Utility of Immobilized Recombinant Carbonic Anhydrase of Bacillus

Feb 7, 2017 - Carbonic anhydrase (CA) based conversion of CO2 to CaCO3 has been identified as a green and economic strategy to sequester CO2 from ...
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Utility of immobilized recombinant carbonic anhydrase of Bacillus halodurans TSLV1 on the surface modified iron magnetic nanoparticles in carbon sequestration Shazia Faridi, Himadri Bose, and Tulasi Satyanarayana Energy Fuels, Just Accepted Manuscript • DOI: 10.1021/acs.energyfuels.6b02777 • Publication Date (Web): 07 Feb 2017 Downloaded from http://pubs.acs.org on February 16, 2017

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Figure 1. rBhCA-Si-MNPs: (A) The rBhCA-Si-MNPs remain suspended in the aqueous medium before application of magnetic field; (B) rBhCA-Si-MNPs attracted towards the magnetic field thus easy separation from the aqueous medium. 86x78mm (96 x 99 DPI)

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Figure 2. Effect of enzyme concentration on loading of enzyme on Si-MNPs. The immobilization increased on increasing the enzyme concentration up to 3 mg/mL, thereafter a plateau was observed. 93x82mm (150 x 146 DPI)

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Figure 3. FTIR spectra of plain MNPs (red), APTES coated MNPs (pink) and rBhCA immobilized APTES coated iron MNPs (blue). 685x914mm (72 x 72 DPI)

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Figure 4. pH stability profile of rBhCA-Si-MNPs: (A) pH stability of free rBhCA after 24 and 48 h; (B) pH stability of immobilized rBhCA after 24 and 48 h. The immobilized rBhCA retained 80 and 45% residual activities at pH 11 after 24 and 48 h, respectively as compared to 65 and 20 % in the free rBhCA. 220x95mm (150 x 150 DPI)

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Figure 5. Temperature stability profile of rBhCA-Si-MNPs and free rBhCA. The immobilized enzyme is much more stable than free enzyme retaining 50 % activity at 70 °C and 15 % activity at 80 °C whereas free enzyme completely got denatured at 70 °C after 30 min of incubation.

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Figure 6. Immobilization of rBhCA on silanized MNPs. The immobilized rBhCA retained 50% of it activity even after 22 cycles of reuse. 120x98mm (74 x 73 DPI)

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Figure 7. Storage stability of free and immobilized rBhCA. The immobilized enzyme started losing its activity after 28 days whereas the free rBhCA stable for years both at room temperature

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Figure 8. Scanning electron microscopic picture of calcium carbonate crystals in the presence of rBhCA-SiMNPs. Rhombohedral and well defined calcite crystals were observed in the presence of immobilized enzyme. 26009x19507mm (1 x 1 DPI)

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Utility of immobilized recombinant carbonic anhydrase of Bacillus halodurans

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TSLV1 on the surface modified iron magnetic nanoparticles in carbon

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sequestration

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Shazia Faridi, Himadri Bose & T. Satyanarayana*

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Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New

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Delhi - 110 021, India

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*Corresponding author

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Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New

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Delhi - 110 021, India

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Tel.: +91-11-24112008, Fax: +91-11-24115270

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E-mail address: [email protected]

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ABSTRACT

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Carbonic anhydrase (CA) based conversion of CO2 to CaCO3 has been identified as a green and

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economic strategy to sequester CO2 from flue gas and industrial emissions. The method is,

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however, cost-intensive as an efficient immobilization method for reusing the enzyme poses a

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major challenge. In this investigation, the recombinant carbonic anhydrase of polyextremophilic

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bacterium Bacillus halodurans TSLV1 (rBhCA) has been immobilized on the surface modified

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magnetic (silanized) iron oxide nanoparticles (Si-MNPs). The immobilized rBhCA exhibited

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improvement in alkalistability and retained significantly high activity at elevated temperatures as

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compared to the free rBhCA. Furthermore, rBhCA immobilized on Si-MNPs could be easily

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isolated from the reaction by magnetic separation. After 22 repeated uses, the immobilized 1 ACS Paragon Plus Environment

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rBhCA retained 50 % of the initial activity and could be stored for 28 days without any loss in

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activity. rBhCA-Si-MNPs accelerated the onset of CaCO3 precipitation over that of the free

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enzyme, but the amount of CaCO3 precipitated was not affected, suggesting that the silanized

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MNPs act as efficient supports for immobilization of CA for utility in CO2 sequestration.

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Keywords: Baillus halodurans, recombinant carbonic anhydrase, immobilization, silanized

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magnetic nanoparticles, carbon sequestration

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1.0. Introduction

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Global warming is leading to increase in the planet’s average surface temperature and is

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adversely affecting our climate, communities and health. The main reason is the rise in CO2

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concentration in the atmosphere, emitted due to burning of fossil fuels for energy generation1.

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Fossil fuels will continue to be the mainstay for energy generation, thus, we are compelled to

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look for developing strategies to reduce carbon emissions in the atmosphere. Biomimetic CO2

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capture by using the enzyme carbonic anhydrase (CA) is one of the efficient method of CO2

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sequestration, which offers greener, economical and safe capture of carbon dioxide from

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industrial emissions, which can also be utilized for the synthesis of several useful products2, 3.

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CA catalyzes the reversible hydration of CO2 forming bicarbonate which can be mineralized into

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calcium carbonate in the presence of Ca2+ at alkaline pH. CAs are mostly zinc containing

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metalloenzymes; these belong to at least 6 different classes which do not share any significant

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amino acid sequence similarity among them4. A moderately thermophilic and alkaliphilic CA is

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preferable for biomineralization based sequestration of CO2.

