JPS6312292A - Production of l-lysine - Google Patents

Abstract

PURPOSE:To produce the titled compound useful as a food additive, etc., on an industrial scale at a low cost, by culturing a microbial strain belonging to glutamic acid-producing coryne-form strain resistant to beta-naphthoquinoline and capable of producing L-lysine in a medium and separating the product from the cultured liquid. CONSTITUTION:A microbial strain belonging to glutamic acid-producing coryne- form strain resistant to beta-naphthoquinoline and capable of producing L-lysine [e.g. Corynebacterium glutamicum H-4412 (FERM BP-1069), etc.] is cultured in a medium containing glucose, etc., as carbon source, urea, etc., as nitrogen source and potassium phosphate, etc., as inorganic component. The produced and accumulated L-lysine is separated from the cultured liquid.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to a method for producing L-lysine by fermentation. More specifically, the present invention involves culturing in a medium a microorganism that belongs to coryneform glutamate-producing bacteria, is resistant to β-naphthoquinoline, and has the ability to produce L-lysine, and L-lysine is collected from the culture. The present invention relates to a method for producing L-lysine by a fermentation method, characterized in that:

The purpose is to provide a method for industrially producing L-IJ lysine, which is one of the essential amino acids and is in high demand as an additive to pharmaceuticals, feeds, and foods.

Conventional technology Conventionally, regarding the production of L-lysine by fermentation, homoserine (or methionine and threonine) auxotrophic mutant strains of bacteria of the genera Corynebacterium, Brevibacterium, Arthrobacterium, Bacillus and other microorganisms have been used. (
Special Publication No. 36-6499, USP 2,979.439
) and other auxotrophic mutants [Special Publication No. 48-28677,
No. 51-21078, No. 51-34477 (LIS
P3,825.472).

No. 55-1040, JP-A No. 56-8692.

No. 55-9784], and various drug-resistant mutant strains [Special Publication No. 48-28078 (USP 3.70)].
7.441), No. 53-1833.

No. 53-43591, No. 59-4993, JP-A-5
No. 3-9394, No. 55-9785 (USP 3,68
7.810):1, and methods using microorganisms having a combination of these properties are known.

Problems to be Solved by the Invention L-'J lysine is one of the essential amino acids and is in great demand as an additive for pharmaceuticals, feed, and food, and it has always been desired to develop a method to produce it industrially at low cost. There is.

Means for Solving the Problems As a result of various studies to obtain a strain with improved productivity of L-lysine, the present inventor has further added β to the L-IJ lysine-producing strain belonging to coryneform glutamic acid-producing bacteria. - It was discovered that L-lysine productivity was dramatically improved by imparting naphthoquinoline resistance, and the present invention was completed. When producing L-lysine by fermentation, it is known that lysine production ability is improved by imparting resistance to quinolines (Japanese Patent Publication No. 4993/1983); however, growth inhibition by these drugs is It has been shown to be released by leucine. Therefore, it is presumed that these quinolines act as L-leucine analogs. However, since the growth inhibition caused by β-naphthoquinoline is not recovered by L-leucine, β-naphthoquinoline is considered to be a substance belonging to a different category from the quinolines described in Japanese Patent Publication No. 59-4993. It was discovered for the first time by the present inventors that l,-+J lysine productivity can be dramatically improved by using a bacterial strain that is resistant to β-naphthoquinoline and has such properties.

The present invention will be explained in detail below.

The present invention involves culturing a microorganism belonging to coryneform glutamate-producing bacteria, having resistance to β-naphthoquinoline, and having L-lysine producing ability in a medium, producing and accumulating L-IJ lysine in the culture, and To provide a method for producing L-lysine, which is characterized by collecting L-lysine from a substance.

As used herein, coryneform glutamate-producing bacteria are
A group of glutamic acid-producing bacteria belonging to the genus Corynebacterium, Brevibacterium, Microbacterium, or Arthrobacterium [see "Fermentation and Industry" Vol. 40, 102, 1982].

The microorganism used in the present invention belongs to coryneform glutamic acid-producing bacteria, and any L-lysine-producing strain that is resistant to β-naphthoquinoline can be used. That is, in the present invention, a strain that belongs to the coryneform glutamate-producing bacteria and has the ability to produce L-lysine may be imparted with β-naphthoquinoline resistance, or a strain that belongs to the coryneform glutamate-producing bacteria and has the ability to produce β-naphthoquinoline may be used. A strain obtained by imparting the ability to produce L-U gin to a resistant strain may also be used.

