A lanM protein-based rare earth ion solid-phase adsorption system and construction and application thereof

Abstract

The application relates to the technical field of green recovery of rare earth resources, in particular to a rare earth ion solid-phase adsorption system based on LanM protein and construction and application thereof, which comprises Ni-NTA affinity chromatography medium as a solid-phase carrier and LanM protein fixed on the surface of the carrier through coordination of nickel ions with a histidine tag. The construction steps include carrier balancing, protein directional fixing and impurity removal; when applied, the EF-hand domain of the LanM protein is used to specifically capture rare earth ions under the condition of pH 4.0-6.0, and the proton desorption is realized through a dilute acid with pH less than 2.5. The application can significantly improve the selectivity to low-concentration rare earth, the rare earth combination rate is more than 92% under a complex high-salt environment, and no organic chelating agent is needed, so that green enrichment of the rare earth ions and recycling of the adsorption system are realized.

Description

Technical Field

[0001] This invention belongs to the field of green recycling technology of rare earth resources, specifically involving a highly selective rare earth ion solid-phase adsorption system based on Lanmodulin (LanM) protein, the preparation method of the system, and its application in the separation and recovery of rare earth ions in low-concentration, high-salt complex environments. Background Technology

[0002] Rare earth elements, as key strategic resources supporting modern high-tech industries and national defense, have irreplaceable application value in fields such as new energy materials, electronic information, and aerospace. Due to the lanthanide contraction effect, rare earth elements have highly similar chemical properties, making the separation and purification of single rare earth elements extremely challenging. In actual rare earth ore leaching solutions, industrial wastewater, and tailings solutions, rare earth ions usually exist in a low-concentration state with many impurities, often accompanied by high concentrations of alkaline earth metal ions such as calcium and magnesium ions. This places extremely high demands on the selectivity and anti-interference capabilities of separation materials.

[0003] Traditional rare earth separation and recovery technologies mainly rely on solvent extraction, chemical precipitation, or ion exchange. However, these technologies generally suffer from poor selectivity, high consumption of chemical reagents, serious organic solvent pollution, complex processes, and high energy consumption in practical applications, making it difficult to achieve efficient and green enrichment of rare earth ions in low-concentration complex matrices.

[0004] In recent years, biosorption technology has become a research hotspot due to its advantages such as mild operating conditions, environmental friendliness, and high selectivity. Among them, Lanmodulin protein, as a specific rare earth binding protein, can achieve high affinity and high selectivity for rare earth ions through its unique structural domains. Moreover, this binding behavior has significant acid-base reversibility, making it an ideal biomaterial for achieving green separation of rare earths.

[0005] However, directly applying such proteins to solution systems presents technical bottlenecks, including difficulty in recovery, inability to be reused, poor stability, and challenges in continuous industrial operation. Therefore, solid-phase immobilization of proteins and the construction of stable solid-phase adsorption systems are essential prerequisites for their industrial application. Although some research has been conducted on protein immobilization and adsorption systems, practical applications still face problems such as unstable protein immobilization, poor recyclability, and weak resistance to environmental interference, failing to meet the processing needs of complex industrial matrices.

[0006] Therefore, developing a protein solid-phase adsorption system with stable immobilization effect, high selectivity, strong resistance to salt interference, and the ability to achieve green desorption and regeneration has become an urgent need for the green, efficient, and highly selective separation and recovery of rare earth ions. This has important social and economic significance for promoting the industrial application of rare earth bioseparation technology. Summary of the Invention

[0007] This invention addresses the shortcomings of existing rare earth recycling technologies, such as low selectivity, high consumption of chemical reagents, serious environmental pollution, and insufficient stability and difficulty in recycling of bioadsorbents. It provides a rare earth ion solid-phase adsorption system based on LanM protein, its construction method, and its application.

