CN116574061A - Method for separating and purifying L-histidine for cell culture medium - Google Patents
Method for separating and purifying L-histidine for cell culture medium Download PDFInfo
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- CN116574061A CN116574061A CN202310481983.4A CN202310481983A CN116574061A CN 116574061 A CN116574061 A CN 116574061A CN 202310481983 A CN202310481983 A CN 202310481983A CN 116574061 A CN116574061 A CN 116574061A
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- histidine
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- clear liquid
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- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 title claims abstract description 65
- 229960002885 histidine Drugs 0.000 title claims abstract description 41
- 238000000034 method Methods 0.000 title claims abstract description 35
- 239000006143 cell culture medium Substances 0.000 title abstract description 4
- 239000007788 liquid Substances 0.000 claims abstract description 54
- 238000001728 nano-filtration Methods 0.000 claims abstract description 28
- 239000000919 ceramic Substances 0.000 claims abstract description 18
- 239000012528 membrane Substances 0.000 claims abstract description 18
- 238000000855 fermentation Methods 0.000 claims abstract description 16
- 230000004151 fermentation Effects 0.000 claims abstract description 16
- 238000000926 separation method Methods 0.000 claims abstract description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 38
- 239000012452 mother liquor Substances 0.000 claims description 33
- 239000013078 crystal Substances 0.000 claims description 25
- 238000001914 filtration Methods 0.000 claims description 22
- 238000013375 chromatographic separation Methods 0.000 claims description 17
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 claims description 17
- 238000002425 crystallisation Methods 0.000 claims description 15
- 230000008025 crystallization Effects 0.000 claims description 15
- 238000001816 cooling Methods 0.000 claims description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 14
- 238000005406 washing Methods 0.000 claims description 12
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 11
- 229910052799 carbon Inorganic materials 0.000 claims description 11
- 238000011084 recovery Methods 0.000 claims description 9
- 238000001291 vacuum drying Methods 0.000 claims description 8
- 241001052560 Thallis Species 0.000 claims description 6
- 238000012258 culturing Methods 0.000 claims description 6
- 238000001035 drying Methods 0.000 claims description 6
- 239000011347 resin Substances 0.000 claims description 5
- 229920005989 resin Polymers 0.000 claims description 5
- 239000000284 extract Substances 0.000 claims description 4
- 239000001963 growth medium Substances 0.000 claims description 4
- 150000002500 ions Chemical class 0.000 claims description 4
- 238000002386 leaching Methods 0.000 claims description 4
- 238000004042 decolorization Methods 0.000 claims description 3
- 239000012535 impurity Substances 0.000 claims description 3
- 239000003480 eluent Substances 0.000 claims description 2
- 238000000605 extraction Methods 0.000 claims description 2
- 238000010438 heat treatment Methods 0.000 claims description 2
- 230000000415 inactivating effect Effects 0.000 claims description 2
- 229920002521 macromolecule Polymers 0.000 claims description 2
- 238000001953 recrystallisation Methods 0.000 claims 1
- 239000000047 product Substances 0.000 abstract description 20
- 239000012043 crude product Substances 0.000 abstract description 11
- 238000004519 manufacturing process Methods 0.000 abstract description 7
- 239000003814 drug Substances 0.000 abstract description 4
- 230000007547 defect Effects 0.000 abstract description 3
- 238000011097 chromatography purification Methods 0.000 abstract description 2
- 230000003287 optical effect Effects 0.000 abstract description 2
- 108090000623 proteins and genes Proteins 0.000 abstract description 2
- 102000004169 proteins and genes Human genes 0.000 abstract description 2
- 239000010865 sewage Substances 0.000 abstract description 2
- 239000002253 acid Substances 0.000 description 39
- 229940024606 amino acid Drugs 0.000 description 11
- 150000001413 amino acids Chemical class 0.000 description 11
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 6
- 239000003610 charcoal Substances 0.000 description 4
- 238000004587 chromatography analysis Methods 0.000 description 4
- 238000000502 dialysis Methods 0.000 description 4
- 238000010828 elution Methods 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 238000002834 transmittance Methods 0.000 description 4
- 229910021529 ammonia Inorganic materials 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 238000005265 energy consumption Methods 0.000 description 2
- 239000000413 hydrolysate Substances 0.000 description 2
- 239000003456 ion exchange resin Substances 0.000 description 2
- 229920003303 ion-exchange polymer Polymers 0.000 description 2
