Preparation method and application of high-purity glutamine dipeptide
By combining enzymatic synthesis with ceramic membrane separation, electrodialysis, decolorization, crystallization, and molecular imprinting separation, the problem of low purity of propionyl-glutamyl dipeptide was solved, achieving efficient and low-cost preparation of high-purity propionyl-glutamyl dipeptide, which meets the needs of cell culture.
Patent Information
- Application Number
- CN202511015632.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-23
- Publication Date
- 2025-12-09
AI Technical Summary
Existing methods for preparing propionyl dipeptide suffer from difficulties in removing impurities, resulting in low purity that fails to meet the requirements of high-level applications such as cell culture. Furthermore, traditional methods are costly and fail to achieve efficient and low-cost purification.
The reaction solution obtained by enzymatic synthesis was subjected to ceramic membrane separation, electrodialysis desalination, decolorization, and crystallization. Combined with molecular imprinting separation and recrystallization, surface molecular imprinted chromatographic materials were prepared using propionyl dipeptide as a template for purification. Finally, high-purity propionyl dipeptide was obtained by crystallization.
This method achieves a purity of over 99.5% for propionyl dipeptide, effectively removing enzyme proteins and salt impurities, reducing production costs, and improving the safety and effectiveness of cell culture.
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Figure CN121085993A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of pharmaceutical and chemical technology, and in particular to a method for preparing and applying high-purity propionylglutamate dipeptide. Background Technology
[0002] Propionylglutamate (PGD) is commonly used in cell culture as a substitute for glutamine. Firstly, PDD has high thermal stability, allowing for heat sterilization, which is a significant advantage in cell culture. Secondly, it can replace L-glutamine, is more stable in aqueous solutions, and does not spontaneously degrade to form ammonia and pyrrolidine carboxylic acids, thus reducing cell damage. During cell culture, PDD is broken down into L-glutamine, an essential nutrient for cells. Because its breakdown process is relatively slow, cells have sufficient time to absorb and utilize these nutrients, thereby promoting cell growth and function.
[0003] The preparation methods for propylglycine dipeptide mainly include chemical synthesis and enzymatic synthesis. While chemical synthesis is a mature method, it suffers from complex steps, low yield, and high cost. Enzymatic synthesis, due to its high efficiency and environmental friendliness, has gradually become the primary method for industrial production. However, the reaction solution obtained by enzymatic synthesis contains a large amount of impurities such as enzyme proteins and inorganic salts. The presence of these impurities not only affects the purity and quality of the propylglycine dipeptide but also its effectiveness and safety in cell culture. Therefore, subsequent separation and purification processes are necessary to obtain high-purity propylglycine dipeptide products.
[0004] Existing processes for separating and purifying propylglutamic acid (PGA) mainly involve activated carbon adsorption, crystallization, and filtration. However, these methods suffer from low yields and low product purity. Furthermore, due to cost constraints, domestically produced PGA has low purity (below 99.0%), failing to meet the requirements of higher-level applications such as cell culture and standards. Traditional methods struggle to remove trace impurities from PGA; while traditional preparative chromatographic purification methods can remove these impurities, their high cost necessitates the development of a low-cost, high-efficiency PGA purification process. Summary of the Invention
[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for preparing and applying high-purity propionyl dipeptide.
[0006] The technical solution adopted by this invention to solve its technical problem is:
[0007] A method for preparing high-purity propionylglutamate dipeptide, the method comprising the following steps:
[0008] The reaction solution obtained by enzymatic synthesis was purified to obtain crude propionyl-glutamic dipeptide; then, through molecular imprinting separation and recrystallization, propionyl-glutamic dipeptide with a purity of over 99.5% was obtained.
[0009] Furthermore, the impurity removal includes: ceramic membrane separation, electrodialysis desalination, decolorization, and crystallization.
[0010] Furthermore, the ceramic membrane has a separation pore size of 50–100 nm, a controlled temperature of 35–50 °C, and inlet and outlet pressures of 0.3 MPa and 0.4 MPa, respectively.
[0011] Furthermore, high-purity pro-glutamic dipeptide was obtained by separation using a chromatographic column made from a surface molecular imprinting material of pro-glutamic dipeptide.
[0012] Furthermore, the specific steps are as follows:
[0013] (1) Enzyme-catalyzed synthesis of alanine dipeptidase: The wet cells of alanine dipeptidase produced by fermentation were mixed with pure water and 0.1% of the weight of the wet cells with a nonionic surfactant. The mixture was stirred at 35°C for 1 h to obtain the treated cells. Pure water, L-glutamine, and L-alanine methyl ester hydrochloride were added to the reaction vessel in sequence. The pH was adjusted to 8.5 with NaOH. The treated cells were added. The reaction temperature was set to 25°C and the reaction time was 30 min to obtain the alanine dipeptidase reaction solution.
