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We have recently presented a sequential treatment method, in which steam explosion (STEX) was followed by hydrotropic extraction (HEX), to selectively fractionate cellulose, hemicellulose, and lignin in hardwood into separate process streams. However, above a treatment severity threshold, the structural alterations in the cellulose-enriched fraction appeared to restrict the enzymatic hydrolyzability and delignification efficiency. To better understand the ultrastructural changes in the cellulose, hardwood chips were treated by single (STEX or HEX) and combined treatments (STEX and HEX), and the cellulose accessibility quantified with carbohydrate-binding modules (CBMs) that bind preferentially to crystalline (CBM2a) and paracrystalline cellulose (CBM17). Fluorescent-tagged versions of the CBMs were used to map the spatial distribution of cellulose substructures with confocal laser scanning microscopy. With increasing severities, STEX increased the apparent crystallinity (CBM2a/CBM17-ratio) and overall accessibility (CBM2aH6 + CBM17) of the cellulose, whereas HEX demonstrated the opposite trend. The respective effects could also be discerned in the combined treatments where increasing severities further resulted in higher hemicellulose dissolution and, although initially beneficial, in stagnating accessibility and hydrolyzability. This study suggests that balancing the severities in the two treatments is required to maximize the fractionation and simultaneously achieve a reactive and accessible cellulose that is readily hydrolyzable., Elucidation of cellulose structural properties that restrict enzymatic hydrolysis in sustainable fractionation processes for hardwoods.

Genome sequencing has revealed substantial variation in the predicted abilities of individual species within animal gut microbiota to metabolize the complex carbohydrates comprising dietary fiber. At the same time, a currently limited body of functional studies precludes a richer understanding of how dietary glycan structures affect the gut microbiota composition and community dynamics. Here, using biochemical and biophysical techniques, we identified and characterized differences among recombinant proteins from syntenic xyloglucan utilization loci (XyGUL) of three Bacteroides and one Dysgonomonas species from the human gut, which drive substrate specificity and access to distinct polysaccharide side chains. Enzymology of four syntenic glycoside hydrolase family 5 subfamily 4 (GH5_4) endo-xyloglucanases revealed surprising differences in xyloglucan (XyG) backbone cleavage specificity, including the ability of some homologs to hydrolyze congested branched positions. Further, differences in the complement of GH43 alpha-l-arabinofuranosidases and GH95 alpha-l-fucosidases among syntenic XyGUL confer distinct abilities to fully saccharify plant species-specific arabinogalactoxyloglucan and/or fucogalactoxyloglucan. Finally, characterization of highly sequence-divergent cell surface glycan-binding proteins (SGBPs) across syntenic XyGUL revealed a novel group of XyG oligosaccharide-specific SGBPs encoded within select Bacteroides. IMPORTANCE The catabolism of complex carbohydrates that otherwise escape the endogenous digestive enzymes of humans and other animals drives the composition and function of the gut microbiota. Thus, detailed molecular characterization of dietary glycan utilization systems is essential both to understand the ecology of these complex communities and to manipulate their compositions, e.g., to benefit human health. Our research reveals new insight into how ubiquitous members of the human gut microbiota have evolved a set of microheterogeneous gene clusters to efficiently respond to the structural variations of plant xyloglucans. The data here will enable refined functional prediction of xyloglucan utilization among diverse environmental taxa in animal guts and beyond.

The rigid structures at the surface of plant cells are some of the most structurally complex & mechanically robust sets of biomaterials in nature. Given the dominance of plants on land, these cell walls are highly abundant and are a major repository of fixed carbon and form the bulk of the Earth's biomass. Plant cell walls and their component polymers are used extensively for food, textiles, paper, lumber and industrial polymers as well as a sustainable form of bioenergy. Our research interests are focused on plant cell walls in relation to plant cell development & growth as well as their use in applied & industrial contexts. We are specifically interested in developing tools to understand primary cell wall structures & architectures in relation to cell adhesion, cell expansion & the differentiation of plant cells. We are also interested in secondary cell wall structures and properties in growing plants as well as in relation to their use as industrial fibres. Our lab maintain, and continue to develop, a panel of rat hybridoma & phage display monoclonal antibodies to plant cell wall glycans to help in our studies of cell walls and for the imaging of cell wall molecular architectures. We also use carbohydrate-binding modules (CBMs) from microbial glycoside hydrolases as molecular probes for cell wall polymers. Immunochemistry is a major tool in our strategies and procedures to explore cell wall properties and functions. Our antibodies and CBMs are also being assembled for use in high throughput, systematic procedures for cell wall analyses including epitope detection chromatography (EDC) which uses our panels of molecular probes and detection tools for chromatographic separations of cell wall matrix glycans. Departamento de Biología Vegetal. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech.

International audience; For more than a century, filamentous fungi have been used for the production of a wide variety of endogenous enzymes of industrial interest. More recently, with the use of genetic engineering tools developed for these organisms, this use has expanded for the production of nonnative heterologous proteins. In this review, an overview is given of examples describing the production of a special class of these proteins, namely chimeric proteins. The production of two types of chimeric proteins have been explored: (a) proteins grafted for a specific substrate-binding domain and (b) fusion proteins containing two separate enzymatic activities. Various application areas for the use of these chimeric proteins are described.

