Kinetics, thermodynamics, and physicochemical properties of 1, 4-α-d-glucan glucohydrolase from Aspergillus oryzae NRRL1560

Abstract

The aim of the study was to produce Ca2+ and Mg2+ independent raw starch hydrolyzing 1,4-α-d-glucan glucohydrolase (GGH) for glucose and fructose syrup production. A thermophilic GGH was produced from Aspergillus oryzae NRRL 1560. Two iso-forms of the enzyme, i.e., GGH1 and GGH2, were purified to homogeneity up to 4.93- and 8.82-fold that showed broad bands of 40 and 48 kDa on SDS-PAGE, respectively. Km and Vmax values of GGH2 were 1.315 mg mL−1 and 70.864 μmol mL−1 min−1, respectively. Enzyme showed optimum activity at 60 °C, pH 5.0. It displayed a broad range of pH (3.0–9.0) and temperature (30–60 °C) stability. Inactivation kinetics parameters were calculated as Kd = 0.014 min−1, t1/2 = 49.5 min, D = 164.5 min, z = 22.23 °C, Tm = 77 °C, and Ea(d) = 337.481 kJ/mol. Thermodynamics parameters for thermal unfolding were ΔG*(d) = 264.323, ΔH*(d) = 334.713 kJ/mol, and ΔS*(d) = 0.211 kJ/mol K. GGH2 was activated by 1 mM Na+ (18%), K+ (15%), Mg2+ (6%), Ca2+ (1%) and inhibited by Cu2+ (21%), Zn2+ (10%), Mn2+ (6%) at the same concentrations. The enzyme was also inhibited by 5 mM SDS (95%), DTT (8%), β-mercaptoethanol (4%), and EDTA (2%). GGH2-hydrolyzed raw starches were in the following order rice > wheat > corn > sweet potato > potato. This is the first report on raw starch digesting enzyme from A. oryzae. All these features make this enzyme a potential candidate for industrial production of glucose/fructose syrups and ethanol from raw starch via biological conversion.