Purification and characterization of glucoamylase from Bacillus sp. isolated from root flora of Prunus mahaleb tree...

Glucoamylases are enzymes that release free glucose by hydrolyzing consecutive α-1,4 bonds at the non-reducing ends of starch molecules. This enzyme was purified using ammonium sulfate precipitation, and its molecular mass was determined to be approximately 65.2 kDa via SDS-PAGE. Using zymogram analysis, the purified sample's active glucoamylase content was verified. Nano-Liquid Chromatography Mass Spectrometry (nLC-MS/MS) characterization identified peptides covering 46 % of the glucoamylase protein sequence, indicating partial characterization of the enzyme. It was found that the ideal pH and temperature for glucoamylase activity were 6.0 and 37 °C, respectively. Using soluble starch as a substrate, the kinetic parameters were calculated: the K_m (substrate concentration at half-maximal velocity) was 30.21 μM, and the V_max (maximum reaction velocity) was 35.59 μmol mg protein^-1 min^-1. The enzyme demonstrated optimal activity with soluble starch, highlighting its specificity for starch hydrolysis. Additionally, the enzymatic activity was enhanced in the presence of CaCl₂, indicating a positive effect of calcium ions. Its optimal conditions and kinetic parameters provide valuable insights for its industrial and biomedical use.