A GFP-based assay for the determination of hydrolytic activity and substrate specificity of subtilisins under washing conditions

The serine protease subtilisin is one of the most important industrial enzymes and is mostly used as a detergent protease. The development of new proteases with improved washing performance is limited by the tedious screening of variants on stained cloths. Here, an easy and flexible green fluorescence protein (GFP) based protease assay is described, which is performed under almost authentic washing conditions. A GFP-substrate–His-tag fusion protein is hydrolyzed by subtilisin and the activity is followed by measurement of fluorescence values from the GFP released. Two detergent proteases variants (Bacillus lentus alkaline protease, BLAP P and BLAP X) were investigated using three model substrates (AAAAPF, AAFAAF and KHDRKD). As expected from the known substrate specificities of these proteases, AAAAPF and AAFAAF were hydrolyzed rapidly, whereas KHDRKD represents a rather stable peptide. The kcat/KM-values determined using this GFP-based assay closely matched the values using p-nitroanilides (pNA) of model peptides. In contrast to the use of pNA-substrates, this format is not restricted to synthetically available substrates and therefore in principle allows the screening of random peptide libraries to determine the substrate specificities of subtilisins and its variants.