Experiment 4.

1
Problem Statement :
What are the effects of temperature on the activity of salivary amylase on starch?
Objective :
To study the effects of temperature on the activity of salivary amylase on starch.
Variables:
(a) Manipulated variable : temperature of medium
(b) Responding variable : rate of enzymatic reaction
(c) Fixed variable : volume of saliva/ volume and concentration of starch suspension
Hypothesis:
The rate of activity of salivary amylase on starch increases with the increase in
temperature until it reaches the optimum temperatue of 37C.
Materials :
1% starch suspension, saliva solution, iodine solution, ice and distilled water.
Apparatus :
Beakers, test tubes, syringes, droppers, glass rods, spotting tiles, thermometers, a
Bunsen burner, a tripod stand, a test-tube rack, a wire gauze and a stopwatch
Technique:
Test for the presence of starch using the iodine test, measure and record t the time
taken for the hydrolysis of starch to be completed with a stopwatch.
Discussion
1. The test tubes are immersed in their respective water baths for 5 minutes at
the beginning of the expriment to allow both solutions to reach the
temperatures set.
2. Starch is hydrolysed by salivary amylase to a reducing sugar (maltose).
3. The iodine solution is used to test for the presence of starch.
4. If the iodine solution changes from brownish-yellow to blue-black, this indicates
that starch is still present in the test tube.
5. At low temperatures, for every 10C increase in temperature, the rate of
enzymatic reaction is doubled.
6. The maximum rate of reaction occurs at 37C, which is the optimum temperature
for salivary amylase.
7. The average human body temperatuer is also 37C.
8. At 0C, the enzyme is not active. The salivary amylase cannot hydrolyse the
starch.
9. As the temperature increases, the rate of enzymatic reaction increases until it
reaches an optimum temperature of 37C.
10. The hydrolysis of starch is completed in the shortest period.
11. Beyond the optimum temperature, the rate of enzymatic reaction decreases and
ceases altogether at 60C.
12. No enzyme activity at 60C. Enzyme has denatured due to high temperature.

Conclusion:
Changes in the temperature affect the activity of salivary amylase on starch.
Salivary amylase is inactive at 0C and denatures at 60C.
The rate of reaction catalysed by salivary amylase is highest at 37C, which is the
optimum temperature.
The hypothesis might be accepted.











Experiment 4.2
Problem statement :
What are the effects of pH on the activity of pepsin?
Objective :
To study the effects of pH on the activity of pepsin.
Variables:
(a) Manipulated variable : pH of medium
(b) Responding variable : rate of enzymatic reaction
(c) Fixed variable : volume and concentration of albume suspension / volume and
concentration
Of pepsin solution
Hypothesis:
The optimum pH for the activity of pepsin is an acidic medium of pH 3.
Materials:
Albumen suspension, 1% pepsin solution, 0.1M hydrochloric acid, 0.1M sodium hydroxide
solution and distilled water.
Apparatus :
Beakers, droppers, thermometers, test tubes, 5ml syringes, pH papers, wire gauge, a
stopwatch and a test-tube rack.
Technique :
Observe and record the condition of mixture before and after 20 minutes.
Procedures:
1. Preapre an albumen suspension by mixing the egg white from an egg with 500ml
of distilled water.
2. Boil and cool down the suspension.
3. Using a glass wool, remove large particles.
4. Prepare three test tubes and label them as P, Q, and R respectively.
5. Using a syringe, add 5ml of albumen suspension to each test tube.
6. Add the solutions below to the test tubes.

P : 1ml of 0.1M hydrochloric acid + 1ml of 1% pepsin solution
Q : 1ml of distilled water + 1ml of 1% pepsin solution
R : 1ml of 0.1M sodium hydroxide solution + 1ml of 1 % pepsin solution
7. Dip a piece of pH paper into each test tube.
8. Determine and record the pH value.
9. Immerse all the test tubes in a water bath maintained at 37C for 20 minutes.
10. Observe the conditions of the mixtures at the beginning of the experiment and
again after 20 minutes.
11. Record the results in a table.
Results
Test tube pH Mixture

At the beginning of
the experiment


After 20 minutes
P 3 Cloudy Clear
Q 7 Cloudy Cloudy
R 8 Cloudy Cloudy

Discussion
1. The test tubes are immersed in the water bath maintained at 37C because this
is the optimum temperature for the action of pepsin.
2. Pepsin hydrolyses albumen ( a protein) into polypeptides in an acidic medium.
3. The solution turns clear because polypeptides are soluble in water.
4. The pH condition in test tube P is optimum for the function of pepsin.
5. This is because the contents of test tube P become clear at the end of the
experiment.
6. This shows that albumen has been completely digested/ hydrolysed by pepsin.
7. The contents of test tubes Q and R are still cloudy at the end of the
experiment.
8. This shows that a neutral and an alkaline pH are not suitable for the activity of
pepsin.

Conclusion :
The activity of pepsin is affected by the pH of its medium.
An acidic medium is the most suitable medium for pepsin to function effeciently.
The hypothesis might be accepted.
























