Protein-containing compositions for rare earth element separation and methods of use thereof

Abstract

This disclosure provides REE-binding proteins for preferential separation of REEs, as well as methods of their use.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Patent Application No. 63 / 210,311, filed June 14, 2021, the disclosure of which is incorporated by reference in its entirety as if fully set forth herein, including all references and appendices filed therewith.

[0002] STATEMENT REGARDING FEDERALLY FUNDED RESEARCH The U.S. Government owns rights in this application pursuant to Contract No. DE-AC52-07NA27344 between the U.S. Department of Energy and Lawrence Livermore National Security, LLC, and Contract No. SC-DE0021007 between the U.S. Department of Energy and The Pennsylvania State University, for the operation of the Lawrence Livermore National Laboratory. [Background technology]

[0003] Rare earth elements ("REE") are mined from the Earth's crust. Due to their unique physical and chemical properties, these elements are important in an increasing number of advanced technology products including high performance magnets, lasers, computer memory, mobile phones, catalytic converters, camera and telescope lenses, and environmental technologies such as wind turbines and hybrid vehicles, to name a few. Although many countries, including the United States, produce REEs, China remains the dominant producer of REEs, accounting for 70-90% of the world's supply. REEs are difficult to mine, in part because they are rarely found in high enough concentrations for economical extraction. The use of GPS-controlled drills and gamma-ray sampling allows geologists to identify ores that contain more REEs. The ores are often contaminated with naturally occurring radioactive materials such as thorium, and current methods for REE extraction and processing require large amounts of carcinogenic toxins such as organic solvents, ammonia salts, alkyl phosphorus-containing extractants, and strong mineral acids. Leaching and separation of the metals has high energy / capital costs, high CO2 emissions, and many unfavorable health and environmental impacts. As demand for REEs continues to soar, there remains a need for tools to help increase and diversify REE supplies, develop clean, low-cost extraction processes, improve efficiency, and recapture REEs through reuse and recycling, particularly those capable of preferentially separating REEs from REE feedstocks with low REE content relative to non-REEs, with an emphasis on maximizing efficiency and minimizing waste. Summary of the Invention

[0004] Methods and materials are provided for preferential separation of rare earth elements (REEs) from non-REEs in an REE-containing material, and for separation of specific REEs from other REEs, both individually and in groups. In certain embodiments, such methods include the steps of: (a) providing a protein capable of selectively binding to one or more REEs; (b) contacting the protein with an REE-containing material, wherein the protein binds to at least a portion of one or more REEs to form one or more protein-REE complexes and an REE-depleted material; (c) separating the one or more protein-REE complexes from at least a portion of the REE-depleted material; and (d) separating the one or more REEs from the protein to produce a purified fraction of the one or more REEs and regenerated the protein. In another embodiment, a method is provided for preparing a material for rare earth element (REE) separation, comprising the steps of: (a) providing a protein capable of selectively binding to REEs and a porous support material functionalized with a conjugation agent; and (b) binding the protein to the porous support material by the conjugation agent.

[0005] In certain embodiments, a method for preferentially separating rare earth elements (REEs) from an REE-containing material is provided, the method comprising the steps of: (a) providing a plurality of proteins capable of selectively binding one or more REEs; (b) contacting the plurality of proteins with an REE-containing material, wherein the plurality of proteins binds to at least a portion of one or more REEs to form a plurality of protein-REE complexes and an REE-depleted material; (c) separating the plurality of protein-REE complexes from at least a portion of the REE-depleted material; and (d) separating the plurality of protein-REE complexes from a first solution comprising a chelating agent or a solution having a first pH. (g) preferentially separating heavy REEs (HREEs) from the plurality of proteins by contacting the plurality of protein-REE complexes with a first solution comprising a chelating agent or a second solution having a second pH; and (h) separating light REEs (LREEs) from the plurality of proteins by contacting the plurality of protein-REE complexes with a third solution comprising a chelating agent or a third solution having a third pH.

[0006] In certain embodiments, a method is provided for preferentially separating scandium and yttrium from an REE-containing material using a chelating agent, the method comprising the steps of: (a) providing a plurality of proteins capable of selectively binding one or more REEs; (b) contacting the plurality of proteins with an REE-containing material, wherein the plurality of proteins binds to at least a portion of one or more REEs to form a plurality of protein-REE complexes and an REE-depleted material; (c) separating the plurality of protein-REE complexes from at least a portion of the REE-depleted material; and (d) preferentially separating scandium from the plurality of proteins by contacting the plurality of protein-REE complexes with a first chelating agent solution. (e) preferentially separating yttrium from the plurality of proteins by contacting the plurality of protein-REE complexes with a second chelator solution; (f) separating heavy (HREE) and medium REE (MREE) from the plurality of proteins by contacting the plurality of protein-REE complexes with a solution comprising a third chelator solution; and (g) separating light REE (LREE, in this case La and Ce) from the plurality of proteins by contacting the plurality of protein-REE complexes with a fourth chelator solution or a solution having a low pH (<1.7). In certain embodiments, the first solution contains malonate at a concentration of 20-50 mM, the second solution contains citrate at a concentration of about 15.0 mM, the third solution contains citrate at a concentration of about 25 mM to about 50 mM, and the fourth solution has a pH of about 1.5.

[0007] In certain embodiments, a method is provided for preferentially separating scandium from an REE-containing material using pH, the method comprising the steps of: (a) providing a plurality of proteins capable of selectively binding one or more REEs; (b) contacting the plurality of proteins with an REE-containing material, wherein the plurality of proteins binds to at least a portion of one or more REEs to form a plurality of protein-REE complexes and an REE-depleted material; (c) separating the plurality of protein-REE complexes from at least a portion of the REE-depleted material; (d) preferentially separating yttrium and HREEs from the plurality of proteins by contacting the plurality of protein-REE complexes with a solution having a first pH, (e) preferentially separating MREEs and LREEs from the plurality of proteins by contacting the plurality of protein-REE complexes with a solution having a second pH; and (f) separating scandium from the plurality of proteins by contacting the plurality of protein-REE complexes with a solution comprising a solution having a third pH.

[0008] In certain embodiments, a method for separating rare earth elements (REEs) from an REE-containing material comprises an adsorption / desorption cycle, in such embodiments, the adsorption / desorption cycle comprises the steps of: loading a column containing a biosorption material with the REE-containing material, the biosorption material comprising a protein capable of adsorbing REEs present in the REE-containing material to form one or more protein-REE complexes and an REE-depleted material; and separating one or more REEs or one or more groups of REEs from the one or more protein-REE complexes using a desorption process comprising eluting a first REE-enriched fraction with a first solution capable of preferentially separating the one or more REEs or one or more groups of REEs from the one or more protein-REE complexes. In some embodiments, the desorption process comprises a second step of eluting a fraction enriched in a second REE from the column with a second solution capable of desorbing one or more REEs from one or more protein-REE complexes. In other embodiments, the desorption process comprises a third step of eluting a fraction enriched in a third REE from the column with a third solution capable of desorbing one or more REEs from one or more protein-REE complexes. Each of the enriched fractions may have a higher concentration of REEs than the feed solution (i.e., the REE-containing material).

[0009] In some embodiments, a second adsorption / desorption cycle may be performed after the first adsorption / desorption cycle. In such embodiments, the second adsorption / desorption cycle comprises: loading a column containing the biosorption material with a fraction enriched in a first REE to form one or more protein-REE complexes; and separating one or more REEs from the one or more protein-REE complexes using a desorption process comprising: eluting the fraction enriched in the first high-purity REE from the column with a first solution. In some embodiments, the desorption process of the second cycle comprises a second step of eluting the fraction enriched in the second high-purity REE from the column with a second solution. In other embodiments, the desorption process further comprises a third step of eluting the fraction enriched in the third high-purity REE from the column with a third solution.

[0010] In certain embodiments, each of the first, second and third solutions is designed to have a specific selected pH, includes a chelating agent, or both, to achieve the claimed preferential separation. In some embodiments, the first solution has a first selected pH. In some aspects, the first selected pH may be 3.0 or less, 2.5 or less, 2.0 or less, or about 1.5 or less. In other embodiments, the first solution includes a chelating agent. In certain aspects, the chelating agent is citrate. In some embodiments, the citrate is present in the first solution at a concentration of about 3.0 mM or about 15.0 mM. When a chelating agent is used, the pH of such a solution is greater than 3.0, and in some embodiments, the chelating agent solution has a pH greater than 4.0, between about 4 and 7, or greater than 7.

[0011] In some embodiments, the second solution comprises a second selected pH. In some embodiments, the second selected pH is lower than the first selected pH. In certain aspects, the second selected pH is 2.5 or less, 2.0 or less, or 1.5 or less. In other embodiments, the second solution comprises a chelating agent. In certain aspects, the chelating agent is citrate. In some embodiments, the second solution comprises a higher concentration of citrate than the first solution. In some embodiments, the citrate is present in the second solution at a concentration of about 15.0 mM citrate. In some embodiments, the citrate is present in the second solution at a concentration of between about 25.0 and about 50.0 mM citrate. In some embodiments, the citrate is present in the second solution at a concentration of about 75.0 mM citrate. When a chelating agent is used, the pH of such a solution is greater than 3.0; in some embodiments, the chelating agent solution has a pH greater than 4.0, greater than 5.0, between about 5 and 7, or greater than 7.

[0012] In some embodiments, the first solution and the second solution have a pH between about 2.5 and 1.5. In other embodiments, the first solution comprises a pH between about 2.0 and about 2.5, and the second solution comprises a pH between about 1.5 and about 2.0. In other embodiments, the first solution comprises a pH of 2.1, and the second solution comprises a pH of 1.7. In some embodiments, the first solution comprises a citrate concentration of about 3.0 mM, and the second solution comprises a citrate concentration of about 15 mM. In other embodiments, the first solution comprises a citrate concentration of about 15.0 mM, and the second solution comprises a citrate concentration of about 25 mM to about 50 mM. In another embodiment, the third solution comprises a third selected pH. In another embodiment, the third selected pH is lower than the second selected pH and the first selected pH is lower than the second selected pH. In another embodiment, the third selected pH is about 1.5 or less or about 1.5. In another embodiment, the third solution comprises a chelating agent selected from EDTA, citrate, dimercaprol, malonate, iminodiacetate, diglycolic acid, hydroxypyridinone, hydroxamate, catechol, polyaminocarboxylate, acetate, nitrilotriacetate, dipicolinic acid, α-hydroxyisobutyric acid, or other mono-, di-, and tricarboxylic acids. In another embodiment, the third solution comprises citrate at a higher concentration than the second solution. In another embodiment, the first solution comprises about 75.0 mM citrate.

[0013] In another embodiment, the first solution comprises citrate at a concentration of about 15.0 mM, the second solution comprises citrate at a concentration of about 25 mM to about 50 mM, and the third solution has a pH of about 1.5. In certain embodiments, the methods described herein also include a step of refolding proteins using a clearing solution after eluting the REE-enriched fraction or fractions or the high purity REE-enriched fraction or fractions. In certain embodiments, the cleaning (or clearing) solution comprises a buffer and / or a solution with a pH of about 1.0 (e.g., HCl or H2SO4 or a HCl / H2SO4 mixture).

[0014] In certain embodiments, the protein used in accordance with the present disclosure is lanmodulin (LanM). In some embodiments, LanM has an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3. In other embodiments, LanM has an amino acid sequence having at least 95% identity to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3. In some embodiments, a biosorption material is provided. The biosorption material may be comprised of microbeads for rare earth element (REE) separation, comprising a protein capable of selectively binding at least one REE, the protein being embedded in the bead or on its surface. In one particular embodiment, the protein is LanM.

[0015] In certain embodiments, the biosorption material is comprised of microbeads. In certain embodiments, the beads comprise agarose. In certain embodiments, the concentration of protein in the beads is at least about 2 μmol, about 3 μmol, about 4 μmol, about 5 μmol, about 6 μmol, about 7 μmol, about 8 μmol, about 9 μmol, or about 10 μmol of protein per mL of total volume of the beads. In some embodiments, LanM comprises a C-terminal cysteine ​​residue, an N-terminal cysteine, or an internal cysteine. In some embodiments, the cysteine ​​is linked to LanM via a glycine-serine-glycine amino acid linker or a hydrophilic linker.

[0016] In some embodiments, the separating step preferentially separates one or more individual REEs, groups of REEs, REEs that are adjacent to each other in the periodic table, REEs that have similar ionic radii, or combinations thereof, hi certain embodiments, the protein does not bind to non-REE metals in the REE-containing material.

[0017] In certain embodiments, the REE material is rare earth ores, geothermal brines, coal, coal by-products, mining tailings, acid mine drainage, phosphogypsum, end of service products, electronic waste, industrial wastewater, or total REE mixtures derived from REE sources, such as leachates derived from salts (e.g., oxides, oxalates, carbonates, etc.) derived from these ores. In some embodiments, the REE material is in an aqueous solution. [Brief description of the drawings]

