CN109628518B - A kind of method for producing and extracting L-glutamine - Google Patents
A kind of method for producing and extracting L-glutamine Download PDFInfo
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- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 title claims abstract description 64
- 229930182816 L-glutamine Natural products 0.000 title claims abstract description 56
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 21
- 238000000855 fermentation Methods 0.000 claims abstract description 79
- 230000004151 fermentation Effects 0.000 claims abstract description 79
- 238000000034 method Methods 0.000 claims abstract description 27
- 238000001914 filtration Methods 0.000 claims abstract description 24
- 239000000919 ceramic Substances 0.000 claims abstract description 22
- 238000000605 extraction Methods 0.000 claims abstract description 14
- 230000008569 process Effects 0.000 claims abstract description 11
- 238000002425 crystallisation Methods 0.000 claims abstract description 5
- 230000008025 crystallization Effects 0.000 claims abstract description 5
- 238000004042 decolorization Methods 0.000 claims abstract description 5
- 238000005374 membrane filtration Methods 0.000 claims abstract description 5
- 239000007788 liquid Substances 0.000 claims description 59
- 239000012528 membrane Substances 0.000 claims description 22
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 22
- 239000004475 Arginine Substances 0.000 claims description 15
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 claims description 15
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 claims description 15
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 claims description 15
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 12
- 239000008103 glucose Substances 0.000 claims description 12
- 239000000047 product Substances 0.000 claims description 12
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 8
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical group [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 8
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 8
- 238000009423 ventilation Methods 0.000 claims description 8
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 7
- 238000001035 drying Methods 0.000 claims description 6
- 241000186226 Corynebacterium glutamicum Species 0.000 claims description 4
- 240000008042 Zea mays Species 0.000 claims description 4
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 4
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 4
- 239000004202 carbamide Substances 0.000 claims description 4
- 235000005822 corn Nutrition 0.000 claims description 4
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 claims description 4
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 claims description 4
- ISPYRSDWRDQNSW-UHFFFAOYSA-L manganese(II) sulfate monohydrate Chemical compound O.[Mn+2].[O-]S([O-])(=O)=O ISPYRSDWRDQNSW-UHFFFAOYSA-L 0.000 claims description 4
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 4
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 4
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 4
- 239000003795 chemical substances by application Substances 0.000 claims description 3
- 239000002054 inoculum Substances 0.000 claims description 3
- 239000000706 filtrate Substances 0.000 claims 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims 1
- 235000003891 ferrous sulphate Nutrition 0.000 claims 1
- 239000011790 ferrous sulphate Substances 0.000 claims 1
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 claims 1
- 229910000359 iron(II) sulfate Inorganic materials 0.000 claims 1
- 210000003205 muscle Anatomy 0.000 claims 1
- 230000007613 environmental effect Effects 0.000 abstract description 2
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 17
- 229960002989 glutamic acid Drugs 0.000 description 16
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 15
- 235000013922 glutamic acid Nutrition 0.000 description 15
- 239000004220 glutamic acid Substances 0.000 description 15
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N Glutamine Chemical compound OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 10
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 description 10
- SQUHHTBVTRBESD-UHFFFAOYSA-N Hexa-Ac-myo-Inositol Natural products CC(=O)OC1C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C1OC(C)=O SQUHHTBVTRBESD-UHFFFAOYSA-N 0.000 description 9
- 229960000367 inositol Drugs 0.000 description 9
- 230000001105 regulatory effect Effects 0.000 description 9
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 description 9
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 8
