Submitted by Ana Guimarães Pereira (agpereir@ucs.br) on 2017-07-14T14:43:43Z No. of bitstreams: 1 Tese Laisa dos Reis.pdf: 202579 bytes, checksum: 65cd12057624b5708899c86c663d936c (MD5) / Made available in DSpace on 2017-07-14T14:43:43Z (GMT). No. of bitstreams: 1 Tese Laisa dos Reis.pdf: 202579 bytes, checksum: 65cd12057624b5708899c86c663d936c (MD5) Previous issue date: 2017-07-14 / Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul, FAPERGS.

A linhagem S1M29 de Penicillium echinulatum é um fungo filamentoso cujo sistema celulolítico tem potencial para aplicação em processos de degradação de materiais lignocelulósicos visando a obtenção de produtos com interesse biotecnológico, como o etanol de segunda geração. A abundância de biomassas lignocelulósicas associada à busca por alternativas aos combustíveis fósseis faz aumentar o interesse em estudos que envolvam a elucidação dos mecanismos de secreção de enzimas lignocelulolíticas. Neste estudo, o P. echinulatum S1M29 foi crescido em condições de indução para a produção de endoglicanases, celobiohidrolases, β-glicosidases, xilanases e swoleninas. Foram avaliados os perfis enzimáticos em géis de poliacrilamida e o padrão de expressão gênica para estas proteínas. Observou-se que a produção enzimática foi favorecida pela presença de celulose Celufloc E® e bagaço de cana-de-açúcar (BCA) no meio de cultivo em comparação à glicose, sendo que o uso de resíduo de biomassa proporcionou a obtenção dos melhores rendimentos. Resultado semelhante foi atingido na análise dos perfis de secreção enzimática em gel de poliacrilamida, sugerindo ainda, a presença de uma endoglicanase constitutiva de aproximadamente 80 kDa. O estudo de qRT-PCR revelou a presença de quatro genes para endoglicanases com padrão de expressão distintos, destacando-se um acúmulo de transcritos 9779,19 vezes superior à amostra calibradora do gene egl1 em 24 h de cultivo no meio formulado com celulose Celufloc E®. Este mesmo gene gerou 1257,58 vezes mais transcritos acumulados que a amostra calibradora em meio suplementado com BCA. Na avaliação da expressão relativa do gene para celobiohidrolase obteve-se expressão superior no meio formulado com BCA em 48 h em relação ao meio com celulose Celufloc E® em 24 h, correspondendo, respectivamente, a 6464,49 e 3093,26 vezes mais transcritos acumulados que a amostra calibradora. O gene para β-glicosidase expressou-se em meio formulado com BCA no início do cultivo, embora a atividade desta enzima tenha ocorrido ao final do cultivo. A expressão de xilanases foi mais significativa com o uso de celulose Celufloc E® no meio de cultivo. Já o gene para swolenina apresenta um perfil de expressão com valores semelhantes em comparação ao uso de celulose Celufloc E® e BCA no meio de cultivo, apesar da presença de BCA ter proporcionado uma expressão mais tardia. Para todos os genes avaliados foram verificados valores muito reduzidos de expressão quando a glicose foi utilizada como fonte de carbono para o P. echinulatum. Observou-se ainda uma expressão coordenada de genes codificadores de endoglicanases, celobiohidrolase, β-glicosidase e swolenina. / Conselho Nacional de Desenvolvimento Científico e Tecnológico, CNPq. / The strain S1M29 of Penicillium echinulatum is a filamentous fungus that presents a cellulolytic system with potential for application in the degradation process of lignocellulosic materials to obtain products of biotechnological interest, such as second-generation ethanol. The availability of lignocellulosic biomass associated with the search for alternatives to fossil fuels increases the interest in studies that involve understanding the secretion mechanisms of lignocellulolytic enzymes. In this study, P. echinulatum S1M29 was grown under inducing conditions for the production of endoglucanases, cellobiohydrolases, β-glucosidases, xylanases and swollenins. The enzyme secretion profiles were evaluated in polyacrylamide gels and the pattern of gene expression for these proteins was also assessed. It was observed that enzyme production was enhanced in the presence of cellulose Celufloc E® and sugar cane bagasse (SCB) in the culture medium compared to glucose, with the use of biomass residue providing the best yields. A similar result was obtained analyzing enzyme secretion profiles in polyacrylamide gels, suggesting the presence of constitutive endoglucanases of approximately 80 kDa. Studies with qRT-PCR revealed the presence of four genes encoding endoglucanases with distinct expression patterns, standing out an accumulation of transcripts from egl1 gene 9779.19 times higher than the calibrator sample in 24 h of growth in medium formulated with cellulose Celufloc E®. The same gene generated 1257.58 more accumulated transcripts than the reference sample in medium supplemented with SCB. Evaluation of gene expression for cellobiohydrolase yielded higher expression levels in the medium formulated with SCB in 48 h, in comparison to the medium containing cellulose Celufloc E® in 24 h, corresponding, respectively, to 6464.49 and 3093.26 more accumulated transcripts than the reference sample. The β-glucosidase gene was expressed in medium formulated with SCB at the beginning of growth, as opposed to that seen for activity of this enzyme, which occurred at the end of cultivation. The xylanase expression was more significant with the use of cellulose Celufloc E® in the culture medium. On the other hand, the gene for swollenin presents an expression profile with similar values compared to using cellulose Celufloc E® and SCB in the culture medium, although the presence of SCB has provided a later expression. Very low values of gene expression have been found for all genes evaluated when glucose was used as carbon source for P. echinulatum. A coordinated expression of endoglucanases, cellobiohydrolase, β-glucosidase and swollenin was was also verified.

Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul, FAPERGS.

Submitted by Marlene Santos (marlene.bc.ufg@gmail.com) on 2014-11-10T18:07:11Z No. of bitstreams: 2 Dissertação - Douglas Endrigo Perez Pereia - 2014.pdf: 1726049 bytes, checksum: 6458dcad45ca242b032db7ff26c4f792 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Jaqueline Silva (jtas29@gmail.com) on 2014-11-10T20:00:00Z (GMT) No. of bitstreams: 2 Dissertação - Douglas Endrigo Perez Pereia - 2014.pdf: 1726049 bytes, checksum: 6458dcad45ca242b032db7ff26c4f792 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2014-11-10T20:00:00Z (GMT). No. of bitstreams: 2 Dissertação - Douglas Endrigo Perez Pereia - 2014.pdf: 1726049 bytes, checksum: 6458dcad45ca242b032db7ff26c4f792 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2013-05-06 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Lignocellulosic biomass is constituted by cellulose, hemicellulose and lignin in the plant cell walls. The conversion of lignocellulose to fermentable sugars mainly depends on the action of a complex of enzymes that hydrolyze cellulose and hemicellulose. This study was to analyze the production of cellulases and xylanases by fungi isolated from soil and decaying material in Cerrado. Initially, the fungi were analyzed for cellulase production capacity among the fungal cellulase-producing fungus capable of producing the highest levels of cellulase was selected for the experiments of xylanase and cellulase production by submerged fermentation and biochemical analysis of the enzyme secreted. Among the isolated fungi, fungus called IFBMD01 was that higher activity of cellulase in the supernatant when cultivated with wheat bran as carbon source. This was identified as Aspergillus cf niger. The enzymes studied with their respective results were: FPase with better production time of 7 days, showing activity of 0.10 U / mL, optimum pH 5.5, optimum temperature of 60 ° C; CMCase with better time production 6 days, showing activity of 8.19 U / mL, pH optimum of 5, optimum temperature of 70 º C; Avicelase with better production time of 9 days, showing activity of 0.01 U / mL, pH optimum of 5.5 and optimum temperature of 60 ° C; xylanase, with better production time 3 days, showing activity of 35.5 U / mL, and the optimum pH of 5 and the optimum temperature of 50 ° C, the β-Glycosidase, with higher production on the sixth day showing activity of 0.98 U / mL, with the optimum pH 5 and optimum temperature of 60 ° C. The activity of β-glucosidase showing in the culture supernatant was influenced by the ions Mn2 +, NH4, K +, Mg2 +, Ca2 +, Ba2 +, Al3 +, Na +, and Co2+, as well as EDTA, glucose, xylose and cellobiose. In the culture supernatant of the fungus A. niger IFBMD01 grown on FT for 3 and 6 days protein bands were visualized with activity against xylan and CMC molecular weight be 30 to be 66 kDa. / A biomassa lignocelulósica é constituída pela celulose, hemicelulose e lignina, presentes na parede das células vegetais. A xilana é o principal componente da fração de hemicelulose da lignocelulose. A conversão da lignocelulose à açúcares fermentáveis é realizada por um complexo de celulases e xilanases: enzimas que hidrolisam as frações de celulose e xilana. O objetivo deste trabalho foi analisar a produção de celulases e xilanases por fungos isolados a partir de solo e material em decomposição do Bioma Cerrado. Inicialmente, os fungos foram analisados quanto à capacidade de produção de celulases por atividade em placa, neste experimento, os fungos produtores de maiores níveis de celulases foram selecionados para os experimentos de produção de celulases e xilanases por fermentação submersa utilizando meio suplementado com resíduos lignocelulósicos como fonte de carbono. Dentre os fungos analisados, o isolado IFBMD01 apresentou maior atividade de celulases e xilanases no sobrenadante de cultura do fungo cultivado em meio com farelo de trigo, sendo que o pico de produção FPAses, CMCases, Avicelases e xilanases foram obtidos após 7, 6, 9 e 3 dias de cultivo respectivamente. A caracterização bioquímica das celulases e xilanases produzidas pelo isolado IFBMD01 demonstrou que: as enzimas com atividade de FPase apresentaram atividade de 0,10 U/mL, pH e temperatura ótimos de 5,5e 60ºC; as CMCases apresentaram atividade de 8,19 U/mL, pH ótimo de 5 e temperatura ótima de 70ºC; as Avicelases apresentaram atividade de 0,01 U/mL, pH ótimo de 5,5 e temperatura ótima de 60ºC; as xilanase apresentaram atividade de 35,5 U/mL, sendo o pH ótimo de 5 e a temperatura ótima de 50ºC e as β-glicosidases apresentaram atividade de 0,98 U/mL, com o pH ótimo de 5 e temperatura ótima de 60ºC. A atividade de β-glicosidase presente no sobrenadante de cultura foi influenciada pelos íons Mn2+, NH4, K+, Mg2+, Ca2+, Ba2+, Al3+, Na+ e Co2+, assim como por EDTA, glicose, xilose e celobiose. No sobrenadante de cultura do isolado IFBMD01 cultivado em FT por 3 e 6 dias foram visualizadas bandas de proteínas com atividade contra xilana e CMC, com massa molecular aproximada de 30 a 66 kDa. A identificação morfológica e molecular do isolado IFBMD01 demonstrou que é um isolado de Aspergillus cf niger.

