|Formulation:||In 3.2 M ammonium sulphate|
|Stability:||> 4 years at 4oC|
|Synonyms:||endo-1,4-beta-xylanase; 4-beta-D-xylan xylanohydrolase|
|Concentration:||Supplied at ~ 10,000 U/mL|
|Expression:||Recombinant from Neocallimastix patriciarum|
|Specificity:||endo-hydrolysis of (1,4)-β-D-xylosidic linkages in xylans.|
~ 600 U/mg (40oC, pH 6.0 on wheat arabinoxylan);
~ 900 U/mg (50oC, pH 6.0 on wheat arabinoxylan)
|Unit Definition:||One Unit of xylanase activity is defined as the amount of enzyme required to release one µmole of xylose reducing-sugar equivalents per minute from wheat arabinoxylan (5 mg/mL) in sodium phosphate buffer (100 mM), pH 6.0.|
|Application examples:||Applications in carbohydrate and biofuels research and in the food and feeds and paper pulping industries.|
High purity recombinant endo-1,4-β-Xylanase (Neocallimastix patriciarum) for use in research, biochemical enzyme assays and in vitro diagnostic analysis.
See our complete list of Carbohydrate Active enZYmes.
(Trichoderma longibrachiatum) E-XYAN4 - endo-1,4-β-Xylanase M4 (Aspergillus niger) E-XYRU6 - endo-1,4-β-Xylanase (rumen microorganism) E-XYNBS - endo-1,4-β-Xylanase
(Bacillus stearothermophilus T6) E-XYNACJ - endo-1,4-β-Xylanase (Cellvibrio japonicus) E-XYLATM - endo-1,4-β-Xylanase (Thermotoga maritima)
Mangan, D., Cornaggia, C., Liadova, A., McCormack, N., Ivory, R., McKie, V. A., Ormerod, A. & McCleary, D. V. (2017). Carbohydrate Research, 445, 14-22.
endo-1,4-β-Xylanase (EC 184.108.40.206) 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.Hide Abstract
McCleary, B. V., McKie, V. A., Draga, A., Rooney, E., Mangan, D. & Larkin, J. (2015). Carbohydrate Research, 407, 79-96.
A range of α-L-arabinofuranosyl-(1-4)-β-D-xylo-oligosaccharides (AXOS) were produced by hydrolysis of wheat flour arabinoxylan (WAX) and acid debranched arabinoxylan (ADWAX), in the presence and absence of an AXH-d3 α-L-arabinofuranosidase, by several GH10 and GH11 β-xylanases. The structures of the oligosaccharides were characterised by GC-MS and NMR and by hydrolysis by a range of α-L-arabinofuranosidases and β-xylosidase. The AXOS were purified and used to characterise the action patterns of the specific α-L-arabinofuranosidases. These enzymes, in combination with either Cellvibrio mixtus or Neocallimastix patriciarum β -xylanase, were used to produce elevated levels of specific AXOS on hydrolysis of WAX, such as 32-α-L-Araf-(1-4)-β-D-xylobiose (A3X), 23-α-L-Araf-(1-4)-β-D-xylotriose (A2XX), 33-α-L-Araf-(1-4)-β-D-xylotriose (A3XX), 22-α-L-Araf-(1-4)-β-D-xylotriose (XA2X), 32-α-L-Araf (1-4)-β-D-xylotriose (XA3X), 23-α-L-Araf-(1-4)-β-D-xylotetraose (XA2XX), 33-α-L-Araf-(1-4)-β-D-xylotetraose (XA3XX), 23 ,33-di-α-L-Araf-(1-4)-β-D-xylotriose (A2+3XX), 23,33-di-α-L-Araf-(1-4)-β-D-xylotetraose (XA2+3XX), 24,34-di-α-L-Araf-(1-4)-β-D-xylopentaose (XA2+3XXX) and 33,34-di-α-L-Araf-(1-4)-β-D-xylopentaose (XA3A3XX), many of which have not previously been produced in sufficient quantities to allow their use as substrates in further enzymic studies. For A2,3XX, yields of approximately 16% of the starting material (wheat arabinoxylan) have been achieved. Mixtures of the α-L-arabinofuranosidases, with specific action on AXOS, have been combined with β-xylosidase and β-xylanase to obtain an optimal mixture for hydrolysis of arabinoxylan to L-arabinose and D-xylose.Hide Abstract
McCleary, B. V. & McGeough, P. (2015). Appl. Biochem. Biotechnol., 177(5), 1152-1163.
