Brooke P.
Carpenter‡
a,
Ben
Rose‡
a,
Elisa M.
Olivas
a,
Mariana X.
Navarro
a,
A. Rain
Talosig
a,
Paul J.
Hurst
a,
Giuseppe
Di Palma
a,
Li
Xing
e,
Rweetuparna
Guha
b,
Stacy M.
Copp
bcd and
Joseph P.
Patterson
*a
aDepartment of Chemistry, University of California, Irvine, Irvine, CA 92697-2025, USA. E-mail: patters3@uci.edu
bDepartment of Materials Science and Engineering, University of California, Irvine, Irvine, CA 92697, USA
cDepartment of Physics and Astronomy, University of California, Irvine, Irvine, CA 92697, USA
dDepartment of Chemical and Biomolecular, Engineering University of California, Irvine, Irvine, CA 92697, USA
eIrvine Materials Research Institute, University of California, Irvine, Irvine, CA 92697, USA
First published on 11th December 2023
Enzymes present a promising green chemistry alternative for accelerating chemical reactions in industry. However, many industrial applications require harsh synthetic conditions (i.e. high temperature and/or organic solvents), causing enzymes to denature. Metal–organic frameworks (MOFs) offer a promising method to overcome the structural limitations of potentially revolutionary enzymes through enzymatic immobilization. However, for high activity of immobilized enzymes to be achieved, a comprehensive understanding of the factors influencing enzyme performance is critical. While extensive efforts have demonstrated that final MOF properties such as crystallinity, porosity, and network defects significantly influence enzyme activity, here we propose a separate, but equally impactful, factor in determining enzyme@MOF activity: enzyme folding in prenucleation clusters. To investigate both MOF structural property and enzyme folding factors, two model enzymes, glucose oxidase and catalase, were encapsulated into ZIF-8. The biocomposite formation and prenucleation clusters were examined through electron microscopy, advanced spectroscopy, and scattering techniques, respectively. Results from these studies indicate enzyme folding with MOF precursors is a crucial aspect to consider when pairing enzymes with selected MOF precursors. To assist in this consideration, we provide alternative strategies for retaining enzyme activity in denaturing conditions. Finally, a roadmap is provided for tuning activity of immobilized enzymes, paving the way for industrial-scale production of enzyme@MOFs.
Zeolitic imidazolate framework-8 (ZIF-8) has emerged as a widely studied MOF for enzyme immobilization due to its precursor availability, ease of synthesis, and environmentally friendly synthetic conditions.13,14 ZIF-8 forms a sodalite (sod) topology consisting of zinc metal nodes connected by 2-methylimidazole (HmIM) linkers. The crystal formation and final crystal properties in ZIF-8 can be tuned through variations in the ligand-to-metal ratio (HmIM:
Zn) with the aim of achieving high enzyme activity and encapsulation efficiency.6 At low ratios of HmIM
:
Zn, enzymes with low isoelectric points (<7) are thought to drive nucleation through attraction of the positively charged metal ions to the negatively charged enzyme surface.3,15,16 This approach, coined biomimetic mineralization, has yielded various ZIF-8 morphologies and network topologies, such as the most observed polymorph, sodalite (sod), along with ZIF-C and ZIF-L.5 Through the biomimetic mineralization process, mixtures of different ZIF-8 polymorphs are often obtained, with the exact polymorphs varying from enzyme to enzyme, indicating that biomolecular differences play significant roles in MOF polymorph determination and enzyme activity.
