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Process Biochemistry 46 (2011) 1010–1018

Contents lists available at ScienceDirect

Process Biochemistry

journa l homepage: www.e lsev ier .com/ locate /procbio

mmobilization of carbonic anhydrase on chitosan beads for enhancedarbonation reaction

nehal Wanjari a, Chandan Prabhua, Renu Yadava, T. Satyanarayanab, Nitin Labhsetwara,adhana Rayalua,∗

Environmental Material Division, National Environmental Engineering Research Institute (NEERI), CSIR, Nehru Marg, Nagpur 440 020, Maharashtra, IndiaDepartment of Microbiology, University of Delhi, South Campus, New Delhi 110 021, India

r t i c l e i n f o

rticle history:eceived 23 September 2010eceived in revised form 13 January 2011ccepted 17 January 2011

a b s t r a c t

Carbonic anhydrase (CA) has been immobilized on chitosan beads and has been tested for its activityusing p-NPA. CA immobilized chitosan beads were further tested for targeted application of carbonationreaction to convert CO2 to CaCO3. It was observed that onset time for precipitation of CaCO3 was 40 sfor immobilized catalyst as compared to non-catalyzed system (100 s). Mechanistic and kinetic aspects

eywords:hitosan beadsarbonic anhydrase

mmobilizationarbonation reactioninetics

have been addressed for immobilized catalyst as well as free CA using p-NPA assay. Km and Vmax of theimmobilized CA was observed to be 2.36 mM and 0.54 �moles/min/ml as compared to Km and Vmax of0.87 mM and 0.93 �moles/min, respectively, for free CA. Storage stability test was conducted up to 20 daysand it was observed that at −20 ◦C, the Half Life Period (HLP) of immobilized CA was 216 h as comparedto 192 h for free CA whereas at 25 ◦C the HLP for immobilized and free CA was observed to be 456 h and408 h, respectively.

tability

. Introduction

Increasing atmospheric concentration of CO2 is resulting inlobal warming and climate change. The emission reduction of CO2an be achieved by: (i) improved energy efficiency (ii) low car-on fuel and (iii) carbon capture and sequestration. Extensive R&Dfforts are now underway to develop new approaches to capturend sequester CO2 to avoid its release in the atmosphere includingineral carbonation, oceanic sequestration, geological sequestra-

ion, etc. The resulting sequestration system would offer severalotential advantages, including avoidance of cost intensive CO2oncentration and transportation steps, an environmental friendlyrocess, performed in aqueous solution at near ambient tempera-ures, a site specific solution to CO2 sequestration.

Biomimetic carbonation reaction is carried out by using CA, isne such approach which is being studied extensively [1–6]. Car-onic anhydrase (CA) is an enzyme with potential as a tool to

equester carbon dioxide from emission sources. Carbonic anhy-rase is a common enzyme found in animals, plants and bacteria.his enzyme catalyzes the conversion of carbon dioxide and waternto bicarbonate, as shown in Fig. 1. The turnover for the CA is one

∗ Corresponding author. Tel.: +91 712 2247828; fax: +91 712 2247828.E-mail address: s rayalu@neeri.res.in (S. Rayalu).

359-5113/$ – see front matter © 2011 Elsevier Ltd. All rights reserved.oi:10.1016/j.procbio.2011.01.023

© 2011 Elsevier Ltd. All rights reserved.

million reactions per second, depending on the type of carbonicanhydrase [2–5].

The mechanism for CO2 hydration, catalyzed by CA, can be cat-egorized into four steps as follows [6].

CO2 (gaseous) → CO2 (aqueous) (1)

CO2 (aqueous) + H2O → H2CO3 (2)

H2CO3 → H+ + HCO−3 (3)

HCO−3 → H+ + CO2−

3 (4)

Ca2+ + CO2−3 → CaCO3 (5)

Biocatalysts have a number of advantages such as high level ofcatalytic efficiency and high degree of selectivity. However, thereare several practical problems such as high cost, limited stability,etc, which needs to be addressed. To overcome these problems,enzyme immobilization on solid support has been regarded as auseful technique to improve the thermal and operational stabilityand recovery of the immobilized enzyme [7,8].

