KR20160139842A - Chimeric carbonic anhydrase from Dunaliella Salina and use thereof - Google Patents

Abstract

The present invention relates to chimeric carbonic anhydrase derived from Dunaliella salina, and to a use thereof. The chimeric carbonic anhydrase derived from Dunaliella salina has excellent activity of decomposing ester and hydrating carbon dioxide, and thus can be used to develop an industrial process of collecting and fixating carbon dioxide. Also, the chimeric carbonic anhydrase derived from Dunaliella salina can be used to prepare a bicarbonate compound, and can be used as a catalyst for synthesizing useful metabolites, thereby increasing synthesis yield of the metabolites.

Description

Chimeric carbonic anhydrase from Dunaliella salinina and its use {Chimeric carbonic anhydrase from Dunaliella Salina and use thereof}

The present invention relates to chimeric carbonic anhydrase derived from Dnaliella salinina and its use.

Concentrations of atmospheric greenhouse gases such as carbon dioxide (CO ₂ ), methane (CH ₄ ), chlorofluorocarbons (CFCs) and nitrogen oxides (NO x) continue to rise as a result of the rapid growth of science and technology . Most of the greenhouse gases produced by fossil fuels such as coal, oil, and natural gas are produced. After the Industrial Revolution, the atmospheric concentration of carbon dioxide was 280 ppm in 2007, but it continued to increase to 400 ppm in 2013. And by 2065, atmospheric carbon dioxide concentrations are expected to double to the pre-industrial era. Therefore, the main objective of many studies has been to develop a method to reduce carbon dioxide emissions. Several techniques have been devised for the capture and storage of carbon dioxide for the purpose of reducing or limiting the emission of excess carbon dioxide. In the field of biology, CA was extensively investigated in order to apply it as an improved clean-up technology to capture a large amount of carbon dioxide in the atmosphere. CA is an enzyme with a metal, including zinc, that acts as a catalyst to turn carbon dioxide into a hydration state capable of reversible reaction, such as bicarbonate or both. This enzyme was found in all living mammals and was also found in plants, archaea, and prokaryotes. CA is an essential protein in the biological processes of eukaryotes such as optics, respiration, carbon dioxide ion transport, calcification, and the maintenance of equilibrium of acid bases. The classification of CA has been classified and defined as alpha, beta, gamma-CA in three evolutionary categories. However, two additional delta and zeta additions have been identified.

Previously, the inventors of the present invention found that each of the amino terminal domain half and the carboxyl terminal domain half of the double-structure CA (Dsp-CA) in which two α-type CAs derived from dunaliella sp. Lt; / RTI > Only the carboxyl terminal domain (Dsp-CA-c) exhibited the enzyme activity, although these two α-type CAs have a similar structure. However, Dsp-CA-c was only one-third of the soluble protein expression of Dsp-CA-n, the amino terminal domain half. The low yield of Dsp-CA-c became a problem when trying to apply this protein as a biocatalyst for carbon dioxide removal and conversion. Therefore, in order to apply CA enzyme system realistically, it was essential to increase the expression level. In some naturally occurring multi-domain proteins, the amino-terminal domain improved the solubility of the remaining domains. In addition, it is known that amino terminal residues play an important role in the function of the protein, such as stability and solubility [Non-Patent Documents 1 to 5].

Kanth, B. K., Min, K., Kumari, S., Jeon, H., Jin, E. S., Lee, J., Pack, S.P., (2012). Expression and characterization of codon-optimized carbonic anhydrase from Dunaliella species for CO2 sequestration application. Applied biochemistry and biotechnology 167, 2341-2356. Ki, M. R., Kanth, B. K., Min, K. H., Lee, J., Pack, S. P., (2012). Increased expression level and catalytic activity of internally-duplicated carbonic anhydrase from Dunaliella species by reconstitution of two separate domains. Process Biochem 47, 1423-1427. Kim, C. W., Han, K.S., Ryu, K.S., Kim, B.H., Kim, K.H., Choi, S.I., Seong, B.L., (2007). N-terminal domains of native multidomain proteins have the potential to assist de novo folding of their downstream domains in vivo by acting as solubility enhancers. Protein science: a publication of the Protein Society 16, 635-643. Gaudry, A., Lorber, B., Neuenfeldt, A., Sauter, C., Florentz, C., Sissler, M., (2012). Re-designed N-terminus enhances expression, solubility and crystallizability of mitochondrial protein. Protein Engineering Design and Selection 25, 473-481. Varshavsky, A., (1997). The N-end rule pathway of protein degradation. Genes to cells: devoted to molecular & cellular mechanisms 2, 13-28.

Accordingly, the present inventors have made efforts to solve the above problems, and as a result, they have found that a part of the amino terminal of a protein having high expression such as Dsp-CA-n is fused to a protein showing low solubility such as Dsp-CA- And the present invention was completed by developing a new CA that induces an enzyme activity similar to a circular protein through fusion of the amino terminal domain and the carboxyl terminal domain of the double structure protein.

It is an object of the present invention to provide a chimeric carbonic anhydrase derived from Dnaliella salinina and its use.

As a means for solving the above problems, the present invention provides a carbonic anhydrase [Dsp-nCA-c] comprising an amino acid sequence represented by SEQ ID NO: 4.

As another means for solving the above problems, the present invention provides a carbonic anhydrase (Dsp-nCA-c (G263S)) comprising an amino acid sequence represented by SEQ ID NO: 5.

In another aspect of the present invention, there is provided a composition for capturing or fixing carbon dioxide comprising the carbonic anhydrase.

The chimeric proteins Dsp-nCA-c and Dsp-nCA-c (G263S) of the present invention show higher water-solubility and CO ₂ hydration activity than the conventional Dsp-CA-c. In addition, the two proteins show a similar pattern in Dsp-CA and CO ₂ mineralization. Since the novel chimeric protein of the present invention exhibits increased solubility and CO ₂ hydration activity, it can be utilized for various industrial raw materials production using CO ₂ removal process using CO catalyst and CO ₂ mineralization.

Fig. 1 is a schematic diagram showing the production process of chimeric proteins Dsp-nCA-c and Dsp-nCA-c (G263S) [A: Chimeric protein Dsp-nCA-c structure, B: Overlap PCR Pattern diagram].
Fig. 2 compares the amino acid sequence of the novel chimeric protein and its modified protein and the sequence with the unmodified proteins.
Figure 3 compares Dsp-CA-n, Dsp-CA-c, Dsp-nCA-c and Dsp-nCA-c (G263S) protein expression by SDS-PAGE analysis [M; marker, lane 1: Dsp-CA-n, lane 2: Ds-CA-c, lane 3: Dsp-nca-c, and lane 4: Dsp-nca-c (G263S). The figure on the left shows the comparison of the amount of purified protein in E. coli producing pre-purified protein, and the figure on the right shows the amount of purified protein after purification using cobalt resin.
Figure 4 compares the esterase activity (A) and CO ₂ hydration activity (B) between each CA.
Figure 5 shows the XRD analysis of CaCO ₃ formed by each CA [C: calcite; v: vaterite peak].
Figure 6 compares SEM images of CaCO ₃ formed by each CA [A: CaCO ₃ formed in the absence of enzyme. B: Dsp-CA, C: Dsp-CA-n, D: Dsp-CA-c, E: Dsp-nCA-c, F: CaCO ₃ formed by Dsp-nCA-c (G263S).

Hereinafter, the configuration of the present invention will be described in detail.

(Dsp-CA-n) and half-carboxyl-terminal domain (Dsp-CA-c), respectively, derived from Dunaliella salina. , And Dsp-CA-c, respectively, have a complete alpha-type CA structure. When they are expressed as a single α-type CA, Dsp-CA-n exhibits good protein expression but little activity and Dsp-CA-c exhibits low enzyme activity. In order to increase the expression rate of active Dsp-CA-c, Dsp-nCA-c, a chimeric protein that replaced the amino-terminal 10 amino acids of Dsp-CA-c with 12 amino acids of the amino terminal of Dsp-CA- Invented. Dsp-nCA-c and Dsp-nCA-c (G263S), a naturally occurring variant of Dsp-nCA-c, produced approximately two-fold increased amounts of CO _{2 and} hydration compared to Dsp-CA-c. In addition, these novel chimeric proteins exhibited mineral forming ability similar to the mineral forming ability by the CO ₂ conversion of the circular double Dsp-CA.

Accordingly, the present invention provides a method for producing Dsp-nCA-c and a modification thereof, Dsp-nCA-c (G263S), which is a novel carbonic anhydrase having increased protein expression and enzyme activity, and uses thereof.

The Dsp-nCA-c carbonic anhydrase according to the present invention can be represented by the amino acid sequence of SEQ ID NO: 4. In addition, the Dsp-nCA-c (G263S) carbonic anhydrase according to the present invention can be represented by the amino acid sequence of SEQ ID NO: 5.

In addition, the carbonic anhydrase has not only the amino acid sequence of SEQ ID NO: 4 or 5 but also a sequence homology of 80% or more, 85% or more, 90% or more, 93% or more, 94% or more, 95% , 96% or more, 97% or more, 98% or more, and 99% or more. In the present invention, the fact that the carbonic anhydrase has a specific ratio (for example, 80%, 85%, 90%, 95%, or 99%) to another sequence means that when the two sequences are aligned Quot; means that the amino acid residues in the ratio are the same in the comparison of the sequences.

In addition, the Dsp-nCA-c carbonic anhydrase may be the one encoded from the nucleotide sequence shown in SEQ ID NO: 10. Further, the Dsp-nCA-c (G263S) carbonic anhydrase may be the one encoded from the nucleotide sequence shown in SEQ ID NO: 11.

