定点突变改善<i>Pv</i>EH1对邻甲基苯基缩水甘油醚的催化特性 <sup>*</sup>

doi:10.13523/j.cb.20190602

中国生物工程杂志

2019, Vol. 39

Issue (6): 9-16 DOI: 10.13523/j.cb.20190602

研究报告

定点突变改善PvEH1对邻甲基苯基缩水甘油醚的催化特性 ^*

阚婷婷¹,宗迅成²,苏永君¹,王婷婷¹,李闯²,胡蝶^3,^**(

),邬敏辰^3,^**(

)

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

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

Ting-ting KAN¹,Xun-cheng ZONG²,Yong-jun SU¹,Ting-ting WANG¹,Chuang LI²,Die HU^3,^**(

),Min-chen WU^3,^**(

)

1 School of Pharmaceutical Science,Jiangnan University,Wuxi 214122, China
2 School of Biotechnology,Jiangnan University,Wuxi 214122, China
3 Wuxi School of Medicine,Jiangnan University, Wuxi 214122, China

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摘要：

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。

关键词： 环氧化物水解酶; 定点突变; 催化活性; 动力学拆分; 邻甲基苯基缩水甘油醚

Abstract:

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.

Key words: Epoxide hydrolase Site-directed mutagenesis Catalytic activity Kinetic resolution Ortho-methylphenyl glycidyl ether

收稿日期: 2018-11-17 出版日期: 2019-07-12

ZTFLH:

Q814.9

基金资助: * 江苏省研究生科研与实践创新计划(JSCX17_0503);国家自然科学基金资助项目(21676117)

通讯作者: 胡蝶,邬敏辰 E-mail: Butterflystudy@163.com;biowmc@126.com

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引用本文:

阚婷婷,宗迅成,苏永君,王婷婷,李闯,胡蝶,邬敏辰. 定点突变改善PvEH1对邻甲基苯基缩水甘油醚的催化特性 ^*[J]. 中国生物工程杂志, 2019, 39(6): 9-16.

Ting-ting KAN,Xun-cheng ZONG,Yong-jun SU,Ting-ting WANG,Chuang LI,Die HU,Min-chen WU. Site-directed Mutagenesis of PvEH1 to Improve Its Catalytic Properties towards ortho-Methylphenyl Glycidyl Ether. China Biotechnology, 2019, 39(6): 9-16.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20190602 或 https://manu60.magtech.com.cn/biotech/CN/Y2019/V39/I6/9

表1 分析条件

表2 突变引物序列

表3 PvEH1的催化活性及对映选择性

图1 分子对接模拟与多序列比对

表4 突变体的催化特性

图2 PvEH1和PvEH1L105I/V106ISDS-PAGE分析

表5 PvEH1和PvEH1L105I/V106I的动力学常数

表6 E.coli/pveh1L105I/V106I水解动力学拆分高浓度1a

图3 E.coli/pveh1L105I/V106I催化1a的水解进程曲线

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