AUSTRALIAN SCIENCE AND MATHEMATICS SCHOOL
Effects of substrate concentration on
enzyme activity of catalase from yeast
Investigation Report
Melanie Bennett
February 2014
Effects of substrate concentration on enzyme activity of catalase from yeast
Melanie Bennett
Name: Melanie Bennett
Group Members: Mary-Jo Sioson, Oliwia Derda
Studio Support Staff: Mary-Anne Eddy
Stage 2 Biology Teacher: Penny Collins
Date: 20th February 2014
Introduction
Enzymes
The following introduction is primarily based on the textbook ‘Biology’ by Alan Crierie and David Grieg (pp. 18–
250). Metabolism is defined as the sum of an organism’s chemical reactions in the body (Campbell, et al,
1999). Enzymes are biological catalysts that speed up and control chemical reactions occurring in body (pp.
19). As shown in figure 1, enzymes work by complementarily binding with a substrate molecule on the active
site of the enzyme (pp. 19). It is the induced fit between the substrate and enzyme which causes the reaction
to activate (pp. 19). Enzymes cause reactions to occur faster by lowering the amount of activation energy
required (pp. 24). The reaction itself does not change the enzyme; therefore, enzymes can constantly speed up
chemical reactions (pp. 25)
Product synthesised
Complementary Binding
Substrates
Active Site
Enzyme
Figure 1: Enzyme catalysing two substrates
There are two types of metabolic reactions that occur in the body. Metabolic reactions are either exergonic or
Endergonic. Exergonic is a breakdown reaction and energy is given off when molecule’s bonds are broken (pp.
249). Endergonic reactions are synthesis reactions that require energy for molecules to join together (pp. 250).
Molecular recognition is an important part of metabolic processes in an organism (pp. 24). All enzymes are
specific to their reaction and their substrate (pp. 24). Therefore, there is one reaction each enzyme will speed
up and there is one specific substrate complementary to that enzyme (pp. 24).
Hydrogen Peroxide and Catalase
Hydrogen peroxide is a by-product produced by chemical reactions in the body. Hydrogen is a substrate to the
enzyme catalase (pp. 25). The enzyme catalase can be found in yeast. Catalase activates an exergonic reaction
when it is bound with hydrogen peroxide to produce the products water and oxygen as shown in the following
formula (pp. 25):
2H2O2  2H2O + O2 (pp.25)
A method called the floating disc method can be used to calculate the enzymes reaction rate (Kok, 2012). By
placing a small amount of filter paper (soaked in yeast) in its substrate solution, the catalase in the yeast will
catalyse the hydrogen peroxide to produce the products oxygen and water (Kok, 2012). Oxygen will stick to the
oxygen, causing the paper to float (Kok, 2012). The time it takes for the filter paper to float back to the surface
can be then used to calculate its reaction rate (Kok, 2012).
Effects of substrate concentration on enzyme activity of catalase from yeast
Melanie Bennett
Factors altering complementary binding between enzymes and substrates
Factors such as pH levels, temperature, inhibitors and cofactors can effect and prevent substrates and
enzymes binding on the active site (pp. 26)
-
pH – Each enzyme has a specific pH level it works efficiently at (pp. 27)
Temperature – can increase or decrease collisions between molecule to change the enzyme activity, it
can also permanently denature an enzymes protein structure (pp. 27)
Due to the concern of these factors preventing substrates and enzymes binding on the active site, these
factors were kept constant.
Investigation Plan
The aim of the investigation was to explore the effects of changing the concentrate of a substrate called
hydrogen peroxide on the activity of the enzyme catalase in yeast.
The independent variable was the concentration of the hydrogen peroxide used. This variable was
independent because when the concentration of hydrogen peroxide changed, there would also be a change in
the rate at which the catalase soaked paper floated to the surface of the beaker filled with Hydrogen peroxide.
There were 6 different concentrations of hydrogen peroxide tested; 0%, 0.1%, 0.2%, 1%, 3% and 5%.
Ultimately, the dependent variable was the time it took for the catalase soaked paper to float which
corresponded to the rate of the catalysts reaction. The time was tested in seconds, rounded off to the nearest
two decimal points.
For the results of the experiment to be highly accurate there were a number of factors needed to be kept
constant:








The size of the beakers
The area and thickness of the paper discs
The measurement of solution in the beakers (40ml)
The concentration of the catalase solution
The stop watch used and it’s decimal placed
The formula used to calculate the rate of the reaction
Where the catalase soaked paper was placed in the solution
The amount of time the paper was soaked in the catalase
Hypothesis:
If the concentration of the hydrogen peroxide solution increases then the rate of the reaction will also
increase.
Effects of substrate concentration on enzyme activity of catalase from yeast
Melanie Bennett
Materials
Container of paper filter disc of the same size
Tweezers
Yeast Sachet
Stop Watch
Hydrogen Peroxide Solutions (0, 0.1, 0.2, 1, 3, 5 (v/v) %)
Distilled water at Room Temperature
Wooden Skewer
½ Teaspoon Sugar
50mL measuring cylinder
100mL measuring cylinder
Small weigh tray
6 X 50mL beakers
Paper Towel
Plastic Spoon
Sticky Labels
Procedure
1.
