JPH051716B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention relates to a method for producing L-amino acids using a novel microorganism. 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 (Flavobacterium aminogenes)
aminogenes) FERM-3133 and its mutants
A method is known in which FERM-3134 and FERM-3135 act on penta-substituted hydantoin. In addition, as a method for enzymatically converting L-N-carbamoyl amino acid to L-amino acid, L- or DL-N-carbamoylmethionine is converted to Corynebacterium cepedonicum (Corynebacterium cepedonicum).
There is a known method of converting the L-N-carbamoylmethionine contained in L-methionine into L-methionine by reacting with IF3306 etc.
No. 29678). However, only the L-form can be converted by this method, and the D-form cannot be converted into 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 reaction solution concentration can be increased and the reaction should be carried out advantageously.
- It was impossible to simplify the procedure because there were no microorganisms that could convert it into amino acids. [Means for Solving the Problems] While researching the decomposition mechanism of penta-substituted hydantoins by microorganisms, the inventors discovered that a microorganism newly isolated from a soil container cleaved the hydantoin compound. It was also discovered that L-N-carbamoyl amino acid and D-N-carbamoyl amino acid are produced when amino acids are produced, and that the amino acids successively produced are in the L form. The present invention is based on this new knowledge and has led to the completion of a method for producing L-amino acids easily and in high yield from 5-substituted hydantoin and N-carbamoyl amino acid. That is, the present invention relates to Arthrobacter sp., which belongs to the genus Arthrobacter and has the ability to convert 5-substituted hydantoins and N-carbamoyl amino acids into L-amino acids.
A culture of the DP-B-1001 strain or its mutant Arthrobacter sp. DP-B-1002 strain was prepared using the following formulas () and (),

【formula】

[Formula] (wherein R represents an N-carbamoyl amino acid residue or a hydantoin residue) (In the formula, R is the same as above.) This is a method for producing an L-amino acid represented by the following formula. The microorganism used in the present invention belongs to the genus Arthrobacter, and contains 5-substituted hydantoin and N-
There is no particular restriction as long as it has the ability to convert carbamoyl amino acids to L-amino acids, and mutant strains thereof may be used. Arthrobacter sp. DP-B-1001 is a microorganism that has the following mycological properties:
(Feikoken Bacterial Serial No. 8190) and the mutant strain derived therefrom, Arthrobacter sp. DP
-B-1002 (Feikoken Bibori No. 8191) is mentioned.

【table】

【table】

【table】

[Effect]

The novel microorganism belonging to the genus Arthrobacter used in the present invention has the ability to cleave 5-substituted hydantoin to produce L-amino acids and the ability to produce L-amino acids from N-carbamoyl amino acids. This ability is exhibited regardless of whether each of the 5-substituted hydantoin and the N-carbamoyl amino acid is in the D, L, or DL form. [Effects of the Invention] The method for producing L-amino acids of the present invention provides the following advantages: (1) D-N-carbamoyl amino acids, which could not be used due to the lack of suitable microorganisms, can also be produced by the corresponding L-amino acids.
-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 production process is extremely simplified, and the desired L-amino acid can be produced in 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. It has various advantages such as [Example] Next, reference examples and examples will be given and explained. Reference example 1 Arthrobacter sp. DP-B-1001
The strain was isolated from a soil dump in Nikko City, Tochigi Prefecture as a result of screening using the following method. Approximately 0.5 g of the 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 O0.04g/, 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. was gotten. Reference example 2 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 trypto-soy agar plate. The bacterium was obtained by culturing at 28°C, and the amino acid production activity was measured by the method described above, and the present bacterium was obtained as a mutant strain with extremely high amino acid production activity. Example 1 (1) Cultivation of seed culture The same liquid medium as used in Reference Example 1 was cultured at 500 g.
Dispense 200 ml into a ml Erlenmeyer flask, and after sterilize Arthrobacter sp. DP obtained in Reference Example 1.
A loopful of -B-1001 was inoculated and cultured with shaking at 28°C for 24 hours. (2) Main culture, bacterial cell preparation Add 0.15% to the same liquid medium as used in Reference Example 1.
% of 5-indolylmethylhydantoin was prepared, and 4 ml of the culture solution obtained in (1) was added.
The cells were inoculated and cultured with shaking at 28°C for 24 hours. Collect bacterial cells from the culture solution by centrifugation at 18,000G for 10 minutes, wash once with physiological saline, and remove wet bacterial cells.
This was 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 M NH ₄ OH−NH ₄ Cl (PH
8.0), 4mM FeSO ₄ 7H ₂ O and 4mM
Add 25 ml of a buffer solution with the composition MnSO ₄ 5H ₂ O,
Furthermore, as raw materials, 200g/D-N-carbamoylphenylalanine, 20g/20g/D-N-carbamoyl-3-O-methyldopa, D-N
-Carbamoyltyrosine 20g/, D-N-
Carbamoyl tryptophan 20g/, D-
N-carbamoylmethionine 20g/, D-
N-carbamoylvaline 20g/, D-N-
Add 25 ml of each sodium salt solution containing 20 g of carbamoylleucine, blow in a stream of _N2 , and heat at 37°C.
It was kept warm 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, 4 mmφ x 150 mm, eluent: sodium citrate buffer 0.4 ml/min, assay: ninhydrin reaction (570 nm) and quantified by bioassay method using Leuconostoc mesenteroides, as shown in Table 1 below. A large amount of L-amino acids had accumulated.

