AbstractBACKGROUNDSince the common protease substrates did not give satisfactory results for the determination of Sunn pest protease activity in damaged wheat, different peptide substrates derived from the repeated sequences of high molecular weight glutenin subunits were synthesized.RESULTSHydrolysis of peptides by pest protease was determined by high‐performance liquid chromatography. Among three peptides having the same consensus motifs, peptide1 (PGQGQQGYYPTSPQQ) showed the best catalytic efficiency. A novel assay was described for monitoring the enzymatic activity of protease extracted from damaged wheat flour. The selected peptide was labeled with a fluorophore (EDANS) and quencher (Dabcyl) to display fluorescence resonance energy transfer. The proteolytic activity was measured by the change in fluorescence intensity that occurred when the protease cleaved the peptide substrate. Furthermore, the assay developed was modified for rapid and easy detection of bug damage in flour. Flour samples were suspended in water and mixed with fluorescence peptide substrate. After centrifugation, the fluorescence intensities of the supernatants, which are proportional to the protease content of the flour, were determined.CONCLUSIONThe total analysis time for the assay developed is estimated as 15 min. The assay developed permits a significant decrease in time and labor, offering sensitive detection of Sunn pest damage in wheat flour. © 2018 Society of Chemical Industry