A process for separating and purifying UMP liquid by sequential continuous ion exchange

Abstract

The invention discloses a process for separating and purifying UMP liquid by using a sequential continuous ion exchange method, wherein the sequential continuous ion exchange method is composed of three or more resin columns, the resin columns are divided into zone I, zone II and zone III, each zone has at least one resin column, the liquid enters from the top of the resin column and flows out from the bottom, and each resin column is filled with anion exchange resin; adsorption, washing, elution and regeneration operations are performed according to the steps. The invention can continuously produce UMP with a yield and purity of up to 99%.

Description

Technical Field

[0001] The invention belongs to the technical field of biological separation, and in particular relates to a process for separating and purifying UMP liquid by using a sequential continuous ion exchange method. Background Art

[0002] 5'-Uracil nucleotide (Uridine monophosphate, also known as monophosphate uridine, hereinafter referred to as UMP), with a molecular weight of 324.18, together with cytidylic acid, constitutes the pyrimidine nucleotide part of RNA. It has a wide range of uses in the food, pharmaceutical and chemical industries, especially in the field of infant food and medicine, where it has irreplaceable functions. Most of the UMP production in my country is produced by enzymatic hydrolysis, which has a long production cycle, cumbersome process, high separation difficulty, low UMP yield and high cost. The biocatalytic production of UMP technology is a new technology for the production of nucleotides. It has the advantages of high efficiency, high selectivity, mild conditions, and environmental friendliness. It shortens the production cycle and greatly increases the yield of UMP. The reaction system is simple, generally requiring only substrates, surfactants, enzyme cofactors (such as magnesium ions) and a certain amount of pH regulator. Therefore, in the production of UMP liquid, uridine-cytidylase (UCK) as an important catalyst in the compensation pathway of nucleotide metabolism in organisms can catalyze the phosphorylation of uridine and cytidine into uridine acid and cytidy acid. The obtained UMP liquid contains Na + Mg 2+ , UMP, and PO4 3- etc., and the pH value of the feed solution is about 2. UMP is mostly in the form of UMP at pH 2. - Since the components in the enzymatic hydrolysate are relatively complex, the traditional method of separating UMP feed is usually to first use a cation exchange column to separate the four mononucleotides. [1] , respectively, to obtain dilute solutions of UMP, cytidine (CMP), adenylate (AMP) and a mixed solution of guanylate (GMP) and CMP; then, after adjusting the pH value of UMP, separate, purify and concentrate it on a hydroxyl anion exchange column. This method has the disadvantages of low resin utilization, difficulty in completely separating the four nucleotides, and the exchange group OH in the purification process of the hydroxyl anion exchange resin. - Easy with Mg 2+ The formation of precipitation causes the column to be blocked, resulting in excessive resource consumption. [2] A new cation exchange resin was studied, which can separate four nucleotides on the cation column, but there is almost no distance between the chromatographic peaks of each product, which makes it difficult to collect the effluent product. [3]A series of cation exchange columns are used to first adsorb AMP, CMP, and GMP under acidic conditions. The unadsorbed UMP flows through a weak alkaline resin column and a strong alkaline resin column, and then is eluted, concentrated, and dried to obtain the UMP product. The process is relatively cumbersome, and the regeneration of the resin will consume too much acid and alkali.

[0003] References:

[0004] DEODA AJ, SINGHAL R S. 5'-Phosphodiesterase (5'-PDE) from germinated barley for hydrolysis of RNA to produce flavor nucleotides[J]. BioresourTechnol, 2003,

[0005] 88(3):245-50.

[0006] Xiao Linping. Research on separation and purification technology of 5'-uridine monophosphate[D]; Nanjing University of Technology, 2003.

[0007] Tanaka,Release of intracellularly stored 5-phosphodiesterase withpreserved plant.

[0008] Biotech Bioengineering 1985. Summary of the invention

[0009] The technical problem to be solved by the present invention is to provide a process for separating and purifying UMP liquid by sequential continuous ion exchange method in view of the shortcomings of the existing process which is relatively complicated and consumes a lot of energy, so as to solve the problem of Ca in UMP liquid. 2+ Mg 2+ ATP + AMP + , UR - PO4 3- The difficult problem of removal can be solved, and finally UMP with a yield and purity of more than 99% can be obtained.

