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  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
  • C12P7/42—Hydroxy-carboxylic acids

Definitions

• the present invention relates to a process for the enantioselective enzymatic reduction of a keto compound to the corresponding chiral hydroxy compound, wherein the keto compound is reduced with an oxidoreductase. Furthermore, the invention relates to new oxidoreductases for use in the enantioselective reduction of keto compounds to chiral hydroxy compounds.
• Optically active hydroxy compounds are valuable chirons with broad applicability for the synthesis of pharmacologically active compounds, aromatic substances, pheromones, agricultural chemicals and enzyme inhibitors.
• chiral compounds and thus chiral synthesis technologies can be noted particularly in the pharmaceutical industry, since, in the future, racemic compounds will hardly be used as pharmaceutical preparations.
• the asymmetric reduction of prochiral keto compounds is a sector of stereoselective catalysis, wherein biocatalysis constitutes a powerful competitive technology versus chemical catalysis.
• the chemical asymmetric hydrogenation requires the use of highly toxic and environmentally harmful heavy metal catalysts, of extreme and thus energy-intensive reaction conditions as well as large amounts of organic solvents. Furthermore, these methods are often characterized by side reactions and insufficient enantiomeric excesses.
• Carbonyl reductase from Candida parapsilosis (U.S. Pat. No. 5,523,223 and U.S. Pat. No. 5,763,236, (Enzyme Microb Technol. 1993 November; 15(11):950-8)) and Pichia capsulata (DET0327454.4).
• Carbonyl reductase from Rhodococcus erythropolis (RECR) (U.S. Pat. No. 5,523,223), Norcardia fusca (Biosci. Biotechnol. Biochem., 63 (10) (1999), pp. 1721-1729), (Appl Microbiol Biotechnol. 2003 September; 62(4):380-6. Epub 2003 Apr. 26), and Rhodococcus ruber (J Org. Chem. 2003 Jan. 24; 68(2):402-6.).
• the subject matter of the present invention is now a number of novel, enantioselective R- and S-specific oxidoreductases characterized by good stability in aqueous/organic two-phase systems as well as by good expressibility in Escherichia coli (>500 units/g E. coli wet biomass), as well as a process for the enantioselective enzymatic reduction of a keto compound to the corresponding chiral hydroxy compound.
• the polypeptide according to SEQ ID No 1 can be obtained from yeasts, in particular from yeasts of the genus Rhodotorula , in particular from Rhodotorula mucilaginosa .
• a further subject matter of the invention is a nucleic acid sequence SEQ ID No 9, which codes for the protein having the amino acid sequence SEQ ID No 1.
• the oxidoreductase from Rhodotorula mucilaginosa reduces, for example, 2-octanone to S-2-octanol and preferably oxidizes S-2-octanol out of the two enantiomers of the 2-octanol.
• the oxidoreductase from Rhodotorula mucilaginosa is, for example, a homodimer having a molecular weight determined in the SDS—gel of 30 ⁇ 2 k Da.
• the pH optimum for the reduction reaction ranges from 7.0 to 8.0 for said oxidoreductase, and the pH optimum for the oxidation reaction is in the range of from 8.5-10.
• the oxidoreductase from Rhodotorula mucilaginosa exhibits good temperature and pH stabilities and shows only minor activity losses in the pH range of from 5.5 to 10 and at temperatures of up to 35° C., even with incubation periods of several hours. Furthermore, the oxidoreductase from Rhodotorula mucilaginosa exhibits high stability in organic solvents.
• Polypeptides according to SEQ ID No 2 or SEQ ID No 8 can be obtained from yeasts, in particular from yeasts of the genera Pichia, Candida, Pachysolen, Debaromyces or Issatschenkia , in particular from Pichia farinosa DSMZ 3316 or Candida nemodendra DSMZ 70647.
• a further subject matter of the invention is a nucleic acid sequence SEQ ID No 10 and a nucleic acid sequence SEQ ID No 16, which code for the amino acid sequences SEQ ID No 2 and SEQ ID No 8, respectively.
• the oxidoreductase preferably reduces 2-butanone to R-2-butanol and preferably oxidizes R-2-butanol out of the two enantiomers of the 2-butanol.
• the oxidoreductase from Pichia farinosa exhibits a significantly higher activity towards R-2-butanol and 2-propanol than towards R-2-octanol, in addition, the enzyme exhibits a significantly higher activity towards acetone and 2-butanone than towards 2-octanone.
• the oxidoreductase from Candida nemodendra exhibits a similar activity towards R-2-butanol, 2-propanol and R-2-octanol, in addition, the enzyme also exhibits an approximately similar activity towards 2-octanone.
• the oxidoreductase from Pichia farinosa is a homodimer having a molecular weight determined in the SDS—gel of 27 ⁇ 2 k Da.
• the pH optimum for the reduction reaction ranges from 5.0 to 6.0 for said oxidoreductase, and the pH optimum for the oxidation reaction ranges from 7.5-10.
• the oxidoreductase from Pichia farinosa exhibits good pH and solvent stabilities and shows only minor activity losses in the pH range of from 5.5 to 10, even with incubation periods of several hours.
• the oxidoreductase from Candida nemodendra is a homomer having a molecular weight determined in the SDS—gel of 27 ⁇ 2 k Da.
• the pH optimum for the reduction reaction is at pH 6 for said oxidoreductase, and the pH optimum for the oxidation reaction ranges from 10-11.
• the oxidoreductase from Candida nemodendra exhibits good pH and solvent stabilities and shows only minor activity losses in the pH range of from 6.5 to 9.5, even with incubation periods of several hours.
• polypeptides according to SEQ ID No 3 or SEQ ID No 7 can be obtained from yeasts, in particular from yeasts of the genera Pichia and Candida , in particular from Pichia stipidis DSMZ 3651 and Pichia trehalophila DSMZ 70391.
• a further subject matter of the invention is a nucleic acid sequence SEQ ID No 11 and a nucleic acid sequence SEQ ID No 15, which encode polypeptides SEQ ID No 3 and SEQ ID No 7, respectively.
• the carbonyl reductases from yeasts of the genera Pichia and Candida which have at least 65% identity to the amino acid sequence SEQ ID No 3 or at least 50% identity to the amino acid sequence SEQ ID No 7, preferably reduce 2-octanone to S-2-octanol and preferably oxidize S-2-octanol out of the two enantiomers of the 2-octanol. They are also particularly suitable for the reduction of 4-haloacetoacetate esters to R-4-halo-3-hydroxybutyric acid esters.
