Method for separating and purifying L-methionine and succinic acid

Abstract

The invention belongs to the technical field of biochemical engineering, and particularly relates to a method for separating and purifying L-methionine and succinic acid. According to the method, the sample loading flow rate of the enzyme catalysis liquid in the sample loading treatment process is optimized, and the cation exchange resin column is washed by using the deionized water with the volume 1.5-4 times of the column volume (BV) after the sample loading treatment, so that the residual succinic acid in the cation exchange resin column can be effectively separated, the loss of L-methionine in the cation exchange resin caused by excessive washing is avoided, and the yield of the L-methionine is improved. And meanwhile, less succinic acid is remained on the cation exchange resin, so that the balance between the purity and the yield of the double products of the L-methionine and the succinic acid is realized.

Description

Technical Field

[0001] The present application belongs to the field of biochemical engineering technology, and specifically relates to a method for separating and purifying L-methionine and succinic acid from an enzyme catalytic solution. Background Art

[0002] L-methionine and succinic acid are both compounds with important application value. Among them, L-methionine is the only sulfur-containing amino acid necessary for the human body and one of the basic components of protein synthesis. It is widely used in various fields such as nutritional supplements, cosmetics and pharmaceutical industries. Succinic acid is an important organic acid and an important 4-carbon intermediate in the raw materials of special chemical products. It was selected by the U.S. Department of Energy as the first of the twelve most commercially valuable platform compounds and has key uses in many fields such as chemical industry, pharmaceuticals, and food.

[0003] At present, microbial fermentation is often used to prepare L-methionine and succinic acid separately. However, in industrial production, a specific enzymatic reaction pathway is used to efficiently synthesize L-methionine. This pathway uses sulfhydryl lyase as a catalyst, O-succinyl-L-homoserine (OSH) as a substrate, and methyl mercaptan or sodium methyl mercaptan as a sulfhydryl donor. In this process, the succinyl group in OSH reacts with the sulfhydryl group on methyl mercaptan (sodium), releasing L-methionine while generating succinic acid (sodium) as a by-product. Therefore, in the process of producing L-methionine, an enzyme catalytic liquid containing two products, L-methionine and succinic acid (sodium), is produced.

[0004] After searching, no mature separation method for this enzyme catalytic liquid containing two components, L-methionine and succinic acid (sodium), has been found. Since the above enzymatic reaction pathway is currently mainly used to prepare L-methionine, and the recovery of the succinic acid component is not considered in the existing L-methionine separation and purification technology, after obtaining the enzyme catalytic liquid, the L-methionine therein is simply separated and purified. In addition, L-methionine is not considered in the existing succinic acid separation and purification technology. Ion exchange chromatography can realize the separation and recovery of the two components of L-methionine and succinic acid in the enzyme catalytic liquid. In order to ensure the yield and purity of the separation and recovery of L-methionine and succinic acid, the amount of flushing liquid needs to be controlled during the chromatographic separation process so that the non-adsorbed component succinic acid is washed away completely, and at the same time, the partial desorption of the L-methionine adsorbed on the resin needs to be reduced to achieve a balance between the yield and purity of the double products of L-methionine and succinic acid. Summary of the invention

[0005] The purpose of the present application is to provide a method for separating and purifying L-methionine and succinic acid from the above-mentioned enzyme catalytic solution, which is specifically achieved through the following technical scheme:

[0006] A method for separating and purifying L-methionine and succinic acid comprises the following steps: using a cation exchange resin column to perform sample loading on an enzyme catalytic solution containing L-methionine and sodium succinate to obtain an effluent solution, namely a succinic acid solution; the cation exchange resin is a strongly acidic cation exchange resin; the sample loading flow rate in the sample loading process is 2-5 BV / h; the sample loading process further comprises, after the sample loading of the enzyme catalytic solution is completed, using 1.5-4 BV of deionized water to rinse the cation exchange resin column; using an eluent to elute the cation exchange resin column to obtain an L-methionine eluent; and performing evaporation, concentration, crystallization and filtration on the succinic acid solution and the L-methionine eluent to obtain succinic acid and L-methionine respectively.

