Biochemical and thermodynamic characterization of de novo synthesized β-amylase from fenugreek

β-Amylase has been de novo synthesized from germinating fenugreek seeds. Enzyme has been isolated and purified from 36 h germinated seeds with 226-fold purification and specific activity of 763 U/mg. Homogeneity of the purified β-amylase has been confirmed with size-exclusion chromatography, SDS-PAGE and MALDI MS/MS analysis. The isoelectric point, optimum pH and temperature of the enzyme were found to be pH 5.2, 5.7 and 57 °C, respectively. The enzyme was specific for soluble starch with Km and Vmax of 2.4 mg/mL and 833.3 U/mg, respectively. Maltose was found to be competitive inhibitor of the enzyme with inhibition constant (Ki) of 14 mM. However, metallic ions like Ag+ and Hg2+ were found to be non-competitive inhibitors of the enzyme. Thermodynamic parameters like Gibbs free energy (ΔG), enthalpy (ΔH) and entropy (ΔS) changes have further revealed that thermal denaturation of the enzyme has followed first-order with the enzyme unfolding rather an aggregation with the process being irreversible. The activation energy of β-amylase during thermal activation and denaturation were 27.5 kJ/mol and 145.23 kJ/mol, respectively at R2 > 0.92. Thus, the enzyme was stable even at higher temperature with ability of undergoing catalysis making it commercially exploitable, particularly in food and pharmaceutical industries.