Method for separating and purifying fructose 1, 6-diphosphate from fructose 1, 6-diphosphate reaction liquid

Abstract

The invention discloses a method for separating and purifying 1, 6-fructose diphosphate from a 1, 6-fructose diphosphate reaction liquid, which comprises the following steps: adjusting the pH value of the 1, 6-fructose diphosphate reaction liquid to be acidic, performing microfiltration, collecting permeate liquid, performing first electrodialysis desalination to obtain a reaction liquid after first desalination, loading the reaction liquid to strongly basic anion exchange resin, and performing adsorption, impurity washing and elution to obtain the 1, 6-fructose diphosphate. The method comprises the following steps: adding a sodium salt solution into an eluent containing 1, 6-fructose diphosphate, collecting the eluent containing 1, 6-fructose diphosphate, carrying out second electrodialysis desalination to obtain a 1, 6-fructose diphosphate solution, carrying out nanofiltration concentration, collecting a concentrated solution, crystallizing, carrying out suction filtration, washing, and drying to obtain the 1, 6-fructose diphosphate existing in a sodium salt form. The electrodialysis technology is high in desalination efficiency and high in target substance yield; the resin in the chromatographic separation section can be recycled after being regenerated, so that the separation cost is low; the purity of the fructose 1, 6-diphosphate existing in the form of sodium salt obtained by the separation and purification method exceeds 99.0%, and the total yield exceeds 95%.

Description

Technical Field

[0001] The present invention relates to the technical field of separation and purification of enzyme-catalyzed products, and specifically relates to a method for separating and purifying fructose 1,6-diphosphate from a fructose 1,6-diphosphate reaction solution. Background Art

[0002] Fructose 1,6-diphosphate is an intermediate product of intracellular energy metabolism and plays a very important role in energy metabolism. It can improve myocardial ischemia and can be used for the adjuvant treatment of coronary heart disease angina, acute myocardial infarction, heart failure, arrhythmia, etc. It can also promote liver metabolism, improve liver function, and can be used in the symptomatic treatment of hepatitis to reduce liver cell damage. In addition, it can regulate blood sugar, promote insulin secretion, and improve the symptoms of diabetes.

[0003] Patent CN 119082230 A discloses a method for synthesizing fructose 1,6-diphosphate by enzymatic method. There are some impurities such as ATP (adenosine triphosphate), ADP (adenosine diphosphate), AMP (adenosine monophosphate) and inorganic salts in the obtained fructose 1,6-diphosphate catalytic reaction solution. At the same time, there are also impurities such as proteins and nucleic acids generated by cell autolysis, and the refining is difficult. Summary of the Invention

[0004] The object of the present invention is to provide a method combining membrane separation technology and chromatographic separation technology to separate and purify high-purity fructose 1,6-diphosphate from a fructose 1,6-diphosphate enzymatic reaction solution in view of the problem that it is difficult to separate and purify high-purity fructose 1,6-diphosphate from a fructose 1,6-diphosphate enzymatic reaction solution in the prior art.

[0005] In order to achieve the above object, the technical solution provided by the present invention is as follows:

[0006] A method for separating and purifying fructose 1,6-diphosphate from a fructose 1,6-diphosphate reaction solution, comprising the following steps:

[0007] (1) Adjust the pH of the fructose 1,6-diphosphate reaction solution to acidic to inactivate the enzymes therein, and perform microfiltration, and collect the permeate;

[0008] (2) Perform first electrodialysis desalination on the permeate obtained in step (1) to obtain a reaction solution after the first desalination;

[0009] (3) Load the reaction solution after the first desalination obtained in step (2) onto a strongly basic anion exchange resin for adsorption, impurity washing and elution, and collect the eluate containing fructose 1,6-diphosphate;

[0010] (4) Perform second electrodialysis desalination on the eluate obtained in step (3) to obtain a fructose 1,6-diphosphate solution;

[0011] (5) The fructose 1,6-diphosphate solution obtained in step (4) is subjected to nanofiltration concentration, the concentrated solution is collected, crystallized, suction filtered, washed, and dried to obtain fructose 1,6-diphosphate in the form of its sodium salt (trisodium fructose 1,6-diphosphate).

