JPH0437718B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention uses a microorganism belonging to the genus Bacillus that has resistance to 5-fluorotryptophan, an anthranilic acid requirement, and has the ability to produce 5-hydroxy-L-tryptophan. 5-hydroxy-L-
Concerning a method for producing tryptophan.

5-Hydroxy-L-tryptophan is a precursor of serotonin, which is known as a physiologically active amine, and is an important amino acid expected to be used as a medicine for treating clonic convulsions.

Prior art and problems to be solved by the invention As a method for producing 5-hydroxy-L-tryptophan, 5-hydroxy-L-tryptophan is produced from 5-hydroxyindole, pyruvic acid and ammonia.
-Enzymatic methods using microorganisms capable of producing tryptophan have been known, but their conversion rates and yields are not necessarily sufficient, and it cannot be said to be a satisfactory industrial production method. .

Means for Solving the Problems In view of the current state of the conventional microbiological production method of 5-hydroxy-L-tryptophan described above, the present inventors have developed a method for producing 5-hydroxy-L-tryptophan at a high concentration in a medium containing a high concentration of 5-hydroxy-L-tryptophan. 5-hydroxy-L-
As a result of intensive research to obtain a microorganism capable of producing tryptophan culture solution, we succeeded in obtaining a 5-hydroxy-L-tryptophan-producing microorganism that is resistant to 5-fluorotryptophan and requires anthranilic acid. This led to the present invention.

That is, according to the present invention, Bacillus amyloliquifaciens SD- has 5-fluorotryptophan resistance and anthranilic acid requirement and has the ability to produce 5-hydroxy-L-tryptophan.
5-hydroxy-L-tryptophan, which consists of culturing 53 in an anthranilic acid-containing nutrient medium by adding 5-hydroxyanthranilic acid or a salt thereof, and collecting 5-hydroxy-L-tryptophan produced in the culture. A manufacturing method is provided. According to this method, 5-hydroxy-L-tryptophan can be accumulated in the culture solution at a high concentration and at a high conversion rate, with almost no accumulation of L-tryptophan in the culture solution. That is, the desired 5-hydroxy-L is added to the culture solution.
- When L-tryptophan accumulates together with tryptophan, it is difficult to separate 5-hydroxy-L-tryptophan and L-tryptophan from the culture solution, and some of the produced 5-hydroxy-L-tryptophan is discarded without being separated. Considering that this is extremely disadvantageous in terms of cost, it is practically desirable that L-tryptophan does not accumulate in the culture solution.

The microorganism Bacillus amyloliquifaciens SD-53 belonging to the genus Bacillus having resistance to 5-fluorotryptophan and anthranilic acid requirement and ability to produce 5-hydroxy-L-tryptophan according to the present invention is as follows. That's how I got it.

That is, the present inventors used the original strain Bacillus amyloliquifaciens.
amyloliquefaciens) IAM1521 according to the conventional method
-Artificial mutagenesis treatment using methyl-N'-nitro-N-nitrosoguanidine (NTG),
This mutant-treated strain was grown on an agar plate medium (spizaizen medium) supplemented with 5-fluorotryptophan (5-FT) at a minimum inhibitory concentration (MIC) or higher.
- The productivity of hydroxy-L-tryptophan is investigated to obtain a 5-FT-resistant strain (that is, the microorganism's inherent feedback suppression mechanism due to accumulation of tryptophan concentration has been released) that has the highest 5-hydroxy-L-tryptophan production ability. This strain is 5-
Resistant to fluorotryptophan of over 5000ppm.

Next, this 5-FT resistance and 5-hydroxy-
A mutant strain capable of producing L-tryptophan was again subjected to artificial mutagenesis treatment using NTG in the same manner as above, and after treatment, centrifugation (10000G x 5 minutes)
Then, suspend the bacterial cells in M/100 Tris buffer (PH7.0) and centrifuge them three times to remove NTG. The bacterial strain is cultured for 1 to 3 hours at 30 to 40°C in a minimal medium for Bacillus subtilis, such as Spizaizen medium, containing 20 to 50 ppm of anthranilic acid.

