The present invention also provides a reaction system that operates in such a way.

Enantiopure D-amino acids and in particular sterically demanding representatives thereof are still rewarding targets for organic synthesis, but they can be useful intermediates for the production of bioactive molecules or catalysts.

A number of chemical and biocatalytic racemate resolutions have been developed to date for the production of enantiopure sterically demanding D-amino acids.

In DE1952911 they are produced by enzymatic racemate resolution using hydantoinases. In this case, however, a second, subsequent chemical reaction is needed to eliminate the N-carbamoyl intermediate that is formed. This increases the total number of reaction steps starting from the racemic amino acid to three, starting with the conversion to the racemic hydantoin, the formation of the optically active N-carbamoyl-D-amino acid and the N-decarbamoylation as already mentioned.

Another method for producing sterically demanding D-amino acids is described by Laumen et al. , using the example of D-tert-leucine [K. Laumen et al., Biosci. Biotechnol.

Biochem. 2001, 65, 1977-1980.]. This involves an enzymatic ester hydrolysis of racemic N-acetyl-tert-leucine chloroethyl ester. The problem with this route, in addition to the potential formation of amides as undesirable

secondary products, which is a general occurrence with ester hydrolysis reactions, is again the total number of process steps, consisting of a (two-stage) derivatisation of the racemic amino acid, followed by enzymatic racemate resolution and elimination of the N-acetyl group.

A reaction system based on the coupled reaction of a leucine dehydrogenase (LeuDH), NAD (H) and an NADH oxidase, which oxidises L-amino acids, was developed by Kiba et al. for analytical purposes. The blood values of branched-chain L-amino acids are measured, and the H202 that is formed is analysed by luminol fluorescence (Kiba et al. Analyt. Chim.

Acta 1998,375, 65-70; Kiba et al. J. Chrom. 1996, 724, 354-7).

The object of the present invention was therefore to provide another process for the production of enantiomer- concentrated, in particular sterically demanding D-amino acids, which helps to overcome the disadvantages of the prior art processes. In particular, when used on an industrial scale the process according to the invention should offer advantages from an economic and ecological perspective, particularly also with regard to the number of synthesis steps.

These and other objects not described in any more detail, but obvious to the person skilled in the art from the prior art, are achieved by a process provided by claim 1. Claims 2 to 8 relate to preferred embodiments of the process according to the invention. Claim 9 focuses on a reaction system. Claim 10 protects a whole-cell catalyst operating according to the invention.

Surprisingly, the stated object is advantageously achieved in that, in a process for the production of D-amino acids, a mixture of D-and L-amino acids is reacted with an enzymatic reaction system displaying a leucine dehydrogenase (LeuDH), NAD (H) or NADP (H) and an agent for

oxidising NADH or NADPH selected from the group containing an NADH oxidase or an electrochemical arrangement.

With the process according to the invention, even sterically demanding D-amino acids can be produced in high enantiopurities in a single step, starting from the readily available racemic mixture. The reaction is illustrated by way of example by the diagram below.

The abbreviation NOX stands for NADH oxidase.

Alternatively, in place of this enzymatic oxidation illustrated in the diagram above, an electrochemical oxidation of the cofactor NADH can be performed, with formation of NAD+.

The fact that the process according to the invention is by no means obvious derives from the surprising fact that-as mentioned in comparative example 1-a similarly acting PheDH from Rhodococcus sp. cannot be used in the present process due to inhibition effects. Inhibition effects can be caused in particular by the D-amino acid component present in the substrate mixture, in a proportion of around 50% in the case of a racemate. In contrast to the biocatalytic oxidation of pure L-amino acids, the use of racemates would thus have been expected to result in a considerable reduction in the reaction rate or an incomplete formation of D-amino acid.

No less advantageous is the fact that no additional auxiliary reagents, possibly forming secondary products and

having to be painstakingly isolated again, are used in the synthesis. This also helps to save on material costs.

The agents that can be used for the oxidation of NADH or NADPH are known to the person skilled in the art. They are in particular the NADH oxidases. Particularly suitable representatives are described in EP1285962 and in the literature cited therein (see also below). Electrochemical processes can also be used here. The suitability in principle of electrochemical oxidation to regenerate NAD+ from NADH is described for example-for oxidation reactions with alcohols-in Kelly, R. M.; Kirwan, D. J., Biotechnol. Bioeng. 19 (1977) 1215-1218 and Schroder, I.; Steckhan, E.; Liese, A. Journal of Electronanalytical Chemistry 541 (2003) 109-115. An overview of electrochemical studies in this regard can be found in Gorton, L. Journal of the Chemical Society, Faraday Transactions 1: Physical Chemistry in Condensed Phases 82 (1986) 1245-1258.

The use of especially sterically demanding amino acids is advantageous. Sterically demanding in the present case denotes amino acids having a branched radical in the a position to the carboxyl group. The compounds mentioned in EP692538 in this respect can be regarded as being sterically demanding, for example. They are in particular amino acids selected from the group comprising tert.- leucine, neopentyl glycine, adamantyl glycine.

