Study On The Expression,Modification And Properties Of GmEH3,an Epoxide Hydrolase From Glycine Max

Optically pure epoxides and 1,2-diols are the important building blocks in chiral chemical and organic synthesis.Epoxide hydrolases（EHs,EC 3.3.2.-）can catalyze the asymmetric hydrolysis（kinetic resolution and enantioconvergent hydrolysis）of racemic（rac-）epoxides,were considered to have broad application prospects.EHs with high enantioselectivity can be applied to the kinetic resolution of rac-epoxides,retaining the single configuration of epoxide.EHs with high and complementary regioselectivity can be used for enantioconvergent hydrolysis of rac-epoxides to generate chiral pure 1,2-diols.The catalytic properties of most reported wild-type EHs were still unsatisfactory,resulting in low yield or chiral purity,cannot reach the industrial requires.In addition,one EH cannot display excellent catalytic properties toward each epoxide.So it’s necessary to explore some novel EHs with excellent catalytic performance and/or modify the existed EHs to improve their catalytic properties.（1）Using the primary structure of a reported Phaseolus vulgaris epoxide hydrolase（Pv EH3,Genbank:ATG22745）as a template,an unknown functional protein（hGmEH,XP＿006604802）with 85.3%similarity to Pv EH3 from Glycine max was searched using the BLAST in the NCBI database.The sequence alignment and analysis of hGmEH with six other known plant EHs revealed that hGmEH had the all necessary domains of EHs,such as a catalytic triad and two proton donors.The total RNA was extracted from soybean germination tissues.Then an about 1.0 kpb nucleic acid sequence（named gmeh3）was obtained by PCR.The gmeh3 was ligated to the cloning vector p UCm-T,and transformed into E.coli JM109 for proliferation and verifying by sequence test.The recombinant plasmid p UCm-T-gmeh3 was extracted.The gene,gmeh3 was cut by double digestion,and connected to the cold shock expression plasmid p Cold II,forming another recombinant plasmid p Cold-gmeh3.Finally,the p Cold-gmeh3 was transformed into the E.coli Rosetta（DE3）,obtaining the recombinant strain E.coli/gmeh3 for inducible expression.（2）Using the 10 epoxides rac-1a～10a（styrene oxide and glycidyl ether derivatives）as substrates to determine the substrate spectrum of GmEH3.Among them,GmEH3 displayed regioselectivity to rac-1a～5a,the ee_p values of the generated 1,2-diols（R）-1b～5b reached72.1%–90.1%.The activities towards rac-1a～5a were between 4.2 and 28.2 U·g^-1 wet cell（wet cell,wc）.On the other hand,GmEH3 showed enantioselectivity towards rac-6a～10a.The E values were 7.4–55.6.When the（S）-6a～10a were just hydrolyzed completely.The yields of（R）-6a～10a were 17.1%–43.5%,and the activities were between 2.6 and 35.1 U·g^-1 wc.Wherein,GmEH3 had the highest regiocomplementary for（R）-and（S）-5a（m-chlorophenyl ethylene oxide）,α_s=93.7%andβ_R=97.2%.At the time of rac-5a was complete hydrolyzing（c>99%）,the ee_p of generated（R）-5b reached 90.1%,and the yield_p reached 94.5%.The activity towards rac-5a was 22.4 U·g^-1 wc.GmEH3 also displayed the highest enantioselectivity to rac-7a（phenyl glycidyl ether）,E=55.6,resulting（R）-7a with the ee_s>99%and yields of 43.5%,and the activity was 35.1 U·g^-1 wc.Using the whole cells of E.coli/gmeh3 as biocatalyst,the maximum allowable concentration（MAC）of enantioconvergent hydrolysis of rac-5a was 150 m M(18.0 g·L^-1).The reaction process of hydrolysis of rac-5a was completed after 1.5 h.The ee_p of（R）-5b was 90.2%,and the yield was 95.4%.The MAC of kinetic resolution of rac-7a was 500 m M(75.1 g·L^-1).The（S）-7a was hydrolyzed completely at the time at 2.5 h,and the ee_s>99%and yields was 43.2%,respectively.The molecular mechanism of the high regiocomplementary of GmEH3 for（R）-and（S）-5a as well as high enantioselectivity towards rac-7a were analyzed by molecular docking simulation.（3）To further broaden the substrate spectrum of GmEH3,rac-11a～15a（aliphatic chain epoxides）were also used as the substrates.Among them,GmEH3 haved the highest activity for rac-11a,16.2 U·g^-1 wc.The hydrolysis reactions of 20 m M rac-11a～15a were conducted using the 40 mg·m L^-1 E.coli/gmeh3 wet cells as biocatalyst.As the results,among the five products（（R）-11b～15b）,（R）-11b had the highest ee_p of 47.2%.In order to further improve the ee_p of generated（R）-11b via the enantioconvergent hydrolysis of rac-11a by GmEH3,five specific amino acid residues(Trp¹⁰²,Ile¹⁰⁵,Ile¹⁷⁸,Pro¹⁸⁷ and Leu¹⁸⁹)in the substrate binding pocket（SBP）of GmEH3 were identified as mutation sites.Each site was replaced by four amino acids（Leu,Ile,Val and Phe）.A total of 17 single site mutants were obtained.After screening,the ee_pvalues of generated（R）-11b from rac-11a catalyzed by E.coli/gmeh3^W102L,/gmeh3^W102I,/gmeh3^W102F and/gmeh3^P187F were significantly enhanced,were 75.9%,76.8%,78.3%and 59.3%respectively.Then three double-site mutants were obtained via separate combining the P187F with W102L,W102I,and W102F.Among them,（R）-11b produced by E.coli/gmeh3^W102V/P187Fhad the highest ee_p of 83.8%.Theα_S of E.coli/gmeh3 for（S）-11a was obviously improved by semi-rational modification of GmEH3.The gram-scale preparation of（R）-11b was conducted by 200 mg·m L^-1 E.coli/gmeh3^W102V/P187F wet cells.The MAC was confirmed as 500 m M.The ee_p of finally prepared（R）-11b was 83.1%,and yield was 91.5%.Finally,the molecular mechanism of enhancedα_S was analyzed by molecular docking simulation.