LAB REPORT 4

Course name (and course’s ID): Practice in Biology S1_2023-24_G06

Instructor: Ms. Phạm Hồng Điệp
Group number: 5 – Saturday afternoon
Group member (name and ID): Phạm Hữu Tuấn Anh BTBTIU23119
Bùi Ngọc Thảo My BTBTIU23134
Trần Bảo Ngọc BTBTIU23145
Trần Vũ Thanh Thảo BTBTIU23115
Date of submission: 11/11/2023

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I/ AMYLASE:
1/- Introduction:
- Amylases belong to the hydrolase group enzyme. Amylases's main function is to
hydrolyze the glycosidic bonds in starch molecules, converting complex carbohydrates
to simple sugars.
- There are 3 types of amylases:
+ ɑ-amylases: α-1,4 glycosidic bonds to yield either maltose and maltotriose
+ 𝛃-amylases: 1,4-α-D-glucan-malto-glycoside linkage.
+ 𝛄-amylases: the last α-1,4 glycosidic bond and the α-1,6 glycosidic bond in the
starch
2/- Procedure:
- Material: green bean sprout, starch suspension, Lugol solution, pasteur pipettes, test
cubes and racks, mortar and pestle, filter paper, two waterbaths.
- Procedure:
1. Select 40-60 green bean sprouts, put all into the mortar, add 20 ml 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:

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.
5. After the time indicated, take tubes out, add 1-2 drops of Lugol solution into each tube
and 2 ml of water into each “S” tube. Mix well.
6. Observe the color of these tubes.

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3/- Results:

Figure I.1: 4-S and 4-E tubes before and after adding Lugol solution (from left to right):
4-S: transparent 4-S: turned blue-violet
4-E: light yellow 4-E: became blue-violet and rapidly
returned to original color

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Figure I.2: RT-S and RT-E tubes before and after adding Lugol solution (from left to right):
RT-S: transparent RT-S: turned dark blue
RT-E: light yellow RT-E: turned brown

Figure I.3: 50-S and 50-E before and after adding Lugol solution (from left to right):
50-S: transparent 50-S: turned dark blue
50-E: light yellow 50-E: turned brown and rapidly
returned to original color

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Figure I.4: 100-S and 100-E tube before and after adding Lugol solution (from left to right):
100-S: transparent 100-S: turned dark blue
100-E: light yellow 100-E: turned brown and rapidly
returned to original 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 4oC:
- The S tube: The solution in the test tube turns blue-violet.
- The E tube: The solution in the test tube turns blue-violet.
- Phenomenon explanation:
+ Because enzyme activity was reduced in low temperatures, Amylase can not break
down starch. As a result, both the 2 test tubes have the same color after adding the Lugol
solution.
+ Because of the existence of Elemental iodine and Potassium iodine in Lugol's
solution, both solutions in the 2 test tubes have blue-violet color.
*At RT:
- The S tube: The solution is transparent. After adding 1-2 drops of Lugol solution, the RT-S
solution turns to dark blue.
- The E tube: The solution has a light yellow color. After adding 1-2 drops of Lugol solution,
the solution in the test tube turns brown.
- Phenomenon explanation:
+ In the RT-S test tube, temperature was increasing, less Iodine molecule was trapped
in the amylase coil => dark blue. Concentrations of 2 solution also affect the color

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 + In the RT-E test tube, because the temperature was increasing so the enzymes are
active and break down starch => the color of the solution is different from the RT-S test
tube.
o
*At 50 C:
-The S tube: The solution in the test tube turns blue-violet.
-The E tube: The solution in the test tube turns brown
- Phenomenon explanation:
+The test tube 50-S has blue-violet color because the more heat we add, the lighter
color we get.
+ In test tube 50-E, the certain temperature has overcome the enzyme’s optimal
temperature, so the solution has a color nearly the same as the “S+Lugol” color.
*At 100oC:
-The S tube: The solution in the test tube turns to light blue-violet
-The E tube: The solution in the test tube turns to brown.
- Phenomenon explanation:
+ Temperature of 100oC is the temperature that causes amylase enzyme to
be denatured (catalyst inactivation). Hence, starch isn’t broken down. When Lugol reagent is
instilled,this amount of starch will cause the solution to have the lighter blue-violet color.
+ Test tube 100-E’s solution, 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 the
phenomenon.
- All the S tubes haven’t changed dramatically because starch has transparent color and there
is no enzyme amylase to catalyze the hydrolysis reaction of starch.
- All the E tubes’ color changes significantly because there is not just starch that affects the
color of the solution but also enzymes. Moreover, enzymes are easily affected by temperature
so all the test tubes bring many results after adding Lugol solution.
c) What is the optimal range of temperature for amylase activity?
The optimal range of temperature for amylase activity is 32 - 40 oC.

