Subscriber access provided by CARLETON UNIVERSITY

Communication
Probing Interactions between Metal-Organic Frameworks
and Freestanding Enzymes in a Hollow Structure
Sheng-Yu Chen, Wei-Shang Lo, Yi-Da Huang, Xiaomeng Si, Fu-Siang Liao, Shang-
Wei Lin, Benjamin P Williams, Ting-Qian Sun, Hao-Wei Lin, Yuanyuan An, Tu Sun,
Yanhang Ma, Hsiao-Ching Yang, Lien-Yang Chou, Fa-Kuen Shieh, and Chia-Kuang Tsung
Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.0c02265 • Publication Date (Web): 05 Aug 2020
Downloaded from pubs.acs.org on August 6, 2020

Just Accepted

“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W.,

Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.
Page 1 of 14 Nano Letters

1
2
3
4
5
6
7
8
Probing Interactions between Metal-Organic
9
10
11
12
Frameworks and Freestanding Enzymes in a Hollow
13
14
15
16
Structure
17
18
19
20
21
Sheng-Yu Chen,%, † Wei-Shang Lo,%, ‡ Yi-Da Huang,# Xiaomeng Si,† Fu-Siang Liao,# Shang-Wei
22 Lin,§ Benjamin P. Williams,‡ Ting-Qian Sun,# Hao-Wei Lin,# Yuanyuan An,† Tu Sun,† Yanhang
23
24 Ma,† Hsiao-Ching Yang,*, § Lien-Yang Chou,*, † Fa-Kuen Shieh,*, # Chia-Kuang Tsung*, ‡
25
26
27
28
29 † School of Physical Science and Technology, ShanghaiTech University, Shanghai 201210,
30
31 China
32
33 ‡
34
Department of Chemistry, Merkert Chemistry Center, Boston College, Chestnut Hill,
35 Massachusetts 02467, United States
36
37
# Department of Chemistry, National Central University, Taoyuan 32001, Taiwan
38
39
40 § Department of Chemistry, Fu Jen Catholic University, New Taipei City 24205, Taiwan
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59 1 Environment
60 ACS Paragon Plus
 Nano Letters Page 2 of 14

1
2
3 Abstract: It has been reported that the biological functions of enzymes could be altered when they
4
5 are encapsulated in metal-organic frameworks (MOFs) due to the interactions between them.
6
7 Herein, we probed the interactions of catalase in solid and hollow ZIF-8 microcrystals. The solid
8
9
sample with confined catalase is prepared through a reported method and the hollow sample is
10 generated by hollowing the MOFs crystal, sealing freestanding enzymes in the central cavities of
11
12 hollow ZIF-8. During the hollowing process, the samples were monitored by small angle X-ray
13
14 scattering (SAXS) spectroscopy, electron microscopy, powder X-ray diffraction (PXRD), and
15
nitrogen sorption. The interfacial interactions of the two samples were studied by infrared (IR) and
16
17 fluorescence spectroscopy. IR study shows that freestanding catalase has less chemical interaction
18
19 with ZIF-8 than confined catalase, and fluorescence study indicates that the freestanding catalase
20
21 has lower structural confinement. We have then carried out the hydrogen peroxide degradation
22 activities of catalase at different stages and revealed that the freestanding catalase in hollow ZIF-
23
24 8 has higher activity.
25
26
27 Keywords: metal-organic frameworks •enzyme immobilization • hollow MOF • interface • small
28
29 angle X-ray scattering
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47 Table of Contents
48
49
50
51
52
53
54
55
56
57
58
59 2 Environment
60 ACS Paragon Plus
Page 3 of 14 Nano Letters

