A new highly enantioselective stable epoxide hydrolase from Hypsibius dujardini: Expression in Pichia pastoris and immobilization in ZIF-8 for asymmetric hydrolysis of racemic styrene oxide

In this study, an epoxide hydrolase gene (hdeh) from extremophile Hypsibius dujardini was expressed heterolo-gously in Pichia pastoris under the optimized conditions. The purified HdEH was immobilized in a metal-organic framework (HdEH@ZIF-8) using zinc nitrate and 2-methylimidazole. The HdEH@ZIF-8 was further treated with different concentration of glutaraldehyde (HdEH@Glu/ZIF-8) to investigate the effect of glutaraldehyde con-centration on the activity and stability. The cross-linking of HdEH@ZIF-8 with glutaraldehyde resulted in decreasing the activity, while increasing the thermal stability. The optimum pH of free and immobilized HdEH samples was all determined as 8.0. The free and both immobilized HdEHs exhibit its optimal activity at 20 degrees C and 30 degrees C respectively. The HdEH@ZIF-8 and HdEH@Glu/ZIF-8 showed around 16-and 18-fold higher stability than free HdEH at 30 degrees C. All the HdEHs were used in the asymmetric hydrolysis of racemic styrene oxide (R/S-SO) and the results showed that R-enantiomer of epoxide was preferentially hydrolyzed by free HdEH, HdEH@ZIF-8 and HdEH@Glu/ZIF-8. After 360 min reaction time, (S)-styrene oxide was obtained with 99% enantiopurity for the all the HdEH preparations. HdEH@ZIF-8 and HdEH@Glu/ZIF-8 showed 1.9-and 1.4-fold higher enantiose-lectivity than that of the free HdEH. HdEH@ZIF-8 and HdEH@Glu/ZIF-8 were remained 91% and 93% of its initial activity after 5 reuses. The results show that HdEH@ZIF-8 and HdEH@Glu/ZIF-8 are a promising stable and enantioselective EH preparations for the asymmetric hydrolysis of racemic styrene oxide.