Different procedures have been developed for enzyme immobi-

lization wherein adsorption on solid support material is the easiestand conventional method for enzyme immobilization. The impor-tant advantage of this method is that the stability of enzyme isretained after immobilization and it can be reused for several cycles[9]. In this connection several materials are being investigated. Chi-

S. Wanjari et al. / Process Biochem

tea

athbp

cdbtc

ca[ctcc

Fig. 1. Mechanism of CO2 hydration catalyze by CA.

osan is one such material which is being extensively studied fornzyme immobilization by virtue of its non-toxicity and advantagess physical and chemical properties.

Chitosan is a linear polyglucosamine of high molecular weightnd is obtained by N-deacetylation of chitin in a strong alkali solu-ion. It has reactive amino (−NH2) and hydroxyl (−OH) groups andas been identified as an ideal support material for enzyme immo-ilization. It is also inexpensive and exhibits high affinity towardsrotein [10].

The positive charge of chitosan reacts with the negativelyharged surface of enzyme thus form the physical adsorption (Van-er Waals attraction and hydrogen bonding) and ionic interactionetween the positively charged surface of the material and nega-ive charge on the enzyme. The mechanism for adsorption of CA onhitosan support is shown in Fig. 2.

There are reports available on immobilization of enzymes onhitosan-based materials, which deals with activities, stabilitiesnd reaction kinetics using different enzymes [10–15]. Chang et al.16] reported immobilization of acid phosphatase on chitosan

omposite beads and activated clay. Chang et al. also reportedhe immobilization of �-amylase and �-amylase on chitosan–clayomposite beads to improve the stability of immobilized enzyme asompared to free enzyme [17]. Altun et al. reported greater thermal

Fig. 2. Bonding between chitosan and enzyme.

istry 46 (2011) 1010–1018 1011

and storage stability in immobilized enzyme on chitosan beads ascompared to free enzyme [18]. Rayalu and coworkers have reportedimmobilization of carbonic anhydrase enriched microorganism ondifferent chitosan based materials [19].

To the best of our knowledge, detailed studies on immobilizationof carbonic anhydrase on chitosan beads have not been reportedso far. In this paper, partially purified CA from Bacillus pumiluswas immobilized on chitosan beads and detailed kinetic parame-ters like, pH, temperature, substrate reuse and storage stability onthe free and immobilized CA has been investigated. Also gas chro-matography technique has been used for quantification of calciumcarbonate.

2. Materials and methods

2.1. Materials

Chitosan with deacetylation degree (DD) of 95% and molecular weight (MW) of360 kDa was purchased from chemchito India Limited, Chennai. All the chemicalsused in this study were of analytical grade and purchased from E-Merck, India Ltd.Tris buffer used in the carbonation study has been purchased from Calbiochem, U.S.The partially purified CA from B. Pumilus was provided by Department of Micro-biology, University of Delhi South campus, New Delhi. One gram of lyophilizedpowder has 6840 units of CA. One unit of enzyme activity was expressed as 1 �molp-nitrophenol released per minute at room temperature.

2.2. Experimental

2.2.1. Preparation of chitosan beadsChitosan solution was prepared by dissolving 3 g of chitosan flakes in 100 ml

of 5% acetic acid. It was stirred for 1 h to obtain a viscous solution of chitosan. Thisviscous chitosan solution was added drop wise into 250 ml of 3.2 M NH4OH solutionwith continuous stirring to form chitosan beads. The beads were allowed to stabilizein NH4OH solution for 1 h at room temperature. The chitosan beads were filteredand rinsed with distilled water for several times till the pH of the supernatant was7.0. The beads were finally dried in an oven at 60 ◦C for 6 h.

2.2.2. Preparation of cross linked chitosan beadsChitosan flakes (3 g) were dissolved in 1 M acetic acid solution and stirred for

an hour. This viscous chitosan solution was added drop wise to 1 M NH4OH solutionwith constant stirring. The solution containing beads were stirred for 30 min at roomtemperature and it was filtered. Wet beads were treated with 10% glutaraldehydesolution for 24 h. The CA treated beads were filtered, washed with distilled waterand dried in an oven at 60 ◦C for 16 h.

