Studies On Purification And Characterization Of Superoxide Dismutase From Thermomyces Lanuginosus

The superoxide dismutase (SOD, EC1.15.1.1) are metallo-enzymes that catalyze the dismutation of superoxide(O2.-)to hydrogen peroxide(H2O2) and molecular oxygen(O2). They have been found in nearly all organisms examined to date and play a major role in the defense against oxidative stress. There are three general classes of SODs in organisms, which differ in their metal cofactor: copper-zinc-containing SOD(Cu-Zn-SOD), manganese containing SOD(Mn-SOD) and iron-containing SOD(Fe-SOD).Cu-Zn-SOD is found exclusively in peroxisomes of eukaryotes. In contrast, Mn-SOD is present in prokaryotes and in mitochondria of eukaryotes, while Fe-SOD is present in prokaryotes and in chloroplasts of eukaryotes. Mn-SOD and Fe-SOD resemble each other with respect to their amino acid sequences, suggesting their common ancestry.Thermomyces lanuginosus, a kind of the thermophilic fungi, is a metatrophic fungus. It canbe thrive at the temperature between 45 degrees C and 50 degrees C while most fungi will die above 40 degrees C soonly. Few report have been published about superoxide dismutase from thermophilic fungi, except from Thermomyces lanuginosus.In this article, a kind of superoxide dismutase in the mycelium of Thermomyces lanuginosus was purified to homogeneity by ammonium sulfate fraction(50%-65%), DEAE-Sepharose anion-exchange chromatography, Phenyl-Sepharose chromatography and Sephacryl S-100 chromatography. The molecular weight of the enzyme is 22.4kDa by SDS-polyacrylamide gel electrophoresis and 89.1 kDa by gel filtration on Sephacryl S-100. These demonstrated that this enzyme is tetrameric composed of four subunits of equal size of 22.4kDa. The enzyme has an optimum temperature of 55 degrees C and an optimum pH of 8.0. It is thermostable at 50 degrees C and 60 degrees C and retains 49% activity after60 minutes at 70 degrees C. The half life time of the enzyme at 80 degrees C is 28 min and even retains 19% activity after 20 min at 90 degrees C. The superoxide dismutase is stable at pH 7.0-8.0 and retains at least 50% activity between pH 6.5 and pH 9.0.Different metal ions show different effect on superoxide dismutase activity. Mn2+,Na+ and Ca2+ enhance the activity of superoxide dismutase, while K+, Mg2+, Zn2+, Fe2+, Ba2+, Cu2+ and NH4+ cause inhibition. And the enzyme activity was strongly inhibited by Ag+, Hg2+ and Al3+. The enzyme shows insensitive in the presence of KCN and H2O2, suggests that the enzyme purified in this article is Mn-SOD.