PRACTICAL 5

Title : Enzymes and Digestion

Objective : (1)To show the action of amylase on starch

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

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

(4) To show coagulation of milk

(5) To show emulsification of fats

Theory : Enzymes are proteins which act as biological catalysts accelerating

specific chemical reactions, such as the digestion of food. Without enzymes,
these reactions often require very high temperatures and pressures. Although
enzymes take part in the reactions, they are not chemically altered by them.
Consequently they are not used up and are required in relatively small
concentrations. The body varies the concentration of a particular enzyme to
regulate a specific activity; generally, the higher the enzyme concentration, the
greater the rate of reaction. Enzymes bind temporarily to one or more of the
reactants of the reaction they catalyze. In doing so, they lower the amount of
activation energy needed and thus speed up the reaction. Enzyme activity can be
affected by other molecules. Inhibitors are molecules that decrease enzyme activity;
activators are molecules that increase activity. Many drugs and poisons are enzyme
inhibitors. The activity of the enzymes is strongly affected by changes in pH,
temperature and the concentration of substrate. The optimum coagulation by calf
rennin is about 40° to 42°C. Below this temperature coagulation is less rapid and
no clotting occurs below 10° to 15°C. Also no clotting occurs above 60° to 65°C.
The clot is softer at low temperatures and tougher and stringy at high
temperatures. By optimum is meant the temperature at which coagulation takes
place most rapidly for a definite concentration of rennin and milk. The Gall
 Bladder is located right underneath the Liver and is stained green by its contents.
The gall bladder hold a production of the liver called Bile within which are Bile
Salts, it does not produce anything itself. The bile salts emulsify fat in kind of the
same way soap does to dirt. It sticks a hydrophobic tail into the fat has a
hydrophilic head in the water. This make the fat globule finds the shape with
lowest surface area, a sphere. The reason the body must do this is to allow the
transport of fat more easily and to increase the overall surface area for enzymes
to act on it. Enzymes are very specific, and it was suggested by Emil Fischer in 1894
that this was because both the enzyme and the substrate possess specific
complementary geometric shapes that fit exactly into one another. This is often referred
to as "the lock and key" model. However, while this model explains enzyme specificity, it
fails to explain the stabilization of the transition state that enzymes achieve

Apparatus : Beaker

Blue and red litmus paper

Measuring cylinder

Thermometer

Test tube

White tile

Dropper

Stopwatch

Material : 1% starch solution

Dilute hydrochloric acid

Dilute sodium hydroxide

Benedict solution
 Iodine solution

Egg

Pepsin

Aim

To show the action of amylase on starch.

Problem statement

What is the suitable medium for the action of the amylase on starch?

Hypothesis

The alkaline medium is the suitable for action of the amylase on starch.

Variables:

Manipulates variables : The content in the test tube (The medium)

Responding variables : The changes in the colour of the solution in iodine

test and benedict test.

Fixed variable : The amount of starch solution, the amount of

Saliva solution used and the temperature.

Procedure

1. Mouth had rinsed.

2. Chewing movements had performed to stimulate the flow of saliva and it had
collected in a test tube.
3. The saliva had tested with litmus paper to find out whether it was acidic or
alkaline.
4. About an equal volume of distilled water had diluted.
 5. The saliva preparation had divided into 3 equal amounts in three test tubes
and labeled them B, C and D. Each tube was filled as follows:
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 had added to each of the test tubes and had stirred
thoroughly.
7. After 30 minutes, one half of the contents of each test tube had tested with
dilute iodine solution and the other half had boiled with an equal amount of
Benedict solution.
8. The results had tabulated as shown in the table.

Results :

Test tube Contents Iodine test Benedict test Inferences

The yellow colour The action of
Starch + distilled
A iodine change to Not dissolved amylase on starch
water
blue. does not occur.
The action of
B Starch + saliva Not dissolved Dark yellow amylase on starch
occurs.
The yellow colour The action of
Starch + saliva +
C iodine change to Not dissolved amylase on starch
dilute HCI
dark blue does not occur.
The action of
Starch + saliva +
D Not dissolved Yellow amylase on starch
dilute NAOH
occurs.
From this experiment, what general conclusion can draw regarding the nature of
amylase activity?

