We evaluated an assay method of proteolytic activity applicable to both commercially available microbial protease and papain, which are used as enzyme preparations for food in Japan.
The optimal pH values for the reaction with casein as a substrate for 10 minutes were 3, 6, or 8 for fungal protease, 7, 8, or 10 for bacterial protease, and 7 or 8 for papain. Therefore, 0.6% casein solution at the optimal pH for each enzyme was used as the substrate. The appropriate reaction temperature was 37°C for each enzyme. As a diluent, 0.01M sodium chloride solution containing 0.002M calcium acetate and 0.002M calcium sulfate prevented decreases in enzyme activity. As a reagent for protein precipitation, 0.11M trichloroacetic acid containing 0.22M sodium acetate and 0.33M acetic acid was appropriate for all the enzyme sample preparations.
The enzyme activities of 35 microbial proteases from 13 companies measured by this method were in the range of 200-1, 132, 100U/g, and those of 3 papain preparations from 3 companies were in the range of 13, 500-167, 400U/g.