Abstract

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 t_1/2 of 102 min and over a pH range of 3.0-11.0 with maximum t_1/2 of 521 min at pH 6.5. V_max for CMC hydrolysis was 250.0 ± 24 U mg^-1 and K_m of 5 mg ml^-1 at 60 °C. The turnover number (K_cat) was 50 sec^-1 at 60°C. The pK_a1 and pK_a2 and heat of ionization of ionizable groups of active site residues controlling V_max 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.

Graphical Abstract