Proteínas tipicamente são capazes de manter a sua conformação funcional somente dentro de um intervalo limitado de temperaturas. A despeito do maquinário sofisticado de manutenção da homeostase celular, é sabido que uma variedade de fenômenos moleculares são capazes de induzir desequilíbrios localizados de energia vibracional, e que a eficiência com que cada proteína dissipa estas perturbações pode estar relacionada com a sua tolerância a altas temperaturas. No entanto, a transferência de energia térmica entre diferentes segmentos de uma cadeia proteica é difícil de caracterizar experimentalmente. Uma alternativa teórica para a investigação destes mecanismos é o emprego de simulações de Dinâmica Molecular, particularmente associadas à técnica de Difusão Térmica Anisotrópica (ATD). Aqui, verificamos a possibilidade de empregar conceitos da teoria de Redes Complexas para construir modelos para estruturas de proteínas, e por meio destes identificar resíduos com capacidade significativa de dissipar perturbações térmicas. Investigamos os diversos protocolos de construção de modelos de rede para proteínas encontrados na literatura, e utilizamos dados experimentais representativos da base SCOP para calcular com rigor os parâmetros numéricos necessários. Produzimos uma definição precisa para o conceito de contato entre resíduos de aminoácidos, e a partir desta calculamos a centralidade de cada resíduo. Com isto, demonstramos que, em um conjunto de Xilanases para as quais dispomos de dados de ATD, a capacidade de difundir perturbações térmicas é fortemente correlacionada com a centralidade de proximidade de cada resíduo, fornecendo argumentos para o uso de modelos de rede para estudar a termoestabilidade de proteínas. / Proteins are typically able to mantain a functional conformation only within a narrow range of temperatures. In spite of the complex cellular homeostatic machinery, it is known that a variety of molecular phenomena can induce localized vibrational imbalances, and that the efficiency with which each protein dissipates these perturbations may be related to its tolerance of higher temperatures. The transference of thermal energy among different sections of a protein chain is, however, hard to characterize experimentally. A theoretical alternative for the investigation of these mechanisms is the use of Molecular Dynamics simulations, particularly when associated with the Anisotropic Thermal Diffusion (ATD) technique. In this work, we verify the possibility of using concepts from Network Theory to construct models for protein structures, and using those to reveal residues with significant ability to dissipate thermal perturbations. We investigate several protocols of network model construction for proteins present in the literature, and we study representative experimental data from the SCOP database to rigorously calculate the necessary parameters. We produce a precise definition for the concept of contact between amino acid residues, and from this we calculate the centrality of each residue. We then show that, in a set of Xylanases for which we have data from ATD experiments, the ability to dissipate thermal perturbations is highly correlated to the closeness centrality of each residue, providing arguments for the use of network models to study protein thermal stability.

