Preparation By Enzymatic Hydrolysis And Preliminary Study On Its Mechanism Of Antithrombotic Peptides From Mytilus Edulis

Mytilus edulis is a kind of marine shellfish with high protein content,China has the largest production of Mytilus edulis in the world,enzymatic hydrolysis of natural proteins to release the antithrombotic active peptides has a lot of advantages such as mild conditions,low cost,high safety,easy to digest and absorb for its hydrolysate,which can make fully effective use of Mytilus edulis resources.At present,antithrombotic peptides in Mytilus edulis with antithrombotic activity are rarely reported.In this study,the Mytilus edulis was hydrolyzed into different peptide fragments by different proteases,and the high antithrombotic activity hydrolysate was screened.The amino acid sequences of peptides in hydrolysate were identified by UPLC-Q-TOF.Molecular docking,structure-activity relationship analysis and homology comparison bioinformatics method were used to predict the antithrombotic activities of peptides.Fmoc solid-phase synthesis and structure identification were used for selected peptides,then the antithrombotic mechanism in vitrowas further elucidated.The principal contents and results were concluded as following.First,the optimum hydrolytic enzyme was determined by protease pepsin,trypsin and neutrase hydrolysis,and its optimal hydrolysis conditions were optimized.Tricine-SDS-PAGE electrophoresis showed that the degradation of trypsin was the most significant,with the highest activity of thrombin inhibition of 72.51 ± 6.80%.Thus the protease species was determined to be trypsin.A total of 39 Mytilus edulis peptides were identified by UPLC-Q-TOF method for the best hydrolysate.All the peptides were tested with no toxicity.The identified peptides and positive control hirudin(peptides 54-65)were semi-flexible docked with thrombin,respectively.The-CDOCKER_Energy results of each polypeptide were obtained then,and the peptides were compared with the antithrombotic peptides in the local database.The peptides with high antithrombotic activity were selected comprehensively by the four kinds of prediction methods.Peptide 26(KNAENELGEVTVR)and peptide 36(NAESLRK)had high antithrombotic activity and research value and were determined as the next research objects in the study.The results showed that the purity of the two antithrombotic Peptide 26(KNAENELGEVTVR)and Peptide 36(NAESLRK)were determined by Fmoc solid-phase synthesis method.The purity of the two samples was as high as 99.21% and 99.27%,respectively,and the synthesis sequence was identified by UPLC-Q-TOF method.The secondary structure of the peptide was analyzed by Fourier transform infrared spectroscopy.It was found that both of them had β-fold secondary structure in the amide I band.The thrombin inhibitory activited at the concentration of 1~7 mg/m L were determined,the inhibitory rates for Peptide 26 and Peptide 36 were 86.96 ± 5.30% and 81.32 ± 7.12% at a concentration of 7 mg/mL,respectively,which showed higher thrombin inhibitory activity.The interaction between Peptide 26 and Peptide 36 and thrombin was determined by isothermal titration calorimetry.The binding of both of them to thrombin was strong and weak,and spontaneously and hydrogen bonds were formed.Hydrophobic interaction was used as the main driving force.Four coagulation indicators of Peptide 26 and Peptide 36 were measured,and found that they have a significant inhibitory effect on thrombosis both in both endogenous and exogenous pathways.