Method for purifying L-homoserine from fermentation liquor

Abstract

The invention provides a method for purifying L-homoserine from fermentation liquor, which adopts a mixed solvent recrystallization method and specifically comprises the following steps: (1) mixing the L-homoserine fermentation liquor with a poor solvent, and separating out precipitate to obtain a solid 1 and a filtrate 1; (2) mixing the solid 1 and a mixed solvent until the solid 1 is dissolved, performing phase splitting, and precipitating an upper-layer phase 1 to obtain a solid 2 and a filtrate 2; (3) mixing the solid 2 with a benign solvent, decoloring, mixing with a non-benign solvent, and carrying out phase splitting, so that an upper-layer phase 2 is separated out and precipitated to obtain a solid 3 and a filtrate 3; wherein the solid 3 is subjected to optional post-treatment such as pulping to obtain the pure product L-homoserine. The purification method established by the invention has the advantages of simple steps, mild conditions, low cost and high product purity, and the mixed solvent can be recycled in a recycling manner, so that the amount of three wastes is reduced, and environmental protection is facilitated.

Description

Technical Field

[0001] The invention belongs to the technical field of purification and relates to a method for purifying L-homoserine fermentation broth. Background Art

[0002] L-homoserine ((S)-2-amino-4-hydroxybutyric acid) is a non-protein amino acid. Its derivatives can not only be converted into other important chemical intermediates through enzymatic reactions, but also have rich biological activities, such as effectively inhibiting sickle cells and antifungal. In addition, L-homoserine is also an important intermediate for the synthesis of chiral herbicide L-phosphinothricin. Therefore, in terms of pharmacy and biology, the research on L-homoserine has received more and more attention from scientific researchers.

[0003] L-homoserine fermentation broth is a complex mixture. In addition to L-homoserine, it also contains a large amount of bacteria, protein, residual sugar, pigments and inorganic salts. The removal of protein, pigments and inorganic salts is the key to extracting L-homoserine. The commonly used decolorization and desalination methods in industry are activated carbon decolorization, ion exchange desalination or electrodialysis desalination. Activated carbon decolorization will produce a large amount of waste carbon, while intercepting part of the fermentation broth and reducing the product yield; ion exchange desalination will produce wastewater and consume a large amount of acid and alkali; electrodialysis technology has the disadvantages of long processing time, low desalination rate and low product purity.

[0004] Reference 1 provides a method for preparing L-homoserine, which uses L-aspartic acid as a raw material to prepare L-aspartic acid methyl ester or ethyl ester, and then uses a metal complex hydrogen compound as a reducing agent to prepare L-homoserine in an organic solvent at normal pressure. However, the purification steps of this method are cumbersome and the environmental pollution is serious.

[0005] Reference 2 provides a method for the biosynthesis of L-homoserine and its derivatives, which uses pyruvate and aldehyde compounds as substrates, reacts under the catalysis of aldolase, and the resulting product is sequentially subjected to chromatographic separation, desalting, decolorization, concentration crystallization, drying and other steps to obtain L-homoserine. In the post-treatment process, ion exchange chromatography is used as a key purification step, which has the advantages of high recovery rate, thorough impurity removal, and large processing capacity. However, a large amount of acid and alkali will be consumed during the resin regeneration process, and a large amount of wastewater will be generated, polluting the environment. In addition, formaldehyde is contained in the raw materials, posing a safety hazard.

[0006] References:

[0007] Reference 1: CN101100438A

[0008] Reference 2: CN105506014A Summary of the invention

[0009] Problem that the invention aims to solve

[0010] Since the L-homoserine fermentation broth product contains a large amount of soluble impurities, it is not conducive to subsequent process development. The existing purification methods often have problems such as low product purity, complex process, long processing time and serious environmental pollution. Based on the above problems, a new method for extracting L-homoserine is urgently needed.

[0011] Solutions for solving problems

[0012] [1] The present invention provides a method for purifying L-homoserine from a fermentation broth, comprising the following steps:

[0013] (1) Obtaining a segment of L-homoserine: mixing L-homoserine fermentation broth and a poor solvent, precipitating a precipitate, and filtering to obtain a solid 1 and a filtrate 1, wherein the solid 1 is a segment of L-homoserine;

[0014] (2) Obtaining the second-stage L-homoserine: mixing the first-stage L-homoserine and a mixed solvent until dissolved, separating the system into two phases, obtaining an upper phase 1 and a lower phase 1 by phase separation, precipitating the upper phase 1, and filtering to obtain a solid 2 and a filtrate 2, wherein the solid 2 is the second-stage L-homoserine;

[0015] (3) Obtaining three-stage L-homoserine: The two-stage L-homoserine is mixed with a good solvent until dissolved, and after decolorization, mixed with a bad solvent and heated to separate the system into two phases, and the phases are separated to obtain an upper phase 2 and a lower phase 2, and the upper phase 2 is precipitated, and filtered to obtain a solid 3 and a filtrate 3, wherein the solid 3 is the three-stage L-homoserine.

