Method for separating and purifying histidine from fermentation liquor

Abstract

The invention discloses a method for separating and purifying histidine from fermentation liquor, and belongs to the technical field of amino acid preparation. The invention provides a method for purifying high-purity low-emission L-histidine, which comprises the following steps of: filtering and sterilizing L-histidine fermentation liquor, adsorbing with sodium cation exchange resin, washing with pure water to remove impurities, eluting resin with sodium hydroxide solution, decoloring collected liquid with activated carbon, and finally concentrating, crystallizing and desalting by utilizing the characteristic of low solubility of the L-histidine to obtain the high-purity low-emission L-histidine. The purity (greater than 98.5%) and the light transmittance (greater than 99.5%) of the L-histidine are effectively improved, and the yield of the histidine is improved. By using the method, the resin does not need to be regenerated, so that the acid-base consumption is reduced, the economic cost is reduced, and the emission of three wastes is reduced.

Description

Technical Field

[0001] The invention relates to a method for separating and purifying histidine from fermentation liquid, belonging to the technical field of amino acid preparation. Background Art

[0002] L-Histidine (His for short), also known as L-α-amino-β-imidazole propionic acid, is a basic amino acid with an imidazole group in the side chain. Its molecular formula is C6H9N3O2, with a relative molecular mass of 155.16. It is a colorless needle-shaped or flaky crystal with a slightly bitter taste and no odor. Its solubility in water at 25°C is 4.25g / 100g. It is insoluble or extremely difficult to dissolve in ethanol and ether. Its isoelectric point pI at 25°C is 7.59.

[0003] L-histidine has multiple physiological functions and is widely used in various fields such as food and medicine. L-histidine is an extremely important component of amino acid preparations and amino acid infusions. It can also be used to make drugs for the treatment of anemia, heart disease, rheumatoid arthritis, etc. L-histidine contains imidazole heterocycles, which can maintain intracellular pH stability. L-histidine and β-alanine form carnosine, which has a series of physiological functions such as delaying cell aging, anti-oxidation, chelating metal ions, and neuromodulation.

[0004] At present, the main methods for preparing L-histidine can be divided into protein hydrolysis, chemical synthesis and microbial fermentation. Microbial fermentation is the mainstream method for producing L-histidine. Ion exchange purification method is usually required to obtain high-purity amino acids from fermentation broth. The traditional ion exchange method is to adjust the pH of the fermentation broth of L-histidine to 2-4, and combine it with a hydrogen-type cation exchange column. The hydrogen-type cation exchange column has a strong binding ability with L-histidine, which can ensure the yield of L-histidine during the purification process. Therefore, the traditional ion exchange method usually uses the above-mentioned ion exchange column to purify L-histidine. However, when the content of other amino acids in the fermentation broth is high, the purity of the product obtained by this method is not ideal (the purity of the fermentation broth produced by different strains and different fermentation conditions is different, usually only 90% to 95%), and the product usually contains a small amount of other amino acids such as alanine, branched-chain amino acids, etc. At the same time, a large amount of strong acid needs to be used to regenerate the ion exchange resin during the purification process; and the use of ammonium-type cation exchange resin requires the use of ammonia water for elution, but the strong volatility of ammonia water is not conducive to environmental protection. The use of these two ion exchange methods to purify L-histidine consumes a large amount of acid and alkali and produces a large amount of waste liquid, which is neither economical nor environmentally friendly.

[0005] Therefore, the prior art lacks a process that can avoid the generation of acidic or alkaline waste liquid during the purification process of L-histidine fermentation broth while ensuring the purity and yield of L-histidine. Summary of the invention

[0006] In view of the defects and shortcomings of the prior art, the present invention provides a high-purity and low-emission L-histidine purification method, which comprises the following steps: filtering and sterilizing the L-histidine fermentation liquid, adsorbing it through a sodium-type cation exchange resin, washing impurities with pure water, eluting the resin with a sodium hydroxide solution, decolorizing the collected liquid through activated carbon, and finally concentrating, crystallizing and desalting the liquid by utilizing the low solubility of L-histidine, thereby effectively improving the purity (>98.5%) and transmittance (>99.5%) of L-histidine, and improving the yield of L-histidine by applying the eluent and recycling the mother liquor. The resin does not need to be regenerated by using the method, thereby reducing the amount of acid and alkali, reducing the economic cost, and reducing the discharge of the three wastes.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] (1) adjusting the pH of the L-histidine fermentation broth, filtering it through a ceramic membrane, and obtaining a filtrate;

