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KMID : 0613820080180010136
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Journal of Life Science
2008 Volume.18 No. 1 p.136 ~ p.142
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Production of Enantiopure Styrene Oxide by Recombinant Pichia pastoris carrying Double Expression cassette of Epoxide Hydrolase Gene
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Kim Hee-Sook
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Abstract
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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 (), EH gene and transcription terminator () in pPICZ C vector. Double expression cassette with RgEH was inserted into the chromosomal DNA of P. pastoris. () on (R)-styrene oxide of P. pastoris with double expression cassette was about 6-fold higher than that () 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 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.
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KEYWORD
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Epoxide hydrolase, enantiopure, styrene oxide, double expression cassette
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