Mitochondria are a key organelle within human cells, with functions ranging from ATP synthesis to apoptosis. Changes in mitochondrial function are associated with many diseases, as well as 'natural' processes like ageing. Mitochondria have a unique evolutionary origin, as the result of an endosymbiotic relationship between a bacterium and an archaeal cell. Therefore, the phylogenetic history of the mitochondrial proteome is also unique within the total human proteome. A new description of the genes encoding the human mitochondrial proteome - IMPI (Integrated Mitochondrial Protein Index) 2017 - provided an opportunity for exploration of mitochondrial proteome history and the application of this knowledge to the understanding of gene function, disease and ageing. To facilitate the exploration of the mitochondrial proteome, I created a manually curated dataset of 190,097 predicted orthologues of the 1,550 IMPI 2017 human genes across 359 species, using reciprocal best hit analysis as the basis for orthologue prediction. I used this to explore gene history and the potential for phylogenetic profiling to predict the function of uncharacterised genes. This inspired the use of phylogenetic profiling within two phyla of animals, to link presence and absence of metabolic genes to the function of mitochondrial transporters. Potential transport substrates were predicted for two groups of uncharacterised mitochondrial carriers. I also used the dataset to identify features of genes associated with monogenetic disease, as well as differences between recessive and dominant disease genes. A similar orthologue identification method was used to explore the total sequenced viral proteome for potential orthologues of mitochondrial proteins. This showed that a range of mitochondrial proteins are shared with viruses, potentially facilitating the co-opting of mitochondrial function during viral infection of eukaryotic cells. I then used orthology to explore the conservation of residues linked to protein acetylation and identify a link with lifespan in warm-blooded vertebrates. In conclusion, I have used orthology to further the understanding of human mitochondrial proteome history and developed applications of this information. For example, phylogenetic features of disease genes are being used as part of a wider pipeline to predict mitochondrial disease genes. Furthermore, predicted substrates of the SLC25A14/30 mitochondrial carriers are being tested. My dataset provides further opportunities to explore the evolution and function of the mitochondrion.

O presente trabalho teve como principal objetivo correlacionar a composição química do bagaço de cana-de-açúcar pré-tratado com H2SO4 diluído com a eficiência da sacarificação enzimática da celulose presente no material. Primeiramente, o bagaço in natura foi extraído com água, etanol ou água seguida de etanol, e as composições químicas determinadas. Posteriormente, o bagaço in natura foi pré-tratado com H2SO4 diluído em êmbolos de 200 mL, utilizando 15% de teor de sólidos (m/v). A temperatura (112,5-157,5 °C), o tempo de residência (5-35 min) e a concentração ácida (0-3,0% m/v) variaram de acordo com um planejamento fatorial 23 completo. Após o pré-tratamento, as amostras foram caracterizadas quimicamente. Em seguida, dois extratos enzimáticos comerciais foram caracterizados quanto às atividades de enzimas hidrolíticas e fenoloxidases, e aos teores de proteínas. As condições adequadas de sacarificação enzimática da celulose para a amostra de bagaço pré-tratada com H2SO4 diluído (15% sólidos, 2% ácido, a 150ºC por 30 min) foram determinadas através de planejamentos fatoriais 23 completos, variando teor de sólidos (1,19-4,81% m/v), carga enzimática (1,91-38,09 FPU/g de bagaço) e carga de surfactante (0-0,1 g/g de bagaço), para os dois extratos enzimáticos. As amostras de bagaço pré-tratadas sob diferentes condições de temperatura, tempo de residência e concentração de H2SO4 (primeiro planejamento fatorial) foram submetidas à sacarificação enzimática com um dos extratos. Por fim, amostras selecionadas foram caracterizadas quanto às alterações morfológicas provocadas pelo pré-tratamento e pela hidrólise enzimática, por microscopia eletrônica de varredura. Os resultados mostraram que água seguida de etanol extraiu maior quantidade de extrativos do bagaço. Os extrativos apresentaram absorção de luz apenas na região do ultravioleta. A porcentagem de lignina nos bagaços extraídos com água, etanol e água seguida de etanol foi menor que aquela encontrada no bagaço in natura. De acordo com a condição de pré-tratamento, os teores de celulose, hemicelulose e lignina nos bagaços pré-tratados diferiram substancialmente. A maior variação foi observada para hemicelulose (3,67-27,27%). Os três fatores avaliados no pré-tratamento influenciaram na composição química do bagaço pré-tratado. Por sua vez, os dois extratos enzimáticos apresentaram um complexo celulolítico completo e considerável atividade de xilanases, porém não foi observada atividade de fenoloxidases. O extrato II apresentou maior quantidade de proteínas (152,45±10,0 mg/mL), comparado ao extrato I (105,2±6,6 mg/mL). Para 24 horas de hidrólise enzimática com o extrato I, as três variáveis independentes influenciaram na digestão do bagaço pré-tratado. Somente os efeitos das cargas de enzima e surfactante foram significantes, utilizando o extrato II. Posteriormente, foi possível aumentar o teor inicial de sólidos sem comprometer o rendimento de sacarificação com o extrato II. Não foi possível correlacionar a conversão de celulose com o fator de severidade do pré-tratamento. Por outro lado, foi observada correlação negativa entre o conteúdo de hemicelulose e a conversão enzimática de celulose, enfatizando a influência da composição química do bagaço de cana na hidrólise enzimática da celulose. Observaram-se diferenças morfológicas entre o bagaço in natura e amostras pré-tratadas sob condições branda e severa, bem como após suas respectivas hidrólises enzimáticas. / This study aimed to correlate the chemical composition of a sugarcane bagasse pretreated with dilute H2SO4 with the efficiency of cellulose enzymatic saccharification of this material. First, the sugarcane bagasse was extracted with water, ethanol or water followed by ethanol, and its chemical composition was determined. Subsequently, the sugarcane bagasse was pretreated with dilute H2SO4 in 200 mL stainless steel containers, using 15% of