The effect of Temperature and pH effect on the enzymatic activity of

salivary amylase
Group 2

Abstract

Enzymes are catalyst involved in chemical reactions and metabolism. Enzymes undergo enzymatic
activity wherein their substrate is converted into a product. One common enzyme known is amylase which
is also present in our saliva that mainly functions as initial digestion for food. Temperature and pH play a
significant role in the enzymatic activity of salivary amylase as temperature in lower temperature slower
enzymatic reaction while higher temperature speed up enzymatic reaction. pH involved in enzymatic
activity changes as it gets higher or lower to the optimum pH level of salivary amylase.

Introduction
Enzymes which are usually proteins are known to be a catalyst that speeds up the
rate of chemical reactions that happens within the cells. It serves as an aid for digestion
and metabolism that are vital for life. Enzymes also function in helping large molecules to
break into smaller pieces to be more easily absorbed by the body and sometimes bind
two molecules together to make up new molecules. (Castor, 2014). Unlike other catalysts,
enzymes catalyze a single reaction in an enzyme substrate. One good example of
enzyme is the salivary amylase which is found in human digestive tract. It is what the food
that we eat first encounter inside the mouth that plays a role in the initial digestion of
starch., it is released by the salivary gland and it the most abundant enzyme in the saliva.
(Mathew, Vazhacharickal, Jose, N K, 2017). Amylase catalyzes only the hydrolysis of
starch that yields to glucose while cellulose and other polysaccharides are unchanged.
(McMurry, 2010).
Enzymatic activity is defined as the rate of enzyme conversion of a substrate into
a product on a biochemical reaction. In catalyzation of reaction the substrate and enzyme
first need to bind onto an active site. An active site is the small three-dimensional portion
in the enzyme structure which is formed from different parts of protein chains by folding
and bending. (Stocker,2010). Another component involved in the experiment is the
presence of starch, Starch is known to be one of the most abundant organic compounds
and a very important biopolymer. (Marini,2006). Hydrolyzation of starch by amylase is
related to the iodine added, it reacts with the starch present in the amylase producing the
intense blue color and the more intense the color the more starch remains but the more
hydrolyzed starch the lighter the color the mixture gets.
In this experiment, salivary amylase enzymatic was investigated in different
conditions which are temperature and pH levels to determine its effect. As it is part of the
four possible factors that can affect enzymatic activities which also includes substrate
concentration and enzyme concentration.
 Methodology
In preparation for the experiment, an enzyme solution was prepared by adding 1
mL saliva to 9 mL distilled water and 30 mL 0.5 % sodium chloride. In a large test tube 2
mL of the enzyme solution was placed and 2 mL buffered solution was in a separate large
test tube. Then, both test tube was incubated in a beaker subjected to different
temperatures (4°C, RT, 24°C, 37°C,50°C,60°C,70°C) for 10 minutes. After 10 minutes,
the 2 mL enzyme solution and 2 mL buffered solution was quickly mixed together.
On a spot plate 3 drops of the mixture was dropped and 2 drops of iodine solution
was added, it was recorded as the zero minute. The mixture is incubated again for one
minute before putting 3 drops of the mixture onto the spot plate and adding 2 drops of
iodine solution, after it was recorded as the one minute. The process was recorded until
a light yellow-colored or colorless solution was observed.
In the experiment involving different pH level, 1mL of acetate buffer in pH
(4,5,6.7,8,10) and 1 mL 2% unbuffered starch was used. In a separate test tube 2 mL of
the enzyme solution was prepared in a large test tube. Both test tubes were incubated in
a water bath at a temperature of 37 °C. After 10 minutes, the 2 mL enzyme solution and
2 mL buffered solution was quickly mixed together.
On a spot plate 3 drops of the mixture was dropped and 2 drops of iodine solution
was added, it was recorded as the zero minute. The mixture is incubated again for one
minute before putting 3 drops of the mixture onto the spot plate and adding 2 drops of
iodine solution, after it was recorded as the one minute. The process was recorded until
a light yellow-colored or colorless solution was observed.
The results were recorded, and the reciprocal time was solved. Both results were
used to plot and determine the optimum temperature and pH.

