Capture and Sequestration of CO2 by Human ... - ACS Publications

ARTICLE pubs.acs.org/JPCC

Capture and Sequestration of CO2 by Human Carbonic Anhydrase Covalently Immobilized onto Amine-Functionalized SBA-15 Mari Vinoba,† Margandan Bhagiyalakshmi,‡ Soon Kwan Jeong,*,† Yeo II Yoon,† and Sung Chan Nam† †

Greenhouse Gas Research Center, Climate Change Technology Research Division, Korea Institute of Energy Research, 102 Gajeong-ro, Yuseong-gu, Daejeon 305-343, Korea ‡ Department of Chemistry, University College of Engineering-Arni, Anna University of Technology Chennai, Arni 632-317, India ABSTRACT: Human carbonic anhydrase (HCA) was immobilized onto mesoporous SBA-15 surfaces that had been covalently functionalized using one of three amine compounds, namely, tris(2-aminoethyl)amine (TAEA), tetraethylenepentamine (TEPA), and octa(aminophenyl)silsesquioxane (OAPS). Amine functionalization over SBA-15 was characterized by XRD, FE-SEM, BET analysis, and 29Si and 13C CP MAS NMR spectroscopy. HCA immobilization was verified by FT-IR spectroscopy. The catalytic activity toward hydrolysis of p-nitrophenylacetate (p-NPA) was calculated for free and immobilized HCA. The kcat values for HCA/TEPA/SBA-15, HCA/TAEA/SBA-15, and HCA/OAPS/SBA-15 were found to be 7182, 7368, and 7569 M
1 s
1, respectively. The activities of immobilized HCA were retained even after long-term storage, exposure to high temperatures, and reuse for 40 cycles. For comparison, CO2 hydration and sequestration were measured in the presence of both free and immobilized HCA. Importantly, the CO2 conversion efficiency was calculated using the ion chromatography method. The CO2 capture efficiency of immobilized HCA was 36 times higher than that of free HCA, and 75% of the initial enzymatic activity was retained through 40 cycles.

1. INTRODUCTION There is a growing concern that anthropogenic CO2 emissions into the atmosphere are contributing to global climate change. Several CO2 capture and storage technologies have been explored to reduce CO2 emissions during the post-, pre-, and oxy-combustion processes.1 CO2 capture and separation technologies frequently involve amine solvents,2 metal oxides,3 nickel complexes,4 surface-modified silicas,5,6 zeolites,7 or metal
organic frameworks,8,9 and each process has advantages and disadvantages. Recently, an enzyme-based system demonstrated good efficiency toward CO2 capture and sequestration by mimicking the mechanism by which the mammalian respiratory system performs the same task.10 The use of free enzymes in solution has several disadvantages, however, including low enzyme stability, limited reusability, and difficulties in recovering enzymes from the reaction environment. Indeed, unsupported enzymes can denature and show limited stability in industrial applications. Fortunately, immobilization onto a solid support increases the thermal and chemical stabilities of enzymes, thereby ensuring their reusability, providing an eco-friendly catalyst, and generally enhancing their biocatalytic activity. Many methods have been developed for carbonic anhydrase immobilization with the goal of improving enzymatic activity.11
16 Among these methods, immobilization by covalent attachment through the amino group of lysine, which is frequently located on the enzyme surface, stands out.17 In the present study, human carbonic anhydrase (HCA) was immobilized onto amine-grafted mesoporous silica through covalent methods using glutaraldehyde as a cross-linking agent. A high density of amine groups promotes dialdehyde covalent r 2011 American Chemical Society