The carbonic anhydrase has esterase activity and CO ₂ And is characterized by having hydration activity.

One embodiment of the present invention is a method for mass production of carbonic anhydrase, comprising the step of preparing a recombinant vector comprising a coding sequence of a carbonic anhydrase represented by the nucleotide sequence shown in SEQ ID NO: 3 or SEQ ID NO: 9 .

The carbonic anhydrase can be prepared by in vitro synthesis through gene recombination and protein expression system. That is, a recombinant vector containing an essential regulatory element operably linked to the expression of the gene insert of the carbonic anhydrase can be used.

Such vectors include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors, and viral vectors. Suitable expression vectors include signal sequence or leader sequences for membrane targeting or secretion in addition to expression control elements such as promoter, operator, initiation codon, termination codon, polyadenylation signal and enhancer, and may be prepared in various ways depending on the purpose. The promoter of the vector may be constitutive or inducible. Further, the expression vector includes a selection marker for selecting a host cell containing the vector, and includes a replication origin in the case of a replicable expression vector.

The recombinant vector of the present invention can be preferably prepared by inserting a nucleic acid encoding said carbonic anhydrase into a vector for expression of a common E. coli strain, pET42b vector. In one embodiment of the present invention, pET42b vector is used as a vector for Escherichia coli expression, but not always limited thereto, and all commonly used Escherichia coli expression vectors can be used without limitation.

Such transformation includes any method of introducing the nucleic acid into an organism, cell, tissue or organ, and can be carried out by selecting a suitable standard technique depending on the host cell as is known in the art. Such methods include electroporation, protoplast fusion, calcium phosphate (CaPO ₄ ) precipitation, calcium chloride (CaCl ₂ ) precipitation, agitation with silicon carbide fibers, Agrobacterium mediated transformation, PEG, dextran sulfate, ≪ / RTI > lipofectamine, and the like.

In addition, since the expression amount of the protein and the expression are different depending on the host cell, the host cell most suitable for the purpose may be selected and used.

Examples of host cells include prokaryotic cells such as Escherichia coli, Bacillus subtilis, Streptomyces, Pseudomonas, Proteus mirabilis or Staphylococcus. Host cells, but are not limited thereto. It is also possible to use fungi (for example Aspergillus), yeast (for example, Pichia pastoris, Saccharomyces cerevisiae, Schizo < (R) > Saccharomyces, Neurospora crassa, etc.), insect cells, plant cells, mammals and the like can be used as host cells. The transformant can be easily prepared by introducing the recombinant vector into any host cell.

According to one embodiment of the present invention, a transformant can be prepared by introducing the recombinant vector pET-Dsp-nCA-c for expressing the carbonic anhydrase obtained by the expression vector into E. coli strain BL21 (DE3) .

The transformant expressing the above-mentioned carbonic anhydrase can be mass-produced by culturing and isolating and purifying the transformant according to a conventional culture method. Although not particularly limited, it can be purified by chromatographic method by culturing in usual LB (Luria broth) and recovering only the soluble fraction from the culture.

The present invention also relates to a composition for trapping or fixing carbon dioxide comprising a carbonic anhydrase represented by the amino acid sequence set forth in SEQ ID NO: 4 or 5.

As described above, the carbonic anhydrase of the present invention has an ester-raising activity and a carbon dioxide hydration activity, and can be effective for capturing and immobilizing carbon dioxide.

The composition for trapping or fixing carbon dioxide of the present invention may include a carbonic anhydrase according to the present invention prepared by a known method in the art for synthesizing peptides. For example, by in vitro synthesis through gene recombination, a protein expression system, or a peptide synthesizer.

In addition, the composition for capturing or fixing carbon dioxide of the present invention may be contained in the form of a culture of a transformant expressing a carbonic anhydrase.

The present invention also relates to an apparatus for collecting and fixing carbon dioxide which comprises a composition for capturing or fixing carbon dioxide according to the present invention.

The apparatus for trapping and fixing carbon dioxide of the present invention may be a conventional apparatus for extracting carbon dioxide from air and converting it into bicarbonate and fixing it. For example, the capture and fixation device of carbon dioxide may include upright towers, a chamber and a source of water, wherein the upright tower includes at least one air inlet at the bottom And at least one air outlet at the top of the tower, the chamber being located between at least one air inlet and at least one air outlet, The water supply unit may have a structure for wetting the resin to release carbon dioxide, and the collector for capturing the carbon dioxide may include the carbonic anhydrase according to the present invention.

In another embodiment, the trapping and anchoring device for carbon dioxide of the present invention may be an indoor air purifier. The indoor air cleaning apparatus may be configured such that a carrier containing an enzyme is fixed in an existing fractional apparatus and has a carbon dioxide abatement function and a humidifying function and is submerged in an aqueous solution of an aqueous solution reservoir so that the enzyme can continuously perform its function . Therefore, the enzyme contained in the carrier containing the enzyme has a catalytic function for rapidly converting dissolved carbon dioxide into bicarbonate, so that the carbon dioxide in the air can be continuously reduced. The enzyme may include a carbonic anhydrase according to the present invention.

The present invention also relates to a sensor for capturing and fixing carbon dioxide in which a composition for trapping or fixing carbon dioxide according to the present invention is immobilized.

In the sensor for trapping and fixing carbon dioxide in the present invention, the enzyme fixing method can be fixed by an adsorption method, an ion-binding method, a crosslinking method, a capturing method, or a covalent bonding method, but the method is not particularly limited thereto.

The present invention also relates to a method for trapping or fixing carbon dioxide comprising the step of reacting a carbon dioxide saturated solution with a composition for trapping or fixing carbon dioxide according to the present invention.

The carbon dioxide saturation solution may be prepared by dissolving carbon dioxide gas in an aqueous carbonate solution, but is not particularly limited thereto.

The alpha-type carbonic anhydrase according to the present invention can rapidly convert carbon dioxide to bicarbonate by dehydration.

The present invention also relates to a kit for producing a bicarbonate or a carbonate compound comprising a composition for capturing or fixing carbon dioxide according to the present invention.

The present invention also relates to a method for producing carbon dioxide, comprising the steps of: reacting a carbon dioxide saturated solution with a composition for trapping or fixing carbon dioxide according to the present invention; And reacting the reactant obtained above with an alkali cationic salt to produce an amorphous bicarbonate or carbonate compound. The present invention also provides a method for producing a bicarbonate or a carbonate compound.

The carbonic anhydrase has reversible reactivity, and bicarbonate as well as carbon dioxide can be used as a substrate. Thus, the bicarbonate converted from carbon dioxide in the carbon dioxide fixation process can be converted to an amorphous bicarbonate or carbonate compound upon treatment of the alkali cation.

The bicarbonate compound may be selected from the group consisting of sodium bicarbonate (Na ₂ HCO ₃ ), potassium bicarbonate (KHCO ₃ ), magnesium bicarbonate (Mg (HCO ₃ ) ₂ ), calcium bicarbonate (Ca (HCO ₃ ) ₂ ) ((NH ₄₎ HCO _3), and the like.

The carbonate compound is a carbonic acid (H ₂ CO _3), lithium carbonate (Li ₂ CO _3), sodium carbonate (Na ₂ CO _3), potassium carbonate (K2CO _3), rubidium carbonate (Rb2CO _3), cesium carbonate (Cs ₂ (BaCO ₃ ), manganese carbonate (MnCO ₃ ), iron carbonate (FeCO ₃ ), barium carbonate (BaCO ₃ ), iron carbonate (FeCO ₃ ), beryllium carbonate (BeCO ₃ ), magnesium carbonate (MgCO ₃ ), calcium carbonate (CaCO ₃ ), strontium carbonate ₃₎ and cobalt carbonate (CoCO _3), nickel carbonate (NiCO _3), copper carbonate (CuCO _3), silver carbonate (Ag ₂ CO _3), zinc carbonate (ZnCO _3), cadmium carbonate (CdCO _3), aluminum carbonate ( Al ₂ (CO ₃ ) ₃ ), thallium carbonate (Tl ₂ CO ₃ ), lead carbonate (PbCO ₃ ), or lanthanum carbonate (La ₂ (CO ₃ ) ₃ ).

The alkali cation salt may be at least one selected from the group consisting of Mg, K, Na, NH4, Ca, Li, Rb, Cs, Be, Sr, Ba, Mn, Fe, Co, Ni, Cu, Zn, Cd, Ag, Al, And the like. For example, calcium chloride (CaCl ₂ ), calcium sulfate (CaSO ₄ ), calcium bicarbonate (Ca (HCO ₃ ) ₂ ), calcium oxide (CaO) and calcium hydroxide (Ca (OH) ₂ ) It is not.

The amorphous bicarbonate or carbonate compound may be converted into a crystal phase bicarbonate or carbonate compound through phase transformation through a precipitation reaction.

The precipitation can be carried out at a pH of 8 to 10 at a temperature of 0 to 50 ° C, but is not particularly limited thereto.

The crystal phase bicarbonate or carbonate compound may be calcite, aragonite or veterite.

The present invention also relates to a kit for the production of metabolites using bicarbonate or carbon dioxide comprising a composition for capturing or fixing carbon dioxide according to the present invention.

The present invention also provides a method for producing a metabolite using a composition for capturing or fixing carbon dioxide according to the present invention as a catalyst in C4 metabolism using bicarbonate or carbon dioxide or lipid metabolism.

The metabolite may be a C4 metabolite or a lipid metabolite. In the C4 metabolism, in the PEP carboxylase reaction proceeding to oxaloacetate by attaching CO2 to PEP (phosphoenol pyruvate), the carbonic anhydrase reacts with the catalyst Coenzymes) to synthesize C4 compounds effectively.

