Isolation,Purification And Structure Identification Of Whey Protein-derived ACE Inhibitory Peptide

Hypertension is the most common global disease.Angiotensin-converting enzyme(ACE)is a key factor in the rise of blood pressure.It can hydrolyze angiotensin I(decapeptide)to angiotensin II(octapeptide).Angiotensin II binds to the AT1 receptor on vascular smooth muscle and endothelial cells,resulting in vasoconstriction.It also inactivates endothelium-dependent bradykinin,which leads to hypertension.ACE inhibitory peptides are a sort of peptides having hypotensive activity obtained from edible proteins,which inhibit the increase in blood pressure by inhibiting the activity of ACE.In this paper,the preparation of ACE inhibitory peptide from whey protein was studied,and the hydrolysis process conditions of pepsin and trypsin were optimized.The hydrolyzate was filtered through a 3 kD ultrafiltration membrane and further purified by Sephadex G-15 gel chromatography.The structures of peptides from component B1 were identified by nano-scale reversed-phase liquid chromatography-tandem mass spectrometry(nano RPLC-MS/MS).The research results are as follows:(1)The process of preparing ACE inhibitory peptide from whey protein which was hydrolized by pepsin and trypsin was optimized.The single factor test and orthogonal test showed that when the hydrolysis temperature was 37 °C,the ratio of enzyme to substrate was 1:100,and the substrate concentration was 6%,the hydrolysate had the highest ACE inhibitory,86%.Then hydrolysate was hydrolyzed by trypsin.When the hydrolysis temperature was 55 °C,the substrate concentration was 6%,and the ratio of enzyme to substrate was 1:500,the hydrolysate had the highest ACE inhibitory,72%.The IC50 value of final hydrolysate by pepsin and trypsin was 425.66 ?g/mL.(2)The separation and purification process of the enzymatic hydrolysate of whey protein was studied.The whey protein hydrolysate was separated by ultrafiltration,and three components,A1(>10 kD),A2(3-10 kD)and A3(<3 kD)were obtained.Among these three components,A3 had the highest ACE inhibition rate of 92% and an IC50 value of 327.28 ?g/mL.Component A3 was separated by Sephadex G-15(dextran gel filtration chromatography)and two components,B1 and B2,were obtained.The ACE inhibition rate of component B1 was 97.5%,which was significantly higher than the ACE inhibition rate of B2 component by 83%(P<0.05).and the IC50 value of B1 was 273.82 ?g/mL.(3)The B1 fraction was identified by nano-scale reversed phase liquid chromatography-tandem mass spectrometry(nano RPLC-MS/MS).73 polypeptide sequences were obtained.Since the ACE inhibitory peptides are generally short peptides,7 polypeptide sequences(VLVLDTDY,LSPNPTQLE,DLEILLQK,DEALEKFD,LDAQSAPLR,MAASDISL,LIVTQTMK)were selected to be synthesized by solid phase synthesis.Only LIVTQTMK showed ACE inhibitory activity with an IC50 of 248.1 ?g/mL.(4)The interaction of ACE inhibitor peptide LIVTQTMK with ACE enzyme was studied using molecular docking technology.The results showed that electrostatic and hydrophobic interactions are the main driving factors for the combination of LIVTQTMK and ACE.Among them,the protonated N atoms of Leu1 and Lys8 of LIVTQTMK form a salt bridge with the carboxyl groups of Asp77 and Asp237 of ACE,respectively.The carboxyl group of Met7 and the backbone oxygen atom of Gln5 form a salt bridge with the sulfhydryl groups of Arg124 and Arg415,respectively.Several hydrogen bonds formed between Gln5 and Tyr231 and Trp235,Thr6 and Thr131,and Met7 and Asn417,respectively(hydrogen bond length is 2.8~3.5 ?).