BIOLOGY  

Factors Affecting Enzyme Activity   

How do different concentrations of substrate solution and different pH levels affect 
the rate of enzyme activity? 
Sara Du 

INTRODUCTION
I read a few recent scientific studies that indicated that low catalase levels could be a factor in
the graying process of human hair. Hydrogen peroxide is produced naturally in the human body
and is a strong oxidizing and bleaching agent. Catalase breaks down hydrogen peroxide into
water and oxygen. However, when there is a drop in catalase levels, hydrogen peroxide cannot
be decomposed. The hydrogen peroxide accumulation bleaches the hair inside out. Therefore,
this came of great interest to me, to understand the process of enzyme activity and the factors
that affect it ​(Sciencing.com, 2019).

RESEARCH QUESTION
How does changing the concentration of substrate solution (hydrogen peroxide) and different
pH levels affect the rate of enzyme(catalase) activity ?

HYPOTHESIS
As the concentration of substrate increases, the rate of enzyme activity will also increase. T​his is
because more ​substrate​ molecules will be colliding with ​enzyme​ molecules, therefore more
product will be formed. While a pH level around 7 will have the highest enzyme activity as the
optimum pH levels for catalase is near neutral.

BACKGROUND INFORMATION
All cells and organisms rely on enzymes to catalyze chemical reactions. An enzyme is
something produced within a living thing that acts as a catalyst to increase the rate of chemical
reactions by lessening the level of energy needed to activate the reaction. Without enzymes,
many of the chemical reactions that organisms require to sustain life would proceed too slowly
to be useful. Enzymes increase the rate of these reactions by bringing the reactants closer and
facilitating their interaction. The catch that comes with the use of enzymes is the fact that they
require consistency within their environment to be able to function properly. When enzymes
are not in their ideal environment for their best functionality they can begin to breakdown and
become ‘denatured ​(Worthington-biochem.com, 2019).

Catalase is a common enzyme found in nearly all living organisms exposed to oxygen. Catalase
is involved in the breakdown of hydrogen peroxide into water and oxygen:
A number of factors are known to affect the rate of enzyme-controlled reactions including:
● pH: Each enzyme has an optimum pH range. Changing the pH outside of this range will
slow enzyme activity. Extreme pH values can also cause enzymes to denature.
● Substrate concentration: Increasing substrate concentration also increases the rate of
reaction to a certain point. Once all of the enzymes have bound, any substrate increase
will have no effect on the rate of reaction, as the available enzymes will be saturated
and working at their maximum rate ​(Khan Academy, 2019)​.

VARIABLES TABLE
VARIABLE IDENTITY MANIPULATION

Dependent Percentage of dissolved oxygen - This will be measured using a

variable (Rate of enzyme activity ) data logger/probe for the initial
and final percentage of
dissolved oxygen. The
difference will be calculated for
each trial and averaged. The
data probe will also be rinsed
before and after each trial

Independent Different concentrations of - Four readings will be obtained

variable substrate solution (hydrogen from four different
peroxide) concentrations of hydrogen
peroxide : 5%, 10%, 12%, 15%
Different pH levels - Four readings will be obtained
from four different pH buffer
solutions : 4, 7, 9, 10

Controlled Used apparatus - The apparatus will be used

variable throughout the experiment and
will not be changed. Apparatus
will be cleaned after each trial
to prevent contamination
Volume of Hydrogen peroxide - Volume of hydrogen peroxide
and pH buffers and pH buffer solution used to
react with each concentration
will remain constant at 20mls
for the peroxide, and 10mls for
the buffer solution for each trial
Concentration of the Hydrogen - 5% substrate concentration of
 peroxide used in the pH factor hydrogen peroxide will be used
experiment throughout the entire pH factor
experiment
Temperature of yeast - The temperature will be
suspension solution and measured to be approximately
substrate solutions 37 °C.

MATERIALS
Apparatus
- 6 x Beakers (4x 100mls, 2x 250mls)
- 2x Measuring cylinder (100cm​3, ​10cm​3,​) (±0.01 cm3)
- Whiteboard markers
- 1 x Dropper
- Stirring rod
- Kettle to boil water
- Stopwatch (±0.01 sec)
- Electronic scale (±0.01g)
- Thermometer (±0.05 °C )
- Data logger/oxygen probe
- Access to tap water
Chemicals
- 4 different concentrations of hydrogen peroxide (60mls per each concentration) and
(extra 300mls of 5% hydrogen peroxide)
➢ 5%, 10%, 12%, 15%
- 4 different pH buffer solutions (30mls per each pH)
➢ 4, 7, 9, 10
- 250mls x 10% Yeast suspension
- 25g Yeast + 250mls Warm water (~37 °C)
Creating the 1% yeast suspension:
Measure out 25g of yeast using the electronic scale and place in the 250 ml beaker
1. Boil water then let cool. Mix the boiled water with tap water in a separate
beaker, and use a thermometer to ensure that the temperature is approximately
37 ​°C.
2. Measure out 250 mls in a separate beaker using the 100cm​3​ measuring cylinder
Dissolve the yeast by adding 100 mls of warm water first and gradually add in
water till 250ml of yeast is made. Stir well with a stirring rod until all yeast is
dissolved
Safety Apparatus
- Lab coat
- Safety goggles
- Gloves

