Enzymatic Assay of XYLANASE (EC 3.2.1.8) Principle

Enzymatic Assay of XYLANASE
(EC 3.2.1.8)
PRINCIPLE:
Xylan + H2O
CONDITIONS:
METHOD:
Xylanase
> Reducing Sugar (measured as Xylose)
T = 30 C, pH = 4.5, A540nm, Light path = 1 cm
Colorimetric
REAGENTS:
A.
50 mM Sodium Acetate Buffer, pH 4.5 at 30 C

(Prepare 50 ml in deionized water using Sodium
Acetate, Trihydrate, Sigma Prod. No. S-8625. Adjust
to pH 4.5 at 30 C with 1 M HCl.)
B.
1.0% (w/v) Xylan Substrate Solution (Xylan)

(Prepare 5 ml in Reagent A using Xylan, Sigma
Prod. No. X-0627.)
C.
0.05% (w/v) Bovine Serum Albumin (Enz Diluent)

(Prepare 25 ml in Reagent A using Albumin, Bovine,
Sigma Prod. No. A-4503.)
D.
Xylanase Enzyme Solution

(Immediately before use, prepare a solution containing
5 - 10 units/ml of Xylanase in cold Reagent C.)
E.
16 mM Copper Sulfate, 1.3 M Sodium Sulfate,

226 mM Sodium Carbonate, 190 mM Sodium Bicarbonate and
43 mM Sodium Potassium Tartrate Solution (Copper Soln)
(Prepare 1 liter in deionized water using Cupric
Sulfate Pentahydrate, Sigma Prod. No. C-7631, Sodium
Bicarbonate, Sigma Prod. No. S-8875, Sodium Sulfate,
Anhydrous, Sigma Prod. No. S-9627, Sodium Carbonate,
Anhydrous, Sigma Prod. No. S-2127, and Sodium
Potassium Tartrate Tetrahydrate, Sigma Prod. No. S2377.)1
F.
40 mM Molybdic Acid, 19 mM Arsenic Acid and

756 mM Sulfuric Acid Solution (Ars-Mol)
(Prepare 1 liter in deionized water using Molybdic
Acid, Ammonium Salt Tetrahydrate, Sigma Prod. No. M0878, Arsenic Acid, Sodium Salt, Sigma Prod. No. A6756 and Sulfuric Acid, Sigma Prod. No. S-1526.)2
Revised:
04/04/94
Page 1 of 4

(EC 3.2.1.8)
REAGENTS: (continued)
G.
1 mg/ml Xylose Standard Solution (Xylose Std)

(Prepare 10 ml in deionized water using D(+)Xylose,
Sigma Prod. No. X-1500.)
PROCEDURE:
Pipette (in milliliters) the following reagents into
suitable containers:
Test
Blank
Std 1
Std 2
Std 3
Std 4
Std
Blank
Reagent B (Xylan)
1.90
Reagent G (Xylose Std) ---Reagent C (Enz Diluent) ----
1.90
-------
---0.02
1.98
---0.05
1.95
---0.07
1.93
---0.10
1.90
------2.00
Mix by swirling and equilibrate to 30 C.

Reagent D (Enzyme Soln) 0.10
Reagent C (Enz Diluent) ----
---0.10
-------
-------
Then add:
-------
-------
-------
Mix by swirling and incubate at 30 C for exactly 10

minutes. Then add:
Reagent E (Copper Soln) 2.00
2.00
2.00
2.00
2.00
2.00
2.00
Mix by swirling. Place a marble over the top of the tubes

and transfer the tubes to a boiling water bath. Incubate
for 10 minutes. Remove the tubes from the boiling water
bath and allow to cool to room temperature. Then add:
Reagent F (Ars-Mol)
2.00
2.00
2.00
2.00
2.00
2.00
2.00
Shake or vortex the tubes until foaming stops and any

precipitate present is dissolved. Centrifuge to clarify.
Transfer the solutions to suitable cuvettes. Obtain the
A540nm for Test, Blank and Standards, using a suitable
spectrophotometer.
Revised:
04/04/94
Page 2 of 4

(EC 3.2.1.8)
CALCULATIONS:
Standard Curve:
A540nm Std = A540nm Std - A540nm Std Blank
Prepare a standard curve by plotting the A540nm Standard vs
the moles of Xylose.
Sample Determination:
A540nm Sample = A540nm Test - A540nm Blank
Determine the moles of xylose using the Standard Curve.
(moles of xylose liberated)(df)
Units/ml enzyme =
(10) (0.1)
df = Dilution factor
10 = Time of assay (in minutes) as per Unit Definition
0.1 = Volume (in milliliters) of enzyme used
units/ml enzyme
Units/mg solid =
mg solid/ml enzyme
units/ml enzyme
Units/mg protein =
mg protein/ml enzyme
UNIT DEFINITION:
One unit will liberate 1.0 mole of reducing sugar
measured as xylose equivalents from xylan (X-0627) per
minute at pH 4.5 at 30 C.
FINAL ASSAY CONCENTRATIONS:
In a 2.00 ml reaction mix, the final concentrations are
50 mM sodium acetate, 0.95% (w/v) xylan, 0.003% (w/v)
bovine serum albumin, and 0.5 - 1.0 unit xylanase.
Revised:
04/04/94
Page 3 of 4

(EC 3.2.1.8)
REFERENCE:
Chen, W.P., Matsuo, M., and Yasui, T. (1986) Agric. Biol.
Chem. 50, 1183-1194.
NOTES:
1.
Sodium Sulfate, Sodium Carbonate, and Sodium Potassium

Tartrate are dissolved in approximately 500 ml of
deionized water. Cupric Sulfate is dissolved in
approximately 100 ml of deionized water and is slowly
added to the above solution to avoid precipitation.
Sodium Bicarbonate is dissolved first in deionized
water and then added to the above solution. Dilute
the solution to 1 liter. If a precipitate forms, it
should be removed by filtration prior to use. Store
in an amber bottle and avoid exposure to direct
sunlight. Store at room temperature.
2.
Molybdic Acid is dissolved in approximately 300 ml of

deionized water. Add Sulfuric Acid slowly. Caution,
this is an exothermic reaction! Arsenic Acid is
dissolved in approximately 300 ml of deionized water
and is added to the above solution. The solution is
diluted to a total volume of 1 liter and incubated at
37 C for
48 - 72 hours. If a precipitate forms, it should be
removed by filtration prior to use. Store in an amber
bottle and avoid exposure to direct sunlight. The
solution expires six months after preparation. Store
at room temperature in an exhaust hood.
3.
This assay is based on the cited reference.
4.
Where Sigma Product or Stock numbers are specified,

equivalent reagents may be substituted.
This procedure is for informational purposes. For a current copy of Sigmas quality control
procedure contact our Technical Service Department.
Revised:
04/04/94
Page 4 of 4

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Enzymatic Assay of XYLANASE

> Reducing Sugar (measured as Xylose)

T = 30 C, pH = 4.5, A540nm, Light path = 1 cm

50 mM Sodium Acetate Buffer, pH 4.5 at 30 C

1.0% (w/v) Xylan Substrate Solution (Xylan)

0.05% (w/v) Bovine Serum Albumin (Enz Diluent)

Xylanase Enzyme Solution

16 mM Copper Sulfate, 1.3 M Sodium Sulfate,

40 mM Molybdic Acid, 19 mM Arsenic Acid and