- 150000002730 mercury Chemical class 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000000638 solvent extraction Methods 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- 239000002699 waste material Substances 0.000 description 2
- RWSOTUBLDIXVET-UHFFFAOYSA-N Dihydrogen sulfide Chemical compound S RWSOTUBLDIXVET-UHFFFAOYSA-N 0.000 description 1
- -1 L-histidine mercury salt Chemical compound 0.000 description 1
- 208000008469 Peptic Ulcer Diseases 0.000 description 1
- 108010009736 Protein Hydrolysates Proteins 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 208000007502 anemia Diseases 0.000 description 1
- 150000001450 anions Chemical class 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003797 essential amino acid Substances 0.000 description 1
- 208000019622 heart disease Diseases 0.000 description 1
- 208000006454 hepatitis Diseases 0.000 description 1
- 231100000283 hepatitis Toxicity 0.000 description 1
- 229910000037 hydrogen sulfide Inorganic materials 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 229910000474 mercury oxide Inorganic materials 0.000 description 1
- UKWHYYKOEPRTIC-UHFFFAOYSA-N mercury(ii) oxide Chemical compound [Hg]=O UKWHYYKOEPRTIC-UHFFFAOYSA-N 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- 208000011906 peptic ulcer disease Diseases 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000003531 protein hydrolysate Substances 0.000 description 1
- 230000007065 protein hydrolysis Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 206010039073 rheumatoid arthritis Diseases 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
- 239000002351 wastewater Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D233/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings
- C07D233/54—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members
- C07D233/64—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members with substituted hydrocarbon radicals attached to ring carbon atoms, e.g. histidine
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
- C07B2200/00—Indexing scheme relating to specific properties of organic compounds
- C07B2200/07—Optical isomers
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to a method for separating and purifying L-histidine for a cell culture medium, belonging to the field of biological medicine. The invention separates the protein from the fermentation liquor through a ceramic membrane, and the obtained clear liquid is subjected to chromatographic purification, nanofiltration and decoloration to obtain a crude product, and the crude product is recrystallized to obtain the product. The specific optical rotation of the product obtained by the method can reach +12.1 degrees to +12.8 degrees, the content can reach 99.8-101.5 percent, and the product can completely reach the imported merck sigma quality standard. The method has the characteristics of mild condition, simple operation, few separation steps, good selectivity, clean production and the like, overcomes the defects of low yield, large sewage discharge and high production strength in the prior art, and obviously improves the yield and quality of the L-histidine.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a method for separating and purifying L-histidine for a cell culture medium.
Background
L-histidine is a human semi-essential amino acid, and is mainly used as a raw material for amino acid transfusion and oral amino acid preparations; in addition, it is also an important medicine for treating heart disease, anemia, rheumatoid arthritis, peptic ulcer, and essential hepatitis, and has been widely used in the field of medicine.
At present, the main separation methods of L-histidine include an electroanalysis adsorption method, a solvent extraction method, an ion exchange method, a special reagent precipitation method and the like.
The electroanalysis adsorption method is used, and the mobility of the basic amino acid and the acidic amino acid reaches more than 90% after 24 hours of electroanalysis, but the method has the advantages of complex equipment, difficult operation control and great histidine extraction yield change, and is rarely applied to industry. The separation of amino acids from protein hydrolysates by solvent extraction is difficult to apply to the separation of complex amino acids due to the low selectivity of the extractant to amino acids. And separating 6 amino acids including L-histidine from the pig blood powder hydrolysate by adopting an ion exchange resin method, wherein the yield of the L-histidine is 5.4%. Separating the mixed amino acid mother liquor obtained by protein hydrolysis method with secondary strong acid cation resin column, neutralizing with hydrochloric acid, decolorizing with active carbon, exchanging with strong base anion resin, and forming L-histidine with yield of 1.2kg/m 3 And (3) a hydrolysate. The ion exchange resin method has the advantages of large resin consumption, low separation efficiency and large amount of wastewater generation.
