Role of the calcium-binding residues Asp231, Asp233, and Asp438 in alpha-amylase of Bacillus amyloliquefaciens as revealed by mutational analysis.

Role of the calcium-binding residues Asp231, Asp233, and Asp438 of Bacillus amyloliquefaciens alpha-amylase (BAA) on the enzyme properties was investigated by site-directed mutagenesis. The calcium-binding residues Asp231, Asp233, and Asp438 were replaced with Asn, Asn, and Gly to produce the mutants D231N, D233N, and D438G, respectively. The mutant amylases were purified to homogeneity and the purified enzymes was estimated to be approximately 58 kDa. The specific activity for the mutant enzyme D233N was decreased by 84.8%, while D231N and D438G showed a decrease of 6.3% and 3.5% to that of the wild-type enzyme, respectively. No significant changes in the K (m) value, thermo-stability, optimum temperature, and optimum pH were observed in the mutations of D231N and D438G, while substitution of Asp233 with Asn resulted in a dramatic reduction in the value of catalytic efficiency (K (cat)/K (m)) and thermo-stability at 60 degrees C. The ranges of optimum temperature and optimum pH for D233N were also reduced to about 10 degrees C and 3-4 units, respectively.