JPH1080297A - Production of d-amino acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To efficiently obtain a D-amino acid for medicine raw materials and reagents for optical resolution, etc., by reacting a specific racemic amino acid with a microorganism, etc., having asymmetric decomposition ability of L type optically active substance of amino acid and then collecting the residual D type optically active substance. SOLUTION: A racemic amino acid selected from racemic ornithine, racemic citrulline, racemic tyrosine, racemic glutamine, etc., is reacted with culture product of a microorganism belonging to the genus Aerobacter, the genus Achromobacter, the genus Bacillus and the genus Pseudomonas, etc. (e.g. Aerobacter aerogenes UT 8017) or its treated material and the residual D type optically active substance is separated and collected to efficiently provide the objective D-amino acid useful as a raw material or a synthetic intermediate for medicines such as antibody or a reagent for optical resolution, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for producing D-amino acids using microorganisms.

[0002]

2. Description of the Related Art D-amino acids are compounds useful as raw materials for pharmaceuticals such as antibiotics, synthetic intermediates or optical resolving agents.

Hitherto, as a method for producing a D-amino acid, a physicochemical method such as a fractional crystallization method of a racemate, an optical resolution method by chromatography, or an organic chemical asymmetric synthesis method has been known. These methods are complicated in operation, and have disadvantages such as low product yield and low optical purity.

As a biochemical method, an asymmetric hydrolysis of α, δ-dichloroacetylamino acid using an enzyme (Journal of Biological) is known.
Chemistry, Vol. 188, 643-646
P. 1951), a method for asymmetric hydrolysis of N-acetyl-DL-amino acids using microbial enzymes (Applie)
d and Environmental Micro
biology, Vol. 54, pp. 984-989, 19
1988), a method of hydrolyzing DN-carbamoyl-α-amino acid (Japanese Patent Publication No. 4-810579), a method of hydrolyzing a hydantoin derivative (Journal of
Fermentation Technology, 56, 492-498 (1978), a method of transferring an amino group to α-keto acid using a microbial enzyme (Jo).
urnal of Biotechnology, 8th
Vol., Pp. 243-248, 1988) and the like. However, in these methods, the substrate α, δ-dichloroacetylamino acid, DN-carbamoyl-α-amino acid and the like are expensive, and There were difficulties such as difficulty in separation.

Further, as a method of producing D-type amino acids by selectively assimilating or decomposing only the L-type optically active substance in racemic amino acids using a microbial enzyme,
Arginine (JP-B-48-5040), D-alanine (JP-A-63-198997), D-serine (JP-A-1-2594), D-glutamic acid (JP-A-1-2595) ), A process for producing D-aspartic acid (JP-A-1-55193), a process for producing D-asparagine (JP-A-1-55194), a process for producing D-phenylalanine (JP-A-2-65797), and a process for producing D-lysine (JP-A-Heisei 2-65797). 8-173188) and the like, but the production method of D-ornithine, D-citrulline, D-tyrosine, D-glutamine is not known, and D-arginine, D-glutamine,
It has been desired to develop a more industrially advantageous method for producing phenylalanine or D-asparagine.

[0006]

DISCLOSURE OF THE INVENTION The present invention relates to the use of D-ornithine, D-citrulline, D-tyrosine, D-glutamine, D-arginine, D-phenylalanine and D-asparagine using a microbial enzyme. -To provide a method for producing an amino acid in an industrially advantageous manner.

[0007]

DISCLOSURE OF THE INVENTION The present inventors have studied racemic ornithine, racemic citrulline, racemic tyrosine,
Microorganisms having the ability to selectively degrade L-type optically active forms (L-forms) among racemic amino acids selected from racemic glutamine, racemic arginine, racemic phenylalanine and racemic asparagine, and utilizing these microorganisms The present inventors have found that a D-type optically active form (D-form) can be efficiently obtained from a racemic amino acid by the asymmetric decomposition reaction described above, and have completed the present invention.

That is, the present invention relates to the ability of asymmetric amino acids selected from racemic ornithine, racemic citrulline, racemic tyrosine and racemic glutamine to asymmetrically degrade the L-type optically active form of the selected amino acid. A method for producing a D-amino acid, which comprises separating and collecting the remaining D-type optically active substance after the culture of a microorganism having the above or a treated product thereof is allowed to act.