2.2.3. Characterization of materialsXRD of the chitosan based material was obtained by using X-ray diffractometer

(Model no. TW 3660/50), with Cu K� radiation (� = 1.54060 A) at 45 kV and 40 mAand scanned in the range of 10–80◦ . Scanning electron microscopy (SEM) imagesof chitosan beads as such, CA immobilized chitosan beads and precipitate of cal-cium carbonate obtained from CA immobilized chitosan beads was taken using aJEOL JED-2300 scanning electron microscope equipped with an Energy DispersiveX-ray (EDX) analyzer. FTIR spectra of chitosan beads (1 wt%) mixed with KBr pelletswere recorded on a Bruker Vertex-70 apparatus by diffused reflectance accessorytechnique. Spectra of the materials were scanned in the range of 400–4000 cm−1.Portable turbidimeter EUTECH (Code no. TN-100/T-100) assess the solution turbid-ity of calcium carbonate. Accuracy: ±2% of reading ±1LSD for 0–500NTU, ±3% ofreading ±1LSD for 501–1000 NTU.

2.2.4. Immobilization procedure and enzyme assay10 mg material was added to 4.8 ml of phosphate buffer (0.1 M pH 7.0) and 0.2 ml

(5 mg/ml) of enzyme solution, in a tube and incubated for 6 h at 25 ◦C. After incu-bation, the sample was centrifuged and CA immobilized enzyme was collected andused for carbonation reaction. The esterase activity of CA in the supernatant was esti-mated spectrophotometerically at 348 nm by measuring the colour intensity due top-NP [19] and protein concentration was determined by Lowry et al. method [20].

2.2.4.1. p-NPA assay. The assay mixture consisting of 1.8 ml phosphate buffer(0.1 M, pH 7.0) and 0.2 ml of enzyme solution (5 mg/ml) or 2 ml sample (super-natant) and 1 ml of 3 mM paranitro phenyl acetate for its conversion to para nitrophenol [19,21]. All the experiments like screening, kinetic parameters and carbon-

ate precipitation were repeated twice for better accuracy and blank experimentswere also performed throughout the studies.

2.2.4.2. Wilbur–Anderson assay. Wilbur–Anderson assay [22] was performed in avessel maintained at 4 ◦C with water-jacket and constant-temperature circulatorby using crushed ice. The vessel was sealed with a rubber-stopper fitted with a pH

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012 S. Wanjari et al. / Process Bi

lectrode. A volume, 50 �l sample was added to 3 ml of 20 mM Tris buffer solutionf pH 8.3. The reaction was started by addition of 2 ml of water saturated with CO2

t about 4 ◦C. CO2 hydration activity of CA was indicated by the time required forhe pH to change from 8.3 to 6.3.

.2.5. Protocol for mineralization of CO2

Carbonation study was carried out by the method reported by Favre et al. withlight modification [5]. In a typical procedure, 1 ml of Tris buffer (1 M, pH 8.0) wasdded to 10 ml of CO2 saturated water (pH 3.57). The mixture was shaken at 25 ◦Cnd then 10 ml of 2% CaCl2 (pH 6.41) was added along with 1 ml (5 mg/ml loading)f the enzyme in phosphate buffer (0.1 M, pH 7.0). The final pH of the mixture was.85. The time required for formation of carbonate with respect to onset of reactionas monitored in the sample as well as control (without enzyme) by turbidometricethod. The precipitate was filtered using Whatmann filter paper-42 and dried at

oom temperature.

.2.6. Estimation of calcium carbonateQuantification of precipitate calcium carbonate was carried by using gas chro-

atographic (GC) method coupled with thermal conductivity detector (TCD). Inhis method, the carbonate precipitate was treated with 0.5 M HCl and evolutionf CO2 was monitored using GC. The reactor used for quantifying CO2 is presentedn Fig. 1 (supplementary information). The reaction was carried out in borosilicatelass reactor in which carbonate precipitate was taken and 0.5 M HCl was added.he evolved gas was collected in the collector and then analyzed in GC/TCD usingorapak Q column.

.3. Kinetic studies

Parameters including incubation time, material dose, enzyme concentration andhaking speed have been varied within feasible parameter ranges to obtain optimalonditions for immobilization of enzyme.

.3.1. Effect of temperature and pHThe stability of the immobilized and free enzyme at different temperature and

H were tested by measuring the activities of the enzymes by incubating for 8 h atifferent temperatures (25–55 ◦C) and solution pH (5.4–9.4).