- 238000007872 degassing Methods 0.000 description 8
- 238000010828 elution Methods 0.000 description 8
- 229940024606 amino acid Drugs 0.000 description 7
- 235000001014 amino acid Nutrition 0.000 description 7
- 150000001413 amino acids Chemical class 0.000 description 7
- 230000001276 controlling effect Effects 0.000 description 6
- 239000000463 material Substances 0.000 description 6
- 238000000926 separation method Methods 0.000 description 6
- 238000003786 synthesis reaction Methods 0.000 description 6
- 238000005516 engineering process Methods 0.000 description 5
- KPGXRSRHYNQIFN-UHFFFAOYSA-N 2-oxoglutaric acid Chemical compound OC(=O)CCC(=O)C(O)=O KPGXRSRHYNQIFN-UHFFFAOYSA-N 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 238000013375 chromatographic separation Methods 0.000 description 4
- 238000004587 chromatography analysis Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 230000037361 pathway Effects 0.000 description 4
- 230000035699 permeability Effects 0.000 description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 238000001704 evaporation Methods 0.000 description 3
- 230000008020 evaporation Effects 0.000 description 3
- 239000012535 impurity Substances 0.000 description 3
- 238000005342 ion exchange Methods 0.000 description 3
- SURQXAFEQWPFPV-UHFFFAOYSA-L iron(2+) sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Fe+2].[O-]S([O-])(=O)=O SURQXAFEQWPFPV-UHFFFAOYSA-L 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 238000003825 pressing Methods 0.000 description 3
- 230000002829 reductive effect Effects 0.000 description 3
- 239000011347 resin Substances 0.000 description 3
- 229920005989 resin Polymers 0.000 description 3
- 150000003839 salts Chemical class 0.000 description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- 238000009825 accumulation Methods 0.000 description 2
- 230000002378 acidificating effect Effects 0.000 description 2
- 230000003213 activating effect Effects 0.000 description 2
- HWXBTNAVRSUOJR-UHFFFAOYSA-N alpha-hydroxyglutaric acid Natural products OC(=O)C(O)CCC(O)=O HWXBTNAVRSUOJR-UHFFFAOYSA-N 0.000 description 2
- 229940009533 alpha-ketoglutaric acid Drugs 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 230000003750 conditioning effect Effects 0.000 description 2
- 238000007599 discharging Methods 0.000 description 2
- 238000003912 environmental pollution Methods 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 239000003797 essential amino acid Substances 0.000 description 2
- 235000020776 essential amino acid Nutrition 0.000 description 2
- 230000002349 favourable effect Effects 0.000 description 2
- 238000011010 flushing procedure Methods 0.000 description 2
- HHLFWLYXYJOTON-UHFFFAOYSA-N glyoxylic acid Chemical compound OC(=O)C=O HHLFWLYXYJOTON-UHFFFAOYSA-N 0.000 description 2
- 229910001385 heavy metal Inorganic materials 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 230000007935 neutral effect Effects 0.000 description 2
- 150000007524 organic acids Chemical class 0.000 description 2
- 239000012466 permeate Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 235000018102 proteins Nutrition 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000005086 pumping Methods 0.000 description 2
- 238000006268 reductive amination reaction Methods 0.000 description 2
- 239000002893 slag Substances 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 238000001308 synthesis method Methods 0.000 description 2
- 230000004102 tricarboxylic acid cycle Effects 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000011001 backwashing Methods 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000005008 domestic process Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
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- 150000003467 sulfuric acid derivatives Chemical class 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/14—Glutamic acid; Glutamine
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- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Wood Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
本发明属于生物发酵技术领域，公开了一种生产和提取L‑谷氨酰胺的方法，其包括如下步骤：步骤1）发酵，步骤2）陶瓷膜过滤，步骤3）脱色，步骤4）精滤，步骤5）色谱提取，步骤6）浓缩结晶。本发明方法发酵产率高，工艺简单、高效环保、生产成本低。The invention belongs to the technical field of biological fermentation, and discloses a method for producing and extracting L-glutamine, which comprises the following steps: step 1) fermentation, step 2) ceramic membrane filtration, step 3) decolorization, and step 4) fine filtration , step 5) chromatographic extraction, step 6) concentration crystallization. The method of the invention has high fermentation yield, simple process, high efficiency and environmental protection, and low production cost.