While final MOF polymorph and crystallinity have been extensively examined as driving factors behind activity of MOF-encapsulated enzymes, a biomolecule-focused approach examining enzyme folding in prenucleation clusters has not previously been offered. This understanding is critical because enzymatic catalysis is dependent on proper enzyme folding. In previous research, we have shown that biomolecule folding affects the crystallization mechanism, kinetics, and structural properties of MOFs synthesized in situ.17 This study also found that the MOF precursors, specifically zinc acetate, can influence the initial protein folding. Other studies have shown that hydrophilic ligands can stabilize enzymes in their active conformation, retaining activity of the enzyme in the final enzyme@MOF,18 whereas more hydrophobic ligands lead to a loss of functionality.19 Thus, a more comprehensive understanding of biomolecular folding during enzyme@MOF synthesis is needed. Here, we reemphasize the role of MOF crystallinity in enzyme@ZIF-8 activity, while introducing a novel factor relevant to activity: enzymatic interactions with MOF building blocks. More specifically, the enzyme interactions with MOF precursors, particularly upon formation of prenucleation clusters consisting of enzymes and MOF precursors, are shown to influence the folding, encapsulation efficiency, and activity of the enzymes glucose oxidase (GOx) and catalase (CAT) within the ZIF-8 system. To exhibit enzyme activity, we show that building units must form with minimal enzyme denaturation. If this requirement is met, the physical properties of the crystal can then be optimized for high activity, as crystallinity and structure influence the diffusion and accessibility of the substrate to the immobilized enzyme. Time-resolved cryogenic-transmission electron microscopy (cryoTEM) is utilized to elucidate two distinct mechanisms resulting in either highly or poorly active enzyme@ZIF-8 biocomposites.
At 4:
1 GOx@ZIF-8 synthesis conditions with water washes, crystals have an encapsulation efficiency (EE%) of ∼100% (ESI Fig. S2†). Powder X-ray diffraction (P-XRD) verifies the product to be a heterogeneous mixture of both an amorphous phase, which can be observed in the broad peak around 14°, and crystalline ZIF-CO3-1 (ZIF-C), a ZIF-8 polymorph which incorporates CO2 into its crystal structure as carbonate ion.5,20 Dry-state TEM shows small, ∼20 nm, spherical particles along with sheets for 4
:
1 GOx@ZIF-8 (Fig. 1b). At 4
:
1 GOx@ZIF-8 synthesis conditions with methanol washes, P-XRD verifies phase transformation of the ZIF-C crystals to ZIF-8 (sod) with no obvious amorphous phase present in the pattern. Dry-state TEM images indicate multiple phases in the final product; specifically, particles of ZIF-8 sod with rhombic dodecahedron morphologies and amorphous spherical particles are present (Fig. 1b). We believe that this phase transformation occurs as a result of methanol being a stronger coordinating solvent and displacing the carbonate ligands in ZIF-C.5,20 As the carbonate ligands are displaced, the coordination environment around the metal nodes changes, causing the structural rearrangement to sodalite topology with minimal coordination defects. At 35
:
1 GOx@ZIF-8 synthesis conditions, crystals have a low EE% of 30% (ESI Fig. S2†). P-XRD verifies the absence of an amorphous phase and the presence of only sodalite (sod) with coordination defects; this is seen in the lower relative intensity of the first peak (visible around 7°) compared to the higher angle peaks. This is in contrast to pure sod, where the first peak is significantly more intense than all subsequent peaks (Fig. 1a). Dry-state TEM images show biocomposites with rhombic dodecahedron morphology, which is characteristic of sod, while also indicating the presence of both defects and polycrystallinity (Fig. 1d and ESI S8†).
At 4:
1 CAT@ZIF-8 conditions with water washes, crystals have an EE% of 100% (ESI Fig. S2†). P-XRD verifies the product to be ZIF-C with a slight amorphous peak present. Dry-state TEM images show a continuous film for CAT@ZIF-8 along with stacked sheets found throughout the film (Fig. 1e). At 4
:
1 CAT@ZIF-8 with methanol washes, P-XRD verifies phase transformation of ZIF-C crystals to ZIF-8 (sod) with no obvious amorphous phase present in the pattern. Dry-state TEM shows multiple phases with concentrated regions of amorphous particles surrounding crystals with rhombic dodecahedron morphology (Fig. 1f). At 35
:
1 CAT@ZIF-8, crystals have an EE% of 100%. P-XRD confirms no amorphous phase present and only sodalite (sod). Dry-state TEM images show biocomposites with rhombic dodecahedron morphology, and confirm the presence of coordination defects (Fig. 1g). SEM of both 4
:
1 and 35
:
1 CAT@ZIF-8 confirm these results (ESI Fig. S3†).
In summary, both CAT and GOx drive similar final crystal structures when varying the HmIM:
Zn ratios. Both enzymes have low isoelectric points (<7); thus, the ability for each enzyme to drive the formation of a kinetic product when below the normal supersaturation conditions for ZIF-8 is expected. However, each enzyme drives the formation of kinetic products with different morphologies at 4
:
1 conditions with water washes. Additionally, CAT consistently becomes encapsulated into ZIF-8 at any HmIM
:
Zn ratio, whereas the EE% for GOx decreases with increasing HmIM
:
Zn.