[0012] In step (1), the acidity has a pH value of 2 to 3; the membrane pore size of the microfiltration membrane used for microfiltration is 10 to 100 nm; the working pressure during microfiltration is 0.25 to 0.5 Mpa.

[0013] Preferably, the microfiltration is carried out using a ceramic membrane with a membrane pore size of 10 to 100 nm;

[0014] More preferably, the microfiltration is carried out using a ceramic membrane with a membrane pore size of 30 to 70 nm;

[0015] Most preferably, the microfiltration is carried out using a ceramic membrane with a membrane pore size of 50 nm.

[0016] To improve the yield of fructose 1,6-diphosphate, after microfiltration, pure water can be repeatedly used to rinse the residual microfiltration permeate in the microfiltration pipeline, and the rinse liquid is combined with the microfiltration permeate.

[0017] Through step (1), the insoluble bacteria and macromolecular proteins in the fructose 1,6-diphosphate reaction solution can be removed.

[0018] In steps (2) and (4), when performing the first electrodialysis desalting or the second electrodialysis desalting, in the electrodialysis equipment, the permeate or the eluate is added to the desalination cell, water is added to the concentration cell, and the volume ratio of the permeate or the eluate to the water is 1:0.5 to 1, preferably 1:1; the electrodialysis working voltage is 1.0 to 2.0 V / couple of membranes (the working voltage is obtained by multiplying the number of membrane couples), and the circulation flow rate is 2.0 to 4.0 m 3 / h, preferably 3 m 3 / h; when the conductivity of the permeate or the eluate in the desalination cell is reduced to less than 2 mS / cm, desalting is stopped, and the reaction solution after the first desalting or the fructose 1,6-diphosphate solution is collected. To improve the yield of fructose 1,6-diphosphate, the desalination cell pipeline can be repeatedly rinsed with pure water, and the liquid for rinsing the pipeline is combined with the reaction solution after the first desalting or the fructose 1,6-diphosphate solution, and the next purification step is continued.

[0019] In step (3), the strongly basic anion exchange resin is an anion exchange resin with a styrene-divinylbenzene copolymer as the inert skeleton and a quaternary amine group as the functional group; wherein, the particle size of the strongly basic anion exchange resin is 0.15 - 0.38 mm, the average pore diameter is 3 - 5 nm, the wet true density is 1.00 - 1.20 g / mL, the wet apparent density is 0.60 - 0.79 g / mL, the quaternary amine group content > 1.0 mmol / L, preferably 0.9 - 1.1 mmol / L, and the water content is 65% - 75%; before use, the strongly basic anion exchange resin should be first rinsed with a 0.10 - 1.0 mol / L sodium hydroxide aqueous solution and then rinsed with pure water until neutral.

[0020] In step (3), the impurity washing is carried out using a 0.01 - 0.05 mol / L sodium chloride aqueous solution, and the elution is carried out using a 0.1 - 0.3 mol / L sodium chloride aqueous solution.

[0021] In step (3), the flow rates of the adsorption, impurity washing, and elution are all 1 - 2 BV / h.

[0022] In step (3), when loading the sample, stop loading when the concentration of fructose 1,6-diphosphate in the effluent is 0.01 - 0.02 g / L; when carrying out the impurity washing, stop washing when the concentration of fructose 1,6-diphosphate in the effluent is 0.1 - 0.2 g / L.

[0023] In step (5), the nanofiltration concentration is to concentrate the fructose 1,6-diphosphate solution to 350 - 400 g / L using a nanofiltration membrane with a molecular weight cut-off of 100 - 200 Da; the working pressure during the nanofiltration is 1 - 3 Mpa, preferably 2 Mpa.

[0024] In step (5), for the crystallization, under continuous stirring, sodium acetate is added to the concentrated solution, after stirring for 20 - 30 min, absolute ethanol is added, and after stirring for 2 - 3.5 h, crystallization occurs and the stirring is stopped; the stirring is carried out under the condition of 10 - 40 °C, and the stirring speed is 100 - 200 rpm; the washing is carried out using absolute ethanol; the drying temperature is 50 - 55 °C and the time is 8 - 12 h.

