CA1215068A - Method of separating l-tryptophan - Google Patents
Method of separating l-tryptophanInfo
- Publication number
- CA1215068A CA1215068A CA000444164A CA444164A CA1215068A CA 1215068 A CA1215068 A CA 1215068A CA 000444164 A CA000444164 A CA 000444164A CA 444164 A CA444164 A CA 444164A CA 1215068 A CA1215068 A CA 1215068A
- Authority
- CA
- Canada
- Prior art keywords
- tryptophan
- reaction mixture
- microorganism
- acid
- alkali
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/22—Tryptophan; Tyrosine; Phenylalanine; 3,4-Dihydroxyphenylalanine
- C12P13/227—Tryptophan
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
- C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
- C07D209/04—Indoles; Hydrogenated indoles
- C07D209/10—Indoles; Hydrogenated indoles with substituted hydrocarbon radicals attached to carbon atoms of the hetero ring
- C07D209/18—Radicals substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
- C07D209/20—Radicals substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals substituted additionally by nitrogen atoms, e.g. tryptophane
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/02—Separating microorganisms from their culture media
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Biomedical Technology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Abstract of the Disclosure When an L-tryptophan-containing reaction mixture produced by a reaction utilizing a micro-organism is adjusted to pH 2 to 5 with a mineral acid and then heated, the microorganism contained in the reaction mixture is flocculated. The flocculated microorganism is removed by either (1) filtering the reaction mixture, or (2) adding an alkali to the reaction mixture to convert L-tryptophan to its alkali salt and then filtering the mixture. L-tryptophan of high purity can be obtained from the filtrate.
Description
16~
This invention relates to a method of isolate in L-tryptophan from a reaction mixture containing 1-tryptophan and a microorganism obtained during the production of L-tryptophan by utilizing the micro-organism.
In reactions utilizing microorganisms, it is necessary to separate the microorganisms from the reaction products. Some methods have been known in the past for removing the microorganisms from the reaction mixtures and isolating the reaction products.
For example, Japanese Patent Publication No. 16460/1963 discloses a method which comprises adding a surface-active agent to an L-glutamic acid fermentation broth, heating the mixture to flocculate and sediment the lo microorganism, and separating it by filtration upon addition of diatomaceous earth, and Japanese Laid-Open Patent Publication No. 29996/1978 discloses a method which comprises filtering a reaction mixture such as an amino acid fermentation broth on an ultra-filtration membrane and then isolating the product by crystallization.
These methods, however, have not proved to be entirely satisfactory for industrial practice because by the method involving adding the surfactant, the microorganism can be easily removed but the surfactant cannot be easily separated and is likely to remain in the reaction product, and the method involving the use I
of the ultrafiltration membrane has difficulty in washing the used apparatus owing to its nature.
It is an object of this invention therefore to provide a method of isolating L-tryptophan from a reaction mixture obtained during the production of L-tryptophan by utilizing a micro organ-is, which comprises effectively removing the microorganism from the reaction mixture.
The present inventors made extensive investigations in order to achieve this object. While it has been commonly known that when heated in an acidic solution, L-tryptophan is unstable, these investigations have led to the surprising discovery that when reaction mixture containing L-tryptophan and a micro organ-is is adjusted to pi 2 to 5 with a mineral acid and then heated, L-tryptophan is stable and the microorganism is modified and floe-quilted to a size which can be easily filtered off.
Thus, according to this invention, there is provided a method of isolating L-tryptophan from a reaction mixture contain-in L-tryptophan and a microorganism which is obtained during the production of L-tryptophan by utilizing the microorganism; said method comprising adjusting said reaction mixture to pi 2 to 5 with a mineral acid selected from sulfuric acid, hydrochloric acid and phosphoric acid and heating it, thereafter separating the flocculated microorganism by either (1) filtering said reaction mixture while L-tryptophan is maintained in the completely dissolved state, or (2) adding an alkali selected from ammonia, sodium hydroxide, potassium hydroxide, sodium hydrogen carbonate and potassium hydrogen carbonate to said reaction mixture to . ., 5~6~
convert L--tryptophan therein to its alkali salt and then filtering said mixture while the alkali salt is maintained in the completely dissolved state, and recovering L-tryptophan from the filtrate.
Examples of the "reaction mixture containing L-trypto-plan and a microorganism which is obtained during the production of L-tryptophan by utilizing the microorganism" (to be sometimes referred to simply as a reaction mixture), as referred to in the present application, are a reaction mixture obtained by reacting L-serine and insole in the presence of Escherichia golf; a react lion mixture obtained by the above method using DL-serine instead of L-serine and Pseudomonas putted (MT-101~2) or Pseudomonas punctata (MT-10243) jointly with Escherichia golf as a shrine rhizomes; a reaction mixture obtained by using anthranilic acid as a precursor in the presence of Bacillus subtilis; and a reaction mixture obtained by using insole, pyruvic acid and ammonia in the presence of Aerobacter archness.
These reaction mixtures counterweighing L-tryptophan contain the used microorganisms in the dissolved or suspended state.
Since it has been very difficult in the prior art to separate the microorganisms from L-tryptophan, the cost of the purifying step in industrial practice has been high.
When the reaction mixture contains a water-immis-cable organic solvent it is desirable to remove the organic solvent prior to applying the method of this invention by suitable means such as liquid separation or distillation.
