Cloning and characterization of a new manganese superoxide dismutase from deep-sea thermophile Geobacillus sp. EPT3

• Published in: World journal of microbiology & biotechnology Vol. 30; no. 4; pp. 1347 - 1357
• Main Authors: Zhu, Yanbing, Wang, Guohong, Ni, Hui, Xiao, Anfeng, Cai, Huinong
• Format: Journal Article
• Language: English
• Published: Dordrecht Springer Netherlands 01.04.2014; Springer Nature B.V
• Subjects: Amino acids; Analysis; Applied Microbiology; Bacteria; Binding sites; Biochemistry; Biomedical and Life Sciences; Biotechnology; Chromatography; Cloning; Cloning, Molecular; Conserved Sequence; Deep sea; Detergents; DNA, Bacterial - chemistry; DNA, Bacterial - genetics; E coli; Engineering; Environmental Engineering/Biotechnology; Enzyme Inhibitors - analysis; Enzyme Stability; Enzymes; Escherichia coli; Escherichia coli - genetics; Gene Expression; Genes; Geobacillus; Geobacillus - classification; Geobacillus - enzymology; Geobacillus - genetics; Hydrogen peroxide; Hydrogen-Ion Concentration; Inhibitors; Life Sciences; Manganese; Marine biology; Microbiology; Microorganisms; Molecular Sequence Data; Molecular Weight; Neurosciences; Open Reading Frames; Original Paper; Protein Multimerization; Proteins; Recombinant; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Seawater - microbiology; Sequence Analysis, DNA; Sod; Sodium; Studies; Superoxide dismutase; Superoxide Dismutase - chemistry; Superoxide Dismutase - genetics; Superoxide Dismutase - isolation & purification; Superoxide Dismutase - metabolism; Temperature; Thermophiles; Urea; IE, East Pacific; Thermostability; Characterization; Superoxide dismutase; Expression; Manganese
• DOI: 10.1007/s11274-013-1536-5
• ISSN: 0959-3993

A new gene encoding a superoxide dismutase (SOD) was identified from a thermophile Geobacillus sp. EPT3 isolated from a deep-sea hydrothermal field in east Pacific. The open reading frame of this gene encoded 437 amino acid residues. It was cloned, overexpressed in Escherichia coli (DE3), and the recombinant protein was purified to homogeneity. Geobacillus sp. EPT3 SOD was of the manganese-containing SOD type, as judged by the insensitivity of the recombinant enzyme to both KCN and H 2 O 2 , and the activity analysis of Fe or Mn reconstituted SODs by polyacrylamide gel electrophoresis. The recombinant SOD was determined to be a homodimer with monomeric molecular mass of 59.0 kDa. In comparison with other Mn–SODs, the manganese-binding sites are conserved in the sequence (His260, His308, Asp392, His396). The recombinant enzyme had high thermostability at 50 °C. It retained 57 % residual activity after incubation at 90 °C for 1 h, which indicated that this SOD was thermostable. The enzyme also showed striking stability over a wide range of pH 5.0–11.0. At tested conditions, the recombinant SOD from Geobacillus sp. EPT3 showed a relatively good tolerance to some inhibitors, detergents, and denaturants, such as β-mercaptoethanol, dithiothreitol, phenylmethylsulfonyl fluoride, Chaps, Triton X-100, urea, and guanidine hydrochloride.