Method for extracting R-2-(4-hydroxyphenoxy) propionic acid from fermentation liquor

Abstract

The invention discloses a method for extracting R-2-(4-hydroxyphenoxy) propionic acid from fermentation liquor, which belongs to the technical field of fermentation engineering and comprises the steps of heating and separating solid and liquid, respectively extracting under different pH values, filtering, recrystallizing and the like. Wherein the viscosity of the fermentation liquid is improved through the heating operation, the heating temperature adapts to the extraction agent, the heating temperature is 45-60 DEG C when the extraction agent is trichloromethane, the heating temperature is 30-45 DEG C when the extraction agent is dichloromethane, and the heating temperature is 60-70 DEG C when the extraction agent is methyl isobutyl ketone. The pH value is adjusted to be 6-7.5 before the first extraction so as to remove organic impurities such as protein, the pH value is adjusted to be 1-1.5 before the second extraction so as to separate out R-2-(4-hydroxyphenoxy) propionic acid in a solid phase, and the purposes of separation and purification are achieved through two times of extraction. According to the purity of R-2-(4-hydroxyphenoxy) propionic acid extracted by the method, the average yield is 85%-95%, and the purity reaches 90% or above.

Description

Technical Field

[0001] The present invention relates to the field of fermentation engineering technology, in particular to a method for separating and purifying a fermentation liquid. R -2-(4-hydroxyphenoxy)propionic acid. Background Art

[0002] Phenoxypropionic acid compounds are a class of acetyl-CoA carboxylase inhibitors that can specifically kill grass weeds, and their synthesis has attracted widespread attention. R -2-(4-Hydroxyphenoxy)propionic acid ( R -HPPA) is a key chiral intermediate in the synthesis of aryloxyphenoxypropionic acid herbicides, and its production and preparation is particularly important.

[0003] Currently, domestic and foreign production R -HPPA routes can be divided into two major directions: traditional chemical synthesis and biocatalytic synthesis. Among them, the biosynthetic catalytic route has the advantages of mild reaction conditions, strict stereoselectivity, few by-products, and environmental friendliness. R -2-(4-hydroxyphenoxy)propionic acid. Authorized invention CN109536392B, authorized invention CN109609389B, and published invention application CN116590153A respectively proposed using Aspergillus versicolor ZJB16085, Penicillium oxalicum ZJB16086, and Beauveria bassiana ZJB23323 to achieve biocatalytic synthesis R -HPPA method.

[0004] The reaction mechanism of biological method is microbial biofilm fermentation R -2-Phenoxypropionic acid ( R -PPA) production R -HPPA, the fermentation broth after the reaction contains incompletely utilized culture medium components, various organic acids and other metabolites produced by bacterial metabolism, incompletely converted substrates and catalytic products. It is a highly complex mixture system, which inevitably faces problems such as difficulty in product separation and purification. Therefore, it is urgent to develop a high-efficiency and low-cost separation and purification process. The prior art has not yet disclosed a method for separating and purifying the fermentation broth from biocatalytic synthesis. R -2-(4-hydroxyphenoxy)propionic acid. Summary of the invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide a method for separating and purifying the fermentation broth with simple process, high yield and stable quality. R -2-(4-hydroxyphenoxy)propionic acid method.

[0006] The present invention provides a method for separating and purifying a fermentation broth R-2-(4-hydroxyphenoxy)propionic acid, comprising the following steps: S1. Fermenting and culturing a fermentation liquid using Beauveria bassiana, heating the fermentation liquid to above 30° C., separating the solid from the liquid, and collecting the liquid phase; S2, taking the liquid phase obtained in S1, adjusting the pH to 6.0-7.5 at the heating temperature in S1, adding an extractant, stirring, and separating to obtain a first organic layer and a first aqueous layer; S3, taking the first water layer, adjusting the pH to 1-1.2, continuing to add the extractant, stirring, and obtaining a reaction system including a second water layer; S4, taking the reaction system, discarding the second aqueous layer, concentrating the remaining components, filtering and drying to obtain R -2-(4-Hydroxyphenoxy)propionic acid crude product; S5, recrystallize the crude product to obtain R -2-(4-Hydroxyphenoxy)propionic acid crystals.

[0007] The fermentation broth contains culture medium components and broken bacteria, and a large amount of sugars, proteins, amino acids, and inorganic ions are suspended or dissolved. The system has a high viscosity and cannot fully contact with the extractant during direct extraction. Experiments have found that heating the fermentation broth can reduce the viscosity and promote the full transfer of the target to the organic phase; but after the temperature is raised to a certain extent, the extraction efficiency is reduced because the extractant evaporates rapidly at this time. Therefore, the heating temperature must be adapted to the selected extractant.

