?-glucosidases são enzimas que catalisam a hidrólise de ligações glucosídicas ?-1,4, ?-1,3 e ?-1,6 a partir da extremidade não redutora de oligossacarídeos de cadeias pequenas, alquil e aril ?-D-glucosídeos e dissacarídeos. Além de serem enzimas chave do complexo celulolítico, apresentam funções importantes como o melhoramento de aromas de vinhos e a hidrólise de antocianinas. Malbranchea pulchella usualmente é encontrado em fragmentos vegetais em decomposição ou material rico em celulose, podendo ser considerado promissor à produção de enzimas de interesse biotecnológico. Neste contexto, o objetivo desse projeto foi a caracterização funcional de uma ?-glucosidase de M. pulchella e sua aplicação na hidrólise de resíduos agroindustriais e de antocianinas. Uma BGL da família GH3 foi purificada com um fator de purificação 6,32 e recuperação de aproximadamente 35 %. A sua massa molecular aproximado foi de 100 kDa, e o Km, Vmáx e Kcat foram calculadas em 0,33 mM, 13,67 U/mg, 26,5 s-1 respectivamente. O dicroísmo circular revelou uma estrutura composta por aproximadamente 25% de ?-hélices e 20% de ?-folhas. A BGL apresentou pH e temperatura ótimos igual a 6,0 e 50 °C; e foi estável a 40 °C e apresentou boa estabilidade nos pH 5,0 a 8,0, por 24 horas. Nenhum dos sais de íons metálicos ativou a enzima e apenas o HgCl2 inibiu a atividade em 90%. A enzima não apresentou inibição em presença de glucose (0,1-1M) por até 24 horas. Além disso, a GH3 mostrou-se glicosilada e a proporção de açúcar corresponde a 15% massa da enzima. O efeito da celobiose (C) e do bagaço de cana-de-açúcar in natura (BCAN) na produção das BGLs foram avaliados em um DCCR, que indicou um modelo reduzido com influência das duas variáveis. A melhor condição de cultivo para a produção de BGLs foi 0,6% de C (p/v) e 4% de BCAN (p/v). Por meio de um planejamento de mistura, os resíduos BCAN, a casca de soja moída (CS) e o bagaço de cevada (BCev), foram avaliados quanto ao potencial de hidrólise a partir das enzimas presentes no extrato enzimático, resultando no maior potencial de hidrólise sobre o BCev, com a produção de aproximadamente 2 mg/mL de açúcares redutores em 48 horas. As BGLs presentes no extrato enzimático otimizado foram imobilizadas em suporte MANAE-agarose, Concanavalina A-Sepharose e BrCN-Sepharose. Os derivados BGL-MANAE e BGL-ConA foram ativados aproximadamente 10 e 3 vezes, respectivamente. BGL-MANAE e BGL-ConA foram mais estáveis que o controle BGLBrCN em todos os pH testados em 24 horas e, além disso, BGL-ConA permaneceu com 100% de sua atividade em temperaturas de 40 °C, 50 °C e 60 °C, já o BGL-MANAE mostrou-se estável a 40 °C permanecendo com 83% de sua atividade, ambos em 24 horas. BGL-MANAE e BGL-ConA apresentaram menor efeito inibitório em presença de diferentes concentrações de glucose e etanol quando comparados ao BGL-BrCN, e esses resultados indicaram que a imobilização de alguma forma colaborou para uma maior estabilidade ao pH e à temperatura, bem como ao aumento da tolerância por glucose e etanol. Os derivados puderam ser reutilizados por até 20 vezes e quando avaliados quanto à capacidade de clarificar vinhos e sucos de uva (hidrólise de antocianinas), BGL-MANAE clarificou 52% o vinho, 71% o vinho diluído, 77% o suco de uva e 56% o suco de uva diluído, e BGL-ConA em contrapartida clarificou 41% o vinho, 46% o vinho diluído, 63% o suco de uva e 23% o suco de uva diluído. BGL-MANAE foi mais eficiente que BGL-ConA na clarificação de vinhos e sucos de uva podendo ser considerado um biocatalisador promissor na hidrólise de antocianinas e, consequentemente, para a produção de vinhos brancos e rose a partir de diferentes variedades de uvas. Este trabalho pelo que sabemos, é o primeiro a usar BGLs imobilizadas com aplicação na clarificação de sucos de uva e vinhos, podendo ser considerado um trabalho inovador, e de grande importância para a indústria de alimentos e bebidas. / ?-glucosidases are enzymes that catalyze the hydrolysis of ?-1,4, ?-1,3 and ?-1,6 glucosidic linkages, from the non-reducing end of short chain oligosaccharides, alkyl and aryl ?-D-glucosides and disaccharides. Besides being complex key cellulolytic enzymes, they have important functions such as the improvement of wine flavors and anthocyanins hydrolysis. Malbranchea pulchella is usually found in decaying plant debris or in material rich in cellulose, for this reason it can be considered promising for the production of enzymes of biotechnological interest. In this context, the aim of this project was the functional characterization of a ?