FULL PAPER

DOI: 10.1002/chem.201201112

Carbonic Anhydrase Immobilized on Encapsulated Magnetic Nanoparticles

for CO2 Sequestration

Mari Vinoba,[a] Margandan Bhagiyalakshmi,[b] Soon Kwan Jeong,*[a]

Sung Chan Nam,[a] and Yeoil Yoon[a]

Abstract: Bovine carbonic anhydrase
(BCA) was covalently immobilized
onto OAPS (octa(aminophenyl)silses-
quioxane)-functionalized Fe3O4/SiO2
nanoparticles by using glutaraldehyde
as a spacer. The Fe3O4 nanoparticles
were coated with SiO2, onto which was
grafted OAPS, and the product was
characterized using SEM, TEM, XRD,
IR, X-ray photoelectron spectroscopy
(XPS), and magnetometer analysis.
The enzymatic activities of the free and
Fe3O4/SiO2/OAPS-conjugated BCA
(Fe CA) were investigated by hydro-
lyzing p-nitrophenylacetate (p-NPA),
and hydration and sequestration of
CO2 to CaCO3. The CO2 conversion ef-
ficiency and reusability of the Fe CA
were studied before and after washing
the recovered Fe CA by applying a
magnetic field and quantifying the un-
reacted Ca2 + ions by using ion chroma-
tography. After 30 cycles, the Fe CA
Keywords: carbon dioxide · green
chemistry · magnetic properties ·
nanoparticles · sequestration
displayed strong activity, and the CO2
capture efficiency was 26-fold higher
than that of the free enzyme. Storage
stability studies suggested that Fe CA
retained nearly 82 % of its activity
after 30 days. Nucleation of the precipi-
tated CaCO3 was monitored by using
polarized light microscopy, which re-
vealed the formation of two phases,
calcite and valerite, at pH 10 upon ad-
dition of serine. The magnetic nanobio-
catalyst was shown to be an excellent
reusable catalyst for the sequestration
of CO2.

Introduction cules to bicarbonate per second.[4] The use of free enzymes

Anthropogenic carbon dioxide (CO2) is a major greenhouse
gas that acts as a blanket to absorb thermal radiation emit-
ted by the earths surface, thereby resulting in global warm-
ing and climate change. The atmospheric CO2 concentration
is predicted to rise by more than 450 ppm coupled with a
global temperature increase of more than 2 8C in 2100 due
to CO2 emissions from the ongoing industrial revolution.[1]
Many studies have introduced new technologies for CO2
capture; however, the quest for feasible capture technolo-
gies continues.[2] To this end, biology-inspired methods pres-
ent innovative model systems for trapping CO2 from emis-
sion sources. Recently, carbonic anhydrase (CA) was used
to convert CO2 to bicarbonate, which was then converted to
CaCO3 in the presence of Ca2 + ions.[3] CA is the fastest
enzyme to catalyze the conversion of one million CO2 mole-
[a] Dr. M. Vinoba, Dr. S. K. Jeong, Dr. S. C. Nam, Dr. Y. Yoon
Climate Change Technology Research Division
Korea Institute of Energy Research
102 Gajeong-ro, Yuseong-gu
Daejeon 305-343 (Korea)
Fax: (+ 82) 42-860-3134
E-mail: jeongsk@kier.re.kr
[b] Dr. M. Bhagiyalakshmi
Department of Chemistry
School of Basic and Applied Sciences
Central University of Tamil Nadu
Thiruvarur 610-004 (India)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201201112.
as biocatalysts for large-scale industrial processes poses sig-
nificant drawbacks because the free enzymes are soluble
and cannot be easily recovered and reused. The catalytic
properties of the enzymes are greatly improved by immobi-
lization on nanomaterials.[5] Several techniques have been
proposed to improve the enzymatic activity of CA upon im-
mobilization.[6] Surface-modified magnetic nanoparticles
provide excellent templates for enzyme immobilization,
thereby enabling their easy recovery from a reaction
medium upon application of a static magnetic field.[7] Cur-
rent reports have demonstrated that SiO2 and Fe2O3/SiO2
nanoparticles are biocompatible, nontoxic, and chemically
stable toward enzyme immobilization.[8] CA immobilization
on SiO2 was achieved by amine functionalization.[9] The
present study describes the immobilization of bovine car-
bonic anhydrase (BCA) onto amine-grafted Fe3O4/SiO2.
Scattered amine groups provide a good distribution of im-
mobilized enzymes over the nanomaterial surface. Accord-
ingly, octa(aminophenyl)silsesquioxane (OAPS), an organic–
inorganic nanostructured hybrid derivative of polyhedral oli-
gomeric silsesquioxanes, was shown to be a good spacer for
enzyme immobilization because it possess eight amine
groups on each edge of a cubic silicon cage structure.[9]
This study demonstrates that the OAPS-functionalized
Fe3O4/SiO2 was synthesized here by using Fe2 + and Fe3 +
salts in combination with tetraethyl orthosilicate (TEOS),
followed by grafting with 3-chloropropyltrimethoxysilane
(CPTMS). BCA was immobilized through covalent bonds
between the amine groups of the enzyme and the OAPS-

