DETERMINATION OF ALPHA -AMYLASE ACTIVITY (89)
Materials required
Note: all chemicals cited below can be obtained from Sigma-Aldrich.
1. KPO4-NaCl buffer; 0.1 M pH 6.9 at 25°C containing 0.01 M NaCl. Dissolve 8.71 gm K2HPO4 in 500 ml H2O. Titrate to pH 6.9 with 0.1 M KH2PO4 (6.8gm/500 ml H2O). Add NaCl to 0.01 M. CAUTION! Use ventilated hood and stirrer/heat plate in steps 2 and 3 below.
2. Substrate solution; 1% soluble starch. Dissolve 1 gm in 80 ml H2O by heating with stirring to ca. 90°C, cool and adjust volume to 100 ml.
3. Color reagent; 44.0 mM 3,5-dinitrosalicylic acid. Dissolve 1 gm 3,5 DNSA in 80 ml 0.5 M NaOH by heating with stirring ca. 70°C. Add 30 gm NaK-tartrate, stir until dissolve. Cool to room temperature and bring to 100 ml with H2O. Store in an amber bottle. Stable 30 days at room temperature.
4. Maltose standard; 0.01 M. Dissolve 36 mg maltose monohydrate in 10 ml KPO4-NaCl buffer.
5. Enzyme solution. Dilute aAmylase to 0.05 mg/ml with KPO4-NaCl buffer.
6. Prepare the following dilutions in tubes suitable for heating at 100°C.
| Tube |
mlPO4-NaCl |
 Amylase |
ml |
μM |
ml H20 |
| 1 Test |
0.50 |
0.01 ml |
----- |
----- |
0.40 |
| 2 Std |
0.5 |
---------- |
0.05 |
0.5 |
0.45 |
Bring all tubes to 25°C. At 0 time add 0.5 ml substrate solution to tubes 1 and 6 and incubate for 2 minutes. Stop the reaction by addition of 1.0 ml color reagent. Heat all tubes at 100° for 10 minutes in a temperature block. Cool to room temperature and add 2.5 ml H2O to each tube. Let the tubes sit at room temperature for 10 minutes.
7. Read Abs vs. the blank at 540 nm. Plot the Abs vs. μMoles of Maltose. Determine the slope 540/μM Maltose.
8. Calculate of specific activity, i.e., μM of maltose released per minute per mg of aAmylase at pH 6.9 and
25°C, from the slope.