JPH0438399B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention relates to a method for producing a corresponding L-amino acid from an N-carbamyl form of an amino acid using a microbial enzyme system. [Prior Art] Conventionally, as a method for producing L-amino acid from DL-N-carbamyl amino acid using a microbial enzyme system, a method for producing L-methionine from DL-N-carbamylmethionine is known. (Special Publication No. 55-29678). [Problems to be solved by the invention] In the conventional method, since DN-carbamylmethionine remains as it is, L from the reaction product solution is
- Requires separation of methionine and DN-carbamylmethionine. The purpose of the present invention is to convert all N-carbamyl amino acids, regardless of D and L forms, into the corresponding L-amino acids using microbial enzyme systems.
An object of the present invention is to provide a method for producing amino acids. [Means for Solving the Problems] To summarize the present invention, the present invention relates to a method for producing an L-amino acid, and the present invention relates to a method for producing an L-amino acid. L-form of carbamyl body,
The D-form or DL-form is treated with a culture solution, bacterial cells, or treated bacterial cells of a microorganism belonging to the genus Arilobacter, and the corresponding L-phenylalanine, L-tryptophan, or L-tyrosine produced is collected. Features. According to the method of the present invention, the D-type N-carbamyl form can also be converted into the corresponding L-amino acid. Examples of raw materials used in the method of the present invention include N-
Examples include carbamylphenylalanine, N-carbamyltryptophan, and N-carbamyltyrosine. These may be any of the D, L and DL forms. The raw material for the method of the present invention is a known substance or can be obtained by a known method,
For example, DL-N-carbamyl amino acid can be obtained by a known method of reacting DL-amino acid with potassium cyanide or the like, or by hydrolyzing a DL-hydantoin derivative of an amino acid under appropriate conditions. Next, as the Arthrobacter microorganism used in the method of the present invention, its culture solution can be used as it is, but live cells, frozen cells, freeze-dried cells, ground bacterial cells, or bacterial cells can be used. A processed product such as an extract may also be used. It goes without saying that mutant strains with improved performance through mutation treatment can also be used more effectively. Among the microorganisms of the genus Arithrobacter mentioned above, a suitable example is Arilobacter genus DK-200, which is a new bacterial species newly isolated from the natural world by the present inventors.
There are strains. In addition, the DK-200 strain is
Arthrobacter sp.DK−200 (Arthrobacter genus)
It has been deposited with the National Institute of Microbiology, Agency of Industrial Science and Technology as FERM P-7472. The present inventors previously discovered that the above-mentioned Arilobacter spp.
A method for producing the corresponding L-amino acid from 5-substituted hydantoin was developed using a culture of the DK-200 strain (Japanese Patent Application No. 70906/1983). The mycological properties of the DK-200 strain described above are shown below. 1 Morphology (1) Cell shape and size: 0.3 to 0.5 x 0.8 to 5.0 μm (2) Presence or absence of pleomorphism: Yes (3) Presence or absence of motility: No (4) Presence or absence of flagella, epiphytic state No (5) Presence of spores: None (6) Gram staining:
Negative to weakly positive, but has Gram-positive particles (7) Acid-fast: None Growth status on each medium (1) Broth agar plate culture Colony color: Pale white Colony shape: Round Colony bulge: Convex Colony Periphery: Entire edge (2) Meat juice agar slant culture Growth condition: Moderate Gloss: Yes Shape: Thread-like (3) Meat juice liquid culture Degree of turbidity: Uniform Sedimentation: Yes Growth on liquid surface: None (4) Meat juice gelatin puncture culture: Liquefaction 3 Physiological properties (1) Nitrate reduction: Yin (2) Denitrification reaction: Yang (3) MR test: Yin (4) VP test: Yin (5) Indole formation: Yin ( 6) Formation of hydrogen sulfide: Yin (7) Hydrolysis of starch: Yin (8) Utilization of citric acid:
Do not use Koser medium or Christensen medium (9) Use of inorganic nitrogen source: Use nitrate and ammonium salts (10) Pigment production: None (11) Urease: Yin (12) Oxidase: Yin (13) Catalase: Positive (14) Attitude towards oxygen: Aerobic (15) Growth range Temperature: 17.8-33.2°C PH: 5-10 (16) OF test: Oxidative (17) Acid from sugars and presence or absence of gas generation.

