Prospecção, isolamento e atividade antimicrobiana de peptídeos de folhas de Mikania sp.

• Autor(a) principal: Magalhães Junior, Marcos Jorge de
• Data de Publicação: 2009
• Tipo de documento: Dissertação
• Idioma: por
• Título da fonte: LOCUS Repositório Institucional da UFV
• Texto Completo: http://locus.ufv.br/handle/123456789/2427
• topic: Atividade antimicrobiana; Peptídeos; Mikania sp.; Antimicrobial activity; Peptide; Mikania sp.; CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::QUIMICA DE MACROMOLECULAS::PROTEINAS

Antimicrobial peptides (AMPs) have being attracting the researcher s attention as potential components of the plant defense, to be used in the agribusiness. The objective of this work was, by using proteomic techniques, bioprospect the antimicrobial potential of peptide-enriched fractions from leaves of Mikania sp. for controling the growth of important plant pathogens, aiming biotechnological applications. Leaves were macerated with Tris buffer added of protease inhibitors, the homogenate was centrifuged, and the supernatant was named soluble extract (ES). The precipitate was resuspended in LiCl solution containing protease inhibitors, the homogenate was centrifuged and the supernatant was named cell wall extract (EP). Both ES and EP were fractionated by ultrafiltration procedures by using membranes of 3 and 30 kDa cut-off. To evaluate the antimicrobial potential of the peptide enriched fractions, the ultrafiltrated samples ES>30kDa, ES3-30kDa, EP>30kDA and EP3-30kDa were evaluated against five bacteria, Ralstonia solanacearum, Pseudomonas syringae pv. tomato, Xanthomonas axonopodis pv. phaseoli, Erwinia caratovora subsp. caratovora and Clavibacter michiganensis subsp. michiganensis, and against the fungus Fusarium oxysporum. ES>30kDa inhibited the growth of the six assayed microorganisms. The best inhibition degrees were obtained for the bacterium Clavibacter michiganensis subsp. michiganensis and the fungus Fusarium oxysporum. Considering the major objective of confirming the presence of AMPs in the ultrafiltered fractions, the extracts were separate by SDS-Tricine-PAGE, and the bands presenting molecular masses similar to AMPs were electroeluted and analyzed by mass spectrometry in a MALDI-TOF/TOF equipment. Also, the ultrafiltrated fractions were separated on a C18-RP-HPLC column, and the peaks were analyzed by mass spectrometry, when various molecular mass values were observed, from 4,367.092 to 9,164.098 kDa. The fractions with molecular masses similar to AMPs were selected for development of limited proteolysis for biochemical characterization of the peptides through peptide mass fingerprint and/or MS/MS. These protein or peptide extracts, presenting antimicrobial activity, could be biotechnological explored for commercial application as defense agents.