The present invention also relates to a genus of Proteus having the ability to asymmetrically degrade an L-type optically active form of a selected amino acid into a racemic amino acid selected from racemic arginine, racemic phenylalanine and racemic asparagine. A method for producing a D-amino acid, comprising separating and collecting the remaining D-type optically active substance after a culture of a microorganism such as described above or a processed product thereof is allowed to act.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, a racemic amino acid as a raw material compound includes not only those containing equal amounts of D-amino acids and L-amino acids but also those containing both optically active forms. Can be used.

The microorganism used in the present invention may be any microorganism that has the ability to asymmetrically degrade L-amino acids, ie, the ability to selectively degrade L-type optically active substances in racemic amino acids. Examples of such microorganisms include bacteria and yeasts. Examples of bacteria include Aerobacterium, Achromobacter, Acinetobacter,
Alcaligenes, Arthrobacter, Escherichia, Enterobacter, Xanthomonas, Kryubera, Klebsiella, Comamonas, Corynebacterium, Sarsina, Citrobacter, Pseudomonas, Serratia, Bacillus, Hafnia , Paracoccus, Brevibacterium, Proteus, Providencia, Micrococcus, Microbacterium, Morganella, Lactobacillus or Rhodococcus. Examples of the yeast include microorganisms belonging to the genera Candida, Saccharomycopsis or Hansenula.

[0012] Specific examples of these microorganisms include Aerobacter aerogenes.
origins) OUT8017, Achromobacter dendritic cam (Achromobacter den)
driticum) OUT8009, Achromobacter spellficialis (Achromobacter)
uppericialis) IAM1420, Achromobacter pestifer (Achromobacte)
r pastifier IAM1446, Achromobacter butyric
ri) OUT8004, Achromobacter liquid O
UT8013, Acinetobacter calcoaceti
cus) IFO12552, Alcaligenes denitrificans
ficans) JCM5490, Alcaligenes eutrophs
us) ATCC 17697, Alcaligenes faecalis OU
T8025, ATCC25094, FERM P-
6745, IFO 12669, Arthrobacter paraffin
fineus) ATCC 21219, Escherichia
Escherichia coli ATCC9
637, ATCC11105, ATCC2716
6. Enterobacter Aerogenes
acter aerogenes) ATCC1304
8, the same IFO 12010, Enterobacter cloacae N
CTC 9394, Xanthomonas maltophilia (X
anthomonas maltophilia) IF
O12020, Xanthomonas oryzae (Xantho)
monas oryzae) IFO 3995, Xanthomonas prun
i) IAM1313, Kluyvera cryoserescens
ATCC 14237, ATCC 21285, Klebsiella pneumoniae (Klebsiella pne
umoniae) IFO3317, ATCC1003
1. Comamonas acidovorans
acidivorans) IFO13582, AT
CC11299a, Corynebacterium alkanolicam (Corynebacterium alkan)
oliticum) ATCC 21511, Corynebacterium primorioxidans (Corynebact)
erium primorioxydans) ATCC
31015, Corynebacterium murisepticum
icum) ATCC 21374, Sarcina albida IAM1012, Citrobacter Freundi
freundii) ATCC 8090, Pseudomonas ovali
s) IAM1049, Pseudomonas gelidicola O
UT8116, Pseudomonas dacne (Pseudo
monas dacunhae) IAM1199, Pseudomonas puti
ida) ATCC 17453, Pseudomonas solanace
arum) IFO3509, Pseudomonas aeruginosa
a) IFO3445, IFO3446, IFO34
52, same IAM1007, same IAM1267, same IFO
3918, ATCC7700, ATCC1014
5. Pseudomonas fluorescens (Pseudom
onas fluorescens) IFO 3081,
IFO3459, ATCC13525, Serratia
Marsessens
s) IFO3736, ATCC14764, IAM
12143, Serratia Primulka
plymutica) IAM1255, Bacillus
Coagulans (Bacillus coaguran)
s) IFO 12714, Bacillus sphaericus (Ba
Cillus sphaericus) FERM P-
6746, Bacillus subtilis
subtilis) OUT8105, OUT810
6, OUT8109, IFO12210, Bacillus pumilus IF
O12088, Bacillus licheniformis (Baci
lluslicheniformis) ATCC217
33, Hafnia alve
i) IFO3731, Paracoccus denitrificans (Paracoccus denitrifican)
s) IFO12442, Brevibacterium ammoniagenes
niagenees) IAM1641, Brevibacterium i.
mperiale) IAM1654, Brevibacterium hebolum
lvolum) IAM1637, Proteus vulgaris OUT814
4, IFO3045, RIMD KS (IAM12
003), Proteus mirabilis (Proteus
mirabilis) IFO3849, Providencia alkaline faciliens (Providencia a)
lcalifaiens) JCM1673, Providencia reiteri (Providencia re)
ttgeri) IFO13501, ATCC2593
2, ATCC 29944, ATCC 9919, Providencia Rustidiani (Providencia)
rusticianii) JCM3953, Micrococcus uree
ae) IAM1010, Micrococcus roseus IFO37
64, Microbacterium sp.
cterium sp. ) ATCC 21376, Morganella morganii
nii) IFO3848, Lactobacillus delbruii
ckii) ATCC7830, Rhodococcus sp. ATCC1559
2. Candida tropicalis
organicis) IFO1402, Candida rugosa ATCC
10571, same IFO0591, Saccharomycopsis fibriguera
fibuligera) IFO0106, Saccharomycopsis lipolytica
sis lipolytica) IFO0717, I
FO1195, IFO1548, IFO1209,
Hansenula polymorpha (Hansenula po
(lymorpha) IFO1024.

