200 assays (manual) / 400 assays (auto-analyser)
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100 assays (manual) / 200 assays (auto-analyser)
200 assays (manual) / 400 assays (auto-analyser)
Short term stability: 2-8oC,
Long term stability: See individual component labels
|Stability:||> 2 years under recommended storage conditions|
|Assay Format:||Spectrophotometer, Auto-analyser|
|Limit of Detection:||5.3 x 10-4 U/mL|
|Reproducibility (%):||~ 3%|
|Total Assay Time:||10 min|
|Application examples:||Fermentation broths, industrial enzyme preparations, animal feed, biofuels research, barley malt analysis.|
|Method recognition:||Novel 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 Documents tab.
- 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
(Trichoderma longibrachiatum) E-XYLAA - endo-1,4-β-Xylanase (Aspergillus aculeatus) E-XYAN4 - endo-1,4-β-Xylanase M4 (Aspergillus niger) E-XYRU6 - endo-1,4-β-Xylanase (rumen microorganism) E-XYNAP - endo-1,4-β-Xylanase (Aeromonas punctata) E-XYNBS - endo-1,4-β-Xylanase
(Bacillus stearothermophilus T6) E-XYNACJ - endo-1,4-β-Xylanase (Cellvibrio japonicus) E-XYNBCM - endo-1,4-β-Xylanase (Cellvibrio mixtus) E-XYLATM - endo-1,4-β-Xylanase (Thermotoga maritima) E-BXSR-1KU - β-D-Xylosidase (Selenomonas ruminantium) E-BXSEBP - β-Xylosidase (Bacillus pumilus)
Prediction of potential malt extract and beer filterability using conventional and novel malt assays.
Cornaggia, C., Evans, D. E., Draga, A., Mangan, D. & McCleary, B. V. (2019). Journal of Institute of Brewing, In Press.
Colourimetric assays were used to measure the activities of six key hydrolases endogenous to barley: β‐glucanase, xylanase, cellulase, α-amylase, beta‐amylase and limit dextrinase. The analysed barley malt samples were previously characterised by 27 conventional malt quality descriptors. Correlations between enzymatic activities and brewing parameters such as extract yield, fermentability, viscosity and filterability were investigated. A single extraction protocol for all six hydrolases was optimised and used for multi‐enzyme analysis using fully automatable assay formats. A regression analysis between malt parameters was undertaken to produce a relationship matrix linking enzyme activities and conventional malt quality descriptors. This regression analysis was used to inform a multi‐linear regression approach to create predictive models for extract yield, apparent attenuation limit, viscosity and filterability using the Small‐scale Wort rapId Filtration Test (SWIFT) and two different mashing protocols – Congress and a modified infusion mash at 65oC (MIM 65oC). It was observed that malt enzyme activities displayed significant correlations with the analysed brewing parameters. Both starch hydrolases and cell wall hydrolase activities together with modification parameters (i.e. Kolbach index) were found to be highly correlated with extract yield and apparent attenuation limit. Interestingly, it was observed that xylanase activity in malts was an important predictor for wort viscosity and filterability. It is envisaged that the automatable measurement of enzyme activity could find use in plant breeding progeny selection and for routine assessment of the functional brewing performance of malt batches. This analytical approach would also contribute to brewing process consistency, product quality and reduced processing times.Hide Abstract
Development of an automatable method for the measurement of <i>endo</i>-1,4-β-xylanase activity in barley malt and initial investigation into the relationship between <i>endo</i>-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). <i>Journal of Cereal Science</i>, 84, 90-94.
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 <i>endo</i>-1,4-β-xylansase activity that is available to degrade these polysaccharides. The development of a novel method for the measurement of <i>endo</i>-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).Hide Abstract
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 18.104.22.168) 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. & 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