JPH0455674B2 - - Google Patents

Description

[Detailed description of the invention]

[Objective of the Invention] <Industrial Application Field> The present invention is directed to biochemically hydrolyzing 5-substituted hydantoins using microorganism cells or a processed product of microorganisms to produce D-N-carbamyl- The present invention relates to a method for obtaining α-amino acids, and the object is to produce substances useful as intermediates for pharmaceuticals and the like in a very advantageous manner. <Prior art> As a method similar to the method of the present invention, when an enzyme (hydropyrimidine hydrolase) collected from calf liver is reacted with DL-form 5-substituted hydantoin, D-form N-carbamyl-α is produced. - It has already been reported that amino acids can be obtained [FEBS LETTERS Vol. 57, 2
No. 192, 1975). Furthermore, as in the present invention, 5-substituted hydantoins can be produced using microorganisms.
JP-A-53-91189 is known as a method for converting into -carbamyl-α-amino acids. <Problems to be solved by the present invention> The problems to be solved by the present invention are different from the conventional D-
The object of the present invention is to develop a manufacturing method that is cheaper and more efficient than the method for manufacturing N-carbamyl-α-amino acids. [Structure of the Invention] <Means for Solving the Problems> The present inventors conducted research to find a more efficient method for producing D-N-carbamyl-α-amino acids than the conventional method for producing D-N-carbamyl-α-amino acids. Results: Flavobacterium, Mycoplana, Lydobium, Colletotrichum, Corynespora, Curvularia, Fusarium, Pestaloteia, Septoria, Colethropsis, Hercoceras, Bysochlamis, Xylaria, Cladosporium, Eupenicillium Genus, Emeryseropsis,
It was discovered for the first time that microorganisms belonging to the genus Sordaria and Wardmyces have the ability to hydrolyze 5-substituted hydantoin and convert it into DN-carbamyl-α-amino acid, leading to the completion of the present invention. That is, the present invention provides 5-substituted hydantoin with D-N
- A microorganism capable of converting into carbamyl-α-amino acid is allowed to act on 5-substituted hydantoin to convert 5-substituted hydantoin into D-N-carbamyl-amino acid.
D characterized in that it is converted into α-amino acid.
- A method for producing N-carbamyl-α-amino acid. Furthermore, it is already known that when an optically active N-carbamyl-α-amino acid is reacted with nitrous acid, an optically active α-amino acid can be obtained without changing the steric position. Therefore, by combining the methods of the present invention, D-amino acids can be advantageously produced industrially. In recent years, the importance of D-form α-amino acids as intermediate raw materials for antibiotics and the like has been increasing, and in this sense, the usefulness of the present invention is extremely large. The intracellular enzyme used in the present invention is 5-
It selectively acts only on the D-form of substituted hydantoins to cleave and hydrolyze it. Since the unhydrolyzed L form is racemized in the reaction medium, the D form is substantially always supplied to the reaction system. Therefore,
Regardless of whether DL, L, or D is used as a starting material for the reaction, D-form N-carbamyl-α-amino acids can be obtained as a product. The main points of the present invention can be expressed as follows. (In the general formulas [] and [], R represents an alkyl group, an aralkyl group, or a group thereof having a substituent.) As described above, the present invention relates to the 5-substituted D-N represented by the general formula [], which is characterized in that hydantoins are treated with a culture solution, bacterial cells, or treated bacterial cells of a microorganism that has the ability to asymmetrically cleave and hydrolyze hydantoin rings.
- A method for producing carbamyl-α-amino acid. <Function> Specifically, the microorganisms of the present invention include the following. Flavobacterium fucatum
AJ-2478 FERM P-7053 Mycoplana Deimorpha
dimorpha) ATCC−4279 Rhizobium meliloti
ATCC-4400 Colletotrichum graminicola ATCC-11870 Corynespora
cordinale) ATCC−13063 Curvularia coicis
ATCC−42585 Fusarium nivale
ATCC−42316 Pestalotia hoedance (Pestalotia
septoria nodorum) ATCC−11817 Septoria nodorum
ATCC−24425 Corethropsis hominis
hominis) AJ-6568 FERM P-8024 Helicoceras oryzae
AJ−6580 FERM P−8025 Byssochlamys fulva
ATCC−24474 Xylaria polymorpha (Xylaria
IFO−30369 Cladosporium reginae
resinae) IFO−6367 Eupenicillium alutaceum IFO−8943 Emericellopsis minita
minta) ATCC−20422 Sordaria equina
ATCC−24019 Wardmys Obalis
ovalis) ATCC-22329 As detailed in the Examples, all of the 5-substituted hydantoins that the inventors were able to obtain and test were converted to the corresponding D-N-carbamyl-α-amino acids by the method of the present invention. It is clear that anything can be converted to a DN-carbamyl-α-amino acid by the method of the present invention. The following are examples of 5-substituents of 5-substituted hydantoins. 1 Straight chain, branched chain or cyclic saturated aliphatic hydrocarbon residue. Examples include methyl group, ethyl group, propyl group, isopropyl group, isobutyl group, 1-methylpropyl group, tertiary butyl group, cyclohexyl group, and cyclobenzyl group. 2 A linear, branched or cyclic aliphatic hydrocarbon residue containing an unsaturated bond. For example, 2-propenyl group, 2-propyl group, 1-cyclohexenyl group, 1,4-dicylhexenyl group, etc. are exemplified. 3 A linear, branched, or cyclic aliphatic hydrocarbon having saturated and unsaturated bonds with one or more substituents. Here, the substituents include hydroxyl group, carboxyl group, sulfhydryl group, alkylmercapto group, amino group, alkylamino group, alkoxy group, carbamoyl group, guanidide group, ureido group, sulfoxyl group, nitro group, halogen atom, and phosphoric acid group. , an acyl group, an aminosulfenyl group, an allylmercapto group, a 4-imidazoyl group, a 2-thienyl group, and the like. 4 Aromatic hydrocarbon group Examples include phenyl group and naphthyl group. 