Cloning, functional expression and biochemical characterization of a stereoselective alcohol dehydrogenase from Pseudomonas fluorescens DSM50106

Appl Microbiol Biotechnol. 2002 Aug;59(4-5):483-7. doi: 10.1007/s00253-002-1036-2. Epub 2002 Jun 26.

Abstract

Sequencing of a genomic library prepared from Pseudomonas fluorescens DSM 50106 identified an orf showing 29% identity to a C alpha-dehydrogenase of Pseudomonas paucimobilis and high homology to several sequences with unknown functions derived from genome projects. The corresponding gene adhF1 encodes a dehydrogenase of 296 amino acids with a calculated molecular mass of 31.997 kDa. The gene was functionally expressed in E. coli using a rhamnose inducible expression system. The resulting recombinant enzyme was active in the pH range 6-10 (best pH 8) and at 5-25 degrees C. This dehydrogenase converts cyclic ketones to the corresponding alcohols utilizing the cofactor NADH. The highest activity was found for cyclohexanone. The enzyme also exhibits high stereoselectivity in the desymmetrization of the prochiral ketone acetophenone, producing optically pure ( R)-alpha-phenyl ethanol (>99%ee) at high conversion (95%).

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alcohol Dehydrogenase / genetics*
  • Alcohol Dehydrogenase / metabolism*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Cloning, Molecular*
  • Escherichia coli / enzymology
  • Escherichia coli / genetics
  • Kinetics
  • Molecular Sequence Data
  • Pseudomonas fluorescens / enzymology*
  • Sequence Analysis, DNA
  • Stereoisomerism
  • Substrate Specificity

Substances

  • Bacterial Proteins
  • Alcohol Dehydrogenase

Associated data

  • GENBANK/AF090329