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.
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Acknowledgment
The authors wish to thank DST-FIST for the financial support for equipment purchase given to Department of Biochemistry and Molecular Biology, Pondicherry University, Puducherry, where the work has been carried out.
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Shyamala, S., Ravikumar, S., Vikramathithan, J. et al. Isolation, Purification, and Characterization of Two Thermostable Endo-1,4-β-d-glucanase Forms from Opuntia vulgaris . Appl Biochem Biotechnol 165, 1597–1610 (2011). https://doi.org/10.1007/s12010-011-9380-2
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DOI: https://doi.org/10.1007/s12010-011-9380-2