盖子环替换及定点突变提高菜豆环氧化物水解酶对邻甲基苯基缩水甘油醚的催化性能

doi:10.16085/j.issn.1000-6613.2019-1727

化工进展 ›› 2020, Vol. 39 ›› Issue (7): 2788-2794.DOI: 10.16085/j.issn.1000-6613.2019-1727

盖子环替换及定点突变提高菜豆环氧化物水解酶对邻甲基苯基缩水甘油醚的催化性能

章晨¹(), 朱秀秀¹, 李闯², 邬敏辰³()

^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

出版日期:2020-07-05 发布日期:2020-07-10
通讯作者: 邬敏辰
作者简介:章晨（1995—），男，硕士研究生。E-mail：2723285191@qq.com。
基金资助:
国家自然科学基金(21676117)

Improvement in catalytic properties of a phaseolus vulgaris epoxide hydrolase towards o-methylphenyl glycidyl ether by replacement of a lid loop and site-directed mutation

Chen ZHANG¹(), Xiuxiu ZHU¹, Chuang LI², Minchen WU³()

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

Online:2020-07-05 Published:2020-07-10
Contact: Minchen WU

摘要/Abstract

摘要：

菜豆环氧化物水解酶1和2（PvEH1、PvEH2）能够动力学拆分外消旋邻甲基苯基缩水甘油醚（rac-oMGE），从而保留(R)-oMGE。基于对PvEH1和PvEH2结构的同源模拟和分析，发现二者分子中的盖子环差异较大，故本文选择盖子环作为研究目标。经融合聚合酶链式反应（FPCR），获得了PvEH2的盖子环区域被PvEH1对应区域替换的杂合酶Pv2Pv1。用全细胞酶E. coli/pv2pv1催化rac-oMGE，当(S)-oMGE刚好水解完全时，产物(S)-3-邻甲苯基-1,2-丙二醇（(S)-oTPD）的ee_p由PvEH2的58.3%提高至75.5%。为进一步提高酶的性质，在Pv2Pv1中选取11个氨基酸位点进行丙氨酸(A)突变，获得最优突变子E. coli/pv2pv1^K176A，活性为E. coli/pv2pv1（4.2U/g）的2.1倍，且当S构型的底物刚好完全水解时，(S)-oTPD的ee_p进一步提高为80.3%。分子对接分析发现，盖子环替换和K176位点突变为A，均使(R)-oMGE环氧环中的C_α更易受到酶中D101位点的攻击。利用E. coli/pv2pv1^K176A催化150mmol/L rac-oMGE水解制备(R)-oMGE（ee_s＞99%）和(S)-oTPD（ee_p=80.4%），二者的产率Y_S和Y_P分别为32.7%和60.1%，时空产率STY_S和STY_P为1.6g/(L·h)和3.3g/(L·h)。本实验为改善EH的催化性质提供了一种有效策略。

关键词: 菜豆环氧化物水解酶, 邻甲基苯基缩水甘油醚, 盖子环, 丙氨酸突变, 分子对接

Abstract:

Phaseolus vulgaris epoxide hydrolase 1 and 2 (PvEH1, PvEH2) can retain (R)-oMGE via kinetic resolution of rac-oMGE. Based on the homologous simulation and analysis of the structures of PvEH1 and PvEH2, we found there were sizable differences between the lid loops of PvEH1 and PvEH2. Therefore, the lid loops were identified as the research objects. Then, a hybrid enzyme, Pv2Pv1, of which the lid domain of PvEH2 was replaced by the corresponding region of PvEH1, was acquired by fusion PCR. Using the E. coli/pv2pv1 as the biocatalyst and compared with the E. coli/pveh2, the ee_p of generated (S)-oTPD was increased from 58.3% to 75.5% at the moment of (S)-oMGE was just hydrolyzed completely. To further improve the catalytic performances, 11 amino residues were selected for alanine (A) mutations. Then a best mutant, E. coli/pv2pv1^K176A was acquired, which activity was 2.1-fold than that of E. coli/pv2pv1 (4.2U/g), and ee_p was further enhanced to 80.3%. According to the results of MD simulation, the replacement of lid loop and substitution A for K176 caused the C_αin epoxy ring of(R)-oMGE more vulnerable to be attacked by D101. Using the E. coli/pv2pv1^K176A to prepare (R)-oMGE (ee_s＞99%) and (S)-oTPD (ee_p=80.4%) by hydrolysis of 150 mMrac-oMGE, the yield of (R)-oMGE (Y_S) and (S)-oTPD (Y_P) were 32.7% and 60.1%, and the space time yield, STY_S and STY_P were 1.6g/(L·h) and 3.3g/(L·h), respectively. Therefore, this study provided an effective strategy for improving the catalytic properties of EHs.

Key words: phaseolus vulgaris epoxide hydrolase, o-methylphenyl glycidyl ether, lid loop, alanine mutation, molecule docking simulation

中图分类号:

章晨, 朱秀秀, 李闯, 邬敏辰. 盖子环替换及定点突变提高菜豆环氧化物水解酶对邻甲基苯基缩水甘油醚的催化性能[J]. 化工进展, 2020, 39(7): 2788-2794.

Chen ZHANG, Xiuxiu ZHU, Chuang LI, Minchen WU. Improvement in catalytic properties of a phaseolus vulgaris epoxide hydrolase towards o-methylphenyl glycidyl ether by replacement of a lid loop and site-directed mutation[J]. Chemical Industry and Engineering Progress, 2020, 39(7): 2788-2794.

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盖子环替换及定点突变提高菜豆环氧化物水解酶对邻甲基苯基缩水甘油醚的催化性能

Improvement in catalytic properties of a phaseolus vulgaris epoxide hydrolase towards o-methylphenyl glycidyl ether by replacement of a lid loop and site-directed mutation

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