新型菜豆环氧化物水解酶的异源表达及对映归一性催化特性

为挖掘新型环氧化物水解酶（EH），探讨其对映归一性催化特性，以菜豆（Phaseolus vulgaris）总RNA为模板，采用RT-PCR扩增了一种菜豆EH（PvEH1）的编码基因pveh1，并将其在大肠杆菌BL21（DE3）中进行了表达。一级和三维结构分析表明，PvEH1与绿豆和苜蓿EHs的同源性分别为85.7和81.1%，其催化三联体为Asp¹⁰¹-His²⁹⁹-Asp²⁶⁴，属于α/β水解酶超家族。当PvEH1催化外消旋环氧苯乙烷水解的转化率达99.1%时，产物（R）-苯乙二醇的对映体纯度为33.6% e.e._p。PvEH1对（S）-和（R）-SO的区域选择性系数α_S和β_R分别为91.1和53.3%。PvEH1的挖掘及其对映归一性催化特性的分析不仅增加了此类植物EHs的数目，而且为其归一性催化机制的研究和区域选择性的改造奠定了基础。

Expression of a Novel Epoxide Hydrolase from Phaseolus vulgaris and Its Enantioconvergent Catalytic Performance

To excavate a novel epoxide hydrolase from Phaseolus vulgaris (PvEH1) and explore its enantioconvergent catalytic performance, a PvEH1-encoding gene (pveh1) was amplified from the P. vulgaris total RNA by RT-PCR technique. Then, pveh1 was heterologously expressed in E. coli BL21(DE3) mediated by an expression plasmid pET28a(+). Analysis of primary and three-dimensional structures indicated that the identities of PvEH1 with Vigna radiata and Medicago truncatula epoxide hydrolases are 85.7 and 81.1%. Its catalytic triad is Asp¹⁰¹-His²⁹⁹-Asp²⁶⁴, belonging to the α/β-hydrolase superfamily. When the conversion rate of racemic styrene oxide catalyzed by PvEH1 reached 99.1%, the product, (R)-1-phenyl-1,2-ethanediol, was obtained with an enantiomeric purity of 33.6% e.e._p. PvEH1 possesses the opposite regioselectivity towards (S)-SO and (R)-SO with regioselectivity coefficients (α_S and β_R) of 91.1 and 53.3%. The discovery of PvEH1 and characterization of its enantioconvergence not only increased the number of plant EHs, but also established a foundation for the study of its catalytic mechanism and the directed modification of its regioselectivity.