cross-linking of enzymes, which, in turn, enables high enzyme loads of >30% g/g.18 Three types of amino compounds, namely, tris(2-aminoethyl)amine (TAEA), tetraethylenepentamine (TEPA), and octa(aminophenyl)silsesquioxane (OAPS), which contain four, five, and eight amine groups, respectively, were grafted onto SBA-15 surfaces. The organic amine compounds TEPA and TAEA are sufficiently small, and OAPS is a derivative of polyhedral oligomeric silsesquioxanes (POSS)19,20 with a molecular diameter of 1
1.5 nm21 that has eight amine groups on each edge of a cubic silicon cage structure. Hence, the immobilization of each of these three materials onto SBA-15 preserves the mesoporous structure. The synthesized large-pore short-channel mesoporous SBA-15 was functionalized with 3-chloropropyltrimethoxysilane (CPTMS) and subsequently grafted with OAPS,22 TAEA, or TEPA,23,24 onto which HCA was immobilized. The catalytic activities of the HCA-immobilized SBA-15 materials were tested in the hydrolysis of p-nitrophenylacetate (p-NPA). The thermal stability, reusability, and storage stability were measured, demonstrating the successful immobilization of HCA onto OAPS-, TAEA-, and TEPA-functionalized mesoporous SBA-15. In particular, the activities toward hydration, sequestration of CO2, reusability, CO2 conversion efficiency, and quantity of CaCO3 generated of the the resulting substrates were measured using ion chromatography (IC) techniques. Received: May 19, 2011 Revised: August 29, 2011 Published: September 11, 2011 20209

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Scheme 1. Grafting of 3-CPTMS Followed by TEPA, TAEA, and OAPS onto SBA-15

2. EXPERIMENTAL SECTION 2.1. Materials. Human carbonic anhydrase (HCA), tetraethylorthosilicate (TEOS), P123 triblock copolymer (MW 5800), 3-chloropropyltrimethoxysilane (CPTMS), glutaraldehyde (GA), trimethyl benzene (TMB), tris(2-aminoethyl)amine (TAEA), tetraethylenepentamine (TEPA), tetrahydrofuran (THF), calcium chloride (CaCl2 3 6H2O), and the Bradford reagent were all purchased from Sigma Aldrich, and octa(aminophenyl)silsesquioxane (OAPS) was obtained from Hybrid Polymer (AM0280); all compounds were used without further purification. All solutions were prepared with deionized water. 2.2. Synthesis and Amine Functionalization of SBA-15. Short-channel SBA-15 was synthesized by the method described in the literature,25,26 with some modification. Typically, 4 g of P123 was dissolved in 150 mL of 1.6 M HCl at 40 °C and stirred until complete dissolution. After addition of a swilling agent (i.e., 0.3 g of TMB), the reaction mixture was stirred for 2 h; then, the silica precursor TEOS (9.2 mL) was added. The mixture was stirred at 40 °C for 10 min, allowed to sit for 24 h at 40 °C under static conditions, and then aged at 120 °C for 24 h. The mixture was then cooled and filtered. The resulting white powder was allowed to dry in air under a vacuum for 24 h and was then calcined in air at 550 °C for 6 h at a heating rate of 1 °C/min. To 1 g of the final product (denoted SBA-15) dispersed in 70 mL of dry toluene was added 50 mM CPTMS with stirring. The reaction mixture was refluxed for 24 h, and the final product was filtered, washed with toluene and then alcohol, and dried under a vacuum at 70 °C for 8 h. This product is denoted CPTMS/SBA-15.22
24 Subsequently, a 20 mM solution of an amine compound (TAEA, TEPA, or OAPS) was added to 1 g of CPTMS/SBA-15 to achieve surface functionalization using the procedure developed for CPTMS grafting. OAPS grafting was performed in THF. These products are denoted TAEA/SBA-15, TEPA/SBA-15, and OAPS/SBA-15 (Scheme 1). 2.3. Immobilization and Biocatalytic Activity of HCA. HCA was immobilized onto TEPA/SBA-15, TAEA/SBA-15, and OAPS/SBA-15 through covalent bonds.27 Briefly, HCA immobilization was achieved by mixing 10 mg of amine-functionalized SBA-15 (TEPA/SBA-15, TAEA/SBA-15, or OAPS/SBA-15) with a 0.1% GA solution in 50 mM sodium phosphate (pH 8.0) for 1 h. Subsequently, the product recovered from the first step

