Biochemical and thermodynamic characterization of a novel α-amylase from Avena fatua for biotechnological applications

This study reports the production, purification, and characterization of a thermostable, raw starch-hydrolysing α-amylase from Avena fatua seeds. The enzyme was purified to a 16.5-fold increase in purity through a series of steps, including pH adjustment, lyophilization, PEG precipitation, and multiple chromatographic techniques, ultimately achieving a specific activity of 90 U/mg. SDS-PAGE analyses confirmed the enzyme's purity and monomeric structure, with a molecular weight of approximately 29 kDa. The enzyme showed high stability at 4 °C, with a half-life of 90 days, extended to 121 days with acetaminophen. Kinetic values included K_M of 0.5 mM, V_max of 119 μmol/min/mg of protein, and kcat of 335 s⁻¹. Activity was enhanced by Co²⁺, Ca²⁺, Mg²⁺, Ni²⁺, NH₄⁺, NAD⁺, glycine, F1,6BP, and phenylalanine, while Mn²⁺, Li⁺, K⁺, NADH, ADP, ATP, citrate, and urea inhibited it. Thermal inactivation studies at 60 °C revealed that the enzyme followed first-order kinetics, with a rate constant of 0.005 min⁻¹. It effectively hydrolysed raw corn and wheat starch (36.7 % and 39.2 % respectively). Hydrolysis products were identified using thin-layer chromatography. The α-amylase also showed potential for industrial use in apple juice clarification and detergent formulations. The enzyme functioned independently of divalent cations but responded variably to different effectors, suggesting potential for industrial starch processing applications.