A mesophilic alpha-amylase (FSA) from Sinomicrobium sp. 5DNS001 is closely related with a thermophilic Pyrococcus alpha-amylase (PWA) in glycoside hydrolase (GH) 13_7 subfamily. In this study, a fusion FSA (FAPBFC) with high hydrolysis efficiency was generated by replacing domain B with corresponding regions of PWA. As a result, the thermostability of FAPBFC was greatly enhanced, with 100% amylolytic activity retained after 3-h incubation at 70 degrees C. Moreover, the pH profile of FAPBFC shifted toward the acidic pH of PWA (pH 5.0-6.0). Isothermal titration calorimetry analyses further verified that FAPBFC had a two metal (Ca, Zn) binding center that is most similar to that of PWA. These variations indicate that domain B replacement is an available and effective strategy for a alpha-amylases engineering: many genetically engineered alpha-amylases with desired catalytic properties can be generated by domain B fusion within various GH13 alpha-amylases.