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Purification, Characterization, and Specificity Determination of a New Serine Protease Secreted by Penicillium waksmanii

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Abstract

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.

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Acknowledgments

This work was supported by grants from Brazilian National Council for Research and Development (CNPq) and Sao Paulo Research Foundation (FAPESP) Sao Paulo, Brazil. All authors have agreed to submit this manuscript to the “Applied Biochemistry and Biotechnology.”

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Authors and Affiliations

  1. Department of Pharmaceutical Sciences, Faculty of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, 14040-903, Brazil

    Eduardo Rezende Graminho, Tatiana Pereira de Freitas Cabral, Eliane Candiani Arantes, Nathalia Gonsales da Rosa & Hamilton Cabral

  2. Institute of Biosciences, Letters and Exact Sciences, Universidade Estadual Paulista, São José do Rio Preto, 15054-000, Brazil

    Ronivaldo Rodrigues da Silva

  3. Department of Biophysics, Universidade Federal de São Paulo, São Paulo, 04023-900, Brazil

    Luiz Juliano, Debora Noma Okamoto, Lilian Caroline Gonçalves de Oliveira, Marcia Yuri Kondo & Maria Aparecida Juliano

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  1. Eduardo Rezende Graminho
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  2. Ronivaldo Rodrigues da Silva
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Correspondence to Hamilton Cabral.

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Graminho, E.R., da Silva, R.R., de Freitas Cabral, T.P. et al. Purification, Characterization, and Specificity Determination of a New Serine Protease Secreted by Penicillium waksmanii . Appl Biochem Biotechnol 169, 201–214 (2013). https://doi.org/10.1007/s12010-012-9974-3

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