Method of obtaining n-carbamyl phenylglycine derivatives

专利摘要:
The way to obtain derivatives of K-Karbamsh1Fensh1gly1 by enzymatic hydrolysis of solutions of the corresponding derivatives of hydanions, characterized in that, in order to ensure the possibility of using concentrated solutions of 1Hydantoins with a high yield of the target products, the flow of programs will be treated with a strain of the flow of hydantoins, with the aim of the flow 50 0. O)
公开号:SU1124889A3
申请号:SU782589348
申请日:1978-03-15
公开日:1984-11-15
发明作者:Вилья Аурелио；Фасчетти Эудженио；Перриконе Елена；Деген Людвиг
申请人:Аник С.П.А.(Фирма)；
IPC主号:

专利说明:

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95 No. Vretmin The invention relates to the microbiological industry, in particular to a method for preparing phenylglycine derivatives, in particular N-carbamylphenylglycine and carbamylparamethoxyphenylglycine, used in the pharmaceutical industry. A known method of obtaining phenylglycine derivatives, including N-carbamylphenylglycine, consists in carrying out the enzymatic hydrolysis of 5- (D, L) -phenylhydantoin by the Pseudomonas sp. 942 i.p. 945 for 24 hours. 13. The yield of the target product is 57-90% of theoretical with a maximum concentration of 20 mm / ml. The purpose of the invention is to provide the possibility of using concentrated solutions of hydantoins with a high yield of the target products. This goal is achieved by the fact that according to the method of obtaining derivatives of N-carbamyl-1-glycine, which involves enzymatic hydrolysis of solutions corresponding to the derivatives of hydantoins, enzymatic hydrolysis is carried out by a strain of Bacillus brevis NRRL 1286 or Bacillus bcearothermophilus NRKL 1287 at 40-50. The method is carried out as follows. Bacterial strains, isolated from soil, vegetables, and garbage, are inoculated at 50 ° C from oblique agar into 250 ml Erlenmeyer flasks, each containing 50 ml of the following nutrient medium, at pH 7.2, g / l: Bulk peptone 10 Yeast extract 10 NaCI3 5-B, L-methyl.hydantion1 The medium is sterilized at 110 ° C for 30 minutes. Incubation is carried out for 18–20 h at 50 ° C with re-generation. In an Erlenmeyer flask with a volume of 500 MP, 100 ml of the same medium are added with the addition of 2 ml of a previously prepared culture. After 1618 h, an additional enzyme reaction is carried out, for which 1 ml of bacterial suspension (dry) is added to a tube containing 10 ml of 0.07 M phosphate buffer at pH 8.5 and 20 µmol / ml 5-B, L-phenylhydantoin. weight 40 mg / ml). After 15 minutes incubation at 40 ° C., the reaction is carried out with b-dimethylaminobenzal. 1 9 dehydro to quantitatively determine the carbamyl derivative thus obtained. The method uses cultures of Bacillus brevis and Bacillus stefrothermophilus, which were deposited in 1977 at the North Regional Research Center in Peori, Illinois, United States. They were assigned the numbers NRRL 11079 for strain 1286 and NRRL-B 11080 for strain 1287. The characteristics of the strains are presented in Table. 1. During the enzymatic hydrolysis of D-hydantoin at pH 7-10, it was established that the resulting carbamine derivatives are in the D-form. The chemical structure and its identity with N-carbamyl-phenylglycine and M-carbamyl-paramethoxy-phenyl-glycine was confirmed after recrystallization of the reaction product using IR spectra of N.M.R., mass spectrometry and elementary analysis. The optical rotation constants are respectively (in 1 n solution) 140 ° (in 1 n solution), which corresponds to the literature data. Hydrolysis can occur with the use of whole cells of microorganisms, spores or extracts of the above mentioned microorganisms with the help of so-called resting cells, for which bacterial cells isolated from the culture medium after rinsing are suspended in buffer with the addition of racemic hydantoin. Preparations containing a hydrolase can be used, such as its extracts or concentrates, purified hydrolases obtained with the use of aforementioned microorganisms. Example 1. Preparing the medium for the bacterial strain B. brevis 1286 of the following composition, g / l: Meat peptone 10 Yeast extract 10 NaCl 3 5-B, hydrohydantoin 1 The pH of the medium is adjusted to 7.2, sterilized at 30 minutes and incubated. 100 kp of culture liquid are taken, placed in a 500 ml Erlenmeyer flask, and an incubation w (using a planetary mixer) is added at 18 o'clock with 100 ml of 0.14 M phosphate buffer (pH 8.5) containing 40 µmol / mp 5- (B, b) -phenylgilltoin. After another 4 hours of incubation under the same conditions, the amount obtained by means of Ncarbamylphenylglycine is determined. From 705 mg of 5- (0, b) -phenylhydantoin, 700 mg of N-carbamylphenylglycine is obtained, which corresponds to a yield of approximately 90%. Example 2. The same as in measure 1, but after 4 hours with and with the help of strain B. stearothermophilus NRRI. 1287 out of 705 mg of 5- (P, b) -phenylgidaction receive 600 mg of L-carbamshphenylglycine, which corresponds to a yield of about 85%. . EXAMPLE 3 A medium is prepared having the above composition and containing 1 g / l of 5- (B, b) -metalsyl hydride. The pH was adjusted to 7.5 by adding soda and the culture fluid was dispensed in 50 ml portions into a 250 ml Erlenmeyer. After sterilization for 30 minutes at 110 ° C, the flasks are inoculated with Bacillus brevis NRKL 1286 strain from canted agar containing the same solid agar medium (DIFCO) at 2% concentration and incubated for 22 hours at s. Use planetary agitator with a speed of 220 rpm. Using the resulting culture (optical density 0.4 at 550 n dilution 1:10) in an amount of 2 mp to inoculate 100 ml of the same strain in 500 ml Erlen meier flasks, the resulting culture was incubated at 40 ° C using a planetary stirrer (220 o6.) for 18 hours. The cells were separated from the broth by centrifugation at g-500 (g is the acceleration of gravity) for 20 minutes, washed three times with isotonic solution at pH 8.0, suspended in 0.07 M phosphate buffer ( pH 8.5), thus obtaining cell counts. To carry out the enzymatic hydrolysis reaction at 40 ° C and stirring (220 rpm) in 250 ml Erlenmeyer flasks, 64 ml of the reaction mixture containing 200 mg of bacteria (dry weight) and 20 µmol / ml 5- (B, L -phenylhydantoin (3.52 mg / mp). At suitable times 9L of time, the content of the product of Grolysis is measured. D-carbamylphenylglycine, using a colorimeter1 {ical method at 438 mmkm. The drawing shows the dependence curve, expressing the percentage of theoretical yield of the carbamyl derivative obtained. Example4. Bacterial cells of strain B. brevis NRRL 1286 are prepared as described in Example 3. Cell suspension (41.5 mg / ml of dry bacterial cells) is placed in phosphate salt buffer at pH 8.5, subjected to mechanical grinding in a Manton laulin homogen at a temperature below 35 ° C and pressure of 650 kgf / cm. Cell debris is separated from the extract by centrifugation (25,000-fold acceleration of gravity for 30 minutes). To 970 ml of 0.2 M phosphate salt buffer (pp 8.5) containing 4.7 g of 5- (B, b) -feiylchantoin, 30 ml of an extract containing 280 units is added. enzyme (one unit corresponds to the amount of enzyme that provides conversion of 1 µmol / ml per minute of the substrate in 0.2 M phosphate buffer, pH 8.5, at 50 ° C, and the buffer contains 20 µmol / ml 5- (B, b) -phenylhydantoin ). After one hour, 3.2 g of N-carcinoyl felix glycine is formed, which corresponds to approximately 63% of the total hydrolysis. Example 5. Preparing a semi-synthetic medium, having the following composition, g / l: NH4Ci5 NajPO 7.05 KH2P042.72 Meat peptone5 Other burn extract 0.5 5- (D, L) -Netnvgdak. tons; 1.0. The medium was distributed in portions of 50 ml in 250-MP Erlenmeyer flasks and 100 liters in 500 ml Erlenmeyer flasks and sterilized for 30 t-nm at. A seed culture is obtained by insulinizing 250 milliliter flasks from beveled agar with a strain of B. brevis strain and incubated as in Example 4 at 40 ° C for 22 hours. For this culture (optical density 0.105 at 550 nm, dilution 1:10) taken in an amount of 2.5 ml
in a 500 ml Erlenmeyer flask containing the same medium. After 15 hours of incubation at 40 ° C, the reaction mixture contains in 64 ml of buffer 100 mg of bacteria (in terms of dry weight and equivalent of 100 ml of broth culture) and 20 µmol / ml of 5 (D, b) -phenylhydanthion.
After 5, 10 and 15 minutes, the amount of N-carbamylphenylglycine obtained is measured, which is 0.6; 1.0 and 1.4 μmol / ml.
Example 6. A nutrient medium is prepared consisting of water from soaking maize in an amount of 2.6% (based on dry weight). The medium was adjusted to pH 7.5 with KOH, distributed in 50 ml portions into 250 ml flasks, and 100 ml portions into 250 and 500 ml flasks, followed by sterilization for 30 minutes at. To obtain a seed culture, inoculate in 250 ml flasks from beveled agar with B.brevis NRRL 1286 culture followed by incubation, as in Example 4, at 40 ° C for 22 hours.
To inoculate the medium in 500 ml flasks, take 2.5 ml of the inoculant culture obtained. After 18 hours of incubation, cells are obtained in the same manner as in Example 3.
Enzymatic hydrolysis is carried out at 40 ° C in a reaction mixture containing 0.07 M phosphate buffer (pH 9.5) in 64 MP bacterial cells obtained from 100 ml of broth culture and 20 µmol / ml 5- (N, L) phenylhydantione; in the reaction mixture containing in 64 MP 0.07 -M phosphate buffer (pH 8.5) bacterial cells obtained from 100 ml of broth culture, and 20 µmol / ml 5- (N, L) -napamethoxyphenylhydanthion.
After 5, 10, 15 and 60 minutes, the amount of N-carbamylphenylglycine thus obtained and N-carbamylparamethoxyphenylglycine obtained in this way is determined;
The results are presented in table 2.
Example 7. Receive, acetone powder cells of the strain Bacillius brevis NRRL 1286, and 80 mg of this powder containing 420 units. enzyme suspended in 1 l of 0.2 M phosphate saline buffer (pH 8.5) containing 5.0 g of 5- (D, L) -phenylhydanion. After one hour, 4.8 g of L-carbamylphenylglydine is obtained, which corresponds to approximately 87% of the total hydrolysis.
Example 8. Using a strain of Bacillus brevis NRRL 1286 get biomass in a 20-liter fermenter Fomel, containing 16 liters of culture medium pH 7.8, having a composition, g / l:
Yeast extract 10 M clear peptone. 10 NaCi3
 0.56
5- (D, L) -Methylhydantoin1
The culture 3 in the fermenter is inoculated using 16 ml of seed culture (optical density, 360 at 550 nm, dilution 1:10) grown in 50 ml flasks containing 100 ml of the same nutrient medium and incubated for 16 hours at 40 C with stirring (220 rpm). Fermentation is carried out at a pH of 7.8 with the addition of 2NSB, while maintaining the O.A.R. about 0.45. By the 16th hour of fermentation (optical density 0.350 at 550 nm. Dilution 1:10), the biomass was separated from the culture liquid by centrifuging in an Alfa Laval separator at room temperature.
Cells are washed with an isotonic solution at pH 8.0. Enzymatic hydrolysis is carried out at 40 ° C in a reaction mixture containing 0.04 M phosphate-buffered saline buffer (pH 8.5), 0.5 g of bacterial suspension (equivalent to 0.120 g of dry cells) and 20 µmol / 5- (D, L) -hydantoin, and in a reaction mixture similar to the above, but containing 20 μmol / ml 5- (D, L) -n-methoxyphenylhydantoin. After 5, 10, 15, and 60 minutes, the amount of N-carbamylphenylglycine and N-carbaminemethoxyphenylglycine obtained is determined.
The results are presented in table 3.
The name of indicators
Table 1
Characteristics of the strain
Bacillus brevis NRRL 1286, and
at
there o)
atri:
Bacillus stearothermophilus NRRL 1287
0.6-1
0.6-0.9 2.0-3.5
1.5-4
t
+ f
+ Ellipsoid Ellipsoid
SubterminalSterminal
40-50
40-50
thirty
+ +
+
+ +
7.5

