Purification and characterization of xylanases from Thermomyces lanuginosus and its mutant derivative possessing novel kinetic and thermodynamic properties

Xylanases produced from a locally isolated strain of Thermomyces lanuginosus and its mutant derivative were purified to a yield of 39.1 and 42.83% with specific activities of 15,501 and 17,778 IU mg⁻¹ protein, respectively. The purification consisted of two steps i.e., ammonium sulphate precipitation, and gel filtration chromatography. The mutant enzyme showed high affinity for substrate, with a K m of 0.098 mg ml⁻¹ as compared to wild type enzyme showing K m of not less than 0.112 mg ml⁻¹. It was found that pH values of 8.1 and 7.3 were best for activity of the mutant and wild-type-derived enzymes, respectively. The values of pK a of the acidic limbs of both enzymes were the same (5.0 and 4.9, respectively) but the pK a value of the basic limb was slightly increased, indicating the participation of a carboxyl group present in a non-polar environment. Temperatures of 70 and 65°C were found optimal for mutant and wild-derived xylanase, respectively. Enzymes displayed a high thermostability showing a half life of 31.79 and 6.0 min (5.3-fold improvement), enthalpy of denaturation (ΔH*) of 146.06 and 166.95 kJ mol⁻¹, entropy of denaturation (ΔS*) of 101.44 and 174.67, and free energy of denaturation (ΔG*) of 110.25 and 105.29 kJ mol⁻¹ for mutant- and wild-organism derived enzyme, respectively at 80°C. Studies on the folding and stability of cellulase-less xylanases are important, since their biotechnological employments require them to function under extreme conditions of pH and temperature. The kinetic and thermodynamic properties suggested that the xylanase from the mutant organism is better as compared to xylanase produced from the wild type and previously reported strains of same species, and may have a potential usage in various industrial fields.