Salivary Amylase

Armin Purificacion, Rina Ramas, Marianne Sayo, Cristina Zurbano*

Department of Biological Sciences, College of Science
University of Santo Tomas, España Street, Manila 1008

Date Submitted: December 12, 2008

Abstract:
In this experiment, the enzymatic activity of the salivary amylase was examined depending on the
changes in pH and temperature. The solution was incubated in an ice bath and in water bath with different
temperatures and iodine test was performed. Also different buffers were used. A light yellow-colored solution
was observed. After observing the results, the reciprocal of time versus pH and temperature were plotted.
The optimum pH and optimum temperature was also noted.

I. Introduction

Enzymes are very efficient catalysts for biochemical reactions inside the body. They
speed up reactions by providing an alternative reaction pathway of lower activation
energy. Since enzymes do not undergo permanent changes by the alternative reaction
pathway, they remain unchanged at the end of the reaction. Most chemical catalysts
catalyze a wide range of reactions so they are not usually very selective however,
enzymes are usually highly selective, catalyzing specific reactions only due to the
different shapes of the enzyme molecules. Many enzymes consist of a protein and a
non-protein known as co-factors. The proteins in enzymes are usually globular. The
intra- and intermolecular bonds that hold proteins in their secondary and tertiary
structures are disrupted by changes in temperature and pH. This affects shapes and so
the catalytic activity of an enzyme is pH and temperature sensitive. Common cofactors
are the prosthetic groups that are permanently bound to the enzyme, the activators that
are cations which temporarily bind to the active site of the enzyme giving an intense
positive charge to the enzyme's protein, and the coenzymes which are the organic
molecules like vitamins which are not permanently bound to the enzyme molecule, but
combine with the enzyme-substrate complex temporarily. In order for an enzyme to
work, the two molecules that will react must collide with one another in the proper
orientation and with sufficient energy. This sufficient energy, commonly known as
activation energy, means that they have enough energy to overcome the energy barrier
to the reaction. A common enzyme that will be used in this experiment is the salivary
amylase that is found in human saliva. Its ability is to hydrolyze or break down starch
molecules to maltose molecules when the pH is almost neutral.
Amylase is a calcium dependent enzyme which hydrolyzes complex carbohydrates
at alpha 1,4-linkages to form maltose and glucose. Amylase is filtered by renal tubules
and resorbed (inactivated) by tubular epithelium. Active enzyme does not appear in
urine. Salivary amylase, a major component of human salivary secretions, possesses
multiple functions in the oral cavity. It is the only enzyme in the saliva capable of
degrading oligosaccharides, which are used by the oral microflora for nutritional
purposes. The structural neighbor, b-amylase, breaks down starch during the
germination of seeds (rich in starch) into sugars. These sugars constitute the chief
energy source in the early development of the plant. b-Amylase is able to break down
the a-1,4 linkages of starch polymers in plants and seeds. The pancreas has the highest
amylase concentration and largest total amount of amylase of any organ in the body.
The objectives for this experiment is to determine the enzymatic activity of salivary
amylase by its effects on pH and temperature and to know the optimum activity
observed by the enzyme from factors such as the narrow range of pH values and the
different temperatures.

II. Materials

The materials used were enzyme solution (1 mL saliva + 9 mL distilled H20 + 30 mL

0.5% NaCl), buffered starch (1% starch in phosphate buffer pH 6.7), 2% unbuffered
starch, 0.001 M iodine solution, acetate buffer solutions with (pH 4 and 5), phosphate
buffer solutions (pH 6.7 and 8) and bicarbonate buffer (pH 10), spot plate, test tubes,
medicine droppers and water bath.

III. Methodology

A. Effect of Temperature

For the effect of temperature on salivary amylase, two milliliters of the enzyme
solution was placed in a test tube and was labeled as four degrees Celsius. In another
test tube, two milliliters of the buffered starch solution was added; then, both of the test
tubes were simultaneously immersed to incubate for 10 minutes in an ice bath at 4
degrees Celsius. The two test tubes were immediately mixed once it reached at the said
time. After that, three drops of the mixture was taken quickly and two drops of the iodine
solution was simultaneously added onto the first well of the spot plate. This first well was
labeled as the zero minute. The same process was repeated after a one-minute interval
until the physical appearance of the solution changed from a violet solution to a light
yellow-colored or colorless solution. The time for this reaction was recorded
After the procedure was repeated following the desired incubation temperature
for the other given temperatures. A graph was made by indicating the temperature as the
x-axis and the reciprocal of time as the y-axis. The optimum pH was determined as well.

B. Effect of pH.

For the effect of pH on salivary amylase, 1 mL of acetate buffer, depending on

the pH, was mixed to 1 mL 2% unbuffered starch in one test tube. In another test tube, 2
mL of the enzyme solution was added. Afterwards, both test tubes were incubated in a
37 degrees Celsius water bath for ten minutes and then were immediately mixed. Three-
drops of the mixture were taken quickly and two drops of the iodine solution was
simultaneously added to the first well on the spot plate labeled as the zero minute.
Again, this process was repeated after a one-minute interval until the physical
appearance had the same observations as that on the effect of temperature.
The procedure was repeated for the other pH (5, 6.7, 8, and 10) using the
appropriate buffer and then a plot were made indicating the pH as the x-axis and the
reciprocal of time as the y-axis. The optimum pH was determined as well.