[0009] (2) the filtrate is purified by sodium cation exchange resin, impurities are washed away with pure water, the resin is eluted with sodium hydroxide solution, and the effluent with pH ≤ 9 is collected, and the effluent with pH > 9 is used as the eluent;

[0010] (3) decolorizing the collected solution in the previous step by means of activated carbon to obtain a decolorized solution;

[0011] (4) pouring the decolorized liquid into a reaction kettle for crystallization, and concentrating under vacuum to obtain wet crystals and mother liquor, and recycling the mother liquor for reuse;

[0012] (5) The wet crystals and the mother liquor are cooled to room temperature, separated by suction filtration, and dried to obtain L-histidine crystals.

[0013] In one embodiment, the pH of the fermentation broth in step (1) is adjusted to 5.5-6.0 using sulfuric acid.

[0014] In one embodiment, the pore size of the ceramic membrane described in step (1) is 50-100 nm, the operating pressure is 0.1-0.2 MPa, the flow rate is 5-10 L / h, and the temperature is controlled to be below 50°C.

[0015] In one embodiment, the sodium type cation exchange resin in step (2) is 732 or HZ016 cation exchange resin, the sodium hydroxide elution concentration is 0.5% to 2%, and the elution flow rate is 0.5 to 1.0 L / h.

[0016] In one embodiment, the flow rate of the filtrate through the column in step (2) is 0.5-1.0 L / h.

[0017] In one embodiment, the activated carbon in step (3) is medicinal carbon, the dosage is 1% to 3%, the decolorization temperature is room temperature, and the time is 0.5 to 1.0 h.

[0018] In one embodiment, the reactor in step (4) is concentrated under vacuum with a vacuum degree of 0.09-0.098 MPa, a concentration temperature of 55-65° C., and a stirring speed of 100-150 r / min. Concentration is stopped when the solid content reaches 30%-40%.

[0019] In one embodiment, the wet crystals in step (5) are cooled to room temperature, which is 20-30°C.

[0020] In one embodiment, the drying method described in step (5) uses a vacuum dryer with a vacuum degree of 0.12±0.01 MPa, a drying temperature of 45-55° C., and a drying time of 3-6 hours.

[0021] Beneficial effects:

[0022] The present invention uses a sodium-type cation exchange resin, which can effectively separate L-histidine from other amino acids with similar isoelectric points from fermentation broth, compared with a hydrogen-type cation exchange resin, and greatly improves the purity (>98.5%) and transmittance (>99.5%) of L-histidine. At the same time, the yield of L-histidine is improved (>89.8%) by applying the eluent and recycling the mother liquor.

[0023] The present invention uses sodium type cation exchange resin and sodium hydroxide as eluent. When elution is completed, the resin has been transformed into sodium type and does not need to be regenerated. Compared with hydrogen type / ammonium type cation exchange resin, no regeneration liquid is used and no acidic or volatile alkaline pollutants are generated. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the pure water (blank) chromatogram.

[0025] Figure 2 This is the chromatogram of L-histidine (His) standard.

[0026] Figure 3 This is the chromatogram of the L-histidine (His) sample of Example 1.

[0027] Figure 4 This is the chromatogram of the L-histidine (His) sample of Example 2.

[0028] Figure 5 This is the chromatogram of the L-histidine (His) sample of Example 3.

[0029] Figure 6This is the chromatogram of the L-histidine (His) sample of Comparative Example 1.

[0030] Figure 7 This is the chromatogram of the L-histidine (His) sample of Comparative Example 2.

[0031] Figure 8 This is the chromatogram of the L-histidine (His) sample of Comparative Example 3.

[0032] Fig. 9 This is the chromatogram of the L-histidine (His) sample of Comparative Example 4.

[0033] Fig.10 This is the chromatogram of the L-histidine (His) sample of Comparative Example 5.

