Characterization of α-amylases isolated from Cyperus esculentus seeds (tigernut): Biochemical features, kinetics and thermal inactivation thermodynamics

Abstract

In this study, α-amylases from fresh and dried Cyperus esculentus seeds were isolated and characterized for biochemical and thermodynamic properties. The α-amylases (FTNE-α-amylase and DTNE-α-amylase) were optimally active at 60–70 °C, pH 7.0 (FTNE-α-amylase) and pH 6.0 (DTNE-α-amylase) with activation energies (Ea) of 3.56 and 6.81 kJ mol⁻¹, respectively. Both enzymes retained above 70% of their hydrolytic activities after 1 h at pH 6.0–8.0. The enzyme activities were more enhanced in the presence of divalent metal ions (Ca²⁺, Mg²⁺, Zn²⁺, Cu²⁺, Ba²⁺, Co²⁺, Pb²⁺, and Hg²⁺) than monovalent ions. SDS, cyclohexanol, ether and diethylamine inhibited them except EDTA. Using soluble starch as substrate, DTNE-α-amylase had higher Vₘₐₓ, Kcₐₜ and lower Kₘ than FTNE-α-amylase. Both enzymes expressed high substrate preferences for yam flour amidst other substrates with catalytic efficiencies (Kcₐₜ/Kₘ) of 2.71 and 4.44 mL. mg⁻¹sec⁻¹, respectively. The higher catalytic efficiency of DTNE-α-amylase was due to its transition state stabilization (ΔGE₋S = -0.90 kJ mol⁻¹ and ΔGE₋T = -2.22 kJ mol⁻¹). Fourier Transformed-Infrared (FTIR) spectroscopy revealed changes in C–O stretching and -N-H bending in the substrate-bound enzymes. Thermal inactivation of the amylases followed first-order rate kinetics and was non-spontaneous (ΔG#>0). FTNE-α-amylase half-lives and D-values at 70 and 80 °C, and Z-value (256.41 °C), were however higher than in DTNE-α-amylase (Z-value = 63.29 °C; t₁/₂ = 22.43 min at 70 °C) indicating that FTNE-α-amylase is more thermally stable than DTNE-α-amylase and would be more industrially applicable as a thermostable starch-hydrolyzing enzyme.