| 摘要 | 第4-6页 |
| ABSTRACT | 第6-8页 |
| CHAPTER 1 INTRODUCTION | 第13-29页 |
| 1.1 PREPARATION OF OPTICALLY PURE COMPOUNDS | 第13-16页 |
| 1.2 SOME IMPORTANT ENZYMES RELATED TO AMINO ACIDS METABOLISM | 第16-24页 |
| 1.2.1 Serine hydroxymethyltransferase | 第16-19页 |
| 1.2.2 The properties of tryptophan synthase | 第19-21页 |
| 1.2.3 The properties of tryptophanase | 第21-23页 |
| 1.2.4 The properties of transaminases | 第23-24页 |
| 1.3 RESPONSE SURFACE METHODOLOGY | 第24-25页 |
| 1.4 THE RESEARCH BACKGROUND AND OBJECTIVES | 第25-29页 |
| 1.4.1 The background of this thesis | 第25-27页 |
| 1.4.2 The objectives of this thesis | 第27-29页 |
| CHAPTER 2 PREPARATION OF OPTICALLY ACTIVE β-HYDROXY-a-AMINO ACID WITH SERINE HYDROXYMETHYL TRANSFERASE | 第29-44页 |
| 2.1 INTRODUCTION | 第29-31页 |
| 2.2 MATERIALS AND METHODS | 第31-35页 |
| 2.2.1 Chemicals | 第31页 |
| 2.2.2 Synthesis of DL-threo-β-phenylserine and its derivatives | 第31-32页 |
| 2.2.3 Bacterial strains and plasmids | 第32页 |
| 2.2.4 General recombinant DNA techniques | 第32-33页 |
| 2.2.5 PCR-amplification of the serine hydroxymethyl transfer ase gene (glyA) | 第33页 |
| 2.2.6 Media and cultivation conditions | 第33-34页 |
| 2.2.7 Immobilization of cells | 第34页 |
| 2.2.8 SHMT activity assays | 第34-35页 |
| 2.2.9 Protein determination and SDS-PAGE analysis | 第35页 |
| 2.2.10 Molecular mass determination | 第35页 |
| 2.2.11 Analytical methods | 第35页 |
| 2.3 RESULTS | 第35-42页 |
| 2.3.1 Cloning and overexpression of the glyA gene | 第35-36页 |
| 2.3.2 SHMT substrate specificity | 第36-38页 |
| 2.3.3 pH and temperature effect | 第38-39页 |
| 2.3.4 Substrate concentration effect | 第39-40页 |
| 2.3.5 Operational stability of immobilized cells | 第40-41页 |
| 2.3.6 Isolation and identification of D-threo-β-(methlsulfonylphenyl)serine | 第41-42页 |
| 2.4 DISCUSSION | 第42-44页 |
| CHAPTER 3 ENZYMATIC SYNTHESIS OF L-TRYPTOPHAN WITHTRYPTOPHAN SYNTHASE FROM KERATIN ACIDIC HYDROLYSISINDUSTRIES | 第44-69页 |
| 3.1 INTRODUCTION | 第44-45页 |
| 3.2 MATERIALS AND METHODS | 第45-51页 |
| 3.2.1 Chemicals | 第45-46页 |
| 3.2.2 Bacterial strains and plasmids | 第46-47页 |
| 3.2.3 General recombinant DNA techiques | 第47页 |
| 3.2.4 PCR-amplification of trpBA and tnaA gene | 第47-48页 |
| 3.2.5 Media and cultivation conditions | 第48页 |
| 3.2.6 Enzyme substrate specificity and activity assay | 第48-49页 |
| 3.2.7 Protein determination and SDS-PAGE analysis | 第49页 |
| 3.2.8 Decolorization and transmittance assay | 第49页 |
| 3.2.9 Rapid detection of tryptophan by TLC | 第49-50页 |
| 3.2.10 Scale up fermentation in fermentor 10 L | 第50页 |
