Metal-Organic Framework Induced Stabilization of Proteins in Polymeric Nanoparticles.

Developing protein confinement platforms is an attractive research area that not only promotes protein delivery but also can result in artificial environment mimicking of the cellular one, impacting both the controlled release of proteins and the fundamental protein biophysics. Polymeric nanoparticles (PNPs) are attractive platforms to confine proteins due to their superior biocompatibility, low cytotoxicity, and controllable release under external stimuli. However, loading proteins into PNPs can be challenging due to the potential protein structural perturbation upon contacting the interior of PNPs. In this work, we developed a novel approach to encapsulate proteins in PNPs with the assistance of the zeolitic imidazolate framework (ZIF). Here, ZIF offers an additional protection layer to the target protein by forming the protein@ZIF composite via aqueous-phase cocrystallization. We demonstrated our platform using a model protein, lysozyme, and a widely studied PNP composed of poly(ethylene glycol)-poly(lactic-co-glycolic acid) (PEG-PLGA). A comprehensive study via standard loading and release tests as well as various spectroscopic techniques was carried out on lysozyme loaded onto PEG-PLGA with and without ZIF protection. As compared with the direct protein encapsulation, an additional layer with ZIF prior to loading offered enhanced loading capacity, reduced leaching, especially in the initial stage, led to slower release kinetics, and reduced secondary structural perturbation. Meanwhile, the function, cytotoxicity, and cellular uptake of proteins encapsulated within the ZIF-bound systems are decent. Our results demonstrated the use of ZIF in assisting in protein encapsulation in PNPs and established the basis for developing more sophisticated protein encapsulation platforms using a combination of materials of diverse molecular architectures and disciplines. As such, we anticipate that the protein-encapsulated ZIF systems will serve as future polymer protein confinement and delivery platforms for both fundamental biophysics and biochemistry research and biomedical applications where protein delivery is needed to support therapeutics and/or nutrients.

A Protocol for Custom Biomineralization of Enzymes in Metal-Organic Frameworks (MOFs).

Enzyme immobilization offers a number of advantages that improve biocatalysis; however, finding a proper way to immobilize enzymes is often a challenging task. Implanting enzymes in metal-organic frameworks (MOFs) via co-crystallization, also known as biomineralization, provides enhanced reusability and stability with minimal perturbation and substrate selectivity to the enzyme. Currently, there are limited metal-ligand combinations with a proper protocol guiding the experimental procedures. We have recently explored 10 combinations that allow custom immobilization of enzymes according to enzyme stability and activity in different metals/ligands. Here, as a follow-up of that work, we present a protocol for how to carry out custom immobilization of enzymes using the available combinations of metal ions and ligands. Detailed procedures to prepare metal ions, ligands, and enzymes for their co-crystallization, together with characterization and assessment, are discussed. Precautions for each experimental step and result analysis are highlighted as well. This protocol is important for enzyme immobilization in various research and industrial fields. Key features • A wide selection of metal ions and ligands allows for the immobilization of enzymes in metal-organic frameworks (MOFs) via co-crystallization. • Step-by-step enzyme immobilization procedure via co-crystallization of metal ions, organic linkers, and enzymes. • Practical considerations and experimental conditions to synthesize the enzyme@MOF biocomposites are discussed. • The demonstrated method can be generalized to immobilize other enzymes and find other metal ion/ligand combinations to form MOFs in water and host enzymes.

Magnetic Multienzyme@Metal-Organic Material for Sustainable Biodegradation of Insoluble Biomass.

Biodegradation of insoluble biomass such as cellulose via carbohydrase enzymes is an effective approach to break down plant cell walls and extract valuable materials therein. Yet, the high cost and poor reusability of enzymes are practical concerns. We recently proved that immobilizing multiple digestive enzymes on metal-organic materials (MOMs) allows enzymes to be reused via gravimetric separation, improving the cost efficiency of cereal biomass degradation [ACS Appl. Mater. Interfaces 2021, 13, 36, 43085-43093]. However, this strategy cannot be adapted for enzymes whose substrates or products are insoluble (e.g., cellulose crystals). Recently, we described an alternative approach based on magnetic metal-organic frameworks (MOFs) using model enzymes/substrates [ACS Appl. Mater. Interfaces 2020, 12, 37, 41794-41801]. Here, we aim to prove the effectiveness of combining these two strategies in cellulose degradation. We immobilized multiple carbohydrase enzymes that cooperate in cellulose degradation via cocrystallization with Ca2+, a carboxylate ligand (BDC) in the absence and presence of magnetic nanoparticles (MNPs). We then compared the separation efficiency and enzyme reusability of the resultant multienzyme@Ca-BDC and multienzyme@MNP-Ca-BDC composites via gravimetric and magnetic separation, respectively, and found that, although both composites were effective in cellulose degradation in the first round, the multienzyme@MNP-Ca-BDC composites displayed significantly enhanced reusability. This work provides the first experimental demonstration of using magnetic solid supports to immobilize multiple carbohydrase enzymes simultaneously and degrade cellulose and promotes green/sustainable chemistry in three ways: (1) reusing the enzymes saves energy/sources to prepare them, (2) the synthetic conditions are "green" without generating unwanted wastes, and (3) using our composites to degrade cellulose is the first step of extracting valuable materials from sustainable biomasses such as plants whose growth does not rely on nonregeneratable resources.

