1,721,125 research outputs found
Mixed Linkage β-1,3/1,4-Glucan Oligosaccharides Induce Defense Responses in Hordeum vulgare and Arabidopsis thaliana
No full textPlants detect conserved microbe-associated molecular patterns (MAMPs) and modified “self” molecules produced during pathogen infection [danger associated molecular patterns (DAMPs)] with plasma membrane-resident pattern recognition receptors (PRRs). PRR-mediated MAMP and/or DAMP perception activates signal transduction cascades, transcriptional reprogramming and plant immune responses collectively referred to as pattern-triggered immunity (PTI). Potential sources for MAMPs and DAMPs are microbial and plant cell walls, which are complex extracellular matrices composed of different carbohydrates and glycoproteins. Mixed linkage β-1,3/1,4-glucan (β-1,3/1,4-MLG) oligosaccharides are abundant components of monocot plant cell walls and are present in symbiotic, pathogenic and apathogenic fungi, oomycetes and bacteria, but have not been detected in the cell walls of dicot plant species so far. Here, we provide evidence that the monocot crop plant H. vulgare and the dicot A. thaliana can perceive β-1,3/1,4-MLG oligosaccharides and react with prototypical PTI responses. A collection of Arabidopsis innate immunity signaling mutants and >100 Arabidopsis ecotypes showed unaltered responses upon treatment with β-1,3/1,4-MLG oligosaccharides suggesting the employment of a so far unknown and highly conserved perception machinery. In conclusion, we postulate that β-1,3/1,4-MLG oligosaccharides have the dual capacity to act as immune-active DAMPs and/or MAMPs in monocot and dicot plant species.Plants detect conserved microbe-associated molecular patterns (MAMPs) and modified “self” molecules produced during pathogen infection [danger associated molecular patterns (DAMPs)] with plasma membrane-resident pattern recognition receptors (PRRs). PRR-mediated MAMP and/or DAMP perception activates signal transduction cascades, transcriptional reprogramming and plant immune responses collectively referred to as pattern-triggered immunity (PTI). Potential sources for MAMPs and DAMPs are microbial and plant cell walls, which are complex extracellular matrices composed of different carbohydrates and glycoproteins. Mixed linkage β-1,3/1,4-glucan (β-1,3/1,4-MLG) oligosaccharides are abundant components of monocot plant cell walls and are present in symbiotic, pathogenic and apathogenic fungi, oomycetes and bacteria, but have not been detected in the cell walls of dicot plant species so far. Here, we provide evidence that the monocot crop plant H. vulgare and the dicot A. thaliana can perceive β-1,3/1,4-MLG oligosaccharides and react with prototypical PTI responses. A collection of Arabidopsis innate immunity signaling mutants and >100 Arabidopsis ecotypes showed unaltered responses upon treatment with β-1,3/1,4-MLG oligosaccharides suggesting the employment of a so far unknown and highly conserved perception machinery. In conclusion, we postulate that β-1,3/1,4-MLG oligosaccharides have the dual capacity to act as immune-active DAMPs and/or MAMPs in monocot and dicot plant species.Open-Access-Publikationsfonds 202
Investigating catalytic diversity in copper radical oxidases for potential bioproduct applications
No full textThe utilization and valorization of biomass has been proposed as a sustainable alternative to petroleum-based energy, chemicals and materials. Microbial enzymes can be used as selective catalysts for the depolymerization and functionalization of plant material. In this regard, the copper radical oxidases (CROs) from Auxiliary Activity Family 5 subfamily 2 (AA5_2) are attractive targets since they oxidize primary alcohols in a chemo-selective manner to the corresponding aldehydes without utilizing complex and expensive organic cofactors. AA5_2 was first defined by the archetypal galactose oxidase from Fusarium graminearum, however, other fungal AA5_2 members have recently been shown to comprise a wide range of specificities for aromatic, aliphatic and furan-based alcohols. This suggests a broader substrate scope of CROs for biocatalytic applications. However, only 10% of the annotated AA5_2 members have been characterized to date.
