CN100509760C - Method for separating and purifying glutamine from fermentation liquor by four-area simulation moving bed - Google Patents
Method for separating and purifying glutamine from fermentation liquor by four-area simulation moving bed Download PDFInfo
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Abstract
The invention discloses a method for separating and purifying glutamine from fermentation liquor by using four- section imitate moving bed. It comprises following steps: feeding glutamine fermentation liquor into PES ultra- filtering device equipped with ultral- filtering membrane, ultral- filtering to remove yeast cell, solid matter and protein under condition of nitrogen gas less than 0.25 Mpa; decoloring filtered fermentation liquior with active charcoal; condensing and compressing decolored fermentation liquor, crystallizing and desalting; preparing feeding sample liquor with concentration being 10- 80 g/l with desalted coarse crystal and feeding it into imitate moving bed, decoloring with deioned water of pH 10- 14, collecting glutamine and aminoglutaric acid from extract discharging port and raffinate discharging port; condensing extract liquor with rotary steaming equipment until waterless, cleaning with methanol and vacuum drying in drying oven and getting final product. The invention is characterized by continuous industrial production, good product purity, high productivity, large yield and low production cost.
Description
Technical field
The invention belongs to the biological products manufacture field, relate to the method for the glutamine in a kind of four-area simulated moving bed separation and purification fermented liquid.
Background technology
L-glutaminate is a seed amino acid of the γ-Carboxylamideization of L-L-glutamic acid, chemical name 2,5-diamino-5-oxopentanoic acid, constitute one of proteinic primary amino acid as 20 kinds, glutamine is rich in amino acid in the blood of human body, also is rich in amino acid in the Skeletal Muscle Cell, accounts for the over half of whole body total free aminoacids, playing a part very importantly in the organism metabolism, is a kind of " conditionality indispensable amino acid ".After nineteen fifty-two has been determined its molecular structure with the X-ray diffraction, its application is just caused people's attention day by day, one of most important amino acid known to being considered at present also is a kind of extremely rising new drug.Glutamine production is mainly used fermentation method at present, and the fermentative Production glutamine is able to suitability for industrialized production and also depends on the glutamine separation and extraction technology.It is the column chromatography of filler that the separation and Extraction of at present domestic and international glutamine nearly all adopts with ion exchange resin, though can carry out mass preparation, isolating efficient is low, and the moving phase waste is serious, and the stationary phase utilising efficiency is low, yields poorly the cost height.
Since the seventies, American UOP company has developed the chromatographic technique of simulation moving-bed (SMB) principle, this technology is not moving after making the absorbent particles filling, by the method that raw material is imported and exported and product liquid flow inlet and outlet constantly switches, the formation absorbent particles flows relative to countercurrent movement with liquid simulates moving of stationary phase.The nineties in 20th century, the simulated moving bed chromatography range of application also constantly enlarged, spread all over a lot of production fields such as oil, fine chemistry industry, biological fermentation, medicine, food, especially in the separating of mixtures such as homologous compound, chiral isomer medicine, carbohydrate, organic acid and amino acid, demonstrate its special performance.Some patent documentations of the U.S. have reported that simulated moving bed chromatography separating chiral medicine such as optical isomer, racemize material separate with enantiomorph and petroleum chemicals.Japan Daicel chemical industrial company has also developed the method for using simulated moving bed chromatography separating chiral isomer.But, do not see open report yet for the method for using simulated moving bed chromatography to separate the purification glutamine.
Summary of the invention
The objective of the invention is to: provide the method for the glutamine in a kind of four-area simulated moving bed separation and purification fermented liquid, industrialization continuous production, purity, yield and the output of raising product.
Technical solution of the present invention may further comprise the steps: a, glutamine yeast fermentation broth are packed in the ultrafiltration apparatus of PES ultra-filtration membrane, carry out ultrafiltration under the nitrogen of 0.25MPa and remove yeast cell thalline, solid substance and protein being no more than, reduce the viscosity of feed liquid; B, the fermented liquid of ultra filtration is passed through activated carbon decolorizing; C, the fermented liquid after will decolouring are by concentrating under reduced pressure, crystal desalination; D, the coarse-grain after the desalination is made into concentration is that the sample introduction liquid of 10-80g/l is packed into and adsorbed among the simulated moving bed system SMB that Zeo-karb is housed, deionized water by pH10-14 carries out wash-out, control the travelling speed of each functional zone sample, glutamine and L-glutamic acid are collected from extracting solution and raffinate outlet respectively; E, simulation moving-bed extraction liquid are concentrated into anhydrous through Rotary Evaporators, by after the methanol-eluted fractions vacuum drying oven dry finished product.
