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Xylanase Assay Kit (XylX6 Method)

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endo-Xylanase Assay Kit XylX6 Method K-XylX6 Scheme
   
Product code: K-XylX6-2V

Content:

€312.00

200 assays (manual) / 400 assays (auto-analyser)

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Content: (K-XylX6-1V)
100 assays (manual) / 200 assays (auto-analyser)
(K-XylX6-2V)
200 assays (manual) / 400 assays (auto-analyser)
Shipping Temperature: Ambient
Storage Temperature: Short term stability: 2-8oC,
Long term stability: See individual component labels
Stability: > 2 years under recommended storage conditions
Analyte: endo-1,4-β-Xylanase
Assay Format: Spectrophotometer, Auto-analyser
Detection Method: Absorbance
Wavelength (nm): 400,
405  
Signal Response: Increase
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).

Need to measure other enzyme activities? View our complete list of assay kits for measurement of enzyme activity.

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.

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
Documents
Certificate of Analysis
Safety Data Sheet
FAQs Booklet Data Calculator Supporting Information Validation Report
Publications
Megazyme publication

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, 125(3), 294-309.

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.

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Megazyme publication

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.

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).

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Megazyme publication
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.

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.

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Megazyme publication
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.

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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Publication

Synbiotic Effects of Enzyme and Probiotics on Intestinal Health and Growth of Newly-Weaned Pigs Challenged with Enterotoxigenic F18+ E. coli. Escherichia coli.

Kim, S. W., Tyus, J. & Duarte, M. E. (2020). Frontiers in Veterinary Science, 7, 573.

This study aimed to investigate the effect of dietary supplementation with xylanase and probiotics on growth performance and intestinal health of nursery pigs challenged with enterotoxigenic Escherichia coli (ETEC). Sixty-four newly weaned pigs (32 barrows and 32 gilts with 7.9 ± 0.4 kg BW) were allotted in a randomized complete block design (2 × 2 factorial). Two factors were ETEC challenge (oral inoculation of saline solution or E. coli F18+ at 6 × 109 CFU) and synbiotics (none or a combination of xylanase 10,000 XU/kg and Bacillus sp. 2 × 108 CFU/kg). All pigs were fed experimental diets following NRC (2012) in two phases (P1 for 10 d and P2 for 11 d). The ETEC was orally inoculated on d 7 after weaning. Feed intake and BW were measured on d 7, 10, 15, and 20. On d 20, pigs were euthanized to collect samples to measure gut health parameters and microbiome. Synbiotics increased (P < 0.05) ADG in phase 1 and ETEC reduced (P < 0.05) ADG and G:F in the post-challenge period. ETEC increased (P < 0.05) the fecal score of pigs from d 7 to 13; however, synbiotics reduced (P < 0.05) it at d 9 and 11 in challenged pigs. ETEC increased (P < 0.05) mucosal MDA, IL-6, Ki-67+, and crypt depth, whereas synbiotics tended to reduce TNFα (P = 0.093), protein carbonyl (P = 0.065), and IL-6 (P = 0.064); reduced (P < 0.05) crypt depth and Ki-67+; and increased (P < 0.05) villus height. ETEC reduced (P < 0.05) the relative abundance of Bacteroidetes and Firmicutes and increased (P < 0.05) the relative abundance of Proteobacteria. In conclusion, ETEC challenge reduced growth performance by affecting microbiome, immune response, and oxidative stress in the jejunum. Synbiotics enhanced growth performance by reducing diarrhea, immune response, and oxidative stress in the jejunum.

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Safety Information
Symbol : Not Applicable
Signal Word : Not Applicable
Hazard Statements : Not Applicable
Precautionary Statements : Not Applicable
Safety Data Sheet
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