-glucosidase from M. pulchella and its application in the organic residues and anthocyanins hydrolysis. A BGL GH3 family produced by M. pulchella was purified with a purification factor and recovery of about 6.32 and 35 times. Its approximate molecular mass was 100 kDa, and Km, Vmax and kcat were 0.33 mM, 13.67 U/mg, 26.5 s-1, respectively. The circular dichroism revealed a structure composed of approximately 25% of ?-helix and 20% of ?-sheets. BGL presented optimum pH and temperature at 6.0 and 50 °C; and it was stable at 40 °C. It also showed good stability at pH 5.0 to 8.0, for 24 hours. None of the metal ions salts activated the enzyme and HgCl2 inhibited the activity by 90%. The enzyme showed no inhibition in the presence of glucose (0,1-1M) for 24 hours. Furthermore, it is glycosylated and the sugar proportion correspondsto 15% of the enzyme mass. The effect of cellobiose (C) and sugarcane bagasse in natura (BCAN) in the production of BGLs were evaluated in a CCRD, which indicated a reduced model of influence of the two variables. The best culture condition for BGLs production was 0.6% of C (w/v) and 4% (w/v) of BCAN. Through a mixture design, using the BCAN, ground soybean hulls (CS) and barley bagasse (BCev) were used to evaluate the potential of hydrolysis of these residues in the presence of enzymes present in the enzymatic extract, resulting in a greater efficiency of (BCev) hydrolysis, producing approximately 2 mg/mL of reducing sugars in 48 hours. The BGLs present in the optimized enzyme extract were also used in the immobilization on ionic support MANAE-agarose and affinity support Concanavalin A-Sepharose (ConA-Sepharose). The BGL-MANAE and BGL-ConA derivatives were activated approximately 10 and 3 times, respectively. BGL-MANAE and BGL-ConA were more stable than BGL-BrCN control in all pH tested within 24 hours. In addition, BGL-ConA remained 100% of its activity at 40 °C, 50 °C and 60 °C , and BGL-MANAE was stable at 40 °C and remained 83% of its activity, both in 24 hours. BGL-MANAE and BGL-ConA showed lower inhibitory effect in the presence of different glucose and ethanol concentrations when compared to BGL-BrCN and these results indicate that the immobilization, somehow, cooperated to a greater pH and temperature stability, as well as to increased tolerance by glucose and ethanol. The derivatives could be reused up to 20 times and when they were tested for their capacity to clarify wine and grape juice (anthocyanins hydrolysis), BGL-MANAE clarified 52% wine, 71% diluted wine, 77% grape juice and 56% diluted grape juice. On the other hand, BGL-ConA clarified 41% wine, 46% diluted wine, 63% grape juice and 23% diluted grape juice. BGL-MANAE was more efficient than BGL-ConA in clarifying wines and grape juices and it may be considered a promising biocatalyst in the anthocyanins hydrolysis, and consequently in the production of white and rose wines from different varieties of grapes. This work as we know, is the first to use immobilized BGLs applied in the clarification of grape juice and wine, for this reason, it can be considered an innovative work, and of great importance to the food and beverage industry

?-glucosidases são enzimas que catalisam a hidrólise de ligações glucosídicas ?-1,4, ?-1,3 e ?-1,6 a partir da extremidade não redutora de oligossacarídeos de cadeias pequenas, alquil e aril ?-D-glucosídeos e dissacarídeos. Além de serem enzimas chave do complexo celulolítico, apresentam funções importantes como o melhoramento de aromas de vinhos e a hidrólise de antocianinas. Malbranchea pulchella usualmente é encontrado em fragmentos vegetais em decomposição ou material rico em celulose, podendo ser considerado promissor à produção de enzimas de interesse biotecnológico. Neste contexto, o objetivo desse projeto foi a caracterização funcional de uma ?-glucosidase de M. pulchella e sua aplicação na hidrólise de resíduos agroindustriais e de antocianinas. Uma BGL da família GH3 foi purificada com um fator de purificação 6,32 e recuperação de aproximadamente 35 %. A sua massa molecular aproximado foi de 100 kDa, e o Km, Vmáx e Kcat foram calculadas em 0,33 mM, 13,67 U/mg, 26,5 s-1 respectivamente. O dicroísmo circular revelou uma estrutura composta por aproximadamente 25% de ?