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 106 kDa. The activation energy for tannic acid hydrolysis was 32.6 kJ mol⿿1 and its temperature quotient (Q10) was 1.0. The pKa1 and pKa2 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: οH* = 30.02 kJ mol⿿1, οG* = 59.75 kJ mol⿿1 οS* = ⿿95.90 J mol⿿1 K⿿1, (οG*E-S) = 3.66 kJ mol⿿1 and οG*E-T ⿿12.61 kJ mol⿿1. The pure enzyme exhibited Km, Vmax and kcat of 4.13 mM, 3507 U mg protein⿿1 and 551.4 s⿿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 2.95 min, 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.