CN109628518A - A method of production and extraction L-Glutamine - Google Patents
A method of production and extraction L-Glutamine Download PDFInfo
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- CN109628518A CN109628518A CN201811576619.1A CN201811576619A CN109628518A CN 109628518 A CN109628518 A CN 109628518A CN 201811576619 A CN201811576619 A CN 201811576619A CN 109628518 A CN109628518 A CN 109628518A
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- glutamine
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- 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
Abstract
The invention belongs to technical field of biological fermentation, disclose the method for a kind of production and extraction L-Glutamine comprising following steps: step 1) fermentation, step 2 ceramic membrane filter, step 3) decoloration, step 4) refined filtration, step 5) chromatography is extracted, step 6) condensing crystallizing.The method of the present invention fermentation production rate is high, and simple process, high-efficiency environment friendly, production cost are low.
Description
Technical field
The invention belongs to the L-Glutamine production field in biofermentation industry, a kind of production and extraction L- are specifically provided
The method of glutamine.
Background technique
L-Glutamine (L-Glutamine, L-Gln) is a kind of amino acid of γ-carboxy amidation by Pidolidone,
Chemical name 2,5- diamino -5- oxopentanoic acid are one of the primary amino acids for constituting protein.L-Glutamine is a kind of white
Orthorhombic system crystals or crystalline powder, it is odorless nontoxic, there is slightly sweet fragrance, is insoluble in the organic solvents such as methanol, ether, chloroform.
L-Glutamine is one of the primary amino acid of 20 kinds of synthetic proteins matter, accounts for about the 60% of human body free amino acid,
Serve in organism metabolism very important.Medical discovery shows that L-Glutamine shortage will cause a variety of diseases in recent years,
It plays an important role in vital movement, is widely used in medicine, health care, cell culture and feedstuff industry.
Currently, the method for production L-Glutamine mainly includes chemical synthesis, enzymic synthesis and biological fermentation process,
Wherein, chemical synthesis has used a large amount of chemical reagent, be easy to cause environmental pollution, and product quality is not high；Enzymatic closes
At method higher cost, can not promote the use of；Biological fermentation process is the main method for producing L-Glutamine, has there is more grind
Study carefully and biological fermentation process is improved, including mutagenic strain, Optimal Medium and condition of culture etc., the patent before applicant
Fermentation method is optimized in technology " a kind of method of Production of L-Glutamine by Microbial Fermentation ", using two kinds of bacterial strain cooperative fermentations
The defects of mode improves fermentation yield, but there are strain culturing and operating parameter are complicated, and yield is unstable.
Currently, the domestic conventional method for extracting L-Glutamine is ion-exchange, there are device structures to answer for this method
The disadvantages such as miscellaneous, the degree of automation is low, and production efficiency and product quality are low.In addition, existing traditional extraction technique institute output
L-Glutamine finished product is extremely difficult to the requirement of certain clients, in order to improve product quality, can use sequential simulated movement
Bed chromatographic technique substitutional ion exchange process, extraction process route in road after readjustment improve market to improve product quality
Competitiveness.
Summary of the invention
To solve the problems, such as that above-mentioned L-Glutamine production and extraction exist, the present invention provides a kind of productions and extraction L-
The method of glutamine, this method fermentation production rate is high, and simple process, high-efficiency environment friendly, production cost are low.
The present invention is achieved by the following technical solution:
A method of production and extraction L-Glutamine comprising following steps: step 1) fermentation, step 2 ceramic membrane mistake
Filter, step 3) decoloration, step 4) refined filtration, step 5) chromatography are extracted, step 6) condensing crystallizing.
