Separation and purification preparation process of gamma-aminobutyric acid
By reacting glutamic acid solution with whole cells of glutamic acid decarboxylase, combined with calcium salt solution and strong acid cationic resin column treatment, the yield and purity of γ-aminobutyric acid were successfully improved, and the preparation of γ- in the prior art was solved. The low efficiency and high cost of aminobutyric acid have achieved high yield preparation.
Patent Information
- Application Number
- CN202411350014.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-26
- Publication Date
- 2025-01-03
AI Technical Summary
In the prior art, the preparation method of γ-aminobutyric acid has problems such as long production cycle, low yield, difficulty in separation and extraction, high cost and long reaction time, which is difficult to meet the needs of industrial applications.
The reaction of glutamic acid solution with whole cells containing glutamic acid decarboxylase is performed, and the pH and temperature is adjusted. The filtration, extraction, strong acid cationic resin column elution and concentration can be achieved. Prepared with high yield, high purity and high yield.
The yield and purity of γ-aminobutyric acid are significantly improved, the preparation process is simplified, the cost is reduced, and the problem of preparing γ-aminobutyric acid in the prior art is solved.
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Figure BDA0005062083090000091
Abstract
Description
Technical Field
[0001] The invention relates to the field of biotechnology, and in particular to a separation, purification and preparation process of gamma-aminobutyric acid. Background Art
[0002] γ-Aminobutyric acid (also known as 4-aminobutyric acid, γ-aminobutyric acid, abbreviated as GABA) is a naturally occurring non-protein amino acid and an important inhibitory neurotransmitter in the central nervous system. It is produced by L-glutamate (L-glutamic acid, L-Glu) catalyzed irreversibly by L-glutamate decarboxylase (glutamic acid decarboxylase, GAD). It has multiple physiological functions, including improving sleep and treating depression, enhancing immunity, lowering blood pressure, improving visual cortex function, resisting obesity, reducing anxiety and menopausal syndrome, and treating epilepsy. However, the content of GABA produced endogenously in the human body is low, and it often needs to be supplemented exogenously to maintain human health.
[0003] The preparation methods of γ-aminobutyric acid mainly include chemical synthesis, plant enrichment, microbial fermentation and biotransformation. Chemical synthesis mainly uses γ-butyrolactone, γ-halogenated butyronitrile, 2-bromopropionic acid and glutaric anhydride as substrates to synthesize GABA. It has a fast reaction speed and high yield, but it has harsh conditions, high energy consumption, high cost, low yield and poor safety, and cannot be used in the fields of food and medicine. Enriching GABA from plants by low temperature or high salt stress treatment is simple, safe and environmentally friendly, but it has the limitations of low enrichment yield and high cost, which cannot meet market demand. The microbial fermentation method has a long production cycle, low production yield and difficult subsequent separation and extraction, which limits its industrial application. The biotransformation method, i.e., the whole-cell catalytic conversion method, is increasingly favored due to its advantages of simple operation, mild conditions, high raw material utilization, high conversion rate and low separation and purification cost. Although the prior art discloses the preparation of γ-aminobutyric acid by bioconversion, the reaction time is long; and coenzymes are required in the reaction process, which is costly; in addition, glutamic acid and γ-aminobutyric acid are not easy to separate. Therefore, it is urgent to develop a new method for extracting γ-aminobutyric acid with a simple production process, low cost and high yield. Summary of the invention
[0004] The technical problem to be solved by the present invention is: in view of the deficiencies of the prior art, a separation and purification process for preparing γ-aminobutyric acid is provided, by which a γ-aminobutyric acid product with high yield, high purity and high yield can be obtained.
