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

Abstract

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.

Purification, characterization and thermodynamics of antifungal protease from Streptomyces sp. A6

“…The study of these thermodynamic parameters at 40 °C suggested that the thermal stability of enzyme was due to higher value of ΔG* and negative value of ΔS* which enabled the enzyme to resist against thermal denaturation. Similar results have been reported for the endoglucanase (CMCase) of Aspergillus oryzae cmc-1 and chitinase of Pantoea dispersa [18,19].…”

Thermostability and thermodynamic characterization of sprouted pearl millet ?-amylases for its biotechnological applications

“…Once the Ea (D) barrier has been overcome, the enzyme is denatured (I) and cannot refold to the native form. This is known as the irreversible thermal denaturation of the enzyme (Siddiqui et al, 1997;Javed et al, 2009). The Ea (D) was calculated from the lines of best fit from the Arrhenius plots of the rate of thermal denaturation (K d ) versus temperature (Fig.…”

Structural characterization, catalytic, kinetic and thermodynamic properties of Aspergillus oryzae tannase

“…The maximum reaction velocity (V max ), Michaelis-Menten constant (K m ), specificity constant (V max /K m ), the turnover number (k cat ), free energy of transition state binding (G* E−T ) and free energy of substrate binding (G* E−S ) were determined as described by Javed et al [19] as follows: …”