Development of a peptide substrate for detection of sunn pest damage in wheat flour

Abstract

BACKGROUND: Since the common protease substrates did not give satisfactory results for the determination of Sunn pestprotease activity in damaged wheat, different peptide substrates derived from the repeated sequences of high molecular weightglutenin subunits were synthesized.RESULTS: Hydrolysis of peptides by pest protease was determined by high-performance liquid chromatography. Among threepeptides having the same consensus motifs, peptide1 (PGQGQQGYYPTSPQQ) showed the best catalytic efficiency. A novel assaywas described for monitoring the enzymatic activity of protease extracted from damaged wheat flour. The selected peptide waslabeled with a fluorophore (EDANS) and quencher (Dabcyl) to display fluorescence resonance energy transfer. The proteolyticactivity 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 weresuspended in water and mixed with fluorescence peptide substrate. After centrifugation, the fluorescence intensities of thesupernatants, which are proportional to the protease content of the flour, were determined.CONCLUSION: The total analysis time for the assay developed is estimated as 15 min. The assay developed permits a significantdecrease in time and labor, offering sensitive detection of Sunn pest damage in wheat flour.