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The Effect of Temperature on the Activity of Rennin in Milk

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The Effect of Temperature on the Activity of Rennin in Milk

Aim:

To find out what effect different temperatures have on the enzyme,
rennin, in milk.

Introduction

An enzyme is a biological catalyst. It speeds up a reaction by
lowering the activation energy required to start the reaction. It
speeds up a reaction, but remains unchanged unless certain limiting
factors are introduced. It is composed of polymers of amino acids. An
enzyme has an optimum pH and temperature. When an enzyme is at its
optimum conditions, the rate of reaction is the fastest. In their
globular structure, one or more polypeptide chains twist and fold,
bringing together a small number of amino acids to form the active
site, or the location on the enzyme where the substrate binds and the
reaction takes place. An enzyme has an active site, which has a unique
shape into which only a substrate of the exact same unique shape can
fit. When this substrate fits into the active site, it forms an
enzyme-substrate complex. This means that an enzyme is specific. The
bonds that hold enzymes together are quite weak and so are easily
broken by conditions that are very different when compared with their
optimum conditions. When these bonds are broken the enzyme, along with
the active site, is deformed, thus deactivating the enzyme. This is
known as a denatured enzyme. The primary structure is the sequence of
amino acids that make up a polypeptide chain. 20 different amino acids
are found in proteins. The exact order of the amino acids in a
specific protein is the primary sequence for that protein.

[IMAGE]

[IMAGE]Protein secondary structure refers to regular, repeated
patterns of folding of the protein backbone. The two most common
folding patterns are the alpha helix and the beta sheet.

[IMAGE]

In this experiment, the enzyme rennin will be used. Rennin is a
coagulating enzyme occurring in the gastric juice of the calf, forming
the active principal of rennet and able to curdle milk. It is formed
in the stomach glands of calves. Rennin is a mixture of enzymes called
chymosin. Chymosin is a proteolytic enzyme that is secreted by the
cells lining the stomach in mammals. Its role in digestion is to
curdle or coagulate milk in the stomach, a process of considerable
importance in a very young animal. If the milk were not coagulated, it
would rapidly flow through the stomach and miss the opportunity for
initial digestion of its proteins. Rennin efficiently converts liquid
milk to a semisolid like cottage cheese allowing it to be retained for
longer periods in the stomach. Rennin secretion is maximal during the
first few days after the animal's birth but it declines thereafter
replaced by the secretion of pepsin as the major gastric protease.
Chymosin is similar to pepsin in the sense that it is secreted as an
inactive proenzyme and is activated on exposure to acid. It is also
similar to pepsin because it is most active in acidic environments,
i.e. at a very low pH.

Rennin is also a very important industrial enzyme because it is widely
used in cheese making. In the past rennin was extracted from dried
calf stomachs for this purpose but the cheese making industry has
expanded beyond the supply of available calf stomachs which have to be
from young calves. Many proteases are able to coagulate milk by
converting casein to paracasein and alternatives re readily available.
'Rennet' is the name given to any enzymatic preparation that clots
milk. In order for milk to coagulate and eventually become cheese,
enzymes must be added to breakdown the proteins that keep milk a
liquid. All of these enzymes are in the protein breaking subclass
known as proteases. The best proteases or coagulants for making cheese
are the types that break a specific protein called kappa casein. When
the kappa casein is broken the milk loses its liquid infrastructure
and begins to coagulate.



Preliminary Experiment:
=======================



Apparatus
=========

3 Test tubes Big Beaker (acts as a water bath)

0.5% rennin Thermometer

Milk Test tube rack

2 Syringes wit measuring guides Stopwatch



§ Three test tubes should be taken and 5ml of milk should be measured
using a syringe into each of them.
=====================================================================



§ 5ml of milk should be put into one test tube and it should be at
room temperature (which should be noted). 1ml of rennin should then be
added. The stopwatch should be started and a bung should be placed on
the test tube to help prevent evaporation. Every 15 seconds, the test
tube should be tipped to approximately 45º. Once the milk has fully
clotted, i.e. it does not move when tipped, the stopwatch should be
stopped and the time should be noted.
======================================================================



§ Hot water should be put into the large beaker. This acts as the
water bath.
=================================================================

§ A thermometer should be placed into pone test tube ands this should
be placed in the water bath until it reaches a temperature of 60ºC.