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Although CAs are one of the fastest known enzymes with Kcat as high as 106 s-1, there exists a

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number of practical problems in using the free enzyme in solution such as low stability and 2 ACS Paragon Plus Environment

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limited reusabilty5. Immobilization of the enzyme onto solid support is the most successful

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method which overcomes the problem posed by free enzymes and this technique is being used

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conventionally for decades. This offers easier separation of products from the enzymes,

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continuous reuse of the enzyme, besides the possibility of improving the stability and

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performance of enzymes under harsh operational conditions. CAs have also been immobilized on

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various solid matrices for improving their resusability and efficiency. For instance, Bovine CA

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(BCA) has been immobilized by encapsulation in chitosan alginate beads by Liu et al.6 Prabhu et

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al.7 tested different chitosan and sodium alginate based materials for entrapment of whole cells

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of Bacillus pumilus (extracellular CA producer) and these were found to enhance the associated

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CA activity as compared to the free cells. The purified CAs from different bacterial species

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(Pseudomonas fragi, Micrococcus lylae, Micrococcus luteus and Bacillus pumilus) have been

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immobilized in chitosan and alginate beads. When the CA of Bacillus subtilis VSG4 was

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entrapped within a chitosan–alginate polyelectrolyte complex (C-A PEC) hydrogel, the

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immobilized enzyme exhibited better sequestration ability than the free CA8. Immobilized CAs

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have been reported to exhibit improved storage stability with almost 50 % retention of initial

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activity for 30 days9. While Wanjari et al.10 immobilized CA of Bacillus pumilus on chitosan

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beads that improved thermostability. When CA was immobilized on silylated chitosan beads,

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improvement in storage stability was recorded11.

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The immobilization of enzymes on nanoparticles (NPs) may lead to reduction in protein

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unfolding, improvement in storage stability and performance12. Due to their unique size and

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physical properties immobilization on NPs offers high surface area, large surface-to-volume ratio

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and easy separation of product from the immobilized enzyme by applying magnetic field13. By

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suitable surface modification, different iron manganese nanoparticles (MNPs) are being 3 ACS Paragon Plus Environment

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synthesized in order to use them for immobilizing protein/enzyme. The CA from B. pumilus TS1

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was immobilized successfully on chitosan stabilized iron MNPs14. While Mikhaylova et al.15

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immobilized BSA on amine functionalized supramagnetic iron oxide nanoparticles. Silanization

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of iron MNPs is emerging as the most widely used technique for introducing functional groups on

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the surface of iron MNPs due to characteristics such as low cytotoxicity, stability under acidic

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conditions, satisfying the response and inertness to redox reactions. In addition, the surface

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modification can be carried out simply in aqueous or organic media at moderate temperatures

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without any need of expensive equipment, thus, an ideal method for protecting the inner magnetic

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core12. We have recently shown the alkali-thermo-stable CA of the polyextremophilic bacterium

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Bacillus halodurans TSLV1 (BhCA) to be suitable for sequestration of CO2 from flue gas16,

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therefore, we cloned and over expressed in Escherichia coli17. In this investigation, we have

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attempted to immobilize rBhCA on silanized iron MNPs and studied their applicability in carbon

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sequestration.

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2.0. Experimental

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2.1. Materials

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Recombinant carbonic anhydrase (rBhCA) from B. haldodurans TSLV1 purified from

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recombinant E. coli was used in this investigation17. Other chemicals used were of analytical

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grade and purchased from Merck, India Ltd.; SRL Chemicals, India and Sigma Chemicals Co.,

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USA.

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2.2. Methods

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2.2.2. Preparation of purified recombinant BhCA

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rBhCA was purified from E. coli clone as described previously17. The recombinant E. coli cells

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were induced by IPTG for expression of rBhCA and were harvested and resuspended in Tris

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buffer (pH 8.3, 20 mM) containing 500 mM NaCl. Sonication was performed to prepare the

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crude cell lysate [cycle conditions: amplitude: 25; total time: 30 min; 2 sec off and 2 sec on with

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1 min pause after 1 min of sonication]. The crude lysate was applied on Ni2+-NTA affinity

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chromatographic column under native conditions. After washing with 0.2 M imidazole, pure

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rBhCA was obtained by elution using 0.3 M imidazole followed by dialysis against 20 mM Tris-

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HCl (pH 8.3) to remove imidazole.

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2.2.3. Preparation of iron MNPs

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Magnetic iron MNPs were prepared by the coprecipitation of Fe3+and Fe2+ according to Mahdavi

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et al.18 2:1 molar ratio of Fe3+and Fe2+ were dissolved separately in Milli-Q water, and the two

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solutions were mixed and stirred vigorously at 45 °C for 30 min at 800 rpm under nitrogen

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sparging. Then aqueous ammonia solution (25%) was added such that the pH of the solution is >

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8.0 and < 11.0. Black magnetic slurry thus formed was collected by applying magnetic field and

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was washed several times with ethanol and then with Milli-Q. MNPs were dispersed by

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sonication for 15 min and large precipitates were removed.

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2.2.4. Surface modification of iron MNPs with silane and immobilization of rBhCA on

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silanized MNPs

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MNPs were coated with silane layer using 3-(aminopropyl) triethoxysilane (APTES) and

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activated by keeping in an aqueous acidic solution (pH 4.0) that acts as a catalyst. APTES and

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4.0 mL glycerol were added to iron MNPs and the mix was heated for 2 h at 90°C under nitrogen

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sparging with stirring. After cooling, the mix was sonicated for 10 min and washed thrice with

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200 mL Milli-Q and then with 100 mL methanol followed by washing with 200 mL Milli-Q. The

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silanized MNPs were stored in Milli-Q.

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For immobilization of the enzyme, the carboxyl groups of rBhCA were treated with 3-(3

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dimethylaminopropyl) N’-ehtylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS). EDC (2

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mg) was added to 5 mL solution of rBhCA and incubated at room temperature with shaking

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followed by the addition of 2.4 mg of NHS and incubation for an hour. The solution was then

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added to 10 mg iron MNPs and incubated by shaking at RT for 3 h and then assayed for CA

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activity.

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2.2.5. Effect of extent of silanization of magnetite particles on the immobilization

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efficiency of rBhCA

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The concentration of APTES (%) was varied to evaluate the effect of silanization of MNPs on

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enzyme loading and interaction of enzyme with the metal ion. To achieve this, silanized MNPs

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were prepared by using varying concentration of APTES (2 %, 5%, 10%, 15 %, 20 %, 30 %, 40

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% and 60 %). After immobilization of the enzyme, the amount of free enzyme present in the

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supernatant was measured by Folin Phenol Ciocaltaue’s reagent. Loading capacity of the MNPs

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with varied silanization was determined by subtracting the concentration of free enzyme after

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immobilization from the initial enzyme concentration.

