Preparation Of ACE Inhibitory Peptide By Enzymatic Hydrolysis Of Sheep Casein And Evaluation Of The Molecular Binding Mechanism

Angiotensin I-converting enzyme(ACE,E.C.3.4.15.1)can catalyze the formation of angiotensin II(a potent vasoconstrictor)and degrade bradykinin(a vasodilator).Therefore,ACE inhibitors can be used to treat hypertension.Sheep milk has high nutritional value and high casein content,which is a good source for preparing ACE inhibitory peptides.Thus,the purpose of this study was to prepare a novel ACE inhibitory peptide by hydrolyzing sheep casein with protease and analyze its molecular inhibitory mechanism.In this experiment,ACE inhibitory peptide was prepared from sheep casein by five proteases enzymatic hydrolysis,and taking molecular weight distribution,degree of hydrolysis and inhibition rate of angiotensin-I-converting enzyme(ACE)as indicators to screening the most suitable protease.The single factor and response tests were used to optimize enzymatic hydrolysis conditions.The amino acid sequences of components less than 3 k Da were identified by Orbitrap Fusion Lumos Tribrid MS,and the potential ACE inhibitory peptides were screened and determining their IC₅₀values after synthesis.The inhibition kinetics of enzyme was determined by Linewaver-Burk mapping,and molecular docking was used to explain the mechanism of peptides inhibition of ACE.The results as following.(1)The order of hydrolysis ability of five proteases to sheep casein was as follows:alkaline protease(J)>trypsin(Y)?protease K(K)>neutral protease(Z)>pepsin(W);The maximum ACE inhibition rate of the sheep casein hydrolysate of the five proteases was in the order of J(94.25%)>K(92.61%)>Z(90.16%)>W(88.22%)>Y(85.54%).(2)Alkaline protease was the most suitable protease,the optimal enzymolysis conditions were as follows:p H 6,substrate concentration of 8%,enzyme/substrate ratio(E/S)of 4%,temperature at 55?and enzymatic hydrolysis time for 90 min.The ACE inhibition rate of casein antihypertensive peptide prepared under this condition was 99.1%.The proportion of peptides with molecular weight of 0.2-1 k Da in sheep casein hydrolysate is the largest,followed by peptides with molecular weight of 1-3 k Da.(3)A total of 484 peptides were identified in the fraction less than 3 k Da,of which 84,116,137 and 74 were derived from?_s1-,?_s2-,?-and?-casein,respectively,and 18 peptides had been verified to have ACE inhibitory activity.The ratio of peptides with molecular weight of 1-1.5 k Da was 33.82%,that of 0.5-1 k Da was 24.57%,that of 1.5-2 k Da was20.44%,and that of 2-3 k Da was 18.9%.(4)There were eight novel ACE inhibitory peptides were screened out:FYPQLFR and LFRQFY were derived from?_s1-casein,with IC₅₀values of 13.95?M and 7.91?M,respectively.Their inhibition on ACE is a mixed inhibition mode.Molecular docking shows that FYPQLFR forms a hydrogen bond and an electrostatic force with Glu384residue in S1 active pocket of ACE,forms two hydrogen bonds with Ala354 residue,and forms a bond with Tyr523 residue.It forms a hydrogen bond with His513 residue in S2active pocket,and simultaneously forms a hydrogen bond and two electrostatic forces with ACE amino acid residues His387,Glu411 and ZN701 in tetrahedral structure with Zn²⁺.LFRQFY can form hydrogen bond interaction with amino acid residues Ala354(active pocket S1)and His353(active pocket S2)of ACE,showing significant antihypertensive activity in vitro.YQKFPQ and KFPQYL were derived from?_s2-casein,with IC₅₀values of64.57?M and 43.98?M respectively,which showed mixed inhibition mode and competitive inhibition mode for ACE;Molecular docking revealed that 13 hydrogen bonds were formed between YQKFPQ and ACE,KFPQYL forms a hydrogen bond with Glu384 residue in S1 active pocket of ACE,and forms an electrostatic force with Glu411amino acid residue in tetrahedral structure of Zn²⁺.These two forces may inhibit the catalytic activity of ACE and make KFPQYL have the effect of lowering blood pressure.YYQQRP,FLPYPY and KYIPIQY were derived from?-casein with IC₅₀values of 7.96?M,148.55?M and 5.73?M,respectively,which are mixed inhibition modes for ACE.The molecular docking shows that YYQQKP forms a hydrogen bond and a hydrophobic force with S1 active pocket residues Ala354 and Tyr523 of ACE,and 6 hydrogen bonds and an electrostatic force with S2 active pocket residues Lys511,Tyr520,His353 and His513,and forms a Metal-Acceptor interaction with ZN701 and a hydrophobic force with His383.FLPYPY forms an electrostatic force with ZN701 and?-alkyl interaction with Met223.KYIPIQY forms two hydrogen bonds and three hydrophobic forces with Ala354 and His353 in the S1 and S2 active pockets of ACE,and three hydrogen bonds and one hydrophobic force with His383,His387 and Glu411 of ACE residues may lead to distortion of tetrahedral coordination Zn²⁺,which leads to inactivation of ACE catalytic activity.RGPFPIL is derived from?-casein with IC₅₀value of 8.15?M,ACE is a mixed inhibition mode;Molecular docking showed that RGPFPIL and ACE amino acid residue His387 formed tetrahedral structure with Zn²⁺formed a hydrophobic force.