Le Mexique se caractérise par la présence sur son territoire de nombreuses espèces d’agave qui peuvent être cultivées ou non. En particulier l’Agave tequilana Weber var. azul a une grande importance économique, car elle constitue la principale matière première pour l’élaboration de la tequila. Les agaves durant leur développement, qui dure plusieurs années, accumulent des réserves de sucres constitués par des fructanes. Actuellement, l’optimisation de l’hydrolyse des fructanes d’agave est surtout importante pour l’industrie de la tequila. Elle permettra d’améliorer les rendements d’extraction des sucres. La méthode classique d’hydrolyse des fructanes est constituée principalement d’un procédé de cuisson des agaves crus. L’utilisation d’enzymes spécifiques pour réaliser ce même procédé d’hydrolyse suscite un récent intérêt industriel, parce qu’il permettrait une réduction de la consommation d’énergie. Les fructanes d’agave présentent des structures complexes, les résidus de fructose sont reliés par des liaisons osidiques de type β (2→1) et β (2→6), et la structure est fortement branchée. Il est nécessaire de comprendre les changements de structure des fructanes en fonction de l’étape de croissance des plantes, pour connaître la variabilité naturelle du substrat utilisé pour l’hydrolyse. D’autre part, il est important de découvrir de nouvelles enzymes susceptibles d’hydrolyser de manière spécifique les fructanes d’agave, et les caractériser biochimiquement, pour arriver à une meilleure connaissance de l’intéraction enzyme-substrat qui permettra le développement de nouvelles applications industrielles possibles pour les fructanes d’Agave tequilana. Dans ce travail, la première partie est consacrée à la détermination de la composition en sucres solubles et à la caractérisation de la structure des fructanes d’Agave tequilana présents dans des plantes d’âges différents. Puis, dans la deuxième partie, la purification et la caractérisation biochimique d’une fructanase isolée d’une souche de levure Kluyveromyces marxianus obtenue à partir du procédé de fermentation du mezcal (boisson d’agave distillée) a été étudiée. L’activité de cette enzyme a été comparée à un cocktail enzymatique commercial le fructozyme®. Finalement, dans une troisième partie, des levures isolées de la fermentation de différents types de mezcal ont été criblées et ont permis la sélection de souches capables de dégrader spécifiquement les fructanes d’Agave / Mexico has a high diversity of Agave plants, which could be cultivated or not. The most economically important is Agave tequilana Weber var. azul, because it is the raw material for the tequila elaboration process. As agaves grow, they accumulate reserve sugars as fructans. Actually, optimizing the A. tequilana fructans hydrolysis, in order to increase the sugar yield, is important to the tequila industry. Traditionally, agaves are cooked to hydrolyze the fructans. However, using enzymes for hydrolysis may reduce energy consumption and increase sugar yields.The fructans of A. tequilana have a complex structure, composed of fructose chains with β (2→1) and β (2→6) linkages with branching points. It is important to understand how the structure of these molecules changes as a function of plant growth, in order to know the natural variability of the substrate that must be hydrolysed. It is also necessary to find new enzymes for the efficient hydrolysis of A. tequilana fructans, and to characterize them biochemically for a better understanding of the enzyme-substrate interaction.The present work has three parts that focuses separately on each of these needs: First, characterizing the water soluble carbohydrates and the structure of the A. tequilana fructans as a function of the plant’s growth (age). Second, purifying and biochemically characterizing a fructanase from Kluyveromyces marxianus yeast isolated from the fermentation of mezcal, and comparing it to a commercial cocktail (Fructozyme®). Third, a screening of enzymes from yeasts used to ferment mezcal, in order to determine their ability to hydrolyze A. tequilana fructans

