Multiple sequence alignment-inspired mutagenesis of marine epoxide hydrolase of Mugil cephalus for enhancing enantioselective hydrolytic activity

The marine fish microsomal epoxide hydrolase (mEH) of Mugil cephalus was engineered to enhance the enantioselective hydrolytic activity by multiple sequence alignment-inspired mutagenesis. The amino acid sequences of Aspergillus niger, Rhodotorula glutinis, zebra fish and human mEH were aligned and analyzed for identifying target amino acids. Single-point mutants (Q170K, E186K, E378D) and double-point mutants (E378D-Q170K, E378D-Y348F, E378D-Y348H) were developed and their hydrolytic activities were compared. The double-point mutant, E378D-Q170K, exhibited an enhanced hydrolytic activity by 4.6-fold, compared to the wild-type M. cephalus mEH. Enantiopure (S)-styrene oxide could be readily prepared with high enantiopurity more than 99%ee by using the double-point mutant.