A Protocol to Depict the Proteolytic Processes Using a Combination of Metal-Organic Materials (MOMs), Electron Paramagnetic Resonance (EPR), and Mass Spectrometry (MS).

Proteolysis is a critical biochemical process yet a challenging field to study experimentally due to the self-degradation of a protease and the complex, dynamic degradation steps of a substrate. Mass spectrometry (MS) is the traditional way for proteolytic studies, yet it is challenging when time-resolved, step-by-step details of the degradation process are needed. We recently found a way to resolve the cleavage site, preference/selectivity of cleavage regions, and proteolytic kinetics by combining site-directed spin labeling (SDSL) of protein substrate, time-resolved two-dimensional (2D) electron paramagnetic resonance (EPR) spectroscopy, protease immobilization via metal-organic materials (MOMs), and MS. The method has been demonstrated on a model substrate and protease, yet there is a lack of details on the practical operations to carry out our strategy. Thus, this protocol summarizes the key steps and considerations when carrying out the EPR/MS study on proteolytic processes, which can be generalized to study other protein/polypeptide substrates in proteolysis. Details for the experimental operation and cautions of each step are reported with figures illustrating the concepts. This protocol provides an effective approach to understanding the proteolytic process with the advantages of offering time-resolved, residue-level resolution of structural basis underlying the process. Such information is important for revealing the cleavage site and proteolytic mechanisms of unknown proteases. The advantage of EPR, probing the target substrate regardless of the complexities caused by the proteases and their self-degradation, offers a practically effective, rapid, and easy-to-operate approach to studying proteolysis. Key features • Combining protease immobilization, EPR, spin labeling, and MS experimental methods allows for the analysis of proteolysis process in real time. • Reveals cleavage site, kinetics of product generation, and preference of cleavage regions via time-resolved SDSL-EPR. • MS confirms EPR findings and helps depict the sequences and populations of the cleaved segments in real time. • The demonstrated method can be generalized to other proteins or polypeptide substrates upon proteolysis by other proteases.

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

Aqueous-phase co-crystallization (also known as biomimetic mineralization or biomineralization) is a unique way to encapsulate large enzymes, enzyme clusters, and enzymes with large substrates in metal-organic frameworks (MOFs), broadening the application of MOFs as enzyme carriers. The crystallinity of resultant enzyme@MOF biocomposites, however, can be low, raising a concern about how MOF crystal packing quality affects enzyme performance upon encapsulation. The challenges to overcome this concern are (1) the limited database of enzyme performance upon biomineralization in different aqueous MOFs and (2) the difficulty in probing enzyme restriction and motion in the resultant MOF scaffolds, which are related to the local crystal packing quality/density, under the interference of the MOF backgrounds. We have discovered several new aqueous MOFs for enzyme biomineralization with varied crystallinity [Jordahl, D.; Armstrong, Z.; Li, Q.; Gao, R.; Liu, W.; Johnson, K.; Brown, W.; Scheiwiller, A.; Feng, L.; Ugrinov, A.; Mao, H.; Chen, B.; Quadir, M.; Pan, Y.; Li, H.; Yang, Z. Expanding the Library of Metal-Organic Frameworks (MOFs) for Enzyme Biomineralization. ACS Appl. Mater. Interfaces 2022, 14 (46), 51619-51629, DOI: 10.1021/acsami.2c12998]. Here, we address the second challenge by probing enzyme dynamics/restriction in these MOFs at the residue level via site-directed spin labeling (SDSL)-electron paramagnetic resonance (EPR) spectroscopy, a unique approach to determine protein backbone motions regardless of the background complexity. We encapsulated a model large-substrate enzyme, lysozyme, in eight newly discovered MOFs, which possess various degrees of crystallization, via aqueous-phase co-crystallization. Through the EPR study and simulations, we found rough connections between (a) enzyme mobility/dynamics and MOF crystal properties (packing quality and density) and (b) enzyme areas exposed above each MOF and their catalytic performance. This work suggests that protein SDSL and EPR can serve as an indicator of MOF crystal packing quality/density when biomineralized in MOFs. The method can be generalized to probing the dynamics of other enzymes on other solid surfaces/interfaces and guide the rational design of solid platforms (ca. MOFs) to customize enzyme immobilization.

