Enzymatic Assay of α-Amylase (EC 3.2.1.1)

Overview: 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 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
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.
   
   

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