JPS62100296A - Production of l-amino acid - Google Patents

Abstract

PURPOSE:To obtain the titled compound useful as drugs industrially advantageously, by reacting fumaric acid with ammonium ion and a 2-ketocarboxylic acid in an aqueous solution in the presence of a bacterium capable of converting the 2-ketocarboxylic acid into an L-amino acid. CONSTITUTION:(A) Fumaric acid is reacted with (B) ammonium ion or urea and (C) a ketocarboxylic acid (e.g., 3-hydroxypyruvic acid, etc.,) in an aqueous solution in the presence of a mold of a bacterium (e.g., Arthrobacter globiformis ATCC8010, etc.,) capable of converting the 2-ketocarboxylic acid to a corresponding L-amino acid, the L-amino acid (e.g., L-serine, etc.,) corresponding to the used 2-ketocarboxylic acid is formed in the aqueous solution and collected to give the aimed compound.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to a process for the production of L-amino acids such as L-serine, L-)ributophane, L-tyrosine, L-methionine and L-isoleucine by enzymatic methods. These L-
Amino acids are widely used in industrial fields such as pharmaceuticals and foods.

BACKGROUND ART The following method is known as a method for producing a corresponding L-amino acid from a 2-ketocarboxylic acid. For example 3
- As a method for producing L-serine from hydroxypyruvic acid, a method using alanine, glutamine, or glutamic acid as an amino donor and causing the action of Ragtobacillus blankrum [:2eszyty Nauk
owe-Tolitechnika shoddzka.

Ser+a: Che+n+a Spozywcza
, Nα20.209 (1972)], a method in which tra-nsaminase is made to act using aspartic acid as an amino donor (Japanese Patent Application Laid-open No. 60-91993), and the like are known. As a method for producing L-)ributophane from indolepyruvic acid, phenylalanine [Agricultural Biological Chemistry (^gric, Bio1. CheI) is used as an amino donor.
11. ), 44. 2013. 1980] Inorganic ammonia (Special Publication No. 38-9886) or As/X
A method using Ilaginic acid (Japanese Unexamined Patent Publication No. 60-91993) is known. From p-hydroxyphenylpyruvate or 2-keto-4-(methylmercapto)-butyrate, a transamination reaction using aspartic acid as the amino donor produces L-tyrosine or L-, respectively.
A method for producing methionine is known (Japanese Unexamined Patent Application Publication No. 1983-1999-
91993). Furthermore, a method for producing dl-α-keto-β-methylvaleric acid mustard-isoleucine in the presence of glucose and ammonium ions [JP-A-5
9-34890, Fin'/ x 'chemica/l/
-L/tar (Finn, Chem, Lett,)
1981, 2l-24) is known.

Problems to be Solved by the Invention and Means for Solving the Problems In the conventional methods described above, the raw materials for the amino donors used are expensive, so there is a need for an excellent method for producing -amino acids using inexpensive amino/donor 5 bodies. ing.

The present inventors have discovered that L-amino acids can be obtained efficiently and at low cost without using expensive amino donors by a method using fumaric acid and ammonium ions or urea.

The present invention will be explained in detail below.

The present invention deals with the following methods: (1) fumaric acid, (2) ammonium ion or urea, and (2) 2-ketocarboxylic acid in an aqueous solution in the presence of microbial cells or bacterial cell-treated products having the ability to convert 2-ketocarboxylic acid into the corresponding L-amino acid. L-amino acid corresponding to the 2-ketocarboxylic acid is produced in the aqueous solution by reacting an acid, and the L-amino acid is collected.
Provides a method for producing amino acids.

As the L-amino acid produced by the method of the present invention, 1% l
Examples thereof include J-N, 1-1-ributophane, L-dyrosine, L-methionine, and L-isolinine. The 2-ketocarboxylic acids used in the method of the present invention include 3-hydroxypyruvic acid, indolepyruvic acid, p-hydroquinphenylpyruvic acid, 2-keto-4-(methylmercapto)-butyrate or dA-α- Examples include keto-β-methylhalerianic acid.

The microorganisms used in the present invention include Arthrobacter, Bacillus, Brevibacterium, which have the ability to convert 2-ketocarboxylic acids into the corresponding L-amino acids in the presence of fumaric acid and ammonium ions or urea;
Citrobacter, Corynebacterium, Flavobacterium, Klebsiella, Kluybella, Micrococcus, Erwinia, Enterobacter, Eschierichia, Proteus, Balacobucus, Serratia, Pseudomonas, Salmonella, Alkali Earth metals, microorganisms belonging to the genus Agrobatherium, Aeromonas, Xanthomonas, or Sarcina, such as wild strains, mutant strains, and recombinant strains induced by cell fusion, genetic manipulation, or other genetic methods. Either can be used. Specifically, the following strains are used.