[0018] [Figure 1]FIG. 1 shows an exemplary method for separation of REEs using immobilized LanM according to an embodiment of the present disclosure. [Figures 2A-2D] 2A-2D show exemplary schemes, plots, and images confirming the immobilization of LanM on agarose microbeads according to embodiments of the present disclosure. Immobilization of LanM on agarose microbeads via thiol-maleimide click chemistry (FIG. 2A) was confirmed by Fourier transform infrared spectroscopy (FTIR) of aminoagarose, N-succinimidyl 4-(maleimidomethyl)cyclohexane-1-carboxylate (SMCC), maleimide agarose, and LanM agarose (FIG. 2B), LanM immobilization kinetics (FIG. 2C), and fluorescence microscopy (FIG. 2D, scale bar is 100 μm, lower image is a split channel of the upper image). [Figure 3A-3G] 3A-3C show exemplary plots confirming that immobilized LanM retains the ability to bind REEs at low pH and is stable for reuse according to embodiments of the present disclosure. The plots include Nd breakthrough curves as a function of pH (FIG. 3A), Nd desorption curves as a function of HCl concentration (FIG. 3B), plots confirming column reusability based on Nd breakthrough curves using 10 consecutive adsorption / desorption cycles (FIG. 3C), independent single element breakthrough curves for Y, La, Nd, Dy and Lu at pH 3 (FIG. 3D), metal ion breakthrough curves using a synthetic feed solution to confirm Nd selectivity of the LanM column over non-REEs (FIG. 3E), desorption profiles of metal ions after treatment with HCl at pH 1.5 (FIG. 3F), and breakthrough experiments using a binary Nd / Fe solution containing citrate to evaluate the ability of LanM to preferentially separate Nd from Fe (FIG. 3G). [Figure 4A-4B] 4A-4B show exemplary plots confirming that LanM in solution binds 3 equivalents of REE in accordance with embodiments of the present disclosure, including a competitive titration assay using xylenol orange as an indicator (FIG. 4A) and a plot showing the structural response of LanM to lanthanum tracked as an increase in absorbance at A275 nm (FIG. 4B). [Figure 5A-5B] 5A-5B show exemplary plots suggesting that ligand competition in solution can cause REE intraspecies separation according to embodiments of the present disclosure, including stoichiometric titration monitored by changes in intrinsic tyrosine fluorescence emission of proteins (FIG. 5A) and REE desorption using citrate (FIG. 5B). [Figure 6A-6F] FIG. 1 shows an exemplary plot confirming the ability of immobilized LanM to separate REE pairs using a two-step pH scheme in accordance with an embodiment of the present disclosure. The ability of immobilized LanM to separate REE pairs was confirmed by analyzing the cumulative desorption profiles of single element loaded columns using stepwise pH schemes (Figure 6A, REE ion desorption normalized to total REE desorbed; experimental conditions: independent single REE solutions were used to load the column to 90% saturation); a feed material composed of a 50:50 mixture of Dy:Nd was loaded to 90% column saturation and then subjected to a two-step pH desorption scheme (2.1 and 1.7) (Figure 6B), a feed material composed of a 5:95 mixture of Dy:Nd was loaded to 90% column saturation and then subjected to a two-step pH desorption scheme (2.2 and 1.7) (Figure 6C); a feed material composed of a 22:78 mixture of Y:Nd was loaded to 90% column saturation and then subjected to a two-step pH desorption scheme (2.3 and 1.7) (Figure 6D). FIG. 6E provides a summary of the REE composition in the feed and the three desorption zones by using a two-stage pH scheme. [Figure 7A-7D]FIG. 1 shows an exemplary plot confirming the ability of immobilized LanM to separate REE pairs using a graded chelator concentration and pH scheme in accordance with an embodiment of the present disclosure. The ability of immobilized LanM to separate pairs of REEs was confirmed by analyzing the cumulative desorption profiles of single-element loaded columns using a stepwise citrate concentration (pH 5) scheme (Figure 7A); a feed material composed of a 5:95 mixture of Dy:Nd was subjected to a two-step desorption scheme using 15 mM citrate (pH 5) followed by pH 1.7 (Figure 7B); a feed material composed of a 50:50 mixture of Dy:Nd was loaded to 90% column saturation and then subjected to a two-step desorption scheme using 10 mM citrate (pH 5) followed by pH 1.7 (Figure 7C), and a feed material composed of a 22:78 mixture of Y:Nd was loaded to 90% column saturation and then subjected to a two-step desorption scheme using 10 mM citrate (pH 5) followed by pH 1.7 (Figure 7D) (the values ​​above each panel indicate the purity of the REEs over each elution zone separated by the vertical dotted line). FIG. 6F provides a summary of the REE composition in the feed and the three desorption zones by using a citrate-pH scheme. [Figure 8A-8F] FIG. 1 shows an exemplary plot confirming the ability of immobilized LanM to separate and extract REEs from low grade feedstock leachate. The ability of immobilized LanM to separate and extract REEs from low-grade feed leachates was confirmed using a LanM column for REE recovery from Powder River Basin (PRB) fly ash leachate, and the results were analyzed by the adsorption profile of metal ions (Figure 8A, adsorption conditions: pH 5, 0.5 mL / min. 1 bed volume = 0.8 mL), metal composition in the PRB feed and recovered biosorption solution (Figure 8B); percentage of metal ions (but excluding monovalent ions) in the PRB feed and recovered biosorption solution (Figure 8C), separation factor of total REEs over selected non-REEs (Figure 8D), selective desorption of HREEs (Tb-Lu+Y) and LREEs (La-Gd) by a two-step pH scheme (Figure 8E), and the REE ratios over total REEs in the PRB feed and three elution zones (Figure 8F). [Figure 9] FIG. 13 shows an exemplary plot illustrating the desorption profile of PRB fly ash leachate by using LanM agarose column (desorption condition: HCl at pH 1.5). [Figure 10A-10B] 10A-10B show exemplary plots demonstrating the major differences in LanM-REE stability among the REEs that form the basis for REE separation according to embodiments of the present disclosure. The plots include breakthrough curves for the lanthanide series using a LanM-agarose column (FIG. 10A, Feed: Concentration of each REE: 13 μM; pH 3.0, 10 mM glycine) and the breakthrough points of individual REEs as a function of ionic radius (FIG. 10B). [Figures 11A-11D] 11A and 11C show exemplary plots demonstrating the effectiveness of a LanM column (loaded to 90% capacity) in allowing separation of total REEs into HREE, MREE, and LREE fractions when subjected to a stepwise pH desorption scheme according to an embodiment of the present disclosure. The plots show normalized concentration of each REE versus bed volume (FIGS. 11A and 11C) and cumulative desorption of each REE versus bed volume (FIGS. 11B and 11D). In Figures 11A and 11B, the feed composition was as follows: Y: 72.7 μM; La: 53.4 μM; Ce: 113.5 μM; Pr: 13.4 μM; Nd: 51.5 μM; Sm: 10.2 μM; Eu: 2.2 μM; Gd: 9.5 μM; Tb: 1.4 μM; Dy: 7.9 μM; Ho: 1.4 μM; Er: 4.0 μM; Tm: 0.5 μM; Yb: 3.0 μM; Lu: 0.4 μM. In Figures 11C and 11D, scandium was added to the feed composition used in Figures 11A and 11B at a concentration equivalent to each lanthanide. [Figure 12] 1 shows an exemplary plot illustrating the affinity of LanM for extract Sc according to an embodiment of the present disclosure. The plot shows that LanM exhibits a thermodynamic dissociation constant (known as Kd) for Sc of 1.3E-13 M (0.13 pM) at pH 5. [Figure 13]FIG. 1 shows an exemplary plot illustrating the highly selective malonate-induced dissociation of Sc and Lu from LanM in solution, suggesting that Sc can be separated from the heaviest REE, Lu, and therefore potentially other lanthanides and Y as well according to embodiments of the present disclosure (experiments performed with 20 μM protein in 100 mM KCl, 30 mM MOPS, pH 5). [Figure 14] FIG. 1 shows an exemplary plot showing citrate-induced dissociation data for Lu, La, and Ce, demonstrating the feasibility of using citrate to separate Lu from Y and MREE in accordance with embodiments of the present disclosure. (The data for Nd, Dy, and Y shown in this figure are the same as in FIG. 5.) [Figure 15A-15B]15A-15C show exemplary plots showing that citrate preferentially desorbs scandium, then HREEs, then MREEs, then LREEs in ascending order of ionic radius from a LanM agarose column according to an embodiment of the present disclosure. The plots show the breakthrough curves of REEs chelated by citrate using a LanM agarose column [FIG. 15A, Feed: equimolar (0.013 mM each) REEs (Sc, Y, La, Ce, Pr, Nd, Sm, Eu, Gd, Yb, Dy, Ho, Er, Tm, Yb, and Lu) in 20 mM citrate at pH 3.5 condition] and the stepwise desorption of REEs using different citrate concentrations (FIG. 15B, plot depicts cumulative desorption of each REE versus bed volume). For Figure 15B, experimental conditions: 21 bed volumes of REE solution were pumped onto the LanM column to achieve approximately 90% column saturation, followed by a 10 bed volume wash with deionized water. Then a pH 5 desorption solution (3-75 mM) including a citrate concentration step was pumped onto the column. Feed: equimolar REEs (Sc, Y, La, Ce, Pr, Nd, Sm, Eu, Gd, Yb, Dy, Ho, Er, Tm, Yb, and Lu) in 10 mM glycine at pH 3.0. We demonstrate the effectiveness of the LanM column (loaded to 90% capacity) in allowing the separation of total REEs into HREE, MREE, and LREE fractions when subjected to a stepwise citrate desorption scheme. [Figure 16A-16B]16A-16C show exemplary plots demonstrating that removal of residues 1-40 of LanM (SEQ ID NO:3) results in a functional protein capable of binding REEs at a 2:1 stoichiometric ratio of REE to protein, in accordance with embodiments of the present disclosure. LanMΔ1-40 binds approximately 2 equivalents of La and Dy as judged by intrinsic tyrosine fluorescence (FIG. 16A), and such LanMΔ1-40 exhibits similar (but not identical) solution metal dissociation properties as wt-LanM (using citrate as a desorbent), suggesting that productive EF-hands are preserved and similar dissociation strategies can be applied to this variant (FIG. 16B). The plots include LanMΔ1-40 fluorimeter experiments in which the LanMΔ1-40 variant, in which its first non-productive EF hand has been removed, was tested for REE binding stoichiometry (Figure 16A) and citrate-induced metal dissociation in 100 mM KCl, 30 mM MOPS at pH 5 (Figure 16B) (all experiments were performed with 20 μM protein). [Figures 17A-17C] 17A-17C show exemplary plots demonstrating stoichiometric REE binding by the double LanM variant (SEQ ID NO: 4) according to embodiments of the present disclosure, including a xylenol orange competition assay suggesting about 5 equivalents of metal binding exhibiting greater than micromolar affinity at pH 6 (FIG. 17A), a tyrosine absorbance assay suggesting about 4 equivalents of REE binding (FIG. 17B), and a tyrosine fluorescence assay suggesting 4 equivalents of REE binding at pH 5 (FIG. 17C). [Figure 18] 1 shows an exemplary plot demonstrating that the metal release properties of the double LanM variant are similar to wt-LanM, suggesting that double LanM is a productive means for increasing column capacity in accordance with embodiments of the present disclosure. The plot includes citrate-induced metal release curves of the double LanM variant (20 μM) tested in 100 mM KCl, 30 mM MOPS, pH 5. [Figure 19]FIG. 1 shows an exemplary method for continuous flow separation of REEs using immobilized LanM with a three column rotation scheme according to an embodiment of the present disclosure. [Figure 20] FIG. 1 shows an exemplary plot of metal ion breakthrough curves using a LanM agarose column in accordance with an embodiment of the present disclosure (Feed: E-waste leachate produced using Biolixivant at pH 3.5. Biolixivant is prepared from the supernatant of a Gluconobacter culture and contains primarily gluconic acid and other organic acids as leaching agents). [Figure 21] Graph showing that LanM allows the production of high purity Nd and Dy after Nd / Dy separation. A solution containing a 5:95 mixture of Dy:Nd (pH 3) was subjected to two coupled adsorption / desorption cycles. The first cycle (left panel) produced a high purity Nd solution (99.9%) and an upgraded Dy (44%Dy / 56%Nd) solution. The upgraded Dy solution was used as a feed solution in the second adsorption / desorption cycle (right panel) to produce high purity Dy and Nd fractions. The duration of each pH step is depicted by the dark grey dashed line and corresponds to the second axis. [Figure 22] Figure 3. REE recovery and separation from e-waste bioleachate. (A) Schematic of the stepwise Nd / Pr and Dy recovery and separation process. (B) Relative concentration of metals in the effluent (compared to the influent). The transitions between the steps shown in panel A are depicted by the vertical grey dotted lines. The pH value of the strip solution is shown by the darker grey horizontal dotted line. (C) Absolute concentration of REEs eluted from the column. (D) Nd breakthrough curves of the column before and after e-waste testing demonstrating column reusability. [Figure 23] 1 is a graph showing an exemplary Dy vs. Nd / Pr separation from an e-waste feedstock. A synthetic solution having the same composition as the Dy-enriched fraction (29% Dy) from the first extraction step was subjected to a two-stage desorption process to produce high purity Dy and Nd fractions. [Figure 24] (A) Graph showing elemental composition of synthetic REE solutions used to test LREE (La+Ce) removal strategies. The REE composition reflects the REE ratios in a typical ore-based leaching solution (e.g., allanite). (B) Cumulative HREE / MREE / LREE desorption profile of a LanM column loaded with the synthetic REE feed material depicted in panel A using a stepwise citrate concentration (pH 5) scheme. REE desorption was normalized to the total REE desorbed in each group. [Diagram 25] (A) Desorption profile of a column loaded with REEs (feed composition depicted in FIG. 2A) using a stepwise citrate concentration (Zone I: 15 mM; Zone II: 50 mM, pH 5) and pH (Zone III: pH 1.5) scheme. (B) Summary of REE distribution in the initial feed and in the three desorption zones relative to the total REEs in the feed solution using the citrate-pH scheme. [Figure 26] 1 is a graph showing the relative REE composition of synthetic solutions similar to the REE composition in Sc-containing ore (e.g., allanite) and waste (e.g., bauxite) feedstocks. [Figure 27] (A) Citrate allows high purity and high yield separation of scandium from the REEs. Desorption profile and (B) cumulative desorption (yield) of a LanM column loaded with REEs after stepwise citrate (3 mM and 15 mM at pH 5) and pH 1.5 desorption schemes. (C) REE distribution in the initial feed and in the three desorption fractions. [Figure 28] We show that malonate allows for high purity and high yield separation of scandium from the REEs. The LanM column was loaded with a synthetic REE solution similar to the REE composition in a typical Sc-containing ore-based feedstock leach solution. (A) Desorption profile and (B) cumulative desorption (yield) after treatment with 30 mM and 50 mM malonate. [Figure 29]Figure 1 shows the use of a stepwise pH desorption scheme to generate Sc-enriched fractions. The LanM column was loaded with a synthetic REE solution similar to the REE composition in a typical Sc-containing ore-based feedstock leach solution. (A) Desorption profile and (B) cumulative desorption (yield) and (C) relative composition after treatment with a stepwise pH gradient. [Diagram 30] Graph showing the partition coefficients of immobilized LanM over the REE series, providing a quantitative measure of selectivity between REEs. The column was first washed with deionized water and then purged by blowing air through the column. Then, 5 mL of feed solution 1 or 2 (15 μmol REEs total) was circulated through the column at 0.5 mL / min for 2 hours and then purged by forcing air through the column. The equilibrated liquid was collected as [M]ad. Then, 4 mL of 0.1 M HCl was passed through the column and the effluent was collected as [M]de. Column: WT-LanM (0.9 mL, Nd adsorption capacity of about 3.5 μmol / mL). The column still contains about 0.8 mL of water when hydrotreated. Free water was removed by blowing air through the column. Solution 1. Equimolar (0.33 mM each, 3 mM total) La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy at pH 5. Solution 2. Equimolar (0.33 mM each, 3 mM total) Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, Y at pH 5. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] Rare earth elements (REEs) are made up of yttrium, scandium, and the lanthanides, and are essential for the transition from a fossil fuel era to a low-carbon era because they are important to clean energy technologies such as electric vehicles, wind turbines, and LEDs. 1 However, current REE extraction and separation processes require high energy consumption, pose significant environmental burdens that hinder the development of a diversified REE supply chain and undermine the environmental benefits of clean energy technologies. 2、3To meet the REE requirements of the emerging clean energy technology market, it is imperative to develop new processing methods that enable environmentally friendly extraction of REEs from contained REE-containing resources, low-grade sources and recyclable waste materials.

[0020] The separation between REEs is due to their similar physicochemical properties and their co-occurrence in REE-bearing sediments. 4、5 This is particularly challenging, accounting for about 30% of the total environmental impact during REE production. 2、6、7 Currently, REE separation is dominated by chemically intensive hydrometallurgical processes involving primary separation of the REEs from impurities in acid leach solutions, followed by separation of groups or individual REEs by liquid-liquid extraction. 5 To achieve acceptable levels of REE enrichment, liquid-liquid extraction may require hundreds of stages (i.e. mixer-settler or pulse columns) that generate large volumes of liquid waste. Furthermore, it is extremely difficult for liquid-liquid extraction to process volatile or dilute REE leachate solutions from non-traditional sources, such as industrial waste and end-of-life consumer electronics (e.g. e-waste), which require contacting vast amounts of leachate solution with organic solvents, resulting in large separation units (i.e. high CAPEX) and losses of solvent dispersed in the aqueous system.

[0021] To reduce solvent losses, liquid-liquid extraction processes have been adapted to column chromatography by dissolving the REE-selective ligands (extraction solvents) in organic solvents and loading them into solid supports. 8、9 However, this physical impregnation strategy still inevitably results in leaching of the stationary liquid phase, causing cross-contamination and limited reusability. 10 Solid-liquid extraction (SLE), in which chemical ligands (extractants) are covalently immobilized on a solid support resin, offers faster phase separation between the solid sorbent and the REE-containing solution, a significant reduction in the amount of organic diluent present in the process, and minimal extraction solvent loss compared to liquid-liquid extraction. 15 It offers a number of advantages, such as11~14 However, most of the SLE sorbents employed for REE separation are based on existing synthetic organic extractants (ligands) (e.g., HDEHP, TBP, TODGA, or Cyanex derivatives) used in liquid-liquid extraction, and their selectivity for REE extraction and separation is usually limited. 16 .

[0022] Incorporating biological ligands into the SLE process offers the potential for the development of novel chemically and environmentally sustainable REE separation processes. 17 For example, lanthanide binding tags (LBTs), short peptides engineered for high affinity and selectivity for REEs, have been presented on biomaterial surfaces (cells, curli fibers, etc.) and employed in SLE for the selective recovery of medium and heavy REEs from various feedstock leachates. 18、19 However, Zn 2+ , Cu 2+ , and large amounts of Ca 2+ There remains a need for a method that has selectivity for REEs, increased REE binding below pH 5, and conditions compatible with the REE feed material. 18 .

[0023] Of particular importance for REE extraction is lanmodulin (LanM), a recently discovered small (12 kDa) periplasmic protein that is part of the lanthanide uptake and utilization pathway in methylotrophic bacteria. 20 Biochemical and biophysical characterization of LanM revealed a remarkable selectivity for REEs over other non-REE cations, an ability to bind REEs down to a pH as low as 2.5, and remarkable robustness to repeated acidification cycles. 16Despite the high affinity of this protein, desorption can be induced by relatively mild treatments (lowering the pH or common chelating agents such as water-soluble carboxylates), and it is able to bind and desorb REEs multiple times. Furthermore, LanM also shows a rare preference for medium-light REEs over heavy REEs; although modest, such a preference may allow for efficient separation of light / heavy REE groups or even important REE pairs. 20 .

[0024] To address the technical, economic, and environmental limitations of current REE separation approaches, a protein-based REE separation method is provided herein. The protein-based REE separation method is a biomaterial-based, all-aqueous REE extraction and separation scheme that uses the REE-selective LanM protein chelator or its derivatives. To allow for easy protein reuse, the immobilized LanM is immobilized on porous agarose microbeads that are biologically renewable and commercially available. 21 The resulting biomaterial enabled the effective extraction of grouped REEs from unconventional low-grade REE feedstocks. Furthermore, by exploiting LanM's preference for medium-light REEs, the REE separation method described herein provides high-purity separations between REE pairs (Nd / Dy, and Y / Nd) and grouped separations between heavy REEs (HREEs) and light REEs (LREEs). Thus, the main advantage of this approach over liquid-liquid extraction is the combination of primary REE extraction from non-REEs and secondary separation between heavy and light REEs within a single, all-aqueous adsorption / desorption cycle.

[0025] After reading this description, it will be apparent to a person skilled in the art how to implement the present invention in various alternative embodiments and alternative applications. However, not all of the various embodiments of the present invention are described herein. It will be understood that the embodiments presented herein are presented by way of example only and are not limiting. Therefore, this detailed description of various alternative embodiments should not be construed as limiting the scope or breadth of the present invention described below. The detailed description is divided into various sections for the convenience of the reader, and disclosure found in any section may be combined with disclosure in another section. Titles or subtitles may be used in the specification for the convenience of the reader and are not intended to affect the scope of the disclosure of the present invention. Unless the context dictates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination. Moreover, the present disclosure also contemplates that in some embodiments, any feature or combination of features described herein can be excluded or omitted. For illustrative purposes, if a composite is described herein as comprising components A, B, and C, it is specifically intended that any of A, B, or C, or combinations thereof, alone or in any combination, can be omitted or discarded.

[0026] definition All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, inclusive of ranges, are approximations that vary (+) or (-) in increments of 1.0 or 0.1, as appropriate, or alternatively, by a variance of + / - 15%, or alternatively 10%, or alternatively 5%, or alternatively 2%. Although not always expressly stated, all numerical designations shall be understood to be preceded by the term "about". It will be understood that such range formats are used for convenience and brevity and should be flexibly understood to include not only the numerical values ​​expressly identified as limits of the range, but also all individual numerical values ​​or subranges contained within the range, as if each numerical value and subrange were expressly identified. For example, a ratio within the range of about 1 to about 200 should be understood to include not only the explicitly recited upper and lower limits of about 1 and about 200, but also the individual ratios, e.g., about 2, about 3, and about 4, as well as subranges, e.g., about 10 to about 50, about 20 to about 100, etc. It is also to be understood, although not always expressly stated, that the reagents described herein are merely exemplary and that equivalents thereof are known in the art.

[0027] It must be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a protein" includes a plurality of proteins. As used herein, the following terms have the following meanings. The term "about," as used herein when referring to a measurable value, e.g., amount or concentration, is meant to encompass a variation of 20%, 10%, 5%, 1%, 0.5%, or even as much as a variation of 0.1% of the specified amount. When the terms "acceptable," "effective," or "sufficient" are used to describe the selection of any ingredients, ranges, dosage forms, etc. disclosed herein, it is intended that the ingredients, ranges, dosage forms, etc. are suitable for the purpose disclosed.

[0028] The phrases "free" or "substantially free" refer to any competing metals being present in an amount of less than about 0.0001%, less than about 0.001%, less than about 0.01%, less than about 0.1%, less than about 1%, less than about 5%, or less than about 10% of the total mass or volume of the purified REE material, composition, or eluted solution. The phrase "click chemistry," as used herein, refers to a family of organic reactions that proceed rapidly and selectively under mild conditions to covalently link molecular building blocks. "And / or," as used herein, also refers to and includes any and all possible combinations of one or more of the associated listed items, as well as the exclusion of combinations when interpreted as an alternative ("or").

[0029] protein Aspects of the present disclosure provide proteins for use in the separation of REEs, including the EE-selective Lanmodulin (LanM) protein.

