Purification And Characterization Of A Novel Antimicrobial Peptide From Brevibacillus Laterosporus Strain A60

Antimicrobial peptides have been studied for approximately fifty years. They are short-sequencepeptides with broad-spectrum activities against bacteria, fungi, viruses and even tumorcells.Antimicrobial peptides are divided into two groups based on the biosynthesis. One group is thenon-ribosomally synthesized peptides, such as cyclopeptides and pseudopeptides, which are mostlyproduced by bacteria; the other group comprises the ribosomally synthesized peptides that are isolatedfrom all organisms, including bacteria. Most antimicrobial peptides have linear or cyclic structures, withhydrophobic or amphipathic properties, and they can lyse microbial cells by forming ion channels ortransmembrane pores and also regulate replication transcription and translation by interacting withcertain intracellular targets.Such peptides derived from bacteria are important antimicrobial compounds due to their inherentability to defend against potential pathogens and their low toxicity in mammalian cells. Brevibacilluslaterosporus, previously classified as Bacillus laterosporus, can produce different virulence factors:parasporal crystalline, extracellular protease and lipopeptide antibiotics. Additionally, B. laterosporusalso secretes short sequence peptides with broad antibiotic spectra, such as loloatin A; however, thesemetabolites have not been extensively studied.We have previously screened a Brevibacillus laterosporus strain A60having antimicrobial activitythat was isolated from the soil of mango plants in Changjiang, Hainan province, China. This studyprovides a potential application prospect in plant diseases protection and novel therapeutics design. Themajor results are as follows:1. We have previously screened a Brevibacillus laterosporus strain A60having antimicrobial activitythat was isolated from the soil of mango plants in Changjiang, Hainan province, China.2. A60was classified to Brevibacillus laterosporus according to phylogenetic tree based on16SrDNA3. Phytophthora capsici is screened out as the sensitive indication microorganism. Bl-A60showed abroad-spectrum antimicrobial activity against the tested bacteria and fungus. The MIC values of theinhibitory activity of the antimicrobial peptide against P. capsici, B. cinerea, and F. omysporum were15,50,25μg ml-1,respectively.4. The purification procedure consisted of ammonium sulfate precipitation, cation exchangechromatography on an HiTrap SP HP column, thin layer chromatography and High Performance LiquidChromatography (HPLC) on C18reversed-phase column. After the four isolation procedures, onepeptide with antimicrobial activity was obtained and named BL-A60.5. Purified BL-A60showed high pH and thermal stability, BL-A60is amphipathic, containing bothhydrophilic and hydrophobic amino acid residues it is a candidate for plant pathogen control products.6. The TOF-MS analysis of fraction showed that the molecular mass of1602.0469Da agreed wellwith the theoretical mass. The determination of the complete amino acid sequences of this peptide showed that it contains eleven amino acid residues, L-Y-K-L-V-K-V-V-L-N-M, and a choline connectedto the N-terminal and a tenuazonic acid modified of the C-terminal. This peptide shows relatively lowidentification to other antimicrobial peptides from bacteria.7. Bioinfromatics analysis showed that the peptide is extracellular product, the secondary structuralprediction analysis showed that BL-A60can form an α-helix, the theoretical pI was9.70, and the totalnumber of positively charged residues (Arg, Lys) was2; thus the total net charge was+2. The instabilityindex (Ⅱ) was computed to be-8.96, classifying the peptide as stable. The GRAVY value was1.209.8. Bl-A60showed a broad-spectrum antimicrobial activity against the tested bacteria and fungus. TheMIC values of the inhibitory activity of the antimicrobial peptide against X．Vesicatoria, B. subtilis, P.capsici, B. cinerea, and F. omysporum were100,5,15,25and50μg ml-1, respectively.9. Purified BL-A60showed a strong inhibition of different stages of the life cycle of Phytophthoracapsici, including mycelial growth, sporangia formation and cystospore germination, with EC50valuesof7.89,0.60and21.96μg ml-1, respectively.