Experiment 4.3
Problem statement :
What are the effects of substrate concentration on the activity of salivary amylase?
Objective:
To study the effects of substrate concentration on the activity of salivary amylase.
Variables:
(a) Manipulated variable : concentration of starch suspension
(b) Responding variable : time taken for the hydrolysis of starch to be completed
(c) Fixed variable : enzyme concentration/ temperature/pH of the medium
Hypothesis :
The rate of enzymatic reaction increases with the increase in substrate concentration
until it reaches a maximum rate.
Materials:
Starch suspensions at various concentrations ( 0.1%, 0.2%, 0.3%, 0.4%, 0.5% and 0.6%) ,
0.1% amylase/saliva suspension, iodine solution and distilled water.
Apparatus :
5ml syringes, 1ml syringes, test tubes, glass rods, a stopwatch, a white spotting tile,
droppers and measuring cylinders
Technique :
Test for the presence of starch using the iodine test. Measure and record the time
taken for the hydrolysis of starch to be completed with stopwatch.
Procedures :
1. Prepare 10ml of 0.1% salivary amylase.
2. Prepare six test tubes, labelled A to F.
3. Pour 4ml of starch suspensions of various concentrations into the following test
tubes using different syringes.
A : 0.1% starch suspension
B : 0.2% starch suspension
C : 0.3% starch suspension
D : 0.4% starch suspension
E : 0.5% starch suspension
F : 0.6% starch suspension
4. Immerse the test tubes in a water bath at 37C.
5. Add drops of iodine solution separately onto the grooves of the white tile.
6. Add 1ml of 0.1% amylase to test tube A using a syringe.
7. Activate the stopwatch immediately ( 0 minute).
8. Stir the contents with a glass rod.
9. Test a drop of the mixture with the iodine solution on the white tile.
10. Repeat the step at 30-seconds intervals until the mixture stops turning blue-
black in colour when tested with iodine solution.
11. Record the time taken for the hydrolysis of starch to be completed.
12. Repeat steps 6 to 11 with test tubes B, C, D,E and F.
13. Record the results in the table below.
Results
Test tube Concentration
of starch
suspension (%)
Time taken for
the hydrolysis
of starch to be
completed

(seconds)





(minutes)
Rate of
reaction =
A 0.1 240 4.0 0.025
B 0.2 240 4.0 0.050
C 0.3 240 4.0 0.075
D 0.4 240 4.0 0.100
E 0.5 300 5.0 0.100
F 0.6 300` 6.0 0.100

Discussion
1. The rate of reaction refers to the total substrate concentration that has been
catalysed per unit time (minute).
2. The graph shows that the enzyme reaches its saturation point at 0.4% of
substrate concentration.
3. At the substrate concentration, the rate of reaction does not increase even
though the substrate concentration is increased because the concentration of
the enzyme has become a limiting factor.
4. The time taken to hydrolyse the starch completely at low substrate
concentrations is a constant, since the enzymes are not yet saturated.
5. However, when the concentration of starch suspension increases, the time taken
for the hydrolysis of starch to be completed also increases as the enzyme
molecules become saturated.
Conclusion :
The rate of enzymatic reaction increases with the increase in substrate
concentration until it is at maximum rate. The hypothesis might be accepted.




















Experiment 4.4
Problem statement :
What are the effects of enzyme concentration on the activity of salivary amylase?
Objective :
To study the effects of enzyme concentration on the activity of salivary amylase.
Hypothesis :
The rate of enzymatic reaction increases with the increase in enzyme concentration
as long as there are no other factors limiting the rate of reaction.
Variables:
(a) Manipulated variable : concentration of enzyme
(b) Responding variable : time taken for the hydrolysis of starch to be completed
(c) Fixed variable : substrate concentration/ temperature /pH of the medium
Materials :
1% starch suspension, 0.8% amylase /saliva suspension, iodine solution, distilled
water
Apparatus :
5ml syringes, 1ml syringes, test tubes, glass rods, a stopwatch, a spotting tile,
measuring cylinders and droppers
Technique :
Test for the presence of starch using iodine test. Measure and record the time
taken for the hydrolysis of starch to be completed with a stopwatch.
Procedures:
1. Prepare six test tubes labelled A to F.
2. The test tubes contain the following mixtures:
A : 0.5ml of 0.8% amylase + 2.5ml distilled water
B : 1.0ml of 0.8% amylase + 2.0 distilled water
C : 1.5ml of 0.8% amylase + 1.5 distilled water
D : 2.0ml of 0.8% amylase + 1.0 distilled water
E : 2.5 ml of 0.8% amylase + 0.5 distilled water
F : 3.0 ml of 0.8% amylase
3. Immerse test tubes A to F in a water bath set at 37C.
4. Meanwhile, add drops of iodine solution separately onto the grooves of the white
tile.
5. Add 4ml of 1% starch suspension to test tube A using a syringe.
6. Activate the stopwatch adn record the time as 0 minute.
7. Stir the mixture in the test tube using a glass rod.
8. Remove a small amount of the mixture and test it with the iodine solution on the
tile.
9. Repeat the iodine test at 30-second intervals until the mixture does not turn-
black when tested with iodine solution.
10. Record the time taken for the hydrolysis of starch to be completed.
11. Record and tabulate all results in a table.
Results
Test tube Concentration
of amylase (%)
Time taken fro
the hydrolysis
of starch to be
completed

(seconds)





(minutes)
Rate of
enzymatic
reaction
A 0.17 330 5.5 0.18
B 0.33 150 2.5 0.40
C 0.50 90 1.5 0.67
D 0.67 60 1.0 1.00
E 0.83 60 1.0 1.00
F 1.00 60 1.0 1.00

Discussion
1. The time taken for amylase to hydrolyse starch decreases as the enzyme
concentration increases.
2. A higher concentration of the enzyme contains more enzyme molecules to
hydrolyse the starch molecules.
3. Hence, it takes less time for the hydrolysis of starch to be completed.
4. A further increase in enzyme concentration will not increase the rate of
reaction.
5. This is because the substrate concentration has become the limiting factor.
Conclusion :
The rate of reaction increases with the increase in enzyme concentration until a certain
concentration of enzyme is achieved. The hypothesis might be accepted.