INTERNATIONAL UNIVERSITY (IU)

VIETNAM NATIONAL UNIVERSITY – HCMC

SCHOOL OF BIOTECHNOLOGY (BT)
-------------------------------------------

ENZYME
LABORATORY REPORT

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TRẦN VĨNH BẢO NGỌC BTBTIU21228
LÊ HOÀNG GIA HÂN BTFTIU21104
NGUYỄN PHAN TƯỜNG VI BTBTIU21273
LÊ HÀ PHƯƠNG LY BTBTIU21220

Contents
I. INTRODUCTION:...........................................................................................3
II. AMYLASE:.......................................................................................................4
1. Introduction:...................................................................................................4
2. Material and procedure:...............................................................................4
2.1. Materials:..................................................................................................4
2.2. Procedure:.................................................................................................4
3. Expecting result:............................................................................................5
4. Discussion:......................................................................................................8
III. PROTEASE:...................................................................................................10
1. Introduction:.................................................................................................10
2. Material and Procedure:.............................................................................11
2.1. Material:..................................................................................................11
2.2. Procedure:...............................................................................................11
3. Expecting result:..........................................................................................12
4. Discussion:....................................................................................................14
IV. CATALASE:...................................................................................................16
1. Introduction:.................................................................................................16
2. Material and procedure:.............................................................................16
2.1. Material:..................................................................................................16
2.2. Procedure:...............................................................................................17
3. Expecting result:..........................................................................................17
4. Discussion:....................................................................................................18
V. REFERENCES...............................................................................................20
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VI. SUMMARY:....................................................................................................21

I. INTRODUCTION:
- Enzymes are a kind of protein present in the cells of living organisms and
can be defined as biological polymers that catalyze biochemical processes.
- Enzymes are defined by their super-active capacity to accelerate chemical
processes in biological and non-biological systems. For instance, whereas
sugar remains stable for years, it may be consumed by humans in seconds
through oxidation to CO2 and H2O. Enzymes have a far higher catalytic
activity than inorganic or synthetic catalysts. Additionally, many enzymes
may cooperate to catalyze a multistep process, with each step catalyzed by a
distinct enzyme. Enzymes catalyze essentially all chemical processes in
biological systems, including energy conversion, food breakdown, and
biomolecule synthesis.
- Enzymes are classified into six functional classes depending on the type of
reaction they are utilized to catalyze:
 Oxidoreductases: e.g: pyruvate dehydrogenase
  Transferases: e.g: transaminase
  Hydrolases: e.g: enzyme pepsin hydrolyzes, lipase, protease
  Lyases: e.g: aldolase
  Isomerases: e.g: phosphoglucomutase
  Ligases: e.g:  DNA ligase
- In this report, we will introduce about 3 types of Hydrolases enzymes:
Amylase; Protease, and Catalase, and show detail about the three enzyme
properties in reactions along with the effect of temperature on the activity of
the enzyme.

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II. AMYLASE:
1. Introduction:
- Amylase is a digestive enzyme. It is a hydrolase group enzyme. Hydrolase
catalyzes the hydrolysis of starch into small molecules. Amylase acts on the
alpha-glycosidic bonds of starch. This leads to the formation of glucans and
glucoses. In the digestive systems of humans and many other mammals, an
alpha-amylase called ptyalin is produced by the salivary glands. Amylase is
found mostly in animal saliva, pancreas, intestine, etc, and sprouting seeds.
- There are three types of amylases:
1. Alpha-amylase acts on 1,2-alpha-d-glucan-glucan glycoside
2. Belta-amylase acts on 1,4-alpha-d-glucan-Malto glycoside linkage
3. Gamma-amylase acts on glucan 1,4-alpha-glucoside and glucan 1-6-alpha-
glycoside linkages
- Amylases are heat sensitive (affected by heat). The change of reaction
temperature causes its change of activities.

2. Material and procedure:

2.1. Materials:
 Green bean sprout
 Starch suspension
 Lugol solution
 Pasteur pipettes
 Test tubes and rack
 Mortar and pestle
 Filter paper
 Two water baths

2.2. Procedure:
- Select 40-60 green bean sprouts, put them all into the mortar, add 20ml of
water and grind till homologous. Filter this suspension and collect the
aqueous phase (enzyme - amylase suspension)
- Prepare 8 test tubes and mark them as indicated below:

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 Temperature 20°C 40°C 50°C 80°C

Marked 20-S 20-E 40-S 40-E 50-S 50-E 80-S 80-E

tubes

- Add into each tube with “E” 2ml amylase suspensions prepared from green
bean sprouts.
- Then, add into all tubes 4 ml of starch suspension.
- After the time indicated, take tubes out, add 1-2 drops of solution from each
tube into Lugol solution.