When the reaction mixture is heated under neutral to alkaline conditions, the microorganism in the reaction mixture cannot be flocculated to a filth-able state. In contrast, when the reaction mixture after the reaction is adjusted to pi 2 to 5 with a mineral acid and then heated in accordance with the method of this invention, the microorganism is floe-quilted very easily. Unexpectedly, L-tryptophan is not decomposed at this time and the flocculated micro-organism can be removed by filtration. When after flocculation of the microorganism in the above-mentioned manner, an alkali is added to the reaction mixture to dissolve L-tryptophan, the flocculated microorganism is still maintained in the filterable state. Accordingly, the method ox this invention is an industrial method by which L-tryptophan produced by utilizing a microorganism can be efficiently separated from the reaction mixture.
Examples of the mineral acid used in the method of this invention are sulfuric acid, hydra-caloric acid and phosphoric acid. The pi of the I I
L-tryptophan-containing reaction mixture is adjusted to 2 to 5, preferably 3 to 4, with such a mineral acid. The pH-adjusted reaction mixture is then heated at a temperature of 60 to 120C, preferably 80 to 105C By this pi adjustment and heat treatment the microorganism is modified and flocculated to a size which can be easily filtered off, whereas L-tryptophan remains stable without a change.
Hence, the heat-treating time is not portico-laxly restricted and the heat-treatment may be term-noted at a time when the microorganism has been floe quilted in a suitable state.
An alcohol may be added as a solvent in order to promote dissolution of L-tryptophan in the reaction mixture. Lower alcohols such as methanol, ethanol and isopropanol are preferred as the alcohol, isopropanol being particularly preferred. The alcohol may be used in such an amount that its concentration in the reaction mixture is not more than 70% by weight, preferably 40 to 60~ by weight. When the reaction mixture contains a water-immiscible organic solvent, a predetermined amount of the alcohol is added after the water-immis-cable organic solvent has been removed.
In one embodiment of the method of this invention, after the aforesaid pi adjustment and heat-treatment, the flocculated microorganism is separated from the reaction mixture by filtration to ~29~5Q613 give an aqueous solution of L-tryptophan, To increase the ratio of L-tryptophan recovered, this filtration operation is carried out while L.-tryptophan in the reaction mixture is in the completely dissolved state namely while the concentration of L-tryptophan in the reaction mixture is below its volubility. Usually, therefore, it is necessary to heat the reaction mix-lure and hot-filter it; or to dilute the reaction mixture fully with water and filter it. From the viewpoint of the operating efficiency, it is preferred to hot-filter it immediately after the pi adjustment and heat-treatment. In performing the filtration, activated carbon, or a silica-type filtration aid, or both may be used.
In another embodiment of the method of this invention, as another method of increasing the ratio of L-tryptophan recovered, an alkali is added to the reaction mixture after adjusting its pew to 2 to S and heating it to flocculate the microorganism. Thus, L-tryptophan is substantially completely dissolved as its alkali salt and thereafter the reaction mixture is filtered to obtain an aqueous solution of L-tryptophan.
according to the method of this embodiment, hot filtration or dilution as in the aforesaid embodiment is not required, and the cost of heat energy can be saved. Or an aqueous solution having a higher L-tryptophan concentration can be handled. Accordingly I
this embodiment is generally advantageous for industrial practice.
The alkali to be added to the reaction mixture which has been subjected to the pit adjustment and heat-treatment may be any alkali which can form a water-sol~ble salt by reaction with L-tryptophan.
Examples include ammonia, sodium hydroxide, potassium hydroxide, sodium hydrogen carbonate and potassium hydrogen carbonate. Industrially, ammonia is preferred because of its high volubility in the aqueous solution and its ease of recovery.
The amount of the alkali is usually that which is required to neutralize the reaction mixture and convert L-tryptophan present in the reaction mixture to its alkali salt. No serious inconvenience is caused even if the alkali is used in excess.
However, since at the time of taking out crystals of L-tryptophan, the pi is adjusted with an acid to its isoelectric point so as to increase the yield of L-tryptophan isolated, the use of an excess of the alkali undesirably results in an increase in the amount of inorganic salts.
Preferably, the alkali is added after the heat-treated reaction mixture is cooled to 0 to 50C, particularly 5 to 20C. When the alkali is added at higher temperatures, L-tryptophan may undergo deco-position or racemization.
When ammonia gas is used as the alkali, it is preferred to blow cooled ammonia gas into the reaction mixture in order to increase the volubility of ammonia in the reaction mixture. When an inorganic base such as sodium hydroxide is used, its addition at room temperature results in the formation of an alkali salt of L-tryptophan, which dissolves very rapidly.
When activated carbon and/or a silica-type filtration aid is added to the reaction mixture contain in the alkali salt of L-tryptophan and it is filtered in the presence of the added filtration aid, the microorganism are easily separated, and an aqueous solution of the alkali salt of L-tryptophan can be obtained.
L-tryptophan can be recovered by subjecting the resulting aqueous solution of the alkali salt of L-tryptophan to an ordinary crystallizing method such as neutralization.
The following Examples illustrate the present invention more specifically.
Example 1 One platinum loopful of Escherichia golf was inoculated in 50 ml of a culture medium having the composition [I] below, and cultivated with shaking at 30C for 20 hours. One liter of the culture broth was centrifuged and the cells were collected and used as a source of tryptophan synthetase.
~Z~5~8 Culture medium composition [If Meat extract OWE% by weight Depone 0.5% by weight Yeast extract 0.1% by weight K~2PO~ 0.2% by weight Initial stage pi 7.0 One platinum loopful of Pseudomonas Utica (IF 12996) was inoculated in 50 ml of a culture medium having the following composition [II], and cultivated with shaking at 30C for 20 hours. One liter of the culture broth was centrifuged and the cells were collected and used as a source of shrine rhizomes.
