Deciphering the effect of microbead size distribution on the kinetics of heterogeneous biocatalysts through single-particle analysis based on fluorescence microscopy

Muñoz-Morales, E. ; Velasco-Lozano, S. ; Benítez-Mateos, A.I. ; Marín, M.J. (Universidad de Zaragoza) ; Ramos-Cabrer, P. ; López-Gallego, F. (Universidad de Zaragoza)

Resumen: 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.

Idioma: Inglés

DOI: 10.3390/catal9110896

Año: 2019

Publicado en: CATALYSTS 9, 11 (2019), 896 [16 pp]
ISSN: 2073-4344

Factor impacto JCR: 3.52 (2019)

Categ. JCR: CHEMISTRY, PHYSICAL rank: 65 / 159 = 0.409 (2019) - Q2 - T2

Factor impacto SCIMAGO: 0.722 - Physical and Theoretical Chemistry (Q2) - Catalysis (Q3)

Financiación: info:eu-repo/grantAgreement/EC/H2020/722361/EU/Cofund on Biotechnologies/ERA CoBioTech
Financiación: info:eu-repo/grantAgreement/ES/MINECO/BIO2014-61838-EXP
Financiación: info:eu-repo/grantAgreement/ES/MINECO/BIO2015-69887-R
Financiación: info:eu-repo/grantAgreement/ES/MINECO/PCI2018-092984

Tipo y forma: Artículo (Versión definitiva)

Área (Departamento): Área Química Orgánica (Dpto. Química Orgánica)
Área (Departamento): Servicios. División Biomédica (Serv.Gral. Apoyo Investigación)