Cloning, expression and characterization of β- and γ‑carbonic anhydrase from Bacillus sp. SS105 for biomimetic sequestration of CO2

Accepted Manuscript Cloning, expression and characterization of β- and γ'carbonic anhydrase from Bacillus sp. SS105 for biomimetic sequestration of CO2

Neha Maheshwari, Madan Kumar, Indu Shekhar Thakur, Shaili Srivastava PII: DOI: Reference:

S0141-8130(19)30048-0 https://doi.org/10.1016/j.ijbiomac.2019.03.082 BIOMAC 11912

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

2 January 2019 4 March 2019 12 March 2019

Please cite this article as: N. Maheshwari, M. Kumar, I.S. Thakur, et al., Cloning, expression and characterization of β- and γ'carbonic anhydrase from Bacillus sp. SS105 for biomimetic sequestration of CO2, International Journal of Biological Macromolecules, https://doi.org/10.1016/j.ijbiomac.2019.03.082

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ACCEPTED MANUSCRIPT Title of the paper Cloning, expression and characterization of β- and γ-carbonic anhydrase from Bacillus sp. SS105 for biomimetic sequestration of CO2 Neha Maheshwari1, Madan Kumar2, Indu Shekhar Thakur2, Shaili Srivastava1, 2*

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1. Amity School of Earth and Environmental Science, Amity University Haryana, India

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2. School of Environmental Sciences, Jawaharlal Nehru University, New Delhi 110067, India

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Running title

calcite production

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*Address of the corresponding author

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Purification and characterization of β- and γ-carbonic anhydrases from Bacillus sp. SS105 for

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Shaili Srivastava

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Amity School of Earth and Environmental Science

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Amity University Haryana

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Telephone: +91-124-2337015 Ext 1406 E.mail: [email protected] The number of text pages:

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Legends and Figure:

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Table:

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ACCEPTED MANUSCRIPT Cloning, expression and characterization of β- and γ-carbonic anhydrase from Bacillus sp. SS105 for biomimetic sequestration of CO2 Abstract Bacterium Bacillus sp. SS105, isolated from Free Air CO2 Enriched (FACE) soil was previously

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screened for carbonic anhydrase activity and CO2 sequestration. In this study, strain was selected

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to amplify carbonic anhydrase encoding genes. The CA genes from Bacillus sp. SS105 were

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found to be homologous with beta-carbonic anhydrase (β-CA) and gamma-carbonic anhydrase (γ-CA). Both types of CA genes was cloned in pET30b (+) and expressed in E coliBL21 (DE3)

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with His-tag at the N-terminus. The recombinant proteins were purified by Ni-NTA affinity

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chromatography. The molecular size of β-CA and γ-CA were approximately 27 kDa and 25 kDa respectively. The optimum pH and temperature were found to be 8.0 and 37 °C respectively. The

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Zn+ was enhancing the CAs enzyme activity. Anions and modulators showed inhibitory effect on

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CAs at specific concentration. Functional domain analysis of both CA proteins showed conserved region of respective proteins. Recombinant enzymes were used for bio-mineralization

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based conversion of atmospheric CO2 into valuable calcite. Calcite formation was evaluated with

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or without use of enzymes and confirmed by SEM and XRD analysis. SEM result confirmed the conversion of flower-shaped unstable form of vaterite to hexagonal cubic stable form of calcite

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in presence of enzymes.

Keywords: CO2 sequestration; Carbonic anhydrase;Bacillus sp. SS105

ACCEPTED MANUSCRIPT 1. Introduction The continuous elevation in the concentration of carbon dioxide (CO2) due to anthropogenic activities had led to several undesirable consequences in the climate. To combat the climate changes, several carbon capture and sequestration (CCS) technologies have been

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deliberated in recent years. Biological CO2 sequestration in form of bicarbonate is the most

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effective methods to reduce rising levels of CO2 into the atmosphere by microorganism [1].The

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conversion of CO2 into bicarbonate within the cell is depending upon the level of CO2 and bicarbonate inside and outside the cell [2, 3]. Since this hydration reaction is very slow, carbonic

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anhydrase used to enhance the reaction with the catalytic rate of 104-106 reactions per second.

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Carbonic anhydrase (CA) is a metalloenzyme, coordinated either with zinc, iron or cadmium via three histidine residues present at the active site. It can facilitate the inter-conversion of CO2 and

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HCO3- by nucleophilic attack of CO2 by metal bounded hydroxide ion, followed by the

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displacement of bicarbonate by a water molecule and removal of a proton from the active site [46]. CAs is present in all three domains of life i.e. bacteria, archaea and eukarya [2]. Based on

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sequence and structure similarities, CAs are classified into wide variety of evolutionarily distinct

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classes i.e alpha (α), beta (β), gamma (γ), epsilon (ε), delta (δ), zeta (ζ), eta (η) and theta (θ) carbonic anhydrases [4, 7-9]. These enzymes play variable physiological roles in different

