Objective To systematically identify potential angiotensin-converting enzyme (ACE) inhibitory peptides from proteins in waste pearl shells of Pteria martensii using an integrated in silico and experimental (“dry-wet”) strategy. Methods Waste pearl shells were subjected to decalcification and dialysis to obtain nacre proteins. The optimal protease was screened using ACE inhibitory activity and degree of hydrolysis as evaluation indicators. The enzymatic hydrolysates were separated and purified via ultrafiltration and gel filtration chromatography, and the peptide sequences of active fractions were characterized by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Bioinformatic tools were applied to predict and screen peptides with high potential ACE-inhibitory activity; candidate peptides were then prepared by solid-phase peptide synthesis (SPPS) and verified for their in vitro ACE inhibitory capacity. Lineweaver-Burk double-reciprocal plots were used to calculate enzyme kinetic parameters and define the inhibition mode of active peptides. An in vitro simulated gastrointestinal digestion assay was performed to evaluate the digestive stability of target peptides. Molecular docking was performed to investigate the interactions between the active peptide and ACE and to compare its interactions with the active sites in the N- and C-domains of ACE. Molecular dynamics (MD) simulations were conducted to assess the conformational stability and dynamic interaction characteristics of peptide-ACE complexes. Results Eight proteases were used to hydrolyze nacre proteins, among which papain hydrolysate showed a degree of hydrolysis of (11.77 ± 0.09)% and a half-maximal inhibitory concentration (IC₅₀) of (2.03 ± 0.14) μg/mL against ACE. After separation and purification, the active fraction F1 was harvested with an IC₅₀ value of (1.56 ± 0.06) μg/mL. A total of 294 unique peptides were identified from F1, and nine peptides with high predicted activity were selected for solid-phase synthesis and in vitro activity validation. Among all synthetic peptides, peptide SPAR exhibited the strongest ACE inhibitory activity with an IC₅₀ of (33.46 ± 0.56) μmol/L, and its inhibitory pattern against ACE was confirmed as mixed-type inhibition. After in vitro simulated gastrointestinal digestion, the retention rate of SPAR was (23.6 ± 4.9) %. Molecular docking results revealed that SPAR formed hydrogen bonds with key amino acid residues located in the S1 and S2 active pockets of ACE, including Ala354, Glu384, Tyr523, Gln281 and Lys511. SPAR was capable of binding to both the N-domain and C-domain of ACE, and displayed stronger binding affinity for the C-domain. MD simulation results demonstrated that the SPAR-ACE complex maintained favorable conformational stability and sustained intermolecular interactions throughout the simulation period. Conclusions Eight novel ACE inhibitory peptides were successfully screened from waste pearl shell proteins via the integrated dry-wet strategy, among which peptide SPAR possessed the optimal ACE inhibitory activity. This work provides a novel research perspective and technical route for high-value reutilization of discarded pearl shells. The complementary combination of in silico prediction and wet-lab experiments enables more efficient excavation of latent bioactive peptides from traditional Chinese medicine waste proteins..

waste shell of Pteria martensii (Dunker)  /  optimal protease  /  bioinformatics  /  ACE inhibitory peptides  /  inhibitory peptides  /  dry-wet strategy