-hélices e 20% de ?-folhas. A BGL apresentou pH e temperatura ótimos igual a 6,0 e 50 °C; e foi estável a 40 °C e apresentou boa estabilidade nos pH 5,0 a 8,0, por 24 horas. Nenhum dos sais de íons metálicos ativou a enzima e apenas o HgCl2 inibiu a atividade em 90%. A enzima não apresentou inibição em presença de glucose (0,1-1M) por até 24 horas. Além disso, a GH3 mostrou-se glicosilada e a proporção de açúcar corresponde a 15% massa da enzima. O efeito da celobiose (C) e do bagaço de cana-de-açúcar in natura (BCAN) na produção das BGLs foram avaliados em um DCCR, que indicou um modelo reduzido com influência das duas variáveis. A melhor condição de cultivo para a produção de BGLs foi 0,6% de C (p/v) e 4% de BCAN (p/v). Por meio de um planejamento de mistura, os resíduos BCAN, a casca de soja moída (CS) e o bagaço de cevada (BCev), foram avaliados quanto ao potencial de hidrólise a partir das enzimas presentes no extrato enzimático, resultando no maior potencial de hidrólise sobre o BCev, com a produção de aproximadamente 2 mg/mL de açúcares redutores em 48 horas. As BGLs presentes no extrato enzimático otimizado foram imobilizadas em suporte MANAE-agarose, Concanavalina A-Sepharose e BrCN-Sepharose. Os derivados BGL-MANAE e BGL-ConA foram ativados aproximadamente 10 e 3 vezes, respectivamente. BGL-MANAE e BGL-ConA foram mais estáveis que o controle BGLBrCN em todos os pH testados em 24 horas e, além disso, BGL-ConA permaneceu com 100% de sua atividade em temperaturas de 40 °C, 50 °C e 60 °C, já o BGL-MANAE mostrou-se estável a 40 °C permanecendo com 83% de sua atividade, ambos em 24 horas. BGL-MANAE e BGL-ConA apresentaram menor efeito inibitório em presença de diferentes concentrações de glucose e etanol quando comparados ao BGL-BrCN, e esses resultados indicaram que a imobilização de alguma forma colaborou para uma maior estabilidade ao pH e à temperatura, bem como ao aumento da tolerância por glucose e etanol. Os derivados puderam ser reutilizados por até 20 vezes e quando avaliados quanto à capacidade de clarificar vinhos e sucos de uva (hidrólise de antocianinas), BGL-MANAE clarificou 52% o vinho, 71% o vinho diluído, 77% o suco de uva e 56% o suco de uva diluído, e BGL-ConA em contrapartida clarificou 41% o vinho, 46% o vinho diluído, 63% o suco de uva e 23% o suco de uva diluído. BGL-MANAE foi mais eficiente que BGL-ConA na clarificação de vinhos e sucos de uva podendo ser considerado um biocatalisador promissor na hidrólise de antocianinas e, consequentemente, para a produção de vinhos brancos e rose a partir de diferentes variedades de uvas. Este trabalho pelo que sabemos, é o primeiro a usar BGLs imobilizadas com aplicação na clarificação de sucos de uva e vinhos, podendo ser considerado um trabalho inovador, e de grande importância para a indústria de alimentos e bebidas. / ?-glucosidases are enzymes that catalyze the hydrolysis of ?-1,4, ?-1,3 and ?-1,6 glucosidic linkages, from the non-reducing end of short chain oligosaccharides, alkyl and aryl ?-D-glucosides and disaccharides. Besides being complex key cellulolytic enzymes, they have important functions such as the improvement of wine flavors and anthocyanins hydrolysis. Malbranchea pulchella is usually found in decaying plant debris or in material rich in cellulose, for this reason it can be considered promising for the production of enzymes of biotechnological interest. In this context, the aim of this project was the functional characterization of a ?-glucosidase from M. pulchella and its application in the organic residues and anthocyanins hydrolysis. A BGL GH3 family produced by M. pulchella was purified with a purification factor and recovery of about 6.32 and 35 times. Its approximate molecular mass was 100 kDa, and Km, Vmax and kcat were 0.33 mM, 13.67 U/mg, 26.5 s-1, respectively. The circular dichroism revealed a structure composed of approximately 25% of ?-helix and 20% of ?