Core practical - investigating the effect of pH on enzyme activity
Aim
To determine the The best or most appropriate - for instance, the conditions under which an enzyme works best (eg temperature and pH). pH at which an enzyme’s activity is greatest.
Method
- Set up a Bunsen burner, heatproof mat, tripod and gauze.
- Place a beaker of water on the gauze and adjust the flame to keep the water at about 35°C.
- Now put two drops of iodine solution into each spot of a spotting tile.
- Add 2 cm3 of amylase enzyme solution to a test tube.
- Place 2 cm3 of starch solution into the same tube.
- Finally add 1 cm3 of pH solution to the tube. This will keep the pH constant.
- Mix the solution in the test tube and place it into the beaker of water on the Bunsen burner.
- Use a pipette to remove a few drops of solution every 20 seconds from the test tube and put them into a different well of the spotting tile.
- Repeat until the iodine solution stops turning black.
- Record the time this takes.
- Repeat with different pH solutions.
Note: this practical uses the temperature of 35°C. This is close to body temperature. Warmer temperatures may denature the enzymes and the reaction will take longer at lower temperatures.
Risks
- Iodine solution is an irritant. If it touches skin it should be washed off.
- Goggles should be worn at all times.
Results
| pH of solution | Time taken before iodine did not change colour (s) |
| 5 | 240 |
| 6 | 120 |
| 7 | 60 |
| 8 | 140 |
| pH of solution | 5 |
|---|---|
| Time taken before iodine did not change colour (s) | 240 |
| pH of solution | 6 |
|---|---|
| Time taken before iodine did not change colour (s) | 120 |
| pH of solution | 7 |
|---|---|
| Time taken before iodine did not change colour (s) | 60 |
| pH of solution | 8 |
|---|---|
| Time taken before iodine did not change colour (s) | 140 |
Conclusions
The enzyme amylase breaks down starch into glucose. If the enzyme is working effectively, this will happen quickly. At pH 7 it took the shortest time before the iodine no longer changed colour. This shows that the starch was broken down more quickly at this pH. The optimum pH for amylase is therefore pH 7.