Caracterização de protease e lipase de Pseudomonas fluorescens e quorum sensing em bactérias psicrotróficas isoladas de leite
Autor(a) principal: | |
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Data de Publicação: | 2007 |
Tipo de documento: | Tese |
Idioma: | por |
Título da fonte: | LOCUS Repositório Institucional da UFV |
Texto Completo: | http://locus.ufv.br/handle/123456789/1571 |
Resumo: | A protease and a lipase produced by Pseudomonas fluorescens were purified and characterized. The aprX and lipM genes were cloned, sequenced, and expressed in Escherichia coli. These genes presented high identity with the sequences available in the GenBank. The molecular mass of both enzymes were 50 kDa. It was verified that calcium is essential to the enzymatic activities since when this ion was not added into the dialysis solution no activity was found. The protease was active in a large range of pH, had highest activity against casein and gelatin, and its temperature optimum was at 37 °C. Besides, this enzyme showed the highest thermal stability at 75 °C for 20 s. In contrast to the protease, the temperature optimum for the lipase was 25 °C and the pH optimum was close to 7.5. This enzyme showed the highest activity against p-nitrophenyl-palmitate, and the thermal treatments of 65 °C for 30 min and 75 °C for 1 min reduced its activity to 13.2% and 25.4%, respectively. The mechanism of quorum sensing (QS) was studied in P. fluorescens and it was verified that although the strains evaluated induced Agrobacterium tumefaciens NTL4 and A136, they do not produce acyl-homoserine lactones (AHLs). However, it was detected production of auto-inducer two (AI-2) and presence of diketopiperizines (DKPs) into the chemical extract obtained from TYEP medium inoculated with P. fluorescens. The 16S rDNAs of strains 039, 059, 067, 068, 071, and 099 isolated from raw milk were sequenced and they were identified as Pantoea sp., Hafnia alvei, Enterobacter sp., Hafnia alvei, Hafnia alvei, and Aeromonas hydrophila, respectively. It was verified that these strains presented different spoilage potentials and resistance against different antibiotics. After cross-streak assays in order to detect AHLs, only Pantoea sp. was not able to induce the monitor strains. The thin layer chromatography and the chemical characterization of the extracts by mass spectrometry confirmed that these strains produce different AHLs. The quorum quenching mechanism was used and the lactonase enzyme was expressed in the Enterobacter sp. transconjugant, which was unable to secrete AHLs into LB medium. This strain was more proteolytic than the wild type, indicating that the QS negatively regulates the proteolytic activity. Of the 32 restriction enzymes used to digest the native plasmid from H. alvei 068 and 071, only DdeI, HinfI, MspI, and RsaI were effective. Moreover, it was not possible to express lactonase in H. alvei 068 and 071 transconjugants which compromised the evaluation of the influence of the QS mechanism on spoilage activity. The halI gene, which encodes the AHL synthase in H. alvei, was identified in the strains 059, 067, 068, and 071. This gene was cloned, sequenced, and expressed in E. coli and it was verified that it encodes a synthase responsible for the production of N-hexanoyl-DL-homoserine lactone (C6-HSL) and N-3-oxohexanoyl-L-homoserine lactone (3-oxo-C6-HSL). Besides producing AHLs, A. hydrophila produced chitinase, AI-2 and was pathogenic against Caenorhabditis elegans. |
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Martins, Maurilio Lopeshttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4794520A8Araujo, Elza Fernandes dehttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4783675E2Mantovani, Hilário Cuquettohttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4727026Z7Vanetti, Maria Cristina Dantashttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4783874H3Ribon, Andréa de Oliveira Barroshttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4727026E6Riedel, Anna Katharina Maria2015-03-26T12:51:01Z2007-07-242015-03-26T12:51:01Z2007-03-23MARTINS, Maurilio Lopes. Characterization of protease and lipase from Pseudomonas fluorescens and quorum sensing in psychrotrophic bacteria isolated from milk. 2007. 