Multi-Point Covalent Immobilization of Enzymes on Supports Activated with Epoxy Groups: Stabilization of Industrial Enzymes

  • Cesar Mateo
  • Olga Abian
  • Gloria Fernandez-Lorente
  • Benevides C. C. Pessela
  • Valeria Grazu
  • Jose M. GuisanEmail author
  • Roberto Fernandez-Lafuente
Part of the Methods in Molecular Biology book series (MIMB, volume 2100)


Commercial epoxy supports may be very useful tools to stabilize proteins via multipoint covalent attachment if the immobilization is properly designed. In this chapter, a protocol to take full advantage of the support’s possibilities is described. The basics of the protocol are as follows: (1) the enzymes are hydrophobically adsorbed on the supports at high ionic strength. (2) There is an “intermolecular” covalent reaction between the adsorbed protein and the supports. (3) The immobilized protein is incubated at alkaline pH to increase the multipoint covalent attachment, thereby stabilizing the enzyme. (4) The hydrophobic surface of the support is hydrophylized by reaction of the remaining groups with amino acids in order to reduce the unfavorable enzyme–support hydrophobic interactions. This strategy has produced a significant increase in the stability of penicillin G acylase compared with the stability achieved using conventional protocols.

Key words

Multipoint covalent attachment Hydrophobic interactions Hydrophylization Enzyme stabilization 


  1. 1.
    Bickerstaff GF (ed) (1997) Immobilization of enzymes and cells, methods in biotechnology, volume 1. Humana Press, Totowa, NJGoogle Scholar
  2. 2.
    Chibata I, Tosa T, Sato T (1986) Biocatalysis: immobilized cells and enzymes. J Mol Catal 37:1–24CrossRefGoogle Scholar
  3. 3.
    Gupta MN (1991) Thermostabilization of proteins. Biotechnol Appl Biochem 4:1–11Google Scholar
  4. 4.
    Hartmeier W (1985) Immobilized biocatalysts from simple to complex systems. Trends Biotechnol 3:149–153CrossRefGoogle Scholar
  5. 5.
    Katchalski-Katzir E (1993) Immobilized enzymes-learning from past successes and failures. Trends Biotechnol 11:471–478CrossRefGoogle Scholar
  6. 6.
    Kennedy JF, Melo EHM, Jumel K (1990) Immobilized enzymes and cells. Chem Eng Prog 45:81–89Google Scholar
  7. 7.
    Klivanov AM (1983) Immobilized enzymes and cells as practical catalysts. Science 219:722–727CrossRefGoogle Scholar
  8. 8.
    Rosevear A (1984) Immobilized biocatalysts: a critical review. J Chem Technol Biotechnol 34B:127–150Google Scholar
  9. 9.
    Royer GP (1980) Immobilized enzymes as catalysts. Catal Rev 22:29–73CrossRefGoogle Scholar
  10. 10.
    Lasch J, Koelsch R (1978) Enzyme leakage and multipoint covalent attachment of agarose-bound enzyme preparations. Eur J Biochem 82:181–186CrossRefGoogle Scholar
  11. 11.
    Kolb HJ, Renner R, Hepp KD, Weiaa L, Wieland O (1975) Reevalution of sepharose-insulin as a tool for the study of insulin action. Proc Natl Acad Sci U S A 72:248–252CrossRefGoogle Scholar
  12. 12.
    Mateo C, Abian O, Fernandez-Lafuente R, Guisán JM (2000) Increase in conformational stability of enzymes immobilized on epoxy-activated supports by favouring additional multipoint covalent attachment. Enzym Microb Technol 26:509–515CrossRefGoogle Scholar
  13. 13.
    Guisán JM (1988) Aldehyde gels as activated support for immobilization-stabilization of enzymes. Enzym Microb Technol 10:375–382CrossRefGoogle Scholar
  14. 14.
    Guisán JM, Bastida A, Cuesta C, Fernandez-Lafuente R, Rosell CM (1991) Immobilization-stabilization of chymotrypsin by covalent attachment to aldehyde agarose gels. Biotechnol Bioeng 39:75–84Google Scholar
  15. 15.
    Mozhaev VV, Klibanov AM, Goldmacher VS, Berezin IV (1990) Operational stability of copolymerized enzymes at elevated temperatures. Biotechnol Bioeng 25:1937–1945CrossRefGoogle Scholar
  16. 16.
    Kramer DM, Lehman K, Pennewiss H, Plainer H (1979) Oxirane acrylic adsorption. 26th International IUPAC Symposium on Macromolecules, Mainz, Germany, Sept. 1979Google Scholar
  17. 17.
    Wheatley JB, Schmidt DE (1993) Salt induced immobilization of proteins on a high-performance liquid chromatographic epoxide affinity support. J Chromatogr A 644:11–16CrossRefGoogle Scholar
  18. 18.
    Wheatley JB, Schmidt DE (1999) Salt induced the immobilization of affinity ligands onto epoxide-activated supports. J Chromatogr A 849:1–1CrossRefGoogle Scholar

Copyright information

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

Authors and Affiliations

  • Cesar Mateo
    • 1
  • Olga Abian
    • 1
  • Gloria Fernandez-Lorente
    • 2
    • 3
  • Benevides C. C. Pessela
    • 1
  • Valeria Grazu
    • 1
  • Jose M. Guisan
    • 1
    Email author
  • Roberto Fernandez-Lafuente
    • 1
  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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