JPH0352959B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention is directed to the production of L. - A method for producing L-tryptophan using a microorganism capable of producing tryptophan.

L-tryptophan is an important compound as an essential amino acid, and is used as an additive for medicines, foods, and feeds.

Problems to be Solved by the Prior Art and the Invention As mentioned above, L-tryptophan is an important compound as an essential amino acid, and is a substance that is particularly susceptible to nutritional deficiency, so an economical industrial production method for it has not been developed. Development is strongly desired. From this point of view, various chemical or microbiological synthesis methods have been proposed. As an example of such a method for synthesizing L-tryptophan, a method is known in which L-tryptophan is produced from precursors such as anthranilic acid and indole using microorganisms.

However, in this case, if a relatively large amount of anthranilic acid is added to the culture system, the biosynthesis of L-tryptophan is inhibited, so it is difficult to obtain a culture solution containing a high concentration of L-tryptophan.
This was an obstacle to practical application. Under these circumstances, we use a mutant strain that is resistant to tryptophan analogues, such as 5-fluorotryptophan and 5-methyltryptophan, to release the inhibition of tryptophan biosynthetic enzyme production by L-tryptophan. A method for producing L-tryptophan by direct fermentation has been proposed. However, until now, there has been no example in which the inhibition of the activity of anthranilate synthase, which is one of the tryptophan biosynthetic enzymes, by L-tryptophan has been removed.

Means for Solving the Problems In view of the current situation in which there is no example of removing the inhibition of the activity of anthranilate synthase by L-tryptophan in the conventional microbiological production method of L-tryptophan described above, the present inventors solved the problem. Moreover, in order to further improve the productivity of L-tryptophan, we believed that it was essential to release this inhibition, and as a result of our intensive research, we discovered that the inhibition of the activity of anthranilate synthase by L-tryptophan was removed. We have succeeded in obtaining a tryptophan-producing microbial strain, leading to the present invention.

That is, according to the present invention, a strain belonging to the genus Bacillus that is resistant to 5-fluorotryptophan and has an anthranilic acid auxotrophy is reversely mutated to become anthranilic acid non-auxotrophic, and has the ability to produce L-tryptophan. Bacillus amyloliquifaciens SD-43 was cultured in a medium and L-
A method for producing L-tryptophan is provided, which comprises producing tryptophan and collecting it.

A microorganism Bacillus belonging to the genus Bacillus having an ability to produce L-tryptophan, which is obtained by reverting a strain belonging to the genus Bacillus having resistance to 5-fluorotryptophan and requiring anthranilic acid according to the present invention to make it non-auxotrophic for anthranilic acid.・Amiroliquifaciens SD-43 was obtained as follows.

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 strain was grown on an agar plate medium (spizaizen medium) supplemented with 5-fluorotryptophan (5-FT) above the minimum inhibitory concentration (MIC) as a tryptophan analog, and several hundred colonies were grown. The L-tryptophan productivity of each strain was examined to obtain a 5-FT-resistant strain with the highest L-tryptophan production ability (that is, the microorganism's inherent feedback suppression mechanism due to accumulation of tryptophan concentration was released). This strain has a resistance of 5000 ppm or more to 5-fluorotryptophan.

Next, this mutant strain having 5-FT resistance and L-tryptophan production ability was treated again in the same manner as above.
Artificial mutagenesis treatment using NTG, after treatment,
Centrifuge (10000G x 5 minutes), suspend the bacterial cells in M/100 Tris buffer (PH7.0), and centrifuge three times to remove NTG. The bacterial cells are 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.

Next, the anthranilic acid auxotrophic strain obtained above was subjected to artificial mutagenesis treatment again using NTG as described above, and the mutated strain was collected by centrifugation as described above and added to Tris buffer. Wash and incubate in nutrient medium for 3 hours at 30-40°C. After culturing, the bacteria were collected by centrifugation as described above, suspended in Tris buffer at a concentration of 1 x 10 ⁸ cells/ml, and mixed with anthranilic acid in a spizizen medium to which 16 g of agar was added. Bacillus amyloliquifaciens SD-43, a strain with high L-tryptophan production, was selected from among the growing colonies by applying it to a minimal plate medium for Bacillus subtilis that does not contain Bacillus subtilis and culturing it for 3 days at 30 to 40°C. Selected.

This strain was identified as Bacillus amyloliquefaciens SD-
43 (Fiber Science and Technology Research Institute No. 7772). The mycological properties of this strain are similar to those of the original strain Bacillus amyloliquifaciens IAM1521 and 5-
They were the same except that they had FT resistance and the feedback inhibition of anthranilate synthase by L-tryptophan was released, resulting in very high L-tryptophan productivity.

To produce L-tryptophan according to the invention, Bacillus amyloliquefaciens SD
-43 in culture 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. In addition, since the microorganism of the present invention does not require anthranilic acid for its growth, it is usually cultured in an anthranilic acid-free medium, but anthranilic acid is added to the medium to improve L-tryptophan productivity. However, there is no problem in its growth, and in that case, anthranilic acid may be added as an aqueous solution of a soda salt, potassium salt, or ammonium salt, or a solution of the free acid in ethyl alcohol or methyl alcohol.

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
This will be carried out for approximately 72 hours.

The desired L-tryptophan can be collected from the culture solution or culture according to a conventional method. For example, L-tryptophan can be isolated and crystallized from a culture supernatant from which bacterial cells have been removed by centrifugation, using an appropriate combination of ion exchange resin treatment, activated carbon treatment, and other operations.

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 (Example) Liquid medium 2 having the following composition was placed in a jar fermenter (No. 5) and sterilized by heating at 115° C. for 15 minutes.

Liquid medium composition Glucose 10%, Ammonium sulfate 0.3%, Na ₂ HPO ₄ .
_12H2O0.5 %, _{KH2PO40.2 %} , _MgSO4・_7H2O0.1
%, _{FeSO4.7H2O5ppm} , and _{MnSO4.4 ~}
6H ₂ O1ppm Next, Bacillus amyloliquifaciens SD-43, which had been previously cultured on a broth agar medium at 30℃ for 16 hours, was inoculated into this culture tank by suspending it in 10ml of sterile water, and the amount of dissolved oxygen was increased. 30 while keeping the glucose concentration at 2% and the pH at 7.0 above 0.5ppm.
Time-aerated agitation culture was performed.

In the culture solution thus obtained, 13 g/L-tryptophan was accumulated.

Example 2 (Comparative Example) Same as Example 1 except that in place of Bacillus amyloquifaciens SD-43 in Example 1, a 5-FT resistant strain, which is the parent strain of Bacillus amyloquifaciens SD-43, was used. The culture was carried out in the same manner with aeration for 30 hours. In the culture solution thus obtained, 9 g/L-tryptophan was accumulated.

Effects of the Invention According to the present invention, a high concentration of L-tryptophan can be accumulated in the medium by suppressing the production of the L-tryptophan synthase system and releasing inhibition of its activity.

Claims (1)

[Claims] 1. Bacillus amyloliquifaciens SD, which has the ability to produce L-tryptophan and is made non-anthranilic acid by backmutating a strain belonging to the genus Bacillus that is resistant to 5-fluorotryptophan and requires anthranilic acid. A method for producing L-tryptophan, which comprises culturing L-43 in a medium to produce L-tryptophan in the culture, and collecting the L-tryptophan.