![]() | ||
Fig. 2 Enzyme activity assays of (a) GOx@ZIF-8 and (b) CAT@ZIF-8 at a variety of synthetic conditions. Standard deviations are shown as highlighted sections. |
As a control, we compared the CD spectra of free CAT and CAT/Zn mixture to CAT in the presence of potassium acetate, to determine that stability of CAT was dependent on the specific metal cation and not the presence of any counterion, potassium is a common biological cation found in high concentrations in most living systems, unlike zinc which is usually found in low concentrations; as such, potassium is expected to lead to very little denaturation.23 Furthermore, CAT activity has been reported in a potassium-based MOF system in a recent publication by Di Palma et al.24 The folding of CAT incubated with zinc acetate and potassium acetate was examined via CD spectroscopy, which showed that potassium acetate altered the secondary structure of the protein minimally compared to zinc acetate (Fig. 3b). Based on these results, we propose that the Zn2+ ions cause unfolding in prenucleation clusters, not the counterions.
While CD studies are useful when determining unfolding in specific enzymatic secondary structural elements, these structural changes do not necessarily correspond to changes in activity. To better understand the effects of MOF precursors on CAT activity, enzymatic assays were performed with CAT incubated with HmIM or zinc acetate, with potassium acetate as a control. CAT incubated with potassium acetate had the best activity, followed by CAT incubated with HmIM, then finally zinc acetate (Fig. 3c). While we note that these studies only deal with prenucleation clusters and not the whole MOF, our findings support previous studies which showed that CAT exhibits excellent activity when confined in a potassium-based MOF.24 Furthermore, the activity and folding of CAT with MAF-7 – which shares the zinc metal ion and sodalite topology of ZIF-8 but uses a more hydrophilic linker, 3-methyl-1H-1,2,4 triazole (HmTz)18 – precursors were also investigated. MAF-7 was investigated specifically because it shares many features with ZIF-8, yet encapsulated CAT shows activity (ESI Fig. S9†). While MAF-7 and ZIF-8 share zinc metal ions, the metal salts used are different, with ZIF-8 being commonly made with zinc acetate and MAF-7 being commonly made with zinc nitrate. Activity assays confirmed CAT incubated with either salt to have similar activity (Fig. 3c). CAT was then incubated with either ligand, HmIM or HmTz, and enzymatic activity was also similar with each ligand.
To understand how the mechanistic and structural properties of the 35:
1 GOx@ZIF-8 differentiated from the previous system, we performed similar in situ time-resolved cryoTEM studies. CryoTEM images reveal that at 1 minute, aggregates similar to the structure found in the 4
:
1 system form and are surrounded by individual particles that are 12 ± 2 nm (Fig. 5a). These particles continue to grow and double in size (21 ± 4 nm) by 10 min (Fig. 5b). At 10 minutes, these particles are localized and surround a highly concentrated region. The amorphous phases continue to aggregate and condense to form dense phases surrounded by a cloud, which is hypothesized to serve as a rich reservoir of MOF precursors for the growing dense phase (Fig. 5c). Outlines of irregular crystals can be observed within these clouds and closer inspection into the regions with high-resolution TEM reveals crystallinity as indicated by the lattice spacing (Fig. 5e) and fast Fourier transform (FFT) (Fig. 5f).13 The crystal appears poorly crystalline at 30 min with lattices appearing in multiple directions along with gaps without lattices (Fig. 5g). These gaps in the lattice are believed to be coordination defects containing enzymes. By 1 hour, well-defined rhombic dodecahedron crystals that are believed to be monocrystalline can be observed along with branched networks of aggregates dispersed throughout the sample (Fig. 5d and ESI S7†). As commonly observed with this system, amorphous phases contributing to the crystal growth undergo solid-state transformation to achieve a monocrystalline structure. While topological and structural characteristics of ZIF-8 (sod) are observed after 24 h, indentions and pores can also be observed on the surface of the crystals (Fig. 5h). Highly porous, irregular crystals can also be observed after 24 h in addition to the lesser porous, regular crystals (ESI Fig. S8†).
