Practical 2: Temperature and enzyme activity

Aim

To investigate how the temperature affects the time taken for amylase to break down starch.

Background

Enzymes are biological catalysts. Amylase is an enzyme that breaks down starch into sugars. Each enzyme has an active site that fits its substrate, and the substrate and enzyme must collide for a reaction to happen.

Raising the temperature gives the particles more kinetic energy, so they collide more often and with more energy, and the reaction speeds up. Above the optimum temperature the enzyme's shape starts to change, so the active site no longer fits the substrate. The enzyme is denatured and stops working.

Iodine solution is orange-brown, and turns blue-black when starch is present. Samples of the reaction mixture are added to iodine solution at regular intervals. The time at which the iodine stays orange-brown shows that all the starch has been broken down. A shorter time means a faster rate.

Hypothesis

As the temperature rises towards the optimum, the starch will be broken down in a shorter time because the particles collide more often and with more energy. Above the optimum the time will increase again because the amylase is denatured.

Variables

Equipment

• Starch solution (1%), 2 cm³ per tube
• Amylase solution (1%), 2 cm³ per tube
• Buffer solution at pH 7, 1 cm³ per tube
• Iodine solution in a dropping bottle
• Spotting tile with 6 or more wells
• Water baths (beakers of water, 250 cm³ or larger) set at 10, 20, 30, 40 and 50 °C, using ice, cold tap water and a kettle
• Thermometers (0–100 °C) or water bath thermostats
• Test tubes, test tube rack and marker pen
• Syringes or graduated pipettes (2 cm³ and 5 cm³)
• Dropping pipettes or glass rods for sampling, one per temperature
• Stopwatch or timer
• Eye protection

Risk assessment

Method

1. Put on eye protection. Set up water baths at 10, 20, 30, 40 and 50 °C and check each temperature with a thermometer.
2. Using a dropping pipette, put one drop of iodine solution into each well of a spotting tile (at least 6 wells per temperature to be tested).
3. Label a test tube for each temperature. Put 2 cm³ of starch solution and 1 cm³ of pH 7 buffer into each of these tubes.
4. Label a second set of tubes and put 2 cm³ of amylase solution in each.
5. Stand one starch tube and one amylase tube in the water bath at the first temperature for 5 minutes so they reach that temperature.
6. Pour the amylase into the starch and buffer tube, mix, and return it to the water bath. Start the stopwatch at once.
7. After 30 seconds, use a clean dropping pipette to transfer one drop of the mixture into the first well of iodine solution. Record whether it is blue-black or orange-brown.
8. Take a further drop every 30 seconds and add it to the next well of iodine solution (or to a cleaned well).
9. Stop sampling when the iodine stays orange-brown. Record this time as the time for all the starch to be broken down.
10. If the iodine is still blue-black after 10 minutes, record the time as '>10 min'.
11. Repeat steps 5–9 for each of the other temperatures, using fresh tubes and a clean pipette each time.
12. Repeat the whole investigation at least twice more, then calculate the mean time at each temperature.
13. Calculate the rate of reaction at each temperature as 1 ÷ mean time (min⁻¹).

Results

Fill this table in as you go. Print the PDF for a copy to write on.

Drawing the graph

Plot rate (1 ÷ mean time, min⁻¹) on the y-axis against temperature (°C) on the x-axis. Mark each point with a cross and draw a smooth curve through the points, or join them with straight lines; do not extend the line beyond the data. Read off the temperature that gives the highest rate: this is the optimum temperature for the amylase, to the accuracy of the temperatures you tested.