Energy and Fuels -CA

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6198r 2010 American Chemical Society pubs.acs.org/EF

Energy Fuels 2010, 24, 6198 – 6207 : DOI:10.1021/ef100750y

Published on Web 11/01/2010

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 ) NagpurUniversity, 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 thecarbonation 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. Theeffect of various parameters, such as pH, temperature, and storage stability, on immobilized CA wasinvestigated using a p-nitrophenyl acetate ( p-NPA) assay. The optimum pH and temperature weredetermined to be 7 and 35 °C, respectively. Kinetic parameters of immobilized and free CA (K m and V max

values) were also evaluated. For immobilized CA, the K m value was 4.547 mM and the V max value was

1.018 mmol min

-1

mg

-1

,whereasforthefreeCA,theK mvaluewas 1.211mM andthe V max value was1.125mmol 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 carbonationreaction. The CO2 sequestration capacity in terms of conversion of CO2 to carbonatewas quantified by gaschromatography (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).

1. Introduction

The increase in the atmospheric level of carbon dioxide(CO2), one of the vital greenhouse gases, causes globalwarming. In the global effort to combat the predicted cata-strophe, several CO2 capture and storage technologies arebeing deliberated. One of the most promising ways is biolo-gical CO2 sequestration, in which CO2 has been sequesteredusing carbonic anhydrase (CA) by converting them intobicarbonates, which is further converted into calcium carbo-nate using calcium chloridesolution. CA is a major zinc-basedmetalloenzyme, which is ubiquitous in nature and found in theprokaryotic as well as eukaryotic domains. Each molecule of the CA isoenzyme can catalyze 1.4 Â 106 molecules of CO2 in1 s.1-3

CO2 þH2OTCA

HþþHCO3-

CA in its native form has certain limitations in its applicationbecause of the short lifetimeof the enzyme. There are different

methods to improve the catalytic stability of the enzyme, suchas enzyme immobilization, enzyme modification, geneticmodification, and medium engineering. Immobilization of the enzyme onto a solid support is currently an active areaof research because of its wide range of applications. There areseveral advantages over theuse of soluble enzyme preparations,

including easier separation of the reaction products from theincubation mixture, the abilityto recoverand reusethe enzyme,stabilization of the tertiary structure of the enzyme, and anincrease of the the enzyme stabilityand operational lifetime.4,5

The success of an immobilized enzyme mainly depends uponthe properties of the carriers employed.

Chitosan is a polysaccharide easily obtained by alkalinehydrolysis of chitin and has been used in pharmaceuticalfields, medicines, drug-delivery carriers, wound-dressing ma-terials, and tissue engineering.6 It is considered to be a suitablesupport for enzyme immobilization because it is biocompa-tible, available in various forms (gel, membrane, fiber, andfilm), 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 bymodification of the chitosan backbone to improve its activityfor immobilization of CA, which have both a hydrophilicsurface and hydrophobic area near the active site. Chang andJuang10 have reported immobilization of acid phosphatase onchitosancompositebeads andactivated clay. Chang andJuang11

have recently reported aboutR- and β-amylase immobilization

*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.

(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.



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Energy Fuels 2010, 24, 6198 – 6207 : DOI:10.1021/ef100750y Yadav et al.

on chitosan-clay composite beads. Pepsin immobilized onchitosan beads has greater thermal and storage stability thanfree pepsin, as reported by Altun and Centinus.9 Prabhu etal.12 have reported immobilization of a CA-enriched micro-organism on biopolymer-based materials. Also, some reportsare available on the immobilization of enzymes on a chitosan-based matrix, with details on activities, stabilities, and reac-tion kinetics using other enzymes.13-17 Surface modificationof chitosan has been reported using a variety of silane-

coupling agents, which have organofunctional groups withdi- or trialkoxy structures, and the reactions are performed ineither the gas or liquid phase.18 The silanol groups condensewith the surface residues to form siloxane linkages. In the caseof trialkoxysilanes, the presence of three silanol residues in thehydrolysis product can lead to multiple surface attachments.Silylated materials are further treated with surfactant-likehexadecyltrimethylammonium bromide (HDTMABr) to in-crease the surface of the carrier by forming the mesh network,which will in turn allow for more enzymes to be embedded inthis bipolar surface layer.19

In the present study, partially purified CA isolated fromBacillus pumilus TS1 was immobilized on the surfactant-modified silylated chitosan (SMSC). The influence of param-

eters, such as pH, temperature, substrate, and storage stabi-lity, on the free and immobilized CAwas studied. This study isalso aimed at validating the carbonation reaction usingpartially purified CA and further quantifying the carbonateprecipitate obtained using free and immobilized CA by gaschromatography (GC).

