Purification, characterization, and synergistic action of phytate-resistant α-amylase and α-glucosidase from Geobacillus thermodenitrificans HRO10

The α-amylase (1, 4-α-d-glucanohydrolase; EC 3.2.1.1) and α-glucosidase (α-d-glucoside glucohydrolase; EC 3.2.1.20) secreted by Geobacillus thermodenitrificans HRO10 were purified to homogeneity (13.6-fold; 11.5% yield and 25.4-fold; 32.0% yield, respectively) through a series of steps. The molecular weight of α-amylase was 58 kDa, as estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The α-amylase activity on potato starch was optimal at pH 5.5 and 80 °C. In the presence of Ca2+, the α-amylase had residual activity of more than 92% after 1 h of incubation at 70 °C. The α-amylase did not lose any activity in the presence of phytate (a selective α-amylase inhibitor) at concentrations as high as 10 mM, rather it retained 90% maximal activity after 1 h of incubation at 70 °C. EGTA and EDTA were strong inhibitory substances of the enzyme. The α-amylase hydrolyzed soluble starch at 80 °C, with a Km of 3.05 mg ml−1 and a Vmax of 7.35 U ml−1. The molecular weight of α-glucosidase was approximately 45 kDa, as determined by SDS-PAGE. The enzyme activity was optimal at pH 6.5–7.5 and 55 °C. Phytate did not inhibit G. thermodenitrificans HRO10 α-glucosidase activity, whereas pCMB was a potent inhibitor of the enzyme. The α-glucosidase exhibited Michaelis–Menten kinetics with maltose at 55 °C (Km: 17 mM; Vmax: 23 μmol min−1 mg−1). Thin-layer chromatography studies with G. thermodenitrificans HRO10 α-amylase and α-glucosidase showed an excellent synergistic action and did not reveal any transglycosylation catalyzed reaction by the α-glucosidase.