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.