Practical: Factors Affecting Enzyme Activity (SQA National 5 Biology): Revision Note

Investigating the effect of temperature on enzyme activity

• Amylase is an enzyme that digests starch into maltose

• Iodine solution is used to test for the presence of starch

  • Iodine solution has a yellow-brown colour

  • In the presence of starch, iodine changes colour from yellow-brown to blue-black

• It is possible to investigate the effect of temperature on the activity of the enzyme amylase

Apparatus

• Measuring syringe / cylinder

• Starch solution

• Test tubes

• Water baths at a range of different temperatures

• Thermometer

• Iodine solution

• Spotting tile

• Amylase solution

• Stopwatch

• Glass rod

• Paper towel

Method

1. Add 5 cm³ starch solution to a test tube and place the tube in a water bath at 20°C; leave the tube in the water until a thermometer shows that its contents have reached 20°C

2. Add a drop of iodine solution to each well of a spotting tile

3. Add 2 cm³ of amylase to the starch solution and start the timer

4. Immediately use a glass rod to transfer a drop of the reaction mixture to the first iodine well and observe any colour change

5. Clean the pipette with a paper towel

6. Repeat steps 4-5 every 30 seconds until the iodine no longer changes colour

7. Record the time taken for the starch to be completely broken down

   • This will be the first well showing no colour change

8. Repeat the test twice more at 20°C to

   • This allows any anomalous results to be identified

9. Repeat steps 1-8 at other temperatures, e.g. 30°C, 40°C, 50°C, 60°C

Expected results

• We would expect this investigation to show that:

  • at the optimum temperature the iodine will stop turning blue-black in the shortest time period

    • This is because the enzyme is working at its fastest rate and has digested all the starch in the solution

  • at lower temperatures the iodine will take longer to stop turning blue-black

    • This is because the amylase enzyme is working slowly as it is below its optimum

  • at high temperatures the iodine will remain blue-black throughout the whole investigation

    • This is because the amylase enzyme has become denatured and so can no longer bind with the starch

Limitations

Investigating the effect of pH on enzyme activity

• It is possible to investigate the effect of pH on the activity of the enzyme amylase

Apparatus

• Iodine

• Spotting tile

• Measuring syringe

• Amylase solution

• Test tube

• Buffer solutions at a range of pH levels

• Starch solution

• Stopwatch

• Glass rod

• Paper towel

Method

1. Add a drop of iodine solution to each well of a spotting tile

2. Use a measuring syringe to place 2 cm³ amylase into a test tube

3. Add 1 cm³ pH 2 buffer solution to the same tube

4. Use a syringe to measure out 2 cm³ starch solution

5. Add the starch solution to the amylase / buffer solution, and start the stopwatch while mixing the new solution

6. Immediately transfer a drop of the reaction mixture to an iodine well using a glass rod; observe any colour change

7. Clean the glass rod with a paper towel

8. Repeat every 30 seconds until the iodine no longer changes colour

9. Record the time taken for the reaction to complete

   • This will be the first well showing no colour change

10. Repeat the test twice more at pH2

    • This allows any anomalous results to be identified

11. Repeat steps 1-10 with buffers of different pH values (e.g. pH4, pH6 and pH8

Expected results

• We would expect this investigation to show that:

  • at the optimum pH the iodine will stop turning blue-black within the shortest amount of time

    • This is because the enzyme is working at its highest rate and digests all the starch quickly

  • At higher or lower pH levels the iodine will take longer to stop turning blue-black, or may continue to turn blue-black for the entire investigation

    • This is because on either side of the optimum pH, the enzymes are starting to become denatured and are unable to bind with the starch

Limitations