Determination of Enzymatic Activity of Salivary Amylase Depending

on the Effect of Temperature and pH

Cuan, Lindane Camille

Dalupang, Ma. Audrey Jannie R.
*Dones, Maria Ronalee
Enriquez, Paula Camille
College of Science, University of Santo Tomas, Espaa Blvd., Manila

Abstract
Salivary amylase is an enzyme that breaks chemical bonds called alpha-bonds that occur in between long
chains of glucose molecules in complex starches. The effect of temperature and pH on the activity of
salivary amylase on starch can be studied by using the Iodine test. The optimum temperature for the
enzymatic activity of salivary amylase ranges from 32 C to 37 C while the optimum pH ranges from 6 to
7. Graphs for the temperature/pH versus the reciprocal of time was plotted and resulted in a bell shaped
curve with the peak indicating the optimum temperature/pH. Ranges below and above this optimum
reduces reaction rate and denatures enzyme.

Introduction
Enzymes are proteins that have catalytic functions indispensable to
maintenance and activity of life. All chemical reactions occurring in a living
organism are dependent on the catalytic actions of enzymes. At present, there
are

about

4,000

kinds

of

enzymes

whose

actions

are

well

known

(amrita.olabs.edu.in, 2015). Enzymes function in a mild environment similar to

the body environment of a living organism. They support life by synthesizing and
degrading materials that constitute the building blocks of the organism and by
creating energy. A specific enzyme used in this experiment is salivary amylase.

It catalyzes the hydrolysis of starch into sugars and is present in the saliva of
humans and some other mammals, where it begins the chemical process of
digestion (Brooker, Widmaier, Graham & Stiling, 2011). Not all amylase works at
the same rate. This is called the activity of the enzyme. The activity of an
enzyme is affected by its environmental conditions, including temperature and
pH. Changing these alter the rate of reaction caused by the enzyme (Brooker et.
al, 2011). In nature, organisms adjust the conditions of their enzymes to produce
an optimum rate of reaction or they may have enzymes which are adapted to
function well in extreme conditions where they live (Talamond, Noirot & de
Kochko, 2005).
All enzymes speed up a reaction to a time scale useful for our bodies.
Starch is a long biopolymer made from thousands of repeating glucose units.
When these glucose units are attached in a straight line, the type of starch is
called amylose. When the units are branched, the type of starch is called
amylopectin (Talamond et. al, 2005). Starch can be broken down over thousands
of years without the aid of other chemicals, or it can be broken down in less than
an hour with the enzyme amylase. Several types of salivary glands in the mouth
produce and secrete a digestive enzyme called salivary amylase. This enzyme is
known as an alpha-amylase. It can break chemical bonds called alpha-bonds
that occur in between long chains of glucose molecules in complex starches. The
main purpose of salivary amylase is to partially digest starches into shorter
chains of glucose called polysaccharides, as well as a disaccharide consisting of
two glucose molecules called maltose (amrita.olabs.edu.in, 2015).

The objectives of this experiment are to examine the enzymatic activity and
specificity of salivary amylase depending on the changes in temperature and pH.
It also aims to determine the narrow range of pH values and different
temperature at which salivary amylase exhibits its optimum activity.

Methodology
A. Effect of Temperature
An enzyme solution was prepared by combining one ml of saliva, nine ml
of distilled water and 30 ml of 0.5% NaCl solution. Two ml of this enzyme solution
was placed in five large test tubes and was labeled 4C. Two ml of the buffered
starch solution containing a 1% starch in phosphate buffer pH 6.7 was placed in
a separate large test tube. Both of the test tubes were incubated at the indicated
temperature for each group for 10 minutes. After 10 minutes, the two solutions
were immediately mixed and three drops of the mixture was quickly taken and
was placed onto a spot plate, simultaneous with an iodine solution. This indicates
the zero minute of the experiment. After a one-minute interval with continued
incubation, three drops of the mixture were again taken simultaneously with the
two drops of iodine solution and was placed onto the second well. This procedure
was repeated until a light-yellow colored solution was observed and the time for
each interval was noted. The same procedure was done for room temperature
(RT), 37C, 60C and 70C. The reciprocal of time (1/time, min -1) versus the
temperature (T) was plotted and the optimum temperature of the amylase was
determined.

