JPH0488991A - Production or l-lysine by fermentation - Google Patents

Abstract

PURPOSE:To obtain L-lysine useful for feed and drug by culturing a specific variant belonging to the genus Brevibacterium. CONSTITUTION:A variant [e.g. Brevibacterium flavum AJ 12,526 (FERM P-11,490)] belonging to the genus Brevibacterium, having resistance to alpha- ketobutyric acid, capable of producing L-lysine, is cultured in a medium containing a carbon source, a nitrogen source, an inorganic salt, etc., at pH 5-9 at 20-40 deg.C for 24-72 hours and formed and accumulated L-lysine is collected.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a method for producing L-lysine by a fermentation method. L
-Lysine is an important amino acid used for feed and medicine.

[Conventional technology]

Conventionally, as a method for producing L-lysine by fermentation method, S-
Method using (2-aminoethyl)-L-cysteine (hereinafter referred to as AEC)-resistant bacteria (Japanese Patent Publication No. 48-28078
method using L-homoserine, L-threonine auxotrophic mutant strain (Japanese Patent Publication No. 1983-55213),
AEC resistant and L-leucine, L-homoserine, L-
Method using proline, L-arginine, or L-alanine auxotrophic mutant strains (Japanese Patent Application Laid-Open No. 49-36888,
Japanese Patent Publication No. 55-1040, equity [51-21078 publications], method using β-fluoropyruvate sensitive mutant strain (Japanese Patent Publication No. 57-14157), method using mutant strain with decreased pyruvate kinase activity (Unexamined Japanese Patent Publication No. 58-1
70487) and a method using a mutant strain with decreased pyruvate kinase activity and pyruvate dehydrogenase activity (Japanese Patent Application Laid-open No. 168393/1983).

[Problem to be solved by the invention]

L produced by an L-lysine producing mutant strain of Brevibacterium sp.
-The aim is to further increase the production yield of lysine.

[Means to solve the problem]

As a result of various studies to solve the above-mentioned problems, the present inventors further imparted resistance to α-ketobutyric acid to a previously known L-lysine producing bacterium of the genus Brevibacterium. - Even more L than lysine-producing bacteria -
discovered that it produces lysine. The present invention was completed as a result of further research based on this knowledge.

The microorganism used in the L-lysine production method of the present invention belongs to the genus Brevibacterium and is a mutant strain that is resistant to α-ketobutyric acid and has the ability to produce L-lysine. In addition to these properties, EC resistance, L-homoserine requirement, L-threonine requirement, L-leucine requirement, L-
Use a strain that is endowed with properties such as alanine auxotrophy, T-methyllysine resistance, α-chlorocaprolactam resistance, β-fluoropyruvate sensitivity, decreased citrate synthase activity, decreased pyruvate kinase activity, and decreased pyruvate dehydrogenase activity. Accordingly, the production capacity of L-lysine can be further increased.

The parent strain of the present invention is a microorganism of the genus Brevibacterium, which is known as a so-called L-glutamic acid producing bacterium. For example, Brevibacterium devaricatum ATCC1402
0 Brevibacterium flavum ATCC140
67 Previbacterium lactofermentum ATC
C13869 Brevibacterium roserum ATCC1382
There is a 5th grade. The mutant strains used in the present invention are obtained by mutating the above-mentioned bacterial strains as parent strains, resulting in α-ketobutyric acid resistance and L
- Obtained by imparting lysine production ability, such as AEC resistance. The mutation operation was performed using ultraviolet irradiation, N-methyl-N'-nitro-N-nitrosoguanidine (hereinafter NG
), treatment with a mutagenic agent such as nitrous acid, and other conventional mutagenesis methods can be used.

Below are two examples of strains used in the present invention, Brevibacterium flavum AJ12526 (FERM P-11490) and Brevibacterium flavum AJ12527 (FER
Examples of experiments showing a specific method for mutagenic isolation of M P-11491) and their degree of resistance to α-ketobutyric acid are shown below.

Brevibacterium flavum FA l-30 (K, 5
ano.

1.5hiio, J, Gen, Appl, Microb
See iol 16+373 (1970), Special Publication Showa 4B
-FAECI-30 (FER?
IP-282) was used as the parent strain.

The derivation of Brevibacterium flavum AJ12526 follows. The parent strain FAI-30 was 1500. cTg
/dNONG for 30″C1s (survival rate 4.1
%), and then applied to a plate medium prepared by adding α-ketobutyric acid at a concentration of 7 μ/i, which is a concentration at which the parent strain cannot grow, to the synthetic medium shown in Table 1. After culturing at 30°C for 8 days, the strain growing as a colony on the ground, that is, the α-ketobutyric acid resistant strain, was collected, and the mutant strain AJ with excellent L-lysine production ability was collected.
12,526 stocks were selected. On the other hand, Previbacterium flavum AJ12527 was induced in the same manner as the AJ12526 strain, but the survival rate in NG treatment was 33%.
, and in addition to 4■/- α-ketobutyric acid in the synthetic medium, 4■/-
DL-aspartic acid hydroxamate of xi was added and cultured at 30° C. for 7 days, and resistant strains that appeared were selected.

