Purification and characterization of two alpha-amylases from wheat

ABSTRACT

Using chromatography on DEAE-cellulose, four forms, A1, A2, A3 and A4 of germinated wheat Sakha 8 alpha-amylases were demonstrated. alpha-Amylases A2 and A3 were purified to homogencity by Sephacryl S-200 column and the homogeneity proved by polyacrylamide gel electrophoresis. The molecular masses of alpha-amylases A2 and A3 were 29,000 and 31,000, respectively. The Km values of alpha-amylases A2 and A3 were 7.8 and 5.0 mg starch/ml, respectively. The affinity between substrate and the two enzymes decreased in the order of potato starch > glycogen > starch > corn starch for alpha-amylase A2 and glycogen > potato starch > starch > corn starch for alpha-amylase A3. The two enzymes were found to have the same pH and temperature optima of 7.5 and 50°C, and were heat stable up to 50°C. The effect of metal ions showed that Ca[+2], Co[+2] and Mg[+2] caused activating effects on alpha-amylases A2 and A3, while Cd[+2], Fe[+2], Cu[+2], Ni[+2] and Hg[+2] caused partial or strong inhibition effects. The three metal chelators, EDTA, citric and oxalic acids, have the same pattern of inhibition toward alpha-amylases A2 and A3, where 1 mM of these chelators caused 50% inhbiition of amylolytic activity and most enzyme activity was lost at 20 mM. This information will have a bearing on their potential use in the food industries