CN109796357A - A method of l-Isoleucine is extracted using chromatographic technique - Google Patents
A method of l-Isoleucine is extracted using chromatographic technique Download PDFInfo
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- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 title claims abstract description 53
- 238000000034 method Methods 0.000 title claims abstract description 21
- 238000004587 chromatography analysis Methods 0.000 title claims abstract description 10
- 238000000855 fermentation Methods 0.000 claims abstract description 36
- 230000004151 fermentation Effects 0.000 claims abstract description 36
- 238000000746 purification Methods 0.000 claims abstract description 6
- 150000003839 salts Chemical class 0.000 claims abstract description 6
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- 239000007788 liquid Substances 0.000 claims description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 claims description 9
- 239000012535 impurity Substances 0.000 claims description 9
- 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 claims description 7
- 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 claims description 7
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- 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 3
- 239000007836 KH2PO4 Substances 0.000 claims description 3
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 3
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 3
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 3
- 239000000919 ceramic Substances 0.000 claims description 3
- 238000002425 crystallisation Methods 0.000 claims description 3
- 230000008025 crystallization Effects 0.000 claims description 3
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 3
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims description 3
- 229910052564 epsomite Inorganic materials 0.000 claims description 3
- 239000008103 glucose Substances 0.000 claims description 3
- 239000002054 inoculum Substances 0.000 claims description 3
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 3
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 claims description 3
- 229910052603 melanterite Inorganic materials 0.000 claims description 3
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- 229910052760 oxygen Inorganic materials 0.000 claims description 3
- 239000001301 oxygen Substances 0.000 claims description 3
- 238000004806 packaging method and process Methods 0.000 claims description 3
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 3
- 238000001035 drying Methods 0.000 claims description 2
- 235000013922 glutamic acid Nutrition 0.000 claims description 2
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- DWNBOPVKNPVNQG-LURJTMIESA-N (2s)-4-hydroxy-2-(propylamino)butanoic acid Chemical compound CCCN[C@H](C(O)=O)CCO DWNBOPVKNPVNQG-LURJTMIESA-N 0.000 claims 1
- 241000894006 Bacteria Species 0.000 claims 1
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- 229960000310 isoleucine Drugs 0.000 description 12
- 229930182844 L-isoleucine Natural products 0.000 description 9
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 8
- 239000000047 product Substances 0.000 description 7
- 238000003786 synthesis reaction Methods 0.000 description 5
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 4
- 239000002253 acid Substances 0.000 description 4
- 230000007423 decrease Effects 0.000 description 4
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- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- AGPKZVBTJJNPAG-AKGZTFGVSA-N (2s)-2-amino-3-methylpentanoic acid Chemical compound CCC(C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-AKGZTFGVSA-N 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
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- 239000011347 resin Substances 0.000 description 2
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- GBKVTQSWZCBVSL-FHAQVOQBSA-N (2s,3s)-2-amino-3-methylpentanoic acid;hydrochloride Chemical compound Cl.CC[C@H](C)[C@H](N)C(O)=O GBKVTQSWZCBVSL-FHAQVOQBSA-N 0.000 description 1
- 101000950981 Bacillus subtilis (strain 168) Catabolic NAD-specific glutamate dehydrogenase RocG Proteins 0.000 description 1
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- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- 206010054949 Metaplasia Diseases 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/10—Process efficiency
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention belongs to l-Isoleucine extraction process fields, disclose a kind of method for extracting l-Isoleucine using chromatographic technique comprising following steps: step 1) fermentation, step 2 filtering, step 3) chromatogram purification, step 4) prepare finished product.Fermentation efficiency of the present invention is high, realizes l-Isoleucine and protein, sugar, inorganic salts, being completely separated between heteroacid in extraction process substantially, extraction process is full-automatic, and labor intensity is low, and production cost reduces, and meets energy conservation and environmental protection theory.
Description
Technical field
The invention belongs to l-Isoleucine extraction process fields, specifically provide a kind of utilization chromatographic technique extraction different bright ammonia of L-
The method of acid.
Background technique
L-Isoleucine, also known as isoleucine, -3 methylvaleric acid of chemical name L-2- amino, 1901, Fischer was by egg
The optical activity substance bigger compared with leucine is had found in the L-Leu component of white matter hydrolyzate separation, this is the different bright ammonia of related L-
The earliest report of acid.L-Isoleucine is the important component of living organism, belongs to essential amino acid, because it is special
Structure and function occupies important function in human life metabolism.If human body long-term lacking l-Isoleucine, will affect body
Physiological function leads to metabolic disorder, resistance decline etc..
