SU984405A3 - Method of producing d-alpha-aminoacids - Google Patents

Abstract

D- alpha -Aminoacids are prepared by the hydrolysis of a 5-substituted hydantoin, a corresponding hydantoic acid or a mixture thereof using an enzyme obtained by cultivating the novel microorganism Pseudomonas sp. HM-40 (CBS 259.79).

Description

The invention relates to an improved method for the preparation of 0-c (amino acid-general formula (I)

R-CH-COOH

Shz

where R-2-methylmercaptoethyl, phenyl. benzyl, 2-m (gtilpropyl, propyl, indolylmethyl, 4-hydroxyphenyl, 3-hydroxyphenyl or 3-cyanopropyl groups used in the pharmaceutical industry.).

The closest to the present invention is a method of obtaining natural 0-c amino acids by microbiological hydrolysis of the corresponding hydantoins using microorganisms, for example. Pseudomonas solanacearum AZ 11149, Pseudomonas caryophHli: A3 11150. The target product output averages 20% 1.

The disadvantage of this method is the low yield of the target products.

The aim of the invention is to increase the yield of the target products.

This goal is achieved by; that according to the method of producing D-oi: -amino acids bx1} of the indicated general form; mules (1), where R - has the above value, by microbiological hydrolysis of hydantoin of the general formula (II)

,, c-jra it- "

ten

 Ns

H 7 II but

where R is the above values by the microorganism Pseudomonas

15 Sp., HM-40 (CBS 259.79) and the resulting O-o /. Amino acid is isolated by chromatography on Dowex-50 ion-exchange resin or by precipitation at an isoelectric point.

20

The yields of the desired products are 80-92.5%.

The distinction of the proposed method is that the hydrolysis is carried out by the microorganism Pseudomonas sp.

25 (CBS 259.79).

The positive effect of the proposed method is a significant increase in the yield of the target products p 20% to 80-92.5%.

Example 1, a. Obtaining an enzyme composition.

Prepare a culture medium of the following composition, g / l:

Glucose20

Potassium monosubstituted phosphate 3 Dv 3 potassium phosphate 7 substituted magnesium sulfate 0.7 Manganese sulfate 0.02. Iron (II) sulfate O, 02 Sodium chloride 1 Hydantoin D, L-methionine 3

The medium is poured into a flask with a capacity of 250 cm of a cost of 50 cm per bottle is sterilized for 15 temperature of 120 s. After seeding the strain Pseudomonas.: Sp. on a selective medium with agar C1 l Nki mix (.250 od / min) for 48 h at a temperature.

Productive growth is carried out in a 2 liter fermenter containing 1 liter of the above culture medium and 1 cm of antifoaming agent. The flasks are seeded with the strain prepared as described above. The culture was grown at a temperature of 30 ° C for 48 hours with turbine stirring at a turbine stirrer (rotation speed of 500 rpm) and with aeration with sterile air at a flow rate of 1 l / min,

By the end of growth, the cell mass is separated by centrifugation and suspended in 50 cm of distilled water. This suspension contains 7, b dry extract per 100 cm.

at. Hydrolysis of D, L-Phenylalaningvdantoina. but

5 g of D, L-phenylalanine idantoin are added to 200CM distilled water. Heated to partially dissolve the hydroin, then the medium is cooled to 37 ° C. The pH is adjusted to 7.8 by the addition of 1N. caustic soda solution. Thereafter, 25 cm of the cell suspension obtained earlier is added. The volume of the reaction mixture was adjusted to 250 cm and the pH was adjusted to 7.8.

The reaction mixture is placed under an atmosphere of nitrogen, the temperature is maintained at 37 ° C, the pH is maintained at 7.8 by gradually adding 1N. caustic soda solution.

After moderate stirring for 24 hours, all the hydantoin goes into solution.

The reaction mixture was clarified by centrifugation, then passed through an ion exchange resin (Dowex 50 resin, Form H), eluting with 2N. ammonia solution .. Alkaline fractions are evaporated to dryness. 3.98 g of D-phenylalani are obtained.

on (yield 92.5%) with the following

properties. 2O

Id3-i3 29.2 ± 2 ° (s 1, water)

(%) 8.63 (according to theory 8.48)

N

Potentiometric titration: 93.3%.

The optical purity of the obtained 0-phenylalanine exceeds 93%.

Example 2. To 150 cm of distilled water, 4 g of D, L-methioningidantoin and 8 cm of cell suspension containing 0.6 g of dry extract (prepared under the conditions described in example 1) are added.

The pH is adjusted to 7.8 by the addition of 1N. caustic soda solution. The volume of the reaction mixture was adjusted to 200 cm by the addition of distilled water. The reaction is carried out for 24 hours at a temperature of 37 ° C, maintaining the pH at 7.8 by gradually adding 1N. caustic soda solution ..

