In this article we will discuss about the tests for estimation of amylase in plants.
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.
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.
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.
A unit of α- or β-amylase is expressed as mg of maltose produced during 5 min incubation with 1% starch.
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.