title: Purification, sequencing, and biochemical characterization of a novel calcium-independent α-amylase AmyTVE from Thermoactinomyces vulgaris.
abstract: α-Amylase from Thermoactinomyces vulgaris was highly purified 48.9-fold by ammonium sulfate precipitation, gel filtration on Sephadex G-50 column, and ion exchange chromatography column of DEAE-cellulose. The molecular weight of the enzyme was estimated to be 135 and 145 kDa by SDS-PAGE. Its high molecular weight is due to high glycosylation. The purified amylase exhibited maximal activity at pH 6.0 to 7.0 and was stable in the range of pH 4.0 to 9.0. The optimum temperature for its activity was 50 °C. The enzyme half-life time was 120 min at 50 °C, suggesting intermediate temperature stable α-amylase. The enzyme was sensitive to different metal ions, including NaCl, CoCl(2), and CaCl(2), and to different concentrations of EDTA. The enzyme activity was inhibited in the presence of 1 mM CaCl(2), suggesting that it is a calcium-independent α-amylase. The TLC showed that the amylase hydrolyzed starch to produce large maltooligosaccharides as the main products. A 1.1-kb DNA fragment of the putative α-amylase gene (amy TVE) from T. vulgaris was amplified by using two specific newly designed primers. Sequencing analysis showed 56.2 % similarity to other Thermoactinomyces α-amylases with two conserved active sites confirming its function.
authors.affiliation: Botany Department, Faculty of Science, Damietta University, P.O. Box 34517, New Damietta, Egypt.
authors.forename: Ahmed K A
authors.initials: AK
authors.lastname: El-Sayed
authors.forename: Mohamed I
authors.initials: MI
authors.lastname: Abou Dobara
authors.forename: Amira A
authors.initials: AA
authors.lastname: El-Fallal
authors.forename: Noha F
authors.initials: NF
authors.lastname: Omar
mesh_terms: D004795:Enzyme Stability; D006031:Glycosylation; D056509:Thermoactinomyces / Q000201:enzymology* / Q000235:genetics; D000516:alpha-Amylases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D000516:alpha-Amylases