JP6459483B2 - Rare earth species capture peptide and use thereof - Google Patents

Description

  The present specification relates to rare earth species capture peptides and uses thereof.

  In recent years, the use of inorganic elements centered on rare earths has become essential. On the other hand, there is concern about the stable supply of rare earths and the environmental impact of rare earths. Therefore, development of technology to efficiently collect rare earths from the natural world and establishment of technology to selectively and efficiently collect and recycle dilute rare earth contained in discarded products and wastewater with low energy are desired. ing.

  For example, neodymium is a rare earth belonging to the lanthanoid series, and is particularly included in the light rare earth series containing elements from La to Gd and Sc. Neodymium is a constituent metal of neodymium oxide used in permanent magnets and motors.

  As such a rare earth recovery technique, a solvent extraction using N, N-dioctyl diglycolamidic acid (DODGAA) or the like and a recovery method using a reducing microorganism have been reported. On the other hand, peptides that bind to rare earth ions such as lanthanoid ions have been reported (Patent Documents 1 and 2).

US Patent Publication No. 2003/0228622 JP 2014-51449 A

  The above-described solvent extraction method and recovery method using a reducing microorganism are difficult to make a profitable cost. Moreover, even if it couple | bonds with a lanthanoid ion by the method of patent documents 1, 2, it cannot capture | acquire a lanthanoid ion as a rare earth mineral (rare earth mineral), and the collection | recovery is still difficult. Furthermore, at the present time, ions belonging to light rare earths such as neodymium ions cannot be captured, nor can they be captured as rare earth minerals.

  The disclosure of the present specification provides a material having excellent efficiency for capturing or recovering rare earth species including light rare earth species such as ions belonging to light rare earth and minerals thereof, and use thereof.

  The present inventors searched for a peptide capable of capturing or recovering neodymium oxide. In addition to binding to neodymium oxide and further capable of capturing neodymium ions, the present inventors obtained characteristics of a peptide useful for capturing neodymium as a neodymium mineral. . Furthermore, the present inventors have found that the peptide has the same effect on rare earths other than neodymium ions and also has the same action on heavy rare earths. The disclosure of the present specification provides the following means based on such findings.

(1) A rare earth species-trapping agent comprising a rare earth species-trapping peptide region having a rare earth species-trapping ability for capturing rare earth species.
(2) The scavenger according to (1), wherein the rare earth species is a light rare earth species.
(3) The scavenger according to (2), which captures the oxide of the light rare earth species.
(4) The scavenger according to (2) or (3), wherein ions of the light rare earth species are mineralized and captured.
(5) The scavenger according to any one of (2) to (4), wherein the light rare earth species is derived from neodymium.
(6) The capture agent according to any one of (1) to (5), wherein the rare earth species capture peptide region contains one or more acidic amino acid residues.
(7) The capture agent according to (6), wherein the rare earth species capture peptide region includes two or more acidic amino acid residues.
(8) The scavenger according to (6) or (7), wherein the acidic amino acid residue is selected from glutamic acid and aspartic acid.
(9) The capture agent according to any one of (1) to (8), wherein the rare earth species capture peptide region has the following amino acid sequence.
(1) X1-X2-X3-X4-X5-A1-X6-X7-A2-X8-X9-X10 (SEQ ID NO: 1)
(However, A1 and A2 are each independently an acidic amino acid, X1 is proline, X2 is valine, X3 is tryptophan, X4 is cephenylalanine, and X5 is serine. Yes, X6 is valine, X7 is glycine, X8 is phenylalanine, X9 is methionine, and X10 is valine.)
(10) The capture according to (9), wherein the amino acid sequence consists of Pro-Val-Trp-Phe-Ser-Asp / Glu-Val-Gly-Asp / Glu-Phe-Met-Val (SEQ ID NO: 2) Agent.
(11) The capturing agent according to any one of (1) to (10), comprising two or more rare earth species capturing peptide regions.
(12) A peptide having the following amino acid sequence.
(1) X1-X2-X3-X4-X5-A1-X6-X7-A2-X8-X9-X10 (SEQ ID NO: 1)
(However, A1 and A2 are each independently an acidic amino acid, X1 is proline, X2 is valine, X3 is tryptophan, X4 is cephenylalanine, and X5 is serine. Yes, X6 is valine, X7 is glycine, X8 is phenylalanine, X9 is methionine, and X10 is valine.)
(13) The peptide according to (12), wherein the acidic amino acid is aspartic acid or glutamic acid.
(14) The amino acid sequence consists of Pro-Val-Trp-Phe-Ser-Asp / Glu-Val-Gly-Asp / Glu-Phe-Met-Val (SEQ ID NO: 2), (12) or (13) The peptide according to 1.
(15) A composite comprising a rare earth species and the scavenger according to any one of (1) to (11).
(16) A contact step of bringing a rare earth species into contact with the scavenger according to any one of (1) to (11),
A method for capturing rare earth species.
(17) The capturing method according to (16), wherein, in the contacting step, the rare earth species is a light rare earth ion, and the rare earth species is captured as a mineral derived from the light rare earth ion.
(18) In the said contact process, the said rare earth seed | species is a light rare earth oxide, The manufacturing method as described in (16).
(19) The capturing method according to (16), wherein in the contacting step, the rare earth species is a light rare earth ion.
(20) The capturing method according to any one of (16) to (19), wherein the rare earth species is derived from neodymium.
(21) contacting the rare earth species with the scavenger according to any one of (1) to (11) to recover the rare earth species;
A method for recovering rare earth species.
(22) a step of contacting the rare earth species with the scavenger according to any one of (1) to (11) to detect the rare earth species;
A method for detecting rare earth species.
(23) A method for screening a peptide having the ability to capture light rare earth species,
A step of contacting the test peptide with a light rare earth oxide and evaluating the ability of the test peptide to capture the light rare earth oxide;
A method comprising:

It is a figure which shows the outline | summary of the biopanning by a phage display method. It is a figure which shows the number of rounds of biopanning, and a neodymium oxide binding phage titer measurement result. It is a figure which shows the binding ability evaluation result of the monoclonal phage with respect to neodymium oxide. It is a figure which shows the evaluation result of the mineralization ability of various lanthanoid ion (La, Nd, Sm) about Lamp-3 peptide. It is a figure which shows the evaluation result of the mineralization ability of various lanthanoid ion (Dy, Lu) about Lamp-3 peptide.

