JPH0439316B2 - - 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 addition, in the present invention, unless otherwise specified, 5
The compound names substituted hydantoin and N-carbamoyl amino acid collectively refer to 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 (Flavpbacterium
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 when 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 if it remains 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 the present inventors were researching the decomposition mechanism of penta-substituted hydantoins by microorganisms, the microorganisms newly isolated by the inventors 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. We discovered that this is a new microorganism with the following characteristics, and filed a separate application. This microorganism belongs to the genus Arthrobacter, and
A new bacterial species Arthrobacter sp. DP-B-1001 (Feikoken Bibori No. 8190) has the ability to convert substituted hydantoins and N-carbamoyl amino acids into their respective L-amino acids. . The microorganism of the present invention is a mutant strain derived from this DP-B-1001 strain, and has the same ability as the DP-B-1001 strain. 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. 1002 (Fiber Engineering Research Institute No. 8191). Next, Arthrobacter sp. DP-B
The mycological properties of -1002 are shown together with those of DP-B-1001.

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[Effect]

Arthrobacter sp. DP- of the present invention
B-1002 contains 5-substituted hydantoin and N-carbamoyl amino acid as 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. (4) Arthrobacter sp. DP-B-
Since the 1002 strain is highly active, it is a mutant strain that does not require the addition of a substance that acts as an inducer of activity to the culture solution to increase cell activity, so the method of the present invention using this strain is different from the conventional method. In comparison, culture costs can be significantly reduced and culture management can be simplified, which is very advantageous from an industrial perspective. [Example] Next, reference examples, working examples, and test examples will be given and explained. Reference example 1 Arthrobacter sp. DP-B-1001
(Feikoken Bacteria No. 8190) strain was isolated from the soil of 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. Example 1 Arthrobacter sp. DP-B-1002
The strain is a mutant strain of the same DP-B-1001 strain obtained in Reference Example 1, and was isolated by the following method. Strain DP-B-1001 was cultured in the same liquid medium as used in Reference Example 1 plus 1 mg/ml of sodium p-dimethylaminobenzenediazosulfonate, and then spread on a tryptoson agar plate. A bacterium was obtained by culturing at 28°C, and the amino acid production activity was measured in the same manner as in Reference Example 1, and the present bacterium was obtained as a mutant strain that had extremely high amino acid production activity. Test example 1 (1) Cultivation of inoculum, preparation of bacterial cells 500 ml of the same liquid medium used in Reference example 1
Dispense 200ml into a Erlenmeyer flask and sterilize it.Example 1
Arthrobacter sp. DP-B- obtained from
1002 strain was inoculated and cultured with shaking at 28°C for 72 hours.
Collect bacterial cells from the culture solution by centrifugation at 18,000G for 10 minutes, wash once with physiological saline to obtain wet bacterial cells,
This was used as washed bacterial cells for use in enzyme reactions. (2) Enzyme reaction Suspend 10g of washed bacterial cells obtained in (1) in 50ml of purified water, add 0.8MNH ₄ OH-Cl (PH8.0), 4mM
Add 25 ml of a buffer solution containing FeSO ₄ .7H ₂ O and 4mM MnSO ₄ .5H ₂ O, and add D-N as a raw material.
-Carbamoylphenylalanine 200g/,
D-N-carbamoyl 3-0-methyldopa 20
g/, D-N-carbamoyltyrosine 20g/
, D-N-carbamoyl tryptophan 20
g/, D-N-carbamoylmethionine 20
g/, D-N-carbamoylvaline 20g/
, 20 g of DN-carbamoylleucine/25 ml of each sodium salt solution were added thereto, a stream of N ₂ was blown into the mixture, 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.

[Table] *Measured values using an amino acid analyzer.
Test Example 2 DP-B- obtained in the same manner as Test Example 1 (1)
10g of washed bacterial cells of strain 1002 were suspended in 50ml of purified water, and _0.4MNH4Cl - _NH4OH (PH9.0), 2mM
50 ml of a buffer solution having a composition of FeSO.7H ₂ O and 2mM MnSO _{4.5H 2} O was added to make 100 ml. DL for this
-5-Benzylhydantoin 10g, DL-5-
(3'-methoxy-4'-hydroxybenzyl)hydantoin 10g, DL-5-(4'hydroxybenzyl)hydantoin 1g, DL-5-indolylmethylhydantoin 1g, DL-5-methylmercaptoethylhydatoin 1g , 1 g of DL-5-isopropylhydantoin, and 1 g of DL-5-isobutylhydantoin were suspended in N ₂
It was kept at 37°C for 48 hours with a stream of air. 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 Test Example 1. As a result, L-amino acids were accumulated in the amounts shown in Table 2 below.

[Table] * Same as Table 1.
Test example 3 Glucose 10g/, cornstarch liquor
A medium containing 20g/adjusted to pH 7.0,
Arthrobacter sp. DP-B-1002 strain was inoculated into a mini jar fermenter, and the mixture was heated at 28°C under aeration of 1/min and stirring at 700 rpm.
The cells were cultured for 36 hours. After the culture was completed, the cells were collected by centrifugation to obtain 78 g of wet cells. Suspend the above wet bacterial cells in 800ml of purified water and add 1.2nM
400ml of cobalt acetate solution and 80g/DL-N-
Add 400ml of carbamoylphenylalanine, _N2
The temperature was maintained at 37°C for 22 hours while the pH was adjusted to 6.7 with hydrochloric acid and ammonia under air flow. After the reaction is complete,
The bacterial cells were collected by centrifugation and used again in a new reaction according to the reaction solution preparation method described above. As a result of performing the above operation seven times, a supernatant containing L-phenylalanine in the amount shown in the table below was obtained.

[Table] Test example 4 Glucose 10g/, corn staple liquor 20
Two 3-volume mini-jar fermenters were charged with PH7.0 culture medium containing 1.5 g/g/DL-5-indolylmethylhydantoin, and Arthrobacter sp. DP-B-1001 strain was inoculated.
Separately, Arthrobacter sp. DP-B-1002 strain was similarly inoculated into a pH 7.0 medium containing 10 g/g of glucose and 20 g/20 g of corn staple liquor. Then, each mini-jar fermenter was aerated for 1/min and stirred at 700 rpm.
Culture was performed at 28°C for 40 hours. Bacterial cells were collected from 50 ml of the culture solution by centrifugation and washed with physiological saline to obtain each washed bacterial cell. The reaction was carried out using 50g/DL-5 benzylhydantoin, 1mM FeSO ₄ 7H ₂ O, 1mM MnSO ₄ 5H ₂
10 mm _and 50 g of reaction solution containing O, 0.2M _NH4Cl - _NH4OH (PH9.0)/D-N-carbamoylphenylalanine, 1mM _FeSO4.7H2O , 1mM
MnSO ₄・5H ₂ O, 0.2M NH ₄ Cl−NH ₄ OH (PH
Prepare two types of reaction solutions (10 ml each) containing 8.0), add an equal amount of the above-mentioned washed bacterial cells of DP-B-1001 strain or DP-B-1002 strain, and seal tightly.
It was kept warm at ℃ for 24 hours. After the reaction was completed, the bacterial cells were removed by centrifugation to obtain a supernatant, and the L-phenylalanine contained therein was quantified.
The results are shown in Table 4. From Table 4, it can be seen that the DP-B-1002 strain has higher activity.

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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.
1002 (Microtechnology Research Institute No. 8191).