Fabricating Covalent Organic Framework Capsules with Commodious Microenvironment for Enzymes

Article abstract of DOI:10.1021/jacs.0c00285

Enzyme immobilization has been demonstrated to be a favorable protocol to promote industrialization of biomacromolecules. Despite tremendous efforts to develop new strategies and materials to realize this process, maintaining enzyme activity is still a fo

Full text of DOI:10.1021/jacs.0c00285

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Article
Fabricate Covalent Organic Framework Capsules
with Commodious Microenvironment for Enzymes
Mingmin Li, Shan Qiao, Yunlong Zheng, Yassin H Andaloussi, Xia Li,
Zhenjie Zhang, Ang Li, Peng Cheng, Shengqian Ma, and Yao Chen
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.0c00285 • Publication Date (Web): 20 Mar 2020
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Fabricate Covalent Organic Framework Capsules with Commodious
Microenvironment for Enzymes
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Mingmin Li,† Shan Qiao,† Yunlong Zheng,† Yassin H. Andaloussi,‡ Xia Li,‡ Zhenjie Zhang,‡,§ Ang Li,†
Peng, Cheng,§ Shengqian Ma⊥ and Yao Chen*,†
† State Key Laboratory of Medicinal Chemical biology, College of Pharmacy, Nankai University, Tianjin 300071, China
§ College of Chemistry, Nankai University, Tianjin, 300071, China
‡ Department of Chemical Sciences, Bernal Institute, University of Limerick, Limerick V94 T9PX, Republic of Ireland
⊥ Department of Chemistry, University of South Florida, 4202 E. Fowler Avenue, Tampa, Florida 33620, United States
KEYWORDS: covalent organic frameworks, capsule, immobilization, biomacromolecules, biocatalysis
ABSTRACT: Enzyme immobilization has demonstrated itself to be a favorable protocol to promote industrialization of
biomacromolecules. Despite tremendous efforts to develop new strategies and materials to realize this process, maintaining
enzyme activity is still of formidable challenge. Herein, we created a sacrificial templating method, using metal-organic
frameworks (MOFs) as sacrificial templates to construct hollow covalent organic framework (COF) capsules for enzyme
encapsulation. This strategy can provide capacious microenvironment to unleash enzyme molecules. The improved
conformational freedom of enzymes, enhanced mass transfer and protective effect against external environment ultimately
boosted the enzymatic activities. We also found that this strategy possesses high versatility that is suitable for diverse
biomacromolecules, MOF templates and COF capsules. Moreover, the dimensions, pore sizes and shell thickness of COF
capsules can be conveniently tuned, allowing for customizing bioreactors for specific functions. For example, co-
encapsulation of different enzymes with synergistic functions were successfully demonstrated using this bioreactor platform.
This study not only opens up a new avenue to overcome the present limitations of enzymatic immobilization in porous
matrixes, but also provides new opportunities for construction of bio-microdevices or artificial organelles based on crystalline
porous materials.
Exploration of new platforms to overcome these challenges
for optimal enzyme performance in confined space is,
therefore, in urgent demand.
INTRODUCTION
As natural catalysts, enzymes possess fascinating
characteristics, such as high efficiency, regio- and
stereoselectivity, allowing for continual advances in
biotechnological applications.1,2 However, the intrinsically
fragile nature of enzymes greatly impedes their
industrialization. Enzyme immobilization has become a
favorable strategy with increased stability and reusability,
easy operation, energy conservation and continuous-flow
production, prompting the application of enzymes.3-11
Despite these merits, most confined enzymatic systems still
confront great challenges. For instance, tightly packed
enzymes within host materials typically lack
conformational freedom that may greatly affects molecular
recognition.12,13 Mass transfer of substrates or products can
also be a limiting factor, and thus internally-adsorbed
enzymes may display substantially reduced activity.14,15
Moreover, traditional incorporation methods such as
adsorption often suffer from low loading or high leaching
due to weak interaction or pore size mismatching.16
Compartmentalization of biomacromolecules is
a
common phenomenon in living systems.17,18 The eukaryotic
cell is a prototypical example whereby the unique
biochemical microenvironment allows the spatial control of
various functions. Such enclosed units confer physical
protection against external disturbance, while maintaining
a certain degree of freedom for the internal contents.19,20
Inspired by nature, the design and construction of
biomimetic ‘smart capsules’ can be a promising approach to
overcome the challenges of confined enzymatic systems.21-
23
Organic polymer and inorganic nanoparticles are
materials commonly used to prepare capsules through
various approaches.18,24,25 However, they may suffer from
irregular structure or lack of chemical functionality.26,27
Covalent organic frameworks (COFs) are an emerging class
of crystalline porous materials with high and well-defined
porosity, high stability, and readily engineered
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functionality, allowing them as excellent hosts for enzyme
(e.g. enzymes) and protect them against harsh conditions
while also being feasibly digested under mildly acidic
conditions.35-37 Thus, ZIF-90 is a perfect candidate to
demonstrate the concept due to the presence of unreacted
aldehyde groups in the ligand 2-imidazolecarbaldehyde
(ICA), which could be anticipated in the amine-aldehyde
condensation reaction involved in a subsequent COF
formation, and therefore allows the MOF particles as
templates in the formation of COF shells (Figure 1). In order
to maintain the activity of biomacromolecules during the
synthesis process, an acid-stable COF that can be
synthesized under mild conditions is then required for the
construction of capsules, for which COF-42 was chosen.39,40
COF-42 was synthesized via the room temperature
condensation of 1,3,5-triformylbenzene (TB) and 2,5-
Bis(ethoxy)terephthalohydrazide (BETH) under acetic acid
catalysis. However, because protic acids could decompose
the enzyme@MOF complexes, a MOF-compatible catalyst,
Sc(OTf)3,41 was used instead of acetic acid. Various
structural analogues of the BETH monomer were screened
to find one that produced a crystalline COF-42 product
(Table S1, Figure S1 and S2). It was found that only 2, 5-
Bis(butenyloxy)terephthalohydrazide (BBTH) succeeded to
afford a crystalline COF-42 analogue (named as COF-42-B)
which was stable under acid condition (e.g. pH = 5,
phosphate buffer (PB) solution, 50 mM) (Figure S3 and S4).