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2.2.6. Effect of enzyme concentration on loading of rBhCA on silanized MNPs (rBhCA-Si-

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MNPs)

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For studying the effect of enzyme concentration on enzyme loading, 5 mg MNPs were mixed

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with 1 to 9 mg/mL rBhCA and the amount of free protein present in the supernatant was

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measured by Folin Phenol Ciocaltaue’s reagent and loading capacity was determined as

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discussed in previous section.

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2.2.7. Enzyme assay

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For determining the activity of CA, hydration of CO2 was measured using electrometric Wilbur–

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Anderson assay according to Khalifah et al.19 with certain modifications. The assay was

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performed at 4 °C by adding 0.1 mL of the enzyme solution to 3.0 mL of 20-mM Tris–HCl

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buffer (pH 8.3). The reaction was initiated by adding 2.0 mL of ice-cold CO2 saturated water.

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The time interval for the pH to drop by 1 unit (from 8.3 to 7.3) due to protons released during

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hydration of CO2 was measured. The reactions were performed in triplicates and average of three

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replicates was used in calculations. One unit of CA activity is defined as the amount of enzyme

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required to bring down the pH of the buffer from 8.3 to 7.3 and expressed as WA units per unit

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volume. The assays were performed with Metrohm pH meter (model 824), Herisau, Switzerland,

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equipped with a sensitive biotrode pH microelectrode, which has the sensitivity of response time

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less than a second in the range of pH 1.0–14.0.

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2.2.8. Characterization of rBhCA-Si-MNPs

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Adsorption of functional groups on the surface of Fe3O4 MNPs was examined by A Nicolet

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Protege 360 Fourier transform infrared spectroscopy. Fourier transform infrared (FT-IR) spectra

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were recorded by KBr method.

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2.2.9. Effect of pH and temperature on rBhCA-Si-MNPs

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To study the effect of different pH on immobilized rBhCA, rBhCA-Si-MNPs were incubated at

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37 °C in 50 mM buffers of varying pH [sodium acetate buffer (0.1 M, pH 4.0 & 5.0), citrate

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phosphate buffer (pH 6.0), sodium phosphate buffer (pH 7.0 & 8.0) and glycine-NaOH buffer

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(pH 9.0 & 10.0)]. Thereafter, residual activities were calculated in comparison with the control

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(rBhCA-Si-MNPs in 50 mM Tris buffer pH 8.3), which was considered as 100.

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Thermal stability of the free and immobilized rBhCA was compared by incubating both

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the free and the immobilized rBhCA in 20 mM Tris buffer at different temperatures (50, 60, 70

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and 80 °C) for 30 min. The relative activities of the free and immobilized rBhCA without

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exposure to heat were considered as controls (considered as 100 % activity).

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2.2.10. Storage stability of rBhCA-Si-MNPs

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For assessing storage stability, immobilized rBhCA was kept at 4 °C for a period of 30 days and

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relative CA activity was determined after every 3 days.

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2.2.11. Effect of anions on the activity of immobilized rBhCA

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Immobilized rBhCA was incubated with varying concentrations of metal ions such as Hg2+,

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Mg2+, Pb2+ (chloride salts), anions ( NO3-, SO42-, SO32- sodium salts) and residual enzyme

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activities were determined.

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2.2.12. Application of rBhCA-Si-MNPs in sequestration of CO2

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determination of carbonate precipitation by free and immobilized rBhCA

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To study the role of MNPs in affecting the initial rate of precipitation of CaCO3 by rBhCA,

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turbidimetric measurement experiment was conducted as described earlier16. Briefly, absorbance

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change at 600 nm using UV/Vis spectrophotometer was used as an indicator of CaCO3

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precipitation rate. CO2 saturated water (500 µl) was added to a reaction cuvette that contained

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500 µl of 1.0 M Tris buffer (20 mM CaCl2, pH 9.5) along with rBhCA-Si-MNPs (corresponding

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to a protein concentration of 0.005 mg); 0.005 mg of free rBhCA in case of positive control and

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Tris buffer in case of blank and mixed thoroughly. The cuvette was closed immediately with a

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plastic cap to prevent CO2 leakage and the reaction was performed at 30°C. The time at which

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precipitation started (an average rate of increase in A600 more than 0.001/s) was recorded.

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Morphology of carbonate crystals formed using free and immobilized rBhCA was confirmed by

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Scanning Electron Microscopy carried out at the Advanced Instrumentation Research Facility

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(AIRF) at Jawaharlal Nehru University (JNU).

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The effect of immobilized rBhCA on the precipitation of CO2 into CaCO3 was studied under

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optimal conditions for CO2 mineralization using rBhCA16. Briefly, 0.15 mg of rBhCA was added

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to 15 mL Tris buffer containing 1.5 % CaCl2.2H2O in 1 M Tris buffer. To initiate the

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mineralization process, 60 mL of CO2 solution was added to the enzyme mix. The reaction was

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performed for 5 min at 37 °C and the amount of CaCO3 formed was determined gravimetrically

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by filtering the solution through 0.2 µm membrane filters (Millipore). The precipitate was dried

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at 80 °C overnight to constant weight.

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3.0. Results

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3.1. Immobilization of rBhCA and characterization of iron rBhCA-Si-MNPs

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The surface of MNPs was successfully coated with silane layer, and rBhCA was immobilized via

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covalent attachment. When magnetic field was applied to the suspension of rBhCA-Si-MNPs in

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an aqueous medium, iron-MNPs got attracted to the magnetic force and were easily separated

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from the reaction mixture. While upon withdrawal of magnetic field and subsequent agitation,

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rBhCA-Si-MNPs got resuspended again in the solution as shown in figure 1.

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3.2. Effect of the extent of silanization on the loading capacity of MNPs

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The initial concentration of APTES used for silanization affects the amount of rBhCA

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immobilized on MNPs. As the initial concentration of APTES increased (2 to 20 %), there was

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an increase in amount of protein loaded onto MNPs (Table 1). A maximum of 5.7 mg protein

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loading was observed with 20 % APTES. No further increase was observed on the loading of

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enzyme beyond 20 %, indicating that a lower concentration of APTES resulted in maximum

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cross linking of the enzyme through its active –NH2 groups, 20 % APTES being the optimum

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concentration for maximum loading of rBhCA on MNPs.

