Interfacial inactivation of epoxide hydrolase in a two-liquid-phase system

Enantioselective epoxide hydrolases are useful biocatalysts for the preparation of enantiopure epoxides and diols. The kinetic resolution of racemic epoxides can be carried out in an organic/aqueous biphasic system to allow use of high epoxide concentrations. Enzyme inactivation in such a system, however, may occur by contact with the interface. In this study, we investigated the factors which influence the interfacial inactivation of Agrobacterium radiobacter epoxide hydrolase in an octane/water biphasic system. Rates of interfacial inactivation were measured both in a stirred-cell, which has a planar interface, and in an emulsion reactor. Interfacial inactivation rates measured in the stirred-cell at a fixed interfacial area increased with mixing intensity. Interfacial inactivation rates per unit area were lower in the emulsion reactor than in the stirred-cell and increased with bulk aqueous enzyme concentration. Circular dichroism measurements showed that during biphasic incubation all unadsorbed soluble enzyme existed in the native conformation. Activity assays showed that the dissolved enzyme was also fully active, indicating that inactivated enzyme precipitated from solution. Using an inactive epoxide hydrolase mutant structurally similar to the wild-type enzyme in order to avoid the conversion of the epoxide, it was found that high concentrations of epoxide in the organic phase increased the rate of interfacial inactivation.