CN109628518B - Method for producing and extracting L-glutamine - Google Patents

Method for producing and extracting L-glutamine Download PDF

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CN109628518B
CN109628518B CN201811576619.1A CN201811576619A CN109628518B CN 109628518 B CN109628518 B CN 109628518B CN 201811576619 A CN201811576619 A CN 201811576619A CN 109628518 B CN109628518 B CN 109628518B
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glutamine
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ceramic membrane
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CN109628518A (en
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包鑫
李德衡
王健
徐庆阳
刘元涛
张宗华
杨瑞丽
李江涛
庄会华
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Xinjiang Fufeng Biotechnology Co ltd
Jilin University
Tianjin University of Science and Technology
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Jilin University
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    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00Preparation of nitrogen-containing organic compounds
    • C12P13/04Alpha- or beta- amino acids
    • C12P13/14Glutamic acid; Glutamine

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Abstract

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, step 4) fine filtration, step 5) chromatographic extraction, and step 6) concentration and crystallization. The method has the advantages of high fermentation yield, simple process, high efficiency, environmental protection and low production cost.

Description

Method for producing and extracting L-glutamine
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.

Claims (7)

1. 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 crystallization, and is characterized in that 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 24 hours, the fermentation temperature is 32-35 ℃, the ventilation volume is 0.4vvm, and the pH is controlled at 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 1-2% 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 feeding ammonia water; after the second stage is finished, collecting L-glutamine fermentation liquor; the regulating solution comprises the following components: proline 50g/L, arginine 50g/L, inositol 10 g/L.
2. The method of claim 1, wherein the components of the fermentor medium are, in 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.
3. The method according to claim 1, wherein the step 2) ceramic membrane filtration comprises the following processes: filtering with ceramic membrane, and collecting ceramic membrane clear solution.
4. The method according to claim 1 or 3, characterized in that the step 3) of decolorizing comprises the following processes: and (2) feeding the clear liquid of the ceramic membrane obtained in the step 1) into a decoloring tank for decoloring, wherein a decoloring agent is activated carbon, and filtering to obtain a decolored clear liquid after decoloring.
5. The method as claimed in claim 4, wherein the step 4) fine filtering: 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.
6. The method according to claim 5, wherein the step 5) of chromatographic extraction comprises the steps of: 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.
7. The method according to claim 6, wherein the step 6) of concentrating the crystals comprises the steps of: and (3) concentrating, crystallizing, centrifuging and drying the L-glutamine extracting solution to obtain the finished product of the L-glutamine.
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