Modified carbonic anhydrase

Abstract

To provide a modified carbonic anhydrase derived from Thermosulfurimonas dismutans.SOLUTION: This problem is solved by a carbonic anhydrase having an amino acid sequence in which an amino acid at a specific position in the amino acid sequence of carbonic anhydrase is substituted with another amino acid.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present disclosure relates to modified carbonic anhydrases. [Background technology]

[0002] Carbonic anhydrase is an enzyme that catalyzes the reaction of converting carbon dioxide and water into hydrogen ions and bicarbonate ions. Carbonic anhydrase can be used, for example, to treat carbon dioxide.

[0003] Patent Document 1 and Non-Patent Document 1 disclose the introduction of the amino acid substitution S82Y for the purpose of improving the productivity of carbonic anhydrase derived from Thermosulfurimonas dismutans.

[0004] Patent Document 2 discloses an amino acid sequence that has 90% or more identity to carbonic anhydrase derived from Thermobibrio ammonificans and has amino acid substitutions at one or more positions selected from the amino acid residues at positions 2, 5, 7, 8, 9, 12, 13, 16, 17, 18, 19, 21, 22, 52, 73, 77, 116, 125, 126, 131, 138, 156, 190, 193, and 206.

[0005] Patent Document 3 discloses a CO2 absorption method using a mutant of Thermobibrio ammonificans-derived carbonic anhydrase in which positions 2 to 6 (GGGAH) on the N-terminus have been deleted, and discloses that the mutant has a residual activity of 50% or more when kept at 80°C for 16 hours.

[0006] Patent Document 4 discloses an amino acid sequence that has 90% or more identity to carbonic anhydrase derived from Thermobibrio ammonificans and has amino acid substitutions at one or more positions selected from the amino acid residues at positions 3, 6, 11, 15, 17, 20, 24, 25, 38, 39, 48, 64, 79, 88, 119, 128, 130, 137, 145, 148, 149, 154, 160, 166, 168, 195, 199, 203, 210, and 223.

[0007] Patent Document 5 discloses an amino acid sequence that has 90% or more identity to carbonic anhydrase derived from Sulfuhydrogenibium sp. and has amino acid substitutions at one or more positions selected from the amino acid residues at positions 18, 20, 38, 52, 57, 82, 100, 130, 150, and 181.

[0008] Patent Document 6 discloses carbonic anhydrase derived from Desulfovibrio vulgaris that has amino acid substitutions.

[0009] However, there have been no reports on improving the thermal stability of the carbonic anhydrase derived from Thermosulfurimonas dismutans, and no mutants with improved stability have been known.Furthermore, further improvements in the productivity of the carbonic anhydrase derived from Thermosulfurimonas dismutans have been desired. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] No. KR10-2021-0120388 [Patent Document 2] WO2017 / 035667 issue [Patent Document 3] WO2016 / 029316 issue [Patent Document 4] WO2020 / 194124 issue [Patent Document 5] WO2014 / 066999 issue [Patent Document 6] WO2012 / 003277 issue [Non-patent literature]

[0011] [Non-Patent Document 1] Byung Hoon Jo et al., Int.J.Mol.Sci.,2020,21,103 Summary of the Invention [Problem to be solved by the invention]

[0012] An objective of the present disclosure is to provide a modified carbonic anhydrase derived from Thermosulfurimonas dismutans. One aspect of the present disclosure is to provide a carbonic anhydrase derived from Thermosulfurimonas dismutans with improved stability. In another aspect, an objective of the present disclosure is to provide a carbonic anhydrase derived from Thermosulfurimonas dismutans with improved thermal stability. In yet another aspect, an objective of the present disclosure is to provide a carbonic anhydrase derived from Thermosulfurimonas dismutans with improved alkali resistance. An object of one aspect of the present disclosure is to provide a carbonic anhydrase derived from Thermosulfurimonas dismutans with improved productivity. [Means for solving the problem]

[0013] The present inventors have discovered amino acid mutations that improve the stability and / or productivity of Thermosulfurimonas dismutans-derived carbonic anhydrase.

[0014] [1] A carbonic anhydrase selected from any one of the following (i) to (iii): (i) A carbonic anhydrase having an amino acid sequence from the 21st glycine residue to the 249th lysine residue in the amino acid sequence set forth in SEQ ID NO: 1, the amino acid sequence including at least one or more amino acid substitutions selected from the following (1) to (22): (1) The amino acid residue corresponding to the lysine residue at position 33 of SEQ ID NO: 1 is substituted with a glutamic acid residue (2) The amino acid residue corresponding to the lysine residue at position 165 of SEQ ID NO: 1 is substituted with a glutamic acid residue (3) The amino acid residue corresponding to the methionine residue at position 182 of SEQ ID NO: 1 is substituted with a leucine residue (4) The amino acid residue corresponding to the 66th isoleucine residue of SEQ ID NO: 1 is substituted with a valine residue (5) The amino acid residue corresponding to the lysine residue at position 229 of SEQ ID NO: 1 is substituted with a glutamic acid residue (6) The amino acid residue corresponding to the 231st methionine residue of SEQ ID NO: 1 is substituted with an isoleucine residue (7) The amino acid residue corresponding to the lysine residue at position 173 of SEQ ID NO: 1 is substituted with an arginine residue (8) The amino acid residue corresponding to the lysine residue at position 214 of SEQ ID NO: 1 is substituted with an arginine residue (9) The amino acid residue corresponding to the lysine residue at position 246 of SEQ ID NO: 1 is substituted with an arginine residue (10) The amino acid residue corresponding to the 26th valine residue of SEQ ID NO: 1 is substituted with an alanine residue (11) The amino acid residue corresponding to the 43rd alanine residue of SEQ ID NO: 1 is substituted with a valine residue (12) The amino acid residue corresponding to the lysine residue at position 226 of SEQ ID NO: 1 is substituted with an arginine residue (13) The amino acid residue corresponding to the 69th glutamine residue of SEQ ID NO: 1 is substituted with an arginine residue (14) The amino acid residue corresponding to the 103rd valine residue of SEQ ID NO: 1 is substituted with an isoleucine residue (15) The amino acid residue corresponding to the serine residue at position 63 of SEQ ID NO: 1 is substituted with a threonine residue (16) The amino acid residue corresponding to the 69th glutamine residue of SEQ ID NO: 1 is substituted with an aspartic acid residue (17) The amino acid residue corresponding to the 169th glutamic acid residue of SEQ ID NO: 1 is substituted with an aspartic acid residue (18) The amino acid residue corresponding to the histidine residue at position 170 of SEQ ID NO: 1 is substituted with a glutamic acid residue (19) The amino acid residue corresponding to the 23rd glycine residue of SEQ ID NO: 1 is substituted with any amino acid residue other than a glycine residue or a threonine residue. (20) The amino acid residue corresponding to the 21st glycine residue of SEQ ID NO: 1 is substituted with a proline residue (21) The amino acid residue corresponding to the 22nd glycine residue of SEQ ID NO: 1 is substituted with a serine residue (22) the amino acid residue corresponding to the histidine residue at position 24 of SEQ ID NO: 1 is replaced with a glutamic acid residue; (ii) a carbonic anhydrase having an amino acid sequence comprising amino acid residues from the 21st glycine residue to the 249th lysine residue in the amino acid sequence set forth in SEQ ID NO: 1, which contains one or more amino acid substitutions selected from (1) to (22), and further including one or more substitutions, deletions, insertions, and additions of one or more amino acid residues at one or more positions in addition to the amino acid substitutions, and which has at least one or more activities selected from the group consisting of decarboxylation activity and CO hydration activity; (iii) A carbonic anhydrase having an amino acid sequence from the 21st glycine residue to the 249th lysine residue in the amino acid sequence set forth in SEQ ID NO: 1, which has an identity of 70% or more to the entire amino acid sequence containing one or more amino acid substitutions selected from (1) to (22), with the proviso that the amino acid sequence maintains the amino acid substitutions and has at least one of the activities of decarboxylation activity and CO2 hydration activity. [2] The carbonic anhydrase according to claim 1, selected from any one of the following (iv) to (vi): (iv) a carbonic anhydrase having an amino acid sequence from the 21st glycine residue to the 249th lysine residue in the amino acid sequence set forth in SEQ ID NO: 1, which contains the amino acid substitution of (1) above and further contains one or more amino acid substitutions selected from (2) to (14) and (19) above; (v) A carbonic anhydrase having an amino acid sequence from the 21st glycine residue to the 249th lysine residue in the amino acid sequence set forth in SEQ ID NO: 1, which contains the amino acid substitution (1) above, and further contains one or more amino acid substitutions selected from (2) to (14) and (19) above, and further contains, in addition to the amino acid substitutions, any one or more of substitution, deletion, insertion, and addition of one or more amino acid residues at one or more positions, and which has at least one or more activities selected from the group consisting of decarboxylation activity and CO hydration activity; (vi) the amino acid sequence of SEQ ID NO: 1 from the 21st glycine residue to the 249th glycine residue a carbonic anhydrase having an amino acid sequence up to the lysine residue of (1), which contains the amino acid substitution of (1) above and which has an identity of 70% or more to the entire amino acid sequence further containing one or more amino acid substitutions selected from (2) to (14) and (19), with the proviso that the amino acid sequence maintains the amino acid substitutions and has at least one of the activities of decarboxylation and CO2 hydration. [3] A polynucleotide encoding the carbonic anhydrase according to [1] or [2]. [4] [3] An expression vector comprising the polynucleotide described in [3]. [5] A transformant obtained by transforming a host with the expression vector according to [4]. [6] The transformant according to [5], wherein the host is Escherichia coli. [7] A method for producing carbonic anhydrase, comprising the steps of: culturing the transformant described in [5] to express carbonic anhydrase; and recovering the expressed enzyme from the resulting culture. [8] A carbon dioxide separation and absorption solution containing the carbonic anhydrase according to [1] or [2]. [9] A method for separating and capturing carbon dioxide, comprising: A method comprising a step of using the carbonic anhydrase according to [1] or [2]. [Effects of the Invention]

[0015] According to the present disclosure, mutant Thermosulfurimonas According to another embodiment, a mutant Thermosulfurimonas dismutans-derived carbonic anhydrase having improved thermostability can be provided. According to another embodiment, a mutant Thermosulfurimonas dismutans-derived carbonic anhydrase having improved alkali resistance can be provided. According to another embodiment of the present disclosure, a mutant Thermosulfurimonas dismutans-derived carbonic anhydrase having improved productivity while maintaining enzyme activity equivalent to that of the wild type can be provided. dismutans-derived carbonic anhydrase can be provided.

[0016] Use of this mutant Thermosulfurimonas dismutans carbonic anhydrase is expected to enable efficient treatment of carbon dioxide under high temperature and / or alkaline conditions. [Brief explanation of the drawings]

[0017] [Figure 1] A comparison of the decarboxylation activity of amino acid-substituted Thermosulfurimonas dismutans carbonic anhydrase (tdCA). [Figure 2] A graph comparing the CO2 hydration activity of tdCA amino acid substitutions. [Figure 3] FIG. 1 is a graph comparing the decarboxylation activity of tdCA amino acid substitutions. [Figure 4] FIG. 1 is a graph comparing the decarboxylation activity of tdCA amino acid substitutions. [Figure 5] FIG. 1 is a graph comparing the decarboxylation activity of tdCA amino acid substitutions. [Figure 6] FIG. 1 is a graph comparing the decarboxylation activity of tdCA amino acid substitutions. [Figure 7]FIG. 1 is a graph comparing the decarboxylation activity of tdCA amino acid substitutions. [Figure 8] A graph comparing the CO2 hydration activity of tdCA amino acid substitutions. [Figure 9] A graph comparing the CO2 hydration activity of tdCA amino acid substitutions. [Figure 10] FIG. 1 is a graph comparing the decarboxylation activity of tdCA amino acid substitutions. [Figure 11] FIG. 1 is a graph comparing the decarboxylation activity of tdCA amino acid substitutions. [Figure 12] A graph comparing the productivity of tdCA amino acid substitution mutants. [Figure 13] FIG. 1 is a graph comparing the decarboxylation activity of tdCA amino acid substitutions. [Figure 14] FIG. 1 is a graph comparing the decarboxylation activity of tdCA amino acid substitutions. [Figure 15] FIG. 1 is a graph comparing the decarboxylation activity of tdCA amino acid substitutions. [Figure 16]

[0033] FIG. 1 shows the results of comparing the purification yield per culture medium between the amino acid-substituted Thermosulfurimonas dismutans-derived carbonic anhydrase (tdCA) mutants prepared in Example 19 and wild-type tdCA (SEQ ID NO: 2). In this figure, the purification yield per culture medium is shown as a relative value, with the purification yield per culture medium for wild-type tdCA set at 1. [Figure 17] 1 shows the results of comparing the decarboxylation activity of the amino acid-substituted tdCA prepared in Example 19 with that of wild-type tdCA (SEQ ID NO: 2). In this figure, the decarboxylation activity is shown as a relative value, with the decarboxylation activity of wild-type tdCA set at 1. [Figure 18]

[0033] Figure 2 shows the results of comparing the purification yield per culture medium between the amino acid-substituted tdCA prepared in Example 23 and wild-type tdCA (SEQ ID NO: 2). In this figure, the purification yield per culture medium is shown as a relative value, with the purification yield per culture medium for wild-type tdCA set at 1. [Figure 19]1 shows the results of comparing the decarboxylation activity of the amino acid-substituted tdCA prepared in Example 23 with that of wild-type tdCA (SEQ ID NO: 2). In this figure, the decarboxylation activity is shown as a relative value, with the decarboxylation activity of wild-type tdCA set at 1. DETAILED DESCRIPTION OF THE INVENTION

[0018] In this specification, the words "include" or "have" include the case of "consisting of." Therefore, the description of "include" or "have" in this specification may be read as "consisting of."

[0019] <1> Carbonic anhydrase The present disclosure provides carbonic anhydrases having the "specific mutations" described herein. More specifically, the present disclosure may provide a carbonic anhydrase selected from any of the following (i) to (iii): (i) A carbonic anhydrase having an amino acid sequence from the 21st glycine residue to the 249th lysine residue in the amino acid sequence set forth in SEQ ID NO: 1, the amino acid sequence including at least one or more amino acid substitutions selected from the following (1) to (22): (1) The amino acid residue corresponding to the lysine residue at position 33 of SEQ ID NO: 1 is substituted with a glutamic acid residue (2) The amino acid residue corresponding to the lysine residue at position 165 of SEQ ID NO: 1 is substituted with a glutamic acid residue (3) The amino acid residue corresponding to the methionine residue at position 182 of SEQ ID NO: 1 is substituted with a leucine residue (4) The amino acid residue corresponding to the 66th isoleucine residue of SEQ ID NO: 1 is substituted with a valine residue (5) The amino acid residue corresponding to the lysine residue at position 229 of SEQ ID NO: 1 is substituted with a glutamic acid residue (6) The amino acid residue corresponding to the 231st methionine residue of SEQ ID NO: 1 is substituted with an isoleucine residue (7) The amino acid residue corresponding to the lysine residue at position 173 of SEQ ID NO: 1 is substituted with an arginine residue (8) The amino acid residue corresponding to the lysine residue at position 214 of SEQ ID NO: 1 is substituted with an arginine residue (9) The amino acid residue corresponding to the lysine residue at position 246 of SEQ ID NO: 1 is substituted with an arginine residue (10) The amino acid residue corresponding to the 26th valine residue of SEQ ID NO: 1 is substituted with an alanine residue (11) The amino acid residue corresponding to the 43rd alanine residue of SEQ ID NO: 1 is substituted with a valine residue (12) The amino acid residue corresponding to the lysine residue at position 226 of SEQ ID NO: 1 is substituted with an arginine residue (13) The amino acid residue corresponding to the 69th glutamine residue of SEQ ID NO: 1 is substituted with an arginine residue (14) The amino acid residue corresponding to the 103rd valine residue of SEQ ID NO: 1 is substituted with an isoleucine residue (15) The amino acid residue corresponding to the serine residue at position 63 of SEQ ID NO: 1 is substituted with a threonine residue (16) The amino acid residue corresponding to the 69th glutamine residue of SEQ ID NO: 1 is substituted with an aspartic acid residue (17) The amino acid residue corresponding to the 169th glutamic acid residue of SEQ ID NO: 1 is substituted with an aspartic acid residue (18) The amino acid residue corresponding to the histidine residue at position 170 of SEQ ID NO: 1 is substituted with a glutamic acid residue (19) The amino acid residue corresponding to the 23rd glycine residue of SEQ ID NO: 1 is substituted with any amino acid residue other than a glycine residue or a threonine residue. (20) The amino acid residue corresponding to the 21st glycine residue of SEQ ID NO: 1 is substituted with a proline residue (21) The amino acid residue corresponding to the 22nd glycine residue of SEQ ID NO: 1 is substituted with a serine residue (22) the amino acid residue corresponding to the histidine residue at position 24 of SEQ ID NO: 1 is replaced with a glutamic acid residue; (ii) a carbonic anhydrase having an amino acid sequence comprising amino acid residues from the 21st glycine residue to the 249th lysine residue in the amino acid sequence set forth in SEQ ID NO: 1, which contains one or more amino acid substitutions selected from (1) to (22), and further including one or more substitutions, deletions, insertions, and additions of one or more amino acid residues at one or more positions in addition to the amino acid substitutions, and which has at least one or more activities selected from the group consisting of decarboxylation activity and CO hydration activity; (iii) A carbonic anhydrase having an amino acid sequence from the 21st glycine residue to the 249th lysine residue in the amino acid sequence set forth in SEQ ID NO: 1, which has an identity of 70% or more to the entire amino acid sequence containing one or more amino acid substitutions selected from (1) to (22), with the proviso that the amino acid sequence maintains the amino acid substitutions and has at least one of the activities of decarboxylation activity and CO2 hydration activity.

[0020] The present disclosure may provide a carbonic anhydrase selected from any one of the following (iv) to (vi): (iv) a carbonic anhydrase having an amino acid sequence from the 21st glycine residue to the 249th lysine residue in the amino acid sequence set forth in SEQ ID NO: 1, which contains the amino acid substitution of (1) above and further contains one or more amino acid substitutions selected from (2) to (14) and (19) above; (v) A carbonic anhydrase having an amino acid sequence from the 21st glycine residue to the 249th lysine residue in the amino acid sequence set forth in SEQ ID NO: 1, which contains the amino acid substitution (1) above, and further contains one or more amino acid substitutions selected from (2) to (14) and (19) above, and further contains, in addition to the amino acid substitutions, any one or more of substitution, deletion, insertion, and addition of one or more amino acid residues at one or more positions, and which has at least one or more activities selected from the group consisting of decarboxylation activity and CO hydration activity; (vi) the amino acid sequence of SEQ ID NO: 1 from the 21st glycine residue to the 249th glycine residue a carbonic anhydrase having an amino acid sequence up to the lysine residue of (1), which contains the amino acid substitution of (1) above and which has an identity of 70% or more to the entire amino acid sequence further containing one or more amino acid substitutions selected from (2) to (14) and (19), with the proviso that the amino acid sequence maintains the amino acid substitutions and has at least one of the activities of decarboxylation and CO2 hydration.

[0021] The present disclosure may provide a carbonic anhydrase selected from any of the following (vii) to (xii): (vii) a carbonic anhydrase having an amino acid sequence from the 21st glycine residue to the 249th lysine residue in the amino acid sequence set forth in SEQ ID NO: 1, which contains all of the amino acid substitutions (1) and (4) to (6) above, and further contains one or more amino acid substitutions selected from the amino acid substitutions (7) to (9) and (19) above; (viii) A carbonic anhydrase having an amino acid sequence from the 21st glycine residue to the 249th lysine residue in the amino acid sequence set forth in SEQ ID NO: 1, which contains all of the amino acid substitutions (1) and (4) to (6) above, and further contains one or more amino acid substitutions selected from (7) to (9) and (19) above, and further contains, in addition to the amino acid substitutions, any one or more of substitution, deletion, insertion, and addition of one or more amino acid residues at one or more positions, and which has at least one or more activities selected from the group consisting of decarboxylation activity and CO hydration activity; (ix) A carbonic anhydrase having an amino acid sequence from the 21st glycine residue to the 249th lysine residue in the amino acid sequence set forth in SEQ ID NO: 1, which contains all of the amino acid substitutions (1) and (4) to (6) above, and which has 70% or more identity to the entire amino acid sequence containing one or more amino acid substitutions selected from the above (7) to (9) and (19), provided that the amino acid sequence maintains the amino acid substitutions and has at least one of the activities of decarboxylation and CO hydration; (x) a carbonic anhydrase having an amino acid sequence from the 21st glycine residue to the 249th lysine residue in the amino acid sequence set forth in SEQ ID NO: 1, which contains all of the amino acid substitutions (1) and (4) to (9) above, and further contains one or more amino acid substitutions selected from the amino acid substitutions (10) to (14) and (19) above; (xi) A carbonic anhydrase having an amino acid sequence from the 21st glycine residue to the 249th lysine residue in the amino acid sequence set forth in SEQ ID NO: 1, which contains all of the amino acid substitutions (1) and (4) to (9) above, and further contains one or more amino acid substitutions selected from (10) to (14) and (19) above, and further contains, in addition to the amino acid substitutions, any one or more of substitution, deletion, insertion, and addition of one or more amino acid residues at one or more positions, and which has at least one activity selected from the group consisting of decarboxylation activity and CO hydration activity; (xii) A carbonic anhydrase having an amino acid sequence from the 21st glycine residue to the 249th lysine residue in the amino acid sequence set forth in SEQ ID NO: 1, which contains all of the amino acid substitutions (1) and (4) to (9) above, and which has an identity of 70% or more to the entire amino acid sequence containing one or more amino acid substitutions selected from the amino acid substitutions (10) to (14) and (19), provided that the amino acid sequence maintains the amino acid substitutions and has at least one of the activities of decarboxylation activity and CO2 hydration activity.

