PRODUCTION OF ASPERGILLUS NIGER GLUCOAMYLASE USING GUINEA CORN STARCH AMYLOPECTIN AS THE ONLY CARBON SOURCE

ABSTRACT

This study was aimed at the production of glucoamylase which can be utilised for starch hydrolysis. A fourteen days experimental study was carried out to determine the day of highest glucoamylase activity. Day five and day twelve of the fourteen days experimental study had the highest glucoamylase activity. The specific activity for the crude enzyme was found to be 729.45 U/mg for glucoamylase isolated from Aspergillus niger in submerged fermentation using amylopectin fractionated from guinea corn starch as the carbon source after five days of fermentation (GluAgGC5), and 1046.82 U/mg for glucoamylase isolated from Aspergillus niger in submerged fermentation using amylopectin fractionated from guinea corn starch as the carbon source after twelve days of fermentation (GluAgGC12).The crude enzyme was purified by ammonium sulphate precipitation and by gel filtration (using sephadex G 100 gel).  Ammonium sulphate saturations of 70% and 20% were found suitable to precipitate proteins with highest glucoamylase activity. After ammonium sulphate precipitation, the specific activities of the enzyme were found to be 65.98 U/mg and 61.51 U/mg for GluAgGC5 and GluAgGC12, respectively. Similarly, after gel filtration, the specific activities of the enzyme were found to be 180.52 U/mg and 272.81 U/mg for GluAgGC5 and GluAgGC12, respectively. The optimum pH for GluAgGC5 were found to be 7.5,7.5 and 6.0 when using tiger nut starch, cassava starch and guinea corn starch as substrates, respectively, while the optimum pH  for GluAgGC12 were found to be 5.0, 8.5  and 7.0 when using tiger nut starch, cassava starch and guinea corn starch as substrates, respectively. The enzyme activity in GluAgGC5 was enhanced by Ca²⁺,Co²⁺, Fe²⁺, Mn²⁺and Zn²⁺ but Pb²⁺ had inhibitory effect on the enzyme. Similarly, the enzyme activity of GluAgGC12 was enhanced by Ca²⁺, Zn²⁺, Co²⁺, Fe²⁺ and Mn²⁺ while  Pb²⁺ had inhibitory effect on the enzyme. The optimum temperatures were found to be 50˚C and 45˚C for GluAgGC5 and GluAgGC12, respectively. The Michaelis Menten’s constant, K_m and maximum velocity V_max of GluAgGC5 obtained from the Lineweaver-Burk plot of initial velocity data at different substrate concentrations were found to be 770.75 mg/ml and 2500µmol/min using cassava starch as substrate, 158.55 mg/ml and 500 µmol/min using guinea corn starch as substrate and 46.23 mg/ml and 454.53µmol/min using tiger nut starch as substrate. Also, the K_m   and  V_max   of  GluAgGC12 were found to be 87.1 mg/ml and 384.61µmol/min using cassava starch as substrate, 29.51 mg/ml and 243.90 µmol/min using guinea corn starch as substrate and 2364 mg/ml and 2500µmol/min using tiger nut starches as substrate.