The special reagent precipitation method is a separation method which adopts certain organic or inorganic reagents to form insoluble derivatives with corresponding amino acids. The L-histidine can react with mercury oxide to form a precipitate of L-histidine mercury salt, which is then treated with hydrogen sulfide to obtain L-histidine (amino acid production technique and its use, 1997). The method has strong selectivity for separating L-histidine. However, the method is difficult to apply in practical production due to the problems of difficult recycling and use of the mercury salt of the precipitant, extremely toxic substances of the mercury salt and the like.
Therefore, a manufacturing process with strong selectivity, simple process, low energy consumption and small waste liquid discharge amount is needed.
Disclosure of Invention
The invention aims to overcome the defects in the prior art, and provides a separation and purification mode with high equipment utilization rate, low energy consumption, high yield and high waste liquid discharge recovery rate, particularly chromatographic separation and nanofiltration decoloration, wherein the purity of the obtained L-histidine clear liquid is high, and the prepared product has high content.
In order to achieve the above purpose, the invention adopts the following specific scheme:
a method for separating and purifying L-histidine for culture medium includes such steps as chromatographic separation to obtain high-purity L-histidine solution, vacuum concentrating for crystallizing, recrystallizing and vacuum drying.
Preferably, the method specifically comprises the following steps:
(1) Fermentation broth ceramic membrane degerming body:
heating and inactivating the fermentation liquor containing L-histidine, filtering by a 50nm ceramic membrane, and removing thalli to obtain fermentation clear liquor;
(2) Chromatographic separation:
separating the fermentation clear liquid obtained in the step (1) in chromatographic separation equipment to obtain an extracting solution, wherein the purity of L-histidine in the extracting solution is more than 99%;
(3) Nanofiltration removes macromolecular substances and heat sources:
sending the extract obtained in the step (2) into a nanofiltration membrane system, carrying out nanofiltration filtration and impurity removal, and adding water for top washing to obtain nanofiltration clear liquid;
(4) Decolorizing with active carbon:
and (3) adding medical grade active carbon into the nanofiltration liquid obtained in the step (3) for decoloring treatment to obtain decolored clear liquid.
(5) Concentrating and crystallizing in vacuum:
vacuum concentrating the decolorized clear liquid obtained in the step (4) to 300-330g/L at 60-70 ℃, sending the decolorized clear liquid into a crystallization tank, cooling to 20 ℃ or less, and culturing the crystals for 1-2 hours to obtain a crystallization liquid;
(6) And (3) centrifugal washing:
sending the crystallization liquid obtained in the step (5) into a centrifugal machine for solid-liquid separation, spin-drying, leaching the crystals with 75% ethanol until no ions are removed, and stopping washing and spin-drying to obtain a crude histidine product and primary mother liquor;
(7) And (5) recrystallizing:
dissolving the crude histidine product obtained in the step (6) in pure water according to a ratio of 1:20, adding 10% of medical grade active carbon for decolorization and filtration to obtain refined clear liquid, concentrating to 300-330g/L at 60-70 ℃ in a vacuum evaporator, cooling and crystallizing, centrifuging, eluting with 75% ethanol until no ions are discharged, and washing to obtain wet histidine crystals and secondary mother liquor; temporarily storing the mother liquor obtained for three times after the leaching and the alcohol recovery;
(8) And (3) drying:
and (3) putting the histidine wet crystal obtained in the step (7) into a vacuum drying oven to be dried for 6-7 hours, and cooling to obtain the high-purity histidine crystal.
