Characterization of a thermostable manganese-containing superoxide dismutase from inshore hot spring thermophile Thermus sp. JM1

A thermostable superoxide dismutase (SOD) from the inshore thermophile Thermus sp. JM1 was purified to homogeneity by steps of fractional ammonium sulfate precipitation, DEAE-Sepharose chromatography and Phenyl-Sepharose chromatography. The specific activity of the purified native enzyme was 1 656 U/mg. A sod gene from this strain was cloned and overexpressed in Escherichia coli (E. coli). The prepared apo-enzyme of the purified recombinant SOD (rSOD) was reconstituted with either Fe or Mn by means of incubation with appropriate metal salts. As a result, only Mn²⁺-reconstituted rSOD (Mn-rSOD) exhibited the specific activity of 1 598 U/mg. SOD from Thermus sp. JM1 was Mn-SOD, judging by the specific activities analysis of Fe or Mn reconstituted rSODs and the insensitivity of the native SOD to both cyanide and H₂O₂. Both the native SOD and Mn-rSOD were determined to be homotetramers with monomeric molecular mass of 26 kDa and 27.5 kDa, respectively. They had high thermostability at 50°C and 60°C, and showed striking stability across a wide pH span from 4.0 to 11.0.