[0022] The present disclosure may provide a carbonic anhydrase selected from any of the following (xiii) to (xv): (xiii) a carbonic anhydrase having an amino acid sequence from the 21st glycine residue to the 249th lysine residue in the amino acid sequence set forth in SEQ ID NO: 1, the amino acid sequence containing at least one amino acid substitution selected from (15) to (18); (xiv) A carbonic anhydrase having an amino acid sequence from the 21st glycine residue to the 249th lysine residue in the amino acid sequence set forth in SEQ ID NO: 1, which contains at least one or more amino acid substitutions selected from (15) to (18) above, and which further contains, in addition to the amino acid substitutions, one or more of substitution, deletion, insertion, and addition of one or more amino acid residues at one or more positions, and which has at least one or more activities selected from the group consisting of decarboxylation activity and CO hydration activity; (xv) A carbonic anhydrase having an amino acid sequence from the 21st glycine residue to the 249th lysine residue in the amino acid sequence set forth in SEQ ID NO: 1, which has an identity of 70% or more to the entire amino acid sequence containing at least one amino acid substitution selected from (15) to (18), wherein the amino acid sequence maintains the amino acid substitution and has at least one of the activities of decarboxylation and CO2 hydration.

[0023] The present disclosure may provide a carbonic anhydrase selected from any of the following (xvi) to (xviii): (xvi) a carbonic anhydrase having an amino acid sequence from the 21st glycine residue to the 249th lysine residue in the amino acid sequence set forth in SEQ ID NO: 1, in which at least the amino acid residue corresponding to the 23rd glycine residue in SEQ ID NO: 1 is substituted with any of an asparagine residue, a serine residue, an alanine residue, an aspartic acid residue, a glutamic acid residue, a valine residue, a tyrosine residue, a histidine residue, a lysine residue, a leucine residue, a methionine residue, a proline residue, a glutamine residue, an arginine residue, and a tryptophan residue; (xvii) A carbonic anhydrase having an amino acid sequence ranging from the 21st glycine residue to the 249th lysine residue in the amino acid sequence set forth in SEQ ID NO: 1, in which at least the amino acid residue corresponding to the 23rd glycine residue in SEQ ID NO: 1 is substituted with any of asparagine, serine, alanine, aspartic acid, glutamic acid, valine, tyrosine, histidine, lysine, leucine, methionine, proline, glutamine, arginine, and tryptophan residues, and which further includes any one or more of substitution, deletion, insertion, and addition of one or more amino acid residues at one or more positions in addition to the amino acid substitution, and which has at least one or more activities selected from the group consisting of decarboxylation activity and CO hydration activity; (xviii) A carbonic anhydrase having an amino acid sequence of 70% or more identity to the entire amino acid sequence set forth in SEQ ID NO: 1, which is an amino acid sequence from the 21st glycine residue to the 249th lysine residue, in which at least the amino acid residue corresponding to the 23rd glycine residue in SEQ ID NO: 1 is substituted with any of asparagine residue, serine residue, alanine residue, aspartic acid residue, glutamic acid residue, valine residue, tyrosine residue, histidine residue, lysine residue, leucine residue, methionine residue, proline residue, glutamine residue, arginine residue, and tryptophan residue, wherein the amino acid sequence maintains the amino acid substitution and has at least one of decarboxylation activity and CO2 hydration activity.

[0024] The present disclosure may provide a carbonic anhydrase selected from any of the following (xix) to (xxi): (xix) a carbonic anhydrase having an amino acid sequence from the 21st glycine residue to the 249th lysine residue in the amino acid sequence set forth in SEQ ID NO: 1, in which at least the amino acid residue corresponding to the 23rd glycine residue in SEQ ID NO: 1 is substituted with any one of a histidine residue, a lysine residue, a leucine residue, a methionine residue, a proline residue, a glutamine residue, an arginine residue, and a tryptophan residue; (xx) an amino acid sequence from the 21st glycine residue to the 249th lysine residue in the amino acid sequence set forth in SEQ ID NO: 1, in which at least the amino acid residue corresponding to the 23rd glycine residue in SEQ ID NO: 1 is a histidine residue, a lysine residue, a leucine residue, or a methionine residue a carbonic anhydrase having an amino acid sequence in which one or more amino acid residues are substituted with any of α, β, β-actin, β-glutamin, β-arginine, β-tryptophan, β-glutamin ... (xxi) A carbonic anhydrase having an amino acid sequence from the 21st glycine residue to the 249th lysine residue in the amino acid sequence set forth in SEQ ID NO: 1, wherein the amino acid residue corresponding to at least the 23rd glycine residue in SEQ ID NO: 1 is substituted with any of a histidine residue, a lysine residue, a leucine residue, a methionine residue, a proline residue, a glutamine residue, an arginine residue, or a tryptophan residue, and wherein the amino acid sequence maintains the amino acid substitution and the carbonic anhydrase has at least one of the activities of decarboxylation and CO2 hydration.

[0025] In this specification, "any amino acid residue other than a glycine residue or a threonine residue" may refer to any amino acid residue excluding glycine residues and threonine residues among the amino acid residues that generally constitute a peptide, and specifically may refer to any amino acid residue of asparagine residue, serine residue, alanine residue, cysteine residue, aspartic acid residue, glutamic acid residue, phenylalanine residue, histidine residue, isoleucine residue, lysine residue, leucine residue, methionine residue, proline residue, glutamine residue, arginine residue, valine residue, tryptophan residue, or tyrosine residue.

[0026] The "one or several" in (ii), (v), (viii), (xi), (xiv), (xvii), or (xx) will vary depending on the position or type of amino acid residue in the three-dimensional structure of the protein, but may specifically be, for example, 1 to 50, 1 to 40, 1 to 30, 1 to 25, 1 to 24, 1 to 23, 1 to 22, 1 to 20, 1 to 10, 1 to 5, or 1 to 3.

[0027] Furthermore, the "identity" of (iii), (vi), (ix), (xii), (xv), (xviii), or (xxi) may be, for example, an amino acid sequence having 50% or more, 65% or more, 70% or more, 80% or more, 90% or more, 95% or more, 97% or more, or 99% or more identity to the entire amino acid sequence.

[0028] The proteins (ii), (iii), (v), (vi), (viii), (ix), (xi), (xii), (xiv), (xv)(xvii), (xviii), (xx) and / or (xxi) may preferably be proteins having both decarboxylation activity and CO2 hydration activity.

[0029] Carbonic anhydrase is a enzyme that converts bicarbonate ions (HCO3 - Carbonic anhydrase may refer to a protein that has the activity of catalyzing the reaction of converting carbon dioxide (CO2) into bicarbonate ions (HCO3 - ) into CO2 hydration activity. This activity is also referred to as "CO2 hydration activity." In other words, carbonic anhydrase may refer to a protein having at least one of decarboxylation activity and CO2 hydration activity. Carbonic anhydrase may preferably be a protein having both decarboxylation activity and CO2 hydration activity. A gene encoding carbonic anhydrase is also referred to as a "carbonic anhydrase gene."

[0030] Specifically, the term "decarboxylation activity" may mean the activity of catalyzing at least the reaction of the following formula (1). The following reaction is also referred to as a "decarboxylation reaction." HCO3 - +H + →CO2+H2O (1)

[0031] Decarboxylation activity can be measured, for example, by measuring the change in pH over time in a buffer solution (e.g., pH 8.3 initially) containing an enzyme and bicarbonate ions in water (the method described in WO2012 / 003277). The phrase "having decarboxylation activity" may mean that the protein has decarboxylation activity measured under at least one appropriate condition. The same applies to the activity of other proteins mentioned in this application.

[0032] Specifically, the term "CO2 hydration activity" may refer to the activity of catalyzing at least the reaction of the following formula (2). The following reaction is also referred to as the "CO2 hydration reaction." CO2+H2O→HCO3 - +H + ···(2)

[0033] CO2 hydration activity can be measured, for example, by adding an enzyme to a buffer solution containing CO2 in water (e.g., pH 8.3 at the start) into which CO2 gas has been bubbled for a certain period of time in an ice bath, and measuring the change in pH over time (the method described in JH Kim et al., Catalysts, 12(11), 1391). The phrase "having CO2 hydration activity" may mean that the protein has CO2 hydration activity measured under at least one appropriate condition.

[0034] The carbonic anhydrase of the present disclosure has a "specific mutation." A carbonic anhydrase having a "specific mutation" is also referred to as a mutant carbonic anhydrase. In other words, the carbonic anhydrase of the present disclosure is a mutant carbonic anhydrase. A gene encoding a mutant carbonic anhydrase is also referred to as a "mutant carbonic anhydrase gene." As an example, a "mutant carbonic anhydrase gene" may refer to a polynucleotide encoding a mutant carbonic anhydrase.

[0035] Carbonic anhydrase that does not have a "specific mutation" is also referred to as a "wild-type carbonic anhydrase." A gene encoding a wild-type carbonic anhydrase is also referred to as a "wild-type carbonic anhydrase gene." Note that the term "wild-type" used here is a convenient description for distinguishing "wild-type" carbonic anhydrase from "mutant" carbonic anhydrase, and is not limited to those obtained in nature, as long as they do not have a "specific mutation." Wild-type carbonic anhydrase may or may not have mutations other than the "specific mutation," as long as it does not have a "specific mutation."

[0036] When a wild-type carbonic anhydrase and a mutant carbonic anhydrase are identical except for the presence or absence of a "specific mutation," the wild-type carbonic anhydrase is also called "the wild-type carbonic anhydrase corresponding to a mutant carbonic anhydrase," and the mutant carbonic anhydrase is also called "the mutant carbonic anhydrase corresponding to a wild-type carbonic anhydrase."

[0037] Wild-type carbonic anhydrase will be described below.

[0038] A wild-type carbonic anhydrase may or may not have decarboxylation activity and CO hydration activity, as long as the corresponding mutant carbonic anhydrase has at least one of these activities. A wild-type carbonic anhydrase may usually have at least one of decarboxylation activity and CO hydration activity. Alternatively, a wild-type carbonic anhydrase may have both decarboxylation activity and CO hydration activity.

[0039] An example of a wild-type carbonic anhydrase is carbonic anhydrase derived from Thermosulfurimonas dismutans. The amino acid sequence of carbonic anhydrase derived from Thermosulfurimonas dismutans is shown in SEQ ID NO: 1. Furthermore, a sequence obtained by removing the second lysine residue to the twentieth alanine residue, which corresponds to the N-terminal signal sequence, from the amino acid sequence shown in SEQ ID NO: 1 is shown in SEQ ID NO: 2. An example of a wild-type carbonic anhydrase is a protein having the amino acid sequence shown in SEQ ID NO: 2, i.e., a sequence obtained by removing the second lysine residue to the twentieth alanine residue from the amino acid sequence shown in SEQ ID NO: 1. The wild-type carbonic anhydrase gene may be a protein having the nucleotide sequence shown in SEQ ID NO: 3. The sequence of the first 20 amino acids in the amino acid sequence shown in SEQ ID NO: 1 is also known as the N-terminal signal sequence. That is, the wild-type carbonic anhydrase may be, for example, a protein having the amino acid sequence of the carbonic anhydrase derived from Thermosulfurimonas dismutans, excluding the N-terminal signal sequence. An example of a nucleotide sequence encoding the amino acid sequence from the 21st glycine residue to the 249th lysine residue in the amino acid sequence shown in SEQ ID NO: 1, among the gene sequences of the carbonic anhydrase derived from Thermosulfurimonas dismutans, is shown in SEQ ID NO: 3. The wild-type carbonic anhydrase gene may be a gene having the nucleotide sequence shown in SEQ ID NO: 3. Unless otherwise specified, the expression "a gene or protein has a nucleotide sequence or amino acid sequence" may mean that the gene or protein contains the nucleotide sequence or amino acid sequence, and may also include cases where the gene or protein consists of the nucleotide sequence or amino acid sequence.

[0040] A wild-type carbonic anhydrase may be a variant of the above-exemplified wild-type carbonic anhydrase (e.g., a protein having the amino acid sequence shown in SEQ ID NO: 2), as long as it does not have a "specific mutation." Similarly, a wild-type carbonic anhydrase gene may be a variant of the above-exemplified wild-type carbonic anhydrase gene (e.g., a gene having the nucleotide sequence shown in SEQ ID NO: 3), as long as the carbonic anhydrase it encodes does not have a "specific mutation." That is, the term "wild-type carbonic anhydrase" may encompass not only the above-exemplified wild-type carbonic anhydrase (e.g., a protein having the amino acid sequence shown in SEQ ID NO: 2), but also variants thereof. Similarly, the term "wild-type carbonic anhydrase gene" may encompass not only the above-exemplified wild-type carbonic anhydrase gene (e.g., a gene having the nucleotide sequence shown in SEQ ID NO: 3), but also variants thereof. Examples of variants include homologs and artificially modified forms of the above-exemplified genes and proteins.

[0041] Wild-type carbonic anhydrase homologs or wild-type carbonic anhydrase gene homologs can be easily determined from public databases by, for example, BLAST or FASTA searches using the amino acid sequences of the above-exemplified wild-type carbonic anhydrases or the nucleotide sequences of the above-exemplified wild-type carbonic anhydrase genes as query sequences. Furthermore, wild-type carbonic anhydrase gene homologs can be obtained, for example, by PCR using the chromosomes of various organisms as templates and oligonucleotides prepared based on the nucleotide sequences of the above-exemplified wild-type carbonic anhydrase genes as primers.

[0042] A wild-type carbonic anhydrase gene may encode a protein having an amino acid sequence in which one or more amino acids at one or more positions in the above amino acid sequence (e.g., the amino acid sequence shown in SEQ ID NO: 2) have been substituted, deleted, inserted, and / or added, as long as the encoded carbonic anhydrase does not have a "specific mutation." For example, the encoded protein may have its N-terminus and / or C-terminus extended or shortened. Note that the term "one or several" varies depending on the position and type of amino acid residue in the three-dimensional structure of the protein, and may specifically be, for example, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10, 1 to 5, or 1 to 3.

[0043] The substitution, deletion, insertion, or addition of one or several amino acids may be a conservative mutation that maintains the original function of the protein. A representative conservative mutation may be a conservative substitution. A conservative substitution is a mutation in which Phe, Trp, and Tyr are substituted when the substitution site is an aromatic amino acid, Leu, Ile, and Val are substituted when the substitution site is a hydrophobic amino acid, Gln and Asn are substituted when the substitution site is a polar amino acid, Lys, Arg, and His are substituted when the substitution site is a basic amino acid, Asp and Glu are substituted when the substitution site is an acidic amino acid, and Ser and Thr are substituted when the substitution site is an amino acid having a hydroxyl group. Specific examples of substitutions that are considered to be conservative substitutions include a substitution of Ala with Ser or Tyr. substitution of hr, substitution of Arg with Gln, His or Lys, substitution of Asn with Glu, Gln, Lys, His or Asp, substitution of Asp with Asn, Glu or Gln, substitution of Cys with Ser or Ala, substitution of Gln with Asn, Glu, Lys, His, Asp or Arg, substitution of Glu with Gly, Asn, Gln, Lys or Asp, substitution of Gly with Pro, substitution of His with Asn, Lys, Gln, Arg or Tyr, substitution of Ile with Leu, Met, Val or P Examples of amino acid substitutions include substitutions of Lys with Lys, substitutions of Leu with Ile, Met, Val, or Phe, substitutions of Lys with Asn, Glu, Gln, His, or Arg, substitutions of Met with Ile, Leu, Val, or Phe, substitutions of Phe with Trp, Tyr, Met, Ile, or Leu, substitutions of Ser with Thr or Ala, substitutions of Thr with Ser or Ala, substitutions of Trp with Phe or Tyr, substitutions of Tyr with His, Phe, or Trp, and substitutions of Val with Met, Ile, or Leu. Furthermore, the above-mentioned amino acid substitutions, deletions, insertions, or additions may include those resulting from naturally occurring mutations (mutants or variants) based on individual differences or differences in species of the organism from which the gene is derived.

[0044] Furthermore, a wild-type carbonic anhydrase gene may be a gene encoding a protein having an amino acid sequence that is, for example, 50% or more, 65% or more, 70% or more, 80% or more, 90% or more, 95% or more, 97% or more, or 99% or more identical to the entire amino acid sequence described above, as long as the encoded carbonic anhydrase does not have a "specific mutation."

[0045] Furthermore, the wild-type carbonic anhydrase gene may be a gene, e.g., DNA, that hybridizes under stringent conditions with a probe that can be prepared from the above-mentioned nucleotide sequence (e.g., the nucleotide sequence shown in SEQ ID NO: 3), e.g., a sequence complementary to all or part of the above-mentioned nucleotide sequence, as long as the encoded carbonic anhydrase does not have a "specific mutation." "Stringent conditions" may refer to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. One example of such conditions includes conditions under which DNAs with high identity, for example, DNAs with identity of 50% or more, 65% or more, 70% or more, 80% or more, 90% or more, 95% or more, 97% or more, or 99% or more, hybridize with each other, but DNAs with lower identity do not hybridize with each other, or conditions for washing once, preferably two to three times, at a salt concentration and temperature equivalent to those used in standard Southern hybridization, such as 60°C, 1×SSC, 0.1% SDS, preferably 60°C, 0.1×SSC, 0.1% SDS, and more preferably 68°C, 0.1×SSC, 0.1% SDS.

[0046] As mentioned above, the probe used in the hybridization may be a portion of the complementary sequence of the gene. Such a probe can be prepared by PCR using oligonucleotides prepared based on a known gene sequence as primers and a DNA fragment containing the gene as a template. For example, a DNA fragment of about 300 bp in length can be used as the probe. When a DNA fragment of about 300 bp in length is used as the probe, washing conditions for the hybridization include 50°C, 2×SSC, and 0.1% SDS.

[0047] Furthermore, since codon degeneracy differs depending on the host, the wild-type carbonic anhydrase gene may be one in which any codon has been replaced with an equivalent codon. That is, the wild-type carbonic anhydrase gene may be a variant of the wild-type carbonic anhydrase gene exemplified above due to the degeneracy of the genetic code. For example, the wild-type carbonic anhydrase gene may be modified to have optimal codons depending on the codon usage frequency of the host used.

[0048] The "identity" between amino acid sequences is calculated using the default Scoring Parameters (Matrix: BLOSUM62; Gap Cost s:Existence=11, Extension=1;Compositional "Identity" refers to the identity between amino acid sequences calculated using the "Conditional Compositional Score Matrix Adjustments" ("Conditional Compositional Score Matrix Adjustment"). Furthermore, "identity" between nucleotide sequences refers to the identity between nucleotide sequences calculated using the default Scoring Parameters (Match / Mismatch Scores = 1, -2; Gap Costs = Linear) by blastn.

[0049] The mutant carbonic anhydrase will be explained below.

[0050] The mutant carbonic anhydrase has at least one of decarboxylation activity and CO hydration activity. Preferably, the mutant carbonic anhydrase may have both decarboxylation activity and CO hydration activity.

[0051] The mutant carbonic anhydrase has a "specific mutation" in the wild-type carbonic anhydrase.

[0052] That is, the mutant carbonic anhydrase may be, for example, a protein having an amino acid sequence with a "specific mutation" in the amino acid sequence shown in SEQ ID NO: 2. Alternatively, the mutant carbonic anhydrase may be, for example, a protein having an amino acid sequence with a "specific mutation" in the amino acid sequence shown in SEQ ID NO: 2 and further including any one or more of substitution, deletion, insertion, and addition of one or more amino acid residues at one or more positions, and having at least one or more activities of decarboxylation activity and CO2 hydration activity, and preferably a protein having both decarboxylation activity and CO2 hydration activity.

[0053] In other words, the mutant carbonic anhydrase may be a protein having the same amino acid sequence as a wild-type carbonic anhydrase except for the "specific mutation." That is, the mutant carbonic anhydrase may be, for example, a protein having the amino acid sequence set forth in SEQ ID NO: 2 except for the "specific mutation." The mutant carbonic anhydrase may be, for example, a protein having an amino acid sequence that includes one or more substitutions, deletions, insertions, and additions of one or more amino acid residues at one or more positions in the amino acid sequence set forth in SEQ ID NO: 2 except for the "specific mutation," and having at least one or more activities of decarboxylation activity and CO2 hydration activity, and preferably a protein having both decarboxylation activity and CO2 hydration activity. Furthermore, the mutant carbonic anhydrase may be, for example, a protein having an amino acid sequence that is 70% or more, preferably 80% or more, more preferably 90% or more, more preferably 95% or more, more preferably 97% or more, and particularly preferably 99% or more identical to the amino acid sequence shown in SEQ ID NO: 2, except for the "specific mutation," and having at least one of decarboxylation activity and CO2 hydration activity, and preferably a protein having both decarboxylation activity and CO2 hydration activity.

[0054] In detail, the mutant carbonic anhydrase may be a protein selected from any one of (i) to (iii) above, a protein selected from any one of (iv) to (vi) above, a protein selected from any one of (vii) to (xv) above, a protein selected from any one of (xvi) to (xviii) above, or a protein selected from any one of (xix) to (xxi) above.

