Enzymatic Assay of α-Amylase (EC 3.2.1.1)

Section Overview

• Determination of Alpha-Amylase Activity
• Reagents and Equipment Required
• Preparation Instructions
• Procedure
• Results
• Related Products

Determination of Alpha-Amylase Activity

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 No. S0751
• Sodium chloride, Product No. S9888
• Starch from potato, Product No. S2004
• Sodium hydroxide, Product No. S5881
• Potassium sodium tartrate, tetrahydrate, Product No. S2377
• 3,5-Dinitrosalicylic acid, Product No. D0550
• D‑(+)‑Maltose, monohydrate, Product No. 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 No. S0751, and 0.39 mg/mL of sodium chloride, Product No. 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 No. 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 No. 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 No. 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 No. 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 No. 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
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:

Reagent	STD1	STD2	STD3	STD4	STD5	STD6	STD7	STD BLK
0.2% (w/v) Maltose Standard	0.05	0.20	0.40	0.60	0.80	1.00	2.00	–
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 ΔA₅₄₀ of each Standard vs. the Standard Blank.
   ΔA_{540 (Standard)} = A_{540 (Standard)} – A_{540 (Standard Blank)}
2. Prepare a standard curve by plotting the ΔA₅₄₀ of the standards vs. mg of maltose using linear regression.
3. Determine the ΔA₅₄₀ of each Sample vs. the Sample Blank.
   ΔA_{540 (Sample)} = A_{540 (Sample)} – A_{540 (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.

Reference

1. Bernfeld P. 1955. [17] Amylases, ? and ?.149-158. https://doi.org/10.1016/0076-6879(55)01021-5