[0030] Lanmodulin (LanM) is a low molecular weight protein of about 12 kDa produced by some methylotrophic organisms and is a naturally occurring lanthanide-regulated protein. The wild-type M. extorquens LanM protein has the sequence of SEQ ID NO: 1 and may be optionally His-tagged at the N- or C-terminus. The methods and compositions of the present disclosure utilize LanM protein. Suitable LanM proteins include wild-type M. extorquens LanM protein or homologs from other organisms that have at least two EF-hand motifs, at least one EF-hand motif having at least three carboxylic acid residues and at least two of the EF-hand motifs separated by a space of 10-15 residues. References herein are generally made to "lanmodulin," "LanM" or "LanM protein" and should be understood to include the wild-type and homologs described herein. "LanM" may include a full length protein having one or more LanM units, or a portion thereof comprising one or more LanM units. A LanM unit comprises at least two EF hand motifs, at least one EF hand motif having at least three carboxylic acid residues, and at least two of the EF hand motifs are separated by a space of 10-15 residues. For ease of reference, discussion will be made by reference to lanmodulin, LanM or LanM protein, which should be understood to include both the full length protein and a portion of the full length protein having a suitable LanM unit.

[0031] The unique features of the EF-hands of this protein have been previously discussed in the initial characterization of the protein (Cotruvo et al., J. Am. Chem. Soc. 2018, 140, 44, 15056-15061). Based on biochemical and structural studies of the AM1 Lanmodulin of M. extroquens as well as homologues from other organisms, the Lanmodulin protein domain has certain key features. In many instances, at least one of the EF-hands contained a proline residue in the second position. However, not all LanM homologues have such a residue; moreover, they exhibit other key features of LanM identified, such as, for example, a spacing between at least two of the adjacent EF-hands (10-15 residues, e.g., 12-13 residues) and an additional carboxylic acid residue in the EF-hand. In particular, the spacing between adjacent EF-hands is considered to be a prominent feature of these proteins that distinguishes them from proteins containing conventional EF-hands, such as calmodulin. Even sequences with very low (<40%) identity to LanM of M. extroquens may have similar properties if other general features mentioned above are conserved. In an embodiment, LanM is wild-type LanM of M. extroquens having the sequence of SEQ ID NO: 1, optionally terminally His-tagged. In an embodiment, LanM may be a homologue of wild-type LanM. A suitable homologue has at least two EF-hand motifs, at least one EF-hand motif having at least three carboxylic acid residues, and at least two of the EF-hand motifs are separated by a spacer of 10-15 residues (including 12-13 residues). For example, a protein may contain at least one, and preferably at least two, EF-hand motifs having the following form: (D / N)-X1-(D / N)-X2-(D / N)-X3-X4-X5-X6-X7-X8-(E / D), where each numbered X is any residue (not necessarily the same residue at each position); X6 and / or X8 are D or E, with glycine being preferred, but not necessarily at X3. In this embodiment, LanM may include any naturally occurring or unnatural amino acid substituted into these EF-hands or elsewhere in the protein. In embodiments, the proteins used in the disclosed methods may include any number of Lanmodulin domains (referred to herein as LanM units) linked together into a single polypeptide unit, together with appropriate amino acid spacers. In embodiments, LanM includes only one LanM protein unit. In embodiments, LanM, as used herein, includes a proline residue at the second position. In embodiments, LanM, as used herein, includes 10-15 residues, e.g., 12-13 residues, between at least two adjacent EF-hands. In some embodiments, LanM, as used herein, includes additional carboxylic acid residues in one or more of the EF-hands, e.g., 3 or more, 4 or more, or 5 or more carboxylic acid residues.

[0032] SEQ ID NO:1 of wild-type M. extroquens LanM (EF-hand motifs are underlined) APTTTTKVDIAAF DPDKDGTIDLKE ALAAGSAAFDKL DPDKDGTLDAKE LKGRVSEADLKKL DPDNDGTLDKKE YLAAVEAQFKAA NPDNDGTIDARE LASPAGSALVNLIR Some examples of wild type homologues include the following (Note: sequences are shown after removal of the predicted signal sequence):

[0033] Example A: RH AL1 AKMDMKAI DPDSDGTVSLAE AQDAAAKKFAAM DPDNDGTIDLKE AKGKMAKAKFKKT DADNDGTVDKAE YSALVESAFKAA DPDGDGTLDAKE LKTPAGQKLLSLIQ In this protein, one of the EF hands lacks a proline, and EF1 lacks carboxylates at positions 9 and 11, yet it still undergoes a structural response at free concentrations of rare earth elements in the picomolar range.

[0034] B: Hansschlegelia sp. ASGADALKAL NKDNDDSLEIAE VIHAGATTFTAI NPDGDTTLESGE TKGRLTEKDWARA NKDGDQTLEMDE WLKILRTRFKRA DANKDGKLTAAE LDSKAGQGVLVMIMK In this protein (31% identity), only one of the EF-hands has a proline in the second position and only one has an Asp residue in the first position, but it still undergoes a structural response to free concentrations of rare earth elements in the picomolar range and a much weaker structural response to other metals (e.g., calcium).

[0035] Example C: Xanthomonas axonopodis AQAQVQVQDSQQYLQRM DTDGDGRVSLDE YLAWMSYAFDQR DTDHDGVLQGDE LPG RRGKPITRAAHRATLIARFARQ DANGDGYLSARE LLAPPR This protein shares about 33% sequence identity with M. extroquens LanM and contains only three EF-hands (underlined; designated EF1, EF2, and EF4 based on sequence alignment with M. extroquens LanM), none of which have prolines, yet it undergoes a structural response to free concentrations of rare earth elements in the picomolar range and a much weaker structural response to other elements (e.g., calcium). It should be noted that although EF1 and EF2 are only 13 residues apart, the distance between adjacent EF2 and EF4 in this sequence is longer (25 amino acids) than in most lanmodulins due to the loss of one of the EF-hands (EF3).

[0036] In certain embodiments, the LanM protein provided herein comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO:1 or a portion thereof, or a sequence having at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:1.

[0037] In certain embodiments, the LanM protein provided herein comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO:2 or a portion thereof, or a sequence having at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:2.

[0038] In certain embodiments, the LanM protein provided herein comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO:3 or a portion thereof, or a sequence having at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:3. In certain embodiments, the LanM protein provided herein comprises, consists of, or consists essentially of an amino acid sequence set forth in SEQ ID NO:3 or a portion thereof, or a sequence having at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:4. In certain embodiments, where the LanM protein comprises an amino acid sequence having less than 100% identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or a portion thereof, all of the substitutions resulting in sequence differences are conservative substitutions. In other embodiments, the LanM protein provided herein may comprise one or more non-conservative substitutions relative to the LanM protein. In certain embodiments, the LanM protein provided herein comprises one, two, three, four, or five or more conservative substitutions relative to the LanM protein.

[0039] Table 1. Exemplary LanM amino acid sequences TIFF2024520862000002.tif99158

[0040] The REEs are a group of 17 chemical elements that include yttrium and 15 of the lanthanide elements. Scandium is found in most REE deposits and is often present therein. Table 2: Rare earth elements TIFF2024520862000003.tif57143

[0041] The REE binding proteins can bind to any of the following elements: lanthanum (La), cerium (Ce), praseodymium (Pr), neodymium (Nd), promethium (Pm), samarium (Sm), europium (Eu), gadolinium (Gd), terbium (Tb), dysprosium (Dy), holmium (Ho), erbium (Er), thulium (Tm), ytterbium (Yb), lutetium (Lu), scandium (Sc), yttrium (Y), or any combination thereof. The REE binding ligands can bind to any element in any oxidation state (e.g., Ln 2+ , Ln 3+ , Ln 4+ etc.)

[0042] In some embodiments, the REE binding protein (e.g., LanM) has a binding affinity (i.e., K d, a thermodynamic dissociation constant commonly used for protein complexes). d In yet other embodiments, K is about 0.1 pM, about 100 pM, about 500 pM, about 1 nM, about 10 nM, about 50 nM, about 100 nM, about 500 nM, or about 1 μM. d is in the pM range. Affinity can be determined by any suitable means known to those skilled in the art. Non-limiting examples include titration with REE and detection using fluorescence, circular dichroism, ICP, NMR or calorimetry. For tight binding sequences, it may be necessary to employ competition experiments.

[0043] In some embodiments, LanM comprises a cysteine ​​residue. In some embodiments, the cysteine ​​residue is a C-terminal cysteine ​​residue, an N-terminal cysteine ​​residue, or an internal cysteine ​​residue. In some embodiments, a short, hydrophilic, flexible linker separates the cysteine ​​residue from LanM. Exemplary linkers include Gly x Ser y wherein x and y are each independently an integer of 0 to 10, provided that x and y are not both 0 (e.g., GlySerGly, (Gly4Ser)2; (Gly3Ser)2; Gly2Ser; or a combination thereof, such as (Gly3Ser)2Gly2Ser).

[0044] In some embodiments, the LanM proteins provided herein comprise, consist of, or consist essentially of the amino acid sequence set forth in SEQ ID NO: 1, or a portion thereof, and a C-terminal cysteine ​​residue, e.g., GSGC. In some embodiments, the LanM proteins provided herein comprise, consist of, or consist essentially of the amino acid sequence set forth in SEQ ID NO: 2, or a portion thereof, and a C-terminal cysteine ​​residue, e.g., GSGC. In some embodiments, the LanM proteins provided herein comprise, consist of, or consist essentially of the amino acid sequence set forth in SEQ ID NO: 3, or a portion thereof, and a C-terminal cysteine ​​residue, e.g., GSGC.

[0045] Biosorption system Also provided is a system for REE extraction and preferential separation (ie, a biosorption / sorption medium) that contains a predetermined amount of proteins, including LanM protein. Biosorption is a chemical process based on various mechanisms such as adsorption, absorption, ion exchange, surface complexation, and precipitation. When coupled with materials from biological origin such as proteins or biomass, the material is referred to as a biosorption material. Biosorption materials can, for example, bind REEs and separate them from the REE-containing material (e.g., feed material). Provided herein are biosorption materials that contain proteins for preferentially separating REEs from REE-containing materials. REE extraction and preferential separation includes a predetermined amount of proteins.

[0046] These biosorption media include, for example, biofilms, microbeads, and carbon nanotube embedded membranes, which can be used for adsorption under continuous flow. Protein immobilization in biosorption media for use in a flow-through setup is expected to enable complete (or substantially complete) separation of REEs from mixed metal solutions containing REEs, including separation of REEs from other REEs in the mixed metal solution in a single step, e.g., without the need for liquid-liquid separation, centrifugation, filtration, or both. In some embodiments, the biosorption material is a bead and / or capsule. In some embodiments, the bead and / or capsule is suitable for separation of REEs. In some embodiments, the bead and / or capsule comprises an REE-selective protein. In some embodiments, the biosorption material is a microbead. As used herein, the term "capsule" is used interchangeably with "bead."

[0047] In some embodiments, the protein is attached to a solid support, e.g., a column, a membrane, a bead, etc. In some embodiments, the solid support is a porous support material. The solid support may be of any suitable composition known to those of skill in the art, including, for example, a polymer, agarose, alginate, acrylamide, regenerated cellulose, cellulose esters, plastic, or glass. In one embodiment, the protein is bound (i.e., embedded) within the bead or on the surface of the bead. In some embodiments, the bead is a polymer. Suitable polymers include PEG (e.g., about 10% PEG), alginate (e.g., about 2% calcium alginate), acrylamide (e.g., about 10% polyacrylamide), and agarose. In other embodiments, the bead is glass, plastic, or steel.

[0048] In other embodiments, the disclosure provides a method for preparing a microbead solution for REE separation. In some embodiments, the method for preparing beads for REE separation comprises the steps of: (a) providing a protein capable of selectively binding to REEs and a porous support material functionalized with a conjugation agent; and (b) binding the protein to the porous support material via the conjugation agent. In some embodiments, the method includes functionalizing the support material with a terminal amine (e.g., -NH2). In some embodiments, the porous support material comprises agarose. In some embodiments, the porous support material is amine-functionalized agarose.

[0049] In some embodiments, the conjugation agent is a maleimide. In such embodiments, the method may further comprise reacting the terminal amine with a maleimide to form a maleimide-functionalized agarose bead. In some embodiments, the maleimide is succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC). In some embodiments, the method further comprises coupling the maleimide-functionalized porous support material with an REE-selective protein. In some embodiments, the protein comprises a cysteine ​​residue. In some embodiments, the porous support material is coupled to the REE-selective protein via the maleimide of the porous support material and the cysteine ​​residue of the protein. In some embodiments, the REE-selective protein comprising a cysteine ​​residue is coupled to the maleimide of the porous support material by "click" chemistry. Coupling the REE-selective protein to the porous support material by "click" chemistry is advantageous as it allows for coupling under biocompatible reaction conditions, i.e., aqueous conditions in the absence of organic solvents.

[0050] In some embodiments, binding a protein to a porous support material may include any bioconjugation approach using a conjugation agent. In some embodiments, the bioconjugation method uses thiol-ene "click" chemistry. In some embodiments, N-hydroxysuccinimide (NHS) can be used as a conjugation agent for bioconjugation of a protein to a porous support structure. Exemplary NHS crosslinkers that can also be used as a conjugation agent include dibenzycyclooctyne-N-hydroxysuccinimide (DBCO-NHS), bicyclononyne-N-hydroxysuccinimide (BCN-NHS), and dibenzocyclooctyne-sulfo-N-hydroxysuccinimide (DBCO-sulfo-NHS). In some embodiments, the bioconjugation method uses alkyne / azide "click" chemistry. For example, alkyne / azide "click" chemistry can be utilized to bind a protein containing an azide moiety to a porous support material containing an alkyne moiety used as a conjugation agent.

[0051] In some embodiments, the beads have a high concentration of REE-selective protein. It is expected that high loading of protein can act, at least in part, to enhance the saturation capacity of the biosorption material by increasing the number of available REE binding sites. An increase in the number of REE binding sites results in a greater percentage of REEs from the REE-containing material that are complexed with the REE-binding ligand to form protein-REE complexes (e.g., increased saturation capacity). In some embodiments, an increase in saturation capacity correlates with an increase in adsorption capacity (i.e., an increase in the number of REEs that are complexed with the REE-selective protein per unit volume or unit mass of REE-containing material). It is expected that the increase in saturation and adsorption capacity will eliminate the need for additional energy-consuming steps, such as centrifugation and filtration, in the REE separation process.

[0052] In some embodiments, a high concentration of protein (e.g., LanM) is about 2 μmol (μmol / mL) protein per mL of total porous support material (e.g., microbeads), 2.2 μmol / mL, 2.4 μmol / mL, 2.6 μmol / mL, 2.8 μmol / mL, 3.0 μmol / mL, 3.2 μmol / mL, 3.4 μmol / mL, 3.6 μmol / mL, 3.8 μmol / mL, 4 μmol / mL, 5 μmol / mL, 10 μmol / mL, 15 μmol / mL, 20 μmol / mL, 25 μmol / mL, 30 μmol / mL, 35 μmol / mL, 40 μmol / mL, 45 μmol / mL, or 50 μmol / mL protein. In some embodiments, the high concentration of protein (e.g., LanM) is from about 2 μmol / mL to about 4 μmol / mL, from about 3 μmol / mL to about 4 μmol / mL, from about 2.2 μmol / mL to about 3 μmol / mL, from about 3.2 μmol / mL to about 4 μmol / mL, from about 2 μmol / mL to about 3.6 μmol / mL, from about 10 μmol / mL to about 30 μmol / mL, from about 20 μmol / mL to about 30 μmol / mL, from about 10 μmol / mL to about 40 μmol / mL, from about 20 μmol / mL to about 40 μmol / mL, or from about 25 μmol / mL to about 50 μmol / mL per mL of total porous support material (e.g., microbeads).

[0053] In some embodiments, the high concentration of protein is at least about 20 weight percent (wt%), at least about 5 wt%, at least about 10 wt%, at least about 15 wt%, at least about 20 wt%, at least about 25 wt%, at least about 30 wt%, at least about 35 wt%, at least about 40 wt%, at least about 45 wt%, at least about 50 wt%, at least about 55 wt%, at least about 60 wt%, at least about 65 wt%, at least about 70 wt%, at least about 75 wt%, at least about 80 wt%, at least about 85 wt%, at least about 90 wt%, at least about 95 wt%, or more of the total weight of the beads, or the total porous At least about 20 volume percent (vol%), at least about 5 vol%, at least about 10 vol%, at least about 15 vol%, at least about 20 vol%, at least about 25 vol%, at least about 30 vol%, at least about 35 vol%, at least about 40 vol%, at least about 45 vol%, at least about 50 vol%, at least about 55 vol%, at least about 60 vol%, at least about 65 vol%, at least about 70 vol%, at least about 75 vol%, at least about 80 vol%, at least about 85 vol%, at least about 90 vol%, at least about 95 vol%, or more of the support material (e.g., microbeads).

[0054] In some embodiments, the high adsorption capacity of a protein is at least about 1 milligram (mg), at least about 2 mg, at least about 3 mg, at least about 4 mg, at least about 5 mg, at least about 6 mg, at least about 7 mg, at least about 8 mg, at least about 9 mg, at least about 10 mg, at least about 11 mg, at least about 12 mg, at least about 13 mg, at least about 14 mg, at least about 15 mg, at least about 16 mg, at least about 17 mg, at least about 18 mg, at least about 19 mg, at least about 20 mg, at least about 21 mg, at least about 22 mg, at least about 23 mg, at least about 24 mg, at least about 25 mg, at least about 26 mg, at least about 27 mg, at least about 28 mg, at least about 29 mg, at least about 30 mg, at least about 31 mg, at least about 32 mg, at least about 33 mg, at least about 34 mg, at least about 35 mg, at least about 36 mg, at least about 37 mg, at least about 38 mg, at least about 39 mg, at least about 40 mg, at least about 41 mg, at least about 42 mg, at least about 43 mg, at least about 44 mg, at least about 45 mg, at least about 46 mg, at least about 47 mg, at least about 48 mg, at least about 49 mg, at least about 50 mg, at least about 51 mg, at least about 52 mg, at least about 53 mg, at least about 54 mg, at least about 55 mg, at least about 56 mg, at least about 57 mg, at least about 58 mg, at least about 59 mg, at least about 60 mg, at least about 61 mg, at least about 62 mg, at least about 63 mg, at least about 24 mg, at least about 25 mg, at least about 26 mg, at least about 27 mg, at least about 28 mg, at least about 29 mg, at least about 30 mg, at least about 31 mg, at least about 32 mg, at least about 34 mg, at least about 35 mg, at least about 36 mg, at least about 37 mg, at least about 38 mg, at least about 39 mg, at least about 40 mg, at least about 41 mg, at least about 42 mg, at least about 43 mg, at least about 44 mg, at least about 45 mg, at least about 46 mg, at least about 47 mg, at least about 48 mg, at least about 49 mg, or at least about 50 mg of REE.