Hazard minimization procedures were put into place. Safety glasses, tied back hair, closed in shoes,
lab coats, gloves and camera were also prepared.
2.
The yeast solution was prepared before the investigation began. The yeast solution was made by
adding one sachet of yeast (7g) and ½ teaspoon sugar to 100mL of distilled water which has been
warmed to 30 degrees Celsius.
3.
40mL of each of the different concentrations (0, 0.1, 0.2, 1, 3, 5 (v/v) %) of hydrogen peroxide solution
was measured into clean separate 50mL beakers.
4.
Using the tweezers, the paper discs were carefully dipped one at a time into the yeast solution for 5
seconds and placed onto a piece of untouched paper towel for 10 seconds to remove any excess
solution. Handling the discs with fingers was avoided.
5.
Using the tweezers, the soaked disc was placed on the bottom of the beaker of hydrogen
peroxide solution. Timing using the stop watch began when the disc was released at the
bottom of the beaker and was stopped when the disc reached the surface of the solution.
The time was recorded to two decimal places.
6.
It was planned that if a disc did not rise, the time would be automatically stopped at 5.00
minutes.
7.
Steps 1 to 5 were repeated for three replicates. All data was recorded in a results table.
8.
Lastly, the reaction rate between the catalase and the hydrogen peroxide was calculated by
taking the mean number of seconds it took the disc to float to the surface and inverting it.
Effects of substrate concentration on enzyme activity of catalase from yeast
Melanie Bennett
Results
Concentration of Hydrogen Peroxide and its effect on the reaction rate of Catalse
Concentration of Hydrogen
Peroxide (%)
Trial 1 (sec)
0
0.1
0.2
1
3
5
64.29
38.19
24.06
7.35
2.37
1.28
Trial 2
(sec)
65.19
40.25
24.34
9.81
1.85
0.60
Trial 3
(sec)
65.03
30.91
23.09
10.82
1.62
1.19
Average Time
recorded (sec)
Rate of Reaction
seconds-1
64.84
36.45
23.83
9.33
1.95
1.02
0.02
0.03
0.04
0.11
0.51
0.98
Figure 2: Table of results of the effects of substrate concentration on enzyme activity
Effect of Substrate Concentration (Hydrogen Peroxide) on
the Rate of Enzyme (Catalase) Reaction
Rate of Reaction (s--1)
1.20
1.00
R² = 0.987
0.80
0.60
Rate of Reaction 1/T
0.40
Linear (Rate of Reaction 1/T)
0.20
Linear (Rate of Reaction 1/T)
0.00
0%
2%
4%
6%
Concentration of Hydrogen Peroxide (%)
Figure 3: Line Graph representing the effect of substrate concentration (Hydrogen Peroxide) on enzyme activity (Catalase)
Figure 3 is a line graph representing the effect of Substrate Concentration on enzyme activity. The xaxis describes the concentration and the y-axis describes the rate of the enzymes reaction. Two lines
are shown distinguishing the rate of the reaction and the line of best fit; correlating closely. A trend
is evident; as the concentration increases from zero the reaction rate increases steadily until the
concentration reaches 5%.
Effects of substrate concentration on enzyme activity of catalase from yeast
Melanie Bennett
Discussion
Interpretation
The discussion will be in relation to figure 3 in the results section of the report.
The trend in figure 3 suggests that as the concentration of hydrogen peroxide increases, the reaction
rate increases and that the hypothesis is supported. The correlation coefficient calculated on figure 3
describes that there is a strong positive correlation between the rate of the enzymes reaction and
the concentration of hydrogen peroxide. Increased concentration increases the rate of the enzymes
reaction involving one particle because there is an increase in the amount of molecules that have
the required activation energy. For any enzyme reaction to occur, molecules must contain an energy
level that is equivalent or larger than that activation energy. An increase in the concentration of the
substrate molecules with the required activation energy will also increase, causing the reaction rate
to also increase.
If the concentration of substrate molecules doubles, than the reaction rate should double because
there is double the amount of molecules with the required activation energy (Kok, 2012). The results
did not support this. The statement also suggests that when the concentration of hydrogen peroxide
solution is at a concentration of 0% then there should be no reaction rate because there are no
molecules with the required activation energy in the solution. However, the results described the
reaction rate to be 0.02 (s-1). Thus, the results including the solution at 0% are invalid and cannot be
taken into account for the trend of the investigation.
Analysis
The investigation had room for systematic and random errors which altered results. The investigations
procedure and aim was not designed; therefore, systematic and random errors were not predicted or
prevented.
Possible Systematic Errors
A systematic error is an error of inaccuracy in observation and measurement, inbuilt in the system. Systematic
errors are easier to correct and may balance throughout the experiment; preventing the results being affected
substantially.


Amount of Hydrogen Peroxide Solution – The specific measurement of the solution for testing may
have been incorrect. However, each solution was tested 3 times each; all the samples reactions would
increase or decrease and not be subjected to variations.