2. Materials and Methods

2.1. Materials. Extracellular CA from B. pumilus TS1 wasprepared by centrifuging the culture broth at 8000 rpm for 20min and concentrating the CA from the supernatant by acetone(20-60% saturation) precipitation. The precipitate was lyoph-ilized. A total of 1 g of the lyophilized powder contained 6840

units of CA. This partially purified CA was provided by theDepartment of Microbiology, University of Delhi, South Cam-pus, New Delhi, India.

Chitosan, used in this work, has a deacetylation degree (DD)of 95% with a molecular weight (MW) of 360 kDa and waspurchased from Chemchito, India Ltd., Chennai, India. 3-Ami-nopropyltriethoxysilane (APTES) was purchased from Sigma-Aldrich, St. Louis, MO. Tris buffer used in the carbonationstudy was purchased from Calbiochem, San Diego, CA. Na2H-PO4 3 2H2O, NaH2PO4 3 2H2O, CaCl2, sodium potassium tarta-rate, Na2CO3, CuSO4, glutaraldehyde (25%), folin reagent, andbovineserumalbumin (BSA) were purchasedfrom Merck,IndiaLtd., Mumbai, India. All other chemicals and reagents usedwere of analytical grade.

Spectrophotometric measurements were carried out with aPerkin-Elmer Lambda 650S UV/vis spectrophotometer. pHmeasurements were carried out with a Eutech pH-meter(model pH 1500). Centrifugation was made using a BeckmanOptima XL-100K model. For immobilization, an orbital shak-ing incubator (model RIS.24BL) by Remi Instruments Ltd. wasused, and the mixing procedure was made using a vortex(VX-200). X-ray diffraction (XRD) patterns of material wasrecorded 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 aJEOL JED-2300 scanning electron microscope equipped withan energy-dispersive X-ray (EDX) analyzer. The calcium car-bonate precipitate was evaluated using GC (model Perkin-Elmer 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 acidand stirred for 1 h to obtained viscous chitosan solution, whichwas then centrifuged, and the insoluble fraction was discarded.To the supernatant, 1 mL of APTES was added and stirred for1 h. The silylated chitosan solution was subjected to treatmentwith 0.25% HDTMABr and stirred for 1 h. Thus, the sampleobtained 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. Thewashed sample was subjected to drying at 110 °C in an oven for6 h. The sample was designated as SMSC.

2.2.2. Immobilization Procedure and Enzyme Assay. The im-mobilization procedure that follows has been reported in ourprevious study.12 The enzyme activity of CA was estimatedspectrophotometrically using p-nitrophenyl acetate ( p-NPA) asa substrate according to the method described by Armstronget al.,20 with slight modification. The assay system contained0.2 mL of enzyme solution (1 mg/mL) in a 1 cm spectrophoto-metric cell, containing 1.8 mL of phosphate buffer (0.1 M, pH7.0) and 1 mL of 3 mM p-NPA. The change in absorbance at348 nm at 25 °C was recorded over the first 5 min, before andafter adding immobilized enzyme. The enzyme activity wascalculated by the following formula:

enzyme activity ðU=mLÞ

¼ðΔA 348 nm=min test-ΔA 348 nm=min blankÞ Â 1000 Â TVÂ DF

millimolar extinction coefficient of p-nitrophenyl acetateÂ V

where 1000 is the conversion to micromoles, TV is the totalvolume of the reaction mixture, V is the volume of the enzymesolution taken, and DF is the dilution factor.