After culturing, the bacteria are collected by centrifugation, the bacterial cells are washed with the above Tris buffer, and then resuspended in Tris buffer and shaken at 30 to 40°C for 1 to 3 hours. After shaking, the bacteria were collected by centrifugation, washed with Tris buffer, and suspended in minimal medium for Bacillus subtilis containing 500 to 2000 ppm of penicillin to concentrate the anthranilic acid auxotrophic bacterial strain. ~30~ at ~40℃
Shake for 90 minutes, preferably 40-60 minutes. After shaking, the bacteria were collected and washed in the same manner as above, and the same operation was repeated 3 to 10 times on the minimal medium for Bacillus subtilis containing penicillin. , to screen for anthranilic acid auxotrophic bacterial strains that do not grow on anthranilic acid-free plate medium. 90 under optimal conditions
% or more.

The thus obtained bacterial strain Bacillus amyloliquifaciens has 5-fluorotryptophan resistance and anthranilic acid requirement and has the highest 5-hydroxy-L-tryptophan production ability.
I chose SD-53.

This strain was identified as Bacillus amyloliquefaciens SD-
53 (Fiber Science and Technology Research Institute No. 7773). In addition,
The mycological properties of this strain are that it has 5-FT resistance and anthranilic acid requirement compared to the original strain Bacillus amyloliquifaciens IAM1521.
The mycological properties were the same as those of the original strain, except for a high L-tryptophan production ability.

According to the present invention, 5-hydroxy-L-tryptophan is produced by culturing Bacillus amyloliquifaciens SD-53 in a medium supplemented with 5-hydroxyanthranilic acid or a salt thereof. do. There is no particular limitation on the concentration of 5-hydroxyanthranilic acid or its salt in the nutrient medium, but it is generally 0.1 to 1000 mg/, preferably from the viewpoint of the desired yield of 5-hydroxy-L-tryptophan, culture conditions, and economics. Culture is carried out while maintaining a concentration of 50 to 300 mg/. In addition, the aforementioned bacterial strain SD
-53 is anthranilic acid auxotrophic, so the minimum amount required for bacterial growth (e.g. 30-300 ppm in the medium)
of anthranilic acid must be included in the medium.

The medium that can be used in the method of the present invention may be any medium as long as the microorganisms can be grown by culturing.For example, carbon sources include glucose, molasses, sucrose, starch, starch saccharification solution, Saccharides such as cellulose decomposition products, acetic acid, ethanol, etc. are used. As the nitrogen source, ammonium salts such as ammonia, ammonium sulfate, ammonium chloride, ammonium nitrate, ammonium phosphorus, urea, nitrates, etc. are used as appropriate. Examples of inorganic salts include salts of phosphoric acid, potassium, magnesium, manganese, etc., such as ammonium phosphate, potassium phosphate,
Usual industrial chemicals such as sodium phosphate, magnesium sulfate, ferrous sulfate, manganese sulfate, and caustic potassium may be used, and trace elements such as salts such as calcium, zinc, boron, copper, cobalt, and molybdenum may also be added. In addition, trace organic nutrients such as vitamins, amino acids, and nucleic acid-related substances are not particularly required for the growth of bacteria, but they can be added to
Corn steep liquor, meat extract, yeast extract,
Organic substances such as peptone may be added. 5-hydroxyanthranilic acid as it is or with methanol,
Although it may be used in the form of a solution in an organic solvent such as ethanol or acetic acid, it is preferably used as an aqueous solution of an alkali salt such as sodium, potassium, or ammonium or an acid salt such as sulfuric acid, hydrochloric acid, or acetic acid.

Culture in the method of the present invention can be carried out under aerobic conditions, for example, by aerated stirring or reciprocating shaking. Culture conditions are not particularly limited, but generally speaking, temperature is 30-40℃, pH 6.0-8.0 and 18
It will be carried out for about 72 hours.