184 f. Tese (Doutorado em Associações micorrízicas; Bactérias láticas e probióticos; Biologia molecular de fungos de interesse) - Universidade Federal de Viçosa, Viçosa, 2007.http://locus.ufv.br/handle/123456789/1571A protease and a lipase produced by Pseudomonas fluorescens were purified and characterized. The aprX and lipM genes were cloned, sequenced, and expressed in Escherichia coli. These genes presented high identity with the sequences available in the GenBank. The molecular mass of both enzymes were 50 kDa. It was verified that calcium is essential to the enzymatic activities since when this ion was not added into the dialysis solution no activity was found. The protease was active in a large range of pH, had highest activity against casein and gelatin, and its temperature optimum was at 37 °C. Besides, this enzyme showed the highest thermal stability at 75 °C for 20 s. In contrast to the protease, the temperature optimum for the lipase was 25 °C and the pH optimum was close to 7.5. This enzyme showed the highest activity against p-nitrophenyl-palmitate, and the thermal treatments of 65 °C for 30 min and 75 °C for 1 min reduced its activity to 13.2% and 25.4%, respectively. The mechanism of quorum sensing (QS) was studied in P. fluorescens and it was verified that although the strains evaluated induced Agrobacterium tumefaciens NTL4 and A136, they do not produce acyl-homoserine lactones (AHLs). However, it was detected production of auto-inducer two (AI-2) and presence of diketopiperizines (DKPs) into the chemical extract obtained from TYEP medium inoculated with P. fluorescens. The 16S rDNAs of strains 039, 059, 067, 068, 071, and 099 isolated from raw milk were sequenced and they were identified as Pantoea sp., Hafnia alvei, Enterobacter sp., Hafnia alvei, Hafnia alvei, and Aeromonas hydrophila, respectively. It was verified that these strains presented different spoilage potentials and resistance against different antibiotics. After cross-streak assays in order to detect AHLs, only Pantoea sp. was not able to induce the monitor strains. The thin layer chromatography and the chemical characterization of the extracts by mass spectrometry confirmed that these strains produce different AHLs. The quorum quenching mechanism was used and the lactonase enzyme was expressed in the Enterobacter sp. transconjugant, which was unable to secrete AHLs into LB medium. This strain was more proteolytic than the wild type, indicating that the QS negatively regulates the proteolytic activity. Of the 32 restriction enzymes used to digest the native plasmid from H. alvei 068 and 071, only DdeI, HinfI, MspI, and RsaI were effective. Moreover, it was not possible to express lactonase in H. alvei 068 and 071 transconjugants which compromised the evaluation of the influence of the QS mechanism on spoilage activity. The halI gene, which encodes the AHL synthase in H. alvei, was identified in the strains 059, 067, 068, and 071. This gene was cloned, sequenced, and expressed in E. coli and it was verified that it encodes a synthase responsible for the production of N-hexanoyl-DL-homoserine lactone (C6-HSL) and N-3-oxohexanoyl-L-homoserine lactone (3-oxo-C6-HSL). Besides producing AHLs, A. hydrophila produced chitinase, AI-2 and was pathogenic against Caenorhabditis elegans.Protease e lipase produzidas por Pseudomonas fluorescens foram purificadas e caracterizadas. Os genes aprX e lipM foram clonados, seqüenciados, e expressos em Escherichia coli e apresentaram alta identidade com as seqüências disponíveis no banco de dados. A massa molecular deduzida de ambas as enzimas foi de 50 kDa. Foi verificado que cálcio é essencial para as atividades enzimáticas, uma vez que quando este íon não foi adicionado à solução de diálise nenhuma atividade foi encontrada. A protease foi ativa em ampla faixa de pH, apresentou temperatura ótima de 37 °C, e maior atividade foi verificada sobre caseína e gelatina. Maior estabilidade térmica da enzima foi a 75 °C por 20 s. A lipase foi mais ativa a 25 °C e em pH próximo de 7,5 e p-nitrofenil-palmitato foi o substrato preferencial. Tratamentos térmicos de 65 °C por 30 min e de 75 °C