Safaa Tatou

How Temperature Affects Enzyme Activity

Introduction:
Introducing the independent and dependant variables: Hydrogen peroxide is the by-product of
numerous biological processes, and to break down this molecule, the body uses an enzyme called
catalase. The key catalase function is protecting cells from hydrogen peroxide (H2O2) molecules by
converting them to oxygen (O2) and water (H2O). H2O2 can damage DNA (Smith, 2018).
How enzymes work: Enzymes are biological catalysts meaning they speed up reactions by lowering
the activation energy. It is important to note that enzymes are reusable and are not reactants and once
an enzyme binds to a substrate and catalyses, the enzyme is released, unchanged to be used for
another reaction. Essentially the activity of an enzyme is the rate of conversion of the substrate to
products. The active site is the region of an enzyme where substrate molecules bind and undergo a
chemical reaction (Liam Garvey, Pers. comm.).
The chemical reaction: The catalase enzyme is comprised of four polypeptide chains, with each chain
containing more than 500 amino acids. As hydrogen peroxide enters the active site of the catalase
enzyme, it interacts with two amino acids, causing a proton to transfer between the oxygen atoms.
This forms a new water molecule, and the freed oxygen atom then goes on to react with another
hydrogen peroxide molecule to form water and an oxygen molecule (Smith, 2018).
Chemical + Word Equation

The activity of catalase is highly dependent on temperature. Catalase is most effective at an optimum
temperature.
Research Question: What is the effect of the temperature (10°C - 50°C, increments of 10°C) on the
rate of reaction to produce oxygen gas in a catalase reaction, with substrate 3% hydrogen peroxide,
measured by a gas syringe?
Hypothesis: Naturally like many chemical reactions, the higher the temperature, the higher the rate of
reaction with an enzyme will generally become because there is more kinetic energy in the system
which translates to more collisions between the substrate and enzyme. However, increasing or
decreasing the temperature outside of an optimal range can affect chemical bonds within the enzyme
and change its shape. If the enzyme changes shape, the active site may no longer bind to the
appropriate substrate and the rate of reaction will decrease. Dramatic changes to the temperature and
pH will eventually cause enzymes to denature (Biology LibreTexts, 2021). Therefore, I predict that
the rate of reaction will continue increasing as the temperature increases until the solutions reach a
certain temperature and then the yeast catalase will denature. According to Mitsuda and Yasumatsu,
the optimum temperature of catalase in spinach leaves is 15°C and in rice-plant leaves is 35°C
(Mitsuda and Yasumatsu, 1995). According to this study, how a catalase enzyme works seems to be
highly dependent on the life history of the organism. On the other hand, Horst, Rueda and Ferreira
found it to be between 40°C and 60°C depending on the catalase type they used during
experimentation (Horst, Rueda, Ferreria, 2006). This leaves me to predict the catalase will denature
between any of these values presenting a rather big but also accurate range of expected optimal
temperatures.

Figure 1: This graph supports my hypothesis in that it displays the increase of rate of reaction as the
temperature reaction particularly during the initial rate but as soon as the enzyme reaches the optimal
temperature the rate of reaction drops and the reaction ends (the enzyme denatures).
Different sources of enzymes have different optimal temperatures and through this experiment, I want
to understand what temperature is optimal for the yeast catalse in this reaction and whether the
general hypothesis of increasing the temperature is indeed valid.
Variables:
Independent Variable: Temperature of catalase and hydrogen peroxide solutions (10°C, 20°C, 30°C,
40°C,50°C)
Dependant Variable: Rate of reaction (cm3/s)

Controlled Variable Why it is being controlled How it is being controlled

Source of catalase
Substrate concentration
Operational stability of yeast
catalase is 3.5-fold higher than
the stability of bovine catalase
and much higher during cyclic
decomposition of 50 mM
H2O2(IP;,IM;Chernikevich,
2019).
Increasing Substrate
Concentration increases the
rate of reaction. This is because
more substrate molecules will
be colliding with enzyme
molecules, so more product will
The source of catalase will be
controlled in that I will only
be using yeast and not
switching between liver and
yeast.
During the reaction the
substrate concentrate will stay
the same. This means that I
will be using only 3% H202
during my experiment.
 be formed (A level notes,
2018).
pH Any change in pH above or belo The pH will stay the same in
w the Optimum will quickly cau that I will not be adding acid
se a decrease in the rate of nor base to any of my
reaction, since more of the solutions.
enzyme molecules will
have Active
Sites, whose shapes are not (or
at least are
less) Complementary to
the shape of their Substrate (A
level notes, 2018).