[0055] An example of a mutant carbonic anhydrase in which the amino acid residue corresponding to the lysine residue at position 33 in SEQ ID NO: 1 (position 14 in SEQ ID NO: 2) is substituted with a glutamic acid residue is shown in SEQ ID NO: 5. Also, the amino acid residue corresponding to the lysine residue at position 165 in SEQ ID NO: 1 (position 146 in SEQ ID NO: 2) is shown in SEQ ID NO: 5. An example of a mutant carbonic anhydrase in which the amino acid residue at position 182 of SEQ ID NO: 1 (position 163 in SEQ ID NO: 2) is substituted with a glutamic acid residue is shown in SEQ ID NO: 7. Furthermore, an example of a mutant carbonic anhydrase in which the amino acid residue corresponding to the methionine residue at position 182 of SEQ ID NO: 1 (position 163 in SEQ ID NO: 2) is substituted with a leucine residue is shown in SEQ ID NO: 9. Furthermore, an example of a mutant carbonic anhydrase in which the amino acid residue corresponding to the lysine residue at position 33 of SEQ ID NO: 1 (position 14 in SEQ ID NO: 2) is substituted with a glutamic acid residue and the amino acid residue corresponding to the lysine residue at position 165 (position 146 in SEQ ID NO: 2) is substituted with a glutamic acid residue is shown in SEQ ID NO: 11. Furthermore, an example of a mutant carbonic anhydrase in which the amino acid residue corresponding to the lysine residue at position 33 of SEQ ID NO: 1 (position 14 in SEQ ID NO: 2) is substituted with a glutamic acid residue and the amino acid residue corresponding to the methionine residue at position 182 (position 163 in SEQ ID NO: 2) is substituted with a leucine residue is shown in SEQ ID NO: 13. An example of a mutant carbonic anhydrase in which the amino acid residue corresponding to the lysine residue at position 165 in SEQ ID NO: 1 (position 146 in SEQ ID NO: 2) has been substituted with a glutamic acid residue and the amino acid residue corresponding to the methionine residue at position 182 (position 163 in SEQ ID NO: 2) has been substituted with a leucine residue is shown in SEQ ID NO: 15. An example of a mutant carbonic anhydrase in which the amino acid residue corresponding to the lysine residue at position 33 in SEQ ID NO: 1 (position 14 in SEQ ID NO: 2) has been substituted with a glutamic acid residue, the amino acid residue corresponding to the lysine residue at position 165 (position 146 in SEQ ID NO: 2) has been substituted with a glutamic acid residue, and the amino acid residue corresponding to the methionine residue at position 182 (position 163 in SEQ ID NO: 2) has been substituted with a leucine residue is shown in SEQ ID NO: 17. In addition, an example of a mutant carbonic anhydrase in which the amino acid residue corresponding to the lysine residue at position 33 in SEQ ID NO: 1 (position 14 in SEQ ID NO: 2) has been replaced with a glutamic acid residue, and the amino acid residue corresponding to the isoleucine residue at position 66 in SEQ ID NO: 1 (position 47 in SEQ ID NO: 2, position 47 in SEQ ID NO: 5) has been replaced with a valine residue is shown in SEQ ID NO: 19.SEQ ID NO: 21 shows an example of a mutant carbonic anhydrase in which the amino acid residue corresponding to the lysine residue at position 33 of SEQ ID NO: 1 (position 14 of SEQ ID NO: 2) has been substituted with a glutamic acid residue, and the amino acid residue corresponding to the lysine residue at position 229 of SEQ ID NO: 1 (position 210 of SEQ ID NO: 2, position 210 of SEQ ID NO: 5) has been substituted with a glutamic acid residue. SEQ ID NO: 23 shows an example of a mutant carbonic anhydrase in which the amino acid residue corresponding to the lysine residue at position 33 of SEQ ID NO: 1 (position 14 of SEQ ID NO: 2) has been substituted with a glutamic acid residue, and the amino acid residue corresponding to the methionine residue at position 231 of SEQ ID NO: 1 (position 212 of SEQ ID NO: 2, position 212 of SEQ ID NO: 5) has been substituted with an isoleucine residue. SEQ ID NO: 25 shows an example of a mutant carbonic anhydrase in which the amino acid residue corresponding to the lysine residue at position 33 of SEQ ID NO: 1 (position 14 of SEQ ID NO: 2) is substituted with a glutamic acid residue, the amino acid residue corresponding to the isoleucine residue at position 66 of SEQ ID NO: 1 (position 47 of SEQ ID NO: 2, position 47 of SEQ ID NO: 5) is substituted with a valine residue, the amino acid residue corresponding to the lysine residue at position 229 of SEQ ID NO: 1 (position 210 of SEQ ID NO: 2, position 210 of SEQ ID NO: 5) is substituted with a glutamic acid residue, and the amino acid residue corresponding to the methionine residue at position 231 of SEQ ID NO: 1 (position 212 of SEQ ID NO: 2, position 212 of SEQ ID NO: 5) is substituted with an isoleucine residue.

[0056] SEQ ID NO: 55 shows an example of a mutant carbonic anhydrase in which the amino acid residue corresponding to the lysine residue at position 33 of SEQ ID NO: 1 (position 14 of SEQ ID NO: 2) is substituted with a glutamic acid residue, the amino acid residue corresponding to the isoleucine residue at position 66 of SEQ ID NO: 1 (position 47 of SEQ ID NO: 2, position 47 of SEQ ID NO: 5) is substituted with a valine residue, the amino acid residue corresponding to the lysine residue at position 229 of SEQ ID NO: 1 (position 210 of SEQ ID NO: 2, position 210 of SEQ ID NO: 5) is substituted with a glutamic acid residue, the amino acid residue corresponding to the methionine residue at position 231 of SEQ ID NO: 1 (position 212 of SEQ ID NO: 2, position 212 of SEQ ID NO: 5) is substituted with an isoleucine residue, and the amino acid residue corresponding to the glycine residue at position 23 of SEQ ID NO: 1 (position 4 of SEQ ID NO: 2) is substituted with a leucine residue. In addition, the amino acid residue corresponding to the lysine residue at position 33 of SEQ ID NO: 1 (position 14 of SEQ ID NO: 2) is substituted with a glutamic acid residue, the amino acid residue corresponding to the isoleucine residue at position 66 of SEQ ID NO: 1 (position 47 of SEQ ID NO: 2, position 47 of SEQ ID NO: 5) is substituted with a valine residue, and the amino acid residue corresponding to the isoleucine residue at position 229 of SEQ ID NO: 1 (position 24 of SEQ ID NO: 2, position 25 of SEQ ID NO: 5) is substituted with a valine residue. SEQ ID NO: 57 shows an example of a mutant carbonic anhydrase in which the amino acid residue corresponding to the lysine residue at position 210 in SEQ ID NO: 2 and position 210 in SEQ ID NO: 5 has been replaced with a glutamic acid residue, the amino acid residue corresponding to the methionine residue at position 231 in SEQ ID NO: 1 (position 212 in SEQ ID NO: 2 and position 212 in SEQ ID NO: 5) has been replaced with an isoleucine residue, and the amino acid residue corresponding to the glycine residue at position 23 in SEQ ID NO: 1 (position 4 in SEQ ID NO: 2) has been replaced with an isoleucine residue.

[0057] SEQ ID NO: 27 shows an example of a mutant carbonic anhydrase in which the amino acid residue corresponding to the lysine residue at position 33 of SEQ ID NO: 1 (position 14 in SEQ ID NO: 2) is substituted with a glutamic acid residue, the amino acid residue corresponding to the isoleucine residue at position 66 of SEQ ID NO: 1 (position 47 in SEQ ID NO: 2) is substituted with a valine residue, the amino acid residue corresponding to the lysine residue at position 229 of SEQ ID NO: 1 (position 210 in SEQ ID NO: 2) is substituted with a glutamic acid residue, the amino acid residue corresponding to the methionine residue at position 231 of SEQ ID NO: 1 (position 212 in SEQ ID NO: 2) is substituted with an isoleucine residue, and the amino acid residue corresponding to the lysine residue at position 173 of SEQ ID NO: 1 (position 154 in SEQ ID NO: 2) is substituted with an arginine residue. SEQ ID NO: 29 shows an example of a mutant carbonic anhydrase in which the amino acid residue corresponding to the lysine residue at position 33 of SEQ ID NO: 1 (position 14 in SEQ ID NO: 2) is substituted with a glutamic acid residue, the amino acid residue corresponding to the isoleucine residue at position 66 of SEQ ID NO: 1 (position 47 in SEQ ID NO: 2) is substituted with a valine residue, the amino acid residue corresponding to the lysine residue at position 229 of SEQ ID NO: 1 (position 210 in SEQ ID NO: 2) is substituted with a glutamic acid residue, the amino acid residue corresponding to the methionine residue at position 231 of SEQ ID NO: 1 (position 212 in SEQ ID NO: 2) is substituted with an isoleucine residue, and the amino acid residue corresponding to the lysine residue at position 214 of SEQ ID NO: 1 (position 195 in SEQ ID NO: 2) is substituted with an arginine residue. SEQ ID NO: 31 shows an example of a mutant carbonic anhydrase in which the amino acid residue corresponding to the lysine residue at position 33 of SEQ ID NO: 1 (position 14 of SEQ ID NO: 2) is substituted with a glutamic acid residue, the amino acid residue corresponding to the isoleucine residue at position 66 of SEQ ID NO: 1 (position 47 of SEQ ID NO: 2) is substituted with a valine residue, the amino acid residue corresponding to the lysine residue at position 229 of SEQ ID NO: 1 (position 210 of SEQ ID NO: 2) is substituted with a glutamic acid residue, the amino acid residue corresponding to the methionine residue at position 231 of SEQ ID NO: 1 (position 212 of SEQ ID NO: 2) is substituted with an isoleucine residue, and the amino acid residue corresponding to the lysine residue at position 246 of SEQ ID NO: 1 (position 227 of SEQ ID NO: 2) is substituted with an arginine residue.SEQ ID NO: 33 shows an example of a mutant carbonic anhydrase in which the amino acid residue corresponding to the lysine residue at position 33 of SEQ ID NO: 1 (position 14 in SEQ ID NO: 2) is substituted with a glutamic acid residue, the amino acid residue corresponding to the isoleucine residue at position 66 of SEQ ID NO: 1 (position 47 in SEQ ID NO: 2) is substituted with a valine residue, the amino acid residue corresponding to the lysine residue at position 229 of SEQ ID NO: 1 (position 210 in SEQ ID NO: 2) is substituted with a glutamic acid residue, the amino acid residue corresponding to the methionine residue at position 231 of SEQ ID NO: 1 (position 212 in SEQ ID NO: 2) is substituted with an isoleucine residue, the amino acid residue corresponding to the lysine residue at position 173 of SEQ ID NO: 1 (position 154 in SEQ ID NO: 2) is substituted with an arginine residue, the amino acid residue corresponding to the lysine residue at position 214 of SEQ ID NO: 1 (position 195 in SEQ ID NO: 2) is substituted with an arginine residue, and the amino acid residue corresponding to the lysine residue at position 246 of SEQ ID NO: 1 (position 227 in SEQ ID NO: 2) is substituted with an arginine residue.

[0058] In addition, the amino acid residue corresponding to the lysine residue at position 33 of SEQ ID NO: 1 (position 14 in SEQ ID NO: 2) is substituted with a glutamic acid residue, the amino acid residue corresponding to the isoleucine residue at position 66 of SEQ ID NO: 1 (position 47 in SEQ ID NO: 2) is substituted with a valine residue, the amino acid residue corresponding to the lysine residue at position 229 of SEQ ID NO: 1 (position 210 in SEQ ID NO: 2) is substituted with a glutamic acid residue, the amino acid residue corresponding to the methionine residue at position 231 of SEQ ID NO: 1 (position 212 in SEQ ID NO: 2) is substituted with an isoleucine residue, the amino acid residue corresponding to the lysine residue at position 173 of SEQ ID NO: 1 (position 154 in SEQ ID NO: 2) is substituted with an arginine residue, and the amino acid residue corresponding to the lysine residue at position 214 of SEQ ID NO: 1 (position 195 in SEQ ID NO: 2) is substituted with a glutamic acid residue. SEQ ID NO:59 shows an example of a mutant carbonic anhydrase in which the amino acid residue at position 33 of SEQ ID NO:1 (position 14 of SEQ ID NO:2) is substituted with a glutamic acid residue, the amino acid residue at position 66 of SEQ ID NO:1 (position 47 of SEQ ID NO:2) is substituted with a valine residue, the amino acid residue at position 229 of SEQ ID NO:1 (position 210 of SEQ ID NO:2) is substituted with a glutamic acid residue, the amino acid residue at position 231 of SEQ ID NO:1 (position 212 of SEQ ID NO:2) is substituted with an isoleucine residue, and the amino acid residue at position 17 of SEQ ID NO:1 is substituted with an arginine residue, the amino acid residue at position 246 of SEQ ID NO:1 (position 227 of SEQ ID NO:2) is substituted with an arginine residue, and the amino acid residue at position 23 of SEQ ID NO:1 (position 4 of SEQ ID NO:2) is substituted with a serine residue. SEQ ID NO: 61 shows an example of a mutant carbonic anhydrase in which the amino acid residue corresponding to the third lysine residue (the 154th in SEQ ID NO: 2) is substituted with an arginine residue, the amino acid residue corresponding to the 214th lysine residue in SEQ ID NO: 1 (the 195th in SEQ ID NO: 2) is substituted with an arginine residue, the amino acid residue corresponding to the 246th lysine residue in SEQ ID NO: 1 (the 227th in SEQ ID NO: 2) is substituted with an arginine residue, and the amino acid residue corresponding to the 23rd glycine residue in SEQ ID NO: 1 (the 4th in SEQ ID NO: 2) is substituted with an asparagine residue.

[0059] In addition, the amino acid residue corresponding to the lysine residue at position 33 of SEQ ID NO: 1 (position 14 in SEQ ID NO: 2) is substituted with a glutamic acid residue, the amino acid residue corresponding to the isoleucine residue at position 66 of SEQ ID NO: 1 (position 47 in SEQ ID NO: 2) is substituted with a valine residue, the amino acid residue corresponding to the lysine residue at position 229 of SEQ ID NO: 1 (position 210 in SEQ ID NO: 2) is substituted with a glutamic acid residue, the amino acid residue corresponding to the methionine residue at position 231 of SEQ ID NO: 1 (position 212 in SEQ ID NO: 2) is substituted with an isoleucine residue, and the amino acid residue corresponding to the methionine residue at position 231 of SEQ ID NO: 1 (position 212 in SEQ ID NO: 2) is substituted with an isoleucine residue. SEQ ID NO: 35 shows an example of a mutant carbonic anhydrase in which the amino acid residue corresponding to the lysine residue at position 73 (position 154 in SEQ ID NO: 2) is substituted with an arginine residue, the amino acid residue corresponding to the lysine residue at position 214 in SEQ ID NO: 1 (position 195 in SEQ ID NO: 2) is substituted with an arginine residue, the amino acid residue corresponding to the lysine residue at position 246 in SEQ ID NO: 1 (position 227 in SEQ ID NO: 2) is substituted with an arginine residue, and the amino acid residue corresponding to the valine residue at position 26 in SEQ ID NO: 1 (position 7 in SEQ ID NO: 2) is substituted with an alanine residue. In addition, the amino acid residue corresponding to the lysine residue at position 33 of SEQ ID NO: 1 (position 14 in SEQ ID NO: 2) is substituted with a glutamic acid residue, the amino acid residue corresponding to the isoleucine residue at position 66 of SEQ ID NO: 1 (position 47 in SEQ ID NO: 2) is substituted with a valine residue, the amino acid residue corresponding to the lysine residue at position 229 of SEQ ID NO: 1 (position 210 in SEQ ID NO: 2) is substituted with a glutamic acid residue, the amino acid residue corresponding to the methionine residue at position 231 of SEQ ID NO: 1 (position 212 in SEQ ID NO: 2) is substituted with an isoleucine residue, and the amino acid residue corresponding to the methionine residue at position 231 of SEQ ID NO: 1 (position 212 in SEQ ID NO: 2) is substituted with an isoleucine residue. SEQ ID NO: 37 shows an example of a mutant carbonic anhydrase in which the amino acid residue corresponding to the lysine residue at position 73 (position 154 in SEQ ID NO: 2) is substituted with an arginine residue, the amino acid residue corresponding to the lysine residue at position 214 in SEQ ID NO: 1 (position 195 in SEQ ID NO: 2) is substituted with an arginine residue, the amino acid residue corresponding to the lysine residue at position 246 in SEQ ID NO: 1 (position 227 in SEQ ID NO: 2) is substituted with an arginine residue, and the amino acid residue corresponding to the alanine residue at position 43 in SEQ ID NO: 1 (position 24 in SEQ ID NO: 2) is substituted with a valine residue.In addition, the amino acid residue corresponding to the lysine residue at position 33 of SEQ ID NO: 1 (position 14 in SEQ ID NO: 2) is substituted with a glutamic acid residue, the amino acid residue corresponding to the isoleucine residue at position 66 of SEQ ID NO: 1 (position 47 in SEQ ID NO: 2) is substituted with a valine residue, the amino acid residue corresponding to the lysine residue at position 229 of SEQ ID NO: 1 (position 210 in SEQ ID NO: 2) is substituted with a glutamic acid residue, the amino acid residue corresponding to the methionine residue at position 231 of SEQ ID NO: 1 (position 212 in SEQ ID NO: 2) is substituted with an isoleucine residue, the amino acid residue corresponding to the lysine residue at position 173 of SEQ ID NO: 1 (position 154 in SEQ ID NO: 2) is substituted with an arginine residue, the amino acid residue corresponding to the lysine residue at position 214 of SEQ ID NO: 1 (position 195 in SEQ ID NO: 2) is substituted with an arginine residue, and the amino acid residue corresponding to the lysine residue at position 246 of SEQ ID NO: 1 (position 227 in SEQ ID NO: 2) is substituted with an arginine residue. SEQ ID NO: 39 shows an example of a mutant carbonic anhydrase in which the amino acid residue corresponding to the lysine residue at position 226 of SEQ ID NO: 1 (position 207 in SEQ ID NO: 2) is substituted with an arginine residue, and the amino acid residue corresponding to the lysine residue at position 33 of SEQ ID NO: 1 (position 14 in SEQ ID NO: 2) is substituted with a glutamic acid residue, the amino acid residue corresponding to the isoleucine residue at position 66 of SEQ ID NO: 1 (position 47 in SEQ ID NO: 2) is substituted with a valine residue, the amino acid residue corresponding to the lysine residue at position 229 of SEQ ID NO: 1 (position 210 in SEQ ID NO: 2) is substituted with a glutamic acid residue, the amino acid residue corresponding to the methionine residue at position 231 of SEQ ID NO: 1 (position 212 in SEQ ID NO: 2) is substituted with an isoleucine residue, and the amino acid residue corresponding to the lysine residue at position 173 of SEQ ID NO: 1 (position 154 in SEQ ID NO: 2) is substituted with an arginine residue. SEQ ID NO: 41 shows an example of a mutant carbonic anhydrase in which the amino acid residues have been replaced with arginine residues, the amino acid residue corresponding to the lysine residue at position 214 of SEQ ID NO: 1 (position 195 in SEQ ID NO: 2) has been replaced with arginine residue, the amino acid residue corresponding to the lysine residue at position 246 of SEQ ID NO: 1 (position 227 in SEQ ID NO: 2) has been replaced with arginine residue, the amino acid residue corresponding to the glutamine residue at position 69 of SEQ ID NO: 1 (position 50 in SEQ ID NO: 2) has been replaced with arginine residue, and the amino acid residue corresponding to the valine residue at position 103 of SEQ ID NO: 1 (position 84 in SEQ ID NO: 2) has been replaced with isoleucine residue.In addition, the amino acid residue corresponding to the lysine residue at position 33 of SEQ ID NO: 1 (position 14 in SEQ ID NO: 2) is substituted with a glutamic acid residue, the amino acid residue corresponding to the isoleucine residue at position 66 of SEQ ID NO: 1 (position 47 in SEQ ID NO: 2) is substituted with a valine residue, the amino acid residue corresponding to the lysine residue at position 229 of SEQ ID NO: 1 (position 210 in SEQ ID NO: 2) is substituted with a glutamic acid residue, the amino acid residue corresponding to the methionine residue at position 231 of SEQ ID NO: 1 (position 212 in SEQ ID NO: 2) is substituted with an isoleucine residue, the amino acid residue corresponding to the lysine residue at position 173 of SEQ ID NO: 1 (position 154 in SEQ ID NO: 2) is substituted with an arginine residue, and the amino acid residue corresponding to the lysine residue at position 214 of SEQ ID NO: 1 (position 195 in SEQ ID NO: 2) is substituted with an arginine residue. An example of a mutant carbonic anhydrase in which the amino acid residue corresponding to the lysine residue at position 246 (position 227 in SEQ ID NO:2) of SEQ ID NO:1 is substituted with an arginine residue, the amino acid residue corresponding to the valine residue at position 26 (position 7 in SEQ ID NO:2) of SEQ ID NO:1 is substituted with an alanine residue, the amino acid residue corresponding to the alanine residue at position 43 of SEQ ID NO:1 (position 24 in SEQ ID NO:2) is substituted with a valine residue, the amino acid residue corresponding to the glutamine residue at position 69 of SEQ ID NO:1 (position 50 in SEQ ID NO:2) is substituted with an arginine residue, the amino acid residue corresponding to the valine residue at position 103 of SEQ ID NO:1 (position 84 in SEQ ID NO:2) is substituted with an isoleucine residue, and the amino acid residue corresponding to the lysine residue at position 226 of SEQ ID NO:1 (position 207 in SEQ ID NO:2) is substituted with an arginine residue is shown in SEQ ID NO:43. An example of a mutant carbonic anhydrase in which the amino acid residue corresponding to the serine residue at position 63 of SEQ ID NO:1 (position 44 in SEQ ID NO:2) is substituted with a threonine residue is shown in SEQ ID NO:45. An example of a mutant carbonic anhydrase in which the amino acid residue corresponding to the glutamic acid residue at position 69 in SEQ ID NO: 1 (position 50 in SEQ ID NO: 2) has been substituted with an aspartic acid residue is shown in SEQ ID NO: 47. An example of a mutant carbonic anhydrase in which the amino acid residue corresponding to the glutamic acid residue at position 169 in SEQ ID NO: 1 (position 150 in SEQ ID NO: 2) has been substituted with an aspartic acid residue is shown in SEQ ID NO: 49.An example of a mutant carbonic anhydrase in which the amino acid residue corresponding to the histidine residue at position 170 of SEQ ID NO: 1 (position 151 in SEQ ID NO: 2) has been substituted with a glutamic acid residue is shown in SEQ ID NO: 51. An example of a mutant carbonic anhydrase in which the amino acid residue corresponding to the serine residue at position 63 of SEQ ID NO: 1 (position 44 in SEQ ID NO: 2) has been substituted with a threonine acid residue, the amino acid residue corresponding to the glutamic acid residue at position 69 of SEQ ID NO: 1 (position 50 in SEQ ID NO: 2) has been substituted with an aspartic acid residue, the amino acid residue corresponding to the glutamic acid residue at position 169 of SEQ ID NO: 1 (position 150 in SEQ ID NO: 2) has been substituted with an aspartic acid residue, and the amino acid residue corresponding to the histidine residue at position 170 of SEQ ID NO: 1 (position 151 in SEQ ID NO: 2) has been substituted with a glutamic acid residue is shown in SEQ ID NO: 53.