Among the microorganisms having the ability to asymmetrically degrade L-ornithine, that is, the ability to selectively degrade L-ornithine in racemic ornithine, of the genus Aerobacterium, Achromobacter and Acinetobacter , Alcaligenes, Escherichia, Enterobacter, Xanthomonas, Kluyvera, Klebsiella, Comamonas, Corynebacterium, Citrobacter, Pseudomonas, Serratia, Bacillus, Hafnia, Paracoccus, Brevibacterium Microorganisms belonging to the genera, Proteus, Providencia, Micrococcus, Morganella, Lactobacillus, Candida, Saccharomycopsis or Hansenula are preferred, of which Proteus (Proteus vulgaris) Microorganisms belonging to the genus Proteus mirabilis, of the genus Providencia (Providencia littigeri, Providencia rustigiani, etc.) or of the genus Hafnia (Hafnia albay, etc.) are preferred in that the reaction time required until the L-form is almost completely degraded is short. Microorganisms belonging to the genus (Proteus vulgaris etc.) or the genus Hafnia (Hafnia albay, etc.) are preferred.

As the microorganism having the ability to asymmetrically degrade L-citrulline, ie, the ability to selectively degrade L-citrulline in racemic citrulline, a microorganism belonging to the genus Proteus (Proteus vulgaris) is preferable.

Microorganisms having the ability to asymmetrically degrade L-tyrosine, ie, the ability to selectively degrade L-tyrosine in racemic tyrosine, include those belonging to the genera Proteus, Providencia, Morganella or Saccharomycopsis. Microorganisms belonging to the genus Proteus (Proteus vulgaris) or Providencia (Providencia littigeri) are preferred in that the reaction time required until the L-form is almost completely decomposed is short. Proteus (Proteus
Microorganisms belonging to Bulgaris) are preferred.

As the microorganism having the ability to asymmetrically degrade L-glutamine, ie, the ability to selectively degrade L-glutamine in racemic glutamine, a microorganism belonging to the genus Proteus (Proteus vulgaris) is preferable.

Microorganisms having the ability to asymmetrically degrade L-arginine, that is, the ability to selectively degrade L-arginine in racemic arginine, include Achromobacter, Arthrobacter, Corynebacterium, Microorganisms belonging to the genus Sarsina, Pseudomonas, Bacillus, Brevibacterium, Proteus, Providencia, Microbacterium, Morganella, Rhodococcus, Candida or Saccharomycopsis are preferred, Proteus (Proteus
Microorganisms belonging to the genus Proteus (such as Proteus vulgaris) are preferable because the reaction time required for the L-form to be almost completely decomposed is short, and microorganisms belonging to the genus Proteus (such as Proteus vulgaris) are preferable. .

