Biology Internal Assessment

A study of the effect of

temperature on the enzyme
catalase from solanum tuberosum
Student: Xinyi Zou
Teacher: Ms. Marin
Date: March 14, 2017
Candidate Number:
Word Count: 3582
Pages: 13
Background information
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.

Catalase converts hydrogen peroxide into water and oxygen gas

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. 1 Manipulating these
limiting factors as individual variables in a lab can determine how it affects the rate
of reaction. Temperature in particular

Aim
This investigation focuses specifically on the affects temperature as an individual
variable on the rate of reaction of catalase from solarum tuberosum commonly
known as the red potato2. All other limiting factors are to be strictly controlled to
ensure that the data recorded is a reflection of the effects of temperature on the
rate of reaction of catalase all other factors held constant. This experiment is
inspired by an in-class practical investigation of similar limiting factors on the
enzyme lactase. Though this investigation, I will gain insight into the impact of
temperature as a limiting factor and how organisms use catalase to effectively
decompose ROS. I was particularly interested in looking at the oxygen gas
production by the enzyme catalase decomposing hydrogen peroxide due to the
potential for the construction of a non-electric aquarium air pump in the event of a
power outage without directly dosing hydrogen peroxide, which has to potential of
harming fish and invertebrates. Diced potatoes and hydrogen peroxide is often
advertised as an effective method of oxygen generation. However, at room
temperature, it does not yield a substantial amount of oxygen. Thus, I was very
interested in exploring how the temperature could affect oxygen yield.

1 http://www.worthington-biochem.com/introbiochem/factors.html
2 http://www.produceoasis.com/ProductDetailPage/TabId/272/pid/407/Vegetables/Red-
Potatoes.aspx
 Research Question
How does temperature affect the rate of reaction of the enzyme catalase from
solanum tuberosum based on the change of oxygen concentration from the
decomposition of hydrogen peroxide?

Hypothesis
As temperature increases, the rate of reaction catalase will increase as well in a
positive correlation until it reaches optimal 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. As enzymes catalyse reactions through collisions with the
substrate, higher temperatures would increase the kinetic energy of the substrate
molecules thus increasing the rate of collisions. However, enzymes are also a
protein and at high temperatures, proteins denature and the substrate would no
longer be able to bind to the active site.
Rate of catalysis

Temperature

Figure 1 Predicted rate of reaction

Variables
Variable Method of control
measured
Independe Temperatur The temperature of the enzyme reaction of catalase was
nt e of manipulated by placing the enzyme and the substrate in
catalase a water bath of the desired temperature for a period of 5
and minutes, monitoring and maintaining the temperature at
hydrogen 10 different increments: 10C, 15C, 20C, 25C, 30C,
peroxide 35C, 40C, 45C, 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.

Dependen Rate of The amount of oxygen gas produced from the reaction
t reaction ( can be measured with Vernier oxygen probes over the
%/minute) set time interval. The rate of oxygen gas production per
minute from reaction can therefore be determined by
subtracting the initial oxygen concentration from the
final oxygen concentration and dividing by the time
interval of t minutes to give the rate

Control Amount of All trials will employ 10 mL of 6% hydrogen peroxide and

substrate 10 mL of catalase solution. The amount of substrate and
(H2O2) and enzyme has a direct impact on the rate of catalysis thus
concentrati this must be carefully controlled by measuring out
on identical amounts of both substrate and enzyme solution
Amount of for each trial. 10 mL of each gives enough of a reaction
enzyme to measure a noticeable difference in oxygen
(catalase) concentration within the time interval while also ensuring
that the oxygen gas sensors do not come into contact
with the foam produced.

Time All trials will have a duration of 5 minutes exactly. The

(minutes) amount of time the oxygen concentration is measured is
important as more time allows the enzyme to react more
with the substrate. The LabQuest can be programed to
record data for a specified time period of 300 seconds.
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 ensure the pH remains constant
Type of All trials will use catalase solution from 30 grams of
catalase skinned red potato with 80 mL distilled water. Different
source species 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 in order 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, skin the potato prior to
 cutting

Temperatur The temperature of the water bath is to remain constant

e for the entire trial so that the overall data can reflect
throughout trends between temperature and reactivity. Glass
a trial thermometers will be used to monitor the temperature

Table 1 List of variables and their controls

Method
Materials:
9 red potatoes
Potato peeler
Magic Bullet Blender
Blender cup
Cross blade
9 coffee filters
Analytical balance (0.01g)
Knife
Cutting board
800 mL of distilled water in 800 mL beaker (5%)
1 200 mL beaker (5%)
Label tape
Marker
2 x 10 mL graduated cylinder (1 for catalase and 1 for hydrogen peroxide) ( 0.2
mL)
500 mL 6% hydrogen peroxide in 800 mL beaker ( 5%)
Vernier Labquest
3 Vernier oxygen gas sensor
(0.01%)
3 Vernier 250 mL container
Large waterproof container at least
25 cm wide and 10 cm tall
2 full Ice cubes
Hot plate
Large 1000 mL beaker (5%)
Tap water
3 glass thermometers (0.5
degree)
Timer
1 stirring rod
2 pipettes
Calculator
Data table
Paper towels
Safety goggles
Beaker tongs
 Safety
Use beaker tongs when handling hot glassware from the hotplate
Hot plate should not be set over 300C to protect the glassware
Hot water added to the water bath should be hot but not boiling as it may melt
plastic containers
Work carefully with the knife when cutting the potatoes
Wear safety goggles as hydrogen peroxide is an irritant
Do not dump hydrogen peroxide down the drain as it may harm sewer treatment
microorganisms
Consult the material safety data sheet of 6% hydrogen peroxide for more
information

