Fusarium sp. BU-011, as a high-yielding strain for D-pantolactone hydrolase production was screened from 19 Fusarium sp. strains. The conditions for enzyme formation were studied. The most suitable carbon source for D-pantolactone hydrolase production was sucrose and the best nitrogen source was corn steep liquor. The optimum temperature and the initial pH for enzyme formation was 26 degrees C and pH 7.0, respectively. When the fungus was cultured in a medium containing 30 g/L sucrose, 100 g/L corn steep liquor in the 10 L fermentor, 30.2 g dry cells/L and 410 U/L lactonohydrolase activity were obtained. For optical resolution, the optimum temperature and the optimum pH for the reaction with free cells was 50 degrees C and pH 7.0, respectively. The optimum substrate concentration was 200 g/L racemic pantolactone. The lactonohydrolase activity of immobilized cells remained at 80% of the original reaction after 20 batches of reuse. (c) 2004 Published by Elsevier Ltd.