[0007] Preferably, before preparing the succinic acid solution, the enzyme catalytic solution is pretreated under the following conditions: temperature 60-100° C., time 10-60 min; then, the solid components in the enzyme catalytic solution are removed to obtain the pretreated enzyme catalytic solution.

[0008] The present application can effectively remove bacterial proteins and other macromolecular impurities by pretreating the enzyme catalytic liquid before preparing the succinic acid liquid, preventing these substances from precipitating in the cation exchange resin column, thereby avoiding the impact on the purity of L-methionine and succinic acid products, reducing the hidden danger of cation exchange resin column blockage, and ensuring the smooth progress of the separation process.

[0009] Preferably, the strongly acidic cation exchange resin is selected from one of HPR650, IR120H, D072, 001×7, D001, D001-cc, LX-S1850, LXT-104, and D061.

[0010] Preferably, the succinic acid solution and the L-methionine eluate are first subjected to an activated carbon decolorization treatment before being subjected to an evaporation concentration treatment.

[0011] Preferably, in the decolorization treatment of succinic acid solution, the amount of activated carbon used is 2.0 to 8.0 g / L based on the volume of the succinic acid solution.

[0012] Preferably, before the L-methionine eluate is decolorized, it is firstly subjected to an acidification treatment so that the pH value of the L-methionine eluate is 3 to 6 to obtain an acidified solution.

[0013] Preferably, in the decolorization treatment of the L-methionine eluate, the amount of activated carbon used is 3.0 to 10.0 g / L based on the volume of the acidified liquid.

[0014] Preferably, the conditions of the evaporation concentration treatment are 55-85°C and -0.1 MPa.

[0015] Preferably, during elution of the cation exchange resin column, an eluent with a concentration of 2 to 5 BV and a concentration of 0.5 to 2.0 mol / L is selected, and the eluent is ammonia water or sodium hydroxide solution; the elution flow rate is 2 to 5 BV / h.

[0016] Preferably, after the L-methionine eluate is evaporated and concentrated, ethanol is added to promote L-methionine crystallization, and the crystallization temperature is 0-10° C.; the amount of ethanol used is 10%-50% by volume of the concentrated solution.

[0017] Compared with the prior art, this application has the following beneficial effects:

[0018] The present application optimizes the sample loading flow rate of the enzyme catalytic solution during the sample loading process, and uses 1.5 to 4 times the column volume (BV) of deionized water to rinse the cation exchange resin column after the sample loading process, which can effectively separate the succinic acid remaining in the cation exchange resin column, avoid the loss of L-methionine in the cation exchange resin caused by excessive rinsing, and ensure that less succinic acid remains on the cation exchange resin, thereby achieving a balance between the purity and yield of L-methionine and succinic acid double products. Specifically, performance tests show that by optimizing the conditions-controlling the sample loading flow rate to 3BV / h, and using 2BV of rinsing water, the succinic acid yield is as high as 89.5%, and the purity reaches 96.3%. Further, in the process of eluting the cation exchange resin column, the present application uses 3BV of ammonia water to elute at a speed of 3BV / h. This strategy ensures the effective recovery of L-methionine. Finally, the yield of L-methionine reaches 90.3%, and its purity is as high as 99.2%, which is the best performance among all embodiments, reflecting unexpected technical effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The present invention is a flow chart of a method for separating and purifying L-methionine and succinic acid. DETAILED DESCRIPTION

[0020] The present application will be further described below in the form of specific embodiments. Those of ordinary skill in the art will be able to implement the present application based on these descriptions. In addition, the embodiments of the present application involved in the following description are generally only embodiments of a part of the present application, rather than all embodiments. Therefore, based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without making creative work should fall within the scope of protection of the present application.

[0021] The sources of the enzyme catalytic solution in the following examples are as follows: in the O-succinyl-L-homoserine fermentation broth, add the crude enzyme solution of sulfhydryl lyase (see patent application number 202110456745.9) and the coenzyme pyridoxal phosphate, keep warm at 30°C and keep stirring, and then slowly add sodium methyl mercaptan solution to the system, the molar amount of sodium methyl mercaptan is 1 to 1.1 times the molar amount of O-succinyl-L-homoserine. After the reaction is completed, an enzyme catalytic solution containing L-methionine and sodium succinate is obtained.

