title: Purification and characterization of a cis-epoxysuccinic acid hydrolase from Nocardia tartaricans CAS-52, and expression in Escherichia coli.
abstract: A highly enantioselective cis-epoxysuccinic acid hydrolase from Nocardia tartaricans was purified to electrophoretic homogeneity. The enzyme was purified 184-fold with a yield of 18.8 %. The purified cis-epoxysuccinic acid hydrolase had a monomeric molecular weight of 28 kDa, and its optimum conditions were 37 °C and pH 7-9. With sodium cis-epoxysuccinate as the substrate, Michaelis-Menten enzyme kinetics analysis gave a Km value of 35.71 mM and a Vmax of 2.65 mM min(-1). The enzyme was activated by Ni(2+) and Al(3+), while strongly inhibited by Fe(3+), Fe(2+), Cu(2+), and Ag(+). The cis-epoxysuccinic acid hydrolase gene was cloned, and its open reading frame sequence predicted a protein composed of 253 amino acids. A pET11a expression plasmid carrying the gene under the control of the T7 promoter was introduced into Escherichia coli, and the cis-epoxysuccinic acid hydrolase gene was successfully expressed in the recombinant strains.
authors.affiliation: National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China.
authors.forename: Ziqiang
authors.initials: Z
authors.lastname: Wang
authors.forename: Yunshan
authors.initials: Y
authors.lastname: Wang
authors.forename: Zhiguo
authors.initials: Z
authors.lastname: Su
mesh_terms: D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D020536:Enzyme Activators / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008670:Metals / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009615:Nocardia / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013696:Temperature
chemical_list: D004269:DNA, Bacterial; D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D008670:Metals; D011994:Recombinant Proteins; D006867:Hydrolases; C492296:cis-epoxysuccinate hydrolase, Nocardia tartaricans