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3.3. Effect of enzyme concentration on loading of rBhCA on silanized MNPs (rBhCA-Si-

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MNPs)

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An increase in immobilization of rBhCA was observed on raising enzyme concentration, with

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maximum loading of enzyme being at 3.0 mg mL-1, beyond that a decline in enzyme loading was

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observed (figure 2).

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3.4.Characterization of rBhCA-Si-MNPs

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3.4.1. Fourier transform infrared (FT-IR) analysis

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The FTIR spectra of MNPs, Si-MNPs and rBhCA-Si MNPs showed spectral lines with

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characteristics peaks (figure 3).

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3.4.2. Effect of pH and temperature on rBhCA-Si-MNPs

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rBhCA-Si-MNPs showed improved pH stability in the alkaline range as compared to the free

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rBhCA. rBhCA-Si-MNPs retained 80 and 45 % residual activities at pH 11.0 after 24 and 48 h,

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respectively (figure 4) as compared to 65 and 20 % residual activities of the free rBhCA under

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the same conditions 17. Both lost activity rapidly below pH 6.0.

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The immobilized rBhCA retained 50 % activity at 70 °C and 15 % activity at 80 °C after 30 min

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exposure, while the stability of free enzyme declined gradually beyond 40 °C, and complete loss

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in activity was recorded at 70 °C (figure 5).

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3.4.3. Reusability study and storage stability of rBhCA-Si-MNPs

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The rBhCA-Si-MNPs permitted reuse of rBhCA over 22 cycles by retaining up to 50 % activity

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(figure 6). rBhCA-Si-MNPs could be stored for 28 days, with a gradual decrease in activity

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thereafter (figure 7), while free rBhCA was stable for years.

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3.4.4. Effect of various ions on the activity of rBhCA-Si-MNPs

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The rBhCA-Si-MNPs showed similar activity profile like the free BhCA with most of the ions

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tested (Table 2); the latter showed stimulation by SO42- and tolerance to SO42- and NO317. In the

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presence of Hg2+, rBhCA-Si-MNPs lost 80 % activity. There is no effect of immobilization on

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tolerance to SOX and NOX and stimulation by sulphate. In presence of Pb2+, a slight increase in 11 ACS Paragon Plus Environment

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the residual activity of rBhCA-Si-MNPs was observed (82 ± 2 %) in comparison with 70 ± 2.7 %

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with free rBhCA (Table 2).

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3.5. Application of rBhCA-Si -MNPs in sequestration of CO2

239

3.5.1. Effect of rBhCA-Si-MNPs on the precipitation rate of CaCO3 by rBhCA

240

comparative determination of carbonate precipitation by free and immobilized

241

rBhCA

242

rBhCA-Si-MNPs caused a significant reduction in the time required for the onset of CaCO3

243

precipitation(10 s) as compared to the free rBhCA (32 s) [Table 3]. For the control reactions

244

(having Si-MNPs instead of enzyme and blank), the time required for the onset of precipitation

245

was similar (138 s). The CaCO3 precipitate with both the free rBhCA (140 mg ±2.9) and rBhCA-

246

Si-MNPs (138 mg ±3) was comparable. In the absence of enzyme, the amount of precipitate

247

formed was far less (28 mg ±1.5). Well defined rhombohedral calcite crystals were seen in the

248

mineral carbonation reaction with free as well as immobilized rBhCA (figure 8).

249

4.0. Discussion

250

Immobilization of enzyme through covalent binding on solid supports comprises the generation

251

of functional groups (hydroxyl, lactone, amino, carboxylic, and carbonyl groups) on the surface

252

of a solid through which the enzyme binds. MNPs can be used for immobilization of enzyme

253

through covalent bonding that affords the advantage of fixing the enzyme tightly to its surface,

254

minimizing leaching of enzyme and also making it easier to separate the enzyme from the

255

reaction medium20. The present investigation has confirmed the successful immobilization of

256

rBhCA on to the silanized MNPs. As shown in figure 1 rBhCA-Si-MNPs were proven to be

257

separable easily by using a magnet field, thus enabling their easy recovery from the product. The

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258

silane layer can provide protection to the magnetic core from etching under harsh application

259

conditions21. In addition, the –NH2 group of APTES participates in chemical bonding with

260

enzyme and may result in higher enzyme immobilization as compared to uncoated MNPs where

261

enzyme is immobilized by static adsorption22. The mechanism of surface modification of MNPs

262

involves the formation of Fe−O−Si bond between the nanoparticles and silane ligand. Silane

263

molecules are first activated by hydrolysis which is followed by polycondensation reactions

264

between the Si–OH groups of the silanol and the OH groups present on the surface of iron MNPs

265

leading to the formation of a stable bond on the surface of MNPs. NHS and EDC aided in

266

covalently linking rBhCA on the silanized MNPs. EDC activated the carboxyl group of rBhCA

267

forming an unstable enzyme–EDC complex, which was stabilized by NHS. EDC and NHS are

268

frequently employed in peptide synthesis, immobilization of enzyme, and the cross-linking of

269

proteins to nucleic acids23, 24. A similar technique has also been employed in immobilizing BSA

270

on iron MNPs15. Optimization of APTES concentration in order to achieve high immobilization

271

of enzyme was done and 20 % APTES proved to be the optimum concentration for achieving

272

maximum enzyme loading (Table 1). As compared to immobilization of enzymes by physical

273

adsorption on porous support, surface immobilization of enzymes on the magnetic nanoparticles

274

offers added advantage of high enzyme loading. As there is no problem of pore size restrictions,

275

surface immobilization on iron MNPs ensures a high loading of enzyme. Diffusion will not limit

276

the kinetics of the enzymes25. An increase in immobilization of rBhCA was observed on

277

increasing the enzyme concentration (figure 2). The initial increase in loading capacity could be

278

simply because the solution containing higher enzyme concentrations has greater chance of

279

reaction with reactive groups of silane, resulting in the enhancement in immobilization of

280

rBhCA. The decline in immobilization recorded after reaching a maximum value could be due to

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281

aggregation and crowding of enzyme at high concentration, resulting in decreased exposure of

282

reactive sites on the enzyme26.

