Purification and characterization of an organic-solvent-tolerant halophilic α-amylase from the moderately halophilic Nesterenkonia sp. strain F.
A halophilic α-amylase produced by Nesterenkonia sp. strain F was purified to homogeneity by 80% ethanol precipitation, Q-Sepharose anion exchange, and Sephacryl S-200 gel filtration chromatography. The purified amylase exhibited specific activity of 357 unit/mg protein that corresponds to twofold purification. The molecular mass of the amylase was determined to be 57 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration chromatography. The optimal pH and temperature for enzyme activity were 6.5 and 45°C, respectively. The amylase was active over a wide range of salt concentrations (0-4 M) with maximum activity at 0.75-1 M NaCl. The α-amylase activity was stimulated by Ca(2+) and inhibited by ethylenediamine tetraacetic acid (EDTA), suggesting that this enzyme is a metalloenzyme. The purified enzyme showed remarkable stability towards surfactants (Tween 20, Tween 80, and Triton X-100), and its activity was increased by β-mercaptoethanol. The halophilic α-amylase was stable in the presence of various organic solvents such as benzene, chloroform, toluene, and cyclohexane. These properties indicate wide potential applications of this α-amylase in starch-processing industries.
Shafiei
Mohammad
M
Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran. shafieimoh@yahoo.com
Ziaee
Abed-Ali
AA
Amoozegar
Mohammad Ali
MA
D004789:Enzyme Activation / Q000187:drug effects*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008670:Metals / Q000494:pharmacology; D008835:Micrococcaceae / Q000201:enzymology*; D012965:Sodium Chloride / Q000494:pharmacology; D012997:Solvents / Q000494:pharmacology*; D013379:Substrate Specificity; D013696:Temperature; D000516:alpha-Amylases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*
D004791:Enzyme Inhibitors; D008670:Metals; D012997:Solvents; D012965:Sodium Chloride; D000516:alpha-Amylases