EP2643467A1 - Procédé de fabrication d'alcools optiquement actifs n-hétérocycliques - Google Patents

Procédé de fabrication d'alcools optiquement actifs n-hétérocycliques

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Publication number
EP2643467A1
EP2643467A1 EP11784705.3A EP11784705A EP2643467A1 EP 2643467 A1 EP2643467 A1 EP 2643467A1 EP 11784705 A EP11784705 A EP 11784705A EP 2643467 A1 EP2643467 A1 EP 2643467A1
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EP
European Patent Office
Prior art keywords
nucleic acid
sequence
alkyl
sequences
azoarcus
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.)
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EP11784705.3A
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German (de)
English (en)
Inventor
Nina Schneider
Melanie Bonnekessel
Michael Breuer
Jürgen Däuwel
Klaus Ditrich
Ulrich Karl
Tobias STÄB
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BASF SE
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BASF SE
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Priority to EP11784705.3A priority Critical patent/EP2643467A1/fr
Publication of EP2643467A1 publication Critical patent/EP2643467A1/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D211/00—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings
    • C07D211/04—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D211/06—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D211/08—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms
    • C07D211/18—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D211/20—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms with substituted hydrocarbon radicals attached to ring carbon atoms with hydrocarbon radicals, substituted by singly bound oxygen or sulphur atoms
    • C07D211/22—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms with substituted hydrocarbon radicals attached to ring carbon atoms with hydrocarbon radicals, substituted by singly bound oxygen or sulphur atoms by oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/24—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D213/28—Radicals substituted by singly-bound oxygen or sulphur atoms
    • C07D213/30—Oxygen atoms
    • 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
    • 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
    • C12P17/12—Nitrogen as only ring hetero atom containing a six-membered hetero ring
    • 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
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/22—Preparation of oxygen-containing organic compounds containing a hydroxy group aromatic

Definitions

  • the present invention relates to a process for the preparation of N-heterocyclic optically active alcohols by dehydrogenases.
  • dehydrogenases as biocatalysts
  • the function of dehydrogenases as biocatalysts is well known [Chemico-Biological Interactions (2003) 143: 247, Journal of Biological Chemistry (2002) 277: 25677].
  • the technical application of this class of enzymes for the preparation of fine chemicals is documented [Tetrahedron (2004) 60: 633, Trends Biotechnol (1999) 17: 487].
  • the known dehydrogenase differ in their activity and specificity. A distinction is made between them in terms of their stereoselectivity in so-called 'Prelog' and 'anti' Prelog enzymes ⁇ Pure and Applied Chemistry, (1964), 9: 119).
  • the present invention relates to a process for the preparation of optically active alcohols of the formula I.
  • R are alkyl groups, which in turn may be mono- or polysubstituted, by alkyl, halogen, SH.SR 3 , OH, OR 3 , NO 2 , CN, CO, COOR 3 , NR 3 R 4 or NR 3 R 3 R 5+ X " , where R 3 , R 4 and R 5 independently of one another are H or a lower alkyl or lower alkoxy radical and X " is a counterion
  • R 2 represents N-containing heteroaryl groups, which in turn may be mono- or polysubstituted, by alkyl, halogen, SH.SR 3 , OH, OR 3 , NO 2 , CN, CO, COOR 3 , NR 3 R 4 or NR 3 R 3 R 5+ X " , wherein R 3 , R 4 and R 5 independently represent H or a lower alkyl or lower alkoxy radical and X " represents a counterion by reduction of the corresponding ketone, wherein the reduction with a dehydrogenase with the polypeptide sequence SEQ ID NO: 2 or NO: 4, or with a Polypeptide sequence in which up to 25% of the amino acid residues to SEQ ID NO: 2 or NO: 4 by deletion; Insertion, substitution or a combination thereof are changed.
  • a particularly good embodiment of the invention consists in a method for producing optically active alcohols of formula I, wherein R 1, C1-C5-alkyl, and R 2 is pyridinyl, in particular 4-pyridinyl, where the radicals R1 and / or R2 optionally substituted by halogen monosubstituted.
  • the process according to the invention gives optically active alcohols having (S) -configuration.
  • This process is also suitable for the preparation of optically active alcohols which contain N-containing non-aromatic heterocycles by subsequently hydrogenating the N-containing heteroaryl radical following the process described above.
  • Another object of the invention is a process for the preparation of optically active alcohols of formula III,
  • R represents alkyl groups, which in turn may be monosubstituted or polysubstituted, by alkyl, halogen, SH.SR 3 , OH, OR 3 , NO 2 , CN, CO, COOR 3 , NR 3 R 4 or NR 3 R 3 R 5+ X " , where R 3 , R 4 and R 5 independently of one another are H or a lower alkyl or lower alkoxy radical and X " is a counterion.
  • [R 2 ] H represents saturated N-containing heterocycles, which in turn may be monosubstituted or polysubstituted, by alkyl, halogen, SH.SR 3 , OH, OR 3 , NO 2 , CN, CO, COOR 3 , NR 3 R 4 or NR 3 R 3 R 5 + X " , where R 3 , R 4 and R 5 independently of one another are H or a lower alkyl or lower alkoxy radical and X " is a counterion.