  The present disclosure relates to a rare earth species scavenger having a rare earth species scavenging ability for capturing rare earth species such as rare earth minerals and / or rare earth ions, and use thereof.

  The rare earth species-trapping agent of the present disclosure can be easily captured by binding to a rare earth species that is a rare earth mineral, which is a rare earth ion or a rare earth mineral, via its peptide region. By separating or collecting the rare earth species scavenger, the rare earth species can be easily separated or collected.

  In addition, the rare earth species scavenger of the present disclosure is excellent in the ability to capture light rare earth species. For example, the light rare earth oxide can be captured, and the light rare earth can be mineralized in contact with the light rare earth ion to be captured as the light rare earth mineral.

  In the present specification, the “rare earth species” includes rare earth ions and rare earth minerals.

  In this specification, “rare earth” means two elements of scandium (Sc) and yttrium (Y), and the lanthanoid lanthanum (La), cerium (Ce), praseodymium (Pr), neodymium (Nd), and promethium (Pm). , Samarium (Sm), europium (Eu), gadolinium (Gd), terbium (Tb), dysprosium (Dy), holmium (Ho), erbium (Er), thulium (Tm), ytterbium (Yb) and lutetium (Lu) Is mentioned.

In this specification, “light rare earth” means lanthanoid lanthanum (La), cerium (Ce), praseodymium (Pr), neodymium (Nd), promethium (Pm), samarium (Sm), europium (Eu) and gadolinium. (Gd) and scandium (Sc). Neodymium is useful as a magnet material. In this specification, “heavy rare earth” means terbium (Tb), dysprosium (Dy), holmium (Ho), erbium (Er), Tm (thulium), ytterbium (Yb), lutetium (Lu) and yttrium ( Y). In the present specification, “medium rare earth” includes samarium (Sm), europium (Eu), and gadolinium (Gd).

  In the present specification, the “rare earth ion” includes a state of an ion having a valence of oxidation possible for each rare earth.

  In the present specification, the “rare earth mineral” includes oxides, hydroxides, inorganic acid salts, and organic acid salts with possible oxidation numbers for each rare earth. Oxides and hydroxides are preferable. The rare earth mineral produced by the mineralization agent of the rare earth ion in the aqueous medium is typically a hydroxide.

(Rare earth species scavenger)
The rare earth species-trapping agent according to the present disclosure (hereinafter simply referred to as “trapping agent”) can include a peptide region having a rare earth species-trapping ability.

(Rare earth species capture ability)
The rare earth species capturing ability refers to the ability to contact or separate rare earth species by separation or retention. The rare earth species capturing ability can include an ability (rare earth mineral capturing ability) capable of being separated or retained as a rare earth mineral as it is in contact with a rare earth mineral such as a rare earth oxide. In addition, the rare earth species trapping ability can include an ability (rare earth ion trapping ability) to contact or separate rare earth ions in contact with rare earth ions. Furthermore, the rare earth species trapping ability can include the ability (mineralization ability) of trapping rare earth ions to produce rare earth minerals such as rare earth hydroxides.

  Hereinafter, unless otherwise specified, when referring to the ability to capture rare earth species or capture rare earth species, it is intended to be based on any one or more of the above-mentioned capabilities. In addition, the term “rare earth mineral capturing ability” or “rare earth mineral capturing ability” is intended to be based on the rare earth mineral capturing ability. In addition, the term “rare earth ion trapping ability” or “rare earth ion trapping” is intended to be based on the rare earth ion trapping ability. Furthermore, when individually referred to as mineralization ability or mineralization of rare earth ions, it is intended to be based on the mineralization ability.

  As will be described later, such a peptide having the ability to capture rare earth species can be obtained by screening using various peptide libraries (including random peptide libraries) for the ability to capture the rare earth species as a target.

(Rare earth species capture peptide region)
A rare earth species capture peptide region (hereinafter simply referred to as a capture region) is a peptide region having a rare earth species capture ability. The capture region includes a peptide chain that exhibits one or more of rare earth mineral capture ability, rare earth ion capture ability, and mineralization ability.

  The capture region can form at least temporarily a complex with the rare earth species to the extent that the rare earth species can be captured by an interaction other than a covalent bond with the rare earth species. Non-covalent interactions include electrostatic bonds, ionic bonds, hydrogen bonds, and the like, but the interactions related to “trapping” in this specification are not limited to these.

  The capture region may be any region that can capture at least one rare earth species. That is, the rare earth species captured by the capture region may be one or more rare earth species (ions and / or minerals). The capture region may capture 3 or more, preferably 4 or more, more preferably 5 or more rare earth species.

  The capture region preferably has a mineralization ability. When the mineralization ability is provided, a rare earth mineral such as a rare earth hydroxide can be generated from a liquid medium containing a rare earth ion. For this reason, while separating the ionized rare earth, a rare earth mineral can be produced at once from the ion, and separation and recovery are facilitated, which can greatly contribute to the efficiency of separation and recovery of rare earth species.

  In the present specification, a peptide is usually a polymer in which several or more natural amino acids and / or unnatural amino acids are linked by acid amide bonds. Peptides generally have about 100 amino acid residues or less. The number of amino acid residues in the capture region is not particularly limited, but the number of amino acid residues is preferably 5 or more, more preferably 7 or more, still more preferably 8 or more, and even more preferably 10 That's it. Also, the upper limit of the number of amino acid residues is not particularly limited, but considering rare earth species capturing ability, it may be 25 or less, 20 or less, or 15 or less. .

  The capture region is preferably a polymer of L-form amino acid residues, but does not exclude a D-form polymer.

  The capture region may be linear or may be cyclized by an intramolecular disulfide bond or the like. When circularized, an amino acid sequence that is considered to potentially contribute to binding to rare earth species in the capture region (hereinafter simply referred to as capture sequence. For example, an appropriate number of upstream and downstream containing acidic amino acid residues. It is preferable that the amino acid sequence is an amino acid sequence comprising the amino acid residues of Such a capture region is typically a capture region comprising a cysteine residue directly on each side of the capture sequence or via one or more appropriate amino acid residues, each having a cysteine residue. A circularized capture region may be mentioned. Cyclization via a cysteine residue is easily realized by adding an oxidizing agent such as iodine or hydrogen peroxide to a peptide containing a cysteine residue.

