Effect of temperature on the enzyme renin

Background Research

An enzyme is a biological catalyst in living organisms which increases the rate of

chemical reactions without being altered in the process and occurs in cells.
Enzymes are usually proteins because they are made of amino acids but can also
be RNA (ribonucleic acid). These proteins travel through the Golgi apparatus
where they are customised into a usable shape. They then emerge from the Golgi
body as enzymes. The sequences and structures the amino acids are customised
into create a specific ideal chemical environment within the active site of the enzyme. Most chemical
reactions in living things are catalyzed by enzymes which also aid in cell metabolism , digestion, etc. An
enzyme only interacts/binds with a group of substances or one substance
called a substrate. Enzymes have the important duty of lowering a reaction’s
activation energy which is the amount of input energy required to start a
reaction. To work, enzymes must bind to reactant molecules and have to hold
them in a particular way in that the chemical bond-breaking and
bond-forming processes take place more readily. Enzymes do not change a
reactions △G value because they do not affect the free energy of the
reactants or products. Enzymes, instead, lower the energy of the transition
state.
Enzymes have an optimum temperature to live in which is 37ºC. When the enzymes are in a temperature
higher or lower than 37ºC, they denature. This means that the enzyme’s active site changes shape
meaning it cannot bind to the substrate. Enzymes can no longer catalyse the reaction, meaning the
bodily systems slow down.

Catalytic Property: Due to enzymes being highly active in small quantities, they have immense catalytic
power. A minute amount of enzyme is enough to convert a large quantity of substrate and the enzymes
remain unchanged after the reaction.
Reversibility: Majority of enzyme catalyzed reactions are reversible but the reversibility of the reaction
depends on the cellular requirements. In particular cases, there are separate enzymes for forward and
reverse reaction. Some enzyme-catalyzed reactions are not reversible.
Highly specific nature of enzymes: certain enzymes can catalyse certain reactions. E.g. The enzyme
sucrase can only catalyse hydrolysis of sucrose. This highly specific nature is evident in the lock and key
and induced fit model.
Lock and Key- Due to the highly specific nature of enzymes, they can be greatly specific with their
substrate and their catalysed reactions. The lock and key model showcases the interactions between the
enzyme is similar to a key (the substrate) that is highly specific to the lock (active site of the enzyme).
This shows how the distinct shape of the active site must complement the shape of a particular substrate
so that they can ideally fit together.

Induced Fit- An enzyme slightly alters its shape when binding to its substrate resulting in a more tighter
fit. This adjustment of the enzyme to closely fit the substrate is called an induced fit. The induced fit
model shows that enzymes are flexible structures in which its interactions with the substrate cause the
active site to continually reshape until the substrate is completely bound.
Some environmental factors that can affect enzyme function due to their sensitivity to changes include
temperature, pH and the substrate concentration.
- Temperature- Generally, the higher the temperature, the higher the rate of reaction and the
lower the temperature the slower the rate of reaction. Enzymes can however lose their shape
(denature) in extremely high temperatures and stop working.
- pH- Every enzyme has an optimum pH range in which changing the pH outside of the range
results in slow enzyme activity. Like temperature, extreme pH values can cause enzymes to
denature.
- Substrate concentration- The reaction rate increases to a certain point as the substrate
concentration increases. When all the enzymes are bound, a substrate increase will not have an
effect on the rate of reaction because the available enzymes will be saturated and working at
maximum rate

This scientific report aims to test the effect of temperature on the enzyme renin with the substrate being
milk consisting of Angiotensinogen (AGT). Rennin, also known as chymosin, is an enzyme in the fourth
stomach of unweaned animals. It is only found in animals who have a fourth stomach such as cows,
goats and sheep. Rennin allows milk to be retained in the mammal’s stomach for long periods of time by
coagulating the milk. Coagulation is the process of which the substance, in this case the milk, is turned
into a solid or semi-solid.k. Rennin is important for young animals as milk is the main component in their
diet and without rennin, these baby mammals would not grow or gain the nutrients they need to
function efficiently. This could cause stunted growth and the baby calves could die without the enzyme.
Therefore, rennin is a necessary enzyme in animal growth and survival. However, rennin is absent in
humans At low temperatures, the rate of renin’s catalysed reactions decreases as the enzyme is inactive
and denatured. The enzyme cannot work efficiently as the temperatures are not in the optimal range for
the enzyme to create the activation energy necessary for a chemical reaction to occur. However, as
temperature increases, the enzyme begins to work efficiently as kinetic energy
Aim:

To determine the effect of different temperatures on the enzyme Renin through the coagulation of milk.

