Development of a peptide substrate for detection of sunn pest damage in wheat flour
Hançerlioğulları, Begüm Zeynep
Dudak, Fahriye Ceyda
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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.