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.Hide Abstract
An integrated approach to obtain xylo-oligosaccharides from sugarcane straw: from lab to pilot scale.
Brenelli, L. B., Figueiredo, F. L., Damasio, A., Franco, T. T. & Rabelo, S. C. (2020). Bioresource Technology, 313, 123637.
Sugarcane straw (SS) is a widely available agricultural processing feedstock with the potential to produce 2nd generation bioethanol and bioproducts, in addition to the more conventional use for heat and/or electrical power generation. In this study, we investigated the operational parameters to maximize the production of xylo-oligosaccharides (XOS) using mild deacetylation, followed by hydrothermal pretreatment. From the laboratory to the pilot-scale, the optimized two-stage pretreatment promoted 81.5% and 70.5% hemicellulose solubilization and led to XOS yields up to 9.8% and 9.1% (w/w of initial straw), respectively. Moreover, different fungal xylanases were also tested to hydrolyze XOS into xylobiose (X2) and xylotriose (X3). GH10 from Aspergillus nidulans performed better than GH11 xylanases and the ratio of the desired products (X2 + X3) increased to 72% due to minimal monomeric sugar formation. Furthermore, a cellulose-rich fraction was obtained, which can be used in other high value-added applications, such as for the production of cello-oligomers.Hide Abstract
Kinetics and regioselectivity of three GH62 α-L-arabinofuranosidases from plant pathogenic fungi.
Sarch, C., Suzuki, H., Master, E. R. & Wang, W. (2019). Biochimica et Biophysica Acta (BBA)-General Subjects, 1863(6), 1070-1078.
Backgound: Xylan is the second most abundant plant cell wall polysaccharide after cellulose with α-L-arabinofuranose (L-Araf) as one of the major side substituents. Capacity to degrade xylan is characteristic of many plant pathogens; and corresponding enzymes that debranch arabinoxylan provide tools to tailor xylan functionality or permit its full hydrolysis. Method: Three GH62_2 family α-arabinofuranosidases (Abfs) from plant pathogenic fungi, NhaAbf62A from Nectria haematococca, SreAbf62A from Sporisorium reilianum and GzeAbf62A from Gibberella zeae, were recombinantly produced in Escherichia coli. Their biochemical properties and substrate specificities were characterized in detail. Particularly with 1H NMR, the regioselectivity and debranching preference of the three Abfs were directly compared. Results: The activities of selected Abfs towards arabinoxylan were all optimal at pH 6.5. Their preferred substrates were wheat arabinoxylan, followed by soluble oat spelt xylan. The Abfs displayed selectivity towards either α-(1 → 2) or α-(1 → 3)-L-Araf mono-substituents in arabinoxylan. Specifically, SreAbf62A and GzeAbf62A removed m-α-(1 → 3)-L-Araf and m-α-(1 → 2)-L-Araf substituents with a similar rates, whereas NhaAbf62A released m-α-(1→ 3)-L-Araf 1.9 times faster than m-α-(1 → 2)-L-Araf. Major conclusions: Building upon the known selectivity of GH62 family α-arabinofuranosidases towards L-Araf mono-substituents in xylans, the current study uncovers enzyme-dependent preferences towards m-α-(1 → 3)-L-Araf and m-α-(1 → 2)-L-Araf substitutions. Comparative sequence-structure analyses of Abfs identified an arginine residue in the xylose binding +2R subsite that was correlated to the observed enzyme-dependent L-Araf debranching preferences. General significance: This study expands the limited pool of characterized GH62 Abfs particularly those from plant pathogenic fungi, and provides biochemical details and methodology to evaluate regioselectivity within this glycoside hydrolase family.Hide Abstract
Endo-xylanases as tools for production of substituted xylooligosaccharides with prebiotic properties.
Karlsson, E. N., Schmitz, E., Linares-Pastén, J. A. & Adlercreutz, P. (2018). Applied Microbiology and Biotechnology, 102(21), 9081-9088.
