DE3018584A1 - PROCESS FOR THE PREPARATION OF D-ALPHA AMINO ACIDS - Google Patents

Description

XT 504 ■ _3_

A.E.C. Societe De Chimie Organique Et Biologique, Commentry / Fr.

Process for the preparation of D-CC amino acids

description

The invention relates to a process for the preparation of D - ^ - amino acids. by enzymatic means. In particular, the invention relates to a method which consists in a 5-position to hydrolyze substituted hydantoins directly to D-cC amino acids.

Those obtained by the process according to the invention are preferred D-cC-amino acids D-phenyl alanine, D-methionine, the D-phenylglycine or the D-p-hydroxyphenylglycine, which as Medicinal products or as intermediates in the manufacture of products, which as such on pharmaceutical or agricultural Area are usable, are particularly useful; however, numerous D-amino acids such as D-leucine, the D-valine, the D-tryptophan when carrying out the invention Procedure can be obtained.

According to the method of the invention, a D-oC-amino acid is prepared, by a hyantoin suitably substituted in the 5-position brought into contact with an enzyme that is able to convert this hydantoin into the corresponding D-tt amino acid will.

The enzyme used in the method of the invention is by cultivating a microorganism as detailed below

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licher is identified, preserved in a suitable milieu. The microorganism, the cultivation of which under suitable conditions provides the enzyme necessary for carrying out the invention Method used must be regarded as a new species belonging to the genus Pseudomonas.

It was isolated from a sample of mud that was found in Commentry (France) according to the usual isolation methods for microorganisms was taken «A sample of this strain was taken in the ' Centraalbureau yoor Schimmmelcultures in Baarn (Netherlands) where it is registered under number CBS 259.79. This laboratory is authorized to identify the strain of the microorganism to be distributed to anyone who is lawfully aware of the foregoing Has knowledge.

The microorganism HM-40 is a Pseudomonas, do not comply with the certain properties of those species described in Bergey's Manual of Determinative Bacteriology ^f, 8th Edition. It is known under the name Pseudomonas spi HM-40.

The breeding characteristics and the morphological characteristics of Pseudomonas sp. HM-40 are the following:

1. Appearance of cultures on different media

a) "Trypticase soy agar" environment at 30 ° C

- After 24 hours of incubation: very small colonies

- After 48 hours of incubation! Larger colonies (about 1 to 1.5 mm in diameter) with distinct and semi-transparent Margins

- After 4 days of incubation: even larger colonies (approx. 4 mm diameter), opaque, mucoid.

b) With nutrient broth at 30 ° C

In test tubes (16 160 mm) with 5 cm medium

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the development occurs within 24 hours, but is not very productive. The development increases in the following days. Formation of a uniform turbidity with rather adherent sediment at the bottom of the tubes. Carbon dioxide gas (10%) increased not the development.

c) Various solid media

On nutrient gel and on "Brain Heart Infusion Agar", the development and appearance of the cultures are identical to those which are described for the "Trypticase soy agar" milieu.

d) Various liquid media

On "Brain Heart Infusion Agar", peptonized water, tryptonized water ,. In nutrient broth with yeast extract, the development and appearance of the cultures are identical to those described for nutrient broth. The development is somewhat weaker on "Trypticase Soy Broth" and on peptonized water with 0.2 % glucose.

2. Morphology

a) Microscopic examination without staining:

After 24 hours of cultivation in a liquid medium at 37 ° C: Bacteria 0.5 to 5 1/2 in length, in boats. The germ is mobile and the mobility is more pronounced when the incubation is carried out at 24 to 30 ° C.

b) Microscopic examination after staining:

The germ is grief-negative; often the central part of the bacterial body is less colored.

