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.

Although β-xylosidases have broad applications in fields such as food and medicine, there is limited research on cold-active β-xylosidases. This study cloned a novel cold-active β-xylosidase XYL13 from Parabacteroides distasonis. The purified XYL13 exhibited the highest activity at 40°C, with 42% and 25% of its maximum activity at 4°C and 0°C, respectively. Meanwhile, XYL13 predominantly produces X1 while degrading X2-X6. Additionally, XYL13 showed a significant synergistic effect (18.5-fold) with endo-xylanase for degrading beechwood xylan at low temperatures. Moreover, the site-directed mutagenesis assay indicated that Ile269 and Glu621 are essential catalytic sites of XYL13. Finally, molecular docking showed that XYL13 has an excellent binding effect with X2-X6, verifying that XYL13 can effectively cut X2-X6 to produce xylose. These results highlight the potential of cold-adapted XYL13 from P. distasonis for application in the food industry.

Acetic acid (HAc) pretreatment has been widely used for the production of xylo-oligosaccharides (XOS), requiring harsh reaction conditions because XOS are intermediates during the xylan degradation process. This complexity makes the pretreatment process difficult to regulate. In this study, a pH-controlled HAc pretreatment using sodium hydroxide (NaOH) was proposed to enhance the yield of XOS and to reduce its degree of polymerization (DP) from corncobs (CC). By employing this method (0.3 M-2.7), 49.7% of XOS with DP 2-6 was obtained, alongside a notable increase in the fraction of XOS with DP 2-4 (10.1 g/L). This performance significantly surpassed that of the HAc alone (0.3 M). Moreover, the glucose yield from CC via pH-controlled HAc pretreatment was as high as 93.1% after 72-h enzymatic hydrolysis. These results suggested that the pH-controlled HAc pretreatment could be a promising strategy for the coproduction of low-DP XOS and fermentable sugars.

Nanofiltration was applied to purify steam explosion hydrolysates obtained from Eucalyptus globulus woodchips. The complex hydrolysate comprised xylooligosaccharides, monosaccharides, glucuronic acid (GluA), and degradation products, including acetic acid, furfural, and 5-hydroxymethylfurfural (HMF). Membranes with 750 and 200 Da molecular weight cut-offs were assessed for their ability to retain oligosaccharides while allowing degradation products to permeate. Using the 750 Da membrane, the average compound retention order was: acetic acid (9.8 %) < furfural (10.8 %) < HMF (31.4 %) << arabinose (52.5 %) < xylose (63.8 %) < glucose (76.0 %) < GluA (90.6 %). The separation mechanism for neutral saccharides and furans was primarily molecular sieving, while Donnan exclusion was the primary retention mechanism responsible for GluA. Separation factors obtained for acetic acid and furfural over xylotriose and xylobiose exceeded 10 and 3, respectively, for both membranes, showing that desired compounds were predominantly retained. Permeability loss during nanofiltration was fully restored after implementing cleaning-in-place for both membranes.

Mandelic acid (MA), a natural and environmentally friendly organic acid, demonstrates high selectivity and efficiency in hydrolyzing hemicellulose, making it an excellent candidate for xylooligosaccharides (XOS) production at low acid dosages. Despite its potential, the application of MA for XOS production has not been evaluated. The study first investigated the effectiveness of MA in hydrolyzing hemicellulose in bamboo into XOS. Under optimized conditions (50 mM MA, 180°C, 45 min), a high XOS yield of 65.9% was achieved, with a total xylobiose and xylotriose yield of 43.5%. Subsequent alkaline pretreatment enabled 92.1% lignin removal from MA-pretreated bamboo. The recovered lignin exhibited a high purity of 95.2% and retained fundamental structure and functional groups of native lignin. The resulting residue displayed enhanced crystallinity and accessibility, with reduced hydrophobicity and surface area lignin compared to untreated bamboo. At high substrate concentration of 20%, cellulase hydrolysis resulted in a glucose conversion efficiency of 83.9%. Overall, this integrated strategy offered an efficient approach for the co-production of valuable XOS, lignin, and glucose from bamboo. The efficient energy utilization and economic viability further highlighted the potential of this method for large-scale industrial applications, making it an attractive option for biomass valorization.

Understanding the relation between enzyme domain structure and catalytic activity is crucial for optimal engineering of novel enzymes for lignocellulose bioconversion. Xylanases with varying specificities are commonly used to valorise the hemicellulose arabinoxylan (AX), yet characterization of specific arabinoxylanases remain limited. Two homologous GH5_34 arabinoxylanases, HhXyn5A and CtXyn5A, in which the two domains are connected by a 40-residue linker, exhibit distinct activity on AX, yielding different reaction product patterns, despite high sequence identity, conserved active sites and similar domain composition. In this study, the carbohydrate binding module 6 (CBM6), or the inter domain linker together with CBM6, were swapped to investigate their influence on hydrolytic activity and oligosaccharide product pattern on cereal AXs. The variants, with only CBM6 swapped, displayed reduced activity on commercial wheat and rye AX, as well as on extracted oat fibre, compared to the original enzymes. Additionally, exchange of both linker and CBM6 resulted in a reduced ratio of enzyme produced in soluble form in Escherichia coli cultivations, causing loss of activity of both HhXyn5A and CtXyn5A variants. Analysis of oligosaccharide product patterns applying HPAEC-PAD revealed a decreased number of reaction products for CtXyn5A with swapped CBM6, which resembled the product pattern of HhXyn5A. These findings emphasize the importance of the CBM6 interactions with the linker and the catalytic domain for enzyme activity and specificity, and underlines the role of the linker in enzyme structure organisation and product formation, where alterations in linker interactions with the catalytic and/or CBM6 domains, influence enzyme-substrate association and specificity.

