WO2009040080A1 - Process for the enantioselective enzymatic reduction of intermediates - Google Patents

Abstract

A process for the enantioselective enzymatic reduction of a keto compound of general formula (I), wherein R may represent any protective group for amino functions (tert. butyloxycarbonyl group (BOC), benzyloxycarbonyl group, 9-fluorenylmethoxycarbonyl group) and X = -Cl, -CN, -OH, Br, F.

Description

Process for the enantioselective enzymatic reduction of intermediates

The invention relates to a process for the enantioselective enzymatic reduction of a keto compound of general formula I

(I )

wherein R may represent any protective group for amino functions (tert. butyloxycarbonyl group, benzyloxycarbonyl group, 9-fluorenylmethoxycarbonyl group) and X = -Cl, -CN, -OH, Br, F,

(III)

to the compounds of formulae II (R,S-alcohol) and III (S,S-alcohol), respectively, with an oxidoreductase in the presence of a co factor.

Preferred compounds of formula I contain the butyloxycarbonyl group or the benzyloxycarbonyl group as an amino protective group and a chlorine atom in place of X. Chiral alcohols of general formulae II and III are important intermediates in the production of protease inhibitors for the therapy of HIV. Such protease inhibitors are, for example, Ritonavir, Amprenavir, Fosamprenavir, Atazanavir or Darunavir.

Intermediates of formulae II (R,S-alcohol) and III (S,S-alcohol), respectively, are obtainable, for example, by enantioselective reduction of the corresponding keto compounds of formula I, which is performed chemically in current production processes. In doing so, the chemically catalyzed reduction has the disadvantage that, on the one hand, it may result in byproducts due to harsh reaction conditions and, on the other hand, yields unsatisfactory enantiomeric and diastereomeric excesses, respectively, and is technically feasible only with very large efforts. Thereby, the intermediate of formula II (R,S-alcohol) in its enantiomerically enriched form is chemically accessible with more difficulty than that of formula III (S,S-alcohol).

For this reason, there have, for quite some time, been endeavours to develop biocatalytic processes which allow for the enantioselective reduction of said intermediates. Biocatalytic processes usually operate under mild conditions, for which reason they can be expected to enable the reduction of the keto compounds of formula I without the formation of further byproducts. So far, however, it has not been possible to find any suitable biocatalysts by means of which the enzymatic reduction is possible with isolated enzymes.

As far as we know, only few publications exist in which reactions of ketones of formula I with strains of Rhodococcus or Streptomyces in whole-cell processes are described (Tetrahedron Asymmetry 14 (2003) 3105-3109, Tetrahedron Asymmetry 8 (1997) p. 2547). However, the reactions have thereby occurred only with whole cells and lysates, respectively, of wild strains and have thus been carried out only at very low concentrations far below 2% and without coenzyme regeneration. Enzymatic reduction processes applicable on an industrial scale have not been available so far, and the enzymes involved in the reaction have neither been isolated nor identified.

It is the object of the invention to provide a process which enables the economic production of enantiomerically pure and, respectively, enantiomerically enriched intermediates of general formulae II and III in high yields and with high enantiomeric purity without any byproducts. According to the invention, said object is achieved by a process of the initially mentioned kind which is characterized in that the oxidoreductase used for the production of the compound of formula II (R,S-alcohol)

a) comprises an amino acid sequence according to SEQ ID NO:1, SEQ ID No 2, SEQ ID No3 or SEQ ID No 4, b) comprises an amino acid sequence in which at least 60% of the amino acids are identical to those of amino acid sequences SEQ ID NO:1, SEQ ID No 2 or SEQ ID No3, SEQ ID No 4, or c) comprises an amino acid sequence in which at least 70% of the amino acids are identical to those of amino acid sequences SEQ ID NO:1, SEQ ID No 2 or SEQ ID No3, SEQ ID No 4, or d) is encoded by the nucleic acid sequence SEQ ID NO: 16, SEQ ID No 17, SEQ ID No 18, SEQ ID No 19, or e) is encoded by a nucleic acid sequence which hybridizes to SEQ ID NO: 16, SEQ ID No 17, SEQ ID No 18, SEQ ID No 19 under stringent conditions, f) has a length of from 220 to 260 amino acids and comprises one or several of the partial sequences selected from the group consisting of sequences SEQ ID NO:31 to SEQ ID NO:51 and reduces the compound of formula I preferably to the compound of formula II.

nalvtgasrgig (31) nalvtggsrgig (32), gysvt (33), gynvt (34), gygitl (35), gygvt (51) vlaklp (36), vkaklp (37) fkgaplpa (38), frgaplpa (39), lkgaplpa (40), spialtk (41), spvaltk (42), sqialtq (43), avysask (44), avysatk (45), gvysatk (46), pikgwi (47), piegwi (48), piggwi (49) and pisgwi (50),

Furthermore, said object is achieved by a process of the initially mentioned kind which is characterized in that the oxidoreductase used for the production of the compound of formula III (S,S-alcohol)

a) comprises an amino acid sequence according to SEQ ID NO:5, SEQ ID No 6, SEQ ID No7, SEQ ID No 8, SEQ ID No9, SEQ ID NoIO, SEQ ID NoI 1, SEQ ID No 12, SEQ ID Nol3, SEQ ID No 14, SEQ ID NoI 5, b) comprises an amino acid sequence in which at least 60% of the amino acids are identical to those of amino acid sequences according to SEQ ID NO:5, SEQ ID No 6, SEQ ID No7, SEQ ID No 8, SEQ ID No9, SEQ ID NoIO, SEQ ID NoI 1, SEQ ID No 12, SEQ ID NoI 3, SEQ ID NoH, SEQ ID No 15, or c) is encoded by the nucleic acid sequence SEQ ID NO:20, SEQ ID No 21 , SEQ ID No22, SEQ ID No 23, SEQ ID No24, SEQ ID No25, SEQ ID No26, SEQ ID No27, SEQ ID No28, SEQ ID No29 or SEQ ID No30, or d) is encoded by a nucleic acid sequence which hybridizes to SEQ ID NO:20, SEQ ID No 21 , SEQ ID No22, SEQ ID No 23, SEQ ID No24, SEQ ID No25, SEQ ID No26, SEQ ID No27, SEQ ID No28, SEQ ID No29 or SEQ ID No30 under stringent conditions, e) has a length of from 220 to 260 amino acids and comprises one or several of the partial sequences selected from the group consisting of sequences SEQ ID NO:31 to SEQ ID NO:66 and reduces the compound of formula I preferably to the compound of formula III.

nalvtgasrgig (31) nalvtggsrgig (32), gysvt (33), gynvt (34), gygitl (35), gygvt (51) vlaklp (36), vkaklp (37) fkgaplpa (38), frgaplpa (39), lkgaplpa (40), fkaaplpa (52), fkgsplpa (53) spialtk (41), spvaltk (42), sqialtq (43), avysask (44), avysatk (45), gvysatk (46), pikgwi (47), piegwi (48), piggwi (49) and pisgwi (50),

gigrat (54), gigrasa (55), gigret (56), nnagig (57), nnagieg (58), irwaiapg (59), irvnaiapg (60), irvnaicpg (61), irwgiapg (62), peqiagav (63), peaianav (64), peevanav (65), peaianav (66)

A polypeptide which reduces the compound of formula I preferably to the compound of formula II is understood to be such a polypeptide in which the maximum enantiomeric excess of the R,S-alcohol achievable under optimum reaction conditions amounts to at least 50%. Optimum reaction conditions are thereby understood to be those reaction conditions of a polypeptide under which a polypeptide yields the highest enantiomeric excess of the R,S- alcohol.

It has been found that the polypeptides comprising amino acid sequences SEQ ID NO: 1 , SEQ ID No 2, SEQ ID NO:3 and SEQ ID No 4 show oxidoreductase activity and can be used for reducing the compound of formula I preferably to the compound of formula II (R,S- compound). The achievable enantiomeric excess of the R,S-alcohol amounts to >50%, preferably to >80% and particularly preferably to >95%. The enantiomeric excess achieved when using SEQ ID NO:1 can, for example, account for up to >99% of the R,S-compound (formula II).

Similarly, it has been found that the polypeptides comprising amino acid sequences SEQ ID NO:5 to SEQ ID No 15 show oxidoreductase activity and can be used for reducing the compound of formula I preferably to the compound of formula III (S,S-compound). The achievable enantiomeric excess of the R,S-alcohol amounts to >80%, preferably to >90% and particularly preferably to >95%. The enantiomeric excess achieved when using SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9 or SEQ ID NO: 12 can account for up to >99% of the R,S-compound (formula II).

A number of the mentioned oxidoreductases such as, e.g., SEQID No 1,3,4,5,6,7 and 15 have the additional advantage that they are able to regenerate the oxidized cofactor formed during the reduction by reducing a secondary alcohol. Thus, a particular economic advantage of said oxidoreductases is also that no further enzyme has to be used for cofactor regeneration in contrast to prior art methods.

A DNA sequence SEQ ID NO:20, which codes for a polypeptide comprising SEQ ID NO:5, is obtainable, for example, from the genome of the organism Rubrobacter xylanophilus DSM 9941.

A DNA sequence SEQ ID NO:21, which codes for a polypeptide comprising SEQ ID NO:6, is obtainable, for example, from the genome of the organism Geobacillus thermodenitήficans DSM 465.

A DNA sequence SEQ ID NO:22, which codes for a polypeptide comprising SEQ ID NO:7, is obtainable, for example, from the genome of the organism Chloroflexus aurantiacus DSM 635.

A DNA sequence SEQ ID NO:23 or a DNA sequence SEQID No 24, which codes for a polypeptide comprising SEQ ID NO:8 or SEQ ID NO:9, respectively, is obtainable, for example, from the organism Candida magnoliae DSMZ 70638.

