Catalytic and Thermodynamic Characterization of Endoglucanase (CMCase) from Aspergillus oryzae cmc-1

Monomeric extracellular endoglucanase (25 kDa) of transgenic koji (Aspergillus oryzae cmc-1) produced under submerged growth condition (7.5 U mg−1 protein) was purified to homogeneity level by ammonium sulfate precipitation and various column chromatography on fast protein liquid chromatography system. Activation energy for carboxymethylcellulose (CMC) hydrolysis was 3.32 kJ mol−1 at optimum temperature (55 °C), and its temperature quotient (Q10) was 1.0. The enzyme was stable over a pH range of 4.1–5.3 and gave maximum activity at pH 4.4. Vmax for CMC hydrolysis was 854 U mg−1 protein and Km was 20 mg CMC ml−1. The turnover (kcat) was 356 s−1. The pKa1 and pKa2 of ionisable groups of active site controlling Vmax were 3.9 and 6.25, respectively. Thermodynamic parameters for CMC hydrolysis were as follows: ΔH* = 0.59 kJ mol−1, ΔG* = 64.57 kJ mol−1 and ΔS* = −195.05 J mol−1 K−1, respectively. Activation energy for irreversible inactivation ‘Ea(d)’ of the endoglucanase was 378 kJ mol−1, whereas enthalpy (ΔH*), Gibbs free energy (ΔG*) and entropy (ΔS*) of activation at 44 °C were 375.36 kJ mol−1, 111.36 kJ mol−1 and 833.06 J mol−1 K−1, respectively.