3. Optimal development temperature: 24 ° C: good development

30 ° C: Very good development 37 ° C: Average development 42 C: Poor development

4. Biochemical and breeding characteristics:

a) Respiratory type: Strictly aerobic

b) Oxidase: positive

c) Catalase: positive

d) "Mac Conkey Agar": Abundant culture

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e) "Salmonella Shigella Agar": Little abundant cultures

f) "Cetrimide Agar": No development

g) Urease (in Ferguson environment): positive h) Indole production: negative

i) Test with methyl red: negative j) Voges-Proskauer test: negative

k) Utilization of citrates (Simmons): positive within 48 hours; Alkalinization

1) Liquefaction of the gelatin: negative

m) Oxidation-fermentation test (Hugh and Leifson): Oxidizing η) Formation of acids from carbohydrates: Glucose: Positive

Lactose: positive arabinose: positive maltose: positive mannitol: positive

Sucrose: positive xylose: positive

o) Reduction of nitrates: positive p) Reduction of nitrites: negative q) Denitrification: negative r) Formation of pigment: negative s) Formation of HpS:

- on "Triple Sugar Iron Agar ⁷¹ - Environment: very slightly positive - paper with lead subacetate: traces of HS t) hemolysis (horse blood): negative u) litmus milk: no change v) / 3-galactosidase: positive w) arginine dihydrolase: negative x ) Lysine decarboxylase: negative y) ornithine decarboxylase: negative

If one considers the strain Pseudomonas ^ p. HM-40 grows in classical culture media, the enzyme that can convert the hydantoins into the corresponding D-oC-amino acids is essentially formed.

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lichen in the cells of the microorganism and weaker in the culture medium.

The growth medium should essentially be assimilable carbon and sources of nitrogen and other .nutrients, which for Development of the HM-40 strain are necessary as mineral salts The enzyme production can be improved by adding a 5-substituted hydantoin to the culture medium as a nitrogen source. In general, the hydantoin of methionine is used for this purpose, but can also other hydantoins, such as the hydantoin of valine, the hydantoin of leucine, hydantoin of isoleucine or 5-cyanopropyl hydantoin can be used advantageously.

Sources of assimilable carbon can be carbohydrates, such as glucose, sucrose, lactose or maltose in pure form or in the form of complex residues such as molasses or Use lactoserum or whey; you can also use alcohols or organic acids. Certain animal or vegetable Oils such as lard or soybean oil can replace or be added to these various carbon sources.

The appropriate sources of assimilable nitrogen are extraordinarily different. They can be chemically very simple substances, such as mineral or organic ammonium salts or urea. They can also be caused by complex substances be taught, which the nitrogen mainly in protein or. Contain protein form.

Among the added mineral elements, certain can have a buffering or neutralizing effect, such as this Alkali or alkaline earth phosphates or calcium or magnesium carbonate · Others provide this for the development of the trunk HM-40 necessary ionic equilibrium, such as the chlorides and sulfates of alkali and alkaline earth metals or the zinc,

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Cobalt, iron, copper or manganese salts.

The pH of the fermentation medium at the beginning of the cultivation should be between 4 and 5 and preferably between 6 and 8. The optimal temperature for fermentation is between 25 and 35 ° C, however it will still produce a satisfactory one for Temperatures obtained between 20 and 37 ° C.

The fermentation aeration can vary within fairly wide limits. However, it has been found that Belüftun9 ^en • 0.3 to 3 1 Air P * x>. Liters of bouillon and per minute are particularly suitable. The maximum yield is obtained after 10 to 72 hours of cultivation, this time depending essentially on the environment used.

Since the enzyme is mainly produced in the cells, the transfer of the hydantoin substituted in the 5-position to the D-cC-amino acid using the containing the cells Culture medium or that from this broth by centrifugation isolated cells} these cells can be used as such after precipitation with acetone or lyophilization will; they can also be used in the form of cell extracts obtained by grinding or ultrasound treatment.

The conversion of the hydantoin substituted in the 5-position into the corresponding D- ^ C-amino acid is generally in aqueous The environment is carried out at a pH between 6 and 9, and preferably at around 7.8, the pH is optionally adjusted by adding an aqueous alkaline solution, especially sodium hydroxide, kept close to this value.

The temperature of the conversion reaction is generally between 20 and 55 ° C and preferably at about 37 ° C.

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The concentration of hydantoin in the reaction medium is a puncture of solubility. It is generally over 5% (weight / volume), but it can also be over 10 % (weight / volume).