encapsulation.28-31 However, the harsh synthetic conditions
required in the fabrication of COFs make it impractical to
directly
make
COF
capsules
encapsulating
biomacromolecules through a “one-pot” strategy.32-34 The
sister material of COFs, metal-organic frameworks (MOFs)
can in-situ encapsulate biomacromolecules to form
biomacromolecule@MOF (@ = encapsulating) with high
loading and good protection.35-38 However, this often results
in a penalty to lose the conformational freedom of enzymes,
leading to a detrimental effect on enzyme activity. Many
reports including our recently work have proved that
biomacromolecule@MOF can be easily digested to release
biomacromolecules without losing their bioactivities which
render MOFs as perfect sacrificial templates to make COF
capsules.37 Combining the benefits of both COFs and MOFs
to form ‘smart capsules’ may open up new opportunities to
greatly enhance the performance of confined enzyme: i) the
MOF encapsulation can protect enzymes from harsh
synthetic conditions, such as those amenable to COF
formation, allowing for COFs to directly coat on
enzyme@MOF particles; ii) the high porosity of COF shells
then allows for subsequent digestion of MOF cores while
facilitating mass transfer of substrates resulting in active,
conformationally unhindered enzymes within a protective
COF capsule.
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Herein, we created a versatile approach to fabricate
biomimetic ‘smart COF capsules’ for the encapsulation of
active biomacromolecules. Biomacromolecules are first
encapsulated within digestible MOFs via in-situ
encapsulation to form biomacromolecules@MOF which can
protect biomacromolecules and act as templates for the
growth of biomarcomolecule@MOF@COF core-shell
structures. Finally, the MOF core was etched away to
liberate the biomacromolecules within the hollow COF
capsule (Scheme 1). Such a commodious microenvironment
in the formed biomacromolecule@COF offers protective
effects while maintaining the conformational freedom of
biomacromolecules and facilitating the mass transfer of
substrates/products, which is essential for high activity or
synergistic effect of different enzymes. This study opens up
a new avenue to overcome the present limitations of
enzymatic immobilization in porous matrixes.
Figure 1. Illustration of the synthetic route of
Scheme 1. Schematic illustration of the stepwise approach to
construct biomacromolecule@capsule.
biomacromolecule@COF
capsules.
(a)
Immobilizing
biomacromolecules in ZIF-90 via an in-situ encapsulation
method; (b) A one-pot reaction of ZIF-90 with COF monomers;
RESULTS AND DISCUSSION
(c)
Illustration
of
the
core-shell
structure
of
Materials Preparation and Characterizations.
A
biomacromolecule@ZIF-90@COF-42-B (middle), structure of
COF-42-B (left), a TEM image of the core-shell structure (right);
supporting matrix is required for COF capsule formation in
order to protect enzymes against harsh synthetic conditions
and to act as a template. ZIF-90 has been previously shown
to efficiently load various biological active macromolecules
(d)
Illustration
of
the
capsule
(middle),
structure
of
biomacromolecule@COF-42-B
EDS
carbon
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distribution (left) and
biomacromolecule@COF-42-B capsule.
a
TEM image (right) of
reaction was monitored by TEM, which showed a clear
transformation from the core-shell structure to a hollow
capsule within 1 h. Tracking characteristic PXRD peaks of
ZIF-90 afforded a result consistent with TEM data (Figure
S8 and S9). Energy dispersive spectrometer (EDS) element
mapping further demonstrated the successful construction
of COF-42-B capsules (Figure 1d). Porosity of these
materials was found to be retained during the synthetic
procedure (Figure S10). N2 sorption data collected at 77 K
revealed the Langmuir surface areas of 1390 m2/g, 610
m2/g, 436 m2/g and 835 m2/g, BET surface areas of 723
m2/g, 367 m2/g, 235 m2/g and 530 m2/g, pore size
distribution centered around 0.9 nm, 1.8 nm, 1.2 nm, and
1.8 nm for BSA@ZIF-90, COF-42-B, BSA@ZIF-90@COF-42-
B and BSA@COF-42-B, respectively. Moreover, it was found
that the thickness of the COF-42-B shell could be readily
adjusted by altering the amount of added BSA@ZIF-90.
