JPH028719B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing L-tryptophan (hereinafter referred to as tryptophan) by a fermentation method. 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 methods using these precursors, tryptophan is produced 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 any precursors. Method (Tokuko Sho 48-18828, Tokko Sho
53-39517) has been developed. Therefore, the present inventors conducted research to develop a method to inexpensively produce tryptophan from carbon sources such as sugars by a direct fermentation method using microorganisms of the genus Bacillus. It has also been found that among microorganisms that are resistant to cinnamic acid or hydrocinnamic acid in addition to resistant to cinnamic acid, there are strains that have the ability to produce tryptophan in larger quantities than previously known. This invention was completed as a result of further research based on this knowledge. The mutant strain used in the method of the present invention is a microorganism belonging to the genus Bacillus that is resistant to tryptophan analogs and cinnamic acid or hydrocinnamic acid, and has the ability to produce tryptophan,
For example, the following mutant strains are used. Bacillus subtilis AJ11906 FERM−
P6562 (5-F-Trp ^r , Cin ^r ) Bacillus subtilis AJ11907 FERM-
P6563 (5-F-Trp ^r , Leu ^- , Cin ^r ) Bacillus subtilis AJ11908 FERM-
P6564 (5-F-Trp ^r , IM ^r , Cin ^r ) Bacillus subtilis AJ11909 FERM-
P6565 (5-F- ^Trrp , ^IMr , ^HCinr ) 5-F- ^Trrp : 5-fluorotryptophan resistant Leu ^- : L-leucine auxotrophic IM ^r : Indolmycin resistant Cin ^r : Cinnamate resistant HCin ^r : Hydrocinnamic acid resistance These mutant strains used in the present invention have tryptophan analog-resistant tryptophan-producing bacteria of the genus Bacillus as the parent strain, and are subjected to conventional mutagenic procedures such as ultraviolet irradiation or N-methyl- N′−
Nitro-N-nitrosoguanidine (hereinafter abbreviated as NG) is treated with chemical agents such as nitrous acid, and the mutated strain is cultured on a plate agar medium containing cinnamic acid or hydrocinnamic acid in an amount that makes it impossible for the parent strain to grow. It is obtained by separating the colonies growing on the plate medium. The above-mentioned parent strain may include tryptophan-producing bacteria that have properties useful for tryptophan production in addition to tryptophan analog resistance, such as tryptophan-producing bacteria that require L-arginine, L-lysine, L-leucine, or L-phenylalanine. Bacteria (Japanese Patent Publication No. 53-39517), and tryptophan-producing bacteria resistant to tryptophan analogs and indolmycin (Japanese Patent Publication No. 56-92796)
etc. are used. As a specific example, the following tryptophan analog-resistant tryptophan-producing bacteria are used. Bacillus subtilis FT−145 FERM−
P1783 (5-F-Trp ^r ) Bacillus subtilis FFL-5 FERM-
P1786 (5-F-Trp ^r +Leu ^- ) Bacillus subtilis AJ11483 FERM-
P5286 (5-F- ^Trrp + IM ^r ) In addition, the mutant strain of the present invention uses a wild strain of the Bacillus genus as a parent strain, and after imparting resistance to cinnamic acid or hydrocinnamic acid to this strain, resistance to tryptophan analogs is imparted. It can be guided even if it is twisted. In this case, properties useful for tryptophan production, such as auxotrophy for amino acids such as L-phenylalanine, L-tyrosine, L-leucine, and L-histidine, or resistance to phenylalanine analogues are imparted. This is desirable. In the following experimental examples, the degree of resistance of the cinnamic acid-resistant tryptophan-producing bacteria of the present invention to cinnamic acid and the degree of resistance of the hydrocinnamic acid-resistant tryptophan-producing bacteria to hydrocinnamic acid will be shown. Experimental Example A minimal medium having the composition shown in Table 1 was dispensed into 4.0 ml test tubes with a diameter of 16.5 mm, and heated at 110° C. for 10 minutes.
A prepared culture medium was prepared by adding a cinnamic acid (hydrocinnamic acid) solution that had been separately filtered (sterilized) to a concentration shown in Table 2. Bacillus subtilis AJ11906, AJ11907, AJ11908 obtained by culturing for 24 hours in a minimal medium containing no cinnamic acid (hydrocinnamic acid) in the above medium.
and AJ11909 culture solution to 0.1ml each,
Culture with shaking at 30°C for 48 hours, and
The absorbance was measured. The results are shown in Table 2. Table 1 Composition of minimal medium (PH7.0) Component concentration Glucose 5.0g/ Ammonium sulfate 1.0 〃 KH ₂ PO ₄ 8.65 〃 MgSO ₄・7H ₂ O 0.2 〃 FeSO ₄・7H ₂ O 10mg/ MnSO ₄・4H ₂ O 10 〃 Sodium citrate 0.5g/ *L-leucine 10mg/dl (*Added only for L-leucine auxotrophic strains)

【table】

【table】

[Table] 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. Table 3 Composition of tryptophan production medium (PH7.0) Component concentration Glucose 80g/ Ammonium chloride 10 〃 KH ₂ PO ₄ 1 〃 KCl 2 〃 MnSO ₄・7H ₂ O 10ml/ FeSO ₄・4H ₂ O 10 〃 Casamino Acid 4g/ MgSO ₄・7H ₂ O 0.4 〃 CaCO ₂ 40 〃 *L-Leucine 20mg/dl (*Added only in case of L-leucine auxotrophic strain) Table 4 Tryptophan production amount Strain (mg/ml) FT-145 1.90 AJ11906 3.5 FEL−5 4.2 AJ11907 6.5 AJ11483 6.2 AJ11908 8.0 AJ11909 7.5

Claims (1)

[Claims] 1. A microorganism belonging to the genus Bacillus, resistant to tryptophan analogs and cinnamic acid or hydrocinnamic acid, and capable of producing L-tryptophan is cultured aerobically in a liquid medium, and L-tryptophan is added to the culture solution.
- A method for producing L-tryptophan, which comprises producing and accumulating tryptophan and collecting it.