• Title/Summary/Keyword: Enantiopure epoxide

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Molecular Engineering of Epoxide Hydrolases for Production of Enantiopure Epoxides (분자공학 기반의 광학활성 에폭사이드 제조용 epoxide hydrolase 생촉매 개발)

  • Kim, Hee-Sook;Lee, Eun-Yeol
    • Journal of Life Science
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    • v.16 no.1
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    • pp.168-174
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    • 2006
  • Enantiopure epoxides are valuable intermediates for the asymmetric synthesis of enantiopure bioactive compounds. Microbial epoxide hydrolases (EHs) are versatile biocatalysts for the preparation of enantiopure epoxides by enantioselective hydrolysis of cheap and easily available racemic epoxide substrates. EHs are commercially potential biocatalysts due to their characteristics such as high enantioselectivity, cofactor-independent catalysis, and easy-to-prepare catalysts. In this paper, recent progresses In molecular engineering of EHs are reviewed to evaluate the commercial feasibility of EH-catalyzed hydrolytic kinetic resolution for the production of enantiopure epoxides.

Enantioconvergent Hydrolysis of Racemic Epoxides for Production of Enantiopure Epoxides and Vicinal Diols using Epoxide Hydrolases (에폭사이드 가수분해효소에 의한 광학수렴반응을 이용한 광학활성 에폭사이드 및 Vicinal Diol 제조)

  • Lee, Eun-Yeol
    • KSBB Journal
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    • v.22 no.3
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    • pp.123-128
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    • 2007
  • One drawback of conventional kinetic resolution of racemic epoxides by epoxide hydrolase (EH) is that the theoretical yield can never exceed 50%. This 50% limitation can be overcome by using enantioconvergent process, in which both enantiomers of the racemic epoxide are transformed via stereochemically matching pathways into a single enantiopure diol as the sole product in 100% theoretical yield. In order to make a single enantiopure vicinal diol, the two enantiomers of the racemic epoxide must be hydrolyzed with retention and inversion of configuration each other. The EHs should be enantio- and regiospecific at the same time. The enantioconvergent hydrolysis with EHs and relevant biotransformation for preparing enantiopure epoxides and vicinal diols with a high yield are reviewed.

Production of enantiopure epoxides by yeast epoxide hydrolase using a two-phase membrane bioreactor (한국생물공학회 정기총회 및 연구논문발표회 발표논문)

  • Choe, Won-Jae;Choe, Cha-Yong
    • 한국생물공학회:학술대회논문집
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    • 2000.04a
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    • pp.145-148
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    • 2000
  • Large-scale resolution of epoxides by the yeast Rhodotorula glutinis was demonstrated in an aqueous/organic two-phase cascade membrane bioreactor. Due to the chemical instability and low solubility of epoxides in aqueous phases, an organic solvent was introduced into the reaction mixture in order to enhance resolution of epoxide. A cascade hollow-fiber membrane bioreactor was used (i) to minimize the toxicity of organic solvents towards the epoxide hydrolase of Rhodotorula glutinis, and (ii) to remove inhibitory amounts of formed diol from the yeast cell containing aqueous phase. Dodecane was selected as a suitable solvent and 1,2-epoxyhexane as a model substrate. By use of this membrane bioreactor, highly concentrated (0.9 M in dodecane) enantiopure (>98% ee) (S)-1,2-epoxyhexane (6.5 g, 30% yield) was obtained from its racemic mixture.

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Asymmetric resolution of racemic styrene oxide using recombinant Escherichia coli harboring epoxide hydrolase of Rhodotorula glutinis (Rhodotorula glutinis 유래의 고효율 재조합 Epoxide Hydrolase를 이용한 라세믹 Styrene Oxide의 비대칭 광학분할)

  • Park, Kyu-Deok;Choi, Sung-Hee;Kim, Hee-Sook;Lee, Eun-Yeol
    • KSBB Journal
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    • v.23 no.5
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    • pp.369-374
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    • 2008
  • The effects of reaction temperature and the addition of various detergents on the enantioselective hyrolysis activity of the recombinant Escherichia coli containing the epoxide hydrolase (EH) gene of Rhodotorula glutinis were investigated for the production of enantiopure styrene oxide. The recombinant E. coli harboring the EH gene from R. glutinis exhibited the enantiopreference toward (R)-styrene oxide with the maximum hydrolytic activity of $165.04{\mu}mol/min/mg$ of dry cell weight (dcw). The addition of 0.5% (w/v) Tween 20 at $10^{\circ}C$ increased the initial hydrolysis rate and enantioselectivity by 1.45-fold and 2.0-fold, respectively. Enantiopure (S)-styrene oxide was prepared with 99% ee enantiopurity and 46.0% yield (theoretical yield=50%) from 20 mM racemic styrene oxide.