Polysaccharide utilization loci (PUL) within the genomes of resident human gut Bacteroidetes are central to the metabolism of the otherwise indigestible complex carbohydrates known as “dietary fiber.” However, functional characterization of PUL lags significantly behind sequencing efforts, which limits physiological understanding of the human-bacterial symbiosis. In particular, the molecular basis of complex polysaccharide recognition, an essential prerequisite to hydrolysis by cell surface glycosidases and subsequent metabolism, is generally poorly understood. Here, we present the biochemical, structural, and reverse genetic characterization of two unique cell surface glycan-binding proteins (SGBPs) encoded by a xyloglucan utilization locus (XyGUL) from Bacteroides ovatus, which are integral to growth on this key dietary vegetable polysaccharide. Biochemical analysis reveals that these outer membrane-anchored proteins are in fact exquisitely specific for the highly branched xyloglucan (XyG) polysaccharide. The crystal structure of SGBP-A, a SusD homolog, with a bound XyG tetradecasaccharide reveals an extended carbohydrate-binding platform that primarily relies on recognition of the β-glucan backbone. The unique, tetra-modular structure of SGBP-B is comprised of tandem Ig-like folds, with XyG binding mediated at the distal C-terminal domain. Despite displaying similar affinities for XyG, reverse-genetic analysis reveals that SGBP-B is only required for the efficient capture of smaller oligosaccharides, whereas the presence of SGBP-A is more critical than its carbohydrate-binding ability for growth on XyG. Together, these data demonstrate that SGBP-A and SGBP-B play complementary, specialized roles in carbohydrate capture by B. ovatus and elaborate a model of how vegetable xyloglucans are accessed by the Bacteroidetes., IMPORTANCE The Bacteroidetes are dominant bacteria in the human gut that are responsible for the digestion of the complex polysaccharides that constitute “dietary fiber.” Although this symbiotic relationship has been appreciated for decades, little is currently known about how Bacteroidetes seek out and bind plant cell wall polysaccharides as a necessary first step in their metabolism. Here, we provide the first biochemical, crystallographic, and genetic insight into how two surface glycan-binding proteins from the complex Bacteroides ovatus xyloglucan utilization locus (XyGUL) enable recognition and uptake of this ubiquitous vegetable polysaccharide. Our combined analysis illuminates new fundamental aspects of complex polysaccharide recognition, cleavage, and import at the Bacteroidetes cell surface that may facilitate the development of prebiotics to target this phylum of gut bacteria.

The breakdown of plant cell wall (PCW) glycans is an important biological and industrial process. Noncatalytic carbohydrate binding modules (CBMs) fulfill a critical targeting function in PCW depolymerization. Defining the portfolio of CBMs, the CBMome, of a PCW degrading system is central to understanding the mechanisms by which microbes depolymerize their target substrates. Ruminococcus flavefaciens, a major PCW degrading bacterium, assembles its catalytic apparatus into a large multienzyme complex, the cellulosome. Significantly, bioinformatic analyses of the R. flavefaciens cellulosome failed to identify a CBM predicted to bind to crystalline cellulose, a key feature of the CBMome of other PCW degrading systems. Here, high throughput screening of 177 protein modules of unknown function was used to determine the complete CBMome of R. flavefaciens. The data identified six previously unidentified CBMfamilies that targeted beta-glucans, beta-mannans, and the pectic polysaccharide homogalacturonan. The crystal structures of four CBMs, in conjunction with site-directed mutagenesis, provide insight into the mechanism of ligand recognition. In the CBMs that recognize beta-glucans and beta-mannans, differences in the conformation of conserved aromatic residues had a significant impact on the topology of the ligand binding cleft and thus ligand specificity. A cluster of basic residues in CBM77 confers calcium-independent recognition of homogalacturonan, indicating that the carboxylates of galacturonic acid are key specificity determinants. This report shows that the extended repertoire of proteins in the cellulosome of R. flavefaciens contributes to an extended CBMome that supports efficient PCW degradation in the absence of CBMs that specifically target crystalline cellulose.

Background Enzymes still comprise a major part of ethanol production costs from lignocellulose raw materials. Irreversible binding of enzymes to the residual substrate prevents their reuse and no efficient methods for recycling of enzymes have so far been presented. Cellulases without a carbohydrate-binding module (CBM) have been found to act efficiently at high substrate consistencies and to remain non-bound after the hydrolysis. Results High hydrolysis yields could be obtained with thermostable enzymes of Thermoascus aurantiacus containing only two main cellulases: cellobiohydrolase I (CBH I), Cel7A and endoglucanase II (EG II), Cel5A. The yields were decreased by only about 10% when using these cellulases without CBM. A major part of enzymes lacking CBM was non-bound during the most active stage of hydrolysis and in spite of this, produced high sugar yields. Complementation of the two cellulases lacking CBM with CBH II (CtCel6A) improved the hydrolysis. Cellulases without CBM were more sensitive during exposure to high ethanol concentration than the enzymes containing CBM. Enzymes lacking CBM could be efficiently reused leading to a sugar yield of 90% of that with fresh enzymes. The applicability of cellulases without CBM was confirmed under industrial ethanol production conditions at high (25% dry matter (DM)) consistency. Conclusions The results clearly show that cellulases without CBM can be successfully used in the hydrolysis of lignocellulose at high consistency, and that this approach could provide new means for better recyclability of enzymes. This paper provides new insight into the efficient action of CBM-lacking cellulases. The relationship of binding and action of cellulases without CBM at high DM consistency should, however, be studied in more detail.

