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

  • Jose M. GuisanEmail author
  • Fernando López-Gallego
  • Juan M. Bolivar
  • Javier Rocha-Martín
  • Gloria Fernandez-Lorente
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2100)

Abstract

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.

Key words

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

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Copyright information

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

Authors and Affiliations

  • Jose M. Guisan
    • 1
    Email author
  • Fernando López-Gallego
    • 1
    • 2
  • Juan M. Bolivar
    • 3
  • Javier Rocha-Martín
    • 1
  • Gloria Fernandez-Lorente
    • 1
    • 4
  1. 1.Department of BiocatalysisInstitute of Catalysis and Petrochemistry (ICP), CSIC, Campus UAMMadridSpain
  2. 2.Departamento de Química OrgánicaInstituto de Síntesis Química y Catálisis Homogénea (ISQCH) CSIC-Universidad de ZaragozaZaragozaSpain
  3. 3.Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, NAWI GrazGrazAustria
  4. 4.Department of Biotechnology and MicrobiologyInstitute of Food Science Research (CIAL), CSIC-UAM, Campus UAMMadridSpain

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