Comparison of amino and epoxy functionalized SBA-15 used for carbonic anhydrase immobilization

Journal of Bioscience and Bioengineering VOL. xx No. xx, 1e8, 2016 www.elsevier.com/locate/jbiosc

Comparison of amino and epoxy functionalized SBA-15 used for carbonic anhydrase immobilization Xiaoyao Fei, Shaoyun Chen, Dai Liu, Chunjie Huang, and Yongchun Zhang* State Key Laboratory of Fine Chemistry, School of Chemical Engineering, Dalian University of Technology, Dalian 116024, China Received 16 September 2015; accepted 8 February 2016 Available online xxx

Two functionalized SBA-15 [amine-functionalized SBA-15 (AFS) and epoxy-functionalized SBA-15 (GFS)] with different types of functional groups were synthesized by a hydrothermal process and post functionalized with 3aminopropyltriethoxysilane (APTES) and 3-glycidyloxypropyltrimethoxysilane (GPTMS), respectively. They were used for the immobilization of carbonic anhydrase (CA). The physicochemical properties of the functionalized SBA-15 were characterized by X-ray powder diffraction (XRD), N2 adsorptionedesorption, 13C, 29Si solid-state nuclear magnetic resonance (NMR) spectroscopy, and scanning electron microscopy (SEM). Before and after CA was immobilized on AFS and GFS, the effects of temperature and pH value on the enzyme activity, storage stability, and reusability were investigated using para-nitrophenyl acetate (p-NPA) assay. CA/GFS showed a better performance with respect to storage stability and reusability than CA/AFS. Moreover, the amount of CaCO3 precipitated over CA/AFS was less than that precipitated over CA/GFS, which was almost equal to that precipitated over the free CA. The results indicate that the epoxy group is a more suitable functional group for covalent bonding with CA than the amino group, and GFS is a promising support for CA immobilization. Ó 2016 The Society for Biotechnology, Japan. All rights reserved. [Key words: Enzyme biocatalysis; Carbonic anhydrase; Functionalized SBA-15; Immobilization; Immobilized enzymes]

The rapid accumulation of carbon dioxide (CO2) in atmosphere from the combustion of fossil fuels has been regarded as the major contributor to global warming (1). It is challenging to stabilize or reduce the concentration of CO2 in the atmosphere without reducing the use of fossil fuels. Biocatalysts are particularly attractive for CO2 capture due to their high specific selectivity and efficiency. Carbonic anhydrase (CA, E.C. 4.2.1.1), a biocatalyst, is responsible for the interconversion of CO2 and bicarbonate in living organisms (2,3). Under ambient conditions, one CA isoenzyme molecule can catalyze 1,400,000 molecules of CO2 in 1 s (4,5). An eco-friendly carbon sequestration method has been developed by transforming CO2 to carbonate via a biomimetic route utilizing CA as the catalyst (6). Bond et al. (7) developed an integrated system in which CA was first employed to accelerate the hydration of CO2 for converting it into carbonate. Mirjafari et al. (8) studied the effect of bovine CA on the hydration of CO2, in which the CO2 was fixed in the form of calcium carbonate. Trachtenberg et al. (9) induced CA in membrane CO2 capture systems that showed significant cost decrease and performance advantages over amine scrubbing systems for the capture of CO2 from combustion flue gases. In addition, post-combustion capture of CO2 using CA as the rate promoter in alkaline solvents such as amines, and potassium carbonate has also been reported (5, 10e12). However, several practical problems such as high cost, limited stability, narrow pH range, and difficulties in recovery and reuse

* Corresponding author. Tel./fax: þ86 411 84986332. E-mail address: [email protected] (Y. Zhang).

