The stereoinversion process involving two sequential stereoselective oxidation and reduction reactions by oxidoreductases was a typical and important one-pot method for chiral secondary alcohol synthesis which has great potential for industrial application. The process to produce (S)-1-phenyl-1,2-ethanediol with whole cell was chosen as a model reaction to investigate the kinetic and thermodynamic properties of key enzymes which catalyzed the single oxidation and reduction step of the whole reaction. The kinetic data from initial rate experiments in the absence of product prove that both oxidation and reduction reactions follow Theorell-Chance BiBi mechanism. Parameters of V(m), and AS suggest that the oxidation reaction was thermodynamically unfavorable and the restrictive step of the one-pot process was inhibited at high concentration of substrate PED. To remove the inhibition of substrate, extractive biocatalytic methods were successfully applied to improve the substrate PED concentration from 15 g/l to 50 g/l with high optical purity of 91.6% and yield of 82.5%. (C) 2010 Elsevier Ltd. All rights reserved.