[0016] [2] The method according to [1], wherein:

[0017] The absolute content of L-homoserine in the fermentation broth is 30-55 wt %.

[0018] [3] The method according to [1] or [2], wherein:

[0019] The bad solvent is alcohol or nitrile;

[0020] and / or,

[0021] The benign solvent is water;

[0022] and / or,

[0023] The mass ratio of the first section of L-homoserine to the mixed solvent is 1:3 to 1:6;

[0024] and / or,

[0025] The mixed solvent is an alcohol aqueous solution.

[0026] [4] The method according to any one of [1] to [3], wherein in step (1),

[0027] The mass ratio of the L-homoserine fermentation broth to the poor solvent is 1:1.0 to 1:2.0;

[0028] and / or,

[0029] The precipitation temperature is 0 to 30°C;

[0030] and / or,

[0031] The L-homoserine is a wet product.

[0032] [5] The method according to any one of [1] to [4], wherein in step (1),

[0033] The filtrate 1 is used to recover the water-containing bad solvent;

[0034] Preferably, the recovery is carried out by distillation and optional concentration.

[0035] [6] The method according to any one of [1] to [5], wherein in step (2),

[0036] The dissolving temperature is 30 to 70°C;

[0037] and / or,

[0038] The temperature of the precipitation is below 20°C.

[0039] [7] The method according to any one of [1] to [6], wherein in step (2),

[0040] The lower phase 1 and / or the filtrate 2 are used to obtain additional second-stage L-homoserine;

[0041] Preferably, the additional two-stage L-homoserine is obtained by the following steps: heating the lower phase 1 and / or the filtrate 2 to separate the system into two phases, separating to obtain an upper phase 1' and a lower phase 1', precipitating the upper phase 1', filtering to obtain a solid 2' and a filtrate 2', wherein the solid 2' is the additional two-stage L-homoserine;

[0042] More preferably, in the step of obtaining the additional second-stage L-homoserine, the target temperature of the heating is 30-70°C; and / or the temperature of the precipitation is below 20°C.

[0043] [8] The method according to any one of [1] to [7], wherein in step (3),

[0044] The mass ratio of the second-stage L-homoserine to the benign solvent is 1:0.8-1:1;

[0045] and / or,

[0046] The dissolving temperature is 20 to 60°C;

[0047] and / or,

[0048] The decolorization temperature is 20-60°C; and / or, the decolorization is performed by activated carbon adsorption; preferably, the relative amount of the activated carbon is 0.5-1.5wt%;

[0049] and / or,

[0050] The mass ratio of the second-stage L-homoserine to the poor solvent is 1:1.5-1:2.5;

[0051] and / or,

[0052] The target temperature of the heating is 30 to 70°C;

[0053] and / or,

[0054] The temperature of the precipitation is below 20°C.

[0055] [9] The method according to any one of [1] to [7], wherein in step (3),

[0056] The lower phase 2 is used to obtain additional three segments of L-homoserine;

[0057] Preferably, the additional three-stage L-homoserine is obtained by the following steps: precipitating the lower phase 2, filtering to obtain a solid 3' and a filtrate 3', wherein the solid 3' is the additional three-stage L-homoserine;

[0058] More preferably, in the step of obtaining the additional three-stage L-homoserine, the temperature of the precipitation is below 20°C.

[0059]

[10] The method according to any one of [1] to [9], wherein the method further comprises the following steps:

[0060] (4) Obtaining pure L-homoserine: adding the three-stage L-homoserine to a poor solvent for slurrying, filtering to obtain a solid 4 and a filtrate 4, wherein the solid 4 is pure L-homoserine;

[0061] Preferably, in step (4), the poor solvent is an alcohol or a nitrile.

[0062] Effects of the Invention

[0063] 1. The present invention establishes a method for purifying L-homoserine fermentation broth, and obtains high-optical-purity L-homoserine with a high yield, and the product purity reaches more than 99%.

[0064] 2. The mixed solvent recrystallization of the present invention greatly reduces the production cost compared with the chemical method (L-homoserine lactone hydrochloride) in the prior art, improves the chiral purity, produces less wastewater than the fermentation bacteria method, and the solvent is recycled through negative pressure distillation, which is beneficial to environmental protection.

