Purification and biochemical characterization of two major thermophilic xylanase isoforms (T ₇₀ and T ₉₀) from xerophytic Opuntia vulgaris plant spp

Thermostable xylanase isoforms T ₇₀ and T ₉₀ were purified and characterized from the xerophytic Opuntia vulgaris plant species. The enzyme was purified to homogeneity employing three consecutive steps. The purified T ₇₀ and T ₉₀ isoforms yielded a final specific activity 134. 0 and 150. 8 U mg ^-1 protein, respectively. The molecular mass of these isoforms was determined to be 27 kDa. The optimum pH for the T ₇₀ and T ₉₀ xylanase isoforms was 5. 0 and the temperature for optimal activity was 70 and 90 °C, respectively. The Km value of T ₇₀ and T ₉₀ enzyme isoforms was 3. 49, 2. 1 mg ml ^-1, respectively when oat spelt xylan was used as a substrate. The T ₇₀ had a Vmax of 10. 4 μmol min ^-1 mg ^-1, and T ₉₀ had a Vmax of 8. 9 μmol min ^-1 mg ^-1, respectively. In the presence of 10 mM Co ²⁺, and Mn ²⁺ the activity of T ₇₀ and T ₉₀ isoforms increased, where as 90 % inhibition was noted with of the use 10 mM Hg ²⁺, Cd ²⁺, Cu ²⁺, Zn ²⁺ while partial inhibition was observed in the presence of Fe ³⁺, Ni ²⁺, Ca ²⁺and Mg ²⁺. The T ₇₀ and T ₉₀ isoforms retained nearly 50 % activity in the presence of 2. 0 M urea, while use of 40 mM SDS lowered the activity nearly 38-41 %. The substrate specificity of both T ₇₀ and T ₉₀ isoforms showed maximum activity for oat spelt xylan. Western blot, immunodiffusion, and in vitro inhibition assays confirmed reactivity of the T ₉₀ isoform with polyclonal anti-T ₉₀ antibody raised in rabbit, as well as cross-reactivity of the antibody with the T ₇₀ xylanase isoform.