[0010] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0011] A process for separating and purifying UMP feed liquid by sequential continuous ion exchange method, wherein the sequential continuous ion exchange method comprises three or more resin columns, wherein the resin columns are divided into zone I, zone II and zone III, each zone has at least one resin column, and each resin column is a chromatography column of the same specification, wherein the feed liquid enters from the top of the resin column and flows out from the bottom, and each resin column is filled with anion exchange resin; the operation is performed according to the following steps ( Figure 1 ):

[0012] (1) Zone I and Zone II are connected in series; the UMP feed liquid enters Zone I for adsorption, and the residual liquid containing impurities is collected at the outlet of Zone II, PO4 3- and UMP undergo ion exchange with the resin, while impurities (Ca 2+ Mg 2+ 、Na + ATP + AMP + , UR - ) does not adsorb, that is, does not retain components; when the resin in zone I is saturated with adsorption, pure water enters zone I for washing and removing the materials in the gaps between the resin particles. The materials in the gaps between the resin particles mainly contain Ca 2+ Mg 2+ 、Na + ATP + AMP + , UR - , containing trace amounts of UMP - , and at the same time, residual impurities are collected at the outlet of zone II until the breakthrough curve of the impurity magnesium ion C / C0≤0.1; the first eluent enters zone I for elution, and at the same time, the weakly retained component UMP is collected at the outlet of zone II until the breakthrough curve of UMP C / C0≤0.1; in step (1), zone III is simultaneously regenerated with pure water;

[0013] (2) Disconnect zone I and zone II, allow the second eluent to enter zone I for elution, and collect the strongly retained component PO4 at the outlet of zone I. 3- ; In step (2), zone II and zone III are simultaneously regenerated with pure water;

[0014] (3) Zone II and Zone III are connected in series to replace Zone I and Zone II in Step (1) and Step (2), and Zone I replaces Zone III in Step (1) and (2), and the operations of Step (1) and (2) are repeated, and the operations of adsorption, washing and regeneration are switched in this order. When the system reaches stability, the resin in each switching cycle is consistent with the state of the previous cycle, for example: each time the switching is performed, the adsorption state of the adsorption section is the same as that of the previous cycle; wherein each stage is carried out simultaneously at room temperature.

[0015] The anion exchange resin is a type I or type II strong alkaline anion exchange resin with styrene as the skeleton, or a polyamine type alkaline anion exchange resin, or a gel type anion exchange resin.

[0016] Wherein, the polyamine comprises triethylenetetramine and / or tetraethylenepentamine.

[0017] Among them, the functional groups of the anion exchange resin are quaternary amine groups and / or tertiary amine groups, the content of the quaternary amine groups is 1.0 to 2.5 mmol / g of the anion exchange resin, and the content of the tertiary amine groups is 1.0 to 2.5 mmol / g of the anion exchange resin; the exchangeable ions of the anion exchange resin are chloride ions, the content of chloride ions is 1.0 to 2.5 mmol / g, and the volumetric total exchange capacity is ≥1.35 mmol / mL; the cross-linking degree of the anion exchange resin is 3 to 10%.

[0018] The anion exchange resin has a particle size of 0.1 to 0.8 mm, a water content of 42 to 48%, and a wet true density of 1.03 to 1.18 g / cm 3 , with a specific surface area of ​​100 to 2000 m 2 / g, pore volume is 0.51~1.33cm 3 / g, pore size is 1~200nm.

[0019] The anion exchange resin includes but is not limited to the anion exchange resins described above.

[0020] The UMP liquid is obtained by enzymatically removing two phosphate groups from UTP. In the preparation process of the UMP liquid, due to the addition of Ca 2+ Mg 2+ After acid adjustment, the pH value of the entire liquid is 1 to 3, basically around pH 2. The UMP liquid contains Mg 2+ 、Na + 、UMP - , Ca 2+ AMP + ATP + , UR - and PO4 3- , pH 1 to 3. The UMP liquid is a process in which UTP is subjected to enzymatic hydrolysis reaction and only needs to be filtered through a plate and frame to remove the bacteria, and resin separation can be performed without ultrafiltration or nanofiltration operation.

