Gene Cloning,Molecular Modification Of An Epoxide Hydrolase From Aspergillus Usamii And Its Application In Synthesis Of Chiral Compounds

Epoxide hydrolases?EHs?-catalyzed enantioselective hydrolysis of rac-epoxides or meso-epoxides provide a simple and green method for the synthesis of chiral epoxides and vicinal diols,which are high value-added synthons for the production of pharmaceuticals,agrichemicals and fine chemicals.Nevertheless,the traditional limitations of EHs,such as low enantioselectivity,poor stability and product inhibition,severely hamper their practical industrial application.Hence,there is a constant need for exploring novel EHs or engineering the existing one to breaking the stringent bottleneck for realizing their industrialization.In this study,a novel EH?AuEH2?was firstly identified and characterized from Aspergillus usamii E001.A biphasic system was constructed to improve the kinetic resolution process.Subsequently,directed modification of AuEH2 was performed to further improve its enantioselectivity and activity based on the semi-rational design.Mutants with excellent enantioselectivity were successfully applied to prepare chiral epoxides and vicinal diols.This study will not only promote the industrial development and application of AuEH2 and its mutants,but also provide a new idea of engineering other EHs and enzymes.?1?An Aueh2 gene was cloned from A.usamii E001 by RT-PCR and THSO-PCR techniques.The open reading frame of Aueh2 was 1188 bp in length,which encodes 395amino acids.Bioinformatic analysis showed the highest identities of AuEH2 with other reported EHs was 58.2%.AuEH2 has several conserved motifs identical to those of?/?hydrolase superfamily,such as HGXP,S_mXNu XS_mS_m and GGHFAALE.The catalytic triad of AuEH2 was predicted to be D¹⁹¹-H³⁶⁹-E³⁴³ and its proton donor residuals Y²⁴⁹ and Y³¹² are also conserved.The three-dimensional structure of AuEH2 consists of an N-terminal meander domain(M^1P84),a core domain(Q^85L212 and P^317K395)and a cap domain(S^247F316),displaying it belongs to microsomal EH subfamily.?2?The Aueh2 gene mediated by pET28a?+?was heterologous overexpressed in E.coli BL21?DE3?.The specific activity reached 1440 U·L^-1?35??and 533 U·g^-1 cdw?25??,which was 400 times higher than that of A.usamii E001.SDS-PAGE analysis showed the apparent molecular weight of re AuEH2 was 44.3 kDa and soluble reAu EH2 was account for35.7%of total E.coli protein.The purified reAuEH2 displayed the maximum activity of 9.7U·mg^-1 at pH 7.0 and 35?.It shows highly activity and stability at pH 6.5⁷.5 and 25⁴0?.The specificity constant[(k^R_cat/K_m^R)/(k^S_cat/K_m^S)=8.9]and regioselectivity coefficients??_R and?_S>98.3%?indicated that AuEH2 preferentially hydrolyze?R?-1a and nucleophilic attack C_?of both?R?-1a and?S?-1a.Molecular docking simulations showed?R?-1a is more easily protonated by Y³¹² via forming a hydrogen bond with O-atom,and C_?easily nucleophilic attacked by D¹⁹¹.?3?The kinetic resolution of rac-1a was performed with E.coli/Aueh2 at different temperatures,affording>99%ee_s?S?-1a in 38%?10??,32%?20??and 27.5%?35??yield.E values were 24.2?10??,19.3?20??and 10.3?35??,indicating that reaction temperature is a key factor affecting the enantioselectivity of AuEH2.E.coli/Aueh2 possess high activity(119¹143 U·g^-1 cdw)and moderate enantioselectivity?E=2.1²2.6,except for 4a with E value of 95.9?towards monosubstituted epoxides 1a¹7a,affording styrene oxide derivatives?S?-1a⁶a and diols?R?-1b⁶b,glycidyl ether derivatives?R?-8a¹4a and diols?S?-8b¹4b,aliphatic oxirane?S?-15a and diol?R?-15b,but inactive for meso-epoxide 18a.Those results indicated reAuEH2 has a broad substrate scope.?4?In a single aqueous system,reAuEH2 shows high substrate tolerance towards rac-1a?>1 M?,but is inactivated at a product concentration of 300 mM.In an optimized n-hexanol/buffer biphasic system,a gram-scale resolution of rac-1a at a high concentration was successfully performed at 25?for 2 h.The substrate concentration,space-time yield and ee_s of?S?-1a?1 M,20.6 g/L/h,98.2%?in a biphasic system significantly increased compared with those?0.2 M,3.15 g/L/h,36.8%?in a single aqueous system.The results demonstrated product inhibition was relieved effectively in the biphasic system,thus significantly improving the substrate concentration and production efficiency.?5?Based on computer-aided design,eight residuals(K¹⁹⁵,A²¹⁴,Y²¹⁶,S²⁴⁷,A²⁵⁰,N³¹⁵,L³⁴⁴ and V³⁴⁵)around the substrate binding pocket of AuEH2 were chosen and subsequently subjected to site-and iterative-saturation mutagenesis.A"smart"mutant library,including about 750 transformants,was constructed.Eleven mutants were obtained from A²¹⁴,S²⁴⁷ and A²⁵⁰ mutations via high-throughput screening,which E values increased from 16.2 to18.7²02.2?25??towards rac-1a.The specific activities of purified AuEH2^A250I?E=48.2?and AuEH2^A250I/A214C?E=202.2?were 10.7 and 16.7 U·mg^-1.The kinetic resolution of rac-1a were performed with Au EH2^A250I and AuEH2^A250I/A214C,affording>99%ee_s?S?-1a in increased yield from 39.8%to 43.8 and 49.1%?theoretical yield was 50%?,and?R?-phenyl glycol in increased ee_p from 65.8%to 78.5 and 95.5%,respectively.Kinetic results showed the improvement of enantioselectivity of AuEH2^A250I and AuEH2^A250I/A214C are mainly derived from the dramatically enhanced k_cat of?R?-1a and K_m of?S?-1a.?6?The substrate scope analysis showed the specific activities of AuEH2 mutants were increased from 119¹143 to 189¹400 U·g^-1 cdw,and E values remarkably improved from2.1⁹5.9 to 16.5³41.7 towards epoxides 1a¹5a?25??,which were 2.1¹8-fold as compared with AuEH2.AuEH2 mutants exhibited high E values exceeding 30 towards 10epoxides of 1a,3a⁶a,8a,11a¹3a and 15a.To our knowledge,E values towards 1a?E>200?,3a?E=39.9?,4a?E>200?,6a?E=37.4?,10a?E=18.3?and 12a?E=35.7?represent the highest enantioselectivities among well-known EHs.Furthermore,the facile preparation of useful chiral epoxides 1a¹5a in 97.1⁹9.9%ee_s and 36.3⁴9.1%yield,and chiral diols in 1b¹5b in 56.7⁹5.5%ee_p demonstrated the great application potential of AuEH2 mutants.