  The rare earth metal captured by the capture region is a group consisting of lanthanoid lanthanum (La), cerium (Ce), praseodymium (Pr), neodymium (Nd), promethium (Pm), samarium (Sm), and europium (Eu). It is preferably a light rare earth species that is one or more selected from

  The rare earth species trapping ability of the trapping region can be obtained with various performances by the screening method described later. A peptide that captures a target rare earth species can be screened by applying various peptide libraries by the phage display method or the like for the rare earth species to be captured. The target rare earth species may be a rare earth ion or a rare earth mineral.

  It is considered that the rare earth species capturing ability of the capture region varies depending on the amino acid sequence. That is, for example, there are capture regions that capture neodymium species with high selectivity, and there are capture regions that capture lanthanum species with high selectivity. There is also a capture region that captures a plurality of rare earth species on average. There is also a capture region that captures both rare earth ions and rare earth minerals.

  As already described, the capture sequence included in the capture region preferably includes one or more acidic amino acid residues. The capture sequence does not exclude the inclusion of three or more acidic amino acid residues, but preferably contains two acidic amino acid residues. Although this is an inference and does not restrict the present disclosure, it is speculated that the rare earth scavenging ability can be ensured by having one or more acidic amino acid residues. It is considered that the capture strength and the type of rare earth species serving as a target nucleic acid to be captured vary to some extent depending on the amino acid sequence containing acidic amino acid residues.

  Examples of the acidic amino acid residue include glutamic acid, aspartic acid, isoaspartic acid, isoglutamic acid, 2-aminoadipic acid, 2-aminosuberic acid and the like. Preferred are aspartic acid and glutamic acid. Two or more acidic amino acid residues may be the same or different.

  When the capture sequence has two or more acidic amino acid residues, the closest acidic amino acid residues may be directly adjacent to each other, or are adjacent via 1 or 2 or more and 4 or less amino acid residues You may do it. More preferably, it is via two amino acid residues. The type of the intervening amino acid residue (intervening amino acid residue) is not particularly limited, but is preferably intervened by a neutral amino acid residue, an aromatic amino acid residue, or the like. The intervening amino acid residue more preferably includes a neutral amino acid residue.

  Here, examples of the neutral amino acid residue include glycine, alanine, valine, leucine, isoleucine, serine, threonine, and norvaline. Examples of basic amino acid residues include lysine, norleucine, 2-aminobutanoic acid, methionine, o-methylserine, t-butylglycine, t-butylalanine, and cyclohexylalanine. Examples of acid amide amino acid residues include asparagine and glutamine. Examples of basic amino acid residues include lysine, arginine, ornithine, 2,4-diaminobutanoic acid, and 2,3-diaminopropionic acid. Examples of cyclic amino acid residues include proline, 3-hydroxyproline, and 4-hydroxyproline. Examples of the OH-containing amino acid residue include serine, threonine, and homoserine. Aromatic amino acid residues include phenylalanine, tyrosine and tryptophan.

The capture region can have, for example, the following capture sequence (1).
(1) X1-X2-X3-X4-X5-A1-X6-X7-A2-X8-X9-X10 (SEQ ID NO: 1)
(However, A1 and A2 are each independently an acidic amino acid, X1 is proline, X2 is valine, X3 is tryptophan, X4 is cephenylalanine, and X5 is serine. Yes, X6 is valine, X7 is glycine, X8 is phenylalanine, X9 is methionine, and X10 is valine. More specifically, the supplementary sequence can consist of Pro-Val-Trp-Phe-Ser-Asp / Glu-Val-Gly-Asp / Glu-Phe-Met-Val (SEQ ID NO: 2).

  In the capture sequence (1), the acidic amino acid is preferably aspartic acid or glutamic acid. More specifically, examples include the following. Among the amino acid sequences shown below, the capture sequence is preferably a sequence excluding 3 amino acid residues at the N-terminus and 2 amino acid residues at the C-terminus, more preferably a sequence excluding SC- and -CS at both ends. . More preferred is a sequence excluding one amino acid group (S) at both ends.

SerCys-Pro-Val-Trp-Phe-Ser-Asp-Val-Gly-Asp-Phe-Met-Val-CysSer (SEQ ID NO: 3)
SerCys-Pro-Val-Trp-Phe-Ser-Asp-Val-Gly-Glu-Phe-Met-Val-CysSer (SEQ ID NO: 4)
SerCys-Pro-Val-Trp-Phe-Ser-Glu-Val-Gly-Asp-Phe-Met-Val-CysSer (SEQ ID NO: 5)
SerCys-Pro-Val-Trp-Phe-Ser-Glu-Val-Gly-Glu-Phe-Met-Val-CysSer (SEQ ID NO: 6)

  The capture sequences are rare earths for the rare earth species of lanthanoids (La), cerium (Ce), praseodymium (Pr), neodymium (Nd), promethium (Pm), samarium (Sm) and europium (Eu) which are light rare earths. It has the ability to capture species, and in particular, it has the ability to capture rare earth species of lanthanum, neodymium, and samarium. The capturing sequence has a rare earth mineral capturing ability and a rare earth ion capturing ability for light rare earth species, and particularly has a mineralization ability for light rare earth species. In addition, the capture sequence has a rare earth species capturing ability for heavy rare earth species. In particular, it has mineralization ability for heavy rare earth species. That is, the capture sequence has a mineralization ability for rare earth ions widely from light rare earth to heavy rare earth.

  One or more amino acid residues may be further added to the N-terminus or C-terminus of the various capture sequences described above. For example, 1 or 2 amino acid residues may be added, 1 or more and 3 or less amino acid residues may be added, or 1 or more and 5 or less amino acid residues may be added. 1 to 7 or less amino acid residues may be added, 1 to 19 amino acid residues may be added, or 1 to 10 amino acid residues may be added. No more than amino acid residues may be added.

  Examples of the amino acid residue added to the end of the capture sequence include serine, cysteine, asparagine and the like. Moreover, this peptide can be cyclized by introducing cysteine at both ends.

  The capture agent can comprise one or more capture regions. For example, one or more capture regions characterized by a single capture sequence can be provided. Further, two or three or more capture regions having different capture sequences may be provided. Depending on the application, capture regions having the same or different rare earth species capture ability can be combined.