[0014] (2) The content of alanine dipeptidase in the reaction solution was 50.2-58.4 mg / ml, and the salt content was 16.5-20.4 mg / ml. The solution was filtered using a 50 nm ceramic membrane filter at a controlled temperature of 40℃ and a pressure of 0.3 MPa. Water was added during the process to wash away residual enzyme protein in the reaction solution, resulting in ceramic membrane dialysis solution.
[0015] (3) Dilute the glutamic acid dipeptide dialysate with deionized water at a mass ratio of 1:1 and perform electrodialysis to remove salt. The initial conductivity was 27300-32200 μS / cm, and the conductivity at the end was 1832-2020 μS / cm. The total time for desalting was 75-89 min. There was no significant temperature rise in the solution during the desalting process.
[0016] (4) After desalination, add 0.5% activated carbon to the solution for decolorization. The final concentration of activated carbon is 23.97–24.89 g / L. pH
[0017] 6.98~7.12, decolorize for half an hour, filter by plate and frame filter to obtain decolorized solution;
[0018] (5) Concentration and crystallization to obtain crude glutathione: The decolorized solution was concentrated in a vacuum concentrator at 65°C and a vacuum of -0.09 MPa until the concentration of glutathione was 230-243 g / L. The concentrated solution was then transferred to a cryogenic crystallizer and cooled to 5°C for 5 hours. The crystallized solution was then separated by centrifugation at 4000 rpm for 20 minutes to obtain a crude crystalline solid with a purity of 98.5%-98.6% glutathione. The crystallized solid was dried at 80°C for later use.
[0019] (6) High-purity propionylglutamate dipeptide was obtained through purification: The preparation method of propionylglutamate dipeptide molecularly imprinted material is as follows: Step 1: Preparation of vinyl-modified porous silica gel: Take 5 μm Spherical porous silica gel was placed in a reaction vessel, and vinyltriethoxysilane and toluene were added. The mixture was heated under reflux for 17 hours, filtered, and vinyl-modified porous silica gel was obtained. The ratio of spherical porous silica gel: vinyltriethoxysilane: toluene (g:g:ml) is 10:6:150;
[0020] Step 2: Preparation of surface molecular imprinted materials: 5μm Vinyl-modified porous silica gel was placed in a reaction vessel, and then acetonitrile, methacrylic acid, ethylene glycol dimethacrylate, and propionyl dipeptide were added. Azobisisobutyronitrile was then added, and the mixture was stirred at 60°C for 24 hours. After filtration, 5 μm of the silica gel was obtained. A surface molecular imprinting material for propionyl dipeptide; wherein the ratio of vinyl-modified porous silica gel: acetonitrile: methacrylic acid: ethylene glycol dimethacrylate: propionyl dipeptide: azobisisobutyronitrile (AIBN) in g: ml: g: g: g: mg is 10:150:6:1.5:0.6:100;
[0021] A chromatographic column was made using the surface molecular imprinting material of glutamic dipeptide, and then the saturated solution of glutamic dipeptide prepared in step (5) after crude extraction was separated by column chromatography. The liquid corresponding to the retention time of the chromatographic peak of glutamic dipeptide was collected.
[0022] (7) Crystallization: The purified propionyl dipeptide solution is concentrated and crystallized to obtain a high-purity propionyl dipeptide product with a purity of 99.84% to 100.00%.
[0023] Furthermore, the nonionic surfactant in step (1) is Triton X-100.
[0024] Further, in step (1), the ratio of wet bacterial cells: pure water: pure water in the reactor: L-glutamine: L-alanine methyl ester hydrochloride (g: ml: ml: g: g) is 16.8~17.30: 50: 700: 80.0~85.0: 102.0~126.0.
[0025] The application of the preparation method described above in the preparation of high-purity propionyl dipeptide.
[0026] Furthermore, the purity of the high-purity propionylglutamate is 99.84% to 100.00%.
[0027] The advantages and positive effects of this invention are as follows:
[0028] 1. The purity of the crude extract of the propionylglutamate prepared by the method of the present invention can reach more than 98.5%, and after purification by the molecular imprinting material specially prepared by the present invention, the purity can reach more than 99.5%.
[0029] 2. The stock solution for preparing propionyl dipeptide by the method of the present invention is a reaction solution obtained by enzymatic synthesis, which can effectively remove enzymes.
[0030] 3. The stock solution for preparing propionyl dipeptide by the method of the present invention is a reaction solution obtained by enzymatic synthesis. A large amount of salt will be generated during the reaction. This method can effectively remove the large amount of salt generated during the reaction, reducing the initial conductivity from 29100 μS / cm to 1920 μS / cm, or from 32200 μS / cm to 2020 μS / cm, or from 27300 μS / cm to 1832 μS / cm.