[0008] Preferably, the strain of Beauveria bassiana is Beauveria bassiana ZJB23323 ( Beauveriabassiana ZJB23323 ), deposited in China Center for Type Culture Collection, deposit number: CCTCC NO: M 2023584, deposit date: April 21, 2023, address: Wuhan University, Wuhan, China, 430072. This strain is a known strain. Beauveriabassiana ZJB16001 As the starting strain, it has been subjected to natural selection, UV mutagenesis, room temperature plasma (A R TP) mutagenesis, nitrosoguanidine mutagenesis and other steps, which have been disclosed in Chinese patent CN116590153A.

[0009] Preferably, the fermentation culture process in S1 is as follows: Activation of strains: Inoculate Beauveria bassiana ZJB23323 into slant medium and culture at 28°C for 15 days to obtain slant bacteria; Seed culture: inoculate the slant bacteria into the fermentation medium and culture at 28℃ for 72 h to obtain seed solution; Fermentation culture: Inoculate the seed liquid into fresh fermentation medium at a volume concentration of 10%, and culture it at 28°C for 15 days. Supplement 10% (v / v) 600 g / L sterile glucose solution on the 3rd and 5th days of the biofilm fermentation process. Supplement 5% (v / v) 200 g / L sterile yeast extract solution on the 3rd and 5th days of the biofilm fermentation process. Take the product on the 15th day of the reaction as the final fermentation product for subsequent separation and purification processes.

[0010] Preferably, the final concentration of the slant culture medium is: R -2-Phenoxypropionic acid 40~60 g / L, glucose 10~30 g / L, yeast extract 5~15 g / L, MgSO4 0.9~1.1 g / L, CaCl2 0.65~0.85 g / L, K2HPO4 1.7~1.9 g / L, KH2PO4 0.65~0.85 g / L, EDTA-2Na·2H2O 1~3 g / L, FeSO4·7H2O 0.4~0.8 g / L, ZnSO4·7H2O 0.1~0.3 g / L, MnSO4·H2O 0.1~0.3 g / L, NiCl·6H2O 0.02~0.06 g / L, CoCl2 0.01~0.03 g / L, H3BO3 0.02~0.04 g / L, Na2MoO4·H2O 0.003~0.007 g / L, agar 1~3 g / L, solvent is water, sterilize at 115℃ for 30 min and then cool before use.

[0011] Preferably, the final concentration of the fermentation medium is: R -2-Phenoxypropionic acid 40~60 g / L, glucose 30~50 g / L, yeast extract 5~15 g / L, MgSO4 0.9~1.1 g / L, CaCl2 0.65~0.85 g / L, K2HPO4 1.7~1.9 g / L, KH2PO4 0.65~0.85 g / L, EDTA-2Na·2H2O 1~3 g / L, FeSO4·7H2O 0.4~0.8 g / L, ZnSO4·7H2O 0.1~0.3 g / L, MnSO4·H2O 0.1~0.3 g / L, NiCl·6H2O 0.02~0.06 g / L, CoCl2 0.01~0.03 g / L, H3BO3 0.02~0.04 g / L, Na2MoO4·H2O 0.003~0.007 g / L, the solvent is water, sterilized at 115℃ for 30 minutes and then cooled before use.

[0012] Preferably, the solid-liquid separation operation in S1 is vacuum rotary evaporation under a vacuum degree of 0.090 to 0.095 MPa, and the conversion liquid is retained in the collection bottle.

[0013] Further preferably, the heating temperature in S1 is 45-60° C., and the extractant is chloroform.

[0014] Further preferably, the heating temperature in S1 is 30-45° C., and the extractant is dichloromethane.

[0015] Further preferably, the heating temperature in S1 is 60-70° C., and the extractant is methyl isobutyl ketone.

[0016] Preferably, the solid-liquid separation operation in S1 is performed by filtering with a 0.22 μm microporous membrane at 30 to 70°C.

[0017] Preferably, the acid used to adjust the pH in S2 and S3 is hydrochloric acid or sulfuric acid.

[0018] Preferably, the concentration operation in S4 is vacuum rotary evaporation at a temperature of 20 to 35° C. and a vacuum degree of 0.090 to 0.095 MPa. The vacuum rotary evaporation endpoint is when the concentration reaches 40% to 70% of the original volume.

