Breaking the wall! Deconstruction of the celullosome - a journey from cohesin-dockerinexpression to novel carbohydrate binding module structures
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2016
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Os Ruminantes estabelecem relações simbióticas, altamente complexas, com bactérias anaeróbias produtoras de celulossomas de forma a degradarem eficientemente os
polissacarídeos da parede celular de vegetal. Os celulossomas são compostos por enzimas
modulares constituídas por módulos catalíticos ligados a módulos não-catalíticos, envolvidos
em ligações proteína:carbohidratos (Carbohydrate-Binding Modules ou CBMs) e interações
proteína:proteína (Coesinas:Doquerinas ou Coh-Doc).
Este projeto visa ampliar o conhecimento dos celulosomas e dos seus componentes,
explorando tanto o complexo Coesina-Doquerina como os CBMs.
Apesar de já se encontrar bem caracterizado, o processo de produção e cristalização
do complexo Coesina-Doquerina de Bacteroides cellulosolvens, ainda necessita de alguma
otimização. Forçando o complexo a adotar apenas uma das suas duas conformações
possíveis, através de mutagénese direta, pretende-se testar e otimizar a sua purificação.
Os mutantes positivos foram testados em diferentes condições de crescimento,
sendo a melhor expressão obtida em células BL21 induzidas a 25ºC em meio LB.
Dos vários CBMs ainda por caraterizar de Ruminococcus flavefaciens estirpe FD-1
selecionámos o Rf2 e o Rf4 para, através de géis de afinidade, determinar a sua afinidade a
diversos substratos: HEC, β-Glucano, Galactomanano e xiloglucano.
Concluímos que a RF2 apresenta maior afinidade para Galactomanano e
xiloglucano variando entre 56 e 49 (w/v)-1, respetivamente. A Rf4 apresenta apenas afinidade
para o xiloglucano com um Ka significativo de 296 (w/v)-1.
Por último, a estrutura deste novo CBM-Rf2 foi resolvida por difração de raios-X
por dispersão anómala de comprimento de onda único.
Ruminant herbivores establish highly complex symbiotic relationships with anaerobic bacteria that produce cellulosomes to efficiently degrade plant cell wall polysaccharides. Cellulosomes are composed of modular enzymes containing catalytic modules which are linked to non-catalytic modules involved in protein:carbohydrate (Carbohydrate-Binding Modules or CBMs) or protein:protein (Cohesins:Dockerins or Coh- Doc) interactions. This project aims to aid in the quest of broadening the knowledge of the cellulosome and its components. Exploring both the Cohesin-Dockerin complex and the CBMs. Despite already well characterized, the production and crystallization process of Bacteroides cellulosolvens’s Cohesin-Dockerin Complex, still needs some optimization. By forcing the complex to adopt only one of the two possible conformations, through direct mutagenesis, we intended to test and optimize protein purification. The resulting positive mutants were tested in different growth conditions from which the best Dockerin expression were obtain for BL21 induced at 25ºC in LB. Among Ruminococcus flavefaciens strain FD-1 uncharacterized CBMs, we selected Rf2 and Rf4 to determine its substrate affinity to: HEC, β-Glucan, Galactomannan and Xyloglucan, using affinity gel electrophoresis. From which we concluded that RF2 presents higher affinities for Galactomannan and Xyloglucan ranging from 56 % to 49% (w/v)-1, respectively, and Rf4 only has affinity for Xyloglucan with a significant Ka of 296% (w/v)-1. Lastly, this novel CBM-Rf2 structure was solved at the As edge by single wavelength anomalous dispersion.
Ruminant herbivores establish highly complex symbiotic relationships with anaerobic bacteria that produce cellulosomes to efficiently degrade plant cell wall polysaccharides. Cellulosomes are composed of modular enzymes containing catalytic modules which are linked to non-catalytic modules involved in protein:carbohydrate (Carbohydrate-Binding Modules or CBMs) or protein:protein (Cohesins:Dockerins or Coh- Doc) interactions. This project aims to aid in the quest of broadening the knowledge of the cellulosome and its components. Exploring both the Cohesin-Dockerin complex and the CBMs. Despite already well characterized, the production and crystallization process of Bacteroides cellulosolvens’s Cohesin-Dockerin Complex, still needs some optimization. By forcing the complex to adopt only one of the two possible conformations, through direct mutagenesis, we intended to test and optimize protein purification. The resulting positive mutants were tested in different growth conditions from which the best Dockerin expression were obtain for BL21 induced at 25ºC in LB. Among Ruminococcus flavefaciens strain FD-1 uncharacterized CBMs, we selected Rf2 and Rf4 to determine its substrate affinity to: HEC, β-Glucan, Galactomannan and Xyloglucan, using affinity gel electrophoresis. From which we concluded that RF2 presents higher affinities for Galactomannan and Xyloglucan ranging from 56 % to 49% (w/v)-1, respectively, and Rf4 only has affinity for Xyloglucan with a significant Ka of 296% (w/v)-1. Lastly, this novel CBM-Rf2 structure was solved at the As edge by single wavelength anomalous dispersion.
Descrição
Orientação: Pedro Sampaio
Palavras-chave
MESTRADO EM ENGENHARIA BIOTECNOLÓGICA, BIOTECNOLOGIA, CELULOSSOMAS, BIOTECHNOLOGY, CELLULOSOMES