CN107382706B - A method of extracting adipic acid from fermentation liquid - Google Patents

A method of extracting adipic acid from fermentation liquid Download PDF

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CN107382706B
CN107382706B CN201710622975.1A CN201710622975A CN107382706B CN 107382706 B CN107382706 B CN 107382706B CN 201710622975 A CN201710622975 A CN 201710622975A CN 107382706 B CN107382706 B CN 107382706B
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liquid
fermentation liquid
gene
adipic acid
tfu
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CN107382706A (en
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邓禹
赵梅
毛银
赵运英
卢伟强
陆春波
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Jiangnan University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
    • C07C51/42—Separation; Purification; Stabilisation; Use of additives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
    • C07C51/42—Separation; Purification; Stabilisation; Use of additives
    • C07C51/43—Separation; Purification; Stabilisation; Use of additives by change of the physical state, e.g. crystallisation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
    • C07C51/42—Separation; Purification; Stabilisation; Use of additives
    • C07C51/47—Separation; Purification; Stabilisation; Use of additives by solid-liquid treatment; by chemisorption
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
    • C07C51/42—Separation; Purification; Stabilisation; Use of additives
    • C07C51/48—Separation; Purification; Stabilisation; Use of additives by liquid-liquid treatment
    • 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
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/44—Polycarboxylic acids

Abstract

The method that the invention discloses a kind of to extract adipic acid from fermentation liquid, belongs to bioengineering field.Method of the invention be by fermentation liquid by separation of solid and liquid, ultrafiltration, concentration, ion exchange, solution extraction, evaporation and crystallization and etc. processing after, obtain finished product adipic acid product.The adipic acid yield and purity extracted using extracting method of the invention all reach higher level, and ultimate yield is up to 71.7%, and purity is up to 95.86%.

Description

A method of extracting adipic acid from fermentation liquid
Technical field
The method that the present invention relates to a kind of to extract adipic acid from fermentation liquid, belongs to bioengineering field.
Background technique
Adipic acid (Adipic acid, adipate) is also known as adipic acid, is a kind of important organic dibasic acid, is widely used in Chemical Manufacture, organic synthesis industry, medicine, lubricant manufacture etc..
The production method of adipic acid mainly uses air oxidation process or nitric acid oxidation method at present, but the product of such method Yield is lower than 60%, and wastewater flow rate discharge amount is larger, and raw material and intermediate product toxicity are very strong, and generate a large amount of N in the process2O Isothermal chamber gas, environmental pollution is serious and unsustainable.Therefore, sight is focused on the road of biosynthesis adipic acid by people On, and done a large amount of element task.By the full biosynthesis adipic acid of substrate of glucose there is process flow simply, always to put into Outstanding advantages of at low cost, Ke Xunhuanliyong, therefore favor by researcher.But these methods are not directed to fermentation liquid The extraction of middle adipic acid, extracting the problem of adipic acid is primarily present in fermentation liquid is that heteroacid is more in fermentation liquid, it is difficult to separate one Kind organic acid, purity not can guarantee.And the factors such as bacterium upgrowth situation, the organic acid extraction comparison also resulted in fermentation liquid are tired It is difficult.
The present invention extracts adipic acid from the fermentation liquid that glucose is the full biosynthesis adipic acid of substrate, and the rate of recovery is up to 60% More than, purity is up to 90% or more.Method is simply green, and the adipic acid purity is high extracted is not necessarily to secondary treatment, can directly make With.
Summary of the invention
The method that the purpose of the present invention is to provide a kind of to extract adipic acid from fermentation liquid, the method includes walking as follows It is rapid:
(1) it is separated by solid-liquid separation: after fermentation liquid removes thallus and impurity, collecting fermentation liquid clear liquid；
(2) ultrafiltration: utilize ultrafiltration membrane system, by fermentation liquid clear liquid high molecular weight protein and macromolecule impurity remove, receive Collect ultrafiltration permeate；
(3) it is concentrated: ultrafiltration concentration permeate；
(4) ion exchange: the fermentation liquid after concentration is added to and is washed till through alkali cleaning, deionization washing, pickling, deionized water In neutral cation exchange resin, stirs evenly, after adsorbing resin sufficiently, filtrate is collected by filtration；
(5) solvent extraction: by the filtrate being collected into using acid for adjusting pH to acidity, then being extracted using organic solvent, is received Collect extract liquor；
(6) it evaporates: the organic solvent in evaporation liquid；
(7) it crystallizes: by the remaining liquid of rotary evaporation in 4-8 DEG C of low temperature crystallization, after crystallization, being carried out in 20-40 DEG C complete Crystallization, obtains adipic acid finished product.