Upon increasing the amount of BSA@ZIF-90, the PXRD
reflection peaks assigned to ZIF-90 exhibited increased
intensity relative to the intensity of the COF-42-B peaks
(Figure S11), and TEM images showed the COF shell varying
from 130 nm to 15 nm (Figure 2b). A control experiment
was further performed to test if BSA can be directly
incorporated into COF-42-B via the traditional pore
adsorption method. The attempt failed because the channel
size of COF-42-B was much smaller than the molecule size
of BSA (Figure S12).
Exploration of the generality of capsule fabrication
strategy. In order to explore the generality of this synthetic
strategy, we examined the use of ZPF-2 (ZPF = Zeolitic
Pyrimidine Framework) as the sacrificial template, instead
of ZIF-90. ZPF-2 with a sod topology was synthesized from
Zn(NO3)2·6H2O and 2-Hydroxy-5-fluoropyrimidine (Figure
S13) in aqueous solution at room temperature.42 Herein, for
the first time, we demonstrated that ZPF-2 can efficiently
incorporate biomacromolecules via in-situ encapsulation
method. As revealed by TEM, PXRD, and FT-IR (Figure S14-
S16), ZPF-2 can efficiently encapsulate BSA and serve as a
sacrificial template to yield hollow BSA@COF-42-B
capsules. Additionally, we found that digesting rate of ZPF-
2 was faster than ZIF-90 and the MOF cores was completely
removed in 10 min (Figure S17). Moreover, we found ZIF-8
can also be applied to fabricate COF capsules via a similar
approach as ZIF-90 and ZPF-2 (Figure S18-S20). However,
only a relatively small amount of ZIF-8 could be
incorporated within COF-42-B because increased amounts
of ZIF-8 inhibited the formation of COF-42-B. We also
explore whether other COF materials can be used to make
COF capsules. A hollow COF capsule containing BSA was
successfully fabricated by COF-43-B (Figure S21 and S22).39
This stepwise strategy has, therefore, proven to be an
adaptable method to fabricate biomacromolecules-
containing hollow COF capsules that could foreseeably be
modified for use in highly specific applications.
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Figure 2. Characterizations of BSA@COF-42-B capsule. (a)
PXRD patterns of products in different synthesis stage.
BSA@ZIF-90 (blue), BSA@ZIF-90@COF-42-B (purple),
BSA@COF-42-B (green), calculated pattern of ZIF-90 (black)
and COF-42-B (red); (b) TEM images showing BSA@ZIF-
90@COF-42-B precursors (upper) and BSA@COF-42-B
capsules (below) with different shell thickness tuned via
adding different amounts of BSA@ZIF-90 (from left to right, 6.0
mg, 9.0 mg, 12.0 mg, 15.0 mg and 18.0 mg). Scale bar: 100 nm.
In order to verify the construction strategy of COF
capsules, BSA (Bovine Serum Albumin) was chose as a
model biomacromolecule on the basis of its low cost and
high solubility. BSA@ZIF-90 was first prepared via the in-
situ encapsulation method according to literature.36 The
encapsulation efficiency of BSA was measured to be as high
as 99%, using a standard Bradford Assay, and the loading
percentage was calculated to be 0.22 g BSA per g ZIF-90
(Figure S5). Subsequently, the as-prepared BSA@ZIF-90
was mixed with TB, BBTH and Sc(OTf)3 in 1, 4-
dioxane/mesitylene (3:1 v/v) for 30 min. PXRD patterns,
TEM images, showed a combination of two crystalline
phases (i.e. ZIF-90 and COF-42-B) (Figure 2 and S6),
indicating the existence of BSA@ZIF-90@COF-42-B. FT-IR
results further verified the successful encapsulation of BSA
(Figure S7). Our previous result has demonstrated that ZIF-
90 can be digested in aqueous solution with pH < 7.37 We
then exposed the as-prepared BSA@ZIF-90@COF-42-B to a
PB solution (pH = 5, 50 mM). The progress of digesting
Biocatalysis application of COF capsules. On the basis
of the successful encapsulation of BSA in COF capsules, we
further explored whether enzymes can be trapped within
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COF capsules. CAT is a well-known multimeric protein with
prepared a series of COF capsules in the range of 350-1500
nm, with a capsule thickness of ~60 nm (Figure S26b-g),
and found that capsules of ~1500 nm exhibited the best
performance (Figure S30). Thus, the optimized COF
capsules of ~1500 nm size and ~60 nm thickness were used
for further study.
four subunits, which can catalyze the breakdown of
hydrogen peroxide (H2O2) to generate O2 and H2O,
protecting cells from the oxidative damage caused by
hydrogen peroxide.43 In order to immobilize CAT within
COF capsules, ZPF-2 was selected as the model sacrificial
MOF. EDS mapping was performed to confirm the successful
encapsulation of CAT within the capsules. As shown in
Figure 3, Fe, the characteristic element present in CAT, was
uniformly dispersed in all samples; indicative of the
successful encapsulation of CAT. Additionally, confocal
images further verified the uniform dispersion of CAT in all
samples (Figure S23), and circular dichroism (CD) and UV-
Vis spectra confirmed that the structure of CAT was
retained during this synthesis process (Figure S24).
