Purification and characterization of alkaline-stable β-amylase in malted African finger millet (Eleusine coracana) seed

Abstract

Alkaline-stable β-amylase (EC 3.2.1.2) was purified to apparent homogeneity from malted African finger millet (Eleusine coracana) seed by ammonium sulfate fractionation and anion exchange and affinity chromatographies. Gel filtration chromatography together with SDS-PAGE revealed that the enzyme is monomeric, with a molecular weight of 59.1kDa. It has a pI value of 5.2 and is optimally active at pH 5.0 and 50°C. 2DE-MS isolated and identified two isoforms of the enzyme with unique amino acid sequences. The purified enzyme was highly selective for soluble potato starch, amylose and amylopectin, and α-cyclodextrin was shown to be a competitive inhibitor with a Gibbs free energy (ΔG°′) of binding of 18.11kJ/mol. The enzyme was stable at a pH range of 4.0–10.0 and temperature range of 30–70°C. It was irreversibly inactivated by heating to 60°C and 70°C, which was often related to aggregation. The apparent KM of the enzyme for p-nitrophenyl maltopentaoside (PPNG-5) was 2.1mM. NH4+, Hg2+, Al3+, EDTA, ascorbic acid, iodoacetamide, iodoacetic acid and gibberellic acid all inhibited the enzyme. The enzyme unfolded after treatment with 4.0M guanidine–HCl and regained 68% of its original activity upon dilution at 4°C in the presence of 5mM trehalose. The kinetic parameters of renaturation were first order. This enzyme could be important in the malting of African finger millet seeds, based on the kinetics and properties reported in this study.