Identify data sources, plan, choose equipment or resources and perform a first-hand investigation

to test the effect of:

increased temperature
change in pH
change in substrate concentrations on the activity of named enzyme(s)

The effect of temperature on enzyme activity

Aim: To demonstrate the effect of temperature on enzyme activity.
Hypothesis: An increase in temperature would reduce enzyme activity, and thus reduce the rate of
reaction. Enzyme of activity is at its highest at 40C
Risk Assessment: Exposure to hot plate can cause 3rd degree burns.
Variables:
Independent: Temperature of milk
Dependent: Rate of reaction
Controlled:
Amount of rennin placed in milk
Quantity of milk
Type of milk
Type of enzyme (rennin)
Equipment:
Pipette and pipette pump
4 test tubes
Test tube rack
300 mL of water
6 ice cubes
Junket tablets
Stirring rod
Fresh milk
Stopwatch
Hotplate
50ml beaker and 500ml beaker
Measuring cylinder
Method:
1. Prepare an ice water bath by filling the 500mL beaker with approximately half a dozen ice cubes
and 300mL of water. Increase or decrease the number of ice cubes.
2. Turn the hotplate on and place another beaker, containing 300 mL of water, onto the hotplate,
letting it boil and reach 100 C. Turn off the hotplate once water has boiled.
3. Attach the pipette to a pump, then fill the pipette with 10mL of milk. Pump this amount of milk
into a test tube. Repeat for the remaining three test tubes.
4. Place one test tube of milk into the hot water beaker, and let it heat up until 75C. Once the test
tube reaches 75C, remove it from the beaker.
5. Place another test tube of milk into the ice water bath, and let it cool down to 5C.
 6. Ensure the remaining two test tubes are 40C. (Since the experiment is performed at room
temperature, the test tubes will roughly be 25C, so to get the test tubes to 40C, place them into
the hot water beaker briefly).
7. Remove the plastic covering from junket tablets and place the powder into a 50mL beaker filled
with 30mL of water and create a rennin solution.
8. Place all the test tubes into a test tube rack in ascending order of temperature (5C,
40C,40C,75C).
9. Measure 2mL of rennin solution using a measuring cylinder and then pour into the coldest test
tube. Repeat for the 40C,75C. Do not put the rennin enzyme into one of the test tubes which is
40 C, as that test tube acts as the control.
10. Stopwatch the time it takes for the milk to completely curdle and become solid. Record these
times.
Results:

Temperature (C) Time taken for milk to curdle

5 No curdle at 7+ min

5 heated to 40 Curdled in 30 seconds

40 (control) No curdle (no enzyme)

40 3.25 mins

80 No curdle at 7+ mins

Analysis:
The time taken for milk to curdle was the least in the test tube of milk that was 40C and was the highest
in the test tube that was 5C. However, when the 5C test tube was heated up to 40C, the time taken for
the milk to become solid and curdle was majorly reduced.
Discussion:
The experiment proved the hypothesis correct, as it was observed that when the milk was 40C, the time
taken for milk to curdle was at its lowest (3.25 seconds). This is because 40C is the average human body
temperature and thus is the optimum temperature for rennin to act upon milk in the stomach. At 5C,
rennin would change shape and its functioning as a result of the excessive cold, and hence there was
minimal curdling observed. At 80C, rennin would have denatured as a result of the high temperatures,
and thus, the time taken for milk to curdle was larger in comparison to the milk that was 40C.

To avoid parallax error, multiple people read the measurements for milk volumes at eye level and from
the meniscus, and hence this allowed for accuracy in the experiments.

Reliability was achieved due to the fact that a variety of groups performed the same experiment and
consistent results were achieved with each group. To improve reliability, different temperatures could be
tested, eg. 20C, 40C, 60C. This would allow for examination of the trend with varying temperatures.

The experiment can be said to be accurate due to the fact that apparatus that was used was not faulty,
examples being thermometers, measuring cylinders and pipette.
Due to the controlled variables that were maintained throughout the experiment, the experiment is said to
be very valid. Such controlled variables include the pH, quantity and concentration of milk.

Conclusion:
Enzyme activity was significantly greatest when the temperature of the milk was 40C.

