Novel Dipeptidyl Peptidase-IV (DPP-IV) Inhibitory Oligopeptides from Bovine Casein: Active Oligopeptides Screening, Enzyme and Cellular Validation, and Oligopeptide-Enzyme Binding Mechanisms

This study identified novel DPP-IV inhibitory oligopeptides from bovine casein hydrolysates for hyperglycemia regulation. Initial in vitro screenings showed that low-molecular-weight fractions (<3 kDa) exhibited superior DPP-IV inhibitory activity. From these fractions, high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) integrated with in silico, in vitro, and in situ validations identified potent oligopeptides, notably FPQYLQYL, which outperformed FVAPF and DAYPSGAWY. Kinetic analysis confirmed a competitive inhibition mode targeting the catalytic pocket. Innovatively, integrating microscale thermophoresis (MST), fluorescence quenching, molecular dynamics simulations, and atomic force microscopy (AFM) proved that the inhibitory potency was driven by high binding affinity and oligopeptide-enzyme aggregation. Crucially, structure-activity relationship analysis identified a critical substrate-mimicking N-terminal Phe-Pro motif, where the proline-driven insertion is stabilized by specific hydrophobic and aromatic interactions within the pocket. Collectively, our findings provided insights into the design and discovery of bioactive oligopeptides with hypoglycemic potential and offered a promising natural alternative to synthetic DPP-IV inhibitors.