Shielding against Unfolding by Embedding Enzymes in Metal–Organic

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Shielding against Unfolding by Embedding Enzymes in Metal− Organic Frameworks via a de Novo Approach Fu-Siang Liao,†,‡,∥ Wei-Shang Lo,‡,∥ Yu-Shen Hsu,‡,∥ Chang-Cheng Wu,‡ Shao-Chun Wang,‡ Fa-Kuen Shieh,*,‡ Joseph V. Morabito,# Lien-Yang Chou,† Kevin C.-W. Wu,§ and Chia-Kuang Tsung*,# †

School of Physical Science and Technology, ShanghaiTech University, Shanghai 201210, China Department of Chemistry, National Central University, Taoyuan 32001, Taiwan # Department of Chemistry, Merkert Chemistry Center, Boston College, Chestnut Hill, Massachusetts 02467, United States § Department of Chemical Engineering, National Taiwan University, Taipei 10617, Taiwan ‡

S Supporting Information *

Scheme 1. De Novo Approach and Structural Confinement

ABSTRACT: We show that an enzyme maintains its biological function under a wider range of conditions after being embedded in metal−organic framework (MOF) microcrystals via a de novo approach. This enhanced stability arises from confinement of the enzyme molecules in the mesoporous cavities in the MOFs, which reduces the structural mobility of enzyme molecules. We embedded catalase (CAT) into zeolitic imidazolate frameworks (ZIF-90 and ZIF-8), and then exposed both embedded CAT and free CAT to a denature reagent (i.e., urea) and high temperatures (i.e., 80 °C). The embedded CAT maintains its biological function in the decomposition of hydrogen peroxide even when exposed to 6 M urea and 80 °C, with apparent rate constants kobs (s−1) of 1.30 × 10−3 and 1.05 × 10−3, respectively, while free CAT shows undetectable activity. A fluorescence spectroscopy study shows that the structural conformation of the embedded CAT changes less under these denaturing conditions than free CAT.

are confined in the tight mesoporous cavities left in the MOF crystals by growth of the framework around the enzyme molecules, and we believe that this confinement reduces the structural changes that lead to unfolding. To test this hypothesis, we exposed the embedded enzymes to chemical agents that specifically denature or inhibit free enzymes and examined their resulting catalytic activity, accompanied by fluorescence spectroscopy to monitor the structural conformation changes of the enzymes. The results indicate that the confinement provided by the de novo approach indeed reduces the structural changes and allows enzymes function under denaturing reaction conditions. We embedded catalase (CAT) into sodalite (SOD) zeolitic imidazolate frameworks (ZIF-8 and ZIF-90)23,24 via a waterbased mild de novo approach previously reported by our group.18,25 The structure of the composite catalysts (CAT@ ZIF) was characterized (Figures S1 and S2) and the loading was determined to be 6.0 wt % (Figures S3 and S4). According to our hypothesis, although the embedded CAT may be less active compared to free CAT under denaturant-free conditions due to confinement,26 the confinement will keep CAT

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mplementing enzymes in industrial applications requires effective immobilization methods to enhance their stability and recyclability.1−10 Previous studies suggest that the use of a proper support can not only enhance the recyclability but also maintain the biological functions of the enzymes under a wider range of reactions conditions.11 Due to this discovery, metal− organic frameworks (MOFs), have been used to immobilize enzymes recently owing to their versatile functions.12−17 The selection of MOFs for this purpose is usually limited to largepore MOFs because, in a typical preparation, the large enzyme molecules are loaded by impregnation into pre-synthesized MOFs; yet, the recently developed de novo approach expands the selection of MOFs.18−20 In the de novo approach, the MOFs are grown around the enzyme molecules under a mild synthetic condition (Scheme 1), and therefore the selection of MOFs is not restricted by the pore sizes and structures. In this work, we hypothesized that using the de novo approach to embed enzymes in MOF crystals could keep enzymes functional under a wider range of reaction conditions.21,22 In the de novo approach, the enzyme molecules © 2017 American Chemical Society

Received: February 20, 2017 Published: May 1, 2017 6530

DOI: 10.1021/jacs.7b01794 J. Am. Chem. Soc. 2017, 139, 6530−6533

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Journal of the American Chemical Society functional under denaturing conditions in which free CAT will lose its biological functions. To test this hypothesis, we exposed free CAT and CAT@ZIF to urea, a chaotropic molecule and a protein-unfolding chemical.27 Urea disrupts hydrophobic interactions by allowing water molecules to solvate nonpolar groups in the interior of proteins, leading to unfolding of the protein and loss of its biological function. Before we performed the catalytic test and spectroscopy study, we confirmed that CAT@ZIF is structurally stable in urea (Figures S5 and S6). In addition, we have designed a control experiment to ensure that urea molecules were able to diffuse into the ZIF-90 (Figure S7). We used the same de novo approach to embed urease, which decomposes urea, into ZIF-90 (Figures S8 and S9). We then measured the decomposition of urea and revealed that urea molecules were able to diffuse into the ZIF-90 (Figure S10). We next measured H2O2 decomposition over free CAT and CAT@ZIF-90 after incubating them in solutions with different concentrations of urea for 30 min. The crystal structures were maintained after the reactions (Figure S11). Apparent rate constants (kobs) were quantified via the FOX assay that measures the disappearance of the reactant H2O2 (Figure 1 and

Figure 2. (a) Maintained activity of free CAT, CAT@MCF, and CAT@ZIF-90 incubated with 0.05 M urea, and CAT@ZIF-90 incubated with 0.1 M 3-AT. (b) Amount of urea diffused into ZIF90 microcrystals (green) and kobs of CAT@ZIF-90 (red) after incubation in 6 M urea for various times. Error bars indicate the standard deviation of three independent measurements.

to change its structural configuration, it is also possible that the maintained catalytic activity of the embedded CAT@ZIF-90 results from the slowed diffusion of urea in ZIF-90 μm-sized crystals, causing the concentration of urea to not reach equilibrium inside the crystals during the 30 min incubation time. To eliminate this possibility, we performed activity assays of CAT@ZIF-90 in 6 M urea for various incubation times. Figure 2b illustrates that the diffusion of urea reaches equilibrium after a 20 min incubation period. To further test our hypothesis that the maintenance of enzyme function comes from structural confinement, we performed a negative control experiment by exposing the catalysts to an inhibitor, 3-amino-1,2,4-triazole (3-AT).29 Instead of denaturing CAT via unfolding, 3-AT covalently binds to the CAT-H2O2 complex30 and inhibits CAT without changing the enzyme’s structural conformation. As predicted by our hypothesis, the biological function of CAT@ZIF-90 was halted after exposure to 3-AT because the MOF confinement cannot stop 3-AT from binding to the embedded CAT molecules (Figure 1). To obtain a more direct measurement of the structural changes of the enzyme molecules, we carried out fluorescence spectroscopy. The fluorescence emission profile of CAT is dependent on the structural conformation of the tryptophan buried in the interior of the enzyme.31,32 Structural changes of CAT such as unfolding lead to a red shift of the wavelength of maximum emission (λmax), which is used to monitor the structural conformation of CAT in situ. Owing to interference from the linker of ZIF-90, 2-imidazolecarboxyaldehyde (ICA),

Figure 1. Kinetic measurements of H2O2 degradation for free CAT, CAT@MCF, and CAT@ZIF-90 incubated with urea and 3-AT. All assays performed in Tris buffer (pH 7.5, 50 mM). (a) k obs measurements. Error bars indicate the standard deviation of three independent measurements. (b) Photograph of the time-dependent assay of H2O2 degradation. Purple indicates high concentrations of H2O2.

Table S1).28 Figure 2a shows that after exposing the catalysts to 0.05 M urea for 30 min, the activity of free CAT decreased to 3.5% of its original activity (without urea), while that of CAT@ ZIF-90 only decreased to 85.5%. When the concentration of urea was increased to 6 M, the activity of free CAT was undetectable; in contrast, CAT@ZIF-90 is still functional with kobs = 1.30 × 10−3 s−1 (Figure 1a). Although this observation strongly supports our hypothesis that the MOF confines the enzyme molecules, and thus reduces the ability of the enzyme 6531

DOI: 10.1021/jacs.7b01794 J. Am. Chem. Soc. 2017, 139, 6530−6533

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Journal of the American Chemical Society

were treated at 80 °C for 3 min. Figure 4 shows that CAT@ ZIF-90 maintains its biological function (kobs = 1.05 × 10−3

ZIF-8 was used instead. ZIF-8 has no emission around the same region. After exposing the samples to 3-AT and urea in different concentrations, the fluorescence measurement was carried out. Both free CAT and CAT@ZIF-8 in a solution without urea and 3-AT show λmax at 337 nm, which indicates that CAT maintains its native structure in ZIF-8 (Figure 3). Free CAT and CAT@

Figure 4. Apparent rate constants after treatment at 80 °C for 3 min; inset shows photographs of the time-dependent FOX assay. Error bars indicate the standard deviation of three independent measurements.

