Amylase Activity Determination Protocol

Amylase activity was assayed according to the DNS method. The amounts of released reducing sugars were determined using the dinitrosalicylic acid method [26 (link)]. The absorbance of the reaction system was measured at 540 nm. Up to 1.5 mL of starch solution was added for amylase activity assays, and the reaction was incubated at 40 °C for 10 min. One unit of enzymatic activity (U) was defined as the amount of enzyme needed to release 1 μmol of glucose equivalent per minute. Protein concentration was measured by using a Bradford reagent kit (Sangon Biotech, China). SDS-PAGE was performed using 12% polyacrylamide to determine protein purity. The protein profile was analyzed by staining gels with Coomassie Brilliant Blue R-250 (Sangon Biotech, China) destaining gels with 10% (w/v) acetate solution.