Energy Fuels 2010, 24, 6198–6207 Published on Web 11/01/2010
: DOI:10.1021/ef100750y
Immobilized Carbonic Anhydrase for the Biomimetic Carbonation Reaction Renu Yadav,† Snehal Wanjari,† Chandan Prabhu,† Vivek Kumar,† Nitin Labhsetwar,† T. Satyanarayanan,‡ Swati Kotwal,§ and Sadhana Rayalu*,† † Environmental Material Unit, National Environmental Engineering Research Institute (NEERI), Council of Scientific and Industrial Research (CSIR), Nehru Marg, Nagpur 440020, India, ‡Department of Microbiology, University of Delhi, South Campus, New Delhi 110021, India, and §University Department of Biochemistry, Rashtrasant Tukadoji Maharaj (RTM) Nagpur University, Nagpur 440001, India
Received June 17, 2010. Revised Manuscript Received October 5, 2010
Carbonic anhydrase (CA) has been immobilized on surfactant-modified silylated chitosan (SMSC) for the carbonation reaction. CA immobilized on SMSC was characterized using a scanning electron microscope, energy-dispersive X-ray and X-ray diffraction spectroscopy, and Fourier transform infrared analysis. The effect of various parameters, such as pH, temperature, and storage stability, on immobilized CA was investigated using a p-nitrophenyl acetate (p-NPA) assay. The optimum pH and temperature were determined to be 7 and 35 °C, respectively. Kinetic parameters of immobilized and free CA (Km and Vmax values) were also evaluated. For immobilized CA, the Km value was 4.547 mM and the Vmax value was 1.018 mmol min-1 mg-1, whereas for the free CA, the Km value was 1.211 mM and the Vmax value was 1.125 mmol min-1 mg-1. It was observed that immobilized CA had longer storage stability and retained 50% of its initial activity up to 30 days. Proof of concept has been established for the biomimetic carbonation reaction. The CO2 sequestration capacity in terms of conversion of CO2 to carbonate was quantified by gas chromatography (GC). It was 10.73 and 14.92 mg of CaCO3/mg of CA for immobilized and free CA, respectively, under a limiting concentration of CO2 (14.5 mg of CO2/10 mL).
including easier separation of the reaction products from the incubation mixture, the ability to recover and reuse the enzyme, stabilization of the tertiary structure of the enzyme, and an increase of the the enzyme stability and operational lifetime.4,5 The success of an immobilized enzyme mainly depends upon the properties of the carriers employed. Chitosan is a polysaccharide easily obtained by alkaline hydrolysis of chitin and has been used in pharmaceutical fields, medicines, drug-delivery carriers, wound-dressing materials, and tissue engineering.6 It is considered to be a suitable support for enzyme immobilization because it is biocompatible, available in various forms (gel, membrane, fiber, and film), nontoxic, and has reactive amino (-NH2) and hydroxyl (-OH) groups amenable to chemical modifications.7-9 Thus, various kinds of chitosan supports have been developed by modification of the chitosan backbone to improve its activity for immobilization of CA, which have both a hydrophilic surface and hydrophobic area near the active site. Chang and Juang10 have reported immobilization of acid phosphatase on chitosan composite beads and activated clay. Chang and Juang11 have recently reported about R- and β-amylase immobilization
1. Introduction The increase in the atmospheric level of carbon dioxide (CO2), one of the vital greenhouse gases, causes global warming. In the global effort to combat the predicted catastrophe, several CO2 capture and storage technologies are being deliberated. One of the most promising ways is biological CO2 sequestration, in which CO2 has been sequestered using carbonic anhydrase (CA) by converting them into bicarbonates, which is further converted into calcium carbonate using calcium chloride solution. CA is a major zinc-based metalloenzyme, which is ubiquitous in nature and found in the prokaryotic as well as eukaryotic domains. Each molecule of the CA isoenzyme can catalyze 1.4 106 molecules of CO2 in 1 s.1-3 CA
