<p>The aim of this work was to develop a production process for the enzyme xyloglucan <i>endo</i>-transglycosylase from <i>Populus tremula x tremuloides</i> (<i>Ptt</i>XET16-34). The natural transglycosylating activity of this enzyme has previously been employed in a XET-Technology. This chemo enzymatic method is useful for biomimetic modification of cellulose surfaces and holds great potential for industrial applications. Thus, it requires that the XET-enzyme can be produced in larger scale.</p><p>This work also shows how the wildtype <i>Ptt</i>XET16-34 was modified into a glycosynthase. By mutation of the catalytic nucleophile into an alanine, glycine or serine residue, enzymes capable of synthesising defined xyloglucan fragments were obtained. These defined compounds are very valuable for further detailed studies of xyloglucan active-enzymes, but are also useful in molecular studies of the structurally important xyloglucan-cellulose interaction.</p><p>A heterologous production system for <i>Ptt</i>XET16-34 was previously developed in the methylotrophic yeast Pichia pastoris. A methanol-limited fed-batch process was also previously established, but the yield of active XET was low due to proteolysis problems and low productivity. Therefore, two alternative fed-batch techniques were investigated for the production of <i>Ptt</i>XET16-34: a temperature-limited fed-batch (TLFB) and an oxygen-limited high-pressure fed-batch (OLHPFB).</p><p>For the initial recovery of XET after the fermentation process, two different downstream processes were investigated: expanded bed adsorption (EBA) and cross-flow filtration (CFF).</p>

As mudanças climáticas estão causando prejuízos em vários setores da economia mundial. Na reunião da COP21, que teve como foco estas mudanças climáticas, participantes do mundo todo decidiram tomar atitudes urgentes para tentar conter aumento da temperatura média global. Dentro deste cenário, a produção e o consumo de energia têm uma importância central, onde fontes de energia renováveis vêm sendo preferidas às fontes de energias fósseis. O Brasil tem uma participação importante na geração de energia renovável mundial aportando um 40% do total de sua matriz energética. A degradação dos componentes da parede celular vegetal tem um vasto potencial na geração de biocombustíveis e outros compostos verdes a partir da celulose, hemicelulose e lignina. Para isto estudos das enzimas capazes de degradas estes componentes vem sendo realizados, com ênfase nas enzimas hidrolases de glicosídeos. Dentre as hidrolases, encontram-se as arabinanases, enzimas capazes de hidrolisar o arabinano, componente polissacídeo da hemicelulose, em L-arabinose. Neste trabalho, estudos envolvendo duas arabinanases de Bacillus licheniformis foram realizados, iniciando na etapa de clonagem dos genes. Os produtos foram transformados em Escherichia coli e expressos e purificados. A avaliação da estabilidade térmica indicou uma afinidade das enzimas por metais divalentes. Tentativas de cristalização resultaram na formação de um cristal, que possibilitou a determinação da estrutura uma das arabinanases. Através de ensaios bioquímicos, foi determinada a especificidade por substrato, temperatura e pH ótimos e a atividade frente a metais. Foi observado que as enzimas são seletivas para arabinano não ramificado, tem temperatura ótima em 45 e 40 graus, para BlAbn-1 e BlAbn-2, respectivamente, e pH ótimo em 8 e 7. Por último, foram realizados ensaios complementares de sinergismo e atividade oxidativa. Embora os ensaios de atividade oxidativa tenham sido inconclusivos, os ensaios de sinergismo mostraram que a enzima BlAbn-1 é capaz de aumentar em 30% a atividade do coquetel enzimático Accellerase 1500 sobre biomassa pré-tratada e sobre celulose pura. Este efeito é ainda maior na presença de sulfato de níquel. / Climate change is causing losses in different sectors of the world economy. At the meeting of COP21, focused on climate changes, participants from around the world decided to take urgent actions to try to halt the increase in global average temperature. Within this scenario, the production and consumption of energy are of central importance, where renewable energy sources have been preferred to fossil fuels. Brazil has an important role in the global renewable energy generation by contributing 40% of its total energy mix. The degradation of the components of plant cell wall has a vast potential in the generation of biofuels and other green chemical from cellulose, hemicellulose and lignin. Thus, studies of enzymes that degrade these components have been carried out, with emphasis on glycoside hydrolases. Among the hydrolases are the arabinanases, enzymes capable of hydrolyzing arabinan, a polysaccharide component of hemicellulose, in L-arabinose. In this work, studies involving two arabinanases from Bacillus licheniformis were carried out, starting in gene cloning step. The products were transformed into Escherichia coli, expressed and purified. The evaluation of the thermal stability of the enzymes showed an affinity for divalent metals. Crystallization attempts resulted in the formation of a single crystal, which made it possible to determine the crystal structure of one arabinanase. Through biochemical assays, it was determined the substrate specificity, optimum temperature and pH and activity against metals. It was observed that the enzymes are selective for non-branched arabinan, have optimum temperature at 45 and 40 degrees, to BlAbn-1 and BlAbn-2, respectively, and optimum pH of 8 and 7. Finally, additional tests were performed to evaluate the possible synergism and oxidative activity. Although the oxidative activity assays were inconclusive, the synergism tests showed that BlAbn-1 is able to increase by 30% the activity of the enzymatic cocktail Accellerase 1500 on pre-treated biomass and on pure cellulose. This effect is even greater in the presence of nickel sulfate.