[0021] Wherein, the UMP liquid, Mg 2+ The content is 0.1mg / L~3g / L, Na + The content is 1g / L~30g / L, UMP- The content is 10g / L~70g / L, Ca 2+ The content of AMP is 0.1mg / L~3g / L, + The content is 2g / L~10g / L, ATP + The content is 2g / L~10g / L, UR - The content is 2g / L~10g / L, PO4 3- The content of Mg is 0.5 g / L to 10 g / L. Preferably, the UMP liquid, Mg 2+ The content is 0.1mg / L~0.3g / L, Na + The content is 10g / L~20g / L, UMP - The content is 35g / L~45g / L, Ca 2+ The content is 60mg / L~100mg / L, AMP + The content is 2.5g / L~3.5g / L, ATP + The content is 2g / L~4g / L, UR - The content is 2g / L~5g / L, PO4 3- The content is 2g / L~3g / L.

[0022] In step (1), the adsorption has a loading rate of 2.5 to 4 BV / h; whether the resin is adsorbed saturated can be determined by measuring the saturation point (C / C0=1) of the penetration curve in the fixed bed penetration experiment; the residual liquid containing impurities, the impurity is Ca 2+ Mg 2+ 、Na + ATP + AMP + and UR - ; The pure water washing rate is 2.5~4BV / h.

[0023] In step (1) and step (2), the first eluent is a mixed aqueous solution of 0.1M-1.5M NaCl and 0.001M-0.1M HCl; the second eluent is a mixed aqueous solution of 1M-3M NaCl and 0.001M-0.1M HCl; and the elution rates are independently selected from 2.5-4BV / h.

[0024] The pure water regeneration flow rate is generally controlled at 3 to 8 BV / h.

[0025] In steps (1) and (2), the switching time of each process section of adsorption, washing and elution is 45 to 200 minutes.

[0026] The C / C0 described in the present invention, wherein C refers to the instantaneous concentration, and C0 refers to the initial concentration of the feed solution.

[0027] Beneficial effects: The present invention utilizes a sequential continuous ion exchange method to separate and purify UMP liquid, does not require complicated operations, achieves the highest resin utilization rate, does not require nanofiltration dephosphorization, and can continuously produce UMP through continuous chromatographic separation with a yield and purity of up to 99%. It only needs to be scaled up in proportion and can be applied to industrial production.

[0028] Compared with the prior art, the present invention has the following advantages:

[0029] (1) The sequential continuous ion exchange method is carried out at room temperature, which greatly reduces energy consumption.

[0030] (2) The resin is easy to regenerate and can be reused, with the highest utilization rate. It also has a certain pigment removal effect. The process is continuous, the operating cost is low, and the process can be directly scaled up.

[0031] (3) No need for nanofiltration dephosphorization, the impurity Ca can be removed in one step through continuous separation. 2+ Mg 2+ ATP + AMP + , UR - PO4 3- Separation from the product UMP ensures the purity and quality of the UMP product, with a yield of more than 99% and HPLC purity of 100%. 2+ Mg 2+ ATP + AMP + , UR - PO4 3- The removal rate of plasma reaches over 99%. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0033] Figure 1 This is the process flow chart of the present invention, switching from step ae in sequence.

[0034] Figure 2 This is the HPLC chart of UMP sodium salt solution (sample diluted 100 times).

[0035] Figure 3 This is the HPLC chart of UMP sodium salt product solution (sample diluted 100 times).

[0036] Figure 4 This is the ion chromatogram of UMP sodium salt solution (sample diluted 100 times).

[0037] Figure 5 This is the ion chromatogram of the UMP sodium salt product solution (the sample was not diluted). DETAILED DESCRIPTION

[0038] In the following examples, the external standard method was used to measure the UMP and Mg in the feed solution. 2+ and PO4 3- Concentration was tested.

[0039] Ca 2+ Mg 2+ 、Na + The ion chromatography conditions were:

[0040] 1) Detector: Thermo Scientific-CD detector;

[0041] 2) Chromatographic column: Dionex Ionpac TM CS12A;

[0042] 3) Suppressor: Dionex TM CDRS 600 4mm;

[0043] 4) Mobile phase: 20mmol / L dimethylsulfonic acid;

[0044] 5) Flow rate: 1 mL / min;

[0045] 6) Column temperature: 25°C;

[0046] 7) Injection volume: 25 μL.

[0047] Ca 2+ Mg 2+ 、Na + Detection methods and steps:

[0048] 1) Equilibration of the chromatographic column: flush the chromatographic column with 20 mmol / L dimethylsulfonic acid at a flow rate of 1 mL / min, turn on the column oven at the same time, and start collecting the baseline. When the CD signal value baseline tends to be below 0.5, the equilibrium is completed.