[0025] The dosage of sodium acetate is 2% - 5% of the mass of fructose 1,6-diphosphate in the concentrated solution; the dosage of absolute ethanol is 3 - 5 times the volume of the concentrated solution.

[0026] In step (5), sodium acetate is used for crystallization. Sodium acetate, a strong electrolyte, can be completely dissociated into Na + and CH3COO -, which serves as a sodium ion source and provides an alkaline environment for neutralizing acidic groups. The molecular structure of fructose 1,6-diphosphate (FDP) contains two phosphate groups and one hydroxyl group. Under alkaline conditions, the phosphate groups in FDP are gradually deprotonated to form phosphate ions (-PO3 - ), and each deprotonated phosphate group binds to a Na + to form a sodium phosphate salt structure. The hydroxyl group in FDP is deprotonated under alkaline conditions and further binds to Na + to finally form trisodium fructose 1,6-diphosphate. Fructose 1,6-diphosphate in the form of sodium salt has higher chemical stability, solubility, and biocompatibility, facilitating preparation, storage, and transportation, and being more suitable for wide applications in the fields of medicine, scientific research, and industry.

[0027] The fructose 1,6-diphosphate reaction solution is an enzyme-catalyzed reaction solution containing fructose 1,6-diphosphate obtained by the method for synthesizing fructose 1,6-diphosphate by enzyme disclosed in Patent CN 119082230 A, which contains impurities such as ATP, ADP, AMP, inorganic salts, enzymes, bacterial cells, and proteins and nucleic acids generated by cell autolysis. The concentration of fructose 1,6-diphosphate therein is 35 - 70 g / L. The concentration of fructose 1,6-diphosphate in the fructose 1,6-diphosphate reaction solution is preferably 65 - 68 g / L.

[0028] Beneficial effects:

[0029] The present invention combines membrane separation technology with chromatographic separation technology, improving the separation efficiency and the yield of the target substance. Among them, compared with the traditional nanofiltration desalination process, the electrodialysis desalination process takes less time, consumes less water, and has a higher yield; among them, the adsorption capacity on the column in the chromatographic separation process can reach 100 mg / g (that is, 1 g of resin can adsorb 100 mg of fructose 1,6-diphosphate), and at the same time, the yield of fructose 1,6-diphosphate in the chromatographic separation section is ≥99.9%, and the purity detected by liquid phase is ≥99.9%, greatly reducing the loss of fructose 1,6-diphosphate. The present invention simplifies the separation and purification process of fructose 1,6-diphosphate, significantly improves the yield of fructose 1,6-diphosphate, and has good separation efficiency. The purity of fructose 1,6-diphosphate in the form of sodium salt obtained by separating and purifying through the above method exceeds 99.0%, and the total yield exceeds 95%. Description of the drawings

[0030] The following further specifically describes the present invention in conjunction with the drawings and specific embodiments, and the above and / or other advantages of the present invention will become clearer.

[0031] Figure 1 It is a photo of fructose 1,6-diphosphate (trisodium fructose 1,6-diphosphate) in the form of sodium salt purified by the method of the present invention. Detailed implementation mode

[0032] The present invention will be further described according to the following embodiments. It should be understood that the following embodiments are only used to illustrate the present invention, rather than limiting the present invention.

[0033] For specific technologies or conditions not specified in the embodiments, they shall be in accordance with the technologies or conditions described in the literature in this field or in accordance with the product specifications. For reagents or instruments whose manufacturers are not specified, they are all conventional products that can be obtained through regular channels.

[0034] The preparation method of the 1,6-fructose diphosphate reaction solution separated and purified in the following examples is as follows: The reaction system (with water as the solvent) contains 200 mM glucose, 2 mM ATP, 100 mM magnesium chloride, and 460 mM acetyl phosphate. After adjusting the pH to 7.5, a crude enzyme solution with a final concentration of 2.5 g / L glucose kinase, 5 g / L glucose-6-phosphate isomerase, 2.5 g / L 6-phosphofructokinase 1, and 0.5 g / L acetate kinase is added at 37°C, and the reaction is carried out for 2 h to obtain the 1,6-fructose diphosphate reaction solution.

