Enzyme Reactions and Temperature

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Enzyme Reactions and Temperature


Aim - To determine the effect of temperature on the rate of reaction
of rennin when it reacts with milk to cause coagulation.

Hypothesis - As rennin is naturally found in the human stomach I
believe that the optimal temperature for reaction will be
approximately 37°C as this is the regular human body temperature. This
is shown by the graph below.


Background Research

Rennin is "an enzyme that catalyses the coagulation of milk, found in
the gastric juice of the fourth stomach of young ruminants and used in
making cheeses and junkets. Also called chymosin, rennet."[2] It is
also found in the gastric juices, or Gastric mucosa, of many other
mammals, including humans. In the human stomach, particularly those of
infants, rennin works to curdle milk so that pepsin, another stomach
enzyme, can further breakdown the proteins into absorbable amino acids
called polypeptides

Several experiments have already been conducted testing similar
hypothesis and having similar aims. All of these experiments had very
similar results. They found that approximately 37°C was the optimal
temperature for rennin, it was at this temperature that the milk
solidified quickest. Below that the reaction would occur far more
slowly, sometimes taking hours to complete, sometimes not reacting at
all. Above 37°C at approximately 45°C the enzyme would become
denatured, and the reaction would never occur, even after the
temperature was lowered back down to 37°C.

Name of enzyme - Rennin

Name of substrate - Milk


- 50ml of full cream milk.

- 10 small test tubes.

- 5 140ml beakers.

- Access to both hot and cold water supplies.

- Pipettes.

- Junket tablets.

- Mortar and Pestle.

- Distilled water.

- Stopwatch.

- Thermometer.



-Using a pipette add 5ml of milk to each of the 10 small test tubes.
Label 5 controls and 5 variables.

-Place 100ml of water into each of the beakers, and using hot and cold
water and thermometers raise the temperature of the first beaker to
20°C, the second to 30°C, the third to 40°C, the fourth to 50°C and
the sixth to 60°C. These will be the water baths.

-Place 2 test tubes, one control and one variable, in each of the
water baths.

-Whilst waiting for the milk to adjust temperature using the mortal
and pestle grind up approx. 2 junket tablets and add 25ml of distilled

-Recheck the temperature of the water baths and test tubes and make
any necessary adjustments by adding hot or cold water to the beakers.

-Add 4 drops of the rennin solution to each of the variable test tubes
and time how long it takes for the milk to solidify.

-Finally check your control test tubes to ensure that they have not
solidified due to the temperature.

Safety precautions that would need to be taken can be seen on included





Did not solidify within time limit


2min 25sec


1min 20sec


Did not solidify (most likely denatured)


Did not solidify (most likely denatured)

Also none of the control test tubes solidified during the experiment.


My conclusion is that my hypothesis was accurate. The optimal
temperature based on the results was 40°C. However it is likely that
this temperature could actually lie somewhere between 30°C and 40°C,
most likely closer to 40°C. The reaction slows down considerably at
20°C taking longer than 15mins to clot. Above 50°C it appears that the
enzyme was denatured as the milk did not coagulate at all. As none of
the control experiments coagulated during the experiment we can safely
assume that the results found were the product of rennin, not simply
the effect of the increase in temperature on the milk.


A control experiment must be performed to eliminate the possibility
that the results are produced by a factor other than the one being
tested. A Control experiment and the variable must be exactly the same
except for on factor, the variable, which is what will be tested. My
controls eliminated the possibility that the milk was simply clotting
because of the increase in temperature. As the milk in the controls
did not clot we can assume that the milk did indeed clot due to the
rennin in the variable experiments.

The variables I controlled were concentration of the rennin, quantity
of substrate (milk) and enzyme, quality of the milk, and temperature.
The concentration of the rennin was controlled by ensuring all rennin
was taken from the same solution. The same quantity of both milk and
rennin were used in order to make sure it is fair. All milk used was
also taken from the same carton to ensure there were no differences in
the quality of the milk. The temperature difference between the
control and variable was also controlled by placing both test tubes in
the same water bath. This ensured that both test tubes were the same

The results were similar to what I expected. I was not sure if I would
get a result for the 50°C experiment as I was not sure if the enzyme
would have been denatured yet, but obviously it had been. The results
supported my hypothesis to a certain degree, as the optimal
temperature shown by the experiment was 40°C I was not able to test a
smaller scale, so I have no way of knowing whether 37°C is the optimal
temperature or another temperature around 40°C is.

If I was able to do the experiment again I would most likely not
conduct the experiment at 20°C, 50°C or 60°C and instead have more
precise measurements in between 30°C and 45°C. If I did this far more
accurate results would be achieved, and a more exact optimal temperate
would be concluded.

Accuracy could perhaps be improved by maybe taking a little more time,
as the experiment was quite rushed and some results may not be
completely accurate because of this.

Safety precautions that would need to be taken can be seen on included










[1] http://www.soton.ac.uk/~plf/ScI-Journal/vol1no2/v1n2k41.htm

[2] Dictionary.com

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