L-Isoleucine is widely used in health care of food, the row such as biological medicine and medical and beauty treatment with its unique effect
Industry.
L-Isoleucine production method has extraction method, chemical synthesis and three kinds of fermentation method.Extraction method is mainly by soybean
Albumen etc. is hydrolyzed, and hydrochloric acid is added then to generate and be settled out l-Isoleucine-hydrochloric acid salt crystal.The method separating effect
Good, extraction operation is simple, with short production cycle, but the yield of l-Isoleucine is lower, causes production cost high, causes
The method is difficult to be promoted in industrialized production.Chemical synthesis has a variety of synthesis modes, but production cost is higher, instead
Answer step more, reaction process is complicated, causes by-product more, it is very difficult to graft in industrial-scale production.
Currently, l-Isoleucine production method is mainly fermentation method, Studies on Fermentation of L-isoleucine is able to industrial metaplasia
Production also relies on l-Isoleucine separation and extraction technology.At present both at home and abroad l-Isoleucine separation and Extraction nearly all use with from
Sub- exchange column partition method, although this method can carry out large scale preparation, product cost is high, and exchanger resin dosage is huge,
Elution and regenerated acid and alkali consumption are too many, cause serious environmental pollution, and in addition l-Isoleucine is easy in highly basic and strong acid environment
Cause yield low, purity is not high.
Summary of the invention
It is at low cost the present invention provides a kind of high income to solve the problems, such as that l-Isoleucine extraction process exists, energy conservation
The high efficiency l-Isoleucine novel technology for extracting of consumption reduction.
The present invention is achieved by the following technical solution:
A method of l-Isoleucine being extracted using chromatographic technique comprising following steps: step 1) fermentation, step 2 mistake
Filter, step 3) chromatogram purification, step 4) prepare finished product.
Further, the step 1) fermentation, includes the following: to connect Corynebacterium glutamicum seed liquor according to 10% inoculum concentration
Kind is into fermentation medium, and fermented and cultured 72 hours, fermentation culture conditions are as follows: 30 DEG C of temperature, dissolved oxygen amount 30%, pH 6.5-
6.8, obtain l-Isoleucine fermentation liquid.
Preferably, the component of the fermentation medium are as follows: glucose 120 g/L, (NH4)2SO4 20g/L, KH2PO4 2.2
G/L, K2HPO4 1.0 g/L, MgSO4·7H2O 0.4 g/L, FeSO4·7H2O 0.015 g/L, MnSO4·H2O 0.015
G/L, VH 80 μ g/L, VB12.5mg/L, Met 30mg/L, Glu 20mg/L, inositol 20mg/L.
Further, the step 2 filtering includes the following: to be retained l-Isoleucine fermentation liquid by ceramic membrane
Object and filtered fluid；Film filtration temperature is 50 DEG C, and operating pressure difference is 1.5bar, is 20-200nm by aperture.
Further, the step 3) chromatogram purification includes the following:
(1) it opens 4 inlet valve feed liquid of chromatographic column and column 4 is entered by inlet valve, open 1 inlet valve of column, open simultaneously the discharging of 5 impurity of column
Valve, the impurity such as protein, sugar, pigment, inorganic salts, miscellaneous amino acid are flowed out from the impurity outlet valve of column 5 at this time.
(2) circulating valve is opened, entire chromatographic system enters the circulatory system at this time.
(3) l-Isoleucine liquid enters column 3, arrives column 2 again, finally arrives column 1 by the circulatory system, opens 1 inlet valve of column
Water inlet, opens simultaneously 1 outlet valve of column.L-Isoleucine product liquid is flowed out by 1 discharge port of column.
(4) step 4 is to step 6, step 7 to step 9, step 10 to step 12, step 13 to step 15, step 16 to step
Rapid 18, it takes steps 1 to carry out to the mode of step 3.The chromatography column valve serial number only opened is changed to next chromatography column valve, until
Step 18 is completed a chromatographic cycles system, is circuited sequentially.
Further, the step 4) prepares finished product, includes the following: the concentrated crystallization of feed liquid, baking by chromatogram purification
Finished product is made in dry, packaging.
Technical solution of the present invention has the advantages that following prominent and uniqueness:
1, the present invention separates l-Isoleucine fermentation liquid using chromatographic separation technology, obtains l-Isoleucine product.With it is traditional
Ion exchange column partition method is improved compared to yield, and isolated l-Isoleucine purity is higher.
