PRACTICAL 5: EMZYMES

Group: 03
Group members:
Huỳnh Thanh Vân - BTBTIU23094
Hồ Bảo Ngọc - BTBTIU23057
Nguyễn Linh Chi - BTBTIU23014
Nguyễn Hồ Mai Anh - BTBTIU23005

Course name: Practice in Biology (BT321IU_2_23-24)

Instructor name: Dr. Phạm Hồng Điệp

Date of submission: April 2 nd, 2023

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REPORT 5:
I/ AMYLASE

1/- Introduction:

- An amylase is an enzyme that catalyzes the hydrolysis of starch (Latin

amylum) into sugars. Amylase is present in the saliva of humans and some
other mammals, where it begins the chemical process of digestion.
- The pancreas and salivary gland make amylase (alpha amylase) to hydrolyze
dietary starch into disaccharides and trisaccharides which are converted by
other enzymes to glucose to supply the body with energy. Plants and some
bacteria also produce amylase. Specific amylase proteins are designated by
different Greek letters. All amylases are glycoside hydrolases and act on
α-1,4-glycosidic bonds.
- There are three types of amylase:
+ 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.
- Like other enzymes, amylases are heat sensitive (affected by heat). In this
experiment, we will observe and demonstrate how the temperature affects
the activity of amylase.

2/- Procedure:

The experiment requires:

- Green bean sprout - Cloth, knife
- Stach suspension - Foil paper
- Blender - Waterbath
- Lugolsolution - Icebox
- Distilled water - Pasteur pipettes
- 8 test tubes, test - Beaker
tube clamps
- 1 test tube rack

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Detailed procedure:

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

Temperature 4 0C RT 500C 1000C

Marked tubes 4-S 4-E RT-S RT-E 50-S 50-E 100-S 100-E

- Add into each tube with “E” 2ml amylase suspensions prepared
from green bean sprouts and place tubes with “4” labels in ice box,
“50” in warm water, “100” in boiled water (waterbath) and “RT” at
room temperature for 5 minutes. *cover the “100” test tubes with
foil paper to prevent the evaporation.
- Then, add 4 ml of starch suspension into all test tubes (8 test
tubes). Continue to keep all reactions for 10 minutes in the same
condition.
- After the time indicated, take 8 test tubes out, add 2ml of
distilled water into each “S” tube (here, we have 4 test tubes
marked with “S”. Then, add 1 drop of Lugol solution into each
tube. Mix very gently, observe the color and take photo quickly.

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3/ Results:

Temp. (o C) Marked tubes Color Observation

Before Lugol After Lugol

4-S

4-E

RT-S

RT

RT-E

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 50-S

50

50-E

100-S

100

100-E

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4/- Discussion:

a) Compare “S” with “E” tubes in each condition and explain the
phenomenon.
At 40C:
- “S”: The color changes to dark blue (This is because amylose in
starch is responsible for the formation of a dark blue color in the
presence of Iodine. The Iodine molecule slips inside of the amylose
coil and forms the “Iodine - amylase” complex. In fact, there was no
reaction happened).
- “E”: The color is dark purple. At this temperature, the enzyme
breaks starch down slowly due to reduced kinetic energy. So the
color changes to blue-purple.
At RT:
- ”S”: Temperature was increasing, less Iodine molecule is trapped in
the amylose coil, which makes the deep blue color.
- ”E”: When the temperature increases, the enzyme starts to react and
the color changes slowly into the Iodine’s original color.
At 500C:
- “S”: At this temperature, it seems to have almost no effect on
Starch’s structure . So, the color changes to deep blue.
- “ E”: When the temperature is higher, the enzyme starts to break
starch down slowly. The color changes slowly into the purple-brown.
At 1000C:
- “S”: The effect of high temperature is not significant. After adding
Lugol, the color changes to dark blue.
- “E”: At this temperature, the enzyme is denatured, the enzyme
almost does not react . So, the color changes to purple-blue.

b) Compare all “S” tubes in all conditions and all “E” tubes in all
conditions. Explain the phenomenon.
- All “S” tubes in all conditions: All the tubes have the same color
because there is no enzyme amylase to catalyze the hydrolysis
reaction of starch and the helical structure is not affected anymore so
that Iodine can fit inside the Starch structure and give the dark blue
color.
- All “E” tubes in all conditions: the 4-E, and 100-E tubes have the
same color because the enzyme cannot catalyze the hydrolysis
reaction, hence the glycoside bond was not affected and Iodine can fit
with Starch to give the dark blue color like the others “S” tube. And
enzyme amylase in RT-E and 50-E can activate and catalyze the

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hydrolysis reaction lead to the different colors in the other 2 tubes. At
room temperature amylase can active well so that we can see the
purple-brown color. At 500C amylase cannot work well as at RT.

c) What is the optimal range of temperature for amylase

activity?
The optimal range of temperature for amylase activity is between 30
to 500C.

II/PROTEASE:

1/ Introduction:
- Proteases, also known as proteinases, are enzymes responsible for
breaking down proteins by cleaving the peptide bonds, converting
them into smaller units called polypeptides or single amino acids.
Proteases play crucial rrolqs in various physiological processes in
organisms, such as digestion, blood clotting, immunity, and cell death.
- Proteases catalyze proteolysis, which is the process of breaking
down proteins into smaller units and creating new protein products.
They accomplish this by hydrolyzing the peptide bonds inside
proteins, which is a process where water breaks bonds.
- Proteases are classified into six distinct groups based on their
catalytic mechanism: aspartic, glutamic, and metalloproteases,
cysteine, serine, and threonine proteases. They are involved in
various biological tasks, including protein digestion, protein
catabolism (the breakdown of old proteins), and cell signaling.
- The aim of this experiment is to observe how proteases break down
proteins at different temperatures. To achieve this, pineapple will be
used as a source of proteases. The pineapple proteases will be set at
two temperatures, 100°C and room temperature, and white eggs will
be added as protein samples.

