DE3400603A1 - METHOD FOR SEPARATING L-TRYPTOPHANE - Google Patents

Description

Case 2131-K73

METHOD FOR SEPARATING L-TRYPTOPHANE

Description

The invention relates to a method for isolating L-tryptophan from a reaction mixture containing L-tryptophan and a microorganism that is used in the production of L-tryptophan using the microorganism.

For reactions using microorganisms, it is necessary to to separate the microorganisms from the reaction products. So far, some methods have been used to remove the microorganisms known from reaction mixtures and for isolating the reaction products. For example, the Japanese Patent Publication No. 16460/1963 a method that the Adding a surfactant to an L-glutamic acid fermentation broth, heating the mixture to flocculate and settle the microorganism and separate it by filtration after adding diatomaceous earth, and Japanese Patent Laid-Open No. 29996/1978 discloses a Process which involves the filtration of a reaction mixture, such as an amino acid fermentation broth, on an ultrafiltration membrane and then isolating the product by crystallization.

However, these methods have not proven to be entirely satisfactory for industrial practice, since the method using the Addition of the surfactant includes the microorganism can be easily removed, but the surfactant Agent cannot be separated in a simple manner and tends to remain in the reaction product, and the process which includes the use of the ultrafiltration membrane, natural Has difficulty washing the equipment used.

The aim of the invention is therefore to provide a process for the isolation of L-tryptophan from a reaction mixture which is involved in the formation of L-tryptophan obtained using a microorganism capable of effective removal of the Microorganism from the reaction mixture.

The inventors made extensive studies in order to achieve this object. While it is common knowledge that L-tryptophan is unstable when heated in an acidic solution, these investigations led to the discovery of the surprising Facts that when setting a L-tryptophan and a reaction mixture containing a microorganism to pH 2 to 5 with a mineral acid and subsequent heating, L-tryptophan is stable and the microorganism is modified and flocculated to a size in which it can be easily filtered off can.

Thus, the invention provides a method for isolating L-tryptophan from an L-tryptophan and a microorganism containing reaction mixture obtained in the production of L-tryptophan using the microorganism, created the adjustment of the reaction mixture with a mineral acid to pH 2 to 5 and its heating, the subsequent Separate the flocculated microorganism by either (1) filtering the reaction mixture while L-tryptophan is in completely dissolved state is maintained, or (2) adding an alkali to it

the reaction mixture / in order to convert the L-tryptophan contained therein into its alkali salt, and the subsequent filtration the mixture, while the alkali salt is kept in a completely dissolved state / and the extraction of L-tryptophan from comprises the filtrate.

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

These L-tryptophan-containing reaction mixtures contain the microorganisms used in dissolved or suspended form State. In the prior art, since it was very difficult to separate the microorganisms from L-tryptophan, the cost was the cleaning level in industrial practice is high.

If the reaction mixture contains a water-immiscible organic solvent, it is desirable to use the organic solvent Solvent prior to the application of the method according to the invention with the help of suitable means, such as liquid separation or Liquid separation or distillation, to remove.

If the reaction mixture is kept under neutral to alkaline conditions

heated conditions, the microorganism in the reaction mixture cannot be flocculated to a filterable state. In contrast, if the reaction mixture according to the process according to the invention after the reaction with a Mineral acid is adjusted to pH 2 to 5 and then heated, the microorganism is very easily flocculated. Unexpectedly L-tryptophan is not decomposed at this point and the flocculated microorganism can be removed by filtration. Used after flocculation of the microorganism in the above Way, an alkali is added to the reaction mixture to dissolve L-tryptophan, the flocculated microorganism becomes still kept in a filterable state. Accordingly, the process according to the invention represents an industrial method with With the aid of which L-tryptophan produced using a microorganism is effectively separated from the reaction mixture can be.

Examples of the mineral acid used in the method of the present 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 of the reaction mixture which has been set is then heated ^{to a temperature of 60 to 120 °} C., preferably 80 to 105 ^{° C.} Through this pH adjustment and heat treatment, the microorganism is modified and flocculated to a size in which it can easily be filtered off, whereas L-tryptophan remains stable without any change.

