Efficient purification and biochemical characterization of a thermostable and organic solvent-stable α-amylase from...

Thermophilic bacteria from four geothermal springs in Türkiye were screened for extracellular α-amylase production, and isolate SO7 was identified as Bacillus licheniformis by phenotypic, biochemical, enzymatic, and 16 S rRNA gene analyses. α-Amylase from B. licheniformis SO7 was evaluated using starch affinity purification, ethanol and acetone precipitation, and three-phase partitioning (TPP). TPP yielded 71-fold purification with 98.6% recovery; the TPP aqueous phase showed 0.1482 U/mL activity and 592.8 U/mg specific activity. The purified enzyme had an apparent molecular mass of approximately 83.17 kDa, an activity optimum at pH 7.0 and 90 °C, and retained substantial activity after alkaline-pH and thermal pre-incubation. It also retained activity after short-term exposure to selected surfactants, inhibitors, metal ions, and organic solvents. Rice starch gave the highest relative activity, with Km = 3.24 mg/mL and Vmax = 204.08 mmol mL⁻¹ min⁻¹. Measurements were obtained from three independent experiments. These in vitro findings indicate potential applicability of SO7 α-amylase in high-temperature and alkaline bioprocesses; however, solvent tolerance was assessed only under the tested short-term conditions, and performance under real industrial process conditions remains to be validated.