The biocatalytic assay revealed that CAT@ZPF-2@COF-
42-B before MOF etching presented 49% lower enzyme
activity than CAT@ZPF-2, which should be ascribed to the
high mass transfer resistance in CAT@ZPF-2@COF-42-B.
After etching treatment, the generated CAT@COF-42-B
capsule demonstrated a dramatic 58% improvement of
activity compared to CAT@ZPF-2, which could be 210%
when compared to CAT@ZPF-2@COF-42-B (Figure 4a).
Negligible leaching of CAT was detected during the etching
process (Figure S31). A further comparison of the kinetics
of CAT@ZPF-2 and CAT@COF-42-B revealed that their Vmax
and KM were 2.73 M/s and 0.0625 mM, and 3.98 M/s and
0.0531 mM, respectively (Figure S32). Although their
activities are lower than free CAT (Figure S33), the results
show significantly boosted biocatalytic activity for
CAT@COF-42-B capsule structure after the removal of
MOFs, indicating the unleashing CAT molecules from the
tightly packed MOF composite to the commodious
microenvironment of COF capsules is beneficial for
enzymatic performance. Additionally, we also investigate
the CAT-loaded COF-43-B capsules which showed a similar
trend as COF-42-B (Figure S34). After optimization, the
activity of CAT@COF-43-B exceeded CAT@COF-42-B and
showed an increase up to 81% compared with that of
CAT@ZPF-2 (Figure 4b) (loading capabilities was 1.49 g/g).
This result is probably due to the larger pore size of COF-
43-B (3.2 nm) compared with COF-42-B (1.9 nm) (Figure
S35) that facilitate the mass transport of substrates and
products. These results indicate that the synthetic strategy
can be applied as a versatile and tunable approach for on-
demand preparation of high efficient bioreactor based on
COF capsules.
Exploration of protection effect and reusability of
COF capsules. The protection effect of the formed COF
capsules was examined under various perturbation
conditions, such as acid, proteases, acetone and high
temperature (Figure 4c). CAT@COF-42-B prepared via
CAT@ZPF-2 was selected as a representative. First, we
assessed its acid tolerance in disodium hydrogen
phosphate-citric acid buffer (100 mM, pH = 4) for 30 min.
CAT@COF-42-B was seen to retain 85% of its original
activity while free CAT retained only 35%, and CAT@ZPF-2
lost almost all activity (>99%) due to the leaching of CAT
resulted from dissolution of ZPF-2 under the acidic
condition (Figure S36a). COF-42-B was shown to protect
CAT with 95% of original activity retained after exposure to
acetone for 60 min. By contrast, free CAT retained only 25%
of activity. A similar result was observed after exposure to
protease trypsin (6 mg/mL) for 60 min: CAT@COF-42-B
retained ~100% of its activity, while free CAT retained only
25% of activity. Next, the influence of heating was evaluated.
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Figure 3. Bright field images (left) and EDS elemental mapping
(right) of Fe, Zn and C for (a) CAT@ZPF-2, (b) CAT@ZPF-
2@COF-42-B and (c) CAT@COF-42-B.
Factors such as particle size and capsule shell thickness
were also investigated to optimize the catalytic activity of
this novel enzyme-incorporated system. A series of CAT-
loaded ZPF-2 crystals with particle sizes ranging from 100
nm to 8000 nm were synthesized (Figure S25). TEM images
were used to confirm particle size uniformity for each
sample (Figure S26a). Catalytic activity towards H2O2 was
evaluated via the FOX method (Figure S27 and S28). With
the same amount of enzymes, CAT@ZPF-2 achieved the
highest Kobs of 0.0473 s-1 with a particle size of ~1500 nm.
COF capsule shell thickness was also tuned by varying the
amount of added CAT@ZPF-2 core (with particle size of
~1500 nm). The results reveal that the activity of
enzyme@COF-42-B capsules obeyed
a
Gaussian
distribution when shell thickness was modified (Figure
S29), and the best performance was achieved with a shell
thickness of ~60 nm (Kobs = 0.0743 s-1) (the loading
capabilities was 1.66 g/g). Generally, the thicker capsule
shell is, the higher mass transfer resistance exists. Besides
thickness, capsule size of COF-42-B also plays a crucial role
in the activity of encapsulated enzymes. Thereafter, we
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After treatment under 60 °C for 10 min, free CAT and
was firstly encapsulated in COF-42-B capsules like CAT. We
found that GOx@COF-42-B using ZPF-2 as a sacrificial
template exhibited an improved activity (~170%)
compared to GOx@ZPF-2 (Figure 5a). We co-encapsulated
GOx and CAT into COF-42-B and examined their cascade
reaction effect. The cascade catalytic efficiency was
evaluated by the detection of glucose consumption using
the 3,5-dinitrosalicylic acid (DNS) method (Figure S41).48
Compared to GOx@ZPF-2, GOx&CAT@ZPF-2 exhibited a
much higher glucose consumption rate attributed to the
participation of CAT. As shown in Figure 5b,
CAT@ZPF-2 retained only 20% and 50% of original activity,
respectively, while CAT@COF-42-B retained 88% of
original enzymatic activity. These results revealed that
CAT@COF-42-B capsules demonstrated superior protective
effect that surpassed its counterparts such as free CAT and
CAT@ZPF-2. Furthermore, we investigated the catalytic
durability and stability of CAT@COF-42-B, which is an
important criterion for heterogeneous biocatalysts.
