JPS6257315B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing L-lysine by fermentation. More particularly, the invention belongs to the genus Corynebacterium or Brevibacterium and requires homoserine or homoserine substitutes (threonine and methionine, threonine and cystathion or threonine and homocysteine), leucine and pantothenic acid for growth;
cultivating an L-lysine-producing microorganism in a nutrient medium that is resistant to lysine analogs, requires homoserine or a homoserine substitute and nicotinic acid for growth, or requires inositol for growth;
The present invention relates to a method for producing L-lysine by a fermentation method, which is characterized by accumulating L-lysine in a culture and collecting L-lysine from the culture. An object of the present invention is to provide an industrially inexpensive method for producing L-lysine, which has a wide range of uses, including as a feed additive, a food additive, a raw material for pharmaceuticals, and more. Conventionally, many methods for producing L-lysine by fermentation are known. Examples include the use of mutant strains of Micrococcus glutamicum (a synonym for Corynebacterium glutamicum) that require homoserine, threonine and methionine, threonine and cystathion, or threonine and homocysteine (i.e., homoserine or a homoserine substitute). (Special Publication No. 36-6499) Method using threonine-, phenylalanine-, histidine-, methionine-, cysteine-, leucine- or isoleucine-requiring mutants of Brevibacterium ammoniagenes (British Patent No. 1118719), Brevibacterium, Brevibacterium Brevibacterium lactofamentum, method using S-2-aminoethyl-L-cysteine (hereinafter abbreviated as AEC) (one of the lysine analogues) resistant mutant strains (Special Publication No. 48-28078), Brevibacterium lactofamentum, Corynebacterium A method using a mutant strain of Bacterium acetoglutamicum resistant to AEC and leucine analogs (Special Publication No. 53-1833) A method that belongs to Brevibacterium lactofamentum and requires nicotinamide or nicotinamide and leucine, etc. A method using a mutant strain that is resistant to AEC (Japanese Patent Laid-Open No. 49-36888), a method that uses a mutant strain that belongs to Brevibacterium lactofamentum and requires pantothenic acid, serine, etc. and is resistant to AEC (Japanese Patent Laid-Open No. 49-1794). In order to cope with the increasing demand for L-lysine, the present inventors have made various attempts to improve the bacteria used.
It has been discovered that when culture is performed using an L-lysine producing bacterium belonging to the genus Corynebacterium or Brevibacterium and having the above-mentioned properties, a significant amount of L-lysine accumulates in the culture. completed. Next, the present invention will be explained in more detail. The microorganisms used in the present invention belong to the genus Corynebacterium or Brevibacterium,
Homoserine or homoserine substitute for growth,
L that requires leucine and pantothenic acid and is resistant to lysine analogs, or that requires homoserine or homoserine substitutes and nicotinic acid for growth, or that requires inositol for growth.
- Any lysine-producing microorganism can be used. The microorganisms used in the present invention can generally be obtained as follows. That is, for example, a microorganism belonging to the genus Corynebacterium is used as a parent strain, and a mutant strain obtained by mutation induction treatment using a conventional method is selected from the mutant strains obtained, which have homoserine or homoserine substitute requirement and L-lysine productivity. do. The selected strain is then used as a parent strain to undergo mutation induction treatment, and a mutant strain having leucine auxotrophy is selected. Next, the mutant strain is used as a parent strain to undergo mutation induction treatment, and a mutant strain having lysine analog resistance is selected. The selected strain is then used as a parent strain to undergo mutation induction treatment, and a mutant strain having pantothenic acid requirement is selected. The order of imparting requirements and resistance is not limited to the above, and may be in any order. For example, using a parent strain as a mutant strain that requires pantothenic acid, this can also be obtained by imparting homoserine auxotrophy, leucine auxotrophy, and AEC resistance. Moreover, in addition to the above-mentioned mutational properties, mutant strains having other types of auxotrophic properties or other mutational properties can also be used in the present invention. Addition of other properties to the microorganism of the present invention and mutation treatment when a microorganism belonging to the genus Brevibacterium is used as a parent strain can also be carried out by the same method as described above. Methods of mutation induction include ultraviolet irradiation,
Methods such as radiation irradiation, nitrogen mustard treatment, and nitrosoguanidine treatment are used. As an example of a specifically suitable strain for use in the present invention, Corynebacterium glutamicum LYC
-3 (Microtechnology Research Institute No. 5039) (NRRL B-
11476) (homoserine requirement, leucine requirement, pantothenic acid requirement, AEC resistance), Brevibacterium flavum HN-103 (Feikoken No. 5040)
(NRRL B-11477) (homoserine auxotrophy, threnion auxotrophy, nicotinic acid auxotrophy), Flevibacterium flavum HI-4 (Feibacterium Bacterial Serial No. 5041
No.) (NRRL B-11478) (homoserine requirement, threnion requirement, inositol requirement). The parent strain of the LYC-3 strain is the L-lysine producing bacterium ATCC21526 (homoserine auxotrophy, leucine auxotrophy, AEC resistance) of Corynebacterium glutamicum, and the HN-103 and HI-4 strains are L-lysine producing bacteria of Brevibacterium flavum. -Lysine producing bacteria
It was derived from ATCC21128 (homoserine auxotrophy, threonine auxotrophy) as a parent strain, treated with N-methyl-N'-nitro-N-nitrosoguanidine, and then treated with conventional methods. In other words, each of these parent stocks was
Slant medium (yeast extract 0.5g/dl, meat extract 1g/dl, peptone 1g/dl, NaCl 0.5
G/dl, agar 2 g/dl, pH 7.2) for 24 hours at 30°C. Then, the bacterial cells were scraped off, and the cells containing 200γ/ml of N-methyl-N′-nitro-N-nitrosoguanidine were collected.
