One-Point Covalent Immobilization of Enzymes on Glyoxyl Agarose with Minimal Physico-Chemical Modification: Immobilized “Native Enzymes”

Jose M. Guisan*, Fernando López-Gallego, Juan M. Bolivar, Javier Rocha-Martín, Gloria Fernandez-Lorente

*Corresponding author for this work

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review


The immobilization of soluble enzymes inside the porous structure of a preexisting support is one of the most interesting techniques to prepare heterogeneous biocatalysts. The main cause of inactivation of these biocatalysts is the distortion of the tridimensional structure of the immobilized enzymes. In some cases, immobilization of enzymes on preexisting supports can be used in order to improve its functional properties: stabilization by multipoint covalent immobilization, hyper-activation, and stabilization of lipases by interfacial adsorption on hydrophobic supports, etc. In other cases, the properties of the enzyme can be modified by additional interactions of the enzyme surface with the support surface: hydrophobic or electrostatic interactions. In all cases, it would be very interesting to evaluate the intrinsic tridimensional stability of native industrial enzymes. Under drastic experimental conditions, soluble enzymes may undergo undesirable aggregations, and the tridimensional stability of one enzyme is more accurately evaluated by using immobilized native enzymes. That is, immobilized derivatives associated to a minimal chemical modification of the enzyme surface placed in the proximity of a fully hydrophilic and inert support surfaces. In this chapter, the immobilization of enzymes with minimal physicochemical modification on glyoxyl agarose supports is proposed. At pH 8.5, the unique reactive amino group on the enzyme surface is the N-terminus. At the end of the immobilization, mild borohydride reduction, the primary amino terminus is simply converted into a secondary amino group, with similar physical properties, and aldehyde groups on the supports are converted into fully inert hydroxyl groups. The preparation of immobilized derivatives of penicillin G acylase (PGA) with identical properties (activity and stability) that one of the soluble enzyme is reported: preparation of immobilized native PGA.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Number of pages10
Publication statusPublished - 1 Jan 2020

Publication series

NameMethods in Molecular Biology
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029


  • 2-picoline borane
  • Covalent immobilization
  • Glyoxyl agarose
  • N-terminus
  • Native enzymes
  • Thiolated compounds

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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