EP2145009A1 - Process for enzymatic reduction of alkene derivatives - Google Patents

Process for enzymatic reduction of alkene derivatives

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Publication number
EP2145009A1
EP2145009A1 EP07822525A EP07822525A EP2145009A1 EP 2145009 A1 EP2145009 A1 EP 2145009A1 EP 07822525 A EP07822525 A EP 07822525A EP 07822525 A EP07822525 A EP 07822525A EP 2145009 A1 EP2145009 A1 EP 2145009A1
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EP
European Patent Office
Prior art keywords
radical
carried out
reaction
reduction
seq
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP07822525A
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German (de)
French (fr)
Inventor
Rainer STÜRMER
Bernhard Hauer
Thomas Friedrich
Kurt Faber
Melanie Hall
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BASF SE
Original Assignee
BASF SE
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Publication date
Priority claimed from EP06126993A external-priority patent/EP1959019A1/en
Application filed by BASF SE filed Critical BASF SE
Priority to EP07822525A priority Critical patent/EP2145009A1/en
Publication of EP2145009A1 publication Critical patent/EP2145009A1/en
Withdrawn legal-status Critical Current

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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
    • 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
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/008—Preparation of nitrogen-containing organic compounds containing a N-O bond, e.g. nitro (-NO2), nitroso (-NO)
    • 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
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/10—Nitrogen as only ring hetero atom
    • 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
    • C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
    • C12P41/002—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by oxidation/reduction reactions
    • 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
    • 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
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • 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
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/44—Polycarboxylic acids
    • C12P7/46—Dicarboxylic acids having four or less carbon atoms, e.g. fumaric acid, maleic acid
    • 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
    • C12P7/62—Carboxylic acid esters

Definitions

  • the invention relates to a process for the enzymatic reduction of alkene derivatives of the general formula (1).
  • the object was to provide a process for the enzymatic preparation of compounds of the general formula (2) from unsaturated alkene derivatives of the general formula (1), which proceeds in particular in high chemical yield and very good stereoselectivity.
  • the present invention relates to a process for the enzymatic preparation of
  • R 1 , R 2 and R 3 independently of one another are H, C 1 -C 6 -alkyl, C 2 -C 6 -alkenyl, carboxyl or an optionally substituted carbo- or heterocyclic, is aromatic or nonaromatic, or R 1 is linked to R 3 to become part of a 4-8 membered cycle, or R 1 is linked to R to become part of a 4-8 membered cycle, with the determination in that R 1 , R 2 and R 3 may not be identical, by reduction of a compound of formula (1) in the presence of a reductase
  • the process according to the invention can be carried out either with purified or enriched enzyme itself or with microorganisms which naturally or recombinantly express this enzyme or with cell homogenates derived therefrom.
  • C 1 -C 6 -alkyl in particular methyl, ethyl, propyl, butyl, pentyl or hexyl, and the corresponding mono- or polysubstituted analogs, such as i-propyl, i-butyl, sec-butyl, tert. Butyl, i-pentyl or neopentyl; in particular, the stated CrC 4 - alkyl radicals are preferred;
  • Carboxyl in particular the group COOH, - carbocyclic and heterocyclic aromatic or non-aromatic rings, in particular optionally fused rings having 3 to 12 carbon atoms and optionally 1 to 4 heteroatoms, such as N, S and O, in particular N or O.
  • cyclopropyl Cyclobutyl, cyclopenty, cyclohexyl, cycloheptyl, the mono- or polyunsaturated analogs thereof, such as cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclohexadienyl, cycloheptadienyl; Phenyl and naphthyl; and 5- to 7-membered saturated or unsaturated heterocyclic radicals having 1 to 4 heteroatoms, which are selected from O, N and S, wherein the heterocycle may optionally be fused with another heterocycle or carbocycle.
  • heterocyclic radicals derived from pyrrolidine, tetrahydrofuran, piperidine, morpholine, pyrrole, furan, thiophene, pyrazole, imidazole, oxazole, thiazole, pyridine, pyran, pyrimidine, pyridazine, pyrazine, cumarone, indole and quinoline.
  • the cyclic radicals but also the abovementioned alkyl and alkenyl radicals one or more times, such as. B. 1-, 2- or 3-fold, be substituted.
  • halogen in particular F, Cl, Br; - OH, -SH, -NO 2 , -NH 3 , -SO 3 H, C r C 4 alkyl and C 2 -C 4 alkenyl, C r C 4 alkoxy; and hydroxy-dC 4 alkyl; wherein the alkyl and alkenyl radicals are as defined above and the alkoxy radicals are derived from the above-defined corresponding alkyl radicals.
  • radicals R 1 and R 3 can also be so linked directly together to a 4-8, preferably together with the reducible double bond a 5 or 6 membered ring, for example a cyclopentene or cyclohexene structure, which may also be optionally substituted, for example by alkyl, preferably methyl radicals.
  • radicals R 1 and R may also be linked directly to one another such that they form a 4-8, preferably a 5 or 6-membered cycle together with the double bond to be reduced, for example a cyclopentene or cyclohexene structure, which may also be optionally substituted, for example by alkyl, preferably methyl radicals.
  • the above-mentioned 4-8-membered cycles can be both carbocycles, ie only C atoms form the cycle, as well as heterocycles, ie heteroatoms such as O, S, N, are included in the cycle. If desired, these carbocycles or heterocycles may also be substituted, ie H atoms are replaced by heteroatoms.
  • N-phenylsuccinimides see Substrate 3 below
  • substituted heterocycles which are formed by cyclization between R 1 and R 3 .
  • Particularly advantageous embodiments of the invention are the enzymatic reduction of the following substrates (compounds of general formula 1) to give the corresponding compounds of general formula (2):
  • the process according to the invention can be carried out in particular with compounds of the general formula (1) in which A is an aldehyde or ketone radical and R 1 or R 2 is methyl.
  • the reductases suitable for the process according to the invention (which are sometimes also referred to as enoate reductases) have a polypeptide sequence according to SEQ ID NO: 1, 2, or 3 or a polypeptide sequence which is at least 80%, such as at least 90%, or at least 95% and especially at least 97%, 98% or 99% sequence identity with SEQ ID NO: 1, 2 or 3.
  • a polypeptide with SEQ ID NO: 1 is known under the name YqjM from Bacillus subtilis. (UniprotKB / Swissprot entry P54550)
  • a polypeptide of SEQ ID NO: 2 is encoded by the OPR1 gene from tomato. (U-niprotKB / Swissprot entry Q9XG54)
  • a polypeptide of SEQ ID NO: 3 is encoded by the OYPR3 gene from tomato. , (UniprotKB / Swissprot entry Q9FEW9).
  • reductases can be obtained starting from SEQ ID NO: 1, 2, or 3 by targeted or randomized mutagenesis methods known to the person skilled in the art.
  • microorganisms preferably in the genera Alishewanella, Alterococcus, Aquamonas, Aranicola, Arsenophonus, Azotivirga, Brenneria, Buchnera (aphid P-endosymbionts), Budvicia, Buttiauxella, Candidatus Phlomobacter, Cedecea, Citrobacter, Dickeya, Edwardsieila, Enterobacter , Erwinia, Escherichia, Ewingella, Grimontella, Hafnia, Klebsiella, Kluyvera, Leclercia, Leminorel Ia, Moellerella, Morganella, Obesumbacterium, Pantoea, Pectobacterium, Photorhabdus, Plesiomonas, Pragia, Proteus, Providencia,
  • the reductase can be used in purified or partially purified form or else in the form of the microorganism itself. Methods for the recovery and purification of dehydrogenases from microorganisms are well known to those skilled in the art.
  • the enantioselective reduction with the reductase preferably takes place in the presence of a suitable cofactor (also referred to as cosubstrate).
  • a suitable cofactor also referred to as cosubstrate.
  • cofactors for the reduction of the ketone is usually NADH and / or NADPH.
  • NADH and / or NADPH cofactors for the reduction of the ketone
  • Reductases are used as cellular systems which inherently contain cofactor or alternative redox mediators are added (A. Schmidt, F. Hollmann and B. Buehler "Oxidation of Alcohols" in K. Drauz and H. Waldmann, Enzyme Catalysis in Organic Synthesis 2002, Vol. III, 991-1032, Wiley-VCH, Weinheim).
  • the enantioselective reduction with the reductase preferably takes place in the presence of a suitable reducing agent which regenerates the oxidized cofactor in the course of the reduction.
  • suitable reducing agents are sugars, in particular hexoses, such as glucose, mannose, fructose, and / or oxidizable alcohols, in particular ethanol, propanol or isopropanol, and formate, phosphite or molecular hydrogen.
  • a second dehydrogenase e.g.
  • Glucose dehydrogenase when using glucose as a reducing agent or formate dehydrogenase in the use of formate as a reducing agent This can be used as a free or immobilized enzyme or in the form of free or immobilized cells. They can be produced either separately or by coexpression in a (recombinant) reductase strain.
  • a preferred embodiment of the claimed process is the regeneration of the cofactors by an enzymatic system in which a second dehydrogenase, more preferably a glucose dehydrogenase, is used.
  • the reductases used according to the invention can be used freely or immobilized.
  • An immobilized enzyme is an enzyme which is fixed to an inert carrier. Suitable support materials and the enzymes immobilized thereon are known from EP-A-1149849, EP-A-1 069 183 and DE-OS 100193773 and from the references cited therein. The disclosure of these documents is hereby incorporated by reference in its entirety.
  • Suitable support materials include, for example, clays, clay minerals such as kaolinite, diatomaceous earth, perlite, silica, alumina, sodium carbonate, calcium carbonate, cellulose powders, anion exchange materials, synthetic polymers such as polystyrene, acrylic resins, phenol-formaldehyde resins, polyurethanes and polyolefins such as polyethylenes and polypropylene.
  • the support materials are usually used in a finely divided, particulate form for the production of the supported enzymes, wherein porous forms are preferred.
  • the particle size of the carrier material is usually not more than 5 mm, in particular not more than 2 mm (grading curve).
  • Carrier materials are, for example, calcium alginate, and carrageenan.
  • Enzymes as well as cells can also be crosslinked directly with glutaraldehyde (cross-linking to CLEAs). Corresponding and further immobilization processes are described, for example, in J. Lalonde and A. Margolin "Immobilization of Enzymes" in K. Drauz and H. Waldmann, Enzyme Catalysis in Organic Synthesis 2002, Vol. III, 991-1032, Wiley-VCH, Weinheim.
  • the reaction can be carried out in aqueous or non-aqueous reaction media or in 2-phase systems or (micro) emulsions.
  • the aqueous reaction media are preferably buffered solutions which generally have a pH of from 4 to 8, preferably from 5 to 8.
  • the aqueous solvent may also contain, besides water, at least one alcohol, e.g. Ethanol or isopropanol or dimethyl sulfoxide.
  • Non-aqueous reaction media are to be understood as meaning reaction media which contain less than 1% by weight, preferably less than 0.5% by weight, of water, based on the total weight of the liquid reaction medium.
  • the reaction can be carried out in an organic solvent.
  • Suitable organic solvents are for example aliphatic hydrocarbons, preferably having 5 to 8 carbon atoms, such as pentane, cyclopentane, hexane, cyclohexane, heptane, octane or cyclooctane, halogenated aliphatic hydrocarbons, preferably having one or two carbon atoms, such as dichloromethane, chloroform, carbon tetrachloride, dichloroethane or Tetrachloroethane, aromatic hydrocarbons, such as benzene, toluene, the xylene, chlorobenzene or dichlorobenzene, aliphatic acyclic and cyclic ethers or alcohols, preferably having 4 to 8 carbon atoms, such as ethanol, isopropanol, diethyl ether, methyl tert-butyl ether, ethyl tert - butyl ether, dipropy
  • the reduction with the reductase in an aqueous-organic reaction medium such as.
  • an aqueous-organic reaction medium such as.
  • water / isopropanol in any mixing ratio such as 1: 99 to 99: 1 or 10:90 to 90:10, or an aqueous reaction medium.
  • the substrate (1) is preferably used in a concentration of 0.1 g / l to 500 g / l, more preferably from 1 g / l to 50 g / l in the enzymatic reduction and can be performed continuously or discontinuously after ,
  • the enzymatic reduction is generally carried out at a reaction temperature below the deactivation of the reductase used and above -10 0 C. It is particularly preferably in the range of 0 to 100 0 C, in particular from 15 to 60 0 C and especially from 20 to 40 0 C, for example at about 30 0 C.