Description
Technical Field
The invention belongs to the field of L-glutamine production in the biological fermentation industry, and particularly provides a method for producing and extracting L-glutamine.
Background
L-Glutamine (L-Gln) is an amino acid amidated with the gamma-carboxyl group of L-glutamic acid, and has the chemical name of 2, 5-diamino-5-oxopentanoic acid, which is one of the essential amino acids constituting proteins. L-glutamine is a white orthorhombic crystal or crystalline powder, has no odor, no toxicity, slightly sweet flavor, and is difficult to dissolve in organic solvents such as methanol, ether, chloroform, etc.
L-glutamine is one of the essential amino acids of 20 synthetic proteins, accounts for about 60% of human free amino acids, and plays a key role in organism metabolism. In recent years, medical findings show that L-glutamine deficiency causes various diseases, plays an important role in life activities, and is widely applied to the industries of medicine, health care, cell culture and feed.
At present, the method for producing L-glutamine mainly comprises a chemical synthesis method, an enzymatic synthesis method and a biological fermentation method, wherein the chemical synthesis method uses a large amount of chemical reagents, easily causes environmental pollution and has low product quality; the enzymatic synthesis method has higher cost and cannot be popularized and used; the biological fermentation method is a main method for producing L-glutamine, and a lot of researches are carried out to improve the biological fermentation method, including mutagenesis strains, optimization of culture medium, culture conditions and the like.
At present, the traditional domestic method for extracting L-glutamine is an ion exchange method, and the method has the defects of complex equipment structure, low automation degree, low production efficiency, low product quality and the like. In addition, the finished L-glutamine product produced by the conventional extraction process hardly meets the requirements of some customers, in order to improve the product quality, a sequential simulated moving bed chromatography technology can be adopted to replace an ion exchange process, and a subsequent extraction process route is readjusted so as to improve the product quality and improve the market competitiveness.
Disclosure of Invention
In order to solve the problems of the production and extraction of the L-glutamine, the invention provides a method for producing and extracting the L-glutamine, which has the advantages of high fermentation yield, simple process, high efficiency, environmental protection and low production cost.
The invention is realized by the following technical scheme:
a method for producing and extracting L-glutamine comprising the steps of: step 1) fermentation, step 2) ceramic membrane filtration, step 3) decolorization, step 4) fine filtration, step 5) chromatographic extraction, and step 6) concentration and crystallization.
Further, the step 1) fermentation comprises the following processes: inoculating corynebacterium glutamicum to a fermentation tank containing a fermentation medium in an inoculation amount of 6-10%, wherein the fermentation time is 48 h; the fermentation time is divided into two stages, the first stage is 24h, the fermentation temperature is 32-35 ℃, the ventilation volume is 0.4vvm, and the pH is controlled at 5.0 by feeding hydrochloric acid or ammonia water; the second stage is 24h, the fermentation temperature is 32 ℃, the ventilation rate is 0.5vvm, when the second stage starts, regulating solution is added into the fermentation tank, the adding amount accounts for 1-2% of the volume of the fermentation liquid, glucose solution is added into the fermentation tank, the concentration of glucose in the fermentation tank is controlled to be not less than 0.8%, and the pH value is controlled to be 7.0-7.2 by adding ammonia water; after the second stage is finished, collecting L-glutamine fermentation liquor.
Further, the components of the fermentation tank culture medium are as follows (mass percent): 10% of glucose, 3% of corn steep liquor, 0.5% of urea, 0.1% of potassium dihydrogen phosphate, 0.01% of ferrous sulfate heptahydrate, 0.01% of magnesium sulfate heptahydrate and 0.005% of manganese sulfate monohydrate.
Further, the components of the conditioning fluid are as follows: proline 50g/L, arginine 50g/L, inositol 10 g/L.
Further, the step 2) of ceramic membrane filtration comprises the following processes: filtering with ceramic membrane, and collecting ceramic membrane clear solution.