  The plurality of capture regions may be linked directly or via an appropriate linker, or may be linked via a region containing an assembly element described later. As such a linker, besides a known linker, a β sheet structure or an α helix structure may be formed.

  When the capture agent is a peptide as a whole, it can be obtained by methods well known to those skilled in the art, such as genetic engineering methods, in addition to known chemical synthesis methods. In addition, in the case of connecting an assembly element to a capture region, a person skilled in the art can connect this type of compound by a known method, for example, when the assembly element is a peptide or a nucleic acid. Can be implemented as appropriate.

  In addition, the capturing agent may include a labeling substance. By providing the labeling substance, it becomes possible to easily identify the capture agent that captured the rare earth species, which is convenient for separation and recovery. The label is not particularly limited, and various known labeling substances can be used. The labeling substance may be visually identifiable or may emit light by light of a predetermined wavelength. In addition to being colored, it may be capable of coloring by reaction with other compounds. The labeling substance may be held on a carrier such as a bead. Examples of such labeling substances include colored beads, colloidal gold, fluorescent compounds, and enzyme proteins. The labeling substance includes label binding substances such as those using an antibody-antibody reaction and those using a biotin-avidin bond. A label-binding substance is a substance that binds a labeling substance, and such a label-binding substance can also function as a labeling substance.

  The labeling substance is bound by a known method. Typically, the peptide is attached to the N-terminus and / or C-terminus of the peptide via an appropriate number of linker peptides.

  When two or more types of capture agents having capture regions having different capture specificities for rare earth species are used in combination, different labeling substances can be provided for each capture agent.

  Furthermore, the capture agent may be provided with a tag so that it can be recovered using affinity chromatography or the like. The tag may be an antigen (epitope) or the like, and in addition to a known His tag, biotin or the like can be used. Such tags may be linked via a suitable linker.

  Furthermore, the peptide can be identified without labeling the capture agent by obtaining an antibody against the capture agent or capture region and using the antibody.

  The scavenger can further comprise other functional peptide chains, and can be a composite material having such other functions in addition to the ability to capture rare earth species. The composite material is composited so that the rare earth species trapping ability of the trapping region is substantially maintained. The fact that the rare earth species capturing ability is substantially maintained means that the specificity of the initial rare earth species capturing ability is maintained, and the degree (amount) of capturing of the specific rare earth species by the initial rare earth species capturing ability is preferably 50% or more, more preferably 60% or more, still more preferably 70% or more, still more preferably 80% or more, and still more preferably 90% or more. Whether or not the initial rare earth species capturing ability of the fusion protein is maintained can be confirmed based on examples and the like described later.

  Other functional peptide chains can have the desired properties and functions. Such a peptide chain can impart functions other than the separation, recovery and identification of the capture agent described above to the capture agent. As such peptide chains, various known proteins such as antibodies, enzymes, membrane proteins, and the like can be selected as necessary. Those skilled in the art can obtain such composite materials by well-known genetic engineering techniques or chemical techniques.

(Capturing rare earth species with scavengers)
The capture of the rare earth species by the capture agent corresponds to the rare earth species capture ability of the capture region of the capture agent. That is, when the capture region has a rare earth mineral capturing ability, the rare earth species can be captured in a form that comes into contact with and captures a rare earth mineral such as a rare earth oxide or hydroxide. Further, when the trapping region has mineralization ability, the rare earth species can be trapped in the form of mineralization that contacts and traps rare earth ions to generate rare earth minerals such as rare earth hydroxides.

(Capturing rare earth species with the ability to capture rare earth minerals)
The conditions for capturing the rare earth mineral by the scavenger are not particularly limited as long as the scavenger can capture the rare earth mineral. For example, the capture agent and the rare earth mineral may be brought into contact under appropriate conditions and incubated as necessary.

  Such trapping conditions can be obtained by varying the conditions of pH, temperature, and salt concentration of the liquid medium, contacting the trapping agent with the rare earth mineral, and checking the trapping state of the rare earth mineral by the trapping agent. For example, the liquid medium may be any liquid medium as long as the scavenger can capture the rare earth minerals. The liquid medium may be an aqueous medium or an organic solvent, or a mixed medium thereof. Typically, a neutral buffer or the like, a mixed solution containing them, or the like can be used. For example, the pH is not particularly limited, but can be about 5 or more and 8 or less, and the salt concentration is not particularly limited, but can be 10 mM or more and 1 M or less. Further, the temperature is not particularly limited, and can be easily bonded without temperature control. Typically, the temperature can be 4 ° C. or higher and 80 ° C. or lower, more preferably 10 ° C. or higher and 40 ° C. or lower. Preferably, it can be set to 15 ° C. or higher and 30 ° C. or lower. For contact between the rare earth mineral and the scavenger, the contact efficiency can be increased by stirring as appropriate. The time for contact is not particularly limited, but can be about 10 minutes to several hours, preferably 30 minutes to 8 hours, and the upper limit time is more preferably 6 hours or less. More preferably, it can be about 4 hours or less. More preferably, it can be about 1 hour or more and 3 hours or less.

  The concentration of the scavenger and the rare earth mineral concentration in the liquid medium for exhibiting the ability to capture the rare earth mineral by the scavenger is not particularly limited. For example, the scavenger concentration is preferably 20 nM or higher, and the rare earth mineral concentration is preferably 100 μM or higher.

(Rare earth species capture by mineralization ability)
The conditions for capturing rare earth ions by the scavenger or producing rare earth minerals are not particularly limited as long as the mineralization ability of the scavenger is exhibited. As in the case of the rare earth mineral scavenging ability, the scavenger and rare earth ions may be brought into contact under suitable conditions and incubated as necessary.

  In the case of capturing rare earth species by the mineralization ability, a rare earth ion for generating a rare earth mineral and an anion such as an inorganic acid ion or a hydroxide ion that forms the mineral with the rare earth ion (a combination of these mineral ions) May be referred to as a raw material for lysation). Among these anions, inorganic acid ions can be present in the liquid medium used for capture by addition of an acid or salt. Further, hydroxide ions can be contained in a liquid medium containing water by ionization of water or the like.

