Publicação: Purification, characterization, and specificity determination of a new serine protease secreted by penicillium waksmanii
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The purpose of this work was to purify a protease from Penicillium waksmanii and to determine its biochemical characteristics and specificity. The extracellular protease isolated that was produced by P. waksmanii is a serine protease that is essential for the reproduction and growth of the fungus. The protease isolated showed 32 kDa, and has optimal activity at pH 8.0 and 35 C towards the substrate Abz-KLRSSKQ-EDDnp. The protease is active in the presence of CaCl2, KCl, and BaCl, and partially inhibited by CuCl2, CoCl2 and totally inhibited by AlCl3 and LiCl. In the presence of 1 M urea, the protease remains 50 % active. The activity of the protease increases 60 % when it is exposed to 0.4 % nonionic surfactant-Triton X-100 and loses 10 % activity in the presence of 0.4 % Tween-80. Using fluorescence resonance energy transfer analysis, the protease showed the most specificity for the peptide Abz-KIRSSKQ-EDDnp with k cat/K m of 10,666 mM-1 s-1, followed by the peptide Abz-GLRSSKQ-EDDnp with a k cat/K m of 7,500 mM -1 s-1. Basic and acidic side chain-containing amino acids performed best at subsite S1. Subsites S2, S3, S′ 2, and S′ 1, S ′ 3 showed a preference for binding for amino acids with hydrophobic and basic amino acid side chain, respectively. High values of k cat/K m were observed for the subsites S2, S3, and S′ 2. The sequence of the N-terminus (ANVVQSNVPSWGLARLSSKKTGTTDYTYD) showed high similarity to the fungi Penicillium citrinum and Penicillium chrysogenum, with 89 % of identity at the amino acid level. © 2012 Springer Science+Business Media New York.
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Fungal enzymes, N-terminal Penicillium, Protease, Purification, Specificity, Biochemical characteristics, Extracellular protease, Fluorescence resonance energy transfer analysis, N-terminals, Nonionic, Optimal activity, Penicillium chrysogenum, Penicillium citrinum, Serine protease, Side-chains, Subsites, Tween 80, Calcium chloride, Copper compounds, Energy transfer, Enzyme activity, Nonionic surfactants, Peptides, Urea, Amino acids, aluminum chloride, barium chloride, calcium chloride, cobalt chloride, copper chloride, fungal enzyme, polysorbate 80, potassium chloride, serine proteinase, triton x 100, urea, amino acid sequence, amino terminal sequence, binding site, controlled study, enzyme activity, enzyme analysis, enzyme inhibition, enzyme kinetics, enzyme purification, enzyme release, enzyme specificity, enzyme substrate, enzyme synthesis, fluorescence resonance energy transfer, fungal reproduction, fungus growth, nonhuman, Penicillium, Penicillium waksmanii, pH, protein cleavage, protein function, species difference, Amino Acid Sequence, Enzyme Stability, Extracellular Space, Fungal Proteins, Kinetics, Molecular Sequence Data, Protein Transport, Serine Proteases, Substrate Specificity, Fungi
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Inglês
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Applied Biochemistry and Biotechnology, v. 169, n. 1, p. 201-214, 2013.
