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올레산으로부터 9-(nonanoyloxy)nonanoic acid 생산을 위한 Baeyer-Villiger monooxygenase 효소의 스크리닝, 발현, 특성화
- Alternative Title
- Screening, expression, and characterization of Baeyer-Villiger monooxygenases for the production of 9-(nonanoyloxy)nonanoic acid from oleic acid
- Author(s)
- 윤주현
- Advisor
- 최권영
- Department
- 일반대학원 환경공학과
- Publisher
- The Graduate School, Ajou University
- Publication Year
- 2018-02
- Language
- eng
- Keyword
- Baeyer-Villiger monooxygenases; soluble expression; whole-cell biotransformation; 9-(nonanoyloxy)nonanoic acid
- Abstract
- Baeyer-Villiger monooxygenases (BVMOs)는 인접한 carbonyl group에 산소를 도입하여 ketone을 ester로 전환하는 BV oxidation 반응을 일으키는 생물학적 촉매이다. BVMO는 미생물 내에서 soluble한 형태로 발현시키기 어렵기 때문에 반응 조건을 최적화하고 BVMO engineering을 통해 안정성을 높이고 soluble하고 active하게 발현될 수 있도록 하였다. Escherichia coli에서 다양한 BVMO를 screening하고 functional expression을 하였고 chaperones 혹은 fusion tags를 이용한 연구를 하였다. 단백질 시퀀스 분석을 통해 BVMO를 채택하고 oleic acid를 기질로 사용하여 9-(nonanoyloxy)nonanoic acid를 생성하는 연구를 진행하였으며 이때 BVMO가 soluble과 active한 형태로 발현될 수 있는지를 평가하였다. Oleic acid의 whole cell biotransformation반응은 OhyA(hydratase), ADH (Alcohol dehydrogenase), 그리고 BVMO효소를 통해 연속적으로 진행되었다. 반응의 최적화를 하기 위해 inducer의 농도를 조절하고, 분자 샤페론을 동시에 발현하거나 media 조건을 사용하였다. 9개의 BVMO를 screen하였고 그 중 3개의 BVMO가 target product를 생성하였고 Di_BVMO3 from Dietzia sp. D5가 가장 좋은 기질 전환 수율을 보였다. Di_BVMO3는 phosphoglycerate kinase를 N-terminal fusion tag로 도입하여 soluble한 발현이 증진되었다. Fusion enzyme을 통한 whole cell biotransformation은 기존 반응보다 3배에서 5배까지 target product의 생성을 증가시켰고 최종 productivity는 105.3 mgL-1 까지 달성되었다. Rh_BVMO4 from Rhodococcus sp. RHA1과 AFL838 from Aspergillus flavus NRRL3357도 이 반응을 위해 screen하였고 다른 long chain ketones의 whole cell biotransformation 반응에 사용될 수 있다.
- Alternative Abstract
- In this study the production of 9-(nonanoyloxy)nonanoic acid from oleic acid using Baeyer-Villiger monooxygenases (BVMOs) was investigated. The whole cell biotransformation of oleic acid includes OhyA (hydratase), ADH (alcohol dehydrogenase), and BVMO Enzymes consecutively. BVMOs are known to catalyze regiospecific Baeyer-Villiger oxygenation of a variety of cyclic and linear ketones to generate the corresponding lactones and esters, respectively. However, the enzymes are difficult to overexpress in a soluble form in microorganisms. Thereby, this study has focused on screening and functional expression of the BVMOs in Escherichia coli. Initially BVMOs were selected by protein sequence analysis and were examined for their ability to express in soluble and active form to generate 9-(nonanoyloxy)nonanoic acid from oleic acid. Secondly various optimization strategies of inducer concentrations, co-expression with molecular chaperones, and different media conditions were investigated. Among the 9 BVMOs screened, three BVMOs were found to produce the target product and among these, Di_BVMO3 isolated from Dietzia sp. D5 was found to be best. Further, the soluble expression of Di_BVMO3 was enhanced by adding phosphoglycerate kinase as N-terminal fusion tag. The whole cell biotransformation with this fusion enzyme resulted in 3 to 5-fold enhancement in product formation compared with the non-fusion counterpart. Final productivity up to 105.3 mgL-1 was achieved. Besides Di_BVMO3, other two new BVMOs of Rh_BVMO4 from Rhodococcus sp. RHA1 and AFL838 from Aspergillus flavus NRRL3357 were screened for production of 9-(nonanoyloxy)nonanoic acid and could be used for whole cell biotransformation reaction of other long chain ketones.
- Fulltext
- Appears in Collections:
- Graduate School of Ajou University > Department of Environmental Engineering > 3. Theses(Master)
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