In one embodiment of the invention, adipic acid content is 10-30g/L in the fermentation liquid in the step (1).
In one embodiment of the invention, the fermentation liquid in the step (1) is with recombination bacillus coli Mad2 △ AtoB ferments to obtain；The recombination bacillus coli Mad2 △ atoB is to have knocked out the e. coli bl21 of atoB gene (DE3) For host, heterologous gene beta-Ketothiolase gene of the sub-module overexpression from thermobifida fusca, 3- hydroxy acyl-coenzyme A dehydrogenase gene, 3- hydroxyl adipyl dehydrogenase gene, penta enoyl CoA reductase gene of 5- carboxyl -2-, adipyl coacetylase Synthase gene segment Tfu_2576, Tfu_2577；Wherein, beta-Ketothiolase gene, 3-hydroxyacyl-CoA dehydrogenase gene Using pRSFDuet-1 as expression vector；3- hydroxyl adipyl dehydrogenase gene, penta enoyl CoA reductase gene of 5- carboxyl -2- Using pTrc99a as expression vector；Adipyl CoA synthetase gene segment Tfu_2576, Tfu_2577 is with pCDFDuet-1 Expression vector.
In one embodiment of the invention, the preparation of the fermentation liquid is: recombination bacillus coli Mad2 △ atoB kind Sub- liquid is seeded in the 5L fermentor equipped with 3L SOB culture medium with 2% inoculum concentration, speed of agitator 400rpm, ventilatory capacity It is 6.8~7.2 that 1vvm, 2M NaOH, which maintain pH, 37 DEG C of fermentation temperature, is cultivated to OD600To add 1mM IPTG when 0.6-0.8, drop Temperature to 30 DEG C induce；Glucose consumption inside fermentation medium adds glycerol when remaining 2g/L or so, to maintain glycerol concentration Feed supplement is carried out in the speed of 4g/L, until glycerol adds total amount and reaches 100g/L.
In one embodiment of the invention, the ingredient of the SOB culture medium is 2g/100ml tryptone, 0.5g/ 100ml yeast powder, 0.05g/100ml NaCl, 2.5mM KCl, 10mM MgCl2, 0.8g/100ml glucose, 50 μ g/ml sulphur Sour kanamycins, 50 μ g/ml ampicillins, 50 μ g/ml streptomysins.
In one embodiment of the invention, the ultrafiltration membrane elements that ultrafiltration membrane system uses in the step (2) is volumes Formula film, the molecular cut off of ultrafiltration membrane are 500-3000.
In one embodiment of the invention, the concentration in the step (3) is that ultrafiltration permeate is concentrated into ultrafiltration The 60-90% of liquid.
In one embodiment of the invention, the cation exchange resin in the step (4) is 732 cation exchanges Resin；The pretreatment mode of cation exchange resin are as follows: cation exchange resin is cleaned 3 times with deionized water first, it is rear to use NaOH adjusting pH to 10 after stirring half an hour, adds H after being rinsed with deionized water until pH is neutral2SO4PH to 1 is adjusted, then again It is rinsed with deionized water to neutrality.
In one embodiment of the invention, the active group of the cation exchange resin in the step (4) is H+。
In one embodiment of the invention, the organic solvent in the step (5) is ethyl alcohol, methanol or acetic acid second Ester.
In one embodiment of the invention, the volume of the organic solvent in the step (5) is 2-4 times of filtrate, Extraction times are 3 times.
In one embodiment of the invention, the evaporation in the step (6) is by organic solvent evaporation to extract liquor 10-20%.
The present invention has the advantages that extracting the adipic acid purity is high in fermentation liquid using the method, yield is high, saves the time And raw material, reduce production cost.Using method of the invention, adipic acid ultimate yield is up to 71.7%, and purity is reachable 95.86%.