Further, the step 1) fermentation, including following technique: by Corynebacterium glutamicum with the inoculation of 6-10% inoculum concentration
Into the fermentor containing fermentation medium, fermentation time 48h；Fermentation time is divided into two stages, the first stage be for 24 hours,
Fermentation temperature is 32-35 DEG C, ventilatory capacity 0.4vvm, controls pH 5.0 by stream plus hydrochloric acid or ammonium hydroxide；Second stage be for 24 hours,
Fermentation temperature is 32 DEG C, ventilatory capacity 0.5vvm, when second stage starts, and into fermentor, addition adjusts liquid, and additive amount accounts for hair
The 1-2% of zymotic fluid volume, stream plus glucose solution into fermentor control concentration of glucose in fermentor and are not less than 0.8%, pass through
Feeding ammonia water controls pH in 7.0-7.2；After the completion of second stage, L-Glutamine fermentation liquid is collected.
Further, the group of the fermentation tank culture medium is divided into (mass percent): glucose 10%, corn pulp 3%, urea
0.5%, potassium dihydrogen phosphate 0.1%, ferrous sulfate heptahydrate 0.01%, epsom salt 0.01%, manganese sulfate monohydrate 0.005%.
Further, the component for adjusting liquid are as follows: proline 50g/L, arginine 50g/L, inositol 10g/L.
Further, the step 2 ceramic membrane filter, including following technique: by ceramic membrane filter, ceramic membrane is collected
Clear liquid.
Further, step 3) decoloration, including following technique: by the ceramic membrane clear liquid of step 2 squeeze into bleacher into
Row decoloration, decolorising agent is active carbon, and after the completion of decoloration, decoloration clear liquid is obtained by filtration.
Further, step 3) decoloration clear liquid access strainer the step 4) refined filtration: is subjected to refined filtration, temperature control
35-40 DEG C, pressure controls 0.1-0.2MPa, obtains refined filtration clear liquid.
Further, the step 5) chromatography is extracted, and includes the following steps: the refined filtration clear liquid of step 4) accessing chromatography chamber
It extracts, controls Extraction technique: chromatography chamber flow 3-5m3/ h, 35-40 DEG C of temperature, pressure difference 0.8-1.0MPa obtains L-
Glutamine extracting solution.
Further, the step 6) condensing crystallizing includes the following steps: L-Glutamine extracting solution passing through concentration knot
Brilliant, centrifugation drying obtains L-Glutamine finished product.
Technical solution of the present invention has the advantages that following prominent and uniqueness:
It in zymotechnique of the present invention, is added to fermentation and adjusts liquid, wherein suitable inositol can strengthen the fixed reaction of CO2, cut
Weak glyoxalic acid circulation, guarantees that tricarboxylic acid cycle is not disrupted and continually supplies α-ketoglutaric acid, anti-by reductive amination
It answers, largely accumulates glutamic acid, improve fermentation conversion rate；And glutamic acid can be metabolized and generate glutamine, proline and arginine,
Proline and arginine are added by the fermentation middle and later periods, feedback inhibition can be generated to corresponding proline and arginine pathway and made
With so that more flowing to glutamine approach.It ferments the first stage, by adjusting the pH slant acidity of fermentation liquid, favorably
In the synthesis of glutamic acid, and cell permeability is deteriorated, glutamic acid will not flow out it is extracellular, be conducive to accumulate paddy ammonia
Acid, second stage of fermenting, the pH for adjusting fermentation liquid by Feeding ammonia water are conducive to intracellular glutamic acid to alkalinity neutral or on the weak side
Glutamine is synthesized, and cell permeability improves, to improve the yield of glutamine.
The present invention extracts L-Glutamine using " sequential type simulated moving bed chromatography " technology, with traditional ion-exchange
It compares, acid and alkali consumption is substantially reduced with water consume, and entire production process energy conservation and environmental protection reduces the pollution of environment.The present invention uses
Feed liquid flash evaporation technology handles L-Glutamine feed liquid, greatly improves the yield of L-Glutamine product, product matter
Amount is also substantially increased.L-Glutamine feed liquid of the present invention through ceramic membrane, decoloration film, the processes such as strainer and by filtering, filters pressing,
The links such as backwash, greatly eliminate big molecular impurity.The present invention proposes L-Glutamine feed liquid using specific resin
It takes, greatly eliminates salinity and other amino acid, product purity is substantially increased with yield.
