A thermally stable Klebsiella oxytoca hydrolase was explored as an enantioselective biocatalyst for the hydrolytic resolution of (R,S)-ethyl mandelate in biphasic media. Effects of various process parameters such as solvent type, temperature, pH, product inhibition, enzyme loading and substrate concentration on the enzyme performance were studied, leading to the high enzyme (S)-enantioselectivity of E=56 at 45 degrees C for the reaction media consisting of iso-octane and pH 7 buffer. The optimal conditions of pH between 7 and 8, temperature 45 degrees C and enzyme loading no less than 0.5 mg/ml were proposed for obtaining optically pure (R)-ethyl mandelate remained in the organic phase. (c) 2005 Elsevier Inc. All rights reserved.