Further, the step 3) of decoloring comprises the following processes: and (3) feeding the clear liquid of the ceramic membrane obtained in the step 2) into a decoloring tank for decoloring, wherein a decoloring agent is activated carbon, and filtering to obtain a decolored clear liquid after decoloring.
Further, the step 4) fine filtration: and (3) connecting the decolorized clear liquid in the step 3) to a fine filter for fine filtration, controlling the temperature to be 35-40 ℃ and the pressure to be 0.1-0.2MPa, and obtaining fine filtered clear liquid.
Further, the step 5) of chromatographic extraction comprises the following steps: introducing the fine filtered clear liquid obtained in the step 4) into a chromatographic chamber for extraction, and controlling extraction process parameters: the flow rate of the chromatographic chamber is 3-5m3At 35-40 deg.C and 0.8-1.0MPa to obtain L-glutamine extractive solution.
Further, the step 6) of concentrating and crystallizing comprises the following steps: and (3) concentrating, crystallizing, centrifuging and drying the L-glutamine extracting solution to obtain the finished product of the L-glutamine.
The technical scheme of the invention has the following outstanding advantages and uniqueness:
in the fermentation process, the fermentation regulating solution is added, wherein a proper amount of inositol can strengthen the CO2 fixed reaction, weaken the glyoxylate cycle, ensure that the tricarboxylic acid cycle is not interrupted and continuously supply alpha-ketoglutaric acid, and accumulate a large amount of glutamic acid through reductive amination reaction, thereby improving the fermentation conversion rate; the glutamic acid can be metabolized to generate glutamine, proline and arginine, and the proline and arginine are added in the middle and later stages of fermentation, so that the feedback inhibition effect on corresponding proline and arginine pathways can be generated, and more glutamic acid flows to the glutamine pathway. The first stage of fermentation is favorable to the synthesis of glutamic acid and the accumulation of glutamic acid because the pH of the fermentation liquor is slightly acidic and the cell permeability is poor, the glutamic acid cannot flow out of the cell, and the second stage of fermentation is favorable to the synthesis of glutamine from intracellular glutamic acid because ammonia water is fed and added to adjust the pH of the fermentation liquor to be neutral or slightly alkaline and improve the cell permeability, thereby improving the yield of glutamine.
The invention adopts the 'sequential simulated moving bed chromatography' technology to extract the L-glutamine, compared with the traditional ion exchange method, the acid-base consumption and the water consumption are greatly reduced, the whole production process is energy-saving and environment-friendly, and the environmental pollution is reduced. The invention adopts the feed liquid flash evaporation technology to process the L-glutamine feed liquid, greatly improves the yield of the L-glutamine product and greatly improves the product quality. The L-glutamine liquid of the invention is subjected to the working procedures of ceramic membrane, decolorizing membrane, fine filter and the like, and is subjected to the links of filtration, filter pressing, back washing and the like, so that macromolecular impurities are greatly removed. The invention adopts specific resin to extract L-glutamine liquid, greatly removes salt and other amino acids, and greatly improves the purity and yield of the product.
Example 1
A method for producing and extracting L-glutamine comprising the steps of:
step 1) seed liquid (concentration 1X 10) of Corynebacterium glutamicum ATCC137619cfu/ml) was inoculated into a fermenter containing a fermentation medium at an inoculum size of 10% for a fermentation time of 48 h; the fermentation time is divided into two stages, the first stage is 24 hours, the fermentation temperature is 32 ℃, the ventilation volume is 0.4vvm, and the pH is controlled to be 5.0; the second stage is 24h, the fermentation temperature is 32 ℃, the ventilation rate is 0.5vvm, when the second stage begins, regulating solution is added into the fermentation tank once, the adding amount accounts for 1.5% of the volume of the fermentation liquid, glucose solution with the concentration of 100g/L is added into the fermentation tank, the concentration of glucose in the fermentation tank is controlled to be not less than 0.8%, and the pH value is controlled to be 7.0 by adding ammonia water; after the second stage is finished, collecting fermentation liquor;
the fermentation tank culture medium comprises the following components in percentage by mass: 10% of glucose, 3% of corn steep liquor, 0.5% of urea, 0.1% of potassium dihydrogen phosphate, 0.01% of ferrous sulfate heptahydrate, 0.01% of magnesium sulfate heptahydrate and 0.005% of manganese sulfate monohydrate.