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organisms, such as respiration, pH homeostasis, CO2/ bicarbonate transport and carbon fixation [4]. The most extensively studied α-class CAs has been found in mammals, protozoa, prokaryotes, fungi, algae and plant cytoplasm. The α-class CAs has been identified in bacterium such as Neisseria gonorrhoeae, Sulfurihydrogenibium yellow stonense, Sulfurihydrogenibium azorense, Ralstonia eutropha etc. [10]. The β-class CA was first identified in plants and has been found in other photosynthetic bacteria (Ralstonia eutropha, Citrobacter freundii, Pseudomonas

ACCEPTED MANUSCRIPT aeruginosa, P. profundum) where they play a role in the inter-conversion of CO2 to bicarbonate [11-14]. The γ-class CAs was initially restricted to archaea, although recently have been identified and characterized in eubacteria (Enterococcus faecalis, Staphylococcus aureus, Serratia sp. etc) [10, 15]. The enzymes of CA family (β-CA and γ-CA) has been previously

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found in gram-negative (Helicobacter pylori, Brucellasuis, Porphyromonas gingivalis, and

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Ralstoniaeutropha) and gram-positive bacteria (Mycobacterium tuberculosis, Clostridium

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perfringens, Streptococcus pneumonia and Bacillus subtilis) [10]. Previously carbonic anhydrase activities have been reported in Helicobacter pylori [16], Pseudomonas fragi [17], Neisseria

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gonorrhoeae [18] and cyanobacteria like Synechocystis [19]. The δ-and ζ-classes have been

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found in marine diatoms [8]. The ε-CA has been found in cyanobacteria. It is a modified form of β-class of CA. The η-class of CA has been found in Plasmodium and most recent θ-class of CA

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has been reported in diatom, chlorophyte and cyanobacterium [8, 20]. Bicarbonate generated by

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CA during CO2 sequestration can be mineralized using divalent metal ions into carbonates of mineral i.e calcium or magnesium carbonate. Microbial induced role of carbonic anhydrase in

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calcium carbonate precipitation has been previously reported [17, 21-23]. Production of γ-

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carbonic anhydrase enzyme by bacterium for the calcification and biomineralization has been reported in Serratia sp. ISTD04 [15]. Therefore, in the present study β-CA and γ-CA genes were

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identified from Bacillus sp. SS105. Identified β-CA and γ-CA enzyme were overexpressed, purified and characterized to study its potential application in carbon sequestration. The enzyme has been found suitable for biomineralization based carbon sequestration. 2. Methods 2.1. Screening and selection of potential strain

ACCEPTED MANUSCRIPT Five bacterial strains isolated from FACE soil were previously screened for CA activity using a hydration assay [24, 25]. A strain exhibiting the highest carbonic anhydrase activity was identified and selected for further study in cloning and expression followed by purification of carbonic anhydrasefor CO2 sequestration in form of calcite production.

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2.2. Cloning of β and γ-CA encoding genes

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The region encoding β-CAand γ-CA were amplified using Bacillus sp. SS105 genomic DNA as a template. PCR was performed using degenerate primers designed with restriction sites Kpn1

and

BamH1

to

amplify

β-carbonic

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for

CGGGGTACCATGAAGTCATTAGAAGAGAT-3';

anhydrase

(β-Ca-Forward

β-Ca-Reverse

5'5'-

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CGCGGATCCTTAATTGTCGTAGCCATTC-3') and γ-carbonic anhydrase (γ-CA-Forward 5'γ-CA-Reverse

5'-

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CGGGGTACCATGATATATCCTTACAAAG-3';

CGCGGATCCTTATTTTTGTAGTGATTTAT-3'). Amplified PCR products were analyzed by

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gel electrophoresis and purified by gel extraction kit (QIAGEN). The vector pET30b(+) contains

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a unique site for Kpn 1 at the initiator ATG position and Bam H1 further at downstream. The vector pET30b (+) was introduced with T7 promoter and a N-terminal hexahistidine (6xHis) tag

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to construct recombinant pET30b(+)-carbonic anhydrase genes. The gel extracted PCR products

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and the pET30b(+) vector were digested overnight with restriction enzymes for Kpn 1 and Bam H1 sites at 37°C. Digested products were purified using purification kit (QIAGEN) and ligated overnightusing T4 DNA ligase. The ligatedplasmids were transformed into E. coli BL21 cells (DE3) containing gene for T7 RNA polymerase under the control of the lac promoter. Positive recombinant clones with inserts of desired genes were selected by PCR screening. Ligated plasmids were purified from the transformed cells using a plasmid purification kit (QIAGEN)

ACCEPTED MANUSCRIPT and the presence of CA genes in the plasmids was confirmed by colony PCR and gene sequencing methods [15, 26, 27]. 2.3. Enzyme induction and expression of CAs enzymes in E.coli strain Over-expression of the carbonic anhydrase genes were performed by transforming the

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recombinant plasmids into the E. coli BL21(DE3) cells. Transformed cells were inoculated in