One way to prevent metal-induced unfolding from occurring is by using a hydrophilic ligand. Previous studies and our own work have shown CAT to remain active in the zinc-based MAF-7 (ESI Fig. S9†),18 which we believe is attributed to the ability of the more hydrophilic ligand to stabilize an active protein conformation. While CAT exhibited activity when incubated with both HmTz or HmIM, we hypothesize that HmTz, with an additional nitrogen group (ESI Fig. S10†), will exhibit enhanced binding strength to the protein complex, while the two nitrogen groups in HmIM are more likely to bind to zinc. Supporting this hypothesis, the solution of HmTz and CAT becomes turbid rapidly, indicating large aggregates, whereas the solution of HmIM and CAT remains clear (ESI Fig. S5†). Importantly, these CAT/HmTz complexes still retain somewhat high activity, meaning that significant protein denaturation is not occurring (Fig. 2c). We suggest that these strong protein/HmTz complexes stabilize CAT in a more active conformation and protect it against denaturation by zinc. Additionally, previous studies have demonstrated that the addition of hydrophilic groups to organic ligands can influence the restoration of the active protein conformation in final MOF crystals. These studies have shown that hydrophilic groups facilitate hydration effects within MOF pores, which reduce chelation interactions between the metal and the protein, thereby preventing unfolding.26
The understanding of how each individual MOF precursor and their combination affects enzyme folding and orientation in initial complexes is indeed crucial when initially designing a biocomposite. This knowledge helps in determining the appropriate enzyme/MOF precursor pairing for a specific application. By studying the interactions between the enzyme and different MOF precursors, researchers can identify compatible combinations that promote favorable enzyme folding and orientation within the MOF framework. However, in some cases, a desired enzyme/MOF precursor pairing may be inherently incompatible. In such situations, extra steps can be employed to induce an active enzyme conformation, like the addition of a hydrophilic ligand,18,26 or protecting polymer,27 along with subjecting the protein to ultrasound waves.28 Whatever the case, all techniques and enzyme@MOF synthesis processes must take into account the effect of MOF precursors on the stability of the enzyme in prenucleation clusters. However, this is only the first step in ensuring enzyme@MOF activity.
Recently, Wu et al. have found that GOx packaged in amorphous MOFs (aMOFs) exhibits high enzymatic activity due to large defects and interconnected mesopores, allowing access to substrate by enzymes inside of the composite.8 While defects are present in our aMOFs, the particles are 5× smaller (∼20 nm) than the aMOFs in the previously mentioned study (100 nm). As GOx has an average length of ∼6 nm and the aMOFs are only ∼20 nm, a large surface of the enzyme is likely to be exposed to the particle surface and accessible to substrate.29 These findings support that the activity of 4:
1 GOx@ZIF-8 is partially due to the lower diffusion barrier that the substrate must overcome to enter the biocomposite and reach the enzyme. While GOx may be present on the surface of 4
:
1 GOx@ZIF-8, it is also immobilized inside the MOF, as confirmed through FTIR analysis of biocomposites washed with sodium dodecyl sulfate, a detergent known to remove surface-bound protein (ESI Fig. S11†). Furthermore, along with EE% measurements that assess the overall enzyme content in an enzyme@MOF, loading capacity (LC%) studies were performed to quantify the concentration of encapsulated enzymes within the MOF interior. LC% studies determined that 4
:
1 GOx@ZIF-8 contains significant GOx content within the amorphous structure, while 35
:
1 GOx@ZIF-8 contains almost no GOx within the crystalline structure. Thus, for 35
:
1 GOx@ZIF-8, most of the enzyme concentration determined by EE% is immobilized either on the surface or just under the surface of the MOF (ESI Table 1†).7
