WO2003044206A2

WO2003044206A2 - Process for the preparation of an enantiomerically enriched a-amino acid

Info

Publication number: WO2003044206A2
Application number: PCT/NL2002/000758
Authority: WO
Inventors: Joannes Gerardus Theodorus Kierkels; Wilhelmus Hubertus Joseph Boesten
Original assignee: Dsm Ip Assets B.V.
Priority date: 2001-11-23
Filing date: 2002-11-22
Publication date: 2003-05-30
Also published as: EP1446492A2; AU2002347652A8; AU2002347652A1; WO2003044206A3; JP2005509439A; CN1592789A; NL1019416C2

Abstract

The invention relates to a process for the preparation of an enantiomerically enriched D-α-amino acid in a reaction carried out in a one pot reaction in the presence of a hydantoinase and a D-carbamoylase, in which an N-carbamoyl-L-α-amino acid is converted into the corresponding D-α-amino acid and in which the N-carbamoyl-L-α-amino acid may be prepared from the corresponding L-α-amino acid. The enzymes used preferably originate from Agrobacterium radiobacter or Arthrobacter aurescens . Examples of very suitable α-amino acids are leucine, methionine, cysteine, threonine and phenylalanine.

Description

PROCESS FOR THE PREPARATION OF AN ENANTIOMERICALLY ENRICHED A-AMINO ACID

The invention relates to a process for the preparation of an enantiomerically enriched D-α-amino acid in a one pot reaction in the presence of a hydantoinase and a D-carbamoylase. D-α-amino acids can be prepared enzymatically from a mixture of the corresponding D- and L-5-substituted hydantoin. Such a process is known from EP 0 775 748. In said process a mixture of D- and L-5-substituted hydantoin is enantioselectively converted into the corresponding D-α-amino acid in a one pot reaction in the presence of a D-hydantoinase and a D-carbamoylase. Enantiomerically enriched α-amino acids are important building blocks for biologically active preparations such as, for example, β-lactam antibiotics, peptide hormones and pesticides.

A drawback of this process is that the starting material for the preparation of the enantiomerically enriched α-amino acid, the corresponding 5- substituted hydantoin, is usually difficult to handle due to the poor water solubility of most 5-substituted hydantoins.

The invention aims to provide a simple process for the preparation of a D-α-amino acid in which use can be made of a starting material that it easier to handle than a 5-substituted hydantoin. According to the invention this is achieved through conversion of an N-carbamoyl-L-α-amino acid into the corresponding D-α- amino acid.

It has been found that the conversion of N-carbamoyl-L-α-amino acids into the corresponding D-α-amino acids proceeds relatively rapid and with a high yield and that the enantioselectivity of the hydantoinase is not critical in the process of the invention. This is surprising, because it is disclosed by Syldatk et al (1999), Appl. Microbiol. Biotechnol., vol. 51 , p 293-309, that a non-selective hydantoinase step would limit the space/time yield, which is a measure for the amount of product prepared per reaction volume per time unit. Since N-carbamoyl-L-α-amino acids can easily be prepared from the corresponding L-α-amino acids the invention also provides a process by means of which D-α-amino acids can be prepared rapidly and in a high yield from the corresponding L-α-amino acids. L-α-amino acids are usually cheap and widely available. In addition, the process according to the invention is eminently suited for preparing D-α-amino acids from L-α-amino acids which, when they are chemically converted into the corresponding hydantoins, yield less stable hydantoins (as a result of which a great deal of by-product formation takes place on chemical conversion of these L-α-amino acids into the corresponding hydantoins). Examples of such α-amino acids are serine, asparagine, glutamine, lysine, threonine and cysteine.

