A study of the effect of temperature on the enzyme catalase from

solanum tuberosum

Aim
A commonly used method of emergency aquarium aeration during power shortages
is to mix hydrogen peroxide with diced potatoes in a sealed container to produce
oxygen gas. However, this method does not yield a substantial amount of oxygen
gas, particularly when refrigerated potatoes were used. As an aquarium owner, I
was very interested in exploring how the temperature of the reaction could affect
yield.

From a in-class lab investigating of limiting factors affecting the catalytic ability of
lactase, I have previous knowledge that enzyme activity is affected by temperature.
I will be adapting similar protocols for my investigation by increasing the
temperature of the enzyme solution in small increments across a range of
temperatures, using a water bath to regulate the temperature of the enzyme-
substrate solution.

Research question
To what extent does temperature affect the rate of reaction of the enzyme catalase
from red potatoes (Solanum tuberosum) as a catalyst in the decomposition of
hydrogen peroxide?

Introduction
Enzymes are globular proteins that speed up chemical reactions by decreasing the
activation energy required. With orientation of appropriate direction and energy,
enzymes are able to bind to substrate molecules and form a reaction (Damon et al,
2014). This makes enzymes very important in a variety of bodily functions. Catalase
is an enzyme produced by aerobic organisms to break down toxic forms of oxygen
known as reactive oxygen species (ROS) such as hydrogen peroxide, which are
produced by various cellular processes including mitochondrial respiration.
Commonly found in the liver, catalase is produced to prevent accumulation and
tissue damage by decomposing hydrogen peroxide into water and oxygen
(McDowall, n.d).

Catalase converts hydrogen peroxide into water and oxygen gas

1
 Thus, the amount of oxygen gas produced over a specified duration of a trial can be
used to determine the rate of reaction of catalase. The rate of reaction is affected
by external factors: temperature, pH, enzyme concentration, substrate
concentration, and the presence of inhibitors and activators (Enzymes, n.d.).
Manipulating these limiting factors as individual variables in a lab can determine
how it affects the rate of reaction. The presence of these external factors can
change the rate at which collision occurs and the shape or charge of the substrate
or active site.

Hypothesis
As temperature increases, the rate of reaction catalase will increase in a positive
correlation until it reaches a temperature after which the enzyme begins to
denature and rate of reaction decreases as the substrate is no longer able to bind to
the enzyme. Effectiveness, for this experiment, will be measured in the change of
oxygen gas concentration from oxygen gas produced during the reaction. Enzymes
catalyse reactions through collisions with the substrate. Thus, higher temperatures
would increase the kinetic energy of the substrate molecules and increase the rate
of successful collisions, and hence rate of reaction increases. However, enzymes are
also a protein structure and at high temperatures the proteins denature and the
secondary and tertiary structures are destroyed, ultimately changing the shape of
Rate of catalysis

TemperatureC

the enzyme. The substrate will no longer be able to bind to the active site, and
hence the rate of reaction decreases.

Figure 1 Predicted rate of reaction

Variables
Independent - Temperature of catalase solution (C)

2
The temperature of the enzyme reaction of catalase was manipulated by placing the
enzyme and the substrate in a water bath of the desired temperature for a period of
5 minutes, monitoring and maintaining the temperature of the water bath at
different increments: 10C, 15C, 20C, 25C, 30C, 35C, 40C, 45C, and 50C. As
the temperature of the water bath will ideally be the same between trials of the
same temperature, three trials of equivalent temperature will be run in the same
water bath in order to simulate an identical temperature.

Dependent rate of change in oxygen gas concentration per minute (%min -1)
The amount of oxygen gas produced from the reaction can be measured with
Vernier oxygen probes over the set time interval. This value will be determined by
subtracting the initial oxygen gas concentration from the final oxygen gas
concentration at the end of the data collection period and dividing by 5 minutes.

