Several individual amino acids served as the sole nitrogen source for growth and extracellular protease synthesis by cells of Aeromonas hydrophila. In the presence of urea, ammonium compounds and glycine, enzyme production was severely repressed although cell growth was only moderately affected.
Twoendoproteases, proteinase-I and proteinase-II, and one aminopeptidase were isolated from the culture supernatant of Aeromonashydrophila 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-/?-nitroanilide showed maximumactivity 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.
Butylated hydroxyanisole (BHA), propylhydroxy parabenzoate (paraben), and sodium tripolyphosphate (TPP) were found to inhibit protease secretion by resting cells of Aeromonas hydrophila at lower concentrations than those required for inhibiting growth. Incorporation of the above compounds, in calcium caseinate agar resulted in colonies surrounded by smaller areas of clear zones indicating inhibition of protease secretion. The results are discussed with respect to the protective influence of the above compounds against spoilage of flesh foods by microbial proteases.
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