Expanding the “Library” of Metal–Organic Frameworks for Enzyme Biomineralization

Abstract

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 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 multiphase 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 pH values. The stability under weakly basic conditions depended upon 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 enzyme stability, functionality, and optimal pH.

Unveiling the orientation and dynamics of enzymes in unstructured artificial compartments of metal–organic frameworks (MOFs)

“…Such a unique enzyme immobilization approach has been observed and confirmed in several of our recent works. 21,[30][31][32][33][34][35][36] In this communication, we report the case of aq-ZIF. Because urea can only unfold the exposed portion of enzymes (the buried portion is protected by the MOF scaffolds), 21,44 column 3 of Table 1 reports the chance for different lys regions to be exposed (13-32% depending on labelled regions).…”

“…16,21,30 We found that the enzyme was confined both on the crystal surface ( partially embedded) and within crystal defects, a condition also discovered in our recent works. 21,[30][31][32][33][34][35][36] We then distinguished the latter from the former using a recently reported urea-perturbation strategy, 21 followed by revealing the backbone dynamics and contact residues of lys in the co-crystal defects. To our best knowledge, this is the first report on experimental unveiling of enzyme dynamics in unstructured artificial compartments.…”

Impact of Crystallinity on Enzyme Orientation and Dynamics upon Biomineralization in Metal–Organic Frameworks

“…We recently discovered several new combinations of metal ions and ligands to biomineralize enzymes in water with various degrees of crystallinity and packing quality. 35 We have also developed an experimental strategy to probe enzyme backbone dynamics in MOFs via site-directed spin labeling (SDSL)−electron paramagnetic resonance (EPR) spectroscopy and tested it in other MOFs. 36,37 SDSL−EPR probes enzyme dynamics and structural information at the residue level regardless of the complexities caused by the background and heterogeneity.…”

“…In this work, for proof of principle, we apply this strategy to reveal the orientation and dynamics of a model large-substrate enzyme, T4 phage lysozyme (T4L), upon encapsulations in eight MOFs via biomineralization. 35 These MOFs possess various degrees of crystallization (single crystals, multiphase crystals, and amorphous, gel-like crystals) based on the combination of four metal ions and two ligands. We found that the restriction in the motional dynamics of the labeled sites on the surface of T4L increases as the packing quality/ density is increased.…”

“…The crystals of the MOFs formed by BPDC and Zn 2+ , Ni 2+ , and Al 3+ display layer-by-layer, two-dimentional (2D) structures, which would allow for less space if a protein is trapped. 35 In fact, these 2D materials should be more accurately named as metal−organic materials (MOMs) instead of MOFs; MOFs are often considered to be three-dimentional (3D) structures formed by the frameworks of metal ions and ligands. 35 In our case, most likely, the 2D, layer-by-layer packing is more intense than the 3D cases.…”