As a further preference, the process also comprises recovery of mother liquor, in particular:
directly sleeving the primary mother liquor obtained in the step (6) back to the step (2) for chromatographic separation;
directly using the secondary mother liquor obtained in the step (8) as the dissolved water in the step (7);
and (3) directly sleeving the tertiary mother liquor obtained in the step (8) back to the step (2) for chromatographic separation.
As a further preferred aspect, the chromatographic separation device in the step (2) is a simulated moving bed loaded with Mg-type homogeneous chromatographic resin, the separation temperature is 40-50 ℃, and the eluent is sterile pure water.
As a further preference, the chromatographic separation described in step (2) is carried out at a feed flow rate of 1.5bv/h and an elution flow rate of 2.0bv/h with a throughput of 1.5L/h.
As a further preference, the nanofiltration membrane of step (3) has a molecular weight cut-off of 500Da, a temperature of 35-40 ℃, a pH of 7-8 and an operating pressure of 2.0-2.2MPa.
Compared with the prior art, the invention uses chromatographic separation to remove most of impurities in fermentation liquor, improves the purity of histidine clear liquid to more than 99%, carries out nanofiltration, decolorization and secondary crystallization to continue purification, and finally separates and extracts high-purity crystals for culture medium. Has the characteristics of simple method, high yield, stable product quality and the like.
The invention separates the protein from the fermentation liquor through a ceramic membrane, and the obtained clear liquid is subjected to chromatographic purification, nanofiltration and decoloration to obtain a crude product, and the crude product is recrystallized to obtain the product. The specific optical rotation of the product obtained by the method can reach +12.1 degrees to +12.8 degrees, the content can reach 99.8-101.5 percent, and the product can completely reach the imported merck sigma quality standard.
The method has the characteristics of mild condition, simple operation, few separation steps, good selectivity, clean production and the like, overcomes the defects of low yield, large sewage discharge and high production strength in the prior art, and obviously improves the yield and quality of the L-histidine.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below in connection with the embodiments of the present invention.
Example 1:
(1) Taking 5L histidine fermentation liquor (40 g/L detected, 200g total acid) and filtering by a ceramic membrane to remove thalli so as to obtain ceramic clear liquid.
(2) The ceramic supernatant was fed to 8L chromatography at a feed flow rate of 1.5bv/h and an elution flow rate of 2.0bv/h with a throughput of 1.5L/h. The volume of the extracting solution is 17L, the acid content is 11.2g/L, the purity of the dry matter is 90.2%, and the total acid content is 190.4g.
(3) And (3) taking all the extracting solution, sending the extracting solution into a nanofiltration membrane with 500 molecular weight, adding dialysis water, dialyzing until the acid content of the concentrated solution is less than 3g/L, and stopping dialyzing. 30L of nanofiltration liquid and 180g of total acid are obtained.
(4) Adding 18g of medical charcoal into the nanofiltration clear liquid, decoloring and filtering, wherein the light transmittance of the clear liquid is more than 99T percent.
(5) Concentrating the decolorized clear liquid to 330g/L under vacuum at 50-60 ℃, sending the decolorized clear liquid into a crystallization tank, cooling to 20 ℃ or less, and culturing for 2 hours.
(6) The resulting crystals were centrifuged in a centrifuge to obtain 154 g of crude product (dried). 0.35L of primary mother liquor and 41.2g/L of acid.
(7) Dissolving the crude product to 3.85L, adding 15g of medical grade active carbon, maintaining the temperature at 50 ℃ for 60min, filtering to obtain decolorized clear liquid, transmitting 99T, continuously concentrating in vacuum to 350g/L, cooling and crystallizing.
(8) The refined crystallization material is centrifugated by a centrifugal machine, and 0.4L of 75 percent ethanol is leached. To obtain refined wet crystal. 0.2L of secondary mother liquor and 40.10g/L of acid. Wash liquor 0.38L.
(9) Filtering to obtain wet crystals, and vacuum drying to obtain 126g of finished product.
