Preparation of the reagent D.N.S.
10 g of 3,5-dinitrosalicylic acid are gradually dissolved under heating conditions, in 700 ml of NaOH solution 0.5 N. Then 300 g of sodium potassium tartrate (NaKC4O6.4H2O) are added and distillated water was added in the mixture until a final volume of 1000 ml. The reagent has a dark orange colour and is stable for several days in room temperature.
Enzymatic reaction and determination of the enzymatic activity
0.5 ml of properly diluted (in acetic acid buffer solution; pH=4.9) crude enzyme are incubated for 15 min at T=40 °C with 0.5 ml of soluble starch solution 1 % w/v. Afterwards, the produced quantity of reducing sugars released from starch is determined as described previously. As a unit of activity (unit, U) of the enzyme a-amylase, is arbitrarily appointed, the quantity of the enzyme required for the production of 1 μmole of maltose in 1 min, when the enzyme is incubated along with the substrate at pH=4,9 and Τ=40 °C.