Le travail présenté dans ce mémoire porte sur la modification d'un polysaccharide extrait d'un tubercule d'espèces d'Amorphophallus konjac sp., une plante pérenne appartenant à la famille des Araceae. Ce polysaccharide, le glucomannane, est un copolymère linéaire soluble dans l'eau, constitué de résidus D-mannopyranosyle (M) et D-glucopyranosyle (G) liés en β-(1→4) (M/G ≅ 1,6) et la chaîne principale présente des groupements acétyles. La masse molaire varie de 4.105 à 2.106 g.mol-1 selon les sources botaniques et les méthodes de purification, et l'étude de sa structure ainsi que ses propriétés physico-chimiques ont fait l'objet de plusieurs travaux de recherche. Pour trouver des nouvelles voies de valorisation de ce polysaccharide la modification de sa structure et/ou la réduction de sa masse molaire ont été envisagées. Le but était d'obtenir, soit des polysaccharides originaux avec des propriétés physico-chimiques différentes de celles du polymère de départ, soit des polymères de faible masse molaire ou des oligosaccharides présentant des activités biologiques particulières. Nous avons étudié la modification chimique de glucomannanes par oxydation avec le dioxyde d'azote et l'obtention d'oligosaccharides de glucomannane par des méthodes classiques en milieu aqueux telles que l'hydrolyse acide et enzymatique. In fine nous avons développé une nouvelle méthode d'obtention d'oligoglucomannanes partiellement oxydés par réaction hétérogène en milieu diphasique solide/gaz avec le dioxyde d'azote et obtenus des molécules chargées de petites tailles. Nous avons généralisé cette méthode à d'autres polysaccharides sélectionnés parmi les plus connus (alginates, agar, cellulose, xanthane, galactomannanes et dextranes) et enfin nous avons évoqué les principales perspectives de valorisation des molécules obtenues, en particulier grâce aux activités antivirales et prébiotiques.

Several lactic acid bacteria contain β-galactosidases. Beta galactosidases catalyze lactose hydrolysis and transfer acceptor sugars onto galactose, producing galactooligosaccharides. The aim of this work was to exploit β-galactosidases of lactic acid bacteria as crude cell extracts to produce novel oligosaccharides with mannose, N-acetylglucosamine, and fucose. Of 17 strains of lactic acid bacteria, transferase activity was the strongest in crude cell extracts of Lactobacillus delbrueckii subsp. bulgaricus, followed by Streptococcus thermophilus, Lactobacillus animalis, and Lactobacillus reuteri in a buffered 19% (w/w) lactose solution. Incorporation of 6 % (w/w) glycerol increased transferase activity and enzyme stability at higher incubation temperatures. Incorporation of 10% (w/w) mannose, N-acetylglucosamine and fucose as acceptor sugars yielded three distinct oligosaccharides with mannose and two with N-acetylglucosamine and fucose, with the composition confirmed using gas chromatography-mass spectrometry. This is the first public report indicating production of oligosaccharides containing N-acetylglucosamine and fucose from β-galactosidases of lactic acid bacteria.

Graduation date: 1993

Carbohydrates play important roles in biological processes. Their function is closely related to their conformation. In this thesis, conformational studies of carbohydrates by NMR spectroscopy and molecular dynamics computer simulations are described. The first two papers discuss the anomalous solubility of β-cyclodextrin compared to other cyclodextrins. Time correlation functions revealed flexibility in all cyclodextrins. Molecular dynamics computer simulations showed that the glycosidic linkages were rather rigid and the flexibility was suggested to be macrocyclic. From spatial distribution functions β-cyclodextrin was found to have greater ability to order the surrounding water than the other cyclodextrins. Paper III deals with some of the difficulties of conformational studies. In Paper IV, a new method, Additative Potential Maximum Entropy, APME, is applied to a disaccharide. Conformational distribution functions are derived from NOEs, J-couplings and residual dipolar couplings and calculated from computer simulations. All distribution functions were found to be in good agreement. In papers V and VI oligosaccharides from human milk are studied. Residual dipolar coupling, J-couplings and cross relaxation rates were measured by NMR spectroscopy and molecular dynamics computer simulations were carried out. Both oligosaccharides showed high flexibility for the β-D-GlcpNAc-(1→3)-β-D-Galp linkage.