TABLE OF CONTENTS

Title page …………………………………………………………………………………………….. i

Certification……………………………………………………………………………………………… ii

Dedication…………………………………………………………………………………………………….. iii

Acknowledgement……………………………………………………………………………………… iv

Abstract……………………………………………………………………………………………………… v

Table of Contents……………………………………………………………………………………… vi

List of Figures………………………………………………………………………………………… x

List of Tables……………………………………………………………………………………………. xii

CHAPTER ONE: INTRODUCTION

1.1 Glucoamylase……………………………………………………………………………….. 3

1.2 Aspergillus niger as a Microbial Source of Glucoamylase……………………… 4

1.2.1 Taxonomy of Aspergillus niger …………………………………………………. 5

1.2.2 Identification of Aspergillus niger …………………………………………………. 5

1.2.3 Morphological Identification of Aspergillus culture …………………………. 5

1.2.4 Uses of Aspergillus niger …………………………………………………………… 8

1.2.5 Safety Aspect of Aspergillus niger ………………………………………………… 9

1.3 Guinea Corn…………………………………………………………………………………… 9

1.3.1 Taxonomy of Guinea Corn  ………………………………………………. 9

1.3.2 Amylopectin from Guinea Corn as Carbon Source for Glucoamylase ……………………….. 11

1.3.3 Other Substrates Commonly Used for Glucoamylase Production…….. 13

1.4 Properties of Glucoamylase …………………………………………… 13

1.5 Sources of Glucoamylase………………………………………………………….. 13

1.6 Forms of Glucoamylase………………………………………………………………… 14

1.7 Types of Glucoamylase………………………………………………………………… 15

1.8 Mechanism of Action of Glucoamylase………………………………………………….. 15

1.8.1 Mechanism of Hydrolysis of Glucoamylase……………………………………… 16

1.9 Structure of Glucoamylase …………………………………………………………….. 17

1.10 Amino acid Sequence of Glucoamylase……………………………………………. 20

1.11 Immobilisation of Glucoamylase…………………………………………………… 23

1.12 Glucoamylase Immobilisation with other Enzyme……………………………. 24

1.13 Inhibitors of Glucoamylase …………………………………………………………… 25

1.14 Activators of Glucoamylase…………………………………………………………….. 27

1.15 Applications of Glucoamylase………………………………………………………….. 27

1.16 Aim and Objectives ………………………………………………………………. 29

1.16.1 Aim of the Study…………………………………………………………….. 29

1.16.2 Specific Objectives of the Study…………………………………………… 29

CHAPTER TWO: MATERIALS AND METHODS

2.1 Materials…………………………………………………… 30

2.1.1 Reagents……………………………………………………………………………….. 30

2.1.2 Apparatus……………………………………………………………………………….. 31

2.2 Methods………………………………………………………………………………………… 31

2.2.1 Collection of Plant Material………………………………………………………… 31

2.2.2 Processing of Cassava Starch……………………………………………………………. 31

2.2.3 Processing of Guinea Corn Starch……………………………………………………. 32

2.2.4 Processing of Tiger Nut Starch…………………………………………………………. 32

2.2.5 Fractionation of Guinea Corn Starch………………………………………….. 32

2.2.6 Isolation of the Glucoamylase Producing Fungi……………………………. 32

2.2.7 Inoculation of Plates and Subculturing…………………………………….. 33

2.2.8 Storage of Pure Fungal Isolates………………………………………………… 33

2.2.9 Microscopic Features of the Isolated Fungi…………………………………….. 33

2.2.10 Fungal Identification………………………………………………………………… 33

2.2.10.1 Fermentation Experiment…………………………………………………….. 33

2.2.10.2 The Fermentation Broth……………………………………………………………. 33

2.2.10.3 Inoculation of the Broth………………………………………………………. 34

2.2.10.4 Harvesting of the Fermented Broth………………………………….. 34

2.2.11 Mass Production of the Enzyme…………………………………………………. 34

2.2.12 Protein Determination……………………………………………………………. 34

2.2.12.1 Procedure Protein Determination………………………………………………. 34

2.2.13 Enzyme Activity…………………………………………………………………….. 35

2.2.13.1 Assay for Amylase Activity…………………………………………………… 35

2.2.13.2 Assay for Glucoamylase activity………………………………………………. 35

2.2.14 Purification of the Enzyme………………………………………………………. 36

2.2.14.1 Determination of Percentage Ammonium Sulphate Saturation… 36

2.2.14.2 Ammonium Sulphate Precipitation……………………… 36

2.2.14.3 Gel Filtration Chromatography…………………………………. 37

2.2.15 Studies on the Partially Purified Enzyme………………………. 37

2.2.15.1 Effect of pH on Glucoamylase Activity………………………….. 37

2.2.15.2 Effect of Temperature on Glucoamylase Activity……………… 37

2.2.15.3 Effect of Substrate Concentration on Glucoamylase Activity….. 37

2.2.15.4 Effect of Ion Concentration on Glucoamylase Activity………………… 38

CHAPTER THREE: RESULTS

3.1 Fractionation of the Guinea corn Starch…………………………………. 39

3.1.1 Percentage Extraction Yield of Amylopectin from Guinea Corn Starch…………………….. 39

3.1.2 Photograph of Pure Culture of Aspergillus niger………………………….. 40

3.2 Production of Glucoamylase via Submerged Fermentation………………… 41

3.2.1 Protein Determination and Enzyme Activity Using Cassava Starch as Substrate………… 41