[0008] This invention provides a rare earth ion solid-phase adsorption system based on LanM protein, comprising a solid support and LanM protein immobilized on the solid support; the solid support is an affinity chromatography medium with nickel-nitrotriacetic acid (Ni-NTA) functional groups modified on its surface; the LanM protein has a histidine tag fused to its N-terminus or C-terminus; the LanM protein forms a coordination bond with nickel ions on the surface of the solid support through the histidine tag, thereby achieving directional immobilization of the LanM protein on the solid support.

[0009] Furthermore, the LanM protein is Lanmodulin protein or its homologous protein derived from Hansschlegelia quercus, and its molecular structure contains four EF-hand domains that specifically bind rare earth ions; the EF-hand domains form coordination chelate centers with rare earth ions through carboxyl oxygen atoms in an environment of pH 4.0 to 8.0.

[0010] Furthermore, the matrix of the solid support is a highly cross-linked agarose, polymethacrylate, or silica gel, with a particle size ranging from 45 μm to 165 μm; the grafting density of the Ni-NTA functional groups on the solid support meets the requirement of binding 5 mg to 40 mg of protein per milliliter of medium.

[0011] This invention also provides a method for constructing the above-mentioned rare earth ion solid-phase adsorption system based on LanM protein, specifically including the following steps: S1. Carrier equilibration treatment: Ni-NTA affinity chromatography medium is packed into the chromatography column and washed with equilibration buffer at a flow rate of 0.5 mL / min to 1.0 mL / min until the pH and conductivity of the effluent are consistent with those of the equilibration buffer. The equilibration buffer consists of a 20 mM to 100 mM phosphate buffer system containing 0.1 M to 0.5 M sodium chloride, and the pH of the equilibration buffer is adjusted to 7.4 to 8.5.

[0012] S2. Protein Immobilization: The crude extract containing LanM protein with histidine tag is passed into an equilibrated chromatography column, and the loading flow rate is controlled at 0.2 mL / min to 1.0 mL / min. During this process, the imidazole ring on the histidine residue undergoes affinity coordination with Ni²⁺ on the surface of the medium, achieving high-density loading of LanM protein on the solid phase surface.

[0013] S3. Impurity Removal and System Stabilization: First, rinse with a equilibration buffer containing 10 mM to 50 mM imidazole to remove physically adsorbed proteins; then rinse with a stabilization buffer to switch the system environment to the acid-tolerant range; the stabilization buffer is a 10 mM to 50 mM sodium acetate buffer with a pH of 4.5 to 5.5 and containing 50 mM to 200 mM potassium chloride.

[0014] This invention also provides a method for applying the above-mentioned rare earth ion solid-phase adsorption system based on LanM protein in rare earth ion recovery, specifically including the following steps: Adsorption stage: The water sample containing rare earth ions is passed through the solid-phase adsorption system. The flow rate of the empty tower or the linear flow rate is controlled to ensure that the rare earth ions are in full contact with the EF-hand domain of the LanM protein. Under the conditions of pH 4.0 to 6.0, the LanM protein undergoes a conformational change and encapsulates the rare earth ions, realizing the transfer of rare earth ions from the liquid phase to the solid phase.

[0015] Selective washing: The washing liquid is flowed through the adsorbed solid phase system, and the pH value of the washing liquid is maintained at 4.5 to 5.5. Under this pH condition, the affinity of LanM protein for calcium and magnesium ions is 3 to 6 orders of magnitude lower than that for rare earth ions, thereby promoting the non-specific binding of alkaline earth metal ions to be discharged with the washing liquid, while rare earth ions are retained on the solid phase support.

[0016] Desorption and enrichment: A desorption solution is introduced into the solid-phase system. The desorption solution is a dilute hydrochloric acid with a concentration of 10 mM to 100 mM and a pH value controlled below 2.5. Under this acidic condition, the carboxyl groups on the LanM protein binding sites are protonated, causing the protein conformation to change from the bound state to the extended state, releasing the bound rare earth ions, thereby obtaining a high-purity rare earth ion enrichment solution in the effluent.

[0017] System regeneration: The solid phase system is reequilibrated using the stabilizing buffer from step S3 to restore the LanM protein to its binding conformation, completing one adsorption-desorption cycle.

