Dissertations / Theses on the topic 'Carbohydrate active enzymes (CAZymes)'

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)

Tese de Doutoramento em Ciências Veterinárias na especialidade Produção Animal
The biodegradation of plant cell wall (PCW) carbohydrates is performed by microbial enzymes that are generally referred to as CAZymes. In animal nutrition, it is now well established that the monogastric animals produce a limited repertoire of CAZymes and as such cannot use efficiently some dietary ingredients that sometimes display antinutritional properties. The dietary supplementation with exogenous CAZymes improves the nutritive value of diets and increases animal’s performance. In particular, this study demonstrated that 1,3-1,4-β-glucanases and not cellulases improve the nutritive value of β-glucan-containing diets for monogastric animals. In addition, it was revealed that exogenous enzyme supplementation with β-xylanases improved the nutritive value of diets incorporating wheat lots with high viscosity and low endogenous endo-1,4-β-xylanase activity. In contrast, when the wheat lot showed lower viscosity and higher levels of endogenous endo-1,4-β-xylanase activity, broiler response was clearly diminished. Moreover, the data revealed that xylo-oligosaccharides released by xylanases acting on cereal arabinoxylans display a pre-biotic and positive effect in broiler chicks. However, although we observe an exponential accumulation of genomic and metagenomic information, knowledge on CAZYmes with potential to be used in animal nutrition is limited. This work also aimed to develop high-throughput (HTP) methodologies to isolate and characterize potentially important enzymes for animal nutrition. Thus, 1476 recombinant enzymes were selected and produced recombinantly. The data revealed that 79% of recombinant proteins were produced in the soluble form in Escherichia coli. Factors, such as, organism of origin, gene production strategy, fusion with solubility tags, protein molecular weight and amino acids composition of primary sequences may be used to justify and predict levels of solubility. The establishment of a high-throughput pipeline for recombinant enzyme production was used to obtain a library of feruloyl esterases (FAEs) and glucuronoyl esterases (GEs), enzymes which remove the side chains and break crosslinks between hemicellulosic carbohydrates and lignin. Thus 480 putative FAEs and 20 GEs were produced and biochemically characterized. Following gene isolation, 372 FAEs and 11 GEs were produced in a soluble form in E. coli. Activity results showed that 50% of the enzymes produced retained significant levels of activity and stability. The library of innovative FAEs and GEs produced during this project will be used to develop a novel generation of enzymes for animal nutrition, in particular to exploit the release of cellulose and hemicellulose from lignin.
RESUMO - Na natureza, a biodegradação dos hidratos de carbono da parede celular vegetal é realizada por enzimas microbianas, geralmente conhecidas como CAZymes. Os animais monogástricos produzem um reportório limitado de enzimas para degradação destes hidratos de carbono, não conseguindo usar eficientemente alguns ingredientes da dieta, que muitas vezes manifestam propriedades anti nutritivas. Assim, é sabido que a suplementação com CAZymes exógenas melhora o valor nutritivo das dietas e aumenta o desempenho produtivo dos animais. Este trabalho revelou que as enzimas mais apropriadas para suplementar dietas ricas em 1,3-1,4-β-glucanos são as 1,3-1,4-β-glucanases e não as celulases. Além disso, verificou-se que a suplementação com β-xilanases melhorou o valor nutritivo de dietas que continham variedades de trigo com maior viscosidade e menor atividade endógena de endo-1,4-β-xilanase. Em oposição, quando o lote de trigo apresentou menor viscosidade e maiores níveis de atividade endógena de endo-1,4-β-xilanase, a resposta dos animais à adição das enzimas foi menor. Este trabalho mostra, igualmente, que os xilo-oligossacarídeos, resultantes da degradação de arabinoxilanos por xilanases exógenas, possuem uma ação pré-biótica na alimentação de frangos, promovendo a melhoria do desempenho zootécnico. Contudo, apesar de estarem descritas uma grande diversidade CAZymes, poucas são as estudadas com potencial para serem usadas em alimentação animal. Portanto, este trabalho pretendeu, também, desenvolver metodologias para isolar e caracterizar enzimas potencialmente importantes em larga escala. Foram selecionadas, produzidas e expressas na forma recombinante 1476 CAZymes. Os dados revelaram que 79% das proteínas recombinantes foram produzidas na forma solúvel em Escherichia coli. Verificou-se, ainda, que fatores como o organismo de origem, a estratégia de produção, a fusão com marcadores de solubilidade, o peso molecular da proteína e composição de aminoácidos das sequências primárias, parecem justificar os resultados da solubilidade. Estes ensinamentos foram utilizados para produzir enzimas, tais como ferulolil esterases (FAEs) e glucuronil esterases (GEs), que removem as cadeias laterais e quebram as ligações cruzadas entre hidratos de carbono hemicelulósicos e a lenhina. Assim sendo, foram selecionadas 480 FAEs e 20 GEs para produção recombinante e caracterização bioquímica. Cerca de 372 FAEs e 11 GEs foram produzidos em forma solúvel em E. coli e aproximadamente 50% das enzimas produzidas mantiveram níveis significativos de atividade e estabilidade. Com isto, foi possível identificar e produzir um número significativo de FAEs e GEs com potencial para alimentação animal, em especial as que libertam celulose e hemicelulose da lenhina.
N/A

The focus of this thesis is a comparative study of approaches in discovery of carbohydrate-active enzymes (CAZymes). CAZymes synthesise, bind to, and degrade all the multitude of carbohydrates found in nature. As such, when aiming for sustainable methods to degrade plant biomass for the generation of biofuels, for which there is a strong drive in society, CAZymes are a natural source of environmentally friendly molecular tools. In nature, microorganisms are the principal degraders of carbohydrates. Not only do they degrade plant matter in forests and aquatic habitats, but also break down the majority of carbohydrates ingested by animals. These symbiotic microorganisms, known as the microbiota, reside in animal digestive tracts in immense quantities, where one of the key nutrient sources is complex carbohydrates. Thus, microorganisms are a plentiful source of CAZymes, and strategies in the discovery of new enzymes from bacterial sources have been the basis for the work presented here, combined with biochemical characterisation of several enzymes. Novel enzymatic activities for the glycoside hydrolase family 31 have been described as a result of the initial projects of the thesis. These later evolved into projects studying bacterial multi-gene systems for the partial or complete degradation of the heterogeneous plant polysaccharide xyloglucan. These systems contain, in addition to various hydrolytic CAZymes, necessary binding-, transport-, and regulatory proteins. The results presented here show, in detail, how very complex carbohydrates can efficiently be degraded by bacterial enzymes of industrial relevance.

