Problem statement:This study reported the purification and characterization of a novel highly thermostable alkaline amylase from a newly isolated Bacillus strain HUTBS71. Approach: The enzyme was purified using ammonium sulfate precipitation, ion exchange and gel filtration chromatography. Results: Maximum amylase activity (72 U mL −1 ) was obtained at 100°C after 10 min of incubation. The enzyme was purified 24 fold with 12.5% yield and showed a monomer band with a molecular weight of 58.8 kDa by SDS-PAGE. This enzyme exhibited maximum activity at pH and temperature, 7.8 and 100°C, respectively. It performed stability over a broad range of pH and temperature, 5.2-10.0 and 80-115°C, respectively. The half-life of the enzyme at 90 and 100°C was estimated to be 3 h. The activation energy of denaturation of purified enzyme was 2.53 kJ moL −1 . The enzyme was activated by 5 mM of CoCl 2 , MgSO 4 , MnCl 2 , ZnSO 4 and MnSO 4 (relative activity was 133, 126, 133, 106.6 and 103%, respectively). It was strongly inhibited by CuSO 4 and CdCl 2 but less affected by NaCl, CaCl 2 , FeCl 3 , ZnCl 2 and EDTA. Conclusion: The present purified amylase therefore could be defined as a highly thermostable, extremely hyperthermophilic and alkalitolerant with new properties make the present enzyme applicable for many starch processing and food industries.

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.

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