JPH0412720B2 - - Google Patents

Description

[Detailed description of the invention]

[Object of the Invention] <Industrial Application Field> The present invention relates to a method for producing L-tryptophan (hereinafter referred to as tryptophan) by a fermentation method. Tryptophan is one of the essential amino acids for animals and is an important substance as a feed additive. <Prior Art> As a conventional method for producing tryptophan, a method for producing tryptophan from anthranilic acid, indole, or 3-indolepyruvic acid, which is a precursor of tryptophan, is known. In contrast to the methods using these precursors, there is a method of producing tryptophan by direct fermentation using a mutant strain belonging to the genus Bacillus that is resistant to tryptophan analogs and using sugars as a carbon source without using precursors. (Special Publication 18828, Special Publication 53-39517)
is being developed. <Problems to be Solved by the Invention> The problems to be solved by the present invention are to develop a method for inexpensively producing tryptophan from carbon sources such as sugars by direct fermentation using microorganisms belonging to the genus Bacillus. It is in. [Structure of the Invention] <Means for Solving the Problems> The present inventors have conducted intensive research to solve the above-mentioned problems, and have found that vitamin B has been added to microorganisms belonging to the genus Bacillus and capable of producing tryptophan. We have discovered that there is a strain that has the ability to produce tryptophan in larger amounts than the previously known tryptophan-producing bacteria by imparting ₁₂ resistance. The present invention was completed based on this knowledge. The microorganism used in the present invention belongs to the genus Bacillus, and is a mutant strain that is resistant to vitamin _B12 and has the ability to produce tryptophan. The mutant strain capable of producing tryptophan in the present invention is, for example, a mutant strain that is resistant to tryptophan analogs, and further has an auxotrophy for L-phenylalanine, L-arginine, L-lysine, or L-leucine. Alternatively, it is referred to as a mutant strain that has drug resistance such as phenylalanine analog resistance and indolmycin resistance. In the present invention, tryptophan analog refers to a chemical substance that has a chemical structure similar to tryptophan and inhibits the growth of microorganisms belonging to the genus Bacillus, but the inhibition is canceled by the presence of tryptophan. For example, 4- Lower alkyl tryptophan, 5-lower alkyl tryptophan, 6-lower alkyl tryptophan, 7-lower alkyl tryptophan, 5-halogeno tryptophan, 6-
Examples include halogenotryptophan, tryptophan hydroxamate, α-lower alkyltryptophan, and 7-azatryptophan. Furthermore, phenylalanine analog refers to a substance that has a chemical structure similar to phenylalanine and whose inhibition is canceled by phenylalanine, which inhibits the growth of microorganisms belonging to the genus Bacillus. For example, 3-thiazolealanine, 2-thiazolealanine, 2-pyridinealanine, 2-naphthalenealanine, 1-naphthalenealanine, β-3-ethylalanine, β-2-thienylalanine, 4-
Nitrophenylalanine, 2-nitrophenylalanine, 4-aminophenylalanine, 3-aminophenylalanine, 2-aminophenylalanine, 5-hydroxy-2-pyridinealanine,
Tyrosine hydroxamate, phenylalanine, hydroxamate, 3-aminotyrosine,
Examples include 3-fluorotyrosine, 3-hydroxytyrosine, 3-nitrotyrosine, and the like. The vitamin B ₁₂ referred to in the present invention is cyanocobalamin isolated from the liver, and vitamin B ₁₂ is also obtained not as cyanide but also as hydroxide, nitrate, chloride, and sulfate. This vitamin is broken down into hydroxycobalamin and aminocobalamin as coenzyme _B12 . There are also coenzymes that contain syudovitamin _B12 , and vitamins including these substances listed above are also available.
It is called B ₁₂ . Examples of the microorganisms used in the present invention include the following mutant strains. Bacillus subtilis AJ12200 (5-FT ^r , VB ₁₂ ^r ) Bacillus subtilis AJ12200 (5-FT ^r , IM ^r , SG ^r , VB ₁₂ ^r ) 5-FT ^r : 5-fluorotryptophan resistance IM ^r : Indole Mycin resistance ^SGr : Sulfaguanidine resistance _VB12r : Vitamin _B12 resistance These mutant strains used in the present invention are derived from ^the parent strain L-tryptophan-producing bacteria of the genus Bacillus that are resistant to tryptophan analogs, and Mutagenic manipulations, such as ultraviolet irradiation or N-methyl-N'-
After treatment with chemical agents such as nitro-N-nitrosoguanidine and nitrous acid, culturing on an agar plate medium containing vitamin _B12 at a concentration such that the parent strain cannot grow, and isolating colonies growing on the plate medium. obtained by. The above parent strain may include tryptophan-producing bacteria that have properties useful for tryptophan production in addition to tryptophan analog resistance, such as L-arginine, L-lysine, L-leucine, or L-phenylalanine auxotrophs. Tryptophan-producing bacteria (Japanese Patent Publication No. 53-39517), and tryptophan-producing bacteria resistant to tryptophan analogs and indolmycin (Japanese Patent Application Laid-Open No. 56-92796)
etc. are used. As a specific example, the following tryptophan analog-resistant tryptophan-producing bacteria are used. Bacillus subtilis FT145FERM-P1783 (5-FT ^r ) Bacillus subtilis AJ12099FERM-P7295 (5-FT ^r , IM ^r , SG ^r ) In addition, the mutant strain of the present invention uses a wild strain of the Bacillus genus as a parent strain, and adds vitamin B. It can also be induced by conferring tryptophan analog resistance after conferring B ₁₂ tolerance. Below, a specific method for inducing Bacillus subtilis AJ12201 (FERM-P8077), which is an example of the strain used in the present invention, and its degree of resistance to vitamin _B12 will be shown in experimental examples. Bacillus subtilis AJ12099 (FERM−
P7295) was used as the parent strain and treated with 500 μg/ml NG for 30 minutes at 0°C. Next, vitamin B ₁₂ was added to the minimal medium shown in Table 1 at a concentration of 100 μg/ml, which is a concentration at which the parent strain cannot grow, to prepare a plate medium, and the mutation-treated AJ12099 was applied to this plate medium and incubated at 30°C.
After standing for 7 days, the strains growing as colonies, that is, the vitamin B ₁₂ resistant mutant strain, were collected.
(FERM-P8077) was collected. As shown in the example, this mutant strain contains 21% more tryptophan than the parent strain.
generated a lot. Next, Table 2 shows the results of examining the degree of resistance of strains AJ12200 and AJ12201 to vitamin _B12 . Dissolve vitamin B ₁₂ in the minimal medium shown in Table 1 to the concentration shown in Table 2 to prepare a plate medium (8.5 cm in diameter). Polypeptone 10g/,
Contains sodium chloride 5g/, agar 20g/PH
7.2) Bacillus subtilis FT145 grown on
Approximately ¹⁰⁷ cells each of AJ12200, AJ12099 and AJ12201 were inoculated and cultured at 30°C for 4 days.
The number of colonies generated was examined. The results are shown in Table 2.

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[Table] Shows the combination.
In the present invention, vitamin B ₁₂ resistance refers to approximately 10 ⁷ microorganisms inoculated into the above medium containing 100 μg/ml of vitamin B ₁₂ under the above culture conditions and incubated at 30°C.
This refers to cases in which 5 x 10 ² or more colonies are produced after 4 days of culture. The medium used in the present invention is a conventional nutrient medium containing a carbon source, a nitrogen source, inorganic salts, and other organic micronutrients such as amino acids and vitamins as necessary. As the carbon source, glucose, sucrose, maltose, starch hydrolyzate, molasses, etc. are used, and ethanol, acetic acid, citric acid, etc. are also used alone or in combination with the other carbon sources mentioned above. As the nitrogen source, organic or inorganic nitrogen sources such as ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium phosphate, nitrates, urea, and peptone are used. Organic micronutrients include amino acids,
Vitamins, fatty acids, nucleic acids, and peptones containing these things, casamino acids, yeast extracts, soybean protein decomposition products, etc. are used, and when using auxotrophic mutant strains that require amino acids etc. for growth, It is necessary to supplement the nutrients. As the inorganic salts, phosphates, magnesium salts, calcium salts, iron salts, manganese salts, etc. are used. Cultivation can be carried out under normal culture conditions, with a pH of 5.
to 9, 1 to 9 while controlling the temperature between 20 and 40℃
A significant amount of tryptophan is accumulated in the culture solution by shaking culture or aeration stirring culture for 4 days.
If the pH decreases during culture, add calcium carbon after sterilization or neutralize with alkali such as aqueous ammonia or ammonia gas. In addition, when an organic acid is used as a carbon source, the increase in pH is neutralized with a mineral acid or an organic acid. How to collect tryptophan from culture fluid:
This may be carried out according to a known method for recovering tryptophan, and the tryptophan can be collected by removing the bacterial cells from the culture solution and then concentrating and crystallizing it, or by ion exchange chromatography. Examples will be described below. Example 1 20 ml of a tryptophan production medium having the composition shown in Table 3 below was dispensed into a 500 ml flask and incubated at 110°C.
After heating for 10 minutes, each microorganism shown in Table 4 was inoculated in a 1/3 slant amount and cultured with shaking at 30°C for 96 hours. The amount of tryptophan produced in each culture solution was as shown in Table 4. Vitamins in FT145
The amount of tryptophan produced by AJ12200 to which _B12 resistance was conferred was 2.3 times higher than that of the parent strain. Similarly, it was revealed that by imparting vitamin B ₁₂ resistance to indolmycin-resistant strain AJ12099, tryptophan production was increased by 1.9 g/.

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Claims (1)

[Claims] 1. A mutant strain belonging to the genus Bacillus that is resistant to vitamin _B12 and has the ability to produce L-tryptophan is cultivated aerobically in a liquid medium to produce and accumulate L-tryptophan in the medium, A method for producing L-tryptophan by a fermentation method, which comprises collecting the L-tryptophan.