Xylobiose

The most commonly used method for the measurement of the level of endo-xylanase in commercial enzyme preparations is the 3,5-dinitrosalicylic acid (DNS) reducing sugar method with birchwood xylan as substrate. It is well known that with the DNS method, much higher enzyme activity values are obtained than with the Nelson-Somogyi (NS) reducing sugar method. In this paper, we have compared the DNS and NS reducing sugar assays using a range of xylan-type substrates and accurately compared the molar response factors for xylose and a range of xylo-oligosaccharides. Purified beechwood xylan or wheat arabinoxylan is shown to be a suitable replacement for birchwood xylan which is no longer commercially available, and it is clearly demonstrated that the DNS method grossly overestimates endo-xylanase activity. Unlike the DNS assay, the NS assay gave the equivalent colour response with equimolar amounts of xylose, xylobiose, xylotriose and xylotetraose demonstrating that it accurately measures the quantity of glycosidic bonds cleaved by the endo-xylanase. The authors strongly recommend cessation of the use of the DNS assay for measurement of endo-xylanase due to the fact that the values obtained are grossly overestimated due to secondary reactions in colour development.

Microarrays are powerful tools for high throughput analysis, and hundreds or thousands of molecular interactions can be assessed simultaneously using very small amounts of analytes. Nucleotide microarrays are well established in plant research, but carbohydrate microarrays are much less established, and one reason for this is a lack of suitable glycans with which to populate arrays. Polysaccharide microarrays are relatively easy to produce because of the ease of immobilizing large polymers noncovalently onto a variety of microarray surfaces, but they lack analytical resolution because polysaccharides often contain multiple distinct carbohydrate substructures. Microarrays of defined oligosaccharides potentially overcome this problem but are harder to produce because oligosaccharides usually require coupling prior to immobilization. We have assembled a library of well characterized plant oligosaccharides produced either by partial hydrolysis from polysaccharides or by de novo chemical synthesis. Once coupled to protein, these neoglycoconjugates are versatile reagents that can be printed as microarrays onto a variety of slide types and membranes. We show that these microarrays are suitable for the high throughput characterization of the recognition capabilities of monoclonal antibodies, carbohydrate-binding modules, and other oligosaccharide-binding proteins of biological significance and also that they have potential for the characterization of carbohydrate-active enzymes.

Rice husk is a readily available residue which can be used for producing bioproducts in a biorefinery context. In this study, the hemicellulose fraction was hydrolyzed in a hydrothermal process to produce xylooligosaccharides (XOS), whereas the cellulosic hydrolysate was used for red pigment production by Monascus ruber Tieghem IOC 2225. The highest XOS (X2-X4) production (24 g per 1 kg of rice husk) was achieved at 180°C for 68 min in a non-stirred Parr reactor (50 mL). Subsequently, using a stirred parr reactor (1 L) at 180 °C for 60 min, 40 g of XOS (42% of xylobiose, 35% of xylobiose, 13% of xylotriose, 7% of xylotetraose, and 3% of xylopentaose) per 1 kg of rice husk were obtained. The XOS was then purified by using ultrafiltration (UF) with two diafiltration membranes at 6.5 pH, recovering approximately 92% of total XOS. Further purification was conducted with nanofiltration (NF) at 3.8 pH, recovering approximately 86.4% of XOS in the retentate. This process yielded XOS with a purity of 77%. Additionally, the enzymatic process yielded 132 g/kg of sugar, and the hydrolysate was used to produce 2.1 UA490nm of red pigment by fungi after 7 days.

The display of enzymes on bacterial surfaces is an interesting approach for immobilising industrially important biocatalysts. In recent years, non-recombinant surface display using food-grade bacteria, such as lactic acid bacteria (LAB), have gained interest because of their safety, simplicity, and cost-effectiveness. β-Xylosidase is one of the many biocatalytic enzymes targeted for immobilisation due to its key role in the complete saccharification of lignocellulosic biomass, including xylan hemicellulose. Recently, the xylose-tolerant β-xylosidase, LfXyl43, was identified in Limosilactobacillus fermentum. LfXyl43 is capable of producing xylose from the degradation of xylo-oligosaccharides (XOS) and beechwood xylan. This study aimed to immobilise this new biocatalyst on the surface of LAB-derived bacteria-like particles (BLP) and investigate its applicability and reusability in the degradation of xylan hemicellulose. Additionally, the influence of the anchor position and the presence of linker peptides on the display and activity of the β-xylosidase was investigated. Four expression vectors were constructed to express different anchor-xylosidase fusion proteins. Upon expression and purification, all anchor-xylosidase fusion proteins were active towards the artificial substrate p-nitrophenyl-β-D-xylopyranoside. In addition, all anchor-xylosidase fusion proteins were successfully displayed on the surface of BLP. However, only the β-xylosidases with linker peptide showed hydrolytic activity after immobilisation on BLP. BLP displaying β-xylosidases demonstrated high activity against XOS and beechwood xylan, thereby producing high amounts of xylose. Moreover, the immobilised enzyme demonstrated reusability across several bioconversion cycles. Overall, this study highlights the potential industrial application of surface-displayed β-xylosidase for the effective degradation of lignocellulosic biomass.