was treated with 2 mL of free HCA in buffer solution (3 mg/mL HCA in 100 mM sodium phosphate, pH 7.0) and incubated at 25 °C with shaking for 1 h. The samples were then washed with copious amounts of Tris-HCl buffer (50 mM pH 8.0) and stored. These materials are denoted HCA/TEPA/SBA-15, HCA/TAEA/ SBA-15, and HCA/OAPS/SBA-15. The quantities of immobilized and unbound HCA were calculated using the Bradford method.28 The biocatalytic activities of HCA/TAEA/SBA-15, HCA/TEPA/ SBA-15, and HCA/OAPS/SBA-15 were estimated using p-NPA as per our recent report.27 Blank experiments were also conducted to estimate the self-dissociation of p-NPA in each assay solution (at various pH values and temperatures). These dissociation rates of p-NPA were subtracted from the enzymatic reaction rates (free and immobilized HCA). 2.4. Hydration and Carbonization of CO2 over HCA/TEPA/ SBA-15, HCA/TAEA/SBA-15, and HCA/OAPS/SBA15. CO2 hydration was conducted in the presence of 5 mg of HCA, either free in solution or immobilized in HCA/TEPA/SBA-15, HCA/ TAEA/SBA-15, or HCA/OAPS/SBA-15 and dispersed in 25 mL of 1.0 M Tris-HCl buffer (pH 8.0). The catalyst solution was then added to 100 mL of a saturated CO2 solution (0.131 wt %).29 CO2 hydration was determined to be complete within 10 min because the pH stabilized at a constant value.30 The hydrated CO2 solution was filtered to recover the HCA/TEPA/SBA-15, HCA/TAEA/SBA-15, or HCA/OAPS/SBA-15 substrate for the next cycle. The recovered substrates were treated with 25 mL of calcium ion solution (360 mg Ca2+, 1.5 M Tris base) at pH 10.0 or 10.5 and were stirred for 1 h. The Tris base solutions of pH 10 and 10.5 were prepared by mixing 2-amino-2-(hydroxymethyl)1,3-propanediol and the desired volume of HCl.31 The solubility of CaCO3 is very low,32 so the precipitated carbonate was quantified indirectly using an ion chromatograph coupled with a conductivity detector (801 Compact IC pro). Data were interpreted using the MagIC Net 2.1 software. After 1 h, the precipitated CaCO3 was filtered, and unreacted calcium ions were quantified in the filtrate using a Metrosep C2-150 (silica gel with carboxyl groups) cationic column with a mixture of tartaric acid/dipicolinic acid (4 mmol/0.75 mmol) as the eluent. Precipitated CaCO3 was dried and characterized. For control correction, the above experiment was also carried out in the absence of enzyme. The sequestration efficiency was determined from the difference in calcium ion concentration (initial and unreacted) in the reaction mixture 20210

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Figure 2. Nitrogen adsorption/desorption isotherms of the samples.

Figure 1. XRD patterns of SBA-15 and functionalized SBA-15.

Table 1. Textural Properties of Amine-Functionalized SBA-15 surface area

pore volume

pore diameter

(m2/g)

(cm3/g)

(nm)

SBA-15

712

1.34

13.4

CPTMS/SBA-15

493

1.11

9.68

TEPA/SBA-15

289

0.86

7.68

TAEA/SBA-15

242

0.91

8.13

OAPS/SBA-15

194

0.73

7.21

sample

as determined using IC. The percentage efficiency of calcium ion utilized was obtained after the control correction. The weight of calcium carbonate and the gram equivalents of CO2 present in CaCO3 were also determined.

3. RESULTS AND DISCUSSION 3.1. Characterization. X-ray diffraction (XRD) patterns showed a well-ordered hexagonal mesophase for the parent SBA-15, whereas CPTMS-functionalized and TAEA-, TEPA-, and OAPS-grafted SBA-15 substrates showed no significant changes or shifts in the XRD peaks position at 2θ = 0.8°, 1.6°, and 1.7° (Figure 1). A decrease in the intensity of the higherorder (hkl) peaks was observed, although this was most likely a function of the scattering contrast between the silica wall and the pore channel, possibly due to pore filling by CPTMS followed by TAEA, TEPA, or OAPS grafting.22,23 The parent SBA-15 exhibited well-resolved diffraction peak at 2θ = 0.8° and two weak peaks at 1.6° and 1.7°, corresponding to (100), (110), and (200) reflections, respectively, indicative of a hexagonal mesophase.33 The surface areas of the amine-grafted SBA-15 materials were evaluated by the Brunauer
Emmett
Teller (BET) method,

Figure 3. SBA-15.

