Table of Contents
Title page i
Certification ii
Dedication iii
Acknowledgments iv
Table of Contents v
List of Tables viii
List of Figures ix
List of Appendices x
Abstract xi
Introduction 1
Literature Review 4
Nature and Structure of Cellulose 4
Major Sources and Distribution of Cellulose 8
Micro-organisms Involved in Cellulose Degradation 9
Enzymes Involved in Cellulose Degradation 10
The Cellulase Complex 10
Cellulase Improvement 13
Production and Properties of Microbial Endo-β-Glucanase 20
Importance of Microbial Endo-β-Glucanase 23
Solid State Fermentation 24
Materials and Methods
Materials 28
Carbon Sources 28
Nitrogen Sources 28
Micro-organism used 28
Methods
Pretreatment of Carbon Substrates 29
Preparation of Cow Blood Meal 29
Screening for Cellulase Production 29
Preparation of Inoculum 30
The SSF System 30
Estimation of Moisture Content of the SSF System 30
Cultural Requirements for production of Endo- β-glucanase
in SSF System 31
Effect of Carbon Sources on Enzyme Production 31
Enzyme Extraction 31
Endo- β-Glucanase Assay 31
Effect of Concentrations of Corn Cob on Enzyme Production 32
Effect of Nitrogen Sources on Enzyme Production 32
Effect of Metal ions on Enzyme Production 33
Effect of Concentration of MnS04.H20 33
vi
Effect of Surfactants on Enzyme Production 34
Effect of Concentrations of SDS on the Production of
Endo- β-Glucanase 34
Effect of initial pH of the medium on Enzyme production 34
Time Course of Enzyme Synthesis 35
Estimation of Biomass 35
Relative Rate of Hydrolysis of Cellulosic Substrates 35
Results
Effect of Carbon (Lignocellulosic wastes) on Enzyme Production 36
Effect of Concentrations of Corn Cob on Enzyme Production 38
Effect of Nitrogen Sources on Enzyme Production 40
Effect of Metal ions on Enzyme Production 42
Effect of Concentration of MnS04.7H20 on Enzyme Production 44
Effect of Surfactant on Enzyme Production 46
Effect of Concentrations of SDS on Enzyme Production 48
Effect of Initial pH of Medium on Enzyme Production 50
Time Course of Endo-β-Glucanase Production 51
Relative Rates of Hydrolysis of Cellulose 54
Discussion 56
Conclusion 59
References 60
vii
List of Table
Page
Table 1: Effect of Carbon Sources on Enzyme Production 37
Table 2: Effect of Concentrations of Corn Cob on Enzyme
Production 39
Table 3: Effect of Nitrogen Sources on Enzyme Activity 41
Table 4: Effect of Metal ions on Enzyme Production 43
Table 5: Effect of Concentration of MnS04.7H20 on Enzyme
Production 45
Table 6: Effect of Surfactant on Enzyme Production 47
Table 7: Effect of Concentration of SDS on Enzyme Production 49
Table 8: Effect of Initial pH of Medium on Enzyme Production 51
Table 9: Relative Rates of Hydrolysis of Cellulose 55
viii
List of figures
Figure 1 : Conformation of Cellulose Molecule 5
Figure 2 : Models of the Structure of Microfibrils 7
Figure 3 : Scheme of Rational Protein Design 17
Figure 4 : Scheme of Directed Protein Evolution 18
Figure 5 : Time course of Endo-β-Glucanase Production 53
ix
List of Appendices
Appendix 1: Statistical analysis of variables represented
in various tables/graphs. 68
x
ABSTRACT
An endo-β-1, 4-glucanase (EC. 3.2.1.4) from a Neurospora sp isolated from a mouldy
corn cob was produced in a solid substrate fermentation (SSF) system of 30.2% moisture
level. The enzyme was optimally produced in 48h at an initial culture pH of 6.0 in a
medium consisting of corn cob, 5g; defatted melon meal, 1g; MnS04.H20, 2.5mM and
sodium dodecyl sulphate (SDS), 0.16%. The relative rates of hydrolysis of
carboxymethyl-cellulose, crystalline cellulose and crude lignocellulosic wastes by the
crude enzyme extract showed the preference of the enzyme to carboxymethyl-cellulose.