^;'A DNA sequence SEQ ID NO:26, which codes for a polypeptide comprising SEQ ID NO:11, is obtainable, for example, from the organism Candida magnoliae DSMZ 70639. A DNA sequence SEQ ID NO: 16, which codes for a polypeptide comprising SEQ ID NO: 1, is obtainable, for example, from the organism Candida magnoliae CBS 6396.

Furthermore, the oxidoreductases of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15 are obtainable, for example, by homology screening from the strains Candida magnoliae CBS 5659, CBS 7318, CBS 2798, JCM 9448, Candida geochares MUCL 29832, Candida spec. MUCL 40660, Candida gropengiesseri MUCL 29836.

Thus, the present invention relates to a process for the reduction of keto compounds of general formula I to compounds of general formulae II and III, respectively, characterized in that one of the compounds II or III is formed clearly in excess, using a polypeptide comprising one of the amino acid sequences SEQ ID NO:1 to SEQ ID NO: 15, or a polypeptide which comprises an amino acid sequence which is identical by at least 50% to one of the amino acid sequences SEQ ID NO:1 to SEQ ID NO: 15, i.e., a polypeptide which can be derived from the sequences SEQ ID NO: 1 to SEQ ID NO: 15 by substitution, insertion, deletion or addition of at least one amino acid, or using a polypeptide which is encoded by one of the nucleic acid sequences SEQ ID NO: 16 to SEQ ID No30 or by nucleic acid sequences which hybridize under stringent conditions to one of the sequences SEQ ID NO: 16 to SEQID No30.

A nucleic acid sequence which hybridizes, for example, to SEQ ID NO: 16 under stringent conditions is understood to be a polynucleotide which can be identified via the colony hybridization method, the plaque hybridization method, the Southern hybridization method or comparable methods, using SEQ ID NO: 16 as a DNA probe.

For this purpose, the polynucleotide immobilized on a filter is hybridized, for example, to SEQ ID NO: 16 in a 0.7-1 M NaCl solution at 60°C. Hybridization is carried out as described, for instance, in Molecular Cloning, A Laboratory Manual, Second Edition (Cold Spring Harbor Laboratory Press, 1989) or in similar publications. Subsequently, the filter is washed with a 0.1 to 2-fold SSC solution at 65°C, wherein a 1-fold SSC solution is understood to be a mixture consisting of 150 mM NaCl and 15 mM sodium citrate.

Furthermore, the present invention relates to polypeptides of amino acid sequences SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15 as well as to polypeptides which are identical by at least 55%, preferably by 65% to 75% , particularly preferably by more than 75%, to one of the amino acid sequences SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 11, SEQ ID NO:12, SEQ ID NO: 13, SEQ ID NO:14 and SEQ ID NO:15, i.e., to polypeptides which can be derived from the sequences SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:1 1, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15 by substitution, insertion, deletion or addition of at least one amino acid. Furthermore, the invention relates to polypeptides which are encoded by the nucleic acid sequences SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29 or SEQ ID NO:30 or by nucleic acid sequences which hybridize under stringent conditions to one of the sequences SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29 or SEQ ID NO:30.

In the process according to the invention, polypeptides comprising the sequences SEQ ID NO: 1 to SEQ ID NO: 15 and polypeptides derivable from said polypeptides, respectively, can be used either in a completely purified state, in a partially purified state or as cells containing one of the polypeptides SEQ ID NO: 1 to SEQ ID NO: 15. The cells used can thereby be provided in a native, permeabilized or lysed state. Preferably, polypeptides comprising the sequences SEQ ID NO:1 to SEQ ID NO: 15 and derivatives derivable therefrom, respectively, are overexpressed in a suitable host organism such as, for example, Escherichia coli, and the recombinant polypeptide is used for reducing the hydroxy ketone of general formula I.

The enzymatic reduction according to the invention proceeds under mild conditions so that the degradation of the unstable compounds of formula I and thus the formation of undesired byproducts can be largely avoided. The process according to the invention has an enantiomeric purity of the compound of formula II (R,S-compound) of up to 99%, at least, however, of 50% of the R,S-compound, depending on the polypeptide used.

For the compound of formula III (S,S-compound), the process according to the invention has an enantiomeric purity of the compound of formula III (S,S-compound) of up to 99%, at least, however, of 80% of the R,S-compound, depending on the polypeptide used.

A preferred embodiment of the invention is characterized in that the cofactor used in the process is continuously reduced with a cosubstrate. Preferably, NAD(P)H is used as the cofactor, with the NAD(P) formed in the reduction being reduced back to NAD(P)H by means of a cosubstrate. In the processes according to the invention, the oxidized cofactor NAD or NADP formed by the oxidoreductase/dehydrogenase is preferably regenerated continuously.

According to a preferred embodiment of all processes according to the invention, the oxidized cofactor NAD or NADP is regenerated by oxidation of an alcohol.

Secondary alcohols such as 2-propanol, 2-butanol, 2-pentanol, 3-pentanol, 4-methyl-2- pentanol, 2-heptanol, 2-octanol or cyclohexanol are preferably used as cosubstrates. According to a particularly preferred embodiment, 2-propanol or 4-methyl-2-pentanol is used for coenzyme regeneration. The amount of cosubstrate for the regeneration can range from 5 to 95% by volume, based on the total volume.

Preferably, a secondary alcohol of general formula RχRγCHOH is used for cofactor regeneration, wherein Rx and Ry independently are hydrogen, a branched or unbranched Q- C₈-alkyl group and C_totai ≥ 3.

According to a further preferred embodiment of the processes according to the invention, an additional oxidoreductase/dehydrogenase is added for the regeneration of the cofactor.

In a further preferred embodiment, a further alcohol dehydrogenase can, in addition, be added for the regeneration of the cofactor. Suitable NADH-dependent alcohol dehydrogenases are obtainable, for example, from baker's yeast, from Candida parapsilosis (CPCR) (US 5,523,223 and US 5,763,236, Enzyme Microb. Technol., 1993, 15(1 1):950-8), Pichia capsulata (DE 10327454.4), from Rhodococcus erythropolis (RECR) (US 5,523,223), Norcardia fusca (Biosci. Biotechnol. Biochem., 63(10), 1999, p. 1721-1729; Appl. Microbiol. Biotechnol,. 2003, 62(4):380-6; Epub 2003, Apr. 26) or from Rhodococcus ruber (J. Org. Chem., 2003, 68(2):402-6). Suitable cosubstrates for those alcohol dehydrogenases are, for example, the already mentioned secondary alcohols such as 2- propanol (isopropanol), 2-butanol, 2-pentanol, 4-methyl-2-pentanol, 2-octanol or cyclohexanol.

Suitable secondary alcohol dehydrogenases for the regeneration of NADPH are, for example, those as described above and isolated from organisms of the order of Lactobacillales, e.g., Lactobacillus kefir (US 5,200,335), Lactobacillus brevis (DE 19610984 Al; Acta Crystallogr. D. Biol. Crystallogr. 2000 Dec; 56 Pt 12:1696-8), Lactobacillus minor (DE 101 19274), Leuconostoc carnosum (A 1261/2005, Kl. C12N) or, as described, those from Thermoanerobium brockii, Thermoanerobium ethanolicus or Clostridium beijerinckii. However, other enzymatic systems can, in principle, also be used for cofactor regeneration. For example, cofactor regeneration can be effected using NAD- or NADP-dependent formate dehydrogenase (Tishkov et al., J. Biotechnol. Bioeng. [1999] 64, 187-193, Pilot- scale production and isolation of recombinant NAD and NADP specific formate dehydrogenase). Suitable cosubstrates of formate dehydrogenase are, for example, salts of formic acid such as ammonium formate, sodium formate or calcium formate.

In the processes according to the invention, the compound of general formula I is used in the reaction batch preferably in an amount of from 10 g/1 to 500 g/1, preferably from 25 g/1 to 300 g/1, particularly preferably from 50 g/1 to 200 g/1, based on the total volume.

The aqueous portion of the reaction mixture in which the enzymatic reduction proceeds preferably contains a buffer, e.g., a potassium phosphate, tris/HCl or triethanolamine buffer, having a pH value of from 5 to 10, preferably a pH of from 6 to 9. In addition, the buffer can contain ions for stabilizing or activating the enzymes such as, for example, zinc ions or magnesium ions.

While carrying out the process according to the invention, the temperature suitably ranges from about 10°C to 70°C, preferably from 20°C to 45°C.

In a further preferred embodiment of the process according to the invention, the enzymatic reaction is carried out in the presence of an organic solvent which is not miscible with water or is miscible with water only to a limited degree. Said solvent is, for example, a symmetric or unsymmetric di(Ci-C₆)alkyl ether, a linear-chain or branched alkane or cycloalkane or a water-insoluble secondary alcohol which, at the same time, represents the cosubstrate. The preferred organic solvents are diethyl ether, tertiary butyl methyl ether, diisopropyl ether, dibutyl ether, butyl acetate, heptane, hexane, 2-octanol, 2-heptanol, 4-methyl-2-pentanol and cyclohexanol. In this case, the solvent can simultaneously also serve as a cosubstrate for cofactor regeneration.

If water-insoluble solvents and cosubstrates, respectively, are used, the reaction batch consists of an aqueous phase and an organic phase. According to its solubility, the compound of the formula is distributed between the organic phase and the aqueous phase. In general, the organic phase has a proportion of from 5 to 95%, preferably from 10 to 90%, based on the total reaction volume. The two liquid phases are preferably mixed mechanically so that, between them, a large surface area is generated. Also in this embodiment, e.g., the NAD(P) formed during the enzymatic reduction can be reduced back to NAD(P)H with a cosubstrate, such as described above.

The concentration of the co factor, in particular of NADH or NADPH, respectively, in the aqueous phase generally ranges from 0.001 mM to 10 mM, in particular from 0.01 mM to 1 mM.

The TTN (total turn over number = mol of reduced compound of formula I / mol of co factor used) achieved in the processes according to the invention normally ranges from 10 to 10 , preferably, however, it is >10³.

