Lab #1: Factors Influencing Enzyme Activity

Kanishka Sikri
 Jefin Matthews, Abisha Kathirkamanathan, Nadiya Memraj, Amaar Hussein
Abstract
Through conducting this experiment, we learnt at which temperature (2C, 10C, 20C,
30C, 40C, or 50C) and enzyme concentration (1mL, 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8
mL, 9 mL, or10 mL) the rate of reaction is the highest. This experiment was done on a reaction
between potato extract and hydrogen peroxide, the two controlled variables in the experiment,
which produced a foam through the release of oxygen. Our group determined that the optimal
temperature the enzyme preformed the fastest in was 20C and the optimal enzyme
concentration level was 5mL. Enzyme activity was determined by the amount of foam produced.
We determined that as the temperature and enzyme concentration levels increased, enzyme
activity also increased. However, once enzyme activity reached its optimal temperature and
enzyme concentration level, the rate of reaction started to decrease. There were three main errors
done in this experiment, which led to us not achieving the optimal temperature and enzyme
concentration levels for potato catalase (40C); an error in taking the bath water measurements
with the thermometer, incorrect recording of results after two minutes, and inconsistent sampling
techniques.
 Introduction

Most enzymes are proteins. Enzymes are catalysts, which

are substances that speed up reactions without being
consumed. The enzymes speed up the reaction by
lowering the activation energy; this can be seen in Figure
1. Activation energy is the minimum amount of energy
required for the reaction to occur. Enzyme activity is the
rate at which the enzyme speeds up the reaction. Factors
like temperature, pH and substrate concentration can
affect enzyme activity.
Figure 1
In this lab, an enzyme called catalase was examined from a potato. The catalase broke down
hydrogen peroxide as follows:
H2O2 2H2O + O2
Hydrogen Peroxide Water + Oxygen

The purpose of this lab was to see at which temperature and enzyme concentration level the
enzyme activity was the highest. Through the study of enzyme activity, it is known that as the
temperature increases, the enzyme activity also increases, however once the enzyme has
achieved its optimal temperature, the enzyme activity starts to decrease. This is shown through
Figure 2 below. As enzyme concentration increases, enzyme activity also increases. This can be
seen in Figure 3.

Figure 2 Figure 3

The use of hydrogen peroxide in this lab requires many safety procedures. While using this
product, one should always wear safety goggles and keep the liquid away from their skin,
particularlytheireyes.Itisalsoimportanttokeeplonghairawayfromtheliquids,andtowear
closedtoeshoes.
 Hydrogen Peroxide (H2O2 ) is a toxic waste product of metabolism. During this reaction, catalase
created a foam from oxygen gas, that was measured to determine how fast catalase produced
oxygen gas. In this experiment, catalase was reacted with H2O2 under different temperature
levels and enzyme concentration levels to find the ideal setting where the catalase worked at a
higher rate. The best temperature/enzyme concentration would be observed when the reaction
produced the largest amount of foam. If the largest amount of foam is not produced, we do not
know the ideal situation.

Materials
Hydrogen Peroxide
Potato Extract
250mL Beaker
Test tube (25mL, 25mL)
50mL Graduated Cylinder
Thermometer
Pasteur pipettes
Hot plate
Water bath
Timer
Ice
Brush
Soap

Experimental Procedure
*I was not here to do the lab*
Effect of Enzyme Concentration on Enzyme Action:
I. Measured 8.0 mL of 3% H2O2 into a 10 mL graduated cylinder.
II. Measured 1.0 mL of the potato extract into another 10 mL graduated cylinder.
III. Combined the H2O2 and potato extract into a 50 mL graduated cylinder.
IV. Recorded the starting volume of hydrogen peroxide and potato extract.
V. Recorded the maximum volume (in mL) of the foam produced after 2 minutes.
VI. Repeated steps 2,3,4,5 and 6 nine times using first 2 mL of potato extract and then 3 mL,
4 mL, etc up to 10 mL.
VII. Disposed of all waste liquid.
VIII. Washed all glassware with a brush, soap, and water.

