Megazyme S-AXBP 木聚糖酶特殊检测底物
英文名：Azo-Xylan (Birchwood)(powder)
货号：S-AXBP
规格：3 g
品牌： Megazyme
用于对- 1 ,4-β-D-木聚糖酶的具体检测.
外观：深蓝色粉末或液体
溶解性：易溶于水
pH值：5.5
气味：无
在室温下稳定，五年或五年以上
成分：
名称 比例
偶氮木聚糖（桦木）Azo-Xylan (Birchwood) 98％
Azo-Xylan (Birchwood)
Substrate for the specific assay of endo-1,4-?-D-xylanase.
Catalogue Number: S-AXBL
Content: 100 mL
Appearance Dark blue powder or liquid.
Specific Gravity Not applicable
Solubility in Water Soluble
pH Value ~ 5.5
Odour None
Form Powder
Stability Stable at room temperature for five or more years
Ingredients Name CAS Proportion
Azo-Xylan (Birchwood) 98%
PRINCIPLE:
This assay procedure is specific for endo-1,4-?-D-xylanase activity.
On incubation of Azo-Xylan (birchwood) with endo-xylanase, the
substrate is depolymerised to produce low-molecular weight dyed
fragments which remain in solution on addition of ethanol to the
reaction mixture. High-molecular weight material is removed by
centrifugation, and the colour of the supernatant is measured. endo-
Xylanase in the sample solution is determined by reference to a
standard curve.
SUBSTRATE:
Birchwood xylan is first purified (to remove starch) and then it is
dyed with Remazolbrilliant Blue R to an extent of approx. one dye
molecule per 30 sugar residues.
DISSOLUTION:
Add 1 g of powdered substrate to 80 mL of boiling and vigorously
stirring water on a hot-plate stirrer. Turn the heat off and continue
stirring until the polysaccharide is completely dissolved (about
20 min). Adjust the volume to 100 mL and add 0.02 g of sodium
azide and dissolve. Store this solution at 4°C between use. Under
these conditions the solution is stable for 12 months if contamination
with enzyme is avoided. Shake the solution container before
removing aliquots for assays. Because the solution is viscous, it should
preferably be dispensed with a positive displacement dispenser
(eg. Eppendorf Multipette? with a 5.0 mL Combitip).
PRECIPITANT SOLUTION:
Industrial methylated spirits (IMS; 95 % v/v) or ethanol (95 % v/v).
BUFFER SOLUTIONS:
1. Sodium acetate buffer, 100 mM, pH 4.5
Add 6.0 g of glacial acetic acid (1.05 g/mL) to 800 mL of distilled
water. Adjust the pH to 4.5 with 5 M (20 g/100 mL) sodium
hydroxide solution. Adjust the volume to 1 litre. Stable for approx.
4 weeks at 4°C.
2. Sodium phosphate buffer, 100 mM, pH 6.0
Add 8.9 g of di-sodium hydrogen phosphate dihydrate
(Na2HPO4?2H2O) to 450 mL of distilled water and dissolve.
Adjust the pH to 6.0 with 1 M hydrochloric. Adjust the volume to
500 mL and add 0.1 g of sodium azide as a preservative. Stable for
approx. 4 weeks at 4°C.
1
ENZYME EXTRACTION AND DILUTION:
Using a positive displacement dispenser, transfer 1.0 mL of liquid
enzyme preparation to 49 mL of buffer 1 (100 mM sodium acetate
buffer, pH 4.5) or buffer 2 (100 mM sodium phosphate buffer, pH 6.0)
and mix thoroughly. This is termed the Original Extract. Dilute
this solution 10-fold by transferring 1.0 mL of diluted enzyme to
9.0 mL of either buffer 1 or buffer 2. Repeat this process until a
dilution of enzyme suitable for assay is obtained.
For powdered enzyme preparations, add 1.0 g of material to 50 mL of
buffer 1 or buffer 2 and gently stir the slurry for 15 min, or until the
sample is completely dispersed or dissolved. Clarify this solution (the
Original Extract) by centrifugation at 1,000 g for 10 min, or by
filtration through Whatman No. 1 (9 cm) filter circles. Dilute this
solution as for the liquid enzyme preparations.
ASSAY PROCEDURE:
Add 0.5 mL of buffered enzyme preparation (pre-equilibrated to
40°C) to 0.5 mL of pre-equilibrated substrate solution (1 % w/v
Azo-Xylan birchwood) with thorough mixing on a vortex stirrer.