Suspend at approximately 10 ⁷ to 10 ⁹ cells/ml in 0.05M Tris-maleate buffer (PH 6.0) and incubate at room temperature.
Leave for 30 minutes. Thereafter, centrifuge at 3000 rpm for 15 minutes to collect bacteria, and wash with sterilized physiological saline. Next, the bacterial cells are suspended in 0.05M tris-maleate buffer (PH6.0) to about 10 ⁶ to 10 ⁸ cells/ml. A small amount (approximately 0.2 ml) of the suspension is added to a bouillon plate medium (total volume: approximately 20 ml) having the same composition as the bouillon slant medium, and the suspension is grown at 30° C. for 24 hours. Scrape off the bacterial cells from each colony produced and transfer to a minimal medium agar plate of the parent strain (for ATCC21526, glucose 0.5 g/dl, ammonium sulfate 0.15 g/dl, KH ₂ PO ₄ 0.1
g/dl, K ₂ HPO ₄ 0.3 g/dl, MgSO ₄・7H ₂ O
0.01g/dl, CoCl ₂・2H ₂ O 0.0001g/dl, Piotin 300μg/, Thiamine・HCl 100μg/
l, MnCl ₂・4H ₂ O 0.0007g/dl, FeSO ₄ .7H ₂ O
0.001g/dl, NaCl 16μg/ml, L-threonine 100μg/ml, L-methionine 33.3μg/ml,
Consists of 100 μg/ml of L-leucine and 2 g/dl of agar. PH6.0. In the case of ATCC21128, in the above composition L-
(without leucine) and 1 mg/dl of pantothenic acid, nicotinic acid or inositol in minimal medium.
Spread on an agar plate containing 100% of the mixture and grow at 30°C for 24 hours. Select bacteria that do not grow on the minimal medium or grow poorly on the medium supplemented with pantothenic acid, nicotinic acid, or inositol. Next, LYC-3 strain obtained as above,
Experimental examples show that strain HN-103 and strain HI-4 require pantothenic acid, nicotinic acid, and inositol, respectively. Experimental example Corynebacterium glutamicum LYC-
3, and Brevibacterium flavum HN-
103 and HI-4 respectively on bouillon agar medium.
The cells were cultured at 30°C for 20 hours. After culturing, the culture solution is centrifuged to collect bacterial cells and washed with physiological saline. Then, the bacterial cells were added to 10 ml of each of the following basic medium or basic medium in a test tube to which pantothenic acid, nicotinic acid, or inositol was added in the amount shown in Table 1.
The cells were inoculated at 10 ⁷ cells/ml and cultured with shaking at the same temperature for 21 hours. Basic medium composition: glucose 0.5g/dl, ammonium sulfate
0.15g/dl, KH ₂ PO ₄ 0.1g/dl, K ₂ HPO ₄ 0.3
g/dl, MgSO ₄・7H ₂ O 0.01g/dl, CoCl ₂・
2H ₂ O 0.0001g/dl, Biotin 300μg/, Thiamin・HCl 100μg/, MnCl ₂・4H ₂ O
0.0007g/dl, FeSO ₄・7H ₂ O 0.001g/dl,
NaCl 16μg/ml, L-threonine 100μg/
ml, L-methionine 33.3μg/ml (PH before sterilization
7.8). The degree of growth (OD ₆₆₀ ) of the bacteria at the end of the culture was measured using a Hitachi Model 101 colorimeter using a cell with a light diameter of 1 cm. The results are shown in Table 1.

[Table] The mycological properties of Corynebacterium and Brevibacterium are shown in the table.