29

Si CP MAS NMR analysis of SBA-15 and functionalized

and the pore sizes were determined using the Barrett
Joyner
Halenda (BJH) method. The textural properties are summarized in Table 1. The nitrogen adsorption/desorption of pristine SBA15 and amine-functionalized SBA-15 showed type IV isotherms, exhibiting a well-defined hysteresis loop (Figure 2). The hysteresis indicated capillary condensation of nitrogen in the mesopores. Successive functionalization of CPTMS and TAEA, TEPA, or OAPS onto SBA-15 resulted in a lower specific surface area, pore volume, and pore diameter. Such significant decreases in the textural properties of porous materials upon grafting have been reported previously.34,35 Figure 3 depicts the 29Si cross-polarization (CP) magic-anglespinning (MAS) nuclear magnetic resonance (NMR) spectra of the parent and CPTMS-functionalized SBA-15 materials. Three peaks at
101.3,
109.3, and
92 ppm were observed in all mesoporous silicas and were attributed to resonance lines representing Q4 [siloxane, (SiO)4Si], Q3 [single silanol, (SiO)3SiOH], and Q2 [geminal silanol, (SiO)2Si(OH)2] silicons, respectively. New peaks emerged at
58 and
67 ppm and were assigned to T2 [Si(OSi)2(OH)R] and T3 [Si(OSi)3R] silicon species, respectively, indicating the formation of new siloxane linkages (Si
O
Si) between the chloropropylsilane silicon and 20211

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Figure 4. 13C CP MAS NMR spectra of (a) CPTMS/SBA-15, (b) TEPA/SBA-15, (c) TAEA/SBA-15, and (d) OAPS/SBA-15.

Figure 6. FT-IR spectra of (a) free HCA, (b) SBA-15, (c) HCA/TEPA/ SBA-15, (d) HCA/TAEA/SBA-15, and (e) HCA/OAPS/SBA-15.

Figure 5. FE-SEM images of (a) SBA-15, (b) HCA/TEPA/SBA-15, (c) HCA/TAEA/SBA-15, and (d) HCA/OAPS/SBA-15.

the surface Si
OH groups of SBA-15 during hydrolysis of Si
OH groups.36,37 OAPS/SBA-15 exhibited peaks at
63.9,
70.8, and
77.4 ppm corresponding to cubic silicon cages.38 These observations were due to the grafting of OAPS to the inside of the pores of mesoporous SBA-15. The 13C CP MAS NMR spectrum of CPTMS/SBA-15 displayed three peaks, corresponding to the Si
CH2,
CH2, and Cl
CH2 carbon atoms of the chloropropyl moiety at 10.2, 26.8, and 49.3 ppm, respectively (Figure 4a). This confirmed the successful grafting of the chloropropyl moiety. The presence of peaks at 18 and 62 ppm indicated a residual ethoxy group.39 Broad peaks at 60
20 ppm were observed, corresponding to TEPA and TAEA grafted by the chloropropyl moiety (Figure 4b,c). OAPS grafting was confirmed by the presence of new peaks at 160
110 ppm, attributed to 13C resonances of the benzene-ring carbon atoms (Figure 4d).40 The surface morphologies of the parent SBA-15, HCA/TEPA/SBA-15, HCA/TAEA/SBA-15, and HCA/OAPS/SBA-15 materials appeared as hexagonal rods in field-emission scanning electron microscopy (FE-SEM) images