Therefore, the heat treatment time is not particularly limited and the heat treatment can be carried out at the time to which the microorganism is flocculated in a suitable state.

You can add an alcohol as a solvent to dissolve it to promote the L-tryptophan in the reaction mixture.

Lower alcohols such as methanol, ethanol and isopropanol are preferred as alcohol, with isopropanol being particularly preferred is. The alcohol can be used in such an amount that its concentration in the reaction mixture does not is more than 70% by weight, preferably 40 to 60% by weight. If the reaction mixture contains something immiscible with water organic solvent, a predetermined amount of alcohol is added after that which is immiscible with water organic solvent has been removed.

According to one embodiment of the method according to the invention After the above pH adjustment and heat treatment, the flocculated microorganism becomes out of the reaction mixture separated by filtration to obtain an aqueous solution of L-tryptophan. To the ratio or the proportion of To increase recovered L-tryptophan, this filtration process is carried out while L-tryptophan is in the reaction mixture in a completely dissolved state, namely while the concentration of L-tryptophan in the reaction mixture is below its solubility. It is common therefore, it is necessary to heat the reaction mixture and subject it to hot filtration; or the reaction mixture to be completely diluted with water and filtered. In view of process efficiency, it is preferable to immediately perform hot filtration after pH adjustment and heat treatment. When carrying out the filtration can be activated Carbon or a silica-type filtration aid or both can be used.

According to another embodiment of the inventive method the ratio or proportion of recovered L-tryptophan alkali to the reaction mixture after adjustment of its pH is ¹ to S 2 to 5 and their heat added to the flocculation of the microorganism as an additional method to increase. Thus, the L-tryptophan is substantially completely dissolved in the form of its alkali salt, and then the reaction mixture is filtered off to obtain an aqueous L-tryptophan solution. Ge

The measure of this embodiment is the hot filtration or the dilution as in the previous embodiment, not necessary and thermal energy costs can be saved or an aqueous solution with a higher L-tryptophan concentration can be used be handled. Accordingly, this embodiment is for industrial practice generally beneficial.

The alkali to be added to the reaction mixture which has been subjected to the pH adjustment and the heat treatment be any alkali that 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. Ammonia is industrial because of it high solubility in the aqueous solution and the ease of its recovery are preferred.

The amount of alkali is usually that required to neutralize the reaction mixture and that in the reaction mixture convert the L-tryptophan present into its alkali salt. There is no serious disadvantage even if the alkali is used in excess. However, since at the time of taking out the L-tryptophan crystals, the pH with an acid adjusted to its isoelectric point to increase the yield of isolated L-tryptophan leads to the use an excess of alkali undesirably leads to an increase in the amount of inorganic salts.

Preferably, the alkali is added after the heat-treated reaction mixture to 0 to 5O ⁰ C, in particular 5 cooled to 20 ⁰ C is. If alkali is added at higher temperatures, the L-tryptophan can suffer from decomposition or racemization.

If gaseous ammonia is used as the alkali, it is preferred to blow cooled ammonia gas into the reaction mixture in order to increase the solubility of the ammonia in the reaction mixture to increase. If an inorganic base, such as sodium hydroxide,

is used, its addition at room temperature leads to the formation of an alkali salt of L-tryptophan, which dissolves very quickly.

Becomes activated carbon and / or a filtration aid of the SiliciumclicKidrl! Vjp or silica type. zu.der Beaktionsmischunq, .. die.Alkalisals of the L-tryptophan is added and if it is filtered in the presence of the added filter aid, the Microorganism is easily separated and an aqueous solution of the alkali salt of L-tryptophan can be obtained.

L-tryptophan can be obtained by adding the resulting aqueous solution of the alkali salt of L-tryptophan to a usual Crystallization method such as neutralization.

The following examples illustrate the invention.

example 1

A platinum loop of Escherichia coli was dissolved in 50 ml of a culture medium having the following composition (I) and cultured with shaking for 20 hours at 3O ⁰ C. One liter of culture broth was centrifuged and the cells were collected and used as a source of tryptophan synthetase.

