Composition for tungsten separation and its applications

Abstract

To provide separation means for tungsten (tungstic acid) with enhanced selectivity for tungsten (tungstic acid).SOLUTION: A tungsten separation composition comprises at least one polypeptide selected from a group comprising (A-1) to (A-4) and (B-1) to (B-2): (A-1) a polypeptide comprising an amino acid sequence of SEQ ID NO. 1; (A-2) a polypeptide comprising an amino acid sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO. 1; (A-3) a polypeptide comprising an amino acid sequence derived from the amino acid sequence of SEQ ID NO. 1 wherein the 176th glutamic acid is substituted with alanine; (A-4) a polypeptide comprising an amino acid sequence having at least 85% sequence identity to the amino acid sequence of the (A-3) polypeptide, while maintaining the substituted amino acids in the (A-3) polypeptide; (B-1) a polypeptide comprising the amino acid sequence of SEQ ID NO. 2; and (B-2) a polypeptide comprising an amino acid sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO. 2.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present disclosure relates to a composition for tungsten fractionation and its use. [Background technology]

[0002] Although rare metals such as molybdenum, tungsten, and vanadium are used in various fields, their distribution and usage are limited due to the relatively small amount present in the earth's crust and the high cost of mining and refining. However, some rare metals exist in the ocean as oxoacids, and the total amount in the ocean is overwhelmingly greater than the land reserves. Therefore, there is a demand for the development of a method to recover rare metals from the ocean.

[0003] Meanwhile, in recent years, the existence of a molybdenum storage protein (Molybdenum Storage Protein; Mosto) capable of storing molybdenum has been revealed (Non-Patent Document 1). Mosto, discovered in Azotobacter vinelandii, has a heterohexamer structure consisting of α and β subunits, with a large cage-like cavity in the center. It has been reported that molybdate is taken up into this cavity, and then stored in the cage in the form of a polyoxometalate formed by polymerization of molybdic acid. However, since A. vinelandii takes up both molybdenum and tungsten, which have similar properties, into its cells, it is problematic that they cannot be separated and recovered. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Proc Natl Acad Sci US A. 2019 Dec 26;116(52):26497-26504. Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present disclosure is to provide a means for separating tungsten (tungstic acid) having excellent tungsten (tungstic acid) selectivity. [Means for solving the problem]

[0006] The present inventors searched for homologues similar to Mosto derived from A. vinelandii, and focused on Mosto (Wsp) derived from Isosphaera pallida and Mosto derived from Desulfococcus multivorans. These Mosto were found to have high tungsten selectivity, and further improved them.

[0007] The present disclosure includes, for example, the subject matter described in the following sections: Section 1. A composition for tungsten fractionation, comprising at least one polypeptide selected from the group consisting of (A-1) to (A-4), (B-1) to (B-2), (D-1) to (D-2), and (E-1) to (E-2) below. (A-1) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 (A-2) A polypeptide having an amino acid sequence that has a sequence identity of 85% or more with the amino acid sequence of SEQ ID NO:1, and that forms a heteromultimer with a polypeptide having the amino acid sequence of SEQ ID NO:2 to exhibit tungsten uptake ability. (A-3) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 in which glutamic acid at position 176 is replaced with alanine. (A-4) A polypeptide comprising an amino acid sequence having 85% or more sequence identity with the amino acid sequence of the polypeptide of (A-3) while maintaining the substituted amino acids, and which forms a heteromultimer with a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 and exhibits the ability to incorporate tungsten. (B-1) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 (B-2) A polypeptide having an amino acid sequence that has a sequence identity of 85% or more with the amino acid sequence of SEQ ID NO:2, and that forms a heteromultimer with a polypeptide having the amino acid sequence of SEQ ID NO:1 to exhibit tungsten uptake ability. (D-1) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 17 (D-2) A polypeptide having an amino acid sequence having 85% or more sequence identity with the amino acid sequence of SEQ ID NO: 17, and forming a heteromultimer with a polypeptide having the amino acid sequence of SEQ ID NO: 18 to exhibit tungsten uptake ability. (E-1) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 18 (E-2) A polypeptide having an amino acid sequence having 85% or more sequence identity with the amino acid sequence of SEQ ID NO: 18, and forming a heteromultimer with a polypeptide having the amino acid sequence of SEQ ID NO: 17 to exhibit tungsten uptake ability. Section 2. A combination of at least one polypeptide selected from the group consisting of (A-1) to (A-4) and at least one polypeptide selected from the group consisting of (B-1) to (B-2), or Item 1, comprising a combination of at least one polypeptide selected from the group consisting of (D-1) to (D-2) and at least one polypeptide selected from the group consisting of (E-1) to (E-2). Section 3. Item 3. A composition for tungsten fractionation according to Item 1 or 2, wherein the 49th histidine in the amino acid sequence of SEQ ID NO: 1 is substituted with alanine. Section 4. A composition for tungsten fractionation comprising the following polypeptides (C-1) and / or (C-2). (C-1) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 3 (C-2) A polypeptide having an amino acid sequence having 85% or more sequence identity with the amino acid sequence of SEQ ID NO: 3, and capable of forming homopolymers and of incorporating tungsten. Section 5. A composition for tungsten fractionation, comprising a transformant in which at least one polypeptide selected from the group consisting of the following (A-1) to (A-4), (B-1) to (B-2), (D-1) to (D-2), and (E-1) to (E-2) is expressed. (A-1) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 (A-2) A polypeptide having an amino acid sequence that has a sequence identity of 85% or more with the amino acid sequence of SEQ ID NO:1, and that forms a heteromultimer with a polypeptide having the amino acid sequence of SEQ ID NO:2 to exhibit tungsten uptake ability. (A-3) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 in which glutamic acid at position 176 is replaced with alanine. (A-4) A polypeptide comprising an amino acid sequence having 85% or more sequence identity with the amino acid sequence of the polypeptide of (A-3) while maintaining the substituted amino acids, and which forms a heteromultimer with a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 and exhibits the ability to incorporate tungsten. (B-1) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 (B-2) A polypeptide having an amino acid sequence that has a sequence identity of 85% or more with the amino acid sequence of SEQ ID NO:2, and that forms a heteromultimer with a polypeptide having the amino acid sequence of SEQ ID NO:1 to exhibit tungsten uptake ability. (D-1) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 17 (D-2) A polypeptide having an amino acid sequence having 85% or more sequence identity with the amino acid sequence of SEQ ID NO: 17, and forming a heteromultimer with a polypeptide having the amino acid sequence of SEQ ID NO: 18 to exhibit tungsten uptake ability. (E-1) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 18 (E-2) A polypeptide having an amino acid sequence having 85% or more sequence identity with the amino acid sequence of SEQ ID NO: 18, and forming a heteromultimer with a polypeptide having the amino acid sequence of SEQ ID NO: 17 to exhibit tungsten uptake ability. Section 6. A transformant expressing at least one polypeptide selected from the group consisting of (A-1) to (A-4) and at least one polypeptide selected from the group consisting of (B-1) to (B-2), or Item 6. A composition for tungsten fractionation according to Item 5, comprising a transformant expressing at least one polypeptide selected from the group consisting of (D-1) to (D-2) and at least one polypeptide selected from the group consisting of (E-1) to (E-2). Section 7. Item 7. The composition for tungsten fractionation according to Item 5 or 6, wherein the 49th histidine in the amino acid sequence of SEQ ID NO: 1 is substituted with alanine. Section 8. A composition for tungsten fractionation comprising a transformant in which the following polypeptide (C-1) and / or (C-2) is expressed: (C-1) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 3 (C-2) A polypeptide having an amino acid sequence having 85% or more sequence identity with the amino acid sequence of SEQ ID NO: 3, and capable of forming homopolymers and of incorporating tungsten. Section 9. At least one polypeptide selected from the group consisting of (A-1) to (A-4), (B-1) to (B-2), (D-1) to (D-2), and (E-1) to (E-2) below, or A transformant expressing at least one polypeptide selected from the group consisting of the following (A-1) to (A-4), (B-1) to (B-2), (D-1) to (D-2), and (E-1) to (E-2); A method for fractionating tungsten, comprising the step of contacting a tungsten-containing aqueous solution with (A-1) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 (A-2) A polypeptide having an amino acid sequence that has a sequence identity of 85% or more with the amino acid sequence of SEQ ID NO:1, and that forms a heteromultimer with a polypeptide having the amino acid sequence of SEQ ID NO:2 to exhibit tungsten uptake ability. (A-3) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 in which glutamic acid at position 176 is replaced with alanine. (A-4) A polypeptide comprising an amino acid sequence having 85% or more sequence identity with the amino acid sequence of the polypeptide of (A-3) while maintaining the substituted amino acids, and which forms a heteromultimer with a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 and exhibits the ability to incorporate tungsten. (B-1) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 (B-2) A polypeptide having an amino acid sequence that has a sequence identity of 85% or more with the amino acid sequence of SEQ ID NO:2, and that forms a heteromultimer with a polypeptide having the amino acid sequence of SEQ ID NO:1 to exhibit tungsten uptake ability. (D-1) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 17 (D-2) A polypeptide having an amino acid sequence having 85% or more sequence identity with the amino acid sequence of SEQ ID NO: 17, and forming a heteromultimer with a polypeptide having the amino acid sequence of SEQ ID NO: 18 to exhibit tungsten uptake ability. (E-1) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 18 (E-2) A polypeptide having an amino acid sequence having 85% or more sequence identity with the amino acid sequence of SEQ ID NO: 18, and forming a heteromultimer with a polypeptide having the amino acid sequence of SEQ ID NO: 17 to exhibit tungsten uptake ability. Section 10. A combination of at least one polypeptide selected from the group consisting of (A-1) to (A-4) and at least one polypeptide selected from the group consisting of (B-1) to (B-2), or A combination of at least one polypeptide selected from the group consisting of (D-1) to (D-2) and at least one polypeptide selected from the group consisting of (E-1) to (E-2), or A transformant expressing at least one polypeptide selected from the group consisting of (A-1) to (A-4) and at least one polypeptide selected from the group consisting of (B-1) to (B-2), or A transformant expressing at least one polypeptide selected from the group consisting of (D-1) and (D-2) and at least one polypeptide selected from the group consisting of (E-1) and (E-2); Item 10. The method for fractionating tungsten according to item 9, comprising a step of contacting a tungsten-containing aqueous solution with the tungsten-containing aqueous solution. Section 11. Item 11. The method for separating tungsten according to Item 9 or 10, wherein the 49th histidine in the amino acid sequence of SEQ ID NO: 1 is substituted with alanine. Section 12. A polypeptide of the following (C-1) and / or (C-2): A method for fractionating tungsten, comprising the step of contacting a transformant in which the following polypeptide (C-1) and / or (C-2) is expressed with a tungsten-containing aqueous solution: (C-1) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 3 (C-2) A polypeptide having an amino acid sequence having 85% or more sequence identity with the amino acid sequence of SEQ ID NO: 3, and capable of forming homopolymers and of incorporating tungsten. Section 13. A nucleic acid consisting of at least one base sequence selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 21, and SEQ ID NO: 22, or A nucleic acid encoding a polypeptide having a base sequence having 85% or more sequence identity with the base sequence of SEQ ID NO: 15 and forming a heteromultimer with a polypeptide having the amino acid sequence of SEQ ID NO: 2 to exhibit tungsten uptake ability; A nucleic acid encoding a polypeptide having a base sequence having 85% or more sequence identity with the base sequence of SEQ ID NO:16 and forming a heteromultimer with a polypeptide having the amino acid sequence of SEQ ID NO:1 to exhibit tungsten uptake ability; A nucleic acid encoding a polypeptide having a base sequence having 85% or more sequence identity with the base sequence of SEQ ID NO: 21 and forming a heteromultimer with a polypeptide having the amino acid sequence of SEQ ID NO: 18 to exhibit tungsten uptake ability; and A nucleic acid encoding a polypeptide having a base sequence having 85% or more sequence identity with the base sequence of SEQ ID NO: 22 and forming a heteromultimer with a polypeptide having the amino acid sequence of SEQ ID NO: 17 to exhibit tungsten uptake ability; At least one nucleic acid selected from the group consisting of: Effect of the Invention