.3.2. Determination of kinetic constantsFor determining rate and kinetic constants of free and immobilized enzyme,

mg/ml of enzyme loading was incubated in 4.8 ml phosphate buffer (0.1 M, pH.0) at temperature of 25 ◦C. The assay of immobilized and free enzyme was carriedut using different concentration of p-nitrophenyl acetate (1, 2, 3, 4 and 5 mM). Km

nd Vmax of free and immobilized enzyme were calculated from Lineweaver–Burklots.

.3.3. Storage stabilityStability of free and immobilized enzyme was measured by determining the

esidual activity on storing at different period up to 20 days. Samples were incubatedith 1 mg/ml of enzyme in 4.8 ml phosphate buffer (0.1 M, pH 7.0) at −20 ◦C and

5 ◦C. Enzyme activity was assayed at an interval of 5 days.

.3.4. Leaching of immobilized enzymeThe CA immobilized chitosan beads were used for leaching studies by monitor-

ng the protein concentration in the leachate. Subsequently to the leaching of theA immobilized chitosan beads were tested for carbonation reaction and amountf calcium carbonate formed was quantified. In the protocol for leaching, 20 mg ofA immobilized chitosan beads were mixed with 4 ml of 0.1 M phosphate bufferpH 7.0) and it was shaken at room temperature (25 ◦C). The supernatant was col-ected and tested for protein concentration by using Lowry et al. [22] as well as for-NPA assay and the CA immobilized chitosan beads was washed and tested forarbonation reaction (Section 2.2.5). This study was repeated up to 5th cycle.

. Results and discussion

.1. Point of zero charge of chitosan beads

The surface charge on chitosan beads has been elucidated on theasis of point of zero charge (IPE) of material. The isoelectric point

IEP) of chitosan beads determined by Balistrieri and Murray [23]s at pH 5.0. The pH of the solution obtained by stirring chitosaneads for 24 h is pH 4.96. As the pH value below the point of zeroharge of material, M-OH2

+ species predominate and the chitosaneads have a positive charge on the surface.

istry 46 (2011) 1010–1018

3.2. Characterization of materials

3.2.1. Shape and sizeThe chitosan beads formed are spherical in shape with approx-

imate particle diameter of 1.1–1.7 mm [19].

3.2.2. XRD analysisTwo major peaks at 2� of 10 ◦ and 20 ◦ have been observed in X-

ray diffraction patterns of chitosan flakes and chitosan beads (Fig. 3)indicating that the degree of crystallinity decreases in chitosanbeads as compared to chitosan flakes.

3.2.3. SEM analysis and EDX spectraThe surface morphology of chitosan and CA immobilized on chi-

tosan beads has been studied using SEM. CA enzyme contains Znatom, which can be used as a marker for confirming the presenceof enzyme on beads. Fig. 4 shows a comparison between bare chi-tosan beads and beads with enzyme. SEM image Fig. 4[A] of chitosanbeads as such shows a spongy surface morphology. The surfacemorphology of chitosan beads with immobilized enzyme is differ-ent and appears to be scaly in nature (Fig. 4[B]). The presence of Znatom on the surface of immobilized beads is confirmed by the EDXspectrum (Fig. 5).

3.2.4. FTIR analysisChitosan is a heteropolymer made up of glucosamine and acetyl

glucosamine units. The functional groups of chitosan are amino andhydroxyl groups which are very important for immobilization ofenzyme. The FTIR spectra of chitosan flakes and beads are givenin Fig. 2 (supplementary information). The band at 3694 cm−1 inchitosan flakes is attributed to stretching vibration of N–H group,which shifted to 3653 cm−1 in chitosan beads. The hydroxyl groupin chitosan flakes detected at 3298 cm−1 is shifted to 3308 cm−1

in chitosan beads. This shift of band may be due to the forma-tion of weak intermolecular hydrogen bonding between aminoand hydroxyl groups of chitosan. As reported by Paulino et al.[24], in chitosan beads and flakes, the band at 1575 cm−1 has alarger intensity than at 1676 cm−1, suggesting effective deacetyla-tion. The peaks at 2918 cm−1 and 1321 cm−1 in chitosan flakes andat 2888 cm−1 and 1407 cm−1 in chitosan beads are attributed toC–H stretching vibration in polymeric backbone and C–H bendingvibration, respectively.