  • a preferred embodiment of the process according to the invention consists in carrying out the reaction in the presence of divalent metal ions, in particular in the presence of Ca, Mg, Mn, Zn, Ni, Fe, Mo ions. It is advantageous to choose the concentration of alkaline earth metal ions about the same as the concentration of the substrate to be used (alkene derivative of general formula I). In particular, if the substrate is capable of complexing metal ions due to its structure, e.g. For dicarboxylic acid derivatives, the addition of divalent metal ions in equimolar concentration as the substrate is recommended.
  • the substrate (1) with the reductase, the solvent and optionally the coenzymes optionally present a second dehydrogenase for the regeneration of the coenzyme and / or other reducing agents and mix the mixture, z. B. by stirring or shaking.
  • a second dehydrogenase for the regeneration of the coenzyme and / or other reducing agents and mix the mixture, z. B. by stirring or shaking.
  • immobilize the reductase in a reactor for example in a column, and to pass through the reactor a mixture containing the substrate and optionally coenzymes and / or cosubstrates.
  • the mixture can be circulated through the reactor until the desired conversion is achieved.
  • the reduction will lead to a conversion of at least 70%, particularly preferably at least 85% and in particular of at least 95%, based on the substrate contained in the mixture.
  • the progress of the reaction ie the sequential reduction of the double bond can be followed by conventional methods such as gas chromatography or high pressure liquid chromatography.
  • "functional equivalents" or analogues of the specifically disclosed enzymes are different polypeptides which furthermore have the desired biological activity, such as substrate specificity.
  • “functional equivalents” are understood as meaning enzymes which catalyze the model reaction and which has at least 20%, preferably 50%, particularly preferably 75%, very particularly preferably 90% of the activity of an enzyme comprising one of the amino acid sequences listed under SEQ ID NO: 1, 2 or 3.
  • functional equivalents are preferably stable between pH 4 to 10 and advantageously have a pH optimum between pH 5 and 8 and a temperature optimum in the range from 20 ° C. to 80 ° C.
  • “functional equivalents” are in particular also understood as meaning mutants which, in at least one sequence position of the abovementioned amino acid sequences, have a different amino acid than the one specifically mentioned but nevertheless have one of the abovementioned biological activities
  • “Functional equivalents” thus include those represented by a or multiple amino acid additions, substitutions, deletions and / or inversions of available mutants, said changes may occur in any sequence position, as long as they lead to a mutant with the property profile according to the invention. Functional equivalence is given in particular even if the reactivity patterns between mutant and unchanged polypeptide match qualitatively, ie, for example, the same substrates are reacted at different rates.
  • Precursors are natural or synthetic precursors of the polypeptides with or without the desired biological activity.
  • “Functional derivatives” of polypeptides of the invention may also be produced at functional amino acid side groups or at their N- or C-terminal end by known techniques
  • Such derivatives include, for example, aliphatic esters of carboxylic acid groups, amides of carboxylic acid groups, obtained by reaction with ammonia or with a primary or secondary amine; N-acyl derivatives of free amino groups prepared by reaction with acyl groups; or O-acyl derivatives of free hydroxy groups prepared by reaction with acyl groups.
  • “functional equivalents” in the case of a possible protein glycosylation, include proteins of the type described above in deglycosylated or glycosylated form as well as modified forms obtainable by altering the glycosylation pattern.
  • “functional equivalents” also include polypeptides that are accessible from other organisms, as well as naturally occurring variants. For example, regions of homologous sequence regions can be determined by sequence comparison and, based on the specific requirements of the invention, equivalent enzymes can be determined.
  • “Functional equivalents” also include fragments, preferably single domains or sequence motifs, of the polypeptides of the invention having, for example, the desired biological function.
  • Fusion equivalents are also fusion proteins which comprise one of the abovementioned polypeptide sequences or functional equivalents derived therefrom and at least one further, functionally different, heterologous sequence in functional N- or C-terminal linkage (ie without substantially mutual functional impairment of the fusion protein portions
  • heterologous sequences are, for example, signal peptides or enzymes.
  • Homologs of the proteins of the invention can be prepared by screening combinatorial libraries of mutants, e.g. Shortening mutants, to be identified.
  • a variegated library of protein variants can be generated by combinatorial mutagenesis at the nucleic acid level, such as e.g. by enzymatic ligation of a mixture of synthetic oligonucleotides.
  • methods that can be used to prepare libraries of potential homologs from a degenerate oligonucleotide sequence. The chemical synthesis of a degenerate gene sequence can be carried out in a DNA synthesizer, and the synthetic gene can then be ligated into a suitable expression vector.
  • degenerate gene set allows for the provision of all sequences in a mixture that encode the desired set of potential protein sequences.
  • Methods of synthesizing degenerate oligonucleotides are known to those skilled in the art (eg, Narang, SA (1983) Tetrahedron 39: 3; Itakura et al. (1984) Annu. Rev. Biochem. 53: 323; Itakura et al., (1984) Science 198: 1056; Ike et al. (1983) Nucleic Acids Res. 1 1: 477).
  • REM Recursive ensemble mutagenesis
  • the invention furthermore relates to nucleic acid sequences (single-stranded and double-stranded DNA and RNA sequences, such as, for example, cDNA and mRNA) which code for an enzyme with reductase activity according to the invention.
  • nucleic acid sequences which are e.g. for amino acid sequences according to SEQ ID NO: 1, 2 or 3 characterizing partial sequences thereof.
  • nucleic acid sequences mentioned herein can be prepared in a manner known per se by chemical synthesis from the nucleotide units, for example by fragment condensation of individual overlapping, complementary nucleic acid units of the double helix.
  • the chemical synthesis of oligonucleotides can be carried out, for example, in a known manner by the phosphoamidite method (Voet, Voet, 2nd edition, Wiley Press New York, pages 896-897).
  • the attachment of synthetic oligonucleotides and filling of gaps with the aid of the Klenow fragment of the DNA polymerase and ligation reactions and general cloning methods are described in Sambrook et al. (1989), Molecular Cloning: A laboratory manual, CoId Spring Harbor Laboratory Press.
  • the pH in the process according to the invention is advantageously maintained between pH 4 and 12, preferably between pH 4.5 and 9, more preferably between pH 5 and 8, min. 98% ee achieved.
  • growing cells can be used which contain nucleic acids encoding the reductase, nucleic acid constructs or vectors.
  • dormant or open cells can be used.
  • open cells are meant, for example, cells which have been rendered permeable by treatment with, for example, solvents, or cells which have been disrupted by enzyme treatment, by mechanical treatment (eg French Press or ultrasound) or by some other method.
  • the crude extracts thus obtained are advantageously suitable for the process according to the invention.
  • Purified or purified enzymes can also be used for the process. Also suitable are immobilized microorganisms or enzymes that can be used advantageously in the reaction.
  • the process according to the invention can be operated batchwise, semi-batchwise or continuously.
  • the operation of the process may advantageously be carried out in bioreactors, e.g. in Biotechnology, Volume 3, 2nd Edition, Rehm et al. Ed., (1993), especially Chapter II.
  • the products prepared in the process according to the invention can be isolated from the reaction medium by methods familiar to the person skilled in the art and, if desired, purified. These include distillation processes, chromatographic processes, extraction processes and crystallization processes. The cleaning of the products can be significantly increased depending on the requirement by combining several of these methods.
  • the asymmetric bioreduction of the substrates was carried out according to the following general procedure using the isolated enzymes YqjM, OPR1, OPR3. leads. Because of poor water solubility, N-phenyl-2-methylmaleimide was added as a 10% DMF solution (1% final concentration).
  • the enzyme preparation (100-200 ⁇ g) was 50 mM pH 1.5 (0.8 ml) with the cofactor NADH or NADPH (15 mM) was added to a solution of the substrate (5 mM) in Tris buffer and the reaction was performed at 30 0 C under Shaking (140rpm) performed. After 48 hours, the reaction mixture was extracted with ethyl acetate and the reaction products were analyzed by GC.
  • NADH / GDH To a mixture of substrate (5 mM) oxidized cofactor NAD + (100 ⁇ M), glucose (20 mM) in Tris buffer 50 mM pH 7.5 (0.8 ml), (D) -GDH (10 ⁇ ) was added after the enzyme ( 100-200 ⁇ g) was added and the reaction was (at 30 0 C 140 rpm) was performed for 48 hours.
  • GC-FID analyzes were performed on a Varian 3800 gas chromatograph with H 2 as the carrier gas (14.5 psi).
  • the enantiomeric excess was determined using a cyclodextrin-bonded dimethylpolysiloxane phase capillary column (CP-Chirasil-DEX CB, 25 m, 0.32 mm, 0.25 ⁇ m film) with a split-phase ratio of 25: 1.
  • Temperature program 105 0 C 5 min stop, 1 ° C / min to 120 ° C, 6 min. Hold, 20 ° C / min to 180 0 C, 2 min. Hold.
  • Retention times were as follows: (S) - and (R) -1-nitro-2-phenylpropane 12.06 and 12.57 min, respectively.
  • the absolute configuration of 1-nitro-2-phenylpropane was determined by coinjection of an independently synthesized reference sample (J. Org. Chem. 1989, 54, 1802-1804).
  • Retention times were as follows: Citronellal 5.21 min, 1-octanol (internal standard) 5.83 min, Geranial 7.53 min Determination of the enumeric excess and the absolute configuration: The enantiomeric excess of citronellal was measured using a modified ⁇ -cyclodextrin Capillary column (Hydrodex-ß-TBDAc, 25 m, 0.25 mm) Temperature program: 40 ° C hold 2 min, 4 ° C / min to 120 0 C, 1 min. Hold, 20 ° C / min to 180 0 C, 3 min. Hold. Retention times were as follows: (S) - and (R) - citronellal 19.84 and 19.97 min resp. The absolute configuration of citronellal was determined by coinjection of a commercially available reference sample of known absolute configuration.

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Abstract

Process for enzymatic production of compounds of the general formula (2) from unsaturated alkene derivatives of the general formula (1) by reduction of a compound of the formula (1) in the presence of a reductase comprising at least one of the polypeptide sequences SEQ ID NO: 1, 2, or 3 or having a functionally equivalent polypeptide sequence which has at least 80% sequence identity with SEQ ID NO: 1, 2, or 3.

Description

Verfahren zur enzymatischen Reduktion von Alkenderivaten Process for the enzymatic reduction of alkene derivatives
Die Erfindung betrifft ein Verfahren zur enzymatischen Reduktion von Alkenderivatender allgemeinen Formel (1 ).The invention relates to a process for the enzymatic reduction of alkene derivatives of the general formula (1).
Aufgabenstellungtask
Es bestand die Aufgabe, ein Verfahren zur enzymatischen Herstellung von Verbindungen der allgemeinen Formel (2) aus ungesättigten Alken-Derivaten der allgemeinen Formel (1 ) bereitzustellen, das insbesondere in hoher chemischer Ausbeute und sehr guter Stereoselektivität abläuft.The object was to provide a process for the enzymatic preparation of compounds of the general formula (2) from unsaturated alkene derivatives of the general formula (1), which proceeds in particular in high chemical yield and very good stereoselectivity.
Kurzfassung der ErfindungSummary of the invention
Obige Aufgabe wurde gelöst durch Verwendung der Reduktasen YqjM, OPR1 , OPR3 und funktionaler Äquivalente davon zur Reduktion von Alkenderivaten der allgemeinen Formel (1 ).The above object has been achieved by using the reductases YqjM, OPR1, OPR3 and functional equivalents thereof for the reduction of alkene derivatives of the general formula (1).
Detaillierte Beschreibung der ErfindungDetailed description of the invention
Die vorliegende Erfindung betrifft ein Verfahren zur enzymatischen Herstellung vonThe present invention relates to a process for the enzymatic preparation of
Verbindungen der allgemeinen Formel (2) aus ungesättigten Alken-Derivaten der allgemeinen Formel (1 )Compounds of the general formula (2) from unsaturated alkene derivatives of the general formula (1)
worinwherein
A für einen Nitrorest (-NO2), einen Ketonrest (-CRO), einen Aldehydrest (-CHO) einen Carboxylrest (-COOR) mit R= H oder gegebenenfalls substituerter CrCβ- Alkylrest, steht,A is a nitro radical (-NO 2 ), a ketone radical (-CRO), an aldehyde radical (-CHO) is a carboxyl radical (-COOR) where R = H or optionally substituted C 1 -C 6 -alkyl radical,
R1 , R2 und R3 unabhängig voneinander für H, C-i-Cβ- Alkyl, C2-C6- Alkenyl, Car- boxyl oder einen gegebenenfalls substituierten carbo- oder heterocyclischen, a- romatischen oder nichtaromatischen Rest steht, oder R1 ist mit R3 so verknüpft, dass sie Teil eines 4-8 gliedrigen Cyclus werden, oder R1 ist mit R so verknüpft, dass sie Teil eines 4-8 gliedrigen Cyclus werden, mit der Bestimmung, daß R1, R2 und R3 nicht identisch sein dürfen, durch Reduktion einer Verbindung der Formel (1 ) in Gegenwart einer ReduktaseR 1 , R 2 and R 3 independently of one another are H, C 1 -C 6 -alkyl, C 2 -C 6 -alkenyl, carboxyl or an optionally substituted carbo- or heterocyclic, is aromatic or nonaromatic, or R 1 is linked to R 3 to become part of a 4-8 membered cycle, or R 1 is linked to R to become part of a 4-8 membered cycle, with the determination in that R 1 , R 2 and R 3 may not be identical, by reduction of a compound of formula (1) in the presence of a reductase
(i) umfassend wenigstens eine der Polypeptidsequenzen SEQ ID(i) comprising at least one of the polypeptide sequences SEQ ID
NO:1 , 2, oder 3 oder (ii) mit einer funktional äquivalenten Polypeptidsequenz, die mindestensNO: 1, 2, or 3, or (ii) having a functionally equivalent polypeptide sequence which is at least
80% Sequenzidentität mit SEQ ID NO:1 , 2, oder 3 aufweist.80% sequence identity with SEQ ID NO: 1, 2, or 3.
