1. NAME_______________________
ID# ______________________
LABORATORY EXERCISE #5
HYDROLYSIS OF STARCH BY AMYLASE
Introduction
The enzyme to be used in this exercise is amylase, which is commonly found in saliva and germinating
seeds. It catalyses the breakdown of starch. When amylase acts on starch, it liberates maltose units
(disaccharides of two glucose molecules linked together). As the reaction progresses, less starch is
present and more maltose is present.
In this exercise, the activity of amylase will be observed by using iodine. Recall that iodine reacts with
starch to form a dark brown / blue-black colour. As amylase breaks down starch, the colour of the
solution (if iodine is added), becomes lighter and lighter. The colour change can be observed by using
spot-plates (see diagram below).
List of Reagents
•
Pancreatic amylase powder, 40,000 SKB units/g. (Concentration to be used in class: 75 g/l)
•
1M Potato starch solution
•
Iodine solution
•
Citric acid – Sodium hydrogen phosphate buffer solutions, pH 5 and 7
•
Sodium carbonate – Sodium bicarbonate buffer solution, pH 9.2
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2. Test 1
Effect of pH
1. Add 1ml of the pH 5 buffer solution to 0.5 ml of starch solution in a test tube. Mix well, and then
remove 1ml of the buffer-starch solution and place in a fresh test tube. Add 1ml of the available
enzyme solution to the buffer-starch mixture and mix well. Start the clock immediately on
adding enzyme to the substrate mixture.
2. At intervals of 1 minute, remove a drop of the reaction mixture and place it on a spot plate.
Immediately add a drop of the iodine solution and note the colour change. Record the time
taken for the colour to change from blue-black to light brown / colourless.
3. Repeat steps 2 and 3 using buffer solutions of pH 7 and 9.2.
Test 2
Effect of substrate concentration
1. Prepare the following substrate concentrations using the 1M starch solution given: 0.2M, 0.4M,
0.6M, 0.8M, 1.0M.
2. Add 0.5ml of the 0.2M starch solution to 1ml of the pH buffer which gave the fastest reaction
rate in test 1. Remove 1 ml of this buffer-starch mixture and place it into a clean test tube. Add
1ml of the available enzyme solution to the buffer-starch solution and mix well. Start the clock
immediately on adding enzyme to the substrate mixture.
3. At intervals of 1 minute, remove a drop of the reaction mixture and place it on a spot plate.
Immediately add a drop of the iodine solution and note the colour change. Record the time
taken for the colour to change from blue-black to light brown / colourless.
4. Repeat steps 2 and 3 using the 0.4M, 0.6M, 0.8M, and 1M substrate concentrations.
Questions
1. Plot a graph of enzyme activity versus pH. From this curve, what is the optimal pH?
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3. 2. Explain why enzyme activity depends on the pH?
3. How does the optimal pH for the amylase enzyme compare with the pH of the stomach? Explain
why this is significant.
4. Plot a graph of enzyme activity versus substrate concentration.
5. Is the enzyme activity directly proportional to the substrate concentration? Explain why / why
not?
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