Structural characterization, catalytic, kinetic and thermodynamic properties of Aspergillus oryzae tannase

Tannase (EC.3.1.1.20) from Aspergillus oryzae was purified using ammonium sulphate precipitation (75%), gel filtration chromatography through Sephadex G-100, and G-200. The purified enzyme was monomeric protein with a molecular mass of 106kDa. The activation energy for tannic acid hydrolysis was 32.6kJmol^-1 and its temperature quotient (Q₁₀) was 1.0. The pK_a1 and pK_a2 values of acidic and basic limbs of the active site residues were 4.6 and 6.4. The calculated values of thermodynamic parameters for tannic acid hydrolysis, were as follows: ΔH*=30.02kJmol^-1, ΔG*=59.75kJmol^-1 ΔS*=-95.90Jmol^-1K^-1, (ΔG*_E-S)=3.66kJmol^-1 and ΔG*_E-T -12.61kJmol^-1. The pure enzyme exhibited K_m, V_max and k_cat of 4.13mM, 3507Umgprotein^-1 and 551.4s^-1_. The calculated half-life time at 40, 45, 50, 55, 60, and 70°C was 955.15, 142.0, 30.28, 17.88, 8.23 and 2.95min, respectively. The thermodynamic parameters for irreversible thermal inactivation at different temperatures (40-70°C) were determined. The enzyme was activated by Ca²⁺, and Mg²⁺ while Hg²⁺, Fe²⁺, and Cu²⁺ strongly inhibited it. Hydrolysis of tannic acid by the pure enzyme indicated that gallic acid was the end-product.