JPH11113592A - Production of d-amino acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing D-amino acid used in the field of medicines, chemicals and cosmetics, in an industrially advantageous way using the corresponding acylated amino acid as raw material through making microorganisms having D-aminoacylase activity or a treatment product thereof act on the acylated amino acid to accumulate the aimed D-amino acid in the resulting reaction liquor. SOLUTION: This method for producing the objective D-amino acid comprises that the corresponding acylamino acid is subjected to a culture liquid, microbial cells or enzyme extract (after microbial cell treatment) of newly found microorganisms high in D-aminoacylase activity and belonging to the genus Arthrobacter, Corynebacterium, Erwinia, Escherichia, Flavobacterium, Nocardia, Protaminobacter, Rhodococcus, or Xanthomonas, and the aimed D-amino acid formed is collected.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing D-amino acids used in the fields of pharmaceuticals, chemicals and cosmetics.

[0002]

2. Description of the Related Art Conventionally, as a method for producing a D-amino acid, D-aminoacylase is allowed to act on an acylated amino acid to produce D-amino acid.
-Methods for obtaining amino acids are known. As a source of D-aminoacylase, Pseudomonas (Pseudomona
s) Bacteria of the genus (JP-A-55-42534), bacteria of the genus Streptomyces (JP-B-53-3603)
5) Also, bacteria belonging to the genus Alcaligenes (JP-A-64-5488) are known.

[0003] However, all of the D-aminoacylases hitherto known have the disadvantage of low activity. In addition, a method of using a recombinant technique to enhance the activity and amplifying the enzyme activity has also been studied (Protein Ex
pression and purification Vol. 7, p. 395 (1996)
However, this is a method for expressing an enzyme of a bacterium belonging to the genus Alcaligenes in Escherichia coli, and is a method based on heterologous gene recombination, so that it is poor in versatility.

[0004]

DISCLOSURE OF THE INVENTION The present invention searches for a microorganism having a high D-aminoacylase activity or a microorganism which is easily self-cloned, and industrially implements the microorganism using those microorganisms having a high D-aminoacylase activity. The present invention provides a method for producing a D-amino acid which is advantageous for the above.

[0005]

Means for Solving the Problems The present inventors have conducted intensive studies on a method for producing a D-amino acid in order to solve the above-mentioned problems. As a result, the present inventors have newly found an arthrobacter.
r) genus, Corynebacterium genus, Erwinia genus, Escherichia genus,
Flavobacterium, Nocardia
(Nocardia) genus, Protaminobacter
Microorganisms belonging to the genus, Rhodococcus or Xanthomonas have D-aminoacylase activity and have the ability to efficiently convert acyl amino acids to D-amino acids. Thus, the present invention has been completed.

That is, the present invention provides an
-Has the ability to convert to amino acids,
rthrobacter), Corynebacteriu
m) genus, Erwinia, Escheria
ichia), Flavobacterium
Genus, Nocardia, Protaminobacter, Rhodococcus
A culture of a microorganism belonging to the genus Xanthomonas or the genus Xanthomonas, a microbial cell isolated from the culture or a processed product of the microbial cell, acting on an acyl amino acid;
Collecting D-amino acids to be produced,
-It relates to a method for producing an amino acid.

[0007]

DETAILED DESCRIPTION OF THE INVENTION The microorganism used in the present invention has a D-aminoacylase activity, that is, a genus of the genus Arthrobacter, Corynebacterium, Erwinia, which has the ability to convert acyl amino acids to D-amino acids. Any microorganisms belonging to the genus Eselicia, the genus Flavobacterium, the genus Nocardia, the genus Protaminobacterium, the genus Rhodococcus, and the genus Xanthomonas may be used.Specifically, the following microorganisms are exemplified. Can be.