The effect of pH on enzyme activity

Aim: To investigate the effect of substrate acidity on rate of reaction

Hypothesis: As pH decreases, enzyme activity also increases, thus the time taken for reaction to occur
decreases.
Equipment:
Test tube rack
3 test tubes
30mL milk
Sodium Hydroxide
Hydrochloric Acid
Thermometer
Hotplate
Pipette and pump
50mL beaker
500mL beaker
Rennin (from Junket tablets)
Stirring rod
Stopwatch
Measuring cylinder
Variables:
Independent: pH of milk
 Dependent: Time taken for milk to curdle
Controlled:
Temperature of milk
Concentration of milk
Amount of milk
Amount of enzyme (rennin) placed into milk
Method:
1. Using a pipette and pump, place 10 mL of milk into a test tube. Repeat for the remaining two test
tubes.
2. Fill the 500mL beaker with 300 mL of water and then place on hotplate. Place all 3 test tubes into
the beaker, and remove them once all test tubes reach a temperature of 40C.
3. Measure 1mL of sodium hydroxide into a measuring cylinder, then pour into one of the test tubes.
4. Measure 1mL of water into a measuring cylinder, then pour into another test tube.
5. Measure 2mL of rennin into a measuring cylinder then pour into a test tube. Repeat for the
remaining test tubes .
6. Using a stopwatch, record the time taken for milk to curdle in the test tube containing milk and
sodium hydroxide and the test tube containing milk and water
7. Measure 1mL of hydrochloric acid into a measuring cylinder, then pour into the last test tube.
8. Start recording the time for milk to curdle in the test tube containing hydrochloric acid.
Results:
pH Time taken for milk to curdle

Acidic (1mL HCl) 0:43

Neutral 2:41

Basic (1mL NaOH) 8+

Analysis:
The time taken for milk to curdle was the least in the test tube that contained 1 mL of HCl. It was also
seen that the test tube containing 1mL of NaOH, that the time taken for milk to curdle was recorded to
exceed 8 minutes. Hence, since HCl is an acid and NaOH is a base, it has been proven that as pH
decreases and the substrate becomes more acidic, enzyme activity is maximised.
Discussion:
Since rennin is an enzyme used in the digestion and breakdown of milk, this enzyme is adapted to be
more effective at higher acidity levels.

The experiment can be deemed to be valid given the controlled variables that were maintained throughout
the entirety of the experiment, such examples are the concentration and quality of milk and the amount of
enzyme. However, although all the test tubes were placed in the hot bath and removed at the same time,
heat would have been lost to surroundings after removal from the hot water bath, and hence, temperature
was not maintained. An improvement for this, is to check the temperatures of all the test tubes after
removal from hot water bath and prior to putting enzyme in.
The experiment was reliable due to the fact that a large number of groups replicated this experiment and
the same trend was evident in the results of each group.
Accuracy was maintained through the experiment through the use of non faulty equipment, especially
with the measuring cylinder and pipette which had small increments to ensure proper measurements of
substances were used.
Conclusion: Enzyme activity was greatest in the test tube that had milk at a higher acidity.

The effect of substrate concentration on enzyme activity

Aim: To investigate the effect of substrate concentration on enzyme activity

Hypothesis: As the concentration of milk increases, rennin activity increases.
Equipment:
Stirring rod
Hotplate
Water
4 test tubes
Milk
Rennin (Junket tablets)
Thermometer
Stopwatch
Measuring cylinder
500mL beaker
50mL beaker
Pipette and pump
Variables:
Independent: Milk concentration
Dependent: Time taken for milk to curdle
Controlled:
Temperature of milk
pH of milk
Amount of enzyme added to milk
 Type of milk
Method:
1. Using a pipette and pump, place 10mL of milk in one test tube, 7.5mL of milk and 2.5mL of
water in another test tube, 5mL of milk and 5 mL of water in another test tube, and 2.5mL of milk
and 7.5mL of water in the final test tube.
2. Prepare a hot water bath by filling the 500mL beaker with 300 mL of water and then placing this
beaker onto a hotplate.
3. Place the 4 test tubes into the hot water beaker until all the test tubes reach a temperature of 40
C, then remove and place onto test tube rack.
4. Measure 2mL of rennin using a measuring cylinder then pouring into a test tube. Repeat this 3
times and ensure that the rennin is stirred before being poured into test tubes which contain milk.
5. Using a stopwatch, record the time it takes the milk to curdle in each test tube.

Results:
Milk concentration Time taken for milk to curdle

100% 0:45

75% 2:42

50% 4:50

25% 9+

Discussion:
The experiment affirmed the hypothesis by proving that as milk concentration increases, enzyme activity
also increases, thus the time taken for milk to curdle decreased. This is because with a higher amount of
substrate, enzymes can work upon more substances.
The experiments reliability could have been improved by repeating the experiment with varying milk
concentrations (10%, 30%, 50%, 70% and 90%), to ensure the trend was evident in other amounts of milk
concentration.

Conclusion: As milk concentration increased, the time taken for milk to curdle decreased, and thus
rennin activity was increased.