The term carbamoylase is known from the literature. The expert understands carbamoylase to be an enzyme with carbamoylase activity, that is, the ability to catalyze the conversion of N-carbamoyl-α-amino acid into the corresponding α-amino acid. The expert understands D-carbamoylase to be an enzyme that preferably catalyzes the conversion of N-carbamoyl-D-α-amino acid into the corresponding D-α-amino acid. The D-carbamoylase involved in the process for the preparation of a D-α-amino acid according to the invention preferably has a selectivity of at least 90%, more in particular at least 95%, even more in particular at least 98% and most particularly at least 99%. In the context of the invention, a 90% selectivity of the D-carbamoylase is understood to mean that the D-carbamoylase converts the N- carbamoyl-(D,L)-α-amino acid into 90% D-α-amino acid and 10% L-α-amino acid at an overall conversion of 50%, which corresponds to an enantiomeric excess (e.e.) of 80% of the D-α-amino acid at 50% conversion.

The term hydantoinase is known from the literature. The expert understands hydantoinase to be an enzyme with hydantoinase activity, that is, the ability to catalyze the reaction of 5-substituted hydantoin to form the corresponding N- carbamoyl-α-amino acid. The hydantoinase activity that is required for the process according to the invention may be provided by one or by more hydantoinases. The enantioselectivity of the hydantoinase(s) involved in the conversion of N-carbamoyl-L- α-amino acid into the corresponding D-α-amino acid is not very critical. For example, a D-hydantoinase, an L-hydantoinase or a non enantioselective hydantoinase may be employed as well as combinations thereof. The expert understands a D-hydantoinase to be a hydantoinase that preferably catalyzes the reversible hydrolysis of a 5- substituted D-hydantoin into the corresponding N-carbamoyl-D-α-amino acid. It is surprising that a D-hydantoinase can be employed in the process of the invention as it is generally assumed that D-hydantoinases are strictly D-selective (Drauz, K., Waldmann, H, Enzyme catalysis in organic synthesis - a comprehensive handbook, second, completely revised and enlarged edition, volume III, Wiley-VCH Verlag GmbH, Weinheim 2002) and therefore the person skilled in the art would not expect that N- carbamoyl-L-α-amino acid can be converted by a D-hydantoinase. Preferably, the conversion of N-carbamoyl-L-α-amino acid into the corresponding D-α-amino acid according to the invention is carried out in the presence of a hydantoin racemase. Hydantoin racemases are known from the literature (for example from WO 01/23582 A1). The expert understands a hydantoin racemase to be an enzyme with hydantoin racemase activity, that is, the ability to catalyze the racemization of the L-5-substituted hydantoin (or D-5 substituted hydantoin).

In the process according to the invention it is of course also possible to use a random mixture of the enantiomers of the N-carbamoyl-α-amino acid in the preparation of the corresponding D-α-amino acid. The temperature and the pH are not very critical in the conversion of

N-carbamoyl-L-α-amino acid into the corresponding D-α-amino acid according to the invention. Preferably, however, the conversion is carried out at a pH between 5 and 10. In particular, the conversion is carried out at a pH between 6.0 and 7.5. The temperature of the conversion of N-carbamoyl-L-α-amino acid into the corresponding D-α-amino acid is preferably chosen between 0 and 80°C, in particular between 10 and 50°C, more in particular between 35 and 45°C. Preferably the reaction conditions in the process according to the invention are chosen such that the formed enantiomerically enriched D-α-amino acid crystallizes out, facilitating the recovery of the enantiomerically enriched D-α-amino acid. Preferably, the process for converting N-carbamoyl-L-α-amino acid into the corresponding D-α-amino acid is used in the preparation of a D-α-amino acid from the corresponding L-α-amino acid. Use is made of the same process as described above, to which an extra step is added, namely the preparation of N-carbamoyl-L-α- amino acid from the corresponding L-α-amino acid. The preparation of N-carbamoyl-α- amino acid from the corresponding α-amino acid can for example be effected chemically by contacting the α-amino acid with, for example, MOCN, where M represents alkali metal, preferably potassium or sodium in the presence of water. The temperature used is not critical: the reaction preferably takes place at temperatures between about -5 and 100°C, preferably at temperatures between 0 and 80^CC; the pH of the chemical reaction is preferably chosen between 8 and 11 , more in particular between 9 and 10.

