KR101884384B1 - Carbonic Anhydrase Mutant Having Enhanced Thermostability and Activity - Google Patents

Abstract

The present invention relates to a carbonic anhydrase mutant having enhanced enzymatic activity and thermostability, and when the enzyme mutant of the present invention is used, carbon dioxide can be removed and fixed in an eco-friendly manner, and thus the carbonic anhydrase mutant can be usefully used in industrial fields of carbon dioxide abatement and immobilization.

Description

{Carbonic Anhydrase Mutant Having Enhanced Thermostability and Activity}

The present invention relates to a carbonic anhydrase mutant, and more particularly, to a carbonic anhydrase mutant derived from Hydrogenobibrio mariners and a method for fixing carbon dioxide using the carbonic anhydrase variant.

Recently, global warming has emerged as the biggest environmental problem, and as the biggest culprit, carbon dioxide has been selected and technologies and policies for reducing carbon dioxide are being developed. For environmentally sustainable social development, a method of reducing carbon dioxide using biologically derived enzymes is more advantageous than chemical methods. The biochemical carbonic anhydrase can remove and immobilize carbon dioxide by converting carbon dioxide to bicarbonate. In order to incorporate these biologically derived enzymes into practical industrial fields such as power plants, high stability and resistance to heat are required.

There have been attempts to induce carbon dioxide reduction in the industry using carbonic anhydrase, but the activity of carbonic anhydrase was low and could not withstand high temperatures. In addition, since the protein expression rate is low, there is a limitation in actual use. Therefore, existing methods have limitations in industrial use.

On the other hand, Hydrogenovibrio Carinic anhydrase derived from marinus not only has a very high protein expression rate compared to other enzymes but also has similar activity when compared with bovine-derived carbonic anhydrase which is sold at high prices in the market And exhibits high stability and residual activity under heat treatment conditions at 60 캜 (Korean Patent Publication No. 10-2015-0122542 (Nov.

Accordingly, the present inventors have made intensive efforts to find a biologically-derived enzyme having high activity and thermal stability that can be usefully used in an environmentally friendly carbon dioxide abatement and immobilization industry. As a result, the present inventors have found that the hydrolyzate of a hydrocarbyl anhydrase gene derived from a Hydrogenobibrio marinus strain It has been confirmed that enzyme activity and thermal stability increase when a mutation of a carbonic anhydrase is produced by a designated mutation, and the present invention has been completed.

It is an object of the present invention to provide a carbonic anhydrase variant having increased enzyme activity and thermal stability.

It is another object of the present invention to provide a gene encoding the carbonic anhydrase variant, a recombinant vector containing the gene, and a recombinant microorganism into which the recombinant vector is introduced.

It is still another object of the present invention to provide a method for producing a carbonic anhydrase variant by culturing the recombinant microorganism.

It is still another object of the present invention to provide a method for fixing carbon dioxide using the carbonic anhydrase mutant or the recombinant microorganism.

In order to achieve the above object, the present invention provides a method for producing a carbonic anhydrase having an amino acid sequence of SEQ ID NO: 1, which has at least one mutation from the group consisting of (i) G94K or (ii) Provide variants.

The present invention also provides a gene encoding the carbonic anhydrase variant, a recombinant vector containing the gene, and a recombinant microorganism into which the recombinant vector is introduced.

(A) culturing the recombinant microorganism to produce a carbonic anhydrase variant; And (b) obtaining the resulting carbonic anhydrase mutant. The present invention also provides a method for producing a carbonic anhydrase mutant.

The present invention also provides a method for fixing carbon dioxide characterized by using the carbonic anhydrase variant or the recombinant microorganism.

The carbonic anhydrase variant of the present invention significantly increases the activity and thermal stability of the carbonic anhydrase, and can be used to remove and fix the carbon dioxide in an environmentally friendly manner, thus being useful in the field of carbon dioxide reduction and immobilization.

Fig. 1 shows the result of SDS-PAGE of the wild type carbonic anhydrase and mutants.
2 shows the results of esterase activity experiments for comparing the enzymatic activity of the carbonic anhydrase wild type and the mutant.
Fig. 3 shows the results of esterase activity test according to the temperature for comparing the thermal stability of the carbonic anhydrase wild type and the mutant.
Fig. 4 shows the long-term thermal stability test results of the carbonic anhydrase wild type and mutants.

In the present invention, a carbonic anhydrase variant was prepared in order to extract biologically-derived enzymes having high activity and thermal stability in order to remove and fix the carbon dioxide.

In order to prepare the above-mentioned carbonic anhydrase mutant, the 94th and 97th amino acids of the carbonic anhydrase derived from the Hydrogenobibrio marinus strain were substituted with the cysteine having the SS bond and the lysine interacting therewith to obtain a carbonic anhydrase variant As a result, it was confirmed that the above carbonic anhydrase mutant has higher activity and thermal stability than the conventional carbonic anhydrase.

Accordingly, in one aspect, the present invention relates to a carbonic anhydrase variant having (i) G94K or (ii) S97C mutation in a carbonic anhydrase having the amino acid sequence of SEQ ID NO: 1.

In the present invention, the carbonic anhydrase is derived from Hydrogenovibrio marinus .