Simulated moving bed system in the inventive method is by many identical cation exchange resin column series connection, switching in order between i.e. absorption by the strong and weak absorbed component in the ion exchange process, refining, desorb, the resin regeneration difference in functionality district, separate according to the different principle of strong and weak absorbed component travelling speed on separating medium, the position that the switching by combined valve changes opening for feed, raffinate outlet, moving phase inlet, extracting liquid outlet obtains target product; Wherein, the standard of switching time is the travelling speed that the radical of flow velocity by adjusting each functional zone elutriant and ion exchange column comes the controlled target thing; The quantity of each functional zone ion exchange column, is switched by functional zone during switching in this switching time of eluted migration length decision according to the target compound of required wash-out.
Simulated moving bed chromatography in the inventive method (SMBC) separation system is connected to form by wash-out pump, sampling pump, extraction pump, chromatographic column, magnetic valve, signal picker, central controller and computer, wash-out pumping capacity 200-800ml/min, pressure 1-10Mpa; Sampling pump flow 10-50ml/min, pressure 1-10Mpa; Extraction pumping capacity 100-500ml/min, pressure 1-10Mpa; Carry surplus pumping capacity 100-500ml/min, pressure 1-10Mpa; Valve switching time is 18-24min; Whole simulation moving-bed temperature is 25-35 ℃, 25-35 ℃ of feeding liquid, elutriant temperature, and the two temperature is consistent.
Simulated moving bed system in the inventive method is connected by 4-16 radical ion exchange column, simulated moving bed system is according to the Fen Si district, position of four mouths, every district is by the identical ion exchange column of 1-4 root, one district is positioned between elutriant ingress and the extracting liquid outlet place, in this district, realize the desorb of strong absorbed component in the glutamine ferment liquid crude extract; Two districts are positioned between extracting liquid outlet place and the opening for feed, and its effect is equivalent to rectifying tower, make in the glutamine crude extract strong absorbed component absorption repeatedly, desorb and concentrate, and obtain L-glutamic acid from extracting liquid outlet; Three districts are positioned between opening for feed and the raffinate outlet, and its effect is as much as possible glutamine and L-glutamic acid to be adsorbed on the stationary phase, obtain containing glutamine from the raffinate outlet; Four districts are positioned between elutriant inlet and the raffinate outlet, and on the one hand, the glutamine in the liquid phase is fixed with L-glutamic acid and adsorbs mutually, and its elutriant enters a district with fresh elutriant, thereby reaches the purpose of recycle; Jiang San district and one separates out on the other hand, pollutes extracting solution in order to avoid glutamine in the raffinate and L-glutamic acid enter a district, plays certain shock absorption.
Method of the present invention adopts the glutamine in the simulated moving bed system separation and purification fermented liquid, the serialization suitability for industrialized production, and good product purity, efficient height, output is big, production cost is low.
Embodiment
Example 1: select glutamine ferment liquid for use, on Hanbon Sci. ﹠ Tech. Co., Ltd. simulation moving-bed, realize the purifying of glutamine:
A, get the glutamine yeast fermentation broth and pack in the ultrafiltration apparatus of PES ultra-filtration membrane, carry out ultrafiltration under the nitrogen of 0.25MPa and remove yeast cell thalline, solid substance and protein being no more than, reduce the viscosity of feed liquid; B, the fermented liquid of ultra filtration is passed through activated carbon decolorizing; C, the fermented liquid after will decolouring are by concentrating under reduced pressure, crystal desalination; D, the coarse-grain after the desalination is made into concentration is that the sample introduction liquid of 10g/l enters simulated moving bed system and carries out wash-out by the deionized water of pH10; E, simulation moving-bed extraction liquid is concentrated into through Rotary Evaporators anhydrous, by after the methanol-eluted fractions vacuum drying oven dry finished product.
Simulated moving bed chromatography in the inventive method (SMBC) separation system is connected to form by wash-out pump, sampling pump, extraction pump, chromatographic column, magnetic valve, signal picker, central controller and computer, eluent flow rate is 200ml/min, the extraction liquid flow velocity is 100ml/min, and the raffinate flow velocity is 100ml/min; Valve switching time is 18min; The feeding liquid flow velocity is 10ml/min; Each pump work pressure 1Mpa; Whole simulation moving-bed temperature is 25 ℃, 25 ℃ of feeding liquid, elutriant temperature.