(10) The washing liquid enters an alcohol recovery tower to recover 0.25L of ethanol with the ethanol content of 83 percent. 0.13L of tertiary mother liquor containing 35g/L of acid is obtained.
The specific rotation of the obtained product is 12.5, the content is 100.5, and the yield is 63%.
Example 2:
(1) Taking 5L histidine fermentation liquor (40.3 g/L detected, 200g total acid) and filtering by a ceramic membrane to remove thalli so as to obtain ceramic clear liquid.
(2) The ceramic supernatant was fed to 8L chromatography with the primary mother liquor (0.35L. Acid containing 41.2 g/L) and the tertiary mother liquor (0.13L, acid containing 35 g/L.) of case 1 at a feed flow rate of 1.5bv/h and an elution flow rate of 2.0bv/h with a throughput of 1.5L/h. The volume of the extracting solution is 19.3L, the acid content is 11.4g/L, the purity of the dry matter is 90.4%, and the total acid content is 220g.
(3) And (3) taking all the extracting solution, sending the extracting solution into a nanofiltration membrane with 500 molecular weight, adding dialysis water, dialyzing until the acid content of the concentrated solution is less than 3g/L, and stopping dialyzing. 35L of nanofiltration solution and 210g of total acid were obtained.
(4) 21g of medical charcoal is put into nanofiltration clear liquid for decoloration and filtration, and the light transmittance of the clear liquid is more than 99T percent.
(5) Concentrating the decolorized clear liquid to 330g/L under vacuum at 50-60 ℃, sending the decolorized clear liquid into a crystallization tank, cooling to 20 ℃ or less, and culturing for 2 hours.
(6) The resulting crystals were centrifuged in a centrifuge to obtain 174 g of crude product (dried). The primary mother liquor is 0.39L, and the acid content is 40.9g/L.
(7) Dissolving the crude product into 4.35L pure water, adding 0.2L secondary mother liquor in case 1 and 40.10g/L acid, stirring thoroughly, adding 17g of medical grade active carbon, maintaining the temperature at 50deg.C for 60min, filtering to obtain decolorized clear solution, transmitting 99T, vacuum concentrating to 350g/L, cooling and crystallizing,
(8) The refined crystallization material is centrifugated by a centrifugal machine, and 0.45L of 75 percent ethanol is leached. To obtain refined wet crystal. The secondary mother liquor is 0.4L, and the acid content is 40.8g/L. Wash liquor 0.42L.
(9) Filtering to obtain wet crystals, and vacuum drying to obtain 140g of finished product.
(10) The washing liquid enters an alcohol recovery tower to recover 0.25L of ethanol with the ethanol content of 83 percent. 0.13L of tertiary mother liquor and 35.4g/L of acid are obtained.
The specific rotation of the obtained product is 12.4, the content is 100.3, and the yield is 70%.
Example 3:
(1) Taking 5L histidine fermentation liquor (39.6 g/L detected, 200g total acid) and filtering by a ceramic membrane to remove thalli to obtain ceramic clear liquid,
(2) The ceramic supernatant was fed to 8L chromatography with the primary mother liquor (0.39L. Acid-containing 40.9 g/L) of case 2 and the tertiary mother liquor (0.13L, acid-containing 35.4 g/L.) at a feed flow rate of 1.5bv/h and an elution flow rate of 2.0bv/h with a throughput of 1.5L/h.
The volume of the extracting solution is 20.3L, the acid content is 11.5g/L, the purity of the dry matter is 90%, and the total acid content is 233.5g.
(3) And (3) taking all the extracting solution, sending the extracting solution into a nanofiltration membrane with 500 molecular weight, adding dialysis water, dialyzing until the acid content of the concentrated solution is less than 3g/L, and stopping dialyzing. 35L of nanofiltration solution and 219g of total acid are obtained.
(4) 21g of medical charcoal is put into nanofiltration clear liquid for decoloration and filtration, and the light transmittance of the clear liquid is more than 99T percent.