In addition, in the present invention, the carbonic anhydrase mutant includes the G94K, S97C, and G94K / S97C mutations in SEQ ID NO: 1.

In one embodiment, the carbonic anhydrase variant of the present invention can be prepared by a variety of methods known in the art, including error-prone PCR, DNA shuffling, Site-directed mutagenesis, and the like, but it is characterized in that it is prepared by a site-directed mutagenesis method in detail.

In the present invention, glycine, which is a 94th amino acid, was substituted with lysine (DNA SEQ ID NO: 6) by using a primer in a carbonic anhydrase gene comprising the nucleic acid sequence of SEQ ID NO: 5, (DNA SEQ ID NO: 7). After this, the DPN1 restriction enzyme was treated to leave only the mutated gene. Since the plasmid directly extracted from Escherichia coli has a methyl group in adenosine, DPN1 restriction enzyme was used to specifically cleave it. The plasmid DNA was cut into small pieces by the restriction enzyme treatment, and then a recombinant expression vector having a mutated gene was constructed by PCR.

In addition, the 94th amino acid-substituted recombinant vector was subjected to site-directed mutagenesis to replace the 97th amino acid serine with cysteine (DNA SEQ ID NO: 8) to construct a double-insert recombinant expression vector.

The recombinant expression vectors prepared in the present invention were transformed into Escherichia coli strain BL21 and then cultured to obtain G94K carbonic anhydrase variant (amino acid sequence number 2), S97C carbonic anhydrase variant (amino acid sequence number 3) and G94K / S97C carbonic acid (Amino acid sequence number 4) was obtained.

In order to compare the activity of the wild-type carbonic anhydrase (amino acid sequence number 1) and the enzyme mutant in the present invention, an esterase activity experiment was performed.

 As a result, it was confirmed that the activity of G94K, S97C and G94K / S97C carbonic anhydrase mutants increased 0.6, 1.3 and 3.5 times, respectively, compared with the wild type carbonic anhydrase (FIG. 2).

In order to compare the protein thermal stability of the wild-type and enzyme mutants of the carbonic anhydrase, the activity of esterase activity was examined according to temperature. As a result, the residual activities of carbonic anhydrase, G94K carbonic anhydrase and S97C carbonic anhydrase mutant were increased up to 50 ℃. This is due to the preference and tolerance of Won-Kyunju for the heat inhabited by the hot heat pipe. Thereafter, as the temperature increased, the residual activity rapidly decreased. However, the G94K carbonic anhydrase variant and the S97C carbonic anhydrase mutant showed higher residual activity than the wild type.

The G94K / S97C carbonic anhydrase variant maintained a residual activity close to 100% up to 90 DEG C (FIG. 3). Therefore, it can be seen that the disulfide bond and the lysine amino acid induce the interaction between the protein and increase the thermal stability.

In the present invention, G94K, S97C, and G94K / S97C carbonic anhydrase variants maintained their residual activity for more than 31 hours as a result of an experiment to compare the long-term activities of the wild-type and enzyme mutants of the carbonic anhydrase.

As a result, it was confirmed that the G94K / S97C carbonic anhydrase mutant exhibits significantly higher activity than the carbonic anhydrase wild type, has a strong resistance to high temperature, and maintains high activity for a long time.

In another aspect, the present invention relates to a gene encoding the carbonic anhydrase variant, a recombinant vector comprising the gene, and a recombinant microorganism into which the recombinant vector is introduced.

As used herein, the term "vector" means a DNA product containing a DNA sequence operably linked to an appropriate regulatory sequence capable of expressing the DNA in the appropriate host. The vector may be a plasmid, phage particle, or simply a potential genome insert. Once transformed into the appropriate host, the vector may replicate and function independently of the host genome, or, in some cases, integrate into the genome itself. Because the plasmid is the most commonly used form of the current vector, the terms "plasmid" and "vector" are sometimes used interchangeably in the context of the present invention. However, the present invention includes other forms of vectors having functions equivalent to those known or known in the art. Protein expression vectors used in E. coli include the pET family from Novagen (USA); PBAD family from Invitrogen (USA); PHCE or pCOLD from Takara (Japan); Genocarpus (USA) pACE series; Etc. may be used. In Bacillus subtilis, protein expression can be realized by inserting a desired gene in a specific part of the genome, or a pHT series vector of MoBiTech (Germany) can be used. Proteins can also be expressed in fungi or yeast using genomic insertions or self-replication vectors. A plant protein expression vector can be used using a T-DNA system such as Agrobacterium tumefaciens or Agrobacterium rhizogenes. Typical expression vectors for mammalian cell culture expression are based on, for example, pRK5 (EP 307,247), pSV16B (WO 91/08291) and pVL1392 (Pharmingen).

The expression " expression control sequence "means a DNA sequence that is essential for the expression of a coding sequence operably linked to a particular host organism. Such regulatory sequences include promoters for carrying out transcription, any operator sequences for regulating such transcription, sequences encoding suitable mRNA ribosome binding sites, and sequences controlling the termination of transcription and translation. For example, regulatory sequences suitable for prokaryotes include promoters, optionally operator sequences, and ribosome binding sites. Eukaryotic cells include promoters, polyadenylation signals and enhancers. The most influential factor on the expression level of the gene in the plasmid is the promoter. As the promoter for high expression, SRα promoter and cytomegalovirus-derived promoter are preferably used.