Preparing catalase solution

1. Using the 200 mL beaker, measure 80 mL of distilled water
2. Skin 1 red potato using the potato peeler
3. Cut the potato using the knife and cutting board into small pieces around 1 cm3
4. Place the blender cup onto the analytical balance
5. Zero the scale
6. Measure out 30 g of potato into the blender cup
7. Add the distilled water to the blender cup
8. Screw on the cross blade tightly to ensure no leakage
9. Blend until smooth for 60 seconds
10.Dry the 200 mL beaker with a paper towel
11.Filter the mixture through a coffee filter into a 200 mL beaker
12.Repeat steps 1 10 for each for each 3 trials

* Potato solution must be remade immediately prior to each test to prevent spoilage
and ensure consistent data

Preparing the LabQuest

1. Turn on the LabQuest
2. Connect 3 Vernier oxygen gas sensors
3. Set the time to 300 seconds

Preparing the water bath

1. Fill the large container with water until tap water reaches at least 5 cm in depth,
place a thermometer into the container
2. Fill a large 1000 mL beaker with water and set on the hot plate at 285C
3. Use ice cubes or hot water from the hot plate to adjust to the appropriate
temperature at 10C, 15C, 20C, 25C, 30C, 35C, 40C, 45C, 50C.
4. Continue to add water to the 1000 mL beaker so that hot water is available when it
is needed

Running the lab

1. Label one graduated cylinder and one pipette for each of catalase and hydrogen
peroxide using label tape and the marker
2. Using the thermometer in the water bath, wait until the water bath reaches 10C
3. Place three Vernier containers into the water, submerge until the vessels touch the
bottom of the container, tape into place using masking tape ensuring that the
containers are fully upright and no water can spill in
4. Place the 800 mL beaker of hydrogen peroxide into the water bath, place a
thermometer to monitor the temperature. Ensure that no water spills into the
hydrogen peroxide, remove amounts of water from the water bath if necessary
5. Stir the catalase solution lightly with the catalase pipette
6. Measure out 10 mL of catalase solution using the pipette into the catalase
graduated cylinder
7. Pour the contents of the graduated cylinder into a Vernier container, repeat three
times for each container
8. Place a thermometer in one of the Vernier containers
9. Wait 5 minutes, continue adjusting the temperature of the water based on readings
from the thermometer placed in the water bath so that it stays at the desired
temperature using ice cubes and water as needed.
10.After 5 minutes, read the temperature of the thermometers in the Vernier container
and the hydrogen peroxide, if the temperature is not yet at 10C, continue
monitoring the temperature until the both temperatures reach 10C
11.Using the pipette for hydrogen peroxide, measure out 10 mL of hydrogen peroxide
from the submerged beaker into graduated cylinder for peroxide
12.Pour the 10 mL of hydrogen peroxide into a Vernier container, stir the solution for 5
seconds using the stirring rod, wipe the stirring rod clean using a paper towel
13.Repeat steps 10-12 two times for each Vernier container
14.Place an oxygen gas sensor into each container
15.Zero the oxygen sensor
16.Begin data collection
17.Continue to monitor the temperature of the water bath and adjust accordingly so
that the temperature remains constant
18.At the end of data collection, after 5 minutes, record the change in oxygen
concentration
19.Remove the sensors and set aside upright on a dry surface
20.Remove the vessels from the container and clean thoroughly with a brush and dish
soap, ensure no residue is left behind
21.Discard any remaining catalase solution
22.Dry the containers as much as possible
23.Repeat steps 5-19 for the same temperature for trial 4-6
24.Repeat steps 4-21, changing the desired temperature to 15C, 20C, 25C, 30C,
35C, 40C, 45C, 50C using ice cubes and/or hot water from the hot plate
 Figure 3 Lab setup for trial at 10C with a cold water bath
Protocol Diagram

Vernier oxygen gas sensor

LabQuest
Vernier container
Water bath

Figure 4 protocol diagram of lab setup

Data collection and processing
Uncertainties
Amount of hydrogen peroxide (analogue) ( 0.5mL)
Amount of potato solution (analogue) ( 0.5mL)
Temperature (analogue) - ( 0.5C)
Oxygen concentration (digital) ( 0.01%)