权利要求:
Claims (1)
[1]
METHOD FOR PRODUCING N-CARBAMYLPHENYLGLICINE DERIVATIVES by enzymatic hydrolysis of solutions of the corresponding derivatives of guadanthions, characterized in that, in order to enable the use of concentrated solutions of <hydantoins at high yield of the target products, enzymatic hydrolysis is carried out with strain Ilus brevis Nrlilus 12Rlilophus 12 or Rlillophilus Nerlilus ratris 86 Nrll86 12Brius Nerlillus brytrilus nobrilus brytrilus nobrilus 12198 Nerlil -50 ^ft C.
. 1124889 ί

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IT987278B|1973-05-11|1975-02-20|Snam Progetti|PROCEDURE FOR THE PREPARATION OF L CARBAMIL AMINO ACIDS AND THE CORRESPONDING L AMINO ACIDS|

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US4065353A|1975-05-12|1977-12-27|Snamprogetti, S.P.A.|Method for the preparation of D-carbamyl aminoacids and the corresponding D-aminoacids|

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法律状态:

优先权:

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申请号 | 申请日 | 专利标题

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IT21232/77A|IT1075132B|1977-03-15|1977-03-15|ENZYMATIC COMPLEXES FOR TRANSFORMING IDANTOINE RACEME IN OPTICALLY ACTIVE AMINO ACIDS AND THEIR APPLICATIONS|

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