PRODUCTION, PARTIAL PURIFICATION AND CHARACTERIZATION OF CELLULASE BY ASPERGILLUS NIGER FROM SUBMERGED FERMENTATION OF GRAPE BAGASSE

ABSTRACT
The increase in agricultural practices has necessitated the judicious use of agricultural wastes into value added products. In this study the ability of selected cellulosic substrate to induce cellulase production by Aspergillus niger and the ability of the induced enzyme to hydrolyze the cellulosic substrate were assessed. The enzyme was produced by submerged fermentation technique in which grape bagasse was the cellulosic substrate which served as a carbon source. Crude enzyme was harvested after 5 days of growth with activity of 8.2μmole/min for enzyme produced by Aspergillus niger. Cellulase produced from Aspergillus niger was subjected to a three step purification process: 50% ammonium sulphate precipitation, dialysis and gel column chromatography for characterization of the cellulase. The gel column chromatography yielded two peaks. Gel elution fractions were assayed for total cellulase activity and protein concentration. The 2 peaks indicate isoforms of the enzyme produced by Aspergillus niger. The total cellulase activity as well as β-glucosidases activity was characterized using filter paper and cellobiose as substrate. The partially purified enzyme showed that total cellulase activity had an optima pH and temperature of 5.5 and 50oC for isoform A and 5.0 and 55oC for isoform B using filter paper as substrate. Similarly, β-glucosidases activity had an optima pH 5.5 and 6.0 with optima temperature of 50oC for both isoforms using cellobiose as substrate. Kinetic parameter showed a Vmax and Km of 90.9μmole/min and 0.09mM cellobiose and 83.3μmole/min and 0.08mM cellobiose for both isoforms respectively. This kinetic study showed that grape bagasse is a good substrate for cellulase from Aspergillus niger and can be utilized as substrate for cellulase production. These results obtained in this study have established suitable conditions for maximizing the production of cellulase which is used for conversion of high cellulosic waste into wealth as found in bioethanol production.

TABLE OF CONTENTS

Cover page i
Title page ii
Approval iii
Dedication iv
Acknowledgement v
Abstract vi
Table of contents vii
List of figures xii
List of tables xiv
List of Appendices xv

CHAPTER ONE: INTRODUCTION
1.0 Introduction 1
1.1 Cellulose 2
1.1.1 History of cellulose 2
1.1.2 Structure of Cellulose 3
1.1.3 Properties of cellulose 4
1.2 Lignocellulosic substrate 4
1.2.1 Methods of lignocellulose substrate pre-treatment 5
1.2.1.1 Mechanical pulverization 5
1.2.1.2 Pyrolysis 5
1.2.1.3 Acid treatment 6
1.2.1.4 Alkali treatment 6
1.2.1.5 Biological treatment 6
1.2.1.6 Ozonolysis 7
1.3 Cellulase 7
1.3.1 Molecular Structure of Cellulases 8
1.3.1.1 Catalytic Binding Domain (CD) 9
1.3.1.2 Cellulose binding Domains (CBD) 9
1.3.2 Properties of Cellulase 10
1.3.3 Microorganisms Producing Cellulases 10
1.3.4 Components of Cellulase 11
1.3.4.1 Endoglucanses (EC 3.2.1.4) 11
1.3.4.2 Exoglucanase (EC 3.2.1.91) 11
1.3.4.3 β-Glucosidase (EC 3.2.1.21) 12
1.3.5 Enzymatic Degradation of Cellulose 13
1.3.6 Adsorption Characteristics 14
1.3.7 Synergism of Cellulases 15
1.3.7.1 Synergy between exoglucanase and β-glucosidase 15
1.3.7.2 Endo-exo synergism 16
1.3.7.3 Exo-exo synergism 16
1.3.8 Cellulase assays 16
1.3.8.1 Substrate for cellulase activity assays 17
1.3.8.1.1 Soluble substrate 17
1.3.8.1.2 Insoluble substrates 17
1.3.9 Cellulase Activities 17
1.3.9.1 Total cellulase activity assay 17
1.3.9.2 Exoglucanase activity assay 18
1.3.9.3 Endoglucanase activity assay 18
1.3.9.4 β-glucosidase activity assay 18
1.3.10 Factors affecting cellulase activities 18
1.3.10.1 Effect of temperature on cellulase activity 18
1.3.10.2 Effect of pH on cellulase activity 18
1.3.11 Application of cellulases in industries 19
1.3.11.1 Textile and laundry biotechnology 19
1.3.11.2 Pulp and paper biotechnology 19
1.3.11.3 Bioethanol industry 20
1.3.11.4 Food processing industry 20
1.3.11.5 Animals feed industry 20
1.3.12 Economic feasibility of cellulase 20
1.4 Aspergillus niger 21
1.4.1 Scientific classification of Aspergillus niger 21
1.4.2 Macroscopic features 22
1.4.3 Preference of fungal over bacterial species 22
1.4.4 Advantages and disadvantages of using Aspergillus niger 23
1.5 Fermentation 23
1.5.1 Solid-state fermentation (SSF) 24
1.5.2 Submerged fermentation 24
1.5.3 Substrates used for fermentation 24
1.6 Grape fruit 25
1.6.1 Scientific classification of grape fruit 25
1.6.2 Grape bagasse 26
1.6.3 Composition of grape bagasse 26
1.6.4 Usefulness of grape bagasse 26
1.7`Aim of research 26
1.8 Research objectives 27