CO2 þ H2 O T Hþ þ HCO3 CA in its native form has certain limitations in its application because of the short lifetime of the enzyme. There are different methods to improve the catalytic stability of the enzyme, such as enzyme immobilization, enzyme modification, genetic modification, and medium engineering. Immobilization of the enzyme onto a solid support is currently an active area of research because of its wide range of applications. There are several advantages over the use of soluble enzyme preparations,
(4) Siso, M. I. G.; Lang, E.; Gomez, B. J.; Becerra, M.; Espinar, F. O.; Mendez, J. B. Process Biochem. 1997, 32, 211–216. (5) Vaillant, F.; Millan, A.; Millan, P.; Dornier, M.; Decloux, M.; Reynes, M. Process Biochem. 2000, 35, 989–996. (6) Ravi Kumar, M. N. V.; Muzzarelli, R. A. A.; Muzzarelli, C.; Sashiwa, H.; Domb, A. J. Chem. Rev. 2004, 104, 6017–6084. (7) Krajewska, B. Enzyme Microb. Technol. 2004, 35, 126–139. (8) Dincer, A.; Telefoncu, A. J. Mol. Catal. B: Enzym. 2007, 45, 10–14. (9) Altun, G. D.; Centinus, S. A. Food Chem. 2007, 100, 964–971. (10) Chang, M. Y.; Juang, R. S. Process Biochem. 2004, 39, 1087– 1090. (11) Chang, M. Y.; Juang, R. S. Enzyme Microb. Technol. 2007, 36, 75–82.
*To whom correspondence should be addressed. Fax: þ91 (712) 2247828. E-mail:
[email protected]. (1) Mirjafari, P.; Asghari, K.; Mahinpey, N. Ind. Eng. Chem. Res. 2007, 46, 921–926. (2) Bond, G. M.; Stringer, J.; Brandvold, D. K.; Simsek, F. A.; Medina, M. G.; Egeland, G. Energy Fuels 2001, 15, 309–316. (3) Bhattacharya, S.; Nayak, A.; Schiavone, M.; Bhattacharya, S. K. Biotechnol. Bioeng. 2004, 86, 37–46. r 2010 American Chemical Society
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on chitosan-clay composite beads. Pepsin immobilized on chitosan beads has greater thermal and storage stability than free pepsin, as reported by Altun and Centinus.9 Prabhu et al.12 have reported immobilization of a CA-enriched microorganism on biopolymer-based materials. Also, some reports are available on the immobilization of enzymes on a chitosanbased matrix, with details on activities, stabilities, and reaction kinetics using other enzymes.13-17 Surface modification of chitosan has been reported using a variety of silanecoupling agents, which have organofunctional groups with di- or trialkoxy structures, and the reactions are performed in either the gas or liquid phase.18 The silanol groups condense with the surface residues to form siloxane linkages. In the case of trialkoxysilanes, the presence of three silanol residues in the hydrolysis product can lead to multiple surface attachments. Silylated materials are further treated with surfactant-like hexadecyltrimethylammonium bromide (HDTMABr) to increase the surface of the carrier by forming the mesh network, which will in turn allow for more enzymes to be embedded in this bipolar surface layer.19 In the present study, partially purified CA isolated from Bacillus pumilus TS1 was immobilized on the surfactantmodified silylated chitosan (SMSC). The influence of parameters, such as pH, temperature, substrate, and storage stability, on the free and immobilized CA was studied. This study is also aimed at validating the carbonation reaction using partially purified CA and further quantifying the carbonate precipitate obtained using free and immobilized CA by gas chromatography (GC).