In this review, the present knowledge on the occurrence of cellulases, with a special emphasis on the presence of carbohydrate-binding modules (CBMs) in various fungal strains, has been summarized. The importance of efficient fungal cellulases is growing due to their potential uses in biorefinery processes where lignocellulosic biomasses are converted to platform sugars and further to biofuels and chemicals. Most secreted cellulases studied in detail have a bimodular structure containing an active core domain attached to a CBM. CBMs are traditionally been considered as essential parts in cellulases, especially in cellobiohydrolases. However, presently available genome data indicate that many cellulases lack the binding domains in cellulose-degrading organisms. Recent data also demonstrate that CBMs are not necessary for the action of cellulases and they solely increase the concentration of enzymes on the substrate surfaces. On the other hand, in practical industrial processes where high substrate concentrations with low amounts of water are employed, the enzymes have been shown to act equally efficiently with and without CBM. Furthermore, available kinetic data show that enzymes without CBMs can desorb more readily from the often lignaceous substrates, that is, they are not stuck on the substrates and are thus available for new actions. In this review, the available data on the natural habitats of different wood-degrading organisms (with emphasis on the amount of water present during wood degradation) and occurrence of cellulose-binding domains in their genome have been assessed in order to identify evolutionary advantages for the development of CBM-less cellulases in nature.

International audience; Microarrays are powerful tools for high throughput analysis, and hundreds or thousands of molecular interactions can be assessed simultaneously using very small amounts of analytes. Nucleotide microarrays are well established in plant research, but carbohydrate microarrays are much less established, and one reason for this is a lack of suitable glycans with which to populate arrays. Polysaccharide microarrays are relatively easy to produce because of the ease of immobilizing large polymers noncovalently onto a variety of microarray surfaces, but they lack analytical resolution because polysaccharides often contain multiple distinct carbohydrate substructures. Microarrays of defined oligosaccharides potentially overcome this problem but are harder to produce because oligosaccharides usually require coupling prior to immobilization. We have assembled a library of well characterized plant oligosaccharides produced either by partial hydrolysis from polysaccharides or by de novo chemical synthesis. Once coupled to protein, these neoglycoconjugates are versatile reagents that can be printed as microarrays onto a variety of slide types and membranes. We show that these microarrays are suitable for the high throughput characterization of the recognition capabilities of monoclonal antibodies, carbohydrate-binding modules, and other oligosaccharide-binding proteins of biological significance and also that they have potential for the characterization of carbohydrate-active enzymes.

Tese de Doutoramento em Ciências Veterinárias. Especialidade de Ciências Biológicas e Biomédicas. The importance of carbohydrate-binding modules (CBMs) and the use of novel enzymes with specific catalytic activities to improve the nutritive value of barley based diets for broilers and the effectiveness of a lipidic supplementation to improve the levels of benefic fatty acids in broilers meat remain to be investigated. In this work we studied the importance of a β-glucan binding domain (CBM11) when appended to three different enzymes (GH26GH5 and GH16, belonging to Clostridium thermocellum, and GH5, belonging to Celvibrio mixtus) to improve the nutritional quality of barley-based diets for broilers. In addition, the crystal structure and biochemical properties of a family 42 carbohydrate binding module (CBM) from Clostridium thermocellum, termed CtCBM42A, were investigated. Data presented here revealed that CBM11 has an important target effect in directing the appended catalytic modules to their target substrates, resulting in an improvement in broiler performance. However, this effect seems to be dependent on the level of supplementation. In addition, barley composition, namely its endogenous β-glucanase activity, influences the response to enzyme supplementation. Thus, exogenous enzymes were shown to be ineffective when used to supplement barleys expressing high endogenous β- glucanase activity. CtCBM42A revealed to be a type C CBM with three subdomains (α, β and γ), with affinity for arabinoxylan (arabinose side chains) and arabinan. The γ subdomain seems to dominate ligand recognition for arabinoxylan while the β and γ subdomains cooperate in arabinan recognition. Thus, CtCBM42A is potentially a good candidate for strategies aimed at improving the nutritive value of wheat-based diets for broilers. In order to improve the fatty acid profile of poultry meat, two different lipidic sources, extruded linseed and a subproduct of a marine alga (DHA goldTM), were used to supplement broiler diets. This experiment allowed the evaluation of the metabolic rates of the biosynthetic pathway of long-chain ómega 3 polyunsaturated fatty acids (LC n-3 PUFA).The supplementation of broiler diets with DHA goldTM and extruded linseed showed that conversion of linolenic acid in LC n-3 PUFA is not effective and, consequently, direct supplementation with LC n-3 PUFA seems to be the best option to enrich and improve LC n-3 PUFA in broilers meat. However, higher incorporation dosages of DHA goldTM could affect meat quality. RESUMO - Efeito da suplementação enzimática e lipídica de dietas para frangos no desempenho produtivo e na qualidade da carne - Uma melhor adequação da qualidade dos produtos animais, em concreto da carne de frango, às necessidades nutricionais dos consumidores, associada a uma maior eficiência de transformação dos alimentos para animais em produtos edíveis, são aspectos da maior importância prática na avicultura moderna e suscitam uma análise científica detalhada. Neste trabalho estudou-se a aplicação de um módulo de ligação ao β-glucano (CBM11), acoplado a três enzimas diferentes (GH26GH5 e GH16, ambas pertencentes ao Clostridium thermocellum, e a GH5, pertencente ao Celvibrio mixtus) na melhoria do valor nutritivo de dietas à base de cevada para frangos de carne. Foram também determinadas as propriedades bioquímicas e a estrutura cristalográfica do CBM da família 42 do Clostridium thermocellum, CtCBM42A. Os resultados demonstraram que o CBM11 tem um efeito importante no direccionamento do módulo catalítico das enzimas ao substrato, que resulta num aumento da performance zootécnica dos frangos de carne. No entanto, esse efeito parece estar dependente da dose enzimática aplicada. Demonstrou-se também que a composição das cevadas, principalmente a actividade endo-β-glucanásica, influencia o efeito da suplementação enzimática. Em cevadas com actividade endo-β-glucanásica alta a suplementação enzimática tem um efeito redundante não se obtendo melhoria da performance dos frangos de carne. O estudo do CBM42 revelou que se trata dum CBM do tipo C, com três subdomínios (α, β e γ), com afinidade para o arabinoxilano (nas suas cadeias laterais de arabinose) e arabinano. O subdomínio γ parece ser o responsável pela afinidade ao arabinoxilano enquanto o subdomínio β juntamente com o γ parecem interagir pela afinidade ao arabinano, revelando-se como um módulo potencialmente interessante para uma futura utilização na suplementação enzimática de dietas à base de trigo para frangos. Foram efectuados ensaios com frangos de carne cujas dietas foram suplementadas com semente de linho extrudida e um subproduto de algas marinhas (DHA goldTM) para estudar os seus efeitos no perfil dos ácidos gordos da carne e na qualidade da carne. Também se avaliou a extensão da bioconversão dos percursores ácidos linoleico (LA) e linolénico (LNA) nos seus homólogos de cadeia longa. Os resultados mostraram que a conversão dos ácidos gordos não é eficiente e por isso a suplementação directa com uma fonte de ácidos gordos de cadeia longa parece ser a melhor opção para melhorar o conteúdo de ácidos gordos ómega-3 de cadeia longa. No entanto, a qualidade da carne pode estar afectada negativamente em doses de incorporação elevadas de DHA goldTM. This work was funded by Fundação para a Ciência e a Tecnologia, grant SFRH/BD/32321/2006, and co-funded by POCI 2010 and FSE from Ministério da Ciência, Tecnologia e Ensino Superior