[0005] In order to solve the above technical problems, the technical solution of the present invention is:
[0006] A separation, purification and preparation process of γ-aminobutyric acid comprises the following steps:
[0007] (1) preparing a glutamate solution, adding whole cells containing glutamate decarboxylase, and then adding a calcium salt solution, adjusting the pH to acidic, controlling the temperature and reaction time of the reaction system, and obtaining a conversion solution containing γ-aminobutyric acid for later use;
[0008] (2) taking the conversion solution containing γ-aminobutyric acid described in step (1), filtering and collecting the filtrate for later use;
[0009] (3) taking the filtrate in step (2), adding an extractant, controlling the stirring speed and time, standing to separate the layers, and collecting the lower organic phase for later use;
[0010] (4) taking the organic phase described in step (3), adjusting the pH to acidic, entering into a strong acid cationic resin column, using a decomposing agent for elution, and collecting the decomposing solution for later use;
[0011] (5) taking the analytical solution described in step (4), concentrating the first concentrated solution collected after the first concentration, and then concentrating the second concentrated solution for later use;
[0012] (6) taking the second concentrated solution in step (5), adding anhydrous ethanol, stirring, cooling, and filtering to collect the crude γ-aminobutyric acid;
[0013] (7) Take the crude γ-aminobutyric acid described in step (6), add purified water, heat and stir until dissolved, then add a decolorizer, continue stirring and filtering, cool the collected decolorized liquid, filter, wash and dry to obtain the finished γ-aminobutyric acid.
[0014] As an improved technical solution, the concentration of the glutamate solution in step (1) is 500-800 g / L, the amount of whole cells containing glutamate decarboxylase added is such that the OD600 value of the reaction system is 10-50 OD / ml, the pH is adjusted to 4-5, the temperature of the reaction system is controlled to be 20-40° C., and the reaction time is 4-6 h; the calcium salt solution is a calcium chloride solution, and the concentration of calcium ions in the calcium salt solution is 6-12 mM.
[0015] As an improved technical solution, a ceramic membrane with a pore size of 50-200 nm is used for filtration in step (2).
[0016] As an improved technical solution, the extractant in step (3) is ethyl acetate, ethyl ether, petroleum ether or n-hexane, preferably n-hexane; the stirring speed is controlled to 100-300 rpm, and the stirring is performed for 0.5-1h.
[0017] As an improved technical solution, the organic phase in step (4) is adjusted to a pH of 1-3 and then enters a strong acid cationic resin column at a flow rate of 1-3 BV / h.
[0018] As an improved technical solution, the analytical agent in step (4) is a 5-15wt% potassium chloride solution.
[0019] As an improved technical solution, in step (4), before the organic phase enters the strong acid cationic resin column, the strong acid cationic resin column needs to be eluted with 20-30wt% sodium chloride solution and water until the conductivity of the effluent is less than 10us / cm, thereby obtaining a transformed cationic resin column.
[0020] As an improved technical solution, in step (5), a nanofiltration membrane with a molecular weight cutoff of 100-200Da is used for the first concentration, and thermal concentration is used for the second concentration. The solid content of the second concentrated liquid is 70-80wt%.
[0021] As an improved technical solution, the anhydrous ethanol in step (6) is added in an amount of 1-4 times the volume of the second concentrated solution, and the temperature is lowered to 20-25°C.
[0022] As an improved technical solution, in step (7), purified water is added and heated to 60-80°C; the decolorizing solution is cooled to 10-15°C at a rate of 3-5°C / h.
[0023] After adopting the above technical solution, the beneficial effects of the present invention are:
[0024] The present invention uses glutamic acid solution and whole cells containing glutamic acid decarboxylase as reaction raw materials, adds calcium salt solution, adjusts pH, controls reaction temperature and time, and reacts to obtain a conversion liquid containing γ-aminobutyric acid, which is filtered through a ceramic membrane, and an extractant is added to the collected filtrate, and after stirring and standing for stratification, the collected lower organic phase is adjusted to pH to acidic, and then enters the transformed strong acid cation resin column, and then an analytical agent is used for analysis, and the collected analytical solution is concentrated once and concentrated twice, and then anhydrous ethanol is added, stirred, cooled, and filtered, and purified water is added to the collected γ-aminobutyric acid crude product, heated, stirred until dissolved, activated carbon is added, and continued stirring and filtering are performed, and the collected decolorized liquid is cooled, filtered, washed, and dried according to a gradient cooling method to obtain a γ-aminobutyric acid product. The above-mentioned process method greatly improves the output, yield and purity of γ-aminobutyric acid. DETAILED DESCRIPTION
[0025] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.
[0026] Example 1
[0027] A separation, purification and preparation process of γ-aminobutyric acid comprises the following steps:
[0028] (1) 40 L of a glutamic acid solution with a concentration of 500 g / L was prepared, whole cells containing glutamic acid decarboxylase were added (the amount added was such that the OD600 value of the reaction system was 10 OD / ml), and then a calcium chloride solution with a calcium ion concentration of 6 mM was added, the pH was adjusted to 4, the temperature of the reaction system was controlled to be 20° C., and the reaction was terminated after reacting for 4 hours to obtain 42 L of a conversion solution containing γ-aminobutyric acid for standby use;
[0029] (2) taking 42 L of the conversion solution containing γ-aminobutyric acid in step (1), filtering it through a ceramic membrane with a pore size of 50 nm, and collecting 57 L of the filtrate for later use;
[0030] (3) Take 57 L of the filtrate in step (2), add 1 times the volume of the filtrate with an extractant (ethyl acetate), control the stirring speed to 100 rpm, stir for 0.5 h, let stand for 30 min, collect 29 L of the lower organic phase for later use;
[0031] (4) Take 29L of the organic phase in step (3), adjust the pH to 1 with hydrochloric acid solution, and then enter a 10L strong acid cationic resin column at a flow rate of 1BV / h (it is necessary to use 20wt% sodium chloride solution and water to elute the strong acid cationic resin column successively until the conductivity of the effluent is less than 10us / cm, and the transformed cationic resin column re-adsorbs γ-aminobutyric acid in the organic phase), then use 20L, 5wt% potassium chloride solution as an analytical agent for elution, and collect 21L of analytical solution for use;
[0032] (5) 21 L of the analytical solution in step (4) was concentrated once using a nanofiltration membrane with a molecular weight cutoff of 100 Da, and the collected 18.5 L of the first concentrated solution was subjected to a second thermal concentration at 50° C. and a vacuum degree of less than -0.090 MPa, and 5.6 L of the second concentrated solution with a solid content of 70 wt % was collected for later use;
[0033] (6) Take 5.6 L of the second concentrated solution in step (5), add anhydrous ethanol in an amount of 1 times the volume of the second concentrated solution, stir, cool to 20° C., filter, and collect 19300 g of crude γ-aminobutyric acid;
[0034] (7) Take 19300 g of the crude γ-aminobutyric acid in step (6), add 19.3 L of purified water, heat to 60 ° C, stir until dissolved, then add 386 g of decolorizer (activated carbon), continue stirring for 20 min, filter, and cool the collected decolorized liquid to 10 ° C at a rate of 3 ° C / h, filter, wash, and dry to obtain the finished γ-aminobutyric acid.
