A highly efficient method for screening dual-target inhibitory peptides against angiotensin I-converting enzyme and α-amylase from Chlorella using an enzyme-immobilized affinity membrane

Food-derived dual-effect peptides exhibit multi-target therapeutic potential and represent a promising strategy for managing comorbid conditions. However, technical challenges in their purification remain major obstacles to practical application. In this study, a nanofibrous membrane immobilized with angiotensin I-converting enzyme (ACE) and α-amylase (PPA) was developed for the efficient screening of dual-target inhibitory peptides. The immobilized enzymes achieved maximum loading capacities of 1.0466 mg/cm 2 (ACE) and 2.2871 mg/cm 2 (PPA), while retaining specific activities of 0.0173 U/mg and 0.0208 U/mg, respectively. This approach markedly reduced the number of peptide candidates from 1714 to 234, while enhancing their quality, as evidenced by increased inhibitory activities of 21.85 % for ACE and 22.22 % for PPA. Two dual-target inhibitory peptides, YDPR and LPDYR, were successfully isolated from Chlorella protein hydrolysates. Enzyme inhibition assays confirmed their potent inhibitory effects on ACE (YDPR: IC 50  = 89.05 ± 0.90 μM; LPDYR IC 50  = 170.50 ± 0.68 μM) and PPA (YDPR: IC 50  = 76.30 ± 1.05 μM; LPDYR: IC 50  = 112.51 ± 3.19 μM). Molecular dynamics simulations revealed that the dual inhibitory activities of these peptides arise from the formation of stable enzyme-peptide complexes involving key catalytic residues of ACE (Glu384, Tyr523, His353, Ala354, and Glu411) and PPA (His305, Asp300, Asp356, and Trp59). In summary, the high-efficiency screening strategy developed here provides a solid theoretical and technical foundation for the discovery of multi-target inhibitors.