Safety precautions:
- This experiment requires the use of corrosive substances hydrogen peroxide, as well as
breakable glassware. Subsequently, gloves, lab coats, safety goggles, and enclosed shoes
were worn. Care was taken when disposing of the chemical and water was provided to
clean the equipment.

METHOD
- Set up one 250ml beaker filled with water on the side and have the data probe in the
water filled beaker. This is used to clean and rinse the data probe after each trial
Method for measuring oxygen production with different pH levels
1. Set up four 100mls beakers and label each beaker with a different pH: 4, 7, 9, 10
2. Measure out 20mls of 5% Hydrogen peroxide and add into the four beakers
3. Add 10 mls of pH buffer solution of pH 4 into beaker labeled pH 4 and repeat for the
other levels of pH buffer solution with their respective beakers, rinsing the measuring
cylinder each time.
4. Place the data probe into the pH 4 beaker and let it sit for a minute, read and record the
oxygen produced at the minute mark. This is the initial oxygen produced.
5. Remove the probe and rinse in the water beaker. Keep the probe stationary at this
beaker when not in trial
6. Measure out 5 mls of yeast suspension using the 10cm​3 ​measuring cylinder and pour
into the pH 4 beaker. At the same time place the rinsed data probe into beaker pH 4 and
start recording oxygen production, timing it for 90 seconds.
7. Once the stopwatch hits 90 seconds, stop and remove the data probe and record the
final oxygen percentage produced.
8. Rinse the data probe in the water rinse beaker
9. Repeat steps 4-8 for the other 3 individual pH levels (7, 9, 10)
10. Do three trials for each pH level.

Method for measuring dissolved oxygen with different substrate concentrations

1. Set up four 100mls beakers and label each beaker with a different concentration of
substrate solution : 5%, 10%, 12%, 15%
2. Measure out 20mls of 5% Hydrogen peroxide and pour into the beaker labelled with the
respective concentration, repeat for the other three concentrations
 3. Place the data probe into the substrate 5% beaker and let sit for a minute, read and
record the oxygen produced at the minute mark. This is the initial oxygen produced
4. Measure out 5 mls of yeast suspension using the 10cm​3 ​measuring cylinder and pour
into the 5% beaker. At the same time place the rinsed data probe into beaker 5% and
start recording oxygen production, while timing it for 90 seconds.
5. Once the stopwatch hits 90 seconds, stop and remove the data probe and record the
final oxygen percentage produced
6. Rinse the data probe in the water beaker
7. Repeat steps 3-6 for the other 3 substrate concentrations (10%, 12%, 15%)
8. Do three trials for each concentration of substrate solution.

Qualitative Data

pH level After adding yeast suspension

4
Frothy thin layer and bubbles present
7

9 Frothy thick layer and large bubbles present

10 Frothy layer and bubbles lower that pH level 9 present

Concentration of substrate solution

After adding yeast suspension
(%)

5
Thin layer of bubbles and foam visible
10

12 Slightly thicker layer of foam and bubbles present

15 Large bubbles and foam that filled beaker

Raw Data

Substrate Initial percentage of dissolved Final percentage of dissolved oxygen (%)

concentration oxygen (%)

5 17.20 14.85

23.69 37.74

21.09 39.84

10 18.16 12.27

24.13 48.38

25.30 52.62

12 19.46 44.08

26.61 85.74

25.42 76.54

15 20.80 13.61

63.14 104.58

27.42 98.27

pH level Initial percentage of dissolved oxygen Final percentage of dissolved oxygen (%)
(%)

4 18.29 15.23

20.59 15.57

21.07 15.96

7 21.74 18.98

23.9 25.15

23.81 26.91

9 32.92 35.18

28.55 38.46
 23.09 24.99

10 40.57 36.67

21.35 27.72

24.29 23.59

Processed Data
Substrate Percentage of Average mass of Time taken (sec) Rate of reaction
concentration (%) oxygen produced oxygen produced (%/sec)
(%) (%)