(Figure 5). This provided evidence that the hexagonal structure was preserved, even after enzyme immobilization. Parts a
e of Figure 6 show Fourier transform infrared (FTIR) spectra of free HCA, SBA-15, HCA/TEPA/SBA-15, HCA/ TAEA/SBA-15, and HCA/OAPS/SBA-15, respectively. The FTIR spectrum of the parent SBA-15 (Figure 6b) showed its characteristic peaks at 1079 and 963 cm
1 due to Si
O
Si and Si
OH stretching, respectively. Upon CPTMS followed by TEPA, TAEA, and OAPS grafting, the peak at 963 cm
1 (Figure 6c
e) disappeared, and new peaks appear at 1567 cm
1 (δN
H), 2931 cm
1 (
CH2 νas), and 2818 cm
1 (
CH2 νs) because of the amine groups present in TEPA, TAEA, and OAPS. These results confirm the successful functionalization of SBA-15. Furthermore, the characteristic peaks of free HCA (Figure 6a) at 2920, 1614, 1524, and 1413 cm
1 were attributed to methyl groups, the CdN stretching mode, N
H bending vibrations, and the presence of an aromatic ring with a low degree of substitution, respectively. FT-IR spectra of HCA/TEPA/SBA-15, HCA/TAEA/ SBA-15, and HCA/OAPS/SBA-15 showed characteristic peaks due to HCA and amine-functionalized SBA-15 (Figure 6c
e). These results confirmed that HCA was immobilized on amine-functionalized SBA-15. 3.2. Biocatalytic Activities of Free HCA, HCA/TEPA/SBA15, HCA/TAEA/SBA-15, and HCA/OAPS/SBA-15. The hydrolysis of p-NPA (reaction substrate) was monitored spectrophotometrically by observing the formation of the p-nitrophenolate anion and acetic acid. The reaction proceeded through an acyl
enzyme intermediate over the free HCA, HCA/TEPA/SBA-15, HCA/TAEA/SBA-15, and HCA/OAPS/SBA-15 materials. Hydrolysis of p-NPA can be described by k1

k2

k3

E þ S h ES sf P1 þ ES1 sf P2 þ E k
1

The acyl
enzyme intermediate of HCA is actually a zinc
acetate complex [ES] that is less stable and dissociates to release p-NP [P1], acetic acid [P2], and HCA [E], releasing HCA for further acteylation.41 The enzymatic activities of free and immobilized 20212

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Figure 7. Performances of HCA/TEPA/SBA-15, HCA/TAEA/SBA-15, and HCA/OAPS/SBA-15: (a) pH profile of free HCA and effects of (b) thermal stability, (c) reusability, and (d) storage stability.

Table 2. Profiles of HCA Samples and CO2 Sequestration Efficiencies at pH 10.0 and pH 10.5a immobilized HCA catalyst

a

mg/(g of silica)

utilization of calcium (IC)

CaCO3 (mg)

mg

efficiency (%)

IC

solidb

CO2 conversion efficiency (%) IC

solidb

free HCA




71.88 (74.57)

19.97 (20.71)

179.69 (186.43)

163.33 (170.11)

60.36 (62.62)

54.86 (57.14)

HCA/TEPA/SBA-15

113

67.88 (70.11)

18.86 (19.48)

169.71 (175.28)

154.51 (159.92)

57.00 (58.87)

51.89 (53.71)

HCA/TAEA/SBA-15 HCA/OAPS/SBA-15

187 291

70.28 (71.65) 71.08 (72.71)

19.52 (19.90) 19.74 (20.20)

175.70 (179.13) 177.70 (181.77)

157.89 (164.53) 160.52 (167.41)

59.01 (60.17) 59.69 (61.05)

53.03 (55.26) 53.91 (56.23)

Values in parentheses are for pH 10.5. b Dried CaCO3.

HCA were determined by holding the enzyme and/or p-NPA concentration constant. The concentration of p-NPA varied from 0.5 to 2.5 mM in the presence of 10% acetonitrile. The kinetic parameters for the hydrolysis of p-NPA were estimated using the Michaelis
Menten equation (eq 1) and the Lineweaver
Burk equation (eq 2) kcat ½E½S km þ ½S