Chem. Eur. J. 2012, 00, 0 – 0  2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim &1&
These are not the final page numbers! ÞÞ
functionalized Fe3O4/SiO2 nanoparticles by using a cross-
linking agent such as glutaraldehyde (GA). The activity of
the free or immobilized BCA toward hydrolysis of p-nitro-
phenylacetate (p-NPA), hydration and sequestration of CO2,
the material reusability, the CO2 conversion efficiency, and
the quantity of CaCO3 generated were evaluated by using
ion chromatography (IC) techniques. The formation of dif-
ferent CaCO3 phases depending on the addition of amino
acid was monitored by polarized light microscopy at a con-
stant pH.
Results and Discussion
Synthesis and morphological characterization of the nano-
materials: The magnetic nanoparticles were synthesized by
co-precipitation, followed by silica coating using a modified
Stçber method. Silica-coating Fe3O4 nanoparticles was chal-
lenging because of the low chemical affinity of silica toward
Fe3O4, which was overcome by using a modified Stçber
method. Isopropanol has a low dielectric constant (19.9),
and its use as a solvent resulted in the ready formation of
larger silica particles, in contrast to the results obtained
when using a high dielectric constant (24.3) solvent, etha-
nol.[10] In this work, the silica coating obtained using isopro-
panol as a solvent was thicker than that obtained using etha-
nol. The chloropropyl moieties were grafted onto the surfa-
ces of the Fe3O4/SiO2 nanoparticles by treating the Si OH
silica groups with CPTMS. OAPS was then treated with the
chloropropyl moieties of the Fe3O4/SiO2/CPTMS to yield
Fe3O4/SiO2/OAPS (Scheme 1).
Figure S1 in the Supporting Information shows the XRD
patterns of pure, silica-coated, and functionalized magnetic
nanoparticles. The characteristic peaks of the pure Fe3O4 at
2q = 30.1, 35.5, 43.1, 53.4, 56.97, and 62.618 were observed,
marked in Figure S1a of the Supporting Information by
their indices (220), (311), (400), (422), (511), and (440), re-
spectively. These peaks coincided with those in the JCPDS
database for magnetic materials (82-1533), thus indicating a
Scheme 1. Schematic illustrations of functionalization and carbonic anhydrase immobilization on encapsulated magnetic nanoparticles.
&2& www.chemeurj.org  2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!
crystalline cubic spinel structure for the Fe3O4 nanoparti-
cles.[11] Fe3O4/SiO2 displayed a broad peak at 2q = 238, which
indicated the presence of an amorphous silica structure. The
remaining XRD peaks corresponded to structural features
of the Fe3O4 particles (Figure S1b in the Supporting Infor-
mation). Even upon functionalization with CPTMS, fol-
lowed by OAPS, the phases of the Fe3O4 nanoparticles re-
mained unchanged (Figure S1 c, d in the Supporting Informa-
tion). The average Fe3O4 nanoparticle size was measured
using the Debye–Scherrer equation and was found to be
10 nm. The XRD results agreed well with the TEM meas-
urements. TEM images of Fe3O4 nanoparticles were quasi-
spherical in shape with an average diameter of 10–12 nm
(Figure 1). The Fe3O4/SiO2 nanoparticles were monodisperse
with well-defined core–shell structures, even though the
silica shells trapped multiple Fe3O4 particles. The Fe3O4/SiO2
nanoparticles used in this case for the production of func-
tionalized Fe3O4/SiO2/OAPS particles had an average diam-
eter of (115  10) nm (Figure 1b).
The surface morphologies of the Fe3O4 and Fe3O4/SiO2/
OAPS particles were deduced on the basis of the SEM
images. Fe3O4 nanoparticles were spherical with smooth sur-
faces, as shown in Figure 2a. The SEM images of Fe3O4/
SiO2/OAPS revealed well-defined spherical nanoparticles
(Figure 2b). The energy-dispersive X-ray diffraction spectro-
scopy (EDS) data of the Fe3O4 and Fe3O4/SiO2/OAPS nano-
particles confirmed the presence of metal ions (Figure S2 in
the Supporting Information). The presence of elemental
carbon, nitrogen, oxygen, silicon, and iron in the Fe3O4/
SiO2/OAPS materials indicated the successful functionaliza-
tion of OAPS over Fe3O4/SiO2, and their respective elemen-
tal contents were 14.96, 6.15, 34.28, 25.57, and 19.04 %. EDS
elemental mapping of the Fe3O4/SiO2/OAPS revealed that
C, Si, O, and N were homogeneously distributed over the
surface of each particle (Figure S3 in the Supporting Infor-
mation), thus illustrating that each particle was surface-func-
tionalized by the nitrogen-containing OAPS groups.
Figure S4 in the Supporting Information shows the FTIR
spectra of pristine and functionalized Fe3O4 particles. The
Chem. Eur. J. 0000, 00, 0 – 0
Carbonic Anhydrase
Figure 1. HRTEM images of a) Fe3O4 and b) Fe3O4/SiO2/OAPS.
Figure 2. FESEM of a) Fe3O4 and b) Fe3O4/SiO2/OAPS.
FTIR spectrum of pure Fe3O4 displayed a characteristic
peak at 580 cm 1 due to Fe O stretching.[12] The peaks at
1423 and 3405 cm 1 were attributed to C O, O H bond vi-
brations, which may have been on account of contact with
air and water (Figure S4a in the Supporting Information).
The silica coating was confirmed by the observation of a
new peak at 956 cm 1, which was assigned to the Si OH
bond, and the vibrational bands at 1086 and 806 cm 1, which
corresponded to the stretching and bending of the Si-O-Si
bonds, respectively (Figure S4b in the Supporting Informa-
tion). The disappearance of the peak at 956 cm 1 and the
emergence of new peaks at 1403 cm 1 (dC,H) and nas =
2925 cm 1 ( CH2), as shown in Figure S4c in the Supporting
Information, indicated CPTMS grafting onto the Fe3O4/SiO2
Chem. Eur. J. 2012, 00, 0 – 0  2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
These are not the final page numbers! ÞÞ
FULL PAPER
nanoparticles. OAPS grafting onto Fe3O4/SiO2/CPTMS was
verified by the presence of the characteristic peaks at 1124,
1598, 3049, and 3384 cm 1, on account of Si-O-Si, phenyl
C=C, phenyl C H, and NH2, respectively (Figure S4d in the
Supporting Information).[13]
Figure 3 shows the X-ray photoelectron spectroscopy
(XPS) patterns of Fe3O4 and Fe3O4/SiO2/OAPS. The charac-
teristic iron oxide peaks in the Fe3O4 nanoparticles, includ-
Figure 3. XPS spectrum of the Fe3O4 and Fe3O4/SiO2/OAPS particles.
Inset: XPS pattern of the Fe 2p, Si 2p, Si 2s, and N 1s binding energies.
ing O 1s, Fe 2p3/2, and Fe 2p1/2, appeared at 530, 712, and
725 eV, respectively, were observed in both the pristine and
functionalized Fe3O4.[14] The XP spectrum of Fe3O4/SiO2/
OAPS revealed two new peaks at 156 and 105 eV, which
corresponded to photoelectrons that originated from the Si
2s and Si 2p energy levels, respectively. The intensity of
characteristic Fe 2p3/2 and Fe 2p1/2 peaks in Fe3O4/SiO2/
OAPS decreases, thus indicating that all Fe3O4 nanoparticles
in the composite were confined within a SiO2 shell. The
grafted OAPS introduced N 1s binding energies at 402 eV.
The XPS and IR data reveal the confinement of Fe3O4
nanoparticles by the silica layer and the presence of amine
groups on the Fe3O4/SiO2/OAPS surfaces, respectively.
Magnetic characterization of the nanomaterials was ob-
served by a vibrating sample magnetometer. Figure 4 shows
that the saturation magnetization values of Fe3O4, Fe3O4/
SiO2, Fe3O4/SiO2/CPTMS, Fe3O4/SiO2/OAPS, and Fe CA
were 77.7, 56.2, 42.3, 29.5, and 24.6 emu g 1, respectively.
This descending order corresponds to silica-layer coating
followed by functionalization and BCA immobilization on
Fe3O4 nanoparticles. The magnetization values suggested
that Fe CA can be easily separated from reaction media in
the presence of an external magnetic field, which is demon-
strated in Scheme 1, and redispersion of the catalysts occur-
red rapidly with a slight shaking as the magnetic field was
removed. These results show the efficacy of magnetic sepa-
ration.
www.chemeurj.org &3&
 S. K. Jeong et al.

