Cloning, Expression And Characterization Of Thermostable Manganese Superoxide Dismutase From Thermoascus Aurantiacus Var. Levisporus

The superoxide dismutases (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 cofactors: copper zinc-containing SOD (Cu,Zn-SOD), manganese-containing SOD (Mn-SOD)and iron-containing SOD (Fe-SOD).Thermoascus aurantiacus var. levisporus is a thermophilic fungus with higher growth temperature, which is shown to produce considerable thermostable enzymes such as lipase, cellucose, endocellulase, xylanase, laccase and so on. However the high culture temperature needed by T. aurantiacus to produce superoxide dismutase greatly is difficult in industrial applications. In recent years most of studies focus on the cloning and expression of gene in different expression systems by gene engineering methods.During growth in a induced medium containing casein, T. aurantiacus produced superoxide dismutases. Based on a reported lipase gene cloned by our lab, we designed special primers and cloned the full length genomic cDNA of superoxide dismutase gene using PCR and RT-PCR methods. We named the lipase gene as mnsod. The full length of the cDNA is 878bp, which contains an ORF of 696bp. The coding sequence predicted a 231 residues protein with a unique 35 amino acids extension at the N-terminus. Registered in GenBank , the accession number was EF428323.The coding region of mnsod without their signal sequences, and expression vector pPIC9K were digested with EcoRⅠand NotⅠ, and ligation was carried out in vitro. The recombinant expression plasmid pPIC9K/mnsod was constructed and sequenced to confirm the correct reading frame. The construct plasmid pPIC9K/mnsod and pPIC9K was linearized with a restriction enzyme SacI (insertion at 5'AOX1), and transformed into Pichia pastoris GS115 competent cell by electroporation methods, and screened for His+Mut+ transformants on MD and MM plates. The parent vector was linearized with the same restriction enzyme and transformed GS115 as a control. PCR analysis of P. pastoris integrants and G418 screening determined the multicopy integrants to induce by methanol. These integrants were used to analyze expression levels of interest protein every 24 hours. The engineering strains with high expression level, named as MS18, were tested and characterized. After seven days induction the transformant strain MS18 had the highest activity and the expression level was 0.92 mg/ml.The expression level of recombinant manganese superoxide dismutase was kept stable. The expression enzyme had temperature optima at 55℃. The recombinant Mn-SOD from T. aurantiacus var. levisporus was a thermostable SOD. The enzyme was thermostable at 50 and 60℃, and retained 65 % activity after 60 min at 70℃. The half-life of the enzyme at 80℃was approximately 40 min. Optimum pH of the enzyme was 7.5. Optimum activity was obtained in the range of buffer from pH 6.5 to 9.0. Outside this range, activity was lost rapidly.The T. aurantiacus manganese superoxide dismutase was secreted into the culture medium by the yeast P. pastoris in an active form, and the recombinant Mn-SOD from T. aurantiacus was a thermostable enzyme. It implied the expressed Mn-SOD could be widely applied in the biological fields and chemical industry.