Recombinant Collagenase-Assisted Production of Bovine Bone-Derived Peptides and EGFR-Guided Discovery of Osteogenic Oligopeptides

Abstract Bovine bone is a collagen-rich byproduct with potential for producing osteogenic peptides. A recombinant collagenase was heterologously expressed in Escherichia coliBL21(DE3), purified, and characterized. The enzyme showed a specific activity of 231.05 U/mg and hydrolyzed bovine bone collagen-containing matrix with a peptide yield of 58.12%. Nano-HPLC-MS/MS identified 1449 peptides in the most active fraction. EGFR-guided virtual screening prioritized GPYGPP and GPLGPA, with docking scores of −8.9 and −8.6 kcal/mol, respectively. At 250 μg/mL, GPYGPP and GPLGPA significantly increased MC3T3-E1 cell proliferation to 138.10% and 139.98% of the untreated control, respectively, and promoted cell-cycle progression, alkaline phosphatase activity, matrix mineralization, and osteogenic marker expression (p < 0.05). Transcriptomic analysis associated GPYGPP-induced osteogenesis with extracellular matrix remodeling and VEGF, HIF-1, and cAMP-related signaling. These findings provide an integrated strategy for producing bovine bone-derived peptides and identifying food-derived osteogenic oligopeptides with potential applications in bone-health functional foods.