solids loading (w/v). The temperature (112.5-157.5°C), time of residence (5-35 min) and acid concentration (0-3.0% w/v) varied according to a 23 full factorial design. After the pretreatments, the chemical compositions of the pretreated bagasses were determined. Then, two commercial enzymatic extracts were characterized regarding the activities of hydrolytic enzymes and phenoloxidases, and protein contents. The enzymatic saccharification conditions for the bagasse sample pretreated with dilute H2SO4 (15% solids, 2% acid, 150°C for 30 min) were determined through 23 full factorial designs, varying the solids loading (1,19-4.81% w/v), enzyme loading (1.91-38.09 FPU/g of bagasse) and surfactant loading (0-0.1 g/g of bagasse) for the two enzymatic extracts. The bagasse samples pretreated under the different conditions of temperature, time of residence and H2SO4 concentration (first factorial design) were subjected to enzymatic saccharification using one of the extracts. Finally, selected samples were analyzed for morphological changes caused by pretreatment and enzymatic hydrolysis, by scanning electron microscopy. The results showed that water followed by ethanol extracted the highest amount of extractives. The extractives showed light absorption only in the ultraviolet region. The percentage of lignin in the bagasse samples extracted with water, ethanol and water followed by ethanol was lower than that found in the raw material. According to the pretreatment conditions, the amount of cellulose, hemicellulose and lignin in the pretreated bagasse differed substantially. The greatest variation was observed for the hemicellulose content (3.67-27.27%). All the three factors evaluated in the pretreatment affected the chemical composition of the pretreated bagasse. In turn, the two enzymatic extracts showed complete cellulolytic complexes and considerable activities of xylanases, without activities of phenoloxidases. The extract II showed higher protein content (152.45±10.0 mg/mL) when compared with the extract I (105.2±6.6 mg/mL). For 24 hours of enzymatic hydrolysis using the extract I, all the three independent variables influenced the saccharification of pretreated bagasse. Only the enzyme and surfactant loadings were significant, when using the extract II. Later, it was possible to increase the initial solids content without hindering the saccharification yield, using the extract II. It was not possible to correlate the cellulose conversion with the pretreatment severity. On other hand, it was possible to observe a negative correlation between the hemicellulose content and the efficiency of enzymatic conversion, emphasizing the influence of the sugarcane bagasse chemical composition in the enzymatic hydrolysis of cellulose. Morphological differences were observed between the raw material and sugarcane bagasse samples pretreated under high or low severity, as well as after their corresponding enzymatic hydrolysis.

O cultivo da cana-de-açúcar é uma das atividades agrícolas mais importantes no Brasil, produzindo após a moagem o caldo, utilizado para a produção de açúcar e etanol, e o bagaço, resíduo lignocelulósico. O bagaço é recalcitrante à hidrólise enzimática, em parte pela baixa porosidade, resultante do recobrimento das fibrilas de celulose com lignina e hemicelulose. Neste estudo, o bagaço foi pré-tratado com sulfito alcalino nas concentrações de 2,5% de NaOH e 5% de Na2SO3 versus 5% de NaOH e 10% de Na2SO3 para produzir substratos para hidrólise enzimática. Ambos pré-tratamentos produziram substratos com teor de hemicelulose, grupos ácidos, grau de retenção de água e área superficial semelhantes. O conteúdo de lignina foi bem diferente nos bagaços pré-tratados com 5% de sulfito (21% de lignina) e 10% de sulfito (13% de lignina). A hidrólise da celulose e hemicelulose do bagaço com alto teor de lignina, utilizando a carga enzimática de 40 FPU/g e 80 U/g de ?-glicosidase foram próximas a 50% em 48 horas e, mesmo no bagaço com pouca lignina a conversão dos polissacarídeos não foi completa (90%). Considerando a importância do tipo de enzimas para a conversão dos polissacarídeos dos bagaços pré-tratados, realizou-se um planejamento experimental 24 com 6 pontos centrais ampliado em estrela, para avaliar uma mistura de enzimas partindo de 5 FPU/g do extrato comercial de Trichoderma reesei (celluclast) combinado com enzimas purificadas comerciais: xilanase de Neocallimastix patriciarum (família 10), xilanase de Thermotoga maritima (família 11), ?-xilosidase de Selelomonas ruminantium e ?-glicosidase de Aspergillus niger. A aplicação da mistura de enzimas otimizada no bagaço pré-tratado com alta carga de sulfito aumentou a conversão de celulose e hemicelulose em 6,6% e 15% respectivamente, comparado com a mistura de referência (5FPU de celluclast e 10UI de Novozyme 188 por grama de bagaço). A suplementação da celluclast com ?-xilosidase e ?- glicosidase foi estatisticamente significativa em 24 horas de hidrólise a um nível de 95% de confiança e a interação da xilanase 10 e 11 foi significativa com um nível de confiança de 90%. Quando foram realizados os mesmos ensaios do planejamento com o substrato com alto teor de lignina, as hidrólises da celulose e hemicelulose com a mistura de enzimas foram inferiores à obtida com a referência. A suplementação com xilanases e ?-xilosidase aumentou a conversão enzimática da hemicelulose apenas do substrato com pouca lignina, entretanto nos hidrolisados de ambos os substratos foi detectada a presença de xilooligossacarídeos, indicando a necessidade de adição de mais ?-xilosidase à mistura enzimática. As velocidades iniciais de hidrólise da celulose e hemicelulose foram pouco alteradas quando a lignina do bagaço reduziu de 21% para 13%, porém a conversão em 48 h de reação foi o dobro. Este estudo mostrou que o acesso das enzimas à hemicelulose foi limitado pelo alto teor de lignina do substrato, e que o benefício do uso de xilanases para a conversão de celulose foi obtido no substrato pré-tratado com alta carga de sulfito. / The cultivation of sugarcane is one of the most important agricultural activities in Brazil. The juice obtained from the crushed stalks of sugarcane is used to produce sugar and ethanol and the dry, fibrous residue remaining is the bagasse. Bagasse is recalcitrant to enzymatic hydrolysis, in part by low porosity due to the partial filling of space between the cellulose microfibrils by lignin and hemicelluloses. In this study, bagasses were pretreated with alkaline sulfite at concentrations of 2.5% NaOH and 5% Na2SO3 NaOH versus 5% and 10% Na2SO3 to produce substrates for enzymatic hydrolysis. Both substrates presented similar hemicellulose content, acid groups, water retention and specific surface area. Lignin content differed between pretreated bagasse with 5% sulfite (21%) and 10% sulfite (13%). The hydrolysis of cellulose and hemicellulose of bagasse with high lignin content, using 40 FPU/g and 80 U/g of ?