The launch of national and international initiatives to reduce pollution, reliance on fossil fuels and increase the beneficiation of agricultural wastes has prompted research into sugar monomer production from lignocellulosic wastes. These sugars can subsequently be used in the production of biofuels and environmentally degradable plastics. This study investigated the use of synergistic combinations of commercial and pure enzymes to lower enzyme costs and loadings, while increasing enzyme activity in the hydrolysis of agricultural waste. Pineapple pomace was selected due to its current underutilisation and the substantial quantities of it produced annually, as a by-product of pineapple canning. One of the primary costs in beneficiating agricultural wastes, such as pineapple pomace, is the high cost of enzyme solutions used to generate reducing sugars. This can be lowered through the use of synergistic combinations of enzymes. Studies related to the inclusion of hemicellulose degrading enzymes with commercial enzyme solutions have been limited and investigation of these solutions in select combinations, together with pineapple pomace substrate, allows for novel research. The use of synergistic combinations of purified cellulosomal enzymes has previously been shown to be effective at releasing reducing sugars from agricultural wastes. For the present study, MannanaseA and XylanaseA from Clostridium cellulovorans were heterologously expressed in Escherichia coli BL21 (DE3) cells and purified with immobilised metal affinity chromatography. These enzymes, in addition to two commercially available enzyme solutions (Celluclast 1.5L® and Pectinex® 3XL), were assayed on defined polysaccharides that are present in pineapple pomace to determine their substrate specificities. The degree(s) of synergy and specific activities of selected combinations of these enzymes were tested under both simultaneous and sequential conditions. It was observed that several synergistic combinations of enzyme solutions in select ratios, such as C20P60X20 (20% cellulose, 60% pectinase and 20% xylanse), C20P40X40 (20% cellulose, 40% pectinase and 40% xylanase) and C20P80 (20% cellulose, 80% pectinase) with pineapple pomace could both decrease the protein loading, while raising the level of activity compared to individual enzyme solutions. The highest quantity of reducing sugars to protein weight used on pineapple pomace was recorded at 3, 9 and 18 hours with combinations of Pectinex® 3XL and Celluclast 1.5L®, but for 27 h it was combinations of both these commercial solutions with XynA. The contribution of XynA was significant as C20P60X20 displayed the second highest reducing sugar production of 1.521 mg/mL, at 36 h from 12.875 μg/mL of protein, which was the second lowest protein loading. It was also shown that certain enzyme combinations, such as Pectinex® 3XL, Celluclast 1.5L® and XynA, did not generate synergy when combined in solution at the initial stages of hydrolysis, and instead generated a form of competition called anti-synergy. This was due to Pectinex® 3XL which had anti-synergy relationships in select combinations with the other enzyme solutions assayed. It was also observed that the degree of synergy and specific activity for a combination changed over time. Some solutions displayed the highest levels of synergy at the commencement of hydrolysis, namely Celluclast 1.5L®, ManA and XynA. Other combinations exhibited the highest levels of synergy at the end of the assay period, such as Pectinex® 3XL and Celluclast 1.5L®. Whether greater synergy was generated at the start or end of hydrolysis was a function of the stability of the enzymes in solution and whether enzyme activity increased substrate accessibility or generated competition between enzymes in solution. Sequential synergy studies demonstrated an anti-synergy relationship between Pectinex® 3XL and XynA or ManA, as well as Pectinex® 3 XL and Celluclast 1.5L®. It was found that under sequential synergy conditions with Pectinex® 3 XL, XynA and ManA, that anti-synergy could be negated and high degrees of synergy attained when the enzymes were added in specific loading orders and not inhibited by the presence of other active enzymes. The importance of loading order was demonstrated under sequential synergy conditions when XynA was added before ManA followed by Pectinex® 3 XL, which increased the activity and synergy of the solution by 50%. This equates to a 60% increase in reducing sugar release from the same concentrations of enzymes and emphasises the importance of removing anti-synergy relationships from combinations of enzymes. It can be concluded that a C20P60X20 combination (based on activity) can both synergistically increase the reducing sugar production and lower the protein loading required for pineapple pomace hydrolysis. This study also highlights the importance of reducing anti-synergy in customised enzyme cocktails and how sequential synergy can demonstrate the order in which a lignocellulosic waste is degraded.

