endo-Xylanase Assay Kit (XylX6 Method)

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The XylX6 assay reagent for the measurement of endo-xylanase (endo-1,4-β-xylanase) contains two components; 1) 4,6-O-(3-Ketobutylidene)-4-nitrophenyl-β-D-45-glucosyl-xylopentaoside and 2) β-xylosidase. The ketone blocking group prevents any hydrolytic action by the β-xylosidase or other exo-acting glycosidases on the XylX6 substrate. Incubation with an endo-xylanase generates a non-blocked colourimetric oligosaccharide that is rapidly hydrolysed by the ancillary β-xylosidase. The rate of formation of 4-nitrophenol is therefore directly related to the hydrolysis of XylX6 by the endo-xylanase. The reaction is terminated and the phenolate colour is developed on addition of Tris buffer solution (pH = 10.0).

Note that standard curves relating the absorbance obtained using the XylX6 assay to endo-xylanase activity on the native substrates, wheat arabinoxylan and beechwood xylan, are provided in the Supporting Information file under the Documentation tab.

endo-Xylanase Assay Kit (XylX6 Method)
Product Code
Content/Size
Stock
Price
Qty
K-XylX6-1V
100 assays (manual) / 200 assays (auto-analyser)
$260.00
K-XylX6-2V
200 assays (manual) / 400 assays (auto-analyser)
$390.00

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Colourimetric method for the determination of endo-1,4-β-xylanase in any
sample type including crude enzyme extracts, fermentation broths or
commercial enzyme preparations

Principle:
                                               (endo-1,4-β-xylanase)
(1) 3-Ketobutylidene-GX5-β-PNP + H2O → Blocked-GXY + X(5-Y)-β-PNP

                             (β-xylosidase)
(2) X(5-Y)-β-PNP + H2O → D-xylose + PNP

       (alkaline solution)
(3) PNP → phenolate ion (yellow colour)
Note: PNP = 4-nitrophenol

Kit size:
K-XylX6-1V                        100 assays (manual) / 200 (auto-analyser)
                                           or
K-XylX6-2V                        200 assays (manual) / 400 (auto-analyser)
Method:                            Spectrophotometric at 400/405 nm
Total assay time:             10 min
Detection limit:                 5.3 x 10-4 U/mL
Application examples: 
Fermentation broths, industrial enzyme preparations, animal feed, biofuels research, barley 
malt analysis
Method recognition:        Novel method

Advantages

  • Very cost effective
     
  • All reagents stable for > 4 years
     
  • Completely specific for endo-1,4-β-xylanase
     
  • Generally applicable and highly sensitive
     
  • Simple format. Well suited to automation
     
  • Excellent reproducibility
     
  • Standard included

Development of an automatable method for the measurement of endo-1,4-β-xylanase activity in barley malt and initial investigation into the relationship between endo-1,4-β-xylanase activity and wort viscosity.

Mangan, D., Cornaggia, C., Liadova, A., Draga, A., Ivory, R., Evans, D. E. & McCleary, B. V. (2018). Journal of Cereal Science, 84, 90-94.
Link to Article
Read Abstract
Stages of the brewing process, such as mash separation to produce wort and beer filtration, can in certain cases prove problematic due to the increased viscosity caused by high levels of the non-starch polysaccharides, primarily β-glucan and arabinoxylan. Of these two polysaccharides, β-glucan has been extensively studied, but arabinoxylan has been somewhat overlooked. The concentration of arabinoxylan present during these process stages is principally expected to be inversely related to the malt endo-1,4-β-xylansase activity that is available to degrade these polysaccharides. The development of a novel method for the measurement of endo-1,4-β-xylansase activity in barley malt extracts is described herein. The method was characterised by two analysts in terms of repeatability (single analyst CVs=2.2% and 2.3%, n=8; interanalyst CV=4.8%, n=16) and sensitivity (LOD=10 U/kg, LOQ=34 U/kg). The assay procedure was then applied to the measurement of xylanase activity in a series of eight standard barley malts and the results obtained were compared with their associated Congress wort viscosities as measured using the conventional EBC Method 4.8, wort viscosity. A highly statistically significant relationship between xylanase activity and wort viscosity was found with a Pearson's correlation coefficient of -0.82 (p-value of 0.007).

Novel substrates for the automated and manual assay of endo-1,4-β-xylanase.

Mangan, D., Cornaggia, C., Liadova, A., McCormack, N., Ivory, R., McKie, V. A., Ormerod, A. & McCleary, D. V. (2017). Carbohydrate Research, 445, 14-22.
Link to Article
Read Abstract
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.

A Comparison of Polysaccharide Substrates and Reducing Sugar Methods for the Measurement of endo-1,4-β-Xylanase

McCleary, B. V. & McGeough, P. (2015). Appl. Biochem. Biotechnol., 177(5), 1152-1163.
Link to Article
Read Abstract
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.

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