Method for separating and purifying AMP, ADP and ATP

Abstract

The invention discloses a method for separating and purifying AMP, ADP and ATP, and belongs to the technical field of biochemical separation and purification. The method comprises the following steps: firstly, adjusting the pH value of a mixed solution containing AMP, ADP and ATP to 2.0-4.0, then adsorbing and separating out electrically neutral or weakly negatively charged AMP by using cation exchange resin, and enabling ADP and ATP with strong negative charges to penetrate and flow out; then, the pH value of the effluent containing ADP and ATP is adjusted to 2.0-4.0, anion exchange resin is used for adsorption, and finally ADP and ATP are collected through isocratic elution. The method is simple in operation step, short in production period, free of toxic and harmful reagents, low in requirement for production equipment, capable of efficiently separating the high-purity AMP, ADP and ATP from complex mixtures such as synthetic biological reaction liquid at a time, good in economical efficiency and amplification performance and suitable for industrial large-scale production.

Description

Technical Field

[0001] This invention belongs to the field of biochemical separation and purification technology, specifically relating to a method for separating and purifying AMP, ADP and ATP. Background Technology

[0002] Adenosine monophosphate (AMP, ADP, and ATP) is a crucial energy molecule and metabolic intermediate in living organisms, with wide applications in pharmaceuticals, health products, cell therapy, and biological reagents. With the rapid development of synthetic biology techniques, it has become possible to produce a mixture of AMP, ADP, and ATP in a one-step process using engineered strains or enzyme catalytic systems. This method has the potential to be highly efficient and cost-effective.

[0003] However, because AMP, ADP, and ATP differ by only one or two phosphate groups in their molecular structure, and their molecular weights and charges are very similar, efficiently separating and obtaining high-purity single components from complex reaction solutions is extremely challenging. Existing separation methods mostly focus on the purification of single components (such as ATP), or employ techniques such as preparative high-performance liquid chromatography (HPLC). While the latter offers high resolution, it suffers from expensive equipment, limited throughput, and high operating costs, making it difficult to meet the needs of large-scale industrial production. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a method for separating and purifying AMP, ADP and ATP, so as to overcome the shortcomings of the existing technology in which the separation process of AMP, ADP and ATP is complicated, inefficient and costly.

[0005] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0006] A method for separating and purifying AMP, ADP, and ATP includes the following steps: (1) Pretreatment of reaction solution: The synthetic biology reaction solution containing AMP, ADP and ATP is centrifuged or filtered to remove bacterial cells or solid impurities and obtain a clear reaction solution; (2) Adjust the pH of the reaction solution containing AMP, ADP and ATP to acidity to obtain the first solution; (3) Load the first sample solution onto a cation exchange resin column for the first elution, collect the first eluent, and use alkaline eluent for the second elution to obtain the AMP enriched sample solution; (4) Adjust the pH of the first eluent to acidity to obtain the second feed solution; (5) Load the second sample solution onto an anion exchange resin column, first elute with a low concentration salt solution to obtain an ADP enriched sample solution, and then elute with a high concentration salt solution to obtain an ATP enriched sample solution. (6) The obtained AMP, ADP and ATP enriched solutions were desalted, alcoholized, separated into solid and liquid and dried to obtain pure AMP, ADP and ATP products respectively.

[0007] In some embodiments, in step (1), the reaction solution includes the following main components: AMP concentration of 5-6 mM, ADP concentration of 8-10 mM, and ATP concentration of 10-12 mM.

[0008] In some embodiments, in step (2), the acidity is achieved by adjusting the pH of the reaction solution to 2-4, preferably 2.5, using an aqueous hydrochloric acid solution; the concentration of the aqueous hydrochloric acid solution is 1-2 M.

[0009] In some embodiments, in step (3), the flow rate of the sample loading is 0.1~0.2 BV / h (preferably 0.1 BV / h); the cation exchange resin column is a strong acid type cation exchange resin with polystyrene as the backbone and sulfonic acid groups as functional groups. Preferably, the cation exchange resin is Dowex 50WX8, H + type.