[0055] In some embodiments, a high adsorption capacity of a protein is at least about 1 milligram (mg), at least about 2 mg, at least about 5 mg, at least about 10 mg, at least about 15 mg, at least about 20 mg, at least about 25 mg, at least about 30 mg, at least about 35 mg, at least about 40 mg, at least about 45 mg, at least about 50 mg, at least about 60 mg, at least about 65 mg, at least about 70 mg, at least about 75 mg, at least about 80 mg, at least about 85 mg, at least about 90 mg, at least about 95 mg, or at least about 100 mg of REE per gram (g) of protein. In some embodiments, the protein has an adsorption capacity of between about 30 and about 70 mg of REE per gram of protein.

[0056] In some embodiments, a high adsorption capacity of a protein is at least about 0.1 milligrams (mg), at least about 2 mg, at least about 5 mg, at least about 10 mg, at least about 15 mg, at least about 20 mg, at least about 25 mg, at least about 30 mg, at least about 35 mg, at least about 40 mg, at least about 45 mg, at least about 50 mg, at least about 60 mg, at least about 65 mg, at least about 70 mg, at least about 75 mg, at least about 80 mg, at least about 85 mg, at least about 90 mg, at least about 95 mg, at least about 100 mg of REE per gram (g) of biosorption media (e.g., system and / or material). In some embodiments, a protein has an adsorption capacity of between about 30 and about 70 mg of REE per gram of biosorption media.

[0057] In another embodiment, the protein is encapsulated within the bead and / or on the surface of the bead. When the protein is encapsulated within the bead and / or on the surface of the bead, the bead can efficiently bind with the REE. When the REE-containing material flows over and / or passes through the bead, the immobilized LanM protein can capture the REE both within the bead and on the surface of the bead, which optimizes the adsorption capacity of the bead by increasing the ratio of available binding sites (i.e., binding ligands) to the total volume of the bead.

[0058] In some embodiments, the support material (e.g., microbeads) is porous. In some embodiments, the support material is a fibrous material. For example, in some embodiments, the support material is a fibrous material produced using electrospun fibers. The porous support material allows the flow of REE-containing material to contact the inner as well as the outer surfaces of the beads, thereby increasing the saturation and adsorption capacity of the support material for the REEs (i.e., increasing the accessibility). In some embodiments, the porous support material has a pore size of at least about 0.10 nm, at least about 1.0 nm, at least about 10 nm, at least about 50 nm, at least about 100 nm, at least about 150 nm, at least about 200 nm, at least about 250 nm, at least about 300 nm, at least about 350 nm, at least about 400 nm, at least about 450 nm, at least about 500 nm, at least about 550 nm, at least about 600 nm, at least about 650 nm, at least about 700 nm, at least about 750 nm, at least about 800 nm, at least about 850 nm, at least about 900 nm, at least about 950 nm, or at least about 1000 nm. In some embodiments, the porous support material has a pore size of about 1.0 nm to about 500 nm, about 0.10 nm to about 10 nm, about 150 nm to about 1000 nm, about 300 nm to about 600 nm, about 200 nm to about 800 nm, about 300 nm to about 500 nm, about 500 nm to about 1000 nm, or about 600 nm to about 800 nm.

[0059] method Also provided are methods of using proteins, such as LanM, to preferentially separate REEs from REE-containing materials, and further to separate REEs from other REEs. Preferential separation of REEs from REE-containing materials is important for the development of technologies such as batteries, magnets, and electronics, but the similar chemical properties of REEs make them extremely difficult to separate from each other and from other non-REEs. Conventional techniques are hampered by low selectivity for REEs, especially when the REEs are in the presence of mixtures of competing metals, such as non-REEs (e.g., alkali, alkaline, and other transition metals). Even more difficult is separating the REEs from competing metals when the competing metals are present in concentrations much higher than the REEs. Due to the inherent difficulties in REE separation, conventional techniques are limited in their ability to separate REEs with high purity and have further hindered the development of systems capable of preferentially separating REEs on an industrial bulk scale. The present disclosure provides methods for preferential separation of REEs with high selectivity, high efficiency, and low cost, each of which is useful for systems capable of separating REEs on a large industrial scale. In some embodiments, a method for preferentially separating REEs from an REE-containing material includes separating an individual REE or group of REEs from other REEs or non-REEs. Preferential separation of an individual REE refers to the isolation of an REE (e.g., La) from either another REE (e.g., Er), group of REEs, or non-REEs such that there is no or substantially no other element, REE, or non-REE present after separation (i.e., in the eluted solution). For example, the preferential separation method provided herein allows for the separation of Nd (i.e., an individual REE) from Y, Dy, and Tb (i.e., group of REEs) and / or from competing metals (i.e., non-REEs). In some embodiments, preferential separation provides high purity separation of an individual REE with no or substantially no other individual REEs, group of REEs, and / or non-REEs present after separation (i.e., in the eluted solution).

[0060] In one aspect, provided herein is a method for preferentially separating REEs from a REE-containing material, the method comprising the steps of: (a) providing a protein capable of selectively binding one or more REEs; (b) contacting the protein with a REE-containing material, wherein the protein binds to at least a portion of one or more REEs to form a protein-REE complex and a REE-depleted material; (c) separating the protein-REE complex from at least a portion of the REE-depleted material; and (d) separating the REEs from the protein to produce a refolded protein. In some embodiments, the steps described are performed once. In other embodiments, steps or portions of steps are performed more than once, e.g., 2, 3, 4, 5 or more times. In some embodiments, steps or portions of steps are performed more than once with more than one REE-containing material, e.g., 1, 2, 3, 4, 5 or more REE-containing materials.

[0061] In some embodiments, a step or portion of a step is repeated until at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, or at least about 10% of the REEs are separated from the protein-REE complex.

[0062] In some embodiments, the protein is added to the column prior to contacting the protein with the REE-containing material. In some embodiments, the protein is bound within or to a solid structure (e.g., beads and / or capsules) prior to adding the protein to the column. Once the protein is added to the column, it is used as the stationary phase, as conventionally defined in column chromatography. This allows for a continuous flow system, where the REE-containing material is introduced to the column and flows through the column. In some embodiments, the flow is from the top of the column to the bottom. In some embodiments, the flow is reversed and the flow is from the bottom to the top of the column.

[0063] In certain embodiments, a method for separating rare earth elements (REEs) from a REE-containing material includes one or more adsorption / desorption cycles. In some embodiments, the methods disclosed herein may include two or more adsorption / desorption cycles. In some embodiments, each cycle includes a step of separately adsorbing and desorbing the REE to the protein. For example, in some embodiments, the REE is adsorbed to the protein (i.e., forming a protein-REE complex) during the step of contacting the protein with the REE-containing material, and then the REE is desorbed from the REE-binding ligand (i.e., dissociating the REE from the protein, thereby breaking apart the protein-REE complex) when a solution is introduced. In some embodiments, the REE-containing material is flowed over the column containing the protein until the column is saturated (i.e., until all or substantially all of the protein binding sites are bound to the REE to form the protein-REE). In some embodiments, at least about 95%, at least about 90%, at least about 85%, at least about 80%, at least about 75%, at least about 70%, at least about 65%, at least about 60%, at least about 55%, or at least about 50% of the protein binding sites are bound to REE. In the first adsorption step, the majority of non-REE (i.e., more than 50%) passes through the column while the REE is retained (i.e., remains bound to the REE-binding ligand). The column is then washed with a solution to wash away or separate remaining unbound REE or non-REE from the protein-REE complex. After saturation and washing, a solution is used to differentially separate (i.e., elute) the REE from the protein. According to embodiments disclosed herein, the differentially separated / eluted REE solution is a REE-enriched solution that may have a higher concentration of REE compared to the feed solution.

[0064] In some embodiments, the REEs are simultaneously adsorbed to and desorbed from the REE-binding ligand during the mass transfer process. For example, in some embodiments, the REE-containing material is flowed over a column containing a protein, and only a portion of the protein binding sites adsorb the REE to form a protein-REE complex. The solution then flows through the column, and as the REEs progress from the top to the bottom of the column, they undergo a series of adsorptions and desorptions from the REE-binding ligand (i.e., this is a dynamic process). The difference in affinity of each REE to the REE-binding ligand (i.e., solid phase) and the adjustable solution (i.e., mobile phase) controls the migration rate of the REEs through the column. Since the complexes formed with the heavier REEs and proteins are weaker compared to the complexes formed with the lighter REEs and proteins, the heavier REEs migrate through the column faster than the lighter REEs, thereby providing a method for preferential separation of the REEs. Specifically, given that ranmodulin forms the strongest complex with Sc, Y migrates with the HREEs, whereas Sc migrates through the column slowest. The simultaneous adsorption and desorption process allows for the separation of individual REEs and / or groups of REEs in high purity. This method offers advantages over conventional REE separation processes that require the use of expensive chemical resins or with harmful solvents to obtain highly purified REEs and / or groups of REEs.

[0065] In some embodiments, the method for preferentially separating REEs from a REE-containing material further comprises the step of introducing a modifiable solution. The modifiable solution is a solution having a different concentration of chelating agent and / or a different pH compared to the initial solution used to introduce the REEs to the protein. The modifiable solution preferentially separates the REEs from the REE-containing material. Preferential separation can be achieved if the solution is modified in a manner that increases the affinity of individual REEs or groups of REEs for the solution (i.e., mobile phase) but decreases the affinity of individual REEs or groups of REEs for the REE-binding ligand (i.e., stationary phase). Conversely, preferential separation can also be achieved if the solution is modified in a manner that increases the affinity of individual REEs or groups of REEs for the REE-binding ligand (i.e., stationary phase) but decreases the affinity of individual REEs or groups of REEs for the REE-solution.

[0066] In some embodiments, to selectively desorb one or more REEs from other REEs, the REEs are adsorbed to and desorbed from the protein separately. For example, in some embodiments, the REE-containing material is flowed over the column containing the protein until the column is saturated (i.e., until all or substantially all of the protein binding sites are bound to the REEs to form protein-REEs). In some embodiments, at least about 95%, at least about 90%, at least about 85%, at least about 80%, at least about 75%, at least about 70%, at least about 65%, at least about 60%, at least about 55%, or at least about 50% of the protein binding sites are bound to the REEs. In some embodiments, once saturated, one or more solutions are used to differentially separate (i.e., elute) one or more REEs from the protein in a sequential manner. For example, in some embodiments, a first solution is used to separate HREEs, a second solution is used to separate MREEs, and a third solution is used to separate LREEs. In other embodiments, one or more solutions are used to preferentially separate one or more REEs from the protein.

[0067] In some embodiments, the method for preferentially separating the REEs is continuous, and the REE separation is not interrupted by additional energy intensive steps such as centrifugation and / or filtration, hi other embodiments, the method for preferentially separating the REEs includes the additional steps of centrifugation, filtration, or both. In some embodiments, the protein-REE complex is formed at a pH of about 2.4 to about 7. In some embodiments, the protein-REE complex is formed at a pH of about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, or about 10. In some embodiments, the protein-REE complex is formed at a pH of about 2.4 to about 10, and the protein does not bind to any non-REE moieties (e.g., non-REE metals).

[0068] In some embodiments, the REE-selective protein binds two REEs per protein. For example, in some embodiments, the stoichiometric ratio of REE to protein is 2:1. In some embodiments, the protein-REE complex comprises two REEs per protein. In some embodiments, the protein is LanM and the stoichiometric ratio of REE to LanM is 2:1. In some embodiments, the REE-selective protein binds three REEs per protein. For example, in some embodiments, the stoichiometric ratio of REE to protein is 3:1. In some embodiments, the protein-REE complex comprises three REEs per protein. In some embodiments, the protein is LanM and the stoichiometric ratio of REE to LanM is 3:1.

[0069] In some embodiments, the REE-selective protein binds four REEs per protein. For example, in some embodiments, the stoichiometric ratio of REE to protein is 4:1. In some embodiments, the protein-REE complex comprises four REEs per protein. In some embodiments, the protein is LanM and the stoichiometric ratio of REE to LanM is 4:1.

[0070] In some embodiments, the REE-selective protein binds 5 REEs per protein. For example, in some embodiments, the stoichiometric ratio of REE to protein is 5:1. In some embodiments, the protein-REE complex comprises 5 REEs per protein. In some embodiments, the protein is LanM and the stoichiometric ratio of REE to LanM is 5:1. In some embodiments, the REE-selective protein binds 6 REEs per protein. For example, in some embodiments, the stoichiometric ratio of REE to protein is 6:1. In some embodiments, the protein-REE complex comprises 6 REEs per protein. In some embodiments, the protein is LanM and the stoichiometric ratio of REE to LanM is 6:1.

[0071] In some embodiments, the REE-selective protein binds 7 REEs per protein. For example, in some embodiments, the stoichiometric ratio of REE to protein is 7:1. In some embodiments, the protein-REE complex comprises 7 REEs per protein. In some embodiments, the protein is LanM and the stoichiometric ratio of REE to LanM is 7:1. In some embodiments, the REE-selective protein binds 8 REEs per protein. For example, in some embodiments, the stoichiometric ratio of REE to protein is 8:1. In some embodiments, the protein-REE complex comprises 8 REEs per protein. In some embodiments, the protein is LanM and the stoichiometric ratio of REE to LanM is 8:1.

[0072] In some embodiments, the REE-selective protein binds 9 REEs per protein. For example, in some embodiments, the stoichiometric ratio of REE to protein is 9:1. In some embodiments, the protein-REE complex comprises 9 REEs per protein. In some embodiments, the protein is LanM and the stoichiometric ratio of REE to LanM is 9:1. In some embodiments, the REE-selective protein binds 10 REEs per protein. For example, in some embodiments, the stoichiometric ratio of REE to protein is 10:1. In some embodiments, the protein-REE complex comprises 10 REEs per protein. In some embodiments, the protein is LanM and the stoichiometric ratio of REE to LanM is 10:1.

[0073] In some embodiments, the REE-selective protein is a LanM protein, which comprises or consists essentially of the amino acid sequence set forth in SEQ ID NO:4, or a portion thereof, and which binds four REEs per protein. For example, in some embodiments, the stoichiometric ratio of REE to protein is 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. In some embodiments, the protein-REE complex comprises four REEs per protein. In some embodiments, the protein is LanM (SEQ ID NO:4), and the stoichiometric ratio of REE to LanM is 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. In some embodiments, the REE is separated from the protein by contacting the protein-REE complex with a solution having a pH of less than about 2.4. In some embodiments, the solution has a pH of less than about 2.4, less than about 2.3, less than about 2.2, less than about 2.1, less than about 2, less than about 1.9, less than about 1.8, less than about 1.7, less than about 1.6, less than about 1.5, or less. According to the embodiments described herein, a solution having a specific or specified pH is produced by using any solution or composition known in the art to produce that pH, such as a HCL solution, a H2SO4 solution, a HNO3 solution, a solution having a mixture of HCl / NaCl, H2SO4 / KHSO4 / Na2SO4, or other mixtures, a solution of glycine, or any other compatible solvent capable of producing the desired solution.

[0074] In some embodiments, the REEs are separated from the protein by contacting the REE-complex with a solution containing a chelator. The chelator can include any compound that contains a functional group capable of binding with a non-REE metal or an REE. For example, in certain embodiments, the chelator or chelating agent can be a monocarboxylic acid, a dicarboxylic acid, or a tricarboxylic acid. Non-limiting examples of chelators or chelating agents include EDTA, citrate, dimercaprol, malonate, iminodiacetate, diglycolic acid, hydroxyisobutyric acid, polyaminocarboxylate, hydroxypyridinone, catechol, hydroxamate, acetate, nitrilotriacetate, dipicolinic acid, or α-hydroxyisobutyric acid.

[0075] In some embodiments, the REE and / or group of REEs are separated with a purity of at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% relative to any other REE and / or group of REEs. In some embodiments, the REE and / or REE group are isolated with a purity of at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100%, relative to any other element.

[0076] In some embodiments, the REE and / or REE group are separated with a purity of at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% relative to any non-REE components. 2+ , Al 3+ , Ca 2+ , Co 2+ , Ni 2+ , Cu 2+ , Fe 2+ , Fe 3+ , Zn 2+ , U, Th, and other alkali, alkaline earth, and transition metals found in the REE-containing feedstock. In some embodiments, the methods provided herein include 3+In some embodiments, LanM allows preferential separation of REE from Fe 3+ In some embodiments, Fe has a higher selectivity (i.e., binding affinity) for the REE compared to 3+ is preferentially separated from the REE by contacting the protein-REE complex with a solution having a pH of about 4 to about 5. In this case, Fe 3+ precipitates from solution and the REE remains bound to the protein, hi some embodiments, the REE is then separated from the protein-REE complex by contacting the protein-REE complex with a solution having a pH of 2.4 or less.

[0077] In some embodiments, the REE and / or group of REE is Fe 3+ with a purity of at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% relative to 。

[0078] In some embodiments, the method provides preferential separation of REEs that are adjacent to each other in the periodic table, for example, in some embodiments, the method provides preferential separation of Ce to La, Pr to Ce, Nd to Pr, Pm to Nd, Sm to Pm, Eu to Sm, Gd to Eu, Tb to Gb, Dy to Tb, Ho to Dy, Er to Ho, Tm to Er, Yb to Tm, and / or Lu to Yb.

[0079] In some embodiments, the method provides preferential separation of REEs from other REEs or non-REE metals with similar ionic radii, for example, in some embodiments, the method provides preferential separation of Y to La, Y to Ce, Y to Pr, Y to Nd, Y to Pm, Y to Sm, Y to Eu, Y to Gb, Y to Tb, Y to Dy, Y to Ho, Y to Er, Y to Tm, Y to Yb, Y to Lu, or Y to Sc. In some embodiments, the method provides preferential separation of REE from Sc. For example, in some embodiments, the method provides preferential separation of Sc from La, Sc from Ce, Sc from Pr, Sc from Nd, Sc from Pm, Sc from Sm, Sc from Eu, Sc from Gb, Sc from Tb, Sc from Dy, Sc from Ho, Sc from Er, Sc from Tm, Sc from Yb, Sc from Lu, or Sc from Y.

