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KMID : 0545120070170010074
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Journal of Microbiology and Biotechnology
2007 Volume.17 No. 1 p.74 ~ p.80
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Molecular Cloning and Functional Expression of esf Gene Encoding Enantioselective Lipase from Serratia marcescens ES-2 for Kinetic Resolution of Optically Active (S)-Flurbiprofen
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Lee Kwang-Woo
Lee Yong-Hyun
Bae Hyun-AE
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Abstract
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An enantioselective lipase gene (esf) for the kinetic resolution of optically active (S)-flurbiprofen was cloned from the new strain Serratia marcescens ES-2. The esf gene was composed of a 1,845-bp open reading frame encoding 614 amino acid residues with a calculated molecular mass of 64,978 Da. The lipase expressed in E. coli was purified by a three-step procedure, and it showed preferential substrate specificity toward the medium-chain-length fatty acids. The esf gene encoding the enantioselective lipase was reintroduced into the parent strain S. marcescens ES-2 for secretory overexpression. The transformant S. marcescens BESF secreted up to 217 kU/ ml of the enantioselective lipase, about 54-fold more than the parent strain, after supplementing 3.0% Triton X-207. The kinetic resolution of (S)-flurbiprofen was carried out even at an extremely high (R,S)-flurbiprofen ethyl ester [(R,S)-FEE] concentration of 500 mM, 130 kU of the S. marcescens ES-2 lipase per mmol of (R,S)-FEE, and 1,000 mM of succinyl β- cyclodextrin as the dispenser at 37oC for 12 h, achieving the high enantiomeric excess and conversion yield of 98% and 48%, respectively.
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KEYWORD
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Enantioselective lipase, esf gene, Serratia marcescens ES-2, molecular cloning and expression, kinetic resolution, (S)-flurbiprofen
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