ð1Þ

1 km 1 1 ¼ þ R Rmax Rmax ½S

ð2Þ

R ¼

In these equations, R is the rate of p-NP formation, Rmax is the maximum rate, kcat is the catalytic rate constant, km is the substrate concentration when the rate is equal to Rmax/2 (also

indicative of the affinity of the enzyme for the reaction substrate), and kcat/km is the kinetic constant. Figure 7a shows the effects of pH for p-NPA hydrolysis with free HCA in Tris-HCl buffer (50 mM) in the presence of 10% acetonitrile. The values of kcat/km were highest at pH 8 for the free HCA. Hence, pH 8 was optimized for further kinetic studies. The km and kcat/km values for free HCA and HCA/TEPA/SBA15, HCA/TAEA/SBA-15, and HCA/OAPS/SBA-15 were found to be 27.29, 25.23, 25.88, and 26.59 mM and 7768, 7182, 7368, and 7569 M
1 s
1, respectively. The kcat/km values for the immobilized HCA materials approached that of the free HCA, and no loss of enzyme activity was observed upon immobilization. Table 2 lists the quantities of HCA loaded onto TEPA/SBA-15, TAEA/SBA-15, and OAPS/SBA-15. Among HCA/TEPA/SBA15, HCA/TAEA/SBA-15, and HCA/OAPS/SBA-15, the highest HCA loading was achieved on HCA/OAPS/SBA-15 because of 20213

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Table 3. Reusability of Immobilized HCA Materials for CO2 Hydrationa HCA/TAEA/

HCA/OAPS/

SBA-15

SBA-15

SBA-15

number of cycles

IC

5

75.55

68.06

78.19

68.96

80.73

73.17

10 15

74.76 71.25

65.80 62.62

76.96 74.67

68.08 65.09

78.54 76.96

70.30 68.59

20

68.44

60.35

72.04

62.44

74.94

66.77

25

66.15

57.95

68.87

60.98

70.02

62.67

30

63.87

56.86

67.47

58.29

68.00

60.28

35

63.08

56.37

64.13

55.78

66.59

59.22

40

59.65

52.94

60.97

53.82

63.60

56.67

total

the eight
NH2 groups on the edges of cubic OAPS moiety. Although HCA/TAEA/SBA-15 and HCA/TEPA/SBA-15 contain four and five amine groups, respectively, the enzymatic environment played a vital role in increasing the kcat/km values. The high kcat/km value for the hydrolysis of p-NPA in the presence of HCA/OAPS/SBA-15 was due to the enzyme molecules immobilized onto the well-distributed amine groups in OAPS. The thermal stabilities of free HCA, HCA/TEPA/SBA-15, HCA/TAEA/SBA-15, and HCA/OAPS/SBA-15 were determined by hydrolysis of p-NPA at 20, 25, 30, 35, 40, 45, and 50 °C (Figure 7b). The highest activities of the free HCA and immobilized HCA were found to occur at 25 and 30 °C, respectively. The relative activity of free HCA decreased (by 30%) for increasing temperatures42,43 as a result of conformational changes.44 For HCA/TEPA/SBA-15, HCA/TAEA/SBA-15, and HCA/OAPS/ SBA-15, such decreases were relatively low, because of the strong binding of HCA inside SBA-15 through covalent attachment. Figure 7c shows 40 cycles of p-NPA hydrolysis in the presence of HCA/TEPA/SBA-15, HCA/TAEA/SBA-15, and HCA/ OAPS/SBA-15. The biocatalyst was washed with 50 mM TrisHCl buffer between each cycle and was separated by centrifugation. The results illustrate that HCA/TEPA/SBA-15, HCA/ TAEA/SBA-15, and HCA/OAPS/SBA-15 retained 82% of their initial enzyme activity up to 40 cycles. Therefore, HCA/TEPA/ SBA-15, HCA/TAEA/SBA-15, and HCA/OAPS/SBA-15 are reusable biocatalysts, one of the main requirements for employing these biocatalysts in huge plants for the capture of CO2. Figure 7d shows the storage stability after incubation of free HCA, HCA/TEPA/SBA-15, HCA/TAEA/SBA-15, and HCA/ OAPS/SBA-15 at 30 °C in Tris-HCl buffer (50 mM, pH 8.0). The activities of the stored free HCA, HCA/TEPA/SBA-15, HCA/TAEA/SBA-15, and HCA/OAPS/SBA-15 after 30 days were checked by measuring the hydrolysis of p-NPA. The activities of HCA/TEPA/SBA-15, HCA/TAEA/SBA-15, and HCA/OAPS/SBA-15 remained high, at 82%, 88%, and 92%, respectively, of the initial activities, even after 30 days. In contrast, the free HCA showed a decrease in catalytic activity to 35% of its original value. 3.3. CO2 Hydration and Sequestration as CaCO3 by HCA/ TEPA/SBA-15, HCA/TAEA/SBA-15, and HCA/OAPS/SBA-15. Carbonic anhydrase is a zinc metalloenzyme that reversibly catalyzes the conversion of CO2 to bicarbonate. Zn2+ ions are present in the active site of carbonic anhydrase. The reaction proceeds through nucleophilic attack by a Zn2+
OH moiety, coordination through three histidine residues, and proton transfer catalyzed by

