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

doi:10.13523/j.cb.20161004

中国生物工程杂志

2016, Vol. 36

Issue (10): 21-27 DOI: 10.13523/j.cb.20161004

研究报告

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

叶慧华¹, 胡蝶², 李闯², 程建青³, 邓超³, 邬敏辰³

1 江南大学药学院无锡 214122;
2 生物工程学院无锡 214122;
3 无锡医学院无锡 214122

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

YE Hui-hua¹, HU Die², LI Chuang², CHENG Jian-qing³, DENG Chao³, WU Min-chen³

1. School of Pharmaceutical Science, Jiangnan University, Wuxi 214122, China;
2. School of Biotechnology, Jiangnan University, Wuxi 214122, China;
3. Wuxi Medical School, Jiangnan University, Wuxi 214122, China

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摘要：

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。

关键词： 菜豆; 对映归一性; 表达; 环氧化物水解酶

Abstract:

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.

Key words: Epoxide hydrolase Expression Phaseolus vulgaris Enantioconvergence

收稿日期: 2016-04-05 出版日期: 2016-10-25

ZTFLH:

Q78

基金资助:

江苏省研究生科研创新计划资助项目（KYLX15-1151；KYLX15-1190）

通讯作者: 邬敏辰,电子信箱:biowmc@126.com E-mail: biowmc@126.com

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引用本文:

叶慧华, 胡蝶, 李闯, 程建青, 邓超, 邬敏辰. 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[J]. 中国生物工程杂志, 2016, 36(10): 21-27.

YE Hui-hua, HU Die, LI Chuang, CHENG Jian-qing, DENG Chao, WU Min-chen. Expression of a Novel Epoxide Hydrolase from Phaseolus vulgaris and Its Enantioconvergent Catalytic Performance. China Biotechnology, 2016, 36(10): 21-27.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20161004 或 https://manu60.magtech.com.cn/biotech/CN/Y2016/V36/I10/21

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