Biochemical Characterization of purified β-amylase from Dioscorea alata (water yam)

Abstract Water yam (Dioscorea alata) β-amylase was isolated and purified to apparent homogeneity. The enzyme was found to be monomeric with a molecular weight of 30.1 kDa based on SDS-PAGE and 31.0 kDa based on non-denaturation gel filtration. The enzyme readily hydrolyzed soluble starch, amylose, amylopectin and glycogen. The enzyme was stable over a wide range of pHs (4 - 8). The enzyme has a Km, of 2.24 mg/ml for soluble starch. Activation energy (Ea) for catalysis by β-amylase at 25 0C was 6.45 kcal/mol. β-amylase Contains high content of both acidic and hydrophobic amino acid but low in arginine and histidine. The activation energy (Ea), half-life, free energy change (ΔG‡), enthalpy change (ΔH‡), and entropy change (ΔS‡) for inactivation were 13.92 kcal/mol, 41.25 min, 20.89 kcal/mol, 13.30 kcal/mol and -24.25 cal/mol/K, respectively. The binding profile of β-amylase with epicatechin, quercetin, rutin and gallic acid were all spontaneous with stoichiometric ratio of 1:1. Hydrophobic bonding played a major role in stabilizing the β-amylase-ligand complex. Sodium alginate immobilization of the enzyme improved the Optimum temperature from 40 to 50 0C and changed the optimum pH from 5.0 to 6.0 with retainment up to 67 % activity after 5 cycles of usage. The enzyme would be of importance in manufacturing company based on the kinetics and other parameters reported in this study.