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Immunoaffinity purification of glucose/xylose isomerase fromStreptomyces

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Abstract

A procedure was developed to purify glucose/xylose isomerase from cell extract ofStreptomyces sp. NCIM 2730 using immunoaffinity chromatography. High-titer polyclonal antibodies were raised in rabbit using electrophoretically homogeneous glucose/xylose isomerase as an antigen. The specificity of antibodies was confirmed by double immunodiffusion, rocket electrophoresis, and Western-blot ELISA, which revealed the presence of a single immunoreactive protein with an Mr of 40,000. The antibodies recognized 2-3 antigenic determinants/mol of enzyme and were found to partially neutralize the enzymatic activity in an immunotitration experiment. The affinity gel was prepared by coupling antibodies at pH 10.0 to divinyl sulfone-activated Sepharose CL-4B. The glucose/xylose isomerase purified by immunoaffinity chromatography yielded 75% recovery with a single enzymatically active protein band on gel electrophoresis and showed specific activity of 16 U/mg. The crossreaction of the antibodies with glucose isomerase from other actinomycetes indicated that they share common epitopes.

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  1. Division of Biochemical Sciences, National Chemical Laboratory, 411 008, Pune, India

    Mohini Ghatge, Yogesh Mawal, Sushama Gaikwad & Vasanti Deshpande

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  1. Mohini Ghatge
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  2. Yogesh Mawal
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  3. Sushama Gaikwad
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  4. Vasanti Deshpande
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Ghatge, M., Mawal, Y., Gaikwad, S. et al. Immunoaffinity purification of glucose/xylose isomerase fromStreptomyces . Appl Biochem Biotechnol 31, 11–20 (1991). https://doi.org/10.1007/BF02922121

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  • DOI: https://doi.org/10.1007/BF02922121

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