Molecular cloning and functional expression of esf gene encoding enantioselective lipase from Serratia marcescens ES-2 for kinetic resolution of optically active (S)-flurbiprofen

J Microbiol Biotechnol. 2007 Jan;17(1):74-80.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Motifs
  • Amino Acid Sequence
  • Base Sequence
  • Cloning, Molecular
  • DNA Primers / genetics
  • DNA, Bacterial / genetics
  • Escherichia coli / genetics
  • Flurbiprofen / chemistry*
  • Flurbiprofen / isolation & purification
  • Gene Expression
  • Genes, Bacterial
  • Hydrogen-Ion Concentration
  • Kinetics
  • Lipase / chemistry
  • Lipase / genetics*
  • Lipase / metabolism*
  • Molecular Sequence Data
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Sequence Homology, Amino Acid
  • Serratia marcescens / enzymology*
  • Serratia marcescens / genetics*
  • Stereoisomerism
  • Substrate Specificity
  • Temperature

Substances

  • DNA Primers
  • DNA, Bacterial
  • Recombinant Proteins
  • Flurbiprofen
  • Lipase

Associated data

  • GENBANK/DQ841349