2/ Procedure:

The experiment requires:

- Pineapple
- White egg
- Pipettes
- Test tubes
- Silver paper

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Detailed procedure:

- Prepare 2 tubes and mark 100 and RT on each tube.

- Grind pineapple with water and filter them.
- Add 4 ml pineapple juice into 2 tubes.
- Wrap the head of the 100-tube with silver paper.
- Put 100-tube in boiled water and RT-tube at room temperature for
15 minutes.
- Take it out and add 2 small pieces of white egg into 2 tubes.
- Cover the tubes with silver paper and put them at room
temperature.
- After 2 days, pour out the liquid on the petri dish and compare the
difference between 2 pieces of white egg.

3/ Results:

Temp.(oC) At the beginning After 2 days

RT

100

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 4/Discussion:

a) Do 2 pieces of egg have different shapes after 2 days of

incubation? Explain.
- Yes. After two days of incubation, the two pieces of boiled egg will
likely have different shapes. The egg in the tube labeled "100" was
boiled before being incubated, which inactivated the protease
enzyme in the extract. This enzyme is responsible for breaking down
egg protein. As a result, the egg piece in the "100" tube is expected to
maintain its original shape.
- The egg in the tube marked "RT" was exposed to protease activity at
room temperature, which is within the optimal range for protease
activity. The protease enzyme is likely to break down the protein in
the egg, causing it to change shape or partially dissolve over time

b) What is the optimal range of temperature for protease

activity?
- Based on the experimental setup and results, it can be concluded
that the optimum temperature range for protease activity is likely
around room temperature. This is because the observed changes in
the egg piece in the "RT" tube show that protease activity was most
effective at room temperature. However, there was no significant
protease activity observed in the "100" tube as the protease enzyme
was denatured at high temperatures (100°C).

III/ CATALASE:

1/ Introduction:
- Catalase is an enzyme present in cells that catalyzes the breakdown of
hydrogen peroxide into water and oxygen. The enzyme plays a major
role in the protection of cells from the harmful effects of oxidizing
agents such as hydrogen peroxide.
- Catalase, also known as peroxide enzyme, is a prominent
oxidoreductase enzyme that catalyzes the breakdown of hydrogen
peroxide into water and oxygen gas. The function of catalase in the body
is primarily associated with hydrogen peroxide metabolism. It protects
cells and tissues from the harmful effects of reactive oxygen species
(ROS) by converting hydrogen peroxide to water and oxygen.
Additionally, catalase helps maintain cellular homeostasis and redox
potential by maintaining an equilibrium state between oxidizing agents

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and reducing the damage caused to cells and tissues. Furthermore,
catalase is involved in several cellular processes such as detoxification,
antioxidant defense, and cellular signaling. Finally, catalase has been
found to be a diagnostic marker for several diseases, including cancer,
diabetes, and viral infections.
- In this experiment, the objective is to use potatoes as a source of
catalase to observe the foaming phenomenon, which is influenced by
various temperature settings (-4oC, room temperature, 50 oC, and 100oC
for 5 minutes) to gain a better understanding of catalase's role in
oxidation and reduction processes.

2/ Procedure:
The experiment requires:
- Potato
- Distilled water
- Pipettes
- Test tubes
- H2 O2 solution

Detailed procedure:
- Prepare 4 tubes and mark 4, RT, 50, and 100 on each tube
- Grind a potato with distilled water and filter them.
- Add 5 ml enzyme suspension into each tube.
- Mark 2 lines 5 cm above the solution and current water level.
- Put all of them into the indicated conditions and wait for 5 minutes.
- Take it out and add 5 drops of H 2O solution into each tube.
- Observe the foaming phenomenon, take the timer, and wait until the
foam rises to 5 cm.

3/ Results: Report the time for the gas column to reach the 5 cm line
and take the photo of these tubes:

Temp (oC) Time recorded (seconds)

4 37 seconds
RT 19 seconds
50 42 seconds
100 No gas column

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 The RT tube reached
the 5cm line The 4 tube reached
the 5cm line

4 tubes after 19 seconds 4 tubes after 37 seconds

The 50 tube reached

the 5cm line

The 100 tube still had no

reaction.

4 tubes after 42 seconds

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4/ Discussion:

a) Why is there different in time when bubbles columns reach the 5

cm line between different conditions?
Catalase is an enzyme that facilitates the breakdown of hydrogen
peroxide into water and oxygen. The rate of enzymatic reactions is
influenced by temperature. The higher the temperature, the faster
the catalase enzyme will break down hydrogen peroxide into water
and oxygen. Therefore, the column of oxygen bubbles will reach the
5-cm line faster in the test tubes at higher temperatures than in the
test tubes at lower temperatures.

b) What is the optimal range of temperature for catalase

activity?
- At 4°C: The reaction is expected to be slower due to the lower enzyme
activity at this temperature.
- At room temperature (RT): This condition is likely to be closer to the
optimal range for catalase activity, resulting in a faster reaction
compared to 4°C.
- At 50°C: Enzyme activity is lower than at room temperature, leading to
a slower reaction.
- At 100°C: The high temperature is likely to denature the enzyme,
resulting in no catalase activity.
- Based on the results, the optimal range of temperature for catalase
activity is likely to be around room temperature. Temperatures lower
than room temperature (4°C) and higher than 50°C (100°C) are less
favorable for catalase activity.

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