In order to express the DNA sequences of the present invention, any of a wide variety of expression control sequences may be used in the vector. Examples of useful expression control sequences include, for example, early and late promoters of SV40 or adenovirus, lac system, trp system, TAC or TRC system, T3 and T7 promoters, major operator and promoter regions of phage lambda, fd A regulatory region of a coding protein, a promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, a promoter of said phosphatase, such as Pho5, a promoter of yeast alpha-mating system and a prokaryotic or eukaryotic cell or a virus And other sequences known to modulate the expression of the gene of < RTI ID = 0.0 > SEQ ID < / RTI > The T7 RNA polymerase promoter < RTI ID = 0.0 > 10 < / RTI > Can be useful for expressing proteins in E. coli.

A nucleic acid is "operably linked" when placed in a functional relationship with another nucleic acid sequence. This may be the gene and regulatory sequence (s) linked in such a way that the appropriate molecule (e. G., Transcriptional activator protein) is capable of gene expression when bound to the regulatory sequence (s). For example, DNA for a pre-sequence or secretory leader is operably linked to DNA for a polypeptide when expressed as a whole protein participating in the secretion of the polypeptide; A promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; Or the ribosome binding site is operably linked to a coding sequence if it affects the transcription of the sequence; Or a ribosome binding site is operably linked to a coding sequence if positioned to facilitate translation. Generally, "operably linked" means that the linked DNA sequences are in contact and, in the case of a secretory leader, are in contact and present in the reading frame. However, the enhancer need not be in contact. The linkage of these sequences is carried out by ligation (linkage) at convenient restriction sites. If such a site does not exist, a synthetic oligonucleotide adapter or a linker according to a conventional method is used.

The expression vector of the present invention is usually a recombinant carrier into which a fragment of a different DNA is inserted, and generally means a fragment of double-stranded DNA. Herein, the heterologous DNA means a heterologous DNA that is not naturally found in the host cell. Once an expression vector is in a host cell, it can replicate independently of the host chromosomal DNA, and several copies of the vector and its inserted (heterologous) DNA can be generated.

As is well known in the art, to increase the level of expression of a transfected gene in a host cell, the gene must be operably linked to a transcriptional and detoxification regulatory sequence that functions in the selected expression host. Preferably the expression control sequence and the gene are contained within an expression vector containing a bacterial selection marker and a replication origin. If the expression host is a eukaryotic cell, the expression vector should further comprise a useful expression marker in the eukaryotic expression host.

Host cells transformed or transfected with the above expression vectors constitute another aspect of the present invention. As used herein, the term "transformation" means introducing DNA into a host and allowing the DNA to replicate as an extrachromosomal factor or by chromosomal integration. As used herein, the term "transfection" means that an expression vector, whether or not any coding sequence is actually expressed, is accepted by the host cell.

It should be understood that not all vectors and expression control sequences function equally well in expressing the DNA sequences of the present invention. Likewise, not all hosts function identically for the same expression system. However, those skilled in the art will be able to make appropriate selections among a variety of vectors, expression control sequences, and hosts without undue experimentation and without departing from the scope of the present invention. For example, in selecting a vector, the host should be considered because the vector must be replicated within it. The number of copies of the vector, the ability to control the number of copies, and the expression of other proteins encoded by the vector, such as antibiotic markers, must also be considered. In selecting the expression control sequence, a number of factors must be considered. For example, the relative strength of the sequence, controllability and compatibility with the DNA sequences of the present invention should be considered in relation to particularly possible secondary structures. The single cell host may be selected from a selected vector, the toxicity of the product encoded by the DNA sequence of the present invention, the secretion characteristics, the ability to fold the protein correctly, the culture and fermentation requirements, the product encoded by the DNA sequence of the invention And ease of purification. Within the scope of these variables, one skilled in the art can select various vector / expression control sequences / host combinations that can express the DNA sequences of the invention in fermentation or in large animal cultures. As a screening method for cloning cDNA by expression cloning, a binding method, a panning method, a film emulsion method, or the like can be applied.

By " substantially pure "in the context of the present invention is meant that the DNA sequence encoding the polypeptides and polypeptides according to the invention is substantially free of other proteins derived from bacteria.

Host cells for expressing recombinant proteins are widely used for prokaryotic cells such as Escherichia coli and Bacillus subtillis, which are capable of culturing high concentration of cells in a short time, easy to genetically manipulate, and well-characterized genetic and physiological characteristics Has come. However, in order to solve problems of post-translational modification, secretion process, active three-dimensional structure, and active state of proteins, recently, single cell eukaryotic cells such as Pichia pastoris, Saccharomyces cerevisiae, Hansenula polymorpha The present invention utilizes the recombinant protein as a host cell for production of recombinant proteins up to higher organisms such as mammalian cells, filamentous fungi, insect cells, plant cells and mammalian cells. Therefore, In addition, the use of other host cells is readily applicable to those of ordinary skill in the art.

In the present invention, the strain includes, but is not limited to, Escherichia coli BL21 strain.

According to another aspect of the present invention, there is provided a method for producing a carbonic anhydrase variant, comprising: (a) culturing the recombinant microorganism to produce a carbonic anhydrase variant; And (b) obtaining the resulting carbonic anhydrase mutant. The present invention also relates to a method for producing a carbonic anhydrase variant.

