US20110020887A1 - Process for the enantioselective enzymatic reduction of secodione derivatives - Google Patents

Process for the enantioselective enzymatic reduction of secodione derivatives Download PDF

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US20110020887A1
US20110020887A1 US12/518,025 US51802507A US2011020887A1 US 20110020887 A1 US20110020887 A1 US 20110020887A1 US 51802507 A US51802507 A US 51802507A US 2011020887 A1 US2011020887 A1 US 2011020887A1
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secodione
oxidoreductase
dehydrogenase
process according
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Antje Gupta
Anke Tschentscher
Maria Bobkova
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Cambrex IEP GmbH
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IEP GmbH
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group
    • C12P7/26—Ketones
    • C12P7/38—Cyclopentanone- or cyclopentadione-containing products
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P33/00—Preparation of steroids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P23/00—Preparation of compounds containing a cyclohexene ring having an unsaturated side chain containing at least ten carbon atoms bound by conjugated double bonds, e.g. carotenes
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the present invention relates to a process for the enantioselective enzymatic reduction of secodione derivatives of general formula I, wherein the secodione derivative is reduced with an oxidoreductase/dehydrogenase in the presence of NADH or NADPH as a cofactor.
  • the industrial preparation of steroid hormones occurs in two ways which are independent of each other, namely, on the one hand, starting out from naturally occurring steriod compounds from plant sources and, on the other hand, in a totally synthetic manner via an enantioselective synthesis from prochiral precursors.
  • the steroid total synthesis is increasingly gaining in importance, particularly since it also allows the introduction of structural elements which are not contained in naturally occurring steriods.
  • a key step in the preparation of enantiomerically pure steroid compounds is the conversion of the compound of formula I (e.g., II and III) into an optically active compound with a preformed asymmetric C-13 by enantioselective reduction of one of the keto groups to the hydroxy group.
  • the resulting optically active hydroxy secosteroid compounds (secoles, Formulae VI to IX) can subsequently be processed further into chiral steroid compounds by cyclization, while chirality is maintained.
  • yeasts of the genus Saccharomyces such as, e.g., S. uvarum can be used advantageously for preparing, for example, the respective 17-beta-hydroxy secosteroids (Kosmol et al; Liebigs Ann. Chem. 701, 199 (1967)).
  • Other yeast strains such as, e.g., Saccharomyces drosophilarum reduce secodione preferably to the corresponding 14-alpha-hydroxy secosteroid (Acta microbiol. Acad. Sci. hung. 22, 463-471 (1975)).
  • said object is achieved according to the invention by a process for the enantioselective enzymatic reduction of secodione derivatives of general formula I,
  • R 1 is hydrogen or a C 1 -C 4 alkyl group
  • R 2 is hydrogen, a C 1 -C 8 alkyl group or a protective group for OH known in prior art, such as an ester
  • R 3 is hydrogen, a methyl group or a halide
  • the secodione derivative represents a benzene ring or a C 6 ring having 0, 1 or 2 C—C double bonds, a double bond is optionally included at positions 6/7 or 7/8, and the carbon at positions 1, 2, 4, 5, 6, 7, 8, 9, 11, 12 and 16 is independently substituted with hydrogen, a C 1 -C 4 alkyl group, a halide or a phenyl group, wherein the secodione derivative is reduced with an oxidoreductase/dehydrogenase in the presence of NADH or NADPH as a cofactor, which process is characterized in that the secodione derivative is used in the reaction batch at a concentration of ⁇ 10 g/l and the oxidized cofactor NAD or NADP formed by the oxidoreductase/dehydrogenase is regenerated continuously.
  • This process represents a significant improvement of the enantioselective enzymatic reduction of secodione derivatives over the prior art.
  • the process according to the invention allows the reduction of secodione derivatives to the different corresponding hydroxy secosteroids with free enzymes at concentration ranges far exceeding those described in the prior art.
