The role of the aspartate residues Asp193, Asp217, Asp228, Asp234, Asp236, Asp238, Asp438, and Asp461 of a truncated Bacillus sp. strain TS-23 α-amylase (BACΔNC) was investigated by site-directed mutagenesis. These residues were replaced with Asn to generate D193N, D217N, D228N, D234N, D236N, D238N, D438N, and D461N, respectively. Parental and mutant amylases were purified to homogeneity by nickel-chelated chromatography, and the purified enzymes had a molecular mass of approximately 54 kDa. The amylolytic activity of purified BACΔNC was abolished completely upon the addition of 15 mM EDTA. D438N and D461N were very similar to BACΔNC in terms of specific activity, temperatureactivity profiles, and kinetic parameters, whereas the remaining mutant enzymes showed a dramatic reduction in both catalytic efficiency (k cat /K M ) and thermostability. Compared with BACΔNC, measurement of intrinsic tryptophan fluorescence revealed the minor alterations of the microenvironment of aromatic amino acid residues in all of mutant enzymes. Far-UV circular dichroism spectra were nearly identical for the parental and mutant enzymes, but D193N, D217N, D228N, D234N, D236N, and D238N exhibited a different sensitivity towards temperature-induced denaturation. This implicates that the rigidity of the enzyme has been changed as the consequence of Asp193, Asp217, Asp228, Asp234, Asp236, and Asp238 mutations.
