In the first chapter, sodium tetrathionate (TT), a sulfhydryl blocking agent, is assessed for its ability to protect the proteolytic activity (PA) of papaya latex during air, sun or vacuum drying, and of crude papain during storage. By means of Taguchi's L₂₇ (3¹³) fractional factorial design, it was found that the addition of 1% TT significantly increased the retention of PA of papaya latex when it was air dried at a temperature of 55°C. This protection of PA was found to be 23% higher than the one given by the addition of 1% sodium metabisulfite, the compound commonly used in the commercial processing of papaya latex. When drying was carried out either under 27 inches vacuum at 50°C or in the sun, the protective effect of TT on the PA was not significantly different from that of metabisulfite. The PA of crude papain during storage at room temperature was also protected by TT. A loss of 20% of the original PA occurred over a period of 13 wk when crude papain contained 1% TT, compared to a loss of 45% when the crude enzyme preparation contained 1% metabisulfite. In the same chapter five different oxidants for synthesis of TT from thiosulfate are compared, namely: iodine, hydrogen peroxide, ferric chloride, cupric sulfate and sodium vanadate. The results indicated that hydrogen peroxide or sodium vanadate were not only effective in the oxidation but also much less expensive than iodine, which is the most popular oxidant for the synthesis of TT. The results obtained in this chapter warrant the use of TT in the commercial production of commercial papain to prevent the destruction of the enzymes during harvesting, storage, transportation and processing. In the second chapter, chemical modification of pure papain by TT is discussed. Optimization techniques were applied for improving the precision of two methods used in this study: circular dichroism (CD) and proteolytic activity determination. Simplex optimization significantly improved repeatability and signal to noise ratio of the CD scan of papain. A new optimization approach, which was a combination of a central composite rotatable design and simplex optimization, was successfully applied to achieve maximum precision for the proteolytic activity assay of papain using casein as a substrate. This approach may also be applied to other analytical methods to improve the reliability of the experimental data. Influential factors in the inactivation of PA of papain by using TT and reactivation of the inactivated papain by cysteine were carried out using two Taguchi's L₁₆ (2¹⁵) fractional factorial designs. The results indicated that when inactivation was carried out at pH 6.8, with a reaction time of 5 min at 22°C, and a molar ratio of TT to papain of 10, the inactivation reaction was highly reversible upon addition of 20 mM cysteine. Although some interactions of the factors were significant, 70% reactivation was achieved in most cases. Analysis of UV absorbance, near-UV and far-UV CD spectra indicated that there were no major changes in the spectra in papain upon the chemical modification of the enzyme with TT. Secondary structure computed from far-UV CD spectra also demonstrated no significant changes upon this modification. Sulfhydryl data and pH-fluorescence profiles of the modified papain support the hypothesis that reversible blocking by TT results from binding with the single reactive cysteine residue present in papain. Quenching of the intrinsic fluorescence of papain when the modification was carried out using high molar ratios of TT to papain was suggestive of modification of tryptophan residues in the enzyme during the oxidation reaction with TT. Precipitation or insolubilization of pure papain, and of the proteins of papaya latex and commercial papain was observed upon the chemical modification with TT under certain conditions. Addition of β-mercaptoethanol and TT at levels of 100 mM and 50 mM, respectively, precipitated 90% of pure papain. Solubility studies together with electrophoretic analysis of the precipitated papain suggested formation of insoluble aggregates due to the insoluble aggregation as a result of inter-molecular disulfide bonds formation. TT was found to be a competitive inhibitor of both reversible and irreversible inhibition of the enzyme action, when carbobenzoxyglycine p-nitrophenyl ester was used as a substrate. The second order inactivation constant in the absence of substrate was computed to be 16,919 M⁻¹sec⁻¹, indicating that the reaction had a high rate. / Land and Food Systems, Faculty of / Graduate

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 number of exoglycosidases have been prepared from bacterial and plant sources using established methods for the separation of enzymes, in conjunction with certain novel purification systems hitherto not described in the literature for these enzymes. The enzyme, beta-galactosidase from E. coli has been prepared using previously described methods of phase separation and ion-exchange chromatography. As a final step in this purification, the use of a new hydroxyl-rich chromatographic support for the isolation of high-grade enzyme suitable for use in enzyme immunoassays was investigated. Methods have also been studied for the recovery of alpha-mannosidase as a by-product of the procedure used for the extraction of urease from jack bean (Canavalia ensiformis). The inclusion of a novel step involving the use of hydrophobic-interaction chromatography on Phenyl-Sepharose led to excellent recoveries of enzyme suitable for commercial use. Studies on a second glycosidase, beta-N-acetylhexosaminidase, from the same source (jack bean) paved the way for an adaptation of existing purification methods to provide increased yields and an improved quality of enzyme. Since the research unit in which this work was performed is associated with commercial organizations responsible for the preparation and marketing of biologically active products, it is important that the methods of purification described in this thesis are compatible with the requirements for largescale purification.

