title: Isolation, purification, and characterization of two thermostable endo-1,4-β-D-glucanase forms from Opuntia vulgaris.
abstract: 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, School of Life Sciences, Pondicherry University, Puducherry 605014, India.
authors.forename: Sivalingam
authors.initials: S
authors.lastname: Shyamala
authors.forename: Sambandam
authors.initials: S
authors.lastname: Ravikumar
authors.forename: Jeyaraman
authors.initials: J
authors.lastname: Vikramathithan
authors.forename: Kotteazeth
authors.initials: K
authors.lastname: Srikumar
mesh_terms: D000818:Animals; D002480:Cellulase / Q000737:chemistry* / Q000302:isolation & purification*; D004795:Enzyme Stability; D007700:Kinetics; D008970:Molecular Weight; D029747:Opuntia / Q000737:chemistry / Q000201:enzymology*; D010940:Plant Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D011817:Rabbits; D013379:Substrate Specificity
chemical_list: D010940:Plant Proteins; D002480:Cellulase