Omar Farha and coworkers have
Omar Farha and coworkers have recently successfully employed metal-organic frameworks (MOFs) to tackle these limitations while retaining the essential characteristics of an immobilization material. MOFs consist of metal ions, or small clusters thereof, coordinated with organic ligands. MOFs are highly porous materials with a high degree of crystallinity, a high surface-to-volume ratio, tunable functional groups, and a strong BEC of guest molecules, ranging from small gases to large biomolecules such as enzymes.5, 6 Many MOF reagents are commercially available, cheap, and easily tailored with different functional groups, which promotes their industrial use. Their uniformity and ordered structures can be finely tuned with precise pore sizes that are superior to common immobilizing supports. Immobilization of enzymes by MOFs has been shown to improve the loading efficiency and stability of the enzymes and to reduce their leaching during reactions. In this issue of Chem, Omar Farha and coworkers propose several design rules for optimal biocatalyst immobilization using MOFs as a support: control of pore and particle size, accessibility and conformation of the supported enzymes, MOF stability, and the diffusion pathways of the reagents and products through the MOFs. This work provides an enzyme-immobilization strategy with high enzyme stability, high substrate accessibility, high enzyme loading, and good catalytic efficiency (Figure 1). The main new design rule proposed and investigated here is the use of a hierarchical pore structure with sufficiently large pores that allow entry of the enzymes resulting in immobilization and small pores that provide orthogonal entry and exit ports for substrates and products. In this way, the channels mediated by the small pores create fast diffusion pathways (“highways”) for the reagent and product molecules. Even at high enzyme loadings, by which the main channels accessed by the large pores get blocked, the reagents can still access the enzymes easily. In an enzymatic hydrolysis example, Farha et al. showed that 93% of the enzyme was accessible and catalytically active in the hierarchical MOF, whereas this was much lower in another MOF (49%) and a commercial support (17%). Confocal microscopy images showed that diffusion of the enzyme occurs in a linear fashion through the main channels of the hierarchical structures along the length of the MOF crystals. The immobilization by in-diffusion is therefore based on spontaneous equilibration, and as a consequence, some extent of leaching can be expected as well. The authors tried to limit this observed leaching effect by matching the MOF’s pore size with the size of the enzyme (cutinase) and by increasing the affinity between the MOF and the enzyme by Coulombic interactions. To this aim, loading experiments were performed at neutral pH, where the MOF had a net negative charge and cutinase had a net positive charge. This strategy was successful in achieving a high loading efficiency (Figure 1B). However, the required enzyme incubation time was long (72 hr), and the enzyme concentration was high. The diffusion process was modeled and tracked experimentally, which showed that cutinase diffused well through the main channels. Molecular-mechanics calculations showed that the enzyme needed to elongate to enter through the pores. Obviously, this close match between pore and enzyme sizes provides a slow release but also slow access. The value of this approach was most clear under denaturing conditions. Whereas the catalytic activity of the immobilized enzyme was the same as that of the free enzyme in optimal buffer, the activity of the MOF-enzyme system was retained under various denaturing conditions, but the free enzyme became much less active. These results underline the added value of using immobilization in terms of stability. The MOF showed enzyme leaching that led 60% of catalytic activity to be retained after five cycles, which compared favorably with some other MOF systems. Yet, there exist other immobilization strategies that have reported better catalytic activities after several cycles.