Tannase from Aspergillus niger NL112 was purified 5.1-fold with a yield of 50.44% via ultrafiltration, DEAE-Sepharose Fast Flow column chromatography, and Sephadex G-100 column chromatography. The molecular weight of the purified tannase was estimated as 45 kDa. The optimum temperature and pH for its activity were 45 °C and 5.0, respectively. The results of circular dichroism, FT-IR (Fourier transform infrared) spectroscopy, and fluorescence spectra indicated that high temperature could lead to the change of tannase secondary and tertiary structures. Tannase had a greater affinity for tannic acid at 40 °C with a Km value of 2.12 mM and the greatest efficiency hydrolysis (Kcat/Km) at 45 °C. The rate of inactivation (k) increased with the increase of temperature and the half-life (t1/2) gradually decreased. It was found to be 1.0 of the temperature quotient (Q10) value for tannic acid hydrolysis by tannase. The thermodynamic parameters of the interaction system were calculated at various temperatures. The positive enthalpy (ΔH) values and decreasing ΔH values with the increase of temperature indicated that the hydrolysis of tannase was an endothermic process. Our results indicated that elevated temperature could change the tertiary structure of tannase and reduce its thermostability, which caused a gradual decrease of tannase activity with an increase in temperature.
Keywords: Catalytic properties; High temperature; Protein structure; Tannase; Thermodynamic properties.