JPH05284984A - Medium for producing l-amino acid and production of l-amino acid using the same - Google Patents

Abstract

PURPOSE:To efficiently produce a large amount of an L-amino acid such as L-glutamic acid, L-lysine or L-glutamine by culturing a bacterium capable of producing an L-amino acid in a medium containing a specific polyglycerol and collecting the L-amino acid from the culture mixture. CONSTITUTION:Glycerol is condensed through dehydration in the presence of an alkali preferably at 200-300 deg.C to prepare a polyglycerol, an alkylene oxide is added to the polyglycerol in the presence of an alkali catalyst under pressure by heating to give an adduct of the polyglycerol with a polyoxyalkylene of the formula (n is 2-50; R<1> to R<3> are H or 2-31C acyl; X is 2-4C alkylene; m1, m2 and m3 are 0-200). Then this compound is added to a medium containing a carbon source, a nitrogen source, a salt, etc., a bacterium (e.g. Corynebacterium glutamicum) capable of producing an L-amino acid is inoculated into the medium, cultured at 30 deg.C for 18 hours and a product is collected from the culture solution by ion exchange resin treatment to efficiently produce a large amount of the L-amino acid.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an L-amino acid producing medium capable of efficiently producing L-amino acid and a method for producing L-amino acid using the medium.

[0002]

BACKGROUND OF THE INVENTION Conventionally, L-glutamic acid, L-lysine, L-glutamine, L-arginine, L-phenylalanine, L-threonine, L-isoleucine, L-histidine, L-proline, L-valine, L-. Serine, L-
L-amino acids such as ornithine, L-citrulline, L-tyrosine, L-tryptophan, and L-leucine are produced by fermentation of microorganisms belonging to the genus Prepibacterium, Corynebacterium, Bacillus, or Escherichia. Are being produced in a regular manner.

However, the conventional methods have not been satisfactory in terms of yield.

As an attempt to increase the yield, there is a method in which a specific surfactant is added to the medium and the fermentation is continued while crystallizing the L-amino acid (JP-A-62-288).

However, although this method is improved as compared with the conventional method, it has not been able to obtain a satisfactory yield.

[0006]

Therefore, an object of the present invention is to provide a medium and a culture method for obtaining L-amino acid in high yield.

[0007]

In view of such circumstances, the present inventors have conducted diligent research, and as a result, by adding a specific polyglycerin to a medium and culturing an L-amino acid-producing bacterium in the medium, The present invention has been completed by finding that L-amino acids can be obtained in high yield.

That is, the present invention has the following general formula (1):

[0009]

[Chemical 3]

[Wherein n represents a number of 2 to 50, R ¹ ,
R ² and R ³ are hydrogen atoms or an acyl group having ^{2 to 31} carbon atoms, X is an alkylene group having 2 to 4 carbon atoms,
m ₁ , m ₂ and m ₃ are each a number from 0 to 200. ] An L-amino acid-producing medium containing a compound represented by: and a method for producing an L-amino acid, comprising culturing an L-amino acid-producing bacterium in this medium and collecting the L-amino acid from the culture. It is a thing.

The compound (1) added to the L-amino acid-producing medium of the present invention includes polyglycerin, a mono-, di- or tri-acyl derivative of polyglycerin, a polyoxyalkylene adduct of polyglycerin and polyglycerin. Mono-, di- or tri-acyl adducts of the polyoxyalkylene adducts of

These compounds (1) can be produced by a known method. For example, polyglycerin
Glycerin in the presence of an alkali catalyst to 200 to 3
A method in which dehydration condensation is performed at a high temperature of 00 ° C. may be mentioned. Alkali catalysts used here include NaOH, KOH,
LiOH, Na ₂ CO ₃ , K ₂ CO ₃ , Li ₂ CO ₃ , Ca
O, MgO, etc. are mentioned. Although the degree of polymerization can be adjusted by changing the reaction conditions, the obtained polyglycerin is not a single component but a mixture having a certain molecular weight distribution. For example, what is called commercialized hexaglycerin has a hydroxyl value that matches the theoretical value, but its component is a mixture of polyglycerin of various degrees of polymerization.

The polyglycerin thus obtained is a highly viscous yellow to blackish brown liquid, and its hue becomes poor and blackish brown as the degree of polymerization increases. Therefore, decolorization treatment with an adsorbent such as activated carbon or activated clay, decatalysis with an ion exchange resin, decolorization treatment and the like are performed. Generally, diglycerin, tetraglycerin, hexaglycerin, and decaglycerin are commercialized.

The polyoxyalkylene adduct of polyglycerin may be, for example, a known method in which the polyglycerin obtained as described above is added with an alkali catalyst and then alkylene oxide is added under pressure and at an elevated temperature. Examples of the alkylene oxide to be added include ethylene oxide having 2 to 4 carbon atoms, propylene oxide, butylene oxide and the like. These may be a single type or two or more types of blocks or random additions.
The number of added moles is 1 to 200 moles, preferably 1 to 5 on average.
It is 0 mol.

