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 991.82 U/mL/min, which was similar to the experimental amylase activity of 973.5 U/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 51 kDa. The purified α-amylase was stable under the following conditions: pH (4-9), temperature (40-80 °C), NaCl (1-4 M), and detergents (1-10 mM). The K_m and V_max values of enzyme were found to be 0.624 mU/mg and 0.836 mg/mL, respectively.

Keywords: Central composite design; Desert actinobacterium; Gel filtration chromatography; Streptomyces fragilis; α-Amylase.

Fig. 1

Colony morphology and microscopic view of Streptomyces fragilis DA7-7. a Isolate DA7-7 cultured on ISP-2 medium. b Phase-contrast microscopic view of isolate DA7-7

Fig. 2

Phylogenetic tree of the 16S rRNA gene sequence of Streptomyces fragilis DA7-7 and other closely related gene sequences. Numbers in the parentheses indicate GenBank accession numbers

Fig. 3

Graphical representation of surface and contour plots. a1, a2 pH and temperature. b1, b2 pH and dextrose. c1, c2 pH and peptone. d1, d2 Temperature and dextrose. e1, e2 Temperature and peptone. f1, f2 Dextrose and peptone

Fig. 4

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) showing the purified α-amylase enzyme produced by Streptomyces fragilis DA7-7. Lane 1 Molecular marker protein. Lane 2 Crude α-amylase enzyme in the culture filtrate. Lane 3 Purified α-amylase enzyme

Fig. 5

Effects of pH, temperature, NaCl, and detergents on stability of purified α-amylase enzyme. Relative activity (%) of purified α-amylase was determined by comparing to the control activity of 100%