Culture medium composition lit]
Meat extract 1.0~ by weight Petunia 0.5% by weight Nail 0.5% by weight Initial stage pi 7.0 A 300 ml flask equipped with a stirrer was charged with 11.3 g of DL-serine, 6 g of ammonium sulfate, 10 my of pyridoxal phosphate and 66 g of water, and they were well stirred. Concentrated aqueous ammonia was added to the resulting aqueous solution to adjust its pi to 8.5. Then, 6~8 g (solids content 1.7 g) of a wet cream cake of Escherichia golf and 3.4 g (solids content 0.85 g) of a wet cream cake of Pseudomonas putted were suspended in water to form US
a suspension having a total volume of 20 ml. The resulting suspension was added to the above aqueous solution.
After maintaining the mixture at 35C, 57.2 9 of a Tulane solution containing 11.5 g of insole was added and reacted at 35C for 48 hours. The reaction yield was quantitative.
The reaction mixture was distilled to remove Tulane, and water was added to adjust the total amount of the mixture to 450 g. It was adjusted to pi 3.5 with sulfuric acid, and 3 9 of activated carbon was added. The mixture was heated to 95 to 98C and maintained at this temperature for 1 hour. It was hot-filtered at the same temperature while L-tryptophan was maintained in the dissolved state to separate activated carbon and the flocculated microorganism.
The filtrate was concentrated to an L-tryptophan concern-traction of 10% by weight. It was cooled to 20C, and the resulting crystals were separated by filtration.
Crystals of L-tryptophan having a purity of 99.5~ were isolated in a yield of 80% based on insole.
Example 2 The same reaction as in Example 1 was carried out in water using cells of Escherichia golf (MT-10232) and cells of _eudomonas punctata (MT-102~3) which were cultivated in the same way as in Example 1. The reaction mixture was centrifugally filtered to separate the L-tryptophan crystals precipitated in it and the microbial jells used in the reaction.
The cream cake was discharged into water to adjust the concentration of L-tryptophan to 4,0% by weight. Then, the pi of the solution was adjusted to 4.0 with phosphoric acid. Two grams of activated carbon and 2 g of Elite 545 (a trade name for a product of Johns~Manville Corporation) were added, and the mixture was heated at 95 to 98C for 1 hour It was hot-filtered at the same temperature to separate activated carbon, Elite and the flocculated microbial cells. The filtrate was concentrated to an L-tryptophan concentration of 15% by weight, and cooled to 20C, The precipitated crystals were recovered by filtration.
The yield of L-tryptophan isolated was 87%, and its purity was 99.7~.
Example The same cream cake consisting of L-tryptophan and microbial cells as obtained in Example 2 was suspended in a 1:1 try volume) mixture of water and isopropanol, and the concentration of L-tryptophan was adjusted to 7% by weight. Concentrated hydrochloric acid was added to adjust the pi of the suspension to 3.5. Activated carbon (3 g) was added, and the mixture was heated at 80 to 84C for 1 hour. The mixture was hot-filtered at the same temperature. The filtrate was cooled to 5C, and the precipitated crystals were 5C16~3 collected by filtration.
The yield of L-tryptophan isolated was 75%, and its purity was 98.5%.
Example 4 The same reaction as in Example 1 was carried out. After removing Tulane, the reaction mixture was diluted with water to an L-tryptophan concentration of 1% by weight. The pi of the reaction mixture was adjusted to 4.0 with sulfuric acid, and it was stirred at room temperature for 2 hours to dissolve L-tryptophan.
Activated carbon (3 g) and 3 y of Standard Super cell (a trade name for a product of Johns-Manville Corporation) as a filtration aid were added, and the solution was filtered at room temperature. The filtrate was concern-treated to an L-tryptophan concentration of 10% by weight, and then cooled to 5C. The precipitated cells were collected by filtration.
The yield of L-tryptophan isolated was 79%, and its purity was 98.8%.
Exhume 5 In the same way as in Example 1, insole and DL-serine were reacted in a Tulane solution. The reaction yield was quantitative. Tulane was removed from the reaction mixture by distillation.
The resulting L-tryptophan-containing reaction mixture was adjusted to pi 4.0 with sulfuric acid, and heated at 95 to 98C for 1 hour. after cooling to say room temperature, ammonia gas was blown into the reaction mixture to dissolve L-tryptophan therein as its ammonium salt. Activated carbon in an amount of 10~ by weight based on L-tryptophan and Elite 545 (a trade name for a product of Johns-Manville Corporation) in an amount of 10~ by weight based on L~tryptophan were added to the solution, and the solution was filtered. The microorganism was separated together with Elite 545 and activated carbon. The filtrate was heated to 100C to remove ammonia, and the concentration of L-tryptophan was adjusted to 10% by weight by adding water. The solution was cooled to 20C, and the precipitated crystals were separated by filtration, washed with water and dried.
The yield of L-tryptophan isolated was 75 based on the resulting tryptophan, and its purity measured by liquid chromatography was 98.5%.