  • the hydrogenation under step (b) can be carried out with all methods known to the person skilled in the art for the hydrogenation of aromatics.
  • Hydrogenation catalysts are preferably used here Use containing the elements Ru, Co, Rh, Ni, Pd or Pt (see Yang, P., eds., The Chemistry of Nanostructured Materials, World Scientific Publishing: Singapore, (2003); 357.)
  • the isolation and work-up of the resulting reaction product can be carried out by all common methods such as distillation, chromatography and crystallization. Usually, a distillation step, preferably a molecular distillation, is followed.
  • the product can normally be obtained in high chemical purity, for example by crystallization.
  • Alkyl stands for straight-chain or branched alkyl radicals 1 to 10, preferably 2-8, in particular 3-6 C-atoms, in particular methyl, ethyl, i- or n-propyl, n-, i-, sec- or tert Butyl, n-pentyl or 2-methyl-butyl, n-hexyl, 2-methyl-pentyl, 3-methyl-pently, 2-ethyl-butyl, 2-ethyl-hexyl.
  • Halogen is fluorine, chlorine, bromine or iodine, in particular fluorine or chlorine.
  • “Lower alkyl” denotes straight-chain or branched alkyl radicals of 1 to 6 C atoms, such as methyl, ethyl, isopropyl or n-propyl, n-, i-, sec- or tert-butyl, n-pentyl or 2-methyl- Butyl, n-hexyl, 2-methyl-pentyl, 3-methyl-pentane, 2-ethyl-butyl.
  • N-containing heteroaryl denotes a mononuclear or polynuclear, preferably mono- or binuclear, optionally substituted heteroaromatic radical which carries at least one nitrogen atom as constituent of the aromatic system, in particular for a pyridinyl bound via an arbitrary ring position
  • the N-containing heteroaryl preferably contains 5 and 6 rings
  • the N-containing heteroaryl may, in addition to the obligatory presence of one N atom, also carry further heteroatoms selected from among N, O and S.
  • These N-containing heteroaryls Ryl radicals may optionally carry 1, 2 or 3 identical or different substituents, for example halogen, lower alkyl, lower alkoxy as defined above or trifluoromethyl.
  • the linkage of the N-containing heteroaryl radical with the other radicals of the compound of the formula I can be effected via any ring position of the N-containing heteroaryl radical.
  • the linking is preferably carried out as 4-pyridinyl.
  • dehydrogenases are, above all, NAD- or NADP-dependent dehydrogenases (EC 1.1.1.x), in particular alcohol dehydrogenases (EC1.1.1.1 or EC1.1.1.2), which facilitate the selective reduction of the ketone to P effect / / og'-alcohol.
  • the dehydrogenase is preferably obtained from a microorganism, more preferably from a bacterium, a fungus, in particular a yeast, each deposited in strain collections or obtainable from isolates of natural source, such as soil samples, biomass samples and the like, or by de novo gene synthesis.
  • the dehydrogenase may be used in purified or partially purified form or in the form of the original microorganism or a recombinant host organism which expresses the dehydrogenase.
  • Methods for recovering and purifying dehydrogenases from microorganisms are well known to those skilled in the art, e.g. from K. Nakamura & T. Matsuda, "Reduction of Ketones” in K. Drauz and H. Waldmann, Enzyme Catalysis in Organic Synthesis 2002, Vol.III, 991-1032, Wley-VCH, Weinheim.
  • "Recombinant methods of production of dehydrogenases are also known, for example from W. Hummel, K. Abokitse, K. Drauz, C. Rollmann and H. Gröger, Adv. Synth. Catal. 2003, 345, No. 1 + 2, pp. 153-159.
  • Suitable bacteria are, for example, those of the orders of the Burkholderiales, Hydrophophilales, Methylophilales, Neisseriales, Nitrosomonadales, Procabacteriales or Rhodocyclales.
  • dehydrogenases from the family of the family Rhodocyclaceae. Particular preference is given to dehydrogenases from the genera Azoarcus Azonexus, Azospira, Azovibrio, Dechloromonas, Ferribacterium, Petrobacter, Propionivibrio, Quadricoccus, Rhodocyclus, Sterolibacterium, Thauera and Zoogloea.
  • dehydrogenases from species of the genera Azoarcus.
  • the reduction with the dehydrogenase in the presence of a suitable cofactor is usually NADH and / or NADPH.
  • a suitable cofactor also referred to as cosubstrate.
  • the cofactor used for the reduction of the ketone is usually NADH and / or NADPH.
  • dehydrogenases can be 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, Wley-VCH, Weinheim).
  • the reduction with the dehydrogenase is usually carried out 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 the hexoses, such as glucose, mannose, fructose, and / or oxidizable alcohols, in particular ethanol, propanol, butanol, pentanol or isopropanol, and also formate, phosphite or molecular hydrogen.
  • a second dehydrogenase e.g.
  • Glucose dehydrogenase when using glucose as a reducing agent phosphite dehydrogenase when using phosphite as a reducing agent or formate dehydrogenase in the use of formate as a reducing agent, are added. This can be used as a free or immobilized enzyme or in the form of free or immobilized cells. Their production can be both separately and through
  • the dehydrogenases 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-1 149849, 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, phenolformaldehyde resins, polyurethanes and polyolefins such as polyethylene and polypropylene.
