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

Abstract

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 (DH*) of 146.06 and 166.95 kJ mol -1 , entropy of denaturation (DS*) of 101.44 and 174.67, and free energy of denaturation (DG*) of 110.25 and 105.29 kJ mol -1 for mutant-and wildorganism 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.

Characterization of a thermostable and alkaline xylanase from Bacillus sp. and its bleaching impact on wheat straw pulp

“…Turnover number and catalytic efficiency values of xylanase suggested that enzyme hydrolyses birchwood xylan more effectively than oat spelt xylan. Gibbs free energy (DG*), enthalpy (DH*), entropy (DS*), Gibbs free energy demands for transition state stabilization (DG Ã EÀT ) and substrate binding (DG Ã EÀS ) were lower for birchwood than those were needed for oat spelt xylan hydrolysis (Table 2) but comparable with those of xylanase derived from a thermophilic culture of Thermomyces lanuginosus (Bokhari et al 2009). …”

“…All other related enzyme activities were determined as described previously (Ghose 1987). The kinetic parameters of purified xylanase such as K m , V max , K cat and K cat /K m , were determined against xylan using concentration range of 0.5-10 mg/ml under optimized assay conditions as described previously (Bokhari et al 2009). …”

“…Enzymatic treatment of pulp for improving the pulp properties and reducing the toxic and mutagenic compounds in mill effluents is a growing technology and is advancing considerably in the world over the past few years (Bokhari et al 2009;Pokhrel and Viraraghavan 2004). The purpose is to reduce the utilization of chlorinebased chemicals (tetrachloro compounds) which although economical and cheap leads to environmental pollution by exerting a harmful impact on biological systems.…”

Evaluation of operational parameters on the precipitation of endoglucanase and xylanase produced by solid state fermentation of Aspergillus niger

“…Xylanase precipitation with ammonium sulfate as an initial purification step was also described in the literature with recoveries varying from 59 to 72%: Bokhari et al (2009) purified xylanases produced from a strain of Thermomyces lanuginosus and its mutant by using a sequence of two steps: an overnight ammonium sulfate precipitation with 30-40% saturation at 0°C (which resulted in recovery yields of 69.80 and 71.64%) and gel filtration chromatography. Purification of xylanases from Streptomyces cyaneus using precipitation up to 60% saturated ammonium sulfate left overnight at 4°C, followed by ion exchange chromatography was also described by Ninawa et al (2008).…”

“…However, most of them describe the precipitation step at a single specific condition (Bokhari et al, 2009;Li et al, 2009;Jabbar et al, 2008;Ninawa et al, 2008;Saha, 2004;Oikawa et al, 1997). Finding the optimum operational condition for precipitation can involve a study of different variables such as the type and concentration of the precipitant agent, temperature, aging time, agitation, pH and so forth.…”