QC 20130826

To understand and modify the secondary cell walls of plants the project group Enzyme Discovery in Hybrid Aspen for Fiber Engineering (EDEN) was founded composed of nine laboratories with funding from the European Commission. The main target of EDEN´s research is to genetically engineer fiber structure in order to produce transgenic trees with modified properties for the pulp and paper industries.
In this target framework, the Populus tremula x tremuloides xyloglucan endotransglycosylase (PttXET16A) was selected for in-depth study of its transglycosylase activity catalyzing cleavage and reconnection of xyloglucan molecules, which is proposed to be involved in secondary cell wall morphogenesis.
The creation of a family 16 carbohydrate active enzyme -glucanase/XET hybrids were attempted in order to design a chimeric enzyme with one or more of the following altered properties: specificity, activity, and or stability.
The two enzymes, Bacillus licheniformis 1,3-1,4--glucanase and Populus tremula x tremuloides xyloglucan endotransglycosylase, are members of the same enzymatic family and have highly homologous 3-dimensional structures. However, the enzymes exhibit different activities, one a hydrolase the other a transferase; different specificities, one accepts only linear glcosydic substrates while the other branched substrates; and different stabilities.
Hybrid enzyme construction represented an investigational challenge in order to understand what physical characteristics of both enzymes attribute to the specific pattern of activity and specificity observed.
Removal of the 1,3-1,4--glucanase major loop resulted in a folded protein which still maintained some β-glucan hydrolase activity. However, no xyloglucan endotransglycosylase-like activity or specificity was observed. Next, point mutations of the β-sheets forming the enzymatic binding site cleft were mutated to resemble PttXET16A residues. The final chimeric protein neither exhibited XET nor β-glucanase activities. Structural analysis by X-ray crystallography revealed a major unexpected structural rearrangement providing a clear insight for further enzyme engineering.
Amb la finalitat d'entendre i modificar la paret cel·lular secundària de les plantes, es va fundar el grup Enzyme Discovery in Hibrid Aspen for Fibern Engineering (EDEN) composat per nou laboratoris amb la finançament de la Comissió Europea. El principal objectiu de la recerca del grup EDEN és enginyar genèticament l'estructura de fibres per tal de produir arbres transgènics amb propietats modificades per les indústries de la polpa i el paper.
En el marc d'aquest projecte, es va seleccionar el Populus tremula x tremuloides xiloglucà endotransglicosilasa (PttXET16A) per estudiar en profunditat la seva activitat transglicosilasa catalitzant el trencament i la reconnexió de molècules de xiloglucà, el qual sembla estar involucrat en la morfogènesi de la paret cel·lular secundària.
D'aquesta manera, s'intentà crear una família 16 d'híbrids de l'enzim actiu amb carbohidrats -glucanasa/XET per tal de dissenyar un enzim quimèric amb una o més de les propietats següents alterades: especificitat, activitat i/o estabilitat.
Els dos enzims, Bacillus licheniformis 1,3-1,4--glucanasa i Populus tremula x tremuloides xiloglucà endotransglicosilasa, són membres de la mateixa família enzimàtica i tenen una gran homologia en les seves estructures en 3-dimensions. Tot i així, aquests enzims presenten diferents activitats, un presenta activitat hidrolasa i l'altre, transferasa; diferents especificitats, un accepta només substrats glicosílics lineals mentre l'altre, substrats ramificats; i diferents estabilitats.
La construcció d'un enzim híbrid representa un repte en la investigació amb la finalitat d'entendre quines característiques físiques dels dos enzims s'atribueixen al model específic de l'activitat i especificitat observada.
L'extracció del llaç més gran de l'1,3-1,4--glucanasa va resultar en l'obtenció d'una proteïna plegada que encara manté certa activitat hidrolasa del -glucà. Tot i això, no s'observà activitat o especificitat similar a la xiloglucà endotransglicosilasa. A partir d'aquí, es realitzaren mutacions puntuals a diferents punts de les fulles  que formen l'escletxa del lloc d'unió de l'enzim per assemblar-se als residus del PttXET16A. La proteïna quimèrica final tampoc presentava activitat XET ni -glucanasa. L'anàlisi de l'estructura per cristal·lografia de raigs X revelà una major reorganització estructural de l'esperada proveint el nou enzim d'un clar espai intern que obra moltes més portes a l'enginyeria de l'enzim.
Con la finalidad de entender y modificar la pared celular secundaria de las plantas, se fundó el grupo Enzyme Discovery in Hibrid Aspen for Fibern Engineering (EDEN) compuesto por nueve laboratorios con la financiación de la Comisión Europea. El principal objetivo de la búsqueda del grupo EDEN es ingeniar genéticamente la estructura de fibras para producir árboles transgénicos con propiedades modificadas para las industrias de la pulpa y el papel.
En el marco de este proyecto, se seleccionó el Populus tremula x tremuloides xiloglucán endotransglicosilasa (PttXET16A) para estudiar en profundidad su actividad transglicosilasa catalizando la rotura y la reconnexión de moléculas de xiloglucán, el cual parece estar involucrado en la morfogénesis de la pared celular secundaria. De esta forma, se intentó crear una familia 16 de híbridos de la enzima activa con carbohidratos -glucanasa/XET con la finalidad de diseñar una enzima quimérica con una o más de las propiedades siguientes alteradas: especificidad, actividad y/o estabilidad.
Las dos enzimas, Bacillus licheniformis 1,3-1,4--glucanasa y Populus tremula x tremuloides xiloglucà endotransglicosilasa, son miembros de la misma familia enzimática y tienen una gran homología en sus estructuras en 3-dimensiones. Aún así, estas enzimas presentan diferentes actividades, una tiene actividad hidrolasa y la otra, transferasa; diferentes especificidades, una acepta sólo sustratos glicosílicos lineales mientras la otra, sustratos ramificados; y diferentes estabilidades.
La construcción de una enzima híbrida representa un reto dentro de la investigación con la finalidad de entender qué características físicas de las dos enzimas se atribuyen al modelo específico de la actividad y especificidad observada. La extracción del lazo más grande de la 1,3-1,4--glucanasa resultó en la obtención de una proteína plegada que todavía mantiene cierta actividad hidrolasa del -glucán. Aún así, no se observó actividad o especificidad similar a la xiloglucán endotransglicosilasa. A partir de este punto, se realizaron mutaciones puntuales a diferentes puntos de las hojas  que forman la brecha del lugar de unión de la enzima por asemejarse a los residuos del PttXET16A. La proteína quimérica final tampoco presentaba actividad XET ni -glucanasa. El análisis de la estructura por cristalografía de rayos X reveló una mayor reorganización estructural de la esperada proveyendo la nueva enzima de un claro espacio interno que obre muchas más puertas a la ingeniería de la enzima.