• the oxidoreductase from Pichia stipidis is a homodimer having a molecular weight determined in the SDS—gel of 36 ⁇ 2 k Da.
• the pH optimum for the reduction reaction ranges from 5.5 to 6.5 for said oxidoreductase, and the pH optimum for the oxidation reaction ranges from 6.5-8.0.
• the oxidoreductase from Pichia stipidis exhibits good pH and solvent stabilities and shows only minor activity losses in the pH range of from 5.5 to 10, even with incubation periods of several hours.
• the oxidoreductase from Pichia trehalophila is a homomer having a molecular weight determined in the SDS—gel of 36 ⁇ 2 k Da.
• the pH optimum for the reduction reaction ranges from 7-7.5 for said oxidoreductase, and the pH optimum for the oxidation reaction ranges from 7-8.
• the polypeptide according to SEQ ID No 4 can be obtained from bacteria of the class Leuconostoc , in particular from Leuconostoc carnosum DSMZ 5576.
• a further subject matter of the invention is a nucleic acid sequence SEQ ID No 12, which codes for a protein having the amino acid sequence SEQ ID No 4.
• the polypeptide is particularly suitable for the reduction of 2-octanone to R-2-octanol and for the oxidation of R-2-octanol. It is also very suitable for the reduction of 4-haloacetoacetate esters to S-4-halo-3-hydroxybutyric acid esters.
• the oxidoreductase from Leuconostoc camosum is a homodimer having a molecular weight determined in the SDS—gel of 27 ⁇ 2 k Da.
• the pH optimum for the reduction reaction ranges from 5.0 to 6.0 for said oxidoreductase, and the pH optimum for the oxidation reaction ranges from 6.0-9.0.
• the oxidoreductase from Leuconostoc camosum exhibits good temperature, pH and solvent stabilities and shows only minor activity losses in the pH range of from 4.5 to 10 and at temperatures of up to 35° C., even with incubation periods of several hours.
• the polypeptide according to SEQ ID No 5 can be obtained from bacteria of the class Actinobacteria, in particular from bacteria of the class Microbacterium , in particular from Microbacterium spec. DSMZ 20028.
• a further subject matter of the invention is a nucleic acid sequence SEQ ID No 13, which codes for the protein having the amino acid sequence SEQ ID No 5.
• the polypeptide is very suitable for the reduction of 2-octanone to S-2-octanol and preferably oxidizes S-2-octanol out of the two enantiomers of the 2-octanol. It is also very suitable for the reduction of 4-haloacetoacetate esters to R-4-halo-3-hydroxybutyric acid esters.
• the oxidoreductase from Microbacterium spec. DSMZ 20028 is, for example, a homotetramer having a molecular weight determined in the SDS—gel of 35 ⁇ 2 k Da.
• the pH optimum for the reduction reaction ranges from 6.0 to 7.5 for said oxidoreductase, and the pH optimum for the oxidation reaction ranges from 7.5-9.5.
• the oxidoreductase from Microbacterium spec exhibits good temperature, pH and solvent stabilities and shows only minor activity losses in the pH range of from 4.5 to 10 and at temperatures of up to 50° C., even with incubation periods of several hours.
• the polypeptide according to SEQ ID No 6 can be obtained from bacteria of the class Actinobacteria, in particular from bacteria of the class Gordonia, in particular from Gordonia rubripertincta DSMZ 43570.
• a further subject matter of the invention is a nucleic acid sequence SEQ ID No 14, which codes for the protein having the amino acid sequence SEQ ID No 6.
• the polypeptide is very suitable for the reduction of 2-octanone to S-2-octanol and preferably oxidizes S-2-octanol out of the two enantiomers of the 2-octanol. It is also very suitable for the reduction of 4-haloacetoacetate esters to R-4-halo-3-hydroxybutyric acid esters.
• the oxidoreductase from Gordonia rubripertincta DSMZ 43570 is a homomer having a molecular weight determined in the SDS—gel of 41 ⁇ 3 k Da.
• the pH optimum for the reduction reaction ranges from 4.5 to 5.5 for said oxidoreductase, and the pH optimum for the oxidation reaction ranges from 7.5 to 9.5.
• the oxidoreductase from Gordonia rubripertincta DSMZ 43570 exhibits good temperature, pH and solvent stabilities and shows only minor activity losses in the pH range of from 4.5-10 and at temperatures of up to 55° C., even with incubation periods of several hours.
• the polypeptide according to SEQ ID No 129 can be obtained from yeasts, in particular from yeasts of the genera Lodderomyces, in particular from Lodderomyces elongisporus DSMZ 70320.
• a further subject matter of the invention is a nucleic acid sequence SEQ ID No 130, which codes for the protein having the amino acid sequence SEQ ID No 129.
• the polypeptide is very suitable for the reduction of 2-octanone to S-2-octanol and preferably oxidizes S-2-octanol out of the two enantiomers of the 2-octanol. It is also very suitable for the reduction of 4-haloacetoacetate esters to R-4-halo-3-hydroxybutyric acid esters.
• the invention relates to fusion proteins which are characterized in that they represent oxidoreductases having the amino acid sequences SEQ ID No 1, SEQ ID No 2, SEQ ID No 3, SEQ ID No 4, SEQ ID No 5, SEQ ID No 6, SEQ ID No 7, SEQ ID No 8, SEQ ID No 129 or homologues thereof, which are peptidically linked to a further polypeptide at the N-terminal or carboxy-terminal end.
• Fusion proteins can, for example, be separated more easily from other proteins or can be recombinantly expressed in larger amounts.
• the invention relates to antibodies which specifically bind to oxidoreductases according to SEQ ID No 1, SEQ ID No 2, SEQ ID No 3, SEQ ID No 4, SEQ ID No 5, SEQ ID No 6, SEQ ID No 7, SEQ ID No 8, SEQ ID No 129 or to homologues thereof.
• SEQ ID No 1 SEQ ID No 2
• SEQ ID No 3 SEQ ID No 4
• SEQ ID No 5 SEQ ID No 6
• SEQ ID No 7, SEQ ID No 8 SEQ ID No 129 or to homologues thereof.
• the production of these antibodies is performed according to known methods by immunization of appropriate mammals and subsequent recovery of the antibodies.
• the antibodies can be monoclonal or polyclonal.
• Comparisons of amino acid sequences can, for example, be conducted in the internet in protein databases such as, e.g., SWISS-PROT, PIR as well as in DNA databases such as, e.g., EMBL, GenBank etc., using the FASTA-program or the BLAST-program.