3.3. Immobilization studies

Studies have been carried out for optimizing conditions forimmobilization of enzymes and are discussed in following sections.

3.3.1. Effect of variation time on immobilization of enzymeThe effect of time on enzyme immobilization is presented in

Fig. 3 (supplementary information). The immobilization time wasvaried from 30 min to 24 h. It can be observed that the amount ofenzyme adsorbed increases up to 8 h and decreases subsequently.This decrease in activity may be attributed to leaching of enzymeor denaturation of enzyme due to stirring beyond 8 h.

3.3.2. Effect of variation of material dose on immobilization ofenzyme

The effect of material dose, on immobilization of enzyme ispresented in Fig. 4 (supplementary information). The material

dose was varied from 1 mg/5 ml to 20 mg/5 ml. The optimal doseappears to be 10 mg/5 ml. Further increase in material dose resultsin marginal decrease in loading of enzyme and may be due to lowerconcentration of enzyme and higher number of active site due toincreased dose of adsorbent.

S. Wanjari et al. / Process Biochemistry 46 (2011) 1010–1018 1013

osan fl

3e

eeotTnl

Fig. 3. XRD spectra of (A) chit

.3.3. Effect of enzyme concentration on immobilization ofnzyme

The effect of enzyme concentration on immobilization ofnzyme is presented in Fig. 5 (supplementary information). Thenzyme concentration was varied from 0.5 to 3.0 mg/ml. It wasbserved that increasing the enzyme loading above the satura-

ion level resulted in gradual decrease in the enzyme activity.his is because a high concentration of enzyme on the exter-al surface can contribute to internal diffusional restriction, thus

eading to the underestimation of the initial activity of the immo-

Fig. 4. Comparison of SEM images of [A] bare chitosan beads, [B] immobilized.

akes and (B) chitosan beads.

bilized enzyme. In the present study, the loading appears to haveincreased up to 1 mg/ml thereafter a consistent decrease has beenobserved. This observation corroborates with that reported [25]. Ithas been reported that, overloading needs to be avoided, and there-fore enzyme loading (1 mg/ml) below saturation level is selected forfurther study.

3.3.4. Effect of shaking on immobilization of enzymeThe effect of shaking speed on immobilization of enzyme on

chitosan beads is shown in Fig. 6 (supplementary information)

Chitosan beads, [C] calcite form of CaCO3 on immobilized chitosan beads.

1014 S. Wanjari et al. / Process Biochemistry 46 (2011) 1010–1018

mmobilized chitosan beads.

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600

700

800

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herein shaking speed was varied from 60 to 160 rpm. The opti-um shaking speed was observed to be 120 rpm. Further increase

n shaking speed decreases its activity, which may be due to theeaching of enzyme. This may be attributed to weakening of thenteraction between enzyme and materials at high speed.

.4. Kinetic studies

.4.1. Effect of temperature on immobilized enzymeThe effect of temperature on immobilized enzyme is presented

n Fig. 6. The activity is observed to be maximum at 25 ◦C, withonsistent decrease on further increasing the temperature up to

5 ◦C for free as well as immobilized enzyme. However, the decline

n activity was more pronounced for free enzyme as compared tommobilized enzyme. This may be due to denaturation of enzymet higher temperature.

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200

300

400

500

600

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Temperature (in degree)

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1098765300

400

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pH

Fig. 7. Effect of pH on immobilized enzyme [�].

3.4.2. Effect of pH on immobilized enzymepH is one of the most important parameter altering enzyme

activity in an aqueous medium. Immobilization is likely to result inconformational change of enzyme leading to inactivity of enzyme.The stability of immobilized enzyme at various pH is presentedin Fig. 7. Maximum activity of free and immobilized enzyme was

observed at pH 7.4 with significant decline on further increase inthe pH. It was observed that CA appears to be stable upto pH 7.4and thereafter shows decline in its activity.

Table 1Kinetic parameter of free and immobilized enzyme.

Enzyme Km (mM) Vmax (�mol/min)

Free enzyme 0.87 0.93Immobilized enzyme 2.36 0.54

S. Wanjari et al. / Process Biochemistry 46 (2011) 1010–1018 1015

Fig. 8. Lineweaver–Burk plots for estimation of Km and Vmax at 25 ◦C

Table 2Half Life Period of free and immobilized enzyme at −20 ◦C and 25 ◦C.

Half Life Period Free CA(hours) Immobilized CA(hours)

−20 ◦C 192 21625 ◦C 408 456

Table 3Comparison with CA immobilized on other reported matrices.

Chitosanbeads

CN–Cavilinka NH2–Cavilinka

Material(in mg) 10 mg 111 mg 111 mgEnzyme used for

immobilization0.4 mg 8.6 mg 8.6 mg

Percent of enzymeimmobilized

95% >99% 82%

3

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reagent blank (the material) forms the precipitate. Free enzyme

TS

S

Capacity (immobilized)(U/mg beads)

2.85 U/mg 1.7 U/mg 1.6 U/mg

a Hsuanyu et al. [24].

.4.3. Substrate studyThe kinetic constants (Km and Vmax) for free and immobi-

ized enzyme were determined by using Lineweaver–Burk plotss shown in Fig. 8. Km and Vmax values of free and immobilized

A were calculated from the intercepts on x and y axes of theineweaver–Burk plots, respectively. The Km and Vmax values forree and immobilized enzyme are summarized in Table 1. Km andmax for free enzyme is 0.87 mM and 0.93 �moles/min/ml used p-

able 4ummary of precipitation of calcium carbonate reaction.

Sr. No. Samples

1. CA (partially purified)2. Immobilized enzyme (CA–Chitosan–NH4OH beads)

D: standard deviation.

of immobilized. Enzyme [�], free enzyme [�] by p-NPA assay.

NPA as substrate. The immobilized enzyme showed an increased Km

value of 2.36 mM. Vmax value is slightly lower (0.54 �moles/min/ml)for the immobilized enzyme at 25 ◦C and may be attributed tomass transfer resistance of the substrate onto the immobilizationmedium [26].

3.4.4. Storage stabilityThe storage stability of free and immobilized carbonic anhydrase

is presented in Table 2. The storage stability investigation werecarried out at −20 ◦C and 25 ◦C up to 20 days. At −20 ◦C the HalfLife Period (HLP) of free and immobilized enzyme was observedto be 192 h and 216 h, respectively. At 25 ◦C the HLP for free andimmobilized enzyme was 408 h and 456 h, respectively. The resultsindicated improved storage stability of immobilized catalyst at boththe temperature of −20 ◦C and 25 ◦C.

4. Precipitation of calcium carbonate

In order to confirm the efficacy of immobilized enzyme for tar-get application of carbonation reaction. Studies were carried outusing the reported method [5]. In the carbonation reaction, it wasobserved that the sample with immobilized material as well as

formed the calcium carbonate precipitation in 20 s; however, theformation of precipitate was rapid in immobilized material (40 s)because of the presence of enzyme on its surface, which helps toaccelerate the carbonation reaction. However, in the blank (only

Time for precipitation of CaCO3 (in s) mg of CaCO3/mg of enzyme

20 33.05 [SD = ±1.2438]40 26 [SD = ±0.6652]

1016 S. Wanjari et al. / Process Biochemistry 46 (2011) 1010–1018

Fig. 9. Leaching of CA on bare chitosan beads.

Fig. 10. Leaching of CA on crosslinked chitosan beads.

S. Wanjari et al. / Process Biochem

Table 5Hydration assay for CA by W–A method.

Sr. No. Samples aWilbur–Andersonactivity (U)

1. Free CA 1600.7 ± 95.562. CA Immobilized chitosan beads 151.2 ± 0.063. CA Immobilized chitosan beads treated 558.7 ± 0.01

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a One WAU = (t0 − t)/t where t0 is the time for uncatalyzed reaction and t is theime for the enzyme catalyzed reaction.

aterial) the time taken for precipitation was observed to be 100 s,hich is approximately 2.5 times higher as compared to carbon-

tion reaction in presence of immobilized enzyme. Further SEMnalysis (Fig. 4[C]) confirmed the formation of calcium carbonaterystals.

