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Glycoside hydrolase (GH) 30 family xylanases are enzymes of biotechnological interest due to their capacity to degrade recalcitrant hemicelluloses, such as glucuronoxylan (GX). This study focuses on a subfamily 7 GH30, TtXyn30A from Thermothelomyces thermophilus, which acts on GX in an "endo" and "exo" mode, releasing methyl‐glucuronic acid branched xylooligosaccharides (XOs) and xylobiose, respectively. The crystal structure of inactive TtXyn30A in complex with 23‐(4‐O‐methyl‐α‐D‐glucuronosyl)‐xylotriose (UXX), along with biochemical analyses, corroborate the implication of E233, previously identified as alternative catalytic residue, in the hydrolysis of decorated xylan. At the −1 subsite, the xylose adopts a distorted conformation, indicative of the Michaelis complex of TtXyn30AEE with UXX trapped in the semi‐functional active site. The most significant structural rearrangements upon substrate binding are observed at residues W127 and E233. The structures with neutral XOs, representing the "exo" function, clearly show the nonspecific binding at aglycon subsites, contrary to glycon sites, where the xylose molecules are accommodated via multiple interactions. Last, an unproductive ligand binding site is found at the interface between the catalytic and the secondary β‐domain which is present in all GH30 enzymes. These findings improve current understanding of the mechanism of bifunctional GH30s, with potential applications in the field of enzyme engineering. [ABSTRACT FROM AUTHOR]

BACKGROUND: Xylobiose, a non‐digestible disaccharide, largely contributes to the beneficial physiological effects of xylooligosaccharides. However, there is insufficient evidence to assess the direct effect of xylobiose on intestinal barrier function. Here, we investigated the intestinal barrier function in human intestinal Caco‐2 cells treated with xylobiose. RESULTS: In total, 283 genes were upregulated and 256 genes were downregulated in xylobiose‐treated Caco‐2 cells relative to the controls. We focused on genes related to intestinal barrier function, such as tight junction (TJ) and heat shock protein (HSP). Xylobiose decreased the expression of the TJ gene Claudin 2 (CLDN2) and increased the expression of the cytoprotective HSP genes HSPB1 and HSPA1A, which encode HSP27 and HSP70, respectively. Immunoblot analysis confirmed that xylobiose suppressed CLDN2 expression and enhanced HSP27 and HSP70 expression. A quantitative reverse transcription‐PCR and promoter assays indicated that xylobiose post‐transcriptionally regulated CLDN2 and HSPB1 levels. Additionally, selective inhibition of phosphatidyl‐3‐inositol kinase (PI3K) inhibited xylobiose‐mediated CLDN2 expression, whereas HSP27 expression induced by xylobiose was sensitive to the inhibition of PI3K, mitogen‐activated protein kinase kinase and Src. CONCLUSION: The results of the present study reveal that xylobiose suppresses CLDN2 and increases HSP27 expression in intestinal Caco‐2 cells via post‐transcriptional regulation, potentially strengthening intestinal barrier integrity; however, these effects seem to occur via different signaling pathways. Our findings may help to assess the physiological role of xylobiose. © 2023 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Endo-xylanase hydrolyzing xylan in cellulosic residues releasing xylobiose as the major product at neutral pH are desirable in the substitute sweeteners industry. In this study, two endo-xylanases were obtained from Streptomyces rochei and Bacillus velezensis. SrocXyn10 showed the highest identity of 77.22%, with a reported endo-xylanase. The optimum reaction temperature and pH of rSrocXyn10-Ec were pH 7.0 and 60°C, with remarkable stability at 45°C or pHs ranging from 4.5 to 11.0. rBvelXyn11-Ec was most active at pH 6.0 and 50°C, and was stable at 35°C or pH 3.5 to 10.5. Both rSrocXyn10-Ec and rBvelXyn11-Ec showed specific enzyme activities on wheat arabinoxylan (685.83 ± 13.82 and 2809.89 ± 21.26 U/mg, respectively), with no enzyme activity on non-xylan substrates. The Vmax of rSrocXyn10-Ec and rBvelXyn11-Ec were 467.86 U mg-1 and 3067.68 U mg-1, respectively. The determined Km values of rSrocXyn10-Ec and rBvelXyn11-Ec were 3.08 g L-1 and 1.45 g L-1, respectively. The predominant product of the hydrolysis of alkaline extracts from bagasse, corncob, and bamboo by rSrocXyn10-Ec and rBvelXyn11-Ec were xylooligosaccharides. Interestingly, the xylobiose content in hydrolysates by rSrocXyn10-Ec was approximately 80%, which is higher than most reported endo-xylanases. rSrocXyn10-Ec and rBvelXyn11-Ec could be excellent candidates to produce xylooligosaccharides at neutral/near-neutral pHs. rSrocXyn10-Ec also has potential value in the production of xylobiose as a substitute sweetener. [ABSTRACT FROM AUTHOR]

