title: Purification, characterization, and specificity determination of a new serine protease secreted by Penicillium waksmanii.
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 CaCl(2), KCl, and BaCl, and partially inhibited by CuCl(2), CoCl(2) and totally inhibited by AlCl(3) 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.
authors.affiliation: Department of Pharmaceutical Sciences, Faculty of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto 14040-903, Brazil.
authors.forename: Eduardo Rezende
authors.initials: ER
authors.lastname: Graminho
authors.forename: Ronivaldo Rodrigues
authors.initials: RR
authors.lastname: da Silva
authors.forename: Tatiana Pereira
authors.initials: TP
authors.lastname: de Freitas Cabral
authors.forename: Eliane Candiani
authors.initials: EC
authors.lastname: Arantes
authors.forename: Nathalia Gonsales
authors.initials: NG
authors.lastname: da Rosa
authors.forename: Luiz
authors.initials: L
authors.lastname: Juliano
authors.forename: Debora Noma
authors.initials: DN
authors.lastname: Okamoto
authors.forename: Lilian Caroline Gonçalves
authors.initials: LC
authors.lastname: de Oliveira
authors.forename: Marcia Yuri
authors.initials: MY
authors.lastname: Kondo
authors.forename: Maria Aparecida
authors.initials: MA
authors.lastname: Juliano
authors.forename: Hamilton
authors.initials: H
authors.lastname: Cabral
mesh_terms: D000595:Amino Acid Sequence; D004795:Enzyme Stability; D005110:Extracellular Space / Q000737:chemistry / Q000201:enzymology / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D010407:Penicillium / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D021381:Protein Transport; D057057:Serine Proteases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D005656:Fungal Proteins; D057057:Serine Proteases