should be solved (13,14). One of the most attractive methods to address these problems is immobilization of enzyme onto porous silica supports (15). Nanostructured mesoporous materials are promising candidates for enzymatic immobilization, since their ideal characteristics such as the large surface area, uniform size, narrow pore size distribution, and excellent thermal, mechanical, and chemical stabilities (16,17). Enzyme immobilization on mesoporous materials has been widely reported. The immobilization of the isolated heme domain of P450 BM-3 on two mesoporous molecular sieves indicated that the matching between pore diameter and protein dimensions plays a key role in the operational stability and activity of the immobilized enzyme (18). Ajitha et al. (19) immobilized a-amylase over well-ordered mesoporous molecular sieve SBA-15 with different pore diameters; SBA-15 with the optimum pore size showed the maximum adsorption of the enzyme. However, a significant leakage was observed from the support under the reaction conditions; it was concluded that the interactions between the enzyme and inorganic surface were not strong enough (16,20). One approach to reduce the leaking of enzymes from mesoporous materials is to partially reduce the pore openings at the external surface by silanation reactions, thus binding the enzyme molecules covalently within the pores but still allowing the reactants and products to diffuse freely (21). Organic functionalization of the surface can efficiently strengthen the interactions between the enzymes and the pore wall. However, the functional groups required for modification mostly depend on the chemical composition and structure of the enzyme immobilized (22). The active site of CA consists of histidine, threonine and glutamic acid residues, and the surface of enzyme contains amino and

1389-1723/$ e see front matter Ó 2016 The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2016.02.004

Please cite this article in press as: Fei, X., et al., Comparison of amino and epoxy functionalized SBA-15 used for carbonic anhydrase immobilization, J. Biosci. Bioeng., (2016), http://dx.doi.org/10.1016/j.jbiosc.2016.02.004

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carboxyl groups, suitable sites for attachment via both physical and chemical bonds (23,24). Numerous functional groups such as amino, chloride, thiol, carboxylic acid, epoxy, and phenyl groups can be attached to the surface of mesoporous molecular sieves via tethering alkyl chains (25e27). These groups provide different interactions between the surface of the support and enzyme molecules. Among these functional groups, the amino group is mostly used to modify supports (14), whereas epoxy-activated supports are presumably the most accessible supports, since the epoxy rings react with the amino groups of the enzymes to form strong covalent linkages without using of cross-linking agents under mild reaction conditions (28e30). Therefore, the amino and epoxy groups were selected to functionalize the support material for CA immobilization. This paper reports a comparison of mesoporous materials modified with amino and epoxy groups. SBA-15 with a uniform and adjustable pore size was functionalized with both amino and epoxy groups and used for CA immobilization. In order to compare the effect of different functional groups on the surface of the support on CA immobilization, the biocatalytic performance of immobilized CA including the pH, thermal, and storage stabilities and recyclability were investigated in detail. Carbonation study was also carried out to prove that the immobilized CA can be effectively used for the sequestration of CO2 through mineral carbonation.

SBA-15 was obtained by refluxing a mixture of 1 g of SBA-15 and 1 mL of APTES in absolute ethanol under N2 atmosphere for 24 h. The product was filtered, washed with ethanol, and dried overnight under vacuum. Epoxy-functionalized SBA-15 was prepared using GPTMS, following the same procedure as described for the preparation of amine-functionalized SBA-15. The SBA-15 functionalized with amino and epoxy groups were denoted as AFS and GFS, respectively. Characterization of SBA-15, AFS and GFS Powder X-ray diffraction (XRD) patterns were collected using a Rigaku Dm/Xa-2400 diffract meter with Cu Ka  radiation (l ¼ 1.5418, 40 kV, 100 mA) from 0.5 to 5 at a scanning rate of 1 /min (0.02 step length). The nitrogen adsorptionedesorption isotherms were recorded on a Quantachrome AUTOSORB-1-MP setup at 196 C. The samples were evacuated at 80 C under vacuum (p < 105 mbar) prior to each adsorption measurement. The specific surface area (SBET) and pore size distribution were calculated using the BET and BarretteJoynereHalenda (BJH) methods, respectively. The 13C and 29Si CP MAS nuclear magnetic resonance (NMR) spectra were recorded on an Agilen 500 MHz NMR spectrometer at 125.7 MHz and 99.3 MHz with a sample spinning frequency of 5 kHz. Field emission scanning electron microscopy (FEeSEM) images were recorded using a QUANTA 450 microscope. Immobilization of CA on AFS and GFS The CA immobilizations over AFS and GFS were performed by crosslinking enzyme aggregation and covalent attachment method, respectively. Approximately 10 mg AFS was mixed with 3.0 mL of CA solution (2 mg/mL CA in 100 mM sodium phosphate, pH 7.0). The mixture was kept in a shaker at 25 C for 1 h, and 0.5 mL of GA was added. After 1 h, the samples were centrifuged and washed with sodium phosphate buffer (100 mM, pH 7.0) at 25 C for 30 min. The immobilization of CA with GFS was conducted by a similar procedure as that for AFS without GA treatment. The filtrate was used for further assays. The final products were denoted as CA/AFS and CA/GFS (Fig. 1). The quantities of immobilized CA were calculated using the Bradford method (32), based on the difference in the enzyme contents in the supernatants before and after the immobilization. Determination of protein concentration The protein concentration was determined by the Bradford method. The assay mixture consisted of 2 mL of CA solution (or supernatants) and 8 mL of Coomassie Brilliant Blue G-250 reagent. The absorbance at 595 nm was measured after 5 min against a reagent blank prepared from 2 mL of the appropriate buffer and 8 mL of Coomassie Brilliant Blue G-250 reagent. The concentration of CA was plotted against the corresponding absorbance providing a standard curve. The loading capacity was calculated using the following formula:

