Characterization of a recombinant amylolytic enzyme of hyperthermophilic archaeon Thermofilum pendens with extremely thermostable maltogenic amylase activity.

A gene (Tpen_1458) encoding a putative alpha amylase from hyperthermophilic archaeon Thermofilum pendens (TfMA) was cloned and expressed in Escherichia coli. The recombinant amylolytic enzyme was purified by Ni-NTA affinity chromatography and its catalytic properties were examined. Purified TfMA was extremely thermostable with a half-life of 60 min at an optimal temperature of 95 degrees C. TfMA activity increased to 136% in the presence of 5 mM CaCl(2). Maximal activity was measured toward gamma-cyclodextrin with a specific activity of 56 U/mg using copper bicinchoninate method. TfMA catalyzed the ring-opening reaction by cleaving one alpha-1,4-glycosidic linkage of cyclodextrin to produce corresponding single maltooligosaccharide at the initial time. The final products from cyclodextrins, linear maltooligosaccharides, and starch were glucose and maltose, and TfMA could also degrade pullulan and amylase inhibitor acarbose to panose and acarviosine-glucose, respectively. These results revealed that TfMA is a novel maltogenic amylase.