[0065] 3. The method of the present invention has simple steps, mild reaction conditions, low equipment requirements, and only needs to react at room temperature, without the need for high-temperature equipment in the prior art. DETAILED DESCRIPTION

[0066] Various exemplary embodiments, features and aspects of the present invention will be described in detail below. The word "exemplary" used here means "used as an example, embodiment or illustrative". Any embodiment described here as "exemplary" is not necessarily interpreted as being superior or better than other embodiments.

[0067] In addition, in order to better illustrate the present invention, numerous specific details are provided in the following specific embodiments. It should be understood by those skilled in the art that the present invention can be implemented without certain specific details. In other examples, methods, means, equipment and steps well known to those skilled in the art are not described in detail in order to highlight the subject matter of the present invention.

[0068] Unless otherwise stated, the units used in this specification are all international standard units, and the numerical values ​​and numerical ranges appearing in the present invention should be understood to include the inevitable systematic errors in industrial production.

[0069] In this specification, the word "may" means both performing a certain process and not performing a certain process.

[0070] In this specification, the references to "some specific / preferred embodiments", "other specific / preferred embodiments", "embodiments", etc., mean that the specific elements (e.g., features, structures, properties and / or characteristics) described in connection with the embodiments are included in at least one embodiment described herein, and may or may not exist in other embodiments. In addition, it should be understood that the elements may be combined in various embodiments in any suitable manner.

[0071] In this specification, the numerical range expressed using "a numerical value A to a numerical value B" means a range including the endpoints numerical values ​​A and B.

[0072] In this specification, when "normal temperature" or "room temperature" is used, the temperature may be 10-40°C.

[0073] The present invention provides a method for purifying L-homoserine from a fermentation broth, comprising the following steps:

[0074] (1) Obtaining a segment of L-homoserine: mixing L-homoserine fermentation broth and a poor solvent, precipitating a precipitate, and filtering to obtain a solid 1 and a filtrate 1, wherein the solid 1 is a segment of L-homoserine;

[0075] (2) Obtaining the second-stage L-homoserine: mixing the first-stage L-homoserine and a mixed solvent until dissolved, separating the system into two phases, obtaining an upper phase 1 and a lower phase 1 by phase separation, precipitating the upper phase 1, and filtering to obtain a solid 2 and a filtrate 2, wherein the solid 2 is the second-stage L-homoserine;

[0076] (3) Obtaining three-stage L-homoserine: The two-stage L-homoserine is mixed with a good solvent until dissolved, and after decolorization, mixed with a bad solvent and heated to separate the system into two phases, and the phases are separated to obtain an upper phase 2 and a lower phase 2, and the upper phase 2 is precipitated, and filtered to obtain a solid 3 and a filtrate 3, wherein the solid 3 is the three-stage L-homoserine.

[0077] In some specific embodiments, in step (1), the method for obtaining a segment of L-homoserine can be mixing L-homoserine fermentation broth with a poor solvent under stirring, controlling the temperature and stirring, precipitating a precipitate, filtering, and obtaining a solid 1 and a filtrate 1, wherein the solid 1 is a segment of L-homoserine.

[0078] In some specific embodiments, in step (1), the absolute content of L-homoserine in the fermentation broth is 30-55%wt%, for example, 32wt%, 35wt%, 39wt%, 40.2wt%, 41.22wt%, 43wt%, 47wt%, 51wt%, etc.

[0079] In some specific embodiments, the absolute content of L-homoserine in the fermentation broth is 35-45 wt %.

[0080] In some specific embodiments, the absolute content of L-homoserine in the fermentation broth is 39-41 wt %.

[0081] In some specific embodiments, the absolute content of L-homoserine in the fermentation broth is 40.2 wt %.

[0082] In some specific embodiments, in steps (1) and (3), the poor solvent is an alcohol or a nitrile, such as methanol, ethanol, acetonitrile, etc.

[0083] In some specific embodiments, the poor solvent is ethanol or acetonitrile.

[0084] In some specific embodiments, the poor solvent is ethanol.

[0085] In some specific embodiments, in step (1), the mass ratio of the L-homoserine fermentation broth to the poor solvent is 1:1.0 to 1:2.0, for example, 1:1.0, 1:1.2, 1:1.3, 1:1.5, 1:1.8, 1:2.0, etc.

[0086] In some specific embodiments, the mass ratio of the L-homoserine fermentation broth to the poor solvent is 1:1.0 to 1:1.5.

