Keap1-Binding Peptides from Ganoderma lucidum Spores by Enzymatic Hydrolysis, In Silico Screening and Mechanism Based on Quantum Chemical Calculation

Given the prevalence of oxidative stress–driven chronic diseases and the poor targeting of synthetic antioxidants, novel natural antioxidant peptides were systematically identified from Ganoderma lucidum spore proteins using an integrated strategy combining peptidomics, bioinformatics, molecular docking, quantum chemical calculations, and in vitro cellular validation. Among the 6369 identified peptide sequences, three candidate peptides, including CDLDIR, FEQPR and PPSDFAK demonstrated superior performance. They exhibited strong binding affinities toward the Keap1 Kelch domain with the lowest binding energies of −75.00, −73.09 and −72.01 kcal/mol, respectively, driven by stable interactions with key residues Arg415, Arg483 and Tyr334. Density functional theory (DFT) calculations revealed that CDLDIR possessed the narrowest HOMO–LUMO energy gap (3.3163 eV) with its Arg-6 guanidinium group acting as the primary active site for radical scavenging. Subsequently, the antioxidant activity of the three peptides was validated, providing robust experimental confirmation for the computational results. In vitro cellular assays confirmed that CDLDIR was non-cytotoxic from 50 up to 500 μg/mL. Moreover, it remarkably reduced intracellular MDA and ROS levels while upregulating SOD, CAT and GPx activities in a concentration-dependent manner. These computational and experimental findings demonstrate that CDLDIR would be a promising natural antioxidant peptide from G. lucidum spores, offering a strong theoretical foundation for further investigation of their antioxidant and cytoprotective potential and for the development of functional food ingredients or nutraceuticals.