. Quantification of carbonate precipitation

The precipitate obtained using CA was quantified by gravimetrics well as by GC method. The interferences in the precipitate wereliminated by quantifying evolution of CO2 from the precipitatey acidification. The amount of CO2 evolved is directly propor-ional to the carbonate in the precipitate. The CO2 concentrationas estimated by using GC. The results of the screening study areresented in Table 3 [27] and Table 4. The CO2 sequestration capac-

ty of enzyme immobilized chitosan beads was 26 mg of CaCO3/mgf enzyme (on the basis of 6.21 mg of CO2 evolved). In compari-on free enzyme shows CO2 sequestration capacity of 33.05 mg ofaCO3/mg of enzyme (on the basis of 6.56 mg of CO2 evolved). Thisay be due to easy access of the substrate to the free enzyme as

ompared to immobilized enzyme.

. Leaching of immobilized enzyme

Esterase activity was monitored in the leachate for differentycles and the immobilized enzyme was studied for the carbona-ion reaction (conversion of CO2 to calcium carbonate). This processas repeated up to 10th cycle. It is apparent from Fig. 9 that

nitial carbonation capacity of immobilized CA on bare chitosaneads is 23 mg of CaCO3/10 mg of immobilized beads (100% enzymeetained) was retained in the 1st cycle and has decreased to10.5 mgf CaCO3/10 mg of immobilized beads (45.65% enzyme retained) inth cycle. This decrease in activity in 5th cycle is proposed to bevercome by functionalization of bare chitosan beads. From Fig. 10t was observed that the initial carbonation capacity of immobi-ized CA on crosslinked chitosan beads is 27.9 mg of CaCO3/10 mgf immobilized beads (90% enzyme retained) was retained upto 4thycle and has decreased to15.2 mg of CaCO3/10 mg of immobilizedeads (54.4% enzyme retained) in 9th cycle (Unpublished work).he specific activity of both bare chitosan beads and crosslinkedhitosan beads was increased in the leachate upto 10th cycle. Thending has been substantiated by our results were increased activ-

ty has been observed in the leachate and hence decrease carbonateormation by the immobilized CA on bare chitosan and crosslinkedhitosan beads.

. Hydration assay for CA

Hydration assay (Wilbur–Anderson, W–A) for carbonic anhy-

rase was also performed by procedure reported [21] toubstantiate the research findings. The results are presented inable 5 which are in link with the activities obtained for free andmmobilized CA using p-NPA and carbonation reaction.

[

[

istry 46 (2011) 1010–1018 1017

8. Conclusions

Immobilization of carbonic anhydrase on chitosan beads hasbeen successfully carried out. Optimum pH and temperature ofimmobilized as well as free enzyme is 7.4 and 25 ◦C, respectively.Increase in Km value (2.36 mM) for the immobilized enzyme ascompared to free enzyme Km value (0.87 mM) may be attributed toa possible change in the enzyme conformation resulting in decreasein the binding of the substrate or lowering the accessibility of theactive site to the substrate. Proof of concept has been establishedfor carbonation reaction which appears to be faster in case of theimmobilized enzyme.

Acknowledgments

This work was carried out under the Department of Biotech-nology (DBT), New Delhi and SIP (4.2) sponsored by CSIR. We arethankful to Director, NEERI for providing the research facility. Weare also thankful to Dr. Peshwe, VNIT for characterization of materi-als. Two of the authors Snehal Wanjari and Chandan Prabhu wouldalso take the opportunity to sincerely acknowledge the Councilof Scientific and Industrial Research (CSIR) India for granting theSenior Research Fellowship.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.procbio.2011.01.023.

References

[1] Mirjafari P, Asghari K, Mahinpey N. Investigating the application of enzymecarbonic anhydrase for CO2 sequestration purpose. Ind Eng Chem Res2007;46:921–6.

[2] Bond GM, Stringer J, Brandvold DK, Simsek FA, Medina MG, Egeland G. Develop-ment of integrated system for biomimetic CO2 sequestration using the enzymecarbonic anhydrase. Energy Fuels 2001;15:309–16.

[3] Bhattacharya S, Nayak A, Schiavone M, Bhattacharya SK. Solubilization and con-centration of carbon dioxide: novel sprays reactors with immobilized carbonicanhydrase. Biotechnol Bioeng 2004;86:37–46.

[4] Bhattacharya S, Schiavone M, Chakrabarti S, Bhattacharya KS. CO2 hydra-tion by immobilized carbonic anhydrase. Biotechnol Appl Biochem 2003;38:111–7.

