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

INTRODUCTION

The digestive system or

gastrointestinal (GI) system is divided

into the upper GI tract and the lower GI

tract consisting of different digestive

organs. It also consists of accessory

glands, which secrete enzymes and

fluids that aid in digestion. Digestion is

the process of breaking down food taken

in the body, to smaller units with the aid

of enzymes. One of these enzymes is

amylase. It is an enzyme produced by

the salivary glands and secreted into the

mouth. It is used to hydrolyze starch to

maltose. During amylase activity, starch

decreases and sugar increases. These

chemical changes are observed with the

use of enzyme assay, specifically IKI

and Benedict’s assay. The IKI assay

detects the presence of starch or

cellulose while the Benedict’s assay tests

for the presence of reducing sugars, such

as glucose or maltose. Plants contain

polysaccharides, which includes starch

and cellulose. Starch is used to store

energy while cellulose provides rigidity

to the plant’s cell wall. Both

polysaccharides are polymers of glucose

but with different linkages.

MATERIALS AND METHODS

In the Activity 2 setup, the test

tubes were dragged into place on top of

the incubator.  Each test tube were then

filled with three substances, as follows:

test tube #1: amylase, starch, pH 7.0

buffer, test tube #2: amylase, glucose,

pH 7.0 buffer, test tube #3: amylase,

cellulose, pH 7.0 buffer, test tube #4:

cellulose, deionized water, pH 7.0

buffer, test tube #5: peptidase, starch, pH

7.0 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

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.

The tubes were lowered into the

incubation unit and incubated at 37C for

1 hour. After incubation, half of the

contents of each test tube were

transferred to the assay tubes. A drop of

IKI reagent was dispensed to each of the

assay tubes and the results were

recorded. Five drops of Benedict’s

reagent were then dispensed to the

remaining contents of each test tube in

the incubation unit. These test tubes

were then boiled. The results were

recorded after boiling. 

RESULTS AND DISCUSSION

Tube Reagent 1 Reagent

2

Reagent

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

Tube IKI Benedict’s

1 - ++

2 - ++

3 + -

4 + -

5 + -

6 - ++

Table 2. Results of each test tube

Enzyme assay is the chemical

method of detecting the presence of

digested substances. It only indicates the

presence or absence of a substance. In

the experiment, IKI assay and Benedict’s

assay were used to determine the

presence of starch / cellulose and

reducing sugars, respectively. For the

IKI test, a blue-black color indicates a

positive test for starch and a yellow

color indicates a negative test. While for

the Benedict’s test, a green, orange, or

reddish color indicates the presence of

maltose, which is a positive result. And a

blue color indicates that no maltose is

present which is a negative result. A (+)

sign was used for a green result, (++) for

a reddish-brown result, and (-) for a blue

result.

In the table, tubes 1 and 2 both

tested positive for the Benedict’s 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 Benedict’s test since

the bacterial suspension contained the

enzyme cellulase, which digested the

cellulose producing glucose. 

CONCLUSION

The salivary amylase breaks

starch polymers down into smaller sugar

units. However, it cannot break down

cellulose polymers. Bacteria produces

enzymes called cellulase that can aid in

the digestion of cellulose. Peptidases

break down peptides into amino acids

but they cannot break down starch.

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/BIOL1020/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.