In the process according to the invention, a stabilizer of oxidoreductase/dehydrogenase can also be used. Suitable stabilizers are, for example, glycerol, sorbitol, 1 ,4-DL-dithiothreitol (DTT) or dimethyl sulfoxide (DMSO).

The process according to the invention is carried out, for example, in a closed reaction vessel made of glass or metal. For this purpose, the components are transferred individually into the reaction vessel and stirred under an atmosphere of, e.g., nitrogen or air.

According to another possible embodiment of the invention, the oxidized cosubstrate (e.g. acetone) can be removed continuously and/or the cosubstrate (e.g. 2-propanol) can be newly added in a continuous manner in order to shift the reaction equilibrium towards the reaction product.

In a further embodiment, the addition of the oxidoreductases according to SEQ ID NO:1 to SEQID No 15 and/or of the cosubstrate may also occur little by little in the course of the process.

After completion of the reduction, the reaction mixture is processed. For this purpose, e.g., the aqueous phase is optionally separated from the organic phase and the organic phase containing the product is filtered. Optionally, the aqueous phase can also be extracted and processed further like the organic phase. Thereupon, the solvent is evaporated from the organic phase and the product of general formula II or III is obtained as a crude product. The crude product can then be purified further or used directly for the synthesis of a resultant product. In the following, the invention is illustrated further by way of examples.

Example 1

Cloning and providing an oxidoreductase from Rubrobacter xylanophilus DSM 9941 (SEQID No 5)

A) Cultivation of Rubrobacter xylanophilus DSM 9941

Cells of Rubrobacter xylanophilus DSM 9941 were cultivated in the following medium at 50°C (pH 7.2) and 140 rpm in a bacteria-shaker: 0.1% yeast extract, 0.1% tryptone, 0.004% CaSO₄ x 2 H₂O, 0.02% MgCl₂ x 6 H₂O, 0.01% nitrilotriacetic acid, 100 ml phosphate buffer [5.44 g/1 KH₂PO₄, 43 g/1 Na₂HPO₄ x 12 H₂O], 500 μl/1 0.01 M Fe citrate, 500 μl/1 trace element [500 μl/1 H₂SO₄, 2.28 g/1 MnSO₄ x H₂O, 500 mg/1 ZnSO₄ x 7 H₂O, 500 mg H₃BO₃, 25 mg/1 CuSO₄ x 5 H₂O, 25 mg/1 Na₂MoO₄ x 2 H₂O, 45 mg/1 CoCl₂ x 6 H₂O]. On day 6 of the cultivation, cells were separated from the culture medium by centrifugation and stored at -80°C.

B) Amplification of the gene coding for selective oxidoreductase

Genomic DNA was extracted according to the method described in ,,Molecular Cloning" by Manniatis & Sambrook. The resulting nucleic acid served as a template for the polymerase chain reaction (PCR) involving specific primers which were derived from the gene sequence published under number 46106817 in the NCBI database. In doing so, the primers were provided in a 5 '-terminal position with restriction sites for the endonucleases Nde I and Hind III or Sph I, respectively (SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69), for subsequent cloning into an expression vector.

Amplification was carried out in a PCR buffer [10 mM Tris-HCl, (pH 8.0); 50 mM KCl; 10 mM MgSO₄; 1 mM dNTP Mix; in each case 20 pMol of primer and 2.5 U of Platinum Pfx DNA Polymerase (Invitrogen)] with 500 ng of genomic DNA and the following temperature cycles:

Cycle 1 : 94°C, 2 min

Cycle 2 x 30: 94°C, 15 sec

54°C, 30 sec 68°C, 60 sec

Cycle 3: 68°C, 7 min

4°C, oo

The resulting PCR product having a size of about 750 bp was restricted after purification over a 1 % agarose gel with the aid of the endonucleases Nde I and Hind III or Sph I and Hind III, respectively, and was ligated into the backbone of the pET21a vector (Novagen) or of the pQE70 vector (Qiagen), respectively, which backbone had been treated with the same endonucleases. After transforming 2 μl of the ligation batch into E.coli Top 10 F' cells (Invitrogen), plasmid DNA of ampicillin-resistant colonies was tested for the presence of an insert having a size of 750 bp by means of a restriction analysis with the endonucleases Nde I and Hind III or Sph I and Hind III, respectively. Plasmid preparations from the clones which were positive for the fragment were subjected to a sequence analysis and subsequently transformed into Escherichia coli BL21 Star (Invitrogen) and E.coli RB791 (genetic stock, Yale), respectively.

C.) Efficient expression of polypeptide SEQ ID NO:5 in Escherichia coli cells

For an efficient expression of the polypeptide SEQ ID NO:5 in Escherichia coli cells, coding DNA SEQ ID NO:70 was used as a template in a PCR reaction for cloning into an expression vector. In the region of the first, this DNA sequence differed in 153 bases from the previously known DNA sequence (SEQ ID NO:20). This modification was conservative and did not result in a change in the amino acid sequence.

Amplification was carried out in a PCR buffer [10 mM Tris-HCl, (pH 8.0); 50 mM KCl; 10 inM MgSO₄; 1 mM dNTP Mix; in each case 20 pMol of primer (SEQ ID NO: 71, SEQ ID NO: 68) and 2,5 U of Platinum Pfx DNA Polymerase (Invitrogen)] with 50 ng of DNA SEQ ID NO:70 as a template and the following temperature cycles: Cycle 1 : 94°C, 2 min

Cycle 2 x 30: 94°C, 40 sec

56°C, 30 sec

68°C, 60 sec Cycle 3: 68°C, 7 min

4°C, oo The resulting PCR product having a size of about 750 bp was ligated after purification over a 1 % agarose gel with the aid of the endonucleases Nhe I and Hind HI into the backbone of the pET21a vector (Novagen), which backbone had been treated with the same endonucleases. After transforming 2 μl of the ligation batch into E.coli Top 10 F' cells (Invitrogen), plasmid DNA of ampicillin-resistant colonies was tested for the presence of an insert having a size of 750 bp by means of a restriction analysis with the endonucleases Nhe I and Hind III. Plasmid preparations from the clones which were positive for the fragment were subjected to a sequence analysis and subsequently transformed into Escherichia coli BL21 Star (Invitrogen).

D.) Preparation of oxidoreductase from Rubrobacter xylanophilus DSM 9941

The Escherichia coli strains BL21 Star (Invitrogen, Karlsruhe, Germany) and RB791 {E.coli genetic stock, Yale, USA), respectively, which had been transformed with the expression construct, were cultivated in a medium (1% tryptone, 0.5% yeast extract, 1% NaCl) with ampicillin (50 μg/ml) until an optical density of 0.5, measured at 550 nm, was reached. The expression of recombinant protein was induced by adding isopropylthiogalactoside (IPTG) at a concentration of 0.1 mM. 16 hours after the induction at 25°C and 220 rpm, the cells were harvested and frozen at -20°C.

For enzyme recovery, 30 g of cells were suspended in 150 ml triethanolamine buffer (100 mM, pH = 7, 2 mM MgCl₂, 10% glycerol) and broken down using a high-pressure homogenizer. Subsequently, the enzyme solution was mixed with 150 ml glycerol and stored at -20°C.

The enzyme solution thus obtained was used for the reduction of compound I (example 3).

In analogy to the procedure mentioned in example 2, the oxidoreductases SEQ ID NO 6 and SEQ ID NO 7 can also be provided.

Example 2

Cloning and providing an oxidoreductase from Candida magnoliae by molecular screening (SEQ ID No I) A) Molecular screening for an oxidoreductase

Genomic DNA isolated from the cells of Candida magnoliae CBS 6396 was used as a template for molecular screening via PCR. In doing so, amplification was carried out in a PCR buffer [16 mM (NH₄)₂SO₄ ; 67 mM Tris-HCl pH 8.3 (at 25°C); 1.5 m MgCl₂; 0.01% Tween 20; 0.2 mM dNTP Mix; in each case 30 pMol of primer (SEQ ID NO: 72, SEQ ID NO: 73) and 1.25 U of Bio Therm Star Polymerase (Genecraft)] with 50 ng of genomic DNA isolated from the cells of Candida magnoliae CBS 6396 as a template and with the following cycles:

Cycle 1 : 95°C, 7 min

Cycle 2 x 28: 94°C, 40 sec

Temperature drop start 63°C -0,5°C / step, 30 sec

68°C, 60 sec x 20: 94°C, 40 sec

53°C, 40 sec

70°C, 60 sec Cycle 3: 70°C, 7 min

4°C, oo

After the fractionation of the entire PCR batch in the 1% agarose gel, a band of about 400 bp was identified and cloned via overhanging adenosine moieties into a Topo-TA vector (Invitrogen) for the determination of the DNA sequence.

The DNA band resulting from the screening reaction exhibited an open reading frame corresponding to the fragment of an oxidoreductase of 137 amino acid residues.

B) Isolation (total and mRNA)

600 mg of fresh cells were resuspended in 2.5 ml of ice-cold LETS buffer. 5 ml (about 20 g) of glass beads washed in nitric acid and equilibrated with 3 ml phenol (pH 7.0) were added to said cell suspension. The entire batch was then alternately treated by 30 sec of vortexing and 30 sec of cooling on ice, in total for 10 min. Subsequently, 5 ml of ice-cold LETS buffer was added, and this was again vigorously vortexed. Said cell suspension was centrifuged at 4°C and with 11000 g for 5 min. The aqueous phase was recovered and extracted twice with an equal volume of phenol: chloroform: isoamyl alcohol (24:24:1). This was subsequently followed by the extraction with chloroform. After the final extraction, the total RNA was precipitated at -20°C for 4 h by adding 1 / 10 vol. of 5 M LiCl₂.

1 mg of total RNA thus obtained was used via Oligo-dT cellulose (NEB Biolabs) for the enrichment of the mRNA molecules.

The determination of the entire sequence coding for the oxidoreductase was accomplished by a RACE (rapid amplification of cDNA ends) according to the method described in

,,Molecular Cloning" by Manniatis & Sambrook.