Any LeuDH that is familiar to the person skilled in the art and can be used for the purpose under consideration can be used in principle as leucine dehydrogenase. LeuDHs can be found for example in A. Bommarius in: Enzyme Catalysis in Organic Synthesis (Editors: K. Drauz, H. Waldmann), Volume III, Wiley-VCH, Weinheim, 2002, chapter 15.3. The LeuDH in accordance with that from Bacillus species can preferably be used. Most particularly preferred is the LeuDH in accordance with that from Bacillus cereus, B.

stearothermophilus or B. sphaericus. It should be noted that the enzymes mentioned here can also be used in the process according to the invention as more highly developed mutants enhanced by mutagenesis. Mutagenesis processes, which can bring about improved stability, for example heat stability, storage stability and/or selectivity of the LeuDH, are known to the person skilled in the art. They are in particular saturation mutagenesis, random mutagenesis, shuffling methods and site-directed mutagenesis (Eigen M. and Gardinger W. (1984) Evolutionary molecular engineering based on RNA replication. Pure & Appl. Chem. 56 (8), 967- 978; Chen & Arnold (1991) Enzyme engineering for nonaqueous solvents: random mutagenesis to enhance activity of subtilisin E in polar organic media. Bio/Technology 9, 1073-1077; Horwitz, M. and L. Loeb (1986)"Promoters Selected From Random DNA-Sequences"Proceedings Of The National Academy Of Sciences Of The United States Of America 83 (19): 7405-7409; Dube, D. and L. Loeb (1989) "Mutants Generated By The Insertion Of Random Oligonucleotides Into The Active-Site Of The Beta-Lactamase Gene"Biochemistry 28 (14): 5703-5707; Stemmer PC (1994).

Rapid evolution of a protein in vitro by DNA shuffling.

Nature. 370; 389-391 and Stemmer PC (1994) DNA shuffling by random fragmentation and reassembly: In vitro recombination for molecular evolution. Proc Natl Acad Sci USA. 91; 10747- 10751). According to the invention the term improved selectivity is understood to mean an increase in enantioselectivity and/or a reduction in substrate selectivity to give a broader substrate spectrum.

NADH oxidases that can be used in the process according to the invention are familiar to the person skilled in the art (Nishiyama, Y. et al. J. Bacteriol. Vol. 183 (2001), 2431- 2438; Lopez de Felipe, F. et al. , J. Bacteriol. Vol. 180 (1998), 3804-08). Examples which yield irreversibly inert water as the reduction product are preferably used, since a formation of secondary products can be avoided in this way,

in contrast to hydrogen peroxide-forming NADH oxidases.

These can come from the Lactobacillus organism, for example. The use of the NADH oxidase in accordance with that from Lactobacillus kefir or Lactobacillus brevis is particularly advantageous. An NADH oxidase that can most particularly preferably be used can be taken from DE1014088. Regarding the use of improved mutants, reference is made to the comments above in respect of LeuDHs.

In the present process the enzymatic reaction system is preferably used in the form of a whole-cell catalyst displaying cloned genes for both enzymes. The whole-cell catalyst according to the invention should preferably display an NADH oxidase or leucine dehydrogenases. The representatives claimed above in particular are especially suitable.

The production of such an organism is familiar to the person skilled in the art (PCT/EP00/08473 ; PCT/US00/08159 ; Sambrook et al. 1989, Molecular cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Balbas P & Bolivar F. 1990; Design and construction of expression plasmid vectors in E. coli, Methods Enzymology 185,14-37 ; Vectors: A Survey of Molecular Cloning Vectors and Their Uses. R. L. Rodriguez & D. T. Denhardt, Eds: 205- 225).

The advantage of such an organism is the simultaneous expression of both enzyme systems, which means that only one rec-organism is required for the reaction. To adjust the expression of the enzymes in respect of their reaction rates, it is possible to accommodate the appropriately coding nucleic acid fragments on different plasmids with different copy numbers and/or to use promoters of different strengths for gene expressions of different strengths. By adjusting enzyme systems in this way, there is advantageously no accumulation of an intermediate possibly having an inhibitory action, and the reaction in question can proceed at an optimum overall rate. This is

sufficiently known to the person skilled in the art, however (PCT/EP00/08473 ; Gellissen et al, . Appl. Microbiol.

Biotechnol. 1996,46, 46-54). The organism from DE10155928 can advantageously be used.

The process is preferably performed at pH values in the range from 4 to 9, particularly preferably 5 to 8. The temperature range in the preferred embodiment is 15 to 100°C, preferably 20 to 50°C and in particular 25 to 40°C.

It can also be advantageous to perform the process in a two-phase mixture consisting of water and an organic phase, in which the keto acid dissolves very readily. The enzymatic reaction takes place in the aqueous phase, wherein the keto acid that is formed concentrates in the organic phase and can therefore be readily separated from the remaining mixture of substances.

The invention also provides an enzymatic reaction system displaying a leucine dehydrogenase (LeuDH), NAD (H) or NADP (H) and an NADH oxidase, together with a mixture of the D-and L-form of an amino acid. Such a system has not yet been described in the literature and can be used most particularly advantageously-as shown-for the production of D-amino acids.