• the optimal alignment is determined by means of the BLAST algorithm (Basic Local Alignement Search Tool) (Altschul et al. 1990, Proc. Natl. Acd. Sci. USA. 87: 2264-2268).
• the PAM30 matrix is used as a scoring matrix for evaluating the sequence similarity. (Dayhoff; M. O., Schwarz R. M., Orcutt B. C. 1978. “ A model of evolutionary change in Proteins” in “Atlas of Protein Sequence and structure” 5(3) M. O. Dayhoff (ed) 345-352, National Biomedical Research foundation).
• the invention relates to protein fragments which are characterized in that they represent fragments of the amino acid sequence SEQ ID No 1, with a number of more than 26 amino acids per fragment.
• a further subject matter of the invention is a microbial carbonyl dehydrogenase which comprises the amino acid sequence MPATLRLDK (SEQ ID No 17) N-terminally and/or the amino acid sequence QALAAPSNLAPKA (SEQ ID No 18) C-terminally and/or one of the internal partial sequences VEIIKTQVQD (SEQ ID No 19), KVAIITGGASGIGL (SEQ ID No 20), SCYVTPEG (SEQ ID No 21), TDFKVDGG (SEQ ID No 22), VMFNNAGIMH (SEQ ID No 23) or VHAREGIRIN (SEQ ID No 24).
• the invention relates to protein fragments which are characterized in that they represent fragments of the amino acid sequence SEQ ID No 2, with a number of more than 15 amino acids per fragment.
• a further subject matter of the invention is a microbial carbonyl dehydrogenase which comprises the amino acid sequence MAYNFTNKVA (SEQ ID No 25) N-terminally and/or the amino acid sequence TTLLVDGGYTAQ (SEQ ID No 26) C-terminally and/or one of the internal partial sequences EYKEAAFTN (SEQ ID No 27), NKVAIITGGISGIGLA (SEQ ID No 28), DVNLNGVFS (SEQ ID No 29), HYCASKGGV (SEQ ID No 30), NCINPGYI (SEQ ID No 31) or LHPMGRLGE (SEQ ID No 32).
• the invention relates to protein fragments which are characterized in that they represent fragments of the amino acid sequence SEQ ID No 3, with a number of more than 15 amino acids per fragment.
• a further subject matter of the invention is a microbial carbonyl dehydrogenase which comprises the amino acid sequence MSIPATQYGFV (SEQ ID No 33) N-terminally and/or the amino acid sequence SAYEGRVVFKP (SEQ ID No 34) C-terminally and/or one of the internal partial sequences CHSDLHAIY (SEQ ID No 35), GYQQYLLVE (SEQ ID No 36), TFDTCQKYV (SEQ ID No 37), LLTPYHAM (SEQ ID No 38), LVSKGKVKP (SEQ ID No 39), GAGGLGVNG (SEQ ID No 40), IQIAKAFGAT (SEQ ID No 41) or LGSFWGTS (SEQ ID No 42).
• the invention relates to protein fragments which are characterized in that they represent fragments of the amino acid sequence SEQ ID No 4, with a number of more than 18 amino acids per fragment.
• a further subject matter of the invention is a microbial carbonyl dehydrogenase which comprises the amino acid sequence MTDRLKNKVA (SEQ ID No 43) N-terminally and/or the amino acid sequence AEFVVDGGYLAQ (SEQ ID No 44) C-terminally and/or one of the internal partial sequences VVITGRRAN (SEQ ID No 45), GGASIINMS (SEQ ID No 46), TQTPMGHI (SEQ ID No 47) or GYIKTPLVDG (SEQ ID No 48).
• the invention relates to protein fragments which are characterized in that they represent fragments of the amino acid sequence SEQ ID No 5, with a number of more than 18 amino acids per fragment.
• a further subject matter of the invention is a microbial carbonyl dehydrogenase which comprises the amino acid sequence MKALQYTKIGS (SEQ ID No 49) N-terminally and/or the amino acid sequence LAAGTVRGRAVIVP (SEQ ID No 50) C-terminally and/or one of the internal partial sequences CHSDEFVMSLSE (SEQ ID No 51), VYGPWGCGRC (SEQ ID No 52), VSLTDAGLTPYHA (SEQ ID No 53), LRAVSAATVIAL (SEQ ID No 54) or DFVGADPTI (SEQ ID No 55).
• the invention relates to protein fragments which are characterized in that they represent fragments of the amino acid sequence SEQ ID No 6, with a number of more than 26 amino acids per fragment.
• a further subject matter of the invention is a microbial carbonyl dehydrogenase which comprises the amino acid sequence MKAIQIIQ (SEQ ID No 56) N-terminally and/or the amino acid sequence DLRGRAVVVP (SEQ ID No 57) C-terminally and/or one of the internal partial sequences TAAGACHSD (SEQ ID No 58), TPYHAIKPSLP(SEQ ID No 59), DFVGLQPT (SEQ ID No 60), VYGAWGCG (SEQ ID No 61), DDARHLVP (SEQ ID No 62), MTLGHEGA (SEQ ID No 63) or GGLGHVGIQLLRHL (SEQ ID No 64).
• the invention relates to a cloning vector comprising one or several nucleic acid sequences coding for the carbonyl reductases according to SEQ ID No 1, SEQ ID No 2, SEQ ID No 3, SEQ ID No 4, SEQ ID No 5, SEQ ID No 6, SEQ ID No 7, SEQ ID No 8, SEQ ID No 129 or homologues thereof.
• the invention comprises a cloning vector which, in addition to the carbonyl reductase, includes a suitable enzyme for the regeneration of NAD(P) such as, e.g., formate dehydrogenases, alcohol dehydrogenases or glucose dehydrogenase.
• the invention relates to an expression vector located in a bacterial, insect, plant or mammalian cell and comprising a nucleic acid sequence which codes for the carbonyl reductases according to SEQ ID No 1, SEQ ID No 2, SEQ ID No 3, SEQ ID No 4, SEQ ID No 5, SEQ ID No 6, SEQ ID No 7, SEQ ID No 8, SEQ ID No 129 or homologues thereof and is linked in an appropriate way to an expression control sequence.
• the invention relates to a recombinant host cell which is a bacterial, yeast, insect, plant or mammalian cell and has been transformed or transfected with such an expression vector as well as to a production process for obtaining a carbonyl reductase based on the cultivation of such a recombinant host cell.
• Suitable cloning vectors are, for example, ppCR-Script, pCMV-Script, pBluescript (Stratagene), pDrive cloning Vector (Quiagen, Hilden, Germany), pS Blue, pET Blue, pET LIC-vectors (Novagen, Madison, USA) and TA-PCR cloning vectors (Invitrogen, Düsseldorf, Germany).