  As in the case of the rare earth mineral scavenging ability, the trapping conditions are different in the pH, temperature, and salt concentration of the liquid medium, and the trapping agent and the mineralization raw material are brought into contact with each other to trap the rare earth ions by the trapping agent. It can be acquired by checking the state (the state of rare earth mineral production). As the capture conditions, typically, the conditions for capturing the rare earth mineral of the capture agent described above can be adopted. In addition, since the production | generation state and precipitation state of rare earth minerals change with pH, temperature, and / or incubation time, it is preferable to adjust pH adjustment, temperature adjustment, and / or incubation time suitably etc. suitably.

  The peptide concentration and the mineralization raw material concentration in the liquid medium for exerting the mineralization ability of the scavenger are not particularly limited. For example, the concentration of the scavenger is preferably 5 μM or more, and the rare earth ion concentration of the mineralization raw material is preferably 100 μM or more. Further, other mineralization raw materials other than the rare earth ions are preferably the same as the rare earth ion concentration.

  In addition, the rare earth mineral produced | generated by mineralization can be acquired as an insoluble matter (precipitate) etc. in the liquid medium in incubation. A rare earth mineral separated from the capture agent can be obtained by recovering the solid phase by a known solid-liquid separation means such as centrifugation and separating the capture agent using a surfactant or the like as necessary. . Moreover, a rare earth mineral can be obtained by drying and / or baking as needed. The firing for drying the rare earth mineral obtained by mineralization and crystallization of the amorphous rare earth mineral will be described later.

  Mineralization with a scavenger is advantageous in that it can be realized under simple low-cost conditions.

  Rare earth minerals generated based on the mineralization ability of the scavenger are generated as particles from which rare earth is detected. The generated rare earth mineral is presumed to be a rare earth hydroxide, a rare earth salt, or a rare earth oxide (or a hydrate thereof). The rare earth mineral produced may be crystalline or amorphous at the time of production. In the case of an amorphous material, it can be crystallized by performing a firing step as necessary.

  According to the present specification, DNA such as nucleotides encoding a capture region, a capture agent and a composite material composed of a peptide, DNA for expressing such DNA as a peptide having a predetermined amino acid sequence, and a vector containing the DNA Is also provided. Such DNA acquisition and expression vector construction can be easily carried out by those skilled in the art by techniques well known in the art. The elements of the expression vector are selected according to the type of host cell for expressing the DNA encoding the capture region, capture agent, and the like.

(Capturing agent-immobilized solid phase carrier)
The capture agent may be held on a solid phase carrier. For example, it may be held in a granular material such as various beads or a sheet-like material made of various materials. Such solid phase carriers are known and can be appropriately selected and used by those skilled in the art. The form and method of immobilizing peptides on the surface of such a solid support are known. A person skilled in the art can appropriately select an immobilization technique, select a desired form (such as an immobilization pattern on a sheet-like solid phase carrier), and obtain a peptide solid phase carrier. The particulate solid phase carrier can typically take a form in which the present peptide is held on the entire surface of the solid phase carrier by dipping or the like. Further, the sheet-like solid phase carrier can take a form in which the present peptide is held in a film form or in an arbitrary pattern by dipping, coating, spotting or the like.

  When two or more capture agents having different capture capacities for rare earth species (capture selectivity, etc.) are immobilized on a solid phase carrier, each capture agent may be immobilized on a particulate solid phase carrier having different discrimination characteristics. Good. In addition, each capture agent may be immobilized on a sheet-like solid phase carrier as a spot based on different position information.

  In addition, the capture agent may be accompanied by a biological carrier. Specifically, the capture agent may be in a state presented on the surface layer of a biological carrier such as a cell or a state constituting the surface layer. Examples of biological carriers include various microorganisms, plant cells, animal cells, viruses and phages. This peptide may be displayed, for example, on the surface of a microorganism such as yeast or Escherichia coli, or may be configured as an outer shell protein in a phage or virus.

  It is also preferred that the capture agent is held on a solid phase carrier in the form of a multimer. This makes it possible to capture rare earth species efficiently on the solid support.

  As described above, the scavenger can be used as a rare earth species scavenger, a rare earth mineral recovery agent, a rare earth ion recovery agent, and a mineralization agent (rare earth mineral production agent). In addition to these, it can also be used as a detection agent (probe) for rare earth species. Furthermore, the capture agent-immobilized solid phase carrier is useful for any of these applications. The capture agent-immobilized solid phase carrier is also useful as a column or array device for chromatography, etc. for recovering, separating, and detecting rare earth species.

(Rare earth species capture method)
The method for capturing rare earth species according to the present disclosure can include a step of bringing a capturing agent into contact with the rare earth species. According to this method, the rare earth species can be separated, recovered and detected by capturing the rare earth species. This method can also capture rare earth species with a scavenger to form a complex, so that the rare earth species can be provided with an index for separation, recovery and detection based on the scavenger. .

  The capture of rare earth species can include two capture forms, as already described. The rare earth species can be captured by applying the capture conditions already described according to each of these modes. As the capture agent, a monomer or multimer of the capture agent can be used, or a capture agent-immobilized solid phase carrier can be used.

  In capturing the rare earth species, the capture agent is brought into contact with a sample that may contain the rare earth species. Examples of such samples include various recycled materials such as coal ash and petroleum ash that may contain rare earth species, and various resource materials such as mine resource materials and marine resource materials.

(Rare earth species recovery method)
(Rare earth mineral recovery method)
The method for recovering rare earth minerals of the present disclosure can recover rare earth minerals based on the ability to capture rare earth minerals in the capture region of the capture agent. That is, the rare earth mineral can be recovered by bringing a capturing agent having a capture region having a rare earth mineral capturing ability with respect to the target rare earth mineral into contact with a sample in which the target rare earth mineral may exist.

  In order to recover the rare earth mineral, it is preferable to use a scavenger (complex) capturing the rare earth mineral. For example, it can be recovered using a capture region in the capture agent, a tag and a label attached to the capture agent, and the like. For example, an appropriate recovery step can be performed using an antibody that specifically binds to these. In this recovery method, the capture agent that captures the rare earth mineral can be used as a recovery index, so that efficient recovery is possible. When a capture agent-immobilized solid phase carrier is used, it is advantageous because the capture agent has already been separated into a solid phase.