-glucosidase was aproximately 50% in 48 hours and even on bagasse with low lignin content, the polysaccharides conversion was not complete (90%). Considering the importance of the type of enzymes for the conversion of polysaccharides of pretreated bagasses, a 24 full factorial experimental design with six central points was performed to evaluate a mixture of enzymes. A load of 5 FPU/g of Trichoderma reesei extract (celluclast) was combined with purified commercial enzymes: Neocallimastix patriciarum xylanase (family 10), Thermotoga maritima xylanase (family 11), Selelomonas ruminantium ?-xylosidase and ?-glucosidase from Aspergillus niger. The optimized mixture improved the conversion of cellulose and hemicellulose of the substrate with low lignin content in 6.6% and 15% respectively, when compared to the reference mixture (5FPU of celluclast and 10 IU of novozyme 188 per gram of bagasse). Supplementation with ?-xylosidase and ?-glucosidase was statistically significant at 24 hours of reaction and also the interaction of xylanases 10 and 11. When the same assays were performed with the substrate with low lignin, hydrolysis of the cellulose and hemicellulose with a mixture of purified enzymes was inferior to that obtained by the reference. Supplementation with xylanase and ?-xylosidase improved the enzymatic conversion only for substrate with low lignin content, however in supernatants of both substrates was detected the presence of xylo-oligosaccharides, suggesting the need for further addition of ?-xylosidase to the enzyme mixture. Initial rate of cellulose and hemicellulose hydrolysis changed very little when the lignin in the bagasse was reduced from 21% to 13%, but the conversion at 48 h conversion time was twice higher. This study showed that access of enzymes to the hemicellulose was limited by the high lignin content of the substrate, and that the benefit of using xylanases for the conversion of cellulose was obtained on the substrate pretreated with high sulfite load.

O presente projeto teve como objetivo selecionar lipases comerciais para a interesterificação enzimática da gordura do leite com óleo de soja visando à obtenção de produto enriquecido com ácidos graxos essenciais. Foram testadas lipases de diferentes fontes microbianas (Aspergillus niger, Mucor javanicus, Rhizopus oryzae, Candida rugosa, Penicillium roqueforti e Rhizomucor miehei) imobilizadas em matriz híbrida polissiloxano-álcool polivinílico (POS-PVA) previamente caracterizada quanto às suas propriedades físicoquímicas e morfológicas. Tanto as enzimas livres como imobilizadas, foram inicialmente caracterizadas com relação às suas atividades hidrolítica e de esterificação. Em função da alta complexidade das matérias-primas \"gordura do leite e óleo de soja\", optou-se pela triagem inicial da enzima em um sistema reacional modelo de interesterificação. Como reagentes de partida, foram escolhidos tripalmitina e trioleína, uma vez que os ácidos graxos palmítico e oléico estão presentes em grande quantidade nos triglicerídeos que compõem as matériasprimas de interesse. Assim, as lipases imobilizadas no suporte POS-PVA foram empregadas na catálise de reações de interesterificação no sistema modelo empregando-se hexano como solvente. O valor mais elevado de rendimento de interesterificação (31%) foi obtido com o emprego da lipase de Rhizopus oryzae (L036P), sendo esta enzima selecionada para continuidade do trabalho. Testes adicionais foram efetuados para verificar a influência da concentração de trioleína e tripalmitina no rendimento da reação. Neste caso, o melhor resultado foi alcançado no substrato equimolar de tripalmitina e trioleína (40mM), para o qual observou-se um rendimento de interesterificação de 45%, em 24 h de reação. O sistema imobilizado selecionado foi caracterizado quanto às suas propriedades físico-químicas e de estabilidade, empregando-se técnicas como difratometria de raios-X e espectroscopia no infravermelho. Foi observada boa estabilidade à estocagem, mantendo-se 100% da atividade hidrolítica inicial após 120 dias de armazenamento. Finalmente, foram realizados testes de interesterificação da gordura do leite com óleo de soja sendo que, de maneira geral, a lipase de Rhizopus oryzae imobilizada em POS-PVA promoveu o aumento na concentração dos triglicerídeos C26-C34 e C46-C50, e diminuição dos triglicerídeos C38, C44 e C54, obtendo-se 12% de rendimento de interesterificação. A análise de textura do meio reacional e do produto interesterificado mostrou que a simples mistura física de óleo de soja com a gordura do leite provocou uma diminuição de mais de 50% na consistência desta gordura. Após a reação de interesterificação, o produto obtido apresentou redução de aproximadamente 24 % na consistência em relação à da mistura física das matérias-primas, devido à incorporação dos ácidos graxos insaturados do óleo de soja nos triglicerídeos presentes na gordura do leite, e à formação de mono e diglicerídeos devida à hidrólise parcial das matérias-primas. Assim, os resultados promissores obtidos com o uso da lipase de Rhizopus oryzae (L036) imobilizada em POS-PVA, tanto na reação modelo de interesterificação de tripalmitina com trioleína quanto nas matérias-primas lipídicas, possibilitaram a seleção deste sistema enzimático visando ao seu emprego no bioprocesso de interesse. / The objective of this work was to select a commercial lipase for enzymatic interesterification of milkfat with soybean to obtain a product enriched with essential fatty acids. Lipases from different microbial sources (Aspergillus niger, Mucor javanicus, Rhizopus oryzae, Candida rugosa, Penicillium roqueforti, Rhizomucor miehei) were immobilized on polysiloxane- polyvinyl alcohol hybrid matrix (POS-PVA) previously characterized in relation to its physico-chemical and morphological properties. All lipase preparations in both free and immobilized forms were initially characterized, regarding their hydrolytic and esterification