1
2
3 To improve recyclability, enzymes have been immobilized on various supports for
4
5 different applications.1-3 The immobilized enzymes have even shown enhanced performance, such
6
7 as increased activity and stability.4, 5 Immobilization has been carried out through many methods,
8
9
including adsorption, entrapment, covalent binding, and cross-linking.6, 7 In general, the enzymes
10 are either fixed on the external surfaces of a solid support, or trapped in a porous material.8, 9 While
11
12 the specific or non-specific interactions between the enzymes and host materials have been
13
14 reported to increase enzymatic stability,10, 11 these interactions often alter the enzymes from their
15
native state and lead to a change in their biological activity.12-15 Therefore, it could be beneficial
16
17 to develop a method to reduce interfacial interactions after the syntheses and allows enzymes to
18
19 work under a less altered state, which could enhance the activities of immobilized enzymes. Using
20
21 metal-organic frameworks (MOFs) as host provides such an opportunity because of their post-
22 synthetic modification strategies.16-18
23
24
25 MOFs have been used as host materials to impart new enzymatic functions.19-21 Farha and
26
27 co-workers have immobilized organophosphorus acid anhydrolase into NU-1000 derivatives.22-24
28
29
Zhou and co-workers have impregnated several enzymes in PCN-333 derivatives.25-27 Falcaro,
30 Doonan, and co-workers have exhibited the embedding of biomolecules in zeolitic imidazolate
31
32 framework-8 (ZIF-8).28-31 Ma and co-workers have incorporated microperoxidase-11 into Tb-
33
34 mesoMOF.32-35 We have encapsulated catalase in ZIF-90 and ZIF-8.36, 37 These studies have
35
shown interesting results; however, like other host materials, the interfacial interactions between
36
37 the enzymes and MOFs impact their biological activities. Herein, to investigate this influence, we
38
39 propose to hollow out the solid MOFs microcrystals with enzymes encapsulated in and compare
40
41 the interactions between enzyme and MOFs before and after the hollowing process. Before
42 hollowing process, the enzymes are confined in the solid MOFs crystals. After hollowing, the
43
44 enzymes are sealed inside of the central cavity of hollow MOFs crystals in a freestanding form
45
46 and the permeable MOFs shell allows reactants to go in without leaching of the enzymes (Figure
47
1).
48
49
50
51
52
53
54
55
56
57
58
59 3 Environment
60 ACS Paragon Plus
 Nano Letters Page 4 of 14

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33 Figure 1. TEM and SEM images of catalase encapsulated in (a-c) solid and (d-f) hollow MOFs.
34
35 (g) The electron diffraction pattern of catalase in hollow ZIF-8. Schematic illustration of catalase
36
encapsulated in (h) solid and (i) hollow MOFs.
37
38
39
40
41
42
43 To demonstrate this concept, catalase was first encapsulated into ZIF-67 microcrystals and
44 then ZIF-8 shells were overgrown on the ZIF-67 cores.36 Due to the epitaxial overgrowth, single-
45
46 crystalline core-shell microcrystals were formed. Then we used a reported method to remove the
47
48 ZIF-67 cores through a mild hollowing process and form a single-crystalline hollow ZIF-8.38, 39
49
50
After the hollowing process, the catalase is held in the central cavity of the hollow ZIF-8 crystal.
51 Both solid and hollow samples were characterized by transmission electron microcopy (TEM) and
52
53 scanning electron microscopy (SEM). Figures 1a-c show the uniform solid core-shell MOFs with
54
55 confined enzymes, and Figures 1d-f show that the central cavities formed with no change to the
56
57
58
59 4 Environment
60 ACS Paragon Plus
Page 5 of 14 Nano Letters

1
2
3 morphology of the MOFs crystals after the hollowing process. The hollow sample shows a
4
5 hysteresis loop in the nitrogen isothermal sorption profiles, indicating central cavity formation
6
7 (Figure S1). Powder X-ray diffraction (PXRD) shows that the crystal structure was maintained
8
9
after the hollowing process (Figure S2). The electron diffraction pattern reveals that the hollow
10 ZIF-8 microcrystals are single crystalline (Figure 1g). To verify enzyme encapsulation in solid
11
12 and hollow MOFs microcrystals, we digested the samples and analyzed them with sodium dodecyl
13
14 sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The gels show a characteristic band of
15
catalase, demonstrating that catalase is indeed encapsulated in both solid and hollow samples
16
17 (Figure S3). To obtain the enzyme loading, Bradford protein assays were carried out. The loading
18
19 amounts of confined catalase in the solid MOFs and freestanding catalase in hollow MOFs crystals
20
21 were quantified to be ~10 wt% (Table S1).
22
23
24
25
26 Table 1. Comparison of enzyme-in-MOFs samples in the early stages of the hollowing process
27
28 per their in situ SAXS profile.
29
30 Macropore[a] Mesopore[a] Micropore[b]
31 Time
32 q = 0.01 ~ 0.04 Å-1 q = 0.04 ~ 0.07 Å-1 q = 0.1 ~ 0.4 Å-1
33
34 0 min N/A (11 × 18 × 4.1) Sphere: r = 1.1
35
36 30 min (41 × 62 × 11) (13 × 19 × 4.8) Sphere: r = 0.9
37
38 [a] An
elliptical cylinder model was used to fit the macropore and mesopore with results indexed
39
40
as Minor × Major × Length. [b] A spherical model was used to fit the micropore structure. All
41 dimensions are in nm.
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59 5 Environment
60 ACS Paragon Plus
 Nano Letters Page 6 of 14

