BACKGROUND Epoxide hydrolase can regioselectively catalyze the oxirane ring-opening hydrolysis of rac-epoxides producing the corresponding chiral diols. In our laboratory, a gene named pveh1 encoding an EH from Phaseolus vulgaris was cloned. Although the directed modification of PvEH1 was carried out, the mutant PvEH1Y3 showed a limited degree of enantioconvergence towards racemic (rac-) m-chlorostyrene oxide (mCSO). RESULTS PvEH1 and PvEH1Y3 were combinatively subjected to laboratory evolution to further enhance the enantioconvergence of PvEH1Y3 towards rac-mCSO. Firstly, the substrate-binding pocket of PvEH1 was identified using a CAVER 3.0 software, and divided into three zones. After all residues in zones 1 and 3 were subjected to leucine scanning, two E. coli transformants, E. coli/pveh1Y149L and /pveh1P184L, were selected, by which rac-mCSO was transformed into (R)-m-chlorophenyl-1,2-ethanediol (mCPED) having 55.1% and 27.2% eep. Secondly, two saturation mutagenesis libraries, E. coli/pveh1Y149X and /pveh1P184X (X: any one of 20 residues) were created at sites Y149 and P184 of PvEH1. Among all transformants, both E. coli/pveh1Y149L (65.8% αS and 55.1% eep) and /pveh1P184W (66.6% αS and 59.8% eep) possessed the highest enantioconvergences. Finally, the combinatorial mutagenesis was conducted by replacements of both Y149L and P184W in PvEH1Y3, constructing E. coli/pveh1Y3Z2, whose αS reached 97.5%, higher than that (75.3%) of E. coli/pveh1Y3. In addition, the enantioconvergent hydrolysis of 20 mM rac-mCSO was performed by E. coli/pveh1Y3Z2, giving (R)-mCPED with 95.2% eep and 97.2% yield. CONCLUSIONS In summary, the enantioconvergence of PvEH1Y3Z2 was successfully improved by laboratory evolution, which was based on the study of substrate-binding pocket by leucine scanning. Our present work introduced an effective strategy for the directed modification of enantioconvergence of PvEH1.

中文翻译：

---

通过实验室改进，大大提高了菜豆环氧化物水解酶PvEH1对间氯苯乙烯的对映体收敛性。

背景技术环氧化物水解酶可以区域选择性催化外消旋环氧化物的环氧乙烷开环水解，产生相应的手性二醇。在我们的实验室中，克隆了一个编码来自菜豆的编码EH的名为pveh1的基因。尽管进行了PvEH1的直接修饰，但突变体PvEH1Y3对外消旋（rac-）间-氯代环氧乙烷（mCSO）的对映体收敛程度有限。结果将PvEH1和PvEH1Y3联合进行实验室研究，以进一步增强PvEH1Y3对rac-mCSO的对映收敛。首先，使用CAVER 3.0软件识别PvEH1的底物结合口袋，并将其分为三个区域。在对1区和3区的所有残基进行亮氨酸扫描后，选择了两个大肠杆菌转化子，E。coli / pveh1Y149L和/ pveh1P184L，通过该方法，rac-mCSO被转化为具有55.1％和27.2％的陡峭度的（R）-间氯苯基-1，2-乙二醇（mCPED）。其次，在PvEH1的位点Y149和P184上创建了两个饱和诱变文库，大肠杆菌/ pveh1Y149X和/ pveh1P184X（X：20个残基中的任何一个）。在所有转化体中，大肠杆菌/pveh1Y149L（65.8%αS和55.1％eep）和/pveh1P184W（66.6%αS和59.8％eep）都具有最高的对映融合度。最后，通过置换PvEH1Y3中的Y149L和P184W进行组合诱变，构建了大肠杆菌/ pveh1Y3Z2，其αS达到97.5％，高于大肠杆菌/ pveh1Y3的（75.3％）。另外，通过大肠杆菌/ pveh1Y3Z2进行20mM rac-mCSO的对映体水解，得到（R）-mCPED，具有95.2％的eep和97.2％的产率。结论总而言之，通过亮氨酸扫描对底物结合口袋的研究，通过实验室改进成功地改善了PvEH1Y3Z2的对映收敛性。我们目前的工作为定向修饰PvEH1的对映融合引入了一种有效的策略。