The Effects of pH and temperature on the enzymatic activity of

Salivary Amylase
Balmes, Angela Rose M.
*Basilio, Moira C.
Borlongan, Gedric Lenar Y.
College of Science, University of Santo Tomas, Espaa Blvd., Manila
Abstract
Salivary amylase is found in human saliva. It is an enzyme used to hydrolyze the starch molecules. The
enzymatic activity can be affected by different factors, such factors are by the temperature and pH. The
rates of the enzymatic activity of salivary amylase in different pH and temperatures were measured. The
optimum temperature for the salivary amylase range is from 32C to 37C. While the optimum pH range is
from 5.6 to 6.9. Two graphs were made that includes the time reciprocal against the pH and temperature
that both produced bell-like shape curves.

I.

Introduction
Enzyme is a catalyst for a biological reaction. Usually a protein molecule,
but some enzymes are RNA molecules or ribonucleic proteins. Enzymes has three
characteristics, the main function of enzymes serves as catalysts that increases the rate
or velocity of a chemical reaction without itself being changed in the overall process.
The second is that the substance that is acted on by an enzyme is called the substrate
of that enzyme. Lastly the rate of enzymatic activity can either be regulated from state of
low activity and high activity or vice versa (Appling, Cahill & Matthews, 2016). The
activity of enzymes are affected by different factors such as temperature and pH that

was used in the experiment. Each enzymes works best at certain temperature and pH.
The values can be lower than the activity or higher to the point of denaturation.
Denaturation for enzymes are involved in the disruption and possible destruction of both
the secondary and tertiary structures (Ophardt, 2003).
Alpha-amylase is the most abundant enzyme of the saliva of man. Salivary
amylase is mainly formed in the parotid gland. Its activity of high inter-individual and
intra-individualistic variability is also visible (Boehlke, Zierau & Hannig, 2015). Amylase,
like other enzymes works as a catalyst. Catalyst is a substance that can increase the
rate of reaction but do not become as the end product. Amylase digests starch via
catalyzing hydrolysis, which is the splitting by addition of water molecule (Sethi, 2009).
The use of buffered and unbuffered starch were used in the effect of temperature and
effect pf pH. In the effect of temperature a buffer was used. Buffer solutions resists
changes in pH when small quantities of an acid or an alkali are added. The use of a
buffer is needed because it controls and maintains the equilibrium of the enzymes even
if at high temperatures and do not change the pH. While an unbuffered solution was
used in the effect of pH because we need to test the different pH, so its not possible to
use a buffered one that can resist changes in pH (Clark, 2016).
The objectives of the study is to examine the enzymatic activity and specificity of
salivary amylase depending on the changes in pH and temperature. Also to do the tests
for the factors such as narrowing the range of pH values and different temperatures at
which the enzyme exhibits its optimum activity.
II.

Methodology
A. Effect of Temperature

2 mL of the enzyme solution was placed in a large test tube and was
labeled 4C. In a separate large test tube, 2 mL of the buffered starch solution was
added. Both test tubes were then incubated in an ice bath (maintained 4C) for ten
minutes. They were mixed immediately. Three drops of the mixture was then taken and
was put onto a spot plate together with the four drops of Iodine solution. It was labeled
as the zero minute.
After a one minute interval, three drops of the mixture was put onto the next well
of the spot plate together with four drops of Iodine solution. It was labeled as one
minute. This was repeated until a light yellow colored solution was observed. Time (t)
was noted. The reciprocal of time (1/time, min-1) was plotted and the optimum
temperature of the amylase was determined.
B. Effect of pH
1 mL of the acetate buffer (pH 4) and I mL of 2% unbuffered starch were mixed in
a large test tube. In a separate large test tube, 2 mL of the enzyme solution was added.
Both test tubes were incubated in a 37C water bath for ten minutes. The solution was
mixed. Three drops of the mixture was quickly added onto a spot plate together with the
four drops of Iodine solution. It was labeled as the zero minute.
After a one minute interval, three drops of the mixture was put onto the next well
on the spot plate together with the four drops of Iodine solution. It was labeled as one
minute. This was repeated until a light yellow colored solution was observed. For the
other values of pH, appropriate buffer was used. Reciprocal of time was plotted versus
the buffer pH. The optimum pH for amylase was determined.

III.

Results and Discussion

The different effects of pH and temperature on the enzymatic activity of salivary
amylase was determined by measuring the rates of reaction of different values given in
temperature and pH. 0.5% NaCl solution was added in the enzyme solution to activate
the salivary amylase to function. In order for it to hydrolyze the starch. The indication of
the breakdown of starch is due to the reaction of salivary amylase that can be seen as a
blue-black coloration to light-yellow solution.
A. Effect of Temperature
Enzymes exhibits an optimum temperature at which it performs best. When the
temperature gradually increases of decreases there is a high chance that it will
denature. Table 1 shows the results obtained on how the temperature affect the
enzymatic activity of salivary amylase.
Table 1. Results of the effect of temperature on salivary amylase
Temperature (T) - C
4C
Room Temperature
37C
60C
70C

Time (t) - min

12
1
1
5

1/t (min -1)

0.083
1
1
0.20
0

Due to the extreme temperature, some proteins tend to uncoil causing them en a
protein denatures, it loses its biological activity. Figure 1 represents the graph of
temperature versus reciprocal of time.

12
10
8
1/t (min -1)

6
4
2
0
0

10

20

30

40

50

60

70

80

Temperature C

Figure 1. Plot of the temperature versus the reciprocal of time for the
enzymatic activity of salivary amylase
At 4C, the reaction of salivary amylase occurs slowly or maybe not at all due to
the lack of heat and energy. Whenever as the temperature rises, the enzymatic reaction
also increases up until the optimum temperature. The figure 1 shows that the range of
the optimum temperature of salivary amylase is from 32C to 37C. Since the salivary
amylase is from a human, it can applied that the human body is suitable to function
within these temperatures also. After the 37C, the graph steeply declines as the result
of the loss of activity. At 60C and 70C, the salivary amylase is denatured already. The
molecular conformation of enzymes becomes altered and changed as the hydrogen
bonds that are responsible for its secondary, tertiary, and quaternary structures are
broken.

Table 2. Results of the change in temperature at different environments

pH
4

Time (min)

1/t (min -1)

0

5
6.7
8
10

12
3
4
14

0.083
0.333
0.25
0.071

Figure 2 shows the graph of the reciprocal of time versus pH based on the data
from Table 2.The graph produced a bell-shaped curve and the highest peak should
indicate the optimum pH for enzymatic activity.

0.35
0.3
0.25
0.2
1/t (min -1)

0.15
0.1
0.05
0
3

10

11

pH
Fi
gure 2. Plot of the pH versus the reciprocal of time for the enzymatic activity
of salivary amylase
We found that different enzymes have different optimal pH values. At pH 4, the
salivary amylase is in a very acidic environment to function. As the pH continue to

decrease certain amino acids are protonated. That causes them to lose the net negative
charge which denatures the enzyme. The optimum pH for salivary amylase ranges from
5.6 to 6.9 (Talwar & Srivastava, 2006). This is evident in the graph within the pH 5 and
6.7 in the figure 2. The value of pH 8 exhibited as the highest peak in the graph. Also
the last value of pH is at 10, it should be inferred that in the values of pH 8 and 10, the
amylase should be denatured. The result should be infinity as the result of pH 4. It may
be said that some inconsistencies were met during the experiment due to timing and
measurement. At pH 10 the salivary amylase should be denatured. As the pH increases,
some amino acids are deprotonated causing them to lose the positive net charge that
results also to enzyme denaturation.
The enzymatic activity of enzymes may be markedly changed by alterations in pH
which in turn alters the charges on the enzyme. Changes in charge can affect the ionic
bonds that contributes to the tertiary and quaternary structures. Therefore changing the
conformations and activities. Moreover, pH-activity relationship of enzymes is
dependent in the amino acid side chains that are present in the enzyme (Caret,
Denniston & Topping, 1997).
IV.

Conclusion
Many factors can affect the enzymatic activities of different enzymes.
Temperature and pH was used in the experiment. At optimum levels, enzymes perform
and function at their best. The optimum temperature and optimum pH are different with
every enzymes. Salivary amylase found in humans are used to hydrolyze starch
molecules. Through the experiment, it was shown that the optimum temperature ranges
from 32C to 37C. While the optimum pH ranges from 5.6 to 6.9. As the optimum levels
are reached it can be inferred that an enzyme can perform its function at very good

conditions. However if the optimum levels increase or decrease exceedingly, enzymes

tend to denature and lose its biological activity.
In the experiment, some inconsistencies in the result occurred. In the values of
pH, the higher value of pH, the more it should be denatured and resulted into an infinity.
But in the experiment it was not fulfilled. Maybe it was because of the measurements
and timing.

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