ITFI980199A1 - PROCESS FOR THE EXTRACTION OF AMINO ACIDS, PRODUCED WITH BIOTECHNOLOGICAL TECHNIQUES, FROM THEIR RESPECTIVE FERMENTATION BROTHS. - Google Patents
PROCESS FOR THE EXTRACTION OF AMINO ACIDS, PRODUCED WITH BIOTECHNOLOGICAL TECHNIQUES, FROM THEIR RESPECTIVE FERMENTATION BROTHS. Download PDFInfo
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- ITFI980199A1 ITFI980199A1 IT98FI000199A ITFI980199A ITFI980199A1 IT FI980199 A1 ITFI980199 A1 IT FI980199A1 IT 98FI000199 A IT98FI000199 A IT 98FI000199A IT FI980199 A ITFI980199 A IT FI980199A IT FI980199 A1 ITFI980199 A1 IT FI980199A1
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- Prior art keywords
- process according
- amino acid
- fermentation broth
- extraction
- extracted
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- 150000001413 amino acids Chemical class 0.000 title claims description 21
- 238000000034 method Methods 0.000 title claims description 21
- 238000000855 fermentation Methods 0.000 title claims description 20
- 230000004151 fermentation Effects 0.000 title claims description 20
- 238000000605 extraction Methods 0.000 title claims description 15
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 27
- 239000000243 solution Substances 0.000 claims description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- 239000002253 acid Substances 0.000 claims description 7
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 claims description 6
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 6
- 238000000926 separation method Methods 0.000 claims description 3
- 239000004475 Arginine Substances 0.000 claims description 2
- 230000001476 alcoholic effect Effects 0.000 claims description 2
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 claims description 2
- 239000003637 basic solution Substances 0.000 claims description 2
- 238000001914 filtration Methods 0.000 claims description 2
- 241000271317 Gonystylus bancanus Species 0.000 claims 2
- 238000002425 crystallisation Methods 0.000 claims 1
- 230000008025 crystallization Effects 0.000 claims 1
- 235000010633 broth Nutrition 0.000 description 16
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 5
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 235000000346 sugar Nutrition 0.000 description 3
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 230000008030 elimination Effects 0.000 description 2
- 238000003379 elimination reaction Methods 0.000 description 2
- 239000002054 inoculum Substances 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 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 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 1
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 1
- 241000282849 Ruminantia Species 0.000 description 1
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- 238000010306 acid treatment Methods 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 238000001953 recrystallisation Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Description
Domanda di brevetto per invenzione industriale dal titolo: Processo per l’estrazione di aminoacidi dai rispettivi brodi di fermentazione Patent application for industrial invention entitled: Process for the extraction of amino acids from the respective fermentation broths
Campo dell’invenzione Field of the invention
La presente invenzione si riferisce ad un processo per l’estrazione di aminoacidi dai rispettivi brodi di fermentazione in cui l aminoacido desiderato viene estratto in forma notevolmente pura e con rese elevate, per trattamento del brode di fermentazione con alcool nbutilico. The present invention refers to a process for the extraction of amino acids from the respective fermentation broths in which the desired amino acid is extracted in a remarkably pure form and with high yields, by treating the fermentation broth with nbutyl alcohol.
Stato dell'arte State of the art
E' noto che gran parte degli aminoacidi oggi largamente utilizzati ai fini industriali viene prodotta per via sintetica ed essenzialmente utilizzando tecniche biotecnologiche. It is known that most of the amino acids widely used today for industrial purposes are produced synthetically and essentially using biotechnological techniques.
in pratica si utilizza un substrato di fermentazione costituito da una soluzione di sali e di zuccheri che vengono miscelati e quindi trasferiti in un fermentatore. in practice, a fermentation substrate is used consisting of a solution of salts and sugars which are mixed and then transferred to a fermenter.
In laboratorio si prepara l’inoculo utilizzando un opportuno ceppo di microrganismi geneticamente modificati in modo da produrre raminoacido desiderato a partire da glucosio o saccarosio e una volta raggiunta la densità ottimale l’inoculo viene alimentato ai fermentatore. Il fermentatore viene quindi alimentato con aria sterile che fornisce l’ossigeno necessario alla crescita dei batteri a spese dei vari nutrienti e dello zucchero presenti. In the laboratory, the inoculum is prepared using an appropriate strain of genetically modified microorganisms in order to produce the desired ruminant acid starting from glucose or sucrose and once the optimal density is reached, the inoculum is fed to the fermenter. The fermenter is then fed with sterile air that provides the oxygen necessary for the growth of bacteria at the expense of the various nutrients and sugar present.