Figure 4. Magnetic hysteresis loop of a) Fe3O4, b) Fe3O4/SiO2, c) Fe3O4/

SiO2/CPTMS, d) Fe3O4/SiO2/OAPS, and e) Fe CA nanoparticles.

CO2 carbonization: CO2 transformation into HCO3 in-

volved the nucleophilic attack of Zn2 + OH (BCA) onto
the C of CO2, the internal proton transfer of Zn2 + -bound
HCO3 , the binding of water to Zn2 + , and the ionization of
Zn-bound water to facilitate the release of HCO3 .[4] Bicar-
bonate ions were converted into CaCO3 through nucleation
with Ca2 + in a basic medium. The present study was con-
ducted in a tris(hydroxymethyl)aminomethane (Tris)-HCl
buffer solution to achieve hydration (pH 8.0) and minerali-
zation (pH 10.0) of CO2. The BCA catalytic turnover rate
was high and, as reported previously, dramatic pH variations
and the large uncertainties in the conversion-rate measure-
ments,[6b] could be modeled using Michaelis–Menten kinetics
with respect to p-NPA hydrolysis.[15] Enzyme loading onto
the Fe3O4/SiO2/OAPS was 213 mg of BCA per gram of solid
materials. The catalytic efficiency (kcat/Km) of the soluble
and immobilized BCA for p-NPA hydrolysis was found to
be 874 and 783 m 1 s 1, respectively. Upon comparison with
free CA, the catalytic activity of Fe CA was found to be
89.5 %, which is higher than that of previously reported CA
encapsulated microcapsules (10 %)[16a] and nanogels
(70 %).[16b] The CO2 conversion efficiency was quantified by
using indirect approaches. BCA catalyzed the hydration of
CO2 to produce bicarbonate ions that subsequently clustered
with Ca2 + ions to from CaCO3. Unreacted calcium ions Figure 5. Optimization of a) control experiments, b) reusability of Fe
were measured by using the IC method applied to the fil- CA, and c) the effect of storage stability.

