Purification of antimicrobial peptides secreted by Saccharomyces cerevisiae and proteomic analysis of cell membrane-associated proteins

Autor: Santos, Lara Andreia Brito Dos
Přispěvatelé: Almeida, Maria Gabriela Machado de, Albergaria, Maria Helena Whytton da Terra Soares de
Jazyk: angličtina
Rok vydání: 2015
Předmět:
Zdroj: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
Agência para a Sociedade do Conhecimento (UMIC)-FCT-Sociedade da Informação
instacron:RCAAP
CIÊNCIAVITAE
Popis: Dissertação para obtenção do grau de mestre na Escola Superior de Saúde Egas Moniz Saccharomyces cerevisiae secretes antimicrobial peptides (AMPs) during mixed culture fermentations that are derived from the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) enzyme and induce the early death of non-Saccharomyces yeasts. Considering the potential of AMPs to be used as natural preservatives in wine, the first aim of the present work was to produce and purify them in a preparative scale so that they could be applied in winery fermentations. Firstly, we produced 3 L of S. cerevisiae fermentation supernatants and subjected those to ion-exchange chromatography using a preparative DEAE-Sephadex column. One bioactive fraction, exhibiting a chromatographic profile similar to that exhibited by the previously found AMPs, was obtained. However, our work showed that the chromatographic system used was not fully efficient to purify the AMPs in a single step, requiring an additional size-exclusion chromatographic step. Several studies have shown that early death of non-Saccharomyces yeasts during wine fermentations is induced by S. cerevisiae through cell-cell contact and secretion of AMPs. Moreover, unpublished work showed that S. cerevisiae cells pre-grown for 48 h are able to induce death of Hanseniaspora guilliermondii by cell-cell contact, while 12 h-grown cells are not. Considering these findings and knowing that GAPDH is a cell wall-associated protein in S. cerevisiae, we hypothesized that these AMPs could be present in the membranes of S. cerevisiae and in this way induce death of non-Saccharomyces by cell-cell contact. The second aim of our work was to analyze membrane proteins of S. cerevisiae cells grown for 12 h and 48 h, using two dimensional polyacrylamide gel electrophoresis. Proteomic analysis revealed the presence of two spots of molecular weight and isoelectric point similar to the previously found AMPs, which were differentially expressed in the two growth stages. The spots were analysed by mass spectrometry, confirming the presence of GAPDH-derived peptides. Thus, our work raised evidence that death of non-Saccharomyces yeasts by cell-cell contact might be due to the presence of GAPDH-derived AMPs in the membranes of S. cerevisiae cells. However, to definitively confirm this hypothesis further work is required. Durante fermentações vínicas efectuadas com culturas mistas, a levedura Saccharomyces cerevisiae liberta péptidos antimicrobianos, que derivam da enzima gliceraldeído 3-fosfato desidrogenase (GAPDH) e provocam a morte prematura de leveduras não-Saccharomyces. Considerando o potencial destes péptidos antimicrobianos para serem usados como conservantes naturais do vinho, o primeiro objectivo deste trabalho consistiu na sua produção e purificação a uma escala preparativa, de forma a poderem ser aplicados em fermentações de adega. Primeiramente, produziram-se 3 L de sobrenadantes de fermentações efectuadas com S. cerevisiae que, em seguida, foram sujeitos a uma cromatografia de troca iónica utilizando um coluna preparativa DEAE-Sephadex. Foi obtida uma fracção bioactiva que exibia um perfil cromatográfico semelhante ao exibido pelos péptidos antimicrobianos previamente identificados. Embora tenha sido possível purificar os referidos péptidos com a coluna cromatográfica preparativa, os resultados deste trabalho mostraram que o sistema cromatográfico utilizado não foi suficientemente eficiente para isolar os péptidos de interesse num único passo. Assim propõe-se que de futuro se utilize um passo adicional de cromatografia de exclusão molecular que permita isolar a fração péptica (proteínas
Databáze: OpenAIRE
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