activities. Owing to the high complexity of the raw materials \"milkfat and soybean oi\", the lipase screening assay was carried out using a model interesterification system consisted of tripalmitin and triolein. These triglycerides were chosen as starting materials since palmitic and oleic fatty acids are present at high amount proportions in the raw materials. Thus, immobilized lipase derivatives on POS-PVA were used to catalyze the interesterification of tripalmitin with triolein in the presence of hexane as solvent. The highest interesterification (31%) was obtained using the lipase from Rhizopus oryzae (L036P) and therefore, this enzyme was selected for continuing the work. Additional tests were performed to verify the influence of the molar ratio between triolein and tripalmitin in the reaction yield. The best result was achieved for the substrate containing equimolar amounts of tripalmitin and triolein (40 mM), attaining 45% of interesterification yield at 24 h reaction. The selected immobilized system was characterized in relation to its physico-chemical and stability properties using techniques as X-ray difractometry and IR spectroscopy. High stability was found for the immobilized lipase that maintained full original activity after 120 days storage at 4?C. Finally, interesterification tests of milkfat with soybean oil were carried out, and the use of Rhizopus oryzae lipase immobilized in POS-PVA increased the concentration of the triglycerides C26- C34 and C46-C50, and decreased the concentration of the triglycerides C38, C44 and C54, which corresponded to 12% of interesterification yield. The texture analysis of both reaction medium and interesterified product showed that simple physical blend of soybean oil with milk fat reduced more than 50% the milkfat consistency. After the interesterification reaction, the obtained product showed even lower consistency (24% reduction) in comparison with the physical raw materials blend, due to the incorporation of unsaturated fatty acids from soybean oil in the triglycerides of milkfat, and to the formation of mono and diglycerides resulting from partial hydrolysis of the raw materials. Thus, the promising results obtained using Rhizopus oryzae lipase (L036P) immobilized on POS-PVA, both in the model interesterification reaction of tripalmitin with triolein and in the lipid raw materials, indicated the suitability of the selected immobilized derivative to be applied in the proposed bioprocess.

Os materiais lignocelulósicos possuem características que limitam a sacarificação enzimática da celulose. Entre essas características pode-se classificar a porosidade como uma das mais importantes, sendo usualmente mensurada pela técnica de exclusão de solutos. A secagem do material também pode aumentar sua recalcitrância por meio do fenômeno de hornificação. Neste contexto, o presente trabalho teve como objetivo avaliar a influência do teor de lignina e da secagem na porosidade e na sacarificação enzimática do bagaço de cana-de-açúcar. A partir de uma amostra in natura quatro amostras com teores de lignina decrescentes foram geradas, utilizando o método de deslignificação por clorito-ácido. Uma fração destas amostras foi seca ao ar em temperatura ambiente e o restante teve seu teor de umidade mantido. A análise das modificações estruturais promovidas pelo tratamento citado foi feita utilizando microscopia eletrônica de varredura (MEV). A porometria das amostras, determinada via técnica de exclusão de solutos, foi realizada utilizando 1 g de massa seca de amostra e 20 g de soluções de sondas moleculares com concentração de 1,5% (m/m); após 24 h sob agitação manual ocasional a 25 °C a determinação da distribuição do volume e área superficial de poros foi realizada com base na redução da concentração inicial da solução. O valor de retenção de água das amostras foi calculado via centrifugação. As amostras foram submetidas à sacarificação com cargas enzimática e de surfactante de 10 FPU e 0,025 g de Tween 20 por grama de bagaço, respectivamente. A reação ocorreu a 45 °C sob agitação de 150 rpm e a conversão de celulose foi medida após 2, 8, 24 e 72 h. As amostras deslignificadas por 1, 2, 3 e 4 horas apresentaram 14,2; 9,2; 8,0 e 5,9% de lignina, respectivamente, enquanto que a amostra in natura foi composta por 20,7%. Por meio das análises feitas por MEV, pôde-se observar que a remoção de lignina acarretou em uma descompactação estrutural dos feixes vasculares. As amostras com maiores teores de lignina apresentaram menores volumes e áreas superficiais de poros e piores conversões enzimáticas de celulose. Para a amostra in natura o volume total de poros foi de 0,89 mL/g de bagaço enquanto que para as amostras deslignificadas por 1, 2, 3 e 4 horas este volume foi de 1,19, 1,77, 1,92 e 2,21 mL/g de bagaço, respectivamente. A secagem reduziu o volume total de poros das amostras deslignificadas por 2, 3 e 4 horas para 1,47, 1,55 e 1,98 mL/g de bagaço respectivamente. Os valores de retenção de água foram similares aos valores de volume total de poros obtidos via técnica de exclusão de solutos. Enquanto cerca de apenas 20% da celulose da amostra in natura foi convertida após 72 h de sacarificação, a amostra com o menor teor de lignina apresentou conversão próxima a 100%. A secagem das amostras deslignificadas não alterou as taxas nem os rendimentos de sacarificação. Pode-se concluir então que o teor de lignina desempenha um papel importante na limitação da sacarificação enzimática da celulose, e que sua remoção implica num aumento do volume de poros do material. / Lignocellulosic materials present characteristics that limit the enzymatic saccharification of cellulose. Among these features, the porosity, usually measured by the solute exclusion technique, can be classified as one of the most important. Drying of the material can also increase its recalcitrance by the hornification phenomenon. In this context, this study aimed to evaluate the influences of lignin content and drying in the porosity and enzymatic saccharification of the sugar cane bagasse. From an in natura sample, other four samples with decreasing lignin contents were generated using the method of delignification by acid chlorite. A fraction of these samples was air dried at room temperature and the remainder one was kept wet. The analysis of the structural changes promoted by the aforementioned treatment was performed using scanning electron microscopy (SEM). The samples