5 -2.92 9.96 90 0.111

14.05

18.75

10 -5.89 15.22 90 0.169

24.25

27.30

12 24.62 44.96 90 0.500

59.13

51.12

15 -7.19 35.03 90 0.389

41.44

70.85

pH level Percentage of Average oxygen Time taken (mins) Rate of reaction

oxygen produced produced (%) (%/sec)
(%)

4 -3.06 -4.40 90 -0.0489

-5.02

-5.11
 7 -2.76 2.37 90 0.0263

1.25

3.10

9 2.26 51.18 90 0.5690

9.91

1.90

10 -3.90 1.77 90 0.0200

6.37

-0.70

Average percentage of oxygen produced was calculated using the formula :

Avg = total of three trials/no.of trials

Eg. average percentage of oxygen produced for pH level 9 = (2.26 + 9.91 +1.90)/3
= 0.01966667
​ ≅ 0.0200 % (rounded off to three significant figures)

RESULTS

(graph 1)
The graph above is a scatter graph showing the percentage of dissolved oxygen against different pH levels. The
graph shows a positive correlation and most of the data are slightly closer to the trendline suggesting slight
accuracy. From the graph it can be derived that the pH level of 9 is the optimum pH level for catalase activity.
 (graph 2)
The graph above is a scatter graph showing the percentage of dissolved oxygen against different concentrations of
substrate solutions. The graph shows a positive correlation and most of the data are slightly closer to the trendline
suggesting slight accuracy. From the graph it can be derived that the 12% substrate solution is the optimum
concentration for catalase activity.

(graph 3)
The graph above is a scatter graph showing the rate of enzyme activity against different concentrations of
substrate solutions. The graph shows a positive correlation and most of the data are slightly closer to the trendline
suggesting slight accuracy. From the graph it can be derived that the 12% substrate solution is the optimum
concentration for catalase activity.
 (gr​ aph 4)
The graph above is a scatter graph showing the rate of enzyme activity against differentpH levels. The graph shows
a positive correlation and most of the data are not close to the trendline suggesting less accuracy. From the graph
it can be derived that the pH level 9 is the optimum pH level for catalase activity.

CONCLUSION
As stated in the hypothesis, as the concentration of substrate increases, the rate of enzyme
activity will also increase. The experimental data partially supports this as the increase of
concentrations from 5 to 12% had an increase in enzyme activity. Although the enzyme activity
increases till 12%, it dropped down when 15% of substrate solution was added. This suggests
that all of the enzymes have bound, therefore any substrate increase after 12% will have no
effect on the rate of reaction, as the available enzymes will be saturated and working at the
maximum rate. The hypothesis also stated that a pH level around 7 would have the highest
enzyme activity, the experimental data disproves this. The experimental data shows that a pH
level of 9 had the highest enzyme activity. Although the optimum pH level of yeast catalase is
considered to be around -4.5-5.5, the experimental data did not support this. This could be due
to random and systematic errors. Finally, although there aren't any sufficient data to prove a
statement, the experiment suggests that the optimum pH level for catalase in yeast is a pH level
of 9 while the optimum concentration of hydrogen peroxide is 12%.

EVALUATION
The experimental data is believed to be fairly accurate as this experiment had controlled
variables to prevent other factors affecting the results and a proper method to carry out the
experiment. The data is reliable as the controlled variable such as the apparatus used and
volume of pH or substrate solution was kept constant, leading to a fair experiment where no
results had been affected or slightly changed. In addition, safety precautions were also carried
out properly and no injuries occurred.
The low accuracy from the graphs suggest that more random errors were present than
systematic. Since there was only one probe, the experiment was separated into multiple days
due to time constraints. Therefore the substrate concentrations used and yeast suspensions
were put in freezers. Taking the yeast and solutions out of the fridge and warming them up may
not have been properly executed, which may affect results as temperature is a factor that
affects enzyme activity. This will result in a lower rate of enzyme activity as catalase works best
at around 37 degrees. Secondly, the rinsing and cleaning of equipment could have been carried
out with distilled water for more accurate results. Lastly, the data probe rinsing between the
trails may have affected the results as the water was changed after each trial of all four pH
levels and substrate solutions. The contaminated water mixed with several different pH levels
could have affected the results.
REFERENCES
Khan Academy. (2019). ​Enzymes review​. [online] Available at:
https://www.khanacademy.org/science/high-school-biology/hs-energy-and-transport/hs
-enzymes/a/hs-enzymes-review [Accessed 15 Sep. 2019].

Sciencing.com. (2019). [online] Available at:

https://sciencing.com/role-catalase-5521462.html [Accessed 15 Sep. 2019].

Worthington-biochem.com. (2019). ​Enzymes and Life Processes (Introduction to Enzymes)​.

[online] Available at:
http://www.worthington-biochem.com/introBiochem/lifeProcesses.html [Accessed 15
Sep. 2019].