Tese de Doutoramento em Ciência e Tecnologia Animal Plant cell wall polysaccharides offer an extraordinary source of carbon and energy that can be used by various microorganisms, thus constituting a central component of the carbon cycle. The anaerobic thermophilic bacterium Clostridium thermocellum is one of the most prolific degraders of plant cell wall polysaccharides. It produces a multi-enzyme extra-cellular complex of cellulases and hemicellulases, the cellulosome, and these enzymes were shown to have a remarkable biotechnological potential. Based on the recently determined genome sequence of Clostridium thermocellum, we aimed to address several unresolved questions concerning the mechanism of plant cell wall hydrolysis by microbial multi-enzyme complexes.The crystal structure and biochemical properties of the N-terminal carbohydrate esterase domain of Clostridium thermocellum CtCes3-1 were determined (chapter 2). The enzyme is a thermostable acetyl-specific esterase that exhibits a strong preference for acetylated xylan. In adition, we report, in Chapter 3, the characterization of four carbohydrate-binding modules (CBMs) of family 35 that display specificity for Δ4,5-anhydrogalacturonic acid (Δ4,5-GalA), although two of the proteins also interact with glucuronic acid (GlcA). X-ray crystallographic data revealed that the ligand binding site is highly conserved in the four CBM35s. In chapter 4, biochemical properties of a CBM6 (CBM6-1) from Cellvibrio mixtus CmCel5A are presented. The data revealed that CBM6-1 recognizes specifically β1,3-glucans through a previous unknown ligand binding plataform. These studies reveal the different mechanisms by which a highly conserved protein platform (CBM6) can recognise a common ligand. Finally, we identified a novel CBM within the large C. thermocellum cellulosomal protein Cthe_2193 (CtCBM55), which is the founder member of a new CBM family. CtCBM55, in contrast to the previously characterized cellulosomal CBMs, binds to D-galactose and L-arabinose in either anomeric configuration in complex polysaccharides. Ligand specificity is conferred through numerous interactions with the axial O4 of the target sugars, a feature that distinguishes galactose and arabinose from the other major sugars located into plant cell walls. RESUMO - Perspectivas Estruturais e funcionais do papel das carbohidrato esterases e dos modulos de ligação a hidratos de carbono na hidrólise da parede cellular vegetal. - Os polissacáridos da parede celular vegetal constituem uma fonte de carbono e energia que pode ser utilizada por diversos microorganismos desempenhando assim um papel de relevo no ciclo do carbono. A bactéria termofílica anaeróbia Clostridium thermocellum é muito eficaz na degradação dos polissacáridos da parede celular das plantas. Esta produz um complexo multi-enzimático extra-celular de celulases e hemicelulases, denominado celulossoma, demonstrando estas enzimas um extraordinário potencial biotecnológico. Neste trabalho foram determinadas as propriedades bioquímicas e a estrutura cristalográfica do domínio catalítico N-terminal da enzima CtCes3 (CtCes3-1) do Clostridium thermocellum (Capítulo 2). Esta enzima é uma esterase termostável específica para grupos acetilo e que demonstra uma forte preferência por xilano acetilado. É também feita, no Capitulo 3, a caracterização de quatro módulos de ligação a hidratos de carbono (CBMs) da familia 35 que demonstram afinidade para o ácido Δ4,5-anidrogalacturónico (Δ4,5-GalA), apesar de duas das proteínas também interagirem com o acido glucurónico (GlcA). Os dados cristalográficos demonstram que o local de ligação é altamente conservado nos quatro CBM35s. No Capítulo 4, são reveladas as propriedades bioquímicas de um CBM6 da enzima CmCel5A (CBM6-1) do Cellvibrio mixtus. Os dados revelaram que o CBM6-1 reconhece especificamente β1,3-glucanos através de uma plataforma de ligação previamente desconhecida. Estes estudos demonstram os diferentes mecanismos pelos quais uma plataforma proteíca altamente conservada (CBM6) pode reconhecer o mesmo ligando. Finalmente, identificámos um novo CBM da enzima Cthe_2193 (CtCBM55), pertencente ao celulossoma do C. thermocellum, estabelecendo-se assim uma nova família de CBMs. O CtCBM55, ao contrário de outros CBMs do celulossoma, liga-se à D-galactose e à L-arabinose em qualquer configuração anomérica em polissacáridos complexos. A sua especificidade é conferida por varias interacções com o O4 axial dos açucares alvo, uma característica que distingue a galactose e a arabinose dos outros hidratos de carbono que compoêm a parede celular vegetal. This work was funded by Fundação para a Ciência e Tecnologia through individual fellowship SFRH/BD/23784/2005. Co-funded by POCTI/CVT/2004/61162, POCTI/BIA-PRO/2004/59118 and FSE from Ministério da Ciência, Tecnologia e Ensino Superior.