283

The Fourier transform infrared spectroscopy (FTIR) generated spectra of unmodified iron

284

MNPs, Si-MNPs and rBhCA-Si-MNPs (figure 3). All the three spectra exhibited a sharp band

285

at 588.17 cm-1 and 640.24 cm-1 characteristic of Fe-O vibration related to the nanomagnetite

286

core27. Peak observed at 1627.6 in the unmodified MNPs and 1633.39 cm-1 is due to the

287

adsorption of water molecules to the surface of MNPs and Si-MNPs via hydrogen bond. A broad

288

peak at 3200-3500 cm-1 is also seen in all the spectra which originate from the hydroxyl group

289

present on the surface of the particles, uncondensed silanol groups present in the coating layer on

290

MNPs, and water on the surfaces of the MNPs adsorbed physically and chemically22.The peaks

291

observed at 2923.52 and 2854.1 cm-1 are because of antisymmetric and asymmetric stretching

292

vibrations of C-H bond and are due to contamination with organic solvent used during synthesis

293

of iron MNPs, indicating that multiple washing did not completely remove all the organic

294

solvent28.

295

The modification of iron MNPs with silane polymer was confirmed by the bands present at

296

1114.8 and 1047.2 cm-1 corresponding to Si-O-H and Si-O-Si groups29 and the absorption of the

297

–NH2 bending/scissoring vibration at 1560.11 cm−1

298

silanized iron MNPs. In the spectra of rBhCA-Si-MNPs, the appearance of peak at 1648 cm-1

299

can be assigned to the amide carbonyl (CO-NH2) group, which confirms the formation of bond

300

between enzyme and Si- MNPs30. Also, the FTIR spectrum of rBhCA-Si-MNPs (figure 3)

301

displayed an intense and broad absorption band at 3410–3450 cm-1, which is the characteristic

302

signal of the stretching vibration of amine (NH2) group31. The band at 795 cm-1 relative to δ(HC

22

.These results indicate the formation of

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303

= CH) out-of-the-plane vibration indicates the presence of histidine groups32. These peaks

304

revealed the successful immobilization of rBhCA on Si-MNPs.

305

The potential of rBhCA-Si-MNPs for industrial applications was further studied by studying the

306

alkalistability, thermostability, reusability and storage properties. It is known that immobilization

307

may lead to a change in conformation of enzyme, which may alter its properties. rBhCA-Si-

308

MNPs showed improved pH stability in the alkaline range as compared to the free rBhCA under

309

the same conditions17 (figure 4). A similar improvement in pH stability has been observed for

310

CA from

311

immobilized CA exhibited high residual activity at different pH as compared to the free enzyme

312

33

313

immobilization led to a decrease in pH stability of the enzyme34. The improved pH stability of

314

rBhCA-Si-MNPs is a good indication as the stability at alkaline pH is important for efficient

315

sequestration of CO2 into CaCO335.

316

The thermal stability of the enzyme is one of the most crucial factors for industrial applications.

317

In particular for CA, stability between temperature range of 40-60 °C is important for CO2

318

sequestration from flue gas36. Immobilization of rBhCA on silanized MNPs augmented the

319

thermostability as compared to the free rBhCA (figure 5). The enhanced stabilization thus

320

attained upon immobilization could be explained by the fact that the multipoint covalent bonding

321

between rBhCA molecule and the solid support, which restricts the enzyme to the local

322

environment of the solid support37, 38. Therefore, immobilized rBhCA exhibited greater rigidity

323

and higher resistance to unfolding at elevated temperatures than the free rBhCA. CA

324

immobilized on Chitosan/SiO2/γ-Fe2O3 composite support also displayed significantly improved

325

thermal stability with 30% activity retention at 70 °C, whereas the free CA lost 91% activity25.

B. pumilus immobilized on mesoporous aluminosilicates (CAImAlK), where the

. While in case of CA immobilized on chitosan-alginate polyelectrolyte complex (CA-PEC),

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Page 24 of 35

326

An improvement in thermostability of CA upon immobilization has also been reported by

327

Kanbar and Ozdemir39 and Bhattacharya et al.40

328

Reusability of enzyme is yet another important parameter for using enzymes for industrial

329

applications, since the cost of enzyme production is expensive. Immobilization can bring down

330

the cost of enzyme-driven processes by allowing the reuse of enzymes multiple times41. rBhCA-

331

Si-MNPs were found to be reasonably reusable up to 22 cycles The loss of activity after each

332

cycle could be due to loss of MNPs and leaching of enzyme from the MNPs (figure 6). The CA

333

immobilized on chitosan-alginate polyelectrolyte complex (CA-PEC) could be used up to 6th

334

and 8th cycle retaining 71 and 53 % activity, respectively. Yadav et al.14 reported reusability up

335

to 6 cycles with 67 % activity retention, for CA immobilized on alginate matrix. While CA

336

immobilized on chitosan based activated alumina–carbon composite beads retained 50 % activity

337

over 4 cycles only42. CA immobilized on chitosan/SiO2/γ-Fe2O3 retained 90 % activity after 10

338

cycles of reuse25. Kanbar and Ozdemir39 reported CA immobilized in polyurethane foam could

339

be used up to 7 cycles with 100 % retention of activity.

340

The economics of industrial applications of enzymes are affected by the cost associated with

341

enzyme production. Industrial enzymes with long-term reusability and storage stability without

342

compromise in their activity are, therefore, preferred. As displayed in figure 7, free BhCA is a

343

robust enzyme which possesses long term storage stability and is stable for years both at 4 °C

344

and room temperature16. Whereas a slight decrease in the activity of rBhCA-Si-MNPs is

345

observed after 28 days of storage. This decline is probably due to leaching of enzyme molecule

346

from the immobilization support. The storage stability of rBhCA-Si-MNPs is comparable to the

347

stability attained by other methods of CA immobilization, such as CA immobilization on

348

surfactant-modified silylated chitosan (SMSC)43 on PU foam39 and chitosan SiO2/γ-Fe2O314. 16 ACS Paragon Plus Environment

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349

The rBhCA-Si-MNPs retained the properties of stimulation by SO42- and tolerance to SO42- and

350

NO32- (Table 2) like free rBhCA 17. This further supports the utility of rBhCA-Si-MNPs for CO2

351

sequestration from flue gas as SOX and NOX in very small amounts are known to exert toxic

352

effect on CA leading to reduction in activity44. Pb2+ present in flue gas also exerts inhibitory

353

effect on the activity of CA enzymes. Interestingly, rBhCA-Si-MNPs showed a slight increase in

354

the residual activity compared to free rBhCA in presence of Pb2+. The slight improvement in

355

tolerance to Pb2+ could be due to masking of some of the Pb2+ interaction site on the enzyme due

356

to immobilization.

