Bacillus amyloliquefaciens α-amylase(BAA) is one of the most widely used industrial enzymes in the starch processing,food,brewing,fermentation,textile,papermaking,and medical industries.To further enhance fermentation level and application property,the mutant with significantly improved catalytic properties was constructed via in vitro molecular evolution and its nature was preliminarily illustrated.The BAA mutation library,with the mutational frequency of 56%,the efficiency of mutation of 2.8 points/kb DNA,and the missense mutation rate of26.8%,was constructed with the capacity of 2×10~4 transformants by using error-prone PCR.The BAA mutants with enzymatic activity were screened using halo-plate assay and shaking flask fermentation test.Six mutants with improved activity were selected out and one of which,mutant BAA28,displayed 37% increased activity.Sequencing results illustrated that mutant BAA28 had four separated mutations:T341 P,P348 L,T356 P,and P362 L and of which,T341 P and T356 P were in the random coil structure,P348 L was in the !-helix structure,and P362 L was in the "-fold structure.The BAA28 was further heterologously prepared and purified and its biochemical properties were examined in comparison to wild BAA.BAA28 displayed a remarkable change in ion-dependence and thermostability.Its specific activity was significantly increased with 1.31 folds of BAA and κcat/Kmwas increased by 92%.BAA28 mutant performed significantly improved catalysis and application properties in comparison to BAA,which is of potential application in BAA-overproducing strain improvement and industrial properties.