The reaction medium can contain surfactants, antioxidants, Coenzymes, which favor the formation of the D-oC ~ amino acid, and thus enable an improvement in the yield. In general, the duration of the reaction is between 2 hours and 4 days. The response will actually take that long followed up until no more hydantoin is converted into B-t <-Ami no acid will.

During the conversion of the hydantoin into the Ό-cL amino acid, a D-hydantoin acid can be formed as an intermediate, which is converted into the D-ct amino acid under the action of the enzymatic system. The process according to the invention can accordingly be carried out either on a hydantoin or a hydantoic acid or on a mixture thereof.

In addition, the strain HM-40 favors the in situ racemization of L-hydantoin, which is next to the desired D-cC-amino acid forms. As a result, the racemic hydantoin used can be converted entirely into D-oC-amino acid.

The O-Oi- amino acid obtained by the process according to the invention can be isolated from the reaction medium by the separation methods customary for amino acids, generally after clarification of the reaction medium by centrifugation and filtration through an ion exchange resin. In general, the amino acids are caused to precipitate at their isoelectric point.

The following examples are intended _to clarify the invention in more detail:

example 1

a) Preparation of the enzymatic composition A culture medium of the following composition is prepared:

- Glucose: 20 g / l

- Monopotassium phosphate: 3 g / l

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⁷ g / i 0.7 g / l 0.02 g / l 0.02 g / l ι g / i 3 g / i

- dipotassium phosphate

- magnesium sulfate

- manganese sulfate

- ferrous sulfate

- sodium chloride

- Hydantoin of D, L-methionine

3

The medium is distributed in flasks of 250 on in an amount of 50 cm per flask and sterilized ^{at 120 ° C. for 15 minutes.} After inoculation with the strain Pseudomonas sp, HM-40, which is kept in a gel environment, the flasks are shaken for 48 hours at 30 ° C. (250 revolutions / minute).

The production culture is effected in a fermentation vessel of 2 1, containing 1 1 of the above culture medium and 1 cm of antifoam agent which was prepared as described above. The culture is at 30 ° C for 48 hours while stirring with a turbine developed at 500 revolutions / minute and aeration with sterile air at a rate of 1 liter / minute.

After the cultivation has ended, the cell bodies are separated off by centrifugation and suspended in 50 ml of distilled water. This suspension contains 7.6 g of dry extr-Jct "per 100 cm ³ .

b) Hydrolysis of the hydantoin of D, L-phenylalanine 5 g of hydantoin of D, L-phenylalanine are added to 200 cm of distilled water. After heating to partially dissolve the hydantoin, the medium is cooled to 37 ° C. The P ^H value is adjusted to 7.8 by adding an N sodium hydroxide solution and then 25 cm of the cell suspension obtained as above is added. The volume of the reaction mixture is adjusted to 250 cm and the pH to 7.8.

The reaction mixture is placed under a nitrogen atmosphere; its temperature is 3 7 ° c, the pH value is increasing by Addition of an N sodium hydroxide solution kept at 7.8.

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After 24 hours of moderate stirring, all of the hydantoin is in Solution gone.

The reaction mixture is clarified by centrifugation and then passed through an ion exchange resin (Dowex 50 H -form resin), eluting with a 2N ammonia solution. The alkaline Fractions are concentrated to dryness. This gives 3.98 g of D-phenylalanine (yield 92.5%), its characteristics the following are:

[α] ²⁰ = 29.2 + 2 ° (c = 1, water)

D ■■■ - ■ ', ■ "
:% N = 8.63 (theory 8.48)

Potentiometric titer: 93.3%. The optical purity of the D-phenylalanine obtained is over

Example 2

3 ■ "
4 g of D, L-methionine hydantoin and 8 cm of a cell suspension which contains 0.6 g of dry extract (prepared under the conditions described in Example A ) are added to 150 cm of distilled water.

The pH is adjusted to 7.8 by adding an N sodium hydroxide solution set. The volume of the reaction mixture is adjusted to 200 cm by adding distilled water. The reaction is continued for 24 hours at 37 ° C., the pH value being reduced by the progressive addition of an N sodium hydroxide solution is held at 7.8.

