Enzymatic Activity of

Salivary Amylase
Arce, Christine Grace S.M.
Cua, Lance A.
Draheim, Kristella M.
Go-Oco, Raquel M.

Introduction

Enzymes
consist of a protein and a non-protein (called
thecofactor)
Biological catalysts
- increase the rate of a reaction
- lowers activation energy of reactions
highly selective, and highly sensitive
- due to the shapes of the enzyme molecules
Globular proteins (with exception of some
RNAs)

Figure 1. Comparison of free energy of activation

profiles for catalyzed and uncatalyzed reactions
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Figure 2. Maltose is made of two glucose molecules bonded

together (1). The maltase enzyme is a protein that is perfectly
shaped to accept a maltose molecule and break the bond (2). The
two glucose molecules are released (3)

Factors affecting catalytic

activity of enzymes
1. Temperature
2. pH
3. Concentration of enzyme and
substrate
4. Inhibition of enzyme activity
5. Effect of time
6. Metal ion activators

1. Temperature
As the temperature rises, reacting
molecules gain more kinetic energy
There is a certain temperature at
which an enzyme's catalytic activity
is at its greatest, called the optimal
temperature

Figure 3. Optimal
temperature of
enzymes found
human body

Optimal temperature is usually

around human body temperature
(37.5oC) for the enzymes in human
in

the

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achers/Resources/cfb/images/0
7A.jpg
Denaturation
- above the

optimal temperature the

enzyme structure begins to break down (denature) since at
higher temperatures intra- and intermolecular bonds are
broken.

Denaturationof proteins involves the

disruption and possible destruction of
both the secondary and tertiary
structures, but not the primary
structure.

2. On pH
Optimal pH: the
pH at which the
enzyme attains
its maximal
activity

Figure 4. Optimum pH of different

enzymes; Pepsin: 1.5 2, Salivary
amylase: 6.8, Arginase: 9 9.5
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ces/cfb/images/07B.jpg

3. On Concentration of enzyme and

substrate

Figure 5. Two graphs show the effect of increasing the

concentration of enzyme and substrate, respectively
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http://www.rsc.org/Education/Teachers/Resources/cfb/images/07D.jpg

As the concentration of either is increased, the rate of reaction

increases (at constant temperature and pH)
The rate of reaction increases with increasing substrate concentration
up to a point, above which any further increase in substrate
concentration produces no significant change in reaction rate

4. Inhibition of enzyme activity

Inhibitors-

molecules that reduce or even stop

the catalytic activity of enzymes in biochemical
reactions. They block or distort the active site.

Types:
1. Active site-directed(competitive) - inhibitors that
occupy the active site and prevent a substrate molecule
from binding to the enzyme
2. Non-active site-directed(non competitive) inhibitors that attach to the enzyme, perhaps distort its
shape

5. Effect of time
at the beginning, the rate of reaction increases
but by time the rate decreases due to:

Depletion of substrate
Accumulation of end products
Change in pH of the reaction

6. Activators
Activators, which temporarily bind to the active site of the
enzyme, switch enzymes from inactive to active state

Chloride ions activate salivary amylase

(anionic activation)
Calcium ions activate thrombokinase
enzyme (cationic activation)

Enzymes

Advantages over chemical catalyst:

1. enzyme-catalyzed reactions often
have much higher rates
2. Enzymes have greater reaction
specifity
3. Enzymes can be regulated

Amylase
Alpha Amylase
Salivary Amylase

Amylase
digests starch by catalyzing
hydrolysis, which is the breaking of a
bond in a molecule by water
Classifications:
-Amylase
-Amylase
-Amylase

Saccharification
- final stages of depolymerization are mainly
the formation of mono-, di-, and trisaccharides

Alpha amylase
- made by the pancreas and salivary glands
to hydrolyse alpha bonds of large, alphalinked polysaccharides, such as starch and
glycogen,
intodisaccharides
and
trisaccharides

Activator:
Chloride is essential

Salivary Amylase
A digestive enzyme secreted by
salivary glands
begins the chemical process of
digestion
Formerly known as ptyalin
utilizes starch as a substrate and
produces sugars as products

Hydrolysis

is a reaction involving the breaking of

a bond in a molecule using water. The reaction mainly
occurs between an ion and water molecules and often
changes thepHof a solution.
Enzymatic hydrolysis of starch breaks large, insoluble
starch molecules into dextrins:
1. Amylodextrin intermediate product of hydrolysis
of starch, soluble in water. Gives color blue with iodine.
2. Eryhtrodextrin gives red color with iodine
3. Achrodextrin gives no color with iodine

Starch

Insoluble in cold water or alcohol

starch molecules are glucose polymers linked together
by the alpha-1,4 and alpha-1,6 glucosidic bonds
Can be separated into two fractions: amylose and
amylopectin
Amylose: 10-20%, unbranched, single chain polymer
of 500 to 2000 glucose subunits with only the alpha1,4 glucosidic bonds (forms colloidal dispersion in hot
water);
Amylopectin: 80-90%, branched, the presence of
alpha-1,6 glucosidic linkages (insoluble)

Amylose in starch is responsible for the formation of a

deep blue color in the presence of iodine.

On Starch

Figure 6. Starch molecule in either branched chains

(amylopectin) or unbranched chains (amylose)
Retrieved from:
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Figure 7.