Plant cell wall-derived oligosaccharides, i.e., damage-associated molecular patterns (DAMPs), could be generated after pathogen attack or during normal plant development, perceived by cell wall receptors, and can alter immunity and cell wall composition. Therefore, we hypothesised that xylo-oligosaccharides (XOS) could act as an elicitor and trigger immune responses. To test this, we treated Arabidopsis with xylobiose (XB) and investigated different parameters. XB-treatment significantly triggered the generation of reactive oxygen species (ROS), activated MAPK protein phosphorylation, and induced callose deposition. The combination of XB (DAMP) and flg22 a microbe-associated molecular pattern (MAMP) further enhanced ROS response and gene expression of PTI marker genes. RNA sequencing analysis revealed that more genes were differentially regulated after 30 min compared to 24 h XB-treated leaves, which correlated with ROS response. Increased xylosidase activity and soluble xylose level after 30 min and 3 h of XB-treatment were observed which might have weakened the DAMP response. However, an increase in total cell wall sugar and a decrease in uronic acid level was observed at both 30 min and 24 h. Additionally, arabinose, rhamnose, and xylose levels were increased in 30 min, and glucose was increased in 24 h compared to mock-treated leaves. The level of jasmonic acid, abscisic acid, auxin, and cytokinin were also affected after XB treatment. Overall, our data revealed that the shortest XOS can act as a DAMP, which triggers the PTI response and alters cell wall composition and hormone level. Key message: Xylobiose treatment in Arabidopsis rosette leaf induces plant-triggered immunity (PTI) responses, alters cell wall polysaccharide composition and influences growth hormone levels. [ABSTRACT FROM AUTHOR]

Xylooligosaccharides (XOS) are non‐digestible oligosaccharides with a significant role as functional food ingredients in the food industry. The present research is undertaken to evaluate water soluble xylan (WSX) extraction from eight different sources using water at ambient temperature (25 ± 2 °C) and structural characterization (FTIR, NMR, XRD, and TGA). Enzymatic production of XOS from extracted substrates and its characterization using HPLC‐RID and FTIR is determined. WSX from orange fleshed sweet potato peel shows highest yield (9%), while the least (2.48%) is from Teff seed coat. FTIR structural identification of xylan confirms the presence of β‐glycosidic linkages and acetyl groups; whereas 1H NMR validates the occurrence of β‐D‐1,4‐xylopyranose backbone with α‐L‐arabinofuranose and 4‐O‐methylglucuronic acid substituents. XRD and TGA characteristics of the extracts indicate similarities in the eight sources in comparison with standard beech wood xylan. WSX is hydrolyzed with endoxylanase to obtain XOS (highest yield of 57.16 ± 1.01% in Cassava cake; lowest yield of 21.72 ± 0.72% in Bambara groundnut). HPLC‐RID reveals xylobiose as the main monomeric sugar component (DP 2) and β‐linkages are identified by FTIR in all the sources. The study suggests the potential commercial production of XOS from the analyzed agricultural by‐products. [ABSTRACT FROM AUTHOR]

Resveratrol is a natural polyphenol with antioxidant activity and numerous health benefits. However, in vivo application of this compound is still a challenge due to its poor aqueous solubility and rapid metabolism, which leads to an extremely low bioavailability in the target tissues. In this work, rXynSOS-E236G glycosynthase, designed from a GH10 endoxylanase of the fungus Talaromyces amestolkiae, was used to glycosylate resveratrol by using xylobiosyl-fluoride as a sugar donor. The major product from this reaction was identified by NMR as 3-O-ꞵ-d-xylobiosyl resveratrol, together with other glycosides produced in a lower amount as 4′-O-ꞵ-d-xylobiosyl resveratrol and 3-O-ꞵ-d-xylotetraosyl resveratrol. The application of response surface methodology made it possible to optimize the reaction, producing 35% of 3-O-ꞵ-d-xylobiosyl resveratrol. Since other minor glycosides are obtained in addition to this compound, the transformation of the phenolic substrate amounted to 70%. Xylobiosylation decreased the antioxidant capacity of resveratrol by 2.21-fold, but, in return, produced a staggering 4,866-fold improvement in solubility, facilitating the delivery of large amounts of the molecule and its transit to the colon. A preliminary study has also shown that the colonic microbiota is capable of releasing resveratrol from 3-O-ꞵ-d-xylobiosyl resveratrol. These results support the potential of mutagenic variants of glycosyl hydrolases to synthesize highly soluble resveratrol glycosides, which could, in turn, improve the bioavailability and bioactive properties of this polyphenol. [ABSTRACT FROM AUTHOR]

