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

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-beta-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.

Carbonyl reductase identification and development of whole-cell biotransformation for highly efficient synthesis of (R)-[3,5-bis(trifluoromethyl)phenyl] ethanol

“…Zhu et al reported that CR-3 also showed the reductive activity toward acetophenone, but producing ( S )-alcohols. Compared to ketones, CR-3 preferred aldehydes as substrates [25]. Together with our study, L. kefir has at least two carbonyl reductases (CR-2 and CR-3) with reductive activity toward ketones but opposite enantioselectivity.…”

“…Previous studies indicated that enhancing the spatial proximity of enzymes through the creation of fusion constructs could regulate catalytic efficiency and enhance product synthesis in multi-enzyme reactions [25, 29, 30, 31]. The common and easiest approach to construct a fusion enzyme is to fuse the sequentially acting enzymes end to end by a linker peptide.…”

Highly enantiomeric reduction of acetophenone and its derivatives by locally isolated Rhodotorula glutinis

“…However, these reactions generally require expensive cofactors such as NADPH. The cofactors can, however, be regenerated with whole cells as catalysts during the reduction processes 14, 18–20. In this study, the method used for the bioreduction is different, because the substrate was directly added to the culture medium without using resting cells, cofactors, and buffers.…”

Enzymatic Bioreactors: An Electrochemical Perspective

“…A variety of oxidoreductases have been used for the asymmetric synthesis of amines, amino acids and alcohols [186][187][188]. Dong et al developed a biphasic bioelectrocatalysis system for the synthesis of (R)-CHCN (81.2%), (R)-3-hydroxy-3-Phenylpropanenitrile (96.8% ee), (S)-3-hydroxy-4phenylbutanenitrile (94.6% ee) using alcohol dehydrogenase and halohydrin dehalogenase.…”