Stability Modification Of Epoxide Hydrolase And Its Application In The Preparation Of (R)-epichlorohydrin

Chiral epichlorohydrin is an important C₃ chiral synthons,has been widely used in synthesis of?-adrenergic blocking drugs,atorvastatin side chain key intermediate?S?-4-chloro-3-hydroxybutyrate ethyl ester,diet pills L-carnitine,narcotics baclofen and other chiral drugs.The molecular modification of epoxide hydrolase was carried out,and the 4 single point mutants were selected to characterize their stability.The results showed that the mutants t_1/2/2 of IIe108Leu,Asp131Ser and Thr247Lys were 2.84 h,2.05 h and 1.93 h,increased 3.94 times,2.85times and 2.68 times higher than that of the wide-type respectively;The T₅₀0 of mutant Asp131Ser increased by 5.1oC compared with wide-type.The tolerance in different volume fraction of methanol and Triton X-100solutions of 4 single point mutants were inverstigated.It was found that the 4 mutants activity in 5%methanol solvent were higher than the wide-type.The t_1/2/2 of IIe108Leu,Asp131Ser and Thr247Lys were increased by 2.21 times,1.53 times,1.43 times than of wide-type;The relative activity of mutant IIe108Leu in 0.8%Triton X-100 was 2.05times higher than the wide-type.Epoxide hydrolase catalyzed hydrolysis of epichlorohydrin in the two phase system was investigated.The results showed that the best organic solvent was isooctane?isooctane:water ratio was 3:7?v/v??,and the yield of?R?-epichlorohydrin was 45.2%at the epichlorohydrin concentration of 800 mM.The ee of?R?-epichlorohydrin was increased from 60%to 99%by using the cell feeding at the epichlorohydrin concentration of 1 M;Epichlorohydrin concentration can be up to more than 1 M by substrate feeding.When the product concentration reach to300 mM,it will observe severe product inhibition,which could make the enzyme activity decreased severely.Immobilized epoxide hydrolase catalyzed hydrolysis of epichlorohydrin was investigated.We found that the polyethylene-glutaraldehyde cross linking was the best immobilization method than other methods.When the amount of polyethylene,glutaraldehyde and diatomite were 3%?v/v?,1%?v/v?,0.6%?m/v?respectively,the immobilized cell activity recovery was 88.4%.The optimum pH and temperature of immobilized cells were 8.0 and 40oC respectively.The immobilized cell showed quite impressive operational stability.After 10 cycle reaction,the immobilized cells still retained 90%initial activity.A gram-scale kinetic resolution of epichlorohydrin using immobilized preparation as biocatalyst was performed and?R?-epichlorohydrin was obtained with 37%yield and 99%ee.