Meat extract 1.0% by weight

Peptone 0.5% by weight

Yeast extract 0.1% by weight

KH ₂ PO ₄ 0.2% by weight

pH in the initial stage 7.0

A platinum loop of Pseudomonas putida (IFO 12996) was inoculated in 50 ml of culture medium having the following composition (II) and cultured for 20 hours with shaking at 3O ⁰ C. One liter of culture broth was centrifuged and the cells were collected and used as a source of serine racemase.

3A00603

Z ^ usmensetz ___ (II)

Meat extract 1.0% by weight

Peptone 0.5% by weight

NaCl 0.5% by weight

Initial pH 7.0

A 300 ml flask equipped with a stirrer became charged with 11.3 g of DL-serine, 6 g of ammonium sulfate, 10 mg of pyridoxal phosphate and 66 g of water and then stirred well. One gave concentrated aqueous ammonia to the resulting aqueous solution to adjust its pH to 8.5. This gave 6.8 g (solid content 1.7 g) of a moist creamy cake of Escherichia coli and 3.4 g (solids content 0.85 g) of a moist one creamy cake of Pseudomonas putida suspended in water to form a suspension with a total volume of 20 ml. The resulting suspension was added to the above aqueous solution.

After the mixture was kept at 35 ⁰ C, was added 57.2 g of a 11.5 g of indole-containing toluene solution and sat for 48 hours at 35 ⁰ C. 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 98 ^° C. and kept at this temperature for one hour. It was filtered hot at the same temperature while keeping L-tryptophan in a dissolved state to separate activated carbon and the flocculated microorganism. The filtrate was concentrated to an L-tryptophan concentration of 10% by weight. It was ^{cooled to 20 °} C. and the crystals formed were separated off by filtration.

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

Example 2

The same reaction was carried out as in Example 1 in water and using cells from Escherichia coli (MT-10232) and Cells of Pseudomonas punctata (MT-10243), which were carried out in in the same manner as in Example 1. The reaction mixture was filtered by centrifugation to obtain the to separate the precipitated L-tryptophan crystals and the microbial cells used in the reaction.

The creamy cake was taken out in water to adjust the L-tryptophan concentration to 4.0% by weight. The pH of the solution was then adjusted to 4.0 with phosphoric acid. 2 g of activated carbon and 2 g of Celite 545 (trade name for a product of Johns-Manville Corporation) were added, and the mixture was heated at 95 to 98 ° C. for one hour. It was filtered hot 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 20 ° C.} The crystal precipitated was collected by filtration.

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

Example 3

The same creamy 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 increased to 7 Wt .-% adjusted. Concentrated hydrochloric acid was added to adjust the pH of the suspension to 3.5. 3 g of activated carbon were added and the mixture was heated to 80 to 84 ° C. for 1 hour. The mixture was filtered hot at the same temperature and the filtrate was cooled ^{to 5 ° C}

and the precipitated crystals were collected by filtration.

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

Example 4

The same reaction as in Example 1 was carried out. After removing the 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. 3 g of activated carbon and 3 g of Standard Supercell (trade name for a product of Johns-Manville Corporation) were added as a filtration aid, 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 isolated yield of L-tryptophan was 79% and its purity was 98.8%.

Example 5

In the same manner as in Example 1, indole and DL-serine implemented in a toluene solution. The reaction yield was quantitative. Toluene was drained from the reaction mixture Distillation removed.

The resulting reaction mixture containing L-tryptophan was adjusted to pH 4.0 with sulfuric acid and ^{heated to 95 to 98 °} C. for one hour. After cooling to room temperature, ammonia gas was blown into the reaction mixture to dissolve L-tryptophan contained therein in the form of its ammonium salt. Activated carbon was added in an amount of

% By weight based on L-tryptophan and Celite 545 (trade name for a product of Johns-Manville Corporation) in an amount of 10% by weight based on L-tryptophan was added to the solution, and the solution was filtered. The microorganism was separated along with Celite 545 and activated carbon. The filtrate was ^{heated to 100 °} C. to remove ammonia and the concentration of the L-tryptophan was adjusted to 10% by weight by adding water. The solution was cooled to 2O ⁰ C and the precipitated crystals separated by filtration / washed with water and dried.