CHAPTER TWO: MATERIALS AND METHODS
2.0 Materials and Methods 28
2.1 Materials 28
2.1.1 Sources of grape fruits 28
2.2 Reagents and Chemicals 28
2.3 Apparatus 29
2.4 Collection of microorganism 29
2.5 Method 29
2.5.1 Preparation of grape bagasse 29
2.5.2 Isolation of Cellulolytic fungi 30
2.5.2.1 Preparation of Liquid Broth 30
2.5.2.2 Inoculation of Plates and Sub culturing 30
2.5.2.3 Storage of Pure Fungal Isolates 30
2.5.2.4 Fungal Identification 30
2.5.3 Fermentation Experiment 31
2.5.3.1 Fermentation Broth 31
2.5.3.2 Inoculation of the Broth 31
2.5.3.3 Mass production of enzyme 31
2.5.3.4 Harvesting of the fermented Broth 31
2.5.4 Protein Determination 31
2.5.4.1 Principle of Protein Determination 31
2.5.4.2 Procedure for Protein Determination 32
2.5.5 Determination of glucose 32
2.5.5.1 Principle of glucose determination 32
2.5.5.2 Procedure for glucose Determination 32
2.6 Cellulase Activity 32
2.6.1 Determination of total cellulase activity 32
2.6.2 Determination of endoglucanase activity (Carboxymethyl cellulose assay) 33
2.6.3 Determination of β – 1, 4-glucosidase activity 33
2.7 Enzyme purification 34
2.7.1 Determination of percentage Ammonium sulphate Saturation suitable for cellulase precipitation 34
2.7.1.1 Ammonium Sulphate Precipitation of Cellulase 34
2.7.2 Dialysis 34
2.7.3 Gel filtration column chromatography 35
2.7.3.1 Principle of gel filtration 35
2.7.3.2 Swelling of gel 35
2.7.3.3 Packing/Filling of the column` 35
2.7.3.4 Application of sample 35
2.7.3.5 Collection of column fractions 35
2.8 Studies on purified enzyme 36
2.8.1 Effect of pH on cellulase activity 36
2.8.2 Effect of pH on β- glucosidase activity 36
2.8.3 Effect of temperature on cellulase activity 36
2.8.4 Effect of temperature on β- glucosidase activity 36
2.8.5 Effect of substrate concentration on cellulase activity 36
2.8.6 Effect of substrate concentration on β- glucosidase activity 37
2.8.7 The effect of incubation time on the total cellulase activity 37
2.8.8 Further Studies with Partially Purified Enzyme 37