[0060] SEQ ID NO: 63 shows an example of a mutant carbonic anhydrase in which the amino acid residue corresponding to the glycine residue at position 21 in SEQ ID NO: 1 (position 2 in SEQ ID NO: 2) is substituted with a proline residue. SEQ ID NO: 67 shows an example of a mutant carbonic anhydrase in which the amino acid residue corresponding to the glycine residue at position 22 in SEQ ID NO: 1 (position 3 in SEQ ID NO: 2) is substituted with a serine residue. SEQ ID NO: 69 shows an example of a mutant carbonic anhydrase in which the amino acid residue corresponding to the glycine residue at position 23 in SEQ ID NO: 1 (position 4 in SEQ ID NO: 2) is substituted with an asparagine residue. SEQ ID NO: 71 shows an example of a mutant carbonic anhydrase in which the amino acid residue corresponding to the glycine residue at position 23 in SEQ ID NO: 1 (position 4 in SEQ ID NO: 2) is substituted with a serine residue. SEQ ID NO: 75 shows an example of a mutant carbonic anhydrase in which the amino acid residue corresponding to the histidine residue at position 24 in SEQ ID NO: 1 (position 5 in SEQ ID NO: 2) is substituted with a phenylalanine residue. Furthermore, SEQ ID NO: 79 shows an example of a mutant carbonic anhydrase in which the amino acid residue corresponding to the glycine residue at position 23 of SEQ ID NO: 1 (position 4 of SEQ ID NO: 2) is substituted with an alanine residue. SEQ ID NO: 80 shows an example of a mutant carbonic anhydrase in which the amino acid residue corresponding to the glycine residue at position 23 of SEQ ID NO: 1 (position 4 of SEQ ID NO: 2) is substituted with a cysteine residue. SEQ ID NO: 81 shows an example of a mutant carbonic anhydrase in which the amino acid residue corresponding to the glycine residue at position 23 of SEQ ID NO: 1 (position 4 of SEQ ID NO: 2) is substituted with an aspartic acid residue. SEQ ID NO: 82 shows an example of a mutant carbonic anhydrase in which the amino acid residue corresponding to the glycine residue at position 23 of SEQ ID NO: 1 (position 4 of SEQ ID NO: 2) is substituted with a glutamic acid residue. SEQ ID NO: 83 shows an example of a mutant carbonic anhydrase in which the amino acid residue corresponding to the glycine residue at position 23 of SEQ ID NO: 1 (position 4 of SEQ ID NO: 2) is substituted with a phenylalanine residue. An example of a mutant carbonic anhydrase in which the amino acid residue corresponding to the glycine residue at position 23 in SEQ ID NO: 1 (position 4 in SEQ ID NO: 2) has been substituted with a histidine residue is shown in SEQ ID NO: 84. An example of a mutant carbonic anhydrase in which the amino acid residue corresponding to the glycine residue at position 23 in SEQ ID NO: 1 (position 4 in SEQ ID NO: 2) has been substituted with an isoleucine residue is shown in SEQ ID NO: 85.SEQ ID NO: 86 shows an example of a mutant carbonic anhydrase in which the amino acid residue corresponding to the glycine residue at position 23 in SEQ ID NO: 1 (position 4 in SEQ ID NO: 2) is substituted with a lysine residue. SEQ ID NO: 87 shows an example of a mutant carbonic anhydrase in which the amino acid residue corresponding to the glycine residue at position 23 in SEQ ID NO: 1 (position 4 in SEQ ID NO: 2) is substituted with a leucine residue. SEQ ID NO: 88 shows an example of a mutant carbonic anhydrase in which the amino acid residue corresponding to the glycine residue at position 23 in SEQ ID NO: 1 (position 4 in SEQ ID NO: 2) is substituted with a methionine residue. SEQ ID NO: 89 shows an example of a mutant carbonic anhydrase in which the amino acid residue corresponding to the glycine residue at position 23 in SEQ ID NO: 1 (position 4 in SEQ ID NO: 2) is substituted with a proline residue. SEQ ID NO: 90 shows an example of a mutant carbonic anhydrase in which the amino acid residue corresponding to the glycine residue at position 23 in SEQ ID NO: 1 (position 4 in SEQ ID NO: 2) is substituted with a glutamine residue. An example of a mutant carbonic anhydrase in which the amino acid residue corresponding to the glycine residue at position 23 in SEQ ID NO: 1 (position 4 in SEQ ID NO: 2) is substituted with an arginine residue is shown in SEQ ID NO: 91. An example of a mutant carbonic anhydrase in which the amino acid residue corresponding to the glycine residue at position 23 in SEQ ID NO: 1 (position 4 in SEQ ID NO: 2) is substituted with a valine residue is shown in SEQ ID NO: 92. An example of a mutant carbonic anhydrase in which the amino acid residue corresponding to the glycine residue at position 23 in SEQ ID NO: 1 (position 4 in SEQ ID NO: 2) is substituted with a tryptophan residue is shown in SEQ ID NO: 93. An example of a mutant carbonic anhydrase in which the amino acid residue corresponding to the glycine residue at position 23 in SEQ ID NO: 1 (position 4 in SEQ ID NO: 2) is substituted with a tyrosine residue is shown in SEQ ID NO: 94.

[0061] The mutant carbonic anhydrase is a protein comprising the amino acid sequence shown in SEQ ID NO: 5 or a variant thereof, a protein comprising the amino acid sequence shown in SEQ ID NO: 7 or a variant thereof, a protein comprising the amino acid sequence shown in SEQ ID NO: 9 or a variant thereof, a protein comprising the amino acid sequence shown in SEQ ID NO: 11 or a variant thereof, a protein comprising the amino acid sequence shown in SEQ ID NO: 13 or a variant thereof, a protein comprising the amino acid sequence shown in SEQ ID NO: 15 or a variant thereof, a protein comprising the amino acid sequence shown in SEQ ID NO: 17 or a variant thereof, a protein comprising the amino acid sequence shown in SEQ ID NO: 19 or a variant thereof, a protein comprising the amino acid sequence shown in SEQ ID NO: 21 or a variant thereof, ant, a protein comprising the amino acid sequence set forth in SEQ ID NO: 23 or a variant thereof, a protein comprising the amino acid sequence set forth in SEQ ID NO: 25 or a variant thereof, a protein comprising the amino acid sequence set forth in SEQ ID NO: 27 or a variant thereof, a protein comprising the amino acid sequence set forth in SEQ ID NO: 29 or a variant thereof, a protein comprising the amino acid sequence set forth in SEQ ID NO: 31 or a variant thereof, a protein comprising the amino acid sequence set forth in SEQ ID NO: 33 or a variant thereof, a protein comprising the amino acid sequence set forth in SEQ ID NO: 35 or a variant thereof, a protein comprising the amino acid sequence set forth in SEQ ID NO: 37 or a variant thereof, a protein comprising the amino acid sequence set forth in SEQ ID NO: 39 or a variant thereof, a protein comprising the amino acid sequence set forth in SEQ ID NO: 41 or a variant thereof, a protein comprising the amino acid sequence set forth in SEQ ID NO: 43 or a variant thereof, a protein comprising the amino acid sequence set forth in SEQ ID NO: 45 or a variant thereof, a protein comprising the amino acid sequence set forth in SEQ ID NO: 47 or a variant thereof, a protein comprising the amino acid sequence set forth in SEQ ID NO: 49 or a variant thereof, a protein comprising the amino acid sequence set forth in SEQ ID NO: 51 or a variant thereof, or a protein comprising the amino acid sequence set forth in SEQ ID NO: 53 or a variant thereof.

[0062] Furthermore, the mutant carbonic anhydrase is a protein comprising the amino acid sequence shown in SEQ ID NO: 55 or a variant thereof, a protein comprising the amino acid sequence shown in SEQ ID NO: 57 or a variant thereof, a protein comprising the amino acid sequence shown in SEQ ID NO: 59 or a variant thereof, a protein comprising the amino acid sequence shown in SEQ ID NO: 61 or a variant thereof, a protein comprising the amino acid sequence shown in SEQ ID NO: 63 or a variant thereof, a protein comprising the amino acid sequence shown in SEQ ID NO: 67 or a variant thereof, a protein comprising the amino acid sequence shown in SEQ ID NO: 69 or a variant thereof, a protein comprising the amino acid sequence shown in SEQ ID NO: 71 or a variant thereof, a protein comprising the amino acid sequence shown in SEQ ID NO: 75 or a variant thereof, a protein comprising the amino acid sequence shown in SEQ ID NO: 79 or a variant thereof, a protein comprising the amino acid sequence shown in SEQ ID NO: 80 or a variant thereof, a protein comprising the amino acid sequence shown in SEQ ID NO: 81 or a variant thereof, an amino acid sequence shown in SEQ ID NO: 82 or a variant thereof, The amino acid sequence may be a protein or variant thereof comprising the amino acid sequence set forth in SEQ ID NO: 83, a protein or variant thereof comprising the amino acid sequence set forth in SEQ ID NO: 84, a protein or variant thereof comprising the amino acid sequence set forth in SEQ ID NO: 85, a protein or variant thereof comprising the amino acid sequence set forth in SEQ ID NO: 86, a protein or variant thereof comprising the amino acid sequence set forth in SEQ ID NO: 87, a protein or variant thereof comprising the amino acid sequence set forth in SEQ ID NO: 88, a protein or variant thereof comprising the amino acid sequence set forth in SEQ ID NO: 89, a protein or variant thereof comprising the amino acid sequence set forth in SEQ ID NO: 90, a protein or variant thereof comprising the amino acid sequence set forth in SEQ ID NO: 91, a protein or variant thereof comprising the amino acid sequence set forth in SEQ ID NO: 92, a protein or variant thereof comprising the amino acid sequence set forth in SEQ ID NO: 93, or a protein or variant thereof comprising the amino acid sequence set forth in SEQ ID NO: 94.

[0063] Specifically, the mutant carbonic anhydrase may be a protein selected from any one of the following (xxii) to (xxiv): (xxii) a protein comprising any one of the amino acid sequences set forth in SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 67, 69, 71, 75, and 79 to 94; (xxiii) an amino acid sequence shown in any one of SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 67, 69, 71, 75, 79 to 94, wherein one or several amino acid residues at one or several positions are present; a protein having an amino acid sequence containing any one or more of substitutions, deletions, insertions, and additions of the above, with the proviso that specific mutations are maintained, and having at least one or more activities of decarboxylation activity and CO2 hydration activity; (xxiv) A protein having an amino acid sequence that is 70% or more identical to any one of the amino acid sequences set forth in SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 67, 69, 71, 75, and 79 to 94, with certain mutations maintained, and having at least one of decarboxylation activity and CO hydration activity.

[0064] The term "one or several" in (xxiii) varies depending on the position and type of amino acid residue in the three-dimensional structure of the protein, but specifically may be, for example, 1 to 50, 1 to 45, 1 to 40, 1 to 30, 1 to 25, 1 to 24, 1 to 23, 1 to 22, 1 to 20, 1 to 15, 1 to 10, 1 to 5, or 1 to 3.

[0065] Furthermore, the "identity" in (xxiv) may be, for example, an amino acid sequence that has 50% or more, 65% or more, 70% or more, 80% or more, 90% or more, 95% or more, 97% or more, or 99% or more identity to the entire amino acid sequence.

[0066] The protein (xxiii) and / or (xxiv) may preferably be a protein having both decarboxylation activity and CO 2 hydration activity.

[0067] A mutant carbonic anhydrase may contain another amino acid sequence in addition to the amino acid sequence of a mutant carbonic anhydrase as exemplified above. Such another amino acid sequence is also referred to as an "additional sequence." That is, a mutant carbonic anhydrase may be a fusion protein with an additional sequence. Furthermore, a mutant carbonic anhydrase may be expressed, for example, in a form containing an additional sequence, i.e., as a fusion protein with an additional sequence, and may ultimately lose part or all of the additional sequence. Unless otherwise specified, "the mutant carbonic anhydrase contains an additional sequence" or "the mutant carbonic anhydrase is a fusion protein with an additional sequence" means that the finally obtained mutant carbonic anhydrase contains the additional sequence. On the other hand, "the mutant carbonic anhydrase is expressed in a form containing an additional sequence" or "the mutant carbonic anhydrase contains the additional sequence upon expression" means that the mutant carbonic anhydrase contains the additional sequence at least upon expression, but does not necessarily mean that the finally obtained mutant carbonic anhydrase contains the additional sequence. In other words, the mutant carbonic anhydrase gene may contain a base sequence encoding an additional sequence in addition to the base sequence of the mutant carbonic anhydrase gene as exemplified above. The same applies to wild-type carbonic anhydrase and wild-type carbonic anhydrase gene. The additional sequence is not particularly limited as long as the mutant carbonic anhydrase has at least one of the activities of decarboxylation and CO hydration. The additional sequence can be appropriately selected depending on various conditions, such as the intended use. Examples of the additional sequence include a peptide tag, a signal peptide (also called a signal sequence), and a protease recognition sequence. The additional sequence may be linked, for example, to the N-terminus, the C-terminus, or both of the mutant carbonic anhydrase. The additional sequence may be a single amino acid sequence, or a combination of two or more amino acid sequences.

[0068] Specific examples of peptide tags include His tags, FLAG tags, GST tags, Myc tags, maltose binding protein (MBP), cellulose binding protein (CBP), thioredoxin (TRX), green fluorescent protein (GFP), horseradish peroxidase (HRP), alkaline phosphatase (ALP), and antibody Fc regions. Examples of His tags include 6xHis tags. The peptide tag can be used, for example, to detect and purify the expressed mutant carbonic anhydrase.

[0069] The signal peptide is not particularly limited as long as it functions in a host in which the mutant carbonic anhydrase is expressed. Examples of signal peptides include signal peptides recognized by the Sec secretory pathway and signal peptides recognized by the Tat secretory pathway. Signal peptides can be used, for example, for the secretory production of mutant carbonic anhydrase. When a signal peptide is used to secrete and produce a mutant carbonic anhydrase, the signal peptide is cleaved during secretion, and a mutant carbonic anhydrase without the signal peptide can be secreted outside the bacterial cell. That is, typically, the mutant carbonic anhydrase obtained finally does not need to have a signal peptide.

[0070] Specific examples of protease recognition sequences include the recognition sequence for Factor Xa protease and the recognition sequence for proTEV protease. Protease recognition sequences can be used, for example, to cleave an expressed mutant carbonic anhydrase. Specifically, when a mutant carbonic anhydrase is expressed as a fusion protein with a peptide tag, a protease recognition sequence can be introduced at the junction between the mutant carbonic anhydrase and the peptide tag, allowing the peptide tag to be cleaved from the expressed mutant carbonic anhydrase using a protease, thereby obtaining a mutant carbonic anhydrase without the peptide tag.

[0071] The mutant carbonic anhydrase gene is not particularly limited as long as it encodes the mutant carbonic anhydrase described above. As used herein, the term "gene" is not limited to DNA and may encompass any polynucleotide as long as it encodes a protein of interest. That is, a "mutant carbonic anhydrase gene" may refer to any polynucleotide encoding a mutant carbonic anhydrase. The mutant carbonic anhydrase gene may be DNA, RNA, or a combination thereof. The mutant carbonic anhydrase gene may be single-stranded or double-stranded. The mutant carbonic anhydrase gene may be single-stranded DNA or single-stranded RNA. The mutant carbonic anhydrase gene may be double-stranded DNA, double-stranded RNA, or a hybrid chain consisting of a DNA strand and an RNA strand. The mutant carbonic anhydrase gene may contain both DNA residues and RNA residues in a single polynucleotide chain. When the mutant carbonic anhydrase gene contains RNA, the descriptions regarding DNA, such as the nucleotide sequences exemplified above, may be interpreted appropriately to refer to RNA. The form of the mutant carbonic anhydrase gene can be appropriately selected depending on various conditions such as the mode of use.

[0072] The "specific mutation" will be explained below.

[0073] In the present disclosure, a "specific mutation" refers to one or more amino acid substitutions selected from the following (1) to (22): (1) The amino acid residue corresponding to the lysine residue at position 33 of SEQ ID NO: 1 is substituted with a glutamic acid residue (2) The amino acid residue corresponding to the lysine residue at position 165 of SEQ ID NO: 1 is substituted with a glutamic acid residue (3) The amino acid residue corresponding to the methionine residue at position 182 of SEQ ID NO: 1 is substituted with a leucine residue (4) The amino acid residue corresponding to the 66th isoleucine residue of SEQ ID NO: 1 is substituted with a valine residue (5) The amino acid residue corresponding to the lysine residue at position 229 of SEQ ID NO: 1 is substituted with a glutamic acid residue (6) The amino acid residue corresponding to the 231st methionine residue of SEQ ID NO: 1 is substituted with an isoleucine residue (7) The amino acid residue corresponding to the lysine residue at position 173 of SEQ ID NO: 1 is changed to an arginine residue. replacement (8) The amino acid residue corresponding to the lysine residue at position 214 of SEQ ID NO: 1 is substituted with an arginine residue (9) The amino acid residue corresponding to the lysine residue at position 246 of SEQ ID NO: 1 is substituted with an arginine residue (10) The amino acid residue corresponding to the 26th valine residue of SEQ ID NO: 1 is substituted with an alanine residue (11) The amino acid residue corresponding to the 43rd alanine residue of SEQ ID NO: 1 is substituted with a valine residue (12) The amino acid residue corresponding to the lysine residue at position 226 of SEQ ID NO: 1 is substituted with an arginine residue (13) The amino acid residue corresponding to the 69th glutamine residue of SEQ ID NO: 1 is substituted with an arginine residue (14) The amino acid residue corresponding to the 103rd valine residue of SEQ ID NO: 1 is substituted with an isoleucine residue (15) The amino acid residue corresponding to the serine residue at position 63 of SEQ ID NO: 1 is substituted with a threonine residue (16) The amino acid residue corresponding to the 69th glutamine residue of SEQ ID NO: 1 is substituted with an aspartic acid residue (17) The amino acid residue corresponding to the 169th glutamic acid residue of SEQ ID NO: 1 is substituted with an aspartic acid residue (18) The amino acid residue corresponding to the histidine residue at position 170 of SEQ ID NO: 1 is substituted with a glutamic acid residue (19) The amino acid residue corresponding to the 23rd glycine residue of SEQ ID NO: 1 is substituted with any amino acid residue other than a glycine residue or a threonine residue. (20) The amino acid residue corresponding to the 21st glycine residue of SEQ ID NO: 1 is substituted with a proline residue (21) The amino acid residue corresponding to the 22nd glycine residue of SEQ ID NO: 1 is substituted with a serine residue (22) The amino acid residue corresponding to the 24th histidine residue in SEQ ID NO: 1 is substituted with a glutamic acid residue.

