Cloning, expression, and characterization of thermostable manganese superoxide dismutase from Thermoascus aurantiacus var. levisporus.
A superoxide dismutase (SOD) gene of Thermoascus aurantiacus var. levisporus, a thermophilic fungus, was cloned, sequenced, and expressed in Pichia pastoris and its gene product was characterized. The coding sequence predicted a 231 residues protein with a unique 35 amino acids extension at the N-terminus indicating a mitochondrial-targeting sequence. The content of Mn was 2.46 microg/mg of protein and Fe was not detected in the purified enzyme. The enzyme was found to be inhibited by NaN(3), but not by KCN or H(2)O(2). These results suggested that the SOD in Thermoascus aurantiacus var. levisporus was the manganese superoxide dismutase type. In comparison with other MnSODs, all manganese-binding sites were also conserved in the sequence (H88, H136, D222, H226). The molecular mass of a single band of the enzyme was estimated to be 21.7 kDa. The protein was expressed in tetramer form with molecular weight of 68.0 kDa. The activity of purified protein was 2,324 U/mg. The optimum temperature of the enzyme was 55 degrees C and it exhibited maximal activity at pH 7.5. The enzyme was thermostable at 50 and 60 degrees C and the half-life at 80 degrees C was approximately 40 min.
Song
Ning-Ning
NN
Department of Environmental Biology, Shandong Agricultural University, Tai'an, Shandong 271018, PR China.
Zheng
Yan
Y
E
Shi-Jin
SJ
Li
Duo-Chuan
DC
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005656:Fungal Proteins / Q000502:physiology*; D006861:Hydrogen Peroxide / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000737:chemistry; D008345:Manganese / Q000737:chemistry; D008969:Molecular Sequence Data; D016877:Oxidants / Q000494:pharmacology; D011190:Potassium Cyanide / Q000494:pharmacology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid; D019810:Sodium Azide / Q000494:pharmacology; D013054:Spectrophotometry, Atomic; D013482:Superoxide Dismutase / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D013696:Temperature; D055327:Thermoascus / Q000201:enzymology* / Q000235:genetics
D004791:Enzyme Inhibitors; D005656:Fungal Proteins; D016877:Oxidants; D011994:Recombinant Proteins; D008345:Manganese; D019810:Sodium Azide; D006861:Hydrogen Peroxide; D007501:Iron; D013482:Superoxide Dismutase; D011190:Potassium Cyanide