Characteristics of a Novel Highly Thermostable and Extremely Thermophilic Alkalitolerant Amylase from Hyperthermophilic Bacillus Strain HUTBS71

Abstract

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 CoCl₂, MgSO₄, MnCl₂, ZnSO₄ and MnSO₄ (relative activity was 133, 126, 133, 106.6 and 103%, respectively). It was strongly inhibited by CuSO₄ and CdCl₂ but less affected by NaCl, CaCl₂, FeCl₃, ZnCl₂ 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.