-sheets. BGL presented optimum pH and temperature at 6.0 and 50 °C; and it was stable at 40 °C. It also showed good stability at pH 5.0 to 8.0, for 24 hours. None of the metal ions salts activated the enzyme and HgCl2 inhibited the activity by 90%. The enzyme showed no inhibition in the presence of glucose (0,1-1M) for 24 hours. Furthermore, it is glycosylated and the sugar proportion correspondsto 15% of the enzyme mass. The effect of cellobiose (C) and sugarcane bagasse in natura (BCAN) in the production of BGLs were evaluated in a CCRD, which indicated a reduced model of influence of the two variables. The best culture condition for BGLs production was 0.6% of C (w/v) and 4% (w/v) of BCAN. Through a mixture design, using the BCAN, ground soybean hulls (CS) and barley bagasse (BCev) were used to evaluate the potential of hydrolysis of these residues in the presence of enzymes present in the enzymatic extract, resulting in a greater efficiency of (BCev) hydrolysis, producing approximately 2 mg/mL of reducing sugars in 48 hours. The BGLs present in the optimized enzyme extract were also used in the immobilization on ionic support MANAE-agarose and affinity support Concanavalin A-Sepharose (ConA-Sepharose). The BGL-MANAE and BGL-ConA derivatives were activated approximately 10 and 3 times, respectively. BGL-MANAE and BGL-ConA were more stable than BGL-BrCN control in all pH tested within 24 hours. In addition, BGL-ConA remained 100% of its activity at 40 °C, 50 °C and 60 °C , and BGL-MANAE was stable at 40 °C and remained 83% of its activity, both in 24 hours. BGL-MANAE and BGL-ConA showed lower inhibitory effect in the presence of different glucose and ethanol concentrations when compared to BGL-BrCN and these results indicate that the immobilization, somehow, cooperated to a greater pH and temperature stability, as well as to increased tolerance by glucose and ethanol. The derivatives could be reused up to 20 times and when they were tested for their capacity to clarify wine and grape juice (anthocyanins hydrolysis), BGL-MANAE clarified 52% wine, 71% diluted wine, 77% grape juice and 56% diluted grape juice. On the other hand, BGL-ConA clarified 41% wine, 46% diluted wine, 63% grape juice and 23% diluted grape juice. BGL-MANAE was more efficient than BGL-ConA in clarifying wines and grape juices and it may be considered a promising biocatalyst in the anthocyanins hydrolysis, and consequently in the production of white and rose wines from different varieties of grapes. This work as we know, is the first to use immobilized BGLs applied in the clarification of grape juice and wine, for this reason, it can be considered an innovative work, and of great importance to the food and beverage industry

Cette étude a pour objectif la mise en évidence, chez les basidiomycetes du genre Pycnoporus, de nouvelles oxydo-réductases impliquées dasn la dégradation de la biomasse végétale: de l'expression des gènes aux applications biotechnologiques. Les champs d'application visés concernent essentiellement le domaine de la chimie verte, dans le cadre du projet européen BIORENEW. Le travail s'est articulé autour de trois axes principaux. Le premier a concerné l'exploration de la biodiversité naturelle en particulier tropicale, pour la sélection de souches productrices de nouvelles laccases de haut potentiel d'oxydo-réduction. Le gène codant pour la laccase Lac1 chez Pycnoporus a été utilisé comme marqueur moléculaire d'identification et de relation phylogénie-fonction, mettant en évidence une distribution des souches fortement corrélée avec leur écozone. Le deuxième axe a porté sur l'isolement de trois nouvelles laccases issues de P.sanguineus et P. coccineus qui exhibent des caractéristiques biochimiques complémentaires: haute thermostabilité, résistance aux solvants, au pH, constantes catalytiques et potentiels rédox élevés. Ces enzymes constituent de bons modèles pour des applications en biotechnologies blanches:décoloration de colorants polyphénoliques, oxydation de composés modèles de type lignine non-phénolique, oligomérisation de flavonoides naturels adaptés aux applications cosmétiques et pharmaceutiques. Enfin, dans le cadre de l'annotation du génome des souches monocaryotiques P. cinnabarinus BRFM 137 et P; sanguineus BRFM 1264, dont le séquençage a été réalisé par notre Unité, un regard tout à fait nouveau est porté sur le système lignolytique du genre Pycnoporus, longtemps décrit comme produisant que de la laccase comme enzyme du système lignolytique. Pour la première fois, nous avons montré la présence de gènes codant pour tout l'arsenal enzymatique de dégradation des lignines, c'est à dire plusieurs laccases mais surtout de nombreuses peroxydases et des enzymes auxilliaires génératrices