>Magister Scientiae - MSc / Second generation biofuels production requires a suite of lignocellulolytic enzymes, acting synergistically to liberate the fermentable monosaccharides contained within agricultural waste materials. The use of thermostable lignocellulosic enzymes in a high-temperature process represents a number of advantages over their respective mesophilic counterparts, including increased solubility of the polymeric lignocellulosic substrates, enhanced enzyme processivity and reduced risk of bacterial and/or phage contamination during fermentation (Turner et al., 2007; Viikari et al., 2007). Alpha-L-arabinofuranosidases (AFases) participate in the deconstruction of lignocellulosic materials by hydrolysing the arabinofuranosyl bonds contained within the hemicellulosic portion of lignocellulose. In this study, three AFases isolated from compost-derived metagenomic DNA were characterised. Three genes derived from the fosmid metagenomic library constructed from 70°C compost were cloned into the pET21a(+) expression vector and expressed in E. coli BL21. The heterologously expressed proteins, AFase_H4, AFase_E3 and AFase_D3, were subsequently purified and their biochemical characteristics determined. All three AFases were shown to be active between pH 4.0 and 6.0. AFase_H4 and AFase_E3 displayed the highest activity at 60oC, while AFase_D3 had an optimum temperature at 25oC. Furthermore, the three AFases had differing thermostability profiles. In particular, AFase_E3 maintains 100% residual activity following 60 min incubation at 80oC and 24 hour incubation at 60oC. All three AFases have activity upon p-Nitrophenyl-arabinofuranoside and none against a range of alternative p-Nitrophenylglycosidic substrates. Phylogenetic analysis of the catalytic domain, identified within the amino acid sequences of the AFases, suggests that these AFases belong to glycoside hydrolase (GH) family 51. The difference in the AFase primary amino acid sequence motifs were used to infer differences in thermostability. All three AFases indicated similar biochemical and biophysical characteristics. AFase_E3 was determined to be the most thermostable amongst all three AFases. In the assessment of the three AFases respective suitability for inclusion in thermogenic bioethanol production processes, AFase_E3 was concluded as a suitable candidate for hydrolysis and synergistic testing on natural substrates. AFase_D3 possess the potential to be included in novel mesophilic industrialized process.

Orientador: Eleni Gomes / Resumo: As leveduras são organismos quimiorganotróficos que utilizam principalmente glicose como fonte de energia e carbono. Além da glicose, outros açúcares fermentescíveis se encontram em abundância na natureza e têm sido subaproveitados na indústria, dos quais se destaca a xilose. Para algumas leveduras, como Saccharomyces cerevisiae, a utilização de pentoses é limitada pela carência de transportadores de membrana específicos e enzimas intracelulares para a metabolização deste açúcar. Entretanto, algumas leveduras são capazes de utilizar xilose como fonte de carbono e bioconverte-la em produtos como etanol, ácidos orgânicos ou peptídeos. Isso implica na existência de um sistema de transporte e enzimas intracelulares para metabolizala. Neste contexto, a prospecção de enzimas auxiliares despolimerizantes do material lignocelulósico, tais como β-glicosidases e α-L-arabinofuranosidases, também assume função importante para obtenção de açúcares fermentescíveis. Além disso, poucos estudos estão disponíveis a respeito da produção de peptídeos bioativos por leveduras, quais podem ser fontes promissoras de produção dos mesmos. Sendo assim, o presente trabalho buscou investigar o consumo de xilose, a produção de peptídeos com atividade biológica e a produção de βglicosidases pelas espécies Pichia ofunaensis e Trichosporon multisporon, assim como a produção de α-L-arabinofuranosidases por Aureobasidium pullulans e A. leucospermi. As enzimas foram prospectadas utilizando farelo de trigo como ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Yeasts are chemorganotrophic organisms that mainly use glucose as a source of energy and carbon. In addition to glucose, other fermentable sugars are found in abundance in nature and have been underutilized in the industry, of which xylose stands out. For some yeasts, such as Saccharomyces cerevisiae, the use of pentoses is limited by the lack of specific membrane transporters and intracellular enzymes for the metabolization of this sugar. However, some yeasts are able to use xylose as a carbon source and bioconvert it into products such as etanol, organic acids or peptides. This implies the existence of a transport system and intracellular enzymes to metabolize it. The fermentation of pentoses is an essential step to improve the yield in the production of ethanol and organic acids. In this context, the prospection of depolymerizing auxiliary enzymes of lignocellulosic material, such as β-glycosidases and α-Larabinofuranosidases, also plays an important role in obtaining fermentable sugars. In addition, few studies are available regarding the production of bioactive peptides by yeasts, which can be promising sources of their production. Thus, the present work sought to investigate the consumption of xylose, the production of peptides with biological activity and the production of β-glycosidases by the species Pichia ofunaensis and Trichosporon multisporon, as well as the production of α-L-arabinofuranosidases by Aureobasidium pullulans and A. leucospermi. The enzymes were pro... (Complete abstract click electronic access below) / Mestre