Tese de Doutoramento em Ciências Veterinárias Xyloglucan (XG) is the major plant cell wall hemicellulose. Biochemical and structural studies on a xyloglucanase, Xgh74A, from Clostridium thermocellum are presented here. In addition, the carbohydrate-binding modules (CBMs) from C. thermocellum Cel9D-Cel44A, CtCBM30 and CtCBM44, which recognize XG, are characterized in this work. CtCel9D-Cel44A’s C-terminal module of unknown function is a CBM, constituting the founder member of family 44. Structural studies revealed that both CtCBM30 and CtCBM44 present Type B binding site topologies where tryptophans play an important role in ligand recognition. Cel44A is an endoglucanase domain displaying some xylanase activity, which action is potentiated by CtCBM44 through a targeting effect. Biochemical and structural studies on CtLic26A-Cel5E are also described here. CtLic26A is a mixed b-1,4-b-1,3-glucanase and Cel5E an endo-b-1,4- glucanase. The three-dimensional structure of CtLic26A provides insights in the mechanism of lichenan recognition by family 26 glycoside hydrolases. Finally, novel biotechnological applications for CBMs were investigated. An experiment was conducted where a barley-based diet for broilers was supplemented with CtLic26ACel5 derivatives, with or without CtCBM11, and with a commercial enzyme. The fusion of four antimicrobial peptides with CipA from CtCBM3 originated recombinants with high affinity for crystalline cellulose, indicating CBMs could fix bioactive molecules to cellulose. RESUMO: O xiloglucano é a principal hemicelulose das paredes celulares vegetais. Neste trabalho apresentam-se estudos bioquímicos e estruturais sobre a xiloglucanase Xgh74A do Clostridium thermocellum. São também estudados módulos de ligação a carbohidratos (CBMs) com afinidade para o xiloglucano, o CtCBM30 e o CtCBM44 da CtCel9DCel44A. O módulo C-terminal revelou-se um CBM, sendo o membro fundador da família 44. As estruturas cristalográficas desses CBMs mostram locais de ligação aos polissacáridos com topologia do Tipo B, nos quais os triptofanos representam um papel importante no reconhecimento dos ligandos. A Cel44A é uma endoglucanase com actividade xilanásica, cuja acção é potenciada pelo CtCBM44. Estudos bioquímicos e estruturais sobre a CtLic26A-Cel5E são também apresentados, sendo a CtLic26A uma b-1,4-b-1,3-glucanase e a Cel5E uma endo-b-1,4-glucanase. A estrutura tridimensional da CtLic26A revelou os mecanismos estruturais que modulam a especificidade da enzima. Neste trabalho são pesquisadas novas aplicações biotecnológicas para os CBMs. Efectuou-se um ensaio com frangos de carne, suplementando dietas à base de cevada com derivados recombinantes da CtLic26A-Cel5, com e sem CtCBM11, e com uma enzima comercial. A fusão de quatro péptidos antimicrobianos com a CipA do CtCBM3 originou recombinantes com elevada afinidade para celulose cristalina, indicando que os CBMs poderão fixar moléculas bioactivas a materiais celulósicos. This work was funded by Fundação para a Ciência e a Tecnologia, grant SFRH/BD/16731/2004, and co-funded by POCI 2010 and FSE from Ministério da Ciência, Tecnologia e Ensino Superior

Tese de Doutoramento em Ciências Veterinárias Submitted by Maria São José Lalanda Ribeiro (mlalanda@fmv.utl.pt) on 2008-12-02T18:12:33Z No. of bitstreams: 2 Resumos_Tese Doutoramento_Catarina Guerreiro.pdf: 22507 bytes, checksum: 7be4a43c0ad9e475f867683c5b2e718f (MD5) Tese Doutoramento_Catarina Guerreiro_Setembro 2008.pdf: 11286475 bytes, checksum: b188ff846c082bc26e63d0d39b7dd4e8 (MD5) Made available in DSpace on 2008-12-02T18:12:33Z (GMT). No. of bitstreams: 2 Resumos_Tese Doutoramento_Catarina Guerreiro.pdf: 22507 bytes, checksum: 7be4a43c0ad9e475f867683c5b2e718f (MD5) Tese Doutoramento_Catarina Guerreiro_Setembro 2008.pdf: 11286475 bytes, checksum: b188ff846c082bc26e63d0d39b7dd4e8 (MD5) Previous issue date: 2008-09-09 This work was funded by Fundação para a Ciência e a Tecnologia, grant SFRH/BD/16731/2004, and co-funded by POCI 2010 and FSE from Ministério da Ciência, Tecnologia e Ensino Superior

