Research Question

At which pH level will the enzyme catalase, found in Saccharomyces Cerevisiae,

display the most activity when decomposing 3% hydrogen peroxide, which is
determined by the volume of oxygen gas produced? O2 H 2 O H 2 O2

Background Information
Catalase is an enzyme that can be found in many natural organisms and products
such as yeast, potatoes, and the human liver (Smith, J., & Johnson, A. (2018).
Catalase is a biological enzyme, meaning its primary function is to speed up the
reaction between specific molecules and atoms, otherwise known as substrates.
This is vital in living organisms, as without enzymes some reactions would be too
slow or may not even be able to occur, which could lead to undesirable outcomes.
The substrate that catalase uses as its substrate is Hydrogen Peroxide ( H 2 O2).

Hydrogen Peroxide is found in the lungs and gut of the human body and is produced
as a result of the breakdown of a different molecule, known as a "super oxide".
Hydrogen peroxide is a natural oxidant and disinfectant, and as a result the body
must implement the use of the catalase enzyme to break down the H 2 O2, before it
does any damage to the body's internals (Joe Schwarcz PhD | 18 Jan 2017). When
catalase catalyzes hydrogen peroxide in the body, water ( H 2 O) and oxygen gas (O2)
are produced, where the water and oxygen are absorbed into the body. The
production of oxygen gas acts as a visual indicator that a reaction is taking place,
and this can be used to measure the activity of the enzyme.

Another common source of catalase can be found in Saccharomyces Cerevisiae or

otherwise known as baker’s yeast. Saccharomyces Cerevisiae contains a large
number of enzymes including catalase, making it suitable for industries where
fermentation is required such as winemaking or bread baking (Vendatu). Thanks to
the presence of catalase in the Saccharomyces Cerevisiae, it makes it a suitable
product to break down hydrogen peroxide.

Although enzymes are not living organisms, they are still susceptible to external
factors such as temperature and the pH concentration that they are present in.
These external factors can alter the effectivness of an enzyme and can cause them
to even lose all function, also known as denaturing. Denaturing occurs as a result of
two primary reasons, high temperature and pH changes. High temperatures can
cause denaturing as the bonds holding the enzyme together break, resulting in the
loss of function of the enzyme. Changes in pH can alter the charge of the enzyme,
meaning it can no longer accept substrates as a result of its differing charge. These
variables can alter the effectivness of an enzyme, and subjecting the enzyme to
different pH's or temperatures can change its effectivness. Another external factor
does not involve the denaturing of the enzyme, but the concentration of substrate
surrounding it and the concentration of itself in a solution. A higher percentage of
substrate will result in a higher chance for a sucsessful catalysis, thus allowing the
enzyme to catalyze a larger volume of substrate. Whereas a larger concentration of
enzyme will allow for a larger number of reactions to occur at the same time, in turn
speeding up the process of catalysis.
 Hypothesis
It is hypothesized that the catalase enzymes activity, measured by the volume of O2
produced, will peak at a pH value of 7 because catalase experiences the least
denaturing at this pH level.

Variables
The independent variable of the experiment was the pH value of the buffer solution
to which the enzyme is placed. The change in pH value allowed for the measuring of
the effectivness of the enzyme when exposed to differing pH values. The
effectivness of the catalase in differing pH values was measured by the volume of O2
produced in a period of 3 min.

The dependent variable of the experiment was the volume of oxygen gas produced
in a period of 3 min. As oxygen is a product of the catalysis of hydrogen peroxide, it
could be collected and used to determine the activity of the enzyme. In order to
ensure the experiment remains fair and consistent, a number of control variables
were identified and mitigated.

Control Variables

Variable to be Possible effect on Data How variable was controlled

Controlled
Changing Changes in temperature could alter the The temperature of the
Temperatures activity of the catalase enzyme, therfore surrounding air was measured
of solutions making it impossible to determine whether using a thermometer and the
the pH or temperature was responsible for temperature of the water bath was
any change in enzyme activity. measured with a thermometer.

Mass of Changes in the mass of enzyme can alter the Precisely 1.00g of Saccharomyces
Catalase used volume of substrate catalyzed and will make Cerevisiae was measured in a
it impossible to tell if a change in pH or weigh boat on an electronic scale
change in Enzyme concentration was prior to being added to the pH
responsible for any changes in Oxygen buffer solution
produced.

Volume of Altered volumes of pH buffer solution can Precisely 25ml of buffer solution
Buffer solution have adverse effects on the catalase was measured in a measuring
enzyme, where more denaturing may be cylinder prior to being added to the
experienced if exposed to a higher volume of conical flask with the enzyme.
buffer.