Amylase is an enzyme that breaks starch down into sugar. Amylase is present in human
saliva, where it begins the chemical process of digestion. Foods that contain much
starch but little sugar, such as rice and potato, taste slightly sweet as they are chewed
because amylase turns some of their starch into sugar in the mouth. The pancreas also
makes amylase (alpha amylase) to break down 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.

Conclusion

When the solution in the test tube which contains saliva, starch and NaOH is tested with
iodine, the solution is does not dissolved and when the Benedict test is carried the
solution change yellow. This shows the starch is digested by the amylase. So that,
alkaline medium is the suitable medium for the amylase activity on starch. Hypothesis is
accepted.
Aim

To study the effect of temperature on enzyme action

Problem statement

What is the optimum temperature for the enzyme activity?

Hypothesis

370C is optimum temperature for the enzyme activity.

Variables:

Manipulates variables : The temperature.

Responding variables : The activity of the enzyme reaction.

Fixed variable : The concentration of the enzyme, the concentration of

The starch solution, the volume of the enzyme, and

The volume of the starch solution

Procedure

1. A solution of saliva had prepared as in experiment (a).

2. 5 cm3 of this saliva solution had placed in a test tube and 5 cm 3 of 1% starch
solution in another.
3. Both test tubes had allowed standing at room temperature.
4. A white tile had taken and a series of drops of dilute iodine solution had
placed on it.
5. The two solutions in the tubes had mixed and the time of mixing had noted.
6. A drop of the properly stirred mixture had removed by means of a clean glass
rod and it had tested with a drop of iodine on the white tile. A deep blue colour
was appeared.
 7. This test had repeated at intervals of one minute, the glass rod was washing
with distilled water between each test until the mixture failed to give a blue
colour with iodine.
8. The total time taken between the mixing of the saliva and the starch solution
and the end of the test had recorded. This was the time for all the starch to be
converted to maltose by amylase at room temperature.
9. The experiment had repeated at different temperature examples at 5 0, 150,
250, 450, 450, 550, 650 and 750.
10. It was important that for each of those experiments the saliva and the starch
solutions be warmed or cooled to the required temperature before they were
allowed to mix. For temperatures higher than room temperature used a water
bath, for temperatures lower than room temperature used ice cubes to bring
the temperature down.
11. The results had tabulated.

Result

Temperature (0C) Time in minutes (t) Activity (1/t)

Room temperature 3 0.30
5 < 30 0.00
15 23 0.09
25 6 0.16
45 8 0.13
55 25 0.04
65 < 30 0.00

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)
Questions:

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

370C.

2. Describe how temperature affects enzyme catalysed biochemical reactions.

All enzymes have optimum temperatures at where its activity is maximmum.
Some are very specific and have a range of only 5 degrees they can function in.
Reactions take place at higher temperatures, because molecules move faster
and thus it is more likely that they collide in the necessary configuration to cause
the reaction. If temperature is too high or too low, an enzyme can be denatured.
That it’s folding or conformation will be compromised and it will cease to function
normally.
High temperatures increase the rate of reaction. This is because temperature
causes molecules to move faster. They can find the proper configuration or
conformation for a reactiondue to this freedom of movement. Catalysts help to
lower the activation energy do this much more efficiently. Both have same effect
on reaction rate.
Conclusion
The activity of the enzyme is maximum in 370C. So the optimum temperature for
the enzyme action is 370C.

Aim : To show the action of pepsin on egg white (protein)

Problem statement

What is the effect of the enzyme on the protein solution?

Hypothesis

The enzyme digests the protein.

Variables:

Manipulates variables : The presence of the enzyme.

Responding variables : The action of pepsin on egg white

Fixed variable : The volume of egg suspension, the temperature.