[0022] Example 1

[0023] This embodiment discloses a method for separating and purifying L-methionine and succinic acid from an enzyme catalytic solution, comprising the following steps:

[0024] Step 1: Take 1L of enzyme catalytic solution, heat it in a 70°C water bath for 30 minutes to denature and precipitate the soluble protein, centrifuge it at 8000rpm for 10 minutes, collect the supernatant, and obtain the enzyme catalytic solution pretreatment solution.

[0025] Step 2: Take 140mL of pretreated IR120H cation exchange resin and load it into a chromatography column (the IR120H cation exchange resin is first washed with deionized water until the immersion water is colorless, and then the column is wet packed with 3BV 1M HCl slowly flowing through the resin, and rinsed with deionized water until the pH of the effluent is greater than 4, and then 3BV 1M NaOH slowly flowing through the resin, and rinsed with deionized water until the pH of the effluent is less than 10, and finally 4BV 1M HCl slowly flowing through the resin, and rinsed with deionized water until the pH of the effluent is greater than 6 for standby use), the height-to-diameter ratio is 10:1, 150mL of the enzyme catalytic liquid pretreatment liquid is loaded onto the IR120H cation exchange resin column at a flow rate of 3BV / h, and after loading, the cation exchange resin column is rinsed with 2BV deionized water, and the effluent containing succinic acid during loading and rinsing is collected to obtain succinic acid solution.

[0026] Step 3: Add 5 g / L of activated carbon powder (200 mesh) to the succinic acid solution, stir and decolorize for 20 min, and filter to obtain the succinic acid decolorized solution.

[0027] Step 4: The succinic acid decolorizing solution is evaporated and concentrated in a 70°C water bath by a rotary evaporator (vacuum degree is -0.1MPa) until a crystal film appears on the liquid surface, then cooled at 0°C for crystallization, filtered, washed with a small amount of ice water, and dried to obtain succinic acid.

[0028] Step 5: Elute the cation exchange resin column rinsed with deionized water with 3BV 1M ammonia water at a flow rate of 3BV / h, and collect the eluate.

[0029] Step 6: Adjust the pH of the eluate to 5 with hydrochloric acid, add 8 g / L of activated carbon powder (200 mesh), stir and decolorize for 20 min, and filter to obtain the methionine decolorized solution.

[0030] Step 7: The methionine decolorizing solution is evaporated and concentrated in a 80°C water bath by a rotary evaporator (vacuum degree is -0.1MPa) until a crystal film appears on the liquid surface, and then 30% anhydrous ethanol is added based on the volume of the concentrated solution. The solution is cooled at 0°C for crystallization, filtered, and the crystals are rinsed with 0°C 50% ethanol, and dried to obtain L-methionine.

[0031] Example 2

[0032] This example discloses a method for separating and purifying L-methionine and succinic acid from an enzyme catalytic solution. The difference from Example 1 is that the sample loading and elution processing parameters are different, and the method specifically comprises the following steps:

[0033] Step 1: Take 1L of enzyme catalytic solution, heat it in a 70°C water bath for 30 minutes to denature and precipitate the soluble protein, centrifuge it at 8000rpm for 10 minutes, collect the supernatant, and obtain the enzyme catalytic solution pretreatment solution. It is understood that in other embodiments, the pretreatment conditions can also be: temperature 60-100°C, time 10-60 minutes.

[0034] Step 2: Take 140mL of pretreated IR120H cation exchange resin and load it into a chromatography column (the IR120H cation exchange resin is first washed with deionized water until the immersion water is colorless, and then the column is wet packed with 3BV 1M HCl slowly flowing through the resin, and rinsed with deionized water until the pH of the effluent is greater than 4, and then 3BV 1M NaOH slowly flowing through the resin, and rinsed with deionized water until the pH of the effluent is less than 10, and finally 4BV 1M HCl slowly flowing through the resin, and rinsed with deionized water until the pH of the effluent is greater than 6 for standby use), the height-to-diameter ratio is 10:1, 150mL of the enzyme catalytic liquid pretreatment liquid is loaded onto the IR120H cation exchange resin column at a flow rate of 2BV / h, and after loading, the cation exchange resin column is rinsed with 1.5BV deionized water, and the effluent containing succinic acid during loading and rinsing is collected to obtain succinic acid solution. It can be understood that in other embodiments, the sample loading flow rate can be selected from 2 to 5 BV / h, and the deionized water used for flushing can be 1.5 to 4 BV.

