Site-directed mutagenesis of the calcium-binding site of [alpha]-amylase of Bacillus licheniformis

Amylases that are active under acidic conditions (pH 70°C) and have less reliance on Ca^sup 2+^ are required for starch hydrolysis. The α-amylase gene of Bacillus licheniformis MTCC 6598 was cloned and expressed in Escherichia coli BL21. The calcium-binding site spanning amino acid residues from 104 to 200 in the loop regions of domain B and D430 in domain C of amylase were changed by site-directed mutagenesis and the resultant mutant amylases were analyzed. Calcium-binding residues, N104, D161, D183, D200 and D430, were replaced with D104 and N161, N183, N200 and N430, respectively. Mutant amylase with N104D had a slightly decreased activity at 30°C but a significantly improved specific activity at pH 5 and 70°C, which is desirable character for a food enzyme. The amylase mutants with D183N or D200N lost all activity while the mutant amylase with D161N retained its activity at 30°C but had significantly less activity at 70°C. On the other hand, the activity of the mutant amylase with D430N was not changed at 30°C but had an improved activity at 70°C.[PUBLICATION ABSTRACT]