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Dans ce travail, nous avons étudié la contribution que les inhibiteurs de glicosidases ont dans la réponse de défense du blé à Fusarium graminearum. Nous avons démontré que les inhibiteurs de xylanases ont la capacité à la fois de contenir l'activité de dégradation de xylanases sécrétées par l'agent pathogène, et de limiter la possibilité de provoquer nécrose dans les tissus du blé. Nous avons démontré que l’expression de la PvPGIP2 dans la lemme, la paléa, les anthères et le rachis détermine une réduction des symptômes de la fusariose de l’épi, au même niveau de l’expression constitutive de cet inhibiteur. Inversement, l'expression de la PvPGIP2 dans l'endosperme ne détermine pas une réduction des symptômes de la maladie. Cela indique que, lorsque l'agent pathogène a atteint ce tissu, l'activité de polygalacturonases de l'agent pathogène n’est pas indispensable pour la propagation fongique. Enfin, la combinaison des différents inhibiteurs de glicosidases, qui renforcent différentes parties de la paroi cellulaire dans le même génotype, a été efficace pour réduire les symptômes de la fusariose, par rapport aux génotypes qui présentent seulment un type d’inhibiteur. Nous avons démontré cet aspect à travers les plantes qui expriment la PvPGIP2 et le TAXI-III. Au contraire, les génotypes qui expriment la PvPGIP2 et l’AcPMEI n’ont pas montré un effect additif sur la résistance, probablement parce que ils renforcent la même partie de la paroi cellulaire, c’est-à-dire la pectine. / In this work we studied the contribution of glycosidase inhibitors in the defense response of wheat against Fusarium graminearum. We demonstrated that xylanase inhibitors are able to limit both the degrading activity of the xylanases secreted by the pathogen and to limit their ability to induce necrosis in wheat cell suspensions and tissues.We demonstrated that the expression of PvPGIP2 in lemma, palea, anthers and rachis causes a reduction in Fusarium head blight symptoms, at the same level of the constitutive expression of this inhibitor. On the contrary, the expression of PvPGIP2 in the endosperm did not result in a reduction of disease symptoms, suggesting that once the pathogen has reached the endosperm, the activity of polygalacturonases secreted by the pathogen is not essential for the progression of symptoms.The pyramiding of glycosidase inhibitors in the same genotype is effective in reducing FHB symptoms although it depends on the specific combination. Pyramiding of PvPGIP2 and AcPMEI does not enhance further wheat resistance against FHB, possibly because they target the same virulent component secreted by the pathogen, that is PG. Conversely, the pyramiding PvPGIP2 and TAXI-III supports a further improvement of resistance compared to plants carrying only PvPGIP2 or TAXI-III.

Foram purificadas através de uma combinação de cromatografias as duas &#946;- glicosidases digestivas (Mr 47.000 e 50.000 - denominadas &#946;47 e &#946;50, respectivamente) encontradas na larva de S. frugiperda. Experimentos de competição entre substratos e modificação química mostraram que a &#946;47 possui dois sítios ativos. Um desses sítios denominado aril&$946;glicosidase apresenta um subsítio -1 que liga galactose mais eficientemente do enquanto ,que o subsítio +1 prefere pequenos grupos hidrofóbicos cíclicos. O segundo sítio, denominado celobiase, possui um subsítio -1 que prefere glicose. Já a região de ligação do aglicone apresenta 4 subsítios, que ligam glicose com afinidade decrescente à medida que afastam-se do ponto de clivagem do substrato. O cDNA que codifica a &#946;50 foi clonado e sequenciado. Alinhamentos de sequência de aminoácidos, experimentos de competição entre substratos e inibição mostraram que esta enzima possui apenas um sítio ativo. O subsítio -1, cuja especificidade é controlada por uma rede de pontes de hidrogênio, foi estudado comparando-se os parâmetros cinéticos (Kcat e KcaUKm) para a hidrólise de NP&#946;glicosídeos. A região de posicionamento do aglicone, uma fenda hidrofóbica composta de 3 subsítios, foi caracterizada utilizando-se alquil &#946;-glucosídeos e oligocelodextrinas como inibidores. O alinhamento da sequência de aminoácidos da &#946;50 com outras glicosil hidrolases sugeriu quais aminoácidos participariam da ligação do substrato e que o GlU187 (doador de prótons - pKa = 7,5) e o GIU399 (nucleófilo - pKa = 4,5) estão diretamente envolvidos na catálise. Além disso, a Arg97 e a Tyr331 participam indiretamente modulando o pKa do GIU399. r . / Two digestive &#946;-glycosidases (MW 47,000 and 50,000, named &#946;gly47 and &#946;gly50, respectively) whose are found in the S. frugiperda larvae were purified by a combination of chromatographic steps. Substrate competition experiments and chemical modification data showed that &#946;gly47 has two active sites. One of them was called aryl &#946;-glycosidase and presents a -1 subsite that prefers galactose while the +1 subsite binds small cyclic hydrophobic groups. The other active site was called cellobiase and presents 4 subsites that bind glucose residues weaker as they get far from the cleavage point. The cDNA that codes the &#946;gly50 was cloned and sequenced. Amino acid sequence alignment, substrate competition experiments and inhibitions proved that this enzyme has just one active site. The -1 subsite specificity is controlled by a hydrogen bond network as it was showed comparing the kinetic parameters (Kcat and KcatlKm) for some NP&#946;glycosides hydrolysis. The aglycone binding region, a hydrophobic cleft, was studied with alkyl &#946;-glucosides and oligocellodextrins as competitive inhibitors. Amino acid sequence alignment between the &#946;gly50 and other glycosil hydrolases showed the amino acids responsible for the substrate binding and that the GIU<SUB.187 (proton donor - pKa = 7.5) and GIU399 (nucleophile - pKa = 4.5) are directly involved in the catalysis. Beside this, Arg97 and Tyr331 participate indirectly in the catalysis, modulating the nucleophile pKa

El objetivo general de la presente tesis doctoral es el desarrollo de herramientas para la obtencion, produccion y aplicacion de dos enzimas glicosidicas: �¿-L-arabinofuranosidasa proveniente del hongo Aspergillus niger (Abf) y �À-D-glucosidasa (Bgl), proveniente de la levadura Candida molischiana. Estas hidrolasas se emplean en la liberacion de azucares en procesos de conversion de biomasa y en la industria alimentaria, pero tambien en la sintesis de aminoglicosidos, glicoconjugados y oligosacaridos, compuestos de alto valor anadido para la industria quimico-farmaceutica. Las enzimas se han expresado en la levadura metilotrofica Pichia pastoris, y se han purificado para caracterizar sus propiedades bioquimicas. Asimismo, se ha comprobado su capacidad para catalizar reacciones de transglicosilacion con alto rendimiento. En relacion a su produccion, se ha establecido y validado un modelo basado en restricciones del metabolismo de Pichia pastoris, evaluando su consistencia mediante analisis de flujos metabolicos posibilistico. El modelo permite estimar la tasa de crecimiento y la distribucion de flujos intracelulares a partir de unos pocos flujos extracelulares medidos experimentalmente. Adicionalmente, el modelo se ha extendido para estimar la productividad de proteina recombinante, y se ha empleado para analizar diferentes condiciones de cultivo de las cepas transgenicas que sobreproducen las enzimas Abf y Bgl. Finalmente, las enzimas se han inmobilizado en organosilicas bimodales de la familia UVM-7. Los biocatalizadores resultantes se han caracterizado bioquimica y fisico-quimicamente y se han evaluado en diferentes aplicaciones de interes biotecnologico. / Tortajada Serra, M. (2012). Process development for the obtention and use of recombinant glycosidases: expression, modelling and immobilisation [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/16800

Proteolytic enzymes and a glycolytic enzyme used in dishwashing detergents and by the starch conversion industry were examined for their ability to remove bacteria attached to black buna-N rubber. Pure culture and mixed culture biofilms of Pseudomonas fluorescens and Listeria monocytogenes were treated with the proteolytic enzymes Purafect® (Genencor International), Durazym™, and Savinase® and the glycolytic enzyme Termamyl® (Novo Nordisk BioChem North America). Compared to controls, none of the enzyme treatments were able to significantly remove P. fluorescens cells adherent in pure culture (p>0.05). Durazym™, Purafect®, and Termamyl® did significantly reduce the number of adherent cells of L. monocytogenes grown in pure culture. Treatment with Purafect® reduced the number of attached cells of both P. fluorescens and L. monocytogenes when grown in mixed culture. Material which absorbs at 280 nm was released from both pure and mixed culture biofilms when all three proteolytic enzymes were used. No survivors remained after planktonic (free floating) cells of both P. fluorescens and L. monocytogenes were incubated with Purafect®. Reduction in overall numbers of P. fluorescens and L. monocytogenes attached in mixed culture by Purafect® appeared to involve a combination of release of proteinaceous material followed by bactericidal effects on exposed cells. / Master of Science