A busca por substâncias benéficas à saúde humana tem impulsionado o desenvolvimento de pesquisas visando o estudo de enzimas e seus produtos através da otimização de bioprocessos. Um dos principais componentes utilizados como base para a indústria de alimentos funcionais são carboidratos denominados frutooligossacarídeos (FOS) derivados da sacarose. Estes são sintetizados por enzimas denominadas frutosiltransferases que podem ser encontradas em plantas, bactérias e fungos. Os FOS têm atraído grande interesse da indústria, devido às suas características fisiológicas e biomoduladoras. Por serem polissacarídeos prebióticos não-digeríveis, têm a capacidade de estimular seletivamente o crescimento de bifidobactérias e lactobacilos, auxiliando na prevenção da cárie dentária e câncer de cólon em humanos. Podem também contribuir na diminuição do colesterol total e triglicerídeos no sangue, promover a reabsorção de cálcio e magnésio e serem utilizados em dietas com restrições alimentares, por serem açúcares de baixo valor calórico e elevado valor nutricional. Tendo em vista a existência de diferentes formas de FOS sintetizados por diferentes mecanismos, o presente trabalho buscou realizar a caracterização estrutural e funcional de um conjunto de 13 frutosiltransferases de bactérias e fungos. Os genes alvo foram clonados e as enzimas expressas e purificadas. Ensaios estruturais e de atividade enzimática, incluindo de hidrólise e polimerização da sacarose, foram conduzidos para a melhor compreensão das bases moleculares envolvidas no reconhecimento do substrato. Oito enzimas, duas β-frutofuranosidases de B. adolescentis, três sucrose-6-phosphate hydrolase de B. licheniformis e L. gasseri e uma invertase de A.niger foram cristalizadas e as enzimas de B. adolescentis e de L. gasseri tiveram suas estruturas resolvidas e seus sítios catalíticos mapeados. Estas apresentam em sua estrutura uma região β-propeller, local identificado como sítio catalítico, conectada à um módulo β-sanduíche. Ambas as enzimas apresentaram atividade hidrolítica da sacarose e a enzima de L. gasseri apresentou a formação dos FOS nistose e 1-cestose com concentrações de 1 M de sacarose bem como em tempos de 8 e 12 horas de incubação. Estes estudos, somados às análises das outras enzimas, permitirão o melhoramento na produção em larga escala, além da otimização e o controle destes processos de obtenção de FOS. / The search for beneficial substances to human health has driven the development of researches on the study of enzymes and their products through bioprocess optimization. One of the main components used as a basis for functional food industry are carbohydrates called fructooligosaccharides (FOS) derived from sucrose. These are synthesized by enzymes called fructosyltransferases which can be found in plants, bacteria and fungi. The FOS has attracted great interest of the industry due to its physiological and biomodulator properties. Because it is non-digestible polysaccharides prebiotics, have the ability to selectively stimulate the growth of bifidobacteria and lactobacilli, assisting in the prevention of tooth decay and colon cancer in humans. They can also contribute to decrease total cholesterol and triglycerides in the blood, to promote the absorption of calcium and magnesium and can be used in diets with dietary restrictions, because they are low-calorie sugars presenting high nutritional value. Considering there are different forms of FOS synthesized by different mechanisms, the present work attempts to make structural and functional characterization of a set of 13 fructosyltransferases of bacteria and fungi. The target genes were cloned and the enzymes were expressed and purified. Structural testing of X-ray crystallography and enzymatic activity, including sucrose hydrolysis and polymerization were carried out for a better understanding of the molecular basis involved in substrate recognition. Eight enzymes, two β-frutofuranosidases B. adolescentis, three sucrose-6-phosphate hydrolase of B. licheniformis and L. gasseri and A. niger invertase, were crystallized and the enzymes from B. adolescentis and from L. gasseri had their structures determined and their catalytic site mapped. These are similar to each other and present in their structure one β-propeller region which was identified as catalytic site, connected to one β-sandwich module. Both enzymes showed hydrolytic activity of the sucrose and L. gasseri showed the formation of FOS, 1-kestose and nystose with 1 M sucrose concentrations and times of 8 and 12 hours of incubation. These studies together with the analysis of other enzymes will enable the improvement in large-scale production, besides the optimization and control of these processes for the production of FOS.

