A novel thermophilic recombinant obligate xylobiohydrolase (AcGH30A) from Acetivibrio clariflavus orchestrates the deconstruction of xylan polysaccharides.

GH30 xylobiohydrolases, an expanding enzyme category, need deeper insights for optimal use. The primary aim of this study was to characterize a new xylobiohydrolase, AcGH30A of GH30 family from Acetivibrio clariflavus. The gene encoding AcGH30A was cloned using pET28a(+) vector and expressed in E. coli BL21(DE3) cells. AcGH30A was purified by immobilized metal-ion affinity chromatography. SDS-PAGE analysis of AcGH30A showed molecular mass of ~58 kDa. AcGH30A showed optimum temperature 80 °C and optimum pH 7.0. AcGH30A was stable (maintaining >80 % of control activity) in pH range, 4-7 and temperature range, 30 °C -70 °C when incubated for 90 min. AcGH30A displayed melting temperature, 72 °C and half-life, 21 days at 4 °C. The enzyme activity of AcGH30A 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. AcGH30A showed activity against various xylan polysaccharides displaying highest Vmax, 139 U.mg-1 and KM, 0.71 mg.ml-1 against 4-O-methyl glucuronoxylan under optimum conditions. TLC, HPLC and LC-MS analyses of AcGH30A hydrolyzed products from xylan substrates revealed the release of sole product, xylobiose, confirming it as an obligate xylobiohydrolase. AcGH30A being a highly thermostable enzyme can be potentially utlilized in various biotechnological applications.

Structural and molecular insights into a bifunctional glycoside hydrolase 30 xylanase specific to glucuronoxylan.

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 ß-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.

Xylobiose treatment strengthens intestinal barrier function by regulating claudin 2 and heat shock protein 27 expression in human Caco-2 cells.

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.

Transformation of corncob into high-value xylooligosaccharides using glycoside hydrolase families 10 and 11 xylanases from Trichoderma asperellum ND-1.

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.

Xylobiose treatment triggers a defense-related response and alters cell wall composition.

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.

The xylobiohydrolase activity of a GH30 xylanase on natively acetylated xylan may hold the key for the degradation of recalcitrant xylan.

Acetyl substitutions are common on the hemicellulosic structures of lignocellulose, which up until recently were known to inhibit xylanase activity. Emerging data, however, suggest that xylanases are able to accommodate acetyl side-groups within their catalytic site. In the present work, a fungal GH30 xylanase from Thermothelomyces thermophila, namely TtXyn30A, was shown to release acetylated xylobiose when acting on pretreated lignocellulosic substrate. The released disaccharides could be acetylated at the 2-OH, 3-OH or both positions of the non-reducing end xylose, but the existence of the acetylation on the reducing end cannot be excluded. The synergy of TtXyn30A with acetyl esterases indicates that particular subsites within its active site cannot tolerate acetylated xylopyranose residues. Molecular docking showed that acetyl group can be accommodated on the 2- or 3-OH position of the non-reducing end xylose, unlike the reducing-end xylose (subsite -1), where only 3-OH decoration can be accommodated. Such insight into the catalytic activity of TtXyn30A could contribute to a better understanding of its biological role and thus lead to a more sufficient biotechnological utilization.

Gene cloning, expression, and characterization of two endo-xylanases from Bacillus velezensis and Streptomyces rochei, and their application in xylooligosaccharide production.

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.

Simultaneous production of cellobiose and xylobiose from alkali-treated bagasse using cellulase secreted by Fe-ion-irradiated Trichoderma reesei mutant.

Cellobiose and xylobiose are disaccharides composed of two glucose or xylose units with ß-1,4 linkages. This study aimed to isolate a Trichoderma reesei mutant that lacks ß-glucosidase and ß-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 ß-glucosidase and ß-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.

Synthesis and Characterization of a Novel Resveratrol Xylobioside Obtained Using a Mutagenic Variant of a GH10 Endoxylanase.

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.

Understanding the Xylooligosaccharides Utilization Mechanism of Lactobacillus brevis and Bifidobacterium adolescentis: Proteins Involved and Their Conformational Stabilities for Effectual Binding.

