International University, Vietnam National University - HCMC 1

BIOLOGY LABORATORY

REPORT 4
PRACTICAL 4: ENZYMES
Instructor : MSc. Nguyen Thien Quang
Class: Wednesday afternoon
Group: 04
Group members:
Hoàng Nguyên Vũ BTBTIU17069
Đặng Thị Kim Sang BTBTIU17156
Nguyễn Thị Như Quỳnh BTBTIU17143
Ngô Hoàng Phương Nhi BTBTIU17109
Date: 24/10/2018

Score: _________

Instructor : MSc. Nguyen Thien Quang

 International University, Vietnam National University - HCMC 2
BIOLOGY LABORATORY

I/-AMYLASE
1/ -Introduction:
An amylase is an enzyme that catalyses the hydrolysis of starch into sugars. Amylase is
present in the saliva of humans and some other mammals, where it begins the chemical
process of digestion. Foods that contain large amounts of starch but little sugar, such
as rice and potatoes, may acquire a slightly sweet taste as they are chewed because amylase
degrades some of their starch into sugar. The pancreas and salivary gland make amylase
(alpha amylase) to hydrolyse 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. All amylases are glycoside hydrolases and act on α-1,4-
glycosidic bonds.
Amylases belong to the hydrolase group enzyme. They all act on α-glycosidic bonds of
starch. The actions of amylases on these linkages result in the formation of glucans and
glucoses. Amylases are found mostly in animal saliva, pancreas, intestine…, and in
sprouting seeds.
There are three types of amylase:
+ γ-amylase acts on glucan 1,4-α-glucoside and glucan 1-6-α- glycoside linkages.
+ β-amylase acts on 1,4-α-D-glucan-malto glycoside linkage.
+ α-amylase acts on 1,4-α-D-glucan-glucan glycoside.
Like other enzymes, amylases are heat sensitive (affected by heat). The change of
reaction temperature causes its change of activities.
2/ -Procedure:
1. Select 40-60 green bean sprouts, put all into the mortar, add 20ml of water and grind
till homogenous. Filter this suspension and collect the aqueous phase (enzyme -
amylase suspension.
2. Prepare 8 test tubes and mark them as indicated below:
Temperature 40C RT 500C 1000C
Marked tubes 4-S 4-E RT-S RT-E 50-S 50-E 100-S 100-E

3. Add into each tube with “E” 2ml amylase suspensions prepared from green bean
sprouts and place tubes with “4” label in ice, “50” in warm water, “100” in boiled water
and “RT” at room temperature for 5 minutes. *cover the “100” test tubes to prevent the
evaporation.
4. Then, add into all test tubes 4 ml of starch suspension. Continue to keep all reactions
for 10 minutes in the same condition.
Instructor : MSc. Nguyen Thien Quang
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5. After the time indicated, take tubes out, add 1-2 drops of Lugol solution into each
tube and 2ml of water into each “S” tube. Mix well.
6. Observe the color of these tubes.

3/-Results: Report the observed color in different test tubes after adding Lugol
solution.

Temp. Marked Color Observation

(0C) Tubes Before After Lugol
4-S White Dark blue
4
4-E White Dark violet
RT-S White Dark blue
RT
RT-E Yellow-white Dark violet
50-S Yellow-white Dark blue
50
50-E Light yellow white Light pink
100-S Yellow white Dark blue
100
100-E Colorless Colorless ( supposed
Light violet)
Explanation of different 100E color change :

The transparent color of 100E is the consequence of adding too much amylases. The
volume of solution is higher than the other so it doesn’t change the color .

4/- Discussion:
a. Compare “S” with “E” tubes in each condition and explain the phenomenon.
The color of both “S” and “E” before adding Lugol solution in all condition is white
( or no color).
After adding Lugol solution:
+ At 40C:

Instructor : MSc. Nguyen Thien Quang

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-The S’s 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 form the “Iodine - amylase” complex. In fact there was
no reation happended).
-The E's color is dark purple. At this temperature, the enzyme is denatured . So the color
stays nearly the same as the “S+Lugol” color.
+At RT:
-”S”: Temperature was increasing, less Iodine molecule is trapped in amylase coil =>
Blue purple. Concentrations of 2 solution also affect the color.
-”E”: When we increase the temperature, the enzyme also starts to reactive againand the
color changes slowly into towards the Iodine’s original color.
+At 500C:
-“S”: I think, the color should not be dark blue. Because the more heat we add, the
lighter color we get. It might be we had left these tubes at RT for a long time so it didn’t
reflect the exactly color.
-“E”: In this stage, the temperature has overcome the optimal temperature, so the
enzyme begins to be denatured again. The color stay nearly the same as the “S+Lugol”
color.
+At 1000C:
-“S”: I think, the color should not be dark blue. Because the more heat we add, the
lighter color we get. It might be we had left these tubes at RT for a long time so it didn’t
reflect the exactly color.
-“E”: In this stage, the temperature has overcome the optimal temperature, so the
enzyme begins to be denatured again. The color stay nearly the same as the “S+Lugol”
color.

b. Compare all “S” tubes in all conditions and all “E” tubes in all condition. Explain
thephenomenon.
All “ S” tubes in all condition: All the tube have the same color because there are no
enzyme amylase to catalyze the hydrolysis reaction of starch and the helical structure
are not affected anymore so that Iodine can fit inside the structure and give the dark
blue color.
All “ E” tubes in all condition: the 50-E, 100-E tube has the same color because
enzyme cannot catalyze the hydrolysis reaction, hence the glycoside bond were not
affect and Iodine can fit withStarch to give the dark blue color like the others “ S” tube.
And enzyme amylase in 4-E and RT-E can active and catalyze the hydrolysis reaction
lead to the different color if the other 2 tubes. But in room temperature amylase can
active well so that we can see the purple pink color, and at 4*C amylase cannot work
well as at RT.
c. What is the optimal range of temperature for amylase activity?

