BACKGROUND: A process combining biocatalytic reaction and cell activity regeneration was designed for the asymmetric reduction of 3-oxo ester. By immobilizing resting baker's yeast (Saccharomyces cerevisiae) in calcium alginate beads, the high yield and long catalyst life were achieved in the aqueous phase in this process with methyl acetoacetate (MAA) as the model substrate. RESULTS: Two combined fixed-bed reactors were able to work steadily for at least 16 days. The activity of immobilized baker's yeast could be retained by re-culture with culture medium regularly. The re-culture time for bead reactivation was optimized to be 30 h. High yield (about 80%) and high enantiomeric excess (>95%) were maintained after 12 batches of asymmetric reduction. The immobilized beads retained their original shapes even after a long reaction time in the fixed-bed reactor, while the beads broke after reaction of five batches in a flask. CONCLUSION: The combined process of biocatalysis and cell activity regeneration was successfully achieved in the asymmetric reduction and decreased the breakage of beads as well as increased the efficiency of catalyst. (C) 2008 Society of Chemical Industry