Grundsätzlich ist das erfindungsgemäße Verfahren sowohl mit gereinigten oder angereicherten Enzym selbst als auch mit Mikroorganismen, welche dieses Enzym natürlich oder rekombinant exprimieren, oder mit davon abgeleiteten Zellhomogenaten durchführbar.In principle, the process according to the invention can be carried out either with purified or enriched enzyme itself or with microorganisms which naturally or recombinantly express this enzyme or with cell homogenates derived therefrom.
Werden keine anderen Angaben gemacht, so bedeutet:If no other information is given, this means:
CrCβ-Alkyl insbesondere Methyl, Ethyl, Propyl, Butyl, Pentyl oder Hexyl, sowie die entsprechenden ein- oder mehrfach verzweigte Analoga, wie i-Propyl, i-Butyl, sec- Butyl, tert. -Butyl, i-Pentyl oder Neopentyl; wobei insbesondere die genannten CrC4- Alkylreste bevorzugt sind;C 1 -C 6 -alkyl, in particular methyl, ethyl, propyl, butyl, pentyl or hexyl, and the corresponding mono- or polysubstituted analogs, such as i-propyl, i-butyl, sec-butyl, tert. Butyl, i-pentyl or neopentyl; in particular, the stated CrC 4 - alkyl radicals are preferred;
C2-C6-Alkenyl insbesondere die einfach ungesättigten Analoga der oben genannten Alkylreste mit 2 bis 6 Kohlenstoffatomen, wobei insbesondere die entsprechenden C2-C4-Alkenylreste bevorzugt sind,C 2 -C 6 -alkenyl, in particular the monounsaturated analogs of the abovementioned alkyl radicals having 2 to 6 carbon atoms, the corresponding C 2 -C 4 -alkenyl radicals in particular being preferred,
Carboxyl insbesonders die Gruppe COOH, - Carbo- und heterocyclische aromatische oder nichtaromatische Ringe insbesondere gegebenenfalls kondensierte Ringe mit 3 bis 12 Kohlenstoffatomen und gegebenenfalls 1 bis 4 Heteroatomen, wie N, S und O, insbesondere N oder O. Als Beispiele können genannt werden Cyclopropyl, Cyclobutyl, Cyclopenty, Cyclohexyl, Cycloheptyl, die ein- oder mehrfach ungesättigten Analoga davon, wie Cyclobutenyl, Cyclopentenyl, Cyclohexenyl, Cycloheptenyl, Cyclohexadienyl, Cycloheptadienyl; Phenyl und Naphthyl; sowie 5- bis 7-gliedrige gesättigte oder ungesättigte heterocyclische Reste mit 1 bis 4 Heteroatomen, die ausgewählt sind unter O, N und S, wobei der Heterocyc- lus gegebenenfalls mit einem weitern Heterocyclus oder Carbocyclus kondensiert sein kann. Insbesondere sind zu nennen heterocyclische Reste abgeleitet von Pyrrolidin, Tetrahydrofuran, Piperidin, Morpholin, Pyrrol, Furan, Thiophen, Pyrazol, Imidazol, Oxa- zol, Thiazol, Pyridin, Pyran, Pyrimidin, Pyridazin, Pyrazin, Cumaron, Indol und Chinolin. Gegebenenfalls können die cyclischen Reste, aber auch die oben genannten Alkyl- und Alkenylreste ein- oder mehrfach, wie z. B. 1-, 2- oder 3-fach, substituiert sein. Als Beispiel für geeignete Substituierte sind zu nennen: Halogen, insbesondere F, Cl, Br; - OH, -SH, -NO2, -NH3, -SO3H, CrC4-Alkyl und C2-C4-Alkenyl, CrC4 -Alkoxy; und Hydro- xy-d-C4-Alkyl; wobei die Alkyl- und Alkenyl-Reste wie oben definiert sind und die Alko- xy-Reste von den oben definierten korrespondierenden Alkylresten abgeleitet sind.Carboxyl in particular the group COOH, - carbocyclic and heterocyclic aromatic or non-aromatic rings, in particular optionally fused rings having 3 to 12 carbon atoms and optionally 1 to 4 heteroatoms, such as N, S and O, in particular N or O. Examples which may be mentioned are cyclopropyl, Cyclobutyl, cyclopenty, cyclohexyl, cycloheptyl, the mono- or polyunsaturated analogs thereof, such as cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclohexadienyl, cycloheptadienyl; Phenyl and naphthyl; and 5- to 7-membered saturated or unsaturated heterocyclic radicals having 1 to 4 heteroatoms, which are selected from O, N and S, wherein the heterocycle may optionally be fused with another heterocycle or carbocycle. Particular mention may be made of heterocyclic radicals derived from pyrrolidine, tetrahydrofuran, piperidine, morpholine, pyrrole, furan, thiophene, pyrazole, imidazole, oxazole, thiazole, pyridine, pyran, pyrimidine, pyridazine, pyrazine, cumarone, indole and quinoline. Optionally, the cyclic radicals, but also the abovementioned alkyl and alkenyl radicals one or more times, such as. B. 1-, 2- or 3-fold, be substituted. As an example of suitable substituents may be mentioned: halogen, in particular F, Cl, Br; - OH, -SH, -NO 2 , -NH 3 , -SO 3 H, C r C 4 alkyl and C 2 -C 4 alkenyl, C r C 4 alkoxy; and hydroxy-dC 4 alkyl; wherein the alkyl and alkenyl radicals are as defined above and the alkoxy radicals are derived from the above-defined corresponding alkyl radicals.
Die Reste R1 und R3 können auch so direkt miteinander verknüpft sein, dass sie zusammen mit der zu reduzierenden Doppelbindung einen 4-8, bevorzugt einen 5 oder 6 gliedrigen Cyclus bilden, beispielsweise eine Cyclopenten oder Cyclohexenstruktur, die auch gegebenenfalls substituiert sein kann, beispielsweise durch Alkyl-, bevorzugt Methylreste.The radicals R 1 and R 3 can also be so linked directly together to a 4-8, preferably together with the reducible double bond a 5 or 6 membered ring, for example a cyclopentene or cyclohexene structure, which may also be optionally substituted, for example by alkyl, preferably methyl radicals.
Die Reste R1 und R können auch so direkt miteinander verknüpft sein, dass sie zusammen mit der zu reduzierenden Doppelbindung einen 4-8, bevorzugt einen 5 oder 6 gliedrigen Cyclus bilden, beispielsweise eine Cyclopenten oder Cyclohexenstruktur, die auch gegebenenfalls substituiert sein kann, beispielsweise durch Alkyl-, bevorzugt Methylreste.The radicals R 1 and R may also be linked directly to one another such that they form a 4-8, preferably a 5 or 6-membered cycle together with the double bond to be reduced, for example a cyclopentene or cyclohexene structure, which may also be optionally substituted, for example by alkyl, preferably methyl radicals.
Die o.g. 4-8 gliedrigen Cyclen können sowohl Carbocyclen sein, d.h. nur C-Atome bil- den den Cyclus, als auch Heterozyklen, d.h. Heteroatome wie O;S;N, sind im Cyclus enthalten. Gewünschtenfalls können diese Carbo- oder Heterozyklen auch noch substituiert sein, d.h. H-Atome sind durch Heteroatome ersetzt. Beispielsweise sind N- Phenylsuccinimide (siehe unten Substrat 3) als solche substituierte Heterozyklen aufzufassen, die durch Cyclusbildung zwischen R1 und R entstehen.The above-mentioned 4-8-membered cycles can be both carbocycles, ie only C atoms form the cycle, as well as heterocycles, ie heteroatoms such as O, S, N, are included in the cycle. If desired, these carbocycles or heterocycles may also be substituted, ie H atoms are replaced by heteroatoms. For example, N-phenylsuccinimides (see Substrate 3 below) are to be understood as substituted heterocycles which are formed by cyclization between R 1 and R 3 .
Besonders vorteilhafte Ausführungsformen der Erfindung sind die enzymatische Reduktion der folgenden Substrate (Verbindungen der allgemeinen Formel 1 ) zu den entsprechenden Verbindungen der allgemeinen Formel (2): Particularly advantageous embodiments of the invention are the enzymatic reduction of the following substrates (compounds of general formula 1) to give the corresponding compounds of general formula (2):
SubstratiSubstrati
Substrat2substrate 2
Substrat3Substrat3
Substrat4 Substrat4
SubstratδSubstratδ
SubstratδSubstratδ
Substrat7 Tabelle 1Substrat7 Table 1
OPR1 OPR3 wt YqjMOPR1 OPR3 wt YqjM
Subst %Subst%
Cofactor E.e. % % E.e. % % E.e. % ratCofactor E.e. %% E.e. %% E.e. % advice
1 NADH >99 (K) 97 69 (S) 82 94 (S) 921 NADH> 99 (K) 97 69 (S) 82 94 (S) 92
1 NADPH >99 (R) 96 72 (S) 87 85 (S) 701 NADPH> 99 (R) 96 72 (S) 87 85 (S) 70
1 NAD+-FDH >90 (R) 95 40 (S) 75 50 (S) 851 NAD + FDH> 90 (R) 95 40 (S) 75 50 (S) 85
1 NADP+-G6PDH 90 (R) 98 75 (S) 93 47 (S) 841 NADP + -G6PDH 90 (R) 98 75 (S) 93 47 (S) 84
2 NADH >99 (S)>95 90 (S)>95 70 (S)>952 NADH> 99 (S)> 95 90 (S)> 95 70 (S)> 95
2 NADPH >99 (S)>95 90 (S)>95 73 (S)>952 NADPH> 99 (S)> 95 90 (S)> 95 73 (S)> 95
2 NAD+-FDH Main product: Citronellol (saturated alcohol)2 NAD + -FDH Main product: Citronellol (saturated alcohol)
2 NAD+-GDH 20 (S)>95 90 (S)>95 3 (S)>952 NAD + -GDH 20 (S)> 95 90 (S)> 95 3 (S)> 95
2 NADP+-G6PDH 15 (S)>95 95 (S)>95 57 (S)>952 NADP + -G6PDH 15 (S)> 95 95 (S)> 95 57 (S)> 95
3 NADH 58 (S) 61 27 (S) 45 50 (S) 553 NADH 58 (S) 61 27 (S) 45 50 (S) 55
3 NADPH 45 (S) 64 19 (S) 45 100 (S) 663 NADPH 45 (S) 64 19 (S) 45 100 (S) 66
3 NAD+-FDH 88 (R) 1 65 (R) 1 100 (R) 23 NAD + FDH 88 (R) 1 65 (R) 1 100 (R) 2
3 NADP+-G6PDH 14 (S) 61 10 (S) 58 72 (S) 943 NADP + -G6PDH 14 (S) 61 10 (S) 58 72 (S) 94
4 NADH n.c. - 1 (S)>99 0.5 (S) 594 NADH n.c. - 1 (S)> 99 0.5 (S) 59
4 NADPH n.c. - 2 (S)>99 n.c. -4 NADPH n.c. - 2 (S)> 99 n.c. -
4 NAD+-FDH - - 5 (S)>99 1 (S) 644 NAD + FDH - - 5 (S)> 99 1 (S) 64
4 NADP+-G6PDH - - 1 (S)>99 - -4 NADP + -G6PDH - - 1 (S)> 99 - -
5 NADH n.c. - 3 (S)>99 n.c. -5 NADH n.c. - 3 (S)> 99 n.c. -
5 NADPH n.c. - 2 (S)>99 n.c. -5 NADPH n.c. - 2 (S)> 99 n.c. -
5 NAD+-FDH - - 11 (S)>99 - -5 NAD + FDH - - 11 (S)> 99 - -
5 NADP+-G6PDH - - 1 (S)>99 - -5 NADP + -G6PDH - - 1 (S)> 99 - -
6 NADH 99 (R)>99 99 (R)>99 99.5 (R)>996 NADH 99 (R)> 99 99 (R)> 99 99.5 (R)> 99
6 NADPH 99 (R)>99 99 (R)>99 98.5 (R)>996 NADPH 99 (R)> 99 99 (R)> 99 98.5 (R)> 99
6 NAD+-FDH 99 (R) 97 99 (R) 92 99 (R) 926 NAD + FDH 99 (R) 97 99 (R) 92 99 (R) 92
6 NADP+-G6PDH 99 (R) 96 99 (R) 97 99 (R) 966 NADP + -G6PDH 99 (R) 96 99 (R) 97 99 (R) 96
7 NADH >99 (R)>99 n.c. -7 NADH> 99 (R)> 99 n.c. -
7 NADPH >99 (R)>99 n.c. -7 NADPH> 99 (R)> 99 n.c. -
7 NAD+ -FDH 2 (R)>99 n.d. -7 NAD + FDH 2 (R)> 99 nd -
7 NADP+ -G6PDH 96 (R)>99 n.d. -7 NADP + -G6PDH 96 (R)> 99 nd -
Das erfindungsgemäße Verfahren kann insbesondere mit Verbindungen der allgemeinen Formel (1 ) durchgeführt werden, in denen A für einen Aldehyd oder Ketonrest steht und R1 oder R2 für Methyl steht.The process according to the invention can be carried out in particular with compounds of the general formula (1) in which A is an aldehyde or ketone radical and R 1 or R 2 is methyl.