• Suitable expression vectors are, for example, pKK223-3, pTrc99a, pUC, pTZ, pSK, pBluescript, pGEM, pQE, pET, PHUB, pPLc, pKC30, pRM1/pRM9, pTrxFus, pAS1, pGEx, pMAL or pTrx.
• Suitable expression control sequences are, for example, trp-lac (tac)-promoter, trp-lac (trc)-promoter, lac-promoter, T7-promoter or ⁇ pL-promoter.
• the oxidoreductases according to SEQ ID No 1, SEQ ID No 2, SEQ ID No 3, SEQ ID No 4, SEQ ID No 5, SEQ ID No 6, SEQ ID No 7, SEQ ID No 8, SEQ ID No 129 or homologues thereof can be obtained in such a manner that the above-mentioned recombinant E. coli cells are cultivated, the expression of the respective oxidoreductase is induced and subsequently, after about 10 to 18 hours (h), the cells are digested by ultrasonic treatment, by wet grinding with glass beads in a globe mill (Retsch, GmbH, Haan Germany 10 min, 24 Hz) or using a high-pressure homogenizer.
• the cell extract obtained can either be used directly or purified further.
• the cell extract is, e.g., centrifuged and the supernatant obtained is subjected to ion exchange chromatography, for example, by ion exchange chromatography on Q-Sepharose Fast Flow® (Pharmacia).
• the invention relates to a process for the enantioselective enzymatic reduction of a keto compound to the corresponding chiral hydroxy compound, wherein the keto compound is reduced with an oxidoreductase in the presence of a cofactor, characterized in that an oxidoreductase is used which has an amino acid sequence in which
• R 1 stands for one of the moieties 1) —(C 1 -C 20 )-alkyl, wherein alkyl is linear-chain or branched, 2) —(C 2 -C 20 )-alkenyl, wherein alkenyl is linear-chain or branched and optionally contains up to four double bonds, 3) —(C 2 -C 20 )-alkynyl, wherein alkynyl is linear-chain or branched and optionally contains up to four triple bonds, 4) —(C 6 -C 14 )-aryl, 5) —(C 1 -C 8 )-alkyl-(C 6 -C 14 )-aryl, 6) —(C 5 -C 14 )-heterocycle which is unsubstituted or substituted one, two or three times by —OH, halogen, —NO 2 and/or —NH 2 , or 7) —(C 3 -C 7 )-cycloalkyl,
• the invention relates to a process for the enantioselective enzymatic reduction of a keto compound to the corresponding chiral hydroxy compound, wherein the keto compound is reduced with an oxidoreductase in the presence of a cofactor, which process is characterized in that an oxidoreductase is used which
• aromatic carbon moieties comprising 6 to 14 carbon atoms within the ring are understood —(C 6 -C 14 )-aryl moieties are, for instance, phenyl, naphthyl, e.g., 1-naphthyl, 2-naphthyl, biphenylyl, e.g., 2-biphenylyl, 3-biphenylyl and 4-biphenylyl, anthryl or fluorenyl.
• Biphenylyl moieties, naphthyl moieties and in particular phenyl moieties are preferred aryl moieties.
• halogen an element from the family of fluorine, chlorine, bromine or iodine is understood.
• —(C 1 -C 20 )-alkyl a hydrocarbon moiety is understood the carbon chain of which is linear-chain or branched and comprises 1 to 20 carbon atoms, for example, methyl, ethyl, propyl, isopropyl, butyl, tertiary butyl, pentyl, hexyl, heptyl, octyl, nonenyl or decanyl.
• —C 0 -alkyl a covalent bond is understood.
• cyclic hydrocarbon moieties such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl are understood.
• —(C 5 -C 14 )-heterocycle stands for a monocyclic or bicyclic 5-membered to 14-membered heterocyclic ring which is partially or completely saturated. N, O and S are examples of heteroatoms.
• Preferred compounds of Formula I are, for example, ethyl-4-chloroacetoacetate, methylacetoacetate, ethyl-8-chloro-6-oxooctanoic acid, ethyl-3-oxovaleriate, 4-hydroxy-2-butanone, ethyl-2-oxovaleriate, ethyl-2-oxo-4-phenylbutyric acid, ethyl pyruvate, ethyl phenyl glyoxylate, 1-phenyl-2-propanone, 2-chloro-1-(3-chlorophenyl)ethane-1-one, acetophenone, 2-octanone, 3-octanone, 2-butanone, 1-[3,5-bis(trifluoromethyl)phenyl]ethane-1-one, 2,5-hexanedione, 1,4-dichloro-2-butanone, acetoxyacetone, phenacy
• the oxidoreductases can be used either in a completely purified or in a partially purified state or the process can be performed with cells containing the oxidoreductases according to the invention. In doing so, the cells used can be provided in a native, permeabilized or lysed state.
• the cloned oxidoreductases according to SEQ ID No 1, SEQ ID No 2, SEQ ID No 3, SEQ ID No 4, SEQ ID No 5, SEQ ID No 6, SEQ ID No 7, SEQ ID No 8, SEQ ID No 129 or homologues thereof, respectively, are preferably used.
• the enzyme unit 1 U corresponds to the enzyme amount which is required for converting 1 ⁇ mol of the compound of Formula I per minute (min).
• the enzymatic reduction itself proceeds under mild conditions so that the alcohols produced will not react further.
• the processes according to the invention exhibit a high residence time and an enantiomeric purity of normally more than 95% of the chiral alcohols produced and a high yield, relative to the amount of keto compounds that is employed.
• the carbonyl compound is used in an amount of from 3% to 50%, based on the total volume, preferably from 5% to 40%, in particular from 10%-30%.
• a preferred embodiment of the invention is characterized in that the NAD or NADP formed during the reduction is continuously reduced to NADH or NADPH, respectively, with a cosubstrate.
• primary and secondary alcohols such as ethanol, 2-propanol, 2-butanol, 2-pentanol, 3-pentanol, 4-methyl-2-pentanol, 2-heptanol, 2-octanol or cyclohexanol are preferably used as the cosubstrate.
• Said cosubstrates are reacted to the corresponding aldehydes or ketones and NADH or NADPH, respectively, with the aid of an oxidoreductase and NAD or NADP, respectively. This results in a regeneration of the NADH or NADPH, respectively.
• the proportion of the cosubstrate for the regeneration thereby ranges from 5 to 95% by volume, based on the total volume.