Arthrobacter paraffineus (Arthrobacter paraffineus) ATCC15590 ATCC15583 Corynebacterium acetoacidphilum ATCC373 Corynebacterium xerosis ATCC13870 Escherichia coli AJ2606 (FERM BP-477) Escherichia coli Eiacherica Escherichia coli Escherichia coli (Escherichia coli) (Escherichia coli) Flavobacterium sewanense AJ2476 (FERM BP-476) Nocartia Nocardia asteroides ATCC3318 Protaminebacterium alboflavus ATCC8458 Rhodococcus erythripolis ATCC11048 Rhodococcus sp. Escherichia coli AJ2606
(FERM BP-477) was deposited on April 25, 1973 in the International Depositary Authority: Institute of Microbial Industry and Technology, Ministry of International Trade and Industry (currently, Institute of Biotechnology and Industrial Technology, Ministry of International Trade and Industry). Strain No. FERM BP-477, which was transferred to an international deposit under the Budapest Treaty on April 25, 1983, and is a strain of Flavobacterium sewanense.
AJ2476 (FERM BP-476) was deposited with the depositary authority on April 25, 1973 and transferred to an international deposit under the Budapest Treaty on April 25, 1983, under accession number FERM BP-47.
6 strains, Xanthomonas citri AJ
2785 (FERM P-3396) was filed with the depositary authority on January 2, 1976.
It is a strain of Accession No. FERM P-3396 deposited on the 7th.

An improved strain in which the activity of contaminating enzymes such as L-aminoacylase has been reduced or inactivated by artificially using the microorganism as a parent strain for mutation treatment or recombinant DNA manipulation, or D-amino acid. It is also possible to use an improved strain having enhanced acylase activity. In particular, a method for obtaining a mutant in which the contaminating enzyme L-aminoacylase has been inactivated by artificial mutation treatment is disclosed in
-126969 or JP-A-62-126976, which can be easily obtained. Therefore, by using a mutant in which L-aminoacylase is inactivated,
Production of D-amino acids having high optical purity from L-amino acids is possible.

In order to obtain the cells of such a microorganism, the microorganism may be cultured and grown in an appropriate medium. Such a medium is not particularly limited, and may be an ordinary medium containing ordinary carbon sources, nitrogen sources, inorganic ions, and, if necessary, organic nutrients such as amino acids and vitamins. Examples of the carbon source include sugars such as glucose, sucrose, fructose, galactose, and lactose; starch saccharified solutions containing these sugars; sweet potato molasses, sugar beet molasses, hightest molasses; and organic acids such as acetic acid; ethanol; Alcohols, glycerin and the like are also used. As a nitrogen source, ammonia gas, aqueous ammonia, ammonium salts, urea, nitrates, and other organic nitrogen sources used as auxiliary substances, such as oil cakes, soybean hydrolysate, casein hydrolyzate, other amino acids, corn steep liquor, Peptides such as yeast or yeast extract and peptone are used. As the inorganic ions, phosphate ions, magnesium ions, calcium ions, iron ions, manganese ions, and the like are appropriately added. If the microorganism of the present invention has a required substance such as an amino acid, the required substance must be added.

There are no particular restrictions on the conditions for culturing microorganisms.
For example, under aerobic conditions, pH and temperature are appropriately controlled within a range of 5 to 8 and a temperature of 25 to 40 ° C.
Culture may be performed for about 2 to 48 hours. PH of culture solution
May be adjusted to a predetermined value with an inorganic or organic acid, an alkaline substance, urea, calcium carbonate, ammonia gas or the like.

The above-mentioned microorganisms can be reacted with acylamino acids by a method using the microorganism culture thus obtained as it is, by separating cells from the microorganism culture by centrifugation or the like, and washing or washing the same with a buffer solution. , Water and the like, and then reacting by adding an acyl amino acid. In addition, as a processed product of the microbial cells, crushed cells, acetone-treated cells, freeze-dried cells, or these cells or the processed cells are treated with a polyacrylamide gel method, a carrageenan method, an alginic acid method, or the like. The cells immobilized by a known method can be used. Further, as the treated microorganism cells, there may be used a cell extract or D-purified and obtained by combining known methods.
-Enzymes having aminoacylase activity can also be used. In this case, D- is obtained from the treated cells obtained by mechanically grinding cells, sonication-crushed cells, or lysozyme.
A fraction having an aminoacylase activity may be purified and used, and the purification method may be a method for purifying a normal enzyme, such as ammonium sulfate fractionation, ion-exchange chromatography, hydrophobic chromatography, affinity chromatography, etc. Can be mentioned.

[0013] The amount of the cells or the treated cells is as follows.
It is sufficient that the amount (effective amount) exerts the intended effect in the case of a normal reaction, and this effective amount can be easily determined by a person skilled in the art by a simple preliminary experiment. In this case, 10 to 40 per liter of the reaction solution is used.
0 g.