Spectrophotometric measurements were carried out with a Perkin-Elmer Lambda 650S UV/vis spectrophotometer. pH measurements were carried out with a Eutech pH-meter (model pH 1500). Centrifugation was made using a Beckman Optima XL-100K model. For immobilization, an orbital shaking incubator (model RIS.24BL) by Remi Instruments Ltd. was used, and the mixing procedure was made using a vortex (VX-200). X-ray diffraction (XRD) patterns of material was recorded using an X-ray diffractometer (model TW 3660/50), and Fourier transform infrared (FTIR) spectra of material were recorded on a Bruker Vertex-70. Scanning electron microscopy (SEM) images were obtained from secondary electrons with a JEOL JED-2300 scanning electron microscope equipped with an energy-dispersive X-ray (EDX) analyzer. The calcium carbonate precipitate was evaluated using GC (model PerkinElmer Clarus 500). 2.2. Methods. 2.2.1. Synthesis of SMSC Material. A total of 1 g of chitosan flakes was dissolved in 40 mL of 5% acetic acid and stirred for 1 h to obtained viscous chitosan solution, which was then centrifuged, and the insoluble fraction was discarded. To the supernatant, 1 mL of APTES was added and stirred for 1 h. The silylated chitosan solution was subjected to treatment with 0.25% HDTMABr and stirred for 1 h. Thus, the sample obtained was subjected to cross-linking with glutaraldehyde (25%) by stirring it for 24 h, which was then filtered and washed with distilled water to remove unreacted glutaraldehyde. The washed sample was subjected to drying at 110 °C in an oven for 6 h. The sample was designated as SMSC. 2.2.2. Immobilization Procedure and Enzyme Assay. The immobilization procedure that follows has been reported in our previous study.12 The enzyme activity of CA was estimated spectrophotometrically using p-nitrophenyl acetate ( p-NPA) as a substrate according to the method described by Armstrong et al.,20 with slight modification. The assay system contained 0.2 mL of enzyme solution (1 mg/mL) in a 1 cm spectrophotometric cell, containing 1.8 mL of phosphate buffer (0.1 M, pH 7.0) and 1 mL of 3 mM p-NPA. The change in absorbance at 348 nm at 25 °C was recorded over the first 5 min, before and after adding immobilized enzyme. The enzyme activity was calculated by the following formula:
2. Materials and Methods 2.1. Materials. Extracellular CA from B. pumilus TS1 was prepared by centrifuging the culture broth at 8000 rpm for 20 min and concentrating the CA from the supernatant by acetone (20-60% saturation) precipitation. The precipitate was lyophilized. A total of 1 g of the lyophilized powder contained 6840 units of CA. This partially purified CA was provided by the Department of Microbiology, University of Delhi, South Campus, New Delhi, India. Chitosan, used in this work, has a deacetylation degree (DD) of 95% with a molecular weight (MW) of 360 kDa and was purchased from Chemchito, India Ltd., Chennai, India. 3-Aminopropyltriethoxysilane (APTES) was purchased from SigmaAldrich, St. Louis, MO. Tris buffer used in the carbonation study was purchased from Calbiochem, San Diego, CA. Na2HPO4 3 2H2O, NaH2PO4 3 2H2O, CaCl2, sodium potassium tartarate, Na2CO3, CuSO4, glutaraldehyde (25%), folin reagent, and bovine serum albumin (BSA) were purchased from Merck, India Ltd., Mumbai, India. All other chemicals and reagents used were of analytical grade.
enzyme activity ðU=mLÞ ¼
ðΔA 348 nm=min test - ΔA 348 nm=min blankÞ 1000 TV DF millimolar extinction coefficient of p-nitrophenyl acetate V
where 1000 is the conversion to micromoles, TV is the total volume of the reaction mixture, V is the volume of the enzyme solution taken, and DF is the dilution factor. A total of 1 unit of CA is defined as the amount of enzyme required for liberation of 1 μmol of p-nitrophenol min-1 mL-1 at 25 °C. All experiments were performed in triplicate. 2.2.3. Protocol for Mineralization of CO2. The carbonation study was performed by the method described in Favre et al.,21 with slight modification, as shown in Figure 1. The time required for the onset of precipitate and carbonate formation was monitored in the sample as well as control (without CA). The carbonate obtained was filtered and dried at 25 °C. 2.2.4. Evaluation of Calcium Carbonate. The quantity of carbonate precipitated was substantiated using the GC method coupled with a thermal conductivity detector (TCD). This eliminated the interference of other precipitates, such as calcium phosphate. The precipitate obtained was treated with 0.5 M HCl, and evolution of CO2 was monitored using GC. The evolved gas was collected in the collector and then analyzed in GC/TCD using the Porapak Q column.
