Isolaiton and characterization of plant peptides with antimicrobial activity

The alarming increase in number of pathogenic microorganisms resistant to most of known antibiotics is the reason for intensive search of new antimicrobial compounds and modification of existing molecules. Natural short peptides belong among the newly discovered antimicrobials. Their use is not assumed only in human or veterinary medicine. Biopesticides based on peptides could increase crop yields without environmental burden. Given that antimicrobial peptides act very rapidly, they can be applied on plants and after destruction the pest, they can be disintegrated by proteolytic enzymes. One of obtainable and cheap sources of antimicrobial peptides the plant residues after harvest could become. The method, that can be used to obtain from plat material pure antimicrobial peptide, could be divided into two basic phases, namely extraction and separation with purification. The extraction was realized in 50% solution of acetonitrile in deionized water and 0.1% trifluoracetic acid. The separation together with purification was done in two steps, the first step was tandem size-exclusion chromatography and second step was RP-HPLC. Presence of peptidic compounds was confirmed by tricine-SDS-PAGE. The most important characteristic of antimicrobial peptides is their activity, antimicrobial activity, possible toxic effect against mammalian cells or hemolysis. For determination of antimicrobial activity, the whole spectrum of microorganisms was tested gram-positive and gram-negative bacteria, yeasts and molds, especially plant and animal pathogens. With described approach tobacco plants (Nicotiana tabacum) were utilized. Noninduced plants were used for the first parallel and plants in which the resistance had been induced by methyljasmonic acid (MeJa) or benzothiodiazole (BTH) for the other two parallels. There were demonstrated the effect of induction by MeJa and/or BTH. By SDS-PAGE were detected proteins present only in induced plants. The extracts from induced plants also exhibited wider spectrum of antimicrobial activity. There were obtained fraction with activity against gram-positive, gram-negative bacteria and yeasts (e.g. Staphylococcus aureus, Bacillus megaterium, Escherichia coli, Pseudomonas aeruginosa, Candida krusei).