The Analysis Of Rapeseed Protein Bioactive Peptides Based On Bioinformatics And The Inhibition Mechanism Of ACE Inhibitory Peptides

With the rapid development of bioinformatics technology,intelligent prediction based on computational methods has become an effective method to identify bioactive peptides.Rapeseed protein is a kind of high-quality plant protein resource.Enzymatic hydrolysis of rapeseed protein can obtain bioactive peptides,which can improve the application value of rapeseed protein.Therefore,in this paper,bioinformatics technology was used to simulate enzymatic hydrolysis of rapeseed protein,then analyze and predict the biological activity of released peptides.Futhermore,five kinds of proteases were used to hydrolyze rapeseed protein,and ACE inhibitory peptides were isolated,purified and identified.Finally,the biological activity of synthetic peptides was determined,and the inhibition mechanism of ACE inhibitory peptides was explored.The main results are as follows:Thirty protein sequences of Napin,Cruciferin and Oleosin were used for homology analysis by Blast tool,among which 12 rapeseed protein sequences with low identity(<80%)were seclected for further research.BIOPEP-UWM analysis showed that there were 24 kinds of bioactive peptides in rapeseed protein,among these peptides,DPP-IV inhibitory peptide and ACE inhibitory peptide appeared more frequently,with the frequency of 0.573-0.749 and 0.350-0.536,respectively.Eight single enzymes and a two-enzyme combination of pepsin + trypsin were used to hydrolyze rapeseed protein in BIOPEP-UWM.The degree of hydrolysis of rapeseed protein by three plant proteases was the highest,while the three animal proteases resulted in the lowest.Thirteen kinds of bioactive peptides were obtained after enzymatic hydrolysis,among which DPP-IV inhibitory peptide and ACE inhibitory peptide had the highest frequency.In the eight single enzymes,papain can release the most number of DPP-IV inhibitory peptide and ACE inhibitory peptide,with the frequency of1.452 and 0.876,respectively.In contrast,the frequency obtained by trypsin was the lowest,which was 0.136 and 0.042,respectively.The possibility of peptides with 3-6 amino acids as bioactive peptides was predicted in Peptide Ranker.The score of 11 peptides was greater than0.95,and none of them was toxic.Among them,8 peptides were non-allergenic.26 new potential AMPs were obtained by enzymatic hydrolysis,and 13 of them were obtained by enzymatic hydrolysis of Cruciferin.Enzymatic hydrolysis of rapeseed protein by trypsin generated the highest number of AMPs compared with other proteases,with 10 different AMPs obtained.The 26 AMPs were all non-toxic,and 14 of them were non-allergenic.The ACE and DPP-IV inhibitory activities of rapeseed protein hydrolyzed by five kinds of proteases(alkaline protease,papain,bromelain,pepsin and trypsin)were compared.The results showed that the ACE inhibitory activity of rapeseed protein hydrolyzed by the same protease was significantly higher than that of DPP-IV.Moreover the degree of hydrolysis and ACE inhibitory rate of hydrolysates obtained by alkaline protease were the highest,which was 65% and 68.44% respectively.The optimum conditions of hydrolysis by alkaline protease were as follows: the ratio of substrate concentration was 1:25,the amount of enzyme was 5%,the hydrolysis time was 6 h,the temperature was 50? and the p H was 8.0.Under these conditions,the degree of hydrolysis and ACE inhibition rate were 11.42% and70.53% respectively.After separation and purification of Sephadex G-15 and DEAE-Sepharose Fast Flow,the ACE inhibition rates of fractions F21 was the highest,which was 76.69%.A total of 101 peptides were identified in F21 by mass spectrometry,and the scores of 17 peptides in peptideranker were higher than 0.95.The ACE inhibition rate of FRP was the highest,which was 92.11%,and the IC50 value was 10.75 ?M.The ACE inhibitory rates of FSW,FQW,FRW and CPF were above 80%,which were92.81%,91.32%,87.55 and 80.85% respectively,and their IC50 values were 45.61 ?M,45.88?M,47.28 ?M and 131.35 ?M respectively.The ACE inhibitory rate of GGF was the lowest,which was 23.56%.Five peptides including CPF,GPF,FSW,RPF and SPF had DPP-IV inhibitory activity.The DPP-IV inhibitory rate of RPF was the highest,which was 67.06%,while FSW had the lowest inhibitory rate of 23.29%.FSW,FQW,FRW and CPF all competitively inhibited ACE.The results of molecular docking showed that FSW had the strongest affinity with ACE,and the binding energy was-11.09 kcal/mol.CPF had the weakest affinity,and the binding energy was-8.70 kcal/mol.CPF and FSW formed two coordination with Zn2+,while CPF had less interaction with other amino acids.FQW and FRW formed only one coordination with Zn2+.Therefore,the order of affinity of four peptides with ACE was FSW > FQW> FRW > CPF.