[0035] The method for measuring the content of 1,6-fructose diphosphate is as follows: High performance liquid chromatography is used, with a refractive index detector and an AMINEX HPX-87H chromatographic column (300 mm × 7.8 mm, 9 μm). The mobile phase is a 5 mM dilute sulfuric acid aqueous solution, with isocratic elution, a flow rate of 0.6 mL / min, a test column temperature of 60°C, and a detector temperature of 35°C.

[0036] The columns used in the following examples are filled with a strongly basic anion exchange resin with a styrene-divinylbenzene copolymer as the inert skeleton and a quaternary amino group as the functional group. Its particle size is 0.15 - 0.38 mm, the average pore size is 3 - 5 nm, the wet true density is 1.00 - 1.20 g / mL, the wet apparent density is 0.60 - 0.79 g / mL, the water content is 65% - 75%, and the quaternary amino group content is 0.9 - 1.1 mmol / L; after the above resin is rinsed with a 0.50 mol / L sodium hydroxide aqueous solution, it is then rinsed with pure water until neutral before use.

[0037] The electrodialysis equipment used in the following examples is purchased from Anhui Zhongke Xinyang Membrane Technology Co., Ltd.

[0038] Example 1

[0039] 3 L of the 1,6-fructose diphosphate reaction solution, in which the concentration of 1,6-fructose diphosphate is 67.4 g / L. The separation and purification steps are as follows:

[0040] (1) Adjust the pH value of the fructose 1,6-diphosphate reaction solution to 2.5 with 12 mol / L hydrochloric acid to inactivate the enzyme therein, and perform microfiltration through a ceramic membrane with a pore size of 50 nm at a working pressure of 0.5 Mpa. Collect the permeate, and repeatedly rinse the residual permeate in the microfiltration pipeline with pure water. The rinsing solution is combined with the permeate to obtain a total of 5.4 L of microfiltered permeate. After detection, the concentration of fructose 1,6-diphosphate in the microfiltered permeate is 37.3 g / L.

[0041] (2) Use an electrodialysis device to perform the first electrodialysis desalination on the permeate. Add the microfiltered permeate to the electrodialysis desalination cell, and add pure water with the same volume as the microfiltered permeate to the concentration cell. The working voltage is 1.0 V / pair of membranes, and the circulation flow rate is 3.5 m 3 / h. When the conductivity of the reaction solution in the desalination cell decreases to 1.6 mS / cm, the desalination ends. Collect the solution in the desalination cell, and rinse the pipeline of the desalination cell 3 times with pure water. The rinsing solution is combined with the solution in the desalination cell to obtain the desalted reaction solution.

[0042] (3) Load the desalted fructose 1,6-diphosphate reaction solution onto the column at a loading flow rate of 1.5 BV / h. The reaction solution enters the chromatographic column for adsorption. When the concentration of fructose 1,6-diphosphate in the adsorption effluent reaches 0.01 g / L, it is considered that the breakthrough point is reached, and the adsorption is stopped. The adsorption capacity of fructose 1,6-diphosphate is calculated to be 106 mg / g; use 0.05 mol / L sodium chloride aqueous solution to elute impurities at a flow rate of 1.5 BV / h; when the concentration of fructose 1,6-diphosphate in the effluent reaches 0.1 g / L, the impurity elution ends. Use 0.1 mol / L sodium chloride aqueous solution to elute fructose 1,6-diphosphate at a flow rate of 1.5 BV / h. Collect the fructose 1,6-diphosphate eluate, measure the volume of the eluate, and use ultra-high performance liquid chromatography to detect the concentration of fructose 1,6-diphosphate therein. After the column chromatography section of this step, the recovery rate of fructose 1,6-diphosphate is calculated to be 99.9%.

[0043] (4) Use an electrodialysis device to perform the second electrodialysis desalination on the fructose 1,6-diphosphate eluate. The operation steps and working parameters are the same as those in step (2) to obtain a fructose 1,6-diphosphate solution.

