Effect of mutation of an amino acid residue near the catalytic site on the activity of Bacillus stearothermophilus alpha-amylase.

Site-directed mutagenesis of a thermostable alpha-amylase from Bacillus stearothermophilus was performed to assess the role of amino acid residues near the catalytic site in catalysis. Asn329 is presumed to be adjacent to the proposed catalytic residue Asp331. Its mutation to Lys, which is found at the corresponding position in pullulanase, resulted in the loss of 99.7% of the activity, while the mutation to Asp or Val did not drastically reduce the activity. The mutation to Val altered the temperature/activity profile so that the activity was reduced to 25% of wild-type alpha-amylase at 60 degrees C but was over twofold greater at 5 degrees C. This effect could be ascribed to a decrease in the activation enthalpy by 32%. The mutation to Asp or Lys altered the pH/activity profile concomitant with possible changes in the ionization state of the groups introduced. These results show the feasibility of altering and possibly improving the enzyme activity by mutagenesis of residues near the catalytic groups.