JPH0314436B2 - - 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, the present inventors have developed a direct fermentation method from carbon sources such as sugars using microorganisms of the genus Brevibacterium that are resistant to tryptophan analogs such as 5-methyltryptophan and 5-fluorotryptophan. developed a method for producing tryptophan (Japanese Patent Publication No. 48-18828, Japanese Patent Publication No. 55-162771), and also developed these strains with auxotrophic properties such as phenylalanine and tyrosine, and resistance to phenylalanine and tyrosine analogs. By granting (Tokukosho
48-18828) or serine analogs (Japanese Patent Application Laid-open No. 57-174096), it was also revealed that the amount of tryptophan accumulated increased. The present inventors conducted research to develop a method for producing the product at a lower cost using these direct fermentation methods, and as a result, they added sulfur, which is known as a sulfur drug, to the previously known tryptophan-producing bacteria of the genus Brevibacterium. By imparting resistance to guanidine, they discovered that they could produce tryptophan in larger amounts than conventional tryptophan-producing bacteria. This invention was completed as a result of further research based on this knowledge. The microorganism used in the tryptophan production method of the present invention belongs to the genus Brevibacterium and has the above-mentioned properties necessary for tryptophan production, such as 5
- It is a mutant strain that is resistant to methyltryptophan and sulfaguanidine. In addition to these properties, L-phenylalanine, L
- It also includes strains endowed with auxotrophy for tyrosine, L-histidine, etc., and drug resistance against phenylalanine analogs, tyrosine analogs, azaserine, etc. The parent strain of the mutant strain of the present invention is a microorganism of the genus Brevibacterium, which is known as a so-called L-glutamic acid producing bacterium. Examples include Brevibacterium flavum ATCC14067, Brevibacterium devaricatam ATCC14020, Brevibacterium lactofamentum ATCC13869, Brevibacterium roseum ATCC13825, and the like.
The mutant strains used in the present invention can be obtained by mutating the above-mentioned bacterial strains as parent strains to impart properties necessary for tryptophan production, such as resistance to 5-methyltryptophan and sulfaguanidine. In this case, the order of imparting both resistances may be arbitrary. The mutation operation is done in the usual way,
For example, this can be carried out by ultraviolet irradiation or chemical treatment with N-methyl-N'-nitro-N-nitrosoguanidine (hereinafter abbreviated as NG), nitrous acid, or the like. Examples of strains used in the present invention, Brevibacterium flavum AJ12022 (FERM-P7034,
An experimental example showing a specific method of inducing FERM BP-475) and its degree of resistance to sulfaguanidine is shown. Brevibacterium flavum AJ11667
(FERM-P5907, FERM BP-114) (5-fluorotryptophan resistance, parafluorophenylalanine resistance, azaserine resistance, L-tyrosine requirement, L-methionine requirement) (JP-A-57-174096)
was used as the parent strain and treated with 200 μg/ml NG at 30°C for 15 minutes (survival rate 11%). Next, if the parent strain cannot grow on the synthetic medium shown in Table 1,

[Table] A plate medium was prepared by adding sulfaguanidine to a concentration of 1200 μg/ml, and mutated AJ 11667 was applied to this medium. After leaving it at 30°C for 9 days,
Bacterial strains growing as colonies, that is, sulfaguanidine-resistant mutant strains, were collected, and among these, the mutant strain AJ12022 (FERM-
P7034, FERM BP-475) (sulfaguanidine resistance, 5-fluorotryptophan resistance, parafluorophenylalanine resistance, azaserine resistance, L
- Tyrosine requirement, L-methionine requirement) were collected. As shown in the Examples, this mutant strain produced 68% more tryptophan than the parent strain. Next, the results of examining the degree of resistance of this AJ12022 strain to sulfaguanidine are shown in Table 2. Dissolve sulfaguanidine in the synthetic medium shown in Table 1 to the concentration shown in Table 2 to prepare a plate medium (diameter 8.5 cm), and add complete medium (yeast extract 10 g/, Polypeptone 10g/
, sodium chloride 5g/, glucose 5g/
, L-methionine 200mg/, L-tyrosine 200
After inoculating approximately 10 ⁷ Brevibacterium flavum AJ11667 and Brevibacterium flavum AJ12022 grown at pH 7.0), culture was performed at 30° C. for 4 days, and the number of colonies produced was determined. The results are shown in Table 2.

[Table] Shows the combination.
In addition, sulfaguanidine resistance as referred to in the present invention means:
Under the above culture conditions, sulfaguanidine was
This refers to the case where approximately 10 ⁷ microorganisms are inoculated into the above culture containing 1200 μg/ml, and 1000 or more colonies are generated after culturing at 30° C. for 4 days. The culture medium for tryptophan production is not particularly limited, and is a conventional medium containing a carbon source, a nitrogen source, inorganic salts and, if necessary, organic micronutrients. Carbohydrates (glucose, fructose or starch, hydrolysates of cellulose, molasses, etc.), organic acids (acetic acid, citric acid, etc.), alcohols (glycerin, ethanol, etc.), or hydrocarbons (Norman paraffin, etc.) are used as carbon sources. can. Nitrogen sources include ammonium sulfate, urea, ammonium nitrate, ammonium phosphate, ammonium chloride, ammonia gas, and others; inorganic salts include phosphates, magnesium salts, calcium salts, iron salts, manganese salts, and other trace metal salts. Use accordingly. As for organic micronutrients, appropriate amounts of amino acids, vitamins, fatty acids, organic basic substances, etc. are added if there is a nutritional requirement.
If necessary, amino acids may be added as growth-promoting substances.
Vitamins, flavor liquid (registered trademark, soybean hydrolyzate), yeast extract, peptone, casamino acids, etc. can be used. The culture may be carried out in a conventional manner under aerobic conditions at a pH of 5 to 9 and a temperature of 20 to 40°C for 24 to 72 hours. If the pH decreases during culture, add calcium carbonate after sterilization or neutralize with alkali such as aqueous ammonia or ammonia gas.
In addition, in a field where an organic acid is used as a carbon source, the increase in pH is neutralized with a mineral acid or an organic acid. Tryptophan can be isolated and collected by conventional methods. The obtained product was confirmed to be tryptophan by Rf value on paper chromatogram, tryptophan-specific reaction using Ehritzg reagent, or biological activity value using microbial quantitative method, and it was confirmed that it matched that of the tryptophan standard and was identified as tryptophan. Tryptophan was quantified according to the microbial quantification method using Leuconostochus mesenteroides (ATCC8042). 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 each microorganism shown in Table 4 was inoculated in 1/3 slant amount and shaken at 30°C for 72 hours. Cultured. The amount of tryptophan produced in each culture solution was as shown in Table 4.

【table】

[Table] Shows.

Claims (1)

[Claims] 1. Cultivating a microorganism belonging to the genus Brevibacterium, resistant to sulfaguanidine, and capable of producing L-tryptophan in a liquid medium aerobically, producing and accumulating L-tryptophan in the culture solution, A method for producing L-tryptophan, which comprises collecting the L-tryptophan.