[0018] Furthermore, during the adsorption stage, the concentration of rare earth ions in the water sample to be treated ranges from 0.2 to 2.0 g / L; the rare earth ions include any one or more of lanthanum (La), cerium (Ce), praseodymium (Pr), neodymium (Nd), samarium (Sm), europium (Eu), gadolinium (Gd), terbium (Tb), dysprosium (Dy), holmium (Ho), erbium (Er), thulium (Tm), ytterbium (Yb), lutetium (Lu), and yttrium (Y).

[0019] Furthermore, in step S4, when the water sample to be treated contains calcium or magnesium ions at a concentration 10 times higher than that of rare earth ions, the binding rate of the solid-phase adsorption system to rare earth ions remains above 70%.

[0020] Furthermore, in step S6, by controlling the volume of the desorption liquid to be 1 / 10 to 1 / 5 of the volume of the water sample to be treated, the rare earth ions are concentrated by 5 to 10 times.

[0021] Advantages and beneficial effects of the present invention: First, this invention achieves directional and stable immobilization of LanM protein through the specific chelation of Ni-NTA with histidine. Compared to traditional covalent bonding methods, this immobilization method protects the core active site of the LanM protein, the EF-hand domain, from being obscured, maintaining the protein's high affinity for rare earth ions. Simultaneously, this binding method exhibits extremely high mechanical strength, with a very low protein detachment rate during multiple washing cycles, solving the technical problems of easy loss and short lifespan of bioadsorbents during use.

[0022] Secondly, this invention utilizes the significant difference in affinity between LanM protein and rare earth ions and alkaline earth metal ions, exhibiting extremely high selectivity. In complex matrices containing high concentrations of calcium and magnesium ions (such as mine leachates), the solid-phase adsorption system can accurately identify and capture rare earth ions. Experimental data show that even in an environment with a rare earth concentration of only 0.2 g / L and the presence of numerous interfering ions, the binding rate of rare earth ions reaches over 92%, achieving highly efficient enrichment of low-concentration rare earth resources.

[0023] Third, this invention constructs a pH-responsive green desorption mechanism. Utilizing the reversible conformational change of the LanM protein under low pH conditions, complete desorption of rare earth ions can be achieved using only a low concentration of dilute hydrochloric acid (pH < 2.5). This process eliminates the need for difficult-to-treat organic chelating agents such as EDTA and DTPA, reducing reagent costs and avoiding secondary pollution, thus aligning with the requirements of green chemistry development.

[0024] Fourth, the solid-phase adsorption system described in this invention exhibits excellent regeneration performance. In more than 10 consecutive adsorption-desorption cycle experiments, the system retains over 95% of its rare earth ion adsorption capacity. This demonstrates that the binding between the Ni-NTA support and the LanM protein is very stable, and the protein can rapidly recover its biological activity after strong acid elution by pH adjustment, possessing the potential for continuous industrial production.

[0025] Fifth, the construction method described in this invention is simple and operates under mild conditions. The entire immobilization and adsorption process is carried out at room temperature and pressure, without the need for complex chemical pretreatment equipment. By adjusting the flow rate, injection volume, and desorption solution ratio, the treatment and enrichment of rare earth wastewater of different concentration levels can be flexibly achieved, demonstrating strong process adaptability.

[0026] Specifically, the present invention achieves the above-mentioned effects through the following technical solutions: Regarding the microscopic mechanism of the solid-phase adsorption system, the nickel ions on the surface of the Ni-NTA medium have six coordination sites, four of which bind to hypozinotriacetic acid, while the remaining two active sites form a stable coordination structure with the imidazole ring in the six consecutive histidine residues at the N-terminus of the LanM protein. This multi-site binding mode ensures the ordered spatial arrangement of the protein on the solid-phase surface, allowing each LanM molecule to fully expose its four rare-earth binding sites.

