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

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

Author Graminho, Eduardo Rezende Google Scholar
Silva, Ronivaldo Rodrigues da Google Scholar
Freitas Cabral, Tatiana Pereira de Google Scholar
Arantes, Eliane Candiani Google Scholar
Rosa, Nathalia Gonsales da Google Scholar
Juliano, Luiz Autor UNIFESP Google Scholar
Okamoto, Debora Noma Autor UNIFESP Google Scholar
Goncalves de Oliveira, Lilian Caroline Autor UNIFESP Google Scholar
Kondo, Marcia Yuri Autor UNIFESP Google Scholar
Juliano, Maria Aparecida Autor UNIFESP Google Scholar
Cabral, Hamilton Google Scholar
Institution Universidade de São Paulo (USP)
Univ Estadual Paulista
Universidade Federal de São Paulo (UNIFESP)
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 A degrees 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 S-1. Subsites S-2, S-3, 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 S-2, S-3, 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.
Keywords Protease
Fungal enzymes
Purification
Specificity
N-terminal Penicillium
Language English
Sponsor Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Date 2013-01-01
Published in Applied Biochemistry and Biotechnology. Totowa: Humana Press Inc, v. 169, n. 1, p. 201-214, 2013.
ISSN 0273-2289 (Sherpa/Romeo, impact factor)
Publisher Humana Press Inc
Extent 201-214
Origin http://dx.doi.org/10.1007/s12010-012-9974-3
Access rights Closed access
Type Article
Web of Science ID WOS:000314023100018
URI http://repositorio.unifesp.br/handle/11600/35787

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