Purification, Cloning And Expression Of Thermostable SOD From The Thermophilic Fungus Thermoascus Aurantiacus Var. Levisporus

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.Thermoascus aurantiacus var.levisporus is a variation of Thermoascus, it can thrive well between 45 and 50℃. Few reports 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 Thermoascus aurantiacus var.levisporus was purified to homogeneity by ammonium sulfate fraction, DEAE-Sepharose anion-exchange chromatography, and Phenyl-Sepharose chromatography. The molecular weight of the enzyme is 16.8 kDa by SDS-polyacrylamide gel electrophoresis and 33.2 kDa by gel filtration on Sephacryl S-100. These demonstrated that this enzyme is tetrameric composed of two subunits of equal size of 16.8kDa. The first 7 amino acids from the N-terminal of the Cu, Zn-SOD were VKAVAVL. The sequence showed a certain similarity with some other fungal Cu, Zn-SOD such as those from Claviceps purpurea, Paecilomyces sinensis and Glomerella cingulata.Degenerate primers based on the N-terminal sequences of purified Cu, Zn-SOD and the conserved domain of other reported Cu, Zn-SOD, and a cDNA fragment encoding the Cu, Zn-SOD gene was obtained through RT-PCR. The RACE was used to generate full-length cDNA clones. The Cu, Zn-SOD gene contained an ORF of 468bp encoding 155 amino acids. The gene has been registered in Genbank with accession number DQ497636.The Cu, Zn-SOD gene and expression vector pET-22b (+) were digested with EcoRⅠand NotⅠ, and ligation was carried out in vitro. The recombinant plasmid pET/czsod was generated, and transformed into E. coli BL21 .The recombinant protein was produced in large amount after IPTG induction, approximately 17 kDa protein was determined by SDS-PAGE, and this size was agreed with the protein molecular weight from the putative amino acid sequence; no interest protein was produced by inducing with IPTG after the pET-22b (+) was transformed into BL21. The solubility analysis showed that the recombinant protein was presented in a fusion form.Degenerate primers based on the conserved domain of other reported Mn-SOD, and a cDNA fragment encoding the gene was obtained through RT-PCR. The RACE was used to generate full-length cDNA clones. The gene contained an ORF of 696 bp encoding 231 amino acids. The Mn-SOD gene has been registered in Genbank with accession number EF428323.