[0010] In some embodiments, in step (3), the first elution uses an aqueous solution with a pH of 2.0 to 4.0 as the eluent, and the elution flow rate is 0.1 to 0.2 BV / h (preferably 0.1 BV / h). Elution is stopped when the OD260 signal drops back to the baseline and remains stable. The first eluent is a mixed solution of ADP and ATP. The alkaline eluent is an aqueous solution of NaOH or NH4OH with a pH of 7.0 to 9.0 (preferably 8.0). The second elution uses a flow rate of 0.1 to 0.2 BV / h (preferably 0.2 BV / h). Elution begins when the OD260 signal starts to rise significantly and is stopped when the signal drops back to the baseline and remains stable.

[0011] In some embodiments, in step (4), the acidity is achieved by adjusting the pH of the first elution solution to 2.0-4.0 (preferably 4.0) using an acidic buffer solution; the acidic buffer solution is hydrochloric acid, phosphate, or citrate buffer solution.

[0012] In some embodiments, in step (5), the flow rate of the sample loading is 0.1~0.2 BV / h (preferably 0.1 BV / h); the anion exchange resin is a strong base anion exchange resin with polystyrene as the backbone and quaternary ammonium groups as functional groups. Preferably, the anion exchange resin is Dowex 1X8, Cl - type.

[0013] In some embodiments, in step (5), the elution is isocratic elution with a flow rate of 0.05~0.06 BV / h (preferably 0.05 BV / h); the low-concentration salt solution is a NaCl solution with a concentration of 0.05-0.2 M (preferably 0.20 M) and a pH value of 2.0-4.0 (preferably 4.0); the high-concentration salt solution is a NaCl solution with a concentration of 2-4 M (preferably 2 M) and a pH value of 2.0-4.0 (preferably 4.0).

[0014] In some embodiments, in step (6), the desalination is carried out by desalination and concentration using a nanofiltration membrane at an operating pressure of 2.0-3.0 MPa, and the molecular weight cutoff of the nanofiltration membrane is 150-200 Da; the alcohol precipitation crystallization is carried out by crystallization using ethanol at a temperature of 5-7°C for 12-24 hours; the solid-liquid separation is carried out by centrifugation; and the drying is carried out at a temperature of 40-50°C (preferably 40°C) until constant weight is achieved.

[0015] In some embodiments, in step (6), the purity of the AMP, ADP and ATP products is all above 98 wt%.

[0016] Beneficial effects:

[0017] (1) Separation of three products in one step: This invention innovatively utilizes the charge difference of AMP, ADP and ATP under different pH conditions, and achieves the separation of three high-purity products from the mixture in one step through the strategy of "removing AMP with cation resin + separating ADP / ATP with anion resin". The process is simple and efficient.

[0018] (2) High purity: By precisely controlling the sample loading pH and elution conditions, the complete separation of AMP and ADP / ATP, as well as the effective separation of ADP and ATP, are achieved. The purity of the final products AMP, ADP and ATP can all reach more than 98%.

[0019] (3) Low cost and easy to scale up: This invention uses conventional ion exchange resin chromatography technology, avoiding the use of expensive preparative HPLC equipment. The resin can be reused and the reagent cost is low, making it very suitable for large-scale industrial production.

[0020] (4) High recovery rate: The separation process has fewer steps, less loss of target product, and a high overall recovery rate (over 90%), resulting in significant economic benefits. Attached Figure Description

[0021] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0022] Figure 1 This is a flow chart of the separation and purification process of the present invention. Detailed Implementation

[0023] The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that the descriptions in the embodiments are for illustrative purposes only and should not, and will not, limit the invention as detailed in the claims.

[0024] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the reagents, methods and equipment used are conventional reagents, methods and equipment in this technical field.

[0025] The adenosine kinase used in this invention was purchased from Merck, catalog number M3003-1KU; the acetate kinase was purchased from Merck, catalog number A7437-1KU.