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Luria-Bertani (LB) broth supplemented with kanamycin (100 µg ml-1) and culture was grown at 37 °C to achieve optical density 0.5-0.6 at 600 nm. E. coli BL21(DE3) was a supporting strain

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for over-expression of CAs from plasmids that were driven by T7 promoter [28]. The E. coli BL21 (DE3) strain has DE3 sequence in its genome to encode T7 RNA polymerase under the

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control of the isopropyl thio-P-D-galactoside (IPTG)-inducible lac promoter. Protein expressions

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were induced by IPTG to a final concentration of 1 mM in culture medium and the cells were grown additional for 4-5h at 37°C. Culture cell pellets were collected after centrifugation at

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14,000xg for 30 min at 4°C. A small amount of cells were lysed by using sonicator with 10 mL

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lysis buffer (10mM DTT, 1mM PMFS, 500 µl of 100 mM Tris-Cl and 140 mM lysozyme) and remaining cell pellet was stored at -20°C for purification purpose. Cell debris was removed by

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centrifugation at 14,000xg for 30 min at 4°C to separate the total expressed proteins. Expressed

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protein samples were denatured with SDS and loaded on a 12% SDS–polyacrylamide gel to check the expression of desired proteins of β-CA and γ-CA [26]. 2.4. Purification of CAs enzymes Purification of the expressed recombinant proteins was performed by using Ni-NTA Fast Start Kit (QIAGEN). Collected frozen pellet of recombinant E.coli BL21 (DE3) cells were thawed on ice for 15 min and resuspended in 10 ml of lysis buffer (pH 8.0) supplemented with 100 µl of lysozyme and 10 µl of Benzonase Nuclease solution. The mixture was incubated on ice

ACCEPTED MANUSCRIPT for 30 min. Cellular debris was removed by centrifugation at 14,000 x g for 30 min at 4°C. Cell lysate supernatant was used to apply on resin containing Fast Start column (0.5 ml). After removing flow through, the column was washed twice with 4 ml wash buffer (pH 8.0). The bound 6x His-tagged protein was eluted two times with 1 ml aliquots of elution buffer. Elution

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fraction was collected in a separate tube and analyzed by SDS-PAGE [29, 30]. Total

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concentrations of bound tagged proteins were determined by the Bradford method [31] and

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enzymatic assay was also performed to determine the total enzymatic activity of purified β-CA and γ-CA. Both samples were loaded on 12% SDS–Polyacrylamide gel to visualize the band size

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of the particular protein [26].

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2.5. Total protein concentration and enzyme assay of CAs

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Total protein concentrations were determined by the Bradford method. Bovine serum albumin (BSA) was used as a standard of protein concentration. Confirmations of the active

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nature of the purified CA were performed on the basis of their enzymatic hydration assay.

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Hydration assay for CA was done with a mixture containing 30 ml Tris–sulfate buffer (pH 8.3), 0.5 mg enzyme and 20 ml of CO2-saturated water to initiate the hydration reaction. CA activity

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was calculated and expressed in Wilbur-Anderson (WA) units per milligram of protein [15, 25,

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32].The Michaelis-Menten constant (Km) and maximum velocity of enzyme (Vmax) values were calculated using Lineweaver–Burk double reciprocal plot of carbonic anhydrase activities. Saturated solution of CO2 ranged from 1 to 20 mM was used as different concentrations of substrate [33]. 2.6. Effect of different pH, temperature, metal ions, anions and modulators on CAs stability The enzyme activity was measured at different pH using various buffers (50 mM Tris–Cl pH 4.0–5.0; 50 mM sodium phosphate, pH 6.0–7.6; 50 mM sodium carbonate, pH 8.0, 9.0, 10.0,

ACCEPTED MANUSCRIPT 11.0 and 12.0). The enzyme sample was incubated in different buffers (pH 3.0–11.0) at 37°C for 1 h and the residual activity was measured to check the pH stability [15]. Effect of temperature on the stability of purified carbonic anhydrase (β-CA and γ-CA) was studied by measuring enzyme activity at 0, 10, 20, 30, 40, 50, 60 °C using phosphate buffer of

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pH 8 for 10 min. The residual enzyme activity was determined after incubation period to check

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the stability [15].

The enzyme was treated with salts of different metal ions (Fe2+, Mg2+, Co2+, Zn2+ and

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Hg2+), anions (HCO3-, CO3-, NO3-, SO4- and Cl-) and modulators (EDTA, DTT, SDS, Tween 20

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and Triton X 100) at pH 8 and 37°C for 1h and the residual enzyme activity was assayed as described previously [15, 23]. The activity of the carbonic anhydrase without pretreatment was

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considered as 100%.

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2.7 Effect of CA specific inhibitors on their activity

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Well known CA specific inhibitors such as acetazolamide and sulphanilamide were used to assess the enzymatic activity of β-CA and γ-CA. The IC50 values of the given inhibitors were

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calculated by determining residual activities at different concentrations of the inhibitor.