357

The ability of the immobilized system in mineralization of CO2 was studied in terms of time

358

required for the onset of precipitation and the final amount of CaCO3 formed. Interestingly, the

359

use of rBhCA-Si-MNPs caused a significant acceleration in the time required for the onset of

360

CaCO3 precipitation as compared to the free rBhCA (Table 3). A similar observation had been

361

made with CA immobilized on activated alumina-carbon composite beads, where immobilized

362

CA showed rapid onset of precipitation in comparison with free CA43 and CA immobilized in

363

alginate bead14. The time required for the onset of precipitation was similar in the control

364

reactions (having Si-MNPs instead of enzyme and blank), indicating that Si-MNPs do not

365

influence the reaction catalyzed by CA, consequently precipitation, suggesting the importance of

366

CA in accelerating mineralization of CO2. The amount of carbonate precipitate attained with

367

either the free or the rBhCA-Si-MNPs was the same which is consistent with earlier report by

368

Yadav et. al14. While, Wanjari et al.10 and Oviya et al.34 reported that the amount of carbonate

369

precipitated by immobilized enzyme was less than that with free enzyme. Moreover, using both

370

the immobilized rBhCA and free rBhCA17, CO2 was sequestered into calcite form of CaCO3

371

which is the most stable, durable and coherent polymorph of CaCO3 (figure 8). 17 ACS Paragon Plus Environment

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Page 26 of 35

372

The rBhCA-Si-MNPs performed better than free rBhCA for mineralization based sequestration

373

of CO2 because of the improved alkalistability and thermal stability, reasonably good reusability

374

and storage properties of the former over the latter.

375

5.0. Conclusions

376

The rBhCA has been successfully immobilized on MNPs. The immobilized rBhCA displayed

377

improved operational stability and reusability in carbon sequestration, thus, the rBhCA-Si-MNPs

378

are quite suitable for use in harsh operational conditions.

379

380

Acknowledgements

381

We are grateful to the University Grants Commission and Indian Council of Medical Research,

382

Govt. of India and University of Delhi for financial assistance and award of fellowship to SF

383

during the course of this investigation. Authors wish to thank Advanced Instrumentation

384

Research Facility (AIRF), Jawaharlal Nehru University, New Delhi for extending help in SEM

385

analysis.

386

Technology, New Delhi) and Miss Neerja (Department of Chemistry, Indian Institute of

387

Technology, New Delhi) for extending help in FTIR analysis.

We also thank Prof. Sunil Khare (Department of Chemistry, Indian Institute of

388

389

390

391

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392

Energy & Fuels

Tables section

393

394

Table 1 Effect of silanization of magnetite particles on the loading capacity of rBhCA

395

APTES (%)

Si-MNPs (mg)

rBhCA (mg/mL)

Bound rBhCA (mg)

396

397

398

399

400

401

402

2.0

10

1

1.9

5.0

10

1

1.2

10.0

10

1

2.2

15.0

10

1

3.8

20.0

10

1

5.7

25.0

10

1

5.7

30.0

10

1

5.7

35.0

10

1

5.7

40.0

10

1

5.7

45.0

10

1

5.7

50.0

10

1

5.7

55.0

10

1

5.7

60.0

10

1

5.7

403

404

405

406

407

408

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409

Table 2. Effect of various ions on the activity of rBhCA-Si-MNPs Anion

Residual activity (%)

Concentration

SO4-

100±0

1.0 M

172 ±3.0

1.25M

100.±0.5

1.0 M

100 ±0

1.25M

100 ±1.5

0.5 M

85.1 ±1.5

1.0 M

77.4±1.8

1.5 M

100±0

1.0 mM

85.0±3.0

5.0 mM

20±2.0

500 µM

0±2.8

1.0 mM

SO32-

NO3-

Pb2+

Hg2+

410

Page 28 of 35

*Data presented as mean±S.D of three readings

411 412 413 414 415 20 ACS Paragon Plus Environment

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416

Table 3. Onset of carbonate precipitation with free rBhCA, rBhCA-Si-MNPs and Si-MNPs.

417

rBhCA-Si-MNPs significantly reduced the time required for the onset of CaCO3 precipitation.

418

Sample

Free rBhCA

rBhCA-SiMNPs

Si-MNPs

Blank

Time (s)

32 ± 1

10.5 ±2.5

138 ± 2.0

139.5 ± 1.5

*Data presented as mean ±S.D of three readings

419

420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436

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References

438

(1) Shakun, J. D.; Clark, P. U.; He, F.; Marcott, S. A.; Mix, A. C.; Liu, Z.; Bard, E. Global

439

warming preceded by increasing carbon dioxide concentrations during the last

440

deglaciation. Nature 2012 484, 49-54.

441

(2) Del Prete, S.; Vullo, D.; Fisher, G. M.; Andrews, K. T.; Poulsen, S. A.; Capasso, C.;

442

Supuran, C. T. Discovery of a new family of carbonic anhydrases in the malaria pathogen

443

Plasmodium falciparum—The η-carbonic anhydrases. Bioorg. Med. Chem. Lett. 2014 24,

444

4389-4396.

445

(3) Di Fiore, A.; Alterio, V.; Monti, S. M.; De Simone, G.; D'Ambrosio, K. (2015).

446

Thermostable carbonic anhydrases in biotechnological applications. Int. J. Mol. Sci. 2015

447

16, 15456-15480.

448

(4) Faridi, S.; Satyanarayana, T. Applicability of carbonic anhydrase in mitigating global

449

warming and development of useful products from CO2. Climate Change and

450

Environmental Sustainability 2015, 3, 77-92.

