JPH0439315B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention relates to Arthrobacter
The present invention relates to a novel microorganism belonging to the genus A. In the present invention, unless otherwise specified, the compounds 5-substituted hydantoin and N-carbamoyl amino acid collectively refer to the D-form, L-form, and DL-form, respectively. [Prior art and problems] Conventionally, a method for enzymatically converting DL-5 substituted hydantoins into L-amino acids was disclosed in
Many patent applications have been filed since No. 42-13850.
In particular, as a method for producing aromatic amino acids, Flavobacterium aminogenes
aminogenes) FERM-3133 and its mutants
A method is known in which FERM-3134 and FERM-3135 act on 5-substituted hydantoin. In addition, as a method for enzymatically converting L-N-carbamoyl amino acid to L-amino acid, L- or
Corynebacterium cepedonicum (DL-N-carbamoylmethionine)
There is a known method of converting the L-N-carbamoylmethionine contained in L-methionine into L-methionine by reacting L-N-carbamoylmethionine with IFO3306 etc.
No. 29678). However, only the L-form can be converted by this method, and the D-form cannot be converted to L-methionine. Furthermore, in the conventional enzymatic method for producing L-amino acids, it is necessary to use different types of microorganisms when using a 5-substituted hydantoin as a raw material and when using an N-carbamoyl amino acid as a raw material. No microorganism has been found that can act on both hydantoin and N-carbamoyl amino acid and convert them into the corresponding L-amino acids. Therefore, when using DL-5-substituted hydantoins as a raw material, by-products during the production of the raw material
It is necessary to remove DL-N-carbamoyl amino acid, and if DL-N-carbamoyl amino acid is used as a raw material, measures must be taken to prevent DL-N-carbamoyl amino acid from being mixed into the reaction system or remaining after the reaction is completed. It was necessary to take complicated steps to separate and racemize the DN-carbamoyl amino acid. Furthermore, if DL-N-carbamoyl amino acid is used as a water-soluble salt, the concentration of the reaction solution can be increased and the reaction should be carried out advantageously. Since there were no microorganisms to obtain, it was impossible to simplify the procedure. [Means for Solving the Problems] While researching the decomposition mechanism of penta-substituted hydantoins by microorganisms, the present inventors discovered that microorganisms newly isolated from soil cleaved hydantoin compounds and produced amino acids. When producing L-N-carbamoyl amino acid and D-N-carbamoyl amino acid, and the amino acids that are sequentially produced are L-amino acids, this microorganism has a previously unknown ability. The present invention was completed based on the discovery that the present invention has the following properties. That is, the present invention relates to a new bacterial species, Arthrobacter sp. DP-B-, which belongs to the genus Arthrobacter and has the ability to convert 5-substituted hydantoins and N-carbamoyl amino acids into their corresponding L-amino acids. 1001 (Fiber Engineering Research Institute No. 8190). Arthrobacter sp. DP-B-1001
is a microorganism newly isolated by the present inventors from the soil of Nikko City, Tochigi Prefecture, and has the following mycological properties.

【table】

【table】

【table】

[Effect]