CAT@COF-42-B can be readily recovered without
significant loss of activity for at least 10 cycles (Figure 4d),
and negligible leakage was detected during recycling
experiments (Figure S36b). In comparison, traditional
porous materials such as SBA-15 and active carbon were
used to immobilize CAT via the conventional adsorption
method. Their loading capabilities were demonstrated to be
0.21 g/g, and 0.09 g/g, respectively (Figure S37 and S38).
The formed CAT composites exhibited much lower enzyme
loading or activity compared with the CAT@COF capsule
(~1.6 g/g, Figure S39), and exhibited no recyclability
(Figure S40).
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GOx&CAT@COF-42-B composite produced
a
130%
consumption rate of glucose compared to its counterpart
(GOx&CAT@ZPF-2). These results demonstrated the
synergistic effect between the two enzymes was
significantly enhanced after unleashed from ZPF-2 to the
commodious COF capsule. To further prove the concept of
cascade reactions in COF capsule, another multi-enzyme
bioreactor based on GOx&Hemin@COF-42-B was evaluated
spectroscopically using 3,3',5,5'-Tetramethylbenzidine
(TMB) and glucose as substrates, accompanying by a
colorimetric change detectable at 650 nm (Figure 5c). The
results revealed that GOx&Hemin@COF-42-B showed an
enhanced activity of 180% compared to its counterpart
(GOx&Hemin@ZPF-2), and further validated COF capsule
can provide the freedom to multiple enzymes for better
cascade performance, which implying potential
applications in the fabrication of bioreactors or advance
microdevices.
Figure 4. Biological activity of CAT@COF capsules. (a) Kinetics
of degradation of H2O2 for CAT@ZPF-2 (black), CAT@ZPF-
2@COF-42-B (blue) and CAT@COF-42-B (red). (b) Kinetics of
degradation of H2O2 for CAT@ZPF-2 (black), CAT@ZPF-
2@COF-43-B (blue) and CAT@ COF-43-B (red). (c) Activity
percentage of CAT, CAT@ZPF-2 and CAT@COF-42-B after
treatment under various harsh conditions. (d) Recycling
experiments of CAT@COF-42-B.
Cascade reactions conducted in COF capsule systems.
In practical applications, two or more enzymes are often
required to cooperate with each other for advanced
functions.44 Fabricating multi-enzyme architectures has
garnered increasing attention with great potential in the
field of biocatalysis and advanced microdevice, such as
‘artificial organelles’.27,45,46 For example, GOx can
spontaneously oxidize glucose into gluconic acid,
meanwhile producing H2O2 which can serve as the substrate
of CAT. Combining these two enzymes represents a model
cascade reaction of a biologically relevant system.47 To
prove the concept of cascade reactions in COF capsule, GOx
Figure 5. Catalytic performance of various systems. (a)
GOx@ZPF-2 and GOx@COF-42-B activity using a colorimetric
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assay based on the oxidation of ABTS by a Cyt c coupled system
The authors declare no competing financial interest.
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(λ = 660 nm). (b) GOx&CAT@ZPF-2 and GOx&CAT@COF-42-B
activity based on the kinetic consumption of glucose. (c)
GOx&Hemin@ZPF-2 and GOx&Hemin@COF-42-B activity
using a colorimetric assay.
ACKNOWLEDGMENT
The authors acknowledge the financial support from the
National Natural Science Foundation of China (21871153) and
Tianjin Natural Science Foundation of China (18JCZDJC37300).
CONCLUSION
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REFERENCES
In conclusion, we created a facile three-step approach to
fabricate novel COF capsules to encapsulate various
biomacromolecules which cannot be easily incorporated by
COFs via traditional approaches (i.e. adsorption method).
Firstly, biomacromolecules are first encapsulated within
digestible MOFs via in-situ encapsulation to form
biomacromolecule@MOF systems. This composite can
further act as templates for the growth of
biomarcomolecule@MOF@COF core-shell structures.
Finally, the MOF cores were etched away to unleash the tied
biomacromolecules within the hollow COF capsule. This
facile, scalable and effective strategy afforded high-
performance COF bioreactors, which can maintain enzyme
conformational freedom, stabilize the enzyme and
meanwhile allow effective diffusion of the substrates and
products. The COF capsules can provide capacious internal
environments for the optimal performance of the
encapsulated biomacromolecules with good protection
effect against external perturbations. The encapsulated
enzymes in COF capsules exhibited excellent reusability
which surpassed the traditional porous materials such as
SBA-15. In addition, this synthetic platform exhibits high
versatility and tunability: different sacrificial MOF
templates (e.g. ZIF-90, ZIF-8 and ZPF-2) and COF shells
(COF-42-B and COF-43-B), as well as various enzymes (e.g.
CAT, GOx) were used in this study; various factors such as
the COF shell thickness and pore size could be fine-tuned to
optimize the performance the bioreactors. We also
demonstrated that multiple enzymes could be easily co-
encapsulated within one COF capsule using this synthetic
strategy and act as advanced bioreactors capable of efficient
cascade reactions. Such diverse advances may inform the
further understanding of enzyme behavior in both in vivo
and in vitro systems, and expand the possibilities for the
construction of microdevices, artificial organelles, and even
artificial cells.