[0074] As used herein, the term "Xth amino acid in SEQ ID NO: 1" refers to the amino acid located at position X, counting from the N-terminus of the amino acid sequence set forth in SEQ ID NO: 1. The term "amino acid residue corresponding to Xth amino acid in SEQ ID NO: 1" in a specific amino acid sequence refers to an amino acid residue in the specific amino acid sequence that is located at the same position as the Xth amino acid in the amino acid sequence set forth in SEQ ID NO: 1, when the specific amino acid sequence is aligned with the amino acid sequence set forth in SEQ ID NO: 1. For example, the term "amino acid residue corresponding to the 33rd lysine residue in SEQ ID NO: 1" in a specific amino acid sequence refers to an amino acid residue in the specific amino acid sequence that is located at the same position as the 33rd lysine in the amino acid sequence set forth in SEQ ID NO: 1, when the specific amino acid sequence is aligned with the amino acid sequence set forth in SEQ ID NO: 1. The term "amino acid residue corresponding to the Xth amino acid in SEQ ID NO: 1" in the amino acid sequence set forth in SEQ ID NO: 1 refers to the Xth amino acid itself in the amino acid sequence set forth in SEQ ID NO: 1. In other words, the positions of the amino acid substitutions exemplified above (i.e., the amino acid substitutions at the specific positions and, optionally, other amino acid substitutions) do not necessarily indicate absolute positions in the protein of the present invention, but rather indicate relative positions based on the amino acid sequence set forth in SEQ ID NO: 1. That is, for example, when the protein of the present invention contains an insertion, deletion, or addition of an amino acid residue on the N-terminal side of the position of the amino acid substitution exemplified above, the absolute position of the amino acid substitution may vary accordingly.

[0075] The positions of the above-exemplified amino acid substitutions in the protein of the present invention can be identified, for example, by aligning the amino acid sequence of the protein of the present invention with the amino acid sequence set forth in SEQ ID NO: 1. The alignment can be performed, for example, using an alignment program such as BLAST or FASTA. The same applies to the positions of the above-exemplified amino acid substitutions in any amino acid sequence, such as a variant sequence of the amino acid sequence set forth in SEQ ID NO: 1.

[0076] Furthermore, the amino acid residues before the amino acid substitutions in the mutant carbonic anhydrases exemplified in the present invention indicate the types of amino acid residues before the substitution in the amino acid sequence set forth in SEQ ID NO: 1, and may or may not be conserved in unmodified amino acid sequences other than the amino acid sequence set forth in SEQ ID NO: 1.

[0077] The mutant carbonic anhydrase may have "specific mutations" that improve its stability, preferably its thermostability and / or alkaline tolerance, compared to wild-type carbonic anhydrase. The mutant carbonic anhydrase may have one or more amino acid substitutions selected from (1) to (19) above, thereby improving its thermostability compared to wild-type carbonic anhydrase. Furthermore, the mutant carbonic anhydrase may have one or more amino acid substitutions selected from (7) to (14) above, thereby improving its alkaline tolerance compared to wild-type carbonic anhydrase.

[0078] The mutant carbonic anhydrase may have improved productivity compared to wild-type carbonic anhydrase by having a "specific mutation." For example, the mutant carbonic anhydrase may have one or more amino acid substitutions selected from (19) to (22) above, thereby improving its productivity compared to wild-type carbonic anhydrase. Furthermore, a mutant carbonic anhydrase having a "specific mutation" may have decarboxylation activity and / or CO hydration activity equivalent to that of a corresponding wild-type carbonic anhydrase that does not have the "specific mutation." Therefore, a mutant carbonic anhydrase having a "specific mutation" may have improved productivity while maintaining decarboxylation activity and / or CO hydration activity equivalent to that of the corresponding wild-type carbonic anhydrase. The improved productivity may be demonstrated, for example, by an improved purification yield per culture medium. Specifically, the mutant carbonic anhydrase may have a purification yield per culture medium that is 1.1 times or more, 1.5 times or more, 1.6 times or more, 2.0 times or more, 2.5 times or more, or 2.7 times or more improved compared to the corresponding wild-type carbonic anhydrase.

[0079] Improved thermostability may be demonstrated, for example, by improved residual activity after heat treatment. Specifically, the decarboxylation activity of the mutant carbonic anhydrase after heat treatment may be improved compared to the corresponding wild-type carbonic anhydrase. More specifically, improved thermostability may be demonstrated by the fact that the remaining decarboxylation activity of the mutant carbonic anhydrase after heating at 90 to 99°C for 10 to 90 minutes is improved compared to the corresponding wild-type carbonic anhydrase. Improved alkali resistance may be demonstrated, for example, by improved residual activity after alkali treatment. Specifically, the decarboxylation activity of the mutant carbonic anhydrase after alkali treatment may be improved compared to the corresponding wild-type carbonic anhydrase. More specifically, improved alkali resistance may be demonstrated by the fact that the remaining decarboxylation activity of the mutant carbonic anhydrase after treatment at pH 8 to 14 for 10 to 90 minutes is improved compared to the corresponding wild-type carbonic anhydrase. Furthermore, a mutant carbonic anhydrase having a "specific mutation" may have decarboxylation activity and / or CO hydration activity equivalent to that of a corresponding wild-type carbonic anhydrase that does not have the "specific mutation." Thus, a mutant carbonic anhydrase having a "specific mutation" may have improved stability, preferably improved thermostability and / or alkaline tolerance, while maintaining decarboxylation activity and / or CO hydration activity equivalent to that of the corresponding wild-type carbonic anhydrase.

[0080] The term "improved alkali resistance" may mean, for example, improved stability under high pH conditions such as in an alkaline aqueous solution. In this specification, "alkali resistance" may also be read as alkaline stability.

[0081] <2> Production of mutant carbonic anhydrase The mutant carbonic anhydrase can be produced, for example, by expressing the mutant carbonic anhydrase gene in a host having the gene.

[0082] The production of mutant carbonic anhydrase using a host carrying a mutant carbonic anhydrase gene will be described in detail below.

[0083] <2-1>Host A host having a mutant carbonic anhydrase gene can be obtained by introducing the mutant carbonic anhydrase gene into a suitable host. "Introducing a mutant carbonic anhydrase gene into a host" may also include modifying a carbonic anhydrase gene, such as a wild-type carbonic anhydrase gene, that the host has so that it encodes a mutant carbonic anhydrase. "Having a mutant carbonic anhydrase gene" is also referred to as "having a mutant carbonic anhydrase."

[0084] The host is not particularly limited as long as it can express a functional mutant carbonic anhydrase. Examples of the host include microorganisms, plant cells, insect cells, and animal cells. Examples of the host include, in particular, microorganisms. Examples of the microorganisms include bacteria and yeast. Examples of the microorganisms include, in particular, bacteria.

[0085] Examples of bacteria include bacteria belonging to the family Enterobacteriaceae, coryneform bacteria, and Bacillus bacteria.

[0086] Examples of bacteria belonging to the Enterobacteriaceae family include bacteria belonging to genera such as Escherichia, Enterobacter, Pantoea, Klebsiella, Serratia, Erwinia, Photorhabdus, Providencia, Salmonella, and Morganella. Specifically, bacteria classified as Enterobacteriaceae according to the classification used in the NCBI (National Center for Biotechnology Information) database (http: / / www.ncbi.nlm.nih.gov / Taxonomy / Browser / wwwtax.cgi?id=91347) can be used. Examples of bacteria belonging to the genus Escherichia include, but are not limited to, bacteria classified as Escherichia according to classifications known to microbiologists. Examples of Escherichia bacteria include those described in the book by Neidhardt et al. (Backmann, BJ 1996. Derivations and Genotypes of some mutant derivatives of Escherichia coli K-12, pp. 2460-2488. Table 1. In F.D. Neidhardt (ed.), Escherichia coli and Salmonella Cellular and Molecular Biology / Second Edition, American Society for Microbiology Press, Washington, DC). Examples of Escherichia bacteria include Escherichia coli, i.e., Escherichia coli. Examples of Escherichia coli include Escherichia coli B strains such as the BL21(DE3) strain; Escherichia coli K-12 strains such as the W3110 strain (ATCC 27325) and the MG1655 strain (ATCC 47076); Escherichia coli K5 strain (ATCC 23506); and their derivatives. Examples of Enterobacter bacteria include Enterobacter agglomerans and Enterobacter aerogenes. Examples of bacteria belonging to the genus Pantoea include Pantoea ananatis, Pantoea stewartii, Pantoea agglomerans, and Pantoea citrea. Examples of bacteria belonging to the genus Erwinia include Erwinia amylovora, Erwinia carotovora, and the like. Examples of bacteria of the genus Klebsiella include Klebsiella planticola.

[0087] Examples of coryneform bacteria include bacteria belonging to genera such as Corynebacterium, Brevibacterium, and Microbacterium.

[0088] The genus Corynebacterium includes bacteria that were previously classified as Brevibacterium but have now been integrated into the genus Corynebacterium (Int. J. Syst. Bacteriol., 41, 255 (1991)). Corynebacterium stationis also includes bacteria that were previously classified as Corynebacterium ammoniagenes but have been reclassified as Corynebacterium stationis based on 16S rRNA sequence analysis and other factors (Int. J. Syst. Evol. Microbiol., 60, 874-879 (2010)).

[0089] Examples of Bacillus bacteria include Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus pumilus, Bacillus licheniformis, Bacillus megaterium, Bacillus brevis, Bacillus polymixa, and Bacillus stearothermophilus. Specific examples of Bacillus subtilis include Bacillus subtilis 168 Marburg strain (ATCC 6051) and Bacillus subtilis PY79 strain (Plasmid, 1984, 12, 1-9). Specific examples of Bacillus amyloliquefaciens include Bacillus amyloliquefaciens T strain (ATCC 23842) and Bacillus amyloliquefaciens N strain (ATCC 23845).

[0090] Examples of yeast include yeasts belonging to the genus Saccharomyces, such as Saccharomyces cerevisiae, the genus Candida, such as Candida utilis, the genus Pichia, such as Pichia pastoris, the genus Hansenula, such as Hansenula polymorpha, and the genus Schizosaccharomyces, such as Schizosaccharomyces pombe.

[0091] These strains can be obtained, for example, from the American Type Culture Collection (12301 Parklawn Drive, Rockville, Maryland 20852, PO Box 1549, Manassas, VA 20108, United States of America). Each strain has a corresponding accession number, which can be used to obtain strains (see http: / / www.atcc.org / ). The accession numbers for each strain are listed in the catalog of the American Type Culture Collection. These strains can be obtained, for example, from the depository institutions where the strains have been deposited.

[0092] A mutant carbonic anhydrase gene can be obtained, for example, by modifying a wild-type carbonic anhydrase gene so that the encoded carbonic anhydrase has a "specific mutation." The wild-type carbonic anhydrase gene that is the source of the modification can be obtained, for example, by cloning from an organism having a wild-type carbonic anhydrase gene or by chemical synthesis. A mutant carbonic anhydrase gene can also be obtained without the intervention of a wild-type carbonic anhydrase gene. A mutant carbonic anhydrase gene may be obtained directly, for example, by chemical synthesis. The obtained mutant carbonic anhydrase gene may be used as is or after further modification. For example, a mutant carbonic anhydrase gene of one embodiment may be modified to obtain a mutant carbonic anhydrase gene of another embodiment.

[0093] Genetic modification can be carried out by known techniques. For example, site-directed mutagenesis can be used to introduce a desired mutation into a target site in DNA. That is, for example, site-directed mutagenesis can be used to modify the coding region of a gene so that the encoded protein contains substitution, deletion, insertion, and / or addition of amino acid residues at a specific site. Site-directed mutagenesis can be performed using PCR (Higuchi, R., 61, in PCR technology, Erlich, H.A. Eds., Stockton Press (1989); Carter, P., Meth. in Enzymol., 154, 382 (1987)) and methods using phage (Kramer, W. and Frits, HJ, Meth. in Enzymol., 154, 350 (1987); Kunkel, TA et al., Meth. in Enzymol., 154, 367 (1987)).

[0094] The method for introducing a mutant carbonic anhydrase gene into a host is not particularly limited, as long as the mutant carbonic anhydrase gene is retained in the host in an expressible manner. The mutant carbonic anhydrase gene can be introduced into a host in the same manner as the method described in detail below in the section "Method for introducing a gene."

[0095] Furthermore, when a host already has a carbonic anhydrase gene such as a wild-type carbonic anhydrase gene in a chromosome or the like, the host can be modified to have a mutant carbonic anhydrase gene by modifying the carbonic anhydrase gene so that it encodes a mutant carbonic anhydrase. Modification of a carbonic anhydrase gene present in a chromosome or the like can be carried out by, for example, natural mutation, mutation treatment, or genetic engineering.

[0096] The host may have any properties as long as it is capable of producing a mutant carbonic anhydrase.

[0097] <2-2> Methods for introducing genes The following describes the method for introducing a gene into a host.

[0098] Introduction of a gene into a host can be achieved by introducing the gene into the host chromosome. Introduction of a gene into a chromosome can be carried out, for example, by using homologous recombination (Miller, J.H. Experiments in Molecular Genetics, 1972, Cold Spring Harbor Laboratory). Examples of gene introduction methods that utilize homologous recombination include a method using linear DNA such as the Red-driven integration method (Datsenko, K. A., and Wanner, B.L. Proc. Natl. Acad. Sci. USA 97:6640-6645 (2000)), a method using a plasmid containing a temperature-sensitive replication origin, a method using a conjugatively transferable plasmid, a method using a suicide vector that does not have a replication origin that functions in the host, Examples of such methods include transduction using phage. A single copy of a gene may be introduced, or two or more copies may be introduced. For example, multiple copies of a gene can be introduced into a chromosome by performing homologous recombination targeting a sequence present in multiple copies on a chromosome. Examples of sequences present in multiple copies on a chromosome include repetitive DNA sequences and inverted repeats at both ends of transposons. Homologous recombination can also be performed targeting an appropriate sequence on a chromosome, such as a gene not required for carbonic anhydrase production. Genes can also be randomly introduced into a chromosome using transposons or Mini-Mu (Japanese Patent Application Laid-Open No. 2-109985, US Pat. No. 5,882,888, EP805867B1).

[0099] The introduction of the target gene into the chromosome can be confirmed by Southern hybridization using a probe having a sequence complementary to all or part of the gene, or by PCR using primers prepared based on the sequence of the gene.

[0100] Introduction of a gene into a host can also be achieved by introducing a vector containing the gene into the host. For example, a DNA fragment containing a target gene can be ligated to a vector that functions in the host to construct an expression vector for the gene, and the host can be transformed with the expression vector to introduce the gene into the host. A host transformed with an expression vector is also called a transformant. A DNA fragment containing a target gene can be obtained, for example, by PCR using the genomic DNA of a microorganism containing the target gene as a template. Vectors that can autonomously replicate within host cells can be used. The vector may be a multicopy vector. Furthermore, the vector may contain a marker such as an antibiotic resistance gene to select transformants. The vector may also contain a promoter or terminator for expressing the inserted gene. The vector may be, for example, a bacterial plasmid-derived vector, a yeast plasmid-derived vector, a bacteriophage-derived vector, a cosmid, or a phagemid. Specific examples of vectors capable of autonomous replication in Enterobacteriaceae bacteria such as Escherichia coli include pUC19, pUC18, pHSG299, pHSG398, pBR322, pSTV29, pCold series vectors (all available from Takara Bio Inc.), pACYC177, pACYC184, pMW219 (Nippon Gene), pTrc99A (Pharmacia), pET series vectors (Merck), and pQE series vectors (Qiagen).

[0101] When a gene is introduced, it is sufficient that the gene can be expressed by the host. Specifically, the gene is maintained so that it is expressed under the control of a promoter that functions in the host. A "promoter that functions in the host" may refer to a promoter that has promoter activity in the host. The promoter may be a promoter derived from the host or a heterologous promoter. The promoter may be a promoter native to the gene to be introduced or a promoter of another gene. Specific examples of promoters include the T7 promoter, trp promoter, lac promoter, thr promoter, tac promoter, trc promoter, tet promoter, araBAD promoter, rpoH promoter, msrA promoter, Bifidobacterium-derived Pm1 promoter, PR promoter, PL promoter, P4 promoter, and P8 promoter.

[0102] A terminator for terminating transcription can be placed downstream of the gene. There are no particular limitations on the terminator as long as it functions in the host. The terminator may be a terminator derived from the host or a heterologous terminator. The terminator may be a terminator inherent to the gene to be introduced or a terminator of another gene. Specific examples of terminators include the T7 terminator, T4 terminator, fd phage terminator, tet terminator, and the trpA terminator.

[0103] <2-3> Host culture The mutant carbonic anhydrase can be expressed by culturing a host having a mutant carbonic anhydrase gene. For example, the mutant carbonic anhydrase may be expressed by transforming a host with an expression vector containing the mutant carbonic anhydrase and culturing the resulting transformant.

[0104] The medium used is not particularly limited as long as it allows the host to grow and a functional mutant carbonic anhydrase to be expressed. For example, a conventional medium used for culturing microorganisms such as bacteria and yeast can be used as the medium. The medium may contain medium components such as a carbon source, a nitrogen source, a phosphate source, a sulfur source, and various other organic and inorganic components as needed. The types and concentrations of medium components may be appropriately determined depending on various conditions such as the type of host.

[0105] Specific examples of carbon sources include sugars such as glucose, fructose, sucrose, lactose, galactose, xylose, arabinose, blackstrap molasses, starch hydrolysates, and biomass hydrolysates; organic acids such as acetic acid, citric acid, succinic acid, and gluconic acid; alcohols such as ethanol, glycerol, and crude glycerol; and fatty acids. Plant-derived materials are preferably used as carbon sources. Examples of plants include corn, rice, wheat, soybeans, sugarcane, beets, and cotton. Examples of plant-derived materials include organs such as roots, stems, trunks, branches, leaves, flowers, and seeds, plants containing these, and decomposition products of these plant organs. The form of use of plant-derived materials is not particularly limited, and they can be used in any form, such as raw products, squeezed juice, pulverized products, or purified products. Furthermore, pentoses such as xylose, hexoses such as glucose, or mixtures thereof can be obtained from plant biomass and used. Specifically, these sugars can be obtained by subjecting plant biomass to treatments such as steam treatment, concentrated acid hydrolysis, dilute acid hydrolysis, hydrolysis with enzymes such as cellulase, and alkali treatment. Because hemicellulose is generally more easily hydrolyzed than cellulose, the hemicellulose in plant biomass may be hydrolyzed in advance to liberate pentoses, and then the cellulose may be hydrolyzed to produce hexoses. Xylose may also be supplied by converting hexoses such as glucose into xylose, for example, by providing the host with a pathway for converting hexoses to xylose. As the carbon source, one type of carbon source may be used, or two or more types of carbon sources may be used in combination.

[0106] The concentration of the carbon source in the medium is not particularly limited, as long as the host can grow and a functional mutant carbonic anhydrase is expressed. The concentration of the carbon source in the medium may be as high as possible, for example, within a range that does not inhibit the production of the mutant carbonic anhydrase. The initial concentration of the carbon source in the medium may be, for example, typically 5 to 30 w / v%, preferably 10 to 20 w / v%. Furthermore, the carbon source may be additionally supplied to the medium as appropriate. For example, the carbon source may be additionally supplied in response to a decrease or depletion of the carbon source as the culture progresses. The carbon source may be temporarily depleted as long as the mutant carbonic anhydrase is ultimately produced, but it may be preferable to carry out the culture so that the carbon source does not become depleted or does not remain depleted.

[0107] Specific examples of nitrogen sources include ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium phosphate; organic nitrogen sources such as peptone, yeast extract, meat extract, and soy protein hydrolysate; ammonia; and urea. Ammonia gas or aqueous ammonia, which are used for pH adjustment, may also be used as a nitrogen source. As the nitrogen source, one type of nitrogen source may be used, or two or more types of nitrogen sources may be used in combination.

[0108] Specific examples of the phosphate source include phosphate salts such as potassium dihydrogen phosphate and dipotassium hydrogen phosphate, and phosphate polymers such as pyrophosphate. As the phosphate source, one type of phosphate source may be used, or two or more types of phosphate sources may be used in combination.

[0109] Specific examples of sulfur sources include inorganic sulfur compounds such as sulfates, thiosulfates, and sulfites, and sulfur-containing amino acids such as cysteine, cystine, and glutathione. As the sulfur source, one type of sulfur source may be used, or two or more types of sulfur sources may be used in combination.

[0110] Specific examples of other various organic and inorganic components include inorganic salts such as sodium chloride and potassium chloride; trace metals such as iron, manganese, magnesium, and calcium; vitamins such as vitamin B1, vitamin B2, vitamin B6, nicotinic acid, nicotinamide, and vitamin B12; amino acids; nucleic acids; and organic components containing these, such as peptone, casamino acids, yeast extract, and soy protein hydrolysate. As other various organic and inorganic components, one type of component may be used, or two or more types of components may be used in combination.

[0111] When an auxotrophic mutant strain that requires nutrients such as amino acids for growth is used, it is preferable to supplement the medium with such required nutrients.

[0112] The culture conditions are not particularly limited as long as the host can grow and a functional mutant carbonic anhydrase can be expressed. The culture can be carried out under normal conditions used for culturing microorganisms such as bacteria and yeast. The culture conditions can be appropriately set depending on various conditions such as the type of host. Furthermore, the expression of the mutant carbonic anhydrase gene can be induced as necessary.

[0113] The culture can be carried out using a liquid medium. For example, the host may be cultured in a solid medium such as an agar medium and then directly inoculated into the liquid medium, or the host may be cultured in a liquid medium as a seed culture and then inoculated into the liquid medium for main culture. That is, the culture may be divided into a seed culture and a main culture. In this case, the culture conditions for the seed culture and the main culture may or may not be the same. The mutant carbonic anhydrase only needs to be expressed in at least the main culture. The amount of the host contained in the medium at the start of the culture is not particularly limited. For example, a seed culture solution with an OD660 of 4 to 100 may be added at the start of the culture in an amount of 0.1% by mass to 100% by mass, preferably 1% by mass to 50% by mass, relative to the medium for main culture.

