Table of Contents 1. CHAPTER I INTRODUCTION 1 [A] GLUCOSE ISOMERASE 1

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1 Table of Contents 1. CHAPTER I INTRODUCTION 1 [A] GLUCOSE ISOMERASE Enzymatic versus chemical isomerization Glucose Isomerase producing microorganisms Structure of Glucose Isomerase Production of Glucose Isomerase Influence of magnesium and cobalt ions on GI production and activity Purification of GI Immobilization of GI Continuous isomerization of glucose Strain improvement by mutagenesis and genetic engineering Applications of Glucose Isomerase High Fructose Corn Syrup production Ethanol production Simulataneous isomerisation and fermentation Cofermentation of Glucose and Xylose Production of fructose syrup from whey permeate Xylitol Purification 27 [B] ACTINOMYCETES Life cycle and morphology of Actinomycetes Identification of Actinomycetes Actinomycetal genes Biotechnological importance of Actinomycetes Enzymes Antibiotics Biotransformation of steroids Agriculture Biocontrol of phytopathogens Bioremediation 41 [C] AIM AND OBJECTIVES 43 v

2 2. CHAPTER II ISOLATION AND DIVERSITY OF ACTINOMYCETES INTRODUCTION MATERIALS AND METHODS Sampling Isolation Procedure Morphological analysis Sugar utilization and enzyme characterization RESULTS AND DISSCUSSION Isolation Aerial spore mass appearance Pigmentation Colony reverse Colony surface appearance Growth response on different media Growth in liquid media Morphological Analysis Biochemical characterization CONCLUSIONS CHAPTER III MOLECULAR CHARACTERIZATION OF STREPTOMYCETAL ISOLATES P1 AND AII INTRODUCTION MATERIALS AND METHODS s rrna Sequencing Phylogenetic analysis and rrna secondary structure determination RESULTS AND DISSCUSSION s rrna Sequence Analysis for P1 and AII Phylogenetic Analysis for Streptomyces sp. SB-P1 and Streptomyces sp. SB-AII Elucidation of rrna Secondary Structure for P1 and AII CONCLUSIONS CHAPTER IV ENZYME PROFILING OF ACTINOMYCETAL ISOLATES INTRODUCTION MATERIALS AND METHODS Qualitative Analysis Glucose Isomerase production 114 vi

3 Amylase Production Cellulase production Protease production Lipase production Pectinase production Quantitative Analysis Screening for Glucose isomerase production by submerged fermentation Screening for Amylase production by submerged fermentation Screening for Cellulase production by submerged fermentation Screening for Protease production by submerged fermentation RESULTS AND DISSCUSSION Qualitative Analysis Glucose Isomerase Production Amylase Production Cellulase production Protease production Lipase production Pectinase production Quantitative Analysis Screening for Glucose isomerase production by submerged Fermentation Screening for Amylase production by submerged fermentation Screening for Cellulase production by submerged fermentation Screening for Protease production by submerged fermentation CONCLUSIONS CHAPTER V GLUCOSE ISOMERASE PRODUCTION BY STREPTOMYCES SP. SB-AII4 AND STREPTOMYCES SP. SB-P INTRODUCTION 134 vii

4 5.2 MATERIALS AND METHODS Comparison for intracellular and extracellular enzyme production Growth Curve for Streptomyces sp. SB - AII Time Scale Analysis of Streptomyces sp. SB - AII Growth Curve for Streptomyces sp. SB - P Time Scale Analysis Streptomyces sp. SB - P Crude enzyme kinetics Effect of Substrate Concentration on GI activity Effect of Enzyme Aliquot on GI activity Effect of Temperature on GI activity Effect of ph on GI activity Effect of Reaction period on GI activity RESULTS AND DISCUSSION Comparison of Intracellular and Extracellular production of Glucose Isomerase Growth Curve for Streptomyces sp. SB - AII Time Scale Analysis for Streptomyces sp. SB - AII Growth curve for Streptomyces sp. SB - P Time scale analysis for Streptomyces sp. SB - P Crude Enzyme Kinetics Effect of Substrate Concentration on GI activity Effect of enzyme aliquot on activity Determination of optimum temperature for GI activity Determination of optimum ph for GI activity Determination of suitable Reaction period for GI activity CONCLUSIONS CHAPTER VI OPTIMIZATION OF FERMENTATION MEDIUM FOR GI PRODUCTION BY STREPTOMYCES SP. SB - P INTRODUCTION MATERIALS AND METHODS Comparison of different media combinations Effect of different Carbon sources on the production of GI Effect of different Nitrogen sources on the production of GI Effect of varying concentration of carbon source on GI production Effect of varying concentration of nitrogen source on GI production 157 viii

5 6.2.6 Effect of varying concentration of xylose on GI production Media optimization using statistical design RESULTS AND DISSCUSSION Comparison of different media combinations Effect of different Carbon sources on the production of GI Effect of different Nitrogen sources on the production of GI Effect of varying concentration of Carbon source on GI production Effect of varying concentration of nitrogen source on GI production Effect of varying concentration of xylose on GI production Media Optimization Using Statistical design CONCLUSIONS CHAPTER VII OPTIMIZATION OF PROCESS PARAMETERS INFLUENCING GI PRODUCTIVITY BY STREPTOMYCES SP. SB - P INTRODUCTION MATERIALS AND METHOD Effect of Inoculum Size on production of Glucose Isomerase Effect of Temperature on production of Glucose Isomerase Effect of ph on production of Glucose Isomerase Effect of Agitation Rate on production of Glucose Isomerase RESULTS AND DISSCUSSION Effect of Inoculum Size on production of Glucose Isomerase Effect of Temperature on production of Glucose Isomerase Effect of ph on production of Glucose Isomerase Effect of Agitation Rate on production of Glucose Isomerase CONCLUSIONS CHAPTER VIII PARTIAL PURIFICATION AND CHARACTERIZATION OF GI PRODUCED BY STREPTOMYCES SP. SB - P INTRODUCTION MATERIALS AND METHODS Ammonium Sulphate Precipitation Gel Filtration for removal of salt on Sephadex G DEAE Cellulose Chromatography Dialysis for Concentration of enzyme Molecular Weight Determination by Sodium Dodecyl Sulphate (SDS) Polyacrylamide Gel Electrophoresis (PAGE) Kinetic characterization of partially purified GI 198 ix

6 Effect of Substrate Concentration on GI activity Effect of Temperature on GI activity Effect of ph on GI activity Effect of Metal ions on GI activity RESULTS AND DISCUSSION Ammonium Sulphate Precipitation8.3.2 Gel Filtration for removal of salt Sephadex G Ion Exchange for separation of proteins Dialysis for Concentration of enzyme Molecular Weight Determination by Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis Kinetic characterization of purified GI Effect of substrate concentration on GI activity Effect of Temperature on GI activity Effect of ph on GI activity Effect of Metal Ions on GI activity CONCLUSIONS CHAPTER IX APPLICATION OF GLUCOSE ISOMERASE FOR HFCS PRODUCTION INTRODUCTION MATERIALS AND METHODS Production of Amylase Hydrolysis of Corn Starch Glucose Isomerase Production from Streptomyces sp. SB - P Isomerisation of Glucose Units RESULTS AND DISCUSSION Production of Amylase Hydrolysis of Corn Starch Isomerisation of Glucose Units CONCLUSIONS CHAPTER X SUMMARY AND CONCLUSIONS 219 APPENDIX I List of Media 231 APPENDIX II List of Reagents 237 REFERENCES 244 x