Mitochondrial malate dehydrogenase from the thermophilic, filamentous fungus <i>Talaromyces emersonii</i>

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  • Purification of the native enzyme, cloning and overexpression of the corresponding gene

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<jats:p>Mitochondrial malate dehydrogenase (m‐MDH; EC 1.1.1.37), from mycelial extracts of the thermophilic, aerobic fungus <jats:italic>Talaromyces emersonii</jats:italic>, was purified to homogeneity by sequential hydrophobic interaction and biospecific affinity chromatography steps. Native m‐MDH was a dimer with an apparent monomer mass of 35 kDa and was most active at pH 7.5 and 52 °C in the oxaloacetate reductase direction. Substrate specificity and kinetic studies demonstrated the strict specificity of this enzyme, and its closer similarity to vertebrate m‐MDHs than homologs from invertebrate or mesophilic fungal sources. The full‐length m‐MDH gene and its corresponding cDNA were cloned using degenerate primers derived from the N‐terminal amino acid sequence of the native protein and multiple sequence alignments from conserved regions of other m‐MDH genes. The m‐MDH gene is the first oxidoreductase gene cloned from <jats:italic>T. emersonii</jats:italic> and is the first full‐length m‐MDH gene isolated from a filamentous fungal species and a thermophilic eukaryote. Recombinant m‐MDH was expressed in <jats:italic>Escherichia coli</jats:italic>, as a His‐tagged protein and was purified to apparent homogeneity by metal chelate chromatography on an Ni<jats:sup>2+</jats:sup>‐nitrilotriacetic acid matrix, at a yield of 250 mg pure protein per liter of culture. The recombinant enzyme behaved as a dimer under nondenaturing conditions. Expression of the recombinant protein was confirmed by Western blot analysis using an antibody against the His‐tag. Thermal stability studies were performed with the recombinant protein to investigate if results were consistent with those obtained for the native enzyme.</jats:p>

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