GB2152031A

GB2152031A - Separating L-tryptophan from fermentation broth

Info

Publication number: GB2152031A
Application number: GB08334071A
Authority: GB
Inventors: Masaharu Ohoka; Yukihiro Yoshikawa; Nobuyuki Kawashima; Nobuhiro Kawashima; Syosuke Nagai; Takao Takano
Original assignee: Mitsui Toatsu Chemicals Inc
Current assignee: Mitsui Toatsu Chemicals Inc
Priority date: 1984-01-10
Filing date: 1983-12-21
Publication date: 1985-07-31
Also published as: AU2274983A; GB2152031B; FR2557873B1; GB8334071D0; DE3400603A1; FR2557873A1; CA1215068A; CH659828A5; NL8304497A; AU566747B2

Abstract

A L-tryptophan-containing reaction mixture produced by a reaction utilizing a microorganism is adjusted to pH 2 to 5 with a mineral acid and then heated whereby the microorganism contained in the reaction mixture is flocculated, and removed by either (1) filtering, or (2) adding an alkali to convert L-tryptophan to its alkali salt and then filtering. L-tryptophan of high purity can be obtained.

Description

SPECIFICATION Method of separating L-tryptophan This invention relates to a method of isolating L-tryptophan from a reaction mixture containing L-tryptophan and a microorganism obtained during the production of L-tryptophan by utilizing the microorganism.

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 microorganism, and separating it by filtration upon addition of diatomaceous earth, and Japanesed 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 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 microorganism, 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 a reaction mixture containing L-tryptophan and a microorganism is adjusted to pH 2 to 5 with a mineral acid and then heated, L-tryptophan is stable and the microorganism is modified and flocculated 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 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 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 to said reaction mixture to 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-tryptophan 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 indole in the presence of Escherichia coli; a reaction mixture obtained by the above method using DL-serine instead of L-serine and Psudomonas putida (MT-10182) or Pseudomonas punctata (MT-10243) jointly with Escherichia coil as a serine racemase; a reaction mixture obtained by using anthranilic acid as a precursor in the presence of Bacillus subtilis; and a reaction mixture obtained by using indole, pyruvic acid and ammonia in the presence of Aerobacteraerogenes.

These reaction mixtures containing 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-immiscible 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 filtrable state. In contrast, when the reaction mixture after the reaction is adjusted to pH 2 to 5 with a mineral acid and then heated in accordance with the method of this invention, the microorganism is flocculated very easily. Unexpectedly, L-tryptophan is not decomposed at this time and the flocculated microorganism can be removed by filtration. When after flocculation of the microorganism in the above-mentioend manner, an alkali is added to the reaction mixture to dissolve L-tryptophan, the flocculated microorganism is still maintained in the filtrable state. Accordingly, the method of 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, hydrochloric acid and phosphoric acid. The pH of the 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 120"C, preferably 80 to 105"C. By this pH 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 particularly restricted and the heat-treatment may be terminated at a time when the microorganism has been flocculated in a suitable state.

An alcohol may be added as a solvent in order to promote dissolution of L-tryptohan 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-immiscible organic solvent has been removed.

In one embodiment of the method of this invention, after the aforesaid pH adjustment and heat-treatment, the flocculated microorganism is separated from the reaction mixture by filtration to 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 solubility. Usually, therefore, it is necessary to heat the reaction mixture 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 pH 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 pH to 2 to 5 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, this embodiment is generally advantageous for industrial practice.

The alkali to be added to the reaction mixture which has been subjected to the pH adjustment and heat-treatment may be any alkali which can form a water-soluble 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 solubility 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 pH 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 toO to 50"C, particularly 5 to 20"C. When the alkali is added at higher temperatures, L-tryptophan may undergo decomposition 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 solubility 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 containing 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 specificaily.

Example 1 One platinum loopful of Escherichia colt was inoculated in 50 ml of a culture medium having the composition [Ij below, and cultivated with shaking at 30"C for 20 hours. One liter of the culture broth was centrifuged and the cells were collected and used as a source of tryptophan synthetase.

Culture medium composition [11 Meat extract 1.0% by weight Peptone 0.5% by weight Yeast extract 0.1% by weight KH2PO4 0.2% by weight Initial stage pH 7.0 One platinum loopful of Pseudomonas putida (IFO 12996) was inoculated in 50 ml of a culture medium having the following composition [II], and cultivated with shaking at 3000 for 20 hours. One liter of the culture broth was centrifuged and the cells were collected and used as a source of serine racemase.

Culture medium composition fil] Meat extract 1.0% by weight Peptone 0.5% by weight NaCI 0.5%byweight Initial stage pH 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 mg 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 pH to 8.5. Then, 6.8 g (solids content 1.7 g) of a wet cream cake of Escherichia coli and 3.4 g (solids content 0.85 g) of a wet cream cake of Pseudomonasputida were suspended in water to form a suspension having a total volume of 20 ml. The resulting suspension was added to the above aqueous solution.