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2.8. Enzymatic precipitation of calcium carbonate and characterization Precipitation of calcium carbonate (CaCO3) was performed using 10 mM NaHCO3 through enzymatic reaction of purified β-CA and γ-CA. To perform the enzymatic reaction, 50 ml solution mixture containing 200 mM Tris buffer (pH10.3), 25 mM CaCl2 solution, and 1 mg purified enzyme (β-CA or γ-CA) was prepared and incubated for 24 h at 30°C with 150 rpm. After incubation, a precipitate solution was filtered through Whatman filter paper and dried in an oven at 50°C. The surface morphologies of the calcium carbonate crystals (calcite, vaterite, and

ACCEPTED MANUSCRIPT aragonite) were measured by SEM using a model Carl Zeiss EVO 40 (Cambridge UK). Procedure to perform SEM was described previously [34]. The polymorphs of the calcium carbonate crystals were also identified and analyzed by X-ray powder diffraction patterns (XRD) for confirming the formation of calcium carbonate crystals by recombinant CAs. Powder X-ray

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diffraction patterns (XRD) were recorded using the X-pert system; PANalytical workstation with

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Cu Kα radiation (λ= 1.5406Å) operated at 40 kV and 25 mA. The diffraction data were recorded

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at scanning angle 2θ range with counting time of 10 s per step over the range of 20-70° [34].

3.1. Selected bacterial strain for CO2 sequestration

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3. Results and discussion

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Based on the previous study on carbonic anhydrase enzymatic assay, identified Bacillus sp.

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SS105 (Accession no.KX379741) was showing maximum activity under the culture condition of MSM with 50mM NaHCO3 (carbon source) at 30°C [25]. SS105 was dominant chemoautotroph

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of a CO2 rich environment of FACE field. This strain was further enriched in the batch culture

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containing 50mM NaHCO3 and used to overexpression, purification and characterization of β-

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CA and γ-CA for CO2 sequestration.

3.2. Cloning and expressionof CAs in E.coli strain

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The total catalytic activity of the β-CA and γ-CA were performed by cloning, heterologous expression and purification of enzymes. PCR amplification using gene-specific degenerated primers has resulted in 564 bp product for β-CA (Accession no. MH157177) and 513 bp product for γ-CA (Accession no.MH157178) with kpn1 and BamH1 restriction site (Fig 1a and 1c). The resulted gene products were purified by gel-extraction and were cloned in pET30b(+). Constructed plasmids were positively verified by DNA sequencing. Protein classifications and architecture study of conserved domains have proved that amplified CAs from Bacillus sp.

ACCEPTED MANUSCRIPT SS105 are a member of β- and γ-CA family (Fig. 1b and 1d). Recombinant plasmids were expressed in E. coli strain BL21 (DE3) at 37°C under the induction period of 1mM IPTG. Expressed recombinant proteins were isolated from a soluble fraction of E. coli and analyzed by SDS–PAGE. Overexpression of β-CA and γ-CA were indicated by SDS-PAGE with an

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approximate molecular weight of 27kDa and 25kDa for β-CA and γ-CA respectively. Previously,

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Lotlikar et al., [13] has cloned and expressed three functional β-CAs encoding genes (PAO102,

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PA2053 and PA4676) from Pseudomonas aeruginosa PAO1. In the previous literature of Gai et al., [35], CA enzymes (α, β, and γ type CA) isolated from R. eutropha have been cloned,

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heterologously expressed and purified using Escherichia coli.

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3.3. Purification of CAs using metal affinity chromatography

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Recombinant proteins were expressed with a 6xHistidine tag at N-terminal. Purification of histidine-tagged β-CA and γ-CA was occurred due to its high affinity to nickel ions immobilized

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with NTA absorbent in affinity column. This property has been exploited in the purification of

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recombinant proteins that are tagged histidine at N- or C-terminal. During purification, binding of the β-CA and γ-CA (histidine-tagged) to Ni-NTA resin was achieved after passing through

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column. Bound proteins were eluted using elution buffer and the success of their purification was

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analyzed by SDS-PAGE. The presence of distinct 27kDa bands for β-CA and 25kDa bands for γ–CA in the elution fractions indicate that both the proteins were successfully purified using NiNTA spin columns (Fig. 2). In previous literature, a molecular weight of three purified β-CA genes (psCA1, psCA2 and psCA3) has been found as 27, 23 and 24 kDa respectively [13]. In our study, total recovery and purification of β-CA was 23% and 17 fold respectively. Total recovery and purification of γ–CA was 31% and 23 fold respectively (Table 1). In the previous study, approximately 55% yield of γ–CA has been reported [15]. Purification of recombinant SspCA

ACCEPTED MANUSCRIPT form Sulfurihydrogenibium sp. YO3AOP1 has been found 27% recovery and enzyme was purified by 16 fold [36]. Ramanan et al., [12] has been reported purified β-CA protein from Bacillus subtilis SA3 strain with 15% relative yield.

