J Nanopart Res (2011) 13:263–271
DOI 10.1007/s11051-010-0026-z
RESEARCH PAPER
Single enzyme nanoparticle for biomimetic CO2
sequestration
Renu Yadav • Nitin Labhsetwar • Swati Kotwal
Sadhana Rayalu
Received: 19 January 2010 / Accepted: 6 July 2010 / Published online: 3 August 2010
Ó Springer Science+Business Media B.V. 2010
Abstract Nanoparticle technology is being increas-
ingly used in environmental sciences. We prepared
single enzyme nanoparticle (SEN) by modifying the
surface of carbonic anhydrase (CA) with a thin layer of
organic/inorganic hybrid polymer. SEN-CA appears to
be improving the stability of free enzyme. CA, as
ubiquitously found enzyme, is involved in gaseous
CO2 sequestration and is being looked as a promising
candidate for combating global warming. We report
here physical characterization of SEN-CA using
transmission electron microscope (TEM), Fourier-
transform infrared analysis (FTIR), X-ray diffraction
analysis (XRD), and energy dispersive X-ray (EDX).
Average size of SEN-CA particles appears to be in the
range of 70–80 nm. We also report the effect of SEN
formation on the kinetic parameters of free CA such as
Michaelis–Menten constant (Km), maximum reaction
velocity (Vmax), and storage stability of free CA and
SEN-CA. The Vmax of SEN-CA (0.02857 mmol/min/
mg) and free enzyme (0.02029 mmol/min/mg) is
almost similar. Km has decreased from 6.143 mM for
SEN-CA to 1.252 mM for free CA. The stabilization of
R. Yadav  N. Labhsetwar  S. Rayalu (&)
Environmental Materials Unit, National Environmental
Engineering Research Institute (NEERI), CSIR,
Nehru Marg, Nagpur 400 020, India
e-mail: s_rayalu@neeri.res.in
S. Kotwal
Department of Biochemistry, R.T.M. Nagpur University,
Nagpur 440 033, India
•
CA by SEN formation results in improved the half-life
period (up to 100 days). The formation of carbonate
was substantiated by using gas chromatography (GC).
The conversion of CO2 to carbonate was 61 mg of
CaCO3/mg of CA and 20.8 mg of CaCO3/mg of CA
using SEN-CA and free CA, respectively.
Keywords Single enzyme nanoparticle  Carbonic
anhydrase  Carbonation reaction  Half-life period 
Environmental remediation
Introduction
Enzymes, being highly specific and effective under
ambient conditions, show great promise in industrial
applications. The major drawback in their use is their
limited life. Various methods have been reported to
improve catalytic stability of enzymes such as immo-
bilization, modification, genetic modification, and
medium engineering (Koeller and Wong 2001; Schmid
et al. 2001; Guilbault 1984; Duran and Esposito 2000;
Burton et al. 2002; Tischer and Wedekind 1999;
Livage et al. 2001; DeSantis and Jones 1999; Govard-
han 1999; Mozhaev 1993). In the recent years, the issue
of enzyme stability has been tackled by creating
modified forms of the enzyme such as single enzyme
nanoparticles (SENs) (Kim et al. 2006a, b). In SENs, an
enzyme molecule is modified by enclosing it in a cage
formed with a porous organic/inorganic structure of
less than a few nanometers thick. SENs of chymotrypsin
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and trypsin have been endorsed with great advantages
such as increased stability and activity, high surface
area, performance at high temperatures, stability to
denaturation and aggregation (Yan et al. 2007), with
minimal mass-transfer limitation on substrates, and
improved half-life (Kim et al. 2004, 2005a, b, 2006a, b;
Lee et al. 2005a, b; Kim and Grate 2003).
Enzymes are being used for various applications in
energy and environment including carbon capture and
sequestration (CCS). Global warming is one of the
most important environmental issues that the world
faces today. The main cause behind this is the increase
in the concentration of CO2 in the atmosphere. Since
the industrial revolution, CO2 concentration has
increased from 280 to 360 ppm leading to global
climate change (Drake 2000). Therefore, there is an
urgent need to devise some strategies for sequestra-
tion of CO2. In nature, CO2 is sequestered by chemical
fixation of CO2 in the form of carbonate minerals
such as calcite, aragonite, magnesite, dolomite, and
dolomitic limestone. The reaction sequence of the
production of calcium carbonate as a result of CO2
sequestration is given below:
CO2 ðgaseousÞ $ CO2 ðaqueousÞ ð1Þ
CO2 ðaqueousÞ þ H2 O $ H2 CO3 ð2Þ
H2 CO3 $ Hþ þ HCO3  ð3Þ
HCO3  $ Hþ þ CO3 2 ð4Þ
Ca2þ þ CO3 2 ! CaCO3 # ð5Þ
Reaction 2 is the rate limiting step in above reaction
scheme and is the slowest. The process of CO2
sequestration is safe and eco-friendly but it works
over a geological time scale. It is also insufficient to
ensure the increased CO2 in air. Scientists are hence
proposing biological sequestration of CO2 by CA to
reduce the atmospheric CO2. We report here the use
of SEN of CA (SEN-CA) to increase the rate limiting
step of this reaction (Bond et al. 1999a, b, 2001a, b).
Since we propose to use carbonic anhydrase (CA), a
zinc enzyme to resolve a non-biological problem, we
are referring it as biomimetic sequestration (Bond
et al. 2001a, b; Bhattacharya et al. 2003, 2004).
CA, a ubiquitous enzyme, catalyzes the reversible
hydration of CO2 to form bicarbonate anion and a
proton.
CO2 þ H2 O $ HCO3  þ Hþ
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With the objective of further improving the stability
of the enzyme we report here a unique protocol for
the synthesis of SEN-CA by caging each enzyme
molecule with a hybrid organic/inorganic biopolymer
silica network by using biopolymer such as chitosan.
The biopolymer is non-toxic and user-friendly and
has protein affinity. This study describes the synthesis
and stability of SEN-CA and compares it to free CA
for their application in conversion of CO2 to mineral
carbonates. To the best of our knowledge, we are
reporting for the first time the use of CA as SEN-CA
for CO2 sequestration.
Experimental
Materials
Partially purified CA isolated from a strain of Bacillus
pumilus (TS1) was processed at University Depart-
ment of Microbiology, Delhi University, South Cam-
pus, New Delhi, India. Chitosan flakes used in this
study were obtained from Chemchito India Limited,
Chennai, India. All chemicals used in the study were
of analytical grade and were purchased from Merck,
India Ltd and Sigma Chemicals Co, USA.
Synthesis of CA nanoparticles
CA (10 mg) dissolved in phosphate buffer (50 mM; pH
7) was stirred with sodium metaperiodate (1.85 mM) for
30 min in dark to convert the hydroxyl group (–OH) to
aldehyde group. An aliquot of ethylene glycol (1.5 mL)
was added to arrest the reaction and it was stirred for 2 h.
The solution was dialyzed overnight against 2.5 L of
200 mM phosphate buffer (pH 7) in dark. CA was
activated with chitosan (6 mg/mL) dissolved in 3%
acetic acid by stirring in the dark for 4 h. This allows
formation of Schiff base between the amino group of
chitosan and aldehyde group of enzyme. The Schiff
complex was then stabilized by converting it into
imine by drop-wise adding of sodium borohydride
(0.74 mM), with continuous stirring for 4 h. The
mixture was then dialyzed overnight against 2.5 L
of 50 mM phosphate buffer (pH 7) in dark to
remove unreacted reagents. The imine complex was
then silylated with 3-aminopropyltriethoxysilane
(APTES) and hexane (1:10), vortexed and shaken at
J Nanopart Res (2011) 13:263–271
22 °C (300 rpm) for 5 min. The lower buffer fraction
was filtered (0.1 lm), and stored in the refrigerator.
To the upper hexane fraction, equal volume of
200 mM phosphate buffer (pH 7) was added and
again vortexed at 22 °C (300 rpm) for 5 min for
extraction of CA. The buffer fraction was filtered
(0.1 lm). This process was repeated till no CA
activity was obtained in upper hexane fraction. The
buffer fractions were pooled and stored in refrigerator
for 3 days for silanol condensation (aging). The
solution was dialyzed for 3 days against 2.5 L of
50 mM phosphate buffer (pH 7) in the dark to remove
the autolytic products of CA and unreacted reagents.
The final clear solution (SENs) was stored in a
refrigerator at 4 °C. The yield of CA activity in the
Fig. 1 Schematic (a) and chemical reaction (b) for the synthesis of single-enzyme nanoparticles
265
form of SEN was 50–60%. Schematic and chemistry
for the synthesis of SENs are shown in Fig. 1a, b.
Characterization of SEN-CA
Transmission electron microscopy image of lyophi-
lized SEN-CA was recorded by using a Tecnai-F20
(FEI, The Netherlands).The lyophilized SEN-CA was
dispersed in ethanol, and after 5 min, clear supernatant
was dropped on a Cu grid covered with carbon.