Preferably, the preparation of D-α-amino acid from the corresponding L-α-amino acid via the corresponding N-carbamoyl-L-α-amino acid is carried out in a single vessel. This has the advantage that no intermediate recovery of the N- carbamoyl-α-amino acid is needed. The invention is in no way limited by the form in which the enzymes are used for the present invention. The different enzymes may each independently be present in a certain form. The enzymes may for example be present as a crude enzyme solution or as purified enzyme. The enzymes may for example also be present in (permeabilized and/or immobilized) cells that naturally or through genetic modification possess the desired enzymes/enzyme activity, or in a lysate of cells with such activity. The enzymes can for example also be used in immobilized form or in chemically modified form. It will be clear to the average person skilled in the art that use can also be made of mutants of naturally occurring (wild type) enzymes with hydantoinase and/or enantioselective carbamoylase and/or hydantoin racemase activity in the process according to the invention. Mutants of wild-type enzymes can for example be made by modifying the DNA encoding the wild type enzymes using the mutagenesis techniques known to the person skilled in the art (random mutagenesis, site-directed mutagenesis, directed evolution, gene shuffling, etc.) so that the DNA encodes an enzyme that differs by at least one amino acid from the wild type enzyme and by effecting the expression of the thus modified DNA in a suitable (host) cell.

Suitable hydantoinases and/or D-carbamoylases and/or hydantoin racemases for the process according to the present invention can be present in, or originate from, for example the microorganisms that usually also supply the enzymes for the hydantoinase-carbamoylase processes, for example the microorganisms of the following genera: Pseudomonas, preferably Pseudomonas sp., more in particular Pseudomonas sp. FERM BP 1900, Hansenula, Agrobacterium, preferably Agrobacterium sp. or Agrobacterium radiobacter. more in particular Agrobacterium sp. IP 1-671 or Agrobacterium radiobacter NRRLB 11291 , Aerobacter, preferably Aerobacter cloacae, more in particular Aerobacter cloacae IAM 1221 , Aeromonas, Bacillus, preferably Bacillus macroides, more in particular Bacillus macroides ATCC 12905. Brevibacterium. Flavobacterium, Serratia, Micrococcus, Arthrobacter, preferably Arthrobacter aurescens. more in particular Arthrobacter aurescens DSM 3747 Nocardia, Corvnebacterium, Mvcobacterium. Actinoplanes, Streptomvces or Paracoccus. The optimum choice of hydantoinases and/or D-carbamoylases and/or hydantoin racemases may vary dependent on the specific substrate/desired D-α-amino acid and can easily be determined by the person skilled in the art. Most preferably, one or more of the enzymes used in the conversion of N-carbamoyl-L-α-amino acid into the corresponding D-α-amino acid according to the invention originate from Agrobacterium radiobacter or Arthrobacter aurescens. The invention can be used in the preparation of both proteinogenic and non-proteinogenic D-enantiomers of α-amino acids having the formula R-CH(NH₂)- COOH, where R represents an amino acid restgroup, for example a substituted or non- substituted alkyl group with for example 1-20 C atoms or a substituted or a non- substituted (hetero)aryl group with for example 1-20 C atoms. Examples of α-amino acids having the formula R-CH(NH₂)-COOH are: alanine, valine, leucine, isoleucine, serine, threonine, methionine, cysteine, asparagine, glutamine, tyrosine, tryptophan, aspartic acid, glutamic acid, histidine, lysine, arginine, citrulline, phenylalanine, 3- fluorophenylalanine. Preferably the invention is used in the preparation of the D- enantiomers of methionine, leucine, cysteine, threonine or phenylalanine.