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3.4. Enzymatic kinetic properties

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The values of km and Vmax were derived using an equation obtained by Lineweaver Burk

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plotting. The values of Km and Vmax were 1.54 mM and 62.75 ×10-2 µM min-1 for β-CA and 1.36 mM and 10.9 ×10-1 µM min-1 for γ-CA respectively at pH 7.6. Gamma CA from the archae

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on Methanosarcina thermophile showed Km value in range of 1.8 mM and Vmax value in range

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of 7.1×104 S-1 [37]. Gamma-CA from the Serratia sp. ISTD4 has been showed Km and Vmax value of 12 mM and 5.2×10-4µM min-1 respectively (Srivastava et al., 2015).

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3.5. Temperature, pH, metal ions, anions and modulators effect on CAs stability

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Recombinant carbonic anhydrases were subjected to various pH using phosphate buffer for 1 h incubation at 37°C to ascertain the extent of their stability. Optimal activities of purified β-

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CA and γ-CA enzyme were found to retain 100% activity at pH 8.0. CAs activity for β-CA and

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γ-CA were determined 92% and 97 % respectively at pH 7.0 (Fig 3a). Enzymes β-CA and γ-CA were sensitive to pH values below 7.0 and above 8.0. Activity was almost declined inacidic pH

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of 3.0 and a basic pH of 11.0 (Fig. 3a). Both β-CA and γ-CA were retained 13 and 16% activity respectively at pH 3.0. Similarly, CAs from Paracoccidioides has been shown 43% and 45% activity at acidic pH 5.5 for rCA1 and rCA4, respectively [38]. In comparison, CA from H. pylori has been showed high acid tolerancein the acidic environment [16]. Thus both recombinant CAs enzymes from Bacillus sp. SS105 were showed highly stable activity at pH 8.0.

ACCEPTED MANUSCRIPT The temperature denaturation effect indicated recombinant CA enzymes were not stable at the ranges of 40 to 60°C. The enzymes were found to retain the maximum activity at the range of 30 to 37°C (Fig. 3b). Ramanan et al., [12] has been also found the optimum activity of β-CA from Bacillus subtilis SA3 at 37°C. There were progressive declines in CAs activity at a

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temperature above 40 °C. The β- and γ-CA were showed only 15% and 23% of residual activity

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at 60°C. Similarly CAs from Paracoccidioides has been shown only 10% residual activity at 60

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°C [38]. In previous study, β-CA from Methano-bacterium and γ-CA from the archaeon Methanosarcina thermophile have been found to be active at 75°C [4, 8]. Thermophilic β-CA

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isolated from Methano-archaeon Methanobacterium thermoautotrophicum has been also shown

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higher temperature stability [12, 39]. In this study, we have found that γ-CA was better in enzymatic stability at various pH and temperature range as compared to β-CA. Therefore, the

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result demonstratedthat β- and γ-CA has maximum stability at pH 8.0 and 37°C as compared to

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other CAs reported earlier.

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Both carbonic anhydrase were showed inhibition to a varied extent by different concentration of metal ions, anions and other modulators(Table 2). In addition to zinc ions, CO2

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hydration activity of β-CA and γ-CAwere stimulated by 14.38% and 22.9% respectively but only

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γ-CA activity was stimulated in the presence of iron ion by 33.59%. Zinc and iron ions are physiologically relevant cofactors and have been observed as a part of the active center in different classes of CAs [40. 41]. These ions could have stabilization effects on enzyme structure and fold, thus enhancing CAs activity.CAs activity was inhibited by Mg2+, Co2+ and Hg2+.The inhibition of the CAs activity by metal ions was possibly due to its interaction with sulphydryl (SH) groups in the active site of the β-CA and γ-CA enzymes. Divalent ions (Mg2+ and Co2+) have shown weak interaction with CAs enzyme [42]. In previous literature, metal inhibition of

ACCEPTED MANUSCRIPT Hg2+ has been also suggested due to presence of thiol group in active site of CAs [40]. This implies the presence of cysteine residue near or at the enzyme active site which could play an important role in enzyme conformation [43]. The increased CAs activity in the presence of Zn2+ suggests metals may contribute to enhance β-CA and γ-CA enzymes function in the Bacillus sp.

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SS105.

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It is noteworthy that β-CA were stimulated by SO4-2 (2.0 M), while it did not affect γ-CA activity at 1.0 M and enhanced the activity at 500 mM (Table 2b). In previous literature, CA

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from Citrobacter freundii has also been showed stimulation effect of SO4 2- [44]. In the presence of 1.0 M nitrate, only 1.5 % inhibition was recorded for β-CA and 20.9 % for γ-CA. CAs from P.

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fragi, M. lylae, and M. luteus have been showed inhibition by SO42- and NO32- to a varied extent

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[45]. The increase in percentage residual activity of β-CA and γ-CA in presence of SO42- and NO32- suggested as a bio-catalyst for CO2 sequestration from flue gas. Both the enzymes retained

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100% stability at 10 mM of bicarbonate ion and maximum stability (95.76% for β-CA and

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80.4% for γ-CA) at 10 mM of carbonate ion (Table 2b).