[0087] In some specific embodiments, the mass ratio of the L-homoserine fermentation broth to the poor solvent is 1:1.2 to 1:1.3;

[0088] In some specific embodiments, in step (1), the precipitation temperature is 0-30°C, for example, 0°C, 5°C, 18°C, 20°C, 30°C, etc.

[0089] In some specific embodiments, the precipitation temperature is 0-20°C.

[0090] In some specific embodiments, the precipitation temperature is 0-5°C.

[0091] In some specific embodiments, in step (1), the filtering method can be centrifugation or suction filtration, and the section of L-homoserine after filtration is a wet product, wherein the target component content is 45-75wt%; when the filtering method is suction filtration, the section of L-homoserine is a wet product with a target component content of 45-65wt%, for example, 45wt%, 50wt%, 55wt%, 60wt%, 65wt%; when the filtering method is centrifugation, L-homoserine is a wet product with a target component content of 50-75wt%, for example, 50wt%, 55wt%, 60wt%, 65wt%, 70wt%, 75wt%.

[0092] In some specific embodiments, the L-homoserine is a wet product having a target component content of 50-65 wt %.

[0093] In some specific embodiments, the L-homoserine is a wet product with a target component content of 50 wt%.

[0094] In some specific embodiments, in step (1), the filtrate 1 is used to recover the water-containing poor solvent.

[0095] In some more specific embodiments, said recovering is performed by distillation and optional concentration.

[0096] In some specific embodiments, in step (2), the method for obtaining the second-stage L-homoserine can be mixing the first-stage L-homoserine with a mixed solvent, heating and maintaining the temperature under stirring to separate the system into two phases, and obtaining an upper phase 1 and a lower phase 1 by phase separation; the upper phase 1 precipitates under stirring and cooling conditions, and is filtered to obtain a solid 2 and a filtrate 2, wherein the solid 2 is the second-stage L-homoserine.

[0097] In some specific embodiments, in step (2), the mixed solvent is an alcohol aqueous solution.

[0098] In some specific embodiments, the mixed solvent is an ethanol-water solution.

[0099] In some specific embodiments, the mixed solvent is an ethanol aqueous solution with a water content of 21-28 wt%, such as 21 wt%, 24 wt%, 26 wt%, 28 wt%, etc.

[0100] In some specific embodiments, the mixed solvent is an ethanol aqueous solution with a water content of 24-26 wt %.

[0101] In some specific embodiments, in step (2), the mass ratio of the section of L-homoserine to the mixed solvent is 1:3 to 1:6, for example, 1:3, 1:4, 1:5, 1:6, etc.

[0102] In some specific embodiments, the mass ratio of the segment of L-homoserine to the mixed solvent is 1:3.

[0103] In some specific embodiments, in step (2), the dissolution temperature is 30-70°C, for example, 40°C, 50°C, 60°C, 70°C, etc.

[0104] In some specific embodiments, the dissolving temperature is 60-70°C.

[0105] In some specific embodiments, the dissolving temperature is 60-64°C.

[0106] In some specific embodiments, in step (2), the temperature of the precipitation is below 20°C, such as 15°C, 10°C, 5°C, etc.

[0107] In some specific embodiments, the precipitation temperature is below 15°C.

[0108] In some specific embodiments, the precipitation temperature is below 5°C and greater than 0°C.

[0109] In some specific embodiments, in step (2), the lower phase 1 and / or the filtrate 2 are used to obtain additional second-stage L-homoserine.

[0110] In some more specific embodiments, the additional two-stage L-homoserine is obtained by the following steps: heating the lower phase 1 and / or the filtrate 2 to separate the system into two phases, separating to obtain an upper phase 1' and a lower phase 1', precipitating the upper phase 1', filtering to obtain a solid 2' and a filtrate 2', wherein the solid 2' is the additional two-stage L-homoserine;

[0111] In some more specific embodiments, in the step of obtaining the additional second-stage L-homoserine, the target temperature of the heating is 30-70°C, such as 40°C, 50°C, 60°C, 70°C.

[0112] In some more specific embodiments, the target temperature of the heating is 60-70°C.

[0113] In some more specific embodiments, the target temperature of the heating is 60-64°C.

[0114] In some more specific embodiments, in the step of obtaining the additional second-stage L-homoserine, the precipitation temperature is below 20°C.

[0115] In some more specific embodiments, the precipitation temperature is below 15°C.

[0116] In some more specific embodiments, the precipitation temperature is below 5°C and greater than 0°C.

