Effect of temperature on amylase activity

Prof. Wilson: You will now test the amylase activity in different temperatures, using the optimal pH 7.0 buffer solution. You will once again be using iodine solution to colour the starch dark blue and you will need to observe for a color change to light-yellow, denoting the presence of maltose. 

You are provided with the following to begin preparing your samples:

   1. Stopwatch

   2. Ice (4°C)

   3. Water bath at 22°C

   4. Water bath at 37°C

   5. Water bath at 100 °C

   6. Glass tubes

   7. Marker

   8. Pasteur pipettes

   9. I2/KI solution

  10. Amylase solution

  11. 1% starch solution

  12. 1M phosphate buffer solutions at pH 7.0

You follow the steps below:

1. Label four glass test tubes as 1S, 2S, 3S, 4S, using a marker. Add 4 ml of 1% starch solution in each respectively, using a Pasteur pipette. Add 0.5 ml of 1M phosphate buffer solutions at pH 7.0 in each test tube. 

2. Place these four test tubes as stated below, for 20 minutes:

1. Tube 1S in water bath 100 °C

2. Tube 2S in water bath 22 °C

3. Tube 3S in water bath 37 °C

4. Tube 4S on ice (4°C)

3. Label another four glass test tubes as 1E, 2E, 3E, 4E, using a marker. Add amylase solution in each respectively. 

4. Place these four test tubes as stated below, for 20 minutes:

1. Tube 1E in water bath 100 °C

2. Tube 2E in water bath 22 °C

3. Tube 3E in water bath 37 °C

4. Tube 4E on ice (4°C)

5. Prepare six new glass tubes and label them as 0 min, 2 min, 4 min, 6 min, 8 min, 10 min, using a marker.  Using a pasteur pipette, add one drop of I2/KI solution.

6. Mix the contents of each 1S and 1E test tube and place them back in the water bath at 100 °C. Start the stopwatch.

7. Immediately collect 0.5 ml of this mixture solution and add it in the glass test tube labelled 0 min. Observe the colour change. 

8. Repeat step 7 for the remaining glass test tubes until 10 minutes have been completed. Observe any changes in the colour of the mixture and record the time it took for the dark blue colour to turn light-yellow for each reaction.

9. Repeat steps 2 – 5 at 37 °C

1. Repeat steps 2 – 5 at 22 °C

2. Repeat steps 2 – 5 at 4 °C

12. Calculate the rate of each reaction using the formula 1/t.