MATERIALS AND METHODS Materials CA (separated from bovine erythrocyte) was purchased from Worthington Biochemical Co. (NJ, USA). Tetraethyl orthosilicate (TEOS), triblock copolymer (poly(ethylene oxide)-block-poly-(propylene oxide)-block-poly(ethylene oxide), EO20-PO70-EO20, MW 5800, P123), 3-glycidyloxypropyltrimethoxysilane (GPTMS), 3-aminopropyltriethoxysilane (APTES), glutaraldehyde (GA), trimethyl benzene (TMB), p-nitrophenyl acetate (p-NPA), p-nitrophenol (p-NP), tris(hydroxymethyl)aminomethane (Tris), acetonitrile, anhydrous calcium chloride, and Bradford reagent were purchased from Aladdin Chemicals (Shanghai, China). All the compounds were used as received without further purification.

Capacity

Activity assay of free CA, CA/AFS, and CA/GFS The catalytic activity of CA was tested using a variation of a published procedure by performing the hydrolysis of p-NPA (33). The Km and kcat/Km values of the free and immobilized CA were investigated by determining the activity of CA in the presence of varying concentrations of p-NPA (1, 2, 3, 4, and 5 mM). MichaeliseMenten and LineweavereBurk plots were used to calculate the Km and kcat/Km of the enzyme. The esterase activity of CA was measured using the method described previously (34). Briefly, the assay system comprised 0.3 mL enzyme in a 1 cm

Modification of SBA-15 SBA-15 was modified by the reaction between the hydroxyl groups and alkoxy groups of APTES and GPTMS. Amine-functionalized

O Si O O

Si

O

H2 H2 H2 C C C NH2

O

OH APTES

H2 H2 H2 C C C NH2

GPTMS O

H2 H2 H2 H2 H C C C O C C

CH2 O

Enz-NH2 (Enzyme) O

O

H2 H2 H2 H2 H2 O Si C C C N C C C CH2 H

C N EnZ H

O

Si O

O H2 H2 H2 H2 H2 O Si C C C N C C C CH2 H CA/AFS

CH2 O

GFS

Enz-NH2 (Enzyme)

O

Si

H2 H2 H2 H2 H C C C O C C

O

AFS GA

Si O

OH Surface of SBA-15

O

(1)

where C0 is the original concentration of the CA solution, V0 is the volume of the CA solution added to the immobilization mixture, CS is the CA concentration of the supernatant, VS is the total volume of the supernatant, and msupport is the quality of the supporting materials.