Embodiment 1
A method of production and extraction L-Glutamine comprising following steps:
By Corynebacterium glutamicum ATCC13761 seed liquor, (concentration is 1 × 10 to step 1)9Cfu/ml it) is inoculated into 10% inoculum concentration
In fermentor containing fermentation medium, fermentation time 48h；Fermentation time is divided into two stages, and the first stage is hair for 24 hours
Ferment temperature is 32 DEG C, ventilatory capacity 0.4vvm, controls pH 5.0；Second stage is that for 24 hours, fermentation temperature is 32 DEG C, and ventilatory capacity is
0.5vvm, when second stage starts, into fermentor, disposably addition adjusts liquid, and additive amount accounts for the 1.5% of fermentating liquid volume, past
The glucose solution that stream plus concentration are 100g/L in fermentor controls concentration of glucose in fermentor and is not less than 0.8%, passes through stream
Water management pH is ammoniated 7.0；After the completion of second stage, fermentation liquid is collected；
The group of the fermentation tank culture medium is divided into (mass percent): glucose 10%, corn pulp 3%, urea 0.5%, biphosphate
Potassium 0.1%, ferrous sulfate heptahydrate 0.01%, epsom salt 0.01%, manganese sulfate monohydrate 0.005%.
The component for adjusting liquid are as follows: proline 50g/L, arginine 50g/L, inositol 10g/L.
Step 2 ceramic membrane separation process receives the L-Glutamine fermentation liquid that fermentation finishes from fermentation procedure at normal temperature,
Feed liquid is accessed in storage tank and is saved.Ceramic membrane 1* and 2* mould group are activated, conveying frequency 38HZ is controlled, produces frequency 40HZ, row
Slag frequency 30HZ, 37 DEG C of filtration temperature, pressure 1.5Mpa, by feed liquid in membrane tube flow at high speed, driven by pressure containing small
Along direction normal thereto, to exosmosis, ceramic membrane clear liquid, the concentrate envelope containing macromolecular components is made in the clear liquid of molecular components
Retention.
The ceramic membrane clear liquid of step 2 is squeezed into bleacher by step 3), and when splicing to bleacher 25% opens agitating device, complete
At self-loopa, every tank is added 40kg active carbon and carries out decolorization to ceramic membrane clear liquid, obtains decoloration clear liquid.
Step 3) decoloration clear liquid is accessed strainer by step 4), passes sequentially through three filtering, filters pressing, backwash links.?
During this, temperature controls 37 DEG C, and pressure controls 0.15MPa, obtains refined filtration clear liquid.
The refined filtration clear liquid of step 4) is accessed amino acid feed liquid separation chamber by step 5), and wherein amino acid feed liquid separation chamber includes
Filtering and degasser and the L-Glutamine chromatographic fractionation system based on sequential type simulated moving bed chromatography technology completely.It is complete
Full filtering and degasser: including heat exchanger, filter, flash tank, water pot, water flash tank, batch can after degassing, after degassing are eluted
Water pot, residual night tank extract flow container.L-Glutamine chromatographic fractionation system: including 6 chromatography chambers, respectively k1, k2, k3, k4,
K5, k6 and chromatography automatic control system.
Step 6) charging prepares link, and steam enters heat exchanger and goes out steam condensate (SC) and filtering steam.
The filtering steam of step 6) is accessed flash tank by step 7), and batch can after access degassing obtains flash-off steam.
The elution water for eluting water pot is passed through water after access degassing after heat exchanger and filter and water flash tank by step 8)
Tank obtains flash distillation water.
Step 4) refined filtration clear liquid, step 7) flash-off steam, step 8) flash distillation water quality standard are accessed mistake in filter chamber by step 9)
Filter, is obtained cleaner liquid.
