JPS6231916B2 - - Google Patents

Description

[Detailed description of the invention]

Technical Field The present invention relates to a method for producing 5-hydroxy-L-tryptophan using a microorganism resistant to 5-hydroxyanthranilic acid and capable of producing 5-hydroxy-L-tryptophan. Prior Art 5-Hydroxy-L-tryptophan is an important compound as a precursor of the physiologically active amine serotonin and as a tryptophan homolog as a raw material for medicine and the chemical industry. It has been proposed. As a method for microbiologically synthesizing 5-hydroxy-L-tryptophan, for example, 5-hydroxy-L-tryptophan
A method for synthesizing from hydroxyindole and serine (Japanese Patent Application Laid-open No. 10955/1982), a method for synthesizing from 5-hydroxyindole and serine (or pyruvic acid and ammonia) (Japanese Patent Application Laid-open No. 81590/1982), 5 - Method of synthesis from hydroxyindole and β-halogenoalanine (Japanese Patent Application Laid-Open No. 50-95484
5-hydroxyindole and X-CH2
-CH(NH2)-COOH (X: lower alkoxy group,
benzyloxy group, thiobenzyl group) (JP-A-51-121597), method of synthesis from 5-hydroxyindole and serine (JP-A-57-83288), and L-tryptophan or ω. A method for producing it from -N-acyl-L-tryptophan (Japanese Unexamined Patent Publication No. 48-44489) is known. However, these methods have problems in that the raw material compounds are expensive or difficult to obtain, and there is a practical need for the development of inexpensive production methods. Purpose and Structure of the Invention The present inventors have also conducted extensive research in view of the current state of the microbiological production method of 5-hydroxy-L-tryptophan, and as a result, in a medium containing a relatively high concentration of 5-hydroxyanthranilic acid. A microorganism capable of efficiently producing 5-hydroxy-L-tryptophan was discovered, and the present invention was accomplished. That is, according to the present invention, it has resistance to 5-hydroxyanthranilic acid and 5-hydroxy-L-
Bacillus subtilis SD-51, which has the ability to produce tryptophan, is cultured in a medium supplemented with 5-hydroxyanthranilic acid or its salt, and 5-hydroxyanthranilic acid or its salt is added to the culture.
A method for producing 5-hydroxy-L-tryptophan is provided, which comprises producing and collecting 5-hydroxy-L-tryptophan. Description of Structure and Effects of the Invention The present inventors have discovered that 5-hydroxy-L
- In order to obtain a microorganism that has the ability to produce and accumulate tryptophan, microorganisms that have an enzyme system that produces anthranilic acid → L-tryptophan are used to produce 5-hydroxyanthranilic acid → 5-hydroxy-L-
Based on the assumption that it may produce tryptophan, we first selected Bacillus subtilis IAM1026 as an original strain from known strains that have the ability to produce L-tryptophan, and released the feedback control mechanism by L-tryptophan that is unique to this microorganism. Therefore, 5, which is an analogue of tryptophan,
- For the purpose of imparting resistance to fluorotryptophan (5-FT), the microorganism is treated with N-
Artificial mutagenesis was performed using methyl-N'-nitro-N-nitrosoguanidine (NTG), and the mutagenized strain was 5- or more than the minimum inhibitory concentration (MIC).
Spread on an agar plate medium containing fluorotryptophan (Spicaizen medium), isolate the grown colonies, and inoculate 5-fluorotryptophan-resistant strains into test tubes with cotton plugs dispensed into a medium with the following composition. After culturing with shaking at 35°C for 30 hours, the amount of 5-hydroxy-L-tryptophan produced was quantified. Medium composition Ammonium sulfate 2g, K2HPO4 14g, KH2PO4 6g, sodium citrate/2H2O 1g, MgSO4/7H2O
0.2g, FeSO4・7H2O 1mg, MnSO4・4~
6H2O 1mg, glucose 50g, and distilled water 900ml
Sterilized at 115°C for 20 minutes, to which was added 0.1g of 5-hydroxyanthranilic acid and 100ml of distilled water filtered through a 0.45μ filter. Approximately 2,000 5-fluorotryptophan-resistant strains were treated with 5-hydroxy-L-
We compared the tryptophan production ability and selected the strain SD-51 with the highest productivity. This strain is from the Showa era.
It was deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology on July 4, 1958, as Microtechnology Research Institute Deposit No. 7136. The mycological properties of this bacterium include a high ability to synthesize 5-hydroxy-L-tryptophan from the original strain Bacillus subtilis IAM1026 and 5-hydroxyanthranilic acid, and 5-FT resistance (feedback control mechanism by L-tryptophan). Cancellation)
The mycological properties were the same as those of the original strain, except for the difference in the following points. Bacillus subtilis used in the present invention
SD-51 and its original strain B. subtilis
The main mycological properties of 1AM1026 are as shown below.

