Induction, Purification And Characterization Of SOD From Halophilic A.pascens DMDC12

The superoxide dismutase (SOD, EC1.15.1.1) are metalloenzymes 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 classes of SODs in organisms, which differ in their metal cofactor: copper-zinc-containing SOD (Cu Zn-SOD), manganese-containing SOD (Mn-SOD), 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.A halophilic Arthrobacter pascens DMDC12 was cultured in media with citraconic acid as carbon resource. SDS-PAGE analysis of its cell-free extract showed a protein of about 24KDa was obviously induced within the fermentational proceeding. This protein was identified as superoxide dimutase(SOD) by N-terminal amino acid analysis. This report first documents the significant enhancement of SOD activity of A. pascens DMDC12 by the growth environmental stress. To increase the understanding of the inducing factor of SOD, A. pascens DMDC12 was cultured in four kinds of media. The SOD activity was significantly enhanced when the cells incubated in citraconate media. This is equivalent to 280%,240%,180%of that incubated in glucose , yeast extract or Citraconic acid media. The result suggest that the induction of SOD in citraconate media was caused by some factor in cultural environment.A kind of SOD from the strain of A. pascens DMDC12 was purified. The crude enzyme was purified by ammonium sulfate fractional pricipitation(50%～75%), DEAE Sepharose FF chromatography and Source 15Q chromatography. The purified enzyme showed homogeneity and subunit molecular weight of 23.3Kda on electrophoresis of sodium dodecyl sulfate polyacylamide slab gel. The purification...