[0080] In some embodiments, the method provides preferential separation of REEs based on the ionic radius, atomic radius, and / or mass of the REEs. In some embodiments, REEs with smaller ionic radii are preferentially separated from REEs with larger atomic radii. In some embodiments, preferential separation of REEs is influenced by the effect of lanthanide contraction, where the ionic radii of the lanthanides decrease significantly moving from left to right of the periodic table (i.e., from La to Lu). In some embodiments, the method provides preferential separation of REEs based on the ionic radius, atomic radius, and / or mass of the REEs using a two-stage desorption process.

[0081] In some embodiments, the two-step desorption process comprises contacting the protein-REE complex with a first solution having a specific pH and a second solution having a specific pH. In some embodiments, the first step of the desorption process comprises introducing a solution having a first pH to the protein-REE complex and the second step of the desorption process comprises introducing a solution having a second pH to the protein-REE complex. In other embodiments, the desorption process comprises more than two steps. In some embodiments, the desorption process comprises a first step of introducing a first solution having a specific pH to the protein-REE complex, a second step of introducing a second solution having a specific pH, and a third step of introducing a third solution having a specific pH to the protein-REE complex. In some embodiments, the desorption process comprises a first step of introducing a first solution having a specific pH to the protein-REE complex, a second step of introducing a second solution having a specific pH, and a third step of introducing a third solution having a specific pH, and a fourth step of introducing a fourth solution having a specific pH to the protein-REE complex. In some embodiments, the first pH is about 1.9 to about 2.4. For example, in some embodiments, the first pH is about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, or about 2.4. In some embodiments, the second pH is about 1.5 to about 1.7. For example, in some embodiments, the second pH is about 1.5, about 1.6, or about 1.7. In other embodiments involving more than two steps, the pH is sequentially lowered to between about 2.4 to about 1.5 or below. In some embodiments, contacting the protein-REE complex with a solution having a first pH (i.e., a higher pH) separates REEs having smaller ionic radii from the REE-complex, but REEs having larger ionic radii are not separated from the REE-complex. In some embodiments, contacting the protein-REE complex with a solution having a second pH (i.e., lowering the pH) separates REEs having smaller ionic radii from the REE-complex. In some embodiments, the REE with the smaller ionic radius is Dy or Y and the REE with the larger ionic radius is Nd or Pr.In some embodiments, the method allows for preferential desorption of Dy from Nd, Dy from Pr, or Y from Nd. In some embodiments, the method allows for preferential separation of LREEs (e.g., La-Nd), HREEs (Ho-Lu plus Y), and medium REEs (MREEs; Sm-Dy).

[0082] In some embodiments, the two-step desorption process comprises contacting the protein-REE complex with a first solution comprising a chelator and another solution (e.g., a second solution) having a particular pH. In some embodiments, the first step of the desorption process comprises introducing a solution comprising a chelator to the protein-REE complex and the second step of the desorption process comprises introducing a solution having a particular pH to the protein-REE complex. In other embodiments, the desorption process comprises more than two steps. In some embodiments, the desorption process comprises a first step of introducing a first solution comprising a chelator to the protein-REE complex, a second step of introducing a second solution comprising a chelator solution, and a third step of introducing a third solution having a particular pH to the protein-REE complex. In some embodiments, the desorption process comprises a first step of introducing a first solution comprising a chelator to the protein-REE complex, a second step of introducing a second solution comprising a chelator solution, a third step of introducing a third solution comprising a chelator solution, and a fourth step of introducing a fourth solution having a specific pH. In some embodiments, the chelator is a citrate or a malonate. In some embodiments, the specific pH is less than 3.0. In some embodiments, the specific pH is less than 2.0. In some embodiments, the specific pH is between about 1.7 and about 2.4. For example, in some embodiments, the pH is about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, or about 2.4. In some embodiments, contacting a solution containing a chelator with the protein-REE complex separates the REE with the smaller ionic radius from the REE-complex, whereas the REE with the larger ionic radius does not separate from the REE-complex. In some embodiments, contacting a solution at a particular pH (e.g., 1.7-2.2) separates the REE with the smaller ionic radius from the REE-complex. In some embodiments, the REE with the smaller ionic radius is Dy or Y and the REE with the larger ionic radius is Nd or Pr.In some embodiments, the method allows for preferential desorption of Dy from Nd, Dy from Pr, or Y from Nd. In some embodiments, the method allows for preferential separation of LREEs (e.g., La-Nd), HREEs (Ho-Lu plus Y), and MREEs (Sm-Dy).

[0083] In some embodiments, LREEs, such as La, Ce, Pr, Nd, Sm, or Eu, are preferentially separated from HREEs, such as Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, Sm, Y, or Sc. In some embodiments, the method provides preferential separation of LREEs from HREEs, comprising: (a) contacting the protein-REE complexes with a first concentration of a chelating agent (e.g., citrate), whereby the HREEs are separated from the protein-REE complexes; and (b) contacting the remaining protein-REE complexes (e.g., proteins bound to the LREEs) with a second concentration of a chelating agent (e.g., citrate) to separate the remaining REEs. In some embodiments, the first concentration of the chelating agent is about 5 mM to about 10 mM. For example, in some embodiments, the first concentration of the chelating agent is about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, or about 10 mM. In some embodiments, the second concentration of the chelating agent is about 15 mM to about 50 mM. For example, in some embodiments, the second concentration of the chelating agent is about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, or about 50 mM.

[0084] In some embodiments, the present disclosure provides methods for preferentially separating REEs in a single step. Single step separation occurs when the REE-containing material is introduced into the protein once, followed by isolation and purification of individual REEs or groups of REEs free or substantially free of other elements from different REE groups and / or non-REEs (i.e., the REE-containing material does not need to be introduced into the column more than once to achieve high purity). This is in contrast to conventional REE separation techniques, which require multiple purifications of the REE-containing material, making them both inefficient and expensive.

[0085] Aspects of the present disclosure also provide a method for preferentially separating Sc from REE-containing materials using an REE-selective protein such as LanM. Sc can be preferentially separated from REE and non-REE containing materials by selectively dissociating Sc from a protein-REE complex (e.g., a protein-Sc complex). In some embodiments, Sc is selectively dissociated from the protein by contacting the protein-REE complex with a solution comprising a chelator that dissociates Sc from the protein but not other REEs. In some embodiments, the solution comprises malonate. In some embodiments, contacting the protein-REE complex with a solution comprising malonate selectively dissociates Sc without disrupting the protein-REE complex (e.g., without dissociating the REE from the protein), where the REE is an HREE. In a non-limiting embodiment, such a solution (e.g., a first solution) comprises malonate at a concentration of 20-50 mM. In another non-limiting embodiment, such a solution (e.g., a first solution) comprises citrate at a concentration of about 3.0 mM. Once Sc has been desorbed using the first solution, one or more additional solutions can be used to selectively desorb additional REEs. For example, in some embodiments, a second solution containing citrate at a concentration of about 15.0 mM, a third solution containing citrate at a concentration of about 25 mM to about 50 mM, and a fourth solution having a pH of about 1.5 can be flowed sequentially over the protein-REE complex to remove the additional REEs of Y, the HREE / MREE, and La / Ce, respectively.

[0086] In some embodiments, a method for preferentially separating Sc from an REE-containing material using REE-selective proteins comprises the steps of: (a) providing a plurality of proteins capable of selectively binding to one or more REEs; (b) contacting the plurality of proteins with an REE-containing material, wherein the plurality of proteins binds to at least a portion of one or more REEs to form a plurality of protein-REE complexes and an REE-depleted material; (c) separating the plurality of protein-REE complexes from at least a portion of the REE-depleted material; (d) separating Sc from the plurality of proteins by contacting the plurality of protein-REE complexes with a solution comprising a chelating agent, e.g., malonate or citrate; and (e) contacting the plurality of proteins with a solution comprising a chelating agent, e.g., malonate or citrate. (f) separating HREE (e.g., Lu, Yb) from said plurality of proteins by contacting said plurality of protein-REE complexes with a solution comprising a low concentration of chelating agent; (g) separating Y from said plurality of proteins by contacting said plurality of protein-REE complexes with a solution comprising a medium concentration of chelating agent (e.g., malonate or citrate) or a solution comprising a pH of about 2.3; (g) separating MREE from said plurality of proteins by contacting said plurality of protein-REE complexes with a solution comprising a high concentration of chelating agent or a solution having a medium pH; and (h) separating LREE from said plurality of proteins by contacting said plurality of protein-REE complexes with a solution comprising a low pH (1.7). In some embodiments, the chelating agent is citrate. In some embodiments, the solution comprising a low concentration of chelating agent comprises a chelating agent at a concentration of about 5 mM. In some embodiments, the chelating agent is citrate. In some embodiments, the solution with a medium concentration of chelating agent comprises a chelating agent at a concentration of about 15 mM. In some embodiments, the solution with a high concentration of chelating agent comprises a chelating agent at a concentration of about 30 mM. In some embodiments, the solution with a higher pH has a pH of about 2.3. In some embodiments, the solution with a medium pH has a pH of about 2.1. In some embodiments, the solution with a low pH has a pH of about 1.7.

[0087] The REE-containing material can be any material known to contain REEs or suspected to contain REEs. In some embodiments, the material is a solid material, a semi-solid material, or an aqueous medium. In a preferred embodiment, the material is an aqueous solution. Non-limiting examples of suitable materials for use in the extraction of REEs include leachates obtained from rare earth ores (e.g., bastnaesite, monazite, loparite, xenotime, allanite, and laterite ion-adsorbed clays), geothermal brines, coal, coal by-products, mining tailings, phosphogypsum, acid leachates of solid source materials, REE solutions extracted from solid materials via ion exchange methods, or any solid / liquid that reacts with other ore materials, such as REE-containing clays, volcanic ash, organic materials, and igneous and sedimentary rocks.

[0088] In some embodiments, the REE-containing material is a low grade material and the REEs are present in less than about 2 wt.% of the total mass of the low grade material, hi other embodiments, the REE-containing material is a high grade material and the REEs are present in more than about 2 wt.% of the total mass of the high grade material. In some embodiments, the REE-containing material contains less than about 5% by weight, less than about 10% by weight, less than about 15% by weight, less than about 20% by weight, less than about 25% by weight, less than about 30% by weight, less than about 35% by weight, less than about 40% by weight, less than about 45% by weight, or less than about 50% by weight of the total weight of the REE-containing material.

[0089] The proteins can also be used to recover REEs from recycled REE-containing products, such as compact fluorescent light bulbs, electroceramics, fuel cell electrodes, NiMH batteries, permanent magnets, catalytic converters, camera and telescope lenses, carbon lighting applications, computer hard drives, wind turbines, hybrid vehicles, X-ray and magnetic imaging systems, television screens, computer screens, fluid cracking catalysts, phosphor powder from recycled lamps, etc. These materials are characterized by containing a quantity of REEs, including, for example, scandium, yttrium, lanthanum, cerium, praseodymium, neodymium, samarium, promethium, europium, gadolinium, terbium, dysprosium, erbium, thulium, ytterbium, lutetium, or any combination thereof. In some embodiments, the REEs are recovered from liquid waste streams from certain industries (e.g., from factories requiring decontamination from their REEs, or from hospital effluents with the potential for gadolinium recovery) or leach solutions derived from these REE-containing materials.

[0090] In some embodiments, the material is pretreated prior to providing the protein. Non-limiting examples of suitable pretreatments include acid leaching, bioleaching, ion exchange extraction, pH adjustment, iron oxide precipitation, temperature cooling (e.g., geothermal brine). In other embodiments, the REE-containing material is purified to remove at least a portion of the non-REE metals prior to providing the protein. In some embodiments, at least a portion of the protein is attached (i.e., immobilized) to the surface of the solid support prior to contacting the REE-containing material. In some embodiments, the attachment of the protein to the surface of the solid support is reversible. Protein immobilization in a biosorption medium for use in a flow-through setup is expected to allow complete (or substantially complete) separation of the REEs from a mixed metal solution containing the REEs in a single step. In one embodiment, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 95%, about 97%, about 98%, about 99%, or 100% of the REEs in the REE-containing material (e.g., mixed metal solution) are extracted in a single step. In some embodiments, about 1%, 5%, 10%, 15%, 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 95%, about 97%, about 98%, about 99%, or 100% more of the REEs in the REE-containing material (e.g., mixed metal solution) are extracted in a single step compared to the amount of REEs extracted in a single step using conventional extraction methods.

[0091] The binding of the REE to the protein may be reversible. In some embodiments, at least a portion of the REE in the protein-REE complex is desorbed (i.e., removed or separated) from the protein. Non-limiting examples of suitable methods include acid treatment (e.g., sulfuric acid / HNO3 and HCl), citrate, acetate, malonate, and gluconate. In a preferred embodiment, the removal step is performed by acid stripping. In another preferred embodiment, the removal step is performed using a predetermined amount of citrate.

[0092] The protein may be reused. In some embodiments, the method further comprises removing the REEs from the protein to regenerate the protein. The protein may be used 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30 or more times. In other embodiments, the protein is disposable. The protein may be regenerated by any means known to those skilled in the art. For example, the protein may be regenerated by cleaning the protein with a buffer to wash off the citrate. In one embodiment, the method further comprises reusing the regenerated protein to perform extraction of REEs from the REE-containing material. The protein can be reused 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30 or more times while still maintaining its high adsorption capacity. In some embodiments, the protein maintains an adsorption capacity of about 10 mg REE, about 15 mg REE, about 20 mg REE, about 30 mg REE, about 40 mg REE, about 50 mg REE, about 60 mg REE, or about 70 mg REE per gram of protein during each of the adsorption cycles. In some embodiments, the protein maintains an adsorption capacity of about 30 to about 70 mg REE per gram of protein over 9 cycles.

[0093] Aspects of the present disclosure provide kits comprising (a) a protein capable of selectively binding to an REE, and (b) instructions for differentially separating the REE from an REE-containing material, in some embodiments, the protein is bound to a porous support material (e.g., microbeads).

[0094] Working Example EXAMPLES

[0095] Protein-based methods for REE isolation Extraction and subsequent separation of individual REEs starting from REE-bound feedstocks represents a challenging yet necessary challenge for the growth and sustainability of renewable energy technologies. As a key step towards overcoming the technical and environmental limitations of current REE processing methods, the following example demonstrates a bio-based, all-aqueous REE extraction and separation scheme using the REE-selective Lanmodulin (LanM) protein. LanM was coupled onto a porous support material using thiol-maleimide click chemistry to enable REE purification and separation under flow-through conditions. The immobilized LanM maintains the attractive properties of the free protein, such as its remarkable REE selectivity and ability to bind REEs at low pH. The example further demonstrates the ability of LanM to achieve high purity separation of Nd / Dy and Nd / Y, REE pairs important for clean energy, and to convert an industrially important low-grade leachate into separate heavy and light REE fractions in a single column run. The main advantages of the following process over the prior art are its compatibility with low grade feed leachates, the lack of organic solvents, and the ability to achieve efficient separation of specific REE pairs while using approximately 90% of the column volume, in contrast to conventional ion exchange chromatography. This technology can be further developed to enable a sustainable, low-cost REE recovery and separation process that is broadly applicable to REE feed materials. Figure 1 provides a simplified schematic depicting the overall process of REE separation outlined in Example 1.

[0096] method Chemicals and Materials REE chloride salts (>99.9%), solvents, and buffers were purchased from Millipore Sigma. Amine-functionalized agarose beads were purchased from Nanocs Inc. N-Succinimidyl 4-(maleimidomethyl)cyclohexane-1-carboxylate (SMCC) was purchased from Chem-Impex International, Inc. without further purification. NHS-Fluorescein (5 / 6-Carboxyfluorescein succinimidyl ester), mixed isomers, was purchased from ThermoFisher Scientific.

[0097] Preparation of LanM A plasmid containing the gene encoding LanM-GSGC was obtained from Twist Bioscience (pET-29b(+)-LanM-GSGC). The gene sequence consisted of the codon-optimized wt-LanM sequence with (ggcagcggctgc) inserted before the stop codon. 20 .OD of 0.6 600nm Proteins were overexpressed in E. coli BL21(DE3) cells (NEB) at 37° C. with induction with 0.2 mM IPTG. Purification was performed as described, except that all buffers contained 5 mM TCEP. 39 Briefly, cells were lysed and loaded onto a 25 mL (2.5 x 5.0 cm) Q-Sepharose Fast Flow column and eluted using a 0.01-1 M NaCl gradient. Fractions containing LanM-GSGC were further purified using gel filtration chromatography (HiLoad 16 / 600 Superdex 75 pg column (120 mL)) in 30 mM MOPS, 100 mM KCl, 5 mM CaCl2, 5% glycerol, 5 mM TCEP, pH 7.0. Fractions containing LanM-GSGC were exchanged by FPLC into 20 mM acetate, 10 mM EDTA, 100 mM NaCl, 5% glycerol, pH 4.0, concentrated to approximately 3 mM, and frozen in liquid N2 for storage.

[0098] Maleimide functionalization of agarose beads The amine-functionalized agarose microbeads (1.2 ml) were transferred to a 5 mL Eppendorf tube, washed three times with phosphate-buffered saline (PBS) pH 7.4, and resuspended in a final volume of approximately 1.7 mL (1.2 mL microbeads and 0.5 mL PBS supernatant). 0.15 g SMCC was dissolved in 3.4 mL DMSO and then combined with the microbeads. After 2.5 h of incubation on a shaking mixer at room temperature, the functionalized agarose microbeads were washed three times with DMSO to remove unreacted SMCC and three times with coupling buffer (50 mM HEPES (pH 7), 50 mM KCl, and 10 mM ethylenediaminetetraacetic acid (EDTA)) to remove the DMSO solvent. The maleimide microbeads were then used for LanM immobilization within 2 h.

[0099] LanM immobilization LanM immobilization was performed using thiol-maleimide click chemistry conjugation reaction. Specifically, immediately before immobilization, the buffer of LanM-GSGC protein was exchanged into coupling buffer at pH 7.0 (50 mM HEPES, 50 mM KCl, and 10 mM EDTA) using a VivaSpin® 2 centrifugal concentrator (3,000 g / mol molecular weight cutoff, GE Healthcare) to obtain a final protein concentration of about 2 mM. Then, 2 mL of LanM solution was combined with 1 mL of maleimide-microbeads, and the conjugation reaction was carried out at room temperature for 16 hours. Unbound LanM protein was removed by washing with coupling buffer at pH 7, and the LanM-microbeads were stored in coupling buffer at pH 7 for subsequent testing. Maleimide-microbeads were also incubated with coupling buffer without LanM protein as an unbound control sample.

