JPH0212560B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing L-histidine by fermentation. Conventionally, methods for producing L-histidine (hereinafter abbreviated as histidine) by fermentation have been carried out using mutations that belong to the genus Brevibacterium and require amino acids such as L-arginine and L-methionine and are resistant to 2-thiazolealanine. A method using a strain (Japanese Patent Publication No. 51-23593) or a method using a 2-thiazolealanine and sulfur drug-resistant mutant strain belonging to the genus Brevibacterium (Japanese Patent Publication No. 51-23594) is known. On the other hand, it is known that 2-thiazolealanine-resistant mutant strains of microorganisms of the genus Pacillus produce histidine (Tokuko Showa).
50−13358), the amount of histidine produced and accumulated is 0.1
g/dl, which was significantly inferior to Brevibacterium mutant strains. The present inventors conducted various studies with the aim of obtaining mutant strains that belong to the genus Bacillus and have the ability to accumulate significant amounts of histidine. As a result, the present inventors conferred resistance to purine analogs on histidine-producing bacteria that are resistant to histidine analogs and belong to the genus Bacillus. Among these mutant strains, we found that some mutant strains have the ability to accumulate significant amounts of histidine. The present invention was completed based on this knowledge. The present invention will be explained below. The histidine analog used in the present invention refers to a chemical substance that inhibits the growth of microorganisms belonging to the genus Bacillus, but this growth inhibition is reversed by the presence of histidine, such as 2-thiazolealanine, α
-methylhistidine, 1,2,4-triazolealanine, 3-amino-1,2,4-triazole, and the like. In addition, purine analogs inhibit the growth of microorganisms of the genus Bacillus, and this growth inhibition is caused by adenine,
Refers to chemical substances that are recovered by the presence of purines such as guanine and inosine, such as 8-azaxanthin,
8-Azahypoxanthine, 6-mercaptopurine, 6-mercaptoguanosine, 6-azathioprine, 6-methoxypurine, 6-methylmercaptopurine, 2-chloroadenosine, 8-bromoguanine, 8-mercaptoguanisine, 2,6 -Diaminopurine, etc. The microorganism used in the present invention belongs to the genus Bacillus,
It is a mutant strain that is resistant to histidine analogs and preanalogs and has the ability to produce histidine,
For example, the following mutant strains are used. Bacillus subtilis AJ 11755 FERM−
P6272 (2TA〓, 8AG〓) Bacillus subtilis AJ 11756 FERM-P
6273 (2-TA〓,
6MP〓) Bacillus subtilis AJ 11757 FERM-P
6274 (2-TA〓,2,
6DAP〓) 2TA〓: 2-thiazolealanine resistant 8AG〓: 8-azaguanine resistant 6MP〓: 6-mercaptopurine resistant 2,6DAP〓: 2,6-diaminopurine resistant All of the above mutant strains are resistant to histidine analog (2TA). This is a mutant strain obtained by conventional mutagenesis operations using Bacillus subtilis T-100 FERM-P3800 (Japanese Patent Publication No. 13358/1983), a resistant histidine-producing bacterium, as the parent strain. The mutant strain of the present invention can be obtained by using a histidine-producing mutant strain resistant to histidine analogs as a parent strain as described above, or can also be obtained by a method of sequentially imparting resistance to these drugs to wild strains of the genus Bacillus. . As a mutation induction method, for example, a conventional method using ultraviolet irradiation or treatment with a chemical agent such as N-methyl-N'-nitroso-N-nitrosoguanidine (hereinafter abbreviated as NG) or nitrous acid may be used. A specific method for inducing mutations in AJ11755, AJ11756, and AJ11757 is shown in the following experimental example. Experimental example Bacillus subtilis T-100FERM-P3800 cells grown on a bouillon agar topslant medium were suspended in 1/30M phosphate buffer (pH 7.0) containing 250γ/ml NG (number of cells). 10 ⁹ /ml), 30 at 30℃
The cells were held for a minute and subjected to mutation processing. Next, the bacterial cells were collected by centrifugation, washed with the same buffer, and then transferred to the minimum agar plate medium shown in Table 1 containing 2.00 μ/ml of 8-azaguanine, 6-mercaptopurine, or 2,6-diaminopurine. The plate was plated and cultured at 30°C for 3 to 10 days.