[0027] In terms of the technical details of the adsorption process, when a solution containing rare earth ions flows through a solid-phase chromatography column, the carboxyl groups of the aspartic acid and glutamic acid side chains in the EF-hand domain of the LanM protein coordinate with the rare earth ions. Because the ionic radius and coordination requirements of the rare earth ions are highly compatible with this pocket, a strong electrostatic attraction and coordination bond are formed. Calcium ions, due to their lower charge density and ionic radius difference, cannot induce the protein to form a tight conformational package and are therefore easily removed in the subsequent selective washing step.

[0028] In terms of the technical details of the desorption process, a high concentration of protons (H⁺) is introduced to protonate the carboxyl residues involved in coordination within the EF-hand domain. This protonation disrupts the ionic bonds and coordination interactions between the carboxyl groups and rare earth ions, while the resulting charge repulsion forces cause the protein structure to transition from a bound state to a loose state. At this point, the rare earth ions are released into the liquid phase. Because the volume of the desorption solution is much smaller than that of the initial sample, the rare earth ions are significantly physically concentrated in the desorption solution.

[0029] In particular, the system described in this invention exhibits extremely strong environmental tolerance when treating acidic mine wastewater with a pH value of 4.0 to 5.0. Within this pH range, most metal oxides or hydroxides do not precipitate, while the LanM protein remains in its active conformation. This allows the present invention to directly treat acidic wastewater that has not undergone neutralization pretreatment, greatly simplifying the industrial treatment process and reducing sludge production.

[0030] In summary, this invention combines the highly efficient biorecognition molecule LanM protein with mature affinity chromatography technology to construct a highly stable, highly selective, and easily regenerable rare earth ion solid-phase adsorption system. This system not only solves the problem of difficult biosorbent recovery but also achieves green enrichment of rare earth ions through precise pH control, providing a novel and industrially viable technical solution for the recycling of low-concentration rare earth resources.

[0031] Furthermore, to verify the reliability of the system described in this invention, adsorption tests were conducted on different types of rare earth ions. The results showed that the system has extremely high capture efficiency for light rare earths (such as La and Ce), medium rare earths (such as Gd and Eu), and heavy rare earths (such as Yb and Lu).

[0032] Meanwhile, the system described in this invention exhibits excellent salt tolerance to monovalent cations such as sodium, potassium, and ammonium commonly found in industrial wastewater. Even with a background electrolyte concentration of 0.5 M, the binding capacity of the LanM protein to rare earth ions remains almost unaffected. This is because the binding of histidine to Ni²⁺ and the binding of LanM to rare earth ions are both strong coordination interactions, rather than simple electrostatic adsorption, thus maintaining stable performance under high ionic strength environments.

[0033] In practice, the above steps can be achieved using automated liquid chromatography systems or industrial-scale fixed-bed adsorption towers. By using online pH and conductivity monitors, the switching points between the adsorption, washing, and desorption zones can be precisely controlled, thus enabling continuous and automated recovery of rare earth ions. This operating mode significantly reduces the cost of manual intervention and improves production efficiency.

[0034] Ultimately, the technical solution described in this invention achieves the transformation of rare earth ions from a complex, sparse matrix into a high-concentration, high-purity recovery solution, opening up a new biotechnological pathway for the recycling of rare earth resources. The solid-phase adsorption system provided by this invention meets all the technical requirements for industrial applications, demonstrating significant economic benefits and environmental significance. Attached Figure Description

[0035] Figure 1This is a comparison curve of the binding rate of La³⁺ in the Ni-NTA-LanM system and the blank support system as a function of concentration in the embodiments of the present invention; Figure 2 This is a graph showing the effect of pH gradient on the binding performance of rare earth ions and the response curve in an embodiment of the present invention. Detailed Implementation

[0036] To make the technical solution and advantages of this invention patent clearer, the following description is in conjunction with the appendix. Figure 1 Appendix Figure 2 The present invention provides a detailed logical description and technical supplement to a rare earth ion solid-phase adsorption system based on LanM protein, its construction and application, along with specific embodiments.