Step 10) passes through control chromatography chamber flow 3m3/ h, 37 DEG C of temperature, pressure difference beats cleaner liquid within 1.0MPa
Enter in chromatography chamber k2, L-Glutamine is extracted by resin, open chromatography chamber k6 discharge port, is automated and controlled by chromatography
System processed cross cleaner liquid by circulation according to this it is secondary enter chromatography chamber k1, k3, k4, k5, k6.Extracting solution is obtained in chromatography chamber k6, point
Separate out organic acid and other salt impurity components.
Extracting solution is obtained L-Glutamine finished product by condensing crystallizing, centrifugation drying by step 11).
L-Glutamine index after measured: L-glutamine yield 85.7%；
L-Glutamine content: 99.3% specific rotation [α] :+6.6
PH value: 4.50 sulfate: < 0.03%
Loss on drying: < 0.3% ash content: < 0.3%
Heavy metal (in terms of pb): < 15mg/kg chloride: < 0.05%
Embodiment 2
A method of production and extraction L-Glutamine comprising following steps:
By Corynebacterium glutamicum ATCC13761 seed liquor, (concentration is 2 × 10 to step 1)9Cfu/ml it) is inoculated into 7% inoculum concentration
In fermentor containing fermentation medium, fermentation time 48h；Fermentation time is divided into two stages, and the first stage is hair for 24 hours
Ferment temperature is 32 DEG C, ventilatory capacity 0.4vvm, controls pH 5.0；Second stage is that for 24 hours, fermentation temperature is 32 DEG C, and ventilatory capacity is
0.5vvm, when second stage starts, into fermentor, disposably addition adjusts liquid, and additive amount accounts for the 2% of fermentating liquid volume, toward hair
Stream adds concentration to be the glucose solution of 100g/L in fermentation tank, controls concentration of glucose in fermentor and is not less than 0.8%, is added by stream
Ammonium hydroxide controls pH 7.0；After the completion of second stage, fermentation liquid is collected；
The group of the fermentation tank culture medium is divided into (mass percent): glucose 10%, corn pulp 3%, urea 0.5%, biphosphate
Potassium 0.1%, ferrous sulfate heptahydrate 0.01%, epsom salt 0.01%, manganese sulfate monohydrate 0.005%.
The component for adjusting liquid are as follows: proline 50g/L, arginine 50g/L, inositol 10g/L.
Step 2 ceramic membrane separation process receives the L-Glutamine fermentation liquid that fermentation finishes from fermentation procedure at normal temperature,
Feed liquid is accessed in storage tank and is saved.Ceramic membrane 1* and 2* mould group are activated, conveying frequency 38HZ is controlled, produces frequency 40HZ, row
Slag frequency 30HZ, 38 DEG C of filtration temperature, pressure 2.0Mpa, by feed liquid in membrane tube flow at high speed, driven by pressure containing small
Along direction normal thereto, to exosmosis, ceramic membrane clear liquid, the concentrate envelope containing macromolecular components is made in the clear liquid of molecular components
Retention.
The ceramic membrane clear liquid of step 2 is squeezed into bleacher by step 3), and when splicing to bleacher 25% opens agitating device, complete
At self-loopa, every tank is added 40kg active carbon and carries out decolorization to ceramic membrane clear liquid, obtains decoloration clear liquid.
Step 3) decoloration clear liquid is accessed strainer by step 4), passes sequentially through three filtering, filters pressing, backwash links.?
During this, temperature controls 38 DEG C, and pressure controls 0.2MPa, obtains refined filtration clear liquid.
The refined filtration clear liquid of step 4) is accessed amino acid feed liquid separation chamber by step 5), and wherein amino acid feed liquid separation chamber includes
Filtering and degasser and the L-Glutamine chromatographic fractionation system based on sequential type simulated moving bed chromatography technology completely.It is complete
Full filtering and degasser: including heat exchanger, filter, flash tank, water pot, water flash tank, batch can after degassing, after degassing are eluted
Water pot, residual night tank extract flow container.L-Glutamine chromatographic fractionation system: including 6 chromatography chambers, respectively k1, k2, k3, k4,
K5, k6 and chromatography automatic control system.
Step 6) charging prepares link, and steam enters heat exchanger and goes out steam condensate (SC) and filtering steam.
