Torreon, Ma. Bhie Jia A.

BSN 1-11
PROF: SIR, Jess Magno.

ENZYMES
Exp No. 2
A. Action of Salivary Amylase
Reagents: Dilute Iodine solution, Benedict’s solution

Procedure:
1. Place 5ml of starch solution in a test tube.
2. Add five (5) drops of filtered saliva.
3. Shake the mixture.
4. Place a drop of the starch-saliva mixture on a spot place and add one (1) drop of dilute iodine
solution to it. Observe the change in color after 15 minutes.
5. Warm the remaining starch-saliva mixture in a water batch at 350c. while performing the next step.
6. Repeat the iodine test at 1 minute, 2 minute, 3 minute and 5 minute intervals, noting the color
change.
7. Transfer five (5) drops of the remaining saliva-starch mixture in a test tube.
8. Add 1 ml of Benedict’s solution.
9. Place in a water bath for 3 minutes. Record the result.1.

Results/Conclusion:
• Maltose consists of two glucose molecules joined together. In humans, the production of
maltose from starch occurs within the mouth in a reaction catalyzed by amylase, an enzyme
found in human saliva.
• Maltose cannot combine with iodine to produce this color. Therefore, in the presence of iodine,
the digestion of starch into maltose will be accompanied by the disappearance of the blue-black
color.

Test for Dilution on Enzymes Activity

Reagent: Iodine Solution, Benedicts Solution

Procedure:
1. Place 5 ml of water in each of 6 test tubes and number the test tube from 1 to 6.
2. To test tube 1, add 1 ml of filtered saliva and shake the mixture. Transfer 1 ml from test tube 1
to test tube 2, then 2 to 3, 3 to 4, 4 to 5, 5 to 6, making 6 different solutions gradually with
decreasing concentrations.
3. To each of the test tubes, add 1 ml of starch solution and shake well.
4. Place the test tube in a water bath at a temperature of 400C for 10 minutes then divide the set
samples into two equal portions.
5. Treat the first portion with 5 drops of Iodine solution and 1 ml Benedict’s solution for the
second portion.
Results/conclusion:
Some enzymes are unstable in dilute solution, and so are readily denatured, resulting in lower activity in
dilute solutions than would otherwise be expected. This is especially a problem with purified enzyme
preparations, where there is very little protein present in the sample.
Effect of Temperature on Enzymes Activity
Reagent: Iodine Solution

Procedure:
1. Place 5 ml of starch solution in each of the 6 test tubes.
2. Label the test tubes 1-6.
3. Place test tube 1 in an ice bath at 5 Celsius, test tube 2 in an ice water at 10 celsius, test tube 3
in a cold water bath at 20 celcius, test tube 4 at 30 celsius, test 5 in a water bath at 40 celcius
and test tube 6 at 60 degree Celsius.
4. Add 5 drops of saliva to test tube 1,2,3,4 and 6, and 5 drops of boiled saliva to test tube 5. Shake
all test tubes well then add one drop of iodine to each test tube. Observe.
In Addition, repeat the procedure of adding a drop of combined solution of saliva and starch to iodine
solution every minute until there is no changes in color occur.

Results/Conclusion:

Iodine turns blue-black or blue if it is in contact with starch and it stays gold or light brown in the
presence of glucose. Blue or blue-black color means that the enzyme activity is slowed down or inactive
while light-brown or gold color means that the enzyme activity is high. As also for the results, the
enzyme salivary amylase will break down starch very quickly at optimum temperature, the enzyme
salivary amylase will break down starch slowly at low temperature due to reduced kinetic energy, and
the enzyme salivary amylase will break down starch very slowly or not at all at high temperature due to
denaturation of enzyme’s active site.

B. Action of Catalase

Effect of concentration
Reagents: 3% H2O2, 1% H2O2

Procedure:
1. Place 1 ml. of potato extract to two (2) separate test tubes.
2. Place the two (2) test tubes in a water bath at 350C for 5 minutes.
3. Add 15 drops of 3% H2O2 on the 1st test tube and 15 drops of 1% H2O2 on the second test tube.
4. Note the time required for bubbles to appear.
Results/Concentration:
Catalase enzyme formed the bubbles in the two tubes. The height of the bubbles for 1% is 4.7cm and for
the 3% of h²0² is 0cm.
Effect of Temperature
Reagents: 3% H2O2

Procedure:
1. Place 1 ml. of liver chunk in three (3) separate test tubes.
2. Place the first test tube in an ice bath, the second test tube to a hot bath at 370C, and the last one is
the normal temperature.
3. Treat the three (3) test tubes with 5 drops of 3% H2O2. Note the time required for bubbles to appear.

Result/conclusion:
Test Tube 1 (Hot Water):
The liver reacted instantly on the first (1) test tube with a hot water bath that has a temperature of 370C
and a 3% of H2O2.

Test Tube 2 (Room Temperature Water):

The second (2) tube with a temperature of 20-220C tube that has 3% of H2O2 had the 2nd best reaction
of bubbles.

Test Tube 3 (Ice Water):

Unlike the first tested tubes, the third one with a temperature of 40C that has a 3% of H2O2 reacted
slowly.

Effect of pH
Reagents: 10% NaOH,3% H2O2

Procedure:
Place 1 ml. of potato extract in a test tube. Determine the pH.
2. Then add five (5) drops of 10% NaOH. Determine the pH.
3. Warm the extract in a water bath at 350C. Add 10 drops of 3% H2O2.
4. Note the time required for bubbles to appear.

Results/Conclusion:
After 3 minutes, at a pH 7, the enzymes are really working well on breaking down the H2O2.