Littérature scientifique sur le sujet « Cello-oligosaccharides »

The modes of action of the five major endo-(1→4)-beta-D-glucanases (I, II, III, IV and V) purified from Penicillium pinophilum cellulase were compared by h.p.l.c. analysis, with normal, 1-3H-labelled and reduced cello-oligosaccharides and 4-methylumbelliferyl glycosides as substrates. Significant differences were observed in the preferred site of cleavage even when substrates with the same number of glycosidic bonds were compared. Thus, although endoglucanase I was unable to attack normal cello-oligosaccharides shorter than degree of polymerization 6, it hydrolysed reduced cellopentaose to yield cellotriose and cellobi-itol, and it produced cellotriose and 4-methylumbelliferyl glucoside from 4-methylumbelliferyl cellotetraoside. Endoglucanase IV hydrolysed [1-3H]cellotriose but did not attack either cellotri-itol or 4-methylumbelliferyl cellobioside. These and other anomalous results indicated clearly that modification of the reducing glycosyl residue on the cello-oligosaccharides induces in an apparent change in the mode of action of the endoglucanases. It is suggested that, although cello-oligosaccharide derivatives are useful for differentiating and classifying endoglucanases, conclusions on the mechanism of cellulase action resulting from these measurements should be treated cautiously. Unequivocal information on the mode of endoglucanase action on cello-oligosaccharides was obtained with radiolabelled cello-oligosaccharides of degree of polymerization 3 to 5. Indications that transglycosylation was a property of the endoglucanases were particularly evident with the 4-methylumbelliferyl cello-oligosaccharides. Turnover numbers for hydrolysis of the umbelliferyl cello-oligosaccharides were calculated, and these, along with the other analytical data collected on the products of hydrolysis of the normal, reduced and radiolabelled cello-oligosaccharides, suggested that the various endoglucanases had different roles to play in the overall hydrolysis of cellulose to sugars small enough to be transported through the cell membrane.

Larger cello-oligosaccharides undergo faster hydrolysis over carbon catalysts. This is attributed to reduction in activation energy caused by conformational change in the structure of oligosaccharides as they adsorb within the micropores of carbon.

Plant colonization by Streptomyces scabiei, the main cause of common scab disease on root and tuber crops, is triggered by cello-oligosaccharides, cellotriose being the most efficient elicitor. The import of cello-oligosaccharides via the ATP-binding cassette (ABC) transporter CebEFG-MsiK induces the production of thaxtomin phytotoxins, the central virulence determinants of this species, as well as many other metabolites that compose the ‘virulome’ of S. scabiei. Homology searches revealed paralogues of the CebEFG proteins, encoded by the cebEFG2 cluster, while another ABC-type transporter, PitEFG, is encoded on the pathogenicity island (PAI). We investigated the gene expression of these candidate alternative elicitor importers in S. scabiei 87-22 upon cello-oligosaccharide supply by transcriptomic analysis, which revealed that cebEFG2 expression is highly activated by both cellobiose and cellotriose, while pitEFG expression was barely induced. Accordingly, deletion of pitE had no impact on virulence and thaxtomin production under the conditions tested, while the deletion of cebEFG2 reduced virulence and thaxtomin production, though not as strong as the mutants of the main cello-oligosaccharide transporter cebEFG1. Our results thus suggest that both ceb clusters participate, at different levels, in importing the virulence elicitors, while PitEFG plays no role in this process under the conditions tested. Interestingly, under more complex culture conditions, the addition of cellobiose restored thaxtomin production when both ceb clusters were disabled, suggesting the existence of an additional mechanism that is involved in sensing or importing the elicitor of the onset of the pathogenic lifestyle of S. scabiei.

The C-terminal 191-residue module of Cel5A from the alkalophilic Bacillus sp. 1139 comprises a carbohydrate-binding module (CBM) belonging to a previously unidentified family that we have classified as CBM family 28. This example, called CBM28, bound specifically to cello-oligosaccharides and mixed β-(1,3)(1,4)-glucans (barley β-glucan) with association constants of approximately (1–4)×104 M−1. Its binding to barley β-glucan was remarkably insensitive to pH between 7.0 and 10.9, in keeping with its alkalophilic source. CBM28 bound to cellulose having a significant non-crystalline content with an association constant similar to that for its binding to soluble glucans. CBM17 (CBM family 17) and CBM28 modules naturally occur as tandems. The CBM17/CBM28 tandem from Cel5A bound with apparent co-operativity to barley β-glucan. The association of CBM28 with cello-oligosaccharides was driven enthalpically and marked by the different thermodynamic contribution of three putative binding subsites that accommodate a cellohexaose molecule.