IV. Data and Results

pH t (in minutes) 1/t (min-1)

4 ∞ 0
5 16 0.063
6.7 6 0.17
8 5 0.20
10 13 0.077
Table 1. Effect of pH
 Effect of pH

Reciprocal of Time 0.8

0.6

0.4

0.2

0
0 2 4 6 8 10 12
pH value

Figure 1. Plot of reciprocal of time versus pH value

Temperature (ºC) t (in minutes) 1/t (min -1)

4° ∞ 0
30° 3 0.33
37° 2 0.50
50° 16 0.063
70° ∞ 0
Table 2. Effect of temperature

Effect of Temperature

0.6
0.5
Reciprocal of Time

0.4
0.3
0.2

0.1
0
0 10 20 30 40 50 60 70 80
Temperature in Celsius

Figure 2. Plot of reciprocal of time versus temperature

V. Discussion
 The enzyme amylase will catalyze the hydrolysis of starch to maltose when the pH is
near 7.0. The iodine serves as an indicator for the presence of starch. The iodine will
react with starch to form a brown color. As the amylase breaks down starch, lesser
starch will be present and the color of the solution will be lighter (light yellow-colored
solution).
α – Amylase contains no coenzymes, but as a calcium metalloenzyme, it requires at
least one gram of calcium per mole of enzyme in order to maintain its activity. This
calcium stabilizes the enzyme against heat denaturation and attack from proteases.
Moreover, the action of amylase on starch is quicker in the presence of chloride ions.
Chloride ions serve as activators that make the enzyme-substrate complex (amylase-
starch complex) from more easily by reducing the activation energy.
Each enzyme works within quite a small pH range. The pH value at which the
enzyme’s activity is maximal is called the optimum pH. At optimum pH, the active site is
at its optimum conformation. If the pH becomes alkaline that the group loses its proton,
the enzyme’s activity may be depressed. In addition, substrates may also be affected.
Also the changes in ionizable groups may change the tertiary structure of the enzyme.
The changes in pH can make and break intra- and intermolecular bonds, changing the
shape of the enzyme and therefore its effectiveness Enzymes may also undergo
changes in conformation when the pH is varied. As the charge group is changed, the
protein may dissociate into protomers resulting to loss of activity. The changes in pH
may not only affect the shape of an enzyme but it may also change the shape or charge
properties of the substrate so that either the substrate cannot bind to the active site or it
cannot undergo catalysis. The pH can affect the enzyme activity by changing the
structure or by changing the charge on a residue functional in substrate binding or
catalysis.
All chemical reactions are affected by temperature. Table 2 shows that the reaction rate
initially increases as temperature rises owing to increased kinetic energy of the reacting
molecule. Each enzyme has an optimum temperature at which it operates at maximal
efficiency. This optimal temperature is usually around human body temperature (37.5°C)
for the enzymes in human cells. If the temperature is raised beyond the optimum
temperature, the activity of many enzymes declines abruptly.

VI. Conclusion
 By means of the said procedures, the group was able to determine the enzymatic
activity and specificity of the salivary amylase. The activity of enzymes is strongly
affected by changes in pH and temperature. Each enzyme works best at a certain pH
and temperature. In addition to temperature and pH there are still other factors which
can affect the enzymatic reaction. Each of these physical and chemical parameters must
be considered and optimized in order for an enzymatic reaction to be accurate.

VII. References

[1] Bettelheim, Frederick A. & Jerry March. Introduction to Organic and Biochemistry.
Saunders College Publishing. 1990. Pages 255-256.
[2] Mckee, Trudy & James R. McKee. Biochemistry: The Molecular Basis of Life, 3rd ed.
McGraw-Hill, New York. 2003. Page 185.
[3] Boyer, Rodney. Concepts in Biochemistry. Brooks/Cole Publishing Company. 1999.
Pages 152-154.
[4] Murray, Robert K., Daryl K. Granner. Peter A. Mayes & Victor W. Rodwell. Harper’s
Biochemistry, 23rd ed. Appleton & Lange. 1993. Pages 76-78.
[5] http://www.woodrow.org/teachers/chemistry/institutes/1988/starch.html. Dec. 10,
2008.
[6] http://www.greatvistachemicals.com/biochemicals/amylase.html. Dec. 10, 2008.
[7] http://www.rsc.org/education/teachers/learnnet/cfb/enzymes.htm. Dec. 10, 2008.
[8] http://www.worthington-biochem.com/introBiochem/tempEffects.html. Dec. 11, 2008.
[8] Uhlig, Helmut & Elfriede M. Linsmaier-Bednar. Industrial Enzymes and Their
Applications.
http://books.google.com.ph/books?id=coenzyme+and+cofactors+of+amylase&source=w
eb&ots=rZ29xWoCr&sig=hcblKYpWVI_ZzPGI_TudMlvKP2M&hl=en&sa=X&oi=book_res
ult&resnum=2&ct=result. December 12, 2008.