§ 1ml of rennin should be added to the test tube and the stopwatch
should be started. A bung should also be placed on this test tube.

§ This should then be repeated except the temperature of the milk in
the test tube should be at 40ºC instead of 60ºC.

§ All the times should be recorded.

§ If a test tube does not coagulate after 12 minutes, then it can be
classified that no time has been obtained.

Temperature/ºC

Time/s

22.5

-

40

240

60

-

Results



Conclusion
==========

No times were obtained for the milk when it was at room temperature
and 60ºC. This shows that the rennin did not have enough kinetic
energy to work when the substrate was at room temperature and it had
denatured when the substrate was at 60ºC. So these temperatures cannot
be used in the main experiment. Therefore it was decided that the
lowest temperature used in the main experiment would be 25ºC and the
highest temperature would be 55ºC. The temperature range was not too
low so that the enzyme would not have too little kinetic energy and it
was not too high so that the enzyme would not denature too much.



Prediction
==========

I think that as the temperature increases, the smaller the amount of
time the rennin will take to clot the milk. Therefore as the
temperature increases, the activity of the rennin will also increase.
As seen in the preliminary experiment, if the temperature is too low
it takes a long time for the rennin to coagulate the milk. This is
because as rennin is an enzyme, it will not be able to have enough
kinetic energy for the reaction to take pace. The rate of reaction is
extremely slow due to the very slow diffusion of enzyme and substrate
molecules. Conversely, as the temperature increases beyond
approximately 37°C, which is its optimum temperature, the active site
of a rennin molecule will start to denature and so the 'lock and key'
method would become less and less effective. Beyond a certain
temperature the enzyme will have fully denatured and so it will no
longer fit into the substrate.

Rennin's optimum temperature is about 37° because it works in the body
of a mammal and a typical mammalian body temperature is around 37°C.
At low temperatures, the reaction would be very slow because the
molecules are moving relatively slowly. The substrate molecules will
not often collide with the active site of the enzyme and so bonding
between the pairs of molecules is rare. As the temperature increases,
the enzyme and substrate molecules gain more kinetic energy, so they
move around at a greater speed. The collisions between the substrate
and enzyme molecules are more frequent and this allows a greater
number of substrate molecules to enter the active site of the enzyme
more often. When they do collide, they do so with more energy. It is
easier for bonds to be broken so that the reaction can occur.

As the temperature increases even further, the speed of movement of
the molecule also increases further. However, when the temperature
reaches a certain value, the structure of the enzyme vibrates so
energetically that some of the bonds holding the enzyme molecule in
its precise shape begin to break. The primary structure of rennin,
which is an enzyme, is made up of proteins that are divided into
sub-units called amino acids. These are called peptides bonds. A
polypeptide or protein molecule may contain several hundred amino
acids. As the temperature ascends to beyond the optimum temperature
required, there are an increased amount of permanent bonds made.
Nonetheless, as these bonds are made the secondary structure of rennin
has an effect on how the enzyme retains its shape. Amino acids in a
polypeptide chain have an effect on each other even if they are not
directly next to each other. The chains bend upon themselves and so
distorting the shape of the molecule. The thermal energy breaks the
hydrogen bonds holding the secondary structure of the enzyme together,
so the enzyme, and especially the active site, loses its shape to
become a random coil. The substrate can no longer bind, and the
reaction is no longer catalysed. At very high temperatures this is
irreversible.

The graph for the time against the temperature should look like this:

At first the rennin takes a long time to coagulate the milk. As the
temperature rises and the molecules begin to gain more kinetic energy,
the time starts to decreases, whereby when it reaches around 40°C, the
time is the smallest throughout the experiment. As the temperature
surpasses the optimum of the rennin, the time begins to increase once
more. At this point the active sites of the rennin molecules begin to
get more denatured until eventually they cannot denature any further,
so the time would remain constant.