[0049] 2) Sample detection: Write the injection sequence and method according to the ion chromatography conditions, place the standard products and samples after membrane treatment in the corresponding positions of the automatic injector according to the injection sequence, start injection and collect spectral information.

[0050] PO4 3- Detection methods and steps:

[0051] Phosphorus determination reagent: 3 mol / L sulfuric acid, water, 2.5% ammonium molybdate, and 10% ascorbic acid are prepared in a ratio of 1:2:1:1 (volume ratio). When preparing, add them in order. Take two hard glass test tubes, add 0.1 ml of sample, 2.9 ml of water, and 3.0 ml of phosphorus determination reagent to one tube; add only 3.0 ml of water and 3.0 ml of phosphorus determination reagent to the other tube as a blank; shake the two tubes well, place them in a 45°C water bath, heat for 25 minutes, then take them out and cool to room temperature, measure the OD value of the sample at 660 nm with the blank as reference, and then obtain the phosphorus content according to the linear relationship of the standard curve.

[0052] The UMP chromatography conditions are:

[0053] 1) Detector: Agilent 1260 high performance liquid chromatograph-ultraviolet detector (254 nm);

[0054] 2) Chromatographic column: ZORBAX SB-AQ liquid chromatography column;

[0055] 3) Mobile phase: triethylamine phosphate buffer with a pH of 6.5: methanol = 91:9;

[0056] 4) Flow rate: 0.7 mL / min;

[0057] 5) Column temperature: 25°C;

[0058] 6) Injection volume: 20 μL.

[0059] Detection methods and steps:

[0060] 1) Balance of the chromatographic column: prepare a mobile phase of triethylamine phosphate buffer with a pH of 6.5: methanol = 91:9, filter with a mixed microporous filter membrane with a pore size of 0.22μm, and then perform ultrasonic treatment for 30 minutes. Rinse the chromatographic column with the treated mobile phase at a flow rate of 0.7mL / min, turn on the column oven at the same time, start collecting the baseline, and when the baseline tends to be a straight line, the balance is completed.

[0061] 2) Sample detection: Write the injection sequence and method according to the chromatographic conditions, place the standard products and samples after membrane treatment in the corresponding positions of the automatic injector according to the injection sequence, start injection and collect chromatographic information.

[0062] The yield of UMP in the product solution was calculated using the following method:

[0063]

[0064] C1: Concentration of UMP in the product solution;

[0065] v2: volume of product liquid;

[0066] C: UMP concentration in the sample solution;

[0067] v: volume of sample solution.

[0068] The Ca content in the product solution was calculated using the following method: 2+ Mg 2+ ATP + AMP + , UR - PO4 3- Plasma removal rate:

[0069]

[0070] C0: Ca in the sample solution 2+ Mg 2+ ATP + AMP + , UR - PO4 3- concentration;

[0071] C2: Ca in product solution 2+ Mg 2+ ATP + AMP + , UR - PO4 3- concentration.

[0072] In the following examples, the chloride-type anion exchange resin (HY06) used is a type I strong alkaline anion resin with styrene as the skeleton. The functional group of the resin is trimethylamine, and the content of the amine group is 1.0-2.5 mmol / g anion exchange resin; the exchangeable ion is chloride ion, and the content of the chloride ion is 1.0-2.5 mmol / g anion exchange resin; the particle size of the ion exchange resin is 0.4-0.7 mm, the water content is 42.00-48.00%, and the wet true density is 1.07-1.10 g / cm 3 , the volumetric total exchange capacity is ≥1.35mmol / mL. Resin manufacturers can synthesize according to the above conditions.