The filtering steam of step 6) is accessed flash tank by step 7), and batch can after access degassing obtains flash-off steam.
The elution water for eluting water pot is passed through water after access degassing after heat exchanger and filter and water flash tank by step 8)
Tank obtains flash distillation water.
Step 4) refined filtration clear liquid, step 7) flash-off steam, step 8) flash distillation water quality standard are accessed mistake in filter chamber by step 9)
Filter, is obtained cleaner liquid.
Step 10) passes through control chromatography chamber flow 5m3/ h, 38 DEG C of temperature, pressure difference is within 1.0MPa, by the mistake of step 9)
Cleaner liquid is squeezed into chromatography chamber k2, is extracted by resin to L-Glutamine, is opened chromatography chamber k6 discharge port, is passed through chromatography
Automatic control system cross cleaner liquid by circulation according to this it is secondary enter chromatography chamber k1, k3, k4, k5, k6.It is mentioned in chromatography chamber k6
Liquid is taken, organic acid and other salt impurity components are isolated.
Extracting solution is obtained L-Glutamine finished product by condensing crystallizing, centrifugation drying by step 11).
L-Glutamine index after measured: L-glutamine yield 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 content: < 0.3%
Heavy metal (in terms of pb): < 15mg/kg chloride: < 0.05%.
Embodiment 3
Influence of each fermentation factor to L-Glutamine yield in fermentation liquid.
1, influence of the liquid component to L-Glutamine yield is adjusted, for fermentation method referring to embodiment 1, concrete outcome is shown in Table 1:
Adjust the component of liquid are as follows: proline 50g/L, arginine 50g/L, inositol 10g/L
Table 1
| Group | Fermentation time h | L-Glutamine yield g/100ml |
| Proline+arginine+inositol | 48 | 9.34 |
| Proline+arginine | 48 | 8.56 |
| Arginine+inositol | 48 | 8.17 |
| Proline+inositol | 48 | 8.34 |
| Do not add regulator | 48 | 7.82 |
Conclusion: as shown in table 1, being compared with the group for not adding regulator, and the L-Glutamine yield of other groups is
It improves, wherein proline+arginine+inositol group L-Glutamine yield highest improves 15.5 percentage points；Reason point
Analysis, suitable inositol can strengthen the fixed reaction of CO2, weaken glyoxalic acid circulation, guarantee that tricarboxylic acid cycle is not disrupted and source
Source is continuing to supply α-ketoglutaric acid, by reduction of amination, largely accumulates glutamic acid, improves fermentation conversion rate；And paddy ammonia
Acid, which can be metabolized, generates glutamine, proline and arginine, adds proline and arginine by the fermentation middle and later periods, can be right
Proline and arginine pathway generate feedback inhibition, so that more flowing to glutamine approach.
2, influence of the change of pH to L-Glutamine yield in fermentation liquid.
It ferments the first stage, the pH slant acidity of fermentation liquid is adjusted by stream plus dilute hydrochloric acid or ammonium hydroxide, is conducive to glutamic acid
Synthesis, and makes cell permeability be deteriorated, and glutamic acid will not flow out extracellular, is conducive to accumulate glutamic acid, fermentation second
Stage, the pH for adjusting fermentation liquid by Feeding ammonia water are conducive to intracellular glutamic acid synthesis glutamy to alkalinity neutral or on the weak side
Amine, and cell permeability improves, to improve the yield of glutamine.Specific data are shown in Table 2:
Table 2
| Group | Glutamic acid yield g/100ml | L-Glutamine yield g/100ml |
| Entire fermentation process pH is 5 | 0.69 | 8.56 |
| It is 7 that 1st stage pH, which was the 5, the 2nd stage, | 0.21 | 9.34 |
| Entire fermentation process pH is 7 | 0.17 | 7.86 |
Listed above is only best specific embodiment of the invention.It is clear that the invention is not restricted to which above embodiments, can also have
Many deformations.All deformations that those skilled in the art directly can export or associate from present disclosure,
It is considered as protection scope of the present invention.