Microwave-assisted autohydrolysis (MAA) has gained attention as an alternative to conventional hydrothermal treatment (CHT) to solubilize hemicellulosic-derived compounds, such as oligosaccharides, within shorter residence times. MAA assays were conducted on Acacia dealbata wood, an invasive species, at severities (S₀) ranging from 3.63 to 4.64 to optimize xylooligosaccharides (XO) recovery and assess the enzymatic susceptibility of the spent solids for bioethanol production. S₀ between 3.77 and 4.15 yielded XO concentrations > 9.8 g/L corresponding to a recovery of > 80% regarding initial xylan. Besides, at S₀= 3.77, a bioethanol yield of 71% was attained (26.25 g/L). Furthermore, CHT was performed at S₀ values of 3.80 and 4.44 to compare the impact of both heating strategies under optimal conditions for (i) XO production and (ii) higher enzymatic susceptibility of the spent solid. MAA resulted in higher bioethanol yields and, particularly under harsher conditions, lower by-products formation and higher oligosaccharides content. Additionally, MAA consumed 2.60–2.75-fold less energy than CHT.

Plant β-xylosidases are less well characterized for hemicellulose degradation than their microbial counterparts. To address this, a broadly expressed rice (Oryza sativa) glycoside hydrolase family 3 (GH3) β-xylosidase designated OsXyl1 was expressed in heterologous Pichia pastoris. OsXyl1 showed maximal enzyme activity at pH 4.0 and 60 °C. It was relatively stable at 30–50 °C. It hydrolyzed 4NP-β-d-xylopyranoside (4NPXyl) and β-1,4-linked xylooligosaccharides (XOS) with degrees of polymerization (DP) of 2–6. OsXyl1 hydrolylsis of 4NPXyl was much more rapid and specific than that of other 4NP glycosides with an apparent k_cat/K_m value of 19.0 mM^–1 s^–1. OsXyl1 had similar specificity toward XOS having DP values of 2–5 with apparent k_cat/K_m values of 2.6–4.2 mM^–1 s^–1. OsXyl1 was also efficient at transglycosylating short alcohols with 4NPXyl and XOS xylosyl donors. Therefore, rice OsXyl1 β-xylosidase may function in recycling of xylans in plant cell wall recycling and it may be applied for transglycosylation of alcohol acceptors.

Ex vivo techniques can provide more physiologically significant insights into prebiotic activity and overcome some limitations of in vitro tests. In this study, an ex vivo model, formed of a large intestine of mice, was tested to assess the effects of the hydrocolloidal natural polymer, xylan (XY), and its hydrolysis product, xylooligosaccharides (XOS). XY and XOS were loaded separately into the cecum, proximal colon, and distal colon. Their utilization and short-chain fatty acid (SCFA) formation by the colonized microflora and levels of dominant phyla and key genera such as Bifidobacterium, Bacteroides, and Lactobacillus were followed. XY and XOS were metabolized in all sections, and SCFAs were released. The results suggest that the slower utilization of XY compared to XOS in the cecum can enable this polysaccharide to move towards distal parts of the large intestine and extend the sites of prebiotic activity. Unlike widely used in vitro models, the ex vivo model allowed testing the utilization pattern and effects of the prebiotics in the natural environment of the microflora and examining the intestinal sections separately.