[0026] Example 1

[0027] (1) Prepare a reaction system with a total volume of 1.1 L: Adenosine (25 mM) was dissolved in 50 mM Tris-HCl buffer (pH 7.5) containing 2% (w / v) MgCl2 as substrate, and 10 mM ATP was added as the initial phosphate donor. Adenosine kinase was added to the system to a final concentration of 100 U / mL, and acetate kinase was added to a final concentration of 20 U / mL. After reacting at 36 °C for 2 h, a mixed reaction solution containing AMP, ADP, and ATP was obtained (the concentrations of each component were determined to be: AMP 5 mM, ADP 8 mM, and ATP 10 mM). The reaction was terminated by adjusting the pH to 2.2-2.5 with hydrochloric acid, and centrifuged at 4 °C and 10000 rpm for 10 minutes. 1 L of supernatant was collected for subsequent purification.

[0028] (2) The pH of the reaction supernatant was precisely adjusted to 2.5 with 1 M HCl, and the solution was loaded at a flow rate of 0.1 BV / h onto a strong acid cation exchange resin column (Dowex 50WX8, H) that had been equilibrated with an aqueous solution of pH 2.5. + The column has a packing volume of 2000 mL, with an inner diameter of 10 cm, a height of 50 cm, and a bed height of 25 cm. By adjusting the pH of the supernatant to 2.5, at this pH, AMP (pKa: 3.8~6.5) molecules are generally electrically neutral or slightly negatively charged; while ADP / ATP, carrying a larger negative charge, can penetrate.

[0029] (3) After the sample loading is completed, the first elution is performed. The column is washed with deionized water at pH 2.5 at the same flow rate until the baseline of the UV detector (260 nm) is stable. All the effluent (breakthrough and washing solution) in this stage is collected to obtain the ADP and ATP mixture. Then, the pH is adjusted to 8.0 with 1 M NaOH solution for the second elution. The cation column is eluted at a flow rate of 0.2 BV / h. The absorption peak of the UV detector (260 nm) (i.e. AMP) is collected. The purity of the AMP enrichment solution is 99.2% according to HPLC.

[0030] (4) Adjust the pH of the collected ADP and ATP mixture to 4.0 with 1 M Tris-HCl, and then load this solution at a flow rate of 0.1 BV / h onto a strong base anion exchange resin column (Dowex 1X8, Cl) that has been equilibrated with 50 mM Tris-HCl buffer (pH 4.0). - The column has a packing volume of 200 mL, with column specifications of 4 cm inner diameter, 30 cm height, and a packing bed height of 16 cm.

[0031] (5) After the sample loading is completed, elute isocratically with 0.20 M NaCl (in 50 mM Tris-HCl, pH 4.0) at a flow rate of 0.05 BV / h. The first UV (260 nm) absorption peak is eluted and collected as ADP enrichment solution (HPLC purity 98.5%). After the first absorption peak (ADP) is completely eluted and the spectrum returns to the baseline, switch the elution buffer to 2.0 M NaCl (in 50 mM Tris-HCl, pH 4.0) and elute the second major UV (260 nm) absorption peak at the same flow rate. The second major UV (260 nm) absorption peak is collected as ATP enrichment solution (HPLC purity 99.0%).

[0032] (6) The obtained AMP, ADP and ATP enriched solutions were desalted, ethanol crystallized, centrifuged and dried respectively. The specific operation was as follows: using a nanofiltration membrane with a molecular weight cutoff of 150 Da, each solution was concentrated to 1 / 10 of its original volume under a pressure of 2.5 MPa, and then 3 times the volume of deionized water was added for washing and filtration; then the desalted concentrate was placed in an ice bath, and 4 times the volume of pre-cooled anhydrous ethanol was slowly added under stirring. The solution was left to stand overnight at 6°C to allow the crystals to precipitate completely. The crystals were then collected by centrifugation at 3000 rpm for 10 min. The crystals were placed in a vacuum drying oven and dried at 40°C to constant weight to obtain a white powdery high-purity product. The total recovery rate of the three products was over 92%.

[0033] Comparative Example 1: Single-column separation using only anion exchange resin

[0034] This comparative example simulates the conventional approach of separation relying solely on anion exchange resins.

