Immobilization of carbonic anhydrase on chitosan beads for enhanced carbonation reaction

Process Biochemistry 46 (2011) 1010–1018

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Immobilization of carbonic anhydrase on chitosan beads for enhanced carbonation reaction Snehal Wanjari a , Chandan Prabhu a , Renu Yadav a , T. Satyanarayana b , Nitin Labhsetwar a , Sadhana Rayalu a,∗ a b

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

a r t i c l e

i n f o

Article history: Received 23 September 2010 Received in revised form 13 January 2011 Accepted 17 January 2011 Keywords: Chitosan beads Carbonic anhydrase Immobilization Carbonation reaction Kinetics Stability

a b s t r a c t Carbonic anhydrase (CA) has been immobilized on chitosan beads and has been tested for its activity using p-NPA. CA immobilized chitosan beads were further tested for targeted application of carbonation reaction to convert CO2 to CaCO3 . It was observed that onset time for precipitation of CaCO3 was 40 s for immobilized catalyst as compared to non-catalyzed system (100 s). Mechanistic and kinetic aspects have been addressed for immobilized catalyst as well as free CA using p-NPA assay. Km and Vmax of the immobilized CA was observed to be 2.36 mM and 0.54 ␮moles/min/ml as compared to Km and Vmax of 0.87 mM and 0.93 ␮moles/min, respectively, for free CA. Storage stability test was conducted up to 20 days and it was observed that at −20 ◦ C, the Half Life Period (HLP) of immobilized CA was 216 h as compared to 192 h for free CA whereas at 25 ◦ C the HLP for immobilized and free CA was observed to be 456 h and 408 h, respectively. © 2011 Elsevier Ltd. All rights reserved.

1. Introduction Increasing atmospheric concentration of CO2 is resulting in global warming and climate change. The emission reduction of CO2 can be achieved by: (i) improved energy efficiency (ii) low carbon fuel and (iii) carbon capture and sequestration. Extensive R&D efforts are now underway to develop new approaches to capture and sequester CO2 to avoid its release in the atmosphere including mineral carbonation, oceanic sequestration, geological sequestration, etc. The resulting sequestration system would offer several potential advantages, including avoidance of cost intensive CO2 concentration and transportation steps, an environmental friendly process, performed in aqueous solution at near ambient temperatures, a site specific solution to CO2 sequestration. Biomimetic carbonation reaction is carried out by using CA, is one such approach which is being studied extensively [1–6]. Carbonic anhydrase (CA) is an enzyme with potential as a tool to sequester carbon dioxide from emission sources. Carbonic anhydrase is a common enzyme found in animals, plants and bacteria. This enzyme catalyzes the conversion of carbon dioxide and water into 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 [email protected] (S. Rayalu). 1359-5113/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2011.01.023

million reactions per second, depending on the type of carbonic anhydrase [2–5]. The mechanism for CO2 hydration, catalyzed by CA, can be categorized into four steps as follows [6]. CO2 (gaseous) → CO2 (aqueous)

(1)

CO2 (aqueous) + H2 O → H2 CO3

(2)

H2 CO3 → H+ + HCO− 3

(3)

HCO− 3

(4)

→H

+

+ CO2− 3

Ca2+ + CO2− 3 → CaCO3

(5)

Biocatalysts have a number of advantages such as high level of catalytic efficiency and high degree of selectivity. However, there are 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 a useful technique to improve the thermal and operational stability and recovery of the immobilized enzyme [7,8]. Different procedures have been developed for enzyme immobilization wherein adsorption on solid support material is the easiest and conventional method for enzyme immobilization. The important advantage of this method is that the stability of enzyme is retained after immobilization and it can be reused for several cycles [9]. In this connection several materials are being investigated. Chi-