Hypothesis:

Test tube B will solidify the quickest in the 38℃ water bath and the state will not change in other water
baths.

Equipment List

- Thermometer
- 2 Junket tablets
- Mortar
- Pestle
- 80ml Milk
- Stopwatch
- 8 Test Tubes
- Cold Water
- Ice cubes
- Paper
- Measuring Cylinder
- Beaker
- Hot water bath
- Test tube holder
- PPE
- Marker
Risk Assessment Table

Hazard Risk Precautions Likelihood Treatment

Beaker falling and Surface level Handle the equipment with Unlikely Use a bandage to
breaking resulting cuts, in severe care and wear leather shoes suppress bleeding,
in broken glass cases deep-level to avoid injury from falling clean/disinfect the
lacerations objects. wound with rubbing
alcohol/ointment.
and notify an
adult/supervisor to
ensure a safe
disposal of the glass.

Pestle and mortar- Bruise, fractures Handle the equipment with Unlikely Apply an ice pack to
The pestle falling , broken bone/s care and wear leather shoes injury and if pain
onto your foot to avoid injury from falling persists for an
objects. extended amount of
time seek medical
attention

Hot water touching Burns, scalds Be aware of surroundings Likely Immediately run
skin and proceed with caution. If burn under
necessary, wear cool/lukewarm
gloves/mitts. water (not cold) for
20 mins. If the burn
is serious or pain
persists, seek
medical attention.

Paper Paper cut Handle with care. Unlikely Apply a band aid to
the wound. Disinfect
if necessary.
 Consumption of Choking Handle equipment with Highly Notify someone
Junket Tablet caution and do not digest unlikely immediately and
any substance. seek help.

Variables

* Independent variable- Temperature ( of water baths that the rennin+milk is placed in)
* Dependent variable- Time taken for milk to solidify
* Controlled variables- environment, amount of milk, amount of enzyme (junket tablets) that goes into
the milk, type of milk

Method

1. Set up four baths in the appropriate temperatures of 0℃, 20℃, 38℃ and 80℃.
2. Set up a beaker with a thermometer
3. Pour cold water and ice into the beaker until it reaches 0℃
4. Repeat steps 2 and 3 to achieve 20℃
5. Set up two hot water baths at 38℃ and 80℃
6. Crush 2 junket tablets using a mortar and pestle
7. Divide the crushed junket tablet into 4 even amounts on four pieces of paper
8. Label 4 test tubes A using a marker
9. Label 4 test tubes B using a marker
10. Place all test tubes in a test tube holder
11. Measure 10ml of milk
12. Pour 10ml of milk into each test tube
13. Place an A and B test tube into each of the different temperature baths
14. Leave test tubes in the baths for 5mins to ensure the milk is at a regulated temperature
15. Pour 1 of the 4 crushed junket tablet into each of the B test tubes
16. Time how long it takes before the milk solidifies using a stopwatch
17. Observe
 18. Record results into a result table

Results

28/03/21 Time Taken for milk to solidify (minutes)

Temperature of water Test Tube A (milk ) Test Tube B (milk+ rennin)

bath (°C)

0°C No noticeable change No noticeable change

20°C No noticeable change No noticeable change

38°C No noticeable change 4 mins 27 secs

80°C No noticeable change No noticeable change

28/03/22 Observations:

- When the enzyme first dissolved in the milk,

the consistency of test tube B was a bit
granular
- The consistency was slightly altered in the
20°C, becoming more dense but was not
significant enough to clot or change its state of
matter
- The 80°C bath kept both test tubes of milk at a
liquid state
- Test tubes A did not change state at all and
showed no sign of doing so
Discussion
Accuracy refers to the precision of a measurement in an experiment and was lacking in this experiment.
The inaccurate measuring of the junket tablet (enzyme) being added to the milk was a weakness in the
experiment. Since the amount of enzyme in each B test tube was inconsistent it will result in
inconsistent/inaccurate results due to the unequal distribution. To ensure accuracy, next time it would be
suggested to measure the enzyme and consistently add the same amount to each test tube to assure the
reliability. Another weakness was measuring the results. There was no form of ensuring an accurate
measurement of the time since it was just what was observed and a test tube could have possibly
solidified beforehand because human error was a high risk as humans do not have instant knowledge of
when a substance solidifies and are limited to just observe.

Reliability refers to the consistency of an experiment and comparing results achieved from repeating an
experiment multiple times. Throughout the experiment, reliability was not ensured as the experiment
was never repeated and the experiment only occurred once with a set range of temperatures. There was
no spread or range of results due to the lack of repetition and small range of temperatures. To improve,
next time the experiment should be repeated at a minimum of 5 trials for reliability and with a wider
range of various temperatures. Overall, the experiment was not completely reliable as the enzyme
rennin measurements or milk measurements may not have been exactly equal. If the equipment was not
cleaned beforehand or had traces of other substances left over, this could have severely affected the
results.

A success in the experiment was the accurate controlled temperatures of the water baths. This was done
by consistently measuring the temperature with a thermometer and adding/removing ice/water to
ensure the colder water baths were at a stagnant temperature. Another success was observing the
enzyme react with the substrate in the optimum temperature of 38°C hence the aim being reached.

The low temperatures did not coagulate the milk due to the low amount of energy forming in the
reaction which doesn’t achieve the activation energy even with an enzyme present. At high
temperatures, the rennin denatures meaning the shape of the active site changes. Hence, the substrate
(milk) can no longer bond to the active site of
the enzyme, meaning the rennin no longer
functions and the milk doesn’t clot. This is
evident in the results as the milk did not
coagulate in the extreme temperature baths of
0°C and 80°C. The controlled variables test tube
A remained unaltered with no/minimal change
in the state of matter also due to the lack of
activation energy required for the enzyme to
catalyse the experiment.
The experiment repeated by another group reinforces the results of this experiment as their optimal
temperature is 30°C and 50°C similar to that of 38°C in this experiment which can be seen in figure 8.
The other groups experiment however tested a wider range of temperatures and had multiple trials
before averaging the results making theirs a more accurate and reliable experiment. In the experiment
(shown in figure 9), only the 38°C coagulated whereas, more extreme temperatures still resulted in the
clotting of milk in the other group’s experiment. In this experiment's graph (figure 9) 0 does not mean
that the milk solidified straight away, rather, it never solidified in the given time whereas the other
group’s graph has a set time period in which all the rennin filled test tubes ended up coagulating.

The results relate to the real world because, as mentioned in the background research, enzymes have an
optimum temperature to live in which is 37°C. When the enzymes are in a temperature higher or lower
than 37°C, they denature. This can be seen in the experiment results of the milk solidifying at 38°C which
is a highly similar temperature and the enzyme had no effect on the extreme temperatures causing them
to remain in a liquid state. Rennin comes from the juxtaglomerular glands, which are the cells in the
kidney, which release the rennin from the storage granules. Rennin’s unique substrate is called
Angiotensinogen (AGT), which is produced by the liver. Rennin allows milk to be retained in the
mammal’s stomach for long periods of time by coagulating the milk.
Conclusion

The results of this experiment concluded that the enzyme rennin catalysed a greater reaction in the 38°C
water bath and had no effect on other temperatures which supports the hypothesis. In conclusion, the
aim was achieved and the hypothesis was correct as enzymes can be seen to work efficiently in a
temperature close to 37°C (38°C).

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