In vitro-in silico screening strategy and mechanism of angiotensin I-converting enzyme inhibitory peptides from α-lactalbumin

This work proposed an in vitro-in silico strategy for the discovery of angiotensin I-converting enzyme (ACE) inhibitory peptides. Alpha-lactalbumin was hydrolyzed by pepsin and pancreatin. The hydrolysate under 3 kDa was separated into five fractions, among which the fractions F3, F4 and F5 exhibited potent ACE inhibition, by ultrafiltration and dextran gel chromatography. Thirty-eight peptides were identified by Nano-HPLC-MS/MS from fractions F3, F4 and F5 to form a peptide library. Five novel ACE inhibitory peptides (SLPEW, VSLPEW, GVSLPEW, VGINYW and LDQWL) were predicted according to the binding energy calculated by molecular docking. The IC50 values of such peptides ranged from 6.94 to 17.5 μmol/L. Lineweaver-Burk plots revealed LDQWL as competitive inhibitor, and SLPEW and VSLPEW as mixed-type, while other peptides as non-competitive ones. Molecular docking showed that the competitive inhibitors interacted with the residues of active pocket, and the non-competitive peptides connected to the residues outside the active site and the mixed-type inhibitors anchored in both entrance channel and active pocket of ACE. Circular dichroism indicated that these peptides significantly changed the secondary structure of ACE. VGINYW binding caused the most reduction of α-helix and the most increase of random coils.