Mandatory Practical - Investigating Amylase Activity
Aim: To determine the optimum pH and temperature for salivary amylase
Hypothesis:
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Background:
Iodine solution (I2/KI) is a yellow/orange colour, but in the presence of starch, it turns into a
blue/black colour. When the iodine solution no loner changes colour after the sample is
added (i.e. it remains yellow), all the starch has been hydrolysed.
Method (PART A: Effect of pH on amylase activity)
1. Perform the experiment at room temperature.
2. Add 1 drop of 0.1M iodine solution to each well of the stopping plates.
3. Add 2mL of 1% amylase solution and 1mL of buffered pH 4 solution to a test tube.
4. The solutions were mixed and 2mL of starch solution was added. Immediately start the
timer.
5. After 10 seconds, use a plastic pipette to add 1 – 2 drops of the buffer solution to one of
the wells.
6. Repeat this at 10 second intervals, adding a drop of the reaction solution into a new well
until the iodine solution no longer changes colour (remains yellow/orange).
7. Repeat the experiment using the buffer solutions of pH 5, 6, 7, 8.
Results
pH
No. of drops until no
No. of seconds until no
colour change occurred colour change occurred
Reaction rate (1÷
seconds)
4
5
6
7
8
Discussion
1. Why was it important to add the buffer and enzyme together before adding the starch?
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2. Complete the results table by calculating the reaction rate for each pH (1 ÷ seconds)
3. a) Graph the reaction rate vs pH on
the grid.
b) Identify the pH where amylase
activity was the highest.
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c) Is this what you had expected?
Explain
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4. The experiment was repeated at pH 1. Each sample turned blue/black when added to the
iodine even after 5 mins of sampling. Explain what has happened here.
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Method (Part B: Effect of temperature on amylase activity)
1. Add 1 drop of 0.1M iodine solution to each well of the spotting plates.
2. To 5 test tubes, add 2mL of 1% amylase solution and 1mL of pH 7 buffer solution.
3. Place the test tubes in temperatures of 10°C, 20°C, 30°C, 40°C and 50°C. (20°C will be
room temperature, 10°C in the fridge and the rest in hot water baths)
4. Once the temperatures are reached, add 2mL of 1% starch solution to the 10°C test tube.
Immediately start the timer.
5. After one minute, use a plastic pipette to add 1 -2 drops of the solution to a well of the
spotting plate.
6. Repeat this at one minute intervals until no colour change is seen.
7. Repeat this process for all the other temperatures.
Results
Temp (°C)
No. of drops until no
No. of mins until no
colour change occurred colour change occurred
10
20
30
40
50
Discussion
1. What would be a suitable control for this experiment?
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2. Why was the temperature investigation experiment carried out at pH7?
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3. Identify the temperature at which amylase shows no activity (enzyme is denatured)
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4. On the grid, plot the time taken for all the starch to be digested against temperature.
5. Identify the optimum temperature for the amylase.
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6. Describe how temperature affects the activity of amylase.
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7. Predict amylase activity below 10°C and give a reason for your prediction.
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