[0037] The core principle of the solid-phase adsorption system constructed in this invention lies in utilizing the specific affinity between biomolecules and inorganic materials to achieve functionalization. This system mainly consists of a solid support, nickel ions loaded on the surface of the solid support, a LanM protein with specific recognition function, and a histidine tag fused to the protein's terminal. During construction, the solid support is a Ni-NTA affinity chromatography medium with high porosity and chemical stability, and its surface is covalently chelated with nickel ions. The LanM protein serves as the core recognition element, with a histidine tag fused to its N-terminus or C-terminus using genetic engineering technology. When a solution containing the LanM protein flows through the solid support, the imidazole ring in the histidine tag forms a stable coordination bond with the nickel ions, thereby directionally immobilizing the LanM protein on the surface of the solid support, forming a stable solid-phase adsorption layer.

[0038] During the adsorption phase, when the solution containing rare earth ions comes into contact with the solid-phase system, the four EF-hand domains in the LanM protein molecule play a crucial role. These EF-hand domains coordinate with rare earth ions at multiple points via the carboxyl groups on their unique amino acid side chains, inducing a conformational shift from a loose to a tightly packed state, thus achieving highly selective capture of rare earth ions. In the desorption phase, the introduction of an acidic eluent containing protons causes protonation of the coordinating residues in the EF-hand domains. This protonation disrupts the electrostatic and coordination balance between the protein and the rare earth ions, prompting the LanM protein to release the rare earth ions and return to its initial conformation, thus completing the recovery of rare earth ions and the regeneration of the system.

[0039] The specific construction steps of the solid-phase adsorption system of this invention are as follows: S1. Support Equilibration: First, prepare a chromatography column with an inner diameter of 1 cm and pack it with 1 mL of Ni-NTA affinity chromatography medium as the solid-phase support. To remove the protective solution stored in the medium and activate the activity of nickel ions, pretreatment is performed using an equilibration buffer. The specific components of the equilibration buffer are 50 mM sodium dihydrogen phosphate and 0.3 M sodium chloride, and the pH is precisely adjusted to 8.0 by adding sodium hydroxide. Turn on the constant flow pump and control the equilibration buffer to wash the solid-phase support at a flow rate of 1 mL / min, with a washing volume of 8 times the column volume, i.e., 8 mL. By monitoring the pH of the effluent, ensure that the internal environment of the solid-phase support reaches chemical equilibrium.

[0040] S2, LanM protein fixation: The LanM protein used in this embodiment is derived from Hansschlegelia quercus. Its encoding gene was cloned into the expression vector after codon optimization and heterologously expressed in the engineered Escherichia coli strain REC2.

[0041] The gene encoding the LanM protein, after codon optimization, has the following sequence: LanM (Sequence ATGGCAAGTGGCGCGGATGCTTTGAAGGCGCTTAACAAAGACAATGACGATTCGCTGGAAATTGCAGAGGTAATCCACGCAGGCGCAACTACGTTCACGGCAATCAACCCGGACGGAGACACAACTTTGGAGAGCGGAGAGACGAAAGGACGCTTGACAGAAAAGGATTGGGCTAGAGCTAATAAAGACGGGGACCAGACGTTGGAAATGGACGAATGGCTGAAGATCCTGCGTACTAGATTTAAAAGAGCCGATGCTAATAAGGATGGCAAATTAACGGCTGCGGAGTTGGATTCCAAAGCGGGGCAAGGGGTATTGGTCATGATCATGAAATGAGCGGCCGCACTCGAGCACCACCACCACCACCACTGA) The fermented cell precipitate was collected and disrupted using an ultrasonic homogenizer under ice bath conditions. The supernatant was then collected by centrifugation at 12,000 rpm for 20 min. This supernatant contained a large amount of LanM protein fused with a histidine tag. The supernatant was pumped into an equilibrated chromatography column at a slow flow rate of 0.5 mL / min. During this process, the histidine tag fully contacts and chelates with nickel ions. By controlling the low flow rate, the residence time of protein molecules on the solid support surface is increased, thereby maximizing the loading of LanM protein; typically, 10 mg to 20 mg of protein can be loaded per milliliter of solid support.