[0117] In some specific embodiments, in step (3), the method for obtaining three-stage L-homoserine can be mixing the two-stage L-homoserine with a benign solvent until dissolved, decolorizing the dissolved solution, stirring, filtering, mixing the filtrate with a bad solvent, heating and maintaining the temperature under stirring to separate the system into two phases, and obtaining an upper phase 2 and a lower phase 2 by phase separation; stirring the upper phase 2, cooling it, and precipitating it, filtering it to obtain a solid 3 and a filtrate 3, wherein the solid 3 is the three-stage L-homoserine.

[0118] In some specific embodiments, in step (3), the benign solvent is water.

[0119] In some specific embodiments, in step (3), the mass ratio of the second-stage L-homoserine to the benign solvent is 1:0.8-1:1, for example, 1:0.8, 1:0.9, 1:1, etc.

[0120] In some specific embodiments, in step (3), the dissolving temperature is 20-60°C.

[0121] In some specific embodiments, the dissolving temperature is 20-30°C.

[0122] In some specific embodiments, in step (3), the decolorization temperature is 20-60°C.

[0123] In some specific embodiments, the decolorization temperature is 20-30°C.

[0124] In some specific embodiments, the decolorization is performed by adsorption on activated carbon.

[0125] In some more specific embodiments, the relative amount of activated carbon is 0.5-1.5 wt%, such as 0.5 wt%, 1 wt%, 1.5 wt%.

[0126] In some specific embodiments, in step (3), the mass ratio of the second-stage L-homoserine to the poor solvent is 1:1.5-1:2.5, for example, 1:1.5, 1:2, 1:2.5, etc.;

[0127] In some specific embodiments, in step (3), the target temperature of the heating is 30-70°C, for example, 40°C, 50°C, 60°C, 70°C.

[0128] In some specific embodiments, the target temperature of the heating is 60-70°C.

[0129] In some specific embodiments, the target temperature of the heating is 60-64°C.

[0130] In some specific embodiments, in step (3), the temperature of the precipitation is below 20°C, such as 15°C, 10°C, 5°C, etc.

[0131] In some specific embodiments, the precipitation temperature is below 15°C.

[0132] In some specific embodiments, the precipitation temperature is below 5°C and greater than 0°C.

[0133] In some specific embodiments, in step (3), the lower phase 2 is used to obtain additional three segments of L-homoserine.

[0134] In some more specific embodiments, the additional three-stage L-homoserine is obtained by the following steps: precipitating the lower phase 2, filtering to obtain a solid 3' and a filtrate 3', wherein the solid 3' is the additional three-stage L-homoserine;

[0135] More preferably, in the step of obtaining the additional three-stage L-homoserine, the temperature of the precipitation is below 20°C.

[0136] In some more specific embodiments, the precipitation temperature is below 15°C.

[0137] In some more specific embodiments, the precipitation temperature is below 5°C and greater than 0°C.

[0138] In some specific embodiments, the method further comprises the steps of:

[0139] (4) Obtaining pure L-homoserine: adding the three-stage L-homoserine to a poor solvent for slurrying, filtering to obtain a solid 4 and a filtrate 4, wherein the solid 4 is pure L-homoserine;

[0140] In some more specific embodiments, in step (4), the poor solvent is an alcohol or a nitrile.

[0141] In some more specific embodiments, in step (4), the poor solvent is ethanol or acetonitrile.

[0142] In some more specific embodiments, in step (4), the poor solvent is ethanol.

[0143] The embodiments of the present invention will be described in detail below in conjunction with the examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If no specific conditions are specified in the examples, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0144] Raw materials: L-homoserine fermentation broth.

[0145] Raw material analysis method: liquid chromatography.

[0146] Standard sample preparation: Accurately weigh 50 mg ± 0.5 mg of L-homoserine standard with a content of 98.5% into a 50 ml volumetric flask, dissolve it with 1:1 acetonitrile and water by ultrasonication, dilute to the scale, shake well, and inject 10 μl.

[0147] Sample preparation: Accurately weigh 125 mg ± 10 mg of L-homoserine sample with a content of about 40% into a 50 ml volumetric flask, dissolve it by ultrasonication with 1:1 acetonitrile and water, dilute to the scale, shake well, and inject 10 μl.

[0148] Moisture detection: Karl Fischer method.

[0149] Table 1 Analysis of components of L-homoserine fermentation broth

[0150]

[0151] Conclusion: There are more organic impurities in the stock solution and the pH value is neutral.

[0152] The fermentation liquid was directly concentrated to solid, and the inspection data after drying were as follows (see Table 2).

[0153] Table 2 Analysis of components of fermentation broth dry product

[0154]

[0155] According to the above detection, the components of the fermentation broth are analyzed as shown in Table 3.

[0156] Table 3 Analysis of fermentation broth components (mass percentage)

[0157]

[0158] According to theoretical analysis, when using this type of analysis method in fermentation broth, the response values ​​of impurities and L-homoserine are different, which is relatively of little reference significance. The L-homoserine stock solution contains about 40% L-homoserine, 10% inorganic salts, 45% water, and 3-4% organic impurities.

[0159] Example 1: Investigation of ethanol dosage and crystallization temperature in the preparation of one-stage L-homoserine

[0160] Preparation of a first-stage L-homoserine: Using ethanol as a poor solvent, the mass ratio of 1000 g of the fermentation broth to ethanol and the crystallization temperature are adjusted as shown in Table 4, and anhydrous ethanol is added to the L-homoserine stock solution under stirring conditions to precipitate a solid product, and stirred for crystallization for 2 hours. The solid in the system is filtered out by centrifugation, and the collected solid is a first-stage L-homoserine. The filtrate is concentrated by distillation to recover the aqueous ethanol, and the concentrated residual liquid is marked and collected as a first-stage residual liquid 1.

[0161] The L-homoserine stock solution used in this embodiment has an absolute L-homoserine content of 40.2%, is from the same batch of materials, and has the same water content.

[0162] The results at different ethanol dosages and different crystallization temperatures are shown in Table 4.

[0163] Table 4 Solvent ratio and crystallization temperature screening

[0164]

[0165] Since the filterability of the product is poor at too low a temperature, the product contains obvious impurities and the product purity is low, the temperature was investigated to be above 0°C. The results showed that the solvent ratio was 1.3 times of anhydrous ethanol, and cooling to 0-5°C could significantly increase the yield of L-homoserine.

[0166] Example 2: Investigation of dissolution temperature in the preparation of two-stage L-homoserine

[0167] Preparation of a solid L-homoserine: Using ethanol as a poor solvent, add anhydrous ethanol to 1000g of L-homoserine stock solution at a mass ratio of 1:1.3, control the temperature at 5°C, add anhydrous ethanol to the L-homoserine stock solution under stirring conditions, precipitate a solid product, and stir and crystallize for 2 hours. Filter out the solid in the system by centrifugation, and the collected solid is a solid L-homoserine. After testing, the purity of the L-homoserine solid is 84% ​​absolute and 2% water;

[0168] Preparation of two-stage L-homoserine: 100g of one-stage L-homoserine solid was mixed with aqueous ethanol (water content 28wt%) in a mass ratio of 1:3, heated to different dissolution temperatures (30°C, 40°C, 50°C, 60°C and 70°C), stirred for 0.5h, and allowed to stand for phase separation. The upper phase was cooled and crystallized at different temperatures (using gradient cooling of 15°C / h), and when cooled to 15°C, kept warm for 8h, and centrifuged to obtain the two-stage L-homoserine solid. The yield results of the two-stage L-homoserine at different dissolution temperatures are shown in Table 5.

[0169] Table 5 Second stage L-homoserine yield at different dissolution temperatures

[0170]

[0171] The results show that when the internal temperature of the solution is 60°C and the stirring is carried out for 0.5h, the yield of the L-homoserine product through phase separation is high. Too high a temperature will cause the system to boil, which is not conducive to the static conditions during phase separation. In addition, during the temperature increase from 60°C to 64°C, there is no obvious change in the dissolution situation in the system. Therefore, the preferred dissolution temperature is 60-64°C.

[0172] Example 3: Investigation of crystallization temperature in the preparation of two-stage L-homoserine

[0173] The preparation method of a section of L-homoserine solid is shown in Example 2.

[0174] 1000g of a first-stage L-homoserine solid was mixed with aqueous ethanol (water content 28wt%) in a mass ratio of 1:3, heated to a dissolution temperature of 60°C and stirred for 0.5h, and allowed to stand for phase separation. The upper phase was cooled and crystallized at different temperatures (gradient cooling 15°C / h was used), cooled to the crystallization temperature, kept warm for 8h, and centrifuged to obtain a second-stage L-homoserine solid and a filtrate. The results of residual L-homoserine in the filtrate at different crystallization temperatures are shown in Table 6.

[0175] Table 6 Cooling and standing two-stage L-homoserine precipitation temperature screening

[0176]

[0177] The results show that, within the optional temperature range above 0°C, the lower the crystallization temperature, the better the effect, preferably 5°C.

