title: Expression and characterization of a recombinant psychrophilic Cu/Zn superoxide dismutase from Deschampsia antarctica E. Desv. [Poaceae].
abstract: We present here the structural modeling and biochemical characterization of a recombinant superoxide dismutase (SOD) from Deschampsia antarctica E. Desv. [Poaceae] produced in Escherichia coli. The recombinant protein was purified by affinity chromatography nickel-nitrilotriacetic acid (Ni-NTA), and its identity was demonstrated by immunoblotting and inhibition by H2O2 and KCN. Inductively coupled plasma optical emission spectroscopy (ICP-OES) analysis confirmed the presence of Cu and Zn. Modeling of the D. antarctica Cu/Zn-SOD (DaSOD) amino acid sequence using the SWISS-MODEL and 2Q2L_B monomer of the psychrophilic Cu/Zu-SOD from Potentilla atrosanguinea (PaSOD) as template produced a structure similar to that of the typical eukaryotic Cu/Zn-SODs. Activity assays using the p-nitro blue tetrazolium chloride (NBT) solution method showed that the purified DaSOD had a specific activity of 5818 U/mg at 25 °C and pH 7.2 and that it was active in a pH interval of 5-8 and a temperature interval of 0-40 °C. Furthermore, DaSOD was still active at -20 °C as observed by a zymogram assay. We found 100 % activity when it was heated at 80 °C for 60 min, indicating a high thermostability. DaSOD properties suggest that this enzyme could be useful for preventing the oxidation of refrigerated or frozen foods, as well as in the preparation of cosmetic and pharmaceutical products.
authors: División de Biología Molecular, Instituto Potosino de Investigación Científica y Tecnológica, A.C. (IPICyT), Camino a la Presa San José 2055, Lomas 4a. Sección, CP 78216, San Luis Potosí, Mexico.
authors: Juan A
authors: JA
authors: Rojas-Contreras
authors: Ana P Barba
authors: AP
authors: de la Rosa
authors: Antonio
authors: A
authors: De León-Rodríguez
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D003080:Cold Temperature; D004926:Escherichia coli / Q000235:genetics; D018506:Gene Expression Regulation, Plant; D006861:Hydrogen Peroxide; D006109:Poaceae / Q000201:enzymology*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013482:Superoxide Dismutase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D011994:Recombinant Proteins; D006861:Hydrogen Peroxide; D013482:Superoxide Dismutase