The D-methionine is in the reaction mixture according to the method von Stein und Moore determined: Its concentration is 17 g / l what. a degree of conversion of 100% based on the amount used Hydantoin des D, L-r-methionine, corresponds. The reaction mixture is clarified by centrifugation and then passed through an ion exchange resin (Dowex 50 resin, Form H J. This gives 2.7 g of D-methionine (yield 80%, based on the certain D-methionine), the characteristics of which are as follows:

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[α] ρ ° = -19.3 ° (c = Λ; 5N hydrochloric acid)

% N = 9.36-9.42 (theory: 9.39).

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

Example 3

a) Preparation of the enzymatic composition

A culture medium is created with the following composition:

- Gluco se 20 g / l

- Yeast extract (DIFCO) 10 g / l.

- Bactopeptone (DIFCO) 10 g / l

3- 3

The medium is in 250 cm flasks in an amount of 50 cm distributed per flask and sterilized for 15 minutes at 120 ° C «. After inoculation with the Pseudomonas sp », HM-40 strain the flask was stirred for 24 hours at 30 ° C (250 revolutions per minute). ■

The contents of a flask are used to inoculate a fermentation vessel of 2 liters containing 1 liter of the above medium. The culture is developed for 24 hours at 30 q , stirring with a turbine at 50o revolutions / minute and aerating with sterile air at a rate of 1 l / minute.

When the cultivation is finished, the microbial Separated body by centrifugation and distilled in 100 cm Suspended water} this suspension contains 8.9 g dry cell extract.

b ) Enzymatic hydrolysis of methionine hydantoin

5 g of D, L-methionine hydantoin are dissolved in 200 cm of distilled water Water. The pH is adjusted by adding an N / 10 sodium solution liquor solution to 7.8, then 20 cm of the solution obtained as above is added Cell suspension too. The volume is adjusted to 250 cm; the reaction mixture is placed under nitrogen, its temperature is 37 ° C.

After 5 hours of reaction, what is present in the medium is determined Methionine by chromatography according to the method of

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Stein and Moore, The Hydantoinase Activity of the Cell Suspension (Number of .1CMoles of methionine, which per mg of dry cell extract and generated per hour of enzymatic reaction) is calculated; it is 4.5.

After 22 hours of reaction, the reaction mixture is through
Centrifugation clarified. The methionine formed is through
Passing through an ion exchange resin (Dowex 50, H -form) isolated, eluting with a 2N ammonia solution. To
Evaporation of the eluate to dryness gives 3.9 g of D-methionine (yield 91 %) with the following characteristics:

% N = 9 ₄ 4 (theory: 9.39)

= -23 ° (c = 1; 5N hydrochloric acid)
Potentiometric titer: 98%

Example 4

Enzymatic hydrolysis of p-hydroxyphenylqlycine hydantoin
100 cm of a suspension containing 8.9 g of dry cell extract is prepared, operating under the conditions described in Example 3a.

5 g of p-hydroxyphenylglycine hydantoin are dissolved in 250 cm of distilled water Water. The pH is adjusted by adding

n / 10 sodium hydroxide solution to 7.5 and add 20 cm of the cell

3
suspension to. The volume is set to 250 cm ^ that

Reaction mixture is placed under nitrogen; its temperature is 37 ° C.

After a 5-hour reaction, the p-hydroxyphenylglycine is determined. The hydrolyzation nase activity is 1.8 yu-M amino acid formed per mg dry cell extract per hour of reaction.
After 22 hours of reaction, it becomes Dp-hydroxyphenylglycine
isolated by passing through an ion exchange resin (Dowex 50, H -form). This gives 3.78 g of Dp-hydroxyphenylglycine (yield 87%), which is homogeneous on chromatography and has the following characteristics:

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% N = 7.6 (theory: 8.4) [α] ρ ° = -135 ° (c = 1; 5N hydrochloric acid) Potentiometric titer: 110 %

Example 5

The Pseudomonas sp, HM-40 strain is cultivated under the following Conditions:

Composition of the cultural environment

- Gluco se 20 g / l

- dipotassium phosphate 7 g / l

- Monopotassium phosphate 3 g / l

Magnesium sulfate 0.7 g / l

Manganese sulfate 0.02 g / l

Iron (II) sulfate 0.02 g / l

Sodium chloride 1 g / l

D, L-methionine hydantoin 3 g / l

Yeast extract 2 g / l

Precultures

A first preculture is contained in a flask of 250 cm

3
tend 50 cm of the milieu, produced during 24 hours »A second preculture, inoculated from the previous flask, is also produced for 24 hours in a 2 l fermentation vessel containing 1 liter of culture medium. Producer culture

The contents of the 2-1 fermentation vessel are used to inoculate a 7.5 liter fermentation vessel containing 5 liters of medium. The culture is developed for 24 hours at 37 ° C. with aeration with sterile air at a rate of 5 l / minute and with stirring with a turbine at 500 revolutions / minute. The biomass obtained under these conditions is 3.8 g dry extract per 1 culture medium. Enzymatic reaction

After the cultivation is finished, the microbial bodies become isolated from the medium by centrifugation and suspended in 250 cm of distilled water.

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10 g of DL-phenylglycine hydantoin are suspended in 400 cm of distilled water Water and adjusts the pH to 7.5 by adding an η 10 sodium hydroxide solution. One gives 50 cm of the above described cell suspension and the reaction volume is

3
filled up to 500 cm by adding water. It is then left under a nitrogen atmosphere at 37 ° C., the pH being kept at 7.5 for 17 hours.

After 17 hours of reaction, the reaction mixture is centrifuged clarified, concentrated by evaporation to 1/5 of its volume and adjusted to pH by adding concentrated hydrochloric acid acidified. The precipitate formed is separated off by filtration and dried. Chromatographic analysis shows that it is phenylglycine hydantoin .. with the following character i stika:

% N = 13.9% (theory: 14.4%)

[a] _D s -137 ° (c = 1; 1 % ammonia)

Yield of D-hydantoic acid obtained: 6.3% based on the used hydantoin.

The mother liquors are concentrated again and then by addition brought to pH 5 by sodium hydroxide ^ the precipitate D-phenylglycine is separated off by filtration, washed with water and ethyl alcohol and then dried. You get so 7,6oD-phenylglycine (yield: 88%) with the following characteristics:

■% N = 9.2 (theory: 9.3)

[α] ρ ° = -448 ° (c - 1; 5N hydrochloric acid)

Example 6 '

The cultivation of the Pseudomonas HM-40 is carried out under the conditions of Example 5 causes. After centrifugation, the microbial bodies suspended in 0.1 M phosphate buffer at pH 7.8; this suspension is used as a hydrolysis catalyst for various hydranties; are the reaction conditions the following:

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The initial concentration of hydantoin is 20 g / l in O ₁ phosphate buffer at pH 7.8. ■

The biomass used is 6.5 g (calculated on dry extract) per 1 reaction mixture.
The reaction temperature is 37 C.
The reaction time is 5 hours.

The amino acid formed is then determined in the reaction mixture and "mai derives from it the enzymatic activity of the cell extract away.

The activities measured in this way are shown in the following table of amino acids formed in JUM per mg of enzymatic dry extract and per hour of reaction.
The results are summarized in the following table:

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Faatur of the substrate

Formula activity / M / mg / hour

Hydantoin from DL-vthionine

CH, -S-CH_-CHp-CH CO '- NH NH - CO

2. *

Hydantoin from DL- ^ nenylalania

CO NH

NH - CO

. 2.0:

^CH

Hydantoin from DL-Leucine

5 \ ^ CO-NH ^ NH -CO

Hydantoin from DL- ^ lin

CH 1 -CH ₀ -CH ₀ -CH 5 Z CO -NH

"NH - CO

1.0

Hydantoin from Di ^ Tryptopban

CH ₂ -CH,

, CO NH

NH - CO

0 * 7

Hydantoin from Dli-Phssylgly c i h

CO -NH

I NH - CO

0.6

Hydantoin 'Y ^on PL · para-hydroxyphenylglycine

CO-NH

i - CO

0.6

Racemic cyanopropyl hydahtoin CO - NH NH - CO

3.5

Hydantoin _from DL-ra et a-hydroxy phenylglycine

CO - NH

CH 'NH - CO

0.7

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Example 7

A cell suspension of Pseudomonas HM-40 in phosphate buffer was obtained as described in Example 6. This suspension contains 6.5 g of dry extract in 100 cm; before use the cells are crushed by ultrasound under the following conditions:

- Apparatus: PONS, equipped with a TC4C probe

- Frequency of the ultrasound: 20 kHz

- Treated volume: 15 cm.