The work presented in this thesis describes the biochemical characterization of fourteen fungal copper radical oxidases, recombinantly produced in Pichia pastoris, encompassing galactose oxidases, raffinose oxidases, broad spectrum alcohol oxidases, non-carbohydrate alcohol oxidases and aryl alcohol oxidases. Detailed product analysis was conducted of carbohydrates, furans and glycerol oxidized by these novel enzymes to further explore their potential applications. Of note, we observed direct oxidation of 5-hydroxymethylfurfural (HMF), an important platform chemical derived from biomass, to 5-formyl-2-furoic acid (FFCA) by six enzymes. One enzyme facilitated the direct one-pot conversion of HMF to 2,5-furandicarboxylic acid (FDCA), a building block for bio-plastic production. Through docking of galactose into homology models of these newly characterized enzymes, and comparing it to the archetypal galactose oxidase, key amino acid residues important for substrate specificity have been proposed. Finally, the potential of enzyme immobilization using the cross-linked enzyme aggregate (CLEA) strategy has been investigated for a specific alcohol oxidase to increase its utilization in biocatalysis.
This work expands our understanding of the catalytic diversity of copper radical oxidases from AA5_2 and doubles the number of characterized AA5_2 members. Detailed sequence and enzymatic analysis highlights their structure-function relationships while product analysis informs their possible biotechnological applications in bio-products manufacturing. Furthermore, immobilization was shown to be a promising avenue for increasing the biocatalytic potential of CROs.Science, Faculty ofChemistry, Department ofGraduat
Understanding the diversity, biochemistry, and biological role of endo-glucanase 16 enzymes in the context of plant evolution
No full textA defining feature of plants, the plant cell wall is an essential structure that provides mechanical support, cell shape, and protection from pathogens and the environment. Cell walls are dynamic structures that are composed of a network of crystalline cellulose microfibrils, amorphous polysaccharides, and structural proteins. The synthesis, maintenance, and modification of the plant cell wall polysaccharides are carried out by carbohydrate active-enzymes (CAZymes). Xyloglucan endotransglycosylase/hydrolase (XTH) are secreted cell wall enzymes that restructure the cell wall matrix glycan, xyloglucan, thereby facilitating cell wall remodelling and growth. The plant endo-glucanase 16 (EG16) enzymes were recently identified as a unique clade that is structurally and functionally intermediate between the bacterial mixed-linkage β(1,3)/(1,4)-glucanases and the plant XTHs in glycoside hydrolase family 16 (GH16). Correspondingly, EG16 members were broadly specific towards the cell wall polysaccharides mixed-linkage β-glucan (MLG) and xyloglucan, as well as soluble cellulosic substrates. Although EG16 activity and protein structure have been demonstrated, their biological role is unknown. This thesis employs a multi-disciplinary approach to the characterization of the newly designated EG16 group.
A comprehensive census of the publicly available genome and transcriptomes showed that EG16 orthologs were found in all plant clades spanning nearly 500 million years of evolution. Notably, the presence of EG16 orthologs observed in the charophyte green algae species that are the most phylogenetically distant from angiosperms, support that EG16-like genes are ancestors to extant EG16 and XTH genes. EG16 substrate specificities, primary hydrolysis site, and digest products are consistent between vastly different plant species. Elucidation of the biological role of EG16 in the moss Physcomitrella patens and a hybrid poplar species, two different distantly related land plants, show gene expression in young tissues and leaderless cell wall localization. In P. patens, EG16 deletion yielded larger plants that senesced earlier than wild-type, whereas, in poplar, downregulation did not affect growth.