Xylooligosaccharides having various degrees of polymerization such as xylobiose, xylotriose, and xylotetraose positively affect human health by interacting with gut proteins. The present study aimed to identify proteins present in gut microflora, such as xylosidase, xylulokinase, etc., with the help of retrieved whole-genome annotations and find out the mechanistic interactions of those with the above substrates. The 3D structures of proteins, namely Endo-1,4-beta-xylanase B (XynB) from Lactobacillus brevis and beta-D-xylosidase (Xyl3) from Bifidobacterium adolescentis, were computationally predicted and validated with the help of various bioinformatics tools. Molecular docking studies identified the effectual binding of these proteins to the xylooligosaccharides, and the stabilities of the best-docked complexes were analyzed by molecular dynamic simulation. The present study demonstrated that XynB and Xyl3 showed better effectual binding toward Xylobiose with the binding energies of - 5.96 kcal/mol and - 4.2 kcal/mol, respectively. The interactions were stabilized by several hydrogen bonding having desolvation energy (- 6.59 and - 7.91). The conformational stabilities of the docked complexes were observed in the four selected complexes of XynB-xylotriose, XynB-xylotetraose, Xyl3-xylobiose, and Xyn3-xylotriose by MD simulations. This study showed that the interactions of these four complexes are stable, which means they have complex metabolic activities among each other. Extending these studies of understanding, the interaction between specific probiotics enzymes and their ligands can explore the detailed design of synbiotics in the future.

Multi-Objective Optimization Through Machine Learning Modeling for Production of Xylooligosaccharides from Alkali-Pretreated Corn-Cob Xylan Via Enzymatic Hydrolysis.

The hemicellulose content present in corn cobs can help in producing a high amount of xylooligosaccharides (XOS) in an eco-friendly manner. In this work, the XOS was produced from alkali pre-treated corn-cobs having a true yield of 38 ± 1.4% via enzymatic hydrolysis with the help of xylanase from T. lanuginosus VAPS-24. The production process was optimized to achieve a high concentration of XOS using innovative multi-objective optimization through machine learning modeling and finding out the most suitable parameters where xylobiose production is higher than xylose. The Multi-objective connected neural networks (MOCNN) model with tangent sigmoid activation function yielded a correlation coefficient of 96.51%; there were six optimal sets where xylobiose concentration was higher than xylose. The best-optimized conditions yielded 3.03 mg/ml of xylobiose and 1.31 mg/ml of xylose. Therefore, this novel approach of machine learning can target the increasing demand for xylooligosaccharides in the growing industrial market of prebiotics.

A New Subfamily of Glycoside Hydrolase Family 30 with Strict Xylobiohydrolase Function.

The Acetivibrio clariflavus (basonym: Clostridium clariflavum) glycoside hydrolase family 30 cellulosomal protein encoded by the Clocl_1795 gene was highly represented during growth on cellulosic substrates. In this report, the recombinantly expressed protein has been characterized and shown to be a non-reducing terminal (NRT)-specific xylobiohydrolase (AcXbh30A). Biochemical function, optimal biophysical parameters, and phylogeny were investigated. The findings indicate that AcXbh30A strictly cleaves xylobiose from the NRT up until an α-1,2-linked glucuronic acid (GA)-decorated xylose if the number of xyloses is even or otherwise a single xylose will remain resulting in a penultimate GA-substituted xylose. Unlike recently reported xylobiohydrolases, AcXbh30A has no other detectable hydrolysis products under our optimized reaction conditions. Sequence analysis indicates that AcXbh30A represents a new GH30 subfamily. This new xylobiohydrolase may be useful for commercial production of industrial quantities of xylobiose.

Deciphering effectual binding potential of xylo-substrates towards xylose isomerase and xylokinase through molecular docking and molecular dynamic simulation.

Xylooligosaccharides (XOS) such as xylobiose and xylotriose are prebiotics with important functions and relevance and the study of interaction mechanism between these substrate and their respective enzymes has scope and applications. Thus, the present study aimed to decipher the interaction mechanisms of xylose isomerase (XylA) and xylokinase (XylB) towards their xylo-substrates namely xylobiose and xylotriose by computational modeling and molecular dynamic simulation studies. The three-dimensional structures of XylA and XylB, not available in their native forms, were predicted, energy minimized and validated by various computational biology tools and software. The binding mechanisms of xylobiose and xylotriose towards XylA and XylB were modeled by molecular docking and the stability of the docked complexes was confirmed by molecular dynamic (MD) simulation. The current study suggested that the theoretical models of XylA and XylB possessed good stereo-chemical validity, structural stabilities and minimum energy conformers. The molecular docking studied showed that xylotriose showed better binding interactions to XylA than xylobiose and xylobiose showed better binding interaction to XylB than xylotriose with ideal root mean square deviation (RMS), minimum binding energy (kcal/mol), hydrogen bonding and weak interactions. The MD simulation confirmed the stabilities of the docked complexes predicted by docking studies. The study suggested that interactions between the probiotics and prebiotics and provides the novel insights in exploring synbiotics as functional foods towards their futuristic applications. [Formula: see text]HighlightsThis study deciphers the interactions of xylosubstrates to XylA and XylB.The XylA and XylB possessed ideal structural stability and stereochemistryXylotriose and Xylobiose showed significant interactionsThe interactions of Xylotriose-XylA and Xylobiose-XylB were found stable in MD studies.Communicated by Ramaswamy H. Sarma.

A novel bacterial GH30 xylobiohydrolase from Hungateiclostridium clariflavum.

