Rapid colorimetric determination of activity of subtilisin enzymes in cleaning products

A new colorimetric method is described for the determination of enzymatic activity of subtilisin in cleaning products. The procedure is more rapid and precise than the casein digestion methods commonly used to assay protease activity. The principle of the colorimetric method depends on the determination rate of p-nitrophenol released on hydrolysis of N-CBZ-L-leucine-p-nitrophenyl ester at pH 8.0 by subtilisin, with correction for any nonenzymatic (spontaneous) hydrolysis of the substrate. Because of the broad range of hydrolytic activity of this enzyme, and the difficulties in predicting its proteolytic activity, this hydrolytic rate was chosen as a general indicator of subtilisin enzyme behavior. The slope for 7 replicate standard curves generated over a 6 week period exhibited a relative standard deviation of 7.5%, and 8.0% for 20 replicates with an enzyme cleaning product. Papain does not interfere with this assay.