Effects of pH , Concentration and

Temperature on -amylase activity

ANANTH A/L MANICKAM WASH

ANEESCH PREETHA VIDA
NAIGAM
ELLVANNDHO JUNNY
LAURA VITALIS
NORAZRINAH RAMON
SYLVIANAH SYLVESTER
ONG XIN ROU
 WHAT ARE ENZYMES?
A protein that acts as a catalysts

More than 2000 enzymes species

Catalysts is a substance that speed up the rate

of reaction without having a chemical change
 HOW ENZYMES WORK?

Enzymes lower the activation energy

Binding with the substrate and formed enzyme-substrate complex
The interaction between the enzymes and substrate where usually Van Der
Waals forces and hydogen bonding forces
 ENZYME-SUBSTRATE COMPLEX
REACTION

Specific site on the enzyme known as active site

The lock-and-key model, represents the lock as the
enzymes and the key is the substrate
 AMYLASE ENZYMES

The major salivary enzyme is alpha amylase

Acts an carbohydrates
The optimum pH of salivary amylase is 6
The parotid gland secretes most of the amylase
 FACTORS THAT AFFECT THE RATE
OF REACTION

1. Effects of temperature
2. Effects of pH
3. Effects of enzyme concentration
Materials

0.01% Amylase 0.05% Amylase 0.1% Amylase

Solution Solution Solution

5% Starch 0.1M Iodine Buffer Solutions

Solution Solution (pH4, 7 , 10)
50mL Conical 50mL Beakers Measuring 250mL Volumetric
Flask Cylinders Flask

100mL Droppers Electronic

Volumetric Flask Magnetic Stirrer Stopwatch
Material Preparation
5% 0.1M 0.01% 0.05% 0.1%
Starch Iodine Amylase Amylase Amylase
Solution Solution Solution Solution Solution
12.5g 1.269g 0.01g 0.05g 0.1g
Potato Iodine Amylase Amylase Amylase
Starch 100ml powder powder powder
250ml water 100ml 100ml 100ml
water water water water
 5 mL Starch Solution
5 mL Buffer Solution

5 mL Amylase Solution 2.5 mL Iodine

Solution
 All conical flasks
and beakers are
placed in the
water bath for 10
minutes to allow
the temperature to
equilibrate.
 When iodine solution is added into the starch solution, the solution turns
dark blue. In the presence of starch, the brown coloured iodine solution
turns dark blue.

If the starch is completely hydrolysed by amylase enzyme, the dark blue

solution becomes colourless due to the absence of starch at the end of
hydrolysis reaction.

When the temperature of the water bath is too high, the colour of the
iodine-starch solution becomes colourless before mixing with amylase
buffer solution within 10 minutes.

The time taken for the dark blue solution to turn colourless at pH4 is the
longest whereas for pH10 is to shortest.
 Temperature: 32 C

pH\ Concentration of
Amylase Solution 0.01 % 0.05 % 0.1 %
4 3.3000 2.3000 0.4833
7 0.2500 0.8625 0.0547
10 0.0383 0.0333 0.0410
 Temperature: 37 C

pH\ Concentration
of Amylase Solution 0.01 % 0.05 % 0.1 %
4 3.6200 2.1200 0.3167
7 0.0360 0.1518 0.0532
10 0.0298 0.0310 0.0212
 Temperature: 42 C
0.01% Amylase Solution
0.05% Amylase Solution
4.00 0.1% Amylase Solution
Time Taken for Hydrolysis of

3.50
50% Starch Solution

3.00
2.50
2.00
1.50
1.00
0.50
0.00
4 7 10 pH
 Graph of Effect of x-amylase Enzyme Concentration,
Temperature and pH on the Time taken for Hydrolysis of Starch
7.0

6.0
TIme taken for Starch Hydrolysis (min)

5.0

4.0

3.0

2.0

1.0

0.0
pH4 pH7 pH10 pH4 pH7 pH10 pH4 pH7 pH10
32 37 42
x-Amylase Enzyme Conditions
0.01% Amylase Solution 0.05% Amylase Solution 0.1% Amylase Solution
 AMYLASE
Differences Alpha Beta
Sources Can be found in humans, Found in plants or fungi
other mammals

Site of hydrolysis Random location on starch Acts from non-reducing of

chain polysaccharides

Activity (Rate of reaction) Faster Slower

Enzyme Catalyse the hydrolysis of starch into

smaller carbohydrate molecules
Two categories Alpha-amylase and Beta-amylase
Three various pH, Concentration, and Temperature
pH : 4 (acidic), 7( neutral) , 10 (base)

Temperature :
32C ( below body tempt)

37 C ( body tempt)
42 C ( above body tempt)

Concn of amylase : (0.01, 0.05, 0.10) M

Amylase
solution Starch
+ Solution
Buffer +
Solution Iodine

Cloudy Solution Blue Black Solution

Blue Black Solution - Starch is present

Higher the intensity of Higher the

blue black solution concentration of starch

Two solution mixed together, blue black solution turns colourless

Blue Black solution Colorless
solution
All the starch has been hydrolysed

No starch is present

Less time taken for blue

black solution to turn
colorless

Higher the enzyme activity

of amylase
 Graph of Effect of x-amylase Enzyme Concentration, Temperature and pH on the Time
7.0
taken for Hydrolysis of Starch

6.0
TIme taken for Starch Hydrolysis (min)

5.0

4.0

3.0

2.0

1.0

0.0
pH4 pH7 pH10 pH4 pH7 pH10 pH4 pH7 pH10
32 37 42
x-Amylase Enzyme Conditions

0.01% Amylase Solution 0.05% Amylase Solution 0.1% Amylase Solution

APPLICATION OF ALPHA-AMYLASE IN INDUSTRY

Liquefaction

Process of dispersion of insoluble starch granules in aqueous solution by

partial hydrolysis

The enzyme provide partially hydrolysed starch suspension of relatively low

viscosity in order to stabilize gradation for further processing

Manufacturing of maltose
Maltose used as sweetener and intravenous sugar supplement

The enzyme help to reduce tendency to be crystallized.

 Removal of starch sizer from textile

In textile weaving, starch used to prevent the loss of string by friction

The enzyme used to remove starch in order for the clothes to undergo scouring
and dyeing.

Fermentation of starch and ethanol (alcohol production)

The enzyme used to increase rate of saccharification, alcohol yield and yeast growth
during the process of production.
 The optimum condition :
37C , 0.1 %, pH 10

Higher Temperature = Higher Reaction

High Temperature causes denaturation too