[0035] (1) The pH of the reaction supernatant obtained in step (1) of the example was directly adjusted to 4.0 with 1 M Tris-HCl;

[0036] (2) Load the sample solution onto a strong base anion exchange resin column (same as in Example 1). Try isocratic elution using a continuous gradient of NaCl solution (0.10 M → 3.00 M) (in 50 mM Tris-HCl, pH 4.0).

[0037] Results and Discussion: Although all three substances were adsorbed onto the anion exchange column, the decrease in pH at the start of elution reduced the negative charge of AMP, weakening its adsorption force. This resulted in AMP being eluted simultaneously with some ADP, leading to severe overlap of elution peaks. The separation between the AMP and ADP elution peaks was very poor, making it impossible to obtain high-purity single components using conventional gradient elution. The collected AMP product contained >15% ADP impurities, with a purity of less than 85%. This indicates that anion exchange resins and gradient elution alone cannot effectively solve the problem of separating the three components.

[0038] Comparative Example 2: Separation effect of changing the pH value of the cation exchange column (pH 6.0)

[0039] This comparative example aims to examine the importance of strictly controlling the loading pH of the cation exchange column within the acidic range (pH 2.0-4.0) described in this invention during the separation and purification method. The effect on separation selectivity and product purity was compared and analyzed by setting the loading pH to 6.0 (closer to the pKa value of AMP).

[0040] The specific steps are as follows:

[0041] (1) Pretreatment of reaction solution: Take 1 L of enzyme-catalyzed reaction supernatant containing AMP, ADP and ATP, which is exactly the same as step (1) in Example 1;

[0042] (2) Pretreatment and loading of the supernatant: The pH of the supernatant was precisely adjusted to 6.0 using 1 M HCl. Then, this pH 6.0 solution was loaded at a flow rate of 0.1 BV / h onto a strong acid cation exchange resin column (Dowex 50WX8, H) that had been equilibrated with deionized water at pH 6.0. + The type, volume, and specifications of the column are the same as those of the cation column in step (2) of Example 1.

[0043] Subsequent separation steps were performed in accordance with Example 1.

[0044] Results and Discussion: At pH 6.0, AMP molecules are partially dissociated and carry a certain negative charge, which weakens their adsorption capacity on the cation exchange resin. Simultaneously, some ADP molecules may also exhibit weak cation exchange characteristics due to changes in the charge environment. The results showed increased AMP breakthrough loss on the cation exchange column (recovery rate decreased to approximately 75%), and a small amount of AMP was found in the breakthrough solution, while AMP impurities were also detected in the ADP product. This demonstrates that strictly controlling the pH of the cation exchange column loading within the acidic range (2.0-4.0) described in this invention is crucial for achieving a complete "all-or-none" separation of AMP and ADP / ATP.

[0045] Comparative Example 3: Change the elution order of the anion column (elute ATP first, then ADP).

[0046] This comparative example aims to investigate the effect of the elution order in the anion exchange step on the separation effect. Compared with the optimized order of "eluting ADP with low salt first, then eluting ATP with high salt" used in Example 1, it demonstrates that the reverse elution order of "strong first, then weak" will destroy the separation selectivity, thus highlighting the importance of the elution order of the present invention.

[0047] The specific steps are as follows:

[0048] (1) Following steps (1)-(4) in Example 1 of the present invention, an anion exchange resin column adsorbed with ADP and ATP is obtained;

[0049] (2) At a flow rate of 0.05 BV / h, the solution was first eluted isocratically with a high-concentration salt solution of 2.0 M NaCl (in 50 mM Tris-HCl, pH 2.0) to wash away the ATP with the strongest binding first.

[0050] Then, ADP was eluted using a low-concentration salt solution, 0.20 M NaCl (in 50 mM Tris-HCl, pH 2.0), at the same flow rate.

[0051] The subsequent steps are the same as in Example 1.

[0052] Results and Discussion: While high-concentration salt elution successfully washed away ATP, the excessive elution intensity also caused some weaker ADP molecules to be carried away, resulting in a decrease in ATP peak purity (approximately 95%). Subsequently, elution with low-concentration salt resulted in the slow elution of a small amount of ATP remaining on the column during the extended elution process, mixing with the ADP main peak and affecting ADP purity (approximately 96%). This confirms that the "weak-to-strong" gradient elution strategy (eluting ADP first, then ATP) employed in this invention conforms to the thermodynamics of ion exchange, achieving clearer and more thorough separation, and is key to obtaining extremely high purity ADP and ATP.