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Due to the high resistance of the spores, the bacterium Paenibacillus larvae is the most dangerous bacterial pathogen of the honey bee (Apis mellifera). Thanks to its biological properties and restricted pathogenicity, this bacterium can be used as a model organism to study gram positive sporulating aerobic rods. This work is focused on completing information about secreted proteases of this bacterium and in a study of proteases bound in a spore structure. MYPGP medium was used for the cultivation of P. larvae. In this medium, lysis of the culture was shown after 40 hours of cultivation. The pH of the medium decreased below 6.4 by lysis. The induction of temperate bacteriophage BLA was detected as a causative agent of this lysis. A new sporulation medium called HCBB agar was proposed for the sporulation of P. larvae. In comparison with HCBB agar with MYPGP agar by 31 strains of P. larvae stored in our collection, HCBB agar was evaluated as an appropriate sporulation medium with a median of sporulatin 4.2 ' 106 spores per cm2 in aerobic conditions and 5.65 ' 106 spores per cm2 in aerobic conditions with 10 % CO2. For purification of the secreted proteases, a one-day culture incubated at room temperature was used. Optimal purification of 87/74 kDa and 42/40 kDa proteases was observed after application of this...

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Titre de l'écran-titre (visionné le 25 septembre 2023) / Dendroctonus ponderosae, appelé le dendroctone du pin ponderosa (DPP), est l'un des insectes ravageurs les plus destructeurs de l'ouest de l'Amérique du Nord. Les champignons qu'il transporte causent de la mortalité chez plusieurs espèces du genre Pinus. L'infestation d'un pin commence par la libération d'une phéromone d'agrégation, le trans-verbénol, par un DPP femelle adulte, initiant ainsi une attaque de masse. En grand nombre, le DPP réussit à surmonter les défenses du pin incluant la production d'oléorésine contenant des composés toxiques comme l'α-pinène. La phéromone est initialement produite par l'insecte au stade larvaire par hydroxylation de l'α-pinène dans un processus de détoxification. Le trans-verbénol ainsi produit est ensuite lié à un acide gras afin d'être entreposé sous forme d'esters. Quand l'adulte colonise un nouveau pin, une carboxylestérase prédite causerait la libération de la phéromone séquestrée. L'hypothèse émise est que cette réaction est catalysée par l'estérase du clone DPO062_I20 du DPP. La production recombinante de l'enzyme a été faite dans Escherichia coli, mais celle-ci formait des corps d'inclusion. Les corps d'inclusion ont été solubilisés et la protéine solubilisée a été repliée, mais aucune activité enzymatique n'a été mesurée lors des essais préliminaires avec les esters de trans-verbénol identifiés dans le DPP. Une analyse des homologues de l'estérase a révélé qu'un organisme eucaryote pourrait être mieux adapté pour l'expression d'une enzyme active. L'estérase a donc été produite dans la levure Pichia pastoris dans laquelle elle a été retrouvée sous forme glycosylée. Cependant, aucune activité enzymatique n'a été mesurée dans les conditions testées. Différentes conditions d'expression et d'essais enzymatiques devront être étudiées afin d'obtenir une conclusion fiable quant à la caractérisation enzymatique de l'estérase. Finalement, un arbre phylogénétique des carboxylestérases du DPP a révélé que la carboxylestérase d'intérêt pourrait être spécifique à l'ordre Coleoptera ou à la sous-famille Scolytinae. / Dendroctonus ponderosae, known as the mountain pine beetle (MPB), is one of the most destructive insect pests in western North America. The fungi carried by MPB cause mortality in several Pinus species. Pine colonization is started by an adult female MPB releasing an aggregation pheromone called trans-verbenol, thus initiating a mass attack. In large numbers, MPB can overcome the host's defenses, which includes the production of oleoresin which contains toxic molecules such as α-pinene. The pheromone is initially produced at the larval stage through hydroxylation of α-pinene as a detoxification process. The resulting trans-verbenol is then conjugated with a fatty acid in order to be stored in the form of esters. When an adult MPB colonizes a new host, a predicted carboxylesterase causes the release of the sequestered pheromone. It is hypothesized that the esterase from the MPB clone DPO062_I20 catalyzes this reaction. The enzyme was recombinantly produced in Escherichia coli but formed inclusion bodies. These inclusion bodies were solubilized and the proteins were refolded, but no enzyme activity was detected in preliminary assays using trans-verbenol esters identified in MPB. Analysis of the esterase's homologs suggested that a eukaryotic organism could be more suitable for the expression of an active enzyme. The esterase was thus produced in the yeast Pichia pastoris, in which it was found to be glycosylated. However, no enzyme activity was measured under the tested conditions. Further exploration of different expression and assay conditions will be necessary to obtain a reliable conclusion regarding the enzymatic characterization of the carboxylesterase. Lastly, a phylogenetic tree of MPB carboxylesterases revealed that the carboxylesterase of interest may be specific to the Coleoptera order or to the Scolytinae subfamily (bark beetles).