Al fermentatore vengono quindi alimentate varie soluzioni di nutrienti e una soluzione di zucchero che viene metabolizzato dai datteri geneticamente modificati con conseguente produzione di aminoacidi. A questo punto è necessario separare e purificare l'aminoacido desiderato separandolo dagli altri componenti del brodo di fermentazione. Fino ad oggi, la separazione e purificazione veniva effettuata con tecniche di ultrafiltrazione su membrane avvolte o di cromatografia industriale che comportano notevoli investimenti per gli impianti necessari e sono molto lunghe e costose anche perché le rese sono spesso insoddisfacenti. The fermenter is then fed various nutrient solutions and a sugar solution which is metabolized by the genetically modified dates resulting in the production of amino acids. At this point it is necessary to separate and purify the desired amino acid by separating it from the other components of the fermentation broth. Until now, separation and purification was carried out with ultrafiltration techniques on wrapped membranes or industrial chromatography which involve considerable investments for the necessary plants and are very long and expensive also because the yields are often unsatisfactory.
E’ quindi evidente l'importanza ai poter disporre di un processo più semplice che consenta di separare gli aminoacidi dal brodo di fermentazione in forma pura e con rese accettabili. It is therefore evident the importance of having a simpler process that allows to separate the amino acids from the fermentation broth in pure form and with acceptable yields.
Descrizione dettagliata del'invenzione Detailed description of the invention
La presente invenzione consente di superare i problemi suddetti per mezzo di un processo in cui il broao di fermentazione viene raffreddato a 2°-3°C ed estratte con opportune quantità di alcool n-butilico, si ottiene così l’estrazione selettiva del’aminoacido voluto che viene poi recuperato in forma notevolmente pura e con ottime rese. The present invention allows to overcome the aforesaid problems by means of a process in which the fermentation broao is cooled to 2 ° -3 ° C and extracted with suitable quantities of n-butyl alcohol, thus the selective extraction of the amino acid is obtained. wanted that it is then recovered in a remarkably pure form and with excellent yields.
Il processo secondo la presente invenzione si è rivelato particolarmente interessante per la separazione dai rispettivi brodi di fermentazione per la produzione di : triptofano, treonina, arginina. The process according to the present invention has proved to be particularly interesting for the separation from the respective fermentation broths for the production of: tryptophan, threonine, arginine.
Secondo il processo oggetto deila presente invenzione, :i brodo di fermentazione prelevato dai fermentatore viene trattato, se la concentrazione di aminoacido presente nel brodo è inferiore a 6-7 gr/lt. con soluzioni basiche (ad esempio NaOH 10%) fino a pH nettamente basico (normalmente pH 12 -13) in modo da stabilizzare la soluzione e quindi concentrato sotto vuoto a temperature non superiori a 52°C fino a circa un quarto del volume iniziale. According to the process object of the present invention, the fermentation broth taken from the fermenter is treated if the amino acid concentration present in the broth is lower than 6-7 g / l. with basic solutions (for example 10% NaOH) up to a clearly basic pH (normally pH 12 -13) in order to stabilize the solution and then concentrated under vacuum at temperatures no higher than 52 ° C up to about a quarter of the initial volume.
Il brodo è quindi trattalo con una soluzione acida (ad esempio una soluzione acquosa di HCI 1 :1 ) fino al raggiungimento del pH del punto isoelettrico dell'aminoacido da estrarre. The broth is then treated with an acid solution (for example an aqueous solution of HCI 1: 1) until the pH of the isoelectric point of the amino acid to be extracted is reached.
Qualora la concentrazione dell’aminoacido nel brodo iniziale sia superiore al valore sopra indicato - si procede direttamente a! trattamento acido sopra descritto continuando poi come segue, il brodo cosi ottenuto viene estratto con alcool n-butilico Preferibilmente l'estrazione è eseguita in più riprese aggiungendo dopo ogni estrazione la quantità di acqua assorbita dall’alcool in modo da mantenere costante il volume del brodo da trattare nelle successive estrazioni. If the amino acid concentration in the initial broth is higher than the value indicated above - proceed directly to! acid treatment described above, then continuing as follows, the broth thus obtained is extracted with n-butyl alcohol Preferably the extraction is carried out in several stages by adding after each extraction the amount of water absorbed by the alcohol in order to keep the volume of the broth constant to be treated in subsequent extractions.
li rapporto fra la quantità di alcool totale utilizzata per le varie estrazioni ed ii liquido di partenza è compresa fra 0.6 i e 1 : 1 (v/v). Le varie fasi alcoliche vengono quindi riunite e concentrate sotto vuoto a temperature comprese fra 52°-65°C, in modo da eliminare l’acqua che distilla come azeotropo. The ratio between the amount of total alcohol used for the various extractions and the starting liquid is between 0.6 i and 1: 1 (v / v). The various alcoholic phases are then combined and concentrated under vacuum at temperatures between 52 ° -65 ° C, in order to eliminate the water that distills as azeotrope.
A seguito dell’eliminazione dell'acqua laminoacido desiderato precipita nell’alcool in cui è insolubile in forma notevolmente pura. Following the elimination of the water, the desired acid precipitates into the alcohol in which it is insoluble in a remarkably pure form.