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and storage stability in immobilized enzyme on chitosan beads as compared to free enzyme [18]. Rayalu and coworkers have reported immobilization of carbonic anhydrase enriched microorganism on different chitosan based materials [19]. To the best of our knowledge, detailed studies on immobilization of carbonic anhydrase on chitosan beads have not been reported so far. In this paper, partially purified CA from Bacillus pumilus was immobilized on chitosan beads and detailed kinetic parameters like, pH, temperature, substrate reuse and storage stability on the free and immobilized CA has been investigated. Also gas chromatography technique has been used for quantification of calcium carbonate. 2. Materials and methods 2.1. Materials

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

tosan is one such material which is being extensively studied for enzyme immobilization by virtue of its non-toxicity and advantages as physical and chemical properties. Chitosan is a linear polyglucosamine of high molecular weight and is obtained by N-deacetylation of chitin in a strong alkali solution. It has reactive amino (−NH2 ) and hydroxyl (−OH) groups and has been identified as an ideal support material for enzyme immobilization. It is also inexpensive and exhibits high affinity towards protein [10]. The positive charge of chitosan reacts with the negatively charged surface of enzyme thus form the physical adsorption (Vander Waals attraction and hydrogen bonding) and ionic interaction between the positively charged surface of the material and negative charge on the enzyme. The mechanism for adsorption of CA on chitosan support is shown in Fig. 2. There are reports available on immobilization of enzymes on chitosan-based materials, which deals with activities, stabilities and reaction kinetics using different enzymes [10–15]. Chang et al. [16] reported immobilization of acid phosphatase on chitosan composite beads and activated clay. Chang et al. also reported the immobilization of ␣-amylase and ␤-amylase on chitosan–clay composite beads to improve the stability of immobilized enzyme as compared to free enzyme [17]. Altun et al. reported greater thermal

Chitosan with deacetylation degree (DD) of 95% and molecular weight (MW) of 360 kDa was purchased from chemchito India Limited, Chennai. All the chemicals used 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 Microbiology, University of Delhi South campus, New Delhi. One gram of lyophilized powder has 6840 units of CA. One unit of enzyme activity was expressed as 1 ␮mol p-nitrophenol released per minute at room temperature. 2.2. Experimental 2.2.1. Preparation of chitosan beads Chitosan 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. This viscous chitosan solution was added drop wise into 250 ml of 3.2 M NH4 OH solution with continuous stirring to form chitosan beads. The beads were allowed to stabilize in NH4 OH solution for 1 h at room temperature. The chitosan beads were filtered and rinsed with distilled water for several times till the pH of the supernatant was 7.0. The beads were finally dried in an oven at 60 ◦ C for 6 h. 2.2.2. Preparation of cross linked chitosan beads Chitosan 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 NH4 OH solution with constant stirring. The solution containing beads were stirred for 30 min at room temperature and it was filtered. Wet beads were treated with 10% glutaraldehyde solution for 24 h. The CA treated beads were filtered, washed with distilled water and dried in an oven at 60 ◦ C for 16 h. 2.2.3. Characterization of materials XRD of the chitosan based material was obtained by using X-ray diffractometer ˚ at 45 kV and 40 mA (Model no. TW 3660/50), with Cu K␣ radiation ( = 1.54060 A) and scanned in the range of 10–80◦ . Scanning electron microscopy (SEM) images of chitosan beads as such, CA immobilized chitosan beads and precipitate of calcium carbonate obtained from CA immobilized chitosan beads was taken using a JEOL JED-2300 scanning electron microscope equipped with an Energy Dispersive X-ray (EDX) analyzer. FTIR spectra of chitosan beads (1 wt%) mixed with KBr pellets were recorded on a Bruker Vertex-70 apparatus by diffused reflectance accessory technique. 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 turbidity of calcium carbonate. Accuracy: ±2% of reading ±1LSD for 0–500NTU, ±3% of reading ±1LSD for 501–1000 NTU. 2.2.4. Immobilization procedure and enzyme assay 10 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 incubation, the sample was centrifuged and CA immobilized enzyme was collected and used for carbonation reaction. The esterase activity of CA in the supernatant was estimated spectrophotometerically at 348 nm by measuring the colour intensity due to p-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 (supernatant) and 1 ml of 3 mM paranitro phenyl acetate for its conversion to para nitro phenol [19,21]. All the experiments like screening, kinetic parameters and carbonate precipitation were repeated twice for better accuracy and blank experiments were also performed throughout the studies.