• an additional alcohol dehydrogenase can be added.
• Suitable NADH-dependent alcohol dehydrogenases are obtainable, for example, from baker's yeast, from Candida boidinii, Candida parapsilosis or Pichia capsulata .
• suitable NADPH-dependent alcohol dehydrogenases are present in Lactobacillus brevis (DE 196 10 984 A1), Lactobacillus minor (DE 101 19 274), Pseudomonas (U.S. Pat. No. 5,385,833) or in Thermoanaerobium brockii .
• Suitable cosubstrates for these alcohol dehydrogenases are the already mentioned secondary alcohols such as ethanol, 2-propanol (isopropanol), 2-butanol, 2-pentanol, 4-methyl-2-pentanol, 2-octanol or cyclohexanol.
• cofactor regeneration can also be effected, for example, 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.
• the processes according to the invention are preferably carried out without such an additional dehydrogenase, i.e., substrate-coupled coenzyme regeneration takes place.
• 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 value of from 6 to 9.
• a buffer e.g., a potassium phosphate, tris/HCl or triethanolamine buffer, having a pH value of from 5 to 10, preferably a pH value of from 6 to 9.
• the buffer can comprise ions for stabilizing or activating the enzymes, for example, zinc ions or magnesium ions.
• the temperature is suitably in the range of from about 10° C. to 70° C., preferably from 20° C. to 40° C.
• the enzymatic conversion is effected in the presence of an organic solvent which is not miscible with water or miscible with water only to a limited extent.
• Said solvent is, for example, a symmetric or unsymmetric di(C 1 -C 6 )alkyl ether, a straight-chain or branched alkane or cycloalkane or a water-insoluble secondary alcohol that is simultaneously representing the cosubstrate.
• the preferred organic solvents are, for example, diethyl ether, tertiary butyl methyl ether, diisopropyl ether, dibutyl ether, butyl acetate, heptane, hexane, 2-octanol, 2-heptanol, 4-methyl-2-pentanol or cyclohexane.
• the solvent can, at the same time, also serve as a cosubstrate for cofactor regeneration.
• the reaction batch consists of an aqueous and an organic phase.
• the substrate is distributed between the organic and aqueous phases according to its solubility.
• the organic phase generally 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 a large surface is produced between them.
• the NAD or NADP, respectively, formed during the enzymatic reduction can be reduced back to NADH or NADPH, respectively, using a cosubstrate, as described above.
• the concentration of the cofactor NADH or NADPH, respectively, in the aqueous phase generally ranges from 0.001 mM to 1 mM, in particular from 0.01 mM to 0.1 mM.
• a stabilizer of the oxidoreductase/dehydrogenase can, in addition, 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.
• the components are transferred individually into the reaction vessel and stirred under an atmosphere of, e.g., nitrogen or air.
• the reaction time ranges from 1 hour to 48 hours, in particular from 2 hours to 24 hours.
• the reaction mixture is processed.
• the aqueous phase is separated, the organic phase is filtered.
• the aqueous phase can optionally be extracted once more and can be processed further like the organic phase.
• the solvent is optionally evaporated from the filtered organic phase.
• the invention relates to a process for obtaining chiral hydroxy compounds of Formula II,
• R 1 and R 2 are as defined above, which is characterized in that
• the reaction conditions are basically the same as in the above-mentioned process for the enantiospecific reduction of the keto compound of Formula I. However, instead of an enantioselective reduction of the keto compound of Formula I from the racemic mixture of the compound of Formula II, only one enantiomer of the hydroxy compound of Formula II is oxidized enantioselectively into the corresponding keto compound. Thus, the opposite enantiomer of the hydroxy compound of Formula II remains and can be isolated.
• the corresponding ketones thereof such as acetone are used in the process for the regeneration of the NAD.
• the acetone and NAD(P)H are converted into NAD and isopropanol by means of the oxidoreductase according to the invention or an additional dehydrogenase.
• the yeast strains Rhodotorula mucilaginosa DSMZ 70825, Pichia farinosa DSMZ 3316, Candida nemodendra DSMZ 70647, Pichia stipidis DSMZ 3651 and Pichia trehalophila DSMZ 70391, Lodderomyces elongisporus DSMZ 70320 were cultivated in the following medium: yeast extract (3), malt extract (3), peptone (5) and glucose (10) (the numbers in brackets are, in each case, g/l).
• the medium was sterilized at 121° C. and the yeasts were cultivated without further pH-adjustment at 25° C. and on a shaker at 160 revolutions per minute (rpm).
• the strain Leuconostoc carnosum DSMZ 5576 was cultivated in the following medium: glucose (20), yeast extract (5), meat extract (10), diammonium hydrogen citrate (2), sodium acetate (5), magnesium sulfate (0.2), manganese sulfate (0.05), dipotassium hydrogen phosphate (2).
• the medium was sterilized at 121° C. and the organism was cultivated at 30° C. without further pH-adjustment or oxygen supply.
• the strain Microbacterium spec. DSMZ 20028 was cultivated on a medium of yeast extract (3) and trypticase soy flour (30) at 30° C. and with 160 revolutions per minute (rpm).
• the strain Gordonia rubripertincta DSMZ 43570 was cultivated on a medium of yeast extract (4), glucose (4), malt extract (10) and CaCO 3 (2) at 37° C. and with 160 revolutions per minute (rpm).
• the supernatant (lysate) obtained after 2 min of centrifugation at 12.000 rpm was used in the following activity screening and for determining the enantiomeric excess (ee-value).
• Different ketones such as 2-butanone, 2-octanone, ethyl-4-chloroacetoacetate, acetophenone or ethyl-2-oxo-4-phenylbutyric acid were used as substrates.
• the reaction was pursued for 1 min at 340 nm.
• ee(%) (( R -alcohol ⁇ S -alcohol)/( R -alcohol+ S -alcohol)) ⁇ 100.
• DSMZ stands for Deutsche Sammlung für Mikroorganismen und Zellkulturen, Mascheroder Weg 1b, 38124 Braunschweig. Definition of enzyme units: 1 U corresponds to the enzyme amount which is required for converting 1 ⁇ mol of substrate per min.
• All oxidoreductases according to the invention could be purified by different combinations of ion exchange chromatography, e.g., on Q-Sepharose Fast Flow (Pharmacia) or Uno Q (Biorad, Kunststoff, Germany), hydrophobic interaction chromatography, e.g., on Octyl-Sepharose Fast Flow or Butyl-Sepharose Fast Flow (Pharmacia), ceramic hydroxylapatite chromatography and gel permeation.
