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.

Expanding the "Library" of Metal-Organic Frameworks for Enzyme Biomineralization.

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.

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).