trate solution. As a control reaction, the CO2 solution was

treated with a buffer solution (pH 8.0) for 10 min, followed
by application of a negative pressure at various time inter-
vals. The above mixture was treated with Ca2 + ions (Fig-
ure 5a). After 10 min, no change in the Ca2 + ion consump-
tion was observed on account of the pH and negative pres-
sure (control reaction). The CO2 solubility was very low at
pH 8; hence, under negative pressure, the insoluble/unstable
CO2 may be removed. This could not be applicable in the
presence of BCA because the enzyme catalyzes the hydra-
tion of CO2 in a fraction of a second upon adding CO2 solu-
tion and converts it into carbonic acid. The gram equivalents
of CO2 present in CaCO3 were obtained after correcting on
the basis of the control reaction. The initial concentration of
CO2 is 30 mm, from which the relative carbonization was
calculated for 100 %. Hence, the relative carbonizations of
control, free BCA, and Fe CA reactions were found to be
24, 70, and 68 % and quantities of CaCO3 produced by the
end of a first cycle: 74, 209, and 202 mg, respectively. Thus,
from the above studies, it was concluded that both free
BCA and Fe CA show approximately 70 % of CO2 hydra-
tion and sequestration.
The Fe CA could be recovered at the end of each CO2
hydration cycle by applying an on/off magnet to separate
the catalyst and subsequently reused after washing with the
buffer solution. The hydrated CO2 was sequestered as

&4& www.chemeurj.org  2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Chem. Eur. J. 0000, 00, 0 – 0

ÝÝ These are not the final page numbers!

Carbonic Anhydrase
FULL PAPER
CaCO3 by Ca2 + ions. The quantity of unreacted Ca2 + ions
was quantified by IC, and the CO2 conversion efficiency rel-
ative to carbonization could be calculated. The CO2 conver-
sion efficiency remained constant over 12 cycles both with
and without washing of the catalyst. After 12 cycles, the
extent of CO2 carbonization and its conversion efficiency
decreased gradually in the presence of unwashed Fe CA.
After 30 cycles without washing, the recovered Fe CA lost
its activity by around 24 % of the initial activity, and the cor-
responding CO2 carbonization was 0.46 mmol. Deactivation
of the enzyme on account of lack of washing led to a de-
crease in Fe CA activity; the HCO3 species produced acts
Figure 6. Polarized-light microscopy images of CaCO3 nucleation.
as an inhibitor by binding with the active site of enzyme
(Zn2 + OH) by means of a Brønsted acid interaction,[17]
which could be solved by washing the Fe CA catalyst with to form a new more stable crystallographic mononuclear
buffer at the end of each cycle. Partial loss of the immobi- phase (solid-line circle in Figure 6). Two or more particles
lized enzyme might have occurred during the washing steps then combine to form a single polynuclear crystal (dotted-
as well, although this loss was relatively minor. The Fe CA line circle in Figure 6). A stable pure calcite phase formed,
was mostly captured by using the magnetic on/off system, and the crystallographic morphology of the particles did not
which prevented extensive loss of Fe CA during washing. subsequently change.[20] The surface morphologies of the cal-
The retained activity of the recovered and reused Fe CA cite crystals exhibited well-defined faceted rhombohedral
after washing with buffer at the end of the 30th cycle was shapes, as confirmed by polarized-light microscopy and
90 %, and 1.84 mmol CO2 was found to be carbonized. Thus field-emission scanning electron microscopy (FESEM; Fig-
Fe CA exhibits excellent reusability and was 26-fold higher ure 7a, b). The organic additives were involved in nucleation
than free BCA (Figure 5b). CA immobilized on chitosan
beads, on the other hand, loses its CO2 sequestration activity
by about 70 % after the seventh cycle.[18] In addition to the
enzyme reusability, the activities of the stored free or immo-
bilized enzymes were monitored for 30 days at 5-day inter-
vals by measuring the CO2 hydration and sequestration. The
residual activities of either the free BCA or Fe CA were 49
or 82 %, respectively, after 30 days (Figure 5c). Fe CA ex-
hibit a greater stability toward CO2 conversion than CA im-
mobilized on iron mesh[19a] and alginate bead[19b] materials,
which retained about 70 % of its activity after 30 days.