Extremozymes are enzymes produced by microorganisms that live in extreme habitats. Due to their higher stability, extremozymes is attracting interest as biocatalysts in various industrial processes. In this context, carbohydrate-active extremozymes can be used in various processes relevant to the paper, food and feed industry. In this thesis, the crystal structure, biochemical characterization and the capacity to synthesize prebiotic galacto-oligosaccharides (GOS) were investigated for a β-glucosidase (HoBGLA) from the halothermophilic bacterium Halothermothrix orenii. The wild-type enzyme displays favorable characteristics for lactose hydrolysis and produces a range of prebiotic GOS, of which β-D-Galp-(1→6)-D-Lac and β-D-Galp-(1→3)-D-Lac are the major products (Paper I). To further improve GOS synthesis by HoBGLA, rational enzyme engineering was performed (Paper II). Six enzyme variants were generated by replacing strategically positioned active-site residues. Two HoBGLA variants were identified as potentially interesting, F417S and F417Y. The former appears to synthesize one particular GOS product in higher yield, whereas the latter produces a higher yield of total GOS. In Paper III, the high-resolution crystal structure and biochemical characterization of a hemicellulase (HoAraf43) from  H. orenii is presented. HoAraf43 folds as a five-bladed β-propeller and displays α-Larabinofuranosidase activity. The melting temperature of  HoAraf43 increases significantly in the presence of high salt and divalent cations, which is consistent with H. orenii being a halophile. Furthermore, the crystal structures of a thermostable tetrameric pyranose 2-oxidase from Phanerochaete chrysosporium (PcP2O) were determined to investigate the structural determinants of thermostability (Paper IV). PcP2O has an increased number of salt links between subunits, which may provide a mechanism for increased stability. The structures also imply that the N-terminal region acts as an intramolecular chaperone during homotetramer assembly.

QC 20150429

Tese de Doutoramento em Ciências Veterinárias, especialidade em Ciências Biológicas e Biomédicas
Carbohydrate-active enzymes (CAZymes) include a range of enzymes that, in nature, make, break or modify glycosidic bonds. CAZymes act on highly recalcitrant polysaccharides, such as cellulose and hemicellulose, and often exhibit a modular architecture including catalytic domains fused through flexible linker regions to non-catalytic domains such as carbohydrate-binding modules (CBMs). In some anaerobic bacteria these enzymes can associate in high molecular mass multi-enzyme complexes termed cellulosomes. Cellulosomal organisms express a vast repertoire of plant cell wall degrading enzymes and constitute a promising source for the discovery of novel CAZymes. Presently, an exponential accumulation of genomic and metagenomic information is observed while the identification of the biological role of both genes and proteins of unknown function is sorely lacking. In addition, for most of the known CAZymes, structure and/or biochemical characterization is missing. In this study we have developed innovative approaches for the discovery of novel CAZymes in cellulosomal bacteria and provide a detailed biochemical characterization of some of those enzymes. A high-throughput platform was designed for cloning, expression and production of recombinant cellulosomal proteins in Escherichia coli, aiming at looking for novel cellulosomal CAZymes encoded in the genomes of Clostridium thermocellum and Ruminococcus flavefaciens. As a result, a series of novel prokaryotic expression vectors (pHTP) were constructed to allow ligation-independent cloning with high levels of soluble recombinant protein production. In addition, to allow total automation of the procedure, both novel cell culture media and protein purification methods have been established. The platform allowed the production of 184 cellulosomal proteins of unknown function that after the implementation of an enzyme discovery screen lead to the discovery of a novel family of α-Larabinofuranosidases. In order to achieve recombinant soluble expression in E. coli, novel fusion tags were designed and incorporated into pHTP-derivatives. Both Rf1 and Rf47 tags, derived from cellulosomal components, were shown to display a high capacity to enhance protein solubility, as fusion proteins containing both these tags were expressed at high levels and in the soluble form in E. coli. CBMs were confirmed to affect the catalytic activity of appended CAZymes, as it was illustrated by the CBM32 of CtMan5A. This work revealed that members of family 35 CBM have the capacity to bind β-mannose-containing polymers. The biochemical characterization of PL1A, PL1B and PL9 reported here describes the pectinolytic activity expressed by C. thermocellum cellulosome. These enzymes are appended to CBMs that display considerable ligand promiscuity. The application of β- glucanases in animal feed supplementation was tested either in the free state or while associated in mini-cellulosomes. This study revealed that β-1,3-1,4-glucanases and not β-1,4-glucanases are necessary to improve the nutritive value of barley-based diets for broilers. In addition, it was shown that mini-cellulosomes designed to improve the efficacy of exogenous enzymes used for feed supplementation require an effective mechanism to protect linker regions from proteolytic cleavage.
RESUMO - Descoberta de novas enzimas celulossomais de bactérias anaeróbias que degradam hidratos de carbono - As enzimas que na natureza degradam os hidratos de carbono (CAZymes) são capazes de construir, quebrar ou modificar ligações glicosídicas. Estas enzimas actuam sobre polissacáridos complexos e recalcitrantes, como a celulose e a hemicelulose, e apresentam geralmente uma estrutura modular, podendo incluir módulos catalíticos fundidos através de sequências de ligação a domínios não catalíticos, sendo os mais comuns os módulos de ligação a hidratos de carbono (CBMs). Em algumas bactérias anaeróbias, estas enzimas podem associar-se em complexos multi-enzimáticos de elevada massa molecular designados de celulossomas. Os organismos que produzem estes complexos apresentam um vasto repertório de enzimas envolvidas na degradação da parede celular vegetal e constituem um bom ponto de partida para a descoberta de novas CAZymes. Actualmente, verifica-se uma crescente acumulação de informação genómica e metagenómica a um ritmo superior à capacidade de identificação da função biológica de uma plêiade de genes e proteínas de funções desconhecidas. Para além disso, para a maioria das CAZymes já conhecidas, não foi ainda efectuada uma caracterização estrutural e/ou bioquímica. Neste estudo foram desenvolvidas metodologias inovadoras para a descoberta de novas CAZymes em bactérias celulossomais, bem como se procedeu a uma caracterização bioquímica detalhada para algumas destas enzimas. Desenvolveu-se uma plataforma de alta capacidade para a clonagem, expressão e produção de proteínas celulossomais recombinantes em Escherichia coli, tendo como objectivo descobrir novas CAZymes codificadas nos genomas de Clostridium thermocellum e Ruminococcus flavefaciens. Como resultado, foi construída uma nova série de vectores de expressão (pHTP) a fim de sustentarem um método de clonagem independente de ligação. Para possibilitar a total automatização do processo foram desenvolvidos novos meios de cultura celulares e métodos de purificação de proteínas adaptados a um esquema de produção de alta capacidade. A pesquisa de novas enzimas nos módulos celulossomais de função desconhecida possibilitou a descoberta de uma nova α-L-arabinofuranosidase em R. flavefaciens, que se constitui como a enzima fundadora de uma nova família de CAZymes. A fim de potenciar a solubilidade de proteínas recombinantes em E. coli, foram desenhadas novas tags de fusão, as quais foram incorporadas em vectores derivados do pHTP. Tanto as tags Rf1 como Rf47, derivadas de componentes celulossomais, mostraram possuir uma capacidade elevada para potenciar a solubilidade de proteínas, uma vez que as proteínas de fusão contendo quer uma quer outra destas tags foram produzidas na forma solúvel em níveis mais elevados do que com parceiros de fusão anteriormente descritos. Confirmou-se que os CBMs afectam a actividade catalítica das CAZymes associadas, tal como ilustrado pelo CBM32 da CtMan5A. Este trabalho forneceu indicações de que os CBMs membros da família 35 têm a capacidade de se ligarem a polímeros de β-manose. A caracterização bioquímica das PL1A, PL1B e PL9 aqui descrita constituiu o primeiro relato de actividade pectinolítica no celulossoma de C. thermocellum. Estas enzimas podem estar associadas a CBMs que revelam pouca especificidade de ligação aos substratos. Testou-se a aplicação de β-glucanases na suplementação alimentar animal, tanto como enzimas isoladas, como associadas em mini-celulossomas. Os dados apresentados aqui revelam que são as β-1,3-1,4-glucanases e não as β-1,4-glucanases as enzimas responsáveis por melhorar o valor nutritivo de dietas à base de cevada para frangos. Por outro lado, os resultados mostram que a eficácia dos mini-celulossomas para melhorar o desempenho das enzimas exógenas usadas na suplementação alimentar requer um mecanismo eficaz para proteger as regiões de ligação entre os componentes celulossomais da degradação por proteases.