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32 Figure 2. Characterization of the hollowing process: (a) Schematic illustration of different pore
33
34 sizes present in enzyme-in-MOFs samples. (b, c) in situ SAXS profile of initial stage and
35
36 intermediate stage, and (d) time-resolved studies of the hollowing process.
37
38
39
40
41
42 To understand the hollowing process in the presence of enzymes, we have used a
43
synchrotron light source to carry out in situ small angle X-ray scattering (SAXS) measurements,
44
45 which are sensitive to structural changes in the mesostructured range. Detailed parameters are
46
47 elaborated in Table 1. Figure 2 shows the X-ray scattering profiles of the hollowing process. The
48
49 features at three regions, q = 0.1 ~ 0.4 Å-1, 0.04 ~ 0.07 Å-1, 0.01 ~ 0.04 Å-1, were profiled by
50 computational model to give detailed microporous, mesoporous, and macroporous structures
51
52 (details are discussed in supporting information). At the initial stage, two structural spaces are
53
54 observed: the intrinsic MOFs micropores with a diameter of 1.1 nm and the space holding the
55
56
encapsulated catalase with dimensions of 11 × 18 × 4.1 nm (Figure 2b). After 30 min, additional
57
58
59 6 Environment
60 ACS Paragon Plus
Page 7 of 14 Nano Letters

1
2
3 space, representing > 50 nm macroporous cavities, was observed, which supports the formation of
4
5 central cavities (Figure 2c). Figure 2d shows the profiles of the whole process. There is a clear
6
7 trend of the intensity increasing in macroporous region (q = 0.01 ~ 0.04 Å-1) and intensity
8
9
decreasing in microporous region (q = 0.1 ~ 0.4 Å-1), indicating the increasing of central
10 macropores and decreasing of micropores because the ZIF-67 cores were being removed gradually
11
12 during the hollowing process.
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36 Figure 3. (a) Infrared spectra of free catalase, confined catalase, and freestanding catalase in Tris
37 buffer (pH 7.5, 50 mM), respectively. (b-d) Fluorescence spectra of free catalase, confined
38
39 catalase, and freestanding catalase in Tris buffer (pH 7.5, 50 mM) before and after exposure to 8
40
41 M urea.
42
43
44
45
46
47 After investigating the structures and hollowing process, we studied the interfacial
48
49 interactions between enzyme and MOFs. First, we used infrared (IR) spectroscopy to study the
50 chemical interactions between the encapsulated catalase and MOFs for the two samples. We
51
52 focused on the amide-I stretches of catalase at 1633 cm-1, which is sensitive to the surrounding
53
54 coordination environment of the enzyme.28 The IR spectra reveals a difference between the
55
56
freestanding and confined samples (Figure 3a). Confined catalase exhibits a blue shift of the
57
58
59 7 Environment
60 ACS Paragon Plus
 Nano Letters Page 8 of 14