Concentration Changes in substrate concentration can Through the experiment, only 5ml
of Substrate result in more substrates being catalyzed, of 3% hydrogen peroxide solution
thus resulting in the O2gas produced being was used.
unreliable
Time Allowing enzymes to catalyze within different Each experiment was run for
time period can result in inconsistent volumes precisely 3 min with the volume of
of O2gas being produced. O2gas being produced being
measured at each minute interval
 for a total of 3 measurements.
Tools
 Electronic scale (+- 0.001g)
 Stopwatch (+- 0.001s)
 Eudiometer (+- 0.025ml)
 Weigh Boats
 Conical Flask
 Rubber Stopper
 Gas Tube
 Water Tray
 Measuring Cylinder (+- 0.5ml)
 pH meter (+- 0.001)
 Thermometer (+- 0.1 degrees)

Materials
 Saccharomyces Cerevisiae (5g) Fig.1
 pH 8 Buffer solution (125ml) The set up of the
 Tap water (around 4 liters) experiment
 Hydrogen Peroxide (25ml)

Method
1. Ensure that the pH meter is calibrated and ready to take measurements.
2. Set up eudiometer by filling with water and attach to retort stand upside down in
water bath. Ensure that there is water in the upside-down eudiometer. Refer to
figure 1.
3. Measure the volume at which the meniscus of the water is at in the eudiometer.
4. Insert gas tube into bottom of eudiometer.
5. Measure out 1 gram of Bakers yeast (Saccharomyces Cerevisiae) and add 25
mL of pH buffer solution in a conical flask.
6. Swirl buffer yeast mixture and allow to mix for 5 minutes.
7. After 5 min, measure the pH of the yeast-buffer solution with a pH meter.
8. Add 5mL of 3% hydrogen peroxide to yeast suspension. Swirl 3 times before
attaching rubber stopper with gas tube to the mouth of the conical flask.
9. Allow gas to be collected for 3 min, swirling every minute.
10. After 3 min record the volume of gas produced by the reaction
11. Remove the rubber stopper from the flask and measure the final pH of the
solution with a pH meter.
12. Repeat the experiment 5 times for each pH buffer.
Ethical Issues

Issue Description of Issue Method of Minimizing

Hydrogen peroxide Hydrogen peroxide can Hydrogen peroxide was not
sourcing be sourced from human derived from human or animal
or animal sources sources but will be derived from
a synthesized sample.

Catalase Enzyme The catalase enzyme can The catalase enzyme was not
sourcing be sourced from human derived from animal or human
or animal sources sources but was derived from
Saccharomyces Cerevisiae
(baker’s yeast) with no harm to
any organisms.

Enzyme The use of biological The enzyme being used is not

Biohazards material could pose a considered a biohazard and is
biohazard if mishandled not dangerous to humans,
animals or the nearby
ecosystem.

Hydrogen Hydrogen peroxide could Appropriate PPE will be worn at

Peroxide Burns spill and cause burns. all times to prevent burns.

Glassware and Glassware could be Effective PPE was worn to

sharps dropped or broken prevent injuries as a result of
resulting in the creation of sharps and a sharps disposal bin
sharps will be nearby in the lab to clean
up the sharps

Analysis

Over the course of the experiment the volumes of gas produced for each pH level
where collected and tabulated. This data was collected to enable the average
volume of gas produced at each pH level to be determined which would be further
used to determine the pH level at which catalase is the most active at.
 Raw Data Table 1: Table displaying the volume of oxygen gas produced (± 0.025 ml)
by the reaction of catalase in Saccharomyces Cerevisiae with a 3% hydrogen
peroxide solution at varying pH levels over 3 min.

pH Trial 1 Trial 2 Trial 3 Trial 4 Trial 5

(±0.025ml) (±0.025ml) (±0.025ml) (±0.025ml) (±0.025ml)
4 19 22 22 23 20
5 27 30 29.5 30 28.5
6 36 35 33 45 34
7 32 34 37 33 40.5
8 30 35 32 33.5 35
9 32 29 30.5 31.5 31

Uncertainty for volume measurement was determined by halving the smallest

increment on the analogue eudiometer (0.5ml / 2)

Qualitative Observations

 The mixture of catalase and hydrogen peroxide would begin to bubble and
froth as a result of oxygen gas being produced, indicating that the reaction
was taking place.
 Reactions taking place in pH6 and pH7 buffer solutions appeared to froth
more than other pH levels.
 Yeast was sourced from store brought containers and mixed to ensure a
homogenous sample.
 The hydrogen peroxide used was refrigerated and at a concentration of 3%
 Enzyme reactions appeared to be taking place after the 3 min time frame had
ended.
 All reactions appeared to start immediatley as soon as hydrogen peroxide
was added to yeast buffer mixture.
 A small volume of liquid was present in the gas tube before the reaction was
started.

