Purification and characterization of a halophilic α-amylase with increased activity in the presence of organic solvents from the moderately halophilic Nesterenkonia sp. strain F.

An extracellular halophilic α-amylase was purified from Nesterenkonia sp. strain F using 80 % ethanol precipitation and Q-Sepharose anion exchange chromatography. The enzyme showed a single band with an apparent molecular weight of 110 kDa by SDS-PAGE. The amylase exhibited maximal activity at pH 7-7.5, being relatively stable at pH 6.5-7.5. Optimal temperature for the amylase activity and stability was 45 °C. The purified enzyme was highly active in the broad range of NaCl concentrations (0-4 M) with optimal activity at 0.25 M NaCl. The amylase was highly stable in the presence of 3-4 M NaCl. Amylase activity was not influenced by Ca²⁺, Rb⁺, Li⁺, Cs⁺, Mg²⁺ and Hg²⁺, whereas Fe³⁺, Cu²⁺, Zn²⁺ and Al³⁺) strongly inhibited the enzyme activity. The α-amylase was inhibited by EDTA, but was not inhibited by PMSF and β-mercaptoethanol. K(m) value of the amylase for soluble starch was 6.6 mg/ml. Amylolytic activity of the enzyme was enhanced not only by 20 % of water-immiscible organic solvents but also by acetone, ethanol and chloroform. Higher concentration (50 %) of the water-miscible organic solvents had no significant effect on the amylase activity. To the best of our knowledge, this is the first report on increased activity of a microbial α-amylase in the presence of organic solvents.