1
2
3 amide-I stretch (1660 cm-1) compared to free catalase (1633 cm-1), indicating a chemical
4
5 interaction between the confined catalase and MOFs.28 We have probed the samples in
6
7 intermediate states during the hollowing process and observed a graduate red-shift (Figure S4),
8
9
revealing that the states of the enzymes were changed gradually from confined to freestanding
10 state. After hollowing process, the amide-I stretch (1647 cm-1) of freestanding catalase is closer to
11
12 that of the free catalase (1633 cm-1), showing that the hollow structure reduces chemical
13
14 interactions between catalase and MOFs. The difference in the amide-Ⅰ stretch frequencies of
15
16 free and freestanding catalase can be attributed to the attachment of catalase to the internal surface
17 of the central cavities.30
18
19
20 Next, we studied the structural confinement of enzymes in solid and hollow MOFs. Urea,
21
22 a universal unfolding agent, has been used as a probe molecule to study structural confinement of
23
24
enzymes.40 When exposed to urea, the conformation of free enzymes without confinement can be
25 altered easily.37 This structural change can be identified by fluorescence spectroscopy because the
26
27 emission of the tryptophan of catalase is sensitive to the conformational change.41 Solid and hollow
28
29 samples were exposed to 8 M urea solutions and monitored by fluorescence spectroscopy (Figures
30
3b-d). The size of urea molecules is smaller than the pore aperture size so it can diffuse into ZIF-
31
32 8.29 After exposure to urea, free catalase shows a redshift (from 330 to 342 nm) of the maximum
33
34 fluorescence emission (λmax) due to change in its conformation. The solid sample shows no
35
36 significant shift, indicating that the conformation of catalase in solid MOFs is confined by
37 interfacial interactions.37 As expected, during the hollowing process, the spectra gradually red-
38
39 shift (Figure S5), and the freestanding catalase shows a large (13 nm) shift after urea exposure,
40
41 similar to free catalase, which suggests that confinement generated by interfacial interactions
42
43
gradually decreased during the process. The lesser confined state benefits activity but could
44 negatively impact stability (Figure S6). This understanding highlights the importance of balancing
45
46 the tradeoff between stability and activity.
47
48
49 With the observation of lesser interfacial interaction between the MOFs and freestanding
50
enzyme, we tested their biological function by carrying out H2O2 degradation over the
51
52 encapsulated samples. Note that catalase loading is kept consistent and we have characterized the
53
54 samples after the reaction to show that the structure was not damaged during the reactions (Figure
55
56 S7). Figures 4 and S8 present the catalytic rate constants (kobs) of encapsulated catalase. The
57
58
59 8 Environment
60 ACS Paragon Plus
Page 9 of 14 Nano Letters

1
2
3 confined catalase shows an observed rate constant of 4.3 × 10-3 s-1, and kinetic study indicates that
4
5 there is no significant mass transport limitation (Figure S9). During the hollowing process, activity
6
7 gradually increased with freestanding catalase showing the highest catalytic rate constant of 1.1 ×
8
9
10-2 s-1, almost three times greater than confined catalase. Correlating the kinetic studies of H2O2
10 catalysis with the spectroscopic studies suggests that the higher activity of freestanding catalase
11
12 could be caused by a decrease in the enzyme-MOF interfacial interactions. The Michaelis-Menten
13
14 constants, KM and Vmax, of the confined catalase, freestanding catalase, and free catalase were
15
compared. Free catalase was reported to decompose H2O2 with a KM of 25.16 mM and a Vmax of
16
17 400.1 µM·s-1 (SI).42 In Table 2, compared to free catalase, the confined catalase showed reduced
18
19 KM (6.45 mM) and Vmax (47.30 µM·s-1), which is in agreement with the previous observations of
20
21 enzymes immobilized in MOFs.19, 25 For the freestanding catalase, both KM (18.71 mM) and Vmax
22 (432.90 µM·s-1) are closer to free catalase compared to confined catalase. This phenomenon
23
24 reasserts that the hollow structure reduces interfacial interactions and allows for better
25
26 performance. To test whether this phenomenon can be observed on other enzymes, we have carried
27
28
out the study over chymotrypsin and lipase. In both cases, the freestanding enzyme shows a higher
29 activity than the confined enzymes (Figure S10).
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52 Figure 4. Kinetic studies of H2O2 degradation for confined and freestanding catalase with and
53
54 without exposure to proteinase-K. All assays were performed in Tris buffer (pH 7.5, 50 mM). Error
55
bars show the standard deviation of three independent measurements.
56
57
58
59 9 Environment
60 ACS Paragon Plus
 Nano Letters Page 10 of 14

