Identification of an Extracellular Thermostable Glycosyl Hydrolase Family 13 α-Amylase from Thermotoga neapolitana

We cloned the gene for an extracellular alpha-amylase, AmyE, from the hyperthermophilic bacterium Thermotoga neapolitana and expressed it in Escherichia coli. The molecular mass of the enzyme was 92 kDa as a monomer. Maximum activity was observed at pH 6.5 and temperature 75 degrees C and the enzyme was highly thermostable. AmyE hydrolyzed the typical substrates for alpha-amylase, including soluble starch, amylopectin, and maltooligosaccharides. The hydrolytic pattern of AmyE was similar to that of a typical alpha-amylase; however, unlike most of the calcium (Ca2+)-dependent alpha-amylases, the activity of AmyE was unaffected by Ca2+. The specific activities of AmyE towards various substrates indicated that the enzyme preferred maltooligosaccharides which have more than four glucose residues. AmyE could not hydrolyze maltose and maltotriose. When maltoheptaose was incubated with AmyE at the various time courses, the products consisting of maltose through maltopentaose was evenly formed indicating that the enzyme acts in an endo-fashion. The specific activity of AmyE (7.4 U/mg at 75 degrees C, pH 6.5, with starch as the substrate) was extremely lower than that of other extracellular alpha-amylases, which indicates that AmyE may cooperate with other highly active extracellular alpha-amylases for the breakdown of the starch or alpha-glucans into maltose and maltotriose before transport into the cell in the members of Thermotoga sp.