Enzymatic Assay of α-AMYLASE (EC 3.2.1.1)
Document History
Based from SSSTAR01. Refer to CR SOP DEK ENZ
1. OBJECTIVE
To standardize a procedure for determining the enzymatic activity of α-Amylase.
2. SCOPE
This procedure applies to all products that have a specification for α-amylase.
3.1 3.1 Purified Water - water from a deionizing system, resistivity > or = 18MΩ•cm @ 25ºC
3.2. Unit Definition - One unit will liberate 1.0 mg of maltose from starch in 3 minutes at pH 6.9 at 20°C.
3.3. STD = Maltose Standard
4. DISCUSSION
Starch + H2O α-Amylase > Reducing Groups (Maltose)
5. RESPONSIBILITIES
It is the responsibility of trained Analytical Services laboratory personnel to follow this procedure as written.
6. SAFETY
Refer to Material Safety Data Sheets (MSDS) for hazards and appropriate handling precautions.
7.1 CONDITIONS:
T = 20°C, pH = 6.9, A540nm, Light path = 1 cm
7.2 METHOD:
Spectrophotometric Stop Reaction
7.3 REAGENTS:
7.3.1 20 mM Sodium Phosphate Buffer with 6.7 mM Sodium Chloride, pH 6.9 at 20°C (Buffer)
Prepare 100 ml in purified water using Sodium Phosphate, Monobasic, Anhydrous, Sigma-Aldrich Product Number S0751 and Sodium Chloride, Sigma Product No S9888. Adjust to pH 6.9 at 20°C with 1 M NaOH.
7.3.2 1.0% (w/v) Soluble Starch Solution (Starch)
Prepare 25 ml in Reagent 7.3.1 using Starch Potato Soluble, Sigma-Aldrich Product Number S2630. Facilitate solubilization by heating the starch solution in a glass beaker directly on a heating/stir plate using constant stirring. Bring to boil and maintain the solution at this temperature for 15 minutes. Allow the starch solution to cool to room temperature with stirring. Return the starch solution to its original volume (25 ml) by the addition of purified water and dispense aliquots for assay with stirring.
7.3.3 Sodium Potassium Tartrate Solution
Dissolve 12.0 g of Sodium Potassium Tartrate, Tetrahydrate, Sigma-Aldrich Product Number S2377, in previously heated 8.0 ml of 2 M NaOH, 50°C - 70°C. Heat directly on a heating/stir plate with constant stirring to dissolve. DO NOT BOIL.
7.3.4 96 mM 3,5-Dinitrosalicylic Acid Solution
Prepare 20 ml in purified water, 50°C - 70°C, using 3,5-Dinitrosalicylic Acid, Sigma-Aldrich Product Number D0550. Heat directly on a heating/stir plate with constant stirring to dissolve. DO NOT BOIL.
7.3.5. Color Reagent Solution (Clr Rgt Soln)
To 12 ml of purified water, 50°C - 70°C, slowly add Reagent 7.3.3 followed by Reagent 7.3.4. If not completely dissolved, the reagents should dissolve when mixed. The solution should be stored in an amber bottle at room temperature. The Color Reagent Solution is stable for 6 months.
7.3.6 0.2% (w/v) Maltose Standard (STD)
Prepare 10 ml in purified water using Maltose, Monohydrate, Sigma-Aldrich Product Number M5885 .
7.3.7 α-Amylase Solution (Enzyme)
7.3.7.1 Immediately before use, prepare a solution containing 1 unit/ml of α- Amylase in 20°C purified water.
7.3.7.2 If test enzyme requires a dilution scheme, the first dilution and subsequent dilutions should be in cold purified water until the last dilution, and the last dilution should be in 20°C purified water.
7.4 ASSAY
7.4.1 Pipette (in milliliters) the following reagents into suitable containers:
| Test1 | Test2 | Test3 | Blank | |
| Reagent 7.3.2 (Starch) | 1.00 | 1.00 | 1.00 | 1.00 |
7.4.2 Mix by swirling and equilibrate to 20°C. Then add:
| Reagent 7.3.7 (Enzyme) | 0.50 | 0.70 | 1.00 | ---- |
7.4.3 Mix by swirling and incubate for exactly 3.0 minutes at 20°C. Then add:
| Reagent 7.3.5 (Clr Rgt Soln) | 1.00 | 1.00 | 1.00 | 1.00 |
7.4.4 Cap with a vented cap and place in a boiling water bath. The add:
| Reagent 7.3.7 (Enzyme) | 0.50 | 0.30 | ----- | 1.00 |
7.4.5 Boil for exactly 15 minutes, then cool on ice to room temperature, approximately 3 minutes, and add:
7.4.6
| Purified water | 9.00 | 9.00 | 9.00 | 9.00 |
7.4.7 Mix by inversion and record the A540nm for both the Test and Blank using a suitable spectrophotometer.
7.4.8 Due to the short enzymatic incubation time of three minutes, each test lot must be run one at a time.
7.5. Standard Curve:
7.5.1 A standard curve is made by pipetting (in milliliters) the following reagents into suitable containers:
| Std1 | Std2 | Std3 | Std4 | Std5 | Std6 | Std7 | Std Blank | |
| Reagent 7.3.6 (STD) | 0.05 | 0.20 | 0.40 | 0.60 | 0.80 | 1.00 | 2.00 | ----- |
| Purified Water | 1.95 | 1.80 | 1.60 | 1.40 | 1.20 | 1.00 | ----- | 2.00 |
| Reagent 7.3.5 (Clr Rgt Soln) | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 |
7.5.2 Place in a boiling water bath for exactly 15 minutes, then cool on ice to room temperature and add:
| Purified water | 9.00 | 9.00 | 9.00 | 9.00 | 9.00 | 9.00 | 9.00 | 9.00 |
7.5.3 Mix by inversion and record the A540nm for the Standards and Standard Blank using a suitable spectrophotometer.
7.6 CALCULATIONS
7.6.1 Standard Curve
ΔA540nm Standard = A540nm Std - A540nm Std Blank
Plot the ΔA540nm of the Standards vs milligrams of Maltose. Calculate and record the slope, y-intercept, and linear regression(r-square).
7.6.2 Sample Determination
ΔA540nm Sample = A540nm Test - A540nm Test Blank
Determine the milligrams of Maltose liberated using the Standard Curve.
| 7.6.3 | Units/Ml enzyme = | mg of Maltose released(df) |
| (1) |
df = Dilution Factor
1 = Volume (in milliliter) of enzyme used
| 7.6.4 | Units/mg solid = | units/mL enzyme |
| mg solid/mL enzyme |
| 7.6.5 | Units/mg protein = | units/mL enzyme |
| mg protein/mL enzyme |
7.7 FINAL ASSAY CONCENTRATION
In a 2.00 ml reaction mix, the final concentrations are 8 mM sodium acetate, 0.50% (w/v) starch and 1 unit β-amylase.
8. REFERENCES & ATTACHMENTS
Bernfeld, P. (1955) Methods in Enzymology 1, 149-158
9. APPROVAL
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