[0042] S3. Impurity Removal and System Stabilization: After protein loading, the system was switched back to equilibration buffer and washed with 8 column volumes at a flow rate of 1 mL / min. The salt ion concentration gradient and flow shear force in the buffer removed impurities and cell debris physically adsorbed on the solid support surface. Subsequently, to adapt the system to a microenvironment suitable for rare earth adsorption, a second equilibration was performed using a stabilization buffer. The stabilization buffer consisted of 20 mM sodium acetate and 100 mM potassium chloride, with the pH adjusted to 5.0. After washing with 8 column volumes, the LanM protein on the solid support surface was in an active state, with the EF-hand domain fully exposed, resulting in a Ni-NTA-LanM solid-phase adsorption system ready for production.

[0043] The application method and performance verification of the solid-phase adsorption system of this invention in rare earth ion recovery are as follows: S4. Rare Earth Ion Adsorption: Prepare a simulated rare earth ore leaching solution containing five rare earth ions: La³⁺, Pr³⁺, Ce³⁺, Gd³⁺, and Y³⁺. The initial total mass concentration is set within the range of 0.2 g / L to 2.0 g / L, and the pH is maintained at 5.0 using sodium acetate buffer. Pass this solution into the prepared solid-phase adsorption system, and control the incubation or flow contact time at room temperature for 10 min. (Refer to...) Figure 1 Taking La³⁺ as an example, under low concentration (0.2 g / L) conditions, the Ni-NTA-LanM system exhibited a high La³⁺ binding rate of up to 92%, demonstrating extremely strong capture ability. With increasing concentration, the binding rate showed a characteristic gradient decrease trend and eventually approached saturation, confirming that the LanM protein and rare earth ions bind via a ligand-receptor mechanism based on specific sites, rather than simple surface physical stacking. In contrast, the unfixed protein-free Ni-NTA carrier showed extremely low La³⁺ binding rates that did not change with concentration, further demonstrating the core recognition role of the LanM protein in the system.

[0044] S5. Selective Washing: After adsorption, the system is washed with a stable buffer solution at pH 5.0. Since the affinity of LanM protein for rare earth ions is much higher than that for alkaline earth metal ions such as calcium and magnesium, this washing step allows impurity ions non-specifically adsorbed in the medium pores or on the protein surface to be removed with the liquid flow, while the rare earth ions remain firmly bound to the EF-hand domain. The binding selectivity of five different rare earth ions was tested. The results showed that the system has extremely high binding activity for light rare earths such as La³⁺, Pr³⁺, and Ce³⁺, and also maintains a significant binding rate for medium and heavy rare earths such as Gd³⁺ and Y³⁺. The blank Ni-NTA column showed only weak non-specific adsorption for all REEs (light-colored column). This broad-spectrum capture characteristic targeting all rare earth components, combined with its high repulsion of non-rare earth ions, achieves high-purity pre-enrichment of the target elements.

[0045] S6. Desorption and Enrichment: 25 mM dilute hydrochloric acid was used as the desorption buffer, with a pH less than 2.5. The solution was pumped into the chromatography column at a flow rate of 1 mL / min. (Refer to...) Figure 2 As the pH decreases, the proton concentration increases and competes with rare earth ions for carboxyl coordination sites on the LanM protein. At pH 2.5, the binding activity of the EF-hand domain is almost completely lost, and rare earth ions are rapidly released into the desorption buffer. By controlling the volume of the desorption buffer to 1 / 10 of the initial loading volume, a 10-fold concentration of rare earth ions in the liquid phase is achieved. This process does not require complex organic chelating agents; efficient desorption is achieved solely through the protonation of a strong inorganic acid.