The second graph of the temperature against the rate of reaction
should look like this:

This is a typical curve which can be applied to the activity of almost
any enzyme taking into account that the optimum temperatures for
certain enzymes are different. This is the opposite of the time
against temperature graph. The curve goes through the origin because
at 0°C, the molecules do not have enough kinetic energy to move and
collide with the other molecules causing a reaction to occur. The rate
then slowly increases until it attains its optimum temperature around
40°C. Here the rate is at its peak. From here it slowly decreases due
to the denaturing active sites of the rennin molecules, whereby it
reaches the point where there is no further reaction at a certain
temperature. This is where the reaction stops and so the rennin is no
longer able to coagulate the milk.



Plan:
=====


Apparatus Diagram of Apparatus
------------------------------

Test tubes

1 Boiling Tube

Stopwatch

Test tube rack

2 Syringes wit measuring guides

Big Beaker (acts as a water bath)

Thermometer

Clamp

Clamp stand

Bungs

0.5% rennin

Milk

· The clamp and the stand should be set up. A boiling tube should be
placed in the clamp and rotated until it is slanted at an angle of
about 45° to ensure that a fair test is carried out. This is so that
all the test tubes are at the same slant height when they are being
tested.

· Three test tubes should be placed in the test tube rack.

· 5ml of milk should be measured out using the syringe and this should
then be poured into a test tube. This should then be repeated twice
more for the other two test tubes.

· The temperature of the milk in each test tube should then be
measured. It should approximately be at room temperature in which case
the milk should be heated, as it is not at the correct temperature
(30°C).

· This can be done by putting quite hot water into a large beaker. A
thermometer should then be placed into one of the test tubes
containing the milk and the all three test tubes should be placed in
the beaker which acts as a water bath. The thermometer should be
placed in each of the test tubes alternately to ensure that the
correct temperature is being attained for each test tube.

· When the temperature reaches 30°C, the test tubes should be taken
out of the water bath. If any of the test tubes exceed the required
temperature, a thermometer should be paced in that particular one and
it should then be left to cool down until it is at the precise
temperature.

· 1ml of rennin should be measured out using another syringe.

· Once they are all at the required temperature, 1ml of the rennin
should be added to the test tube. At the same time, the stopwatch
should be started and a bung should be placed on the test tube.

· This should then be repeated ten seconds later with the second test
tube, i.e. when the initial test tube has had ten seconds to
coagulate. Once again, this should be repeated with the third test
tube of milk at 30°C, but this time the rennin has to be added twenty
seconds after the rennin has been added to the first test tube.

· The test rubes should be placed in the boiling tube in the clamp
every 15s. Once it can be seen that the milk is thickening
substantially but not quite fully coagulated, the test should be
carried out every 10s. This will be seen when the milk only slightly
tips when it is placed in the boiling tube. The test tubes should be
checked until the time has exceeded 12 minutes whereby it has to be
stated in the results that there was no reading obtained for that
particular experiment. Once the milk has fully clotted, it will no
longer 'slant' when it is put at an angle.

· The readings and repeats for the milk at 30°C have now been
completed.

· The whole process should be repeated with repeat readings to ensure
accuracy, except the temperature of the milk should be changed because
this is the variable in this investigation. The temperatures that the
milk has to be heated to are 30ºC, 35ºC, 40ºC, 45ºC, 50ºC, and 55ºC.

· All the readings should be noted in a results table.

Factors to keep constant

Factor to change

The type of milk.

The temperature of the milk.

The concentration of rennin (0.5%).

The amount of rennin put into each test tube.

The amount of milk put into each test tube.

The angle at which the boiling tube is set at (the clamp will ensure
this).


Precautions to undertake

§ Care has to be taken to ensure that the two samples of rennin and
milk do not get cross-contaminated. This can be done by using separate
syringes to measure out both substances.

§ Care should be taken when handling hot water. Goggles may be worn
although they are not always necessary.