While the precise mechanism of ZIF-C formation remains somewhat elusive throughout the in situ cryoTEM studies, it is hypothesized to occur through the initial aggregation followed by the subsequent rearrangement of amorphous particles. While the inclusion of ZIF-C, a polymorph that has been reported in solvothermal synthesis,20 in biomimetically mineralized CAT and GOx ZIF-8 composites is a novel result, we hypothesize that the ZIF-C composites are not active, and activity comes from aMOFs present in the sample. Previous studies have shown that ZIF-C is not porous to N2, making it unlikely that the composite is porous to glucose, the substrate of GOx, which is larger and more polar.5 Thus, we believe the smaller size of the aMOFs, coupled with the disordered aMOF, to be the major factors in driving the high activity for this system, and the ZIF-C samples to be much less relevant in conferring activity. This is confirmed by comparing enzymatic activity assays of 4:
1 GOx@ZIF-8 aged at 1 h (before ZIF-C has formed) and 24 h (after some ZIF-C has formed). The 24 h aged 4
:
1 GOx@ZIF-8 exhibited lower activity than the 1 h aged 4
:
1 GOx@ZIF-8 (ESI Fig. S12†), which supports a direct correlation between amorphous character and enzyme activity. We believe such amorphous characteristics also influence the activity of the 35
:
1 GOx@ZIF-8 biocomposite. Irregular crystals observed through TEM are hypothesized to have remained polycrystalline with coordination defects within them. As the size of glucose (∼10 Å) is much larger than the 3.4 Å window typically observed in ZIF-8 (sod),6,13,30 such defects are required for accessibility of enzymes to substrate. By increasing the enzyme loading concentration, we can expect to have an increase in amorphous character and defects in the crystal resulting in greater activity.6,15 Furthermore, such a system is expected to have some enzymatic activity as a result of the defects, but not as high as the free enzyme or at 4
:
1 GOx@ZIF-8 biocomposites. This claim is supported by our enzymatic assay results, which show low but present activity for the 35
:
1 GOx@ZIF-8 sample (Fig. 2a). However, when enzymatic activity assays of 35
:
1 GOx@ZIF-8 were replicated in Tris–HCl buffer, the biocomposites showed a marked decrease in activity, as opposed to 4
:
1 GOx@ZIF-8 which retained high activity (ESI Fig. S13†). This observation can be supported by previous studies which have reported the phosphate anions in PBS to degrade crystalline ZIF-8.31 Additionally, these enzyme activities studies, along with the aforementioned loading capacity studies (ESI Table 1†), indicate that 35
:
1 GOx@ZIF-8 requires defects to showcase activity of enzymes that are immobilized just under the MOF surface.15 On the other hand, this data suggests 4
:
1 GOx@ZIF-8 does not require PBS degradation to showcase activity as the biocomposite contains pores large enough for glucose to reach the enzyme.8
While outside of the scope of this study, another factor that could influence the activity of 35:
1 GOx@ZIF-8 is the orientation of the enzymes located within or near the defects. A study by Pan et al. used electron paramagnetic resonance spectroscopy to investigate the orientation of metalloenzymes within MOF surfaces.10 Although these enzymes were not immobilized onto the MOF in a similar mechanism, the idea of being able to control the orientation and exposure of enzymes to substrates would allow enhanced selectivity of substrates of varying sizes.
This work focuses on the effect that MOF precursors have on enzymatic folding and activity. Moreover, biomolecular unfolding from precursor interactions can additionally affect the formation mechanism and final MOF structure, which has been demonstrated in an FITC-BSA@ZIF-8 model.17,32 Furthermore, while the amorphous structure that we report in 4:
1 GOx@ZIF-8 aligns with some reports that have also observed amorphous ZIF-8 at this condition,8 other studies have also reported crystalline sod products at the same L
:
M ratio.6 We suggest that these differences may be due to the differing stabilities, structures, and purities of GOx obtained by different laboratories. Inherent to any biomolecular study is the understanding that many biomolecules – even those purchased from the same retailer and produced by the same organism line – are not structurally and compositionally identical, and studies on protein folding offer an opportunity to bridge this gap. The study of enzyme@MOFs is an inherently interdisciplinary field, and while our study only focuses on two enzymes, we believe that we have established a new protocol to take into consideration when designing and producing an enzyme@MOF.
Footnotes |
† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d3ta05397k |
‡ Indicates equal contributions to this manuscript. |
This journal is © The Royal Society of Chemistry 2024 |