[5] Favre N, Christ ML, Pierre A. C. Biocatalytic capture of CO2 with carbonicanhydrase and its transformation to solid carbonate. J Mol Catal B: Enzyme2009;60:163–70.

[6] Raymond D. Development of catalysts for fast, energy efficient post combustioncapture of CO2 into water; an alternative to monoethanolamine (MEA) solvents.Energy Procedia 2009;1:885–92.

[7] Fereshteh A, Saman H, Mohsen NG. Use of reversible denaturation for adsorp-tive immobilization of urase. Appl Biochem Biotechnol 2001;94:265–77.

[8] Saman H, Mohsen N-G. Partial unfolding of carbonic anhydrase providesa method for its immobilization on hydrophobic adsorbents and pro-tects it against irreversible thermoinactivation. Enzyme Microb Technol2003;33:179–84.

[9] Akgol S, Bereli N, Denizli A. Magnetic dye affinity beads for the adsorption ofbeta-casein macromol. Bioscience 2005;5(8):786–94.

10] Krajewska B. Application of chitin- and chitosan-based materials forenzyme immobilizations: a review. Enzyme Microb Technol 2004;35:126–39.

11] Dutta PK, Ravikumar MNV, Dutta J. Chitin and chitosan for versatile application.J Macromol Sci 2002:307–54. C42.

12] Tang ZX, Qian JQ, Shi LE. Characterization of immobilized neutral lipase onchitosan nano-particles. Mater Lett 2007;61:37–40.

13] Cetinus SK, Sahin E, Saraydin D. Preparation of Cu (II) adsorbed chitosan beadsfor catalase immobilization. Food Chem 2009;114:962–9.

14] Gomez L, Ramirez HL, Cabrera G, Simpson BK, Villalonga R. Immobilizationof invertase-chitosan conjugate on hyaluronic-acid-modified chitin. J FoodBiochem 2008;32:264–77.

15] Dhananjay SK, Mulimani VH. Optimization of immobilization process oncrab shell chitosan and its application in food processing. J Food Biochem2008;32:521–35.

16] Chang MY, Juang RS. Stability and catalytic kinetics of acid phosphatase immo-bilized on composite beads of chitosan and activated clay. Proc Biochem2004;39:1087–91.

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018 S. Wanjari et al. / Process Bi

17] Chang MY, Juang RS. Activities Stabilities, and reaction kinetics of three freeand chitosan–clay composite immobilized enzyme. Enzyme Microb Technol2007;36:75–82.

18] Altun GD, Cetinus SA. Immobilization of pepsin on chitosan beads. Food Chem2007;100:964–71.

19] Prabhu C, Wanjari S, Gawande S, Das S, Labhsetwar N, Kotwal S, Puri AK,Satyanarayana T, Rayalu S. Immobilization of carbonic anhydrase enrichedmicroorganism on biopolymer based materials. J Mol Catal B: Enzyme

2009;60:13–21.

20] Lowry OH, Rosenbrough NJ, Forr AL, Randall RJ. Protein estimation with thefolin phenol reagent. J Biol Chem 1951;193:265–75.

21] Armstrong JM, Myers DV, Verpoorte JA, Edsall JT. Purification and prop-erties of human erythrocyte carbonic anhydrases. J Biol Chem 1966;241:5137–49.

[

[

istry 46 (2011) 1010–1018

22] Wilbur KM, Anderson NG. Electrometric and colorimetric determination ofcarbonic anhydrase. J Biol Chem 1948;176:147–54.

23] Balistrieri LS, Murray JW. Effect of silicon on the crystallization and adsorptionproperties of ferric oxides. Am J Sci 1981;281:788–96.

24] Paulino AT, Simionato JI, Garcia JC, Nozaki J. Characterization of chi-tosan and chitin produced from silkworm chrysalides. Carbohydr Polym2006;64:98–103.

25] Mansour EH, Dawoud FM. Immobilization of invertase on celite and on poly-

acrylamide by an absorption procedure. J Sci Food Agric 2003;83:446–50.

26] Vu TKH, Le VVM. Biochemical studies on the immobilization of the enzymeinvertase (EC 3. 2. 1. 26) in alginate gel and its kinetics. ASEAN Food J2008;15(1):73–8.

27] Hsuanyu Y, Benson JR, Li NH. A Functionalized highly porous polymer forenzyme immobilization. Am Labor News 2007;June/July.