(5) Concentrating the decolorized clear liquid to 330g/L under vacuum at 50-60 ℃, sending the decolorized clear liquid into a crystallization tank, cooling to 20 ℃ or less, and culturing for 2 hours.
(6) The resulting crystals were centrifuged in a centrifuge to obtain 183 g of crude product (dried). The primary mother liquor is 0.4L, and the acid content is 40.7g/L.
(7) Dissolving the crude product into 4.35L pure water, adding 0.4L secondary mother liquor in case 2, containing 40.8g/L acid, adding 18g of medical grade active carbon, maintaining the temperature at 50deg.C for 60min, filtering to obtain decolorized clear solution, concentrating under vacuum to 350g/L, cooling to crystallize,
(8) The refined crystal material is centrifugated by a centrifugal machine, and 0.47L of 75 percent ethanol is leached. To obtain refined wet crystal. The secondary mother liquor is 0.43L, and the acid content is 40.4g/L. Wash liquor 0.44L.
(9) Filtering to obtain wet crystals, and vacuum drying to obtain 146g of finished product.
(10) The washing liquid enters an alcohol recovery tower to recover 0.25L of ethanol with the ethanol content of 83 percent. 0.15L of tertiary mother liquor and 35g/L of acid are obtained.
The specific rotation of the obtained product is 12.5, the content is 100.2, and the yield is 73 percent.
Control example 4 (Ammonia type chromatography)
(1) Taking 5L histidine fermentation liquor (40 g/L detected, 200g total acid) and filtering by a ceramic membrane to remove thalli so as to obtain ceramic clear liquid. (fermentation broth and in the case of the same batch)
(2) The ceramic clear liquid is sent to 8L ammonia type chromatographic separation, the feeding flow rate is 1.5bv/h, the eluting flow rate is 2.0bv/h, and the treatment capacity is 1.5L/h. The volume of the extracting solution is 18.1L, the acid content is 10.3g/L, the purity of the dry matter is 85.4%, and the total acid content is 187.5g.
(3) And (3) taking all the extracting solution, sending the extracting solution into a nanofiltration membrane with 500 molecular weight, adding dialysis water, dialyzing until the acid content of the concentrated solution is less than 3g/L, and stopping dialyzing. 30L of nanofiltration liquid and 171g of total acid are obtained.
(4) Adding 18g of medical charcoal into the nanofiltration clear liquid, decoloring and filtering, wherein the light transmittance of the clear liquid is more than 99T percent.
(5) Concentrating the decolorized clear liquid to 330g/L under vacuum at 50-60 ℃, sending the decolorized clear liquid into a crystallization tank, cooling to 20 ℃ or less, and culturing the crystals for 1-2 hours.
(6) The resulting crystals were centrifuged in a centrifuge to obtain 148 g of crude (dried) product. 0.33L of primary mother liquor and 40.9g/L of acid.
(7) Dissolving the crude product to 3.85L, adding 15g of medical grade active carbon, maintaining the temperature at 50 ℃ for 60min, filtering to obtain decolorized clear liquid, transmitting 99T, continuously concentrating in vacuum to 350g/L, cooling and crystallizing.
(8) The refined crystallization material is centrifugated by a centrifugal machine, and 0.4L of 75 percent ethanol is leached. To obtain refined wet crystal. 0.18L of secondary mother liquor and 40.33g/L of acid. Wash liquor 0.38L.
(9) Filtering to obtain wet crystals, and vacuum drying to obtain 119g of finished product.
(10) The washing liquid enters an alcohol recovery tower to recover 0.22L of ethanol with the ethanol content of 83 percent. 0.13L of tertiary mother liquor containing 35g/L of acid is obtained.
The specific rotation of the obtained product is 12.1, the content is 99.1 percent, and the yield is 59.5 percent.
The purity of the separated chromatographic extract is greatly improved by separating by using different chromatographs, and the product yield is at least 5 percent higher than that of the ammonia type chromatograph.
