Gene sequence, modeling, and enzymatic characterization of α-amylase AmyLa from the thermophileLaceyellasp. DS3

α-Amylase (AmyLa) from Laceyella sp. DS3 was purified 13.8-fold by ammonium sulfate precipitation, gel filtration, and ion exchange chromatography. The purified AmyLa showed an optimum pH of 7 and an optimum temperature 55°C. It had activation energy, Km, and Vmax of 196.1 kJ, 0.81 mg/mL, and 2.85 mg/min, respectively. Co2+, Cu2+, Hg2+, and Na+ were strong inhibitors of AmyLa whereas Cl− was an activator. Although AmyLa is a Ca2+ independent α-amylase, it was activated by Ca2+. The AmyLa gene encoding α-amylase activity was sequenced. It revealed putative triple promoters and an open reading frame of 1449 bp (483 amino acids) including a 29 amino acid putative signal peptide. AmyLa was shown to possess four conserved sequence regions of the GH13 family. Polyacrylamide gel electrophoresis identified 69.2 and 72.5 kDa proteins whose α-amylase activities were confirmed by native gel staining. Modeling of AmyLa revealed three domains A–C with domain A containing a catalytic (β/α)8 barrel. Phylogenetic and evolutionary tree analysis suggests that AmyLa might be have been transferred from fungi to an ancient thermoactinomycete ancestor, then adapted to bacterial genome features.