Purification and Characterization of an Endoglucanase of Scytalidium thermophilum Possessing Novel Catalytic and Thermodynamic Properties
Muhammad Ibrahim Rajoka,
Muhammad Imran Qadir,
Shazia A. Bukhari,
Endo-glucanase (CMCase) is widely used in detergents, textile and biofuel industry as color caring agent,
smooth surfacing agent of cotton fabrics and biofuels production from plant biomass. Extracellular CMCase of
Scytalidium thermophilum grown on 2.0% rice polish medium (pH 5.5) for 96 h at 45°C was purified to homogeneity
level by sequential application of ammonium sulphate precipitation, ion exchange and gel filtration chromatography.
CMCase was monomeric with 49 kDa as molecular mass. Temperature- and pH-dependent kinetic parameters for CMC
hydrolysis were determined. Enzyme exhibited stability up to 85°C with t1/2 of 102 min and over a pH range of 3.0-11.0
with maximum t1/2 of 521 min at pH 6.5. Vmax for CMC hydrolysis was 250.0 ± 24 U mg-1 and Km of 5 mg ml-1 at 60 °C.
The turnover number (Kcat) was 50 sec-1 at 60°C. The pKa1 and pKa2 and heat of ionization of ionizable groups of active
site residues controlling Vmax indicated the involvement of Asp or Glu on acidic limb and imidazole group on basic limb.
Thermodynamic parameters (ΔH* = 43.26 kJ mol-1, ΔS* = -161.46 J mol-1.K-1) of irreversible inactivation in a
temperature range of 50-85 °C were also determined.
Keywords: Activation energy, enthalpy, entropy, ionizable amino acids, kinetics, Scytalidium thermophilum, endoglucanase,
thermodynamics, turn over number.
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