INTERNATIONAL UNIVERSITY – HCMC

School of Biotechnology

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BIOLOGY
LABORATORY
REPORT FOR PRACTICAL 4
ENZYMES

Course name: Biology

Course ID: BT155IU
Instructor: Mr. Quang
Group number: 2
Group members: Nguyễn Ngọc Phương Anh BTBCIU19034
Nguyễn Thị Ánh Hồng BTBTIU19015
Nguyễn Anh Thư BTBTIU19041
Phan Hải Nam BTBCIU19050
Date of submission: 08/01/2021

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I/ AMYLASE
1. Introduction
Amylases is an enzyme that catalyses the hydrolysis of starch into sugars. They
act on 𝛂-glycosidic bonds of starch. They hydrolyse dietary starch into
disaccharides and trisaccharides then converted by other enzymes to glucose
and glucans to supply the body with energy. They are found in animal saliva,
pancreas, intestine…, and in sprouting seeds.
Amylase has 3 types:
‒ 𝛂-amylase acts on 1,4-𝛂-D-glucan-glucan glycoside.
‒ 𝛃-amylase acts on 1,4-𝛂-D-glucan-malto glycoside linkage.
‒ 𝛄-amylase acts on glucan 1,4-𝛂-glucoside and glucan 1,6-𝛂-glycoside
linkages.
In this lab, we observed the change of temperature reaction lead to the change of
color and their activities.
2. Procedure
- Prepare 8 identical test tubes and mark as follows:
Temperature 4oC RT 50oC 100oC
Marked 4-S 4-E RT-S RT-E 50-S 50-E 100-S 100-
tubes E
- Add to each tube 2 ml of the prepared “E” amylase suspension from green
bean sprouts and place tubes labeled “4” in ice, “50” in warm water, “100”
in boiled water, and “RT” in on tray for 5 minutes.
- After 5 minutes, add to all test tubes 1 ml of the starch suspension. Continue
holding all the tubes for 10 minutes under the same conditions as initially.
- After the indicated time, remove the tubes, add 2 ml of water to the S tubes,
then add 1-2 drops of Lugol's solution to each tube and shake well.
- Observe the colors of the tubes and take pictures.

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 3. Results
Temp. Marked Color Observation
(0C) Tubes Before Lugol After Lugol

4-S Transparent Dark blue

Dark blue
4-E Opaque
(lighter)

RT-S Transparent Dark blue

RT

RT-E Opaque Dark purple

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 50-S Transparent Dark blue

50

50-E Opaque Purple

100 100-S Transparent Dark blue

Opaque
100-E Opaque
(darker)

4. Discussion
a) Compare “S” with “E” tubes in each condition and explain the
phenomenon.
Marked tubes Before add After add Explain
Lugol Lugol
4-S Colorless The solution Starch does not decompose, so
solution turns dark blue when adding Lugol the solution
purple turns dark green

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 4-E The solution is The solution At low temperatures, the
light white turns dark Amylase enzyme decomposes
green starch very slowly, so after
adding Lugol, the solution turns
dark green.
RT-S Colorless The solution Starch does not decompose, so
solution turns dark blue when adding Lugol the solution
purple turns dark green
RT-E The solution is The solution At room temperature, in this
light white turns blue condition the Amylase eyzyme is
purple the most active and it
( Lighter than decomposes starch very slowly,
4-E) so after the addition of Lugol,
the solution becomes blue
purple.
50-S Colorless The solution Starch does not decompose, so
solution turns dark blue when adding Lugol the solution
purple turns dark green
50-E The solution is The solution At 50oC, the Amylase eyzyme
light white turns light decomposes starch, but not
purple much, so the starch still remains
in the test tube, so after adding
Lugol, the solution turns pale
purple.
100-S Colorless The solution Starch does not decompose, so
solution turns dark blue when adding Lugol the solution
purple turns dark green
100-E The solution is The solution At 100oC, the rate of Amylase
light white turns light enzyme action decreased, which
yellow is alters the active site of the
enzyme, the enzyme resolves all
the starch so the solution has
light yellow color.

b) Compare all “S” tubes in all conditions and all “E” tubes in all condition.
Explain the phenomenon.
Explain the phenomenon.