Arthrobacter globiformis
) ATCC8010 Arthrobacter citreus (Arthrobacter citreus) AT
C: C11624 Bacillus sphaericus (Bac+1lus 5phaericus) 8TCC10208 Bacillus subtilis (Bacillus 5ubtilis) ATCC6
051 Bacillus megaterium ^TC'
C19380 Brevibacterium lactofermentum
Mentum) ATCC13655 Brevibacterium ammoniagenes
ammon+agenes) ATCC6871 Brevibacterium flavum A
TCC13826 Brevibacterium divaricat
um) ATCC14020'/) Citrobacter freundii AT
CC6750 Corynebacterium hydrocarboclusters (Corynebacterium hydro
carbolastus) ATCC15108 Corynebacterium glutamicum
um) ATCC13032 Eschericha coli ATCC11
303 Escherichia coli ATC[:
9637 Enterobacter cloacae AT [
:C13047 Erwinia herbicola ATCC2
1434 Flavobacterium suaveolens
s) ATCC958 Klebsiella oxytoca ATCC
8724 Klebsiella pneumoniae I
AM 1183 Kluyvera cryocrescens (Kluyvera cryocrescens) A
TCC14237 Micrococcus luteus (Micrococcus 1uteus) ATCC
10240 Micrococcus luteus (Micrococcus 1uteus) ATCC
4698 Serratia mareescens ATC
C13880 Pseudomonas putida NRRL
-8-11064 Pseudomonas putida ATCC1
5175 Pseudomonas fluoresens
IAM 1051 Pseudomonas boreopolis
ATCC15452 Pseudomonas dacunhae, A
T(: [21192 Salmonella typhimurium) A
TCC19585 Proteus mirabilis (Proteus m1rabilis) IFo 3
849 Proteus vulgaris ATCC191
81 Paracoccus denitrificans
) ATCC19367 Alcaligenes faecalis (Alcaligenes faecalis) AT
CC8750 Agrobacterium tumefaciens (
Agrobacterium tumefaciens
) ATCC4452 Aeromonas hydrophila ATC
C13137 Xanthomonas citri ATCC1
5923 Xanthomonas citri IFO
12213 Xanthomonas oryzae> IFO3
995 Sarcina aurantiaca IFO3
064 Media for culturing these microorganisms include natural media, as long as they contain carbon sources, nitrogen sources, inorganic salts, etc.
Any artificial medium may be used.

Carbon sources include glucose and fructose.

1 such as sucrose, maltose, I & powder, blackstrap molasses etc.
1 Sugar alcohols such as glycerol, sorbitol, and mannitol, organic acids such as acetic acid, formic acid, fumaric acid, malic acid, citric acid, and gluconic acid, and alcohols such as methanol, ethanol, and propatool can be used.

Nitrogen sources include ammonium compounds such as aqueous ammonia, ammonium chloride, ammonium sulfate, ammonium carbonate, ammonium acetate, and ammonium phosphate, nitrogen compounds such as urea, glutamic acid, aspartic acid, methionine, and glycine.

Amino acids such as lysine, arginine, ornithine, peptone, yeast extract, casein hydrolyzate.

Natural nutrients such as defatted soybeans or their digests can be used.

As the inorganic substance, potassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, carbonate, etc. can be used.

If the microorganisms used in the present invention require specific nutrients for growth, appropriate amounts of those nutrients must be present in the culture medium, but these substances are not included in the natural products exemplified as nitrogen sources. Sometimes it is added.

Cultivation is performed at a temperature of 20 to 40°C and a pH of 5 to 8 for 1 to 5 days. The obtained microbial cells can be used for the reaction as they are, or the processed products obtained by subjecting the microorganisms to various treatments may be used for the reaction.

Examples of bacterial cell-treated products include mechanically ground bacterial cells, ultrasonic-treated products, freeze-dried products, solvent-treated products, enzyme-treated products, dry-processed products, surfactant-treated products, and bacterial cell protein components. For example, microorganisms, microbial cells, and immobilized products of the processed microbial cells can be used.

In the reaction, the bacterial cells obtained above or the processed product thereof are mixed with 3-
Hydroxypyruvate, indolepyruvate, p-hydroquinphenylpyruvate, 2-keto-4-(methylmercapto)-butyrate or dβ-α-keto-β-
It is carried out by reaction in an aqueous solution containing methylvaleric acid, fumaric acid and ammonium ions or urea.

3-Hydroquinepyruvate, indolepyruvate, p-hydroquine phenylpyruvate, 2-hydroquinepyruvate used in the reaction
Keto-4-(methylmercapto)-butyre), dA-
α-keto-β-methylvaleric acid and fumaric acid can be used in the form of various salts such as ammonium salt, sodium salt, potassium salt, and calcium salt.

