Cloning, expression, and characterization of an (R)-specific alcohol dehydrogenase from Lactobacillus kefir

Lactobacillus kefir DSM 20587 produces an (R)-specific NADP-dependent alcohol dehydrogenase (ADH) with a broad substrate specificity. The gene of this ADH was isolated and the complete nucleotide sequence determined. The adh gene comprises 759 bp and encodes a protein of 252 amino acids with a calculated molecular weight of 26 781 Da. The deduced amino acid sequence indicated a high degree of similarity to short-chain dehydrogenases. After cloning and expression in Escherichia coli the enzyme was purified and characterized. For the reduction of acetophenone the specific activity of the homogeneous recombinant ADH was 558 U mg−1. The enzyme shows its maximum activity at 50°C while the pH optimum was at pH 7.0. In order to demonstrate its preparative application, purified ADH was used for the stereoselective reduction of several aliphatic and aromatic ketones as well as β-keto esters. Glucose dehydrogenase was added for the regeneration of NADPH. All prochiral ketones were stereoselectively reduced to the c...