The calcium-binding residues, Tyr302 and His235, and the sodium-binding residue, Asp194, on the activity of alpha-amylase were investigated using site-directed mutagenesis. Tyr302 and His235 were replaced by Asn and Asp, respectively, to produce the mutants Y302N and H235D; Asp194 was replaced by Ala to produce D194A. The mutant amylases were purified to homogeneity; each was 53 kDa. The specific activity of the D194A was 236 U mg(-1), lower than the specific activity of the wild-type enzyme by 55%. No significant changes of thermostability, optimum temperature, and optimum pH level were observed in D194A. Mutant amylases with H235D and Y302N significantly improved their specific activity by 43% (754 U mg(-1)) and 7% (563 U mg(-1)), respectively, compared with the wild-type enzyme. H235D substitution decreased its optimum pH by approx. 0.5-1 pH unit.