Improving the thermostability and enhancing the Ca2+ binding of the maltohexaose-forming α-amylase from Bacillus stearothermophilus

The thermostability of the maltohexaose-forming α-amylase from Bacillus stearothermophilus (AmyMH) without added Ca2+ was improved through structure-based rational design in this study. Through comparison of a homologous model structure of AmyMH with the crystal structure of the thermostable α-amylase from Bacillus licheniformis, Ser242, which located at the beginning of fourth α-helix of the central (β/α)8 barrel was selected for mutation to improve thermostability. In addition, an amide-containing side chain (Asn193) and a loop in domain B (ΔIG mutation), which have been proven to be important for thermostability in corresponding position of other α-amylases, were also investigated. Five mutants carrying the mutations ΔIG, N193F, S242A, ΔIG/N193F, and ΔIG/N193F/S242A were generated and their proteins characterized. The most thermostable mutant protein, ΔIG/N193F/S242A, exhibited a 26-fold improvement in half-life at 95 °C compared to the wild-type enzyme without added Ca2+. Mutant ΔIG/N193F/S242A also exhibited substantially better activity and stability in the presence of the chelator EDTA, demonstrating enhanced Ca2+ binding. These results suggest that mutant ΔIG/N193F/S242A has potential for use in the industrial liquefaction of starch.