3. Expecting result:

Amylase 30s 60s 90s 120s 150s

with Starch

Lugol Dark Dark blue Dark blue A little bit of No more

solution blue color change color change

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Amylase with 0-10s 30s 60s
Starch

Lugol solution Dark blue Dark blue No color change

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Amylase with 0-10s 30s 60s
Starch

Lugol solution Dark blue Dark blue No color change

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 Amylase with Starch 0-10s 30s

Lugol solution Dark blue No color change

4. Discussion:
1. Explain the phenomenon:
- Lugol solution is used to detect starch. The enzyme amylase has managed
the break of the bonds within starch to convert it to glucose.
- If the Lugol solution turns black blue, it means that there is starch. No more
blue color means there is no starch left.
- Besides, the act of enzyme amylase on starch depends on the temperature.
At higher temperatures, the enzymes are denatured, while at a lower
temperature, the enzymes are deactivated, so this takes more time at low and
high temperatures to digest the starch. At optimum temperature (30–60°C),
the enzyme is active and therefore consumes less time for starch digestion. [1]
2. Compare each condition:
- At 20°C, the enzyme amylase is deactivated. It takes a long time to break
the bonds within starch. Therefore, there is still black blue color for 90
seconds.

- At 40°C, the enzyme amylase is active. It takes 60 seconds to break all the
bonds within starch.

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 - At 50°C, the enzyme amylase is at its best activity. There is starch for 30
seconds. Then starch is broken down all by the enzyme amylase. Thus, the
yellow-brown color which is the Lugol solution’s color left.

- At 80°C, the enzyme amylase is denatured. There is starch for about only 10
seconds. Then starch is quickly broken down all by high temperature.
Therefore, after 30 seconds, there is a Lugol solution’s color left.

- At the optimum temperature, the amylase will break down starch very
quickly. At low temperatures, the amylase will break starch down slowly. At
high temperatures, the amylase will break starch down slowly or not at all
due to the denaturation of the enzyme's active site.
3. What is the optimal range of temperature for amylase activity?
- The optimum temperature for amylase activity is from 30–60°C where the
enzyme amylase is well active.
- The amylase activity at different temperature points was significantly
different. The figure shows that the activity of α-amylase increased with an
increase in temperature and reached a maximum at 50°C followed by a
sharp decline in activity with increasing of the temperature. [2]

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 Effect of the temperature on the amylase activity. [2]

III. PROTEASE:
1. Introduction:
- Enzyme Protease is an enzyme with the nature of protein synthesized by
organisms, participating in biological chemical reactions. Enzyme protease
has the following properties:
 Made in living cells, non-toxic, environmentally friendly.
 Participates in reactions both in living cells and when separated from living
cells.
 Can participate in catalyzing reactions inside and outside the body from the
beginning to the end, completely releasing the energy stored in chemical
compounds. In an open or closed chain, the product of the former is the
substrate for the next.
 A reaction can be made occurs extracellularly (as in a test tube).
 Enzyme reaction consumes very little energy, high efficiency, and speed
 Highly selective.
 Controlled by genes and response conditions.

- Protease is necessary for living organisms, is very diverse in function from

the level of cells, organs to the body, so it is very widely distributed on
many objects from microorganisms (bacteria, fungi, and viruses) to plants
(papaya, pineapple…) and animals (liver, calf stomach…). Compared with
animal and plant proteases, microbial proteases have different

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 characteristics. First of all, the microbial protease system is a very complex
system consisting of many enzymes that are very similar in structure, mass,
and molecular shape, so it is difficult to separate in the form of
homogeneous crystals. Also because of the complexity of many different
enzymes, microbial proteases often have broad specificity for complete and
diverse hydrolysis products.

Structure of Protase [3]

2. Material and Procedure:
- Enzyme Protease properties in reactions and understanding the effect of
temperature on the activity of the enzyme.
2.1. Material:
 One pineapple
 Two packs of Gelatin
 Beakers
 Pot
 Spoon
 Boiling water
2.2. Procedure:
- Mix two packs of Gelatin with 700ml of boiling water.

