POST-LABORATORY GUIDE QUESTIONS

1 What is the effect of boiling o the enzyme activity?

At temperatures around boiling, the chemical bonds that hold together the structure of
enzymes begin to break down.

2. What is the optimum temperature for amylase activity?

the optimum temperature of salivary amylase ranges from 32C to 37C.This applies to the
human body since salivary amylase is suitable to function within these temperatures.

3. What do you think is the influence of temperature on the three-dimensional structure of the enzyme
to affect its activity?

The resulting loss of three-dimensional structure causes enzymes to no longer fit their target
substrate molecules, and enzymes entirely stop functioning. This loss of structure, known as
denaturation, is irreversible once enzymes are heated so much that the chemical bonds
holding them together break down, they will not spontaneously form again if temperatures
decrease.

4. Describe and rationalize the shape of the curve from the plot of 1/t versus pH. How is it related to
the enzymatic activity of amylase?

the salivary amylase is in a too acidic environment to function. As pH decreases, certain

amino acids like aspartate and glutamate are protonated, causing them to lose their net
negative charge which consequently denatures the enzyme. The optimum pH for the action of
salivary amylase ranges from 5.6 to 6.9

5. What is the optimum pH for amylase activity? List factors on which the pH activity relationship
of enzymes depends in general.

the optimum temperature of Salivary Amylase is 37C(the body temperature). Any

temperature higher than that, the structure of Salivary Amylase is denatured because more
kinetic energy is applied. Kinetic energy makes the molecules vibrate and collide with each
other. The more the kinetic energy, the more times the molecules collide with each other and
this destabilizes the structure.

6. What is the role of sodium chloride added to the enzyme solution?

Enzymes are proteins. Protein function is dictated by its tertiary structure that is determined
by its environment. If amylase, an enzyme, is in a high salt environment, it would be outside
of its optimal environmental conditions. The excess ions would denature the protein, change
its shape, and affects its function.

ENZYMATIC ACTIVITY OF SALIVARY AMYLASE

Experiment no. 4

INTRODUCTION
Enzymes are biologically important molecules
Salivary amylase is a common enzyme found in digestive system
There are several factors affecting enzymatic activities (i.e. pH & temperature).

In this experiment, the enzymatic activity and specificity of salivary amylase will be examined
depending on changes in pH and temperature. Factors such as narrow range of pH values and
different temperatures at which the enzyme exhibits its optimum activity will be done.

A. EFFECT OF TEMPERATURE
B.
MATERIALS
Enzyme solution
Buffered starch
1.1 M Iodine Solution
Spot plates
Test tubes
Medicine Droppers
Constant Temperature bath (4 Room temperature 37,50,60 and 70 C)
C. EFFECT OF pH
MATERIALS
Enzyme solution
2 % Buffered starch
1.2 M Iodine Solution
Spot plates
Large Test tubes
Medicine Droppers
Water bath set at 37 C

METHODOLOGY (effect oF temperature)

Acetate Buffer Solutions

Phosphate Buffer solution
Bicarbonate buffer

2mL
enzyme
sltn.
Label as 4C; separate
test tube + 2mL

Incubate both (ice bath)for

10mins.; mix, take 3 drops
+ 2 drops iodine sltn. onto

Repeat
steps
until
light
yellow-colored
solution is observed.

After
1
min.
(continue
incubation), repeat earlier
step onto the next well (note

Repeat steps for other

temps (RT, 37, 50, 60,
70) to obtain other

METHODOLOGY (effect oF pH)

Mix
1mL
acetate
buffer (pH 4) + 1mL
2% unbuffered starch
solution separate test
tube + 2mL enzyme

Incubate both (37C

water bath) for 10mins.;
mix, take 3 drops + 2
drops iodine solution.

Repeat steps until

light yellow-colored
solution
is
observed. Note the

After
1
min.
(continue
incubation), repeat earlier
step onto the next well (note

Repeat steps for other

pH (5, 6.7, 8, 10) to
obtain other results

RESULTS
EFFECT OF TEMPERATURE

Reciprocal Time
0.15
0.1
0.05
0
4C 37C50C60C70C
Reciprocal Time
Figure 1. Reciprocal time of enzymatic activity at various temperatures

Table 1. Summary of results of the effect of temperature on enzymatic activity of amylase

A. 4C (NVR)
50C (Rxn. at 9mins)

B. 37C (Rxn. at 11mins

C.

D. 60C (Rxn. at 11mins)

RESULTS:
EFFECT OF pH

E. 70C (Rxn. at 11mins)

Reciprocal Time
0.15
0.1
0.05
0
5

6.7

10

Reciprocal Time
Figure 7. Reciprocal time of enzymatic activity at various pH levels

Table 2. Summary of results of the effect of pH on enzymatic activity of amylase

A. pH 5 (Rxn at 8 mins)

B. pH 6.7 (Rxn at 15 mins)

C. at pH 8 (NVR)
D. pH 10 (NVR)