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Effect of Temperature on Enzyme Activity

Aim: To investigate the effect of temperature on the activity of an enzyme (rennin).

Hypothesis: The optimum temperature for the enzyme, rennin, works best at 37C (i.e. body temperature), as
it is found within the stomach of an organism.
Apparatus: Junket tablet (rennin enzyme which is produced in the stomach of many mammals to cause the
clotting of milk), mortar and pestle, distilled water, pipette, 8 test tubes, test tube rack, measuring cylinder,
water baths, thermometers, stopwatch, milk.
Independent variable: Temperature of the water bath. This temperature is constantly changed
throughout the experiment to test its effect on rennin. The temperatures used were 20C, 30C, 40C
and 50C, and were measured using a thermometer.
Dependent variable: Enzyme activity or rate of reaction, measured as average time it takes for milk
to curdle. The time it took for the milk to curdle was measured using a stopwatch.
Controlled variable: Amount of milk placed into each of the test tubes (3mL), the amount and
concentration of rennin used (5 drops), the time test tubes were left in the water bath (5-10 mins)
and the same amount and type of milk.
Risk Assessment/Safety Procedures:
1. The milk and the enzyme may be contaminated and should not be consumed. Safety gloves should be
worn when handling them.
2. Glassware is fragile and if broken, can cause cuts. The glassware was placed in the centre of the table
and handled carefully to avoid breakages.
3. Hot plate/water bath can cause burns. Care should be taken while handling the hot water bath.
4. Care was taken when using mercury thermometers as if they break the glass can cut and mercury is
poisonous and in contact with the skin can cause an allergic rash.
1. Prepare four water baths by half-filling Styrofoam cups with different amounts of tap water, hot
water and ice to give each one a different temperature: 20C, 30C, 40C and 50C. Label each cup
with its temperature.
2. Add 3mL of milk to each of 8 test tubes using a pipette. Label 4 test tubes A and 4 B.
3. Place one A test tube and one B test tube into each of the water baths, and leave for 5-10
minutes to allow the contents of both tubes to come to the temperature of the water bath.
4. While you are waiting, crush a junket tablet in 10mL of water in a small beaker. Ensure the junket and
water are fully mixed before doing the next step.
5. After the test tubes have reached the required temperature, add 5 drops of junket solution to each of
the test tubes labelled A. Shake each tube to mix the contents, and replace in the water bath.
Record the time when the rennin (i.e. junket solution) was added in each case.
6. Examine the tubes each minute for 20 minutes by tilting them gently, but not shaking them. Record
the time taken for the contents of each tube to clot, and enter it in the results table. If any tubes show
no clotting in the 20 minutes allowed, record a negative sign in the table.

It was found that the fastest time was at 40C. The rate increased steadily until 40C and then dramatically
stopped. The milk at the higher temperatures did not clot.
Temperature Clotting time Enzyme Activity (1 Clotting time)
10C - -
20C 12 minutes (720) 0.00138
30C 6 minutes (360) 0.00278
40C 2 minutes and 36 seconds (156) 0.00641

Conclusion: Enzymes work best at an optimum temperature. In the experiment, the optimum temperature
was 40C. This is because rennin is produced by the walls of the stomach where the temperature is about 37C.
As temperature increase beyond the optimum, the enzyme activity decreases till it finally stops because
enzyme is denatured. If temperature is decreased lower than the optimum, the rate of enzyme activity slows.
1. a) The contents of the tubes labelled B remained unchanged throughout the duration of the tests.
b) The purpose of the B Test tubes was as a control to determine if it was in fact the enzyme causing
the reactions to take place and that it did not already happen naturally at the given temperatures.
2. 40A was the most active enzyme activity as evidenced in the results documented as it clotted the
fastest it was more active at reacting with its substrate and was thus able to react with all of the milk
protein in the shortest amount of time.
3. There was no activity from 10-20 degrees. There was a gradual increase in activity from 30-40 and
then no activity from temperatures 50-70. 40 degrees was observed to be the optimum temperature
as the highest rate of activity was observed at 40 degrees.
4. a) At low temperatures there is less heat energy to collide and activate the enzyme (rennin) with the
substrate (milk protein) that is why nothing appears to be happening.
b) At high temperatures the heat energy damages the fragile and very complex structure of the
enzyme and so the enzyme is said to denature. This denaturisation deforms the complex protein
structure and the activation site on the enzyme so that it cannot bind with substrates anymore that is
why there is no enzyme activity because they are all damaged and not working.

The temperatures 30C and 40C were the only 2 samples that clotted under the initial 10 minutes yet the
controls (milk by itself) did not clot at all. This suggests that the enzyme rennin played the role in clotting the
milk at these temperatures. The 40C sample clotted the fastest at ______ seconds and thus shown to be the
optimal temperature within the test. After the initial 10 minutes, the optimal temperature had been
established at 40C and the samples that had not clotted were added to the 40C sample to determine if they
would clot.
The result was positive for the samples 10C and 20C yet negative for the 50C sample. This suggests that
something has happened to the enzymes exposed to the higher temperatures that caused them to
malfunction when returned to the optimal operating temperatures yet nothing had damaged the colder
samples. A gradual increase in activity was demonstrated between the 30-40C range however this range is too
unspecific to record an accurate optimal temperature as we may have overshot the optimal temperature or
undershot it at 40C.
For a more accurate representation of the activity of the enzyme, recordings should be made at intervals of
1C between 30 and 45C to better map the rise and fall of enzyme activity over its optimal operating range.
There may have been complications to the experiment due to the rennin solution not getting to the milk and
instead adhering to the walls of the test tube which would have affected the enzyme-substrate
concentrations. Inaccurate temperature readings or an inability for students to maintain their set
temperatures effectively will have caused errors in the data collected. A further complication is the factor of
human judgement and deciding exactly when to determine if the milk has completely clotted which may affect
clotting times. For better accuracy this experiment must be repeated.