[Table] When we investigated the histidine-producing ability of colonies that appeared on plates containing these purine analogs, we found that there were many mutant strains that clearly had a higher ability to accumulate histidine than the parent strain, and among them, mutant strains that were resistant to 8-azaquanine. AJ11755, a mutant strain AJ 11756 showing resistance to 6-mercaptopurine, and a mutant strain AJ 11757 showing resistance to 2,6-diaminopurine showed high histidine production ability. Table 2 shows the degree of resistance of the resistant mutant strains thus obtained to various drugs.

[Table] The growth rates in Table 2 are determined by washing the cells of each mutant strain thoroughly with a minimal medium, and then adding 2-thiazolealanine, 8-azaguanine, 6-mercaptopurine, or 2,6-diaminopurine. Minimal medium containing 5
Inoculated at ×10 ⁶ /ml, cultured with shaking at 30°C for 40 hours,
This is a value obtained by measuring the absorbance of the obtained culture solution at 562 hm, and is shown as a relative value. There are no particular difficulties in producing histidine using these mutant strains, as they can be cultured using a normal nutrient medium containing carbon sources, nitrogen sources, inorganic salts, and, if necessary, organic micronutrients. . Carbon sources include sugars such as glucose, sucrose, molasses, and starch hydrolysates, organic acids such as benzoic acid, acetic acid, and propionic acid, alcohols such as ethanol and propanol, and, depending on the bacteria, hydrocarbons. can. Nitrogen sources include ammonium sulfate, ammonium nitrate, ammonium chloride, ammonium phosphorus,
Urea, aqueous ammonia, ammonia gas and other common sources of nitrogen can be used. The main fermentation conditions are aeration culture, and the fermentation temperature is 24℃.
to 37°C, and the number of fermentation days is usually 2 to 7 days. The pH at the start of fermentation and during culture is preferably 5.0 to 9.0, and inorganic or organic acidic or alkaline substances, such as urea, calcium carbonate, ammonia, etc., can be used to adjust the pH. Collection of histidine from the fermentation liquor is usually carried out by a combination of the ion exchange resin method and other known methods. When cultured in this manner, histidine is accumulated in the culture medium at 0.4 to 0.8 g/dl. This accumulation amount is significantly increased compared to the conventional accumulation amount of about 0.1 g/dl for mutant strains of the genus Bacillus, and the method of the present invention uses microorganisms of the genus Bacillus. It is important as it makes industrial production possible. In addition, the quantitative determination of L-histidine was performed using Kapeiler-
A colorimetric method using Alder reaction [Biochem Z., 264 131 (1933)] was used. The present invention will be specifically explained below using Examples. Example 1 Glucose 10g/dl, KH ₂ PO ₄ 0.1g/,
NH ₄ Cl 2.0g/dl, MgSO ₄・7H ₂ O 0.04g/dl,
KCl 0.2g/dl, Fe and Mn ions each
Contains 2ppm CaCO ₃ 4.0g/dl (separately sterilized), pH 7.0
A regulated medium was prepared, and 20 ml of it was dispensed into a 500 ml shake flask. After sterilization, one platinum loop of each of the various bacterial strains shown in Table 3, which had been grown on a bouillon slant in advance, was inoculated and cultured with shaking at 31°C for 96 hours. The amount of histidine accumulated in the obtained culture solution was as shown in Table 3.

[Table] Supernatant liquid 1 obtained by removing bacterial cells and calcium salts from the culture solution of AJ11755 by centrifugation ^is
type) to adsorb histidine. Next, the adsorbed histidine was eluted with 3% aqueous ammonia, and the eluate was concentrated under reduced pressure. When the concentrated solution was cooled and left to stand, histidine crystals precipitated. The crystals were dried to obtain 5.0 g of crystals.

Claims (1)

[Claims] 1 A mutant strain belonging to the genus Bacillus that is resistant to histidine analogs and purine analogs and has the ability to produce L-histidine is cultured in a liquid medium to produce L-histidine.
- A method for producing L-histidine by a fermentation method, which comprises producing and accumulating histidine and collecting it.