Journal of General and Applied Microbiology
13 , 279–301 (1967). Carbon sources for the culture medium used in the present invention include sugars such as blackstrap molasses, starch hydrolyzate, sucrose, glucose, galactose, trehalose, maltose, cellobiose, and arabinose, sugar alcohols such as glycerin, acetic acid, gluconic acid, succinic acid, Organic acids such as formic acid, citric acid, fumaric acid, glutamic acid, and lactic acid, and their salts, and alcohols such as methanol, ethanol, and propanol can be used alone or in combination. These carbon sources can also be used by adding the entire amount to the culture medium or by dividing them into portions. As the nitrogen source, organic and inorganic ammonium compounds such as ammonia, ammonium chloride, ammonium sulfate, ammonium carbonate, ammonium phosphate, ammonium acetate, and urea can be used. As the inorganic substance, salts of metals such as sodium, potassium, manganese, magnesium, calcium, cobalt, nickel, zinc, and copper, and salts of acids such as hydrochloric acid, sulfuric acid, phosphoric acid, and nitric acid can be used. Amino acids such as homoserine or homoserine substitutes, leucine, and threonine, which are required by the bacteria used for growth, are added to the culture medium at a concentration of 100 to 1000 μg/ml, and pantothenic acid, nicotinic acid, inositol, biotin, thiamine, etc. are added to the culture medium at a concentration of 100 to 10000 μg/ml. added to. Each of the above nutrients, especially nitrogen sources, inorganic substances, and required nutrients are natural nutrients that contain them, such as bacterial cell hydrolysates of various microorganisms, yeast extracts, sake lees extracts, meat extracts, peptones, soybean meal decomposition products, and corn steep.・Can be substituted with liquor, etc. Furthermore, the amount of L-lysine accumulated may be increased by adding various substances, such as nucleic acid-related substances, amino acids other than those mentioned above, and vitamins, to the medium. Cultivation is carried out under aerobic conditions such as shaking culture or aerated agitation culture, and the pH during culturing is preferably about 5 to 9. Moreover, the culture temperature is preferably 23 to 40°C. The culture period is usually 3 to 7 days, and the amount of L accumulated in the culture solution is
-Produced by lysine. L-lysine can be collected by removing the bacterial cells from the culture solution and subjecting the resulting solution to a known method such as treatment with an ion exchange resin or other precipitation method. Next, examples will be shown. Example 1 Corynebacterium glutamicum LYC-3 is used as the inoculum. Glucose 4
g/dl, polypeptone 2g/dl, KH ₂ PO ₄ 0.15
g/dl, K ₂ HPO ₄ 0.05g/dl, MgSO ₄・7H ₂ O
0.05g/dl, biotin 50μg/, urea 0.3g/
dl, yeast extract 0.5g/dl (PH7.2) was cultured with shaking at 30℃ for 24 hours, and then added to a fermentation medium with the following composition at a ratio of 5% (v/v), and incubated at 30℃. The amount of L-lysine produced during shaking culture for 4 days is
It was 42mg/ml. Fermentation medium composition: Molasses (converted to glucose) 10g/
dl, soybean meal decomposition product (in terms of soybean meal dry matter before decomposition) 2
g/dl, KH ₂ PO ₄ 0.07g/dl, MgSO ₄ 7H ₂ O 0.05
g/dl, urea 0.3g/dl, ammonium sulfate 0.5g/dl, biotin 50μg/, CaCO ₃ 3g/dl (PH7.2). After culturing, the bacterial cells were removed, and the solution 1 was passed through a column of Diaion SK1A (trade name of strong acidic cation exchange resin, manufactured by Mitsubishi Kasei Corporation), followed by L adsorbed on the resin.
-Elute lysine with aqueous ammonia. The eluate was concentrated, decolorized with activated carbon, concentrated again, and alcohol was added to obtain 31 g of precipitated L-lysine set crystals. In addition, the amount of L-lysine produced by Corynebacterium glutamicum ATCC21526, which was used as a control, was 37.5.
It was mg/ml. Example 2 Brevibacterium flavum HN as inoculum
-103 and HI-4 were used, and 200μ of each was added to the seed medium and fermentation medium shown in Example 1.
When carried out in the same manner as in Example 1 except for using a medium supplemented with thiamine at
L-lysine was produced at 23.6 mg/ml and 24.1 mg/ml at HI-4. In Brevibacterium flavum ATCC21128, which was used as a control, L-
produced by lysine.

Claims (1)

[Claims] 1. Cultivating L-lysine-producing microorganisms that belong to the genus Brevibacterium and requiring inositol for growth in a nutrient medium, accumulating L-lysine in the culture, and collecting L-lysine from the culture. A method for producing L-lysine using a characteristic fermentation method.