[0031] 4. This invention relates to a method for preparing high-purity propionyl-glutamyl dipeptide. The method involves obtaining a reaction solution via enzymatic synthesis, followed by separation using a ceramic membrane, electrodialysis for desalting, decolorization, and crystallization to obtain crude propionyl-glutamyl dipeptide. Then, using propionyl-glutamyl dipeptide as a template, a surface molecularly imprinted chromatographic material is prepared and packed into a chromatographic column. The purified crude propionyl-glutamyl dipeptide is then purified using column chromatography to remove impurities. Finally, the purified propionyl-glutamyl dipeptide is crystallized to obtain a high-purity solid product. Attached Figure Description
[0032] Figure 1 This is the chromatogram of the propionylglutamate sample in Example 1 of this invention before purification.
[0033] Figure 2 This is the chromatogram of the propionylglutamate sample in Example 2 of this invention before purification.
[0034] Figure 3 This is the chromatogram of the propionylglutamate sample in Example 3 of this invention before purification.
[0035] Figure 4 This is the chromatogram of the purified propionylglutamate sample from Example 1 of this invention.
[0036] Figure 5 This is the chromatogram of the purified propionylglutamate sample from Example 2 of this invention.
[0037] Figure 6 This is the chromatogram of the purified propionylglutamate sample from Example 3 of this invention.
[0038] Figure 7 This is a chromatogram of the purified propionylglutamate sample from Example 4 of this invention. Detailed Implementation
[0039] The present invention will be further described below with reference to the embodiments. The following embodiments are descriptive and not limiting, and should not be used to limit the scope of protection of the present invention.
[0040] The various experimental operations involved in the specific embodiments are all conventional techniques in the field. For parts not specifically annotated in this document, those skilled in the art can refer to various commonly used reference books, scientific and technological documents or related instructions and manuals prior to the filing date of this invention to carry out the operations.
[0041] A method for preparing high-purity propionylglutamate dipeptide, the method comprising the following steps:
[0042] The reaction solution obtained by enzymatic synthesis was purified to obtain crude propionyl-glutamic dipeptide; then, through molecular imprinting separation and recrystallization, propionyl-glutamic dipeptide with a purity of over 99.5% was obtained.
[0043] Preferably, the impurity removal includes: ceramic membrane separation, electrodialysis desalination, decolorization, and crystallization.
[0044] Preferably, the ceramic membrane has a separation pore size of 50–100 nm, a controlled temperature of 35–50 °C, and inlet and outlet pressures of 0.3 MPa and 0.4 MPa, respectively.
[0045] Preferably, high-purity pro-glutamic dipeptide is obtained by separation using a chromatographic column made from a surface molecular imprinting material of pro-glutamic dipeptide.
[0046] Preferably, the specific steps are as follows:
[0047] (1) Enzyme-catalyzed synthesis of alanine dipeptidase: The wet cells of alanine dipeptidase produced by fermentation were mixed with pure water and 0.1% of the weight of the wet cells with a nonionic surfactant. The mixture was stirred at 35°C for 1 h to obtain the treated cells. Pure water, L-glutamine, and L-alanine methyl ester hydrochloride were added to the reaction vessel in sequence. The pH was adjusted to 8.5 with NaOH. The treated cells were added. The reaction temperature was set to 25°C and the reaction time was 30 min to obtain the alanine dipeptidase reaction solution.
[0048] (2) The content of alanine dipeptidase in the reaction solution was 50.2-58.4 mg / ml, and the salt content was 16.5-20.4 mg / ml. The solution was filtered using a 50 nm ceramic membrane filter at a controlled temperature of 40℃ and a pressure of 0.3 MPa. Water was added during the process to wash away residual enzyme protein in the reaction solution, resulting in ceramic membrane dialysis solution.
[0049] (3) Dilute the glutamic acid dipeptide dialysate with deionized water at a mass ratio of 1:1 and perform electrodialysis to remove salt. The initial conductivity was 27300-32200 μS / cm, and the conductivity at the end was 1832-2020 μS / cm. The total time for desalting was 75-89 min. There was no significant temperature rise in the solution during the desalting process.
[0050] (4) Add 0.5% activated carbon to the desalted solution for decolorization. The final concentration of activated carbon is 23.97-24.89 g / L, pH is 6.98-7.12, decolorize for half an hour, filter by plate and frame filter to obtain the decolorized solution.
[0051] (5) Concentration and crystallization to obtain crude glutathione: The decolorized solution was concentrated in a vacuum concentrator at 65°C and a vacuum of -0.09 MPa until the concentration of glutathione was 230-243 g / L. The concentrated solution was then transferred to a cryogenic crystallizer and cooled to 5°C for 5 hours. The crystallized solution was then separated by centrifugation at 4000 rpm for 20 minutes to obtain a crude crystalline solid with a purity of 98.5%-98.6% glutathione. The crystallized solid was dried at 80°C for later use.
