Optimization of the Reaction Conditions for (R)-Phenyl-1,2-ethanediol Preparation by Recombinant Epoxide Hydrolase from Caulobacter crescentus

재조합 Caulobacter crescentus 에폭사이드 가수분해효소를 이용한 광학활성 Diol 제조 조건의 최적화

  • Lee, Ok-Kyung (Department of Food Science and Biotechnology, Kyungsung University) ;
  • Kim, Hee-Sook (Department of Food Science and Biotechnology, Kyungsung University)
  • 이옥경 (경성대학교 공과대학 식품생명공학과) ;
  • 김희숙 (경성대학교 공과대학 식품생명공학과)
  • Published : 2007.12.28

Abstract

Enantioconvergent hydrolysis process for the preparation of chiral diol from racemic epoxides by using the recombinant Caulobacter crescentus epoxide hydrolase (CcEH) in Escherichia coli BL21 (DE3) was optimized. For the optimization, the effects of detergent, temperature and product inhibition on the enantiopurity and the yield of diol were investigated. (R)-phenyl-1,2-ethanediol with 92% enantiomeric excess and 56% yield from 20 mM racemic styrene oxide was obtained by using the recombinant CcEH at the optimal condition of $10^{\circ}C$ and the addition of 2% (w/v) Tween 80. At 50 mM racemic styrene oxide was used as a substrate, (R)-phenyl-1,2-ethanediol was obtained with 87% enantiomeric excess and 77% yield. Racemic phenyl-1,2-ethanediol, (R)-phenyl-1,2-ethanediol and (S)-phenyl-1,2-ethanediol dramatically inhibited the hydrolytic activity of the recombinant CcEH. These results suggested that another EH with the regioselectivity on ${\beta}$-position of (R)-enantiomer and without feedback inhibition by products would be needed as the partner EH of C. crescentus EH.

대장균 BL21(DE3)에 Caulobacter crescentus epoxide hydrolase (CcEH)를 발현시킨 제조합균주를 이용하여 광학수렴 가수분해(enantioconvergent hydrolysis) 반응을 수행하였으며 라세믹 에폭사이드 기질로부터 광학활성 diol을 생합성하는 조건을 최적화하였다. 반응최적화를 위하여, 계면활성제의 첨가와 반응온도가 생성물인 diol의 광학순도 및 수율에 미치는 영향을 분석하였으며 또한 생성물인 diol에 의한 EH의 가수분해활성 저해효과를 측정하였다. 재조합 CcEH를 생촉매로 사용한 광학수렴 반응에서 Tween 80을 2%(w/v)첨가하여 $10^{\circ}C$로 반응시켰을 때 20 mM 라세믹 styrene oxide로부터 광학순도 92%의 (R)-phenyl-1,2-ethanediot을 수율 56%로 얻을 수 있었다. 기질인 라세믹 styrene oxide를 50 mM 농도로 사용한 경우, 광학순도 87% (R)-phenyl-1,2-ethanediol을 77% 얻을 수 있었다. 생성물인 diol의 저해효과를 실험한 경우, 라세믹 phenyl-1,2-ethanediol, (R)-phenyl-1,2-ethanediol 및 (S)-phenyl-1,2-ethanediol은 10 mM 농도에서부터 재조합 CcEH의 가수분해활성을 현저하게 저해하는 것으로 나타났다. 위의 결과들로 볼 때 CcEH를 사용하여 높은 광학순도의 (R)-phenyl-1,2-ethanediol을 생성하기 위해서는 (R)-styrene oxide의 린치를 선택적으로 공격하는 동시에 생성물에 의해 저해를 받지 않는 partner EH를 개발하는 것이 중요할 것이다.

Keywords

References

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