Дисертації з теми "Cell wall degradation"

Thesis (Ph. D.) -- University of Maryland, College Park, 2005.
Thesis research directed by: Marine-Estuarine-Environmental Sciences. Title from t.p. of PDF. Includes bibliographical references. Published by UMI Dissertation Services, Ann Arbor, Mich. Also available in paper.

京都大学
新制・課程博士
博士(エネルギー科学)
甲第23395号
エネ博第422号
新制||エネ||80(附属図書館)
京都大学大学院エネルギー科学研究科エネルギー社会・環境科学専攻
(主査)教授 河本 晴雄, 教授 亀田 貴之, 教授 杉山 淳司
学位規則第4条第1項該当
Doctor of Energy Science
Kyoto University
DFAM

Uma alternativa para aumentar a produção de bioetanol por área de cana plantada no Brasil seria utilizar os resíduos de sua biomassa para conversão em etanol. O conhecimento de como processos de degradação da parede celular se dão em plantas usadas para a produção de bioenergia e a compreensão de como eles funcionam pode ser de grande utilidade para esta tecnologia. Na investigação da anatomia de cana encontramos evidências da formação de um aerênquima lisígeno na raiz de cana, espaços gasosos no córtex da raiz decorrentes da morte celular e degradação da parede. Assim, decidiu-se aprofundar os estudos neste sistema através de técnicas de bioquímica de parede celular, microscopia de luz e transmissão e imunolocalização. A formação do aerênquima nas raízes de cana-de-açúcar tem início com a morte celular programada e a degradação de β-glucano e pectinas, principalmente daquelas associadas às lamelas médias, resultando na separação das células. As hemiceluloses arabinoxilano e xiloglucano mostram apenas modificações em suas estruturas finas, mas permanecendo nas paredes. Além disto, foram observados em microscopia de transmissão alguns pontos onde houve a degradação completa de parede celular, porém a presença de diversas paredes celulares colapsadas nas lamelas entre o aerênquima e ao seu redor parece ser mais importante para a formação do aerênquima. As modificações dos polissacarídeos estão possivelmente associadas com a alteração de características físicas das paredes, tornando-as mais suscetíveis a dobras e colapsos, gerando os espaços de gás e lamelas resistentes, que sustentam estes espaços. Mais do que a \"degradação da parede celular\", como é tratado em definições de aerênquima, pudemos observar que este fenômeno é resultado de uma sequência de eventos que permitem modificações da parede celular, e não necessariamente a sua completa degradação, resultando na abertura dos espaços gasosos
An alternative to increase bioethanol production per area of sugarcane plantation in Brazil would be to use its biomass residue for conversion into ethanol. The knowledge of how cell wall degradation processes occur in plants used for bioenergy production and understanding how they work can be of great use for this technology. Studying the sugarcane anatomy, we found evidences for the formation of a lysigenous aerenchyma in the roots, gas spaces in the root cortex originated from cell death and cell wall degradation. Thus, we decided to deepen the studies in this system using cell wall biochemistry, light and transmission microscopy and immunolabeling. The aerenchyma formation in sugarcane roots starts with programmed cell death and degradation of β-glucan and pectins, especially those from middle lamellae, resulting in cell separation. The hemicelluloses arabinoxylan and xyloglucan only show modifications in fine structure, but they remain in the cell wall. Besides, complete cell wall degradation was observed in a few spots through transmission electron microscopy, although the collapsing of cell walls seems to be more important for aerenchyma formation. Modifications in the polysaccharides are possibly associated with changes in cell wall physical properties, making them more susceptible to folding and collapsing, generating gas spaces and resistant lamellae that support these spaces. Described as \"cell wall degradation\" in aerenchyma definition in literature, we observed that this phenomenon is the result of a series of events that allow cell wall modifications, and not necessarily its complete degradation, resulting in the formation of gas spaces

Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第21808号
農博第2321号
新制||農||1066(附属図書館)
学位論文||H31||N5180(農学部図書室)
京都大学大学院農学研究科応用生命科学専攻
(主査)教授 植田 充美, 教授 渡邊 隆司, 教授 栗原 達夫
学位規則第4条第1項該当