The isolated yield of L-tryptophan was 75% based on the resulting tryptophan and its purity, measured by liquid chromatography, were "98.5%.

Example 6

The same L-tryptophan-containing reaction mixture as obtained in Example 5 was centrifuged to obtain a mixture of L-tryptophan crystals and the microorganisms in the form of a creamy cake. The creamy cake was taken out in 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 for 2 hours at 95 to 98 ⁰ C, the microorganisms used in the reaction to flocculate. After cooling to room temperature, aqueous ammonia was added to dissolve L-tryptophan in the reaction mixture in the form of 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 off at room temperature. Gaseous nitrogen was blown into the filtrate at an elevated temperature to remove ammonia. After cooling to 5 ° C., precipitated L-tryptophan was separated off by a centrifugal dehydrator.

The isolated yield of L-tryptophan was 88.0% by weight

on the resulting tryptophan and its purity was 98.0%.

Example 7

A t2 wt .-% Ige. aqueous L-tryptophan solution was obtained in the same manner as in Example 5, and was adjusted with phosphoric acid to pH 4.0 heated at 95 to 98 ⁰ C. After cooling to 25 ° C., a 20% aqueous sodium hydroxide solution was added in order to adjust the pH of the solution to 10. To the aqueous L-tryptophan solution, 10% by weight based on tryptophan of activated carbon, 10% by weight based on tryptophan of Standard Supercell (trade name for a product of Johns-Manville Corporation) was added as a filtration aid, and the solution was added at 20 ⁰ C filtered. The filtrate was neutralized to pH 6 with acetic acid, and the precipitated L-tryptophan crystals were separated by filtration and dried. The isolated yield of L-tryptophan was 75% based on the resultant tryptophan, and its purity was 99.2%. "

Example 8

A creamy cake of L-tryptophan as obtained 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 heated to 80-84 ° C for 2 hours. After cooling to 5 ^° C., gaseous ammonia was blown into the reaction mixture in order to convert L-tryptophan into the solvent in the form of its
Dissolve ammonium salt. To the solution was added 20% by weight of activated carbon based on tryptophan, and the solution was suction-filtered to give a pale yellow clear L-tryptophan solution. Gaseous nitrogen was bubbled 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 isolated yield of L-tryptophan was 83% based on the resultant tryptophan, and its purity was 98.8%.

The microorganisms Pseudomonas putida (ΜΤ-1Ό182), Pseudomonas punctata (MT-10243) and Escherichia coli (MT-10232) in the Fermentation Research Institute, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, Japan, and the microorganism Pseudomonas putida (IFO-12996) was found in the Institute of Fermentation, Osaka, Japan, deposited.

Claims (2)

Dr. F. Zumstein Sr. - Dr. E. Assmann - Dr. R. Koenigsberger Dipl.-Ing. F. Klingseisen - Dr. F. Zumstein jun. q / Π fi C. Π ¹ ? PATENT LAWYERS APPROVED REPRESENTATIVES AT THE EUROPEAN PATENT OFFICE REPRESENTATIVES BEFORE THE EUROPEAN PATENT OFFICE Case 2131-K73 PATENT CLAIMS A process for separating L-tryptophan from L-tryptophan and a microorganism reaction mixture containing _r which is obtained in the production of L-tryptophan using the microorganism / characterized in that adjusting the reaction mixture with a mineral acid to a pH of 2 to 5 / it is heated and then the heated reaction mixture is filtered / in order to remove the microorganism. 2. Process for the separation of L-tryptophan from a reaction mixture containing L-tryptophan and a microorganism which is used in the production of L-tryptophan the microorganism is obtained, characterized in that the reaction mixture with a mineral acid Adjusts the pH to 2 to 5, heats it, adds an alkali to the heated reaction mixture to add the L-tryptophan contained therein to be converted into its alkali metal salt, and thereafter the Reaction mixture filtered to remove the microorganism.