| 3.2.11 Rapid analytic methods of tryptophan and cysteine in enzymatic conversion solution | 第50-51页 |
| 3.3 RESULTS AND DISCUSSION | 第51-68页 |
| 3.3.1 Cloning and overexpression of the trpBA and tnaA gene | 第51-53页 |
| 3.3.2 Enzyme substrate specificity and activity assay | 第53-56页 |
| 3.3.3 pH and temperature effect | 第56-57页 |
| 3.3.4 Ammonium chloride effect | 第57-58页 |
| 3.3.5 Surfactant effect | 第58-59页 |
| 3.3.6 Decolorization by intact cell | 第59-60页 |
| 3.3.7 Thin layer chromatography assay | 第60-61页 |
| 3.3.8 Scale up fermentation, isolation and identification of tryptophan | 第61-62页 |
| 3.3.9 Concentration determination of cysteine in enzymatic conversion solution | 第62-67页 |
| 3.3.10 Concentration determination of tryptophan in enzymatic conversion solution | 第67-68页 |
| 3.4 CONCLUSIONS | 第68-69页 |
| CHAPTER 4 ASYMMETRIC SYNTHESIS OF UNNATURAL L-AMINOACIDS AND D-AMINO ACIDS USING AROMATIC AMINO ACIDTRANSAMINASE | 第69-80页 |
| 4.1 INTRODUCTION | 第69-70页 |
| 4.2 MATERIALS AND METHODS | 第70-73页 |
| 4.2.1 Chemicals | 第70-71页 |
| 4.2.2 Synthesis of phenylpyruvic acid and its derivatives | 第71页 |
| 4.2.3 Bacterial strains and plasmids | 第71-72页 |
| 4.2.4 General recombinant DNA techniques | 第72页 |
| 4.2.5 PCR-amplification of tyrB gene | 第72-73页 |
| 4.2.6 Media and cultivation conditions | 第73页 |
| 4.2.7 Protein determination and SDS-PAGE analysis | 第73页 |
| 4.2.8 AroAT activity assays | 第73页 |
| 4.3 RESULTS | 第73-78页 |
| 4.3.1 Cloning and over expression of the tyrB gene | 第73-75页 |
| 4.3.2 AroAT substrate specificity | 第75-76页 |
| 4.3.3 pH and temperature effect | 第76-78页 |
| 4.3.4 Isolation and identification of D-trp and L-methlsulfonyl-phenylalanine | 第78页 |
| 4.4 DISCUSSION | 第78-80页 |
| CHAPTER 5 PROCESS OPTIMIZATION OF RECOMBINANT E. COLISTRAIN CULTURE | 第80-95页 |
| 5.1 INTRODUCTION | 第80-81页 |
| 5.2 MATERIALS AND METHODS | 第81-86页 |
| 5.2.1 Chemicals and Microorganism | 第81页 |
| 5.2.2 Shake flask fermentation | 第81页 |
| 5.2.3 Aspartase activity assay | 第81-82页 |
| 5.2.4 Optimization procedure | 第82-85页 |
| 5.2.5 Scale up fermentation in fermentor 10 L | 第85-86页 |
| 5.3 RESULTS AND DISCUSSION | 第86-93页 |
| 5.3.1 Optimization of RSM | 第86-91页 |
| 5.3.2 Temperature and substrate concentration effect | 第91-92页 |
| 5.3.3 Metallic ions effect | 第92-93页 |
| 5.4 DISCUSSION | 第93-95页 |
| CHAPTER 6 SUMMARY | 第95-97页 |
| 6.1 MAIN CONCLUSIONS | 第95页 |
| 6.2 MAIN INNOVATION | 第95-96页 |
| 6.3 PROSPECTS | 第96-97页 |
| 总结 | 第97-98页 |
| Reefrenees | 第98-123页 |
| APPendixes | 第123-129页 |
| Research achievements | 第129-131页 |
| Aeknowledgements | 第131-132页 |