Enzyme concentration Likewise, to the substrate The enzyme concentration

concentration, increasing will ensured to stay the same
Enzyme in that I will only be using
1cm3 of yeast during the
Concentration will increase the
experiment.
rate of reaction, as more
enzymes will
be colliding with substrate mole
cules (A level notes, 2018)

Materials:
3% catalase solution (enzyme source), heating plate, beaker, graduated cylinder, conical flask + stand
+clamp, gas syringe, 3% H202 supply, thermometer, recording device
Set-Up Diagram

(chemix.org)
Method:
The first step to the experiment is of course to review any safety precautions necessary to be taken.
Refer to the table (Table 1) below the method. Once, I collected all the materials shown above, I
started with the 20° temperature experiment as it is simply room temperature, and I didn’t need to
use the heat plate nor ice cubes here (I used a thermometer to make sure that the solutions were
indeed 20°). I poured the hydrogen peroxide into the conical flask and as soon as I poured in the
yeast solution, I closed it using the stopper (and made sure it was properly closed) started the timer
and continuously stirred the mixture using a regular hand movement. This was to ensure the
enzymes were well distributed with the substrate. I kept in mind (especially when using higher
temperatures) that the reaction is not too fast in that the syringe pops off. As soon as the catalase
was added to the H2O2, I started recording the gas syringe retrieving the data of gas produced. Once
I reached 30 seconds of moving the conical flask, I removed the stopper, pushed the syringe back in
and started cleaning up and preparing for the next round. I simply rinsed the conical flask with water
and ensured cleanliness by using distilled water. Once I refilled the measuring cylinders with the
hydrogen peroxide and yeast, wiped down the surface, and began the next round. This time I used
the heat plate. I plugged it into an outlet and turned it on to about roughly three. I grabbed a large
beaker and filled it with tap water about halfway. Then placed it onto the heat plate and put a
thermometer inside. Once it reached about 50°C (which shouldn’t take long) I took out the
thermometer. Then I put the hydrogen peroxide (in the conical flask) and the yeast (in the measuring
cylinder) both inside. Each had a thermometer inside and I monitored it until both reached 30°C.
Once they did, I took them out and as soon as I did that, I poured the yeast into the conical flask,
closed the stopper, and began monitoring the results on the gas cylinder. This is because the
temperature would decrease if I waited too long. I repeated the same process of monitoring the gas
cylinder until the time reaches 30 seconds. I repeated the same steps, the only difference being the
temperatures of the hydrogen peroxide and the yeast.

Table 1: Safety Rules to be reviewed before starting the experiment

Hazard Possible harm Possible Precaution

Hydrogen peroxide solution
Heat Plate
Causes skin and eye irritation
Burns damaging your skin
Wear eye protection (ex.
Safety glasses). If you get any
of it on your skin, immediately
wash your hands. Don’t touch
your face while completing the
experiment.
Just make sure you are not
touching the actual surface of
the heat plate and make sure
no clothing items touches the
part that warms up!

Table 2: Three trials of gas collected using a gas syringe during a hydrogen peroxide yeast reaction at
temperatures 10°C, 20°C, 30°C, 40°C and 50°C over 30 seconds

Gas collected over 30 seconds (10s , 15s , 20s , 25s , 30s) (cm3)
 Temp (°C) Time (s)
10 15 20 25 30
12 20 25 30 35
10 10 20 25 27 38
13 18 23 32 37
17 23 32 39 47
20 20 30 39 42 52
18 26 37 40 54
24 39 44 58 66
30 26 37 50 60 70
28 35 52 63 68
21 35 45 55 66
40 32 43 51 60 71
35 44 52 61 73
23 38 40 54 61
50 26 36 42 52 58
20 30 39 50 59