Thermal processing has been used as a means of modifying the sensory aspects of wine. Cabernet Sauvignon wines were heated prior to dejuicing (3C per day from 25C to 42C) or after bottling (42C for 21 days) to determine the effects on total glycosides and glycosidic fractions. Total and phenol-free glycosidic concentrations in the wine and skins were quantified by analysis of glycosyl-glucose. Pre-dejuicing thermal vinification resulted in higher total glycosides (12%), phenol-free glycosides (18%), total hydroxycinnamates (16%), large polymeric pigments (LPP) (208%) small polymeric pigments (SPP) (41%), and lower monomeric pigments (42%) in wines. Skins had lower total glycosides (-16%), and no significant difference in phenol-free glycosides. Post-bottling heat treatment resulted in lower total (-15%) and phenol-free (-16%) glycosides, increased hue (25%), a 62% increase in LPP and a 29% decrease in monmeric pigments. A second study investigated the potential of enological spoilage microorganisms to affect wine aroma, flavor, and color. The activities of b-glucosidase were determined in model systems for fourteen strains of Brettanomyces bruxellensis yeast and nine strains of lactic acid bacteria (Oenococcus oeni). All Brettanomyces strains and seven Oenococcus strains exhibited enzymatic activity. B. bruxellensis b-glucosidase activity was primarily intracellular; O. oeni showed some extracellular activity. Yeasts and bacteria showing activity greater than 1000 nmole mL-1 g -1 for Brettanomyces, or 100 nmole mL-1 g -1 for Oenococcus, were evaluated for their effect on Viognier grape glycosides. Neither was active on native grape glycosides. / Master of Science

Thesis (Msc (Viticulture and Oenology))--University of Stellenbosch, 2007. / Among the factors contributing to wine complexity and quality, wine aroma is one of the most important factors. Wine aroma is the outcome of interaction among different compounds produced from the grapes, during fermentation as well as during the ageing process. Apart from its origin from grapes, fungi and yeasts, wine aroma can also be derived from the metabolic activity of wine lactic acid bacteria (LAB). These microorganisms are usually associated with malolactic fermentation (MLF) which normally occurs after alcoholic fermentation. MLF is beneficial to wine due to its contribution to deacidification, microbiological stabilisation and wine aroma formation, with the latter being the most important area of interest in our study. The production of volatile aromatic components in wine can, in part, be achieved through the hydrolytic action of enzymes produced by LAB associated with wine. These enzymes include β-glucosidase, protease, esterase, lipase and glucanase. Most of the work done on bacterial enzymes has been on LAB from food sources other than wine, in which these enzymes contribute to the flavour development of some cheeses, yoghurt and other fermented foods. The activity of these enzymes during wine fermentation has mostly been concerned with β-glucosidase from Oenococcus oeni. Only in recent years has there been a renewed interest in evaluating the activity of β-glucosidase in other genera of wine LAB. The overriding goal of this study was to screen and characterise wine-related enzymes produced by LAB associated with wine. All the LAB isolates tested in this study were obtained from IWBT culture collection and were previously isolated from five different wineries situated in the Western Cape region, South Africa. We first screened isolates using classical methods. The isolates were grown on agar medium supplemented with appropriate substrate analogues in order to evaluate the activity of enzymes (i.e. β- glucosidase, glucanase, lipase and esterase). The colonies exhibiting enzymatic activity ...