Example 6 The same L~tryptophan-containing reaction mixture as obtained in Example 5 was centrifuged and a mixture of L-tryptophan crystals and the microorganisms was obtained as a cream cake. The cream cake was disk charged into water to form a slurry having an L-trypto-plan concentration of 30% by weight. The pi of the slurry was adjusted to 3.5, and it was heated at 95 to 98C for 2 hours to flocculate the microorganisms used in the reaction. After cooling to room temperature, S~68 aqueous ammonia was added to dissolve L-tryptophan in the reaction mixture as its ammonium salt. To the solution was added 20~ by weight, eased on L-tryptophan, of activated carbon, and the flocculated microbial cells were separated at room temperature, Nitrogen gas was blown into the filtrate at an elevated temperature to remove ammonia. After cooling to 5C, the precipi-toted L-tryptophan was separated by a centrifugal dehydrator The yield of L-tryptophan isolated was 88~
based on the resulting tryptophan, and its purity was 98.0%.
Example 7 12% by weight aqueous solution of L-tryptophan obtained in the same way as in Example 5 was adjusted to pi 4.0 with phosphoric acid, and heated to 95 to 98C. After cooling to 25C, a 20~ aqueous solution of sodium hydroxide was added to adjust the pi of the solution to 10. To the aqueous L-tryptophan solution were added 10% by weight, base on teyptophan, of activated carbon and 10% by weight, based on tryptophan, of Standard Super cell (a trade name for a product of Johns-Manville Corporation) as a filtration aid, and the solution was filtered at 20C. The filtrate was neutralized to a pi of 6 with acetic acid, and the precipitated crystals of L-tryptophan were separated by filtration and dried The yield of L-tryptophan So isolated was 75 % based on the resulting tryptophan, and its purity was 99.2 %.
Exhume 8 A cream cake of L-tryptophan obtained in the same way as in Example 5 was dispersed in a 1:1 (by volume) mixture of water and isopropanol to form a slurry having an L-tryptophan concentration of 20% by weight. The slurry was adjusted to pi 3.5 with sulfuric acid, and it was heated at 80 to 84C for 2 hours.
After cooling to 5C, ammonia gas was blown into the reaction mixture to dissolve L-tryptophan in the solvent as its ammonium salt. To the solution was added 20% by weight, based on tryptophan, of activated carbon, and the solution was suction-filtered to give a pale yellow clear L-tryptophan solution. Nitrogen gas was blown into the solution under heating to remove ammonia. The solution was cooled, and the precipitated Lrtryptophan crystals were separated by filtration, and dried.
The yield of L-tryptophan isolated was 83%
based on the resulting tryptophan, and its purity was 98.8~
This invention relates to a method of isolate in L-tryptophan from a reaction mixture containing 1-tryptophan and a microorganism obtained during the production of L-tryptophan by utilizing the micro-organism.
In reactions utilizing microorganisms, it is necessary to separate the microorganisms from the reaction products. Some methods have been known in the past for removing the microorganisms from the reaction mixtures and isolating the reaction products.
For example, Japanese Patent Publication No. 16460/1963 discloses a method which comprises adding a surface-active agent to an L-glutamic acid fermentation broth, heating the mixture to flocculate and sediment the lo microorganism, and separating it by filtration upon addition of diatomaceous earth, and Japanese Laid-Open Patent Publication No. 29996/1978 discloses a method which comprises filtering a reaction mixture such as an amino acid fermentation broth on an ultra-filtration membrane and then isolating the product by crystallization.
These methods, however, have not proved to be entirely satisfactory for industrial practice because by the method involving adding the surfactant, the microorganism can be easily removed but the surfactant cannot be easily separated and is likely to remain in the reaction product, and the method involving the use I
of the ultrafiltration membrane has difficulty in washing the used apparatus owing to its nature.
It is an object of this invention therefore to provide a method of isolating L-tryptophan from a reaction mixture obtained during the production of L-tryptophan by utilizing a micro organ-is, which comprises effectively removing the microorganism from the reaction mixture.
The present inventors made extensive investigations in order to achieve this object. While it has been commonly known that when heated in an acidic solution, L-tryptophan is unstable, these investigations have led to the surprising discovery that when reaction mixture containing L-tryptophan and a micro organ-is is adjusted to pi 2 to 5 with a mineral acid and then heated, L-tryptophan is stable and the microorganism is modified and floe-quilted to a size which can be easily filtered off.
Thus, according to this invention, there is provided a method of isolating L-tryptophan from a reaction mixture contain-in L-tryptophan and a microorganism which is obtained during the production of L-tryptophan by utilizing the microorganism; said method comprising adjusting said reaction mixture to pi 2 to 5 with a mineral acid selected from sulfuric acid, hydrochloric acid and phosphoric acid and heating it, thereafter separating the flocculated microorganism by either (1) filtering said reaction mixture while L-tryptophan is maintained in the completely dissolved state, or (2) adding an alkali selected from ammonia, sodium hydroxide, potassium hydroxide, sodium hydrogen carbonate and potassium hydrogen carbonate to said reaction mixture to . ., 5~6~
convert L--tryptophan therein to its alkali salt and then filtering said mixture while the alkali salt is maintained in the completely dissolved state, and recovering L-tryptophan from the filtrate.
Examples of the "reaction mixture containing L-trypto-plan and a microorganism which is obtained during the production of L-tryptophan by utilizing the microorganism" (to be sometimes referred to simply as a reaction mixture), as referred to in the present application, are a reaction mixture obtained by reacting L-serine and insole in the presence of Escherichia golf; a react lion mixture obtained by the above method using DL-serine instead of L-serine and Pseudomonas putted (MT-101~2) or Pseudomonas punctata (MT-10243) jointly with Escherichia golf as a shrine rhizomes; a reaction mixture obtained by using anthranilic acid as a precursor in the presence of Bacillus subtilis; and a reaction mixture obtained by using insole, pyruvic acid and ammonia in the presence of Aerobacter archness.