The way in which the preparation of a D-α-amino acid is assumed to work in a one pot reaction in the presence of a hydantoinase and a D-carbamoylase starting from N-carbamoyl-L-α-amino acid/L-α-amino acid is illustrated in Figure 1. Figure 1 shows the conversion of L-α-amino acid to D-α-amino acid; the compounds 1, 2 and 3 represent compounds with the L-configuration and the compounds 4, 5, and 6 compounds with the D-configuration. R represents an amino acid restgroup as defined above.

The conversion of N-carbamoyl-L-α-amino acid from the corresponding L-α-amino acid is shown in the reaction from 1 to 2. A possible route for the conversion of N-carbamoyl-L-α-amino acid into the corresponding D-α-amino acid is shown in the reactions from 2 to 6. According to figure 1 , the N-carbamoyl-L-α-amino acid (2) is converted into the corresponding L-5-substituted hydantoin (3) in the presence of a hydantoinase and a D-carbamoylase. The L-5-substituted hydantoin is then racemized in situ. Racemization of L-5-substituted hydantoin may occur spontaneously, but can also be effected for example by means of heating or by using a hydantoin racemase. D-5 substituted hydantoin present in the mixture of enantiomers of 5-substituted hydantoin (4) formed, is subsequently converted into the corresponding N-carbamoyl-D-α-amino acid (5), after which the N-carbamoyl-D-α-amino acid is converted to form the corresponding D-α-amino acid (6), use being made of the presence of a hydantoinase and a D-carbamoylase.

The invention is further explained below with reference to the following examples, without however being limited to these. Examples

Example I

17.4 g (0.10 mol) N-carbamoyl-L-leucine was added to 100 ml water, after which the pH was adjusted to 7.2 by means of 10 N NaOH. The volume was then made up to 150 ml with water. This mixture was heated to 40°C. Subsequently, under a nitrogen atmosphere, the enzymatic reaction was started by the addition of 18 g of an Agrobacterium radiobacter cell suspension. The pH of the reaction mixture was kept constant at pH 7.2 by means of titration with 3 mol/l H₃PO₄. The composition of the reaction mixture was monitored by means of HPLC analysis. After a reaction time of 43 hours a D-leucine concentration of 67 g/l was measured, which corresponds to a conversion of > 95% on the basis of the N-carbamoyl-L-leucine. The enantiomeric excess of the formed D-leucine was > 98%.

Example II

A slurry of 17.9 g (0.093 mol) N-carbamoyl-D,L-methionine in 115 ml water was transferred to a double-walled glass reactor. The mixture was heated to 40°C and its pH was adjusted to 7.2 by means of 10 N NaOH, after which the mixture was made up to a volume of 170 ml. Then 12 g of an Agrobacterium radiobacter cell suspension was added, after which the pH of the reaction mixture was kept constant at pH 7.2 by addition of 3 mol/l H₃PO . The reaction was carried out under a nitrogen atmosphere. The composition of the reaction mixture was analyzed by means of HPLC analysis. After a reaction time of about 29 hours a conversion of > 88% on the basis of N-carbamoyl-D,L-methionine was reached. The enantiomeric excess of the formed D- methionine was > 96%.

Example III

24.0 g (0.12 mol) N-carbamoyl-L-leucine was added to 100 ml water, after which the pH was adjusted to 7.2 by means of 10 N NaOH. The volume was then made up to 170 ml with water. This mixture was heated to 40°C. Subsequently, under a nitrogen atmosphere, the enzymatic reaction was started by the addition of 14.2 g of an Agrobacterium radiobacter cell suspension. The pH of the reaction mixture was kept constant at pH 7.2 by means of titration with 3 mol/l H₃PO₄. The composition of the reaction mixture was monitored by means of HPLC analysis. After a reaction time of 44 hours the reaction was stopped. The results of this experiment are presented in Figure 2, the conversion (c) in % being given as a function of the time in hours (t(h)). Fig. 2 shows that after over 40 reaction hours a conversion of about 65% on the basis of N- carbamoyl-L-methionine (S) is reached. The enantiomeric excess of the formed D- methionine (P) was > 97.8%.

