Measurement of Microbial Protease Activity Using a pH-Stat Titration

The activity of purified and crude microbial proteases was measured by titration at pH 9 using an automatic pH-stat instrument. The ability of the pH-stat titration method and the trinitrobenzenesulfonic acid (TNBS) method to detect protease activity was compared. The pH-stat titration produced higher measurements of activity than the TNBS method when purified protease from Bacillus amyloliquefaciens was tested. Protease activity determination using the two methods resulted in a linear correlation of R² = 0.985 and the same repeatability (C.V.=2.6%). The pH-stat titration method was more sensitive than the TNBS method in measuring activity of the purified protease, and it indicated greater proteolytic activity than the TNBS method when culture filtrates from five Pseudomonas spp. which produced proteolytic enzyme with greater activity at pH 9 than pH 7.5 were tested. When culture filtrates from four Pseudomonas spp. with similar but low proteolytic activity at pH 9 and 7.5 were tested, the two methods produced similar results. For proteases with optimum activity at or above pH 9, the pH-stat titration method was simplier, faster, and more sensitive than the TNBS method in determining activity.