[0019] Preferably, the drying operation in S4 is forced air drying at 55-65° C. for 10 hours.

[0020] Beneficial effects of the present invention: The present invention provides a R Extracted from 2-(4-hydroxyphenoxy)propionic acid fermentation broth R -2-(4-hydroxyphenoxy) propionic acid, the present invention screens multiple solvent systems and explores purification conditions to improve R -2-(4-Hydroxyphenoxy)propionic acid fermentation broth R -2-(4-hydroxyphenoxy)propionic acid yield and purity. First, heat the fermentation broth to above 30°C to reduce the viscosity of the fermentation broth to increase the speed of separation and extraction steps, while avoiding excessive system temperature that may cause the extractant to boil violently or accelerate volatilization. Adjust the system pH to 6.0-7.5, R -HPPA dissociates and dissolves in the water phase. At this time, an extractant is added to separate the metabolic waste of the strain, protein and other impurities. Adjust the pH of the water phase to 1-1.2, and dissolve the R -HPPA is partially precipitated in solid form, and partially transferred into the organic phase after adding an extractant. R The purity of 2-(4-hydroxyphenoxy)propionic acid is 85% to 95% on average, and the purity is more than 90%. DETAILED DESCRIPTION

[0021] The present invention is further described below with specific embodiments. Those of ordinary skill in the art will be able to implement the present invention based on these descriptions. In addition, the embodiments of the present invention involved in the following description are generally only embodiments of a part of the present invention, rather than all embodiments. Therefore, based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work should belong to the scope of protection of the present invention.

[0022] The strains of Beauveria bassiana used in the examples are all Beauveria bassiana ZJB23323 ( Beauveriabassiana ZJB23323 ), deposited in China Center for Type Culture Collection, deposit number: CCTCC NO: M2023584, deposit date: April 21, 2023, address: Wuhan University, Wuhan, China, 430072. This strain is a known strain. Beauveriabassiana ZJB16001 As the starting strain, it has been subjected to natural selection, UV mutagenesis, room temperature plasma (A R TP) mutagenesis, nitrosoguanidine mutagenesis and other steps, which have been disclosed in Chinese patent CN116590153A.

[0023] Example 1: Biofilm fermentation production R -2-(4-Hydroxyphenoxy)propionic acid S1. Beauveria bassiana ZJB23323 Inoculate into slant culture medium, culture at 28°C for 15 days, and obtain single bacterial colonies on slant; the slant culture medium is prepared by adding R -2-Phenoxypropionic acid 50 g / L, glucose 20 g / L, yeast extract 10 g / L, MgSO4 0.977 g / L, CaCl2 0.755 g / L, K2HPO4 1.8 g / L, KH2PO4 0.75 g / L, EDTA-2Na·2H2O 2 g / L, FeSO4·7H2O 0.6 g / L, ZnSO4·7H2O 0.2 g / L, MnSO4·H2O 0.15 g / L, NiCl·6H2O 0.04 g / L, CoCl2 0.02 g / L, H3BO3 0.03 g / L, Na2MoO4·H2O 0.005 g / L, agar 2 g / L, sterilize at 115℃ for 30 min and use after cooling.

[0024] S2, inoculating the single bacterial colony obtained in S1 into the fermentation medium, culturing at 28°C for 72 hours, and obtaining seed liquid; the fermentation medium is prepared by adding R-2-Phenoxypropionic acid 50 g / L, glucose 40 g / L, yeast extract 10 g / L, MgSO4 0.977 g / L, CaCl2 0.755 g / L, K2HPO4 1.8 g / L, KH2PO4 0.75 g / L, EDTA-2Na·2H2O 2 g / L, FeSO4·7H2O 0.6 g / L, ZnSO4·7H2O 0.2 g / L, MnSO4·H2O 0.15 g / L, NiCl·6H2O 0.04 g / L, CoCl2 0.02 g / L, H3BO3 0.03 g / L, Na2MoO4·H2O 0.005 g / L, sterilize at 115℃ for 30 min and use after cooling.

[0025] S3. The seed solution obtained in S2 is inoculated into fresh fermentation medium at a volume concentration of 10%, and biofilm fermentation culture is carried out at 28°C.

[0026] S4. On the 3rd and 5th days of biofilm fermentation culture, 10% (v / v) 600 g / L sterile glucose solution was supplemented.

[0027] S5: On the 3rd and 5th days of biofilm fermentation culture, 10% (v / v) 200 g / L sterile yeast extract solution was supplemented.