[0044] (5) The fructose 1,6-diphosphate solution obtained after the second desalination was concentrated using a nanofiltration membrane with a molecular weight cut-off of 200 Da. The working pressure was set at 2.7 MPa and concentrated until the concentration of fructose 1,6-diphosphate in the concentrated solution reached 369.9 g / L. Sodium acetate with a mass of 3% of the mass of fructose 1,6-diphosphate in the concentrated solution was added to the concentrated solution and then stirring was started at a stirring speed of 170 rpm. After stirring for 40 min, the system became stable. Anhydrous ethanol with a volume three times that of the concentrated solution was added, and after continuous stirring for 3.0 h, crystallization was complete. Stirring was stopped, and after suction filtration, washing with anhydrous ethanol, and vacuum drying at 55 °C for 8 h, fructose 1,6-diphosphate trisodium salt with a purity of 99.5% was obtained, and the yield was 93.5%.

[0045] Figure 1 This is a photograph of fructose 1,6-diphosphate (fructose 1,6-diphosphate trisodium) in the form of sodium salt obtained by separation and purification in this example.

[0046] Example 2

[0047] 5 L of the fructose 1,6-diphosphate reaction solution, in which the concentration of fructose 1,6-diphosphate was 66.9 g / L. The separation and purification steps were as follows:

[0048] (1) The pH value of the fructose 1,6-diphosphate reaction solution was adjusted to 2.0 using 12 mol / L hydrochloric acid to inactivate the enzymes therein, and microfiltration was carried out through a ceramic membrane with a pore size of 50 nm at a pressure of 0.25 Mpa. The permeate was collected, and the permeate remaining in the microfiltration pipeline was repeatedly rinsed with pure water. The rinsing solution was combined with the permeate, and a total of 8.7 L of microfiltration permeate was obtained. After detection, the concentration of fructose 1,6-diphosphate in the microfiltration permeate was 38.1 g / L.

[0049] (2) The permeate was subjected to the first electrodialysis desalination using an electrodialysis device. The microfiltration permeate was added to the electrodialysis desalination cell, and pure water with the same volume as the microfiltration permeate was added to the concentration cell. The working voltage was 1.5 V / pair of membranes, and the circulation flow rate was 3.0 m 3 / h. The desalination was ended when the conductivity of the reaction solution in the desalination cell decreased to 2.0 mS / cm. The solution in the desalination cell was collected, and the desalination cell pipeline was rinsed 3 times with pure water. The rinsing solution was combined with the solution in the desalination cell to obtain the reaction solution after desalination.

[0050] (3) The desalted fructose 1,6 - diphosphate reaction solution was loaded onto a column at a sample loading flow rate of 1.7 BV / h. The reaction solution entered the chromatographic column for adsorption. When the concentration of fructose 1,6 - diphosphate in the adsorption effluent reached 0.01 g / L, it was considered that the breakthrough point was reached, and the adsorption was stopped. The adsorption amount of fructose 1,6 - diphosphate was calculated to be 115 mg / g. Impurities were eluted with a 0.05 mol / L sodium chloride aqueous solution at a flow rate of 1.7 BV / h. When the concentration of fructose 1,6 - diphosphate in the effluent reached 0.12 g / L, the impurity elution was completed. Fructose 1,6 - diphosphate was eluted with a 0.1 mol / L sodium chloride aqueous solution at a flow rate of 1.7 BV / h. The fructose 1,6 - diphosphate eluate was collected, the volume of the eluate was measured, and the concentration of fructose 1,6 - diphosphate in it was detected by ultra - high performance liquid chromatography. After the treatment of this step of the column chromatography section, the recovery rate of fructose 1,6 - diphosphate was calculated to be 100%.

[0051] (4) An electrodialysis device was used to perform a second electrodialysis desalination on the fructose 1,6 - diphosphate eluate. The operating steps and working parameters were the same as those in step (2) to obtain a fructose 1,6 - diphosphate solution.

[0052] (5) The fructose 1,6 - diphosphate solution obtained after the second desalination was concentrated using a nanofiltration membrane with a cut - off molecular weight of 200 Da. The working pressure was set at 2.5 MPa and concentrated until the concentration of fructose 1,6 - diphosphate in the concentrated solution reached 398.4 g / L. Sodium acetate with a mass of 2.5% of the mass of fructose 1,6 - diphosphate in the concentrated solution was added and then stirring was started at a stirring speed of 180 rpm. After stirring for 35 min, the system was stable. Four times the volume of anhydrous ethanol of the concentrated solution was added, and after continuous stirring for 3.5 h, the crystallization was complete. Stirring was stopped, and after suction filtration, washing with anhydrous ethanol, and vacuum drying at 55 °C for 8 h, fructose 1,6 - diphosphate trisodium salt with a purity of 99.8% was obtained, and the recovery rate was 93.0%.