[0073] In the following examples, the continuous separation device used is composed of 3 resin columns, which are divided into zone I, zone II and zone III, with 1 resin column in each zone, each resin column is a chromatography column of the same specification, the feed enters from the top of the resin column and flows out from the bottom, and each resin column is filled with anion exchange resin; the operation is carried out according to the following steps ( Figure 1 ):

[0074] (1) Zone I and Zone II are connected in series; the UMP feed liquid enters Zone I for adsorption, and the residual liquid containing impurities is collected at the outlet of Zone II, PO4 3-and UMP undergo ion exchange with the resin, while impurities (Ca 2+ Mg 2+ 、Na + ATP + AMP + , UR - ) does not adsorb, that is, does not retain components; when the resin in zone I is saturated with adsorption, pure water enters zone I for washing and removing the materials in the gaps between the resin particles. The materials in the gaps between the resin particles mainly contain Ca 2+ Mg 2+ 、Na + ATP + AMP + , UR - , containing trace amounts of UMP - , and at the same time, residual impurities are collected at the outlet of zone II until the breakthrough curve of the impurity magnesium ion C / C0≤0.1; the first eluent enters zone I for elution, and at the same time, the weakly retained component UMP is collected at the outlet of zone II until the breakthrough curve of UMP C / C0≤0.1; in step (1), zone III is simultaneously regenerated with pure water;

[0075] (2) Disconnect zone I and zone II, allow the second eluent to enter zone I for elution, and collect the strongly retained component PO4 at the outlet of zone I. 3- ; In step (2), zone II and zone III are simultaneously regenerated with pure water;

[0076] (3) Zone II and Zone III are connected in series to replace Zone I and Zone II in steps (1) and (2), and Zone I replaces Zone III in steps (1) and (2), and the operations of steps (1) and (2) are repeated, and the adsorption, washing and regeneration operations are repeated in this order. Figure 1 a~ Figure 1 d, the state of the resin column is changed by switching the inlet and outlet positions of the feed liquid. The three areas switch the inlet and outlet of each feed liquid in sequence to maintain the state of continuous injection. When the system reaches stability, the resin in each switching cycle is consistent with the state of the previous cycle. For example, each time it is switched, the adsorption state of the adsorption section is the same as the previous cycle; among them, each stage is carried out simultaneously at room temperature.

[0077] Example 1: Obtaining UMP solution

[0078] The UMP solution was provided by Nanjing Tongkai Zhaoye Biotechnology Co., Ltd. The preparation method is that UTP is enzymatically hydrolyzed to generate UMP solution. Due to the addition of Mg in the process 2+ After acid adjustment, the pH value of the entire liquid is about 2, so the entire UMP liquid contains Ca 2+ Mg 2+ 、Na+ ATP + AMP + , UR - PO4 3- 、UMP - .

[0079] The experiment was repeated three times according to the above method to obtain three batches of UMP solution.

[0080] In the first batch of liquid, Mg 2+ The content is 0.17g / L, PO4 3- The content is 2.2g / L, UMP - The content is 38g / L, Na + The content is 13g / L, Ca 2+ The content is 0.08g / L, AMP + The content is 2.7g / L, ATP + The content is 2.9g / L, UR - The content is 3.7g / L;

[0081] In the second batch of liquid, Mg 2+ The content is 0.19g / L, PO4 3- The content is 2.1g / L, UMP - The content is 43g / L, Na + The content is 17g / L, Ca 2+ The content is 0.07g / L, AMP + The content is 3.1g / L, ATP + The content is 3g / L, UR - The content is 4.1g / L;

[0082] In the third batch of liquid, Mg 2+ The content is 0.18g / L, PO4 3- The content is 2.3g / L, UMP - The content is 41g / L, Na + The content is 16g / L, Ca 2+ The content is 0.06g / L, AMP + The content is 3.5g / L, ATP + The content is 2.7g / L, UR - The content is 2.8g / L;

[0083] Example 2: Continuous Chromatographic Separation of UMP Liquid

[0084] Each resin column was filled with 74 mL of specific anion exchange resin, and the resin column had a diameter of 2.2 cm and a height of 27 cm.

[0085] The liquid phase diagram of UMP liquid (first batch of liquid) is as follows Figure 2 As shown, the loading amount of UMP liquid was 2.7 BV and the adsorption flow rate was 3.5 BV / h.

[0086] The pure water washing flow rate is 3.5BV / h.

[0087] The first eluent was a mixed aqueous solution of 0.4 M NaCl and 0.01 M HCl, with a dosage of 8.1 BV and a flow rate of 3.5 BV / h.

[0088] The second eluent was a mixed aqueous solution of 1.5 M NaCl and 0.01 M HCl, with a dosage of 6.8 BV and a flow rate of 3.5 BV / h.

[0089] The experiment ended after 6.25 hours.

[0090] The collected product effluent was tested for UMP by HPLC. - ATP + AMP + , UR - Concentration, Ca was measured by ion chromatography 2+ Mg 2+ Plasma concentration, PO4 detected by UV spectrophotometer 3- concentration, the yield of UMP sodium salt product reached 99%, the purity of HPLC reached 100%, and the Mg 2+ PO4 3- The plasma removal rate reached 99.6%. From the HPLC and ion chromatograms, Figure 2 This is the HPLC chromatogram of the UMP solution diluted 100 times. Figure 2 It can be seen that in addition to the UMP product peak, there are also some impurity peaks (such as ATP + AMP + , UR - ), these impurity peaks slightly overlap with the UMP product peaks, causing a slight deviation in the automatic integration of the chromatographic peaks. Figure 3 The chromatogram of UMP sodium salt product shows that the purity of UMP reaches 100%, and the impurities (ATP + AMP + , UR - ) is completely removed. At the same time, Figure 4 This is the ion chromatogram of the UMP solution diluted 100 times, and Figure 5 This is the ion chromatogram of the undiluted UMP sodium salt product. Figure 4 and Figure 5 In contrast, the Mg content in UMP sodium salt products is 2+ Plasma (except Na +The content of the 5-amino-2-nitropropene (2-amino-2-nitropropene) was almost 0, which also showed that the purification effect of this experiment was good.

[0091] Example 3: Continuous Chromatographic Separation of UMP Liquid

[0092] Each resin column was filled with 74 mL of specific anion exchange resin, and the resin column had a diameter of 2.2 cm and a height of 27 cm.

[0093] The loading amount of UMP solution (second batch of solution) was 2.7 BV, and the adsorption flow rate was 3.1 BV / h.

[0094] The pure water washing flow rate is 3.5BV / h.

[0095] The first eluent was a mixed aqueous solution of 0.4 M NaCl and 0.01 M HCl, with a dosage of 8.1 BV and a flow rate of 3.5 BV / h.

[0096] The second eluent was a mixed aqueous solution of 1.5 M NaCl and 0.01 M HCl, with a dosage of 6.8 BV and a flow rate of 4 BV / h.

[0097] The experiment ended after 5.9 hours.

[0098] The collected product effluent was tested for UMP by HPLC. - ATP + AMP + , UR - Concentration, Ca was measured by ion chromatography 2+ Mg 2+ Plasma concentration, PO4 detected by UV spectrophotometer 3- Concentration, Ca 2+ Mg 2+ The concentration was below the detection limit of ion chromatography, ATP + AMP + , UR - The concentration was below the detection limit of liquid chromatography, PO4 3- The concentration was below the detection limit of the UV spectrophotometer, the yield of the UMP sodium salt product reached 99%, the purity of HPLC reached 100%, and the Ca 2+ Mg 2+ PO4 3- The plasma removal rate reached 99.4%.

[0099] Example 4: Continuous Chromatographic Separation of UMP Liquid

[0100] Each resin column was filled with 74 mL of specific anion exchange resin, and the resin column had a diameter of 2.2 cm and a height of 27 cm.

[0101] The loading amount of UMP solution (the third batch of solution) was 2.7 BV, and the adsorption flow rate was 3.6 BV / h.

[0102] The pure water washing flow rate is 3.5BV / h.

[0103] The first eluent was a mixed aqueous solution of 0.41 M NaCl and 0.01 M HCl, with a dosage of 8.1 BV and a flow rate of 3 BV / h.

[0104] The second eluent was a mixed aqueous solution of 1.5 M NaCl and 0.01 M HCl, with a dosage of 6.8 BV and a flow rate of 3.4 BV / h.

[0105] The experiment ended after 6.7 hours.

[0106] The collected product effluent was tested for UMP by HPLC. - ATP + AMP + , UR - Concentration, Ca was measured by ion chromatography 2+ Mg 2+ Plasma concentration, PO4 detected by UV spectrophotometer 3- Concentration, Ca 2+ Mg 2+ The concentration was below the detection limit of ion chromatography, ATP + AMP + , UR - The concentration was below the detection limit of liquid chromatography, PO4 3- The concentration was below the detection limit of the UV spectrophotometer, the yield of the UMP sodium salt product reached 99%, the purity of HPLC reached 100%, and the Ca 2+ Mg 2+ PO4 3- The plasma removal rate reached 99.8%.