(1) Schmid, A.; Dordick, J. S.; Hauer, B.; Kiener, A.; Wubbolts, M.;
Witholt, B. Industrial Biocatalysis Today and Tomorrow. Nature
2001, 409 (6817), 258-268.
(2) Prasad, S.; Roy, I. Converting Enzymes into Tools of
Industrial Importance. Recent Pat. Biotechnol. 2018, 12 (1), 33-56.
(3) Kuchler, A.; Yoshimoto, M.; Luginbuhl, S.; Mavelli, F.; Walde,
P., Enzymatic Reactions in Confined Environments. Nat.
Nanotechnol. 2016, 11 (5), 409-420.
(4) Sheldon, R. A.; van Pelt, S. Enzyme Immobilisation in
Biocatalysis: Why, What and How. Chem. Soc. Rev. 2013, 42 (15),
6223-6235.
(5) Sirisha, V. L.; Jain, A.; Jain, A. Enzyme Immobilization: An
Overview on Methods, Support Material, and Applications of
Immobilized Enzymes. Adv. Food Nutr. Res. 2016, 79, 179-211.
(6) Lian, X.; Fang, Y.; Joseph, E.; Wang, Q.; Li, J.; Banerjee, S.;
Lollar, C.; Wang, X.; Zhou, H.-C. Enzyme-MOF (metal-organic
framework) Composites. Chem. Soc. Rev. 2017, 46 (11), 3386-3401.
(7) Li, P.; Modica, J. A.; Howarth, A. J.; Vargas L, E.; Moghadam, P.
Z.; Snurr, R. Q.; Mrksich, M.; Hupp, J. T.; Farha, O. K. Toward Design
Rules for Enzyme Immobilization in Hierarchical Mesoporous
Metal-Organic Frameworks. Chem 2016, 1 (1), 154-169.
(8) Huang, S.; Kou, X.; Shen, J.; Chen, G.; Ouyang, G. Armoring the
Enzymes with Metal-Organic Frameworks. Angew. Chem. Int. Ed.
2020. http://dx.doi.org/10.1002/anie.201916474.
(9) Yang, X. Y.; Li, Z. Q.; Liu, B.; Klein-Hofmann, A.; Tian, G.; Feng,
Y. F.; Ding, Y.; Su, D. S.; Xiao, F. S. “Fish-in-Net” Encapsulation of
Enzymes in Macroporous Cages as Stable, Reusable, and Active
Heterogeneous Biocatalysts. Adv. Mater. 2006, 18, 410-414.
(10) Fernandez-Lafuente, R. Stabilization of Multimeric
Enzymes: Strategies to Prevent Subunit Dissociation. Enzyme
Microb. Technol. 2009, 45 (6-7), 405-418.
(11) Pang, Y.; Li, H.; Farmakes, J.; Xiao, F.; Chen, B.; Ma, S.; Yang,
Z. How Do Enzymes Orient When Trapped on Metal-Organic
Framework (MOF) Surfaces? J. Am. Chem. Soc. 2018, 140 (47),
16032-16036.
(12) Johnson, B. J.; Algar, W. R.; Malanoski, A. P.; Ancona, M. G.;
Medintz, I. L. Understanding Enzymatic Acceleration at
Nanoparticle Interfaces: Approaches and Challenges. Nano Today
2014, 9 (1), 102-131.
(13) Sun, Q.; Pan, Y.; Wang, X.; Li, H.; Farmakes, J.; Aguila, B.;
Yang, Z.; Ma, S. Mapping Out The Degree of Freedom of Hosted
Enzymes in Confined Spatial Environments. Chem 2019, 5, 1-12.
(14) Klermund, L.; Castiglione, K. Polymersomes As
Nanoreactors for Preparative Biocatalytic Applications: Current
Challenges and Future Perspectives. Bioproc. Biosyst. Eng. 2018, 41
(9), 1233-1246.
(15) Hanefeld, U.; Gardossi, L.; Magner, E. Understanding
Enzyme Immobilisation. Chem. Soc. Rev. 2009, 38 (2), 453-468;
(16) An, H. D; Li, M. M.; Ga, J.; Zhang, Z. J.; Ma, S. Q.; Chen, Y.
Incorporation of Biomolecules in Metal-Organic Frameworks for
Advanced Applications. Coordin. Chem. Rev. 2019, 384, 90-106.
(17) Rodriguez-Arco, L.; Kumar, B. V. V. S. P.; Li, M.; Patil, A. J.;
Mann, S. Modulation of Higher-Order Behaviour in Model Protocell
Communities by Artificial Phagocytosis. Angew. Chem. Int. Ed.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website. Detailed experimental procedures,
material, FT-IR, 1H NMR, PXRD, TEM, and so on.
AUTHOR INFORMATION
Corresponding Author
*chenyao@nankai.edu.cn
Notes
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1
2
3
4
5
6
7
8
9
2019, 58 (19), 6333-6337.
Halder, A.; Verma, S.; Banerjee, R. Self-Templated Chemically Stable
Hollow Spherical Covalent Organic Framework. Nat. Commun.
2015, 6, 6786-6795.
(34) Feng, X.; Ding, X.; Jiang, D. Covalent Organic Frameworks.
Chem. Soc. Rev. 2012, 41 (18), 6010-6022.
(35) Liang, K.; Ricco, R.; Doherty, C. M.; Styles, M. J.; Bell, S.; Kirby,
N.; Mudie, S.; Haylock, D.; Hill, A. J.; Doonan, C. J.; Falcaro, P.
Biomimetic Mineralization of Metal-Organic Frameworks as
Protective Coatings for Biomacromolecules. Nat. Commun. 2015, 6,
7240-7247.
(36) Shieh, F.-K.; Wang, S.-C.; Yen, C.-I.; Wu, C.-C.; Dutta, S.; Chou,
L.-Y.; Morabito, J. V.; Hu, P.; Hsu, M.-H.; Wu, K. C. W.; Tsung, C.-K.
Imparting Functionality to Biocatalysts via Embedding Enzymes
into Nanoporous Materials by A De Novo Approach: Size-Selective
Sheltering of Catalase in Metal-Organic Framework Microcrystals.
J. Am. Chem. Soc. 2015, 137 (13), 4276-4279.
(37) Feng, Y.; Wang, H.; Zhang, S.; Zhao, Y.; Gao, J.; Zheng, Y.;
Zhao, P.; Zhang, Z.; Zaworotko, M. J.; Cheng, P.; Ma, S.; Chen, Y.
Antibodies@MOFs: An In Vitro Protective Coating for Preparation
and Storage of Biopharmaceuticals. Adv. Mater. 2019, 31 (2),
1805148-1805154.
(38) Wu, X.; Ge, J.; Yang, C.; Hou, M.; Liu, Z. Facile Synthesis of
Multiple Enzyme-Containing Metal-Organic Frameworks in A
Biomolecule-Friendly Environment. Chem. Commun. 2015, 51 (69),
13408-13411.
(39) Uribe-Romo, F. J.; Doonan, C. J.; Furukawa, H.; Oisaki, K.;
Yaghi, O. M. Crystalline Covalent Organic Frameworks with
Hydrazone Linkages. J. Am. Chem. Soc. 2011, 133 (30), 11478-
11481.
(40) Ding, S.-Y.; Cui, X.-H.; Feng, J.; Lu, G.; Wang, W. Facile
Synthesis of -C=N-Linked Covalent Organic Frameworks under
Ambient Conditions. Chem. Commun. 2017, 53 (87), 11956-11959.
(41) Matsumoto, M.; Valentino, L.; Stiehl, G. M.; Balch, H. B.;
Corcos, A. R.; Wang, F.; Ralph, D. C.; Marinas, B. J.; Dichtel, W. R.
Lewis-Acid-Catalyzed Interfacial Polymerization of Covalent
Organic Framework Films. Chem 2018, 4 (2), 308-317.
(42) Galli, S.; Masciocchi, N.; Tagliabue, G.; Sironi, A.; Navarro, J.
A. R.; Salas, J. M.; Mendez-Linan, L.; Domingo, M.; Perez-Mendoza,
M.; Barea, E. Polymorphic Coordination Networks Responsive to
CO2, Moisture, and Thermal Stimuli: Porous Cobalt(II) and Zinc(II)
Fluoropyrimidinolates. Chem-Eur. J. 2008, 14 (32), 9890-9901.
(43) Tehrani, H. S.; Moosavi-Movahedi, A. A. Catalase and Its
Mysteries. Prog. Biophys. Mol. Bio. 2018, 140, 5-12.
(44) Shi, J.; Wu, Y.; Zhang, S.; Tian, Y.; Yang, D.; Jiang, Z.
Bioinspired Construction of Multi-Enzyme Catalytic Systems.
Chem. Soc. Rev. 2018, 47 (12), 4295-4313.
(45) Graefe, D.; Gaitzsch, J.; Appelhans, D.; Voit, B. Cross-Linked
Polymersomes As Nanoreactors for Controlled and Stabilized
Single and Cascade Enzymatic Reactions. Nanoscale 2014, 6 (18),
10752-10761.
(46) Chang, F. P.; Chen, Y. P.; Mou, C. Y. Intracellular Implantation
of Enzymes in Hollow Silica Nanospheres for Protein Therapy:
Cascade System of Superoxide Dismutase and Catalase. Small
2014, 10 (22), 4785-4795.
(47) Ma, Y.; Zhao, Y.; Bejjanki, N. K.; Tang, X.; Jiang, W.; Dou, J.;
Khan, M. I.; Wang, Q.; Xia, J.; Liu, H.; You, Y.-Z.; Zhang, G.; Wang, Y.;
Wang, J. Nanoclustered Cascaded Enzymes for Targeted Tumor
Starvation and Deoxygenation-Activated Chemotherapy without
Systemic Toxicity. ACS Nano 2019, 13 (8), 8890-8902.
(48) Yong, Y.; Su, R.; Liu, X.; Xu, W.; Zhang, Y.; Wang, R.; Ouyang,
P.; Wu, J.; Ge, J.; Liu, Z. Lectin Corona Enhances Enzymatic Catalysis
on The Surface of Magnetic Nanoparticles. Biochem. Eng. J. 2018,
129, 26-32.
