Purification and Characterization of an Extracellular Trypsin-Like Protease of Fusarium oxysporum var. lini

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概要

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• An alkaline serineprotease, capable of hydrolyzing N★-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★ and showed a rapid decrease of activity at 48★. The optimum pH was around 8.0. Characterization of the protease showed that Ca^<2+> and Mg^<2+> cations increased the activity, which was not inhibited by EDTA or 1,10-phenanthroline. The enzyme activity on N★-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 chioromethyl ketone. The enzyme is also inhibited by substrate concentrations higher than 2.5★10^<-4> M. The protease had a Michaelis-Menten constant of 0.16 mM and a V_<max> of 0.60 ★mol released product★min^<-1> mg^<-1> enzyme when assayed in a non-inhibiting substrate concentration. The activity on N★-benzoyl-DL arginine p-nitroanilide was competitively inhibited by p-aminobenzamidine dihydrochoride. A K_i value of 0.04 mM was obtained.

• 公益社団法人日本生物工学会の論文
• 2002-10-25

著者

• Barata Ricardo

  Laboratorio De Bioquimica E Fisiologia De Microorganismos Nucleo De Pesquisa Em Ciencias Biologicas

• Rodrigues Roberta

  Laboratorio De Bioquimica E Fisiologia De Microorganismos Nucleo De Pesquisa Em Ciencias Biologicas

• ANDRADE MILTON

  de Enzimologia e Planejamento de Fafrmacos, Nucleo de Pesquisa em Ciencias Bioldgicas, Universidade

• CASTRO IESO

  Laboratorio de Bioquimica e Fisiologia de Microorganismos, Nucleo de Pesquisa em Ciencias Biologicas

• Andrade Milton

  De Enzimologia E Planejamento De Fafrmacos Nucleo De Pesquisa Em Ciencias Bioldgicas Universidade Fe

• Castro Ieso

  Laboratorio De Bioquimica E Fisiologia De Microorganismos Nucleo De Pesquisa Em Ciencias Biologicas

関連論文

• Purification and Characterization of an Extracellular Trypsin-Like Protease of Fusarium oxysporum var. lini

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