Keap1-Inhibitory Peptides from Ganoderma lucidum Spores: Virtual Enzymolysis, Fragmentomics and Antioxidant Mechanism

Ganoderma lucidum spores protein (GLSP) holds significant potential for providing antioxidant peptides. We employed in silico enzymatic hydrolysis to generate small peptide fragments by specific proteins. Through fast computer screening and molecular docking with Keap1 receptor, we identified two potential antioxidant peptides, KAF (Lys-Ala-Phe) and NDSF (Asn-Asp-Ser-Phe), from 1171 candidates after efficient hydrolysis by pepsin and proteinase K. Molecular docking result showed both of them could bind onto the Leu557, Ala 510 and Val512 of bioactive pockets of Keap1 through hydrogen bonds and NDSF had lower docking energy (-85.6073 kcal/mol). The in vitro antioxidant validation indicated both of them could eliminate DPPH and ABTS radicals dramatically, and NDSF had a stronger scavenging capacity on DPPH (IC₅₀ = 35.1 μg/mL) and ABTS (IC₅₀ = 55.9 μg/mL), respectively. Quantitative chemical analysis further revealed that the key antioxidant active sites of NDSF were located at O18 of Ser amino side chain, and N9 of Lys terminal amino residue for KAF. Furthermore, in the cellular experiments, NDSF and KAF effectively increased the activities of antioxidant enzymes such as SOD, CAT, and GPx, while also reducing the level of MDA. Together, these findings highlight the potential of Ganoderma lucidum spore proteins as a source for the rapid identification of antioxidant peptides. The two selected peptides, therefore, s hold promising prospects for applications in functional foods and health products.