(18) Godoy-Gallardo, M.; York-Duran, M. J.; Hosta-Rigau, L.
Recent Progress in Micro/Nanoreactors toward the Creation of
Artificial Organelles. Adv. Healthcare Mater. 2018, 7 (5), 1700917-
1700951.
(19) Lu, A. X.; Oh, H.; Terrell, J. L.; Bentley, W. E.; Raghavan, S. R.
A New Design for An Artificial Cell: Polymer Microcapsules with
Addressable Inner Compartments That Can Harbor Biomolecules,
Colloids or Microbial Species. Chem. Sci. 2017, 8 (10), 6893-6903.
(20) Einfalt, T.; Witzigmann, D.; Edlinger, C.; Sieber, S.; Goers, R.;
Najer, A.; Spulber, M.; Onaca-Fischer, O.; Huwyler, J.; Palivan, C. G.
Biomimetic Artificial Organelles with In Vitro and In Vivo Activity
Triggered by Reduction in Microenvironment. Nat. Commun. 2018,
9, 1127-1138.
(21) Borodina, T.; Markvicheva, E.; Kunizhev, S.; Moehwald, H.;
Sukhorukov, G. B.; Kreft, O. Controlled Release of DNA from Self-
Degrading Microcapsules. Macromol. Rapid Comm. 2007, 28, 1894-
1899.
(22) Huo, J.; Aguilera-Sigalat, J.; El-Hankari, S.; Bradshaw, D.
Magnetic MOF Microreactors for Recyclable Size Selective
Biocatalysis. Chem. Sci. 2015, 6, 1938-1943.
(23) Zyzin, M. V.; Ramos-Cabrer, P.; Carregal-Romero, S.
Encapsulation of Enzymes in Porous Capsules via Particle
Templating. Methods Mol. Biol. 2020, 2100, 227-241.
(24) Renggli, K.; Baumann, P.; Langowska, K.; Onaca, O.; Bruns,
N.; Meier, W. Selective and Responsive Nanoreactors. Adv. Funct.
Mater. 2011, 21 (7), 1241-1259.
(25) Xu, Z.; Xiao, G.; Li, H.; Shen, Y.; Zhang, J.; Pan, T.; Chen, X.;
Zheng, B.; Wu, J.; Li, S.; Zhang, W.; Huang, W.; Huo, F.
Compartmentalization Within Self-Assembled Metal-Organic
Framework Nanoparticles for Tandem Reactions. Adv. Funct.
Mater. 2018, 28 (34), 1802479 -1802488.
(26) Shi, J.; Jiang, Y.; Wang, X.; Wu, H.; Yang, D.; Pan, F.; Su, Y.;
Jiang, Z. Design and Synthesis of Organic-Inorganic Hybrid
Capsules for Biotechnological Applications. Chem. Soc. Rev. 2014,
43 (15), 5192-5210.
(27) Schoonen, L.; van Hest, J. C. M. Compartmentalization
Approaches in Soft Matter Science: From Nanoreactor
Development to Organelle Mimics. Adv. Mater. 2016, 28 (6), 1109-
1128.
(28) Sun, Q.; Tang, Y.; Aguila, B.; Wang, S.; Xiao, F.-S.; Thallapally,
P. K.; Al-Enizi, A. M.; Nafady, A.; Ma, S. Enhancement Reaction
Environment Modification in Covalent Organic Frameworks for
Catalytic Performance. Angew. Chem. Int. Ed. 2019, 58 (26), 8670-
8675.
(29) Sun, Q.; Fu, C.-W.; Aguila, B.; Perman, J.; Wang, S.; Huang, H.-
Y.; Xiao, F.-S.; Ma, S. Pore Environment Control and Enhanced
Performance of Enzymes Infiltrated in Covalent Organic
Frameworks. J. Am. Chem. Soc. 2018, 140 (3), 984-992.
(30) Sun, Q.; Aguila, B.; Lan, P. C.; Ma, S. Tuning Pore
Heterogeneity in Covalent Organic Frameworks for Enhanced
Enzyme Accessibility and Resistance against Denaturants. Adv.
Mater. 2019, 31 (19), 1900008- 1900014.
(31) Zhang, S.; Zheng, Y.; An, H.; Aguila, B.; Yang, C.-X.; Dong, Y.;
Xie, W.; Cheng, P.; Zhang, Z.; Chen, Y.; Ma, S. Covalent Organic
Frameworks with Chirality Enriched by Biomolecules for Efficient
Chiral Separation. Angew. Chem. Int. Ed. 2018, 57 (51), 16754-
16759.
(32) Ma, T.; Kapustin, E. A.; Yin, S. X.; Liang, L.; Zhou, Z.; Niu, J.; Li,
L.-H.; Wang, Y.; Su, J.; Li, J.; Wang, X.; Wang, W. D.; Wang, W.; Sun, J.;
Yaghi, O. M. Single-Crystal X-Ray Diffraction Structures of Covalent
Organic Frameworks. Science 2018, 361 (6397), 48-52
(33) Kandambeth, S.; Venkatesh, V.; Shinde, D. B.; Kumari, S.;
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