Production, Purification, and Characterization of Thermostableα-Amylase Produced byBacillus licheniformisIsolate AI20

Abstract

An optimization strategy, based on statistical experimental design, is employed to enhance the production of thermostableα-amylase by a thermotolerantB. licheniformisAI20 isolate. Using one variant at time (OVAT) method, starch, yeast extract, and CaCl2were 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 andα-amylase activity. The optimal combination of the major constituents of media forα-amylase production was 1.0% starch, 0.75% yeast extract, and 0.02% CaCl2. The predicted optimumα-amylase activity was 384 U/mL/min, which is two folds more than the basal medium conditions. The producedα-amylase was purified through various chromatographic techniques. The estimated enzyme molecular mass was 55 kDa and theα-amylase had an optimal temperature and pH of 60–80°C and 6–7.5, respectively. Values ofVmaxandKmfor the purified enzyme were 454 mU/mg and 0.709 mg/mL. Theα-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.