Substrate selectivity and kinetic studies of (S)-specific alcohol dehydrogenase purified from Candida parapsilosis ATCC 7330

Abstract

Biocatalytic reduction catalysed by alcohol dehydrogenases is a valuable tool for asymmetric synthesis of chiral alcohols. This study reports the broad substrate specificity of NADH dependent (S) - specific alcohol dehydrogenase (S-ADH) purified from Candida parapsilosis ATCC 7330. The substrates for this enzyme include aliphatic and aromatic ketones, cyclic and diketones, aldehydes, ketoesters, primary and secondary alcohols. The kinetic studies of different substrates indicate that ketones and secondary alcohols are the most preferred substrates of S-ADH with highest catalytic efficiencies reported for reduction of acetone (4153 s−1mM−1) and oxidation of 2-propanol (1358 s−1mM−1). The double reciprocal plots obtained for varied concentrations of acetophenone (0.2–16 mM) at a fixed concentration of NADH (0.05, 0.1 and 0.2 mM) and with varied concentrations of NADH (0.01–0.2 mM) at a fixed concentration of acetophenone (1, 4, 8 and 16 mM) showed intersecting lines indicating sequential kinetic mechanism. S-ADH follows Prelog's stereopreference in reducing prochiral carbonyl substrates to yield (S)-alcohols with >99% enantiomeric excess using a simple coupled substrate approach for cofactor recycling.