[Table] Aggregation form Single or chain Same as left

[Table] Circular, wavy edges Circular, wavy edges Juicy agar Good growth, grayish white Good growth, grayish white slope Spreading Spreading

[Table] Milk Peptonization Peptonization

【table】

【table】

[Table] To produce 5-hydroxy-L-tryptophan according to the present invention, Bacillus subtilis
This is carried out by culturing SD-5 in a medium supplemented with 5-hydroxyanthranilic acid or its salt. 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 from the viewpoint of the desired yield of 5-hydroxy-L-tryptophan, culture conditions, and economics.
mg/, preferably 50 to 300 mg/. 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 saccharified solution, Saccharides such as cellulose decomposition products, acetic acid, ethanol, etc. are used. As the storage 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 as 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. It is particularly preferable to use the acid salt as an aqueous solution. 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. According to the present invention, (1) the strain can be inoculated with 5-hydroxyanthranilic acid added to the medium before the start of culture, (2) 5-hydroxyanthranilic acid can be added in the fermenter during mass culture. (3) In 5-hydroxyanthranilic acid-sensitive bacteria, 5-hydroxyanthranilic acid is Although there may be a shortage of anthranilic acid supply, in the case of the bacteria of the present invention, oversupply culture can always be performed, so the shortage of 5-hydroxyanthranilic acid can be resolved. effect can be achieved. 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 5%, ammonium sulfate 0.2%, K2HPO4 1.4
%, KH2PO4 0.6%, sodium citrate.
2H2O 1g, MgSO4・7H2O 0.02%, FeSO4・
Bacillus subtilis SD-51 was inoculated into an Erlenmeyer flask containing 50 ml of a liquid medium (PH7.0) containing 1 ppm of 7H2O and 1 ppm of MnSO4.4 to 6H2O and cultured with shaking at 35°C. After inoculation, 25 mg of 5-hydroxyanthranilic hydrochloride was added and cultured with shaking for 48 hours. As a result, the desired 5-hydroxy-L-tryptophan was produced and accumulated at a concentration of 315 ppm. Example 2 (Production example of 5-hydroxy-L-tryptophan) Put liquid medium 2 with the following composition into the jar fermenter No. 5, and then use the medium of Example 2 at 35°C for 18
Bacillus subtilis SD pre-shaking cultured for an hour
-51 was inoculated into the above Jiahua Armentor at 5%.
Maintain the amount of dissolved oxygen above 0.5pm at 35℃, and maintain the pH level.
7.0±0.2 using 1N caustic soda with PH controller
Aerated agitation culture was performed while maintaining the temperature. Liquid medium composition Glucose 10%, Ammonium sulfate 0.3%, Na2HPO4・
12H2O0.5%, KH2O4 0.2%, MgSO4・7H2O
0.05%, FeSO4・7H2O 5ppm, MnSO4・4~
6H2O 1ppm and 5-hydroxyanthranilic acid
0.05% 10 hours after the start of the culture, 5-hydroxyanthranilic hydrochloride was added to the fermenter at a rate of 1 g for 1 hour. 25 g of 5-hydroxyanthranilic acid was added 34 hours after the start of the culture, and the culture was continued for 6 hours. The concentration of 5-hydroxy-L-tryptophan in the culture solution after culturing for 40 hours was 14.3 g/. The tryptophan concentration at this time was 0.55 g. After centrifuging the culture solution and removing the bacterial cells, the supernatant 1.8 was lowered to pH 2.5 with hydrochloric acid and applied to a strongly acidic cation exchange resin column (30 x 500 mm) to adsorb 5-hydroxy-L-tryptophan. ,1
After washing with pure water, it was eluted with 3% ammonia.
Fractions of 5-hydroxytryptophan were collected, adsorbed on activated carbon, eluted with hot ethanol, and concentrated under reduced pressure to recover crystals of 5-hydroxytryptophan. The yield of the obtained 5-hydroxytryptophan crystal was 7.5 g, and when the optical rotation of this crystal was measured, [α] ²⁰ _D = -32.1, and from this value, the produced 5-hydroxytryptophan was It turned out to be L-form.

Claims (1)

[Claims] 1 Bacillus subtilis SD-51, which is resistant to 1,5-hydroxyanthranilic acid and has the ability to produce 5-hydroxy-L-tryptophan, was
- 5-hydroxy-L in the culture by culturing in a medium supplemented with hydroxyanthranilic acid or its salt.
- A method for producing 5-hydroxy-L-tryptophan, which comprises producing tryptophan and collecting it.