Synthesis of SBA-15 Mesoporous silica SBA-15 was synthesized following a literature procedure (31). Typically, 4 g of P123 was dissolved in 120 mL of 2.0 M HCl and 30 mL of deionized water at 40 C. The mixture was stirred till the surfactant completely dissolved; then, 0.3 g of TMB was slowly added. The stirring was then continued for 2 h and 9.2 mL of TEOS was added drop wise to the solution. The mixture was stirred at 40 C for 10 min, allowed to stand for 24 h at 40 C, and aged at 120 C for 24 h. The solid material was then cooled, filtered and washed with deionized water until neutral, and dried under vacuum at 100 C for 24 h. The recovered as-synthesized sample was calcined in air at 550 C for 6 h.

O

  mg C V  Cs Vs ¼ 0 0 g msupport

C N EnZ H

O

Si

H2 H2 H2 H2 H C C C O C C

H2 H C N

EnZ

H2 H C N

EnZ

OH H2 H2 H2 C C C O

H2 H C C OH

O CA/GFS

FIG. 1. Synthesis of AFS and GFS, and followed by CA immobilization.

Please cite this article in press as: Fei, X., et al., Comparison of amino and epoxy functionalized SBA-15 used for carbonic anhydrase immobilization, J. Biosci. Bioeng., (2016), http://dx.doi.org/10.1016/j.jbiosc.2016.02.004

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spectrophotometric cell containing 2.4 mL of Tris buffer (50 mM, pH 8.5) and 0.3 mL of a solution of p-NPA in acetonitrile. The change in the absorbance at 348 nm, which is the characteristic absorption wavelength of p-nitrophenoxide ion and p-nitrophenol (35), was measured using a UVevisible spectrophotometer for 5 min at 30 s intervals before and after adding the enzyme. Different concentrations of p-NPA (1.0, 2.0, 3.0, 4.0, and 5.0 mM) were tested at a fixed enzyme concentration (CA ¼ 0.05 mg/mL) to determine the kinetic parameters. The catalytic activities of CA/AFS and CA/GFS were estimated using the following procedure. Appropriate amounts of immobilized CA were dispersed in 9.0 mL of TriseHCl buffer (50 mM, pH 8.5), and 1.0 mL of p-NPA dissolved in acetonitrile was added to a final solution of 10% acetonitrile. The hydrolysis process was activated by adding p-NPA. After 3 min, the solution was filtered, and the absorbance of the filtrate at 348 nm was measured by UVevisible spectrometry. The samples were washed with TriseHCl buffer (50 mM, pH 8.5) and stored at 4 C. One unit of enzyme activity was expressed as 1 mmol p-NP released per minute at room temperature. Blank experiments were also conducted to exclude the self-dissociation of p-NPA in each assay solution. Thermal stability of free CA, CA/AFS, and CA/GFS The influence of temperature on the activity of free and immobilized CA was examined by incubating the p-NPA solution and enzyme in TriseHCl buffer (50 mM, pH 8.5) in temperature range between 15 C and 55 C for 4 h. The enzyme activity was tested in the same manner as described previously. pH stability of free CA, CA/AFS, and CA/GFS The free and immobilized CA were incubated in TriseHCl buffer (50 mM) at different pH values (5.0, 6.0, 7.0, 7.5, 8.0, 8.5, 9.0, 10.0, and 10.5) for 4 h to investigate the effect of pH values on the activity of the enzyme. The enzyme activity was calculated using the method described above. Reusability of CA/AFS and CA/GFS The reusability of CA/AFS and CA/GFS was assayed for 20 cycles. The immobilized CA was filtered and washed with TriseHCl buffer (50 mM, pH 8.5) after each cycle, and the enzyme activity was calculated for p-NPA hydrolysis as described above. Storage stability of free CA, CA/AFS and CA/GFS The stability of free and immobilized CA was tested by determining the residual activity after storing in TriseHCl buffer (50 mM, pH 8.5) at different periods for up to 30 days at 4 C. The activity was assayed at an interval of 5 days using the same assay procedure described previously. Carbonation study The hydration of CO2 and precipitated as CaCO3 catalyzed by the free and immobilized CA were performed as follows. CA (1 mg), both in the form of free and immobilized CA, was dispersed in 10 mL of TriseHCl buffer (0.5 M, pH 8.5), and 40 mL of CO2 saturated water was added. The mixture was filtered after 10 min to recover the immobilized CA for the next cycle prior to treating with 25 mL of 5% CaCl2 solution in TriseHCl buffer (0.5 M, pH 10.0) incubated at 25 C for 10 min. The precipitate was filtered and dried at 70 C under vacuum. The quantification of the precipitated calcium carbonate was carried by the organic elemental analysis.

