Gene Cloning,Expression And Directed Modification Of Phaseolus Vulgaris Epoxide Hydrolase

Chiral epoxides and vicinal diols are versatile building blocks for the synthesis of functional materials,pharmaceuticals and pesticides.Epoxide hydrolases?EHs?catalyze the enantioselective ring opening of racemic epoxides,affording chiral vicinal diols and/or retaining unreacted epoxide enantiomers.The enantioconvergent hydrolysis of racemic epoxides by a single enzyme is considered to an ideal way to prepare enantiopure vicinal diols,but few natural EHs possess the catalytic characteristic.In this study,a PvEH3-encoding gene,pveh3,was amplified from Phaseolus vulgaris and expressed successfully in E.coli BL21?DE3?.Then,the gene pveh3 was modified by site-directed and iterative mutagenesis to obtain PvEH3 variants with high activity and enantioconvergence.Finally,an optimized biphasic catalytic system was used to relieve substrate inhibition of PvEH3^{G170E/F187L/P237L} in enantioconvergent hydrolysis of p-chlorostyrene oxide?pCSO?.pveh3 was amplified from Phaseolus vulgaris total RNA by reverse transcription PCR and nested PCR techniques.The sequence was 957 bp,which encodes a protein of 318 amino acid residues.Then pveh3 mediated by expression vector pET-28a?+?was heterologously expressed in E.coli BL21?DE3?.Primary and three-dimensional structures analysis indicated that PvEH3 belongs to?/?-hydrolase superfamily.The catalytic triad of PvEH3 is D101-H297-D262,and its two conservative tyrosine residues served as proton donor are Y150 and Y232.PvEH3 can catalyze the enantioconvergent hydrolysis of p-chlorostyrene oxide?pCSO?to afford?R?-p-chlorophenyl-1,2-ethanediol?pCPED?with an enantiomeric purity of 85.1%ee_p.PvEH3 showed an opposite regioselectivity towards?R?-and?S?-pCSO,the regioselectivity coefficients of?_S and?_R were 87.0%and 98.0%.PvEH3 was purified by Ni-NTA columns,the activity,K_m and k_cat/K_m of purified PvEH3 towards p CSO were the 1.36U·mg^-1,6.60 mM and 0.583 mM^-1·s^-1,respectivety.Acoording to the conformation of PvEH3 docking with?R?-pCSO,as well as the multiple alignment of PvEH3 with five plant EHs,seven nonconservative sites were selected to be modified by site-directed mutagenesis.Among the seven mutants,PvEH3^G170E showed the highest specific activity of 12.86 U·g^-1 wcw,PvEH3^F187L and PvEH3^P237L showed the best enantioconvergence of seven mutants with 90.0%and 91.2%ee_p,respectively.In addition,PvEH3^{G170E/F187L/P237L} were obtained by multi-site mutagenesis,which showed the highest specific activity of 20.31 U·g^-1 wcw.Its regioselectivity coefficients of?_S and?_R were 95.0%and 98.0%.The K_m and k_cat/K_m towards pCSO were 4.86 mM and 1.81 m M^-1·s^-1,which were more excellent than those of PvEH3.The phase volume ratio in biphasic system has remarkable effects on activity and stability of enzyme.In the optimal cyclohexane/buffer biphasic system,200 mM pCSO was hydrolyzed by PvEH3^{G170E/F187L/P237L} in 5.5 h with 6.16 g·L^-1·h^-1 of space-time-yield,and the ee_p and yield of?R?-pCPED were 96.1%and 98.0%respectivety.The substrate inhibition could be relieved effectively but the product inhibition still exists in the cyclohexane/buffer biphasic system.PvEH3 shows different activity and enantioconvergence towards styrene oxide and its four derivatives.Compared with PvEH3,PvEH3^{G170E/F187L/P237L} shows higer specific activity towards five substrates,increased by 2.1⁵.9 fold.