[Table] *Measured values using an amino acid analyzer.
Example 2 DP- obtained in the same manner as in Example 1 (1) and (2)
10 g of washed bacterial cells of strain B-1001 were suspended in 50 ml of purified water, and 2 m of 0.4 M NH ₄ Cl-NH ₄ OH (PH9.0) was added.
M FeSO ₄・7H ₂ O and 2mM MnSO ₄・5H ₂ O
Add 50 ml of a buffer solution having the following composition to make 100 ml. Add to this 10g of DL-5-benzylhydantoin, 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 while 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, L-amino acids were accumulated in the amounts shown in Table 2 below.

[Table] * Same as Table 1.
Example 3 D-N-carbamoylphenylalanine 60g/
, DP-B-1001 strain wet bacterial cells 100g/0.2M
_NH4Cl - _NH4OH (PH8.0), 1mM _FeSO4 .
A reaction solution 1 with a composition of 7H ₂ O and 1mM MnSO ₄ .5H ₂ O was prepared and heated at 37°C while blowing N ₂ gas.
It was kept warm for 48 hours. After the reaction was completed, the bacterial cells were removed by centrifugation to obtain a supernatant. The supernatant contains L-phenylalanine 46
g (yield 96%) was accumulated. This supernatant was concentrated, treated with activated carbon, and cooled to obtain crystals. When the specific rotation of the crystal was measured, it was found to be [α] ²⁰ _D
-34.1° and all were L-forms. Example 4 (1) Cultivation of inoculum and preparation of bacterial cells The same liquid medium used in Reference Example 1 was used for 500
Dispense 200 ml into a ml Erlenmeyer flask, and after sterilization, collect Arthrobacter sp. DP obtained in Reference Example 2.
-B-1002 strain was inoculated and cultured with shaking at 28°C for 72 hours. Bacterial cells were collected from the culture solution by centrifugation at 18,000G for 10 minutes, and washed once with physiological saline to obtain wet microbial cells, which were used as washed microbial cells for use in enzyme reactions. (2) Enzyme reaction 10 g of washed bacterial cells obtained in (1) were suspended in 50 ml of purified water, and 0.8 M NH ₄ OH−NH ₄ Cl (PH
8.0), 4mM FeSO ₄ 7H ₂ O and 4mM
Add 25 ml of a buffer solution with the composition MnSO ₄ 5H ₂ O,
Further, as raw materials, 200g/D-N-carbamoylphenylalanine, 20g/20g/D-N-carbamoyl 3-O-methyldopa, DN-
Carbamoyltyrosine 20g/, D-N-carbamoyltryptophan 20g/, D-N
-Carbamoylmethionine 20g/, D-N
- Add 25 ml of each sodium salt solution of 20 g of carbamoylvaline/20 g of D-N-carbamoylleucine, blow in a stream of _N2 , and heat to 37°C.
It was kept warm for 48 hours. 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, L-amino acids were accumulated in the amounts shown in Table 3 below.

[Table] * Same as Table 1.
Example 5 DP-B-1002 obtained in the same manner as Example 4 (1)
Suspend 10g of washed bacterial cells of the strain in 50ml of water,
0.4M _NH4Cl - _NH4OH (PH9.0), 2mM
50 ml of a buffer solution having a composition of FeSO ₄ .7H ₂ O and 2 mMMnSO 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-5-
Suspend 1 g of isobutylhydantoin,
Bubbled with a stream of _N2 and incubated at 37°C for 48 hours. 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, L-amino acids were accumulated in the amounts shown in Table 4 below.

[Table] * Same as Table 1.
Example 6 Glucose 10g/cornstap 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 1.2mM
400ml of cobalt acetate solution and 80g/DL-N
−Add 400ml of carbamoylphenylalanine,
pH 6.7 with hydrochloric acid and ammonia under _N2 stream
The temperature was maintained at 37°C for 22 hours. After the reaction was completed, 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】