  • the support materials are used to prepare the supported Enzymes are usually used in a finely divided, particulate form, with porous forms being 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 cross-linked directly with glutaraldehyde (cross-linking to CLEAs). Corresponding and further immobilization methods 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, Wley-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 contain, in addition to water, at least one alcohol, e.g. Contain ethanol or isopropanol or dimethyl sulfoxide.
  • Non-aqueous reaction media are 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 reaction medium.
  • the reaction is carried out in an organic solvent.
  • Suitable 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 xylenes, chlorobenzene or dichlorobenzene, aliphatic acyclic and cyclic ethers or
  • the reduction with the dehydrogenase is carried out in an aqueous-organic, in particular aqueous reaction medium.
  • the ketone to be reduced is preferably used in a concentration of 0, 1 g / l to 500 g / l, more preferably from 1 g / l to 200 g / l in the enzymatic reduction and can be followed continuously or discontinuously.
  • the enzymatic reduction is generally carried out at a reaction temperature below the deactivation temperature of the dehydrogenase used, and preferably at least -10 ° C. More preferably, it is in the range of 0 to 100 ° C, in particular from 15 to 60 ° C and especially from 20 to 40 ° C, for example at about 30 ° C.
  • the ketone with the dehydrogenase, 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.
  • the mixture can be circulated through the reactor until the desired conversion is achieved.
  • the keto group of the ketone is reduced to an OH group, wherein essentially one of the two enantiomers of the alcohol is formed.
  • 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 ketone contained in the mixture.
  • the sequential reduction of the ketone can be followed by conventional methods such as gas chromatography or high pressure liquid chromatography.
  • a first subject of the invention relates to functional phenylethanol dehydrogenase mutants derived from the phenylethanol dehydrogenase EbN1 from Azoarcus sp. with an amino acid sequence according to SEQ ID NO: 2.
  • Particularly preferred dehydrogenases used in the process according to the invention are alcohol dehydrogenases having the following properties:
  • Alcohol dehydrogenase from Azoarcus having an amino acid sequence according to SEQ ID NO: 2 and alcohol dehydrogenases with amino acid sequences in which up to 25%, preferably up to 15%, more preferably up to 10, in particular up to 5% of the amino acid residues compared to SEQ ID NO : 2 by deletion; Insertion, substitution or a combination thereof are changed.
  • the invention relates to functional alcohol dehydrogenase mutants derived from the alcohol dehydrogenase EbN1 from Azoarcus sp. having an amino acid sequence as shown in SEQ ID NO: 2, wherein the mutant comprises at least one mutation in at least one sequence region selected from
  • Sequence area 142 to 153 also referred to as Loop 2
  • Sequence region 190 to 21 1 also referred to as helix alpha FG1
  • the invention relates to functional alcohol dehydrogenase mutants which additionally comprise at least one further mutation in a further sequence region, chosen from
  • Sequence region 138 to 141 hydrophilic region binding pocket, also referred to as Loop 2
  • the invention relates to functional alcohol dehydrogenase mutants derived from the alcohol dehydrogenase EbN1 from Azoarcus sp. with an amino acid sequence according to SEQ ID NO: 2, wherein the mutant is selected from among the mutants listed in Table 1. Particular mention should be made of mutants, where at least one of the following residues is mutated:
  • mutants according to the invention are selected from mutants containing at least one of the following mutations:
  • Y151X A A, R, N, E, Q, G, H, I, L, M, T or V;
  • “Functional equivalents” or analogues of the specifically disclosed enzymes are, in the context of the present invention, different polypeptides which furthermore have the desired biological activity, such as substrate specificity "Functional equivalents” enzymes which reduce from the ketone to the corresponding "anti-Prelog” alcohol and which contain at least 20%, preferably 50%, more preferably 75%, most preferably 90% of the activity of an enzyme comprising one of Seq ID
  • 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 to mean mutants which have a different amino acid than the one specifically mentioned in at least one sequence position of the abovementioned amino acid sequences but nevertheless possess one of the abovementioned biological activities.
  • “Functional equivalents” thus include those by one or more Amino acid additions, substitutions, deletions and / or inversions available mutants, said changes can occur in any sequence position, as long as they lead to a mutant with the property profile according to the invention.
  • Functional equivalence is especially given when the Reactivity pattern between mutant and unchanged polypeptide match qualitatively, ie, for example, the same substrates are reacted at different speeds.
  • Val lle; Leu "Functional equivalents” in the above sense are also “precursors" of the described polypeptides and “functional derivatives” and “salts” of the polypeptides.
  • Precursors are natural or synthetic precursors of the polypeptides with or without the desired biological activity.
  • Salts are understood as meaning both salts of carboxyl groups and acid addition salts of amino groups of the protein molecules of the invention
  • Salts of carboxyl groups can be prepared in a manner known per se and include inorganic salts such as, for example, sodium, calcium, ammonium, iron and zinc salts, and salts with organic bases such as amines such as triethanolamine, arginine, lysine, piperidine and the like, acid addition salts such as salts with mineral acids such as hydrochloric acid or sulfuric acid and salts with organic acids such as acetic acid and oxalic acid
  • “Functional derivatives" of polypeptides according to the invention can also be prepared at functional amino acid side groups or at their N- or C-terminal end using known techniques.
