Molecular Cloning and Functional Expression of esf Gene Encoding Enantioselective Lipase from Serratia marcescens ES-2 for Kinetic Resolution of Optically Active (S)-Flurbiprofen

• Published in: Journal of microbiology and biotechnology Vol. 17; no. 1; pp. 74 - 80
• Main Authors: Lee, Kwang-Woo, Bae, Hyun-Ae, Lee, Yong-Hyun
• Format: Journal Article
• Language: Korean
• Published: 한국미생물생명공학회 01.01.2007; 한국미생물·생명공학회
• Subjects: (S)-flurbiprofen; Enantioselective Lipase; esf gene; kinetic resolution; molecular cloning and expression; Serratia marcescens ES-2; 생물학; kinetic resolution; (S)-flurbiprofen; esf gene; Enantioselective lipase; Serratia marcescens ES-2; molecular cloning and expression
• ISSN: 1017-7825; 1738-8872

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 217kU/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 ${\beta}-cyclodextrin$ as the dispenser at $37^{\circ}C$ for 12h, achieving the high enantiomeric excess and conversion yield of 98% and 48%, respectively.