Cellobiose and xylobiose are disaccharides composed of two glucose or xylose units with β-1,4 linkages. This study aimed to isolate a Trichoderma reesei mutant that lacks β-glucosidase and β-xylosidase activities for the simultaneous production of these disaccharides. Mutagenesis using Fe-ion beam resulted in a mutant strain, T. reesei T1640; the cellulase production in this strain was as high as that in the parent strain. Genomic analysis revealed that T1640 lost both the β-glucosidase and β-xylosidase activities owing to the translocation of the responsible genes. Hydrolysis of alkali-treated bagasse using the enzymes from T1640 leads to high yields (365 mg/g-biomass) and ratios (72.7% of the total sugars) of cellobiose and xylobiose. [Display omitted] • Trichoderma reesei T1640 was developed using Fe ion irradiation mutagenesis. • T1640 lost both β-glucosidase and β-xylosidase activities through translocation. • T1640 showed a high cellulase production similar to that of the wild parent strain. • One-step co-production of cellobiose and xylobiose from bagasse was achieved. [ABSTRACT FROM AUTHOR]

GH30 xylobiohydrolases, an expanding enzyme category, need deeper insights for optimal use. The primary aim of this study was to characterize a new xylobiohydrolase, Ac GH30A of GH30 family from Acetivibrio clariflavus. The gene encoding Ac GH30A was cloned using pET28a(+) vector and expressed in E. coli BL21(DE3) cells. Ac GH30A was purified by immobilized metal-ion affinity chromatography. SDS-PAGE analysis of Ac GH30A showed molecular mass of ~58 kDa. Ac GH30A showed optimum temperature 80 °C and optimum pH 7.0. Ac GH30A was stable (maintaining >80 % of control activity) in pH range, 4–7 and temperature range, 30 °C -70 °C when incubated for 90 min. Ac GH30A displayed melting temperature, 72 °C and half-life, 21 days at 4 °C. The enzyme activity of Ac GH30A was enhanced by 10 mM Ca2+ and Mg2+ ions by 25 % and 21 %, respectively, whereas 10 mM Co2+, Zn2+, Fe2+, and Cu2+ ions significantly reduced it. Ac GH30A showed activity against various xylan polysaccharides displaying highest V max , 139 U.mg−1 and K M , 0.71 mg.ml−1 against 4- O -methyl glucuronoxylan under optimum conditions. TLC, HPLC and LC-MS analyses of Ac GH30A hydrolyzed products from xylan substrates revealed the release of sole product, xylobiose, confirming it as an obligate xylobiohydrolase. Ac GH30A being a highly thermostable enzyme can be potentially utlilized in various biotechnological applications. [Display omitted] • Recombinant Ac GH30A cloned from Acetivibrio clariflavus showed molecular size 58 kDa. • Thermophilic Ac GH30A showed optimum temperature 80 °C and optimum pH 7.0. • Ac GH30A displayed melting temperature, 72 °C and half-life, 21 days at 4 °C. • Ac GH30A gave V max , 139 U.mg−1 and K M , 0.71 mg.ml−1 with 4- O -methyl glucuronoxylan. • Xylobiohydrolase, Ac GH30A released only xylobiose from xylan polysaccharides. [ABSTRACT FROM AUTHOR]

