Caracterização de protease e lipase de Pseudomonas fluorescens e quorum sensing em bactérias psicrotróficas isoladas de leite

• Ano de defesa: 2007
• Autor(a) principal: Martins, Maurilio Lopes
• Orientador(a): Vanetti, Maria Cristina Dantas
• Banca de defesa: Ribon, Andréa de Oliveira Barros , Riedel, Anna Katharina Maria
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de Viçosa
• Programa de Pós-Graduação: Doutorado em Microbiologia Agrícola
• Departamento: Associações micorrízicas; Bactérias láticas e probióticos; Biologia molecular de fungos de interesse
• País: BR
• Palavras-chave em Português: Protease; Lipase; Quorum sensing; Bactérias psicrotróficas proteolíticas; Leite cru; Pseudomonas fluorescens
• Palavras-chave em Inglês: Protease; Lipase; Quorum sensing; Proteolytic psychrotrophic bacteria; Raw milk; Pseudomonas fluorescens
• Área do conhecimento CNPq: CNPQ::CIENCIAS AGRARIAS::CIENCIA E TECNOLOGIA DE ALIMENTOS::CIENCIA DE ALIMENTOS::MICROBIOLOGIA DE ALIMENTOS
• Link de acesso: http://locus.ufv.br/handle/123456789/1571

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.