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 - Stir the solution until all the Gelatin has been dissolved.
- Leave the solution in the fridge until it becomes semi-solid.
- Boil some pieces of pineapple for 5 to 10 minutes.
- Prepare three bakers. The first beaker is gelatin and fresh pineapple. The
second beaker contains gelatin with boiled pineapple (cooked pineapple).
The last beaker just contains gelatin.
- Observe the phenomenon in the beakers over time at room temperature.
3. Expecting result:

Bea
kers
Fresh pineapple Cooked No Pineapple
Pineapple
Phenomenon

Gelatin is Gelatin turn Gelatin is not

liquefied from clear but the liquefied and
State of gelatin
the top to the surface of remains the semi-
bottom gelatin is not solid
broken down
into the liquid

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 Compare the phenomenon between the beaker with fresh pineapple and the beaker without pineapple [4]

  

Compare the phenomenon between the beaker with fresh pineapple and the beaker with cooked pineapple [4]

4. Discussion:
a. Do 2 pieces of egg have different shapes after 2 days of
incubation? Explain.

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 Experiment with Protease enzyme and white egg

● Two pieces of egg in two tubes will have different shapes after 2 days of
incubation:
The pieces of egg in tube 1 that are not boiled are completely dissolved.
The pieces of egg in tube 2 boiled in 10 minutes remains the original shape.
● Explanation:
- The Proteolytic enzyme (belongs to the Protease enzyme group) in pineapple
can decompose protein by breaking down the peptide bonds linking the
long-chain protein together.
- So when the Proteolytic enzyme comes to contact with protein (albumin) in
a white egg, the enzyme cuts down the peptide bonds in the white egg so the
shape of the white egg in tube 1 is destroyed and the piece is dissolved in the
solution.
- Tube 2 is boiled for 10 minutes, the structure of the Proteolytic enzyme
breaks down and is no longer able to digest protein. So in tube 2, the enzyme
is inactive so the long-chain protein in egg white remains which means the
shape of the piece of white egg keeps the original shape.

b. What is the optimal range of temperature for protease activity?

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 Optimal range of temperature for protease activity [5]

- As we can see in the diagram, the optimal range of temperature for protease activity
is from 10˚C to 70˚C.
c. Explain the experiment with pineapple:
- Gelatin is made out of an animal protein known as collagen. Collagen’s
structure is a triple helix, when it is heated, the chain is unwound and when
the Gelatin cools, the chain is solidified, tangled, and traps water in the
middle of the chain. So the Gelatin from the liquid turns into semi-solid at
room temperature. So basically, Gelatin is also one type of protein and will
react with the Proteolytic enzyme in different conditions.
- The activity of the Proteolytic enzyme in Pineapple is explained in question
a.

Origin and molecular

structure of gelatin [6]

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 IV. CATALASE:
1. Introduction:
- Catalase is an antioxidant enzyme that can be found in almost every type of
living tissue. The catalase belongs to the group of oxidoreductase. This
enzyme functions to convert peroxide into less toxic substances. To be more
specific, with the help of catalase, hydrogen peroxide (H2O2) which is a very
toxic compound is broken down into oxygen (O2) and water (H2O).

Water and oxygen are formed after the breakdown of hydrogen peroxide by the catalase enzyme.

- Thus, this enzyme plays an important role in the cell using its detoxication
ability.
- However, catalase, like other enzymes, is heat-inactivated. When an enzyme
loses activity, the amount of oxygen and water produced decreases. Catalase
is known to be prevalent in potatoes. In mammals, catalase is
found predominantly in the liver. In addition, many fruits such as bananas,
watermelon, or pineapple have high amounts of catalase.

- Yeast is a microscopic fungus containing catalase enzyme so, in this section,

we will use yeast to test for the reaction rate of catalase in different
temperatures by measuring the volume of foam produced in 20 seconds.

