Laminaripentaose

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.

β-1,3-Glucan is a dietary fiber with microbiome-modulating potential, yet the role of polymerization degree on microbial composition and metabolism remains unclear. Curdlan is an insoluble linear β-1,3-glucan widely used as a food hydrocolloid that provides a model to examine structure-dependent microbiome responses. We compared glucose, curdlan (CBG), and curdlan-derived β-1,3-glucan oligosaccharides (OBG) using anaerobic in vitro fermentation of pooled human feces. A multi-omics approach integrating 16S rRNA sequencing, shotgun metagenomics, short-chain fatty acid (SCFA) quantification, and untargeted metabolomics was applied to resolve taxonomic, functional, and metabolic shifts. Both CBG and OBG enriched Bacteroidota and enhanced carbohydrate metabolism relative to glucose, but through distinct pathways. CBG promoted polymer-associated carbohydrate processing, with enrichment of β-1,3-glucan-degrading enzymes such as GH16, Sus-like transport systems, and polysaccharide-specialized taxa (e.g., Prevotella and Bifidobacterium), and butyrate production. In contrast, OBG favored transporter-mediated substrate uptake, broader microbial participation (e.g. Parabacteroides and Enterobacteriaceae), and a metabolite profile enriched in amino acid-derived compounds and branched-chain SCFAs. These findings demonstrate that β-1,3-glucan polymerization degree governs microbiome functional routing and highlight polymerization degree as a tunable parameter for designing microbiome-directed carbohydrate ingredients with predictable metabolic outcomes.

In the CAZy classification of carbohydrate-active enzymes, the GH-Q clan regroups three distantly related glycoside hydrolase (GH) families: GH94, GH149, and GH161. These families contain glycoside phosphorylases (GPs) that mainly use α–d-glucose-1-phosphate as a glucosyl donor. In the present study, we investigated the structure–function relationships within family GH161, which exhibits β-1,3-glucan phosphorylase activity. Using cryo-electron microscopy (cryo-EM), we determined the dimeric structure of a GH161 enzyme. Comparison between the unliganded form, the acceptor-bound form, and finally a catalytically active complex revealed conformational changes during catalysis that involve coordinated rearrangements within the active site, including loops held by the adjacent protomer. These structural insights, combined with biochemical characterization, deepen our understanding of the molecular determinants driving catalysis in GH-Q enzymes.

This study assessed mango seed husk (MSH) fractions for producing glucose and phenolic compounds using commercial enzymes. We focused on cleaving lignin-carbohydrate linkages, specifically feruloyl and glucuronoyl esters, to decrease biomass recalcitrance and enhance product extraction. For saccharification studies, we used Sigma’s C1184 cellulase from Aspergillus niger. Characterisation results of ground MSH using phloroglucinol and scanning electron microscopy revealed that it could be separated into a fine fraction, containing less lignin and cellulose fibres with parallel orientation, and a coarse fraction, with higher lignin content and cellulose fibres at an angled orientation. Activity assays and zymogram analysis of the C1184 preparation prior to saccharification studies revealed diverse CAZyme activities associated with distinct proteins, with xylanolytic activity dominating. Saccharification studies with ground MSH found that the C1184 preparation supplemented with feruloyl or glucuronoyl esterases was suitable for extracting phenolic compounds (0.4-1.7% w/w) from MSH while converting up to 20% of the total biomass as glucose. Interestingly, when replacing 50% (w/w) of the C1184 preparation with glucuronoyl esterase, glucose release nearly doubled from both MSH fractions. Additionally, phenolics attached to carbohydrates may be less condensed in the fine fraction, as all three esterases released three-to-five times more phenolics from the fine fraction compared to the coarse fraction with higher lignin content. Saccharification trials with alkali-pretreated ground MSH showed that the C1184 preparation supplemented with β-glucosidase produced low glucose levels (170-250 mg/g dry biomass) from the substrate after 24 h, even at 50 mg/g biomass protein loading. Overall, this work advances our understanding of the importance of lignin–carbohydrate linkages formed via glucuronoyl esters in biomass recalcitrance. Furthermore, our study corroborates the potential of MSH as a valuable feedstock for producing value-added products in the biorefinery sector.

Fungal cell walls undergo continual remodeling that generates β-1,3-glucan fragments as products of endo-glycosyl hydrolases (GHs), which can be recognized as pathogen-associated molecular patterns (PAMPs) and trigger plant immune responses. How fungal pathogens suppress those responses is often poorly understood. Here, we study mechanisms underlying the suppression of β-1,3-glucan-triggered plant immunity by the blast fungus Magnaporthe oryzae. We show that an exo-β-1,3-glucanase of the GH17 family, named Ebg1, is important for fungal cell wall integrity and virulence of M. oryzae. Ebg1 can hydrolyze β-1,3-glucan and laminarin into glucose, thus suppressing β-1,3-glucan-triggered plant immunity. However, in addition, Ebg1 seems to act as a PAMP, independent of its hydrolase activity. This Ebg1-induced immunity appears to be dampened by the secretion of an elongation factor 1 alpha protein (EF1α), which interacts and co-localizes with Ebg1 in the apoplast. Future work is needed to understand the mechanisms behind Ebg1-induced immunity and its suppression by EF1α.