Simulated moving bed system SMB in the inventive method is connected by 4 identical ion exchange columns, divide 4 districts according to the position of four mouths: a district is positioned between elutriant ingress and the extracting liquid outlet place, in this district, realize the desorb of strong absorbed component in the glutamine ferment liquid crude extract; Two districts are positioned between extracting liquid outlet place and the opening for feed, and its effect is equivalent to rectifying tower, make in the glutamine crude extract strong absorbed component absorption repeatedly, desorb and concentrate, and obtain L-glutamic acid from extracting liquid outlet; Three districts are positioned between opening for feed and the raffinate outlet, and its effect is as much as possible glutamine and L-glutamic acid to be adsorbed on the stationary phase, obtain containing glutamine from the raffinate outlet; Four districts are positioned between elutriant inlet and the raffinate outlet, and on the one hand, the glutamine in the liquid phase is fixed with L-glutamic acid and adsorbs mutually, and its elutriant enters a district with fresh elutriant, thereby reaches the purpose of recycle; Jiang San district and one separates out on the other hand, pollutes extracting solution in order to avoid glutamine in the raffinate and L-glutamic acid enter a district, plays certain shock absorption.
Finished product column front derivation RPLC (HPLC) method detects: PerkinElmer Series 200 highly effective liquid phase chromatographic systems; UV-detector detects wavelength 254nm; Hedera ODS-24.6mm * 250mm analytical column, packing material size 5 μ m; 1%2, the acetonitrile solution of 4-dinitrofluorobenzene is as derivating agent, and sample thief 9.86mg puts in the brown measuring bottle of 10ml, adds 0.5mol/L pH=9.0NaHCO
3Solution 1ml adds 1% 2,4-dinitrofluorobenzene acetonitrile solution 1ml, puts in 60 degrees centigrade of water-baths the dark place heating and takes out after 1 hour, puts coldly, adds the pH=7.0 phosphate buffered saline buffer to scale; Elutriant is by A, B two-pack composition gradient wash-out, and A is CH
3CN:H
2O=1:1, B are 0.03mol/l NaAC-HAC, flow velocity 1ml/min, sample introduction concentration 1mg/ml, sample size 20 μ l, 25 ℃ of detected temperatures; Use external standard method finished product glutamine content 98% according to parameter in the following form.
| T(min) | A | B |
| 0 | 16 | 84 |
| 1 | 30 | 70 |
| 4 | 34 | 66 |
| 16 | 34 | 66 |
Example 2: select glutamine ferment liquid for use, on Hanbon Sci. ﹠ Tech. Co., Ltd. simulation moving-bed, realize the purifying of glutamine:
A, get the glutamine yeast fermentation broth and pack in the ultrafiltration apparatus of PES ultra-filtration membrane, carry out ultrafiltration under the nitrogen of 0.25MPa and remove yeast cell thalline, solid substance and protein being no more than, reduce the viscosity of feed liquid; B, the fermented liquid of ultra filtration is passed through activated carbon decolorizing; C, the fermented liquid after will decolouring are by concentrating under reduced pressure, crystal desalination; D, the coarse-grain after the desalination is made into concentration is that the sample introduction liquid of 45g/l enters simulated moving bed system and carries out wash-out by the deionized water of pH12; E, simulation moving-bed extraction liquid is concentrated into invariably through Rotary Evaporators, by after the methanol-eluted fractions vacuum drying oven dry finished product.
Simulated moving bed chromatography in the inventive method (SMBC) separation system is connected to form by wash-out pump, sampling pump, extraction pump, chromatographic column, magnetic valve, signal picker, central controller and computer, and eluent flow rate is 500ml/min, pressure 5Mpa; The sample introduction flow velocity is 30ml/min, and pressure 5Mpa, extraction liquid flow velocity are 300ml/min, pressure 5Mpa; The raffinate flow velocity is 300ml/min; Valve switching time is 30 ℃ of 21min working temperatures, 30 ℃ of feeding liquid, elutriant temperature.
Simulated moving bed system SMB in the inventive method is by 12 identical ion exchange column series connection, divide 4 districts according to the position of four mouths, every district is by 3 exchange columns, one district is positioned between elutriant ingress and the extracting liquid outlet place, in this district, realize the desorb of strong absorbed component in the glutamine ferment liquid crude extract; Two districts are positioned between extracting liquid outlet place and the opening for feed, and its effect is equivalent to rectifying tower, make in the glutamine crude extract strong absorbed component absorption repeatedly, desorb and concentrate, and obtain L-glutamic acid from extracting liquid outlet; Three districts are positioned between opening for feed and the raffinate outlet, and its effect is as much as possible glutamine and L-glutamic acid to be adsorbed on the stationary phase, obtain containing glutamine from the raffinate outlet; Four districts are positioned between elutriant inlet and the raffinate outlet, and on the one hand, the glutamine in the liquid phase is fixed with L-glutamic acid and adsorbs mutually, and its elutriant enters a district with fresh elutriant, thereby reaches the purpose of recycle; Jiang San district and one separates out on the other hand, pollutes extracting solution in order to avoid glutamine in the raffinate and L-glutamic acid enter a district, plays certain shock absorption.
