Method for separating and purifying glutamic acid

Abstract

The invention belongs to the technical field of amino acid production, and discloses a method for separating and purifying glutamic acid, which comprises the following steps: separating and purifying a glutamic acid crude product by using a weak cation exchanger to obtain a medicinal glutamic acid refined product; the weak cation exchanger is functionalized polystyrene microspheres, and the glutamic acid crude product contains heteroamino acid. The method can effectively separate and remove heteroamino acid, is simple in process and low in cost, and completely meets the standard of medicinal glutamic acid.

Description

Technical Field

[0001] This invention belongs to the field of amino acid production technology, and relates to a method for separating and purifying glutamic acid, specifically a method for separating and purifying glutamic acid using an ion exchange process. Background Technology

[0002] Glutamic acid is an important amino acid with multiple functions in living organisms and is widely used in medicine, food, and chemical industries. As a medicinal substance, glutamic acid and its derivatives play an important role in the treatment of nervous system diseases, metabolic regulation, and nutritional support. Medicinal glutamic acid requires extremely high purity, needing to reach at least 99%.

[0003] The main production method for glutamic acid is microbial fermentation. Using glucose as the carbon source, appropriate amounts of inorganic salts and biotin are added, and fermentation is carried out by glutamic acid-producing bacteria (such as Corynebacterium) to ultimately produce glutamic acid, which is then purified by centrifugation, filtration, and isoelectric crystallization. During microbial fermentation, small amounts of other amino acids are produced, which are difficult to completely remove during the separation and purification process, preventing most products from being used in the pharmaceutical field. Currently, ion chromatography is a key technology for separating and purifying amino acids. Its core principle is based on the amphoteric electrolyte properties of amino acids and the differences in isoelectric points among different amino acids. Separation is achieved through the selective adsorption and exchange of amino acids by ion exchange resins. Specifically, amino acids in solution exist as cations, anions, or zwitterions depending on the pH value. Ion exchange resins (such as strong acid-type and weak acid-type cation exchange resins or strong base-type and weak base-type anion exchange resins) utilize the difference in affinity between functional groups and amino acid ions to specifically adsorb the target amino acid, which is then desorbed and separated by an eluent.

[0004] The study, "Performance Study of Ion Exchange Fiber in Separating Mixed Amino Acids, Polymer Materials Science and Engineering, 2005," tested the adsorption and separation performance of various mixed amino acids using ion exchange fiber resin. A commercially available strong cation exchange resin was used as a comparison. The study found that the separation effect of amino acids is highly dependent on the structure and performance of the ion exchange material. The resolution of PP-g-St-SO3H fiber in separating histidine and glutamic acid was 1.87.

[0005] "Study on the separation of lysine and histidine by ion exchange method, Applied Chemical Industry, 2008", based on 001×7 cation exchange resin, using 0.4% ammonia water as the eluent, the separation of lysine and histidine was tested on the ion exchange resin.

[0006] Existing ion exchange separation methods suffer from drawbacks such as easy corrosion of ion exchange materials, low adsorption capacity, and poor purification efficiency. The following are examples of research efforts aimed at improving ion exchangers.

[0007] "Preparation of a Novel Anion Exchange Fiber, Leather Science and Engineering, 2009" describes a process where cotton fibers are alkalized and aged, and the resulting alkali fibers are then reacted with intermediates to produce anion exchange cellulose. The effects of aging time on the alkali resistance of the alkali fibers were investigated, and the optimal aging time for the alkali fibers was determined. The effects of sodium hydroxide dosage, the mass ratio of intermediates to cellulose, reaction temperature, and reaction time on the product quality and exchange capacity were also studied.

[0008] Patent CN202211213909 discloses a method for preparing a high-capacity anion exchange chromatography medium. First, a vinyl monomer with tertiary or quaternary amines is mixed with a functional compound with amino, carboxyl, or thiol groups at the end groups. Then, an initiator is added to polymerize the mixture to obtain a prepolymerized long-chain functional ligand with reactive polytertiary or quaternary amine end groups. The obtained prepolymerized long-chain functional ligand is then chemically bonded to the surface of porous microspheres to prepare a high-capacity anion exchange chromatography medium.