RESULTS XRD analysis The XRD patterns of SBA-15, AFS and GFS are shown in Fig. 2. All the samples showed three well-defined typical peaks of hexagonal SBA-15 around 2q ¼ 0.95 , 1.61 and

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1.83 (36), indicating the presence of (100), (110), and (200) planes. This indicates that the long range structure ordering of SBA-15 was not disrupted after the functionalization with APTES and GPTMS. Surface area All the samples exhibited typical type IV isotherms with well-defined H1 type hysteresis loops associated with capacity condensation at P/P0 from 0.60 to 0.75 according to the IUPAC classification (37), indicating the presence of mesoporous phase. The textural properties of SBA-15, AFS, and GFS are summarized in Table 1. The AFS and GFS samples displayed a lower specific area, pore volume, and pore diameter than SBA-15 after the grafting of APTES and GPTMS. Such a decrease in the textural properties of the materials was due to the pore filling and structural construction of APTES and GPTMS (31). The suitability of AFS and GFS for enzyme immobilization can be attributed to the appropriate environment required for the immobilization of large enzyme molecules such as CA with an average diameter of 3.5 nm (38) and the diffusion of substrate. 29 Si and 13C CP MAS NMR Fig. 3 shows the solid-state 29Si and 13C CP MAS NMR spectra of SBA-15, AFS, and GFS. The chemical shifts, d ¼ 93, e102, and 111 ppm of the 29Si CP MAS NMR spectrum (Fig. 3a) of SBA-15 can be assigned to the Si species of Q2(Si(OSi)2(OH)2), Q3(Si(OSi)3(OH)), and Q4(Si(OSi)4), respectively, which are commonly observed in SBA-15 (39). After the functionalization with APTES/GPTMS, the Q4 signal was strengthened at the expense of the Q2 and Q3 signals; this can be attributed to the replacement of the germinal and isolated silanol groups by amino/epoxy groups. The new peaks at 58 and 68 ppm can be assigned to the T2(Si(OSi)2R(OH)1) and T3(Si(OSi)4) silicon species (40,41), which formed during the reaction between the defective SieOH groups of mesoporous silica and APTES/GPTMS. In summary, Fig. 3a provides the evidence for the chemisorption of silane and the effective substitution of silanol groups with the modifier molecules (41,42). The solid-state 13C CP MAS NMR spectra of SBA-15, AFS, and GFS shown in Fig. 3b further verified the grafting of amino/epoxy groups on SBA-15. In the 13C CP MAS NMR spectrum of AFS, all the observed signals at 9.8, 27.4, and 43.8 ppm were assigned to the presence of eSieCH2e, eCH2eCH2e, and eCH2eNH2 groups, respectively, confirming that amino groups grafted onto SBA-15 (41). The 13C CP MAS NMR spectrum of GFS shows six main resonance peaks at 6.5, 22.6, 43.8, 51.2, and 72.1 ppm, attributed to the labeled carbon atoms C1, C2, C6, C5, C3, and C4 of GFS (39,43,44). Moreover, the two signals at 59.2 and 15.8 ppm can be attributed to the carbons in the alkoxy groups of either incompletely hydrolyzed GPTMS or ethanol solvent used during the functionalization (45). Therefore, the 29Si and 13C CP MAS NMR spectra verified the amino and epoxy functionalization of SBA-15.

SEM images Fig. 4 shows the FEeSEM images of parent SBA15, CA/AFS, and CA/GFS. A clear hexagonal rod-like structure and channel morphology were observed. The surface morphologies of CA/AFS and CA/GFS also appeared as hexagonal rods, and the pore structure was still observed, indicating that the pore and hexagonal rod-like structure was retained, even after the functionalization and enzyme immobilization.

