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Identification and Characterization of a Novel Intracellular Alkaline [alpha]-Amylase from the Hyperthermophilic Bacterium Thermotoga maritima MSB8

Ballschmiter, Meike, Fütterer, Ole, Liebl, Wolfgang
Applied and environmental microbiology 2006 v.72 no.3 pp. 2206-2211
EDTA (chelating agent), Escherichia coli, Thermotoga maritima, adenosine triphosphate, amino acid sequences, bacteria, dithiothreitol, enzyme activity, enzymes, genes, hydrolysis, nucleotide sequences, pH, proteins
The gene for a novel [alpha]-amylase, designated AmyC, from the hyperthermophilic bacterium Thermotoga maritima was cloned and heterologously overexpressed in Escherichia coli. The putative intracellular enzyme had no amino acid sequence similarity to glycoside hydrolase family (GHF) 13 [alpha]-amylases, yet the range of substrate hydrolysis and the product profile clearly define the protein as an [alpha]-amylase. Based on sequence similarity AmyC belongs to a subgroup within GHF 57. On the basis of amino acid sequence similarity, Glu185 and Asp349 could be identified as the catalytic residues of AmyC. Using a 60-min assay, the maximum hydrolytic activity of the purified enzyme, which was dithiothreitol dependent, was found to be at 90°C. AmyC displayed a remarkably high pH optimum of pH 8.5 and an unusual sensitivity towards both ATP and EDTA.