[0035] Example 2
[0036] A separation, purification and preparation process of γ-aminobutyric acid comprises the following steps:
[0037] (1) preparing 40 L of a glutamic acid solution having a concentration of 580 g / L, adding whole cells containing glutamic acid decarboxylase (the amount added is such that the OD600 value of the reaction system is 20 OD / ml), then adding a calcium chloride solution having a calcium ion concentration of 8 mM, adjusting the pH to 4.2, controlling the temperature of the reaction system to 25° C., reacting for 4.5 h, and ending the reaction to obtain 43 L of a conversion solution containing γ-aminobutyric acid for standby use;
[0038] (2) taking 43 L of the conversion solution containing γ-aminobutyric acid in step (1), filtering it through a ceramic membrane with a pore size of 80 nm, and collecting 58 L of the filtrate for later use;
[0039] (3) Take 58 L of the filtrate in step (2), add 1.5 times the volume of the filtrate with an extractant (ether), control the stirring speed to 150 rpm, stir for 0.6 h, let stand for 32 min, and collect 44 L of the lower organic phase for later use;
[0040] (4) Take 44 L of the organic phase in step (3), adjust the pH to 1.5 with hydrochloric acid solution, and then enter a 20 L strong acid cationic resin column at a flow rate of 1.5 BV / h (it is necessary to use 22 wt % sodium chloride solution and water to elute the strong acid cationic resin column successively until the conductivity of the effluent is less than 10 us / cm, and the transformed cationic resin column re-adsorbs γ-aminobutyric acid in the organic phase), then use 40 L, 8 wt % potassium chloride solution as an analytical agent for elution, and collect 41 L of analytical solution for use;
[0041] (5) 41 L of the analytical solution in step (4) was concentrated once using a nanofiltration membrane with a molecular weight cutoff of 120 Da, and the collected 19.0 L of the first concentrated solution was subjected to a second thermal concentration at 55° C. and a vacuum degree of less than -0.090 MPa, and 5.7 L of the second concentrated solution with a solid content of 72 wt % was collected for later use;
[0042] (6) Take 5.7 L of the second concentrated solution in step (5), add anhydrous ethanol in an amount twice the volume of the second concentrated solution, stir, cool to 21° C., filter, and collect 19400 g of crude γ-aminobutyric acid;
[0043] (7) Take 19400 g of the crude γ-aminobutyric acid in step (6), add 19.4 L of purified water, heat to 65 ° C, stir until dissolved, then add 388 g of decolorizer (activated carbon), continue stirring for 25 min, filter, and cool the collected decolorized liquid to 11 ° C at a rate of 3.5 ° C / h, filter, wash, and dry to obtain the finished γ-aminobutyric acid.
[0044] Example 3
[0045] A separation, purification and preparation process of γ-aminobutyric acid comprises the following steps:
[0046] (1) 40 L of a glutamic acid solution with a concentration of 640 g / L was prepared, whole cells containing glutamic acid decarboxylase were added (the amount added was such that the OD600 value of the reaction system was 30 OD / ml), and then a calcium chloride solution with a calcium ion concentration of 10 mM was added, the pH was adjusted to 4.5, the temperature of the reaction system was controlled to be 35° C., and the reaction was terminated after reacting for 5 h to obtain 43 L of a conversion solution containing γ-aminobutyric acid for standby use;
[0047] (2) taking 43 L of the conversion solution containing γ-aminobutyric acid in step (1), filtering it through a ceramic membrane with a pore size of 100 nm, and collecting 58 L of the filtrate for later use;
[0048] (3) Take 58 L of the filtrate in step (2), add 2.0 times the volume of the filtrate with an extractant (n-hexane), control the stirring speed to 200 rpm, stir for 0.7 h, let stand for 35 min, and collect 58 L of the lower organic phase for later use;
[0049] (4) Take 58L of the organic phase in step (3), adjust the pH to 2 with hydrochloric acid solution, and then enter a 25L strong acid cation resin column at a flow rate of 2BV / h (it is necessary to use 25wt% sodium chloride solution and water to elute the strong acid cation resin column successively until the conductivity of the effluent is less than 10us / cm, and the transformed cation resin column re-adsorbs γ-aminobutyric acid in the organic phase), then use 50L, 10wt% potassium chloride solution as an analytical agent for elution, and collect 51L of analytical solution for use;
[0050] (5) 51 L of the analytical solution in step (4) was concentrated once using a nanofiltration membrane with a molecular weight cutoff of 150 Da. The collected 18.5 L of the first concentrated solution was subjected to a second thermal concentration at 60° C. and a vacuum degree of less than -0.090 MPa. 5.8 L of the second concentrated solution with a solid content of 75 wt % was collected for later use;
[0051] (6) Take 5.8 L of the second concentrated solution in step (5), add anhydrous ethanol in an amount 3 times the volume of the second concentrated solution, stir, cool to 22° C., filter, and collect 19600 g of crude γ-aminobutyric acid;
[0052] (7) Take 19600 g of the crude γ-aminobutyric acid in step (6), add 19.6 L of purified water, heat to 70° C., stir until dissolved, then add 392 g of decolorizer (activated carbon), continue stirring for 30 min, filter, and cool the collected decolorized liquid to 12° C. at a rate of 4° C. / h, filter, wash, and dry to obtain the finished γ-aminobutyric acid.