  Furthermore, for the recovery of the rare earth mineral from the complex of the rare earth mineral and the scavenger, for example, a mixed liquid of isopropanol, methanol / acetone (for example, 1: 1), or a surfactant solution can be used. That is, using these various liquids, the rare earth is separated from the capture agent and recovered by a treatment such as denaturation of the capture agent or contact with a solvent that blocks the interaction between the capture agent and the rare earth species. be able to. The treatment for eliminating the interaction between the scavenger and the rare earth species in the complex and the degree thereof can be appropriately selected according to the bond strength with the rare earth species and the scavenger.

  Moreover, the recovery of the rare earth mineral from the composite may be such that the scavenger disappears by firing the composite.

(Rare earth ion recovery method and rare earth mineral production method)
The method for recovering rare earth ions and the method for producing rare earth minerals of the present disclosure can separate and recover rare earth ions and can produce rare earth minerals based on the mineralization ability of the capture region of the capture agent. That is, rare earth ions are recovered or rare earth minerals by contacting a capture agent having a capture region having a mineralization ability with respect to the target rare earth ions and a sample in which the target rare earth ions may exist. Can produce.

  Both of these methods are based on the mineralization ability of the capture region. Rare earth ions are finally recovered from the sample as rare earth minerals. Rare earth minerals can be recovered by the method described above. The recovered rare earth mineral can be dissociated as ions, for example, by appropriately dissolving it as necessary.

  As described above, the production of the rare earth mineral can include a firing step for recovering the rare earth mineral from the composite. In addition, when the rare earth mineral is amorphous, a firing step of the composite or the collected rare earth mineral for promoting crystallization can be provided. In the case of a rare earth mineral hydroxide or the like, a firing step for dehydrating the mineral to convert it into an oxide or the like can be provided.

  The firing step for crystallization can be performed based on known conditions for crystallizing a known amorphous compound. For example, the heating temperature can be 300 ° C. or higher and 1500 ° C. or lower. When the rare earth mineral is an inorganic salt such as carbonate, a heating temperature for crystallization while maintaining the state of the inorganic salt can be set. In the case where an oxide is obtained by desorbing an inorganic acid such as dehydration from a hydroxide or decarboxylation from a carbonate, the temperature at which the desorption occurs can be appropriately selected.

  This production method also has the advantage that it can produce particles of rare earth minerals at the nm level.

  Note that the various methods including the rare earth species capturing method described above can be implemented as a rare earth species detection method by including a rare earth species detection step. As a detection process, in addition to specifically detecting rare earth species captured by the capture agent, based on the selectivity of the capture agent with respect to the rare earth species, the capture region or tag of the capture agent is detected as a detection index. Can do.

(Screening method for peptides having ability to capture rare earth species)
The method for screening a peptide having the ability to capture rare earth species according to the present disclosure comprises contacting a rare earth species with one or more subject peptides and evaluating the binding ability of the one or more test peptides to the rare earth species. The process to perform can be provided. According to this screening method, a peptide having a capturing ability for rare earth species can be screened. In other words, the capture sequence or capture region can be screened or identified.

  As already described, the rare earth species includes rare earth ions and rare earth minerals. Each of these can be a screening target independently. In the case of rare earth ions, a solution of nitrate or hydrochloride can be used. Moreover, in the case of a rare earth mineral, it can be set as a dispersion liquid etc.

  The rare earth mineral can be a rare earth oxide or the like. By evaluating the capture ability using an oxide as a target, it is possible to screen a capture sequence or capture region having a capture ability for the oxide, a rare earth ion capture ability, and a mineralization ability. For example, when screening for the capture ability of light rare earth species (including mineralization ability), by screening with the capture ability for neodymium oxide, a capture sequence having mineralization ability for neodymium ions and other light rare earths or The capture area can be screened. Consequently, it is possible to screen a capture sequence having a mineralization ability for heavy rare earths.

  The test peptide is not particularly limited and includes a peptide having an artificial amino acid sequence in addition to a natural amino acid sequence. The peptide length is not particularly limited, but as already described, the number of amino acid residues can generally be about 100 or less, and typically, the number of amino acid residues is preferably 5 or more, and more Preferably it is 7 or more, more preferably 8 or more. Further, it may be 25 or less, 20 or less, or 15 or less. The test peptide may be a cyclic peptide. Cyclic peptides can be formed by disulfide bonds with two or more cysteine residues.

  The test peptide may be a natural L-form polymer or a non-natural D-form polymer. Furthermore, the test peptide may contain an artificial amino acid residue.

  The test peptide can be subjected to screening by a method of displaying various peptides using, for example, a phage display method, a ribosome display method, an in vitro virus method and the like. In addition, it can be obtained by chemical synthesis, genetic engineering synthesis including a cell-free protein synthesis system, etc., and used for screening. The test peptide may also belong to a mutant library prepared by a known technique based on a certain amino acid sequence. Furthermore, the test peptide may be in the form of being held on a solid phase carrier or a biological carrier, similar to the capture agent.

  As already described for the capture region, the test peptide can be provided with a labeling substance or a tag so that it is convenient for evaluating the capture ability of rare earth species. Alternatively, when the amino acid sequence of the test peptide is already known, an antibody that specifically binds to the test peptide can be prepared in advance. Use of a test peptide having such an adduct is suitable for secondary screening for further evaluating the binding ability with rare earth species.

  As described above, the test peptide and the rare earth species may be contacted with the rare earth species in a state where denaturation of the test peptide is suppressed. Typically, the test peptide and the rare earth species are brought into contact in a liquid medium such as an aqueous medium having the capture conditions as described above.

  The contact form of one or more rare earth species and the test peptide is not particularly limited. In particular, in the secondary screening for evaluating the capture ability of rare earth species with high accuracy, for example, a rare earth species-immobilized solid phase carrier in which rare earth species are immobilized on a solid phase carrier in an array can be used. By using such a rare earth species-immobilized solid phase carrier, it is possible to evaluate the capture ability of a plurality of rare earth species and a plurality of test peptides all at once. In the rare earth species-immobilized solid phase carrier, for example, the rare earth species may be immobilized in a well formed on a sheet (plate) solid phase carrier. The rare earth species is fixed by supplying a dispersion of rare earth species to the surface of glass or plastic, for example, by drying in a vacuum or the like.

