Uncovering Novel DPP-IV Inhibitory Peptides from Amphibian (Lithobates catesbeiana) Skin via Peptidomics and Molecular Simulation

As an emerging natural source of DPP-IV inhibition strategy, we report for the first time the use of <i>Lithobates catesbeianus</i> skin gelatin (LSG) as a novel source for DPP-IV inhibitory peptides in this study. Through enzymatic hydrolysis with multiple proteases, the papain-treated hydrolysate exhibited superior performance in hydrolysis degree, protein recovery, and DPP-IV inhibition, with 93.47% of peptides under 1 kDa. Subsequent separation and peptidomics analysis identified 13 previously unreported peptides. Molecular docking and <i>in silico</i> screening pinpointed four candidate peptides, i.e., LGPQR, RGFDQ, RGPVGP, and RLDDVT, which were then synthesized and functionally validated. Enzyme kinetic studies revealed that these peptides acted via competitive or mixed-type inhibition mechanisms. Notably, this study uncovered the bio-functional potential of amphibian-derived gelatin and provided a new strategy for natural DPP-IV inhibitor discovery through integrated enzymatic, computational, and biochemical approaches. This work pioneered the use of amphibian skin gelatin in antidiabetic peptide discovery and laid the foundation for its application in functional foods.