BACKGROUND: A simple procedure was employed to covalently immobilize a Klebsiella oxytoca hydrolase (SNSM-87) onto epoxy-activated supports of Eupergit C 250L via multipoint covalent attachment. The resultant biocatalyst was explored for the hydrolytic resolution of a variety of (R,S)-2-hydroxycarboxylic acid ethyl esters. RESULTS: With the hydrolytic resolution of (R,S)-ethyl mandelate in biphasic media as the model system, optimal conditions of 55 degrees C, pH 6 buffer and isooctane as the organic phase were selected for improving the enzyme stability (activity retained from 10% to 50% at 96h) and enantioselectivity (V(S)V(R)(-1)value enhanced from 44 to 319) in comparison to the performance of free enzyme. Moreover, the immobilized enzyme retained its activity and enantioselectivity after eight cycles of hydrolysis at 55 degrees C. When applying the resolution process to other (R,S)-2-hydroxycarboxylic acid ethyl esters, 2.4- to 4.0-fold enhancements of the enantioselectivity in general were obtainable. CONCLUSIONS: The enantioselectivity enhancement, good reusability and easy recovery after reaction indicate that the immobilized SNSM-87 may have the potential as an industrial biocatalyst for the preparation of optically pure 2-hydroxycarboxylic acids. (C) 2008 Society of Chemical Industry