Xylobiohydrolase plays a crucial role in the hydrolysis of xylan, a complex polysaccharide present in the cell walls of plants. This study focuses on the solution structure and substrate binding analysis of a novel xylobiohydrolase, Ac GH30A, from Acetivibrio clariflavus . Secondary structure analysis of Ac GH30A in an aqueous environment using Circular Dichroism and in silico modeling revealed an α/β/α sandwich structure with a central β-barrel comprising eight β-strands. Superposition of the homology-modelled structure of Ac GH30A with its closest homolog showed that the active-site contains Glu175 and Glu268 as the catalytic residues. Molecular docking confirmed xylobiose as the preferred ligand, showcasing polar interactions with the catalytic amino acids, indicating its xylobiohydrolase activity. Ac GH30A displayed a high binding affinity with xylobiose with an association constant ( K a ) of 7.83 × 10 5 M -1 , as determined by isothermal titration calorimetry. Molecular dynamics (MD) simulations of Ac GH30A and Ac GH30A-xylobiose complex in solution showed reduced RMSD, R g and SASA values, confirming the stability and compactness of the complex. MD simulations further highlighted the crucial role of Glu175 in hydrogen bonding with the ligand, which acts as an acid or base. Small-angle X-ray scattering (SAXS) analysis of Ac GH30A showed its molecular shape as an earbud with a globular structure existing in a monodispersed state, which was corroborated by dynamic light scattering (DLS). The hydrodynamic radius ( R h ) of Ac GH30A, determined by DLS, was 3.7 nm. This study significantly contributed valuable insights into the structure and functional aspects of Ac GH30A.

[Display omitted] • Two novel xylanase genes from T. asperellum were expressed for the first time. • XynTR10 and XynTR11 displayed high halophilic and ethanol tolerant. • XynTR11 hydrolyzed xylan/XOS into xylobiose without xylose via transglycosylation. • Glu153/Glu258 and Glu161/Glu252 were the key active sites for XynTR10 and XynTR11. • XynTR11 holds great potential for XOS production from agricultural waste. Effective production of xylooligosaccharides (XOS) with lower proportion of xylose entails unique and robust xylanases. In this study, two novel xylanases from Trichoderma asperellum ND-1 belonging to glycoside hydrolase families 10 (XynTR10) and 11 (XynTR11) were over-expressed in Komagataella phaffii X-33 and characterized to be robust enzymes with high halotolerance and ethanol tolerant. Both enzymes displayed strict substrate specificity towards beechwood xylan and wheat arabinoxylan. (Glu153/Glu258) and (Glu161/Glu252) were key catalytic sites for XynTR10 and XynTR11. Notably, XynTR11 could rapidly degrade xylan/XOS into xylobiose without xylose via transglycosylation. Direct degradation of corncob using XynTR10 and XynTR111 displayed that while XynTR10 yielded 77% xylobiose and 25% xylose, XynTR11 yielded much less xylose (11%) and comparable amounts of xylobiose (63%). XynTR10 or XynTR111 has great potential as a catalyst for bioconversion of xylan-containing agricultural waste into high-value products (biofuel or XOS), which is of significant benefit for the economy and environment. [ABSTRACT FROM AUTHOR]

Resveratrol is a natural polyphenol with antioxidant activity and numerous health benefits. However, in vivo application of this compound is still a challenge due to its poor aqueous solubility and rapid metabolism, which leads to an extremely low bioavailability in the target tissues. In this work, rXynSOS-E236G glycosynthase, designed from a GH10 endoxylanase of the fungus Talaromyces amestolkiae, was used to glycosylate resveratrol by using xylobiosyl-fluoride as a sugar donor. The major product from this reaction was identified by NMR as 3- O -ꞵ-d-xylobiosyl resveratrol, together with other glycosides produced in a lower amount as 4'- O -ꞵ-d-xylobiosyl resveratrol and 3- O -ꞵ-d-xylotetraosyl resveratrol. The application of response surface methodology made it possible to optimize the reaction, producing 35% of 3- O -ꞵ-d-xylobiosyl resveratrol. Since other minor glycosides are obtained in addition to this compound, the transformation of the phenolic substrate amounted to 70%. Xylobiosylation decreased the antioxidant capacity of resveratrol by 2.21-fold, but, in return, produced a staggering 4,866-fold improvement in solubility, facilitating the delivery of large amounts of the molecule and its transit to the colon. A preliminary study has also shown that the colonic microbiota is capable of releasing resveratrol from 3- O -ꞵ-d-xylobiosyl resveratrol. These results support the potential of mutagenic variants of glycosyl hydrolases to synthesize highly soluble resveratrol glycosides, which could, in turn, improve the bioavailability and bioactive properties of this polyphenol.