Fig. 2. Bonding between chitosan and enzyme.

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

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electrode. A volume, 50 ␮l sample was added to 3 ml of 20 mM Tris buffer solution of pH 8.3. The reaction was started by addition of 2 ml of water saturated with CO2 at about 4 ◦ C. CO2 hydration activity of CA was indicated by the time required for the pH to change from 8.3 to 6.3. 2.2.5. Protocol for mineralization of CO2 Carbonation study was carried out by the method reported by Favre et al. with slight modification [5]. In a typical procedure, 1 ml of Tris buffer (1 M, pH 8.0) was added to 10 ml of CO2 saturated water (pH 3.57). The mixture was shaken at 25 ◦ C and then 10 ml of 2% CaCl2 (pH 6.41) was added along with 1 ml (5 mg/ml loading) of the enzyme in phosphate buffer (0.1 M, pH 7.0). The final pH of the mixture was 6.85. The time required for formation of carbonate with respect to onset of reaction was monitored in the sample as well as control (without enzyme) by turbidometric method. The precipitate was filtered using Whatmann filter paper-42 and dried at room temperature. 2.2.6. Estimation of calcium carbonate Quantification of precipitate calcium carbonate was carried by using gas chromatographic (GC) method coupled with thermal conductivity detector (TCD). In this method, the carbonate precipitate was treated with 0.5 M HCl and evolution of CO2 was monitored using GC. The reactor used for quantifying CO2 is presented in Fig. 1 (supplementary information). The reaction was carried out in borosilicate glass reactor in which carbonate precipitate was taken and 0.5 M HCl was added. The evolved gas was collected in the collector and then analyzed in GC/TCD using Porapak Q column. 2.3. Kinetic studies Parameters including incubation time, material dose, enzyme concentration and shaking speed have been varied within feasible parameter ranges to obtain optimal conditions for immobilization of enzyme. 2.3.1. Effect of temperature and pH The stability of the immobilized and free enzyme at different temperature and pH were tested by measuring the activities of the enzymes by incubating for 8 h at different temperatures (25–55 ◦ C) and solution pH (5.4–9.4). 2.3.2. Determination of kinetic constants For determining rate and kinetic constants of free and immobilized enzyme, 1 mg/ml of enzyme loading was incubated in 4.8 ml phosphate buffer (0.1 M, pH 7.0) at temperature of 25 ◦ C. The assay of immobilized and free enzyme was carried out using different concentration of p-nitrophenyl acetate (1, 2, 3, 4 and 5 mM). Km and Vmax of free and immobilized enzyme were calculated from Lineweaver–Burk plots. 2.3.3. Storage stability Stability of free and immobilized enzyme was measured by determining the residual activity on storing at different period up to 20 days. Samples were incubated with 1 mg/ml of enzyme in 4.8 ml phosphate buffer (0.1 M, pH 7.0) at −20 ◦ C and 25 ◦ C. Enzyme activity was assayed at an interval of 5 days.

3.2. Characterization of materials 3.2.1. Shape and size The chitosan beads formed are spherical in shape with approximate particle diameter of 1.1–1.7 mm [19]. 3.2.2. XRD analysis Two major peaks at 2 of 10 ◦ and 20 ◦ have been observed in Xray diffraction patterns of chitosan flakes and chitosan beads (Fig. 3) indicating that the degree of crystallinity decreases in chitosan beads as compared to chitosan flakes. 3.2.3. SEM analysis and EDX spectra The surface morphology of chitosan and CA immobilized on chitosan beads has been studied using SEM. CA enzyme contains Zn atom, which can be used as a marker for confirming the presence of enzyme on beads. Fig. 4 shows a comparison between bare chitosan beads and beads with enzyme. SEM image Fig. 4[A] of chitosan beads as such shows a spongy surface morphology. The surface morphology of chitosan beads with immobilized enzyme is different and appears to be scaly in nature (Fig. 4[B]). The presence of Zn atom on the surface of immobilized beads is confirmed by the EDX spectrum (Fig. 5). 3.2.4. FTIR analysis Chitosan is a heteropolymer made up of glucosamine and acetyl glucosamine units. The functional groups of chitosan are amino and hydroxyl groups which are very important for immobilization of enzyme. The FTIR spectra of chitosan flakes and beads are given in Fig. 2 (supplementary information). The band at 3694 cm−1 in chitosan flakes is attributed to stretching vibration of N–H group, which shifted to 3653 cm−1 in chitosan beads. The hydroxyl group in 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 formation of weak intermolecular hydrogen bonding between amino and hydroxyl groups of chitosan. As reported by Paulino et al. [24], in chitosan beads and flakes, the band at 1575 cm−1 has a larger intensity than at 1676 cm−1 , suggesting effective deacetylation. The peaks at 2918 cm−1 and 1321 cm−1 in chitosan flakes and at 2888 cm−1 and 1407 cm−1 in chitosan beads are attributed to C–H stretching vibration in polymeric backbone and C–H bending vibration, respectively. 3.3. Immobilization studies

2.3.4. Leaching of immobilized enzyme The CA immobilized chitosan beads were used for leaching studies by monitoring the protein concentration in the leachate. Subsequently to the leaching of the CA immobilized chitosan beads were tested for carbonation reaction and amount of calcium carbonate formed was quantified. In the protocol for leaching, 20 mg of CA immobilized chitosan beads were mixed with 4 ml of 0.1 M phosphate buffer (pH 7.0) and it was shaken at room temperature (25 ◦ C). The supernatant was collected and tested for protein concentration by using Lowry et al. [22] as well as for p-NPA assay and the CA immobilized chitosan beads was washed and tested for carbonation reaction (Section 2.2.5). This study was repeated up to 5th cycle.

3. Results and discussion 3.1. Point of zero charge of chitosan beads The surface charge on chitosan beads has been elucidated on the basis of point of zero charge (IPE) of material. The isoelectric point (IEP) of chitosan beads determined by Balistrieri and Murray [23] is at pH 5.0. The pH of the solution obtained by stirring chitosan beads for 24 h is pH 4.96. As the pH value below the point of zero charge of material, M-OH2 + species predominate and the chitosan beads have a positive charge on the surface.

Studies have been carried out for optimizing conditions for immobilization of enzymes and are discussed in following sections. 3.3.1. Effect of variation time on immobilization of enzyme The effect of time on enzyme immobilization is presented in Fig. 3 (supplementary information). The immobilization time was varied from 30 min to 24 h. It can be observed that the amount of enzyme adsorbed increases up to 8 h and decreases subsequently. This decrease in activity may be attributed to leaching of enzyme or denaturation of enzyme due to stirring beyond 8 h. 3.3.2. Effect of variation of material dose on immobilization of enzyme The effect of material dose, on immobilization of enzyme is presented in Fig. 4 (supplementary information). The material dose was varied from 1 mg/5 ml to 20 mg/5 ml. The optimal dose appears to be 10 mg/5 ml. Further increase in material dose results in marginal decrease in loading of enzyme and may be due to lower concentration of enzyme and higher number of active site due to increased dose of adsorbent.

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Fig. 3. XRD spectra of (A) chitosan flakes and (B) chitosan beads.

3.3.3. Effect of enzyme concentration on immobilization of enzyme The effect of enzyme concentration on immobilization of enzyme is presented in Fig. 5 (supplementary information). The enzyme concentration was varied from 0.5 to 3.0 mg/ml. It was observed that increasing the enzyme loading above the saturation level resulted in gradual decrease in the enzyme activity. This is because a high concentration of enzyme on the external surface can contribute to internal diffusional restriction, thus leading to the underestimation of the initial activity of the immo-

bilized enzyme. In the present study, the loading appears to have increased up to 1 mg/ml thereafter a consistent decrease has been observed. This observation corroborates with that reported [25]. It has been reported that, overloading needs to be avoided, and therefore enzyme loading (1 mg/ml) below saturation level is selected for further study.

3.3.4. Effect of shaking on immobilization of enzyme The effect of shaking speed on immobilization of enzyme on chitosan beads is shown in Fig. 6 (supplementary information)

Fig. 4. Comparison of SEM images of [A] bare chitosan beads, [B] immobilized. Chitosan beads, [C] calcite form of CaCO3 on immobilized chitosan beads.

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Fig. 5. EDX spectra of immobilized chitosan beads.

3.4. Kinetic studies 3.4.1. Effect of temperature on immobilized enzyme The effect of temperature on immobilized enzyme is presented in Fig. 6. The activity is observed to be maximum at 25 ◦ C, with consistent decrease on further increasing the temperature up to 55 ◦ C for free as well as immobilized enzyme. However, the decline in activity was more pronounced for free enzyme as compared to immobilized enzyme. This may be due to denaturation of enzyme at higher temperature.

800

Specific activity (U/mg of protein)

wherein shaking speed was varied from 60 to 160 rpm. The optimum shaking speed was observed to be 120 rpm. Further increase in shaking speed decreases its activity, which may be due to the leaching of enzyme. This may be attributed to weakening of the interaction between enzyme and materials at high speed.

700

600

500

400

300 5

6

7

8

9

10

pH 800

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

Specific activity (U/mg of protein)

700

3.4.2. Effect of pH on immobilized enzyme pH is one of the most important parameter altering enzyme activity in an aqueous medium. Immobilization is likely to result in conformational change of enzyme leading to inactivity of enzyme. The stability of immobilized enzyme at various pH is presented in Fig. 7. Maximum activity of free and immobilized enzyme was observed at pH 7.4 with significant decline on further increase in the pH. It was observed that CA appears to be stable upto pH 7.4 and thereafter shows decline in its activity.

600 500 400 300 200 100

Table 1 Kinetic parameter of free and immobilized enzyme.

0 25

30

35

40

45

50

Temperature (in degree) Fig. 6. Effect of temperature profile on immobilized enzyme [].

55

Enzyme

Km (mM)

Vmax (␮mol/min)

Free enzyme Immobilized enzyme

0.87 2.36

0.93 0.54

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Fig. 8. Lineweaver–Burk plots for estimation of Km and Vmax at 25 ◦ C of immobilized. Enzyme [], free enzyme [] by p-NPA assay. Table 2 Half Life Period of free and immobilized enzyme at −20 ◦ C and 25 ◦ C. Half Life Period ◦

−20 C 25 ◦ C

Free CA(hours)

Immobilized CA(hours)

192 408

216 456

Table 3 Comparison with CA immobilized on other reported matrices.

Material(in mg) Enzyme used for immobilization Percent of enzyme immobilized Capacity (immobilized) (U/mg beads) a

Chitosan beads

CN–Cavilinka

NH2 –Cavilinka

10 mg 0.4 mg

111 mg 8.6 mg

111 mg 8.6 mg

95%

>99%

82%

2.85 U/mg

1.7 U/mg

1.6 U/mg

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 to mass transfer resistance of the substrate onto the immobilization medium [26]. 3.4.4. Storage stability The storage stability of free and immobilized carbonic anhydrase is presented in Table 2. The storage stability investigation were carried out at −20 ◦ C and 25 ◦ C up to 20 days. At −20 ◦ C the Half Life Period (HLP) of free and immobilized enzyme was observed to be 192 h and 216 h, respectively. At 25 ◦ C the HLP for free and immobilized enzyme was 408 h and 456 h, respectively. The results indicated improved storage stability of immobilized catalyst at both the temperature of −20 ◦ C and 25 ◦ C. 4. Precipitation of calcium carbonate

Hsuanyu et al. [24].