Preparation of different CaCO3 phases: Several reports

have focused on the crystallographic structure of CaCO3(s)
obtained during precipitation from a hydrated CO2 solution.
We attempted to induce formation of different CaCO3
phases, calcite or vaterite, by adding a serine at pH 10. The
mechanism reported for the nucleation and growth of each
CaCO3 phase was observed by using polarized-light miACHTUNGREcros-
ACHTUNGREcopy, and images are presented in Figure 6. Here, two
CaCO3 phases were obtained by the addition of serine,
which interacted with the mineral-forming ions in solution
to promote nucleation or to modify the crystal growth and
alter the growth rates at crystal faces at a given pH. Nuclea-
tion was favored only at a critical cluster size, which was di-
rectly proportional to the ratio of the energy required to
grow clusters and the energy released during formation of
bonds in the aggregates. Ca2 + and CO32 ions are densely
packed. The most energetically stable atomic planes in the
rectangular rhombohedral calcite lattice result in metastable
solutions that do not undergo phase transformations over
long periods of time. Nucleation of CaCO3 involves an ini-
tial burst nucleation of particles followed by reprecipitation
Figure 7. Polarized-light microscopy and SEM images of CaCO3 precipi-
tate: a, b) calcite and c, d) coexistence of calcite and vaterite.
sites and induced the precipitation of stable porous spherical
vaterite particles.[21] The addition of serine influenced the
geometric shape of the CaCO3 and resulted in the formation
of both vaterite and calcite phases in porous spherical
CaCO3 particles. Ca2 + ions bound to the anionic COO moi-
eties and the cationic NH3 + groups of the zwitterionic serine
to produce partially nucleated vaterite CaCO3 crystal struc-
tures. Calcite and vaterite coexisted in the spherical porous
CaCO3 particles, as shown in Figure 7c, d. Both CaCO3 mor-
phologies were hexagonal in structure, but the vaterite was
more complex with a packing structure that led to the
growth of aggregates. The vaterite particles were porous,

Chem. Eur. J. 2012, 00, 0 – 0  2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.chemeurj.org &5&
These are not the final page numbers! ÞÞ