[Table] Example 7 Glucose 10g/, cornstarch liquor
A medium containing 20g/adjusted to pH 7.0,
Fill 20 jars in a container and inoculate with Arthrobacter sp. DP-B-1002 strain.
28℃, aeration at 10/min, stirring at 400rpm,
Culture was performed for 40 hours. An average of 642 g of wet bacterial cells was obtained from the 20 culture solutions. For the reaction, cells were added to a reaction solution containing 20g/DL-N-carbamoylphenylalanine and 0.3mM cobalt acetate so that the bacterial concentration was as shown in Figure 1, a stream of _N2 was blown in, and the pH was adjusted with hydrochloric acid and ammonia.
The temperature was maintained at 37°C for 22 hours while adjusting the temperature to 6.7°C. After the completion of the reaction, the bacterial cells were collected by centrifugation, and together with the newly obtained bacterial cells from the culture solution, a reaction solution was prepared according to the above reaction method, and the operation of increasing the amount of reaction solution was repeated three times. A reaction solution was obtained in which 540 g of L-phenylalanine was produced.
Thereafter, 18 reactions were carried out while newly replenishing bacteria in the 10th and 14th reactions, and a total of 8.4 kg of L-phenylalanine was obtained as shown in FIG. Example 8 Glucose 10g/cornstap liquor
Two 3-volume mini-jar fermenters were charged with two PH7.0 media containing 20g/DL-5-indolylmethylhydantoin and 1.5g/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 fermentor was aerated at 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
_MnSO4・_5H2O , 0.2M _NH4Cl − _NH4OH (PH9.0)
and 50 g/D-N-carbamoylphenylalanine, 1mM _FeSO4 .
_7H2O , 1mM _MnSO4・_5H2O , 0.2M _NH4Cl
- Two types of reaction solutions (10 ml) containing NH ₄ OH (PH8.0) were prepared, and DP-B-1001 strain or DP-
Equal amounts of the above-mentioned washed bacterial cells of strain B-1002 were added, the tubes were sealed tightly, and the mixture was kept at 37° C. 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 6.

[Table] Example 9 Glucose 10g/, cornstarch liquor
A medium containing 20g/adjusted to pH 7.0,
Fill 20 jars in a container and inoculate with Arthrobacter sp. DP-B-1002 strain.
28℃, aeration at 10/min, stirring at 400rpm,
Cultured for 40 hours. After the culture was completed, the cells were collected by centrifugation to obtain 828 g of wet cells. This wet bacterial body was suspended in 1.5% purified water, and added with 50% acrylamide monomer, 1.25% N,N'-
Add 1.5 methylene bisacrylamide solution,
The mixture was homogenized, and 3 ml of N,N,N',N'-tetramethylethylenediamine and 150 ml of 2% ammonium persulfate solution were added and mixed to solidify into a thin film with a thickness of 2 mm. After completely solidifying, cut into 2mm squares,
An immobilized bacterial cell gel fragment was obtained and packed into a 6 volume column. Next, 0.1M phosphate buffer (PH6.7) containing 4.49g/DL-5-indolylmethylhydantoin, 4.84g/DL-N-carbamoyltryptophan, and 0.3mM cobalt acetate was added to about 900%
The mixture was introduced into the column kept at 37° C. at a flow rate of ml/hour. As a result of carrying out the above reaction operation over 15 days, a reaction mixer 346 containing L-tryptophan in the amount shown in Table 7 was obtained.

[Table] Example 10 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 1.2mM
400ml of cobalt acetate solution and 80g/DL-N
-Carbamoylphenylalanine 200ml and 16g
Purified water in which DL-benzylhydantoin is suspended
200 ml was mixed and kept at 37° C. for 22 hours under a N ₂ stream while adjusting the pH to 6.7 with hydrochloric acid and ammonia. After the reaction was completed, the bacterial cells were collected by centrifugation to obtain a reaction liquid supernatant. 25.26 g (yield 95%) of L-phenylalanine was accumulated in the supernatant.
This supernatant was concentrated, treated with activated carbon, and cooled to obtain crystals. When the specific optical rotation of this crystal was measured, it was [α] ²⁰ _D -34.1°, and it was all in the L form. Example 11 Glucose 10g/cornstap 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 1.2mM
400ml of cobalt acetate solution and 20g/DL-N
-carbamoyl-3-O-methyldopa 200ml
200 ml of purified water with 28 g of DL-5-(3'-methoxy-4'-hydroxybenzyl)hydantoin suspended in it
The mixture was mixed and kept at 37°C for 22 hours under a N ₂ stream while adjusting the pH to 6.7 with hydrochloric acid and ammonia. After the reaction was completed, the bacterial cells were collected by centrifugation to obtain a reaction liquid supernatant. 26.09 g (yield 92%) of L-3-O-methyldopa was accumulated in the supernatant.
The supernatant was concentrated, treated with activated carbon, and cooled to obtain crystals of L-3-O-methyldopa monohydrate. When we measured the optical rotation of this crystal, we found that [α]
²⁰ _D -25.5°, all were L-forms.

[Brief explanation of the drawing]

Figure 1 shows DL-N-carbamoylphenylalanine and Arthrobacter sp. DP-B-
1002 is a diagram showing the amount of L-phenylalanine produced and the bacterial concentration in the reaction solution for each reaction.

Claims (1)

[Scope of Claims] 1. Arthrobacter sp. DP-B-1001 strain belonging to the genus Arthrobacter and having the ability to convert 5-substituted hydantoins and N-carbamoyl amino acids into L-amino acids or its mutant strain Arthrobacter sp. The culture of the S.P. DP-B-1002 strain was prepared using N- The following formula () is characterized by acting on a carbamoyl amino acid or/and a 5-substituted hydantoin, (In the formula, R is the same as above.) A method for producing an L-amino acid represented by: