JPH0435156B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for isolating L-tryptophan from a reaction solution of L-tryptophan produced using a microorganism.

In reaction methods using microorganisms, it is necessary to separate the reaction product from the microorganism, and several methods have been known to remove the microorganism from the reaction solution and isolate the reaction product. There is. For example, a method in which a surfactant is added to an L-glitamic acid fermentation solution and heated to facilitate coagulation, and diatomaceous earth is added to separate the solution (Japanese Patent Application Laid-Open No. 16460/1973), an amino acid fermentation solution, etc. is subjected to ultrafiltration membrane. There is a method of crystallization isolation after passing through a filter (Japanese Patent Application Laid-open No. 53-29996).

However, these methods are still far from being industrially fully satisfactory. That is, in the method of adding a surfactant, although microorganisms can be easily removed, the surfactant cannot be easily separated from the reaction product, and there is a possibility that the surfactant may be mixed into the product. Furthermore, the method using an ultrafiltration membrane has difficulties in cleaning due to the nature of the device, and cannot be considered an industrial isolation method.

The inventors of the present invention have conducted intensive studies to solve these problems regarding the isolation method of L-tryptophan. As a result, L-tryptophan, which was thought to be unstable when heated in an acidic solution, has surprisingly been found to be PH of L-tryptophan reaction solution containing microorganisms with mineral acid
It is stable even when heated after being made into an acidic solution of 2 to 5,
At the same time, it was discovered that the microorganisms used in the reaction denatured and aggregated to a size that could be easily separated, and that the microorganisms could be easily removed and L-tryptophan isolated by passing the microorganisms under the solubility of L-tryptophan. I have completed the method.

That is, the present invention provides a method for producing L-tryptophan using microorganisms, in which a reaction solution containing L-tryptophan and microorganisms is treated with a mineral acid.
After adjusting the pH to 3 to 4 and heat-treating this reaction solution, L
- A method for separating L-tryptophan, characterized by separating L-tryptophan based on its solubility or lower.

The L-tryptophan reaction solution produced using microorganisms to which the method of the present invention is applied includes, for example,
synthesized from L-serine and indole in the presence of Escherichia coli;
- one synthesized using DL-serine instead of serine in combination with Pseudomonas puteida (MT-10182) or Pseudomonas punctata (MT-10243) as a serine racemase;
In addition, in the presence of Bacillus subtilis, it is synthesized from anthranilic acid, and in the presence of Aerobacter aerogenes, indole and pyruvic acid,
There are some that are synthesized from ammonia.

L-tryptophan synthesized by these production methods exists in an aqueous solution in which microorganisms used in the reaction are suspended, and it has been extremely difficult to separate the microorganisms and L-tryptophan in the industrial purification process. The costs involved were significant.

Further, in these methods, an organic solvent may be used in combination as a reaction medium. In the case of such a reaction method, the organic solvent is removed by appropriate means such as separation or distillation prior to application to the method of the present invention.

The acids used in the method of the invention include sulfuric acid,
Mineral acids such as hydrochloric acid and phosphoric acid. Using these mineral acids, adjust the pH of the L-tryptophan reaction solution to 2 to 5.
Adjust to ~4.

This pH-adjusted L-tryptophan reaction solution is heated to 60 to 120°C, preferably 80 to 105°C.
It will be carried out.

Through this pH adjustment and heat treatment, L-tryptophan does not change and exists stably.
On the other hand, microorganisms denature and aggregate to a size that can be easily separated.

Therefore, the heat treatment time is not particularly limited, and the heat treatment may be terminated when the microorganisms are appropriately aggregated.

In the method of the present invention, alcohol may be added as a solvent to promote dissolution of L-tryptophan in the reaction solution. As the alcohol, lower alcohols such as methanol, ethanol, and isopropanol are preferred, particularly isopropanol.
These alcohols may be used at an alcohol concentration of 70 wt% or less, preferably 40 to 60 wt% in the reaction solution.

When alcohol is used in combination, the method of the present invention is carried out in a water-alcohol mixed solvent, so when an L-tryptophan-containing reaction solution and an organic solvent are used in the reaction, the L-tryptophan-containing reaction solution is removed. The method of the invention is carried out by adding the required amount of alcohol to the liquid.

In the method of the present invention, after the above treatment, the aggregated microorganisms and the aqueous L-tryptophan solution are separated. In order to increase the acquisition rate of L-tryptophan, this separate operation is performed in a state where L-tryptophan in the reaction solution is dissolved, that is, when the concentration of L-tryptophan in the solution is below the solubility. Therefore, the L-tryptophan solution is usually heated or sufficiently diluted and separated. Generally, from the viewpoint of work efficiency, it is preferable to heat immediately after pH adjustment and heat treatment.

Alternatively, activated carbon or silica-based super-aids may be used alone or in combination.

The present invention will be explained below with reference to Examples.

Example 1 One platinum loop of Escherichia coli MT-10242 was inoculated into 50 ml of a medium having the following medium composition, and cultured with shaking at 30°C for 20 hours.

Medium composition Meat extract 1.0wt% Peptone 0.5wt% Yeast extract 0.1wt% KH ₂ PO ₄ 0.2wt% Initial pH 7.0 Culture solution 1 was centrifuged to collect bacterial cells, which were used as an enzyme source for tryptophan synthetase.

One platinum loop of Pseudomonas putida IFO12996 was inoculated into 50 ml of the medium composition, and cultured with shaking at 30°C for 20 hours. Culture solution 1 was centrifuged to collect bacterial cells, which were used as an enzyme source for serine racemase.

Medium composition Meat extract 1.0wt% Peptone 0.5wt% NaCl 0.5wt% Initial PH 7.0 DL-Serine in a 300ml flask equipped with a stirrer
Add 11.3g, 6g of ammonium sulfate, 10mg of pyridoxal phosphate and 66mg of water and stir well. Adjust the pH to 8.5 with concentrated ammonia water, suspend 6.8 g of E. coli wet mass (1.7 g of solid content) and 3.4 g of wet mass of Pseudomonas putida (0.85 g of solid content) in water to make the total volume 20 ml. Add.

After keeping the temperature at 35°C, 57.2g of a toluene solution containing 11.5g of indole was added and reacted at 35°C for 48 hours. The reaction yield was quantitative. Toluene was removed by distillation, water was added to make the total amount 450g,
Adjust the pH to 3.5 with sulfuric acid, add 3 g of activated carbon, and bring to 95
Heat to ~98°C. The mixture was kept warm for 1 hour, and hot filtration was performed at the same temperature in a state in which L-tryptophan was dissolved. The time required for filtration was 4 minutes. Concentrate this solution to make the L-tryptophan concentration 10 wt%. Cool to 20°C and separate the crystals that have crystallized. Crystals of L-tryptophan with a purity of 99.5% were obtained in an isolated yield of 80% (vs. indole).

Example 2 Escherichia spp. cultured in the same manner as in Example 1.
A reaction was carried out in the same manner as in Example 1 using an aqueous solution using cultured cells of coli (MT-10232) and cultured cells of Pseudomonas punctata (MT-10243). The reaction solution is centrifuged to separate the L-tryptophan crystals precipitated in the reaction solution and the bacterial cells used in the reaction.

The reaction mass is drained into water and the L-tryptophan concentration is adjusted to 4.0 wt%. Next, use phosphoric acid to pH
4.0, add 2 g of activated carbon and 2 g of Celite 545 (manufactured by John Manville), and heat at 95 to 98° C. for 1 hour. Heat aging is performed at the same temperature to separate activated carbon, celite, and aggregated bacterial cells. Concentrate this solution to reduce the L-tryptophan concentration to 15wt%.
Then, cool to 20°C and filter the precipitated crystals. L
-Isolated yield of tryptophan is 87%, purity is 99.7
It was %.

Example 3 A mass consisting of L-tryptophan and bacterial cells obtained in the same manner as in Example 2 was suspended in a solution of water and isopropyl alcohol mixed at a volume ratio of 1:1, and the L-tryptophan concentration was adjusted to 7 wt%. do. Add concentrated hydrochloric acid to adjust the pH to 3.5, add 3 g of activated carbon, and heat at 80-84°C for 1 hour. After heating at the same temperature, the liquid was cooled to 5°C and the precipitated crystals were filtered. The yield of L-tryptophan isolation was 75% and the purity was 98.5%.

Example 4 A reaction was carried out in the same manner as in Example 1, and the reaction solution from which toluene was removed was diluted with water to give an L-tryptophan concentration of 1 wt%. Adjust the pH to 4.0 with sulfuric acid and stir at room temperature for 2 hours to dissolve L-tryptophan. Add 3 g of activated charcoal and 3 g of Standard Supercel as a supernatant and filter at room temperature. Concentrate the liquid to reduce the concentration of L-tryptophan.
10wt% and cooled to 5℃. Filter the precipitated crystals. The isolated yield of L-tryptophan is 79
%, and the purity was 98.8%.

Comparative Example 1 Crystals of L-tryptophan were obtained in exactly the same manner as in Example 1, except that the pH of the reaction solution was adjusted to 4.5 with sulfuric acid. Note that the time required for filtration was 35 minutes. Tryptophan purity is 99.5%, yield is 78%
It was hot.

Claims (1)

[Claims] 1. In a method for producing L-tryptophan using microorganisms, a reaction solution containing L-tryptophan and microorganisms is adjusted to pH 3 to 4 with mineral acid, then heated, and then filtered at a temperature below the solubility of L-tryptophan. A method for separating L-tryptophan, characterized in that: