Purification and biochemical characterization of a novel SDS and surfactant stable, raw starch digesting, and halophilic α-amylase from a moderately halophilic bacterium, Nesterenkonia sp. Strain F

摘要

An extracellular halophilic α-amylase from Nesterenkonia sp. Strain F was purified to homogeneity by 80% ethanol precipitation, Q-Sepharose anion exchange and Sephacryl S-200 gel filtration chromatog-raphy, with a 10.8-fold increase in specific activity. The molecular mass of the amylase was estimated to be 100kDa and 106 kDa by SDS-PAGE and gel filtration chromatography, respectively. The enzyme showed maximal activity at pH 7.5 and 45 ℃. The amylase was active in a wide range of salt concentrations (0-4 M) with its maximum activity at 0.5 M NaCl or 1 M KCl and was stable at the salts concentrations between 1 M and 4 M. Fe~(3+), Cu~(2+), Zn~(2+) and Al~(3+) strongly inhibited the enzyme, whereas Ca~(2+) stimulated the amylase activity. The a-amylase was inhibited by EDTA, but was not inhibited by PMSF and β-mercaptoethanol. The enzyme showed remarkable stability towards 0.5% SDS and sarcosyl, and 2% each of Triton X-100, Tween 80 and Tween 20. K_m value of the amylase for soluble starch was 4.5 mg/ml. The amylase hydrolyzed 38% of raw wheat starch and 20% of corn starch in a period of 48 h. The major products of soluble starch hydrolysis were maltose, maltotriose and maltotetraose, indicating an a-amylase activity.