Control Variables
What is controlled Method of control
Amount of All trials will employ 10 mL of 6% hydrogen peroxide and 10
substrate mL of catalase solution. The concentration of hydrogen
hydrogen peroxide peroxide is the ratio of the amount pure hydrogen peroxide
(mL) and to the amount of the solution, in this case 6%. The greater
concentration (%) the hydrogen peroxide, the more substrate molecules are
Amount of present, thus the concentration must be kept constant. The
catalase enzyme catalase solution must be prepared exactly as per the
(mL) instructions to ensure consistency in the concentration of the
potato. If a solution is more concentrated, there will be more
catalase enzyme present which will change the rate of
reaction. The amount of substrate and enzyme also has a
direct impact on the rate of catalysis thus this must be
carefully controlled by measuring out identical amounts of
both substrate and enzyme solution for each trial.
Temperature of Hydrogen peroxide is an oxidizing agent that decomposes at
hydrogen peroxide higher temperatures. In order to ensure that results are
accurate, hydrogen peroxide should be kept at room
temperature, away from heat sources.
Time (seconds) All trials will last 300 seconds. This will be controlled by
programming the LabQuests data collection period to 300
seconds. This will also remove the uncertainty of human
reaction time. The amount of time the oxygen concentration
is measured is important as more time allows the enzyme to
react more with the substrate. This provides enough time to
record the difference in oxygen production in different
temperature environments while still ensuring that
temperature of the water bath is controllable.
pH All preparations of catalase solution will use distilled water to

3
 ensure the pH remains constant as pH is a limiting factor of
enzyme activity.
Type of catalase All trials will use a catalase solution from 30 g of skinned red
source potato. Different types of potato contain varying amounts of
catalase. From prior research, it is determined that red
potatoes contain a high level of catalase, thus red potatoes
should be used in the experiment to clearly show the
difference in oxygen production. The potato will be
thoroughly washed to remove dirt and impurities.
Furthermore, to ensure that equal amounts of potato are
used in each catalase solution, the potato is to be skinned
prior to cutting
Table 1 List of variables and their controls

Method
Materials:
18 red potatoes
Potato peeler
Magic Bullet blender (base, blender cup, and cross blade)
18 coffee filters
Electronic balance (0.01g)
Knife
Cutting board
800 mL of distilled water
200 mL beaker (10 mL)
2 x 10 mL graduated cylinder (0.2 mL)
1 labeled for catalase
1 labeled for hydrogen peroxide
600 mL of 6% hydrogen peroxide
Vernier LabQuest
3 Vernier oxygen gas sensor (0.01%)
3 Vernier containers
Large waterproof container at least 25 cm wide and 10 cm tall
Ice cubes
Hot plate
1000 mL beaker (50 mL)
Tap water
3 glass thermometers (0.5C)
Timer (0.005 s)
Stirring rod
2 x plastic pasteur pipettes (0.25 mL)
1 labeled for catalase
1 labeled for hydrogen peroxide
Paper towels
Safety goggles
Beaker tongs

4
 Safety
Electronics
Cords were kept away from water.
Heated materials
Beaker tongs were used when handling hot glassware from the hot plate.
Hot plate was not set over 300C to protect the glassware.
Water temperature was monitored to prevent melting of the plastic containers.
Sharp objects
Work was done carefully with the knife and peeler when cutting the potatoes.
Care was used with the blender.

Hydrogen peroxide
Safety goggles were worn as hydrogen peroxide is an irritant.
Contact was avoided with skin and eyes, inhalation and ingestion was avoided.
Environmental damage was prevented by decomposing hydrogen peroxide into
water with a catalyst prior to disposal as it is toxic to microorganisms (including
beneficial microorganisms in soil and sewage treatment).
Sources of heat were avoided as it is a strong oxidizing agent.
The material safety data sheet of 6% hydrogen peroxide was consulted for more
information.

Programming the LabQuest

1. The LabQuest was turned on and three Vernier oxygen gas sensors were connected.
2. The data collection period was set to 300 seconds.

Preparing the water bath

1. The plastic container was filled with tap water until water level reached at least 5
cm in depth, a thermometer was placed into the container.
2. The 1000 mL beaker was filled with water and set on the hot plate at a surface
temperature of 285C, a thermometer was placed in the beaker to monitor the
water temperature.
3. Ice cubes or hot water from the hot plate was used to adjust the water bath
temperature to 10C, 15C, 20C, 25C, 30C, 35C, 40C, 45C, or 50C.
4. Water was continuously added to the beaker on the hot plate so that hot water was
available when needed.

