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

The alpha-amylase (1, 4-alpha-D-glucanohydrolase; EC 3.2.1.1) and alpha-glucosidase (alpha-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 alpha-amylase was 58 kDa, as estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The alpha-amylase activity on potato starch was optimal at pH 5.5 and 80 degrees C. In the presence of Ca2+ the alpha-amylase had residual activity of more than 92% after 1 h of incubation at 70 degrees C. The alpha-amylase did not lose any activity in the presence of phytate (a selective alpha-amylase inhibitor) at concentrations as high as 10 mM, rather it retained 90% maximal activity after 1 h of incubation at 70 degrees C. EGTA and EDTA were strong inhibitory substances of the enzyme. The alpha-amylase hydrolyzed soluble starch at 80 degrees C, with a K. of 3.05 mg ml(-1) and a V-max of 7.35 U ml(-1). The molecular weight of a-glucosidase was approximately 45 kDa, as determined by SDS-PAGE. The enzyme activity was optimal at pH 6.5-7.5 and 55 degrees C. Phytate did not inhibit G. thermodenitrificans HRO 10 alpha-glucosidase activity, whereas pCMB was a potent inhibitor of the enzyme. The alpha-glucosidase exhibited Michaelis-Menten kinetics with maltose at 55 degrees C (K-m: 17 mM; V-max: 23 mu mol min(-1) mg(-1)). Thin-layer chromatography studies with G. thermodenitrificans HRO 10 alpha-amylase and alpha-glucosidase showed an excellent synergistic action and did not reveal any transglycosylation catalyzed reaction by the alpha-glucosidase. (c) 2006 Elsevier B.V. All rights reserved.