Example IV

66.0 g (0.50 mol) L-leucine and 44.0 g (0.525 mol) KOCN was added to 300 ml water. The mixture was stirred overnight at room temperature under a nitrogen atmosphere. By means of HPLC the conversion was determined to be >99%. The volume of this solution was 350 ml. After cooling of the reaction mixture, the pH of 70 ml of this mixture was adjusted to 7.2 by means concentrated phosphoric acid, after which heating to 40°C took place under a nitrogen atmosphere. The reaction mixture was made up to a volume of 150 ml. Then 19 g of an Agrobacterium radiobacter cell suspension was added, after which the pH of the reaction mixture was kept constant at pH 7.2 by addition of 3 mol/l H₃PO₄.

The composition of the reaction mixture was analyzed by means of HPLC analysis. After a reaction time of 42 hours a D-leucine concentration of 67 g/l was measured, which corresponds to a conversion of > 95% on the basis of the N- carbamoyl-L-leucine. The enantiomeric excess of the formed D-leucine was > 98%.

Example V: Conversion of N-carbamoyl-L-cysteine and N-carbamoyl-L-threonine

To 100 mL water, N-carbamoyl-L-amino acid (in case of N- carbamoyl-L-cysteine 18.4 g (80 mmol) and in case of N-carbamoyl-L-threonine 17.8g (110 mmol)) was added. The pH of the mixture was brought to 7.2 using 5 mol/L NaOH, after which the volume was adjusted to 150 mL. The mixture was transferred to a thermostated reaction vessel and the temperature was adjusted to 40°C. The enzymatic reaction was started by adding 22 g of an Agrobacterium radiobacter celsuspension. The pH was kept constant at pH 7.2 by means of pH-stat titration using 3 mol/L H₃PO₄. Periodically samples were taken and analysed by means of HPLC. In was shown that in all cases the formed D-amino acid had an enantiomeric excess of >98%. Example VI: Conversion of N-carbamoyl-L-phenylalanine

Cloning of the L-hvdantoinase from Arthrobacter aurescens DSM 3747 into E. coli General procedures Standard molecular cloning techniques such as plasmid DNA isolation, gel electrophoresis, enzymatic restriction modification of nucleic acids, E. coli transformation etc. were performed as described by Sambrook et al., 1989, "Molecular Cloning: a laboratory manual", Cold spring Harbor Laboratories, Cold Spring Harbor, NY or according to the supplier's manual. Synthetic oligodeoxynucleotides were obtained from Invitrogen LifeTechnologies (Paisley, Scotland, UK).

Example VI A. Construction of plasmid pET101/D-TOPOhyuH

Isolation of the gDNA of Arthrobacter aurescens DSM 3747 Strain Arthrobacter aurescens DSM 3747 was used to isolate the hydantoinase hyuH using the pET101 Directional TOPO Expression Kit from Invitrogen.

The genomic DNA from A. aurescens was isolated by the following protocol. A. aurescens DSM 3747 was cultivated at 28°C in K2 medium (4 g bacto peptone, 4 g yeast extract, 7.5 g glycin, 2 g KH₂PO , 3.62 g K₂HPO₄, in H₂O to 1 liter and adjusted to pH 7.0). 10 ml from a preculture was added to 75 ml K2 medium and incubated at 28°C. After 3 hours of incubation, 75 μl lysozyme (100 mg/ml) and 1.5 ml carbenicillin (50 mg/ml) were added and incubation was continued for another hour at 28°C. The cells were isolated by centrifugation and resuspended in 2.5 ml solution consisting of 125μl 1 M Tris-HCI, pH 8, 250 μl 0.5 M EDTA pH 8 and 2.125 ml Milli-Q. Then, 50 μl lysozyme (100 mg/ml) and 20 μl proteinase K (20 mg/ml) were added. This suspension was incubated at 37°C for 30 minutes. After adding 3 ml of Nuclei Lysis Solution of Promega (Leiden, The Netherlands), the solution was incubated again for 15 minutes at 80°C. To the solution, 5 μl RNase A (100 mg/ml, Promega) was added which was followed by incubation for 30 minutes at 37°C. After adding 1 ml of Protein Precipitation Solution of Promega, the solution was vortexed for 20 seconds and transferred to ice for 15 minutes. After centrifugation at 4,500 rpm at 4°C for 10 minutes, the supernatant was transferred to 0.1 volume NaAc (3 M, pH 5) and 2 volumes of absolute ethanol. The DNA was fished out and redissolved in 1xTE. The purity and concentration was determined by spectrophotometric assay and agarose gel electrophoresis. The gDNA was used for PCR.

