title: Purification and characterization of an extracellular trypsin-like protease of Fusarium oxysporum var. lini.
abstract: An alkaline serineprotease, capable of hydrolyzing Nalpha-benzoyl- dl arginine p-nitroanilide, was secreted by Fusarium oxysporum var. lini 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, affinity 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 45 degrees C and showed a rapid decrease of activity at 48 degrees C. The optimum pH was around 8.0. Characterization of the protease showed that Ca2+ and Mg2+ cations increased the activity, which was not inhibited by EDTA or 1,10-phenanthroline. The enzyme activity on Nalpha-benzoyl-DL arginine p-nitroanilide was inhibited by 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride, p-aminobenzamidine dihydrochloride, aprotinin, 3-4 dichloroisocoumarin, and N-tosyl-L-lysine chloromethyl ketone. The enzyme is also inhibited by substrate concentrations higher than 2.5 x 10(-4)M. The protease had a Michaelis-Menten constant of 0.16 mM and a V(max) of 0.60 mumol released product.min(-1).mg(-1) enzyme when assayed in a non-inhibiting substrate concentration. The activity on Nalpha-benzoyl- dl arginine p-nitroanilide was competitively inhibited by p-aminobenzamidine dihydrochoride. A K(i) value of 0.04 mM was obtained.
authors.affiliation: Laboratório de Bioquímica e Fisiologia de Microorganismos, Núcleo de Pesquisa em Ciências Biológicas, Universidade Federal de Ouro Preto, CEP 35.400.000, Ouro Preto, MG, Brazil.
authors.forename: Ricardo Andrade
authors.initials: RA
authors.lastname: Barata
authors.forename: Milton Hercules Guerra
authors.initials: MH
authors.lastname: Andrade
authors.forename: Roberta Dias
authors.initials: RD
authors.lastname: Rodrigues
authors.forename: Ieso Miranda
authors.initials: IM
authors.lastname: Castro