Biochemistry Laboratory Formal Report

Enzymatic Activity of Salivary

Amylase

CHEMISTR
Y

60
0L

John Felipe, Mariah Mae Fredeluces, Katrina Lee Gagan*, Nadia

Giliberte
Department of Biological Sciences, College of Science
Date submitted: 8 September 2015
*katrinalee_gagan@yahoo.com

Abstract
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 32C to 37C
and its optimum pH ranges from 6 to 7.

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. 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

Biochemistry Laboratory Formal Report

tertiary structure in enzyme function and noncovalent forces in determining the shape of
enzymes.
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, Benedicts and Fehlings test. In
general, a blue-black color indicates the presence of starch.
In the experiment, the enzymatic activity and specificity of salivary amylase was
examined depending on changes in pH and temperature. Factors such as narrow range
of pH values and different temperatures at which the enzyme exhibits its optimum
activity were done.
Results and discussion
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. The Table below shows the results
obtained on how enzyme activity of salivary amylase is affected by temperature.

Temp. C
4
25
37

Time (min.)

11 mins.
1 min.

1/t (min-1)
0
0.0909
1
2

Biochemistry Laboratory Formal Report

50
70

0
0

The graph below shows the reciprocal of time against Temperature based on the data
from Table above.

Chart Title
2

10

15

20

25

30

35

40

45

50

55

60

65

70

Column1

Plot of the reciprocal of time against temperature for the enzymatic activity
of salivary amylase

The graph produced a mountain like curve with the highest peak indicating the optimum
temperature for enzymatic activity. At 37C, 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. The graph shows that
the optimum temperature of salivary amylase was 37C.This applies to the human body
since salivary amylase is suitable to function within these temperatures. After 37C, the
graph then steeply declines as a result of loss of activity. At 50C and 70C, salivary
3

Biochemistry Laboratory Formal Report

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.

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. The table below shows the results obtained on
how enzyme activity of salivary amylase is affected by pH.

pH
4
6.7
8
10

Time (min.)

1 min.

1/t (min-1)
0
1 min.
0
0

The graph below shows the reciprocal of time against pH based on the data from Table
above.

Biochemistry Laboratory Formal Report

Chart Title
1.2
1
0.8
0.6
0.4
0.2
0

10

Series 3

Plot of the reciprocal of time against pH for the enzymatic activity of salivary amylase

The graph produced a mountain like curve with the highest peak indicating the optimum
pH for enzymatic activity at pH 6 the graph was at its highest peak. At pH 8 & 10,
salivary amylase is denatured due to high alkalinity which is ideal. 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.

Biochemistry Laboratory Formal Report

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 up to 37C and its optimum pH ranges from 5-6.
Experimental methodology
This procedure was for the determination of the optimum temperature of the amylase,
where we put 2 ml of the enzyme solution in a large test tube and labeled it as 4C.
Then in a separate large test tube we added 2 ml of the buffered starch solution and
incubated both test tubes for 10 mins. in an ice bath. Next, we immediately mixed and
quickly took three drops of the mixture and simultaneously added two drops of the
iodine solution onto a spot plate (first plate) which then was our zero minute. After one
minute interval, we took three drops again of the mixture and simultaneously added two
drops of the iodine solution onto the second plate which then was our one minute. We
repeated step 5 until a light yellow colored solution was observed and noted the time.
This procedure was for the determination of the optimum pH of the amylase, where we
mixed

ml

of

acetate

buffer

(pH

4)

and

ml

2%

un

buffered starch in a large test tube. Then in a separate large test tube we added 2 ml of
the enzyme solution. Next, we incubated both test tubes for 10 mins. in a 37C water
bath. Then, we immediately mixed and quickly took three drops of the mixture and

Biochemistry Laboratory Formal Report

simultaneously added two drops of the iodine solution onto a spot plate (first well) which
then was our zero minute. Lastly, after one minute interval, we took three drops again of
the mixture and simultaneously added two drops of the iodine solution onto the second
well which then was our one minute. We repeated the last step until a light yellow
colored solution was observed and noted the time.
References
(1)

Enzymes.

(2011).

Retrieved

on

Sept.

5,

2015

from

http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/E/Enzymes.html#pHan
dTemp
(2) Role of Enzymes in Biochemical Reactions. (2003). Retrieved on Sept.
5,2015 from http://www.elmhurst.edu/~chm/vchembook/570enzymes.html
(3) Sethi, R. (2009). Biology. Rachna Sagar Pvt. Ltd.: New Delhi