Natural cellulose exists as a composite of cellulose forms, which can be broadly characterized as crystalline or non-crystalline. The recognition of both of these forms of cellulose by the CBMs (carbohydrate-binding modules) of microbial glycoside hydrolases is important for the efficient natural and biotechnological conversion of cellulosic biomass. The category of CBM that binds insoluble non-crystalline cellulose does so with an affinity approx. 10–20-fold greater than their affinity for cello-oligosaccharides and/or soluble polysaccharides. This phenomenon has been assumed to originate from the effects of changes in configurational entropy upon binding. The loss of configurational entropy is thought to be less profound upon binding to conformationally restrained insoluble non-crystalline cellulose, resulting in larger free energies of binding. However, using isothermal titration calorimetry, it is shown that this is not the case for the high-affinity interactions of CcCBM17 (the family 17 CBM from EngF of Clostridium cellulovorans) and BspCBM28 (the family 28 CBM from Cel5A of Bacillus species 1139) with regenerated cellulose, an insoluble preparation of primarily non-crystalline cellulose. The enhanced free energy of binding of non-crystalline cellulose relative to cello-oligosaccharides is by virtue of improved enthalpy, not entropy.

Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第17074号
農博第1957号
新制||農||1005(附属図書館)
学位論文||H24||N4713(農学部図書室)
29794
京都大学大学院農学研究科森林科学専攻
(主査)教授 髙野 俊幸, 教授 西尾 嘉之, 教授 木村 恒久
学位規則第4条第1項該当