Results and Discussion

A. Effect of Temperature
 Group No. Temperature No. of minutes 1/t
(°C)
1 4 20 0.05
2 24 5 0.2
3 37 2 0.5
4 50 2 0.5
5 60 3 0.3
6 70 ∞ 0

Chart Title
0.6

0.5

0.4
A (1/time)

0.3

0.2

0.1

0
20 40 60 80
Temperature

Temperature at 24 °C

Number of Minutes Color

0 Dark Blue
1 Purple
2 Red
3 Light Red
4 Light orange
5 Light yellow

Salivary amylase and other enzymes show characteristic change in activity when
exposed to different temperatures. Temperature is related to the kinetic energy of
molecules wherein temperature increases the molecules movement and colliding. This
can be applied to idea of collision between substrates and enzymes therefore increasing
the rate of reaction in high temperature. (Stocker, 2010). The experiment showed rate of
reactions of salivary amylase in reaction to iodine solution. The optimum temperature of
salivary amylase in this experiment is 37°C -50°C, in which the enzyme exhibits maximum
activity is observed. Salivary amylase has the characteristic to have an enzymatic
reaction that deactivates in lower temperature that are 24 °C room temperature and 4 °C
in an ice bath in the experiment. Lower temperature resulted a slower reaction rate
causing it to have longer minute before obtaining its optimum temperature at 5 minutes
and 20 minutes, respectively. Higher temperature beyond the optimum temperature of
37°C -50°C causes changes in the structure or denaturation. Denaturation is defined as
the reversable result of the unnatural alteration of the ionic state or conformation a protein
due to the loss of biological activity (Wade & Simek, 2017) and usually changes in the
tertiary structure that impedes some catalytic activities during denaturation. The salivary
amylase incubated in higher temperature 60°C -70°C showed slower or no reaction at all
after showing a blue color, since it was able to catalyze any of the starch present. High
temperature causes the denaturation of enzymes that causes it to be completely
destroyed.
B. Effects of pH level
Group No. pH No. of minutes 1/t
7 4 15 0.07
8 5 ∞ 0
9 6.7 3 0.33
10 8 5 0.2
11 10 6 0.17
 Chart Title
0.35

0.3

0.25
A (1/time)

0.2

0.15

0.1

0.05

0
2 4 5 6.7 10 12 14
pH

Another factor related to enzyme activity of salivary amylase is pH. Changes in pH is

change in the basicity or acidity of the amino acids that are present in the active site. The
different/changing pH can affect the protonation or deprotonation of the substrates and
cause enzyme denaturation. (Stocker, 2010). In the experiment, the optimum pH or the
pH where maximum activity of an enzyme is observed is at pH 6.7. The rate of reaction
of pH 6.7 yielded a significantly faster rate than the rest of pH. Changes in pH may it be
higher or lower changes the rate of catalysis. The extremes of the pH show significantly
changes in the catalytic rate of reaction of the enzyme as the charge within the molecule
changes, this causes denaturation or subsequent loss of catalytic activity for the enzyme.
Results of the experiment that the farther the pH change from the optimum pH the greater
the change in the rate of reaction the enzyme encounters.

Conclusion
Salivary amylase is affected by the change in temperature in which lower
temperature in comparison to the optimum temperature deactivates enzymatic activities
that lowers the rate of reaction and higher temperature results to denaturation that
destroys the enzyme present in the salivary amylase making reaction not possible. In the
pH levels involving salivary amylase it is observed that changes in the pH level may it be
lower or higher than the optimum pH creates a significant change that affects the structure
of the enzyme or denaturation of the enzyme which eventually cause it to lose enzymatic
activity to react to the starch present.
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