The microorganism having the ability to asymmetrically degrade L-phenylalanine, ie, the ability to selectively degrade L-phenylalanine in racemic phenylalanine, includes microorganisms belonging to the genus Proteus, Morganella or Saccharomycopsis. Microorganisms belonging to the genus Proteus (Proteus vulgaris and the like) are particularly preferable.

As the microorganism having the ability to asymmetrically degrade L-asparagine, that is, the ability to selectively degrade L-asparagine in racemic asparagine, a microorganism belonging to the genus Proteus (such as Proteus vulgaris) is preferable.

Also, among the microorganisms that can be used in the present invention,
Microorganisms belonging to the genus Proteus (such as Proteus vulgaris) also have the activity of selectively degrading L-lysine, as found by the present inventors (Japanese Patent Laid-Open No. 8-1731).
88), and D-ornithine, D-citrulline, D-
It is particularly preferable because it can be widely applied to the production of various D-amino acids such as tyrosine, D-glutamine, D-arginine, D-phenylalanine, and D-asparagine.

These microorganisms may be any strains as long as they have the necessary ability for the present invention.
The strain may be a strain newly isolated from soil, foods, animals, or the like, or may be a mutant strain obtained by artificial treatment with ultraviolet irradiation or treatment with a mutagen. Furthermore, those derived from these microorganisms by recombinant DNA, genetic engineering such as cell fusion, or biotechnological techniques may be used.

The culture in the present invention includes, for example,
Examples include a culture solution or viable cells of the microorganism. Examples of the processed product of the culture include a processed product of a culture solution (such as a culture supernatant) and a processed product of cells. For example, the culture is subjected to various physicochemical methods, for example, ultrasonic wave, French press, and the like. , Osmotic pressure, freeze-thaw, freeze-drying, alumina destruction, bacteriolytic enzyme, bacteria treated with a means such as a surfactant or an organic solvent, and the above-mentioned culture (culture solution, viable cells, etc.)
Or, from the processed product (culture supernatant, processed bacterial cell, etc.), a known method (ammonium sulfate fractionation, ion exchange chromatography,
Gel purification chromatography) or a partially purified enzyme which has the ability to selectively degrade L-amino acids in racemic amino acids.

The microbial cells, treated cells or enzymes of the present invention may be immobilized by, for example, a polyacrylamide method, a sulfur-containing polysaccharide gel method (such as a carrageenan gel method), an alginic acid gel method, or an agar gel method. Can also be used.

In the present invention, the cultivation of the microorganism is carried out, for example, by culturing the microorganism in a medium usually used in this field (such as a conventional medium containing a carbon source, a nitrogen source and inorganic salts).
Medium, normal temperature or under heating (preferably about 20 to about 40
C) and under aerobic conditions, at a pH of about 4 to about 10. When culturing, the amino acid is added to the medium at about 0.0
The enzyme activity can also be increased by adding more than 01%, especially about 0.1 to about 1%.

In the asymmetric degradation reaction according to the present invention, a culture of a microorganism having the ability to asymmetrically degrade an L-amino acid or a processed product thereof is brought into contact with a racemic amino acid as a raw material compound in a solution and incubated. It can be implemented by doing.

The reaction of the present invention can be suitably carried out in an aqueous solution, and the reaction is carried out at room temperature to under heating, preferably from about 10 to about 6
It preferably proceeds at 0 ° C., particularly preferably at about 25 to about 40 ° C. The reaction solution has a pH of about 3 to about 11, especially about 4 to about 9.
It is preferable to adjust so that

The feed concentration (w / v) of the racemic amino acid, which is a raw material compound serving as a reaction substrate, is usually about 0.05 to
Preferably it is about 50%, especially about 1 to about 20%. At that time, the reaction substrate may be added all at once, or may be added in several portions during the reaction.

When viable cells are used in the present invention, the reaction time can be reduced by adding a surfactant to the reaction solution. Examples of the surfactant used for this purpose include cetylpyridinium bromide, cetyltrimethylammonium bromide, p-isooctylphenyl ether (trade name: Triton X-100, manufactured by Rohm and Haas Co., USA). , About 0.000
It is preferable to use about 1 to about 0.1%.

