Abstract
The immobilization of proteins on heterofunctional amino-epoxy and amino-glyoxyl supports is described in this chapter. Immobilization on both supports is performed through a two-step mechanism: in the first step, the enzyme is physically adsorbed to the support, and in the second step, the intramolecular covalent attachment between the adsorbed enzyme and the support is promoted. On the one hand, amino-epoxy supports present a ratio between amino and epoxy groups of 1:1 to allow the rapid adsorption of the enzyme and promote a strong multipoint covalent linkage. On the other hand, amino-glyoxyl supports contain the highest concentration of glyoxyl groups capable of reacting covalently with primary amino groups on the enzyme surface to promote increased rigidification. The intensity of the covalent enzyme–support interaction can be modulated by modifying the ratio between glyoxyl and amino groups of the support. These heterofunctional supports are able to immobilize and rigidify proteins through different orientations, leading to biocatalysts with different enzyme properties (activity, stability, and selectivity).
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Mateo C, Fernandez-Lorente G, Rocha-Martin J, Bolivar JM, Guisan JM (2013) Oriented covalent immobilization of enzymes on heterofunctional-glyoxyl supports. In: Immobilization of enzymes and cells, 3rd edn. Springer, New York, pp 73–88
Fernandez-Lorente G, Lopez-Gallego F, M Bolivar J, Rocha-Martin J, Moreno-Perez S, M Guisan J (2015) Immobilization of proteins on highly activated glyoxyl supports: dramatic increase of the enzyme stability via multipoint immobilization on pre-existing carriers. Curr Org Chem 19:1719–1731
Garcia-Galan C, Berenguer-Murcia Á, Fernandez-Lafuente R, Rodrigues RC (2011) Potential of different enzyme immobilization strategies to improve enzyme performance. Adv Synth Catal 353:2885–2904
Bolivar JM, Wilson L, Ferrarotti SA, Guisan JM, Fernandez-Lafuente R, Mateo C (2006) Improvement of the stability of alcohol dehydrogenase by covalent immobilization on glyoxyl-agarose. J Biotechnol 125:85–94
Mateo CPJM, Fernandez-Lorente G, Guisan JM, Fernandez-Lafuente R (2007) Improvement of enzyme activity, stability and selectivity via immobilization techniques. Enzym Microb Technol 40:1451–1463
Mateo C, Bolivar JM, Godoy CA, Rocha-Martin J, Pessela BC, Curiel JA, Muñoz R, Guisan JM, Fernández-Lorente G (2010) Improvement of enzyme properties with a two-step immobilizaton process on novel heterofunctional supports. Biomacromolecules 11:3112–3117
Bolivar Juan MR-MJ, Cesar M, Felipe C, Jose B, Daniel V, Roberto F-L, Guisan Jose M (2009) Purification and stabilization of a glutamate dehydrogenase from Thermus thermophilus via oriented multisubunit plus multipoint covalent immobilization. J Mol Catal B Enzym 58:158–163
Santos JCS, Barbosa O, Ortiz C, Berenguer-Murcia A, Rodrigues RC, Fernandez-Lafuente R (2015) Importance of the support properties for immobilization or purification of enzymes. ChemCatChem 7:2413–2432
Bolivar JMMC, Grazu V, Carrascosa AV, Pessela BC, Guisan JM (2010) Heterofunctional supports for the one-step purification, immobilization and stabilization of large multimeric enzymes: amino-glyoxyl versus amino-epoxy supports. Process Biochem 45:1692–1698
Mateo C, Torres R, Fernández-Lorente G, Ortiz C, Fuentes M, Hidalgo A, López-Gallego F, Abian O, Palomo JM, Betancor L et al (2003) Epoxy-amino groups: a new tool for improved immobilization of proteins by the epoxy method. Biomacromolecules 4:772–777
Batalla P, Fuentes M, Mateo C, Grazu V, Fernandez-Lafuente R, Guisan JM (2008) Covalent immobilization of antibodies on finally inert support surfaces through their surface regions having the highest densities in carboxyl groups. Biomacromolecules 9:2230–2236
Mateo C, Abian O, Bernedo M, Cuenca E, Fuentes M, Fernandez-Lorente G, Palomo JM, Grazu V, Pessela BCC, Giacomini C et al (2005) Some special features of glyoxyl supports to immobilize proteins. Enzym Microb Technol 37:456–462
Pedroche J, del Mar Yust M, Mateo C, Fernández-Lafuente R, Girón-Calle J, Alaiz M, Vioque J, Guisán JM, Millán F (2007) Effect of the support and experimental conditions in the intensity of the multipoint covalent attachment of proteins on glyoxyl-agarose supports: correlation between enzyme-support linkages and thermal stability. Enzym Microb Technol 40:1160–1166
Orrego AH, Trobo-Maseda L, Rocha-Martin J, Guisan JM (2017) Immobilization-stabilization of a complex multimeric sucrose synthase from Nitrosomonas europaea. Synthesis of UDP-glucose. Enzym Microb Technol 105:51–58
Bungaruang L, Gutmann A, Nidetzky B (2013) Leloir glycosyltransferases and natural product glycosylation: biocatalytic synthesis of the C-glucoside nothofagin, a major antioxidant of redbush herbal tea. Adv Synth Catal 355:2757–2763
Lepak A, Gutmann A, Kulmer ST, Nidetzky B (2015) Creating a water-soluble resveratrol-based antioxidant by site-selective enzymatic glucosylation. Chembiochem 16:1870–1874
Fernandez-Lafuente R, Rosell CM, Rodriguez V, Santana C, Soler G, Bastida A, Guisán JM (1993) Preparation of activated supports containing low pK amino groups. A new tool for protein immobilization via the carboxyl coupling method. Enzym Microb Technol 15:546–550
Mukherjee J, Gupta MN (2015) Increasing importance of protein flexibility in designing biocatalytic processes. Biotechnol Rep 6:119–123
Orrego H, Romero-Fernández M, Millán-Linares MDC, MDM Y, Guisán JM, Rocha-Martin J (2018) Stabilization of enzymes by multipoint covalent attachment on aldehyde-supports: 2-picoline borane as an alternative reducing agent. Catalysts 8:333
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2020 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Trobo-Maseda, L., Orrego, A.H., Romero-Fernández, M., Guisan, J.M., Rocha-Martín, J. (2020). Immobilization of Enzymes on Hetero-Functional Supports: Physical Adsorption Plus Additional Covalent Immobilization. In: Guisan, J., Bolivar, J., López-Gallego, F., Rocha-Martín, J. (eds) Immobilization of Enzymes and Cells. Methods in Molecular Biology, vol 2100. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0215-7_10
Download citation
DOI: https://doi.org/10.1007/978-1-0716-0215-7_10
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-0214-0
Online ISBN: 978-1-0716-0215-7
eBook Packages: Springer Protocols