A rapid quantitative assay for bacterial protease activity



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A rapid quantitative stained hide powder assay was studied as a possible replacement for the gelatin film test, used to detect bacterial enzymes within hide juice of delayed cured hides. The ability of the method was investigated by employing different kinds of substrates and enzymes. Substrates from dogchew and rawhide were hydrolyzed by Proleather FG-F, Protex 6L and Alcalase enzyme under optimum conditions. Reproducibility of the dogchew substrate was higher but hydrolysis efficiency was less than for the rawhide substrate. To improve reproducibility and hydrolysis efficiency, a stable, neutralized stained hide powder was produced and served as a substrate for calibration. Analytical figures of merit are described. The relations between the efficiency of hydrolysis and enzyme concentration or enzyme activity were linear. The effect of salt shifted the calibration toward lower hydrolysis efficiency. The slopes did not change. The method could detect the Proleather FG-F enzyme concentration as low as 0.01 ìg/ml or enzyme activity of 1.01x10-3 LVU/ml without salt and 0.05 ìg/ml or enzyme activity of 5.05x10-3 LVU/ml with salt.