(12) Prabhu, C.; Wanjari, S.; Gawande, S.; Das, S.; Labhsetwar, N.; Kotwal, S.; Puri, A. K.; Satyanarayana, T.; Rayalu, S. J. Mol. Catal. B: Enzym. 2009, 60, 13–21. (13) Tang, Z. X.; Qian, J. Q.; Shi, L. E. Mater. Lett. 2007, 61, 37–40. (14) Cetinus, S. K.; Sahin, E.; Saraydin, D. Food Chem. 2009, 114, 962–969. (15) Gomez, L.; Ramirez, H. L.; Cabrera, G.; Simpson, B. K.; Villalonga, R. J. Food Biochem. 2008, 32 (2), 264–277. (16) Dhananjay, S. K.; Mulimani, V. H. J. Food Biochem. 2008, 32, 521–535. (17) Mansour, E. H.; Dawoud, F. M. J. Sci. Food Agric. 2003, 83, 446–450. (18) Airoldi, C.; Monteiro, A. O. C., Jr. J. Appl. Polym. Sci. 2000, 77, 797–804. (19) Paetzold, E.; Oehme, G.; Fuhrmann, H.; Richter, M.; Eckelt, R.; Pohl, M. M.; Kosslick, H. Microporous Mesoporous Mater. 2001, 44-45, 517–522.
(20) Armstrong, J. M.; Myers, D. V.; Verpoorte, J. A.; Edsall, J. T. J. Biol. Chem. 1966, 241, 5137–5149. (21) Favre, N.; Christ, M. L.; Pierre, A. C. J. Mol. Catal. B: Enzym. 2009, 60, 163–170.
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Table 1. Comparison to CA Immobilized on Different Matrices chitosan beads
silylated chitosan
SMSC
CN-Cavilinka NH2-
Cavilinka
10 0.4 58.33 2.8
10 0.4 62.50 3
10 0.4 72.91 3.5
111 8.6a >99 1.7
111 8.6a 82 1.6
material (mg) enzyme used for immobilization (mg) percentage of enzyme immobilized (%) capacity (immobilized) (U/mg beads) a
From Hsuanyu et al.26
Figure 2. Comparison between partially purified CA activities after immobilization on different materials.
Figure 1. Protocol for mineralization of CO2.
2.3. Characterization of Materials. XRD of the SMSC was obtained using a (PANalytical) X-ray diffractometer, with Cu KR radiation (λ = 1.540 60 A˚) at 45 kV and 40 mA and scanned over the range of diffraction angle 2θ = 10-80°, and the step size for the XRD measurement is 2θ = 0.0170. The SEM image of SMSC and CA-immobilized SMSC was obtained using a JEOL scanning electron microscope equipped with an EDX analyzer. FTIR spectra of SMSC (1 wt %) mixed with KBr pellets were recorded by a diffused reflectance accessory technique. Spectra of the chitosan flask and SMSC were scanned in the range of 400-4000 cm-1. The resolution is 2 cm-1, and the scan number is 16 for FTIR spectra. 2.4. Kinetic Studies. 2.4.1. Effect of the Temperature and pH on CA Activity. The influence of the temperature on the activity of free and immobilized CA was studied by incubating the reaction mixtures, followed during the immobilization procedure, at different temperatures (15, 25, 35, 45, and 55 °C). The effect of pH on the activity of free and immobilized CA was investigated using phosphate buffer solutions of different pH (6, 7, 8, 9, and 10) at 35 °C. 2.4.2. Kinetic Constants of CA Isolated from B. pumilus. Determination of Km and Vmax values of free and immobilized enzyme was carried out by measuring the activity of CA in the presence of various p-NPA (substrate) concentrations (1, 2, 3, 4, and 5 mM). Michaelis-Menten constant (Km) values and the maximum velocities (Vmax) were determined using the LineweaverBurk double-reciprocal plot,22 in which the reciprocals of the
Figure 3. XRD spectra of (A) chitosan flask and (B) SMSC material.
initial velocities of the CA activity were plotted against the reciprocals of the concentration of p-NPA used. 2.4.3. Storage Stability of CA. For storage stability, study samples were incubated with 3 mg/5 mL of enzyme (free and immobilized) loaded in 4.8 mL of phosphate buffer (0.1 M, pH 7.0) at -20 °C for 30 days, and then the relative activity was determined after intervals of every 5 days.