[0008] The present invention provides a composition for tungsten fractionation that has excellent tungsten selectivity, and also provides an efficient means for fractionating tungsten. [Brief description of the drawings]

[0009] [Figure 1] Schematic diagram of the expression construct of Mosto (WspA01 and WspB01) derived from I. pallida. [Diagram 2] An SDS-PAGE photograph of Mosto (Wsp) derived from I. pallida is shown. [Diagram 3] The results of gel filtration chromatography of Wsp are shown. [Figure 4] The alignment results of WspA (alpha) and AvMosto (beta) are shown. [Diagram 5] Schematic diagram of the expression construct of Mosto (dmMosto) derived from D. multivorans. [Figure 6] An SDS-PAGE photograph of dmMosto is shown. [Figure 7] The results of gel filtration chromatography of dmMosto are shown. [Figure 8] A molecular phylogenetic tree of Mosto derived from various organisms is shown. [Figure 9]The results of SDS-PAGE performed in Test 3 are shown. In the figure, from the left, the lanes are 1: WspAB01 soluble fraction, 2: WspAB01 insoluble fraction, 3: WspAB01' soluble fraction, 4: WspAB01' insoluble fraction, and M: molecular weight marker. The band of the target protein is indicated by an arrow. [Figure 10] The results of SDS-PAGE performed in Test 3 are shown. In the figure, from the left, lanes are 1: WspAB02 soluble fraction, 2: WspAB02 insoluble fraction, and M: molecular weight marker. The band of the target protein is indicated by an arrow. [Figure 11] The amino acid sequence of SEQ ID NO:1 is shown. [Figure 12] The amino acid sequence of SEQ ID NO:2 is shown. [Figure 13] The amino acid sequence of SEQ ID NO:3 is shown. [Figure 14] The base sequence of SEQ ID NO:4 is shown. [Figure 15] The base sequence of SEQ ID NO:5 is shown. [Figure 16] The base sequence of SEQ ID NO:12 is shown. [Figure 17] The base sequence of SEQ ID NO:15 is shown. [Figure 18] The base sequence of SEQ ID NO:16 is shown. [Figure 19] The amino acid sequence of SEQ ID NO:17 is shown. [Figure 20] The amino acid sequence of SEQ ID NO:18 is shown. [Figure 21] The base sequence of SEQ ID NO:19 is shown. [Figure 22] The base sequence of SEQ ID NO:20 is shown. [Figure 23] The base sequence of SEQ ID NO:21 is shown. [Figure 24] The base sequence of SEQ ID NO:22 is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] Each embodiment included in the present disclosure will be described in further detail below.

[0011] The tungsten fractionation composition included in the present disclosure preferably contains at least one polypeptide selected from the group consisting of (A-1) to (A-4), (B-1) to (B-2), (D-1) to (D-2), and (E-1) to (E-2) described below. The tungsten fractionation composition included in the present disclosure preferably contains the polypeptides (C-1) and / or (C-2) described below.

[0012] In this specification, these compositions may be referred to as "tungsten fractionation compositions of the present disclosure."

[0013] In addition, in the present specification, each polypeptide may be referred to as "polypeptide (A-1)", "polypeptide (A-2)", "polypeptide (A-3)", "polypeptide (A-4)", "polypeptide (B-1)", "polypeptide (B-2)", "polypeptide (C-1)", "polypeptide (C-2)", "polypeptide (D-1)", "polypeptide (D-2)", "polypeptide (E-1)", "polypeptide (E-2)", etc. These polypeptides may be collectively referred to as "polypeptides of the present disclosure". In the present disclosure, the term "polypeptide" includes proteins.

[0014] Most (Wsp) from I. pallida is predicted to form a heterohexamer composed of WspA (α subunit) and WspB (β subunit). In this disclosure, the α subunit of Most from I. pallida is sometimes referred to as WspA01, and the β subunit of Most from I. pallida is sometimes referred to as WspB01.

[0015] Mosto from D. multivorans is predicted to form a homohexamer composed of a single subunit.

[0016] The polypeptide (A-1) is a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1. The amino acid sequence of SEQ ID NO: 1 is an amino acid sequence constituting WspA01 (α subunit) of Mosto derived from I. pallida. The amino acid sequence of SEQ ID NO: 1 is shown in FIG.

[0017] The polypeptide (A-2) is a polypeptide consisting of an amino acid sequence having a sequence identity of 85% or more with the amino acid sequence of SEQ ID NO: 1. The sequence identity may be, for example, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. It is preferable that the polypeptide (A-2) forms a heteromultimer with the polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 to exhibit tungsten uptake ability.

[0018] In addition, the fact that a certain polypeptide forms a heteromultimer with another polypeptide and exerts tungsten uptake ability can be confirmed by a known method or a method that can be easily conceived from a known method. For example, the fact that a certain polypeptide forms a heteromultimer with another polypeptide can be confirmed by analyzing the molecular size of the polypeptide complex by performing gel filtration chromatography on a sample containing both polypeptides after grasping the molecular weight of each polypeptide from SDS-PAGE or amino acid sequence, etc. In addition, the fact that a certain polypeptide forms a heteromultimer with another polypeptide can be predicted from the homology with a known polypeptide, and can also be predicted using a commonly available polypeptide structure prediction program such as AlphaFold.

[0019] The tungsten uptake ability can be confirmed, for example, by reacting Na2WO4 with the polypeptide complex, removing free tungstic acid, and then detecting tungstic acid in a solution containing the polypeptide complex by inductively coupled plasma atomic emission spectrometry (ICP-AES).

[0020] Amino acid sequence identity can be calculated using the National Center for Biotechnology Information (NCBI) homology algorithm BLAST (Basic local alignment search tool) http: / / www.ncbi.nlm.nih.gov / BLAST / with default parameters.

[0021] The polypeptide (A-2) may be a polypeptide consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 1. The number of deleted, substituted, or added amino acids is not particularly limited as long as the effect of the present disclosure is achieved. The upper or lower limit of the range may be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50. Specifically, it may be 1 to 50, or 1 to 45.

[0022] The substitution may be a conservative substitution of an amino acid residue. In this specification, the term "conservative substitution" means replacing an amino acid with an amino acid having a side chain with similar properties to the side chain of the amino acid. Specific examples of conservative substitution include substitution of amino acid residues having basic side chains such as lysine, arginine, and histidine; substitution of amino acid residues having acidic side chains such as aspartic acid and glutamic acid; substitution of amino acid residues having non-charged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; substitution of amino acid residues having non-polar side chains such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; substitution of amino acid residues having β-branched side chains such as threonine, valine, and isoleucine; substitution of amino acid residues having aromatic side chains such as tyrosine, phenylalanine, tryptophan, and histidine; and the like.

[0023] Techniques for introducing mutations such as deletion, substitution, or addition of amino acids into a specific amino acid sequence are known in the art and can be carried out by any method, such as restriction enzyme treatment, treatment with exonuclease or DNA ligase, site-directed mutagenesis, random mutagenesis, etc.

[0024] The polypeptide of (A-2) may conserve glycine at position 86, arginine at position 90, tyrosine at position 93, methionine at position 156, or arginine at position 175 of the amino acid sequence of SEQ ID NO: 1. It is presumed that these amino acid residues may form a gate into a protein cage (a cavity formed by the multimerization of polypeptides). Only one, two, three, or all four of these amino acid residues may be conserved.

[0025] In addition, in the polypeptide of (A-2), the histidine at position 49 in the amino acid sequence of SEQ ID NO: 1 may or may not be conserved. It is speculated that this amino acid residue may form a gate into a protein cage (a cavity formed by the multimerization of polypeptides).

[0026] For example, the polypeptide (A-2) may have the 49th histidine in the amino acid sequence of SEQ ID NO:1 substituted with alanine.

[0027] The polypeptide (A-3) is a polypeptide consisting of the amino acid sequence of SEQ ID NO:1 in which glutamic acid at position 176 has been replaced with alanine.

[0028] The polypeptide (A-4) is a polypeptide consisting of an amino acid sequence having a sequence identity of 85% or more with the amino acid sequence of the polypeptide (A-3) while maintaining the substituted amino acids in the polypeptide (A-3). The sequence identity may be, for example, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. It is preferable that the polypeptide (A-4) forms a heteromultimer with the polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 to exhibit tungsten uptake ability.

[0029] The polypeptide (A-4) may be a polypeptide consisting of an amino acid sequence in which one or two or more amino acids are deleted, substituted, or added in the polypeptide (A-3) while maintaining the substituted amino acids in the polypeptide (A-3). The number of deleted, substituted, or added amino acids is not particularly limited as long as the effect of the present disclosure is exhibited. The upper or lower limit of the range may be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50. Specifically, it may be 1 to 50, or 1 to 45. The substitution may be a conservative substitution of amino acid residues.

[0030] The polypeptide of (A-4) may conserve glycine at position 86, arginine at position 90, tyrosine at position 93, methionine at position 156, or arginine at position 175 of the amino acid sequence of SEQ ID NO: 1. It is presumed that these amino acid residues may form a gate into a protein cage (a cavity formed by the multimerization of polypeptides). Only one, two, three, or all four of these amino acid residues may be conserved.

[0031] In addition, in the polypeptide of (A-4), the histidine at position 49 in the amino acid sequence of SEQ ID NO: 1 may or may not be conserved. It is speculated that this amino acid residue may form a gate into a protein cage (a cavity formed by the multimerization of polypeptides).

