Preparation,Identification Of ?-glucosidase Inhibitory Peptides And Design Sequences Based On The Molecular Docking From Paeonia Ostii Seed Meal

Type 2 diabetes has become a chronic disease threatening human health worldwide.?-glucosidase is a hydrolase with dual functions of hydrolyzing sugar and transglycosidase,which could delay the process of converting polysaccharides and oligosaccharides into absorbable monosaccharides.The development of natural and healthy?-glucosidase inhibitors has become a hot research field in functional foods.Peony for oil is one of the important varieties cultivated in woody oil crops in recent years.The seed meal after oil extraction is rich in dietary fiber and protein,and can be used as a natural source of plant protein.Its amino acid composition is rich and the cost is low and easy to obtain.At present,seed meal is mainly discarded directly as waste,and a small amount is used as animal feed or other purposes.Therefore,the development and utilization of the protein in oil peony seed meal has great prospects and application significance.In this study,oil peony seed meal was used as raw material to prepare antidiabetic peptides through continuous double-enzyme hydrolysis of proteins.The enzymatic hydrolysis products were continuously separated,purified and identified.A total of six new antidiabetic peptides were identified.In addition,through enzyme inhibition kinetic analysis,molecular docking research and peptide sequence design,the potential mechanism of peptide hypoglycemia was explored.The specific research content is as follows:1.Protein extraction and preparation of enzymatic hydrolysis products.The oil peony seed meal was defatted by n-hexane,and the peony seed meal protein(PSMP)was prepared by the alkali-soluble acid precipitation method.The hydrolysis degree of protease at five different enzyme cleavage sites was compared,and the alkaline protease and trypsin had the highest levels.The degree of hydrolysis was 12.84±0.80%and 9.77±0.04%,respectively.Continuous enzymolysis method was used to enzymolyze protein,determine and compare the amino acid composition of alkaline protease+trypsin digestion product and trypsin+alkaline protease,both of which are rich in glutamate(16.9 g/100 g and 16.2 g/100 g)and essential amino acids,but the former has higher amino acid content(60.6 g/100 g)and stronger antidiabetic activity(IC₅₀=0.115±0.026 mg/m L).2.Separation,purification and identification of antidiabetic peptides.Using?-glucosidase inhibitory activity as an indicator,the PSMP enzymatic hydrolysis product was separated once by ultrafiltration,and the part with a molecular weight of<1 k Da was separated twice by reversed-phase high performance liquid chromatography to obtain the antidiabetic peptide component with the highest activity.Through nano LC-ESI-MS/MS to identified the amino acid sequence and determined the molecular weight of this component,a total of six antidiabetic peptides were obtained:YFFM(Tyr-Phe-Phe-Met,622.246),TYPLL(Thr-Tyr-Pro-Leu-Leu,605.343),SLLPF(Ser-Leu-Leu-Pro-Phe,575.332),YSPAPL(Tyr-Ser-Pro-Ala-Pro-Leu,647.340),WLLDPM(Trp-Leu-Leu-Asp-Pro-Met,774.386),SLLGDFM(Ser-Leu-Leu-Gly-Asp-Phe-Met,797.363).In vitro?-glucosidase inhibition experiments proved that the peptide YFFM has the highest antidiabetic activity with IC₅₀value of 1.099±0.037 mg/m L.3.Study on the inhibition kinetics of YFFM and?-glucosidase.By measuring the enzymatic reaction speed of different sample concentrations under different substrate concentrations,then changing the peptide concentration and substrate concentration when the enzyme concentration does not change,a Lineweaver-Burk double reciprocal curve was drawn.We found that the peptide YFFM is a reversible mixed inhibitor of?-glucosidase,with the calculated inhibition constant K_i=0.8247 m M and apparent coefficient?=0.5132.4.Structure-activity relationship research and peptide sequence design.Use Peptide Ranker,Toxin Pred,Innovagen and admet SAR online tools to predicted the biological activity,toxicity,water solubility and ADMET properties of the six peptides identified,combined with computer molecular docking simulation,to clarify the receptor-ligand interaction site.Exploring the relationship between the activity of?-glucosidase and the inhibitor may be related to the special amino acid residues on the peptide.In addition,with YFFM as the basic sequence,combined with the interactions generated on the amino acid groups,the antidiabetic peptides FFFM(Phe-Phe-Phe-Met,590.733)and YYFM(Tyr-Tyr-Phe-Met,622.732)with stronger?-glucosidase inhibitory activity were designed,with IC₅₀of 0.145±0.026 mg/m L and 0.191±0.030 mg/m L,respectively.In the binding molecule docking experiment,the two peptides formed 5 and 4 hydrogen bonds with the residues on the?-glucosidase,respectively.This result confirmed that these two peptide molecules can enter the active pocket of the enzyme molecule and produce a stable binding,and can be used as potential?-glucosidase inhibitors.