JPS6234399B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing L-tryptophan by fermentation. Regarding the production method of L-tryptophan by fermentation method, it has been conventionally known that mutant strains of the genus Brevibacterium and Corynebacterium that are resistant to tryptophan analogs have the ability to produce and accumulate L-tryptophan in the culture medium. It has been known. The present inventors applied indolmycin (hereinafter referred to as IM) or N-demethyl-C to a previously known microorganism capable of producing L-tryptophan.
- A mutant strain that has been conferred resistance to demethyl-indolmycin (hereinafter referred to as DM) produces L-tryptophan at a higher yield than its parent strain, that is, the previously known mutant strain that has the ability to produce L-tryptophan. I learned. Furthermore, mutant strains of microorganisms of the genus Bacillus, Brevibacterium, Corynebacterium, Arthrobacter, Microbacterium, or Escherichia that do not have the ability to produce L-tryptophan are given IM or DM resistance. We found a larger number of mutant strains with higher L-tryptophan production ability. That is, the present invention provides a method for producing a mutant strain belonging to the genus Bacillus, Brevibacterium, Corynebacterium, Arthrobacter, Microbacterium, or Escherichia that is resistant to at least IM or DM in a liquid medium. This is a method for producing L-tryptophan, which is characterized by culturing it aerobically and collecting L-tryptophan produced and accumulated in the culture medium. A mutant strain resistant to IM or DM refers to a mutant strain whose growth is inhibited to a lesser extent by IM or DM than its parent strain. The mutant strain of the present invention is
It may have only IM resistance or DM resistance, but
Mutant strains that are resistant to both IM or DM are also included. In addition to IM or DM resistance, tryptophan analog resistance, phenylalanine analog resistance, phenylalanine requirement, telosin requirement,
In many cases, a strain with a higher production ability can be obtained than a mutant strain that has a property known to impart or enhance L-tryptophan production ability, such as histidine auxotrophy. Specific examples of the mutant strains of the present invention include the following. Bacillus subtilis AJ 11488
(FERM-P 5290) (IM resistant) Brevibacterium flavum AJ 11490
(FERM-P 5292) (IM resistant) Corynebacterium glutamicum AJ
11492 (FERM-P 5294) (IM resistant) Arthrobacter globiformis AJ
11494 (FERM-P 5296) (IM resistant) Microbacterium ammonia film
AJ 11496 (FERM-P 5298) (IM resistant) Escherichia coli AJ 11498
(FERM-P 5300) (IM resistant) Bacillus zubitilis AJ 11489
(FERM-P 5291) (DM resistant) Brevibacterium flavum AJ 11491
(FERM-P 5293) (DM resistant) Corynebacterium glutamicum AJ
11493 (FERM-P 5295) (DM resistant) Arthrobacter globiformis AJ
11495 (FERM-P 5297) (DM resistant) Microbacterium ammonia film
AJ 11497 (FERM-P 5299) (DM resistant) Escherichia coli AJ 11500
(FERM-P 5302) (DM resistant) Bacillus subtilis AJ 11483
(FERM-P 5286) (5-fluorotryptophan resistance, IM resistance) Bacillus subtilis AJ 11484
(FERM-P 5287) (5-fluorotryptophan resistance, DM resistance) Bacillus subtilis AJ 11486
(FERM-P 5288) (4-fluorotryptophan resistance, IM resistance) Bacillus subtilis AJ 11487
(FERM-P 5289) (4-fluorotryptophan resistance, DM resistance) Parent strains used to induce such mutant strains include Bacillus, Brevibacterium, Corynebacterium, and Arthrobacter. Microorganisms belonging to the genus Microbacterium or Escherichia may have superior or inferior efficiencies, but there is no need to use a specific microorganism. Mutation induction method is N-methyl-
Conventional methods such as contacting with N'-nitro-N-nitrosoguanidine can be applied. The medium for culturing such mutant strains contains a carbon source,
It is a conventional medium containing a nitrogen source, inorganic salts, and, if necessary, organic micronutrients such as amino acids and vitamins. Carbon sources include carbohydrates such as glucose, sucrose, fructose, maltose, starch hydrolysates containing these, and molasses;
Organic acids such as acetic acid and citric acid, and alcohols such as ethanol can be used. As the nitrogen source, ammonium salts, nitrates, aqueous ammonia, ammonia gas, urea, etc. can be used. As inorganic salts, inorganic salts such as potassium salts, phosphates, and magnesium salts are added to the medium as appropriate. Culture is best under aerobic conditions. During culture, reduce the pH of the medium to 5.
9. Better results can be obtained by controlling the culture temperature between 20°C and 40°C. In addition to ammonia water, alkalis such as ammonia gas, or acids, urea, calcium carbonate, etc. can be used to adjust the pH. If the culture is carried out for 1 to 4 days, a significant amount of L-
Tryptophan is produced and accumulated. L-tryptophan may be isolated by any known method. For example, after removing the bacterial cells from the culture solution, it is concentrated appropriately, and the pH is adjusted to the isoelectric point of L-tryptophan.
may be adjusted or purified using an ion exchange resin. Example 1 [Tolerance test] The minimum agar medium shown in Table 1 was used as the basic medium,
After adding IM or DM to this to make it IM 100 μg/ml or DM 500 μg/ml and heat sterilizing,
It was dispensed into a sieve. On the other hand, the following bacterial strains were cultured in a bouillon liquid medium in advance, and after thoroughly washing the bacterial cells, they were inoculated into the above medium at a rate of 10 ⁵ to 10 ⁶ cells per shear. The growth rate was observed after culturing at 30°C for 48 hours. Table-1 Glucose 0.5g/dl Ammonium sulfate 0.1 〃 KH ₂ PO ₄ 0.865 〃 Sodium citrate 0.05 〃 KOH 0.218 〃 MgSO ₄・7H ₂ O 0.02 〃 FeSO ₄・7H ₂ O 0.001 〃 MnSO ₄・4H ₂ O 0.00 1 〃 Casamino Acid 0.02 d-Biotin 30μg/dl Thiamine 10 Agar 2g/dl (PH7.0) As a result, Bacillus subtilis AJ11488, Brevibacterium flavum AJ11490, Corynebacterium glutamicum AJ11492, Arthrobacter globiformis AJ11494, Microbacterium ammoniaphilum AJ11496 and Escherichia coli AJ11498 can grow in a medium containing 100 μg/ml IM, Bacillus subtilis AJ11489, Brevibacterium flavum
AJ11491, Corynebacterium glutamicum
AJ11493, Arthrobacter globiformis
AJ11495, Microbacterium ammoniaphilum AJ11497 and Escherichia coli AJ11500
The parent strains of these mutants, Bacillus subtilis AJ11481 and Brevibacterium
flavum ATCC14067, Corynebacterium glutamicum ATCC13060, Arthrobacter globiformis ATCC8010, Microbacterium
Ammoniaphilum ATCC15354 and Escherichia coli ATCC10798 are IM100μg/ml or
It could not grow in any of the media containing 500 μg/ml of DM. Bacillus subtilis AJ11483 is 2.0 mg/ml 5-fluorotryptophan, 500 μg/ml IM
Bacillus subtilis in a medium containing both
AJ11486 was able to grow in a medium containing both 2.0 mg/ml 4-fluorotryptophan and 500 μg/ml IM. Also, Bacillus subtilis AJ11484 is 2.0mg/
5-fluorotryptophan 1000μg/ml
Bacillus subtilis AJ11487 was able to grow in a medium containing both 2.0 mg/ml 4-fluorotryptophan and 1000 μg/ml DM, but these parent strains AJ11481, which is AJ11481, could not grow on any of the media. [Productivity test] Glucose 12g/dl, NH ₄ Cl2g/dl, KCl0.2
g/dl, KH ₂ PO ₄ 0.1g/dl, MgSO ₄・7H ₂ O0.04
g/dl, FeSO ₄・7H ₂ O0.001g/dl, MnSO ₄・
20 ml of a medium containing 0.001 g/dl of 4H ₂ O and 0.4 g/dl of casamino acids and adjusted to pH 7.0 was placed in a 500 ml Sakaguchi flask and sterilized at 115° C. for 10 minutes. After adding 0.8g of separately sterilized calcium carbonate to this,
One platinum loop of Bacillus subtilis shown in 2 was inoculated and cultured with shaking at 30°C for 90 hours. Also, glucose 8g/dl, NH ₄ Cl 1g/dl,
KCl0.2g/dl, KH ₂ PO ₄ 0.1g/dl, MgSO ₄・
_7H2O0.04g /dl, _FeSO4・_7H2O0.001g /dl,
MnSO ₄・4H ₂ O0.001g/dl, casamino acid 0.4g/
dl, d-biotin 30μg/dl, thiamine 10μ
20 ml of a medium containing g/dl and adjusted to pH 7.0 was placed in a 500 ml Sakaguchi flask and sterilized at 115°C for 10 minutes. After adding 0.8 g of separately sterilized calcium carbonate, one platinum loop of each strain other than Bacillus subtilis shown in Table 2 was inoculated and cultured with shaking at 30°C for 72 hours. Table 2 shows the amount of L-tryptophan accumulated.
It was shown to. 【table】

Claims (1)

[Claims] 1. A mutant strain belonging to the genus Bacillus, Brevibacterium, Corynebacterium, Arthrobacter, Microbacterium, or Escherichia and resistant to at least indolmycin or N-demethyl-C-demethyl-indolmycin. 1. A method for producing L-tryptophan, which comprises aerobically cultivating L-tryptophan in a liquid medium and collecting L-tryptophan produced and accumulated in the medium.