In this study, the family GH10 xylanase AnXylA10 derived from Aspergillus niger JL15 strain was expressed in Pichia pastoris X33. The recombinant xylanase, reAnXylA10 exhibited optimal activity at 40°C and pH 5.0. The hydrolysates generated from beechwood xylan using reAnXylA10 primarily consisted of xylobiose (X2) to xylohexaose (X6) and demonstrated remarkable antioxidant capacity. Furthermore, the rice xylanase inhibitory protein (riceXIP) was observed to competitively inhibit reAnXylA10, exhibiting an inhibition constant (K_i) of 140.6 nM. Molecular dynamics (MD) simulations of AnXylA10-riceXIP complex revealed that the α-7 helix (Q225-S238) of riceXIP intruded into the catalytic pocket of AnXylA10, thereby obstructing substrate access to the active site. Specifically, residue K226 of riceXIP formed robust interactions with E136 and E242, the two catalytic sites of AnXylA10, predominantly through high-occupied hydrogen bonds. Based on QTAIM, electron densities for the atom pairs K226_riceXIP@HZ1-E136_AnXylA10@OE2 and K226_riceXIP@HZ3-E242_AnXylA10@OE1 were determined to be 0.04628 and 0.02914 a.u., respectively. Binding free energy of AnXylA10-riceXIP complex was −59.0±7.6 kcal/mol, significantly driven by electrostatic and van der Waals forces. Gaining insights into the interaction between xylanase and its inhibitors, and mining the inhibition mechanism in depth, will facilitate the design of innovative GH10 family xylanases that are both highly efficient and resistant to inhibitors.

Xylooligosaccharides (XOS) are important oligomers of xylose. XOS have received particular interest in recent years from the food and pharmaceutical industries due to their functional properties associated with fighting diseases and maintaining health. Therefore, the objective of this work was to evaluate the in vitro digestibility, prebiotic, and antioxidant effects, as well as the production of short-chain fatty acids from short-chain XOS obtained from enzymatic hydrolysis of the bamboo culm. The xylan extracted from the bamboo culm was subjected to enzymatic hydrolysis at an endoxylanases concentration of 7.5 mg protein/g xylan at 50 °C and pH 5.0 for 24 h. The extract obtained showed a high concentration of XOS, approximately 5 g/L, composed of xylobiose (2.39 g/L), xylotriose (1.31 g/L), xylotetraose (0.567 g/L), xylopentose (0.223 g/L) and xylohexose (0.18 g/L). The in vitro digestibility tests indicated that the XOS presented a high and desirable resistance to digestion, considering that 97.65% of the XOS remained intact in the simulated intestinal phase. The XOs showed an expressive prebiotic effect, promoting the growth of Lactobacillus acidophilus and Bifidobacterium bifidum, fermenting the XOS and producing short-chain fatty acids, highlighting acetate, propionate, and butyrate. These organic acids play important roles in fighting inflammation, protecting brain functions, and inhibiting the development of cancer cells and pathogens. XOS showed a high total antioxidant capacity, eliminating 73.5% of the DPPH free radicals. Finally, the XOS produced from bamboo culm has great prebiotic potential and excellent antioxidant activity and can be applied to food and pharmaceutical products.

This study aimed to enhance the resistance of bread to gastrointestinal digestion by partially substituting wheat flour with starch spherulites. Three types of starch spherulites, specifically A-type (exhibiting an A-type crystalline pattern with mostly positive birefringence), B(−)-type (B-type crystalline with negative birefringence), and B(+)-type (B-type crystalline with positive birefringence), were investigated. The A-, B(−)-, and B(+)-type spherulites showed significantly higher resistant starch contents of 63.5, 63.8, and 89.2 %, respectively, compared to the control wheat flour (7.4 %). The melting temperatures of A-type and B(+)-type spherulites were notably higher than those of the control wheat flour, suggesting the potential preservation of certain enzyme-resistant starch during the baking process. The partial substitution of wheat flour with spherulites resulted in a denser crumb structure, increased bread hardness and chewiness, and a pale brown color in the case of B(+)-type spherulite. However, these variations in physicochemical properties did not significantly impact consumer acceptability. Remarkably, in bread containing A- or B(+)-type spherulite, residual ordered spherulite structures were present after baking, as confirmed by differential scanning calorimetry. This resulted in significantly lower digestibility during in vitro gastrointestinal digestion. These findings are useful for the rational design of bread with sustained glucose release during gastrointestinal digestion.