Purification and partial characterization of Cu, Zn containing superoxide dismutase from entomogenous fungal species Cordyceps militaris

Cordyceps militaris mycelium produced mainly Cu, Zn containing superoxide dismutase (Cu, Zn-SOD). Cu, Zn-SOD activity was detectable in the culture filtrates, and intracellular Cu, Zn-SOD activity as a proportion protein was highest in early log phase culture. The effects of Cu2+, Zn2+, Mn2+ and Fe2+ on enzyme biosynthesis were studied. The Cu, Zn-SOD was isolated and purified to homogeneity from C. militaris mycelium and partially characterized. The purification was performed through four steps: (NH4)2SO4 precipitation, DEAE-sepharose™ fast flow anion-exchange chromatography, CM-650 cation-exchange chromatography, and Sephadex G-100 gel filtration chromatography. The purified enzyme had a molecular weight of 35070 ± 400 Da and consisted of two equal-sized subunits each having a Cu and Zn element. Isoelectric point value of 7.0 was obtained for the purified enzyme. The N-terminal amino acid sequence of the purified enzyme was determined for 12 amino acid residues and the sequences was compared with other Cu, Zn-SODs. The optimum pH of the purified enzyme was obtained to be 8.2-8.8. The purified enzyme remained stable at pH 5.8-9.8, 25 °C and up to 50 °C at pH 7.8 for 1.5 h incubation. The purified enzyme was sensitive to H2O2, KCN. 2.5 mM NaN3, PMSF, Triton X-100, β-mercaptoethanol and DTT showed no significant inhibition effect on the purified enzyme within 5 h incubation period.