[0053] Example 3

[0054] 15 L of the fructose 1,6 - diphosphate reaction solution, with the concentration of fructose 1,6 - diphosphate being 67.4 g / L. The separation and purification steps are as follows:

[0055] (1) The pH value of the fructose 1,6 - diphosphate reaction solution was adjusted to 2.7 using 12 mol / L hydrochloric acid to inactivate the enzymes in it. Microfiltration was carried out through a ceramic membrane with a pore size of 50 nm at a working pressure of 0.3 Mpa. The permeate was collected, and the residual permeate in the microfiltration pipeline was repeatedly rinsed with pure water. The rinsing solution was combined with the permeate to obtain a total of 26.8 L of microfiltration permeate. After detection, the concentration of fructose 1,6 - diphosphate in the microfiltration permeate was 37.6 g / L.

[0056] (2) Use an electrodialysis device to perform the first electrodialysis desalination on the permeate. Add the microfiltration permeate to the electrodialysis desalination cell, add pure water with a volume 0.8 times that of the microfiltration permeate to the concentration cell, the working voltage is 1.7 V / couple of membranes, and the circulation flow rate is 3.5 m 3 / h. When the conductivity of the reaction solution in the desalination cell is reduced to 1.9 mS / cm, the desalination ends. Collect the solution in the desalination cell, rinse the pipeline of the desalination cell 3 times with pure water, and combine the rinsing solution with the solution in the desalination cell to obtain the reaction solution after desalination.

[0057] (3) Load the 1,6-fructose diphosphate reaction solution after desalination onto the column, with a loading flow rate of 1.5 BV / h. The reaction solution enters the chromatographic column for adsorption. When the concentration of 1,6-fructose diphosphate in the adsorption effluent is 0.01 g / L, it is considered that the breakthrough point has been reached, stop the adsorption, and calculate that the adsorption capacity of 1,6-fructose diphosphate is 109 mg / g; use a 0.05 mol / L sodium chloride aqueous solution to elute impurities at a flow rate of 1.5 BV / h; when the concentration of 1,6-fructose diphosphate in the effluent is 0.1 g / L, the impurity washing ends, and use a 0.1 mol / L sodium chloride aqueous solution to elute 1,6-fructose diphosphate at a flow rate of 1.7 BV / h. Collect the 1,6-fructose diphosphate eluate, measure the volume of the eluate, and use ultra-high performance liquid chromatography to detect the concentration of 1,6-fructose diphosphate in it. After the column chromatography section of this step, the recovery rate of 1,6-fructose diphosphate is calculated to be 99.9%.

[0058] (4) Use an electrodialysis device to perform the second electrodialysis desalination on the 1,6-fructose diphosphate eluate. The operation steps and working parameters are the same as in step (2) to obtain a 1,6-fructose diphosphate solution.

[0059] (5) Concentrate the 1,6-fructose diphosphate solution obtained after the second desalination using a nanofiltration membrane with a molecular weight cut-off of 200 Da. Set the working pressure to 1.5 MPa and concentrate until the concentration of 1,6-fructose diphosphate in the concentrated solution is 382.8 g / L. Add sodium acetate with a mass 2% of the mass of 1,6-fructose diphosphate in the concentrated solution to the concentrated solution and start stirring. The stirring speed is 180 rpm. After stirring for 40 min, the system is stable. Add anhydrous ethanol with a volume 3 times that of the concentrated solution and continuously stir for 4.0 h until crystallization is complete. Stop stirring, perform suction filtration, wash with anhydrous ethanol, and vacuum dry at 55 °C for 8 h to obtain 1,6-fructose diphosphate trisodium salt with a purity of 99.7% and a recovery rate of 92.6%.