[0053] Example 4
[0054] A separation, purification and preparation process of γ-aminobutyric acid comprises the following steps:
[0055] (1) preparing 40 L of glutamate solution with a concentration of 720 g / L, adding whole cells containing glutamate decarboxylase (the amount added is such that the OD600 value of the reaction system is 40 OD / ml), then adding a calcium chloride solution with a calcium ion concentration of 12 mM, adjusting the pH to 4.8, controlling the temperature of the reaction system to 30° C., reacting for 5.5 h and then terminating the reaction to obtain 44 L of a conversion solution containing γ-aminobutyric acid for standby use;
[0056] (2) taking 44 L of the conversion solution containing γ-aminobutyric acid in step (1), filtering it through a ceramic membrane with a pore size of 150 nm, and collecting 59 L of the filtrate for later use;
[0057] (3) Take 59 L of the filtrate in step (2), add 2.5 times the volume of the filtrate volume of the extractant (ether), control the stirring speed to 250 rpm, stir for 0.8 h, let it stand for 38 min, collect 73.7 L of the lower organic phase for later use;
[0058] (4) Take 73.7 L of the organic phase in step (3), adjust the pH to 2.5 with hydrochloric acid solution, and then enter a 30 L strong acid cation resin column at a flow rate of 2.5 BV / h (it is necessary to use 28 wt % sodium chloride solution and water to elute the strong acid cation resin column successively until the conductivity of the effluent is less than 10 us / cm, and the transformed cation resin column is obtained to adsorb γ-aminobutyric acid in the organic phase), and then use 60 L, 12 wt % potassium chloride solution as an analytical agent for elution, and collect 61 L of analytical solution for use;
[0059] (5) 61 L of the analytical solution in step (4) was concentrated once using a nanofiltration membrane with a molecular weight cutoff of 180 Da, and the collected 18.5 L of the first concentrated solution was subjected to a second thermal concentration at 65° C. and a vacuum degree of less than -0.090 MPa, and 5.6 L of the second concentrated solution with a solid content of 78 wt % was collected for later use;
[0060] (6) Take 5.6 L of the second concentrated solution in step (5), add anhydrous ethanol in an amount 3 times the volume of the second concentrated solution, stir, cool to 23° C., filter, and collect 19100 g of crude γ-aminobutyric acid;
[0061] (7) Take 19100 g of the crude γ-aminobutyric acid in step (6), add 19.1 L of purified water, heat to 75 ° C, stir until dissolved, then add 382 g of decolorizer (activated carbon), continue stirring for 35 min, filter, and cool the collected decolorized liquid to 13 ° C at a rate of 4.5 ° C / h, filter, wash, and dry to obtain the finished γ-aminobutyric acid.
