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.

An arabinogalactan (SGP80-A1) was isolated for the first time from the pulp of Siraitia grosvenorii, with an apparent Mw of 81.3 kDa. Its structure was initially characterized using an integrated strategy combining NMR spectroscopy with database matching, allowing non-destructive identification of sugar residue types directly within intact polysaccharides. Monosaccharide analysis by PMP-HPLC further revealed that SGP80-A1 was composed of arabinose, galactose, and glucuronic acid in a molar ratio of 49.8:46.6:3.6, which was consistent with 1D ¹H qNMR analysis, where arabinose accounted for approximately 49.6% and galactose together with glucuronic acid accounted for approximately 50.4%. Methylation analysis (GC-MS) was subsequently employed to determine the glycosidic linkage patterns, which supported the 2D NMR-based structural assignment. Additionally, Yariv reactivity indicated that SGP80-A1 contains β-1,3-galactan fragments. SGP80-A1 features a backbone of →6)-β-Galp-(1→, which is substituted at the O-3 position primarily by various α-arabinofuranosyl side chains, alongside a small proportion of β-Galp-(1 → 3)-β-Galp-(1 → branches. For the critical 1,3,6-β-Gal residues exhibiting highly overlapped H6b and H5 signals, a ¹³C-coupled HSQC-TOCSY experiment was employed to obtain accurate chemical shift assignments. In vitro bioactivity assays indicated that SGP80-A1 (40-160 μg/mL) potently stimulated immune responses in RAW264.7 macrophages following effective endotoxin removal, as evidenced by significantly enhanced production of NO, IL-6 and TNF-α, thereby confirming the reliability of the observed immunomodulatory potential of SGP80-A1. Collectively, these results not only characterize the structural features of SGP80-A1 but also highlight its potential as an immunostimulant, providing a foundation for further investigation of its in vivo efficacy and underlying mechanisms.

β-Mannanases are involved in the hydrolysis of mannan, a major component of hemicellulose. The genome of Glutamicibacter halophytocola sp., an endophytic bacterium isolated from Crithmum maritimum, a halophilic plant from the Island of Crete, encodes a putatively secreted β-mannanase (Gh_GH26) belonging to glycoside hydrolase 26 family (GH26). This sequence occupies a poorly explored region of the GH26 sequence space. Gh_GH26 consists of three domains, including a C-terminal carbohydrate-binding module 23 (CBM23), which is uncommon among GH26s and has a peculiar amino acidic composition at the −3/−4 substrate subsites. Gh_GH26 displays halophilic behavior, retaining activity up to 2.5 M NaCl, and exhibits endo-mannanase activity. Functional analyses combined with structural predictions suggest that Gh_GH26 coordinates Ca²⁺ ions through a single binding site located within the CBM23 domain. The residues involved in Ca²⁺ binding are conserved in the CBMs of other characterized GH26s, despite their low similarity with Gh_GH26. Site-directed mutagenesis shows that Ca2+ ions contribute to both enzyme activity and stability. These results suggest that Ca²⁺ may play an evolutionary conserved activation role, modulating CBM flexibility and its orientation relative to the catalytic domain. Overall, this work expands our understanding of the functional and structural diversity of the GH26 family, highlighting its potential for application in the degradation of mannan-containing biomass under saline conditions.

This study systematically investigated the regulatory mechanisms of two types of arabinoxylans (AX) with distinct molecular weights, namely low-molecular-weight AX (AXL) and high-molecular-weight AX (AXH), on the structure, interfacial behavior, and foam properties of the ethanol-soluble protein fraction (ES) isolated from the wheat aqueous phase. Both AX fractions induced ES aggregation and charge redistribution through non-covalent interactions, forming composite particles with larger particle sizes and enhanced surface potential. Interfacial studies revealed that ES possesses rapid adsorption capacity and forms predominantly elastic films, whereas AX addition reduced interfacial pressure, diffusion rate, and adsorbed protein content. Quartz crystal microbalance with dissipation monitoring (QCM-D) results indicated that AX dynamically interacted with the ES layer, inducing structural rearrangement and promoting the formation of a stable composite adsorption layer. AX reduced the foaming capacity of ES but significantly improved foam stability. This enhancement in stability does not arise from increased interfacial elasticity, instead, it relies on a synergistic mechanism in which AX increases the viscosity of the continuous phase to retard drainage and enhances interfacial viscous dissipation to inhibit liquid film rupture. The AXL and AXH exhibited distinct mechanisms for regulating interfacial steady-state behavior: AXH contributed primarily through interfacial elasticity, while AXL mainly via viscous dissipation. In summary, AXL and AXH improve the foam stability of ES through the synergistic effect of bulk phase structural rearrangement, dynamic interfacial regulation, and continuous phase rheological properties, providing a theoretical basis for understanding the role of polysaccharide-protein interactions in food foam systems.