Practical 5: Enzymes and Digestion

Objective: a) To show the action of amylase on starch

b) To study the effect of temperature on enzyme action
c) To show the action of pepsin on egg white (protein)
d) To show coagulation of milk
e) To show emulsification of fats

Theoretical Knowledge:
The enzyme is an organic catalytic agent produced by living cell. An enzyme,
even in small amounts, has the unique capacity of speeding up a biochemical reaction
in a cell without itself being altered by the reaction. It accelerates the velocity of the
reaction without necessarily initiating it. Although all enzymes are initially are produced
in cell, some are excreted through the cell wall and function in the cell’s surrounding
environment.

Material and apparatus:

 Beaker
 Blue and red litmus paper
 Measuring cylinder
 Thermometer
 Test tube
 White tile
 Dropper
 Stopwatch
 1% starch solution
 Dilute hydrochloric acid
 Dilute sodium hydroxide
 Benedict solution
  Iodine solution
 Egg
 Pepsin
 Rennin
 Milk
 Coconut oil
 Bile from guinea pig gall bladder

Procedure:

(a) To show the action of amylase on starch.

1. The mouth is rinse

2. Chewing movements is performing to stimulate the flow of saliva and it is
collected in test tube.
3. The saliva is tested with litmus paper to find out whether it is acidic or alkaline.
4. The saliva is diluted about an equal volume of distilled water.
5. The saliva preparation is divided into 3 equal amounts in three test tube and it is
labeled with B, C and D. The tube is filled as follow:
A – 3 cm³ of distilled water (control experiment)
B – 3 cm³ of the saliva preparation
C – 3 cm³ of the saliva preparation and 3 cm³ dilute hydrochloric acid
D – 3 cm³ of the saliva preparation and 3 cm³ dilute sodium hydroxide
6. 5 cm³ of starch solution is put to each test tube and stir thoroughly.
7. One half of the content for each tube is tested with dilute iodine after 30 minute.
The other half is boiled with an equal amount of Benedict solution.
8. The result is tabulated.

(b) To study the effect of temperature on enzyme action

1. The solution saliva is prepared as experiment A

2. 5 cm³ of this saliva solution is placed in a test tube of 1 % starch solution in
another.
3. Both of test tubes are allowed to stand in room temperature.
4. White tile is taking and a series of dilute iodine drop is placed on it.
5. The two solution is mix in the tube and the time of mixing is note.
6. By means of a clean glass rod, a drop of the properly stirred mixture is removed
and tested with a drop of iodine on the white tile. A deep blue colour appears.
7. This test is repeated in interval of one minute, the glass rod is wash with distilled
water between each test until the mixture fails to give blue colour with iodine.
8. The total time is recorded between mixing of the saliva and the starch solution
and the end of the test. This is the time for all the starch to be converted to
maltose by amylase at room temperature.
9. The total time taken between the mixing of saliva and the starch solution is
recorded at the end of the test.
10. The experiment is repeated at different temperature; 5º, 15º, 25º, 45º, 55º, 65º
and 75º.
11. It is important that for each of these experiments the saliva and the starch
solutions be warmed or cooled to the required temperature before they are
allowed to mix. For temperatures higher than room temperature a water bath is
used, for temperatures lower than room temperature ice cubes are used to bring
the temperature down.
12. For the above experiments, it may be necessary to work in groups, each group
working at a particular temperature.
13. The results are tabulate.

(c) To show the action of pepsin on egg white (protein).

1. A suspension of egg white is prepared by beating up a little raw egg white with
boiling water in a small beaker and the suspension is allowed to cool.
2. To each of the three test tubes A, B and C, a few cm³ of the cooled egg white
suspension is added.
3. The tubes are placed in a water bath at about body temperature (about 37º C).
Each tube is filled as follows:
A – 5 cm³ of 0.2 % hydrochloric acid
B – 5 cm³ of ‘artificial gastric juice’ (prepared by dissolving 3.5 g of pepsin in
100 cm³ of 0.2 % hydrochloric acid)
C – 5 cm³ of ‘artificial gastric juice’ and 1 cm³ of dilute sodium hydroxide
4. The mouth of each tube is plugged with a piece of cotton wool after adding a little
thymol to prevent decay.
5. The tubes are left to stand for about one hour and they are shaken periodically.
6. The burette test is carried out on the contents of each tube at the end of one
hour.
7. The results are tabulated.
Results:

(a) To show the action of amylase on starch.

Test tube Contents Iodine test Benedict test Inferences

A Starch + distilled Yellowish Blue colour Starch is
water brown turn present, no
to blue enzyme
black reaction occur
B Starch + saliva Yellowish Red block Starch is
brown precipitate convert to
glucose
C Starch + saliva + Yellowish Blue colour Starch is
dilute HCl brown turn present, no
to blue enzyme
black reaction occur
D Starch + saliva + Yellowish Red block Starch is
dilute NAOH brown precipitate convert to
glucose

(b) To study the effect of temperature on enzyme action

 Temperature Time in minutes (t) Activity (1/t)
Room temperature 4 0.25
5º 20 0.05
15º 10 0.10
25º 5 0.20
35º 3 0.33
45º 5 0.20
55º 10 0.10
65º 20 0.05
75º - -

Since the shorter the time taken the more active the enzyme will be, the activity (rate of
reaction) is denoted by 1/t (the reciprocal of the time taken).