This thesis presents the application of different protein engineering methods on enzymes and non-catalytic proteins that act upon xyloglucans. Xyloglucans are polysaccharides found as storage polymers in seeds and tubers, and as cross-linking glucans in the cell wall of plants. Their structure is complex with intricate branching patterns, which contribute to the physical properties of the polysaccharide including its binding to and interaction with other glucans such as cellulose. One important group of xyloglucan-active enzymes is encoded by the GH16 XTH gene family in plants, including xyloglucan endo-transglycosylases (XET) and xyloglucan endo-hydrolases (XEH). The molecular determinants behind the different catalytic routes of these homologous enzymes are still not fully understood. By combining structural data and molecular dynamics (MD) simulations, interesting facts were revealed about enzyme-substrate interaction. Furthermore, a pilot study was performed using structure-guided recombination to generate a restricted library of XET/XEH chimeras. Glycosynthases are hydrolytically inactive mutant glycoside hydrolases (GH) that catalyse the formation of glycosidic linkages between glycosyl fluoride donors and glycoside acceptors. Different enzymes with xyloglucan hydrolase activity were engineered into glycosynthases, and characterised as tools for the synthesis of well-defined homogenous xyloglucan oligo- and polysaccharides with regular substitution patterns. Carbohydrate-binding modules (CBM) are non-catalytic protein domains that bind to polysaccharidic substrates. An important technical application involves their use as molecular probes to detect and localise specific carbohydrates in vivo. The three-dimensional structure of an evolved xyloglucan binding module (XGBM) was solved by X-ray diffraction. Affinity-guided directed evolution of this first generation XGBM resulted in highly specific probes that were used to localise non-fucosylated xyloglucans in plant tissue sections.
QC 20100902