Processed Data
The data collected from each individual trial at the specified pH levels was compiled
and averaged to produce a representative figure for the volume of oxygen produced
at each specific pH value.
Processed Data Table 1: Table displaying the average volume of oxygen gas
produced (± 0.025 ml) by the reaction of catalase in Saccharomyces Cerevisiae with a
3% hydrogen peroxide solution in varying pH solutions over 3 min.
pH Mean Vol Standard
Buffer (±0.025ml) Deviation
4 21.2 1.643167673
5 29 1.274754878
6 36.6 4.827007354
7 35.3 3.456877203
8 33.1 2.133072901
9 30.8 1.151086443

Calculations
To calculate the mean Volume of gas produced, the following calculation was
performed:

Total ∑ of Volume produced at Specified pH

= Average Volume produced at specific pH
Total Number of Trials at specified pH

Sample Calculation:
36+35+33+ 45+34
=36.60(± 0.025 ml)
5
Rounded t0 2 D.P. In order to satisfy the uncertainty of the measurement which was
± 0.025 ml

Analysis 1
The results from the 5 trials from each of the pH values were averaged and placed
on the processed data graph 1 with their standard deviations being represented by
the error bars. It was found that the pH6 value pertained the highest average oxygen
volume produced with a volume 36.6 mL (±0.025ml). Although this was the highest
volume produced it was also noted that the pH6 value also had a large standard
deviation of 4.83 indicating low precision. The lowest average volume of gas
produced was from the pH4 trials with an average of 21.2 ml (±0.025ml) being
produced. Although this data showed which pH level produced the highest volume of
oxygen gas, the large standard deviations values, especially for the pH 6,7 and 8
values, did not allow for definite conclusions to be drawn. As a result, a single factor
ANOVA test was performed to determine whether there was a significant statistical
difference between the values.

ANOVA Test
This test allowed for the determination of a significant statistical difference in the
data set and would indicate whether a difference was present. (Will Kenton, 2022)
Null Hypothesis
There is no statistical difference/relationship between the reaction of catalase and
hydrogen peroxide in varying pH buffer Solutions.

Alternate hypothesis
There is a statistical difference/relationship between the reaction of catalase and
hydrogen peroxide in varying pH buffer Solutions.

Processed data table 2: A table showing the results of the ANOVA test performed
on the averaged Data.

Analysis 2
Since the F value (20.3749) is greater than the F Critical Value (2.62065) at a p
value of 6.261E-8 , the null hypothesis can be rejected as the test is deemed as
significant. This allows for the alternative hypothesis to be accepted, which states
that there is a significant difference between the differing pH buffer solutions. With
the determination that a significant statistical difference is present, the values
obtained from the pH value averages can be used as points on a graph.
It is important to note that the ANOVA test only tests for whether a significant
difference is present and cannot determine the extent of the differences between the
different values
Processed data graph 1: Graph displaying the average volumes of oxygen gas (
± 0.025 ml) produced by the reaction of catalase in Saccharomyces Cerevisiae with a
3% hydrogen peroxide solution in various Buffer Solution over 3 min.
40
Avg. Vol. of O2 Gas Produced (±0.025ml)

35
R² = 0.964429696547099
30

25

20

15

10

0
3 4 5 6 7 8 9 10
pH Value

Analysis 3
Upon observation of the graph, many points of reference can be drawn. Thanks to
the performance of the ANOVA test and its determination that significant difference
is present, it can be confidently stated that the highest average volume of oxygen
gas produced was at a pH level of 6 with 36.6mL (±0.025ml) being produced.
In addition to this with the inclusion of a trend line it is clear to see how the volume of
gas produced differed over the varying pH levels. The trend line clearly rises from
pH5 to pH7 representing an increase in oxygen produced before falling again from
pH7 to pH9. Thanks to the trendlines high R2value of 0.9644 it is considered
representative of the data points present on the graph and shows an accurate
representation of the change in volumes of O2 gas produced.