  • Such derivatives include, for example, aliphatic esters of carboxylic acid groups, amides of carboxylic acid groups, obtainable by reaction with ammonia or with a primary or secondary amine; N-acyl derivatives of free amino groups, provided by reaction with acyl groups; or O-acyl derivatives of free hydroxy groups prepared by reaction with acyl groups.
  • “functional equivalents” include proteins of the abovementioned type 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, it is possible to determine regions of homologous sequence regions by sequence comparison and to determine equivalent enzymes on the basis of the specific requirements of the invention.
  • “Functional equivalents” also include fragments, preferably single domains or sequence motifs, of the polypeptides of the invention which, for example, have the desired biological function.
  • Fusion equivalents are also fusion proteins which have one of the above-mentioned polypeptide sequences or functional equivalents derived therefrom and at least one further functionally distinct heterologous sequence in functional N- or C-terminal linkage (ie without substantial substantial functional impairment of the fusion protein moieties)
  • heterologous sequences are, for example, signal peptides or enzymes.
  • homologs to the specifically disclosed proteins which have at least 75%, in particular at least 85%, such as 90%, 95%, 97% or 99%, homology to one of the specifically disclosed amino acid sequences, calculated according to Acyl, Sci. (USA) 85 (8), 1988, 2444-2448
  • a percent homology of a homologous polypeptide of the invention specifically means percent identity of the amino acid residues relative to the total length of one of the herein specifically described amino acid sequences.
  • Homologs of the proteins or polypeptides according to the invention can be produced by mutagenesis, for example by point mutation or truncation of the protein.
  • Homologs of the proteins of the invention can be identified by screening combinatorial libraries of mutants such as truncation mutants.
  • a variegated library of protein variants can be generated by combinatorial mutagenesis at the nucleic acid level, such as by enzymatic ligation of a mixture of synthetic Oligonucleotides.
  • methods can be used to prepare libraries of potential homologs from a degenerate oligonucleotide sequence.
  • 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 for the synthesis of degenerate oligonucleotides are known to the person 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 relates to the use of nucleic acid sequences (single-stranded and double-stranded DNA and RNA sequences, such as cDNA and mRNA) which code for an enzyme with dehydrogenase activity according to the invention.
  • nucleic acid sequences which are e.g. for amino acid sequences according to Seq ID 2 or SEQ ID 4 or characteristic partial sequences thereof, or nucleic acid sequences according to Seq ID 1 or SEQ ID 3 or characteristic 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, Cold Spring Harbor Laboratory Press.
  • the invention also relates to nucleic acid sequences (single- and double-stranded DNA and RNA sequences, such as cDNA and mRNA) coding for one of the above polypeptides and their functional equivalents, which are e.g. are accessible using artificial Nukleototidanaloga.
  • the invention relates both to isolated nucleic acid molecules which code for polypeptides or proteins or biologically active portions thereof according to the invention, as well as nucleic acid fragments which are e.g. for use as hybridization probes or primers for the identification or amplification of coding nucleic acids of the invention.
  • nucleic acid molecules of the invention may also contain untranslated sequences from the 3 'and / or 5' end of the coding gene region
  • the invention further comprises the nucleic acid molecules complementary to the specifically described nucleotide sequences or a portion thereof.
  • the nucleotide sequences of the invention enable the generation of probes and primers useful for the identification and / or cloning of homologous sequences in other cell types and organisms.
  • probes or primers usually comprise a nucleotide sequence region which is under "stringent" conditions (see below) at least about 12, preferably at least about 25, such as about 40, 50 or 75 consecutive nucleotides of a sense strand of a nucleic acid sequence of the invention or a corresponding nucleic acid sequence Antisense strands hybridizes.
  • nucleic acid molecule is separated from other nucleic acid molecules present in the natural source of the nucleic acid and, moreover, may be substantially free of other cellular material or culture medium when produced by recombinant techniques, or free from chemical precursors or other chemicals if it is synthesized chemically.
  • a nucleic acid molecule according to the invention can be isolated by means of standard molecular biological techniques and the sequence information provided according to the invention.
  • cDNA can be isolated from a suitable cDNA library by using one of the specifically disclosed complete sequences, or a portion thereof, as a hybridization probe and standard hybridization techniques (such as described in Sambrook, J., Fritsch, EF and Maniatis, T.
  • nucleic acid molecule comprising one of the disclosed sequences or a portion thereof can be isolated by polymerase chain reaction, using the oligonucleotide primers prepared on the basis of this sequence.
  • the thus amplified nucleic acid can be cloned into a suitable vector and characterized by DNA sequence analysis.
  • the oligonucleotides according to the invention can also be prepared by standard synthesis methods, for example with an automatic DNA synthesizer.
  • the nucleic acid sequences according to the invention can be identified and isolated in principle from all organisms.
  • the nucleic acid sequences according to the invention or the homologues thereof can be isolated from fungi, yeasts, archees or bacteria.
  • bacteria are called gram-negative and gram-positive bacteria.
  • the nucleic acids of Gram-negative bacteria according to the invention are preferably advantageously from a-proteobacteria, ⁇ -proteobacteria or ⁇ -proteobacteria, more preferably from bacteria of the orders Burkholderiales, Hydrogenophilales, Methylophilales, Neisseriales, Nitro-somonadales, Procabacteriales or Rhodocyclales ..