[0060] The present invention provides an idea and method for separating and purifying fructose 1,6-diphosphate from a fructose 1,6-diphosphate reaction solution. There are many methods and ways to specifically implement this technical solution. The above description is only a preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention. Each component not clearly defined in this embodiment can be implemented by using the prior art.

Claims

1. A method for separating and purifying fructose 1,6-diphosphate from a fructose 1,6-diphosphate reaction solution, characterized in that, It includes the following steps: (1) Adjust the pH of the fructose 1,6-diphosphate reaction solution to acidic, perform microfiltration, and collect the permeate; (2) Perform first electrodialysis desalination on the permeate obtained in step (1) to obtain the reaction solution after the first desalination; (3) Load the reaction solution after the first desalination obtained in step (2) onto a strongly basic anion exchange resin for adsorption, impurity washing, and elution, and collect the eluate containing fructose 1,6-diphosphate; (4) Perform second electrodialysis desalination on the eluate obtained in step (3) to obtain a fructose 1,6-diphosphate solution; (5) Perform nanofiltration concentration on the fructose 1,6-diphosphate solution obtained in step (4), collect the concentrated solution, crystallize, perform suction filtration, washing, and drying to obtain fructose 1,6-diphosphate in the form of sodium salt.

2. The method according to claim 1, wherein In step (1), for the acidic condition, the pH value is 2-3; the membrane pore size of the microfiltration membrane used for microfiltration is 10-100 nm; the working pressure during microfiltration is 0.25-0.5 Mpa.

3. The method according to claim 1, characterized in that In steps (2) and (4), when performing the first electrodialysis desalination or the second electrodialysis desalination, in the electrodialysis equipment, add the permeate or the eluate to the diluate chamber, add water to the concentrate chamber, and the volume ratio of the permeate or the eluate to the water is 1:0.5-1; the electrodialysis working voltage is 1.0-2.0 V / pair of membranes, and the circulation flow rate is 2.0-4.0 m3 / h; when the conductivity of the permeate or the eluate in the diluate chamber is reduced to below 2 mS / cm, stop desalination.

4. The method according to claim 1, wherein In step (3), the strongly basic anion exchange resin is an anion exchange resin with a styrene-divinylbenzene copolymer as the inert skeleton and a quaternary amine group as the functional group; among them, the particle size of the strongly basic anion exchange resin is 0.15-0.38 mm, the average pore size is 3-5 nm, the wet true density is 1.00-1.20 g / mL, the wet apparent density is 0.60-0.79 g / mL, the quaternary amine group content > 1.0 mmol / L, and the water content is 65%-75%.

5. The method according to claim 1, characterized in that, In step (3), the impurity washing is performed using a 0.01-0.05 mol / L sodium chloride aqueous solution, and the elution is performed using a 0.1-0.3 mol / L sodium chloride aqueous solution.

6. The method according to claim 1, characterized in that In step (3), the flow rates of adsorption, impurity washing, and elution are all 1-2 BV / h.

7. The method according to claim 1, characterized in that In step (3), when loading the sample, stop loading when the concentration of fructose 1,6-diphosphate in the effluent is 0.01-0.02 g / L; when performing impurity washing, stop impurity washing when the concentration of fructose 1,6-diphosphate in the effluent is 0.1-0.2 g / L.

8. The method according to claim 1, wherein In step (5), the nanofiltration concentration is performed using a nanofiltration membrane with a cut-off molecular weight of 100-200 Da to concentrate the fructose 1,6-diphosphate solution to 350-400 g / L; the working pressure during nanofiltration concentration is 1-3 Mpa.

9. The method according to claim 1, wherein In step (5), the crystallization is as follows: under the condition of continuous stirring, sodium acetate is added to the concentrated solution, and after stirring for 20 to 40 minutes, absolute ethanol is added, and then stirred for 2 to 4 hours; the washing is carried out using absolute ethanol; the drying temperature is 50 to 55 °C and the time is 8 to 12 hours.

10. The method according to claim 9, characterized in that, The dosage of the sodium acetate is 2% to 5% of the mass of fructose 1,6-diphosphate in the concentrated solution; the dosage of the absolute ethanol is 3 to 5 times the volume of the concentrated solution; the rotation speed of the stirring is 100 to 200 rpm.

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