Xylan (Beechwood)

endo-1,4-β-Xylanase (EC 3.2.1.8) is employed across a broad range of industries including animal feed, brewing, baking, biofuels, detergents and pulp (paper). Despite its importance, a rapid, reliable, reproducible, automatable assay for this enzyme that is based on the use of a chemically defined substrate has not been described to date. Reported herein is a new enzyme coupled assay procedure, termed the XylX6 assay, that employs a novel substrate, namely 4,6-O-(3-ketobutylidene)-4-nitrophenyl-β-4⁵-O-glucosyl-xylopentaoside. The development of the substrate and associated assay is discussed here and the relationship between the activity values obtained with the XylX6 assay versus traditional reducing sugar assays and its specificity and reproducibility were thoroughly investigated.

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.

There is growing interest in members of the genus Segatella (family Prevotellaceae) as members of a well-balanced human gut microbiota (HGM). Segatella are particularly associated with the consumption of a diet rich in plant polysaccharides comprising dietary fiber. However, understanding of the molecular basis of complex carbohydrate utilization in Segatella species is currently incomplete. Here, we used RNA sequencing (RNA-seq) of the type strain Segatella copri DSM 18205 (previously Prevotella copri CB7) to define precisely individual polysaccharide utilization loci (PULs) and associated carbohydrate-active enzymes (CAZymes) that are implicated in the catabolism of common fruit, vegetable, and grain polysaccharides (viz. mixed-linkage β-glucans, xyloglucans, xylans, pectins, and inulin). Although many commonalities were observed, several of these systems exhibited significant compositional and organizational differences vis-à-vis homologs in the better-studied Bacteroides (sister family Bacteroidaceae), which predominate in post-industrial HGM. Growth on β-mannans, β(1, 3)-galactans, and microbial β(1, 3)-glucans was not observed, due to an apparent lack of cognate PULs. Most notably, S. copri is unable to grow on starch, due to an incomplete starch utilization system (Sus). Subsequent transcriptional profiling of bellwether Ton-B-dependent transporter-encoding genes revealed that PUL upregulation is rapid and general upon transfer from glucose to plant polysaccharides, reflective of de-repression enabling substrate sensing. Distinct from previous observations of Bacteroides species, we were unable to observe clearly delineated substrate prioritization on a polysaccharide mixture designed to mimic in vitro diverse plant cell wall digesta. Finally, co-culture experiments generally indicated stable co-existence and lack of exclusive competition between S. copri and representative HGM Bacteroides species (Bacteroides thetaiotaomicron and Bacteroides ovatus) on individual polysaccharides, except in cases where corresponding PULs were obviously lacking.

In this study, we report the molecular and enzymatic characterisation of Spg103, a novel bifunctional β-glucanase from the marine bacterium Streptomyces sp. J103. Recombinant Spg103 (rSpg103) functioned optimally at 60 °C and pH 6. Notably, Spg103 exhibited distinct stability properties, with increased activity in the presence of Na+ and EDTA. Spg103 displays both lichenase and cellobiohydrolase activity. Despite possessing a GH5 cellulase domain, FN3 and CBM3 domains characteristic of cellulases and CBHs, biochemical assays showed that rSpg103 exhibited higher activity towards mixed β-1,3-1,4-glucan such as barley β-glucan and lichenan than towards beta-1,4-linkages. The endolytic activity of the enzyme was confirmed by TLC and UPLC-MS analyses, which identified cellotriose as the main hydrolysis product. In addition, Spg103 exhibited an exo-type activity, selectively releasing cellobiose units from cellooligosaccharides, which is characteristic of cellobiohydrolases. These results demonstrate the potential of Spg103 for a variety of biotechnological applications, particularly those requiring tailor-made enzymatic degradation of mixed-linked β-glucans. This study provides a basis for further structural and functional investigations of the bifunctional enzyme and highlights Spg103 as a promising candidate for industrial applications.

Thermophilic xylanases catalyzing the cleavage of β-1,4-glycosidic bonds in xylan have applications in food, feed, biorefinery, and pulp industries. In this study, a hyperthermophilic endo-xylanase was obtained by further enhancement of thermal tolerance of a thermophilic GH11 xylanase originated from metagenome of bagasse pile based on rational design. Introducing N13F and Q34L to the previously reported X11P enzyme shifted the optimal working temperature to 85°C and led to 20.7-fold improvement in thermostability at 90°C along with a marked increase in T_m to 93.3°C. X11PNQ enzyme converted xylan to prebiotic xylooligosaccharides with high specificity on xylobiose to xylohexaose and high operational stability at 85°C, resulting in 10.3-folds yield improvement compared to the parental enzyme. Molecular dynamic simulation and quantum mechanical analysis revealed improved H-bonding networks within GH11 xylanase principal domains and greater dynamic cross-correlations. A novel thermostabilization mechanism by π-amide interaction with slightly lower interaction energy than the native H-bond, but compensated by increased occurrence frequency was firstly demonstrated for thermophilic enzymes. The enzyme represents one of the most thermostable xylanases ever reported with biotechnological potential.

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.