[0032] For example, the polypeptide (A-4) may have the 49th histidine in the amino acid sequence of SEQ ID NO:1 substituted with alanine.

[0033] The polypeptide (B-1) is a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2. The amino acid sequence shown in SEQ ID NO: 2 is the amino acid sequence constituting WspB (β subunit) of Mosto derived from I. pallida. The amino acid sequence of SEQ ID NO: 2 is shown in FIG.

[0034] The polypeptide (B-2) is a polypeptide consisting of an amino acid sequence having a sequence identity of 85% or more with the amino acid sequence of SEQ ID NO: 2. The sequence identity may be, for example, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. It is preferable that the polypeptide (B-2) forms a heteromultimer with the polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 to exhibit tungsten uptake ability.

[0035] The polypeptide (B-2) may be a polypeptide consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 2. The number of deleted, substituted, or added amino acids is not particularly limited as long as the effect of the present disclosure is achieved. The upper or lower limit of the range may be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50. Specifically, it may be 1 to 50, or 1 to 45. The substitution may be a conservative substitution of amino acid residues.

[0036] In the polypeptide of (B-2), the 45th lysine, the 82nd glycine, the 86th arginine, the 89th tyrosine, the 152nd methionine, the 171st arginine, or the 172nd threonine in the amino acid sequence of SEQ ID NO: 2 may or may not be conserved. It is presumed that these amino acid residues may form a gate into a protein cage (a cavity formed by the multimerization of polypeptides). Only one of these amino acid residues may be conserved, two may be conserved, three may be conserved, four may be conserved, five may be conserved, or all six may be conserved.

[0037] For example, the polypeptide (B-2) may have the 45th lysine in the amino acid sequence of SEQ ID NO: 2 substituted with alanine. For example, the polypeptide (B-2) may have the 172nd threonine in the amino acid sequence of SEQ ID NO: 2 substituted with alanine.

[0038] Meanwhile, the present inventors performed molecular phylogenetic analysis of Most derived from various organisms and classified Most into three groups, Type I to III. Among these three groups, it was found that Most belonging to Type II may have particularly high tungsten selectivity (FIG. 8). WspA (α subunit) and WspB (β subunit) derived from I. pallida are Most belonging to Type II. However, in the course of further investigation, it was revealed that Most belonging to Type II has a relatively low polypeptide expression efficiency. Therefore, the present inventors attempted to identify Most belonging to Type II and having a high polypeptide expression efficiency. As a result of the investigation, it was revealed that Most derived from Planctomycetota bacterium, which belongs to Type II, has a relatively high polypeptide expression efficiency. It is presumed that Most derived from Planctomycetota bacterium, which belongs to Type II, forms a heterohexamer composed of α subunits and β subunits, similar to Most derived from I. pallida. In the present disclosure, the α subunit of Mosto derived from Planctomycetota bacterium may be referred to as WspA02, and the β subunit of Mosto derived from Planctomycetota bacterium may be referred to as WspB02.

[0039] The polypeptide (D-1) is a polypeptide consisting of the amino acid sequence of SEQ ID NO: 17. The amino acid sequence of SEQ ID NO: 17 is an amino acid sequence constituting WspA02 (α subunit) of Mosto derived from Planctomycetota bacterium. The amino acid sequence of SEQ ID NO: 17 is shown in FIG.

[0040] The polypeptide (D-2) is a polypeptide consisting of an amino acid sequence having a sequence identity of 85% or more with the amino acid sequence of SEQ ID NO: 17. The sequence identity may be, for example, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. It is preferable that the polypeptide (D-2) forms a heteromultimer with the polypeptide consisting of the amino acid sequence of SEQ ID NO: 18 to exhibit tungsten uptake ability.

[0041] The polypeptide (D-2) may be a polypeptide consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 17. The number of deleted, substituted, or added amino acids is not particularly limited as long as the effect of the present disclosure is exhibited. The upper or lower limit of the range may be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50. Specifically, it may be 1 to 50, or 1 to 45.

[0042] The (E-1) polypeptide is a polypeptide consisting of the amino acid sequence of SEQ ID NO: 18. The amino acid sequence of SEQ ID NO: 18 is an amino acid sequence constituting WspB02 (β subunit) of Most derived from Planctomycetota bacterium. The amino acid sequence of SEQ ID NO: 18 is shown in FIG.

[0043] The polypeptide (E-2) is a polypeptide consisting of an amino acid sequence having a sequence identity of 85% or more with the amino acid sequence of SEQ ID NO: 18. The sequence identity may be, for example, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. It is preferable that the polypeptide (E-2) forms a heteromultimer with the polypeptide consisting of the amino acid sequence of SEQ ID NO: 17 to exhibit tungsten uptake ability.

[0044] The polypeptide (E-2) may be a polypeptide consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 18. The number of deleted, substituted, or added amino acids is not particularly limited as long as the effect of the present disclosure is achieved. The upper or lower limit of the range may be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50. Specifically, it may be 1 to 50, or 1 to 45.

[0045] The tungsten fractionation composition of the present disclosure preferably contains at least one polypeptide selected from the group consisting of (A-1) to (A-4) in combination with at least one polypeptide selected from the group consisting of (B-1) to (B-2), or at least one polypeptide selected from the group consisting of (D-1) to (D-2) in combination with at least one polypeptide selected from the group consisting of (E-1) to (E-2), and more preferably contains at least one polypeptide selected from the group consisting of (D-1) to (D-2) in combination with at least one polypeptide selected from the group consisting of (E-1) to (E-2). The types of polypeptides contained may be two, three, four, five, or six.

[0046] In the case of two types, the combination may be, for example, a polypeptide (A-1) and a polypeptide (B-1), a polypeptide (A-1) and a polypeptide (B-2), a polypeptide (A-2) and a polypeptide (B-1), a polypeptide (A-2) and a polypeptide (B-2), a polypeptide (A-3) and a polypeptide (B-1), a polypeptide (A-3) and a polypeptide (B-2), a polypeptide (A-4) and a polypeptide (B-1), or a polypeptide (A-4) and a polypeptide (B-2). Among them, it is preferable to include a polypeptide (A-1) and a polypeptide (B-1), or a polypeptide (A-3) and a polypeptide (B-1). In addition, the tungsten fractionation composition of the present disclosure may include, for example, a polypeptide (A-1), a polypeptide (A-3), and a polypeptide (B-1).

[0047] Furthermore, the tungsten fractionation composition of the present disclosure more preferably comprises a combination of the polypeptide (D-1) and the polypeptide (E-1), the polypeptide (D-1) and the polypeptide (E-2), the polypeptide (D-2) and the polypeptide (E-1), or the polypeptide (D-2) and the polypeptide (E-2).

[0048] The polypeptide (C-1) is a polypeptide consisting of the amino acid sequence of SEQ ID NO: 3. The amino acid sequence of SEQ ID NO: 3 is an amino acid sequence constituting Mosto derived from D. multivorans. The amino acid sequence of SEQ ID NO: 3 is shown in FIG.

[0049] The polypeptide (C-2) is a polypeptide consisting of an amino acid sequence having a sequence identity of 85% or more with the amino acid sequence of SEQ ID NO: 3. The sequence identity may be, for example, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. It is preferable that the polypeptide (C-2) forms a homomultimer to exhibit tungsten uptake ability.

[0050] The polypeptide (C-2) may be a polypeptide consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 3. The number of deleted, substituted, or added amino acids is not particularly limited as long as the effect of the present disclosure is achieved. The upper or lower limit of the range may be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50. Specifically, it may be 1 to 50, or 1 to 45. The substitution may be a conservative substitution of amino acid residues.

[0051] The polypeptide of (C-2) may have conserved lysine at position 50, glycine at position 87, arginine at position 91, tyrosine at position 94, isoleucine at position 158, arginine at position 177, or threonine at position 178 in the amino acid sequence of SEQ ID NO: 3. It is presumed that these amino acid residues may form a gate into a protein cage (a cavity formed by the multimerization of polypeptides). Only one of these amino acid residues may be conserved, two of them may be conserved, three of them may be conserved, four of them may be conserved, five of them may be conserved, or all six of them may be conserved.

[0052] The composition for tungsten fractionation of the present disclosure may contain either the polypeptide (C-1) or the polypeptide (C-2) alone or both.

[0053] The polypeptide of the present disclosure can be produced by genetic engineering techniques using a polynucleotide encoding the polypeptide of the present disclosure. The base sequence of the polynucleotide encoding the polypeptide of the present disclosure is not particularly limited. For example, SEQ ID NO: 4 and SEQ ID NO: 15 are examples of base sequences encoding the amino acid sequence of SEQ ID NO: 1. SEQ ID NO: 5 and SEQ ID NO: 16 are examples of base sequences encoding the amino acid sequence of SEQ ID NO: 2. SEQ ID NO: 12 is an example of a base sequence encoding the amino acid sequence of SEQ ID NO: 3. SEQ ID NO: 19 and SEQ ID NO: 21 are examples of base sequences encoding the amino acid sequence of SEQ ID NO: 17. SEQ ID NO: 20 and SEQ ID NO: 22 are examples of base sequences encoding the amino acid sequence of SEQ ID NO: 18. The base sequence of the polynucleotide encoding the polypeptide of the present disclosure preferably has a base sequence having a certain level of identity with at least one base sequence selected from the group consisting of SEQ ID NOs: 4, 5, 12, 15, 16, and 19 to 22. The certain level of identity is, for example, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0054] The base sequence of SEQ ID NO:4 is shown in Figure 14. The base sequence of SEQ ID NO:5 is shown in Figure 15. The base sequence of SEQ ID NO:12 is shown in Figure 16. The base sequence of SEQ ID NO:15 is shown in Figure 17. The base sequence of SEQ ID NO:16 is shown in Figure 18. The base sequence of SEQ ID NO:17 is shown in Figure 19. The base sequence of SEQ ID NO:18 is shown in Figure 20. The base sequence of SEQ ID NO:19 is shown in Figure 21. The base sequence of SEQ ID NO:20 is shown in Figure 22. The base sequence of SEQ ID NO:21 is shown in Figure 23. The base sequence of SEQ ID NO:22 is shown in Figure 24.