[0034] Fig.11 This is the chromatogram of the L-histidine (His) sample of Comparative Example 6.

[0035] Fig.12 This is the chromatogram of the L-histidine (His) sample of Comparative Example 7. DETAILED DESCRIPTION

[0036] The present invention is further described below in conjunction with specific embodiments and comparative examples.

[0037] The percentages used in the present invention, unless otherwise specified, represent W / V, such as 2% NaOH solution means that the content of sodium hydroxide is 20 g / L.

[0038] The test method involved in the present invention is:

[0039] 1. Determination method of L-histidine concentration: refer to the pre-column OPA and FMOC derivatized amino acid determination method (high performance liquid chromatography, HPLC) in the 9120 Amino Acid Analysis Guidelines in the First Supplement to the 2020 Edition of the Pharmacopoeia of the People's Republic of China.

[0040] 2. Calculation method of L-histidine purity: L-histidine purity = C × V / m × 100%

[0041] Where C is the L-histidine concentration (g / L), V is the volume of the sample solution (L), and m is the mass of the sample.

[0042] 3. Transmittance detection method: Referring to the 2020 edition of the "Pharmacopoeia of the People's Republic of China", take 0.60g of this product, add 20mL of water to dissolve it, and then measure the transmittance at a wavelength of 430nm according to the UV-visible spectrophotometry (General Rule 0401), which shall not be less than 98.0%.

[0043] 4. pH detection method: Referring to the 2020 edition of the "Pharmacopoeia of the People's Republic of China", take 1.0g of this product, add 50mL of water to dissolve it, and determine it according to the law (General Rule 0631). The pH value should be 7.0-8.5.

[0044] 5. Detection method of loss on drying: Refer to the 2020 edition of the "Pharmacopoeia of the People's Republic of China", take this product, dry it at 105°C for 3 hours, and the weight loss shall not exceed 0.2% (General Rule 0831).

[0045] The HZ016, 732 and D155 cation exchange resins used in the following embodiments are purchased from Shanghai Huazhen Technology Co., Ltd. (similar resin products from other companies are also applicable to the present invention).

[0046] The main components contained in the L-histidine (His) fermentation broth of the following examples are shown in Table 1.

[0047] Table 1 L-histidine fermentation broth components

[0048] name Concentration (g / L) Isoelectric point (pI) L-Histidine (His) 20.23～25.35 7.59 L-Glutamic Acid (Glu) 0.5～2.8 3.22 L-Alanine (Ala) 0.8～2.1 6.02 L-Valine (Val) 0.23～1.15 5.96 L-Leucine (Leu) 0.12～0.22 5.98 L-Isoleucine (Ile) 0.05～0.07 6.02 L-Methionine (Met) 0.8～1.2 5.74 Inorganic salt 4.0～5.0 —

[0049] Example 1

[0050] (1) Fermentation broth pretreatment

[0051] Add dilute sulfuric acid to 10L of sterilized histidine fermentation broth to adjust the pH to 5.53 and mix well. Add the fermentation broth to a 50nm ceramic membrane filtration device, control the pressure at 0.1-0.2MPa, the flow rate at 5-10L / h, and the temperature below 80°C, and collect the filtrate.

[0052] (2) Ion exchange purification

[0053] The filtered histidine fermentation broth is passed through a sodium-type 732 strong acid cation exchange column, with a resin volume of about 2L and a column flow rate of about 1.0L / h. When indolequinone is detected to be permeable to histidine, the column is stopped, and the unadsorbed amino acid fermentation broth in the column is washed out and collected with 1 column volume of pure water, the pH is adjusted and the above process is repeated, and the unadsorbed impurities are washed out with 2 column volumes of pure water, and the water washing liquid flow rate is 1.0L / h. After the water washing is completed, 2% NaOH eluent is used for elution, and the eluent flow rate is 0.5L / h. When the refractive index is ≥0.5%, collection begins, and elution is performed until no amino acid is permeated by indolequinone detection. The collected liquid with pH≤9 is selected for the next step of purification, and the collected liquid with pH>9 is recycled as an eluent for reuse. At this time, the 732 strong acid cation exchange resin has been converted to sodium type, and no regeneration is required. It can be reused by simply rinsing with pure water until it is close to neutral.