This thesis serves as a foundation for the EG16 clade and informs their evolutionary history, conserved activity, cell wall localization, and involvement in growth. The research opens the door to investigate unexplored topics such as the EG16 specific secretion mechanism and their role in MLG-containing cell walls.Medicine, Faculty ofBiochemistry and Molecular Biology, Department ofGraduat
Synthesis and analysis of glycocaged prodrugs for the treatment of intestinal inflammation
No full textThe full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.Science, Faculty ofChemistry, Department ofGraduat
Functional characterization of a mixed-linkage beta-glucan utilization locus from the human gut microbe Prevotella copri
No full textThe human gut microbiota (HGM) is a complex microbial community that possesses a huge capacity for carbohydrate degradation in contrast to humans and is dominated by the phylum Bacteroidetes comprising Bacteroides and Prevotella. While Bacteroides species are associated with people consuming protein/fat-rich diets, members of Prevotella including Prevotella copri are highly abundant in people consuming plant-based diets. Here, I present a comprehensive genetic, biochemical, and structural-biological investigation of a recently identified gene cluster of P. copri upregulated in response to mixed-linkage β-glucan (MLG), a polysaccharide commonly found in cereals. The MLG utilization locus (MLGUL) contains an SGBP-A that interacted with MLG specifically and an SGBP-B that recognized 1,4-linked β-glucans promiscuously. The tertiary structure of SGBP-A revealed an aromatic binding platform and flexible loop that interact with cellopentaose or potential MLG-breakdown products. Similarly, a structural model suggested a binding platform for the SGBP-B. Furthermore, the MLGUL encodes a GH5_4, a highly specific, endo-acting MLGase, that produced oligosaccharides by preferential hydrolysis of β-1,3-linkages. The product analysis and tertiary structure revealed that β-1,4-linked glucose units in the negative subsites and occupation of the +2 subsite facilitate this cleavage. In addition, the MLGUL includes an exo-acting β-glucosidase from the GH3 family that degraded longer oligosaccharides into glucose and cellobiose. The latter will be further cleaved to glucose and glucose 1-phosphate by a predicted cellobiose phosphorylase, a member of GH94. Together, these enzymological and biochemical studies support a model for MLG degradation by P. copri in which SGBPs bind substrate at the cell surface to facilitate cleavage by the endo-MLGase GH5_4 and import by the TonB-dependent transporter. The generated oligosaccharides will be sequentially cleaved by a GH3 β-glucosidase and a GH94 cellobiose phosphorylase, in the periplasm and cytosol, respectively, generating glucose and glucose 1-phosphate as final products for the metabolism of P. copri. Thus, the work provides insights into the molecular mechanism of MLG utilization by P. copri and might indicate factors affecting the fitness of P. copri within the HGM. This knowledge might advance our understanding of diet-host-microbe interactions and may result in innovative therapeutic approaches for prevalent diseases linked to the human gut.Science, Faculty ofChemistry, Department ofGraduat
Heterologous production and functional characterization of Auxiliary Activity family 10 lytic polysaccharide monooxygenases for fiber functionalization
No full textThe efficient depolymerization and/or utilization (valorization) of residual waste biomass is crucial for establishing a sustainable bio-based economy. Cellulose and chitin constitute the most abundant polymer components of biomass and represent renewable feedstocks for the production of biofuels, biomaterials, and other bio-based chemicals. The recalcitrance and low chemical reactivity of these polysaccharides limit their viability in numerous industrial applications. Lytic polysaccharide monooxygenases (LPMOs) are oxidative enzymes secreted by saprotrophic organisms to assist in the depolymerization of cellulose and chitin fibers. The recent discovery of LPMOs has shifted the fundamental understanding of microbial biomass utilization while also potentiating many avenues for the commercial valorization of cellulose and chitin. This dissertation presents a thorough investigation of the production, functional analysis, and fiber-modifying application of bacterial LPMOs from Auxiliary Activity family 10 (AA10). Signal peptide-mediated periplasmic secretion through the general secretion pathway (Sec) in Escherichia coli was the most suitable approach for producing active AA10 LPMOs. Five AA10s encoded by the historically significant Cellulomonas flavigena and Cellulomonas fimi bacteria were purified and characterized. Four of these Cellulomonas AA10 homologs were cellulose-active and one was chitin-active. Thermostability studies confirmed only cellulose-active Cellulomonas AA10s could reversibly unfold. Three non-Cellulomonas AA10 catalytic domains were produced in E. coli using the Sec signal peptide derived from the lone AA10 encoded by C. fimi. Notably, Xylanimonas cellulosilytica LPMO10A demonstrated dual activity on cellulose and chitin, while Streptomyces pratensis LPMO10D and Legionella longbeachae LPMO10 were found to be strictly active on chitin or cellulose substrates, respectively. Time course kinetic assays show high initial LPMO-oxidizing activity commonly coincides with rapid inactivation from oxidation. These results highlight the importance of optimizing abiotic parameters such as pH and reductant type to maximize the catalytic potential of LPMOs. Lastly, as a proof-of-concept, a C1-oxidizing cellulose-active AA10 encoded by C. flavigena was used to chemo-enzymatically conjugate several fluorescent probes to cellulose fibers. The work presented here informs future studies on heterologous LPMO production, LPMO activity screening and characterization, as well as LPMO-mediated fiber defibrillation and chemical modification.Medicine, Faculty ofBiochemistry and Molecular Biology, Department ofGraduat
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
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