Raw data
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

Figure 5 Change in oxygen concentration over time for 9 different temperatures

signifying the rate of reaction between catalase and hydrogen peroxide

Qualitative data
Potato catalase solution is light pink and opaque thin liquid with the viscosity of
orange juice. Has white bubbles on the surface after it is blended and smells
lightly of mashed potatoes. The mixture separates when still; small white pieces
of potato settles to the bottom of the container
Hydrogen peroxide is colourless and transparent. Looks very similar to water and
is odorless
During the reaction, the mixture becomes a murky light pink due to the fizzing as
oxygen gas is produced. A layer of dense foam collects at the top of the mixture
as it traps the oxygen gas. At lower temperatures, the production of oxygen gas
is slower and in lesser quantities than that of warmer temperatures
Condensation appears on the sides of the container during all reactions
At high 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
 Figure 6 enzyme before reaction (left) and after reaction at 10C (right)

Sample Calculations
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 =
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

Where
SD: standard deviation of sample
: sum
x : individual measurements in the sample
x : mean value
n : the number of individuals in the sample

SD =
0.0240
5

SD=0.069 3

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=0.30 2

Processed data
Rounded to 3 significant figures

Temperature ( Mean change in Standard deviation Rate of catalysis

0.5C) oxygen (% min-1) (% min-1)
concentration (%)
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 2 Processed data of final calculated result

Impact of Temperature on Rate of Catalysis

2

1.8

1.6

1.4
1.2

Rate of Catalysis (% min-1) 1

0.8
0.6

0.4

0.2

0
10 15 20 25 30 35 40 45 50

Temperature (C)

Figure 7 final graph showing the affect of temperature on rate of reaction of

catalase

T-Test
In order to prove the data has a significant different between two temperature
levels, a t-test is employed. This is needed between the interval of 35C and 40C
as the error bar shows a possible overlap. Below is a sample calculation used to
calculate whether the difference in rate of catalysis is significant between 35C and
40C.
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=

SD 12 SD 22
n1
+
n3
 (1.791.66)
t=

0.02142 0.08112
6
+
6

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

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

ure
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 Reject Reject Reject Reject Reject Reject Reject Reject
hypothesi ed ed ed ed ed ed ed ed
s

Table 3 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.

Evaluation
The results from this experiment answers the research question and demonstrates a
strong positive relationship 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. Both the
processed data represented in the Figure 7 and the raw data in Table 5 support the
predicted hypothesis. The amount of foam produced in each trial also supports this
correlation. 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
substrate and enzyme can occur to catalyse the reaction. However, at higher
temperatures, the protein enzyme denatures and the active site becomes
misshaped so that the substrate can no longer bind to the enzyme. The positive
correlation is most clearly seen in the graph 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 correlation suggests a possible reason why most living
ectothermic organisms maintain a body temperature of around 35C to 40C.

Limitations
The numerous controlled variables in this experiment ensure that there were not
many limitations. All external limiting factors of enzymes were controlled in each
trial so as to isolate temperature as the independent variable. This allowed for the
data to reflect accurately

Due to there being three trials run simultaneously, between the Vernier containers
could not be sealed immediately after oxygen production would begin. This would
result in some data being not recorded thus the entire reaction is not reflected in
the final rates of reaction, which may be inconsistent or lower than the actual rate.
This limitation was considered in the process of experimentation, all sensors were
zeroed prior to data collection, and all tests revealed a linear increase in oxygen
concentration, thus this would not greatly affect the results of the experiment.
However, possible methods to improve this limitation are to use larger quantities so
the data is less influenced by errors or to find a lab partner so that all three trials
can be handled simultaneously.

When combining catalase and hydrogen peroxide in the Vernier container, both
come into contact with the sides of the container. Some molecules could have stuck
to the sides of the container in the form of droplet. This would mean that there is a
lesser and variable amount of each reacting at the bottom of each container. These
errors are significant because substrate amount and concentration and amount of
enzyme are limiting factors that directly impact enzyme activity. This problem can
be fixed by inverting the container so that all the enzyme solution comes into
contact with all substrate molecules.

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

to each set of trials, however this introduced water droplets 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. A possible method of improvement is to use multiple
sets of three Vernier containers which would allow time for each set to dry in
between trials.

The potato solution used to obtain the enzyme catalase was made from red
potatoes from a local supermarket and whilst the potatoes were of the same type
and the amount for each trial, the potatoes may have slightly differing amounts of
enzyme depending on growing condition or even where the potato sample was cut
which may have resulted in a rate of catalysis that is slightly higher or lower than
normal. This is important as a different potato is to be used for each preparation of
catalase solution. To improve this a possibility is to prepare a large sample of
catalase solution and run all the trials simultaneously or so that the catalase
solution remains the same for all trials.

These improvements can help to increase the accuracy and precision of results
obtained. 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 more concrete evidence as to a
possible link between body temperature and optimal temperature of the enzyme
catalase.

Bibliography

Worthington Biochemical Corporation. New Jersey, United States, Introduction to Enzymes,

http://www.worthington-biochem.com/introbiochem/factors.html Last visited March 13 2017

Product Oasis, Red Potatoes,

http://www.produceoasis.com/ProductDetailPage/TabId/272/pid/407/Vegetables/Red-
Potatoes.aspx, Last visited March 13 2017