[0062] Example 5
[0063] A separation, purification and preparation process of γ-aminobutyric acid comprises the following steps:
[0064] (1) 40 L of 800 g / L glutamic acid solution was prepared, whole cells containing glutamic acid decarboxylase were added (the amount added was such that the OD600 value of the reaction system was 50 OD / ml), and then a calcium chloride solution with a calcium ion concentration of 12 mM was added, the pH was adjusted to 5, the temperature of the reaction system was controlled to 40° C., and the reaction was terminated after 6 h to obtain 43 L of conversion solution containing γ-aminobutyric acid for standby use;
[0065] (2) taking 43 L of the conversion solution containing γ-aminobutyric acid in step (1), filtering it through a ceramic membrane with a pore size of 200 nm, and collecting 58 L of the filtrate for later use;
[0066] (3) Take 58 L of the filtrate in step (2), add 3.0 times the volume of the filtrate volume of the extractant (petroleum ether), control the stirring speed to 300 rpm, stir for 1 hour, let it stand for 40 minutes, and then collect 87 L of the lower organic phase for later use;
[0067] (4) Take 87L of the organic phase in step (3), adjust the pH to 3 with hydrochloric acid solution, and then enter a 40L strong acid cation resin column at a flow rate of 3BV / h (it is necessary to use 30wt% sodium chloride solution and water to elute the strong acid cation resin column successively until the conductivity of the effluent is less than 10us / cm, and the transformed cation resin column is obtained to adsorb γ-aminobutyric acid in the organic phase), then use 80L, 15wt% potassium chloride solution as an analytical agent for elution, and collect 78L of analytical solution for use;
[0068] (5) 78 L of the analytical solution in step (4) was concentrated once using a nanofiltration membrane with a molecular weight cutoff of 200 Da, and the collected 19.0 L of the first concentrated solution was subjected to a second thermal concentration at 70° C. and a vacuum degree of less than -0.090 MPa, and 5.7 L of the second concentrated solution with a solid content of 80 wt % was collected for later use;
[0069] (6) Take 5.7 L of the second concentrated solution in step (5), add anhydrous ethanol in an amount 4 times the volume of the second concentrated solution, stir, cool to 25° C., filter, and collect 19200 g of crude γ-aminobutyric acid;
[0070] (7) Take 19200 g of the crude γ-aminobutyric acid in step (6), add 19.2 L of purified water, heat to 80 ° C, stir until dissolved, then add 384 g of decolorizer (activated carbon), continue stirring for 40 min, filter, and cool the collected decolorized liquid to 15 ° C at a rate of 5 ° C / h, filter, wash, and dry to obtain the finished γ-aminobutyric acid.
[0071] In order to better prove that the process of the present invention can obtain a high-yield, high-yield and high-purity γ-aminobutyric acid product, 13 comparative examples are given with reference to Example 3, as shown in the following content.
[0072] Comparative Example 1
[0073] The difference from Example 3 is that magnesium chloride solution is added in step (1), and the remaining operations are the same.
[0074] Comparative Example 2
[0075] The difference from Example 3 is that no calcium salt solution is added in step (1), and the remaining operations are the same.
[0076] Comparative Example 3
[0077] The difference from Example 3 is that the extractant in step (3) is ethyl acetate, and the remaining operations are the same.
[0078] Comparative Example 4
[0079] The difference from Example 3 is that the extractant in step (3) is ether, and the remaining operations are the same.
[0080] Comparative Example 5
[0081] The difference from Example 3 is that the stirring speed in step (3) is 350 rpm, and the other operations are the same.
[0082] Comparative Example 6
[0083] The difference from Example 3 is that in step (4), the pH of the organic phase is adjusted to 3.5 using hydrochloric acid solution, and the remaining operations are the same.
[0084] Comparative Example 7
[0085] The difference from Example 3 is that in step (4), 20 wt % sodium hydroxide solution is used as the analytical agent, and the remaining operations are the same.
[0086] Comparative Example 8
[0087] The difference from Example 3 is that the temperature is lowered to 15° C. in step (6), and the remaining operations are the same.
[0088] Comparative Example 9
[0089] The difference from Example 3 is that the temperature is lowered to 28° C. in step (6), and the remaining operations are the same.
[0090] Comparative Example 10
[0091] The difference from Example 3 is that the heating to 85° C. is performed in step (7), and the remaining operations are the same.
[0092] Comparative Example 11
[0093] The difference from Example 3 is that in step (7), the decolorizing solution is directly cooled to 12° C., and the remaining operations are the same.
[0094] Comparative Example 12
[0095] The difference from Example 3 is that in step (7), the decolorizing solution is cooled to 7° C. at a rate of 4° C. / h, and the remaining operations are the same.
[0096] Comparative Example 13
[0097] The difference from Example 3 is that in step (7), the decolorizing solution is cooled to 18° C. at a rate of 4° C. / h, and the remaining operations are the same.
[0098] Table 1
[0099]
[0100] It can be found from the data in Table 1 that by using the process of the present invention, by comparing Example 3 with other examples and comparative examples 1-13, it can be found that the output, yield and purity of γ-aminobutyric acid obtained in Example 3 are better than those in other examples and comparative examples.
