Purification and characterization of an extracellular trypsin-like protease of Fusarium oxysporum var. lin.

• Autor(a) principal: Barata, Ricardo Andrade
• Data de Publicação: 2002
• Outros Autores: Andrade, Milton Hércules Guerra de, Rodrigues, Roberta Dias, Castro, Ieso de Miranda
• Tipo de documento: Artigo
• Idioma: eng
• Título da fonte: Repositório Institucional da UFOP
• Texto Completo: http://www.repositorio.ufop.br/handle/123456789/8915; http://www.sciencedirect.com/science/article/pii/S1389172302801682; https://doi.org/10.1016/S1389-1723(02)80168-2
• topic: Trypsin-like protease; Fusarium; Inhibitor effects

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.