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Applied Biochemistry and Biotechnology

, Volume 31, Issue 1, pp 11–20 | Cite as

Immunoaffinity purification of glucose/xylose isomerase fromStreptomyces

  • Mohini Ghatge
  • Yogesh Mawal
  • Sushama Gaikwad
  • Vasanti Deshpande
Article

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.

Keywords

Streptomyces Apply Biochemistry Isomerase Divinyl Cobalt Chloride 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Humana Press Inc. 1991

Authors and Affiliations

  • Mohini Ghatge
    • 1
  • Yogesh Mawal
    • 1
  • Sushama Gaikwad
    • 1
  • Vasanti Deshpande
    • 1
  1. 1.Division of Biochemical SciencesNational Chemical LaboratoryPuneIndia

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