定点突变改善PvEH1对邻甲基苯基缩水甘油醚的催化特性

Site-directed Mutagenesis of PvEH1 to Improve Its Catalytic Properties towards ortho-Methylphenyl Glycidyl Ether

环氧化物水解酶能够对外消旋环氧化物进行动力学拆分保留单构型的环氧化物。测定了菜豆环氧化物水解酶(PvEH1)针对苯基缩水甘油醚及其甲基衍生物的催化特性,并基于分子对接及多序列比对分析确定7个突变位点,通过单点和组合突变对PvEH1进行改造,以期改善PvEH1对邻甲基苯基缩水甘油醚(1a)的催化特性。底物谱分析表明PvEH1对1a的催化活性(157.2U/g湿细胞)和对映选择性(E=5.6)最高。单点突变结果显示E.coli/pveh1 L105I和E.coli/pveh1^V106I对1a的催化活性和对映选择性均有明显提高;L105I和V106I位组合突变菌株E.coli/pveh1^L105I/V106I的催化活性(493.8U/g湿细胞)是E.coli/pveh1的3.1倍,对映选择性(E=8.3)也提高至E.coli/pveh1的1.5倍。纯化后PvEH1 L105I/V106I的催化活性为17.6U/mg,是PvEH1的1.5倍,对1a的催化效率提高至PvEH1的2.1倍。SDS-PAGE分析表明提高了蛋白质的可溶性表达量。利用E.coli/pveh1^L105I/V106I全细胞催化100mmol/L 1a水解动力学拆分获得手性纯(R)-1a (ee>96%)的产率和时空产率分别为31.2%和5.12g/(L·h),因此,在手性纯(R)-1a的制备中,E.coli/pveh1^L105I/V106I是一种颇具潜力的生物催化剂。

Epoxide hydrolases can catalyze the kinetic resolution of racemic epoxides,retaining enantiopure single enantiomers of epoxides. The catalytic properties of Phaseolus vulgaris epoxide hydrolase (PvEH1) towards phenyl glycidyl ether and its methyl derivates were assayed.Seven residues of PvEH1 were then selected for site-directed mutagenesis based on the results of molecular docking simulation and multiple sequence alignment,followed by single-site and combinatorial mutagenesis to obtain mutants possessing enhanced catalytic properties towards ortho-methylphenyl glycidyl ether (1a). The substrate spectrum analysis showed that PvEH1 displayed both the highest activity (157.2U/g wet cell) and enantioselectivity (E=5.6)towards 1a.Thus, 1a was selected as the model substrate.Among the constructed seven E.coli transformants expressing single-site mutant of PvEH1,E.coli/pveh1^L105I and E. coli/pveh1^V106I exhibited notably improved EH activity and E value.Compared with E.coli/pveh1, the EH activity and E value of E.coli/pveh1^L105I/V106I were improved by 2.1 times and 50%, respectively.Additionally,the specific activity (17.6U/mg) and the catalytic efficiency [17.7L/(mmol·s)]of purified PvEH1^L105I/V106I were 1.5-and 2.1-fold those of PvEH1. SDS-PAGE analysis indicated that the soluble expression level of target protein was enhanced by the combinatorial mutagenesis.The kinetic resolution of 100mmol/L 1a by E.coli/pveh1^L105I/V106I whole cells afforded (R)-1a (ee>96%)with 31.2% yield and a space-time yield of 5.12g/(L·h). Therefore, the superior enzymatic properties will make E.coli/pveh1 L105I/V106I a promising biocatalyst for the preparation of optically pure (R)-1a.