Tese de Doutoramento em Ciências Veterinárias. Especialidade em Ciência e Tecnologia Animal As glicósido hidrolases modulares, que atacam polissacáridos estruturais, possuem módulos não catalíticos de ligação a hidratos de carbono (CBMs) que desempenham uma função crucial na acção destas enzimas por possibilitarem um contacto prolongado da enzima com o substrato. Neste trabalho descrevem-se as características bioquímicas e estruturais de dois CBMs: o módulo C-terminal da Cel5B do Cellvibrio mixtus, o CmCBM6-2, e um CBM da família 11 do Clostridium thermocellum, o CtCBM11. Os resultados indicam que o CmCBM6-2 pode ligar-se a glucanos de ligações β-1,4 ou β-1,3 na fenda A e na fenda B, e que ambas as fendas são necessárias para a ligação à celulose insolúvel. Contudo, apenas a fenda B interage com as regiões internas dos glucanos de ligações β-1,3-1,4, suportando a hipótese de que a variação da especificidade em membros da família CBM6 reflecte a funcionalidade diferenciada das duas fendas de ligação. O CtCBM11 possui uma preferência específica para glucanos de ligações mistas β-1,3-1,4 e revelou possuir uma única fenda de ligação que pode acomodar glucanos contendo ligações tanto mistas β-1,3-β-1,4 como ligações exclusivas β-1,4. No C. thermocellum, tal como noutros microrganismos anaeróbios, o celulossoma consiste num complexo multi-enzimático de celulases e hemicelulases que é organizado pela interacção do módulo coesina com os módulos doquerina presentes nas subunidades catalíticas. Neste trabalho apresenta-se a estrutura tridimensional da coesina do tipo II da SdbA do C. thermocellum, que possui uma elevada homologia com as coesinas do tipo I. Propõe-se que o local de ligação à doquerina está conservado em ambas as proteínas. Finalmente, estudou-se o efeito da suplementação enzimática de uma dieta à base de Lupinus albus para leitões. A suplementação enzimática não apresentou efeitos significativos na digestibilidade ileal aparente da proteína bruta e na digestibilidade ileal verdadeira dos aminoácidos, mas a digestibilidade ileal da fracção fibra neutro detergente aumentou, o que sugere uma actividade residual das hemicelulases presentes nos preparados de alfagalactosidases utilizados. ABSTRACT: Modular glycoside hydrolases that attack recalcitrant polymers generally contain noncatalytic carbohydrate-binding modules (CBMs), which play a critical role in the action of these enzymes by directing the appended catalytic domains onto the surface of their substrates. Here we report the biochemical and structural properties of two CBMs. The CmCBM6-2, located at the C-terminus of Cellvibrio mixtus endoglucanase 5B was shown to contain two binding sites that display differences in ligand specificity, supporting the view that distinct binding clefts with different specificities can contribute to the variation in ligand recognition displayed by family 6 CBMs. In addition, the family 11 CBM (CtCBM11) from Clostridium thermocellum bifunctional lichenase-celulase enzyme Lic26A-Cel5E, demonstrated a preference for mixed linked glucans and contains a single ligand-binding site that displays affinity for both β-1,4- and β-1,3-1,4- mixed linked glucans. This enzyme is part of the cellulosome which represents an efficient multi-enzyme complex of cellulases and hemicellulases for the hydrolysis of cellulose. Cellulosome assembly results from the interaction of cohesin with dockerin modules present in the catalytic units. Here we report the crystal structure of the Type II cohesin (CohII) from C. thermocellum cell surface anchoring protein SdbA. The structure of CohII is very similar to the Type I cohesin and the dockerin binding site is likely to be conserved in the two proteins. Finally, in this study the effect of supplementing a lupin based diet for piglets with α-galactosidases was evaluated. Enzyme supplementation had no significant effect on the apparent ileal digestibility of crude protein and true ileal digestibility of amino acids, but the ileal digestibility of NDF (neutral detergent fibre) was increased, which suggests a residual hemicellulase activity in the α-galactosidases used. A candidata foi financiada pela Fundação para a Ciência e Tecnologia, Ministério da Ciência, Tecnologia e Ensino Superior, através do programa POCI, no âmbito do III Quadro Comunitário de Apoio, comparticipado pelo Fundo Social Europeu e por fundos nacionais do MCTES, referência: SFRH/BD/12562/2003 SFRH/BD/12562/2003

Cellulases and xylanases display a modular architecture that comprises a catalytic module linked to one or more non-catalytic carbohydrate-binding modules (CBMs). CBMs have been classified into 52 different families, based on primary structure similarity. These non-catalytic modules mediate a prolonged and intimate contact of the enzyme with the target substrate eliciting efficient hydrolysis of the target polysaccharides. 2. A study was undertaken to investigate the importance of a family 11 CBM, displaying high affinities for barley -glucans, in the function of recombinant derivatives of cellulase CtLic26A-Cel5E of Clostridium thermocellum used to supplement a barley-based diet for broiler chicken. 3. The results showed that birds fed on diets containing the recombinant CtLic26A-Cel5E modular derivatives or the commercial enzyme mixture RovabioTM Excel AP displayed improved performance when compared with birds fed on diets not supplemented with exogenous enzymes. 4. It is suggested that the enzyme dosage used in this study (30 U/kg of basal diet), was probably too high for the efficacy of the family 11 CBM to be noticed. It remains to be established if the targeting effect resulting from the incorporation of CBMs in plant cell wall hydrolases may be effective at lower exogenous enzyme dosages.

