NL8304497A - METHOD FOR SEPARATING L-TRYPTOPHANE - Google Patents

Description

* N.0. 32228 *

Method for separating L-tryptophan.

The invention relates to a process for isolating L-tryptophan from an L-tryptophan and a microorganism-containing reaction mixture obtained in the production of L-tryptophan using the microorganism.

In conversions using micro-organisms, it is necessary to separate the micro-organisms from the reaction products. A number of methods are known for removing the microorganisms from the reaction mixtures and isolating the reaction products. For example, Japanese Patent Publication 16460/1963 discloses a method in which a surfactant is added to a fermentation broth of L-glutamic acid, the mixture is heated to flocculate the microorganism and the separation is effected found by filtration after addition of diatomaceous earth; and Japanese Patent Laid-Open Publication 29996/1978 describes a method in which a reaction mixture, such as an amino acid fermentation broth, is filtered through an ultrafiltration membrane and the product is then isolated by crystallization.

However, these methods appear not to be entirely satisfactory for industrial use because, according to the manner in which the surfactant is used, the microorganism can be easily removed, but the surfactant cannot be easily separated. likely to remain in the reaction product, and the process using the ultrafiltration membrane 25 presents difficulties in washing the equipment used, due to its nature.

Therefore, the object of the invention is to provide a method for isolating L-tryptophan from a reaction mixture obtained in the production of L-tryptophan using a microorganism, the microorganism being effectively removed from the reaction mixture is removed.

The present invention has undergone extensive research to achieve this goal. Since it is well known that L-tryptophan is unstable when heated in acidic solution, | Surprisingly, it has been found that when an L-tryptophan and a microorganism-containing reaction mixture with an inorganic acid is adjusted to a pH of 2-5 and then heated, L-tryptophan is stable and the microorganism becomes modified and flocculated to a 8304497 2. <* size * that can be easily filtered off. ~ ..

Therefore, according to the present invention, a process for isolating L-tryptophan from an L-tryptophan and a microorganism-containing reaction mixture obtained in the production of L-tryptophan using the microorganism is provided the process being characterized by adjusting the reaction mixture to a pH of 2-5 with an inorganic acid and heating it, then separating the flocculated microorganism by (1) filtering the reaction mixture while L-tryptophan in completely dissolved or (2) adding an alkali to the reaction mixture to convert L-tryptophan contained therein into its alkali metal salt and then filtering this mixture, while keeping the alkali metal salt in completely dissolved state, and recovering L -tryptophan from the filtrate.

Examples of the "L-tryptophan and a microorganism-containing reaction mixture obtained from the production of L-tryptophan using the microorganism" (sometimes referred to simply as a reaction mixture), as used herein indicated are a reaction mixture obtained by reacting L-serine and indole in the presence of Escherichia coli, a reaction mixture obtained according to the above procedure using DL-serine instead of L-serine and Pseudomonas putida (MT -10182) or Pseudomonas punctata (MT-10243) together with Escherichia coli as a serine racemase; a reaction mixture obtained using anthranilic acid as a precursor in the presence of Bacillus subtilis and a reaction mixture obtained using indole, pyruvic acid and ammonia in the presence of Aerobacter aerogenes.

These L-tryptophan-containing reaction mixtures contain the microorganisms used in the dissolved or suspended state. Since the prior art has found it very difficult to separate the microorganisms from L-tryptophan, the cost of the purification step on an industrial scale is high.

When the reaction mixture contains a water-immiscible organic solvent, it is desirable to remove the organic solvent suitably, such as by liquid separation or distillation, before the process of the invention.

When the reaction mixture is heated under neutral to alkaline conditions, the microorganism in the reaction mixture 40 cannot be flocculated to a material which is in the filterable state 8 3 0 4 4 9 7 4% 3. The microorganism, on the other hand, is highly flocculated when the reaction mixture is adjusted to a pH of 2-5 after reaction with an inorganic acid and then heated (according to the method of the invention). Surprisingly, L-trypto-5 phane is not decomposed and the flocculated microorganism can be easily removed by filtration. When an alkali is added to the reaction mixture after flocculating the microorganism as described above to dissolve L-tryptophan, the flocculated microorganism is still kept in the filterable state.

