Arabinoxylan (Wheat Flour; Low Viscosity)

endo-1,4-β-Xylanase (EC 3.2.1.8) is employed across a broad range of industries including animal feed, brewing, baking, biofuels, detergents and pulp (paper). Despite its importance, a rapid, reliable, reproducible, automatable assay for this enzyme that is based on the use of a chemically defined substrate has not been described to date. Reported herein is a new enzyme coupled assay procedure, termed the XylX6 assay, that employs a novel substrate, namely 4,6-O-(3-ketobutylidene)-4-nitrophenyl-β-4⁵-O-glucosyl-xylopentaoside. The development of the substrate and associated assay is discussed here and the relationship between the activity values obtained with the XylX6 assay versus traditional reducing sugar assays and its specificity and reproducibility were thoroughly investigated.

A range of α-L-arabinofuranosyl-(1-4)-β-D-xylo-oligosaccharides (AXOS) were produced by hydrolysis of wheat flour arabinoxylan (WAX) and acid debranched arabinoxylan (ADWAX), in the presence and absence of an AXH-d3 α-L-arabinofuranosidase, by several GH10 and GH11 β-xylanases. The structures of the oligosaccharides were characterised by GC-MS and NMR and by hydrolysis by a range of α-L-arabinofuranosidases and β-xylosidase. The AXOS were purified and used to characterise the action patterns of the specific α-L-arabinofuranosidases. These enzymes, in combination with either Cellvibrio mixtus or Neocallimastix patriciarum β -xylanase, were used to produce elevated levels of specific AXOS on hydrolysis of WAX, such as 3²-α-L-Araf-(1-4)-β-D-xylobiose (A³X), 2³-α-L-Araf-(1-4)-β-D-xylotriose (A²XX), 3³-α-L-Araf-(1-4)-β-D-xylotriose (A³XX), 2²-α-L-Araf-(1-4)-β-D-xylotriose (XA²X), 3²-α-L-Araf (1-4)-β-D-xylotriose (XA³X), 2³-α-L-Araf-(1-4)-β-D-xylotetraose (XA²XX), 3³-α-L-Araf-(1-4)-β-D-xylotetraose (XA³XX), 2³ ,3³-di-α-L-Araf-(1-4)-β-D-xylotriose (A²⁺³XX), 2³,3³-di-α-L-Araf-(1-4)-β-D-xylotetraose (XA²⁺³XX), 2⁴,3⁴-di-α-L-Araf-(1-4)-β-D-xylopentaose (XA²⁺³XXX) and 3³,3⁴-di-α-L-Araf-(1-4)-β-D-xylopentaose (XA³A³XX), many of which have not previously been produced in sufficient quantities to allow their use as substrates in further enzymic studies. For A^2,3XX, yields of approximately 16% of the starting material (wheat arabinoxylan) have been achieved. Mixtures of the α-L-arabinofuranosidases, with specific action on AXOS, have been combined with β-xylosidase and β-xylanase to obtain an optimal mixture for hydrolysis of arabinoxylan to L-arabinose and D-xylose.

Background: Previous studies have revealed that some Auxiliary Activity family 9 (AA9) lytic polysaccharide monooxygenases (LPMOs) oxidize and degrade certain types of xylans when incubated with mixtures of xylan and cellulose. Here, we demonstrate that the xylanolytic activities of two xylan-active LPMOs, TtLPMO9E and TtLPMO9G from Thermothielavioides terrestris, strongly depend on the presence of xylan substitutions. Results: Using mixtures of phosphoric acid-swollen cellulose (PASC) and wheat arabinoxylan (WAX), we show that removal of arabinosyl substitutions with a GH62 arabinofuranosidase resulted in better adsorption of xylan to cellulose, and enabled LPMO-catalyzed cleavage of this xylan. Furthermore, experiments with mixtures of PASC and arabinoglucuronoxylan from spruce showed that debranching of xylan with the GH62 arabinofuranosidase and a GH115 glucuronidase promoted LPMO activity. Analyses of mixtures with PASC and (non-arabinosylated) beechwood glucuronoxylan showed that GH115 action promoted LPMO activity also on this xylan. Remarkably, when WAX was incubated with Avicel instead of PASC in the presence of the GH62, both xylan and cellulose degradation by the LPMO9 were impaired, showing that the formation of cellulose-xylan complexes and their susceptibility to LPMO action also depend on the properties of the cellulose. These debranching effects not only relate to modulation of the cellulose-xylan interaction, which influences the conformation and rigidity of the xylan, but likely also affect the LPMO-xylan interaction, because debranching changes the architecture of the xylan surface. Conclusions: Our results shed new light on xylanolytic LPMO9 activity and on the functional interplay and possible synergies between the members of complex lignocellulolytic enzyme cocktails. These findings will be relevant for the development of future lignocellulolytic cocktails and biomaterials.