5 Aromatic hydrocarbon group having a substituent Here, the substituent includes an alkyl group, an alkenyl group, an alicyclic group, a hydroxyl group, an alkoxy group, a halogen atom, a benzyloxy group, a benzyloxymethyloxy group, and a methoxymethyl group. Examples include oxy group, acyloxy group, acyl group, aryloxy group, aminosulfonyl group, trifluoromethyl group, alkylmercapto group, amino group, acylamino group, alkylamino group, nitro group, carboxyl group, carbamoyl group, etc. Ru. In addition, one or more of these substituents may be substituted with the same aromatic hydrocarbon group. 6 Heterocyclic group Examples include 2-thienyl group, 5-thiazole group, 4-imidazole group, and 2-furyl group. 7 Heterocyclic group having a substituent The substituent herein is almost the same as shown on page 5. The method of causing the microorganism of the present invention to act on 5-substituted hydantoin may be carried out by culturing the microorganism in a medium containing 5-substituted hydantoin, or by treating the cells or cells of these microorganisms. The product may be contacted with the 5-substituted hydantoin in an aqueous solution. The method for converting 5-substituted hydantoin into DN-carbamyl-α-amino acid by culturing the present microorganism may involve culturing the present microorganism in a medium containing 5-substituted hydantoin from the beginning of the culture, or Furthermore, 5-substituted hydantoin may be added to the medium during the culture. The medium used for culturing this microorganism is 5-
It is a conventional medium containing a conventional carbon source, nitrogen source, and inorganic ions except for the substituted hydantoin.
Additionally, desirable results can often be obtained by adding organic micronutrients such as vitamins and amino acids. As the carbon source, carbohydrates such as glucose and sucrose, organic acids such as acetic acid, alcohols, and others are used as appropriate. As the nitrogen source, ammonia gas, aqueous ammonia, ammonium salt, and others are used. As the inorganic ions, magnesium ions, phosphate ions, potassium ions, iron ions, and others are used as appropriate. Desired results can be obtained if the culture is carried out under aerobic conditions, with pH 4 and temperature controlled within an appropriate range of 25 to 40°C. Thus, if the culture is carried out for 1 to 10 days, 5-
Substituted hydantoins are efficiently converted to only D-α-amino acids. On the other hand, the cells of this microorganism or the processed material of the cells,
When the 5-substituted hydantoin is brought into contact with the aqueous solution, an aqueous solution prepared by dissolving or suspending the 5-substituted hydantoin and bacterial cells or a processed product of the bacterial cells is prepared at a pH of 6 to 11 and an appropriate temperature of 10 to 70°C. What is necessary is to temporarily leave it still or stir it while adjusting the temperature. As the reaction progresses, the pH of the medium decreases, so
It is desirable to maintain the optimum pH by adding a neutralizing agent at appropriate times during the reaction. Suitable neutralizing agents include ammonia, caustic soda, caustic potash, and soda carbonate. As the bacterial cells, a culture solution containing the bacterial cells may be used as is. Alternatively, the powder may be separated from the culture medium and washed or used without washing. Examples of bacterial cell treatments include mechanically ground bacterial cells, ultrasonic-treated bacterial cells, freeze-dried bacterial cells, acetone-dried bacterial cells, bacterial cells treated with enzymes such as lysozyme, surfactants,
Bacterial cells treated with toluene or the like, protein fractions of microbial cells, and others are used as appropriate. As a method for obtaining such bacterial cells, the above-described culture medium and culture method can be used as they are. If a small amount of the 5-substituted hydantoin of the present invention is further added to the medium, the 5-substituted hydantoin can be further converted into D-N-carbamyl-
Bacterial cells with high activity of converting into α-amino acids can be obtained. In this case, the culture may be completed in about 123 to 48 hours, as long as the microorganisms sufficiently proliferate. Antioxidants, surfactants, etc. are added to the aqueous solution as necessary.
Addition of coenzyme, hydroxylamine, etc. may improve the reaction yield. Thus, after 5 to 100 hours have elapsed, a large amount of DN-carbamyl-α-amino acid is produced and accumulated in the aqueous solution. DN-carbamyl thus obtained
According to the method of the present invention, α-amino acid is collected from a culture solution or an aqueous solution, and L-N-carbamyl-α-amino acid is not produced as a by-product. Ordinary methods can be used. The produced DN-carbamyl-α-amino acid was quantified using a conventional colorimetric method using p-dimethylaminobenzaldehyde and a method using liquid chromatography. Optical isomers are determined using an optical separation column (chiral pack).
By measuring the specific rotation of the crystal and liquid chromatography using WH...manufactured by Daicel, D, L
has been established. Example 1 Glucose 0.5g/dl, (NH ₄ ) ₂ SO ₄ 0.5g/dl,
KH ₂ PO ₄ 0.1g/dl, K ₂ HPO ₄ 0.3g/dl,
MgSO ₄ 7H ₂ O0.05g/dl. _FeSO4・_7H2O1mg /
dl, MnSO ₄・4H ₂ O1mg/dl, yeast extract 1.0g/
dl, peptone 1.0 g/dl, and a medium (PH7.0) containing DL-5-isopropylhydantoin 0.2 g/dl.
Pour 50ml into a 500ml flask and heat to 120°C.120
Sterilize for 15 minutes at °C. Add to this a bouillon agar medium.
One platinum loop of Flavobacterium fucatum AJ-2478, Mycoplana deimorpha ATCC-4279, or Rhizobium melilotei ATCC-4400, which had been cultured at 30°C for 24 hours, was inoculated and cultured at 30°C for 20 hours. Bacterial strains were collected from this culture solution by centrifugation, washed once with physiological saline in the same amount as the culture solution, and the bacterial cells were collected. This bacterial cell was added to 0.1 M Tris buffer (PH 9.0) (final volume: 5 ml) containing 2 g/dl of DL-5-isopropylhydantoin at a concentration of 5 g/dl, and the reaction was maintained at 30°C for 16 hours. N-carbamylvaline produced in the reaction solution was measured by the colorimetric method described above, and is shown in Table 1. Also N-
As a result of measuring the optical rotation of the separated carbamylvaline (Table 1), it was found that it was in the D form.