The least hydrolyzed cellulose was sigmacel.
1
INTRODUCTION
Cellulose is the most abundant renewable natural organic resource found in plants, some
micro-organisms, and industrial and agricultural residues. Pure cellulose does not occur
in nature but is always associated with varying amounts of a variety of non-cellulosic
materials especially hemicellulose and lignin (Lutzen et al., 1983).
Cellulose is a high molecular weight linear polymer containing glucose molecules
linked by β-1, 4-glycosidic bonds. It is degraded by a complex of three enzymes called
cellulases. These are:
i. Cellobiohydrolase or exoglucanase or exo-1,4-β-glucan cellobiohydrolase (EC.
3.2. 1.91)
ii. Endo-β-glucanase, also called endoglucanase or endo-1, 4-β-D-glucan 4-
glucanohydrolase (EC.3.2.1.4)
iii. Cellobiase or β-glucosidase (EC. 3.2.1.21)
The endo-β-glucanase is an endo-enzyme that attacks cellulose polymers
randomly resulting in rapid reduction in chain length and increase in reducing groups and
reduction in viscosity.
The cellobiase (β-glucosidase), however, does not attack cellulose but rather the
cello-oligosaccharides and cellobiose which inhibit the activities of cellobiohydrolase and
endo-β-glucanase. Thus its presence is essential in the complete hydrolysis of cellulose
(Halliwell, 1979). Plant biomass and agricultural wastes are in abundance, especially in
the tropical savannah and rainforest zones of Africa. Of fundamental importance is the
fact that many chemicals and materials must be produced from plant biomass once
petroleum, natural gas and coal are no longer affordable or acceptable for use in view of
their role in climate change (van Beihen,2008).
2
Cellulose degradation in nature ensures the maintenance of the carbon cycle and energy
transfer among living systems. Without it, dead vegetations would pile up and suppress
the formation of new ones causing the atmospheric carbon dioxide pool not to be
consumed by photosynthesis (Demain et al., 2005).
Microbial degradation of cellulose has the potential of providing food and energy. For
instance, the glucose resulting from cellulose degradation can be fermentatively
converted to ethanol and butanol (biofuels), acetone, isopropanol, and biomass in form of
single cell protein. In this way, they serve in the production of value-added goods as well
as serving as effective means of disposal of such wastes (Sauer, 2008).
Cellulose is degraded by a large number of organisms including the fungi,
actinomycetes, myxobacteria and the true bacteria. Most research on cellulase –
producing microorganisms has involved fungi’
Neurospora is a perfect ascomycete that is most thoroughly characterized
genetically and produces a complete set of cellulases. It is argued that Neurospora is a
most favourable organism for the degradation of hemicellulose. For instance,
Deshphande et al.(1986) reported the direct conversion of hemicellulose and cellulose to
ethanol by Neurospora and Phadtare et al.(1997) reported of Neurospora’s unique ability
to convert biomass to ethanol.
Neurospora is peculiarly non-pathogenic, perhaps because it normally has the luxury of
growing in a fire scoured landscape, without competitors. So far, no infection or
intoxication by Neurospora has ever been reported in human beings, including immune
compromised people, or in any live animal or plant (Zhao et al., 1998).
Neurospora is closely related to several industrially important organisms such as
Hypocrea jecorina. It is widely consumed as a foodstuff in parts of southeast Asia as the
microbial agent in a cultured pressed peanut or soybean cake called “oncham” (The
3
Scientist, 1996). There are reports on the production of cellulase degrading enzymes by
species of Neurospora ( Oguntimien et al., 1991).
These studies were carried out by submerged fermentation, a process which is
cost-intensive and requires strict asepsis.
In order to reduce the cost of cellulase production, this study aimed at :
i. Optimizing the cultural conditions for endo-β-glucanase production by a
Neurospora sp in a solid substrate fermentation (SSF) system, and
ii. studying the kinetics of the degradation of some cellulosic wastes using the crude
endo-β-glucanase.
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