porometry, carried out using the solute exclusion technique, was performed using 1 g of sample (dry weight) and 20 g of solutions of molecular probes with concentration of 1.5% (w/w); after 24 h under occasional manual agitation at 25 °C, determination of the volume and surface area distribution of pores was carried out based on the reduction of the initial concentration of the solution. The water retention value of the samples was calculated by centrifugation. The samples were subjected to enzymatic saccharification with enzyme and surfactant loads of 10 FPU and 0.025 g of Tween 20 per gram of bagasse, respectively. The reaction was carried at 45 °C under agitation of 150 rpm and the cellulose conversion was measured after 2, 8, 24 and 72 h. Sample delignified by 1, 2, 3 and 4 hours showed 14.2, 9.2, 8.0 and 5.9% of lignin content, respectively, while the in natura sample was composed of 20.7%. Through SEM analysis, it was observed that the lignin removal resulted in a material with the vascular bundles structurally less compact and ordered. The samples with higher contents of lignin had lower volumes and surface areas of pores and worse enzymatic cellulose conversions. For the in natura sample the total pore volume was 0.89 mL/g of bagasse while for the samples delignified by 1, 2, 3 and 4 hours this volume was 1.19, 1.77, 1.92 and 2.21 mL/g of bagasse, respectively. Drying reduced the total pore volume of samples delignified by 2, 3 and 4 hours to 1.47, 1.55 and 1.98 mL/g of bagasse, respectively. The water retention values were similar to the total pore volume obtained by the solute exclusion technique. While only about 20% of the cellulose in the in natura sample was converted after 72 h of saccharification, the sample with the lowest lignin content showed a conversion close to 100%. Drying of the delignified samples did not change rates and yields of saccharification. It can be concluded then that the lignin content plays an important role on limiting the enzymatic saccharification of cellulose, and that its removal implies in increase in the pore volume of the material.

Atualmente, o aumento da preocupação com a sustentabilidade ambiental, alinhado às perspectivas de esgotamento das reservas de petróleo, tem direcionado às buscas por fontes renováveis de energia. O emprego de resíduos agroindustriais, principalmente de usinas sucroalcooleiras destaca-se como sendo uma alternativa para a produção de etanol de segunda geração. Dentre as metodologias aplicadas para disponibilização dos açúcares fermentescíveis está a hidrólise enzimática. Ainda, para facilitar esta etapa e torná-la mais acessível, submete-se, previamente, o material lignocelulósico a um pré-tratamento, com o objetivo de contribuir com a susceptibilidade da celulose a ataques enzimáticos. No entanto, devido à complexidade das estruturas lignocelulósicas, os processos de hidrólise e pré-tratamento precisam se tornar mais eficientes e economicamente viáveis. Desta forma, o objetivo desse trabalho foi avaliar e caracterizar os pré-tratamentos hidrotérmico e organossolve (etanol 50%), isoladamente, e estes combinados em diferentes condições, assim como a influência destes procedimentos na estrutura e composição da biomassa, bem como na hidrólise enzimática. Os resultados demonstraram que os pré-tratamentos hidrotérmicos a 160 ºC nas condições analisadas foram pouco efetivos na melhora do acesso enzimático durante a etapa de hidrólise, pois atuaram de maneira branda na parede celular, pouco solubilizando a hemicelulose e lignina, conforme as análises físicas comprovaram. Os tratamentos combinados hidrotérmico 30 min e 60 min a 160 ºC seguidos pelo organossolve por 150 min apresentaram semelhança morfológica e alta solublização da lignina e hemicelulose, justificando os valores de hidrólise. Nossos resultados abrem perspectivas de novos estudos que visam a otimização dos pré-tratamentos hidrotérmicos e organossolve, além da compreensão das alterações composicionais e morfológicas que levam à melhoria da hidrólise enzimática na biomassa lignocelulósica. / The concerns with environmental sustainability and perspectives of petroleum reserves depletion motivated exploration of new and sustainable energy sources. In this context, renewable energies start to receive significant attention in the world´s energy matrix, with biofuels playing a special role. The use of agro-industrial residues, mainly from the sugarcane industry, stands out as a viable alternative for the production of second-generation ethanol. The enzymatic hydrolysis of the biomass has a number of advantages for polysaccharides depolimerization, such as high substrate specificity, low environmental impact and lack of corrosion issues. To further facilitate this procedure and to make biomass more accessible, the lignocellulosic material has to be previously submitted to a pretreatment in order to increase the cellulose accessibility and susceptibility to the enzymatic action. This process aims at the disorganization of the chemical structure of the lignocellulosic matter, facilitating the further steps of hydrolysis and fermentation. Due to the complexity of the lignocelluloses structures, their pretreatment and hydrolysis processes have to become more efficient and economically viable to be efficiently applied at an industrial scale. Therefore, the objective of this work is to evaluate the hydrothermal and organosolv (50% ethanol) pretreatments, separately and combined in different conditions, and the influence of these procedures on the structure and composition of the biomass and on the efficiency of enzymatic hydrolyses. Our results demonstrated that the hydrothermal pretreatments at 160ºC within the analyzed reaction conditions had minor effects on improving the enzymatic efficiency, being not harsh enough to introduce significant modifications of the cell wall composition and structure, as demonstrated by our physical and chemical analyses. The combined hydrothermal treatments lasting 30 min and 60 min at 160ºC followed by the organosolv step for 150 min resulted in significant morphological changes and high lignin and hemicelluloses solubilization, resulting in an efficient enzymatic hydrolysis. Our results open perspectives of further studies aimed at optimization of hydrothermal and organosolv pretreatments and comprehension of compositional and morphological changes which lead to improved enzymatic hydrolysis of the lignocelulosic biomass.

