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Table of contents
CHAPTER I. INTRODUCTION
CHAPTER II. EPOXIDE HYDROLASES AND THEIR APPLICATION IN ORGANIC SYNTHESIS
II.1 INTRODUCTION
II.2 SOURCES AND REACTION MECHANISM OF EHS
II.2.1 Sources of EHs
II.2.2 Heterologous expression of EHs
II.2.3 Reaction mechanisms of EHs
II.3 MONO-FUNCTIONAL EPOXIDES AS CHIRAL BUILDING BLOCKS FOR THE SYNTHESIS OF BIOLOGICALLY ACTIVE COMPOUNDS
II.3.1 Mono-substituted aromatic epoxides
II.3.2 Di-substituted aromatic epoxides
II.3.3 Non aromatic epoxides
II.3.4 meso-Epoxides
II.4 PREPARATION OF VALUABLE CHIRAL BUILDING BLOCKS FOR THE SYNTHESIS OF BIOLOGICALLY ACTIVE COMPOUNDS STARTING FROM BI-FUNCTIONAL EPOXIDES
II.4.1 Halogenated epoxides
II.4.2 Epoxyamide
II.4.3 Protected epoxy-alcohols
II.4.4 Epoxy-ester
II.4.5 Epoxy-aldehyde
II.5 CONCLUSIONS
CHAPTER III. RESULTS
III.1 INTRODUCTION OF KAU2-EH
III.1.1 Cloning of Kau2-EH from metagenomic DNA and properties
III.1.2 Biocatalytic properties of Kau2-EH
III.1.3 Purposes of this PhD work
III.2 MODELISATION STUDY OF KAU2-EH
III.2.1 Introduction
III.2.2 Preparation of CDU and CIU
III.2.3 Determination of kinetic parameters of Kau2-EH
III.2.4 Determination of Ki and CDU & CIU type of inhibition with Kau2-EH
III.2.5 Determination of Ki and CDU & CIU type of inhibition of with potato EH
III.2.6 Summary
III.2.7 Conclusion
III.3 BIOCONVERSION STUDIES
III.3.1 Introduction
III.3.2 Chemical Synthesis
III.3.3 Kau2-EH production in fermentor
III.3.4 Bioconversion using Kau2-EH
III.3.5 Overall conclusion
III.4 KINETIC STUDIES
III.4.1 Introduction
III.4.2 Construction of Kau2-HisTag and mutants
III.4.3 Heterologous expression of Kau2-His6 and the corresponding mutants
III.4.4 Purification of Kau2-His6 and the corresponding mutants
III.4.5 Preparation of (SS)- and (RR)-TSO
III.4.6 Kinetic study
III.4.7 Conclusion
CHAPTER IV. CONCLUSION
CHAPTER V. MATERIAL AND METHODS
V.1 GENERAL
V.1.1 Reagents, solvents and chromatography
V.1.2 Buffer and culture media
V.2 INSTRUMENTS
V.2.1 Nuclear Magnetic Resonance (NMR)
V.2.2 Gas chromatography (GC)
V.2.3 High Pressure Liquid Chromatography (HPLC)
V.2.4 5 L Fermentor
V.2.5 Chromatography
V.2.6 Others
V.3 CHEMICAL SYNTHESIS
V.3.1 Synthesis of N-cyclohexyl-N′-decylurea (CDU)-2
V.3.2 Synthesis of N-cyclohexyl-N’-(4-iodophenyl) urea (CIU)-3
V.3.3 Synthesis of rac-trans-methyl phenylglycidate 4
V.3.4 Synthesis of rac-trans-ethyl-3-phenylglycidate-5
V.3.5 Synthesis of cis and trans-2, 3-epoxy-3-phenylpropanenitrile 7 and 8
V.3.6 Synthesis of rac-trans-2,3-epoxy-3-phenyl-1-bromo propane-9 and rac-trans-2,3-epoxy-3 phenyl-1-chloro propane-10
V.3.7 Synthesis of rac-cis-methyl phenylglycidate-13
V.3.8 Chemical hydrolysis of rac-trans-methyl phenylglycidate-4
V.4 KAU2-EH PRODUCTION
V.4.1 Cells expressing Kau2-EH production in flask
V.4.2 Cells production in fermentor
V.4.3 Determination of EH activity
V.5 MODELISATION STUDY OF KAU2-EH
V.5.1 Determination of EH activities and kinetic parameters (Km, Vmax)
V.5.2 Determination of Ki and type of inhibition using CDU and Kau2-EH
V.5.3 Determination of Ki and type of inhibition using CIU and Kau2-EH
V.5.4 Determination of Ki and type of inhibition using CDU and Potato-EH
V.5.5 Determination of Ki and the type of inhibitor of CIU with Potato EHs
V.6 BIOCONVERSIONS
V.6.1 Bioconversion of rac-trans-methyl-phenylglycidate-4
V.6.2 Bioconversion of rac-trans-ethyl-3-phenylglycidate-5
V.6.3 Bioconversion of rac-methyl-trans-3-(4-methoxyphenyl)-glycidate-6
V.6.4 Bioconversion of rac-trans-2,3-epoxy-3-phenylpropanenitrile-7
V.6.5 Bioconversion of rac-cis-2,3-epoxy-3-phenylpropanenitrile-8
V.6.6 Bioconversion of rac-trans-2,3-epoxy-3-phenylpropyl bromide 9
V.6.7 Bioconversion of rac-trans-2,3-epoxy-3-phenylpropyl-chloride 10
V.6.8 Bioconversion of rac-trans-stilbene oxide-11
V.6.9 Bioconversion of cis-stilbene oxide-12
V.7 KINETIC STUDIES
V.7.1 Protein purification