- Temperature kept at around OC

- Crushing time: 10 minutes' - _.

The suspension thus treated is used to hydrolyze the hydantoins of methionine, phenylalanine and phenylglycine to catalyze under the conditions described in Example 6. The activities measured are as follows:

Substrate Activity
(/ iM formed amino
acid e / S tund e / mg enz y-
Niatal extract Hydantoin from DL-methionine 1.7 Hydantoin from DL-Phenylaianin 1.0 Hydantoin from DL-phenylglycine 0.8

Example 8

A cell suspension of Pseudomonas sp, HM-40 in water, obtained as described in Example 5 and containing 7.3 g of dry extract in 100 cm was lyophilized before use under the following conditions.

120 ctn of suspension are distributed in 4 flasks of 500 cm, frozen and for 16 hours under reduced pressure

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301858A

_2 -, ■■> ■ '■: ■ - "** -'

(0.5.10 mm Hg). Man- gets hurt £ _ri 8? 8th ¹ g of lyophilized enzymatic powder, which is used under the conditions described in.
The activities measured are as follows:

Substrate activity
{/ M formed amino
acid / hour / mg dryness
substance Hydantoin from 'DL-methionine
• •. 2.4 Hydantoin from DL-phenylglycine 0.4 Hydantoin from DL-phenylalanine 1.5

In summary, a process for the preparation of D-α-amino acids by hydrolysis of a hydantoin substituted in the 5-position in the presence of an enzyme obtained by culturing the microorganism Pseudomonas sp, HM-40 (CBS 259.79).

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ORIGINAL INSPECTiQ

Claims (11)

Dr. F. Zumstein is-. - Dr. E. Assmann - Dr. R. Koenigsberger Dipl.-Phys. R. Holzbauer - Dipl.-Irg. F Klingsefsen - Dr. F. Zumstein jun. 80OO Munich 2 · BräuhausstraQe 4 · Telephone collection no. 225341 Telegrams Zumpat Telex 529979 XT 504
10 / We A.E ο C. Societe "De Chimie Organique Et Biolögique, Commentry / Fr. P_a_t_e_n_t_a_n_s_p__r_ü_c_h_e_ \ 1 · Process for the production of D-ÖC-amino acids, characterized / that one is substituted in the 5-position. Hydantoin, hydrolyzed in the presence of an enzyme produced by culturing a Microorganism with the designation Pseudomonas sp, HM-40 (CBS 259.79) was obtained and that the obtained D - <? (- amino acid isolated. 2. The method according to claim 1, characterized in that the Enzyme by cultivating the microorganism Pseudomonas sp, HM-40 little one in a medium containing Y an assimilable carbon source and an assimilable nitrogen source. 3. The method according to claim 2, characterized in that the Milieu also contains a racemic hydantoin. 4. The method according to claim 1, characterized in that the hydantoin substituted in the 5-position is brought into contact with the enzyme in the culture medium of the microorganism. 5. The method according to claim 1, characterized in that the hydantoin substituted in the 5-position is brought into contact with an enzymatic composition. 030048/0785 6. The method according to claim 1, characterized in that the L-hydantoin remaining in the medium becomes D, L-hydantoin under the action of the microorganism Pseudomonas sp, HM-40 is racemized. 7. The method according to claim 1, characterized in that the enzyme also hydrolyzes the hydantoin acid, which is formed as an intermediate in the course of the hydrolysis of the hydantoin substituted in the 5-position, to the corresponding O-Oi- amino acid. 8. The method according to claim 1, characterized in that the obtained amino acid is D-phenylalanine. 9. The method according to claim 1, characterized in that the amino acid obtained is D-methionine. 10. The method according to claim 1, characterized in that the obtained amino acid is D-phenylglycine. 11. The method according to claim 1, characterized in that - ¹ Xe obtained amino acid is Dp-hydroxyphenylglycine. 030048/0785