[0052] (6) High-purity propionylglutamate dipeptide was obtained through purification: The preparation method of propionylglutamate dipeptide molecularly imprinted material is as follows: Step 1: Preparation of vinyl-modified porous silica gel: Take 5 μm Spherical porous silica gel was placed in a reaction vessel, and vinyltriethoxysilane and toluene were added. The mixture was heated under reflux for 17 hours, filtered, and vinyl-modified porous silica gel was obtained. The ratio of spherical porous silica gel: vinyltriethoxysilane: toluene (g:g:ml) is 10:6:150;
[0053] Step 2: Preparation of surface molecular imprinted materials: 5μm Vinyl-modified porous silica gel was placed in a reaction vessel, and then acetonitrile, methacrylic acid, ethylene glycol dimethacrylate, and propionyl dipeptide were added. Azobisisobutyronitrile was then added, and the mixture was stirred at 60°C for 24 hours. After filtration, 5 μm of the silica gel was obtained. A surface molecular imprinting material for propionyl dipeptide; wherein the ratio of vinyl-modified porous silica gel: acetonitrile: methacrylic acid: ethylene glycol dimethacrylate: propionyl dipeptide: azobisisobutyronitrile (AIBN) in g: ml: g: g: g: mg is 10:150:6:1.5:0.6:100;
[0054] A chromatographic column was made using the surface molecular imprinting material of glutamic dipeptide, and then the saturated solution of glutamic dipeptide prepared in step (5) after crude extraction was separated by column chromatography. The liquid corresponding to the retention time of the chromatographic peak of glutamic dipeptide was collected.
[0055] (7) Crystallization: The purified propionyl dipeptide solution is concentrated and crystallized to obtain a high-purity propionyl dipeptide product with a purity of 99.84% to 100.00%.
[0056] Preferably, the nonionic surfactant in step (1) is Triton X-100.
[0057] Preferably, in step (1), the ratio of wet bacterial cells: pure water: pure water in the reactor: L-glutamine: L-alanine methyl ester hydrochloride (g): ml: ml: g: g is 16.8~17.30: 50: 700: 80.0~85.0: 102.0~126.0.
[0058] The application of the preparation method described above in the preparation of high-purity propionyl dipeptide.
[0059] Preferably, the purity of the high-purity propionyl dipeptide is 99.84% to 100.00%.
[0060] Specifically, the relevant preparation and testing methods are as follows:
[0061] A method for preparing high-purity propionyl-glutamic dipeptide involves separating the reaction solution obtained by enzymatic synthesis through a ceramic membrane, followed by electrodialysis desalination, decolorization, and crystallization to obtain crude propionyl-glutamic dipeptide; then, through molecular imprinting separation and recrystallization, high-purity propionyl-glutamic dipeptide with a purity of over 99.5% is obtained.
[0062] The analytical methods used for the content of propionylglutathione in the following examples are as follows:
[0063] Column A: Packed with amino-bonded silica gel (5 μm, 4.6 × 250 mm);
[0064] Column B: Surface molecularly imprinted column for propionylglutamic dipeptide (5 μm, 4.6 × 250 mm);
[0065] Mobile phase: 0.05 mol / L potassium dihydrogen phosphate buffer (pH adjusted to 4.0 with phosphoric acid) - acetonitrile (35:65, volume ratio) as the mobile phase;
[0066] Detection wavelength: 215nm;
[0067] Flow rate: 0.7 ml / min;
[0068] Column temperature: 30℃;
[0069] Injection volume: 20 μl.
[0070] Example 1
[0071] A method for preparing high-purity propionylglutamate includes the following steps:
[0072] (1) Enzyme-catalyzed synthesis of alanine dipeptidase: 16.8 g of wet cells of alanine dipeptidase produced by fermentation were mixed with 50 ml of pure water and 0.1% of the weight of the wet cells of nonionic surfactant Triton X-100. The mixture was stirred at 35 °C for 1 h to obtain the treated cells. 700 ml of pure water, 80.0 g of L-glutamine, and 120.0 g of L-alanine methyl ester hydrochloride were added to the reaction vessel in sequence. The pH was adjusted to 8.5 with NaOH. The treated cells were then added. The reaction temperature was set to 25 °C and the reaction time was 30 min to obtain the alanine dipeptidase reaction solution.
[0073] (2) The alanine dipeptidase reaction solution contained 55.6 mg / ml of alanine dipeptide and 19.7 mg / ml of salt. It was filtered using a 50 nm ceramic membrane filter at a controlled temperature of 40 °C and a pressure of 0.3 MPa. Water was added during the process to wash away residual enzyme protein in the reaction solution, and ceramic membrane dialysate was obtained.
[0074] (3) The dialysis solution of propionyl dipeptide was diluted with deionized water at a mass ratio of 1:1 and desalted by electrodialysis. The initial conductivity was 29100 μS / cm and the final conductivity was 1920 μS / cm. The total desalting time was 80 min. There was no significant temperature rise in the solution during the desalting process.
