Purification and Characterization of Thermostable and Detergent-Stable α-Amylase from Anoxybacillus sp AH1

A thermostable and detergent-stable alpha-amylase from a newly isolated Anoxybacillus sp. AH1 was purified and characterized. Maximum enzyme production (1874.8 U/mL) was obtained at 24 h of incubation. The amylase was purified by using Sephadex G-75 gel filtration, after which an 18-fold increase in specific activity and a yield of 9 % were achieved. The molecular mass of the purified enzyme was estimated at 85 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH and temperature values of the enzyme were 7.0 and 60 degrees C, respectively. The enzyme was highly stable in the presence of 30 % glycerol, retaining 85 % of its original activity at 60 degrees C within 120 min. K-m and v(max) values were 0.102 mu mol and 0.929 mu mol/min, respectively, using Lineweaver-Burk plot. The enzyme activity was increased by various detergents, but it was significantly inhibited in the presence of urea. Mg2+ and Ca2+ also significantly activated alpha-amylase, while Zn2+, Cu2+ and metal ion chelators ethylenediaminetetraacetic acid (EDTA) and 1,10-phenanthroline (phen) greatly inhibited the enzyme activity. alpha-Amylase activity was enhanced by beta-mercaptoethanol (beta-ME) and dithiothreitol (DTT) to a great extent, but inhibited by p-chloromercuribenzoic acid (PCMB). Iodoacetamide (IAA) and N-ethylmaleimide (NEM) had a slight, whereas phenylmethylsulfonyl fluoride (PMSF) had a strong inhibitory effect on the amylase activity.