TABLE 1. Textural properties of SBA-15, AFS and GFS.

FIG. 2. XRD patterns of SBA-15, AFS, and GFS.

Sample

SBET (m2/g)

VP (cm3/g)

DP (nm)

SBA-15 AFS GFS

642 331 539

1.07 0.58 0.82

9.6 7.8 7.7

SBET, surface area; VP, pore volume; DP, pore diameter.

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FIG. 3.

29

Si CP MAS NMR spectra (a) and

13

C CP MAS NMR spectra (b) of SBA-15, AFS and GFS.

FIG. 4. FEeSEM images of SBA-15 (a), CA/AFS (b) and CA/GFS (c).

Please cite this article in press as: Fei, X., et al., Comparison of amino and epoxy functionalized SBA-15 used for carbonic anhydrase immobilization, J. Biosci. Bioeng., (2016), http://dx.doi.org/10.1016/j.jbiosc.2016.02.004

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Biocatalytic activity of free CA, CA/AFS, and CA/GFS for p-NPA hydrolysis The biocatalytic activities of the free and immobilized CA were assayed by measuring the hydrolysis of p-NPA through an acyl-enzyme intermediate, zinc-acetate complex, which easily dissociated to release p-NP, acetic acid, and CA; the released CA could be used for further acetylation (46). The activities of the free and immobilized CA were measured at different concentrations of p-NPA; the kinetic parameters for the hydrolysis of p-NPA were estimated using the MichaeliseMenten equation (Eq. 2) and the LineweavereBurk equation (Eq. 3). V ¼

kcat ½E½ p-NPA Km þ ½ p-NPA

1 Km 1 1 ¼ þ V Vmax ½ p-NPA Vmax

(2)

(3)

where V is the rate of p-NP formation, kcat is the catalytic rate constant, Vmax is the maximum rate, Km is the substrate concentration when the rate is equal to Vmax/2, exhibiting the affinity of the enzyme for the substrate, and kcat/Km is the kinetic constant. The kinetic constants of the free and immobilized CA were calculated from the plot shown in Fig. 5. Table 2 shows that the Km of free CA (2.4 mM) is lower than those of CA/AFS (3.0 mM) and CA/GFS (3.1 mM). This indicates that the affinity of CA for its substrate decreased. The change in the affinity of CA to its substrate is probably caused by the structural changes in the enzyme introduced by the immobilization procedure. Moreover, the lower accessibility of the substrate to the active site of the immobilized enzyme and the lower transportation of the substrate and products

FIG. 5. LineweavereBurk plots for the estimation of Km and kcat/Km of free CA, CA/AFS, and CA/GFS by p-NPA assay. Experiment conditions: [p-NPA] ¼ 1.0e5.0 mM, free [CA] ¼ 0.05 mg/mL, and [CA] ¼ 0.05 mg/mL in CA/AFS and CA/GFS. Hydrolysis was performed in 50 mM TriseHCl buffer, pH 8.5.

TABLE 2. Amount of enzyme loaded, Km and kcat/Km of p-NPA hydrolysis, and CaCO3 precipitation over free and immobilized CA. Catalyst Free CA CA/AFS CA/GFS

Immobilized CA (mg/g of supports) e 180  4 222  3

Km (mM)a

kcat/Km (M1 s1)a

CaCO3 (mg)a

2.4  0.3 3.0  0.3 3.1  0.2

896.4  6.5 728.2  3.4 757.4  5.9

247  4 209  3 231  6

Data points represent the mean  S.D (n ¼ 3). a p-NPA hydrolysis.