Arthrobacter sp. DP- of the present invention
B-1001 contains 5-substituted hydantoin and N-carbamoyl amino acid in the D form and L form, as shown in the test example below.
Each corresponding L regardless of body or DL body
-Has the effect of converting into amino acids. [Effects of the Invention] Since the novel microorganism of the present invention has the above-mentioned abilities, if it is used in the production of L-amino acids, the productivity can be dramatically improved. That is, (1) D-N-carbamoyl amino acids, which could not be used due to the lack of suitable microorganisms, can also be treated with the corresponding L.
-Can be used as a starting material for amino acid production. (2) Since there is no need to remove DL-N-carbamoyl amino acid, which is a by-product when chemically producing 5-substituted hydantoin, from the reaction system, DL-5-substituted hydantoin and DL can be easily and inexpensively obtained industrially. L-amino acids can be quantitatively produced from a mixture of -N-carbamoyl amino acids in a single reaction. Therefore, measures to prevent the contamination of DN-carbamoyl amino acids into DL-5-substituted hydantoins, which were necessary in the conventional L-amino acid production process using DL-5-substituted hydantoins as a starting material, and those remaining after the completion of the reaction. D-
The complicated process of separating and racemizing N-carbamoyl amino acids is completely unnecessary, and the manufacturing process is extremely simplified and the desired L-
It has become possible to produce amino acids with high yield. (3) Since the reaction can be carried out using a water-soluble salt of DL-N-carbamoyl amino acid as a raw material using an immobilized cell column, the production process can be easily made continuous. [Example] Next, examples and test examples will be given and explained. Example 1 Arthrobacter sp. DP-B-1001
The strain was isolated from soil in Nikko City, Tochigi Prefecture as a result of screening using the following method. Approximately 0.5 g of soil was suspended in 5 ml of sterilized water, diluted, spread on a trypto-soy agar plate, and cultured at 28°C to obtain bacteria. The bacteria thus obtained were mixed with glucose 10g/, yeast extract 5g/, polypeptone 5g/, meat extract 2g/, _MgSO4 .
7H ₂ O0.4g/, FeSO ₄・7H ₂ O0.01g/,
It was inoculated into a liquid medium containing 0.01 g of MnSO ₄ .5H ₂ O and adjusted to pH 7.0, and cultured with shaking at 28° C. for 2 days to obtain bacterial cells. The bacterial cells were added to a 0.2 M ammonium buffer (PH 9.0) containing 10 g/N-carbamoyl amino acid (e.g. DL-N-carbamoyltryptophan) or 5-substituted hydantoin (e.g. DL-5-indolylmethylhydantoin). and kept at 37°C for 24 hours. After incubation, L-amino acids in the buffer supernatant were measured by bioassay using Leuconostoc mesenteroides P-60 and thin layer chromatography using silica gel plates, and this strain was found to have particularly high amino acid production activity. Obtained. Test example 1 (1) Cultivation of seed culture 500 ml of the same liquid medium used in Example 1
Dispense 200ml into a Erlenmeyer flask and sterilize it.Example 1
Arthrobacter sp. DP-B- obtained from
A loopful of 1001 strains was inoculated and cultured with shaking at 28°C for 24 hours. (2) Main culture, bacterial cell preparation 0.15% in the same liquid medium as used in Example 1
Prepare a medium supplemented with 5-indolylmethylhydatoin, inoculate 4 ml of the culture solution obtained in (1),
Culture was performed with shaking at 28°C for 24 hours. From the culture solution
Bacterial cells were collected by centrifugation at 18,000 G for 10 minutes and washed once with physiological saline to obtain wet bacterial cells, which were used as washed bacterial cells for use in enzyme reactions. (3) Enzyme reaction 10 g of washed bacterial cells obtained in (2) were suspended in 50 ml of purified water, and 0.8 MNH ₄ OH-Cl (PH8.0),
Add 25 ml of buffer solution with the composition of 4mMFeSO ₄ .7H ₂ O and 4mMnSO ₄ .5H ₂ O, and add D as a raw material.
-N-carbamoylphenylalanine 200g/,
D-N-carbamoyl-3-0-methyldopa
20g/, D-N-carbamoyltyrosine 20
g/, D-N-carbamoyltryptophan
20g/, D-N-carbamoylmethionine 20
g/, D-N-carbamoylvaline 20g/
, 20 g/25 ml of each sodium salt solution of DN-carbamoylleucine was added, a stream of N ₂ was blown into the solution, and the mixture was kept at 37° C. for 48 hours. After the reaction, the bacterial cells were removed by centrifugation, and the amino acids in the centrifuged supernatant were analyzed using Hitachi Model 638-50, reactive liquid chromatography (column: Hitachi Gel No. 2619,
4mmφ×150mm, eluent: sodium citrate buffer 0.4ml/min, assay: ninhydrin reaction
570nm) and a bioassay method using Leuconostocmesenteroides,
L-amino acids were accumulated in the amounts shown in Table 1 below. Note that similar results were obtained for the DL body.

【table】

[Table] *Measured values using an amino acid analyzer.
Test Example 2 DP- obtained in the same manner as Test Example 1 (1) and (2)
10 g of washed bacterial cells of strain B-1001 were suspended in 50 ml of purified water, and 0.4MNH ₄ Cl-NH ₄ OH (PH9.0),
50 ml of a buffer solution having a composition of 2mMFeSO ₄ .7H ₂ O and 2mMnSO ₄ .5H ₂ O was added to make the total volume to 100 ml. to this
DL-5-benzylhydantoin 10g, DL-
5-(3'-methoxy-4'-hydroxybenzyl)
Hydantoin 10g, DL-5-(4'-hydroxybenzyl)hydantoin 1g, DL-5-indolylmethylhydantoin 1g, DL-5-methylmercaptoethylhydantoin 1g, DL
-5-isopropylhydantoin 1g, DL-
1 g of 5-isobutylhydantoin was suspended in each suspension and kept at 37° C. for 48 hours by blowing in a N ₂ stream. After the reaction was completed, the bacterial cells were removed by centrifugation, and the amino acids in the centrifuged supernatant were quantified in the same manner as in Example 1. As a result, the amounts of L-amino acids shown in Table 2 below were accumulated.

[Table] * Same as Table 1.

Claims (1)

[Claims] 1. A new bacterial species, Arthrobacter sp. DP-B-, which belongs to the genus Arthrobacter and has the ability to convert 5-substituted hydantoins and N-carbamoyl amino acids into their respective L-amino acids.
1001 (Microtechnical Research Institute No. 8190).