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- ACS applied materials interfaces
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- National Science Foundation
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null (Ed.)Metal–organic frameworks/materials (MOFs/MOMs) are advanced enzyme immobilization platforms that improve biocatalysis, materials science, and protein biophysics. A unique way to immobilize enzymes is co-crystallization/co-precipitation, which removes the limitation on enzyme/substrate size. Thus far, most enzyme@MOF composites rely on the use of non-sustainable chemicals and, in certain cases, heavy metals, which not only creates concerns regarding environmental conservation but also limits their applications in nutrition and biomedicine. Here, we show that a dimeric compound derived from lignin, 5,5′-dehydrodivanillate (DDVA), co-precipitates with enzymes and low-toxicity metals, Ca2+ and Zn2+, and forms stable enzyme@Ca/Zn–MOM composites. We demonstrated this strategy on four enzymes with different isoelectric points (IEPs), molecular weights, and substrate sizes. Furthermore, we found that all enzymes displayed slightly different but reasonable catalytic efficiencies upon immobilization in the Ca–DDVA and Zn–DDVA MOMs, as well as reasonable reusability in both composites. We then probed the structural basis of such differences using a representative enzyme and found enhanced restriction of enzymes in Zn–DDVA than in Ca–DDVA, which might have caused the activity difference. To the best of our knowledge, this is the first aqueous-phase, one-pot synthesis of a lignin-derived “green” enzyme@MOF/MOM platform that can host enzymes without any limitations on enzyme IEP, molecular weight, and substrate size. The different morphologies and crystallinities of the composites formed by Ca–DDVA and Zn–DDVA MOMs broaden their applications depending on the problem of interest. Our approach of enzyme immobilization not only improves the sustainability/reusability of almost all enzymes but also reduces/eliminates the use of non-sustainable resources. This synthesis method has a negligible environmental impact while the products are non-toxic to living things and the environment. The biocompatibility also makes it possible to carry out enzyme delivery/release for nutritional or biomedical applications via our “green” biocomposites.more » « less
Farha, Omar (Ed.)Metal-Organic Frameworks (MOFs) are advanced platforms for enzyme immobilization. Enzymes can be entrapped via either diffusion (into pre-formed MOFs) or co-crystallization. Enzyme co-crystallization with specific metals/ligands in the aqueous phase, also known as biomineralization, minimizes the enzyme loss as compared to organic phase co-crystallization, removes the size limitation on enzymes and substrates, and can potentially broaden the application of enzyme@MOF composites. However, not all enzymes are stable/functional in the presence of excess metal ions and/or ligands currently available for co-crystallization. Furthermore, most current biomineralization-based MOFs have limited (acid-) pH stability, making it necessary to explore other metal-ligand combinations that can also immobilize enzymes. Here, we report our discovery on the combination of five metal ions and two ligands that can form biocomposites with two model enzymes differing in size and hydrophobicity in the aqueous phase under ambient conditions. Surprisingly, most of the formed composites are single- or multi- phase crystals even though the reaction phase is aqueous, with the rest as amorphous powders. All 20 enzyme@MOF composites showed good to excellent reusability, and were stable under weakly acidic pHs. The stability under weakly basic conditions depended on the selection of enzyme and metal-ligand combinations, yet for both enzymes, 3-4 MOFs offered decent stability under basic conditions. This work initiates the expansion of the current “library” of metal-ligand selection for encapsulating/biomineralizing large enzymes/enzyme clusters, leading to customized encapsulation of enzymes according to enzymes stability, functionality, and optimal pH.more » « less
Enzymes are catalysts in biochemical reactions that, by definition, increase rates of reactions without being altered or destroyed. However, when that enzyme is a protease, a subclass of enzymes that hydrolyze other proteins, and that protease is in a multiprotease system, protease-as-substrate dynamics must be included, challenging assumptions of enzyme inertness, shifting kinetic predictions of that system. Protease-on-protease inactivating hydrolysis can alter predicted protease concentrations used to determine pharmaceutical dosing strategies. Cysteine cathepsins are proteases capable of cathepsin cannibalism, where one cathepsin hydrolyzes another with substrate present, and misunderstanding of these dynamics may cause miscalculations of multiple proteases working in one proteolytic network of interactions occurring in a defined compartment. Once rates for individual protease-on-protease binding and catalysis are determined, proteolytic network dynamics can be explored using computational models of cooperative/competitive degradation by multiple proteases in one system, while simultaneously incorporating substrate cleavage. During parameter optimization, it was revealed that additional distraction reactions, where inactivated proteases become competitive inhibitors to remaining, active proteases, occurred, introducing another network reaction node. Taken together, improved predictions of substrate degradation in a multiple protease network were achieved after including reaction terms of autodigestion, inactivation, cannibalism, and distraction, altering kinetic considerations from other enzymatic systems, since enzyme can be lost to proteolytic degradation. We compiled and encoded these dynamics into an online platform (
https://plattlab.shinyapps.io/catKLS/) for individual users to test hypotheses of specific perturbations to multiple cathepsins, substrates, and inhibitors, and predict shifts in proteolytic network reactions and system dynamics.