Further, a mono-, di- or tri-acyl derivative of polyglycerin and a mono-, di- or tri-acyl derivative of a polyoxyalkylene adduct of polyglycerin (both may be referred to as polyglycerin fatty acid ester) Is
Usually, it can be produced by a direct esterification reaction. A large number of esters from hydrophilic to lipophilic can be obtained by combining polyglycerin having various degrees of polymerization, types of fatty acids, and degree of esterification, and an ester having a desired HLB value can be prepared.

The esterification reaction is carried out at a temperature of 200 ° C. or higher in the presence of a catalyst or an alkali catalyst. There are methods such as adding sulfite during the reaction to obtain products with good hue and flavor, using fatty acids with good heat stability, and synthesizing with lipase, etc. Has been done. In order to obtain high quality polyglycerin fatty acid ester, it is important to use high quality polyglycerin. In particular, a polyglycerin fatty acid ester having a high degree of polymerization has this tendency, and the quality of the ester obtained greatly changes depending on the quality of decaglycerin, and thus it is necessary to sufficiently purify polyglycerin. The polyglycerin condensed ricinoleic acid ester is synthesized by esterifying polyglycerin with ricinoleic acid (castor oil fatty acid) that has been dehydrated by heating and precondensed for 3 to 6 minutes.
The reaction conditions are almost the same as in the case of polyglycerin fatty acid ester.

The medium of the present invention is prepared by adding the compound (1) thus obtained. The amount of addition depends on the medium, but generally 0.01 to 5% by weight (hereinafter simply referred to as " % "), Preferably 0.05 to 2.5%. The addition may be performed before culturing or after culturing is started, and the number of times of addition may be once or several times.

In addition to the seed culture, it can be added to the main fermentation medium.

As the medium to which the above-mentioned compound (1) is added, those usually used for culturing L-amino acid-producing bacteria can be used. That is, it is a medium containing a carbon source, a nitrogen source, salts and the like. As the carbon source used here, sugars such as glucose, dextrose, sucrose, fructose, maltose, crude sugar, fructose, butter sugar, liquid sugar, cane molasses, sugar beet molasses, molasses waste, tapioca, starch saccharified liquid and the like; Fatty acids such as acetic acid and propionic acid;
Organic acids such as pyruvic acid, citric acid, succinic acid and malic acid; alcohols such as ethyl alcohol and butyl alcohol may be used alone or in combination. As the nitrogen source, ammonium salts such as ammonium sulfate, ammonium chloride and ammonium acetate, urea, aqueous ammonia, corn steep liquor, yeast extract, soybean hydrolyzate, peptone, polypeptone and meat extract can be used.

As salts, if necessary, phosphates, magnesium salts, calcium salts, potassium salts, sodium salts, iron salts, manganese salts, zinc salts, copper salts and other trace metal salts may be added. Good.

The yield of L-amino acid can be increased by adding a surfactant to the medium of the present invention. Examples of the surfactant used here include anionic activators such as higher alcohols, sulfates, alkylbenzene sulfonates, alkyl phosphates and dialkyl sulfosuccinates; cationic activators such as alkylamines, quaternary ammonium salts and the like; polyoxy. Nonionic activators such as ethylene alkyl ether, polyoxyethylene sorbitan monoalkyl ether, sorbitan monolaurate, alkyl glucoside, ester amide; zwitterionic activators such as imidazoline, betaine, etc., among which alkyl glucoside, ester Amides are preferred. These surfactants may be used alone or in combination of two or more, and the addition amount is 0.01 to 5
It is preferably in the range of 0%.

If necessary, antibiotics, vitamins, etc. may be added. Examples of the antibiotics include penicillin, chloramphenicol, erythromycin, streptomycin, kanamycin, oleandomycin, kasugamycin, tetracycline, mitomycin, actinomycin, cycloserine, among which penicillin is preferable. Also, biotin as a vitamin,
Examples include niacin.

The microorganism to which the medium of the present invention is applied is not particularly limited, and any microorganism can be used as long as it is a microorganism that produces an L-amino acid. Specifically, the following are exemplified. Corynebacterium (Corynebacterium)
m) Corynebacterium glutamicum (Coryneba
cterium glutamicum Corynebacterium acetoglutamicum (Coryn)
ebacteriumacetoglutamicu
m) Corynebacterium acetoacidophilum (Cory
nebacterium acetoacidophi
lum) Microbacterium genus (Mrcrobacterium)
m) Microbacterium Ammonia Philum (Mrcro
bacterium genus Brevibacterium
m) Brevibacterium flavum (Brevibacte
rium flavum) Brevibacterium lactofermentum (Brev
ibacterium lactofermentum Brevibacterium saccharoriticum (Brev
ibacterium saccharolyticu
m) Brevibacterium roseum (Brevibacte)
riom roseum) Brevibacterium divaricatum (Breviba)
cterium divaricatum Arthrobacter genus (Arthobacterium) Arthrobacter citrus (Arthobacte)
r cltreus Bacillus Bacillus subtilis
lis Bacillus sph
aericus)

The L-amino acids obtained are L-glutamic acid, L-lysine, L-glutamine, L-arginine, L-phenylalanine, L-threonine, L-isoleucine, L-histidine, L-proline and L-valine. , L-tyrosine, L-tryptophan, L-leucine, L-serine, L-ornithine, L-citrulline and the like, all of which are produced by the fermentation method.

