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Enzymatic Assay of α-Amylase (EC 3.2.1.1)

Description
This procedure may be used for the determination of α-Amylase activity.
The spectrophotometric stop reaction determination (A540, Light path = 1 cm) is based on the following reaction:

Unit Definition: One unit will liberate 1.0 mg of maltose from starch in 3 minutes at pH 6.9 at 20 °C.

Reagents and Equipment Required

Sodium phosphate, monobasic, Product Number S0751
Sodium chloride, Product Number S9888
Starch from potato, Product Number S2004
Sodium hydroxide, Product Number S5881
Potassium sodium tartrate, tetrahydrate, Product Number S2377
3,5-Dinitrosalicylic acid, Product Number D0550
D
(+)
Maltose, monohydrate, Product Number M9171
Precautions
Please consult the Safety Data Sheet for information regarding hazards and safe handling practices.

Preparation Instructions
Use ultrapure water (≥18 MΩ×cm resistivity at 25 °C) for the preparation of reagents.
Buffer solution (20 mM Sodium Phosphate with 6.7 mM Sodium Chloride, pH 6.9 at 20 °C) – Prepare a solution containing 2.4
mg/mL of sodium phosphate, monobasic, Product Number S0751, and 0.39 mg/mL of sodium chloride, Product Number
S9888, in ultrapure water. Adjust to pH 6.9 at 20 °C using 1 M NaOH/1 M HCl.
Starch solution [1.0% (w/v) Soluble Starch Solution] – Prepare a 10 mg/mL solution using starch from potato, Product Number
S2004, in Buffer:
Solubilize the solution by boiling on a heating/stir plate for 15 minutes with mixing.
Remove from heat. Continue to mix the solution and allow to cool to room temperature.
Bring the solution to final volume with ultrapure water.
Continue mixing the solution throughout the assay procedure.

2 M Sodium Hydroxide (NaOH) solution – Prepare a 80 mg/mL solution using sodium hydroxide, Product Number S5881, in
ultrapure water.
5.3 M potassium sodium tartrate, tetrahydrate solution – Prepare a 1,496 mg/mL solution of potassium sodium tartrate,
tetrahydrate, Product Number S2377, in 2 M Sodium Hydroxide (NaOH) solution. Dissolve solids by heating on a heating/stir
plate with mixing. Do not heat to a boil!
96 mM 3,5-Dinitrosalicylic acid solution – Prepare a 21.9 mg/mL solution using 3,5
Dinitrosalicylic acid, Product Number
D0550, in ultrapure water. Dissolve solids by heating on a heating/stir plate with mixing. Do not heat to a boil!
Color Reagent solution – For preparation of 40 mL, add:
12.0 mL of warm (50–70 °C) ultrapure water to an appropriate size amber bottle.
With mixing, slowly add:
8.0 mL of warm 5.3 M potassium sodium tartrate, tetrahydrate solution
20 mL of warm 96 mM 3,5-Dinitrosalicylic acid solution

This solution is stable for 6 months at ambient temperature if protected from light. Volume prepared may be adjusted if
needed.
0.2% (w/v) Maltose Standard – Prepare a 2 mg/mL solution in a volumetric flask using D
(+)
maltose, monohydrate, Product
Number M9171, in ultrapure water.
α-Amylase Sample solution – Immediately before use, prepare a solution containing 0.75
1.5 units/mL of α-Amylase in 20 °C
ultrapure water.

Procedure
Final assay concentration – In a 2.00 mL reaction volume, the final concentration is 0.50% (w/v) starch and ~1 unit of
α-amylase.
1. α-Amylase Sample Assay
a. Pipette (in mL) the following reagents into suitable containers:
Reagent Sample 1 Sample 2 Sample 3 Sample Blank

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Starch solution 1.00 1.00 1.00 1.00

b. Mix by swirling and equilibrate to 20 °C. Then add (in mL):

Reagent Sample 1 Sample 2 Sample 3 Sample Blank
α-Amylase Sample 0.50 0.70 1.00 –

c. Mix by swirling and incubate for exactly 3.0 minutes at 20 °C. Then add:
Reagent Sample 1 Sample 2 Sample 3 Sample Blank
Color Reagent 1.00 1.00 1.00 1.00

d. Cover containers with a vented cap and place in a boiling water bath for exactly 15 minutes.
e. Remove containers from boiling water bath. Then add (in mL):
Reagent Sample 1 Sample 2 Sample 3 Sample Blank
α-Amylase Sample 0.50 0.30 – 1.00

f. Cool solutions on ice to room temperature.

