α-Amylase assay.
The amylase assay was based on reduction of blue color intensity resulting from enzyme hydrolysis of starch
(1). The reaction contained 1 ml enzyme (cell free supernatant) and 10 ml of 1% starch solution incubated at 50°C for 10 min.
The reaction was stopped by adding 10 ml of 0.1N HCl. One millilitre of this acidified solution was added to 10 ml of 0.1N HCl.
1 ml of this was added to 10 ml iodine solution (0.05% iodine in 0. 5% KI).
The optical density of the blue-colored solution was determined at 660 nm.
The same procedure was repeated using 1 ml distilled water instead of the enzyme sample in order to measure the optical density without the enzyme.
One unit of enzyme activity (defined unit number) is defined as DUN and the quantity of enzyme that causes 1% reduction of blue color intensity of starch-iodine solution a 50°C in 1 min (6).