Oligosaccharides are important biomolecules that are targets and also components of many medical treatments, including treatments for cancer, HIV, and inflammation. While the demand for medically-relevant oligosaccharides is increasing, these compounds have proven difficult to synthesize. Whole-cell oligosaccharide synthesis is a promising method that requires relatively inexpensive substrates and can complete the synthesis in just one step. However, whole-cell oligosaccharide synthesis employing common microorganisms like E. coli have been plagued by low yields. This dissertation investigates an alternative microorganism for oligosaccharide production: Agrobacterium sp. ATCC 31749. This Agrobacterium strain produces high levels of curdlan polysaccharide, demonstrating its natural ability to produce the sugar nucleotide precursor for oligosaccharide production. The two main objectives of this dissertation are 1) to develop biocatalysts for oligosaccharide synthesis by engineering ATCC 31749 and 2) to determine what factors affect poly- and oligosaccharide production in this Agrobacterium strain. ATCC 31749 was engineered to produce two oligosaccharides of medical importance: N-acetyllactosamine and galactose-α 1,3-lactose. Oligosaccharide production in the biocatalyst was further improved with additional metabolic engineering. Substrate uptake was increased through expression of a lactose permease, and availability of the sugar nucleotide substrate improved with gene knockout of the curdlan synthase gene. Both of these engineering efforts led to increased oligosaccharide synthesis in the Agrobacterium biocatalyst. Overall, the engineered Agrobacterium strains synthesized gram-scale quantities of the oligosaccharide products in just one step and requiring only a few inexpensive substrates and cofactors. Additional improvement of the oligosaccharide-producing biocatalysts required further investigation of the factors influencing poly- and oligosaccharide production in ATCC 31749. In this dissertation, several environmental and intracellular factors are identified that affect both oligosaccharide and curdlan production. Sucrose was the preferred carbon source for oligosaccharide synthesis, and the addition of citrate to the synthesis reaction led to significant improvement in oligosaccharide production. To identify the genetic factors and possible mechanisms regulating curdlan production, the genome of ATCC 31749 was sequenced. The genome sequence was utilized for transcriptome analysis of ATCC 31749. In the transcriptome analysis, genes significantly up- and down-regulated during curdlan production were identified. Subsequent gene knockout experiments showed several factors to be important for curdlan synthesis, namely the nitrogen signaling cascade, polyphosphate, and the GTP-derived second messengers (p)ppGpp and c-di-GMP. In addition to the development of biocatalysts for oligosaccharide production, this investigation provides insight into the complex mechanisms regulating exopolysaccharide synthesis.

La chimie combinatoire est un moyen efficace pour découvrir des molécules biologiquement actives. Dans ce travail, nous avons exploré deux approches combinatoires différentes pour synthétiser des bibliothèques de cyclopeptides. Tout d'abord, nous avons développé une stratégie basée sur l'assemblage aléatoire et chimiosélectif de biomolécules sur un châssis peptidique. Cette approche réalisée en solution a permis de générer des bibliothèques d'hétéroglycoclusters et de sélectionner les meilleurs ligands par colonne d'affinité portant une lectine modèle, la Concanavaline A. Dans une seconde approche, des bibliothèques de peptides cycliques ont été préparées par la méthode split and mix. Celles-ci ont été conçues et décodées selon l'algorithme mis au point dans le groupe du Pr. Reymond (Berne, Suisse) qui permet d'attribuer la séquence de chaque peptide sans marquage et de manière quasi-unique. Ces bibliothèques ont été criblées sur le support avec la vitamine B12 et la Caseine Kinase 2.

Reproduction de : Habilitation à diriger des recherches : Sciences naturelles. Biologie : Lille 1 : 2006. / N° d'ordre (Lille 1) : 510. Titre provenant de la page de titre du document numérisé. Bibliogr. p. 41-56.

Thesis (Ph. D.)--Institute of Paper Chemistry, 1961. / Includes bibliographical references (p. 85-86).

Thesis (Ph. D.)--Institute of Paper Chemistry, 1975. / Bibliography: leaves 101-103.

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