Instructor : MSc. Nguyen Thien Quang

 International University, Vietnam National University - HCMC 5
BIOLOGY LABORATORY

The optimum temperature of the Amylase varies by species. As it is inside the human
body it works at about 37.50C; the optimum temperature of amylase solution is about
370C; the bacterial amylase has the optimum temperature of 38 0C; the optimum
temperature of fungal amylase is about 500C, etc.

II /PROTEASE:
1/-Introduction:
Protease are enzymes that break peptide bonds between amino acids of protein. The
enzymes use a molecule of water for this and are thus classified as hydrolases.
Proteases occur naturally in all organisms and constitute 1-5% of the gene content.
These enzymes are involved in a multitude of physiological reactions from simple
digestion of food proteins to highly regulated cascades. Peptidases can break either
specific peptide bonds (limited proteolysis), depending on the amino acid sequence of a
protein, or break down a complete peptide to amino acids (unlimited proteolysis).
2/ -Procedure:
+ Prepare two test tubes and put into each tube 5 ml enzyme extract.
+ Label these 2 tubes: one tube (marked 100) is then put in boiling water for 15
minutes, the other (marked RT) left at room temperature.
+ Add into each tube the small piece of boiled white egg.
+ Cover tubes, shake lightly and then incubate these tubes at room temperature.
+ After 2 days, pour out the liquid and compare 2 pieces of boiled egg on a petri dish.
3/-Results : Report the observation of egg piece’s shape after 2 days.
Temp.(0C) At the beginning After 2 days
RT Not change in shape Change in shape ( lose many
small pieces of egg)
100 Change in shape Change in shape complete
( lose many small pieces of egg) decompose

Instructor : MSc. Nguyen Thien Quang

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At the begining

RT on the left & 1000C on the right

After 2 days

1000C on the left & RT on the right

4/- Discussion:
a. Why do 2 pieces of egg have different shape after two days of incubation?
Egg white is made up of long protein molecules. At RT, the protease enzyme in
pineapple extract acts to cut up the egg white proteins making the fragments soluble,
and so they dissolve. After 2 days, the egg white piece appeared to be much smaller and
easily to be broken into pieces. However, after the pineapple extract was boiled, the

Instructor : MSc. Nguyen Thien Quang

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protease enzyme was changed and no longer exist. That is why the egg white piece in
100 degree tube remain unchanged since there was no protease enzymes to react.
b. What is the optimal range of temperature for protease activity.
-Enzyme activity was stable with temperature within the range of 300C to 500C (room
temperature).
III/CATALASE:
1/-Introduction:
-Catalase is a common enzyme found in nearly all living organisms exposed to oxygen
(such as vegetables, fruit or animals). It catalyzes the decomposition of hydrogen
peroxide to water and oxygen. It is a very important enzyme in protecting the cell from
oxidative damage by reactive oxygen species (ROS).
-Like other enzyme, catalase is also heat inactivated. When enzyme loses activity, the
production of oxygen and water is decreased. Potato is known as a source of catalase.
2/-Procedure:
-Collect the aqueous phase (enzyme - catalase suspension) from the mixture potato-
water.
-Prepare 4 tubes. Mark as following:
Temperature 40C RT 500C 1000C
Marked tubes 4 RT 50 100

-Add 5ml of enzyme suspension to each test tube, mark a line at 5cm above the
solution. Then bring these tubes to suitable temperatures and incubate for 5 minutes.
-After that, add 5 drops of H2O2 into each tube, observe what happens.
3/-Results: Report the time for gas column reach the 5-cm line.
Temp.(0C) Time Recorded (seconds)
4 Cannot reach

RT Cannot reach

50 Cannot reach

100 Cannot reach

Instructor : MSc. Nguyen Thien Quang

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Cannot reach the 5cm point after 10 minutes:

the distance between 5cm point and the level of bubbles is 2 cm of 40C, 3.5 cm of RT, 1
cm of 500C, 0.7 cm of 1000C.

The bubbles level cannot reach to the point due to the qualities and concentrations of
hydro peroxide .

4/-Discussion:
a.Why is there different in time when bubbles colume reach the 5cm line between
different conditions.

-Because the enzyme has a optimal range of temperature for its activity. So, different
temperatures lead to different time intervals.

b.What is the optimal range of the temperature for catalase activity.

-Enzyme activity was stable with temperature within the range of 200C to 400C.

Instructor : MSc. Nguyen Thien Quang