The regulating solution comprises the following components: proline 50g/L, arginine 50g/L, inositol 10 g/L.
And step 2) receiving fermented L-glutamine fermentation liquor from the fermentation process in the ceramic membrane separation process at normal temperature, and storing the feed liquid in a storage tank. Activating ceramic membrane modules 1 and 2, controlling the material conveying frequency of 38HZ, the production frequency of 40HZ, the slag discharging frequency of 30HZ, the filtering temperature of 37 ℃ and the pressure of 1.5Mpa, enabling the clear liquid containing the small molecular components to flow in the membrane tube at a high speed through the feed liquid, and enabling the clear liquid to permeate outwards along the direction vertical to the clear liquid under the driving of the pressure to prepare the clear liquid of the ceramic membrane, wherein the concentrated liquid containing the large molecular components is intercepted by the membrane.
And 3) pumping the ceramic membrane clear liquid obtained in the step 2) into a decoloring tank, opening a stirring device when the material is received to 25% of the decoloring tank, completing self circulation, and adding 40kg of active carbon into each tank to decolor the ceramic membrane clear liquid to obtain the decolored clear liquid.
And 4) connecting the decolorized clear liquid obtained in the step 3) into a fine filter, and sequentially performing three links of filtering, filter pressing and back flushing. In the process, the temperature is controlled at 37 ℃ and the pressure is controlled at 0.15MPa, and the fine filtered clear liquid is obtained.
And 5) inoculating the fine filtered clear liquid obtained in the step 4) into an amino acid liquid separation chamber, wherein the amino acid liquid separation chamber comprises a complete filtering and degassing device and an L-glutamine chromatographic separation system based on a sequential simulated moving bed chromatographic technology. Complete filtration and degassing apparatus: comprises a heat exchanger, a filter, a flash tank, an elution water tank, a water flash tank, a post-degassing material tank, a post-degassing water tank, a residual liquid tank and an extraction liquid tank. L-glutamine chromatographic separation system: comprises 6 chromatographic chambers, namely k1, k2, k3, k4, k5 and k6, and a chromatographic automatic control system.
And 6) a feeding preparation link, wherein steam enters the heat exchanger to discharge steam condensate and filtered steam.
And 7) connecting the filtered steam in the step 6) to a flash tank, and connecting the filtered steam to a degassed charging bucket to obtain flash steam.
And 8) passing the elution water in the elution water tank through a heat exchanger, a filter and a water flash tank, and then connecting the elution water to a degassed water tank to obtain flash water.
And 9) performing filtration on the fine filtered clear liquid obtained in the step 4), performing flash evaporation on steam obtained in the step 7) and performing standard filtration on flash evaporated water obtained in the step 8) in a filter chamber to obtain filtered clear liquid.
Step 10) controlling the flow rate of the chromatographic chamber to be 3m3And/h, the temperature is 37 ℃, the pressure difference is within 1.0MPa, the filtered clear liquid is pumped into a chromatographic chamber k2, L-glutamine is extracted through resin, a discharge port of the chromatographic chamber k6 is opened, and the filtered clear liquid is circularly pumped into the chromatographic chambers k1, k3, k4, k5 and k6 in sequence through a chromatographic automatic control system. The extract was obtained in chromatography chamber k6 and the organic acid and other salt impurity components were separated.
And 11) concentrating, crystallizing, centrifuging and drying the extracting solution to obtain the finished product of the L-glutamine.