451

(5) Faridi, S.; Satyanarayana, T. Prospects in biomimetic carbon sequestration. In Carbon

452

Capture, Storage and Utilization: a possible climate change solution for energy industry,

453

Goel, M. Sudhakar, M., Sahi, R.V. Eds., TERI Press: New Delhi, 2015; pp 167-187.

454

(6) Liu, N.; Bond, G. M.; Abel, A.; McPherson, B. J.; Stringer, J. Biomimetic sequestration

455

of CO2 in carbonate form: Role of produced waters and other brines. Fuel. Process.

456

Technol. 2005, 86, 1615-625.

457

(7) Prabhu, C.; Wanjari, S.; Gawande, S.; Das, S.; Labhsetwar, N.; Kotwal,S.;

458

Satyanarayanana, T.; Puri, A.; Rayalu, S. Immobilization of carbonic anhydrase enriched

459

microorganisms on biopolymer based materials. J. Mol. Catal. B Enzym. 2009, 60, 13-21. 22 ACS Paragon Plus Environment

Page 31 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Energy & Fuels

460

(8) Oviya, M.; Giri, S.S.; Sukumaran, V.; Natarajan, P. Immobilization of carbonic

461

anhydrase enzyme purified from Bacillus subtilis VSG-4 and its application as CO2

462

sequesterer. Prep. Biochem. Biotech. 2012, 42, 462-475.

463

(9) Prabhu, C.; Wanjari, S.; Puri, A.; Bhattacharya,A.;Pujari,R.; Yadav,R.; Das, S.;

464

Labhsetwar, N.; Sharma, A.; Satyanarayanana, T.; Rayalu, S. Region-specific bacterial

465

carbonic anhydrase for biomimetic sequestration of carbon dioxide. Energy. Fuels. 2011,

466

25, 1327-1332.

467

(10) Wanjari, S.; Prabhu, C.; Yadav,R.; Satyanarayana, T.; Labhsetwar, N.; Rayalu, S.

468

Immobilization of carbonic anhydrase on chitosan beads for enhanced carbonation

469

reaction. Proc. Biochem. 2011, 46, 1010-1018.

470

(11) Yadav, R.; Wanjari, S.; Prabhu, C.; Vivek, K.; Labhsetwar, N.; Satyanarayana, T.;

471

Kotwal, S.; Rayalu, S. Immobilized carbonic anhydrase for the biomimetic carbonation

472

reaction. Energy. Fuels. 2010, 24, 6198-6207.

473 474 475

(12) Xu, J.; Sun, J.; Wang, Y.; Sheng, J.; Wang, F.; Sun, M. Application of iron magnetic nanoparticles in protein immobilization. Molecules. 2014, 19, 11465-11486. (13) Laurent, S.; Forge, D.; Port, M.; Roch, A.; Robic, C.; Vander Elst, L.; Muller, R. N.

476

Magnetic

iron

oxide

nanoparticles:

synthesis,

stabilization,

vectorization,

477

physicochemical characterizations, and biological applications. Chem. Rev. 2008, 108,

478

2064-2110.

479

(14) Yadav, R.; Joshi, M.; Wanjari, S.; Prabhu, C.; Kotwal, S.; Satyanarayana, T.; Rayalu, S.

480

Immobilization of carbonic anhydrase on chitosan stabilized iron nanoparticles

481

for the carbonation reaction. Water. Air. Soil. Pollut. 2012, 223, 5345-5356.

23 ACS Paragon Plus Environment

Energy & Fuels

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

482

(15) Mikhaylova, M.; Kim, D.K.; Berry, C.C.; Zagorodni, A.; Toprak, M.; Curtis, A.S.G.;

483

Muhammed, M. BSA immobilization on amine-functionalized superparamagnetic iron

484

oxide nanoparticles. Chem. Mater. 2004, 16, 2344–2354.

485

(16) Faridi, S.; Satyanarayana, T. Novel alkalistable α-carbonic anhydrase from the

486

polyextremophilic bacterium Bacillus halodurans: Characteristics and applicability in

487

flue gas CO2 sequestration. Environ. Sci. Pollut. Res. 2016a, 23, 15236–15249.

488

(17) Faridi, S.; Satyanarayana, T. Characteristics of recombinant α-carbonic anhydrase of

489

polyextremophilic bacterium Bacillus halodurans TSLV1. Int. J. Biol. Macromolec.

490

2016b, 89, 659-68

491

(18) Mahdavi, M.; Ahmad, M.B.; Haron, M.J.; Namvar, F.; Nadi, B.; Rahman, M.Z.A.; Amin,

492

J. Synthesis, surface modification and characterisation of biocompatible magnetic iron

493

oxide nanoparticles for biomedical applications. Molecules. 2013, 18, 7533-7548.

494

(19) Khalifah, R.G.; Silverman, D.N. In The carbonic anhydrases: Cellular physiology and

495

molecular genetics, Dodgson, S.J., Tashian, R.E., Gros, G., Carter, N.D. Eds., Plenum

496

Press: New York, 1991, pp 49-70.

497 498

Page 32 of 35

(20) Tran, D.N.; Balkus, K.J. Perspective of recent progress in immobilization of enzymes. ACS Catal. 2011, 956–96.

499

(21) Deng, Y.; Qi, D.; Deng, C.; Zhang, X.; Zhao, D. Superparamagnetic high-magnetization

500

microspheres with an Fe3O4@SiO2 core and perpendicularly aligned mesoporous SiO2

501

shell for removal of microcystins. J. Am. Chem. Soc. 2008, 130, 28-29.

502

(22) Ma, M.; Zhang, Y.; Yu, W.; Shen, H.Y.; Zhang, H.Q.; Gu, N. Preparation and

503

characterization of magnetite nanoparticles coated by amino silane. Colloids Surf. A.

504

2003, 212, 219-226.

24 ACS Paragon Plus Environment

Page 33 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Energy & Fuels

505

(23) Hong, J.; Xu, D.; Gong, P.; Sun, H.; Dong, L.;Yao, S. Covalent binding of α-

506

chymotrypsin on the magnetic nanogels covered by amino groups. J. Mol. Catal. B:

507

Enzym. 2007, 45(3-4), 84-90.

508

(24) Thomson, S. A.; Josey, J. A.; Cadilla, R.; Gaul, M. D.; Fred Hassman, C.; Luzzio, M. J.