Preparing catalase solution*

1. The red potato was washed thoroughly and wiped dry, skinned using the potato
peeler and cut into small pieces around 1 cm 3.
2. 30 g of potato was measured out with the electronic balance and added to the
blender cup.
3. 80 mL of distilled water was added to the blender cup
4. The cross blade was screwed on, the contents were blended for 60 seconds.
5. The mixture was filtered through a coffee filter into the 200 mL beaker.
6. Any remaining potato was discarded

5
 * Potato solution must be remade immediately prior to each set of three trials to
prevent spoilage and ensure consistent data.

Conducting a trial
1. Three Vernier containers were submerged into the water bath until they touch the
bottom of the container and taped into place using masking tape ensuring that the
containers are upright
2. 10 mL of catalase solution was measured using the catalase graduated cylinder with
the pipette
3. The contents of the graduated cylinder were poured into a Vernier container, steps
3-4 were repeated for each container
4. A thermometer was placed into one of the Vernier containers
5. The solution was left in the water bath for until the temperature of the catalase
solution reached the desired temperature, approximately 5 minutes, the
temperature of the water bath was continuously adjusted based on readings from
the thermometer so that it stayed at the desired temperature using ice cubes and
hot water as needed.
6. Once the desired temperature was reached, 10 mL of hydrogen peroxide was
measured using the hydrogen peroxide graduated cylinder with the pipette
7. The hydrogen peroxide was poured into a Vernier container and the mixture was
stirred for 5 seconds using the stirring rod
8. The stirring rod was wiped clean using a paper towel
9. Steps 9-10 were repeated for each Vernier container
10.An oxygen gas sensor was placed on each Vernier container and zeroed prior to
data collection
11.The temperature of the water bath was monitored and adjusted accordingly so that
the temperature remained constant
12.After 300 seconds, the change in oxygen concentration of each container was
recorded.
13.The sensors were removed and set aside upright on a dry surface
14.The Vernier containers were removed from the water bath, rinsed thoroughly, and
dried with paper towels
15.Any remaining catalase solution was discarded and the 200 mL beaker was rinsed
clean
16.Preparation of catalase solution and steps 2-20 were repeated at the same
temperature for trial 4-6
17.Steps 2-22 was repeated at each temperature interval of 10C, 15C, 20C, 25C,
30C, 35C, 40C, 45C, and 50C for a total of 9 intervals with 6 trials each.

6
Protocol diagram of experimental setup

Vernier oxygen gas sensor

LabQuest
Vernier container
Water bath

Figure 2 protocol diagram of lab setup

7
Data collection
Temperatu Trial 1 Trial 2 Trial 3 Trial 4 Trial 5 Trial 6
re ( (change in (change in (change in (change in (change in (change in
0.5C) O2%) O2%) O2%) O2%) O2%) O2%)
0.01% 0.01% 0.01% 0.01% 0.01% 0.01%
Initi Fin Initi Fin Initi Fin Initi Fin Initi Fin Initi Fin
al al al al al al al al al al al al
10C - 1.4 - 1.5 0.00 1.6 0.02 1.4 0.01 1.5 - 1.4
0.01 8 0.01 0 1 7 8 0.02 1
15C 0.01 3.0 - 3.1 - 2.9 - 3.0 0.01 3.0 0.00 2.9
2 0.02 2 0.01 7 0.03 9 9 7
20C - 4.4 0.01 4.5 0.02 4.4 0.00 4.4 0.02 4.5 - 4.4
0.02 1 3 1 1 3 0.01 6
25C 0.01 6.8 0.00 6.9 - 6.8 - 6.7 0.00 6.8 0.00 6.9
5 1 0.03 6 0.02 9 6 0
30C 0.00 7.3 0.04 7.5 0.02 7.4 0.01 7.2 - 7.3 0.00 7.2
4 2 4 7 0.02 4 7
35C - 9.0 0.00 9.0 0.00 9.1 0.02 8.3 - 9.1 - 8.8
0.02 2 9 1 9 0.01 4 0.02 7
40C 0.00 8.2 - 8.2 0.00 8.3 0.00 8.2 0.03 8.3 - 8.2
7 0.03 9 1 8 3 0.01 6
45C 0.00 4.5 0.01 4.7 0.00 4.6 - 4.3 0.00 4.8 0.03 4.4
4 9 5 0.03 3 6 2
50C 0.01 2.3 0.00 2.4 - 2.1 - 2.3 - 2.3 0.00 2.1
3 1 0.03 7 0.01 4 0.03 0 9
Raw data
Table 2 Initial and final values of oxygen concentration over 300 seconds for 9
different temperatures
Observations
Potato catalase solution is light pink and opaque
During the reaction, the mixture becomes a murky light pink due to the fizzing of
oxygen gas being produced. A layer of dense foam collects at the top of the
mixture as it traps the oxygen gas.
At lower temperatures and temperatures above 40 C, the production of oxygen
gas is slower; the rate of change in oxygen concentration per second is smaller
and the layer of foam inside the Vernier container is low.
At higher temperatures up to 40C, the production of oxygen gas is faster, the
rate of change of oxygen concentration per second is greater and the layer of
foam collected nears the Vernier container neck.
Condensation appears on the sides of the container during all reactions, vessels
became slightly heated; suggests catalase reaction generates heat.
At temperatures over 40C, the catalase solution becomes darker in colour as it
sits in the water bath becoming a pink-ish brown and oxygen production
decreases. This is likely an indication of the denaturing of the enzyme.