Finished product column front derivation RPLC (HPLC) method detects: PerkinElmer Series 200 highly effective liquid phase chromatographic systems; UV-detector detects wavelength 254nm; Hedera ODS-24.6mm * 250mm analytical column, packing material size 5 μ m; The acetonitrile solution of 1% 2,4-dinitrofluorobenzene is as derivating agent, and sample thief 9.86mg puts in the brown measuring bottle of 10ml, adds 0.5mol/L pH=9.0NaHCO
3Solution 1ml adds 1% 2,4-dinitrofluorobenzene acetonitrile solution 1ml, puts in 60 degrees centigrade of water-baths the dark place heating and takes out after 1 hour, puts coldly, adds the pH=7.0 phosphate buffered saline buffer to scale; Elutriant is by A, B two-pack composition gradient wash-out, and A is CH
3CN:H
2O=1:1, B are 0.03mol/l NaAC-HAC, flow velocity 1ml/min, sample introduction concentration 1mg/ml, sample size 20 μ l, 25 ℃ of detected temperatures; Use external standard method finished product glutamine content 98.68% according to parameter in the following form.
| T(min) | A | B |
| 0 | 16 | 84 |
| 1 | 30 | 70 |
| 4 | 34 | 66 |
| 16 | 34 | 66 |
Example 3: select glutamine ferment liquid for use, on Hanbon Sci. ﹠ Tech. Co., Ltd. simulation moving-bed, realize the purifying of glutamine:
A, get the glutamine yeast fermentation broth and pack in the ultrafiltration apparatus of PES ultra-filtration membrane, carry out ultrafiltration under the nitrogen of 0.25MPa and remove yeast cell thalline, solid substance and protein being no more than, reduce the viscosity of feed liquid; B, the fermented liquid of ultra filtration is passed through activated carbon decolorizing; C, the fermented liquid after will decolouring are by concentrating under reduced pressure, crystal desalination; D, the coarse-grain after the desalination is made into concentration is that the sample introduction liquid of 80g/l enters simulated moving bed system and carries out wash-out by the deionized water of pH14; E, simulation moving-bed extraction liquid is concentrated into invariably through Rotary Evaporators, by after the methanol-eluted fractions vacuum drying oven dry finished product.
Simulated moving bed chromatography in the inventive method (SMBC) separation system is connected to form by wash-out pump, sampling pump, extraction pump, chromatographic column, magnetic valve, signal picker, central controller and computer, and eluent flow rate is 800ml/min, pressure 10Mpa; The sample introduction flow velocity is 50ml/min, and pressure 10Mpa, extraction liquid flow velocity are 500ml/min, pressure 10Mpa; Raffinate flow velocity 500ml/min; Valve switching time is 24min; 35 ℃ of working temperatures, 35 ℃ of feeding liquid, elutriant temperature.
Simulated moving bed system SMB in the inventive method is by 16 identical ion exchange column series connection, divide 4 districts according to the position of four mouths, 4 exchange columns in every district, one district is positioned between elutriant ingress and the extracting liquid outlet place, in this district, realize the desorb of strong absorbed component in the glutamine ferment liquid crude extract; Two districts are positioned between extracting liquid outlet place and the opening for feed, and its effect is equivalent to rectifying tower, make in the glutamine crude extract strong absorbed component absorption repeatedly, desorb and concentrate, and obtain L-glutamic acid from extracting liquid outlet; Three districts are positioned between opening for feed and the raffinate outlet, and its effect is as much as possible glutamine and L-glutamic acid to be adsorbed on the stationary phase, obtain containing glutamine from the raffinate outlet; Four districts are positioned between elutriant inlet and the raffinate outlet, and on the one hand, the glutamine in the liquid phase is fixed with L-glutamic acid and adsorbs mutually, and its elutriant enters a district with fresh elutriant, thereby reaches the purpose of recycle; Jiang San district and one separates out on the other hand, pollutes extracting solution in order to avoid glutamine in the raffinate and L-glutamic acid enter a district, plays certain shock absorption.
