Anju Chadha

Alcohol dehydrogenases (ADHs) catalyses reversible reduction of carbonyl group to its corresponding alcohols and have been widely employed as versatile biocatalyst due to its high enantioselectivity to produce chiral alcohols. In this study, the (S) - specific alcohol dehydrogenase (S-ADH) enzyme was purified from Candida parapsilosis ATCC 7330. It asymmetrically reduced acetophenone to (S)-1-phenylethanol with > 99% ee with an effective substrate coupled approach for cofactor recycling. In the reverse reaction, i.e., oxidation of 1-phenylethanol, S-ADH produced (R)-1-phenylethanol with > 99% ee via kinetic resolution. S-ADH is a zinc dependent medium chain dehydrogenase/reductase. It was found to be a tetramer in nature with subunit molecular mass of 40 kDa catalysing oxidation and reduction reactions only using NAD+ and NADH as the cofactors respectively with higher affinity for NAD+. Further biochemical characterisation indicated that His and Cys residues play a crucial role in the enzymatic catalysis and requires a reducing environment at the active site. S-ADH was stable at pH 6.0 in reducing acetophenone and pH 8.0 in oxidising 1-phenylethanol at 45 °C with the t1/2 of 6.8 h and 9.0 h respectively. Therefore, purified S-ADH would be a useful biocatalyst in the synthesis of enantiopure (S)- and (R)-1-phenylethanol which are used in the fragrance preparation.

Asymmetric reduction of ethyl-4-chloro-3-oxobutanoate to (S)-ethyl-4-chloro-3-hydroxybutanoate in aqueous medium by resting cells of Candida parapsilosis ATCC 7330 was optimized. The influence of culture parameters (inoculum size, inoculum age and biocatalyst harvest time) and reaction parameters (co-substrate, resting cell, pH and substrate concentrations) on the asymmetric reduction were studied. It was found that these parameters significantly influenced the rate of the asymmetric reduction. Under the optimum conditions, the final concentration of (S)-ethyl-4-chloro-3- hydroxybutanoate, enantiomeric excess and the isolated yield of (S)-ethyl-4-chloro-3-hydroxybutanoate were 1.38 M (230 g/l), >99 and 95%, respectively. The space time yield was 115 mmol/lh, which is significantly higher than other whole cell biocatalysts reported so far. © 2009 Society for Industrial Microbiology.