Amylose
The structure of amylose consists of long polymer
chains of glucose units connected by analpha
acetallinkage
All of the monomer units are alpha -D-glucose,
and all the alpha acetal links connect C1 of one
glucose and to C4 of the next glucose.
As a result of the bond angles in theacetal
linkage, amylose actually forms a spiral orcoiled
spring
The iodine molecule slips inside of the amylose
coil

On the Iodine Test

Figure 8. Iodine inside amylose coil

Retrieved from: http://www.elmhurst.edu/~chm/vchembook/548starchiodine.html

Iodine Test
Blue-black color indicates the presence of
starch
Amylose is responsible for the blue-black
color in the presence of iodine
If starch amylose is not present, then the color will stay orange or
yellow. Starch amylopectin does not give the color, nor does
cellulose

The iodine molecule slips inside of the

amylose coil
KI Reagent Iodine is not very soluble in
water. Reagent is made by dissolving iodine
in water in the presence of potassium iodide

Buffers - used as a means of keeping pH at a nearly

constant value in a wide variety of chemical applications

Acetic Acid Buffer Solutions (pH 3.75.61)

Phosphate buffer solutions(pH 5.8 8.0)
active in unstimulated saliva
acid-base pair has a pKa value in the range 6.8-7.2 ,
which has a maximum buffering capacity that is
relatively close to the salivary pH range: 6-8

Bicarbonate buffer(pH 9.2 10.8)

major buffer in stimulated saliva

Objectives

1. To determine the optimum

enzymatic activity of salivary
amylase depending on the different
changes of pH
2. To determine the optimum
enzymatic activity of salivary
amylase depending on the different
changes of temperature

Methodology

Preparing the Solution

1mL
saliva

30 mL
0.5% NaCl

40mL Salivary Amylase

9 mL
distilled
H2O

Part A: Determining the Optimum

Temperature
2 mL of enzyme
solution

2mL of buffered starch

solution in a large test tube

Incubate for 5 minutes in a 4, 28, 37, 50,

and 70C
water bath
Mix immediately

Mixture

(2mL buffered starch solution + 2 mL

enzyme solution)

Quickly place 2 drops of iodine and 3

drops of solution into well 1 of the spot
plate, labeled as 0 time
Place 2 drops of iodine and 3 drops of
solution after 1 minute.
Repeat for 20 minutes until a yellow colored solution appears

Yellow-Colored
Solution

Part B: Determining the Optimum pH

1mL acetate buffer (pH 4, 5, 6.7, 8,
10) + 1mL 2% unbuffered starch in a
large test tube

2 mL of enzyme
solution

Incubate for 5 minutes in a 37C water b

Mix immediately

Mixture

(2mL buffered starch solution + 2 mL

enzyme solution)

Quickly place 2 drops of iodine and 3

drops of solution into well 1 of the spot
plate, labeled as 0 time
Place 2 drops of iodine and 3 drops of
solution after 1 minute.
Repeat for 20 minutes until a yellow colored solution appears

Yellow-Colored Solution

RESULTS

EFFECTS OF TEMPERATURE
TEAM 1
TEMPERATURE

TIME

1/t , min -1

4oC

28oC (room
temperature)

37oC

50oC

70oC

EFFECTS OF TEMPERATURE
TEAM 2
TEMPERATURE

TIME

1/t , min -1

4oC

28oC (room
temperature)

37oC

18

1/18 = 0.06

50oC

20

1/20 = 0.05

70oC

EFFECTS OF pH
TEAM 1
pH

TIME

1/t , min -1

16

1/16 = 0.07

6.7

1/5 = 0.20

11

1/11 = 0.09

10

EFFECTS OF pH
TEAM 2
pH

TIME

1/t , min -1

20

1/20= 0.05

6.7

1/5 = 0.20

1/5 = 0.20

10

Discussion

A. Effect of Temperature
For most chemical reactions, the rate of enzyme
catalyzed reactions generally increases with
temperature
There is also a temperature range wherein the
enzyme is most stable and retains its full
activity capacity
Shape of the graph is Sigmoidal
Rate of most enzymatic reactions approximately
doubles for each 10C raised in temperature
Varies per enzyme based on their energy of
activation value

B. Effect of pH
Most enzymes have a pH value wherein it reaches its optimal
conditions
Any pH lower or greater than this would decline the enzymes activity

Optimum pH of an enzyme may not be identical with the pH of

its normal intracellular surroundings
Average optimum pH recorded by the group = pH 7
The bell shape is the shape of the graph
- The enzymatic activity has already started at pH 4
- It then reaches to its optimum level, around pH 6.7 to 7
- Once optimum pH has been reached, the enzymatic activity
steadily decreases.
Unlike the acidic or basic environments, amylase works best at
a more neutral pH.

Conclusion

1. The optimum temperature of

enzymatic activity and specificity of
salivary amylase has a value of
37C
2. The optimum pH level of the
enzymatic activity and specificity of
salivary amylase has a pH value of
7.

References

 Chua-Suba, S. (2011). Laboratory manual in biochemistry.

Quezon City: C&E Publishing, Inc.
Garret, R., & Grisham, C. (2012). Biochemistry. Boston,
Massachusetts: Cengage Learning.
Lehninger, A. (1975). Biochemistry. Quezon City: JMC Press,
Inc.
Mckee, T., & Mckee, J. (2003). Biochemistry: the molecular
basis of life. 1221 Avenue of the Americans, New York:
McGraw-Hill Company.
Gaughan, R. What are the Effects of Boiling & Freezing on
Enzyme Activity?. Received from
http://education.seattlepi.com/effects-boiling-freezing-enz
yme-activity-4327.html
ukessays.com (n.d.). Retrieved February 2015, from
http://www.ukessays.com/essays/biology/optimal-temperat
ure-for-enzyme-amylase-biology-essay.php