• the oxidoreductase-containing fractions were combined and concentrated to an appropriate volume by means of ultrafiltration (exclusion limit 10 kDa).
• the concentrated fractions of the oxidoreductase were further purified by Uno Q.
• Catalase (232 kDa), aldolase (158 kDa), albumin (69.8 kDa) and ovalbumin (49.4 kDa) were used as molecular weight standards.
• the enzyme activity of the oxidoreductase was determined in the test system according to Example 1 (batch activity screening), and the determination of the protein amount was performed according to Lowry et al. Journal of Biological Chemistry, 193 (1951): 265-275 or Peterson et al., Analytical Biochemistry, 100 (1979): 201-220). The quotient of enzyme activity to protein amount yields the specific activity, wherein the conversion of 1 ⁇ mol per min corresponds to 1 unit (U).
• the oxidoreductase-containing fractions were combined and concentrated to an appropriate volume by means of ultrafiltration (exclusion limit 10 kDa).
• the concentrated fractions of the oxidoreductase were further purified by Uno Q.
• PVDF-membrane After gel permeation in a 10% sodium dodecyl sulfate (SDS) gel, the enzyme preparations according to Example 2 were separated and transferred onto a polyvinylidene difluoride membrane (PVDF-membrane).
• the conspicuous band was subjected to N-terminal sequencing via Edman degradation (Procise 492 (PE-Biosystems)).
• Chromosomal DNA is extracted according to the method described in “Molecular Cloning” by Manniatis & Sambrook.
• the resulting nucleic acid serves as a template for the polymerase chain reaction (PCR) with degenerate primers.
• PCR polymerase chain reaction
• 5′-primers are derived from the amino acid sequence (SEQ ID No. 66; 72; 80) and 3′-primers are derived from the amino acid sequence (SEQ ID No. 67; 73, 81), involving the genetic code specific for the organism (SEQ ID No. 68; 69; 74; 75; 82; 83).
• Amplification is carried out in a PCR buffer [67 mM Tris-HCl (pH 8.3), 16 mM (NH 4 ) 2 SO 4 , 115 mM MgCl 2 , 0.01% Tween 20], 0.2 mM desoxynucleotide triphosphate mix (dNTPs), 40 pMol of each primer and 2.5 U BioTherm Star Polymerase (Genecraft, Lüdingshausen, Germany)].
• dNTPs desoxynucleotide triphosphate mix
• the specific fragment resulting from the polymerase chain reaction is ligated into the TA-cloning vecor pCR2.1 (Invitrogen, Düsseldorf, Germany) and sequenced with the primers M13 rev (SEQ ID NO 65) and M13 uni (SEQ ID NO 128) with the aid of an ABI DNA sequencer.
• the 5′- and 3′-terminal regions of the gene-coding sequence are determined using the RACE method (rapid amplification of cDNA ends). Based on the nucleic acid sequence of the specific fragment, oligonucleotides for 3′-RACE and 5′-RACE are constructed. Total RNA prepared from the cells serves as a template for the synthesis of the first cDNA strand using the 3′-RACE system (Invitrogen, Düsseldorf, Germany). This is followed by an amplification and a reamplification of the specific cDNA with the aid of 3′-RACE oligonucleotides (SEQ ID No. 76; 77; 84; 85). Subsequently, the batch is applied onto a 1% agarose gel for analysis. The specific fragment carrying the missing 3′-flanking sequence information is isolated, ligated into a TA-cloning vector pCR2.1 and sequenced.
• RACE rapid amplification of cDNA ends
• the coding and non-coding 5′-terminal sequences are determined using the 5′-RACE system (Invitrogen).
• mRNA from the total RNA obtained previously is enriched with the aid of Oligo dT-cellulose (NEB, Beverly, USA) and employed for the synthesis of the first cDNA strand with the gene-specific oligonucleotides (SEQ ID No. 70; 71; 78; 79; 86; 87).
• the subsequent amplification and reamplification of the specific cDNA results in a fragment which is ligated into a pCR2.1 TA-cloning vector (Invitrogen) for analysis.
• the plasmid containing the fragment is analyzed with the aid of an ABI DNA sequencer.
• the missing sequence information about the 5′-end of the gene is obtained.
• Rhodotorulla Protein mucilaginosa Pichia farinosa Pichia stipitis Partially VATAVETFGR LLTQTLALEQAK ADQVLLK sequenced (SEQIDNo 66) (SEQIDNo 72) (SEQIDNo 80) peptides FGEAVEQAR YNFTNKVAIITGGI ISFNLGDLALR (SEQIDNo 67) (SEQIDNo 73) (SEQIDNo 81) Primer for CCRAAYTCVACVGCVGTSGC YTGYTCYAANGCYAADGTYTG GCYGAYCARGTNTTRTTRAAR Touch-Down (SEQIDNO 68) (SEQIDNo 74) (SEQIDNo 82) PCR GCCTGYTCGACVGCYTCRCC CHAAYAARGTNGCHATHATYACHGG CTYAARGCYAARTCDCCYAAR (SEQIDNo 69) (SEQIDNo 75) (SEQIDNo 83) Primer for CAACGTTCTGAA
• chromosomal DNA serves as the template.
• the DNA section coding for the respective oxidoreductase is amplified with the aid of Platinum pfx Polymerase (Invitrogen). After purification over 1% agarose gel, the resulting PCR product is treated with appropriate DNA endonucleases and ligated into the backbone of the pET21a vector (Novagen, Madison, USA) or into the backbone of the pQE70 vector (Qiagen, Hilden, Germany), respectively, which backbone has been treated with the same endonucleases.
• the expression construct formed is taken into the expression strain BL21 Star (Invitrogen) or RB791 ( E. coli genetic stock, Yale, USA), respectively.
• chromosomal DNA was, for example, extracted from the fresh cells of Pichia Farinosa according to the method described in “Molecular cloning” by Manniatis & Sambrook.
• the resulting nucleic acid served as a template for a Touch-Down PCR with oligonucleotides SEQ ID No. 74; 75.
• After 8 minutes of activating the Biotherm Star Polymerase in a PCR Cycler BioRad, Hercules, USA, the following 30 temperature cycles were programmed for an identification of the specific DNA fragment:
• a sequence analysis of the gene fragment having a length of 550 bp showed an open reading frame of 174 amino acid residues, in which the two sequence fragments of the N-terminus and of the internal peptide could also be found.
• oligonucleotides for a 3′-RACE SEQ ID No 76; 77
• a 5′-RACE SEQ ID No 78; 79; 88
• 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 vortexed in each case for 30 sec, in total for 10 min, and was cooled on ice for 30 sec.