Die für das erfindungsgemäße Verfahren geeigneten Reduktasen (welche zuweilen0 auch als Enoat-Reduktasen bezeichnet werden) besitzen eine Polypeptidsequenz ge- mäss SEQ ID NO:1 , 2, oder 3 oder eine Polypeptidsequenz, die mindestens 80%, wie z.B. mindestens 90%, oder mindestens 95% und insbesondere mindestens 97%, 98% oder 99% Sequenzidentität mit SEQ ID NO: 1 , 2 oder 3 aufweist. Ein Polypeptid mit der SEQ ID NO:1 ist bekannt unter der Bezeichnung YqjM aus Bacil- lus subtilis. (UniprotKB/Swissprot entry P54550)The reductases suitable for the process according to the invention (which are sometimes also referred to as enoate reductases) have a polypeptide sequence according to SEQ ID NO: 1, 2, or 3 or a polypeptide sequence which is at least 80%, such as at least 90%, or at least 95% and especially at least 97%, 98% or 99% sequence identity with SEQ ID NO: 1, 2 or 3. A polypeptide with SEQ ID NO: 1 is known under the name YqjM from Bacillus subtilis. (UniprotKB / Swissprot entry P54550)
Ein Polypeptid mit der SEQ ID NO:2 wird kodiert vom OPR1 Gen aus Tomate. (U- niprotKB/Swissprot entry Q9XG54)A polypeptide of SEQ ID NO: 2 is encoded by the OPR1 gene from tomato. (U-niprotKB / Swissprot entry Q9XG54)
Ein Polypeptid mit der SEQ ID NO:3 wird kodiert vom OYPR3 Gen aus Tomate. . (UniprotKB/Swissprot entry Q9FEW9).A polypeptide of SEQ ID NO: 3 is encoded by the OYPR3 gene from tomato. , (UniprotKB / Swissprot entry Q9FEW9).
Die Ermittlung der Sequenzidentität soll für die hier beschriebenen Zwecke durch das Computerprogramm „GAP" der Genetics Computer Group (GCG) der University of Wisconsin erfolgen, wobei die Version 10.3 unter Verwendung der von GCG empfohlenen Standardparameter zum Einsatz kommen soll.Sequence identity determination for the purposes described herein is to be carried out by the GAP computer program of the University of Wisconsin's Genetics Computer Group (GCG) using version 10.3 using the standard parameters recommended by GCG.
Solche Reduktasen können ausgehend von SEQ ID NO: 1 , 2, oder 3 durch dem Fachmann bekannte gezielte oder randomisierte Mutageneseverfahren erhalten werden. Alternativ kann jedoch auch in Mikroorganismen , bevorzugt in solchen der Gattungen Alishewanella, Alterococcus, Aquamonas, Aranicola, Arsenophonus, Azotivirga, Brenneria, Buchnera (aphid P-endosymbionts), Budvicia, Buttiauxella, Candidatus Phlomobacter, Cedecea, Citrobacter, Dickeya, Edwardsieila, Enterobacter, Erwinia, Escherichia, Ewingella, Grimontella, Hafnia, Klebsiella, Kluyvera, Leclercia, Leminorel- Ia, Moellerella, Morganella, Obesumbacterium, Pantoea, Pectobacterium, Photorhab- dus, Plesiomonas, Pragia, Proteus, Providencia, Rahnella, Raoultella, Salmonella, Samsonia, Serratia, Shigella, Sodalis, Tatumella, Trabulsiella, Wigglesworthia, Xe- norhabdus, Yersinia oder Yokenella, nach Reduktasen gesucht werden, die die o.g. Modellreaktion katalysieren und deren Aminosäuresequenz die geforderte Sequenzidentität zu SEQ ID NO: 1 , 2 oder 3 bereits aufweist oder durch Mutageneseverfahren erhalten wird.Such reductases can be obtained starting from SEQ ID NO: 1, 2, or 3 by targeted or randomized mutagenesis methods known to the person skilled in the art. Alternatively, however, in microorganisms, preferably in the genera Alishewanella, Alterococcus, Aquamonas, Aranicola, Arsenophonus, Azotivirga, Brenneria, Buchnera (aphid P-endosymbionts), Budvicia, Buttiauxella, Candidatus Phlomobacter, Cedecea, Citrobacter, Dickeya, Edwardsieila, Enterobacter , Erwinia, Escherichia, Ewingella, Grimontella, Hafnia, Klebsiella, Kluyvera, Leclercia, Leminorel Ia, Moellerella, Morganella, Obesumbacterium, Pantoea, Pectobacterium, Photorhabdus, Plesiomonas, Pragia, Proteus, Providencia, Rahnella, Raoultella, Salmonella, Samsonia , Serratia, Shigella, Sodalis, Tatumella, Trabulsiella, Wigglesworthia, Xenorhabdus, Yersinia or Yokenella, are searched for reductases which are the above mentioned Catalyze model reaction and whose amino acid sequence already has the required sequence identity to SEQ ID NO: 1, 2 or 3 or is obtained by mutagenesis.
Die Reduktase kann in gereinigter oder teilweise gereinigter Form oder auch in Form des Mikroorganismus selbst verwendet werden. Verfahren zur Gewinnung und Aufreinigung von Dehydrogenasen aus Mikroorganismen sind dem Fachmann hinreichend bekannt.The reductase can be used in purified or partially purified form or else in the form of the microorganism itself. Methods for the recovery and purification of dehydrogenases from microorganisms are well known to those skilled in the art.
Bevorzugt erfolgt die die enantioselektive Reduktion mit der Reduktase in Gegenwart eines geeigneten Cofaktors (auch als Cosubstrat bezeichnet). Als Cofaktoren für die Reduktion des Ketons dient üblicherweise NADH und/oder NADPH. Daneben können Reduktasen als zelluläre Systeme eingesetzt werden, die inherent Cofaktor enthalten, oder alternative Redoxmediatoren zugesetzt werden (A. Schmidt, F. Hollmann und B. Bühler „Oxidation of Alcohols" in K. Drauz und H. Waldmann, Enzyme Catalysis in Or- ganic Synthesis 2002, Vol. III, 991-1032, Wiley-VCH, Weinheim).The enantioselective reduction with the reductase preferably takes place in the presence of a suitable cofactor (also referred to as cosubstrate). As cofactors for the reduction of the ketone is usually NADH and / or NADPH. In addition, you can Reductases are used as cellular systems which inherently contain cofactor or alternative redox mediators are added (A. Schmidt, F. Hollmann and B. Buehler "Oxidation of Alcohols" in K. Drauz and H. Waldmann, Enzyme Catalysis in Organic Synthesis 2002, Vol. III, 991-1032, Wiley-VCH, Weinheim).
Bevorzugt erfolgt die enantioselektive Reduktion mit der Reduktase außerdem in Gegenwart eines geeigneten Reduktionsmittels, welches den im Verlauf der Reduktion oxidierten Cofaktor regeneriert. Beispiele für geeignete Reduktionsmittels sind Zucker, insbesondere Hexosen, wie Glucose, Mannose, Fructose, und/oder oxidierbare Alko- hole, insbesondere Ethanol, Propanol oder Isopropanol, sowie Formiat, Phosphit oder molekularer Wasserstoff. Zur Oxidation des Reduktionsmittels und damit verbunden zur Regeneration des Coenzyms kann eine zweite Dehydrogenase, wie z.B. Glucose- dehydrogenase bei Verwendung von Glucose als Reduktionsmittel oder Formiat- Dehydrogenase bei der Verwendung von Formiat als Reduktionsmittel, zugesetzt wer- den. Diese kann als freies oder immobilisiertes Enzym oder in Form von freien oder immobilisierten Zellen eingesetzt werden. Ihre Herstellung kann sowohl separat als auch durch Coexpression in einem (rekombinanten) Reduktase-Stamm erfolgen.In addition, the enantioselective reduction with the reductase preferably takes place in the presence of a suitable reducing agent which regenerates the oxidized cofactor in the course of the reduction. Examples of suitable reducing agents are sugars, in particular hexoses, such as glucose, mannose, fructose, and / or oxidizable alcohols, in particular ethanol, propanol or isopropanol, and formate, phosphite or molecular hydrogen. For oxidation of the reducing agent and, concomitantly, for the regeneration of the coenzyme, a second dehydrogenase, e.g. Glucose dehydrogenase when using glucose as a reducing agent or formate dehydrogenase in the use of formate as a reducing agent. This can be used as a free or immobilized enzyme or in the form of free or immobilized cells. They can be produced either separately or by coexpression in a (recombinant) reductase strain.
Eine bevorzugte Ausführungsform des beanspruchten Verfahrens ist die Regenerie- rung der Cofaktoren durch ein enzymatisches System, bei dem eine zweite Dehydrogenase, besonders bevorzugt eine Glucosedehydrogenase, verwendet wird.A preferred embodiment of the claimed process is the regeneration of the cofactors by an enzymatic system in which a second dehydrogenase, more preferably a glucose dehydrogenase, is used.
Weiterhin kann es zweckmäßig sein, weitere die Reduktion fördernde Zusätze, wie z. B. Metallsalze oder Chelatbildner, wie. z. B. EDTA, zu zusetzten.Furthermore, it may be appropriate to further promoting the reduction additives such. As metal salts or chelating agents, such as. z. As EDTA, zuzusetzten.