In addition, in the lipid metabolism, in the Acetyl CoA carboxylase reaction proceeding to malonyl CoA by attaching bicarbonate (HCO ₃ ^- ) in acetyl CoA, (Or coenzyme) and can effectively synthesize it on malonylcohols.

Thus, the present invention provides a system useful for the synthesis of useful metabolites.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the scope of the present invention is not limited by the following examples.

[ Example ]

1. Strain, plasmid, reagent preparation

GenBank AAC49378.1 Dunaliella Salina, matched with the mature protein amino acid sequence, was used.

E. coli BL21 (DE3) was used as an expression strain.

As the expression vector, Novagen pET42b was used.

Sigma-aldrich purchased antibiotics, IPTG (isopropyl β-D-1-thiogalactopyranoside), p-NPA ( p- nitrophenolacetate), PMSF (phenylmethylsulfonyl fluoride), lysozyme and DNase.

EDTA-free protease inhibitors and protein assay reagents were purchased from Thermo Fisher scientific company.

All reagents used in the experiments were analytical grade and all aqueous solutions were prepared with deionized distilled water.

2. Chimeric Dsp - nCA Manufacture of -c

The gene with the cDNA sequence (SEQ ID NO: 7) obtained from Dsp-CA (SEQ ID NO: 1) was obtained from the vector pET-Dsp-CA which the team of the present invention has.

A primer for gene amplification was prepared with the complementary sequence of the cDNA sequence (SEQ ID NO: 9) encoding Dsp-CA-c (SEQ ID NO: 3) and the fusion gene was amplified by overlap extension PCR. Three forward primers and one reverse primer were used.

F1: CATATGGTTTCTGAACCGCACGACTACAAC (SEQ ID NO: 12)

F2: CGCACGACTACAACTACGAAAAACACGGT (SEQ ID NO: 13)

F3: CTACGAAAAACACGGTTTCGACTGGCGT (SEQ ID NO: 14)

R1: AAGCTTAGCAGCAGCACCGTTGTAACCGTA (SEQ ID NO: 15).

The first PCR reaction was carried out using a pair of F3 and R1 primers, and a plasmid containing the Dsp-CA-c gene (SEQ ID NO: 9) was used as a template. In the second reaction, the PCR product of the first reaction was amplified using F2 and R1 as a template. Finally, the PCR product of the second reaction was used as a template and PCR was performed using F1 and R1 to obtain the final fusion gene I got it.

The PCR method was repeated 30 times at 94 ° C for 3 minutes, at 94 ° C for 30 seconds, at -64 ° C for 1 minute, and at 72 ° C for 1 minute, and at 72 ° C for 6 minutes once. The purified PCR product was pGEM-T Easy vector [Promega co. USA). DNA (SEQ ID NO: 10) synthesized by gene sequence analysis was confirmed. A DNA fragment of 825 bp (SEQ ID NO: 10) was isolated from the T vector cloned using the restriction enzyme Nde I / Hind III, and the gene fragment was inserted into the pET42b vector treated with the same pair of restriction enzymes. Using the T4 ligase After cloning, E. coli XL1-blue was transformed to obtain a cloning vector, which was again confirmed by gene sequencing to complete the expression vector pET-Dsp-nCA-c. (G263S)] (SEQ ID NO: 11) in which the glycine of amino acid sequence 263 was changed to serine while analyzing the gene sequence was also found and the vector pET-Dsp-nCA-c (G263S Respectively.

3. Recombination Dsp - nCA -c expression and purification

Dsp-nCA-c (SEQ ID NO: 4) and Dsp-nCA-c (G263S) (SEQ ID NO: 5) were expressed in E. coli BL21 and purified using Hiso-tagged cobalt resin from Thermo. The Dsp-CA (SEQ ID NO: 1), Dsp-CA-n (SEQ ID NO: 2) and Dsp-CA-c (SEQ ID NO: 3) previously cloned were also used for comparison of the biochemical characteristics of the modified protein. Protein was analyzed by SDS-PAGE and its concentration was quantified by Bradford assay.

4. Esther Reyes ( esterase ) Active measurement

Verpoorte et al., J Biol Chem. 1967; 242 (18): 4221-9], CA esterase activity was analyzed spectrophotometrically at 348 nm using p-NPA (para-nitrophenylacetate).

The reaction was initiated by the addition of 100 μl of 3 mM p-NPA prepared directly to 200 μl of 20 mM Tris-SO ₄ with or without CA. The reaction was continuously monitored at 5 minute intervals for 30 minutes using a multiplate reader. Activity was calculated as the amount of p-NP released in p-NPA. One unit of enzyme was defined as the amount of enzyme producing 1 nmol p-NP per minute.

5. Carbon dioxide hydration ability

The CO ₂ hydration activity of each CA was calculated as the time T spent in the pH decrease to 7.0 as the CO ₂ dissolved in the pH 8.3 buffer solution was converted to bicarbonate by the catalytic action of these proteins, The time required to hydrate naturally without reaction is calculated as T ₀ and expressed as the enzyme activity WAU (Wilbur-Anderson unit) according to the following equation (1) and divided by the amount of protein used in the reaction: 2) were calculated.

[Equation 1]

WAU = (T0 - T) / T

&Quot; (2) "

Activity = WAU / mg protein

6. CO ₂ Mineralization

The conversion of CaCO _{3 to} carbon dioxide by CA in the presence of CaCl ₂ is described in the paper by Mi-Ran Ki et al., Process Biochem. 2012; 47 (9): 1423-27). CO ₂ was sufficiently dissolved in pure dDW (deionized distilled water) obtained through miliQ-grade at 4 ° C for 1 hour, and the mixture was stored in ice for 30 minutes. 10 ml of cold CO ₂ solution was added to 1 ml of Tris-SO ₄ buffer (1M, pH 8.3) containing 100 μg of CA enzyme and kept for 15 minutes. 10 ml of CaCl ₂ solution in another bottle was added to the solution to give a final concentration of 10 mM , And 2 ml of 1 M Tris-SO ₄ buffer (pH 9.5) was immediately added thereto and mixed. The mixture reaction was carried out at 35 ° C for 5 minutes and at 45 ° C for 5 minutes. The precipitated CaCO ₃ was filtered off and dried at 60 ° C. The amount of CO ₂ present in CaCO ₃ was calculated through the dry weight. As a control, mineralization of CO ₂ was carried out in the absence of enzyme.

The CaCO ₃ results were analyzed by SEM image and XRD measured with Cu Ka radiation (λ = 0.154 nm) on D / MAX-2500 / PC. The 2? Range of the diffraction pattern is 20 ° to 70 °, and the 2θ value of each step size is 0.02 °. The obtained XRD results were compared with those of the Joint Committee on Powder Diffraction Standards (JCPDS).

7. chimera  Protein production

Although the dual α-type CA of Dnalariella salina can be expressed in the E. coli system, the amount of product is not sufficient for practical application. When the respective domains of Dsp-CA are separately cloned and expressed, the amino terminal domain, Dsp-CA-n (SEQ ID NO: 2) differs from the carboxyl terminal domain Dsp-CA-c (SEQ ID NO: 3) The amount is between 3 and 10 times, but little or no enzyme activity. In order to increase the expression rate of the active form of Dsp-CA-c, the protein tertiary structure analysis using Pymol revealed that residues 275 to 536 of Dsp-CA (SEQ ID NO: 1) And a chimeric protein Dsp-nCA-c (SEQ ID NO: 4) consisting of the first 12 amino acids of the amino terminal (Fig. 1). The chimeric protein-producing gene was obtained by overlap PCR as shown in Fig. A new Dsp-nCA-c (SEQ ID NO: 4) chimeric protein was prepared by replacing the amino terminal 10 amino acids of Dsp-CA-c (SEQ ID NO: 3) with the amino terminal 12 amino acids of Dsp- CA-n Respectively. (SEQ ID NO: 2), Dsp-CA-c (SEQ ID NO: 3), D. salina (PDB ID: 1y7w) (SEQ ID NO: 6), α-type CAs and novel chimeric proteins Were compared (Figure 2).

8. chimera  Protein production

The expression level of Dsp-nCA-c (G263S), a mutant protein produced during the process of preparing new chimeric Dsp-nCA-c and fusion protein, was 2.1 to 2.4 fold higher than that of existing Dsp-CA-c , Table 1).

Expression yields were compared using the highest protein concentration obtained after affinity chromatography. It was confirmed that the N-terminal residue of Dsp-CA-n influenced the increase in soluble expression of the fused Dsp-CA-c.

Comparison of Protein Production
α-CA source Dsp-CA-n Dsp-CA-c Dsp-nca-c Dsp-nCA-c (G263S) Protein yield mg / L 83.3 ± 1.5 10.2 ± 1.1 21.7 ± 2.7 24.3 ± 0.8

9. chimera  The esterolytic activity of the protein and CO ₂  Hydration activity

The esterase activity of the Dsp-nCA-c protein was 290.9 U / mg, which was about 80% of that of the same amount of Dsp-CA-c. In the case of Dsp-nCA-c (G263S) CA-c < / RTI > (Figure 4A).

In the case of CO ₂ hydration activity, Dsp-nCA-c and Dsp-nCA-c (G263S) showed 2766WAU and 3305WAU activity per mg, respectively, which was twice the amount of Dsp-CA-c (Fig. 4B).

