Production, Purification, and Characterization of Thermostable α-Amylase Produced by Bacillus licheniformis Isolate AI20

An optimization strategy, based on statistical experimental design, is employed to enhance the production of thermostable alpha-amylase by a thermotolerant B. licheniformis AI20 isolate. Using one variant at time (OVAT) method, starch, yeast extract, and CaCl2 were observed to influence the enzyme production significantly. Thereafter, the response surface methodology (RSM) was adopted to acquire the best process conditions among the selected variables, where a three-level Box-Behnken design was employed to create a polynomial quadratic model correlating the relationship between the three variables and alpha-amylase activity. The optimal combination of the major constituents of media for alpha-amylase production was 1.0% starch, 0.75% yeast extract, and 0.02% CaCl2. The predicted optimum alpha-amylase activity was 384 U/mL/min, which is two folds more than the basal medium conditions. The produced alpha-amylase was purified through various chromatographic techniques. The estimated enzyme molecular mass was 55 kDa and the alpha-amylase had an optimal temperature and pH of 60-80 degrees C and 6-7.5, respectively. Values of V-max and K-m for the purified enzyme were 454 mU/mg and 0.709 mg/mL. The alpha-amylase enzyme showed great stability against different solvents. Additionally, the enzyme activity was slightly inhibited by detergents, sodium dodecyl sulphate (SDS), or chelating agents such as EDTA and EGTA. On the other hand, great enzyme stability against different divalent metal ions was observed at 0.1 mM concentration, but 10 mM of Cu2+ or Zn2+ reduced the enzyme activity by 25 and 55%, respectively.