Preparation And Inhibition Mechanism Of Angiotensin Converting Enzyme-Inhibitory Peptide From Yeast

Bioactivity peptide has been widely applied to industries such as medicine, health care,food and makeup, because of its extensive physiological and medical functions.Angiotensin-converting enzyme (ACE)-inhibitory peptide has become potential alternative tohealth care products and hypotension drugs for its natural attribute.In this research, preparation technology of producing ACE from yeast throughmulti-enzyme hydrolysis was studied systematically. Based on these studies, purity, aminoacid sequence and stability of an ACE peptide with high ACE-inhibitory activity weredetermined. On the basis of these studies, the inhibition mechanism and model of reactionsbetween the ACE-inhibitory peptide and ACE were obtained using knowledge on enzymekinetics and thermodynamics, molecular simulation, designated chemical modification,structure biological, etc. The main experimental results are as follows:1. Preparation of ACE-inhibitory peptide by hydrolysis of yeast hydrolytic enzyme. Thehighest yeast protein extraction efficiency of81.32%±1.27%and the degree of yeast proteinhydrolysis of23.38%±1.26%were obtained when solid-liquid ratio is1:10, enzyme loading is1%(w/w), extraction temperature is40℃and extraction time is2hours. Yeast hydrolysatewas then roughly purified with4times (V/V) ethanol by ethanol precipitation method. Afterremoving impurities, the removal rates of polysaccharide and nucleic acid were64.2±2.15%and87.9±1.74%respectively, and the loss rate of protein was only6.88±1.28%. Furthermore,the component with MWCO-10kDa has the highest ACE-inhibitory activity withultrafiltration separation, and was purified with gel filtration chromatography and anionexchange chromatography. An ACE-inhibitory peptide AP1was purified.2. Structure identification and performance test of AP1. A hexapeptide,Thr-Pro-Thr-Gln-Gln-Ser, with a calculated molecular weight of660Da, was identified byMALDI-TOF-MS and automated edman degradation. The hexapeptide showed remarkableACE-inhibitory activity, with an IC50of0.11μmol/mL. The level of ACE-inhibitory activityof the hexapeptide is higher than other ACE-inhibitory peptides food-derived. Based on theresearch of enzyme inhibition kinetics and thermodynamics, the type of ACE inhibited by AP1is noncompetitive inhibition, and AP1could not change Kmof ACE, but change VmaxofACE. RP-HPLC determination method of AP1was established, and thermal stability andresistant of digestion were researched. The results showed that AP1demonstrated goodthermal stability at37-60℃, and resistant of digestion was good.3. ACE-Inhibitory mechanism of AP1. The docking model of AP1and ACE wasestablished. Based on the docking results, the key amino acids which affected the inhibitoryactive of AP1were concerned, and modified peptides with different length and amino acidcomposition were researched. The results showed that the hexapeptide bound to ACE viainteractions of the N-terminal Thr1, Thr3, and Gln4residues with the residues on the lidstructure of ACE, and the C-terminal Ser6attracted the zinc ion, which is vital for ACEcatalysis. The displacement of the zinc ion from the active site resulted in the inhibition ofACE activity. The structural model based on the docking simulation was supported byexperiments in which the peptide was modified. This study provides a new inhibitorymechanism of ACE by a peptide which broads our knowledge for drug designing againstenzyme targets.