Tese de doutoramento em Ciências Veterinárias. Especialidade de Ciências Biológicas e Biomédicas
ABSTRACT - Plant cell wall polysaccharides offer an abundant energy source efficiently utilized by a large repertoire of micro-organisms, which thus play a central role in carbon re-cycling. Aerobic micro-organisms secrete Carbohydrate-Active Enzymes (CAZymes) as free-standing proteins, whereas anaerobic bacteria organize a diverse enzyme consortium in a multi-component complex, the cellulosome, which performs a more efficient deconstruction of this composite structure. CAZymes are modular enzymes containing, in addition to catalytic domains, non-catalytic Carbohydrate-Binding Modules (CBMs). CBMs direct the appended enzymes to their target substrates thus potentiating catalysis. Here we show that the CBMs of Eubacterium cellulosolvens endoglucanase 5A (EcCel5A), designated as CBM65A and CBM65B, display a significant preference for xyloglucan. The crystal structure of CBM65B in complex with a xyloglucan-derived oligosaccharide, in combination with mutagenesis studies on CBM65A, revealed the mechanism by which these proteins display a preference for xyloglucan by establishing hydrophobic interactions with xyloglucan xylose side chains (Chapter 2). The genome of the ruminal cellulolytic bacterium Ruminococcus flavefaciens strain FD-1 encodes a large number of putative novel cellulosomal proteins. Here, genes encoding cellulosomal modules of unknown function were cloned and their corresponding proteins expressed at high levels in Escherichia coli. Complementary techniques combining affinity gel electrophoresis, a microarray platform and isothermal titration calorimetry were used to identify novel CBMs in cellulosomal-modules of unknown function. This strategy allowed the identification of 8 novel CBM families. The structures of representative members of two of these families (CBM-A and CBM-B1) have been solved and detailed functional characterization of these CBMs was performed. CBM-A and CBM-B1 comprise β-sandwich folds. CBM-A binds decorated β-1,4-glucans at a shallow binding cleft and displays preference for xyloglucan. In contrast, CBM-B1 displays a flat surface complementary to an open cleft that allows binding to a range of β-glucans including insoluble cellulose recognition (Chapter 3). Finally, the structure of CBM46 derived from BhCel5B, a Bacillus halodurans endoglucanase, was solved. BhCel5B is a multi-modular enzyme composed of a GH5_4 N-terminal catalytic domain, followed by an internal immunoglobulin-like module (Ig) and a C-terminal CBM46. BhCBM46 does not bind soluble or insoluble polysaccharides. However, the crystal structure of BhCel5B revealed that CBM46 is integral to the GH5_4 enzyme catalytic cleft and thus plays an important role in substrate recognition (Chapter 4).
RESUMO - Estrutura e Função de novas glucosil hidrolases (CAZymes) e de Módulos de Ligação a Hidratos de Carbono (CMBs) envolvidos na degradação da Parede Celular Vegetal - Os polissacarídeos da parede celular vegetal são uma fonte de energia abundante, eficientemente utilizada por um vasto número de microrganismos, os quais desempenham um papel central na recilagem do carbono. As enzimas secretadas pelos microrganismos aeróbicos, que promovem a hidrólise de hidratos de Carbono (CAZymes), funcionam de froma individualizada, ao passo que as bactérias anaeróbicas organizam essas enzimas num complexo multi-enzimático designado por Celulossoma, o qual efetua uma degradação mais eficiente da parede celular vegetal. As CAZymes são enzimas modulares que contêm, além de domínios catalíticos, módulos de ligação a hidratos de Carbono (CBMs) com função não catalítica. Os CBMs direcionam as enzimas a eles ligadas para os substratos-alvo, potenciando assim a catálise. Neste trabalho mostra-se que os CBMs associado à endoglucanase 5A (EcCel5A) da Eubacterium cellulosolvens designados por CBM65A e CBM65B, possuem uma significativa preferência por xiloglucano. A estrutura tridimensional do CBM65B, em complexo com um derivado oligossacárido do xiloglucano e os estudos de mutagenese realizados no CBM65A, revelaram que o mecanismo de preferência destas proteínas pelo xiloglucano se deve ao estabelecimento de interações hidrofóbicas com as cadeias laterais (xilose) deste substrato (capítulo 2). O genoma da bactéria celulolítica do rúmen Ruminococcus flavifaciens, estirpe FD1, codifica um vasto número de putativas proteínas celulosomais, ainda não estudadas. Neste estudo, os genes que codificam os módulos celulosomais de funções desconhecidas foram clonados e as proteínas por eles codificadas foram expressas em níveis elevados em Escherichia coli. Técnicas complementares, combinando eletroforese em gel nativo, uma plataforma de matriz de alta densidade (microarray) e calorimetria de titulação isotérmica, foram usados para identificar novos CBMs em módulos celulosomais de função desconhecida. Esta estratégia permitiu a identificação de 8 novas famílias de CBMs. Foram determinadas as estruturas tridimensionais representativas de duas destas famílias (CBM-A e CBM-B1), e efectuada a sua caracterização funcional detalhada. O CBM-A e o CBM-B1 apresentam um enrolamento em sanduiche β. O CBM-A liga-se ao β-1,4-glucano ramificado através de uma fenda superficial, revelando preferência por xiloglucano. Em contraste, o CBM-B1 revela uma superfície plana complementar a uma fenda aberta que permite a ligação a uma série de glucanos de tipo β, incluindo o reconhecimento de celulose insolúvel (capítulo 3). Por último, a estrutura do CBM46 derivado de uma endoglucanase do Bacillus halodurans designada por BhCel5B, foi determinada. A BhCel5B é uma enzima multi-modular composta por um domínio catalítico da família GH5_4 no terminal N, seguida por um módulo interno do tipo da imunoglobulina (lg) e o CBM46 no terminal C. O BhCBM46 não se liga a polissacarídeos solúveis ou insolúveis. Porém, a estrutura tridimensional da BhCel5B revelou que o CBM46 é parte integrante da fenda onde se alojam os resíduos responsáveis pela catálise da enzima GH5_4 e, por conseguinte, desempenha um papel importante no reconhecimento do substrato (capítulo 4)