These reaction mixtures counterweighing L-tryptophan contain the used microorganisms in the dissolved or suspended state.
Since it has been very difficult in the prior art to separate the microorganisms from L-tryptophan, the cost of the purifying step in industrial practice has been high.
When the reaction mixture contains a water-immis-cable organic solvent it is desirable to remove the organic solvent prior to applying the method of this invention by suitable means such as liquid separation or distillation.
When the reaction mixture is heated under neutral to alkaline conditions, the microorganism in the reaction mixture cannot be flocculated to a filth-able state. In contrast, when the reaction mixture after the reaction is adjusted to pi 2 to 5 with a mineral acid and then heated in accordance with the method of this invention, the microorganism is floe-quilted very easily. Unexpectedly, L-tryptophan is not decomposed at this time and the flocculated micro-organism can be removed by filtration. When after flocculation of the microorganism in the above-mentioned manner, an alkali is added to the reaction mixture to dissolve L-tryptophan, the flocculated microorganism is still maintained in the filterable state. Accordingly, the method ox this invention is an industrial method by which L-tryptophan produced by utilizing a microorganism can be efficiently separated from the reaction mixture.
Examples of the mineral acid used in the method of this invention are sulfuric acid, hydra-caloric acid and phosphoric acid. The pi of the I I
L-tryptophan-containing reaction mixture is adjusted to 2 to 5, preferably 3 to 4, with such a mineral acid. The pH-adjusted reaction mixture is then heated at a temperature of 60 to 120C, preferably 80 to 105C By this pi adjustment and heat treatment the microorganism is modified and flocculated to a size which can be easily filtered off, whereas L-tryptophan remains stable without a change.
Hence, the heat-treating time is not portico-laxly restricted and the heat-treatment may be term-noted at a time when the microorganism has been floe quilted in a suitable state.
An alcohol may be added as a solvent in order to promote dissolution of L-tryptophan in the reaction mixture. Lower alcohols such as methanol, ethanol and isopropanol are preferred as the alcohol, isopropanol being particularly preferred. The alcohol may be used in such an amount that its concentration in the reaction mixture is not more than 70% by weight, preferably 40 to 60~ by weight. When the reaction mixture contains a water-immiscible organic solvent, a predetermined amount of the alcohol is added after the water-immis-cable organic solvent has been removed.
In one embodiment of the method of this invention, after the aforesaid pi adjustment and heat-treatment, the flocculated microorganism is separated from the reaction mixture by filtration to ~29~5Q613 give an aqueous solution of L-tryptophan, To increase the ratio of L-tryptophan recovered, this filtration operation is carried out while L.-tryptophan in the reaction mixture is in the completely dissolved state namely while the concentration of L-tryptophan in the reaction mixture is below its volubility. Usually, therefore, it is necessary to heat the reaction mix-lure and hot-filter it; or to dilute the reaction mixture fully with water and filter it. From the viewpoint of the operating efficiency, it is preferred to hot-filter it immediately after the pi adjustment and heat-treatment. In performing the filtration, activated carbon, or a silica-type filtration aid, or both may be used.
In another embodiment of the method of this invention, as another method of increasing the ratio of L-tryptophan recovered, an alkali is added to the reaction mixture after adjusting its pew to 2 to S and heating it to flocculate the microorganism. Thus, L-tryptophan is substantially completely dissolved as its alkali salt and thereafter the reaction mixture is filtered to obtain an aqueous solution of L-tryptophan.
according to the method of this embodiment, hot filtration or dilution as in the aforesaid embodiment is not required, and the cost of heat energy can be saved. Or an aqueous solution having a higher L-tryptophan concentration can be handled. Accordingly I
this embodiment is generally advantageous for industrial practice.
The alkali to be added to the reaction mixture which has been subjected to the pit adjustment and heat-treatment may be any alkali which can form a water-sol~ble salt by reaction with L-tryptophan.
Examples include ammonia, sodium hydroxide, potassium hydroxide, sodium hydrogen carbonate and potassium hydrogen carbonate. Industrially, ammonia is preferred because of its high volubility in the aqueous solution and its ease of recovery.
The amount of the alkali is usually that which is required to neutralize the reaction mixture and convert L-tryptophan present in the reaction mixture to its alkali salt. No serious inconvenience is caused even if the alkali is used in excess.
However, since at the time of taking out crystals of L-tryptophan, the pi is adjusted with an acid to its isoelectric point so as to increase the yield of L-tryptophan isolated, the use of an excess of the alkali undesirably results in an increase in the amount of inorganic salts.
Preferably, the alkali is added after the heat-treated reaction mixture is cooled to 0 to 50C, particularly 5 to 20C. When the alkali is added at higher temperatures, L-tryptophan may undergo deco-position or racemization.
When ammonia gas is used as the alkali, it is preferred to blow cooled ammonia gas into the reaction mixture in order to increase the volubility of ammonia in the reaction mixture. When an inorganic base such as sodium hydroxide is used, its addition at room temperature results in the formation of an alkali salt of L-tryptophan, which dissolves very rapidly.
When activated carbon and/or a silica-type filtration aid is added to the reaction mixture contain in the alkali salt of L-tryptophan and it is filtered in the presence of the added filtration aid, the microorganism are easily separated, and an aqueous solution of the alkali salt of L-tryptophan can be obtained.
L-tryptophan can be recovered by subjecting the resulting aqueous solution of the alkali salt of L-tryptophan to an ordinary crystallizing method such as neutralization.
The following Examples illustrate the present invention more specifically.
