Improvement of the thermal stability of a calcium-free, alkaline alpha-amylase by site-directed mutagenesis

Alkaline α-amylase from Bacillus sp. strain KSM-K38 (AmyK38) is a calcium-free enzyme that is stable against chelating and oxidative reagents. Recently, the thermostability of this enzyme was improved by replacement of its amino-terminal 11 amino acid residues with the corresponding residues of α-amylase from Bacillus sp. strain KSM-1378. In this study, to further improve the thermal stability, we compared the three-dimensional structure of AmyK38 with that of a hyper-thermostable α-amylase from Bacillus licheniformis. Using site-directed mutagenesis, we created a new possible ionic interaction in the flexible loop region from Gln167 to Gln170 of AmyK38 because the corresponding region in α-amylase from B. licheniformis has an ionic interaction between Glu167 and Lys170. Substitution of Gln167 or Tyr169 with Glu or Lys, respectively, was found to enhance the thermostability of AmyK38. Combination of both substitutions with replacement of the amino-terminal 11 residues further improved the thermostability of the enzyme.