HCA/TEPA/

solid

IC

solid

IC

solid

2713.70 2404.72 2816.48 2467.19 2896.87 2588.36

mg/(mg of HCA)

542.74

480.94

563.30

493.44

579.37

517.67

Average values of five cycles reported as CO2 (mg) in terms of CaCO3 at pH 10.0. a

a non-Zn2+-liganded histidine. The CO2 hydration mechanism is as depicted in Scheme 2: conversion of CO2 to HCO3
by nucleophilic attack of a Zn2+-bound OH
moiety on the carbon of CO2, internal proton transfer of the Zn2+-bound HCO3
, binding of water to Zn2+, and ionization of the Zn-bound water to facilitate release of HCO3
. The coordinated transfer of a proton from the Zn2+-bound water, to a proton-transfer group and then to the buffer (the proton transfer of step III) is catalyzed by carbonic anhydrase. Nucleophilic attack of Zn2+-bound OH
on CO2 leaves a proton on a Zn2+-bound oxygen, and the reverse reaction is possible, in which a HCO3
molecule can bind to an unprotonated oxygen coordinated to Zn2+.45
47 The bicarbonate ion is easily converted to a carbonate ion at higher pH (eq 3), which, in the presence of a Ca2+ cation, can be readily precipitated in the form of calcium carbonate (eq 4). HCO3
T Hþ þ CO3 2


ð3Þ

Ca2þ þ CO3 2
f CaCO3 V

ð4Þ

Different calcium carbonate polymorphs can form depending on the degree of agitation or mixing. The pH can determine the formation of two different CaCO3 solid phases, calcite and vaterite.31 Single particles can form small nanosized subunits. Therefore, pH was found to be important for controlling the properties of the final product of a pure calcite phase (pH 10.0) or of combinations of vaterite and calcite (pH 10.5). CaCO3 precipitation was carried out after CO2 hydration at pH 10.0 or 10.5 at room temperature. At pH 8, HCA can accelerate the hydration of CO2 to H2CO3, whereas in the absence of HCA, transformation of CO2 through the equilibrium reaction generates fewer carbonate ions.48 Therefore, about ∼9.8% of the calcium ion from the reaction mixture was used, and this value was excluded as the control correction from the quantities of CaCO3 precipitated over free HCA, HCA/TEPA/SBA-15, HCA/ TAEA/SBA-15, and HCA/OAPS/SBA-15 in Table 2. In the IC method, the quantity of CaCO3 measured was higher than the amount of precipitated CaCO3 collected because of losses associated with filtration and recovery. The results suggest that the free HCA, HCA/TEPA/SBA-15, HCA/TAEA/SBA-15, and HCA/OAPS/SBA-15 provided nearly 20214

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Figure 9. XRD patterns of CaCO3 precipitates obtained at pH 10.5 after (a) 15, (b) 30, and (c) 60 min and (d) obtained at pH 10.0 after 60 min. Peaks marked C and V are characteristic of calcite and vaterite, respectively. Figure 8. FE-SEM images of CaCO3 precipitates obtained at pH 10.5 after (a) 15, (b) 30, and (c) 60 min and (d) obtained at pH 10.0 after 60 min.

