Characterization of Extracellular Proteases of Aeromonas hydrophila

Abstract

Two endoproteases, proteinase-I and proteinase-II, and one aminopeptidase were isolated from the culture supernatant of Aeromonas hydrophila by ammonium sulfate precipitation, gradient elution on diethylaminoethyl-Sephadex A-50 and Sephadex G-75 column chromatography. The molecular weights of the proteinases and aminopeptidase ranged between 30, 000 to 48, 000. The proteinases had comparable pH (8.5) and temperature (48-50°C) optima. The aminopeptidase assayed using L-leucine-p-nitroanilide showed maximum activity at pH 8.0 and at a temperature of 70°C. The aminopeptidase was remarkably heat stable, while the proteinases were heat sensitive. Both the proteinases hydrolysed several proteins including fish myofibrillar proteins. Divalent cations like Fe²⁺, Cu²⁺ and Zn²⁺ inhibited the activities of the two proteinases while Ca²⁺ and Mg²⁺ did not affect either the activities of the proteinases or that of the aminopeptidase. The aminopeptidase was inhibited by metal chelating agents as well as thiol reagents.