Interestingly, both fusion proteins showed increased CO ₂ hydration as well as yield of water-soluble proteins. These results demonstrate that the amino-terminal amino acids of the substituted Dsp-CA-n play an important role, indicating that these sequences also have a positive effect on the enzyme activity of the carboxyl terminal domain. When the total amount of enzyme activity obtained by 1 L culture was calculated, the ester-raising activity by Dsp-nCA-c and serine mutation was 2 to 4 times that of Dsp-CA-c and the activity of CO ₂ hydration was 4 to 5 times. The amino-terminal domain half is expected to function to stabilize the structure and improve the water-soluble expression while not being active in the body's double Dsp-Cas. Assuming that the two domains are stabilized by mutual binding with a portion of the sequence, Dsp-nCA-c may be similar to the original double protein structure rather than Dsp-CA-c alone. The major difference between the N-terminal sequence between Dsp-CA-c and Dsp-nCA-c is that Dsp-CA-c is valine (V10) and glutamine (Q11) )to be. In the tertiary structure obtained using SWISS-MODEL with 1y7w A as template, these two sequences are present on the surface of Dsp-nCA-c (data not shown). Glutamate and lysine are water-soluble amino acids capable of ionic bonding. These sequences are expected to increase the acceptability of this Dsp-nCA-c. The mutant protein Dsp-nCA-c (G263S) is a 263-mer glycine (a non-polar, hydrophobic amino acid) that has been replaced by a point mutation in a nonpolar, soluble amino acid, serine. The position of the serine residue is remote from the active catalytic site or the N-terminal sequence of Dsp-nCA-c. However, amino acid 263 is also found on the surface of the protein. Therefore, the serine residue of Dsp-nCA-c (G263S) is also expected to contribute to the increased solubility of Dsp-nCA-c. Amino acids with a charged and polar surface are capable of ionic bonding with ionic residues, It is anticipated that it will help stabilize the protein in the state. This may have had some effect on the expression of Dsp-nCA-c and serine mutants and the increase in enzyme activity.

10. CO ₂ Mineralization

CA can be used for CO ₂ removal as a CO ₂ mineralization catalyst. CA can convert CO ₂ to HCO ₃ ^- in aqueous solution and CaCO ₃ in the presence of Ca ^{2 +} . CaCO ₃ has three types of crystals: rhombic calcite, needle aragonite, and spherical vaterite. The weight of dry CaCO ₃ formed with the same amount of Dsp-CA-n, Dsp-CA-c, Dsp-nCA-c and Dsp-nCA-c (G263S) was almost 8, 21, 17 and 20 mg. It was found that Dsp-CA-c, Dsp-nCA-c and Dsp-nCA-c (G263S) remove almost 4 mg CO ₂ / mg protein. The CaCO ₃ formed by these CAs was analyzed by XRD and the calcite and vaterite ratios were shown in FIG. 5 and Table 2 below. Dsp-CA forms 96% of CaCO ₃ with calcite. Similarly, CaCO ₃ by chimeric protein mainly formed calcite at a ratio of 86% and 92%. Dsp-CA-n and Dsp-CA-c, which had little enzyme activity, accounted for 37% and 67%, respectively. The ratio of calcite to Dsp-CA-c and Dsp-CA-n was increased to 79%. SEM images of the formed CaCO ₃ of FIG. 6 also show the differences in the CaCO ₃ structure formed according to different CA's. CaCO ₃ is converted to a calcite phase after vaterite, beginning with a non-crystalline phase. These two CaCO ₃ ratios differed depending on the enzyme activity and type of protein. The total amount of CaCO ₃ formed in the absence of catalyst is about 1/5 of that formed using functional CA, but the calcite ratio is about 85%. This suggests that CA regulates the formation of CaCO ₃ crystals with special structures as well as CO ₂ mineralization catalysts.

Calcite versus vaterite composition of CaCO ₃ crystals formed by CAs division Calcite (%) Vaterite (%) Noce 85 15 Dsp-CA 96 4 Dsp-CA-n 37 63 Dsp-CA-c 67 33 Dsp-CA-n + Dsp-CA-c 79 21 Dsp-nca-c 86 14 Dsp-nCA-c (G263S) 92 8

SEQ ID NO: 1 Dsp-CA

MVSEPHDYNYEKVGFDWTGGVCVNTGTSKQSPINIETDSLAEESERLGTADDTSRLALKG

LLSSSYQLTSEVAINLEQDMQFSFNAPDEDLPQLTIGGVVHTFKPVQIHFHHFASEHAID

GQLYPLEAHMVMASQNDGSDQLAVIGIMYKYGEEDPFLKRLQETAQSNGEAGDKNVELNS

FSINVARDLLPESDLTYYGYDGSLTTPGCDERVKWHVFKEARTVSVAQLKVFSEVTLAAH

PEATVTNNRVIQPLNGRKVYEYKGEPNDKYNYVQHGFDWRDNGLDSCAGDVQSPIDIVTS

TLQAGSSRSDVSSVNLNDLNTDAFTLTGNTVNIGQGMQINFGDPPAGDLPVIRIGTRDVT

FRPLQVHWHFFLSEHTVDGVHYPLEAHIVMKDNDNLGDSAGQLAVIGYYKYGDADPFIT

DMQKRVSDKIASGAITYGQSGVSLNNPDDPFNVNIKNNFLPSELGYAGYDGSLTTPPCSE

IVKWHVFLEPRTVSVEQMEVFADVTLNSNPGATVTTNRMIQPLEGRTVYGYNGAAA

SEQ ID NO: 2 Dsp-CA-n

MVSEPHDYNYEKVGFDWTGGVCVNTGTSKQSPINIETDSLAEESERLGTADDTSRLALKG

LLSSSYQLTSEVAINLEQDMQFSFNAPDEDLPQLTIGGVVHTFKPVQIHFHHFASEHAID

GQLYPLEAHMVMASQNDGSDQLAVIGIMYKYGEEDPFLKRLQETAQSNGEAGDKNVELNS

FSINVARDLLPESDLTYYGYDGSLTTPGCDERVKWHVFKEARTVSVAQLKVFSEVTLAAH

PEATVTNNRVIQPLNGRKVYEYKG

SEQ ID NO: 3 Dsp-CA-c

≪ RTI ID = 0.0 >

FTLTGNTVNIGQGMQINFGDPPAGDLPVIRIGTRDVTFRPLQVHWHFFLSEHTVDGVHYP

SEK

LNNPDDPFNVNIKNNFLPSELGYAGYDGSLTTPPCSEIVKWHVFLEPRTVSVEQMEVFAD

VTLNSNPGATVTTNRMIQPLEGRTVYGYNGAAA

SEQ ID NO: 4 Dsp-nCA-c

MVSEPHDYNYEKHGFDWRDNGLDSCAGDVQSPIDIVTSTLQAGSSRSDVSSVNLNDLNTD

AFTLTGNTVNIGQGMQINFGDPPAGDLPVIRIGTRDVTFRPLQVHWHFFLSEHTVDGVHY

PLEAHIVMKDNDNLGDSAGQLAVIGIMYKYGDADPFITDMQKRVSDKIASGAITYGQSGV

SLNNPDDPFNVNIKNNFLPSELGYAGYDGSLTTPPCSEIVKWHVFLEPRTVSVEQMEVFA

DVTLNSNPGATVTTNRMIQPLEGRTVYGYNGAAA

SEQ ID NO: 5 Dsp-nCA-c (G263S)

MVSEPHDYNYEKHGFDWRDNGLDSCAGDVQSPIDIVTSTLQAGSSRSDVSSVNLNDLNTD

AFTLTGNTVNIGQGMQINFGDPPAGDLPVIRIGTRDVTFRPLQVHWHFFLSEHTVDGVHY

PLEAHIVMKDNDNLGDSAGQLAVIGIMYKYGDADPFITDMQKRVSDKIASGAITYGQSGV

SLNNPDDPFNVNIKNNFLPSELGYAGYDGSLTTPPCSEIVKWHVFLEPRTVSVEQMEVFA

DVTLNSNPGATVTTNRMIQPLESRTVYGYNGAAA

SEQ ID NO: 6

1Y7W: A | PDBID | CHAIN | SEQUENCE

MASMTGGQQMGRGSEEPNPNDGYDYMQHGFDWPGLQEGGTTKYPACSGSNQSPIDINTNQLMEPSSRSGTSAVSLNGLNVDGAQADGITLTNAKVDLEQGMKVTFDQPAANLPTIEIGGTTKSFVPIQFHFHHFLSEHTINGIHYPLELHIVMQEQDPADVATAQLAVIGIMYKYSENGDAFLNSLQTQIEGKIGDGTASYGDTGVSIDNINVKTQLLPSSLKYAGYDGSLTTPGCDERVKWHVFTTPREVTREQMKLFVDVTMGAHAGADVVNNRMIQDLGDREVYKYNY

SEQ ID NO: 7 Dsp-CA

TGACCCGCCGGCTGGTGACCTGCCGGTTATCCGTATCGGTACCCGTGACGTTACCTTCCGTCCGCTGCAGGTTCACTGGCACTTCTTCCTGTCTGAACACACCGTTGACGGTGTTCACTACCCGCTGGAAGCTCACATCGTTATGAAAGACAACGACAACCTGGGTGACTCTGCTGGTCAGCTGGCTGTTATCGGTATCATGTACAAATACGGTGACGCTGACCCGTTCATCACCGACATGCAGAAACGTGTTTCTGACAAAATCGCTTCTGGTGCTATCACCTACGGTCAGTCTGGTGTTTCTCTGAACAACCCGGACGACCCGTTCAACGTTAACATCAAAAACAACTTCCTGCCGTCTGAACTGGGTTACGCTGGTTACGACGGTTCTCTGACCACCCCGCCGTGCTCTGAAATCGTTAAATGGCACGTTTTCCTGGAACCGCGTACCGTTTCTGTTGAACAGATGGAAGTTTTCGCTGACGTTACCCTGAACTCTAACCCGGGTGCTACCGTTACCACCAACCGTATGATCCAGCCGCTGGAAGGTCGTACCGTTTACGGTTACAACGGTGCTGCTGCTTAA

