Molecular analysis of Bacillus velezensis KB 2216, purification and biochemical characterization of alpha-amylase

Alpha-amylase producing strain KB 2216 was identified as Bacillus velezensis. The growth pattern showed that 72 h is the optimum incubation period of amylase production, which is a stationary period for the strain. By the purification process, maximum alpha-amylase activity was achieved up to 418.25 U/mL at 72 h of incubation, which was purified with 4.74 folds, 4230.32 U/mg specific activity, with 31.35% yield. The strain was found to produce an oligomeric alpha-amylase, namely Amy3. Amy3 was a trimeric macromolecule of 195 kDa with 62, 64, and 66 kDa subunits, as revealed by zymogram and SDS PAGE analyses. Amy3 was highly active at 55 °C and pH 5.5. It had shown the highest stability at pH 5.0-5.5 and between 0 ̊C and 4 ̊C. It did not require any metal cofactors, but it was inhibited by Ag²⁺, Hg²⁺ and Cd²⁺ divalent cations. Glucose and maltose were shown to be the end products of soluble starch digestion by Amy3. These interesting properties of Amy3 may be useful for many biotechnological applications in the future.