RESUMO
An extracellular lethal toxin produced by Aeromonas hydrophila strain RT860715K originally isolated from diseased rainbow trout (Oncorhynchus mykiss) was purified by using Fast Protein Liquid Chromatography system with hydrophobic interaction chromatography and anion exchange columns. The toxin was a cysteine protease, inhibited by L-cysteine, iodoacetic acid, N-ethylamleimide, P-chloromercuibenzene-sulfonic acid and N-α-p-tosyl-1-lysine-chloromethyl ketone (TLCK), and showed maximal activity at pH 6.0. The molecular weight of the purified enzyme proved to be 94 kDa as estimated by SDS-PAGE. In addition, the toxin was also completely inhibited by HgCl2 but partially inhibited by ethylenediamine tetraacetic acid (EDTA) and CuCl2. Both the extracellular products of Aeromonas hydrophila RT860715K and the purified protease were lethal to rainbow trout (weighing 18 g) with LD50 values of 2.87 and 0.93 µg protein g-1 fish body weight, respectively. The addition of L-cysteine completely inhibited the lethal toxicity of the purified protease, indicating that this cysteine protease was a lethal toxin produced by the bacterium.
Assuntos
Aeromonas hydrophila/enzimologia , Toxinas Bacterianas/isolamento & purificação , Cisteína Proteases/isolamento & purificação , Doenças dos Peixes/microbiologia , Infecções por Bactérias Gram-Negativas/veterinária , Oncorhynchus mykiss , Aeromonas hydrophila/metabolismo , Animais , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/toxicidade , Cromatografia em Agarose/veterinária , Cromatografia por Troca Iônica/veterinária , Cisteína Proteases/metabolismo , Cisteína Proteases/toxicidade , Eletroforese em Gel de Poliacrilamida , Inibidores Enzimáticos/farmacologia , Infecções por Bactérias Gram-Negativas/microbiologia , Concentração de Íons de Hidrogênio , Dose Letal Mediana , Peso MolecularRESUMO
An extracellular lethal toxin produced by Aeromonas hydrophila strain RT860715K originally isolated from diseased rainbow trout (Oncorhynchus mykiss) was purified by using Fast Protein Liquid Chromatography system with hydrophobic interaction chromatography and anion exchange columns. The toxin was a cysteine protease, inhibited by L -cysteine, iodoacetic acid, N -ethylamleimide, P-chloromercuibenzene-sulfonic acid and N-α-p-tosyl-1-lysine-chloromethyl ketone (TLCK), and showed maximal activity at pH 6.0. The molecular weight of the purified enzyme proved to be 94 kDa as estimated by SDS-PAGE. In addition, the toxin was also completely inhibited by HgCl(2) but partially inhibited by ethylenediamine tetraacetic acid (EDTA) and CuCl2. Both the extracellular products of Aeromonas hydrophila RT860715K and the purified protease were lethal to rainbow trout (weighing 18 g) with LD50 values of 2.87 and 0.93 µg protein g⻹ fish body weight, respectively. The addition of L-cysteine completely inhibited the lethal toxicity of the purified protease, indicating that this cysteine protease was a lethal toxin produced by the bacterium.