B. Effect of pH
One ml of an acetate buffer with pH 4 was mixed with a one ml of 2%
unbuffered starch in a large test tube. Two ml of the enzyme solution was placed
in a separate large test tube. Both test tubes were incubated for 10 minutes at
room temperature. After 10 minutes, the two solutions were immediately mixed
and three drops of the mixture was quickly taken and was placed onto a spot
plate, with an added two drops of iodine solution. This indicates the zero minute.
After a one-minute interval with continued incubation of the solutions, another
three drops of the mixture were taken and it was simultaneously added with
another two drops of iodine solution onto the seconds well of the spot plate. This
indicates the one minute. The time for each interval was noted and the previous
steps were repeated until a light yellow-colored solution was observed. The same
procedure was done for acetate buffer solution with pH 5, 6.7, 8 and 10. The
reciprocal of time (1/time, min-1) versus the buffer pH was plotted and the
optimum temperature of the amylase was determined.
Results and Discussion
Enzymes are affected by changes in temperature and pH. The effect of
these two components in the enzymatic activity of salivary amylase was
determined by the rates of reaction measured in varying temperatures and pH. In
the enzyme solution, 0.5% NaCl was added because salivary amylase, a
chloride-dependent enzyme, requires the binding of a chloride ion to be
allosterically activated. This would now enable the salivary amylase to hydrolyze
starch.

The effect of temperature and pH on the activity of salivary amylase on

starch can be studied by using the Iodine test. If salivary amylase is added on
starch, the salivary amylase present gradually acts on starch and converts it into
maltose.

Starch keeps on giving blue colour with iodine till it is completely

digested into maltose. At this point, a yellow-colored solution is formed.

The hydrolysis or breakdown of starch due to the action of salivary
amylase is indicated by the change in color of the starch solution from a blueblack color to a light yellow-colored solution.
A. Effect of Temperature
A buffered starch solution was used to maintain the enzyme environment
with pH 6.7. Buffers stabilizes the hydrogen ion concentration by neutralizing the
added acid or alkali. Phosphate Buffer (Sorenson's buffer) have a pH value
ranging from 5.8-8. It is the most physiological of common buffers and mimics
certain components of extracellular fluids. It is used in the experiment under the
determination of the effect of temperature since its pH will have minimal changes
with varying temperature.
Incubating the solution 10 minutes before mixing was also necessary to
properly introduce the temperature to the enzyme solution and buffered starch
solution.
On Table 1, the results obtained in how varying temperature affects the
enzymatic activity of salivary amylase was shown.

Table 1. Results for the effect of temperature on salivary amylase activity

Temperature (C)

Time (min)

4
Room temperature (32.8)
37
60
70

12
1
1
5

1
t (min)
0.083
1
1
0.20
0

(min)

Figure 1. Reciprocal of time against temperature for the enzymatic activity of salivary amyl

Temperature (C)
Figure 1 shows the graph of the reciprocal of time against temperature based on
the data from Table 1.

The graph formed a bell-shaped curve with the highest peak indicating the
optimum temperature for enzymatic activity. This is the temperature at 32C and
37C produced both at one-minute reciprocal of time of reaction. Optimum
temperature for the enzymatic activity of salivary amylase ranges from 32 C to
37 C. This means that the temperature at which the enzyme shows the
maximum activity. At this optimum temperature, the enzyme is most active and
hence, takes less time to digest the starch. At a lower temperature, the enzyme
salivary amylase is deactivated which is shown at 4C with 0.083-minute
reciprocal of time of reaction. Enzymatic reaction of salivary amylase at this rate
occurs slowly or not at all due to lack of energy and heat. Increasing temperature
increases the kinetic energy that molecules possess. Its enzymatic activity also
increases up until the optimum temperature. This was shown in the 60C with
0.20 minute and 70C with a 0-minute reciprocal of time of reaction indicating an
infinite time since the starch was still not hydrolyzed after the 20 th minute of
interval. In a fluid, this means that there are more random collisions between
molecules per unit time. Since enzymes catalyze reactions by randomly colliding
with substrate molecules, increasing temperature increases the rate of reaction,
forming more product. However, increasing temperature also increases the

