Journal of Microbiology

Research Support, Non-U.S. Gov't

Cloning, Expression, and Characterization of Thermostable Manganese Superoxide Dismutase from Thermoascus aurantiacus var. levisporus: Ning-Ning Song , Yan Zheng , Shi-Jin E , Duo-Chuan Li; J. Microbiol. 2009;47(1):123-130. Published online February 20, 2009; DOI: https://doi.org/10.1007/s12275-008-0217-9

Abstract: 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 ug/mg of protein and Fe was not detected in the purified enzyme. The enzyme was found to be inhibited by NaN3, but not by KCN or H2O2. 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 55oC and it exhibited maximal activity at pH 7.5. The enzyme was thermostable at 50 and 60oC and the half-life at 80oC was approximately 40 min.

Molecular Cloning and Characterization of Mn-Superoxide Dismutase Gene from Candida sp.: Hong, Yun Mi , Nam, Yong Sik , Choi, Soon Yong; J. Microbiol. 1997;35(4):309-314.

Abstract: The manganese-containing superoxide dismutase (MnSOD) is a major component of the cellular defence mechanisms against the toxic effects of the superoxide radical. Within the framework of studies on oxidative stress=responsible enzymes in the Candida sp., the gene encoding the MnSOD was isolated and examined in this study. A specific primer was designed based on conserved regions of MnSOD sequences from other organisms, and was used to isolate the gene by PCR on reverse-transcribed Candida poly(A^+) RNA. The PCR product was used to screen a Candida genomic lambda library and the nucleotide wequence of positive clone was determined. The deduced primary sequence encodes a 25kDa protein which has the conserved residues for enzyme activity and metal binding. The 28 N-terminal amino acids encoded by the Candida cDNA comprise a putatice mitochondrial transit peptide. Potential regulatory elements were identified in the 5' flanking sequences. Northern blot analysis showed that the transcription of the MnSOD gene is induced 5-to 10-fold in response to mercury, cadmium ions and hydrogen peroxide.

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