Experiment No: 5

ESTIMATION OF AMYLASE ACTIVITY

Aim: To determine amylase activity in the given sample.
Principle: -Amylase is an enzyme that hydrolyses alpha-bonds of large alpha-linked
polysaccharides, such as starch and glycogen, yielding glucose and maltose. It is the
major form of amylase found in humans and other mammals. It is also present in seeds
containing starch as a food reserve, and is secreted by many fungi.
Starch + H2O a-Amylase
Reducing Groups (Maltose)
Reagents required:
1. 20 mM sodium phosphate buffer with 6.7 mM Sodium Chloride, pH 6.9 at 20C:
Prepare 100 ml in deionized water using anhydrous monobasic sodium phosphate.
Adjust to pH 6.9 at 20C with 1 M NaOH.)
2. 1.0% (w/v) Soluble Starch Solution (Starch): Prepare 25 ml in sodium phosphate
buffer with 6.7 mM Sodium Chloride, pH 6.9 at 20C using starch. Facilitate
solubilization by heating the starch solution in a glass beaker directly on a
heating/stir plate using constant stirring. Bring to boil and maintain the solution
at this temperature for 15 minutes. Allow the starch solution to cool to room
temperature with stirring. Return the starch solution to its original volume (25
ml) by the addition of water and dispense samples for assay while stirring.
3. Dinitrosalicylic Acid Reagent (DNS Reagent): Dissolve by stirring 1g
Dinitrosalicylic acid, 200mg crystalline phenol and 50mg sodium sulphite in
100mL 1% NaOH.
4. 40% Rochelle salt solution (Potassium sodium tartrate)
5. Standard maltose solution: 0.1% maltose solution.
6. -Amylase Solution: Prepare a solution containing 1 unit/ml of -Amylase in cold
deionized water.
Procedure:
1. Pipette out 1ml of starch solution into test tubes labelled test and blank.
2. Mix by swirling and equilibrate to 20C. Then add 1ml of enzyme solution.
3. Mix by swirling and incubate for exactly 3.0 minutes at 20C.
4. Add 2mL of DNS reagent.
5. Heat the contents in a boiling water bath for 5-10 minutes.
6. When the contents of the tubes are still warm, add 1mL of 40% Rochelle salt
solution.
7. Mix by inversion and measure the OD at 540nm for both the test and blank using
a suitable spectrophotometer. One unit of amylase activity is defined as 1.0 mg of
maltose liberated from starch in 3 minutes at pH 6.9 at 20C.
Standard Curve Preparation:
1. Label test tubes 0, 1, 2,....10 and add 0,0.1,0.2,....1ml of standard glucose solution
in the test tubes respectively.
2. Make up the volume to 5ml using distilled water.

3. Take 2ml from each tube and follow the procedure from step-4 as described
above.
4. Plot a standard curve with sucrose concentration along X-axis and O.D against Yaxis.

CALCULATIONS:
Units/ml enzyme =

(mg of Maltose released)(Dilution factor)

Volume of enzyme taken

OBSERVATIONS:
1
2
3
4
5

REAGENTS
Starch solution
Enzyme
DNS reagent
Rochelle solution
Enzyme
O.D at 540nm

BLANK
1ml
2ml
1ml
1ml

TEST
1ml
1ml
2ml
1ml
-

STANDARD CURVE DETERMINATION:

Volume of
Standard
Maltose(m
l)
0
0.2
0.4
0.6
0.8
1.0

Volume
of
distilled
water
(ml)
5
4.8
4.6
4.4
4.2
4

Maltose
Sample
Concentrati volume
on (mg)
(ml)
0
0.2
0.4
0.6
0.8
1.0

2
2
2
2
2
2

DNS
Cap
Reagen the
t (ml)
tube
s
and
boil
2
for
2
5-10
2
min
2
2
2

Rochelle
salt
solution
(ml)
1
1
1
1
1
1

Report:
-Amylase activity in the given sample is found to be = __________ Units/ml.

Optical
Density
at
540nm