whereas the thin planar calcite crystals were not porous and
grew on the external surfaces of the vaterite particles.
The XRD patterns of the CaCO3 precipitates varied for a
given pH 10.0, depending on the preparation conditions. A
pure calcite phase (JCPDS 05-0586) was observed in the ab-
sence of additives. The addition of a serine altered the mor-
phologies of the CaCO3. Vaterite (JCPDS 33-0268) and cal-
cite were found to coexist, as shown in Figure 8. Diffraction
Figure 8. XRD patterns of pure calcite, and coexistence of calcite and va-
terite.
peaks were observed at 2q = 24.8, 27.02, 32.74, 43.8, and
49.988, which correspond to the vaterite crystal faces (100),
(101), (102), (110), and (104), respectively. After 60 min, a
pure calcite phase was observed on account of the phase re-
configuration of the vaterite. Diffraction peaks were ob-
served at 2q = 29.5, 36.1, 39.5, and 43.38, which correspond
to the calcite crystal faces (104), (110), (113), and (202), re-
spectively.[22] The results described above indicated that
Fe CA satisfied important criteria for green materials: reus-
ability and stability during storage. These materials are
promising candidate catalysts for the hydration of CO2 and
its sequestration as CaCO3.
Conclusion
BCA was immobilized onto SiO2-coated Fe3O4 magnetic
nanoparticles through OAPS grafting. XPS and EDS ele-
mental mapping of the Fe3O4/SiO2/OAPS indicated the suc-
cessful grafting of the nitrogen-containing OAPS groups.
The biocatalytic activities of free BCA and Fe CA toward
the hydrolysis of p-NPA were found to be 874 and
783 m 1 s 1, respectively. The control study showed that the
Ca2 + ion consumption did not vary with pH or the applica-
tion of a negative pressure, which indicated that relative car-
bonization of 68 % is on account of CO2 hydrated by
Fe CA. Equal quantities of free or Fe CA enzymes pro-
duced equal quantities of CaCO3 by the end of the first
&6& www.chemeurj.org  2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!
S. K. Jeong et al.
cycle: 209 or 202 mg, respectively. The reusability studies of
Fe CA indicated that the activity of the materials was 26-
fold higher than the activity of the free BCA. The washing
protocol during recovery and reuse of Fe CA suggested
that the decrease in activity was on account of the lack of a
washing step. Interestingly, different phases of CaCO3, in-
cluding calcite and vaterite, formed upon addition of serine
at pH 10, as shown by polarized-light microscopy. Fe CA
was shown to be a reusable and stable nanobiocatalyst
toward CO2 hydration and sequestration.
Experimental Section
Synthesis of OAPS magnetic nanoparticles: Magnetic nanoparticles were
synthesized by means of co-precipitation, as described in the literature,[23]
with a slight modification. Typically, the iron salt mixture in a molar ratio
of 1:2 (FeCl2/FeCl3) was stirred by using a Teflon rod for 30 min at 80 8C.
After addition of ammonia, the reaction mixture was stirred for 4 h. N2
was bubbled throughout the reaction. The Fe3O4 nanoparticles were fil-
tered, washed, and dried at 80 8C. Thereafter, silica-coated Fe3O4 particles
were prepared by using a modified Stçber method through hydrolysis
and condensation of TEOS in isopropanol with ammonium hydroxide as
the base catalyst.[24] Typically, Fe3O4 (100 mg) was dispersed in a mixture
of water and isopropanol (1:5), after which TEOS (1 mL) and ammonia
(2 mL) were added to the reaction solution with constant mechanical stir-
ring for 24 h at 25 8C. The obtained spherical Fe3O4/SiO2 particles were
washed with water and dried. CPTMS (50 mmol) was grafted onto Fe3O4/
SiO2 particles (200 mg) dispersed in dry toluene under reflux for 24 h.
The final product was filtered, washed with toluene then alcohol, and
dried under vacuum at 70 8C for 8 h. The product is denoted Fe3O4/SiO2/
CPTMS. Finally, OAPS was grafted onto Fe3O4/SiO2/CPTMS as per the
procedure reported previously.[9] The product was denoted Fe3O4/SiO2/
OAPS.
Immobilization of BCA on Fe3O4/SiO2/OAPS and enzymatic activity
assays: BCA was immobilized onto Fe3O4/SiO2/OAPS particles, as de-
ACHTUNGREscribed previously, with slight modification.[9] Briefly, Fe3O4/SiO2/OAPS
particles (10 mg) were dispersed in a 0.1 m sodium phosphate buffer
(pH 8.0) and stirred with a GA solution for 1 h. The GA-activated parti-
cles were separated by magnetic decantation and subsequently washed
with water. GA acted as a spacer to produce reactive aldehyde groups on
the surface of the Fe3O4/SiO2/OAPS, which then reacted with the amino
groups of BCA to form covalent bonds. The recovered product was treat-
ed with a BCA solution (2 mL) at an enzyme concentration of 3 mg mL 1