  Next, the rare earth species and the test peptide come into contact with each other, and the complex in which the test peptide captures the rare earth species is recovered. The complex can be identified and recovered through the peptide itself or a labeling substance attached thereto.

  When recovering the complex, the rare earth species and test peptide that do not form the complex and the complex should be removed as the supernatant by a separation method using a mass difference such as centrifugation because the mass is smaller than the complex. Thus, the complex can be purified as a centrifuge deposit.

  In recovering the complex, a surfactant solution is added to the recovered complex to release the weak or nonspecific capture state between the test peptide and the rare earth species, and then centrifuged again. It can be separated and removed as a supernatant or the like. Thereby, the complex by the test peptide which captured the rare earth species more strongly and / or with higher specificity can be selected and recovered.

  By performing such a washing operation, it is possible to screen a test peptide that captures the rare earth species strongly and / or specifically with higher accuracy. In addition, by conducting a plurality of screenings combined with these washing operations, the concentration of the test peptide for the rare earth species is increased, so that a test peptide having a stronger and / or more specific capture ability for the rare earth species can be screened. become. Such a screening set is preferably performed 3 times or more, more preferably 4 times or more, still more preferably 5 times or more, and still more preferably 6 times or more. The effect of the washing operation can be generally recognized up to about 10 times.

  The evaluation of the rare earth species capturing ability is performed by specifying the complex or the test peptide that has formed the complex and measuring the capture amount of the rare earth species. The amount of rare earth species captured is obtained by a quantitative method according to the turbidity of rare earth minerals and the type of rare earth species. Thereby, the test peptide which has a capture ability with respect to the target rare earth species can be screened. When the amino acid sequence of the test peptide has already been specified, the amino acid sequence can be identified or screened as a capture sequence by affirming the capture ability for the targeted ionic species. In some cases, such as when the test peptide is obtained by the phage display method, the amino acid sequence may be unknown. In this case, by performing the sequence analysis of the test peptide, the capturing ability can be affirmed and identified or screened for the ion species targeting the amino acid sequence.

  For example, as a rare earth species, a rare earth mineral such as a rare earth oxide can be used as a target, and a test peptide having a strong capturing ability for the target can be screened. Thereby, in addition to the rare earth mineral capturing ability of the rare earth, it is possible to screen a capture sequence or a capture region having the mineralization ability of the rare earth ion.

  The screening method as described above can be performed, for example, as a primary screening method for a specific rare earth species. In particular, the primary screening is preferably performed using a phage display method or the like that can use peptides having various amino acid sequences as test peptides. Moreover, it is preferable to involve repetition of the set of cleaning operations as described above (panning in the examples).

  This screening method is also effective for secondary screening of test peptides and their mutants that have been confirmed to be capable of capturing rare earth species by primary screening. In addition, secondary screening such as evaluating the capture ability for a specific rare earth species or a plurality of rare earth species is performed using a solid phase carrier (array or the like) in which the target rare earth species is immobilized on a solid phase carrier or the like. This is useful for evaluating accuracy.

  According to this screening method, it is possible to identify and screen a capture sequence or a capture region having a capture ability for a specific rare earth species or two or more rare earth species. Further, it is possible to identify and screen a capture sequence or a capture region having a strong and / or specific capture ability for a specific rare earth species.

  Hereinafter, the disclosure of this specification will be specifically described by way of examples. In addition, the following examples do not limit the present invention.

(Construction of random peptide display type T7 phage library)
Using two kinds of oligonucleotide primers T7-Libup (ATG ATT ACC AGG ATC CGA ATT CAG GTG GAG GTT CG, SEQ ID NO: 7), T7-Libdownt (ACT ATC GTC GGC CGC AAG CTT TTA GCT, SEQ ID NO: 8) A PCR reaction was carried out to amplify the template DNA (CGA ATT CAG GTG GAG GTT CGT GT (SEQ ID NO: 9) + (NNK) 9-12 + TGT AGC TAA AAG CTT GCG GCC GA (SEQ ID NO: 10)).
N = A25%, T25%, G25%, C25% (A / T / G / C equal amount mixed base)
K = A 0%, T50%, G50%, C0% mixed base

  The amplified DNA fragment was subjected to phenol treatment and butanol concentration according to a general protocol, and then purified using QIAquick PCR Purification kit (QIAGEN). The purified DNA was treated with restriction enzymes Hind III and Eco RI (Takara Bio), and then ligated to T7 select 10-3 vector arm (Novagen) to construct a T7 phage genome.

The constructed phage genome was mixed with a T7select packaging solution (Novagen) to construct a T7 phage having a random peptide gene-introduced T7 genomic DNA. At this time, when the number of phage populations constructed using a part was counted, it was confirmed that a phage library having a sequence diversity of 1.0 × 10 ^{6 to} 4.0 × 10 ⁷ was constructed. .

Phage populations constructed, amplified E. coli is infected with E. coli BLT5403 strain was cultured until OD _660nm = 0.6~1.0, 8% polyethylene glycol, and concentrated and purified by performing a 0.22μm filter. As a result of counting the number of phages after purification, each library has about 1.0 × 10 ¹² pfu / ml of phage, and is amplified 100,000 to 1,000,000 times per one kind of peptide phage. It was confirmed.

(Biopanning for neodymium oxide using random peptide-displaying T7 phage library)
Using the random peptide-displaying T7 phage library prepared in Example 1, an attempt was made to isolate a T7 phage that displays a peptide that binds to neodymium oxide (Nd ₂ O ₃ ) (Sigma-Aldrich). A series of schemes is shown in FIG.

  The neodymium oxide was washed with a methanol: acetone mixture (1: 1), then washed with isopropanol, and then dispersed in TBS (50 mM Tris, 150 mM NaCl).

  A solution in which 500 µg of neodymium oxide is dispersed is mixed with the T7 phage library, reacted for 1 hour at room temperature, and then the particles are precipitated by centrifugation (6000 rpm, 3 minutes), and the supernatant is removed to remove unbound phage. did.

  After removing the supernatant, TBST was added to disperse the neodymium oxide, and centrifugation was performed again to remove the phage nonspecifically bound to the particles. This operation (washing operation) was repeated 3 to 10 times to remove peptide phage that nonspecifically bound to neodymium oxide.

