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.