Isolation,Purification And Identification Of ACE Inhibitory Peptide Of Paramisgurnus Dabryanus

Paramisgurnus dabryanus,a kind of freshwater fish with high protein and low fat,has various medicinal values,which have adjuvant therapy effect on patients with cardiovascular diseases.Hypertension is the main cause of cardiovascular disease,the prevention and treatment of hypertension can greatly reduce the mortality and disability rate of cardiovascular disease,and has important social value.Therefore,the preparation of angiotensin converting enzyme(ACE)inhibitor peptides from loach protein is of great significance In this work,the potential of loach protein as a source of ACE inhibitor peptides was first evaluated by computer simulation method.Then,computer simulated protease hydrolysis was carried out to explore the ability of ACE inhibitory peptide produced by different protease combinations.Subsequently,using protease preparation to enzymolyze loach protein,the complex enzymolysis method was studied;Finally,the ACE inhibitory peptides in the enzymatic hydrolysates of the loach protein were isolated and purified,and their structures were identified.The main results of the research are as follows:(1)The potential of loach protein as a source of ACE inhibitory peptide was evaluated by computer simulation method.First of all,42 species of leptin loach were identified by amino acid sequence alignment using UniProt database.Then,28 kinds of biological activities in the loach squama protein were determined by using the computer prediction methods and tools in BIOPEP,among which ACE inhibitory activity was one of the main activities.Then,three proteins,matrix metalloproteinase 9,?-actin and ?-defensin with the highest production quantity,occurrence frequency and potential activity of ACE inhibitory peptides were selected.Peptide and ExPASy were used to study the physicochemical properties,ToxinPred and Predicted Antigenic Peptid were used to predict toxicity and sensitization.The results showed that ?-actin and matrix metalloproteinase 9 proteins are acidic proteins with better hydrophilicity and higher binding potential to other proteins.?-defensins are basic proteins,which have hydrophobicity and low binding potential to other proteins.None of the three proteins was toxic,but there was some sensitization.At the same time,the primary structure,physicochemical properties and toxicity characteristics of 27 ACE inhibitory peptides released by simulated digestion of the gastrointestinal tract were studied.The results showed that most of the ACE inhibitory peptides were acidic and negatively charged,while a small amount was alkaline and positively charged,most of which were hydrophilic peptides or hydrophobic peptides,and a small part were amphiphilic peptides.Most peptides had good binding ability to proteins,and the 27 ACE inhibitory peptides were non-toxic.Finally,two kind of tripeptides,GPL(Gly-Pro-Leu)and DGL(Asp-Gly-Leu),were selected for molecular docking;the results showed that GPL and ACE active sites S1,S2 and S'1 had hydrogen bond interactions,while DGL and ACE active site S1 and S2 formed hydrogen bond interaction.Therefore,loach protein is a potential source of ACE inhibitory peptide.(2)Computer simulation analyzed the ability which loach protein produce ACE inhibitory peptides through enzymatic hydrolysis.According to the specificity of the protease cleavage site,select ?-chymotrypsin,alkaline protease and prolyl oligopeptidase.Computer simulation is used to hydrolyze the protein ?-defensin,?-actin,and matrix metalloproteinase 9 of loach,and analyze the number,frequency and ACE inhibitory activity of ACE inhibitory peptides according to different single and different compound enzymatic hydrolysis.The results showed that the use of prolyl oligopeptidase and alkaline protease to hydrolyze loach protein matrix metalloproteinase 9,?-actin and ?-defensin protein to produce ACE inhibitory peptides with a maximum number of 38,14,1;the highest frequency ACE inhibitory peptides is 0.0569,0.0373,0.0290;the highest ACE inhibitory potential is 0.0338,0.0052,0.0049 ?M-1.The combination of prolyl oligopeptidase and alkaline protease enzymatically hydrolyze loach serrata can produce ACE inhibitory peptides more efficiently.(3)Optimization of the preparation of ACE inhibitory peptides by protease enzymatic hydrolysis of loach protein.Firstly,reversed-phase high-performance liquid chromatography was used to determine the amino acid composition of loach protein;sceondly,analyzed the effects of single enzymatic hydrolysis and compound enzymatic hydrolysis method on the production of ACE inhibitory peptides from loach;Tricine-SDS-PAGE further confirmed the hydrolysis effect of different enzymatic hydrolysis methods.The results of the study showed that loach protein is rich in hydrophobic amino acids(39.823%),branched chain amino acids(18.102%)and aromatic amino acids(8.216%).propyl endopeptidase,?-chymotrypsin and alkaline protease were used to hydrolyze loach squamatus.The highest ACE inhibition rates of the enzymatic hydrolysates were 81.01%,64.91%,and 87.84%,and the highest degree of hydrolysis was 1.48%,respectively.9.04%and 28.29%.Select the combination of alkaline protease and propyl endopeptidase for step-by-step digestion and simultaneous digestion.The highest ACE inhibition rates obtained by the two sequential stepwise digestion methods were 79.14%and 78.24%,respectively,and the highest ACE obtained by the simultaneous digestion method The inhibition rate was 90.14%,and the IC50 was 0.491 mg/mL.Therefore,simultaneous enzymatic hydrolysis can produce higher activity ACE inhibitory peptides.(4)Separation,purification and structure identification of ACE inhibitory peptides from the proteolytic hydrolysate of loach.Using ultrafiltration,Sephadex G-15 gel column chromatography and preparative reversed-phase high performance liquid chromatography multi-stage separation technology,the ACE inhibitory peptide in the enzymatic hydrolysis product was purified step by step.The results showed that the enzymatic hydrolysis of loach After ultrafiltration separation,the liquid can be divided into 5 components,namely PDH-?(>10 KDa),PDH-?(10?5 KDa),PDH-?(5?3 KDa),PDH-?(3?1 KDa)and PDH-?(<1 KDa).Among them,the PDH-V component has a strong inhibitory ability,and its IC50 value is 154.70 ± 7.35 ?g/mL;the PDH-? component can be separated by Sephadex G-15 gel chromatography to obtain 3 elution peaks,respectively Labeled as F1,F2,and F3.By measuring the inhibitory activity of each elution peak,it can be seen that the F2 component has the strongest inhibitory ability,with an IC50 value of 98.52± 5.67?g/mL;F2 is further divided into 5 by preparative RP-HPLC Peak component,F2-2 peak component has a strong inhibitory ability,and its IC50 value is 17.89 ± 3.28?g/mL.Then F2-2 was purified twice by RP-HPLC and contained a peak.Through LC-MS/MS mass spectrometry technology,the amino acid sequence of the target peptide was identified as EGH(Glu-Gly-His)with a relative molecular mass of 341.2 Da.The final IC50 value of the target peptide was 17.89 ± 3.28 ?g/mL,and the purification factor was 27.47.Through inhibition kinetic analysis,EGH is a competitive ACE inhibitory peptide.By biologically active database analysis and computer simulation of a protease digestion,it was confirmed loach is a potential precursor of ACE inhibiting peptides.Then,the compound and simultaneous enzymatic hydrolysis of the protein of loach was carried out to prepare a high-activity ACE inhibitory peptide,which was separated and purified to identify its primary structure.The results of the study provide a theoretical basis for the production of ACE inhibitory peptides using loach protein as a raw material,and lay a foundation for the development of functional products for lowering blood pressure.