The desired 5-hydroxy-L-tryptophan can be collected from the culture solution or culture according to a conventional method. For example, the culture supernatant after removing bacterial cells by centrifugation may be treated with ion exchange resin, activated carbon, etc., as appropriate.
-Hydroxy-L-tryptophan can be isolated and crystallized.

Examples Examples of the present invention will be described below, but it goes without saying that the scope of the present invention is not limited to these examples.

Example 1 (Production example of 5-hydroxy-L-tryptophan) Glucose 10%, Ammonium sulfate 0.2%, Na ₂ SO ₄ 0.1%,
KH ₂ PO ₄ 0.5%, MgSO ₄・4~6H ₂ O 0.05%, Fe ^+
+ 1ppm, Mn ⁺⁺ 1ppm and anthranilic acid 200ppm
Liquid medium 2 having the following composition was placed in a 5-jar fermenter and sterilized by heating at 115°C for 20 minutes.

In this culture tank, pre-incubate in bouillon medium at 30℃.
Bacillus amyloliquifaciens SD-53 cultured for 12 hours with shaking was inoculated at 5%, and the amount of dissolved oxygen was reduced.
Aerated agitation culture was performed at 35°C while maintaining the concentration above 0.5 ppm. For culturing, 10% 5-hydroxyanthranilic acid ammonium salt solution was continuously added so that the concentration of 5-hydroxyanthranilic acid in the culture solution was 50 to 100 ppm, while maintaining the pH of the culture solution at 7.0. It was conducted for 30 hours.

21.0 g/5-hydroxy-L-tryptophan was produced and accumulated in the obtained culture solution. At this time, the yield of 5-hydroxy-L-tryptophan based on consumed sugar was 16.0%, and the molar yield relative to 5-hydroxyanthranilic acid was 92%. Note that the concentration of L-tryptophan in the medium was 5 ppm or less.

In addition, in parallel with the above culture experiment, when a non-anthranilic acid auxotrophic strain, which is the parent strain of Bacillus amyloliquifaciens SD-53 described above, was cultured for 30 hours in the same manner as above, 5-hydroxy in the culture solution was -L-tryptophan accumulation concentration is 20.3g/
The yield of 5-hydroxy-L-tryptophan based on consumed sugar was 15.55%, and the molar yield based on 5-hydroxyanthranilic acid was 93%, but the accumulated concentration of L-tryptophan was 2 g/g/.

Example 2 (Production example of 5-hydroxy-L-tryptophan) Bacillus amyloliquefaciens SD-53
was cultured in the medium used in Example 1 for 20 hours in a 5-jar fermenter, and this culture 2 was cultured in the same medium.
Inoculate a 500 jar fermentor containing 200 ml and inoculate at 35°C for 30 min while keeping dissolved oxygen above 0.5 ppm.
Time-aerated agitation culture was performed. During culture, the concentration of 5-hydroxyanthranilic acid in the culture solution is 50~
A 20% 5-hydroxyanthranilic acid ammonium salt solution was added continuously to give a concentration of 100 ppm, and the pH of the culture solution was maintained at 7.0. After completion of the culture, the concentration of 5-hydroxy-L-tryptophan produced and accumulated in the solution was 23.5 g/, and the yield of 5-hydroxy-L-tryptophan based on consumed sugar was 17.5%.
Molar yield of 5-hydroxyanthranilic acid is 93%
The accumulated concentration of L-tryptophan in the culture solution was 3 ppm or less.

Effects of the Invention According to the present invention, since L-tryptophan, which is difficult to separate from 5-hydroxy-L-tryptophan, does not accumulate in the culture solution, the desired 5-hydroxy-L-tryptophan can be obtained in high yield. It is possible to obtain it with.

Claims (1)

[Claims] 1 5-fluorotryptophan resistance and anthranilic acid requirement, and 5-hydroxy-L-
Bacillus amyloliquifaciens SD-53, which has the ability to produce tryptophan, was cultured in a nutrient medium containing anthranilic acid with the addition of 5-hydroxyanthranilic acid or its salt, and the 5-hydroxy-L- produced in the culture was cultured. A method for producing 5-hydroxy-L-tryptophan, which comprises collecting tryptophan.