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 degrees C), and its temperature quotient (Q (10)) was 1.0. The enzyme was stable over a pH range of 4.1-5.3 and gave maximum activity at pH 4.4. V (max) for CMC hydrolysis was 854 U mg(-1) protein and K (m) was 20 mg CMC ml(-1). The turnover (k (cat)) was 356 s(-1). The pK (a1) and pK (a2) of ionisable groups of active site controlling V (max) were 3.9 and 6.25, respectively. Thermodynamic parameters for CMC hydrolysis were as follows: DeltaH* = 0.59 kJ mol(-1), DeltaG* = 64.57 kJ mol(-1) and DeltaS* = -195.05 J mol(-1) K(-1), respectively. Activation energy for irreversible inactivation 'E (a(d))' of the endoglucanase was 378 kJ mol(-1), whereas enthalpy (DeltaH*), Gibbs free energy (DeltaG*) and entropy (DeltaS*) of activation at 44 degrees C were 375.36 kJ mol(-1), 111.36 kJ mol(-1) and 833.06 J mol(-1) K(-1), respectively.