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

• Published in: Methods in molecular biology (Clifton, N.J.) Vol. 2100; p. 309
• Main Author: Benítez-Mateos, Ana I
• Format: Journal Article
• Language: English
• Published: United States 01-01-2020
• Subjects: Biocatalysis; Enzyme Activation; Enzymes, Immobilized - chemistry; Flavin-Adenine Dinucleotide - chemistry; Kinetics; Microscopy, Fluorescence; Microspheres; NAD - chemistry; Oxidation-Reduction; Porosity; Cofactor co-immobilization; Immobilized enzymes; Enzyme kinetics; Heterogeneous biocatalysts; Single-particle analysis; Fluorescence microscopy
• ISSN: 1940-6029
• DOI: 10.1007/978-1-0716-0215-7_20

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 FADH
, 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.