Background

Enzyme immobilization is receiving increased interest in both research and industry due to the (potential) promise of enhanced cost efficiency and catalytic performance control [1–4]. The biggest challenge is still maintaining enzymatic function without disturbance to the enzyme itself [5,6]. Metal–organic frameworks (MOFs) are extended 3-dimensional crystalline networks formed by the coordination bonds between certain metal ions and ligands. MOFs typically contain supreme porosity and highly tunable properties, given the high diversity in metal ions and ligands that can form such a 3D network. MOFs are advanced enzyme immobilization platforms but are mostly limited to smaller enzymes and substrates [6–13]. Co-crystallization of enzymes and certain metal ions/ligands is a unique way to host enzymes in MOF crystal scaffolds, being adaptable to large enzymes and enzyme clusters [14,15]. This strategy also allows for a small portion of enzymes to be implanted at the surface of MOF crystals, thus being partially exposed to the reaction medium, allowing contact with substrates larger than MOF apertures [16]. This strategy can therefore be applied to biocatalytic reactions—involving large substrates such as proteins/polypeptides, polysaccharides, and cells—to be carried out while reusing/recycling immobilized enzymes [17–25].

Commonly, the co-crystallization process is performed in an organic solvent such as methanol, which is a challenging condition for most enzymes [15]. There is a natural co-crystallization process called biomineralization, which is essentially co-crystallization but takes place in water phase under ambient conditions [26–28]. Although the reaction can be slow in nature, the formed biominerals are sufficiently stable with immobilized and possibly functional proteins/enzymes. Inspired by nature, biomineralization has also been carried out on lab benches [29,30]. Although nature might find its own sophisticated way to biomineralize proteins or other biomacromolecules, on-bench biomineralization in a reasonable timeframe for enzymes needs additional planning. Furthermore, although natural biomineralization could generate enzyme@MOF composites with distinct morphology and crystallinity, certain considerations are needed to optimize the on-bench strategy in order to better preserve enzyme activity and reusability and the stability of co-crystals. Lastly, aqueous phase co-crystallization may generate imperfect crystals but retains enzyme activity and reusability, which would still improve biocatalysis and thus be worth pursuing. Focusing on aqueous phase co-crystallization, we have acquired extensive experience in immobilizing various enzymes on MOFs [16,20,21]. Our recent work has revealed a combination of 10 metal ions/ligands to carry out biomineralization for effective enzyme immobilization; broadening the spectrum of available metal/ligand pairs allows for custom immobilization of enzymes according to their characteristics and stability (in certain metal ions and ligands) [19]. However, there is a lack of detailed experimental protocols to carry out such a sophisticated mission.

In this protocol, we detail the biomineralization procedures, highlight the precautions, potential pitfalls, and suggested solutions, and summarize the assessment of successful enzyme biomineralization based on our recent experience. We will cover MOF preparation in aqueous phase (metal/ligand selection and preparation and criteria of a useful MOF) to obtain a background without enzymes, enzyme@MOF composite formation (enzyme preparation and activity assessment on MOFs), and additional experimental conditions that may help the formation of co-crystals without damaging the enzymes. The strategy and methods can be applicable to immobilizing other enzymes and searching for other metal ion/ligand combinations for custom immobilization of other enzymes.