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 and its temperature quotient (Q) was 1.0. The pK and pK 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, ΔG*=59.75kJmol ΔS*=-95.90JmolK, (ΔG*)=3.66kJmol and ΔG* -12.61kJmol. The pure enzyme exhibited K, V and k of 4.13mM, 3507Umgprotein and 551.4s 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 Ca, and Mg while Hg, Fe, and Cu strongly inhibited it. Hydrolysis of tannic acid by the pure enzyme indicated that gallic acid was the end-product.