[0028] S6. Take the product of the 15th day of biofilm fermentation culture as the final fermentation product for subsequent separation and purification process.

[0029] Example 2: Chloroform extraction of 1L reaction system R -2-(4-Hydroxyphenoxy)propionic acid S1. Take 1L of fermentation broth and measure the content of R -The total content of HPPA is about 50.61g; S2. Vacuum the fermented liquid for 4 h under the conditions of 0.095 MPa vacuum and 50-55°C heating temperature, and collect the yellow clear filtrate in the collection bottle; S3, take the filtrate obtained in S2, adjust the pH to 6 with hydrochloric acid (pH test paper turns yellow), add 1L of chloroform while hot, stir for 30 minutes after addition, and separate the organic layer and the aqueous layer; S4. Take the water layer obtained in S3, and continue to adjust the pH to 1 with hydrochloric acid (the pH test paper turns red), and a large amount of solid precipitates; continue to add 1L of chloroform, stir for 30 minutes after the addition, and discard the water layer; S5, take the organic layer obtained in S4, and perform rotary evaporation at 20-25°C, 80rpm, and 0.095MPa vacuum. Stop the rotary evaporation after 50% of the volume of liquid is removed, and filter to obtain R-2-(4-Hydroxyphenoxy)propionic acid crude product; S6. Add the crude product to 100 mL of pure water, heat until the solution is clear, filter while hot, cool and crystallize; filter, and dry the obtained solid at 60±5°C with forced air for 10 h; get R The yield of -HPPA was 46.26 g, the recovery rate was 91.42%, and the purity measured by HPLC was 90.90%.

[0030] Example 3: Chloroform extraction of 1.5L reaction system R -2-(4-Hydroxyphenoxy)propionic acid S1, take 1.5L fermentation broth, and measure the content of R -The total content of HPPA is about 74.86g; S2-S6 are the same as Example 2, except that the vacuum rotary evaporation time of S2 is replaced with 6 hours; get R The yield of -HPPA was 67.91 g, the recovery rate was 90.71%, and the purity measured by HPLC was 90.21%.

[0031] Example 4: Chloroform extraction of 2L reaction system R -2-(4-Hydroxyphenoxy)propionic acid S1, take 2L of fermentation broth, and measure the content of R -The total content of HPPA is about 100.27g; S2-S6 are the same as Example 2, except that the vacuum rotary evaporation time of S2 is replaced with 8 hours; get R The yield of -HPPA was 90.22 g, the recovery rate was 89.98%, and the purity measured by HPLC was 90.78%.

[0032] Example 5: Dichloromethane extraction of 1L reaction system R -2-(4-Hydroxyphenoxy)propionic acid S1. Take 1L of fermentation broth and measure the content of R -The total content of HPPA is about 49.86g; S2-S6 are the same as Example 2, except that the heating temperature of S2 is changed to 30-35°C. get R The yield of -HPPA was 43.1 g, the recovery rate was 86.45%, and the purity measured by HPLC was 90.95%.

[0033] Example 6: Dichloromethane extraction of 1.5L reaction system R -2-(4-Hydroxyphenoxy)propionic acid S1, take 1.5L fermentation broth, and measure the content of R -The total content of HPPA is about 75.98g; S2-S6 are the same as Example 5, except that the vacuum rotary evaporation time of S2 is replaced with 6 hours; get R The yield of -HPPA was 65.32 g, the recovery rate was 85.97% (85.9699%), and the purity measured by HPLC was 90.75%.

[0034] Example 7: Dichloromethane extraction of 2L reaction system R -2-(4-Hydroxyphenoxy)propionic acid S1, take 2L of fermentation broth, and measure the content of R -The total content of HPPA is about 98.75g; S2-S6 are the same as Example 5, except that the vacuum rotary evaporation time of S2 is replaced with 8 hours; get R The yield of -HPPA was 84.89 g, the recovery rate was 85.97% (85.9645%), and the purity measured by HPLC was 90.92%.

[0035] Example 8: Extraction of 1L of reaction system with methyl isobutyl ketone R -2-(4-Hydroxyphenoxy)propionic acid S1. Take 1L of fermentation broth and measure the content of R -The total content of HPPA is about 51.82g; S2-S6 are the same as in Example 2, except that the heating temperature of S2 is replaced with 60-70°C; get R The yield of -HPPA was 48.34 g, the recovery rate was 93.28%, and the purity measured by HPLC was 91.10%.