The gene sequence coding for the oxidoreductase included 720 base pairs and was equivalent to a length of 239 amino acid residues.

C) Synthesis of a full-length transcript coding for a short-chain ADH from Candida magnoliae CBS 6396 by PCR

Specific primers were constructed for subsequent cloning of the full-length transcript into the appropriate expression systems. In doing so, a 5 '-primer with a recognition sequence for Nde /and a 3 '-primer with a recognition sequence for Hind III were modified (SEQ ID NO:74, SEQ ID NO:75). Genomic DNA isolated from the cells of Candida magnoliae CBS 6396 served as a template for the polymerase chain reaction. Amplification was carried out in a PCR buffer [1O mM Tris-HCl (pH 8.0); 50 mM KCl; 10 mM MgSO₄; 1 mM dNTP Mix; in each case 20 pMol of primer and 2.5 U of Platinum Pfx DNA Polymerase (Invitrogen)] with 50 ng of template and the following temperature cycles: Cycle 1 : 94°C, 2 min

Cycle 2 x 30: 94°C, 15 sec

58°C, 30 sec

68°C, 75 sec Cycle 3: 68°C, 7 min

4°C, ∞

The resulting PCR product was restricted after purification over a 1% agarose gel with the aid of the endonucleases Nde I and Hind III and was ligated into the backbone of the pET21a vector (Novagen), which backbone had been treated with the same endonucleases. After transforming 2 μl of the ligation batch into E.coli Top 10 F' cells (Invitrogen), plasmid DNAs of ampicillin- (or kanamycin) resistant colonies were tested for the presence of an insert having a size of 750 bp by means of a restriction analysis with the endonucleases Nde l and Hind. The expression constructs pET21-MIX were sequenced. The gene from Candida magnoliae coding for a short-chain oxidoreductase had an open reading frame of a total of 720 bp (SEQ ID NO: 16), which corresponded to a protein of 239 amino acids (SEQ ID NO:1).

D) Expression of recombinant oxidoreductase in E.coli cells

Competent Escherichia coli StarBL21(De3) cells (Invitrogen) and RB791 cells (E.coli genetic stock, Yale, USA), respectively, were transformed with the expression constructs pET21-MIX coding for the oxidoreductase. The Escherichia coli colonies transformed with the expression constructs were then cultivated in 200 ml of LB medium (1% tryptone, 0.5% yeast extract, 1% NaCl) with 50 μg/ml of ampicillin or 40 μg/ml of kanamycin, respectively, until an optical density of 0.5, measured at 550 nm, was reached. The expression of recombinant protein was induced by adding isopropylthiogalactoside (IPTG) with a concentration of 0.1 mM. After 16 hours of induction at 25°C and 220 rpm, the cells were harvested and frozen at -20⁰C. For the activity test, 10 mg of cells were mixed with 500 μl of 100 mM TEA buffer pH 7.0, 1 mM MgCl₂ and 500 μl glass beads and digested for 10 min using a globe mill. The lysate obtained was then used in a diluted state for the respective measurements.

The activity test was made up as follows: 960 μl of 100 mM TEA buffer pH 7.0, 1 mM MgCl₂, 160 μg NADPH, 10 μl of diluted cell lysate. The reaction was started by adding 10 μl of a 100 mM substrate solution to the reaction mixture.

For enzyme recovery in large amounts, 30 g of cells were resuspended in 150 ml of triethanolamine buffer (100 mM, pH 7, 2 mM MgCl₂, 10% glycerol) and digested using a high-pressure homogenizer. Subsequently, the enzyme solution was mixed with 150 ml glycerol and stored at -20°C.

In analogy to the procedure mentioned in example 2, the oxidoreductases SEQ ID NO: 2, 3, 4, 8, 9, 10, 1 1, 12, 13, 14, 15 can also be provided.

Example 3:

Characterization of oxidoreductases SEO ID No 1 to SEQ ID No 15 with regard to their reduction properties of the compound of formula I The oxidoreductases of sequences SEQID NoI to SEQ ID No 15 were examined as follows for the conversion of the compound of formula I.

Reaction batch A (without coenzyme regeneration)

160 μl buffer (triethanolamine 100 mM pH = 7, 1 mM MgCl₂, 10% glycerol)

150 μl NAD(P)H (40 mg /ml)= 6 mg

20 μl 2-propanol

2 mg compound of formula I

50 μl enzyme solution according to example ID

Reaction batch B (with coenzyme regeneration)

400 μl buffer (triethanolamine 100 mM pH = 7, ImM MgCl₂, 10% glycerol)

0.05 mg NAD(P)H

50 μl 2-propanol

10 mg compound of formula I

50 μl enzyme solution according to example ID

After 24 h of incubating samples A and B, 1 ml of acetonitrile was in each case added to the complete reaction batches, the reaction batch was centrifuged off and transferred into a HPLC analysis vessel (lmg/ml).

The reaction batches were analyzed via HPLC (Nucleodur 100 5 Cl 8 ec, 125 mm, diameter 4 mm, Macherey-Nagel). A flow of lml/min and a solvent system of acetonitrile (B) and water (A) were used. The compounds of formulae I, II and III could be separated within 10 min with an increasing linear gradient from 40% to 80% of acetonitrile.

The retention times were (ketone formula I) 10.0 min; (R,S-compound formula II) 9.3 min and (S,S-compound formula III) 8.5 min. Results

Example 4:

Conversion of the compound of formula I to the compound of formula II (R,S-compound) via oxidoreductase SEQ ID NO: 1

For the conversion of the compound of formula I to the compound of formula II (R₅S- compound), 2.25 ml of an enzyme suspension of SEQID NoI (see example ID) and 75 units (= 2 ml) of the overexpressed alcohol dehydrogenase from Thermoanerobium brockii were in each case added to a mixture of 3ml of a buffer (100 mM TEA, pH = 8, 10% glycerol), 1.5 g of the compound of formula I, 0.3 mg NADP and 7 ml 4-methyl-2-pentanol. The reaction mixture was incubated at room temperature under constant thorough mixing. After 48 h, more than 95% of the compound of formula I used had been reduced to the compound of formula II. The enantiomeric excess amounted to > 98%.

Example 5:

Conversion of the compound of formula I to the compound of formula HI (S,S-compound) via oxidoreductase SEQ ID NO:5

For a further conversion of the compound of formula I to the compound of formula III (S, S- compound), a mixture of 600 μl of a buffer (100 mM TEA, pH = 9), 200 μl 2-propanol, 50 mg of the compound of formula I, 0.1 mg NAD and 200 μl of enzyme suspension SEQ ID NO:5 (see example 1 D) was incubated in an Eppendorf reaction vessel. The reaction mixture was incubated at room temperature under constant thorough mixing. After 48 h, more than 90% of the compound of formula I used had been reduced to the compound of formula III (S, S). The enantiomeric excess amounted to > 98%.

Sequence listing Amino acid sequences

SEQ ID No 1 :

Candida magnoliae CBS 6396 protein sequence carbonyl reductase

1 mnalvtggsr gigeaiatkl aedgysvtia srgidqlnkv kaklpvvreg qthhvwqldl

61 sdaeaassfk gaplpassyd vlvnnagvtd pspiakqsds eihklfsvnl lspvaltkty

121 vqavtgkpre tpahiifiss gvairgypnv avysatksgl dgfmrslare lgpegvhvnt

181 vspgltktem asgvslddfp pspiggwiqp eaiadavryl vksknitgti lsvdngitv

SEQ ID No 2:

Candida magnoliae JCM 9448 protein sequence carbonyl reductase

1 mpstlnalvt ggsrgigeat avklaeegyg itlaardikk lndvkaklpt ikqgqehhvw

61 qldladvqaa lelkgaplpa skydllvana gvsahvptae hddahwqnvi tinlssqial

121 tqalvraige rsdeapfhiv yvssiaalrg npmsavysas kagldgfars isrelgpkgi

181 hvntvhpglt ktdmtvrmrp aedqpikgwv lpdaiadavv flaksknitg tnivvdngrv

241 v

SEQ ID No 3:

Candida geochares MUCL 29832 protein sequence carbonyl reductase

1 mssvpassss ssptlnalvt gasrgigeat aiqlasqgys vtlasrgleq lkavkaklpl

61 vrqgqthhvw qldladvaaa gsfkgaplpa ssydvlvsna gvalfspigd qadedwqrml

121 avnltspial tkalvkaiad kprenpahii fvssavslrg yplvgvysat kagldgftrs

181 lahelgpkri hvntvnpglt ktemakdvel dsfggnvpis gwiqvdaiad avsflvnskn 41 itgtslvvdn gisv SEQ ID No 4:

Candida magnoliae CBS 5659 protein sequence carbonyl reductase

1 mnalvtggsr gigeataiql aqegygvtlv argarqlnev laklpvvrdg qthhiwqldl

61 sdpeaaaafr gaplpassyd vlinnagvss lspfvaqsde vqktilavnl lspialtkaf

121 vkaavgkpre rpahiifiss gaalrgfanm avysatkggl dsfmrslare lgpqgihvns

181 vnpgftetem tattdlndyp ptpiegwiqp raiadailfl lksrnitgtn vtvdngitv

SEQ ID No 5:

Rubrobacterxylanophilus DSM9941 protein sequence carbonyl reductase

1 mlegkvavit gagsgigrat alrfaregar vvvaelderr geevvreile sggeavfvrt

61 dvsefeqvea averaveeyg tldvmfnnag ighyaplleh dpehydrvvr vnqygvyygi

121 laagrkmael enpgviinta svyaflaspg vigyhaskga vkmmtqaaal elaphgirvv

181 aiapggvdtp iiqgykdmgl gerlargqmr rrlqtpeqia gavvllatee adaingsvvm

241 tddgyaefk

SEQ ID No 6:

Geobacillus kaustophilus JCM 12893 (HTA426) protein sequence carbonyl reductase

1 mrlkgkaaiv tggasgigra tairfaeega kvavsdinee ggeetvrlir ekggeaifvq

61 tdvadskqvs rlvqtavdaf gglhilfnna gighsevrst dlseeewdrv invnlkgvfl

121 gikyavpvmk qcgggaivnt ssllgikgkk yesaynaska gvilltknaa leygkfnirv

181 naiapgvidt niitpwkqde rkwpiiskan algrigtpee vanavlflas deasfitgat

241 lsvdgggltf

SEQ ID No 7:

Chloroβexus auratiacus DSMZ 635 (J-10-fl) protein sequence carbonyl reductase

1 meppfigkva Ivtgaaagig rasalafare gakvvvadvn veggeetial cralntdamf 61 vrcdvsqrde verlialavd tfgridfahn nagiegvqam ladypeevwd rvieinlkgv

121 wlcmkyeirh mlkqgggaiv ntssvaglag srgvsayvas khgivgitka aaleyarngi

181 rvnaicpgti htamidrftq gdpqllaqfa egepigrlgs peevanaviw lcsdkasfvt 241 gatlavdggr Ia

SEQ ID No 8:

Candida magnoliae DSM 70638 protein sequence carbonyl reductase

1 mtstpnalit ggsrgigasa aiklaqegys vtlasrdlek ltevkdklpi vrggqkhyvw

61 qldladveaa ssfkaaplpa ssydlfvsna giaqfsptae htnsewlnim tinlvspial

121 tkallqavsg rssenpfqiv fissvaalrg vaqtavysas kagtdgfars larelgpqgv

181 hvnvvnpgwt ktdmtegvet pkdmpikgwi qpeaiadavv flarsknitg anivvdngfs

241 t

SEQ ID No 9:

Candida magnoliae DSM 70638 protein sequence carbonyl reductase

1 mtttsnalvt ggsrgigaas aiklaqegyn vtlasrsvdk lnevkaklpi vqdgqkhyiw

61 eldladveaa ssfkgaplpa rsydvfvsna gvaafsptad hddkewqnll avnlsspial

121 tkallkdvse rpvdkplqii yissvaglhg aaqvavysas kagldgfmrs varevgpkgi

181 hvnsinpgyt ktemtagiea lpdlpikgwi epeaiadavl flaksknitg tnivvdngli

241 a

SEQIDNo 10:

Candida magnoliae ATCC 12573 protein sequence carbonyl reductase

1 msyqmsssap sstslnalvt ggsrgigeat aiklaeegys vtiasrglkq leavkaklpi

61 vkqgqvhhvw qldlsdvdaa aafkgsplpa srydvlvsna gvaqfspfie hakqdwsqml

121 ainlaapial aqtfakaigd kprntpahiv fvssnvslrg fpnigvytat kagidgfmrs

181 varelgpsgi nvnsvnpgpt rtemtkgidv gtidmpikgw iepeaiadav lfvvksknit 41 gttvvvdngs sa SEQ ID No 1 1 :

Candida magnoliae DSM 70639 protein sequence carbonyl reductase

1 mttsstssst ssrslnalvt gasrgigeat aiklasegys vtlasrsleq lkalkeklpv

61 vkqgqthhvw qldlsdvdaa atfkgsplpa ssydavisna gvaqfsplse haredwsqml

121 tinlaapial aqafvkaigd kkrdipaqiv fvssnvvmrg lpylgiytas kagidgfmrs

181 aarelgpkgi nvnsvnpgat qtemtkgvdv naldlpikgw iqleavadav lfvvqsknit

241 gttivvdngs va

SEQ ID No 12:

Candida magnoliae CBS 2798 protein sequence carbonyl reductase

1 mnalvtgasr gigeaiavkl aedgysvtla srsleklesl kkglpvvkdg qahhvweldl

61 gdvdaassfk gaplpaeayd vfvsnagmak stlmvdhpid elqdminvnl vspialtqgl

121 vkalteskrd kpahivfmss irsfrgipng avysatksgl dgfmrsiare lgpqgihvns

181 vcpgfvqtem trkvdmeskk dqlpiagwiq pdaiadtvlf fvksknitgq aivvdngitv

SEQ ID No 13:

Candida gropengoesseri MUCL 29836 protein sequence carbonyl reductase

1 mpsglnalvt ggsrgigaaa atklaaagyn vtvasrgvea lnkvkaslpv vkegqqhhvw

61 qldvsdlaav sgfkgsplpa ksydvvvvna gvanlsplaa qdddviqniv tvnllspial

121 vkslikayge gpratpahiv fvssvaairg fpngavysst ksaldgltrs lakelgpqni

181 rvnsvnpgft rtelasgvdi davtqsspik gwvepeaigd ailflatsnh itgtitvidn

241 gtsa

SEQIDNo 14:

Candida sp. MUCL 40660 protein sequence carbonyl reductase 1 msssssstpl nalvtgasrg igevislqla negynvtlaa rslddlnavk aklpivrdaq

61 khsvwpldis didavtnfkg splpaekydl fvsnagvvdf aplvhqspes isslfnvnli

121 apvaltkall kafgdsprkt tthfiyvssv valrgfpnva vysssksgld gfvrslaaev

181 aplnirvnsi npgptktemt asldveafta gnpikgwiyp daiadgvvyl aksknitgit

241 lqvdngagi

SEQ ID No 15:

Candida vaccinii CBS 7318 protein sequence carbonyl reductase

1 mrstpnalvt ggsrgigaaa aiklaeagys vtlasrgldk lnevkaklpv vkqgqehhvw

61 qldlsdvqaa lefkgaplpa skydlfvsna gvatfsptae hddkdwqnii avnltspiai

121 tkalvkavge rsndnpfqia flssaaalrg vpqtavysat kagldgftrs lakelgpkgi

181 hvnivhpgwt qtemtagvde prdtpipgwi qpeaiaeaiv ylaksknitg tnivvdnglt

241 i

SEQ ID No 16:

Candida magnoliae CBS 6396 nucleic acid sequence carbonyl reductase

1 atgaacgctc tagtgaccgg tggtagccgt ggcattggcg aggcgatcgc gaccaagctg

61 gccgaagatg gctacagcgt gacaatcgcc tcgcgcggaa tcgatcagct caacaaggta

121 aaggctaaac ttccggttgt gagggagggc cagacccacc acgtgtggca gcttgatttg

181 agcgacgccg aggccgcgtc gtccttcaag ggcgctcctt tgccagcaag cagctacgat

241 gtccttgtca acaacgccgg agtaacggat ccgagtccca ttgcgaagca gtcggatagc

301 gagattcaca agctgtttag cgtgaatctg ctgtcaccag ttgctttgac aaagacgtac

361 gtccaggcgg ttaccggaaa gcctcgtgag acgccagctc acattatttt tatctcgtca

421 ggcgttgcca ttcgaggcta cccaaacgtc gctgtatact cggctactaa gagcgggctc

481 gacggtttca tgaggtctct ggcgcgcgag cttggccccg agggcgtcca tgtgaacact

541 gtcagcccgg gtctcaccaa aaccgagatg gccagcggcg tcagcctcga cgacttcccg

601 ccatcgccga ttgggggctg gatccagccc gaggccatcg ctgatgcagt gaggtacctg

661 gtgaagtcga agaacatcac aggcacgatt ctgtcagttg acaacggaat cacggtttaa

SEQIDNo 17: Candida magnoliae JCM 9448 nucleic acid sequence carbonyl reductase

1 mpstlnalvt ggsrgigeat avklaeegyg itlaardikk Indvkaklpt ikqgqehhvw

61 qldladvqaa lelkgaplpa skydllvana gvsahvptae hddahwqnvi tinlssqial

121 tqalvraige rsdeapfhiv yvssiaalrg npmsavysas kagldgfars isrelgpkgi

181 hvntvhpglt ktdmtvrmrp aedqpikgwv lpdaiadavv flaksknitg tnivvdngrv

241 v

SEQIDNo 18:

Candida geochares MUCL 29832 protein sequence carbonyl reductase

1 atgccttcta ctctgaacgc tcttgtcact ggcggcagtc gcggtattgg cgaggctacc

61 gcagtgaagc tcgccgagga gggctacggt atcacacttg ctgcgcgcga tatcaaaaaa

121 ctgaatgacg tgaaggccaa actacccaca atcaagcagg gtcaagagca ccacgtctgg

181 cagcttgact tggccgatgt gcaggctgcg cttgagctca agggcgcacc actgcctgcg

241 agcaagtacg acctgttggt cgcgaatgcg ggcgtttccg cacacgttcc tacggccgag

301 cacgacgatg cgcactggca gaacgtcata actatcaact tgagctcgca gattgcgctc

361 acgcaggccc tagttagggc cattggcgag aggtctgatg aagcgccttt ccacattgtg

421 tatgtgtcct cgatcgccgc cctgcgcggt aaccccatga gcgcggtgta cagtgcctcg

481 aaggccggac ttgatggatt tgctcgttcc atctctcgcg agctcggccc gaagggtatt

541 catgtgaata cggtgcaccc gggactcacg aagacggaca tgaccgttcg catgcggcct

601 gctgaggacc agccgatcaa gggctgggta ctgcccgatg caattgctga tgccgttgtg

661 ttcctcgcga agtctaaaaa catcacgggc acaaacatcg ttgtcgacaa cggccgggtg

721 gtctaa

SEQ ID No 19:

Candida magnoliae CBS5659 protein sequence carbonyl reductase

1 atgaacgcgt tagtgaccgg cggaagccgc gggatcggcg aggccacggc catacagctg

61 gctcaggagg gctacggtgt gacattggtt gcgcgaggag cccgccagct caatgaagtg

121 ttggcaaagc taccagttgt gagagacgga cagacgcacc acatttggca gctagatctg

181 agcgatcctg aggcggccgc tgccttcagg ggtgctcctt tgcccgccag cagctacgac 241 gtgctgatca ataacgcagg tgttagtagt ctcagcccgt tcgtcgcgca gtctgatgag

