In Silico Rational Design And Protein Engineering Of Disulfide Bridges Of An Alpha-Amylase From Geobacillus Sp. To Improve Thermostability

A thermostable alpha-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 degrees C. Through site-directed mutation, the optimum temperature of the mutant AmyS2 (A27C-A90C) is increased from 75 to 80 degrees C and shows enhanced T-1/2 of 104.9 min at 90 degrees 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 T-1/2 (204.9 min at 90 degrees C), compared with free enzyme AmyS2 (104.9 min). Enzyme activity could remain above 90% after 6 reactions. In maize starch saccharification (100 degrees 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.