[0075] (4) Add 0.5% activated carbon (concentration of 24.68 g / L) to the desalted solution for decolorization, pH 7.12, decolorize for half an hour, filter by plate and frame filter to obtain decolorized solution.
[0076] (5) Concentration and crystallization to obtain crude glutathione: The decolorized solution was concentrated in a vacuum concentrator at 65℃ and a vacuum of -0.09 MPa until the glutathione concentration reached 230 g / L. The concentrated solution was then transferred to a cryogenic crystallizer, cooled to 5℃, and crystallized for 5 hours. The crystallized solution was separated by centrifugation at 4000 rpm for 20 min to obtain a crystalline solid with a glutathione purity of 98.6% (area normalization composition percentage: Table 1, spectrum:). Figure 1 The crystallized solid is dried at 80°C for later use.
[0077] (6) High-purity propionyl dipeptide was obtained through purification: The preparation method of propionyl dipeptide molecularly imprinted material is as follows: Step 1: Preparation of vinyl-modified porous silica gel: Take 10g 5μm Spherical porous silica gel was placed in a 250ml reaction vessel, and then 6g of vinyltriethoxysilane and 150ml of toluene were added. The mixture was heated under reflux for 17 hours and filtered to obtain vinyl-modified porous silica gel. Step 2: Preparation of surface molecular imprinted materials: 10g of 5μm Vinyl-modified porous silica gel was placed in a 250 ml reactor, and then 150 ml of acetonitrile, 6 g of methacrylic acid, 1.5 g of ethylene glycol dimethacrylate, and 0.6 g of propionyl dipeptide were added. 100 mg of azobisisobutyronitrile was added, and the mixture was stirred at 60 °C for 24 hours. The mixture was then filtered to obtain 5 μm porous silica gel. Surface molecular imprinting material of glutamic dipeptide; a chromatographic column was made using the surface molecular imprinting material of glutamic dipeptide, and then the glutamic dipeptide obtained in step (5) after crude extraction was separated by column chromatography, and the liquid corresponding to the retention time of the chromatographic peak of glutamic dipeptide was collected.
[0078] (7) Crystallization: The purified propionyl-glutamyl dipeptide solution was concentrated and crystallized to obtain a high-purity solid propionyl-glutamyl dipeptide product. Its purity was measured to be 99.84% (area normalization component percentage: Table 4, spectrum:). Figure 4 The surface molecular imprinting separation and crystallization of glutathione increased the purity of glutathione from 98.6% to 99.84%, and reduced the number of impurities from 6 to 2. The removal effect was particularly good for impurities (relative retention time of 1.08) that were close to the peak of glutathione on the reversed-phase chromatography column.
[0079] Example 2
[0080] A method for preparing high-purity propionylglutamate includes the following steps:
[0081] (1) Enzyme-catalyzed synthesis of alanine dipeptidase: 17.0 g of wet cells of alanine dipeptidase produced by fermentation were mixed with 50 ml of pure water and 0.1% of the weight of the wet cells of nonionic surfactant Triton X-100. The mixture was stirred at 35 °C for 1 h to obtain the treated cells. 700 ml of pure water, 85.0 g of L-glutamine, and 126.0 g of L-alanine methyl ester hydrochloride were added to the reaction vessel in sequence. The pH was adjusted to 8.5 with NaOH. The treated cells were then added. The reaction temperature was set to 25 °C and the reaction time to 30 min to obtain the alanine dipeptidase reaction solution.
[0082] (2) The alanine dipeptidase reaction solution contained 58.4 mg / ml of alanine dipeptide and 20.4 mg / ml of salt. It was filtered using a 50 nm ceramic membrane filter at a controlled temperature of 40 °C and a pressure of 0.3 MPa. Water was added during the process to wash away residual enzyme protein in the reaction solution, and ceramic membrane dialysate was obtained.
[0083] (3) The dialysis solution of propionyl dipeptide was diluted with deionized water at a mass ratio of 1:1 and desalted by electrodialysis. The initial conductivity was 32200 μS / cm and the final conductivity was 2020 μS / cm. The total desalting time was 89 min. There was no significant temperature rise in the solution during the desalting process.
[0084] (4) Add 0.5% activated carbon (concentration of 24.89 g / L) to the desalted solution for decolorization, pH 7.05, decolorize for half an hour, filter by plate and frame filter to obtain decolorized solution.
[0085] (5) Concentration and crystallization to obtain crude glutathione: The decolorized solution was concentrated in a vacuum concentrator at 65℃ and a vacuum of -0.09 MPa until the glutathione concentration reached 238 g / L. The concentrated solution was then transferred to a cryogenic crystallizer, cooled to 5℃, and crystallized for 5 hours. The crystallized solution was then separated by centrifugation at 4000 rpm for 20 min to obtain a crystalline solid with a glutathione purity of 98.5% (area normalization composition percentage: Table 2, spectrum:). Figure 2 The crystallized solid is dried at 80°C for later use.