  • the above-mentioned object is achieved according to the invention by a process for the enantioselective enzymatic reduction of secodione derivatives of general formula I, wherein the secodione derivative is reduced with an oxidoreductase/dehydrogenase in the presence of NADH or NADPH as a cofactor, which process is characterized in that the oxidoreductase/dehydrogenase
  • a) comprises an amino acid sequence in which at least 50% of the amino acids are identical to those of amino acid sequence SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5, b) is encoded by the nucleic acid sequence SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10, or c) is encoded by a nucleic acid sequence which hybridizes to SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10 under stringent conditions.
  • the inventors have identified oxidoreductases which are capable of reducing secodione derivatives to hydroxy secosteroids and which can be produced recombinantly on an industrial scale. Significantly higher substrate concentrations can be achieved by the process according to the invention than with the currently used whole-cell processes.
  • the oxidoreductase having the sequence SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5 or a polypeptide derivable from those polypeptides, respectively can be used either in a completely purified state, in a partially purified state or as cells containing the polypeptide SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.
  • the cells used can be provided in a native, permeabilized or lysed state.
  • the oxidoreductases and derivatives derivable therefrom, respectively are overexpressed in a suitable host organism such as, e.g., Escherichia coli , and the recombinant polypeptide is used for the reduction of secodione derivatives of general formula I.
  • a suitable host organism such as, e.g., Escherichia coli
  • the recombinant polypeptide is used for the reduction of secodione derivatives of general formula I.
  • a DNA sequence SEQ ID NO:6 which codes for a polypeptide with SEQ ID NO:1 is obtainable, for example, from the genome of the organism Chloroflexus aurantiacus DSM 635.
  • a DNA sequence SEQ ID NO:7 which codes for a polypeptide with SEQ ID NO:2 is obtainable, for example, from the genome of the organism Rubrobacter xylanophilus DSM 9941.
  • a DNA sequence SEQ ID NO:8 which codes for a polypeptide with SEQ ID NO:3 is obtainable from a yeast Candida magnoliae CBS 6396.
  • Oxidoreductases of SEQ ID NO:4 and SEQ ID NO:5 are obtainable, for example, from Candida magnoliae DSMZ 70638 by homology screening.
  • a nucleic acid sequence which hybridizes, for example, to SEQ ID NO:6 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:6 or partial sequences of SEQ ID NO:6 as a DNA probe.
  • the polynucleotide immobilized on a filter is hybridized, for example, to SEQ ID NO:6 in a 0.7-1 M NaCl solution at 60° C.
  • Hybridization is carried out as described, e.g., in Molecular Cloning, A Laboratory Manual, Second Edition (Cold Spring Harbor Laboratory Press, 1989) or in similar publications.
  • a 1-fold SSC solution is understood to be a mixture consisting of 150 mM NaCl and 15 mM sodium citrate.
  • a polynucleotide which hybridizes to the polynucleotides SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10 from the sequence list under the above-mentioned stringent conditions should exhibit at least 60% sequence identity to the polynucleotide sequences SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10, better an identity of at least 80%, even better an identity of 95%.
  • the above-mentioned object is achieved according to the invention by a process for the enantioselective enzymatic reduction of secodione derivatives of general formula I, wherein the secodione derivative is reduced with an oxidoreductase/dehydrogenase in the presence of NADH or NADPH as a cofactor, which process is characterized in that the oxidoreductase/dehydrogenase has a length of from 230 to 260 amino acids and comprises one or several of the partial sequences selected from the group consisting of [sequences SEQ ID NO:18 to SEQ ID NO:42]
  • NADH or NADPH is used as the cofactor.
  • NADP nicotinamide adenine dinucleotide phosphate
  • NADPH reduced nicotinamide adenine dinucleotide phosphate
  • NAD means nicotinamide adenine dinucleotide
  • NADH means reduced nicotinamide adenine dinucleotide.