The cultivation of the transformed recombinant microorganism is carried out according to well-known methods, and the conditions such as the culture temperature and time, the pH of the culture medium and the like can be appropriately adjusted.

In another aspect, the present invention relates to a method for fixing carbon dioxide, which uses the carbonic anhydrase variant or the recombinant microorganism.

The enzyme mutant and the recombinant microorganism according to the present invention can remove and fix carbon dioxide by converting carbon dioxide into bicarbonate.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for illustrating the present invention and that the scope of the present invention is not construed as being limited by these embodiments.

Example  One: Carbonic anhydride  Construction of recombinant vector through position mutagenesis of enzyme No. 94 And gene  Amplification

Based on the existing gene sequence of carbonic anhydrase (DNA SEQ ID NO: 5), the site-directed mutagenesis was carried out using the primers shown in Table 1. Through this site-directed mutagenesis, glycine, the 94th amino acid of the existing carbonic anhydrase, was replaced with lysine. To obtain only the mutated gene, the plasmid DNA was cut into small pieces by treating with DPN1 restriction enzyme. Then, only the recombinant expression vector having the mutated gene was obtained by PCR (polymerase chain reaction) to construct the G94K recombinant vector.

The nucleotide sequences of the primers used are shown in Table 1.

SEQ ID NO: 9 CTA CCG GGC TTT GAT GTG CAC TAC GTT GAT ACG GTT CTT AAA G SEQ ID NO: 10 CTT TAA GAA CCG TAT CAA CGT AGT GCA CAT CAA AGC CCG GTA G

Example  2: Carbonic anhydride  Construction of recombinant vector through positional mutagenesis of enzyme No. 97 And gene  Amplification

Based on the existing gene sequence of carbonic anhydrase (DNA SEQ ID NO: 5), the site-directed mutagenesis was carried out using the primers shown in Table 2. Through the site-directed mutagenesis, the 97th amino acid serine of the existing carbonic anhydrase was replaced with cysteine. To obtain only the mutated gene, the plasmid DNA was cut into small pieces by treating with DPN1 restriction enzyme. Then, only the recombinant expression vector having the mutated gene was obtained using PCR to construct the S97C recombinant vector substituted with the 97th amino acid.

The nucleotide sequences of the primers used are shown in Table 2.

SEQ ID NO: 11 GTT GGC ACC ACT TGT CTA CCG GGC TTT GAT GTG CAC SEQ ID NO: 12 GTG CAC ATC AAA GCC CGG TAG ACA AGT GGT GCC AAC

Example  3: Carbonic anhydride  Through position 97 mutagenesis of the enzyme 2 overlap  Construction of recombinant vectors and gene amplification

Using the vector constructed in Example 1 as a template, the site-directed mutagenesis was carried out using the primers shown in Table 2. Through the site-directed mutagenesis, the 97th amino acid, serine, was replaced with cysteine. To obtain only the mutated gene, the plasmid DNA was cut into small pieces by treating with DPN1 restriction enzyme. Then, only the recombinant expression vector having the mutated gene was obtained using PCR to construct a G94K / S97C recombinant vector in which all the 94th and 97th amino acids were substituted.

Example 4: Protein expression and purification of wild-type and mutant carbonic anhydrase

Plasmid DNA was obtained from the recombinant E. coli DH5α prepared in Example 1, Example 2 and Example 3, and then transformed into E. coli strain BL21, which is advantageous for protein expression. The transformed Escherichia coli BL21 was inoculated on a Luria-Butani (LB) medium containing ampicillin (50 mg / L) and cultured at 37 ° C until OD = 0.6. After the isopropyl-β-D- Lactopyranoside (IPTG) was added to a total concentration of 1 mM. And then cultured at 16 DEG C for 16 hours. Then, the supernatant and the cells were separated by centrifugation at 4000 rpm and 4 ° C for 10 minutes, and then the cells were well dissolved in a lysis buffer (50 mM NaH ₂ PO ₄ , 400 mM NaCl, 10 mM Imidazole, pH 8.0) Cells were sonicated. After that, centrifugation was carried out at 13,000 rpm and 4 ° C for 30 minutes to remove insoluble proteins, and affinity chromatography was performed using a histidine tag for the water-soluble proteins. Subsequently, the carbonic anhydrase wild type, G94K, S97C and G94K / S97C mutants were confirmed by SDS PAGE, respectively (Fig. 1).

Example 5: Comparison of wild-type and mutant protein quantification and enzymatic activity of carbonic anhydrase

Quantitation of the carbonic anhydrase wild-type, G94K, S97C, and G94K / S97C variants was determined by Bradford assay prior to the carbonic anhydrase activity assay. For the esterase activity, 20 mM Tris-sulfate (pH 7.5) was used as a basic buffer and 30 mM p-NPA as a substrate. After 20 μg of the enzyme was added, OD _{( 405 nm)} was measured. The degree of activity of wild-type was defined as 1 U and the degree of relative activity against mutants was compared. The activities of G94K, S97C and G94K / S97C mutants were increased 0.6, 1.3 and 3.5 times, respectively, compared to wild-type. Through this, it is confirmed that the activity of the enzyme is increased by adding the SS bond and the lysine amino acid interacting therewith (FIG. 2).

