KSBB Journal

The Korean Society for Biotechnology and Bioengineering (한국생물공학회)
권호 표지

Bioinformatics based Identification and Characterization of Epoxide Hydrolase of Gordonia westfalica for the Production of Chiral Epoxides

Bioinformatics를 활용한 토양미생물인 Gordonia westfalica Epoxide Hydrolase 생촉매 개발 및 Chiral Epoxides 제조 특성 분석

  • Lee Soo Jung (Department of Food Science and Technology, Kyungsung University) ;
  • Lee Eun Jung (Department of Food Science and Technology, Kyungsung University) ;
  • Kim Hee Sook (Department of Food Science and Technology, Kyungsung University) ;
  • Lee Eun Yeol (Department of Food Science and Technology, Kyungsung University)
  • 이수정 (경성대학교 공과대학 식품공학과) ;
  • 이은정 (경성대학교 공과대학 식품공학과) ;
  • 김희숙 (경성대학교 공과대학 식품공학과) ;
  • 이은열 (경성대학교 공과대학 식품공학과)
  • Published : 2005.08.01
Download PDF
⟨ Previous Next ⟩

Abstract

Epoxide hydrolases (EHs) are versatile biocatalysts for the preparation of chiral epoxides by enantioselective hydrolysis from racemic epoxides. Various microorganisms were identified to possess a EH activity by multiple sequence alignment and analysis of conserved domain sequence from genomic and megaplasmid sequence data. We successfully isolated Gordonia westfalica possessing EH activity from various microbial strains from culture type collections. G. westfalica exhibited (R)-styrene oxide preferred enantioselective hydrolysis activity. Chiral (S)-styrene oxide with high optical purity $(>\;99\%)\;ee)$ and yield of $36.5\%$ was obtained from its racemate using whole-cell of G. westfalica.

EH의 catalytic nucleophile residue, His-Asp로 구성된 charge relay system, oxyanion hole 등의 EH 관련 conserved domain의 아미노산 공통 서열을 참고로 하여 G. westfalica megaplasmid로부터 putative EH를 선별할 수 있었다. Bioinformatics를 기반으로 스크리닝한 G. westfalica에 의한 라세믹 styrene oxide 기질에 대한 입체선택성 가수분해 반응에 있어 중요 반응 parameter들인, pH 및 온도 등이 초기 가수분해반응속도에 미치는 영향을 분석하고, 최적 회분식 반응조건을 결정하였다. 최적 반응조건인 pH 7, 반응 온도 $30^{\circ}C$, 생촉매량 40 mg의 조건에서 약 5시간 20분간 반응을 통해 20 mM 라세믹 기질로부터 광학순도 $100\%$ ee인 (S)-styrene oxide를 $36.5\%$의 수율로 얻을 수 있었다.

Keywords

References

  1. Sheldon, R. A. (1993), Chirotechnology, Marcel Dekker, New York
  2. Besse, P. and H. Veschambre (1994), Chemical and biological synthesis of chiral epoxides, Tetrahedron 50, 8885-8927 https://doi.org/10.1016/S0040-4020(01)85362-X
  3. Archelas A. and R. Furstoss (2001), Synthetic applications of epoxide hydrolases, Current Opinion in Chem. Biology 5, 112-119 https://doi.org/10.1016/S1367-5931(00)00179-4
  4. Steinreiber, A. and K. Faber (2001), Microbial epoxide hydrolases for preparative biotransformations, Current Opinion in Biotechnol. 12, 552-558 https://doi.org/10.1016/S0958-1669(01)00262-2
  5. Weijers, C. A. G. M., and J. A. M. de Bont (1999), Epoxide hydrolases from yeasts and other sources: versatile tools in biocatalysis, J. Mol. Catal. B: Enzym., 6, 199-214 https://doi.org/10.1016/S1381-1177(98)00123-4
  6. Lee, E. Y. (2002), Epoxide hydrolase-catalyzed hydrolytic kinetic resolution for the production of chiral epoxides, Kor. J. Biotechnol. Bioeng. 17, 321-325
  7. de Vries, E. J. and D. B. Janssen (2003), Biocatalytic conversion of epoxides, Current Opinion Biotechnol. 14, 1-7 https://doi.org/10.1016/S0958-1669(02)00013-7
  8. Lee, E. Y., S.-S. Yoo, H. S. Kim, S. J. Lee, Y.-K. Oh, and S. Park (2004), Production of (S)-styrene oxide by recombinant Pichia pastoris containing epoxide hydrolase from Rhodotorula glutinis, Enzyme Microbial Technol. 35, 624-631 https://doi.org/10.1016/j.enzmictec.2004.08.016
  9. Hellstrom, H., A. Steinreiber, S. F. Mayer, and K. Faber (2001), Bacterial epoxide hydrolase-catalyzed resolution of a 2,2-disubstituted oxirane: optimization and upscaling, Biotechnol. Letters 23, 169-173 https://doi.org/10.1023/A:1005636121060
  10. Hernandez-Perez, G., F. Fayolle, and J. P. Vandecasteele (2001), Biodegradation of ethyl t-butyl ether, methyl t-butyl ether and t-amyl methyl ether by Gordonia terrae, Appl. Microbiol. Biotechnol. 55, 117-121 https://doi.org/10.1007/s002530000482
  11. Broker, D., M. Arenskotter, A. Legatzki, D. H. Nies, and A. Steinbuchel (2004), Characterization of the 101-kilobase-pair megaplasmid pKBl, isolated from the rubber-degrading bacterium Gordonia westfalica Kbl, J. Bacteriol. 186, 212-225 https://doi.org/10.1128/JB.186.1.212-225.2004
  12. Arand, M., D. F. Grant, J. K. Beetham, T. Friedberg, F. Oesch, and B. D. Hammock (1994), Sequence similarity of mammalian epoxide hydrolases to the bacterial haloalkane dehalogenase and other related proteins. Implication for the potential catalytic mechanism of enzymatic epoxide hydrolysis, FEBS Lett. 338, 251-256 https://doi.org/10.1016/0014-5793(94)80278-5
  13. Lewis, D. F. V., B. G. Lake, and M. G. Bird (2005), Molecular modeling of human microsomal epoxide hydrolase (EH) by homology with a fungal (Aspergillus niger) EH crystal structure of 1.8 $\AA$ resolution: structure-activity relationships in epoxide inhibiting EH activity, Toxicology in Vitro 19, 517-522 https://doi.org/10.1016/j.tiv.2004.07.001
  14. Schwede, T., J. Kopp, N. Guex; and M. C. Peitsch (2003), SWISS-MODEL: an automated protein homology-modeling server, Nucleic Acids Research 31, 3381-3385 https://doi.org/10.1093/nar/gkg520