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

Abstract 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 K m and V max of 2.4 mg/mL and 833.3 U/mg, respectively. Maltose was found to be competitive inhibitor of the enzyme with inhibition constant ( K i ) of 14 mM. However, metallic ions like Ag + and Hg 2+ 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 R 2  > 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.