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.

Process:

• Standard solutions of maltose (0-10 μmoles/l) are prepared in test tubes.
• 1 ml of D.N.S. reagent is added in each tube and the mixture is agitated for a few seconds on vortex mixer.
• The samples are placed in a water bath (T=100°C) for 5 min and then they are left to cool at room temperature.

• 5 ml of deionized water are added in each sample, followed by agitation.
• The absorbance (A) of the samples is measured at λ=540 nm.
• A standard curve is being drawn
• Then the absorbance of each one of the unknown samples is measured and the concentration of the converting sugars is determined, based on the standard curve.

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.