Wood and wood products are of great economical and environmental importance, both in Sweden and globally. Biotechnology can be used both for achieving raw material of improved quality and for industrial processes such as biobleaching. Despite the enormous amount of carbon that is fixed as wood, the knowledge about the enzymes involved in the biosynthesis, re-organization and degradation of plant cell walls is relatively limited. In order to exploit enzymes more efficiently or to develop new biotechnological processes, it is crucial to gain a better understanding of the function and mechanism of the enzymes. This work has aimed to increase the knowledge about some of the enzymes putatively involved in the wood forming processes in Populus. Xyloglucan endotransglycosylases and a putative xylanase represent transglycosylating and hydrolytic enzymes, respectively. Carbohydrate binding modules represent non-catalytic modules, which bind to the substrate. Among 24 genes encoding for putative xyloglucan endotransglycosylases or xyloglucan endohydrolases that were identified in the Populus EST database, two were chosen for further studies (PttXTH16-34 and PttXTH16-35). The corresponding proteins, PttXET16-34 and PttXET16-35, were expressed in P. pastoris, purified and biochemically characterized. The importance of the N-glycans was investigated by comparing the recombinant wild-type proteins with their deglycosylated counterparts. In order to obtain the large amounts of PttXET16-34 that were needed for crystallization and development of biotechnological applications, the conditions for the large-scale production of PttXET16-34 in a fermenter were optimized. In microorganisms, endo-(1,4)-β-xylanases are important members of the xylan degrading machinery. These enzymes are also present in plants where they might fulfill a similar, but probably more restrictive function. One putative endo-(1,4)-β-xylanase, denoted PttXYN10A, was identified in the hybrid aspen EST library. Sequence analysis shows that this protein contains three putative carbohydrate-binding modules (CBM) from family 22 in addition to the catalytic module from GH10. Heterologous expression and reverse genetics were applied in order to elucidate the function of the catalytic module as well as the binding modules of PttXYN10A. Just as in microorganisms, some of the carbohydrate active enzymes from plants have one or more CBM attached to the catalytic module. So far, a very limited number of plant CBMs has been biochemically characterized. A detailed bio-informatic analysis of the CBM family 43 revealed interesting modularity patterns. In addition, one CBM43 (CBM43PttGH17_84) from a putative Populus b-(1,3)-glucanase was expressed in E. coli and shown to bind to laminarin (β-(1,3)-glucan), mixed-linked β-(1,3)(1,4)-glucans and crystalline cellulose. Due to their high specificity for different carbohydrates, CBMs can be used as probes for the analysis of plant materials. Generally, they are more specific than both staining techniques and carbohydrate-binding antibodies. We have used cellulose- and mannan binding modules from microorganisms as tools for the analysis of intact fibers as well as processed pulps.
QC 20100903

Enzymes have attracted the attention of chemists and biologists since long ago due to their molecular complexity and tremendous efficiency. They are highly specific catalysts that make chemical reactions possible at mild conditions with an astonishing rate enhancement. For this reason there have been many efforts to understand how these macromolecules work, trying to find out crucial factors that influence their activity. Whereas it is difficult to settle on how enzymes work in general, with hot debates over many years, significant progress has been made in elucidating specific catalytic mechanisms by means of experimental and computational approaches. In this thesis we have focused on the study of glycoside hydrolases and transferases, enzymes that are known as “carbohydrate-active enzymes” (CAZymes) and that are essential for the processing and remodeling of carbohydrates in living organisms. In spite of the enormous advances in the understanding of how these enzymes work, with several factors that are known or presumed to enhance their reaction rates (such as certain sugar conformations, enzyme-substrate interactions or the flexibility of the enzyme fold), there are still many aspects that remain poorly understood due to the lack of atomistic insights. Given this, in the present thesis we have used cutting-edge computational methods, including all-atom molecular dynamics simulations, hybrid quantum mechanics/molecular mechanics approaches and enhanced sampling techniques, to unveil some of the essential enzymatic interactions and conformations that influence catalysis in CAZymes. With these techniques we have provided proofs for general concepts that are usually assumed, as well as insights for the specific enzymes that have been studied. In particular, we have evaluated the reaction free energy contribution of sugar conformations to catalysis in β-xylanases, the influence of crucial hydrogen bond interactions in β-glucosidases, the importance of enzymatic residues that bind water in the active site of inverting β-mannanases, and the structural flexibility of a human enzyme called glycogenin.
Desde los primeros descubrimientos en el campo de la enzimología, las enzimas han atraído la atención de numerosos químicos y biólogos debido a su gran complejidad molecular y su elevada eficiencia. Estas macromoléculas son catalizadores altamente específicos que hacen posible reacciones químicas en condiciones suaves y a velocidades asombrosas. Por esta razón se han destinado muchos esfuerzos en tratar de comprender su funcionamiento, intentando descubrir los factores fundamentales que influyen en su actividad. En la presente tesis doctoral hemos ahondado en la comprensión de una clase de enzimas llamadas “glicosil hidrolasas” y “glicosil transferasas”, englobadas bajo la denominación de “enzimas activas en carbohidratos”. Estas enzimas están encargadas de la degradación y de la síntesis de carbohidratos, moléculas que debido a su diversidad y flexibilidad añaden un grado de complejidad adicional en su estudio. Aún a pesar de los grandes avances en la comprensión general de estas enzimas, habiéndose destacado distintos factores involucrados en su rendimiento catalítico (e.g. ciertas conformaciones del sustrato, interacciones enzima-sustrato o la flexibilidad de la estructura enzimática), la falta de información a nivel molecular dificulta la racionalización de muchas de estas evidencias. Debido a ello, en esta tesis hemos hecho uso de técnicas computacionales de vanguardia, incluyendo simulaciones atomísticas de dinámica molecular, enfoques híbridos de mecánica cuántica/mecánica molecular y técnicas de exploración avanzada del espacio de fases, para revelar el origen molecular de ciertas interacciones y conformaciones esenciales para la catálisis de enzimas activas en carbohidratos. Con estas técnicas hemos proporcionando pruebas que refuerzan concepciones generalmente asumidas, así como detalles específicos para cada una de las enzimas que hemos estudiado. En particular, hemos analizado la contribución de las conformaciones del sustrato en la catálisis de β-xilanasas, la contribución de puentes de hidrógeno en la catálisis de β-glucosidasas, la importancia de residuos que enlazan agua en β-mananasas y la flexibilidad estructural de una enzima humana llamada glicogenina.

Renewable fibers produced by forest trees provide excellentraw material of high economic value for industrialapplications. Despite this, the genes and corresponding enzymesinvolved in wood fiber biosynthesis in trees are poorlycharacterized. This thesis describes a functional genomicsapproach for the identification of carbohydrate-active enzymesinvolved in secondary cell wall (wood) formation in hybridaspen.

First, a 3' target amplification method was developed toenable microarray-based gene expression analysis on minuteamounts of RNA. The amplification method was evaluated usingboth a smaller microarray containing 192 cDNA clones and alarger microarray containing 2995 cDNA clones that werehybridized with targets isolated from xylem and phloem.Moreover, a gene expression study of phloem differentiation wasperformed to show the usefulness of the amplificationmethod.

