Enzyme rate of reaction required practical

Purpose

Investigate how pH affects the rate of reaction of amylase, an enzyme that breaks down starch into maltose.

Enzymes have an optimum pH at which they work fastest.

At pH levels too high or low, enzymes denature (the active site changes shape), reducing the reaction rate.

Method

Prepare the Solutions:

1. Add 5 cm³ of buffer solutions (at pH 3, 5, 7, 9, and 11) to five test tubes.

2. Place these test tubes in a water bath set to 25°C.

Prepare the Enzyme and Starch Solutions:

3. Add 5 cm³ of starch solution and 5 cm³ of amylase solution to separate test tubes.

4. Place these test tubes in the same water bath.

Wait and Set Up:

5. Wait 5 minutes for all solutions to reach the water bath temperature.

6. While waiting, add iodine solution (1 drop per well) to a spotting tile.

Start the Reaction:

7. Add 1 cm³ of starch solution and 1 cm³ of amylase solution to the test tube containing the pH 3 buffer.

8. Start a timer.

Test for Starch:

9. After 30 seconds, remove a small sample from the test tube with a pipette.

10. Add this sample to a well in the spotting tile containing iodine solution.

11. Observe the color:

    • Black: Starch is still present.

    • No color change: Starch has been broken down.

12. Return any unused solution to the water bath.

13. Repeat Sampling:

    • Continue testing every 30 seconds until the iodine no longer turns black.

    • Record the time when this happens.

14. Repeat for All pH Buffers:

    • Perform the experiment with buffer solutions at pH 5, 7, 9, and 11.

    • If the iodine still turns black after 10 minutes, record “no change after 10 minutes.”

Typical results for an amylase enzyme test

Analysis