Typical bacterial GH30 xylanases are glucuronoxylanases requiring 4-O-methylglucuronic acid (MeGlcA) substitution of a xylan main chain for their action. They do not exhibit a significant activity on neutral xylooligosaccharides, arabinoxylan (AraX), or rhodymenan (Rho). In this work, the biochemical characterization of the bacterial Clocl_1795 xylanase from Hungateiclostridium (Clostridium) clariflavum DSM 19732 (HcXyn30A) is presented. Amino acid sequence analysis of HcXyn30A revealed that the enzyme does not contain amino acids known to be responsible for MeGlcA coordination in the -2b subsite of glucuronoxylanases. This suggested that the catalytic properties of HcXyn30A may differ from those of glucuronoxylanases. HcXyn30A shows similar specific activity on glucuronoxylan (GX) and Rho, while the specific activity on AraX is about 1000 times lower. HcXyn30A releases Xyl2 as the main product from the non-reducing end of different polymeric and oligomeric substrates. Catalytic properties of HcXyn30A resemble the properties of the fungal GH30 xylobiohydrolase from Acremonium alcalophilum, AaXyn30A. HcXyn30A is the first representative of a prokaryotic xylobiohydrolase. Its unique specificity broadens the catalytic diversity of bacterial GH30 xylanases. KEY POINTS: • Bacterial GH30 xylobiohydrolase from H. clariflavum (HcXyn30A) has been characterized. • HcXyn30A releases xylobiose from the non-reducing end of different substrates. • HcXyn30A is the first representative of bacterial xylobiohydrolase.

Production and characterization of functional wheat bran hydrolysate rich in reducing sugars, xylooligosaccharides and phenolic acids.

The aim was to enhance production of functional hydrolysate from wheat bran (WB). WB was hydrolyzed with 3000 U/mL ɑ-amylase and 1200 U/mL alkaline protease to prepare WB insoluble dietary fibre (WBIDF). Functional hydrolysate production from the extract containing crude xylan of WBIDF by xylanase was optimized by Taguchi method. The optimal condition for xylan degradation and functional substances production was 78.50 U/mL xylanase, pH 10.0, 50 °C, and reaction time 6 h. The maximum yield of reducing sugars was 614.0 µg/mL, xylobiose increased from 12.9 µg/mL to 213.3 µg/mL, xylotriose increased from 34.9 µg/mL to 174.0 µg/mL, ferulic acid 13.1 µg/mL made up 57.5 % of the total identifiable phenolic pool in the hydrolysate. The total antioxidant activity of hydrolysate was 141.8 mg ascorbic acid equivalents g-1 crude xylan, and the highest 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity reached 92.7 %. The hydrolysate exhibited great potential in agricultural and food industry application.

An endoxylanase rapidly hydrolyzes xylan into major product xylobiose via transglycosylation of xylose to xylotriose or xylotetraose.

Here, we proposed an effective strategy to enhance a novel endoxylanase (Taxy11) activity and elucidated an efficient catalysis mechanism to produce xylooligosaccharides (XOSs). Codon optimization and recruitment of natural propeptide in Pichia pastoris resulted in achievement of Taxy11 activity to 1405.65 ±â€¯51.24 U/mL. Analysis of action mode reveals that Taxy11 requires at least three xylose (xylotriose) residues for hydrolysis to yield xylobiose. Results of site-directed mutagenesis indicate that residues Glu119, Glu210, and Asp53 of Taxy11 are key catalytic sites, while Asp203 plays an auxiliary role. The novel mechanism whereby Taxy11 catalyzes conversion of xylan or XOSs into major product xylobiose involves transglycosylation of xylose to xylotriose or xylotetraose as substrate, to form xylotetraose or xylopentaose intermediate, respectively. Taxy11 displayed highly hydrolytic activity toward corncob xylan, producing 50.44 % of xylobiose within 0.5 h. This work provides a cost-effective and sustainable way to produce value-added biomolecules XOSs (xylobiose-enriched) from agricultural waste.

A novel GH30 xylobiohydrolase from Acremonium alcalophilum releasing xylobiose from the non-reducing end.

Xylanases of the GH30 family are grouped to subfamilies GH30-7 and GH30-8. The GH30-8 members are of bacterial origin and well characterized, while the GH30-7 members are from fungal sources and their properties are quite diverse. Here, a heterologous expression and characterization of the GH30-7 xylanase AaXyn30A from a cellulolytic fungus Acremonium alcalophilum is reported. From various polymeric and oligomeric substrates AaXyn30A generates xylobiose as the main product. It was proven that xylobiose is released from the non-reducing end of all tested substrates, thus the enzyme behaves as a typical non-reducing-end acting xylobiohydrolase. AaXyn30A is active on different types of xylan, exhibiting the highest activity on rhodymenan (linear ß-1,3-ß-1,4-xylan) from which also an isomeric xylotriose Xyl-ß-1,3-Xyl-ß-1,4-Xyl is formed. Production of xylobiose from glucuronoxylan is at later stage accompanied by a release of aldouronic acids differing from those liberated by the bacterial GH30-8 glucuronoxylanases.

