KOLEJ MATRIKULASI JOHOR MINISTRY OF EDUCATION 84900, TANGKAK JOHOR

MARKS:

NAME RIESHI KEISHI A/L SELVADURAI

MATRIC NUMBER MS2316115458
PRACTICUM H16
LECTURER’S NAME MISS AFIQAH BINTI MOHAMED
DATE OF 6/2/2024
EXPERIMENT

TITLE BIOCATALYSIS 1 mark

OBJECTIVE 1. To extract catalase from liver tissue 2 marks
2. To observe the qualitative activity of catalase
3. To measure the quantitative activity of catalase
4. To determine the factors affecting the catalase activity

INTRODUCTION Define enzyme.

Enzyme is a macromolecule that acts as a catalyst which increase the
rate of biochemical reaction in living things.
1 mark
How enzyme works?
By lowering the activation energy and it remains unchanged at the end
of reaction
1 mark

State 2 factor affect enzymatic

reactions. Temperature and pH 1 mark

Name the substrate for catalase.

Hydrogen peroxide 1 mark

PROCEDURE Experiment 9.1: Estimation of catalase activity 4 marks

9.1.1 Preparation of catalase extract

1. 10 – 15 g fresh liver had been cut into small pieces and the
tissue was macerated inmortar and pestle.

2. 20 ml of water was gradually added.

3. The mixture had been filtered into a beaker using the muslin
cloth.

4. The filterate was the enzyme stock solution in the

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 experiment.

9.1.2 Qualitative test for catalase activity

1. Two boiling tube had been labelled as A and B

2. 1 ml H2O2 solution had been poured into boiling tube A

3. 1 ml enzyme stock solution had been poured into boiling

tube B

4. Stock solution in boiling tube B was added into boiling tube

A using a dropper. The boiling tube had been labelled as
boiling tube C.

5. The activity of the enzyme had been observed and

explained. This boiling tube C had been used for the
following.

9.1.3 Estimation of catalase activity

1. 1 ml of H2SO4 had been poured into the boiling tube D.

2. 1 ml of enzyme-H2O2 mixture had been transferred from C
into D. The boiling tube was shake well.
3. The left over H2O2 solution that does not react had been
measured using KMnO4 solution.
4. KMnO4 solution reacts with H2O2 in acid medium.
5H2O2 + 2KMnO4 + 4H2SO4  2KHSO4 + 2MnSO4 +
8H2O + 5O2
5. The syringe had been filled with KMnO4.
6. Drops of KMnO4 had been added into test tube D until the
red colour remains unchanged for 10 seconds.
7. The amount of KMno4 used was determined. The value
shows the activity of catalase.
8. The more KMnO4 used indicates that more H2O2 is present
in the mixture. It means that the H2O2 is not fully broken
down by catalyst to oxygen molecules.

Experiment 9.2: Factors affecting the activity of catalase

9.2.1 Temperature
1. The following boiling tube had been placed in a beaker
containing chilled water (20 C).
a) Boiling tube A containing 2 ml of enzyme stock solution
b) Boiling tube B containing 3 ml of H2O2
c) Empty boiling tube labelled C
2. 1 ml H2SO4 had been prepared in boiling tube C.
3. The chilled H2O2 had been poured into the boiling tube C
when the temperature in the boiling tube B containing H2O2
dropped to 20 C.
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 4. The cooled enzyme stock had been poured from boiling
tube A had been poured into boiling tube C. The reaction must
take in the iced-chilled beaker. The time was recorded.
5. 1 ml of solution from boiling tube C had been transferred
into boiling tube 5 after 4 minutes. And had been plugged with
rubber stopper. Shake well and estimate the activity of catalase
as conducted in Exercise 15.1C.
6. Step 1 to 5 had been repeated. (Set at different temperature:
30 C, 40 C, and 50 C). Use different boiling tubes.
7. The values obtained in Table 9.1 had been recorded and the
graph of the approximate catalase activity against temperature
had been plotted.

9.2.2 pH
1. Four boiling tubes had been labelled as 6, 7, 8 and 9.
2. 1 ml of H2SO4 had been poured into each boiling tube.
3. Four boiling tubes had been labelled as F, G, H and I.
4. 1 ml of H2O2 had been poured into each boiling tube.
5. 2 ml phosphate buffer solution with pH 5, pH 7, pH 9 and
pH 11 had been added to boiling tube F, G, H and I,
respectively. Shake them well.
6. 1 ml enzyme stock solution had been poured into boiling
tube F. The time had been recorded for 4 minutes.
7. 1 ml of solution from boiling tube F had been transferred
into boiling tube 6 after 4 minutes. Shake well the boiling tube
and the activity of catalase had been estimated as conducted in
Exercise 15.1C.
8. The above step had been repeated for boiling tubes G, H
and I using boiling tubes 7, 8, and 9 respectively.
9. The values obtained in Table 9.2 had been recorded and the
graph of the approximate catalase activity against pH had been
plotted.
RESULTS Results
1. Qualitative test for catalase activity
(9.1.2) State the observation. 1 mark

2. Measuring the activity of catalase (9.1.3) 1 mark

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 3. Temperature (Table 9.1)
Temperature 20°C 40°C 50°C

30°C 2.2 3.0

Amounts (ml) of 3.2 2.8

0.45 0.33 1 mark
KMnO4 used

Approximate 0.31 0.36

catalase activity 1 mark
(1/ Amounts
(ml) of KMnO4
used)
1 mark

Plot graph (complete with title, axis, units)

1 mark

1 mark

1 mark

4. pH (Table 9.1)
pH 5 7 9 11

Amounts (ml) 5.0 2.3 2.9 2.8

of KMnO4 used

Approximate 0.20 0.43 0.34 0.36

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 catalase activity
(1/ Amounts

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 (ml) of
KMnO4
used)

Plot graph (complete with title, axis, units)

DISCUSSION Discussion

1. For catalase activity (9.1.2) 1 mark

4 marks

2. Explain the effects of the temperature on the enzymatic

4 marks
reaction. At low temperatures, the rate of reaction is very slow.
Kinetic energy of molecules is low and moving slowly. When
temperature increases, the rate of reaction increase. Kinetic
energy of the enzyme and substrate increases. More collisions
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 occur. The rate of

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 reaction increases. Beyond the optimum temperature enzyme will
be denatured.

3. Explain the effects of pH on the enzymatic reaction.

Changes in pH above or below the optimal pH value will change
the hydrogen ion concentration. This ion changes the ionic charge
and interrupt the bond that hold the 3D shape of enzyme. Active
site are destroyed and enzyme denatured. The rate of reaction
decrease quickly.

CONCLUSION State the optimum temperature and pH for catalase

Optimum temperature for catalase is 40°C while optimum pH for
catalase is pH 7. 2 marks

QUESTION 1. What is the role of H2SO4 in the reaction?

1 mark
To decomposed slowly and forming oxygen and water
2. What is the gas that will be released, when hydrogen
peroxide is broken down catalyze by catalase? How can we test
the gas it releases?
2 marks
Oxygen gas.The gas can be check by using smoldering test wood.
Reference 1 mark
Malaysia National Library,Biologi Laboratory Manual,semester I &
II,SB015 & SB025 Thirteenth Edition

Submission date 1 mark

(not late)
Total /35 marks

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