and incubated with shaking for 1 h at 25 8C. Unbound BCA was removed
by applying a magnetic field and washing the BCA-immobilized particles
until no BCA was detected in the supernatant Tris-HCl buffer. The prod-
uct was denoted Fe CA. The quantities of immobilized and unbound
BCA were calculated by using the Bradford method, and the biocatalytic
activity of the free and immobilized BCA were estimated by using
p-NPA as described.[15]
Biomimetic sequestration of CO2 : The CO2 hydration was carried out as
per our recent report with modification.[9] Typically, a 30 mm CO2 solu-
tion was added to the free BCA- or Fe CA-dispersed Tris-HCl buffer
(30 mL, 1.0 m, pH 8). The reaction was monitored by recording the pH
change, and completion of the CO2 hydration was determined by the ab-
sence of pH changes.[6b] These studies were conducted in a temperature-
controlled mechanically stirred batch reactor. Upon completion, the Fe
CA nanoparticles were collected by using an on/off magnet (Kanetec,
MB-BV) placed close to the wall of the reactor. The hydrated CO2 solu-
tion was collected by decanting, and the recovered Fe CA was used in
subsequent reaction cycles. The hydrated CO2 solution was treated under
vacuum (BCHI R-215, V-700) for 10 min to remove any unstable or un-
dissolved CO2. The solution was then treated with a 100 mm calcium-ion-
containing buffer solution (100 mL, CaCl2·6 H2O), with or without serine
(10 mm) at pH 10.0 to enable CaCO3 precipitation. The formation of
Chem. Eur. J. 0000, 00, 0 – 0
Carbonic Anhydrase
CaCO3 crystals was observed by polarized-light microscopy and quanti-
fied by ion chromatography coupled with a conductivity detector.[9] As a
control, the above experiment was also carried out in the absence of
enzyme. The percent efficiency of calcium-ion precipitation was obtained
after correcting the experimental results according to the control experi-
ment. The weight of calcium carbonate and gram equivalents of CO2
present in CaCO3 were also determined. Precipitated CaCO3 was dried
and characterized.
Characterization of materials: The surface morphology and elemental
mapping of the amine-functionalized magnetic nanoparticles were meas-
ured by FESEM by using a Hitachi S-4800 and EDS, respectively. The
size distribution of the silica-coated magnetic nanoparticles was meas-
ured by high-resolution TEM using a JEOL-2000EX operated at 120 kV.
The XRD were recorded using a Rigaku Miniflex diffractometer with
CuKa radiation (l = 0.154 nm). The FTIR spectra of BCA, Fe3O4, and the
functionalized nanomaterials were recorded by the KBr pellet method
using a Nicolet 6700 spectrometer. X-ray photoelectron spectroscopy was
used to confirm the silica coating, surface functionalization, and oxida-
tion state of the iron in the oxide nanoparticles. These measurements
were recorded using a Thermo Scientific Multilab 2000 spectrometer
with a MgKa source. The ester (p-NPA) hydrolysis kinetics were moni-
tored using an Optizen 2120F UV/Vis spectrophotometer. The calcium
ions were quantified by using an ion chromatograph (IC) with a Metro-
sep C2-150 cationic column coupled to a conductivity detector (Metrohm
801 Compact IC pro). CaCO3 crystal nucleation was observed by polar-
ized-light microscopy, and the images were captured using an Olympus
BX51M coupled with a megapixel Firewire camera and bright-field ob-
jective (Olympus MPlanFL N 100  0.90 BD).
Acknowledgements
This work was supported by the Korea CCS R&D Center (KCRC) with
a grant funded by the Korean Government (Ministry of Education, Sci-
ence and Technology) (No. 2012-0008933).
[1] IPCC Fourth Assessment Report: Climate Change, 2007 (AR4).
[2] a) D. M. DAlessandro, B. Smit, J. R. Long, Angew. Chem. 2010, 122,
6194 – 6219; Angew. Chem. Int. Ed. 2010, 49, 6058 – 6082; b) G. A.
Olah, G. K. S. Prakash, A. Geoppert, J. Am. Chem. Soc. 2011, 133,
12881 – 12898; c) J. D. Figueroa, T. Fout, S. Plasynski, H. McIlvried,
R. D. Srivastava, Int. J. Greenhouse Gas Control 2008, 2, 9 – 20.
[3] a) H. Miyamoto, T. Miyashita, M. Okushima, S. Nakano, T. Morita,
A. Matsushiro, Proc. Natl. Acad. Sci. USA 1996, 93, 9657 – 9660;
b) P. Mirjafari, K. Asghari, N. Mahinpey, Ind. Eng. Chem. Res. 2007,
46, 921 – 926.
[4] J. M. Berg, J. L. Tymoczko, L. Stryer, Biochemistry, 6th ed., W. H.
Freeman and Company, New York, 2007, pp. 254 – 259.
[5] a) Y. Xiao, F. Patolsky, E. Katz, J. F. Hainfeld, I. Willner, Science
2003, 299, 1877 – 1881; b) U. T. Bornscheuer, Angew. Chem. 2003,
115, 3458 – 3459; Angew. Chem. Int. Ed. 2003, 42, 3336 – 3337.
[6] a) M. Vinoba, K. S. Lim, S. K. Jeong, S. H. Lee, M. Alagar, Lang-
muir 2011, 27, 6227 – 6234; b) M. Vinoba, M. Bhagiyalakshmi, S. K.