Claims (9)
1. a kind of method of production and extraction L-Glutamine comprising following steps: step 1) fermentation, step 2 ceramic membrane mistake
Filter, step 3) decoloration, step 4) refined filtration, step 5) chromatography are extracted, step 6) condensing crystallizing.
2. the method according to claim 1, wherein the step 1) is fermented, including following technique: by glutamic acid
Bar bacterium is inoculated into the fermentor containing fermentation medium with 6-10% inoculum concentration, fermentation time 48h；Fermentation time is divided into
Two stages, first stage are that for 24 hours, fermentation temperature is 32-35 DEG C, ventilatory capacity 0.4vvm, control pH 5.0；Second stage
For for 24 hours, fermentation temperature is 32 DEG C, ventilatory capacity 0.5vvm, when second stage starts, and into fermentor, disposably addition is adjusted
Liquid, additive amount account for the 1-2% of fermentating liquid volume, while stream plus glucose solution into fermentor, and it is dense to control glucose in fermentor
Degree is not less than 0.8%, and controls pH in 7.0-7.2 by Feeding ammonia water；After the completion of second stage, L-Glutamine hair is collected
Zymotic fluid.
3. according to the method described in claim 2, it is characterized in that, the group of the fermentation tank culture medium is divided into (quality percentage
Than): glucose 10%, corn pulp 3%, urea 0.5%, potassium dihydrogen phosphate 0.1%, ferrous sulfate heptahydrate 0.01%, epsom salt
0.01%, manganese sulfate monohydrate 0.005%.
4. according to the method described in claim 2, it is characterized in that, the component for adjusting liquid are as follows: proline 50g/L, smart ammonia
Sour 50g/L, inositol 10g/L.
5. the method according to claim 1, wherein the step 2 ceramic membrane filter, including following technique: it is logical
Ceramic membrane filter is crossed, ceramic membrane clear liquid is collected.
6. method according to claim 2 or 5, which is characterized in that the step 3) decoloration, including following technique: will walk
Rapid ceramic membrane clear liquid 2) squeezes into bleacher and decolourizes, and decolorising agent is active carbon, and after the completion of decoloration, it is clear that decoloration is obtained by filtration
Liquid.
7. according to the method described in claim 6, it is characterized in that, the step 4) refined filtration: the decoloration clear liquid of step 3) is connect
Enter strainer and carry out refined filtration, temperature controls 35-40 DEG C, and pressure controls 0.1-0.2MPa, obtains refined filtration clear liquid.
8. including the following steps: to walk the method according to the description of claim 7 is characterized in that the step 5) chromatography is extracted
Rapid refined filtration clear liquid access chromatography chamber 4) extracts, and controls Extraction technique: chromatography chamber flow 3-5m3/ h, temperature 35-40
DEG C, pressure difference 0.8-1.0MPa obtains L-Glutamine extracting solution.
9. according to the method described in claim 8, it is characterized in that, the step 6) condensing crystallizing, includes the following steps: L-
Glutamine extracting solution obtains L-Glutamine finished product by condensing crystallizing, centrifugation drying.
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| CN111057727A (en) * | 2019-12-16 | 2020-04-24 | 新疆阜丰生物科技有限公司 | Method for producing, separating and extracting L-glutamine |
| CN111057727B (en) * | 2019-12-16 | 2021-10-08 | 新疆阜丰生物科技有限公司 | Method for producing, separating and extracting L-glutamine |
| CN112812985A (en) * | 2020-11-11 | 2021-05-18 | 新疆阜丰生物科技有限公司 | Method for improving fermentation acid production of glutamine |
| CN112812985B (en) * | 2020-11-11 | 2023-01-10 | 新疆阜丰生物科技有限公司 | Method for improving acid production of glutamine fermentation |
| CN113828157A (en) * | 2021-11-01 | 2021-12-24 | 同舟纵横（厦门）流体技术有限公司 | Method for high-pressure concentration of glutamine extracting solution |
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