In Silico Rational Design and Protein Engineering of Disulfide Bridges of an α‐Amylase from Geobacillus sp. to Improve Thermostability

A thermostable α‐amylase gene (AmySN) from Geobacillus stearothermophilus DMSZ 456 with an open reading frame of 1446 bp, encoding 481 amino acids is cloned. The optimal pH and temperature of AmySN are 6.0 and 75 °C. Through site‐directed mutation, the optimum temperature of the mutant AmyS2 (A27C‐A90C) is increased from 75 to 80 °C and shows enhanced T1/2 of 104.9 min at 90 °C in comparison to the wild type (52.9 min). The mutant AmyS2 is immobilized on epoxy‐functionalized supports to obtain immobilized enzyme: I‐AmyS2. Additionally, I‐AmyS2 shows an enhanced T1/2 (204.9 min at 90 °C), compared with free enzyme AmyS2 (104.9 min). Enzyme activity could remain above 90% after 6 reactions. In maize starch saccharification (100 °C), AmyS2 shows better starch hydrolytic efficiency. The final concentration of reducing sugar is 25.9 mg mL−1, which is higher than the final concentration of wild‐type (22.6 mg mL−1), reducing cost and improving efficiency. A thermostable α‐amylase gene AmySN from Geobacillus stearothermophilus DMSZ 456 is cloned. Through site‐directed mutation, the optimum temperature of the mutant AmyS2 (A27C‐A90C) is increased from 75 to 80 °C and shows enhanced T1/2 of 104.9 min at 90 °C in comparison to the wild type (52.9 min).