The measured L-glutamine index is as follows: the yield of L-glutamine is 85.7%;
L-Glutamine content: 99.3% specific rotation [ α ]: +6.6
pH value: 4.50 sulfate salt: <0.03%
Loss on drying: <0.3% ash: <0.3%
Heavy metals (in pb): <15mg/kg chloride: <0.05%
Example 2
A method for producing and extracting L-glutamine comprising the steps of:
step 1) seed liquid (concentration of 2X 10) of Corynebacterium glutamicum ATCC137619cfu/ml) was inoculated in a fermentation tank containing fermentation medium at an inoculum size of 7%, the fermentation time was 48 h; the fermentation time is divided into two stages, the first stage is 24 hours, the fermentation temperature is 32 ℃, the ventilation volume is 0.4vvm, and the pH is controlled to be 5.0; the second stage is 24 hours, the fermentation temperature is 32 ℃, the ventilation volume is 0.5vvm, when the second stage begins, regulating solution is added into the fermentation tank once, the adding amount accounts for 2% of the volume of the fermentation liquid, glucose solution with the concentration of 100g/L flows into the fermentation tank, the concentration of glucose in the fermentation tank is controlled to be not less than 0.8%, and the pH value is controlled to be 7.0 by flowing ammonia water; after the second stage is finished, collecting fermentation liquor;
the fermentation tank culture medium comprises the following components in percentage by mass: 10% of glucose, 3% of corn steep liquor, 0.5% of urea, 0.1% of potassium dihydrogen phosphate, 0.01% of ferrous sulfate heptahydrate, 0.01% of magnesium sulfate heptahydrate and 0.005% of manganese sulfate monohydrate.
The regulating solution comprises the following components: proline 50g/L, arginine 50g/L, inositol 10 g/L.
And step 2) receiving fermented L-glutamine fermentation liquor from the fermentation process in the ceramic membrane separation process at normal temperature, and storing the feed liquid in a storage tank. Activating ceramic membrane modules 1 and 2, controlling the material conveying frequency of 38HZ, the production frequency of 40HZ, the slag discharging frequency of 30HZ, the filtering temperature of 38 ℃ and the pressure of 2.0Mpa, enabling the clear liquid containing the small molecular components to flow in the membrane tube at a high speed through the feed liquid, and enabling the clear liquid to permeate outwards along the direction vertical to the clear liquid under the driving of the pressure to prepare the clear liquid of the ceramic membrane, wherein the concentrated liquid containing the large molecular components is intercepted by the membrane.
And 3) pumping the ceramic membrane clear liquid obtained in the step 2) into a decoloring tank, opening a stirring device when the material is received to 25% of the decoloring tank, completing self circulation, and adding 40kg of active carbon into each tank to decolor the ceramic membrane clear liquid to obtain the decolored clear liquid.
And 4) connecting the decolorized clear liquid obtained in the step 3) into a fine filter, and sequentially performing three links of filtering, filter pressing and back flushing. In the process, the temperature is controlled at 38 ℃ and the pressure is controlled at 0.2MPa, and the fine filtered clear liquid is obtained.
And 5) inoculating the fine filtered clear liquid obtained in the step 4) into an amino acid liquid separation chamber, wherein the amino acid liquid separation chamber comprises a complete filtering and degassing device and an L-glutamine chromatographic separation system based on a sequential simulated moving bed chromatographic technology. Complete filtration and degassing apparatus: comprises a heat exchanger, a filter, a flash tank, an elution water tank, a water flash tank, a post-degassing material tank, a post-degassing water tank, a residual liquid tank and an extraction liquid tank. L-glutamine chromatographic separation system: comprises 6 chromatographic chambers, namely k1, k2, k3, k4, k5 and k6, and a chromatographic automatic control system.
And 6) a feeding preparation link, wherein steam enters the heat exchanger to discharge steam condensate and filtered steam.
And 7) connecting the filtered steam in the step 6) to a flash tank, and connecting the filtered steam to a degassed charging bucket to obtain flash steam.
