Study On Preparation,Identification And Inhibition Mechanism Of Soybean Derived Thrombin Inhibitory Peptide

Soybean protein not only provides essential amino acids for the growth and development of organisms,but also prepares a series of biologically active peptides with important physiological functions after enzymatic hydrolysis.Therefore,soybean protein has a wide application in food industry.At present,soybean peptides have not been studied in terms of antithrombotic bioactivity.In this paper,soybean thrombin inhibitory peptides were prepared by enzymolysis and separation,and multiple peptide compounds with anticoagulant effect were identified,and the related mechanism of their inhibitory effect on thrombin was studied.The main research contents and results are as follows:?1?Preparation of soybean thrombin inhibitory peptides by enzymatic hydrolysis.The thrombin inhibitory activity of soybean protein hydrolyzed by different proteases was studied,and pepsin was determined as best enzyme for hydrolysis,and its hydrolysis conditions were optimized.When enzymolysis time was 3 h,added enzyme amount was 4000 U/g,and material-liquid ratio was 3%?w/v?,the thrombin inhibitoy effect of soybean peptides was the highest which was 38.02±3.26%at 10 mg/m L.Molecular weight distribution studies had shown that pepsin significantly degraded peptides with molecular weights greater than 3 k Da.?2?Identification of soybean thrombin inhibitory peptide compounds.Ultrafiltration membrane separation and gel column chromatography were used to separate peptides mixture to obtain peptide components with high thrombin inhibitory activity which IC₅₀ of thrombin activity was 5.41 mg/m L.2176 peptide compounds were identified from this component by LC-MS/MS.66 peptide compounds were screened through biological activity prediction website,and further semi-flexible molecular docking with thrombin,and the peptides QPLPPPI,GNWGPLV and FFPDIPKIK were predicted to have high thrombin inhibitory activity.Solid-phase synthesis of the above three peptide compounds and then detection of their thrombin inhibitory activity revealed that nonapeptide FFPDIPKIK?Pep-3?activity was highest,and its IC₅₀ of thrombin activity was 9.44 m M?ie 10.42 mg/m L?.?3?Pep-3 stability and inhibitory mechanism research.Reverse phase-high performance liquid chromatography was used to investigate concentration change of Pep-3 under different treatment conditions.Studies had shown that Pep-3 had good stability at 37?and 60?,and Pep-3 could resist the digestion of simulated gastrointestinal fluid.The inhibitory mechanism of Pep-3 on thrombin was explored using enzyme kinetics,fluorescence spectroscopy and circular dichroism spectroscopy,indicating that Pep-3 was a mixed inhibitor of thrombin,and it exerted its inhibitory effect by affecting thrombin structure.?4?Study on anti-thrombin activity of Pep-3 truncated peptides and its related mechanism.Using solid-phase synthesis to prepare multiple peptide compounds with truncated Pep-3,and studied their thrombin inhibitory activity.Studies had shown that the truncated peptide C-2?FPD?had higher thrombin inhibitory activity and its IC50 was 8.40 m M?ie 3.17 mg/m L?.The inhibitory mechanism of C-2 on thrombin was investigated using enzyme kinetics and fluorescence spectroscopy and circular dichroism spectroscopy.Enzymatic kinetic studies had shown that C-2 was a mixed inhibitor of thrombin.Fluorescence spectroscopy results indicated that the mechanism of C-2 quenching thrombin was a dynamic-static combined quenching mechanism,and its binding force with thrombin was mainly hydrophobic.