Molecular cloning and functional expression of esf gene encoding enantioselective lipase from Serratia marcescens ES-2 for kinetic resolution of optically active (S)-flurbiprofen.
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 beta-cyclodextrin as the dispenser at 37 degrees C for 12 h, achieving the high enantiomeric excess and conversion yield of 98% and 48%, respectively.
Department of Genetic Engineering, College of Natural Sciences, Kyungpook National University, Daegu 702-701, Korea.
Kwang-Woo
KW
Lee
Hyun-Ae
HA
Bae
Yong-Hyun
YH
Lee
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005480:Flurbiprofen / Q000737:chemistry* / Q000302:isolation & purification; D015870:Gene Expression; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008049:Lipase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012706:Serratia marcescens / Q000201:enzymology* / Q000235:genetics*; D013237:Stereoisomerism; D013379:Substrate Specificity; D013696:Temperature
D017931:DNA Primers; D004269:DNA, Bacterial; D011994:Recombinant Proteins; D005480:Flurbiprofen; D008049:Lipase