Die erfindungsgemäß verwendeten Reduktasen können frei oder immobilisiert eingesetzt werden. Unter einem immobilisierten Enzym versteht man ein Enzym, das an einen inerten Träger fixiert ist. Geeignete Trägermaterialien sowie die darauf immobilisierten Enzyme sind aus der EP-A-1149849, EP-A-1 069 183 und der DE-OS 100193773 sowie aus den darin zitierten Literaturstellen bekannt. Auf die Offenbarung dieser Schriften wird diesbezüglich in vollem Umfang Bezug genommen. Zu den geeigneten Trägermaterialien gehören beispielsweise Tone, Tonmineralien, wie Kaolinit, Diatomeenerde, Perlit, Siliciumdioxid, Aluminiumoxid, Natriumcarbonat, Calciumcarbonat, Cellulosepulver, Anionenaustauschermaterialien, synthetische Polymere, wie Po- lystyrol, Acrylharze, Phenolformaldehydharze, Polyurethane und Polyolefine, wie PoIy- ethylen und Polypropylen. Die Trägermaterialien werden zur Herstellung der geträger- ten Enzyme üblicherweise in einer feinteiligen, partikelförmigen Form eingesetzt, wobei poröse Formen bevorzugt sind. Die Partikelgröße des Trägermaterials beträgt üblicherweise nicht mehr als 5 mm, insbesondere nicht mehr als 2 mm (Sieblinie). Analog kann bei Einsatz der Dehydrogenase als Ganzzell-Katalysator eine freie oder immobili- serte Form gewählt werden. Trägermaterialien sind z.B. Ca-Alginat, und Carrageenan. Enzyme wie auch Zellen können auch direkt mit Glutaraldehyd vernetzt werden (Cross- linking zu CLEAs). Entsprechende und weitere Immobilisierungsverfahren sind beispielsweise in J. Lalonde und A. Margolin „Immobilization of Enzymes" in K. Drauz und H. Waldmann, Enzyme Catalysis in Organic Synthesis 2002, Vol. III, 991-1032, Wiley- VCH, Weinheim beschrieben.The reductases used according to the invention can be used freely or immobilized. An immobilized enzyme is an enzyme which is fixed to an inert carrier. Suitable support materials and the enzymes immobilized thereon are known from EP-A-1149849, EP-A-1 069 183 and DE-OS 100193773 and from the references cited therein. The disclosure of these documents is hereby incorporated by reference in its entirety. Suitable support materials include, for example, clays, clay minerals such as kaolinite, diatomaceous earth, perlite, silica, alumina, sodium carbonate, calcium carbonate, cellulose powders, anion exchange materials, synthetic polymers such as polystyrene, acrylic resins, phenol-formaldehyde resins, polyurethanes and polyolefins such as polyethylenes and polypropylene. The support materials are usually used in a finely divided, particulate form for the production of the supported enzymes, wherein porous forms are preferred. The particle size of the carrier material is usually not more than 5 mm, in particular not more than 2 mm (grading curve). Similarly, when using the dehydrogenase as a whole-cell catalyst, a free or immobilized form can be selected. Carrier materials are, for example, calcium alginate, and carrageenan. Enzymes as well as cells can also be crosslinked directly with glutaraldehyde (cross-linking to CLEAs). Corresponding and further immobilization processes are described, for example, in J. Lalonde and A. Margolin "Immobilization of Enzymes" in K. Drauz and H. Waldmann, Enzyme Catalysis in Organic Synthesis 2002, Vol. III, 991-1032, Wiley-VCH, Weinheim.
Die Umsetzung kann in wässrigen oder nichtwässrigen Reaktionsmedien oder in 2- Phasensystemen oder (Mikro-)Emulsionen erfolgen. Bei den wässrigen Reaktionsmedien handelt es sich vorzugsweise um gepufferte Lösungen, die in der Regel einen pH- Wert von 4 bis 8, vorzugsweise von 5 bis 8, aufweisen. Das wässrige Lösungsmittel kann neben Wasser außerdem wenigstens einen Alkohol, z.B. Ethanol oder Isopropa- nol oder Dimethylsulfoxid enthalten.The reaction can be carried out in aqueous or non-aqueous reaction media or in 2-phase systems or (micro) emulsions. The aqueous reaction media are preferably buffered solutions which generally have a pH of from 4 to 8, preferably from 5 to 8. The aqueous solvent may also contain, besides water, at least one alcohol, e.g. Ethanol or isopropanol or dimethyl sulfoxide.
Unter nicht-wässrigen Reaktionsmedien werden Reaktionsmedien verstanden, die weniger als 1 Gew.-%, vorzugsweise weniger als 0,5 Gew.-% Wasser, bezogen auf das Gesamtgewicht des flüssigen Reaktionsmediums, enthalten. Insbesondere kann die Umsetzung in einem organischen Lösungsmittel durchgeführt werden.Non-aqueous reaction media are to be understood as meaning reaction media which contain less than 1% by weight, preferably less than 0.5% by weight, of water, based on the total weight of the liquid reaction medium. In particular, the reaction can be carried out in an organic solvent.
Geeignete organische Lösungsmittel sind beispielsweise aliphatische Kohlenwasserstoffe, vorzugsweise mit 5 bis 8 Kohlenstoffatomen, wie Pentan, Cyclopentan, Hexan, Cyclohexan, Heptan, Octan oder Cyclooctan, halogenierte aliphatische Kohlenwasserstoffe, vorzugsweise mit einem oder zwei Kohlenstoffatomen, wie Dichlormethan, Chloroform, Tetrachlorkohlenstoff, Dichlorethan oder Tetrachlorethan, aromatische Kohlenwasserstoffe, wie Benzol, Toluol, die XyIoIe, Chlorbenzol oder Dichlorbenzol, aliphatische acyclische und cyclische Ether oder Alkohole, vorzugsweise mit 4 bis 8 Kohlen- stoffatomen, wie Ethanol, Isopropanol, Diethylether, Methyl-tert-butylether, Ethyl-tert- butylether, Dipropylether, Diisopropylether, Dibutylether, Tetrahydrofuran oder Ester wie Ethylacetat oder n-Butylacetat oder Ketone wie Methylisobutylketon oder Dioxan oder Gemische davon. Besonders bevorzugt werden die vorgenannten Ether, insbesondere Tetrahydrofuran, verwendet.Suitable organic solvents are for example aliphatic hydrocarbons, preferably having 5 to 8 carbon atoms, such as pentane, cyclopentane, hexane, cyclohexane, heptane, octane or cyclooctane, halogenated aliphatic hydrocarbons, preferably having one or two carbon atoms, such as dichloromethane, chloroform, carbon tetrachloride, dichloroethane or Tetrachloroethane, aromatic hydrocarbons, such as benzene, toluene, the xylene, chlorobenzene or dichlorobenzene, aliphatic acyclic and cyclic ethers or alcohols, preferably having 4 to 8 carbon atoms, such as ethanol, isopropanol, diethyl ether, methyl tert-butyl ether, ethyl tert - butyl ether, dipropyl ether, diisopropyl ether, dibutyl ether, tetrahydrofuran or esters such as ethyl acetate or n-butyl acetate or ketones such as methyl isobutyl ketone or dioxane or mixtures thereof. Particular preference is given to using the abovementioned ethers, in particular tetrahydrofuran.
Beispielsweise kann die Reduktion mit der Reduktase in einem wässrig-organischen Reaktionsmedium, wie z. B. Wasser/Isopropanol, in beliebigem Mischungsverhältnis wie z.B. 1 :99 bis 99:1 oder 10: 90 bis 90:10, oder einem wässrigen Reaktionsmedium durchgeführt werden.For example, the reduction with the reductase in an aqueous-organic reaction medium, such as. As water / isopropanol, in any mixing ratio such as 1: 99 to 99: 1 or 10:90 to 90:10, or an aqueous reaction medium.
Das Substrat (1 ) wird vorzugsweise in einer Konzentration von 0,1 g/l bis 500 g/l, be- sonders bevorzugt von 1 g/l bis 50 g/l in die enzymatische Reduktion eingesetzt und kann kontinuierlich oder diskontinuierlich nach geführt werden.The substrate (1) is preferably used in a concentration of 0.1 g / l to 500 g / l, more preferably from 1 g / l to 50 g / l in the enzymatic reduction and can be performed continuously or discontinuously after ,
Die enzymatische Reduktion erfolgt in der Regel bei einer Reaktionstemperatur unterhalb der Desaktivierungstemperatur der eingesetzten Reduktase und oberhalb von -10 0C. Besonders bevorzugt liegt sie im Bereich von 0 bis 100 0C, insbesondere von 15 bis 60 0C und speziell von 20 bis 40 0C, z.B. bei etwa 30 0C.The enzymatic reduction is generally carried out at a reaction temperature below the deactivation of the reductase used and above -10 0 C. It is particularly preferably in the range of 0 to 100 0 C, in particular from 15 to 60 0 C and especially from 20 to 40 0 C, for example at about 30 0 C.
Eine bevorzugte Ausführung des erfindungsgemäßen Verfahrens besteht darin, die Reaktion in Gegenwart von zweiwertigen Metallionen, insbesondere in Gegenwart von Ca-, Mg-, Mn-, Zn-, Ni- Fe-, Mo-Ionen durchzuführen. Vorteilhaft ist es, die Konzentration der Erdalkaliionen etwa gleich hoch wie die Konzentration des einzusetzenden Substrates (Alken — Derivat der allgemeinen Formel I) zu wählen. Insbesondere wenn das Substrat aufgrund seiner Struktur in der Lage ist, Metallionen zu komplexieren, z.B. bei Dicarbonsäurederivaten, empfiehlt sich die Zugabe von zweiwertigen Metallionen in äquimolarer Konzentration wie das Substrat.A preferred embodiment of the process according to the invention consists in carrying out the reaction in the presence of divalent metal ions, in particular in the presence of Ca, Mg, Mn, Zn, Ni, Fe, Mo ions. It is advantageous to choose the concentration of alkaline earth metal ions about the same as the concentration of the substrate to be used (alkene derivative of general formula I). In particular, if the substrate is capable of complexing metal ions due to its structure, e.g. For dicarboxylic acid derivatives, the addition of divalent metal ions in equimolar concentration as the substrate is recommended.
Zur Durchführung kann man beispielsweise das Substrat (1 ) mit der Reduktase, dem Lösungsmittel und gegebenenfalls den Coenzymen, gegebenenfalls einer zweiten Dehydrogenase zur Regenerierung des Coenzyms und/oder weiteren Reduktionsmitteln vorlegen und das Gemisch durchmischen, z. B. durch Rühren oder Schütteln. Es ist aber auch möglich, die Reduktase in einem Reaktor, beispielsweise in einer Säule, zu immobilisieren, und durch den Reaktor eine das Substrat und gegebenenfalls Coenzy- me und/oder Cosubstrate enthaltende Mischung zu leiten. Hierzu kann man die Mischung im Kreislauf durch den Reaktor leiten bis der gewünschte Umsatz erreicht ist.To carry out, for example, the substrate (1) with the reductase, the solvent and optionally the coenzymes, optionally present a second dehydrogenase for the regeneration of the coenzyme and / or other reducing agents and mix the mixture, z. B. by stirring or shaking. However, it is also possible to immobilize the reductase in a reactor, for example in a column, and to pass through the reactor a mixture containing the substrate and optionally coenzymes and / or cosubstrates. For this purpose, the mixture can be circulated through the reactor until the desired conversion is achieved.
In der Regel wird man die Reduktion bis zu einem Umsatz von wenigstens 70 %, besonders bevorzugt von wenigstens 85 % und insbesondere von wenigstens 95%, bezogen auf das in der Mischung enthaltene Substrat führen. Das Fortschreiten der Reaktion, d. h. die sequentielle Reduktion der Doppelbindung kann dabei durch übliche Methoden wie Gaschromatographie oder Hochdruckflüssigkeitschromatographie verfolgt werden. „Funktionale Äquivalente" oder Analoga der konkret offenbarten Enzyme sind im Rahmen der vorliegenden Erfindung davon verschiedene Polypeptide, welche weiterhin die gewünschte biologische Aktivität, wie z.B. Substratspezifität, besitzen. So versteht man beispielsweise unter „funktionalen Äquivalenten" Enzyme, die die Modellreaktion kata- lysieren und die mindestens 20 %, bevorzugt 50 %, besonders bevorzugt 75 %, ganz besonders bevorzugt 90 % der Aktivität eines Enzyms, umfassend eine der unter SEQ ID NO:1 , 2 oder 3 aufgeführten Aminosäuresequenzen, aufweist. Funktionale Äquivalente sind außerdem vorzugsweise zwischen pH 4 bis 10 stabil und besitzen vorteilhaft ein pH-Optimum zwischen pH 5 und 8 sowie ein Temperaturoptimum im Bereich von 200C bis 800C.In general, the reduction will lead to a conversion of at least 70%, particularly preferably at least 85% and in particular of at least 95%, based on the substrate contained in the mixture. The progress of the reaction, ie the sequential reduction of the double bond can be followed by conventional methods such as gas chromatography or high pressure liquid chromatography. In the context of the present invention, "functional equivalents" or analogues of the specifically disclosed enzymes are different polypeptides which furthermore have the desired biological activity, such as substrate specificity. For example, "functional equivalents" are understood as meaning enzymes which catalyze the model reaction and which has at least 20%, preferably 50%, particularly preferably 75%, very particularly preferably 90% of the activity of an enzyme comprising one of the amino acid sequences listed under SEQ ID NO: 1, 2 or 3. In addition, functional equivalents are preferably stable between pH 4 to 10 and advantageously have a pH optimum between pH 5 and 8 and a temperature optimum in the range from 20 ° C. to 80 ° C.