[0053] This invention provides a method for separating and purifying AMP, ADP, and ATP. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A method for separating and purifying AMP, ADP, and ATP, characterized in that, Includes the following steps: (1) Adjust the pH of the reaction solution containing AMP, ADP and ATP to acidity to obtain the first solution; (2) Load the first sample solution onto a cation exchange resin column for the first elution, collect the first eluent, and use alkaline eluent for the second elution to obtain the AMP enriched sample solution; (3) Adjust the pH of the first eluent to acidity to obtain the second feed solution; (4) Load the second solution onto an anion exchange resin column, first elute with a low concentration salt solution to obtain an ADP enriched solution, and then elute with a high concentration salt solution to obtain an ATP enriched solution. (5) The obtained AMP, ADP and ATP enriched solutions were desalted, alcoholized, separated into solid and liquid and dried to obtain pure AMP, ADP and ATP respectively.

2. The method according to claim 1, characterized in that, In step (1), the reaction solution is centrifuged or filtered beforehand to remove bacterial cells or solid impurities, and a clear reaction solution is obtained, which includes the following main components: AMP concentration of 5-6 mM, ADP concentration of 8-10 mM, and ATP concentration of 10-12 mM.

3. The method according to claim 1, characterized in that, In step (1), the acidity is achieved by adjusting the pH of the reaction solution to 2-4 using hydrochloric acid aqueous solution; the concentration of the hydrochloric acid aqueous solution is 1-2 M.

4. The method according to claim 1, characterized in that, In step (2), the sample loading rate is 0.1~0.2 BV / h; the cation exchange resin column is a strong acid type cation exchange resin with polystyrene as the backbone and sulfonic acid groups as functional groups.

5. The method according to claim 1, characterized in that, In step (2), the first elution uses an aqueous solution with a pH of 2.0 to 4.0 as the eluent, and the elution flow rate is 0.1 to 0.2 BV / h. Elution is stopped when the OD260 signal drops back to the baseline and remains stable. The first eluent is a mixed solution of ADP and ATP. The alkaline eluent is an aqueous solution of NaOH or NH4OH with a pH of 7.0 to 9.

0. The second elution uses a flow rate of 0.1 to 0.2 BV / h. Elution is started when the OD260 signal begins to rise significantly, and collection is stopped when the signal drops back to the baseline and remains stable.

6. The method according to claim 1, characterized in that, In step (3), the acidity is achieved by adjusting the pH of the first elution solution to 2.0-4.0 using an acidic buffer solution; the acidic buffer solution is hydrochloric acid, phosphate, or citrate buffer solution.

7. The method according to claim 1, characterized in that, In step (4), the sample loading rate is 0.1~0.2 BV / h; the anion exchange resin is a strong base anion exchange resin with polystyrene as the backbone and quaternary ammonium groups as functional groups.

8. The method according to claim 1, characterized in that, In step (4), the elution is isocratic elution with a flow rate of 0.05~0.06 BV / h; the low-concentration salt solution is a NaCl solution with a concentration of 0.05-0.2 M and a pH value of 2.0-4.0; the high-concentration salt solution is a NaCl solution with a concentration of 2-4 M and a pH value of 2.0-4.

0.

9. The method according to claim 1, characterized in that, In step (5), the desalination is carried out by desalination and concentration using a nanofiltration membrane at an operating pressure of 2.0-3.0 MPa, and the molecular weight cutoff of the nanofiltration membrane is 150-200 Da; the alcohol precipitation crystallization is carried out by crystallization using ethanol at a temperature of 5-7℃ for 12-24 hours; the solid-liquid separation is carried out by centrifugation; and the drying is carried out at a temperature of 40-50℃ until constant weight is achieved.

10. The method according to claim 1, characterized in that, In step (5), the purity of the AMP, ADP and ATP products is all above 98wt%.

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