Single-Particle Kinetics of Immobilized Enzymes by Harnessing the Autofluorescence of Co-Immobilized Cofactors

  • Ana I. Benítez-MateosEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2100)


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.

Key words

Immobilized enzymes Cofactor co-immobilization Heterogeneous biocatalysts Enzyme kinetics Fluorescence microscopy Single-particle analysis 



The author wish to thank the supporting of COST action CM1303-Systems Biocatalysis and the Spanish MINECO (BIO2014-61838-EXP).


  1. 1.
    Liese A, Hilterhaus L (2013) Evaluation of immobilized enzymes for industrial applications. Chem Soc Rev 42:6236–6249CrossRefGoogle Scholar
  2. 2.
    Weltz JS, Schwartz DK, Kaar JL (2016) Surface-mediated protein unfolding as a search process for denaturing sites. ACS Nano 10:730–738CrossRefGoogle Scholar
  3. 3.
    Kato A, Yanagisawa M, Sato YT, Fujiwara K, Yoshikawa K (2012) Cell-sized confinement in microspheres accelerates the reaction of gene expression. Sci Rep 2:283CrossRefGoogle Scholar
  4. 4.
    Benítez-Mateos AI, Bernd N, Bolivar JM, López-Gallego F (2018) Single-particle studies to advance the characterization of heterogeneous biocatalysts. ChemCatChem 10:654–665CrossRefGoogle Scholar
  5. 5.
    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 101:4261–4268CrossRefGoogle Scholar
  6. 6.
    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 1:154–169CrossRefGoogle Scholar
  7. 7.
    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 9:539–545CrossRefGoogle Scholar
  8. 8.
    Rocha-Martín 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 4:1279–1288CrossRefGoogle Scholar
  9. 9.
    Peter RJ, Marguet M, Marais S, Fraaije MW, Van Hest JC, Lecommandoux S (2014) Cascade reactions in multicompartmentalized polymersomes. Angew Chem Int Ed Engl 53:146–150CrossRefGoogle Scholar
  10. 10.
    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 50:12465–12468CrossRefGoogle Scholar
  11. 11.
    Velasco-Lozano S, Benítez-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 56:771–775CrossRefGoogle Scholar
  12. 12.
    Benítez-Mateos AI, San Sebastian E, Ríos-Lombardía N, Morís F, González-Sabín J, López-Gallego F (2017) Asymmetric reduction of prochiral ketones by using self-sufficient heterogeneous biocatalysts based on NADPH-dependent ketoreductases. Chemistry 23:16843–16852CrossRefGoogle Scholar
  13. 13.
    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 39:274–280CrossRefGoogle Scholar

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

Authors and Affiliations

  1. 1.Heterogeneous Biocatalysis Laboratory, CIC-biomaGUNEDonostia-San SebastianSpain

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