Table 3: The rate of reaction calculated by finding the average of 3 slopes (calculated by dividing the
gas produced during the first 20 seconds by its corresponding time and dividing the results by 3) per
temperature from the 3 trials.

rate of
reaction
temp (°C) (cm3s-1)
10 1,27
20 1,77
30 2,27
40 2,00
50 1,73
Figure 2: The Rate of Reaction (cm3s-1) of a hydrogen peroxide yeast reaction at different
temperatures (°C) with maximum error bars calculated by decreasing the mean from the highest
slope value of the three trials and the minimum error bars calculated by decreasing the lowest slope
from the mean.

The data processing that I carried out is directly related to my research question because I went
through a series of refining the data to calculate the rate of reaction. First of all, I found the slope of
each trial by taking the first three results of each trial and divided it by the first three time
increments (10°C,15°C,20°C). This is because I am interested in the initial reaction so the first 20
seconds or so of each trial. Once I have found the slope of all trials, I then calculated the average of
them. This gave me the data values present in graph 2. This is because this is the rate of reaction.
Naturally, I also calculated the maximum and minimum values to generate error bars. This provides a
range or spread of data that represents a built-in uncertainty.

Slope Example: An example is for 10°C, I divided 12 by 10, 20 by 15 and 25 by 20. This calculated the
slope of my first try. This is because I divided the gas produced by the time taken.

Uncertainty Example: An example is for 20°C, I decreased 1,00 (the lowest slope from the temp. 10)
from 1,27 (the mean). This gave me 0,27. This is the minimum uncertainty for the first point on my
graph.

The different graphs and tables provide us with different and essential information in understanding
the effect of temperature on the rate of reaction. Table 3 provides the direct answer to the research
question and supports my hypothesis to a certain extent. Not only does the table display the
increase in the rate of reaction as the temperature increases but it also shows how the rate of
reaction began to decrease as soon as the optimum temperature was reached. In my hypothesis, I
had predicted that the optimum temperature of catalase to be anywhere from 15°C - 60°C but my
results display 30°C as the optimum temperature. This is because the rate of reaction was 2 cm3s-1 at
40°C but at 30°C it was 2,77 cm3s-1 (refer to table 3) . This data tells us that the catalase denatured at
40°C and the reaction stopped. One can see that although an increase in temperature, increases the
rate of reaction, it is only to a certain degree. This is due to the enzyme beginning to lose its three-
dimensional shape because the intra-molecular bonds are being stressed and broken. When the
enzyme loses its shape including the shape of the active site, it has denatured. The trend shown on
graph 2 displays a constant and gradual increase and then a sharp decrease. One should note the
steepness of the line in the first 10 seconds and how it slightly flattens out as we move past the
initial reaction. Were I to have collected more data in terms of another temperature, then we would
see the line continue downwards in a fast pace. It is interesting to note the difference between the
rates of reaction of the temperatures in the graph. Naturally at the initial reaction, we would expect
the points to be doubled but instead they increase at a rather steady and regular pace.

Although the method, shown above, was effective in that I did achieve data that can be understood
and that does explicit enzyme activity in a reaction, there are many improvements that could have
been made to my apparatus to not only attain a larger range of data but to also ensure the reliability
of my data. Although I was able to get adequate data of the initial reaction, the first 10 seconds
could not be recorded because I was conducting this experiment alone and so I had to put the
stopper into the conical flask with two hands and then start stirring it with one hand as I take my
phone to start filming with the other. An improvement that ought to be made to this methodological
step is by utilizing another retort stand and adjust my recording device on the claws of the stand.
This would mean that I will get the amount of gas produced exactly from start to finish leaving no
data out. Moreover, the process of pouring the yeast into the conical flask and then placing the
stopper inside the conical flask meant that I lost some gas in the process. Although it may be
minimal, it still effects both the initial reaction and in general the reliability of the data produced.
Therefore, were I to do this experiment again I would use a syringe to inject the catalase into the
conical flask which would at the same time be connected to the gas syringe through another tube
ensuring no gas is escaping the conical flask. An example can be recognized in figure 3.