Application of membrane separation technology into adipic acid extraction, is effectively removed the high molecular weight protein and sugar in fermentation liquid by the present invention Substance has the extraction using ethyl acetate, effectively prevents forming emulsion after ethyl acetate is added, can not be layered, so that Extract the impure problem of product.With high degree of automation, low energy consumption, pollutes the advantages such as small.
The present invention carries out ion exchange using resin cation, can utmostly remove the salinity introduced in fermentation liquid, It effectively prevents adjusting pH repeatedly during the extraction process.
The present invention is filtered and is concentrated using film before ion exchange, alleviates the pollution of resin, and is extended resin and used the longevity Life；On the other hand the adsorbance and treatment effeciency of resin are also effectively improved.
Specific embodiment
Embodiment 1: the acquisition of recombination bacillus coli Mad2 △ atoB
The sequence of Tfu_0875, Tfu_2399, Tfu_0068, Tfu_1648, Tfu_2576, Tfu_2577 are in the applying date It is preceding to be announced in NCBI.
EcoR I and HindIII double digestion plasmid pRSFDuet-1, gel extraction target gene fragment (3798bp), with same The enzyme digested plasmid pUC57-Tfu_0875 of sample, gel extraction obtain target gene fragment Tfu_0875, then by two purposes Segment T4DNA ligase connection, change turn JM109, bacterium colony PCR picking positive transformant, and extract plasmid enzyme restriction verifying, verifying Plasmid after correct is named as pRSF-Tfu_0875.Bgl II and Kpn I digested plasmid pRSF-Tfu_0875, gel extraction The target gene fragment of 4936bp, with same enzyme digested plasmid pUC57-Tfu_2399, gel extraction target gene fragment, so Afterwards by two target fragment T4DNA ligase connection, change turns JM109, bacterium colony PCR picking positive transformant, and extracts plasmid Digestion verification, the plasmid after verifying is correct are named as pAD-1.
Other plasmids are constructed using same method, and final segment Tfu_0068, Tfu_1648 passes through Nco I, Hind III is connected on plasmid pTrc99a and forms pAD-4 plasmid；Tfu_2576, Tfu_2577 are connected to plasmid by Nco I, Hind III PAD-6 plasmid is formed on pCDFDuet-1.
PAD-1, pAD-4, pAD-6 are transferred in the e. coli bl21 (DE3) for having knocked out atoB gene, weight is prepared Group Escherichia coli Mad2 △ atoB.Referring to patent CN106834200A.
Embodiment 2: upper tank fermentation and interpretation of result
3L culture medium: SOB culture medium, ingredient are+0.5% yeast powder+0.05%NaCl+2.5mM KCl of 2% tryptone +10mM MgCl2+ 50 μ g/ml streptomysin of+50 μ g/ml ampicillin of+50 μ g/ml kanamycin sulfate of+8g/L glucose is mended Material plus 100g/L glycerol.
Seed liquor preparation: for the strain of glycerol stocks in flat lining out, picking single colonie is inoculated in the LB liquid for filling 50ml In the 250ml conical flask of body culture medium, 37 DEG C, 250rpm/min shaking flask stay overnight.Next day takes the switching of 500 μ l bacterium solutions in 60ml LB In fluid nutrient medium, 37 DEG C, 250rpm cultivates to OD600When reaching 0.6-0.8, it is inoculated in 5L fermentor.
Fermentation condition: 2% inoculum concentration, 37 DEG C of cultures to OD600To add 30 DEG C of 1mM IPTG inductions when 0.6-0.8 or so, It is 6.8~7.2 that speed of agitator 400rpm, ventilatory capacity 1vvm, 2M NaOH, which maintain pH,.Disappear to the glucose inside fermentation medium Glycerol is added when consuming surplus 2g/L or so, to maintain glycerol concentration to carry out feed supplement in the speed of 4g/L, until glycerol is added total amount and reached To 100g/L.
Interpretation of result: every 4h takes a sample in fermentation process, and 10000r/min centrifugation 2min separates fermentation liquid with thallus, 0.22 μm of filter membrane handles fermentation liquid, for carrying out HPLC (high performance liquid chromatography, the primary Bio-Rad Bole Aminex HPX- in the U.S. 87H organic acid column) it detects, mobile phase is 5mM H in HPLC detection2SO4, column temperature is 30 DEG C, UV detector 210nm.Upper tank hair Ferment adipic acid yield is 25.57g/L.