[0046] S7. System Regeneration: After desorption, the chromatography column is flushed again with a stable buffer at pH 5.0 until the pH of the effluent rises back to 5.0. At this point, the LanM protein regains its active conformation for binding rare earth ions. Experiments show that steps S4 to S7 can be repeated more than 10 times without significant decrease in adsorption capacity, achieving the recycling of the solid-phase adsorption system.

[0047] To address the complex high-salt environments in practical applications, this invention validated its anti-interference performance. Using a 0.5 g / L mixed solution of REEs as the substrate, Mg²⁺ and Ca²⁺ were doped at 10 and 100 times their mass concentrations, respectively. Experimental results showed that even with 10 times the concentration of alkaline earth metal ions, the Ni-NTA-LanM system maintained a rare earth ion binding rate of over 70%, exhibiting excellent selectivity. When the interfering ion concentration reached 100 times, although the binding rate decreased to some extent, it was still significantly superior to traditional chemical ion exchange materials. This anti-interference capability stems from the high degree of matching between the EF-hand structural domain and the radius and charge density of rare earth ions, making it difficult for impurity ions to crowd out rare earth binding sites.

[0048] In stability tests under low pH conditions, the system exhibited excellent adsorption stability in a neutral to slightly acidic environment ranging from pH 4.0 to 5.0. The binding rate of Ni-NTA-LanM to REEs remained at a high level with no significant difference, demonstrating good low pH tolerance. This is significant for treating acidic rare earth ore leaching solutions, meaning that rare earth capture can be carried out directly without large-scale neutralization adjustments. However, when the pH dropped below 3.0, the binding rate to REEs decreased significantly. Under strongly acidic conditions at pH 2.5, the binding rate of Ni-NTA-LanM to REEs was not significantly different from that of the blank Ni-NTA control group, indicating that specific binding was almost completely inhibited at this point, providing precise control logic for subsequent automated recovery processes.

[0049] In practical industrial applications, such as the recovery of rare earth tailings, the concentration of rare earth elements in the wastewater is often extremely low, and it contains a large amount of silt particles. In this case, pre-filtration can be performed using a 0.22μm filter membrane, followed by continuous pumping of the wastewater into a fixed-bed adsorption tower filled with Ni-NTA-LanM media. Through multi-tower series operation, continuous capture of rare earth ions is achieved. When the first tower reaches adsorption saturation, the process switches to desorption, using dilute hydrochloric acid to recover the rare earth elements, while the other towers continue to maintain adsorption. This operating mode utilizes the high affinity of LanM protein to concentrate rare earth elements from extremely dilute solutions onto the solid surface, followed by elution with a small volume of acid, significantly reducing the energy consumption of subsequent evaporation and concentration.

[0050] The specific embodiments described in this invention combine the molecular recognition advantages of biological proteins with the engineering advantages of solid-phase chromatography, providing a novel rare earth recovery solution that is low-energy, highly selective, and environmentally friendly. Compared with traditional solvent extraction methods, this solution completely eliminates the use of kerosene and phosphoric acid-based organic solvents, thus eliminating the risk of organic pollution. Compared with conventional biomass adsorption, the immobilization on the Ni-NTA support solves the problems of difficult protein recovery and easy inactivation.

[0051] Furthermore, the construction process of the system of this invention is highly scalable. By changing the length or position of the histidine tag, or by performing directed evolutionary modification of the LanM protein, its affinity for a specific single rare earth element can be further tuned, thereby achieving the goal of directly separating a high-purity single rare earth element from a mixture of rare earth elements. Regarding the choice of solid-phase support, in addition to agarose matrix, materials such as magnetic microspheres, cellulose membranes, or porous ceramics can also be used to adapt to industrial requirements with different flow rates and pressures.

[0052] In summary, this invention, through precise control of process parameters S1 to S7 and leveraging the specific chemical structure of Ni-NTA-LanM, successfully constructed a green rare earth recovery system capable of handling challenges such as low concentration, high salt content, and complex matrices. Its beneficial effects are not only reflected in the high binding efficiency of up to 92% and the concentration factor of over 10 times, but also in its simple regeneration mechanism based on pH adjustment, providing solid technical support for the industrial-scale bioseparation of rare earth resources. The technical solution described in this embodiment has undergone multiple experimental verifications, demonstrating high repeatability and practical application value.