2. Material and procedure:

2.1. Material:
 Yeast
 Distilled water
 Buffer
 Graduated cylinders

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 Test tubes
 Rack
 Water bath
 Thermometer
2.2. Procedure:
- Add 5g yeast to 10ml of water in three test tubes.
- Add 20ml of buffer to 20ml of hydrogen peroxide in three graduated
cylinders.
- Place each pair of the test tube and graduated cylinder in a water bath at 5°C,
30°C, and 70°C, in turn.
- Leave them to stay for 10 minutes before being tested.
- Remove all the test tubes and graduated cylinders from the water bath.
- Add the yeast solution into the cylinders which contain hydrogen peroxide
and buffer solution.
- Wait for 20 seconds and observe the change in the foam range. Then, record
the foam volume in each tube.
3. Expecting result:

Temperature 5°C 30°C 70°C

Foam volume 80ml 160ml 50ml

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 The change in foam range as catalase converts hydrogen peroxide into oxygen and
water at different temperatures. [7]

The experiment’s result presented in a graph. [7]

 Conclusion
- Based on the results of the experiment, we may conclude that the amount of
foam in tube two is the greatest. This demonstrates that the action of catalase
produces more oxygen and water in this tube than in the other two tubes. As
a result, it can be deduced that catalase performs best at 30°C and gradually
decreases at 5 and 70°C in this experiment. As a result, temperature
influences catalase activity: At high temperatures, the enzyme can be
degraded. On the other hand, the reaction rate of enzymes is low at low
temperatures. Finally, each enzyme has an optimum temperature where its
activity is best.

4. Discussion:
a) Why is there a difference in the foam volume between different
conditions?
- Temperature influences catalase activity. The rate of an enzyme-catalyzed
reaction rises with temperature, as it does with many other chemical
processes. At high temperatures, however, the rate slows again. This is
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 because each enzyme has its optimum temperature where it can function
best. When the temperature rises toward the optimum, hydrogen bonds
weaken, making catalase's action on hydrogen peroxide molecules simpler.
[8]
When the temperature rises over the optimum, the enzyme denatures and
its structure is disturbed. As a result, the enzyme no longer functions.

b) What is the optimal range of temperature for catalase activity?

- The optimum temperature for catalase activity is from 30 to 40oC and
catalase enzymes can work best at temperatures around body temperature
(37.5°C). [9]

Catalase enzyme’s temperature range and optimum range. [8]

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 V. REFERENCES
Captured images: Effect of temperature on digestion of starch by amylase
https://www.youtube.com/watch?v=1Fa2sSIt4_I
[1] Study the Effect of Temperature on Salivary Amylase Activity
https://link.springer.com/protocol/10.1007/978-1-4939-9861-6_53#:~:text=At
%20higher%20temperature%20the%20enzymes,less%20time%20for%20starch
%20digestion
[2] Effect of temperature on the activity and stability of α-amylase; page 41
https://www.researchgate.net/publication/
321050249_Production_purification_and_characterization_of_thermostable_a-
amylase_from_soil_isolate_Bacillus_sp_strain_B-10#pf5
[3] https://sciencevietnam.com/enzyme-protease
[4]https://www.youtube.com/watch?
v=7t7v8w7EqTM&list=RDCMUC5VY0z4PsTMHeqgA4HdWZxQ&start_radio=
1&rv=7t7v8w7EqTM&t=259
[5]https://www.researchgate.net/figure/Effect-of-temperature-on-protease-activity-
The-optimum-temperature-was-determined-for_fig2_51227641
[6] https://theory.labster.com/gelatin/
[7] Effect of temperature catalase action (2015). Youtube. Retreived from
https://www.youtube.com/watch?v=KvQyM6plOgk.
[8] Thilindu Peiris (2020). How does temperature affect catalase activity? Quora.
Retreived from https://www.quora.com/How-does-temperature-affect-catalase-
activity.
[9] Serena K.T. Low (2016). How does temperature affect catalase activity?
Retreived from https://csef.usc.edu/History/2016/Projects/J0513.pdf.

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VI. SUMMARY:
- To sum up, the human body is made up of several cell kinds, tissues, and
other intricate organs. Our body produces chemicals to expedite biological
functions such as breathing, digestion, and excretion, as well as a few other
metabolic activities necessary for healthy existence. Thus, enzymes are
essential in all living beings since they regulate all biological processes.
- Through 3 experiments, we can find out the best temperature condition that
each enzyme performs:
 Amylase: The optimum temperature for its activity is from 30-60°C.
 Protease: The optimal temperature for its activity is from 10-70˚C.
 Catalase: The optimum temperature for its activity is from 30-40oC.

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