Figure 3: Although the instrument of measurement is different here (I used a gas syringe attached to
a retort stand), the idea of injecting the yeast into the conical flask is the improvement that I would
conduct were I to do this experiment again.

In terms of how I chose to collect the data, I would include more of a range in the temperatures so
possibly going up by increments of 5°C. This is because although I can deduct from my data that at
40, the yeast catalase denatured, it could have honestly been anywhere between 30 and 40 degrees,
and I do not have an exact value to support my hypothesis.

Naturally, there were also many strengths of my apparatus and method that I ought to mention.
Using a handheld thermometer turned out to be especially useful in that it was quite easy to operate
and monitor. Simply by sticking it into any solution, I was able to tell the temperature in a matter of
seconds. I used it in water, acid and yeast and it worked very well! Moreover, the heat plate was
efficient in that it heated up very quickly. I would simply place a beaker of water onto the heat plate
and place a thermometer inside until it reached 50°C, then I would turn off the heat plate to save
energy. The heat plate would continue to stay very warm and keep the water at the regular
temperature of 50°C which aided me in completing as many trials as possible in a short amount of
time. Also using ice cubes to cool down the water was a genius idea as it was also easy to operate
with. I would empty out the beaker with warm water, fill it with tap water, place handful of ice cubes
and thus the temperature would decrease quickly also monitored with the handheld thermometer.

In conclusion, my data collection was highly successful in that I was able to get an insight on enzyme
activity and how temperature really effects how fast of a rate of reaction an experiment can have.
Specifically, I got to understand how enzyme denaturation functions and the ideology behind an
optimum temperature at which enzymes work best and the reasoning behind different optimum
temperatures of the enzyme catalase in different organisms.

Bibliography
How Does Temperature Affect Catalase Enzyme Activity? (2018). Sciencing.
https://sciencing.com/temperature-affect-catalase-enzyme-activity-7776025.html

2.7.2: Enzyme Active Site and Substrate Specificity. (2017, May 6). Biology LibreTexts.
https://bio.libretexts.org/Bookshelves/Microbiology/Book%3A_Microbiology_(Boundless)/2%3A_Ch
emistry/2.7%3A_Enzymes/2.7.2%3A__Enzyme_Active_Site_and_Substrate_Specificity

Factors affecting enzyme action - What happens in cells and what do cells need? - OCR Gateway -
GCSE Combined Science Revision - OCR Gateway - BBC Bitesize. (2022). BBC Bitesize.
https://www.bbc.co.uk/bitesize/guides/z9jrng8/revision/3#:~:text=Higher temperatures disrupt the
shape,enzyme will have been denatured .&text=The enzyme%2C including its active,or the reaction
will stop.

Factors affecting Enzyme Activity | A Level Notes. (2018). Alevelnotes.com; Alevelnotes.com.

https://alevelnotes.com/notes/biology/biological-molecules/enzymes/factors-affecting-enzyme-
activity#:~:text=Increasing Substrate Concentration increases the,more product will be formed.

Mitsuda, Hisateru, and Katsuharu Yasumatsu. “Studies on Plant Catalase.” Bulletin of the
Agricultural Chemical Society of Japan, vol. 19, no. 3, July 1955, pp. 208–213,
10.1080/03758397.1955.10857290.

Horst, F., et al. “Activity of Magnetite-Immobilized Catalase in Hydrogen Peroxide

Decomposition.” Enzyme and Microbial Technology, vol. 38, no. 7, May 2006, pp. 1005–
1012, www.sciencedirect.com/science/article/abs/pii/S0141022905004059,
10.1016/j.enzmictec.2005.08.035. Accessed 21 Feb. 2022.

IP;, IM;Chernikevich. “Comparative Kinetic Characterization of Catalases from Candida

Boidinii Yeast and Bovine Liver.” Biochemistry. Biokhimiia, vol. 62, no. 4, 2015,
pubmed.ncbi.nlm.nih.gov/9275276/. Accessed 21 Feb. 2022.