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Mrs. Jayasekara
BIO-101-054
Kaylie Pace
Catalase Enzyme Lab
1. Abstract
In this lab, we studied catalase, an enzyme extracted from potatoes that will break down
hydrogen peroxide into water and oxygen. During this study, we tested the enzyme extract under
different environmental changes to see if it would affect productivity. The objective of this
experiment was to understand how different factors influence enzyme reactions/activity,
including enzyme concentration, temperature, pH levels, and oxygen exposure. Compared to
other studies, we focused on multiple factors that can affect an enzyme while most other studies
would focus on just one of these factors. During the study, we used Hydrogen Peroxide (H2O2)
as our substrate to cause chemical reactions with different factors. The results state that enzymes
work best in a neutral pH state (7), enzymes work best in normal temperatures, and will denature
if it gets too hot, and increasing enzyme concentration will cause a higher reaction.
2. Introduction
This study is focused on how pH, temperature, oxygen exposure, and enzyme concentration can
result in alterations in the activity of the enzyme. The enzyme concentration for this experiment
consists of 160 grams of a peeled potato that is then blended for 2 minutes with 800 ml of water.
In the study we used an enzyme, catalase hydrogen peroxide (H2O2) which comes from the
following reaction: 2H2O2 -> O2 + 2H2O. Hydrogen peroxide is generated from a variety of
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metabolic reactions and can also result from oxygen exposure. Throughout the study a positive
reaction that shows bubbling was the result of catalase from cells breaking down the H2O2 into
water and oxygen.
Enzymes are binding agents that bind substrates or reactants, catalyze the reaction, release the
products, and proceed to repeat the process over and over again. To regulate enzyme levels cells
will control which genes are expressed and the amount of enzymes synthesized. Throughout the
experiment the enzyme concentration was increased to see if adding more enzyme concentration
will increase the reaction. The hypothesis of this study was that if the concentration of the
catalase is increased, then hydrogen peroxide decomposition will increase and the null
hypothesis is that there would be no change when the concentration is increased.
This study shows how enzyme concentration can speed up or be impacted by different external
changes such as temperature or pH. This is important because it shows how enzymes drive
chemical reactions that cause digestion and metabolism.
3. Materials and Methods
Exercise 6. How Does Exposure to Oxygen Affect the Enzyme Activity?
Firstly, you will cut three cubes of a peeled potato roughly 2 gm (1.3x1.3x1.3). Place one under
50 ml of water in a 100 ml beaker. Place another one aside dry in a weight boat, and for the third
cube potato divide it into small pieces roughly 2mm thick and leave it dry in a second weight
boat. Leave to incubate for at least 45 minutes. Take the low oxygen potato sample (the one
under water) and place it in the mortar. Slice it into several pieces and then add 10 ml of water
and crush and grind with the pestle for 2 minutes. Carefully decant 5 ml of the liquid into a 10 ml
graduated cylinder and then do the same process with the whole piece incubated dry/sliced, add
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10 ml of water, and crush and grind for 2 minutes and decant 5 ml and transfer into test tube #2
repeat with the final high oxygen sample. In 3 separate tubes, measure 5 ml of H2O2. When all
tubes are prepared, then pour the H2O2 into each tube of the potato extract. Over a span of 5
minutes, you will see a bubble reaction reaching up to 5 cm.
Exercise 2. The Catalase Reaction
Start by using a 10 ml graduated cylinder or pipet, and then add 5 ml of enzyme extract to a
clean 20 ml test tube. Next, use a 5 ml pipet and measure 5 ml of hydrogen peroxide (H2O2) and
add that to the enzyme extract in the test tube. Cover the top of the tube with a small piece of
parafilm, hold your thumb over the top, and invert the tube once. Measure the height of the
bubbles in the tube and record the height in centimeters every 30 seconds for 5 minutes or until
the bubbles are greater than 5 cm.
Exercise 3. How Does the Amount of Enzymes Affect the Rate of Reaction?
Start by labeling 4 test tubes #1-#4 and place the following measurements in each Test Tube #1:
0ml enzyme extract + 5 ml water, Test Tube #2: 1 ml enzyme extract + 4 ml water, Test Tube #3:
3ml enzyme extract + 2ml water, Test Tube #4: 5ml enzyme extract + 0ml water. Then
pre-measure into 4 clean test tubes 5 ml of Hydrogen Peroxide (H2O2). Prepare a 2x2 cm square
of parafilm for each of the 4 tubes. For each tube simultaneously add 1 tube of H2O2 to each
tube. Put the parafilm on each tube, then invert once. Monitor the rate of reaction in each tube
closely. When one tube reaches 5 cm high, measure all 4 tubes.
