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

Crassulacean acid metabolism (CAM) provides desert plants with distinct advantages over the C3 and C4 photosynthetic pathways in harsh climates where water is scarce. CAM is, however more metabolically costly than C3 or C4 photosynthesis, and some plants, such as Mesembryanthemum crystallinum, facultatively utilize CAM when water is abundant, and water conservation unnecessary. In such situations, these plants behave akin to a C3 plant when photosynthesizing. CAM is divided into four phases, with each phase displaying unique metabolic processes. Certain changes, including changes in the timing of CO2 fixation, stable carbon isotope ratios, and tissue malic acid content accumulation patterns can indicate that a plant has shifted from CAM to C3 photosynthesis. Such shifts have been observed to be regulated primarily by water availability and ontogenic development. While facultative CAM is well documented in species like Mesembryanthemum crystallinum, and it has not been studied extensively in Agave with the exception of Agave deserti, and Agave angustifolia. A better understanding of this phenomenon would apply to the agricultural growth of this genus. This study aimed to trigger C3 to CAM shifts in Agave sensu lato species, in order to expand upon the findings of previous studies, and better understand the prevalence of facultative CAM expression in the genus. Gas exchange and stable carbon isotope measurements were taken from 2-month-old, 10-month-old, and mature agaves grown in controlled ocnditions. Tissue acid content measurements were taken from mature plants. Despite the Agave sensu lato species in this study being subjected to moisture applications ranging from dry to saturated, we were unable to observe any distinct shifts from CAM to C3 photosynthesis in any of the species tested for both seedlings and mature plants. Diel net CO2 fixation rates also increased with age, and water applications for seedlings, and decreased with heavy irrigation in mature plants. Stable carbon isotope ratios revealed that some carbon in the plant tissues was fixed by rubisco, and that some species (Polianthes tuberosa, Prochnyanthes mexicana) had carbon isotope ratios of a C3 plant, but these ratios did not change with different irrigation treatments. Malic acid accumulation remained typical of CAM plants for the species tested as well, with one exception in Polianthes tuberosa. As such, we conclude that the Agave sensu stricto species tested in this study are obligate CAM plants, and that they perform poorly mature individuals are over-watered. Additionally, the Agave sensu lato species P. mexicana, and P. tuberosa appear to be C3 plants given the results of this study.