[0107] Comparative Example 1

[0108] The same batch of feed solution, the same injection volume, flow rate and other conditions were used as in Example 3. The concentration of the first eluent NaCl was changed to 0.45 M. PO4 was detected in the product solution collected at the outlet of zone II in step (1). 3- The presence of PO4 3- The removal rate is 75%, indicating that the eluent cannot purify the UMP product in the UMP liquid well.

[0109] The present invention provides a process idea and method for separating and purifying UMP liquid by sequential continuous ion exchange. There are many methods and ways to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the protection scope of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. A process for separating and purifying UMP liquid by sequential continuous ion exchange, characterized in that: The sequential continuous ion exchange method is composed of 3 or more resin columns, which are divided into zone I, zone II and zone III, with at least 1 resin column in each zone, and the feed enters from the top of the resin column and flows out from the bottom, and each resin column is filled with anion exchange resin; the operation is carried out according to the following steps: (1) Zone I and Zone II are connected in series; the UMP feed liquid enters Zone I for adsorption, and the residual liquid containing impurities is collected at the outlet of Zone II; When the resin in zone I is saturated with adsorption, pure water enters zone I for washing, and at the same time, residual impurities are collected at the outlet of zone II until the breakthrough curve of the impurity magnesium ion C / C0≤0.1; the first eluent enters zone I for elution, and at the same time, UMP is collected at the outlet of zone II until the breakthrough curve of UMP C / C0≤0.1; in step (1), zone III is simultaneously regenerated with pure water; (2) Disconnect Zone I and Zone II, allow the second eluent to enter Zone I for elution, and collect PO4 at the outlet of Zone I. 3- ; In step (2), zone II and zone III are simultaneously regenerated with pure water; (3) replacing zone I and zone II in steps (1) and (2) in series with zone II and zone III, and replacing zone III in steps (1) and (2) with zone I, and repeating the operations of steps (1) and (2), switching and repeating the operations of adsorption, washing and regeneration in this order; The anion exchange resin is a type I strong alkaline anion exchange resin with styrene as the skeleton; the functional group of the resin is trimethylamine, and the content of tertiary amine is 1.0-2.5 mmol / g anion exchange resin; the exchangeable ion is chloride ion, and the content of chloride ion is 1.0-2.5 mmol / g anion exchange resin; In step (1) and step (2), the first eluent is a mixed aqueous solution of 0.1M~1.5M NaCl and 0.001M~0.1M HCl; the second eluent is a mixed aqueous solution of 1M~3M NaCl and 0.001M~0.1M HCl.

2. The process according to claim 1, characterized in that The anion exchange resin has a volumetric total exchange capacity of ≥1.35 mmol / mL; and the cross-linking degree of the anion exchange resin is 3-10%.

3. The process according to claim 1, characterized in that The anion exchange resin has a particle size of 0.1-0.8 mm, a water content of 42-48%, and a wet true density of 1.03-1.18 g / cm 3 , with a specific surface area of ​​100~2000m 2 / g, pore volume is 0.51~1.33cm 3 / g, pore size is 1~200nm.

4. The process according to claim 1, characterized in that The UMP feed solution is obtained by enzymatically removing two phosphate groups from UTP, and the UMP feed solution contains Mg 2+ 、Na + 、UMP - , Ca 2+ AMP + ATP + , UR - and PO4 3- , pH1~3.

5. The process according to claim 4, characterized in that The UMP liquid, Mg 2+ The content is 0.1mg / L~3g / L, Na + The content is 1g / L~30g / L, UMP - The content is 10g / L~70g / L, Ca 2+ The content is 0.1mg / L~3g / L, AMP + The content is 2g / L~10g / L, ATP + The content is 2g / L~10g / L, UR - The content is 2g / L~10g / L, PO4 3- The content is 0.5g / L~10g / L.

6. The process according to claim 1, characterized in that In step (1), the adsorption has a loading rate of 2.5-4 BV / h; the impurity-containing residual liquid is Ca 2+ Mg 2+ 、Na + ATP + AMP + and UR - ; The pure water washing rate is 2.5~4BV / h.

7. The process according to claim 1, characterized in that In step (1) and step (2), the elution rates of the first eluent and the second eluent are independently selected from 2.5 to 4 BV / h.

8. The process according to claim 1, characterized in that In steps (1) and (2), the switching time of the adsorption, washing and elution sections is 45 to 200 minutes.

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