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1. Expanding the “library” of Metal-Organic Frameworks (MOFs) for enzyme biomineralization

   Jordahl, Drew Jordahl; Armstrong, Zoe; Li, Qiaobin; Gao, Runxiang; Liu, Wei; Johnson, Kelley; Brown, William; Scheiwiller, Allison; Feng, Li; Ugrinov, Angel; et al (October 2022, ACS applied materials interfaces)

   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. 

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2. Water as an Eco-friendly Liquid-Assisted Grinding Agent (ecoLAGent) to Expand the Metal–Organic Framework “Library” for Green Enzyme Encapsulation

   https://doi.org/10.1021/acsami.5c14214  

   Dipti, Tyeaba Tasnim; Hasan, Md Zahidul; Armstrong, Zoe; Quadir, Mohiuddin; Liu, Liu; Wan, Caixia; Baker, Gary A; Feng, Li; MacRae, Austin; Yang, Zhongyu (October 2025, ACS Applied Materials & Interfaces)

   Enzymes are advanced biocatalysts, while immobilizing enzymes on solid supports, particularly metal–organic frameworks (MOFs), enhances enzyme stability, reusability, and substrate selectivity. One-pot cocrystallization (CC) of enzymes in MOFs in the aqueous phase avoids enzyme size limitation and leaching; our recent CC MOF “library” offers a collection of metal–ligand combinations for customizable enzyme immobilization [ACS Appl. Mater. Interfaces 2022, 14, 46, 51619–51629]. However, CC generally suffers from low yield and stability. Mechanochemical or liquid-assisted grinding (LAG) synthesis overcomes this issue, yet most current approaches use metals, ligands, and enzymes in powder states with organic solvents, limiting eco-friendliness and enzyme compatibility. Water as an eco-friendly LAG synthesis agent (eco-LAGent) offers a greener medium to preserve protein folding states, hydration shells, and conformational flexibility, essential for activity. However, only one MOF has been reported to be synthesized this way. Due to the different metal–ligand contact mechanisms, MOFs by CC or eco-LAGent synthesis may not have the same crystal structures either, yet there is a lack of data to compare. Here, we expand the “library” of enzyme@MOF biocatalysts synthesized in an eco-LAGent, water, using abundant, low-toxicity metal ions and ligands, achieving improved thermal and pH stability and significantly enhanced enzyme@MOF yields while proving the high chance of scaling up and eventually industrial applications. Grinding also impacted the interface of metal–ligand contact─compared to aqueous-phase CC, eco-LAGent synthesis increased the crystallinity in 3 MOFs, generated new MOF structures in another 3, and improved the thermal and pH stability across most resultant enzyme@MOF cocrystals. This study generalizes the eco-LAGent synthesis to more MOFs and opens a new avenue to encapsulate enzymes in high-quality crystals with a customized selection of metal ions and ligands (according to enzymes and applications) under green and scalable conditions. 

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

   https://doi.org/https://doi.org/10.1021/acsami.1c12209  

   Li, Q.; Pan, Y.; Li, H.; Lenertz, M.; Reed, K.; Jordahl, D.; Bjerke, T.; Ugrinov, A.; Chen, B.; Yang, Z. (September 2021, ACS applied materials interfaces)

   null (Ed.)

   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. 

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

   https://doi.org/10.21769/BioProtoc.4930  

   Armstrong, Zoe; Jordahl, Drew; MacRae, Austin; Li, Qiaobi; Lenertz, Mary; Shen, Patrick; Botserovska, Anastasiia; Feng, Li; Ugrinov, Angel; Yang, Zhongyu (January 2024, BIO-PROTOCOL)

   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. 

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5. One-pot synthesis of enzyme@metal–organic material (MOM) biocomposites for enzyme biocatalysis

   https://doi.org/DOI https://doi.org/10.1039/D1GC00775K  

   Pan, Y.; Li, H.; Lenertz, M.; Han, Y.; Ugrinov, A.; Kilin, D.; Chen, B.; Yang, Z. (March 2021, Green chemistry)

   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. 

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