Free and immobilized Aspergillus niger epoxide hydrolase-catalyzed hydrolytic kinetic resolution of racemic p-chlorostyrene oxide in a neat organic solvent medium

摘要

An enantio- and regioselective hydrolytic kinetic resolution (HKR) of racemic p-chlorostyrene oxide (rac-pCSO) was achieved by epoxide hydrolase (EH) from recombinant Aspergillus niger in a selected neat organic solvent medium. The solid free EH was reused four times in repeated-batch reactors; however, the relative activity as well as the enantiomeric ratio (E-value) of this EH decreased from 100 to 20% and from 68 to 23, respectively. In order to overcome the diffusion hindrance, due to the accumulation of the hydrophilic diol in the enzyme micro-environment, and thereby to improve the operational stability of EH after recycling, strategies consisting of the immobilization of EH and the use of a binary organic solvent as the reaction medium were successfully applied. Although the highest protein immobilization yield (82%) and retention of EH activity (142%) in heptane were obtained upon the immobilization of EH on Accurel EP, the £-value and the operational stability of the resulted EH immobilizate after recycling were reduced as compared to the free EH. In contrast, the nonporous DEAE-cellulose improved the operational stability of EH by more than twofold. On the other hand, both the HKR efficiency and the operational stability of A niger EH were found to be modest to excellent in various binary organic solvent mixtures of heptane and dioxane, depending on their ratio resulting in different Log P.