菜豆环氧化物水解酶的表达及其催化特性研究  

Expression and characterization of Phaseolus vulgaris epoxide hydrolase

作　　者：石小玲 阚婷婷 李闯[2] 宗迅成 邬敏辰 SHI Xiao-Ling;KAN Ting-Ting;LI Chuang;ZONG Xun-Cheng;WU Min-Chen(School of Pharmaceutical Sciences, Jiangnan University, Wuxi, Jiangsu 214122, China;School ofB iotechnology, Jiangnan University, Wuxi, Jiangsu 214122, China;Wuxi Medical School, Jiangnan University, Wuxi, Jiangsu 214122, China)

机构地区：[1]江南大学药学院,江苏无锡214122 [2]江南大学生物工程学院,江苏无锡214122 [3]江南大学无锡医学院,江苏无锡214122

出　　处：《微生物学通报》2018年第4期797-804,共8页Microbiology China

基　　金：国家自然科学基金(21676117);江苏省普通高校研究生科研创新计划项目(SJZZ16_0217;KYLX16_0804)~~

摘　　要：【背景】光学纯环氧化物及邻二醇是一类多功能手性砌块。与化学合成法相比,环氧化物水解酶(EHs)介导的生物转化法因环境友好而成为当前的研究热点。【目的】从菜豆(Phaseolus vulgaris)中克隆一种EH基因并进行原核表达,研究重组酶催化环氧苯乙烷(Styrene oxide,SO)的水解特性。【方法】通过计算机辅助分析EHs的一级结构,推测一种菜豆来源的未知功能蛋白(PvEH4)可能具有EH活性。利用RT-PCR技术,以菜豆总RNA为模板,扩增编码PvEH4的基因pveh4,并实现其在Escherichia coli BL21(DE3)中的表达。利用重组菌E.coli/pveh4全细胞催化SO水解,分析PvEH4的对映选择性和区域选择性。【结果】一级结构分析表明,PvEH4具有α/β折叠型EH特有的保守区域。SDS-PAGE结果显示PvEH4的表观分子量为39.4 kD。PvEH4针对SO的对映选择率(E值)为10.1,区域选择性系数αS和βR分别为99.5%和82.5%。当外消旋SO转化率达到68.1%时,可同时获得99.9%ees的(R)-SO和92.3%eep的(R)-苯乙二醇(Phenyl-1,2-ethanediol,PED),两者的产率分别为31.9%和65.6%。【结论】PvEH4的挖掘不仅增加了植物类EHs的数量,同时也为EHs的蛋白分子改造提供参考。[Background] Optically pure epoxides and vicinal diols are versatile chiral building blocks. Compared with chemical synthesis, biotransformation mediated by epoxide hydrolases（EHs）, an environmental-friendly way, has become the current research focus. [Objective] A gene encoding EH was cloned from Phaseolus vulgaris and heterologously expressed in Escherichia coli. The catalytic characteristics of recombinant EH towards SO were studied. [Methods] By means of computer-aided analysis of EH primary structure, a hypothetical protein from Phaseolus vulgaris（PvEH4） was predicted to have EH activity. Using P. vulgaris total RNA as the templet, the PvEH4-encoding gene, pveh4, was amplified by RT-PCR technique and expressed in E. coli BL21（DE3）. To assay catalytic characteristics of PvEH4, asymmetric hydrolysis of styrene oxide（SO） was conducted by E. coli/pveh4 whole cells. [Results] The primary structure analysis showed that PvEH4 has the typical characteristics of conserved α/β fold EH motifs. SDS-PAGE analysis displayed that the apparent molecular weight of PvEH4 was 39.4 kD. The enantioselectivity（E value） of PvEH4 towards SO was 10.1, while regioselectivity coefficients, αS and βR were 99.5% and 82.5%, respectively. As the conversion ratio reached 68.1%,（R）-SO with 99.9% ees and 31.9% yield as well as（R）-PED with 92.3% eep and 65.6% yield were simultaneously obtained. [Conclusion] The excavated PvEH4 not only increases the number of plant EHs, but also provides a good reference for EH modification.

关 键 词：菜豆 环氧化物水解酶 对映选择性 区域选择性 不对称水解 

分 类 号：Q946.5[生物学—植物学]