Single‐Particle Studies to Advance the Characterization of Heterogeneous Biocatalysts

Abstract

Immobilized enzymes have been widely exploited because they work as heterogeneous biocatalysts, allowing their recovery and reutilization and easing the downstream processing once the chemical reactions are completed. Unfortunately, we suffer a lack of analytical methods to characterize those heterogeneous biocatalysts at microscopic and molecular levels with spatio‐temporal resolution, which limits their design and optimization. Single‐particle studies are vital to optimize the performance of immobilized enzymes in micro/nanoscopic environments. In this Concept article, we review different analytical techniques that address single‐particle studies to image the spatial distribution of the enzymes across the solid surfaces, the sub‐particle substrate diffusion, the structural integrity and mobility of the immobilized enzymes inside the solid particles, and the pH and O2 internal gradients. From our view, such sub‐particle information elicited from single‐particle analysis is paramount for the design and fabrication of optimal heterogeneous biocatalyst.

Synthesis of Ibuprofen Monoglyceride in Solventless Medium with Novozym®435: Kinetic Analysis

“…The high viscosity of the liquid phase in combination with the high enzyme concentration in the solid can lead to mass transfer hindrances, which can be reduced by a wise selection of agitation speeds or flow rates and enzyme loadings [31]. From a more in-depth perspective, in-situ techniques can render local and particle information on the enzyme distribution, local diffusion, structural features and local mobility of the enzyme, which is critical to the optimization of immobilized biocatalysts in a multiscale approach [32].…”

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

“…Thus far, techniques for the molecular characterization of solid-supported enzymes are scarce, unlike chemical catalysis, where molecular characterization drives the design and optimization of heterogeneous catalysts [3,4]. Recently, several authors have deeply reviewed different techniques for the advanced characterization of heterogeneous biocatalysts [5][6][7]. They summarized an analytical toolbox that provides valuable information about the function, the structure, and the dynamics of the immobilized proteins.…”

The Microenvironment in Immobilized Enzymes: Methods of Characterization and Its Role in Determining Enzyme Performance

“…Various analytical techniques are available for the characterization of the internal microenvironment in solid-supported catalysts (e.g., NMR, Raman and IR) [12,20,26,27,28,29,30,31,32]. Among these techniques, opto-chemical sensing offers the specific advantage that both the external (Figure 2) and the overall (space-averaged) internal concentrations of the analyte of interest can be measured at the same time, using a single analytical device [12,14].…”

“…These latter effects are considered herein, only insofar as their quantitative assessment, unmasked from the effects of the milieu, is concerned. The suitable detection of structural distortions relevant for enzyme function, ideally at single-molecule resolution, remains a significant challenge for immobilized enzymes [12,17,18,19,20].…”

“…It follows from the above that the characterization of the microenvironment of an immobilized enzyme is essential for the understanding of the overall performance of the catalyst [12,13,20]. Kinetic theory accounts for the effect of coupled reactions and diffusion and is a useful predictive tool [23].…”