Cascade/Parallel Biocatalysis via Multi-enzyme Encapsulation on Metal-Organic Materials for Rapid and Sustainable Biomass Degradation.

Multiple-enzyme cooperation simultaneously is an effective approach to biomass conversion and biodegradation. The challenge, however, lies in the interference of the involved enzymes with each other, especially when a protease is needed, and thus, the difficulty in reusing the enzymes; while extracting/synthesizing new enzymes costs energy and negative impact on the environment. Here, we present a unique approach to immobilize multiple enzymes, including a protease, on a metal-organic material (MOM) via co-precipitation in order to enhance the reusability and sustainability. We prove our strategy on the degradation of starch-containing polysaccharides (require two enzymes to degrade) and food proteins (require a protease to digest) before the quantification of total dietary fiber. As compared to the widely adopted "official" method, which requires the sequential addition of three enzymes under different conditions (pH/temperature), the three enzymes can be simultaneously immobilized on the surface of our MOM crystals to allow for contact with the large substrates (starch), while MOMs offer sufficient protection to the enzymes so that the reusability and long-term storage are improved. Furthermore, the same biodegradation can be carried out without adjusting the reaction condition, further reducing the reaction time. Remarkably, the simultaneous presence of all enzymes enhances the reaction efficiency by a factor of ∼3 as compared to the official method. To our best knowledge, this is the first experimental demonstration of using aqueous-phase co-precipitation to immobilize multiple enzymes for large-substrate biocatalysis. The significantly enhanced efficiency can potentially impact the food industry by reducing the labor requirement and enhancing enzyme cost efficiency, leading to reduced food cost. The reduced energy cost of extracting enzymes and adjusting reaction conditions minimize the negative impact on the environment. The strategy to prevent protease damage in a multi-enzyme system can be adapted to other biocatalytic reactions involving proteases.

Protocol for resolving enzyme orientation and dynamics in advanced porous materials via SDSL-EPR.

Enzyme encapsulation in metal-organic frameworks (MOFs)/covalent-organic frameworks (COFs) provides advancement in biocatalysis, yet the structural basis underlying the catalytic performance is challenging to probe. Here, we present an effective protocol to determine the orientation and dynamics of enzymes in MOFs/COFs using site-directed spin labeling and electron paramagnetic resonance spectroscopy. The protocol is demonstrated using lysozyme and can be generalized to other enzymes. For complete information on the generation and use of this protocol, please refer to Pan et al. (2021a).

Enzyme Immobilization on Graphite Oxide (GO) Surface via One-Pot Synthesis of GO/Metal-Organic Framework Composites for Large-Substrate Biocatalysis.

Although enzyme immobilization has improved many areas, biocatalysis involving large-size substrates is still challenging for immobilization platform design because of the protein damage under the often "harsh" reaction conditions required for these reactions. Our recent efforts indicate the potential of using Metal-Organic Frameworks (MOFs) to partially confine enzymes on the surface of MOF-based composites while offering sufficient substrate contact. Still, improvements are required to expand the feasible pH range and the efficiency of contacting substrates. In this contribution, we discovered that Zeolitic Imidazolate Framework (ZIF) and a new calcium-carboxylate based MOF (CaBDC) can both be coprecipitated with a model large-substrate enzyme, lysozyme (lys), to anchor the enzyme on the surface of graphite oxide (GO). We observed lys activity against its native substrate, bacterial cell walls, indicating lys was confined on composite surface. Remarkably, lys@GO/CaBDC displayed a stronger catalytic efficiency at pH 6.2 as compared to pH 7.4, indicating CaBDC is a good candidate for biocatalysis under acidic conditions as compared to ZIFs which disassemble under pH < 7. Furthermore, to understand the regions of lys being exposed to the reaction medium, we carried out a site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy study. Our data showed a preferential orientation of lys in GO/ZIF composite, whereas a random orientation in GO/CaBDC. This is the first report on immobilizing solution-state large-substrate enzymes on GO surface using two different MOFs via one-pot synthesis. These platforms can be generalized to other large-substrate enzymes to carry out catalysis under the optimal buffer/pH conditions. The orientation of enzyme at the molecular level on composite surfaces is critical for guiding the rational design of new composites.