It should be noted that the above-mentioned embodiments are to be understood as illustrative, and not limiting, the scope of the invention, which is defined by the appended claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made to the present invention without departing from its spirit or scope.
Claims (6)
1. A method for separating and purifying L-histidine for a culture medium is characterized in that the method adopts chromatographic separation and extraction to obtain high-purity L-histidine solution, and the high-purity L-histidine solution is subjected to vacuum concentration crystallization, recrystallization and vacuum drying to obtain a product.
2. The method for purifying histidine for culture medium according to claim 1, wherein: the method specifically comprises the following steps:
(1) Fermentation broth ceramic membrane degerming body:
heating and inactivating the fermentation liquor containing L-histidine, filtering by a 50nm ceramic membrane, and removing thalli to obtain fermentation clear liquor;
(2) Chromatographic separation:
separating the fermentation clear liquid obtained in the step (1) in chromatographic separation equipment to obtain an extracting solution, wherein the purity of L-histidine in the extracting solution is more than 99%;
(3) Nanofiltration removes macromolecular substances and heat sources:
sending the extract obtained in the step (2) into a nanofiltration membrane system, carrying out nanofiltration filtration and impurity removal, and adding water for top washing to obtain nanofiltration clear liquid;
(4) Decolorizing with active carbon:
adding medical grade active carbon into the nanofiltration solution obtained in the step (3) for decoloring treatment to obtain decolored clear liquid;
(5) Concentrating and crystallizing in vacuum:
vacuum concentrating the decolorized clear liquid obtained in the step (4) to 300-330g/L at 60-70 ℃, sending the decolorized clear liquid into a crystallization tank, cooling to 20 ℃ or less, and culturing the crystals for 1-2 hours to obtain a crystallization liquid;
(6) And (3) centrifugal washing:
sending the crystallization liquid obtained in the step (5) into a centrifugal machine for solid-liquid separation, spin-drying, leaching the crystals with 75% ethanol until no ions are removed, and stopping washing and spin-drying to obtain a crude histidine product and primary mother liquor;
(7) And (5) recrystallizing:
dissolving the crude histidine product obtained in the step (6) in pure water according to a ratio of 1:20, adding 10% of medical grade active carbon for decolorization and filtration to obtain refined clear liquid, concentrating to 300-330g/L at 60-70 ℃ in a vacuum evaporator, cooling and crystallizing, centrifuging, eluting with 75% ethanol until no ions are discharged, and washing to obtain wet histidine crystals and secondary mother liquor; temporarily storing the mother liquor obtained for three times after the leaching and the alcohol recovery;
(8) And (3) drying:
and (3) putting the histidine wet crystal obtained in the step (7) into a vacuum drying oven to be dried for 6-7 hours, and cooling to obtain the high-purity histidine crystal.
3. The method according to claim 2, characterized in that: the method also comprises the recovery of mother liquor, and specifically comprises the following steps:
directly sleeving the primary mother liquor obtained in the step (6) back to the step (2) for chromatographic separation;
directly using the secondary mother liquor obtained in the step (8) as the dissolved water in the step (7);
and (3) directly sleeving the tertiary mother liquor obtained in the step (8) back to the step (2) for chromatographic separation.
4. The method according to claim 2, characterized in that: the chromatographic separation device in the step (2) is a simulated moving bed filled with Mg-type homogeneous chromatographic resin, the separation temperature is 40-50 ℃, and the eluent is sterile pure water.
5. The method according to claim 2, characterized in that: the chromatographic separation in the step (2) has a feeding flow rate of 1.5bv/h, an eluting flow rate of 2.0bv/h and a throughput of 1.5L/h.
6. The method according to claim 2, characterized in that: the molecular weight cut-off of the nanofiltration membrane in the step (3) is 500Da, the temperature is 35-40 ℃, the pH is 7-8, and the operating pressure is 2.0-2.2Mpa.
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| CN202310481983.4A CN116574061A (en) | 2023-04-28 | 2023-04-28 | Method for separating and purifying L-histidine for cell culture medium |
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