단백질공학을 이용한 밀치 epoxide hydrolase의 입체선택적 가수분해활성 향상에 대한 연구 = (The)effect of group art therapy by cognitive behavior on self-esteem and stress of sibings with the disabled child

다국어 초록 (Multilingual Abstract)

The marine fish microsomal epoxide hydrolase (mEH) of Mugil cephalus was engineered to enhance the enantioselective hydrolytic activity by multiple sequence alignment-inspired mutagenesis. The amino acid sequences of Aspergillus niger, Rhodotorula glutinis, zebra fish and humanm EH were aligned and analyzed for identifying target amino acids. Single-point mutants (Q170K, E186K and E378D) and double-point mutants (E378D-Q170K, E378D-Y348F and E378D-Y348H) were developed and their hydrolytic activities were compared. The double-point mutant, E378D-Q170K, exhibited an enhanced hydrolytic activity by 4.6-fold, compared to the wild-type M. cephalus mEH. Enantiopure (S)-styrene oxide could be readily prepared with high enantiopurity more than 99%ee by using the double-point mutant.
The epoxide hydrolase (EH) of a marine fish, Mugil cephalus, was engineered to improve the catalytic activity based on comparative homology modeling. The 3-D crystal structure of the EH from Aspergillus niger was used as the template. A triple point mutant, F193Y for spatial orientation of the nucleophile (D199), W200L for removing electron density overlap between W200 and Y348, and E378D for good charge relay in the active site, was developed. The initial hydrolysis rate, the reaction time to reach 98%ee, and yield of triple-point mutant were enhanced up to 35-fold, 26-fold and 32%, respectively, by homology modeling-inspired site-directed mutagenesis of M. cephalus EH.
A triple-point mutated fish mEH gene from Mugil cephalus was functionally expressed in Escherichia coli in the presence of various chaperones to prevent protein aggregations. The enantioselective hydrolytic activity was enhanced more than 2-fold by co-expressing the EH mutant gene with pGro7 plasmid. The highly active EH mutant with His-tag was immobilized onto magnetic silica assembled with NiO nanoparticles. The immobilized mEH mutant was re-used more than 10 times with less than 10% activity loss. (S)-styrene oxide with 98% enantiopurity could be repetitively obtained with more than a half of the theoretical yield by the magnetically separable high-performance mEH mutant.

국문 초록 (Abstract)

광학활성 epoxide는 입체적으로 순수한 생화학적 활성물질을 만들기 위한 중요한 중간체이다. Epoxide hydrolase (EH, EC 3.3.2.3)는 라세믹 epoxide로부터 입체선택적 가수분해능을 이용하여 카이랄 epoxide를 만들기 위한 잠재적 유용성이 있는 생촉매이다. 본 논문에서는 단백질 공학을 이용하여 해양 어류인 Mugil cephalus의 epoxide hydrolase를 개량하여 가수분해능을 향상시켰고, 고정화 기술을 이용하여 생촉매를 재사용하였다.
첫째, Comparative homology modeling을 기반으로 해양어류 M. cephalus의 epoxide hydrolase 단백질의 3차 구조를 분석하고 예측한 결과를 바탕으로 확보한 다양한 분자개량 target site를 site-directed mutagenesis를 활용하여 개량하고 촉매 활성을 평가하였다. 분자 공학적으로 epoxide hydrolase 생촉매 활성을 50% 이상 향상시킨 varient를 총 5종 이상 개발 하였으며, 그 중 F193Y-W200L-E378D triple mutant의 최대 활성은 wild-type 대비 약 35배 이상 향상시켰다.
둘째, 고정화 해양생물공정 최적화를 위하여 해양 epoxide hydrolase triple mutant의 발현조건을 최적화하였다. Soluble 형태의 단백질 발현이 용이하지 않은 microsomal protein인 해양어류 M. cephalus EH triple mutant를 단백질 folding에 관여하는 여러 종류의 chaperone 단백질과의 co-expression 조건을 최적화시켜 활성을 2배 이상 향상시켰다. 결과적으로 해양 생촉매 활성을 70배 증가시킨 최적화 결과를 얻었다.
셋째, 표면에 Ni^(2+)를 부착시킨 자성나노입자를 이용하여 his-tag이 붙어있는 M. cephalus EH triple mutant 단백질을 고정화시킨 나노고정화 생촉매를 개발하였다. 자성나노입자 고정화 해양 EH 효소 생촉매를 이용하여 10회 이상의 재사용을 통해 98%ee 고순도의 (S)-styrene oxide를 제조하였다. 나노고정화 EH 효소 생촉매는 free enzyme보다 활성유지 기간이 2배 연장되었다.