[0100] Breakthrough column experiments An Econo-Column glass chromatography column (Bio-Rad; 5 cm × 0.5 cm) was filled with MilliQ water (18.2 MΩ cm ) before gravimetric addition of LanM-microbeads. -1 The column was packed with 1.0 mL of HCl (Homo-PIPES). The column was washed with 25 mM HCl, MilliQ water and conditioned with buffer solution before breakthrough experiments (same buffer as for breakthrough experiments, see below). REE stock solutions were prepared by dissolving individual REE chloride salts in 1 mM HCl. The stock solutions were diluted at the desired pH with either 10 mM buffer (pH 5: homopiperazine-1,4-bis(2-ethanesulfonic acid, Homo-PIPES); pH 4: acetate acid; pH 3.5-2.2: glycine) or HCl. The REE solutions were pumped at 0.5 mL / min unless otherwise stated and the column effluent was collected in 1.0 mL aliquots. For single REE ion solutions, the REE ion concentrations were quantified by the Arsenazo III assay. Specifically, 40 μL of sample was combined with 40 μL of 12.5 wt% trichloroacetic acid (TCA) and then added to 120 μL of filtered 0.1 wt% arsenazo in 6.25 wt% TCA. The absorbance at 652 nm was measured and compared to standards to determine the REE metal ion concentrations. The accuracy of the colorimetric assay has also been confirmed in previous studies by ICP-MS. For experiments with REE mixtures or leachates, the metal ion concentrations were determined by ICP-MS.

[0101] For REE pair separation experiments, metal ion purity is defined as follows:

number

number

[0102] To determine the selectivity between the REEs of Lanmodulin, the REE distribution coefficient (D) between the LanM phase (bound) and the liquid phase (unbound) is calculated as follows:

number

number

number

[0103] LanM labeling with fluorescein LanM (0.3 mL, 2 mM) was desalted in 100 mM KCl and 8 mM Nd using a spin column (Zeba™ Spin Desalting Columns, 7K MWCO, ThermoFisher Scientific). 3+The LanM solution was exchanged into 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (10 mM; pH 7) containing 10 mM fluorescein NHS ester (1 mg) was dissolved in dimethyl sulfoxide (DMSO, 30 μL) and added to the LanM solution at a final concentration of approximately 3 mg / mL. After 3 h of incubation at room temperature, the untagged dye was removed using a spin column (Zeba™ Spin Desalting Columns, 7K MWCO, ThermoFisher Scientific).

[0104] Confocal microscopy For confocal microscopy, fluorescein-labeled LanM (F-LanM) was used for immobilization on agarose microbeads. The microbeads were then dropped onto a microscope glass coverslip with a drop of VECTASHIELD® anti-fade mounting medium (Vectorlabs). Samples were imaged using a Zeiss LSM710 confocal microscope equipped with a 40×NA1.1 water-immersion microscope objective. 3D fluorescent images of LanM agarose were acquired using Airyscan mode.

[0105] Arsenazo III assay for Sc and Nd determination Sc and Nd concentrations and breakthrough curves in batch experiments were determined using the Arsenazo III assay. Specifically, for Sc, 80 μL of sample was combined with 100 μL of 0.2 M pH 2.8 glycine buffer and then added to 20 μL of 0.3 wt.% filtered Arsenazo in 0.2 M glycine. For Nd, 40 μL of sample was combined with 40 μL of 12.5 wt.% trichloroacetic acid (TCA) and then added to 120 μL of 0.1 wt.% filtered Arsenazo in 6.25 wt.% TCA. Absorbance (Nd at 652 nm; Sc at 675 nm) was measured and compared to standards to determine metal ion concentrations. The accuracy of the colorimetric assay was also confirmed in a previous study by ICP-MS.

[0106] Spectrofluorimetric titration of wt-LanM A solution of 20 μM chelexed LanM was prepared in 20 mM acetate, 100 mM KCl, pH 5.0. In experiments starting with metallated LanM, 40 μM (2 equivalents) of metal was also added. Protein solutions (600 μL) were placed in 10 mm quartz spectrofluorimetry cuvettes (Starna Cells, 18F-Q-10-GL14-S) and assayed using a PerkinElmer FL6500 fluorescence spectrometer in kinetic mode (80 kW power, 278 nm excitation, 2.5 nm excitation slit width, 307 nm emission, 5 nm emission slit width). Titrations were performed by addition of at least 0.6 μL of titrant (either 10 mM metal or 1 mM citrate, pH 5.0), followed by approximately 1 min of signal equilibration. For each data point, 10 seconds of signal was averaged after equilibration. These values ​​were corrected for dilution and F for the apoprotein. 307nm Normalized to =1.

[0107] Inductively coupled plasma mass spectrometry (ICP-MS) The elemental composition of the multi-element synthesis and leach solutions was determined using ICP-MS. For the Dy / Nd binary synthesis solutions, ICP-MS analysis was performed on the ICP-MS. All samples and calibration standards were acidified using concentrated nitric acid (70% by mass, trace metals basis with purity ≥ 99.999%, Sigma-Aldrich) to 2.5% (v / v) and spiked with In, Rh, and Bi internal standards to adjust for shifts in signal intensity during analysis. To reduce doubly charged and polyatomic interferences, samples were analyzed in both hydrogen (for Ca and Si) and helium (for all other elements) reactive gas modes. Due to the complex matrix of the coal ash leachate, 45 Sc and 153 It was necessary to use a correction equation to adjust for mass interference with Eu. Considering the high sampling frequency across the breakthrough curve, a single replicate was used for the column breakthrough test. All feed solutions were run in triplicate. To account for the experimental uncertainty of metal ion quantification in complex synthetic and leachate solutions, the coefficient of variation was determined based on triplicate feed solutions for each metal ion concentration.

[0108] Leaching and pH adjustment of PRB coal ash. Leaching and pH adjustment of PRB coal ash was carried out according to previous studies. 34、35 Briefly, a PRB coal fly ash sample from a pulverized coal-fired power plant collected in 2017 was leached in 40 mL of 1 M HCl solution at 85 °C for 4 h at a pulp density of 100 g / L. The leachate was cooled to room temperature and centrifuged at 3000 g for 15 min to remove all undissolved particles. The supernatant of the leachate solution was then collected and 10 M NaOH stock solution was added dropwise (while being monitored by a combination pH electrode) until the desired endpoint pH value was achieved. The pH-adjusted leachate was centrifuged again to remove any precipitate formed during pH adjustment and then filtered using a 0.2 μm polypropylene filter.

[0109] Results and Discussion Immobilization of LanM on agarose microbeads To facilitate the application of LanM for REE recovery in a flow-through format, a LanM variant containing a C-terminal cysteine ​​residue along with a GSG spacer (hereafter, "LanM") was immobilized on agarose microbeads using thiol-maleimide click chemistry (Figure 2A). 22、23Compared to other immobilization strategies such as physical binding or encapsulation, site-specific covalent attachment allows stable and surface-accessible protein presentation, which is desirable for repeated cycles of adsorption / desorption under harsh conditions (e.g., low pH and high ionic strength). Given the ease of coding of terminal cysteine ​​residues and their stability at low pH (see below), thiol-maleimide chemistry was followed. Purified LanM was operated at low pH (see Methods) to eliminate sulfur oxidation while avoiding the use of reducing agents (data not shown), which were found to have a detrimental effect on LanM immobilization. Maleimide and LanM functionalization of the agarose beads was confirmed by FT-IR (Figure 2B), and the immobilization kinetics was monitored by quantifying the free LanM concentration in the conjugation solution over a 16-h conjugation reaction. As shown in Figure 2B, the 1060 cm -1 The peak at 1200 cm was assigned to the glycosidic bond that forms the backbone of the agarose polymer. -1 and 1706 cm -1 The peaks located at 1706 cm were assigned to succinimide and maleimide groups, respectively. After reaction of amine-functionalized agarose with SMCC, the peaks located at 1706 cm -1 The presence of a peak at 1200 cm -1 The absence of a peak at 1,650 cm indicates successful maleimide functionalization. Finally, after thiol-maleimide click chemistry, the LanM agarose spectrum shows two peaks at 1,650 cm, which correspond to amide I and amide II, respectively. -1 and 1520cm -1 The results showed two peaks at 100 nm, suggesting immobilization of the LanM protein.

[0110] Approximately 97% of the added LanM was loaded within 3 h (Figure 2C), resulting in an immobilization density of 2.89 ± 0.34 μmol LanM / mL agarose. To visualize the distribution of LanM in the agarose beads, a fluorescently tagged variant of LanM (FITC-LanM, green) was incorporated during immobilization. Confocal microscopy imaging confirmed the homogenous distribution of FITC-LanM in the agarose microbeads (Figure 2D).

[0111] Immobilized LanM retains the ability to bind REEs at low pH and is stable for reuse. To test the effectiveness of immobilized LanM for REE extraction under flow-through conditions, the LanM conjugate was packed into a fixed-bed column and the influent breakthrough behavior was evaluated using a solution containing synthetic REEs. As a representative model REE system, Nd 3+ was selected. As shown in Figure 3A, the effect of pH on Nd breakthrough was evaluated (experimental conditions: 0.2 mM Nd in 10 mM glycine buffer (pH > 2.2 conditions; for pH < 2.1 conditions, Nd was diluted with HCl solution), flow rate of 0.5 mL / min; control experiment was performed using a column packed with maleimide-functionalized agarose at pH 3). The Nd breakthrough (at pH 5) occurred after about 25 bed volumes in contrast to 1 bed volume with unbound agarose beads due to passage of void volume, indicating that LanM retains high affinity for REEs when immobilized (Figure 3A).

[0112] The adsorption capacity of the LanM column was 5.78 μmol / mL, corresponding to a 2:1 stoichiometry of Nd per immobilized LanM. This is in contrast to the absorption capacity of LanM in solution, as shown in Figures 4A and 4B. In particular, the stoichiometric titration of LanM-Cys with La(III) demonstrated the binding of three equivalents of REE (Figures 4A and 4B), as evidenced by competitive titration using xylenol orange as indicator (Figure 4A, titrations were performed in chelexed 20 mM MES, 100 mM KCl, pH 6.0), and the structural response of LanM to La(III) was followed as a shift in A284 nm (Figure 4B, titrations were performed in chelexed 30 mM MOPS, 100 mM KCl, pH 7). Interestingly, these results show that in solution LanM binds three equivalents of REE (Figures 4A and 4B), suggesting that one metal site is destabilized when immobilized or in a column format. Based on this finding from previous studies, which revealed that solubilized LanM can bind REEs at a pH as low as 2.5–3, we tested the effect of influent pH on Nd extraction performance over the pH range of 1.7–5 (Figure 3A). The results showed that immobilized LanM can effectively bind Nd even at pH down to 2.4, which is consistent with the results of solubilized LanM. 16 Consistent with the behavior observed in solution, binding of Nd to immobilized LanM is reduced by 50% at pH 2.2 and becomes insignificant at pH ≦ 1.7.

[0113] Taking advantage of the pH dependence of REE binding, Nd desorption was tested by pumping HCl solutions through a Nd-saturated column. The effect of pH (HCl concentration) on Nd breakthrough was evaluated (column pre-adsorbed with 40 bed volumes of 0.2 mM Nd at pH 3), as shown in Figure 3B. Below pH 1.7, sharp desorption peaks were observed from 1 to 6 bed volumes, with Nd enriched by more than an order of magnitude compared to the feed solution (Figure 3B). In contrast, an HCl solution at pH 2.0 led to a progressively smaller desorption profile, with 16.5 bed volumes required to desorb >95% of the Nd. Importantly, the LanM-based adsorbent is robust to repeated exposure to low pH, given that 10 consecutive adsorption / desorption cycles (at pH 3.0 and 1.5, respectively) did not result in a reduction in the adsorption capacity (Figure 3C, experimental conditions: flow rate of 0.5 mL / min; desorption conditions: 10 bed volumes of HCl at pH 1.5). To further determine the affinity of immobilized LanM for the entire REE series, breakthrough curves with other representative REEs (i.e., Y, La, Dy, and Lu; Figure 3D, experimental conditions: feed: 0.2 mM) were plotted at pH 3.0, yielding results indistinguishable from those of Nd. Overall, the results demonstrate effective and reversible REE binding by immobilized LanM under low pH conditions.

[0114] LanM enables high purity recovery and enrichment of REEs To test the REE selectivity of immobilized LanM, Nd (0.2 mM) breakthrough experiments were performed using millimolar levels of Mg enriched in REE-containing feed. 2+ , Al 3+ , Ca 2+ , Co 2+ , Ni 2+ , Cu 2+ , and Zn 2+ The experiment was carried out using a synthetic feed solution of pH 3.0 containing 24Nd breakthrough occurred after 24 bed volumes (Fig. 3E, metal ion breakthrough curves using a synthetic feed solution containing 14 mM Na, 3.3 mM Mg, 9.1 mM Al, 2.3 mM Ca, 1.9 mM Co, 1.9 mM Ni, 1.8 mM Cu, 2.1 mM Zn, and 0.2 mM Nd, pH 3), whereas all non-REEs appeared at the void volume. Importantly, the Nd breakthrough curve and subsequent desorption curves were indistinguishable from those observed using a synthetic solution lacking non-REEs (Fig. 3F, grey diamonds indicate Nd breakthrough / desorption profile without competing non-REE ions. Experimental conditions: metal ion desorption profile collected after treatment with HCl at pH 1.5), indicating that REE and non-REE behaviors are completely decoupled during the process due to the selectivity of LanM. Finally, Fe 3+ Since the limited solubility of Nd precludes its use in multi-element experiments, breakthrough experiments using binary Nd / Fe solutions containing citrate to maintain Fe solubility by coordination revealed that Fe 3+ Selectivity to Nd exceeds 3+ The selectivity of LanM for β-actin was confirmed (Figure 3G, experimental conditions: Feed: Nd+Citrate: 0.2 mM Nd, 20 mM citrate, pH 3; Nd+Fe+Citrate: 0.2 mM Nd, 0.9 mM Fe, 20 mM citrate, pH 3). Table 3 below lists the ion concentrations in the synthetic feed for the data collected in Figure 3G.

[0115] Table 3. Ion concentrations (mM) of the synthetic feed solution. TIFF2024520862000009.tif21138

[0116] Fe 3+ The selectivity of LanM for Nd over its selectivity for ions is due to (1) their low solubility at pH 3 (<100 μM) and (2) Fe(OH) in the pH range 3–4, which can form colloidal particles in the form of Fe(OH). 3+Due to the instability of the Nd / Fe ions, they were studied separately. Previous studies have shown that such colloidal particles can exist stably as suspensions in solutions for up to 2 years, but cannot be effectively removed even by 0.22 μm filters due to their small size. In addition, such colloidal particles can physically attach to solid sorbent materials and columns during flow-through experiments. To address this, 20 mM citrate was added to the Nd / Fe synthesis solution to improve the stability of Fe at pH 3 conditions. 3+ Improves the solubility of Fe ions 3+ This allowed us to study the REE selectivity of LanM over Nd. As shown in Figure 3G, the Nd breakthrough curves were 3+ LanM was still almost identical in the presence and absence of Fe after immobilization. 3+ It is shown that the REE selectivity is high for Fe at higher pH. 3+ Most of the Fe impurities can be removed by increasing the pH to the pH 4-5 range, due to their lower solubility (<10 μM at pH 4 and <0.01 μM at pH 5).

[0117] Taken together, these results suggest that immobilized LanM retains the high REE selectivity of LanM in solution and can be employed to separate REEs from non-REEs.

[0118] Ligand competition in solution reveals possible separation between REEs Initial characterization of LanM revealed that the protein exhibits an inverse affinity trend compared to most REE ligands, La 3+ -Nd 3+ The highest and almost similar affinity for Ho 3+ showed approximately one-fifth the affinity to 20 However, in-solution methods to exploit these differences in separation between REEs are 16 And previous studies of on-column REE extraction (see above) have not yet attempted. Competition with milder chelators, such as citrate, which have heavy REE preferences. 25It is anticipated that this could enable enhanced selectivity in the desorption process. To establish this principle, we first investigated ligand competition with LanM in solution. We exploited the observation that the fluorescence of tyrosine residues alone in LanM is quenched upon REE binding, which may be due to different solvent exposure and hydrogen bonding in the two protein conformations. 26 Titration of LanM with REE showed the largest change in tyrosine fluorescence at two equivalents. The REE was negated upon binding of the third equivalent (Figure 5A). It was hypothesized that the REE binding site involved in this third equivalent also corresponds to the site that is destabilized upon immobilization. This change in fluorescence provides a convenient clue to assay the conditions for REE detachment from LanM. These studies using individual titrations with Y-, Dy-, or Nd-bound LanM revealed that the apparent K d We reveal that citrate selectively outcompetes LanM for the HREE before the LREE (Figure 5B), as predicted given the HREE-dependent binding of LanM to the LREE. For example, 6 mM citrate is sufficient to almost completely desorb Dy from LanM, whereas Nd is still fully bound to the protein. The sharp desorption profile (occurring over a four-fold concentration range) is consistent with cooperativity in the LanM metal binding behavior. 20 These results demonstrate that despite the high REE affinity of LanM, mild chelators can be used to desorb the REEs. Moreover, importantly, adjustments to the conditions can be used to selectively desorb the HREEs from LanM without destabilizing the LREE-LanM complex, setting the stage for on-column separation. Table 4 below lists the ion concentrations in the synthetic feed for the data collected in Figures 5A and 5B.

[0119] Table 4. Ion concentrations (mM) in the synthetic feed solutions in Figures 5A-5B TIFF2024520862000010.tif57141

[0120] Separation between REE pairs The next experiment first examined whether careful selection of desorption conditions would allow on-column separation between REEs using LanM columns loaded with individual REEs over a range of pH (Figure 6A, REE ion desorption normalized to total REEs desorbed; experimental conditions: columns loaded to 90% saturation using independent single REE solutions) and a citrate step (Figure 7A, pH 5). To cover the entire ionic radius range of the lanthanides (La and Lu), we further tested a representative set of REEs (Y, La, Pr, Nd, Dy, and Lu) independently based on their criticality for renewable energy technologies (Y, Pr, Nd, and Dy). Distinct elution profiles were observed under both desorption conditions for each REE (Figures 6A and 7A). In the case of citrate, the order of REE elution correlated with ionic radius and closely matched the relative affinities of the LanM-REE and citrate-REE complexes. Similar trends with respect to pH were observed, with the notable exception that Nd required a lower pH for desorption compared to La, but this result suggests a greater relative stability for the LanM-Nd complex compared to LanM-La, consistent with a local maximum in stability for LanM in the Nd / Pr range. 16、20 It is anticipated that differences in stability between the LanM REE complexes tested can be exploited to achieve separation between specific REE pairs.

