Xylopentaose

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.

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.

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.

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.

A primary substitution of the plant cell wall hemicellulosic polysaccharide xylan is (4-O-methyl-)d-glucuronic acid, which hinders the endoxylanases (XLNs) degradation of xylan for the production of valuable xylooligosaccharides (XOS). In this context, α-1,2-glucuronidase (AGU) plays a critical role in hydrolyzing the α-(1→2)-glycosidic linkages between 4-O-methyl-d-glucuronic acid and xylosyl residues in xylan, thereby enhancing XOS production by XLNs. However, AGUs have been relatively poorly studied, and insufficient and incomplete data on their biochemical properties, substrate specificity, and product profiling has limited their application. Here, we cloned, heterologously produced, purified and functionally characterized an AGU from Aspergillus niger (AnAguA) and another AGU from Penicillium subrubescens (PsAguB), belonging to Glycoside Hydrolase family 67 (GH67) and 115 (GH115), respectively, in the Carbohydrate-Active enZyme database. Results showed that neither AGU released 4-O-methyl-d-glucuronic acid from polymeric beech wood glucuronoxylan (BeWX). However, we found that from BeWX pre-digested with GH10 or GH11 XLNs from P. subrubescens (PsXlnA and PsXlnF, respectively), AnAguA released 4-O-methyl-d-glucuronic acid only from the non-reducing end of glucuronoxylan oligosaccharide, whereas PsAguB released 4-O-methyl-d-glucuronic acid from glucuronoxylan oligosaccharides regardless of the xylosyl substitution position. Furthermore, we demonstrated that enhancement of XOS release by adding AGUs to various combinations of GH10 (PsXlnA–C) and GH11 (PsXlnD–F, PsXlnH–I) XLNs from P. subrubescens varied based on the AGU-XLN combination. The combination of AnAguA with PsXlnA was the most effective, achieving at least a 3-fold increase in the release of XOS with a degree of polymerization of 5-7 compared to using PsXlnA alone.

The biorefinery of hemicellulose is being explored to produce bioethanol and xylooligosaccharides from lignocellulosic materials. Among the various methods, enzymatic hydrolysis of xylans is recognized as an environmentally friendly approach.Utilizing by-products from the pulp and paper industry as substrates for xylanase enhances sustainability and reduces waste, providing a cost-effective raw material source. However, the direct application of xylanase on these substrates often yields low amounts of xylooligosaccharides. To address this, xylopentaose was used to evaluate reaction extent and accessibility, achieving a 61% conversion and producing 57% xylobiose (X2) and xylotriose (X3). Improvements in yield were observed when substrates were modified (by pretreatments), doubling the yield of X2 and X3 in commercial xylan to 44%. In Kraft pulp treatment, ozonation increased yields from 18 % to 27%. Different substrates required tailored pretreatments, such as ball milling for solid commercial xylan and ozone treatment for soluble xylan from Kraft pulp. Additionally, direct treatment of high-hemicellulose-content pulp converted 25% of hemicellulose, with 44% yielding X2 and X3, resulting in an overall yield of 11%. These findings support the potential for industrial applications in biotechnological processes.

Barley malt residue (BMR) was subjected to microwave-assisted enzymatic hydrolysis to evaluate its potential as a raw material to produce xylooligosaccharides (XOS) suitable for use as a prebiotic. The influent factors on XOS production, microwave power, exposure time, and xylanase dosage were ascertained with response surface methodology based on Box–Behnken design (BBD). The fitted models of XOS and xylose yields were in good agreement with the experimental results. Using a microwave power of 1235.1 W, a 6 min exposure time, and a xylanase concentration of 89.12 U/g substrate gave the highest yield of XOS: 208.05 mg/g substrate at 4 h of enzyme incubation time. Based on the product composition, BMR-XOS purification by Saccharomyces cerevisiae treatment was superior to the process of activated carbon adsorption and ethanol precipitation treatment and was selected for further experiments. Thin-Layer Chromatography (TLC) and high-performance liquid chromatography (HPLC) clearly elucidated the oligosaccharide compositions, and the result of Fourier Transform Infrared Spectroscopy (FTIR) confirms the molecular structure and sugar components of achieved BMR-XOS. In vitro fermentation of BMR-XOS obtained from this study by the selected probiotics, Lactococcus lactis TISTR 1401, Levicaseibacillus brevis FS 2.1, Lactobacillus casei TISTR 1463, showed similar prebiotic activity compared with the commercial XOS, galactooligosaccharides (GOS), xylose, and glucose (control). In conclusion, the present study was successful in establishing the use of barley malt residue for the extraction of xylan and XOS, which could be further used as a prebiotic.

Arabinoxylan is a prevalent hemicellulose type, notably heterogeneous and resistant to biodegradation. Arabinofuranosidases (Abfs) remove arabinofuranosyl decorations of arabinoxylan, thus enabling hydrolysis by xylanases. However, a variety of Abf and xylanase specificities have emerged in recent years, necessitating a deeper understanding of their role in biomass degradation. This work investigates the biochemical features of TtAbf43 from Thermothelomyces thermophila, which specifically removes the O-3-linked arabinofuranose moieties from di-substituted xylopyranoses. The enzyme also exhibited secondary hydrolytic activity on o-nitrophenyl-β-d-xylopyranoside and arabinan. The hydrolysis of pretreated wheat and corn bran substrates was assessed using TtAbf43 and AnAbf51, two enzymes with distinct catalytic specificities. The Abfs enhanced the performance of endo-xylanases TmXyn10 and AnXyn11, promoting the release of xylo-oligomers, while the xylanases, in turn, stimulated arabinose release by the Abfs. Additionally, the Abfs facilitated the endo- and exo-activities of the bifunctional xylobiohydrolase/glucuronoxylanase TtXyn30A for the release of xylobiose and short aldouronic acids from biomass. AnAbf51 also acted in synergy with the acetyl xylan esterase OCE6 and the exo-deacetylase TtCE16B in debranching enzymatically derived oligomers from lignocellulose, whereas TtAbf43 remained unaffected by the esterases. These diverse synergistic relationships among different hemicellulases could assist the development of new enzymatic approaches for efficient biomass valorization.