  • the oxidoreductase/dehydrogenase is used in the reaction batch at a concentration of ⁇ 10 g/l and the oxidized cofactor NAD or NADP formed by the oxidoreductase/dehydrogenase is regenerated continuously, the oxidoreductase/dehydrogenase
  • a) comprises an amino acid sequence in which at least 50% of the amino acids are identical to those of amino acid sequence SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5, b) the oxidoreductase/dehydrogenase is encoded by the nucleic acid sequence SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10, or c) the oxidoreductase/dehydrogenase is encoded by a nucleic acid sequence which hybridizes to SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10 under stringent conditions.
  • the oxidoreductase/dehydrogenase has a length of from 230 to 260 amino acids and comprises one or several of the partial sequences selected from the group consisting of [sequences SEQ ID NO:18 to SEQ ID NO:42] nalvtgasrgig, nalvtggsrgig, nalitggsrgig, nalitgasrgig, nalitggsrgmg, halvtgasrgig, gysvtla, gynvtla, gysvtlv, gynvtlv, fkgaplpa, fkaaplpa, fvsnag, ffsnag
  • the oxidized cofactor NAD or NADP formed by the oxidoreductase/dehydrogenase is preferably regenerated continuously.
  • the oxidized cofactor NAD or NADP is regenerated by oxidation of an alcohol.
  • primary and secondary alcohols such as ethanol, 2-propanol, 2-butanol, 2-pentanol, 3-pentanol, 4-methyl-2-pentanol, 2-hexanol, 2-heptanol, 2-octanol or cyclohexanol are preferably used as cosubstrates.
  • the proportion of the cosubstrate for the regeneration may range from 5 to 95% by volume, based on the total volume.
  • a secondary alcohol having the general formula R X R Y CHOH is preferably used for cofactor regeneration, wherein R X and R Y independently of each other are hydrogen, a branched or unbranched C 1 -C 8 alkyl group and C total ⁇ 3.
  • an oxidoreductase/dehydrogenase is additionally added for the regeneration of the cofactor.
  • Suitable NADH-dependent alcohol dehydrogenases are, for example, obtainable from baker's yeast, from Candida parapsilosis (CPCR) (U.S. Pat. No. 5,523,223 and U.S. Pat. No. 5,763,236, Enzyme Microb. Technol., 1993, 15(11):950-8), Pichia capsulata (DE 10327454.4), from Rhodococcus erythropolis (RECR) (U.S. Pat. No. 5,523,223), Norcardia fusca (Biosci. Biotechnol. Biochem., 63(10), 1999, p. 1721-1729; Appl. Microbiol.
  • 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 (U.S. Pat. No. 5,200,335), Lactobacillus brevis (DE 19610984 A1; Acta Crystallogr. D. Biol. Crystallogr. 2000 December; 56 Pt 12:1696-8), Lactobacillus minor (DE 10119274), Leuconostoc carnosum (A 1261/2005, K1. C12N) or, as described, those from Thermoanerobium brockii, Thermoanerobium ethanolicus or Clostridium beijerinckii.
  • 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.
  • the processes according to the invention are carried out in an aqueous organic two-phase system.
  • the conversion of the secodione derivative occurs in a two-phase system containing, for example, a 2-alcohol for cofactor regeneration, an oxidoreductase, water, cofactor and the secodione compound.
  • a 2-alcohol for cofactor regeneration for example, an oxidoreductase, water, cofactor and the secodione compound.
  • additional organic solvents which are not involved in the cofactor regeneration, i.e., do not contain any oxidizable hydroxy groups, can also be included.
  • Diethyl ether, tertiary butyl methyl ether, diisopropyl ether, dibutyl ether, ethyl acetate, butyl acetate, heptane, hexane, toluene, dichloromethane, cyclohexane or mixtures thereof are preferably used as additional organic solvents.
  • the amount of non-water-miscible organic components of the two-phase system may range from 10% to 90%, preferably from 20% to 80%, based on the total volume of the reaction batch.