The aim of this study was to evaluate the effect of glycosylation on exogenous xylanase stability when incubated under proteolytic conditions. Xylanase produced by Trichoderma longibrachiatum, was purified using gel filtration chromatography, ammonium sulfate salt precipitation and dialysis. A partially purified xylanase with Mr of 20- and 10 kDa was identified and contained >65% of the original xylanase activity. Glycoproteins present in the xylanase were identified by thymol sulfuric acid staining or by the FITC-Iabeled lectin method, specific for glycoproteins. This naturally glycosylated xylanase was enzymatically deglycosylated with one of two endo-N-glycosidases: PNGase F or Endo H. Efficiency of deglycosylation was determined with electrophoresis by observing protein mobility shifts or by staining with FITC-Iabeled lectin. The effect of glycosylation on the stability of the exogenous xylanase was tested by incubating the glycosylated or deglycosylated xylanase with rumen fluid (Rf), Prevotella ruminicola culture supernatant (Pr) or a commercial protease from Bacillus subtilis (Bs) for 0, 3, 6, 9 and 24h at 37°C. Results indicated that glycosylated xylanase was significantly more stable (P<0.05) against proteolytic inactivation under the relatively low protease conditions of Rf and Pr (0.018 and 0.046 mg azocasein degraded/ml/h, respectively), but not under high proteolytic conditions of Bs (1.009 mg azocasein/mllh). Also, the glycosylation effect was observed earlier when incubated with the numerous proteases of Rf (3h), than with Pr (9h). These results indicate that glycosylation enhances xylanase stability and therefore is an important characteristic for exogenous enzyme supplements for ruminants. / Dissertation (MSc (Agric))--University of Pretoria, 2005. / Animal and Wildlife Sciences / unrestricted

Orientador: Fabio Marcio Squina, Roberto Ruller / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-22T02:46:06Z (GMT). No. of bitstreams: 1 Hoffmam_ZairaBruna_M.pdf: 4922807 bytes, checksum: df6edae9e54a10424f07783fcf889151 (MD5) Previous issue date: 2013 / Resumo: O etanol de segunda geração, produzido a partir dos polissacarídeos da biomassa lignocelulósica, apresenta-se como uma alternativa promissora para tornar a matriz energética brasileira sólida e, em grande parte, renovável. A biomassa é composta basicamente por celulose, hemicelulose e lignina. Celulose e hemicelulose devem ser hidrolisadas a açúcares monoméricos fermentescíveis para produzir etanol. Entretanto, a tarefa de propor um processo enzimático de hidrólise eficiente e de baixo custo com a finalidade de sacarificar a biomassa é desafiadora, pois os polissacarídeos que a compõem são complexos e recalcitrantes. Embora as hemiceluloses representem cerca de 30% dos açúcares do bagaço da cana-de-açúcar, uma das biomassas mais promissoras para a produção de etanol de segunda geração no Brasil, ainda poucos estudos são direcionados ao estudo da sacarificação dessa fração de polissacarídeos. Hemicelulases são glicosídeo hidrolases que catalisam a hidrólise dos polissacarídeos hemicelulósicos, e que em atuação sinérgica com celulases promove a hidrólise do bagaço de cana-de-açúcar de maneira mais eficiente que as classes de enzimas isoladamente. A primeira parte dessa dissertação apresenta a caracterização bioquímica e biofísica de uma arabinofuranosidase GH51 do micro-organismo Bacillus subtilis 168. ?-L-arabinofuranosidases são hemicelulases que hidrolisam terminais não redutores de polissacarídeos contendo arabinose. A AbfA mostrou-se robusta, atuando numa ampla faixa de temperatura, e capaz de liberar monômeros de arabinose hidrolisando 1,5-?-L-arabinoheptaose a partir da extremidade não-redutora. A análise dos dados de SAXS combinada com simulações de dinâmica molecular mostrou que a AbfA em solução é hexamérica, com cada subunidade contendo uma molécula com domínio catalítico em enovelado na forma de barril (?/?)8 seguido por um domínio acessório ?-sandwich. Esses dados evidenciam que a estrutura quaternária pode ser importante para o desempenho de atividade das arabinofuranosidases da família 51. A segunda parte da dissertação aborda uma avaliação da influência dos domínios de ligação a carboidratos na atividade de xilanases mesofílicas e termofílicas. Xilanases são hemicelulases que catalisam a hidrólise de ligações endo-1,4-?-D-xilosídicas na molécula de xilano. Fusões gênicas uniram end-to-end um CBM6 de Clostridium thermocellum aos domínios catatalíticos xilanásicos das enzimas GH10 termofílica (de Thermotoga petrophila) e GH11 mesofílica (de Bacillus subtilis 168). O CBM6 alterou o padrão de hidrólise das quimeras termofílicas e aumentou a constante de eficiência da quimera mesofílica, quando comparadas as enzimas nativas respectivas. Além disso, na hidrólise da biomassa da cana-de-açúcar a xilanase quimérica mesofílica suplementou o coquetel comercial Accellerase 1500, liberando mais açúcares redutores. Dessa forma a fusão ao CBM6 mostrou-se uma alternativa para construir enzimas com melhor desempenho cinético / Abstract: The second generation ethanol, produced from lignocellulosic biomass polysaccharides, presents itself as a promising alternative to make the Brazilian energy matrix solid and largely renewable. Biomass is composed mainly of cellulose, hemicellulose and lignin. Cellulose and hemicellulose must be hydrolyzed in fermentable monomeric sugars to produce ethanol. However, the task of proposing a process for enzymatic hydrolysis efficient and low cost in order to saccharify the biomass is challenging because its polysaccharides are complex and recalcitrant, so it requires the presence of several different catalytic activities for hydrolysis. Although hemicelluloses represent about 30% of sugars from sugarcane bagasse, one of the most promising biomass for the production of second generation ethanol in Brazil, few studies are directed to the saccharification study of this polysaccharidic fraction. Hemicellulases are glycoside hydrolases that catalyze the hydrolysis of hemicellulosic polysaccharides and works synergistically with cellulases promoting the hydrolysis of sugarcane bagasse more efficiently than each one class of enzymes alone. The first part of this dissertation presents the biochemistry and biophysics characterization of a GH51 arabinofuranosidase from microorganism Bacillus subtilis 168. ?