• 5 ml of an ice-cold LETS buffer were added and thoroughly vortexed once again. Said cell suspension was centrifuged at 11000 g and at 4° C. for 5 min.
• RNA was precipitated at ⁇ 20° C. for 4 h by adding 1/10 vol. of 5 M LiCl 2 .
• the synthesis of the first cDNA strand was carried out using the 3′RACE system (Invitrogen, Düsseldorf, Germany).
• the specific cDNA was amplified with the oligonucleotides SEQ ID No 76 and AUAP (Invitrogen, Düsseldorf, Germany) in the reaction: 67 mM Tris-HCl (pH 8.3), 16 mM (NH 4 ) 2 SO 4 , 115 mM MgCl 2 , 0.01% Tween 20], 0.2 mM desoxynucleotide triphosphate mix (dNTPs), 10 pMol of each primer and 2.5 U BioTherm Star Polymerase (Genecraft, Lüdingshausen, Germany) and with the following 30 temperature cycles: 94° C. 40 sec, 55° C. 40 sec, 72° C. 1 min.
• the PCR signal was increased via a nested PCR with primer SEQ ID No 77 and primer UAP (Invitrogen, Düsseldorf, Germany) with 30 temperature cycles: 94° C. 40 sec, 55° C. 40 sec, 72° C. 50 sec.
• the result was a specific DNA fragment having a size of approximately 400 bp, which was ligated after isolation into the vector pCR2.1 (Invitrogen) from the 1% agarose gel.
• the sequence analysis of the DNA section having a length of 382 bp yielded sequence information about the 3′-extension up to the stop codon and the poly-A loop of the cDNA coding for the oxidoreductase from Pichia farinosa.
• RNA prepared from the cells of Pichia farinosa were used.
• the synthesis of gene-specific cDNA was performed using the 5′RACE system (Invitrogen, Düsseldorf, Germany) and the oligonucleotide SEQ ID No 78.
• the resulting gene-specific cDNA was subjected to a homopolymeric dCTP addition reaction.
• the PCR signal was increased via a nested PCR with primer SEQ ID No 88 and primer UAP (Invitrogen, Düsseldorf, Germany) with 30 temperature cycles: 94° C. 40 sec, 55° C. 40 sec, 72° C. 1 min.
• the result was a specific DNA fragment having a size of approximately 350 bp, which was ligated after elution into the vector pCR2.1 (Invitrogen) from the 1% agarose gel.
• the sequence analysis of the DNA segment having a length of 352 bp yielded sequence information about the 5′-end of the cDNA coding for the alcohol dehydrogenase/reductase.
• the DNA segment coding for the protein has a total length of 765 bp (SEQ ID No 10) and an open reading frame of 254 amino acids (SEQ ID No 2).
• Chromosomal DNA of the Pichia farinosa cells was used as a template for the generation of the full-length DNA in a polymerase chain reaction [10 mM Tris-HCl, (pH 8.0); 50 mM KCl; 10 mM MgSO 4 ; 0.2 mM dNTP Mix; 20 pMol Primer SEQ ID No 91 or, respectively, 20 pMol Primer SEQ ID No 92, 20 pMol Primer SEQ ID No 93 and 2 U Platinum pfx Polymerase (Invitrogen)] and with temperature cycles:
• Cycle 1 94° C., 2 min Cycle 2 ⁇ 30 94° C., 15 sec 56° C., 20 sec 68° C., 1 min 15 sec.
• the resulting PCR product was treated with Nde I and Hind III, or with Sph I and Hind III, respectively, and was ligated into the backbone of the vector pET21a (Novagen, Madison, USA) or pQE70 (Qiagen, Hilden, Germany), respectively, which backbone had been treated with the same endonucleases.
• plasmid DNAs of ampicillin-resistant colonies were checked for the correctness of the ligation that had been performed by means of a restriction analysis with the endonucleases Nde I or Sph I and Hind III, respectively.
• the DNA of the vectors positive for the insert was transformed into the expression strain BL21 Star (Invitrogen) and RB791 ( E. coli genetic Stock, Yale, USA), respectively.
• Genomic DNA is extracted according to the method described in “Molecular cloning” by Manniatis & Sambrook.
• the resulting nucleic acid serves as a template for the polymerase chain reaction (PCR) with degenerate primers.
• PCR polymerase chain reaction
• 5′-primers are derived from the amino acid sequence (SEQ ID No 104; 112) and 3′-primers are derived from the amino acid sequence (SEQ ID No 105; 113), involving the genetic code specific for the organism (SEQ ID No 106; 107; 114; 115).
• Amplification is carried out in a PCR buffer [67 mM Tris-HCl (pH 8.3), 16 mM (NH 4 ) 2 SO 4 , 115 mM MgCl 2 , 0.01% Tween 20], 0.2 mM desoxynucleotide triphosphate mix (dNTPs), 40 pMol of each primer and 2.5 U BioTherm Star Polymerase (Genecraft, Lüdingshausen, Germany)].
• dNTPs desoxynucleotide triphosphate mix
• the specific fragment resulting from the polymerase chain reaction is ligated into the TA-cloning vecor pCR2.1 (Invitrogen, Düsseldorf, Germany) and sequenced with the primers M13 rev (SEQ ID No 65) and M13 uni (SEQ ID No 128) with the aid of an ABI DNA sequencer.
• the 5′- and 3′-terminal regions of the gene-coding sequence are determined using the inverse polymerase chain reaction method (iPCR). Based on the nucleic acid sequence of the specific internal fragment, oligonucleotides SEQ ID No 100; 101; 102; 103; 108; 109; 110; 111; 116; 117; 118; 119 are constructed. Genomic DNA is digested by means of a restriction endonuclease and used in a religation so that smaller DNA sections can circulate. Said religation mixture is then used as a template for an iPCR and primers SEQ ID No 100; 102; 108; 110; 116; 118.
• iPCR inverse polymerase chain reaction method
• the PCR signal is increased by a subsequent nested PCR with primers SEQ ID No 101; 103; 109; 111; 117; 119.
• the resulting specific fragment is ligated after elution into the vector pCR2.1 (Invitrogen) from the 1% agarose gel.
• sequence analysis of the vector pCR2.1 containing the fragment yields the missing sequence information about 3′- and 5′-coding regions of the alcohol dehydrogenase/reductase gene.
• genomic DNA was, for example, extracted from the fresh cells of Microbacterium sp. according to the method described in “Molecular cloning” by Manniatis & Sambrook.