[0055] The present disclosure relates to a nucleic acid comprising at least one base sequence selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 21, and SEQ ID NO: 22, and A nucleic acid encoding a polypeptide having a base sequence having 85% or more sequence identity with the base sequence of SEQ ID NO: 15 and forming a heteromultimer with a polypeptide having the amino acid sequence of SEQ ID NO: 2 to exhibit tungsten uptake ability; A nucleic acid encoding a polypeptide having a base sequence having 85% or more sequence identity with the base sequence of SEQ ID NO:16 and forming a heteromultimer with a polypeptide having the amino acid sequence of SEQ ID NO:1 to exhibit tungsten uptake ability; A nucleic acid encoding a polypeptide having a base sequence having 85% or more sequence identity with the base sequence of SEQ ID NO: 21 and forming a heteromultimer with a polypeptide having the amino acid sequence of SEQ ID NO: 18 to exhibit tungsten uptake ability; and A nucleic acid encoding a polypeptide having a base sequence having 85% or more sequence identity with the base sequence of SEQ ID NO: 22 and forming a heteromultimer with a polypeptide having the amino acid sequence of SEQ ID NO: 17 to exhibit tungsten uptake ability; The sequence identity may be, for example, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0056] The nucleotide sequence of SEQ ID NO: 15 encodes the same amino acid sequence (SEQ ID NO: 1) as the nucleotide sequence of SEQ ID NO: 4, but has improved polypeptide expression efficiency compared to SEQ ID NO: 4. The nucleotide sequence of SEQ ID NO: 16 encodes the same amino acid sequence (SEQ ID NO: 1) as the nucleotide sequence of SEQ ID NO: 5, but has improved polypeptide expression efficiency compared to SEQ ID NO: 5. The nucleotide sequence of SEQ ID NO: 21 encodes the same amino acid sequence (SEQ ID NO: 17) as the nucleotide sequence of SEQ ID NO: 19, but has improved polypeptide expression efficiency compared to SEQ ID NO: 19. The nucleotide sequence of SEQ ID NO: 22 encodes the same amino acid sequence (SEQ ID NO: 18) as the nucleotide sequence of SEQ ID NO: 20, but has improved polypeptide expression efficiency compared to SEQ ID NO: 20.

[0057] The identity of nucleotide sequences can be calculated using an analytical tool that is commercially available or available via an electric communication line (Internet), for example, software such as FASTA, BLAST, PSI-BLAST, SSEARCH, etc. Specifically, the main initial conditions generally used for BLAST searches are as follows: In Advanced BLAST 2.1, the blastn program is used to perform a search with various parameters set to default values, and the identity value (%) of nucleotide sequences can be calculated.

[0058] In addition, the polypeptides of the present disclosure can also be produced using general protein chemical synthesis methods (e.g., liquid phase and solid phase methods) based on the information of the amino acid sequences shown in SEQ ID NOs: 1 to 3, and SEQ ID NOs: 17 and 18.

[0059] The polypeptide of the present disclosure may also include a tag, such as maltose binding protein (MBP), glutathione-S-transferase (GST), thioredoxin (TRX), SUMO, NusA, ubiquitin, polyhistidine, and the like.

[0060] The method for adding the tag is not particularly limited, and conventional methods and reaction conditions can be used.

[0061] In the tungsten fractionation composition of the present disclosure, the polypeptide of the present disclosure is preferably in a refolded state. The refolding method is not particularly limited, and a known method can be adopted. For example, a method of solubilizing the polypeptide in an aqueous solution containing a protein denaturant, diluting or dialysis, etc. can be mentioned. After solubilization, a chaperone protein can be added as necessary.

[0062] Examples of protein denaturants include urea, guanidine hydrochloride, etc. These may be used alone or in combination of two or more.

[0063] Examples of chaperone proteins include groESL, dnaK, dnaJ, clpB, grpE, IbpA, IbpB, trigger factor, etc. These may be used alone or in combination of two or more.

[0064] The content of the polypeptide of the present disclosure in the composition for tungsten fractionation of the present disclosure is not particularly limited and can be appropriately set, for example, to about 1 to 99.99 mass %.

[0065] The composition for tungsten fractionation included in the present disclosure preferably contains a transformant in which at least one polypeptide selected from the group consisting of the above-mentioned (A-1) to (A-4), (B-1) to (B-2), (D-1) to (D-2), and (E-1) to (E-2) is expressed.

[0066] Furthermore, the composition for tungsten fractionation included in the present disclosure preferably contains a transformant in which the above-mentioned polypeptides (C-1) and / or (C-2) are expressed.

[0067] In this specification, these compositions are also included in the "tungsten fractionation composition of the present disclosure."

[0068] The host cell used for transformation is not particularly limited as long as it can produce the polypeptide of the present disclosure, and may be either a prokaryotic cell or a eukaryotic cell. Specifically, prokaryotic cells such as Escherichia bacteria (e.g., BL21(DE3), Rosetta(DE3), HB101, MC1061, JM109, CJ236, MV1184, etc.), coryneform bacteria such as Corynebacterium glutamicum, actinomycetes such as Streptomyces bacteria, Bacillus bacteria such as Bacillus subtilis, Streptococcus bacteria, Staphylococcus bacteria, etc., yeasts such as Saccharomyces, Piscia, and Kluyveromyces, fungal cells such as Aspergillus, Penicillium, Talaromyces, Trichoderma, Hypocrea, and Acremonium, etc. can be mentioned.

[0069] Furthermore, the polypeptide of the present disclosure can be produced in the medium by utilizing the protein secretion ability of Bacillus subtilis, yeast, fungi, actinomycetes, and the like.

[0070] The transformant is preferably a vector incorporating a polynucleotide encoding a polypeptide of the present disclosure. The vector preferably contains the polynucleotide encoding the polypeptide of the present disclosure in an expressible manner. In the transformant, the vector may exist autonomously in the host cell, or may exist by being integrated into the genome via homologous recombination or non-homologous recombination.

[0071] Furthermore, a polynucleotide encoding a tag may be added to the polynucleotide encoding the polypeptide of the present disclosure, such as maltose binding protein (MBP), glutathione-S-transferase (GST), thioredoxin (TRX), SUMO, NusA, ubiquitin, polyhistidine, etc.

[0072] The type of vector used in the present disclosure can be appropriately selected in consideration of the type of host cell, and examples thereof include plasmid vectors, cosmid vectors, phage vectors, and viral vectors.

[0073] Examples of vectors that can be expressed in E. coli include pRSFDuet-1, pBB540, pBB550, pET, pUC19, pUC18, pBR322, pHSG299, pHSG298, pHSG399, pHSG398, RSF1010, pMW119, pMW118, pMW219, pMW218, pQE, etc. Examples of vectors that can be expressed in yeast include pBR322, pJDB207, pSH15, pSH19, pYepSec1, pMFa, pYES2, pHIL, pPIC, pAO815, pPink, etc.

[0074] When eukaryotic cells are used as host cells, the expression vector to be used may contain a promoter, an RNA splice site, a polyadenylation site, a transcription termination sequence, etc., upstream of the polynucleotide to be expressed, and may further contain a replication origin, a secretion signal, an enhancer, and / or a selection marker, as necessary.

[0075] The transformant may express a chaperone protein.

[0076] For example, a vector incorporating a gene encoding a chaperone protein may be introduced into the transformant. The vector preferably contains the gene encoding the chaperone protein in an expressible manner. In the transformant, the vector may exist autonomously in the host cell, or may be incorporated into the genome by homologous recombination or non-homologous recombination. The gene encoding the chaperone protein may be incorporated into a vector incorporating a polynucleotide encoding the polypeptide of the present disclosure, or may be incorporated into another vector.

[0077] Examples of chaperone proteins include groESL, dnaK, dnaJ, clpB, grpE, IbpA, IbpB, and trigger factor. These may be used alone or in combination of two or more. Genes encoding these chaperone proteins may be incorporated into the same vector or different vectors.

[0078] For example, the transformant may be introduced with a vector incorporating the groESL, dnaK, and dnaJ genes, and a vector incorporating the clpB and grpE genes.

[0079] The transformant may express a tungstate uptake protein.

[0080] For example, the transformant may be introduced with a vector incorporating a gene expressing a tungstic acid uptake protein. The vector preferably contains a gene expressible for expressing the tungstic acid uptake protein. In addition, in the transformant, the vector may exist autonomously in the host cell, or may be incorporated into the genome by homologous recombination or non-homologous recombination. In addition, the gene expressing the tungstic acid uptake protein may be incorporated into a vector incorporating a polynucleotide encoding the polypeptide of the present disclosure, or may be incorporated into another vector.

[0081] Examples of genes expressing proteins that can take up tungstic acid include modA, modB, modC, modF, wtpA, wtpB, wtpC, tupA, tupB, tupC, vupA, vupB, vupC, mot1, mot2, and mfsd5. These can be used alone or in combination of two or more. Furthermore, these genes expressing proteins that can take up tungstic acid can be incorporated into the same vector or different vectors.

[0082] The expression vector can be introduced into a host cell by a conventional method, including, but not limited to, the competent cell method, the protoplast method, the electroporation method, the microinjection method, the liposome fusion method, and the like.

[0083] The transformant preferably expresses at least one polypeptide selected from the group consisting of (A-1) to (A-4) and at least one polypeptide selected from the group consisting of (B-1) to (B-2), or at least one polypeptide selected from the group consisting of (D-1) to (D-2) and at least one polypeptide selected from the group consisting of (E-1) to (E-2). The number of types of polypeptides to be expressed may be two, three, four, five, six, or more. These polypeptides may be incorporated into the same vector or different vectors.

[0084] When two types of polypeptides are expressed, the combinations include, for example, the (A-1) polypeptide and the (B-1) polypeptide, the (A-1) polypeptide and the (B-2) polypeptide, the (A-2) polypeptide and the (B-1) polypeptide, the (A-2) polypeptide and the (B-2) polypeptide, the (A-3) polypeptide and the (B-1) polypeptide, the (A-3) polypeptide and the (B-2) polypeptide, the (A-4) polypeptide and the (B-1) polypeptide, or the (A-4) polypeptide and the (B-2) polypeptide, etc. Among them, it is preferable to express the combination of the (A-1) polypeptide and the (B-1) polypeptide, or the (A-3) polypeptide and the (B-1) polypeptide.

[0085] The transformant may also express, for example, the polypeptide (A-1), the polypeptide (A-3), and the polypeptide (B-1).

[0086] Furthermore, it is more preferable that the transformant expresses a combination of the (D-1) polypeptide and the (E-1) polypeptide, the (D-1) polypeptide and the (E-2) polypeptide, the (D-2) polypeptide and the (E-1) polypeptide, or the (D-2) polypeptide and the (E-2) polypeptide.

[0087] The transformant may express either the polypeptide (C-1) or the polypeptide (C-2) alone or both of them.

[0088] The content of the transformant expressing the polypeptide of the present disclosure in the composition for tungsten fractionation of the present disclosure is not particularly limited and can be appropriately set, for example, to about 1 to 99.99 mass%.

[0089] The tungsten fractionation composition of the present disclosure may contain other components as long as the tungsten (tungstic acid) retention ability is not impaired.

