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Title: Purification and characterization of an extracellular trypsin-like protease of Fusarium oxysporum var. lin.
Authors: Barata, Ricardo Andrade
Andrade, Milton Hércules Guerra de
Rodrigues, Roberta Dias
Castro, Ieso de Miranda
Keywords: Trypsin-like protease
Inhibitor effects
Issue Date: 2002
Citation: BARATA, R. A. et al. Purification and characterization of an extracellular trypsin-like protease of Fusarium oxysporum var. lin. Journal of Bioscience and Bioengineering USA, v. 94, n. 4, p. 304-308, 2002. Disponível em: <>. Acesso: 10 jan. 2017.
Abstract: An alkaline serineprotease, capable of hydrolyzing Nu-benzoyl-DL arginine p-nitroanilide, was secreted by Fusurium oxysporum var. hi grown in the presence of gelatin as the sole nitrogen and carbon source. The protease was purified 65-fold to electrophoretic homogenity from the culture supernatant in a three-step procedure comprising QSepharose chromatography, aMnity chromatography, and FPLC on a MonoQ column. SDS-PAGE analysis of the purified protein indicated an estimated molecular mass of 41 kDa. The protease had optimum activity at a reaction temperature of 45OC and showed a rapid decrease of activity at 48OC. The optimum pH was around 8.0. Characterization of the protease showed that Ca*+ and MgZ+ cations increased the activity, which was not inhibited by EDTA or l,lO-phenanthroline. The enzyme activity on Nubenzoyl-DL arginine p-nitroanilide was inhibited by 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride,p-aminobenzamidine dihydrochloride, aprotinin, 3-4 dichloroisocoumarin, and IVtosyl-L-lysine chloromethyl ketone. The enzyme is also inhibited by substrate concentrations higher than 2.5x lo-4 M. The protease had a Michaelis-Menten constant of 0.16 mM and a V,, of 0.60 pm01 released product .min-‘.mg’ enzyme when assayed in a non-inhibiting substrate concentration. The activity on Nu-benzoyl-DL arginine p-nitroanilide was competitively inhibited by p-aminobenzamidine dihydrochoride. A Ki value of 0.04 mM was obtained.
ISSN: 1389-1723
metadata.dc.rights.license: O periódico Journal of Bioscience and Bioengineering concede permissão para depósito deste artigo no Repositório Institucional da UFOP. Número da licença: 3266010491564.
Appears in Collections:DEFAR - Artigos publicados em periódicos

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