Isolation, Purification, and Characterization of Two Thermostable Endo-1,4-β- d-glucanase Forms from Opuntia vulgaris.

Shyamala, Sivalingam; Ravikumar, Sambandam; Vikramathithan, Jeyaraman; Srikumar, Kotteazeth

Four endoglucanase temperature isoforms ( T, T, T, and T) were identified and purified from the cladodes of the xerophytic plant Opuntia vulgaris. These isoforms exhibited optimum catalytic activity at 30 °C, 50 °C, 70 °C, and 90 °C and yielded an apparent molecular mass of 150, 20, 74, and 45 kDa, respectively, on gel filtration chromatography. These isoforms were purified 24-, 25-, 29-, and 27-fold with a yield of 15%, 12%, 17%, and 19% and having a specific activity of 120, 125, 144, and 136 U/mg, respectively. The thermostable T and T isoforms exhibited optimum activity at pH 4.5 and 7 and also yielded a molecular weight of 66 and 36 kDa, respectively, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The T had a K of 43 mM and a V of 12.5 μmol min μg of protein, and the T isoform had a K of 40 mM, with an apparent V of 10 μmol min μg of protein. Western blot, immunodiffusion, and in vitro inhibition assays established the reactivity of the T isoform with polyclonal anti- T antibody raised in rabbit. Cross-reactivity of this antibody with the T endoglucanase isoform was also noted.

ANTIGENS; IMMUNITY; IMMUNOGLOBULINS; OPUNTIA; IMMUNE response; MONOCLONAL antibodies

Applied Biochemistry & Biotechnology, 2011, Vol 165, Issue 7/8, p1597

0273-2289

Academic Journal

10.1007/s12010-011-9380-2