Effect of temperature and pH on the enzymatic activity of salivary amylase
Gae Khalil Rodillas, Nonia Carla Ysabel Samson, Raphael Jaime Santos* and Brylle Tabora
Department of Biological Sciences, College of Science, University of Santo Tomas, Manila,
Philippines
Salivary amylase, found in human saliva, is an enzyme used to hydrolyze starch molecules. Its
enzymatic activity is affected by several factors, such as temperature and pH. The rates of enzymatic
activity of salivary amylase in different temperatures and pH were measured. Optimum temperature for
the enzymatic activity of salivary amylase ranges from 32°C to 37°C and its optimum pH ranges from 6 to
7. A graph of the time reciprocal against temperature and pH both produced bell-shaped curves.
I.
Introduction
An enzyme is a protein molecule that is a biological catalyst with three characteristics.
First, the basic function of an enzyme is to increase the rate of a reaction. Second, most
enzymes act specifically with only one reactant, called a substrate, to produce products. The
third and most remarkable characteristic is that enzymes are regulated from a state of low
activity to high activity and vice versa [1]. The activity of enzymes is strongly affected by
changes in pH and temperature. Each enzyme works best at a certain pH and temperature, its
activity decreasing at values above and below that point due to denaturation. For enzymes,
denaturation can be defined as the loss of enough structure rendering the enzyme inactive. This
is not surprising considering the importance of tertiary structure in enzyme function and noncovalent forces in determining the shape of enzymes [2].
Salivary amylase is the enzyme produced by the salivary glands. Formerly known as
ptyalin, it breaks down starch into maltose and isomaltose. Amylase, like other enzymes, works
as a catalyst. All catalysts are enzymes, but not all enzymes are catalysts. A catalyst is a
substance that hastens a chemical reaction but does not become part of the end product.
Amylase digests starch by catalyzing hydrolysis, which is splitting by the addition of a water
molecule. The presence and absence of starch can be confirmed by several tests such as the
iodine test, Benedict’s and Fehling’s test. In general, a blue-black color indicates the presence
of starch [3].
The objectives of this experiment are to examine the enzymatic activity and specificity of
salivary amylase depending on changes temperature and pH. This experiment also aims to
determine the narrow range of temperature and pH values at which salivary amylase exhibits its
optimum activity.
II.
Materials and Methodology
A. Effect of Temperature
An enzyme solution was prepared by mixing one mL saliva with nine mL distilled water and
thirty mL 0.5% sodium chloride. Two mL of the enzyme solution was put in a large test tube and
labeled as 4°C. Two mL of the buffered starch solution (1% stach in phosphate buffer pH 6.7)
was added in a separate large test tube. Both the test tubes were incubated for ten minutes in
an ice bath (4°C). The solutions were immediately mixed. Three drops of the mixture was taken
quickly and two drops of the 0.001 M iodine solution was added simultaneously onto a spot
plate (first well). This was the zero minute. After one minute interval (incubation continued),
three drops of the mixture was taken again and two drops of the iodine solution was added
simultaneously onto the second well. This was the one minute. Step 5 was repeated until a light
yellow-colored solution was observed. The time (t) was noted. For the other temperatures (room
temperature, 37, 50 & 70 °C), steps 1 to 6 were repeated following the desired incubation
temperature. The reciprocal of time (1/time, min-1) in step 6 versus the temperature (T) was
plotted. The optimum temperature of the amylase was determined.
B. Effect of pH
One mL of acetate buffer (pH 4) and one mL 2% unbuffered starch were mixed in a large
test tube. Two mL of the enzyme solution was added in a separate large test tube. Both the test
tubes were incubated for ten minutes in a 37°C water bath. The solutions were immediately
mixed. Three drops of the mixture was taken quickly and two drops of the 0.001 M iodine
solution was added simultaneously onto a spot plate (first well). This was the zero minute. After
one minute interval (incubation continued), three drops of the mixture was taken again and two
drops of the iodine solution was added simultaneously onto the second well. This was the one
minute. Step 5 was repeated until a light yellow-colored solution was observed. The time (t) was
noted. For the other pH (5, 6.7, 8 & 10), steps 1 to 6 were repeated using the appropriate buffer.
Acetate buffer solution for pH 5, phosphate buffer solution for pH 6.7 and 8, and bicarbonate
buffer for pH 10 were used. The reciprocal of time (1/time, min-1) in step 6 versus the buffer pH
was plotted. The optimum pH of the amylase was determined.
III.
Results and Discussion
The effect of temperature and pH on the enzymatic activity of salivary amylase was
determined by measuring the rates of reaction in varying temperatures and pH. The 0.5% NaCl
added in the enzyme solution activates the salivary amylase to perform its function – to
hydrolyze starch. 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 blue-black color to a light yellowcolored solution.