por 1 min reduziram sua atividade para 13,2% e 25,4%, respectivamente. O mecanismo de quorum sensing (QS) em P. fluorescens foi estudado e verificou-se que as estirpes avaliadas, apesar de induzirem Agrobacterium tumefaciens NTL4 e A136, não produziram acil homoserinas lactonas (AHLs). Entretanto, a produção de auto-indutor dois (AI-2) foi detectada e a presença de dicetopiperazinas (DKPs) nos extratos químicos obtidos a partir de meio TYEP inoculado com P. fluorescens foi constatada. O rDNA 16S de seis bactérias gram-negativas isoladas de leite cru foi seqüenciado e o isolado 039 foi identificado como Pantoea sp., 059, 068 e 071 foram identificados como Hafnia alvei, 067 como Enterobacter sp. e 099 como Aeromonas hydrophila. Verificou-se que esses isolados apresentam diferenças no potencial deteriorador, na resistência a antibióticos e, após ensaios de estria cruzada em superfície de meio sólido para detecção de AHLs, constatou-se que somente Pantoea sp. não foi capaz de induzir nenhuma das estirpes monitoras utilizadas. Os ensaios de cromatografia em camada fina e a caracterização química dos extratos por espectrometria de massa confirmaram que essas bactérias produzem diferentes AHLs. O mecanismo de inibição de quorum foi utilizado e a enzima lactonase foi expressa em Enterobacter sp. transconjugante a qual foi incapaz de acumular AHLs em sobrenadante de caldo LB. O transconjugante se mostrou mais proteolítico do que a estirpe selvagem, indicando que o mecanismo de QS regula negativamente a atividade proteolítica neste isolado. Das 32 enzimas de restrição utilizadas para restringir o plasmídeo nativo (pMLM) presente nos isolados de H. alvei 068 e 071, apenas DdeI, HinfI, MspI, e RsaI foram efetivas. Além disso, não foi possível expressar a enzima lactonase em H. alvei 068 e 071 transconjugantes o que impossibilitou a avaliação da influência do mecanismo de QS sobre a atividade proteolítica desses isolados. O gene halI, que codifica a sintase de AHLs produzidas por estirpes de H. alvei, foi identificado nos isolados 059, 067, 068 e 071. Esse gene foi clonado, seqüenciado e expresso em E. coli e verificou-se que codifica uma sintase responsável pela produção de N-hexanoil-DL-homoserina lactona (C6-HSL) e N-3-oxohexanoil-L-homoserina lactona (3-oxo-C6-HSL). Das seis estirpes psicrotróficas proteolíticas avaliadas, apenas A. hydrophila foi capaz de produzir quitinase, AI-2 e de ser patogênica contra Caenorhabditis elegans.Conselho Nacional de Desenvolvimento Científico e Tecnológicoapplication/pdfporUniversidade Federal de ViçosaDoutorado em Microbiologia AgrícolaUFVBRAssociações micorrízicas; Bactérias láticas e probióticos; Biologia molecular de fungos de interesseProteaseLipaseQuorum sensingBactérias psicrotróficas proteolíticasLeite cruPseudomonas fluorescensProteaseLipaseQuorum sensingProteolytic psychrotrophic bacteriaRaw milkPseudomonas fluorescensCNPQ::CIENCIAS AGRARIAS::CIENCIA E TECNOLOGIA DE ALIMENTOS::CIENCIA DE ALIMENTOS::MICROBIOLOGIA DE ALIMENTOSCaracterização de protease e lipase de Pseudomonas fluorescens e quorum sensing em bactérias psicrotróficas isoladas de leiteCharacterization of protease and lipase from Pseudomonas fluorescens and quorum sensing in psychrotrophic bacteria isolated from milkinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/doctoralThesisinfo:eu-repo/semantics/openAccessreponame:LOCUS Repositório Institucional da UFVinstname:Universidade Federal de Viçosa (UFV)instacron:UFVORIGINALtexto completo.pdfapplication/pdf1723018https://locus.ufv.br//bitstream/123456789/1571/1/texto%20completo.pdf9f9fdbc014a6a40353aae85802485722MD51TEXTtexto completo.pdf.txttexto completo.pdf.txtExtracted texttext/plain344218https://locus.ufv.br//bitstream/123456789/1571/2/texto%20completo.pdf.txtcef06ceb5ff5d3dc4624e249f680f138MD52THUMBNAILtexto completo.pdf.jpgtexto completo.pdf.jpgIM Thumbnailimage/jpeg3557https://locus.ufv.br//bitstream/123456789/1571/3/texto%20completo.pdf.jpg590d6f1521e7362ab87328b0e1aad49bMD53123456789/15712016-04-07 23:05:30.666oai:locus.ufv.br:123456789/1571Repositório InstitucionalPUBhttps://www.locus.ufv.br/oai/requestfabiojreis@ufv.bropendoar:21452016-04-08T02:05:30LOCUS Repositório Institucional da UFV - Universidade Federal de Viçosa (UFV)false |
