Prospecção, isolamento e atividade antimicrobiana de peptídeos de folhas de Mikania sp.
Autor(a) principal: | |
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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 |
Resumo: | 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. |
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Magalhães Junior, Marcos Jorge dehttp://lattes.cnpq.br/4959519873817929Ribon, Andréa de Oliveira Barroshttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4727026E6Carvalho, Claudine Márciahttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4794965T6Pereira, Maria Cristina Baracathttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4780021E6Leite, João Paulo Vianahttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4763897U8Oliveira, Leandro Licursi dehttp://lattes.cnpq.br/05782313922181622015-03-26T13:07:31Z2013-11-082015-03-26T13:07:31Z2009-07-31MAGALHÃES JUNIOR, Marcos Jorge de. Prospection, isolation and antimicrobial activity of Mikania sp. leaves peptides. 2009. 67 f. Dissertação (Mestrado em Bioquímica e Biologia molecular de plantas; Bioquímica e Biologia molecular animal) - Universidade Federal de Viçosa, Viçosa, 2009.http://locus.ufv.br/handle/123456789/2427Antimicrobial 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.Peptídeos antimicrobianos (AMPs) têm atraído a atenção de pesquisadores como pontenciais compostos de defesa para serem usados no agronegócio. O objetivo deste trabalho foi, por técnicas proteômicas, bioprospectar o potencial antimicrobiano de frações enriquecidas em peptídeos de folhas de guaco (Mikania sp.) contra fitopatógenos de importância comercial, visando aplicação biotecnológica. Folhas frescas de guaco foram maceradas em tampão Tris acrescido de inibidores de proteases, o homogenato foi centrifugado, e o sobrenadante foi nomeado extrato solúvel (ES). O precipitado foi ressuspendido em solução de LiCl contendo inibidores de proteases, o homogenato foi centrifugado e o sobrenadante foi denominado extrato de parede celular (EP). ES e EP foram fracionados por ultrafiltração em membranas com limite de exclusão de 3 e 30 kDa. Para avaliar o potencial antimicrobiano das frações protéicas de guaco, as frações ultrafiltradas ES>30kDa, ES3-30kDa, EP>30kDA e EP3-30kDa foram testadas contra cinco bactérias, Ralstonia solanacearum, Pseudomonas syringae pv. tomato, Xanthomonas axonopodis pv. phaseoli, Erwinia caratovora subsp. caratovora e Clavibacter michiganensis subsp. michiganensis, e o fungo Fusarium oxysporum. O ES>30kDa inibiu o crescimento de todos os seis microrganismos testados. Os microrganimos mais susceptveis às fraçoes protéicas foram a bactéria Clavibacter michiganensis subsp. michiganensis e o fungo Fusarium oxysporum. O procedimento de ultrafiltração foi essencial para a recuperação de grande quantidade de amostra parcialmente fracionada enriquecida em peptídeos. Com o objetivo inicial de confirmar a presença de AMPs nas frações ultrafiltradas, os extratos de guaco foram separados em SDSTricina-Page e as bandas de massa molecular sugestivamente enriquecidas em AMPs foram eletroeluídas e analisadas por espectrometria de massa em MALDITOF/ TOF. Com o mesmo objetivo, as frações ultrafiltradas foram separadas em C18-RP-HPLC e os picos analisados por espectrometria de massa, quando foram detectadas diversas moléculas com massas moleculares entre 4.367,092 e 9.164,098 kDa. Os picos com massas moleculares semelhantes aos AMPs foram selecionados e estão sendo submetidos à proteólise limitada para a caracterização bioquímica dos peptídeos via peptide mass fingerprint e/ou, MS/MS. Estes extratos protéicos ou peptídicos, com atividade antimicrobiana, podem ser biotecnologicamente explorados para aplicação comercial como compostos de defesa.application/pdfporUniversidade Federal de ViçosaMestrado em Bioquímica AgrícolaUFVBRBioquímica e Biologia molecular de plantas; Bioquímica e Biologia molecular animalAtividade antimicrobianaPeptídeosMikania sp.Antimicrobial activityPeptideMikania sp.CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::QUIMICA DE MACROMOLECULAS::PROTEINASProspecção, isolamento e atividade antimicrobiana de peptídeos de folhas de Mikania sp.Prospection, isolation and antimicrobial activity of Mikania sp. leaves peptidesinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisinfo:eu-repo/semantics/openAccessreponame:LOCUS Repositório Institucional da UFVinstname:Universidade Federal de Viçosa (UFV)instacron:UFVORIGINALtexto completo.pdfapplication/pdf2758973https://locus.ufv.br//bitstream/123456789/2427/1/texto%20completo.pdf18ea18e16565ad3553642db4581530f3MD51TEXTtexto completo.pdf.txttexto completo.pdf.txtExtracted texttext/plain99933https://locus.ufv.br//bitstream/123456789/2427/2/texto%20completo.pdf.txt4a47029a2bd8134b31ccbb7bb963b7b7MD52THUMBNAILtexto completo.pdf.jpgtexto completo.pdf.jpgIM Thumbnailimage/jpeg3676https://locus.ufv.br//bitstream/123456789/2427/3/texto%20completo.pdf.jpgafadf49ec1f514baddfd6e58a70bcfefMD53123456789/24272016-04-08 23:02:14.531oai:locus.ufv.br:123456789/2427Repositório InstitucionalPUBhttps://www.locus.ufv.br/oai/requestfabiojreis@ufv.bropendoar:21452016-04-09T02:02:14LOCUS Repositório Institucional da UFV - Universidade Federal de Viçosa (UFV)false |