If desired, the reaction may be carried out in parallel with the cultivation of the microorganism. When the reaction is carried out in parallel with the cultivation of the microorganism, the reaction may be carried out in the same manner as the cultivation using a medium to which racemic amino acids have been added in advance. The reaction may be performed under the following conditions.

After the completion of the reaction, D-amino acid can be easily collected and isolated from the reaction solution according to a conventional method. For example, a D-amino acid crystal can be obtained by removing insoluble substances such as bacterial cells from the reaction solution by centrifugation, concentrating the treated solution obtained by adsorbing and removing a dye or the like with activated carbon under reduced pressure, and crystallization by cooling. .

The determination of whether or not the culture of the microorganism or the processed product thereof has the ability to selectively degrade the L-amino acid in the racemic amino acid is carried out according to the above reaction method, for example, as follows. Can be easily implemented. That is, a culture of a microorganism to be assayed or a treated product thereof is added to a medium or an aqueous solution containing a racemic amino acid, and the mixture is treated at 30 ° C. for 2 to 1 hour.
Incubate for 68 hours. An optically active column (for example, when quantifying ornithine, glutamine or asparagine, is manufactured by Daicel Chemical Industries, CROWNP
When AKCR, tyrosine, arginine or phenylalanine is quantified, SUMI manufactured by Sumika Chemical Analysis Service, SUMI
When quantifying CHIRAL OA-5000 and citrulline, use SUMICHIRAL manufactured by Sumika Chemical Analysis Service, Ltd.
OA-6100), which is analyzed and quantified by high performance liquid chromatography, and the content of each of the D-amino acid and L-amino acid is measured. By measurement, for example,
When the number of L-amino acids in the racemic amino acid decreases and the number of D-amino acids remains, it is determined that the compound has the ability to asymmetrically decompose L-amino acids.

Hereinafter, the present invention will be described in more detail by way of examples, but the present invention is not limited thereto.

[0033] In this specification, "%" means "weight / volume (g / dl)". Further, in this example, the quantification of the optically active form of the amino acid was performed by the above-described high performance liquid chromatography.

[0034]

【Example】

Example 1 DL-ornithine 2%, ammonium sulfate 0.5%, potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.0
Medium containing 5% and 0.02% yeast extract (pH 7.
0) 3 ml was placed in a test tube and sterilized at 120 ° C. for 10 minutes. This medium was inoculated with the microorganisms shown in Table 1 below,
After shaking culture at ℃ for 168 hours, D-ornithine remaining in the culture solution was quantified. The content of D-ornithine is shown in Table 1 below, and its enantiomer, L-ornithine, was hardly detected in the culture solution.

[0035]

[Table 1]

Example 2 A 500-ml shake flask was charged with 50 ml of a medium (pH 7.0) consisting of 1.0% of DL-ornithine, 1.0% of polypeptone, 1.0% of yeast extract, and 0.5% of sodium chloride. Sterilized at 120 ° C. for 10 minutes. Proteus vulgaris (Proteus vulgari) is added to this medium.
s) One loopful of RIMD KS (IAM12003) was inoculated and cultured at 30 ° C. for 20 hours. The above culture solution 1600
The bacterial cells collected by centrifugation from a ml (32 flasks) were suspended in physiological saline, and then collected by centrifugation. 50 mM containing 48 g of DL-ornithine in the cells
800 ml of phosphate buffer (pH 7.0) was added,
For 90 hours. After the reaction, the bacteria were removed by centrifugation to obtain a supernatant. The supernatant was subjected to ultrafiltration to remove proteins and the like to obtain a filtrate. The filtrate was decolorized with activated carbon to obtain a decolorized liquid. The decolorized solution was concentrated under reduced pressure to obtain 10.5 g of crude D-ornithine crystals. Further, the crude crystals were added to water 10
0-ml and recrystallized by adding 400 ml of ethanol to obtain 8.1 g of D-ornithine crystals.
I got The optical rotation and optical purity of this crystal were as follows. Optical rotation: [α] _D ²⁰ = −22.8 ° (C = 4,6N
HCl) Optical purity: 100%.