Le rese sono normalmente dell'ordine del 75 - 80% (calcolato sulla quantità di prodotto inizialmente presente nel brodo -di fermentazione, misurata con HPLC) e ia purezza del’aminoacido cosi raccolto è ael 98.5% e oltre. The yields are normally of the order of 75 - 80% (calculated on the amount of product initially present in the fermentation broth, measured with HPLC) and the purity of the amino acid thus collected is 98.5% and above.
Se necessario si può proceaere aa una ulteriore purificazione aei prodotto raccolto per ricristallizzazione utilizzando gli adatti solventi. La presente invenzione verrà ora meglio illustrata alla luce dei seguente esempio. If necessary, further purification of the product collected by recrystallization can be carried out using suitable solvents. The present invention will now be better illustrated in the light of the following example.
Esempio Example
Prodotto di partenza: brodo di fermentazione per la produzione di triptofano ottenuto per trattamento per circa 40 h. di saccarosio con ceppi di Escherichia coli modificato (ceppo K12W3110). Starting product: fermentation broth for the production of tryptophan obtained by treatment for about 40 h. of sucrose with modified Escherichia coli strains (strain K12W3110).
Liquido marrone, maleodorante. Brown, foul-smelling liquid.
Un litro dei brodo di fermentazione sopra indicato è stato portato a pH 12.4 aggiungendo NaGH 10% sotto agitazione One liter of the fermentation broth indicated above was brought to pH 12.4 by adding 10% NaGH while stirring
La soluzione è stata quindi concentrata sotto vuoto (25-30 mm Hg) con una pompa ad anello liquido e a temperatura inferiore ai 52°C fino ad un quarto circa del suo volume (circa 250 mi). The solution was then concentrated under vacuum (25-30 mm Hg) with a liquid ring pump and at a temperature below 52 ° C to about a quarter of its volume (about 250 ml).
Il pH è stato quindi portato a pH 5.9 (punto isoelettrico del triptofano) con una soluzione acquosa (1 :1 ) di HCI. The pH was then brought to pH 5.9 (isoelectric point of tryptophan) with an aqueous solution (1: 1) of HCI.
Il brodo cosi trattato è stato estratto con 250 mi di alcool n-butilico (due volte, 125 mi di alcool per ogni estrazione) aggiungendo l acqua assorbita nella prima estrazione in modo da tenere costante il volume di soluzione da estrarre. The broth thus treated was extracted with 250 ml of n-butyl alcohol (twice, 125 ml of alcohol for each extraction) by adding the water absorbed in the first extraction in order to keep the volume of solution to be extracted constant.
Le due frazioni di alcool n-butilico sono state riunite e concentrate sotto vuoto (circa 20 mmHg). The two n-butyl alcohol fractions were combined and concentrated under vacuum (about 20 mmHg).
La miscela azeotropica alcool n-butilico/acqua distilla tra 52°-62°C e via via che l’acqua è eiiminata precipita il triptofano. The azeotropic n-butyl alcohol / water mixture distills between 52 ° -62 ° C and as the water is eliminated, the tryptophan precipitates.
Dopo eliminazione totale dell'acqua, si distilla ancora fino a lasciare circa 100 cc di liquido e si raccoglie per filtrazione il triptofano precipitato (6,5 gr). After total elimination of the water, it is still distilled to leave about 100 cc of liquid and the precipitated tryptophan (6.5 g) is collected by filtration.
Resa: circa 80%, titolo: 98,5% (vedi NMR allegato, Figura 1 ). Yield: about 80%, titer: 98.5% (see attached NMR, Figure 1).
Claims (10)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| IT1998FI000199A IT1304709B1 (en) | 1998-09-04 | 1998-09-04 | PROCESS FOR THE EXTRACTION OF AMINO ACIDS, PRODUCED WITH BIOTECHNOLOGICAL TECHNIQUES, FROM THE RESPECTIVE FERMENTATION BROTHS. |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| IT1998FI000199A IT1304709B1 (en) | 1998-09-04 | 1998-09-04 | PROCESS FOR THE EXTRACTION OF AMINO ACIDS, PRODUCED WITH BIOTECHNOLOGICAL TECHNIQUES, FROM THE RESPECTIVE FERMENTATION BROTHS. |
Publications (3)
| Publication Number | Publication Date |
|---|---|
| ITFI980199A0 ITFI980199A0 (en) | 1998-09-04 |
| ITFI980199A1 true ITFI980199A1 (en) | 2000-03-04 |
| IT1304709B1 IT1304709B1 (en) | 2001-03-29 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| IT1998FI000199A IT1304709B1 (en) | 1998-09-04 | 1998-09-04 | PROCESS FOR THE EXTRACTION OF AMINO ACIDS, PRODUCED WITH BIOTECHNOLOGICAL TECHNIQUES, FROM THE RESPECTIVE FERMENTATION BROTHS. |
Country Status (1)
| Country | Link |
|---|---|
| IT (1) | IT1304709B1 (en) |
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1998
- 1998-09-04 IT IT1998FI000199A patent/IT1304709B1/en active
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| Publication number | Publication date |
|---|---|
| ITFI980199A0 (en) | 1998-09-04 |
| IT1304709B1 (en) | 2001-03-29 |
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