Random Mutagenesis of Ketol-acid Reductoisomerase for Switching its Cofactor Preference
조효소 친화도 전환을 위한 케토산 리덕토아이소머레이즈의 랜덤돌연변이 라이브러리 구축 및 분리
- 주제(키워드) Metabolic engineering , Random mutagenesis , Cofactor preference
- 발행기관 고려대학교 대학원
- 지도교수 김경헌
- 발행년도 2016
- 학위수여년월 2016. 8
- 학위구분 석사
- 학과 대학원 생명공학과
- 세부전공 시스템식품생명공학전공
- 원문페이지 49 p
- 실제URI http://www.dcollection.net/handler/korea/000000069121
- 본문언어 영어
- 제출원본 000045953511
초록/요약
In this study, random mutagenesis of β2αβ-loop region (36 base pair) responsible for NADPH affinity were applied to switch the cofactor preference from NADPH to NADH with a model protein, ketol-acid reductoisomerase (IlvC). For the convenient screening of the mutants with high affinity to NADH, mutated IlvC was overexpressed in the E. coli membrane using pATLIC system. After the initial screening, 42 of E. coli mutants were obtained from the library and 14 mutatednts were further screened by in vitro reaction with the crude enzyems. Then, 2 mutants named as IlvC_IS11 and IlvC_IS12 with the highest affinity to NADH were finally selected. For the kinetic study, 2 mutants were overexpressed in pET expression system and purified using Ni2+-NTA chromatography. In results, kinetic parameters indicated that 1) the cofactor preference of two mutants was switched from NADPH to NADH and 2) catalytic efficiency of two mutants for NADH significantly increased up to 290% in comparison with IlvC wild type. Furthermore, stuctural analysis by computational modeling showed that amino acids alternation in position of Arg68, Arg76, and Ser78 of the flexible loop region is a vital role on swiching the cofactor preference. In conclusion, this study suggests that random mutagenesis of specific region of enzyme can be an alternative way to switch the confactor preference.
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LIST OF FIGURES..........................................................................viii
LIST OF TABLES..............................................................................x
ABSTRACT OF THE THESIS............................................................xi
1. Introduction..............................................................................1
2. Materials and methods
2.1. General.......................................................................................3
2.2. ilvC mutant library construction and cloning for E. coli surface display.4
2.3. 2-Acetolactate preparation and identification of 2-acetolactate by gas chromatography/time-of-flight mass spectrometry (GC/TOF MS)..............7
2.4. Screening using E. coli surface displayed IlvC mutants.....................10
2.5. Heterologous protein expression, purification and kinetic study..........12
2.6. Kinetic assays.............................................................................13
2.7. Sequence alignment and structural modeling of wild type and mutated IlvC................................................................................................14
3. Results and discussion
3.1. ilvC mutant library construction....................................................15
3.2. Screening of NADH-dependent IlvC mutants by cell surface display method............................................................................................17
3.3. Kinetic study of screened IlvC mutants...........................................19
3.4. Enzymatic conversion of 2-AL into 2,3-DHIV by the selected IlvC mutants...........................................................................................22
3.5. Sequence alignment and structural modeling of wild type and two IlvC mutants...........................................................................................24
4. Conclusion...............................................................................27
References....................................................................................28
ABSTRACT IN KOREAN.........................................................32
APPENDICES
Appendix 1: Construction of ilvC mutant library for cloning...................A1
Appendix 2: Lineweaver-Bulk plot with varing NAD(P)H concentration..A2
Appendix 3: Calculation of kinetic parameters of IlvC_IS13 determined using NADH, with 2-AL as a substrate................................................A3
