Extremozymes are enzymes produced by microorganisms that live in extreme habitats. Due to their higher stability, extremozymes is attracting interest as biocatalysts in various industrial processes. In this context, carbohydrate-active extremozymes can be used in various processes relevant to the paper, food and feed industry. In this thesis, the crystal structure, biochemical characterization and the capacity to synthesize prebiotic galacto-oligosaccharides (GOS) were investigated for a β-glucosidase (HoBGLA) from the halothermophilic bacterium Halothermothrix orenii. The wild-type enzyme displays favorable characteristics for lactose hydrolysis and produces a range of prebiotic GOS, of which β-D-Galp-(1→6)-D-Lac and β-D-Galp-(1→3)-D-Lac are the major products (Paper I). To further improve GOS synthesis by HoBGLA, rational enzyme engineering was performed (Paper II). Six enzyme variants were generated by replacing strategically positioned active-site residues. Two HoBGLA variants were identified as potentially interesting, F417S and F417Y. The former appears to synthesize one particular GOS product in higher yield, whereas the latter produces a higher yield of total GOS. In Paper III, the high-resolution crystal structure and biochemical characterization of a hemicellulase (HoAraf43) from  H. orenii is presented. HoAraf43 folds as a five-bladed β-propeller and displays α-Larabinofuranosidase activity. The melting temperature of  HoAraf43 increases significantly in the presence of high salt and divalent cations, which is consistent with H. orenii being a halophile. Furthermore, the crystal structures of a thermostable tetrameric pyranose 2-oxidase from Phanerochaete chrysosporium (PcP2O) were determined to investigate the structural determinants of thermostability (Paper IV). PcP2O has an increased number of salt links between subunits, which may provide a mechanism for increased stability. The structures also imply that the N-terminal region acts as an intramolecular chaperone during homotetramer assembly. / <p>QC 20150429</p>

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Orientador: Gláucia Maria Pastore / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-21T02:44:12Z (GMT). No. of bitstreams: 1 Lemos_AdrianeCristinaGarcia_D.pdf: 1535863 bytes, checksum: be0f41cf63bbd79b46b680df59cba450 (MD5) Previous issue date: 2012 / Resumo: Galacto-oligosacarídeos (GOS) são prebióticos obtidos via transgalactosilação enzimática da lactose. Dentre os vários benefícios associados ao consumo de GOS destaca-se a capacidade de estimular o crescimento e atividade de bactérias benéficas no cólon. O objetivo deste estudo foi avaliar as propriedades prebióticas dos GOS sintetizados, a partir da lactose, por ß-galactosidase de Scopulariopsis sp. A digestibilidade e a fermentabilidade foram avaliadas in vitro, enquanto os efeitos prebióticos foram avaliados in vivo em um conjunto de experimentos com ratos Wistar. Os resultados observados in vitro demonstraram que os GOS produzidos neste estudo são indigeríveis, altamente fermentáveis e convertidos em ácidos graxos de cadeia curta (acetato, propionato e butirato). Estudos in vivo demonstraram que o consumo de diferentes doses de GOS por 42 dias não produziu efeitos tóxicos nos animais, evidenciado a partir de avaliações clínicas, exames hematológicos, bioquímicos, necroscópicos e histológicos. Os ratos suplementados com GOS apresentaram maior (p<0.05) população cecal de bifidobactérias (log10 10,05 ± 0,27 UFC/g) e lactobacilos (log10 8,92 ± 0,16 UFC/g). Para os ratos não suplementados com GOS estas proporções foram de log10 8,22 ± 0,33 e 7,2 ± 0,15 UFC/g, para bifidobacterias e lactobacilos,respectivamente. Por outro lado, a população de Escherichia coli foi significativamente reduzida (p<0.05), sendo 24,75% menor, quando comparada ao grupo controle sem GOS. Além disso, a fermentação dos GOS pelas bactérias intestinais resultou em um aumento na produção de ácidos graxos de cadeia curta de 2,73 vezes, em relação aos animais sem acréscimo de GOS na dieta.Observou-se, ainda, que o grupo suplementado com GOS apresentou maiores valores de espessura total da mucosa, altura dos vilos e profundidade das criptas,evidenciado pela maior relação altura de vilosidades:profundidade de cripta em relação ao grupo controle / Abstract: Galacto-oligosaccharides (GOS) are prebiotics obtained via transgalactosylation enzymatic of lactose. Among the many benefits associated with consumption of GOS stands the ability to stimulate growth and activity of beneficial bacteria in the colon. The objective of this study was to evaluate the properties of prebiotic GOS synthesized from lactose by ß-galactosidase from Scopulariopsis sp. The digestibility and fermentability were evaluated in vitro, while the prebiotic effects were evaluated in vivo in a series of experiments with Wistar rats. The results observed in vitro showed that the GOS produced by this study are indigestible highly fermentable and converted into short-chain fatty acids (acetate,propionate and butyrate). In vivo studies have showed that consumption of different doses of GOS for 42 days produced no toxic effects in animals, as evidenced from clinical, hematological, biochemical, and histological necropsy. The rats supplemented with GOS had higher (p<0.05) cecal populations of bifidobacteria (log10 10.05 ± 0.27 UFC/g) and lactobacillus (log10 8.92 ± 0.16 UFC/g). For rats not supplemented with GOS these proportions were log10 8.22 ± 0.33 and 7.2 ± 0.15 UFC/g, for bifidobacteria and lactobacillus, respectively.Furthermore, the population of Escherichia coli was significantly reduced (p<0.05) and 24.75% less when compared to controls without GOS. Furthermore, the GOS fermentation by intestinal bacteria resulted in an increase in the production of short chain fatty acids from 2.73 times in compared with those without the addition of GOS diet.It was observed also the supplemented group with GOS showed higher values of total mucosal thickness, villous height and crypt depth, evidenced by the higher ratio of villus height: crypt depth in the control group / Doutorado / Ciência de Alimentos / Doutora em Ciência de Alimentos