Bacterial strains that belong to coryneform glutamate-producing bacteria and have the ability to produce L-lysine contain various nutrients (e.g., homoserine, methionine, threonine, histidine,
alanine, loinine, isoL1 isine, valine, serine, glutamic acid, pantothenic acid, nicotinamide, acetic acid, adenine, hypoxanthine, inositol and combinations thereof), various amino acid analogs (e.g. lysine, threonine, methionine, leucine) , inleucine, valine, aspartic acid, tryptophan or histidine analogs, and combinations thereof), and other drugs (e.g., various antibiotics, sulfa drugs, various organic acids, quinone compounds).

quinoline compounds and combinations thereof) resistant to one or a combination thereof.

Therefore, the strain used in the present invention can be obtained by further imparting β-naphthoquinoline resistance to the above-mentioned IJ lysine-producing strain, or it is possible to obtain the strain used in the present invention by further imparting β-naphthoquinoline resistance to the above-mentioned IJ lysine-producing strain. L-lysine-producing strains obtained by imparting various auxotrophic requirements, various amino acid analog resistances, or other drug resistances as described above to the L-lysine-producing strains can also be used in the present invention. In addition to the above-mentioned properties, the strain used in the present invention may have any properties that contribute to L-U gin production.

Parent strains used to induce the mutant strain of the present invention include the following microorganisms known as coryneform glutamate-producing bacteria. For example, Corynebacterium glutamicum ATCC13032゜Corynebacterium glutamicum
Glutamicum H-3149 (FERM BP-158
), Corynebacterium acetoacidophyllum ATC
C13870° Brevibacterium lactofermentum ATCC13869, Brevibacterium flavum ATCC14067, etc.

Induction of mutant strains in the present invention can be carried out by commonly used mutation treatment methods such as ultraviolet irradiation and chemical treatment with N-methyl-N'-nitro-N-nitroguanidine.

To isolate the mutant strain of the present invention from the mutation-treated strain,
β-naphthoquinoline at a concentration that inhibits the growth of the parent strain (40
What is necessary is to obtain a mutant strain that grows on a minimal medium agar plate containing (u/ml or more).

Among the microorganisms used in the present invention, an example of obtaining Corynebacterium glutamicum H-4412 is shown below as a specific example of a β-naphthoquinoline-resistant strain.

Corynebacterium glutamicum H-3149 (FE
RM BP-158) (thiarisin resistance.

Rifampicin resistance, streptomycin resistance, 6
-Adiuracil resistant) is used as the parent strain. This parent stock is 0. IN)! J Sous-leate buffer (pH
N-methyl-N'-nitro N-nitrosoguanidine was added to the suspension at a final concentration of 0.62 cm/ml, and the suspension was incubated at room temperature for 3.
Let stand for 0 minutes. The mutant strain is selected from the growing colonies by spreading it onto a minimal medium agar plate having the composition shown below, which contains β-naphthoquinoline (for example, 40 g/ml) at a concentration that inhibits the growth of the parent strain. One of them is H-4
412, and as shown in Table 1, it can be clearly distinguished from the parent strain H-3149 in that it has β-naphthoquinoline resistance.

Minimal medium agar plate medium composition Gelcose 10g/l, NH1Cj! Ig/j! .

urine? , 2g/l, KH2PO41g/1. KtHPOs
3g/j! , Mg5On.7H200, 4g/l.

Fe50.・7Hz0 10mg/l, MnS○, ・4
H2O 4mg/l, biotin 50px/1. Nicotinic acid 5mg/j! , ZnSO4・7H*O1mg/f
.

Cu5O* ・5H201mg/1. (NH*)sMO
to2<・48201■/l, agar 2%, pH 7.0 Table 1 H-3149++ + - H4412++ ++ + ++: Sufficient growth, +: Slight growth, -2 No growth H
H-4412, which is resistant to β-naphthoquinoline and was obtained by mutating H-3149, was developed in 1986.
As of May 27, 2015, the Institute of Microbial Technology, Agency of Industrial Science and Technology (
It has been deposited as FERMBP-1069 at the Microtechnical Research Institute.

The culture medium composition used in the present invention is based on the strain used.
``Both synthetic and natural media can be used as long as they contain appropriate amounts of usable carbon sources, nitrogen sources, inorganic substances, and other necessary nutrients.

That is, carbon sources include glucose, fructose, sorbitol, glycerol, and am.

Various carbohydrates such as starch hydrolysates, molasses, and fruit juices, organic acids such as acetic acid, fumaric acid, lactic acid, and succinic acid, and alcohols such as ethanol and methanol can also be used.

Nitrogen sources include ammonium salts of various inorganic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphate, urea, amines,
The formed nitrogen compounds, peptone, meat extract, yeast extract, corn steep liquor, casein hydrolyzate, soybean meal hydrolyzate, various fermented microbial cells and digested products thereof, etc. can be used.

Furthermore, potassium phosphate is an inorganic substance.

Potassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate,
Calcium carbonate etc. can be used.