[0009] Based on the existing technologies and practical experience mentioned above, the technical problem we need to solve is how to separate high-purity pharmaceutical-grade glutamic acid products in a simpler, faster, and cheaper way. Summary of the Invention

[0010] The purpose of this invention is to provide a method for separating and purifying glutamic acid, addressing the existing requirements for higher purity in glutamic acid.

[0011] The present invention is achieved through the following technical solution.

[0012] A method for separating and purifying glutamic acid includes the following steps: The crude glutamic acid was separated and purified using a weak cation exchanger to obtain pharmaceutical-grade refined glutamic acid; the weak cation exchanger was functionalized polystyrene microspheres, and the crude glutamic acid contained heteroamino acids.

[0013] Furthermore, the heteroamino acid is selected from any one of isoleucine, threonine, lysine, and arginine, or any combination of two or more of the above.

[0014] Furthermore, the weak cation exchanger is prepared according to the following process: Styrene cross-linked microspheres and chloroform were added to the reactor. Acetone, acid anhydride and ferric chloride were added sequentially under stirring. The reaction was stirred for 3-12 hours. After the reaction was completed, the reactants were washed with anhydrous ethanol and distilled water sequentially until the filtrate was found to be free of chloride ions. The filtrate was then dried under vacuum to constant weight to obtain functionalized polystyrene microspheres, which are weak cation exchangers.

[0015] Specifically, the weak cation exchanger is prepared according to the following process: Styrene cross-linked microspheres and chloroform were added to the reactor at a ratio of 1g:5ml. The mixture was stirred at 100rpm for 30min and then allowed to stand for 1h. Then, acetone, acid anhydride and ferric chloride were added sequentially under stirring at 100rpm, and the reaction was stirred for 6h. After the reaction was completed, the reactants were washed with anhydrous ethanol and distilled water sequentially until the filtrate was free of chloride ions. The filtrate was then dried under vacuum to constant weight to obtain functionalized polystyrene microspheres, which are weak cation exchangers.

[0016] Preferably, the amounts of acetone, acid anhydride, and ferric chloride added are 0.5-2 ml, 2-3 g, and 0.5-1 g per gram of styrene cross-linked microspheres, respectively.

[0017] More preferably, the acetone, acid anhydride and ferric chloride are added in amounts of 1 ml, 2.7 g and 0.8 g per gram of styrene cross-linked microspheres, respectively.

[0018] Preferably, the anhydride is a mixture of maleic anhydride and acetic anhydride.

[0019] More preferably, the anhydride is a mixture of maleic anhydride and acetic anhydride in a mass ratio of 1:3 to 3:1.

[0020] Most preferably, the anhydride is a mixture of maleic anhydride and acetic anhydride in equal mass ratio.

[0021] Furthermore, the styrene cross-linked microspheres can be commercially available microspheres, or they can be obtained by the following preparation method: acetonitrile, styrene, divinylbenzene, and dimethyl azobisisobutyrate are mixed in a ratio of 50 ml: 1 ml: 1 ml: 0.5 g, stirred and mixed thoroughly, sealed under nitrogen gas, and then placed under a constant temperature of 75°C with shaking reaction for 12 h. After separation, the microspheres are washed successively with acetone and ethanol, and finally dried to obtain styrene cross-linked microspheres.

[0022] The beneficial effects achieved by this invention mainly include, but are not limited to, the following points.

[0023] This invention uses a single elution process to separate and remove impurities in amino acids. The process is simple and inexpensive, and the purity of glutamic acid can reach over 99.9%, fully meeting the standards for pharmaceutical-grade glutamic acid, resulting in high industrial added value.

[0024] This invention employs a novel ion exchanger with high protein loading and excellent adsorption performance. During the preparation of the ion exchanger, experiments with different weak acids revealed that maleic anhydride performed best. Surprisingly, a mixture of maleic anhydride and acetic anhydride in equal proportions showed the best effect, improving adsorption efficiency by 40% and 50% respectively compared to using maleic anhydride and acetic anhydride alone.

[0025] In the preparation of styrene copolymer microspheres, chloroform can effectively disperse the microspheres, while acid anhydride and ferric chloride can form a complex. This complex has poor solubility in chloroform, but acetone can dissolve the complex. Therefore, acetone is added as a solvent aid.

[0026] In this invention, a weakly acidic cation exchange resin is most suitable for ion exchange within a pH range of 5-10 during the ion exchange purification process. The carboxylic acid functional groups of this resin ionize to a negative charge above pH 5, effectively binding cations; when the pH is below 5, the groups deprotonate, leading to a decrease in exchange capacity. Considering that the isoelectric point of glutamic acid is around 3.2, a pH of 5 for the eluent is most suitable. Attached Figure Description

[0027] Figure 1 Electron micrograph of styrene cross-linked microspheres.

[0028] Figure 2 The effect of different ion exchangers on the purification rate of glutamic acid.

[0029] Figure 3 The effect of different ion exchangers on the loss rate of glutamic acid. Detailed Implementation

[0030] To enable those skilled in the art to better understand the technical solutions in this application, the present invention will be described more clearly and completely below in conjunction with specific embodiments of this application. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this invention.

[0031] Example 1 Preparation of cation exchangers Acetonitrile, styrene, divinylbenzene, and dimethyl azobisisobutyrate were mixed in a ratio of 50 ml: 1 ml: 1 ml: 0.5 g, stirred until homogeneous, sealed under nitrogen gas, and then subjected to a constant temperature of 75°C with shaking for 12 hours. The mixture was then separated, washed successively with acetone and ethanol, and dried to obtain styrene cross-linked microspheres. Figure 1 As shown, scanning electron microscopy revealed that the microspheres had a diameter between 30 and 50 μm, were spherical, and had a smooth surface.

[0032] Styrene cross-linked microspheres and chloroform were added to the reactor at a ratio of 1g:5ml. The mixture was stirred at 100rpm for 30min and then allowed to stand for 1h. Then, acetone, maleic anhydride and ferric chloride were added sequentially under stirring at 100rpm, and the mixture was stirred for 6h. After the reaction was completed, the reactants were washed sequentially with anhydrous ethanol and distilled water until the filtrate was found to be free of chloride ions. The filtrate was then dried under vacuum to constant weight to obtain functionalized polystyrene microspheres, which are cation exchangers.

[0033] The proportions of acetone, maleic anhydride, and ferric chloride added were 1 ml, 2.7 g, and 0.8 g per gram of styrene cross-linked microspheres, respectively.

[0034] Example 2 Preparation of cation exchangers The preparation of styrene cross-linked microspheres is the same as in Example 1.

[0035] Styrene cross-linked microspheres and chloroform were added to the reactor at a ratio of 1g:5ml. The mixture was stirred at 100rpm for 30min and then allowed to stand for 1h. Then, acetone, succinic anhydride and ferric chloride were added sequentially under stirring at 100rpm, and the reaction was stirred for 6h. After the reaction was completed, the reactants were washed with anhydrous ethanol and distilled water sequentially until the filtrate was found to be free of chloride ions. The filtrate was then dried under vacuum to constant weight to obtain functionalized polystyrene microspheres, which are cation exchangers.

[0036] The proportions of acetone, succinic anhydride, and ferric chloride added were 1 ml, 2.7 g, and 0.8 g per gram of styrene cross-linked microspheres, respectively.

[0037] Example 3 Preparation of cation exchangers The preparation of styrene cross-linked microspheres is the same as in Example 1.

[0038] Styrene cross-linked microspheres and chloroform were added to the reactor at a ratio of 1g:5ml. The mixture was stirred at 100rpm for 30min and then allowed to stand for 1h. Then, acetone, acetic anhydride and ferric chloride were added sequentially under stirring at 100rpm, and the mixture was stirred for 6h. After the reaction was completed, the reactants were washed with anhydrous ethanol and distilled water sequentially until the filtrate was found to be free of chloride ions. The filtrate was then dried under vacuum to constant weight to obtain functionalized polystyrene microspheres, which are cation exchangers.