No description available.

The aim of this work was to develop a production process for the enzyme xyloglucan endo-transglycosylase from Populus tremula x tremuloides (PttXET16-34). The natural transglycosylating activity of this enzyme has previously been employed in a XET-Technology. This chemo enzymatic method is useful for biomimetic modification of cellulose surfaces and holds great potential for industrial applications. Thus, it requires that the XET-enzyme can be produced in larger scale. This work also shows how the wildtype PttXET16-34 was modified into a glycosynthase. By mutation of the catalytic nucleophile into an alanine, glycine or serine residue, enzymes capable of synthesising defined xyloglucan fragments were obtained. These defined compounds are very valuable for further detailed studies of xyloglucan active-enzymes, but are also useful in molecular studies of the structurally important xyloglucan-cellulose interaction. A heterologous production system for PttXET16-34 was previously developed in the methylotrophic yeast Pichia pastoris. A methanol-limited fed-batch process was also previously established, but the yield of active XET was low due to proteolysis problems and low productivity. Therefore, two alternative fed-batch techniques were investigated for the production of PttXET16-34: a temperature-limited fed-batch (TLFB) and an oxygen-limited high-pressure fed-batch (OLHPFB). For the initial recovery of XET after the fermentation process, two different downstream processes were investigated: expanded bed adsorption (EBA) and cross-flow filtration (CFF). / <p>QC 20101108</p>

<p>Xyloglucans are polysaccharides found as storage polymers in seeds and tubers, and as cross-linking glycans 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 glycans such as cellulose.</p><p>Xyloglucan is widely used in bulk quantities in the food, textile and paper making industries. With an increasing interest in technically more advanced applications of xyloglucan, such as novel biocomposites, there is a need to understand and control the properties and interactions of xyloglucan with other compounds, to decipher the relationship between xyloglucan structure and function, and in particular the effect of different branching patterns. However, due to the structural heterogeneity of the polysaccharide as obtained from natural sources, relevant studies have not been possible to perform in practise. This fact has stimulated an interest in synthetic methods to obtain xyloglucan mimics and analogs with well-defined structure and decoration patterns.</p><p>Glycosynthases are hydrolytically inactive mutant glycosidases that catalyse the formation of glycosidic linkages between glycosyl fluoride donors and glycoside acceptors. Since its first conception in 1998, the technology is emerging as a useful tool in the synthesis of large, complex polysaccharides. This thesis presents the generation and characterisation of glycosynthases based on xyloglucanase scaffolds for the synthesis of well-defined homogenous xyloglucan oligo- and polysaccharides with regular substitution patterns.</p>

Wood formation or xylogenesis is a fundamental process for so diverse issues as industry, shelter and a sustainable environment. Wood is comprised of secondary xylem, rigid large cells with thick cell walls that are lignified. The basis for the sturdy cells is an advanced composite made up of cellulose fibers cross-linked by hemicelluloses and finally embedded in lignin. This fiber-composite is the secondary cell walls of woody plants. Cell division and differentiation is regulated by switching on and off genes. Proteins encoded by these genes execute the major functions in the cells. They steer the entire machinery operating the structure and function of the cells, maintaining growth and synthesising essential products such as the cell wall carbohydrates.   Here we describe the investigation of genes and proteins involved in xylan formation as well as the development of a model system that will aid the functional analysis of wood formation. Xylan is the main hemicellulose or cross linking glycan in dicot wood and thereby one of the most abundant carbohydrates on earth. We demonstrate that hybrid aspen cell suspension cultures can be used as a model system for secondary cell wall formation. We have also examined glycosyltransferases from CAZy family 43 that play a part in secondary cell wall formation. We have focused on one of these, Pt×tGT43A, a likely ortholog of Arabidopsis IRX9, which plays a crucial role in xylan formation. The protein was transiently expressed in Nicotiana benthamiana and its function and localization is described. Also, we investigate a glycoside hydrolase, Pt×tXyn10A, involved in wood formation. Its role is not clear but it most likely modifies xylan as it gets incorporated into the secondary cell wall after secretion from the Golgi. This influences the interaction between cellulose, xylan and lignin in the finished wood cell. We have also cloned a transcription factor, Pt×tMYB021, a likely ortholog of Arabidopsis MYB46 and we show that it activates GT43A, GT43B and Xyn10A. By analysis of the promoter sequences we identify a CA-rich motif putatively important for xylem-specific genes.   By mastering proteins involved in xylogenesis we will acquire the tools to improve and develop the wood product market. Xylan is an immense unexploited source of renewable carbohydrate. New products envisioned include e.g. faster growing trees, changed fiber characteristics, optimised utilization of wood carbohydrates for biofuels and biomaterials as well as invention of intelligent materials by biomimetic engineering. / Vedbildning, eller xylogenes, är en grundläggande mekanism för så skilda områden som industri, boende och en hållbar miljö. Ved består av sekundärt xylem som är starka, stora celler med tjocka cellväggar som är lignifierade. Grunden för de starka cellerna är en avancerad komposit bestående av cellulosafibrer tvärbundna av hemicellulosa och slutligen ingjutet i lignin. Denna fiberkomposit är den sekundära cellväggen i vedartade växter. Celldelning och differentiering regleras genom att sätta igång och stänga av gener. Proteiner som kodas av dessa gener utför de viktigaste funktionerna i cellerna. De styr hela maskineriet som upprätthåller cellernas struktur och funktion, underhåller tillväxt samt tillverkar nödvändiga produkter såsom cellväggskolhydraterna. Här beskriver vi utforskningen av gener och proteiner som är inblandade i xylanbildning liksom utvecklandet av ett modellsystem som kommer vara en hjälp i den funktionella analysen av vedbildning. Xylan är den vanligaste hemicellulosan, eller korsbindande glykanen, i lövträd och därför en av de vanligaste kolhydraterna på jorden. Vi demonstrerar att hybridaspcellkulturer i suspension kan användas som ett modellsystem för sekundär cellväggsbildning. Vi har också undersökt glykosyltransferaser från CAZy-familj 43 som tycks spela en viktig roll i bildandet av sekundär cellvägg. Vi har fokuserat på en av dessa, Pt×tGT43A, en trolig ortolog till Arabidopsis IRX9 som spelar en viktig roll i xylanbildning. Proteinet har uttryckts övergående i Nicotiana benthamiana och dess funktion och lokalisering beskrivs. Dessutom undersöker vi ett glykosidhydrolas, Pt×tXyn10A, involverad i vedbildning. Dess roll är oklar men högst sannolikt modifierar det xylan medan det inkorporeras i sekundära cellväggen efter sekretion från Golgi. Detta influerar interaktionen mellan cellulosa, hemicellulosa och lignin i den slutliga vedcellen. Vi har också klonat en transkriptionsfaktor, Pt×tMYB021, en trolig ortolog till Arabidopsis MYB46 och vi visar att den aktiverar GT43A, GT43B och Xyn10A. Genom analys av promotorsekvenserna har vi identifierat ett CA-rikt motiv förmodat viktigt för xylemspecifika gener.Genom att bemästra proteinerna som är ansvariga för vedbildning får vi verktyg att utveckla skogsproduktsmarknaden. Xylan är en ofantligt stor outnyttjad källa till förnyelsebara kolhydrater. En vision är nya produkter som till exempel snabbväxande träd, ändrade fiberegenskaper, optimerat användande av vedkolhydrater för biobränsle och biomaterial såväl som utvecklandet av intelligenta material genom biomimetisk ingenjörskonst. / QC20100730