1
2
3
4
5
6
7 Furthermore, the freestanding catalase is protected by MOFs that could shield the enzymes
8 from inhibitors with a large molecule sizes, such as proteinase-K. When exposed to proteinase-K,
9
10 free catalase shows no detectable activity.36 While we exposed the freestanding catalase in hollow
11
12 ZIF-8 to proteinase-K. The proteinase-K treated freestanding catalase exhibits a similar rate
13
14
constant (1.02 × 10-2 s-1) as before the treatment, indicating that the enzyme is shielded from
15 protease.
16
17
18 Table 2. Summary of KM and Vmax of confined catalases and freestanding catalases.
19
20 Confined catalase[a] Freestanding catalase[a]
21
22 KM (mM) 6.45 18.71
23
24 Vmax (µM·s-1) 47.30 432.90
25
26 [a] All measurements were conducted using the same amount of catalase in each test (0.232 mg).
27
28
29 In summary, we have developed a new strategy to encapsulate enzymes into hollow MOFs
30
31 with reduced interfacial interaction between enzyme and MOFs. It allows us to investigate the
32
relationship between the interfacial interactions and biological function. Our study indicates that
33
34 freestanding enzymes have less chemical interaction with the MOFs and that their structure is less
35
36 confinement. The hollow structure reduces interfacial interactions and is inspired by the cell
37
38 environment, such as glycolysis enzymes in the cytoplasm of living cells. This study not only
39 highlights the importance of the freestanding state for the biological function of encapsulated
40
41 enzymes but also presents a new way to immobilize freestanding enzymes in MOFs.
42
43
44
45
46
47 ASSOCIATED CONTENT
48
49
50 Supporting Information.
51
52
53
This material is available free of charge via the internet at http://pubs.acs.org.
54
55 Experimental section, characterization, and figures.
56
57
58
59 10 Environment
60 ACS Paragon Plus
Page 11 of 14 Nano Letters

1
2
3 AUTHOR INFORMATION
4
5
6 Corresponding Author
7
8 * frank.tsung@bc.edu
9
10
11
* fshieh@ncu.edu.tw
12
13 * zhuoly@shanghaitech.edu.cn
14
15 * hcyang_chem@mail.fju.edu.tw
16
17 Author Contributions
18
19 % These authors contributed equally.
20
21
22 ACKNOWLEDGMENT
23
24
This work made use of the resources of the Instrumental Analysis Center of the SPST at
25
26 ShanghaiTech University for XRD analysis and gas sorption measurements. The SEM and TEM
27
28 analysis was supported by the Center of High-Resolution Electron Microscopy (CℏEM) of the
29
30 SPST at ShanghaiTech University. S.-Y. C, Y. A and L.-Y. C would like to thank the funding from
31 ShanghaiTech University and Shanghai Natural Science Fund support (Grant No. 18ZR1425300).
32
33 W.-S. L, B.P. W and C.-K. T acknowledge the support from Boston College. Y.-D. H, F.-S. L
34
35 T.-Q. S, H.-W. L and F.-K. S would like to thank Ministry of Science and Technology, Taiwan for
36
37 the funding support (MOST 107-2113-M-008 -008 -MY2). S.-W. L and H.-C. Y would like to
38 thank Ministry of Science and Technology, Taiwan for the funding support (MOST 105-2119-M-
39
40 030-002-MY2). We also thank the BL23A1 National Synchrotron Radiation Research Center of
41
42 Taiwan for providing beam time and technical support.
43
44
45
46 ABBREVIATIONS
47
48
49 MOFs, metal-organic frameworks; SEM, scanning electron microscopy; PXRD, Powder X-ray
50
51 diffraction; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SAXS,
52 small angle X-ray scattering; IR spectroscopy, infrared spectroscopy; λmax, maximum fluorescence
53
54 emission.
55
56
57
58
59 11 Environment
60 ACS Paragon Plus
 Nano Letters Page 12 of 14

