|Product Overview:||The Xylanase (Azo-Wax) test kit is suitable for the measurement and analysis of endo-1,4-β-D-xylanase in enzyme preparations, bread improver mixtures and animal feeds. Containing Azo-wheat arabinoxylan and a Trichoderma sp. xylanase control.|
|Kit Components:||1. Azo-wheat arabinoxylan (100 mL, 1% w/v).
2. Trichoderma sp. control xylanase [3700 milli-Units (mU)/mL; pH 6.0 and 40°C] in 50% glycerol.
|Materials Required but Not Supplied:||EXTRACTION BUFFERS (not enclosed):
(A) Sodium acetate buffer (100 mM, pH 4.7)
Add 6.0 g of glacial acetic acid (1.05 g/mL) to 800 mL of distilled water. Adjust the pH to 4.7 with 2 M (8 g/100 mL) sodium hydroxide solution, and the volume to 1 litre. Add 0.2 g of sodium azide and dissolve. Stable at room temperature for > 12 months.
(B) MES buffer (100 mM) plus SDS (1% w/v)
Add 19.5 g of MES free acid (Sigma M-8250) to 800 mL of distilled water and dissolve. Adjust the pH to 6.0 with 1 M sodium hydroxide. Add 10 g of SDS (sodium lauryl sulphate; Sigma L-4509) and dissolve. Adjust the volume to 1 litre and add 0.2 g of sodium azide and dissolve. Stable at room temperature for > 12 months.
1. Glass test tubes (round bottomed; 16 x 100mm and 16 x 120mm).
2. Micro-pipettor, e.g. Gilson Pipetman® (200 μL and 500 μL).
3. Positive displacement pipettor e.g. Eppendorf Multipette®- with 5.0 mL Combitip® (to dispense 0.5 mL aliquots of Azo-WAX solution).
- with 25 mL Combitip® (to dispense 2.5 mL aliquots of IMS or ethanol).
4. Analytical balance.
5. Spectrophotometer set at 590 nm.
6. Vortex mixer (e.g. IKA® Yellowline Test Tube Shaker TTS2).
7. Stop clock.
8. Whatman No.1 (9 cm) filter papers.
|Detection method:||Based on use of Azo-WAX reagent (590 nm)|
|Compatible Sample Types:||Animal feeds, enzyme preparations, bread improver mixtures and other materials|
|Assay Protocol:||EXTRACTION AND ASSAY OF XYLANASE IN FEED SAMPLES:
Trichoderma sp. Xylanases:
1. Mill feed samples (approx. 50 grams) to pass a 0.5 mm screen and mix thoroughly.
2. Weigh 0.5 g ± (0.01 g) of each sample in duplicate into glass testtubes (16 x 120 mm).
3. Add 5 mL of 100 mM sodium acetate buffer (pH 4.7) to each sample and stir on a vortex mixer. Add 0.2 mL of water to one tube and 0.2 mL of xylanase (~ 740 mU) to the other. Immediately stir the slurries on a vortex mixer.
4. Incubate the slurries at room temperature and stir occasionally over the following 20 min.
5. Centrifuge the tubes at 1,500 g for 10 min in a bench centrifuge and use the supernatant directly in assays. Start assays within 30 min of obtaining these extracts to minimise loss of enzyme activity.
1. Accurately transfer 0.5 mL aliquots of supernatant solutions (in duplicate) to glass test-tubes (16 x 100 mm) at room temperature.
2. Add 0.5 ml of Azo-WAX (1% w/v) to each tube and stir the tube vigorously. Immediately place the tubes in a water bath and incubate at 50°C ± 0.1°C for exactly 30 min.
3. Add 2.5 mL of IMS or ethanol and stir the tube vigorously on a vortex mixer. Store the tube at room temperature for 5 min. 3 This treatment terminates the reaction and precipitates non-depolymerised dyed substrate.
4. Centrifuge the tubes at 1,500 g for 10 min.
5. Measure the absorbance of the supernatant solutions at 590 nm against a reaction blank.
Prepare a Reaction Blank by adding 2.5 mL of IMS or ethanol to a mixture of 0.5 mL of Azo-WAX and 0.5 mL of 100 mM sodium acetate buffer (pH 4.7). Stir the slurry and store at room temperature for 5 min before centrifugation (1,500 g, 10 min). A single reaction blank is required for each feed sample.
|Assay time:||~ 45 min|
|Analysis:||The level of xylanase in the flour sample is calculated as follows:
Activity in feed sample (0.5 g) = Added activity x [SA/(TA - SA)]
Added activity = the amount of xylanase added to the feed slurry at the time of assay e.g.: 740 mU in the control xylanase solution (0.2 mL).
SA = the reaction absorbance obtained for extracts of the feed to which no control xylanase was added.
TA = the total absorbance, i.e. the absorbance of extracts of the sample to which the control xylanase was added.
|Sensitivity:||0.2 U/mL of assay solution|