title: 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.
abstract: 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 °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 Ni(2+), Cu(2+), Fe(2+), or Co(2+). 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 Cα) in contrast to (R)-pNSO (87% to Cβ). 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.
authors.affiliation: State Key Laboratory of Bioreactor Engineering, School of Biotechnology, East China University of Science and Technology, Shanghai, 200237, People's Republic of China.
authors.forename: Qing-Qing
authors.initials: QQ
authors.lastname: Zhu
authors.forename: Wan-Hong
authors.initials: WH
authors.lastname: He
authors.forename: Xu-Dong
authors.initials: XD
authors.lastname: Kong
authors.forename: Li-Qiang
authors.initials: LQ
authors.lastname: Fan
authors.forename: Jian
authors.initials: J
authors.lastname: Zhao
authors.forename: Su-Xia
authors.initials: SX
authors.lastname: Li
authors.forename: Jian-He
authors.initials: JH
authors.lastname: Xu
mesh_terms: D002846:Chromatography, Affinity; D020536:Enzyme Activators / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000378:metabolism; D004795:Enzyme Stability; D004851:Epoxide Hydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004852:Epoxy Compounds / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D007887:Fabaceae / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D006018:Glycols / Q000378:metabolism; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009578:Nitrobenzenes / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013696:Temperature
chemical_list: D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D004852:Epoxy Compounds; D006018:Glycols; D009578:Nitrobenzenes; D011994:Recombinant Proteins; C033332:4-nitrostyrene oxide; D004851:Epoxide Hydrolases