Purification, Gene Cloning And Expression Of Thermostable SOD From The Thermophilic Fungus Chaetomium Thermophilum

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 critical 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 (CuZn-SOD), manganese- containing SOD (Mn-SOD) and iron-containing SOD (Fe-SOD).Chaetomium thermophilum is a widely distributed soil-inhibiting fungus of considerable interest producer of thermostable enzymes, including cellulase, endocellulase, xylanase and laccase which have been purified and well studied. However, the superoxide dismutase from the genus C. thermophilum has not yet been studied. During growth in a medium containing Casein, C. thermophilum also produced superoxide dismutase. The enzyme was purified to homogeneity from the culture supernatant of the strain by fractional ammonium sulphate precipitation, DEAE-Sepharose chromatography and Phenyl-Sepharose chromatography.The molecular mass of a single band of the enzyme was estimated to be 23.5 kDa, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using gel filtration on Sephacryl S-100, the molecular mass was estimated to be 94.4 kDa, indicating that this enzyme was composed of four identical subunits of 23.5 kDa each. The SOD exhibited maximal activity at pH 7.5 and optimum temperature at 60oC. It was thermostable at 50 and 60oC and retained 60% activity after 60 min at 70oC. The half-life of the SOD at 80oC was approximately 25 min.The first 10 amino acids from the N-terminal of the Mn-SOD were KATLPDLKYD. The sequence showed a certain similarity with some other fungal manganese superoxide dismutase such as those from Aspergillus fumigatus and Paracoccidioides brasiliensis.Degenerate primers based on the conserved domain of other reported superoxide dismutase, and the cDNA fragments encoding the copper zinc superoxide dismutase and manganese superoxide dismutase were obtained through RT-PCR. The RACE-PCR was used to generate full-length cDNA clones. Then partial DNA encoding the copper zinc superoxide dismutase was cloned and it contained three introns. They have been registered in GenBank with accession number DQ493760, EF569987 and DQ683184.The cz1 gene and expression vector pPIC9K were digested with EcoR I and Not I, then ligationed in vitro to construct the expression plasmid pPIC9K/cz1. The pPIC9K/cz1 was transformed to Pichia GS115 competent cell after linearized with restriction enzyme Sal I. The recombinant Pichia GS-CZ-10 was got and the expressed superoxide dismutase was purified and characterized. The expression level was up to 0.56 mg/mL.