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研究生(外文):LOW, PIK KUAN
論文名稱(外文):Expression of Recombinant Glutamate Decarboxylase (GAD) to Heighten Gamma-aminobutyric Acid (GABA) Production in Beer Yeast
指導教授(外文):John, Chi-Wei, Lan
口試委員(外文):John, Chi-Wei, LanJo-Shu, ChangBor-Yann, ChenYu-Kaung, ChangYu-Hong, Wei
外文關鍵詞:gabagadbeer yeast
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Table of Contents
摘 要 ii
Abstract iii
Acknowledgements iv
Table of Contents v
List of Tables viii
List of Figures ix
List of Symbols x
Chapter One: Introduction 1
1.1 Research Background 1
1.2 Research Scope and Objective 2
Chapter Two: Literature Review 3
2.1 Beer 3
2.1.1 Components 3
2.1.2 Health Benefits 4
2.1.3 Produced by Yeasts 5
2.2 GABA 6
2.2.1 Role of GABA in the Human Brain 7
2.2.2 GABA Synthesis 7
2.2.3 Factors Contributing to GABA Production 8
2.3 GAD 10
2.3.1 Biological GAD enzyme for GABA production 13
2.4 Microorganisms with GAD 14
2.4.1 Lactic Acid Bacteria (LAB) 14
2.4.2 Escherichia coli 16
2.4.3 Saccharomyces cerevisiae 17
2.4.4 Other microorganisms and plants 18
2.5 Aqueous Two Phase System in the Recovery of GABA 19
2.5.1 Aqueous Two Phase System 19
2.5.2 Polymer-polymer and Polymer-salt based ATPS 19
2.5.3 Alcohol-salt based ATPS 20
2.5.4 Applications of ATPS 21
2.6 Quantity Analysis of GABA 22
Chapter Three: Materials and Methods 24
3.1 Experimental design 24
3.2 Microbial Strains 25
3.3 Instruments 25
3.4 Media 26
3.5 Other Chemicals 27
3.6 Yeast Fermentation and Analysis 27
3.7 FDNB Derivatization (GABA Analysis) 28
3.7.1 Preparation of Standard Solutions 28
3.7.2 Derivatization Procedures and Preparation for HPLC Analysis 29
3.7.3 HPLC Gradient Setup for GABA detection 29
3.8 Construction of Recombinant Escherichia coli strain 30
3.8.1 Primer Design 30
3.8.2 GAD Cloning 30
3.8.3 Restriction Enzyme Digestion 31
3.8.4 Construction of Recombinant pIYC08 vector plasmid 32
3.9 GAD Enzymatic Analysis 33
3.9.1 Extraction of GAD from Recombinant Escherichia coli DH5α 33
3.9.2 GAD Protein Analysis 33
3.9.3 GAD Activity Assay 34
3.9.4 Data Analysis 34
Chapter Four: Results and Discussion 35
4.1 Yeast Fermentation 35
4.2 GABA Analysis (FDNB Derivatization) 36
4.3 Construction of Recombinant Vector Plasmid 38
4.3.1 GAD Cloning 38
4.3.2 Restriction Enzyme Digestion 39
4.3.3 Expression of GAD in Escherichia coli DHα 40
Chapter Five: Conclusions and Future Work Plan 42
5.1 Conclusions 42
5.2 Future Work Plan 42
References 43
Appendices 58
Appendix 1: List of Abbreviations 58

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