Finished product column front derivation RPLC (HPLC) method detects: PerkinElmer Series 200 highly effective liquid phase chromatographic systems; UV-detector detects wavelength 254nm; Hedera ODS-24.6mm * 250mm analytical column, packing material size 5 μ m; The acetonitrile solution of 1% 2,4-dinitrofluorobenzene is as derivating agent, and sample thief 9.86mg puts in the brown measuring bottle of 10ml, adds 0.5mol/L pH=9.0NaHCO
3Solution 1ml adds 1%2, and 4-dinitrofluorobenzene acetonitrile solution 1ml puts in 60 degrees centigrade of water-baths the dark place heating and takes out after 1 hour, puts coldly, adds the pH=7.0 phosphate buffered saline buffer to scale; Moving phase is by A, B two-pack composition gradient wash-out, and A is CH
3CN:H
2O=1:1, B are 0.03mol/l NaAC-HAC, flow velocity 1ml/min, sample introduction concentration 1mg/ml, sample size 20 μ l, 25 ℃ of detected temperatures; Use external standard method finished product glutamine content 99.10% according to following form parameter mode.
| T(min) | A | B |
| 0 | 16 | 84 |
| 1 | 30 | 70 |
| 4 | 34 | 66 |
| 16 | 34 | 66 |
Claims (1)
1. the method for glutamine in the simulated four-area moving bed chromatographic separation and purification fermented liquid, it is characterized in that: a, glutamine yeast fermentation broth are packed in the ultrafiltration apparatus of PES ultra-filtration membrane, carry out ultrafiltration under the nitrogen of 0.25MPa and remove yeast cell thalline, solid substance and protein being no more than, reduce the viscosity of feed liquid; B, the fermented liquid of ultra filtration is passed through activated carbon decolorizing; C, the fermented liquid after will decolouring are by concentrating under reduced pressure, crystal desalination; D, the coarse-grain after the desalination is made into concentration is that the deionized water that the sample introduction liquid of 10-80g/l is packed among the simulated moving bed system SMB that Zeo-karb is housed by pH10-14 carries out wash-out, control the travelling speed of each functional zone sample, glutamine and L-glutamic acid are collected from extracting solution and raffinate outlet respectively; E, simulation moving-bed extraction liquid are concentrated into anhydrous through Rotary Evaporators, by after the methanol-eluted fractions vacuum drying oven dry finished product; Separation system of simulated moving bed chromatography is connected to form by wash-out pump, sampling pump, extraction pump, chromatographic column, magnetic valve, signal picker, central controller and computer, wash-out pumping capacity 200-800ml/min, pressure 1-10Mpa; Sampling pump flow 10-50ml/min, pressure 1-10Mpa; Extraction pumping capacity 100-500ml/min, pressure 1-10Mpa; Carry surplus pumping capacity 100-500ml/min, pressure 1-10MPa; Valve switching time is 18-24min; The temperature 25-35 that whole simulation is moved ℃, 25-35 ℃ of feeding liquid, elutriant temperature; Simulated moving bed system is by the series connection of the identical ion exchange column of 4-16 root, and simulated moving bed system is according to the Fen Si district, position of four mouths, every district 1-4 root exchange column, and a district is positioned between elutriant ingress and the extracting liquid outlet place; Two districts are positioned between extracting liquid outlet place and the opening for feed; Three districts are positioned between opening for feed and the raffinate outlet; Four districts are positioned between elutriant inlet and the raffinate outlet.
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| KR102027596B1 (en) * | 2010-12-06 | 2019-10-01 | 타폰 바이오시스템즈, 인코포레이티드 | Continuous processing methods for biological products |
| CN102952034A (en) * | 2011-08-31 | 2013-03-06 | 江苏汉邦科技有限公司 | Method for separating chiral compound metalaxyl by adopting simulated moving bed in fourth region |
| CN102924321B (en) * | 2012-11-30 | 2015-12-09 | 通辽梅花生物科技有限公司 | A kind of method extracting glutamine from fermented liquid |
| CN103159643B (en) * | 2013-03-06 | 2014-09-03 | 山东阜丰发酵有限公司 | Technology for whole membrane extraction of L-glutamine fermentation broth |
| CN109646999A (en) * | 2018-11-23 | 2019-04-19 | 吉林中粮生化有限公司 | It is a kind of for separating the Simulation moving bed and method of glucide |
| CN109628518B (en) * | 2018-12-23 | 2021-11-02 | 新疆阜丰生物科技有限公司 | Method for producing and extracting L-glutamine |
| CN110003039A (en) * | 2019-03-28 | 2019-07-12 | 新泰市佳禾生物科技有限公司 | The isolation and purification method of altheine in a kind of conversion fluid |
| 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 |
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