The process according to the invention therefore relates to a process suitable for industrial use, in which L-tryptophan produced using a micro-organism can be efficiently separated from the reaction mixture.

Examples of inorganic acid used in the process according to the invention are sulfuric acid, hydrogen chloride and phosphoric acid. The pH of the L-tryptophan-containing reaction mixture is adjusted to 2-5, preferably 3-4 with such an inorganic acid. The reaction mixture, whose pH is adjusted, is then heated to a temperature of 60-120 ° C, preferably 80-105 ° C. By adjusting the pH and heat treatment, the microorganism is modified and flocculated to a size which can be easily filtered off, while L-tryptophan remains stable without any change.

The time of the heat treatment is not particularly limited and the heat treatment can be terminated at a time when the microorganism has flocculated in a suitable state.

To aid the dissolution of the L-tryptophan in the reaction mixture, an alcohol as a solvent can be added. Low molecular weight alcohols such as methanol, ethanol and isopropanol are preferred alcohols, with isopropanol being particularly preferred. The alcohol can be used in an amount such that its concentration in the reaction mixture is not more than 70% by weight, preferably 40-60% 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 is removed.

According to an embodiment of the method according to the invention, after the above-mentioned adjustment of the pH and the heat treatment, the flocculated microorganism is separated by filtration of the reaction mixture, which is a water. containing solution of L-tryptophan. To increase the amount of L-tryptophan recovered, this filtration 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 . Therefore, it is usually necessary to heat the reaction mixture and filter hot, or dilute the reaction mixture completely with water and filter it. From the viewpoint of the efficiency of the process, it is preferable to carry out the hot filtration immediately after the adjustment of the pH and the heat treatment. Activated carbon and / or a silicon dioxide-type filtration aid may be used in the filtration operation.

In another embodiment of the process of the invention, as another method of increasing the amount of the recovered L-tryptophan, an alkali is added to the reaction mixture after adjusting its pH to 2-5 and heating it to flocculate the microorganism. Thus, L-tryptophan is completely dissolved as its alkali salt and then the reaction mixture is filtered to yield an aqueous solution of L-tryptophan. According to the method of this embodiment, no hot filtration or dilution as in the above-described embodiment is required and the cost of the energy required for heating can be saved or an aqueous solution having a higher L-tryptophan concentration can be treated.

Therefore, this embodiment is generally advantageous for industrial scale application.

The alkali to be added to the reaction mixture subjected to pH adjustment and heat treatment can be any alkali which can form a water-soluble salt by reaction with L-tryptophan. Examples are ammonia, sodium hydroxide, potassium hydroxide, sodium hydrogen carbonate and potassium hydrogen carbonate. In the industry, ammonia is preferred because of its high solubility in the aqueous solution and its easy recovery.

The amount of the alkali is usually the amount required to neutralize the reaction mixture and convert the L-tryptophan present in the reaction mixture into its alkali salt. There are no serious drawbacks when the alkali is used in excess. However, since at the time of removing 8304497 the 9% 5 L-tryptophan crystals, the pH is adjusted to its isoelectric point with an -acid to increase the yield of the isolated L-tryptophan. 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 has cooled to 0-50 ° C, especially 5-20 ° C. When the alkali is added at higher temperatures, the L-tryptophan may undergo decomposition or racemization.

When ammonia gas is used as the alkali, it is preferable 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 produces an al-15 potassium metal salt of L-tryptophan, which dissolves very quickly.

When activated carbon and / or a silica-type filtration aid is added to the L-tryptophan-containing reaction mixture alkali salt and it is filtered in the presence of the added filtration aid, the microorganisms are easily separated and a water can be containing solution of the alkali salt of L-tryptophan.

L-tryptophan can be recovered by subjecting the obtained aqueous solution of the L-tryptophan alkali salt to a conventional crystallization method, such as neutralization.

In the following examples the invention is further elucidated.