Example 3 Bacteria shown in Table 2 below were placed in 3 ml of a medium (pH 7.0) consisting of 1.0% DL-ornithine, 1.0% polypeptone, 1.0% yeast extract, and 0.5% sodium chloride. Was inoculated and cultured at 30 ° C. for 24 hours. The cells collected by centrifugation from 3 ml of the above culture solution were suspended in physiological saline, and then collected by centrifugation. 2 mM of 50 mM phosphate buffer (pH 7.0) containing 4% DL-ornithine
was added, and the mixture was subjected to an asymmetric decomposition reaction at 30 ° C. for 120 hours. The content of D-ornithine in this reaction solution is shown in Table 2 below, and its enantiomer, L-ornithine, was hardly detected in the reaction solution.

[0038]

[Table 2]

[0039]

[Table 3]

Example 4 Bacteria shown in Table 3 below were placed in 3 ml of a medium (pH 7.0) consisting of 1.0% DL-ornithine, 1.0% polypeptone, 1.0% yeast extract, and 0.5% sodium chloride. Was inoculated and cultured at 30 ° C. for 24 hours. The cells collected by centrifugation from 3 ml of the above culture solution were suspended in physiological saline, and then collected by centrifugation. 50 mM phosphate buffer (pH 7.0) containing 10% DL-ornithine in the cells
Then, the mixture was subjected to an asymmetric decomposition reaction at 30 ° C. for 168 hours.
The content of ornithine in this reaction solution was as shown in Table 3 below.

[0041]

[Table 4]

Example 5 50 ml of a medium (pH 7.0) containing 1.0% of polypeptone, 1.0% of yeast extract and 0.5% of sodium chloride was placed in a 500 ml shake flask, and sterilized at 120 ° C. for 10 minutes. Proteus vulgaris (Pro)
teus vulgaris) RIMD KS (IAM
12003) was inoculated with one platinum loop and shake-cultured at 30 ° C. for 20 hours. The cells collected from 6 ml of the above culture solution by centrifugation were suspended in physiological saline, and then collected by centrifugation. The cells contain 30 mg of DL-citrulline 50
mM phosphate buffer (pH 8.0) 3 ml,
After 5 hours of asymmetric decomposition reaction, citrulline remaining in the reaction solution was quantified. As a result of the quantification, 13 mg of D-citrulline remained in the reaction solution, and the enantiomer L-citrulline was completely decomposed.

Example 6 3 ml of a medium (pH 7.0) consisting of 0.2% of DL-tyrosine, 1.0% of yeast extract, 1.0% of polypeptone and 0.5% of sodium chloride was placed in a test tube, and heated at 120 ° C. At 10
Sterilized for a minute. The microorganisms shown in Table 4 below were inoculated into this medium, and cultured with shaking at 30 ° C. for 24 hours, and then tyrosine remaining in the culture solution was quantified. The content of tyrosine was as shown in Table 4 below.

[0044]

[Table 5]

Example 7 A 500 ml shake flask was charged with 50 ml of a medium (pH 7.0) containing 1.0% of polypeptone, 1.0% of yeast extract, and 0.5% of sodium chloride, and sterilized at 120 ° C. for 10 minutes. Proteus vulgaris (Pro)
teus vulgaris) RIMD KS (IAM
12003) was inoculated with one platinum loop and shake-cultured at 30 ° C. for 20 hours. The cells collected from 6 ml of the above culture solution by centrifugation were suspended in physiological saline, and then collected by centrifugation. The cells contain 30 mg of DL-glutamine 50
mM phosphate buffer (pH 8.0) 3 ml,
After 10 hours of asymmetric decomposition reaction, glutamine remaining in the reaction solution was quantified. As a result of the quantification, 10.3 mg of D-glutamine remained in the reaction solution, and the enantiomer L-glutamine was completely decomposed.

Example 8 DL-arginine 2%, ammonium sulfate 0.5%, potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.0
Medium containing 5% and 0.02% yeast extract (pH 7.
0) 3 ml was placed in a test tube and sterilized at 120 ° C. for 10 minutes. This medium was inoculated with the microorganisms shown in Table 5 below,
After shaking culture at 168 hours at ℃, arginine remaining in the culture solution was quantified. The content of arginine was as shown in Table 5 below.