3.3 Protein Determination and Enzyme Activity Using Guinea Corn Starch as Substrate…….. 43

3.4 Protein Determination and Enzyme Activity Using Tiger Nut Starch as Substrate…………. 45

3.5 Mass Production of the Enzyme……………………………………………. 47

3.6 Partial Purification of the Enzyme  ……………………………………. 47

3.6.1 Ammonium Sulphate Precipitation Profile for Glucoamylase Harvested on Day Five…. 47

3.6.2 Ammonium Sulphate Profile for Glucoamylase Harvested on Day Twelve………………… 49

3.6.3 Gel Filtration for Glucoamylase Harvested on Day Five………………….. 51

3.6.4 Gel Filtration for Glucoamylase Harvested on Day Twelve……………… 53

3.7 Protein Concentration, Enzyme Activity of the Enzyme Harvested on Day Five ………….. 55

3.7.1 Protein Concentration, Enzyme Activity of the Enzyme Harvested on Day Twelve……. 57

3.8 Purification Table………………………………………………………………………. 59

3.9 Characterisation of Glucoamylase…………………………………………………….. 61

3.9.1 Effect of pH on The Enzyme Activity for the Enzyme Harvested on Day Five …………. 61

3.9.2 Effect of pH on The Enzyme Activity for the Enzyme Harvested on Day Twelve……… 63

3.9.3 Effect of Temperature on Enzyme Activity for Enzyme Harvested on Day Five………… 65

3.9.4 Effect of Temperature on Enzyme Activity for Enzyme Harvested on Day Twelve……. 67

3.9.5 Effect of Substrate Concentration on Enzyme Activity for Day Five Enzyme……………. 69

3.9.6 Effect of Substrate Concentration on Enzyme Activity for Day Twelve Enzyme……….. 71

3.9.7 Effect of Ion concentration on Enzyme Activity for Day Five Enzyme…………………….. 74

3.9.8 Effect of Ion concentration on Enzyme Activity for Day Twelve Enzyme………………… 76

CHAPTER FOUR: DISCUSSION

4.1 Discussion …………………………………………………………………………….. 78

4.2 Conclusion………………………………………………………………………………….. 84

4.3 Suggestions for Further Studies……………………………………………….. 84

References……………………………………………………………………………………… 85

Appendices………………………………………………………………………………………. 91

LIST OF FIGURES

Figure 1: Schematic presentation of the action of amylases

Figure 2: Photographs of colonies of Aspergillus Species in czapek yeast agar (CYA) and Malt extract agar (MEA).

Figure 3:  Structure of Amylopectin and Amylose

Figure 4:  Mechanism of action of glucoamylase

Figure 5: The catalytic mechanism of glucoamylase illustrating the action of the catalytic base and acid

Figure 6: Stereoview of the catalytic domain of Aspergillus niger and Aspergillus awamori glucoamylase

  Figure7:  Stereoview of the starch binding domain (SBD) from Aspergillus niger glucoamylase.

Figure 8: The linker region, the catalytic part and starch binding domain of glucoamylase

Figure 9: Sequence comparison of the three glucoamylases

Figure 10: Predicted secondary structure for glucoamylase

Figure 11: Structure of Acarbose and its mode of action

Figure 12: Competitive inhibitors of glucoamylase: acarbose

Figure 13: Schematic flow diagram for the ethanol

Figure 14: Pure culture of Aspergillus niger used for the inoculation

Figure15: Glucoamylase activity, alpha amylase activity and protein concentration on various days of incubation when cassava starch was used as substrate.

Figure16: Glucoamylase activity, alpha amylase activity and protein concentration of various days of incubation when guinea corn starch was used as substrate.

Figure17: Glucoamylase activity, alpha amylase activity and protein concentration of various days of incubation when tiger nut starch was used as substrate.

Figure18: Glucoamylase activity of ammonium sulphate precipitation profile for glucoamylase from Aspergillus niger, harvested on day five of submerged fermentation.

Figure19: Glucoamylase activity of ammonium sulphate precipitation profile for glucoamylase from Aspergillus niger, harvested on day twelve of submerged fermentation.

Figure 20: Elution profile of glucoamylase harvested on day five of submerged fermentation.

Figure 21: Elution profile of glucoamylase harvested on day twelve of submerged fermentation

Figure22: Protein concentration, enzyme activity and specific activity of the glucoamylase harvested on day five.

Figure 23: Protein concentration, enzyme activity and specific activity of the glucoamylase harvested on day twelve.

Figure 24: Effect of pH on glucoamylase from Aspergillus niger harvested on day five of submerged fermentation.

Figure 25: Effect of pH on glucoamylase from Aspergillus niger harvested on day twelve of submerged fermentation

Figure 26:Effect of temperature on glucoamylase activity for glucoamylase harvested on day five of submerged fermentation.

Figure 27:Effect of temperature on glucoamylase activity for glucoamylase harvested on day twelve of submerged fermentation.