[0054] (3) Decolorization

[0055] The collected liquid was decolorized with 1% activated carbon and stirred at 200 r / min for 0.5 h at room temperature, and then filtered through a Buchner funnel to remove the activated carbon.

[0056] (4) Concentration and crystallization

[0057] Adjust the pH of the decolorized liquid to 7.59 and add it to the reactor for concentration and crystallization. The vacuum degree is 0.09-0.098MPa, the concentration temperature is 60-65℃, and the stirring speed is 120r / min. Stop the concentration when the solid content is 40%. Pour out the wet crystals, cool to room temperature and filter to obtain histidine wet crystals and mother liquor. The mother liquor contains a high salt content, which is recovered to adjust the pH and repeat the column loading process.

[0058] (5) Drying

[0059] The wet crystals were placed in a vacuum dryer and dried at a vacuum degree of 0.12±0.01 MPa at 50°C for 3-4 h to obtain histidine crystals.

[0060] The final yield of histidine in this method was 91.5%. The concentration of histidine crystals was determined by the HPLC method of pre-column OPA and FMOC-derived amino acids. The purity of histidine was calculated to be 98.8%, the transmittance was measured to be 99.6%, the acidity was 7.78, the loss on drying was 0.05%, and no other amino acids were detected, such as Figure 3 .

[0061] Example 2

[0062] (1) Fermentation broth pretreatment

[0063] Add dilute sulfuric acid to 10L of sterilized histidine fermentation broth to adjust the pH to 6.00 and mix well. Add the fermentation broth to a 50nm ceramic membrane filtration device, control the pressure at 0.1-0.2MPa, the flow rate at 5-10L / h, the temperature at below 80°C, and collect the filtrate.

[0064] (2) Ion exchange purification

[0065] The filtered histidine fermentation broth is passed through the sodium-type HZ016 strong acid cation exchange column, the resin volume is about 2L, and the column flow rate is about 1.0L / h. When the histidine is detected to be leaking out by indolequinone, the column is stopped, and the unadsorbed amino acid fermentation broth in the column is washed out and collected with 1 column volume of pure water, the pH is adjusted and the above process is repeated, and the unadsorbed impurities are washed out with 2 column volumes of pure water, and the water washing liquid flow rate is 1.0L / h. After the water washing is completed, eluted with 0.5% NaOH eluent, the eluent flow rate is 0.5L / h, and the collection begins when the refractive index is ≥0.5%, and eluted until no amino acid is leaked by indolequinone detection. The collected liquid with pH≤9 is selected for the next step of purification, and the collected liquid with pH>9 is recovered as the eluent for reuse. At this time, the HZ016 strong acid cation exchange resin has been converted to sodium type, and no regeneration is required. It can be reused by simply rinsing with pure water until it is close to neutral.

[0066] (3) Decolorization

[0067] The collected liquid was decolorized with 3% activated carbon and stirred at 200 r / min for 0.5 h at room temperature, and then filtered through a Buchner funnel to remove the activated carbon.

[0068] (4) Concentration and crystallization

[0069] Adjust the pH of the decolorized liquid to 7.59 and add it to the reactor for concentration and crystallization. The vacuum degree is 0.09-0.098MPa, the concentration temperature is 60-65℃, and the stirring speed is 120r / min. Stop the concentration when the solid content reaches 30%. Pour out the wet crystals, cool to room temperature and filter to obtain histidine wet crystals and mother liquor. The mother liquor contains a high salt content, which is recovered to adjust the pH and repeat the column loading process.

[0070] (5) Drying

[0071] The wet crystals were placed in a vacuum dryer and dried at a vacuum degree of 0.12±0.01 MPa at 45°C for 6 h to obtain histidine crystals.

[0072] The final yield of histidine in this method was 89.8%. The concentration of histidine crystals was determined by the HPLC method of pre-column OPA and FMOC-derived amino acids. The purity of histidine was calculated to be 99.2%, the transmittance was 99.8%, the acidity was 7.70, the loss on drying was 0.03%, and no other amino acids were detected, such as Figure 4 .