8
Analysis
Change in O2 concentration (%)
The change shows the amount increase of oxygen concentration from the reaction
of catalase and hydrogen peroxide. Below is a sample calculation used to determine
the change in oxygen concentration when the reaction took place in 10C
environment.
Changeoxygen concentration=final concentrationinitial concentration
Changeoxygen concentration=1.48(0.01)
Changeoxygen concentration=1.49
Temperatur Trial 1 ( Trial 2 ( Trial 3 ( Trial 4 ( Trial 5 ( Trial 6 (
e ( 0.5C) %) %) %) %) %) %)
10C 1.49 1.51 1.61 1.45 1.57 1.43
15C 3.01 3.14 2.98 3.12 3.08 2.97
20C 4.43 4.52 4.39 4.41 4.51 4.47
25C 6.84 6.91 6.89 6.81 6.86 6.90
30C 7.34 7.48 7.42 7.26 7.36 7.27
35C 9.04 9.09 9.11 8.37 9.15 8.89
40C 8.27 8.32 8.31 8.28 8.30 8.27
45C 4.54 4.78 4.65 4.36 4.86 4.39
50C 2.32 2.41 2.20 2.35 2.33 2.19
Table 3 Change in oxygen concentration

Mean
The mean shows the central tendency of the dataset and represents the average of
the data given. Below is a sample calculation used to determine the average rate of
oxygen production when the reaction took place in 10C environment.

x=
changeoxygen concentrationof trials
number of trials
1.49+ 1.51+1.61+1.45+1.57 +1.43
x=
6
x=1.51

Standard deviation
The standard deviation shows the spread and variation of the obtained data
values around the mean. The standard deviation shows the precision of the
collected data. Below is a sample calculation used to determine the standard
deviation of the rate of oxygen production at 10C.

SD=
(xx)2
n1

9
 SD =
0.0240
5
SD=0.0693

Rate of oxygen production

The rate of catalysis is the dependent variable. It is given by dividing the
percentage change in oxygen concentration by the duration of the trial in minutes.
Below is a sample calculation used to determine the rate for 10C.
average change oxygen concentration
Rate=
timeminutes
1.51
Rate=
5
Rate=0.302 %min1
Processed data
Rounded to 3 significant figures
Temperature ( Mean change in Standard deviation Rate of catalysis
0.5C) oxygen (% min-1) ( (% min-1)
concentration (%) 0.00005)
( 0.005%)
10C 1.51 0.0693 0.302
15C 3.02 0.0732 0.604
20C 4.37 0.0536 0.874
25C 6.35 0.0387 1.27
30C 7.36 0.0853 1.47
35C 8.94 0.0811 1.79
40C 8.30 0.0214 1.38
45C 4.60 0.0740 0.920
50C 2.30 0.0216 0.460
Table 4 Processed data of final calculated result

10
 = standard deviation to quantify amount of dispersion of data values
Figure 3 final graph showing the affect of temperature on rate of reaction of
catalase