(c) To show the action of pepsin on egg white (protein).

Test Contents Observation Biuret test Inferences

tube
A Egg white + dilute transparent No changes Absence of
HCl solution is protein
formed
B Egg white + artificial transparent No changes Absence of
gastric juice solution is protein
formed
C Egg white + artificial White Colour turn to Presence of
gastric juice + dilute precipitate can purple protein
NaOH be seen

Question:

(a) To show the action of amylase on starch.

1. From this experiment, what general conclusion can you draw regarding the
nature of amylase activity?
(b) To study the effect of temperature on enzyme action

1. Plot a graph of 1/t against temperature. What is the optimum temperature?

2. Describe how temperature affects enzyme catalysed biochemical reactions.
Enzymes are less active at lower temperature. Increasing the temperature
increase the reaction rate until the optimum reach the maximum temperature at
37. After 37 the temperature will decrease rapidly.

(c) To show the action of pepsin on egg white (protein).

1. What happens to the egg white suspension in tube B? Does it turn yellow,
transparent and then gradually dissolve?
2. Is there any change in the other tubes?

Answer:

(a) To show the action of amylase on starch.

1. From this experiment, amylase can be react with starch and converted the starch
to maltose in alkaline condition and in room temperature.

(b) To study the effect of temperature on enzyme action

1. The optimum temperature is at 37.
2. Enzymes are less active at lower temperature, so at lower temperature the
reaction rate enzyme to starch is slow. But when the temperature is increase, the
reaction of enzyme to starch also increase until reach maximum temperature at
 37. At 37, reaction enzyme with starch is faster. This is the optimum stage to
enzyme react with starch. If the temperature still increase, the enzyme will
denatured. So the reaction will decrease rapidly.

(c) To show the action of pepsin on egg white (protein).

1. The egg white suspension in tube B is turns yellow, transparent and then
gradually dissolves. This is because; the pepsin in gastric juice is reacting with
egg white suspension.
2. In tube A the changes occur, the protein is catalysed same as in tube B while in
tube C there are no reaction because of presenting NaOH. The pepsin cant react
with protein in alkaline condition.

Discussion:

1. Amylase from our salivary glands and pancreas digests starch to maltose in our
mouth and small intestine. Pepsin is a protease that begins digestion of proteins,
breaking them into peptides and amino acids. Pepsinogen, is secreted by gastric
glands of the stomach into the stomach. There, in the acid environment of the
stomach, pepsinogen is converted into pepsin.

2. If the iodine retains its yellow-brown colour, starch is absent. If a purple or blue-
black colour forms, starch is present, and the deeper the colour, the greater the
amount of starch.

3. The sugar test is performed by adding a small amount of Benedict's reagent to

the sample and bringing the mixture to a boil. If the Benedict's reagent retains its
clear blue colour and no precipitate is formed, sugar is absent. The presence of
sugar is indicated by the development of a precipitate which may range in colour
(indicating increasing amounts of sugar) from green, through yellow and orange,
to red. This test depends upon the ability of certain sugars to reduce the cupric
(Cu++) copper present in the Benedict's reagent to the cuprous (Cu+) form.
4. Fat digestion is facilitated by the presence of bile salts (a component of bile
produced in the liver), which bring about a physical (not enzymatic) breakdown of
large fat droplets into smaller particles. This process is called emulsification.

5. As temperature increases, the speed of the substrate and the enzyme increase.
This increases the enzyme activity - its simply more probable that that the
enzyme and the substrate will meet and bond. The activity increases till an
optimum is reached (optimum temperature). From that point on, the activity of the
enzyme decreases as it starts to denature (i.e. loses its tertiary structure -
remember that for enzymes structure and function are highly related!). It will
eventually reach zero when the enzyme has lost its shape completely and can no
longer work.

6. The rate of the reaction is increased if the enzyme and substrate mixture is
brought to body temperature (370 C). The progress of the reaction can be
visualized by testing the reaction mixture for (1) the disappearance of the
substrate (starch) or (2) the appearance of product (maltose). Two simple tests,
iodine test for starch and Benedict's test for sugar are used for this purpose.

Conclusion:
Amylase enzyme is use to breaks down starches into sugars. Pepsin function is to
break down proteins in the stomach. The rate of reaction increase as the temperature
increases. However enzymes denatures at very high temperature. In human, the rate of
enzyme reaction is the highest at 37 degree celcius.

References:
Longman. PRE-U TEXT STPM BIOLOGY. Volume 1. Pearson Malaysia (2006).

Sylvia S. Mader (2007). Biology, Ninth Edition. McGraw Hill.

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