s−1), and both CAT@MCF and free CAT exhibited a much lower apparent activity after 80 °C treatment. Fluorescence spectroscopy shows that the protein structure of CAT@ZIF-8 is maintained after 80 °C treatment (Figure S18). Previous reports have shown that MOF-immobilized proteins also have better resistance to organic solvents;19 therefore, we have tested our CAT@ZIF-90 by exposing it to dimethylformamide (DMF). Fluorescence spectroscopy shows that the protein structure was maintained, and in catalysis, some resistance against DMF was observed but it was not great (Figures S19 and S20). This phenomenon can be explained by the fact that the organic solvent molecules denature proteins not only via unfolding but also via penetrating into the active sites and changing the local environment.36 The confinement provided by our method specifically decreases the structural unfolding but not the penetrating of organic molecules. In summary, we have demonstrated that CAT maintains its biological function under unfolding conditions after being embedded in ZIF microcrystals via a de novo approach. The CAT molecules are confined in the mesoporous cavities in the ZIFs and that the tight confinement limited the structural changes. We compared the activities of free CAT, CAT embedded in MOFs, and CAT physisorbed in MCF after exposing them to different denaturing or inhibiting conditions including urea, high temperature, and 3-AT. Most importantly, we have performed fluorescence spectroscopy to provide an in situ observation that the structural conformation of CAT in ZIFs is mostly maintained.

Figure 3. Fluorescence spectra of (a) free CAT and (b) CAT@ZIF-8, and after exposure to 6 M urea, 8 M urea, and 0.1 M 3-AT.

ZIF-8 show no λmax shift after exposure to 3-AT because there is no conformation change from this covalent inhibitor. Figure 3a shows the fluorescence spectra of the free CAT exposed to 6 or 8 M urea. The values of λmax red-shifted to 343 and 348 nm, respectively, due to structural unfolding which also leads to a great decrease of enzymatic activity.33 In contrast, Figure 3b shows that λmax shifts with a significantly lower degree for CAT@ZIF-8 after exposure to 6 and 8 M urea, which indicates a lower degree of structural change for the embedded enzyme (Figure S12). This result provides spectroscopic evidence of the structural confinement within the MOF. It worth mentioning that although the fluorescence spectroscopy shows that confinement greatly reduces the structural change of CAT in the presence of urea, the activity of CAT@ZIF-90 still drops when exposed to a higher concentration of urea (Figure S13). It can be explained by that even a slight change of residues near the active sites would affect the activity of an enzyme (Table S3 and Figures S14−S17). Nevertheless, the degree of activity drop is much lower compared to that of free CAT, so CAT@ ZIF maintains the enzyme’s biological functions under a wider range of conditions. To assess the importance of the tightness of the enzyme confinement in MOFs provided by the de novo approach in preserving catalytic function, we compared CAT@ZIF-90 with CAT@MCF, in which CAT is loosely confined in the mesopores of a siliceous mesocellular foam (MCF) via physical adsorption. CAT has a total molecular size of 7−8 nm34 and the pore size of MCF is about 13 nm.35 As shown in Figure 2a, the activity drop of CAT@MCF is lower compared to that of free CAT after exposure to 0.05 M urea, indicating a certain degree of confinement; however, the activity drop is much higher compared to that of CAT@ZIF-90. CAT@MCF shows no activity after exposure to 6 M urea (Figure 1), similar to free CAT. This indicates that the protection against enzyme structural conformation changes is more pronounced for CAT@ZIF because of the tight confinement.19 This was further tested by heat-induced unfolding denaturation. Samples

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.7b01794. Detailed experimental procedures, characterizations of the synthesized samples, examinations of enzyme activities, measurements of enzyme kinetic parameters, fluorescence spectroscopy, and SDS-PAGE analysis, including Figures S1−S22 and Tables S1−S3 (PDF) 6532

DOI: 10.1021/jacs.7b01794 J. Am. Chem. Soc. 2017, 139, 6530−6533

Communication

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AUTHOR INFORMATION

Corresponding Authors

*[email protected] *[email protected] ORCID

Joseph V. Morabito: 0000-0002-1598-9427 Kevin C.-W. Wu: 0000-0003-0590-1396 Chia-Kuang Tsung: 0000-0002-9410-565X Author Contributions ∥

F.-S.L., W.-S.L., and Y.-S.H. contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS F.-K.S. would like to thank the Ministry of Science and Technology, Taiwan, for the funding support (MOST 1042119-M-008-010- and MOST 105-2628-M-008-001-MY2) J.V.M. and C.-K.T acknowledge the support from Boston College and the NSF (CHE 1566445).

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DOI: 10.1021/jacs.7b01794 J. Am. Chem. Soc. 2017, 139, 6530−6533