[0114] Cultivation can be carried out by batch culture, fed-batch culture, continuous culture, or a combination of these. The medium at the start of cultivation is also called the "initial medium." The medium supplied to a culture system, for example, a fermenter, in fed-batch or continuous cultivation is also called the "fed-batch medium." Supplying a fed-batch medium to a culture system in fed-batch or continuous cultivation is also called "fed-batch." When cultivation is carried out separately into seed culture and main culture, the culture form of the seed culture and the main culture may or may not be the same. For example, both the seed culture and the main culture may be carried out by batch culture, or the seed culture may be carried out by batch culture and the main culture may be carried out by fed-batch or continuous culture.

[0115] Various components such as a carbon source may be contained in the initial medium, the feed medium, or both. That is, various components such as a carbon source may be additionally supplied to the medium during the culture process, either alone or in any combination. All of these components may be supplied once or multiple times, or continuously. The types of components contained in the initial medium may or may not be the same as the types of components contained in the feed medium. Furthermore, the concentrations of each component contained in the initial medium may or may not be the same as the concentrations of each component contained in the feed medium. Furthermore, two or more feed media containing different types and / or concentrations of components may be used. For example, when multiple feeds are carried out intermittently, the types and / or concentrations of components contained in each feed medium may be the same, It doesn't have to be that way.

[0116] The culture can be carried out under aerobic conditions, for example. "Aerobic conditions" may mean that the dissolved oxygen concentration in the medium is 0.33 ppm or higher, preferably 1.5 ppm or higher. Specifically, the dissolved oxygen concentration may be controlled to, for example, 1 to 100% of the saturated oxygen concentration, preferably about 20 to 100%. The culture can be carried out, for example, by aeration culture or shaking culture. The pH of the medium may be, for example, 3 to 10, preferably 4.0 to 9.5. During culture, the pH of the medium can be adjusted as needed. The pH of the medium can be adjusted using various alkaline or acidic substances such as ammonia gas, ammonia water, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, or an aqueous phosphoric acid solution. The culture temperature may be, for example, 20 to 45°C, preferably 25 to 37°C. The culture period may be, for example, 10 to 120 hours. Cultivation may be continued, for example, until the carbon source in the medium is consumed or until the activity of the host is lost.

[0117] By culturing the host in this manner, a culture containing the mutant carbonic anhydrase can be obtained. The mutant carbonic anhydrase can be accumulated, for example, within the host's bacterial cells. The term "bacterial cells" may be appropriately interpreted as "cells" depending on the type of host. Depending on the host and / or the design of the mutant carbonic anhydrase gene used, it may be possible to cause the mutant carbonic anhydrase to accumulate in the periplasm or to produce and secrete the mutant carbonic anhydrase outside the bacterial cells.

[0118] <2-4> Recovery of mutant carbonic anhydrase The mutant carbonic anhydrase may be recovered as it is contained in the culture, or may be recovered from the culture. In one embodiment of the present disclosure, the "culture" may be, for example, a medium or bacterial cells, or a mixture of a medium and bacterial cells. The mutant carbonic anhydrase may be purified during the process of recovering it from the culture. Purification can be carried out to the desired extent. That is, the mutant carbonic anhydrase includes purified mutant carbonic anhydrase and fractions containing mutant carbonic anhydrase. In other words, the mutant carbonic anhydrase may be recovered in the form of a purified enzyme, in the form of such a fraction, i.e., in the form contained in such a fraction, or in a combination thereof. Such fractions are not particularly limited, as long as they contain the mutant carbonic anhydrase so that it can act on its substrate. Such fractions include cultures of a host harboring a mutant carbonic anhydrase gene, i.e., a host harboring a mutant carbonic anhydrase, bacterial cells recovered from the culture, culture supernatant recovered from the culture, processed products thereof, partially purified products thereof, and combinations thereof. The above-mentioned "processed product thereof" may be, for example, a processed product of the bacterial cells, such as a bacterial cell disruption product, a bacterial cell lysate, a bacterial cell extract, or other products described below. The above-mentioned "partially purified product thereof" may be, for example, a crudely purified product. Note that "purified mutant carbonic anhydrase" may also include crudely purified products. These fractions can be recovered alone or in appropriate combinations. Mutant carbonic anhydrase recovered in any manner, including these, can be used for any application, including the "method for separating and recovering carbon dioxide" described below. Furthermore, the manner in which the mutant carbonic anhydrase is recovered may be appropriately determined depending on the application.

[0119] As an example, the mutant carbonic anhydrase may be recovered in a form contained in bacterial cells. The method for recovering bacterial cells from the culture medium is not particularly limited, and known methods can be used, for example. Such methods include, for example, natural sedimentation, centrifugation, and filtration. A flocculant may also be used. These methods can be used alone or in appropriate combination. The recovered bacterial cells can be washed appropriately using an appropriate medium. The recovered bacterial cells can also be resuspended appropriately using an appropriate medium. Examples of media that can be used for washing and suspension include aqueous media (aqueous solvents) such as water and aqueous buffer solutions. can be done.

[0120] As another example, the mutant carbonic anhydrase may be recovered from the form contained in the bacterial cells by subjecting the bacterial cells to appropriate treatment. Examples of bacterial cell treatment include immobilization on a carrier such as acrylamide or carrageenan, freeze-thaw treatment, treatment to increase membrane permeability, and physical disruption using ultrasonic disruption or a pressure homogenizer. Membrane permeability can be increased by using, for example, a surfactant or organic solvent. These treatments can be used alone or in appropriate combination.

[0121] The mutant carbonic anhydrase may be produced alone or in combination with other proteins.

[0122] <2-5> Production of mutant carbonic anhydrase The mutant carbonic anhydrase may be produced by the method described above.

[0123] For example, a mutant carbonic anhydrase may be produced by a method comprising the steps of expressing a mutant carbonic anhydrase gene in a host harboring the gene and recovering the expressed enzyme. Such a method is also referred to as a "method for producing carbonic anhydrase."

[0124] The method for producing carbonic anhydrase preferably comprises: a step of transforming a host with an expression vector having a mutant carbonic anhydrase gene and culturing the resulting transformant to express carbonic anhydrase; recovering the expressed enzyme from the resulting culture; The method may include:

[0125] The expression vector is as described above. The expression vector having a mutant carbonic anhydrase gene may be an expression vector containing a nucleotide encoding the mutant carbonic anhydrase.

[0126] The host and its culture are as described above.

[0127] The expressed mutant carbonic anhydrase can be recovered as described above. The mutant carbonic anhydrase may be purified during the recovery process from the culture. The purification of the enzyme is also as described above.

[0128] <3> Use of mutant carbonic anhydrase The use of the mutant carbonic anhydrase is not particularly limited. The mutant carbonic anhydrase can be used, for example, for separating and recovering carbon dioxide. Separation and recovery of carbon dioxide may be carried out by any known method other than using the mutant carbonic anhydrase of the present disclosure, and may be carried out, for example, by the method described in Japanese Patent Application No. 2023-026820. Separation and recovery of carbon dioxide may be carried out, for example, by a method for separating and recovering carbon dioxide including a step of using the mutant carbonic anhydrase.

[0129] The separation and recovery of carbon dioxide will be exemplified below.

[0130] <3-1> Carbon dioxide separation and capture method A method for separating and recovering carbon dioxide in one embodiment of the present disclosure includes a step of using a mutant carbonic anhydrase. In other words, the method for separating and recovering carbon dioxide in one embodiment of the present disclosure may include a step of using a carbonic anhydrase selected from any of the above-mentioned (i) to (iii), or a step of using a carbonic anhydrase selected from any of the above-mentioned (iv) to (vi). It may comprise a step of using a carbonic anhydrase selected from any of (vii) to (xii) above, it may comprise a step of using a carbonic anhydrase selected from any of (xiii) to (xv) above, it may comprise a step of using a carbonic anhydrase selected from any of (xvi) to (xviii) above, it may comprise a step of using a carbonic anhydrase selected from any of (xix) to (xxi) above, or it may comprise a step of using a carbonic anhydrase selected from any of (xxii) to (xxiv) above.

[0131] Furthermore, a method for separating and recovering carbon dioxide in one embodiment of the present disclosure may include a step of contacting a composition containing a mutant carbonic anhydrase with a treatment target containing carbon dioxide to allow the composition to absorb the carbon dioxide (absorption step), and a step of dissipating the carbon dioxide from the composition that has absorbed carbon dioxide in the absorption step by heating the composition to a higher temperature and / or reducing the pressure than in the absorption step (dissipation step). Note that the absorption step may be considered an embodiment of a step of using a mutant carbonic anhydrase, since it uses a composition containing a mutant carbonic anhydrase. In other words, the step of using a mutant carbonic anhydrase may, for example, be a step of contacting a composition containing a mutant carbonic anhydrase with a treatment target containing carbon dioxide to allow the carbon dioxide to be absorbed by the composition. Specifically, the composition containing a mutant carbonic anhydrase may be, for example, a "carbon dioxide separation / absorption liquid" described below.

[0132] The carbon dioxide separation method may include any step that uses a mutant carbonic anhydrase, and may include, for example, the absorption step and the diffusion step described above, or may include other steps in addition to these. Examples of other steps include a cooling step, a heating step, a washing step, an extraction step, an ultrasonic treatment step, a distillation step, and other steps involving treatment with a chemical solution.

[0133] Carbon dioxide is separated and recovered from a treatment target by a carbon dioxide separation and recovery method. The carbon dioxide separation method according to one embodiment of the present disclosure can be applied to, for example, the separation of carbon dioxide from combustion exhaust gas generated in thermal power plants, steel plants, cement factories, and the like, and the separation of carbon dioxide from steam-reformed gas obtained in a steam reforming process.

[0134] The target to be treated may be a gas, a liquid, a solid, or a combination thereof, but preferably contains at least a gas. The gas fraction of the target to be treated may be referred to as the "target gas." Furthermore, when the target to be treated contains a liquid, the liquid is preferably an aqueous solution.

[0135] The gas to be treated preferably contains carbon dioxide. The gas to be treated may be pure carbon dioxide gas, or a mixed gas containing carbon dioxide and other gases. The other gases are not particularly limited. Specific examples of the other gases include air, nitrogen, oxygen, hydrogen, argon, neon, helium, carbon monoxide, water vapor, methane, and nitrogen oxides (NOX). In consideration of improving the separation performance between carbon dioxide and other gases, the concentration of carbon dioxide contained in the mixed gas is preferably 5% or more, and more preferably 10% or more.

[0136] Carbon dioxide may refer to carbon dioxide present in the gas fraction of the target to be treated. Furthermore, when the target to be treated contains an aqueous solution, it may refer to carbon dioxide present in the aqueous solution. Note that carbon dioxide present in the air and / or carbon dioxide present in the aqueous solution may typically be in the gaseous state.

[0137] In the step of using the mutant carbonic anhydrase, the mutant carbonic anhydrase may be used as an aqueous solution. In other words, the step of using the mutant carbonic anhydrase may be a step of using an aqueous solution containing the mutant carbonic anhydrase. The composition containing carbonic anhydrase may be an aqueous solution containing a mutant carbonic anhydrase. The mutant carbonic anhydrase may be produced and recovered by the method described herein.

[0138] The step of using a mutant carbonic anhydrase may be carried out by exposing the target to be treated, particularly the gas to be treated, to an aqueous solution containing the mutant carbonic anhydrase, or by dissolving the mutant carbonic anhydrase in an aqueous solution contained in the target to be treated. In other words, the step of using a mutant carbonic anhydrase may be carried out by contacting a composition containing the mutant carbonic anhydrase with a target to be treated containing carbon dioxide. That is, the step of using a mutant carbonic anhydrase may be carried out by the absorption step described above. There are no particular limitations on the method for contacting a composition containing a mutant carbonic anhydrase with a target to be treated containing carbon dioxide, and specifically, known methods such as a bubbling method or a head-on contact method using a packed column or a plate column can be used.

[0139] The mutant carbonic anhydrase of the present disclosure has improved stability, preferably improved thermal stability and / or alkaline resistance. Therefore, the method for separating and recovering carbon dioxide according to one embodiment of the present disclosure may be carried out under high-temperature and / or alkaline conditions at least in the step using the mutant carbonic anhydrase, e.g., the absorption step. Furthermore, the method for separating and recovering carbon dioxide according to one embodiment of the present disclosure may be carried out entirely at high temperatures, or at least in some steps, including the step using the mutant carbonic anhydrase, at high temperatures. Note that "carried out at high temperatures" may mean carrying out the method under conditions of 40°C or higher, or 50°C or higher, 60°C or higher, 70°C or higher, 80°C or higher, 90°C or higher, or 95°C or higher. Furthermore, the method for separating and recovering carbon dioxide according to one embodiment of the present disclosure may be carried out under alkaline conditions at least in the step using the mutant carbonic anhydrase, e.g., the absorption step. Furthermore, the method for separating and recovering carbon dioxide according to one embodiment of the present disclosure may be carried out entirely at high temperatures, or at least in some steps, including the step using the mutant carbonic anhydrase, at high temperatures. In addition, "carrying out under alkaline conditions" may mean carrying out under conditions of pH 8, or may mean carrying out under conditions of pH 9 or higher, pH 10 or higher, pH 11 or higher, pH 12 or higher, pH 13 or higher, or pH 14 or higher.

[0140] By using an aqueous solution containing a mutant carbonic anhydrase, carbon dioxide is separated and recovered from the target to be treated and absorbed into the aqueous solution as bicarbonate ions. The aqueous solution containing a mutant carbonic anhydrase may be considered as a carbon dioxide separation / absorption liquid. The carbon dioxide separation / absorption liquid will be described in detail below.

[0141] <3-2> Carbon dioxide separation and absorption liquid The "liquid for separating and absorbing carbon dioxide" in one embodiment of the present disclosure may contain a mutant carbonic anhydrase. The liquid for separating and absorbing carbon dioxide is preferably an aqueous solution. In other words, the "liquid for separating and absorbing carbon dioxide" in one embodiment of the present disclosure may be an aqueous solution containing a mutant carbonic anhydrase.

[0142] The carbon dioxide separation / absorption liquid may further contain a substance for treating and recovering bicarbonate ions. It may also contain a substance that increases the amount of carbon dioxide absorbed into the aqueous solution, specifically, an amine, K2CO3, an alkali such as NaOH, or the like. The bicarbonate ions may be recovered, for example, as carbonates. An example of an ion that forms a salt with bicarbonate ions is calcium ions. Therefore, the carbon dioxide separation / absorption liquid may further contain calcium ions and / or a substance that releases calcium ions.

[0143] The carbon dioxide separation / absorption liquid may be a buffer solution, such as a buffer solution containing HEPES (2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid). [Example]

[0144] The present disclosure will be described in more detail below using examples, but the present disclosure is not limited to these examples.

[0145] Example 1 Introduction of amino acid substitutions into carbonic anhydrase derived from Thermosulfurimonas dismutans (1) A polynucleotide (SEQ ID NO: 3) encoding a polypeptide (SEQ ID NO: 2) was synthesized by deleting the amino acid residues from the second lysine residue to the twentieth alanine residue, which correspond to the signal peptide, from the amino acid sequence of natural Thermosulfurimonas dismutans carbonic anhydrase (tdCA) (GenBank No. OAQ21602, SEQ ID NO: 1). During synthesis, a recognition sequence for the restriction enzyme NdeI (CAT) was added to the 5'-end, and an oligonucleotide sequence (SEQ ID NO: 4) encoding six histidine residues (histidine tag), a stop codon, and a recognition sequence for the restriction enzyme HindIII (TAAGCTT) were added to the 3'-end.

[0146] (2) The pET-26b(+) plasmid vector (Novagen) pre-digested with NdeI and HindII and the polynucleotide synthesized in (1) (pre-digested with NdeI and HindIII) were inserted, and the ligation product was used to transform Escherichia coli strain BL21(DE3) (Nippon Gene) to prepare a transformant.

[0147] (3) The transformants prepared in (2) were selected and cultured in LB (Luria-Bertani) medium. The recombinant plasmid was purified using a QIAprep Spin Miniprep Kit (Qiagen) to obtain a plasmid capable of expressing wild-type tdCA (designated pET-tdCA_WT).

[0148] (4) Polynucleotides encoding tdCA having the amino acid substitutions listed below (A) to (G) were prepared by PCR using the pET-tdCA_WT obtained in (3) as a template. (A) Substitution of the lysine residue at position 14 of SEQ ID NO: 2 (position 33 of SEQ ID NO: 1) with a glutamic acid residue (hereinafter also referred to as K33E). (B) Substitution of the lysine residue at position 146 of SEQ ID NO: 2 (position 165 of SEQ ID NO: 1) with a glutamic acid residue (hereinafter also referred to as K165E). (C) Substitution of the methionine residue at position 163 of SEQ ID NO: 2 (position 182 of SEQ ID NO: 1) with a leucine residue (hereinafter also referred to as M182L). (D) Amino acid substitutions K33E and K165E (E) Amino acid substitutions K33E and M182L (F) Amino acid substitutions K165E and M182L (G) Amino acid substitutions K33E, K165E, and M182L

[0149] (5) A transformant was prepared from the polynucleotide prepared in (4) by the method described in (2), and a plasmid capable of expressing the tdCA amino acid substitution was obtained by the method described in (3). The base sequence of the site where the amino acid substitution was introduced and the surrounding region of the plasmid were analyzed, and it was confirmed that each sequence was the desired one.

[0150] Table 1 shows the amino acid sequences of tdCA having the amino acid substitutions described in (A) to (G) above and the sequence numbers of the nucleotide sequences encoding the tdCA.

[0151] [Table 1]

[0152] Example 2 Preparation of tdCA (1) Escherichia coli BL21(DE3) strain (Nippon Gene Co., Ltd.) was transformed with pET-tdCA_WT prepared in Example 1 or a plasmid capable of expressing tdCA having any of the amino acid substitutions (A) to (G) above, and then cultured overnight at 37°C on LB agar medium (10 g / L tryptone, 5 g / L yeast extract, 5 g / L NaCl, 15 g / L purified agar, 0.05 mg / mL kanamycin sulfate) to form colonies.

[0153] (2) The colonies formed on the agar medium in (1) were collected and inoculated into test tubes containing 2 mL of 2xYT medium (16 g / L tryptone, 10 g / L yeast extract, 5 g / L NaCl, 0.05 mg / mL kanamycin sulfate), followed by pre-culture at 37°C and 150 rpm overnight with shaking.

[0154] (3) 1 mL of the preculture solution from (2) was inoculated into a 500 mL baffled Erlenmeyer flask containing 100 mL of 2xYT medium (16 g / L tryptone, 10 g / L yeast extract, 5 g / L sodium chloride, 0.05 mg / mL kanamycin sulfate) and cultured at 37°C and 130 rpm for 3 hours with shaking. Then, 50 μL of 100 mmol / L IPTG (Isopropyl β-D-thiogalactopyranoside) was added (final concentration: 50 μmol / L), and the cultured mixture was cultured at 25°C and 150 rpm for 24 hours with shaking.

[0155] (4) After the incubation, the mixture was centrifuged at 10,000 rpm at 4°C for 30 minutes, and the supernatant was removed to recover the wet bacterial cells. The wet bacterial cells were stored at -30°C until use.

[0156] (5) 5 mL of extraction solution (25 mmol / L HEPES (2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid) (pH 8.3) containing BugBuster (trade name) (Merck) solution, 0.2 mg / mL lysozyme, and 25 U / mL Benzonase (Merck)) was added per gram of wet cells, and the mixture was shaken at 25°C and 150 rpm for 30 minutes. The mixture was then centrifuged at 4°C and 10,000 rpm for 30 minutes, and the supernatant was collected.

[0157] (6) The supernatant collected in (5) was sterilized using a filter with a pore size of 0.22 μm, and then Ni The mixture was loaded onto an open column packed with 1 mL of ckel resin (Takara Bio), washed with 10 mL of 25 mmol / L HEPES (pH 8.3) containing 20 mmol / L imidazole, and then eluted with 6 mL of 25 mmol / L HEPES (pH 8.3) containing 300 mmol / L imidazole.

[0158] (7) The tdCA protein contained in the fraction eluted in (6) was adjusted to a concentration of 8 μmol / L, dispensed into a PCR plate at 30 μL / well, and heated at 95°C for 60 minutes using a thermal cycler. After heating, the plate was kept at 4°C.

[0159] Example 3 Evaluation of decarboxylation activity of heat-treated tdCA The decarboxylation activity of the heat-treated tdCA was measured by the following method.

[0160] (1) 5 μL of the heat-treated tdCA solution obtained in Example 2(7) was dispensed into a microplate.

[0161] (2) A reaction solution was prepared based on the reaction solution composition listed in Table 2, and the change in pH accompanying the progress of the decarboxylation reaction was detected by measuring the time-dependent change in absorbance at a wavelength of 550 nm derived from phenolphthalein using a microplate reader.

[0162] [Table 2]

[0163] (3) The decarboxylation activity of tdCA was calculated from the change in absorbance according to the method described in Proc. Natl. Acad. Sci. USA, 111, 16436. Specifically, the magnitude of the slope of the change in absorbance over time was defined as the decarboxylation activity of tdCA, and the decarboxylation activity of each tdCA was measured.

[0164] Example 4: Evaluation of CO2 hydration activity of heat-treated tdCA The CO2 hydration activity of the heat-treated tdCA was measured using the following method.