L'extension de l'espace moléculaire chimique accessible à partir de la biomasse végétale par des méthodes douces et propres est un sujet d'actualité qui stimule la communauté scientifique afin de développer des produits biosourcés à faible impact environnemental et d'élargir le champ d'exploitation de la biomasse. La fonctionnalisation de la cellulose, le polysaccharide le plus abondant sur la planète, et/ou des cello-oligosaccharides telle que décrite dans cette thèse s'inscrit dans cette démarche. Notre objectif était de développer une méthode chimio-enzymatique impliquant l'action d'une laccase assistée par un médiateur pour oxyder des cello-oligosaccharides ou des fibres cellulosiques, suivie d'une amination réductrice pour greffer des composés aminés sur le matériau cellulosique. Dans ce but, nous avons d'abord démontré l'oxydation du cellobiose et du méthyl cellobiose en utilisant la laccase de Trametes versicolor et le TEMPO comme médiateur. Les conditions d'oxydation ont été optimisées avec le méthyl cellobiose et appliquées à un mélange de cello-oligosaccharides et au cellopentaose. En utilisant l'analyse LC/MS, nous avons montré qu'une large gamme de composés oxydés est obtenue et que la méthode est efficace pour produire des cello-oligosaccharides acides potentiellement intéressants pour les domaines biomédical et nutraceutique. Ensuite, nous avons montré que la réactivité du cellopentaose oxydé avec deux molécules aminées, la p-toluidine et la rhodamine 123 (un colorant aminé), permettait la liaison du composé aminé aux oligosaccharides. À l'aide des techniques LC/MS et MS/MS, nous avons mis en évidence la présence d'une liaison amine forte et covalente entre les colorants et le cellopentaose, élargissant ainsi l'espace chimique accessible par ce procédé hybride. Après avoir réalisé cette preuve de concept, nous avons tenté la teinture de fils de coton. Les fibres cellulosiques sont l'un des principaux matériaux textiles biosourcés et biodégradables. Cependant, le traitement chimique des textiles et notamment les méthodes chimiques utilisées pour fixer les colorants de manière covalente sont extrêmement polluants et nocifs pour la santé. Proposer des alternatives plus respectueuses de l'environnement est un défi mais d'un intérêt primordial pour une entreprise comme PILI, impliquée dans le projet de thèse, qui développe des colorants naturels en utilisant la biologie de synthèse. Ainsi, le potentiel du procédé hybride à deux étapes a été utilisé pour greffer avec succès la p-toluidine, la rhodamine 123 et le rouge acide 33 sur des fils de coton. La liaison covalente établie entre ces colorants et la fibre de coton a été prouvée pour la première fois. De plus, une bonne homogénéité et une bonne résistance au lavage ont été observées pour la teinture avec l'acid red 33, démontrant la robustesse et l'applicabilité de l'approche en situation réelle. Ces résultats originaux ont été brevetés. En testant d'autres colorants aminés, nous avons également montré que la solubilité, la réactivité et la structure du colorant aminé sont des paramètres importants à prendre en compte pour l'optimisation de la teinture, ce qui ouvre la voie à la synthèse à façon de nouveaux colorants aminés adaptés à ce procédé hybride prometteur
The extension of the chemical molecular space accessible from plant biomass by soft and clean methods is a timely topic that stimulates the scientific community in order to develop biobased products with low environmental impact and to widen the field of biomass exploitation. The functionalization of cellulose, the most abundant polysaccharide on the planet, and/or cello-oligosaccharides as described in this thesis is part of this approach. Our objective was to develop a chemo-enzymatic method involving the action of a mediator-assisted laccase to oxidize cello-oligosaccharides or cellulosic fibers, followed by reductive amination to graft amino compounds onto the cellulosic material. To this end, we first demonstrated the oxidation of cellobiose and methyl cellobiose using the laccase from Trametes versicolor and TEMPO as a mediator. Oxidation conditions were optimized with methyl cellobiose and applied to a cello-oligosaccharide mixture and cellopentaose. Using LC/MS analysis, we showed that a wide range of oxidized compounds is obtained and that the method is effective in producing acidic cello-oligosaccharides potentially of interest for the biomedical and nutraceutical fields. Then, we showed that the reactivity of oxidized cellopentaose with two aminated molecules, p-toluidine and rhodamine 123 (an aminated dye), allowed the binding of the amino compound to the oligosaccharides. Using LC/ MS and MS/MS techniques, we provided evidence for the presence of a strong, covalent amine bond between the dyes and cellopentaose, thus enlarging the chemical space accessible through this hybrid process. After completed this proof of concept, we attempted the dyeing of cotton threads. Cellulosic fibers are one of the main biosourced and biodegradable textile materials. However, chemical processing of textiles and especially the chemical methods used to covalently fix dyes are extremely polluting and harmful to health. Providing more eco-friendly alternatives is a challenge but of prime interest for a company like PILI, which was involved in the thesis project and is developing natural dyes using synthetic biology. Thus, the potential of the two-pot/two-step hybrid process was used to successfully graft p-Toluidine, rhodamine 123 and Acid Red 33 onto cotton thread. The covalent bond established between these dyes and the cotton fiber was proven for the first time. In addition, good homogeneity and wash-fastness were observed for acid Red 33 dyeing, demonstrating the robustness and applicability of the approach in real life. These original results have been patented. By testing other amino dyes, we also showed that the solubility, reactivity and structure of the aminated dye are important parameters to be addressed for dyeing optimization, which opens the way to the custom synthesis of new amino dyes suitable for this promising hybrid process

