Results
Title

Cloning, Expression, and Characterization of a Novel (S)-Specific Alcohol Dehydrogenase from Lactobacillus kefir.

Authors

Qilei Chen; Wenjie Zhao; Chunbao Zhu; Baoquan Zhu

Abstract

Abstract  A gene encoding a novel (S)-specific NADH-dependent alcohol dehydrogenase (LK-ADH) was isolated from the genomic DNA of Lactobacillus kefir DSM 20587 by thermal asymmetric interlaced-polymerase chain reaction. The nucleotide sequence of (S)-LK-ADH gene (adhS) was determined, which consists of an open reading frame of 1,044 bp, coding for 347 amino acids with a molecular mass of 37.065 kDa. After a BLAST similarity search in GenBank database, the amino acid sequence of (S)-LK-ADH showed some homologies to several zinc containing medium-chain alcohol dehydrogenases. This novel gene was deposited into GenBank with the accession number of EU877965. adhS gene was subcloned into plasmid pET-28a(+), and recombinant (S)-LK-ADH was successfully expressed in E. coli BL21(DE3) by isopropyl-β-d-1-thiogalactopyranoside induction. Purified enzyme showed a high enantioselectivity in the reduction of acetophenone to (S)-phenylethanol with an ee value of 99.4%. The substrate specificity and cofactor preference of recombinant (S)-LK-ADH were also tested.

Subjects

ALCOHOL dehydrogenase; MOLECULAR cloning; GENE expression; LACTOBACILLUS; GENETIC code; POLYMERASE chain reaction; NUCLEOTIDE sequence; ACETOPHENONE

Publication

Applied Biochemistry & Biotechnology, 2010, Vol 160, Issue 1, p19

ISSN

0273-2289

Publication type

Academic Journal

DOI

10.1007/s12010-008-8442-6