Figure 28:Line- Weaver Burk plot (for glucoamylase harvested on day five of the submerged fermentation)

Figure 29:Line-Weaver Burk plot (for glucoamylase harvested on day twelve of the submerged fermentation)

Figure 30:Effect of various ion concentrations on glucoamylase activity for glucoamylase harvested on day five of submerged fermentation.

Figure 31:Effect of various ion concentrations on glucoamylase activity for glucoamylase harvested on day twelve of submerged fermentation

LIST OF TABLES

Table 1: Microscopic characteristics used for identification of Aspergillus isolates

Table 2: Macroscopic characteristics used for identification of Aspergillus isolates

Table 3: Chemical Composition of Guinea Corn

Table 4: Essential amino acid composition (mg/g) of sorghum and finger millet proteins

Table 5: Glucoamylase immobilisation with other enzymes

Table6: Purification table for glucoamylase harvested on day twelve of submerged fermentation

Table7: Purification table for glucoamylase harvested on day twelve of submerged fermentation

Table 8: The characterization of the glucoamylase obtained from Aspergillus niger

CHAPTER ONE

                                   INTRODUCTION

Starch degrading enzymes are currently becoming and gaining more importance among the industrial enzymes because of the importance of starch, sugars and other products in modern biotechnological era (Omemu et al., 2008). Majority of these starch degrading enzymes are carbohydrases (that is, the amylases or starch converting enzymes), and they can be grouped into four types; the endoamylases, the exoamylases, the debranching enzymes and the transferases (Siew et al., 2012).

The endoamylases otherwise referred to as the endoacting enzymes are able to cleave α-1, 4 glucosidic bonds present in the inner part (endo-) of the amylose or amylopectin chain, the enzyme, α-amylase (EC3.2.1.1) is a well-known endoamylases (Van Der Maare et al., 2002 ).  Similarly, the exoamylases cleave either or both the α-1, 4 and α-1, 6 bonds on the external glucose residues of amylose or amylopectin from the nonreducing end and thus produce only glucose (Bertoldo et al., 2002), glucoamylases (EC3.2.1.3) and α-glucosidases (EC 3.2.1.20) are very good examples of the exoamylases. The transferases are another group of starch-converting enzymes that cleave an α-1, 4 glucosidic bond of the donor molecule and transfer part of the donor to a glucosidic acceptor with the formation of a new glucosidic bond (Tharanathan and Mahadevamma, 2003). Enzymes such as amylomaltase (EC 2.4.1.25) and cyclodextrin glycosyltransferase (EC 2.4.1.19) form a new α-1, 4 glucosidic bond while branching enzyme (EC 2.4.1.18) forms a new α-1, 6 glucosidic bond. The debranching enzymes catalyse the hydrolysis of α-1, 6-glucosidic bonds in amylopectin and/or glycogen and related polymers. The affinity of debranching enzymes for the α-1, 6-bond distinguishes these enzymes from other amylases which have primary affinity for α-1, 4-glucosidic linkages (Siew et al., 2012). The enzyme pullulanase and isoamylase are well known examples of the debranching enzymes.

Carbohydrases, therefore, are those groups of enzymes which catalyses the breakdown of carbohydrates (e.g. starch, oligosaccharides as well as polysaccharides), into simple sugars. Examples of the carbohydrases include α – amylase, glucoamylase, etc. Alpha -amylase (E.C.3.2.1.1) hydrolyses α-l, 4- glycosidic bonds randomly in amylose, amylopectin and glycogen in an endo fashion. All α-amylases bypass α-1, 6-glycosidic bonds, but do not cleave them. Hydrolysis of amylose by α -amylase causes its conversion into maltose and maltotriose, followed by a second stage in the reaction, the hydrolysis of maltotrioses. Glucoamylase (EC 3.2.1.3)  is the exo-acting enzyme that hydrolyzes both 1,4-alpha- and 1,6-alpha-glucosidic linkages in amylose, amylopectin, glycogen as well as other related oligo and polysaccharides, yielding β-D-glucose as the end product. Hence, glucoamylases can serve as an industrially useful enzyme (Siddhartha et al., 2012).

Currently, amylases are of great importance in biotechnology with a wide spectrum of applications, such as in textile industry, cellulose, leather, detergents, liquor, bread, children cereals, ethanol production, and high fructose syrups production and in various strategies in the pharmaceutical and chemical industries such as the synthesis of optically pure drugs and agrochemicals (Mervat, 2012).