A busca por substâncias benéficas à saúde humana tem impulsionado o desenvolvimento de pesquisas visando o estudo de enzimas e seus produtos através da otimização de bioprocessos. Um dos principais componentes utilizados como base para a indústria de alimentos funcionais são carboidratos denominados frutooligossacarídeos (FOS) derivados da sacarose. Estes são sintetizados por enzimas denominadas frutosiltransferases que podem ser encontradas em plantas, bactérias e fungos. Os FOS têm atraído grande interesse da indústria, devido às suas características fisiológicas e biomoduladoras. Por serem polissacarídeos prebióticos não-digeríveis, têm a capacidade de estimular seletivamente o crescimento de bifidobactérias e lactobacilos, auxiliando na prevenção da cárie dentária e câncer de cólon em humanos. Podem também contribuir na diminuição do colesterol total e triglicerídeos no sangue, promover a reabsorção de cálcio e magnésio e serem utilizados em dietas com restrições alimentares, por serem açúcares de baixo valor calórico e elevado valor nutricional. Tendo em vista a existência de diferentes formas de FOS sintetizados por diferentes mecanismos, o presente trabalho buscou realizar a caracterização estrutural e funcional de um conjunto de 13 frutosiltransferases de bactérias e fungos. Os genes alvo foram clonados e as enzimas expressas e purificadas. Ensaios estruturais e de atividade enzimática, incluindo de hidrólise e polimerização da sacarose, foram conduzidos para a melhor compreensão das bases moleculares envolvidas no reconhecimento do substrato. Oito enzimas, duas β-frutofuranosidases de B. adolescentis, três sucrose-6-phosphate hydrolase de B. licheniformis e L. gasseri e uma invertase de A.niger foram cristalizadas e as enzimas de B. adolescentis e de L. gasseri tiveram suas estruturas resolvidas e seus sítios catalíticos mapeados. Estas apresentam em sua estrutura uma região β-propeller, local identificado como sítio catalítico, conectada à um módulo β-sanduíche. Ambas as enzimas apresentaram atividade hidrolítica da sacarose e a enzima de L. gasseri apresentou a formação dos FOS nistose e 1-cestose com concentrações de 1 M de sacarose bem como em tempos de 8 e 12 horas de incubação. Estes estudos, somados às análises das outras enzimas, permitirão o melhoramento na produção em larga escala, além da otimização e o controle destes processos de obtenção de FOS. / The search for beneficial substances to human health has driven the development of researches on the study of enzymes and their products through bioprocess optimization. One of the main components used as a basis for functional food industry are carbohydrates called fructooligosaccharides (FOS) derived from sucrose. These are synthesized by enzymes called fructosyltransferases which can be found in plants, bacteria and fungi. The FOS has attracted great interest of the industry due to its physiological and biomodulator properties. Because it is non-digestible polysaccharides prebiotics, have the ability to selectively stimulate the growth of bifidobacteria and lactobacilli, assisting in the prevention of tooth decay and colon cancer in humans. They can also contribute to decrease total cholesterol and triglycerides in the blood, to promote the absorption of calcium and magnesium and can be used in diets with dietary restrictions, because they are low-calorie sugars presenting high nutritional value. Considering there are different forms of FOS synthesized by different mechanisms, the present work attempts to make structural and functional characterization of a set of 13 fructosyltransferases of bacteria and fungi. The target genes were cloned and the enzymes were expressed and purified. Structural testing of X-ray crystallography and enzymatic activity, including sucrose hydrolysis and polymerization were carried out for a better understanding of the molecular basis involved in substrate recognition. Eight enzymes, two β-frutofuranosidases B. adolescentis, three sucrose-6-phosphate hydrolase of B. licheniformis and L. gasseri and A. niger invertase, were crystallized and the enzymes from B. adolescentis and from L. gasseri had their structures determined and their catalytic site mapped. These are similar to each other and present in their structure one β-propeller region which was identified as catalytic site, connected to one β-sandwich module. Both enzymes showed hydrolytic activity of the sucrose and L. gasseri showed the formation of FOS, 1-kestose and nystose with 1 M sucrose concentrations and times of 8 and 12 hours of incubation. These studies together with the analysis of other enzymes will enable the improvement in large-scale production, besides the optimization and control of these processes for the production of FOS.