Unter „funktionalen Äquivalenten" versteht man erfindungsgemäß insbesondere auch Mutanten, welche in wenigstens einer Sequenzposition der oben genannten Aminosäuresequenzen eine andere als die konkret genannte Aminosäure aufweisen aber trotz- dem eine der oben genannten biologischen Aktivitäten besitzen. „Funktionale Äquivalente" umfassen somit die durch eine oder mehrere Aminosäure-Additionen, - Substitutionen, -Deletionen und/oder -Inversionen erhältlichen Mutanten, wobei die genannten Veränderungen in jeglicher Sequenzposition auftreten können, solange sie zu einer Mutante mit dem erfindungsgemäßen Eigenschaftsprofil führen. Funktionale Äquivalenz ist insbesondere auch dann gegeben, wenn die Reaktivitätsmuster zwischen Mutante und unverändertem Polypeptid qualitativ übereinstimmen, d.h. beispielsweise gleiche Substrate mit unterschiedlicher Geschwindigkeit umgesetzt werden. According to the invention, "functional equivalents" are in particular also understood as meaning mutants which, in at least one sequence position of the abovementioned amino acid sequences, have a different amino acid than the one specifically mentioned but nevertheless have one of the abovementioned biological activities "Functional equivalents" thus include those represented by a or multiple amino acid additions, substitutions, deletions and / or inversions of available mutants, said changes may occur in any sequence position, as long as they lead to a mutant with the property profile according to the invention. Functional equivalence is given in particular even if the reactivity patterns between mutant and unchanged polypeptide match qualitatively, ie, for example, the same substrates are reacted at different rates.
Beispiele für geeignete Aminosäuresubstitutionen sind folgender Tabelle zu entnehmen:Examples of suitable amino acid substitutions are shown in the following table:
Ursprünglicher Rest Beispiele der SubstitutionOriginal rest Examples of substitution
AIa SerAIa Ser
Arg LysArg Lys
Asn GIn; HisAsn GIn; His
Asp GIuAsp Glu
Cys SerCys Ser
GIn AsnGIn Asn
GIu AspGiu Asp
GIy ProGIy Pro
His Asn ; GInHis Asn; Gin
Ne Leu; VaINe Leu; Val
Leu Ne; VaILeu Ne; Val
Lys Arg ; GIn ; GIuLys Arg; GIn; Glu
Met Leu ; NeMet Leu; ne
Phe Met ; Leu ; TyrPhe Met; Leu; Tyr
Ser ThrSer Thr
Thr SerThr Ser
Trp TyrTrp Tyr
Tyr Trp ; PheTyr Trp; Phe
VaI Ne; LeuVaI Ne; Leu
„Funktionale Äquivalente" im obigen Sinne sind auch „Präkursoren" der beschriebenen Polypeptide sowie „funktionale Derivate" ."Functional equivalents" in the above sense are also "precursors" of the described polypeptides and "functional derivatives".
„Präkursoren" sind dabei natürliche oder synthetische Vorstufen der Polypeptide mit oder ohne der gewünschten biologischen Aktiviät."Precursors" are natural or synthetic precursors of the polypeptides with or without the desired biological activity.
„Funktionale Derivate" erfindungsgemäßer Polypeptide können an funktionellen Aminosäure-Seitengruppen oder an deren N- oder C-terminalen Ende mit Hilfe bekannter Techniken ebenfalls hergestellt werden. Derartige Derivate umfassen beispielsweise aliphatische Ester von Carbonsäuregruppen, Amide von Carbonsäuregruppen, erhält- lieh durch Umsetzung mit Ammoniak oder mit einem primären oder sekundären Amin; N-Acylderivate freier Aminogruppen, hergestellt durch Umsetzung mit Acylgruppen; oder O-Acylderivate freier Hydroxygruppen, hergestellt durch Umsetzung mit Acylgruppen."Functional derivatives" of polypeptides of the invention may also be produced at functional amino acid side groups or at their N- or C-terminal end by known techniques Such derivatives include, for example, aliphatic esters of carboxylic acid groups, amides of carboxylic acid groups, obtained by reaction with ammonia or with a primary or secondary amine; N-acyl derivatives of free amino groups prepared by reaction with acyl groups; or O-acyl derivatives of free hydroxy groups prepared by reaction with acyl groups.
Im Falle einer möglichen Proteinglykosylierung umfassen erfindungsgemäße „funktionale Äquivalente" Proteine des oben bezeichneten Typs in deglykosylierter bzw. glyko- sylierter Form sowie durch Veränderung des Glykosylierungsmusters erhältliche abgewandelte Formen. "Funktionale Äquivalente" umfassen natürlich auch Polypeptide welche aus anderen Organismen zugänglich sind, sowie natürlich vorkommende Varianten. Beispielsweise lassen sich durch Sequenzvergleich Bereiche homologer Sequenzregionen festlegen und in Anlehnung an die konkreten Vorgaben der Erfindung äquivalente Enzyme ermit- teln.In the case of a possible protein glycosylation, "functional equivalents" according to the invention include proteins of the type described above in deglycosylated or glycosylated form as well as modified forms obtainable by altering the glycosylation pattern. Of course, "functional equivalents" also include polypeptides that are accessible from other organisms, as well as naturally occurring variants. For example, regions of homologous sequence regions can be determined by sequence comparison and, based on the specific requirements of the invention, equivalent enzymes can be determined.
„Funktionale Äquivalente" umfassen ebenfalls Fragmente, vorzugsweise einzelne Domänen oder Sequenzmotive, der erfindungsgemäßen Polypeptide, welche z.B. die gewünschte biologische Funktion aufweisen."Functional equivalents" also include fragments, preferably single domains or sequence motifs, of the polypeptides of the invention having, for example, the desired biological function.
„Funktionale Äquivalente" sind außerdem Fusionsproteine, welche eine der oben genannten Polypeptidsequenzen oder davon abgeleitete funktionale Äquivalente und wenigstens eine weitere, davon funktionell verschiedene, heterologe Sequenz in funktioneller N- oder C-terminaler Verknüpfung (d.h. ohne gegenseitigen wesentliche funktio- nelle Beeinträchtigung der Fusionsproteinteile) aufweisen. Nichtlimitierende Beispiele für derartige heterologe Sequenzen sind z.B. Signalpeptide oder Enzyme."Functional equivalents" are also fusion proteins which comprise one of the abovementioned polypeptide sequences or functional equivalents derived therefrom and at least one further, functionally different, heterologous sequence in functional N- or C-terminal linkage (ie without substantially mutual functional impairment of the fusion protein portions Nonlimiting examples of such heterologous sequences are, for example, signal peptides or enzymes.
Homologe des erfindungsgemäßen Proteine können durch Screening kombinatorischer Banken von Mutanten, wie z.B. Verkürzungsmutanten, identifiziert werden. Beispiels- weise kann eine variegierte Bank von Protein-Varianten durch kombinatorische Muta- genese auf Nukleinsäureebene erzeugt werden, wie z.B. durch enzymatisches Ligieren eines Gemisches synthetischer Oligonukleotide. Es gibt eine Vielzahl von Verfahren, die zur Herstellung von Banken potentieller Homologer aus einer degenerierten Oligo- nukleotidsequenz verwendet werden können. Die chemische Synthese einer degene- rierten Gensequenz kann in einem DNA-Syntheseautomaten durchgeführt werden, und das synthetische Gen kann dann in einen geeigneten Expressionsvektor ligiert werden. Die Verwendung eines degenerierten Gensatzes ermöglicht die Bereitstellung sämtlicher Sequenzen in einem Gemisch, die den gewünschten Satz an potentiellen Proteinsequenzen kodieren. Verfahren zur Synthese degenerierter Oligonukleotide sind dem Fachmann bekannt (z.B. Narang, S.A. (1983) Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al., (1984) Science 198:1056; Ike et al. (1983) Nucleic Acids Res. 1 1 :477).Homologs of the proteins of the invention can be prepared by screening combinatorial libraries of mutants, e.g. Shortening mutants, to be identified. For example, a variegated library of protein variants can be generated by combinatorial mutagenesis at the nucleic acid level, such as e.g. by enzymatic ligation of a mixture of synthetic oligonucleotides. There are a variety of methods that can be used to prepare libraries of potential homologs from a degenerate oligonucleotide sequence. The chemical synthesis of a degenerate gene sequence can be carried out in a DNA synthesizer, and the synthetic gene can then be ligated into a suitable expression vector. The use of a degenerate gene set allows for the provision of all sequences in a mixture that encode the desired set of potential protein sequences. Methods of synthesizing degenerate oligonucleotides are known to those skilled in the art (eg, Narang, SA (1983) Tetrahedron 39: 3; Itakura et al. (1984) Annu. Rev. Biochem. 53: 323; Itakura et al., (1984) Science 198: 1056; Ike et al. (1983) Nucleic Acids Res. 1 1: 477).
Im Stand der Technik sind mehrere Techniken zum Screening von Genprodukten kom- binatorischer Banken, die durch Punktmutationen oder Verkürzung hergestellt worden sind, und zum Screening von cDNA-Banken auf Genprodukte mit einer ausgewähltenSeveral techniques for screening gene products of combinatorial libraries made by point mutations or truncation and for screening cDNA libraries for gene products with a selected one are known in the art
Eigenschaft bekannt. Diese Techniken lassen sich an das schnelle Screening der Genbanken anpassen, die durch kombinatorische Mutagenese erfindungsgemäßer Homologer erzeugt worden sind. Die am häufigsten verwendeten Techniken zum Screening großer Genbanken, die einer Analyse mit hohem Durchsatz unterliegen, umfassen das Klonieren der Genbank in replizierbare Expressionsvektoren, Transfor- mieren der geeigneten Zellen mit der resultierenden Vektorenbank und Exprimieren der kombinatorischen Gene unter Bedingungen, unter denen der Nachweis der gewünschten Aktivität die Isolation des Vektors, der das Gen kodiert, dessen Produkt nachgewiesen wurde, erleichtert. Recursive-Ensemble-Mutagenese (REM), eine Technik, die die Häufigkeit funktioneller Mutanten in den Banken vergrößert, kann in Kombination mit den Screeningtests verwendet werden, um Homologe zu identifizieren (Arkin und Yourvan (1992) PNAS 89:781 1-7815; Delgrave et al. (1993) Protein Engineering 6(3):327-331 ).Property known. These techniques can be applied to the rapid screening of Adapt gene libraries generated by combinatorial mutagenesis of homologs of the invention. The most commonly used techniques for screening large libraries that are subject to high throughput analysis include cloning the library into replicable expression vectors, transforming the appropriate cells with the resulting vector library, and expressing the combinatorial genes under conditions such as detection of the desired activity facilitates the isolation of the vector encoding the gene whose product has been detected. Recursive ensemble mutagenesis (REM), a technique that increases the frequency of functional mutants in the banks, can be used in combination with the screening assays to identify homologs (Arkin and Yourvan (1992) PNAS 89: 781 1-7815; Delgrave et al., (1993) Protein Engineering 6 (3): 327-331).
Gegenstand der Erfindung sind weiterhin Nukleinsäuresequenzen (einzel- und dop- pelsträngige DNA- und RNA-Sequenzen, wie z.B. cDNA und mRNA), die für ein Enzym mit erfindungsgemäßer Reduktase -Aktivität kodieren. Bevorzugt sind Nukleinsäuresequenzen, welche z.B. für Aminosäuresequenzen gemäß SEQ ID NO:1 , 2 oder 3 charakteristische Teilsequenzen davon kodieren.The invention furthermore relates to nucleic acid sequences (single-stranded and double-stranded DNA and RNA sequences, such as, for example, cDNA and mRNA) which code for an enzyme with reductase activity according to the invention. Preferred are nucleic acid sequences which are e.g. for amino acid sequences according to SEQ ID NO: 1, 2 or 3 characterizing partial sequences thereof.
Alle hierin erwähnten Nukleinsäuresequenzen sind in an sich bekannter Weise durch chemische Synthese aus den Nukleotidbausteinen, wie beispielsweise durch Fragmentkondensation einzelner überlappender, komplementärer Nukleinsäurebausteine der Doppelhelix herstellbar. Die chemische Synthese von Oligonukleotiden kann beispielsweise, in bekannter Weise, nach der Phosphoamiditmethode (Voet, Voet, 2. Auf- läge, Wiley Press New York, Seiten 896-897) erfolgen. Die Anlagerung synthetischer Oligonukleotide und Auffüllen von Lücken mit Hilfe des Klenow-Fragmentes der DNA- Polymerase und Ligationsreaktionen sowie allgemeine Klonierungsverfahren werden in Sambrook et al. (1989), Molecular Cloning: A laboratory manual, CoId Spring Harbor Laboratory Press, beschrieben.All nucleic acid sequences mentioned herein can be prepared in a manner known per se by chemical synthesis from the nucleotide units, for example by fragment condensation of individual overlapping, complementary nucleic acid units of the double helix. The chemical synthesis of oligonucleotides can be carried out, for example, in a known manner by the phosphoamidite method (Voet, Voet, 2nd edition, Wiley Press New York, pages 896-897). The attachment of synthetic oligonucleotides and filling of gaps with the aid of the Klenow fragment of the DNA polymerase and ligation reactions and general cloning methods are described in Sambrook et al. (1989), Molecular Cloning: A laboratory manual, CoId Spring Harbor Laboratory Press.
