Expression And Stereoselectivity Of SfEH,Epoxide Hydrolase From Streptomyces Fradiae

Chiral epoxides and o-diols can be used as important intermediates in the synthesis of pesticides,fine chemicals,drugs and other active substances,and have a broad market demand and application prospects.EHs have unique stereoselectivity for epoxides and can catalyze the hydrolysis of racemic epoxides to retain single-configuration epoxides or to generate o-diols,which makes EHs a hot topic of research in biocatalysis.In this study,three novel EHs were mined from Streptomyces fradiae,bioinformatically analyzed,recombinant expression engineering strains were constructed,preliminary substrate profiles and enzymatic properties were characterized,and possible reasons for their stereoselectivity were explained by kinetic assays and molecular docking.The main findings are as follows:（1）Three novel EHs,SfEH1,SfEH2 and SfEH3,were identified in Streptomyces fradiae.1308 bp,948 bp and 816 bp open reading frames were found for the three EHs with amino acid numbers of 435,318 and273,respectively.The conserved regions and catalytic triplets of SfEH1,SfEH2 and SfEH3 were identified by sequence comparison as Asp²⁸⁸-His¹²⁷-Glu²⁸²,Asp¹¹⁶-His¹¹⁵-Asp²⁶⁴and Asp³⁰-His²⁵²-Asp⁹⁶,respectively,and the conserved tyrosine residues providing protons for the reaction were found to be Tyr⁵⁰and Tyr⁶²,Tyr²²⁹and Tyr²¹²,Tyr⁶⁹and Tyr¹⁴⁰,respectively,in the cap structure.And the genes encoding SfEH1,SfEH2 and SfEH3,sfeh1,sfeh2 and sfeh3,were successfully amplified.（2）With the help of expression plasmid pCold II,sfeh1,sfeh2 and sfeh3 were transformed into E.coli BL21,and the optimal induction conditions were determined as OD₆₀₀of 0.6,IPTG final concentration of0.4 m M,induction temperature of 15℃and induction time of 24 h.The expressed proteins were purified by chromogenic assay,and their apparent molecular weight was 47.2 k Da,35.2 k Da and 29.6 k Da,respectively.The expressed proteins were purified and their apparent molecular weights were 47.2 k Da,35.2 k Da and 29.6 k Da,respectively.SfEH3 preferentially hydrolyzed（S）-1a and retained（R）-1a,and the selectivity of SfEH3 was better.The optimum temperatures for the determination of SfEH1,SfEH2 and SfEH3 were 30℃,25℃and 30℃,respectively,and the optimum pH was 7.（3）The substrate profiles of SfEH1,SfEH2 and SfEH3 were determined and analyzed for enantioselectivity and regioselectivity.SfEH1 has high and complementary regioselectivity for rac-3a（or 4a）with 98.7%and 93.8%（or 95.2%and 98.9%）forα_Sandβ_R;SfEH2 has poor enzymatic activity and selectivity for substrates;SfEH3 has high enantioselectivity for rac-1a with preference for hydrolysis（R）-1a and retention（S）-1a.The kinetic parameters and molecular docking analysis of SfEH1 and SfEH3 also confirmed the experimental results,with K_mand V_maxof SfEH1 for（R）-3a（or 4a）being 4.58（or 4.29）m M and 5.32（or5.29）μmol/min/mg;for（S）-3a（or 4a）being 4.58（or 4.29）m M and 5.32（or 5.29）μmol/min/mg;and for（S）-1a（or 4a）being 4.58（or 4.29）m M and 5.32（or 5.29）μmol/min/mg.K_mand V_maxfor（S）-3a（or 4a）were 4.02（or 3.98）m M and 5.32（or 6.11）μmol/min/mg;K_mand V_maxfor SfEH3were 2.72 m M and 25.22μmol/min/mg for（R）-1a;K_mand V_maxfor SfEH3were 11.02 m M and 2.45μmol/min/mg for（S）-1a m M and 2.45μmol/min/mg.（4）The maximum reaction limit of SfEH3 for rac-1a was 200 m M.SfEH3 was immobilised with BC membranes and the immobilised enzyme activity was 22.84 U/g of carrier.Reusability was measured at the limiting concentration of SfEH3.SfEH3 without immobilization could only be reacted once and BC-SfEH3 could be reused three times with an enzyme activity of 51.4%of the initial enzyme activity...