Few aerobic hyperthermophilic microorganisms degrade polysaccharides. Here, we describe the genome-enabled enrichment and optical tweezer-based isolation of an aerobic polysaccharide-degrading hyperthermophile, Fervidibacter sacchari, previously ascribed to candidate phylum Fervidibacteria. F. sacchari uses polysaccharides and monosaccharides for growth at 65-87.5 °C and expresses 191 carbohydrate-active enzymes (CAZymes) according to RNA-Seq and proteomics, including 31 with unusual glycoside hydrolase domains (GH109, GH177, GH179). Fluorescence in-situ hybridization and nanoscale secondary ion mass spectrometry confirmed rapid assimilation of ¹³C-starch in spring sediments. Purified GHs were optimally active at 80-100 °C on ten different polysaccharides. Finally, we propose reassigning Fervidibacteria as a class within phylum Armatimonadota, along with 18 other species, and show that a high number and diversity of CAZymes is a hallmark of the phylum, in both aerobic and anaerobic lineages. Our study establishes Fervidibacteria as hyperthermophilic polysaccharide degraders in terrestrial geothermal springs and suggests a broad role for Armatimonadota in polysaccharide catabolism.

Xylanases require thermal stability to withstand the pelleting process, pH stability to function in the gastrointestinal tract, and resistance to xylanase inhibitors in raw materials to be effective in animal feed. A GH11 family xylanase originating from an anaerobic fungus, Orpinomyces sp. strain PC-2, has high specific activity and resistance to xylanase inhibitors intrinsically. It was engineered using rational protein design methods to obtain a thermal and pH stable enzyme, OXynA-M. OXynA-M showed resistance to three types of xylanase inhibitors, Triticum aestivum xylanase inhibitors TAXI-IB and TAXI-IIA and xylanase inhibitor protein XIP and showed melting temperature of 87.2°C when measured using differential scanning calorimetry. It was stable at all pH between 2.0–10.0 incubated up to 4 h. Xylo-oligosaccharides (XOS) production profile using a wheat arabinoxylan substrate revealed the production of xylobioses up to xylohexaoses, which are known to have prebiotic functionalities. An animal trial was conducted in broiler chickens to evaluate the in vivo efficacy of the xylanase. In total, 600 1-day-old chickens were divided into six dietary treatments, including a positive control (PC) (T1) without the addition of exogenous enzyme and the rest where exogenous xylanase was added at the rates of 1200, 2400, 4800, 9600, and 240000 U/kg of feed from T2–T6. An increase in OXynA-M xylanase improved the performance parameters in the enzyme-treated groups compared with the control. The viscosity of ileal digesta decreased with increasing enzyme dosage. A significantly lower viscosity of 6.54 cP was determined for the minimum dose in T2 (1200 U/kg), and the viscosity was further reduced in T6 (240000 U/kg) (P<0.05) compared to the control treatment. The apparent ileal digestibility of crude protein, fat, and starch improved as the xylanase dosage increased. The application of OXynA-M xylanase improved the apparent ileal digestibility of crude protein when the dose was higher than that of T2 (1200 U/kg). Furthermore, the AME_n of the diets improved when xylanase was supplemented at a rate of 9600 U/kg (T5) compared with the control treatment (P<0.05).

Enzymatic hydrolysis of arabinoxylan is of cost-effective strategy to yield valuable macromolecules, e.g., xylooligosaccharides (XOS). A novel halophilic GH10 xylanase (TaXYL10) from Trichoderma asperellum ND-1 was over-expressed in Pichia pastoris and migrated as a single band (~36 kDa) in SDS-PAGE. TaXYL10 displayed >80 % activity in the presence of 4.28 M NaCl and 10 % ethanol. Moreover, TaXYL10 exhibited optimal activity at pH 6.0 and 55 °C, and remarkable pH stability (>80 % activity at pH 4.0–6.0). K⁺ and Al³⁺ could remarkably promote TaXYL10 activity, while the presence of 10 mM Fe²⁺, Zn²⁺, Cu²⁺ and Fe³⁺ decreased its activity. TaXYL10 possesses the highest catalytic activity towards beechwood xylan. TLC analysis revealed that it could rapidly degrade xylan and XOS with DP ≥ 3, yielding xylotriose and xylobiose. Site-directed mutagenesis indicated that Glu¹⁵⁴ and Glu²⁵⁹ are crucial active residues for TaXYL10, while Asp²⁹⁵ and Glu⁶⁹ played auxiliary roles in xylan hydrolysis. Additionally, TaXYL10 acted cooperatively with a commercial α-L-arabinofuranosidase (AnAra) towards arabinoxylan degradation (583.5 μg/mL), a greater synergy degree of 1.79 was obtained after optimizing enzymatic ratios. This work not only expands the diversity of Trichoderma GH10 xylanases, but also reveals the promising potential of TaXYL10 in various industrial applications.

The primary plant cell wall (PCW) is a specialized structure composed predominantly of cellulose, hemicelluloses and pectin. While the role of cellulose and hemicelluloses in the formation of the PCW scaffold is undeniable, the mechanisms of how hemicelluloses determine the mechanical properties of PCW remain debatable. Thus, we produced bacterial cellulose–hemicellulose hydrogels as PCW analogues, incorporated with hemicelluloses. Next, we treated samples with hemicellulose degrading enzymes, and explored its structural and mechanical properties. As suggested, difference of hemicelluloses in structure and chemical composition resulted in a variety of the properties studied. By analyzing all the direct and indirect evidences we have found that glucomannan, xyloglucan and arabinoxylan increased the width of cellulose fibers both by hemicellulose surface deposition and fiber entrapment. Arabinoxylan increased stresses and moduli of the hydrogel by its reinforcing effect, while for xylan, increase in mechanical properties was determined by establishment of stiff cellulose–cellulose junctions. In contrast, increasing content of xyloglucan decreased stresses and moduli of hydrogel by its weak interactions with cellulose, while glucomannan altered cellulose network formation via surface deposition, decreasing its strength. The current results provide evidence for structure–dependent mechanisms of cellulose–hemicellulose interactions, suggesting the specific structural role of the latter.