[0073] Example 3

[0074] The only difference from Example 1 is that the pore size of the ceramic membrane is 100 nm, the pH of the fermentation liquid is adjusted to 5.5 before loading, the decolorization time is 1 hour, and the wet crystals are dried at 55° C. for 3 hours.

[0075] By the above method, the final yield of histidine was 90.5%. The concentration of histidine crystals was detected by the HPLC method of pre-column OPA and FMOC-derived amino acids. The purity of histidine was calculated to be 99.3%, the transmittance was measured to be 99.9%, the acidity was 7.71, the loss on drying was 0.02%, and no other amino acids were detected, such as Figure 5 .

[0076] Comparative Example 1

[0077] The only difference from Example 1 is that the unadsorbed amino acid fermentation liquid in the column is not washed out and collected and re-loaded on the column, the collected liquid with pH>9 is not recovered, and the mother liquor is not recovered.

[0078] The final yield of histidine in this method was 65.8%. The concentration of histidine crystals was determined by the HPLC method of pre-column OPA and FMOC-derived amino acids. The purity of histidine was calculated to be 99.4%, the transmittance was measured to be 99.5%, the acidity was 7.60, the loss on drying was 0.03%, and no other amino acids were detected, such as Figure 6 .

[0079] Comparative Example 2

[0080] The only difference from Example 1 is that the ion exchange purification step uses a hydrogen-type HZ016 strong acid cation exchange column, and after elution, 2 to 3 column volumes of 4% to 4.5% dilute hydrochloric acid are required to regenerate the resin, and then rinse with pure water until it is close to neutral.

[0081] By the above method, the final yield of histidine was 86.5%. The concentration of histidine crystals was detected by the HPLC method of pre-column OPA and FMOC-derived amino acids. The purity of histidine was calculated to be 91.5%, the transmittance was 91.3%, the acidity was 7.46, the loss on drying was 0.03%, and the sample was found to contain 2.5% alanine (Ala). Figure 7 .

[0082] Comparative Example 3

[0083] The only difference from Example 1 is that the ion exchange purification step uses a hydrogen-type 732 strong acid cation exchange column, and after elution, 2 to 3 column volumes of 4% to 4.5% dilute hydrochloric acid are required to regenerate the resin, and then rinse with pure water until it is close to neutral.

[0084] By the above method, the final yield of histidine was 88.3%. The concentration of histidine crystals was detected by the HPLC method of pre-column OPA and FMOC-derived amino acids. The purity of histidine was calculated to be 90.3%, the transmittance was measured to be 90.2%, the acidity was 7.54, the loss on drying was 0.10%, and the sample was detected to contain 4.5% alanine (Ala). Figure 8 .

[0085] Comparative Example 4

[0086] The only difference from Example 1 is that the ion exchange purification step uses a hydrogen-type D155 weakly acidic cation exchange column, and after elution, 3 to 5 column volumes of 4% to 4.5% dilute hydrochloric acid are required to regenerate the resin, and then rinse with pure water until it is close to neutral.

[0087] By the above method, the final yield of histidine was 80.1%. The concentration of histidine crystals was detected by the HPLC method of pre-column OPA and FMOC-derived amino acids. The purity of histidine was calculated to be 95.4%, the transmittance was measured to be 93.5%, the acidity was 7.65, the loss on drying was 0.06%, and the sample contained 2.4% alanine (Ala). Fig. 9 .

[0088] Comparative Example 5

[0089] The only difference from Example 1 is that the ion exchange purification step uses an ammonium-type HZ016 strongly acidic cation exchange column, and the eluent is 0.8 mol / L ammonia water. After the elution, the resin has been converted to an ammonium type and does not need to be regenerated. It can be reused by simply washing with pure water until it is close to neutral. However, the volatility of the eluent using ammonia water is relatively large, which is not conducive to environmental protection.

[0090] By the above method, the final yield of histidine was 86.5%. The concentration of histidine crystals was detected by the HPLC method of pre-column OPA and FMOC-derived amino acids. The purity of histidine was calculated to be 98.2%, the transmittance was measured to be 98.9%, the acidity was 7.70, the loss on drying was 0.08%, and no other amino acids were detected, such as Fig.10 .