[0178] Example 4: Investigation of solvent ratio and solvent composition in the preparation of two-stage L-homoserine

[0179] The preparation method of a section of L-homoserine solid is shown in Example 2.

[0180] 1000g of the first-stage L-homoserine solid was mixed with water and ethanol in the mass ratio shown in Table 7, heated to the dissolution temperature of 60°C and stirred for 0.5h, and allowed to stand for phase separation. The upper phase was cooled and crystallized at 5°C (using a gradient cooling of 15°C / h), cooled to the crystallization temperature of 5°C, kept warm for 8h, and centrifuged to obtain the second-stage L-homoserine solid and filtrate. The yield of the second-stage L-homoserine and the phase separation results at different mass ratios of L-homoserine, water and ethanol are shown in Table 7.

[0181] Table 7 Screening of solvent ratio and solvent composition

[0182]

[0183] The results show that when the ratio of one section of L-homoserine to the solvent is 1:3 and the proportion of water in the solvent is 28wt%, the yield of the product is higher and the quality is better.

[0184] Example 5: Investigation of solvent components in the three-stage L-homoserine preparation

[0185] The preparation method of the second-stage L-homoserine is shown in Example 4. The obtained second-stage crystallization product is dried at 70° C. and vacuum negative pressure of 0.95 MPa for 4-6 hours, and the moisture content is controlled below 0.5%. The absolute content of L-homoserine is detected, and the purity is 92%-95%.

[0186] The second stage L-homoserine was dissolved in water at a mass ratio of 1:0.84, 1:0.9, 1:1, and 1:1.08, respectively, and the dissolution temperature was 20-30°C. 1wt% activated carbon was added to the system, and the temperature was kept at 20-30°C for constant decolorization for 0.5h, and the filtrate was filtered. The ratio of the second stage L-homoserine to the mixed solvent was kept at 1:3, and anhydrous ethanol was added to the filtrate at a mass ratio of 1:2.16, 1:2.1, 1:2, and 1:1.92, respectively, and the system was heated to 60°C, kept at a constant temperature for 0.5h, and the upper phase 1 and the lower phase 1 were obtained by constant temperature phase separation. The upper phase 1 was slowly cooled to 5°C and kept at a constant temperature for 8 hours, and then centrifuged and filtered to obtain crystals 1 and filtrate 1; the lower phase 1 was naturally crystallized and filtered to obtain crystals 2 and filtrate 2. The three-stage L-homoserine yields and phase separation results are shown in Table 8.

[0187] Among them, filtrate 1 can be used as the second-stage solvent.

[0188] Table 8 Three-stage solvent moisture screening

[0189]

[0190] The results showed that when the solvent water ratio was 33% (i.e., when the ratio of second-stage L-homoserine: water: ethanol was 1:1:2), the lower phase was less and the third-stage L-homoserine yield was higher.

[0191] When the ratio of second-stage L-homoserine: water: ethanol is 1:1:2, the collected crystals 1 and 2 are mixed and dried under negative pressure vacuum drying (-0.95 MPa, 60-70°C) to obtain third-stage L-homoserine solid. After testing, the purity is 97% and the moisture content is 0.1%.

[0192] It should be noted that although the technical solutions of the present invention are described with specific examples, those skilled in the art will appreciate that the present invention should not be limited thereto. The various embodiments of the present invention have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Without departing from the scope and spirit of the described embodiments, many modifications and changes are obvious to those of ordinary skill in the art. The choice of terms used herein is intended to best explain the principles of the embodiments, practical applications, or technical improvements in the market, or to enable other persons of ordinary skill in the art to understand the embodiments disclosed herein.

Claims

1. A method for purifying L-homoserine from a fermentation broth, comprising the following steps: (1) Obtaining a segment of L-homoserine: mixing L-homoserine fermentation broth and a poor solvent, precipitating a precipitate, and filtering to obtain a solid 1 and a filtrate 1, wherein the solid 1 is a segment of L-homoserine; (2) Obtaining the second-stage L-homoserine: mixing the first-stage L-homoserine and a mixed solvent until dissolved, separating the system into two phases, obtaining an upper phase 1 and a lower phase 1 by phase separation, precipitating the upper phase 1, and filtering to obtain a solid 2 and a filtrate 2, wherein the solid 2 is the second-stage L-homoserine; (3) Obtaining three-stage L-homoserine: The two-stage L-homoserine is mixed with a good solvent until dissolved, and after decolorization, mixed with a bad solvent and heated to separate the system into two phases, and the phases are separated to obtain an upper phase 2 and a lower phase 2, and the upper phase 2 is precipitated, and filtered to obtain a solid 3 and a filtrate 3, wherein the solid 3 is the three-stage L-homoserine.

2. The method according to claim 1, characterized in that The absolute content of L-homoserine in the fermentation broth is 30-55 wt %, preferably 35-45 wt %, more preferably 39-41 wt %, and further preferably 40.2 wt %.

3. The method according to claim 1 or 2, characterized in that: The poor solvent is an alcohol or a nitrile, preferably ethanol or acetonitrile, more preferably ethanol; and / or, The benign solvent is water; and / or, The mass ratio of the first section of L-homoserine to the mixed solvent is 1:3 to 1:6, preferably 1:3; and / or, The mixed solvent is an alcohol aqueous solution, preferably an ethanol aqueous solution, more preferably an ethanol aqueous solution with a water content of 21-28 wt %, and further preferably an ethanol aqueous solution with a water content of 24-26 wt %.

4. The method according to any one of claims 1 to 3, characterized in that: In step (1), The mass ratio of the L-homoserine fermentation broth to the poor solvent is 1:1.0 to 1:2.0, preferably 1:1.0 to 1:1.5, and more preferably 1:1.2 to 1:1.3; and / or, The precipitation temperature is 0 to 30°C, preferably 0 to 20°C, more preferably 0 to 5°C; and / or, The L-homoserine is a wet product, preferably a wet product with a target component content of 45-75wt%, more preferably a wet product with a target component content of 50-65wt%, and further preferably a wet product with a target component content of 50wt%.

5. The method according to any one of claims 1 to 4, characterized in that: In step (1), The filtrate 1 is used to recover the water-containing bad solvent; Preferably, the recovery is carried out by distillation and optional concentration.

6. The method according to any one of claims 1 to 5, characterized in that: In step (2), The dissolving temperature is 30-70°C, preferably 60-70°C, more preferably 60-64°C; and / or, The temperature of the precipitation is below 20°C, preferably below 15°C, more preferably below 5°C, and greater than 0°C.

7. The method according to any one of claims 1 to 6, characterized in that: In step (2), The lower phase 1 and / or the filtrate 2 are used to obtain additional second-stage L-homoserine; Preferably, the additional two-stage L-homoserine is obtained by the following steps: heating the lower phase 1 and / or the filtrate 2 to separate the system into two phases, separating to obtain an upper phase 1' and a lower phase 1', precipitating the upper phase 1', filtering to obtain a solid 2' and a filtrate 2', wherein the solid 2' is the additional two-stage L-homoserine; More preferably, in the step of obtaining the additional second-stage L-homoserine, the target temperature of the heating is 30-70°C, preferably 60-70°C, more preferably 60-64°C; and / or the temperature of the precipitation is below 20°C, preferably below 15°C, more preferably below 5°C, and greater than 0°C.

8. The method according to any one of claims 1 to 7, characterized in that: In step (3), The mass ratio of the second-stage L-homoserine to the benign solvent is 1:0.8-1:1; and / or, The dissolving temperature is 20 to 60°C, preferably 20 to 30°C; and / or, The decolorization temperature is 20-60°C, preferably 20-30°C; and / or, the decolorization is performed by activated carbon adsorption; preferably, the relative amount of the activated carbon is 0.5-1.5wt%; and / or, The mass ratio of the second-stage L-homoserine to the poor solvent is 1:1.5-1:2.5; and / or, The target temperature of the heating is 30 to 70°C, preferably 60 to 70°C, more preferably 60 to 64°C; and / or, The temperature of the precipitation is below 20°C, preferably below 15°C, more preferably below 5°C, and greater than 0°C.

9. The method according to any one of claims 1 to 7, characterized in that: In step (3), The lower phase 2 is used to obtain additional three segments of L-homoserine; Preferably, the additional three-stage L-homoserine is obtained by the following steps: precipitating the lower phase 2, filtering to obtain a solid 3' and a filtrate 3', wherein the solid 3' is the additional three-stage L-homoserine; More preferably, in the step of obtaining the additional three-stage L-homoserine, the temperature of the precipitated precipitate is below 20°C, preferably below 15°C, more preferably below 5°C, and greater than 0°C.

10. The method according to any one of claims 1 to 9, characterized in that: The method further comprises the steps of: (4) Obtaining pure L-homoserine: adding the three-stage L-homoserine to a poor solvent for slurrying, filtering to obtain a solid 4 and a filtrate 4, wherein the solid 4 is pure L-homoserine; Preferably, in step (4), the poor solvent is an alcohol or a nitrile, preferably ethanol or acetonitrile, more preferably ethanol.

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