[Table] Example 2 Seuculose 0.1g/dl, peptone 1.0g/
dl, yeast extract 0.5g/dl, malt extract 0.5g/
dl, KH ₂ PO ₄ 0.2g/dl, NaCl0.3g/dl, DL−
5-Methylmercaptoethylhydantoin 0.2
150 ml of a medium (PH7.0) containing g/dl was placed in a 300 ml Erlenmeyer flask and sterilized at 120°C for 15 minutes. In addition to this, yeast extract 0.3g/dl, malt extract 0.3g/dl
The strains shown in Table 2 were cultured in an agar slant medium (PH 6.0) containing 0.5 g/dl, peptone, 0.5 g/dl, and glucose 1.0 g/dl at 26°C for 5 days. Bacterial cells were prepared by washing once with physiological saline in the same amount as the culture solution. DL-
5-methylmercaptoethylhydantoin 1g/
The mixture was added to 0.1 M Tris buffer (PH9.0) (5 ml final) containing dl at a concentration of 5 g/dl, and the reaction was maintained at 30°C for 16 hours. N-carbamylmethionine produced in the reaction solution was measured by the colorimetric method described above and is shown in Table 2. Furthermore, as a result of examining the produced N-carbamylmethionine by high-performance liquid chromatography using an optical resolution column, it was found that all N-carbamylmethionine was in the D-form no matter which strain was used.