[0039] The proportions of acetone, acetic anhydride, and ferric chloride added were 1 ml, 2.7 g, and 0.8 g per gram of styrene cross-linked microspheres, respectively.

[0040] Example 4 Same as in Example 1, except that ferric chloride is replaced with aluminum chloride.

[0041] Example 5 Same as in Example 2, except that ferric chloride is replaced with aluminum chloride.

[0042] Example 6 Same as in Example 3, except that ferric chloride is replaced with aluminum chloride.

[0043] Example 7 The protein loading of the ion exchangers in Examples 1-6 was tested, with commercially available D113 cation exchange resin as a control: Resin pretreatment: Equilibrate to pH 6.0 with potassium dihydrogen phosphate buffer. Accurately weigh the pretreated wet resin (converted to dry resin based on moisture content), add bovine serum albumin solution of known concentration (2 mg / ml), and shake at 25°C and 150 rpm for 12 hours until equilibration. Centrifuge to separate the supernatant, and determine the residual protein concentration using the Bradford method.

[0044] Resin loading (mg / g) = (Co - Ce) × V / m Where (Co) is the initial protein concentration (mg / mL), (Ce) is the equilibrium concentration (mg / mL), (V) is the solution volume (mL), and (m) is the resin dry weight (g).

[0045] Table 1

[0046] As shown in Table 1, the protein loading in Examples 1-6 ranged approximately from 70-110 mg / g, with significant differences between groups. Examples 4-6 used aluminum chloride as a complexing agent to replace ferric chloride, resulting in a reduction in protein loading to a degree similar to that of commercially available D113 cation exchange resin. Comparing the effects of maleic anhydride, acetic anhydride, and succinic anhydride as carboxylating agents, maleic anhydride was superior to acetic anhydride and succinic anhydride. Therefore, Example 1 was selected for further optimization.

[0047] Example 8 Based on Example 1, the effects of different types and ratios of acid anhydrides on ion exchangers were compared.

[0048] Group 1: Example 1; Group 2: Replace the maleic anhydride in Example 1 with an equal mass of mixed acid (the mass ratio of maleic anhydride to acetic anhydride is 1:1). Group 3: Replace the maleic anhydride in Example 1 with an equal mass of mixed acid (the mass ratio of maleic anhydride and succinic anhydride is 1:1). Group 4: Replace the maleic anhydride in Example 1 with an equal mass of mixed acid (succinic anhydride and acetic anhydride in a mass ratio of 1:1).

[0049] The protein loading of groups 1-4 was measured, following the same procedure as in Example 7. The test results are shown in Table 2.

[0050] Table 2

[0051] As shown in Table 2, the mixed acid of maleic anhydride and acetic anhydride in equal proportions showed the best effect, improving the performance by approximately 40% and 50% respectively compared to using maleic anhydride and acetic anhydride alone. However, the mixed acid of maleic anhydride and succinic anhydride, and the mixed acid of succinic anhydride and acetic anhydride, did not show significant improvement. This may be because maleic anhydride, acetic anhydride, and ferric chloride can more effectively form complexes, resulting in more complete functionalization of the styrene copolymer microspheres and a correspondingly enhanced loading capacity.

[0052] Example 9 When fermenting glutamic acid using bacterial strains, amino acids such as isoleucine, threonine, lysine, and arginine are often present. Current techniques for separating and purifying glutamic acid through centrifugation, filtration, concentration, and crystallization typically result in products containing these impurities, failing to meet pharmaceutical-grade standards.

[0053] Taking the crude glutamic acid produced by our company as an example, its composition (by weight %) is as follows: glutamic acid 98.02%, isoleucine 0.65%, threonine 0.46%, lysine 0.39%, arginine 0.23%, histidine 0.16%, with the remainder being other amino acids and impurities. Prepare a 20 mg / ml amino acid solution before use.

[0054] The ion exchanger was wet-packed onto a 15cm column. 5ml of a 20mg / ml amino acid solution was loaded onto the column, and eluted with a pH 5.0 potassium dihydrogen phosphate buffer at a flow rate of 1ml / min. The content of each amino acid was determined by high-performance liquid chromatography (HPLC), and the purity and loss rate of glutamic acid were calculated. Regeneration can be achieved by eluting the resin with a high-pH buffer solution.

[0055] The ion exchangers used were those from groups 1-4 of Examples 8. Figure 2-3 As shown, the purity of glutamic acid in group 2 reached 99.97%, with a glutamic acid loss rate of 1.36%. No other amino acids were detected, indicating that the impurities in the crude glutamic acid were completely separated and removed. The purity of glutamic acid in groups 1, 3, and 4 was between 99.4% and 99.8%, significantly lower than that in group 2. The glutamic acid loss rate was between 0.9% and 1.6%. Since glutamic acid itself is not expensive and the loss rate is relatively low, the difference in glutamic acid loss rate between groups does not need to be considered too much from a cost perspective.

[0056] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for separating and purifying glutamic acid, comprising the following steps: separating and purifying a crude glutamic acid product with a weak cation exchanger to obtain a pharmaceutical-grade glutamic acid product; the weak cation exchanger is a functionalized polystyrene microsphere, and the crude glutamic acid product contains heteroamino acids.

2. The method of claim 1, wherein, The heteroamino acids are selected from isoleucine, threonine, lysine, arginine, or a combination of any two or more of the above.

3. The method of claim 1, wherein, The weak cation exchanger is prepared according to the following process: The reactor is added with styrene cross-linked microspheres and chloroform, and then acetone, acid anhydride and ferric chloride are sequentially added under stirring for 3-12 h; after the reaction is completed, the reaction product is sequentially washed and filtered with anhydrous ethanol and distilled water until the filtrate is determined to be free of chloride ions, and then vacuum dried to constant weight to obtain the functionalized polystyrene microspheres, which are the weak cation exchanger.

4. The method of claim 3, wherein, The weak cation exchanger is prepared according to the following process: The reactor is added with styrene cross-linked microspheres and chloroform in a ratio of 1 g:5 ml, stirred at 100 rpm for 30 min, and then left to stand for 1 h, and then acetone, acid anhydride and ferric chloride are sequentially added under stirring at 100 rpm for 6 h; after the reaction is completed, the reaction product is sequentially washed and filtered with anhydrous ethanol and distilled water until the filtrate is determined to be free of chloride ions, and then vacuum dried to constant weight to obtain the functionalized polystyrene microspheres, which are the weak cation exchanger.

5. The method of claim 4, wherein, The addition amount of the acetone, acid anhydride and ferric chloride is 0.5-2 ml, 2-3 g and 0.5-1 g per gram of the styrene cross-linked microspheres, respectively.

6. The method of claim 5, wherein, The addition amount of the acetone, acid anhydride and ferric chloride is 1 ml, 2.7 g and 0.8 g per gram of the styrene cross-linked microspheres, respectively.

7. The method according to any one of claims 4-6, characterized in that, The acid anhydride is a mixture of maleic anhydride and acetic anhydride.

8. The method according to any one of claims 4-6, characterized by, The acid anhydride is a mixture of maleic anhydride and acetic anhydride in a mass ratio of 1:3-3:

1.

9. The method according to any one of claims 4-6, characterized by, The acid anhydride is a mixture of maleic anhydride and acetic anhydride in an equal mass ratio.

10. The method according to any one of claims 4-6, characterized in that, The preparation method of the styrene cross-linked microspheres comprises the following steps: acetonitrile, styrene, divinylbenzene and azobisdimethylisobutyrate are mixed in a ratio of 50 ml:1 ml:1 ml:0.5 g, stirred and mixed uniformly, sealed with nitrogen, then placed in a constant temperature condition of 75°C for oscillation reaction for 12 h, separated, then sequentially washed with acetone and ethanol, and finally dried to obtain the styrene cross-linked microspheres.

Images