[0090] Examples of other components include solvents such as water, ATP, condensed phosphates (e.g., pyrophosphate, polyphosphate, etc.), magnesium salts (e.g., magnesium chloride, magnesium sulfate, magnesium nitrate, magnesium carbonate, magnesium acetate, etc.), chaperone proteins (e.g., GroESL, DnaK, DnaJ, ClpB, GrpE, IbpA, IbpB, trigger factor, etc.), dispersion media (e.g., glycerol, DMSO, etc.), buffer solutions, preservatives, excipients, activators, antiseptics, pH adjusters, stabilizers, etc. These may be used alone or in combination of two or more.

[0091] The tungsten fractionation composition of the present disclosure may be in a solid form (e.g., powder, granules, gel, paste, etc.) or in a liquid form (e.g., solution, suspension, etc.).

[0092] The tungsten fractionation composition of the present disclosure can be prepared by a conventional method by combining the polypeptide of the present disclosure or a transformant in which the polypeptide of the present disclosure is expressed, and other components as necessary.

[0093] As shown in the following examples, it is presumed that the tungsten fractionation composition of the present disclosure can form a multimer (homomultimer or heteromultimer) of polypeptide and specifically retain tungsten (tungstic acid) at its center.In other words, the tungsten fractionation composition of the present disclosure can have tungsten (tungstic acid) retention ability.

[0094] Examples of the multimer include 4-10 mers etc. For example, it may be a pentamer, hexamer, heptamer, octamer, or nonamer.

[0095] In addition, in this specification, "specific" means that the ability to retain tungsten is high compared with early transition metals other than tungsten, such as molybdenum and vanadium. Therefore, tungsten (tungstic acid) can be separated by contacting the tungsten fractionation composition of the present disclosure with, for example, an aqueous solution containing tungsten (tungstate salt). In other words, it is possible to specifically recover or remove tungsten (tungstic acid).

[0096] In this specification, the tungsten retention is measured by ICP atomic emission spectrometry. Specifically, as described in the examples below, when the subject of tungsten retention is, for example, a composition for tungsten fractionation containing the polypeptide of the present disclosure, the composition for tungsten fractionation of the present disclosure is incubated with buffer D (50 mM MOPS, pH 6.5, 50 mM NaCl, 1 mM ATP, 1 mM MgCl2) containing 1 mM Na2WO4 at 30°C for 30 minutes. Centrifuge at 10000 rpm for 1 minute, and the resulting supernatant is gel-filtered on a PD-10 column to separate excess tungstate (meaning free ions not bound to the polypeptide), and the tungsten oxoanion concentration in the aqueous polypeptide solution is measured by inductively coupled plasma atomic emission spectrometry (ICP-AES) using an ICPS-9000 device (SHIMADZU, Kyoto, Japan).

[0097] In addition, when the subject to be evaluated for tungsten retention ability is, for example, a tungsten fractionation composition containing a transformant in which the polypeptide of the present disclosure is expressed, the tungsten fractionation composition of the present disclosure is incubated with an aqueous solution containing 20 mM potassium phosphate buffer pH 7.0, 1 mM glycerol and 1 mM Na2WO4 at 30°C for 30 minutes. Centrifuged at 10,000 rpm for 1 minute, the resulting supernatant is filtered to separate excess tungstate (meaning free ions not bound to the polypeptide), and the tungsten oxoanion concentration in the aqueous polypeptide solution is measured by inductively coupled plasma atomic emission spectrometry (ICP-AES) using an ICPS-9000 device (SHIMADZU, Kyoto, Japan).

[0098] The present disclosure also includes a method for fractionating tungsten, comprising a step of contacting a polypeptide of the present disclosure, or a transformant in which a polypeptide of the present disclosure is expressed, with a tungsten-containing aqueous solution.

[0099] Specifically, the present disclosure also encompasses a method for fractionating tungsten, comprising the step of contacting at least one polypeptide selected from the group consisting of the above-described (A-1) to (A-4), (B-1) to (B-2), (D-1) to (D-2), and (E-1) to (E-2), or a transformant in which at least one polypeptide selected from the group consisting of the above-described (A-1) to (A-4), (B-1) to (B-2), (D-1) to (D-2), and (E-1) to (E-2) is expressed, with a tungsten-containing aqueous solution.

[0100] The present disclosure also encompasses a method for fractionating tungsten, comprising the step of contacting the above-described polypeptides (C-1) and / or (C-2), or a transformant in which the above-described polypeptides (C-1) and / or (C-2) are expressed, with a tungsten-containing aqueous solution.

[0101] In this specification, the tungsten fractionation method may be referred to as the "tungsten fractionation method of the present disclosure."

[0102] In the tungsten fractionation method of the present disclosure, it is preferable to contact at least one polypeptide selected from the group consisting of (A-1) to (A-4) and at least one polypeptide selected from the group consisting of (B-1) to (B-2) with a tungsten-containing aqueous solution. It is more preferable to contact at least one polypeptide selected from the group consisting of (D-1) to (D-2) and at least one polypeptide selected from the group consisting of (E-1) to (E-2) with a tungsten-containing aqueous solution.

[0103] In addition, the tungsten fractionation method of the present disclosure preferably includes contacting a transformant expressing at least one polypeptide selected from the group consisting of (A-1) to (A-4) and at least one polypeptide selected from the group consisting of (B-1) to (B-2) with a tungsten-containing aqueous solution. It is more preferable to contact a transformant expressing at least one polypeptide selected from the group consisting of (D-1) to (D-2) and at least one polypeptide selected from the group consisting of (E-1) to (E-2) with a tungsten-containing aqueous solution.

[0104] The number of types of polypeptides may be two, three, four, five, or six.

[0105] In the case of two kinds, the combination may be, for example, a combination of the polypeptide (A-1) and the polypeptide (B-1), the polypeptide (A-1) and the polypeptide (B-2), the polypeptide (A-2) and the polypeptide (B-1), the polypeptide (A-2) and the polypeptide (B-2), the polypeptide (A-3) and the polypeptide (B-1), the polypeptide (A-3) and the polypeptide (B-2), the polypeptide (A-4) and the polypeptide (B-1), or the polypeptide (A-4) and the polypeptide (B-2), etc. Among them, the combination of the polypeptide (A-1) and the polypeptide (B-1), or the polypeptide (A-3) and the polypeptide (B-1) is preferred. Further preferred examples include combinations of polypeptide (D-1) and polypeptide (E-1), polypeptide (D-1) and polypeptide (E-2), polypeptide (D-2) and polypeptide (E-1), or polypeptide (D-2) and polypeptide (E-2). Also, for example, it may be a polypeptide of (A-1), a polypeptide of (A-3), and a polypeptide of (B-1).

[0106] In the tungsten fractionation method of the present disclosure, the polypeptide (C-1) and the polypeptide (C-2) may be used alone or both may be used.

[0107] Furthermore, the tungsten fractionation method of the present disclosure can be used in combination with a polypeptide of the present disclosure and a transformant in which the polypeptide of the present disclosure is expressed.

[0108] The tungsten-containing aqueous solution is not particularly limited as long as it contains tungsten (e.g., tungstate). For example, it may be seawater. The tungsten-containing aqueous solution may also contain early transition metals other than tungsten, such as molybdenum and vanadium (e.g., oxoacid salts of early transition metals).

[0109] The concentration of tungsten (for example, tungstate) in the tungsten-containing aqueous solution is not particularly limited. For example, the concentration of tungstate may be about 0.001 μM to 10 mM.

[0110] The method of contacting the polypeptide of the present disclosure or the transformant in which the polypeptide of the present disclosure is expressed with the tungsten-containing aqueous solution is not particularly limited. For example, the method of mixing the polypeptide of the present disclosure or the transformant in which the polypeptide of the present disclosure is expressed with the tungsten-containing aqueous solution, the method of passing the tungsten-containing aqueous solution through a column packed with a resin or carrier that adsorbs the polypeptide of the present disclosure or the transformant in which the polypeptide of the present disclosure is expressed, etc. can be mentioned.

[0111] The concentration of the polypeptide of the present disclosure when contacting the polypeptide with a tungsten-containing aqueous solution is not particularly limited and may be, for example, about 0.001 μM to 10 mM.

[0112] When the transformant in which the polypeptide of the present disclosure is expressed is contacted with a tungsten-containing aqueous solution, the concentration of the transformant in which the polypeptide of the present disclosure is expressed is not particularly limited, and may be, for example, about 0.001 μM to 10 mM.

[0113] The temperature during contact is not particularly limited and can be, for example, about 4 to 50°C, or about 25 to 35°C, etc. The contact time is not particularly limited and can be, for example, about 10 seconds to 3 hours, or about 3 minutes to 1 hour. During the contact, for example, stirring or shaking can be performed as necessary.

[0114] In the step of contacting the polypeptide of the present disclosure or the transformant in which the polypeptide of the present disclosure is expressed with a tungsten-containing aqueous solution, for example, ATP and / or condensed phosphate can be added. Examples of condensed phosphate include pyrophosphate and polyphosphate. Among them, ATP is preferable. Although not wishing to be bound by theory, it is speculated that tungsten (tungstic acid) can be more efficiently retained by utilizing hydrolysis energy by adding ATP and / or condensed phosphate.

[0115] The concentration of ATP and / or condensed phosphate is not particularly limited and may be, for example, about 0.0001 μM to 100 mM.

[0116] In the step of contacting the polypeptide of the present disclosure or the transformant in which the polypeptide of the present disclosure is expressed with a tungsten-containing aqueous solution, for example, a magnesium salt can be added. Examples of magnesium salts include magnesium chloride, magnesium sulfate, magnesium nitrate, magnesium carbonate, magnesium acetate, etc. These can be used alone or in combination of two or more. Among them, magnesium chloride is preferable. Although not wishing to be bound by theory, it is presumed that the addition of a magnesium salt promotes hydrolysis of ATP and / or condensed phosphate, and tungsten (tungstic acid) can be more efficiently retained.

[0117] The concentration of the magnesium ion salt is not particularly limited and may be, for example, about 0.0001 μM to 100 mM.

[0118] When the tungsten fractionation method of the present disclosure includes a step of contacting the polypeptide of the present disclosure with a tungsten-containing aqueous solution, for example, the method may include a step of refolding the polypeptide of the present disclosure before the step. The refolding method is not particularly limited, and a known method can be adopted. For example, a method of solubilizing the polypeptide in an aqueous solution containing a protein denaturant, followed by dilution or dialysis, etc. may be mentioned. In addition, after solubilization, a chaperone protein may be added as necessary.

[0119] Examples of protein denaturants include urea, guanidine hydrochloride, etc. These may be used alone or in combination of two or more.

[0120] The concentration of the protein denaturant is not particularly limited and may be, for example, about 10 mM to 7 M.

[0121] Examples of chaperone proteins include GroESL, DnaK, DnaJ, ClpB, GrpE, IbpA, IbpB, and trigger factor.

