Purification, characterization, and statistical optimization of a thermostable α-amylase from desert actinobacterium Streptomyces fragilis DA7-7.

Abstract

In this study, preliminary screening revealed that of 134 desert soil actinobacterial isolates, only 43 isolates produced amylase. Among these, an isolate DA7-7, which was identified as Streptomyces fragilis DA7-7, showed a prominent zone of clearance and significant amount of α-amylase production. The pre-optimization studies showed varying physicochemical and nutrients properties of the medium influenced the enzyme production significantly. Consequently, central composite design was employed with the selected variables (pH, temperature, dextrose, and peptone) for α-amylase production. The optimum fermentation conditions were 3.07% dextrose, 1.085% peptone, pH 6.0, and incubation temperature 27.27°C. The predicted optimum α-amylase activity was 991.82U/mL/min, which was similar to the experimental amylase activity of 973.5U/mL/min. The crude α-amylase produced by S. fragilis DA7-7 was purified with ammonium sulfate precipitation, followed by gel filtration chromatography, and the estimated molecular mass was 51kDa. The purified α-amylase was stable under the following conditions: pH (4-9), temperature (40-80°C), NaCl (1-4M), and detergents (1-10mM). The Km and Vmax values of enzyme were found to be 0.624mU/mg and 0.836mg/mL, respectively.