A. Effect of Temperature
Each enzyme has an optimum temperature at which it performs best. Below or above this
temperature, the enzyme loses its functionality. Table 1 shows the results obtained on how
enzyme activity of salivary amylase is affected by temperature.
Table 1. Results for the effect of temperature on salivary amylase activity
Temperature (T) °C
4
Room temperature, 32
37
50
70
time (t) min
1.00
11.0
6.00
1/t (min-1)
0
1.000
0.091
0.167
0
Extreme temperatures cause the native folded structure of proteins to uncoil into random
configuration. As a result, the protein loses its biological enzymatic activity. This denaturization
consequently leads to loss of activity. Figure 1 shows the graph of the reciprocal of time against
temperature based on the data from Table 1.
1.2
1/time (min-1)
1
0.8
0.6
0.4
0.2
0
0
10
20
30
40
50
60
70
80
temperature (°C)
Figure 1. Plot of the reciprocal of time against temperature for the
enzymatic activity of salivary amylase
The graph produced a bell-shaped curve with the highest peak indicating the optimum
temperature for enzymatic activity. At 4°C, enzymatic reaction of salivary amylase occurs slowly
or not at all due to lack of energy and heat. As the temperature increases, its enzymatic also
increases up until the optimum temperature. Figure 1 shows that the optimum temperature of
salivary amylase ranges from 32°C to 37°C.This applies to the human body since salivary
amylase is suitable to function within these temperatures. After 37°C, the graph then steeply
declines as a result of loss of activity. At 50°C and 70°C, salivary amylase is denatured. The
molecular conformation of the enzyme becomes altered as the hydrogen bonds responsible for
its secondary, tertiary and quaternary structures are broken [4].
B. Effect of pH
Most enzymes are active only over a narrow pH range and have an optimal pH, at which
reaction is the fastest. An increase or decrease in pH also causes denaturation in enzymes,
thereby affecting their activity. Table 2 shows the results obtained on how enzyme activity of
salivary amylase is affected by pH.
Table 2. Results for the effect of pH on salivary amylase activity
pH
4
5
6.7
8
10
time (t) min
11.0
19.0
10.0
6.00
1/t (min-1)
0
0.091
0.053
0.100
0.167
Figure 2 shows the graph of the reciprocal of time against 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.
1/time (min-1)
0.2
0.15
0.1
0.05
0
0
2
4
6
8
10
12
pH
Figure 2. Plot of the reciprocal of time against pH for the enzymatic
activity of salivary amylase
At pH 4, 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 (Talwar & Srivastava, 2006). This is
consistent with the peak found between pH 4 and 6 in Figure 2. However, the curved peaked
highest at pH 10. Inconsistencies with the results obtained can be attributed to human error
such as inaccuracies in measurement and timing during the experiment. Ideally at pH 10,
salivary amylase is denatured due to high alkalinity. As pH increases, certain amino acids such
as lysine and arginine are deprotonated, causing them to lose their net positive charge which
also results to enzyme denaturation.
The activity of enzymes may be markedly changed by any alteration in pH, which in turn,
alters electrical charges on the enzyme. Changes in charge affect the ionic bonds that
contribute to the enzymes tertiary and quaternary structure, thereby changing the proteins
conformation and activity. Thus, pH-activity relationship of enzymes is dependent on the amino
acid side chains present in the enzyme [5].
IV.
Conclusion
Several factors affect the activity of enzymes. Among these are the temperature and pH.
At optimum levels of these factors, enzymes perform their function best. Optimum temperature
and pH differ from one enzyme to another. Salivary amylase is an enzyme found in human
saliva which functions to break down starch to simpler compounds. Through the experiment, it
was found out that the optimum temperature of salivary amylase ranges from 32°C to 37°C and
its optimum pH ranges from 5.6 to 6.9.
Inconsistencies of the results obtained with literature on the topic were met. These errors
can be attributed to inaccuracies in measurement and timing. Due to time constraints, the
experiment was not repeated to be able to compare and obtain more accurate results.
V.
References
[1] Role of Enzymes in Biochemical Reactions. (2003). Retrieved on January 3, 2012
from http://www.elmhurst.edu/~chm/vchembook/570enzymes.html
[2] Enzymes. (2011). Retrieved on January 3, 2012 from
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/E/Enzymes.html#pHandTemp
[3] Sethi, R. (2009). Biology. Rachna Sagar Pvt. Ltd.: New Delhi
[4] Solomon E., Berg, L. & Martin, D. (2005). Biology. Thomson Learning Inc.: USA
[5] Talwar G.P. & Srivastava L.M. (2006). Textbook of biochemistry and human biology,
3rd Ed. Prentice-Hall:New Delhi