Research On Structure And Characterization Of Antimicrobial Peptides From Brevibacillus Laterosporus

Antimicrobial peptide(AMP)is a kind of peptide that has antimicrobial activity,because of its safety,efficiency,no resistance,there is a wide application prospect in medical,food and animal and plant disease and pest resistance,etc.Up to now,there are more than two thousand AMPs been found,but most of AMPs have a narrow antibacterial spectrum and low stability,which is badly restricted its application.So,it is significant to exploit AMPs which are broad-spectrum and stable.We have previously screened a Brevibacillus laterosporus strain with broad antimicrobial activity,but,structure and properties of peptides are unknown.For this reason,this study firstly established the isolation and purification methods of AMP to take prepares high purity AMP for studying on the structure and properties.Moreover,the structures were investigated to provide theoretical guidance for the inhibitory mechanism.Finally,the properties were investigated to provide theories for the application in food product.This study established the isolation and purification methods of the AMPs from Brevibacillus laterosporus S62-9.The peptides were firstly purified by activated carbon adsorption and ethanol desorption method,then by preparative RP-HPLC.After the two isolation procedures,five peptides with antimicrobial activity were obtained.The chromatographic conditions: the flow rate was 3 mL/min,the sample volume was 400 μL,the detection wavelength was 220 nm,the column temperature was 30℃,acetonitrile and water as gradient eluting solvent.The molecular mass,amino acid sequence and secondary structure of AMPs were analyzed by MALDI-TOF/TOF mass spectrometry and FT-IR.The results indicated that the molecular mass of the peptides are 1 556.0334 Da,1 602.0044 Da,1 570.0189 Da,1 584.0344 Da,1 584.0946 Da,respectively,and the primarty structures of the peptides contain eleven amino acid residues,those are L-Y-K-L-V-K-V-V-V-N-V,L-Y-K-L-V-KV-V-L-N-M,L-Y-K-L-V-K-V-V-L-N-V,L-Y-K-L-V-K-V-V-L-K-V,L-Y-K-L-V-K-VV-L-N-N with a choline and a tenuazonic acid connected to the N-terminal and the C-terminal,respectively.The secondary structures mainly consist of β-turn and random coil.Antimicrobial assay indicated that peptides from Brevibacillus laterosporus S62-9are able to inhibit the growth both Gram-positive and Gram-negative bacteria with theMIC values of 3.91-7.81 μg/mL,7.81-125 μg/mL,respectively.Besides,peptides were also able to inhibit the growth of the Penicillium sp and Aspergillus niger with the MIC value of 31.25 μg/mL and 62.5 μg/mL.In addition,peptides showed high thermal and pH stability and good resitance to proteinases.The hemolytic results showed that the second component and third component of that peptides exhibited hemolytic activity below 10% at 62.5 μg/mL,and more than 80% at125 μg/mL.The fifth component of that peptides exhibited hemolytic activity below 10%at 15.63 μg/mL,but more than 90% at 62.5 μg/mL.The cytotoxicity of those peptides on NIH-3T3 and B16 cells were investigated by MTT assay,and the results showed that the viability of NIH-3T3 and B16 cells were 45% and 25% when the concentration of those peptides reached 62.5 μg/mL.