Weitere Ausgestaltungen zur Durchführung des erfindungsgemäßen enzymatischen Reduktionsverfahrens:Further embodiments for carrying out the enzymatic reduction process according to the invention:
Der pH-Wert im erfindungsgemäßen Verfahren wird vorteilhaft zwischen pH 4 und 12, bevorzugt zwischen pH 4,5 und 9, besonders bevorzugt zwischen pH 5 und 8 gehalten, min. 98 %ee erreicht. Für das erfindungsgemäße Verfahren können wachsende Zellen verwendet werden, die für die Reduktase kodierenden Nukleinsäuren, Nukleinsäurekonstrukte oder Vektoren enthalten. Auch ruhende oder aufgeschlossene Zellen können verwendet werden. Unter aufgeschlossenen Zellen sind beispielsweise Zellen zu verstehen, die über eine Behandlung mit beispielsweise Lösungsmitteln durchlässig gemacht worden sind, oder Zellen die über eine Enzymbehandlung, über eine mechanische Behandlung (z.B. French Press oder Ultraschall) oder über eine sonstige Methode aufgebrochen wurden. Die so erhaltenen Rohextrakte sind für das erfindungsgemäße Verfahren vorteilhaft geeignet. Auch gereinigte oder angereinigte Enzyme können für das Verfahren ver- wendet werden. Ebenfalls geeignet sind immobilisierte Mikroorganismen oder Enzyme, die vorteilhaft in der Reaktion Anwendung finden können.The pH in the process according to the invention is advantageously maintained between pH 4 and 12, preferably between pH 4.5 and 9, more preferably between pH 5 and 8, min. 98% ee achieved. For the method according to the invention, growing cells can be used which contain nucleic acids encoding the reductase, nucleic acid constructs or vectors. Also dormant or open cells can be used. By open cells are meant, for example, cells which have been rendered permeable by treatment with, for example, solvents, or cells which have been disrupted by enzyme treatment, by mechanical treatment (eg French Press or ultrasound) or by some other method. The crude extracts thus obtained are advantageously suitable for the process according to the invention. Purified or purified enzymes can also be used for the process. Also suitable are immobilized microorganisms or enzymes that can be used advantageously in the reaction.
Das erfindungsgemäße Verfahren kann batchweise, semi-batchweise oder kontinuierlich betrieben werden.The process according to the invention can be operated batchwise, semi-batchwise or continuously.
Die Durchführung des Verfahrens kann vorteilhafterweise in Bioreaktoren erfolgen, wie z.B. beschrieben in Biotechnology, Band 3, 2. Auflage, Rehm et al Hrsg., (1993) insbesondere Kapitel II.The operation of the process may advantageously be carried out in bioreactors, e.g. in Biotechnology, Volume 3, 2nd Edition, Rehm et al. Ed., (1993), especially Chapter II.
Die in dem erfindungsgemäßen Verfahren hergestellten Produkte können aus dem Reaktionsmedium mit dem Fachmann geläufigen Verfahren isoliert und gewünschten- falls aufgereinigt werden. Hierzu zählen Destillationsverfahren, chromatographische Verfahren, Extraktionsverfahren und Kristallisationsverfahren. Die Reinigung der Produkte kann je nach Anforderung durch Kombination mehrerer dieser Verfahren deutlich erhöht werden.The products prepared in the process according to the invention can be isolated from the reaction medium by methods familiar to the person skilled in the art and, if desired, purified. These include distillation processes, chromatographic processes, extraction processes and crystallization processes. The cleaning of the products can be significantly increased depending on the requirement by combining several of these methods.
Die nachfolgenden Beispiele sollen die Erfindung veranschaulichen, ohne sie jedoch einzuschränken. Hierbei wird auf beiliegende Abbildungen Bezug genommen, dabei zeigt:The following examples are intended to illustrate the invention without, however, limiting it. Reference is made to the accompanying drawings, in which:
Experimenteller TeilExperimental part
Allgemeine Vorschrift zur asymmetrischen BioreduktionGeneral regulation for asymmetric bioreduction
Die asymmetrische Bioreduktion der Substrate wurde gemäß der folgenden allgemei- nen Vorschrift unter Verwendung der isolierten Enzyme YqjM, OPR1 , OPR3 durchge- führt. Wegen der schlechten Wasserlöslichkeit wurde N-Phenyl-2-methylmaleimid als 10% DMF Lösung zugesetzt ( 1 % Endkonzentration).The asymmetric bioreduction of the substrates was carried out according to the following general procedure using the isolated enzymes YqjM, OPR1, OPR3. leads. Because of poor water solubility, N-phenyl-2-methylmaleimide was added as a 10% DMF solution (1% final concentration).
Die Enzympräparation (100-200μg) wurde zu einer Lösung des Substrats (5mM) in Tris Puffer 50 mM ph 1,5 (0,8ml) mit dem Cofaktor NADH oder NADPH (15 mM) zugesetzt und die Reaktion wurde bei 300C unter Schütteln (140rpm) durchgeführt. Nach 48 Stunden wurde das Reaktionsgemisch mit Ethylacetat extrahiert und die Reaktionsprodukte über GC analysiert.The enzyme preparation (100-200μg) was 50 mM pH 1.5 (0.8 ml) with the cofactor NADH or NADPH (15 mM) was added to a solution of the substrate (5 mM) in Tris buffer and the reaction was performed at 30 0 C under Shaking (140rpm) performed. After 48 hours, the reaction mixture was extracted with ethyl acetate and the reaction products were analyzed by GC.
Bei Verwendung des Cofaktor-Recycling-Systems, wurde folgende Vorgehensweise gewählt:When using the cofactor recycling system, the following procedure was chosen:
NADH/FDH SystemNADH / FDH system
Zu einer Mischung aus Substrat (5mM) oxidiertem Cofaktor NAD+ (100μM), Ammoni- umfomat (2OmM) in Tris Puffer 5OmM pH 7,5 (0,8ml) wurde FDH (10u) zugegeben nachdem das Enzym (100-200μg) zugegeben worden war und die Reaktion wurde bei 300C (140 rpm) für 48 Stunden durchgeführt.To a mixture of substrate (5 mM) oxidized cofactor NAD + (100 μM), ammonium (2OmM) in Tris buffer 5OmM pH 7.5 (0.8 ml) was added FDH (10u) after the enzyme (100-200μg) was added and the reaction was carried out at 30 ° C. (140 rpm) for 48 hours.
NADH/GDH Zu einer Mischung aus Substrat (5mM) oxidiertem Cofaktor NAD+ (100μM), Glucose (2OmM) in Tris Puffer 5OmM pH 7,5 (0,8ml) wurde (D)-GDH (10u) zugegeben nachdem das Enzym (100-200μg) zugegeben worden war und die Reaktion wurde bei 300C (140 rpm) für 48 Stunden durchgeführt.NADH / GDH To a mixture of substrate (5 mM) oxidized cofactor NAD + (100 μM), glucose (20 mM) in Tris buffer 50 mM pH 7.5 (0.8 ml), (D) -GDH (10 μ) was added after the enzyme ( 100-200μg) was added and the reaction was (at 30 0 C 140 rpm) was performed for 48 hours.
NADPH/G6PDHNADPH / G6PDH
Zu einer Mischung aus Substrat (5mM) oxidiertem Cofaktor NADP+ (10μM), Glucose-6- Phosphat (2OmM) in Tris Puffer 5OmM pH 7,5 (0,8ml) wurde G6PDH (10u) zugegeben nachdem das Enzym (100-200μg) zugegeben worden war und die Reaktion wurde bei 30°C (140 rpm) für 48 Stunden durchgeführt.To a mixture of substrate (5 mM) oxidized cofactor NADP + (10 μM), glucose-6-phosphate (2OmM) in Tris buffer 50 mM pH 7.5 (0.8 ml) was added G6PDH (10μ) after the enzyme (100-200μg ) and the reaction was carried out at 30 ° C (140 rpm) for 48 hours.
GC-FID Analysen wurden auf einem Varian 3800 Gas Chromatographen mit H2 als Carriergas (14,5psi) durchgeführt.GC-FID analyzes were performed on a Varian 3800 gas chromatograph with H 2 as the carrier gas (14.5 psi).
1-Nitro-2-phenylpropen: Bestimmung der Umsetzung: Die erhaltenen Produkte wurden durch GC-FID analysiert unter Verwendung einer 6% Cyanopropylphenylpolysiloxane Phasenkapillarsäule (Varian CP-1301 , 30m, 0,25 mm, 0,25μm film) mit einem split-ratio von 30:1. Programm: 120°C/min bis 1800C, 20°C/min bis 2200C, 2 Min. Halt. Retentionszeiten waren folgende: Limonen (interner Standard) 3,81 min, 1-nitro-2-phenylpropan 8,87 Min, 1-Nitro-2- phenylproen Z/E 9,55/ 10,27 min resp.1-Nitro-2-phenylpropene: Determination of Reaction: The products obtained were analyzed by GC-FID using a 6% cyanopropylphenylpolysiloxanes phase capillary column (Varian CP-1301, 30m, 0.25mm, 0.25μm film) with a split ratio of 30: 1. Program: 120 ° C / min to 180 0 C, 20 ° C / min to 220 0 C, 2 min hold.. Retention times were as follows: limonene (internal standard) 3.81 min, 1-nitro-2-phenylpropane 8.87 min, 1-nitro-2-phenylpropene Z / E 9.55 / 10.27 min resp.
Bestimmung des enationmeren Überschuss und der absoluten Konfiguration: Der E- nantiomerenüberschuss wurde unter Verwendung einer Cyclodextrin-gebundenen Di- methylpolysiloxan Phasenkapillarsäule (CP-Chirasil-DEX CB, 25 m, 0,32 mm, 0,25 μm Film) mit einem split-ratio von 25:1. Temperaturprogramm: 1050C 5 min Halt, 1 °C/min bis 120°C, 6 min. Halt, 20°C/min bis 1800C, 2 Min. Halt. Retentionszeiten waren folgende: (S)- und (R) — 1-Nitro-2-phenylpropan 12,06 und 12,57 min, resp. Die absolute Konfiguration von 1-Nitro-2-phenylpropan wurde durch Coinjection einer unabhängig synthetisierten Referenzprobe (J. Org. Chem. 1989, 54, 1802-1804) ermittelt.Determination of the Enantiomeric Excess and the Absolute Configuration: The enantiomeric excess was determined using a cyclodextrin-bonded dimethylpolysiloxane phase capillary column (CP-Chirasil-DEX CB, 25 m, 0.32 mm, 0.25 μm film) with a split-phase ratio of 25: 1. Temperature program: 105 0 C 5 min stop, 1 ° C / min to 120 ° C, 6 min. Hold, 20 ° C / min to 180 0 C, 2 min. Hold. Retention times were as follows: (S) - and (R) -1-nitro-2-phenylpropane 12.06 and 12.57 min, respectively. The absolute configuration of 1-nitro-2-phenylpropane was determined by coinjection of an independently synthesized reference sample (J. Org. Chem. 1989, 54, 1802-1804).
CitralCitral
Bestimmung der Umsetzung: Die erhaltenen Produkte wurden durch GC-FID analysiert unter Verwendung einer Polyethylenglykol Phasenkapillarsäule (Varian CP-Wax 52CB, 30m, 0,25 mm, 0,25μm film) mit einem split-ratio von 20:1. Programm: 100°C Halt für 2 min., 15°C/min bis 2400C, 10 Min. Halt. Retentionszeiten waren folgende: Citronellal 5,21 min, 1-Octanol (interner Standard) 5,83 min, Geranial 7,53 min Bestimmung des enationmeren Überschuss und der absoluten Konfiguration: Der E- nantiomerenüberschuss von Citronellal wurde unter Verwendung einer modifizierten ß- Cyclodextrin Kapillarsäule (Hydrodex-ß-TBDAc, 25 m, 0,25 mm) Temperaturprogramm: 40°C Halt 2 min, 4°C/min bis 1200C, 1 min. Halt, 20°C/min bis 1800C, 3 Min. Halt. Retentionszeiten waren folgende: (S)- und (R) — Citronellal 19,84und 19,97 min resp. Die absolute Konfiguration von Citronellal wurde durch Coinjection einer kom- merziell erhältlichen Referenzprobe mit bekannter absoluter Konfiguration ermittelt.Determination of Reaction: The resulting products were analyzed by GC-FID using a polyethylene glycol phase capillary column (Varian CP-Wax 52CB, 30m, 0.25mm, 0.25μm film) with a split ratio of 20: 1. Program: 100 ° C hold for 2 min, 15 ° C / min to 240 0 C, 10 min hold... Retention times were as follows: Citronellal 5.21 min, 1-octanol (internal standard) 5.83 min, Geranial 7.53 min Determination of the enumeric excess and the absolute configuration: The enantiomeric excess of citronellal was measured using a modified β-cyclodextrin Capillary column (Hydrodex-ß-TBDAc, 25 m, 0.25 mm) Temperature program: 40 ° C hold 2 min, 4 ° C / min to 120 0 C, 1 min. Hold, 20 ° C / min to 180 0 C, 3 min. Hold. Retention times were as follows: (S) - and (R) - citronellal 19.84 and 19.97 min resp. The absolute configuration of citronellal was determined by coinjection of a commercially available reference sample of known absolute configuration.