Carbohydrate active enzymes are valuable tools in cereal processing to valorise underutilized side streams. By solubilizing hemicellulose and modifying the fibre structure, novel food products with increased nutritional value can be created. In this study, a novel GH5_34 subfamily arabinoxylanase from Herbinix hemicellulosilytica, HhXyn5A, was identified, produced and extensively characterized, for the intended exploitation in cereal processing to solubilize potential prebiotic fibres; arabinoxylo-oligosaccharides (AXOS). The purified two-domain HhXyn5A (catalytic domain and CBM6) demonstrated high storage stability, showed a melting temperature T_m of 61 °C and optimum reaction conditions were determined to 55°C and pH 6.5 on wheat arabinoxylan (WAX). HhXyn5A demonstrated activity on various commercial cereal arabinoxylans and produced prebiotic AXOS, while the sole catalytic domain of HhXyn5A did not demonstrate detectable activity. HhXyn5A demonstrated no side activity on oat β-glucan. In contrast to the commercially available homologue CtXyn5A, HhXyn5A gave a more specific HPAEC–PAD oligosaccharide product profile when using WAX and alkali extracted oat bran fibres as substrate. Results from multiple sequence alignment of GH5_34 enzymes, homology modelling of HhXyn5A and docking simulations with ligands XXXA³, XXXA³XX, and X⁵, concluded that the active site of HhXyl5A catalytic domain is highly conserved and can accommodate both shorter and longer AXOS ligands. However, significant structural dissimilarities between HhXyn5A and CtXyn5A in the binding cleft of CBM6, due to lack of important ligand interacting residues, is suggested to cause the observed differences in substrate specificity and product formation.

In the human gut microbiota, Bacteroidetes break down dietary and endogenous glycosides through highly specific polysaccharide utilization loci (PULs). PULs encode a variety of sensor regulators, binding proteins, transporters, and carbohydrate-active enzymes (CAZymes). Surface glycan-binding proteins (SGBPs) are essential for the efficient capture of the glycosides present on the cell surface, providing Bacteroidetes with a competitive advantage in colonizing their habitats. Here, we present the functional and structural characterization of a SusD-like protein encoded by a xylooligosaccharide (XOS) PUL from an uncultured human gut Bacteroides strain. This locus is also conserved in Bacteroides vulgatus, thereby providing new mechanistic insights into the role of SGBPs in the metabolism of dietary fiber of importance for gut health. Various in vitro analyses, including saturation transfer difference nuclear magnetic resonance (STD-NMR) spectroscopy, revealed that the SusD-like protein cannot bind to the cognate substrate of the XOS PUL, although its presence is essential for the PUL to function. Analysis of the crystal structure of the SusD-like protein reveals an unfolded binding surface and the absence or inappropriate orientation of several key residues compared with other known SusD-like structures. These results highlight the critical role of the SusD-like protein in the transport of oligosaccharides and provide fundamental knowledge about the structure-function of SusC/D-like transporters, revealing that the binding specificity of SusD-like SGBPs does not necessarily reflect the uptake specificity of the transporter.

The molecular mobility of water and biopolymers in wheat dough and the influence of xylanases thereon was investigated with time domain proton nuclear magnetic resonance relaxometry. To reduce the complexity, model systems containing starch, gluten and/or water-unextractable arabinoxylan (WU-AX) were used. In the starch-WU-AX-water model, starch binds water fast but less strong compared to WU-AX, resulting in water withdrawal from starch during resting. In contrary, WU-AX did not affect the water distribution in a gluten-WU-AX-water system, despite the higher water retention capacity (WRC) of WU-AX compared to gluten. In a starch-gluten-WU-AX-water model and in wheat flour, water was distributed over the different constituents including WU-AX. Addition of xylanase reduced the WRC of WU-AX, resulting in a release of water. Therefore, the beneficial effect of xylanase on dough and bread quality can, in part, be attributed to the redistribution of water, initially bound by WU-AX, between the other flour constituents.

The aim of this study was to evaluate the effects of dietary levels of xylanase on production performance, egg quality, nutrient digestibility, and excreta microbiota shedding of laying hens in a 12-week trial. Two-hundred-forty Hy-Line brown laying hens (44 wk old) were distributed according to a randomized block experimental design into one of 4 dietary treatments with 10 replicates of 6 birds each. The 4 dietary treatments were corn-soybean-meal-wheat-based diets supplemented with 0, 225, 450, or 900 U/kg xylanase. Daily feed intake, egg production, egg weight, egg mass, feed conversion ratio, and damaged egg rate showed no significant response to increasing xylanase supplementation during any phase (P > 0.05). No significant responses were observed for apparent total tract digestibility of dry matter, nitrogen, or gross energy (P > 0.05). A significant linear increase to increasing xylanase supplementation was seen for lactic acid bacteria numbers, although coliforms and Salmonella counts were not affected. Increasing the dietary xylanase resulted in a significant linear increase in eggshell thickness in wk 3, 6, 9, and 12 (P < 0.05). In addition, a significant linear increase occurred for Haugh unit and albumen height in wk 12 (P < 0.05). In summary, the inclusion of xylanase in corn-soybean-meal-wheat-based diets increased eggshell thickness, Haugh unit, albumen height, and excreta lactic acid bacteria count but had no effect on production performance or nutrient digestibility.