Dans le contexte actuel, il devient nécessaire de rendre les alternatives au pétrole, tel que le bioéthanol 2G, disponibles à grande échelle. Cependant, l’étape d’hydrolyse par les enzymes de Trichoderma reesei reste un verrou à un procédé économiquement stable et rentable. Ces travaux de thèse, s'intègrent dans le cadre du projet Futurol et ont pour objectifs d'identifier et de caractériser de nouvelles enzymes fongiques pour améliorer l'hydrolyse de la biomasse lignocellulosique. A partir des données protéomiques disponibles pour Podospora anserina et Fusarium verticillioides, une douzaine d'enzymes candidates ont été identifiées dans leurs sécrétomes. Ce travail de thèse s'est plus particulièrement focalisé sur les AA9s « Lytic Polysaccharide Monooxygenases » (LPMOs) de P. anserina. Parmi les LPMOs étudiées, PaLPMO9A, PaLPMO9E et PaLPMO9H, qui possèdent un CBM1, sont les plus actives sur la cellulose. La détermination de la régiosélectivité d'action a mis en évidence que PaLPMO9A et PaLPMO9H clivent la cellulose en position C1 et C4 alors que la PaLPMO9E génère uniquement des produits oxydés en C1. La PaLPMO9H est la plus versatile puisqu’elle est active sur les cello-oligosaccharides solubles et sur les polysaccharides hémicellulosiques liés en β-(1,4) (i.e., xyloglucane, glucomannane). La supplémentation du cocktail de T. reesei avec PaLPMO9E ou PaLPMO9H a permis de doubler les rendements d'hydrolyse du miscanthus prétraité. Les travaux réalisés au cours de cette thèse ont permis de démontrer l'importance de ces enzymes oxydatives dans les phénomènes de déconstruction de la lignocellulose chez les champignons filamenteux
In the current context, it becomes essential to make alternative to oil, such as the 2G bioethanol, available at large scale. However, the hydrolysis step by Trichoderma reesei enzymes remains the major bottleneck for an economically sustainable process. The present work is part of the Futurol project, and aims at identifying and characterizing new fungal enzymes to improve the hydrolysis of lignocellulosic biomass. From the proteomic data available for Podospora anserina and Fusarium verticillioides, a dozen of interesting enzymes were identified in their secretomes. This work focuses, mainly, on the AA9s « Lytic Polysaccharide Monooxygenases » (LPMOs) from P. anserina. Among all the LPMOs studied, PaLPMO9A, PaLPMO9E and PaLPMO9H that harbored a CBM1 were the most active on cellulose. Investigation of their regioselective mode of action revealed that PaLPMO9A and PaLPMO9H oxidatively cleaved at both C1 and C4 positions while PaLPMO9E released only C1-oxidized products. PaLPMO9H that was the most versatile in terms of substrate specificity as it also displayed activity on cello-oligosaccharides and β-(1,4)-linked hemicellulose polysaccharides (e.g., xyloglucan, glucomannan). The hydrolysis yield of the pretreated miscanthus was significantly improved up to 2 fold, when the PaLPMO9E, or PaLPMO9H were supplemented to the T. reesei cocktail. This work demonstrated the importance of these oxidative enzymes for lignocellulose deconstruction by fungi. These biocatalysts open new prospects to improve the enzymatic conversion of plant biomass for 2G bioethanol production

碩士
大同大學
生物工程研究所
89
Since the last decade, enzymatic methods have become accepted as complementary procedures in carbohydrate synthesis. Synthesis of gluco- conjugates or its derivatives was carried out in two methods, chemical synthesis and enzymatic synthesis. In order to avoid the inconveniences, such as protection and deprotection processes, thus enzymatic synthesis of cello- oligosaccharides (COS) and alkyl glucosides (AG) would be imperatively. β-glucosidase II is available by combined chromatography column purification, and has about 40 % yields. The purity of the modified purified enzyme is similar to the complete purified enzyme. The procedure makes it possible to apply in large-scale purification of the enzyme. Free state enzyme successfully catalyzes the transglucosylation in methanol, ethanol, propanol, butanol, amyl alcohol, hexanol, heptanol, but except octanol. While the immobilized enzyme can catalyze transglucosylation in all test primary alcohols including octanol. The batch mode and continuous mode of the immobilized enzyme for synthesizing cellooligosaccharides are both investigated in this study. In batch mode, while the concentrations of substrate are various from 5 % to 15 %, the yield of cellotriose are increased from 6.59 mg/mL to 25 mg/mL, and the yield of gentiobiose are also increased from 10.9 mg/mL to 27.22 mg/mL. In the highest concentration of substrate (15% cellobiose), free state enzyme yield only 0.449 mg/mL. In continuous mode, while the concentrations of substrate are various from 5 % to 15 %, the yield of cellotriose are increased from 0.41 mg/mL to 1.63 mg/mL, and the yield of gentiobiose are also increased from 2.67 mg/mL to 7.97 mg/mL, respectively. Continuous reactor might realize the non-stop reaction, which may be applied in production of specific oligosaccharides or their derivatives.