[0047]

[Table 6]

Example 9 Bacteria shown in Table 6 below were placed in 3 ml of a medium (pH 7.0) consisting of 1.0% DL-arginine, 1.0% polypeptone, 1.0% yeast extract, and 0.5% sodium chloride. Was inoculated and cultured at 30 ° C. for 24 hours. The cells collected by centrifugation from 3 ml of the above culture solution were suspended in physiological saline, and then collected by centrifugation. A 50 mM phosphate buffer (pH 7.0) containing 2% of DL-arginine was added to the cells.
1 was added, and an asymmetric decomposition reaction was performed at 30 ° C. for 168 hours. The content of arginine in this reaction solution was as shown in Table 6 below.

[0049]

[Table 7]

Example 10 3 ml of a medium (pH 7.0) consisting of 1% of DL-phenylalanine, 1.0% of yeast extract, 1.0% of polypeptone and 0.5% of sodium chloride was placed in a test tube, and the mixture was heated at 120 ° C. for 10 minutes. Sterilized for a minute. The microorganisms shown in Table 7 below were inoculated into this medium, and cultured with shaking at 30 ° C. for 24 hours, and phenylalanine remaining in the culture solution was quantified. The content of phenylalanine was as shown in Table 7 below.

[0051]

[Table 8]

Example 11 1.0% of DL-phenylalanine, 1.0 of polypeptone
%, Yeast extract 1.0%, and sodium chloride 0.5%, 3 ml of a medium (pH 7.0) was inoculated with the bacteria shown in Table 8 below and cultured at 30 ° C. for 24 hours. 3m of the above culture solution
The cells collected by centrifugation were suspended in physiological saline and collected by centrifugation. 2% DL in the cells
-50 mM phosphate buffer containing phenylalanine (pH
7.0) 2 ml was added, and asymmetric decomposition reaction was carried out at 30 ° C for 168 hours. The content of phenylalanine in this reaction solution was as shown in Table 8 below.

[0053]

[Table 9]

Example 12 50 ml of a medium (pH 7.0) consisting of 1.0% polypeptone, 1.0% yeast extract and 0.5% sodium chloride was placed in a 500 ml shake flask and sterilized at 120 ° C. for 10 minutes. Proteus vulgaris (Pro)
teus vulgaris) RIMD KS (IAM
12003) was inoculated with one platinum loop and shake-cultured at 30 ° C. for 20 hours. The cells collected from 6 ml of the above culture solution by centrifugation were suspended in physiological saline, and then collected by centrifugation. The cells contain 30 mg of DL-asparagine 5
Add 3 ml of 0 mM phosphate buffer (pH 8.0), and add 30 ml.
After the asymmetric decomposition reaction at 10 ° C. for 10 hours, asparagine remaining in the reaction solution was quantified. As a result of the quantification, 9.9 mg of D-asparagine remained in the reaction solution, and the enantiomer L-asparagine was completely decomposed.

[0055]

According to the method of the present invention, D-amino acids can be produced extremely efficiently and with high optical purity from industrially inexpensive racemic amino acids.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification number Office reference number FI Technical indication (C12P 41/00 C12R 1:37) (C12P 41/00 C12R 1: 385) (C12P 41/00 (C12R 41:39) (C12P 41/00 C12R 1:13) (C12P 41/00 C12R 1:85) (C12P 41/00 C12R 1:74) (C12P 41/00 C12R 1:01) (C12P 41/00 (C12R 41:05) (C12P 41/00 C12R 1:19) (C12P 41/00 C12R 1:22) (C12P 41/00 C12R 1:64) (C12P 41/00 C12R 1:38) (C12P 41/00) (C12P 41/00 C12R 1: 425) (C12P 41/00 C12R 1: 425) (C12P 41/00 C12R 1:07) (C12P 41/00 C12R 1: 265) (C12P 41/00 C12R 1: 225 ) (C12P 41/00 C12R 1:10) (C12P 41/00 C12R 1:78) (C12P 41/00 C12R 1:06) (C12P 41/00 C12R 1:15) (C12P 41/00 C12R 1:72) )