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

Protein-carbohydrate interactions are pivotal to many biological processes, from plant cell wall degradation to host-pathogen interactions. Many of these processes require the deployment of carbohydrate-active enzymes in order to achieve their intended effects. One such class of enzymes, glycoside hydrolases, break down carbohydrate substrates by hydrolyzing the glycosidic bond within polysaccharides or between carbohydrates and non-carbohydrate moieties. The catalytic efficiency of glycoside hydrolases is often enhanced by carbohydrate-binding modules (CBMs) which are part of the modular structure of these enzymes. Understanding the carbohydrate binding function of these modules is often key to studying the catalytic properties of the enzyme. This thesis investigates the molecular determinants of carbohydrate recognition by CBMs that share similar amino acid sequences and overall three-dimensional structures and thus fall within the same CBM family. Specifically this research focused on two families; plant cell wall binding family 6 CBMs and the alpha-glucan binding family 41 CBMs. Through X-ray crystallography, isothermal titration calorimetry and other biochemical experiments, the structural and biophysical properties of CBMs were analyzed. Studying members of CBM family 6 allowed us to establish the overall picture of how similar CBMs interact with a diverse range of polysaccharide ligands. This was found to be due to changes in the topology of the binding site brought about by changes in amino acid side chains in very distinct regions of the binding pocket such that it adopted a three-dimensional shape that is complementary to the shape of the carbohydrate ligand. Members of CBM family 41 were shown to have nearly identical modes of starch recognition as found in starch-binding CBMs from other families. However family 41 CBMs are distinct as they are found mainly in pullulanases (starch debranching enzymes) and have developed binding pockets which are able to accommodate alpha-1,6-linkages, unlike other starch-binding CBM families. These are the first studies comparing multiple CBMs from within a given CBM family at the molecular level whose results allow us to examine the distinct modes of carbohydrate recognition within a CBM family. Analysis of the family 41 CBMs revealed that these CBMs are mainly found in pullulanases from pathogenic bacteria. Members from Streptococcal species were shown to specifically interact with glycogen stores within mouse lung tissue, leading us to investigate the role of alpha-glucan degradation by the pullulanase SpuA in the pathogenesis of Streptococcus pneumoniae. SpuA targets the alpha-1,6-branches in glycogen granules, forming alpha-1,4-glucan products of varying lengths. The overall three-dimensional structure of SpuA in complex with maltotetraose was determined by X-ray crystallography and showed that its active site architecture is optimal for interacting with branched substrates. Additionally, the N-terminal CBM41 module participates in binding substrate within the active site, a novel feature for CBMs. This is the first study of alpha-glucan degradation by a streptococcal virulence factor and aids in explaining why it is crucial for full virulence of the organism.

Clostridium perfringens is a gram positive spore forming anaerobe and a causative agent of gas gangrene, necrotic enteritis (pig-bel) and food poisoning in humans and other animals. This organism secretes a battery of exotoxins during the course of infection as well as a variety of virulence factors which may help to potentiate the activities of the toxins. Among these virulence factors is the μ-toxin, a family 84 glycoside hydrolase which acts to degrade hyaluronan, a component of human connective tissue. C. perfringens has 53 open reading frames encoding glycoside hydrolases. About half of these glycoside hydrolases are predicted to be secreted. Among these are CpGH84C, a paralogue of the μ-toxin, and CpGH89. CpGH89 shares sequence similarity to the human α-N-acetylglucosaminidase, NAGLU, in which mutations can cause a devastating genetic disease called mucopolysaccharidosis IIIB. One striking feature of the secreted glycoside hydrolase enzymes of C. perfringens is their modularity, with modules predicted to be dedicated to catalysis, carbohydrate-binding, protein-protein interactions and cell wall attachment. The extent of the modularity is remarkable, with some enzymes containing up to eight ancillary modules. In order to help understand the role of carbohydrate-active enzymes produced by bacterial pathogens, this thesis will focus on the structure and function of the modular extracellular glycoside hydrolase enzymes secreted by the disease causing bacterium, C. perfringens. These structure function studies examine two family 32 CBMs (carbohydrate-binding modules), one from the μ-toxin and the other from CpGH84C. As well we examine the complete structure of CpGH84C in order to help further our understanding of the structure of carbohydrate-active enzymes as a whole. Finally, the catalytic module of CpGH89 is characterized and its relationship to the human NAGLU enzyme is discussed.