[0091] Comparative Example 6

[0092] The only difference from Example 1 is that the ion exchange purification step uses an ammonium type 732 strong acid cation exchange column, and uses sodium hydroxide of the same concentration as the eluent. After the elution, the resin has been converted to the sodium type. To convert the resin to the ammonium type, 3 to 5 column volumes of 5% to 10% ammonia water must be used for regeneration. The ammonia water used as the regeneration liquid is highly volatile and is not conducive to environmental protection.

[0093] By the above method, the final yield of histidine was 88.2%. The concentration of histidine crystals was detected by the HPLC method of pre-column OPA and FMOC-derived amino acids. The purity of histidine was calculated to be 98.8%, the transmittance was measured to be 99.5%, the acidity was 7.66, the loss on drying was 0.03%, and no other amino acids were detected, such as Fig.11 .

[0094] Comparative Example 7

[0095] The only difference from Example 1 is that dilute sulfuric acid is added to the sterilized 10 L histidine fermentation broth to adjust the pH to 6.5.

[0096] By the above method, the final yield of histidine was 82.2%. The concentration of histidine crystals was detected by the HPLC method of pre-column OPA and FMOC-derived amino acids. The purity of histidine was calculated to be 98.9%, the transmittance was measured to be 99.8%, the acidity was 7.65, the loss on drying was 0.03%, and no other amino acids were detected, such as Fig.12 .

[0097] The present invention is disclosed as above with preferred embodiments, and these embodiments are not used to limit the protection scope of the present invention. Any modification made by any person skilled in the field of separation and purification without departing from the spirit of the present invention shall fall within the protection scope of the present invention.

Claims

1. A method for separating L-histidine from a fermentation broth, characterized in that: The pH of the fermentation liquid is adjusted to 5.0-6.0, passed through a sodium-type strongly acidic cation exchange column, eluted with a sodium hydroxide solution, and the mother liquor is recovered and reused.

2. The method according to claim 1, characterized in that: The method comprises the following steps: (1) Fermentation broth pretreatment: adjusting the pH of the fermentation broth, filtering, and collecting the filtrate; (2) Ion exchange purification: the filtrate of step (1) is passed through a sodium-type strongly acidic cation exchange column, the column is washed with pure water, and then eluted with a sodium hydroxide solution, and the effluent with a pH of ≤9 is collected as the collection liquid, and the effluent with a pH of >9 is used as the eluent; (3) Decolorization: Add activated carbon to the collected solution in step (2), and stir at room temperature for 0.5 to 1.0 h to obtain a decolorized solution; (4) Concentration and crystallization: adjusting the pH of the decolorized solution in step (3) to 7.55-7.60, and then concentrating and crystallizing to separate wet crystals and mother liquor, and recovering the mother liquor for repeated use; (5) Drying: Drying the wet crystals in step (4) to obtain histidine crystals.

3. The method according to claim 2, characterized in that: In step (1), the pH of the fermentation broth is adjusted to 5.5-6.0 and then filtered through a ceramic membrane.

4. The method according to claim 2, characterized in that: The sodium-type strongly acidic cation exchange column in step (2) comprises sodium-type HZ016 strongly acidic cation exchange resin or sodium-type 732 cation exchange resin.

5. The method according to claim 2, characterized in that: The flow rate of the filtrate through the column in step (2) is 0.5 to 1.0 L / h.

6. The method according to claim 2, characterized in that: In the elution of step (2), the mass concentration of the sodium hydroxide solution is 0.5% to 2%; and the flow rate of the sodium hydroxide solution is 0.5 to 1.0 L / h.

7. The method according to claim 2, characterized in that: The use of the effluent with pH>9 in step (2) as an eluent means that the effluent is modulated into a 0.5% to 2% sodium hydroxide solution as an eluent.

8. The method according to claim 2, characterized in that: The amount of activated carbon added in step (3) is 1% to 3% W / V.

9. The method according to claim 2, characterized in that: In the wet crystallization of step (4), the solid content is 30% to 40% W / V.

10. The method according to claim 2, characterized in that: The drying described in step (5) includes vacuum drying; the vacuum degree of the vacuum drying is 0.12±0.01MPa, the drying temperature is 45-55°C, and the drying time is 3-6h.

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