Construction of plasmid pET101/D-TOPOhvuH

A 1377 bp fragment comprising the open reading frame (ORF) for hydantoinase HyuH was amplified by PCR from the chromosomal DNA from Arthrobacter aurescens DSM 3747 (nucleotides 4651-6027 of accession number AF146701) using the following primers: ΛyuH-forward:

5'-CACCATGTTTGACGTAATAGTTAAGAACTGCCGTATGG-3' [SEQ. ID: No.1], (overhang for directional cloning into the pET101/D-TOPO vector underlined), and tψuH-reverse:

5'-TCACTTCGACGCCTCGTAGTGGTGACG-3' [SEQ. ID: No.2]

The start-codon was changed from GTG into ATG for better expression in E. coli. The Platinum Pfx DNA Polymerase of Invitrogen was used to amplify the ORF.

Correct size of the amplified fragment (1381 bp) was confirmed by agarose gel electrophoresis. A total of 4 independent PCR's were executed and the resulting product of these reactions was pooled and used for directional TOPO cloning according to the manual of Invitrogen.

After plasmid isolation from several randomly chosen transformants and digestion with restriction enzyme H/ndlll, 5 plasmids showed the correct restriction pattern. These were named pET101/D-TOPOHyuH and used in further experiments.

VI B. Expression of hydantoinase gene hvuH from A. aurescens

According to the manual of Invitrogen, the 5 pET101/D-TOPOHyuH plasmids were transformed to E. coli BL21 STAR (DE3) One Shot cells (Invitrogen). The transformants were cultivated at 28°C in LB medium containing 100 mg/l carbenicillin and induced with 0.5 mM IPTG (final concentration) according to Invitrogen's protocol. After overnight incubation, the cells were harvested by centrifugation and washed with 0.2 M Tris-HCI pH 7.

Crude extracts were prepared by sonification in 0.2 M Tris-HCI buffer pH 7 with 1 mM MnCI₂ (10 ml buffer per 1 gram of wet weight cells). These samples were centrifuged at 20,000 rpm and 4°C for 15 minutes and the supernatants (cell free extracts) were stored at -20°C and used in the activity assay.

VI C. Analysis of hydantoinase HvuH from A. aurescens Activity towards phenylalanine hydantoin and tryptophan hydantoin

The assay mixture of 10 ml contained 0.1 M Tris-HCI buffer pH 8.5 with a) 4 mM phenylalanine hydantoin, or b) 1.8 mM tryptophan hydantoin.

The assay was performed at 37°C and started by the addition of 0.75 ml cell free extract. Several samples of 0.5 ml were taken in time and the reaction was stopped by adding 5 μl 85% phosphoric acid to these samples. Next they were analyzed by HPLC and UV detection at a wavelength of 220 nm. A Nucleosil-120-5 C18 column (50x4 mm, 5μ from Macherey-Nagel, Dϋren, Germany) was used. The column was eluted with eluent A (50mM H₃PO₄ pH 2.7) and eluent B (50 v/v% eluent A and 50 v/v% acetonitril). Gradient: 0-1.5 min, 0 % B; 1.5-6.5 min, 0 % to 50 % B; 6.5- 7.0 min, 50 % B; 7.0-7.1 min, 50 % to 0 % B; 7.1-12 min, 0 % B. The flow was 1.0 ml/min, the column temperature was set at 40°C and the injection volume was 2 μl.