The cellulosome is a multimeric enzyme complex that has the ability to metabolise a wide variety of carbonaceous compounds. Cellulosomal composition may vary according to the microbe’s nutritional requirement and allows for the anaerobic degradation of complex substrates. The complex substrates of interest in this research study were sugarcane bagasse and pineapple fibre waste, as they represent two important lignocellulosic, South African agricultural crops. The effective degradation of complex plant biomass wastes may present a valuable source of renewable compounds for the production of a variety of biofuels, for example bioethanol, and a variety of biocomposites of industrial importance. The identification of renewable energy sources for the production of biofuels is becoming increasingly important, as a result of the rapid depletion of the fossil fuels that are traditionally used as energy sources. An effective means of completely degrading lignocellulose biomass still remains elusive due to the complex heterogeneity of the substrate structure, and the fact that the effective degradation of the substrate requires a consortium of enzymes. The cellulosome not only provides a variety of enzymes with varying specificities, but also promote a close proximity between the catalytic components (enzymes). The close proximity between the enzymes promotes the synergistic degradation of complex plant biomass for the production of valuable energy products. Previous synergy studies have focused predominantly on the synergistic associations between cellulases; however, the synergy between hemicellulases has occasionally been documented. This research project established the synergistic associations between two Clostridium cellulovorans hemicellulases that may be incorporated into the cellulosome and a cellulosomal endoglucanase that is conserved in all cellulosomes. This research study indicated that there was indeed a synergistic degradation of the complex plant biomass (sugarcane bagasse and pineapple fibre). The degrees of synergy and the ratio of the enzymes varied between the two complex substrates. The initial degradation of the bagasse required the presence of all the enzymes and proceeded at an enhanced rate under sulphidogenic conditions; however, there was a low production of fermentable sugars. The low quantity of fermentable sugars produced by the degradation of the bagasse may be related to the chemical composition of the substrate. The sugarcane contains a high percentage of lignin forming a protective layer around the holocellulose, thus the glycosidic bonds are shielded extensively from enzymatic attack. In comparison, the initial degradation of the pineapple fibre required the action of hemicellulases, and proceeded at an enhanced rate under sulphidogenic conditions. The initial degradation of the pineapple fibre produced a substantially larger quantity of fermentable sugars in comparison to the bagasse. The higher production of fermentable sugars from the degradation of the pineapple fibre may be explained by the fact that this substrate may have a lower percentage of lignin than the bagasse, thus allowing a larger percentage of the glycosidic bonds to be exposed to enzymatic attack. The data obtained also indicated that the glycosidic bonds from the hemicellulosic components of the pineapple fibre shielded the glycosidic bonds of the cellulose component. The identification of the chemical components of the different substrates may allow for the initial development of an ideal enzyme complex (designer cellulosome) with enzymes in an ideal ratio with optimal synergy that will effectively degrade the complex plant biomass substrate.