Production of Enantiopure Styrene Oxide by Recombinant Pichia pastoris carrying Double Expression cassette of Epoxide Hydrolase Gene (에폭사이드 가수분해효소 유전자의 double expression cassette 재조합 Pichia pastoris를 이용한 enantiopure styrene oxide의 제조)

  • Kim, Hee-Sook
    • Journal of Life Science
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    • v.18 no.1
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    • pp.136-142
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    • 2008
  • A recombinant Pichia pastoris carrying double expression cassette of Rhodotorula glutinis epoxide hydrolase(RgEH) gene was developed and used for preparing enantiopure (S)-styrene oxide from racemic mixture of styrene oxide. BglII restriction site of original RgEH gene (pPICZ B/RgEH #2) of previous report was mutated using PCR technique for the construction of double expression cassette containing promoter ($P_{AOX1}$), EH gene and transcription terminator ($TT_{AOX1}$) in pPICZ C vector. Double expression cassette with RgEH was inserted into the chromosomal DNA of P. pastoris. $V_{max}$ ($2.2{\mu}mol\;min^{-1}mg\;dcw^{-1}$) on (R)-styrene oxide of P. pastoris with double expression cassette was about 6-fold higher than that ($0.4{\mu}mol\;min^{-1}mg\;dcw^{-1}$) of P. pastoris with single expression cassette. For the determination of the optimal condition, the effects of detergent and temperature on the enantioselective hydrolytic activity and yield of the enantiomer were investigated. When the reaction was performed at $10^{\circ}C$ for 10 min in the presence of 0.5% Tween 20, enantiopure (S)-styrene oxide with 99.9% ee was obtained as the yield 43.4 % from 20 mM racemic sustrate.

Cloning and Characterization of Zebrafish Microsomal Epoxide Hydrolase Based on Bioinformatics (생물정보학을 이용한 Zebrafish Microsomal Epoxide Hydrolase 클로닝 및 특성연구)

  • Lee Eun-Yeol;Kim Hee-Sook
    • Microbiology and Biotechnology Letters
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    • v.34 no.2
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    • pp.129-135
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    • 2006
  • A gene encoding for a putative microsomal epoxide hydrolase (mEH) of a zebrafish, Danio rerio, was cloned and characterized. The putative mEH protein of D. rerio exhibited sequence similarity with mammalian mEH and some other bacterial EHs. A structural model for the putative mEH was constructed using homology modeling based on the crystallographic templates, 1 qo7 and 1 ehy. The catalytic triad consisting of $Asp^{233}$, $Glu^{413}$, and $His^{440}$ was identified, and the characteristic features such as two tyrosine residues and oxyanion hole were found to be highly conserved. Based on bioinformatic analysis together with EH activity assay, the putative protein was annotated as mEH of D. rerio. Enantiopure styrene oxide with enantiopurity of 99%ee and yield of 33.5% was obtained from racemic styrene oxide by the enantioselective hydrolysis activity of recombinant mEH of D. rerio for 45 min.

Production of Chiral (S)-styrene Oxide by Rhodosporidium sp. SJ-4 which has an Epoxide Hydrolase Activity (에폭사이드 가수분해효소를 갖는 Rhodosporidium sp. SJ-4를 이용한 광학활성 (S)-styrene Oxide의 생산)

  • Yoo, Seung-Shick;Lee, Eun-Yeol;Kim, Hee-Sook;Kim, Jung-Sun;Oh, You-Kwan;Park, Sung-Hoon
    • Journal of Institute of Control, Robotics and Systems
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    • v.11 no.10
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    • pp.857-863
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    • 2005
  • A yeast strain utilizing styrene epoxide as a sole carbon and energy source was isolated from soil samples for the production of enantiopure of styrene epoxide by kinetic resolution. The strain, identified as a Rhodosporidium toruloides SJ-4, expressed an epoxide hydrolase which preferentially converted (R)-styrene epoxide into the corresponding diol. A maximum activity of 135 U/L was observed when biomass (dry cell mass) reached 6.7 g/L at 21 h of batch culture. Under the partially optimized reaction conditions ($35^{\circ}C$ and pH 8.0), the optically pure (S)-styrene epoxide was obtained with the yield of 21% when the initial substrate concentration was 100 mM. The reaction was completed at 9 h.