[0101] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.
Claims
1. A separation, purification and preparation process of γ-aminobutyric acid, characterized in that: The following steps are involved: (1) preparing a glutamate solution, adding whole cells containing glutamate decarboxylase, and then adding a calcium salt solution, adjusting the pH to acidic, controlling the temperature and reaction time of the reaction system, and obtaining a conversion solution containing γ-aminobutyric acid for later use; (2) taking the conversion solution containing γ-aminobutyric acid described in step (1), filtering and collecting the filtrate for later use; (3) taking the filtrate in step (2), adding an extractant, controlling the stirring speed and time, standing to separate the layers, and collecting the lower organic phase for later use; (4) taking the organic phase described in step (3), adjusting the pH to acidic, entering into a strong acid cationic resin column, using a decomposing agent for elution, and collecting the decomposing solution for later use; (5) taking the analytical solution described in step (4), concentrating the first concentrated solution collected after the first concentration, and then concentrating the second concentrated solution for later use; (6) taking the second concentrated solution in step (5), adding anhydrous ethanol, stirring, cooling, and filtering to collect the crude γ-aminobutyric acid; (7) Take the crude γ-aminobutyric acid described in step (6), add purified water, heat and stir until dissolved, then add a decolorizer, continue stirring and filtering, cool the collected decolorized liquid, filter, wash and dry to obtain the finished γ-aminobutyric acid.
2. The separation, purification and preparation process of γ-aminobutyric acid according to claim 1, characterized in that: The concentration of the glutamate solution in step (1) is 500-800 g / L, the amount of whole cells containing glutamate decarboxylase added is such that the OD600 value of the reaction system is 10-50 OD / ml, the pH is adjusted to 4-5, the temperature of the reaction system is controlled to be 20-40° C., and the reaction time is 4-6 h; the calcium salt solution is a calcium chloride solution, and the concentration of calcium ions in the calcium salt solution is 6-12 mM.
3. The separation, purification and preparation process of γ-aminobutyric acid according to claim 1, characterized in that: In step (2), a ceramic membrane with a pore size of 50-200 nm is used for filtration.
4. The separation, purification and preparation process of γ-aminobutyric acid according to claim 1, characterized in that: In step (3), the extractant is ethyl acetate, ethyl ether, petroleum ether or n-hexane, preferably n-hexane; the stirring speed is controlled to be 100-300 rpm, and the stirring is performed for 0.5-1 h.
5. The separation, purification and preparation process of γ-aminobutyric acid according to claim 1, characterized in that: The organic phase in step (4) is adjusted to pH 1-3 and then enters the strongly acidic cationic resin column at a flow rate of 1-3 BV / h.
6. The separation, purification and preparation process of γ-aminobutyric acid according to claim 1, characterized in that: The analytical agent in step (4) is a 5-15wt% potassium chloride solution.
7. The separation, purification and preparation process of γ-aminobutyric acid according to claim 1, characterized in that: In step (4), before the organic phase enters the strong acid cationic resin column, the strong acid cationic resin column needs to be eluted with 20-30 wt % sodium chloride solution and water until the conductivity of the effluent is less than 10 us / cm, thereby obtaining a transformed cationic resin column.
8. The separation, purification and preparation process of γ-aminobutyric acid according to claim 1, characterized in that: In step (5), the first concentration is carried out by using a nanofiltration membrane with a molecular weight cutoff of 100-200Da, and the second concentration is carried out by using thermal concentration. The solid content of the second concentrated solution is 70-80wt%.
9. The separation, purification and preparation process of γ-aminobutyric acid according to claim 1, characterized in that: In step (6), the anhydrous ethanol is added in an amount of 1-4 times the volume of the second concentrated solution, and the temperature is lowered to 20-25°C.
10. The separation, purification and preparation process of γ-aminobutyric acid according to claim 1, characterized in that: In step (7), purified water is added and heated to 60-80° C.; the decolorizing solution is cooled to 10-15° C. at a rate of 3-5° C. / h.