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into and out the supporting materials also might be the reason for the affinity change (38,47e51). However, the kcat/Km values of CA/ AFS and CA/GFS (728.2 and 757.4 M1 s1, respectively), are lower than free CA (896.4 M1 s1). This is probably due to the effect of covalent bond between CA and support materials, causing the structure changes in the enzyme active site. The CA immobilization over AFS and GFS was occurred via cross-linked aggregation (CLEA) (31) and covalent attachment methods, respectively. Further, the CLEA method is an extreme case of covalent bond using a bifunctional agent (52). During the immobilization of CA on AFS, the support was mixed with the enzyme solution to allow CA adsorption. The cross-linking agent GA was added later to link the CA and AFS adequately. The mechanism of CA immobilization over GFS can be divided into two steps: (i) The enzyme molecules are first adsorbed on the epoxy supports; (ii) a covalent attachment between the nucleophilic groups of the enzyme (amino, thiol, and hydroxyl groups) and the nearby epoxy groups in the same support is strongly favored (53,54). In addition, CA/GFS afforded a higher kcat/Km value than CA/AFS. This is probably because the covalent bond between CA and the surface of GFS without cross-linking agent offered a larger space for the diffusion of the reactant molecules and the increased loading of CA onto GFS (222 mg/g of supports), with a larger surface area and pore volume compared to AFS. GFS might be a more suitable candidate than AFS for CA immobilization due to the high loading capacity, kinetic parameter, and simple immobilization procedure. Stability of free CA, CA/AFS and CA/GFS Fig. 6a presents the thermal stability of the free and immobilized CA. The free CA showed maximum activities at 35 C, whereas both CA/AFS and CA/GFS showed maximum activities at a higher temperature of 40 C. At 55 C, CA/AFS and CA/GFS retained 82% and 85% of the original activity, whereas the free enzyme lost almost 30% of its activity. The improved thermal stability upon immobilization, which is well documented (55), particularly for CAesilica supports (31), can be attributed to the steric constraints that the support imposes on the structure of the enzyme, making denaturation more difficult (56). Therefore, the covalent bond between CA and the functionalized SBA-15 protects CA from denaturation. In other words, the thermal stability of CA increased after the immobilization on AFS and GFS. pH value is one of the most important parameters that alter the enzyme activity in an aqueous medium. Fig. 6b shows the pH stability of the free and immobilized CA. The maximum activity of the free and immobilized CA were observed at pH 8.5 and pH 10.0, respectively. Clearly, the immobilized CA showed an excellent stability in a higher alkaline solution than free CA. This can be attributed to the immobilization, resulting in the conformational change of the enzyme. This probably leads to a better resistance to the alkaline environment. Fig. 6c shows the reusability of CA/AFS and CA/GFS. As observed in Fig. 4c, after 20 cycles, the activities of CA/AFS and CA/GFS were maintained at 80% and 87% of the initial activity, respectively. The immobilization of CA on functionalized SBA-15 overcame the recovery problem of free CA, a critical requirement for the application of enzyme. Moreover, the reusability of CA/GFS was better than CA/ AFS. The storage stabilities of the free and immobilized CA are shown in Fig. 6d. After 30 days storage, CA/AFS and CA/GFS retained 88% and 91% of the initial activity, respectively. In contrast, the free CA showed a 70% decrease of the original value. The high remained activity of CA/AFS and CA/GFS provided the evidence of the covalent bond between CA and supports, since it has been reported that, when enzyme is immobilized directly on AFS and GFS, leaching of enzyme due to the electrostatic interaction and hydrogen bonding is possible (57). The CA immobilized on functionalized SBA-15

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FIG. 6. Performance of free CA, CA/AFS, and CA/GFS for the hydrolysis of p-NPA: (a) thermal stability, (b) pH stability, (c) reusability and (d) storage stability, where [p-NPA] ¼ 3.0 mM, and [CA] ¼ 0.05 mg/mL in free CA, CA/AFS, and CA/GFS, respectively. Hydrolysis was performed in 50 mM TriseHCl buffer. Data points represent the mean  S.D. (n ¼ 3) and error bars show the standard deviation.

