Biosynthesis of (R)‐epichlorohydrin at high substrate concentration by kinetic resolution of racemic epichlorohydrin with a recombinant epoxide hydrolase

The substrate concentration and yield were shown to be very low in the production of ( R )‐epichlorohydrin by hydrolysis of racemic epichlorohydrin using epoxide hydrolases in previous studies. In this work, we synthesized an epoxide hydrolase gene from Agrobacterium radiobacter and expressed it in Escherichia coli by the PCR assembly method. The recombinant A. radiobacter epoxide hydrolase (ArEH) was applied in the preparation of ( R )‐epichlorohydrin and, a yield of 42.7% with ≥99% enantiomeric excess ( ee ) from 25.6 mM racemic epichlorohydrin was obtained. However, the ee of ( R )‐epichlorohydrin was not able to reach 99% due to substrate and product inhibition when the substrate concentration was over 320 mM. Inhibition studies revealed that ( S )‐3‐chloro‐1,2‐propanediol displayed non‐competitive inhibition in the conversion of ( S )‐epichlorohydrin but non‐significant inhibition for ( R )‐epichlorohydrin. Moreover, ArEH was successfully applied in the preparation of ( R )‐epichlorohydrin at high substrate concentration by eliminating the substrate inhibition. The substrate concentration increased to 448 mM by intermittent feeding of the substrate and to 512 mM by using a two‐phase reaction system, with a high yield (>27%) and ee (>98%) of ( R )‐epichlorohydrin. This is the first report of high‐yield production of ( R )‐epichlorohydrin at high substrate concentration, laying the foundations for its application on the industrial scale.