[0035] Step 3: Add 5 g / L of activated carbon powder (200 mesh) to the succinic acid solution, stir and decolorize for 20 min, and filter to obtain a succinic acid decolorized solution. It is understood that in other embodiments, the amount of activated carbon can also be 2.0 to 8.0 g / L based on the volume of the succinic acid solution.

[0036] Step 4: The succinic acid decolorizing solution is evaporated and concentrated in a 70°C water bath by a rotary evaporator (vacuum degree is -0.1MPa) until a crystal film appears on the liquid surface, then cooled and crystallized at 0°C, filtered, washed with a small amount of ice water, and dried to obtain succinic acid. It is understood that in other embodiments, the conditions for evaporation and concentration treatment can also be 55-85°C and -0.1MPa.

[0037] Step 5: Elute the cation exchange resin column rinsed with deionized water with 4BV 0.5M ammonia water at a flow rate of 2BV / h, and collect the eluent. It is understood that in other embodiments, during the elution of the cation exchange resin column, an eluent of 2 to 5BV and a concentration of 0.5 to 2.0 mol / L can also be selected, and the eluent is ammonia water or sodium hydroxide solution; the elution flow rate is 2 to 5BV / h.

[0038] Step 6: Adjust the pH of the eluent to 5 with hydrochloric acid, add 8 g / L of activated carbon powder (200 mesh), stir and decolorize for 20 min, and filter to obtain a methionine decolorized solution. It is understood that in other embodiments, the pH can also be adjusted to 3-6.

[0039] Step 7: The methionine decolorizing solution is evaporated and concentrated in a water bath at 80°C by a rotary evaporator (vacuum degree is -0.1MPa) until a crystal film appears on the surface of the liquid, and then 30% anhydrous ethanol is added based on the volume of the concentrated solution, cooled at 0°C for crystallization, filtered, and then the crystals are rinsed with 50% ethanol at 0°C, and dried to obtain L-methionine. It is understood that in other embodiments, the conditions for evaporation and concentration treatment can also be 55-85°C, -0.1MPa; crystallization temperature 0-10°C; the amount of ethanol used is 10%-50% based on the volume of the concentrated solution.

[0040] Example 3

[0041] This example discloses a method for separating and purifying L-methionine and succinic acid from an enzyme catalytic solution. The difference from Example 1 is that the sample loading and elution processing parameters are different, and the method specifically comprises the following steps:

[0042] Step 1: Take 1L of enzyme catalytic solution, heat it in a 70°C water bath for 30 minutes to denature and precipitate the soluble protein, centrifuge it at 8000rpm for 10 minutes, collect the supernatant, and obtain the enzyme catalytic solution pretreatment solution.

[0043] Step 2: Take 140mL of pretreated IR120H cation exchange resin and load it into a chromatography column (the IR120H cation exchange resin is first washed with deionized water until the immersion water is colorless, and then the column is wet packed with 3BV 1M HCl slowly flowing through the resin, and rinsed with deionized water until the pH of the effluent is greater than 4, and then 3BV 1M NaOH slowly flowing through the resin, and rinsed with deionized water until the pH of the effluent is less than 10, and finally 4BV 1M HCl slowly flowing through the resin, and rinsed with deionized water until the pH of the effluent is greater than 6 for standby use), the height-to-diameter ratio is 10:1, 150mL of the enzyme catalytic liquid pretreatment solution is loaded onto the IR120H cation exchange resin column at a flow rate of 5BV / h, and after loading, the cation exchange resin column is rinsed with 4BV deionized water, and the effluent containing succinic acid during loading and rinsing is collected to obtain succinic acid solution.