Xylan has a main chain consisting of β-1,4-linked xylose residues with diverse substituents. Endoxylanases cleave the xylan chain at cleavage sites determined by the substitution pattern and thus give different oligosaccharide product patterns. Most known endoxylanases belong to glycoside hydrolase (GH) families 10 and 11. These enzymes work well on unsubstituted xylan but accept substituents in certain subsites. The GH11 enzymes are more restricted by substituents, but on the other hand, they are normally more active than the GH10 enzymes on insoluble substrates, because of their smaller size. GH5 endoxylanases accept arabinose substituents in several subsites and require it in the -1 subsite. This specificity makes the GH5 endoxylanases very useful for degradation of highly arabinose-substituted xylans and for the selective production of arabinoxylooligosaccharides, without formation of unsubstituted xylooligosaccharides. The GH30 endoxylanases have a related type of specificity in that they require a uronic acid substituent in the -2 subsite, which makes them very useful for the production of uronic acid substituted oligosaccharides. The ability of dietary xylooligosaccharides to function as prebiotics in humans is governed by their substitution patterns. Endoxylanases are thus excellent tools to tailor prebiotic oligosaccharides to stimulate various types of intestinal bacteria and to cause fermentation in different parts of the gastrointestinal tract. Continuously increasing knowledge on the function of the gut microbiota and discoveries of novel endoxylanases increase the possibilities to achieve health-promoting effects.Hide Abstract
Mathew, S., Karlsson, E. N. & Adlercreutz, P. (2017). Journal of Biotechnology, 260, 53-61.
The enzymatic, ecofriendly pretreatment of wheat bran with α-amylase from Bacillus amyloliquifaciens or B. licheniformis at 90°C for 1.5 h followed by Neutrase at 50°C for 4 h, aqueous liquefaction at 121°C for 15 h and ethanol precipitationenabled the production of soluble arabinoxylan (AX) with purity of 70.9% and 68.4% (w/w) respectively. Process alternatives tried, to simplify the process and curtail the cost resulted in AX products with different purities, yields and arabinose to xylose ratio (A/X). Among the two glycoside hydrolase (GH) family endoxylanases evaluated, GH10 family hydrolysed soluble AX more efficiently with xylanase from Geobacillus stearothermophilus T-6 (GsXyn10A) producing maximum amount of quantifiable short xylo-oligosaccharides (XOS) and arabinoxylo-oligosaccharides (AXOS) (53% w/w) followed by the catalytic module of Rhodothermus marinus Xyn10A (RmXyn10A-CM) with 37% (w/w) conversion. The GH11 family endoxylanases, from Thermomyces lanuginosus (Pentopan Mono BGTM) and Neocallimastix patriciarum (NpXyn11A) gave conversions of 21% and 22% (w/w) of the soluble AX, respectively (major AXOS products were not quantified). In addition to the XOS formed such as X2, X3, and X4, the AXOS products identified were A3X and A2XX in the case of GsXyn10A and RmXyn10A-CM while Pentopan Mono BG and NpXyn11A produced XA3XX as the major AXOS product.Hide Abstract
Zhang, Z., Smith, C., Li, W. & Ashworth, J. (2016). Journal of Agricultural and Food Chemistry, 64(43), 8128-8137.
The ingestion of foods and food-derived substances that may mediate the immune system is widely studied. Evidence suggests cereal arabinoxylans (AXs) have immunomodulatory activities that may impart health benefits in terms of immune enhancement. This study extracted AXs from corn bran using alkali and developed a modification process using three endoxylanases to obtain fractions of lower molecular weight ranges. In vitro studies showed extracted and modified AXs significantly (P < 0.05) elevated nitric oxide (NO) synthesis by the human U937 monocytic cell line (ranging from 53.7 ± 1.1 to 62.9 ± 1.2 µM per million viable cells) at all concentrations tested (5–1000 µg/mL), indicative of immune enhancement compared to an untreated control (43.7 ± 1.9 µM per million viable cells). The study suggested the dose range and Mw distribution of AXs are key determinants of immune-modulatory activity. AXs in the low Mw range (0.1–10 KDa) were the most effective at inducing NO secretion by U937 macrophages at low AX concentration ranges (5–50 µg/mL /mL), with NO production peaking at 62.9 ± 1.2 µM per million viable cells with 5 µg/mL of AX (P = 0.0009). In contrast, AXs in the high Mw range (100–794 kDa) were most effective at inducing NO at high AX concentration ranges (500–1000 µg/mL /mL) with NO production reaching a maximum of 62.7 ± 1.3 µM per million viable cells at 1000 µg/mL of AX (P = 0.0011). The findings suggest that dietary AXs from corn bran may heighten innate immune responses in the absence of infection or disease.Hide Abstract