• the resulting nucleic acid served as a template for a PCR with 30 pMol each of oligonucleotides SEQ ID No. 106; 107.
• After 10 minutes of activating the Biotherm Star Polymerase in a PCR Cycler BioRad, Hercules, USA
• the following 30 temperature cycles were programmed for an identification of the specific DNA fragment:
• a sequence analysis of the gene fragment having a length of 1002 bp showed an open reading frame of 334 amino acid residues, in which the two sequence fragments of the N-terminus and of the internal peptide could also be found.
• oligonucleotides SEQ ID NO 108; 109; 110; 111 for an inverse PCR (iPCR) were constructed.
• Genomic DNA (2.5 ⁇ g) from the cells of Microbacterium sp. was treated in a 50 ⁇ l batch with 20 U restriction endonuclease Sac I for 25 min. After the phenol:chloroform:isoamyl:alcohol (25:24:1) extraction of the entire batch and after precipitation with 1/10 vol. of 3M Na-acetate (pH5.2) and 2.5 vol. of ethanol, the DNA thus digested was transferred into 25 ⁇ l H 2 O. 5 ⁇ l (200 ng) thereof were used in a religation reaction in a total volume of 40 ⁇ l and 2 U of T4 ligase (Fermentas).
• the amplification was conducted with the following cycles:
• Cycle 1 95° C., 10 min Cycle 2 ⁇ 30 95° C., 1 min 56° C., 1 min 72° C., 2 min
• the amplification signal was increased in a nested PCR with the oligonucleotides SEQ ID No 109 and SEQ ID No 111.
• the amplification reaction was cooled down to 4° C. and applied as a whole onto a 1% agarose gel.
• the result was a specific fragment having a size of approximately 1000 bp.
• the fragment was ligated into the pCR2.1 vector (Invitrogen, Düsseldorf, Germany).
• the sequence analysis of the plasmid containing the fragment yielded information about the 5′- and 3′-flanking sequences.
• the DNA segment coding for the protein has a total length of 1044 bp ending in a stop codon (SEQ ID No 13) and exhibits an open reading frame of 347 amino acids (SEQ ID No 5).
• Genomic DNA of Microbacterium sp. cells was used as a template for the generation of the full-length DNA coding for the protein in a polymerase chain reaction using the GC-Rich PCR system (Roche, Mannheim, Germany) and 30 pMol oligonucleotides SEQ ID No 123 or SEQ ID No 124, respectively, with 30 pMol oligonucleotide SEQ ID No 125 and temperature cycles:
• Cycle 1 95° C., 3 min Cycle 2 ⁇ 30 95° C., 30 sec 59° C., 30 sec 72° C., 45 sec.
• the resulting PCR product was treated with Nde I and Hind III, or with Sph I and Hind III, respectively, and was ligated into the backbone of the vector pET21a (Novagen, Madison, USA) or pQE32 (Qiagen, Hilden, Germany), respectively, which backbone had been treated with the same endonucleases.
• plasmid DNAs of ampicillin-resistant colonies were checked for the correctness of the ligation that had been performed by means of a restriction analysis with the endonucleases Nde I or Sph I and Hind III, respectively.
• the DNA of the vectors positive for the insert was transformed into the expression strain BL21 Star (Invitrogen) and RB791 ( E. coli genetic Stock, Yale, USA), respectively.
• the expression of recombinant protein was induced by the addition of isopropyl thiogalactoside (IPTG) at a concentration of 0.1 mM. After 8 hours or after 16 hours, respectively, of induction at 25° C.
• IPTG isopropyl thiogalactoside
• the cells were harvested and frozen at ⁇ 20° C.
• 10 mg of cells were mixed with 500 ⁇ l of 100 mM TEA buffer pH 7.0 and 500 ⁇ l of glass beads and were digested for 10 min using a globe mill. The lysate obtained was then used for the respective measurements in a diluted state.
• the activity test was composed as follows: 870 ⁇ l of 100 mM TEA buffer pH 7.0, 160 ⁇ g NAD(P)H, 10 ⁇ l diluted cell lysate. The reaction was started with the addition of 100 ⁇ l of a 100 mM substrate solution to the reaction mixture.
• the buffers listed in Table 4 were produced. The concentration of the respective buffer components in each case amounted to 50 mM.
• 2-butanone or 2-octanone was used as the substrate.
• the reaction was pursued for 1 min at 340 nm.
• the enzymatic reaction in the respective buffer listed in Table 4 was analyzed.
• NAD(P) was used as the cofactor and 2-propanol or 2-octanol was used as the substrate.
• the determination of the activity of the recombinant oxidoreductases was examined by storing them in the buffer systems mentioned in Table 4.
• the different buffers 50 mM
• the oxidoreductase produced according to Example 4 was diluted therewith. After 30, 60 and 120 minutes of incubation, 10 ⁇ l were taken from the batch and used in the activity test according to Example 1.
• the enzyme activity for the oxidoreductases according to the invention was measured in the standard measuring batch in a temperature range of from 15° C. to 70° C.
• the temperature stability was determined for the range of from 15° C. to 70° C.
• a dilution of the oxidoreductases according to the invention was in each case incubated at the respective temperature for 60 min and 180 min and was subsequently measured at 30° C. with the above-mentioned test batch.
• Table 8 the temperature ranges in which the enzymes exhibited no less than 50% of the initial activity with an incubation lasting for 120 min are compiled for the oxidoreductases according to the invention.
• the substrate spectrum of the oxidoreductases according to the invention was determined by measuring the enzyme activity for reduction and oxidation with a number of ketones and alcohols. For this purpose, the standard measuring batch according to Example 1 was used with different substrates.
• the activity with methyl acetoacetate was set to 100% for all enzymes and all the other substrates were put in relation thereto.
• the stability of the novel oxidoreductases in aqueous/organic two-phase systems was examined by diluting the lysates obtained in Example 6 (from a recombinant expression) in an aqueous buffer suitable for the respective oxidoreductase (approx. 10 units/ml buffer). Then, the same volume of an organic solvent not miscible with water was added to the oxidoreductase diluted in the buffer and the batch was incubated at room temperature with constant thorough mixing (thermomixer at 170 rpm).
• the reaction mixture was reprocessed by first removing the 2-propanol using a rotary evaporator. Subsequently, the reaction mixture was extracted with ethyl acetate, the solvent was removed using a rotary evaporator, and the crude product was obtained.
• the crude product ethyl-(S)-4-chloro-3-hydroxybutyric acid obtained in this manner exhibited an enantiomeric excess of >99.5%.

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