-Larabinofuranosidases are hemicellulases that hydrolyze linkages in terminal nonreducing ends of polysaccharides containing arabinose. The AbfA showed itself robust, working in a wide temperature range, and able to release arabinose monomers hydrolyzing 1,5-?-L-arabinoheptaose from the non-reducing end. The analysis of SAXS data in combination with molecular dynamics simulations showed that AbfA solution is hexameric, with each subunit containing a molecule with catalytic domain coiled in the form of barrel (?/?)8 followed by a ?-sandwich domain accessory. These results evidence and corroborate data that the quaternary structure may be important for the activity of GH51 arabinofuranosidases family. The second part of the thesis adresses an evaluation of the influence of carbohydratebinding modules, non-catalytic domains of polysaccharides recognition, in the activity of mesophilic and thermophilic xylanases. Xylanases are hemicellulases that catalyze the hydrolysis of endo-1 ,4-?-D-xylosidic linkages in xylan molecule. Gene fusions joined end-to-end a CBM6 from Clostridium thermocellum to catalytic xylanasic domains of enzymes thermophilic GH10 (from Thermotoga petrophila) and mesophilic GH11 (from Bacillus subtilis 168). The CBM6 changed the hydrolysis pattern of thermophilic chimeras increased the efficiency constant of mesophilic chimera, when compared to the respective native enzymes. Furthermore, in the sugarcane biomass hydrolysis mesophilic chimeric xylanase supplemented the commercial cocktail Accellerase 1500, releasing more reducing sugars. In conclusion, the CBM6 fusion proved itself an alternative to construct enzymes with better kinetic performance / Mestrado / Bioquimica / Mestra em Biologia Funcional e Molecular

Orientador: Yoon Kil Chang / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-08T15:39:27Z (GMT). No. of bitstreams: 1 Silva_CamilaBatistada_M.pdf: 8457423 bytes, checksum: 91ab09cb0265813f8e1d7894f28eee5a (MD5) Previous issue date: 2007 / Resumo: A demanda por produtos integrais está crescendo a cada dia no Brasil e no mundo. Isso se deve principalmente ao fato destes alimentos estarem relacionados com a saúde. A relação entre dieta e incidência de doenças crônicodegenerativas tem levado os consumidores a se preocuparem mais com a alimentação. Porém, apesar da grande procura por esses alimentos funcionais, a população prefere aqueles que mantenham as características sensoriais dos produtos originais. O objetivo deste trabalho foi estudar a influência da adição de xilanase, glicose oxidase e ácido ascórbico na qualidade do pão de forma, utilizando farinha de trigo de grão inteiro. Nesta farinha foram realizadas análises de composição centesimal, granulometria, teor e índice de glúten, farinografia, extensografia, viscosidade de pasta e falling number. Foi elaborado um delineamento composto central rotacional com três variáveis independentes: xilanase (x1), glicose oxidase (x2) e ácido ascórbico (x3). O delineamento incluiu dezessete ensaios: oito pontos fatoriais, seis pontos axiais e três repetições do ponto central. Os resultados foram analisados por Metodologia de Superfície de Resposta. As variáveis dependentes foram as propriedades reológicas da farinha e as características do pão. Os pães de forma foram analisados quanto ao volume específico, atividade de água, umidade, textura e cor do miolo. Duas formulações, selecionadas na faixa ótima encontrada no delineamento e a formulação padrão foram submetidas a testes de aceitação e de intenção de compra por 37 provadores, que avaliaram os atributos aparência, cor, aroma, sabor e textura. Os resultados mostraram que a adição de xilanase, glicose oxidase e ácido ascórbico interferiu nas características do pão. Quanto ao volume dos pães, houve diferença significativa entre o pão padrão e apenas duas formulações do delineamento. Em relação à umidade dos pães, verificou-se que maiores valores dessa resposta foram encontrados nos pães onde menores níveis de xilanase e maiores níveis de glicose oxidase e ácido ascórbico foram adicionados. O teor de umidade foi reduzido ao longo do período de armazenamento, tanto para o pão padrão, quanto para os pães dos ensaios. Verificou-se que a textura dos pães de forma foi afetada pela adição das enzimas e do ácido ascórbico, ao longo da vida de prateleira. Menores valores de firmeza dos miolos dos pães de forma foram obtidos quando formulações com maiores concentrações de ácido ascórbico e glicose oxidase e níveis intermediários de xilanase foram utilizadas. Em relação à análise sensorial, os consumidores apresentaram boa aceitação e intenção de compra para os pães de forma. Todas as formulações obtiveram médias localizadas entre os termos ¿gostei moderadamente¿ e ¿gostei muito¿ e a maioria dos provadores indicou que provavelmente comprariam os pães. Os resultados mostram que a farinha de trigo de grão inteiro, adicionada de xilanase, glicose oxidase e ácido ascórbico, possui grande potencial na elaboração de pães de forma com alto valor nutritivo e funcional / Abstract: The demand for whole products is increasing day by day both in Brazil and in the world, mainly because such foods are related to health. The relationship between diet and the incidence of chronic-degenerative diseases has led consumers to think more about eating healthy foods. However, despite the great search for such functional foods, the consumers prefer those that maintain the sensory characteristics of the original ones. The objective of this research was to study the effect of adding xylanase, glucose oxidase and ascorbic acid on the quality of loaf bread made using whole-wheat flour. The proximate composition, granule size, gluten content and index, the farinographic and extensographic parameters, pasting viscosity and falling number of the whole-wheat flour were determined. A central