  • the aqueous amount may range from 90% to 10%, preferably from 80% to 20%, based on the total volume of the reaction batch.
  • a buffer can also be added to the water, for example, a potassium phosphate, tris/HCl, glycine or triethanolamine buffer, having a pH value of from 5 to 10, preferably from 6 to 9.
  • the buffer can comprise ions for stabilizing or activating both enzymes, for example, magnesium ions or zinc ions.
  • additives for stabilizing the enzymes used can be used in the processes according to the invention, for example, glycerol, sorbitol, 1,4-DL-dithiothreitol (DTT) or dimethyl sulfoxide (DMSO).
  • DTT 1,4-DL-dithiothreitol
  • DMSO dimethyl sulfoxide
  • the concentration of the cofactor NAD(P)H ranges from 0.001 mM to 10 mM, in particular from 0.01 mM to 1.0 mM.
  • the temperature can be from 10° C. to 70° C., preferably from 20° C. to 35° C.
  • the secodione derivatives to be reduced are poorly soluble in water. Therefore, the substrate can be provided in a completely or also incompletely dissolved state during the reaction. If the substrate is not dissolved completely in the reaction mixture, a portion of the substrate is present in a solid form and can thus form a third solid phase. The reaction mixture may also temporarily form an emulsion during the conversion.
  • the secodione derivative of general formula I is used in the reaction batch preferably in an amount of from 10 g/l to 500 g/l, preferably from 25 g/l to 300 g/l, particularly preferably from 50 g/l to 200 g/l, based on the total volume.
  • Preferred embodiments of the invention are furthermore characterized in that 13-ethyl-3-methoxy-8,14-seco-gona-1,3,5 (10),9(11)-tetraene-14,17-dione (ethyl secodione—Formula III) or 13-methyl-3-methoxy-8,14-seco-gona-1,3,5(10),9(11)-tetraene-14,17-dione (methyl secodione—Formula II) is used as the secodione derivative.
  • 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 76258197 in the NCBI database.
  • 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:11, SEQ ID NO:12, SEQ ID NO:13), 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 MgSO 4 ; 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:
  • the resulting PCR product with a size of about 750 by was restricted after purification over a 1% agarose gel with the aid of the endonucleases Nde I and Hind III or endonucleases 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 ⁇ l 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 by means of a restriction analysis with the endonucleases Nde I and Hind III or endonucleases 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.
  • the Escherichia coli strains BL21 Star (Invitrogen, Düsseldorf, Germany) and RB791 ( E. coli genetic stock, Yale, USA), respectively, transformed with the expression construct were cultivated in 200 ml LB-medium (1% tryptone, 0.5% yeast extract, 1% NaCl) with ampicillin (50 ⁇ g/ml) or carbenicillin (50 ⁇ g/ml), respectively, until an optical density (OD) 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. After 8 hours or 16 hours of induction at 25° C.
  • IPTG isopropylthiogalactoside
  • 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 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: 870 ⁇ l of 100 mM TEA buffer pH 7.0, 160 ⁇ g NADH, 10 ⁇ l of diluted cell lysate. The reaction was started by adding 100 ⁇ l of a 100 mM substrate solution to the reaction mixture.
  • yeast strains Pichia farinosa DSM 70362, Candida gropengiesseri MUCL 29836, Candida vaccinii CBS 7318, Pichia farinosa DSM 3316, Saccharomyces cerevisiae CBS 1508 and Candida magnoliae CBS 6396 were cultivated in the following medium: yeast extract (5), peptone (5) and glucose (20) (the numbers in brackets are, in each case, g/l).
  • the medium was sterilized at 121° C. and the yeasts were cultivated at 25° C. on a shaker at 140 revolutions per minute without further pH-adjustment.
  • Strain CBS 6396 displayed the highest conversion of ethyl secodione and was thus chosen as the starting organism for the preparation of a cDNA library.
  • 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 minutes.
