Journal of Microbiology

Duo-Chuan Li

2 Articles

Cloning, Expression, and Characterization of Serine Protease from Thermophilic Fungus Thermoascus aurantiacus var. levisporus: An-Na Li , Chen Xie , Jie Zhang , Jia Zhang , Duo-Chuan Li; J. Microbiol. 2011;49(1):121-129. Published online March 3, 2011; DOI: https://doi.org/10.1007/s12275-011-9355-6

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The serine protease gene from a thermophilic fungus Thermoascus aurantiacus var. levisporus, was cloned, sequenced, and expressed in Pichia pastoris and the recombinant protein was characterized. The full-length cDNA of 2,592 bp contains an ORF of 1,482 bp encoding 494 amino acids. Sequence analysis of the deduced amino acid sequence revealed high homology with subtilisin serine proteases. The putative enzyme contained catalytic domain with active sites formed by three residues of Asp183, His215, and Ser384. The molecular mass of the recombinant enzyme was estimated to be 59.1 kDa after overexpression in P. pastoris. The activity of recombinant protein was 115.58 U/mg. The protease exhibited its maximal activity at 50°C and pH 8.0 and kept thermostable at 60°C, and retained 60% activity after 60 min at 70°C. The protease activity was found to be inhibited by PMSF, but not by DTT or EDTA. The enzyme has broad substrate specificity such as gelatin, casein and pure milk, and exhibiting highest activity towards casein.

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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

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Abstract PDF

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.

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