A quantitative starch–iodine method for measuring alpha-amylase and glucoamylase activities

Abstract

Alpha-amylase (EC 3.2.1.1), which cleaves internal -1,4glycosidic linkages in starch to produce glucose, maltose, or dextrins, and glucoamylase (EC 3.2.1.3), which cuts -1,4and -1,6-glycosidic linkages to release glucose from the nonreducing ends of starch, are widely used in the industrial conversion of starch into sugars. The characterization of -amylases and glucoamylases generally needs to use diVerent chromatography techniques such as paper chromatography [1,2], high-performance liquid chromatography [3,4], and thin-layer chromatography [5,6]. There are mainly two types of assays that are used to determine the activity of -amylase and glucoamylase. One is based on measuring the amount of reducing sugars by the dinitrosalicylic acid (DNS) assay [2,4,5,7–10] or the Nelson–Somogyi [1,11,12] method, whereas the other is based on the decreased staining value of blue starch–iodine complexes [13]. The second method, which was developed by Fuwa [13] and is widely used [10–12,14–16], is based on color development that results from iodine binding to starch polymers. However, the starch–iodine assays reported by diVerent researchers are quite diverse with iodine concentrations ranging from 3 M [12] to 0.25 mM [15] and with the wavelength used to measure color development varying from 550 nm [15] to 700 nm [13]. Moreover, -amylase activity is calculated as relative activity according to the following equation. Dextrinizing activityD (D0-D)¥ D0£ 100¥10, where D is the absorbance of the enzyme sample and D0 is the absorbance of the amylose control without the addition of enzyme [13]. Dextrinizing activity