Généralement, diagnostiquer une pathologie donnée à un stade de développement précoce favorise le pronostic vital du patient atteint. Une telle performance nécessite de détecter des marqueurs présents à des seuils de concentrations bas dans des fluides biologiques souvent complexes. Pour détecter ces concentrations extrêmement faibles en analyte donné, la stratégie employée au cours de ce travail est l’amplification moléculaire du signal. Pour cela, différentes approches sont possibles (i) amplifier le signal issu de l’évènement de reconnaissance cible/sonde, (ii et iii) amplifier le signal par régénération ou réplication de la cible. Les stratégies conçues au cours de ce travail de thèse se focalisent principalement sur la détection de petites molécules, telles que l’eau oxygénée ou encore l’anion fluorure, mais avec à terme l’idée de les étendre à la détection indirecte de biomarqueurs ou protéines d’intérêts. La première partie de cette thèse se focalise sur l’amplification moléculaire d’un signal par une catalyse allostérique en utilisant la réaction de reconstitution d’une apoenzyme donnée avec son cofacteur tandis que la seconde partie de cette thèse repose sur la mise en place de systèmes d’amplification catalytique et auto-catalytique pour la détection d’H2O2, grâce à des dérivés pro-quinoniques porteurs de groupement acide/ester boronique. La distinction entre les systèmes catalytique et auto-catalytique se fait selon qu’H2O2 est régénéré ou amplifié au cours de la réaction. / Generally, diagnosing a given pathology at an early stage of development promotes the patient's prognosis. Such a performance requires the detection of specific markers which are present in complex biological fluids at low concentration level. To detect these extremely low analyte concentrations, the strategy employed in this work is the molecular amplification of the signal. To this end, different approaches are possible (i) amplifying the signal resulting from the target / probe recognition event, (ii and iii) amplifying the signal by regeneration or replication of the target. The strategies conceived during this thesis work mainly focus on the detection of small molecules, such as hydrogen peroxide or fluoride anion, but with the idea of extending them to the indirect detection of biomarkers or proteins of interest. The first part of this thesis focuses on the molecular amplification of a signal by allosteric catalysis using the reconstitution reaction of a given apoenzyme with its cofactor. The second part of this thesis is based on the implementation of catalytic and auto-catalytic amplification systems for the detection of H2O2, thanks to pro-quinonic derivatives bearing boronic acid/ester group. The distinction between catalytic and auto-catalytic systems is based on whether H2O2 is regenerated or amplified during the reaction.

L’extrusion biocatalytique, ou bioextrusion, est une technique d’extrusion réactive utilisant des enzymes comme catalyseurs. Cette technique est considérée en temps qu’étape intermédiaire, subséquente au prétraitement physico-chimique et précédente à l’hydrolyse enzymatique enréacteur fermé. L’utilisation de l’extrusion permet un procédé continu, facilement modulable et adaptable à des conditions de hautes consistances, de nombreuses biomasses et facilement transférable à l’échelle industrielle. Néanmoins, les données bibliographiques font ressortir la complexité des entrants et leurs interactions lors de la bioextrusion de biomasses lignocellulosiques. Les conclusions des bioextrusions de biomasses amidonnées soulignent l’importance de l’étude de l’influence de la concentration en substrat et en enzymes. Les résultats obtenus à partir de la bioextrusion des biomasses lignocellulosiques valident l’existence d’une activité enzymatique en extrudeuse malgré la contrainte thermomécanique et le temps de séjour limité. Lors de cette étape, l’hydrolyse de la fraction cellulosique est favorisée pour des milieux concentrés en substrat et en enzymes. Des modifications significatives des fractions cellulosiques cristallines et amorphes en surface, des réductions des tailles de particules, une dégradation visuelle des structures de la biomasse et l’augmentation de la sensibilité à la décomposition thermique, sont aussi observées sur la fraction solide. L’hydrolyse enzymatique des bioextrudats est prolongée en réacteur fermé. La bioextrusion permet des améliorations significatives des taux et vitesses de conversion des sucres sur le long terme, jusqu’à 48 h. Les gains observés sont relativement constants pour la paille de blé et augmentent avec le temps pour les écorces de bouleau et les résidus de maïs. Post-extrusion, la concentration en substrat influence négativement la conversion des sucres. Cependant, les plus-values de conversion du glucose lié à la bioextrusion de paille de blé sont principalement observables pour des concentrations en substrat et en enzymes élevées. À partir de 4 h, des baisses significatives de la conversion du xylose sont observées après bioextrusion. Les déstructurations de la fraction solide, déjà observées au cours la bioextrusion, se poursuivent en réacteur fermé. Les meilleurs résultats hydrolytiques aux niveaux des hautes charges en enzymes et en substrat sont associables aux bonnes conditions de mélanges caractéristiques des éléments bilobes. L’ensemble enzymatique est probablement réparti de façon plus homogène (mélange distributif) pour cibler plus de sites disponibles. De plus, le mélangé dispersif limite la proximité entre enzymes de même type et les gênes associées. Le procédé d’extrusion permet une agitation efficace, un bon transfert de masse et probablement un meilleur contact entre enzymes et substrat. Les moins bons résultats de conversion du xylose sont probablement à relier à des phénomènes d’adsorption non-spécifique, ou encore de désactivation des hémicellulases, provoqués par l’intensité des contraintes thermomécaniques et les résidus ligneux. Les bons résultats de déstructuration après bioextrusionsont associables à une action synergétique des contraintes mécanique et biochimique. Les analyses d’autofluorescence montrent l’évolution de la fraction ligneuse dans le processus de déconstruction de la fraction solide. Une production progressive de particules très fines,visiblement associée à la fraction ligneuse, est observée. Des complexes lignine-carbohydratessont aussi détectés dans la fraction