SITE-DIRECTED MUTAGENESIS OF A THERMOSTABLE ALPHA-AMYLASE FROM BACILLUS-STEAROTHERMOPHILUS - PUTATIVE ROLE OF 3 CONSERVED RESIDUES

The relationship between structure, activity, and stability of the thermostable Bacillus stearothermophilus α-amylase was studied by site-directed mutagenesis of the three most conserved residues. Mutation of His-238 to Asp involved in Ca2+and substrate binding reduced the specific activity and thermal stability, but did not affect the pH and temperature optima. Replacement of Asp-331 by Glu in the active site caused almost total inactiva-tion. Interestingly, in prolonged incubation this mutant enzyme showed an altered end-product profile by liberating only maltose and maltotriose. Conservative mutation of the conserved Arg-232 by Lys, for which no function has yet been proposed, resulted in lowered specific activity: around 12% of the parental enzyme. This mutant enzyme had a wider pH range but about the same temperature optimum and thermal stability as the wild-type enzyme. Results obtained with different mutants were interpreted by computer aided molecular modeling. © 1990 COPYRIGHT, 1990 BY THE JOURNAL OF BIOCHEMISTRY.