Cloning And Characterization Of Enantioconvergent Epoxide Hydrolase(s)

Chiral epoxide and its corresponding diol are important building block for many pharmaceuticals. Epoxide hydrolases catalyze the hydrolysis of epoxides to corresponding vicinal diols ????? with the addition of a H2O molecule, without any prosthetic group or cofactors. Two novel epoxide hydrolases have been found in Vigna radiate and one of them ?VrEH1? has been cloned and overexpressed in E. coli. In this thesis, another two potential epoxide hydrolase genes, vreh2 and vrehg, were successfully cloned from Vigna radiate. The ORF of vreh2 was 957 bp, encoding 318 amino acids and a terminator. It has high homology to GmEH ?Glycine max, XP₀03554634.1,87%? and MtEH ?Medicago truncatula, XP₀03626245.1,86%?, and only shows 72% and 57% homology to VrEH1 ?ADP68585.1? and StEH ?Solanum tuberosum, AAA81892.1?, respectively. The ORF of vrehg encodes 319 aa,98% similar to VrEHl ?ADP68585.1?.The vreh2 and vrehg were successfully choned into pET28a?+? and expressed in E. coli BL21?DE3?. After induction with IPTG 0.1mM at 16? for 12 h, expressed VrEH2 and VrEHg were about 25-35% total bacterial proteins and mainly as inclusion body. Optimization by adjusting the concentration of inducer IPTG, the pH of buffers or changing plasmid and host had no apparent effects on the soluble expression of VrEH2. VrEH2 was one-step purified by nickel affinity chromatography with its N-terminal 6×His tag. The yield was 40% and the purification fold was 925.The optimum reaction temperature and optimum pH of VrEH1 and VrEH2 were 45?,7.0 and 35?,6.5, respectively. Chemicals showed no obvious effect on the activity of VrEH2, while 10% isooctane increased the eep from 81 to 89. Both VrEH1 and VrEH2 could enanconvergently hydrolyze pNSO, but showed opposite enantioselectivity towards two enantiomers. VrEH1 prefers S-pNSO, while VrEH2 prefers R-pNSO. The KM,S, KM,R, kcat,S, kcat,R of VrEH1 were 5.5,1.4 mM,6.2,0.42s-1, and that of VrEH2 were 1.44,0.019 mM,0.13, 1.76 s-1, respectively. The regioselectivities of VrEHl and VrEH2 were determined using S-and R-pNSO as substrates. The coefficient for VrEHl were a?R?/??R?=13/87 and a?S?/??S? =83/17, while that for VrEH2 were a?R?/??R?=7/93 and a?S?/??S?=72/28, respectively.VrEH1 and VrEH2 showed higher activity to BGE or PGE than to pNSO. Except for 3M-PGE, both VrEH1 and VrEH2 showed enantioselectivity to the tested substrates.