Ammonium ions are supplied as salts such as ammonium sulfate, ammonium phosphate, ammonium nitrate, ammonium chloride, ammonium acetate, ammonium formate, ammonium fumarate, ammonium malate, or as ammonia water or ammonia gas.

3-hydroxypyruvic acid, indolepyruvic acid, p-hydroxyphenylpyruvic acid, 2-hydroxypyruvic acid used in the reaction
Keto-4-(methylmercapto)-butyrate and d
The concentration of p-α-keto-β-methylvaleric acid is 0.0
Preferably, the concentration of fumaric acid is 0.01 to 2 mol, and the concentration of ammonium ion or urea is 0.01 to 10 mol. These raw materials are supplied in bulk or intermittently.

The working conditions include a temperature of 20 to 60°C, preferably 25°C.
~45°C, pH 6-10, preferably 7-9, 10-45°C
The reaction is carried out for 48 hours.

In this way, L-amino acids are produced in the reaction solution.

As a method for recovering L-amino acids from the reaction solution, an ion exchange resin method, a precipitation method, etc. are used.

Below is an example of fruitfulness.

Example 1 Trobacter freundii ATCC 675 was placed in a test tube containing 10 ml of a medium (pH 7) with a composition of 0.5% glucose, 0.3% yeast extract, and 1.5% minced meat extract.
0 was inoculated with 1 lactose, shaken at 21 Orpm, 28°C.
The culture was incubated with shaking for 18 hours at a temperature of 1.

After culturing, the cells were cultured by centrifugation, and the cells were cryopreserved (
-20°C). - days after cryopreservation, 10 ml of 0.85
% saline solution was added to the test tube, and after thawing, 3000 rpm,
The mixture was centrifuged for IQ minutes, and the precipitate was washed once with 0.8% saline. The obtained bacterial cells were treated with 3-hydroxypyruvate, indolepyruvate, p-hydroxyphenylpyruvate or 2-ke)i-(methylmercapto)-
When suspended in 1.5 ml of a reaction solution with the following composition containing any one of butyrate as a substrate and reacted at 40°C for 18 hours under static conditions, each microorganism added 5
L-amino acids corresponding to the substrates were produced at concentrations as shown in Table 1.

The composition of the reaction solution is as follows: 2-ketocarboxylate (see Table 1) 100mM, pyridoxal phosphate 200μ
M,) Lys buffer 100mM (pH 8,4), ammonium fumarate 200mM0 Table 1 Example 2゜The microorganisms shown in Table 2 were used as inoculum, and 2-ke)-4-(methylmercapto) was used as the substrate for the enzyme reaction. )-butyrate or indolepyruvic acid was used, but the same procedure as in Example 1 was performed. The results are shown in Table 2.

As shown in Table 2, mustard monomethionine or L-IJ butophane was produced from each substrate.

Table 2 Example 3 The same procedure as in Example 1 was carried out except that the microorganisms shown in Table 3 were used as the inoculum and 2-keto-4-(methylmercapto)-butyrate was used as the substrate for the enzyme reaction. 3rd result
Shown in the table.

As shown in Table 3, -methionine was produced.

Table 3 Example 4 The microorganisms shown in Table 4 were used as seed bacteria, and d
In addition to using β-α-keto-β-methylvaleric acid,
It was carried out in the same manner as in Example 1. As a result, L-inleucine was produced as shown in Table 4.

Table 4 Effects of the Invention According to the method of the present invention, L-amino acids can be supplied efficiently and at low cost.

Claims (4)

[Claims] (1) [1] fumaric acid, [2] ammonium ion or urea and [ 3] A method for producing an L-amino acid, which comprises reacting a 2-ketocarboxylic acid to produce an L-amino acid corresponding to the 2-ketocarboxylic acid in the aqueous solution, and collecting the L-amino acid. (2) The 2-ketocarboxylic acid is 3-hydroxypyruvic acid, indolepyruvic acid, p-hydroxyphenylpyruvic acid, 2-keto-4-(methylmercapto)-butyrate or dl-α-keto-β-methylvalerian. The method according to claim 1, characterized in that the acid is an acid. (3) The method according to claim 1, wherein the L-amino acid is L-serine, L-tryptophan, L-tyrosine, L-methionine or L-isoleucine. (4) The microorganism is of the genus Arthrobacter, the genus Bacillus,
Brevibacterium, Citrobacter, Corynebacterium, Flavobacterium, Klebsiella,
Belongs to the genus Kluybella, Micrococcus, Erwinia, Enterobacter, Escherichia, Proteus, Paracoccus, Serratia, Pseudomonas, Salmonella, Alcaligenes, Agrobacterium, Aeromonas, Xanthomonas or Sarcina A method according to claim 1, characterized in that: