Article

Cite This: J. Agric. Food Chem. 2019, 67, 3372−3379 pubs.acs.org/JAFC

Carbonic Anhydrase@ZIF‑8 Hydrogel Composite Membrane with

Improved Recycling and Stability for Efficient CO2 Capture
Sizhu Ren, Conghai Li, Zhilei Tan, Ying Hou, Shiru Jia,* and Jiandong Cui*
Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin Engineering Research Center of Microbial
Metabolism and Fermentation Process Control, Tianjin University of Science and Technology, 29 13th Avenue, Tianjin Economic
and Technological Development Area (TEDA), Tianjin 300457, People’s Republic of China
*
S Supporting Information
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ABSTRACT: In this study, carbonic anhydrase (CA, EC 4.2.1.1) molecules were embedded into metal−organic frameworks
(MOFs) via co-precipitation (CA@ZIF-8), and then these CA@ZIF-8 nanocomposites were encapsulated in the poly(vinyl
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alcohol) (PVA)−chitosan (CS) hydrogel networks to prepare CA@ZIF-8−PVA−CS composite hydrogels (PVA/CS/CA@
ZIF-8) with high activity, stability, and reusability. The immobilization efficiency of CA was greater than 70%, suggesting the
high immobilization efficiency. The prepared PVA/CS/CA@ZIF-8 composite membranes displayed excellent higher stability
against a high temperature, denaturants, and acid than free CA and CA@ZIF-8. Furthermore, these membranes exhibited an
excellent performance for CO2 capture. The amount of calcium carbonate obtained by PVA/CS/CA@ZIF-8 hydrogel
membranes was 20- and 1.63-fold than free CA and CA@ZIF-8 composites, respectively. Furthermore, the hydrogel
membranes exhibited superior reusability and mechanical strength. The hydrogel membrane maitained 50% of its original
activity after 11 cycles. However, CA@ZIF-8 completely lost activity. These results indicated that the PVA/CS/CA@ZIF-8
membranes can be efficiently applied to capture CO2 sequestration.
KEYWORDS: ZIF-8, carbonic anhydrase, hydrogel membrane, CO2 sequestration, immobilization enzyme

■ INTRODUCTION
Global climate change and CO2 pollution have caused climate
change, which threatens human existence.1,2 In recent years,
CO2 capture has been proposed to solve environmental
problems of the excess emissions in the carbon cycle.3
Generally, capturing CO2 methods include the chemical and
physical absorption processes and enzymatic reactions.
However, chemical and physical methods require large
quantities of energy and are time-consuming.4,5 In contrast,
enzymatic reactions are considered as a promising approach as
a result of them being environmentally friendly and low-cost.6
Carbonic anhydrase (CA, EC 4.2.1.1) is a zinc metalloenzyme
that has been accepted widely for its catalysis of reversible
conversion of CO2 to bicarbonate.7,8 However, its application
is difficult for CO2 capture in industry as a result of its poor
reusability and sensitivity in the harsh conditions.9 Therefore,
reasonable CA immobilization might meet the needs of
enzymatic CO2 conversion. Over the past few decades,
immobilization technology has provided a highly desirable
method to improve catalytic properties of enzymes, including
stability, activity, and tolerance to denaturants.10 Generally, the
immobilization is able to prevent subunit dissociation,
aggregation, and autolysis or proteolysis.11,12 Meanwhile,
multipoint covalent immobilization can also increase rigid-
ification of the enzyme.13 Furthermore, a series of carriers have
been designed and used to immobilize the enzyme, including
magnetic nanoparticles, sol−gel, polymer,14−16 etc. In addition,
as a carrier-free immobilized enzyme method, cross-linked
enzyme aggregate (CLEA) technology was regarded as an
efficient and simple technology.17,18 However, CLEAs are still
considered too soft for industrial application. To improve
mechanical properties of CLEAs, CLEAs were encapsulted into
various materials, including a polymeric matrix based on
poly(vinyl alcohol) (PVA, LentiKats),19 mesoporous silica,20
microporous polymeric membrane,21 and hydrogel composite
beads.22
In recent years, CA was immobilized successfully on/into
various support materials,23 including nanofibers,24 nano-
particles,25,26 biopolymer,27 membrane,28 microspheres,29
beads,30 and hollow nanofiber.31 However, these support
materials for CA have poor homogeneity and amorphous
dimensions. Meanwhile, the immobilization process is time-
consuming, thus affecting enzyme activity. Recently, hybrid
nanoparticle materials are attractive due as a result of their
multifunctional properties.32,33 Among hybrid nanoparticle
materials, metal−organic frameworks (MOFs) have caused
substantial research interests as a result of their controllable
pore distribution and large surface area, thermal stability, and
chemical functionality, serving as a promising immobilization
support material for enzyme immobilization.34,35 Especially,
zeolitic imidazolate framework 8 (ZIF-8) has been widely used
as an immobilization support.36,37 However, ZIF-8 is easy to
damage as a result of its low mechanical strength. To solve this
problem, catalase/ZIF-8 composites were embedded into
mesoporous silica. The micrometer-sized biocatalyst can be
easily recovered by centrifugation, indicaing superior mechan-
ical properties.16 Recently, CA molecules have been success-
Received: November 8, 2018
Revised: January 6, 2019
Accepted: February 26, 2019
Published: February 26, 2019

© 2019 American Chemical Society 3372 DOI: 10.1021/acs.jafc.8b06182

J. Agric. Food Chem. 2019, 67, 3372−3379
Journal of Agricultural and Food Chemistry
Figure 1. Schematic illustration of the preparation process for the PVA/CS/CA@ZIF-8 composite membrane.
fully immobilized into ZIF-8 (CA@ZIF-8 composites).38,39
The biocatalyst displayed superior CO2 capture capacity
compared to free CA. However, it is difficult to recover as a
result of their nanometer size and easy to damage as a result of
low mechanical strength during the stirring process.
Furthermore, ZIF-8 can be easily destroyed and easily
denatured under acidic conditions, which limits its practical
applications. Meanwhile, although it has been demonstrated
that ZIF-8 has an adsorption capacity for CO2,40,41 the capture
ability of ZIF-8 for CO 2 still needs to improve for
industrialized application. Recent years, biopolymers have
been regarded as an alternative membrane material for enzyme
immobilization.42 Among various biopolymers, chitosan (CS)/
PVA hydrogels have been given much attention as a result of
biocompatibility, biodegradability, and antimicrobial activity of
CS and PVA.43,44 Moreover, PVA/CS hydrogels exhibit
excellent mechanical strength.45,46 Therefore, PVA/CS hydro-
gels were used as immobilization supports. For instance, lipase
hybrid nanoflowers were encapsulated in PVA/CS hydrogel
networks and the composite hydrogels exhibited good
mechanical properties and could be separated from the
reaction fluid easily.47
In this study, CA molecules were encapsulated into ZIF-8
via co-precipitation, and then these CA@ZIF-8 nanocompo-
sites were encapsulated in the PVA/CS hydrogel networks to
prepare a novel biocatalyst (PVA/CS/CA@ZIF-8). The newly
designed PVA/CS/CA@ZIF-8 showed excellent recyclability
and stability characteristics against denaturants and heat.
Moreover, the stability of PVA/CS/CA@ZIF-8 was found to
be significantly improved in low pH solution, which might
greatly increase acid tolerance of CA@ZIF-8 composites, with
even the reaction medium becoming acidic as a result of the
hydration of CO2. Importantly, the biocatalytic composite
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membranes showed excellent CO2 capture capacity compared
to conventional CA@ZIF-8 and free CA.
■ MATERIALS AND METHODS
Chemicals. CA (from bovine) was obtained from Sigma Aldrich
Co., Ltd. (Shanghai, China). PVA was purchased from Macklin. CS
(molecular weight of about 635 000 with 85% deacetylation) was
purchased from Beijing Solarbio Science and Technology Co., Ltd.
