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

Abstract

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.

Intraparticle Kinetics Unveil Crowding and Enzyme Distribution Effects on the Performance of Cofactor-Dependent Heterogeneous Biocatalysts

“…Despite this narrow range, we still observed that the V o of immobilized Bs-ADH slightly decreased as the particle size increased ( Figure S11A ). This is consistent with the previous bulk 48 and single-particle studies 23 that show similar effects. Remarkably, the immobilized Bs-ADH concentration negatively correlated with the particle radius; therefore, smaller microbeads loaded higher enzyme concentrations, resulting in higher single-particle initial rates ( V o ) ( Figure S11B ).…”

“…The functional dispersion among beads found in Figures 4 and 5 encouraged us to investigate the particle-to-particle functional dispersion in commercial carriers like agarose porous microbeads. 23 Through monitoring single-particle reaction courses, we study the effects of protein concentration on the apparent kinetic parameters of immobilized enzymes toward their confined cofactors. As a model system for these studies, we selected RhB-labeled Bs-ADH co-immobilized with NADH on AG-Co 2+ /E-PAH microbeads with particle radius size ranging from 40 to 60 μm.…”

“…The shape of the time courses varied depending on the coordinate of the radius where they were recorded. Using the image analytics tool described in the Materials and Methods section together with the volumetric correction previously developed in our group, 23 we were able to locally estimate the apparent kinetic parameters of the immobilized enzymes toward the confined cofactor in areas as small as 2.8 μm 2 . Furthermore, we calculated the enzyme concentration immobilized on those small areas.…”

“… 13 , 14 Harnessing that many of the industrially relevant cofactors are phosphorylated and display fluorescence properties, our group has developed a time-lapse fluorescence microscopy method coupled with a routine for image processing and analysis to assess the activity of enzymes confined within porous materials. 23 However, this methodology misses multiplex information about the cofactor diffusion (thermodynamics), the enzyme and cofactor local concentrations, and their localization within a single particle.…”

Self-sufficient asymmetric reduction of β-ketoesters catalysed by a novel and robust thermophilic alcohol dehydrogenase co-immobilised with NADH

“…In the last years, our groups have been able to co-immobilise different alcohol dehydrogenases and ketoreductases with their corresponding redox cofactors. 26,27,62 As result, we obtain self-sufficient heterogeneous biocatalysts, which require no exogenous supply of NAD(P)H. Harnessing the PEI layer needed to stabilise the quaternary structure of Tt27-HBDH, we co-immobilised the NADH with the enzyme through ionic interactions (AG-G@Tt27-HBDH(PEI-NADH)). The cofactor was effectively bound to the agarose microbeads in less than 15 min (Fig.…”