Tese de Doutoramento em Ciências Veterinárias. Especialidade em Ciência e Tecnologia Animal Submitted by Maria São José Lalanda Ribeiro (mlalanda@fmv.utl.pt) on 2008-11-28T17:24:36Z No. of bitstreams: 4 (Microsoft Word - Dissertação de doutoramento.pdf: 5163765 bytes, checksum: 1a07dc593277671eff2ef0ddb822eee9 (MD5) (Microsoft Word - resumo ingl.pdf: 76538 bytes, checksum: 22ce42652c5a3c6c7ff1ad8de3d1f4d1 (MD5) (Microsoft Word - resumo portugu.pdf: 76032 bytes, checksum: a01d7ae2c291fbcdbb235ee059c0c893 (MD5) (Microsoft Word - titulo da tese com respectiva.pdf: 36214 bytes, checksum: f1c008ea321d345603f80c0415f8e048 (MD5) Made available in DSpace on 2008-11-28T17:24:36Z (GMT). No. of bitstreams: 4 (Microsoft Word - Dissertação de doutoramento.pdf: 5163765 bytes, checksum: 1a07dc593277671eff2ef0ddb822eee9 (MD5) (Microsoft Word - resumo ingl.pdf: 76538 bytes, checksum: 22ce42652c5a3c6c7ff1ad8de3d1f4d1 (MD5) (Microsoft Word - resumo portugu.pdf: 76032 bytes, checksum: a01d7ae2c291fbcdbb235ee059c0c893 (MD5) (Microsoft Word - titulo da tese com respectiva.pdf: 36214 bytes, checksum: f1c008ea321d345603f80c0415f8e048 (MD5) Previous issue date: 2008-05-13 A candidata foi financiada pela Fundação para a Ciência e Tecnologia, Ministério da Ciência, Tecnologia e Ensino Superior, através do programa POCI, no âmbito do III Quadro Comunitário de Apoio, comparticipado pelo Fundo Social Europeu e por fundos nacionais do MCTES, referência: SFRH/BD/12562/2003 SFRH/BD/12562/2003

We have compared heterologous expression of two types of carbohydrate binding module (CBM) in tobacco cell walls. These are the promiscuous CBM29 modules (a tandem CBM29-1-2 and its single derivative CBM29-2), derived from a non-catalytic protein1, NCP1, of the Piromyces equi cellulase/hemicellulase complex, and the less promiscuous tandem CBM2b-1-2 from the Cellulomonas fimi xylanase 11A. CBM-labelling studies revealed that CBM29-1-2 binds indiscriminately to every tissue of the wild-type tobacco stem whereas binding of CBM2b-1-2 was restricted to vascular tissue. The promiscuous CBM29-1-2 had much more pronounced effects on transgenic tobacco plants than the less promiscuous CBM2b-1-2. Reduced stem elongation and prolonged juvenility, resulting in delayed flower development, were observed in transformants expressing CBM29-1-2 whereas such growth phenotypes were not observed for CBM2b-1-2 plants. Histological examination and electron microscopy revealed layers of collapsed cortical cells in the stems of CBM29-1-2 plants whereas cellular deformation in the stem cortical cells of CBM2b-1-2 transformants was less severe. Altered cell expansion was also observed in most parts of the CBM29-1-2 stem whereas for the CBM2b-1-2 stem this was observed in the xylem cells only. The cellulose content of the transgenic plants was not altered. These results support the hypothesis that CBMs can modify cell wall structure leading to modulation of wall loosening and plant growth.

Resistance against the leaf mold fungus Cladosporium fulvum is mediated by the tomato Cf proteins which belong to the class of receptor-like proteins and indirectly recognize extracellular avirulence proteins (Avrs) of the fungus. Apart from triggering disease resistance, Avrs are believed to play a role in pathogenicity or virulence of C. fulvum. Here, we report on the avirulence protein Avr4, which is a chitin-binding lectin containing an invertebrate chitin-binding domain (CBM14). This domain is found in many eukaryotes, but has not yet been described in fungal or plant genomes. We found that interaction of Avr4 with chitin is specific, because it does not interact with other cell wall polysaccharides. Avr4 binds to chitin oligomers with a minimal length of three N-acetyl glucosamine residues. In vitro, Avr4 protects chitin against hydrolysis by plant chitinases. Avr4 also binds to chitin in cell walls of the fungi Trichoderma viride and Fusarium solani f. sp. phaseoli and protects these fungi against normally deleterious concentrations of plant chitinases. In situ fluorescence studies showed that Avr4 also binds to cell walls of C. fulvum during infection of tomato, where it most likely protects the fungus against tomato chitinases, suggesting that Avr4 is a counter-defensive virulence factor.

