ISOLATION, PURIFICATION AND CHARACTERIZATION OF FREE AND IMMOBILIZED ALPHA-AMYLASE FROM BACILLUS LICHENIFORMIS

ABSTRACT

The bacteria Bacillus licheniformis was cultured in nutrient agar and then incubated for 15h at 35oC. The bacteria cells were harvested by centrifugation after incubation. The cell free supernatant was used to estimate alpha-amylase activity. The alpha-amylase obtained was isolated and purified using ammonium sulfate precipitation, gel filtration and ion exchange chromatography. It was purified up to 15.5 fold and a yield of 20.2% on DEAE- Sephadex column with a final specific activity of 12.14 u/mg. The alpha-amylase was immobilized by entrapment in calcium alginate beads. The free and immobilized enzyme had broad temperature ranges from 20oC to 70oC with optima of 60oC and 70oC respectively and optimum pH of 7.0 and 8.0 respectively. Initial velocity studies for the determination of kinetic constants with maltose as substrate revealed a KM value of 2.5 mg/ml and 1.0 mg/ml for the free and immobilized enzyme respectively and a Vmax value of 0.4unit/mg/min and 0.95unit/mg/min for the free and immobilized enzyme respectively. Both the free and immobilized enzyme activity were enhanced by Ca2+ , Mn2+ , and Na+ while Hg2+ and Zn2+ were found to be strong inhibitors of both the free and immobilized enzyme.

TABLE OF CONTENT

Abstract

Table of Contents

List of Abbreviations

CHAPTER ONE

1.0       Introduction

1.1       Background of Study

1.2       Statement of Research Problem

1.3       Justification

1.4       Aim

1.4.2    Objectives

1.5       Research Hypothesis

CHAPTER TWO

2.0       LITERATURE REVIEW

2.1       Enzymes

2.2       Uses of Enzymes in the Industries

2.3       Microorganisms

2.3.1    Sources of Microorganisms

2.3.2    Uses of Microorganisms

2.4       Industrial Microorganisms

2.5       Bacteria

2.6       Bacillus species

2.6.1    Industrial Significance

2.6.2    Clinical Significance

2.6.3    Cell Wall

2.6.4    Phylogeny

2.7       Bacillus licheniformis

2.7.1    Uses of Bacillus licheniformis

2.8       Alpha Amylase

2.8.1    Types of Amylases

2.8.2    Sources of Alpha – Amylase

2.8.3    Uses of Alpha Amylase

2.9       Isolation and Purification of Protein/Enzymes

2.9.1    Tissues Disintegration and Extraction

2.9.2    Protein Purification

2.9.3    Purpose of Protein Purification

2.9.4    Evaluation of Purification Yield

2.9.5   Methods of Protein Purification

2.9.5.1 Extraction

2.9.5.2 Precipitation

2.9.5.3 Centrifugation

2.9.5.4 Chromatographic Methods

2.9.5.4.1 Size Exclusion Chromatography

2.9.5.4.2 Ion Exchange Chromatography

2.9.5.4.3Reverse Phase Liquid Chromatography

2.10. Properties of Enzymes

2.10.1. PH Optimum and Stability

2.10.2 Temperature Optimum and Stability

2.10.3 KM and Vmax Values

2.10.4. Substrate Specificity

2.10.5 Effects of Metal Ions for example Ca2+, Mg2+ and Hg2+

2.10.6 Enzyme Assay

2.11. Immobilization of Enzymes

2.11.1 Background of Enzyme Immobilization

2.11.2 History of Enzyme Immobilization

2.11.3 The Biology of Enzyme Immobilization

2.11.4 Choice of Supports for Enzyme Immobilization

2.11.5 Methods of Irreversible Enzyme Immobilization

2.11.5.1 Formation of Covalent Bonds

2.11.5.2 Entrapment

2.11.6 Methods of Reversible Immobilization

2.11.6.1. Adsorption

2.11.6.2 Ionic Binding

2.11.6.3 Hydrophobic Adsorption

2.11.6.4 Chelation Or Metal Binding

2.11.6.5 Formation of Disulfide Bonds

2.11.7 Properties of Immobilized Enzymes

2.11.8 Uses of Immobilized Enzyme

2.11.8.1 Use of Immobilized Enzyme as Biosensors

2.11.8.2 Use of Immobilized Enzyme in Medicine

2.11.8.3 Use of Immobilized Enzyme for Antibiotic Production

2.11.8.4 Use of Immobilized Enzyme in Food Industry

2.11.8.5 Use of immobilized Enzyme for Biodiesel production

2.11.8.6 Use of Immobilized Enzyme for Bioremediation

2.11.8.7 Use of Immobilized Enzyme in Textile Industry

2.11.8.8 Use of Immobilized Enzyme in Detergent Industry

Chapter Three

3.0 Materials and Methods

3.1 Materials

3.1.1 Microorganism Sample

3.1.2 Chemicals and Reagents

3.1.3 Equipment

3.2 Methods

3.2.1 Production of Alpha – Amylase

3.2.2 Extraction of Alpha – Amylase from the Fermentation Media

3.2.3 Partial Purification of Alpha – Amylase

3.2.3.1 Purification with Ammonium Sulfate

3.2.3.2 Gel Filtration on CM – Sephadex G–25 Column

3.2.3.3 Ion – Exchange on DEAE – Sephadex Column

3.2.4 Determination of Total Protein

3.2.5    Alpha- Amylase Assay

3.2.6    Enzyme Immobilization

3.2.7    Characterization of the Partially Purified Alpha – Amylase

3.2.7.1 Determination of Optimum Temperature

3.2.7.2 Determination of Optimum pH

3.2.7.3 Determination of KM and Vma x

3.2.7.4Determination of Effect of Metal Ions

3.3       Data Analysis

Chapter Four

4.0       Results

Chapter Five

5.0       Discussion

Chapter Six

6.0       Summary and Conclusions

6.1       Summary

6.2       Conclusions

6.3       Recommendations

References

Appendix

CHAPTER ONE

1.0.            INTRODUCTION

1.1.            Background of Study

Amylase is a digestive enzyme classified as a saccharidase (an enzyme that cleaved poly-saccharides). It is mainly a constituent of pancreatic juice and saliva, needed for the breakdown of long-chain carbohydrate (such as starch) into smaller units like disaccharides and trisaccharides.

