Isolation, purification, and characterization of two thermostable endo-1,4-β-d-glucanase forms from Opuntia vulgaris

摘要

Four endoglucanase temperature isoforms (T _(30), T _(50), T _(70), and T _(90)) 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 _(70) and T _(90) 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 _(70) had a K _m of 43 mM and a V _(max) of 12.5 μmol min ~(-1) μg~(-1) of protein, and the T _(90) isoform had a K _m of 40 mM, with an apparent V _(max) of 10 μmol min~(-1) μg~(-1) of protein. Western blot, immunodiffusion, and in vitro inhibition assays established the reactivity of the T _(90) isoform with polyclonal anti-T _(90) antibody raised in rabbit. Cross-reactivity of this antibody with the T _(70) endoglucanase isoform was also noted.