Development of novel bioreaction platforms for the highly efficient process of cytochrome P450 = 시토크롬 P450의 고효율 공정을 위한 신규 생물 반응 플랫폼의 개발

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Cytochrome P450 (CYP or P450) belong to a superfamily of multifunctional monooxygenase concerning oxidative metabolism of endogenous or exogenous molecules. P450s can catalyze various regioselective and stereospecific oxidation reactions of non-functionalized hydrocarbons. P450s containing heme molecule as a prosthetic group have been found from most living organisms. Since discovery of P450s in 1958, P450s have been interested from various industrial fields because of its various catalytic activity to large range of substrates. While there are many interests and a lot of researchers have studied about P450s, however, several characteristics have made application of P450s difficult. Briefly, major reasons for limitations of P450s application are dependence on expensive electron donor cofactor (such as NADPH or NADH), additional electron transfer domain (reductase) and heme molecule on their active site. Also, they need to be engineered for industrial uses and enzyme engineering have been difficult. To overcome these limitations, various techniques are even now being developed and some successful cases were reported. In this study, I have focused to improve P450s utilization by following experiments. First, cell surface display of P450s on the E. coli was developed to efficient whole-cell reaction. As P450s were displayed on cell membrane, they could freely use the electron donor and showed normal activity in stable. As a result, it could be free from limitation of NAD(P)H supplying. Furthermore, I changed conventional electron donor to inexpensive eosin Y (EY). In contrast with NAD(P)H, EY has good permeability to cell membrane and could easily penetrate into E. coli. Also, it can directly transfer electrons to active site, thus more efficient whole-cell reaction can be possible without conventional electron donor and reductase. Also, EY-mediated P450 reactions can directly transfer the electron in absence of heme. It means P450 reactions should not be affected by the amount of heme molecule bound to P450s and harsh handling of P450 enzyme for efficient production can be applied. Finally, general high-throughput screening method was developed to enhance the activity or the regioselectivity from wild-type of P450s. This engineering tool was initially designed to be applied regardless of kind of P450s and substrates. Therefore, following experiments should increase the value of P450s in industrial uses.
Advisors
Jeong, Ki Junresearcher정기준researcher
Description
한국과학기술원 :생명화학공학과,
Publisher
한국과학기술원
Issue Date
2017
Identifier
325007
Language
eng
Description

학위논문(박사) - 한국과학기술원 : 생명화학공학과, 2017.2,[xv, 157 p. :]

Keywords

Escherichia coli▼aCytochrome P450 (P450)▼acell surface display▼alight-driven P450▼aheme-free P450▼ahigh-throughput screening (HTS); 대장균▼a시토크롬 P450▼a세포 표면 발현▼a빛 유래 P450 반응▼a힘 비의존적 P450▼a초고속 스크리닝

URI
http://hdl.handle.net/10203/264928
Link
http://library.kaist.ac.kr/search/detail/view.do?bibCtrlNo=866946&flag=dissertation
Appears in Collection
CBE-Theses_Ph.D.(박사논문)
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