Enzyme assay
Amylase was determined by using soluble starch, 1%
(w/v), as substrate in 0.05 M Sodium phosphate buffer
(pH 6.5) essentially according to Gomes et al. (2001).
The reaction mixture containing 1.8 ml substrate solution
and 0.2 ml suitably diluted enzyme solution was incubated
at 50°C for 10 min. The reaction was stopped
by adding 3 ml dinitrosalicylic acid (DNS). The reducing
sugar released was determined by the method of Miller
(1959). The absorbance was measured at 540 nm with
spectrophotometer (Jenway 6305, USA). One unit (U) of
enzyme activity is defined in all cases as the amount of
enzyme releasing 1 μg of reducing sugar as maltose per
minute, under assay conditions.