Confining proteins in synthetic nanoscale spatial compartments has offered a cell-free avenue to understand enzyme structure–function relationships and complex cellular processes near the physiological conditions, an important branch of fundamental protein biophysics studies. Enzyme confinement has also provided advancement in biocatalysis by offering enhanced enzyme reusability, cost-efficiency, and substrate selectivity in certain cases for research and industrial applications. However, the primary research efforts in this area have been focused on the development of novel confinement materials and investigating protein adsorption/interaction with various surfaces, leaving a fundamental knowledge gap, namely, the lack of understanding of the confined enzymes (note that enzyme adsorption to or interactions with surfaces differs from enzyme confinement as the latter offers an enhanced extent of restriction to enzyme movement and/or conformational flexibility). In particular, there is limited understanding of enzymes' structure, dynamics, translocation (into biological pores), folding, and aggregation in extreme cases upon confinement, and how confinement properties such as the size, shape, and rigidity affect these details. The first barrier to bridge this gap is the difficulty in “penetrating” the “shielding” of the confinement walls experimentally; confinement could also lead to high heterogeneity and dynamics in the entrapped enzymes, challenging most protein-probing experimental techniques. The complexity is raised by the variety in the possible confinement environments that enzymes may encounter in nature or on lab benches, which can be categorized to rigid confinement with regular shapes, rigid restriction without regular shapes, and flexible/dynamic confinement which also introduces crowding effects. Thus, to bridge such a knowledge gap, it is critical to combine advanced materials and cutting-edge techniques to re-create the various confinement conditions and understand enzymes therein. We have spearheaded in this challenging area by creating various confinement conditions to restrict enzymes while exploring experimental techniques to understand enzyme behaviors upon confinement at the molecular/residue level. This review is to summarize our key findings on the molecular level details of enzymes confined in (i) rigid compartments with regular shapes based on pre-formed, mesoporous nanoparticles and Metal–Organic Frameworks/Covalent-Organic Frameworks (MOFs/COFs), (ii) rigid confinement with irregular crystal defects with shapes close to the outline of the confined enzymes via co-crystallization of enzymes with certain metal ions and ligands in the aqueous phase (biomineralization), and (iii) flexible, dynamic confinement created by protein-friendly polymeric materials and assemblies. Under each case, we will focus our discussion on (a) the way to load enzymes into the confined spaces, (b) the structural basis of the function and behavior of enzymes within each compartment environments, and (c) technical advances of our methodology to probe the needed structural information. The purposes are to depict the chemical physics details of enzymes at the challenging interface of natural molecules and synthetic compartment materials, guide the selection of enzyme confinement platforms for various applications, and generate excitement in the community on combining cutting-edge technologies and synthetic materials to better understand enzyme performance in biophysics, biocatalysis, and biomedical applications.
null (Ed.)Co-precipitation of enzymes in metal-organic frameworks is a unique enzyme-immobilization strategy but is challenged by weak acid-base stability. To overcome this drawback, we discovered that Ca2+ can co-precipitate with carboxylate ligands and enzymes under ambient aqueous conditions and form enzyme@metal-organic material composites stable under a wide range of pHs (3.7–9.5). We proved this strategy on four enzymes with varied isoelectric points, molecular weights, and substrate sizes—lysozyme, lipase, glucose oxidase (GOx), and horseradish peroxidase (HRP)—as well as the cluster of HRP and GOx. Interestingly, the catalytic efficiency of the studied enzymes was found to depend on the ligand, probing the origins of which resulted in a correlation among enzyme backbone dynamics, ligand selection, and catalytic efficiency. Our approach resolved the long-lasting stability issue of aqueous-phase co-precipitation and can be generalized to biocatalysis with other enzymes to benefit both research and industry.more » « less