[0086] (6) Purification to obtain high-purity propionyl dipeptide: 5 μm of propionyl dipeptide was prepared using the method in step (6) of Example 1. The surface molecular imprinting material of glutamic dipeptide was used to make a chromatographic column, and then the glutamic dipeptide obtained in step (5) after crude extraction was separated by column chromatography. The liquid corresponding to the retention time of the chromatographic peak of glutamic dipeptide was collected.
[0087] (7) Crystallization: The purified propionyl-glutamyl dipeptide solution was concentrated and crystallized to obtain a high-purity solid propionyl-glutamyl dipeptide product. Its purity was measured to be 100.00% (area normalization component percentage: Table 5, spectrum:). Figure 5 The surface molecular imprinting separation and crystallization of glutathione increased the purity of glutathione from 98.5% to 100.00%, and reduced the number of impurities from 7 to 0. In particular, the removal effect was better on impurities (relative retention time of 1.08) that were close to the peak of glutathione on the reversed-phase chromatography column.
[0088] Example 3
[0089] A method for preparing high-purity propionylglutamate includes the following steps:
[0090] (1) Enzyme-catalyzed synthesis of alanine dipeptidase: 17.30 g of wet cells of alanine dipeptidase produced by fermentation were mixed with 50 ml of pure water and 0.1% of the weight of the wet cells of nonionic surfactant Triton X-100. The mixture was stirred at 35 °C for 1 h to obtain the treated cells. 700 ml of pure water, 85.0 g of L-glutamine, and 102.0 g of L-alanine methyl ester hydrochloride were added to the reaction vessel in sequence. The pH was adjusted to 8.5 with NaOH. The treated cells were then added. The reaction temperature was set to 25 °C and the reaction time was 30 min to obtain the alanine dipeptidase reaction solution.
[0091] (2) The alanine dipeptidase reaction solution contained 50.2 mg / ml of alanine dipeptide and 16.5 mg / ml of salt. It was filtered using a 50 nm ceramic membrane filter at a controlled temperature of 40 °C and a pressure of 0.3 MPa. Water was added during the process to wash away residual enzyme protein in the reaction solution, and ceramic membrane dialysate was obtained.
[0092] (3) The dialysis solution of propionyl dipeptide was diluted with deionized water at a mass ratio of 1:1 and desalted by electrodialysis. The initial conductivity was 27300 μS / cm and the final conductivity was 1832 μS / cm. The total desalting time was 75 min. There was no significant temperature rise in the solution during the desalting process.
[0093] (4) Add 0.5% activated carbon (concentration of 23.97 g / L) to the desalted solution for decolorization, pH 6.98, decolorize for half an hour, filter by plate and frame filter to obtain decolorized solution.
[0094] (5) Concentration and crystallization to obtain crude glutathione: The decolorized solution was concentrated in a vacuum concentrator at 65℃ and a vacuum of -0.09 MPa until the glutathione concentration reached 243 g / L. The concentrated solution was then transferred to a cryogenic crystallizer, cooled to 5℃, and crystallized for 5 hours. The crystallized solution was separated by centrifugation at 4000 rpm for 20 min to obtain a crystalline solid with a glutathione purity of 98.5% (area normalization composition percentage: Table 3, spectrum:). Figure 3 The crystallized solid is dried at 80°C for later use.
[0095] (6) Purification to obtain high-purity propionyl dipeptide: 5 μm of propionyl dipeptide was prepared using the method in step (6) of Example 1. The surface molecular imprinting material of glutamic dipeptide was used to make a chromatographic column, and then the glutamic dipeptide obtained in step (5) after crude extraction was separated by column chromatography. The liquid corresponding to the retention time of the chromatographic peak of glutamic dipeptide was collected.
[0096] (7) Crystallization: The purified propionyl-glutamyl dipeptide solution was concentrated and crystallized to obtain a high-purity solid propionyl-glutamyl dipeptide product. Its purity was measured to be 99.89% (area normalization component percentage: Table 6, spectrum:). Figure 6 The surface molecular imprinting separation and crystallization of glutathione increased the purity of glutathione from 98.54% to 99.89%, and reduced the number of impurities from 7 to 2. The removal effect was particularly good for impurities (relative retention time of 1.08) that were close to the peak of glutathione on the reversed-phase chromatography column.
[0097] Example 4
[0098] A method for preparing high-purity propionylglutamate includes the following steps:
[0099] Steps (1)-(5) are the same as in Example 1.
[0100] (6) Purification to obtain high-purity propionyl dipeptide: The crude propionyl dipeptide was dissolved in 0.05 mol / L potassium dihydrogen phosphate buffer (pH adjusted to 4.0 with phosphoric acid)-acetonitrile (35:65, volume ratio) solution, passed through a reverse-phase C18 preparative column (30 x 150 mm, 5 μm, commercially available), and the liquid corresponding to the retention time of the propionyl dipeptide chromatographic peak was collected.