(22) Lineweaver, H.; Burk, D. J. Am. Chem. Soc. 1934, 56, 658–660.
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Figure 4. Comparison of SEM images of (A) SMSC and (B) immobilized SMSC and (C) EDX spectra of immobilized SMSC.
immobilization of the enzyme (Figure 2). The question of steric hindrance dose not arise because it is an open structure, wherein one HDTMABr ion is coordinating per monomer. CA contains 18 positively charged arginines and 29 negatively charged residues (glutamic and aspartic acids). Histidine, with an average pKa of 6.5 is largely neutral at pH 8. The net charge of BCA is about -3, as measured by capillary electrophoresis.23 Therefore, enhanced adsorption of CA on a positively charged surface because of HDTMA ion and protonated amines has been observed. This may be attributed to the positively charged HDTMA ion coordinating with the free amino group of chitosan through the lone pair of electrons available. This, in turn, enhances entrapment of the enzyme in the bipolar surface layer, by increasing the electrostatic interaction between charged enzyme surfaces
3. Results and Discussion 3.1. Screening of Materials for Immobilization of Partially Purified CA. Figure 2 and Table 1 show the comparison between different materials, viz. SMSC, silylated chitosan, and chitosan beads, for immobilization of partially purified CA. Immobilized catalyst had been tested for its activity using a p-NPA assay. However, almost all materials showed reasonably good activity for enzyme immobilization, with the highest activity observed for SMSC material, followed by silylated chitosan and chitosan beads. Also, the leaching of CA from SMSC is lower, as compared to the other two materials. The mechanism of formation of the chitosan-organosilane hybrid is very well-illustrated by Airoldi and Monteiro18 for immobilization of the enzyme, which is very well-substantiated in our work as well (Figure 2). However, in our study, the material is being modified with surfactant, which further enhances its amphiphillic property, showing higher
(23) Gitlin, I.; Gudiksen, K. L.; Whitesides, G. M. ChemBioChem 2006, 7, 1241–1250.
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using SEM. Panels A and B of Figure 4 show a comparison between bare and immobilized SMSC. Figure 4A of bare SMSC shows a uniform surface morphology with small protruding structures, whereas Figure 4B of the immobilized SMSC shows smooth surface morphology, which may be due to the adsorption of CA on the surface of SMSC. The presence of CA has been further confirmed by EDX spectra (Figure 4C), which shows Zn on the surface. The CA enzyme contains Zn atom, which is used as a marker to confirm the presence of CA on SMSC. 3.2.3. FTIR Analysis of SMSC. The functional groups of chitosan, viz. amino and hydroxyl groups, are very important for adsorption. The FTIR spectra of chitosan flakes and SMSC are given in panels A and B of Figure 5. The band at 3695 cm-1 in chitosan is attributed to the stretching vibration of the N-H group, which is shifted to 3647 cm-1 in SMSC. This shifting of the band may be due to the formation of weak intermolecular hydrogen bonding between the amino and hydroxyl groups of chitosan with the incorporation of the silanol group. The bands at 2924 and 1376 cm-1 in chitosan and 2918 and 2850 cm-1 in SMSC are attributed to the C-H stretching vibration in the polymeric backbone and C-H bending, respectively. This significant decrease in the band intensity is due to the interaction between chitosan and the silanol group of SMSC. The bands at 1155 and 1075 cm-1 in chitosan may be due to the stretching vibration of C-O groups, whereas SMSC shows bands at 1175 and 1058 cm-1 attributed to Si-O-Si stretching. IR spectra of SMSC also contain bands at 1340 and 799 cm-1, which are characteristics of Si-CH3. 