Embodiment 3: organic solvent extracts adipic acid in fermentation liquid
The adipic acid in the fermentation liquid of embodiment 2 is extracted in accordance with the following methods:
(1) tank fermentation liquid is centrifuged removal thallus and impurity by centrifuge first on, collects fermentation liquid clear liquid；
(2) by fermentation liquid clear liquid by molecular cut off be 2500 wound membrane filtration to remove big albumen and macromolecule Impurity collects ultrafiltration permeate；
(3) by concentrated by rotary evaporation at 80 DEG C of ultrafiltration permeate, ultrafiltration permeate is concentrated into the 80% of ultrafiltrate, collects concentration Liquid；
(4) concentrate is adsorbed using 732 cation exchange resins, by the Na in fermentation liquid+It is adsorbed, is replaced At H+, 732 cation exchange resins are cleaned 3 times with deionized water first, after with NaOH adjust pH to 10, stir half an hour Afterwards, add H after handing over deionized water to rinse until pH is neutral2SO4It is rinsed with deionized water to neutrality after adjusting pH to 1.It then will concentration Liquid is added in resin cation, is stirred evenly, and after adsorbing resin sufficiently, filtrate is collected by filtration by funnel；
(5) filtrate collected uses H2SO4Adjust pH to 3 or so, the rear ethyl acetate extraction 3 with 3 times of volumes of filtrate It is secondary, collect extract liquor；
(6) then 60 DEG C of revolving extract liquors, rotate to the 10% of extract liquor；
(7) liquid after revolving is put in 4 DEG C of refrigerators and carries out low temperature crystallization, after crystallization, be put in 30 DEG C of baking ovens and carried out Holocrystalline extracts the calculating of rate using liquid phase after crystallization.
Interpretation of result: the liquid of every step purification is for carrying out HPLC (high performance liquid chromatography, the primary Bio-Rad Bole in the U.S. Aminex HPX-87H organic acid column) it detects, mobile phase is 5mM H in HPLC detection2SO4, column temperature is 30 DEG C, UV detector 210nm detects every step loss.Ultimate yield is up to 71.7%, and purity is up to 95.86%.
Embodiment 4: organic solvent extracts adipic acid in fermentation liquid
The adipic acid in the fermentation liquid of embodiment 2 is extracted in accordance with the following methods:
(1) tank fermentation liquid is centrifuged removal thallus and impurity by centrifuge first on, collects fermentation liquid clear liquid；
(2) by fermentation liquid clear liquid by molecular cut off be 500 wound membrane filtration to remove big albumen and macromolecule Impurity collects ultrafiltration permeate；
(3) by concentrated by rotary evaporation at 80 DEG C of ultrafiltration permeate, ultrafiltration permeate is concentrated into the 90% of ultrafiltrate, collects concentration Liquid；
(4) concentrate is adsorbed using 732 cation exchange resins, by the Na in fermentation liquid+It is adsorbed, is replaced At H+, 732 cation exchange resins are cleaned 3 times with deionized water first, after with NaOH adjust pH to 10, stir half an hour Afterwards, add H after handing over deionized water to rinse until pH is neutral2SO4It is rinsed with deionized water to neutrality after adjusting pH to 1.It then will concentration Liquid is added in resin cation, is stirred evenly, and after adsorbing resin sufficiently, filtrate is collected by filtration by funnel；
(5) filtrate collected uses H2SO4Adjust pH to 3 or so, the rear ethyl acetate extraction 3 with 3 times of volumes of filtrate It is secondary, collect extract liquor；
(6) then 60 DEG C of revolving extract liquors, rotate to the 20% of extract liquor；
(7) liquid after revolving is put in 8 DEG C of refrigerators and carries out low temperature crystallization, after crystallization, be put in 40 DEG C of baking ovens and carried out Holocrystalline extracts the calculating of rate using liquid phase after crystallization.
Interpretation of result: the liquid of every step purification is for carrying out HPLC (high performance liquid chromatography, the primary Bio-Rad Bole in the U.S. Aminex HPX-87H organic acid column) it detects, mobile phase is 5mM H in HPLC detection2SO4, column temperature is 30 DEG C, UV detector 210nm detects every step loss.Ultimate yield is up to 62.2%, and purity is up to 96.1%.
