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Purification and some properties of an extracellular acid protease from Aspergillus clavatus

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Abstract

Acid proteases represent an important group of enzymes, widely used in food, beverage and pharmaceutical industries. For most of these applications the enzymatic preparation must be at least partially purified and free of substances that could change the characteristics of the product or the process. Fungal proteases have replaced other sources because they are easily obtained mainly from Mucor, Rhizopus, Penicillium and Aspergillus species. A strain of Aspergillus clavatus was selected by producing high level of acid protease activity. An extracellular aspartatic protease from this strain was purified 37.2 times with 37% recovery using (NH4)2SO4 fractionation and ion-exchange chromatography. The enzyme was found to be monomeric having a molecular mass of 30.4 kDa. The purified enzyme is an acid protease with optimum pH of 5.5 and temperature for optimum activity of 50 °C. Its high pH stability was verified in the range of 3.5–6.5. The acid protease was strongly inhibited by Hg+2 and partially inhibited by Cu+2, Zn+2 and Mn+2. The enzyme was sensitive to denaturing agent SDS and activated by thiol-containing reducing agent dithiotreitol (DTT). The protease activity was not influenced by iodoacetic acid, E-64 and PMSF, while it was lightly actived by EDTA and totally inhibited by pepstatin, with a Ki of 7.8 μM, indicating that is an aspartic protease. A. clavatus acid protease presents interesting characteristics for biotechnological process, such as cheese and flavor manufacture and dietary supplements, in which activity and stability in acid pH are required.

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Acknowledgments

This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP). We thank Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and FAPESP for the scholarships awarded to the first and third authors, respectively.

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Authors and Affiliations

  1. Departament of Botany, Federal University of São Carlos, UFSCar. Rodov. Washington Luis km 235, São Carlos, São Paulo State, 13565-905, Brazil

    Talita A. Sampaio e Silva

  2. Departament of Biology, State University Midwestern, UNICENTRO. Camargo Varela de Sá Street, 03, Guarapuava, Paraná State, 85040-080, Brazil

    Adriana Knob

  3. Phytopatology Laboratory, Embrapa Amazônia Oriental, Trav. Dr. Enéas Pinheiro s/n, Belém, Pará State, 66095-100, Brazil

    Célia R. Tremacoldi

  4. Department of Biology, São Paulo State University, UNESP. 24-A Avenue, 1515, Rio Claro, São Paulo State, 13506-900, Brazil

    Marcia R. Brochetto-Braga

  5. Department of Biochemistry and Microbiology, São Paulo State University, UNESP. 24-A Avenue, 1515, Rio Claro, São Paulo State, CEP 13506-900, Brazil

    Eleonora Cano Carmona

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  1. Talita A. Sampaio e Silva
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  2. Adriana Knob
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  3. Célia R. Tremacoldi
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  4. Marcia R. Brochetto-Braga
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  5. Eleonora Cano Carmona
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Correspondence to Eleonora Cano Carmona.

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Sampaio e Silva, T.A., Knob, A., Tremacoldi, C.R. et al. Purification and some properties of an extracellular acid protease from Aspergillus clavatus . World J Microbiol Biotechnol 27, 2491–2497 (2011). https://doi.org/10.1007/s11274-011-0717-3

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  • DOI: https://doi.org/10.1007/s11274-011-0717-3

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