3.3. Optimization Study for CA Immobilization. Studies were carried out for optimizing the conditions for immobilization of CA. The conditions studied are being discussed in the following sections. 3.3.1. Effect of the Time Variation on Immobilization of CA. The effect of the time on immobilization of CA is shown in Figure 6. The amount of enzyme adsorbed onto the material increased with an increasing contact time up to 8 h. Subsequently, an increase of the contact time resulted in a decrease of the activity of the immobilized enzyme or an increase of the activity in the supernatant of the immobilized enzyme. The activity of the immobilized enzyme can be calculated by the difference in the free enzyme activity and the activity in the supernatant of the immobilized enzyme. This decrease in the activity of immobilized CA beyond 8 h is being attributed to the leaching of the enzyme or denaturation of the enzyme. 3.3.2. Effect of the Material Dose Variation on Immobilization of CA. The material dose varied between 1 and 10 mg/5 mL. The optimal dose appeared to be 4 mg/5 mL, as shown in Figure 7. A further increase in the dose resulted in decreased enzyme loading, probably because of a lower concentration of enzyme and higher number of active sites on the material as a result of the increased dose of adsorbent. 3.3.3. Effect of the Variation of the CA Concentration on Immobilization. The enzyme concentration varied from 1 to 5 mg/5 mL, and the optimal enzyme concentration appeared to be 3 mg/5 mL, as shown in Figure 8. However, a further increase of the enzyme loading above 3 mg/5 mL resulted in the gradual decrease in the enzyme activity onto the material. This is probably due to optimal adsorption of the enzyme on the matrix surface at 3 mg/5 mL. Mansour and Dawoud17 suggest that the gradual decrease in the enzyme activity is probably due to hindrance between the
Figure 5. FTIR spectra of (A) chitosan flask and (B) SMSC material.
and surfactant ions attached to chitosan through amino groups. Evidence of a positive charge of the SMSC material is calculated using point of zero charge (IPE) of material. The isoelectric point (IEP) of material is pH 5. For the pH values above the point of zero charge of material, the predominate surface species is M-O-, while at the pH value below the point of zero charge of material, M-OH2þ species predominate. SMSC has pH of 4.5, which is below the point of zero charge of material. SMSC is therefore having a positive charge on the surface.24 The mechanism for formation of SMSC is illustrated below
Considering the leaching property along with enhanced enzyme interaction sites, SMSC material had been selected for further studies. 3.2. Characterization of Materials. 3.2.1. XRD Analysis of SMSC. The XRD patterns of bare chitosan and SMSC are shown in panels A and B of Figure 3. The XRD pattern of bare chitosan exhibited its characteristic crystalline peaks at 2θ = 10.2° and 19°. However, these characteristic crystalline peaks of bare chitosan were diminished in XRD analysis of SMSC because of the bonding of the silanol group of silylated chitosan with bare chitosan chains, leading to a decrease in crystallinity. The XRD analysis is not showing sharp peaks of silica nuclei by virtue of its amorphous nature. 3.2.2. SEM Analysis and EDX Spectra of SMSC. Surface morphology of bare and immobilized SMSC had been studied (24) Balistrieri, L. S.; Murray, J. W. Am. J. Sci. 1981, 281 (6), 788–806.
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Figure 6. Effect of the time variation on immobilization of CA.
Figure 7. Effect of the material dose variation on immobilization of CA.