Λ/-carbamoyl-phenylalanine or Λ/-carbamoyl-tryptophan was produced within 1 hour with cell free extracts derived from all 5 clones of £ co/ pET101 /D-TOPOhyuH tested.

Use of Agrobacterium radiobacter and the cell free extracts from £ co///pET101/D- TOPOhyuH L-hvdantoinase clone in the preparation of D-phenyl-alanine

A 45 mL solution containing 1.8 g (8.6 mmol) N-carbamoyl-L- phenylalanine was brought to pH 7.2 using 5 mol/L NaOH. The reaction was thermostated at 40°C. The enzymatic reaction was started by adding 2 mL of an Agrobacterium radiobacter celsuspension and 2 mL of a cellfree extract from the £ co/ ET101/D-TOPOhyuH L-hydantoinase clone, which contained the L- hydantoinase from Arthrobacter aurescens DSM 3747. The pH of the reaction was kept at pH 7.2. Periodically, samples were taken and analysed by means of HPLC in order to follow the conversion and the optical purity of the formed D-amino acid. It was shown that after 4 hours of incubation at 40°C, 46% of the N-carbamoyl-L-phenylalanine was converted to D-phenylalanine. The enantiomeric excess of the formed D-phenylalanine, was > 98%. SEQUENCE LISTING

<110> DSM NN.

<120> Process for the preparation of an enantiomerically enriched a-amino acid

<130> WO 20383

<160> 2

<170> Patentln version 3.1 <210> 1

<211> 38

<212> DΝA

<213> Artificial

<400> 1 caccatgttt gacgtaatag ttaagaactg ccgtatgg 38

<210> 2 <211> 27 <212> DΝA <213> Artificial <400> 2 tcacttcgac gcctcgtagt ggtgacg 27

Claims

1. Process for the preparation of an enantiomerically enriched D-α-amino acid in a one pot reaction in the presence of a hydantoinase and a D-carbamoylase, characterized in that an N-carbamoyl-L-α-amino acid is converted into the corresponding D-α-amino acid.

2. Process according to claim 1, characterized in that the process takes place in the presence of a D-hydantoinase.

3. Process according to claim 1 or claim 2, characterized in that the process takes place in the presence of a D-hydantoinase and an L-hydantoinase.

4. Process according to any one of claims 1-3, characterized in that a mixture of the enantiomers of N-carbamoyl-amino acid is converted into the corresponding D-α-amino acid.

5. Process according to any one of claims 1-4, characterized in that the N- carbamoyl-α-amino acid is prepared from the corresponding α-amino acid.

6. Process according to claim 5, characterized in that the preparation of D-α- amino acid from the corresponding L-α-amino acid is carried out in a single vessel.

7. Process according to any one of claims 1-6, characterized in that the D-carbamoylase has a selectivity of at least 99%.

8. Process according to any one of claims 1-7, characterized in that the conversion takes place in the presence of a hydantoin racemase.

9. Process according to any one of claims 1-8, characterized in that the conversion is carried out at a pH between 6.0 and 7.5. 10. Process according to any one of claims 1-9, characterized in that the conversion is carried out at a temperature between 35 and 45°C. 11. Process according to any one of claims 1-10, characterized in that the reaction conditions are chosen such that the formed enantiomerically enriched D-α- amino acid crystallizes out. 12. Process according to any one of claims 1-11 , characterized in that for the conversion use is made of one or more enzymes originating from Agrobacterium radiobacter or Arthrobacter aurescens.

3. Process according to any one of claims 1-12, characterized in that the

D-α-amino acid is D-leucine, D-methionine, D-cysteine, D-threonine or D- phenylalanine.