Example 1 One platinum loopful of Escherichia golf was inoculated in 50 ml of a culture medium having the composition [I] below, and cultivated with shaking at 30C for 20 hours. One liter of the culture broth was centrifuged and the cells were collected and used as a source of tryptophan synthetase.
~Z~5~8 Culture medium composition [If Meat extract OWE% by weight Depone 0.5% by weight Yeast extract 0.1% by weight K~2PO~ 0.2% by weight Initial stage pi 7.0 One platinum loopful of Pseudomonas Utica (IF 12996) was inoculated in 50 ml of a culture medium having the following composition [II], and cultivated with shaking at 30C for 20 hours. One liter of the culture broth was centrifuged and the cells were collected and used as a source of shrine rhizomes.
Culture medium composition lit]
Meat extract 1.0~ by weight Petunia 0.5% by weight Nail 0.5% by weight Initial stage pi 7.0 A 300 ml flask equipped with a stirrer was charged with 11.3 g of DL-serine, 6 g of ammonium sulfate, 10 my of pyridoxal phosphate and 66 g of water, and they were well stirred. Concentrated aqueous ammonia was added to the resulting aqueous solution to adjust its pi to 8.5. Then, 6~8 g (solids content 1.7 g) of a wet cream cake of Escherichia golf and 3.4 g (solids content 0.85 g) of a wet cream cake of Pseudomonas putted were suspended in water to form US
a suspension having a total volume of 20 ml. The resulting suspension was added to the above aqueous solution.
After maintaining the mixture at 35C, 57.2 9 of a Tulane solution containing 11.5 g of insole was added and reacted at 35C for 48 hours. The reaction yield was quantitative.
The reaction mixture was distilled to remove Tulane, and water was added to adjust the total amount of the mixture to 450 g. It was adjusted to pi 3.5 with sulfuric acid, and 3 9 of activated carbon was added. The mixture was heated to 95 to 98C and maintained at this temperature for 1 hour. It was hot-filtered at the same temperature while L-tryptophan was maintained in the dissolved state to separate activated carbon and the flocculated microorganism.
The filtrate was concentrated to an L-tryptophan concern-traction of 10% by weight. It was cooled to 20C, and the resulting crystals were separated by filtration.
Crystals of L-tryptophan having a purity of 99.5~ were isolated in a yield of 80% based on insole.
Example 2 The same reaction as in Example 1 was carried out in water using cells of Escherichia golf (MT-10232) and cells of _eudomonas punctata (MT-102~3) which were cultivated in the same way as in Example 1. The reaction mixture was centrifugally filtered to separate the L-tryptophan crystals precipitated in it and the microbial jells used in the reaction.
The cream cake was discharged into water to adjust the concentration of L-tryptophan to 4,0% by weight. Then, the pi of the solution was adjusted to 4.0 with phosphoric acid. Two grams of activated carbon and 2 g of Elite 545 (a trade name for a product of Johns~Manville Corporation) were added, and the mixture was heated at 95 to 98C for 1 hour It was hot-filtered at the same temperature to separate activated carbon, Elite and the flocculated microbial cells. The filtrate was concentrated to an L-tryptophan concentration of 15% by weight, and cooled to 20C, The precipitated crystals were recovered by filtration.
The yield of L-tryptophan isolated was 87%, and its purity was 99.7~.
Example The same cream cake consisting of L-tryptophan and microbial cells as obtained in Example 2 was suspended in a 1:1 try volume) mixture of water and isopropanol, and the concentration of L-tryptophan was adjusted to 7% by weight. Concentrated hydrochloric acid was added to adjust the pi of the suspension to 3.5. Activated carbon (3 g) was added, and the mixture was heated at 80 to 84C for 1 hour. The mixture was hot-filtered at the same temperature. The filtrate was cooled to 5C, and the precipitated crystals were 5C16~3 collected by filtration.
The yield of L-tryptophan isolated was 75%, and its purity was 98.5%.
Example 4 The same reaction as in Example 1 was carried out. After removing Tulane, the reaction mixture was diluted with water to an L-tryptophan concentration of 1% by weight. The pi of the reaction mixture was adjusted to 4.0 with sulfuric acid, and it was stirred at room temperature for 2 hours to dissolve L-tryptophan.
Activated carbon (3 g) and 3 y of Standard Super cell (a trade name for a product of Johns-Manville Corporation) as a filtration aid were added, and the solution was filtered at room temperature. The filtrate was concern-treated to an L-tryptophan concentration of 10% by weight, and then cooled to 5C. The precipitated cells were collected by filtration.
The yield of L-tryptophan isolated was 79%, and its purity was 98.8%.
Exhume 5 In the same way as in Example 1, insole and DL-serine were reacted in a Tulane solution. The reaction yield was quantitative. Tulane was removed from the reaction mixture by distillation.
The resulting L-tryptophan-containing reaction mixture was adjusted to pi 4.0 with sulfuric acid, and heated at 95 to 98C for 1 hour. after cooling to say room temperature, ammonia gas was blown into the reaction mixture to dissolve L-tryptophan therein as its ammonium salt. Activated carbon in an amount of 10~ by weight based on L-tryptophan and Elite 545 (a trade name for a product of Johns-Manville Corporation) in an amount of 10~ by weight based on L~tryptophan were added to the solution, and the solution was filtered. The microorganism was separated together with Elite 545 and activated carbon. The filtrate was heated to 100C to remove ammonia, and the concentration of L-tryptophan was adjusted to 10% by weight by adding water. The solution was cooled to 20C, and the precipitated crystals were separated by filtration, washed with water and dried.
