Enzymes and the effect that Ph

1. Lab Report:
Title: Enzymes and the effect that
Ph and concentration has on their
activity
Section
6
Preparation by
‫احمد‬ ‫شهد‬
Suppression by
Dr. Mohammed Nauman Zahid

2. Introduction and background
• Certain macromolecules serve as catalyst and speed up chemical reactions,
these arecalled enzymes.
• They increase reaction rates by lowering the activation energy.Activation
energy is the energy that is required to change molecules so that the bonds
canbreak.
• Enzymes are typically proteins are they are not consumed by the reaction.
• Enzymes lower the activation energy when the substrate bonds to the
enzyme which putsa strain on the bonds and allows them to be modified
with less energy .
• The experiment was performed in order to see the effects that pH and
concentration have on the particular enzyme, catecholase.
• The group’s hypothesis forexperiment A was that the more basic the pH, the
faster the enzyme activity would takeplace.
• The null hypothesis stated that a change in pH would have no effect on the
enzymeactivity. For experiment B, the groups hypothesis was that the lower
the concentration ofa substrate, the faster the rate of enzyme activity.
• The groups null hypothesis was that thechange in the pH would have no
effect on the enzyme activity. For experiment B, thegroups hypothesis was
that the lower the concentration of a substance, the faster the rateof the
enzyme reaction. The null hypothesis stated that the concentration of
Introduction

3. Experiment 1
Catalase activity

4. actors affecting enzymatic reactions
nzyme Catalase
ch of these enzymes is responsible for one particular reaction
at occurs in the cell. In this lab, you will study an enzyme that is
und in the cells of many living tissues. The name of the enzyme
catalase; it speeds up a reaction which breaks down hydrogen
roxide, a toxic chemical, into two harmless substances → water
d oxygen.
The reaction is: 2H2O2 → 2H2O + O2
This reaction is important to cells because hydrogen peroxide (H2O2 ) is
produced as a byproduct ofmany normal cellular reactions. If the cells
did not break down the hydrogen peroxide, they would be poisoned and
die. In this lab, you will study the catalase found in liver cells. You will be
using chicken or beef liver. It might seem strange to use dead cells to
study the function of enzymes. This is possible because when a cell
dies, the enzymes remain intact and active for several weeks, as long as
the tissue is kept refrigerated.
Enzyme/substrate concentration

5. Objectives
• Measure the effects of changes in
temperature, pH, and enzyme
concentration on reaction rates of an
enzyme
• Explain how environmental factors affect
the rate of enzyme-catalyzed reactions

6. Experiment methods and materials
Fresh liver, Apple,
and Potato, Yeast
Straight-edged
razor Pipettes
6 Test tubes
HCL and NaOH
blade Scissors and
Forceps Measuring
Test tube holders
pH paper (optional)
Stirring rod Fresh
3% Hydrogen
peroxide
Materials

7. Procedure 1: Effect of enzyme concentration
or this experiment you will use potato as a source of catalase. When the potato
atalase reacts with hydrogen peroxide, oxygen is released and bubbling should
ccur. Use the following key to record catalase activity:
1. Predict what will happen in each test tube in the “Predicted” column
2. Label 3 test tubes with the numbers 1, 2, and 3.
3. Add 4.5 ml of hydrogen peroxide to each tube.
4. Then add the following to each tube and observe the degree of
bubbling.
Tube #1: Add a pinch of sand
Tube #2: Add a whole potato cube
Tube #3: Add a cube of potato that you have diced into small
pieces
5. Record your results (observations) below and explain these results
based on the above Background

8. Procedure 1: Effect of enzyme concentration

9. Results
Enzymes will work best if there is plenty of
substrate.
As the concentration of the substrate increases,
so does the rate of enzyme activity. However, the
ate of enzyme activity does not increase forever.
This is because a point will be reached when the
enzymes become saturated and no more
substrates can fit at any one time even though
here is plenty of substrate available.

10. Results
Table
Average Height of
bubble layer (cm)
Test
tube
content
No.
4.5
Catalase +
H2O2
1
1.7
H2O + H2O2
2
3
Catalase + sucrose
3

11. Procedure 2: Effect of Temperature
1. Predict what you will see in each of the 3 test tubes you will
prepare (i.e. will/will not solidify).
2. Using your wax pencil label 3 test tubes with the numbers:
1, 2, and
3. Add the following to each tube:
Tube #1: Add 3 ml of cold 2% milk and 3 drops of cold
rennin (both found on ice). Place this tube in the ice
bath tube rack.
Tube #2: Add 3 ml of 2% milk and 3 drops of Rennin.
Place this tube in the 37o C water bath tube rack.
Tube #3: Add 3 ml of 2% milk and 3 drops of BOILED
rennin. Place this tube in the 37o C water bath tube
rack.
NOTE: Your instructor has already boiled the rennin for
you.)After 30 minutes, examine the tubes, and record and
explain your results below.

12. Procedure 2: Effect of Temperature

13. Results
Enzymes will work best if there is plenty of
substrate.
As the concentration of the substrate increases,
so does the rate of enzyme activity. However, the
ate of enzyme activity does not increase forever.
This is because a point will be reached when the
enzymes become saturated and no more
substrates can fit at any one time even though
here is plenty of substrate available.