Vibrational Energy that molecules have, specifically in this case enzyme

molecules, which puts strain on the bonds that hold them together. As
temperature increases, more bonds, especially the weaker Hydrogen and Ionic
bonds, will break as a result of this strain. Breaking bonds within the enzyme will
cause the active site to change shape. This change in shape means that the
active site is less complementary to the shape of the substrate, so that it is less
likely to catalyze the reaction. Eventually, the enzyme will become denatured and
will no longer function.
Therefore, as temperature increases, the rate of reaction will increase,
because of increased kinetic energy. However, the effect of bond breaking will
become greater and the rate of reaction will begin to decrease.
B. Effect of pH
pH changes in buffered and unbuffered solutions The unbuffered starch
solution was used to examine the effect of pH since unbuffered solution changes
quickly when acid or base is added. This will attain the desired pH for each
reaction. Acetate buffer is often used as a buffer solution for protein studies at
acidic pH (from 3.6 to 5.6). Acetate buffers are used in biochemical studies of
enzymes and other chemical components of cells to prevent pH changes that
might change the biochemical activity of these compounds.
Incubating the solution 10 minutes before mixing was also necessary to
properly introduce the pH to the enzyme solution and unbuffered starch solution.
On Table 2, the results obtained in how varying pH affects the enzymatic
activity of salivary amylase was shown.

Table 2. Results for the effect of pH on salivary amylase activity

pH

Time (min)

1
t (min)

4
5
6.7
8
10

(infinite)
12
3
4
14

0
0.0833
0.33
0.25
0.07

Figure 2 shows the graph of the reciprocal of time against temperature

based on the data from Table 2.
The graph formed an approximately bell-shaped curve with the highest
peak indicating the optimum pH for enzymatic activity. This is the pH at 6.7 with
three minutes reaction time. The optimum pH for the enzymatic activity of salivary
amylase ranges from 6 to 7. At the optimum pH, the rate of reaction is at an
optimum. This means that the bonds within them are influenced by H+ and OHIons in such a way that the shape of their active site is the most complementary
to the shape of their substrate thus making the enzyme most active and takes
less time to digest the starch. Any change in pH above or below the optimum will
quickly cause a decrease in the rate of reaction, since more of the enzyme

molecules will have active sites whose shapes are not complementary to the
shape of their substrate. Below the optimum pH was shown in the rate with lower
pH value indicated by pH 4 with a zero reciprocal of time denoting an infinity
since the starch was still not hydrolyzed after the 20 th minute of interval and pH 5
with a 0.0833-minute reciprocal of time. While above the optimum pH was
exhibited by the pH 8 and 10 with 0.25 and 0.07 minutes reciprocal of time,
respectively. In reactions below and above the optimum pH, H+ and OH- ions are
charged and therefore interfere with Hydrogen and Ionic bonds that hold together
an enzyme, since they will be attracted or repelled by the charges created by the
bonds. This interference causes a change in shape of the enzyme, and
importantly, its active site. And these changes in pH can cause enzymes to
denature and permanently lose their function.
Conclusion
All enzymes are proteinaceous in nature. At a lower temperature, the
enzyme salivary amylase is deactivated and at the higher temperature, the
enzyme is denatured. Therefore, more time will be taken by an enzyme to digest
the starch at lower and higher temperatures. Optimum temperature for the
enzymatic activity of salivary amylase ranges from 32 C to 37 C. At optimum
temperature, the enzyme is most active and hence, takes less time to digest the
starch. Change in pH can affect the shape of an enzyme and the shape of the
substrate the enzyme reacts with. If this happens, the substrate may not be able
to bind to the active site or undergo catalysis reaction. The optimum pH for the

enzymatic activity of salivary amylase ranges from 6 to 7. Below this range, the
reaction rate reduces and above this rate the enzymes gets denatured.

References
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