Claims (17)

[Claims] 1. A microorganism having the ability to asymmetrically degrade an L-type optically active form of a selected amino acid into a racemic amino acid selected from racemic ornithine, racemic citrulline, racemic tyrosine and racemic glutamine. A method for producing a D-amino acid, comprising separating and collecting a remaining D-type optically active substance after allowing a culture or a processed product thereof to act. 2. The microorganism is of the genus Aerobacterium, Achromobacter, Acinetobacter, Alcaligenes,
Arthrobacter, Escherichia, Enterobacter, Xanthomonas, Kluyvera, Klebsiella, Comamonas, Corynebacterium, Sarsina, Citrobacter, Pseudomonas, Serratia, Bacillus, Hafnia, Paracoccus A microorganism belonging to the genus Brevibacterium, Proteus, Providencia, Micrococcus, Microbacterium, Morganella, Lactobacillus or Rhodococcus, Candida, Saccharomycopsis or Hansenula. 1. The production method according to 1. 3. The method according to claim 1, wherein the racemic amino acid is racemic ornithine. 4. The microorganism according to claim 1, wherein the microorganism is of the genus Aerobacterium, Achromobacter, Acinetobacter, Alcaligenes,
Escherichia, Enterobacter, Xanthomonas, Kluyvera, Klebsiella, Comamonas, Corynebacterium Citrobacter, Pseudomonas, Serratia, Bacillus, Hafnia, Paracoccus, Brevibacterium, Proteus , Providencia, Micrococcus, Morganella,
4. A microorganism belonging to the genus Lactobacillus, Candida, Saccharomycopsis or Hansenula.
The manufacturing method described. 5. The method according to claim 4, wherein the microorganism is a microorganism belonging to the genus Proteus, Providencia or Hafnia. 6. The method according to claim 1, wherein the racemic amino acid is racemic citrulline. 7. The method according to claim 6, wherein the microorganism is a microorganism belonging to the genus Proteus. 8. The method according to claim 1, wherein the racemic amino acid is racemic tyrosine. 9. The method according to claim 8, wherein the microorganism is a microorganism belonging to the genus Proteus, Providencia, Morganella or Saccharomycopsis. 10. The method according to claim 1, wherein the racemic amino acid is racemic glutamine. 11. The method according to claim 10, wherein the microorganism is a microorganism belonging to the genus Proteus. 12. A racemic arginine, wherein the genus Achromobacter, the genus Arthrobacter, the genus Corynebacterium,
Belonging to the genera Sarsina, Pseudomonas, Bacillus, Brevibacterium, Proteus, Providencia, Microbacterium, Morganella, Rhodococcus, Candida or Saccharomycopsis;
A method for producing D-arginine, which comprises reacting a culture of a microorganism having the ability to asymmetrically degrade arginine or a processed product thereof, and separating and collecting the remaining D-type optically active substance. 13. The method according to claim 12, wherein the microorganism is a microorganism belonging to the genus Proteus or Providencia. 14. A culture of a microorganism belonging to the genus Proteus, Morganella or Saccharomycopsis and having the ability to asymmetrically degrade L-phenylalanine, or a treated product thereof is allowed to act on racemic phenylalanine. Wherein the D-type optically active substance is separated and collected.
-Preparation of phenylalanine. 15. A racemic asparagine is treated with a culture of a microorganism belonging to the genus Proteus and capable of asymmetrically degrading L-asparagine or a processed product thereof, and the remaining D-type optically active substance is separated. A method for producing D-asparagine, which comprises collecting. 16. A racemic amino acid selected from racemic ornithine, racemic citrulline, racemic tyrosine, racemic glutamine, racemic arginine, racemic phenylalanine and racemic asparagine, and the L-type optics of the selected amino acid A method for producing a D-amino acid, comprising separating and collecting a remaining D-type optically active substance after a culture of a microorganism belonging to the genus Proteus or a processed product thereof having the ability to asymmetrically decompose the active substance is allowed to act thereon. . 17. The method according to claim 16, wherein the microorganism belonging to the genus Proteus is Proteus vulgaris.