  • bacteria of the family Rhodocyclaceae Particularly preferred from the genus Azoarcus.
  • Azoarcus sp. EbN Azoarcus sp. FL05
  • Azoarcus sp. HA Azoarcus sp. HxN1
  • Azoarcus sp. mXyN Azoarcus sp.
  • PbN Azoarcus sp. PH002
  • Azoarcus sp. T Azoarcus sp. ToN 1.
  • Nucleic acid sequences according to the invention can be isolated from other organisms, for example via genomic or cDNA libraries, by conventional hybridization methods or the PCR technique, for example. These DNA sequences hybridize under standard conditions with the sequences according to the invention. For hybridization, it is advantageous to obtain short oligonucleotides of the conserved regions, for example from the active center, which are determined by comparisons with a dehydrogenase according to the invention in a manner known to the person skilled in the art can, used. However, it is also possible to use longer fragments of the nucleic acids according to the invention or the complete sequences for the hybridization.
  • nucleic acid hybrids are about 10 ° C lower than those of DNA: RNA hybrids of the same length.
  • the hybridization conditions for DNA are 0.1X SSC and temperatures between about 20 ° C to 45 ° C, preferably between about 30 ° C to 45 ° C.
  • the hybridization conditions are advantageously 0.10X SSC and temperatures between about 30 ° C to 55 ° C, preferably between about 45 ° C to 55 ° C.
  • temperatures for the hybridization are exemplary calculated melting temperature values for a nucleic acid with a length of about 100 nucleotides and a G + C content of 50% in the absence of formamide.
  • the experimental conditions for DNA hybridization are described in relevant textbooks of genetics, such as Sambrook et al., "Molecular Cloning", Cold Spring Harbor Laboratory, 1989, and can be determined by formulas known to those skilled in the art, for example, depending on the length of the nucleic acids that calculate type of hybrid or G + C content. Further information on hybridization can be found in the following textbooks by the person skilled in the art: Ausubel et al.
  • the invention also relates to derivatives of the specifically disclosed or derivable nucleic acid sequences.
  • nucleic acid sequences according to the invention can be derived from Seq ID 1 or Seq ID 3 and differ therefrom by addition, substitution, insertion or deletion of individual or several nucleotides, but furthermore code for polypeptides having the desired property profile.
  • nucleic acid sequences which comprise so-called silent mutations or, corresponding to the codon usage, a special source or vector Host organism, compared to a specific sequence mentioned are changed, as well as naturally occurring variants, such as splice variants or allelic variants thereof.
  • Articles are also available through conservative nucleotide substitutions (i.e., the amino acid in question is replaced by an amino acid of like charge, size, polarity, and / or solubility).
  • the invention also relates to the molecules derived by sequence polymorphisms from the specifically disclosed nucleic acids. These genetic polymorphisms may exist between individuals within a population due to natural variation. These natural variations usually cause a variance of 1 to 5% in the nucleotide sequence of a gene.
  • Examples of derivatives of a nucleic acid sequence according to the invention are allelic variants which have at least 40% homology at the derived amino acid level, preferably at least 60% homology, very particularly preferably at least 80, 85, 90, 93, 95 or 98% homology over the entire sequence range (for homology at the amino acid level, see the above discussion of the polypeptides). About partial regions of the sequences, the homologies may be advantageous higher.
  • derivatives are also to be understood as meaning homologs of the nucleic acid sequences according to the invention, for example fungal or bacterial homologs, truncated sequences, single-stranded DNA or RNA of the coding and noncoding DNA sequence.
  • promoters upstream of the indicated nucleotide sequences may be altered by one or more nucleotide exchanges, insertions, inversions and / or deletions, but without impairing the functionality or efficacy of the promoters.
  • the promoters can be increased in their effectiveness by changing their sequence or completely replaced by more effective promoters of alien organisms.
  • Derivatives are also to be understood as variants whose nucleotide sequence ranges from -1 to -1000 bases upstream of the start codon or 0 to 1000 bases downstream the stop codon were changed so that the gene expression and / or protein expression is changed, preferably increased.
  • the invention also encompasses nucleic acid sequences which hybridize with the abovementioned coding sequences under "stringent conditions.”
  • These polynucleotides can be found in the screening of genomic or cDNA libraries and, if appropriate, multiply therefrom with suitable primers by means of PCR and subsequently isolated, for example, with suitable probes
  • polynucleotides according to the invention can also be chemically synthesized, a property which is understood to be the ability of a poly- or oligonucleotide to bind under stringent conditions to a nearly complementary sequence, while under these conditions unspecific binding between non-complementary partners is avoided the sequences should be complementary to 70-100%, preferably 90-100%, The property of complementary sequences to be able to specifically bind to one another, for example, in the northern or S use outhern blot technique or in the primer binding in PCR or RT-PCR.
  • oligonucleotides are used from a length of 30 base pairs.
  • stringent conditions is meant, for example, in the Northern blot technique, the use of a 50 - 70 ° C, preferably 60 - 65 ° C warm wash, for example, 0.1x SSC buffer with 0.1% SDS (20x SSC: 3M NaCl , 0.3 M Na citrate, pH 7.0) for the elution of unspecifically hybridized cDNA probes or oligonucleotides.
