Cloning, Expression And Characterization Of Manganese 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 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).Thermomyces lanuginosus is a widely distributed soil-inhibiting fungus of considerable interest producer of thermostable enzymes, including protease, chitinase and endocellulase which have been purified and well studied. During growth in a medium containing Casein, T. lanuginosus also produces superoxide dismutase.In this study, a Mn-SOD gene mnsod was isolated for the fist time from T. lanuginosus using RACE method. The full length of mnsod cDNA gene is 791bp, which contained an ORF of 654bp encoding 217 amino acids. The nucleotide sequence has been deposited in the GenBank database under accession number EU364837.The mnsod gene and expression vector pPIC9K were digested with EcoR I and Not I, then ligationed in vitro to construct the expression plasmid pPIC9K/mnsod. The pPIC9K/mnsod was transformed to Pichia pastoris GS115 competent cell . The engineering strain with expression level, named as MS18, were tested and characterized.Induced with methanol, the supernatant was concentrated by ammonium sulfate precipitation. The precipitated proteins were collected by centrifugation and purified using gel filtration. SDS-PAGE analyse showed a 27 kDa single band of Mn-SOD. The recombinant Mn-SOD exhibited optimal activity at 55℃and pH8.0. It's stable at 50℃and between pH7.0～9.0.