The discovery and creation of biocatalysts for plant biomass conversion are essential for industrial demand and scientific research of the plant cell wall. α-1,2 and α-1,3-L-arabinofuranosidases are debranching enzymes that catalyzing hydrolytic release of α-L-arabinofuranosyl residues in plant cell wall. Gene database analyses shows that GH62 family only contains specific α-L-arabinofuranosidases that play an important role in the degradation and structure of the plant cell wall. At present, there are only 22 enzymes in this group has been characterized. In this study, we cloned a novel α-1,3-arabinofuranosidase gene (poabf62a) belonging to glycoside hydrolase family 62 from Penicillium oxalicum sp. 68 and expressed it in Pichia pastoris. The molecular mass of recombinant PoAbf62A was estimated to be 32.9 kDa. Using p-nitrophenyl-α-L-arabinofuranoside (pNPαAbf) as substrate, purified PoAbf62A exhibited an optimal pH of 4.5 and temperature of 35°C. Results of methylation and ¹³C NMR analyses showed that PoAbf62A was exclusively α-1,3-arabinofuranosidase, specific for cleavage of α-1,3-arabinofuranosyl residues, and with the absence of activity towards α-1,2-arabinofuranose and α-1,5-arabinofuranose. Therefore, PoAbf62A exhibits high activity on sugar beet arabinan and wheat arabinoxylan, because their branched side chain are decorated with α-1,3-arabinofuranose. On the other hand, there is a lack of activity with linear-α-L-1,5-arabinan and xylan that only contained α-L-1,5-arabinofuranose or β-1,4-xylose. The α-1,3-arabinofuranosidase activity identified here provides a new biocatalytic tool to degrade hemicellulose and analyze the structure of plant cell walls.

Arabinoxylans (AX) are a type of dietary fiber present in cereal grains. Recent studies have shown consuming water-extractable AX (WE-AX) can reduce blood glucose levels and prevent the growth of pathogenic bacteria in the human gut. WE-AX can affect dough quality by increasing baking absorption and reducing gluten formation. Historically, WE-AX has been quantified using the phloroglucinol assay, however, this method is labor intensive and not amendable to large sample sizes. Enzyme-linked immunosorbent assays (ELISAs) quantify molecules through specific antigen-antibody binding. The monoclonal antibody LM11 specifically binds to wheat WE-AX and can be used in an ELISA based quantification. In this study, an ELISA was developed to quantify WE-AX in whole grain flour. Flour WE-AX content was evaluated using ELISA and the phloroglucinol assay in five varieties and two milling methods using a Retsch osilating mill and a Thomas Wiley mill. Moderate correlations were found between assays and milling methods. The ELISA assay was found to reduce sample processing time by 16.5 min. Twenty soft winter wheat varieties were evaluated for WE-AX content using ELISA. The ELISA developed in this study was found to be a highly accurate method of quantifying WE-AX in large sample sizes.

In previous reports, we characterized four endo-xylanases produced by Streptomyces sp. strain SWU10 that degrade xylans to several xylooligosaccharides. To obtain a set of enzymes to achieve complete xylan degradation, a β-D-xylosidase gene was cloned and expressed in Escherichia coli, and the recombinant protein, named rSWU43A, was characterized. SWU43A is composed of 522 amino acids and does not contain a signal peptide, indicating that the enzyme is an intracellular protein. SWU43A was revealed to contain a Glyco_hydro_43 domain and possess the three conserved amino acid residues of the glycoside hydrolase family 43 proteins. The molecular mass of rSWU43A purified by Ni-affinity column chromatography was estimated to be 60 kDa. The optimum reaction conditions of rSWU43A were pH 6.5 and 40°C. The enzyme was stable up to 40°C over a wide pH range (3.1-8.9). rSWU43A activity was enhanced by Fe²⁺ and Mn²⁺ and inhibited by various metals (Ag⁺, Cd²⁺ , Co²⁺, Cu²⁺, Hg²⁺, Ni²⁺, and Zn²⁺), D-xylose, and L-arabinose. rSWU43A showed activity on p-nitrophenyl-β-D-xylopyranoside and p-nitrophenyl-α-L-arabinofuranoside substrates, with specific activities of 0.09 and 0.06 U/mg, respectively, but not on any xylosidic or arabinosidic polymers. rSWU43A efficiently degraded β-1,3-xylooligosaccharides to produce xylose but showed little activity towards β-1,4-xylobiose, with specific activities of 1.33 and 0.003 U/mg, respectively. These results demonstrate that SWU43A is a β-1,3-D-xylosidase (EC 3.2.1.72), which to date has only been described in the marine bacterium Vibrio sp. Therefore, rSWU43A of Streptomyces sp. is the first β-1,3-xylosidase found in gram-positive bacteria. SWU43A could be useful as a specific tool for the structural elucidation and production of xylose from β-1,3-xylan in seaweed cell walls.