endo-Xylanase Assay Kit (XylX6 Method)
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.
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
(1) 3-Ketobutylidene-GX5-β-PNP + H2O → Blocked-GXY + X(5-Y)-β-PNP
(2) X(5-Y)-β-PNP + H2O → D-xylose + PNP
(3) PNP → phenolate ion (yellow colour)
Note: PNP = 4-nitrophenol
K-XylX6-1V 100 assays (manual) / 200 (auto-analyser)
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
Fermentation broths, industrial enzyme preparations, animal feed, biofuels research, barley
Method recognition: Novel method
- 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.
Novel substrates for the automated and manual assay of endo-1,4-β-xylanase.
A Comparison of Polysaccharide Substrates and Reducing Sugar Methods for the Measurement of endo-1,4-β-Xylanase
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