[0044] Step 3: Add 5 g / L of activated carbon powder (200 mesh) to the succinic acid solution, stir and decolorize for 20 min, and filter to obtain the succinic acid decolorized solution.

[0045] Step 4: The succinic acid decolorizing solution is evaporated and concentrated in a 70°C water bath by a rotary evaporator (vacuum degree is -0.1MPa) until a crystal film appears on the liquid surface, then cooled at 0°C for crystallization, filtered, washed with a small amount of ice water, and dried to obtain succinic acid.

[0046] Step 5: Elute the cation exchange resin column rinsed with deionized water with 2BV 2M ammonia water at a flow rate of 5BV / h, and collect the eluate.

[0047] Step 6: Adjust the pH of the eluate to 5 with hydrochloric acid, add 8 g / L of activated carbon powder (200 mesh), stir and decolorize for 20 min, and filter to obtain the methionine decolorized solution.

[0048] Step 7: The methionine decolorizing solution is evaporated and concentrated in a 80°C water bath by a rotary evaporator (vacuum degree is -0.1MPa) until a crystal film appears on the liquid surface, and then 30% anhydrous ethanol is added based on the volume of the concentrated solution. The solution is cooled at 0°C for crystallization, filtered, and the crystals are rinsed with 0°C 50% ethanol, and dried to obtain L-methionine.

[0049] Comparative Example 1

[0050] The difference between the method for separating and purifying L-methionine and succinic acid disclosed in this comparative example and Example 1 is that in step 2, the sample is loaded onto the IR120H cation exchange resin column at a flow rate of 8 BV / h, and after the loading is completed, the cation exchange resin column is rinsed with 0.5 BV of deionized water, and the effluent containing succinic acid during the loading and rinsing is collected to obtain a succinic acid solution.

[0051] Comparative Example 2

[0052] The difference between the method for separating and purifying L-methionine and succinic acid disclosed in this comparative example and Example 1 is that in step 2, the sample is loaded onto the IR120H cation exchange resin column at a flow rate of 1 BV / h, and after the loading is completed, the cation exchange resin column is rinsed with 8 BV of deionized water, and the effluent containing succinic acid during the loading and rinsing is collected to obtain a succinic acid solution.

[0053] Comparative Example 3

[0054] The difference between the method for separating and purifying L-methionine and succinic acid disclosed in this comparative example and Example 1 is that in step 2, the sample is loaded onto the IR120H cation exchange resin column at a flow rate of 8BV / h, and after the loading is completed, the cation exchange resin column is rinsed with 1.5BV of deionized water, and the effluent containing succinic acid during the loading and rinsing is collected to obtain a succinic acid solution. In step 5, the ion exchange resin column after the sample is loaded is eluted with 2BV of 0.5M ammonia water at a flow rate of 5BV / h, and the eluate is collected.

[0055] Performance Testing

[0056] This performance test uses high performance liquid chromatography to detect the purity of L-methionine and succinic acid separated and purified in Examples 1 to 3 and Comparative Examples 1 to 3, and the yield = (mass of the harvested product / amount of the product contained in the sample solution) * 100%, which is summarized in the following table:

[0057] Table 1. Separation and purification status

[0058] Group Succinic acid(g) Yield (%) purity(%) L-Methionine(g) Yield (%) purity(%) Example 1 8.06 89.5 96.3 6.41 90.3 99.2 Example 2 7.35 81.7 95.9 5.76 81.1 96.4 Example 3 8.03 89.2 91.3 5.95 83.8 98.7 Comparative Example 1 4.82 53.6 85.4 5.45 76.7 90.3 Comparative Example 2 7.91 87.9 92.2 4.71 66.4 98.9 Comparative Example 3 7.13 79.2 96.1 3.34 47.1 94.4