SEQ ID NO: 8 Dsp-CA-n

ATGGTTTCTGAACCGCACGACTACAACTACGAAAAAGTTGGTTTCGACTGGACCGGTGGTGTTTGCGTTAACACCGGTACCTCTAAACAGTCTCCGATCAACATCGAAACCGACTCTCTGGCTGAAGAATCTGAACGTCTGGGTACCGCTGACGACACCTCTCGTCTGGCTCTGAAAGGTCTGCTGTCTTCTTCTTACCAGCTGACCTCTGAAGTTGCTATCAACCTGGAACAGGACATGCAGTTCTCTTTCAACGCTCCGGACGAAGACCTGCCGCAGCTGACCATCGGTGGTGTTGTTCACACCTTCAAACCGGTTCAGATCCACTTCCACCACTTCGCTTCTGAACACGCTATCGACGGTCAGCTGTACCCGCTGGAAGCTCACATGGTTATGGCTTCTCAGAACGACGGTTCTGACCAGCTGGCTGTTATCGGTATCATGTACAAATACGGTGAAGAAGACCCGTTCCTGAAACGTCTGCAGGAAACCGCTCAGTCTAACGGTGAAGCTGGTGACAAAAACGTTGAACTGAACTCTTTCTCTATCAACGTTGCTCGTGACCTGCTGCCGGAATCTGACCTGACCTACTACGGTTACGACGGTTCTCTGACCACCCCGGGTTGCGACGAACGTGTTAAATGGCACGTTTTCAAAGAAGCTCGTACCGTTTCTGTTGCTCAGCTGAAAGTTTTCTCTGAAGTTACCCTGGCTGCTCACCCGGAAGCTACCGTTACCAACAACCGTGTTATCCAGCCGCTGAACGGTCGTAAAGTTTACGAATACAAAGGTTAA

SEQ ID NO: 9 Dsp-CA-c

ATGGAACCGAACGACAAATACAACTACGTTCAGCACGGTTTCGACTGGCGTGACAACGGTCTGGACTCTTGCGCTGGTGACGTTCAGTCTCCGATCGACATCGTTACCTCTACCCTGCAGGCTGGTTCTTCTCGTTCTGACGTTTCTTCTGTTAACCTGAACGACCTGAACACCGACGCTTTCACCCTGACCGGTAACACCGTTAACATCGGTCAGGGTATGCAGATCAACTTCGGTGACCCGCCGGCTGGTGACCTGCCGGTTATCCGTATCGGTACCCGTGACGTTACCTTCCGTCCGCTGCAGGTTCACTGGCACTTCTTCCTGTCTGAACACACCGTTGACGGTGTTCACTACCCGCTGGAAGCTCACATCGTTATGAAAGACAACGACAACCTGGGTGACTCTGCTGGTCAGCTGGCTGTTATCGGTATCATGTACAAATACGGTGACGCTGACCCGTTCATCACCGACATGCAGAAACGTGTTTCTGACAAAATCGCTTCTGGTGCTATCACCTACGGTCAGTCTGGTGTTTCTCTGAACAACCCGGACGACCCGTTCAACGTTAACATCAAAAACAACTTCCTGCCGTCTGAACTGGGTTACGCTGGTTACGACGGTTCTCTGACCACCCCGCCGTGCTCTGAAATCGTTAAATGGCACGTTTTCCTGGAACCGCGTACCGTTTCTGTTGAACAGATGGAAGTTTTCGCTGACGTTACCCTGAACTCTAACCCGGGTGCTACCGTTACCACCAACCGTATGATCCAGCCGCTGGAAGGTCGTACCGTTTACGGTTACAACGGTGCTGCTGCTTAA

SEQ ID NO: 10 Dsp-nCA-c

ATGGTTTCTGAACCGCACGACTACAACTACGAAAAACACGGTTTCGACTGGCGTGACAACGGTCTGGACTCTTGCGCTGGTGACGTTCAGTCTCCGATCGACATCGTTACCTCTACCCTGCAGGCTGGTTCTTCTCGTTCTGACGTTTCTTCTGTTAACCTGAACGACCTGAACACCGACGCTTTCACCCTGACCGGTAACACCGTTAACATCGGTCAGGGTATGCAGATCAACTTCGGTGACCCGCCGGCTGGTGACCTGCCGGTTATCCGTATCGGTACCCGTGACGTTACCTTCCGTCCGCTGCAGGTTCACTGGCACTTCTTCCTGTCTGAACACACCGTTGACGGTGTTCACTACCCGCTGGAAGCTCACATCGTTATGAAAGACAACGACAACCTGGGTGACTCTGCTGGTCAGCTGGCTGTTATCGGTATCATGTACAAATACGGTGACGCTGACCCGTTCATCACCGACATGCAGAAACGTGTTTCTGACAAAATCGCTTCTGGTGCTATCACCTACGGTCAGTCTGGTGTTTCTCTGAACAACCCGGACGACCCGTTCAACGTTAACATCAAAAACAACTTCCTGCCGTCTGAACTGGGTTACGCTGGTTACGACGGTTCTCTGACCACCCCGCCGTGCTCTGAAATCGTTAAATGGCACGTTTTCCTGGAACCGCGTACCGTTTCTGTTGAACAGATGGAAGTTTTCGCTGACGTTACCCTGAACTCTAACCCGGGTGCTACCGTTACCACCAACCGTATGATCCAGCCGCTGGAAGGTCGTACCGTTTACGGTTACAACGGTGCTGCTGCTTAA

SEQ ID NO: 11 Dsp-nCA-c (G263S)

ATGGTTTCTGAACCGCACGACTACAACTACGAAAAACACGGTTTCGACTGGCGTGACAACGGTCTGGACTCTTGCGCTGGTGACGTTCAGTCTCCGATCGACATCGTTACCTCTACCCTGCAGGCTGGTTCTTCTCGTTCTGACGTTTCTTCTGTTAACCTGAACGACCTGAACACCGACGCTTTCACCCTGACCGGTAACACCGTTAACATCGGTCAGGGTATGCAGATCAACTTCGGTGACCCGCCGGCTGGTGACCTGCCGGTTATCCGTATCGGTACCCGTGACGTTACCTTCCGTCCGCTGCAGGTTCACTGGCACTTCTTCCTGTCTGAACACACCGTTGACGGTGTTCACTACCCGCTGGAAGCTCACATCGTTATGAAAGACAACGACAACCTGGGTGACTCTGCTGGTCAGCTGGCTGTTATCGGTATCATGTACAAATACGGTGACGCTGACCCGTTCATCACCGACATGCAGAAACGTGTTTCTGACAAAATCGCTTCTGGTGCTATCACCTACGGTCAGTCTGGTGTTTCTCTGAACAACCCGGACGACCCGTTCAACGTTAACATCAAAAACAACTTCCTGCCGTCTGAACTGGGTTACGCTGGTTACGACGGTTCTCTGACCACCCCGCCGTGCTCTGAAATCGTTAAATGGCACGTTTTCCTGGAACCGCGTACCGTTTCTGTTGAACAGATGGAAGTTTTCGCTGACGTTACCCTGAACTCTAACCCGGGTGCTACCGTTACCACCAACCGTATGATCCAGCCGCTGGAATCTCGTACCGTTTACGGTTACAACGGTGCTGCTGCTTAA

