绿豆环氧化物水解酶基因的克隆、分析及表达

Cloning，Analysis and Expression of a Novel Epoxide Hydrolase Gene（Vreh3） from Vigna radiata

DOI：10.3969/j.issn.1673-1689.2018.02.004

中文关键词: 绿豆 环氧化物水解酶 基因克隆 对映归一性水解 （R）-苯基乙二醇

英文关键词: Vigna radiata,epoxide hydrolase,gene cloning,enantioconvergent hydrolysis,（R）-1-phenyl-1,2-ethanediol

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中文摘要:

环氧化物水解酶可催化制备高光学纯度的环氧化物及邻二醇等高价值手性药物中间体。本研究采用RT-PCR和THSO-PCR扩增侧翼未知DNA序列技术从绿豆（Vigna radiata） 中克隆了一种新型环氧化物水解酶VrEH3的基因Vreh3，Vreh3编码区DNA序列长度为1 178 bp，包含2个142 bp和79 bp的内含子，以及957 bp的开放阅读框，编码318个氨基酸。VrEH3的理论相对分子质量和等电点分别为36.2×103和5.59；保守的催化三联体为Asp101-Asp262-His297。将Vreh3与表达载体pET-28a（+） 连接，转化大肠杆菌BL21（DE3） 获得重组VrEH3。该酶可对映归一性水解外消旋环氧苯乙烷（（R，S）-SO） 产生（R）-苯基乙二醇，得率高达79.4%，对映体过量值（e.e.值） 为94.7%。

英文摘要:

Epoxide hydrolase can be effectively used in the preparation of optically active epoxides and vicinal diols，which are versatile intermediates for the preparation of highly value-added bioactive compounds such as pharmaceuticals. In this study，a gene encoding a novel epoxide hydrolase（VrEH3） was successfully cloned from Vigna radiata by RT-PCR and T-hairpin structure-mediated PCR amplification（THSO-PCR） techniques. The cloned coding region DNA sequence of Vreh3 is 1 178 bp in length，harboring two introns with sizes of 142 bp and 79 bp and a 957 bp open reading frame that encodes a 318-aa VrEH3. The theoretical molecular weight and calculated pI of VrEH3 were 36.2×103 and 5.59，respectively. The catalytic active center was constituted by a strictly conserved catalytic triad of Asp101-Asp262-His297. Subsequently，the expression plasmid pET-28a（+）-Vreh3 was constructed by ligated Vreh3 with pET-28a（+） and transformed into Escherichia coli BL21（DE3），forming a recombination strain E. coli/Vreh3. Using the E. coli/Vreh3 as the catalysis，the（R）-1-phenyl-1，2-ethanediol was obtained with a yield of 79.4% and an enantiomeric excess value of 94.7% by enantioconvergent hydrolysis of（R，S）-styrene oxide.

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