The content of D-methionine in the reaction mixture is determined by the method of Stein and Moore: its concentration is 17 g / l, which corresponds to 100% conversion calculated on the basis of taken D, 1-methioningidantoin.

The reaction mixture was clarified by centrifuging the gel and then passed through an ion exchange resin (Dowex 50 resin, Form H); 2.7 g of D-methionine are obtained (yield 80%) with the following properties:

2, -19.3 ° (with 1; 5n. Hydrochloric acid) N 1%) 9.36-9.42 (theoretical: 9.39)

The optical purity of the obtained D-methionine is close to 90%.

Example 3. a. Obtaining an enzyme composition.

Prepare the culture medium of the following composition, g / l:

Glucose .20

Yeast Extract 10 Baktopepton 10

The medium is poured into 250 cm bottles at a rate of 50 cm per bottle and sterilized for 15 minutes at a temperature of 120 C. After sowing, the Pseudomoinas Sp. HM-40 Flasks are stirred (25Ob / min) for 24 hours at temperature.

The contents of one flask are used to seed a 2-liter fermenter containing 1 l of the above medium. The culture was grown for 24 hours at a temperature with stirring with a turbine mixer with a rotation speed of 500 rpm and aeration with sterile air at a flow rate of 1 l / min.

By the end of the growth, the microbial mass is separated by centrifugation and suspended in 100 cm of distilled water, this suspension contains 8.9 g of dry cell extract. at. Enzyme hydrolysis of methioningidantoin. 5 g of D-r-methioningidine ton are dissolved in 200 cm of distilled water. The pH is adjusted to 7.5 by the addition of 0.1 n. sodium hydroxide solution, then 20 cm of the obtained wound of the cell suspension is added, the volume is brought to 250 cm, the reaction mixture is placed in a nitrogen atmosphere, the temperature of the reaction mixture is 37 ° C. After carrying out the process for 5 hours, the content of methionine in the media is determined by the chromatographic method according to the method of Stein and Moore. The hydanthionase activity of the cell suspension (the amount of mole of methionine produced per 1 mg of the dry cell extract per 1 hour of the enzymatic reaction) is determined to be 4, 5 ... 1 After the process has been performed; for 22 hours, the reaction mixture is clarified by centrifugation. The formed methionine is separated by passing through an ion exchange resin (Dowex 50, Form N), eluting with 2N. ammonia solution. The eluate is evaporated to dryness and dried, to obtain 3.9 g of D-methionine (yield 91%) with the following properties: N (%) 9.4 (theoretical content 9.33% J 23 ° (C 1, 5 n. Hydrochloric acid). Potentiometric titration of 98%. Example 4. Enzymatic hydrolysis of L-hydroxyphenylglycene hydantoin Prepared 100 cm, suspensions containing 8.9 g of dry cell extract under the conditions described in Example 3a. Dissolve 5 g of 4-hydroxyphenylglycine rantoin in 250 cm distilled water. The pH is adjusted to 7.5 by adding 0.1N sodium hydroxide solution and 20 cm of the cell suspension is added. The volume was adjusted to 250 cm and the reaction mixture was placed under a nitrogen atmosphere, the temperature of the reaction mixture was 31 ° C. After the process was carried out for 5 hours, the content of 4-hydroxyphenylglycine was determined.Hydantoinase activity was 1.8 M amino acid per mg dry cell extract per 1 hour enzymatic reaction. After carrying out the process for 22 hours, 4-hydroxyphenylglycine is separated by passing through an ion exchange resin (Dowex 50, Form H). 3.78 g of D- (4-hydroxyphenyl) -glycine are obtained (yield 87%). homogeneous by chromatography, with the following properties: N (%) 7.6 (theoretical content 8.4) N 3% -135 (C 1, 5 n. hydrochloric acid) Potentiometric titration 110%. Example 5. The strain Pseudomonas Sp, HM-40 is grown in the following conditions: Composition of culture medium, g / l: Glucose 20 Disubstituted potassium phosphate 7 Monosodium potassium phosphate 3 Magnesium sulfate 0.7 Manganese sulfate 0.02 Ferrous sulfate 0.02 sodium chloride . 1 D-L-methioningidantoin3 Yeast extract 2 Pre-cultivation. The first pre-cultivation was carried out in a 250 cm bottle containing 50 cm of medium for 24 hours. The second pre-cultivation of the sowing made from the previous bottle was made. carried out. also within 24 hours in a fermentation tank with a capacity of 2. l containing 1 l of culture medium. Production culture. The contents of a two-liter fermenter are used to seed a 7.5-liter fermenter containing 5 liters of medium. The culture is grown for 24 hours at a temperature with aeration of sterile air at a rate of 5 liters / min and stirring with a turbine mixer at a speed of 500 rpm. The biomass obtained under such conditions is 3.8 g of dry extract per liter of culture medium. Enzyme reaction. At the end of the growth, the microbial mass is separated from the medium by centrifugation and suspended in 250 cm of distilled water. 10 g of D, L-phenylglyzing of hydantoin are suspended in 400 cm of distilled water and the pH is adjusted to 7.5 by adding 0.1 n. caustic soda solution. 50 cm of the above-described cell suspension is added and the reaction volume is brought to 500 cm by the addition of water. Placed in a nitrogen atmosphere at a temperature, maintaining a pH of 7.5 for 17 ". After proceeding the process for 17 hours, the reaction mixture was clarified by centrifugation, evaporated to 1/5 of the original volume and acidified to pH 1 by the addition of concentrated hydrochloric acid. The precipitate formed is filtered off and dried. Chromatographic analysis shows that it represents phenylglycinhydrogenic acid with the following properties: N {%) 13.9% (theoretical content 14.4%} Dose 15 ° -1370 (with 1; 1% amYet output of Oggidantoic acid 6.3% with respect to hydantoin.The mother liquor is again evaporated and the pH soaked to pH 5 by adding caustic soda, the precipitated D-phenylglycine is separated by filtration, washed with water and ethyl alcohol, and dried. -O-phenylglycine (yield 88%) with the following properties: .N (%) 9.2 (theoretical content 9.3) Hd - -148 (C 1; 5N hydrochloric acid) Example 6. Pseudomonas Sp., HM-40 culture was grown under conditions of Example 5. After centrifugation, the microbial mass was suspended in O, 1 M phosphate buffer at pH 7.8, this suspension is used as a catalyst for the hydrolysis of various hydantoins, the reaction conditions are as follows: the initial concentration of hydantoin is 20 g / l in 0.1 M phosphate buffer at pH 7.8; The applied biomass is 6.5 g (calculated as dry extract) per 1 liter of the reaction mixture. The reaction time is 5 hours. Then the content in the reaction mixture is determined by the resulting amino acid and the enzymatic activity of the cell extract is calculated. The activity thus calculated is presented in the table in micromolar amino acid formed per 1 mg of the dry enzyme extract per 1 hour of reaction time, i The results are presented in the table.

