Applied Biochemistry and Biotechnology

, Volume 31, Issue 1, pp 11–20

Immunoaffinity purification of glucose/xylose isomerase fromStreptomyces

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

DOI: 10.1007/BF02922121

Cite this article as:
Ghatge, M., Mawal, Y., Gaikwad, S. et al. Appl Biochem Biotechnol (1991) 31: 11. doi:10.1007/BF02922121

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.

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