509

Fmoc mediated synthesis of peptide nucleic acids. Tetrahedron. 1995, 51(22), 6179–

510

6194.

511

(25) Sahoo, P.C.; Jang, Y.N.; Lee, S.W. Immobilization of carbonic anhydrase and an

512

artificial Zn(II) complex on a magnetic support for biomimetic carbon dioxide

513

sequestration. J. Mol. Catal. B Enzym. 2012, 82, 37-45.

514

(26) Jiang, D.S.; Long, S.Y.; Huang, J., Xiao, H.Y., Zhou, J.Y. Immobilization of Pycnoporus

515

sanguineus laccase on magnetic chitosan microspheres. Biochem. Eng. J. 2005, 25, 15–

516

23.

517

(27) Durdureanu-Angheluta, A.; Uritu, C.M.; Coroaba, A.; Minea, B.; Doroftei, F.; Calin, M.;

518

Maier, S.S.; Pinteala, M.; Simionescu, M.; Simionescu, B.C. Heparin-anthranoid

519

conjugates associated with nanomagnetite particles and their cytotoxic effect on cancer

520

cells. J. Biomed. Nanotechnol. 2014,10,131-142.

521

(28) Olsson, R.T.; Hedenqvist, M.S.; Ström, V.; Deng, J.; Savage, S.J.; Gedde, U.W.

522

Core‐shell

structured

ferrite‐silsesquioxane‐epoxy

nanocomposites:

Composite

523

homogeneity and mechanical and magnetic properties. Polym. Eng. Sci. 2011, 51, 862-

524

874.

525

(29) Bumajdad, A.; Ali, S.; Mathew, A. Characterization of iron hydroxide/oxide

526

nanoparticles prepared in microemulsions stabilized with cationic/non-ionic surfactant

527

mixtures. 2011, J. Colloid. Interface. Sci. 355, 282-292.

25 ACS Paragon Plus Environment

Energy & Fuels

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Page 34 of 35

528

(30) Swarnalatha, V.; Esther, R.A.; Dhamodharan, R. Immobilization of α-amylase on gum

529

acacia stabilized magnetite nanoparticles, an easily recoverable and reusable support. J.

530

Mol. Catal. B. 2013, 96, 6–13.

531

(31) Roberge, M.; Lewis, R.N.; Shareck, F.; Morosoli, R.; Kluepfel, D.; Dupont, C.;

532

McElhaney, R.N. Differential scanning calorimetric, circular dichroism, and Fourier

533

transform infrared spectroscopic characterization of the thermal unfolding of xylanase A

534

from Streptomyces lividans. Proteins 2003, 50 341–354.

535

(32) Costa, V.M.; de Souza, M.C.M.; Fechine, P.B.A.; Macedo, A.C.; Gonçalves, L.R.B.

536

Nanobiocatalytic systems based on lipase-Fe3O4 and conventional systems for isoniazid

537

synthesis: a comparative study. Braz. J. Chem. Eng. 2016, 33, 661-673.

538

(33) Wanjari, S.; Prabhu, C.; Satyanarayana, T.; Vinu, A.; Rayalu, S. Immobilization of

539

carbonic

anhydrase

on

mesoporous

aluminosilicate

540

Microporous Mesoporous Mat. 2012, 160, 151-158.

for

carbonation

reaction.

541

(34) Oviya, M.; Sukumaran, V.; Giri, S.S. Immobilization and characterization of carbonic

542

anhydrase purified from E. coli MO1 and its influence on CO2 sequestration. World J.

543

Microbiol. Biotechnol. 2013, 29, 1813-1820.

544

(35) Faridi, S.; Satyanarayana, T. Bioconversion of industrial CO2 emissions into utilizable

545

products by employing microbes and their enzymes, In Chandra R (ed) Environmental

546

waste management, CRC Press: New York, 2016c, pp 111–156.

547 548

(36) Bonra, K.; Ekrem, O. Thermal stability of carbonic anhydrase immobilized within polyurethane foam. Amer. Inst. Chem. Engs. 2010, 26, 1474-1480.

26 ACS Paragon Plus Environment

Page 35 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Energy & Fuels

549

(37) Li, T.P.; Li, S.H.; Wang, N.; Tain, L. Immobilization and stabilization of pectinase by

550

multipoint attachment onto an activated agar-gel support. Food. Chem. 2008, 109, 703–

551

708.

552 553 554 555 556 557

(38) Chiou, S.H.; Wu, W.T. Immobilization of Candida rugosa lipase on chitosan with activation of the hydroxyl groups. Biomaterials. 2004, 25, 197–204. (39) Kanbar, B,; Ozdemir, E. Thermal stability of carbonic anhydrase immobilized within polyurethane foam. Biotechnology. Progress. 2010, 26, 1474-1480. (40) Bhattacharya, S.; Schiavone, M.; Chakrabarti, S.; Bhattacharya, S.K. CO2 hydration by immobilized carbonic anhydrase. Biotechnol. Appl. Biochem. 2003, 38, 111-117.

558

(41) Camacho-Rubio, F.; Jurado-Alameda, E.; Gonzalez-Tello, P.; Luzon Gonzalez, G.A.

559

Comparative study of the activity of free and immobilized enzyme and its application to

560

glucose isomerase. Chem. Eng. Sci. 1996, 51, 4159–65.

561

(42) Prabhu, C.; Valechha, A.; Wanjari, S.; Labhsetwar, N.; Kotwal, S.; Satyanarayana, T.;

562

Rayalu, S. Carbon composite beads for immobilization of carbonic anhydrase. J. Mol.

563

Catal. B. Enzym. 2011, 71, 71-78.

564

(43) Yadav, R.; Wanjari, S.; Prabhu, C.; Kumar, V.; Labhsetwar, N.; Satyanarayanan, T.;

565

Kotwal, S.; Rayalu, S. Immobilized carbonic anhydrase for the biomimetic carbonation

566

reaction. Energy. Fuels. 2010, 24, 6198-6207.

567 568

(44) Li, L.; Fu, M.; Zhaou, Y.; Zhu, Y. Characterization of carbonic anhydrase II from

Chlorella vulgaris in bio-CO2 capture. Environ. Sci. Pollut. Res. 2012, 19, 4227-4232.

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