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研究生:裴維恩
研究生(外文):Pervin Akter
論文名稱:應用熱穩定天冬胺酸轉胺酶以麩胺酸合成同苯丙胺酸
論文名稱(外文):Application of Thermostable Aspartate aminotransferase (TtAspAT) for biosynthesis of homophenylalanine by using L-Glutamate
指導教授:黃贊勳
指導教授(外文):Hwang, Tzann-Shun
口試委員:黃鵬林許德賢黃贊勳
口試委員(外文):Huang, Pung-LingSheu, Der-ShyanHwang, Tzann-Shun
口試日期:2013-06-26
學位類別:碩士
校院名稱:中國文化大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2013
畢業學年度:101
語文別:英文
論文頁數:64
外文關鍵詞:TtAspATEnzymatic assayL-GlutamateL-HomophenylalnineBioconversion
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ABSTRACT
Aspartate aminotransferases (AspAT) can catalyze the reversible transamination reaction from aspartate to α-ketoglutarate to form glutamate and oxaloacetate. T. thermophilus AspAT (TtAspAT) has been cloned from HB8 by Okamoto et al. (67) and crystal structure has been resolved by Nakai et al.(64). It presents a substrate preference of amino acids with carboxyl group (aspartate and glutamate) and also found to possess significant activity against aromatic amino acids and branched-chain amino acids. Our previous study showed that TtAspAT have the ability to catalyze the synthesis of L-homophenylalanine, which is an important compound in the synthesis of anti-hypertensive drugs, the angiotensin-converting enzyme (ACE) inhibitors. TtAspAT is of advantage in high thermostabilty and having better activity than E. coli AspAT.
The objective of this study was to investigate the best condition to synthesis L-homophenylalnine (L-HPA) using TtAspAT and L-Glutamate as a substrate. This enzyme was also test for its activity against monosodium glutamate (MSG) in synthesizing L-homophenylalanine (L-HPA), since MSG is the cheapest amino acid in food industry. In this study, TtAspAT showed the better activity on Tris-HCl buffer than Bis-Tris Propane against L-Glutamate. Significant effect was observed in terms of Tm, pH, L-Glutamate and D-Alanine. Bioconversion of homophenylalanine was conducted at 60°C at different intervals and showed the highest rate after 15-hour incubation.


TABLES OF CONTENTS
ACKNOWLEDGEMENTS ii
TABLES OF CONTENTS iii-iv
LIST OF TABLES v
LIST OF FIGURES vi
APPENDIX vii
ABSTRACT vii
CHAPTER – 1 1
GENERAL INTRODUCTION 1
1.1 Aspartate Aminotransferase; An essential enzyme for all organisms. 1
1.2: Thermus thermophilus Aspartate Aminotransferase (TtAspAT) 2
1.3: Hypertension (High Blood Pressure); a common disease for adult 4
1.4 Angiotensin Converting Enzyme (ACE) 5
1.5 ACEIs (Angiotensin-converting-enzyme inhibitors) 7
1.6. Homophenylalanine and its relationship in Biopharmaceuticals 10
1.7 Examples of biotransformation of L-homophenylalanine 13
1.7.1. The hydantoinase or carbamoylase process in synthesizing of L-homophenylalanine: 14
1.8. Previus study and Research purpose 16
CHAPTER – 2 18
MATERIALS AND METHODS 18
2.1. Bacterial strains, plasmids and reagents 18
2.2. Overexpression and purification of recombinant enzyme (pET-TtAspAT) 18
2.3. Determination of protein concentration 19
2.4. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis 19
2.5.1. Activity of TtAspAT using Aspartate 20
2.5.2. Activity of TtAspAT using L-Glutamate 20
2.6. Bioconversion of Homophenylalanine 21

TABLES OF CONTENTS

CHAPTER – 3 22
RESULTS 22
3.1. Purification of protein by Ni-NTA affinity Chromatography 22
3.2. Enzymatic activities of TtAspAT by using L-Aspartate as an substrate 22
3.3.1. Effect of Temperature 22
3.3.2. Effect of pH 23
3.3.3. Effect of D-Alanine and L-Glutamate 23
3.4. Synthesis of Homophenylalanine 23
CONCLUSION 30
REFERENCES 31-39


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