Abstract
Kinetic analysis and modeling for asymmetric reduction of ethyl-4- chloroacetoacetate to produce S-4-chloro-3-hydroxybutanoic acid ethyl ester catalyzed by Saccharomyces cerevisiae was conducted. The effect of temperature and pH on the activity and the stability of the biocatalyst were examined. The change of the kinetic behavior when XAD 2 resin was added to the reaction solution was also investigated. When the substrate concentration was 73.5 mM, relative high product’s optical purity (87.5% ee) and reaction yield (74.5%) were obtained at 30℃, pH 8.5. A significant decomposition effect for substrate in the reaction solution without yeast cells was observed. At the operation condition, the substrate hydrolysis rate constant was determined as 8.31×10-5 min-1.51．mM-1.51 and the reaction followed a 2.51-order kinetics. On the other hand, the product could exist steadily in the reaction solution. A substrate inhibition was found in this biocatalytic reaction, while no product inhibition was observed. From the initial rate data obtained from the experiments performed at different initial substrate concentrations, Lineweaver-Burk plot, Hanes plot and Eadie-Hofstee plot techniques were used to determine the values of the apparent kinetic parameters. The Km, Ki and vm were estimated to be 17.84 mM, 193.76 mM and 11.66 mM/g cell□ min, respectively. In addition, the apparent Km, Ki and vm for the S-enzymes in S. cerevisiae cells were estimated to be 29.00 mM, 119.23 mM and 12.41 mM/g cell□ min, respectively. The apparent Km, Ki and vm for the R-enzymes were estimated to be 35.91 mM, 96.29 mM and 0.9 mM/g cell□ min, respectively. Under the simultaneous consideration of the effects of substrate inhibition and substrate hydrolysis, a kinetic model for describing the apparent reaction behavior was established. The good consistency between the experimental data and the theoretically predicted values verifies that the proposed model can successfully simulate the behavior of this reaction. Finally, the supplement of XAD 2 resin could effectively reduce the substrate inhibition effect. The reaction yield and the product’s ee could also be enhanced.