2, the present invention using resin to the adsorptivities of the impurity such as l-Isoleucine and protein, sugar, pigment, inorganic salts not
Together, being kept completely separate for the impurity such as l-Isoleucine, protein, sugar, pigment, inorganic salts is realized substantially.
3, production process of the present invention is full-automatic, and labor intensity is low, save the cost.
4, any chemicals is not used in production process of the present invention, does not generate ion exchange, only using water as eluant, eluent, is not had
There is pollution to generate.
5, culture medium is optimized in the present invention, appropriate glutamic acid is added in the medium, to Corynebacterium glutamicum
It produces glutamate pathway and generates feedback inhibition, reduce glutamate dehydrogenase enzymatic activity, so that facilitating metabolism flows to isoleucine production
Raw approach；Cause methionine superfluous by adding methionine, the synthesis of feedback inhibition homoserine transacetylase, and then promote different
The synthesis of leucine；Suitable inositol can strengthen the fixed reaction of CO2, promote the accumulation of amino acid, improve fermentation conversion rate；
Many factors mutually cooperate with, and improve the fermentation yield of tryptophan.
Detailed description of the invention
Figure of description
Influence of Fig. 1: the Glu additive amount to L-Isoleucine in Fermentation concentration；
Fig. 2: influence of the incubation time to L-Isoleucine in Fermentation concentration.
Specific embodiment
In order to make those skilled in the art better understand the technical solutions in the application, having below in conjunction with the application
Body embodiment more clearly and completely describes the present invention, it is clear that described embodiment is only the application one
Divide embodiment, instead of all the embodiments.Based on the embodiment in the application, those of ordinary skill in the art are not making
Every other embodiment obtained, should fall within the scope of the present invention under the premise of creative work.
Embodiment 1
A method of l-Isoleucine is extracted using chromatographic technique comprising following steps:
Step 1) by Corynebacterium glutamicum (CGMCC NO.12153, University Of Science and Technology Of Tianjin gift use) culture to concentration be 5 ×
108Then the seed liquor of CFU/mL is inoculated into fermentation medium according to 10% inoculum concentration, and fermented and cultured 72 hours, fermented and cultured
Condition are as follows: 30 DEG C of temperature, dissolved oxygen amount 30%, pH 6.5-6.8 obtains l-Isoleucine fermentation liquid；The fermentation medium
Component are as follows: glucose 120 g/L, (NH4)2SO4 20g/L, KH2PO4 2.2 g/L, K2HPO4 1.0 g/L, MgSO4·7H2O
0.4 g/L, FeSO4·7H2O 0.015 g/L, MnSO4·H2O 0.015 g/L, VH 80 μ g/L, VB12.5mg/L, Met
30mg/L, Glu 20mg/L, inositol 20mg/L；
L-Isoleucine fermentation liquid is removed mycoprotein by ceramic membrane (50nm) by step 2；Membrane filtration operation temperature: 50 DEG C,
Operating pressure difference is 1.5bar, obtains trapped substance and filtered fluid；
Step 3) concentration be 5.5%(solute weight content) filtered fluid (wherein, miscellaneous amino acid 1 2%, l-Isoleucine 73%,
Other impurities 15%) through inlet valve enter chromatographic column 4, feed liquid inlet amount is 0.1m3/ h, water enter chromatographic column 1 by inlet valve, into
Water flow is 0.2m3/h；
Step 4) chromatographic system is that one connected into from beginning to end by 6 root chromatogram columns (column 1, column 2, column 3, column 4, column 5, column 6) follows certainly
Loop system, every column have feed inlet, water inlet, discharge port, circulation port.Entire chromatographic system uses computer controlled automatic side
Formula controls the opening and closing of feed inlet, water inlet, discharge port, circulation port, to realize the continuous operation behaviour of charging, water inlet, discharging
Make；
Circulating valve is opened, 6 chromatographic columns enter the circulatory system；The impurity outlet valve of column 5 flows out protein, sugar, pigment, nothing at this time
The impurity such as machine salt, miscellaneous amino acid, dirt solution concentration are about 1.5%, wherein l-Isoleucine purity 3%, miscellaneous amino acid purity
62%, other carbohydrate purity are about 12%；
Step 5) opens 1 water intake valve of column, opens simultaneously 1 outlet valve of column；It is water-soluble that 1 discharge port of column is collected into l-Isoleucine
Liquid, concentration are about 38%, and l-Isoleucine purity is 97%；
Step 6) is recycled by whole system, and l-Isoleucine fermentation liquid is successively through column 4, column 3, column 2, column 1, column 6, column 5, last
Return to column 4；
Finished product is made through processes such as subsequent concentration crystallization, drying, packagings in the molten feed liquid of the isolated l-Isoleucine of step 7), passes through
Detection calculate l-Isoleucine yield be 92%, purity 99.5%.
