Alcohol dehydrogenase from Lactobacillus kefir (LKADH)is a versatile catalyst for the conversion of prochiral ketones to chiral alcohols. However, the enzyme itself as well as its essential cofactor are highly unstable in the presence of organic solvents. Thus, the conversion of substrates with low solubility in water is hardly possible despite LKADH accepting a rather broad range of such substrates. In this work, this drawback was overcome by entrapping the enzyme and its cofactor in polyvinyl alcohol gel beads which were applied to pure hexane. The entrapped enzyme successfully transformed a number of hydrophobic ketones to the corresponding enantiomerically pure (R)-alcohols. Enzyme productivity and stability after immobilisation were investigated with the stereoselective conversion of acetophenone into (R)-phenylethanol as a representative example. Almost independent of the bead sizes (circle divide 500-1000 mum), the productivity was 8.35 x 10(-2) g/(L min). Cofactor regeneration was accomplished within the hydrogel matrix using isopropanol as co-substrate. The total turnover number of the cofactor was 10(2)-10(3). The entrapped LKADH showed an improved thermostability and long-term activity under controlled temperature conditions. Also, the storage half-life time of the LKADH at 4degreesC was enhanced to 26.6 days in the presence of Mg2+. (C) 2004 Elsevier Inc. All rights reserved.