Fourier-transform infrared analysis (FTIR) spectra of
lyophilized SEN-CA (1 wt%) mixed with KBr pellets
were recorded on Bruker Vertex-70 by diffused
reflectance accessory technique. Spectra of SEN-CA
were scanned in the range 400–4000 cm-1. X-ray
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diffraction pattern of the SEN-CA was obtained by
using an X-ray diffractometer (PANalytical Model no.
TW 3660/50), with Cu Ka radiation (2h = 1.54060 A°)
at a voltage of 45 kV and 40 mA and scanned over the
range of diffraction angle 2h = 2–80°. The zinc content
of lyophilized SEN-CA was confirmed by energy
dispersive X-ray (EDX).
Assay of enzyme activity
Enzyme activity was measured spectrophotometrically
using p-nitro phenyl acetate (p-NPA) as a substrate
according to the method described by Armstrong et al.
(1966) with a slight modification. In brief, the reaction
was started by adding 0.2 mL enzyme (free CA or
SEN-CA) in a 1-cm spectrophotometric cell containing
1.8 mL phosphate buffer (100 mM; pH 7) and 1 mL of
3 mM p-NPA. The change in absorbance at 348 nm
was recorded over first 5 min at 25 °C. For control,
enzyme was replaced by buffer. One unit of enzyme
activity was expressed as 1 lmol p-nitro phenol
formed per minute per mg protein at 25 °C.
Protein determination
Protein concentrations were determined (Lowry et al.
1951).
Kinetic constant of SEN-CA and free CA
Kinetic studies were carried out by measuring the
enzyme activities at 348 nm in the presence of
varying substrate concentrations (1, 2, 3, 4, and
5 mM), at different time (0, 20, 40, 60, and 80 min),
and the temperature was maintained in shaking
incubator at 25 °C and 120 rpm. Michaelis–Menten
constant (Km) values and the maximum velocities
(Vmax) were determined using the Lineweaver–Burk
double-reciprocal plot (Lineweaver and Burk 1934),
in which the reciprocals of the initial velocities of the
CA activity were plotted against the reciprocals of the
concentration of p-NPA (substrate) used.
Protocol for mineralization of CO2
To study the efficiency of enzymatic carbonation
reaction to convert CO2 to calcium carbonate (CaCO3),
1 mL of the free CA (1 mg/mL) or SEN-CA was added
to 10 mL of water saturated with CO2. The sample was
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shaken at 25 °C and then CaCl2 was added along with
Tris buffer (1 M, pH 10.4). The time required for the
onset of carbonation reaction with respect to control
(without enzyme) was recorded (Liu et al. 2005).
Characterization of carbonate precipitate
FTIR spectra of carbonate precipitate (1 wt%) mixed
with KBr pellets were recorded on Bruker Vertex-70 by
diffused reflectance accessory technique. Spectra of all
the carbonate precipitates were scanned in the range
400–4000 cm-1. Scanning electron microscope (SEM)
study of the carbonate precipitate of SEN-CA was
carried out by using a JEOL JED-2300 SEM. X-ray
diffraction pattern of the carbonate precipitates were
obtained by using an X-ray diffractometer (PANalytical
Model no. TW 3660/50), with Cu Ka radiation
(2h = 1.54060°) at a voltage of 45 kV and 40 mA and
scanned over the range of diffraction angle 2h = 2–80°.
Estimation of carbonated precipitate
Evaluation of the carbonate precipitate was carried
out by a gas chromatographic method, using a
thermal conductivity detector (TCD). In this method,
the carbonate precipitate was treated with 0.5 M HCl
and CO2 evolved was monitored using gas chroma-
tography (GC). The reaction was carried out in
borosilicate glass reactor in which carbonate precip-
itate was taken and 0.5 M HCl was added. The
evolved gas was collected in the collector and then
analyzed in GC/TCD using Porapak Q column.
Storage studies
For storage stability study, free enzyme and SEN-CA
solution were prepared in bulk and stored as aliquots,
and were incubated at -20, 4, and 30 °C for a period
of 100 days, and the enzyme activity was determined
after every 5-day interval.
Results and discussion
Characterization of SEN-CA
Transmission electron microscopy (TEM) of SEN-CA
High resolution TEM images confirmed the presence
of individual nanoparticles with a contrasting outer
J Nanopart Res (2011) 13:263–271
Fig. 2 TEM image of SEN-CA
structure (Fig. 2). The seemingly hollow centers of
the nanoparticles result from the transparency of the
core protein. The dark image surrounding the trans-
parent core is the composite cross-linked polymer;
the presence of silicon was confirmed by EDX
analysis. Average size of these SEN particle appears
to be in the range of 70–80 nm.
FTIR of SEN-CA
The functional groups of SEN-CA is represented in
FTIR spectra shown in Fig. 3. These spectra reveal
functional groups in SEN-CA. FTIR spectra of SEN-
CA show the peak at wave number of 2932–2866 and
1472–1341 cm-1. These bands are attributed to the
presence of alkyl groups between CA and chitosan
layer. The weak band at 3840 cm-1 is attributed to
the presence of silanol (Si–OH) groups shown in the
Fig. 3 FTIR image of SEN-CA
267
spectra. The sharp peak at 1145 cm-1 in SEN-CA
layer is corresponding to siloxane bond (Si–O–Si).
The shift of band may due to weak binding of Si-
groups with chitosan.
X-ray diffraction analysis (XRD) of SEN-CA
The XRD pattern of SEN-CA indicated the charac-
teristics peaks of chitosan at 2h of 10 and 20°, SEN-
CA also shows the intense peaks at 72 and 88°
corresponding to silica groups as shown in Fig. 4.
EDX analysis of SEN-CA
The CA is a zinc-containing enzyme. EDX spectrum
of SEN-CA in Fig. 5 shows the presence of Zn atom
confirming the presence of CA in the particles.
Kinetic constants of SEN-CA and free CA
The kinetic constants (Km and Vmax values) for free
CA and SEN-CA are shown in Table 1 and Fig. 6.
For the free CA Km was 1.252 mM, and apparent Km
value of SEN-CA was 6.143 mM. The Km of SEN-
CA is almost five times as that of free CA and maybe
due to the mass-transfer limitation for the substrate
(p-NPA) through the organic/inorganic hybrid layer
formed around the single enzyme by multipoint
attachment between each CA molecule and the
biopolymeric silica network. In view of this result,
efforts are in progress to reduce the biopolymeric
silica network for possible decrease in Km values.
The turnover number, or kcat, is micromoles of
Fig. 4 XRD image of SEN-CA
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Fig. 5 EDX image of

SEN-CA

Table 1 Kinetic parameters of free CA and SEN-CA Further studies are in progress to use other bioassay
tools such as CO2 hydration (Mirjafari et al. 2007) to
Enzyme Vmax Km kcat kcat/Km
(mmol/min/mg) (mM) (s-1) (M-1/s) substantiate these findings. Also studies are in
progress to substitute chitosan with other polymer,
Free CA 0.02029 1.252 0.6093 486.66 which shall not act as promoter, and also to optimize
SEN-CA 0.02857 6.143 8.579 1396.5 the minimum level of APTES used in the organic/
inorganic hybrid layer, which will not interfere in
enhancing the catalytic efficiency.
product made per minute per micromole of enzyme
(Vmax/Et). The increased kcat for SEN-CA by a factor Mineralization of CO2
of 14 in spite of higher Km maybe attributed to
increased reactivity of SEN-CA with p-NPA mole- In the precipitation reaction, the time recorded for the
cules. The catalytic efficiency (kcat/Km) of SEN-CA formation of precipitate in SEN-CA is 37 s as
has increased by factor of 3, and it may be attributed compared to 170 s in blank without CA (reagent
to the presence of promote reagents in the organic/ blank), whereas for the free CA it is 45 s, as shown in
inorganic hybrid layer, which seem to be functioning Table 2. The time taken for precipitation in the blank
as a promoter and enhancing the catalytic efficiency. without CA (reagent blank) is 170 s, which is

Fig. 6 Lineweaver–Burk
plots: a free CA, b SEN-CA

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Table 2 Summary of precipitation of calcium carbonate
reaction
S.N. Samples Time (s)
1 SEN-CA (partially purified) 37
2 CA (partially purified) 45
3 Blank without CA (reagent blank) 170
Table 3 Comparison of FTIR spectra of SEN-CA and free CA
S.N. Peak for SEN-CA Free CA
CaCO3 (cm-1) (cm-1) (cm-1)
1 712 714 712
2 874 869 874
approximately three times higher as compared to
carbonation reaction in presence of free CA and SEN-
CA. The results establish the proof of concept of
SEN-CA being instrumental in accelerating carbon-
ation reaction. Table 3 shows the comparison with
the spectra of calcium carbonate. It has been observed
that prominent two peaks at 712 and 874 cm-1 for
CaCO3 are present in the spectra of the SEN-CA and
free CA, respectively.