liquide. Etant peu, voire pas hydrolysable par voie enzymatique, ces fractions hétéropolymériques sont un frein à la déconstruction. Si la déstructuration des lignines est probablement majoritairement liée au prétraitement alcalin, le procédé de bioextrusion provoque une diminution de la teneur en hétéropolymères de plus hautes masses moléculaires. / Biocatalytic extrusion, also named bioextrusion, is a reactive extrusion technique using enzymes as catalysts. Bioextrusion is considered as a link between the previous physico-chemical pretreatment (like alkaline extrusion) and the subsequent enzymatic hydrolysis in batch conditions. The extrusion allows a continuous, flexible and versatile process for high consistency media, easily transferable to the industrial level. However, complexity of both lignocellulosic biomass and lignocellulolytic enzymes and their interactions during the extrusion process are underlined by the literature. Numerous response surface methodology experiments with starchy biomass indicate that bioextrusion efficiency is mainly influenced by substrate and enzymes loading. Enzymatic activity during the bioextrusion process of lignocellulosic biomass is confirmed by the experiments despite the mechanical constraints and the limited residence time. During bioextrusion, best holocellulosic fraction hydrolysis results were obtained with high substrate and enzymes loadings. Significant modifications of the solid fraction like particule size reduction, visual deconstruction of the biomass structure, increased sensibility to thermal decomposition and the evolution of the surface exposure of crystalline and amorphous cellulose were observed. Enzymatic hydrolysis of the bioextrdates is prolonged in batch conditions. Clear improvements of speeds and rates of sugars conversion up to 48 h indicate a long term influence of the bioextrusion. Gain observed are steady for the pretreated wheat straw whereas it increases with time for corn residues and birch barks. Post-extrusion, a negative influence of the substrate loading is measured. However, best enhancements for the glucose conversion of pretreated wheat straw are detected for high substrate and enzymes loadings. From 4 to 48 h, significant losses in xylose conversion are measured with previous bioextrusion. Indicators of the solid fraction deconstruction, observed during the bioextrusion step, indicate a stronger biomass degradation after 48 h. Improvements of glucose conversion rates can be associated with good mixing conditions of the extruder, especially due to the use of kneading elements. Enzymes are probably more homogeneously distributed (distributive mixing) and can access more catalytic sites available. Moreover, dispersive mixing limits the enzyme jamming due to the biocatalysts concentration. Extrusion process permits an better agitation efficiency, good mass transfer conditions and probably a higher contact between substrate and enzymes. Lower xylose conversion results may be attributed to non-specific adsorptions or inactivation phenomena due to mechanical constraints and lignin residues. Good deconstruction results on the solid fraction may be associable with a synergetic action between mechanical and biochemical constraints. Autofluorescent signal analysis of the lignin fraction show its evolution during the deconstruction of the solid residue. During the hydrolysis, a progressive production of very small particles, appearing to be associated with the lignin fraction is observed. Lignin-carbohydrate complexes are also detected in the liquid fraction. These heteropolymeric complexes, difficult or even impossible for the enzymes to hydrolyze, are an obstacle to the biomass valorization. If lignin deconstruction is mainly due to the alkaline pretreatment, bioextrusion process seems to reduce the proportion of these heteropylymers with high molecular weights.

The employment of enzymes and whole cells has been important in many industries for centuries. However, it is only in the last 30 years that the use of enzymes for the synthesis of high-value fine chemicals has enjoyed increasing popularity. In fact, esterases and lipases are used almost routinely these days to provide optically active building blocks for the construction of imaginative new routes to chiral target molecules. The major topic of this work describes the utilization of enzymes (namely lipases) in the synthesis and modification of natural and non-natural compounds. Chapter 1 outlines the strengths and weaknesses of the most widely used enzyme systems and a description of a brief summary on the state of the art of biotransformations with special emphasis on the general applicability and reliability of various reaction types is described. Chapter 2 describes the enzymatic resolution of various 3-acetoxy-4-aryl-substituted azetidin-2-ones. Following screening of enzymes, such as Novozym-435, PS-30, PPL and AYS the best conditions were a phosphate buffer with PS-30 as the enzyme. The resulting products were the (3S, 4R)-3-hydroxy-4-aryl-substituted azetidin-2-ones and the unreacted (3R, 4S)-3-acetoxy-4-aryl-substituted azetidin-2-ones. Reactions generally occurred with high conversion and high selectivity. In Chapter 3, the regioselective transesterifications and hydrolysis of peracylated sophorolipid (SL) derivatives catalyzed by lipases was investigated. It was confirmed from the detailed spectral analysis of the products that transesterification failed to furnish any free hydroxyls on the sophorose ring. Instead, transesterification took place on the methyl ester located at the carboxylic end of the 17-hydroxyoctadecenoic acid chain attached to the C-1' position of the sophorose ring. In Chapter 4, the chemo-enzymatic syntheses of enantiomerically pure R and S imperanene from vanillin are described. The key step entails the asymmetrization of a prochiral diol using lipase PS-30. The resulting monoacetate has enantiomeric excesses of >97%. Biocatalysts represent a new class of chiral catalysts useful for a broad range of selective organic transformations. It is stating the obvious to say that biocatalysis is not a panacea for synthetic organic chemistry. However, advances over the past thirty years mean that it would be a serious mistake not to consider the employment of a biocatalyst, in, perhaps, the key step in a sequence of transformations that turn a cheap starting material into an expensive fine chemical.