<110> KOREAN UNIVERSITY RESEARCH AND BUSINESS FOUNDATION <120> Chimeric carbonic anhydrase from Dunaliella Salina and use          the <130> P15U13C0579 <160> 15 <170> Kopatentin 2.0 <210> 1 <211> 536 <212> PRT <213> Dunaliella Salina <400> 1 Met Val Ser Glu Pro His Hisp Tyr Asn Tyr Glu Lys Val Gly Phe Asp   1 5 10 15 Trp Thr Gly Gly Val Cys Val Asn Thr Gly Thr Ser Lys Gln Ser Pro              20 25 30 Ile Asn Ile Glu Thr Asp Ser Leu Ala Glu Glu Ser Glu Arg Leu Gly          35 40 45 Thr Ala Asp Asp Thr Ser Arg Leu Ala Leu Lys Gly Leu Leu Ser Ser      50 55 60 Ser Tyr Gln Leu Thr Ser Glu Val Ala Ile Asn Leu Glu Gln Asp Met  65 70 75 80 Gln Phe Ser Phe Asn Ala Pro Asp Glu Asp Leu Pro Gln Leu Thr Ile                  85 90 95 Gly Gly Val Val His Thr Phe Lys Pro Val Gln Ile His Phe His His             100 105 110 Phe Ala Ser Glu His Ala Ile Asp Gly Gln Leu Tyr Pro Leu Glu Ala         115 120 125 His Met Val Met Ala Ser Gln Asn Asp Gly Ser Asp Gln Leu Ala Val     130 135 140 Ile Gly Ile Met Tyr Lys Tyr Gly Glu Glu Asp Pro Phe Leu Lys Arg 145 150 155 160 Leu Gln Glu Thr Ala Gln Ser Asn Gly Glu Ala Gly Asp Lys Asn Val                 165 170 175 Glu Leu Asn Ser Phe Ser Ile Asn Val Ala Arg Asp Leu Leu Pro Glu             180 185 190 Ser Asp Leu Thr Tyr Tyr Gly Tyr Asp Gly Ser Leu Thr Thr Pro Gly         195 200 205 Cys Asp Glu Arg Val Lys Trp His Val Phe Lys Glu Ala Arg Thr Val     210 215 220 Ser Val Ala Gln Leu Lys Val Phe Ser Glu Val Thr Leu Ala Ala His 225 230 235 240 Pro Glu Ala Thr Val Thr Asn Asn Arg Val Ile Gln Pro Leu Asn Gly                 245 250 255 Arg Lys Val Tyr Glu Tyr Lys Gly Glu Pro Asn Asp Lys Tyr Asn Tyr             260 265 270 Val Gln His Gly Phe Asp Trp Arg Asp Asn Gly Leu Asp Ser Cys Ala         275 280 285 Gly Asp Val Gln Ser Pro Ile Asp Ile Val Thr Ser Thr Leu Gln Ala     290 295 300 Gly Ser Ser Arg Ser Ser Val Ser Ser Val Asn Leu Asn Asp Leu Asn 305 310 315 320 Thr Asp Ala Phe Thr Leu Thr Gly Asn Thr Val Asn Ile Gly Gln Gly                 325 330 335 Met Gln Ile Asn Phe Gly Asp Pro Pro Ala Gly Asp Leu Pro Val Ile             340 345 350 Arg Ile Gly Thr Arg Asp Val Thr Phe Arg Pro Leu Gln Val His Trp         355 360 365 His Phe Phe Leu Ser Glu His Thr Val Asp Gly Val His Tyr Pro Leu     370 375 380 Glu Ala His Ile Val Met Lys Asp Asn Asp Asn Leu Gly Asp Ser Ala 385 390 395 400 Gly Gln Leu Ala Val Ile Gly Ile Met Tyr Lys Tyr Gly Asp Ala Asp                 405 410 415 Pro Phe Ile Thr Asp Met Gln Lys Arg Val Val Ser Asp Lys Ile Ala Ser             420 425 430 Gly Ala Ile Thr Tyr Gly Gln Ser Gly Val Ser Leu Asn Asn Pro Asp         435 440 445 Asp Pro Phe Asn Val Asn Ile Lys Asn Asn Phe Leu Pro Ser Glu Leu     450 455 460 Gly Tyr Ala Gly Tyr Asp Gly Ser Leu Thr Thr Pro Pro Cys Ser Glu 465 470 475 480 Ile Val Lys Trp His Val Phe Leu Glu Pro Arg Thr Val Ser Val Glu                 485 490 495 Gln Met Glu Val Phe Ala Asp Val Thr Leu Asn Ser Asn Pro Gly Ala             500 505 510 Thr Val Thr Thr Asn Arg Met Ile Gln Pro Leu Glu Gly Arg Thr Val         515 520 525 Tyr Gly Tyr Asn Gly     530 535 <210> 2 <211> 264 <212> PRT <213> Dunaliella Salina <400> 2 Met Val Ser Glu Pro His Hisp Tyr Asn Tyr Glu Lys Val Gly Phe Asp   1 5 10 15 Trp Thr Gly Gly Val Cys Val Asn Thr Gly Thr Ser Lys Gln Ser Pro              20 25 30 Ile Asn Ile Glu Thr Asp Ser Leu Ala Glu Glu Ser Glu Arg Leu Gly          35 40 45 Thr Ala Asp Asp Thr Ser Arg Leu Ala Leu Lys Gly Leu Leu Ser Ser      50 55 60 Ser Tyr Gln Leu Thr Ser Glu Val Ala Ile Asn Leu Glu Gln Asp Met  65 70 75 80 Gln Phe Ser Phe Asn Ala Pro Asp Glu Asp Leu Pro Gln Leu Thr Ile                  85 90 95 Gly Gly Val Val His Thr Phe Lys Pro Val Gln Ile His Phe His His             100 105 110 Phe Ala Ser Glu His Ala Ile Asp Gly Gln Leu Tyr Pro Leu Glu Ala         115 120 125 His Met Val Met Ala Ser Gln Asn Asp Gly Ser Asp Gln Leu Ala Val     130 135 140 Ile Gly Ile Met Tyr Lys Tyr Gly Glu Glu Asp Pro Phe Leu Lys Arg 145 150 155 160 Leu Gln Glu Thr Ala Gln Ser Asn Gly Glu Ala Gly Asp Lys Asn Val                 165 170 175 Glu Leu Asn Ser Phe Ser Ile Asn Val Ala Arg Asp Leu Leu Pro Glu             180 185 190 Ser Asp Leu Thr Tyr Tyr Gly Tyr Asp Gly Ser Leu Thr Thr Pro Gly         195 200 205 Cys Asp Glu Arg Val Lys Trp His Val Phe Lys Glu Ala Arg Thr Val     210 215 220 Ser Val Ala Gln Leu Lys Val Phe Ser Glu Val Thr Leu Ala Ala His 225 230 235 240 Pro Glu Ala Thr Val Thr Asn Asn Arg Val Ile Gln Pro Leu Asn Gly                 245 250 255 Arg Lys Val Tyr Glu Tyr Lys Gly             260 <210> 3 <211> 273 <212> PRT <213> Dunaliella Salina <400> 3 Met Glu Pro Asn Asp Lys Tyr Asn Tyr Val Gln His Gly Phe Asp Trp   1 5 10 15 Arg Asp Asn Gly Leu Asp Ser Cys Ala Gly Asp Val Gln Ser Pro Ile              20 25 30 Asp Ile Val Thr Ser Thr Leu Gln Ala Gly Ser Ser Arg Ser Serp Val          35 40 45 Ser Ser Val Asn Leu Asn Asp Leu Asn Thr Asp Ala Phe Thr Leu Thr      50 55 60 Gly Asn Thr Val Asn Ile Gly Gln Gly Met Gln Ile Asn Phe Gly Asp  65 70 75 80 Pro Pro Ala Gly Asp Leu Pro Val Ile Arg Ile Gly Thr Arg Asp Val                  85 90 95 Thr Phe Arg Pro Leu Gln Val His Trp His Phe Phe Leu Ser Glu His             100 105 110 Thr Val Asp Gly Val His Tyr Pro Leu Glu Ala His Ile Val Met Lys         115 120 125 Asp Asn Asp Asn Leu Gly Asp Ser Ala Gly Gln Leu Ala Val Ile Gly     130 135 140 Ile Met Tyr Lys Tyr Gly Asp Ala Asp Pro Phe Ile Thr Asp Met Gln 145 150 155 160 Lys Arg Val Ser Asp Lys Ile Ala Ser Gly Ala Ile Thr Tyr Gly Gln                 165 170 175 Ser Gly Val Ser Leu Asn Asn Pro Asp Asp Pro Phe Asn Val Asn Ile             180 185 190 Lys Asn Phe Leu Pro Ser Glu Leu Gly Tyr Ala Gly Tyr Asp Gly         195 200 205 Ser Leu Thr Thr Pro Pro Cys Ser Glu Ile Val Lys Trp His Val Phe     210 215 220 Leu Glu Pro Arg Thr Val Ser Val Glu Gln Met Glu Val Phe Ala Asp 225 230 235 240 Val Thr Leu Asn Ser Asn Pro Gly Ala Thr Val Thr Thr Asn Arg Met                 245 250 255 Ile Gln Pro Leu Glu Gly Arg Thr Val Tyr Gly Tyr Asn Gly Ala Ala             260 265 270 Ala     <210> 4 <211> 274 <212> PRT <213> Artificial Sequence <220> <223> Dsp-nCA-c <400> 4 Met Val Ser Glu Pro His Hisp Tyr Asn Tyr Glu Lys His Gly Phe Asp   1 5 10 15 Trp Arg Asp Asn Gly Leu Asp Ser Cys Ala Gly Asp Val Gln Ser Pro              20 25 30 Ile Asp Ile Val Thr Ser Thr Leu Gln Ala Gly Ser Ser Ser Ser Serp          35 40 45 Val Ser Ser Val Asn Leu Asn Asp Leu Asn Thr Asp Ala Phe Thr Leu      50 55 60 Thr Gly Asn Thr Val Asn Ile Gly Gln Gly Met Gln Ile Asn Phe Gly  65 70 75 80 Asp Pro Pro Ala Gly Asp Leu Pro Val Ile Arg Ile Gly Thr Arg Asp                  85 90 95 Val Thr Phe Arg Pro Leu Gln Val His Trp His Phe Phe Leu Ser Glu             100 105 110 His Thr Val Asp Gly Val His Tyr Pro Leu Glu Ala His Ile Val Met         115 120 125 Lys Asp Asn Asp Asn Leu Gly Asp Ser Ala Gly Gln Leu Ala Val Ile     130 135 140 Gly Ile Met Tyr Lys Tyr Gly Asp Ala Asp Pro Phe Ile Thr Asp Met 145 150 155 160 Gln Lys Arg Val Val Ser Asp Lys Ile Ala Ser Gly Ala Ile Thr Tyr Gly                 165 170 175 Gln Ser Gly Val Ser Leu Asn Asn Pro Asp Asp Pro Phe Asn Val Asn             180 185 190 Ile Lys Asn Asn Phe Leu Pro Ser Glu Leu Gly Tyr Ala Gly Tyr Asp         195 200 205 Gly Ser Leu Thr Thr Pro Pro Cys Ser Glu Ile Val Lys Trp His Val     210 215 220 Phe Leu Glu Pro Arg Thr Val Ser Val Glu Gln Met Glu Val Phe Ala 225 230 235 240 Asp Val Thr Leu Asn Ser Asn Pro Gly Ala Thr Val Thr Thr Asn Arg                 245 250 255 Met Ile Gln Pro Leu Glu Gly Arg Thr Val Tyr Gly Tyr Asn Gly Ala             260 265 270 Ala Ala         <210> 5 <211> 274 <212> PRT <213> Artificial Sequence <220> &Lt; 223 > Dsp-nCA-c (G263S) <400> 5 Met Val Ser Glu Pro His Hisp Tyr Asn Tyr Glu Lys His Gly Phe Asp   1 5 10 15 Trp Arg Asp Asn Gly Leu Asp Ser Cys Ala Gly Asp Val Gln Ser Pro              20 25 30 Ile Asp Ile Val Thr Ser Thr Leu Gln Ala Gly Ser Ser Ser Ser Serp          35 40 45 Val Ser Ser Val Asn Leu Asn Asp Leu Asn Thr Asp Ala Phe Thr Leu      50 55 60 Thr Gly Asn Thr Val Asn Ile Gly Gln Gly Met Gln Ile Asn Phe Gly  65 70 75 80 Asp Pro Pro Ala Gly Asp Leu Pro Val Ile Arg Ile Gly Thr Arg Asp                  85 90 95 Val Thr Phe Arg Pro Leu Gln Val His Trp His Phe Phe Leu Ser Glu             100 105 110 His Thr Val Asp Gly Val His Tyr Pro Leu Glu Ala His Ile Val Met         115 120 125 Lys Asp Asn Asp Asn Leu Gly Asp Ser Ala Gly Gln Leu Ala Val Ile     130 135 140 Gly Ile Met Tyr Lys Tyr Gly Asp Ala Asp Pro Phe Ile Thr Asp Met 145 150 155 160 Gln Lys Arg Val Val Ser Asp Lys Ile Ala Ser Gly Ala Ile Thr Tyr Gly                 165 170 175 Gln Ser Gly Val Ser Leu Asn Asn Pro Asp Asp Pro Phe Asn Val Asn             180 185 190 Ile Lys Asn Asn Phe Leu Pro Ser Glu Leu Gly Tyr Ala Gly Tyr Asp         195 200 205 Gly Ser Leu Thr Thr Pro Pro Cys Ser Glu Ile Val Lys Trp His Val     210 215 220 Phe Leu Glu Pro Arg Thr Val Ser Val Glu Gln Met Glu Val Phe Ala 225 230 235 240 Asp Val Thr Leu Asn Ser Asn Pro Gly Ala Thr Val Thr Thr Asn Arg                 245 250 255 Met Ile Gln Pro Leu Glu Ser Arg Thr Val Tyr Gly Tyr Asn Gly Ala             260 265 270 Ala Ala         <210> 6 <211> 291 <212> PRT <213> Dunaliella Salina <400> 6 Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg Gly Ser Glu Glu   1 5 10 15 Pro Asn Pro Asn Asp Gly Tyr Asp Tyr Met Gln His Gly Phe Asp Trp              20 25 30 Pro Gly Leu Gln Glu Gly Gly Thr Thr Lys Tyr Pro Ala Cys Ser Gly          35 40 45 Ser Asn Gln Ser Pro Ile Asp Ile Asn Thr Asn Gln Leu Met Glu Pro      50 55 60 Ser Ser Arg Ser Gly Thr Ser Ala Val Ser Leu Asn Gly Leu Asn Val  65 70 75 80 Asp Gly Ala Gln Ala Asp Gly Ile Thr Leu Thr Asn Ala Lys Val Asp                  85 90 95 Leu Glu Gln Gly Met Lys Val Thr Phe Asp Gln Pro Ala Ala Asn Leu             100 105 110 Pro Thr Ile Glu Ile Gly Gly Thr Thr Lys Ser Phe Val Pro Ile Gln         115 120 125 Phe His Phe His His Phe Leu Ser Glu His Thr Ile Asn Gly Ile His     130 135 140 Tyr Pro Leu Glu Leu His Ile Val Met Gln Glu Gln Asp Pro Ala Asp 145 150 155 160 Val Ala Thr Ala Gln Leu Ala Val Ile Gly Ile Met Tyr Lys Tyr Ser                 165 170 175 Glu Asn Gly Asp Ala Phe Leu Asn Ser Leu Gln Thr Gln Ile Glu Gly             180 185 190 Lys Ile Gly Asp Gly Thr Ala Ser Tyr Gly Asp Thr Gly Val Ser Ile         195 200 205 Asp Asn Ile Asn Val Lys Thr Gln Leu Leu Pro Ser Ser Leu Lys Tyr     210 215 220 Ala Gly Tyr Asp Gly Ser Leu Thr Thr Pro Gly Cys Asp Glu Arg Val 225 230 235 240 Lys Trp His Val Phe Thr Thr Pro Arg Glu Val Thr Arg Glu Gln Met                 245 250 255 Lys Leu Phe Val Asp Val Thr Met Gly Ala His Ala Gly Ala Asp Val             260 265 270 Val Asn Asn Arg Met Ile Gln Asp Leu Gly Asp Arg Glu Val Tyr Lys         275 280 285 Tyr Asn Tyr     290 <210> 7 <211> 1611 <212> DNA <213> Dunaliella Salina <400> 7 atggtttctg aaccgcacga ctacaactac gaaaaagttg gtttcgactg gaccggtggt 60 gtttgcgtta acaccggtac ctctaaacag tctccgatca acatcgaaac cgactctctg 120 gctgaagaat ctgaacgtct gggtaccgct gacgacacct ctcgtctggc tctgaaaggt 180 ctgctgtctt cttcttacca gctgacctct gaagttgcta tcaacctgga acaggacatg 240 cagttctctt tcaacgctcc ggacgaagac ctgccgcagc tgaccatcgg tggtgttgtt 300 cacaccttca aaccggttca gatccacttc caccacttcg cttctgaaca cgctatcgac 360 ggtcagctgt acccgctgga agctcacatg gttatggctt ctcagaacga cggttctgac 420 cagctggctg ttatcggtat catgtacaaa tacggtgaag aagacccgtt cctgaaacgt 480 ctgcaggaaa ccgctcagtc taacggtgaa gctggtgaca aaaacgttga actgaactct 540 ttctctatca acgttgctcg tgacctgctg ccggaatctg acctgaccta ctacggttac 600 gacggttctc tgaccacccc gggttgcgac gaacgtgtta aatggcacgt tttcaaagaa 660 gctcgtaccg tttctgttgc tcagctgaaa gttttctctg aagttaccct ggctgctcac 720 ccggaagcta ccgttaccaa caaccgtgtt atccagccgc tgaacggtcg taaagtttac 780 gaatacaaag gtgaaccgaa cgacaaatac aactacgttc agcacggttt cgactggcgt 840 gt; accctgcagg ctggttcttc tcgttctgac gtttcttctg ttaacctgaa cgacctgaac 960 accgacgctt tcaccctgac cggtaacacc gttaacatcg gtcagggtat gcagatcaac 1020 ttcggtgacc cgccggctgg tgacctgccg gttatccgta tcggtacccg tgacgttacc 1080 ttccgtccgc tgcaggttca ctggcacttc ttcctgtctg aacacaccgt tgacggtgtt 1140 cactacccgc tggaagctca catcgttatg aaagacaacg acaacctggg tgactctgct 1200 ggtcagctgg ctgttatcgg tatcatgtac aaatacggtg acgctgaccc gttcatcacc 1260 gacatgcaga aacgtgtttc tgacaaaatc gcttctggtg ctatcaccta cggtcagtct 1320 ggtgtttctc tgaacaaccc ggacgacccg ttcaacgtta acatcaaaaa caacttcctg 1380 ccgtctgaac tgggttacgc tggttacgac ggttctctga ccaccccgcc gtgctctgaa 1440 atcgttaaat ggcacgtttt cctggaaccg cgtaccgttt ctgttgaaca gatggaagtt 1500 ttcgctgacg ttaccctgaa ctctaacccg ggtgctaccg ttaccaccaa ccgtatgatc 1560 cagccgctgg aaggtcgtac cgtttacggt tacaacggtg ctgctgctta a 1611 <210> 8 <211> 795 <212> DNA <213> Dunaliella Salina <400> 8 atggtttctg aaccgcacga ctacaactac gaaaaagttg gtttcgactg gaccggtggt 60 gtttgcgtta acaccggtac ctctaaacag tctccgatca acatcgaaac cgactctctg 120 gctgaagaat ctgaacgtct gggtaccgct gacgacacct ctcgtctggc tctgaaaggt 180 ctgctgtctt cttcttacca gctgacctct gaagttgcta tcaacctgga acaggacatg 240 cagttctctt tcaacgctcc ggacgaagac ctgccgcagc tgaccatcgg tggtgttgtt 300 cacaccttca aaccggttca gatccacttc caccacttcg cttctgaaca cgctatcgac 360 ggtcagctgt acccgctgga agctcacatg gttatggctt ctcagaacga cggttctgac 420 cagctggctg ttatcggtat catgtacaaa tacggtgaag aagacccgtt cctgaaacgt 480 ctgcaggaaa ccgctcagtc taacggtgaa gctggtgaca aaaacgttga actgaactct 540 ttctctatca acgttgctcg tgacctgctg ccggaatctg acctgaccta ctacggttac 600 gacggttctc tgaccacccc gggttgcgac gaacgtgtta aatggcacgt tttcaaagaa 660 gctcgtaccg tttctgttgc tcagctgaaa gttttctctg aagttaccct ggctgctcac 720 ccggaagcta ccgttaccaa caaccgtgtt atccagccgc tgaacggtcg taaagtttac 780 gaatacaaag gttaa 795 <210> 9 <211> 822 <212> DNA <213> Dunaliella Salina <400> 9 atggaaccga acgacaaata caactacgtt cagcacggtt tcgactggcg tgacaacggt 60 ctggactctt gcgctggtga cgttcagtct ccgatcgaca tcgttacctc taccctgcag 120 gctggttctt ctcgttctga cgtttcttct gttaacctga acgacctgaa caccgacgct 180 ttcaccctga ccggtaacac cgttaacatc ggtcagggta tgcagatcaa cttcggtgac 240 ccgccggctg gtgacctgcc ggttatccgt atcggtaccc gtgacgttac cttccgtccg 300 ctgcaggttc actggcactt cttcctgtct gaacacaccg ttgacggtgt tcactacccg 360 ctggaagctc acatcgttat gaaagacaac gacaacctgg gtgactctgc tggtcagctg 420 gctgttatcg gtatcatgta caaatacggt gacgctgacc cgttcatcac cgacatgcag 480 aaacgtgttt ctgacaaaat cgcttctggt gctatcacct acggtcagtc tggtgtttct 540 ctgaacaacc cggacgaccc gttcaacgtt aacatcaaaa acaacttcct gccgtctgaa 600 ctgggttacg ctggttacga cggttctctg accaccccgc cgtgctctga aatcgttaaa 660 tggcacgttt tcctggaacc gcgtaccgtt tctgttgaac agatggaagt tttcgctgac 720 gttaccctga actctaaccc gggtgctacc gttaccacca accgtatgat ccagccgctg 780 gaaggtcgta ccgtttacgg ttacaacggt gctgctgctt aa 822 <210> 10 <211> 825 <212> DNA <213> Artificial Sequence <220> <223> Dsp-nCA-c <400> 10 atggtttctg aaccgcacga ctacaactac gaaaaacacg gtttcgactg gcgtgacaac 60 ggtctggact cttgcgctgg tgacgttcag tctccgatcg acatcgttac ctctaccctg 120 caggctggtt cttctcgttc tgacgtttct tctgttaacc tgaacgacct gaacaccgac 180 gctttcaccc tgaccggtaa caccgttaac atcggtcagg gtatgcagat caacttcggt 240 gacccgccgg ctggtgacct gccggttatc cgtatcggta cccgtgacgt taccttccgt 300 ccgctgcagg ttcactggca cttcttcctg tctgaacaca ccgttgacgg tgttcactac 360 ccgctggaag ctcacatcgt tatgaaagac aacgacaacc tgggtgactc tgctggtcag 420 ctggctgtta tcggtatcat gtacaaatac ggtgacgctg acccgttcat caccgacatg 480 cagaaacgtg tttctgacaa aatcgcttct ggtgctatca cctacggtca gtctggtgtt 540 tctctgaaca acccggacga cccgttcaac gttaacatca aaaacaactt cctgccgtct 600 gaactgggtt acgctggtta cgacggttct ctgaccaccc cgccgtgctc tgaaatcgtt 660 aaatggcacg ttttcctgga accgcgtacc gtttctgttg aacagatgga agttttcgct 720 gacgttaccc tgaactctaa cccgggtgct accgttacca ccaaccgtat gatccagccg 780 ctggaaggtc gtaccgttta cggttacaac ggtgctgctg cttaa 825 <210> 11 <211> 825 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > Dsp-nCA-c (G263S) <400> 11 atggtttctg aaccgcacga ctacaactac gaaaaacacg gtttcgactg gcgtgacaac 60 ggtctggact cttgcgctgg tgacgttcag tctccgatcg acatcgttac ctctaccctg 120 caggctggtt cttctcgttc tgacgtttct tctgttaacc tgaacgacct gaacaccgac 180 gctttcaccc tgaccggtaa caccgttaac atcggtcagg gtatgcagat caacttcggt 240 gacccgccgg ctggtgacct gccggttatc cgtatcggta cccgtgacgt taccttccgt 300 ccgctgcagg ttcactggca cttcttcctg tctgaacaca ccgttgacgg tgttcactac 360 ccgctggaag ctcacatcgt tatgaaagac aacgacaacc tgggtgactc tgctggtcag 420 ctggctgtta tcggtatcat gtacaaatac ggtgacgctg acccgttcat caccgacatg 480 cagaaacgtg tttctgacaa aatcgcttct ggtgctatca cctacggtca gtctggtgtt 540 tctctgaaca acccggacga cccgttcaac gttaacatca aaaacaactt cctgccgtct 600 gaactgggtt acgctggtta cgacggttct ctgaccaccc cgccgtgctc tgaaatcgtt 660 aaatggcacg ttttcctgga accgcgtacc gtttctgttg aacagatgga agttttcgct 720 gacgttaccc tgaactctaa cccgggtgct accgttacca ccaaccgtat gatccagccg 780 ctggaatctc gtaccgttta cggttacaac ggtgctgctg cttaa 825 <210> 12 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 12 catatggttt ctgaaccgca cgactacaac 30 <210> 13 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 13 cgcacgacta caactacgaa aaacacggt 29 <210> 14 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 14 ctacgaaaaa cacggtttcg actggcgt 28 <210> 15 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 15 aagcttagca gcagcaccgt tgtaaccgta 30