II/ PROTEASE:
1/- Introduction:
Protease are the enzymes use a molecule of water to break protein molecules down to peptides
and eventually to free amino acids. They are able to catalyze hydrolytic reactions, which means
that the enzyme use a molecule of water for this and are thus classified as hydrolases. They are
divided into acid, neutral, and alkaline proteases. These enzymes can be obtained from plants,
animals, and microorganisms in several conditions, such as high salt concentrations. The
function of protease is to catalyze the hydrolysis of proteins, which has been exploited for the
production of high-value protein hydrolysates from different sources of proteins such as casein,
whey, soy protein and fish meat.

2/- Procedure:
- Take one eight of the pineapple fruit, peel off the cover, and cut into small pieces.
- Put pineapple pieces into mortar, add 15ml water and grind till homogenous.
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 - Filter this mixture and collect the aqueous phase (enzyme-protease extraction).
- Prepare two test tubes and put into each tube 5ml enzyme extract.
- Label these 2 tubes: 1 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 two days, pour out the liquid and compare two pieces of boiled egg on a petri
dish.

3/- Results:

Temperature (degree At the beginning After 2 days

Celcius)

RT Does not change in shape Changes in shape (becomes

smaller)

100 Does not change in shape Does not change much in

shape

Figure II.1: Egg piece in RT tube and 100 tube after 2 days (from left to right):
RT tube egg piece: becomes smaller than original one
100 tube egg piece: remains unchanged

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4/- Discussion:
a) Do 2 pieces of egg have different shape after 2 days of incubation? Explain.
- Two pieces of egg have different shapes after 2 days:
+ At room temperature, enzyme Protease breaks down protein Albumin in white egg and
makes its shape smaller.
+ The piece of white egg in the 100°C test tube stays unchanged since the enzyme was
deactivated at high temperatures.
b) What is the optimal range of temperature for protease activity?
The optimal temperature for the protease activity is investigated from 30°C to 50°C.

III/ CATALASE:
1/- Introduction:
Enzyme catalases, an antioxidant enzyme, are produced naturally in plants, animal and
microbial cells. This enzyme functions to convert H2O2 into less toxic substances.
The catalase belongs to the group of oxidoreductase. When the body is infected with high
level of H2O2, the catalase stored in peroxisome (in plant cell) or mitochondria (in animal cell)
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 cell by means of its detoxification ability.
Like other enzymes, catalase is also heat inactivated. When an enzyme loses activity, the
production of oxygen and water is decreased.
2/- Procedure:
1. Put 100 g potato and 250 ml water into the blender. Blend this mixture until smooth. Filter
this suspension and collect the aqueous phase (this is enzyme - catalase suspension).
2. Prepare 4 test tubes. Mark these tubes as followings:

Temperature 4oC RT 50oC 100oC

Marked tubes 4 RT 50 100

3. Add 5ml of enzyme suspension to each test tube, and mark a line at 5cm above the solution
surface. Then bring these tubes to the indicated temperatures, and incubate for 5 minutes.
4. After get tubes out, add 5 drops of hydrogen peroxide into each tube; observe what
happens in each tube. Note down the time needed in each tube for the column of oxygen
bubbles forming and reaching to the marked line.

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3/- Results:

Figure III.1. Observing bubbles forming in 4 tube, RT tube, 50 tube, 100 tube (from
left to right):
Oxygen bubbles formed and reached to the marked line in 4 tube after 12 minutes
Oxygen bubbles formed in RT tube more slowly than 4 tube
Oxygen bubbles did not form much in 50 tube
Oxygen bubbles did not form 100 tube
4/- Discussion:
a) Why is there different in time when bubbles columns reach the 5-cm line between different
conditions?
Because enzyme has its own optimum temperature, different temperatures lead to different
time needed for bubbles to reach the 5-cm line.
b) What is the optimal range of temperature for catalase activity?
The optimal pH of catalase is approximately 7 pH and the optimal range of temperature is at
30-37°C.