Production, purification, and characterization of thermostable ?-amylase from thermophilic Geobacillus stearothermophilus

Abstract

The -amylase (-1-4-D-glucan glucanohydrolase; EC 3.2.1.1) secreted by Geobacillus stearotermophilus was purified and characterized. Maximum enzyme production was achieved after 24h cultivation at pH 7.0 and 55 degrees C. The enzyme was active in a broad temperature range, between 50 and 80 degrees C, with an optimum at 70 degrees C; and maximum activity was at pH 7.0. The enzyme was purified using 80% ammonium sulfate precipitation, dialysis, Sephadex G-100 gel filtration, and DEAE-cellulose column chromatography, with a 46-fold and 65% recovery and showed a MW of 63kDa by SDS-PAGE. It was determined that the purified enzyme was stable at 50 and 60 degrees C, and pH 7.0. It was determined that the purified enzyme was stable at 50 and 60 degrees C at the end of 2h. The enzyme retained 100% activity pH 7.0 at the end of 3h. The enzyme was activated by Ca2+, Mn2+, and Triton X-100, but strongly inhibited by Cu2+, Zn2+, Fe2+, and Hg2+. The enzyme follows Michaelis-Menten kinetics with K-m and V-max values of 0.051mM and 1.424mol/min, respectively.