The yield of L-tryptophan isolated was 75 based on the resulting tryptophan, and its purity measured by liquid chromatography was 98.5%.
Example 6 The same L~tryptophan-containing reaction mixture as obtained in Example 5 was centrifuged and a mixture of L-tryptophan crystals and the microorganisms was obtained as a cream cake. The cream cake was disk charged into water to form a slurry having an L-trypto-plan concentration of 30% by weight. The pi of the slurry was adjusted to 3.5, and it was heated at 95 to 98C for 2 hours to flocculate the microorganisms used in the reaction. After cooling to room temperature, S~68 aqueous ammonia was added to dissolve L-tryptophan in the reaction mixture as its ammonium salt. To the solution was added 20~ by weight, eased on L-tryptophan, of activated carbon, and the flocculated microbial cells were separated at room temperature, Nitrogen gas was blown into the filtrate at an elevated temperature to remove ammonia. After cooling to 5C, the precipi-toted L-tryptophan was separated by a centrifugal dehydrator The yield of L-tryptophan isolated was 88~
based on the resulting tryptophan, and its purity was 98.0%.
Example 7 12% by weight aqueous solution of L-tryptophan obtained in the same way as in Example 5 was adjusted to pi 4.0 with phosphoric acid, and heated to 95 to 98C. After cooling to 25C, a 20~ aqueous solution of sodium hydroxide was added to adjust the pi of the solution to 10. To the aqueous L-tryptophan solution were added 10% by weight, base on teyptophan, of activated carbon and 10% by weight, based on tryptophan, of Standard Super cell (a trade name for a product of Johns-Manville Corporation) as a filtration aid, and the solution was filtered at 20C. The filtrate was neutralized to a pi of 6 with acetic acid, and the precipitated crystals of L-tryptophan were separated by filtration and dried The yield of L-tryptophan So isolated was 75 % based on the resulting tryptophan, and its purity was 99.2 %.
Exhume 8 A cream cake of L-tryptophan obtained in the same way as in Example 5 was dispersed in a 1:1 (by volume) mixture of water and isopropanol to form a slurry having an L-tryptophan concentration of 20% by weight. The slurry was adjusted to pi 3.5 with sulfuric acid, and it was heated at 80 to 84C for 2 hours.
After cooling to 5C, ammonia gas was blown into the reaction mixture to dissolve L-tryptophan in the solvent as its ammonium salt. To the solution was added 20% by weight, based on tryptophan, of activated carbon, and the solution was suction-filtered to give a pale yellow clear L-tryptophan solution. Nitrogen gas was blown into the solution under heating to remove ammonia. The solution was cooled, and the precipitated Lrtryptophan crystals were separated by filtration, and dried.
The yield of L-tryptophan isolated was 83%
based on the resulting tryptophan, and its purity was 98.8~
Claims (6)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A method of separating L-tryptophan from a reaction mixture containing L-tryptophan and a microorganism which is obtained during the production of L-tryptophan by utilizing the microorganism; said method comprising adjusting said reaction mixture to pH 2 to 5 with a mineral acid selected from sulfuric acid, hydrochloric acid and phosphoric acid, heating it, and thereafter filtering the heated reaction mixture to remove the microorganism.
2. A method of separating L-tryptophan from a reaction mixture containing L-tryptophan and a microorganism which is obtained during the production of L-tryptophan by utilizing the microorganism; said method comprising adjusting said reaction mixture to pH 2 to 5 with a mineral acid selected from sulfuric acid, hydrochloric acid and phosphoric acid, heating it, adding an alkali selected from ammonia, sodium hydroxide, potassium hydroxide, sodium hydrogen carbonate and potassium hydrogen car-bonate to the heated reaction mixture to convert L-tryptophan contained therein to its alkali metal salt, and thereafter filtering the reaction mixture to remove the microorganism.
3. A method according to claim 1 or 2 wherein the reaction mixture is obtained by reacting L-serine and indole in the presence of Escherichia coli.
4. A method according to claim 1 or 2 wherein the reaction mixture is obtained by reacting DL-serine and indole in the presence of Pseudomonas putida (MT-10182) or Pseudomonas punctata (MT-10243) jointly with Escherichia coli.
5. A method according to claim 1 or 2 wherein the reaction mixture is obtained by using anthranilic acid as a precursor in the presence of Bacillus subtilis.