dc.title.por.fl_str_mv |
Caracterização de protease e lipase de Pseudomonas fluorescens e quorum sensing em bactérias psicrotróficas isoladas de leite |
dc.title.alternative.eng.fl_str_mv |
Characterization of protease and lipase from Pseudomonas fluorescens and quorum sensing in psychrotrophic bacteria isolated from milk |
title |
Caracterização de protease e lipase de Pseudomonas fluorescens e quorum sensing em bactérias psicrotróficas isoladas de leite |
spellingShingle |
Caracterização de protease e lipase de Pseudomonas fluorescens e quorum sensing em bactérias psicrotróficas isoladas de leite Martins, Maurilio Lopes Protease Lipase Quorum sensing Bactérias psicrotróficas proteolíticas Leite cru Pseudomonas fluorescens Protease Lipase Quorum sensing Proteolytic psychrotrophic bacteria Raw milk Pseudomonas fluorescens CNPQ::CIENCIAS AGRARIAS::CIENCIA E TECNOLOGIA DE ALIMENTOS::CIENCIA DE ALIMENTOS::MICROBIOLOGIA DE ALIMENTOS |
title_short |
Caracterização de protease e lipase de Pseudomonas fluorescens e quorum sensing em bactérias psicrotróficas isoladas de leite |
title_full |
Caracterização de protease e lipase de Pseudomonas fluorescens e quorum sensing em bactérias psicrotróficas isoladas de leite |
title_fullStr |
Caracterização de protease e lipase de Pseudomonas fluorescens e quorum sensing em bactérias psicrotróficas isoladas de leite |
title_full_unstemmed |
Caracterização de protease e lipase de Pseudomonas fluorescens e quorum sensing em bactérias psicrotróficas isoladas de leite |
title_sort |
Caracterização de protease e lipase de Pseudomonas fluorescens e quorum sensing em bactérias psicrotróficas isoladas de leite |
author |
Martins, Maurilio Lopes |
author_facet |
Martins, Maurilio Lopes |
author_role |
author |
dc.contributor.authorLattes.por.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4794520A8 |
dc.contributor.author.fl_str_mv |
Martins, Maurilio Lopes |
dc.contributor.advisor-co1.fl_str_mv |
Araujo, Elza Fernandes de |
dc.contributor.advisor-co1Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4783675E2 |
dc.contributor.advisor-co2.fl_str_mv |
Mantovani, Hilário Cuquetto |
dc.contributor.advisor-co2Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4727026Z7 |
dc.contributor.advisor1.fl_str_mv |
Vanetti, Maria Cristina Dantas |
dc.contributor.advisor1Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4783874H3 |
dc.contributor.referee1.fl_str_mv |
Ribon, Andréa de Oliveira Barros |
dc.contributor.referee1Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4727026E6 |
dc.contributor.referee2.fl_str_mv |
Riedel, Anna Katharina Maria |
contributor_str_mv |
Araujo, Elza Fernandes de Mantovani, Hilário Cuquetto Vanetti, Maria Cristina Dantas Ribon, Andréa de Oliveira Barros Riedel, Anna Katharina Maria |
dc.subject.por.fl_str_mv |
Protease Lipase Quorum sensing Bactérias psicrotróficas proteolíticas Leite cru Pseudomonas fluorescens |
topic |
Protease Lipase Quorum sensing Bactérias psicrotróficas proteolíticas Leite cru Pseudomonas fluorescens Protease Lipase Quorum sensing Proteolytic psychrotrophic bacteria Raw milk Pseudomonas fluorescens CNPQ::CIENCIAS AGRARIAS::CIENCIA E TECNOLOGIA DE ALIMENTOS::CIENCIA DE ALIMENTOS::MICROBIOLOGIA DE ALIMENTOS |
dc.subject.eng.fl_str_mv |
Protease Lipase Quorum sensing Proteolytic psychrotrophic bacteria Raw milk Pseudomonas fluorescens |
dc.subject.cnpq.fl_str_mv |
CNPQ::CIENCIAS AGRARIAS::CIENCIA E TECNOLOGIA DE ALIMENTOS::CIENCIA DE ALIMENTOS::MICROBIOLOGIA DE ALIMENTOS |
description |
A protease and a lipase produced by Pseudomonas fluorescens were purified and characterized. The aprX and lipM genes were cloned, sequenced, and expressed in Escherichia coli. These genes presented high identity with the sequences available in the GenBank. The molecular mass of both enzymes were 50 kDa. It was verified that calcium is essential to the enzymatic activities since when this ion was not added into the dialysis solution no activity was found. The protease was active in a large range of pH, had highest activity against casein and gelatin, and its temperature optimum was at 37 °C. Besides, this enzyme showed the highest thermal