Purification And Inhibition Mechanism Of ACE Inhibitory Peptides From Abalone Skirt

Hypertension is a common chronic disease that has become a major global health problem.Angiotensin I-converting enzyme?EC 3.4.15.1,ACE?plays an important role in the hypertension regulatory system.It is a key enzyme in the screening of hypertension treatment drugs.In this study,ACE was isolated from pig lungs by acid precipitation,ammonium sulfate fractionation and HiTrap Q HP column chromatography.SDS-PAGE showed that the molecular weight of ACE was 180 kDa.ACE thus prepared revealed higher purity and specific activity than commercial one.PAS staining showed that ACE is a glycoprotein with a sugar content of 34.37%.Metal ion Zn²⁺can strongly inhibit the enzyme activity of ACE from porcine lung and commercially.Moreover,the inhibitory effect of Zn²⁺on ACE was concentration-dependent.Enzymatic properties showed that porcine lung ACE maintains a high enzyme activity in the range of 30-50°C and pH 6.0-10.0.Circular dichroism chromatography results indicated that the thermal denaturation temperature?T_m?of ACE is 58.5±0.4°C.Enzyme kinetics studies showed that the K_m,K_cat and K_cat/K_m values of ACE towards HHL were 3.1×10^-4 mol/L,17050 min^-1 and 5.5×10⁷?mol/L?^-1 min^-1,respectively.This study provides a certain theoretical reference for the industrial production of porcine lung ACE.Trypsin and prolyl endopeptidase?PEP?were used as tool enzymes to perform stepwise enzymatic hydrolysis of abalone skirt collagen for preparation of peptides.Tricine-SDS-PAGE and reversed-phase high-performance liquid chromatography?RP-HPLC?analysis exhibited that trypsin-PEP stepwise hydrolysis can effectively degrade the collagen from abalone skirt.ACE inhibitory peptide component was obtained by 3 kDa ultrafiltration membrane,Superdex^TMM Peptide10/300 GL gel column and ZORBAX SB-C₁₈ column.Three small peptides?SGEVGQ,GPPGPAGAR and QRGPAGAQGPQ?derived from collagen were identified by mass spectrometry.Using molecular docking technology,GPPGPAGAR was screened for follow-up research based on the docking energy value and interaction mode of ACE inhibitory peptide and ACE.In vitro activity assay of ACE inhibitory peptide revealed that GPPGPAGAR is a new type of ACE inhibitory peptide that show mixed inhibition mode.Its ACE inhibitory activity?IC50?was 177.13?mol/L,which revealed good digestive stability and is non-toxic to Caco-2 cells.GPPGPAGAR cannot be transported to the base compartment in the form of intact peptide during cell transport whilethe transport product after enzymolysis by brush border peptidase has ACE inhibitory activity.Further research found that ACE can hydrolyze GPPGPAGAR to produce a new ACE inhibitor peptide GPPGP.GPPGP is a novel ACE inhibitory peptide that exhibits competitive inhibition of ACE.Its ACE inhibitory activity(IC₅₀)is 190.43?mol/L.It is stable against digestion and is non-toxic to Caco-2 cells.GPPGP is transported to the basement in the form of a complete pentapeptide,and the transport products have ACE inhibitory activity.Therefore,the small peptides GPPGPAGAR and GPPGP derived from abalone skirt collagen are potential antihypertensive peptides.This study not only provides ideas for the high-value utilization of abalone skirts,but also provides technical support and theoretical reference for the preparation of food-derived protein-derived ACE inhibitory peptides.