Discussion
Several conclusions can be drawn from the data obtained from this experiment.
By analysing the data presented in the processed data table and processed data
graph that the catalase enzyme found in the Saccharomyces Cerevisiae is most
active in a pH environment of pH6. The average volume of gas produced by the
catalase enzyme at pH6 was 36.6 mL (±0.025ml) which was the largest average
volume of gas produced. This result was concurrent with external research
performed which stated that the optimum pH level for the catalase enzyme to work in
is around pH 6-7. The use of an ANOVA test to measure the statistical significance
of the data ensured that all results were valid and that the difference in values was a
result of the independent variable and not chance.
In addition to the highest average value, the trend line displayed how the enzymes
activity changed as it was subjected to differing pH levels. The inclusion of the
trendline into the graph allowed for an easier visualization of the change in activity
over differing pH levels, and its high R2 value of 0.9644 allowed it to be considered
accurate and fair. A clear trend upwards from pH 5 to 7 can be observed with a clear
trend downwards being observed from pH7 to 9. This trend is evident of the enzyme
experiencing Denaturing as it was exposed to more extreme pH values (Joanna
Tatomir, 2021). Enzymes have an optimal pH and altering that pH can result in an
enzyme losing its ability to function which can be seen by the trend on the processed
data graph. As the enzymes denatured the average volume of oxygen produced
decreased as less of the hydrogen peroxide substrate was able to be catalyzed. This
can clearly be seen via the trendline by the drops in the volume of gas produced
from pH7 to pH5 and 9.
From the data collected and the analysis of results it can be concluded that the pH
level at which the catalase enzyme is most active is pH6. This conclusion partially
supports the stated hypothesis that the catalase enzymes activity will peak at a pH
level of 7. Thanks to the accurate collection of data and the statistical analysis
performed to ensure it is accurate, these conclusions can be drawn.

Strengths
Overall, it is believed that the experiment conducted is reliable and representative of
the activity of the catalase enzyme in differing pH environments. The process
allowed for a large of raw data to be gathered, processed and analysed in a
relatively short period of time. This is evident by the large number of trials performed
at each varying pH level which can be seen in the raw data table. This method was
effective at collecting a large amount of accurate data, however there where a
number of flaws regarding its process.
Weaknesses
Error Impact on Data Improvements
Lack of control of Changes in heat could A solution to this issue
surrounding air affect the activity of the could be to use air
temperature catalase enzyme. This conditioners to keep the
could potentially alter ambient room
results as changes in heat temperature constant, or
can cause enzymes such to perform multiple trials
as catalase to behave at once to ensure they are
differently or potentially all subjected to the same
even cease to function temperature changes.
thanks to denaturing
Small Sample size of During the course of the Multiple different brands
Saccharomyces experiment, it was noted of yeast could be used
Cerevisiae that only 1 brand of yeast and tested allowing for
was used. This could differences in the catalase
potentially skew results as behaviour to be
 different Saccharomyces disseminated and a more
Cerevisiae from other rounded conclusion to be
brands may behave created
differently

Extensions
An extension to the current experiment could be the use of smaller pH buffer
increments to gather a more populated data set. For the experiment performed, large
jumps of 1 between the pH values used where present. As a result, there was a large
gap between data points which could potentially cause the omission of more precise
data. With a decrease in the increments to which the pH levels are increased, more
data will be able to be collected at a wider range of pH values. This would allow for a
larger, more accurate data set that can be used to draw a more conclusive and
definitive conclusion

Conclusion
The research question and hypothesis where both answered and supported
respectively from the data received from the experiment. The pH level at which the
enzyme catalase was most active in was determined to be pH6 and this conclusion
was able to be supported by a large range of data and statistical checks. Overall, the
investigation was successfully able to answer and support the research question and
hypothesis with a high degree of accuracy and precision.
References
1. National Center for Biotechnology Information. (2019). Catalase: A
Molecular Target of Signaling Pathways in Health and Disease.
Antioxidants, 8(12), 634. Retrieved from
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6885225/

2. Encyclopedia Britannica. (2023). Catalase. In Encyclopedia Britannica.

Retrieved from https://www.britannica.com/science/catalase

3. Encyclopedia Britannica. (2022). Denaturation. In Encyclopedia

Britannica. Retrieved from
https://www.britannica.com/science/denaturation

4. Conduct Science. (2021). Factors That Affect Enzyme Activity.

Retrieved from https://conductscience.com/factors-that-affect-enzyme-
activity/
5. Study.com. (2021). Denatured Enzyme: Overview & Causes. Retrieved
from https://study.com/learn/lesson/denatured-enzyme-overview-
causes.html