T-Test
To prove the data has a significant different between two temperature levels, a t-
test is employed. This is particularly relevant between the interval of 35C and 40C
as the error bars show a possible overlap. Below is a sample calculation used to
calculate whether the difference in rate of catalysis is significant between 35C and
40C. A probability of 0.05 was used to evaluate statistical significance.
Null hypothesis (H0) = There is no difference in the rate of catalysis
between temperatures of 10C and 15C
Alternative hypothesis (HA) = There is a difference in the rate of catalysis
between temperatures of 10C and 15C
( x 2 x 1)
t=

2 2
SD 1 SD 2
+
n1 n3

(1.791.66)
t=

0.02142 0.08112
6
+
6

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 t=3.79 6
Degrees of freedom is given by n1 + n2 2 = 6 + 6 2 = 10
Tcrit for 10 degrees of freedom (p = 0.05) = 2.23

Temperature 10-15 15-20 20-25 25-30 30-35 35-40 40-45 45-50

intervals (C)
tstat 7.34 7.29 18.4 2.62 6.66 3.80 23.5 14.6
tcrit 2.23 2.23 2.23 2.23 2.23 2.23 2.23 2.23
Significant Yes Yes Yes Yes Yes Yes Yes Yes
difference in
rate of reaction
Null hypothesis Reject Reject Reject Reject Reject Reject Reject Reject
ed ed ed ed ed ed ed ed
Table 5 t-test evaluation of significance in difference in rate of reaction

Therefore, the null hypothesis is rejected for all temperature intervals of this
experiment and there is a significant difference in the rate of catalysis between all
temperatures.

Uncertainties
Uncertainty Degree of uncertainty
Timer Reaction time 0.05 s
Thermometer 0.5C
Electronic balance 0.01g
10 mL graduated cylinder for hydrogen 0.2 mL
peroxide and catalase solution
Vernier oxygen gas sensor 0.01%

The materials used were kept constant throughout the experiment to the level of
uncertainty of each trial is the same. For example, the amount of hydrogen peroxide
added to the catalase solution in each trial was kept constant by using identical
graduated cylinders. The uncertainty with the greatest impact on experimental
results would have been temperature as it was not only the independent variable
but also a variable with the highest degree of uncertainty. This proves to be a
limitation of the data. However, as the t-test rejects the null hypothesis in all
temperature intervals, the data is still significant.

Conclusion
It was hypothesized that increasing temperatures of catalase and hydrogen
peroxide would increase the rate of reaction. It was also thought that at high
temperatures, the protein structure of the enzyme would denature and prevent
catalysing of the reaction. As illustrated in Figure 1, there is both a positive
correlation and a negative correlation. This premise was supported by the similarly
to the graph of rate of catalysis in Figure 3 derived from raw data collected from this
experiment shown in Table 2 and the values of rate of reaction in Table 4. At 10C,

12
the production of oxygen gas is very limited, showing only a mean increase of
1.51% in oxygen concentration at a rate of 0.302%min -1. However at 15C, the
mean change in oxygen concentration increases to 3.02% at a rate of 0.604%min -1.
This trend of increasing oxygen production continues to 35C, where the oxygen gas
concentration increases 8.94% and a rate of 1.79%min -1. Between 35C and 40C,
the rate of catalysis decreases from 1.79%min-1 to 1.38%min-1. This trend is of
decreasing oxygen production is observed at 45C and 50C as well. While there is
slight variation in the change of oxygen gas concentration and rate of catalysis
between individual samples, the trend of increasing then decreasing oxygen
production is observed consistently as illustrated in Figure 3 and the standard
deviation and t-test further prove the significance of this data.

The quantitative data reflects a similar result whereby the reaction between
catalase and hydrogen peroxide is progressively more vigorous at higher
temperature intervals between 10C and 35C, evidenced by the fizzing of the
solution and the amount of foam produced inside the Vernier containers as a result
of this reaction. The colouration of the catalase solution while a light slightly
translucent pink at temperatures below 40C, darkens with exposure to higher
temperatures which can be a possible physical evidence of the denaturation of the
enzyme.

