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

Abstract

Multidimensional kinetic analysis of immobilized enzymes is essential to understand the enzyme functionality at the interface with solid materials. However, spatiotemporal kinetic characterization of heterogeneous biocatalysts on a microscopic level and under operando conditions has been rarely approached. As a case study, we selected self-sufficient heterogeneous biocatalysts where His-tagged cofactor-dependent enzymes (dehydrogenases, transaminases, and oxidases) are co-immobilized with their corresponding phosphorylated cofactors [nicotinamide adenine dinucleotide phosphate (NAD(P)H), pyridoxal phosphate (PLP), and flavin adenine dinucleotide (FAD)] on porous agarose microbeads coated with cationic polymers. These self-sufficient systems do not require the addition of exogenous cofactors to function, thus avoiding the extensive use of expensive cofactors. To comprehend the microscopic kinetics and thermodynamics of self-sufficient systems, we performed fluorescence recovery after photobleaching measurements, time-lapse fluorescence microscopy, and image analytics at both single-particle and intraparticle levels. These studies reveal a thermodynamic equilibrium that rules out the reversible interactions between the adsorbed phosphorylated cofactors and the polycations within the pores of the carriers, enabling the confined cofactors to access the active sites of the immobilized enzymes. Furthermore, this work unveils the relationship between the apparent Michaelis–Menten kinetic parameters and the enzyme density in the confined space, eliciting a negative effect of molecular crowding on the performance of some enzymes. Finally, we demonstrate that the intraparticle apparent enzyme kinetics are significantly affected by the enzyme spatial organization. Hence, multiscale characterization of immobilized enzymes serves as an instrumental tool to better understand the in operando functionality of enzymes within confined spaces.

Intraparticle Macromolecular Migration Alters the Structure and Function of Proteins Reversibly Immobilized on Porous Microbeads

“…To this aim, we performed enzyme activity studies through singleparticle measurements, which have shown to reveal valuable information on the enzyme activity at the sub-micrometer level. [13] Interestingly, in operando single-particle kinetics showed an increment of the specific activity of the immobilized enzyme after the migration (Figure 9c), even in the case of storage at 4 °C, where we observed less passive migration. Remarkably, we found a positive correlation between the infiltration radius and the specific activity of His-BsADH, which meant that the specific Figure 9.…”

“…These findings agree with a recent work from our group where we showed that the kinetics of heterogeneous biocatalysts strongly depend on the spatial distribution and density of the immobilized enzymes. [ 13 ] Based on those recent insights we suggest that the intraparticle migration of His‐BsADH leads to a lower enzyme density within the agarose porous microbeads that explains the higher activity of such immobilized enzyme upon migration. Despite the observed changes in activity of His‐BsADH after incubation for 1 day at 4 and 37 °C, we did not observe changes in the protein Trp fluorescence thus in its structure (Figure S12, Supporting Information).…”

“…[11] Likewise, recently, our group demonstrated through singleparticle studies that the spatial distribution of immobilized enzymes has a clear effect on their final catalytic performance. [12,13] Under certain storage and usage conditions, proteins are known to suffer conformational changes that deteriorate the functionality of materials functionalized with proteins. For instance, enzymes and recognition proteins (i.e., antibodies) While migration of reversibly immobilized proteins across the volume of supports is investigated in conditions where an external force is applied or under fluid flow conditions, their passive migration upon sample storage and its effect on the protein functionality remain unexplored.…”

“…To this aim, we performed enzyme activity studies through single‐particle measurements, which have shown to reveal valuable information on the enzyme activity at the sub‐micrometer level. [ 13 ]…”

“…[ 11 ] Likewise, recently, our group demonstrated through single‐particle studies that the spatial distribution of immobilized enzymes has a clear effect on their final catalytic performance. [ 12,13 ]…”