adsorbed enzyme molecules on the matrix, at higher concentrations of enzyme. 3.3.4. Effect of the Variation of the Shaking Speed on Immobilization of CA. The effect of the shaking speed on immobilization of the enzyme on SMSC is shown in Figure 9, wherein the shaking speed varied from 60 to 160 rpm. The optimal shaking speed is 120 rpm. A further increase in shaking speed ultimately lowered the activity of immobilized enzyme probably because of the weakening of the interaction between CA and SMSC at high speed. Thus, a summary from the above studies is that the optimal conditions for enzyme immobilization are as follows: (a) shaking time, 8 h; (b) material dose, 4 mg/5 mL; (c) enzyme concentration, 3 mg/5 mL; and (d) shaking speed, 120 rpm. 3.4. Kinetic Study of the Immobilized Enzyme. 3.4.1. Effect of the Temperature Variation on CA Immobilization. The
variation of the temperature on the immobilized CA activity is shown in Figure 10. The activity of immobilized CA increased up to 35 °C and thereafter decreased with a further increase in the temperature. A similar trend was observed for free CA. However, the decline was more pronounced for free CA. This may happen because of the denaturation of the enzyme at higher temperatures. The optimum temperature for the free enzyme is 25 °C, as compared to 35 °C for the immobilized enzyme. The immobilized enzyme appears to be more thermostable than the free enzyme. The immobilization procedure probably helps to maintain the oligomeric forms of the enzyme prevailing in the free enzyme. Thus, the stability of the immobilized enzyme is better than that of the free enzyme. 3.4.2. Effect of the pH Variation on CA Immobilization. The stability of immobilized CA at various pH values is shown in Figure 11. The effect of pH was restricted from pH 6203
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Figure 8. Effect of the variation of the CA concentration on immobilization.
Figure 9. Effect of the variation of the shaking speed on immobilization of CA.
enzyme are summarized in Table 2. Km and Vmax values of both enzymes were calculated from the intercepts on the x and y axes of the Lineweaver-Burk plots for the free and immobilized CA, respectively. For the free CA, Km and Vmax were 1.211 mM and 1.125 mmol min-1 mg-1, with p-NPA as the substrate. The catalytic efficiency (Kcat) of free CA was found to be 1.875 10-2 s-1. The immobilized CA showed an increase of the Km value to 4.547 mM, which may be due to mass resistance of the substrate into the immobilization medium. The increase of the Km value in the immobilized enzyme may be due to the possible change in the enzyme structure, resulting in a decrease in the binding of the substrate or lowering the accessibility of the active site to the substrate. The Vmax value decreased to 1.018 mmol min-1 mg-1 for the immobilized enzyme at 35 °C, whereas the catalytic efficiency (Kcat) was found to be 1.696 10-2 s-1.
6 to 10, because CA is not stable below pH 6.0 because of the possible denaturation of the enzyme envisaged.25 The maximum activity of the free and immobilized enzyme was observed at pH 10 and is attributed to the fact that, at pH above 8, the substrate (p-NPA) itself shows the activity.20 Therefore, the optimum pH for free and immobilized enzyme is pH 7. 3.4.3. Determination of Km and Vmax. The changes of product concentrations against time at different substrate concentrations are shown in Figure 12. The kinetic constants (Km and Vmax values) for free and immobilized enzyme were determined using Lineweaver-Burk plots, as shown in Figure 13. The Km and Vmax values for free and immobilized (25) Saman Hosseinkhani, S.; Nemat-Gorgani, M. Enzyme Microb. Technol. 2003, 33 (2-3), 179–184. (26) Hsuanyu, Y.; Benson, J. R.; Li, N. H. Am. Lab. 2007, June/July.
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Figure 10. Effect of the temperature variation on CA immobilization.
Figure 11. Effect of the pH variation on CA immobilization.
3.4.4. Storage Stability of Immobilized and Free CA. The storage stability of immobilized and free CA is shown in Figure 14. The storage stability experiments were investigated at -20 °C. From the figure, it was observed that the percentage loss of the activity in immobilized CA was 18% and the percentage loss of the activity in free CA was 25% after the 10th day, whereas the immobilized CA showed 40% and the free CA showed 55% activity loss on the 20th day. The enzyme immobilization provides higher shelf-life compared to that of the free enzyme because the covalent bonds formed between the enzyme and support enhance the conformational stability of the immobilized enzyme. From the above, we conclude that the stability of the immobilized enzyme has improved and retained its 50% initial activity during 30 days, as compared to free enzyme.