01-methioningidinntoin

DL-2-phenyl-alaningidantoin

DL-leucine hydantoin

DL-valingidantoin

DL-tryptophan-dantoin

DL-14-hydroxyphenyl) -glycinhydantoin Example 7. According to the method described in Example 6, a Pseudomonas HM-40 cell suspension is obtained. 60 buffer phosphate solution. This suspension contains 6.5 g of dry extract in 100 cm, n-before using the cells are crushed by ultrasound at. conditions: 65

2.4

2.0 1.6

1.0

D-valine

ABOUT,. 6

D-phenylglycine

D- (4-hydroxyphenyl) -glycine, 6

D- 1-amino- (4-cyan propionic acid 3, 5

Claims (1)

D- (3-hydroxyphenyl) -glycine0, 7 / up PONS device equipped with zonTS4C. Ultrasonic frequency, kHz Treated volume, cm. Duration of grinding, min. The suspension treated in this way was used to catalyze the hydrolysis of methanine, phenylalanine and phenylglycine hydantoins in experimental experiments described in Example 6. The measured activity was as follows, µmol / mg / h: OB-methioningidantoin 1/7 D1- (2-phenyl) -alanine-; hydantoin1,0 DL-phenylglycine-. hydantoin 0.8 PRI me R 8. Cell suspension of Pseudomonas.SP HM-40 in water, prepared as described in Example 5, and containing 7.3 g of dry extract per 100 cm, is lyophilized before use in the following conditions 120 cm of the suspension is poured into 4 flasks with a capacity of 500 cm, frozen and kept under reduced pressure (0, 5 Hg) for 16 h. 8.8 g of lyophilized enzyme powder are obtained, which is used in the conditions described in the example 6. The following activity was determined, µmol / mg / h: pL-methioningidantoin2, 4 DL-phenylglycinhydantoin0, 4 DL-12-phenyl) -alaningidantoin 1.5 Formula of the invention Method of obtaining T) -li-amino acids of the general formula R-OH-COQH where R-2-methylmercaptoethyl, phenyl, benzyl, 2-methylpropyl, propyl, indolylmethyl, 4-hydroxyphenyl , 3-hydroxyphenyl or 3-cyanopropyl group, f microbiological hydrolysis of hydantoin of the general formula, C-NH -s has the indicated values, followed by the target product, characterized in that, in order to increase the yield of the target products, the hydrolysis is carried out by the Pseudomonas Sp microorganism ., NM-40 {CBS 259,79), Sources of information taken into account in examination 1, French Patent No. 7817199, cl. C 12 D 13/06 published 05.01.79 (prototype).