[0101] (7) Crystallization: The purified propionyl-glutamyl dipeptide solution was concentrated and crystallized to obtain a high-purity solid propionyl-glutamyl dipeptide product. Its purity was measured to be 99.14% (area normalization component percentage: Table 7, spectrum:). Figure 7 The preparation and crystallization of glutathione using a reversed-phase C18 column increased the purity of glutathione from 98.6% to 99.14% and reduced the number of impurities from 6 to 2. However, the impurities that were adjacent to the peak of glutathione on the reversed-phase column (with relative retention times of 1.08 and 1.17) did not show significant removal effects.
[0102] Table 1. Proportion of components in the normalized chromatogram area of the pyrogallol sample before purification in Example 1.
[0103] peak Retention time (min) %area 1 9.260 0.07 2 14.319 98.60 3 15.501 0.55 4 16.706 0.31 5 18.113 0.17 6 19.154 0.05 7 24.336 0.25
[0104] Table 2. Component percentages in the chromatogram area normalization method of the propylglutamate sample before purification in Example 2.
[0105]
[0106]
[0107] Table 3 shows the component proportions of the purified chromatogram area of the glutathione sample in Example 3, determined by the normalization method.
[0108] peak Retention time (min) %area 1 14.188 0.15 2 14.871 0.05 3 16.728 98.54 4 18.335 0.83 5 20.026 0.25 6 21.932 0.05 7 27.025 0.10 8 29.905 0.03
[0109] Table 4 shows the component proportions of the purified chromatogram area of the glutathione sample in Example 1, determined by the normalization method.
[0110] peak Retention time (min) %area 1 9.518 0.10 2 14.299 99.84 3 23.010 0.06
[0111] Table 5 shows the component percentages of the purified chromatogram area of the glutathione sample in Example 2, determined by the normalization method.
[0112] peak Retention time (min) %area 1 14.312 100.00
[0113] Table 6 shows the component proportions of the purified chromatogram area of the glutathione sample in Example 3, determined by the normalization method.
[0114] peak Retention time (min) %area 1 9.518 0.07 2 14.299 99.89 3 23.010 0.04
[0115] Table 7 shows the component proportions of the purified chromatogram area of the glutathione sample in Example 4, determined by the normalization method.
[0116] peak Retention time (min) %area 1 14.319 99.14 2 15.501 0.76 3 16.706 0.10
[0117] On a reversed-phase chromatography column, impurities eluting immediately adjacent to the propylglycine peak (with a relative retention time of 1.08) are relatively difficult to remove, indicating they possess similar physicochemical properties. General purification methods (such as recrystallization and activated carbon adsorption) are insufficient to separate them without significant loss of propylglycine. While traditional reversed-phase preparative chromatography can achieve separation, its low resolution between propylglycine and impurities results in a small yield of high-purity sample per preparation, leading to high costs and limiting the industrial production of high-purity propylglycine (greater than 99.5%). Using surface molecularly imprinted materials for propylglycine can leverage their specific selective adsorption capacity for propylglycine, increasing the resolution between propylglycine and impurities, improving the sample yield per preparation, and significantly reducing the production cost of high-purity propylglycine. Chromatographic columns made using surface molecularly imprinted adsorption materials for propylglutamic dipeptide can easily increase the purity of propylglutamic dipeptide from 98.5% to over 99.5%.
[0118] As can be seen from the examples, traditional extraction methods can achieve a purity of over 98.5% for glutamic-peptide, demonstrating good application value and potential applications. Combining molecular imprinting separation can further enhance the purity of glutamic-peptide to over 99.5%.
[0119] Although embodiments of the invention have been disclosed for illustrative purposes, those skilled in the art will understand that various substitutions, variations, and modifications are possible without departing from the spirit and scope of the invention and the appended claims. Therefore, the scope of the invention is not limited to the contents disclosed in the embodiments.
Claims
1. A method for preparing high purity propylvaline dipeptide, characterized by: The method includes the following steps: The reaction solution obtained by enzymatic synthesis was purified to obtain crude propionyl-glutamic dipeptide; then, through molecular imprinting separation and recrystallization, propionyl-glutamic dipeptide with a purity of over 99.5% was obtained.
2. The method for removing impurities from crude propionylglutamate according to claim 1, characterized in that: The impurity removal process includes: ceramic membrane separation, electrodialysis desalination, decolorization, and crystallization.
3. The method for removing impurities from crude propionylglutamate according to claim 2, characterized in that: The ceramic membrane has a separation pore size of 50–100 nm, a controlled temperature of 35–50 °C, and inlet and outlet pressures of 0.3 MPa and 0.4 MPa, respectively.