The increasing concern about water quality and energy demand promotes the development of innovative and low-cost processes to improve the nutrient uptake and energy efficiency of existing wastewater treatments (WWT). In this context, the inclusion of a microalgae system (MAS) in the flowsheet of a WWT plant represents a sustainable alternative to conventional technologies, as it combines a low-cost nutrient uptake system with the production of biomass suitable for biofuel production. However, at present, the energy required to cultivate and process the algae cells is often too high to justify their use. The adoption of a low energy harvesting system and an efficient energy conversion process are the sine qua non requirements to guarantee the sustainability of the process. In this thesis, current and innovative harvesting technologies for large scale applications have been reviewed to identify the optimal working conditions of each system and their link to the main characteristics of the algae suspension. In particular, the performance of the Ballasted Dissolved Air Flotation (BDAF) system was investigated using different algae and compared to the conventional Dissolved Air Flotation (DAF). BDAF was demonstrably a very viable harvesting method where the use of floating microspheres as ballasting agents allowed significant coagulant savings, reduced the level of energy dissipation within the flotation chamber, and lowered the overall carbon emissions and the process costs. Cont/d.

Les procédés de transformation de biomasse lignocellulosique en bioéthanol de seconde génération sont actuellement des sujets de recherche très répandus mais ne sont toujours pas compétitifs avec ceux de la première génération. Les facteurs clés limitants sont : l’efficacité et les coûts du prétraitement, les rendements de l’hydrolyse enzymatique, et la co-fermentation C5-C6. Un procédé continu de déconstruction de la matière végétale, compactant un prétraitement thermo-mécano-chimique utilisant un agent alcalin avec une introduction d’enzymes en extrusion bi-vis, appelé bioextrusion, est développé dans cette étude. Il permet de préparer la matière cellulosique à un haut taux de matière sèche (>20%), à une saccharification et une fermentation pouvant être simultanées (SSF). Le traitement continu peut extraire une grande part des hémicelluloses (jusqu’à 97%) et des lignines (>50%) et améliorer l’accessibilité de la cellulose tout en initiant sa dépolymérisation par des cocktails enzymatiques pendant la bioextrusion. Plusieurs matières premières (Résidu de maïs doux, Bagasse d’agave bleue, Résidu d’huilerie de palme, Paille d’orge, Résidu d’Eucalyptus, Sarments de vigne et Bagasse de canne à sucre) ont été caractérisées et leurs comportements vis-à-vis du procédé ont été comparés. L’évolution de la composition de ces matières à travers le procédé et leur hydrolysabilité ont été étudiées. Suite au traitement, une augmentation du rendement de saccharification dans un réacteur (24h de temps de réaction à 20% de consistance) a été obtenue pour ces matières (jusqu’à 85% des C6 théoriques et 70% des C5-C6 théoriques). Les rendements de fermentation non optimisés atteignent un maximum de 85% théorique des sucres C6 convertis, 65% théorique des C5-C6 convertis, et une concentration d’éthanol de 15g/100g extrudat sec. Le procédé de production d’éthanol dans son ensemble (avec addition de l’énergie de la valorisation des coproduits) atteint un ratio « énergie consommée/produite » de 0.5-0.6. Le nouveau procédé présente ainsi les avantages de minimiser la consommation d’énergie par l’application de faibles températures, de minimiser la consommation d’eau par l’utilisation de faibles ratios liquide/solide, de ne pas produire d’inhibiteurs de fermentation et d’être rapide, compact, continu et adaptable sur différentes biomasses
Lignocellulosic biomass transformation processes in order to produce second generation bioethanol are actually widely studied all around the world but still not yet competitive compare to the first generation. The limiting key factors of the different processes are: the pre-treatment efficiency and costs, the enzymatic hydrolysis yields, and the co-fermentation C5-C6. A continuous plant matter deconstruction process, compacting a thermo-mechanico-chemical pre-treatment using alkali solution with an enzymes injection in twin-screw extruder, called bioextrusion, is developed in this study. It allows preparing the cellulosic material at a high dry matter content (>20%), to a possible simultaneous saccharification and fermentation (SSF). This continuous treatment may extract a big part of hemicelluloses (until 97%) and lignin (>50%) and configures cellulose to a better accessibility and a start of its depolymerisation by enzymes cocktail during the bioextrusion. Several raw matters (Sweet Corn Cob and Spathe, Blue Agave Bagass, Oil Palm Empty Fruit Bunch, Barley Straw, Eucalyptus Residue, Grape Pruning Residue and Sugarcane Bagass) have been characterized and theirs behaviours toward to the process were compared. Evolutions of these matters compositions throughout the process and their hydrolysability have been studied. Further to the treatment, an improvement of the saccharification yields in reactor (24h reaction time at 20% consistency) has been obtained on these matters (until 85% of theoretical C6 and 70% of theoretical C5-C6). The not optimized fermentation yields reach a maximum of 85% of theoretical converted C6 sugars, 65% of theoretical converted C5-C6 sugars, and an ethanol concentration of 15g/100g dry matter extrudate. The whole ethanol production process (with addition of energy from the recovery of the by-products) is achieved with a “consumed/produced energy” ratio of 0.5-0.6. The new process presents the advantages to minimize the energy consumption by operating low temperatures, to minimize water consumption by working at low liquid/solid ratio, to not produce fermentation ‘s inhibitors and to be quick, compact, continuous and adaptable to different biomasses