provides much better storage stability compared to free CA, especially on GFS, since the covalent bond between the enzyme and functional groups on the surface of SBA-15 improved the conformational stability of enzyme. Biomimetic carbonation by free CA, CA/AFS and CA/ GFS The CA catalyzed CO2 hydration mechanism is reported by Lingdskog et al. (58). The process includes two steps: (i) the nucleophilic attack of the zinc-bound hydroxide ion on carbon dioxide to form an intermediate bond with the zinc ion and formation of bicarbonate, and (ii) the recovery of CA and the release of bicarbonate ion. The hydration experiments of CO2 over free CA, CA/AFS and CA/ GFS were performed in Tris base, and the product was precipitated by the addition of 5% CaCl2 solution. After the filtration, the precipitated CaCO3 was recovered and weighed. The results are summarized in Table 2. It can be seen that the amount of CaCO3 precipitated over CA/AFS (209  4 mg) was lower than that precipitated over CA/GFS (231 1 mg), almost equal to that precipitated over free CA (247  6 mg). Moreover, as CA/GFS was found to be thermally stable, reusable, and stable during the storage, it could be a promising catalyst for the continuous hydration of CO2 and its sequestration as CaCO3.

The FEeSEM images of CaCO3 formed from CA/AFS and CA/GFS (Fig. 7a) exhibit well-defined calcite crystal morphologies, similar to the morphology described previously by Li et al. (59). The corresponding EDS spectrum (Fig. 7b) presents the existence of calcium ion. DISCUSSION The major challenges in enzyme application are limited stability and the difficulties in recovery and reuse (12,13). The immobilization of the enzyme onto appropriate support materials is an attractive way to address those problems. Since the amino and epoxy groups are two typical functional groups on the matrix, two representative support materials, AFS and GFS with amino and epoxy groups on the surface, respectively, were prepared for the loading of CA. The characteristics of these two immobilized CA were analyzed and compared in detail to evaluate the influence of amino and epoxy groups on CA immobilization. Both AFS and GFS were synthesized by post synthesis method and characterized by XRD, N2 adsorptionedesorption analysis and SEM analyses. The results demonstrate that the textural structure of SBA-15 remained undamaged; only the pore size, pore volume, and surface area

Please cite this article in press as: Fei, X., et al., Comparison of amino and epoxy functionalized SBA-15 used for carbonic anhydrase immobilization, J. Biosci. Bioeng., (2016), http://dx.doi.org/10.1016/j.jbiosc.2016.02.004

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FIG. 7. SEM images and EDS spectra of CaCO3 over CA/AFS (a) and CA/GFS (b).

decreased due to the modification. The amino and epoxy functionalization were verified by 13C and 29Si CP MAS NMR spectroscopy. The enzyme immobilization capacity of GFS (222 mg/g of supports) was higher than that of AFS (180 mg/g of supports), since GFS has a larger surface area and pore volume compared to AFS. The Km and kcat/Km values of CA/GFS were higher than those of CA/AFS, which probably due to the covalent bond between CA and the surface of GFS without cross-linking agent offered a larger space for the diffusion of reactant molecules. The immobilization increased the stability of CA at a higher pH, as suggested by the maximum activity of immobilized CA at pH 10.0, which is required for the target application. Calcium carbonate generally forms in an alkaline environment. The results also reveal that CA/GFS processed better reusability and storage stability than CA/AFS. In addition, the amount of CaCO3 obtained by hydrated CO2 over CA/GFS was higher than that of CA/AFS, almost the same as for free CA. Compared to AFS, GFS has a larger pore size, pore volume, surface area and enzyme immobilization capacity. After the immobilization of CA, CA/GFS had a higher Km and k cat /Km values, better recyclability and storage stability than CA/AFS, and CA/GFS also had a higher yield of CaCO3 from CO2 hydration. In addition, the procedure for the CA immobilization on GFS was simpler than that on AFS. CA/GFS could be used as a biocatalyst to convert CO2 to calcium carbonate, which might be an efficient eco-friendly method for CO2 sequestration. In conclusion, GFS is a more suitable matrix for CA immobilization. This is because it could provide an attractive way to overcome the drawbacks such as limited stability, reusability, and difficulties in the recovery of CA. GFS is much worth studying for CO2 sequestration applications. Further studies on the CA/GFS for CO2 sequestration associated with amine solution are under investigation.

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Please cite this article in press as: Fei, X., et al., Comparison of amino and epoxy functionalized SBA-15 used for carbonic anhydrase immobilization, J. Biosci. Bioeng., (2016), http://dx.doi.org/10.1016/j.jbiosc.2016.02.004