Purification,Characterization, and Molecular Cloning of a Thermostable Superoxide Dismutase from Thermoascus aurantiacus

A thermostable superoxide dismutase [(SOD) EC 1.15.1.1] from a Thermoascus aurantiacus var. levisporus was purified to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) homogeneity by a series of column chromatographies. The molecular mass of a single band of the enzyme was estimated to be 16.8 kDa by SDS–PAGE. The molecular mass was estimated to be 33.2 kDa by gel filtration on Sephacryl S-100, indicating that the enzyme was composed of two identical subunits of 16.8 kDa each. N-terminal amino acid sequencing (seven residues) yielded VKAVAVL. Using RACE-PCR, a Cu, Zn-SOD gene was cloned from T. aurantiacus var. levisporus. The sequence was 705 bp and contained a 468 bp ORF encoding a Cu, Zn-SOD of 155 amino acid residues.