Fu Lai Hong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 106-110). / Abstracts in English and Chinese. / Thesis/Assessment Committee --- p.ii / Statement --- p.iii / Acknowledgements --- p.iv / Abstract --- p.v / 摘要 --- p.vi / Lists of Figures --- p.x / Lists of Tables --- p.xiii / List of Abbreviations --- p.xiv / Amino acid abbreviation --- p.xvi / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- Human α-L-iduronidase (hIDUA) --- p.2 / Chapter 1.1.1 --- Lysosomal storage disease --- p.2 / Chapter 1.1.2 --- Treatments of MPS 1 --- p.4 / Chapter 1.2 --- Plant cells as bioreactors --- p.5 / Chapter 1.3 --- The Plant secretary pathway --- p.7 / Chapter 1.3.1 --- Transport of soluble proteins --- p.9 / Chapter 1.3.2 --- Transport of integral membrane proteins --- p.10 / Chapter 1.4 --- Differences between plant and human proteins --- p.11 / Chapter 1.5 --- Reducing the differences between plant and human proteins --- p.12 / Chapter 1.6 --- Previous study: Expression of IDUA in transgenic tobacco plant --- p.13 / Chapter 1.7 --- Project objectives --- p.14 / Chapter 1.8 --- Long term significance --- p.14 / Chapter Chapter 2 --- Materials and Methods --- p.15 / Chapter 2.1 --- Introduction --- p.16 / Chapter 2.2 --- Materials --- p.18 / Chapter 2.2.1 --- Chemical --- p.18 / Chapter 2.2.2 --- Plant materials --- p.18 / Chapter 2.2.3 --- Plasmid vectors and bacterial strains --- p.18 / Chapter 2.2.4 --- Human a-iduronidase (hIDUA) cDNA --- p.19 / Chapter 2.2.5 --- Primers --- p.20 / Chapter 2.3 --- Methods --- p.22 / Chapter 2.3.1 --- Generation of IDUA antibodies --- p.22 / Chapter 2.3.1.1 --- Synthetic peptide raised IDUA antibodies --- p.23 / Chapter 2.3.1.1.1 --- Design of synthetic peptides --- p.23 / Chapter 2.3.1.1.2 --- Immunization of rabbits --- p.25 / Chapter 2.3.1.2 --- E. coli-derived rhIDUA protein --- p.25 / Chapter 2.3.1.2.1 --- Cloning and expression of rhIDUA --- p.25 / Chapter 2.3.1.2.2 --- Western analysis of E. coli-derived rhIDUA --- p.29 / Chapter 2.3.1.2.3 --- MS/MS analysis of rhIDUA protein --- p.29 / Chapter 2.3.1.2.4 --- Immunization of rabbits --- p.31 / Chapter 2.3.2 --- Affinity-purified antibodies --- p.33 / Chapter 2.3.3 --- Characterization of affinity-purified IDUA antibodies --- p.33 / Chapter 2.3.4 --- Construction of chimeric gene constructs --- p.34 / Chapter 2.3.5 --- Expression of IDUA in tobacco BY-2 cells --- p.39 / Chapter 2.3.5.1 --- Electropoartion of Agrobacteria --- p.39 / Chapter 2.3.5.2 --- Agrobacterium-mediated transformation --- p.39 / Chapter 2.3.5.3 --- Screening of positive trans formants --- p.40 / Chapter 2.3.6 --- Characterization of transgenic BY-2 cell expressing IDUA fusion --- p.40 / Chapter 2.3.6.1 --- Genomic DNA polymerase chain reaction (Genomic DNA PCR) --- p.40 / Chapter 2.3.6.1.1 --- Genomic DNA extraction from BY-2 callus --- p.40 / Chapter 2.3.6.1.2 --- Genomic DNA PCR of tobacco BY-2 callus --- p.41 / Chapter 2.3.6.2 --- Reverse transcription-PCR (RT-PCR) --- p.42 / Chapter 2.3.6.2.1 --- Total RNA extraction from BY-2 cell --- p.42 / Chapter 2.3.6.2.2 --- RT-PCR of BY-2 cell --- p.42 / Chapter 2.3.6.3 --- Western blot analysis of BY-2 cell and medium --- p.43 / Chapter 2.3.6.3.1 --- Protein extraction from tobacco BY-2 cells and culture medium --- p.43 / Chapter 2.3.6.3.2 --- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) --- p.44 / Chapter 2.3.6.3.3 --- Immunodetection and Coomassie blue stain --- p.44 / Chapter 2.3.7 --- Purification of IDUA from culture media --- p.46 / Chapter Chapter 3 --- Results --- p.47 / Chapter 3.1 --- Generation of IDUA antibodies --- p.48 / Chapter 3.1.1 --- Cloning and expression of rhIDUA in E. coli --- p.48 / Chapter 3.1.2 --- Characterization of IDUA antibodies --- p.51 / Chapter 3.1.2.1 --- Specificity of IDUA antibodies towards hIDUA protein. --- p.51 / Chapter 3.1.2.2 --- Cross-reactivity of IDUA antibodies with wild type tobacco BY-2 cell --- p.55 / Chapter 3.2 --- Chimeric gene constructs construction and confirmation --- p.58 / Chapter 3.3 --- Screening of transformed tobacco BY-2 callus with kanamycin-resistance --- p.66 / Chapter 3.4 --- Genomic DNA PCR screening of transformed tobacco BY-2 callus . --- p.67 / Chapter 3.5 --- RT-PCR screening of transformed BY-2 cells --- p.70 / Chapter 3.6 --- Western blot analysis of transformed tobacco BY-2 cells and culture media --- p.72 / Chapter 3.6.1 --- Tobacco BY-2 cells --- p.72 / Chapter 3.6.2 --- Tobacco BY-2 cell culture media --- p.76 / Chapter 3.7 --- Purification of IDUA protein in culture media --- p.81 / Chapter Chapter 4 --- Discussion --- p.82 / Chapter Chapter 5 --- Summary and Future Perspectives --- p.89 / Chapter 5.1 --- Summary --- p.90 / Chapter 5.2 --- Future perspectives --- p.92 / Appendix Identification and Characterization of an Unknown Protein by 1B Antibody --- p.93 / Chapter 6.1 --- Introduction --- p.94 / Chapter 6.2 --- Objectives --- p.94 / Chapter 6.3 --- Materials and Methods --- p.95 / Chapter 6.3.1 --- Western blot analysis of different plant species --- p.95 / Chapter 6.3.2 --- Subcellular localization of the unknown protein --- p.95 / Chapter 6.3.3 --- Affinity-purification of the unknown protein --- p.95 / Chapter 6.4 --- Results --- p.97 / Chapter 6.4.1 --- Western blot analysis of different plant species --- p.97 / Chapter 6.4.2 --- Subcellular localization of an unknown protein --- p.98 / Chapter 6.4.3 --- Affinity-purification of 1B protein --- p.104 / Chapter 6.5 --- Summary and Future Perspectives --- p.105 / Chapter 6.5.1 --- Summary --- p.105 / Chapter 6.5.2 --- Future Perspectives --- p.105 / References --- p.106

Les glycosidases sont des enzymes impliquées dans un grand nombre de processus biologiques. Leurs inhibiteurs présentent un potentiel thérapeutique en tant qu'agents antidiabétiques, agents antiviraux ou agents pour traiter des maladies génétiques. Au cours de ce travail, nous nous sommes attachés à développer une voie de synthèse chimioenzymatique permettant d'accéder à des analogues de la valiolamine, puissant inhibiteurs d'alpha-glucosidases. Les aminocyclitols que nous avions pour objectif de préparer diffèrent de la valiolamine par la position du groupement amino et des stéréochimies des hydroxyles. Nous avons synthétisé ces aminocyclitols selon une méthode originale, basée sur un procédé en un seul pot mettant en jeu deux enzymes et trois réactions : une aldolisation catalysée par la fructose-1,6- biphosphate aldolase, une déphosphorylation catalysée par une phytase et une nitroaldolisation intramoléculaire. Ainsi deux liaisons C-C sont formées de façon hautement stéréosélective en une seule étape. Nous avons synthétisé neuf nouveaux aminocyclitols et deux nouveaux zwitterions et dérivé N-alkylé. Quelque aminocyclitols se sont révélés des inhibiteurs sélectifs de la béta-glucosidase d'amandes douces et de la béta-galactosidase d'Aspergillus oryzae.

Nous avons réalisé la synthèse de nouvelles indolizidines polyhydroxylées, analogues de la swainsonine, inhibiteurs potentiels de glycosidases et d'urées cycliques de conformation restreinte portées par une pipéridine, agents permettant de contrôler la prolifération de cellules tumorales. Ces deux familles de composés ont été préparées à partir d'une pipéridine clé, obtenue par réaction de Mannich intramoléculaire entre le trans-4-oxo-buténoate d'éthyle et une bétâ-aminocétone protégée sous forme d'acétal. Dans un premier temps, nous avons synthétisé une indolizidine tri-hydroxylée en série racémique et en série optiquement pure en 10 étapes. En vue d'accroître l'activité inhibitrice, nous avons mis au point la synthèse d'une indolizidine tétra-hydroxylée possédant un groupement hydroxyle supplémentaire sur le cycle pipéridinique, nécessitant la préparation de la bétâ-aminocétone protégée optiquement pure correspondante. Les activités de ces deux indolizidines précédemment préparées en série racémique et en série optiquement pure ont été évaluées sur six enzymes commerciales. Seule l'indolizidine racémique tétra-hydroxylée s'est révélée être un inhibiteur sélectif de la bétâ-galactosidase d'Aspergillus oryzae (Ki 157 µM). Dans un deuxième temps, nous avons synthétisé des urées cycliques de conformation restreinte par réaction de l'intermédiaire clé, sur différents isocyanates commerciaux, suivie d'une cyclisation de Michael hautement stéréosélective. L'activité antiproliférative de ces nouveaux composés a fait l'objet d'une évaluation sur trois lignées cellulaires humaines, mais ils ne se sont malheureusement pas révélés cytotoxiques. Enfin, nous avons utilisé la réactivité de l'intermédiaire clé pour synthétiser différents types de molécules : des thiourées cycliques et des composés comportant une guanidine cyclique à 5 ou 6 chaînons constituant le squelette de base d'un produit naturel, la cylindrospermopsine