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. DEFINITIONS

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. PROCEDURE

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