Example I

An inoculum of platinum with Escherichia coli was inoculated into 50 ml of a culture medium of the following composition (I), 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 (I) meat extract 1.0 wt% 35 peptone 0.5 wt% yeast extract 0.1 wt% KH2PO4 0.2 wt% pH in the initial phase 7.0 40 An inoculum of platinum with Pseudomonas putida (IFO 12996) was inoculated into 50 ml of a culture medium of the following composition (II) and cultivated with shaking at 30 ° C for 20 hours. One liter of the broth was centrifuged and the cells were collected and used as a source of serine racemase.

5

Culture medium composition (II) meat extract 1.0 wt% peptone 0.5 wt%

NaCl 0.5 wt% 10 pH in the initial phase 7.0

11.3 g DL-serine, 6.0 g ammonium sulfate, 10 mg pyridoxal phosphate and 66 g water were placed in a 300 ml flask equipped with a stirrer and stirred well. Concentrated ammonia was added to the resulting aqueous solution to adjust its pH to 8.5. Then 6.8 g (solid content 1.7 g) of a wet cream-like cake of Escherichia coli and 3.4 g (solid content of 0.85 g) of a wet cream-like cake of Pseudomonas putida were suspended in water , whereby a suspension with a total volume of 20 ml was formed. The resulting suspension was added to the above aqueous solution.

After the mixture was kept at 35 ° C, 57.2 g of an 11.5 g indole-containing solution in toluene was added and the reaction was carried out at 35 ° C for 48 hours. The yield of the reaction 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 a pH of 3.5 with sulfuric acid and 3 g of activated charcoal were added. The mixture was heated at 95-98 ° C and held at this temperature for 1 hour. It was filtered hot at the same temperature while keeping L-tryptophan in the dissolved state to separate activated charcoal and the flocculated microorganism. The filtrate was concentrated until the L-tryptophan concentration was 10% by weight. It was cooled to 20 ° C and the crystals obtained were separated by filtration.

L-tryptophan crystals of 99.5% purity were isolated in 80% yield based on indole.

Example II

The same conversion as in Example I was carried out in water using Escherichia call cells (MT-10232) and Pseudomonas punctata cells (MI-10243), which were in the same manner as in Example I. grown. The reaction mixture was filtered by centrifugation to separate the L-tryptophan crystals precipitated therein and the 5 microbial cells used in the conversion *

The creamy cake was placed in water, the concentration of L-tryptophan being adjusted to 4.0 wt. Then the pH of the solution was adjusted to 4.0 with phosphoric acid. 2 g of activated charcoal and 2 g of Celite 545 (a trade name for a product of John8-10 Manville Corporation) were added and the mixture was heated at 95-98 ° C for 1 hour. It was filtered hot at the same temperature to separate activated charcoal, Celite and flocculated microbial cells. The filtrate was concentrated to an L-tryptophan concentration of 15 wt% and cooled to 20 ° C. The precipitated crystals were collected by filtration.

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

Example III

The same cream-like filter cake, which included L-tryptophan and microbial cells, as obtained in Example II was suspended in a mixture of water of isopropanol with a volume ratio of 1: 1 and the concentration of L-tryptophan was adjusted to 7 wt .Z. Concentrated hydrochloric acid was added to adjust the pH of the suspension to 3.5. Activated charcoal (3 g) was added and the mixture was heated at 80-84 ° C for 1 hour. The mixture was filtered hot at the same temperature. The filtrate was cooled to 5 ° C and the precipitated crystals were collected by filtration.

The yield of isolated L-tryptophan was 75% and the purity was 98.5%.

Example IV

The same conversion as in Example I was performed. After removing toluene, the reaction mixture was diluted with water until the L-tryptophan concentration was 1 wt.%. The pH of the reaction mixture was adjusted to 4.0 with sulfuric acid and stirred at room temperature for 2 hours to dissolve L-tryptophan. Activated charcoal (3 g) and 3 g Standard Supercell (a trade name for a product of Johns-Manville Corporation) as a filter aid were added and the solution filtered at room temperature. The filtrate was concentrated to L-tryptophan concentration 10 wt. Z was 40 and then cooled to 5 ° C. The precipitated cells were 830 ^ 497

a

»9 8 collected by filtration *

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

Example V

In the same manner as in Example 1, indole and DL-serine were converted into a toluene solution. * The yield of the reaction 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 95-98 ° C for 1 hour.