composite rotational design was elaborated, with three independent variables: xylanase (x1), glucose oxidase (x2) and ascorbic acid (x3), resulting in seventeen assays: eight factorial points, six axial points and three repetitions at the central point. The results were analyzed using Response Surface Methodology, where the dependent variables were the rheological properties of the flour and the bread quality characteristics. The loaf features evaluated were: specific volume, water activity, moisture content, texture and crumb color. Two formulas, selected in the range of the design designated as excellent and the standard bread formula, were submitted to sensory acceptance and buying intention tests with 37 consumers, who evaluated the attributes of appearance, color, aroma, flavor and texture. The results showed that the addition of xylanase, glucose oxidase and ascorbic acid changed the bread characteristics. With respect to loaf volume, a statistical difference was only found between the standard loaf and two of the formulations, and for moisture content, the highest values were found in the bread with lower concentrations of xylanase and larger amounts of glucose oxidase and ascorbic acid. The moisture content decreased during the shelf life for both the standard bread and all the assay samples. The addition of the enzymes and ascorbic acid was shown to affect the texture of the bread during the shelf life. Lower values for breadcrumb firmness were obtained for the formulas with higher concentrations of ascorbic acid and glucose oxidase and intermediate levels of xylanase, and in the sensorial analysis, good scores were obtained for consumer acceptance and buying intention. All the formulas obtained average scores located between the terms " liked moderately" and "liked very much" and the majority of the consumers indicated they would probably buy the bread. The results showed that the whole-wheat flour used, with the addition of xylanase, glucose oxidase and ascorbic acid, presented considerable potential for the elaboration of loaf bread with high nutritional and functional value / Mestrado / Mestre em Tecnologia de Alimentos

[ES] Las plantas están emergiendo como una alternativa atractiva a los sistemas convencionales de producción heteróloga, como bacterias, cultivos celulares de mamíferos, levaduras u hongos, para la síntesis de productos de alto valor, como metabolitos secundarios y proteínas. La demanda de estos productos a menudo se ve limitada por la capacidad de producción y los costos asociados. Sin embargo, utilizar las plantas como plataformas de producción ofrece ventajas en términos de accesibilidad económica, sostenibilidad, escalabilidad y la ausencia de patógenos humanos y de animales, lo que las convierte en una opción cada vez más atractiva. A pesar de las limitaciones en la capacidad de carga, la expresión génica transitoria utilizando vectores virales proporciona un método eficiente y reproducible para la producción de proteínas recombinantes en plantas, a diferencia de la transformación estable, que requiere más mano de obra y tiempo. Los vectores derivados del virus del mosaico del tabaco (TMV), particularmente aquellos en los que el gen de la proteína de la cubierta viral (CP) se reemplaza principalmente por el gen de interés, son clásicos en la biotecnología vegetal y se utilizan con frecuencia para la producción a gran escala de proteínas recombinantes. En este trabajo, nuestro primer objetivo fue optimizar un vector de TMV para mejorar la producción. La fusión traduccional del extremo amino-terminal de la CP del TMV con la proteína recombinante de interés mostró un aumento en la acumulación de la proteína verde fluorescente, interferón alfa-2a y un nanobody contra la proteína Spike del SARS-CoV-2 en hojas de Nicotiana benthamiana. La inserción de un sitio de clivaje específico, basado en las proteasas de inclusión nuclear (NIaPro) de dos potyvirus, junto con la expresión de las proteasas correspondientes, además de la producción de la proteína recombinante madura, aumentó aún más la acumulación de las proteínas recombinantes mencionadas anteriormente. En segundo lugar, nuestro objetivo fue establecer estrategias para producir proteínas recombinantes de interés industrial y farmacéutico en N. benthamiana utilizando un vector de TMV. Logramos producir grandes cantidades de una xilanasa termofílica, activa en condiciones extremas de temperatura y pH alcalino, en N. benthamiana utilizando un vector de TMV. La enzima que se acumuló rápidamente en los tejidos de la planta se dirigió al apoplasto, lo que facilitó enormemente la purificación y evitó cualquier efecto adverso en el crecimiento de la planta. Se demostró que esta enzima producida en planta es útil para la producción de xilooligosacáridos probióticos. También produjimos grandes cantidades de una glucosa oxidasa (GOX) modificada en hojas de N. benthamiana utilizando un vector de TMV. La GOX producida en planta, que también se purificó fácilmente a partir de fluidos apoplásticos, exhibió potentes propiedades antimicrobianas contra Staphylococcus aureus y Escherichia coli. / [CA] Les plantes están emergint com una alternativa atractiva als sistemes convencionals de producció heteròloga, com ara bacteries, cultius cel·lulars de mamífers, llevats o fongs, per a la síntesi de productes d'alt valor, com son metabòlits secundaris i proteïnes d'interés industrial i farmacéutic. La demanda d'aquests productes sovint es veu limitada per la capacitat de producció i els costos associats. No obstant això, utilitzar les plantes com a plataformes de producció ofereix avantatges en termes d'accessibilitat econòmica, sostenibilitat, escalabilitat i l'absència de patògens humans i animals, la qual cosa les