Embodiment 5: organic solvent extracts adipic acid in fermentation liquid
The adipic acid in the fermentation liquid of embodiment 2 is extracted in accordance with the following methods:
(1) tank fermentation liquid is centrifuged removal thallus and impurity by centrifuge first on, collects fermentation liquid clear liquid；
(2) by fermentation liquid clear liquid by molecular cut off be 3000 wound membrane filtration to remove big albumen and macromolecule Impurity collects ultrafiltration permeate；
(3) by concentrated by rotary evaporation at 80 DEG C of ultrafiltration permeate, ultrafiltration permeate is concentrated into the 85% of ultrafiltrate, collects concentration Liquid；
(4) concentrate is adsorbed using 732 cation exchange resins, by the Na in fermentation liquid+It is adsorbed, is replaced At H+, 732 cation exchange resins are cleaned 3 times with deionized water first, after with NaOH adjust pH to 10, stir half an hour Afterwards, add H after handing over deionized water to rinse until pH is neutral2SO4It is rinsed with deionized water to neutrality after adjusting pH to 1.It then will concentration Liquid is added in resin cation, is stirred evenly, and after adsorbing resin sufficiently, filtrate is collected by filtration by funnel；
(5) filtrate collected uses H2SO4Adjust pH to 3 or so, the rear ethyl acetate extraction 3 with 3 times of volumes of filtrate It is secondary, collect extract liquor；
(6) then 60 DEG C of revolving extract liquors, rotate to the 15% of extract liquor；
(7) liquid after revolving is put in 4 DEG C of refrigerators and carries out low temperature crystallization, after crystallization, be put in 20 DEG C of baking ovens and carried out Holocrystalline extracts the calculating of rate using liquid phase after crystallization.
Interpretation of result: the liquid of every step purification is for carrying out HPLC (high performance liquid chromatography, the primary Bio-Rad Bole in the U.S. Aminex HPX-87H organic acid column) it detects, mobile phase is 5mM H in HPLC detection2SO4, column temperature is 30 DEG C, UV detector 210nm detects every step loss.Ultimate yield is up to 72.5%, and purity is up to 91.3%.
Embodiment 6: organic solvent extracts adipic acid in fermentation liquid
The ultrafiltration step of step (2) is omitted, other steps and embodiment 3 are consistent, as a result, it has been found that ethyl acetate extraction is added When form emulsion, can not be layered or be layered unobvious, need to be centrifuged further separation, if centrifugation is also not all right, lower step general It can not carry out, extraction cannot be completed.This step, which is omitted, not only doubles workload, time-consuming, wastes ethyl acetate.
Embodiment 7: organic solvent extracts adipic acid in fermentation liquid
The concentration step of step (3) is omitted, other steps and embodiment 3 are consistent, as a result, it has been found that the dosage of all reagents increases Add, work disposal amount increases, and since every step treating capacity is more, loss also be will increase.
Interpretation of result: the liquid of every step purification is for carrying out HPLC (high performance liquid chromatography, the primary Bio-Rad Bole in the U.S. Aminex HPX-87H organic acid column) it detects, mobile phase is 5mM H in HPLC detection2SO4, column temperature is 30 DEG C, UV detector 210nm detects every step loss.Ultimate yield is up to 45.3%, and purity is up to 87.6%.
Embodiment 8: half preparation liquid phase extracts adipic acid in fermentation liquid
(1) tank fermentation liquid is centrifuged removal thallus and impurity by centrifuge first on, only leaves fermentation liquid.
(2) wound membrane filtration is to remove big albumen and macromolecule impurity.
(3) filtered fermentation liquid liquid phase is prepared by waters1525 half to be collected.
Interpretation of result: fermentation liquid carries out HPLC (high performance liquid chromatography, Waters Xbrige C18) detection, HPLC inspection Mobile phase is A:MeOH (80%), B:H in survey2O (20%), gradient elution runing time are 25min, and column temperature is 40 DEG C, ultraviolet Detector 210nm detects every step loss.Ultimate yield is up to 83%, and purity is up to 100%.But the method is time-consuming, needs to make With more methanol, higher cost.And partly preparation liquid phase needs manual operation, general manual, labor intensive.
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention Enclosing subject to the definition of the claims.

