Screening Of Enantioselectively Microbial Epoxide Hydrolase And Study On Its Fermentation And Resolution Conditions Of Glycidyl Phenyl Ethers

Optically pure epoxides and their corresponding vicinal diols are recognized as important building blocks for the synthesis of various bioactive and chiral products.More than 380 strains which could use 1,2-Epoxy-3-(2-methoxyphenyloxy)-propane(EMPP) as the sole carbon source were obtained from soil samples. Then 25 strains with epoxide hydrolase were screened by TLC and 5 with enantioselective activity by HPCL. A fungi with best enantioselectivity was further studied and identified as Botryosphaeria dothidea based on morphology and ITS sequence. It's named ZJUZQ007.Based on single factor experiments, the fermentation conditions for the strain ZJUZQ007 were investigated, including carbon and nitrogen sources, metal ions and the concentrations, the initial pH of fermentation medium, temperature and inducement. The best fermentation conditions for ZJUZQ007 were: lactose 2.0%, corn flour 2.0%, NaCl 1.0%, FeSO4·7H2O 2mmol/L,temperature 30℃, pH 4.0-7.0, time 30h. The epoxide hydrolase is not inducible.Furthermore, the conditions for kinetic resolution of racemic EMPP and glycidyl (o, m, p)-methylphenyl ethers by ZJUZQ007 were studied, including reaction temperature, pH of potassium phosphate buffer and reaction time. It showed that the best resolution conditions as follows: racemic epoxides (12 mg) in DMSO (1:10 v/v) was hydrolyzed in 10ml KPB (100 mmol/L, pH 7.6) using wet mycelium of ZJUZQ007 (0.8 g) by shaking the mixture (220 rpm) at 30℃for 40 minutes.Under this optimized conversion conditions, the e.e.s and E value for (R)-EMPP was 99.9% and 50.4, 99.9% and 48.6 for (R)-o-GMPE, 91.2 and 18.1 for (R)-m-GMPE, 98.0% and 29.5 for (S)-p-GMPE.We found that by using Botryosphaeria dothidea ZJUZQ007, the sort of substituent group had an effect on the enantioselectivity that the e.e.s and E value for (R)-EMPP were higher than those for (R)-o-GMPE. While the position of the methyl group effected not only on the enantioselectivity but also the configuration of the remained epoxides: the (R)-enantiomer was obtained from rac-glycidyl (o or m)-methylphenyl ethers whereas the (S)-epoxides was obtained from glycidyl p-methylphenyl ether. The observations are explained by enzyme-substrate docking studies.