Xylooligosaccharides chemical stability after high-intensity ultrasound processing of prebiotic orange juice.

The effects of the high-intensity ultrasound (HIUS) technology at the nominal powers of 300, 600, 900, and 1200 W were evaluated on the chemical stability of xylooligosaccharides (XOS) used to enrich orange juice. The ultrasound energy performance for each nominal power applied to the XOS-enriched orange juice was determined by calculating acoustic powers (W), HIUS intensity (W/cm2), and energy density (kJ/mL). Physicochemical properties (pH and soluble solid content), organic acid content (ascorbic, malic, and citric acids), total phenolic content (TPC), antioxidant activity by the FRAP (Ferric reducing ability of plasma) method, sugar (glucose, fructose, and sucrose), and XOS (xylobiose, xylotriose, xylotetraose, xylopentaose, and xylohexaose) content were determined. The pH and soluble solid content did not change after all HIUS treatments. The HIUS process severity was monitored by quantifying ascorbic acid content after the treatments. A significant linear decrease in the ascorbic acid content was observed in prebiotic orange juice with the HIUS process intensification by increasing nominal power. The malic acid and citric acid contents had similar behavior according to the HIUS process intensification. The nominal power increase from 300 to 600 W increased the concentration of both organic acids, however, the intensification up to 1200 W reduced their concentration in the functional beverage. The TPC and FRAP data corroborated with the results observed for the ascorbic acid content. However, the HIUS processing did not alter sugar and XOS contents. The XOS chromatographic profiles were not modified by the HIUS treatment and presented the same amount of all prebiotic compounds before and after the HIUS treatment. Overall, HIUS technology has been evaluated as a promising stabilization technique for prebiotic beverages enriched with XOS due to their high chemical stability to this emerging technology under severe process conditions.

Production, separation, and characterization of high-purity xylobiose from enzymatic hydrolysis of alkaline oxidation pretreated sugarcane bagasse.

The production of high-purity xylobiose from lignocellulose is an expensive and tedious process. In this work, the production of xylobiose from enzymatic hydrolysis of alkaline oxidation pretreated sugarcane bagasse was investigated. Furthermore, a simple process for the separation of xylobiose from enzymatic hydrolysate by activated carbon absorption, water washing, and ethanol-water desorption was developed. Under the optimized separation conditions, 96.77% xylobiose was adsorbed at 16% activated carbon loadings. Moreover, xylose and acetate could not be detected after washing by 3-fold volume of water. Xylobiose with 80.16% yield was eluted by 5-fold volume of 5% (v/v) ethanol-water. The reusability of activated carbon was evaluated by 5 cycles of adsorption-desorption process, suggesting that the activated carbon exhibited good reusability. The separated xylobiose sample with high-purity (97.29%) was confirmed by HPLC, ESI-MS, and NMR. Overall, this study provided a low-cost and robust technology for the production and separation of high-purity xylobiose from lignocellulose.

Purification and characterization of an endo-xylanase from Trichoderma sp., with xylobiose as the main product from xylan hydrolysis.

Fungal endo-ß-1,4-xylanases (endo-xylanases) can hydrolyze xylan into xylooligosaccharides (XOS), and have potential biotechnological applications for the exploitation of natural renewable polysaccharides. In the current study, we aimed to screen and characterize an efficient fungal endo-xylanase from 100 natural humus-rich soil samples collected in Guizhou Province, China, using extracted sugarcane bagasse xylan (SBX) as the sole carbon source. Initially, 182 fungal isolates producing xylanases were selected, among which Trichoderma sp. strain TP3-36 was identified as showing the highest xylanase activity of 295 U/mL with xylobiose (X2) as the main product when beechwood xylan was used as substrate. Subsequently, a glycoside hydrolase family 11 endo-xylanase, TXyn11A, was purified from strain TP3-36, and its optimal pH and temperature for activity against beechwood xylan were identified to be 5.0 and 55 °C, respectively. TXyn11A was stable across a broad pH range (3.0-10.0), and exhibited strict substrate specificity, including xylan from beechwood, wheat, rye, and sugarcane bagasse, with Km and Vmax values of 5 mg/mL and 1250 µmol/mg min, respectively, toward beechwood xylan. Intriguingly, the main product obtained from hydrolysis of beechwood xylan by TXyn11A was xylobiose, whereas SBX hydrolysis resulted in both X2 and xylotriose. Overall, these characteristics of the endo-xylanase TXyn11A indicate several potential industrial applications.