1. A suspension of egg white was prepared by beating up a little raw egg white
with boiling water in a small beaker and allow the suspension to cool.
2. A few cm3 of the cooled egg white suspension added to the each of the three
test tubes A,B and C.
3. The test tubes were placed in a water bath at about body temperature (about
370C) and each tube filled as follows:
A- 5 cm3 of 0.2 % hydrochloric acid
B- 5 cm3 of artificial gastric juice (prepared by dissolving 3.5g of pepsin in
100cm3 of 0.2% hydrochloric acid.
C- 5 cm3 of artificial gastric juice and 1 cm3 of dilute natrium hydroxide.
4. The mouth of each tube was plugged with a piece of cotton wool after adding
a little thymol to prevent decay.
5. The tubes were leaved to stand for about one hour and shaked them
periodically.
6. Biuret test were carry out on the contents of each tube at the end of one hour.
7. The result had tabulated.
 Result

Test tube Contents Observation Biuret test Inferences

The action of pepsin
Egg white + dilute White White
A on egg white does
HCI suspension suspension
not occur.
The action of pepsin
Egg white + artificial White
B Clear solution on egg white occurs.
gastric juice suspension

Egg white + artificial The action of pepsin

White White on egg white does
C gastric juice + dilute
suspension suspension not occur.
NaOH

Conclusion.

The enzyme pepsin digest the protein. Hypothesis accepted.

DISCUSSION
 .

Firstly, an experiment to show the action of amylase on starch was carried out.
Four test tubes was prepared which were filled with starch in each every tube and
distilled water in tube A, saliva preparation in tube B, saliva preparation and dilute
hydrochloric acid in tube C and the saliva preparation and dilute sodium hydroxide in
tube D.. The saliva which was mixed with more water can’t give an accurate result. So
is important that make sure saliva was in the correct concentration. After thirty
minutes, one half of the contents of each test tube were tested with dilute iodine solution
and the other half was boiled an equal amount of Benedict solution. Only the solution in
test tube A and test tube C turned into blue colour while there were no any changes in
test tube B and test tube D. This is because the starch was catalysed immediately by
the saliva solution which contains the amylase enzymes. Wherever, amylase enzymes
which known as an alkaline stops catalyzing the starch when the presence of HCI which
reacts as an acid. So, it shows that amylase enzymes can only catalyse the hydrolysis
of starch into smaller maltose molecules in the mouth.

Then, an experiment on the effect of temperature on enzyme action was carried

out. The saliva solution was prepared and starch solution differently. The solutions were
mixed and tested with a drop of iodine at intervals of one time. The total time was taken
until the mixture of the saliva and the starch solution failed to give a blue colour with
iodine. The experiment is repeated at different temperatures such as 5 0C, 150C, 250C,
450C, 450C, 550C, and 650C. This is the time for all the starch to be converted to
maltose by amylase. Therefore, even for an enzyme-controlled reaction, increasing the
temperature will increase the rate of reaction, but only up to peak. At about 65 0 C,
enzymatic activity stops altogether. Any further increase of temperature beyond the
optimum temperature, about 370 C to 400 C will cause a rapid decrease in the reaction.

Lastly, an experiment to show the action of pepsin on egg white (protein) was
carried out. Three test tubes was prepared which contain suspension of white egg in
each and test tube A filled with hydrochloric acid, test tube B filled with artificial gastric
juice and test tube C filled with the artificial gastric juice and dilute natrium hydroxide.
The results were taken after leave the mixture at optimum temperature, 37 0C for five
minutes. The mixture in test tube B is clear at the end of the experiment because the
albumen has been hydrolysed. Pepsin does not catalyse the hydrolysis of albumen in
neutral and in alkaline conditions. This is because it acts as an acid and it only
catalyses the hydrolysis of protein in acidic conditions. The pH for most enzymes is
about pH 7-8 (normal body pH), but a few enzymes can work at extreme pH, such as
gastric protease which known as pepsin in our stomach, which has an optimum of pH 1.