Cloning and characterization of a new manganese superoxide dismutase from deep-sea thermophile Geobacillus sp. EPT3.
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 H₂O₂, 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.
School of Biotechnology Engineering, Jimei University, 43 Yindou Rd., Xiamen, 361021, People's Republic of China.
Yanbing
Y
Zhu
Guohong
G
Wang
Hui
H
Ni
Anfeng
A
Xiao
Huinong
H
Cai
D003001:Cloning, Molecular; D017124:Conserved Sequence; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004791:Enzyme Inhibitors / Q000032:analysis; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D056506:Geobacillus / Q000145:classification / Q000201:enzymology* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012623:Seawater / Q000382:microbiology*; D017422:Sequence Analysis, DNA; D013482:Superoxide Dismutase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013696:Temperature
D004269:DNA, Bacterial; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D013482:Superoxide Dismutase