Example 6: Thermal stability of wild-type and mutant proteins of carbonic anhydrase

50 ° C, 60 ° C, 70 ° C, 80 ° C and 90 ° C for 30 minutes to measure the enzymatic activity and thermal stability of the carbonic anhydrase wild type, G94K, S97C and G94K / S97C mutants, For 10 minutes to remove the modified protein, and then the activity of carbonic anhydrase esterase activity was measured. The thermal stability was measured by comparing the residual activity against each temperature based on the room temperature of each enzyme performed in Example 5. The carbonic anhydrase wild type, G94K and S97C variants increased the residual activity up to 50 ℃, but then decreased sharply. However, the G94K and S97C mutants showed higher residual activity than the wild type. In addition, it can be seen that the residual activity of the Example 3 mutant is maintained close to 100% up to 90 占 폚. Through this, it was confirmed that the added disulfide bond and the lysine amino acid induce the interaction between the proteins to increase the thermal stability (FIG. 3).

Example 7: Long-term activity comparison between wild-type and mutant proteins of carbonic anhydrase

The enzymes obtained from the carbonic anhydrase wild type, G94K, S97C, and G94K / S97C mutants were subjected to long-term exposure at 70 ° C, and residual activity over time was confirmed. The wild type showed almost no activity within 1 hour of treatment, while the G94K and S97C variants retained 40 to 60% of residual activity for more than 31 hours. Whereas the G94K / S97C mutant maintained nearly 100% residual activity for more than 31 hours (FIG. 4).