Unbound phage was removed by washing the phage non-specifically bound, mixed with E. coli E. coli BLT 5403 solution 10ml cultured until OD _{660 nm} = 0.6 to 1.0, E. coli totally lysis The culture was performed at 37 ° C. until

  After E. coli was completely lysed, 1/10 volume of 5M NaCl was added to the culture solution and centrifuged (3500 rpm, 15 minutes) to precipitate insoluble fractions such as cell walls of E. coli, and the supernatant was collected. .

  A 1/6 amount of 50% polyethylene glycol 6000 solution was added to the collected supernatant, and after stirring, centrifuged at 3500 rpm for 15 minutes to precipitate T7 phage. The precipitated T7 phage population was dissolved in TBS solution, 0.22 μm filtered, and stored at 4 ° C. until use.

(Confirmation of enrichment of neodymium oxide binding phage by biopanning)
After repeating the series of operations of Example 2 5 times, the number of phages bound to neodymium oxide was measured for the phages after each round.

  First, neodymium oxide was washed with a methanol / acetone mixed solution (1: 1), then washed with isopropanol and then dispersed in TBS.

  A solution in which 500 μg of neodymium oxide was dispersed and the phage pool after each round were mixed, reacted for 1 hour at room temperature, and then centrifuged (6000 rpm for 3 minutes) to remove the supernatant. Next, a washing operation was performed 10 times with TBST.

  After washing, the number of phage bound to the neodymium oxide particles was measured by plaque assay. The results are shown in FIG. As shown in FIG. 2, as the number of panning rounds increased, an increase in the number of bound phages against neodymium oxide particles was confirmed. From these results, it was found that by repeating rounds, it was possible to screen for phages displaying peptides excellent in binding ability to neodymium oxide.

(Screening of monoclonal phage displaying peptides that bind to neodymium oxide)
The phage pool after 5 rounds of panning was subjected to single cloning, and the resulting 96 types of phages were measured for binding to neodymium oxide particles.

  Each phage was mixed with 500 μg of neodymium oxide washed and dispersed in TBS, reacted for 1 hour at room temperature, centrifuged (6000 rpm, 3 minutes), and the supernatant was removed. Subsequently, washing operation was performed 10 times with TBST. After washing, the number of phage bound to neodymium oxide particles was measured by plaque assay. The results are shown in FIG. As shown in FIG. 3, 11 types of clones were confirmed to bind to neodymium oxide.

  These clones were subjected to sequence analysis of the presented peptides. As a result, clones 72, 79, 85, 88, 89 and 95 shown in FIG. 3 have the same amino acid sequence SerCys-Pro-Val-Trp-Phe-Ser-Asp-Val-Gly-Asp-Phe-Met-Val- It was found to have CysSer (SEQ ID NO: 3). This sequence was named Lamp-3.

(Preparation of synthetic peptide)
A synthetic peptide was prepared by Fmoc solid phase synthesis for the peptide (Lamp-3) displayed by each phage that was confirmed to bind to neodymium oxide. The N-terminus of the synthetic peptide was biotinylated via GGG derived from the g10 sequence.

  The peptide cut out from the resin and deprotected was formed with intramolecular disulfide bonds with an appropriate oxidizing agent such as iodine, then purified by reverse phase HPLC and lyophilized.

(Evaluation of mineralization phenomenon of synthetic peptides)
Mineralization ability was evaluated using the synthetic peptide (Lamp-3) prepared in Example 5. The Lamp-3 peptide was diluted to 10 μM (DMSO 3%) in HEPES buffer (HEPES 1 mM, pH 6.2), pH was adjusted, and 5 types of lanthanoid ions were added individually and incubated at room temperature with shaking. . After 5 hours, the mixture was centrifuged at 15000 rpm for 10 minutes. After removing the supernatant, 100 μl of ultrapure water was added and the mixture was thoroughly stirred and washed. Centrifugation and agitation washing were repeated twice, and then the precipitate was dispersed with 20 μl of ultrapure water, and 10 μl was dropped on the carbon tape and left to dry completely in the clean bench. The analysis results by SEM / EDX (Hitachi High Technologies) are shown in FIG. 4 and FIG.

  As shown in FIGS. 4 and 5, it is confirmed that the Lamp-3 peptide recognizes any of the five types of lanthanoid ions (La, Nd, Sm, Dy, Lu) and generates particles of each rare earth mineral. did. From the above results, it was found that Lamp-3 peptide can be widely mineralized widely from light rare earth to heavy rare earth ions.

Sequence number 1-6: Synthetic peptide Sequence number 7-10: Synthetic nucleotide

Claims (16)

A capture agent for a rare earth species comprising a peptide region containing the following amino acid sequence: SerCysProValTrpPheSerAspValGlyAspPheMetValCysSer .   The scavenger according to claim 1, wherein the rare earth species is a light rare earth species.   The scavenger according to claim 2, which captures the oxide of the light rare earth species.   The scavenger according to claim 2 or 3, wherein the light rare earth species ions are mineralized and captured.   The scavenger according to any one of claims 2 to 4, wherein the light rare earth species is derived from neodymium. The capturing agent according to any one of claims 1 to 5 , comprising two or more peptide regions . A peptide comprising the following amino acid sequence: SerCysProValTrpPheSerAspValGlyAspPheMetValCysSer . A complex comprising a rare earth species and the scavenger according to any one of claims 1 to 6 . And rare earth species, the contacting step of contacting a capture agent according to any one of claims 1 to 6
A method for capturing rare earth species. The capturing method according to claim 9 , wherein in the contacting step, the rare earth species is a light rare earth ion, and the rare earth species is captured as a mineral derived from the light rare earth ion. The capturing method according to claim 9 , wherein in the contacting step, the rare earth species is an oxide of light rare earth. The capturing method according to claim 9 , wherein in the contacting step, the rare earth species is a light rare earth ion. The capturing method according to claim 9 , wherein the rare earth species is derived from neodymium. Contacting the rare earth species with the scavenger according to any one of claims 1 to 6 to recover the rare earth species;
A method for recovering rare earth species. Contacting the rare earth species with the scavenger according to any one of claims 1 to 6 to detect the rare earth species;
A method for detecting rare earth species. A method for screening peptides having the ability to capture light rare earth species,
A step of contacting the test peptide with a light rare earth oxide and evaluating the ability of the test peptide to capture the light rare earth oxide;
A method comprising: