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

Abstract

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. Understanding such intraparticle macromolecular migration is essential to develop protein functionalized biomaterials with a longer life span. This work investigates the spatiotemporal migration of His‐tagged immobilized fluorescent proteins inside porous agarose microbeads under different storage conditions. A tool that assesses the intraparticle protein migration across the surface of the porous supports is developed. Differences in migration patterns between different proteins suggest that binding dynamics between proteins and their supports play a key role in their migration. The effect of macromolecular migration on the functional and structural properties of bound proteins and enzymes is also explored. Therefore, single‐particle measurements to understand how the migration process affects the functionality of immobilized enzymes are performed. Evaluating protein migration and understanding the reason behind such phenomena allows gaining control over immobilization processes and design immobilization chemistries that either prevent or promote intraparticle macromolecular diffusion upon storage, depending on the desired final application.

Is enzyme immobilization a mature discipline? Some critical considerations to capitalize on the benefits of immobilization

“…[299][300][301][302][303] These movements of the enzyme on the support surface have been recently confirmed. 304 These movements may be relevant when the researcher tries to get some order in the immobilization, e.g., when trying to get concentric crowns of co-immobilized enzymes. [305][306][307][308][309] This may be more relevant as some recent reports show that the distribution of some immobilized enzymes on the support surface has a clear effect on their final catalytic performance (Fig.…”

“…310 The possibility of enzyme migration on the support surface may alter these effects. 304 Such enzyme migration would be hampered if using fully loaded biocatalysts, although the spaces between the immobilized enzymes may still permit some enzyme mobility, even in this situation. As this research is very recent, it may be expected that some exciting phenomena, with significant implications for designed enzyme features, will be forthcoming in the future.…”

“…120 This may be more important if the intermediate product is unstable or if the formed by-product may react with it, as the changed enzyme activity ratio may drive a decrease in the final yield and the presence of the undesired by-product that one intends to prevent using co-immobilized enzymes. [303][304][305][306][307][308][309][310][311]366 Using enzymes co-immobilized in concentric crowns, the problem is much serious, as it is possible that in some of the biocatalyst fragments only one of the co-immobilized enzymes is present, and in any case the ''distribution'' and ''activity optimization'' effects will also be missed.…”

“…299–303 These movements of the enzyme on the support surface have been recently confirmed. 304 These movements may be relevant when the researcher tries to get some order in the immobilization, e.g. , when trying to get concentric crowns of co-immobilized enzymes.…”

Multi-enzyme co-immobilization on tri-heterofunctional supports

“…The resulting micrographs were analyzed with FIJI 25 using an image analytical routine previously reported. 26 From confocal images, we obtained an average and normalized fluorescence radius profile, using FIJI software and its plugin module for radial profile generation (developed by Paul Baggethun). Subsequently, a Gaussian fit was applied to the obtained profiles of at least 10 single beads.…”