Discovery And Identification Of Antimicrobial Peptide From Zanthoxylum Bungeanum Seeds By Bioinformatics And Peptidomics And Its Mechanisms Against E.coli And S.aureus

Zanthoxylum bungeanum seeds which are rich in oil,protein,melanin and dietary fiber are the main by-product of Zanthoxylum bungeanum Maxim production.They have not been fully utilized and often been regarded as a waste for a long time.Two unpurified antimicrobial peptides（A-b-Ⅳand B-b-Ⅲ）had been isolated from the hydrolysate of Zanthoxylum bungeanum seeds protein in our former work.However,it was difficult to carry out the following work due to the poor purification efficiency.In the present work,the potential antimicrobial peptides in the hydrolysate of Zanthoxylum bungeanum seeds protein were firstly screened and identified by assist of bioinformatics and peptidomic.And then their activities were further verified by the antimicrobial tests.Next,the physicochemical properties of the obtained antimicrobial peptides were investigated.Finally,the action mode of the antimicrobial peptide against Escherichia coli and Staphylococcus aureus were determined.The results of the work will provide a new idea for the rapid screening and identification of new natural antimicrobial peptides,as well as for the application of Zanthoxylum bungeanum seeds and the antimicrobial peptides from them.The main results of the work are as follows:1.Peptides in the hydrolysate of Zanthoxylum bungeanum seed protein were identified and predicted by nano LC-ESI-MS/MS and online software CAMP_R3,APD3 and AntiBP2.It was found that sixteen peptides were predicted to be potential antimicrobial peptides.The verifying assays including the determination of inhibition zone and minimum inhibitory concentration（MIC）were performed with E.coli ATCC 25922,S.aureus ATCC 25923,P.aeruginosa ATCC 27853,B.subtilis ATCC 6633,and S.cerevisiae CGMCC 2.3973 as the tested strains.It was found that four peptides（NP-2,NP-5,NP-6 and NP-7）were confirmed to be active.Among them,NP-6 showed the highest activity against E.coli with the MIC being 64μg/mL and S.aureus being 512μg/mL.Moreover,it was a fragment of glyceraldehyde-3-phosphate dehydrogenase according to the mass spectrum analysis.The time-killing curve indicated that the bactericidal rate of the four peptides against E.coli was NP-6>NP-7>NP-2/NP-5 at the concentration of 1 mg/mL.2.The physicochemical properties and simulated 3D structure of NP-2,NP-5,NP-6 and NP-7 were predicted by ExPASy,HemoPI and I-TASSER sever.The results showed that all the four peptides were positively charged,hydrophobic,amphipathic and hemolytic.All the peptides showedα-helix except for NP-5,who showed aβ-sheet conformation.After that,the hemolytic activities of the four peptides（1024 ug/mL）were determined with the results being 4.55%for NP-2,10.32%for NP-5,4.27%for NP-6,and 4.29%for NP-7.Finally,the effect of different pre-treatment such as thermal,acid/alkaline,ionic,and protease on the antimicrobial activities of these peptides were evaluated.It was found that all the four peptides thermal stable in the range of 60¹00℃.All the four peptides could keep their activity in pH 6.0⁸.0.Besides,monovalent cation（K⁺and Na⁺）could inhibit the antimicrobial activity of NP-5,but failed to NP-2,NP-6 and NP-7,unfortunately,divalent cations(Mg²⁺,Ca²⁺)could significantly inhibit the activity of all the four peptides in a dose-dependent manner.Finally,all the four peptides could resist the hydrolysis of pepsin,but failed to resist trypsin and protease K,both of whom could significantly inhibite their antimicrobial activities.3.Scanning electron microscopy（SEM）and Zeta potential analyzer were employed to evaluate the effect of NP-6 on the surface characteristics of bacterial cells,it showed that bacterial cells treated by NP-6 exhibited pits and holes on the cell surface,as well as the enhanced negative electricity of the cell membrane.The efflux of K⁺confirmed that NP-6could increase the permeability of cell membrane.And the transmission electron microscopy（TEM）,confocal laser microscopy,and flow cytometry observation indicated that NP-6could cause the disruption of bacterial cell membrane,leading to the leakage of intracellular contents.In addition,the peptide-DNA/RNA gel retardation assays were performed,as well as the fluorescence spectra analysis of competitive combination of peptide and ethidium bromide（EB）with bacterial genome DNA.It was found that NP-6 could be combined with bacterial nucleic acid.Finally,it was also found that NP-6 could inhibit the activities ofβ-galactosidase and alkaline phosphatase,but had no effect on T-ATPase.