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

  • Qilei Chen
  • Youjia Hu
  • Wenjie Zhao
  • Chunbao Zhu
  • Baoquan Zhu
Article

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.

Keywords

Lactobacillus kefir TAIL-PCR (S)-specific alcohol dehydrogenase NADH dependent 

Notes

Acknowledgements

This research was supported by Science and Technology Commission of Shanghai Municipality (Grant No. 07dz22002 & 04DZ05902).

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Copyright information

© Humana Press 2008

Authors and Affiliations

  • Qilei Chen
    • 1
  • Youjia Hu
    • 1
    • 2
  • Wenjie Zhao
    • 1
  • Chunbao Zhu
    • 1
  • Baoquan Zhu
    • 1
  1. 1.State Key Laboratory of New Drug and Pharmaceutical ProcessShanghai Institute of Pharmaceutical IndustryShanghaiPeople’s Republic of China
  2. 2.ShanghaiPeople’s Republic of China

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