After cooling to room temperature, ammonia gas was blown into the reaction mixture to dissolve the L-tryptophan contained therein as its ammonium salt. Activated carbon in an amount of 10 wt%, based on L-tryptophan and Celite 545 (a trade name for a product of Johns-Manville Corporation) in an amount of 10 wt%, based on L-tryptophan, were added to the solution added and the solution was filtered. The microorganism was separated together with Celite 545 and activated charcoal. The filtrate was heated to 100 ° C to remove ammonia, and the concentration of L-tryptophan was adjusted to 10 wt% 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 isolated L-tryptophan was 75% based on the tryptophan obtained, and the purity measured by liquid chromatography was 98.5%.

Example VI

The same L-tryptophan-containing reaction mixture as obtained in Example V was centrifuged and a mixture of L-tryptophan crystals and the microorganisms was obtained as a crime-like cake. The creamy cake was placed in water to form a slurry with an L-tryptophan concentration of 30% by weight. The pH of the slurry was adjusted to 3.5 and the slurry was heated at 95-98 ° 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. 20% by weight of activated charcoal based on L-tryptophan was added to the solution and the flocculated microbial cells were separated at room temperature. Nitrogen gas was blown into the filtrate at elevated temperature to remove ammonia. After cooling to 5 ° C, the precipitated 40 L-tryptophan was separated by a centrifugal dewaterer 83% 4 9 7%.

The yield of isolated L-tryptophan was. 88%, based on the tryptophan obtained, and its purity was 98.0%.

Example VII

A 12% by weight aqueous solution of L-tryptophan, obtained in the same manner as in Example V, was adjusted to pH 4.0 with phosphoric acid and heated to 95-98 ° C. After cooling to 25 ° C, a 20% strength aqueous sodium hydroxide solution was added to adjust the pH of the solution to 10. Asm. the aqueous L-tryptophan solution containing 10% by weight of activated charcoal based on tryptophan and 10% by weight of Standard Supercell (a trade name for a product of Johns-Manville Corporation) based on tryptophan were added as a filter aid and the solution was filtered at 20 ° C. The filtrate was neutralized to a pH of 15 with acetic acid and the precipitated crystals of L-tryptophan were separated by filtration and dried. The yield of isolated L-tryptophan was 75% based on the tryptophan obtained, and its purity was 99.2%.

Example VIII

A crime-like cake of L-tryptophan, which was obtained in the same manner as in Example V, was dispersed in a mixture of warmer and isopropanol in a volume ratio of 1: 1, whereby a suspension with an L-tryptophan concentration of 20 wt% was formed. The slurry was adjusted to pH 3.5 with sulfuric acid and then heated at 80-84 ° 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, 20% by weight of activated charcoal based on tryptophan was added and the solution was filtered under suction to give a light yellow clear L-tryptophan solution. Nitrogen gas was blown into the solution with heating to remove ammonia. The solution was cooled and the precipitated L-tryptophan crystals were separated by filtration and dried.

The yield of isolated L-tryptophan was 83% based on the tryptophan obtained, and its purity was 98.8%.

8304497 _ - ___— ^

Claims (2)

1. A method for separating L-tryptophan from an L-tryptophan and an antimony-containing reaction mixture obtained in the production of L-tryptophan using the microorganism, characterized in that: adjusting the reaction mixture with inorganic acid to a pH of 2-5, heating it and then filtering the heated reaction mixture to remove the microorganism. 2. A method of separating L-tryptophan from an L-tryptophan and a microorganism-containing reaction mixture obtained in the production of L-tryptophan using the microorganism, characterized adjusting the reaction mixture with inorganic acid to a pH of 2-5, heating it, adding an alkali to the heated reaction mixture to convert the L-tryptophan contained therein into its alkali metal salt and then filtering the reaction mixture to microorganism. 11I II l-l 8304497