Exploring Amylase Substrate Specificity: Physioex Exercise 8: Activity 2

PhysioEx Exercise 8: Activity 2
Exploring Amylase Substrate Specificity

C. Evangelista, M. Garcia, L. Guillermo, A. Jimenez
Department of Biological Sciences, College of Science, University of Santo Tomas,
Espana, Manila
Keywords:
Amylase
Starch
Cellulose
Peptidase
decreases and sugar increases. These
INTRODUCTION
chemical changes are observed with the

use of enzyme assay, specifically IKI
The
digestive
system
or
and Benedicts assay. The IKI assay
gastrointestinal (GI) system is divided
detects the presence of starch or
into the upper GI tract and the lower GI
cellulose while the Benedicts assay tests
tract consisting of different digestive
for the presence of reducing sugars, such
organs. It also consists of accessory
as glucose or maltose. Plants contain
glands, which secrete enzymes and
polysaccharides, which includes starch
fluids that aid in digestion. Digestion is
and cellulose. Starch is used to store
the process of breaking down food taken
energy while cellulose provides rigidity
in the body, to smaller units with the aid
to
of enzymes. One of these enzymes is
polysaccharides are polymers of glucose
amylase. It is an enzyme produced by
but with different linkages.
the salivary glands and secreted into the

mouth. It is used to hydrolyze starch to
maltose. During amylase activity, starch
the
plants
cell
wall.
Both
MATERIALS AND METHODS
assay tubes
and
the results
were
recorded. Five drops of Benedicts

In the Activity 2 setup, the test
reagent were then dispensed to the
tubes were dragged into place on top of
remaining contents of each test tube in
the incubator. Each test tube were then
the incubation unit. These test tubes
filled with three substances, as follows:
were then boiled. The results were
test tube #1: amylase, starch, pH 7.0
recorded after boiling.
buffer, test tube #2: amylase, glucose,

pH 7.0 buffer, test tube #3: amylase,
RESULTS AND DISCUSSION
cellulose, pH 7.0 buffer, test tube #4:

cellulose, deionized water, pH 7.0 buffer,
Tube
test tube #5: peptidase, starch, pH 7.0
2
3
1
Amylase
Starch
pH 7.0
2
Amylase
Glucose
pH 7.0
3
Amylase Cellulose pH 7.0
4
Cellulose
Water
pH 7.0
5
Peptidase
Starch
pH 7.0
6
Bacteria Cellulose pH 7.0
Table 1. Reagents in each test tube
buffer, and test tube #6: bacteria,

cellulose, pH 7.0 buffer.
Each test tube were added with
pH 7.0 buffer which is a solution used to
Reagent 1
Reagent
Reagent
set the pH of the test tube solution.

Deionized water was added to test tube
4, which was used to adjust the test tube
solution volume so it is the same for
each reaction. Amylase and peptidase
were also used to break down starch and
peptides, respectively.
Tube
IKI
Benedicts
1
++
2
++
3
+
4
+
5
+
6
++
Table 2. Results of each test tube
The tubes were lowered into the
Enzyme assay is the chemical
incubation unit and incubated at 37C for
method of detecting the presence of
1 hour. After incubation, half of the
digested substances. It only indicates the
contents
were
presence or absence of a substance. In
transferred to the assay tubes. A drop of
the experiment, IKI assay and Benedicts
IKI reagent was dispensed to each of the
assay were used to determine the
of
each
test
tube
presence of starch / cellulose and
enzyme cellulase, which digested the
reducing sugars, respectively. For the
cellulose producing glucose.
IKI test, a blue-black color indicates a

positive test for starch and a yellow
CONCLUSION
color indicates a negative test. While for

the Benedicts test, a green, orange, or
The salivary amylase breaks
reddish color indicates the presence of
starch polymers down into smaller sugar
maltose, which is a positive result. And a
units. However, it cannot break down
blue color indicates that no maltose is
cellulose polymers. Bacteria produces
present which is a negative result. A (+)
enzymes called cellulase that can aid in
sign was used for a green result, (++) for
the digestion of cellulose. Peptidases
a reddish-brown result, and (-) for a blue
break down peptides into amino acids
result.
but they cannot break down starch.

In the table, tubes 1 and 2 both
tested positive for the Benedicts test

since in tube 1, the amylase hydrolyzed
the starch to glucose while in tube 2,
glucose was already present. Tube 3,
however, tested positive for IKI, since
cellulose is present and amylase cannot
digest cellulose. Same concept applies to
tube 5 which tested positive for IKI.
Peptidases
can
only
breakdown
peptides/proteins into amino acids, but it

cannot breakdown starch. Lastly, tube 6
tested positive to Benedicts test since
the bacterial suspension contained the
REFERENCES
Fincher, G. B. & Hrmova, M. (2001).
Plant
Enzyme
Structure.
Explaining Substrate Specificity
and the Evolution of Function.
Plant Physiology, 125, 54-57.
The Molecules of Life: Biochemistry.
Retrieved
from
http://umanitoba.ca/Biology/BIO
L1020/lab2/biolab2_2.html
Griff, E., Lokuta, A., Smith, L. A.,
Stanle, T. N., & Zao, P. (2011).
PhysioEx
9.0:
Laboratory
Simulations
in
Physiology.
Pearson.

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PhysioEx Exercise 8: Activity 2

Exploring Amylase Substrate Specificity

chemical changes are observed with the

and Benedicts assay. The IKI assay

gastrointestinal (GI) system is divided

detects the presence of starch or

into the upper GI tract and the lower GI

cellulose while the Benedicts assay tests

tract consisting of different digestive

for the presence of reducing sugars, such

organs. It also consists of accessory

as glucose or maltose. Plants contain

glands, which secrete enzymes and

polysaccharides, which includes starch

fluids that aid in digestion. Digestion is

and cellulose. Starch is used to store

the process of breaking down food taken

energy while cellulose provides rigidity

in the body, to smaller units with the aid

of enzymes. One of these enzymes is

polysaccharides are polymers of glucose

amylase. It is an enzyme produced by

but with different linkages.

the salivary glands and secreted into the

MATERIALS AND METHODS

recorded. Five drops of Benedicts

reagent were then dispensed to the

tubes were dragged into place on top of

remaining contents of each test tube in

the incubator. Each test tube were then

the incubation unit. These test tubes

filled with three substances, as follows:

were then boiled. The results were

test tube #1: amylase, starch, pH 7.0

recorded after boiling.

buffer, test tube #2: amylase, glucose,

RESULTS AND DISCUSSION

cellulose, pH 7.0 buffer, test tube #4:

test tube #5: peptidase, starch, pH 7.0

buffer, and test tube #6: bacteria,

set the pH of the test tube solution.

The tubes were lowered into the

Enzyme assay is the chemical

incubation unit and incubated at 37C for

method of detecting the presence of

1 hour. After incubation, half of the

digested substances. It only indicates the

presence or absence of a substance. In

transferred to the assay tubes. A drop of

the experiment, IKI assay and Benedicts

IKI reagent was dispensed to each of the

assay were used to determine the

presence of starch / cellulose and

enzyme cellulase, which digested the

reducing sugars, respectively. For the

cellulose producing glucose.

IKI test, a blue-black color indicates a

color indicates a negative test. While for

The salivary amylase breaks