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


Content

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


 

 

LIST OF ABREVIATIONS

Spp

-

Specie

KM

-

Michaelis Menten Constant

Vmax

-

Maximal Velocity

PGA

-

Penicillin G Acylase

CM-Sephadex

-

Carboxylmethyl Sephadex

DEAE- Sephadex

-

Diethylaminoethyl Sephadex

μl

-

Microliter

ml

-

Milliliter

Mg

-

Milligram

nm

-

Nanometer

w/V

-

Weight per volume

g

-

Gram

CBRT

-

Center for Biotechnology and Research Training

DTT

-

Dithiothreitol

EDTA

-

Ethylenediaminetetracetic acid

G.M.O.

-

Genetically modified organism

SPR

-

Surface Plasmon Resonance

ELISA

-

Enzyme – linked Immunosorbent assay

 

 

 

 

 

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

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