If the microorganisms used in the present invention require specific nutrients for growth, the nutrients must be present in appropriate amounts in the culture medium, but these substances are not included in the natural products exemplified as nitrogen sources. It may be added as a result. Furthermore, the amount of L-lysine produced may be increased by adding various additives to the culture medium, such as various antibiotics, α-aminobutyric acid, cysteine, leucine, leucine fermentation broth, aspartic acid, and glutamic acid.

Cultivation is performed under aerobic conditions such as shaking culture or deep aeration agitation culture. The culture temperature is usually in the range of 20 to 40°C. Although it is desirable to maintain the pH of the culture medium in the range of 3 to 9, preferably near neutrality, it can be carried out under other conditions as long as the strain used grows. The pH of the medium was adjusted using calcium carbonate and urea.

This is done using acid or alkaline solutions, pH buffers, etc. The culture time is usually 1 to 6 days, and L-lysine is produced and accumulated in the culture solution.

After the culture is completed, precipitates such as bacterial cells are removed from the culture solution, and lysine is recovered from the culture solution by using a combination of known ion exchange treatment methods, concentration methods, adsorption methods, salting-out methods, etc. Can be done.

Hereinafter, the present invention will be specifically illustrated by giving examples.

Example 1 Corynebacterium glutamicum H-44 as inoculum
12 (FERM BP-1069) was used.

This strain has 40g/j of glucose! , urea 3g/CKH
aPO41,5g#! , KaHPOa 0.5g/A.

Mg5O＜・7Hz○ 0.5g/! , biotin 50■
/l, peptone 20g/l, yeast extract 5g/j! It was inoculated into a 303 ml Erlenmeyer flask containing 20 ml of a seed medium (pH 7.2) consisting of the following, and cultured at 30°C for 24 hours. This culture solution 2+111 is mixed with blackstrap molasses 100g/j! (converted to glucose), ammonium sulfate 40g/β, K H2P
040.5g/CMg SO40.5g/j! ,
Fermentation medium (pH 7.2) consisting of CaCO 530g/12
) It was inoculated into a 3,0Q+nl Erlenmeyer flask containing 20ml, and cultured with shaking (22Orpm) at 32°C for 3 days. At this time, 47 g of L-IJ lysine hydrochloride/
j! Accumulated.

As a control, the parent strain H-3 was cultured at the same time under the same conditions.
The amount of LIJ gin hydrochloride produced in 149 was 43 g/l.

Effects of the Invention According to the method of the present invention, L-lysine can be supplied industrially at low cost.

Procedural amendment (spontaneous) July ls'' day 1, 1985, case description 1988 Patent Application No. 156775 2, title of invention: Process for producing L-lysine 3, person making the amendment Relationship with the case Patent application Postal code 100 Address 6-1 Otemachi-chome, Chiyoda-ku, Tokyo Name (1
02) In Column 5 of the detailed description of the invention in the specification of Kyowa Hakko Kogyo Co., Ltd., next to the contents of the amendment, ``, Japanese Patent Publication No. 55-44597J is added.

(2) riJg rMgsOnJ on page 10, line 22 of the same book.
Corrected to sO4・7tlzOj.

(3) "3rd" on the bottom line of page 10 of the same book is corrected to "4th".

(4) On page 11 of the same book, lines 1-5, “At this time...
...It was 43g/β. ” to the following sentence.

[After the cultivation was completed, the amount of L-1 gin produced was measured by a colorimetric method using acidic copper ninhydrin reaction. As a result, the amount of l, IJ lysine in the culture solution was 52g as hydrochloride.
/Il. As a control, the L-IJ lysine production of the parent strain H-3149, which was similarly cultured under the same conditions, was 44 g/j as hydrochloride! Met.

Centrifuging the culture solution 11 obtained using the H-4412 strain,
After adjusting the obtained supernatant liquid to pH 1.5 with sulfuric acid, strongly acidic ion exchange resin Diaion 5K-IB (H" type) (
The mixture was passed through a column (manufactured by Mitsubishi Chemical Industries, Ltd.) to adsorb L-lysine, and after washing the column with water, it was eluted with 2N aqueous ammonia to collect the L-lysine fraction. Both collected fractions were concentrated, and the obtained tank a solution was adjusted to pH 2.0 with hydrochloric acid, and then cooled while adding ethanol to crystallize L-lysine hydrochloride to obtain 41 g of crystals. ”

Claims (1)

[Claims] A microorganism belonging to coryneform glutamate-producing bacteria, resistant to β-naphthoquinoline, and capable of producing L-lysine is cultured in a medium, L-lysine is produced and accumulated in the culture, and L-lysine is produced and accumulated in the culture. - A method for producing L-lysine, which comprises collecting lysine.