Immediately return the mixture to the water bath and incubate at
40°C for exactly 10 min from the time of addition of the enzyme
solution. Terminate the reaction by adding 2.5 mL of ethanol
(95 % v/v) with vigorous stirring on a vortex mixer to the reaction
solution. This will precipitate high-molecular weight, non-hydrolysed
substrate. Store the incubation tubes at room temperature for 5 min
and stir them again. Centrifuge the tubes at 1,000 g (approx. 3,000
rpm for 10 min). Pour the supernatant solution directly into a
spectrophotometer cuvette and measure the absorbance of the blank
and reaction solutions at 590 nm against water. Determine the
activity by reference to a standard curve. Alternatively, enter the
absorbance values into the appropriate MegaCalcTM available from the
Megazyme website (www.megazyme.com).
Prepare a reaction blank by adding 2.5 mL of ethanol (or IMS) to
0.5 mL of the substrate solution (1 % w/v) with vigorous stirring.
Immediately add 0.5 mL of the enzyme solution and stir the mixture
vigorously for 10 sec. Because the diluted enzyme preparations are
essentially colourless, a single blank only, is required with each set of
determinations. Typically, blank absorbance values at 590 nm
are ~ 0.07.
2
A standard curve for A. niger endo-b-xylanase (pH optima 4.5) is
shown in Figure 1. Curves for Humicola insolens and Trichoderma
longibrachiatum endo-b-xylanases (pH optima 6.0) are shown in Figures
2 and 3. In each case, the activity of the enzyme preparations
employed were determined using wheat arabinoxylan (Lot 20401)
(10 mg/mL) as substrate, in either 100 mM sodium acetate buffer
(pH 4.5) or sodium phosphate buffer (pH 6.0). The Nelson-Somogyi
reducing sugar method, with D-xylose as standard, was used to
measure activity.
One unit of enzyme activity is defined as the amount of
enzyme required to release one μmole of D-xylose reducingsugar
equivalents from arabinoxylan, at pH 4.5 (or pH 6.0)
per minute at 40°C.
另有爱尔兰可溶性呈色底物如下:
Azo-Barley Glucan 100124000 麦芽粉中葡聚糖酶检测底物 100 ml S-ABG100
Azo-Carob Galactomannan 100124001 甘露聚糖酶检测底物 4g S-ACGLM
Azo-Casein (Sulphanilamide Dyed) 100124002 蛋白内切酶活性高灵敏度检测可溶性底物[相比同类产品其灵敏度高5倍] 10g S-AZCAS
Azo-CM-Cellulose 100124003 葡聚糖酶检测底物 100ml S-ACMCL
Azo-CM-Cellulose 100124004 葡聚糖酶检测底物 4g S-ACMCL
Azo-Fructan 100124005 内切型菊粉酶检测底物 5g S-AZFR5
Azo-Fructan plus exo-Inulinase 100124006 内切型菊粉酶特殊检测底物 2g+4 KU S-AZFRXOI
Azo-Galactan(Potato) 100124007 半乳聚糖酶特殊检测底物 4g S-AGALP
Azo-Wheat Arabinoxylan (Azo-Waz) 100124008 木聚糖酶检测底物 100ml S-AWAXL
Azo-Wheat Arabinoxylan (Azo-Waz) 100124009 木聚糖酶检测底物 3g S-AWAXP
Azo-Xylan(Birchwood) 100124010 木聚糖酶特殊检测底物 100ml S-AXBL
Azo-Xylan(Birchwood) 100124011 木聚糖酶特殊检测底物 3g S-AXBP
Azo-Xylan(Oat Spelt) 100124012 木聚糖酶特殊检测底物 3g S-AXYO
Azo-Xyloglucan(Tamarind) 100124013 纤维素酶可溶性发色检测底物 4g S-AZXG
AZ-Rhamnogalacturonan 100124014 鼠李聚糖半乳糖醛酸内切酶检测底物 2g S-AZXG
Red Debranched Arabinan(Sugar Beet) 100124015 阿拉伯聚糖检测底物 2g S-AZRH
Red Pullulan 100124016 酶制备中的普鲁兰酶和麦粉中的极限糊精的检测底物 3g S-RDAR
Red Starch 100124017 Alpha淀粉酶活性检测底物 5g S-RPUL