Table 1 Synthetic medium composition components Concentration Glucose 20 g / 1 Ammonium sulfate 10 g/l Monopotassium phosphate 1 g/l Magnesium sulfate
Heptahydrate 0.4 g / Ferrous sulfate heptahydrate
10■/l manganese sulfate tetrahydrate
8■/Itd-Biotin 300
μg/l Vitamin Bl - HCl1100μg/l! 2
(N-morpholino) 19.52g/
It ethanesulfonic acid L-serine L-alanine-isoleucine L-cysteine L-proline L-threonine L-lysine L-arginine-methionine-valine agar (neutralized to pfi 7°0 with caustic soda) 50■/1 50 ■/1 50■/1 50■/E 50■/1 50■/1 50■/1 50■/1 200■/1 100■/1 20 g/1 Next, α- of these AJ12526 and AJ12527 strains
The results of examining the degree of tolerance to ketoacetic acid are shown in Table 2 together with the results for DL-aspartic acid hydroxamate and its coexistence with α-ketobutyric acid.

A medium (51d
FAI cultured at 30°C for 16 hours (dispense into test tubes)
-30 stocks, AJ12526 stocks and AJ12527 stocks,
A plate medium (diameter 8.5 CII) prepared by dissolving α-ketobutyric acid and DL-aspartic acid hydroxamate in the synthetic medium shown in Table 1 to the concentrations shown in Table 2 was added to the synthetic medium shown in Table 2. After inoculating the indicated amount of bacteria, the cells were cultured at 30°C for 3 days. Table 2 shows the growth status of each strain.

In the present invention, α-ketobutyric acid resistance refers to approximately 107 microorganisms inoculated into the above medium containing α-ketobutyric acid at 5μ/d under the above culture conditions, and after culturing at 30°C for 3 days, medium or higher growth is observed. This is the case when it shows.

In addition, the parent strain FAI-30 is resistant to DL-aspartate hydroxamate at 5■/-, but coexistence with this increases the sensitivity to α-ketobutyric acid, and the mutant strain AJ12527 isolated under this condition is DL-aspartate hydroxamate. It showed strong resistance to α-ketobutyric acid in the presence of aspartate hydroxamate.

The culture medium for L-lysine production is not particularly limited (it is a conventional medium containing a carbon source, a nitrogen source, inorganic salts and, if necessary, organic micronutrients).

Carbon sources include carbohydrates (glucose, fructose or starch, hydrolysates of cellulose, molasses, etc.);
Organic acids (acetic acid, citric acid, etc.), alcohols (glycerin, ethanol, etc.), or hydrocarbons (normal paraffin, etc.) can be used. Ammonium sulfate as a nitrogen source,
Urea, ammonium nitrate, ammonium phosphate, ammonium chloride, ammonia gas, and others are used as necessary, and as inorganic salts, phosphates, magnesium salts, calcium salts, iron salts, manganese salts, and other trace metal salts are used. As organic micronutrients, appropriate amounts of amino acids, vitamins, fatty acids, organic basic substances, etc. are added if there is a nutritional requirement, and if necessary, amino acids, vitamins, and flavor liquid (registered trademark) are added as growth-promoting substances. , soybean hydrolysate), yeast extract, peptone, casamino acids, etc. can be used.

The culture conditions may be as follows: pH 5 to 9, temperature 20 to 40° C., and aerobic conditions for 24 to 72 hours. If the pH decreases during culture, calcium carbonate should be separately sterilized and added, or neutralized with an alkali such as aqueous ammonia or ammonia gas. Further, when an organic acid is used as a carbon source, the increase in pH is neutralized with a mineral acid or an organic acid.

Collection of L-lysine from the culture solution is usually carried out by a combination of ion exchange resin methods and other known methods, and depending on the type of medium, it can also be carried out by direct crystallization. The obtained product was confirmed to be L-lysine based on the Rf value on the thin layer chromatogram, the elution time in liquid high-performance chromatography, and the biological activity value determined by microbial quantitative method, and was confirmed to match that of the L-lysine sample. .

The present invention will be further explained below with reference to Examples.

〔Example〕

L-lysine production medium 2 (l) having the composition shown in Table 3 below
d was dispensed into a 500-capacity flask with a shoulder, and each strain shown in Table 4 was seeded with two platinum ear plugs, and cultured with shaking at 30° C. for 72 hours. The amount of L-lysine produced in each culture solution was as shown in Table 4. The amount of lysine was determined by acidic copper ninhydrin colorimetry.

Table 3 Medium components for L-lysine production Concentration Glucose 100 g/I
Ammonium sulfate 90g/l Potassium phosphate 1g/J Magnesium sulfate heptahydrate 0.4g/l Ferrous sulfate heptahydrate 10/1 Manganese sulfate tetrahydrate
8■/ld-biotin 300
μg/l vitamin B+-HCj! 200
μg/1 soy protein hydrolyzate” 55w
1/1 (pH 7,0) 1 Semi-finished product of taste liquid J (total nitrogen content 3.38 g/d!>Table 4 Lysine production amount AJ12526 AJ12527 Yes 29.4 36.6

Claims (1)

[Claims] A fermentation method characterized by culturing a mutant strain belonging to the genus Brevibacterium and having resistance to α-ketobutyric acid and the ability to produce L-lysine, and collecting L-lysine produced and accumulated. -Method for producing lysine