equal quantities of CaCO3. The CO2 conversion efficiency was 57
61% (Table 2). Even though free HCA yielded equal quantity of CaCO3, difficulties are encountered in reusing it. In the present study, after 40 cycles, the efficiencies of CO2 sequestration (pH 10.0, determined by IC) on HCA/TEPA/SBA-15, HCA/TAEA/SBA-15, and HCA/OAPS/SBA-15 were 34, 35.6, and 36.6 times higher, respectively, than that on free HCA (Table 3). The amount of CaCO3 precipitated and the gram equivalent mass of CO2 present in CaCO3 over HCA/TEPA/SBA-15, HCA/TAEA/SBA-15, and HCA/OAPS/SBA-15 were nearly equal to the corresponding values over free HCA. Even after 40 cycles, the immobilized HCA activity retained 75
79% of its initial value. FE-SEM images of the vaterite and calcite particles obtained at different pH values are presented in Figure 8. The reaction time affected the preferred particle shape, which rapidly changed during the crystallization process at pH 10.5 (Figure8a
c). Both types of CaCO3 were hexagonal in structure, but the vaterite was more complex, with a packing geometry that promoted aggregation (Figure 8c). On the other hand, the calcite crystals displayed well-defined faceted rhombohedral characteristics at pH 10.0 (Figure8d). The vaterite particles were porous, whereas the thin planar calcite crystals were nonporous. The calcite crystals grew on the external surfaces of the vaterite particles.31 XRD analysis of CaCO3 precipitated at pH 10.5 indicated the presence of vaterite and calcite phases. Rapid changes in the structure with reaction time were observed in the diffraction patterns after 15, 30, and 60 min at pH 10.5 (Figure 9a
c). Under these conditions, the amorphous phase clearly formed first, followed by rapid crystallization of a mixture of vaterite and calcite. Diffraction peaks occurred at 2θ = 25.04°, 27.16°, 32.8°, 44.1°, and 49.9°, corresponding to the vaterite crystal faces (100), (101), (102), (110), and (104), respectively.31 Figure 8d indicates the presence of a pure calcite phase at pH 10.0. Diffraction peaks occurred at 2θ = 29.5°, 36.1°, 39.5°, and 43.3°, corresponding to the calcite crystal faces (104), (110), (113), and (202), respectively.49,50 Thus, HCA/TEPA/SBA-15, HCA/TAEA/SBA-15, and HCA/ OAPS/SBA-15 were found to be green materials, that are thermally stable, reusable, and stable during storage. These compounds

are promising candidate catalysts for the hydration of CO2 and its sequestration as CaCO3.

4. CONCLUSIONS HCA was immobilized over various amine compounds grafted onto mesoporous SBA-15. Three amine compounds, Namely, TAEA, TEPA, and OAPS, were chosen for grafting onto the mesoporous SBA-15 surface, and these compounds differed in the number and nature of amine groups present. Amine functionalization was verified by 29Si and 13C CP MAS NMR spectroscopy. The biocatalytic activities of HCA/TAEA/SBA-15, HCA/ TEPA/SBA-15, and HCA/OAPS/SBA-15 were investigated for the hydrolysis of p-NPA. The kcat/km values for HCA/OAPS/ SBA-15 were higher than those for HCA/TAEA/SBA-15 and HCA/TEPA/SBA-15 because of the greater number of HCA molecules immobilized on the OAPS/SBA-15 surface. The presence of eight amine groups distributed at the edges of the cubic OAPS structure stabilized the HCA molecules to a large extent. Moreover, HCA/TAEA/SBA-15, HCA/TEPA/SBA-15, and HCA/OAPS/SBA-15 were found to be thermally stable and reusable and to display good storage stability, as determined by p-NPA hydrolysis. The CO2 conversion efficiency was calculated using the IC method. CaCO3 precipitation was conducted at different pH values, 10.0 and 10.5, and both calcite and vaterite phases were observed. The immobilized HCA materials displayed conversion efficiencies (conversion of CO2 to CaCO3) that were ∼36 times higher than that of free HCA. The amount of CaCO3 precipitated over HCA/OAPS/SBA-15 was higher than the amounts precipitated over HCA/TAEA/SBA-15 and HCA/TEPA/SBA-15. ’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected].

’ ACKNOWLEDGMENT This research was supported by a grant (CK3-101-1-0-0) from the Carbon Dioxide Reduction & Sequestration Research Center, one of the 21st Century Frontier Programs funded by the Ministry of Education Science and Technology of the Korean government. 20215

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