Using multiple site-directed modification of epoxide hydrolase to significantly improve its enantioselectivity in hydrolysis of rac-glycidyl phenyl ether

The epoxide hydrolase gene (SpEH) from Sphingomonas sp. HXN-200 was synthesized and expressed in robust Escherichia coli cells that had a dual protection system. The enantioselectivity (E-value) of the recombinant SpEH was 7.7 and the yield of the remaining (R)-PGE was 24.3% for the hydrolysis of racemic phenyl glycidyl ether (rac-PGE). To improve the catalytic properties of SpEH, the site-directed mutagenesis was carried out based on homology modeling, sequence alignment and molecular docking. Six residues (V195, V196, F218, N226, Q312, and M332) near the active site were mutated to hydrophobic amino acids and the positive mutations were selected for combinatorial mutation. The optimal mutant SpEHV196A/N226A/M332A had an enhanced E-value of 21.2 and a specific activity of 4.57 U·mg−1-wet cells, which were 2.8-, and 2.3-fold higher than those of wild-type SpEH. The optimal temperature and pH for purified SpEHV196A/N226A/M332A to catalyze the hydrolysis of rac-PGE were 25 °C and 7.0 with 200 U·mg−1. The enantioselectivity and yield of the remaining (R)-PGE of E. coli_SpEHV196A/N226A/M332A increased from 7.7 to 21.2 and 24.3% to 40.9%, respectively. The molecular docking and kinetic parameter analyses showed that SpEHV196A/N226A/M332A has a greater affinity toward (S)-PGE than (R) - PGE, and that it was more difficult for the O-atom of ASP170 to achieve the nucleophilic attack on the Cα of (R)-PGE, resulting in its improved enantioselectivity.