Pressing the stop watch – In the experiment, the time it took the filter paper disc to reach the surface
of the solution was tested using a stop watch. The stop watch was started when the disc was released
in solution and stopped when it reached the surface. Firstly, the time it took would never be exactly
64.29 seconds or 38.19 seconds because of the ‘zero error’; it would either be rounded up or down.
This error will not affect the results because each of the samples being tested was put through the
same conditions. Secondly, the person timing would never exactly start or stop the timer at exactly
the right time; the results would also be rounded up or down. However, this error will also not affect
the results substantially because each of the samples being tested was put through the same
conditions.
Effects of substrate concentration on enzyme activity of catalase from yeast
Melanie Bennett
Possible Random Errors
Random errors are caused by irregular changes in the experiment. The changes are inconsistent,
uncontrollable and can occur in measuring equipment or in environmental conditions.
 Inconsistent amount of time disc was soaked in yeast solution – The amount of time the disc was soaked in
yeast solution was inconsistent. Some of the discs were not submerged for a long time compared to other
discs. Therefore, the amount of enzymes on the disc was inconsistent. Thus, the effect of the substrates
concentration on the enzyme could not be tested efficiently. To prevent the error from affecting results,
many samples at each of the solutions concentrations were tested.
 Cross contamination – Cross contamination will affect the results and they will not be reliable. To prevent
cross contamination, new and clean equipment were used for each sample being tested. More than one
sample was also tested to average out these random variations and decrease the likelihood of the
limitation of precision by random errors.
Accuracy relates to how close data is to the true value. The solution at a concentration of 0% was used as a
controlled sample because it was expected that there would be enzyme reaction if there was no substrate
(hydrogen peroxide) to bind to. The trend shows that the experiment results were not accurate. The sample
controlled did not match the true value and showed enzyme activity.
Precision relates to how close the consistency of the given results are to one another. The trend showed that
the results were precise because there was a high correlation between the concentration and reaction rate.
Figure 3 shows that the correlation coefficient of the relationship equalled 0.987; describing a highly strong
relationship between the two variables. However, this precision does not relate to its accuracy due to outliers
in the results.
Evaluation
The controlled samples were inaccurate and were far away from their true values. Therefore, there was no
way to conclude how accurate the changed samples were. The main factor contributing to the unreliability of
the data was the precision of the technique used. Many variables were not kept constant to ensure that the
effect between the concentration and reaction rate could be tested.
Increasing the accuracy of the reaction rate
Changing the type of measurement used would increase the accuracy of the results because the results would
have a higher resolution. A more accurate way of timing could be used such as videoing the reaction itself;
therefore a person will not stop and start the timer too slow or too fast. The time of the reaction could be
calculated accurately from the video to more decimal places. However, this would not improve the results
because the results would still be invalid unless the random errors were prevented and because less resolution
has still shown a trend between substrate concentration and reaction rate.
Reliability of Results
Reliability relates to ensuring the experiment has the same results if it were repeated. To increase the
reliability of the experiment, random errors could be minimised so that there are less outliers affecting the
results. To decrease the effect of random errors, the sample size could be increased; each concentration
tested more times. Cross contamination errors can also be decreased by setting up new equipment for each
sample. Lastly, the controlled variable needs to be tested first to ensure errors are not affecting results.
Effects of substrate concentration on enzyme activity of catalase from yeast
Melanie Bennett
Conclusion
The aim of the investigation was to explore the effects of changing the concentrate of a substrate called
hydrogen peroxide on the activity of the enzyme catalase in yeast. To investigate the effects, the floating disc
method was used to calculate the enzymes reaction rate when it was submerged into different concentrations
of hydrogen peroxide. The stop watch and a calculation could determine the time it took for the floating disc
to reach the surface of the solution and from this; the reaction rate could be calculated. Variables such as
temperature were kept constant, while the concentration of hydrogen peroxide was changed.
The trend in the results did not support the hypothesis. For this experiment it was hypothesised that ‘If the
concentration of the hydrogen peroxide solution increases then the rate of the reaction will also increase’.
When testing the reaction rate of the enzyme, figure 3 clearly showed that the reaction rate increased when
the concentration increased. However, the results were inaccurate to the true values due to the inaccuracy of
the techniques used. If the investigate was to be repeated, cross contamination would need to be decreased
by setting up new equipment for each sample and the controlled variable needs to be tested first to ensure
errors are not affecting results.
References
Campbell, NA. Reece, JB. & Mitchell, LG. 1999, ‘Biology’, 5th edn, Banjamin/Cummings, an imprint of
Addison Wesley Longman, Inc. California, pp. G14
Crierie, A. & Greig, D. 2010, ‘Biology: Key Ideas’, 3rd edn, SASTA, pp. 18-250
Kok, L. 2012, ‘Biology on decomposition of Hydrogen Peroxide by enzyme catalase’, accessed 22nd
February 2014, http://www.slideshare.net/wkkok1957/ib-biology-on-decomposition-of-hydrogenperoxide-by-enzyme-catalase