Alpha-amylase is the major form of amylase found in humans and other mammals. It is also present in seeds containing starch as food reserve and it is secreted by many fungi. Although found in many tissues, alpha-amylase is most prominent in pancreatic juice and saliva. Alpha-amylase found in saliva breaks starch down to maltose and dextrin. It breaks large insoluble starch molecules into soluble forms e.g. amylodextrin, erythrodextrin and achrodextrin producing successively smaller starches and ultimately maltose. The pancreas produces alpha-amylase which hydrolyses dietary starch into disaccharides and trisaccharides which are converted by other enzyme to glucose to supply the body with energy (Alistair et al., 2006).

Although amylase can be derived from several sources such as plants, animals and microbes, the microbial amylase meet industrial needs and demands. Large numbers of microbial amylase have completely replaced chemical hydrolysis of starch in starch processing industries (Pandey et al., 2000).

Nowadays the use of enzyme in industrial sector is increasing due to the increase of industries, especially in food, beverages, textile, leather and paper industries. Besides its uses in industry, it can also be used in treatment of industrial waste such as cellulase which is able to convert cellulose of wood and paper wastes to ethanol (Vielle and Zeikus., 1996). One of the enzymes widely used in industrial sectors is alpha-amylase. Alpha-amylase from Bacillus species has found application in many industries such as pharmaceutical, textile, paper, detergent and chemical industries. Therefore, these enzymes account for about 39% of the world‟s enzymes production (Gomes and Steiner, 2004).

A biocatalyst is termed immobilized, if its mobility has been restricted by chemical means. Immobilized enzymes are used in food technology, biotechnology, biomedicine and analytical chemistry. Immobilized enzymes offers variety of advantages over free enzyme catalysis including increased stability of enzyme, easy recovery of enzyme, easy separation of reactant and product, repeated or continuous used of a single batch of enzyme which will ultimately save the enzyme, labor and overhead costs (Gerhatz, 1990).

Enzymes can be immobilized to a multitude of different carriers by entrapment, adsorption, ionic binding and covalent binding (Varavinit et al., 2002). Entrapment is taken as the most preferable method because it prevents excessive loss of enzyme activity after immobilization, increases enzyme stability and protects enzyme from microbial contamination (Kennedy and Cabral, 1987).

Physical entrapment of alpha-amylase in calcium alginate beads has shown to be a relatively easy, rapid and safe technique (Dey et al., 2003) in comparison with other immobilization methods.

1.2              Statement of Research Problem

a.       Amylases possess important applications in the production of syrup with high glucose content, sweetener manufacture, detergent and ethanol (Pandey et al.,

2000).

b.      The annual sale of alpha-amylase in global market is estimated to be eleven million dollars (Kilara and Desai, 2002).

c.       There is a need to discover more bacterial sources of alpha-amylase that will produce alpha-amylase with better properties e.g. thermostability that will be of greater use to the industries.

d.      There is a need to discover more ways of producing alpha-amylase in bulk and that will be economically viable.

e.       Most of the enzymes used in the industrial sector in Nigeria including food industries are still imported enzymes and economically, this is not favorable to the nation because Nigeria is rich in natural resources especially the microbial which can be use as enzymes producer for example alpha-amylase enzyme.

f.       There is a need to immobilize alpha-amylase in order to explore the various benefits that can come from the immobilization process.

1.3  Justification

a.       Bacterial alpha-amylase is preferred for the application in starch processing and textile industries due to it‟s stability at higher temperature (75-105ºC) and its neutral to alkaline pH (Shah and Kothari, 1991).

b.      B. licheniformis, B. coagulans, B. polymyxa, B. vulgarus have been used for alpha amylase production in solid state fermentation (Babu and Satyanarayana, 1995).

c.       Due to the increase in the demand for these enzymes in various industries, there is therefore a need to discover more strains of bacteria that can produce alpha-amylase with better properties in terms of thermo-stability, mass production of the enzyme and consistency.

d.      There is also a need to compare the kinetics and physico-chemical properties of the free and immobilized alpha amylase to discover various benefits that immobilization of alpha amylase can offer to the industries.

e.       The strain of B. licheniformis used for this research work is a local strain isolated from Kaduna metropolis soil in Kaduna State, Nigeria and it is different from imported strains of the bacteria that are usually used for other research work.

f.       Also there is no documented work on the kinetic studies and effect of metal ions on immobilized alpha amylase.

1.4 Aim

To  isolate,  purify  and  characterize  free  and  immobilized  alpha-amylase  from  Bacillus licheniformis.

1.4.1          Objectives

a.       Isolation of the enzyme alpha-amylase in fermentation media from the cell of the Bacteria isolate of Bacillus licheniformis.

b.      Purification of alpha-amylase obtained from the Bacillus licheniformis

c.       Immobilization of the enzyme by entrapment in calcium alginate beads.

d.      Characterization of the free and immobilized alpha amylase.