Claims

1. A rare earth ion solid-phase adsorption system based on LanM protein, characterized in that, The invention includes a solid support and a LanM protein immobilized on the solid support; the solid support is an affinity chromatography medium with a surface modified with nickel-nitrotriacetic acid functional groups; the LanM protein has a histidine tag fused to its N-terminus or C-terminus; the LanM protein forms a coordination bond with nickel ions on the surface of the solid support through the histidine tag.

2. The rare earth ion solid-phase adsorption system based on LanM protein as described in claim 1, characterized in that, The LanM protein is a Lanmodulin protein derived from Hansschlegelia quercus, and its molecular structure contains four EF-hand domains that bind rare earth ions.

3. The rare earth ion solid-phase adsorption system based on LanM protein as described in claim 2, characterized in that, The matrix of the solid support is cross-linked agarose, polymethacrylate, or silica gel, with a particle size ranging from 45 μm to 165 μm; the grafting density of the nickel triacetic acid functional group on the solid support is such that 5 mg to 40 mg of the LanM protein are bound per milliliter of medium.

4. A method for constructing a rare earth ion solid-phase adsorption system based on LanM protein as described in claim 1, characterized in that, Includes the following steps: S1. Carrier equilibration treatment: The solid-phase carrier is packed into the chromatography column and washed with equilibration buffer at a flow rate of 0.5 mL / min to 1.0 mL / min until the pH and conductivity of the effluent are consistent with those of the equilibration buffer. S2, Protein Immobilization: Pass the solution containing the histidine-tagged LanM protein into the equilibrated chromatography column, and control the loading flow rate to be 0.2 mL / min to 1.0 mL / min; S3. Impurity removal and system stabilization: First, rinse with an equilibration buffer containing imidazole, then rinse with a stabilization buffer to change the system environment to pH 4.5 to 5.

5.

5. The construction method as described in claim 4, characterized in that, The equilibration buffer in step S1 consists of a phosphate buffer system of 20 mM to 100 mM and contains 0.1 M to 0.5 M sodium chloride. The pH of the equilibration buffer is 7.4 to 8.

5.

6. The construction method as described in claim 5, characterized in that, The concentration of imidazole used for rinsing in step S3 is 10 mM to 50 mM; the stabilizing buffer is a 10 mM to 50 mM sodium acetate buffer with a pH of 4.5 to 5.5 and containing 50 mM to 200 mM potassium chloride.

7. A method for applying the LanM protein-based rare earth ion solid-phase adsorption system as described in claim 1 in rare earth ion recovery, characterized in that, Includes the following steps: Adsorption stage: The water sample containing rare earth ions is passed through the solid-phase adsorption system and the rare earth ions are captured by the EF hand domain of the LanM protein under the condition of pH 4.0 to 6.

0. Selective washing: The washing liquid is flowed through the adsorbed solid phase system, and the pH value of the washing liquid is maintained between 4.5 and 5.5; Desorption and enrichment: A desorption solution is introduced into the solid-phase system. The desorption solution is a dilute hydrochloric acid with a concentration of 10 mM to 100 mM and its pH value is controlled below 2.5, so that the LanM protein releases the bound rare earth ions. System regeneration: Reequilibrate the solid-phase system using the stable buffer solution described in claim 6.

8. The application method as described in claim 7, characterized in that, During the adsorption stage, the concentration of rare earth ions in the water sample to be treated ranges from 0.2 to 2.0 g / L; the rare earth ions include any one or more of lanthanum, cerium, praseodymium, neodymium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, ytterbium, lutetium, and yttrium.

9. The application method as described in claim 7, characterized in that... Further, during the desorption and enrichment stage, the volume of the desorption liquid is controlled to be 1 / 10 to 1 / 5 of the volume of the water sample to be treated.

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