1
2
3
4
5
6 REFERENCES
7
8 1. Kuchler, A.; Yoshimoto, M.; Luginbuhl, S.; Mavelli, F.; Walde, P. Enzymatic reactions in
9 confined environments. Nat. Nanotechnol. 2016, 11, 409-20.
10 2. DiCosimo, R.; McAuliffe, J.; Poulose, A. J.; Bohlmann, G. Industrial use of immobilized
11 enzymes. Chem. Soc. Rev. 2013, 42, 6437-6474.
12 3. Franssen, M. C.; Steunenberg, P.; Scott, E. L.; Zuilhof, H.; Sanders, J. P. Immobilised
13 enzymes in biorenewables production. Chem. Soc. Rev. 2013, 42, 6491-533.
14
15
4. Palla, K. S.; Hurlburt, T. J.; Buyanin, A. M.; Somorjai, G. A.; Francis, M. B. Site-Selective
16 Oxidative Coupling Reactions for the Attachment of Enzymes to Glass Surfaces through
17 DNA-Directed Immobilization. J. Am. Chem. Soc. 2017, 139, 1967-1974.
18 5. Frenkel-Mullerad, H.; Avnir, D. Sol−Gel Materials as Efficient Enzyme Protectors: 
19 Preserving the Activity of Phosphatases under Extreme pH Conditions. J. Am. Chem. Soc.
20 2005, 127, 8077-8081.
21
6. Chapman, R.; Stenzel, M. H. All Wrapped up: Stabilization of Enzymes within Single
22
23 Enzyme Nanoparticles. J. Am. Chem. Soc. 2019, 141, 2754-2769.
24 7. Sheldon, R. A.; van Pelt, S. Enzyme immobilisation in biocatalysis: why, what and how.
25 Chem. Soc. Rev. 2013, 42, 6223-6235.
26 8. Zou, X.; Wei, S.; Badieyan, S.; Schroeder, M.; Jasensky, J.; Brooks, C. L.; Marsh, E. N. G.;
27 Chen, Z. Investigating the Effect of Two-Point Surface Attachment on Enzyme Stability and
28 Activity. J. Am. Chem. Soc. 2018, 140, 16560-16569.
29
30
9. Yiu, H. H. P.; Wright, P. A.; Botting, N. P. Enzyme immobilisation using SBA-15
31 mesoporous molecular sieves with functionalised surfaces. J. Mol. Catal. B Enzym. 2001,
32 15, 81-92.
33 10. Yan, M.; Ge, J.; Liu, Z.; Ouyang, P. Encapsulation of Single Enzyme in Nanogel with
34 Enhanced Biocatalytic Activity and Stability. J. Am. Chem. Soc. 2006, 128, 11008-11009.
35 11. Mateo, C.; Abian, O.; Fernandez–Lafuente, R.; Guisan, J. M. Increase in conformational
36
37
stability of enzymes immobilized on epoxy-activated supports by favoring additional
38 multipoint covalent attachment☆. Enzyme Microb. Technol. 2000, 26, 509-515.
39 12. Hanefeld, U.; Gardossi, L.; Magner, E. Understanding enzyme immobilisation. Chem. Soc.
40 Rev. 2009, 38, 453-68.
41 13. Coscolín, C.; Beloqui, A.; Martínez-Martínez, M.; Bargiela, R.; Santiago, G.; Blanco, R. M.;
42
Delaittre, G.; Márquez-Álvarez, C.; Ferrer, M. Controlled manipulation of enzyme
43
44 specificity through immobilization-induced flexibility constraints. Applied Catalysis A:
45 General 2018, 565, 59-67.
46 14. Böhm, A.; Trosien, S.; Avrutina, O.; Kolmar, H.; Biesalski, M. Covalent Attachment of
47 Enzymes to Paper Fibers for Paper-Based Analytical Devices. Front. Chem. 2018, 6, 214.
48 15. Hoarau, M.; Badieyan, S.; Marsh, E. N. G. Immobilized enzymes: understanding enzyme -
49 surface interactions at the molecular level. Org. Biomol. Chem. 2017, 15, 9539-9551.
50
51
16. Wang, Z.; Cohen, S. M. Postsynthetic modification of metal–organic frameworks. Chem.
52 Soc. Rev. 2009, 38, 1315-1329.
53 17. Kaneti, Y. V.; Dutta, S.; Hossain, M. S. A.; Shiddiky, M. J. A.; Tung, K.-L.; Shieh, F.-K.;
54 Tsung, C.-K.; Wu, K. C.-W.; Yamauchi, Y. Strategies for Improving the Functionality of
55
56
57
58
59 12 Environment
60 ACS Paragon Plus
Page 13 of 14 Nano Letters