A microarray containing 2995 cDNA clones representing aunigene set of a cambial region EST library was used to studygene expression during wood formation. Transcript populationsfrom thin tissue sections representing different stages ofxylem development were hybridized onto the microarrays. It wasdemonstrated that genes encoding lignin and cellulosebiosynthetic enzymes, as well as a number of genes withoutassigned function, were differentially expressed across thedevelopmental gradient.

Microarrays were also used to track changes in geneexpression in the developing xylem of transgenic, GA-20 oxidaseoverexpressing hybrid aspens that had increased secondarygrowth. The study revealed that a number of genes encoding cellwall related enzymes were upregulated in the transgenic trees.Moreover, most genes with high transcript changes could beassigned a role in the early events of xylogenesis.

Ten genes encoding putative cellulose synthases (CesAs) wereidentified in our ownPopulusESTdatabase. Full length cDNA sequences wereobtained for five of them. Expression analyses performed withreal-time PCR and microarrays in normal wood undergoingxylogenesis and in tension wood revealed xylem specificexpression of four putative CesA isoenzymes.

Finally, an approach combining expressionprofiling,bioinformatics as well as EST and full length sequencing wasadopted to identify secondary cell wall related genes encodingcarbohydrate-active enzymes, such as glycosyltransferases andglycoside hydrolases. As expected, glycosyltransferasesinvolved in the carbohydrate biosynthesis dominated thecollection of the secondary cell wall related enzymes that wereidentified.

Key words:Populus, xylogenesis, secondary cell wall,cellulose, hemicellulose, microarrays, transcript profiling,carbohydrate-active enzyme, glycosyltransferase, glycosidehydrolase

Microalgae are gaining more attention for several reasons such as being potential producers of sustainable fuel, for use as health supplements and in skincare. Simris Alg is a Swedish company that produces Omega 3 supplements from a primary producer of these fatty acids - the algal diatom Phaeodactylum tricornutum, which is a sustainable alternative to Omega 3 derived from fish. Omega 3 fatty acids constitute a small fraction of the total algal biomass, and to increase profitability and utilize all of the biomass, the purpose of this thesis project is to present potential applications for the residual material that is left after oil extraction. A general composition study was made of Simris Alg algal residue material, and results are compared to those found in previous studies of P. tricornutum biomass. An optimization of the fractionation is needed to separate the storage carbohydrate chrysolaminarin and cell wall component glucuronomannan, followed by analysis for confirmation. Also, it would be interesting to separate chitin, if there is any, since despite the presence of chitin synthases, it is unclear whether the diatom actually produces chitin. When gathering information, no actual experimental characterization of carbohydrate active enzymes involved in synthesis of the main carbohydrates investigated were found. Such information would be useful to increase production of the carbohydrate of interest, if valuable applications are found. Potential applications of various cell components, such as carbohydrates, in skincare would be interesting to investigate, as well as optimizing fucoxanthin extraction for use as an additional high value product next to Omega 3.

La présence de sucres complexes constitue une source nutritive importante pour le microbiote qui assure leur dégradation via des CAZymes. Dans le cadre de cette thèse, nous avons construit in silico un modèle de type minimicrobiome contenant 177 génomes représentatifs des communautés bactériennes dans un microbiote intestinal conventionnel. L’analyse du contenu de ce minimicrobiome nous a permis d’estimer leur abondance et leur diversité. De plus, la comparaison du contenu CAZymes par groupe bactérien de type « phylum » a révélé une variabilité inter-phylum, notamment une diversité de familles CAZymes et une abondance en gènes bien plus élevées chez les Bacteroidetes. Dans un deuxième temps, nous avons développé une puce à ADN sur laquelle nous avons greffé des sondes non redondantes ciblant plus de 6500 gènes codant des CAZymes. Nous avons ensuite testé la "puce CAZyme" par hybridation d’ADN bactérien extrait d’échantillons de selles. Nos résultats suggèrent que cette méthode serait plus sensible dans la détection de CAZymes provenant de bactéries rares par rapport à la métagénomique. Ainsi, il est intéressant de noter qu’en utilisant la puce CAZyme, nous avons pu détecter un gène codant pour une famille GH6, alors que les études métagénomiques n’ont jamais réussi à détecter ce gène dans le microbiome intestinal humain et animal. Enfin, l’examen de huit échantillons de selles a permis l’identification d’un noyau CAZome contenant 46 familles de GHs et PLs, ce qui suggérerait que le microbiote intestinal est caractérisé par une stabilité fonctionnelle en dépit de variations taxonomiques importantes entre les individus testés et indépendamment de leur état de santé
The bacterial communities that inhabit our gut ensure their growth and survival by extracting their carbon source from the food that transits through the intestines. The complex carbohydrates included in the human diet are almost exclusively degraded by the gut microbiota using CAZymes. We built a minimicrobiome model using 177 genomes associated to gut microbiota. The CAZyme content analysis revealed their huge diversity and abundance in our minimicrobiome model. At the phylum level, the Bacteroidetes genomes showed the greatest CAZyme diversity and abundance. Interestingly, as most of CAZymes found in Bacteroidetes genomes contain a signal peptide allowing their secretion in the intestinal lumen and/or in periplasmic space, members of this phylum are suggested to be the primary degraders of complex carbohydrates. Further, we developed a microarray containing probes to target more than 6,500 CAZyme genes. We then validated the CAZyme microarray by the hybridization of bacterial DNA extracted from the stool samples of individuals. Our results suggest that a microarray-based study can detect genes from low-abundance bacteria better than metagenomic-based studies. A striking example was the detection of gene encoding a GH6-family in all subjects examined, whereas metagenomic studies have consistently failed to detect this gene in both human and animal gut microbiomes. In addition, an examination of eight stool samples allowed the identification of a corresponding core CAZome containing 46 CAZymes families that suggests a functional stability of the gut microbiota despite large taxonomical variations between individuals and independently of health state

