Molecular Modification Of Epoxide Hydrolase And Its Application In Catalyzing Kinetic Resolution Of Phenyl Glycidyl Ether

Chiral epoxides are important intermediates in organic synthesis and widely used in the synthesis of medicines,pesticides and fine chemicals.For example,chiral phenyl glycidyl ether（PGE）is an important intermediate in the preparation of chiral drugs,such as neuroprotective molecules andβ-blockers.The chemical method for preparing chiral epoxides involves harsh reaction conditions and is environmentally unfriendly.These problems may be well overcome in biocatalytic upgrading of chiral epoxides.Epoxide hydrolases（EHs）can selectively catalyze the kinetic resolution of racemic epoxides to obtain chiral epoxides.EHs derived from Sphingomonas sp.HXN-200（SpEH）has the advantages of wide substrate spectrum,high substrate concentration tolerance,high activity and high selectivity,and can catalyze the hydrolysis of racemic PGE to prepare chiral（R）-PGE,but its enantioselectivity（E-value）is insufficient and the inhibition of catalyzing the hydrolysis of high concentration PGE in the aqueous phase is obvious.To solve those problems,the SpEH gene has been cloned and over-expressed in E.coli by gene engineering methods and optimize its induced expression conditions.Then the molecular modification is carried out with site-directed and combined mutation to increase its enantioselectivity,then the optimal mutant enzyme is purified and followed by investigation of its enzymatic properties.The biphasic system is introduced and key factor is optimized for improved substrate concentration and production efficiency to establish a highly efficient and highly selective preparation ofbiocatalysis system.The amount of SpEH was estimated to account for 28.8%of the total amount of robust E.coli protein,the molecular weight of SpEH was close to 45 k Da.The optimal induction conditions for recombinant SpEH were:the initial p H of the culture medium was 7.0,the isopropylβ-D-thiogalactoside（IPTG）concentration was 0.05 m M,the induction temperature was 25°C,and the induction time was 8 h.Under these conditions,the activity of recombinant enzyme was 1.96 U/mg dcw.The regioselectivity coefficients（β_R=99.3%,β_S=98.7%）indicated that the recombinant SpEH nucleophilic attack C_βof bothand.The E-value of E.coli＿SpEH catalyzes kinetics resolution of rac-PGE was 7.7 at 25°C and p H 7.0.Based on computer-aided calculation,six amino acids near the active pocket were selected for site-directed and combined mutations.The E-value and specific activity of the best mutant SpEH^{V196A/N226A/M332A} catalyzes hydrolysis of rac-PGE was 21.2 and 200 U/mg,are 2.8-fold and 2.3-fold that of wild type.The analysis of enzymatic properties showed that the optimal reaction temperature and p H of SpEH^{V196A/N226A/M332A}were 25°C and 7.0,SpEH^{V196A/N226A/M332A}retains high activity and stability under low temperature and weakly alkaline conditions;Cu²⁺,SpEH^{V196A/N226A/M332} were 14.7 m M and 95.4 s^-1towards（R）-PGE,were 2.6 m M and 275.3 s^-1SpEH^{V196A/N226A/M332A} preferentially catalyzes hydrolysis of（S）-PGE.The optimal temperature,buffer p H and substrate concentration for E.coli＿SpEH^{V196A/N226A/M332A} catalyzes hydrolysis of20 m M rac-PGE were 25°C,7.0 and 20 m M.The yield and ee_s value of（R）-PGE were 44.1%and 82.5%respectively,the E-value was 19.6.The main factors of restricting the hydrolysis of high concentration substrate catalyzed by E.coli＿SpEH^{V196A/N226A/M332A} is product inhibition.Butyl acetate/buffer biphasic system can alleviate product inhibition.The optimal process conditions for E.coli＿SpEH^{V196A/N226A/M332A}catalyzes hydrolysis of rac-PGE in the butyl acetate/buffer biphasic system were:the volume ratio of the two phases is 7:3（v/v）,the buffer p H is 7.0,the reaction temperature is 25oC,the S/E ratio is 9（w/w）,and an additional 5%Tween-60（w/w）,the substrate concentration increased to 300 m M.Compared with before optimization,the ee_sof（R）-PGE increased from96.1%to 99.2%,the yield increased from 30.3%to 31.6%,STY increased from 9.64 g/L/h to18.93 g/L/h,and a TOF increased from 2.68 g/h/g to 3.79 g/h/g.This study improves activity and enantioselectivity of SpEH by molecular modification,and provides practical experience for the molecular modification of other enzymes.Also,this study establishes a biocatalytic system for the highly efficient preparation of（R）-PGE,and establishs the basis for the highly efficient and highly selective preparation of chiral epoxides.