Results

Temperature/ºC

Time taken for milk to coagulate/s

Time (1)

Time (2)

Time (3)

Average Time

30

-

-

-

-

35

630

600

*540heat

615

40

*180heat

220

210

215

45

175

185

190

183 (3sf)

50

*210cool

270

250

260

55

*520heat

465

480

473 (3sf)


KEY
---

* = An anomalous value so it is not used to calculate the average
value.

- = Result not obtained because it took over twelve minutes



(3sf) = Value has been rounded to three significant figures
===========================================================



(2sf) = Value has been rounded to two significant figures
=========================================================

Temperature/ºC

Rate of Reaction (2sf)

30

No rate obtained as no time was obtained

35

0.0016

40

0.0047

45

0.0055

50

0.0038

55

0.0021

NB: The rounded values for the time at 45ºC and 55ºC were used to
calculate the rate of reaction.



Conclusion
==========

The results obtained are not very similar. However, the graph for the
time against the temperature agrees with my prediction, even though
the predicted peak point was at a lower temperature than the actual
graph obtained. At 45ºC, the time taken for the milk to fully
coagulate was the least and therefore the rate of reaction was the
greatest. The shape and trends of the graphs support my prediction;
however as some times went above 12 minutes, no results were obtained
and this was not accounted for in the prediction. Consequently, there
were fewer points on the graph which give less accurate overall
curves. There are trends in the results which show that the time
decreases to a certain point, but after this point (in this case it
was at 45ºC) the time starts to increase once more as the temperature
begins to travel past the optimum required for this reaction. In this
aspect the results did support the original prediction, even though
the prediction did not state that same optimum temperature and in
reality the optimum temperature was higher than expected. As
temperature increases, the molecules, according to the 'Kinetic
Theory' move faster, due to increased energy. Therefore, the enzyme
and substrate molecules will meet more often and the rate at which the
product is formed will increase. However, as the temperature continues
to rise the hydrogen and ionic bonds, which hold the enzyme in shape,
break and the active site will no longer accommodate the substrate.
The enzyme is then said to be denatured. If the temperature falls
below 37°C, according to the kinetic theory, the enzyme and substrate
molecules will not be receiving as much energy, and therefore their
movement will slow down as the reduced heat reduces the amount of
energy the molecules are receiving. This will reduce the number of
collisions between the rennin and the milk particles, and there is a
higher probability that these collisions will be unsuccessful due to
the lack of energy. Therefore the rate of reaction will reduce.

Once higher temperatures are restored, the rennin will regain its
catalytic influence. Above 37°C, which is rennin's optimum working
temperature, as this is body temperature, and rennin is found within
the stomach of young mammals; the rate of reaction will decrease. This
is because the rising temperature affects the hydrogen and ionic bonds
which determine the shape of the enzyme. As these bonds are broken the
shape of the active site changes and the molecules of caseinogen (in
the milk) can no longer occupy them. The rennin is now denatured and
the rate of reaction will become zero. If the temperature is increased
above the optimum level, then a decrease in the rate of reaction
occurs despite the increasing frequency of collisions. However, the
molecules vibrate so energetically that certain bond that make up the
secondary and tertiary structure begin to break and so the shape of
the enzyme is no longer exact. The secondary and tertiary structures
of the enzyme are disrupted and as many weak bonds break, the enzyme's
tertiary structure unfolds.

This concludes that increasing the temperature from a low temperature
to the optimum temperature decreases the time taken for rennin to
coagulate milk and so increases the rate of reaction. Nevertheless, as
the temperature ascends above the optimum required, the rennin
molecules start to denature and so the time taken to coagulate
increases. As a result of this the rate of reaction decreases.

