Hu_2016_J.Biotechnol_236_152

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 (50mM, pH 7.0), 200mM 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 750mM rac-SO. The analytical results verified that reAuEH2 shows tolerance towards high substrate concentration but is inactivated at a product concentration of 300mM. 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 degrees C, respectively. In an optimized biocatalytic system, a gram-scale resolution of rac-SO at a high concentration of 1M (120g/L) was performed at 25 degrees C for 2h, obtaining (S)-SO with 98.2% e.e., 34.3% yield (maximum yield of 50%). The substrate concentration and volumetric productivity (1M, 20.6g/L/h) in a biphasic system significantly increased compared with those (0.2M, 3.1g/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.