3.4.3. Substrate study The kinetic constants (Km and Vmax ) for free and immobilized enzyme were determined by using Lineweaver–Burk plots as shown in Fig. 8. Km and Vmax values of free and immobilized CA were calculated from the intercepts on x and y axes of the Lineweaver–Burk plots, respectively. The Km and Vmax values for free and immobilized enzyme are summarized in Table 1. Km and Vmax for free enzyme is 0.87 mM and 0.93 ␮moles/min/ml used p-

In order to confirm the efficacy of immobilized enzyme for target application of carbonation reaction. Studies were carried out using the reported method [5]. In the carbonation reaction, it was observed that the sample with immobilized material as well as reagent blank (the material) forms the precipitate. Free enzyme formed the calcium carbonate precipitation in 20 s; however, the formation of precipitate was rapid in immobilized material (40 s) because of the presence of enzyme on its surface, which helps to accelerate the carbonation reaction. However, in the blank (only

Table 4 Summary of precipitation of calcium carbonate reaction. Sr. No.

Samples

Time for precipitation of CaCO3 (in s)

mg of CaCO3 /mg of enzyme

1. 2.

CA (partially purified) Immobilized enzyme (CA–Chitosan–NH4 OH beads)

20 40

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

SD: standard deviation.

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

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

S. Wanjari et al. / Process Biochemistry 46 (2011) 1010–1018 Table 5 Hydration assay for CA by W–A method.

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8. Conclusions

Sr. No.

Samples

a

1. 2. 3.

Free CA CA Immobilized chitosan beads CA Immobilized chitosan beads treated with glutaraldehyde

1600.7 ± 95.56 151.2 ± 0.06 558.7 ± 0.01

Wilbur–Anderson activity (U)

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

material) the time taken for precipitation was observed to be 100 s, which is approximately 2.5 times higher as compared to carbonation reaction in presence of immobilized enzyme. Further SEM analysis (Fig. 4[C]) confirmed the formation of calcium carbonate crystals. 5. Quantification of carbonate precipitation The precipitate obtained using CA was quantified by gravimetric as well as by GC method. The interferences in the precipitate were eliminated by quantifying evolution of CO2 from the precipitate by acidification. The amount of CO2 evolved is directly proportional to the carbonate in the precipitate. The CO2 concentration was estimated by using GC. The results of the screening study are presented in Table 3 [27] and Table 4. The CO2 sequestration capacity of enzyme immobilized chitosan beads was 26 mg of CaCO3 /mg of enzyme (on the basis of 6.21 mg of CO2 evolved). In comparison free enzyme shows CO2 sequestration capacity of 33.05 mg of CaCO3 /mg of enzyme (on the basis of 6.56 mg of CO2 evolved). This may be due to easy access of the substrate to the free enzyme as compared to immobilized enzyme. 6. Leaching of immobilized enzyme Esterase activity was monitored in the leachate for different cycles and the immobilized enzyme was studied for the carbonation reaction (conversion of CO2 to calcium carbonate). This process was repeated up to 10th cycle. It is apparent from Fig. 9 that initial carbonation capacity of immobilized CA on bare chitosan beads is 23 mg of CaCO3 /10 mg of immobilized beads (100% enzyme retained) was retained in the 1st cycle and has decreased to10.5 mg of CaCO3 /10 mg of immobilized beads (45.65% enzyme retained) in 5th cycle. This decrease in activity in 5th cycle is proposed to be overcome by functionalization of bare chitosan beads. From Fig. 10 it was observed that the initial carbonation capacity of immobilized CA on crosslinked chitosan beads is 27.9 mg of CaCO3 /10 mg of immobilized beads (90% enzyme retained) was retained upto 4th cycle and has decreased to15.2 mg of CaCO3 /10 mg of immobilized beads (54.4% enzyme retained) in 9th cycle (Unpublished work). The specific activity of both bare chitosan beads and crosslinked chitosan beads was increased in the leachate upto 10th cycle. The finding has been substantiated by our results were increased activity has been observed in the leachate and hence decrease carbonate formation by the immobilized CA on bare chitosan and crosslinked chitosan beads. 7. Hydration assay for CA Hydration assay (Wilbur–Anderson, W–A) for carbonic anhydrase was also performed by procedure reported [21] to substantiate the research findings. The results are presented in Table 5 which are in link with the activities obtained for free and immobilized CA using p-NPA and carbonation reaction.