Cette thèse a pour objectif de développer une méthode électrochimique permettant de cribler à haut débit des inhibiteurs d'enzymes. Pour cela, une enzyme cible a été sélectionnée pour son intérêt thérapeutique, la transcétolase (TK) : elle est impliquée dans de nombreuses pathologies (cancer, maladies neurodégénératives, diabète…) et dans la survie de certains parasites pathogènes. Afin de mesurer l'activité de la TK, deux systèmes rapporteurs ont été développés, à l'aide de plaques de criblage électrochimique (PCE) constituées de 96 électrodes indépendantes. Le premier système rapporteur repose sur un système bienzymatique nécessitant l'intervention d'une enzyme auxiliaire, la galactose oxydase (GAOx) sous forme libre ou immobilisée dans la laponite. Cette enzyme est capable d'oxyder les produits de réaction de la TK et de produire du peroxyde d'hydrogène. Le format 96 des PCE a permis d'optimiser rapidement la détection électrochimique par ampérométrie pulsée par intermittence (IPA) d'activité oxydase et seulement 10 minutes sont nécessaires pour réaliser 96 mesures simultanées. Parallèlement, afin d'exploiter au maximum le système à 96 électrodes, cette détection d'activité oxydase a également été réalisée, en 10 minutes par électrochimiluminescence. Cette méthode offre l'avantage d'être plus sensible et moins variable que par IPA, mais limite l'utilisation de matrice d'immobilisation d'enzymes. La GAOx a ensuite été utilisée pour mesurer l'activité TK, cependant, les conditions réactionnelles n'étant pas optimales en présence du système bienzymatique (TK-GAOx), des temps d'incubation longs sont nécessaires et sont peu adaptées au criblage d'inhibiteurs de la TK. Un second système rapporteur ne nécessitant plus d'enzyme auxiliaire et faisant intervenir uniquement un seul substrat de la TK a été optimisé. Ce système repose sur l'oxydation de l'intermédiaire réactionnel formé par la fixation du cofacteur (la thiamine pyrophosphate) et du substrat sur l'enzyme par le ferricyanure. Cette méthode permet de mesurer 96 activités TK en seulement 7 minutes et a aisément été utilisée pour cribler une bibliothèque de molécules. Le criblage de 1360 molécules a permis d'identifier un nouvel inhibiteur de cet enzyme, présentant une CI50 de 63 μM. Le système électrochimique a également été utilisé pour déterminer le mécanisme d'inhibition (non compétitive pure partielle) et la constante d'inhibition associée (3,4 μM). Ces résultats sont inédits dans le domaine de l'électrochimie et offrent un large éventail d'applications que ce soit pour le criblage d'activité enzymatiques ou d'inhibiteurs d'enzymes / This thesis focuses on the development of a new electrochemical method allowing the high throughputscreening of enzyme inhibitors. For this purpose, a target enzyme has been selected for its therapeutic interest,transketolase (TK): this enzyme is involved in many diseases (cancer, neurodegenerative diseases, diabetes ...) and inthe survival of pathogenic parasites. To measure TK activity, two reporter systems have been developed by usingelectrochemical plates, composed of 96 independent electrodes.The first one is based on a bienzymatic system, requiring the use of an auxiliary enzyme, galactose oxidase(GAOx), in its soluble form or immobilized in laponite. This enzyme is able to oxidize TK products and producehydrogen peroxide. The 96-well format allowed to quickly optimize the electrochemical detection of oxidase activityby intermittent pulse amperometry (IPA) of oxidase activity and only 10 minutes are required to perform 96simultaneous measurements. In parallel, in order to harness the 96-well electrochemical system, this detection ofoxidase activity was also carried out in 10 minutes using electrochemiluminescence. This method is more sensitiveand less variable than IPA but is limited to the use of soluble enzymes. However, the reaction conditions are notoptimal for the bienzymatic system (TK-GAOx): long incubation times are required and are poorly adapted for thescreening of TK inhibitors.A second reporter system no longer requiring an auxiliary enzyme and involving only one TK substrate hasbeen optimized. This system relies on the oxidation by ferricyanide of the reactional intermediate resulted of thebinding of the cofactor (thiamine pyrophosphate) and the substrate. This method allows to measure 96 TK activitiesin only 7 minutes and was easily used to screen an in-house chemical library. The screening of 1360 molecules leadto the identification of a new TK inhibitor with an IC50 of 63 μM. This electrochemical system was also used todetermine the mechanism of inhibition (partial non-competitive mechanism) and the associated inhibition constant(3.4 μM). These results are innovative in the field of electrochemistry and offer a wide range of applications forenzymatic activity screening or the screening of enzymes inhibitors