The effects of temperature

and pH on the enzymatic

activity of salivary amylase
Amiel Joseph D. Custodio
Mary Joyce G. Gabac
Kenneth Patrick T. Bautista
John Raphael T. Missiona
Introduction
Enzymes
Three dimensional macromolecules formed by spontaneous
folding of a linear polypeptide chain (Bugg, 2012)

Function as a catalyst in biochemical reactions, increasing the

rate of such reactions without being consumed in the process
and altering the equilibrium constants which they catalyse

Thesubstance acted on by an enzyme is called the substrate

(Appling, 2016)
General Properties of Enzymes

1. Higher
reaction rates: enzyme-catalyzed
reactions are 106-1012 times faster than
uncatalyzed reactions
2. Reaction
conditions: enzyme-driven reactions
are below 100oC, 1 atm and occurs in neutral pH
3. Great
specificity: with respect to identities of
both the substrate and products (induced fit &
lock-and-key)
Factors affecting enzymatic activity

Enzyme concentration
Substrate concentration
pH
Temperature
Classification of enzyme: according to reaction type

Oxidoreductase: catalyze the transfer of electron from one

molecule to another
Ex: dehydrogenase, oxidase

Transferase: transfers one functional group form one molecule

to another
Ex: transaminase, kinase

Ligase: catalyze joining of two molecules accompanied by ATP

hydrolysis
Ex: citric acid synthethase
 Lyase: catalyze removal or addition of a functional group
to substrate
Ex: aldolase, carboxylase

Isomerase: catalyze converison of one molecule from an

isomer
Ex: fumarase

Hydrolase: catalyze the addition of water which results

in the cleavage of bonds
Ex: digestive enzymes (amylase)
Amylase

A subclass of hydrolase which catalyze

breakdown of carbohydrate bonds between
sugar molecules.
-amylase: responsible for hydrolysis of starch
(ex. Salivary amylase & pancreatic amylase)
-amylase: used by lower forms of organism
like bacteria, molds and fungi
Salivary Amylase

Structure

1. single polypeptide chain which consists of

496 amino acids
2. has a calcium binding protein
3. has chloride ion
 Structure of salivary amylase

Retrieved from. https://www.google.com.ph/search?

q=salivary+amylase&biw=1280&bih=809&source=lnms&tbm=isch&sa=X&ved=0ahUKEwih6820jJbPAhWBkZQKHY4DDWcQ_AUIBigB#tbm=isch&q=salivary+amylase+structu
re&imgrc=f4VJAe9kh7JfUM%3A (September 17, 2016)
Domains in the salivary amylase (Ragunath, 2003)

Domain A
residues 1-99; 170-404
has the 3 catalytic residues Asp197, Glu233, Asp300
Domain B
residues 100-169
Calcium binding site
Domain C
residues 405-496
Functions (Luo, 2005)

Hydrolytic
activity responsible for the initial
breakdown of the polymeric starch
Insolution, it binds to oral streptococcus which
may result to clearing
Bacteria-bound amylase is capable of hydrolyzing
starch to glucose source and be metabolized to
lactic acid
Bound to tooth enamel, it can initiate plaque
formation
 Industrial use of amylase

Corn syrup production

Alcohol production
Digestible feed for animals
Bread making

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q=salivary+amylase&biw=1280&bih=809&source=lnms&tbm=isch&sa=X&ved=0ahUKEwih6820jJbPAhWBkZQKHY4DDWcQ_AUIBigB#tbm=isch&q=corn+syrup&imgrc=rfaFx
AjfNbUUWM%3A (September 17, 2016)
Starch

A white, granular, organic chemical that is produced by all

green plants.
Soft, white, tasteless powder that is insoluble in cold
water, alcohol, or other solvents.
Basic chemical formula of a starch molecule (C6H10O5)n
A polysaccharide comprising glucose monomers in 1,4
linkages.
The simplest form of starch is the linear polymer amylose;
amylopectin
Objectives of the Experiment

To determine
the optimum pH and optimum
temperature of salivary amylase
Methodology
A) Effect of Temperature

Enzyme solution Buffered starch solution

Put 2ml in Test tube Put 2ml in Test tube

Incubate both test tubes for

5 minutes on a 4C water
bath

Mix
Take 3 drops of mixture onto spot plate
Add 2 drops of Iodine solution. Record as zero minute
Repeat step every minute until a yellow solution is
observed. Note the time (t)
Follow same procedure for mixtures incubated at
other temperatures (RT, 37C, 50C, 70C)
Plot reciprocal of time (1/t) over temperature (C)