O material lignocelulósico, presente na biomassa vegetal, representa uma importante fonte de energia, entretanto necessita da ação das enzimas lignocelulolíticas para sua degradação. A busca por novas enzimas que atuam na quebra da parede celular da planta em comunidades microbianas evoluídas naturalmente em um ambiente de degradação de biomassa como o rúmen oferece uma estratégia promissora para a prospecção de genes. Com isso, o projeto teve como objetivo a identificação de genes degradarores de biomassa vegetal em microrganismos do rúmen de ovinos usando a abordagem metagenômica. Para tanto, foram coletadas amostras da fase sólida do rúmen de 6 animais fistulados (Ovis aries) divididos em dois grupos e submetidos a duas dietas por 60 dias: tratamento controle e tratamento com dieta contendo bagaço de cana-de-açúcar. O DNA metagenômico total das amostras foi extraído e sequenciado na plataforma MiSeq Personal Sequencer (Illumina®). A análise dos dados para a anotação taxônomica e funcional foi realizada no software MG-RAST. A caracterização dos genes degradadores de biomassa vegetal foi feita na plataforma CLC Genomic Workbench v.5.5.1(CLC Bio, Denmark) e a anotação de 4,68 gigabases de dados foi feita no banco de dados CAZy. A análise taxonômica mostrou uma predominância do domínio Bacteria compondo mais de 96% de todas as amostras, sendo os filos mais abundantes Bacteroidetes, Firmicutes, seguido de Proteobacteria. Entre todos os filos anotados, cinco tiveram a abundância aumentada no tratamento com adição de bagaço de cana-de-açúcar na dieta, Firmicutes, Proteobacteria, Actinobacteria, Spirochaetes e Verrucomicrobia, e dois filos foram mais abundantes no tratamento controle, Bacteroidetes e Synergistetes. De modo geral, a análise de ordenação não mostrou correlação entre a composição do microbioma e o tipo de dieta, porém, na análise funcional, essa correlação foi observada uma vez que houve separação entre os tratamentos. A abundância relativa das famílias de enzimas relacionadas à degradação de carboidratos segue um padrão similar em todas as amostras metagenômicas. O módulo catalítico da família de Glycoside Hydrolases (GH), o qual foi anotado em 129 subfamílias diferentes, foi o mais abundante em todas as amostras (45,5%), seguido da família GT (Glicosyl Tranferase), anotada em 97 subfamílias diferentes e CBM (Carbohydrete-Bining Module), em 78 subfamílias. A montagem do metagenoma resultou em aproximadamente 110.000 contigs e possibilitou a identificação de 15 diferentes genes completos codificados nas subfamílias GH1, GH2, GH3, GH16, GH20, GH25, GH32, GH97 e GH127. A análise comparativa dos diferentes tratamentos mostrou uma maior abundância dessas enzimas no rúmen dos animais alimentados com a dieta enriquecida com bagaço de cana-de-açúcar. Em conclusão, a manipulação da dieta de ovinos por meio da substituição de parte da fração fibrosa da dieta por bagaço de cana-de-açúcar promove o enriquecimento de enzimas que degradam a biomassa vegetal no rúmem, favorecendo a prospecção e identificação de genes ativos em carboidratos.
The lignocellulose present in the plant biomass is a promising source of energy generation. However, the breakdown of plant biomass into simple sugars for bioethanol production is still inefficient and costly due to the recalcitrant nature of the plant fiber. The sheep rumen microbiome is specialized in degradation of plant material, but most members of this complex community are uncultured in the laboratory. Therefore, the search for new lignocellulolytic enzymes in microbial communities naturally evolved in biomass degradation environments, such as the rumen, using the exploration of the metagenome, is a promising strategy for identifying new genes. In this context, this study aimed to prospect plant biomass-degrading genes, selected from the sheep rumen microorganisms. The rumen samples were collected from 6 fistulated animals (Ovis aries), divided into two groups and subjected to two diets: control treatment and a treatment with a diet amended with sugarcane bagasse. The animals were fed for 60 days before sampling. To characterize the composition and functions of the rumen microbiome followed by the search of biomass-degrading genes, the metagenomic DNA was extracted from the solid contents of rumen and sequenced in MiSeq Personal Sequencer platform (Illumina®). The taxonomic and functional data were performed using MG-RAST software. For the characterization of the plant biomass degrading genes, they were analyzed on the CLC platform Genomic Workbench v.5.5.1 (CLC Bio, Denmark) and 4.68 gigabases of data was annotated against the CAZy database. The taxonomic analysis showed a predominance of the Bacteria domain composing more than 96% of all the samples, being the most abundant phyla Bacterioidetes, Firmiutes, followed by Proteobacteria. Five bacterial phyla were significantly more abundant in the treatment were sugarcane bagasse was added, Firmicutes, Proteobacteria, Actinobacteria, Spirochaetes and Verrucomicrobia, and two phyla were more abundant in the control treatment, Bacteroidetes and Synergistetes. In general, the ordination analysis did not show correlation between diet type and rumen microbiota, but in the functional analysis, this correlation was observed since there was separation between the treatments. The relative abundance of enzyme families related to carbohydrate degradation follows a similar pattern of abundance across all metagenomic samples. The catalytic module of the GH (Glycoside Hydrolases) family, which was annotated in 129 different subfamilies, was the most abundant in all samples (45.5%), followed by the GT family (Glycosyltransferase), annotated in 97 different subfamilies and CBM (Carbohydre-Bining Module) in 78 sub-families. Metagenome assembly resulted in ~110,000 contigs enabled the retrieval of 15 complete different genes encoded in the subfamilies GH1, GH2, GH3, GH16, GH20, GH25, GH32, GH97 and GH127. A comparative analysis between the groups of animals in the different treatments showed a greater abundance of enzymes, with no metagenome of the fiber proven from the group of animals fed a diet enriched with sugarcane bagasse. These results show the sheep rumen microbiome as an untapped source of potential new fibrolytic enzymes. Using a diet amended with sugarcane bagasse increases the abundance of CAE and provide a substantially expanded catalog of genes participating in the deconstruction of plant biomass.