The results from this experiment demonstrates a strong positive correlation

between temperature and enzyme activity. This was determined by recording the
change in concentration of oxygen gas over the time interval of 5 minutes at
different temperatures while keeping other limiting factors such as pH, substrate
concentration, enzyme concentration, and presence of inhibitors, activators
constant. As temperature increases, the rate of reaction also increases. However,
past 35C, the correlation becomes negative and further increases to temperature
results in a decrease of enzyme activity. The scientific reason why this trend works
is the increased kinetic energy of substrate particles as temperature increases. As
there is more kinetic energy, the molecules move around more freely and more
collisions between enzyme-substrate collisions can successfully occur to catalyse
the reaction. However, at higher temperatures over 35C, the protein enzyme
denatures and the active site becomes misshapen so that the substrate can no
longer bind to the enzyme thus the rate of reaction decreases. The positive
correlation is most clearly seen in the Figure 3 between (10, 0.302) and (35, 1.79)
where there is almost a six-fold increase in rate of reaction. The decrease in
temperature can be most clearly seen in the graph at the turning point of (35, 1.79)
where left of that point there is a positive correlation and right of the point there is a
negative correlation. This investigation shows that maximum oxygen gas production
from decomposition of hydrogen peroxide by catalase occurs at 35C. Furthermore,
this correlation suggests a possible reason why most living ectothermic organisms
maintain a body temperature of around 35C to 40C; to maximize enzymatic
reactions.

13
Limitations
Although the temperature reading on the glass thermometer was kept consistent for
the duration of the trial, there may have been fluctuations in the actual temperature
of the water bath that was not visible in thermometer readings due to a slight lag
between a change in temperature and a visible change in temperature readings,
this could negatively effect the data obtained. A possible method of improvement,
as stated above would be to use digital thermometers to reduce the time delay in
temperature readings.

Due to there being three trials run simultaneously, the addition of hydrogen
peroxide and the placement of oxygen sensors occurred at different times for each
sample, thus there is unrecorded oxygen production occurring prior to data
collection. This would result in the data collected not reflecting the entire reaction
process. This limitation was considered in the process of experimentation, all
sensors were zeroed prior to data collection. However, the control test revealed a
logarithmic increase in oxygen concentration, thus this may affect the results of the
experiment. Possible methods to improve this limitation are to run each trial one at
a time or to find a lab partner so that all three trials can be handled simultaneously.

In order to prevent contamination of results, Vernier containers were washed prior

to each set of trials, however this introduced water and possible contaminants into
the container. Due to the narrow neck of these containers, it was not possible to
thoroughly dry them out using paper towels. This could affect enzyme activity by
changing the concentration of the substrate as well as introducing possible
inhibitors. A possible method of improvement is to use multiple sets of Vernier
containers which would allow time for each set to dry in between trials.

The catalase solution was made from red potatoes from a local supermarket and
while the potatoes were of the same type and amount for each preparation, the
potatoes may have slightly differing amounts of the enzyme depending on growing
conditions or differ depending on the section of potato used. This may have resulted
in a rate of catalysis that is slightly higher or lower than normal. This is important as
a different potato was used for each preparation of catalase solution. A possible
method of improvement is to create a solution with distilled water and pure catalase
which would remove impurities and variability.

A further extension could be to find the exact optimal temperature of catalase by

changing the temperature at smaller intervals between the local maxima of 35C to
40C. This would also provide greater accuracy into the temperature at which
enzyme activity is maximized.

References

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Damon, A., McGonegal, R., Tosto, P., & Ward, W. (2014). Standard Level Biology (2nd ed.). Harlow,

Essex: Pearson Education Limited.

Enzymes. (n.d.). Retrieved March 30, 2017, from

http://www.rsc.org/Education/Teachers/Resources/cfb/enzymes.htm

McDowell, J. (n.d.). Catalase. Retrieved March 31, 2017, from

http://www.ebi.ac.uk/interpro/potm/2004_9/Page1.htm

Spychalla, J. P., & Desborough, S. L. (1990). Superoxide Dismutase, Catalase, and -Tocopherol

Content of Stored Potato Tubers. Plant Physiology,94(3), 1214-1218.

doi:10.1104/pp.94.3.1214. Retrieved from

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1077364/pdf/plntphys00812-0372.pdf

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