• Title, Summary, Keyword: (S)-styrene oxide

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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.

Characterization of Styrene Catabolic Genes of Pseudomonas putida SN1 and Construction of a Recombinant Escherichia coli Containing Styrene Monooxygenase Gene for the Production of (S)-Styrene Oxide

  • Park Mi-So;Bae Jong-Won;Han Ju-Hee;Lee Eun-Yeol;Lee Sun-Gu;Park Sung-Hoon
    • Journal of Microbiology and Biotechnology
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    • v.16 no.7
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    • pp.1032-1040
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    • 2006
  • Some Pseudomonas species can grow on styrene as a sole carbon and energy source. From the new isolate Pseudomonas putida SN1, the genes for styrene catabolism were cloned and sequenced. They were composed of four structural genes for styrene monooxygenase (styA and styB), styrene oxide isomerase (styC), and phenylacetaldehyde dehydrogenase (styD), along with two genes for the regulatory system (styS and styR). All the genes showed high DNA sequence (91% to 99%) and amino acid sequence (94% to 100%) similarities with the corresponding genes of the previously reported styrene-degrading Pseudomonas strains. A recombinant Escherichia coli to contain the styrene monooxygenase from the SN1 was constructed under the control of the T7 promoter for the production of enantiopure (S)-styrene oxide, which is an important chiral building block in organic synthesis. The recombinant E. coli could convert styrene into an enantiopure (S)-styrene oxide (ee >99%) when induced by IPTG The maximum activity was observed as 140 U/g cell, when induced with 1 mM IPTG at $15^{\circ}C$.

Production of Chiral Styrene Oxide by Microbial Enantioselective Hydrolysis Reaction (미생물 입체선택성 가수분해 반응을 이용한 광학활성 Styrene Oxide 생산)

  • 윤성준;이은열
    • KSBB Journal
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    • v.15 no.6
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    • pp.630-634
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    • 2000
  • Chiral epoxides are useful chiral synthons in organic synthesis, and various biological methods have been investigated for their production. In this work, the enantioselective resolution of racemic styrene oxide was investigated using Aspergillus niger sp. for the production of optically pure (S)-styrene oxide. The enantioselectivity and initial hydrolysis rates of the racemic substrate were highly dependent of the pH, temperature, and the volume ratio of cosolvent. Experimental sets of pH, temperature, and the volume ratio of cosolvent were investigated using a central composite experimental design, and reaction conditions were optimized by response surface analysis. The optimal conditions of pH, temperature, and the volume ratio of cosolvent were determined to be 7.78, $28.32^{\circ}C$, and 2.4%(v/v), respectively, and optically pure (S)-styrene oxide (>99% ee) was obtained at 35% yield using this microbial enantioselective hydrolysis reaction.

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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.

Enantioselective Kinetic Resolution of Racemic Styrene Oxide using Recombinant Marine Fish Epoxide Hydrolase of Mugil cephalus (해양 어류 Mugil cephalus 유래의 에폭사이드 가수분해효소를 이용한 라세믹 styrene oxide의 입체선택적 분할 반응)

  • Choi, Sung Hee;Kim, Hee Sook;Lee, Eun Yeol
    • Applied Chemistry for Engineering
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    • v.19 no.5
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    • pp.491-496
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    • 2008
  • The microsomal epoxide hydrolase gene (referred to as mMCEH) of Mugil cephalus was cloned by PCR, and then inserted to pColdI and pET-21b(+) vector, respectively. The recombinant E. coli possessing the recombinant plasmids exhibited the enantioperference toward (R)-styrene oxide. When enantioselective kinetic resolutions were conducted with 20 mM racemic styrene oxide, enantiopure (S)-styrene oxide was obtained with high enantiopurity more than 99% enantiomeric excess (ee) and 24.50% yield by using the recombinant E. coli harboring pET-21b(+)/mMCEH.

Development of Asymmetric Resolution System for the Production of Chiral Styrene Oxide by Microbial Epoxide Hydrolase (미생물 유래의 Epoxide Hydrolase를 이용한 Chiral Styrene Oxide 생산용 비대칭 광학분할시스템개발)

  • 이지원;윤여준;이은열
    • Journal of Life Science
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    • v.12 no.5
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    • pp.584-588
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    • 2002
  • Asymmetric enantioselective resolution system using epoxide hydrolase activity of Aspergillus niger LK was developed and operated for the production of optically pure styrene oxide. Two-phase hollow-fiber reactor system was employed for the enhanced solubility of racemic styrene oxide in organic phase and protection of epoxide hydrolase activity in aqueous phase. For the removal of phenyl-1,2-ethandiol, the inhibitor of epoxide hydrolase, cascade hollow-fiber reactor system was also developed. Chiral (S)-styrene oxide (39 mM in dodecane) could be asymmetrically resolved with high enantiopurity (> 99% ee) using these reactor system.

