation of Buffers
The standard buffers used in study were pH 4.0, pH 7.0 and pH 9.2. These buffers were used
to standardize the pH meter. The working buffers were prepared as thus: 0.05 M sodium
acetate and 0.05 M Tris-HCl buffers were prepared by dissolving 4.10 g sodium acetate salt
and 6.01 g Tris base, respectively in 1000ml of distilled water and stirred with a magnetic
stirrer till a homogenous solution was formed. The solutions were titrated againt acetic acid
and HCl, respectively till the required pHs were obtained. Also 0.05 M phosphate buffer was
prepared by dissolving 7.10g disodium hydrogen phosphate salt in 1000 ml stirred as for
sodium acetate and phosphate buffers and then titrated against the solution of its conjugate
acid, sodium dihydrogen phosphate till the required pHs were obtained.
1.1 Preparation of Dinitrosalicylic Acid (DNS) Reagent
A modification of DNS reagent method of Miller (1959) as contained in wang et al. (1997)
was used in the assay. The reagent contains 44 mM dinitrosalicylic acid, 4 mM sodium
sulphite, and 375 mM sodium hydroxide.
1.2 Preparation of Cellobiose
15 mM Cellobiose was prepared by dissolving 5.13 g cellobiose in distilled water and made
up to 1 litre
1.3 Preparation of Carboxymethyl Cellulose (CMC)
2% Carboxymethyl Cellulose was prepared by dissolving 2 g of carboxymethyl cellulose in
distilled water and made up to 100 ml.
102
1.4 Preparation of 50mM glucose
50 mM solution of glucose was prepared by weighing 0.9 g of industrial grade glucose and
dissolving in 100 ml of distilled water.
1.5 Calibration Curve for Glucose
A method described by Miller 1959 with little modifications by Wang et al., 1997 was used.
Ten test tubes were arranged in duplicate containing 0.0-1.0 ml of 50 mM glucose. Each tube
was made up to 1ml using 0.05 M sodium acetate buffer of pH 5.5. 1 ml DNS reagent was
added to each of the tubes and placed in boiling water bath for 10 mins. The tubes were then
removed and allowed to cool to room temperature. Na-K tartarate was added to the different
tubes to stabilize the colour, after which the absorbance was read at 540 nm. The
concentration of reducing sugar in each of them was calculated using the formula
“C1V1=C2V2” where:
C1= initial concentration of reducing sugar (mM)
V1= initial volume of the 50mM preparation measured into the tubes
C2= final concentration of reducing sugar (mM)
V2= final volume of the preparation measured in the tube
Using the value obtained from the table described above, the plot of optical density against
concentration was constructed.
1.4 Preparation of the Component Reagents For Protein Determination
Solution A: An alkaline sodium carbonate (Na2CO3) was prepared by dissolving 2 g of
Na2CO3 in 100ml of 0.1 M NaOH (0.4g of sodium hydroxide pellets were dissolved in 100
ml of distilled water).
Solution B: A copper tetraoxosulphate IV - sodium potassium tartarate solution was prepared
by dissolving 0.5 g of CuSO4 in 1 g of sodium potassium tatarate, all in 100 ml of distilled
water. It was prepared fresh by mixing stock solution, and so was done whenever required.
Solution C: Folin-Ciocalteau phenol reagent was made by diluting the commercial reagent
with water in a ratio of 1:1 on the day of use.
Solution D: Standard protein (Bovine Serum Albumin, BSA) solution.
103
Solution E: Freshly prepared alkaline solution was made by mixing 50 ml of solutions A and
1 ml of solution B.
1.6 Preparation of 2 mg/ml Bovine Serum Albumin (BSA) Standard Protein
0.2 g of BSA was dissolved in 100 ml of distilled water and then used as a protein stock
solution.
104
Protein Standard Curve, Using Bovine Serum Albumin (BSA)
105
Glucose Standard Curve
106