ISOLATION, PURIFICATION AND CHARACTERIZATION OF FREE AND IMMOBILIZED ALPHA-AMYLASE FROM BACILLUS LICHENIFORMIS | Blazingprojects Postgraduate Thesis
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ISOLATION, PURIFICATION AND CHARACTERIZATION OF FREE AND IMMOBILIZED ALPHA-AMYLASE FROM BACILLUS LICHENIFORMIS

 

Table Of Contents


Chapter ONE

INTRODUCTION

  • 1.1Introduction
  • 1.2Background of Study
  • 1.3Problem Statement
  • 1.4Objective of Study
  • 1.5Limitation of Study
  • 1.6Scope of Study
  • 1.7Significance of Study
  • 1.8Structure of the Research
  • 1.9Definition of Terms

Chapter TWO

LITERATURE REVIEW

  • 2.1Overview of Alpha-Amylase
  • 2.2Sources of Alpha-Amylase
  • 2.3Enzyme Production
  • 2.4Enzyme Purification Techniques
  • 2.5Enzyme Characterization Methods
  • 2.6Role of Alpha-Amylase in Biotechnology
  • 2.7Industrial Applications of Alpha-Amylase
  • 2.8Factors Affecting Alpha-Amylase Activity
  • 2.9Enzyme Immobilization Techniques
  • 2.10Comparison of Free and Immobilized Alpha-Amylase

Chapter THREE

RESEARCH METHODOLOGY

  • 3.1Research Methodology Overview
  • 3.2Selection of Bacillus licheniformis Strain
  • 3.3Enzyme Extraction and Separation
  • 3.4Enzyme Purification Process
  • 3.5Immobilization Techniques Used
  • 3.6Characterization Methods Employed
  • 3.7Experimental Design and Setup
  • 3.8Data Collection and Analysis Methods

Chapter FOUR

DATA PRESENTATION AND ANALYSIS

  • 4.1Analysis of Enzyme Production Yield
  • 4.2Purification Efficiency Evaluation
  • 4.3Comparison of Free and Immobilized Enzyme Properties
  • 4.4Enzyme Stability Studies
  • 4.5Substrate Specificity Analysis
  • 4.6Effect of pH and Temperature on Enzyme Activity
  • 4.7Industrial Applicability Assessment
  • 4.8Discussion on Research Findings

Chapter FIVE

SUMMARY, CONCLUSION AND RECOMMENDATIONS

  • 5.1Summary of Research Findings
  • 5.2Conclusion and Implications
  • 5.3Recommendations for Future Research
  • 5.4Contribution to the Field
  • 5.5Closing Remarks

Thesis 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.

Thesis Overview

<p>INTRODUCTION<br><br>1.1.Background to Study<br><br>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.<br><br>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).<br></p>

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