The acylamino acid may be used as it is, or may be dissolved in water, or dissolved in an organic solvent that does not affect the reaction, or dispersed in a surfactant or the like. It may be used after adding.

The reaction pH is pH 3 to 9, preferably pH 5
-8, reaction temperature is 10-60 ° C, preferably 20-40 ° C
And under stirring or standing for about 1 to 120 hours. The concentration of the substrate used is not particularly limited, but is 1% to
About 30% is preferable.

After the completion of the reaction, the accumulated D-amino acid is separated from the reaction solution by crystallization such as concentration crystallization and cooling crystallization. The separated crude D-amino acid is redissolved again, and impurities are removed using activated carbon or the like, and then purified by reconstitution.

Hereinafter, the present invention will be described in detail with reference to examples.

【Example】

Example 1: Production of D-phenylalanine (D-Phe) by a microorganism having a novel D-aminoacylase activity N-acetyl-D-phenylalanine (N-Ac-D-Phe) 2 g / L,
Yeast extract S (Nippon Pharmaceutical) 10 g / L, polypeptone (Nippon Pharmaceutical) 10 g / L, KH2PO4 1 g / dL, K2HPO4 3 g / L, MgSO4
・ 7H20 0.5 g / L was adjusted to pH 7.0 with KOH, agar 20 g / L was added and sterilized at 120 ° C. for 15 minutes, and the prepared plate medium was previously treated with bouillon agar medium at 30 ° C. for 24 hours. Microorganisms shown in Table 1 were inoculated and cultured for 24 hours. After the culture, the cells were scraped and collected.

The cells were treated with 150 mM (31.1 mg / L) N-Ac-D-Ph
e in 0.1M phosphate buffer (pH 7.0), 5% by wet weight
(w / v) and reacted at 30 ° C. for 42 hours. The produced D-Phe in the reaction solution was quantified by high performance liquid chromatography (HPLC), and the results are shown in Table 1. As a comparative example, a known D-amino acid producing bacterium Streptomyces turius IFO13418 (Japanese Patent Application Laid-Open No. 62-12969, or Agric. Biol. Chem. 44: 1089)
Page, 1980), the results show that all of the newly found bacteria have higher D-Phe productivity than the comparative examples. The analysis conditions were column
3. nacalaitesque COSMOSIL 5C18 4.6 × 50 mm and Mitsubishi Kasei MCI GEL CRS10W (DLAA)
6 x 50 mm serial flow; detection UV 254 nm; mobile phase 2 mM
CuSO4 aqueous solution: methanol = 85:15; flow rate, 1.0 ml / mi
n .; temperature was 50 ° C. Retention time is D-Phe
7.5 min. And L-Phe 8.8 min.

[0019]

【table 1】

Example 2: Production of various D-amino acids N-acetyl-D-phenylalanine 2 g / L, yeast extract S
(Nippon Pharmaceutical) 10 g / L, Polypeptone (Nippon Pharmaceutical) 10
g / L, KH2PO4 1g / L, K2HPO4 03g / L, MgSO4.7H200.5g / L,
A medium (pH 7.0) containing 20 g / L of fumaric acid is placed in a 500 ml flask.
0 ml was added and sterilized at 120 ° C. for 15 minutes. This was inoculated with Arthrobacter hydrocarboglutamicus ATCC15583, which had been cultured at 30 ° C. for 24 hours on a bouillon agar medium, and cultured for 24 hours. After culturing, use the same medium
2 liters were poured, and the solution was transferred to a similarly sterilized small fermenter (5 liter volume), and cultured for 30 hours at a temperature of 30 ° C., a stirring speed of 350 rpm, aeration of 1 liter per minute, and no culture pH control. After the culture, the cells were collected by centrifugation (10,000 G, 15 minutes).

9 ml of 100 mM phosphate buffer (pH 6.0) was added to the cells (wet weight: 1 g) to obtain a concentrated cell suspension. Then each one
0 g / L N-acetyl-D-alanine, N-acetyl-D-valine,
N-acetyl-D-leucine, N-acetyl-D-isoleucine,
0.5 ml of substrate solution in which N-acetyl-D-tryptophan and N-acetyl-D-methionine are dissolved in 100 mM phosphate buffer (pH 6.0)
0.5 ml of a concentrated cell suspension was added to the mixture, and the mixture was reacted at 30 ° C. for 24 hours. After the reaction, the D-amino acids generated in the reaction solution were analyzed by HPLC in the same manner as in Example 1. As a result, the results were as shown in Table 2, and D-amino acids better corresponding to each acetyl amino acid were obtained.