Etude de la composition osidique d'alginate des genres pelvetia, fucus, arcophyllum, sargassum et laminaria. Preparation et caracterisation des alginates-lyases a partir de divers mollusques marins et d'une bacterie marine. Etude comparee de la regeneration de la paroi du protoplaste et de la mise en place normale de la paroi du zygote de fucus distichus par marquage avec des anticorps monoclonaux

O etanol celulósico a partir da palhada de cana pode elevar a produção do bioetanol, porém esta é normalmente decomposta no campo. A degradação da parede celular no campo não foi elucidada e compreender este processo auxiliará na produção de etanol celulósico. O objetivo deste trabalho foi caracterizar a degradação da palhada de cana de açúcar no campo durante um ano. Foi analisada a composição da parede celular por fracionamento e composição dos monossacarídeos. Na parede celular, observou-se redução de 26% no teor de celulose enquanto houve aumento de 13% na fração de hemiceluloses mais solúveis. Mudanças na composição dos monossacarídeos das frações mostraram que o arabinoxilano (AX) foi o primeiro polímero a ser solubilizado (após 3 meses) seguido dos b-glucanos e celulose (após 6 meses). Isto sugere que o AX é a hemicelulose mais exposta e sua solubilização permitiu a degradação da celulose após 6 meses. A partir dos dados obtidos, sugeriu-se a utilização de xilanases seguidas de glucanases numa possível ordem de enzimas para produção de etanol celulósico.
The sugarcane straw cellulosic ethanol can increase bioethanol production, but the straw is usually degraded in the field. However, the process that leads the cell wall disassembly under field conditions is unknown and understanding how this happens can improve cellulosic ethanol production. In the present work we aimed at studying how sugarcane straw is degraded in the field during a year. Cell wall composition was determined by fractioning and determination of monosaccharide composition. Results showed a decrease (ca.26%) in cellulose content and an increase of 13% in high solubility hemicelluloses fraction. Changes in monosaccharide composition showed that the first polymer to be solubilised is the arabinoxylan (AX) (after 3 months) followed by b-glucans and cellulose (after 6 months). This suggests that AX is the most exposed hemicelullose and its solubilisation allowed cellulose degradation after 6 months. Our data suggest the use of xylanases followed by glucanases as an enzyme order to be used in cellulosic ethanol production from sugarcane straw.