1
2
3 Zeolitic Imidazolate Frameworks: Tailoring Nanoarchitectures for Functional Applications.
4
5
Adv. Mater. 2017, 29, 1700213.
6 18. Gumilar, G.; Kaneti, Y. V.; Henzie, J.; Chatterjee, S.; Na, J.; Yuliarto, B.; Nugraha, N.;
7 Patah, A.; Bhaumik, A.; Yamauchi, Y. General synthesis of hierarchical sheet/plate-like M-
8 BDC (M = Cu, Mn, Ni, and Zr) metal–organic frameworks for electrochemical non-
9 enzymatic glucose sensing. Chem. Sci. 2020, 11, 3644-3655.
10 19. Lian, X.; Fang, Y.; Joseph, E.; Wang, Q.; Li, J.; Banerjee, S.; Lollar, C.; Wang, X.; Zhou,
11
H. C. Enzyme-MOF (metal-organic framework) composites. Chem. Soc. Rev. 2017, 46,
12
13
3386-3401.
14 20. Doonan, C.; Ricco, R.; Liang, K.; Bradshaw, D.; Falcaro, P. Metal-Organic Frameworks at
15 the Biointerface: Synthetic Strategies and Applications. Acc. Chem. Res. 2017, 50, 1423-
16 1432.
17 21. Li, P.; Modica, J. A.; Howarth, A. J.; Vargas, E.; Moghadam, P. Z.; Snurr, R. Q.; Mrksich,
18 M.; Hupp, J. T.; Farha, O. K. Toward design rules for enzyme immobilization in hierarchical
19
mesoporous metal-organic frameworks. Chem. 2016, 1, 154-169.
20
21 22. Li, P.; Moon, S.-Y.; Guelta, M. A.; Lin, L.; Gómez-Gualdrón, D. A.; Snurr, R. Q.; Harvey,
22 S. P.; Hupp, J. T.; Farha, O. K. Nanosizing a metal–organic framework enzyme carrier for
23 accelerating nerve agent hydrolysis. ACS nano 2016, 10, 9174-9182.
24 23. Li, P.; Moon, S.-Y.; Guelta, M. A.; Harvey, S. P.; Hupp, J. T.; Farha, O. K. Encapsulation
25 of a Nerve Agent Detoxifying Enzyme by a Mesoporous Zirconium Metal–Organic
26 Framework Engenders Thermal and Long-Term Stability. J. Am. Chem. Soc. 2016, 138,
27
28
8052-8055.
29 24. Chen, Y.; Li, P.; Modica, J. A.; Drout, R. J.; Farha, O. K. Acid-Resistant Mesoporous Metal–
30 Organic Framework toward Oral Insulin Delivery: Protein Encapsulation, Protection, and
31 Release. J. Am. Chem. Soc. 2018, 140, 5678-5681.
32 25. Feng, D.; Liu, T.-F.; Su, J.; Bosch, M.; Wei, Z.; Wan, W.; Yuan, D.; Chen, Y.-P.; Wang, X.;
33 Wang, K.; Lian, X.; Gu, Z.-Y.; Park, J.; Zou, X.; Zhou, H.-C. Stable metal-organic
34
frameworks containing single-molecule traps for enzyme encapsulation. Nat. Commun.
35
36
2015, 6, 5979.
37 26. Lian, X.; Chen, Y.-P.; Liu, T.-F.; Zhou, H.-C. Coupling two enzymes into a tandem
38 nanoreactor utilizing a hierarchically structured MOF. Chem. Sci. 2016, 7, 6969-6973.
39 27. Lian, X.; Erazo-Oliveras, A.; Pellois, J.-P.; Zhou, H.-C. High efficiency and long-term
40 intracellular activity of an enzymatic nanofactory based on metal-organic frameworks. Nat.
41 Commun. 2017, 8, 2075.
42
28. Liang, K.; Ricco, R.; Doherty, C. M.; Styles, M. J.; Bell, S.; Kirby, N.; Mudie, S.; Haylock,
43
44 D.; Hill, A. J.; Doonan, C. J. Biomimetic mineralization of metal-organic frameworks as
45 protective coatings for biomacromolecules. Nat. Commun. 2015, 6, 7240.
46 29. Liang, K.; Coghlan, C. J.; Bell, S. G.; Doonan, C.; Falcaro, P. Enzyme encapsulation in
47 zeolitic imidazolate frameworks: a comparison between controlled co-precipitation and
48 biomimetic mineralisation. Chem. Commun. 2016, 52, 473-476.
49 30. Liang, W.; Xu, H.; Carraro, F.; Maddigan, N. K.; Li, Q.; Bell, S. G.; Huang, D. M.; Tarzia,
50
51
A.; Solomon, M. B.; Amenitsch, H.; Vaccari, L.; Sumby, C. J.; Falcaro, P.; Doonan, C. J.
52 Enhanced Activity of Enzymes Encapsulated in Hydrophilic Metal–Organic Frameworks. J.
53 Am. Chem. Soc. 2019, 141, 2348-2355.
54
55
56
57
58
59 13 Environment
60 ACS Paragon Plus
 Nano Letters Page 14 of 14

1
2
3 31. Liang, W.; Carraro, F.; Solomon, M. B.; Bell, S. G.; Amenitsch, H.; Sumby, C. J.; White, N.
4
5
G.; Falcaro, P.; Doonan, C. J. Enzyme Encapsulation in a Porous Hydrogen-Bonded Organic
6 Framework. J. Am. Chem. Soc. 2019, 141, 14298-14305.
7 32. Lykourinou, V.; Chen, Y.; Wang, X.-S.; Meng, L.; Hoang, T.; Ming, L.-J.; Musselman, R.
8 L.; Ma, S. Immobilization of MP-11 into a Mesoporous Metal–Organic Framework, MP-
9 11@mesoMOF: A New Platform for Enzymatic Catalysis. J. Am. Chem. Soc. 2011, 133,
10 10382-10385.
11
33. Chen, Y.; Lykourinou, V.; Vetromile, C.; Hoang, T.; Ming, L.-J.; Larsen, R. W.; Ma, S. How
12
13
Can Proteins Enter the Interior of a MOF? Investigation of Cytochrome c Translocation into
14 a MOF Consisting of Mesoporous Cages with Microporous Windows. J. Am. Chem. Soc.
15 2012, 134, 13188-13191.
16 34. Chen, Y.; Han, S.; Li, X.; Zhang, Z.; Ma, S. Why does enzyme not leach from metal–organic
17 frameworks (MOFs)? Unveiling the interactions between an enzyme molecule and a MOF.
18 Inorg. Chem. 2014, 53, 10006-10008.
19
35. Pan, Y.; Li, H.; Farmakes, J.; Xiao, F.; Chen, B.; Ma, S.; Yang, Z. How Do Enzymes Orient
20
21 When Trapped on Metal–Organic Framework (MOF) Surfaces? J. Am. Chem. Soc. 2018,
22 140, 16032-16036.
23 36. Shieh, F.-K.; Wang, S.-C.; Yen, C.-I.; Wu, C.-C.; Dutta, S.; Chou, L.-Y.; Morabito, J. V.;
24 Hu, P.; Hsu, M.-H.; Wu, K. C.-W. Imparting functionality to biocatalysts via embedding
25 enzymes into nanoporous materials by a de novo approach: size-selective sheltering of
26 catalase in metal–organic framework microcrystals. J. Am. Chem. Soc. 2015, 137, 4276-
27
28
4279.
29 37. Liao, F.-S.; Lo, W.-S.; Hsu, Y.-S.; Wu, C.-C.; Wang, S.-C.; Shieh, F.-K.; Morabito, J. V.;
30 Chou, L.-Y.; Wu, K. C.-W.; Tsung, C.-K. Shielding against Unfolding by Embedding
31 Enzymes in Metal–Organic Frameworks via a de Novo Approach. J. Am. Chem. Soc. 2017,
32 139, 6530-6533.
33 38. Liu, X. Y.; Zhang, F.; Goh, T. W.; Li, Y.; Shao, Y. C.; Luo, L.; Huang, W.; Long, Y. T.;
34
Chou, L. Y.; Tsung, C. K. Using a Multi‐Shelled Hollow Metal–Organic Framework as a
35
36
Host to Switch the Guest‐to‐Host and Guest‐to‐Guest Interactions. Angew. Chem. Int. Ed.
37 2018, 130, 2132-2136.
38 39. Yang, J.; Zhang, F.; Lu, H.; Hong, X.; Jiang, H.; Wu, Y.; Li, Y. Hollow Zn/Co ZIF Particles
39 Derived from Core–Shell ZIF-67@ZIF-8 as Selective Catalyst for the Semi-Hydrogenation
40 of Acetylene. Angew. Chem. Int. Ed. 2015, 54, 10889-10893.
41 40. Monera, O. D.; Kay, C. M.; Hodges, R. S. Protein denaturation with guanidine hydrochloride
42
or urea provides a different estimate of stability depending on the contributions of
43
44 electrostatic interactions. Protein Sci. 1994, 3, 1984-1991.
45 41. Vallée-Bélisle, A.; Michnick, S. W. Visualizing transient protein-folding intermediates by
46 tryptophan-scanning mutagenesis. Nat. Struct. Mol. Biol. 2012, 19, 731-736.
47 42. Çetinus, Ş. A.; Öztop, H. N. Immobilization of catalase on chitosan film. Enzyme Microb.
48 Technol. 2000, 26, 497-501.
49
50
51
52
53
54
55
56
57
58
59 14 Environment
60 ACS Paragon Plus