【table】

〔Example〕

EXAMPLES Hereinafter, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited thereto. In addition, unless otherwise specified, % in each example is weight %. Example 1 Glucose 0.5%, yeast extract 0.2%, disodium hydrogen phosphate 0.1%, potassium dihydrogen phosphate
0.05%, magnesium sulfate 0.05% (PH
7.0) 5 ml was dispensed into test tubes and sterilized at 120°C for 15 minutes. After separately sterilized DL-N-carbamyltryptophan was added to the final concentration of 0.1%, Arilobacter DK-200 was inoculated and cultured with shaking at 30°C for 24 hours. The bacterial cells were collected by centrifugation from this culture solution, washed with the same amount of physiological saline, and then separated into L-N cells listed in Table 1 below.
- Carbamyl amino acid 0.1M and manganese chloride
0.1M Tris-HCl buffer (PH7.5) containing 0.1mM
ml and reacted at 35°C for 20 hours. Bioassay the desired product [Lactobacillus arabinosus ATCC]
Table 1 shows the results of quantitative determination by HPLC. Since both analytical values matched, the target product was confirmed to be the corresponding L-amino acid in both cases.

[Table] Example 2 Bacterial cells obtained by culturing in the same manner as in Example 1 except that a 500 ml Erlenmeyer flask containing 100 ml of medium was used.
D-N-carbamyl amino acids listed in Table 2 below
0.1M, cobalt chloride 0.5mM, and manganese chloride
0.1M Tris-HCl buffer (PH7.0) containing 1mM 100
ml and reacted at 35°C for 60 hours. According to HPLC analysis of the reaction product liquid, the reaction was almost complete. Concentrate the supernatant after sterilization to 25 ml by centrifugation, add approximately 5 ml of ethanol, and cool (5°C).
and obtained crystals. This is recrystallized and dried,
HPLC, TLC, bioassay, NMR, and optical rotation analysis were performed, and as shown in Table 2, it was confirmed that each sample corresponded to the corresponding L-amino acid standard.

[Table] Example 3 The same procedure as in Example 2 was performed except that DN-carbamyl amino acid in Example 2 was changed to DL-N-carbamyl amino acid to obtain crystals of the corresponding L-amino acid. Ta. These analysis results were consistent with the standard L-amino acid. The results are shown in Table 3 below.

[Table] Example 4 Washed bacterial cells obtained in the same manner as in Example 2 were mixed with 0.1M solution containing 0.5mM cobalt chloride and 1mM manganese chloride.
It was suspended in 20 ml of Tris-HCl buffer (PH7.0).
This suspension was treated with 20 kHz ultrasound three times for 3 minutes under ice cooling to prepare a bacterial cell disruption solution.
Add 0.1M DL-N-carbamyl amino acid to this solution.
The mixture was added so as to give a reaction temperature of 30.degree. C. and reacted at 30.degree. C. for 20 hours.
After the reaction, the supernatant obtained by centrifuging to remove insoluble matter,
As a result of HLPC and bioassay, the product is
It was confirmed to be the corresponding L-amino acid. The analysis results,
It is shown in Table 4 below.

[Table] [Effects of the Invention] As explained above, according to the present invention, the raw material is D-, L- or DL-form from N-carbamyl amino acid using microorganisms of the genus Arilobacter. Regardless, the remarkable effect of being able to efficiently produce the corresponding L-amino acid can be achieved.

Claims (1)

[Claims] 1. A culture solution, bacterial cells, or treated bacterial cells of a microorganism belonging to the genus Arilobacter is applied to the D, L, or DL form of the N-carbamyl form of an amino acid selected from the group consisting of phenylalanine, tryptophan, and tyrosine. 1. A method for producing an L-amino acid, which comprises collecting the corresponding L-phenylalanine, L-tryptophan, or L-tyrosine produced.