The epoxide hydrolase from Aspergillus niger was immobilized by adsorption onto DEAE-Cellulose. The immobilization yield was near 100% and retention of activity was 50%. Conditions to avoid internal and external mass transfer limitations were defined. The enzymatic properties of free and immobilized enzyme preparations were compared with respect to the effect of temperature and pH on both activity and stability, the effect of substrate concentration on activity, and the enantio- and regio-selectivity were compared. Both enzyme preparations were used for the deracemization of p-chlorostyrene oxide in both batch and continuous reactors.

The kinetics of thermal inactivation of free and immobilized epoxide hydrolases were studied at temperatures ranging from 5 to 47°C. They all followed first order kinetics. The half-lives increased from 4 h at 47°C to 333 h at °C. Surprisingly, the immobilized preparation showed half-lives two to six fold lower than the free enzyme showing a lower stability after immobilization. However, the half-life was increased by a factor of 100-200 by decreasing the temperature from 47 to 5°C. Thus, the reaction should be run at low temperature to obtain a high stability in addition to the beneficial increase of enantioselectivity at low temperature.

Operational stability was assayed by repeated use in batch reactor and in backed-bed reactor. By repeated use at 0°C with 4 mM substrate, more than 90% activity of the immobilized epoxide hydrolase was recovered after 7 batches. In continuous reactor, the column was flushed with the 4 mM substrate concentration at 0°C. The decrease in substrate conversion, in a range were the conversion was limiting, followed first order kinetics from which a half-life of around 600 h was calculated. This value is four times higher than the half-life determined in test tube without substrate. Thus, there is a strong positive effect of the substrate on the enzyme stability.

This high stability allowed the use of immobilized epoxide hydrolase for the enantiomeric resolution of chiral epoxides.