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

Vikramathithan, Jeyaraman; Ravikumar, Sambandam; Muthuraman, Pandurangan; Nirmalkumar, Gali; Shayamala, Sivalingam; Srikumar, Kotteazeth

Thermostable xylanase isoforms T and T were purified and characterized from the xerophytic Opuntia vulgaris plant species. The enzyme was purified to homogeneity employing three consecutive steps. The purified T and T isoforms yielded a final specific activity 134.0 and 150.8 U mg protein, respectively. The molecular mass of these isoforms was determined to be 27 kDa. The optimum pH for the T and T xylanase isoforms was 5.0 and the temperature for optimal activity was 70 and 90 °C, respectively. The Km value of T and T enzyme isoforms was 3.49, 2.1 mg ml, respectively when oat spelt xylan was used as a substrate. The T had a Vmax of 10.4 μmol min mg, and T had a Vmax of 8.9 μmol min mg, respectively. In the presence of 10 mM Co, and Mn the activity of T and T isoforms increased, where as 90 % inhibition was noted with of the use 10 mM Hg, Cd, Cu, Zn while partial inhibition was observed in the presence of Fe, Ni, Caand Mg. The T and T 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 and T isoforms showed maximum activity for oat spelt xylan. Western blot, immunodiffusion, and in vitro inhibition assays confirmed reactivity of the T isoform with polyclonal anti-T antibody raised in rabbit, as well as cross-reactivity of the antibody with the T xylanase isoform.

IMMUNODIFFUSION; HYDROGEN-ion concentration; ANTIGEN-antibody reactions; PLANT species; OPUNTIA

Cellulose, 2012, Vol 19, Issue 4, p1373

0969-0239

Academic Journal

10.1007/s10570-012-9690-8