dc.title.por.fl_str_mv |
Prospecção, isolamento e atividade antimicrobiana de peptídeos de folhas de Mikania sp. |
dc.title.alternative.eng.fl_str_mv |
Prospection, isolation and antimicrobial activity of Mikania sp. leaves peptides |
title |
Prospecção, isolamento e atividade antimicrobiana de peptídeos de folhas de Mikania sp. |
spellingShingle |
Prospecção, isolamento e atividade antimicrobiana de peptídeos de folhas de Mikania sp. Magalhães Junior, Marcos Jorge de Atividade antimicrobiana Peptídeos Mikania sp. Antimicrobial activity Peptide Mikania sp. CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::QUIMICA DE MACROMOLECULAS::PROTEINAS |
title_short |
Prospecção, isolamento e atividade antimicrobiana de peptídeos de folhas de Mikania sp. |
title_full |
Prospecção, isolamento e atividade antimicrobiana de peptídeos de folhas de Mikania sp. |
title_fullStr |
Prospecção, isolamento e atividade antimicrobiana de peptídeos de folhas de Mikania sp. |
title_full_unstemmed |
Prospecção, isolamento e atividade antimicrobiana de peptídeos de folhas de Mikania sp. |
title_sort |
Prospecção, isolamento e atividade antimicrobiana de peptídeos de folhas de Mikania sp. |
author |
Magalhães Junior, Marcos Jorge de |
author_facet |
Magalhães Junior, Marcos Jorge de |
author_role |
author |
dc.contributor.authorLattes.por.fl_str_mv |
http://lattes.cnpq.br/4959519873817929 |
dc.contributor.author.fl_str_mv |
Magalhães Junior, Marcos Jorge de |
dc.contributor.advisor-co1.fl_str_mv |
Ribon, Andréa de Oliveira Barros |
dc.contributor.advisor-co1Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4727026E6 |
dc.contributor.advisor-co2.fl_str_mv |
Carvalho, Claudine Márcia |
dc.contributor.advisor-co2Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4794965T6 |
dc.contributor.advisor1.fl_str_mv |
Pereira, Maria Cristina Baracat |
dc.contributor.advisor1Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4780021E6 |
dc.contributor.referee1.fl_str_mv |
Leite, João Paulo Viana |
dc.contributor.referee1Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4763897U8 |
dc.contributor.referee2.fl_str_mv |
Oliveira, Leandro Licursi de |
dc.contributor.referee2Lattes.fl_str_mv |
http://lattes.cnpq.br/0578231392218162 |
contributor_str_mv |
Ribon, Andréa de Oliveira Barros Carvalho, Claudine Márcia Pereira, Maria Cristina Baracat Leite, João Paulo Viana Oliveira, Leandro Licursi de |
dc.subject.por.fl_str_mv |
Atividade antimicrobiana Peptídeos Mikania sp. |
topic |
Atividade antimicrobiana Peptídeos Mikania sp. Antimicrobial activity Peptide Mikania sp. CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::QUIMICA DE MACROMOLECULAS::PROTEINAS |
dc.subject.eng.fl_str_mv |
Antimicrobial activity Peptide Mikania sp. |
dc.subject.cnpq.fl_str_mv |
CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::QUIMICA DE MACROMOLECULAS::PROTEINAS |
description |
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. |
publishDate |
2009 |
dc.date.issued.fl_str_mv |
2009-07-31 |
dc.date.available.fl_str_mv |
2013-11-08 2015-03-26T13:07:31Z |
dc.date.accessioned.fl_str_mv |
2015-03-26T13:07:31Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/masterThesis |
format |
masterThesis |
status_str |
publishedVersion |
dc.identifier.citation.fl_str_mv |
MAGALHÃES JUNIOR, Marcos Jorge de. Prospection, isolation and antimicrobial activity of Mikania sp. leaves peptides. 2009. 67 f. Dissertação (Mestrado em Bioquímica e Biologia molecular de plantas; Bioquímica e Biologia molecular animal) - Universidade Federal de Viçosa, Viçosa, 2009. |
dc.identifier.uri.fl_str_mv |
http://locus.ufv.br/handle/123456789/2427 |
identifier_str_mv |
MAGALHÃES JUNIOR, Marcos Jorge de. Prospection, isolation and antimicrobial activity of Mikania sp. leaves peptides. 2009. 67 f. Dissertação (Mestrado em Bioquímica e Biologia molecular de plantas; Bioquímica e Biologia molecular animal) - Universidade Federal de Viçosa, Viçosa, 2009. |
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http://locus.ufv.br/handle/123456789/2427 |
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Universidade Federal de Viçosa |
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Mestrado em Bioquímica Agrícola |
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UFV |
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BR |
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Bioquímica e Biologia molecular de plantas; Bioquímica e Biologia molecular animal |
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Universidade Federal de Viçosa |
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reponame:LOCUS Repositório Institucional da UFV instname:Universidade Federal de Viçosa (UFV) instacron:UFV |
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