The conditions for culturing the L-amino acid-producing bacterium using the medium of the present invention are the same as the conditions for ordinary amino acid fermentation, and although there are some differences depending on the target L-amino acid and the strain to be used, the culturing is performed. The temperature is preferably 20-40 ° C,
28-37 degreeC is especially suitable. It is better to control the pH to near neutral during the culture. The culturing period for culturing under aerobic conditions such as aeration, stirring, and shaking culturing is usually 1 to 7 days, but the period can be extended by continuous culturing or the like. A method for isolating each amino acid from each amino acid fermentation liquor is recovered by an ion exchange resin treatment method or another known method.

[0026]

The L-amino acid-producing bacterium is cultured in a medium containing the compound represented by the general formula (1) to obtain L-amino acid.
Large amounts of amino acids can be obtained.

[0027]

The present invention will be described in more detail with reference to examples, but the present invention is not limited thereto. Example 1 The following composition: Molasses molasses 4% KH ₂ PO ₄ 0.2% MgSO ₄ 0.05% Urea 0.8% Biotin 5 μg / l Water in a medium having a pH of 7.2 with KOH. , 30
ml was put in a 500 ml Sakaguchi flask and sterilized by heating. This was inoculated with Corynebacterium and glutamicum grown on glucose-pepton slant and cultured for 18 hours while maintaining at 30 ° C.

On the other hand, a medium having the following composition: molasses 10% (sugar concentration conversion) urea 1.0% K ₂ HPO ₄ 0.1% MgSO ₄ 0.05% Water remaining amount (pH 7 to 8) in 30 ml increments 500 ml Sakaguchi flask 4
And sterilized by heating to prepare mediums A, B, C and D. Add 3 to each of these A to D media.
Each of them was subcultured in an amount of 00 μl, and to A, 0.2% of lauric acid monoester of polyglycerin and 0.2% of polyoxyethylene sorbitan monostearate were added,
B is polyglycerin lauric acid monoester 0.3
%, Polyoxyethylene sorbitan monostearate was added to C in an amount of 0.3%, and D was not added.

Each of A to D was subjected to shaking culture at 30 ° C. for 48 hours, and the amount of L-glutamine in the medium was quantified. The results are shown in Table 1.

[0030]

[Table 1]

Example 2 The following media A and B A: molasses 10% (in terms of glucose) (NH ₄ ) ₂ SO ₄ 4.5% KH ₂ PO ₄ 0.1% peptone 1% water balance B: glucose 10% biotin 50 μg / l thiamine hydrochloride 200 μg / l (NH ₄ ) ₂ SO ₄ 4.5% KH ₂ PO ₄ 0.1% peptone 1% water Remaining volume is adjusted to 40 ml of each 500 ml Sakaguchi flask 2
It was dispensed into individual pieces and sterilized. Brevibacterium SP, which is an L-lysine-producing bacterium, was inoculated into this and cultured at 30 ° C. for 18 hours. 400 μl of this culture was again used for A and B, respectively.
After subculture at 30 ° C. for 8 hours with shaking, the stearic acid ester of polyglycerin was added in an amount of 0.15%, and shaking culture was continued for further 24 hours. As a control
The same culture was performed in the case where polyglycerin stearate was not added. The amount of L-lysine obtained is shown in Table 2.

[0032]

[Table 2]

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical indication C12R 1:01) (C12P 13/04 C12R 1:13) (C12P 13/04 C12R 1:06) (C12P 13/04 C12R 1: 125) (C12P 13/04 C12R 1:07) (C12P 13/08 C12R 1:15)

Claims (5)

[Claims] 1. The following general formula (1): Wherein, n represents a number of 2~50, R ^1, R ² and R ³
Is a hydrogen atom or an acyl group having 2 to 31 carbon atoms, X is an alkylene group having 2 to 4 carbon atoms, and m ₁ , m ₂ and m
Each ₃ is a number from 0 to 200. ] The L-amino acid production medium containing the compound represented by these. 2. An L-amino acid production medium containing the compound (1) according to claim 1 and a surfactant and / or an antibiotic. 3. The L-amino acid-producing medium according to claim 1, wherein one or more selected from molasses, tapioca and molasses is added as a carbon source. 4. A method for producing an L-amino acid, which comprises culturing an L-amino acid-producing bacterium using the medium according to claim 1, 2 or 3, and collecting the L-amino acid from the culture. 5. A compound represented by the general formula (1): Wherein, n represents a number of 2~50, R ^1, R ² and R ³
Is a hydrogen atom or an acyl group having 2 to 31 carbon atoms, X is an alkylene group having 2 to 4 carbon atoms, and m ₁ , m ₂ and m
Each ₃ is a number from 0 to 200. ] The L-amino acid production medium additive which consists of the compound represented by these.