A total of 1 unit of CA is defined as the amount of enzymerequired 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 slightmodification, as shown in Figure1. Thetime requiredfor the onset of precipitate and carbonate formation was

monitored in the sample as well as control (without CA). Thecarbonate 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 methodcoupled with a thermal conductivity detector (TCD). Thiseliminated the interference of other precipitates, such as calciumphosphate. The precipitate obtained was treated with 0.5 MHCl, and evolution of CO2 was monitored using GC. Theevolved gas was collected in the collector and then analyzed inGC/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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2.3. Characterization of Materials. XRD of the SMSC wasobtained using a (PANalytical) X-ray diffractometer, with CuKR radiation ( λ = 1.540 60 A ˚ ) at 45 kV and 40 mA and scannedover the range of diffraction angle 2θ = 10-80°, and the stepsize for the XRD measurement is 2θ = 0.0170. The SEM imageof SMSC and CA-immobilized SMSC was obtained using aJEOL scanning electron microscope equipped with an EDXanalyzer. FTIR spectra of SMSC (1 wt %) mixed with KBrpellets were recorded by a diffused reflectance accessory tech-nique. Spectra of the chitosan flask and SMSC were scanned inthe range of 400-4000 cm-1. The resolution is 2 cm-1, and thescan 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 activityof free and immobilized CA was studied by incubating the

reaction mixtures, followed during the immobilization proce-dure, at different temperatures (15, 25, 35, 45, and 55 °C). Theeffect of pH on the activity of free and immobilized CA wasinvestigated 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 K m and V max values of free and immobilizedenzyme was carried out by measuring the activity of CA in thepresence of various p-NPA (substrate) concentrations (1, 2, 3, 4,and 5 mM). Michaelis-Menten constant (K m) values and themaximum velocities (V max) were determined using theLineweaver-Burk double-reciprocal plot,22 in which the reciprocals of the

initial velocities of the CA activity were plotted against thereciprocals of the concentration of p-NPA used.

2.4.3. Storage Stability of CA. For storage stability, studysamples were incubated with 3 mg/5 mL of enzyme (free andimmobilized) 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 wasdetermined after intervals of every 5 days.

Figure 1. Protocol for mineralization of CO2.

Figure 2. Comparison between partially purified CA activities afterimmobilization on different materials.

Table 1. Comparison to CA Immobilized on Different Matrices

chitosan beads silylated chitosan SMSC CN-Cavilinka NH2- Cavilinka

material (mg) 10 10 10 111 111enzyme used for immobilization (mg) 0.4 0.4 0.4 8.6a 8.6a

percentage of enzyme immobilized (%) 58.33 62.50 72.91 >99 82capacity (immobilized) (U/mg beads) 2.8 3 3.5 1.7 1.6

a From Hsuanyu et al.26

Figure 3. XRD spectra of (A)chitosan flask and(B) SMSC material.

(22) Lineweaver, H.; Burk, D. J. Am. Chem. Soc. 1934, 56, 658 – 660.



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3. Results and Discussion

3.1. Screening of Materials for Immobilization of Partially

Purified CA. Figure 2 and Table 1 show the comparisonbetween different materials, viz. SMSC, silylated chitosan,and chitosan beads, for immobilization of partially purifiedCA. Immobilized catalyst had been tested for its activityusing a p-NPA assay. However, almost all materials showedreasonably good activity for enzyme immobilization, withthehighest activity observed forSMSC material,followedbysilylated chitosan and chitosan beads. Also, the leaching of CA from SMSC is lower, as compared to the other twomaterials.

The mechanism of formation of the chitosan-organosilanehybrid is very well-illustrated by Airoldi and Monteiro18 forimmobilization of the enzyme, which is very well-substan-tiated in our work as well (Figure 2). However, in ourstudy, the material is being modified with surfactant, whichfurther enhances its amphiphillic property, showing higher

immobilization of the enzyme (Figure 2). The question of steric hindrancedose notarisebecause it is an open structure,wherein one HDTMABr ion is coordinating per monomer.CA contains 18 positively charged arginines and 29 nega-tively charged residues (glutamic and aspartic acids). Histi-

dine, with an average pK a of 6.5 is largely neutral at pH 8.The net chargeof BCA is about-3, as measured by capillaryelectrophoresis.23 Therefore, enhanced adsorption of CA ona positively charged surface because of HDTMA ion andprotonated amines has been observed. This may be attrib-uted to the positively charged HDTMA ion coordinatingwith the free amino group of chitosan through the lone pairof electrons available. This, in turn, enhances entrapment of the enzyme in the bipolar surface layer, by increasing theelectrostatic interaction between charged enzyme surfaces

Figure 4. Comparison of SEM images of (A) SMSC and (B) immobilized SMSC and (C) EDX spectra of immobilized SMSC.

(23) Gitlin, I.; Gudiksen, K. L.; Whitesides, G. M. ChemBioChem2006, 7 , 1241 – 1250.