Claims (6)

1. a kind of method for extracting adipic acid from fermentation liquid, which is characterized in that described method includes following steps:
(1) it is separated by solid-liquid separation: after fermentation liquid removes thallus and impurity, collecting fermentation liquid clear liquid；
(2) ultrafiltration: utilizing ultrafiltration membrane system, by fermentation liquid clear liquid high molecular weight protein and macromolecule impurity remove, collect super Filter permeate；
(3) it is concentrated: ultrafiltration concentration permeate；
(4) ion exchange: the fermentation liquid after concentration is added to and is washed till neutrality through alkali cleaning, deionization washing, pickling, deionized water Cation exchange resin in, stir evenly, after adsorbing resin sufficiently, filtrate be collected by filtration；
(5) solvent extraction: by the filtrate being collected into using acid for adjusting pH to acidity, then being extracted using organic solvent, and extraction is collected Take liquid；
(6) it evaporates: the organic solvent in evaporation liquid；
(7) it crystallizes: by the remaining liquid of rotary evaporation in 4-8 DEG C of low temperature crystallization, after crystallization, being fully crystallized in 20-40 DEG C, Obtain adipic acid finished product；
For the ultrafiltration membrane elements that ultrafiltration membrane system uses in the step (2) for rolled film, the molecular cut off of ultrafiltration membrane is 500- 3000；Organic solvent in the step (5) is ethyl alcohol, methanol or ethyl acetate；
Adipic acid content is 10-30g/L in fermentation liquid in the step (1)；
Fermentation liquid in the step (1) is to ferment to obtain with recombination bacillus coli Mad2 △ atoB；The recombination bacillus coli Mad2 △ atoB is to have knocked out the e. coli bl21 of atoB gene (DE3) as host, and sub-module overexpression comes from brown Like the heterologous gene beta-Ketothiolase gene of hot tearing spore bacterium, 3-hydroxyacyl-CoA dehydrogenase gene, 3- hydroxyl adipyl dehydrogenation Enzyme gene, penta enoyl CoA reductase gene of 5- carboxyl -2-, adipyl CoA synthetase gene segment Tfu_2576, Tfu_ 2577；Wherein, beta-Ketothiolase gene, 3-hydroxyacyl-CoA dehydrogenase gene are using pRSFDuet-1 as expression vector；3- hydroxyl Base adipyl dehydrogenase gene, penta enoyl CoA reductase gene of 5- carboxyl -2- are using pTrc99a as expression vector；Adipyl is auxiliary Enzyme A synthase gene segment Tfu_2576, Tfu_2577 is using pCDFDuet-1 as expression vector.
2. method according to claim 1, which is characterized in that concentration is to be concentrated into ultrafiltration permeate in the step (3) The 60-90% of ultrafiltrate.
3. method according to claim 1, which is characterized in that cation exchange resin in the step (4) be 732 sun from Sub-exchange resin；The pretreatment mode of cation exchange resin are as follows: cation exchange resin is cleaned 3 with deionized water first It is secondary, after with NaOH adjust pH to 10, after stirring half an hour, rinsed with deionized water until after pH is neutral plus H2SO4PH to 1 is adjusted, Then it is rinsed again with deionized water to neutrality.
4. method according to claim 1, which is characterized in that the activity of cation exchange resin described in the step (4) Group is H+。
5. method according to claim 1, which is characterized in that the volume of the organic solvent in the step (5) is filtrate 2-4 times, extraction times are 3 times.
6. method according to claim 1, which is characterized in that the evaporation in the step (6) be by organic solvent evaporation extremely The 10-20% of extract liquor.
CN201710622975.1A 2017-07-27 2017-07-27 A method of extracting adipic acid from fermentation liquid Active CN107382706B (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102958893A (en) * 2010-06-25 2013-03-06 蒂森克虏伯伍德公司 Process for removing, isolating and purifying dicarboxylic acids
CN106834200A (en) * 2017-03-01 2017-06-13 江南大学 A kind of method for improving adipic acid yield in Escherichia coli

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102958893A (en) * 2010-06-25 2013-03-06 蒂森克虏伯伍德公司 Process for removing, isolating and purifying dicarboxylic acids
CN106834200A (en) * 2017-03-01 2017-06-13 江南大学 A kind of method for improving adipic acid yield in Escherichia coli

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