Claims (21)

A carbonic anhydrase comprising an amino acid sequence represented by SEQ ID NO: 4.
The method according to claim 1,
Carbonic anhydrase from Dunaliella Salina.
The method according to claim 1,
The carbonic anhydrase encoded from the nucleotide sequence shown in SEQ ID NO: 10.
A carbonic anhydrase comprising an amino acid sequence represented by SEQ ID NO: 5.
5. The method of claim 4,
Carbonic anhydrase from Dunaliella Salina.
5. The method of claim 4,
The carbonic anhydrase encoded from the nucleotide sequence shown in SEQ ID NO: 11.
A composition for the capture or fixation of carbon dioxide comprising the carbonic anhydrase according to claim 1.
A composition for the capture or fixation of carbon dioxide comprising the carbonic anhydrase according to claim 4.
An apparatus for trapping and fixing carbon dioxide, comprising a composition for capturing or fixing carbon dioxide according to claim 7 or 8.
A sensor for capturing and fixing carbon dioxide in which a composition for capture or fixation of carbon dioxide according to claim 7 or 8 is immobilized.
A method for trapping or fixing carbon dioxide comprising the step of reacting a saturated solution of carbon dioxide with a composition for capturing or fixing carbon dioxide according to claim 7 or 8.
A kit for the production of a bicarbonate or carbonate compound comprising a composition for capture or fixation of carbon dioxide according to claim 7 or 8.
13. The method of claim 12,
The bicarbonate compound is selected from the group consisting of sodium bicarbonate (Na ₂ HCO ₃ ), potassium bicarbonate (KHCO ₃ ), magnesium bicarbonate (Mg (HCO ₃ ) ₂ ), calcium bicarbonate (Ca (HCO ₃ ) ₂ ) or ammonium bicarbonate (NH ₄₎ HCO ₃₎ a bicarbonate or carbonate compound for preparing the kit.
13. The method of claim 12,
The carbonate compound may be selected from the group consisting of carbonic acid (H ₂ CO ₃ ), lithium carbonate (Li ₂ CO ₃ ), sodium carbonate (Na ₂ CO ₃ ), potassium carbonate (K ₂ CO ₃ ), rubidium carbonate (Rb ₂ CO ₃ ) (Cs ₂ CO _3), beryllium carbonate (BeCO _3), magnesium carbonate (MgCO _3), calcium carbonate (CaCO _3), strontium carbonate (SrCO _3), barium carbonate (BaCO _3), manganese carbonate (MnCO _3), iron carbonates (FeCO _3), cobalt carbonate (CoCO _3), nickel carbonate (NiCO _3), copper carbonate (CuCO _3), silver carbonate (Ag ₂ CO _3), zinc carbonate (ZnCO _3), cadmium carbonate (CdCO _3), A kit for the production of a bicarbonate or carbonate compound of aluminum carbonate (Al ₂ (CO ₃ ) ₃ ), thallium carbonate (Tl ₂ CO ₃ ), lead carbonate (PbCO ₃ ) or lanthanum carbonate (La ₂ (CO ₃ ) ₃ ).
Reacting a carbon dioxide saturated solution with a composition for capture or fixation of carbon dioxide according to claim 7 or 8; And
Reacting the reactant obtained above with an alkali cationic salt to prepare an amorphous bicarbonate or carbonate compound
&Lt; / RTI &gt; or a carbonate compound.
16. The method of claim 15,
The alkaline cationic salt is a salt of an alkali metal such as Mg, K, Na, NH4, Ca, Li, Rb, Cs, Be, Sr, Ba, Mn, Fe, Co, Ni, Cu, Zn, Cd, Ag, Al, Wherein the salt is a bicarbonate or a carbonate compound.
16. The method of claim 15,
Wherein the crystalline bicarbonate or carbonate compound is calcite, aragonite or veterite. &Lt; RTI ID = 0.0 &gt; 8. &lt; / RTI &gt;
A kit for the production of metabolites using bicarbonate or carbon dioxide comprising a composition for capture or fixation of carbon dioxide according to claim 7 or 8.
19. The method of claim 18,
Metabolites include C4 metabolism or a kit for the production of metabolites using bicarbonate or carbon dioxide, which is a lipid metabolite.
A method for producing a metabolite using a composition for capturing or fixing carbon dioxide according to claim 7 or 8 from a C4 metabolism using bicarbonate or carbon dioxide or a lipid metabolism as a catalyst.
Comprising the step of preparing a recombinant vector comprising the coding sequence of a carbonic anhydrase represented by the nucleotide sequence set forth in SEQ ID NO: 10 or 11.

Images