In this article we will discuss about the tests for estimation of amylase in plants.

Principle:

The reducing sugars produced by the action of α- and or β-amylase react with di-nitro-salicylic acid and reduce it to a brown coloured product, nitro-amino-salicylic acid.

Materials:

i. Sodium acetate buffer, 0.1 M pH 4.7

ii. Starch 1% Solution:

Prepare a fresh solution by dissolving 1 g starch in 100 ml acetate buffer. Slightly warm, if necessary.

iii. Di-nitro-salicylic Acid Reagent

iv. 40% Rochelle salt Solution (Potassium Sodium Tartrate)

v. Maltose Solution:

Dissolve 50 mg maltose in 50 ml distilled water in a standard flask and store it in a refrigerator.

vi. Extraction of Amylases:

Extract 1 g of sample material with 5-10 volumes of ice-cold 10 mM calcium chloride solution overnight at 4°C or for 3 h at room temperature. Centrifuge the extract at 54,000 g at 4°C for 20 min. The supernatant is used as enzyme source.

vii. Extraction of β-Amylases (free and bound):

The free β-amylase is extracted from acetone defatted sample material in 66 mM phosphate buffer (pH 7.0) containing 0.5 M NaCl. The extract is centrifuged at 20,000 rpm for 15 min. The supernatant is used as a source of free β-amylase. The pellet is then extracted with phosphate buffer containing 0.5% 2-mercaptoethanol. The clear extract is used as source of bound β-amylase. All operations are carried out at 4°C.

Test Procedure:

1. 1 ml of starch solution and 1 ml of properly diluted enzyme is pipetted in a test tube.

2. It is incubated at 27°C for 15 min.

3. The reaction is stopped by the addition of 2 ml of di-nitro-salicylic acid reagent.

4. The solution is then heated in a boiling water-bath for 5 min.

5. While the tubes are warm, add 1 ml potassium sodium tartrate solution.

6. Then cool it in running tap water.

7. The volume is made to 10 ml by addition of 6 ml water.

8. The absorbance is read at 560 nm.

9. Terminate, the reaction at zero time in the control tubes.

10. A standard graph with 0-100 µg maltose is prepared.

Calculation:

A unit of α- or β-amylase is expressed as mg of maltose produced during 5 min incubation with 1% starch.

Note:

The extraction procedure given is suitable for cereal grains. There are a variety of extraction procedures used for the purpose depending upon the source material. For instance, the plant tissue is extracted in precooled 20% aqueous glycerol and the filtrate is used as enzyme source of amylases.

Home››Plants››