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Both the enzymes (β-CA and γ-CA) exhibited enhance activity in presence of Triton X 100 and Tween 20 and retained 100% activity in the presence of 1 mM of DTT (Table 2c). Similar

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effect of anionic surfactants has been reported previously [23]. A strong anionic detergent (10% SDS) showed 100% inhibition. EDTA and SDS showed inhibitory effect on both type of CAs activity in all concentration (Table 2c). EDTA is a well-known inhibitor of all metalloenzymes which can chelates metal ions from carbonic anhydrase enzymes. 3.6. Effect of CA specific inhibitors on rBhCA activity

ACCEPTED MANUSCRIPT Both type of carbonic anhydrase (β-CA and γ-CA) showed strong inhibition in presence of acetazolamide with IC50 value of 0.29 µM and 0.27 µM. The second inhibitor of carbonic anhydrase was sulphanilamide, which was showed inhibitory effect with IC50 value of 0.47 µM and 0.34 µM respectively.The classes of CA (α, β and γ) have been already known to show

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different inhibition rate with different inhibitors at varying concentration [46].

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3.7. Characterization of Calcium carbonate crystals

Differentiation of calcium carbonate polymorphs by SEM and XRD were analyzed though

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previous literature [47]. The crystal phase compositions of the CaCO3 precipitates formed in the presence or absences of CAs enzymes were compared. The yield of CaCO3 in absence of enzyme

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(control) was lower than precipitated CaCO3 achieved in presence of the enzyme. The purified

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enzyme β-CA and γ-CA at a concentration of 1 mg enzyme were produced 95.2 and 79.8 mg of calcium carbonate respectively that were higher in amountas compared to control (25.1 mg) in

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24 h. There was not further effect of increase in enzyme concentration for calcite production.

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The presences of biocatalyst could increase the saturation point for CaCO3 in solution and reduce the nucleation activation energy during spontaneous precipitationthus deposition of calcium in

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presence of CAs was significantly faster than control [15, 18]. Comparative differences in the

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size and morphology of CaCO3 crystals phases induced byenzymatic reactions were showed by SEM (Fig. 4d, 4e and 4f). The crystal phase i.e. calcite and vaterite of CaCO3 were obtained in absence of enzymes under control condition. A control sample of CaCO3 showed calcite phase with cubic structure and vaterite phase with a 3D structure of flower-like symmetry (spherules shaped) (Fig. 4d). The CaCO3 crystals induced by CAs were identified in cubic and polyhedralhexagonal shape of calcite structure (Fig. 4e and 4f). The calcite crystals showed well defined faceted rhombohedral characteristics in our study similar to earlier reported [48]. In this study,

ACCEPTED MANUSCRIPT the transformation of the thermodynamically unstable vaterite phase into the stable calcite phase was occurred during the precipitation of calcium carbonate in presence of both types of CAs (βCA and γ-CA). The transformation was occurred through the dissolution of vaterite into small crystals then followed by the crystallization of calcite [48].

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Calcite and vaterite phase formed in control condition (without CAs) were obtained from

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different biological factors by XRD analysis (Fig. 4a, 4b and 4c). Diffraction peaks patterns for calcite and vaterite phase was analyzed by previously studied data of Hu et al., [49]. In control

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sample,the representative crystal surface of calcite face was (1 0 4). Diffraction peaks occurred at 2θ = 29.9◦, 36.4◦, 39.0◦ and 43.3◦ corresponding to calcite crystal face (1 0 4), (1 1 0), (1 1 3) and

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(2 0 2) respectively. The diffraction peaks occurred at 2θ= 27.3◦, 32.1◦ and 43.6◦, respectively,

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corresponding to vaterite crystal face (1 1 2) (1 1 4), (3 0 0) respectively (Fig. 4a). The XRD patterns of crystals formed by β-CA and γ-CA were showed calcite phase as a major form of

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crystals. In case of β-CA, diffraction peaks occurred at 2θ= 29.6◦, 36.1◦, 39.5◦ and 43.3◦

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corresponding to calcite crystal face (1 0 4), (1 1 0) (1 1 3) and (2 0 2) respectively (Fig. 4b). In case of γ-CA, diffraction peaks occurred at 2θ= 29.5◦, 36.1◦, 39.5◦ and 43.5◦ corresponding to

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calcite crystal face (1 0 4), (1 1 0) (1 1 3) and (2 0 2) respectively (Fig. 4c). Previously, similar

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study on X-ray diffraction analysis of standard calcite phase of CaCO3 has performed by Rahman et al., [50]. CaCO3 precipitation in a specific calcite phase has also been previously observed using bovine carbonic anhydrase [51, 52]. During pre-nucleation stage, carbonate ions react with calcium ion and form metastable CaCO3 clusters. In presence of enzyme CAs,the growing clusters of stable calcite particles were formed in a post-nucleation stage. Thus CAs enhanced the nucleation rate during mineralization of CaCO3 [52]. Mineralization of CO2 is a method for biomimetic synthesis of CaCO3 materials. The slow rate of CO2 hydration has been a

ACCEPTED MANUSCRIPT limiting factor for carbonate precipitation. Our results suggested an approach of the biomimetic synthesis of CaCO3 materials using bacterial β-CA and γ-CA. 4. Conclusion The CAs genes, isolated from the Bacillus sp. SS105 have cloned and expressed in E. coli.

