Introduction about amylase, types of Amylases- Alpha amylase and Beta Amylase and those role, Aim of estimation of Amylase, Principle of estimation of Amylase, Materials required for estimation of Amylase- Dinitrosalicylic acid reagent reparation, 1% starch solution preparation , Sodium Phosphate buffer preparation in 0.5M pH7.0, Sodium potassium tartrate preparation, Standard Maltose preparation, Procedure for estimation of Amylase from Germinating seeds, Calibration Curve, Result.

1. Estimation of Amylases

2. Assay of Amylase
Aim:
To determine the activity of amylase present in
food.

3. Principle
Amylase catalyses the hydrolysis of starch
and glycogen into maltose sugars.
α- Amylase is endo-hydrolase which
hydrolyses α-1,4, linkage whereas β- amylase
is exo-hydrolase hydrolyses non-reducing end
to remove β-maltose and limit dextrin at end.

4. Principle
 α- Amylase is present in human saliva, whereas
β- amylase is present in germinating seeds.
 The progress and extend of hydrolysis of starch is
determined by amount of reducing sugar
produced.

7. Materials required
1) Dinitrosalicylic acid : Dissolve 1g of
dinitrosalicylic acid, 200mg crystalline phenol
and 50mg sodium sulphite in 100ml final
volume of 1% NaOH.
2) Sodium potassium tartarate : Dissolve 40g of
sodium potassium tartarate in 100ml final
volume of distilled water.

9. Materials required
3) Starch solution: Dissolve 1.0 g of soluble
starch in 10ml of boiling water.
o Slowly add this solution with constant stirring
in 90ml of boiling water.
o Boil for 20 minutes and cool to room
temperature.

11. Materials required
4) Sodium phosphate buffer (0.5M, pH 7.0):
(a) Dissolve 1.39g NaH2PO4 in 100ml distilled water
(b) Dissolve 2.68g Na2HPO4.7H2O in 100ml distilled
water
o Mix 39ml of (a) and 61.0ml of (b), add 5.8g NaCl.
o Mix and dilute to 200ml with water in a volumetric flask.
o Measure the pH in pH meter and adjust to 7.0 by adding
(a) or (b)

13. Materials required
5) Standard Maltose solution: Dissolve 50mg
of maltose in 500ml distilled water (100μg
/ml)
6) Germinated seeds

14. Procedure - Extraction
 Transfer 500mg of washed germination seeds in
chilled pestle and mortar.
 Homogenize with 5ml of chilled phosphate
buffer.
 Centrifuge at 1000RPM in refrigerated
centrifuge (below 4°C) for 15 minutes.
 Decant and use the supernatant.

15. Procedure - Estimation
 Pipette 1.0ml starch solution (fresh) and 1.0 ml
of enzyme extract in two test tubes.
 In one test tube add 2ml dinitrosalicylic acid
reagent (control, 0 min).
 Incubate other experimental tube at 37°C for
15minutes and stop reaction by adding 2ml
dinitrosalicylic acid reagent.

16. Procedure - Estimation
 Keep both the tubes in boiling water bath for
15 minutes and add 1.0 ml Sodium potassium
tartrate.
 Cool the tube in running tap water and dilute
to 10ml by adding 5ml distilled water.

18. • Read the absorbance at 570nm, setting zero with zero minute control
(first tube) prepare standard curve by taking aliquots 0, 0.2, 0.4, 0.6,
0.8 and 1.0 ml Sodium potassium tartarate.
• Cool and add 6.0ml distilled water, mix and read the absorbance at
570nm.
• Compare the absorbance of experimental tube (second tube) and amount
of maltose in experimental tube.
• The activity of enzyme was calculated in terms of micromoles of
maltose produced per gm fresh weight of sample per minutes.

20. Answer the following questions
1) Define: Enzyme .
2) Define: Enzyme activity.
3) Write the Michaelis-Menton equation.
4) Name the factors which affect the enzyme
activity.
5) Differentiate α amylase from β amylase.