After maintaining the mixture at 35"C, 57.2 g of a toluene solution containing 11.5 g of indole was added and reacted at 35"C for 48 hours. The reaction yield was quantitative.

The reaction mixture was distilled to remove toluene, and water was added to adjust the total amount of the mixture to 450 g. It was adjusted to pH 3.5 with sulfuric acid, and 3 g of activated carbon was added. The mixture was heated to 95 to 980C 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 fluocculated microorganism. The filtrate was concentrated to an L-tryptophan concentration of 10% by weight. It was cooled to 20"C, 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 indole.

Example 2 The same reaction as in Example 1 was carried out in water using cells of Escherichia coli (MT-10232) and cells of Pseudomonas punctata (MT-10243) 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 cells 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 pH of the solution was adjusted to 4.0 with phosphoric acid. Two grams of activated carbon and 2 g of Celite 545 (a tradename for a product of Johns-Manville Corporation) were added, and the mixture was heated at 95 to 98"C for 1 hour. It was hot-filtered at the same temperature to separate activated carbon, Celite and the flocculated microbial cells. The filtrate was concentrated to an L-tryptophan concentration of 15% by weight, and cooled to 200C. The precipitated crystals were recovered by filtration.

The yield of L-tryptophan isolated was 87%, and its purity was 99.7%.

Example 3 The same cream cake consisting of L-tryptophan and microbial cells as obtained in Example 2 was suspended in a 1:1 (by volume) mixture of water and isopropanol, and the concentration of L-tryptophan was adjusted to 7% by weight. Concentrated hydrochloric acid was added to adjujst the pH of the suspension to 3.5. Activated carbon (3 g) was added, and the mixture was heated at 80 to 84"C for 1 hour. The mixture was hot-filtered at the same temperature. The filtrate was cooled to 5"C, and the precipitated crystals were 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 toluene, the reaction mixture was diluted with water to an L-tryptophan concentration of 1% by weight. The pH 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 g of Standard Supercell (a tradename for a product of Johns-Manville Corporation) as a filtration aid were added, and the solution was filtered at room temperature. The filtrate was concentrated to an L-tryptophan concentration of 10% by weight, and then cooled to 5 C. The precipitated cells were collected by filtration.

The yield of L-tryptophan isolated was 79%, and its purity was 98.8%.

Example 5 In the same way as in Example 1, indole and DL-serine were reacted in a toluene solution. The reaction yield was quantitative. Toluene was removed from the reaction mixture by distillation.

The resulting L-tryptophan-containing reaction mixture was adjusted to pH 4.0 with sulfuric acid, and heated at 95 to 98"C for 1 hour. After cooling to 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 Celite 545 (a tradename 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 Celite 545 and activated carbon. The filtrate was heated to 1 OO"C to remove ammonia, and the concentration of L-tryptophan was adjusted to 10% by weight by adding water.The solution was cooled to 20"C, 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 discharged into water to form a slurry having an L-tryptophan concentration of 30% by weight. The pH of the slurry was adjusted to 3.5, and it was heated at 95 to 98"C for 2 hours to flocculate the microorganisms used in the reaction. After cooling to room temperature, aqueous ammonia was added to dissolve L-tryptophan in the reaction mixture as its ammonium salt. To the solution was added 20% by weight, based 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 eievated temperature to remove ammonia. After cooling to 5"C, the precipitated 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 A 12% by weight aqueous solution of L-tryptophan obtained in the same way as in Example 5 was adjusted to pH 4.0 with phosphoric acid, and heated to 95 to 98 C. After cooling to 25"C, a 20% aqueous solution of sodium hydroxide was added to adjust the pH of the solution to 10. To the aqueous L-tryptophan solution were added 10% by weight, base on tryptophan, of activated carbon and 10% by weight, based on tryptophan, of Standard Supercell (a tradename for a product of Johns-Manville Corporation) as a filtration aid, and the solution was filtered at 20"C. The filtrate was neutralized to a pH of 6 with acetic acid, and the precipitated crystals of L-tryptophan were separated by filtration and dried.The yield of L-tryptophan isolated was 75 % based on the resulting tryptophan, and its purity was 99.2 %.

Example 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 pH 3.5 with sulfuric acid, and it was heated at 80 to 84"C for 2 hours. After cooling to 5"C, 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 L-tryptophan 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

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, 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, heating it, adding an alkali to the heated reaction mixture to convert L-tryptophan contained therein to its alkali metal or ammonium salt, and thereafter filtering the reaction mixture to remove the microorganism.

3. A method according to Claim 1 or Claim 2 wherein the heating of the reaction mixture at a pH in the range 2 to 5 is to a temperature in the range 60 to 120"C.

4. A method of separating L-tryptophan from a reaction mixture substantially as herein described in any one of the Examples.