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The recombinants CAs were purified and used for conversion of CO2 into calcium carbonate

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crystals. The enzymatic reaction for β-CA and γ-CA were stable at pH 8.0 and 30°C. Enzyme

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activity was enhanced in presence of Fe2+ and Zn2+. Both enzymes exhibited tolerance to SO42-

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and NO32- and higher stability in presence of Triton X 100 and Tween 20. Other anions and modulators showed variable inhibition effect at different concentrations. Acetazolamide and

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sulphanilamide were the strongest inhibitor for both enzymes. Biomimetic synthesis of CaCO3

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materials was identified as calcite phase of crystals through SEM and XRD pattern. Thus provide a challenging use of β-CA and γ-CA enzymes for conversion of atmospheric CO2 into stable,

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Acknowledgment

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eco-friendly calcite.

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The author is grateful to Department of Science and Technology, Government of India, New Delhi, for providing start-up research grant, (Srivastava, S), research facility, JRF and

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CSIR, New Delhi for providing Junior Research fellowship (Maheshwari, N). We give special thanks to Prof B. C Tripathi (JNU, New Delhi) for providing soil sample of FACE. We are also thankful for Advanced Instrumentation Research Facility (AIRF), Jawaharlal Nehru University, New Delhi for SEM and XRD analysis. Conflict of interest The authors declare no conflict of interest.

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anhydrase from Pseudomonas fragi, Process Biochem 44 (2009)1293-1297. [41] M. G. Lionetto, R. Caricato, M. H. Giordano, E. Erroi, T. Schettino, Carbonic anhydrase and heavy metals, In: Ekinci D (ed) Biochemistry. 1st edition.Intech, Rijeka, (2012) 205-224. [42] C. D. Boone, A. Habibzadegan, S. Gill, R. McKenna, Carbonic Anhydrases and Their Biotechnological Applications, Biomolecules. 3 (2013) 553-562.

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anhydrase and its transformation to solid carbonate, J. Mol. Catal. B Enzym. 60 (2009) 163–

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ammonia-induced vaterite, Am. Mineral. 97 (2012) 1437–1445.

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[50] M.Vinoba, D. H. Kim, K. S. Lim, S. K.Jeong, S. W. Lee, M.Alagar, Biomimetic Sequestration of CO2 and Reformation to CaCO3 Using Bovine Carbonic Anhydrase Immobilized on SBA-15. Energy Fuels. 25 (2011) 438–445. [51] D. H. Chu, M. Vinoba, M. Bhagiyalakshmi, H. Baek, S. C. Nam, Y. Yoon, S. H. Kim, S. K. Jeong, CO2 mineralization into different polymorphs of CaCO3 using an aqueous-CO2 system. RSC Advances. 3(44) (2013) 21722-21729.

ACCEPTED MANUSCRIPT

Legends Fig. 1 (a) Functional domain analysis of beta-carbonic anhydrase generepresentative, (b) architecture of conserved domain in beta-carbonic anhydrase protein, (c) Functional domain analysis of gamma-carbonic anhydrase gene representative, and (d) architecture of conserved

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domain in gamma-carbonic anhydraseof Bacillus sp. SS105.

Fig. 2 SDS-PAGE of carbonic anhydrase of Bacillus sp. SS105; (a) Purified β-CA of

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approximately 27kDa analyzed by affinity chromatography; (b) Purified γ-CA of approximately 25kDa analyzed by affinity chromatography.

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Fig. 3 Effects of pH and temperature on stability of purified recombinant CAs. (a) The β-CA and γ-CA enzymes were incubated in buffer of different pH range (3.0 to 11.0) for 1h at 37°C. (b)

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The reaction mixture of phosphate buffer (pH 8.0) containing purified β-CA and γ-CA was

according to the standard enzyme assay.

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incubated at different temperatures (30 °C to 60 °C) for 1 h. The residual activity was measured

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Fig. 4 The XRD chart of carbonate crystals (a) precipitated in absence of CAs, (b) precipitated by purified β-CA and (C) precipitated by purified γ-CA; Scanning Electron Microscopy of

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calcium carbonate crystals (d) under control condition; (e) induced by purified β-CA and (f) induced by purified γ-CA of Bacillus sp. SS105 in presence of 50 mM NaHCO3.

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Table 1 Purification of recombinant β-CA and γ-CA from Bacillus sp. SS105 in E. coli. Table 2 Effect of metal ions, anions and modulators on CAs activity from Bacillus sp. SS105.