Evaluation

The results seem fairly reliable; conversely the replicates obtained
were not always similar to the initial values acquired. At 40ºC, 55ºC
and 55ºC, the first readings obtained were quite dissimilar to the
replicates. There were substantial differences between these values
and the repeats, so these were not involved when the average time was
being calculated (these values have been highlighted as anomalies on
the results table). To obtain a more accurate rate of reaction, the
average time was used to calculate the rate. The anomalies were at
35ºC, 40ºC, 50ºC and 55ºC and there was one anomaly for nearly every
temperature measured. The anomalous value at 55ºC was the only anomaly
that was higher than its replicates. This may have been because the
temperature may have been slightly higher and so, as rennin is an
enzyme, it may have denatured a little more. Therefore the 'lock and
key' mechanism between the substrate and the enzyme would be less
effcetive and so the reaction would be slower. Also the time may have
been longer owing to the time intervals between measurements. After
the test tube had been put in the boiling tube after 15 seconds, it
may have started coagulating a few seconds after it had been tested so
it would have taken longer for the reaction to be noticed and thus the
timing period being adjusted to 10 seconds. Hence, the time would have
been longer. In addition to this, there may have been a delay between
the time when the test tube was in the water bath to when the rennin
was added and the milk may have heated up a few degrees because there
may have been some excess hot water left on the test tube as they were
not dried. As a result of this, heat may have been transferred from
the hot water to the test tube which then have been transferred to the
milk this could also be held responsible for the lower anomalies such
as those at 35ºC and 40ºCwhich could have been heated, making then
closer to the optimum temperature of 45ºC in this experiment. Some of
the water my have evaporated in between the time when the time when
the test tube was taken out of the water bath to when the bung was
placed on the test tube after the rennin had been added. Furthermore,
the water on the bottom of the test tube may have evaporated using the
heat from the milk which may have reduced the temperature by a few
degrees. Therefore, this may have resulted in an increase in kinetic
energy which would have led to an increase in the rate of reaction
depending on the temperature. The anomaly at 35ºC and 40ºC may have
risen a few degrees and so the kinetic energy would be greater
resulting in a more rapid rate of reaction. The milk in the anomaly at
50ºC may have been slightly under the actual temperature so the enzyme
would be less denatured as it was closer to the optimum temperature.
The optimum temperature was at 45ºC which is slightly different to the
actual optimum temperature which is about 37ºC - body temperature, but
this may have been because of the pH of the milk. Rennin is produced
and used in the stomach and so its optimum pH would be about 2 since
there is hydrochloric acid in the stomach. However the pH of milk is
about 8 as it is slightly alkaline because it contains bicarbonate
ions.

If I was able to do this experiment again I would like to make the
range of temperatures even smaller, especially by breaking down the
range 20°C to 40°C into maybe 5°C intervals. This would enable me to
achieve an even more specific and accurate temperature at which rennin
works to its optimum and I would do four repeats rather than two so
that the average time can be more accurate. I would also keep timing
the results that exceed 12 minutes. This method was not extremely
reliable as many things could have potentially gone wrong. Firstly,
the problem of heat loss or gain could have considerably changed the
results by lowering or raising the temperature by a few degrees. Some
kind of insulation should have been placed around the test tube
constantly. This could have been in the form of cotton wool or bubble
wrap. Also the rennin may have cooled down or heated up whilst it was
in the beaker owing to the room temperature and the temperature of the
rennin at the start (without the milk was never measured). This may
have also affected the actual test tubes which contained both the
rennin and the milk. Again, to improve this, some form of insulation
should be used and the temperature of the rennin should be taken
before it is added to the milk. One thermometer had to be used when
there were three test tubes in the water bath, so this may have made
some of the temperatures of the repeats slightly different. One
thermometer per test tube should have been used or perhaps some kind
of computer software should have been used.

I would also vary the types of milk used in this experiment. For
example full fat, semi skimmed, and skimmed milk could be used and
these results can be compared. Also milk from other animals such as
goat and buffalo milk can be used. Perhaps even milk such as soy can
be tested. The ratio of substrate to enzyme can also be varied as well
as the concentration of each substance. To provide extensional
evidence for this experiment, different levels of pH should be used,
such as the optimum pH that is about 2. This could be achieved by
adding acidic or alkaline compounds to the milk. However, at certain
levels of ph the rate of reaction (the acidic pHs) would be so fast
that it would be too fast to time. Nevertheless, this could be done
using computerised equipment. This could also be done in conjunction
with varying the temperature.

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