CHAPTER THREE: RESULTS
3.0 Results 38
3.1 Grape Bagasse 38
3.1.1 Studies on Crude Enzyme 38
3.1.1.1 Protein concentration of crude enzyme 38
3.1.1.2 Enzyme Activity 38
3.1.1.2.1 Cellulase activities of crude enzymes 38
3.2 Effect of incubation period 39
3.3 Ammonium sulphate precipitation profile of cellulase 40
3.4 Dialysis 42
3.5 Gel filtration column chromatography 42
3.6 Characterization of partially purified enzyme 53
3.6.1 Effect of incubation time on cellulase activity at 50oC 53
3.6.2 Effect of pH using filter paper as substrate 56
3.6.3 Effect of pH using cellobiose as substrate 59
3.6.4 Effect of temperature change using filter paper as substrate 62
3.6.5 Effect of temperature change using cellobiose as substrate 66
3.6.6 Effect of substrate concentration using cellobiose as substrate 70
3.6.7 Determination of Kinetic Parameter (Vmax and Km) using cellobiose as substrate 72

CHAPTER FOUR: DISCUSSION AND CONCLUSION
5.0 DISCUSSION 78
5.1 CONCLUSION 82

REFERENCES 84
APPENDICES 95

LIST OF FIGURES

Figure 1: Diagram of Plant cell wall 2
Figure 2: Structure of cellulose showing Amorphous and crystalline areas 3
Figure 3: Mechanism of Cellulolysis 8
Figure 4: A 3D illustration of the quaternary structure of the endoglucanase complex with cellulose in its active site 11
Figure 5: Exoglucanase 12
Figure 6: Structural of β-glucosidases from bacterium Clostridium cellulovorans 12
Figure 7: Aspergillus niger 22
Figure 8: Pictorial Representation of Grape Fruit 25
Figure 9: Effect of incubation period on cellulase production 39
Figure 10: Ammonium sulphate precipitation profile 41
Figure 11: Elution profile of gel chromatography 43
Figure 12: Change in Protein Concentration (per ml) after Purification Steps 45
Figure 13: Change in Total Protein Concentration after Purification Steps 47
Figure 14: Change in Cellulase Activities (per ml) after Purification Steps 48
Figure 15: Change in Total Cellulase Activities after Purification Steps 50
Figure 16: Change in Specific Activities of Cellulase after Purification Steps. 51
Figure 17: Effect of Incubation Time on Cellulase Activity of Aspergillus niger using filter paper as substrate 54
Figure 18: Effect of incubation time on β-glucosidase activity of Aspergillus niger using cellobiose as substrate 55
Figure 19: pH profile for Cellulase Activity for isoform A 57
Figure 20: pH Profile for Cellulase Activity for isoform B 58
Figure 21: Effect of pH on β-glucosidase Activity on isoform A 60
Figure 22: Effect of pH on β-glucosidase Activity on isoform B 61
Figure 23: Temperature Profile for Cellulase Activity for isoform A 63
Figure 24: Temperature Profile for Cellulase Activity for isoform B 65
Figure 25: Effect of temperature on β-glucosidase activity for isoform A 67
Figure 26: Effect of temperature on β-glucosidase activity for isoform B 69
Figure 27: Effect of substrate concentration of cellulase from Aspergillus niger for isoform A 71
Figure 28:Effect of substrate concentration of cellulase from Aspergillus niger for isoform B 72
Figure 29: Lineweaver-Burk Plot for isoform A 74
Figure 30: Lineweaver-Burk Plot for isoform B 76

LIST OF TABLES
Table 1: Purification Profile of total Cellulase of Aspergillus niger 44

LIST OF APPENDIX
Appendix One
1.0 Preparation of Buffers 95
1.1 Preparation of Dinitrosalicylic Acid (DNS) Reagent 95
1.2 Preparation of 50mM glucose 95
1.3 Calibration Curve for Glucose 95
1.4 Preparation of the Component Reagents for Protein Determination 96
1.5 Preparation of 2mg/ml Bovine Serum Albumin (BSA) Standard Protein 96
Appendix Two
2.0 Glucose Standard Curve using 50mM Industrial Glucose 97
Appendix Three
3.0 Protein Standard Curve, Using 2mg/ml Bovine Serum Albumin (BSA) 98