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- S tubes: Because there is no Amylase enzyme in the test tube, the starch is almost
not degraded, the torsion structure of starch is not affected, so when adding Lugol
the solution turns dark blue.
- E tubes: Because there are Enzyme Amylase in the tube, at different
temperatures, enzymes act on each tube to catch different colors. The higher the
temperature, the more the starch is decomposed, so when the Lugol is added, the
color of the solution fades.
c) What is the optimal range of temperature for amylase activity
- Starch was broken down fastest at 100oC which is the optimal range of
temperature for amylase

II. PROTEASE
1. Introduction
Proteolytic enzyme, also called protease, proteinase, or peptidase, any of a
group of enzymes that break the long chainlike molecules of proteins into shorter
fragments (peptides) and eventually into their components, amino acids. The
enzymes use a molecule of water for this and are thus classified as hydrolases. A
type of Enzymes that are capable of splitting A type of proteins.
Protease helps break down protein in food into amino acids, which the body
can then use for energy, but where proteases stand apart is the fact that they also
play a number of other roles in essential processes, such as blood clotting, cell
division, recycling of proteins, immune support.
2. Procedure
Grind pineapple fruit and collect aqueous phase of enzyme. Separate them
into two test tubes, one (marked RT) put in room temperature environment, one
(marked 100) put in boiling water for 15 minutes. After that, put a piece of egg
white in both test tubes, cover them and leave for 2 days. After two days, take them
out and observe what happened.
3. Results: Report the observation of egg pieces’s shape after 2 days.
Temp. (0C) At the beginning After 2 days
RT Normal shape and size Become smaller

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 100 Normal shape and size Still normal

4. Discussion
a. Do 2 pieces of egg have different shape after 2 days of incubation? Explain.
Yes, they do. Because protease in RT tube broke the peptide bonds between
the protein molecules in it into amino acids, thus changing its shape whereas in the
100-tube, the protease has been denaturized. Because of that, it didn’t react with
the protein in the. boiled egg white so the egg white kept its shape.
b. What is the optimal range of temperature for protease to activate?
The optimal range of temperature for protease activity is between 30 oC and
37oC.

III. CATALASE
1. Introduction
Enzyme catalase, an anti-oxidant enzyme, is produced naturally in plants,
animals and microbial cells. This enzyme functions to convert peroxide into less
toxic substances. The catalase belongs to the group of oxidoreductase. The
enzyme can be monitored by its ability to convert hydrogen peroxide (H 2O2) into
oxygen (O2) and water (H2O). When the body is infected with a high level of

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 hydrogen peroxide, the catalase stored in peroxisome (in plant cells) or
mitochondria (in animal cells) will be activated and contribute in the process to
convert this toxic compound to oxygen and water. Thus the enzymes play an
important role in the cells by means of its detoxication ability. Like other
enzymes, catalase is also heat inactivated. When the enzyme loses activity, the
production of oxygen and water is decreased. Potato is known to be the abundant
source of catalase.
2. Procedure
‒ Prepare 4 test tubes and mark temperatures of them as 4 0C, Room
temperature (RT), 500C and 1000C.
‒ Add 5ml of potato extract in each test tube.
‒ Let these test tubes at designated temperature and incubate for 5 minutes.
‒ After 5 minutes, bring these test tubes out and add 5 drops H 2O2 in each of
them.
‒ Wait for 3 minutes and record the bubble column.
3. Results
Temp. Bubble column Observation
0
( C)
4 Bubbles appear very little and slowly

RT Many bubbles appear

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50 Bubbles appear a lot and fast
( The solution turns lighter)

100 There are a little oxygen bubbles

( The solution turns light yellow)

4. Discussion
a) Why is there different in time when bubble columns reach the 5-cm line
between different conditions?
Catalase is an enzyme. Like other enzymes, when the temperature rises to a
certain point, it breaks down the enzyme's structure. Once the enzyme is denatured,
it cannot be repaired. If the enzyme is inactivated, oxygen and water's production
also decreases.
b) What is the optimal range of temperature for catalase activity?
The effects of catalase, like those of all enzymes, are influenced by the
surrounding temperature. Temperature has an effect on both the structure of the
catalase itself and the hydrogen bonds it is designed to cleave. As the temperature
increases toward the optimum point, hydrogen bonds loosen, making it easier for
catalase to act on hydrogen peroxide molecules. If the temperature increases
beyond the optimum point, the enzyme denatures, and its structure is disrupted.
The optimal temperature range for catalase activity is probably around 30-40
degrees Celsius. In humans, the optimum temperature for catalase is 37 degrees
Celsius.