Determine optimum
temperature of salivary
amylase base on the graph
Methodology
B) Effect of pH
Acetate buffer pH 4 and
Enzyme solution 2% unbuffered starch
Mix 1 ml acetate buffer
Put 2ml in Test tube And 1ml 2% unbuffered
Incubate both test tubes for Solution
5 minutes at 37C water
bath

Mix
Take 3 drops of mixture onto spot plate
Add 2 drops of Iodine solution. Record as zero minute
Repeat step every minute until a yellow solution is
observed. Note the time (t)
Follow same procedure for mixtures of different pH
(5, 6.7, 8, 10)
Plot reciprocal of time (1/t) over pH

Determine optimum pH
of salivary amylase base
on the graph
Results and Discussion
Results of Groups 1 4

A) Effect of Temperature

Temp (C) Time (mins) 1/t (mins-1)

4C 0

RT (30) 3 0.33

37C 2 0.50

50C 3 0.33

70C 0
Results of Groups 5 9

A) Effect of Temperature

Temp (C) Time (mins) 1/t (mins-1)

4C 0

RT (28) 4 0.25

37C 2 0.5

50C 3 0.33

70C 0
.6 Groups 1 4 (Effect of Temperature) 0.6 Groups 5 9 (Effect of Temperature)
0.5 0.5
.5 0.5

.4 0.4
0.33 0.33 0.33
1/t (mins-1) 1/t (mins-1)
.3 0.3
0.25

.2 0.2

.1 0.1

0 0
0 0
0
0 10 20 30 40 50 60 70 80 0
0 10 20 30 40 50 60 70 80
Temperature (C)
Temperature (C)

Optimum Temperature : 37C Optimum Temperature : 37C

Effect of Temperature

NaCl
NaCl binds to the ionic side chains of amino acids and speeds up the reaction
The activity of salivary amylase increases with increasing salt concentration

Boiling
Enzyme are composed of 2o 4o structures maintained by H bonds, these
weak bonds can be broken down at elevated temperatures which causes
enzymes to unwind
Iodine
To test for the presence of starch
Yellow (indicates the breakdown of starch into glucose monomers )
Effects of Temperature

Buffered Starch
We use buffered starch to keep the pH constant while performing the
experiment
The more concentrated, the higher its capacity to stabilize pH; most
enzymes accept between 0.05% - 2%. (Veille, 2001)
With pKa correspond to the optimum pH or the enzyme assay
Phosphate buffer
Also know as phosphate buffered saline which has NaCl, Na 2PO4, and some
K2PO4
As molarity and ion concentrations match those as the human body
(isotonic)
Results of Groups 1 4

A) Effect of pH

Ph Time (mins) 1/t (mins-1)

4 0

5 3 0.33

6.7 1 1

8 1 1

10 5 0.20
Results of Groups 5 9

A) Effect of pH

Temp (C) Time (mins) 1/t (mins-1)

4 0

5 0

6.7 2 0.50

8 0

10 5 0.20
1.2 Groups 1 4 (Effect of pH) 0.6 Groups 5 9 (Effect of pH)
1 1 0.5
1 0.5

0.8 0.4

1/t (mins-1) 1/t (mins-1)

0.6
0.3

0.4 0.2
0.33 0.2

0.2
0.2
0.1

0
0 0 0 0
3 4 5 6 7 8 9 10 11 0
3 4 5 6 7 8 9 10 11
pH
pH

Optimum pH : 7.4 Optimum pH : 6.7

Effects of pH

On the other hand, for the testing of enzymatic activity of salivary

amylase in different pH, unbuffered starch was used instead of buffered
starch because the pH will be variable due to the buffers of different pH
that will be tested.
Instead, the constant factor on this test is the temperature, which is
administered by the 37 water bath, the normal body temperature.
pH affects both the enzyme and substrate, where the enzyme loses the
ionic bonds that holds the protein together, losing its shape, more
importantly the active site where the substrate will bind.
Drastic change in pH also affects the charges of the amino acids present
in the enzyme, halting the formation of an enzyme-substrate matrix.
Effects of pH

The optimum pH of salivary amylase is 6.7, near physiological pH

With acetate buffer at pH 4 and 5, the enzyme is deactivated ,
catalysing starch in longer periods,
With phosphate buffer at pH 6.7 to 8, the enzyme fully catalyzes
starch in less than two minutes under near physiological pH
With bicarbonate buffer at pH 10, the enzyme is almost fully
deactivated, catalysing starch with the longest duration.
Conclusion

The optimum temperature for enzymatic activity in

salivary amylase is 37oC and the optimum pH for
enzymatic activity in salivary amylase is 6.7
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