Development of Recombinant Pseudomonas putida Containing Homologous Styrene Monooxygenase Genes for the Production of (S)-Styrene Oxide

  • Bae, Jong-Wan;Han, Ju-Hee;Park, Mi-So;Lee, Sun-Gu;Lee, Eun-Yeol;Jeong, Yong-Joo;Park, Sung-Hoon
    • Biotechnology and Bioprocess Engineering:BBE
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    • v.11 no.6
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    • pp.530-537
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    • 2006
  • Recently isolated, Pseudomonas putida SN1 grows on styrene as its sole carbon and energy source through successive oxidation of styrene by styrene monooxygenase (SMO), styrene oxide isomerase (SOI), and phenylacetaldehyde dehydrogenase. For the production of (S)-styrene oxide, two knockout mutants of SN1 were constructed, one lacking SOI and another lacking both SMO and SOI. These mutants were developed into whole-cell biocatalysts by transformation with a multicopy plasmid vector containing SMO genes (styAB) of the SN1. Neither of these self-cloned recombinants could grow on styrene, but both converted styrene into an enantiopure (S)-styrene oxide (e.e. > 99%). Whole-cell SMO activity was higher in the recombinant constructed from the SOI-deleted mutant (130 U/g cdw) than in the other one (35 U/g cdw). However, the SMO activity of the former was about the same as that of the SOI-deleted SN1 possessing a single copy of the styAB gene that was used as host. This indicates that the copy number of styAB genes is not rate-limiting on SMO catalysis by whole-cell SN1.

Biosynthesis of (R)-phenyl-1,2-ethanediol by using Single Recombinant Epoxide Hydrolase from Caulobacter Crescentus (재조합 epoxide hydrolase를 단일 생촉매로 사용한 광학수렴 가수분해반응을 통한 광학활성 (R)-phenyl-1,2-ethanediol 생합성)

  • Lee, Ok Kyung;Lee, Eun Yeol
    • Applied Chemistry for Engineering
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    • v.18 no.3
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    • pp.279-283
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    • 2007
  • Epoxide hydrolase (EH) gene of Caulobacter crescentus was cloned by PCR and expressed in Escherichia coli. The C. crescentus EH (CcEH) primarily attacked at the benzylic carbon of (S)-styrene oxide, while the CcEH preferentially attacked at the terminal carbon of (R)-styrene oxide, thus leading to the formation of (R)-phenyl-1,2-ethanediol as the main product. (R)-phenyl-1,2-ethanediol was obtained with 85% enantiomeric excess and yield of 69% from racemic styrene oxide via enantioconvergent hydrolysis by using recombinant CcEH as the single biocatalyst.

Optimization of Epoxide Hydrolase-Catalyzed Enantioselective Hydrolysis of Racemic Styrene Oxide (Rhodotorula sp. CL-83 유래의 에폭사이드 가수분해효소를 이용한 라세믹 Styrene Oxide 입체특이성 가수분해 조건 최적화)

  • 이은열
    • Journal of Life Science
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    • v.12 no.6
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    • pp.765-768
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    • 2002
  • Enantioselective hydrolysis of racemic styrene oxide by Rhodotorula sp. CL-82 was investigated. Reaction conditions including pH, temperature, and volume ratio of organic cosolvent were optimized using response surface methodology, and the optimal conditions of pH, temperature, and the volume ratio of cosolvent were determined to be 7.64, $33.26^{\circ}C$, and 3.09 %(v/v), respectively. Chiral (S)-phenyl oxirane could be obtained with high enantiomeric purity (ee > 99%) and 20% yield (theoretical yield = 50%) at the optimal rendition.

Enhanced Heterologous Expression of Aspergillus niger Epoxide Hydrolase and Its Application to Enantioselective Hydrolysis of Racemic Epoxides (Aspergillus niger의 Epoxide Hydrolase 고효율 발현 및 라세믹 에폭사이드의 입체선택적 가수분해)

  • Lee, Soo Jung;Kim, Hee Sook;Lee, Eun Yeol
    • Applied Chemistry for Engineering
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    • v.17 no.5
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    • pp.557-560
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    • 2006
  • The epoxide hydrolase (EH) of Aspergillus niger LK was expressed to high levels in Escherichia coli based on codon usage. E. coli, Rosetta (DE3)PLysS, containing a large number of tRNAs for rare-codons was employed as a host strain. The recombinant E. coli expressing A. niger EH showed an enhanced enantioselective hydrolysis activity toward racemic styrene oxide. Enantiopure (S)-styrene oxide with a high enantiopurity of 99% ee was obtained from racemic substrates.