[0165] (1) The heat-treated tdCA solution obtained in Example 2(7) was diluted two-fold with 25 mmol / L HEPES (pH 8.3).

[0166] (2) A reaction solution was prepared based on the composition of the reaction solution shown in Table 3, and the change over time in absorbance at a wavelength of 570 nm derived from phenol red was measured.

[0167] [Table 3]

[0168] (3) The CO2 hydration activity of tdCA was calculated from the absorbance change according to the method described by JH Kim et al., Catalysts, 12(11), 1391. Specifically, the CO2 hydration activity of tdCA was defined as the slope of the absorbance change over time, and the CO2 hydration activity of each tdCA was measured.

[0169] The results of Example 3 are shown in Figure 1, and the results of Example 4 are shown in Figure 2. "buf" indicates the results for 25 mmol / L HEPES (pH 8.3) alone (so-called blank), and "WT" indicates the wild-type (SEQ ID NO: 2). All of the tdCAs into which the amino acid substitutions (A) to (G) were introduced maintained higher activity after heat treatment than the wild-type. These results demonstrate that introducing at least one of the amino acid substitutions K33E, K165E, and M182L into wild-type tdCA improves thermal stability.

[0170] Example 5 Introduction of amino acid substitutions into carbonic anhydrase derived from Thermosulfurimonas dismutans (part 2) (1) The K33E amino acid substitution was selected from the mutations involved in improving thermostability identified in Examples 3 and 4, and further mutations were added. Specifically, polynucleotides encoding tdCAs with the amino acid substitutions (H) to (K) below were prepared by PCR using the plasmid capable of expressing tdCA with the K33E amino acid substitution obtained in Example 1(5) as a template. (H) Substitution of the isoleucine residue at position 47 of SEQ ID NO: 5 (position 66 of SEQ ID NO: 1) with a valine residue (hereinafter also referred to as I66V). (I) Substitution of the lysine residue at position 210 of SEQ ID NO: 5 (position 229 of SEQ ID NO: 1) with a glutamic acid residue (hereinafter also referred to as K229E). (J) Substitution of the methionine residue at position 212 of SEQ ID NO: 5 (position 231 of SEQ ID NO: 1) with an isoleucine residue (hereinafter also referred to as M231I). (K) Amino acid substitutions I66V, K229E, and M231I

[0171] (2) Using the polynucleotide prepared in (1), a transformant was prepared by the method described in Example 1(2) and (3), and a plasmid capable of expressing the tdCA amino acid substitution was obtained. The base sequence of the site of amino acid substitution and its surrounding region in the plasmid was analyzed, and it was confirmed that each sequence was the desired sequence. Amino acid sequence of tdCA into which any of the amino acid substitutions described in (H) to (K) has been introduced The sequence numbers of the sequences and the base sequences encoding the tdCA are shown in Table 4.

[0172] [Table 4]

[0173] Example 6 Preparation of tdCA and evaluation of decarboxylation activity (part 2) (1) Using the plasmid capable of expressing tdCA having any of the amino acid substitutions (H) to (K) prepared in Example 5, tdCA was prepared according to the methods described in Example 2(1) to (6).

[0174] (2) The tdCA protein contained in the fraction eluted in (1) was adjusted to a concentration of 8 μmol / L or 40 μmol / L, dispensed into a PCR plate at 30 μL / well, and heated at 95°C for 60 minutes using a thermal cycler. After heating, the plate was kept at 4°C.

[0175] (3) The decarboxylation activity of the heat-treated tdCA was measured by the method described in Example 3. The results at a tdCA protein concentration of 8 μmol / L are shown in Figure 3, and the results at 40 μmol / L are shown in Figure 4. In Figures 3 and 4, the decarboxylation activity of each mutant after heat treatment is expressed as a relative value (residual activity) with the decarboxylation activity before heat treatment set to 100%. Figure 3 shows that the residual activity of all tdCA mutants (m2-d to m2-f) into which the amino acid substitutions (H) to (J) were introduced was improved compared to the tdCA mutant (m1-a) before the introduction of the mutations (H) to (J). This indicates that the introduction of at least one of the amino acid substitutions I66V, K229E, and M231I improves thermostability. Furthermore, Figure 4 shows that tdCA (m4) with the amino acid substitution (K) introduced has improved residual activity compared to tdCA (m2-d to m2-f) with the amino acid substitutions (H) to (J), and that introducing all of the I66V, K229E, and M231I mutations improves thermal stability compared to the individual amino acid substitutions.

[0176] Example 7 Introduction of amino acid substitutions into carbonic anhydrase derived from Thermosulfurimonas dismutans (part 3) (1) Using the plasmid obtained in Example 5(2) capable of expressing tdCA (m4) having the amino acid substitution (K) as a template, polynucleotides encoding tdCA in which one of the amino acid substitutions (L) to (O) below was further added to m4 were prepared. (L) Substitution of the lysine residue at position 154 of SEQ ID NO: 25 (position 173 of SEQ ID NO: 1) with an arginine residue (hereinafter also referred to as K173R). (M) Substitution of the lysine residue at position 195 of SEQ ID NO: 25 (position 214 of SEQ ID NO: 1) with an arginine residue (hereinafter also referred to as K214R). (N) Substitution of the lysine residue at position 227 of SEQ ID NO: 25 (position 246 of SEQ ID NO: 1) with an arginine residue (hereinafter also referred to as K246R). (O) Amino acid substitutions of K173R, K214R, and K246R.

[0177] (2) Using the polynucleotide prepared in (1), a transformant was prepared by the method described in Example 1(2) and (3), and a plasmid capable of expressing the tdCA amino acid substitution was obtained. The base sequence of the site of amino acid substitution and its surrounding region in the plasmid was analyzed, and it was confirmed that each sequence was the desired sequence. Table 5 shows the sequence numbers of the amino acid sequences of tdCA into which any of the amino acid substitutions described in (L) to (O) above has been introduced, and the sequence numbers of the base sequences encoding the tdCA.

[0178] [Table 5]

[0179] Example 8 Preparation of tdCA and evaluation of decarboxylation activity (part 3) (1) tdCA was prepared according to the methods described in Example 2(1) to (6) using a plasmid capable of expressing tdCA into which any of the amino acid substitutions described in (L) to (O) above, which was prepared in Example 7, was introduced.

[0180] (2) The tdCA protein contained in the fraction eluted in (1) was adjusted to a concentration of 40 μmol / L, and dispensed into a PCR plate at a volume ratio of 1:1 or 1:4 (tdCA:K2CO3 aqueous solution) with 1.45 mol / L K2CO3 aqueous solution (adjusted to pH 10 with 1.45 mol / L KHCO3 aqueous solution). The plate was then subjected to heat and alkali treatment at 75°C for 60 minutes using a thermal cycler. After treatment, the plate was kept at 4°C.

[0181] (3) The decarboxylation activity of heat- and alkali-treated tdCA was measured by the method described in Example 3. Figure 5 shows the results for a 1:1 volume ratio of tdCA to K2CO3 aqueous solution, and Figure 6 shows the results for a 1:4 volume ratio. Figure 5 shows that all tdCAs (m5-a to m5-c) containing the amino acid substitutions (L) to (N) exhibited improved decarboxylation activity after heat and alkali treatment compared to the tdCA mutant (m4) before the introduction of the (L) to (N) mutations. This indicates that the introduction of at least one of the amino acid substitutions K173R, K214R, and K246R improves heat and alkali stability. Furthermore, Figure 6 shows that tdCA (m7) containing the amino acid substitution (O) exhibited improved decarboxylation activity after heat and alkali treatment compared to tdCAs (m5-a to m5-c) containing the amino acid substitutions (L) to (N). This indicates that the introduction of all the mutations K173R, K214R, and K246R improves heat and alkali stability compared to the individual amino acid substitutions.

[0182] Example 9 Introduction of amino acid substitutions into carbonic anhydrase derived from Thermosulfurimonas dismutans (part 4) (1) Using the plasmid obtained in Example 7(2) capable of expressing tdCA (m7) having the amino acid substitution (O) as a template, polynucleotides encoding tdCA in which one of the amino acid substitutions (P) to (T) below was further added to m7 were prepared. (P) Substitution of the valine residue at position 7 of SEQ ID NO: 33 (position 26 of SEQ ID NO: 1) with an alanine residue (hereinafter also referred to as V26A). (Q) Substitution of the alanine residue at position 24 of SEQ ID NO: 33 (position 43 of SEQ ID NO: 1) with a valine residue (hereinafter also referred to as A43V). (R) Substitution of the lysine residue at position 207 of SEQ ID NO: 33 (position 226 of SEQ ID NO: 1) with an arginine residue (hereinafter also referred to as K226R). (S) Substitution of the glutamine residue at position 50 of SEQ ID NO: 33 (position 69 of SEQ ID NO: 1) with an arginine residue (hereinafter also referred to as Q69R), and substitution of the valine residue at position 84 of SEQ ID NO: 33 (position 103 of SEQ ID NO: 1) with an isoleucine residue (hereinafter also referred to as V103I). (T) Amino acid substitutions of V26A, A43V, Q69R, V103I, and K226R.

[0183] (2) Using the polynucleotide prepared in (1), a transformant was prepared by the method described in Example 1(2) and (3), and a plasmid capable of expressing the tdCA amino acid substitution was obtained. The base sequence of the site of amino acid substitution and its surrounding region in the plasmid was analyzed, and it was confirmed that each sequence was the desired sequence. Table 6 shows the sequence numbers of the amino acid sequences of tdCA into which any of the amino acid substitutions described in (P) to (T) above has been introduced, and the sequence numbers of the base sequences encoding the tdCA.

[0184] [Table 6]

[0185] Example 10: Preparation of tdCA and evaluation of decarboxylation activity (part 4) and CO2 hydration activity (part 2) (1) Introduce the amino acid substitution described in any one of (P) to (S) prepared in Example 9 Using the introduced plasmid capable of expressing tdCA, tdCA was prepared according to the method described in Example 2 (1) to (6).

[0186] (2) The concentration of the tdCA protein contained in the fraction eluted in (1) was adjusted to 200 μmol / L.

[0187] (3) The tdCA concentration adjusted in (2) was dispensed into a PCR plate at a volume ratio of 1:4 (tdCA:HEPES buffer) with 25 mmol / L HEPES buffer adjusted to pH 12 with sodium hydroxide, and subjected to heat and alkali treatment at 90°C for 10 minutes using a thermal cycler. After treatment, the plate was kept at 4°C.

[0188] (4) The decarboxylation activity of the tdCA treated with heat and alkali in (3) was measured by the method described in Example 3.

[0189] (5) The tdCA protein contained in the fraction eluted in (1) was adjusted to a concentration of 4 μmol / L, dispensed into a PCR plate, and heat-treated at 90°C for 60 minutes using a thermal cycler. After treatment, the plate was kept at 4°C.

[0190] (6) The CO2 hydration activity of the heat-treated tdCA in (5) was measured by the method described in Example 4. The results of the decarboxylation activity measurement are shown in Figure 7, and the results of the CO2 hydration activity measurement are shown in Figure 8. As shown in Figures 7 and 8, the tdCAs (m8-a to m9-a) into which the amino acid substitutions (P) to (S) were introduced all showed improved decarboxylation activity after heat and alkali treatment and / or improved CO2 hydration activity after heat treatment compared to the tdCA mutant (m7) before the introduction of the (P) to (S) mutations. This indicates that the introduction of at least one amino acid substitution from V26A, A43V, Q69R, V103I, and K226R improves heat and / or alkali stability.

[0191] Example 11 Evaluation of CO2 hydration activity of tdCA (part 3) (1) Using the plasmid capable of expressing tdCA into which the amino acid substitutions described in (T) above, which was prepared in Example 9, tdCA was prepared according to the method described in Example 2(1) to (6).

[0192] (2) The concentration of the tdCA protein contained in the fraction eluted in (1) was adjusted to 20 μmol / L.

[0193] (3) The tdCA concentration adjusted in (2) was mixed with 5 mmol / L aqueous sodium hydroxide solution at a volume ratio of 1:4 (tdCA:sodium hydroxide solution) (pH after mixing: 11.4). The mixed tdCA solution was subjected to alkali treatment by incubating at 25°C for 60 minutes. After treatment, it was kept at 4°C.

[0194] (4) Phenol red dissolved in 20 mmol / L Tris buffer (pH 8.3) was added to the tdCA solution treated with alkali in (3) to a final concentration of 0.1 mmol / L.

[0195] (5) The tdCA solution containing phenol red prepared in (4) and ice-cooled carbonate solution were rapidly mixed in equal amounts using a stopped-flow spectrophotometer (Applied Photophysics), and the time-dependent change in absorbance at a wavelength of 570 nm due to phenol red was measured.

[0196] (6) The CO2 hydration activity of tdCA was calculated from the absorbance change according to the method described by JH Kim et al., Catalysts, 12(11), 1391. Specifically, the CO2 hydration activity of tdCA was defined as the slope of the absorbance change over time, and the CO2 hydration activity of each tdCA was measured.

[0197] The measurement results are shown in Figure 9. As can be seen from Figure 9, tdCA (m12) into which the amino acid substitution (T) was introduced had improved CO2 hydration activity after alkaline treatment compared to the tdCA mutant (m7) before the introduction of the amino acid substitution (T) and the wild-type tdCA (WT), and it can be seen that the introduction of all of the mutations V26A, A43V, Q69R, V103I, and K226R improved alkaline stability.

[0198] Example 12 Introduction of amino acid substitutions into carbonic anhydrase derived from Thermosulfurimonas dismutans (part 5) (1) Polynucleotides encoding tdCA containing any of the following amino acid substitutions (U) to (Y) were prepared by PCR using the wild-type tdCA-expressing plasmid (pET-tdCA_WT) obtained in Example 1(3) as a template: (U) Substitution of the serine residue at position 44 of SEQ ID NO: 2 (position 63 of SEQ ID NO: 1) with a threonine residue (hereinafter also referred to as S63T). (V) Substitution of the glutamine residue at position 50 of SEQ ID NO: 2 (position 69 of SEQ ID NO: 1) with an aspartic acid residue (hereinafter also referred to as Q69D). (W) Substitution of the glutamic acid residue at position 150 of SEQ ID NO: 2 (position 169 of SEQ ID NO: 1) with an aspartic acid residue (hereinafter also referred to as E169D). (X) Substitution of the histidine residue at position 151 of SEQ ID NO: 2 (position 170 of SEQ ID NO: 1) with a glutamic acid residue (hereinafter also referred to as H170E). (Y) Amino acid substitutions of S63T, Q69D, E169D, and H170E

[0199] (2) Using the polynucleotide prepared in (1), a transformant was prepared by the method described in Example 1(2) and (3), and a plasmid capable of expressing the tdCA amino acid substitution was obtained. The base sequence of the site of amino acid substitution and its surrounding region in the plasmid was analyzed, and it was confirmed that each sequence was the desired sequence.

[0200] Table 7 shows the amino acid sequences of tdCA having the amino acid substitutions (U) to (Y) and the sequence numbers of the base sequences encoding the tdCA.

[0201] [Table 7]

[0202] Example 13 Preparation of tdCA and evaluation of decarboxylation activity (part 5) (1) Using the plasmid capable of expressing tdCA having any of the amino acid substitutions (U) to (Y) prepared in Example 12, tdCA was prepared according to the methods described in Example 2(1) to (6).

[0203] (2) The tdCA protein contained in the fraction eluted in (1) was adjusted to a concentration of 8 μmol / L, dispensed into a PCR plate at 50 μL / well, and heated at 80°C or 85°C for 60 minutes using a thermal cycler. After heating, the plate was kept at 4°C.

[0204] (3) 10 μL of the tdCA solution before heat treatment obtained in (1) or the heat-treated tdCA solution obtained in (2) was dispensed into a microplate.

[0205] (4) A reaction solution was prepared based on the reaction solution composition shown in Table 8, and the change in pH accompanying the progress of the decarboxylation reaction was detected by measuring the change in absorbance at a wavelength of 550 nm derived from phenolphthalein over time using a microplate reader.

[0206] [Table 8]

[0207] (5) The decarboxylation activity of tdCA was calculated in the same manner as in Example 3(3). The residual decarboxylation activity after heat treatment at 80°C and 85°C (decarboxylation activity after heat treatment divided by decarboxylation activity before heat treatment) is shown in Figure 10. In Figure 10, the residual decarboxylation activity of each mutant is expressed as a relative value, with the residual decarboxylation activity of wild-type tdCA set to 1. All tdCAs with the amino acid substitutions (U) to (Y) maintained higher activity after heat treatment than the wild-type. These results demonstrate that introducing at least one of the amino acid substitutions S63T, Q69D, E169D, and H170E into wild-type tdCA improves thermostability.

[0208] Example 14 Preparation of tdCA and evaluation of decarboxylation activity (part 6) (1) The tdCA prepared in Example 10(1) or Example 11(1) and having the amino acid substitutions described in any of (P) to (T) above introduced therein was adjusted to a protein concentration of 40 μmol / L.

[0209] (2) The tdCA concentration adjusted in (1) was mixed with 25 mmol / L HEPES buffer solution adjusted to pH 12 with sodium hydroxide at a volume ratio of 1:4 (tdCA:HEPES buffer solution). The mixture was dispensed into PCR plates and subjected to heat and alkali treatment at 80°C for 10 minutes using a thermal cycler. After treatment, the mixture was kept at 4°C.

[0210] (3) The decarboxylation activity of the tdCA treated with heat and alkali in (2) was measured by the method described in Example 3. The measurement results are shown in Figure 11. Figure 11 shows that tdCA (m12) into which the amino acid substitution (T) was introduced had improved decarboxylation activity after heat and alkali treatment compared to tdCA (m8-a to m9-a) into which the amino acid substitutions (P) to (S) were introduced, and that the introduction of all of the mutations V26A, A43V, Q69R, V103I, and K226R improved heat and alkali stability compared to the individual amino acid substitutions.

[0211] Example 15 Introduction of amino acid substitutions into carbonic anhydrase derived from Thermosulfurimonas dismutans (part 6) (1) Using the plasmid capable of expressing tdCA (m4) having the amino acid substitution (K) obtained in Example 5(2) as a template, polynucleotides encoding tdCA in which either the amino acid substitution (Z) or (AA) below was further added to m4 were prepared. (Z) Substitution of the glycine residue at position 4 of SEQ ID NO: 25 (m4) (position 23 of SEQ ID NO: 1) with a leucine residue (hereinafter also referred to as G23L) (AA) Substitution of the glycine residue at position 4 of SEQ ID NO: 25 (m4) (position 23 of SEQ ID NO: 1) with an isoleucine residue (hereinafter also referred to as G23I)

[0212] (2) Using the polynucleotide prepared in (1), a transformant was prepared by the method described in Example 1(2) and (3), and a plasmid capable of expressing the tdCA amino acid substitution was obtained. The base sequence of the site of amino acid substitution and its surrounding region in the plasmid was analyzed, and it was confirmed that each sequence was the desired sequence. Table 9 shows the sequence numbers of the amino acid sequences of tdCA into which any of the amino acid substitutions described in (Z) or (AA) above has been introduced, and the sequence numbers of the base sequences encoding the tdCA.

[0213] [Table 9]

[0214] Example 16 Preparation of tdCA and evaluation of productivity and decarboxylation activity (Part 7) (1) Using the plasmid capable of expressing tdCA having the amino acid substitutions described in either (Z) or (AA) prepared in Example 15, tdCA was prepared according to the methods described in Example 2(1) to (6).

[0215] (2) The purified tdCA contained in the fraction obtained in (1) was quantified using a NanoDrop microspectrophotometer (Thermo Fisher Scientific) to evaluate productivity (purification yield per culture medium).

[0216] (3) The tdCA protein contained in the fraction eluted in (1) was adjusted to a concentration of 8 μmol / L, dispensed into a PCR plate at 50 μL / well, and heated at 90°C for 60 minutes using a thermal cycler. After heating, the plate was kept at 4°C.

[0217] (4) The decarboxylation activity of the tdCA solution before heat treatment obtained in (1) or the heat-treated tdCA solution obtained in (3) was measured by the method described in Example 3.

[0218] (5) The residual activity was calculated by dividing the decarboxylation activity of the heat-treated tdCA measured in (4) by the decarboxylation activity of the tdCA before heat treatment.

[0219] Figure 12 shows the productivity (purification yield per culture medium) of mutants in which either the amino acid substitution (Z) or (AA) was introduced into tdCA (m4) having the amino acid substitution (K). In Figure 12, the productivity of the mutants in which either the amino acid substitution (Z) or (AA) was introduced is shown as a relative value, with the productivity of the mutant (m4) in which no amino acid substitution was introduced being set at 1. Both tdCA mutants in which either the amino acid substitution (Z) or (AA) was introduced (m4-G23L and m4-G23I) showed higher productivity than the tdCA mutant (m4) in which no amino acid substitution was introduced.

[0220] The decarboxylation activity of tdCA mutants (m4) containing the amino acid substitution (K) introduced with either the amino acid substitution (Z) or (AA) is shown in Figure 13 before heat treatment. In Figure 13, the decarboxylation activity of the mutants containing either the amino acid substitution (Z) or (AA) is expressed relative to the decarboxylation activity of the mutant (m4) without the mutation, which is set to 1. The tdCA mutants (m4-G23L and m4-G23I) containing either the amino acid substitution (Z) or (AA) showed higher decarboxylation activity than the tdCA mutant (m4) without the mutation.

[0221] The residual activity after heat treatment of mutants in which either the amino acid substitution (Z) or (AA) was introduced into tdCA (m4) having the amino acid substitution (K) is shown in Figure 14. In Figure 14, the residual activity of the mutants in which either the amino acid substitution (Z) or (AA) was introduced is expressed as a relative value, with the residual activity of wild-type tdCA set to 1. Both tdCA mutants (m4-G23L and m4-G23I) in which either the amino acid substitution (Z) or (AA) was introduced showed higher residual activity than wild-type tdCA.

[0222] In summary, the above results indicate that the tdCA mutant (m4), which has improved thermostability due to the introduction of the amino acid substitution (K) described above, can be further improved by introducing either the amino acid substitution (Z) or (AA) described above. Furthermore, the mutants (m4-G23L and m4-G23I) with either the amino acid substitution (Z) or (AA) described above also show improved decarboxylation activity before heat treatment.

[0223] Example 17 Introduction of amino acid substitutions into carbonic anhydrase derived from Thermosulfurimonas dismutans (part 7) (1) By PCR using as a template the plasmid capable of expressing tdCA (m7) having the amino acid substitution (O) obtained in Example 7(2), polynucleotides encoding tdCA in which either the amino acid substitution (AB) or (AC) below was further added to m7 were prepared. (AB) Substitution of the glycine residue at position 4 of SEQ ID NO: 33 (m7) (position 23 of SEQ ID NO: 1) with a serine residue (hereinafter also referred to as G23S) (AC) Substitution of the glycine residue at position 4 of SEQ ID NO: 33 (m7) (position 23 of SEQ ID NO: 1) with an asparagine residue (hereinafter also referred to as G23N)

[0224] (2) Using the polynucleotide prepared in (1), a transformant was prepared by the method described in Example 1(2) and (3), and a plasmid capable of expressing the tdCA amino acid substitution was obtained. The base sequence of the site of amino acid substitution and its surrounding region in the plasmid was analyzed, and it was confirmed that each sequence was the desired sequence.

[0225] Table 10 shows the sequence numbers of the amino acid sequences of tdCA into which any of the amino acid substitutions described in (AB) or (AC) above has been introduced, and the sequence numbers of the base sequences encoding the tdCA.

[0226] [Table 10]

[0227] Example 18 Preparation of tdCA and evaluation of decarboxylation activity (part 8) (1) tdCA was prepared according to the method described in Example 2(1) to (6) using the plasmid capable of expressing tdCA having the amino acid substitutions described in either (AB) or (AC) prepared in Example 17.

[0228] (2) The tdCA protein contained in the fraction eluted in (1) was adjusted to a concentration of 8 μmol / L, dispensed into a PCR plate at 50 μL / well, and heated at 90°C for 60 minutes using a thermal cycler. After heating, the plate was kept at 4°C.

[0229] (3) The decarboxylation activity of the tdCA solution before heat treatment obtained in (1) or the heat-treated tdCA solution obtained in (2) was measured by the method described in Example 3.

[0230] (4) The residual activity was calculated by dividing the decarboxylation activity of the heat-treated tdCA measured in (3) by the decarboxylation activity of the tdCA before heat treatment.

[0231] The measurement results are shown in Figure 15. In Figure 15, the residual activity of the mutants (m7-G23S and m7-G23N) into which either the amino acid substitution (AB) or (AC) was introduced is expressed as a relative value, with the residual activity of a tdCA mutant (m7) into which no such mutation was introduced being set at 1. Both of the tdCA mutants (m7-G23S and m7-G23N) into which either the amino acid substitution (AB) or (AC) was introduced showed improved residual activity after heat treatment compared to the tdCA mutant (m7) into which no such mutation was introduced, indicating that the introduction of either the G23S or G23N mutation improved thermostability.

[0232] Example 19 Introduction of amino acid substitutions into carbonic anhydrase derived from Thermosulfurimonas dismutans (part 8) (1) A polynucleotide was synthesized by adding an oligonucleotide (CAT) for cleavage with the restriction enzyme NdeI to the 5' end of a polynucleotide (SEQ ID NO: 3) encoding a wild-type tdCA polypeptide (SEQ ID NO: 2) in which the amino acid residues from the second lysine to the 20th alanine, which correspond to the signal peptide, of the amino acid sequence of natural Thermosulfurimonas dismutans-derived carbonic anhydrase (hereinafter also referred to as tdCA) (GenBank No. OAQ21602, SEQ ID NO: 1) were removed, and an oligonucleotide (CAT) for cleavage with the restriction enzyme NdeI was added to the 5' end of the polynucleotide, and an oligonucleotide (SEQ ID NO: 4) encoding six histidine residues (histidine tag) and an oligonucleotide (TAAGCTT) corresponding to a stop codon and a restriction enzyme HindIII cleavage site were added to the 3' end of the polynucleotide.

[0233] (2) The wild-type tdCA expression plasmid pET-tdCA_WT was constructed by inserting the polynucleotide synthesized in (1) (previously digested with NdeI and HindIII) into the pET26b(+) plasmid vector (Novagen) previously digested with NdeI and HindIII.

[0234] (3) A plasmid for expressing tdCA amino acid substitutions was prepared by introducing nucleotide substitutions into the polynucleotide (SEQ ID NO: 3) encoding wild-type tdCA (SEQ ID NO: 2) in the pET-tdCA_WT prepared in (2). Specifically, the following amino acid substitutions were made based on information on the physicochemical properties (hydrophilicity / hydrophobicity, charge, bulkiness, etc.) of the amino acid residues in the N-terminal region of tdCA, which have a large effect on the expression level and solubility. from <h>Nucleotide substitutions were introduced to result in the amino acid substitutions described below. < / h> Substitution of glycine at the second position in SEQ ID NO: 2 (the 21st position in SEQ ID NO: 1) with proline (hereinafter also referred to as G21P). Substitution of glycine at position 3 of SEQ ID NO: 2 (position 22 of SEQ ID NO: 1) with alanine (hereinafter also referred to as G22A). <c>Substitution of glycine at position 3 of SEQ ID NO: 2 (position 22 of SEQ ID NO: 1) with serine (hereinafter also referred to as G22S). <d>Substitution of glycine at position 4 of SEQ ID NO: 2 (position 23 of SEQ ID NO: 1) with asparagine (hereinafter also referred to as G23N). <e>Substitution of glycine at position 4 of SEQ ID NO: 2 (position 23 of SEQ ID NO: 1) with serine (hereinafter also referred to as G23S). <f>Substitution of histidine at position 5 of SEQ ID NO: 2 (position 24 of SEQ ID NO: 1) with arginine (hereinafter also referred to as H24R). <g>Substitution of histidine at position 5 of SEQ ID NO: 2 (position 24 of SEQ ID NO: 1) with glutamic acid (hereinafter also referred to as H24E). <h>Substitution of valine at position 6 of SEQ ID NO: 2 (position 25 of SEQ ID NO: 1) with methionine (hereinafter also referred to as V25M).

[0235] The aforementioned< / h> < / g> < / f> < / e> < / d> < / c> from <h>The amino acid sequence of tdCA having the amino acid substitutions described above and the sequence number of the base sequence encoding said tdCA are shown in Table 11.

[0236] [Table 11]

[0237] Example 20 Evaluation of tdCA productivity (1) pET-tdCA_WT prepared in Example 1, or< / h> from <h>The tdCA-expressing E. coli (transformant) was prepared by transforming E. coli BL21(DE3) strain (Nippon Gene Co., Ltd.) with a plasmid capable of expressing tdCA having any of the amino acid substitutions described in any one of the above.

[0238] (2) The tdCA-expressing E. coli prepared in (1) was inoculated into test tubes containing 2 mL of antibiotic-containing 2xYT medium (16 g / L tryptone, 10 g / L yeast extract, 5 g / L sodium chloride), and then pre-cultured overnight at 37°C and 150 rpm with shaking.

[0239] (3) 1 mL of the preculture solution from (2) was inoculated into a baffled flask containing 100 mL of antibiotic-containing 2xYT medium and cultured at 37°C with shaking at 130 rpm for 3 hours. IPTG (isopropyl β-D-thiogalactopyranoside) was then added to a final concentration of 50 μmol / L, and the culture was further cultured at 25°C with shaking at 150 rpm for 24 hours to induce tdCA expression.

[0240] (4) The culture medium from (3) was centrifuged at 10,000 rpm at 4°C for 30 minutes, and the supernatant was removed to recover the wet bacterial cells. The recovered wet bacterial cells were stored at -30°C until use.

[0241] (5) Five mL of extraction buffer containing BugBuster Reagent (Merck) was added per gram of wet bacterial cells recovered in (4), and the mixture was stirred at 25°C and 150 rpm for 30 minutes. The mixture was then centrifuged at 4°C and 10,000 rpm for 30 minutes, and the supernatant was collected to prepare a bacterial cell extract.

[0242] (6) The bacterial extract prepared in (5) was sterilized using a filter with a pore size of 0.22 μm, and then loaded onto an open column packed with 1 mL of Ni-NTA agarose (Fujifilm Wako Pure Chemical Industries, Ltd.). After washing with 20 mL of a buffer solution containing 20 mmol / L of imidazole, the column was eluted with 6 mL of a buffer solution containing 300 mmol / L of imidazole.

[0243] (7) The purified tdCA contained in the fraction obtained in (6) was quantified using a NanoDrop microspectrophotometer (Thermo Fisher Scientific) to evaluate productivity (purification yield per culture medium).

[0244] The results are shown in Figure 16. In Figure 16, the purification yield per culture medium of each tdCA amino acid substitution is shown as a relative value divided by the purification yield per culture medium of wild-type tdCA (SEQ ID NO: 2). It can be seen that the introduction of the amino acid substitutions G23N (SEQ ID NO: 69) or G23S (SEQ ID NO: 71) improves the purification yield per culture medium by approximately 2.7-fold compared to wild-type tdCA. It can also be seen that the introduction of the amino acid substitutions G21P (SEQ ID NO: 63), G22S (SEQ ID NO: 67), or H24E (SEQ ID NO: 75) increases the purification yield per culture medium compared to wild-type tdCA.

[0245] Example 21 Evaluation of tdCA decarboxylation activity (part 9) The decarboxylation activity of each tdCA was evaluated based on the method described in WO2012 / 003277.

[0246] (1) The fraction containing purified tdCA obtained in Example 20(7) was appropriately diluted to a protein concentration of 8 μmol / L, and then 5 μL of the diluted fraction was dispensed into a 96-well microplate.

[0247] (2) A reaction solution was prepared based on the reaction solution composition shown in Table 12, and the pH change accompanying the progress of the decarboxylation reaction was detected by measuring the absorbance at a wavelength of 550 nm derived from phenolphthalein over time using a microplate reader. The absorbance measurement began 6 minutes after mixing the measurement reagents (start of the reaction) and continued thereafter every 90 seconds until 15 minutes after the start of the reaction (a total of 7 measurements).

[0248] [Table 12]

[0249] (3) A graph was created by plotting the results of (2) with the horizontal axis representing reaction time and the vertical axis representing absorbance, and the reaction rate (decarboxylation activity) was evaluated by calculating the slope of the graph for the reaction using each purified tdCA.

[0250] The results are shown in Figure 17. In Figure 17, the decarboxylation activity of each tdCA amino acid substitution mutant is shown as a relative value obtained by dividing it by the decarboxylation activity of wild-type tdCA (SEQ ID NO: 2). All of the tdCA amino acid substitution mutants had decarboxylation activity equivalent to that of wild-type tdCA, indicating that the activity was maintained even after the amino acid substitutions were introduced.

[0251] Example 22: Thermosulfurimonas dismutans derived carbon dioxide decarboxylation Introduction of amino acid substitutions into water enzymes (part 9) As in Example 19(3), a plasmid for expressing tdCA amino acid substitutions was prepared by introducing nucleotide substitutions at predetermined positions in a polynucleotide (SEQ ID NO: 3) encoding wild-type tdCA (SEQ ID NO: 2). Specifically, nucleotide substitutions were introduced so that the glycine residue at position 4 of SEQ ID NO: 2 (position 23 of SEQ ID NO: 1) was replaced with one of 17 amino acid residues, excluding asparagine and serine. The amino acid sequences of tdCA amino acid substitutions in which one of the 17 amino acid residues, i.e., alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, histidine, isoleucine, lysine, leucine, methionine, proline, glutamine, arginine, valine, tryptophan, tyrosine, or threonine, was substituted are shown in SEQ ID NOs: 79 to 95 in the order of appearance.

[0252] Example 23 Evaluation of tdCA productivity (part 2) and decarboxylation activity (part 10)

[0253] (1) Using the tdCA amino acid substitution expression plasmid prepared in Example 22, various tdCA amino acid substitutions in which the glycine residue at position 4 of SEQ ID NO: 2 (position 23 of SEQ ID NO: 1) was substituted were prepared according to the method described in Example 20(1) to (7).

[0254] (2) The purified tdCA contained in the fraction obtained in (1) was quantified using a NanoDrop microspectrophotometer (Thermo Fisher Scientific) to evaluate productivity (purification yield per culture medium).

[0255] (3) The decarboxylation activity of each tdCA amino acid substitution mutant prepared in (1) was evaluated according to the method described in Example 21. The protein concentration of the purified tdCA was changed to 20 μmol / L, and the final concentration of tdCA was changed to 0.001 mmol / L.

[0256] The results of productivity evaluation are shown in Figure 18. In Figure 18, the purification yield per culture medium of each tdCA amino acid substitution is shown as a relative value divided by the purification yield per culture medium of wild-type tdCA (SEQ ID NO: 2). Figure 18 shows that the purification yield per culture medium is improved by about 1.2-fold or more compared to wild-type tdCA for all amino acid substitutions except for the asparagine residue (G23N) and serine residue (G23S) substitutions shown in Example 20 and the threonine residue substitution (G23T). In particular, substitutions with alanine residue (G23A), aspartic acid residue (G23D), glutamic acid residue (G23E), valine residue (G23V), and tyrosine residue (G23Y) resulted in a purification yield per culture medium that was more than 1.6 times higher than that of wild-type tdCA, while substitutions with histidine residue (G23H), lysine residue (G23K), leucine residue (G23L), methionine residue (G23M), proline residue (G23P), glutamine residue (G23Q), arginine residue (G23R), and tryptophan residue (G23W) resulted in a purification yield that was more than twice that of wild-type tdCA.

[0257] The results of the evaluation of decarboxylation activity are shown in Figure 19. Figure 19 shows the relative values obtained by dividing the decarboxylation activity of each tdCA amino acid substitution by the decarboxylation activity of wild-type tdCA (SEQ ID NO: 2). All tdCA amino acid substitutions had decarboxylation activity equal to or greater than that of wild-type tdCA, demonstrating that this activity was maintained even after the amino acid substitutions. In particular, the substitution of the glycine residue at position 4 of SEQ ID NO: 2 (position 23 of SEQ ID NO: 1) with an aspartic acid residue (G23D), phenylalanine residue (G23F), isoleucine residue (G23I), leucine residue (G23L), methionine residue (G23M), proline residue (G23P), arginine residue (G23R), valine residue (G23V), tryptophan residue (G23W), or tyrosine residue (G23Y) resulted in decarboxylation activity that was 1.2-fold or more higher than that of wild-type tdCA. [Industrial Applicability]

[0258] The disclosed Thermosulfurimonas dismutans-derived carbonic anhydrase (tdCA) has improved stability and / or productivity compared to conventional tdCA. Therefore, it is expected that the use of this mutant carbonic anhydrase derived from Thermosulfurimonas dismutans will enable efficient treatment of carbon dioxide under high-temperature and / or alkaline conditions. Furthermore, it will enable industrial mass production of carbonic anhydrase.< / h>

Claims

1. A carbonic anhydrase selected from any of the following (i) to (iii): (i) A carbonic anhydrase having an amino acid sequence from the 21st glycine residue to the 249th lysine residue in the amino acid sequence set forth in SEQ ID NO: 1, which contains at least one or more amino acid substitutions selected from the following (1) to (22): (1) The amino acid residue corresponding to the lysine residue at position 33 of SEQ ID NO: 1 is substituted with a glutamic acid residue (2) The amino acid residue corresponding to the lysine residue at position 165 of SEQ ID NO: 1 is substituted with a glutamic acid residue (3) The amino acid residue corresponding to the methionine residue at position 182 of SEQ ID NO: 1 is substituted with a leucine residue. (4) The amino acid residue corresponding to the isoleucine residue at position 66 of SEQ ID NO: 1 is substituted with a valine residue. (5) The amino acid residue corresponding to the lysine residue at position 229 of SEQ ID NO: 1 is substituted with a glutamic acid residue (6) The amino acid residue corresponding to the methionine residue at position 231 of SEQ ID NO: 1 is substituted with an isoleucine residue (7) The amino acid residue corresponding to the lysine residue at position 173 of SEQ ID NO: 1 is substituted with an arginine residue. (8) The amino acid residue corresponding to the lysine residue at position 214 of SEQ ID NO: 1 is substituted with an arginine residue. (9) The amino acid residue corresponding to the lysine residue at position 246 of SEQ ID NO: 1 is substituted with an arginine residue. (10) The amino acid residue corresponding to the 26th valine residue in SEQ ID NO: 1 is substituted with an alanine residue (11) The amino acid residue corresponding to the 43rd alanine residue in SEQ ID NO: 1 is substituted with a valine residue (12) The amino acid residue corresponding to the lysine residue at position 226 of SEQ ID NO: 1 is substituted with an arginine residue (13) The amino acid residue corresponding to the 69th glutamine residue of SEQ ID NO: 1 is substituted with an arginine residue (14) The amino acid residue corresponding to the 103rd valine residue of SEQ ID NO: 1 is substituted with an isoleucine residue (15) The amino acid residue corresponding to the serine residue at position 63 of SEQ ID NO: 1 is substituted with a threonine residue (16) The amino acid residue corresponding to the 69th glutamine residue of SEQ ID NO: 1 is substituted with an aspartic acid residue (17) The amino acid residue corresponding to the glutamic acid residue at position 169 of SEQ ID NO: 1 is substituted with an aspartic acid residue (18) The amino acid residue corresponding to the histidine residue at position 170 of SEQ ID NO: 1 is substituted with a glutamic acid residue (19) The amino acid residue corresponding to the 23rd glycine residue of SEQ ID NO: 1 is substituted with any amino acid residue other than a glycine residue or a threonine residue. (20) The amino acid residue corresponding to the 21st glycine residue of SEQ ID NO: 1 is substituted with a proline residue (21) The amino acid residue corresponding to the 22nd glycine residue of SEQ ID NO: 1 is substituted with a serine residue (22) the amino acid residue corresponding to the histidine residue at position 24 of SEQ ID NO: 1 is substituted with a glutamic acid residue; (ii) an amino acid sequence comprising amino acid residues from the 21st glycine residue to the 249th lysine residue in the amino acid sequence set forth in SEQ ID NO: 1, which contains one or more amino acid substitutions selected from (1) to (22), and further comprising one or more substitutions, deletions, insertions, and additions of one or more amino acid residues at one or more positions in addition to the amino acid substitutions; and having decarboxylation activity and CO ２ carbonic anhydrase having at least one of the following hydration activities; (iii) an amino acid sequence from the 21st glycine residue to the 249th lysine residue in the amino acid sequence set forth in SEQ ID NO: 1, which has 70% or more identity to the entire amino acid sequence containing one or more amino acid substitutions selected from (1) to (22), provided that the amino acid sequence maintains the amino acid substitutions and has decarboxylation activity and CO ２ Carbonic anhydrase having at least one of the hydration activities.

2. The carbonic anhydrase of claim 1, selected from any one of the following (iv) to (vi): (iv) a carbonic anhydrase having an amino acid sequence from the 21st glycine residue to the 249th lysine residue in the amino acid sequence set forth in SEQ ID NO: 1, which contains the amino acid substitution of (1) above and further contains one or more amino acid substitutions selected from (2) to (14) and (19) above; (v) An amino acid sequence from the 21st glycine residue to the 249th lysine residue in the amino acid sequence set forth in SEQ ID NO: 1, which contains the amino acid substitution of (1), and further contains one or more amino acid substitutions selected from (2) to (14) and (19), and further contains any one or more of substitution, deletion, insertion, and addition of one or several amino acid residues at one or several positions other than the amino acid substitution, and which has decarboxylation activity and CO ２ carbonic anhydrase having at least one of the following hydration activities; (vi) An amino acid sequence from the 21st glycine residue to the 249th lysine residue in the amino acid sequence set forth in SEQ ID NO: 1, which contains the amino acid substitution of (1) above, and which has an identity of 70% or more to the entire amino acid sequence further containing one or more amino acid substitutions selected from (2) to (14) and (19), provided that the amino acid sequence maintains the amino acid substitution and has decarboxylation activity and CO ２ Carbonic anhydrase having at least one of the hydration activities.

3. A polynucleotide encoding the carbonic anhydrase of claim 1 or 2.

4. An expression vector comprising the polynucleotide of claim 3.

5. A transformant obtained by transforming a host with the expression vector according to claim 4.

6. The transformant according to claim 5, wherein the host is Escherichia coli.

7. A method for producing carbonic anhydrase, comprising the steps of: culturing the transformant according to claim 5 to express carbonic anhydrase; and recovering the expressed enzyme from the resulting culture.

8. A solution for separating and absorbing carbon dioxide, comprising the carbonic anhydrase according to claim 1 or 2.

9. A method for separating and capturing carbon dioxide, comprising: A method comprising the step of using the carbonic anhydrase of claim 1 or 2.

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