Purification And Characterization Of A Microbial Antimicrobial Peptide

Microbial antimicrobial peptides are a class of small-molecule proteins or peptides produced by microorganism,which displays antimicrobial activity against food-borne spoilage and pathogenic microorganisms.And now microbial antimicrobial peptides are hot spots in the field of biopreservatives over the world.Preparation of high purity antimicrobial peptide(AMP)from B.laterosporus S62-9,and determination of its physichemical properties and biological antibacterial activity were investigated in the present work.Activated carbon adsorption-desorption method was chosen as the preliminary separation method of the peptide,and SP-Sepharose FF cation exchange chromatography was most suitable to purify the AMP furtherly.Adsorption and desorption of the AMP onto/from activated carbon increased the purification fold 19 times,a yield of about 80%.SP-Sepharose FF cation exchange chromatography resulted in 63.9% of total activity and a 119.2-fold increase in specific activity.Finally the AMP was polished by RP-HPLC,and chromatographically pure sample of the AMP was prepared.Molecular weight of the peptide produced by B.laterosporus S62-9 was determined by LC-MS,and the result is about 1570.13 Da.The AMP has outstanding stability.After treated at 100℃ for 30 min,the residual antimicrobial activity of the AMP was about 70%.When the peptide was incubated at 121℃,the residual activity was 50% after 30 min.The AMP was active over a p H range of 2-11,when it was incubated under these p H at 37℃ for 1 h,the activity recovered were all above 80%,but its optimum p H was neutral condition.The peptide was sensitive to pronase E,pepsin and lipase also had an effect on the activity of the AMP,but it was not sensitive to proteinase K and catalase.Antimicrobial activity was not affected by treatment with organic solvents,such as actone,methanol and ethanol,but the peptide lost its activity after treatment with trichloroacetic acid,and lost 60% after treatment with glacial acetic acid.Inhibitory activity was observed on several Gram-positive bacteria,including important pathogenic and spoilage microorganisms in food,such as B.cereus,S.aureus and L.monocytogenes etc.Among the Gram-negative bacteria tested,only one of the Salmonella spp.was inhibited.P.sanguineus tested was not inhibited.Minimum inhibitory concentration and minimum bactericidal concentration of the AMP from B.laterosporus S62-9 against S.aureus ATCC 14458 were 6.25 AU/m L and 12.5 AU/m L respectively,and the action mode of the peptide is both bactericidal and bacteriostatic.According to the nature of the AMP produced by B.Laterosporus S62-9,it has interesting potential for practical application in food biopreservation.