In 1990, the classification of carbohydrate-active enzymes (CAZymes) was introduced by the scientist Bernard Henrissat. According to sequence similarity, these enzymes were separated into families with conserved structures and reaction mechanisms. One interesting class of CAZymes is the group of glycoside hydrolases (GHs) containing more than 138000 modules divided into 131 families as of February 2013. One of the most versatile and the largest of these GH families, containing enzymes with numerous biomass-deconstructing activities, is glycoside hydrolase family 5 (GH5). However, for large and diverse families like the GH5 family, another layer of classification is required to get a better understanding of the evolution of diverse enzyme activities. In Paper I, a new subfamily classification of GH5 is presented in order to sort the family members into distinct groups with predictive power. In total, 51 subfamilies were defined. Despite the fact that several hundred GH5 enzymes have been characterized, 20 subfamilies lacking biochemically characterized enzymes and 38 subfamilies without structural data were identified. These highlighted subfamilies contain interesting targets for future investigation. The GH5 family includes endo-β-mannanases catalyzing the hydrolysis of the β-1,4-linked backbone of mannan polysaccharides, which are common hemicelluloses found as storage and structural polymers in plant cell walls. Mannans are commonly utilized as raw biomaterials in food, feed, paper, textile and cosmetic industries, and mannanases are often applied for modifying and controlling the property of mannan polysaccharides in such applications. The overwhelming majority of characterized mannanases are from microbial origin. The situation for plant mannanases is quite different, as the catalytic properties for only a handful have been determined. Paper II describes the first characterization of a heterologously expressed Arabidopsis β-mannanase. / År 1990 introducerade forskaren Bernard Henrissat en klassificering av kolhydrataktiva enzymer (CAZymer), enligt vilken enzymerna - baserat på sekvenslikhet - delades in i familjer med konserverade strukturer och reaktionsmekanismer. En intressant CAZym-klass är glykosidhydrolaserna (GH), en klass som i februari 2013 innehöll fler än 138000 katalytiska moduler indelade i 131 olika familjer. En av de största och mest varierade av GH-familjerna är glykosidhydrolasfamilj 5 (GH5), vilken innehåller en mångfald av identifierade enzymaktiviteter relevanta för nedbrytning av biomassa. För stora och diversifierade familjer som GH5 krävs det dock ytterligare en klassificeringsnivå för att bättre förstå evolutionen och uppkomsten av de många förekommande enzymaktiviteterna. I manuskript I presenteras en ny uppdelning av GH5 enzymer i subfamiljer med syfte att dela upp familjemedlemmarna i distinkta grupper som representerar olika funktioner. Utifrån denna klassificering kan sedan ett enzyms funktion förutsägas baserat på vilken subfamilj det tillhör. Totalt definierades 51 subfamiljer. Trots att hundratals GH5 enzymer har karaktäristerats så visade det sig att 20 av subfamiljerna helt saknar biokemiskt karaktäriserade enzymer och 38 av dem saknar publicerade proteinstrukturer. Dessa subfamiljer är särskilt intressanta för framtida studier. GH5-familjen inkluderar endo-β-mannanaser som katalyserar hydrolysen av den β-1,4-länkade huvudkedjan i mannanpolysackarider. Dessa växtpolymerer som ingår i hemicellulosagruppen är vanligt förekommande i cellväggarna, där de fungerar som energilagringsmolekyler eller har en strukturell funktion. Mannaner används ofta som råmaterial för industriell livs- och djurfodersproduktion, papper, textilier och kosmetika. I dessa processer behövs ofta mannanaser för modifiering och kontroll av egenskaperna hos dessa polysackarider. Den överväldigande majoriteten av alla karaktäriserade mannanaser kommer från mikroorganismer. Endast för ett fåtal växtmannanaser har de katalytiska egenskaperna analyserats. Manuskript II beskriver den första karaktäriseringen av ett heterologt uttryckt β-mannanas från Arabidopsis. / <p>QC 20130506</p>