And 8) passing the elution water in the elution water tank through a heat exchanger, a filter and a water flash tank, and then connecting the elution water to a degassed water tank to obtain flash water.
And 9) performing filtration on the fine filtered clear liquid obtained in the step 4), performing flash evaporation on steam obtained in the step 7) and performing standard filtration on flash evaporated water obtained in the step 8) in a filter chamber to obtain filtered clear liquid.
Step 10) controlling the flow rate of the chromatographic chamber to be 5m3At 38 ℃ and under a pressure difference of 1.0MPa, the filtered clear solution obtained in the step 9) is injected into a chromatographic chamber k2, L-glutamine is extracted by resin,the discharge port of the chromatographic chamber k6 is opened, and the clear liquid is filtered by a chromatographic automatic control system and sequentially enters the chromatographic chambers k1, k3, k4, k5 and k6 by circulation. The extract was obtained in chromatography chamber k6 and the organic acid and other salt impurity components were separated.
And 11) concentrating, crystallizing, centrifuging and drying the extracting solution to obtain the finished product of the L-glutamine.
The measured L-glutamine index is as follows: the yield of L-glutamine is 85.5%;
L-Glutamine content: 99.4% specific rotation [ α ]: +6.8
pH value: 4.60 sulfate: <0.03%
Loss on drying: <0.3% ash: <0.3%
Heavy metals (in pb): <15mg/kg chloride: < 0.05%.
Example 3
Influence of various fermentation factors on the yield of L-glutamine in fermentation liquor.
1. The effect of the composition of the conditioning solution on the production of L-glutamine in the fermentation mode according to example 1, the specific results are shown in Table 1:
the components of the regulating solution are as follows: proline 50g/L, arginine 50g/L, inositol 10g/L
TABLE 1
| Group of | Fermentation time h | L-Glutamine yield g/100ml |
| Proline, arginine and inositol | 48 | 9.34 |
| Proline + arginine | 48 | 8.56 |
| Arginine + inositol | 48 | 8.17 |
| Proline + inositol | 48 | 8.34 |
| Without addition of regulators | 48 | 7.82 |
And (4) conclusion: as shown in Table 1, compared with the group without the regulator, the L-glutamine yield of other groups is improved, wherein the L-glutamine yield of the proline, arginine and inositol group is the highest and is improved by 15.5 percent; reason analysis shows that a proper amount of inositol can strengthen the CO2 immobilization reaction, weaken the glyoxylate cycle, ensure that the tricarboxylic acid cycle is not interrupted and continuously supply alpha-ketoglutaric acid, and greatly accumulate glutamic acid through reductive amination reaction to improve the fermentation conversion rate; the glutamic acid can be metabolized to generate glutamine, proline and arginine, and the proline and arginine can be added in the middle and later stages of fermentation to generate a feedback inhibition effect on the proline and arginine pathway, so that more glutamic acid flows to the glutamine pathway.
2. Influence of pH changes on the L-glutamine production in the fermentation broth.
The first stage of fermentation, the pH of the fermentation liquor is regulated to be acidic by feeding diluted hydrochloric acid or ammonia water, so that the synthesis of glutamic acid is facilitated, the permeability of cells is reduced, the glutamic acid cannot flow out of the cells, the accumulation of the glutamic acid is facilitated, the second stage of fermentation, the pH of the fermentation liquor is regulated to be neutral or slightly alkaline by feeding the ammonia water, the synthesis of glutamine by the glutamic acid in the cells is facilitated, the permeability of the cells is improved, and the yield of the glutamine is increased. Specific data are shown in table 2:
TABLE 2
| Group of | Glutamic acid yield g/100ml | L-Glutamine yield g/100ml |
| The pH of the whole fermentation process is 5 | 0.69 | 8.56 |
| pH 5 in stage 1 and 7 in stage 2 | 0.21 | 9.34 |
| The pH of the whole fermentation process is 7 | 0.17 | 7.86 |
The foregoing list is only illustrative of the preferred embodiments of the present invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.
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