Immobilization of carbonic anhydrase on chitosan beads has been successfully carried out. Optimum pH and temperature of immobilized as well as free enzyme is 7.4 and 25 ◦ C, respectively. Increase in Km value (2.36 mM) for the immobilized enzyme as compared to free enzyme Km value (0.87 mM) may be attributed to a possible change in the enzyme conformation resulting in decrease in the binding of the substrate or lowering the accessibility of the active site to the substrate. Proof of concept has been established for carbonation reaction which appears to be faster in case of the immobilized enzyme. Acknowledgments This work was carried out under the Department of Biotechnology (DBT), New Delhi and SIP (4.2) sponsored by CSIR. We are thankful to Director, NEERI for providing the research facility. We are also thankful to Dr. Peshwe, VNIT for characterization of materials. Two of the authors Snehal Wanjari and Chandan Prabhu would also take the opportunity to sincerely acknowledge the Council of Scientific and Industrial Research (CSIR) India for granting the Senior Research Fellowship. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.procbio.2011.01.023. References [1] Mirjafari P, Asghari K, Mahinpey N. Investigating the application of enzyme carbonic anhydrase for CO2 sequestration purpose. Ind Eng Chem Res 2007;46:921–6. [2] Bond GM, Stringer J, Brandvold DK, Simsek FA, Medina MG, Egeland G. Development of integrated system for biomimetic CO2 sequestration using the enzyme carbonic anhydrase. Energy Fuels 2001;15:309–16. [3] Bhattacharya S, Nayak A, Schiavone M, Bhattacharya SK. Solubilization and concentration of carbon dioxide: novel sprays reactors with immobilized carbonic anhydrase. Biotechnol Bioeng 2004;86:37–46. [4] Bhattacharya S, Schiavone M, Chakrabarti S, Bhattacharya KS. CO2 hydration by immobilized carbonic anhydrase. Biotechnol Appl Biochem 2003;38: 111–7. [5] Favre N, Christ ML, Pierre A. C. Biocatalytic capture of CO2 with carbonic anhydrase and its transformation to solid carbonate. J Mol Catal B: Enzyme 2009;60:163–70. [6] Raymond D. Development of catalysts for fast, energy efficient post combustion capture 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 adsorptive immobilization of urase. Appl Biochem Biotechnol 2001;94:265–77. [8] Saman H, Mohsen N-G. Partial unfolding of carbonic anhydrase provides a method for its immobilization on hydrophobic adsorbents and protects it against irreversible thermoinactivation. Enzyme Microb Technol 2003;33:179–84. [9] Akgol S, Bereli N, Denizli A. Magnetic dye affinity beads for the adsorption of beta-casein macromol. Bioscience 2005;5(8):786–94. [10] Krajewska B. Application of chitin- and chitosan-based materials for enzyme 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 on chitosan nano-particles. Mater Lett 2007;61:37–40. [13] Cetinus SK, Sahin E, Saraydin D. Preparation of Cu (II) adsorbed chitosan beads for catalase immobilization. Food Chem 2009;114:962–9. [14] Gomez L, Ramirez HL, Cabrera G, Simpson BK, Villalonga R. Immobilization of invertase-chitosan conjugate on hyaluronic-acid-modified chitin. J Food Biochem 2008;32:264–77. [15] Dhananjay SK, Mulimani VH. Optimization of immobilization process on crab shell chitosan and its application in food processing. J Food Biochem 2008;32:521–35. [16] Chang MY, Juang RS. Stability and catalytic kinetics of acid phosphatase immobilized on composite beads of chitosan and activated clay. Proc Biochem 2004;39:1087–91.

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