[0036] Example 9: Extraction of 1.5 L of the reaction system with methyl isobutyl ketone R -2-(4-Hydroxyphenoxy)propionic acid S1, take 1.5L fermentation broth, and measure the content of R -The total content of HPPA is about 74.18g; S2-S6 are the same as Example 8, except that the vacuum rotary evaporation time of S2 is replaced with 6 hours; get R The yield of -HPPA was 70.14 g, the recovery rate was 94.55%, and the purity measured by HPLC was 90.96%.

[0037] Example 10: Methyl isobutyl ketone extraction of 2L reaction systemR -2-(4-Hydroxyphenoxy)propionic acid S1, take 2L of fermentation broth, and measure the content of R -The total content of HPPA is about 102.75g; S2-S6 are the same as Example 8, except that the vacuum rotary evaporation time of S2 is replaced with 8 hours; get R The yield of -HPPA was 96.37 g, the recovery rate was 93.79%, and the purity measured by HPLC was 90.85%.

[0038] Example 1 is a method for preparing a fermentation broth, and Examples 2-4, Examples 5-7, and Examples 8-10 respectively investigate the effects of chloroform, dichloromethane, and methyl isobutyl ketone on the fermentation broth of different volumes. R -2-(4-hydroxyphenoxy)propionic acid extraction effect. There is no obvious difference in the purity of the products obtained by the three extractants, and the overall value falls within the range of 90.21% to 91.10%; with the increase of the volume of the fermentation broth, the recovery rate of the same extractant fluctuates slightly, but the maximum amplitude does not exceed 1.5%. Compared with the three extractants, methyl isobutyl ketone has the highest requirements for heating conditions, but the recovery rate is relatively the highest; dichloromethane has the lowest requirements for heating conditions and the recovery rate is relatively the lowest, but it is still above 85%. The above data show that the extraction method of the present invention is suitable for large-volume extraction, and the obtained product has stable purity, high yield and controllable quality.

Claims

1. A method of separating from fermentation broth R -2-(4-hydroxyphenoxy)propionic acid extraction method, characterized in that, The following steps are involved: S1. Fermentation broth is obtained by fermentation culture of Beauveria bassiana, and the fermentation broth is heated to above 30°C, and then the solid and liquid are separated, and the liquid phase is collected; S2. The liquid phase obtained in S1 is taken, and the pH is adjusted to 6.0-7.5 at the heating temperature described in S1, and an extractant is added, stirred, and separated to obtain a first organic layer and a first aqueous layer; S3. The first aqueous layer is taken, and the pH is adjusted to 1-1.2, and the extractant is continued to be added, and stirred to obtain a reaction system including a second aqueous layer; S4. The reaction system is taken, the second aqueous layer is discarded, and the remaining components are concentrated, filtered, and dried to obtain R -2-(4-hydroxyphenoxy)propionic acid crude product; S5, recrystallizing the crude product to obtain R -2-(4-Hydroxyphenoxy)propionic acid crystals.

2. The extraction method according to claim 1, characterized in that: The heating temperature in S1 is 45-60° C., and the extractant is chloroform.

3. The extraction method according to claim 1, characterized in that: The heating temperature in S1 is 30-45° C., and the extractant is dichloromethane.

4. The extraction method according to claim 1, characterized in that: The heating temperature in S1 is 60-70° C., and the extractant is methyl isobutyl ketone.

5. The extraction method according to claim 1, characterized in that: The solid-liquid separation operation in S1 is to perform reduced pressure rotary evaporation under a vacuum degree of 0.090-0.095 MPa to collect the liquid phase.

6. The extraction method according to claim 1, characterized in that: The reagent used to adjust the pH in S2 and S3 is one of hydrochloric acid and sulfuric acid.

7. The extraction method according to claim 1, characterized in that: The concentration operation in S4 is vacuum rotary evaporation at a temperature of 20-35° C. and a vacuum degree of 0.090-0.095 MPa. The vacuum rotary evaporation endpoint is when the concentrate reaches 40%-70% of the original volume.

8. The extraction method according to claim 1, characterized in that: The drying operation in S4 is forced air drying at 55-65° C. for 10 hours.

9. The extraction method according to claim 1, characterized in that: The strain of Beauveria bassiana is Beauveria bassiana ZJB23323 ( Beauveria bassiana ZJB23323 ), deposited in China Center for Type Culture Collection, deposit number: CCTCC NO: M 2023584, deposit date: April 21, 2023, address: Wuhan University, Wuhan, China, 430072.

10. A R -2-(4-hydroxyphenoxy)propionic acid product, characterized in that: The extract is prepared by the extraction method according to any one of claims 1 to 9.