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Open AccessArticle

Deciphering the Effect of Microbead Size Distribution on the Kinetics of Heterogeneous Biocatalysts through Single-Particle Analysis Based on Fluorescence Microscopy

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Heterogeneous Biocatalysis Laboratory, Instituto de Síntesis Química y Catálisis Homogénea (iSQCH), CSIC-Universidad de Zaragoza, C/Pedro Cerbuna 12, 50009 Zaragoza, Spain
2
Heterogeneous Biocatalysis Laboratory, CICbiomaGUNE, Edificio Empresarial “C”, Paseo de Miramón, 182, 20014 Donostia-San Sebastián, Spain
3
Servicio General de Apoyo a la Investigación—SAI, Servicio de Microscopía Electrónica de Sistemas Biológicos, Universidad de Zaragoza, C/Pedro Cerbuna 12, 50009 Zaragoza, Spain
4
Magnetic Resonance Imaging Lab, CICbiomaGUNE, Edificio Empresarial “C”, Paseo de Miramón, 182, 20014 Donostia-San Sebastián, Spain
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IKERBASQUE, Basque Foundation for Science, Maria Diaz de Haro 3, 48013 Bilbao, Spain
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ARAID, Aragon foundation for Science, 50009 Zaragoza, Spain
*
Author to whom correspondence should be addressed.
Catalysts 2019, 9(11), 896; https://doi.org/10.3390/catal9110896
Received: 2 October 2019 / Revised: 24 October 2019 / Accepted: 24 October 2019 / Published: 28 October 2019
Understanding the functionality of immobilized enzymes with spatiotemporal resolution and under operando conditions is an unmet need in applied biocatalysis, as well as priceless information to guide the optimization of heterogeneous biocatalysts for industrial purposes. Unfortunately, enzyme immobilization still relies on trial-and-error approximations that prevail over rational designs. Hence, a modern fabrication process to achieve efficient and robust heterogeneous biocatalysts demands comprehensive characterization techniques to track and understand the immobilization process at the protein–material interface. Recently, our group has developed a new generation of self-sufficient heterogeneous biocatalysts based on alcohol dehydrogenases co-immobilized with nicotinamide cofactors on agarose porous microbeads. Harnessing the autofluorescence of NAD+(P)H and using time-lapse fluorescence microscopy, enzyme activity toward the redox cofactors can be monitored inside the beads. To analyze these data, herein we present an image analytical tool to quantify the apparent Michaelis–Menten parameters of alcohol dehydrogenases co-immobilized with NAD(P)+/H at the single-particle level. Using this tool, we found a strong negative correlation between the apparent catalytic performance of the immobilized enzymes and the bead radius when using exogenous bulky substrates in reduction reactions. Therefore, applying image analytics routines to microscopy studies, we can directly unravel the functional heterogeneity of different heterogeneous biocatalyst samples tested under different reaction conditions. View Full-Text
Keywords: protein immobilization; alcohol dehydrogenase; NAD(P)H; biocatalysis; agarose; bio-redox protein immobilization; alcohol dehydrogenase; NAD(P)H; biocatalysis; agarose; bio-redox
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Muñoz-Morales, E.; Velasco-Lozano, S.; Benítez-Mateos, A.I.; Marín, M.J.; Ramos-Cabrer, P.; López-Gallego, F. Deciphering the Effect of Microbead Size Distribution on the Kinetics of Heterogeneous Biocatalysts through Single-Particle Analysis Based on Fluorescence Microscopy. Catalysts 2019, 9, 896.

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