converteix en una opció cada vegada més atractiva. Malgrat les limitacions en la capacitat de càrrega, l'expressió gènica transitoria mitjançant vectors virals proporciona un mètode eficient i reproducible per a la producció de proteïnes recombinants en plantes, a diferència de la transformació estable, que requereix més mà d'obra i temps. Els vectors derivats del virus del mosaic del tabac (TMV), particularment aquells en els quals el gen de la proteïna de la coberta viral (CP) és principalment reemplaçat pel gen d'interès, són clàssics en la biotecnologia vegetal i s'utilitzen sovint per a la producció a gran escala de proteïnes recombinants. En aquest treball, el nostre primer objectiu va ser optimitzar un vector de TMV per a millorar la producció. La fusió traduccional de l'extrem amino-terminal de la CP del TMV amb la proteïna recombinant d'interès va mostrar un augment en l'acumulació de la proteïna verda fluorescent, interferó alfa-2a i un nanobody contra la proteïna Spike del SARS-CoV-2 en fulles de Nicotiana benthamiana. La inserció d'un lloc de tall específic, basat en les proteases d'inclusió nuclear (NIaPro) de dos potivirus, juntament amb l'expressió de les proteases corresponents, a més de la producció de la proteïna recombinant madura, va augmentar encara més l'acumulació de les proteïnes recombinants esmentades anteriorment. En segon lloc, el nostre objectiu va ser establir estratègies per a produir proteïnes recombinants d'interés industrial i farmacèutic en N. benthamiana utilitzant un vector de TMV. Vam aconseguir produir grans quantitats d'una xilanasa termofílica, activa en condicions extremes de temperatura i pH alcalí, en N. benthamiana utilitzant un vector de TMV. El enzim que es va acumular ràpidament en els teixits de la planta es va dirigir a l'apoplast, la qual cosa va facilitar enormement la purificació i va evitar qualsevol efecte advers en el creixement de la planta. Es va demostrar que aquesta enzima produïda en planta és útil per a la producció de xilooligosacàrids probiòtics. També vam produir grans quantitats d'una glucosa oxidasa (GOX) modificada en fulles de N. benthamiana utilitzant un vector de TMV. La GOX produïda en planta, que també es va purificar fàcilment a partir de fluids apoplàstics, va exhibir potents propietats antimicrobianes contra Staphylococcus aureus i Escherichia coli. / [EN] Plants are emerging as an attractive alternative to conventional heterologous production systems, including bacteria, mammalian cell cultures, yeast, or fungi, for the synthesis of high-value products, such as secondary metabolites and proteins. The demand for these products is often limited by production capacity and associated costs. However, using plants as production platforms offers advantages in terms of affordability, sustainability, scalability, and the absence of human and livestock pathogens, making them an increasingly appealing choice. Despite limitations in cargo capacity, transient gene expression using viral vectors provides an efficient and reproducible method for producing recombinant proteins in plants, unlike stable transformation, which is more labor- intensive and time-consuming. Vectors derived from tobacco mosaic virus (TMV), particularly those in which the viral coat protein (CP) gene is mostly replaced with the gene of interest, are classic in plant biotechnology and frequently use for large-scale production of recombinant proteins. In this work, we first aimed to optimize a TMV vector to improve production. Translational fusion of the amino- terminal end of TMV CP to the recombinant protein of interest led to increased accumulation of the green fluorescent protein, interferon alfa-2a and a nanobody against the Spike protein of SARS-CoV-2 in Nicotiana benthamiana leaves. Insertion of a specific cleavage site, based on the nuclear inclusion a proteases (NIaPro) form two potyviruses, along expression of the cognate proteases led to, in addition to production of the mature recombinant protein, a further increase in the accumulation of the aforementioned recombinant proteins. Second, we aimed to set up strategies to produce recombinant proteins of industrial and pharmaceutical interest in N. benthamiana using a TMV vector. We successfully produced large amounts of a thermophilic xylanase, active under extreme temperature and alkaline pH conditions, in N.benthamiana using a TMV vector. The enzyme that accumulated rapidly in plant tissues was targeted the apoplast, which enormously facilitated purification, and avoided any adverse effect on plant growth. This plant-made enzyme was shown to be useful for the production of probiotic xylooligosaccharides. We also produced large amounts of an engineered glucose oxidase (GOX) in N. benthamiana leaves using a TMV vector. The plant-made GOX that was also easily purified from apoplastic fluids exhibited potent antimicrobial properties against Staphylococcus aureus and Escherichia coli. / This work was supported Generalitat Valenciana, grant INNEST/2021/7 from Agència Valenciana de la Innovació, and by grant PID2020-114691RB-I00 from the Spanish Ministerio de Ciencia e Innovación, through the Agencia Estatal de Investigación (co-financed European Regional Development Fund), and by the Bio Based Industries Joint Undertaking, under the European Union’s Horizon 2020 research and innovation program (Project WOODZYMES, Grant Agreement H2020-BBI- JU-792070). M.N.-S. is the recipient of a predoctoral contract from the Spanish Ministerio de Ciencia e Innovación (PRE2018-084771). / Nicolau Sanus, M. (2023). Production of Recombinant Proteins with Pharmaceutical and Industrial Applications in Plants Using a Tobacco Mosaic Virus-Derived Vector [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/200381