Exercise 4. How Does pH Affect the Rate of Reaction?
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Starts by labeling 3 test tubes #1-#3 with the following mixtures in each tube, Test Tube #1: 4ml
enzyme extract + 1ml pH 2.0 phosphate buffer, Test Tube #2: 4ml enzyme extract + 1ml pH 7.0
phosphate buffer, Test Tube #3: 4ml enzyme + 1ml 12.0 phosphate buffer. Use a 10ml graduated
cylinder/pipet for each solution. Prepare 3 separate tubes as before with 5 ml H2O2 in each. Cut
3 small pieces of parafilm to use for mixing each of the tubes. When all tubes are ready,
simultaneously add the H2O2 to tubes #1-#3, cover with the parafilm, hold with your thumb, and
invert. Monitor the rate of reaction in each tube closely. When one tube reaches 5 cm high,
measure all 3 tubes.
Exercise 5. How Does Temperature Affect the Rate of Reaction?
Start by preparing 3 test tubes with 5 ml enzyme extract in each tube. Then prepare 3 separate
tubes with 5 ml H2O2 in each. Place tube #1 and one of the H2O2 tubes into a beaker of ice
water. Leave test tube #2 and one H2O2 tube at room temperature. Then place test tube #3 and
one H2O2 tube into a boiling water bath. Wait 5 minutes for all temperatures to equilibrate, then
mix each H2O2 tube with its enzyme tube. Return each tube to its prescribed temperature. When
one test tube reaches 5 cm high, measure all 3 tubes.
4. Result
TUBE OXYGEN EX 6 VOLUMES FOR EACH TUB AVG SD SEM
LEVEL
1 LOW 9.956667 6.305485 2.574204
12.5 13.6 9.53 4.54 18.43 1.14
2 MEDIUM 4.045 4.017944 1.640319
7.94 10.2 2.27 1.14 2.27 0.45
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3 HIGH 5.86 5.335462 2.178193
4.5 11.4 13.2 0.57 4.81 0.68
As the charts above prove that there is a negative correlation between oxygen exposure and the
reaction. As the exposure to oxygen was increased, the reaction started to decrease.
As the temperature was increased, the reaction increased, but if the temperature becomes too
high then the enzymes will denature causing a rapid decrease in the reaction.
Enzymes have an optimal pH of 5.5-8.5 with the experiment testing with a pH buffer of 2.0, 7.0,
and 12.0 the results prove that the reaction was most active at a pH buffer of 7.0.
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5. Discussion
Throughout the experiment, the hypothesis is that if the concentration of the catalase is
increased, then the hydrogen peroxide decomposition will increase. Was proven right because
higher enzyme concentrations provide more active sites for the reaction to occur. This data is
important to show with different variables, the productivity of the enzyme will change, and
shows how much this enzyme reaction changes. In the study Factors Affecting Reaction Rate and
Bubble, their results were compatible with my results regarding optimal pH for an enzyme. Their
results show that the bubble activity was most active at a pH of 7 with 2.6ml volume of bubble,
while the lowest reaction was at a pH of 5 with activity reaching 0.6ml. With the study we
conducted, the optimal pH was 7 and our lowest reaction was a pH of 2, showing the closer to
neutral pH, the higher the reaction while the farther it is from neutral pH, the lower the reaction
will be. In the same study conducted above, they also tested for temperatures on the enzyme
catalase. Their results are compatible with my final conclusion, as the temperature was increased
in their experiment, they found that the enzyme reaction rapidly decreased. This is caused by the
denaturing of enzymes caused by the rapid heat increase. This is how our enzymes reacted when
we rapidly increased the temperature, they started to denature and the reaction came to a rapid
stop. Both experiments showed a higher reaction at normal temperatures.
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Work Cited
Enzyme Action: Testing Catalase Activity,
[Link]/site/handlers/[Link]?moduleinstanceid=4134&dataid
=5433&FileName=Enzyme_Activity_Lab.pdf. Accessed 16 Mar. 2025.
Single‐step Purification of Catalase Enzyme from Human Blood Erythrocytes Using
Affinity Chromatography Technique - Çıkrıkcı - 2024 - Biomed Research International -
Wiley Online Library, [Link]/doi/10.1155/2024/2222098. Accessed 16
Mar. 2025.
“Enzyme Lab Report: Factors Affecting Reaction Rate and Bubble - Course Sidekick.”
Coursesidekick, [Link]/biology/2088667. Accessed 16 Mar. 2025.
