Enhancing thermostabilization of a newly discovered α-amylase from Bacillus cereus GL96 by combining computer-aided directed evolution and site-directed mutagenesis.

This study has described the characterization of a new a-amylase from the recently isolated Bacillus cereus GL96. Subsequently, an in-silico approach was taken into account to redesign the enzyme to meet higher thermal stability. Finally, the engineered enzyme was constructed experimentally using side-directed mutagenesis (SDM) and characterized accordingly. The enzyme was stable over pH 4-11, with the highest activity at 9.5. The temperature profile of the wild-type enzyme showed optimum activity at 50 °C plus 40% of stability at temperatures up to 70 °C. The in-silico result was indicated D162W, D162R, and D162K as the three stabilizing mutations. Among them, D162K showed better results, especially in the molecular dynamics simulation, and therefore, it was constructed by SDM. This variant was shown 5 °C higher optimum temperature (55 °C) with increasing activity than the native enzyme. In addition, it was significantly more stable than the native form. For example, while the latter almost wholly lost its function at a temperature above 70 °C, the D162K can retain more than 40% of its initial activity up to 80 °C. Considering the promising properties that the mutant enzyme showed, it can be considered for further investigation to meet the industrial requirement completely.