Screening Of Epoxide Hydrolase Producing Strains And Preparation β-Blocks By Biotransformation

Strain which could enantiomeric stereoselectively hydrolysis the racemic glycidyl m-methylphenyl ether has been isolated from the soil and was named Bacillus megaterium ZJUZQ-002. The analysis of chiral purities was based on the chiral HPLC results.Optimization the fermentation conditions of the Bacillus megaterium ZJUZQ-002 revealed that the pH value and temperature have major impacts on biomass and epoxide hydrolase activity. The best result were obatined under the following conditions: pH 7.2, temperature 30℃. Substrate induction also has an impact on the enzyme activity, and substrate 6 mmol/L results the highest enzyme activity, whereas the 12 mmol/L of substrate would lead to the death of bacteria. The best fermentation condition: sucrose 1.0 %,yeast extract 1.0 %,NaCl 1.0 %, MgSO4·7H2O 5mmol/L,K2HPO4·3H2O 0.3 %,KH2PO4 0.1 %,substrate 6 mmol/L,temperature 30℃,pH 7.2,time 30 h, could give the activity of enzyme 58.5 U/L.The best resolution condition: 0.1 M pH 8.0 potassium phosphate buffer, temperature 30℃, time 16 h, cosolvent DMSO, the ration of catalyst/substrate (g/g) = 9/1, substrate concentration 20 mmol/L, could give the e.e. value of substrate is 99.9 %, the E value 37.The study towards the substrate specificity of this enzyme show that, by using Bacillus megaterium ZJUZQ-002, the position of the methyl group exerted effect not only on the enantioselectivities increasing from 2′≈3′> 4′, but also the configuration of the remained epoxides: for glycidyl o or m-methylphenyl ethers, the remaining epoxide was (R)-enantiomer, while p-methylphenyl ether gave (S)-configuration, and then we explain it with docking study.In the optimal resolution conditions, the effect of different magnification factor of the raction system on resolution was studied. It was found that the best magnification factor is five, and then the e.e. value of m-GMPE was obatined in 99.9 %, the conversion rate in 68.6 % after 16 h. After further purification and amination,β-blocks—(S)- Toliprolol was obtained in e.e. value of 98 % and yield of 16.5 % yield.