d'H2O2 comme les glyoxal oxydases. Ces nouvelles enzymes ont été caractérisées in silico. Pour la première fois également, la sécrétion effective de peroxydases, de glyoxal oxydases et d'autres FOLymes dans nos conditions de culture a également pu être démontrée par analyse protéomique. / The purpose of this work was to prospect, in the genus Pycnoporus, for new oxido-reductases involved in the degradation of lignocellulosic biomass: from gene expression to biotechnological applications. This research was conducted in the framework of green chemistry applications according to BIORENEW European Project. The study was divided in three main research axes. Firstly, the exploration of natural biodiversity, especially tropical biodiversity, for the selection of new high redox potential-laccase producing strains. These strains were repositionned in a context of phylogenomic/function through the lac1 gene. Molecular clustering based on lac1 sequences enabled the distribution of P. sanguineus and P. coccineus through four distinct, well supported clades and subclades. This distribution was highly correlated with ecozones. The second part of the work deals with the biochemical and molecular characterization of three novel laccases from P. coccineus and P. sanguineus, and their applicability on natural or model phenolic substrates. The three laccases showed complementary biochemical features: high thermo- and pH stability, high catalytic efficiency and resistance to organic solvents. The three novel laccases proved to be suitable models for white biotechnology processes: polyphenolic dye decolourization, non-phenolic lignin model compound oxidation, and synthesis of new oligomers from natural flavonoids suitable for cosmetic or pharmaceutical applications. Finally, annotation of genomic data from the monocaryotic strains P. cinnabarinus BRFM 137 and P. sanguineus BRFM 1264 (genomes sequenced by the UMR1163 BCF ) was performed for lignolytic enzymes. For the first time, new oxidases (peroxidases, glyoxal oxidases and other FOLymes) were evidenced in Pycnoporus and in silico characterized. Moreover, the active secretion of several of these enzymes has been demonstrated in our culture conditions by 1D-proteomic analysis

Living cells accumulate compensatory solutes for protection against the harmful effects of extreme environmental conditions such as high salinity, temperature and desiccation. Even at high concentrations these solutes do not disrupt the normal cellular functions and at times counteract by stabilizing the cellular components. These properties of compensatory solutes have been exploited for stabilizing proteins and cells in vitro. Betaines are widespread natural compensatory solutes that have also been used in other applications such as therapeutic agents and polymerase chain reaction (PCR) enhancers. Some biomedical applications of novel synthetic analogues of natural betaines were investigated. Natural compensatory solutes are either dipolar zwitterionic compounds or polyhydroxyl compounds, and the physical basis of compensation may differ between these, so one focus was on synthetic betaines with hydroxyl substituents. The majority of the synthetic solutes stabilized different model proteins against stress factors such as high and low temperatures. The presence of hydroxyl groups improved protection against desiccation. The observed stabilization effect is not just on the catalytic activity of the enzyme, but also on its structural conformation. Synthetic compensatory solutes have a potential application as protein stabilizers. Dimethylthetin was evaluated as a therapeutic agent and found to be harmful in a sheep model. However, from the study we were able to generate a large-animal continuous ambulatory peritoneal dialysis (CAPD) model and showed that glycine betaine could be added to the dialysis fluid in chronic renal failure. Some synthetic compensatory solutes reduce the melting temperatures of DNA better than most natural solutes. Synthetic solutes were identified that have potential to enhance PCR and could replace some reagents marketed by commercial suppliers. Density, viscosity and molecular model data on the solutes showed correlations with the biochemical effects of the solutes, but no physical measurements were found that reliably predicted their potential for biotechnological applications.