Zn(NO3)2·6H2O (AR, 99%) and fluorescein isothiocyanate (FITC)
were obtained from Aladdin Industrial Corporation.
Preparation of CA@ZIF-8 and PVA/CS/CA@ZIF-8. CA@ZIF-8
was prepared according to a previously reported method.46 Briefly, 1
mL of Zn(NO3)2·6H2O solution [containing CA (10 mg/mL)] and
10 mL of 2-methylimidazole (1 M) were mixed under magnetic
stirring for 30 min. Then, the resultant precipitates were obtained by
centrifugation, washed with 0.2 M Na2HPO4−NaH2PO4 buffer
solution 4 times, and redispersed in the same buffer. For PVA/CS/
CA@ZIF-8, the CA@ZIF-8 composites (10 mg), PVA (10 wt %), and
CS (5 wt %) were mixed. Subsequently, the product was repeatedly
frozen and thawed 3 times to obtain PVA/CS/CA@ZIF-8. In
addition, immobilization conditions, including the amount of CA,
immobilization time, temperature, and ratios (w/w) of PVA/CS were
optimized. The Bradford method was used to determine the CA
amounts in solution. The immobilization efficiency was calculated
according to eq 1
(m − C1V1)
immobilization efficiency (%) = × 100%
m (1)
where m is the initial mass of CA in the solution (mg), C1 (mg/mL) is
the CA concentration, and V1 (mL) is the volume of the supernate.
Characterization Techniques. CA@ZIF-8 and PVA/CS/CA@
ZIF-8 were characterized by scanning electron microscopy (SEM), X-
ray diffraction (XRD), confocal laser scanning microscopy (CLSM),
and Fourier transform infrared spectroscopy (FTIR). SEM was taken
on a HITACHI SU-1510. TEM was taken by JEOL JEM2100.
Powder X-ray diffraction (PXRD) was collected by a diffractometer
(XRD-6100, Shimadzu, Japan). The FTIR spectra was obtained using
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Figure 2. SEM images of (a) pure ZIF-8, (b) CA@ZIF-8, (c) blank PVA/CS hydrogel membrane without CA@ZIF-8, and (d) PVA/CS/CA@
ZIF-8 composite membrane.
a Bruker TENSOR 27 FTIR spectrometer made by the German
Bruker Corporation. The CA molecules were labeled by FITC and
characterized by CLSM (Leica Camera AG, Germany). For the
mechanical properties of PVA/CS/CA@ZIF-8, a compression test
was taken using a certain size column (25 × 4 mm) at 25 mm/min
rate. Meanwhile, the tensile test was determined at 10 mm/min.
Activity Assay. CA activity was measured by the hydrolysis of 4-
nitrophenyl acetate (p-NPA) to p-nitrophenol (p-NP) according to a
previous report.48 In detail, the CA sample was mixed with 0.2 M of
Na2HPO4−NaH2PO4 buffer solution (4.6 mL, pH 7.0) and 0.2 mM
p-NPA (0.2 mL) and incubated at 25 °C for 3 min. The p-NP
concentration in the reaction solutions was determined at 400 nm. A
total of 1 CA unit is defined as the hydrolysis of p-NPA per minute.
Stability Measurements. Thermal stability of all CA samples
were determined by incubating them at 65 °C for 30 min. The
samples were collected and assayed for residual activity. For acid
tolerance, all of CA samples were incubated in 10 mM phosphate
buffer solution (pH 4) for 30 min and the activity was measured. The
stability of CA samples against denaturants [30 mM sodium dodecyl
sulfate (SDS), 80 mM sulfourea, 30 mM ethylenediaminetetraacetic
acid (EDTA), and 5 M urea] were also checked by measuring the
residual activity after treatment for 30 min. The reusability of the
immobilized CA was measured by sequential cycles for the hydrolysis
of p-NPA. Immobilized CA was taken out and washed by Milli-Q
water 3 times and then added to the fresh reaction solutions.
CO2 Sequestration. For free CA and CA@ZIF-8, the capture of
CO2 in the form of CaCO3 was carried out according to our previous
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reports.39 For the PVA/CS/CA@ZIF-8 composite membrane, 50 mg
of PVA/CS/CA@ZIF-8 composite membrane with the same protein
content as the free CA and CA@ZIF-8 was added to glycine−sodium
hydroxide buffer solution (200 mM, pH 10.5). Subsequently, CO2 gas
was charged into the above solution at a constant rate for 30 min.
Then, PVA/CS/CA@ZIF-8 composite membrane was taken out with
a tweezer, washed with buffer solution, and then repeated with a fresh
reaction solution. The reaction solutions were gathered from all 20
cycles and mixed with 10 mL of CaCl2 (pH 10, 100 mM) solution for
2 min. The resultant precipitates (the size of formed CaCO3 is >1
μm) were recovered and dried.
■
RESULTS AND DISCUSSION
Preparation and Characterization of the CA@ZIF-8
Composites and PVA/CS/CA@ZIF-8 Composite Mem-
brane. CA@ZIF-8 was synthesized and encapsulated by
PVA−CS hydrogels, and the synthetic route is shown in Figure
1. The morphology of pure ZIF-8, CA@ZIF-8, and PVA/CS/
CA@ZIF-8 composite membrane was observed. The results
were shown in Figure 2. Both pure ZIF-8 and CA@ZIF-8
composites exhibited a standard polyhedron shape with good
monodispersity (panels a and b of Figure 2), suggesting that
the presence of CA did not cause changes of the crystal
structure of ZIF-8.49,50 The obtained PVA/CS hydrogel
membrane displayed an interlaced microporous structure
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(Figure 2c). This interlaced porous structure acts as a key role
for improving mass transfer of the substrate and products.
Meanwhile, the CA@ZIF-8 composites maintained a intact
polyhedron structure in the interlaced microporous structure
(Figure 2d). Furthermore, CLSM images showed that CA
molecules were embedded successfully into ZIF-8 (Figure 3a).
Figure 3. CLSM of (a) CA@ZIF-8 and (b) PVA/CS/CA@ZIF-8
composite membrane.
In addition, abundant green fluorescence was observed in the
PVA/CS/CA@ZIF0−8 hydrogel membrane, indicating the
presence of CA@ZIF-8 in the hydrogel membrane (Figure
3b). The XRD patterns illustrated that the crystalline structure
of ZIF-8 was well kept after CA immobilization (Figure 4a).
Meanwhile, the CA@ZIF-8 composites held high crystallinity
after being immobilized in the hydrogel membrane. This result
is consistent with the SEM results. The FTIR spectra of ZIF-8,
CA@ZIF-8, PVA/CS, and PVA/CS/CA@ZIF-8 hydrogel
membrane were shown in Figure 4b. The characteristic
peaks at 1310 and 1450 cm−1 corresponded to C−H and
CC stretching of the benzene group of the imidazole ring in
the PVA/CS/CA@ZIF-8 hydrogel membrane, suggesting the
presence of ZIF-8 in the hydrogel membrane.51,52 The
characteristic peak at 1650 cm−1 was associated with amide I
of the CA protein, indicating the presence of CA in the
composite.53 The characteristic peaks at 1286 and 3450 cm−1
could be observed, attributed to the vibrations of the −OH
stretching vibration peak of CS and PVA, respectively.54,55 The
above results showed that CA@ZIF-8 was embedded into the
hydrogel membrane. In the process of the immobilized enzyme
reaction, it is inevitable to bear various external shear forces;
thus, immobilized enzymes must have excellent mechanical
strength and toughness.56,57 An excellent hydrogel membrane
system should cushion the external pressure, thus protecting
the integrity of CA@ZIF-8 composites. The mechanical
properties of the PVA/CS/CA@ZIF-8 hydrogel membrane
were determined by tension and compression tests. The results
illustrated that the PVA/CS/CA@ZIF-8 hydrogel membrane
possessed very good tenacity and anti-deformation ability. The
tensile strength and tensile yield stress were 8.44 ± 0.28 and
1.72 ± 0.04 MPa, respectively (data not shown). This excellent
mechanical property might be due to the introduction of the
non-covalent interactions in cross-linked PVA and CS. Besides,
the formation of hydrogen between CS with amino groups and
PVA with hydroxyl resulted in the increase of the viscosity and
toughness of the PVA/CS/CA@ZIF-8 hydrogel membrane.58
In short, the excellent mechanical properties of the PVA/CS/
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Figure 4. (a) XRD pattern and (b) FTIR spectra of CA/ZIF-8 and
PVA/CS/CA@ZIF-8 composite membrane and (c) XRD pattern of
calcium carbonate crystals obtained in the presence of free CA, CA@
ZIF-8, and PVA/CS/CA@ZIF-8 composite membrane.
CA@ZIF-8 hydrogel membrane are very important for long-
lasting stability of immobilized enzymes in practical
application.
Stability Measurement. The thermostability of free CA,
CA@ZIF-8, and PVA/CS/CA@ZIF-8 hydrogel membrane
was determined via using the residual enzyme activity after
incubating at 65 °C for 30 min. The results were shown in
Figure 5a. Free CA lost most of its activity. However, CA@
ZIF-8 composites and PVA/CS/CA@ZIF-8 hydrogel mem-
brane retained 79 and 98% of their intital activities,
respectively. This difference in thermal satbility may be due
to restriction of the movement of enzyme molecules and
rigidity of the enzyme conformation after immobilization.59 It
is well-known that the structure of ZIF-8 crystals was easily
denatured under acidic conditions, thus limiting the applied
perspective range of hybrid composites under acidic
conditions.60 Therefore, to assess whether the hydrogel
membrane could improve acid resistance of CA@ZIF-8, we
examined the catalytic activity and pH tolerance of free CA
and immobilized CA in an acidic environment (pH 4.0). The
results were shown in Figure 5b. Although the activity of CA
samples decreased under an acidic environment, the PVA/CS/
CA@ZIF-8 hydrogel membrane exhibited a more stable
performance under the acidic conditions than free CA and
CA@ZIF-8. Free CA and CA@ZIF-8 composites maintained
37 and 44% of their initial activities, respectively. However, the
PVA/CS/CA@ZIF-8 hydrogel membrane maintained 72% of
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Figure 5. Stability of free CA, CA@ZIF-8, and PVA/CS/CA@ZIF-8 composite membrane: (a) thermostability, (b) acid resistance, (c) stability
against chemical denaturants, and (d) reusability.

its orginal activity. This result could also be due to the carbonates.39 To determine whether the PVA/CS/CA@ZIF-
protection of the hydrogel membrane, which can provide an 8 hydrogel membrane could promote the transfer of CO2, we
appropriate microenvironment for the enzyme and prevent CA carried out the formation of CaCO3 from CO2 using CA@ZIF-
from structural changes under an acidic environment.61 The 8 and PVA/CS/CA@ZIF-8 hydrogel membrane. As expected,
tolerance of CA samples against different denaturants was in comparison to free CA, the amount of calcium carbonate
shown in Figure 5c. Obviously, the presence of a hydrogel
obtained using CA@ZIF-8 and PVA/CS/CA@ZIF-8 hydrogel
membrane significantly increased tolerance of CA against
denaturants. For instance, the hydrogel membrane maintained membrane was 20- and 32.6-fold (Figure 6), respectively. This
67% of its original activity in 80 mM sulfourea solution for 30 results suggesting that the PVA/CS/CA@ZIF-8 hydrogel
min. However, free CA and CA@ZIF-8 kept 19 and 42% of membrane had better CO2 capture capacity than free CA
their initial activities. These results indicated that the presence
of the hydrogel membrane significantly increased the tolerance
of CA to extreme environments. In addition, the reusability of
immobilized CA was investigated as a result of its important
role in reducing costs.62 As shown in Figure 5d, the hydrogel
membrane maintained 50% of its original activity after 11
cycles. However, CA@ZIF-8 lost activity. This result indicated
that the presence of the hydrogel membrane accelerated
recovery of CA@ZIF-8, improving reusability. Furthermore,
after the repeated catalytic reaction, the PVA/CS/CA@ZIF-8
hydrogel membrane could be taken out directly from the
reaction medium. However, the CA@ZIF-8 composites must
be recovered from the reaction medium via centrifugation or
filtration. Therefore, the PVA/CS/CA@ZIF-8 hydrogel
membrane is more suitable for industrial application than
CA@ZIF-8 composites.
