Abstract
The co-immobilized enzymes and cofactors onto porous microparticles work as self-sufficient heterogeneous biocatalysts whose catalytic activity can be easily monitored by means of the cofactors autofluorescence. The reduction step of some cofactors as NAD+ and FAD+ to NADH and FADH2, respectively, involves an increase of its autofluorescence. This phenomenon is harnessed to image and analyze the enzymatic reactions catalyzed by cofactor-dependent enzymes at real time and single-particle level during the operational process. Due to the universality and highly accessibility of fluorescence microscopy, the strategy described here allows a straightforward and more accurate analysis at micro-scale of heterogeneous biocatalysts. These studies promote and support the rational design and optimization of biocatalysts toward highly efficient heterogeneous biocatalytic reactions.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Liese A, Hilterhaus L (2013) Evaluation of immobilized enzymes for industrial applications. Chem Soc Rev 42:6236–6249
Weltz JS, Schwartz DK, Kaar JL (2016) Surface-mediated protein unfolding as a search process for denaturing sites. ACS Nano 10:730–738
Kato A, Yanagisawa M, Sato YT, Fujiwara K, Yoshikawa K (2012) Cell-sized confinement in microspheres accelerates the reaction of gene expression. Sci Rep 2:283
BenÃtez-Mateos AI, Bernd N, Bolivar JM, López-Gallego F (2018) Single-particle studies to advance the characterization of heterogeneous biocatalysts. ChemCatChem 10:654–665
Hormigo D, de la Mata I, Acebal C, Arroyo M (2010) Immobilized aculeacin A acylase from Actinoplanes utahensis: characterization of a novel biocatalyst. Bioresour Technol 101:4261–4268
Li P, Modica JA, Howarth AJ, Vargas E, Moghadam PZ, Snurr RQ et al (2016) Toward design rules for enzyme immobilization in hierarchical mesoporous metal-organic frameworks. Chem 1:154–169
Torres P, Datla A, Rajasekar VW, Zambre S, Ashar T, Yates M et al (2008) Characterization and application of a sterol esterase immobilized on polyacrylate epoxy-activated carriers (Dilbeads™). Catal Commun 9:539–545
Rocha-MartÃn J, De las Rivas B, Muñoz R, Guisán JM, López-Gallego F (2012) Rational co-immobilization of bi-enzyme cascades on porous supports and their applications in bio-redox reactions with in situ recycling of soluble cofactors. ChemCatChem 4:1279–1288
Peter RJ, Marguet M, Marais S, Fraaije MW, Van Hest JC, Lecommandoux S (2014) Cascade reactions in multicompartmentalized polymersomes. Angew Chem Int Ed Engl 53:146–150
Li Z, Zhang Y, Su Y, Ouyang P, Ge J, Liu Z (2014) Spatial co-localization of multi-enzymes by inorganic nanocrystal-protein complexes. Chem Commun 50:12465–12468
Velasco-Lozano S, BenÃtez-Mateos AI, López-Gallego F (2017) Co-immobilized phosphorylated cofactors and enzymes as self-sufficient heterogeneous biocatalysts for Chemical Processes. Angew Chem Int Ed Engl 56:771–775
BenÃtez-Mateos AI, San Sebastian E, RÃos-LombardÃa N, MorÃs F, González-SabÃn J, López-Gallego F (2017) Asymmetric reduction of prochiral ketones by using self-sufficient heterogeneous biocatalysts based on NADPH-dependent ketoreductases. Chemistry 23:16843–16852
Mateo C, Palomo JM, Fuentes M, Betancor L, Grazú V, López-Gallego F et al (2006) Glyoxyl agarose: a fully inert and hydrophilic support for immobilization and high stabilization of proteins. Enzym Microb Technol 39:274–280
Acknowledgment
The author wish to thank the supporting of COST action CM1303-Systems Biocatalysis and the Spanish MINECO (BIO2014-61838-EXP).
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
BenÃtez-Mateos, A.I. (2020). Single-Particle Kinetics of Immobilized Enzymes by Harnessing the Autofluorescence of Co-Immobilized Cofactors. In: Guisan, J., Bolivar, J., López-Gallego, F., Rocha-MartÃn, 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_20
Download citation
DOI: https://doi.org/10.1007/978-1-0716-0215-7_20
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