Is there a simple and specific method for determination of fungal and bacterial proteases acting on peptide bond at tyrosine and asparitic acid residues respectively
when trying to estimate its activity using Folin method but the reading of bot test and control are the same and the activity could be seen in culture broth or solid media in form of inhibition zone if media with skimmed milk which confirm the presence of proteases in both
Thanks for your consideration
Getting you right, you are interested to measure specific proteases acting in the direct neighborhood of tyrosin or aspartic acid residues in a screening of fungi and bacteria species or isolates? My suggestion is working first on agar plates supplemented with denatured protein (e.g. casein or ovalbumin or the like). Proteolytic activity is evident by clear zones surround colonies on the plates. In the next step you can inocculate liquid medium with the test protein and try to identify peptides by mass spectrometry. Likewise, medium concentarates can be applied in zymography experiments or you can extract beptides from the clear zones of agarplates directly. MS helps to identify specificities in these cases, too.
Thanks for answers but I need a spectrophotometric method in the form enzyme assay
Hi Donia
If you assume that your expected protease might be endopeptidases then you can -X-X-D-MCA and -X-X-Y-MCA fluorescent synthetic peptide where X is any amino acid. If the proteases are dipeptidyl peptidases (DPP) then you can measure using X-D-MCA or X-Y-MCA dipeptide. The secreted proteases will cleave the AA-MCA bond and the fluorescent intensity will raised when compared with the control medium!
Download protocol for protease assay. Use substrate casein at 0.65% w/v. in some neutral buffer system. Prepare reaction mixture with cell free supenatant and substrate. incubate for 15 min.Stop the reaction with
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