The enzyme under consideration can be used for the application in free form as a homogeneously isolated compound. In addition, the enzyme can also be used as a component of an intact guest organism or in conjunction with the digested cell mass, isolated to the desired level, of the host organism. The enzymes can also be used in immobilised form (Bhavender P. Sharma, Lorraine F. Bailey and Ralph A. Messing,"Immobilisierte Biomaterialien- Techniken und Anwendungen", Angew. Chem. 1982,94, 836- 852). The immobilisation is advantageously performed by lyophilisation (Dordick et al. J. Am. Chem. Soc. 194,116, 5009-5010; Okahata et al. Tetrahedron Lett. 1997, 38, 1971- 1974; Adlercreutz et al. Biocatalysis 1992,6, 291-305).

Most particularly preferable is lyophilisation in the presence of surface-active substances, such as Aerosol OT or polyvinyl pyrrolidone or polyethylene glycol (PEG) or Brij 52 (diethylene glycol monocetyl ether (Goto et al.

Biotechnol. Techniques 1997,11, 375-378). Use as CLECs is likewise conceivable (St Clair et al. Angew Chem Int Ed Engl 2000 Jan, 39 (2), 380-383).

In a general embodiment of the present invention, a racemic mixture of a preferably aliphatic amino acid is dissolved in water at a pH of around 8.5 and brought into contact with an appropriately buffered medium displaying NAD+, LeuDH and NADH oxidase. This initiates the reaction of the L-amino acid to the corresponding keto acid. The enantiopure D-amino acid remains, which can easily be separated from the reaction mixture and isolated by common methods.

An extremely advantageous embodiment of the invention is provided if the keto acid produced in the reaction undergoes reductive amination again. This process can either be non-selective or as strongly D-selective as required. In this way up to 100% of the D-amino acid can be obtained from a batch of racemate if the cycle described is executed several times in succession (diagram below). reductive amination NH2 L-LeuDH HS NH2 COOH + NAD pOH +-- COOH + NADH + NH3 I NOX H20 1/202

This reductive amination, which takes place in situ, can be performed by methods familiar to the person skilled in the art. Chemocatalytic hydrogenation or enzymatic processes are possible.

Within the meaning of the invention, the term optically concentrated (enantiomer-concentrated, enantiopure) compounds is understood to refer to the presence of an optical antipode combined with the other in >50 mol%.

The organism Lactobacillus, brevis DSM 20054 or Lactobacillus kefir DSM 20587 is stored in the Deutsche Sammlung fur Mikroorganismen und Zellkulturen under the corresponding number and is available to the public.

The references cited in this specification are deemed to be included in the disclosure.

The process is illustrated by the examples below.

Example 1 (comparative example) When amino acid dehydrogenases were used for racemate separation to obtain enantiopure D-amino acids, it was striking that, unlike the case with LeuDH, no conversion was observed with phenylalanine dehydrogenase (PheDH) (detection by HPLC in each case). Photometer tests were therefore performed to test the influence of D-Phe and phenyl pyruvate on the oxidation capability of L-Phe. The assay was performed in accordance with the following photometric assay for Phe-DH (Rhodococcus sp. ): 800 ul buffer (50mM tris pH 8.0) 180 pi D-or L-phenylalanine or phenyl pyruvate (L-Phe: 5-0.5 mM in drops.; D-Phe or phenyl pyruvate: 5 mM) 10 ul 20 mM NAD (in drops 0.2 mM) 10 ul Phe-DH E 1. 0 ml The initial reaction rates, which are plotted as activity in Figure 1 below, were measured. The figure shows that the oxidation of L-Phe was greatly inhibited by 5 mM of D-Phe.

The oxidation product phenyl pyruvate also inhibits the oxidation of L-Phe. PheDH can therefore not be used for racemate separation (Figure 1).

Example 2 The volume of the reaction mixture for enzymatic oxidation is 1 ml. This reaction mixture, containing 290 ul tris buffer (50 mM, pH 8.0), 100 zl D, L-tert-leucine (100 mM), 10 pi NAD+ (20 mM), 100ul NADH oxidase (42 U-ml~l), und 500 pi leucine dehydrogenase (0.5 U-ml-1), is incubated at 30°C. One unit of leucine dehydrogenase corresponds here to the amount of leucine dehydrogenase that catalyses the oxidation of 1 pmol of L-tert-leucine per minute in the presence of 20 mM D, L-tert-leucine at a pH of 8.0 (0.1 M

potassium phosphate buffer). The samples are taken at the time intervals indicated in Figure 2 below and then measured by HPLC (Kromasil 100 C18; 250x4 mm; 5 um ; buffer A: 23 mM sodium acetate, pH 6.00 ; buffer B: acetonitrile/H20 10/1.5) after derivatisation with o- phthalaldehyde and N-isobutyryl-L-cysteine (0.1 M sodium borate buffer, pH 10.4). The reaction is terminated by heating the sample to 95°C for 5 minutes. The results are shown in Figure 2 below.