[0059] By referring to the succinic acid yield and purity data obtained by the separation methods of Examples 1 to 3 and Comparative Examples 1 to 3 in the above table, it can be clearly seen that the present application can significantly improve the yield and purity of succinic acid by accurately controlling the sample flow rate and the subsequent flushing water volume. In particular, in Example 1, by optimizing the conditions-controlling the sample flow rate to 3BV / h and using 2BV of flushing water, the yield of succinic acid was as high as 89.5%, and the purity reached 96.3%. Excellent results. Further, in the process of eluting the cation exchange resin column, the present application uses 3BV of ammonia water to elute at a rate of 3BV / h. This strategy ensures the effective recovery of L-methionine. Finally, the yield of L-methionine reached 90.3%, and its purity was as high as 99.2%, which is the best performance among all embodiments. These results show that the present application can not only simply improve the yield and purity of succinic acid by precisely controlling the sample loading flow rate and the amount of flushing water, but more importantly, the method can achieve effective separation of succinic acid and L-methionine in a cation exchange resin column, avoiding the technical problems of low final yield and low purity of succinic acid and L-methionine caused by excessive or insufficient flushing of L-methionine. This optimized separation process is of great significance for industrial production, not only improving the yield and quality of the target product, but also simplifying the production process, reducing costs, and enhancing the economy and environmental friendliness of production.

Claims

1. A method for separating and purifying L-methionine and succinic acid, characterized in that: The following steps are involved: The enzyme catalytic solution containing L-methionine and sodium succinate is subjected to sample loading treatment by using a cation exchange resin column to obtain an effluent solution, namely, a succinic acid solution; the cation exchange resin is a strongly acidic cation exchange resin; the sample loading flow rate in the sample loading treatment is 2-5 BV / h; the sample loading treatment further comprises, after the sample loading of the enzyme catalytic solution is completed, washing the cation exchange resin column with 1.5-4 BV of deionized water; eluting the cation exchange resin column with an eluent to obtain an L-methionine eluent; and evaporating and concentrating the succinic acid solution and the L-methionine eluent and performing crystallization and filtration treatments respectively to obtain succinic acid and L-methionine.

2. A method for separating and purifying L-methionine and succinic acid according to claim 1, characterized in that: Before preparing the succinic acid solution, the enzyme catalytic solution is pretreated under the following conditions: temperature of 60-100° C. and time of 10-60 min. Then, the solid components in the enzyme catalytic solution are removed to obtain the pretreated enzyme catalytic solution.

3. A method for separating and purifying L-methionine and succinic acid according to claim 1, characterized in that: The strongly acidic cation exchange resin is selected from one of HPR650, IR120H, D072, 001×7, D001, D001-cc, LX-S1850, LXT-104, and D061.

4. A method for separating and purifying L-methionine and succinic acid according to claim 1, characterized in that: Before the succinic acid solution and the L-methionine eluate are evaporated and concentrated, they are first decolorized with activated carbon.

5. A method for separating and purifying L-methionine and succinic acid according to claim 4, characterized in that: In the decolorization treatment of succinic acid solution, the amount of activated carbon used is 2.0-8.0 g / L based on the volume of succinic acid solution.

6. A method for separating and purifying L-methionine and succinic acid according to claim 4, characterized in that: Before decolorizing the L-methionine eluate, the L-methionine eluate is firstly subjected to an acidification treatment so that the pH value of the L-methionine eluate is 3-6 to obtain an acidified solution.

7. A method for separating and purifying L-methionine and succinic acid according to claim 6, characterized in that: In the decolorization treatment of the L-methionine eluate, the amount of activated carbon used is 3.0 to 10.0 g / L based on the volume of the acidified liquid.

8. A method for separating and purifying L-methionine and succinic acid according to claim 1, characterized in that: The conditions of the evaporation concentration treatment are 55-85°C and -0.1 MPa.

9. A method for separating and purifying L-methionine and succinic acid according to claim 1, characterized in that: During the elution of the cation exchange resin column, an eluent with a volume of 2 to 5 BV and a concentration of 0.5 to 2.0 mol / L is selected, and the eluent is ammonia water or sodium hydroxide solution; the elution flow rate is 2 to 5 BV / h.

10. A method for separating and purifying L-methionine and succinic acid according to claim 1, characterized in that: After the L-methionine eluate is evaporated and concentrated, ethanol is added to promote L-methionine crystallization, and the crystallization temperature is 0-10°C; the amount of ethanol used is 10%-50% based on the volume of the concentrated solution.

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