Organic acid hydrolysis from lignocelluloses to produce xylooligosaccharides (XOS) is efficient, but the uncertainty of the distribution of the XOS lead to low bioactivity of XOS. In the present work, XOS was produced from moso bamboo by acetic acid/sodium acetate (AC/SA) system, and the XOS composition and distribution was adjusted by xylanase (XYL). The maximum XOS yield was 42.1% under the optimal conditions of 2.0 M AC/SA, pH 4.0, 180°C, 40 min. The subsequent XYL hydrolysis successfully transformed XOS with a degree of polymerization (DP) higher than 6 to low-DP XOS and the final XOS yield reached 65.7%. The total yield of xylobiose and xylotriose reached a high level of 40.9%, accounting for 62.2% of the total XOS. After removing most of lignin from the solid residue with sodium hydroxide, glucose was produced by cellulolytic hydrolysis. When the cellulase load was 15 FPU/g dry matter, the yield of glucose was 93.4%. This work suggested that XYL hydrolysis after AC/SA hydrolysis of bamboo could achieve a high XOS yield with high bioactivity components from moso bamboo.

Quinoa (Chenopodium quinoa), a widely consumed grain, generates quinoa stalks (QS), a byproduct rich in valuable components such as carbohydrates. This study explores the use of citric acid-assisted hydrothermal pretreatment (CA-HTP) to produce glucose through enzymatic hydrolysis of pretreated QS and to recover xylooligosaccharides (XOS). Additionally, QS was converted into activated charcoal to remove phenolic compounds, and XOS were subsequently purified using ultrafiltration (UF) and nanofiltration (NF). After CA-HTP treatment (at 180°C for 60 minutes with 3.5% citric acid concentration), followed by enzymatic hydrolysis, 43.5 g of glucose per 100 g of pretreated material was obtained. Furthermore, a secondary hydrothermal treatment (HTP) step using water increased glucan hydrolysis yield by 26%. Optimization of CA-HTP conditions (180°C for 20 minutes, 7.1% solid-to-liquid ratio, and 2% citric acid) resulted in the production of 17.1 mg of XOS per gram of washed QS. Activated charcoal treatment of the CA-HTP hydrolysate removed 87.62% of phenolic compounds, recovering 62% of XOS at 47 % purity after membrane filtration. These results demonstrate the potential of quinoa stalks for sustainable valorization in biorefinery applications.

Background: Xylan, the second most abundant polysaccharide in plant biomass, requires endoxylanases for its hydrolysis into xylooligosaccharides (XOS). Xylanases have been widely used in industries such as animal feed, bakery, juice production, and paper pulp. Recently, XOS have gained attention for their health benefits, including improved digestion, reduced cholesterol, and antioxidant effects. The cold-adapted GH10 xylanase of Antarctic origin Xyl-L was previously expressed in Escherichia coli, showing promising low-temperature activity. However, Pichia pastoris is currently a preferred host for industrial xylanase production due to its ability to express complex proteins and secrete them into the culture medium. This study explored the expression of Xyl-L in P. pastoris and evaluated its potential for XOS production using common flours as substrates, aiming for applications in the food and nutraceutical industry. Results: Comparison between AOX1 () and GAP () promoters for recombinant Xyl-L production in P. pastoris showed that the  promoter resulted in higher activity per wet-cell weight. Co-transforming -Xyl strains with plasmids encoding genes aiding in protein folding (HAC1 or PDI1) did not enhance Xyl-L catalytic activity compared to the parental strain. Thus, -Xyl was cultivated in 3 L bioreactors in fed-batch cultures; it is presumed that the enzyme is produced with glycosylations within its structure, given its migration within the SDS-PAGE gels. The produced Xyl-L was purified from the culture supernatant, resulting in peak xylanase activity after 90 h, with specific activity of 5.10 ± 0.21 U/mg, at pH 7.5 and C, using beechwood xylan. It also showed a Km of 3.5 mg/mL and a kcat of 9.16 . Xyl-L maintained over 80% of relative activity between pH 5.68.6 and C, and was activated by and , but inhibited by . Xyl-L was tested using several flours (whole wheat, rye, oatmeal and all-purpose) as substrates, where XOS with a polymerization degree (DP) of 2 were obtained from each substrate, whole wheat flour generated XOS with DP 3, and XOS with DP 2, 3 and 4 were produced when beechwood xylan was used as substrate. Conclusions: The xylanase Xyl-L was successfully expressed in P. pastoris and proved to be able to degrade various flour substrates, producing XOS with DP ranging from 2 to 4, indicating its potential applications in the nutraceutical and food industries. Further studies must be performed to optimize its production in bioreactors.