Characteristics of a novel, highly acid- and thermo-stable amylase from thermophilic Bacillus strain HUTBS62 under different environmental conditions

A novel, highly acid- and thermo-stable amylase was detected from culture medium of thermophilic Bacillus strain HUTBS62 isolated from a hot spring located near the Dead Sea, Jordan. The enzyme was purified by precipitation with 60% ammonium sulfate, gel filtration on Sephadex G-100 and DEAE ion exchange chromatography. The enzyme was purified 22.7-fold with 11.6% yield. Purified enzyme was a monomer with a molecular mass of 54.2 kDa. The optimum pH and temperature for catalytic activity was pH 4.4 and 90°C, respectively. Roughly 50% of amylase activity remained even after heat treatment for 90 min at 100°C; moreover 90% of the activity was retained after heat treatment for 2 h at 60°C. The half-life of the enzyme at 70°C, 80°C and 90°C was estimated to be 5, 4 and 2 h, respectively. The activation energy of denaturation of the purified enzyme was 3.29 kJ mol−1. The presence of 5 mM metal ion affected amylase activity variably; for example: the presence of cobalt, magnesium, cadmium, and manganese increased amylase activity. On the other hand, iron and sodium decreased residual activity to different extents, while calcium, zinc and copper inhibited amylase activity. The enzyme was active in the presence of 1 and 2 mM EDTA at pH 4.4 and 90°C. The purified amylase was acid- and thermo-stable with novel properties making it suitable for many industrial food purposes.