【table】

【table】

[Table] Example 3 Mycoplana deimorpha ATCC4279 was cultured in the same manner as in Example 1, and bacterial cells were prepared in the same manner as in Example 1. The cells were injected into 0.1M Tris buffer (PH) containing 3g/dl of 5-substituted hydantoin shown in Table 3.
9.0) at 5g/dl and kept at 30℃ for 16 hours.
Retention reaction occurred. The N-carbamyl amino acid produced at that time was quantified using the method described above, and the results are shown in Table 3.
It was shown to. Furthermore, optical activity measurement using an optical resolution column revealed that all of the produced N-carbamyl amino acids were in the D form.

【table】

[Table] Example 4 Glucose 2.0g/dl, (NH ₄ ) ₂ SO ₄ 0.5g/dl,
KH ₂ PO ₄ 0.1g/dl, K ₂ HPO ₄ 0.3g/dl,
MgSO ₄ 7H ₂ O0.05g/dl, FeSO ₄ 7H ₂ O1mg/dl,
A medium (PH7.0) containing 1 mg/dl of MnSO ₄ H ₂ O, 1.0 g/dl of yeast extract, 1.0 g/dl of peptone, 0.2 g/dl of DL-5-isopropylhydantoin, and 2 g/dl of CaCO ₃ (separately) was used. Pour 50ml into a 500ml flask 120
Sterilize for 15 minutes at °C. Rhizobium melilotei ATCC-4400 cultured on bouillon agar medium
One platinum loop of was inoculated and cultured at 30°C. After 16 hours of culture, 2 ml of 50 g/dl DL-5-isopropylhydantoin was added aseptically to the culture solution, and culture was continued for an additional 24 hours. The amount of produced DN-carbamylvaline in the culture solution was measured and found to be 1.29 g/dl. Example 5 Flavobacterium fucatum AJ-2478, Mycoplana deimorpha ATCC-4279, Rhizobium melilotei prepared in the same manner as in Example 1
Add 1 g of ATCC-4400 bacterial cells to 4 ml of deionized water, suspend, cool on ice, add 750 mg of acrylamide and 45 ml of methylenebisacrylamide to dissolve, and after expelling oxygen through nitrogen gas, 3.5 mg of ammonium persulfate. After 1 hour of adding 8μ of N,N'-dimethylaminopropionitrile and letting it stand under ice-cooling, the gel containing the bacterial cells was strained through a 50-mesh wire mesh, washed with physiological saline, and the gel was fixed. The compound was adjusted. 2g of these immobilized substances
0.1M phosphate buffer (PH8.0) containing DL-5-(p-hydroxyphenyl)hydantoin 1g/dl 5
ml and reacted at 30°C for 24 hours. When this reaction solution was measured, it was 0.59 g/dl for Flavobacterium fucatum AJ-2478 and 0.49 g/dl for Mycoplanadeimorpha ATCC-4279.
In the case of Rhizobium melilotei ATCC-4400, 0.44 g/dl of DN-carbamyl-P-hydroxyphenylglycine was produced.

Claims (1)

[Claims] 1. General formula (In the formula, R represents an alkyl group, an aralkyl group, or a group thereof having a substituent.) Flavobacterium which has the ability to asymmetrically cleave and hydrolyze the hydantoin ring is added to the 5-substituted hydantoin represented by Genus, Mycoplana, Rhizobium, Colletotrichum, Corynespora, Curvularia, Fusarium, Pestaloteia, Septoria, Colethropsis, Helicoceras, Bysochlamys, Xylaria, Cladosporium, Eupenicillium, Emeryceropsis,
A general formula characterized by allowing bacterial cells or treated bacterial cells of microorganisms belonging to the genus Sordaria and Wardmyces to act in an aqueous medium with a pH of 6 to 11. (In the formula, R is the same as the above formula.) D
-Method for producing N-carbamyl-α-amino acids. 2. The production method according to claim 1, wherein live or dried microbial cells are used as the microbial cells. 3. The production method according to claim 1, wherein a microbial cell grind or a microbial cell extract is used as the microbial cell-treated product. 4. The production method according to claim 1, 2, or 3, which uses immobilized microbial cells or processed microbial cells as a form of using microbial cells or processed microbial cells. 5. Claim 1, which uses a microorganism that is cultured by adding hydantoins to a medium and has an enhanced ability to asymmetrically cleave and hydrolyze hydantoin rings.
The manufacturing method according to item 2, item 3, or item 4. 6. The production method according to claim 1, wherein the 5-substituted hydantoin is a DL form. 7. The manufacturing method according to claim 1, wherein the 5-substituted hydantoin is in L-form or D-form.