[0122] The concentration of the chaperone protein is not particularly limited and may be, for example, about 0.0001 μM to 10 mM.

[0123] When the tungsten fractionation method of the present disclosure includes a step of contacting a transformant in which a polypeptide of the present disclosure is expressed with a tungsten-containing aqueous solution, for example, the tungsten fractionation method of the present disclosure may include a step of crushing the transformant in which a polypeptide of the present disclosure is expressed after contact with a tungsten-containing aqueous solution. Methods for crushing transformants are known in the art and can be performed using any method. For example, physical crushing methods such as ultrasonic treatment, French press, and glass bead crushing, and crushing methods using lytic enzymes such as lysozyme can be mentioned.

[0124] When the tungsten fractionation method of the present disclosure includes a step of disrupting the transformant in which the polypeptide of the present disclosure is expressed after contact with the tungsten-containing aqueous solution, for example, the tungsten fractionation method of the present disclosure may include a step of recovering the polypeptide of the present disclosure from the bacterial cell disruption solution. Examples of the method for recovering the polypeptide of the present disclosure include centrifugation, ultracentrifugation, ultrafiltration, salting out, dialysis, ion exchange column chromatography, adsorption column chromatography, affinity chromatography, gel filtration column chromatography, reverse phase chromatography, etc.

[0125] The tungsten fractionation method of the present disclosure may include a step of adjusting the pH of the polypeptide-containing aqueous solution of the present disclosure after contact with the tungsten-containing aqueous solution to 8 to 12. The upper or lower limit of the range may be, for example, 9, 10, or 11. Specifically, it may be 9 to 11. Without wishing to be bound by theory, it is speculated that by making the pH of the aqueous solution alkaline, tungsten (tungstic acid) held by the polypeptide of the present disclosure may be dissociated from the polypeptide.

[0126] The method of adjusting the pH is not particularly limited. For example, a method of adding a pH adjuster can be mentioned. Examples of the pH adjuster include sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, etc. These may be used alone or in combination of two or more.

[0127] In addition, in this specification, the term "comprising" includes "consisting essentially of" and "consisting of." In addition, the present disclosure includes any combination of the constituent elements described in this specification.

[0128] The various characteristics (properties, structures, functions, etc.) described for each embodiment of the present disclosure above may be combined in any way to specify the subject matter included in the present disclosure. In other words, the present disclosure includes all subject matter consisting of all combinations of each combinable characteristic described in this specification. EXAMPLES

[0129] The contents of the present disclosure will be specifically explained using the following experimental examples. However, the present disclosure is not limited to these in any way. In the following, unless otherwise specified, the experiments are performed under atmospheric pressure and room temperature conditions. Furthermore, unless otherwise specified, "%" means "% by mass".

[0130] Homologs similar to the α subunit (mosA, Accession No. AGK13730) or β subunit (mosB, Accession No. AGK13731) of the molybdenum storage protein (sometimes referred to as AvMosto in this specification) of A. vinelandii were searched in the genome database. Specifically, the sequence was identified by sequential iterative search using FASTA (https: / / www.genome.jp / kegg / kegg_ja.html) from the KEGG database (https: / / www.genome.jp / tools / fasta / ). As a result, Mosto derived from I. pallida (sometimes referred to as Wsp in this specification) and Mosto derived from D. multivorans (sometimes referred to as dmMosto in this specification) were focused on. In this embodiment, when simply written as "WspA", it refers to "WspA01". In this embodiment, when simply written as "WspB", it refers to "WspB01".

[0131] I. pallida has an operon linked with WspA01 (α subunit) and WspB01 (β subunit), which are homologues similar to the β subunit of Mosto derived from A. vinelandii, and the sequence identities of these are as follows: WspA01 (identity to mosA: 29%, identity to mosB: 44%) WspB01 (identity to mosA: 33%, identity to mosB: 47%) Furthermore, the sequence identity between WspA and WspB was 49.2%.

[0132] D. multivorans has one homologue similar to the β subunit of Mosto from A. vinelandii, with sequence identity of 28% to mosA and 41% to mosB.

[0133] Test 1. Mosto (Wsp) from I. pallida Preparation of Wsp expression construct The expression plasmid was constructed by homologous recombination. The DNA sequences shown in SEQ ID NO: 4 (WspA01) and SEQ ID NO: 5 (WspB01) were cloned into RSFDuet-1 vector (Novagen) by the following method to prepare a plasmid (wsp plasmid). Specifically, 2 μL of Wsp gene, 1 μL of pRSFDuet-1 vector, 1 μL of 10×SLICE buffer, 1 μL of SLICE solution, and 5 μL of MilliQ water were mixed and left to stand at 37° C. for 15 minutes. Protein expression of Wsp was enhanced in E. coli BL21(DE3) by coexpressing it with molecular chaperones GrpE and ClpB (pBB540 plasmid), as well as DnaK, DnaJ, and GroESL (pBB550 plasmid) (each plasmid is available from Addgene).

[0134] The expression plasmid for AvMosto was also constructed in a similar manner. For expression, only the plasmid (RSFDuet-1 vector) that did not contain the molecular chaperone plasmid was transformed into BL21(DE3).

[0135] Affinity tag introduction PCR A tag sequence was introduced into the α subunit of Wsp. Specifically, inverse PCR was performed using the primers shown in SEQ ID NOs: 6 and 7, followed by DpnI treatment and ligation to introduce an affinity tag sequence.

[0136] Transformation of Strep-tagged Wsp plasmids To isolate Wsp, E. coli BL21(DE3) was co-transformed with the three plasmids. The pBB550 plasmid, pBB540 plasmid, and wsp plasmid were introduced in that order. 5 μL of the plasmid solution with the tag sequence was added to 50 μL of E. coli BL21(DE3) strain competent cells in a clean bench. The cells were left on ice for 20 minutes and then heat-shocked in a 42°C water bath for 45 seconds. After 2 minutes on ice, 500 μL of SOC medium was added to the competent cells and incubated at 37°C for 45 minutes. This was dropped onto 100 μL of LB medium plate containing 15 mg / L kanamycin sulfate, 17 mg / L chloramphenicol, and 25 mg / L spectinomycin, spread, and then cultured at 37°C for 16 hours.

[0137] Purification of Wsp The transformants were cultured in LB medium supplemented with 25 mg / L kanamycin, 20 mg / L chloramphenicol, and 20 mg / L spectinomycin at 37°C, 135 rpm, and until the OD600 reached 0.5. The resulting cells (15 g) were suspended in buffer A (100 mM 2-amino-2-hydroxymethyl-1,3-propanediol (Tris)-HCl, pH 8.0, 1 M NaCl, 1 mM TCEP, 5% glycerol, 0.2% Tween 20, 1 mM ATP, 1 mM MgCl2, pH 7.0), and lysozyme (final, 0.2 mg / mL) and PMSF (final, 0.2 mg / mL) were added. The suspension was then sonicated at 20 W for 15 min and centrifuged at 30,000 × g for 20 min at 4°C to obtain a cell-free lysate. Purification was performed by affinity chromatography using a Strep-tactin 5mL column, with buffer A as the equilibration / wash buffer and buffer B (100mM Tris-HCl, pH 8.0; 1M NaCl, 1mM TCEP, 5% Glycerol, 0.2% Tween 20, 1mM ATP, 1mM MgCl2, 25mM D-biotin, pH 8.0) as the elution buffer. To remove the Strep tag region from the protein, HRV3C protease was added to the elution fraction and incubated overnight at 4°C. Size exclusion chromatography was then performed using Superdex 200 (16 / 60 column, Cytiva) and buffer C (containing 50mM phosphate and 150mM KCl, pH 7.0). Protein samples were stored at -80°C until further use. Purity was assessed using SDS-PAGE. Protein concentration was determined by measuring absorbance at 660 nm using Pierce 660 nm Protein Assay (Thermo Scientific).

[0138] Analytical Size Exclusion Chromatography Gel filtration was performed using Superdex 200 Increase (10 / 300 GL column, Cytiva) and buffer C as the eluent.

[0139] The results of SDS-PAGE are shown in FIG. 2, and the results of gel filtration chromatography are shown in FIG.

[0140] These results suggest that the molecular weight of Wsp is 210,000 and that Wsp forms a heterohexamer in solution.

[0141] Metal uptake by purified Wsp A 200 μg / mL Wsp (apo-form) sample was incubated with buffer D (50 mM MOPS, pH 6.5, 50 mM NaCl, 1 mM ATP, 1 mM MgCl2) containing various concentrations of Na2WO4 or Na2MoO4 (0.5-1 mM) for 30 min at 30°C, and then centrifuged at 10,000 rpm for 1 min. The resulting supernatant was gel-filtered on a PD-10 column to separate excess tungstate or molybdate (representing free ions not bound to Wsp in the aqueous polypeptide solution). Protein concentration was determined by measuring absorbance at 660 nm using the Pierce 660 nm Protein Assay (Thermo Scientific).

[0142] We also evaluated AvMosto in the same way.

[0143] Analysis of metal ion content in Wsp The transition metal oxoanion concentrations in the aqueous polypeptide solutions were measured by inductively coupled plasma atomic emission spectrometry (ICP-AES) using an ICPS-9000 instrument (SHIMADZU, Kyoto, Japan). Calibration curves for metal ions were prepared using standard solutions of metal ions (Mo and W, 1000 ppm).

[0144] The same evaluation was performed for AvMosto. The results are shown in Table 1.

[0145] [Table 1]

[0146] When 1 mM Na2WO4 or Na2MoO4 was evaluated, it was confirmed that Wsp incorporated 6 atoms of molybdenum (Mo) or 168 atoms of tungsten (W), while AvMosto incorporated 143 atoms of Mo or 142 atoms of W, as shown in Table 1. This suggests that Wsp specifically retains tungstic acid.

[0147] The amino acid sequences of WspA01 and AvMosto (β subunit) were compared. The results are shown in FIG. 4. In AvMosto (β subunit), Lys42 is located near the magnesium ion important for hydrolysis and the β- and γ-phosphates of ATP, and Gly79, Arg83, Tyr86, Arg168, and Thr169 are said to form a molybdate binding site, and it has been reported that these sites serve as gates into the protein cage (Non-Patent Document 1). Therefore, mutations were introduced (replacement of each amino acid residue with alanine) into His49 (the 49th histidine residue in the amino acid sequence of SEQ ID NO: 1) and Glu176 (the 176th glutamic acid residue in the amino acid sequence of SEQ ID NO: 1) of WspA01 by the following method. Specifically, the procedure is as follows.

[0148] Mutagenesis experiments were carried out to introduce site-specific mutations into expression vectors containing DNA fragments of the wspA01 and wspB01 genes. Mutagenic oligonucleotide pairs of approximately 30 nucleotides in length were purchased from Macrogen Japan Co., Ltd. (Table 2). Mutagenic inverse PCR was carried out using these pairs. After digestion with DpnI, the resulting products were transformed into Escherichia coli DH5α to obtain plasmids. The mutants were confirmed to have no undesirable mutations in the genes by DNA sequence analysis using a 3730xl DNA analyzer (Applied Biosystems). The uptake abilities of the obtained mutants were compared using the same method as in Table 1. The results are shown in Table 3.

[0149] [Table 2]

[0150] [Table 3]

[0151] As shown in Table 3, no change was observed in the ability to take up molybdate in either the H49A or E176A mutant. On the other hand, the amount of tungstate taken up was confirmed to be reduced by 25 atoms in the H49A mutant and increased by 22 atoms in the E176A mutant compared to the wild type. In the H49A mutant, the His to Ala mutation may have weakened the interaction with magnesium ions, resulting in a decrease in ATP hydrolysis ability. In addition, in the E176A mutant, the Glu to Ala mutation may have widened the gate, leading to an increase in the amount of tungstate taken up.

[0152] Next, the uptake ability of each mutant was compared in the presence of molybdic acid and tungstic acid. Specifically, the uptake ability was compared in the same manner as in Table 1, except that 500 μM of molybdic acid and tungstic acid were added. The results are shown in Table 4.

[0153] [Table 4]

[0154] As shown in Table 4, the H49A mutant and the E176A mutant increased the amount of tungstic acid taken up compared to the wild type. When the results were compared in terms of the ratio of tungstic acid to molybdic acid, the E176A mutant showed nearly two-fold improvement in selectivity compared to the wild type.

[0155] Test 2. Mosto from D. multivorans Preparation of dmMosto expression construct For the expression of dmMosto, a T7 promoter-containing plasmid pET30 was used (Novagen Inc.). The codon-optimized DNA fragment of dmMosto (SEQ ID NO: 12) was purchased from GenScript Japan Inc. This DNA fragment was transformed into E. coli iVEC3 strain with the PCR-amplified linearized pET30 vector to prepare the Mosto plasmid. A pair of mutated oligonucleotides was purchased from Macrogen Japan (Tokyo, Japan). Mutation was performed by inverse PCR using the primers shown in SEQ ID NO: 13 and 14, followed by DpnI digestion and self-ligation to synthesize strep-dmMosto. The resulting gene sequence was determined by DNA sequencing using a 3730xl DNA Analyzer (Applied Biosystems) after vector construction. The expression plasmid thus constructed was transformed into E. coli BL21 (DE3) competent cells, and the transformants were cultured overnight at 37°C in an autoinduction medium (ZY-5052) containing 30 mg / L kanamycin.

[0156] Purification of dmMosto protein The harvested cells (10 g) were suspended in buffer A (100 mM KH2PO4, 150 mM NaCl, 1 mM TCEP, pH 7.0), and lysozyme [0.2 mg / mL (final concentration)] and PMSF [1 mM (final concentration)] were added. The suspension was sonicated at 20 W for 15 min. The cell-free lysate was centrifuged at 30,000 × g for 20 min at 4 °C. Purification was performed using a strep-tactin XT column (5 mL Strep-tactin XT 4Flow high capacity, IBA GmbH, Göttingen, Germany) with buffer A as the equilibration and washing buffer and buffer B (100 mM KH2PO4, 150 mM NaCl, 25 mM D-biotin, 1 mM TCEP, pH 7.0) as the elution buffer. The eluted sample solution was concentrated by ultrafiltration using Vivaspin turbo 15 (Sartorius AG, Göttingen, Germany) and purified by gel filtration chromatography using a Superdex 200 Increase column (16 / 600 GL, Cytiva, Marlborough, USA) in buffer C (100 mM KH2PO4, 50 mM KCl, 1 mM TCEP, pH 7.0). Fractions containing purified protein were collected and concentrated again by ultrafiltration using Vivaspin turbo 15, and the protein solution was desalted using a PD-10 column (Cytiva, Marlborough, USA) and exchanged into buffer D (20 mM KH2PO4, 1 mM TCEP, pH 7.0). The collected fractions were concentrated to 15 mg / mL by ultrafiltration using Vivaspin turbo 15 and stored at -80 °C until use. Purity was assessed using SDS-PAGE. Protein concentration was determined by measuring the absorbance intensity at 280 nm.

[0157] The results of SDS-PAGE are shown in FIG. 6, and the results of gel filtration chromatography are shown in FIG.

[0158] These results suggest that dmMosto forms a homohexamer in solution.

[0159] Metal uptake was evaluated for 200 μg / mL dmMosto in the same manner as in Table 1, except that buffer D (50 mM MOPS, pH 7.0, 50 mM NaCl, 10 μM ATP, 1 mM MgCl2) containing 10 μM Na2WO4, Na2MoO4, Na2SeO4, VOSO4·nH2O, HfO(NO3)2·2H2O, or ZrO(NO3)2·2H2O was used. The results are shown in Table 5.

[0160] [Table 5]

[0161] As shown in Table 5, dmMosto was confirmed to incorporate 15 tungsten atoms and 1 selenium atom, suggesting that it specifically retains tungstic acid.

[0162] Metal uptake was evaluated using Na2WO4 in the same manner as in Table 5, except that CTP, GTP, or UTP was used instead of ATP. The results are shown in Table 6.

[0163] [Table 6]

[0164] As shown in Table 6, it was confirmed that a dozen or so atoms of tungsten were incorporated only when ATP was used.

[0165] Metal uptake was evaluated in the same manner as in Table 5, except that the concentrations of ATP and Na2WO4 were changed to 1 μM and 10 μM, 10 μM and 10 μM, or 100 μM and 100 μM, respectively. The results are shown in Table 7.

[0166] [Table 7]

[0167] As shown in Table 7, when ATP was reduced to 1 μM, the amount of tungstic acid taken up decreased, and when ATP and Na2WO4 were increased to 100 μM, it was confirmed that a maximum of 30 atoms of tungsten were taken up.

[0168] Metal uptake was evaluated using Na2WO4 in the same manner as in Table 5, except that MgCl2 was not included. The results are shown in Table 8.

[0169] [Table 8]

[0170] As shown in Table 8, it was confirmed that the amount of tungstic acid taken up was decreased in the absence of magnesium ions. From these results, it is speculated that dmMosto takes up tungstic acid in an ATP-dependent manner, using the energy of ATP hydrolysis.

[0171] Test 3. Improving protein expression efficiency Next, the present inventors investigated how to improve the expression efficiency of the Mosto protein. Specifically, first, they designed base sequences (SEQ ID NO:15 and SEQ ID NO:16) that encode the same amino acid sequences as the WspA01 and WspB01 proteins evaluated in Test 1, but with some codons modified from the base sequences of SEQ ID NO:4 and SEQ ID NO:5. The sequence identity between SEQ ID NO:15 and SEQ ID NO:4 was 43%. The sequence identity between SEQ ID NO:16 and SEQ ID NO:5 was 31%.

[0172] For the designed base sequences of SEQ ID NO: 15 and SEQ ID NO: 16, plasmids were prepared in the same manner as in Test 1, and proteins were expressed in an E. coli expression system. After purifying the proteins from E. coli, SDS-PAGE was performed. The electrophoretic images of SDS-PAGE were analyzed using the image processing software ImageJ, and the total protein amount and the target protein amount on each lane were estimated from the density of the bands. The ratio of the target protein to the total protein was also calculated.

[0173] The results of SDS-PAGE are shown in FIG. 9. The protein complex translated from the base sequences of SEQ ID NO: 4 and SEQ ID NO: 5 is designated as WspAB01, and the protein complex translated from the base sequences of SEQ ID NO: 15 and SEQ ID NO: 16 is designated as WspAB01'. In the figure, the lanes from left to right are 1: WspAB01 soluble fraction, 2: WspAB01 insoluble fraction, 3: WspAB01' soluble fraction, 4: WspAB01' insoluble fraction, and M: molecular weight marker. The band of the target protein is indicated by an arrow. The functional protein complex is contained in the soluble fraction. As is clear from a comparison of lanes 1 and 3, the base sequences of SEQ ID NO: 15 and SEQ ID NO: 16 had higher protein expression efficiency than the base sequences of SEQ ID NO: 4 and SEQ ID NO: 5. In addition, the ratio of the target protein to the total protein calculated by image analysis using ImageJ was 5.1% in lane 1, while it was 14% in lane 3.

[0174] Furthermore, the present inventors investigated the protein expression efficiency of Most from Planctomycetota bacterium, which belongs to the same Type II as WspA01 and WspB01 in the molecular phylogenetic tree (Figure 8) for Most derived from various organisms. It is presumed that Most, which belongs to Type II, has particularly high tungsten selectivity. The α subunit of Most from Planctomycetota bacterium is represented as WspA02, and the β subunit of Most from Planctomycetota bacterium is represented as WspB02. WspA02 consists of the amino acid sequence of SEQ ID NO: 17, and the sequence identity between SEQ ID NO: 1 and SEQ ID NO: 1 was 63%. In addition, the sequence identity between SEQ ID NO: 17 and mosA was 27%, and the sequence identity between SEQ ID NO: 17 and mosB was 41%. WspB02 consists of the amino acid sequence of SEQ ID NO: 18, and the sequence identity between SEQ ID NO: 18 and SEQ ID NO: 2 was 59%. Furthermore, the sequence identity between SEQ ID NO:18 and mosA was 31%, and the sequence identity between SEQ ID NO:18 and mosB was 48%. The base sequence of SEQ ID NO:17 is shown in Figure 19. The base sequence of SEQ ID NO:18 is shown in Figure 20.

[0175] The base sequence (SEQ ID NO: 19) encoding (native) WspA02 isolated from the genome of Planctomycetota bacterium is shown in FIG. 21. The base sequence (SEQ ID NO: 20) encoding (native) WspB02 isolated from the genome of Planctomycetota bacterium is shown in FIG. 22. The present inventors designed base sequences in which some codons were modified from the base sequences of SEQ ID NO: 19 and SEQ ID NO: 20 while encoding the same amino acid sequence as the protein translated from the base sequences of SEQ ID NO: 19 and SEQ ID NO: 20 (SEQ ID NO: 21 and SEQ ID NO: 22). The sequence identity between SEQ ID NO: 21 and SEQ ID NO: 19 was 38%. The sequence identity between SEQ ID NO: 22 and SEQ ID NO: 20 was 28%.

[0176] For the designed base sequences of SEQ ID NO: 22 and SEQ ID NO: 23, plasmids were prepared in the same manner as in Test 1, and proteins were expressed in an E. coli expression system. After purifying the proteins from E. coli, SDS-PAGE was performed. The electrophoretic images of SDS-PAGE were analyzed using the image processing software ImageJ, and the total protein amount and the target protein amount on each lane were estimated from the density of the bands. The ratio of the target protein to the total protein was also calculated.