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The β-CA and γ-CA enzymes were incubated with (a) different metal ions (Fe2+, Mg2+, Zn2+, Co2+and Hg2+), (b) different anions (HCO3-, CO32-, SO42- and NO32-), (c) different modulators (EDTA, DTT, SDS, Tween 20, Triton X 100) for 1h at 37°C. The residual activity was measured according to the standard enzyme assay.

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Fig. 1

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Fig. 2

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Fig. 3

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(a)

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120 Beta-carbonic anhydrase Gamma-Carbonic anhydrase

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Enzyme activity (%)

100

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80

60

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40

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20

0 5

7

9

11

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3

pH

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(b)

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120

80

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Enzyme activity (%)

100

Beta-Carbonic anhydrase Gamma-Carbonic anhydrase

60

40

20

0 25

30

35

40

45

50

Temperature (°C)

55

60

65

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Fig. 4

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Crude lysate

(U)

276.38

22912.42

3.60

5216.78

304.08

13556.43

3.92

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Purified Protein

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γ-CA

(mg)

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Purified Protein

Recovery

Purification

(%)

fold

82.90

100

1

1449.10

23

17

44.58

100

1

1067.94

31

23

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Specific activity (U/mg)

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Crude lysate

Total activity

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β-CA

Total proteins

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Protein sample

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Table 1

4186.36

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Table 2 (a)

Zn2+ Hg2+

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2 (b)

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Co2+

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Mg2+

Concentration

HCO3-

10 mM 50 mM 100 mM

β-CA Residual activity (%) 100±0 82.6±1.2 62.3±0.7

10 mM 50 mM 100 mM 100 mM 500 mM 1000mM 2000mM

95.76±0.4 78.6±0.3 55.4±0.5 236.9±0.5 191.1±1.4 187.6±1.1 105.4±0.2

SO42-

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CO32-

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Anions

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γ-CA Residual activity (%) 133.59±1.4 85.7±0.8 27.9±1.7 61.72±1.22 31.6±1.4 25.8±1.5 71.79±1.61 45±0.3 24.5±1.8 122.9±1.9 105.4±0.9 94.7±1.4 51.54±1.46 32±0.2 0.8±0.4

IC50 value

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1mM 2mM 5Mm 1mM 2mM 5mM 1mM 2mM 5mM 1mM 2mM 5mM 1mM 2mM 5mM

IC50 value

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Fe2+

β-CA Residual activity (%) 74.84±0.7 45.6±1.1 23.1±0.6 51.15±0.7 37.2±0.3 21.7±1.65 50.77±1.8 30.6±1.4 5.4±1.4 114.38±1.72 89.4±1.2 45.4±0.8 42.3±1.4 38±0.4 12.0±1.5

2.46mM

0.73 Mm

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Concentration

0.68mM

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Metal ions

4.65mM

0.21mM

IC50 Value

128.9mM

112.5mM

2.9 M

γ-CA Residual activity (%) 100±0 72.8±0.4 60.5±1.2 80.4±0.6 75±1.4 54.2±0.7 145.6±0.7 121.2±0.2 100±0 100±0

3.94mM

1.27mM

2.35mM

11.9mM

0.87mM

IC50 Value

117.24mM

120.4mM

2.1 M

ACCEPTED MANUSCRIPT NO32-

145.6±0.8 134.61±0.5 123.8±0.1 98.5±0.7

2.1 M

112±1.2 104±0.4 97±0.5 79.1±1.7

1.9 M

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50 mM 100 mM 500 mM 1000mM

57.1±1.5

1M

18.9±1.2

34.2±1.4

100±0.3

100±1.6

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1%

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100mM

10 mM

Triton X 100

Residual activity (%)

75.3±0.4

5 mM

Tween 20

Value

γ-CA

50mM

1 mM

SDS

Residual activity (%)

ED

DTT

IC50

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EDTA

β-CA

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Concentration

391.83

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Modulators

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2 (c)

75±1.4

8.91mM

67.9±0.5

64±1.1 35±0.8

53±1.4

43.8±0.9

5%

12±0.4

10%

0±0

0±0

0.10%

120.4±0.5

100±1.5

0.20%

100±1.6

0.50%

71.9±0.2

65.7±1.2

0.10%

111.53±0.3

100±0.3

0.20%

100±0.7

0.50%

76.53±1.1

0.68%

0.81%

Value

85.3±0.9

43±0.6

0.40%

IC50

0.8±0

100±1.6

95±0.7 46.7±1.6

651.41

7.16 mM

2.38 mM

0.68%

0.48%

ACCEPTED MANUSCRIPT

Highlights

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  

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

Free air CO2 enriched (FACE) bacterium, Bacillus sp. SS105 was selected for biomimetic sequestration of CO2. Protein encoding genes for β-CA and γ-CA of Bacillus sp. SS105 were amplified and cloned in expression vector. CA enzymes were expressed in E.coli and purified by affinity chromatography. Enzymes (β-CA and γ-CA) were characterized and utilized for CaCO3 precipitation. Calcite phase of CaCO3 was observed by SEM and XRD pattern.

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