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Stabilization of Multimeric Enzymes via Immobilization and Further Cross-Linking with Aldehyde-Dextran

  • Cesar Mateo
  • Benevides C. C. Pessela
  • Manuel Fuentes
  • Rodrigo Torres
  • Lorena Betancor
  • Aurelio Hidalgo
  • Gloria Fernandez-Lorente
  • Roberto Fernandez-Lafuente
  • Jose M. GuisanEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2100)

Abstract

Subunit dissociation of multimeric proteins is one of the most important causes of inactivation of proteins having quaternary structure, making these proteins very unstable under diluted conditions. A sequential two-step protocol for the stabilization of this protein is proposed. A multisubunit covalent immobilization may be achieved by performing very long immobilization processes between multimeric enzymes and porous supports composed of large internal surfaces and covered by a very dense layer of reactive groups. Additional cross-linking with polyfunctional macromolecules promotes the complete cross-linking of the subunits to fully prevent enzyme dissociation. Full stabilization of multimeric structures has been physically shown because no subunits were desorbed from derivatives after boiling them in SDS. As a functional improvement, these immobilized preparations no longer depend on the enzyme.

Key words

Solid-phase chemical modification Aldehyde dextran cross-link Stabilization of quaternary structures Multisubunit immobilization 

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Cesar Mateo
    • 1
  • Benevides C. C. Pessela
    • 1
  • Manuel Fuentes
    • 1
  • Rodrigo Torres
    • 1
  • Lorena Betancor
    • 1
  • Aurelio Hidalgo
    • 1
  • Gloria Fernandez-Lorente
    • 2
    • 3
  • Roberto Fernandez-Lafuente
    • 1
  • Jose M. Guisan
    • 1
    Email author
  1. 1.Institute of Catalysis, CSIC, Campus UAM-CantoblancoMadridSpain
  2. 2.Department of Biotechnology and MicrobiologyInstitute of Food Science Research (CIAL), CSIC-UAM, Campus UAMMadridSpain
  3. 3.Department of BiocatalysisInstitute of Catalysis and Petrochemistry (ICP) CSIC, Campus UAMMadridSpain

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