[0121] Single adsorption / desorption cycle for Dy / Nd separation. Guided by the above results, we tested the effectiveness of REE separation of LanM by loading the column with a 50:50 molar ratio solution of Nd and Dy to 90% saturation, followed by a stepwise desorption process (Figure 6B, feed material composed of a 50:50 mixture of Dy:Nd was loaded to 90% column saturation and then subjected to a two-step pH desorption scheme (2.1 and 1.7); Figure 7B, feed material composed of a 50:50 mixture of Dy:Nd was subjected to a two-step desorption scheme using 15 mM citrate (pH 5) followed by pH 1.7). Notably, Dy and Nd were eluted in high purity in separate fractions by either employing a two-step pH desorption scheme or by combining an initial citrate-mediated Dy desorption with a subsequent pH-mediated elution of Nd. For the two-step pH scheme, 76.2% of Dy was eluted in the first pH 2.1 desorption with 99.9% purity, while 76.8% of Nd was eluted in the second pH 1.7 desorption with 99.9% purity. Less than 24% of the loaded REE material was present in the peak overlap region; considering the identical Nd / Dy composition as the feed solution (50.8% Dy; Figure 8B), this fraction can be combined with the first feed solution and processed during future purification cycles (thus avoiding any loss of REEs). Desorption using 15 mM citrate eluted 94.2% of the adsorbed Dy with 99.1% purity, while the subsequent pH desorption step eluted the remaining Nd (99.2%) with 94.6% purity. Taken together, these results show that high purity Dy / Nd products can be obtained when starting from an equimolar mixture of both metal ions.

[0122] To test with a feed composition reflective of electronic waste containing NdFeB magnets, the column was loaded to 90% saturation with a feed solution composed of 95% Nd and 5% Dy. 27The use of Nd as a surrogate for the combined Nd / Pr content (typically 3:1 Nd:Pr) is supported by the nearly identical desorption profiles of both metal ions as a function of pH or citrate concentration (Figures 6A and 7A). Successful separation of Dy from Nd / Pr is expected to enable the production of high-value dysprosium and didymium oxide products that can be fed back into the magnet manufacturing supply chain. As shown in Figure 6C (feed material composed of a 5:95 mixture of Dy:Nd was loaded to 90% column saturation and then subjected to a two-step pH desorption scheme (2.2 and 1.7)) and Figure 7C (feed material composed of a 50:50 mixture of Dy:Nd was loaded to 90% column saturation and then subjected to a two-step desorption scheme using 10 mM citrate (pH 5) followed by pH 1.7), both the two-step pH and citrate desorption schemes resulted in highly pure Nd fractions (99.8% and 98.7%, respectively) with a significant upgrade in Dy purity. For example, the pH 2.2 elution step resulted in 88.6% Dy content with a purity of 46.1%, while the citrate desorption step resulted in 73.9% Dy with a purity of 48.9%.

[0123] Two adsorption / desorption cycles for Dy / Nd separation. Given the successful Dy / Nd separation results using the 50:50 mixture (Figure 6B), the approximately 50% pure Dy fractions (Figure 6C) generated in the first column step (i.e., the first adsorption / desorption cycle) from the 95%Nd / 5%Dy feed material were tested to determine whether these fractions could be upgraded to even higher purity by performing a second adsorption / desorption cycle. To this end, the Nd / Dy separation using the 95%Nd / 5%Dy feed was repeated five times to accumulate a sufficient volume of the 44% pure Dy desorbed fraction to allow for a second separation step (or cycle) (Figure 21). Despite the dilute nature of the Nd / Dy feed solution, the LanM column exhibited high loading yields (>99%), achieving 88% Dy at 99.2% purity and 82% Nd at 99.9% purity after the pH desorption step, respectively. These results suggest that the LanM column can effectively separate Dy from Nd starting from low purity Dy solutions typical of e-waste leachates. Even higher yields and product purity may be achieved using higher operating volumes. In addition, further REE separations may be realized by carefully adjusting the column operating conditions, such as pH, identity of the competing chelating agent, and concentration, flow rate, and column geometry.

[0124] Dy / Nd separation using e-waste samples. Results using synthetic solutions suggested that LanM could effectively separate a 95%Nd / 5%Dy feed into high purity Nd and Dy products in two adsorption / desorption cycles. To demonstrate the recovery and separation of REEs from real e-waste, a stepwise separation scheme was tested using bio-leachate prepared from HDD scrap (INL). After loading the e-waste bio-leachate onto the LanM column (adsorption step), a washing step with distilled water was used to remove unbound metal ions and bioliquidants. A two-step desorption at pH 2.2 and pH 1.5 was then used to selectively desorb Dy and produce high purity Nd / Pr, respectively. Finally, a 1M HCl treatment step was used to remove Fe precipitates and regenerate the LanM column (Figure 22A-B).

[0125] Using this scheme, a Dy concentrate (28.9%; relative to total REEs) and a high purity Nd / Pr (99.3%) solution were obtained. Based on previous results with synthetic solutions, it is possible to further upgrade the Dy fraction to high purity using a second column step. Moderate levels of Fe impurity were co-extracted in each desorption fraction, but previous studies have shown that this Fe can be effectively removed by precipitation induced by adjusting the solution to pH 6. Residual Fe in the column may be removed by a 1 M HCl (pH 0) cleaning step (Figure 22C). Finally, the column stability was tested using a breakthrough curve with a 0.4 mM Nd solution (pH 3) and the regenerated column (Figure 22D). No loss of REEs in the adsorption capacity was observed. This study showed that the LanM column can be used to treat e-waste bioleachate to produce a Dy concentrate and high purity Nd / Pr.

[0126] To test the effectiveness of a second column step (i.e., a second adsorption / desorption cycle) to further purify the 28.9% Dy fraction to high-purity Dy and Nd, a synthetic 71% Nd / 29% Dy feed solution was loaded onto a Ranmodulin column and subjected to a two-step pH desorption process. High-purity Dy and Nd solutions were obtained (Figure 23). Compared to the separation and purification process of immobilized LanM discussed above, other extraction methods are not as effective. For example, in a recent liquid-liquid extraction study using bis(2,4,4-trimethylpentyl)phosphinic acid (Cyanex272) for Nd / Dy separation, a feed solution containing 6.7% Dy was concentrated to about 47% pure Dy using a single extraction step, resulting in a Dy extraction yield of 85.8%. 28 Another study employing a countercurrent extraction configuration with 2-ethylhexylphosphonic acid mono-2-ethylhexyl ester (PC88A) ligand required three and four extraction stages to upgrade a 20% Dy / 80% Nd feed solution to 76.7% and 95.2% pure Dy solutions, respectively. 29 Therefore, immobilized LanM can be used as an effective and environmentally friendly alternative to liquid-liquid extraction to produce high purity (up to 99.9%) Nd and Dy separations.

[0127] We next tested the ability of LanM to separate Y from Nd (Figure 6D, a feed consisting of a 22:78 mixture of Y:Nd was loaded to 90% column saturation and then subjected to a two-step pH desorption scheme (2.3 and 1.7). Figure 7D, a feed consisting of a 22:78 mixture of Y:Nd was loaded to 90% column saturation and then subjected to a two-step desorption scheme using 10 mM citrate (pH 5) followed by pH 1.7). Both REEs are abundant in primary REE deposits (e.g., those containing monazite, xenotime, and / or allanite) and are considered important for green energy technologies. The ratios of Y and Nd in the feed solution were set to 22% and 78%, respectively, to resemble the ratio of HREEs to LREEs in typical coal by-product leachates. 30、31Two distinct peaks were collected after the two-step pH desorption, achieving 95.6% Y and 99.8% Nd purity, respectively. A small overlapping region (<25% of the adsorbed metals) of nearly identical composition to the influent feed solution (79.1% Nd + 20.9% Y) may be collected and reused as the feed solution in subsequent adsorption / desorption cycles. Notably, the citrate / pH combination resulted in nearly complete Y / Nd separation; 95.8% of Y eluted with 99.4% purity, while 99.7% of Nd eluted with >99.9% purity. Thus, using a single adsorption / desorption cycle, Y can be separated from Nd. A summary of the REE composition in the feed and the three desorption zones by using the two-step pH scheme and the citrate-pH scheme is provided in Figures 6E and 6F, respectively. Table 5 below lists the ion concentrations in the synthetic feed for the data collected in Figures 6A-6F.

[0128] Table 5. Ion concentrations (μM) in the synthetic feed solutions in Figures 6A-6F TIFF2024520862000011.tif41141

[0129] Extraction and separation of grouped REEs from low-grade feedstock leachates. Recovery of REEs from abundant waste materials such as coal fly ash and red mud offers a promising means to diversify REE supplies while avoiding the pollution inherent in mining. However, leach solutions produced from such low-grade REE-containing waste materials contain high concentrations of Al. 3+ , Ca 2+ , and Fe 3+ These contain high levels of metal ion impurities such as 5、24 For example, Al 3+ are commonly co-extracted with the REEs in liquid-liquid extractions, resulting in low REE purity and the formation of gel-like hydroxide-mediated emulsions. 24 Similarly, other impurities, such as Ca 2+ can cause contamination during the liquid-liquid extraction process via gypsum formation32 Therefore, the leach solution is generally subjected to a pre-treatment precipitation step to remove impurities before feeding it to the liquid-liquid extraction unit. Selective precipitation is carried out to remove Fe 3+ It is effective in removing certain impurities such as Al 3+ Complete removal of the REE hydroxides is difficult due to their coprecipitation with aluminum hydroxide. 24、33 Therefore, non-REE impurities, especially Al, 3+ and Ca 2+ REE extraction methods that have high REE selectivity compared to conventional methods are highly desirable.

[0130] As an industrially relevant performance test, the LanM column-based REE extraction and separation concept was demonstrated using a low-grade leachate (0.043% REEs, monovalent ions were rejected) prepared from subbituminous coal (PRB) fly ash. 34、35 The leachate contains about 150 μM total REEs, as well as significant transition metal content (e.g., Zn, Ni, Cu, and Mn), compared to mM levels of Na, Mg, Al, Ca, and Sr (Tables 6A-6B). Using the LanM-based column, breakthrough of the REEs occurred after 30 bed volumes, whereas non-REEs eluted in the void volume (Figure 8A, metal ion adsorption profile; adsorption conditions: pH 5, 0.5 mL / min. 1 bed volume = 0.8 mL). To assess the REE purity of the adsorbed metal content, the metal ion composition was determined after non-selective desorption using a pH 1.5 solution. More than 96.5% of the REEs were desorbed within the most concentrated fraction (3.9 bed volumes total), with an average enrichment factor of 6.9 (Figures 8B and 9, metal composition in the PRB feed and recovered biosorption solution). A total REE purity of 88.2% (Figure 8C, percentage of metal ions (monovalent ions excluded) in the PRB feed and recovered biosorption solution) was observed, representing a significant 2050-fold increase in purity compared to the feed solution (0.043% REEs, monovalent ions excluded). Importantly, radionuclide uranium was not enriched from the PRB leachate.

[0131] Table 6A. Ion concentrations (μM) in PRB feed solution at pH 5. TIFF2024520862000012.tif213146

[0132] Table 6B. Ion concentrations (μM) in the three most concentrated fractions of the PRB desorption solution. TIFF2024520862000013.tif213146 *Uncertainty for each element was assumed based on triplicate tests from Table 5A.

[0133] To evaluate the REE selectivity of the LanM column-based method using a conventional liquid-liquid extraction approach ( Figure 8D , values ​​above the dotted line suggest higher REE affinity over non-REEs), the separation factors of total REEs relative to base metal ions were compared to data generated using a widely available commercially available extraction solvent, di-2-ethyl-hexyl phosphoric acid (DEHPA), with a coal fly ash leachate of comparable composition. 36 The LanM-based approach exhibited high selectivity against all non-REE impurities except for Fe and Si, whereas Mg 2+ , Al 3+ , and Ca 2+ The selectivity to ethanol over several orders of magnitude higher is particularly compelling considering their abundance in low-grade feedstock leachates. 31、37 The lower relative selectivities for Si and Fe are expected to reflect the formation of unfilterable colloidal particles that accumulate on the column and dissolve during the desorption step at low pH. Thus, the current column-based approach requires a pre-column method to remove the Fe / Si content to maximize the action of LanM. In summary, these results highlight the ability of immobilized LanM to selectively enrich REEs from low-grade leachates containing a variety of metal ion impurities.

[0134] After demonstrating effective removal of most of the non-REE impurities during the adsorption step, the ability of LanM to enable group separation of the adsorbed REEs from the PRB leachate using a two-step pH desorption scheme (LREEs (La-Gd): 72%; HREEs (Tb-Lu+Y): 28%). Separation of the REEs grouped into HREE and LREE fractions is a critical initial step during liquid-liquid extraction and requires multiple extraction and stripping steps to enrich the HREEs, typically involving the use of different extraction solvents compared to those used in the non-REE impurities removal step. 5 Two distinct peaks composed mainly of either HREEs or LREEs were observed (Figure 8E); 82% of the HREEs eluted at 72.6% purity at pH 2.3, while 80% of the LREEs eluted at 98.8% purity at pH 1.7 desorption. Sc was also co-extracted with the other REEs and co-desorbed with the LREE group; however, its concentration was notably low (0.02 μM) in the PRB leachate. To achieve similar purity of LREE separation from the acid leachate generated from ion adsorption, 11 countercurrent extraction steps were required via a stepwise liquid-liquid extraction process, in which two common extraction solvents, 2-ethyl-hexylphosphonic acid mono-2-ethylhexyl ester (HEH(EHP), P507) and di-(2-ethylhexyl) phosphoric acid (HDEHP, P204), were sequentially utilized. 38Taken together, these results highlight the main advantage of the LanM column: the ability to achieve both removal of non-REE impurities and separation of grouped REEs in a single adsorption / desorption step, with low grade leachates, without the use of organic solvents or hazardous chemicals. Based on the separation data with Nd / Dy and Nd / Y pairs, more precise separation within the HREE and LREE groups is possible by coupling multiple adsorption / desorption steps and / or by judicious incorporation of organic chelators in the desorption step. Selective desorption of HREEs (Tb-Lu+Y) and LREEs (La-Gd) by a two-step pH scheme is shown in FIG. 8E (the upper values ​​in panel D indicate the purity of LREEs or HREEs relative to the total REE content, and the three elution zones are divided by vertical dotted lines), and the REE ratios relative to the total REEs in the PRB feed and the three elution zones are shown in FIG. 8F (experimental conditions: 29.1 bed volumes of PRB fly ash leachate were pumped into the column at 0.94 mL, followed by a wash step with 10 bed volumes of water at pH 3.5. Desorption was carried out sequentially with 16 bed volumes of HCl solution at pH 2.3 and 10 bed volumes of HCl solution at pH 1.7). The ion concentrations in the synthetic feed for the data collected in Figures 8A-8D are listed in Table 7 below, and the ion concentrations (μM) in the PRB leachate feed and the three desorption zones in Figures 8E and 8F are listed in Table 8 below.

[0135] Table 7. Ion concentrations (μM) of synthetic feed solutions in Figures 8A-8D TIFF2024520862000014.tif21138

[0136] Table 8. Ion concentrations (μM) in the PRB leachate feed and the three desorption zones in Figures 8E and 8F TIFF2024520862000015.tif229152

[0137] Separation of REEs using pH-based desorption Differential affinity of LanM among REEs. To identify the REE selectivity preference of immobilized LanM, a synthetic solution containing all REEs (except Pm) was used for column breakthrough tests (Figures 10A and 10B). The REEs did not elute until the column reached the breakthrough point determined by single element experiments. Breakthrough occurred in the following order: Lu, Yb, Tm, Y, Er, Ho, Dy, Tb, Gd, La, Ce, Eu, Nd, Pr, Sm, and Sc, revealing a preference for medium-light REEs over heavy REEs, which is consistent with the binding affinity determination of solubilized proteins. Interestingly, in the breakthrough region, the effluent concentration of heavy rare earths was higher than that of their feed concentrations, suggesting competitive displacement by REEs with higher affinity for LanM. This differential affinity of LanM among REEs can be exploited for REE separation.

[0138] The selectivity of ranmodulin between the REEs was also quantified. Separation factors were determined by quantifying the equilibrium distribution of REEs between solution and immobilized ranmodulin (Figure 30). More specifically, the REE series was split into two solutions with equimolar concentrations of REEs, which were run in a circular fashion until equilibrium was achieved on the column containing immobilized ranmodulin. Metal ion concentrations in solution before and after adsorption were then quantified. Overall, the data revealed a strong preference of ranmodulin for light-medium REEs over heavy REEs, which is in strong agreement with the competitive breakthrough curve data in Figure 10 and the binding affinity determination of the free protein. The significant differences in partition coefficients observed between adjacent REEs and medium REEs (SFs of 1.4-1.8 for adjacent metal ions; Table 1 below) highlight the potential of employing ranmodulin for the separation of these elements. In contrast, there is minimal difference in partition coefficients from Ce to Eu (SF is less than or equal to 1.3 for any pair in this region), suggesting that a separation process relying solely on the affinity of ranmodulin for the REEs is unlikely to be effective in separating these REEs.

[0139] Table 1. Separation factors for the REE series after one adsorption cycle. (1 / 2). Determined from the partition coefficients in Figure 30. TIFF2024520862000016.tif171166

[0140] Guided by the differential breakthrough of REEs, the feasibility of a stepwise pH-dependent selective desorption process for REE groups from an REE-loaded LanM column was investigated. To perform such experiments under practical conditions, the LanM column was preloaded with a synthetic REE solution similar to the REE composition in coal fly ash leachate, and then a multi-step pH desorption was employed (Figures 11A-11D). In particular, 13 μM of each REE (Lu, Yb, Tm, Y, Er, Ho, Dy, Tb, Gd, La, Ce, Eu, Nd, Pr, Sm, and Sc) was dissolved in a 10 mM glycine solution at pH 3 per equimolar REE ion solution. The REE solution was pumped through the LanM column at 0.5 mL / min, and the column effluent was collected in 1.0 mL aliquots. The REE concentrations in the effluent were determined by ICP-MS. The REE eluate was separated into three groups: heavy REE (Ho-Lu+Y), medium REE (Gd-Dy)+La, and light REE (Ce-Eu). Heavy REE were desorbed first in the pH range of 2.3-2.5, whereas all light REE except La were retained on the column until the pH reached pH 2.1 and below. Medium REE were eluted in the intermediate pH range. These results suggest that REE co-extracted from coal fly ash leachate (or other ore-based feedstock leachate) can be separated into three subgroups in a single adsorption / desorption cycle. It is conceivable that the REEs in the separated groups can be further separated using additional adsorption / desorption cycles on the LanM column.

