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

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 [<i>ACS Appl. Mater. Interfaces</i> <b>2022,</b> <i>14,</i> 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.