N-Pheny-2-methylmaleimid:N-Phenyl-2-methylmaleimide:
Bestimmung der Umsetzung: Die erhaltenen Produkte wurden durch GC-FID analysiert unter Verwendung einer 6% Cyanopropylphenylpolysiloxane Phasenkapillarsäule (Va- rian CP-1301 , 30m, 0,25 mm, 0,25μm film) mit einem split-ratio von 30:1. Programm: 1 10°C Halt 2 min, 30°C/min bis 210°C, 6 Min. Halt. Retentionszeiten waren folgende: Limonen (interner Standard) 3,69 min, N-Phenyl-2-methylmaleimid 8,77min, N-phenyl- 2-methyl-succinimid 9,89 min. Bestimmung des enationmeren Überschuss und der absoluten Konfiguration: Der E- nantiomerenüberschuss wurde an einer Shimadzu Chiral HPLC unter Verwendung einer Chiralcel OD-H Säule und einer Lösung aus n-Heptan/Ethanol 95:5 als Eluent bestimmt. 80μl wurden isokratisch bei 18°C über 33 min eluiert. Retentionszeiten wa- ren folgende: (S)- und (R) — N-Phenyl-2-methylsuccinimid 27,0 und 29,1 min resp. Die absolute Konfiguration von 1 -N-Phenyl-2-methylsuccinimid wurde durch CD Spektroskopie (J. Mol. Catal. B:Enzym 2005, 32, 131-134) ermittelt.Determination of Reaction: The resulting products were analyzed by GC-FID using a 6% cyanopropylphenylpolysiloxane phase capillary column (Varian CP-1301, 30m, 0.25mm, 0.25μm film) with a split ratio of 30: 1. Program: 1 10 ° C stop 2 min, 30 ° C / min to 210 ° C, 6 min stop. Retention times were as follows: limonene (internal standard) 3.69 min, N-phenyl-2-methylmaleimide 8.77 min, N-phenyl-2-methyl-succinimide 9.89 min. Determination of the Enantiomeric Excess and the Absolute Configuration: The enantiomeric excess was determined on a Shimadzu Chiral HPLC using a Chiralcel OD-H column and a solution of n-heptane / ethanol 95: 5 as the eluent. 80μl was eluted isocratically at 18 ° C over 33 min. Retention times the following: (S) - and (R) - N-phenyl-2-methylsuccinimide 27.0 and 29.1 min resp. The absolute configuration of 1-N-phenyl-2-methylsuccinimide was determined by CD spectroscopy (J. Mol. Catal. B: Enzyme 2005, 32, 131-134).
Cyclische Enone:Cyclic enones:
Bestimmung der Umsetzung: Die erhaltenen Produkte wurden durch GC-FID analysiert unter Verwendung einer 6% Cyanopropylphenylpolysiloxane Phasenkapillarsäule (Va- rian CP-1301 , 30m, 0,25 mm, 0,25μm film) mit einem split-ratio von 30:1. Programm: 800C Halt für 10 min, 30°C/min bis 2000C, 3 Min. Halt. Retentionszeiten waren folgende: 2-Methylcyclopentanon 4,27 min, 3-Methylcyclopentanon 4,44 min, 2-Methyl-2- cyclo-penten-1-on 5,82min,3-Methylcylclohexanon 7,27 min, (R)-Limonen (interner Standard) 8,59 min, 3-Methyl-2-cyclopenten-1 on 8,77 min, 3-Methyl-2-cyclohexen-1-on 1 1 ,77 min. resp. Bestimmung des enationmeren Überschuss und der absoluten Konfiguration: Der E- nantiomerenüberschuss wurd unter Verwendung einer modifizierten ß-Cyclodextrin- Kapillarsäule (Chiraldex B-TA, 40 m, 0,25 mm) mit einem split-ratio von 25:1. Temperaturprogramm: 800C 17 min Halt, 30°C/min bis 180°C, 2 Min. Halt. Retentionszeiten waren folgende: (S)- und (R)- 3-Methylcyclopentanon 8,08 und 8,29 min, (R)- und (S)- 2-Methylcyclopentanon 6,55 und 6,77 min, (R)- und (S)- 3-Methylcyclohexanon 13,96 und 15,09 min. Die absolute Konfiguration von 3-Methylcylcopentanon und 3- Methylcyclohexanon wurde durch Coinjection einer kommerziell erhältlichen Referenzprobe mit bekannter absoluter Konfiguration ermittelt. . Die absolute Konfiguration von 2-Methylcylcopentanon wurde durch Coinjection einer unabhängig synthetisierten Re- ferenzprobe mit bekannter absoluter Konfiguration ermittelt (Tetrahedron :Asymmety 2001 , 12, 1479-1483). Determination of Reaction: The resulting products were analyzed by GC-FID using a 6% cyanopropylphenylpolysiloxane phase capillary column (Varian CP-1301, 30m, 0.25mm, 0.25μm film) with a split ratio of 30: 1. Program: 80 0 C hold for 10 min, 30 ° C / min to 200 0 C, 3 min hold.. Retention times were as follows: 2-methylcyclopentanone 4.27 min, 3-methylcyclopentanone 4.44 min, 2-methyl-2-cyclopenten-1-one 5.82 min, 3-methylcyclohexanone 7.27 min, (R) -Limones (internal standard) 8.59 min, 3-methyl-2-cyclopenten-1 on 8.77 min, 3-methyl-2-cyclohexen-1-one 1 1, 77 min. respectively. Determination of the Enantiomeric Excess and the Absolute Configuration: The enantiomeric excess was calculated using a modified β-cyclodextrin capillary column (Chiraldex B-TA, 40 m, 0.25 mm) with a split ratio of 25: 1. Temperature program: 80 0 C 17 min stop, 30 ° C / min to 180 ° C, 2 min stop. Retention times were as follows: (S) - and (R) - 3-methylcyclopentanone 8.08 and 8.29 min, (R) - and (S) -2-methylcyclopentanone 6.55 and 6.77 min, (R) - and (S) - 3-methylcyclohexanone 13.96 and 15.09 min. The absolute configuration of 3-methylcylcopentanone and 3-methylcyclohexanone was determined by coinjection of a commercially available reference sample of known absolute configuration. , The absolute configuration of 2-methylcylcopentanone was determined by coinjection of an independently synthesized reference sample of known absolute configuration (Tetrahedron: Asymmety 2001, 12, 1479-1483).

Claims

Patentansprüche claims
1. Verfahren zur enzymatischen Herstellung von Verbindungen der allgemeinen Formel (2) aus ungesättigten Alken-Derivaten der allgemeinen Formel (1 )1. Process for the enzymatic preparation of compounds of the general formula (2) from unsaturated alkene derivatives of the general formula (1)
a worin A für einen Nitrorest (-NO2), einen Ketonrest (-CRO), einen Aldehydrest (-CHO), einen Carboxylrest (-COOR) mit R= H oder gegebenenfalls substituerter CrCβ- a in which A is a nitrorest (-NO 2 ), a ketone radical (-CRO), an aldehyde radical (-CHO), a carboxyl radical (-COOR) with R = H or optionally substituted CrCβ-
Alkylrest, steht,Alkyl radical,
R1 , R2 und R3 unabhängig voneinander für H, C-i-Cβ- Alkyl, C2-C6- Alkenyl, Car- boxyl oder einen gegebenenfalls substituierten carbo- oder heterocyclischen, a- romatischen oder nichtaromatischen Rest steht, oder R1 ist mit R3 so verknüpft, dass sie Teil eines 4-8 gliedrigen Cyclus werden, oder R1 ist mit R so verknüpft, dass sie Teil eines 4-8 gliedrigen Cyclus werden, mit der Bestimmung, daß R1,R 1 , R 2 and R 3 independently of one another are H, C 1 -C 6 -alkyl, C 2 -C 6 -alkenyl, carboxyl or an optionally substituted carbo- or heterocyclic, aromatic or nonaromatic radical, or R 1 is with R 3 linked to form part of a 4-8 membered cycle, or R 1 is linked to R as part of a 4-8 membered cycle, with the proviso that R 1 ,
R2 und R3 nicht identisch sein dürfen, durch Reduktion einer Verbindung der Formel (1 ) in Gegenwart einer Reduktase (i) umfassend wenigstens eine der Polypeptidsequenzen SEQ IDR 2 and R 3 may not be identical, by reduction of a compound of formula (1) in the presence of a reductase (i) comprising at least one of the polypeptide sequences SEQ ID
NO:1 , 2, oder 3 oderNO: 1, 2, or 3 or
(ii) mit einer funktional äquivalenten Polypeptidsequenz, die mindestens 80% Sequenzidentität mit SEQ ID NO:1 , 2, oder 3 aufweist.(ii) with a functionally equivalent polypeptide sequence having at least 80% sequence identity with SEQ ID NO: 1, 2, or 3.
2. Verfahren nach Anspruch 1 , dadurch gekennzeichnet, dass die Reduktion mit NADPH oder NADH als Cofaktor durchgeführt wird.2. The method according to claim 1, characterized in that the reduction is carried out with NADPH or NADH as cofactor.
3. Verfahren nach Anspruch 2, dadurch gekennzeichnet, dass der verwendete Cofaktor enzymatisch regeneriert wird.3. The method according to claim 2, characterized in that the cofactor used is regenerated enzymatically.
Seq Seq
4. Verfahren nach Anspruch 3, dadurch gekennzeichnet, dass die Cofaktor-4. The method according to claim 3, characterized in that the cofactor
Regenerierung durch Glucose-Dehydrogenase oder Formiatdehydrogenase oder einen sekundären Alkohol erfolgt.Regeneration is carried out by glucose dehydrogenase or formate dehydrogenase or a secondary alcohol.
5. Verfahren nach einem der vorhergehenden Ansprüche, dadurch gekennzeichnet, dass die Reduktion in einem wässrigen, wässrig-alkoholischen oder alkoholischen Reaktionsmedium erfolgt.5. The method according to any one of the preceding claims, characterized in that the reduction is carried out in an aqueous, aqueous-alcoholic or alcoholic reaction medium.
6. Verfahren nach einem der vorhergehenden Ansprüche, dadurch gekennzeichnet, dass die Reduktase immobilisiert vorliegt.6. The method according to any one of the preceding claims, characterized in that the reductase is present immobilized.
7. Verfahren nach einem der vorhergehenden Ansprüche, wobei das Enzym ausgewählt ist unter Reduktasen aus Bacillus subtilis und Lycopersicum esculen- tum.7. The method according to any one of the preceding claims, wherein the enzyme is selected from reductases from Bacillus subtilis and Lycopersicum esculentum.
8. Verfahren nach einem der vorhergehenden Ansprüche, wobei eine Verbindung der Formel (1 ) umgesetzt wird, worin8. The method according to any one of the preceding claims, wherein a compound of formula (1) is reacted, wherein
R1 für Methyl und A für einen Ketonrest steht.R 1 is methyl and A is a ketone radical.
9. Verfahren nach einem der vorhergehenden Ansprüche, wobei die Reaktion bei eeiinneerr TTeemmppeerraattuurr iim Bereich von 0 bis 450C und/oder einem pH-Wert im Bereich von 6 bis 8 erfolgt.9. The method according to any one of the preceding claims, wherein the reaction is carried out in eggeierrert TTeemmppeerraattuurr in the range of 0 to 45 0 C and / or a pH in the range of 6 to 8.
10. Verfahren nach einem der vorhergehenden Ansprüche, wobei die Reaktion in- Gegenwart von zweiwertigen Metallionen durchgeführt wird.10. The method according to any one of the preceding claims, wherein the reaction is carried out in the presence of divalent metal ions.
1 1. Verwendung einer Verbindung der Formel (2), hergestellt nach einem Verfahren nach einem der vorhergehenden Ansprüche als Zwischenprodukt für die chemische oder enzymatische Wirkstoffsynthese. 1 1. Use of a compound of formula (2), prepared by a process according to any one of the preceding claims as an intermediate for the chemical or enzymatic synthesis of active ingredient.
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