Experiment No: 5

ESTIMATION OF AMYLASE ACTIVITY

Aim: To determine amylase activity in the given sample.

Principle: α-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 20°C: Prepare 100 ml in deionized water using anhydrous monobasic sodium phosphate. Adjust to pH 6.9 at 20°C 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 20°C 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. α-Amylase Solution: Prepare a solution containing 1 unit/ml of α-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 20°C. Then add 1ml of enzyme solution. 3. Mix by swirling and incubate for exactly 3.0 minutes at 20°C. 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 20°C.

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 Y-axis.

CALCULATIONS:

Units/ml enzyme = (mg of Maltose released)(Dilution factor) Volume of enzyme takenOBSERVATIONS:

REAGENTS BLANK TEST1 Starch solution 1ml 1ml2 Enzyme - 1ml3 DNS reagent 2ml 2ml4 Rochelle solution 1ml 1ml5 Enzyme 1ml -

O.D at 540nm

STANDARD CURVE DETERMINATION:

Volume of Standard Maltose(ml)

Volume of distilled water (ml)

Maltose Concentration (mg)

Sample volume (ml)

DNS Reagent (ml)

Cap the tubes and boil for 5-10 min

Rochelle salt solution (ml)

Optical Density at 540nm

0 5 0 2 2 10.2 4.8 0.2 2 2 10.4 4.6 0.4 2 2 10.6 4.4 0.6 2 2 10.8 4.2 0.8 2 2 11.0 4 1.0 2 2 1

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