[0177] The results of SDS-PAGE are shown in FIG. 10. The protein complex translated from the base sequences of SEQ ID NO: 22 and SEQ ID NO: 23 is designated as WspAB02. In the figure, from the left, the lanes are 1: WspAB02 soluble fraction, 2: WspAB02 insoluble fraction, and M: molecular weight marker. The band of the target protein is indicated by an arrow. The functional protein complex is contained in the soluble fraction. In addition, the ratio of the target protein to the total protein calculated by image analysis using ImageJ was 14% in lane 1, while it was 17% in lane 2.

[0178] The above tests suggest that according to the present disclosure, polypeptides having particularly high tungsten selectivity can be efficiently obtained.

Claims

1. A composition for tungsten fractionation comprising at least one polypeptide selected from the group consisting of (A-1) to (A-4), (B-1) to (B-2), (D-1) to (D-2), and (E-1) to (E-2) below. (A-1) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 (A-2) A polypeptide having an amino acid sequence having 85% or more sequence identity with the amino acid sequence of SEQ ID NO:1 and forming a heteromultimer with a polypeptide having the amino acid sequence of SEQ ID NO:2 to exhibit tungsten uptake ability. (A-3) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 in which glutamic acid at position 176 is replaced with alanine. (A-4) A polypeptide having an amino acid sequence that has 85% or more sequence identity with the amino acid sequence of the polypeptide of (A-3) while maintaining the substituted amino acids in the polypeptide of (A-3), and that forms a heteromultimer with a polypeptide having the amino acid sequence of SEQ ID NO: 2 to exhibit tungsten uptake ability. (B-1) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 (B-2) A polypeptide having an amino acid sequence having 85% or more sequence identity with the amino acid sequence of SEQ ID NO:2, and forming a heteromultimer with a polypeptide having the amino acid sequence of SEQ ID NO:1 to exhibit tungsten uptake ability. (D-1) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 17 (D-2) A polypeptide having an amino acid sequence having 85% or more sequence identity with the amino acid sequence of SEQ ID NO: 17 and forming a heteromultimer with a polypeptide having the amino acid sequence of SEQ ID NO: 18 to exhibit tungsten uptake ability. (E-1) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 18 (E-2) A polypeptide having an amino acid sequence having 85% or more sequence identity with the amino acid sequence of SEQ ID NO: 18 and forming a heteromultimer with a polypeptide having the amino acid sequence of SEQ ID NO: 17 to exhibit tungsten uptake ability.

2. At least one polypeptide selected from the group consisting of (A-1) to (A-4) and at least one polypeptide selected from the group consisting of (B-1) to (B-2) in combination; or The tungsten fractionation composition according to claim 1, comprising in combination at least one polypeptide selected from the group consisting of (D-1) to (D-2) and at least one polypeptide selected from the group consisting of (E-1) to (E-2).

3. The tungsten fractionation composition according to claim 1 or 2, wherein the 49th histidine in the amino acid sequence of SEQ ID NO: 1 is substituted with alanine.

4. A composition for tungsten fractionation comprising the following polypeptides (C-1) and / or (C-2). (C-1) A polypeptide consisting of the amino acid sequence of SEQ ID NO:3 (C-2) A polypeptide consisting of an amino acid sequence having a sequence identity of 85% or more with the amino acid sequence of SEQ ID NO: 3, and capable of forming homopolymers and of incorporating tungsten.

5. A composition for tungsten fractionation, comprising a transformant in which at least one polypeptide selected from the group consisting of the following (A-1) to (A-4), (B-1) to (B-2), (D-1) to (D-2), and (E-1) to (E-2) is expressed. (A-1) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 (A-2) A polypeptide having an amino acid sequence having 85% or more sequence identity with the amino acid sequence of SEQ ID NO:1 and forming a heteromultimer with a polypeptide having the amino acid sequence of SEQ ID NO:2 to exhibit tungsten uptake ability. (A-3) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 in which glutamic acid at position 176 is replaced with alanine. (A-4) A polypeptide having an amino acid sequence that has 85% or more sequence identity with the amino acid sequence of the polypeptide of (A-3) while maintaining the substituted amino acids in the polypeptide of (A-3), and that forms a heteromultimer with a polypeptide having the amino acid sequence of SEQ ID NO: 2 to exhibit tungsten uptake ability. (B-1) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 (B-2) A polypeptide having an amino acid sequence having 85% or more sequence identity with the amino acid sequence of SEQ ID NO:2, and forming a heteromultimer with a polypeptide having the amino acid sequence of SEQ ID NO:1 to exhibit tungsten uptake ability. (D-1) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 17 (D-2) A polypeptide having an amino acid sequence having 85% or more sequence identity with the amino acid sequence of SEQ ID NO: 17 and forming a heteromultimer with a polypeptide having the amino acid sequence of SEQ ID NO: 18 to exhibit tungsten uptake ability. (E-1) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 18 (E-2) A polypeptide having an amino acid sequence having 85% or more sequence identity with the amino acid sequence of SEQ ID NO: 18 and forming a heteromultimer with a polypeptide having the amino acid sequence of SEQ ID NO: 17 to exhibit tungsten uptake ability.

6. A transformant expressing at least one polypeptide selected from the group consisting of (A-1) to (A-4) and at least one polypeptide selected from the group consisting of (B-1) to (B-2), or The tungsten fractionation composition according to claim 5, comprising a transformant in which at least one polypeptide selected from the group consisting of (D-1) to (D-2) and at least one polypeptide selected from the group consisting of (E-1) to (E-2) is expressed.

7. The composition for tungsten fractionation according to claim 5 or 6, wherein the 49th histidine in the amino acid sequence of SEQ ID NO: 1 is substituted with alanine.

8. A composition for tungsten fractionation comprising a transformant in which the following polypeptide (C-1) and / or (C-2) is expressed: (C-1) A polypeptide consisting of the amino acid sequence of SEQ ID NO:3 (C-2) A polypeptide consisting of an amino acid sequence having a sequence identity of 85% or more with the amino acid sequence of SEQ ID NO: 3, and capable of forming homopolymers and of incorporating tungsten.

9. At least one polypeptide selected from the group consisting of (A-1) to (A-4), (B-1) to (B-2), (D-1) to (D-2), and (E-1) to (E-2) below; or A transformant expressing at least one polypeptide selected from the group consisting of (A-1) to (A-4), (B-1) to (B-2), (D-1) to (D-2), and (E-1) to (E-2) below; A method for fractionating tungsten, comprising the step of contacting a tungsten-containing aqueous solution with (A-1) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 (A-2) A polypeptide having an amino acid sequence having 85% or more sequence identity with the amino acid sequence of SEQ ID NO:1 and forming a heteromultimer with a polypeptide having the amino acid sequence of SEQ ID NO:2 to exhibit tungsten uptake ability. (A-3) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 in which glutamic acid at position 176 is replaced with alanine. (A-4) A polypeptide having an amino acid sequence that has 85% or more sequence identity with the amino acid sequence of the polypeptide of (A-3) while maintaining the substituted amino acids in the polypeptide of (A-3), and that forms a heteromultimer with a polypeptide having the amino acid sequence of SEQ ID NO: 2 to exhibit tungsten uptake ability. (B-1) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 (B-2) A polypeptide having an amino acid sequence having 85% or more sequence identity with the amino acid sequence of SEQ ID NO:2, and forming a heteromultimer with a polypeptide having the amino acid sequence of SEQ ID NO:1 to exhibit tungsten uptake ability. (D-1) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 17 (D-2) A polypeptide having an amino acid sequence having 85% or more sequence identity with the amino acid sequence of SEQ ID NO: 17 and forming a heteromultimer with a polypeptide having the amino acid sequence of SEQ ID NO: 18 to exhibit tungsten uptake ability. (E-1) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 18 (E-2) A polypeptide having an amino acid sequence having 85% or more sequence identity with the amino acid sequence of SEQ ID NO: 18 and forming a heteromultimer with a polypeptide having the amino acid sequence of SEQ ID NO: 17 to exhibit tungsten uptake ability.

10. A combination of at least one polypeptide selected from the group consisting of (A-1) to (A-4) and at least one polypeptide selected from the group consisting of (B-1) to (B-2), or A combination of at least one polypeptide selected from the group consisting of (D-1) to (D-2) and at least one polypeptide selected from the group consisting of (E-1) to (E-2), or A transformant expressing at least one polypeptide selected from the group consisting of (A-1) to (A-4) and at least one polypeptide selected from the group consisting of (B-1) to (B-2), or A transformant expressing at least one polypeptide selected from the group consisting of (D-1) to (D-2) and at least one polypeptide selected from the group consisting of (E-1) to (E-2); The method for fractionating tungsten according to claim 9, comprising the step of contacting a tungsten-containing aqueous solution with the tungsten-containing aqueous solution.

11. The method for separating tungsten according to claim 9 or 10, wherein the 49th histidine in the amino acid sequence of SEQ ID NO: 1 is substituted with alanine.

12. The following polypeptides (C-1) and / or (C-2): A method for fractionating tungsten, comprising the step of contacting a transformant in which the following polypeptide (C-1) and / or (C-2) is expressed with a tungsten-containing aqueous solution: (C-1) A polypeptide consisting of the amino acid sequence of SEQ ID NO:3 (C-2) A polypeptide consisting of an amino acid sequence having a sequence identity of 85% or more with the amino acid sequence of SEQ ID NO: 3, and capable of forming homopolymers and of incorporating tungsten.

13. A nucleic acid consisting of at least one base sequence selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 21, and SEQ ID NO: 22, or A nucleic acid encoding a polypeptide having a base sequence having 85% or more sequence identity with the base sequence of SEQ ID NO:15 and forming a heteromultimer with a polypeptide having the amino acid sequence of SEQ ID NO:2 to exhibit tungsten uptake ability; A nucleic acid encoding a polypeptide having a base sequence having 85% or more sequence identity with the base sequence of SEQ ID NO:16 and forming a heteromultimer with a polypeptide having the amino acid sequence of SEQ ID NO:1 to exhibit tungsten uptake ability; A nucleic acid encoding a polypeptide having a base sequence having 85% or more sequence identity with the base sequence of SEQ ID NO:21 and forming a heteromultimer with a polypeptide having the amino acid sequence of SEQ ID NO:18 to exhibit tungsten uptake ability; and A nucleic acid encoding a polypeptide having a base sequence having 85% or more sequence identity with the base sequence of SEQ ID NO:22 and forming a heteromultimer with a polypeptide having the amino acid sequence of SEQ ID NO:17 to exhibit tungsten uptake ability; At least one nucleic acid selected from the group consisting of:

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