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

Paper ID Volume ID Publish Year Pages File Format Full-Text
25233 43562 2006 12 PDF Available
Title
Purification, characterization, and synergistic action of phytate-resistant α-amylase and α-glucosidase from Geobacillus thermodenitrificans HRO10
Abstract

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.

Keywords
α-Amylase; α-Glucosidase; Phytate; Geobacillus thermodenitrificans HRO10
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Purification, characterization, and synergistic action of phytate-resistant α-amylase and α-glucosidase from Geobacillus thermodenitrificans HRO10
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 125, Issue 1, 20 August 2006, Pages 27–38
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us