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

<110> Korea University Research and Business Foundation <120> Carbonic Anhydrase Mutant Having Enhanced Thermostability and          Activity <130> P17-B037 <160> 12 <170> KoPatentin 3.0 <210> 1 <211> 304 <212> PRT <213> Hydrogenovibrio marinus <400> 1 Met His Ser Asn Ala Pro Leu Ile Asp Leu Gly Ala Glu Met Lys Lys   1 5 10 15 Gln His Lys Glu Ala Ala Pro Glu Gly Ala Ala Pro Ala Gln Gly Lys              20 25 30 Ala Pro Ala Ala Glu Ala Lys Lys Glu Glu Ala Pro Lys Pro Lys Pro          35 40 45 Val Val His Asn Pro His Trp Ser Tyr Ser Gly Glu Glu Gly Pro Asp      50 55 60 His Trp Gly Asp Leu Ser Pro Asp Tyr Ala Thr Cys Lys Thr Gly Lys  65 70 75 80 Asn Gln Ser Pro Ile Asn Leu Met Ala Asp Asp Ala Val Gly Thr Thr                  85 90 95 Ser Leu Pro Gly Phe Asp Val His Tyr Arg Asp Thr Val Leu Lys Val             100 105 110 Ile Asn Asn Gly His Thr Leu Gln Ala Asn Val Pro Leu Gly Ser Tyr         115 120 125 Ile Lys Ile Lys Asn Gln Arg Tyr Glu Leu Leu Gln Tyr His Phe His     130 135 140 Thr Pro Ser Glu His Gln Leu Asn Gly Phe Asn Tyr Pro Met Glu Leu 145 150 155 160 His Leu Val His Arg Asp Gly Arg Gly His Tyr Leu Val Ile Gly Ile                 165 170 175 Leu Phe Arg Glu Gly Lys Glu Asn Asp Ala Leu Gln Thr Ile Leu Asn             180 185 190 His Leu Pro Lys Lys Val Gly Lys Gln Glu Ile Phe Asn Gly Ile Glu         195 200 205 Phe Asn Pro Asn Val Phe Phe Pro Glu Ser Lys Lys Phe Phe Lys Tyr     210 215 220 Ser Gly Ser Leu Thr Thr Pro Pro Cys Thr Glu Gly Val Tyr Trp Met 225 230 235 240 Val Phe Lys Gln Pro Ile Glu Ala Ser Ala Glu Gln Leu Glu Lys Met                 245 250 255 Asn Glu Leu Met Gly Ala Asn Ala Arg Pro Val Gln Asp Leu Glu Ala             260 265 270 Arg Ser Leu Leu Lys Ser Trp Ser Asn Pro Lys Asn Asp Ser Gln Asp         275 280 285 His Arg Tyr Tyr Gln Tyr Tyr Leu Glu His His His His His ***     290 295 300 <210> 2 <211> 304 <212> PRT <213> Artificial Sequence <220> <223> G94K mutant <400> 2 Met His Ser Asn Ala Pro Leu Ile Asp Leu Gly Ala Glu Met Lys Lys   1 5 10 15 Gln His Lys Glu Ala Ala Pro Glu Gly Ala Ala Pro Ala Gln Gly Lys              20 25 30 Ala Pro Ala Ala Glu Ala Lys Lys Glu Glu Ala Pro Lys Pro Lys Pro          35 40 45 Val Val His Asn Pro His Trp Ser Tyr Ser Gly Glu Glu Gly Pro Asp      50 55 60 His Trp Gly Asp Leu Ser Pro Asp Tyr Ala Thr Cys Lys Thr Gly Lys  65 70 75 80 Asn Gln Ser Pro Ile Asn Leu Met Ala Asp Asp Ala Val Lys Thr Thr                  85 90 95 Ser Leu Pro Gly Phe Asp Val His Tyr Arg Asp Thr Val Leu Lys Val             100 105 110 Ile Asn Asn Gly His Thr Leu Gln Ala Asn Val Pro Leu Gly Ser Tyr         115 120 125 Ile Lys Ile Lys Asn Gln Arg Tyr Glu Leu Leu Gln Tyr His Phe His     130 135 140 Thr Pro Ser Glu His Gln Leu Asn Gly Phe Asn Tyr Pro Met Glu Leu 145 150 155 160 His Leu Val His Arg Asp Gly Arg Gly His Tyr Leu Val Ile Gly Ile                 165 170 175 Leu Phe Arg Glu Gly Lys Glu Asn Asp Ala Leu Gln Thr Ile Leu Asn             180 185 190 His Leu Pro Lys Lys Val Gly Lys Gln Glu Ile Phe Asn Gly Ile Glu         195 200 205 Phe Asn Pro Asn Val Phe Phe Pro Glu Ser Lys Lys Phe Phe Lys Tyr     210 215 220 Ser Gly Ser Leu Thr Thr Pro Pro Cys Thr Glu Gly Val Tyr Trp Met 225 230 235 240 Val Phe Lys Gln Pro Ile Glu Ala Ser Ala Glu Gln Leu Glu Lys Met                 245 250 255 Asn Glu Leu Met Gly Ala Asn Ala Arg Pro Val Gln Asp Leu Glu Ala             260 265 270 Arg Ser Leu Leu Lys Ser Trp Ser Asn Pro Lys Asn Asp Ser Gln Asp         275 280 285 His Arg Tyr Tyr Gln Tyr Tyr Leu Glu His His His His His ***     290 295 300 <210> 3 <211> 304 <212> PRT <213> Artificial Sequence <220> <223> S97C mutant <400> 3 Met His Ser Asn Ala Pro Leu Ile Asp Leu Gly Ala Glu Met Lys Lys   1 5 10 15 Gln His Lys Glu Ala Ala Pro Glu Gly Ala Ala Pro Ala Gln Gly Lys              20 25 30 Ala Pro Ala Ala Glu Ala Lys Lys Glu Glu Ala Pro Lys Pro Lys Pro          35 40 45 Val Val His Asn Pro His Trp Ser Tyr Ser Gly Glu Glu Gly Pro Asp      50 55 60 His Trp Gly Asp Leu Ser Pro Asp Tyr Ala Thr Cys Lys Thr Gly Lys  65 70 75 80 Asn Gln Ser Pro Ile Asn Leu Met Ala Asp Asp Ala Val Gly Thr Thr                  85 90 95 Cys Leu Pro Gly Phe Asp Val His Tyr Arg Asp Thr Val Leu Lys Val             100 105 110 Ile Asn Asn Gly His Thr Leu Gln Ala Asn Val Pro Leu Gly Ser Tyr         115 120 125 Ile Lys Ile Lys Asn Gln Arg Tyr Glu Leu Leu Gln Tyr His Phe His     130 135 140 Thr Pro Ser Glu His Gln Leu Asn Gly Phe Asn Tyr Pro Met Glu Leu 145 150 155 160 His Leu Val His Arg Asp Gly Arg Gly His Tyr Leu Val Ile Gly Ile                 165 170 175 Leu Phe Arg Glu Gly Lys Glu Asn Asp Ala Leu Gln Thr Ile Leu Asn             180 185 190 His Leu Pro Lys Lys Val Gly Lys Gln Glu Ile Phe Asn Gly Ile Glu         195 200 205 Phe Asn Pro Asn Val Phe Phe Pro Glu Ser Lys Lys Phe Phe Lys Tyr     210 215 220 Ser Gly Ser Leu Thr Thr Pro Pro Cys Thr Glu Gly Val Tyr Trp Met 225 230 235 240 Val Phe Lys Gln Pro Ile Glu Ala Ser Ala Glu Gln Leu Glu Lys Met                 245 250 255 Asn Glu Leu Met Gly Ala Asn Ala Arg Pro Val Gln Asp Leu Glu Ala             260 265 270 Arg Ser Leu Leu Lys Ser Trp Ser Asn Pro Lys Asn Asp Ser Gln Asp         275 280 285 His Arg Tyr Tyr Gln Tyr Tyr Leu Glu His His His His His ***     290 295 300 <210> 4 <211> 304 <212> PRT <213> Artificial Sequence <220> <223> G94K / S97C mutant <400> 4 Met His Ser Asn Ala Pro Leu Ile Asp Leu Gly Ala Glu Met Lys Lys   1 5 10 15 Gln His Lys Glu Ala Ala Pro Glu Gly Ala Ala Pro Ala Gln Gly Lys              20 25 30 Ala Pro Ala Ala Glu Ala Lys Lys Glu Glu Ala Pro Lys Pro Lys Pro          35 40 45 Val Val His Asn Pro His Trp Ser Tyr Ser Gly Glu Glu Gly Pro Asp      50 55 60 His Trp Gly Asp Leu Ser Pro Asp Tyr Ala Thr Cys Lys Thr Gly Lys  65 70 75 80 Asn Gln Ser Pro Ile Asn Leu Met Ala Asp Asp Ala Val Lys Thr Thr                  85 90 95 Cys Leu Pro Gly Phe Asp Val His Tyr Arg Asp Thr Val Leu Lys Val             100 105 110 Ile Asn Asn Gly His Thr Leu Gln Ala Asn Val Pro Leu Gly Ser Tyr         115 120 125 Ile Lys Ile Lys Asn Gln Arg Tyr Glu Leu Leu Gln Tyr His Phe His     130 135 140 Thr Pro Ser Glu His Gln Leu Asn Gly Phe Asn Tyr Pro Met Glu Leu 145 150 155 160 His Leu Val His Arg Asp Gly Arg Gly His Tyr Leu Val Ile Gly Ile                 165 170 175 Leu Phe Arg Glu Gly Lys Glu Asn Asp Ala Leu Gln Thr Ile Leu Asn             180 185 190 His Leu Pro Lys Lys Val Gly Lys Gln Glu Ile Phe Asn Gly Ile Glu         195 200 205 Phe Asn Pro Asn Val Phe Phe Pro Glu Ser Lys Lys Phe Phe Lys Tyr     210 215 220 Ser Gly Ser Leu Thr Thr Pro Pro Cys Thr Glu Gly Val Tyr Trp Met 225 230 235 240 Val Phe Lys Gln Pro Ile Glu Ala Ser Ala Glu Gln Leu Glu Lys Met                 245 250 255 Asn Glu Leu Met Gly Ala Asn Ala Arg Pro Val Gln Asp Leu Glu Ala             260 265 270 Arg Ser Leu Leu Lys Ser Trp Ser Asn Pro Lys Asn Asp Ser Gln Asp         275 280 285 His Arg Tyr Tyr Gln Tyr Tyr Leu Glu His His His His His ***     290 295 300 <210> 5 <211> 912 <212> DNA <213> Hydrogenovibrio marinus <400> 5 atgcatagca atgccccatt gattgacttg ggcgcggaaa tgaaaaaaca gcacaaggag 60 gcagctcccg aaggcgctgc gccggctcaa ggtaaggcac ctgccgcgga agccaaaaaa 120 gaagaagcac ctaaaccaaa acccgttgtg cataacccac attggtctta ttcgggagaa 180 gaaggccccg accattgggg agacttgtcg cctgattatg caacctgtaa aaccggcaaa 240 aatcagtcac caattaactt gatggcagat gatgccgttg gcaccacttc actaccgggc 300 tttgatgtgc actaccgtga tacggttctt aaagtcatca acaacggcca cacgctgcaa 360 gccaacgtgc ctttgggtag ctatatcaaa atcaaaaatc agcgttatga gctgttgcag 420 tatcattttc ataccccctc agaacatcag ttgaacggtt tcaattatcc gatggagttg 480 catttggttc accgagatgg tcgtgggcat tatctggtaa ttggtatttt gttcagagag 540 ggtaaagaga acgatgcgtt gcaaactatc ctgaaccact tgcctaaaaa agtcggtaaa 600 caggaaattt ttaatggcat tgaatttaat ccaaatgtct ttttccctga aagtaaaaaa 660 ttctttaaat acagcggctc tttaaccaca ccgccttgta cggaaggggt ttattggatg 720 gtgttcaaac aaccaatcga agcgtcggcg gagcaacttg aaaagatgaa cgaattaatg 780 ggggcgaatg ctcgtcctgt tcaggatttg gaagctcgct cgttgttgaa atcttggagc 840 aatcctaaaa acgatagtca ggatcaccgt tactatcaat attacctcga gcaccaccac 900 caccaccact ga 912 <210> 6 <211> 912 <212> DNA <213> Artificial Sequence <220> <223> G94K mutant <400> 6 atgcatagca atgccccatt gattgacttg ggcgcggaaa tgaaaaaaca gcacaaggag 60 gcagctcccg aaggcgctgc gccggctcaa ggtaaggcac ctgccgcgga agccaaaaaa 120 gaagaagcac ctaaaccaaa acccgttgtg cataacccac attggtctta ttcgggagaa 180 gaaggccccg accattgggg agacttgtcg cctgattatg caacctgtaa aaccggcaaa 240 aatcagtcac caattaactt gatggcagat gatgccgtta agaccacttc actaccgggc 300 tttgatgtgc actaccgtga tacggttctt aaagtcatca acaacggcca cacgctgcaa 360 gccaacgtgc ctttgggtag ctatatcaaa atcaaaaatc agcgttatga gctgttgcag 420 tatcattttc ataccccctc agaacatcag ttgaacggtt