Kinetic resolution of racemic styrene oxide at a high concentration by recombinant Aspergillus usamii epoxide hydrolase in an n-hexanol/buffer biphasic system

- An n-hexanol/buffer biphasic system was built to improve biocatalytic efficiency.
- Kinetic resolution of 1 M rac-SO was successfully achieved by reAuEH2.
- (S)-SO was obtained in a 98.2% e.e. and a 34.3% yield within only 2 h.
- It showed high volumetric productivity in the gram-scale resolution of rac-SO.

Using the cell-free extract of engineered E. coli/Aueh2, expressing the recombinant Aspergillus usamii epoxide hydrolase (reAuEH2), as a biocatalyst, the kinetic resolution technique of racemic styrene oxide (rac-SO) was examined. In a phosphate buffer system (50 mM, pH 7.0), 200 mM rac-SO was efficiently resolved, obtaining (S)-SO with 98.1% enantiomeric excess (e.e.), whereas (S)-SO only with 45.2% e.e. was obtained from 750 mM rac-SO. The analytical results verified that reAuEH2 shows tolerance towards high substrate concentration but is inactivated at a product concentration of 300 mM. To produce (S)-SO with the high concentration, e.e. and volumetric productivity, n-hexanol was selected from a variety of water-miscible and water-immiscible organic solvents to construct an n-hexanol/buffer biphasic system. The optimal phase volume ratio, substrate over enzyme ratio and temperature were 1:1 (v/v), 6:1 (w/w) and 25 °C, respectively. In an optimized biocatalytic system, a gram-scale resolution of rac-SO at a high concentration of 1 M (120 g/L) was performed at 25 °C for 2 h, obtaining (S)-SO with 98.2% e.e., 34.3% yield (maximum yield of 50%). The substrate concentration and volumetric productivity (1 M, 20.6 g/L/h) in a biphasic system significantly increased compared with those (0.2 M, 3.1 g/L/h) in a phosphate buffer system. The efficient resolution of rac-SO at a high concentration in a biphasic system makes it a promising technique for preparing a highly value-added enantiopure (S)-SO with high volumetric productivity.