stability at 75 °C for 20 s. In contrast to the protease, the temperature optimum for the lipase was 25 °C and the pH optimum was close to 7.5. This enzyme showed the highest activity against p-nitrophenyl-palmitate, and the thermal treatments of 65 °C for 30 min and 75 °C for 1 min reduced its activity to 13.2% and 25.4%, respectively. The mechanism of quorum sensing (QS) was studied in P. fluorescens and it was verified that although the strains evaluated induced Agrobacterium tumefaciens NTL4 and A136, they do not produce acyl-homoserine lactones (AHLs). However, it was detected production of auto-inducer two (AI-2) and presence of diketopiperizines (DKPs) into the chemical extract obtained from TYEP medium inoculated with P. fluorescens. The 16S rDNAs of strains 039, 059, 067, 068, 071, and 099 isolated from raw milk were sequenced and they were identified as Pantoea sp., Hafnia alvei, Enterobacter sp., Hafnia alvei, Hafnia alvei, and Aeromonas hydrophila, respectively. It was verified that these strains presented different spoilage potentials and resistance against different antibiotics. After cross-streak assays in order to detect AHLs, only Pantoea sp. was not able to induce the monitor strains. The thin layer chromatography and the chemical characterization of the extracts by mass spectrometry confirmed that these strains produce different AHLs. The quorum quenching mechanism was used and the lactonase enzyme was expressed in the Enterobacter sp. transconjugant, which was unable to secrete AHLs into LB medium. This strain was more proteolytic than the wild type, indicating that the QS negatively regulates the proteolytic activity. Of the 32 restriction enzymes used to digest the native plasmid from H. alvei 068 and 071, only DdeI, HinfI, MspI, and RsaI were effective. Moreover, it was not possible to express lactonase in H. alvei 068 and 071 transconjugants which compromised the evaluation of the influence of the QS mechanism on spoilage activity. The halI gene, which encodes the AHL synthase in H. alvei, was identified in the strains 059, 067, 068, and 071. This gene was cloned, sequenced, and expressed in E. coli and it was verified that it encodes a synthase responsible for the production of N-hexanoyl-DL-homoserine lactone (C6-HSL) and N-3-oxohexanoyl-L-homoserine lactone (3-oxo-C6-HSL). Besides producing AHLs, A. hydrophila produced chitinase, AI-2 and was pathogenic against Caenorhabditis elegans. |
publishDate |
2007 |
dc.date.available.fl_str_mv |
2007-07-24 2015-03-26T12:51:01Z |
dc.date.issued.fl_str_mv |
2007-03-23 |
dc.date.accessioned.fl_str_mv |
2015-03-26T12:51:01Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/doctoralThesis |
format |
doctoralThesis |
status_str |
publishedVersion |
dc.identifier.citation.fl_str_mv |
MARTINS, Maurilio Lopes. Characterization of protease and lipase from Pseudomonas fluorescens and quorum sensing in psychrotrophic bacteria isolated from milk. 2007. 184 f. Tese (Doutorado em Associações micorrízicas; Bactérias láticas e probióticos; Biologia molecular de fungos de interesse) - Universidade Federal de Viçosa, Viçosa, 2007. |
dc.identifier.uri.fl_str_mv |
http://locus.ufv.br/handle/123456789/1571 |
identifier_str_mv |
MARTINS, Maurilio Lopes. Characterization of protease and lipase from Pseudomonas fluorescens and quorum sensing in psychrotrophic bacteria isolated from milk. 2007. 184 f. Tese (Doutorado em Associações micorrízicas; Bactérias láticas e probióticos; Biologia molecular de fungos de interesse) - Universidade Federal de Viçosa, Viçosa, 2007. |
url |
http://locus.ufv.br/handle/123456789/1571 |
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por |
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Universidade Federal de Viçosa |
dc.publisher.program.fl_str_mv |
Doutorado em Microbiologia Agrícola |
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UFV |
dc.publisher.country.fl_str_mv |
BR |
dc.publisher.department.fl_str_mv |
Associações micorrízicas; Bactérias láticas e probióticos; Biologia molecular de fungos de interesse |
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Universidade Federal de Viçosa |
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