Hierarchical Pore Structures as Highways for Enzymes and Substrates

Abstract

The use of enzymes in chemical reactions needs packaging inside a porous matrix. In this issue of Chem, Farha et al. show that a hierarchical metal-organic framework with large and small pores allows orthogonal access of enzymes and their substrates. The use of enzymes in chemical reactions needs packaging inside a porous matrix. In this issue of Chem, Farha et al. show that a hierarchical metal-organic framework with large and small pores allows orthogonal access of enzymes and their substrates. Enzymes constitute nature’s way of catalyzing a wide range of reactions with highly selective products. Their high rates, mild operating conditions, and renewable composition make them a highly valuable class of catalysts, particularly now that their production is more standard and cost effective. Yet, various aspects of enzymes, particularly their stability, recovery, and recyclability, require improvement if enzyme work is to become more reproducible, economically feasible, and applicable under conditions normally not found in living organisms. The singular solution to these limitations—stability, recovery, and recyclability—is immobilization, although chemical modification and protein engineering can also contribute. Immobilization is the attachment of an enzyme to a support or its inclusion in a matrix. The use of various immobilization strategies has shown that enzymes are better stabilized and can retain their activity when working in organic solvents, at extreme pH, and under mechanical stress.1Zhang Y. Ge J. Liu Z. ACS Catal. 2015; 5: 4503-4513Crossref Scopus (306) Google Scholar Immobilization of enzymes onto micrometer-sized particles also gives the possibility of easy separation of the catalyst from the products and thus provides access to recovery and recyclability. The ideal support matrix used for immobilization needs to be cheap, be controllable in structure, and provide high enzyme loading with minimal compromise to the activity. Substrate selectivity by the matrix could be an additional benefit. In practice, immobilization has been performed with a wide range of materials such as natural and synthetic polymers, as well as inorganic materials, such as zeolites, ceramics, celites, silica, glass, and activated carbon.2Mohamad N.R. Marzuki N.H. Buang N.A. Huyop F. Wahab R.A. Biotechnol. Biotechnol. Equip. 2015; 29: 205-220Crossref PubMed Scopus (863) Google Scholar, 3Datta S. Christena L.R. Rajaram Y.R.S. Biotech. 2013; 3: 1-9Google Scholar The choice of a particular material is predominantly dictated by the process conditions under which the supported enzyme has to work. Drawbacks of immobilization are an increase in the production costs and the incompatibility of materials with certain enzymes or applications. In addition, the reaction rate is often diminished as a result of mass-transfer limitations occurring in the system, in particular for the substrate reaching the enzyme and for the product diffusing out from the matrix. This behavior is due to enzyme-support interactions, which can block the accessibility of the active site. 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.4Li P. Modica J.A. Howarth A.J. Vargas E.L. Moghadam P.Z. Snurr R.Q. Mrksich M. Hupp J.T. Farha O.K. Chem. 2016; 1: 154-169Abstract Full Text Full Text PDF Scopus (233) Google Scholar 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 absorption of guest molecules, ranging from small gases to large biomolecules such as enzymes.5Cheetham A.K. Rao C.N.R. Science. 2007; 318: 58-59Crossref PubMed Scopus (329) Google Scholar, 6Li H. Eddaoudi M. O’Keeffe M. Yaghi O.M. Nature. 1999; 402: 276-279Crossref Scopus (6550) Google Scholar 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.7Wu X. Hou M. Ge J. Catal. Sci. Technol. 2015; 5: 5077-5085Crossref Google Scholar In this issue of Chem, Omar Farha and coworkers4Li P. Modica J.A. Howarth A.J. Vargas E.L. Moghadam P.Z. Snurr R.Q. Mrksich M. Hupp J.T. Farha O.K. Chem. 2016; 1: 154-169Abstract Full Text Full Text PDF Scopus (233) Google Scholar 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%).7Wu X. Hou M. Ge J. Catal. Sci. Technol. 2015; 5: 5077-5085Crossref Google Scholar 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.7Wu X. Hou M. Ge J. Catal. Sci. Technol. 2015; 5: 5077-5085Crossref Google Scholar The MOF system described by Farha and coworkers combines rational design of the support and good experimental results regarding activity and stability.4Li P. Modica J.A. Howarth A.J. Vargas E.L. Moghadam P.Z. Snurr R.Q. Mrksich M. Hupp J.T. Farha O.K. Chem. 2016; 1: 154-169Abstract Full Text Full Text PDF Scopus (233) Google Scholar However, a further increase in the speed of enzyme loading into the MOF and a decrease in leaching are still required. How can the design rules proposed here provide further leads to these improvements? Of the two parameters mentioned to influence enzyme uptake and release, i.e., pore-size matching and matrix-enzyme interactions, the former influences only the kinetics, whereas the latter can provide a stronger thermodynamic affinity. A possible solution, therefore, is replacing the organic linkers with more optimal hydrophobicity or using stronger covalent and non-covalent binding strategies to make the system function more as a sponge for the enzyme. However, an optimum can also be expected in this strategy, given that strong binding can decrease the total loading capacity as a result of premature blocking of the channels. Other alternatives could be the immobilization and anchoring of the enzyme during MOF synthesis or after in-diffusion in the form of physical or chemical entrapment after enzyme loading. The latter can possibly be achieved by end-capping the crystal with other ligands or by encapsulating the whole MOF-enzyme particle in yet another matrix that does not permit out-diffusion of the enzyme. The strategy developed here could provide future practical handles for new functionalities of immobilized enzymes. Multiple enzyme systems could be applied together within MOFs, possibly providing local reaction-rate enhancement by channeling of the substrate diffusion. To this aim, the MOF composition can be changed to facilitate the immobilization of different enzymes within the same particles while maintaining good activity of the enzymes. Alternatively, the selectivity of the catalysts might be altered by the immobilization material, which can be turned into an advantage by tuning the MOF’s properties. Overall, this study opens a new avenue of investigations where the influence of metal nodes, organic linkers, and connectivities, combined with molecular simulations, sheds new light on the performance of immobilized enzymes. In general, employing MOFs as enzyme supports yields the promise of stabilizing enzymes, thus making catalytic reactions performed by enzymes function more reproducibly. This can give significant improvements for enzyme assays or for biocatalysis in industry. Think of, for example, using this system to stabilize horse radish peroxidase during blotting experiments. This method could also pave the way for enzyme-MOFs as reusable catalysts in organic synthesis or in sensors, in which enzymes often play a role in signal amplification.8Liu G. Qi M. Hutchinson M.R. Yang G. Goldys E.M. Biosens. Bioelectron. 2016; 79: 810-821Crossref PubMed Scopus (97) Google Scholar When the current challenges are overcome, the versatile and functionalizable structures of MOFs hold great promise for improving enzymes, making them ever more useful catalysts also outside living systems. Toward Design Rules for Enzyme Immobilization in Hierarchical Mesoporous Metal-Organic FrameworksLi et al.ChemJune 8, 2016In BriefMetal-organic frameworks (MOFs) are porous, crystalline materials comprised of metal nodes and organic linkers. Here, a Zr-based MOF named NU-1000 is used to encapsulate and protect an enzyme. The encapsulation and subsequent protection of enzymes in solid supports is important for the potential industrialization of enzymes as chemical catalysts. NU-1000 is shown to be capable of stabilizing the enzyme under harsh conditions, and in addition, the encapsulated enzyme is shown to maintain full functionality. Full-Text PDF Open Archive