301 gtccagaaaa ctattttagc ggtgaatctt ttgtcgccaa tcgcgttgac gaaggcgttc

361 gtgaaggcag cggtgggcaa gccgcgtgag aggccggcgc atatcatttt catctcttcg

421 ggcgctgccc tgcgcggttt cgcgaacatg gcagtgtata gtgcaacgaa aggcggcctt

481 gacagtttca tgcgctcgct agctagagag ctaggtcccc agggcatcca cgtcaactca

541 gtcaatccgg gctttactga aacagaaatg acagccacta cagatttgaa tgactacccc

601 ccgaccccca ttgagggctg gattcagcct cgcgcaatcg ccgacgctat acttttccta

661 ctgaagtcca gaaacatcac tggcacaaat gtgaccgtcg acaacggcat cactgtttga

SEQ ID No 20:

Rubrobacterxylanophilus DSM9941 protein sequence carbonyl reductase

1 atgctcgagg ggaaggtcgc ggtcatcacg ggggccggca gcggcatagg ccgggccacc

61 gcgctcaggt tcgcccgcga aggggcccgg gtggtcgtgg cggagctcga cgagcggagg

121 ggggaggagg tcgtccggga gatcctcgag tccggcgggg aggccgtctt cgtgaggacg

181 gacgtctcgg agttcgagca ggttgaggcc gccgtcgagc gcgccgtcga ggagtacggg

241 acgctggacg tcatgttcaa caacgccggc atcgggcact acgcccccct gctggagcac

301 gacccggagc actacgaccg ggtggtccgg gtgaaccagt acggcgtcta ctacgggata

361 ctcgccgccg gcaggaagat ggccgagctg gagaaccccg gcgtgatcat caacaccgcc

421 tcggtctacg ctttcctggc ctcccccggt gtgatcggct atcacgcttc caagggggcg

481 gtgaagatga tgacccaggc cgcagccctg gagctcgccc cccacggcat acgggtcgtc

541 gccatcgccc cgggcggggt ggacaccccg atcatccagg gctacaagga catgggcctc

601 ggtgagcggc tggcccgcgg ccagatgcgt cgcaggctcc agacccccga gcagatcgcc

661 ggcgccgtcg tcctgctcgc caccgaggag gcagacgcca taaacggctc ggtggtgatg

721 accgacgacg gctacgcgga gttcaagtaa

SEQ ID No 21:

Geobacillus kaustophilusJCM12893 (HTA426) protein sequence carbonyl reductase

1 atgaggctaa aaggaaaagc ggcgattgtc accggcggcg cgagcggcat cggccgggcg

61 acggcgattc gctttgcgga agaaggcgcc aaagtggcgg tgagcgacat caatgaggaa

121 ggaggggaag aaacggtccg cctgattcgg gaaaaaggag gggaggcgat ttttgtccaa

181 acggacgtag ccgattccaa gcaagtgagc cgccttgtcc aaacggcggt tgatgccttt 41 ggcggcctac atattctctt taacaatgcc ggcatcggcc attcggaagt gcggagcacc 301 gacttgtctg aagaagagtg ggaccgggtc atcaacgtta atttgaaagg agtgttcctt

361 ggcatcaaat acgcggtgcc cgtgatgaag caatgcggtg gcggggccat tgtcaacaca

421 tcgagcctgc ttggaatcaa agggaaaaag tacgaatcgg cctacaacgc ctcgaaggcc

481 ggggtgattt tgttgacgaa aaatgcagca ttggaatatg ggaagtttaa cattcgcgtc

541 aatgccattg caccgggggt cattgatacg aacatcatca cgccgtggaa acaagatgag

601 cgcaaatggc cgatcatttc gaaagcgaac gccctcggcc gcatcgggac gccagaggaa

661 gtggcgaacg cggtgttgtt tttggcgtcc gatgaagcgt cgtttatcac cggcgcgaca

721 ttgtcggtcg acggcggcgg gctgacgttt tag

SEQ ID No 22:

Chloroβexus auratiacus DSMZ635 (J-10-fl) protein sequence carbonyl reductase

1 atggagccac ctttcattgg gaaggttgcg ctggtcaccg gcgcagcagc cggtattggt

61 cgtgcttcag cactggcgtt tgcccgtgag ggtgccaagg ttgtcgttgc tgatgtgaat

121 gtcgagggcg gggaagagac gattgcgctg tgtcgggctt tgaataccga tgcaatgttc

181 gtgcgttgtg atgtttcgca acgcgatgaa gtggagcgat taattgctct ggcagttgac

241 acgttcggtc ggatcgactt tgcgcacaac aacgccggga ttgaaggcgt gcaggcaatg

301 ctggccgatt atcccgaaga ggtctgggat cgggtgatcg agatcaacct caaaggggtc

361 tggttgtgta tgaagtacga aatccggcac atgctcaagc agggtggcgg tgcgattgtg

421 aatacctcat cggtcgccgg tctggccgga tcacgtggcg tttcggcgta tgtagccagc

481 aagcacggta ttgttggtat taccaaagcg gcagcccttg agtatgcgcg taacggtatt

541 cgtgtcaacg caatctgtcc aggtacgatt catactgcga tgatcgaccg ctttacccag

601 ggtgatcccc aactgcttgc ccagttcgct gagggtgaac cgattggtcg gctcggctcg

661 cctgaagagg tcgccaatgc ggtgatctgg ctctgctcag ataaggcttc gtttgtgacc

721 ggagcgacac tggcggttga tggtggccgc ctggcgtaa

SEQ ID No 23:

Candida magnoliaeDSM 70638 protein sequence carbonyl reductase

1 atgacatcta cacctaatgc cctcatcacg ggaggcagcc gcggcattgg cgcttccgcc

61 gccatcaaac tggctcaaga agggtacagc gtcacgctgg cgtcccgcga ccttgagaaa

121 cttactgagg tcaaggacaa gctgccaatc gtgagaggtg gacagaaaca ctacgtttgg

181 cagctcgatc ttgccgatgt ggaggctgca tcgtctttca aggcggctcc tctgccggcc 41 agcagctacg atttgtttgt ttcgaacgcc ggaattgccc agttctcgcc tacggcagag 301 catactaata gtgagtggct gaacattatg accattaact tagtgtcccc gattgccctg

361 acgaaggctc ttttgcaggc cgtttctggg aggtcgagcg agaacccgtt tcagatcgtc

421 ttcatctcgt cggttgcagc actacgtggc gttgcacaaa cggccgtcta cagtgcgtcg

481 aaggctggta ctgatggatt cgcacgctca cttgctcgcg aactaggtcc tcaaggtgtt

541 catgtgaacg tggtgaaccc tggctggact aagacagaca tgacggaagg agtcgaaacc

601 ccaaaggaca tgcccattaa gggctggatc cagcctgagg caattgctga tgctgtagta

661 ttccttgcga ggtcgaaaaa cattaccggc gcgaatattg tagtggacaa tggtttctcg 721 acgtaa

SEQ ID No 24:

Candida magnoliae DSM 70638 protein sequence carbonyl reductase

1 atgacgacta cttcaaacgc gcttgtcact ggaggcagcc gcggcattgg cgctgcctcc

61 gccattaagc tggctcagga gggctacaat gttacgctgg cctctcgcag tgttgataaa

121 ctgaatgaag taaaggcgaa actcccaatt gtacaggacg ggcagaagca ctacatttgg

181 gaactcgatc tggctgatgt ggaagctgct tcgtcgttca agggtgctcc tttgcctgct

241 cgcagctacg acgtctttgt ttcgaacgcg ggcgtcgctg cgttctcgcc cacagccgac

301 cacgatgata aggagtggca gaacttgctt gccgtgaact tgtcgtcgcc cattgccctc

361 acgaaggccc tcttgaagga tgtctccgaa aggcctgtgg acaagccact gcagattatc

421 tacatttcgt cggtggccgg cttgcatggc gccgcgcagg tcgccgtgta cagtgcatct

481 aaggccggtc ttgatggttt tatgcgctcc gtcgcccgtg aggtgggccc gaagggcatc

541 catgtgaact ccatcaaccc cggatacacg aagactgaaa tgaccgcggg cattgaagcc

601 cttcctgatt tgcctatcaa ggggtggatc gagcccgagg caattgctga cgcggttctg

661 tttctggcaa agtccaagaa tatcaccggc acaaacattg tggtcgacaa tggcttgatt

721 gcttaa

SEQ ID No 25:

Candida magnoliaeATCC 12573 protein sequence carbonyl reductase

1 atgtcttatc aaatgtcttc ttctgctcca tcctccacct ccctgaatgc gcttgtcacg

61 ggcggcagcc gcggcattgg cgaagccact gccattaagc tcgccgagga gggctacagc

121 gtcacgattg cgtctcgcgg ccttaagcag ctcgaggctg tgaaggccaa actacccatt

181 gtgaagcagg gacaggttca ccacgtgtgg cagcttgatc tcagtgatgt cgacgctgcg 41 gccgccttca aagggtcgcc gctacctgcc agccgctacg acgtgctcgt cagcaatgct 301 ggcgtggccc agtttagccc gttcatcgag catgcgaagc aggactggtc gcagatgctt

361 gccatcaatc tggcggcacc cattgcgctg gcccagacat ttgctaaggc cattggcgac

421 aagccgcgca acacaccggc ccacattgtg tttgtctcgt cgaacgtctc gttgcgaggc

481 ttcccgaaca tcggcgtcta cacggccacg aaagccggca ttgacggctt catgcgctcg

541 gtcgcacgcg aactggggcc cagcggcatt aacgtgaact ccgtgaaccc cgggcccacg

601 cggacggaga tgacgaaggg cattgacgtc ggcacgatcg atatgccgat caagggctgg

661 atcgagcccg aggcgattgc ggatgccgtg ctcttcgtgg tcaagtcgaa gaacattacg 721 ggcacgaccg ttgttgtcga caacggctcc tccgcttga

SEQ ID No 26:

Candida magnoliae DSM 70639 protein sequence carbonyl reductase

1 atgaccacct cctccacctc ctcctccacc tcctcccgct ctctaaacgc tcttgtcacc

61 ggcgctagcc gcggcattgg cgaggccact gcaatcaagc tagcatctga gggatacagc

121 gttacgcttg catctcgtag cctcgagcag ctcaaggctt tgaaggagaa gttgcccgtt

181 gtgaagcagg gccagacgca ccacgtctgg cagctcgact tgagcgacgt cgacgccgct

241 gccacgttca agggctcccc cttgccggcc agcagctacg acgccgtcat cagcaatgcc

301 ggtgttgctc agttctctcc gttgtcggaa cacgccaggg aggactggtc tcagatgctg

361 acgatcaacc tcgcggctcc cattgccctc gcgcaggcgt ttgtgaaggc cattggcgac

421 aagaagcgcg acatcccggc ccaaattgtc tttgtttcgt cgaatgtcgt gatgcgtggc

481 ctcccttacc tcggcatcta cacggcttcg aaggctggta tcgatggctt catgcgctcg

541 gccgcccgcg agctgggacc caagggtatc aacgtgaact cagtaaaccc gggcgccacg

601 cagaccgaga tgacgaaggg cgttgatgtc aacgccctcg acctgccgat caagggatgg

661 attcagctcg aggctgtcgc ggacgccgtg ctcttcgtgg tccagtcgaa gaacattacc

721 ggcacgacga ttgttgtcga caacggctcc gtcgcttga

SEQ ID No 27:

Candida magnoliae CBS2798protein sequence carbonyl reductase

1 atgaatgcct tagttactgg tgcgagccgc gggatcggcg aagcaattgc ggtgaagctg

61 gccgaggacg ggtacagcgt gacactggcc tcgcgctctc ttgaaaagct ggagtcgctc

121 aagaaagggc tgccggtcgt gaaggacggc caagcacatc atgtatggga gcttgatctc 81 ggtgatgttg atgccgcgtc atccttcaag ggggcgcctc tgcctgccga ggcctatgac 241 gtgttcgtca gtaacgctgg aatggccaaa tccaccttga tggtagacca tcccattgac

301 gagctgcagg acatgattaa cgtgaatctt gtgtcgccaa ttgcactcac acagggcctt

361 gtcaaggctc tgacagaatc taagcgagac aagcctgcgc atatcgtgtt catgtcgtcc

421 atccgctcgt tcaggggcat tccgaatggc gcggtgtaca gcgccacaaa gagtggtctt

481 gacggattca tgcgatccat tgcgcgagag ctgggccctc agggcatcca cgtcaactct

541 gtgtgccccg gattcgtgca aacggaaatg acgcgcaagg ttgatatgga gtcgaagaaa

601 gaccagctac ccatcgccgg ctggatccag cccgacgcga ttgctgacac cgttctgttt

661 tttgtgaaat cgaagaacat cacgggccag gcaattgtcg ttgacaatgg catcactgtc 721 tga

SEQ ID No 28:

Candida gropengoesseriMUCL 29836protein sequence carbonyl reductase

1 atgccctctg gactcaatgc tcttgtcact ggcggcagcc gcggaatcgg cgctgccgct

61 gctaccaagc tcgccgctgc aggatacaac gtcacggttg cgtcccgcgg ggtcgaggct

121 ctgaataagg tcaaggcctc cttgcctgtt gtcaaggagg gccagcagca ccatgtctgg

181 cagctcgacg taagcgatct cgcagcggtg tctggcttca agggatctcc gctgccggct

241 aagagctacg atgttgttgt tgttaacgcc ggcgtcgcga acctgagccc gctggctgcc

301 caggacgacg acgtcattca gaacattgtg accgtgaacc tgctgtcgcc gattgcgctg

361 gtgaagtcgc tgatcaaggc gtacggcgag ggtcctcgcg cgacaccggc ccacattgtg

421 tttgtgtcgt cggtggccgc gatccgtggg ttccccaacg gcgccgtcta tagctcgacg

481 aagagtgcgc tcgacgggct gacgcggtcg ctggcgaagg agctggggcc ccagaacatc

541 cgggtcaact ccgtgaaccc cggcttcacg aggaccgagc tggccagcgg cgtcgacatt

601 gacgccgtga cgcagagctc tccgatcaag gggtgggttg agccggaggc gattggcgat

661 gcgattttgt ttctcgcgac gtcgaaccac atcacgggca cgatcaccgt catcgacaac

721 ggcactagcg cgtag

SEQ ID No 29:

Candida sp. MUCL 40660 protein sequence carbonyl reductase

1 atgtcctcct cttcctcctc gactcctctc aacgctctcg tcaccggtgc cagccgcggc

61 atcggtgagg tcatctctct ccagctcgcc aacgagggct acaatgttac cctcgcagcc

121 cgcagtcttg acgacctcaa tgcggtgaag gctaagctcc ctatcgtaag ggatgcccag 181 aagcactctg tctggccgct cgacattagc gatatcgacg ccgtgacgaa cttcaaggga

241 tcgcccctgc cggccgagaa gtacgatctg ttcgtcagca acgccggcgt ggtcgacttc

301 gctccgcttg tccaccagag ccccgagagc atcagcagcc tgttcaatgt gaacctaatc

361 gcgcctgttg ccttgacaaa agctcttctt aaggcgttcg gtgacagccc tcgcaagact

421 acgactcact ttatctacgt ttcgtccgtt gttgccctcc gcggcttccc caatgttgcg

481 gtttacagct cctccaagag cggcctcgac gggtttgtgc gctcccttgc cgccgaggtt

541 gctccgctca acatccgcgt caactccatt aacccaggcc ctaccaagac tgagatgacc

601 gcttccctgg atgttgaggc gtttactgcg ggcaacccca tcaagggttg gatttacccc

661 gatgctattg ctgatggagt ggtgtacctg gcgaagtcga agaacattac tggtatcacc 721 ctccaagtcg acaacggcgc cggcatctaa

SEQ ID No 30:

Candida vaccina CBS 7318 protein sequence carbonyl reductase

1 atgaggtcga cacctaacgc ccttgtgact ggcggcagcc gcggcattgg cgcggccgct

61 gcaattaaac tcgccgaggc aggctacagc gtgacgctcg cgtcgcgcgg tctcgacaag

121 ctcaacgagg tgaaggccaa gcttcctgtc gtgaagcagg gccaggagca ccatgtatgg

181 cagcttgatc tcagcgacgt gcaggccgcg ctcgagttca agggcgcacc gctgcccgcg

241 agtaagtacg atttgtttgt ctcgaacgcc ggcgtggcta ctttctcgcc aacggctgag

301 catgacgaca aggactggca gaacattatt gccgtgaact tgacatcgcc cattgccatt

361 acgaaggcgc tcgttaaggc cgttggcgag cgctcaaacg ataacccgtt tcagatcgcg

421 ttcctgtcat cggcggccgc cctgcgcggt gtgccgcaga ccgctgttta cagcgctacg

481 aaggccggcc tcgacggctt cacgcgctcg ctcgccaagg agctcggccc aaagggcatc

541 catgtgaaca tcgtacaccc tggatggacg cagaccgaga tgactgcggg tgtagatgag

601 cctagggata cgcccatccc gggctggatc cagccggaag ccatcgccga ggccattgtg

661 tatctcgcga agtcaaagaa catcacggga acgaacatcg ttgtcgacaa cggcctgact

721 atttaa

SEQ ID No 31 :

Amino acid sequence partial nalvtgasrgig SEQ ID No 32:

Amino acid sequence partial nalvtggsrgig

SEQ ID No 33:

Amino acid sequence partial gysvt

SEQ ID No 34:

Amino acid sequence partial gynvt

SEQ ID No 35:

Amino acid sequence partial gygiti

SEQ ID No 51 :

Amino acid sequence partial gygvt SEQ ID No 36:

Amino acid sequence partial vlaklp

SEQ ID No 37:

Amino acid sequence partial vkaklp

SEQ ID No 38:

Amino acid sequence partial fkgaplpa

SEQ ID No 39:

Amino acid sequence partial frgaplpa

SEQ ID No 40:

Amino acid sequence partial lkgaplpa SEQ ID No 41 :

Amino acid sequence partial spialtk

SEQ ID No 42:

Amino acid sequence partial spvaltk

SEQ ID No 43:

Amino acid sequence partial sqialtq

SEQ ID No 44:

Amino acid sequence partial avysask

SEQ ID No 45:

Amino acid sequence partial avysatk SEQ ID No 46:

Amino acid sequence partial gvysatk

SEQ ID No 47:

Amino acid sequence partial pikgwi

SEQ ID No 48:

Amino acid sequence partial piegwi

SEQ ID No 49:

Amino acid sequence partial piggwi

SEQ ID No 50:

Amino acid sequence partial pisgwi SEQ ID No 52:

Amino acid sequence partial fkaaplpa

SEQ ID No 53:

Amino acid sequence partial fkgsplpa

SEQ ID No 54:

Amino acid sequence partial gigrat

SEQ ID No 55:

Amino acid sequence partial gigrasa

SEQ ID No 56:

Amino acid sequence partial gigret SEQ ID No 57:

Amino acid sequence partial nnagig

SEQ ID No 58:

Amino acid sequence partial nnagieg

SEQ ID No 59:

Amino acid sequence partial irwaiapg

SEQ ID No 60:

Amino acid sequence partial irvnaiapg

SEQ ID No 61 :

Amino acid sequence partial irvnaicpg SEQ ID No 62:

Amino acid sequence partial irwgiapg

SEQ ID No 63:

Amino acid sequence partial peqiagav

SEQ ID No 64:

Amino acid sequence partial peaianav

SEQ ID No 65:

Amino acid sequence partial peevanav

SEQ ID No 66:

Amino acid sequence partial peaianav SEQ ID No 67: GGGAATTCCATATGATGCTCGAGGGGAAGGTCG

