JPH0212559B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing L-histidine by fermentation. Conventionally, L-histidine (hereinafter simply referred to as histidine) has been produced by fermentation, which belongs to the genus Brevibacterium and requires amino acids such as L-arginine and L-methionine, and is resistant to 2-thiazolealanine. A method using a mutant 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)
etc. are known. On the other hand, it is known that 2-thiazolealanine-resistant mutant strains of microorganisms of the genus Pacillus produce histidine (Japanese Patent Publication No. 50-13358), but the amount of histidine produced and accumulated is 0.1
g/dl, which was significantly inferior to Brevibacterium mutant strains. The present inventors have conducted various studies with the aim of obtaining mutant strains belonging to the genus Bacillus that have the ability to accumulate significant amounts of histidine. We also found that among the mutant strains conferred tryptophan analog resistance, there are mutant strains that have the ability to accumulate a significant amount 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-methyl Examples include histidine, 1,2,4-triazolealanine, and 3-amino-1,2,4-triazole. In addition, tryptophan analogs inhibit the growth of microorganisms of the genus Bacillus, but this growth inhibition
Refers to chemicals that are recovered in the presence of tryptophan, such as DL-5-methyltryptophan, DL-5-fluorotryptophan, DL-5-
There are 4-fluorotryptophan, DL-6-fluorotryptophan, DL-7-azatryptophan, etc., and the sulfur drugs referred to in the present invention include (1) chemical structures similar to p-aminobenzoic acid; (2) Substances that inhibit the growth of microorganisms, but are inhibited by the addition of p-aminobenzoic acid, such as sulfadiazine, sulfisoxazole, sulfamerazine, sulfaguanidine, and sulfaguanidine. Examples include fuenazole, sulfamethoxyviridazine, and sulfathiazole. The microorganism used in the present invention belongs to the genus Bacillus,
Microorganisms that are resistant to histidine analogs and sulfur drugs or tryptophan analogs and have the ability to produce histidine are used, such as Bacillus subtilis AJ 11680 FERM-
P5967, AJ11684 FERM-P 6005 and
AJ11681FERM-P5968 is mentioned. This AJ11680 is Bacillus subtilis T-100FERM- which is resistant to histidine analogs.
It is a mutant strain obtained by mutagenesis using P3800 (Special Publication No. 50-13358) as the parent strain, and is resistant to 2-thiazolealanine and sulfaguanidine, and AJ11684 is resistant to 2-thiazolealanine and 5-thiazolelanine. This is a mutant strain that is resistant to fluorotryptophan. Furthermore, AJ11681 is
This mutant strain was obtained using AJ11680 as the parent strain, and is resistant to 2-thiazolealanine, sulfaguanidine, and 5-fluorotryptophan. 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 AJ11680 and AJ11681 is shown in the following 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 treated with sulfaguanidine or 5-fluorotryptophan.
The mixture was plated on a minimal agar plate medium containing 500 μg/ml shown in Table 1, and plated at 30° C. for 3 to 10 days.

[Table] Colonies with good internal growth that appeared on a plate containing sulfaguanidine were examined for their ability to produce histidine.
It accumulated 0.5 g/dl, showing clearly higher accumulation ability than the parent strain. Among these, the mutant strain AJ11680 with the highest histidine-producing ability was selected. Similarly, among the colonies that appeared on the plate containing 5-fluorotryptophan, AJ11684, a strain with high histidine production ability, was selected.
I chose. Next, do the same NG using AJ11680 as the parent strain.
A mutation operation was performed by treatment, and colonies appearing on a minimal agar medium containing 500 μg/ml of 5-fluorotryptophan and having the composition shown in Table 1 were isolated. Among these, the histidine-producing ability of those with good growth was investigated, and strain AJ11681, which had a particularly high histidine-producing ability, was isolated. Table 2 shows the degree of resistance of the thus obtained resistant mutant strains to various drugs.

[Table] The growth rates in Table 2 are determined by washing the cells of each mutant strain thoroughly in a minimal medium, and then placing them in a minimal medium containing 2-thiazolealanine, sulfuranidine, or DL-5-fluorotryptophan. 5×10 ⁶ /ml
The value was obtained by inoculating the culture solution with shaking at 30°C for 40 hours and measuring the absorbance at 562 nm of the resulting culture solution, 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. . As carbon sources, sugars such as glucose, molasses, and starch hydrolysates, organic acids such as benzoic acid, acetic acid, and propionic acid, alcohols such as ethanol and propanol, and even hydrocarbons can be used if the bacteria are selected. 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 cultivation is preferably 5.0 to 9.0, and inorganic or organic acidic or alkaline substances, such as urea, calcium carbonate, ammonia gas, 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 0.5 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 is suitable for the industrial production of histidine using microorganisms of the genus Bacillus. It is important as it makes possible production. 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/
A medium containing 0.2 g/dl of KCl, 2 ppm each of Fe and Mn ions, 4.0 g/dl of CaCO ₃ (separately sterilized) and adjusted to pH 7.0 was prepared, and 20 ml of the medium was dispensed into 500 ml shaking flasks. After sterilization, one platinum loop of each of the strains shown in Table 3, which had been grown in advance on a bouillon slant, 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 obtained by removing bacterial cells and calcium salts from the culture solution of AJ11681 by centrifugation 1 Strongly acidic ion exchange resin "Amberlite" IR-120 (H ⁺
type) to adsorb histidine. Thereafter, 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 5.7 g.

Claims (1)

[Claims] 1 A microorganism belonging to the genus Bacillus, resistant to (1) histidine analogs and (2) sulfur drugs or tryptophan analogs, and capable of producing L-histidine is cultivated in a liquid medium to produce L-histidine. - A method for producing L-histidine, which is characterized by accumulating it and collecting it.