Purification and characterization of a cis-epoxysuccinic acid hydrolase from Nocardia tartaricans CAS-52, and expression in Escherichia coli.
Summary of "Purification and characterization of a cis-epoxysuccinic acid hydrolase from Nocardia tartaricans CAS-52, and expression in Escherichia coli."
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.
Affiliation
National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing, 100190, China.
Journal Details
This article was published in the following journal.
Name: Applied microbiology and biotechnology
ISSN: 1432-0614
Pages:
Links
- PubMed Source: http://www.ncbi.nlm.nih.gov/pubmed/22552902
- DOI: http://dx.doi.org/10.1007/s00253-012-4102-4
Medical and Biotech [MESH] Definitions
Nocardia Infections
Infections with bacteria of the genus NOCARDIA.
Gamma-glutamyl Hydrolase
Catalyzes the hydrolysis of pteroylpolyglutamic acids in gamma linkage to pterolylmonoglutamic acid and free glutamic acid. EC 3.4.19.9.
Pteroylpolyglutamic Acids
Derivatives of folic acid (pteroylglutamic acid). In gamma-glutamyl linkage they are found in many tissues. They are converted to folic acid by the action of pteroylpolyglutamate hydrolase or synthesized from folic acid by the action of folate polyglutamate synthetase. Synthetic pteroylpolyglutamic acids, which are in alpha-glutamyl linkage, are active in bacterial growth assays.
Allophanate Hydrolase
An enzyme that catalyzes the hydrolysis of allophanic acid to two molecules of ammonia plus two molecules of "active carbon dioxide". EC 3.5.1.54.
Pyroglutamate Hydrolase
Hydrolyzes pyroglutamic acid in the presence of ATP to glutamate plus ADP and inorganic phosphate. Deficiency leads to pyroglutamic acidurea.
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