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and surfactant ions attached to chitosan through aminogroups. Evidence of a positive charge of the SMSC material

is calculated using point of zero charge(IPE)of material.Theisoelectric point (IEP) of material is pH 5. For the pH valuesabove the point of zero charge of material, the predominatesurface species is M-O-, while at the pH value below thepoint of zero charge of material, M-OH2

þ species predom-inate. SMSC has pH of 4.5, which is below the point of zerocharge of material. SMSC is therefore having a positivecharge on the surface.24 The mechanism for formation of SMSC is illustrated below

Considering the leaching property along with enhancedenzyme interaction sites, SMSC material had been selectedfor further studies.

3.2. Characterization of Materials. 3.2.1. XRD Analysis of SMSC. The XRD patterns of bare chitosan and SMSC areshown in panels A and B of Figure 3. The XRD pattern of bare chitosan exhibited its characteristic crystalline peaks at2θ = 10.2° and 19°. However, these characteristic crystallinepeaks of bare chitosan were diminished in XRD analysis of SMSC because of the bondingof the silanol groupof silylatedchitosan with bare chitosan chains, leading to a decrease incrystallinity. The XRD analysis is not showing sharp peaksof silica nuclei by virtue of its amorphous nature.

3.2.2. SEM Analysis and EDX Spectra of SMSC. Surfacemorphology of bare and immobilized SMSC had beenstudied

using SEM. Panels A and B of Figure 4 show a comparisonbetween bare and immobilized SMSC. Figure 4A of bareSMSC shows a uniform surface morphology with smallprotruding structures, whereas Figure 4B of the immobilizedSMSC shows smooth surface morphology, which may bedue to the adsorption of CA on the surface of SMSC. Thepresence of CA has been further confirmed by EDX spectra(Figure 4C), which shows Zn on the surface. The CA enzymecontains 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 impor-tant for adsorption. The FTIR spectra of chitosan flakes andSMSC are given in panels A and B of Figure 5. The band at3695 cm-1 in chitosan is attributed to the stretching vibra-tion of the N-H group, which is shifted to 3647 cm-1 inSMSC. This shiftingof the band may be due to the formationof weak intermolecular hydrogen bonding between the ami-no and hydroxyl groups of chitosan with the incorporationof the silanol group. The bands at 2924 and 1376 cm-1 inchitosan and 2918 and 2850 cm-1 in SMSC are attributed tothe C-H stretching vibration in thepolymeric backbone andC-H bending, respectively. This significant decrease in the

band intensity is due to the interaction between chitosan andthesilanol group of SMSC. Thebandsat 1155 and1075cm-1

in chitosan may be due to the stretching vibration of C-Ogroups, whereas SMSC shows bands at 1175 and 1058 cm-1

attributed to Si-O-Si stretching. IR spectra of SMSC alsocontain bands at 1340 and 799 cm-1, which are character-istics of Si-CH3.

3.3. Optimization Study for CA Immobilization. Studieswere carried out for optimizing the conditions for immobi-lization of CA. The conditions studied are being discussed inthe following sections.

3.3.1. Effect of the Time Variation on Immobilization of

CA. The effect of the time on immobilization of CA is shownin Figure 6. The amount of enzyme adsorbed onto the

material increased with an increasing contact time up to8 h. Subsequently, an increase of the contact time resulted ina decrease of the activity of the immobilized enzyme or anincrease of the activity in the supernatant of the immobilizedenzyme. The activity of the immobilized enzyme can becalculated by the difference in the free enzyme activity andthe activity in the supernatant of the immobilized enzyme.This decrease in the activity of immobilized CA beyond 8 h isbeing attributed to the leaching of the enzyme or denatura-tion of the enzyme.

3.3.2. Effect of the Material Dose Variation on Immobiliza-

tion of CA. The material dose varied between 1 and 10 mg/5mL. The optimal doseappearedto be 4 mg/5mL, asshown inFigure 7. A further increase in the dose resulted in decreasedenzyme loading, probably because of a lower concentrationof enzyme and higher number of active sites on the materialas 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 to5 mg/5 mL, and the optimal enzyme concentration ap-peared to be 3 mg/5 mL, as shown in Figure 8. However,a further increase of the enzyme loading above 3 mg/5 mLresulted in the gradual decrease in the enzyme activity ontothe material. This is probably due to optimal adsorption of the enzyme on the matrix surface at 3 mg/5 mL. Mansourand Dawoud17 suggest that the gradual decrease in theenzyme activity is probably due to hindrance between the

Figure 5. FTIR spectra of (A) chitosan flask and (B) SMSCmaterial.

