Immobilization conditions of ketoreductase on enantioselective reduction in a gas-solid bioreactor.

The immobilization conditions of commercial ketoreductase for continuous enantioselective reduction in the gas-phase reaction were investigated with respect to the immobilization efficiency (residual activity and protein loading) and the gas-phase reaction efficiency (initial reaction rate, half-life, and enantioselectivity). For the analyses, ketoreductase was first immobilized by physical deposition on glass supports and the reduction of 2-butanone to (S)-2-butanol with the concomitant regeneration of NADH by 2-propanol was used as a model reaction. The optimal conditions of enzyme immobilization were obtained using an absolute pressure of 100 hPa for drying, a pH between 6.5 and 7.0, and a buffer concentration of 50 mM. The buffer concentration in particular had a strong effect on both the enzyme activity and enantioselectivity. Under optimal immobilization conditions, the thermostability of ketoreductase in the gas-phase system was enhanced compared to the aqueous-phase system, while the enantioselectivity was successfully maintained at a level identical to that of the native enzyme. These results indicate that the gas-phase reaction has a great potential for industrial production of chiral compounds, but requires careful optimization of immobilization conditions for the reaction to progress effectively.