Estimation of carbonated precipitate
The carbonated precipitates were subjected to evolu-
tion of carbon dioxide after the carbonation reaction
by using GC. The carbonated precipitate formed is 61
and 20.8 mg of CaCO3/mg of CA for SEN-CA as
compared to free CA, respectively. The enhanced
efficiency of enzyme may be attributed to the
presence of promoters in the hybrid layer. Alterna-
tively, it may be as a consequence of a specific
conformation of enzyme. Further studies are in
progress to elucidate the reason and mechanism of
enhanced reactivity.
Characterization of carbonate precipitate
of SEN-CA
Scanning electron microscope (SEM)
SEM image of carbonate precipitate of SEN-CA
indicates well-defined faceted rhombohedral charac-
teristics of calcite crystals as shown in Fig. 7.
269
Fig. 7 SEM image of carbonate precipitate of SEN-CA
Storage stability studies
For the storage stability of free CA and SEN-CA, the
enzyme preparations were stored at -20, 4, and
30 °C, and the activity was measured for a period of
100 days. Results are shown in Fig. 8. The initial
activity of free CA was 82.5 U/mL. However, when
stored at -20 °C the enzyme activity shows decline
pattern till 10th day after that activity remained stable
up to 15th day, further it shows complete loss of
enzyme activity on 20th day. In the case of SEN-CA
stored at -20, 4, and 30 °C the enzyme activity
increased slowly for 50 days and then declined. Early
rise in activity may be due to aging and stabilization
of organic/inorganic hybrid layer formed around
enzyme molecule. The method thus provides higher
shelf-life for the enzyme as compared to free enzyme.
It is probably because the multipoint attachment
between CA molecule and the biopolymeric silica
network enhances the conformational stability of CA.
When aging is completed, the activity starts declining
very slowly. From Fig. 8b it has been observed that
the time required for aging at 4 °C is more than other
temperatures (-20, 30 °C), and there is increase in
activity. It appears to be safe to store at 4 °C.
Activities decrease slowly after 35 days (52 U/mL at
4 °C) and continue to remain almost constant up to
100th day (35 U/mL at 4 °C).
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Fig. 8 The storage stabilities: a free CA (solid line -20 °C), b SEN-CA at different temperature (solid line 30 °C, dashed line
-20 °C, dotted line 4 °C)
Conclusions
We have developed a new form of stabilized enzyme
using CA. Individual enzyme molecules are stabilized
within a biopolymeric silica network of nanometer-
scale thickness. A key result is that stabilization of the
activity was achieved with minimal substrate mass-
transfer limitation compared to CA entrapped in larger
scale materials. The development of stabilized SEN-
CA as soluble individual SEN-CA particles also
provides the opportunity to further process them into
other forms. We are optimistic that other enzymes that
are compatible can be converted into SEN with the
described synthetic procedure. This study illustrates
the synthesis of SEN-CA by a simpler method and
corroborates the findings of Kim et. al. of armored
single-enzyme nanoparticles, namely, stabilization of
the activity, reduced mass-transfer limitation relative
to large-scale immobilizations, and processability into
additional forms including hierarchical architectures.
Kinetic properties of free SEN-CA entrapped in
chitosan layer were studied. SEN-CA and free CA
showed a typical Michaelis–Menten profile, but the
entrapment altered the affinity of the enzyme to its
substrate and the maximum rate of reaction. However,
it is required to significantly improve the SEN synthe-
sis by decreasing the thickness of organic/inorganic
matrix. Further, studies are in progress to optimize the
condition for carbonation reaction and elucidating the
kinetics and mechanistic aspects.
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Acknowledgments This study was carried out under the Supra
Institutional Project [SIP-16 (4.2)], Council of Scientific and
Industrial Research (CSIR) and the Department of Biotechnology
(DBT), New Delhi, sponsored project. We are grateful to The
Director, National Environmental Engineering Research Institute
(NEERI), Nagpur for providing constant encouragement and help.
We are thankful to Dr. T. Satyanarayanan, University Department
of Microbiology, New Delhi, for providing partially purified
carbonic anhydrase and Dr. Peshwe, Visvesvaraya National
Institute of Technology (VNIT), Nagpur for characterization of
materials. I acknowledge the help of Prof. Hata, Kyushu
University, Chikushi Campus, Fukuoka, Japan in providing
TEM image of SEN-CA.
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