4. The method for preparing high-purity propionyl-glutamyl dipeptide according to claim 1, characterized in that: High-purity propionyl dipeptide was obtained by separation using a chromatographic column made from a surface molecular imprinting material of propionyl dipeptide.
5. The method for preparing high-purity propionylglutamate according to any one of claims 1 to 4, characterized in that: The specific steps are as follows: (1) Enzyme-catalyzed synthesis of alanine dipeptidase: The wet cells of alanine dipeptidase produced by fermentation were mixed with pure water and 0.1% of the weight of the wet cells with a nonionic surfactant. The mixture was stirred at 35°C for 1 h to obtain the treated cells. Pure water, L-glutamine, and L-alanine methyl ester hydrochloride were added to the reaction vessel in sequence. The pH was adjusted to 8.5 with NaOH. The treated cells were added. The reaction temperature was set to 25°C and the reaction time was 30 min to obtain the alanine dipeptidase reaction solution. (2) The content of alanine dipeptidase in the reaction solution was 50.2-58.4 mg / ml, and the salt content was 16.5-20.4 mg / ml. The solution was filtered using a 50 nm ceramic membrane filter at a controlled temperature of 40℃ and a pressure of 0.3 MPa. Water was added during the process to wash away residual enzyme protein in the reaction solution, resulting in ceramic membrane dialysis solution. (3) Dilute the glutamic acid dipeptide dialysate with deionized water at a mass ratio of 1:1 and perform electrodialysis to remove salt. The initial conductivity was 27300-32200 μS / cm, and the conductivity at the end was 1832-2020 μS / cm. The total time for desalting was 75-89 min. There was no significant temperature rise in the solution during the desalting process. (4) Add 0.5% activated carbon to the desalted solution for decolorization. The final concentration of activated carbon is 23.97-24.89 g / L, pH is 6.98-7.12, decolorize for half an hour, filter by plate and frame filter to obtain the decolorized solution. (5) Concentration and crystallization to obtain crude glutathione: The decolorized solution was concentrated in a vacuum concentrator at 65°C and a vacuum of -0.09 MPa until the concentration of glutathione was 230-243 g / L. The concentrated solution was then transferred to a cryogenic crystallizer and cooled to 5°C for 5 hours. The crystallized solution was then separated by centrifugation at 4000 rpm for 20 minutes to obtain a crude crystalline solid with a purity of 98.5%-98.6% glutathione. The crystallized solid was dried at 80°C for later use. (6) High-purity propionylglutamate dipeptide was obtained through purification: The preparation method of propionylglutamate dipeptide molecularly imprinted material is as follows: Step 1: Preparation of vinyl-modified porous silica gel: Take 5 μm Spherical porous silica gel was placed in a reaction vessel, and vinyltriethoxysilane and toluene were added. The mixture was heated under reflux for 17 hours, filtered, and vinyl-modified porous silica gel was obtained. The ratio of spherical porous silica gel: vinyltriethoxysilane: toluene (g:g:ml) is 10:6:150; Step 2: Preparation of surface molecular imprinted materials: 5μm Vinyl-modified porous silica gel was placed in a reaction vessel, and then acetonitrile, methacrylic acid, ethylene glycol dimethacrylate, and propionyl dipeptide were added. Azobisisobutyronitrile was then added, and the mixture was stirred at 60°C for 24 hours. After filtration, 5 μm of the silica gel was obtained. A surface molecular imprinting material for propionyl dipeptide; wherein the ratio of vinyl-modified porous silica gel: acetonitrile: methacrylic acid: ethylene glycol dimethacrylate: propionyl dipeptide: azobisisobutyronitrile (AIBN) in g: ml: g: g: g: mg is 10:150:6:1.5:0.6:100; A chromatographic column was made using the surface molecular imprinting material of glutamic dipeptide, and then the saturated solution of glutamic dipeptide prepared in step (5) after crude extraction was separated by column chromatography. The liquid corresponding to the retention time of the chromatographic peak of glutamic dipeptide was collected. (7) Crystallization: The purified propionyl dipeptide solution is concentrated and crystallized to obtain a high-purity propionyl dipeptide product with a purity of 99.84% to 100.00%.
6. The method for preparing high-purity propionyl-glutamyl dipeptide according to claim 5, characterized in that: The nonionic surfactant used in step (1) is Triton X-100.
7. The method for preparing high-purity propionyl-glutamyl dipeptide according to claim 5, characterized in that: In step (1), the ratio of wet bacterial cells: pure water: pure water in the reactor: L-glutamine: L-alanine methyl ester hydrochloride (g): ml: ml: g: g is 16.8~17.30: 50: 700: 80.0~85.0: 102.0~126.
0.
8. The application of the preparation method according to any one of claims 1 to 7 in the preparation of high-purity propionyl dipeptide.
9. The application according to claim 8, characterized in that: The purity of the high-purity propionyl dipeptide is 99.84% to 100.00%.