JPS6351678B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing L-histidine by fermentation. As a method for producing L-histidine by a fermentation method, a method using a mutant strain of the genus Brevibacterium or Corynebacterium that is resistant to histidine analogs such as 2-thiazolealanine (US Pat. No. 3,716,453) is known. ing. The present inventors conducted research to find a more efficient L-histidine producing bacterium, and as a result, a mutant strain derived from a microorganism of the genus Brevibacterium or Corynebacterium, which has an improved ability to assimilate gluconic acid compared to the parent strain, was found. It was found that L-histidine was produced in higher yield. This invention has been completed based on this knowledge. The microorganism used in the present invention is a mutant strain of the genus Brevibacterium or Corynebacterium that has an improved ability to assimilate gluconic acid compared to the parent strain and has the ability to produce L-histidine. It is known that in order for microorganisms of the genus Brevibacterium and Corynebacterium to have the ability to produce L-histidine, they only need to have resistance to histidine analogs such as 2-thiazolealanine. To obtain the mutant strain of the present invention, L-
After imparting the histidine production ability, the gluconic acid assimilation ability may be improved, or conversely, the gluconate assimilation ability may be improved first. The wild strains used as parent strains for inducing this mutant strain are those of the genus Brevibacterium or Corynebacterium that are particularly known as coryneform L-glutamate-producing bacteria, such as the following: be. Brevibacterium deivalicatum
ATCC 14020 Brevibacterium flavum ATCC 14067 Brevibacterium lactofermentum
ATCC 13869 Brevibacterium satucaroliticum
ATCC 14066 Corynebacterium acetoacid film
ATCC 13870 Corynebacterium glutamicum
ATCC 13032 To induce mutagenesis of the mutant strain of the present invention from these parent strains, conventional mutagenesis methods such as contacting with N-methyl-N'-nitro-N-nitrosoguanidine can be appropriately applied. The method of isolating the mutant strain of the present invention from a mutation-treated strain is carried out by collecting a mutant strain that can grow better than the parent strain using gluconic acid as the only carbon source. Specifically, Brevibacterium flavum
AJ11611 (FERM-P 5653) and Corynebacterium acetoacidophyllum AJ 11612
(FERM-P 5654) was obtained by performing the following mutation treatment. Brevibacterium flavum AJ 3596
(FERM-P 2262) (2-thiazolealanine resistance, sulfamethomidine resistance, 2-aminobenzothiazole resistance) or Corynebacterium acetoacidophyllum AJ 3390 (ERM-P 1569)
(2-thiazolealanine resistance, lysine requirement)
cells were suspended in 0.1N phosphate buffer, N-methyl-N'-nitro-N-nitrosoguanidine was added to the suspension to a final concentration of 250 μg/ml, and the suspension was incubated at 31.5°C for 10 Shake for a minute. The strain after mutation treatment was treated with gluconic acid 2g/dl and ammonium sulfate.
0.2g/dl, L-glutamic acid 0.3g/dl,
KH ₂ PO ₄ 0.1g/dl, urea 0.2g/dl, MgSO ₄・
7H ₂ O 0.04g/dl, FeSO ₄・7H ₂ O 0.001g/dl,
_MnSO4・_nH2O 0.001g/dl, thiamine hydrochloride
10μg/dl, biotin 50μg/dl, agar 2.0g/dl
The cells were coated on a minimal medium (PH7.2) consisting of the following, and incubated at 31.5°C for 2 to 10 days. Colonies that have grown on the medium are fished out using a conventional method, and then added to a medium containing gluconic acid (gluconic acid 2
g/dl, ammonium sulfate 0.2g/dl, L-glutamic acid 0.3
g/dl, KH ₂ PO ₄ 0.1g/dl, urea 0.2g/dl,
MgSO ₄・7H ₂ O 0.04g/dl, FeSO ₄・7H ₂ O
0.001g/dl, MnSO ₄・nH ₂ O 0.001g/dl, VB ₁
-HCl 10μg/dl, biotin 5.0μg/dl, PH
7.2) Dispense 5 ml into a test tube and incubate for 1 to 2 days to obtain Brevibacterium flavum AJ 11611 with improved gluconic acid assimilation ability than the parent strain.
and Corynebacterium acetoacidoflame
AJ 11612 was selected. Selected mutant strain with improved gluconate assimilation ability
As shown in Table 1, AJ 11611 and AJ 11612 have improved gluconic acid assimilation ability compared to parent strains AJ 3596 and AJ 3390, and can be clearly distinguished. The test method was to use the medium containing gluconic acid and culture in the same manner as described above.

[Table] In addition, the mutant strain of the present invention has requirements for methionine, tryptophan, phenylalanine, etc.
-41592), sulfur drugs (Japanese Patent Application Laid-open No. 50-49490), 5-
Resistance to drugs such as methyltryptophan, α-aminohydroxyvaleric acid, imidazole, 3-amino 1 ^H -1.2.4-triazole (Japanese Patent Publication No. 50-49491), cobalamin (Japanese Patent Application Publication No. 50-70591), etc. The yield is often improved by further adding properties that are already known to improve the productivity of L-histidine, such as. The medium in which these microorganisms are cultured is a conventional medium containing a carbon source, a nitrogen source, inorganic ions and, if necessary, other organic micronutrients. Carbon sources include glucose, sucrose,
Carbohydrates such as starch hydrolysates, organic acids such as acetic acid, and others can be used. Suitable nitrogen sources include ammonia gas, aqueous ammonia, ammonium salts, urea, and the like. It is preferable to culture under aerobic conditions, and the culture medium should be
More favorable results can be obtained by adjusting the pH to 4 to 8 and the temperature to 25 to 37°C. Thus, if the culture is continued for 1 to 7 days, a significant amount of L-histidine will be produced and accumulated in the medium. L-histidine can be collected from the culture solution by a conventional method such as a method using an ion exchange resin. Example 1 Glucose 10g/dl, (NH ₄ ) ₂ SO ₄ 5g/dl,
KH ₂ PO ₄ 0.1g/dl, MgSO ₄・7H ₂ O 0.04g/
dl, FeSO ₄・7H ₂ O 1mg/dl, MnSO ₄・4H ₂ O71
mg/dl, biotin 50μg/dl, thiamine hydrochloride 50μg/dl, soy protein hydrolyzate (total nitrogen 7%) 0.3ml/dl, CaCO 3.5g/dl, (separate sterilization added) PH7.2 adjusted medium 20ml was dispensed into a 500ml shake flask. After sterilization, Brevibacterium grown on bouillon slant in advance.
Inoculate one platinum loop of Corynebacterium acetocidophilum AJ 11611 or Corynebacterium acetoacidophyllum AJ 11612,
They were cultured at 31°C for 72 hours and the results are shown in Table 1. Cells and calcium salts are removed from the culture solution of AJ 11611 and AJ 11612 by centrifugation, and the resulting supernatant 1 is passed through a strongly acidic ion exchange resin "Amberlite IR-120" (H ⁺ type). let me, 2
-Adsorbed histidine. Thereafter, the adsorbed L-histidine was eluted with 3% aqueous ammonia, and the eluate was concentrated under reduced pressure. When the concentrated liquid was cooled and left to stand, crystals of L-histidine were precipitated.

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Claims (1)

[Claims] 1. Cultivate a mutant strain that belongs to the genus Brevibacterium or Corynebacterium and has an improved gluconate assimilation ability compared to the parent strain and the ability to produce L-histidine, and produce L-histidine in the culture solution. A method for producing L-histidine, which comprises accumulating it and collecting it.