ISOLATION, PURIFICATION AND CHARACTERIZATION OF FREE AND IMMOBILIZED ALPHA-AMYLASE FROM BACILLUS LICHENIFORMIS
Table Of Contents
Chapter ONE
1.1 Introduction1.2 Background of Study
1.3 Problem Statement
1.4 Objective of Study
1.5 Limitation of Study
1.6 Scope of Study
1.7 Significance of Study
1.8 Structure of the Research
1.9 Definition of Terms
Chapter TWO
2.1 Overview of Alpha-Amylase2.2 Sources of Alpha-Amylase
2.3 Enzymatic Properties of Alpha-Amylase
2.4 Applications of Alpha-Amylase
2.5 Immobilization Techniques
2.6 Immobilized Alpha-Amylase
2.7 Comparison between Free and Immobilized Alpha-Amylase
2.8 Factors Affecting Alpha-Amylase Activity
2.9 Stability of Alpha-Amylase
2.10 Industrial Importance of Alpha-Amylase
Chapter THREE
3.1 Research Design3.2 Sampling Method
3.3 Data Collection Techniques
3.4 Experimental Setup
3.5 Immobilization Procedure
3.6 Enzyme Assay Methods
3.7 Data Analysis Techniques
3.8 Quality Control Measures
Chapter FOUR
4.1 Analysis of Free Alpha-Amylase Properties4.2 Analysis of Immobilized Alpha-Amylase Properties
4.3 Comparison of Free and Immobilized Alpha-Amylase
4.4 Effect of Immobilization on Enzyme Activity
4.5 Stability Studies
4.6 Kinetic Parameters Determination
4.7 Industrial Applications Evaluation
4.8 Future Research Directions
Chapter FIVE
5.1 Summary of Findings5.2 Conclusion
5.3 Recommendations
5.4 Implications of the Study
5.5 Contribution to the Field
Project 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.
Project Overview
INTRODUCTION
1.1.Background to 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).
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