g. Then add (in mL):
Reagent Sample 1 Sample 2 Sample 3 Sample Blank
Ultrapure water 9.00 9.00 9.00 9.00

h. Mix by inversion. Blank a suitable spectrophotometer against air at 540 nm and record the A540 for the Samples and
Sample Blank.
2. Standard Curve Preparation
a. Prepare a standard curve by pipetting (in mL) the following reagents into suitable containers:
STD
Reagent STD1 STD2 STD3 STD4 STD5 STD6 STD7
BLK
0.2% (w/v) Maltose
0.05 0.20 0.40 0.60 0.80 1.00 2.00 –
Standard
Ultrapure water 1.95 1.80 1.60 1.40 1.20 1.00 – 2.00
Color Reagent 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00

Note: Additional standard levels may be added as needed.

b. Cover containers with a vented cap and place in a boiling water bath for exactly 15 minutes.
c. Remove containers from boiling water bath. Cool solutions on ice to room temperature.
d. Then add (in mL):
Reagent STD1 STD2 STD3 STD4 STD5 STD6 STD7 STD BLK
Ultrapure water 9.00 9.00 9.00 9.00 9.00 9.00 9.00 9.00

e. Mix by inversion. Blank a suitable spectrophotometer against air at 540 nm and record the A540 for the Standards and
Standard Blank.

Results
Calculations
1. Determine the ∆A540 of each Standard vs. the Standard Blank.
∆A540 (Standard) = A540 (Standard) – A540 (Standard Blank)
2. Prepare a standard curve by plotting the ∆A540 of the standards vs. mg of maltose using linear regression.
3. Determine the ∆A540 of each Sample vs. the Sample Blank.
∆A540 (Sample) = A540 (Sample) – A540 (Sample Blank)
4. Determine the mg of Maltose released using the standard curve.

5.

where:
df = dilution factor
mL enzyme = mL of Sample added in step 1b.
6.

Materials

Product # Image Description Molecular Formula Add to Cart

α-Amylase from Aspergillus oryzae aqueous solution,
A8220 pricing
≥800 FAU/g

α-Amylase from Aspergillus oryzae powder, ≥150 units/mg

A9857 pricing
protein (biuret)

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Product # Image Description Molecular Formula Add to Cart

α-Amylase from Bacillus amyloliquefaciens liquid, ≥250 units/g
A7595 pricing

α-Amylase from Bacillus licheniformis Type XII-A, saline

A3403 pricing
solution, ≥500 units/mg protein (biuret)

α-Amylase from Bacillus licheniformis lyophilized powder,

A4551 pricing
500-1,500 units/mg protein, 93-100% (SDS-PAGE)

α-Amylase from Bacillus sp. powder, ≥400 units/mg protein

A6814 pricing
(Lowry)

α-Amylase from Bacillus sp. Type II-A, lyophilized powder,

A6380 pricing
≥1,500 units/mg protein (biuret)

α-Amylase from human pancreas lyophilized powder,

A9972 pricing
≥100 units/mg protein

α-Amylase from human saliva Type XIII-A, lyophilized powder,

A1031 pricing
300-1,500 units/mg protein

α-Amylase from human saliva Type IX-A, lyophilized powder,

A0521 pricing
1,000-3,000 units/mg protein

α-Amylase from porcine pancreas Type VI-B, ≥5 units/mg

A3176 pricing
solid

α-Amylase from porcine pancreas DFP Treated, Type I-A,

A6255 pricing
saline suspension, ≥1000 units/mg protein (E1%/280)

α-Amylase from porcine pancreas Type I-A, PMSF treated,

A4268 pricing
saline suspension, 700-1400 units/mg protein (E1%/280)

A4862 α-Amylase from Bacillus sp. liquid pricing

M9171 D-(+)-Maltose monohydrate BioXtra, ≥99% C12H22O11 · H2O pricing

S2377 Potassium sodium tartrate tetrahydrate ReagentPlus®, ≥99% C4H4KNaO6 · 4H2O pricing

S0751 Sodium phosphate monobasic ReagentPlus®, ≥99.0% H2NaO4P pricing

S2004 Starch from potato Soluble pricing

References
1. Bernfeld, P., Methods in Enzymology, 1, 149-158 (1955).

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