CHAPTER ONE
INTRODUCTION AND LITERATURE REVIEW

Grape fruit (Citrus paradisi) is a subtropical citrus tree known for its sour to semi-sweet fruit. It has been part of human diet for ages due to its nutritional and medicinal values. The frequent use of grape fruits for production of juices, nectars, concentrates, jams, jelly powders and flakes generates wastes in the form of grape peel and bagasse which could bring about environmental pollution if not properly handled. Agricultural wastes and in fact all lignocellulosics can be converted into products that are of commercial interest such as ethanol, glucose, and single cell protein such as in the conversion of grape bagasse to cellulase. There is great interest in utilising cellulose wastes as feedstocks for fermentation processes, thereby converting low cost starting materials into products of greater value (Ojumu et al., 2003). Substantial efforts are going into investigations on refining biomass to derive liquid fuel, chemical feed stock and improved animal feeds to meet global bioenergy demand through the biorefinery concept, since agricultural food processes generate millions of tons of waste each year (Xeros and Christakopoulos, 2009) such as grape bagasse, sugar cane bagasse, wheat straw and rice straw. Cellulose, a basic structural component of plant cell wall (Dewey and Mary, 1980) is a polymer of β-D-Glucose which links successfully through a beta-configuration between carbon 1 and carbon 4 of adjacent units to form a long chain 1,4 glucans. Cellulase refers to a class of enzymes produced by fungi, bacteria and protozoans and it causes hydrolysis of cellulose (Bhat, 2000; Sherief et al., 2010). They are widely distributed throughout the biosphere and are most manifest in fungal and microbial organisms (Chinedu et al., 2011). A cellulosic enzyme system consists of three major components: endo-β-glucanase (EC 3.2.1.4), exo-β-glucanase (EC 3.2.1.91) and β-glucosidase (EC 3.2.1.21) (Knowles et al., 1987). These components act synergistically in the conversion of cellulose to glucose (Chen et al., 1992; Begum and Lemaire, 1996; Chirico and Brown, 1987). Cellulase production has been described for many Aspergillus species (Lockington et al., 2002; Kang et al., 2004; Wang et al., 2006; Gao et al., 2008) under submerged fermentation. The submerged cultivation is carried out by using rotary shaker (Bakare et al., 2005). In enzyme production, purification is important to study the function and expression of the enzyme and to remove any contaminants (other proteins or completely different molecules) that are present in the mixture. The ability to secrete large amounts of extra cellular protein is characteristic of certain fungi and such strains are most studied for production of higher levels of extracellular cellulases. Fungal cellulases are preferred for industrial application because they are inducible enzymes which can produce large quantities of cellulase (Immanuela et al., 2007). This process reflects well the fact that filamentous fungi are naturally excellent protein secretors and can produce industrial enzymes in feasible amounts (Bergquist et al., 2002). Cellulase are used in the textile industry for cotton softening; in laundry detergents for colour care, cleaning, and anti-deposition; in the food industry for mashing; in the pulp and paper industries for deinking, drainage improvement, and fibre modification and they are even used for pharmaceutical applications.
1.1 History of Cellulose
Cellulose, a complex carbohydrate or polysaccharide consisting of 3000 or more glucose units and a basic structural component of plant cell wall (Dewey and Mary, 1980) was discovered in 1838 by the French chemist Anselme Payen, who isolated it from plant matter and determined its chemical formula. Cellulose was used to produce the first successful thermoplastic polymer, celluloid, by Hyatt Manufacturing Company in 1870. Hermann Staudinger determined the polymer structure of cellulose in 1920. The compound was first chemically synthesized (without the use of any biologically derived enzymes) in 1992 (Klemm et al., 2005).
1.1.1 Cellulose