文献类型：期刊文献

英文题名：Heterologous overexpression of Vigna radiata epoxide hydrolase in Escherichia coli and its catalytic performance in enantioconvergent hydrolysis of p-nitrostyrene oxide into (R)-p-nitrophenyl glycol

作者：Zhu, Qing-Qing[1];He, Wan-Hong[1];Kong, Xu-Dong[1];Fan, Li-Qiang[1];Zhao, Jian[1];Li, Su-Xia[1];Xu, Jian-He[1]

机构：[1]E China Univ Sci & Technol, Sch Biotechnol, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China

年份：2014

卷号：98

期号：1

起止页码：207

外文期刊名：APPLIED MICROBIOLOGY AND BIOTECHNOLOGY

收录：;EI(收录号：20140317202346);WOS:【SCI-EXPANDED(收录号:WOS:000329093600019)】；

基金：This work was financially supported by the National Natural Science Foundation of China (Nos. 21276082 and 81071252), Ministry of Science and Technology, People's Republic of China (Nos. 2011CB710800 and 2011AA02A210), and China National Special Fund for State Key Laboratory of Bioreactor Engineering (No. 2060204).

语种：英文

外文关键词：RT-PCR; Vigna radiata epoxide hydrolase; p-Nitrostyrene oxide; Enantioconvergency; Characterization

摘要：Two native epoxide hydrolases (EHs) were previously discovered from mung bean powder (Vigna radiata), both of which can catalyze the enantioconvergent hydrolysis of p-nitrostyrene oxide (pNSO). In this study, the encoding gene of VrEH1 was successfully cloned from the cDNA of V. radiata by RT-PCR and rapid amplification of cDNA ends (RACE) technologies. High homologies were found to two putative EHs originated from Glycine max (80 %) and Medicago truncatula (79 %). The vreh1 gene constructed in pET28a(+) vector was then heterologously overexpressed in Escherichia coli BL21(DE3), and the encoded protein was purified to homogeneity by nickel affinity chromatography. It was shown that VrEH1 has an optimum activity at 45 A degrees C and is very thermostable with an inactivation energy of 468 kJ mol(-1). The enzyme has no apparent requirement of metal ions for activity, and its activity was strongly inhibited by 1 mM of Ni2+, Cu2+, Fe2+, or Co2+. By adding 0.1 % Triton X-100, the enzyme activity could be significantly increased up to 340 %. VrEH1 shows an unusual ability of enantioconvergent catalysis for the hydrolysis of racemic pNSO, affording (R)-p-nitrophenyl glycol (pNPG). It displays opposite regioselectivity toward (S)-pNSO (83 % to C-alpha) in contrast to (R)-pNSO (87 % to C-beta). The K (M) and k (cat) of VrEH1 were determined to be 1.4 mM and 0.42 s(-1) for (R)-pNSO and 5.5 mM and 6.2 s(-1) for (S)-pNSO. This thermostable recombinant VrEH1 with enantioconvergency is considered to be a promising biocatalyst for the highly productive preparation of enantiopure vicinal diols and also a good model for understanding the mechanism of EH stereoselectivity.