View : 142 Download: 0

Full metadata record

DC Field Value Language
dc.description.abstractThe Baeyer-Villiger monooxygenases (BVMOs) catalyze regiospecific insertion of an oxygen atom next to keto-functional group of the substrates. However, the BVMOs generally have poor stability. Hydrogen peroxide can be produced through uncoupling reactions, especially in the absence of a substrate. This hydrogen peroxide may oxidize amino acids in the enzymes. Therefore research was conducted to increase the oxidative stability of the BVMOs by replacing the residues that are easily oxidized with relatively stable residues. The H2O2 migration path was investigated to identify engineering targets by using the protein energy landscape exploration (PELE), a ligand migration simulation program. First, a study on BVMO from Thermobifida fusca (Tf-BVMO) was conducted. Among the various structures discovered, PDB code 2YLR was applied to PELE. In particular, it was confirmed that H2O2 escapes into a funnel-shaped cavity, a structure that Tf-BVMO has specially. Engineering targets were selected using the Tf-BVMOC65D single mutant as a control. M446, Y495 and Y517 were selected and replaced with isoleucine to increase oxidative stability. As a result, a variant with improved oxidation stability was obtained. The results indicated that the approach can be used to improve oxidation stability of the BVMOs. The approach was applied for the industrially relevant BVMO from Pseudomonas putida (Pp-BVMO). The modeled structure was used for the PELE analysis, resulting in two H2O2 migration paths. Engineering targets M380, M381, and M466 were replaced by leucine. As a result, the variants with improved oxidative stability and thermal stability were obtained. Furthermore, the variants showed the greater total turnover number for oxygenation of 4-decanone and 10-ketooctadecanoic acid, as compared to the previous study. In summary, a new research strategy has been proposed to increase the oxidative stability of BVMOs.;Baeyer-Villiger Monooxygenase(BVMO)는 NADPH와 공기 중 산소를 보조인자로 이용하여 카보닐 형태의 기질을 에스터 또는 락톤으로 전환하는 반응을 촉매합니다. 이 과정에서 부산물로는 물만을 생성하는 친환경 적인 반응을 보이는 효소입니다. 그러나 BVMO는 일반적으로 안정성이 매우 떨어지며 특히 기질이 반응 위치에 적절히 위치하지 않는 경우, uncoupling reaction을 통해 활성산소종의 일종인 과산화수소를 생성할 수 있습니다. 이 과산화수소는 효소 내부에서 생성되어 밖으로 빠져나가는 동안 경로 내의 아미노산 잔기를 산화시켜 효소를 불활성화 시킵니다. 따라서 본 연구에서는 protein energy landscape exploration(PELE) 프로그램을 이용하여 과산화수소의 이동경로를 예측하였습니다. Thermobifida fusca 유래의 BVMO는 알려져 있는 다른 BVMO에 비해 안정한 효소이며 구조적으로 연구가 많이 되어왔습니다. 구조상에서 특이적으로 존재하는 funnel-shaped cavity를 따라 과산화수소가 이동할 것이라고 예측되었습니다. 산화안정성이 낮은 Tf-BVMOC65D 단일 돌연변이체에서 M446, Y495, Y517을 엔지니어링 표적으로 선정하였고 아이소류신으로 대체하여 성공적으로 산화안정성을 높힐 수 있었습니다. 또한, 지방산 생물전환에 활성을 나타내는 Pseudomonas putida 유래의 BVMO에 대해서도 동일하게 PELE 프로그램에 적용하여 M380, M381, M466를 엔지니어링 표적으로 선정하였고 류신으로 대체하였습니다. 그 결과 산화 안정성과 열 안정성이 개선되었을 뿐 아니라 이전 연구에 비해 4-decanone 과 10-ketooctadecanoic acid에 대해 총 전환수가 더 큰 variant를 얻을 수 있었습니다. 본 연구에 사용된 접근 방법은 산화안정성에 문제가 있는 여러 효소에서 효율적으로 활용될 수 있을 것입니다.-
dc.description.tableofcontentsI. Introduction 1 II. Materials and Methods 4 A. Microbial Strains and Culture Media 4 B. Chemicals and Reagents 6 C. Engineering targets 7 D. Site-directed Mutagenesis 9 E. Purification of Enzymes 10 F. NADPH assay 10 1. Activity and kinetics 10 2. Oxidative stability. 11 3. Thermal stability . 11 G. Total turnover number 12 H. Whole-cell Biotransformations 12 I. Gas Chromatography/Mass Spectrometry Analysis 13 III. Results and Discussion 14 A. Beayer-Villiger monooxygenase from Thermobifida fusca 14 1. Tf-BVMO C65D single mutant 14 2. Engineering target of Tf-BVMO C65D 16 3. Oxidative stability. 19 B. Beayer-Villiger monooxygenase from Pseudomonas putida 21 1. The importance of Pp-BVMO industry 21 2. Engineering target of of Pp-BVMO 22 3. Oxidative stability 25 4. Thermal stability 28 5. kinetics 30 6. Total turnover number of enzyme reaction 34 7. Whole-cell biotransformation 37 IV. Summary and Conclusions 41 V. Reference 42 Abstract (in Korean) 46-
dc.format.extent1664488 bytes-
dc.publisher이화여자대학교 대학원-
dc.titleImproving oxidative stability of the bacterial Baeyer-Villiger monooxygenases by engineering H2O2 migration paths-
dc.typeMaster's Thesis-
dc.format.pagevi, 47 p.-
dc.identifier.major대학원 식품공학과- 2-
Appears in Collections:
일반대학원 > 식품공학과 > Theses_Master
Files in This Item:
There are no files associated with this item.
RIS (EndNote)
XLS (Excel)


Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.