목차 (Table of Contents)

• CONTENTS Ⅰ
• ABSTRACT Ⅵ
• LIST OF TABLES Ⅷ
• LIST OF FIGURES Ⅸ
• ABBREVIATIONS Ⅻ
• INTRODUCTION 2
• Introduction 2
• Biological ways for the preparation of chiral epoxides 4
• Epoxide hydrolase-catalyzed enantioselective hydrolysis 5
• Structure and catalytic mechanism of epoxide hydrolase 8
• Molecular engineering of EH 11
• Reference 14
• CHAPTER 1 21
• Abstract 21
• 1. 1 Introduction 22
• 1. 2 Materials and methods 24
• 1.2. 1 Multiple sequence alignment, bioinformatics analysis and comparative modeling 24
• 1.2. 2 Site-directed mutagenesis 24
• 1.2. 3 Recombinant cell culture and batch enantioselective hydrolysis of racemic epoxides 25
• 1.2. 4 Analysis 26
• 1. 3 Results and discussion 29
• 1.3. 1 Multiple sequence alignment and identification of target amino acid sequences for site-directed mutation 29
• 1.3. 2 Expression and characterization of the mEH mutants 36
• 1.3. 3 Enantioselective hydrolysis of racemic styrene oxide by the recombinant E. coli expressing the mutated mEH gene of M. cephalus 38
• 1. 4 Conclusion 42
• Reference 43
• CHAPTER 2 47
• Abstract 47
• 2. 1 Introduction 48
• 2. 2 Materials and methods 50
• 2.2. 1 Protein sequence homology analysis and homology modeling of M. cephalus EH 50
• 2.2. 2 Strains, plasmids, site-directed mutagenesis and PCR 50
• 2.2. 3 Kinetic resolution of racemic styrene oxide by the recombinant E. coli possessing the mutated M. cephalus EH gene 53
• 2.2. 4 Analysis 53
• 2. 3 Results and discussion 54
• 2.3. 1 Homology modeling of M. cephalus EH 54
• 2.3. 2 Correction of spatial orientation of nucleophile for effective catalysis 56
• 2.3. 3 Replacement of glutamate by aspartate in charge relay system 62
• 2.3. 4 Development of triple point mutant 62
• 2. 4 Conclusion 68
• Reference 69
• CHAPTER 3 74
• 3. 1 Introduction 75
• 3. 2 Materials and methods 78
• 3.2. 1 Strains and culture conditions 78
• 3.2. 2 Partial purification of the mutated EH proteins 80
• 3.2. 3 Immobilization of the EH mutant on the hybrid Fe3O4- silica-NiO nanoparticles 81
• 3.2. 4 Enantioselective resolution of racemic styrene oxide 85
• 3.2. 5 Effects of pH and temperature on the activity and stability of the free and immobilized EH mutants 85
• 3.2. 6 Re-use of the immobilized EH mutant in a repeated batch operation 86
• 3.2. 7 Analysis 86
• 3. 3 Results and discussion 87
• 3.3. 1 Functional expression of the triple-point mutated fish microsomal EH gene in E. coli via molecular chaperones co-expression 87
• 3.3. 2 Characterization of the triple-point mutated EH protein co-expressed with groES-groEL 92
• 3.3. 3 Immobilization of the triple-point mutated EH enzyme onto magnetic silica nanoparticles 102
• 3. 4 Conclusion 105
• Reference 106
• CONCLUSIONS 110
• Concluding Remarks 110
• Industrial attempts to produce enantiopure epoxides using biocatalysts 110
• Future researches 111
• ABSTRACT IN KOREAN 113
• ACKNOWLEDGEMENT 116