(24) Balistrieri, L. S.; Murray, J. W. Am.J. Sci. 1981, 281 (6), 788 – 806.



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adsorbed enzyme molecules on the matrix, at higher con-centrations of enzyme.

3.3.4. Effect of the Variation of the Shaking Speed on

Immobilization of CA. The effect of the shaking speed onimmobilization of theenzyme on SMSC is shown in Figure 9,wherein the shaking speed varied from 60 to 160 rpm. Theoptimal shaking speed is 120 rpm. A further increase inshaking speed ultimately lowered the activity of immobilizedenzymeprobablybecause of theweakening of theinteractionbetween CA and SMSC at high speed.

Thus, a summary from the above studies is that theoptimal conditions for enzyme immobilization are as fol-lows: (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 activityis shown in Figure 10. The activity of immobilized CAincreased up to 35 °C and thereafter decreased with a furtherincrease in the temperature. A similar trend was observed forfree CA. However, the decline was more pronounced forfree CA. This may happen because of the denaturation of the enzyme at higher temperatures. The optimum tempera-ture for the free enzymeis 25°C, ascompared to35 °C for theimmobilized enzyme. The immobilized enzyme appears to bemore thermostable than the free enzyme. The immobiliza-tion procedure probably helps to maintain the oligomericforms of the enzyme prevailing in the free enzyme. Thus, thestability of the immobilized enzyme is better than that of thefree enzyme.

3.4.2. Effect of the pH Variation on CA Immobilization.

The stability of immobilized CA at various pH values isshown in Figure 11. The effect of pH was restricted from pH

Figure 6. Effect of the time variation on immobilization of CA.

Figure 7. Effect of the material dose variation on immobilization of CA.



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6 to 10, because CA is not stable below pH 6.0 because of thepossible denaturation of the enzyme envisaged.25 The max-imum activity of the free and immobilized enzyme was

observed at pH 10 and is attributed to the fact that, at pHabove 8, the substrate ( p-NPA) itself shows the activity.20

Therefore, theoptimum pH forfree andimmobilizedenzymeis pH 7.

3.4.3. Determination of K m and V max. The changes of product concentrations against time at different substrateconcentrations areshown in Figure 12. The kinetic constants(K m and V max values) for free and immobilized enzyme weredetermined using Lineweaver-Burk plots, as shown inFigure 13. The K m and V max values for free and immobilized

enzyme are summarized in Table 2. K m and V max 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, K m and V max

were 1.211 mM and 1.125 mmol min-1 mg-1, with p-NPA asthe substrate. The catalytic efficiency (K cat) of free CA was

found to be 1.875 Â 10-2 s-1. The immobilized CA showed

an increase of the K m value to 4.547 mM, which may be dueto mass resistance of the substrate into the immobilization

medium. The increase of the K m value in the immobilizedenzyme 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 tothe substrate. The V maxvaluedecreased to 1.018 mmol min-1

mg-1 for the immobilized enzyme at 35 °C, whereas thecatalytic efficiency (K cat) was found to be 1.696 Â 10-2 s-1.

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.

(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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3.4.4. Storage Stability of Immobilized and Free CA. Thestorage stability of immobilized and free CA is shown inFigure 14. The storage stability experiments were investi-gated at -20 °C. From the figure, it was observed that thepercentage loss of the activity in immobilized CA was18% and the percentage loss of the activity in free CA was25% after the 10th day, whereas the immobilized CAshowed 40% and the free CA showed 55% activity losson the 20th day. The enzyme immobilization provideshigher shelf-life compared to that of the free enzymebecause the covalent bonds formed between the enzymeand support enhance the conformational stability of theimmobilized enzyme. From the above, we conclude thatthe stability of the immobilized enzyme has improved andretained its 50% initial activity during 30 days, as com-pared to free enzyme.