6. A method according to claim 1 or 2 wherein the reaction mixture is obtained by using indole, pyruvic acid and ammonia in the presence of Aerobacter aerogenes.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19843400603 DE3400603A1 (en) | 1984-01-10 | 1984-01-10 | METHOD FOR SEPARATING L-TRYPTOPHANE |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1215068A true CA1215068A (en) | 1986-12-09 |
Family
ID=6224602
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000444164A Expired CA1215068A (en) | 1984-01-10 | 1983-12-22 | Method of separating l-tryptophan |
Country Status (7)
| Country | Link |
|---|---|
| AU (1) | AU566747B2 (en) |
| CA (1) | CA1215068A (en) |
| CH (1) | CH659828A5 (en) |
| DE (1) | DE3400603A1 (en) |
| FR (1) | FR2557873B1 (en) |
| GB (1) | GB2152031B (en) |
| NL (1) | NL8304497A (en) |
Families Citing this family (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0648990B2 (en) * | 1987-01-14 | 1994-06-29 | 味の素株式会社 | Method for purifying tryptophan |
| BR8803497A (en) * | 1987-07-13 | 1989-01-31 | Mitsui Toatsu Chemicals | PROCESS FOR THE PREPARATION OF L-TRYPTOPHANE AND PROCESS FOR THE PREPARATION OF AQUEOUS ENZYMATIC SOLUTION OF TRIPTOPHANE SYNTHESIS OUTRIPTOPHANESE |
| KR950013858B1 (en) * | 1987-10-12 | 1995-11-17 | 미쓰이 도아쓰 가가쿠 가부시기가이샤 | Process for producing l-tryptophane |
| DE3915616C1 (en) * | 1989-05-12 | 1990-06-21 | Gesellschaft Fuer Biotechnologische Forschung Mbh (Gbf), 3300 Braunschweig, De | |
| JPH0489479A (en) * | 1990-08-01 | 1992-03-23 | Ajinomoto Co Inc | Recovery of optically active tryptophan |
| BR9605094A (en) * | 1995-10-13 | 1998-07-07 | Ajinomoto Kk | Process for removing cells from a fermentation broth |
| EP0770676A3 (en) * | 1995-10-23 | 1999-05-19 | Ajinomoto Co., Ltd. | Method for treating fermentation broth |
| DE19540788A1 (en) * | 1995-11-02 | 1997-05-07 | Degussa | Use of aqueous L-tryptophan and / or L-threonine salt solutions |
| CA2569204A1 (en) | 2006-11-28 | 2008-05-28 | Apotex Technologies Inc. | Crystalline d-isoglutamyl-d-tryptophan and the mono ammonium salt of d-isoglutamyl-d-tryptophan |
| CA2571645A1 (en) | 2006-12-19 | 2008-06-19 | Apotex Technologies Inc. | Pharmaceutically acceptable salts of thymodepressin and processes for their manufacture |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US2973304A (en) * | 1958-10-02 | 1961-02-28 | Pfizer & Co C | Fermentation process |
| FR1343804A (en) * | 1962-10-25 | 1963-11-22 | Sumitomo Chemical Co | Process for the production of l-amino acids by fermentation |
| US4411991A (en) * | 1980-10-07 | 1983-10-25 | Kanegafuchi Chemical Industry Company, Limited | Process for fermentative production of amino acids |
-
1983
- 1983-12-21 AU AU22749/83A patent/AU566747B2/en not_active Expired
- 1983-12-21 GB GB08334071A patent/GB2152031B/en not_active Expired
- 1983-12-22 CA CA000444164A patent/CA1215068A/en not_active Expired
- 1983-12-30 NL NL8304497A patent/NL8304497A/en active Search and Examination
-
1984
- 1984-01-10 CH CH10984A patent/CH659828A5/en not_active IP Right Cessation
- 1984-01-10 DE DE19843400603 patent/DE3400603A1/en not_active Ceased
- 1984-01-10 FR FR8400301A patent/FR2557873B1/en not_active Expired
Also Published As
| Publication number | Publication date |
|---|---|
| GB8334071D0 (en) | 1984-02-01 |
| AU2274983A (en) | 1985-06-27 |
| FR2557873A1 (en) | 1985-07-12 |
| GB2152031A (en) | 1985-07-31 |
| GB2152031B (en) | 1987-07-22 |
| CH659828A5 (en) | 1987-02-27 |
| DE3400603A1 (en) | 1985-07-18 |
| FR2557873B1 (en) | 1986-05-16 |
| NL8304497A (en) | 1985-07-16 |
| AU566747B2 (en) | 1987-10-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CA1215068A (en) | Method of separating l-tryptophan | |
| CA1215069A (en) | Method of isolating l-trytophan | |
| JP2646343B2 (en) | Method for producing L-aspartic acid from ammonium aspartate | |
| KR870001146B1 (en) | L-tryptophan isolation method | |
| RU2094461C1 (en) | Method of preparing 5-hydroxypyrazine carboxylic acid and/or their salts | |
| JPH0623182B2 (en) | Method for separating L-cysteine hydrochloride monohydrate | |
| JP5092743B2 (en) | Separation and recovery method of optically active amino acid amide | |
| JPS60169451A (en) | Separation and purification of l-phenylalanine | |
| HU221639B1 (en) | Process for producing l-aspartic acid | |
| KR900005773B1 (en) | Process for preparing l-tryptophan and process for preparing a stable agueous enzyme solution | |
| JPS5945896A (en) | Separation of l-tryptophan | |
| JPS5945897A (en) | Separation of l-tryptophan | |
| US5329014A (en) | Method for recovering optically active tryptophan | |
| JPH01320991A (en) | Production of d-homophenylalanines | |
| EP0140713A2 (en) | Method for production and recovery of L-Phenylalanine | |
| JP2804005B2 (en) | Method for producing L-aspartic acid | |
| JP2575403B2 (en) | Method for producing L-tryptophan | |
| JPH04304893A (en) | Production of hydroxide of nitrogen-containing heterocyclic compound by microorganism | |
| JP2931629B2 (en) | Crystallization method of L-tryptophan | |
| US6503739B1 (en) | Processes for producing S,S-2-hydroxypropylenediamine-N-N′-disuccinic acid | |
| JPH0344759B2 (en) | ||
| JP2801689B2 (en) | Method of inhibiting intracellular serine degrading enzyme activity | |
| GB2151634A (en) | Improvement in enzyme reaction | |
| JPH05178801A (en) | Method for crystallizing l-phenylalanine | |
| US20070213559A1 (en) | Method Of Separately Collecting Optically Active Amino Acid Amide And Optically Active Amino Acid |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| MKEX | Expiry |