[0022]

[Table 2]

Example 3 Production of D-Phenylalanine by Purified Enzyme, a Treated Cell Material As in Example 2, Arthrobacter hydrocarboglutamicus ATCC15583 was cultured, and a 100 g wet cell weight was collected. After adding 40 ml of 20 mM phosphate buffer to the paste to form a concentrated cell suspension, the cells were crushed for 5 minutes using a bead beater (manufactured by BioSpec, using a glass bead diameter of 0.1 mm). , 20 mM phosphate buffer (pH 7.
0) was added to make a total volume of 100 ml. Remove the cell lysate from which the glass beads have been removed by decantation, and further centrifuge (1
(0,000 G, 15 minutes) to remove uncrushed residue. Thereafter, 0.1 g of protamine was added to the obtained centrifuged supernatant,
After the nucleic acid component was removed by precipitation, this was used as a crude enzyme solution.

Ammonium sulfate was added to 50 ml of the crude enzyme solution under ice-cooling so as to be 40% saturated. Thereafter, the solution was passed through a butyltoyopearl 650M column (φ2.5 × 30 cm) equilibrated with a 40% ammonium sulfate saturated 20 mM phosphate buffer (pH 7.0) to adsorb the enzyme. 500m
After washing the column with l of 40% ammonium sulfate saturated buffer, (NH
4) The enzyme was eluted with a linear gradient of 2SO4 saturation of 50 to 0% (flow rate 70 ml / min, fractionated 10 ml each). Active fraction (2
200 ml of a fraction eluted with 0% to 0% ammonium sulfate saturated solution) was obtained. afterwards,
The active fraction was concentrated to 10 ml using an ultrafiltration membrane (excluded molecular weight MW 10,000), and then reconstituted in 20 mM phosphate buffer (pH 6.0).
Dialyzed to obtain a purified enzyme solution.

9 ml of a substrate solution obtained by dissolving 20.7 g / L of N-acetyl-D, L-phenylalanine in 100 mM phosphate buffer (pH 6.0)
Then, 1 ml of the purified enzyme solution was added thereto, and incubated at 30 ° C. for 40 hours. Thereafter, the reaction was stopped by boiling the reaction mixture at 100 ° C. for 5 minutes, and the amount of phenylalanine produced in the reaction mixture was analyzed by HPLC in the same manner as in Example 1. As a result, 6.4 g / L of D-phenylalanine was generated in the reaction solution. L-phenylalanine was hardly produced.

[0026]

As described above, according to the present invention, a corresponding D-amino acid can be easily and efficiently produced from an acyl amino acid.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI C12P 13/22 C12P 13/22 AC // (C12P 13/06 C12R 1:06) (C12P 13/06 C12R 1:06) (C12P 13/06 C12R 1:06) (C12P 13/08 C12R 1:06) (C12P 13/12 C12R 1:06) (C12P 13/22 C12R 1:06) (C12P 13/22 C12R 1:15) (C12P 13/22 C12R 1:19) (C12P 13/22 C12R 1:18) (C12P 13/22 C12R 1:20) (C12P 13/22 C12R 1: 365) (C12P 13/22 C12R 1:64) (C12P 13/22 C12R 1:01) (C12P 13/22 C12R 1:06) (72) Inventor Kenzo Yokoseki 1-1 Suzukicho, Kawasaki-ku, Kawasaki-ku, Kawasaki-shi, Kanagawa Prefecture Moto Research Co., Ltd. Inside

Claims (1)

[Claims] The present invention has the ability to convert an acyl amino acid to a D-amino acid, and has the genus Arthrobacter, Corynebacterium, and Erbinia (E).
rwinia, Escherichia, Flavobacterium, Nocardia
rdia) genus, Protaminobacter
Genus, a culture of a microorganism belonging to the genus Rhodococcus or the genus Xanthomonas, a microbial cell isolated from the culture or a processed product of the microbial cell, acting on an acyl amino acid to produce a D-amino acid A method for producing D-amino acids.