Biochemical and Biophysical Characterization of Purified Thermophilic Xylanase Isoforms in Cereus pterogonus Plant Spp

Two thermostable xylanase isoforms T ₆₀ and T ₈₀ were purified to homogeneity from the cladodes of the xerophytic Cereus pterogonus plant species. After three consecutive purification steps, the specific activity of T ₆₀ and T ₈₀ isoforms were found to be 178.6 and 216.2 U mg ^-1 respectively. The molecular mass of both isoforms was determined to be 80 kDa. The optimum temperature for T ₆₀ and T ₈₀ xylanase isoforms were 60 and 80°C respectively. The pH was 5.0 for both isoforms. The presence of divalent metal ions (10 mM Co ²⁺) showed stimulatory effects of both catalytic activities, where as in the presence of Hg ²⁺, Cd ²⁺, Cu ²⁺ showed inhibitory effect on these activities at all concentrations studied. The thermodynamic analysis of xylanase activity using denaturation kinetics and the presence divalent cations at 30-100°C, showed lower ΔH, ΔS, and ΔG values at all the temperatures investigated. The melting temperature of purified T ₈₀ xylanase isoform as determined by TG/DTA analysis and it showed the unfolding temperature was 80°C. The g value and hyperfine (A) value purified xylanase T ₈₀ isoform was 2.017 and 10.80 respectively. Immunoblot analysis with antiserum raised against the purified T ₈₀ xylanase isoforms revealed single immunolgically related polypeptides of 80 kDa, identical with the polypeptide band produced on SDS-PAGE. The results of double immunodiffusion against the T ₈₀ isoforms showed a single precipitin line indicating that the serum used was specific to these xylanase isoforms. The kinetic and thermodynamic properties suggested that xylanase from C. pterogonus may have a potential usage in various industries.