Purification, kinetic and thermodynamic studies of a new ribonuclease from a mutant of Aspergillus niger

Ribonuclease was purified from Aspergillus niger SA-13-20 to homogeneity level by using (NH4)2SO4 precipitation, DEAE-cellulose anion-exchange chromatography, ultrafiltration and Sephacryl HR-200 chromatography. The molecular weight and isoelectric point of the enzyme was 40.1 kDa and 5.3, respectively. The pH- and temperature-dependent kinetic parameters were determined. The RNase showed the strongest affinity with RNA as the substrate, and the highest catalytic efficiency for hydrolysis of the substrate at pH 3.5 and 65 °C. It exhibited Michaelis-Menten Kinetics with kcat of 118.1 s−1 and Km of 57.0 μg ml−1, respectively. Thermodynamic parameters for catalysis and thermal denaturation were also determined. Activation energy (Ea) for catalysis of A. niger SA-13-20 RNase was 50.31 kJ mol−1 and free energy (ΔG#), enthalpy (ΔH#) and entropy (ΔS#) of activation for catalysis of the enzyme at 65 °C were 69.76, 47.50 and −65.83 J mol−1 K−1, respectively. Activation energy (Ea,d) for denaturation of the enzyme was 200.53 kJ mol−1 and free energy (ΔGd#), enthalpy (ΔHd#) and entropy (ΔSd#) of activation for denaturation of the enzyme at 45 °C were 79.18 kJ mol−1, 197.88 and 373.09 J mol−1 K−1, respectively.