SEQ ID No 68: CACATGCATGCGAATGCTCGAGGGGAAGGTC

SEQ ID No 69: CCCAAGCTTATTACTTGAACTCCGCGTAGCCGTC

SEQ ID No 70:

Rubrobacter xylanophilus DSM 9941 nucleic acid sequence carbonyl reductase semisynthetic

1 atgctggaag gtaaagtggc agtcatcacc ggtgcaggca gcggcattgg gcgtgccact

61 gcgctgcgtt ttgcgcgtga aggcgctcgc gtcgttgtgg ccgagctgga tgaacgtcgc

121 ggtgaggaag ttgtacgtga gattctggaa tctggcgggg aggccgtctt cgtgaggacg

181 gacgtctcgg agttcgagca ggttgaggcc gccgtcgagc gcgccgtcga ggagtacggg 41 acgctggacg tcatgttcaa caacgccggc atcgggcact acgcccccct gctggagcac 01 gacccggagc actacgaccg ggtggtccgg gtgaaccagt acggcgtcta ctacgggata 61 ctcgccgccg gcaggaagat ggccgagctg gagaaccccg gcgtgatcat caacaccgcc 21 tcggtctacg ctttcctggc ctcccccggt gtgatcggct atcacgcttc caagggggcg 81 gtgaagatga tgacccaggc cgcagccctg gagctcgccc cccacggcat acgggtcgtc 41 gccatcgccc cgggcggggt ggacaccccg atcatccagg gctacaagga catgggcctc 01 ggtgagcggc tggcccgcgg ccagatgcgt cgcaggctcc agacccccga gcagatcgcc 61 ggcgccgtcg tcctgctcgc caccgaggag gcagacgcca taaacggctc ggtggtgatg 21 accgacgacg gctacgcgga gttcaagtaa SEQ ID No 71 : CCTAGCTAGCATGCTGGAAGGTAAAGTGGC

SEQ ID No 72: CCTTTRCCTGCHAGCAGCTAYG

SEQ ID No: 73 GGCTGGATCCAGCCCTTRATSGG

SEQ ID No: 74 GGAATTCCATATGATGAACGCTCTAGTGACCGGTGGTAG

SEQ ID No: 75 CCCAAGCTTATTAAACCGTGATTCCGTTGTCAACTGAC

Claims

Claims:

1. A process for the enantioselective enzymatic reduction of a keto compound of general formula I

(D

wherein R may represent any protective group for amino functions (tert. butyloxycarbonyl group (BOC), benzyloxycarbonyl group, 9-fluorenylmethoxycarbonyl group) and X = -Cl, -CN, -OH, Br, F, to the hydroxy compound of general formula II (R,S-compound)

wherein R and X have the same meaning as in formula I, with the keto compound being reduced with an oxidoreductase in the presence of a cofactor, characterized in that the oxidized cofactor NAD or NADP formed is regenerated continuously by oxidation of a secondary alcohol of general formula RχRγCHOH, wherein Rx, R_Y independently represent hydrogen, a branched or unbranched Cl-C8-alkyl group and

Qota_l ≥ 3.

2. A process for the enantioselective enzymatic reduction of a keto compound of general formula I

(I)

wherein R may represent any protective group for amino functions (tert. butyloxycarbonyl group (BOC), benzyloxycarbonyl group, 9-fluorenylmethoxycarbonyl group) and X = -Cl, -CN, -OH, Br, F, to the hydroxy compound of general formula II (R,S-compound)

OH (H)

wherein R and X have the same meaning as in formula I, with the keto compound being reduced with an oxidoreductase in the presence of a co factor, characterized in that the oxidoreductase

a) comprises an amino acid sequence according to SEQ ID NO:1, SEQ ID No 2, SEQ ID No3 or SEQ ID No 4, or b) comprises an amino acid sequence in which at least 65% of the amino acids are identical to those of amino acid sequences SEQ ID NO:1, SEQ ID No 2 or SEQ ID No3, SEQ ID No 4, or c) comprises an amino acid sequence in which at least 72% of the amino acids are identical to those of amino acid sequences SEQ ID NO: 1, SEQ ID No 2 or SEQ ID No3, SEQ ID No 4, or d) is encoded by the nucleic acid sequence SEQ ID NO: 16, SEQ ID No 17, SEQ ID No 18, SEQ ID No 19, or e) is encoded by a nucleic acid sequence which hybridizes to SEQ ID NO: 16, SEQ ID No 17, SEQ ID No 18, SEQ ID No 19 under stringent conditions, or f) has a length of from 220 to 260 amino acids and comprises one or several of the partial sequences selected from the group consisting of sequences SEQ ID NO:31 to SEQ ID NO:51 and reduces the compound of formula I preferably to the compound of formula II.

3. A process for the enantioselective enzymatic reduction of a keto compound of general formula I

(I)

wherein R may represent any protective group for amino functions (tert. butyloxycarbonyl group (BOC), benzyloxycarbonyl group, 9-fluorenylmethoxycarbonyl group) and X = -Cl, -CN, -OH, Br, F,

(III)

to the hydroxy compound of general formula III (S,S-compound), wherein R and X have the same meaning as in formula I, with the keto compound being reduced with an oxidoreductase in the presence of a co factor, characterized in that the oxidized cofactor NAD or NADP formed is regenerated continuously by oxidation of a secondary alcohol of general formula RχRγCHOH, wherein

Rx, Ry independently represent hydrogen, a branched or unbranched Cl-C8-alkyl group and

Qotal ≥ 3.

4. A process for the enantioselective enzymatic reduction of a keto compound of general formula I

(I)

wherein R may represent any protective group for amino functions (tert. butyloxycarbonyl group, benzyloxycarbonyl group, 9-fluorenylmethoxycarbonyl group) and X = -Cl, -CN, -OH, Br, F,

(III)

to the hydroxy compound of general formula III (S,S-compound), wherein R and X have the same meaning as in formula I, with the keto compound being reduced with an oxidoreductase in the presence of a cofactor, characterized in that the oxidoreductase

a) comprises an amino acid sequence according to SEQ ID NO:5, SEQ ID No 6, SEQ ID No7, SEQ ID No 8, SEQ ID No9, SEQ ID NoIO, SEQ ID NoI 1, SEQ ID No 12, SEQ ID No 13, SEQ ID No 14, SEQ ID NoI 5, or b) comprises an amino acid sequence in which at least 65% of the amino acids are identical to those of the amino acid sequences according to SEQ ID NO:5, SEQ ID No 6, SEQ ID No7, SEQ ID No 8, SEQ ID No9, SEQ ID NoIO, SEQ ID NoI 1, SEQ ID NoI 2, SEQ ID Nol3, SEQ ID No 14, SEQ ID NoI 5, or c) is encoded by the nucleic acid sequence SEQ ID NO:20, SEQ ID No 21 , SEQ ID No22, SEQ ID No 23, SEQ ID No24, SEQ ID No25, SEQ ID No26, SEQ ID No27, SEQ ID No28, SEQ ID No29 or SEQ ID No30, or d) is encoded by a nucleic acid sequence which hybridizes to SEQ ID NO:20, SEQ ID No 21 , SEQ ID No22, SEQ ID No 23, SEQ ID No24, SEQ ID No25, SEQ ID No26, SEQ ID No27, SEQ ID No28, SEQ ID No29 or SEQ ID No30 under stringent conditions, or e) has a length of from 220 to 260 amino acids and comprises one or several of the partial sequences selected from the group consisting of sequences SEQ ID NO:31 to SEQ ID NO:66 and reduces the compound of formula I preferably to the compound of formula III.

5. A process according to any of claims 2 or 4, characterized in that the oxidized cofactor NAD or NADP formed is regenerated continuously.

6. A process according to any of claims 2, 4 or 5, characterized in that the oxidized cofactor NAD or NADP formed is regenerated continuously by oxidation of a secondary alcohol of general formula RχRγCHOH.

7. A process according to any of claims 1 to 6, characterized in that 2-propanol, 2- butanol, 2-pentanol, 4-methyl-2-pentanol, 2-heptanol or 2-octanol is used as a cosubstrate or secondary alcohol, respectively.

8. A process according to any of claims 1 to 7, characterized in that an additional oxidoreductase/dehydrogenase is added for the regeneration of the cofactor.

9. A process according to any of claims 1 to 8, characterized in that the compound of formula 1 is present in the reaction batch at a concentration of >20 g/1, preferably of >50 g/1 and particularly preferably of > 100 g/1.

10. A process according to any of claims 1 to 9, characterized in that the TTN (total turn over number = mol of reduced compound of formula I / mol of cofactor used) is >10³.

11. A process according to any of claims 1 to 10, characterized in that it is carried out in an aqueous organic two-phase system.

12. A process according to any of claims 1 to 1 1, characterized in that, in addition, an organic solvent such as, for example, diethyl ether, tertiary butyl methyl ether, diisopropyl ether, dibutyl ether, ethyl acetate, butyl acetate, heptane, hexane or cyclohexane is used.

13. A process according to any of claims 1 to 1 1, characterized in that the specific compound of formula IV is used as a keto compound.

(IV)

14. Polypeptides of the amino acid sequences SEQ ID NO. l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:1 1, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15.

15. Polypeptides which are identical by at least 65%, preferably by 70% and particularly preferably by more than 75%, to one of the amino acid sequences SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 1 1, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15.

16. Polypeptides which can be derived from the sequences SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:1 1, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15 by substitution, insertion, deletion or addition of at least one amino acid.

17. Polypeptides which are encoded by the nucleic acid sequences SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29 or SEQ ID NO:30.

18. Polypeptides which are encoded by nucleic acid sequences which hybridize under stringent conditions to one of the sequences SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29 or SEQ ID NO:30.

19. Polypeptides which have a length of from 220 to 260 amino acids, comprise one or several of the partial sequences selected from the group consisting of sequences SEQ ID NO:31 to SEQ ID NO:51 and reduce the compound of formula I preferably to the compound of formula II.