Jeong, Y. Yoon, S. C. Nam, Colloids Surf. B 2012, 90, 91 – 96; c) S.
Wanjari, C. Prabhu, R. Yadav, T. Satyanarayana, N. Labhsetwar, S.
Rayalu, Process Biochem. 2011, 46, 1010 – 1018; d) A. Z. M. Badrud-
doza, K. Hidajat, M. S. Uddin, J. Colloid Interface Sci. 2010, 346,
Chem. Eur. J. 2012, 00, 0 – 0  2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
FULL PAPER
337 – 346; e) J. D. Badjić, N. M. Kostić, Chem. Mater. 1999, 11, 3671 –
3679; f) E. Ozdemir, Energy Fuels 2009, 23, 5725 – 5730.
[7] a) A. K. Mukherjee, T. Satish Kumar, S. K. Rai, J. K. Roy, Biotech-
nol. Bioprocess Eng. 2010, 15, 984 – 992; b) J. Huang, X. Li, Y.
Zheng, Y. Zhang, R. Zhao, X. Gao, H. Yan, Macromol. Biosci.
2008, 8, 508 – 515; c) N. Schultz, C. Syldatk, M. Franzreb, T. J.
Hobley, J. Biotechnol. 2007, 132, 202 – 208.
[8] a) A. Kros, M. Gerritsen, V. Sprakel, N. Sommerdijk, J. Jansen, R.
Nolte, Sens. Actuators B 2001, 81, 68 – 75; b) L. Wang, K. G. Neoh,
E. T. Kang, B. Shuter, Biomaterials 2011, 32, 2166 – 2173; c) C. Yang,
J. Wu, Y. Hou, Chem. Commun. 2011, 47, 5130 – 5141.
[9] M. Vinoba, M. Bhagiyalakshmi, S. K. Jeong, Y. Yoon, S. C. Nam, J.
Phys. Chem. C 2011, 115, 20209 – 20216.
[10] a) X. D. Wang, Z. X. Shen, T. Sang, X. B. Cheng, M. F. Li, L. Y.
Chen, Z. S. Wang, J. Colloid Interface Sci. 2010, 341, 23 – 29; b) H. C.
Wang, C. Y. Wu, C. C. Chung, M. H. Lai, T. W. Chung, Ind. Eng.
Chem. Res. 2006, 45, 8043 – 8048.
[11] R. Chen, G. Song, Y. Wei, J. Phys. Chem. C 2010, 114, 13 409 –
13 413.
[12] W. Xie, N. Ma, Energy Fuels 2009, 23, 1347 – 1353.
[13] a) J. C. Huang, C. B. He, Y. Xiao, K. Y. Mya, J. Dai, Y. P. Siow, Poly-
mer 2003, 44, 4491 – 4499; b) J. Gao, X. Li, W. Wu, H. Lin, Polym.
Compos. 2011, 32, 829 – 836.
[14] a) A. L. Morel, S. I. Nikitenko, K. Gionnet, A. Wattiaux, J. Lai-Kee-
Him, C. Labrugere, B. Chevalier, G. Deleris, C. Petibois, A. Brisson,
M. Simonoff, ACS Nano 2008, 2, 847 – 856; b) Y. Qin, H. Ren, F.
Zhu, L. Zhang, C. Shang, Z. Wei, M. Luo, Eur. Polym. J. 2011, 47,
853 – 860.
[15] M. Vinoba, D. H. Kim, K. S. Lim, S. K. Jeong, S. W. Lee, M. Alagar,
Energy Fuels 2011, 25, 438 – 445.
[16] a) R. C. Boguslaski, A. M. Janik, Biochim. Biophys. Acta Enzymol.
1971, 250, 266 – 269; b) M. Yan, Z. Liu, D. Lu, Z. Liu, Biomacromo-
lecules 2007, 8, 560 – 565.
[17] P. Pocker, T. L. Deits, J. Am. Chem. Soc. 1983, 105, 980 – 986.
[18] A. Sharma, A. Bhattacharya, A. Shrivastava, Enzyme Microb. Tech-
nol. 2011, 48, 416 – 426.
[19] a) S. Bhattacharya, M. Schiavone, S. Chakrabarti, S. K. Bhattachar-
ya, Biotechnol. Appl. Biochem. 2003, 38, 111 – 117; b) R. R. Yadava,
S. N. Mudliarb, A. Y. Shekha, A. B. Fulkea, S. S. Devia, K. Krishna-
murthia, A. Juwarkarc, T. Chakrabartia, Process Biochem. 2012, 47,
585 – 590.
[20] a) L. A. Estroff, I. Cohen, Nat. Mater. 2011, 10, 810 – 811; b) D. Ge-
bauer, A. Volkel, H. Colfen, Science 2008, 322, 1819 – 1822; c) J.
Xiao, Z. Wang, Y. Tang, S. Yang, Langmuir 2010, 26, 4977 – 4983.
[21] a) S. Mann, Nature 1988, 332, 119 – 124; b) S. Mann, R. Heywood, S.
Rajam, J. D. Bichall, Nature 1988, 334, 692 – 695; c) H. Wei, Q. Shen,
Y. Zhao, D.-J. Wang, D.-F. Xu, J. Cryst. Growth 2003, 250, 516 – 524;
d) A. Ahmad, D. Rautaray, M. Sastry, Adv. Funct. Mater. 2004, 14,
1075 – 1080; e) Q. Shen, Y. Chen, H. Wei, Y. Zhao, D. Wang, D. Xu,
Cryst. Growth Des. 2005, 5, 1387 – 1391; f) X. An, C. Cho, J. Phys.
Chem. C 2008, 112, 6526 – 6530; g) S. Lee, S. G. Lee, D. Kwak, J. H.
Park, K. Cho, J. Phys. Chem. C 2011, 115, 2026 – 2029.
[22] Q. Shen, L. Wang, Y. Huang, J. Sun, H. Wang, Y. Zhou, D. Wang, J.
Phys. Chem. B 2006, 110, 23148 – 23153.
[23] X. Wang, P. Dou, P. Zhao, C. Zhao, Y. Ding, P. Xu, ChemSusChem
2009, 2, 947 – 950.
[24] a) W. Stçber, A. Fink, J. Colloid Interface Sci. 1968, 26, 62 – 69; b) T.
Ung, L. M. Liz-Marzn, P. Mulvaney, J. Phys. Chem. B 2001, 105,
3441 – 3452.
Received: April 2, 2012
Published online: && &&, 0000
www.chemeurj.org &7&
These are not the final page numbers! ÞÞ
 S. K. Jeong et al.

CO2 Sequestration

M. Vinoba, M. Bhagiyalakshmi,
S. K. Jeong,* S. C. Nam,
Y. Yoon . . . . . . . . . . . . . . . . . . . . . . . . &&&&—&&&&
Carbonic Anhydrase Immobilized on
Encapsulated Magnetic Nanoparticles
for CO2 Sequestration
Thinking green: Bovine carbonic anhy- enzymatic activities of the free and
drase (BCA) was covalently immobi- Fe CA were investigated by hydrolyz-
lized onto OAPS (octa(aminophenyl)- ing p-nitrophenylacetate, and by
silsesquioxane)-functionalized Fe3O4/ hydration and sequestration of CO2 to
SiO2 nanoparticles (Fe CA). The CaCO3 (see figure).

&8& www.chemeurj.org  2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Chem. Eur. J. 0000, 00, 0 – 0

ÝÝ These are not the final page numbers!