The enzymatic degradation of plant polysaccharides is a process of fundamental importance in nature which involves a wide range of enzymes. In this work, the structure and function of hemicellulose-degrading enzymes was investigated. The focus was on a beta-mannanase (TrMan5A) produced by the filamentous fungus Trichoderma reesei. This enzyme is composed of a catalytic module and a carbohydrate-binding module (CBM). In this thesis, the enzyme-polysaccharide interaction in both these modules was investigated. The results demonstrate that the CBM of TrMan5A is important for hydrolysis of complex mannan substrates containing cellulose. Furthermore, the increase in activity could be linked to binding of the CBM to the complex substrate. Binding studies revealed that the CBM binds to cellulose, but not to mannan. Studies of the enzyme/polysaccharide interaction in the active site cleft of the catalytic module of TrMan5A showed that a mutant of Arg 171 displayed activity in the same range as the wild-type enzyme toward polymeric substrates. However, the Arg171 mutant was impaired in hydrolysis of small substrates. Interestingly, this mutant also appears to have a more alkaline activity pH-optimum than the wild-type. The low or abolished activity observed with mutants of the predicted catalytic glutamates (Glu169 and Glu276) support their importance in hydrolysis. In addition to TrMan5A, the properties of a beta-mannanase (MeMan5A) from blue mussel and a beta-mannosidase (AnMan2A) from Aspergillus niger, were studied in this work. Investigations on the catalytic properties of the enzymes showed that all three enzymes are capable of degrading polymeric mannan. Furthermore, analysis by transmission electron microscopy revealed that TrMan5A and AnMan2A degrades highly crystalline mannan. Degradation of glucomannan and galactoglucomannan by several polysaccharide-degrading enzymes shows that these substrates can be hydrolysed by both mannoside- and glucoside-hydrolases. Furthermore, the results presented show that cellulases potentially are able to hydrolyse other components in the plant cell wall. Altogether, the results presented demonstrates the need to use complex substrates in order to reveal the mechanisms of plant polysaccharide degradation. In conclusion, this work has shown that the enzyme/polysaccharide interaction in the two modules of TrMan5A is important in determining the overall enzymatic activity and specificity.

The family 1 cellulose-binding modules (CBM1) form a groupof small, stable carbohydrate-binding proteins. These modulesare essential for fungal cellulosedegradation. This thesisdescribes both functional studies of the CBM1s as well asprotein engineering of the modules for several objectives. The characteristics and specificity of CBM1s from theTrichoderma reeseiCel7A and Cel6A, along with severalother wild type and mutated CBMs, were studied using bindingexperiments and transmission electron microscopy (TEM). Datafrom the binding studies confirmed that the presence of onetryptophan residue on the CBM1 binding face enhances itsbinding to crystalline cellulose. The twoT. reeseiCBM1s as well as the CBM3 from theClostridium thermocellumCipA were investigated by TEMexperiments. All three CBMs were found to bind in lineararrangements along the sides of the fibrils. Further analysesof the bound CBMs indicated that the CBMs bind to the exposedhydrophobic surfaces, the so called (200) crystalline face ofValoniacellulose crystals. The function and specificity of CBM1s as a part of an intactenzyme were studied by replacing the CBM from the exo-actingCel7A by the CBM1 from the endoglucanase Cel7B. Apart fromslightly improved affinity of the hybrid enzyme, the moduleexchange did not significantly influence the function of theCel7A. This indicates that the two CBM1s are analogous in theirbinding properties and function during cellulosehydrolysis. The CBM1 was also used for immobilization studies. Toimprove heterologous expression of a CBM1-lipase fusionprotein, a linker stability study was carried out inPichia pastoris. A proline/threonine rich linker peptidewas found to be stable for protein production in this host. Forwhole bacterial cell immobilization, theT. reeseiCel6A CBM1 was expressed on the surface of thegram-positive bacteria,Staphylococcus carnosus. The engineeredS. carnosuscells were shown to bind cellulosefibers. To exploit the stable CBM1 fold as a starting point forgenerating novel binders, a phage display library wasconstructed. Binding proteins against an amylase as well asagainst a metal ion were selected from the library. Theamylase-binding proteins were found to bind and inhibit thetarget enzyme. The metal binding proteins selected from thelibrary were cloned on the surface of theS. carnosusand clearly enhanced the metal bindingability of the engineered bacteria. Keywords: cellulose-binding, family 1carbohydrate-binding module, phage display, bacterial surfacedisplay, combinatorial protein library, metal binding, proteinengineering,Trichoderma reesei, Staphyloccus carnosus. NR 20140805

In an effort to characterise the whole transcriptome of the fungus Hypocrea jecorina, cDNA clones of this fungus were identified that encode for previously unknown proteins that are likely to function in biomass degradation. One of these newly identified proteins, found to be co-regulated with the major H. jecorina cellulases, is a protein that was denoted Cellulose induced protein 1 (Cip1). This protein consists of a glycoside hydrolase family 1 carbohydrate binding module connected via a linker region to a domain with yet unknown function. After cloning and expression of Cip1 in H. jecorina, the protein was purified and biochemically characterised with the aim of determining a potential enzymatic activity for the novel protein. No hydrolytic activity against any of the tested plant cell wall components was found. The proteolytic core domain of Cip1 was then crystallised, and the three-dimensional structure of this was determined to 1.5 angstrom resolution utilising sulphur single-wavelength anomalous dispersion phasing (sulphor-SAD). A calcium ion binding site was identified in a sequence conserved region of Cip1 and is also seen in other proteins with the same general fold as Cip1, such as many carbohydrate binding modules. The presence of this ion was found to have a structural role. The Cip1 structure was analysed and a structural homology search was performed to identify structurally related proteins. The two published structures with highest overall structural similarity to Cip1 found were two poly-lyases: CsGL, a glucuronan lyase from H. jecorina and vAL-1, an alginate lyase from the Chlorella virus. This indicates that Cip1 may be a lyase. However, initial trials did not detect significant lyase activity for Cip1. Cip1 is the first structure to be solved of the 23 currently known Cip1 sequential homologs (with a sequence identity cut-off of 25%), including both bacterial and fungal members.