Abstract
Subunit dissociation of multimeric proteins is one of the most important causes of inactivation of proteins having quarternary 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.
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References
Bickerstaff G.F. (ed). (1997) Immobilization of Enzymes and Cells, Humana Press, Totowa, NJ.
Guisán J. M., Blanco R. M., Fernandez-Lafuente R., et al. (eds). (1993) Enzyme Stabilization by Multipoint Covalent Attachment on Activated Supports. Protein Stability and Stabilization of Enzymes, Elsevier, Amsterdam, pp. 55–62.
Fernández-Lafuente R., Rodriguez. V. Mateo, C., et al. (1999) Stabilization of multimeric enzymes via immobilization and post-immobilization techniques. J. Mol. Catal. B: Enzymatic. 7, 181–189.
Torchilin V. P., Trubetskoy V. S., Omelyananko V. G., and Martinek K. J. (1983) Stabilization of subunit enzyme by intersubunit crosslinking with bifunctional reagents: studies with glyceraldehyde-3-phosphate dehydrogenase. J. Mol. Catal. 19, 291–301.
Fernández-Lafuente R., Rodríguez V., Mateo C., et al. (1999) Stabilization of multimeric enzymes via immobilization and post-immobilization techniques. J. Mol. Catal B: Enzymatic. 7, 181–189.
Fernández-Lafuente R., Hernández-Justiz O., Mateo C., et al. (2001) Biotransformations catalyzed by multimeric enzymes: stabilization of tetrameric ampicillin acylase permits the optimization of ampicillin synthesis under dissociation conditions. Biomacromolecules 2, 95–104.
Pessela B. C. C., Mateo C., Fuentes M., et al. (2004) Stabilization of a multimeric β-galactosidase from Thermus sp. strain T2 by immobilization on novel heterofunctional epoxy supports plus aldehyde-dextan cross-linking. Biotechnol. Prog. 20, 388–392.
Betancor, L., Lopez-Gallego F., Hidalgo A., et al. (2004) Prevention of interfaction inactivation of enzymes by coating the enzyme surface with dextranaldehyde. J. Biotechnol., 110, 201–207.
Guisan J. M., Penzol G., Armisen P., et al. (1997) Immobilization of enzymes acting on macromolecular substrates. Reduction of steric problems. In: Immobilization of Enzymes and Cells, Methods in Biotechnology, vol. 1, (Bickerstaff G. F., ed.), Human Press Inc., Totowa, NJ, pp. 261–275.
Betancor L., Hidalgo A., Fernández-Lorente G., Mateo C., Fernández-Lafuente R., and Guisán J. M., (2003) Preparation of a stable biocatalyst of bovine liver catalase using immobilization and postimmobilization techniques. Biotechnol. Prog. 19, 763–767.
Fuentes M., Segura R., Abian O., et al. (2004) Stabilization of protein-protein interaction by specific crosslink with aldehyde-dextran. Proteomics 9, 2602–2607.
Hidalgo A., Betancor L., Lopez-Gallego F., et al. (2003) Desing of an immobilized preparation of catalase from Thermus thermophilus to be used in a wide range of conditions. Structural stabilization of a multimeric enzyme. Enzyme Microb. Technol. 33, 278–285.
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Mateo, C. et al. (2006). Stabilization of Multimeric Enzymes Via Immobilization and Further Cross-Linking With Aldehyde-Dextran. In: Guisan, J.M. (eds) Immobilization of Enzymes and Cells. Methods in Biotechnology™, vol 22. Humana Press. https://doi.org/10.1007/978-1-59745-053-9_12
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DOI: https://doi.org/10.1007/978-1-59745-053-9_12
Publisher Name: Humana Press
Print ISBN: 978-1-58829-290-2
Online ISBN: 978-1-59745-053-9
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