[0141] Separation of REEs using strategic selection of organic chelating agents LanM allows for the separation of Sc from the REE. LanM binds Sc with an apparent dissociation constant of sub-picomolar concentrations (Figure 12). During this experiment, CD spectroscopy was used to measure the solubility of free Sc in various malonate-buffered solutions. III Concentration in [θ] 222nmwas monitored and the data was fitted to the Hill equation using the Hypspec computer program. 40 Speciation diagrams of various Sc-malonate systems in 20 mM acetate, 100 mM KCl, pH 5 were calculated using the CD titration method. The resulting free metal concentrations were used for affinity determination using standard CD titration protocols. Only one blank was needed since Sc-malonate did not contribute to the signal. Impressively, the data with free LanM show that the chelator malonate can strip Sc from LanM-Sc complexes at concentrations that do not affect the heavy REE-LanM binding (Figure 13). Thus, malonate can be employed to separate Sc in high purity from the REE feed leachate by loading the LanM column with REE in an initial adsorption step and then using malonate to selectively recover Sc. Subsequent desorption steps with other chelators or via pH modulation can then be employed for further REE separation.

[0142] Effect of citrate on REE breakthrough and desorption. Based on the encouraging citrate desorption data for free LanM (Figure 14), the effect of citrate on REE breakthrough was investigated. A synthetic solution containing all REEs (except Pm) was prepared in 20 mM citrate at pH 3.5 for column breakthrough testing. The breakthrough order of the REE eluates correlated with the ionic radius (Figure 15A). HREEs (Lu-Tb, Y, Sc) broke through at 5 times the first bed volume, whereas LREEs (La-Nd) did not break through until 25 times the bed volume. Guided by the equimolar REE breakthrough experiments, the possibility of using a citrate step to selectively desorb REEs from a REE-loaded LanM column was investigated (Figure 15B). As expected, the REE desorption order of the REE eluates also followed the ionic radius. Surprisingly, however, the majority (80%) of Sc was desorbed by dilute citrate (3 mM) before all other REEs, including Lu. This data, together with previous studies with free LanM, suggests that co-extracted Sc can be effectively separated from all other REEs by using dilute chelator solutions. Using malonate instead of citrate allows for even cleaner separation of Sc from the REEs (Figures 15A and 15B). The data also suggest that a stepwise desorption process with citrate can be employed to generate separate HREE, MREE, and LREE pools.

[0143] Sc / Y Separation from Synthetic Ore Leach Solutions by Citrate-pH Stepwise Desorption. A desorption-based process was developed for the separation of Sc and Y from lanthanides using a synthetic REE solution similar to the REE composition of ore-based feedstock leach solutions (e.g., bauxite and its waste residues, allanite, coal and coal combustion products; FIG. 26). Considering the preferential binding of citrate to Sc and heavy REEs in combination with the preferential binding of light-medium REEs by lanmodulin (LanM), a desorption scheme using multiple citrate steps was tested to determine whether it could be used to separate Sc and Y from L-MREEs. The study presented in FIG. 15B revealed that citrate (pH 5) concentrations of 3 mM and 15 mM were effective in desorption of Sc and Y from the LanM column, respectively. Furthermore, the 3 mM citrate desorption step resulted in an Sc solution with 96.4% purity and 77% recovery yield. Figure 27 shows the desorption profile (A) and cumulative yield (B). Subsequent desorption steps using 15-30 mM citrate yielded a Y solution with 88% purity and >90% recovery yield. 75 mM citrate was then used to generate a highly useful MREE+Nd / Pr enriched fraction. A pH 1.5 solution was used to desorb the remaining REEs, yielding a La / Ce enriched solution. These results indicate that Sc and Y can be effectively removed from the adsorbed lanthanide fraction by using a citrate-pH desorption strategy.

[0144] Sc / Y separation from synthetic ore leachate by malonate desorption. Considering the encouraging Sc / Lu separation results using free Lanmodulin protein and malonate chelator depicted in Figure 13, the effectiveness of malonate to achieve high purity Sc separation from REEs was tested. LanM-columns were loaded with ore-based feedstock leach solutions (e.g., bauxite and its waste residues, allanite, coal and coal combustion products; Figure 26) and subjected to desorption using increasing concentrations of malonate. The data suggest that 30 mM malonate is sufficient to separate Sc with high purity and yield (>99% purity; >99% yield) from the REE-loaded Lanmodulin column (Figure 28). Based on the data in Figures 25 and 27, a subsequent citrate desorption step could well be employed to obtain Y-enriched, MREE-enriched and La / Ce-enriched fractions. In summary, these data highlight the ability of ranmodulin to yield highly pure Sc solutions when coupled with a desorbing agent such as a carboxylate-containing chelating agent.

[0145] pH-based desorption for Sc separation. Given the preferential binding of Lanmodulin to Sc over REEs, a desorption scheme using pH was tested to determine whether it could be used for Sc separation. To test this, the LanM-column was loaded with a synthetic REE solution similar to the REE composition in typical Sc-containing ore-based feedstock leaching solutions (e.g., bauxite and its waste residues, allanite, coal and its resulting combustion products). Using a stepwise decrease in pH, Sc was observed to elute in the last fraction with a purity of 30% (Figure 29). Since the impurities are LREEs and MREEs rather than HREEs, Sc can be easily separated in a subsequent step with high purity using chelating agents (e.g., malonate or citrate) according to the logic logic described above.

[0146] Separation of grouped REEs using organic chelating agents. Based on the differential desorption experiments presented above (Figure 7A and Figure 15A-B), we identified citrate as a promising candidate for separating light / medium / heavy REEs. To determine the most effective strategy for desorption-based REE separation among LREEs / MREEs / HREEs, the LanM-column was loaded with a synthetic REE solution similar to the REE composition in a typical ore-based feedstock leach solution (Figure 24A; LREE(La+Ce):MREE(Pr-Gd):HREE(Y,Tb-Lu)=approximately 50%:25%:25%) and a stepwise citrate desorption scheme was employed (Figure 24B). LanM has a similar high affinity for medium and light REEs, precluding the use of a pH-based desorption gradient for the separation of MREEs from LREEs. Given the higher affinity of citrate for MREEs than LREEs, a desorption scheme using citrate was tested to determine whether it could be used to separate LREEs / MREEs / HREEs into separate groups. It was found that HREEs could be selectively separated from MREEs and LREEs using 15 mM citrate, while citrate concentrations of 25-50 mM could effectively separate MREEs from LREEs.

[0147] After determining the optimal citrate concentration for REE separation, follow-up experiments were performed to demonstrate grouped REE separation via a stepwise citrate-pH scheme (Figure 25A-B). HREEs were nearly quantitatively desorbed in high purity using a 15 mM citrate desorption step (93.6% HREE purity). A 50 mM citrate step then produced an MREE-enriched solution at 70.7% purity. Finally, a non-selective pH 1.5 desorption step desorbed the remaining REEs and produced a concentrated La / Ce solution (90.6% purity). This result suggests that when starting from a synthetic REE composition (representative of that in the primary ore), the light REEs (La and Ce) can be effectively depleted from the HREEs and MREEs in a single citrate-based desorption step. Thus, the LanM-based approach allows for the separation of REEs versus non-REEs (adsorption step) and the separation of grouped HREEs / MREEs / LREEs (desorption step) in a single adsorption / desorption process.

[0148] It is expected that this sequestering effect may also be observed with other soluble organic chelators, such as iminodiacetic acid (IDA), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), [S,S]-ethylenediamine-N,N'-diglutaric acid (EDDG), etc. Chelators such as malonate and HIBA are effective in Sc and HREE desorption, but have insufficient MREE affinity to compete with LanM at reasonable concentrations.

[0149] conclusion The following examples demonstrate a biomaterial-based, all-aqueous REE extraction and separation platform using REE-selective protein chelators. This concept represents an important step towards sustainable REE production and minimizing the global reliance on primary REE resources. LanM, a recently discovered REE-binding protein chelator, was immobilized on a biologically renewable agarose support resin to enable flow-through REE extraction and easy reuse. The immobilized LanM retained its remarkable REE selectivity and facilitated near quantitative REE separation from non-REE impurities. Following sequential pH gradient or mild chelator treatment, co-extracted Nd / Dy, the most important REE pair for e-waste recovery, was separated to high purity (99.9% purity) within one or two adsorption / desorption cycles depending on the feed ratio. The study further demonstrated the application of LanM for REE extraction and separation into heavy and light REE groups in a single adsorption / desorption cycle. The main advantages of the disclosed process over the prior art are compatibility with low grade feed leachates, lack of organic solvents, ability to achieve high purity separation of specific REEs while using the entire column volume, compatibility with acidic feed materials, high selectivity for REEs during the first adsorption step, allowing for minimization of downstream processing, and no enrichment of radioactive impurities (e.g. U and Th). EXAMPLES

[0150] Improving carbon economy and loading capacity of LanM columns As discussed in Example 1, a 2:1 stoichiometry of Nd versus immobilized LanM was observed. Interestingly, in solution LanM binds 3 equivalents of REE, suggesting that one metal site is destabilized upon immobilization or in a column format. This result suggested that it may be possible to shorten the protein (i.e., by removing non-functional binding sites) without perturbing the 2:1 stoichiometry. Reducing the length of the protein without perturbing the 2:1 stoichiometry could increase the overall immobilization rate of LanM onto the column, which in turn could increase the amount of REE that can be loaded onto the column, thereby increasing the effectiveness of REE separation from REE-containing materials.

[0151] The amino acid sequence of LanM can be shortened without perturbing the REE:LanM stoichiometry of immobilized LanM. Removal of the first 40 amino acids of LanM (i.e., 19 amino acids from the N-terminus of SEQ ID NO:1) resulted in a functional protein with a 10% reduction in molecular weight and a 2:1 stoichiometry of REE to LanM (Figures 16A and 16B) and, unexpectedly, improved the likelihood of Dy / La separation (Figures 16A and 16B); Dy could be completely recovered from LanM at citrate concentrations that did not cause dissociation of the LanM-La complex. This highlights the possibility of separating MREE and LREE with high purity using LanMΔ1-40 (Table 1, SEQ ID NO:2).

[0152] Dual LanM proteins To increase the REE adsorption capacity of the LanM column, a double LanM construct (termed LanM-double(2-1); Table 1, SEQ ID NO: 3) was generated and the stoichiometry of REE binding was tested. The stoichiometry is approximately 4:1-5:1 in solution (i.e., the third binding site in one of the LanM units is considered functional), but is likely to be 4:1 when immobilized, based on the 2:1 stoichiometry of immobilized single LanM (Figure 17A-17C). The LanM-double protein is expected to have a terminal cysteine ​​added and be immobilized on agarose resin for subsequent testing. Importantly, the Nd / Dy dissociation properties of double LanM were quite similar to WT LanM (Figure 18). This suggests that double LanM allows for on-column Dy separation from Nd using citrate as a desorbent. EXAMPLES

[0153] Sequential HREE / LREE separation by using a three-column rotation scheme The following example describes a sequential method for REE separation by using three LanM columns in rotation as shown in Figure 19. Such a scheme involves four steps (steps A-D) to complete a separation cycle and allows for highly efficient, high yielding REE separation (i.e., single REE product with purity >99.9%, yield >99%).

[0154] method An Econocolumn glass chromatography column (Bio-Rad; 5 cm x 0.5 cm) was packed with deionized water before gravimetric addition of LanM-microbeads. The column was washed with 25 mM HCl, deionized water, and conditioned with deionized water before breakthrough experiments. REE feed solution (39 μM Al, 2.3 μM Mn, 3.1 mM Fe, 54 μM Co, 2.5 μM Cu, 5.6 μM Zn, 34.9 μM Pr, 371 μM Nd, and 21.8 μM Dy) was prepared by using Biolixivivant to leach REEs from electronic waste (E-waste). The feed was pumped into the column at 0.5 mL / min and the effluent was collected in 1.0 mL aliquots. REE concentrations in the effluent were determined by ICP-MS.

[0155] Results and Discussion In step A, REE solution, e.g., Nd and Dy at pH>2.5, is pumped into column i and the effluent is collected by column ii. Before reaching the full capacity of column i, both Nd and Dy are extracted by column i, which is supported by previous breakthrough experiments (1-10 bed volumes) shown in Figure 20. However, once column i is empty (after 10 bed volumes), Dy ions are expected to be selectively replaced by Nd due to the higher LREE selectivity over HREE of LanM. Step 1 is expected to end when all Dy ions are replaced by Nd ions in column i, enabling Nd enrichment in column i. It is noteworthy that Dy ions are collected in column ii. In step B, Nd extracted so far in column i will be desorbed by either low pH or mild chelating agent to recover high purity Nd (99.9%) and regenerate column i for subsequent adsorption. Meanwhile, columns ii and iii are connected in series to repeat Nd enrichment in column ii and Dy enrichment in column iii until column ii is completely loaded with Nd. In step C, the same desorption and Nd / Dy enrichment process is repeated as described in step B by rotating the order of the columns. In step D, Dy and Nd are expected to be recovered, respectively. Then step A is expected to be repeated to start a new separation cycle. Of note, this process may be fully continuous if column iv is added, in which case the Dy-loaded column may be replaced with column iv to perform Dy desorption independently, which may be performed less frequently depending on the Nd / Dy ratio.

[0156] From the foregoing, it will be appreciated that specific embodiments of the invention have been described herein for purposes of illustration, but that various modifications may be made without deviating from the scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

[0157] References TIFF2024520862000017.tif129154 TIFF2024520862000018.tif215151 TIFF2024520862000019.tif228153 TIFF2024520862000020.tif189159

Claims

1. A biosorption system comprising the following: (A) A protein comprising the following: (1) A Lanmodulin (LanM) portion having at least two EF-hand motifs, wherein said at least two EF-hand motifs each have at least three carboxylic acid residues, and each of said at least two EF-hand motifs has the form (D / N)-X1-(D / N)-X2-(D / N)-X3-X4-X5-X6-X7-X8-(E / D), where each numbered X is independently any residue, provided that X6 and / or X8 of each of said at least two EF-hand motifs is D or E, said LanM portion; and, (2) A linker portion; and, (B) A solid support to which said protein is operably bound.

2. The biosorption system according to Claim 1, wherein at least one EF-hand motif comprises the amino acid sequence DPDKDGTLDAKE.

3. The biosorption system according to Claim 1, wherein at least one EF-hand motif comprises the amino acid sequence DPDNDGTLDKKE.

4. The biosorption system according to Claim 1, wherein the LanM portion comprises the amino acid sequence PTTTTKVDIAAFDPDKDGTIDLKEALAAGSAAFDKLDPDKDGTLDAKEKGRSSEADLKKLDPDNDGTLDKKEYLAAVEAQFKAAANPDNDGTIDARELASPAGSLVNLI.

5. The biosorption system according to any one of Claims 1 to 4, wherein the linker portion comprises a glycine-serine amino acid chain having 1 to 10 repeats of GlyxSery (where x and y are each independently an integer from 0 to 10, provided that x and y are not both 0).

6. The biosorption system according to any one of Claims 1 to 4, wherein the linker comprises GlySerGly, (Gly4Ser)2, (Gly3Ser)2, Gly2Ser, or a combination thereof. **Claim 7**: The biosorption system according to claim 1, wherein the solid support comprises a porous support. **Claim 8**: The biosorption system according to claim 1, wherein the solid support comprises a polymer, agarose, alginate, acrylamide, regenerated cellulose, cellulose ester, plastic, or glass. **Claim 9**: The biosorption system according to claim 1, wherein the solid support comprises beads. **Claim 10**: The biosorption system according to any one of claims 7 to 9, wherein the solid support is functionalized. **Claim 11**: The biosorption system according to claim 10, wherein the solid support is functionalized with maleimide. **Claim 12**: The biosorption system according to claim 10, wherein the solid support comprises maleimide-functionalized agarose beads. **Claim 13**: The biosorption system according to claim 11, wherein the maleimide is succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC). **Claim 14**: The biosorption system according to claim 1, wherein the linker moiety comprises a C-terminal cysteine residue, an N-terminal cysteine residue, or an internal cysteine residue. **Claim 15**: The protein has the amino acid sequence PTTTTKVVDIAAFDPDKDGTIDLKEAALAAGSAAFDKLDPDKDGTLDAKELKGRVSEADLK KLDPDNDGTLDKKEYLAAVEAQFKAA NPDNDGTIDARELASPAGSALVN LIRGSGC and the biosorption system according to claim 14 comprises the same. **Claim 16**: The biosorption system according to claim 14 or 15, wherein the solid support is functionalized with maleimide, and the solid support is bound to the protein via the maleimide of the solid support and a cysteine residue of the protein. **Claim 17**: The biosorption system according to claim 1, further comprising a column for introducing the protein. **Claim 18**: (A) A protein comprising the amino acid sequence PTTTTKVDIAAFDPDKDGTIDLKELAAGSAAFDKLDPDKDGTLDAKELLGRVSEADLKKLDPDNDGTLDKKELYAAVEAQFKAA NPDNDGTIDARELASPAGSALVNLRGSGC, and (B) A solid support comprising succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate functionalized agarose beads, A biosorption system comprising the same, wherein the solid support is bound to the protein via the maleimide of the solid support and the C-terminal cysteine residue of the protein. The biosorption system. **Claim 19**: The biosorption system according to claim 18, further comprising a column for introducing the protein. **Claim 20** A kit comprising: (A) A protein comprising: (1) A Lanmodulin (LanM) moiety having at least two EF-hand motifs, wherein the at least two EF-hand motifs have at least three carboxylic acid residues, and each of the at least two EF-hand motifs has the form of (D / N)-X1-(D / N)-X2-(D / N)-X3-X4-X5-X6-X7-X8-(E / D), where each numbered X is independently any residue, provided that X6 and / or X8 of each of the at least two EF-hand motifs is D or E; the LanM moiety; and (2) A linker moiety; and (B) A solid support to which the protein is operably bound; (C) A column for introducing the protein and the solid support; (D) Instructions for differentially separating rare earth elements from a sample.

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