Gene Cloning,Expression And Directed Modification Of The Rhodotorula Paludigena Epoxide Hydrolase

Epoxide hydrolases?EHs,EC 3.3.2.-?,a kind of cofactor-independent biocatalysts and existing ubiquitously in nature,can catalyze the enantioselective or enantioconvergent hydrolysis of rac-epoxides or meso-epoxides and prepare chiral epoxides and vicinal diols.Optically pure epoxides and vicinal diols are versatile building blocks in synthesis of various pharmaceuticals,fine chemicals and agrochemicals.In this study,a Rhodotorula sp.was identified by 26S rDNA sequence analysis;the RpEH coding gene was excavated through bioinformatics and successfully expressed in E.coli;the substrate spectrum and catalytic properties of RpEH were studied;the system for preparing?R?-8a was optimized;the regioselectivity of RpEH for rac-1a was improved by directed evolution.?1?We identified a new red yeast R.paludigena JNU001 by analysing its D1/D2 domain of 26S rDNA.A 1236-bp coding sequence and a 1600-bp DNA sequences of Rpeh were amplified from the total RNA and the genomic DNA of R.paludigena JNU001,respectively,using primers Rp-F and Rp-R.?2?The RpEH gene mediated by pET28a?+?was heterologous overexpressed in E.coli BL21?DE3?.The activity of recombinant E.coli/Rpeh toward rac-1a was 2132 U/g wcw.The purification of RpEH was achieved by nickel–nitrilotriacetic acid column.Additionally,the optimum reaction temperature and pH of RpEH were determined to be 30? and pH 7.0.The kinetic parameters of purified RpEH toward rac-1a were determined.The resultant K_m,V_max,k_cat,and k_cat/K_m of purified RpEH toward rac-1a were 7.87 mM,26.2 U/mg,21.1 s^-1,2.7 mM^-1 s^-1,respectively.?3?E.coli/Rpeh displayed high activities of 743?9081 U/g wcw toward 1a and 5a?12a,while relative low activity toward nitro-substituted styrene oxide 2a?4a?24?276 U/g wet cells?.The determination of enantiomeric ratio towards given rac-epoxides displayed that E.coli/Rpeh possessed the low enantioselectivity towards 1a?E=3.5?,11a?E=1.6?and 12a?E=2.3?,moderate enantioselectivity towards 4a?E=19?,and high enantioselectivity toward 2a?E>200?,3a?E=64?,and 5a?10a?E=49?160?.?4?Using only 10 mg wet cells/mL of E.coli/Rpeh,the near-perfect kinetic resolution of rac-8a at a high concentration?1000 mmol/L?was achieved within 2.5 h,giving?R?-8a with more than 99%enantiomeric excess?ee_s?and 46.7%yield and producing?S?-8b with 93.2%ee_p and 51.4%yield with high space-time yield?STY?for?R?-8a and?S?-8b were 30.6 and 37.3g/L/h.And we achieved efficient scale preparation of optically pure?R?-8a?1.89 g,isolated yield 46.0%;ee_s>99.0%?and?S?-8b?2.2g,isolated yield 48.2%;ee_p 92.8%?in 25 mL phosphate buffer?pH 7.0?at high substrate concentration?1000 mmol/L?by using a small amount of E.coli/Rpeh wet cells?0.25g?.?5?Based on computer-aided design,nine residuals?Y152?I194?M235?N266?P335?H336?F351?L360 and F361?around the substrate binding pocket of RpEH were chosen and subsequently subjected to quadruple-code saturation mutagenesis.Herein,each of the four residues was replaced with a set of amino acids?Val,Asn,Phe,and Trp?.Four excellent mutants(RpEH^Y152F?RpEH^P335W?RpEH^H336V and RpEH^L360V)were obtained by screening the mutation library.In addition,RpEH^L360V-H336V was obtained by double site mutagenesis,which showed the highest specific activity of 2814 U/g wcw.Its regioselectivity coefficients of ?_S and?_R were 94.6% and 99.9%.