  • 5 ml of ice-cold LETS buffer was added, and this was again vigorously vortexed. Said cell suspension was centrifuged at 4° C. with 11000 g for 5 minutes.
  • RNA was precipitated at ⁇ 20° C. for 4 h by adding 1/10 vol. of 5 M LiCl 2 .
  • RNA 1 mg was used via Oligo-dT cellulose (NEB Biolabs) for the enrichment of the mRNA molecules. After the subsequent precipitation, 5 ⁇ g mRNA was used for the cDNA synthesis (pBluescript IIXR cDNA Library Construction kit, Stratagene). The library constructed according to the manufacturer's instructions was transformed into XL-10 Gold E. coli and screened for the activity of an ADH. A clone (cM4) was identified and isolated based on the decrease in absorbance with NADPH or NADH, respectively, as the cofactor and ethyl secodione (Formula III) as the substrate.
  • cM4 A clone (cM4) was identified and isolated based on the decrease in absorbance with NADPH or NADH, respectively, as the cofactor and ethyl secodione (Formula III) as the substrate.
  • Amplification was carried out in a PCR buffer [10 mM Tris-HCl (pH 8.0); 50 mM KCl; 10 mM MgSO 4 ; 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:
  • the resulting PCR product was restricted after purification over a 1% agarose gel with the aid of the endonucleases Nde I and Xho I or the endonucleases Sph I and Sac I, respectively, and was ligated into the backbone of the pET21a vector (Novagen) or of the pQME70 vector, respectively, which backbone had been treated with the same endonucleases. After transforming 2 ⁇ l 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 by means of a restriction analysis with the endonucleases Nde I and XhoI or the endonucleases Sph I and SacI, respectively.
  • the expression constructs pET21-MgIV and pQME70-MgIV were sequenced.
  • the gene from Candida magnoliae coding for a short-chain oxidoreductase had an open reading frame of a total of 729 by (contained in SEQ ID NO:8), which corresponded to a protein of 243 amino acids (SEQ ID NO:3).
  • Competent Escherichia coli StarBL21(De3) cells (Invitrogen) and RB791 cells ( E. coli genetic stock, Yale, USA), respectively, were transformed with the expression constructs pET21-MgIV and pQME70-MgIV, respectively, 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 ⁇ g/ml of ampicillin or 40 ⁇ g/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) at a concentration of 0.1 mM. After 16 hours of induction at 25° C. and 220 rpm, the cells were harvested and frozen at ⁇ 20° C. For the activity test, 10 mg of cells were mixed with 500 ⁇ l of 100 mM TEA buffer pH 7.0, 1 mM MgCl 2 and 500 ⁇ l 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.
  • IPTG isopropylthiogalactoside
  • the activity test was made up as follows: 960 ⁇ l of 100 mM TEA buffer pH 7.0, 1 bmM MgCl 2 , 160 ⁇ g NADPH, 10 ⁇ l of diluted cell lysate. The reaction was started by adding 10 ⁇ l of a 100 mM substrate solution in 70% methanol to the reaction mixture.
  • reaction mixture was reprocessed by extraction with dichloromethane, the organic phase containing the product was separated and the 17-beta-hydroxy compound (ethylloid) was obtained by evaporating/distilling off the solvent.
  • reaction mixture was reprocessed by extraction with dichloromethane, the organic phase containing the product was separated and the 17-beta-hydroxy compound (ethylloid) was obtained by evaporating/distilling off the solvent.

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Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
ATA2027/2006 2006-12-07
AT0202706A AT504542B1 (de) 2006-12-07 2006-12-07 Verfahren zur enantioselektiven enzymatischen reduktion von secodionderivaten
PCT/EP2007/010640 WO2008068030A2 (de) 2006-12-07 2007-12-07 Verfahren zur herstellung von secolderivaten durch enantioselektive enzymatische reduktion von secodionderivaten unter verwendung einer oxidoreduktase/dehydrogenase in gegenwart von nadh oder nadph

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