With its unique consortium of microorganisms from all domains of life, termite gut is considered one of the most efficient lignocellulose degrading systems in nature. Recently, host diet and taxonomy as well as gut microenvironmental conditions have emerged as main factors shaping microbial communities in termite guts. The aim of this thesis was to investigate this highly efficient lignocellulolytic system at holobiont level, with a particular focus on gut microbiome function and composition in relation to the host diet. As a starting point, we optimised a complete framework for an accurate termite gut prokaryote-oriented metatranscriptomics, which was at the basis of all subsequent sequencing assay designs and analyses performed in the course of the work. Afterwards, we characterised the compositions and functions of biomass-degrading bacterial communities in guts of plant fibre- and soil-feeding higher termites, proving the existence of functional equivalence across microbial populations from different termite hosts. We also showed that each termite is a reservoir of unique microorganisms and their accompanying genes. We further extended above approach to metagenomics and bacterial genomes reconstruction and we applied it to explore the process of biomass digestion in the different sections of the highly compartmented gut of soil feeding Labiotermes labralis. We showed that primarily cellulolytic activity of the termite host was restricted to foregut and midgut, while bacterial contribution was most pronounced in P1 and P3 hindgut compartments and included activities targeting broad range of lignocellulose components. Finally, we investigated the adaptation of a laboratory-maintained grass-feeding higher termite colony of Cortaritermes spp. to Miscanthus diet at host and symbiont levels. A natural system of a termite gut was shown to progressively change in composition to yield a consortium of microbes specialised in degradation of a specific biomass. Overall, the integrative omics approach proposed here provide a framework for a better understanding of a complex lignocellulose degradation by a higher termite gut system and pave a road towards its future bioprospecting.
Doctorat en Sciences
info:eu-repo/semantics/nonPublished

Lignocellulose is an ubiquitous source of fixed carbon that is presently underexploited for renewable energy technologies. Currently, producing enzyme cocktails that robustly degrade these feedstocks is a significant economic bottleneck. Anaerobic gut fungi native to the digestive tracts of ruminants and hindgut fermenters are widely understudied despite their inherent ability to degrade a significant portion (~50%) of the lignocellulose in herbivorous animals. Challenges in cultivation due to their strict oxygen sensitivity, and the lack of a central repository to maintain axenic stocks substantially impede the progress with anaerobic fungi. Yet, these microbes have evolved elegant strategies and may harbor novel biomass degrading enzymes that could be used to more efficiently hydrolyze lignocellulose. Developing these organisms through characterization and genome engineering will yield significant contributions to the bioenergy community by improving hydrolysis technologies.

In this work, we report the isolation of four novel species of anaerobic gut fungi. A more complete characterization of one of our four fungal isolates is investigated, whereby the effects of substrate composition and the corresponding fungal growth rates are compared. I also explore the growth of one of our fungal isolates on transgenic poplar to understand how fungal growth and enzyme secretion adapt to variable lignin composition. Notably, no significant reductions in growth were observed highlighting the ability of anaerobic fungi to degrade diverse feedstocks regardless of lignin composition. I have additionally included preliminary work intended to identify what epigenetic regulational strategies exist for anaerobic fungi, and how they relate to carbohydrate active enzyme expression. We hope to leverage this knowledge to engineer base enzyme cocktails that release significant portions of the fermentable sugars in untreated or mildly treated plant biomass as a means to make bioenergy technologies more efficient.

The field of proteomics is facing many challenges, one of which is the assignment of structure and function to all of the proteins encoded and expressed by prokaryotic and eukaryotic genomes. One approach to this involves the development of new methodologies for the activity-based protein profiling (ABPP) of enzymes. Glycoside hydrolases (GH) are one group of enzymes that still lack an efficient and specific mass spectrometry-based ABPP (MS-ABPP) method. A probe was synthesized which consists of a biotin tag, a disulfide-bonded spacer, and a 2-deoxy-2-fluoro-xylobioside inactivator that covalently labels retaining xylanases and mixed-function cellulases. The MS-ABPP methodology was demonstrated at three levels of increasing complexity. Firstly, the labelled active-site peptides of a Bacillus circulans endo-xylanase (Bex) and a Cellulomonas fimi mixed-function endo-xylanase/cellulase (Cex) were affinity-isolated from their proteolytic digests and sequenced by ESI-MS/MS, leading to the identification of the enzymes' catalytic nucleophiles, Glu₇₈ (Bex) and Glu₂₃₃ (Cex). Secondly, the method was applied to several model enzyme mixtures including either Bex or Cex or both as targets. Finally, the method was used to analyze the secreted proteome of the soil bacterium, Cellulomonas fimi. The labelled active-site peptide (VQITEL) of a new GH family 10 glycanase was affinity-isolated from the proteolytic digest of the proteome and sequenced by ESI-MS/MS and Edman degradation. The glycanase gene was cloned using inverse PCR techniques and the protein was found to comprise a catalytic domain that shares about 70% sequence identity with those of xylanases from Streptomyces sp. and a family 2b carbohydrate-binding module. The new glycanase hydrolyzes natural and artificial xylo-configured substrates more efficiently than their cello-configured counterparts. Carbohydrate O-esterases and N-deacylases (CE) are a class of carbohydrateactive enzymes whose catalytic mechanisms have not been vigorously investigated. A detailed mechanistic investigation was conducted on Colletotrichum lindemuthianum endo-type chitin deacetylase (CDA) from CE family 4. Steady-state kinetic and mass spectrometric analyses showed that CDA has four sugar-binding subsites (-2, -1, 0, and +1). Subsites -2 and +1 make major contributions to the overall substrate-binding free energy change (A₋₂ = -11 and A₊₁ = -13 kJ.mol⁻¹) and subsites -2 and 0 (reaction site) recognize the acetamido group (ΔΔG[sub (N-acetyl)] = 3 and 4 kJ.mol⁻¹, respectively). Hammett linear free energy correlations using α-haloacetamido substrate analogues demonstrated the presence of an oxyanion intermediate (positive slope) and significant negative charge development in the transition state (ρ = 2). Since this oxyanion intermediate cannot have an enolate structure (an elimination/addition reaction was rejected), the only other possibility is that of a tetrahedral structure in a substitution reaction. A classical bellshaped pH dependence of k[sub cat]/K[sub m] (pK[sub al] = 7.5, pK[sub a]₂ = 10) revealed an active site environment far different from those of carboxyl proteases (both pK[sub a] values in the acidic pH range). Kinetic and ICP-MS results indicated the lack of a structural/catalytic divalent metal cofactor. The lack of covalent inactivation of CDA by a carbamate ester and by α -haloacetamido substrate analogues is consistent with direct water attack in a single-step substitution but these results do not conclusively rule out the possibility of a double-step substitution. Structural information is required to elucidate the exact role of the conserved residues in the active site of CDA.
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