tcaattatcc gatggagttg 480 catttggttc accgagatgg tcgtgggcat tatctggtaa ttggtatttt gttcagagag 540 ggtaaagaga acgatgcgtt gcaaactatc ctgaaccact tgcctaaaaa agtcggtaaa 600 caggaaattt ttaatggcat tgaatttaat ccaaatgtct ttttccctga aagtaaaaaa 660 ttctttaaat acagcggctc tttaaccaca ccgccttgta cggaaggggt ttattggatg 720 gtgttcaaac aaccaatcga agcgtcggcg gagcaacttg aaaagatgaa cgaattaatg 780 ggggcgaatg ctcgtcctgt tcaggatttg gaagctcgct cgttgttgaa atcttggagc 840 aatcctaaaa acgatagtca ggatcaccgt tactatcaat attacctcga gcaccaccac 900 caccaccact ga 912 <210> 7 <211> 912 <212> DNA <213> Artificial Sequence <220> <223> S97C mutant <400> 7 atgcatagca atgccccatt gattgacttg ggcgcggaaa tgaaaaaaca gcacaaggag 60 gcagctcccg aaggcgctgc gccggctcaa ggtaaggcac ctgccgcgga agccaaaaaa 120 gaagaagcac ctaaaccaaa acccgttgtg cataacccac attggtctta ttcgggagaa 180 gaaggccccg accattgggg agacttgtcg cctgattatg caacctgtaa aaccggcaaa 240 aatcagtcac caattaactt gatggcagat gatgccgttg gcaccacttg tctaccgggc 300 tttgatgtgc actaccgtga tacggttctt aaagtcatca acaacggcca cacgctgcaa 360 gccaacgtgc ctttgggtag ctatatcaaa atcaaaaatc agcgttatga gctgttgcag 420 tatcattttc ataccccctc agaacatcag ttgaacggtt tcaattatcc gatggagttg 480 catttggttc accgagatgg tcgtgggcat tatctggtaa ttggtatttt gttcagagag 540 ggtaaagaga acgatgcgtt gcaaactatc ctgaaccact tgcctaaaaa agtcggtaaa 600 caggaaattt ttaatggcat tgaatttaat ccaaatgtct ttttccctga aagtaaaaaa 660 ttctttaaat acagcggctc tttaaccaca ccgccttgta cggaaggggt ttattggatg 720 gtgttcaaac aaccaatcga agcgtcggcg gagcaacttg aaaagatgaa cgaattaatg 780 ggggcgaatg ctcgtcctgt tcaggatttg gaagctcgct cgttgttgaa atcttggagc 840 aatcctaaaa acgatagtca ggatcaccgt tactatcaat attacctcga gcaccaccac 900 caccaccact ga 912 <210> 8 <211> 912 <212> DNA <213> Artificial Sequence <220> <223> G94K / S97C mutant <400> 8 atgcatagca atgccccatt gattgacttg ggcgcggaaa tgaaaaaaca gcacaaggag 60 gcagctcccg aaggcgctgc gccggctcaa ggtaaggcac ctgccgcgga agccaaaaaa 120 gaagaagcac ctaaaccaaa acccgttgtg cataacccac attggtctta ttcgggagaa 180 gaaggccccg accattgggg agacttgtcg cctgattatg caacctgtaa aaccggcaaa 240 aatcagtcac caattaactt gatggcagat gatgccgtta agaccacttg tctaccgggc 300 tttgatgtgc actaccgtga tacggttctt aaagtcatca acaacggcca cacgctgcaa 360 gccaacgtgc ctttgggtag ctatatcaaa atcaaaaatc agcgttatga gctgttgcag 420 tatcattttc ataccccctc agaacatcag ttgaacggtt tcaattatcc gatggagttg 480 catttggttc accgagatgg tcgtgggcat tatctggtaa ttggtatttt gttcagagag 540 ggtaaagaga acgatgcgtt gcaaactatc ctgaaccact tgcctaaaaa agtcggtaaa 600 caggaaattt ttaatggcat tgaatttaat ccaaatgtct ttttccctga aagtaaaaaa 660 ttctttaaat acagcggctc tttaaccaca ccgccttgta cggaaggggt ttattggatg 720 gtgttcaaac aaccaatcga agcgtcggcg gagcaacttg aaaagatgaa cgaattaatg 780 ggggcgaatg ctcgtcctgt tcaggatttg gaagctcgct cgttgttgaa atcttggagc 840 aatcctaaaa acgatagtca ggatcaccgt tactatcaat attacctcga gcaccaccac 900 caccaccact ga 912 <210> 9 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 ctaccgggct ttgatgtgca ctacgttgat acggttctta aag 43 <210> 10 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 ctttaagaac cgtatcaacg tagtgcacat caaagcccgg tag 43 <210> 11 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 gttggcacca cttgtctacc gggctttgat gtgcac 36 <210> 12 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 gtgcacatca aagcccggta gacaagtggt gccaac 36

Claims (8)

A carbonic anhydrase variant having (i) G94K or (ii) S97C mutation in a carbonic anhydrase having the amino acid sequence of SEQ ID NO: 1.
The carbonic anhydrase mutant according to claim 1, wherein the carbonic anhydrase is derived from Hydrogenovibrio marinus .
The carbonic anhydrase variant according to claim 1, wherein the carbonic anhydrase variant comprises G94K / S97C in SEQ ID NO: 1.
A gene encoding the carbonic anhydrase variant of claim 1.
A recombinant vector comprising the gene of claim 4.
A recombinant microorganism into which the recombinant vector of claim 5 is introduced.
A method for producing a carbonic anhydrase variant comprising the steps of:
(a) culturing the recombinant microorganism of claim 6 to produce a carbonic anhydrase variant; And
(b) obtaining the resulting carbonic anhydrase variant.
A method for fixing carbon dioxide, which comprises using the carbonic anhydrase variant of claim 1 or the recombinant microorganism of claim 6.

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