Effect of Temperature on Enzyme Action:

I. Ensured the temperature of the water bath was correct.
II. Measured 5mL of potato extract using the potato cylinder.
III. Put 8 mL of 3% hydrogen peroxide into peroxide cylinder.
IV. Placed each cylinder in the water bath and waited 10 minutes.
 V. Added hydrogen peroxide and potato extract to the reaction cylinder and recorded the
starting volume.
VI. Placed the cylinder to the water bath and observed for 2 minutes.
VII. Took the graduated cylinder out of the water an measured the maximum volume of foam
and liquid in the graduated cylinder.
VIII. Repeated steps 1 to 7 and put the test tubes into different temperature baths of 2C, 10C,
20C, 30C,40C, and 50C.
IX. Disposed of all waste liquid.
X. Washed all glassware with a brush, soap, and water.

Observations and Results

 Temperature and Enzyme Activity
Volume of Foam per Temperature (Our Results)

Temperature () Starting Final volume Change in Class Average

volume (mL) Volume (mL) (mL)
(mL)

2 13 18 5 1.428571429

10 13 16 3 3.285714286

20 13 19 6 4

30 13 18 5 4.571428571

40 13 18 5 4.857142857

50 13 11 2 -2

Volume of Foam per Temperature (Class Comparison)

Groups: 2oC 10oC 20oC 30oC 40oC 50oC
Jefin, Amaar, Nadiya, Abisha 6 3 6 5 5 -2
Sweeter mans 1 2 2 1 7 -2
Vivian & Harani 3 6 7 2 4 -2
Miranda, Prinka, Sameeha -2 3 2 5 5 -2
L.A.B. 2 3 3 10 5 -2
A.P.P. 2 3 6 5 4 -2
Sera -2 3 2 4 4 -2
CLASS AVERAGE 1 3.29 4 4.57 4.86 -2
 Volume of Foam per Temperature

2
Volume of Foam (mL)

-1

-2

-3
0 10 20 30 40 50 60

Temperature (C)

Jefin's Group Class Average

 Figure 4

This graph (Figure 4) compares the volume of foam versus temperature. The dependent
variable in this graph is the volume of foam and the independent variable is the
temperature. The enzyme's optimal temperature that it reacted the fastest in is 20 C. In
the class average, the optimal temperature that the enzyme reacted the fastest in is 40C.
Enzyme Concentration and Enzyme Activity
Volume of Foam per Enzyme Concentration (Our Results)

Amount of Starting Final volume Change in Class average (mL)

extract (mL) volume (mL) (mL) volume (mL)
 1 9 8 -1 1.4285714285

2 10 9 -1 2.1428571428

3 11 11 0 2.357142857

4 12 13 1 2.285714286

5 13 16 3 2.5714285714

6 14 16 2 4

7 15 17 2 3.1428571428

8 16 17 1 3

9 17 16 -1 2.28571428657

10 18 20 2 2.8571428571

Volume of Foam per Enzyme Concentration (Class Comparison)

Group Names: 1mL 2mL 3mL 4mL 5mL 6mL 7mL 8mL 9mL 10mL
Jefin, Amaar, Nadiya,
Abisha -1 -1 0 1 3 2 2 1 -1 2
Harani and Vivian 3 4 3 2 4 10 5 3 3 4
Sweeter mans 1 3 3 3 2 4 3 3 2 1
L.A.B. 1 1 2.5 1 2 2 2 3 2 2
Miranda, Prinka,
Sameeha 1 2 2 3 2 4 3 3 3 4
A.P.P. 3 4 4 3 2 2 3 4 3 3
Sera 2 2 2 3 3 4 4 4 4 4
CLASS AVERAGE 1.43 2.14 2.36 2.29 2.57 4 3.14 3 2.29 2.86
 Volume of Foam per Enzyme Concentration

Volume of Foam (mL)

1

-1

-2
0 2 4 6 8 10 12

Enzyme Concentration (mL)