3.5. Precipitation of Calcium Carbonate from Immobilized

CA. In the precipitation reaction, the time recorded for theonset of the formation of the precipitate in free CA was 35 s,thetime recorded forthe onset of theformation of theprecip-itate in immobilized enzyme was 42 s, and the time recordedfor the onset of the formation of the precipitate in blankwithout 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 timeshigher, as compared to the carbonation reaction in thepresence of free and immobilized CA. The results establishthe concept that immobilized CA is being instrumental inaccelerating the carbonation reaction. Further studies are inprogress to optimize the conditions for the carbonationreaction, elucidating the kinetics and mechanistic aspects.Table 4 shows the FTIR peaks obtained for precipated

Figure 10. Effect of the temperature variation on CA immobilization.

Figure 11. Effect of the pH variation on CA immobilization.



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carbonate, which is compared to standard CaCO3. Theprominent two peaks of precipated carbonate obtained from

immobilized andfree CAwere observed at 712 and874 cm-1,which coincided with the spectra of standard CaCO3. Figure 15showsthe SEMimage of thewell-defined facetedrhombohedral,which is characteristic of calcite crystals of CaCO3, obtainedfrom immobilized SMSC.

3.6. Evaluation of Precipated Calcium Carbonate of Im-

mobilized and Free CA. The carbonated precipitates werequantified by evolution of carbon dioxide after the carbon-ation reaction using GC. The CO2 sequestration capacity of immobilized CA was 10.73 mg of CaCO3/mg of CA, ascompared to 14.92 mg of CaCO3/mg of CA for free CA,under a limiting concentration of CO2 (14.5 mgof CO2/10mL).

Figure 12. Time profiles of the catalytic reaction of the enzyme at different substrate concentrations: (A) free CA and (B) immobilized CA.

Figure 13. Lineweaver-Burk plots for estimation of K m and V max:(A) free CA and (B) immobilized CA.

Table 2. Kinetic Parameters of Free and Immobilized Enzyme

enzyme V max (mmol min-1 mg-1) K m (mM) K cat (s-1)

free CA 1.125 1.211 1.875Â10-2

immobilized CA 1.018 4.547 1.696Â10-2



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It was concluded that the immobilized CA could be used toaccelerate the hydration of CO2 in biomimetic CO2 seques-tration in an aqueous solution.

4. Conclusions

The use of SMSC has been successfully applied to enzymeimmobilization, which is further being used for sequestrationof carbon dioxide. The surfactant (HDTMABr) treatment onmaterial provides the interaction of the positively chargedHDTMA ion, which coordinates withthe free amino group of chitosan through the lone pair of electrons available. This, inturn, enhances the enzyme immobilization on SMSC, ascompared to the chitosan beads. The immobilized enzymeshows better activity, as compared to free enzyme with respectto the pH and temperature. Kinetic parameters of immobi-lized and free CA (K m and V max values) were also evaluatedfrom the Lineweaver-Burk plot. For immobilized CA, theK m value was 4.547 mM and the V max value was 1.018 mmolmin-1 mg-1, whereas for the free CA, the K m value was 1.211mMandthe V maxvalue was 1.125 mmol min-1 mg-1. There isan improvement in the stability of immobilized CA, ascompared to free CA. It was observed that the immobilizedCAhad retained 50% of itsinitial activity up to 30days. It wasfurther concluded that the immobilized CA could be used toaccelerate the hydration of CO2 in biomimetic CO2 sequestra-tion in an aqueous solution.

Acknowledgment. This work was carried out under the SupraInstitutional Project [SIP-16 (4.2)], Council of Scientific andIndustrial Research (CSIR), New Delhi, India, and the Depart-ment of Biotechnology (DBT), New Delhi, India, sponsoredproject. We are thankful to the Director of the National Envi-ronmental Engineering Research Institute (NEERI) for provid-ing the research facility. We are also thankful to Dr. Peshwe,Visvesvaraya National Institute of Technology (VNIT), forcharacterization of materials.

Figure 14. Storage stabilities of free and immobilized CA.

Table 3. Summary of Precipitation of the Calcium CarbonateReaction

number samples

time forprecipitationof CaCO3 (s)

mg of CaCO3/mgof enzyme

1 free CA (partially purified) 35 14.922 immobilized CA 42 10.73

Figure 15. SEM images of the CaCO3 precipitate obtained fromimmobilized CA.

Table 4. Comparison of FTIR Spectra of Free and Immobilized CA

numberpeak for

CaCO3 (cm-1)free CA(cm-1)

immobilizedCA (cm-1)

1 712 712 7112 874 874 870

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Energy and Fuels -CA