Embodiment 2
The present invention has detected the influence of culture medium and incubation time to L-Isoleucine in Fermentation concentration.
Using embodiment 1 as experimental group, the Glu concentration being arranged in culture medium is 0,10,20,30,40,50(mg/L), such as
Shown in Fig. 1, with the increase of Glu concentration, the yield of l-Isoleucine increases obviously, after Glu concentration increases to 20mg/L, L-
The concentration of isoleucine does not have significant change, it is contemplated that volume increase and economic factor select the content of 20mg/L most suitable.
Be arranged fermented and cultured time be 24,36,48,60,72,84(h), by Fig. 2 as it can be seen that with fermentation time increasing
Add, the concentration amplification of L-Isoleucine in Fermentation is obvious, and when fermentation time reaches 60h, amplification slows down, and fermentation time is in 72h
When reach peak value, continue growing fermentation time, l-Isoleucine concentration does not change.
The present invention also has detected the synergistic effect of two kinds of regulatory factors of Met and inositol and produces the shadow of l-Isoleucine to fermentation
It rings, wherein do not add inositol, the content decline 3.3% of l-Isoleucine；Do not add Met, the content decline of l-Isoleucine
4.6%；Do not add inositol and Met, the content decline 9.7% of l-Isoleucine.
Above-mentioned, although specific embodiments of the present invention have been described in conjunction with the embodiments, not protects to the present invention
The limitation of range, those skilled in the art should understand that, based on the technical solutions of the present invention, those skilled in the art
The various modifications or changes that can be made are not needed to make the creative labor still within protection scope of the present invention.
Claims (6)
1. a kind of method for extracting l-Isoleucine using chromatographic technique comprising following steps: step 1) fermentation, step 2 mistake
Filter, step 3) chromatogram purification, step 4) prepare finished product.
2. the method according to claim 1, wherein the step 1) fermentation, includes the following: by glutamic acid rod
Bacterium seed liquor is inoculated into fermentation medium according to 10% inoculum concentration, and fermented and cultured 72 hours, fermentation culture conditions are as follows: temperature 30
DEG C, dissolved oxygen amount 30%, pH 6.5-6.8 obtains l-Isoleucine fermentation liquid.
3. according to the method described in claim 2, it is characterized in that, the component of the fermentation medium are as follows: 120 g/ of glucose
L, (NH4)2SO4 20g/L, KH2PO4 2.2 g/L, K2HPO4 1.0 g/L, MgSO4·7H2O 0.4 g/L, FeSO4·7H2O
0.015 g/L, MnSO4·H2O 0.015 g/L, VH 80 μ g/L, VB12.5mg/L, Met 30mg/L, Glu 20mg/L,
Inositol 20mg/L.
4. according to the method in claim 2 or 3, which is characterized in that the step 2 filtering, includes the following: that L- is different bright
Propylhomoserin fermentation liquid obtains trapped substance and filtered fluid by ceramic membrane；Film filtration temperature is 50 DEG C, and operating pressure difference is 1.5bar, is led to
Crossing aperture is 20-200nm.
5. according to the method described in claim 4, it is characterized in that, the step 3) chromatogram purification, includes the following:
Filtered fluid enters chromatographic column 4 through inlet valve, and feed liquid inlet amount is 0.1m3/ h, water enter chromatographic column 1, feed water flow by inlet valve
Amount is 0.2m3/h；Circulating valve is opened, 6 chromatographic columns enter the circulatory system；The impurity outlet valve outflow of column 5 contains albumen at this time
Matter, sugar, pigment, inorganic salts and miscellaneous amino acid waste liquid；1 water intake valve of column is opened, 1 outlet valve of column is opened simultaneously；Column 1 goes out
Material mouth is collected into l-Isoleucine aqueous solution；Recycled by whole system, filtered fluid successively through column 4, column 3, column 2, column 1, column 6,
Column 5 eventually passes back to column 4.
6. according to the method described in claim 5, including the following: by chromatography it is characterized in that, the step 4) prepares finished product
Finished product is made in the concentrated crystallization of the feed liquid of purifying, drying, packaging.
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