3.5. Precipitation of Calcium Carbonate from Immobilized CA. In the precipitation reaction, the time recorded for the onset of the formation of the precipitate in free CA was 35 s, the time recorded for the onset of the formation of the precipitate in immobilized enzyme was 42 s, and the time recorded for the onset of the formation of the precipitate in blank without CA (only material) was 100 s, as shown in Table 3. The time taken for precipitation in the blank without CA (only material) was 100 s, which is approximately 2.5 times higher, as compared to the carbonation reaction in the presence of free and immobilized CA. The results establish the concept that immobilized CA is being instrumental in accelerating the carbonation reaction. Further studies are in progress to optimize the conditions for the carbonation reaction, elucidating the kinetics and mechanistic aspects. Table 4 shows the FTIR peaks obtained for precipated 6205
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Figure 12. Time profiles of the catalytic reaction of the enzyme at different substrate concentrations: (A) free CA and (B) immobilized CA.
Table 2. Kinetic Parameters of Free and Immobilized Enzyme enzyme free CA immobilized CA
Vmax (mmol min-1 mg-1) Km (mM) 1.125 1.018
1.211 4.547
Kcat (s-1) 1.875 10-2 1.696 10-2
immobilized and free CA were observed at 712 and 874 cm-1, which coincided with the spectra of standard CaCO3. Figure 15 shows the SEM image of the well-defined faceted rhombohedral, which is characteristic of calcite crystals of CaCO3, obtained from immobilized SMSC. 3.6. Evaluation of Precipated Calcium Carbonate of Immobilized and Free CA. The carbonated precipitates were quantified by evolution of carbon dioxide after the carbonation reaction using GC. The CO2 sequestration capacity of immobilized CA was 10.73 mg of CaCO3/mg of CA, as compared to 14.92 mg of CaCO3/mg of CA for free CA, under a limiting concentration of CO2 (14.5 mg of CO2/10 mL).
Figure 13. Lineweaver-Burk plots for estimation of Km and Vmax: (A) free CA and (B) immobilized CA.
carbonate, which is compared to standard CaCO3. The prominent two peaks of precipated carbonate obtained from 6206
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Figure 14. Storage stabilities of free and immobilized CA.
It was concluded that the immobilized CA could be used to accelerate the hydration of CO2 in biomimetic CO2 sequestration in an aqueous solution. 4. Conclusions The use of SMSC has been successfully applied to enzyme immobilization, which is further being used for sequestration of carbon dioxide. The surfactant (HDTMABr) treatment on material provides the interaction of the positively charged HDTMA ion, which coordinates with the free amino group of chitosan through the lone pair of electrons available. This, in turn, enhances the enzyme immobilization on SMSC, as compared to the chitosan beads. The immobilized enzyme shows better activity, as compared to free enzyme with respect to the pH and temperature. Kinetic parameters of immobilized and free CA (Km and Vmax values) were also evaluated from the Lineweaver-Burk plot. For immobilized CA, the Km value was 4.547 mM and the Vmax value was 1.018 mmol min-1 mg-1, whereas for the free CA, the Km value was 1.211 mM and the Vmax value was 1.125 mmol min-1 mg-1. There is an improvement in the stability of immobilized CA, as compared to free CA. It was observed that the immobilized CA had retained 50% of its initial activity up to 30 days. It was further concluded that the immobilized CA could be used to accelerate the hydration of CO2 in biomimetic CO2 sequestration in an aqueous solution.
Figure 15. SEM images of the CaCO3 precipitate obtained from immobilized CA. Table 3. Summary of Precipitation of the Calcium Carbonate Reaction
samples
time for precipitation of CaCO3 (s)
mg of CaCO3/mg of enzyme
free CA (partially purified) immobilized CA
35 42
14.92 10.73
number 1 2
Acknowledgment. This work was carried out under the Supra Institutional Project [SIP-16 (4.2)], Council of Scientific and Industrial Research (CSIR), New Delhi, India, and the Department of Biotechnology (DBT), New Delhi, India, sponsored project. We are thankful to the Director of the National Environmental Engineering Research Institute (NEERI) for providing the research facility. We are also thankful to Dr. Peshwe, Visvesvaraya National Institute of Technology (VNIT), for characterization of materials.
Table 4. Comparison of FTIR Spectra of Free and Immobilized CA number
peak for CaCO3 (cm-1)
free CA (cm-1)
immobilized CA (cm-1)
1 2
712 874
712 874
711 870
6207