14. Results
Table
Average Height of
bubble layer (cm)
Temperature °C
No.
0.2
4
1
5.5
20
2
1.2
37
3
3.8
100
4

15. Procedure 3: Effect of pH on Enzyme Activity
Aim
To determine the optimum pH at which an enzyme’s activity is
greatest
Method
1. Set up a Bunsen burner, heatproof mat, tripod and gauze.
2. Place a beaker of water on the gauze and adjust the flame to keep
the water at about 35°C.
3. Now put two drops of iodine solution into each spot of a spotting
tile.
4. Add 2 cm3 of amylase enzyme solution to a test tube.
5. Place 2 cm3 of starch solution into the same tube.
6. Finally add 1 cm3 of pH solution to the tube. This will keep the pH
constant.
7. Mix the solution in the test tube and place it into the beaker of
water on the Bunsen burner.
8. Use a pipette to remove a few drops of solution every 20 seconds
from the test tube and put them into a different well of the spotting
tile.
9. Repeat until the iodine solution stops turning black.
10. Record the time this takes.

16. Procedure 3: Effect of pH on Enzyme Activity
Note: this practical uses the temperature of 35°C. This is
close to body temperature. Warmer temperatures may
denature the enzymes and the reaction will take longer at
lower temperatures.
Risks
Iodine solution is an irritant. If it touches skin it should be
washed off.
Goggles should be worn at all times.

17. Procedure 3: Effect of pH on Enzyme Activity

18. Results
Table
Average Height of
bubble layer (cm)
PH
No.
1.1
3
1
3.2
5
2
6.7
7
3
1.2
9
4
3.4
11
5

19. onclusions
• The enzyme amylase breaks down starch into
glucose. If the enzyme is working effectively,
this will happen quickly. At pH 7 it took the
shortest time before the iodine no longer
changed colour. This shows that the starch
was broken down more quickly at this pH. The
optimum pH for amylase is therefore pH 7.

20. rocedure 4: Effect of concentration on Enzyme Activity
• TEACHERS GUIDE TO:
ENZYME ACTIVITY FACTORS AFFECTING ENZYME TYPICAL
CLASSROOM USE. Recommended use of this lab for Gifted
First year Biology, Advanced Biology and Chemistry (first year)
STRATEGY FOR CONDUCTING LAB 1. Have students work in
groups of 3 or 4. 2. Assign each group only one factor to
investigate in the lab. a. Only one group need do the "Boiled"
enzyme section. b. Duplicate groups for temperature could be
assigned temperatures of 25°, 35°, 45°, and 55°. c. Duplicate
groups for concentration could include 1.5, 2.5, and 3.0 mL of
extract. *Remember to reduce the amount of buffer to maintain
the 4 mL total liquid volume in the even numbered trial test
tubes. d. Duplicate groups for pH could use different pH buffers
depending on availability.

21. rocedure 4: Effect of concentration on Enzyme Activity

22. Results
Table
Avenge Height of bubble
layer (cm)
Enzyme amount (ml)
No.
15.1
1
1
12.5
2
2
5.8
3
3
4.1
4
4
1.3
10
5
3.1
15
6
15.1
20
7
15.2
25
8
15.3
30
9
15.1
35
10

23. Discussion:
1. When looking at the graphs and data on pages seven through ten it
was evident that there wasroom for error in many respects. First off
the, the most apparent error seemed to be in the temperature lab. As
the temperature was increased the rate of the reaction should have
directly increased as well.
2. This was not completely apparent, because as the temper rose from
five degrees Celsius to ten degrees Celsius the rate of the reaction
decreased from 0.118 (1/s) to 0.066 (1/s). This could be a result of many
errors or the fact that potatoes do not normally grow in high
temperatures.
• This is an apparent discrepancy because it would be expected for the
rate to increase as the temperature increased. The second place for
errors could be basic human errors, while diluting the hydrogen
peroxide from 6% to 1%. However, all the results followed a trend that
was expected and agreed with the hypothesis that was made. Due to
this fact, none of the errors could have been that significant to
completely skew the meaning and significance of the analysis of the
data. From this we can conclude that the results are relatively valid.

24. Conclusion:
• Overall this lab was relatively successful in determining the effects of
enzyme concentration,substrate concentration, temperature, and an
inhibitor on the rate of the reaction.
• The objective of the lab was met, which was the rate of the reaction for
all four sections of the lab.
• In doing so the time for the disc to rise to the surface was measured
and then calculated into a rate. All this data that was collected was
used to agree with not only the objectives of the experiment but also
the hypotheses made on each individual lab.
• The results were relatively valid and showed a correct trend in data. To
make these results even more valid, it would be necessary to complete
the experiment multiple moreLee 14times because some sections did
not have multiple trial to verify the results completely. All in all the
results were reliable and showed the basic properties of enzymes and
their role in chemical reactions.

25. REFERENCES:
• 1. International Association for Soaps, Detergents, Maintenance
products “Guidelines for the Safe Handling of Enzymes in Detergent
Manufacturing” https://www.aise.eu/our-activities/standards-and-
industry-guidelines/safe-handling-ofenzymes.aspx2.
• The Soap and Detergent Association “Work Practices for Handling
Enzymes in the Detergent Industry”
http://www.aciscience.org/docs/Work_Practices_for_Handling_Enzyme
s.pdf3.
• ETA Position Paper: Hazard Classification of Enzymes Under the 2012
OSHA Hazard Communication Standard (29 C.F.R. § 1910.1200).
http://www.enzymeassociation.org/wp-content/uploads/2013/12/ETA-
GHS-Enzymes.pdf4. Basketter DA, Broekhuizen C, Fieldsend M,
Kirkwood S, Mascarenhas R, Maurer K, Pedersen C, Rodriguez C,
Schiff HE : Defining occupational and consumer exposure limits for
enzyme protein respiratory allergens under REACH.
• Toxicology 268 (2010) 165–170. 2009 Dec 21.5. The Soap and Detergent
Association “Risk Assessment Guidance for Enzyme-Containing
Products”
http://www.aciscience.org/docs/SDA_Enzyme_Risk_Guidance_October
_2005.pdf

26. Thank you