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Carbohydrate-active enzymes have multiple biological roles and industrial applications. Advances in genome and transcriptome sequencing together with associated bioinformatics tools have identified vast numbers of putative carbohydrate-degrading and -modifying enzymes including glycoside hydrolases and lytic polysaccharide monooxygenases. However, there is a paucity of methods for rapidly screening the activities of these enzymes. By combining the multiplexing capacity of carbohydrate microarrays with the specificity of molecular probes, we have developed a sensitive, high throughput, and versatile semiquantitative enzyme screening technique that requires low amounts of enzyme and substrate. The method can be used to assess the activities of single enzymes, enzyme mixtures, and crude culture broths against single substrates, substrate mixtures, and biomass samples. Moreover, we show that the technique can be used to analyze both endo-acting and exo-acting glycoside hydrolases, polysaccharide lyases, carbohydrate esterases, and lytic polysaccharide monooxygenases. We demonstrate the potential of the technique by identifying the substrate specificities of purified uncharacterized enzymes and by screening enzyme activities from fungal culture broths., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Summary: The Bacteroidetes phylum is renowned for its ability to degrade a wide range of complex carbohydrates, a trait that has enabled its dominance in many diverse environments. The best studied species inhabit the human gut microbiome and use polysaccharide utilization loci (PULs), discrete genetic structures that encode proteins involved in the sensing, binding, deconstruction, and import of target glycans. In many environmental species, polysaccharide degradation is tightly coupled to the phylum‐exclusive type IX secretion system (T9SS), which is used for the secretion of certain enzymes and is linked to gliding motility. In addition, within specific species these two adaptive systems (PULs and T9SS) are intertwined, with PUL‐encoded enzymes being secreted by the T9SS. Here, we discuss the most noteworthy PUL and non‐PUL mechanisms that confer specific and rapid polysaccharide degradation capabilities to the Bacteroidetes in a range of environments. We also acknowledge that the literature showcasing examples of PULs is rapidly expanding and developing a set of assumptions that can be hard to track back to original findings. Therefore, we present a simple universal description of conserved PUL functions and how they are determined, while proposing a common nomenclature describing PULs and their components, to simplify discussion and understanding of PUL systems. [ABSTRACT FROM AUTHOR]

The enzymatic conversion of acetylated hardwood glucuronoxylan to functional food oligomers, biochemicals or fermentable monomers requires besides glycoside hydrolases enzymes liberating acetic acid esterifying position 2 and/or 3 in xylopyranosyl (Xyl p ) residues. The 3- O -acetyl group at internal Xyl p residues substituted by MeGlcA is the only acetyl group of hardwood acetylglucuronoxylan and its fragments not attacked by acetylxylan esterases of carbohydrate esterase (CE) families 1, 4, 5 and 6 and by hemicellulolytic acetyl esterases classified in CE family 16. Monoacetylated aldotetraouronic acid 3″-Ac 3 MeGlcA 3 Xyl 3 , generated from the polysaccharide by GH10 endoxylanases, appears to be one of the most resistant fragments. The presence of the two substituents on the non-reducing-end Xyl p residue prevents liberation of MeGlcA by α-glucuronidase of family GH67 and blocks the action of acetylxylan esterases. The Ac 3 MeGlcA 3 Xyl 3 was isolated from an enzymatic hydrolysate of birchwood acetylglucuronoxylan and characterized by 1 H NMR spectroscopy as a mixture of two positional isomers, 3″-Ac 3 MeGlcA 3 Xyl 3 and 4″-Ac 3 MeGlcA 3 Xyl 3 , the latter being the result of acetyl group migration. The mixture was used as a substrate for three members of CE16 family of fungal origin. Trichoderma reesei CE16 esterase, inactive on polymeric substrate, deacetylated both isomers. Podospora anserina and Aspergillus niger esterases, active on acetylglucuronoxylan, deesterified effectively only the 4″-isomer. The results indicate catalytic diversity among CE16 enzymes, but also their common and unifying catalytic ability to exo-deacetylate positions 3 and 4 on non-reducing-end Xyl p residues, which is an important step in plant hemicellulose saccharification. [ABSTRACT FROM AUTHOR]

Abstract: Even if carbohydrate preparations from plant/fungal sources have a high degree of purity, observed immune-stimulation may be caused by minute sample contaminations. Using the example of different β-glucans we present a range of analytical tools crucial for validation of possible immune-stimulatory effects. Two yeast (MacroGard and Zymosan) and one cereal β-glucan (CBG40) increased IL-8 secretion by HT-29 cells considerably. Degradation of the β-glucan samples with β-glucan specific enzymes did hardly influence the effect of Zymosan and CBG40 but significantly decreased the effect of MacroGard. Stimulation of IL-8 secretion by CBG40 and Zymosan was hence not due to their β-glucan content. Instead, the effect of the CBG40 sample was due to low levels of LPS despite the inability of the known LPS inhibitor Polymyxin B to supress its stimulatory effect. We conclude that targeted enzymatic degradation of samples is a powerful validation tool to investigate carbohydrate specific immune-modulation. [Copyright &y& Elsevier]