Formation of CaCO3 from CO2. For CO2 sequestration
and utilization, it is well-known that CA@ZIF-8 was used to
accelerate the catalyzation of carbon dioxide to mineral Figure 6. Comparison of conversion of carbon dioxide to CaCO3.

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and CA@ZIF-8. The decreased amount of calcium carbonate
could be due to denaturation of enzymes and the leaching
under CO2 bubbling (the reaction solution becomes acidic
with CO2 bubbling). However, the PVA/CS/CA@ZIF-8
hydrogel membrane could maintain activity under recycled
uses as a result of the prevention of both enzyme leaching and
denaturation. In addition, SEM images showed that most
CaCO3 by free CA and CA@ZIF-8 exhibited vaterite particles
(panels a and b of Figure 7). However, CaCO3 by the hydrogel
Figure 7. SEM images of calcium carbonate crystals obtained in the
presence of (a) free CA, (b) CA@ZIF-8, and (c) PVA/CS/CA@ZIF-
8 composite membrane.
membrane formed calcite particles (Figure 7c). This
phenomenon can be due to the fact that vaterite is a
metastable state, which is easily turned into calcite in water.63
Furthermore, PXRD patterns further demonstrated this result.
CaCO3 by free CA and CA@ZIF-8 had consistent crystal
structures of vaterite, whereas the crystal structures of obtained
CaCO3 by the PVA/CS/CA@ZIF-8 hydrogel membrane were
calcite crystals (Figure 4c).
In summary, we immobilized CA@ZIF-8 composites into
the PVA−CS hydrogels to gain the PVA/CS/CA@ZIF-8
hydrogel membrane with improved CO2 capture capacity.
These PVA/CS/CA@ZIF-8 hydrogel membranes exhibited
higher CO2 transfer performance than CA@ZIF-8 composites.
Furhtermore, after the catalysis cycles, the PVA/CS/CA@ZIF-
8 hydrogel membranes can be recovered from the reaction
fluid easily without high-speed centrifugation or filtration,
which can simplify the separation process and decrease the
production cost for industrial application. Meanwhile, the
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PVA/CS/CA@ZIF-8 hydrogel membranes exhibited increased
thermostability, acid resistance, tolerance toward denaturants,
and good mechanical properties. These results showed that the
PVA/CS/CA@ZIF-8 hydrogel membranes can efficiently
capture CO2.
■
*
ASSOCIATED CONTENT
S Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jafc.8b06182.
Mechanical properties (Table 1) (PDF)
■ AUTHOR INFORMATION
Corresponding Authors
*Telephone: +86-022-60601598. E-mail: jiashiru@tust.edu.cn.
*Telephone: +86-022-60601598. E-mail: cjd007cn@163.com.
ORCID
Jiandong Cui: 0000-0002-2809-530X
Funding
This work was supported by the National Natural Science
Foundation of China under Grant 21676069. Jiandong Cui
also thanks support from the Foundation of Tianjin Engineer-
ing Research Center of Microbial Metabolism and Fermenta-
tion Process Control (17PTGCCX00190).
Notes
The authors declare no competing financial interest.
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3379 DOI: 10.1021/acs.jafc.8b06182

J. Agric. Food Chem. 2019, 67, 3372−3379