Jefin's Group Class Average

 Figure 5

This graph (Figure 5) compares the volume of foam versus enzyme concentration. The
dependent variable in this graph is the volume of foam and the independent variable is the
enzyme concentration level (mL). The enzyme's optimal enzyme concentration level that it
reacted the fastest in is 5mL. In the class average, the optimal enzyme concentration level that
the enzyme reacted the fastest in is 6mL.
Discussion
This experiment was done to determine the optimal temperature and enzyme
concentration that the catalase worked the fastest in. To determine this, hydrogen peroxide and
potato extract was tested with different temperatures of 2C, 10C, 20C, 30C, 40C, and 50C
as well as different enzyme concentration levels of 1mL, 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL,
8 mL, 9 mL, and 10 mL. During both of these experiments a foam was produced as oxygen was
being released. Enzyme activity can be determined through looking at the amount of foam
produced. The temperature or enzyme concentration level that produced the most foam has the
highest rate of reaction.
 Through analyzing the data we learned that there were many errors that happened during
our lab. As we know through the study of enzyme activity, as the temperature increases, the
enzyme activity also increases, however once the enzyme has achieved its optimal temperature,
the enzyme activity starts to decrease. The optimal temperature level for the catalase being 40C,
which was the same as the class average. Yet, during our lab the temperature fluctuated up and
down, without reaching the optimal temperature level for catalase (40C). Instead, our optimal
temperature was 20C.
This is because of two main errors. Firstly, there was an error while taking the bath water
measurements. When the thermometer was dipped into the hot water to take the measurement, it
caused the temperature of the liquid to cool slightly. This lead to the wrong temperature levels
being tested which contributed to the results fluctuating instead of continuously increasing until
it reached its peak, and not achieving the optimal temperatue of catalase (40C). Another error
that occurred was the incorrect recording of results. The reaction had to be recorded after exactly
two minutes however the time that it took us to actually record the results was a little bit past the
two minute mark. This led to us recording the wrong volume produced, which led to inaccurate
results. This was another error that led to our temperature results fluctuating up and down, as
well as us not having our optimal temperature level at 40C, like the class average and optimal
temperature for catalase.
Furthermore, through the study of enzyme activity, we know that as enzyme concentration
levels increase, enzyme activity also increases. Our enzyme concentration levels fluctuated, like
the temperature levels, instead of steadily increasing. Our optimal enzyme concentration level
was 5mL. The class average also fluctuated instead of continuously increasing, the average being
6mL. One of the main errors that caused this was inconsistent sampling techniques. Every time
the experiment was done, each step must have been repeated the exact same way as it was
previously. The slightest change in temperature and enzyme concentration from the previous
experiment led to inaccurate results, which contributed to the fluctuating levels of enzyme
concentration versus enzyme activity in our results, as well as the class average.
The independent variable in the testing of the amount of foam produced versus temperature
is the temperature and the dependent variable is the amount of foam produced. This is because
the amount of foam produced only changed as the temperature changed. This can be observed
through the graph above (Figure 4) which focuses on amount of foam produced versus
temperature.
The independent variable in the testing of the amount of foam produced versus enzyme
concentration is the enzyme concentration and the dependent variable is the amount of foam
produced. The reasoning behind this is the same as the amount of foam produced versus
temperature, as the amount of foam that is produced only changed as the enzyme concentration
changed. This can also be observed through the graph above ( Figure 5) which focuses on
amount of foam produced vs. enzyme concentration.
There were two controlled variables in this experiment: hydrogen peroxide and potato
extract. These two are controlled variables because they remained constant throughout the entire
experiment. While the temperature and enzyme concentration changed throughout, the amount of
hydrogen peroxide and potato extract that was tested never changed.
Moreover, a way to modify this experiment to make it more accurate in a quantitative way
is to repeat specific parts of the experiment to get more precise results. For example, when trying
to find enzyme activity at 20C, you could repeat the process two times instead of one and then
average the results. This gives you a more accurate quantitative measurement.

Conclusion
Ultimately, the optimal temperature for the enzyme was 20C, as the amount of foam
produced was the largest at that temperature. The class average was 40C, which is the same as
the optimal temperature level for potato catalase. The optimal enzyme concentration level was
5mL, the class average being 6mL. Therefore, as the temperature increases the enzyme activity
also increases. However, once the enzyme reaches its optimal temperature (peak), the enzyme
activity begins to decrease.