JP2995816B2 - Production method of L-lysine by fermentation method - Google Patents

Description

The present invention relates to a method for producing L-lysine by a fermentation method.
L-lysine is an important amino acid used for feeds and pharmaceuticals.

[Conventional technology]

Conventionally, as a method for producing L-lysine by fermentation,
A method using a-(2-aminoethyl) -L-cysteine (hereinafter referred to as AEC) -resistant bacterium (JP-B-48-28078), a method using L-homoserine and an L-threonine-requiring mutant ( JP-B-42-55213), AEC-resistant and L-leucine, L-homoserine, L-proline, L-
Methods using arginine or L-alanine auxotrophs (JP-A-49-36888, JP-B-55-1040 and JP-B-51-21078), use of β-fluoropyruvic acid-sensitive mutants (Japanese Patent Publication No. 57-14157)
A method using a mutant strain having reduced pyruvate kinase activity (JP-A-58-170487) and a method using a mutant strain having reduced pyruvate kinase activity and pyruvate dehydrogenase activity (JP-A-60-168393) Etc. are known.

[Problems to be solved by the invention]

An object of the present invention is to further increase the production yield of L-lysine by an L-lysine-producing mutant of the genus Brevibacterium.

[Means for solving the problem]

The present inventors have conducted various studies in order to solve the above-mentioned problems, and as a result of further imparting α-ketobutyric acid resistance to a conventionally known L-lysine-producing bacterium belonging to the genus Brevibacterium, -Larger amounts of L than lysine-producing bacteria
-Found to produce lysine. The present invention has been completed as a result of further research based on this finding.

The microorganism used in the method for producing L-lysine of the present invention is a mutant strain belonging to the genus Brevibacterium, resistant to α-ketobutyric acid, and capable of producing L-lysine. In addition to these properties, AEC resistance, L-homoserine requirement, L-threonine requirement, L-leucine requirement,
L-alanine auxotrophy, γ-methyllysine resistance, α-chlorocaprolactam resistance, β-fluoropyruvate sensitivity,
By using a strain imparted with properties such as reduced citrate synthase activity, reduced pyruvate kinase activity, reduced pyruvate dehydrogenase activity, the ability to produce L-lysine can be further increased.

The parent strain of the present invention is a microorganism of the genus Brevibacterium known as a so-called L-glutamic acid producing bacterium.
For example, Brevibacterium debaricatum ATCC14020 Brevibacterium flavum ATCC14067 Brevibacterium lactofermentum ATCC13869 Brevibacterium roseum ATCC13825 and the like. The mutant strain used in the present invention is subjected to a mutation operation using these strains as a parent strain to obtain α-ketobutyric acid resistant and L-
It can be obtained by imparting lysine-producing ability, for example, AEC resistance. The mutation operation was performed by ultraviolet irradiation, N-methyl-
It can be performed by a conventional mutation treatment method such as treatment with a mutagenic agent such as N'-nitro-N-nitrosoguanidine (hereinafter referred to as NG), sulfurous acid and the like.

The following are two examples of strains used in the present invention:
An experimental example showing specific methods for mutagenesis isolation of Flavam AJ12526 (FERM P-11490) and Brevibacterium flavum AJ12527 (FERM P-11491) and the degree of their resistance to α-ketone butyric acid is shown.

Brevibacterium flavum FA1-30 (K.Sano, I.Sh
Microbiol, 16 , 373 (1970), which is the same strain as FAECI-30 (FERMP-282) described in Japanese Patent Publication No. 48-28078). The induction of Brevibacterium flavum AJ12526 is as follows. Parent strain FA1−
30 was treated with 1500 μg / m of NG at 30 ° C. for 15 minutes (survival rate 4.1
%), And a plate medium prepared by adding 7 mg / m α-ketobutyric acid, a concentration at which the parent strain cannot grow, to the synthetic medium shown in Table 1 was applied. After cultivation at 30 ° C. for 8 days, a strain that grows as a colony on the medium, that is, an α-ketobutyric acid-resistant strain was collected, and a mutant AJ1252 excellent in L-lysine producing ability was obtained therefrom.
Six strains were selected. Brevibacterium flavum AJ1
Induction of 2527 was induced in the same manner as AJ12526 strain, but NG
The production rate in the treatment was 33%, and 4 mg / m DL-aspartic acid hydroxamate was added to the synthetic medium in addition to 4 mg / m α-ketobutyric acid, and the resistance appeared in the culture at 30 ° C. for 7 days. Selected from among the strains.

Next, the results of examining the degree of resistance of these AJ12526 strains and AJ12527 strains to α-ketobutyric acid are shown in Table 2 together with the results for DL-aspartate hydroxamate and the coexistence thereof with α-ketobutyric acid. A medium with a composition excluding agar from the synthetic medium shown in Table 1 (dispensed into test tubes in 5 ml aliquots) at 30 ° C and 16 ° C
The FA1-3 strain, AJ12526 strain and AJ12527 strain cultured for
Table 2 shows a plate medium (diameter 8.5 cm) prepared by dissolving α-ketobutyric acid and DL-aspartate hydroxamate in the synthetic medium shown in Table 1 so as to have the concentrations shown in Table 2. After inoculation, the cells were cultured at 30 ° C. for 3 days. Table 2 shows the growth status of each strain.

The term "α-ketobutyric acid resistance" as used in the present invention means that about 10 ⁷ microorganisms were inoculated into the above-mentioned medium containing 5 mg / ml of α-ketobutyric acid under the above-mentioned culture conditions, and grown at a medium or higher level after culturing at 30 ° C for 3 days. To indicate the case.

The parent strain FA1-30 is resistant to 5 mg / m of DL-aspartic acid hydroxamate.
-Increased sensitivity to ketobutyric acid, and mutant AJ12527 isolated under these conditions showed strong resistance to α-ketobutyric acid in the presence of DL-aspartate hydroxamate.

The culture medium for producing L-lysine is not particularly limited, and is a usual medium containing a carbon source, a nitrogen source, an inorganic salt, and if necessary, an organic micronutrient. Carbon sources include carbohydrates (glucose, fructose or starch, hydrolysates such as cellulose, molasses, etc.), organic acids (acetic acid, citric acid, etc.), alcohols (glycerin, ethanol, etc.)
Alternatively, hydrocarbons (such as normal paraffin) can be used. Ammonium sulfate, urea, ammonium nitrate, ammonium phosphate, ammonium chloride, ammonia gas, etc. are required as nitrogen sources, and phosphates, magnesium salts, calcium salts, iron salts, manganese salts, and other trace metal salts are required as inorganic salts. Use according to. As the organic trace nutrients, when there is an auxotrophy, appropriate amino acids, vitamins, fatty acids, organic base substances, etc. are added in an appropriate amount, and if necessary, amino acids, vitamins, taste liquids (registered trademark) are further added as growth promoting substances. , Soybean hydrolyzate), yeast extract, peptone, casamino acid and the like.

The cultivation may be carried out in a usual manner at pH 5 to 9, at a temperature of 20 to 40 ° C. under aerobic conditions for 24 to 72 hours. If the pH drops during the cultivation, calcium carbonate is separately sterilized and added, or neutralized with an alkali such as aqueous ammonia or ammonia gas. When organic acid is used as carbon source, pH
Neutralized with mineral or organic acids.

The collection of L-lysine from the culture solution is usually performed by combining an ion exchange resin method or other known methods,
Depending on the type of culture medium, it can be carried out by the direct crystallization method. The result is Rf on the thin layer chromatogram.
The L-lysine was identified as L-lysine by confirming its agreement with those of the L-lysine sample by the value, elution time in liquid high-performance chromatography, or biological activity value by microbial quantification. Hereinafter, the present invention will be further described with reference to Examples.

〔Example〕

Medium for production of L-lysine having the composition shown in Table 3 below
Was dispensed into 500-m shoulder flasks, and two platinum loops of each of the strains shown in Table 4 were inoculated and cultured with shaking at 30 ° C. for 72 hours. The amount of L-lysine produced in each culture was as shown in Table 4. The quantification of L-lysine was performed by an acid-copper ninhydrin colorimetric method.

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Claims (1)

(57) [Claims] 1. A method of culturing a mutant strain belonging to the genus Brevibacterium, which is resistant to α-ketobutyric acid and has the ability to produce L-lysine, and collecting L-lysine produced and accumulated. For producing L-lysine by a continuous fermentation method