  • SSC buffer with 0.1% SDS 20x SSC: 3M NaCl , 0.3 M Na citrate, pH 7.0
  • only highly complementary nucleic acids remain bound to each other.
  • the setting of stringent conditions is known to the person skilled in the art and is described, for example, in US Pat. in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. described.
  • the invention also relates to expression constructs comprising, under the genetic control of regulatory nucleic acid sequences, a nucleic acid sequence coding for a polypeptide according to the invention; and vectors comprising at least one of these expression constructs.
  • Such constructs according to the invention preferably comprise a promoter 5'-upstream of the respective coding sequence and a terminator sequence 3'-downstream and optionally further customary regulatory elements, in each case operatively linked to the coding sequence.
  • Operaational linkage is understood to mean the sequential arrangement of promoter, coding sequence, terminator and optionally further regulatory elements in such a way, that each of the regulatory elements can fulfill its function in the expression of the coding sequence as intended.
  • operably linked sequences are targeting sequences as well as enhancers, polyadenylation signals and the like.
  • Other regulatory elements include selectable markers, amplification signals, origins of replication, and the like. Suitable regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).
  • a nucleic acid construct according to the invention is in particular to be understood as meaning those in which the gene for a dehydrogenase according to the invention differs with one or more regulatory signals for the control, e.g. Increased, the gene expression was operatively or functionally linked.
  • the natural regulation of these sequences may still be present before the actual structural genes and possibly have been genetically altered so that the natural regulation is switched off and the expression of the genes has been increased.
  • the nucleic acid construct can also be simpler, ie no additional regulatory signals have been inserted before the coding sequence and the natural promoter with its regulation has not been removed. Instead, the natural regulatory sequence is mutated so that regulation stops and gene expression is increased.
  • a preferred nucleic acid construct advantageously also contains one or more of the already mentioned “enhancer” sequences, functionally linked to the promoter, which allow increased expression of the nucleic acid sequence. Additional advantageous sequences can also be inserted at the 3 'end of the DNA sequences, such as further regulatory elements or terminators.
  • the nucleic acids of the invention may be contained in one or more copies in the construct.
  • the construct may also contain further markers, such as antibiotic resistance or auxotrophic complementing genes, optionally for selection on the construct.
  • Advantageous regulatory sequences for the process according to the invention are, for example, in promoters such as cos-, tac-, trp-, tet-, trp-tet-, Ipp-, lac-, Ipp-lac-, laclq " T7-, T5-, T3- , gal, trc, ara, rhaP (rhaP B AD) SP6, lambda P R - or contained in the lambda P L promoter, which are advantageously used in gram-negative bacteria in the yeast or fungal promoters ADC1, MFalpha, AC, P-60, CYC1, GAPDH, TEF, rp28, ADH.
  • promoters of pyruvate decarboxylase and methanol oxidase are also included. See, for example, Hansenula advantageous. It is also possible to use artificial promoters for regulation.
  • the nucleic acid construct, for expression in a host organism is advantageously inserted into a vector, such as a plasmid or a phage, which allows for optimal expression of the genes in the host.
  • a vector such as a plasmid or a phage
  • vectors include all other vectors known to those skilled in the art, eg viruses such as SV40, CMV, baculovirus and adenovirus, transposons, IS elements, phasmids, cosmids, and linear or circular DNA. These vectors can be autonomously replicated in the host organism or replicated chromosomally. These vectors represent a further embodiment of the invention. Suitable plasmids are described, for example, in E.
  • plasmids mentioned represent a small selection of the possible plasmids. Further plasmids are well known to the person skilled in the art and can be found, for example, in the book Cloning Vectors (Eds. Pouwels PH et al., Elsevier, Amsterdam-New York-Oxford, 1985, ISBN 0 444 904018 ).
  • nucleic acid construct for expression of the further genes contained additionally 3'- and / or 5'-terminal regulatory sequences for increasing expression, which are selected depending on the selected host organism and gene or genes for optimal expression.
  • genes and protein expression are intended to allow the targeted expression of genes and protein expression. Depending on the host organism, this may mean, for example, that the gene is only expressed or overexpressed after induction, or that it is expressed and / or overexpressed immediately.
  • the regulatory sequences or factors can thereby preferably influence the gene expression of the introduced genes positively and thereby increase.
  • enhancement of the regulatory elements can advantageously be done at the transcriptional level by using strong transcription signals such as promoters and / or enhancers.
  • an enhancement of the translation is possible by, for example, the stability of the mRNA is improved.
  • the vector containing the nucleic acid construct according to the invention or the nucleic acid according to the invention can also advantageously be introduced in the form of a linear DNA into the microorganisms and integrated into the genome of the host organism via heterologous or homologous recombination.
  • This linear DNA can consist of a linearized vector such as a plasmid or only of the nucleic acid construct or of the nucleic acid according to the invention.
  • An expression cassette according to the invention is produced by fusion of a suitable promoter with a suitable coding nucleotide sequence and a terminator or polyadenylation signal.
  • common recombination and cloning techniques are used, as described, for example, in T. Maniatis, E.F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989) and T.J. Silhavy, M.L. Berman and L.W. Enquist, Experiments with Gene Fusion, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984) and Ausubel, F.M. et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley Interscience (1987).
  • the recombinant nucleic acid construct or gene construct is advantageously inserted into a host-specific vector for expression in a suitable veterinary organism, which enables optimal expression of the genes in the word.
  • Vectors are well known to those skilled in the art and can be found, for example, in "Cloning Vectors" (Pouwels P.H. et al., Eds. Elsevier, Amsterdam-New York-Oxford, 1985).
  • recombinant microorganisms can be produced, which are transformed, for example, with at least one vector according to the invention and can be used to produce the polypeptides according to the invention.
  • the recombinant constructs according to the invention described above are introduced into a suitable Wrtssystem and expressed.
  • familiar cloning and transfection methods known to those skilled in the art, such as, for example, co-precipitation, protoplast fusion, electroporation, retroviral transfection and the like, are used in order to express the stated nucleic acids in the respective expression system. Suitable systems are described, for example, in Current Protocols in Mo- lecular biology, F.
  • a vector which contains at least a portion of a gene or a coding sequence according to the invention, wherein optionally at least one amino acid deletion, addition or substitution has been introduced in order to modify the sequence according to the invention, eg functionally to disrupt it
  • the introduced sequence may also be a homologue from a related microorganism or derived from a mammalian, yeast or insect source
  • the vector used for homologous recombination may be designed to mutate the endogenous gene upon homologous recombination or otherwise modified, but the functional protein is still coded (eg the upstream regulatory region can be altered in such a way that this alters the expression of the endogenous protein.)
  • the altered segment of the gene according to the invention is in the homologous recombination vector
  • suitable vectors for homologous recombination is described, for example, in Thomas, KR and Capecchi, MR (1987) Cell 51: 503.
  • prokaryotic or eukaryotic organisms are suitable as recombinant host organisms for the nucleic acid according to the invention or the nucleic acid construct.
  • microorganisms such as bacteria, fungi or yeast are used as host organisms.
  • Gram-positive or gram-negative bacteria preferably bacteria of the families Enterobacteriaceae, Pseudomonadaceae, Rhizobiaceae, Streptomycetaceae or Nocardiaceae, particularly preferably bacteria of the genera Escherichia, Pseudomonas, Streptomyces, Nocardia, Burkholderia, Salmonella, Agrobacterium or Rhodococcus are advantageously used , Very particularly preferred is the genus and species Escherichia coli. Further advantageous bacteria are also found in the group of ⁇ -proteobacteria, ⁇ -proteobacteria or ⁇ -proteobacteria.
  • the Wrtsorganismus or Wrtsorganismen according to the invention preferably contain at least one of the nucleic acid sequences described in this invention, nucleic acid constructs or vectors which code for an enzyme with inventive dehydrogenase activity.
  • the organisms used in the method according to the invention are grown or grown, depending on the host organism, in a manner known to those skilled in the art.
  • Microorganisms are usually produced in a liquid medium, which is a carbon source mostly in the form of sugars, a nitrogen source usually in the form of organic nitrogen sources such as yeast extract or salts such as ammonium sulfate, trace elements such as iron, manganese, magnesium salts and optionally containing vitamins, at temperatures between 0 ° C and 100 ° C, preferably between 10 ° C to 60 ° C. attracted under oxygen fumigation.
  • the pH of the nutrient fluid can be kept at a fixed value, that is regulated during the cultivation or not.
  • the cultivation can be done batchwise, semi-batchwise or continuously. Nutrients can be presented at the beginning of the fermentation or fed in semi-continuously or continuously.
  • the ketone can be given directly for cultivation or advantageously after cultivation.
  • the enzymes may be isolated from the organisms by the method described in the Examples or used as crude extract for the reaction.
  • the invention furthermore relates to processes for the recombinant production of polypeptides according to the invention or functional, biologically active fragments thereof, which comprises cultivating a polypeptide-producing microorganism, optionally inducing the expression of the polypeptides and isolating them from the culture.
  • the polypeptides can thus also be produced on an industrial scale, if desired.
  • the recombinant microorganism can be cultured and fermented by known methods. Bacteria can be propagated, for example, in TB or LB medium and at a temperature of 20 to 40 ° C and a pH of 6 to 9. Specifically, suitable cultivation conditions are described, for example, in T. Maniatis, E.F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989).
  • the cells are then disrupted if the polypeptides are not secreted into the culture medium and the product recovered from the lysate by known protein isolation techniques.
  • the cells may optionally be treated by high frequency ultrasound, high pressure, e.g. in a French pressure cell, by osmolysis, by the action of detergents, lyti- see enzymes or organic solvents, by homogenizers or by combining several of the listed methods are digested.
  • Purification of the polypeptides can be accomplished by known chromatographic techniques such as molecular sieve chromatography (gel filtration) such as Q-Sepharose chromatography, ion exchange chromatography and hydrophobic chromatography, as well as other conventional techniques such as ultrafiltration, crystallization, salting out, dialysis and native gel electrophoresis. Suitable methods are described, for example, in Cooper, F.G., Biochemische Harvey Methoden, Verlag Walter de Gruyter, Berlin, New York or in Scopes, R., Protein Purification, Springer Verlag, New York, Heidelberg, Berlin.
  • vector systems or oligonucleotides for the isolation of the recombinant protein, which extend the cDNA by certain nucleotide sequences and thus code for altered polypeptides or fusion proteins which serve, for example, for a simpler purification.
  • suitable modifications include, for example, what are termed anchor tags, such as the modification known as hexa-histidine anchors, or epitopes that can be recognized as antigens of antibodies (described, for example, in Harlow, E. and Lane, D., et al. 1988, Antibodies: A Laboratory Manual, Cold Spring Harbor (NY) Press).
  • anchors can be used to attach the proteins to a solid support such as a poly mermatrix, which may for example be filled in a chromatography column, or may be used on a microtiter plate or on another carrier.
  • these anchors can also be used to detect the proteins.
  • conventional markers such as fluorescent dyes, enzyme markers which form a detectable reaction product after reaction with a substrate, or radioactive markers, alone or in combination with the anchors, can be used to identify the proteins for the derivation of the proteins.
  • the dehydrogenases can be used in the process according to the invention as a free or immobilized enzyme or as a catalyst still present in the recombinant production organism.
  • the inventive method is advantageously carried out at a temperature between 0 ° C to 95 ° C, preferably between 10 ° C to 85 ° C, more preferably between 15 ° C to 75 ° C.
  • the pH in the process according to the invention is advantageously maintained between pH 4 and 12, preferably between pH 4.5 and 9, particularly preferably between pH 5 and 8.
  • Enantiomerically pure or chiral products in the process according to the invention are enantiomers which show an enantiomeric enrichment.
  • enantiomeric purities of at least 70% ee preferably from min. 80% ee, more preferably from min. 90% ee, most preferably min. 98% ee achieved.
  • Growing cells containing the nucleic acids, nucleic acid constructs or vectors according to the invention can be used for the method according to the invention.
  • dormant or open cells can be used.
  • open cells are meant, for example, cells that have been rendered permeable through treatment with, for example, solvents, or cells that have been disrupted by enzyme treatment, mechanical treatment (eg French Press or ultrasound), or any other method.
  • the crude extracts thus obtained are advantageously suitable for the process according to the invention.
  • purified or purified enzymes can be used for the process.
  • 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 method can advantageously be carried out in bioreactors, as described, for example, in Biotechnology, Volume 3, 2nd Edition, Rehm et al. Ed., (1993), in particular Chapter II.
  • the sequence of the dehydrogenase gene EbN2 from Azoarcus sp. EbN1 is stored in databases (SEQ ID NO: 1, [Genbank ID 56475432, Region: 2797788..2798528]). From the nucleic acid sequence of the gene oligonucleotides were derived with which by known methods, the gene from genomic DNA of Azoarcus sp. EbN1 was amplified. The sequence obtained corresponds to the published sequence.
  • the PCR product (about 751 bp) was digested with the restriction endonucleases Nde and ßhamHI and cloned in appropriately digested pDHE19.2 vector (DE19848129).
  • the ligation mixtures were transformed into E. coli XL1 Blue (Stratagene).
  • the resulting plasmid pDHE-PDH-L was transformed into strain E. coli TG10 pAgro4 pHSG575 (TG10: a RhaA " derivative of E.
  • E. coli TG1 (Stratagene); pAgro4: Takeshita, S.; Sato, M; Toba, M Masahashi, W; Hashimoto-Gotoh, T (1987) Gene 61, 63-74; pHSG575: T. Tomoyasu et al (2001) Mol. Microbiol. 40 (2), 397-413) .
  • the recombined E. coli are designated LU 13151.
  • Example 2 Cloning of the alcohol dehydrogenase ChnA from Azoarcus sp. EbN1.
  • the sequence of the dehydrogenase gene ChnA from Azoarcus sp. EbN1 is stored in databases ([Genbank ID 56475432, Region: (complement) 192247..192993]). From the nucleic acid sequence of the gene oligonucleotides were derived with which by known methods, the gene from genomic DNA of Azoarcus sp. EbN 1 was amplified. The sequence obtained corresponds to the published sequence.
  • the PCR product (about 743bp) was digested with the restriction endonucleases Nde ⁇ and BglW and cloned into a Nde ⁇ and ßmHI restricted pDHE19.2 vector (DE19848129).
  • the ligation mixtures were transformed into E. coli XL1 Blue (Stratagene).
  • the resulting plasmid pDHE-PDH-L was transformed into strain E. coli TG 10 pAgro4 pHSG575 (TG10: a RhaA " derivative of E. coli TG 1 (Stratagene); pAgro4: Takeshita, S.; Sato, M; Toba, M; Masahashi, W; Hashimoto-Gotoh, T (1987) Gene 61, 63-74; pHSG575: T. Tomoyasu et al (2001) Mol. Microbiol. 40 (2), 397-413).
  • LU 13151 or LU 13283 were incubated in 20mL LB-Amp / Spec / Cm (100 ⁇ g / l ampicillin; 100 ⁇ g / l surfactinomycin; 20 g / l chloramphenicol), 0.1 mM IPTG, 0.5 g / L rhamnose in 100 ml Erlenmeyerkol - Ben (harassment) 18 h at 37 ° C attracted (alternatively, you can also first make a preculture with the same antibiotics, but without IPTG and rhamnose.This is incubated for 5 h at 37 ° C and then inoculated with 1% in the main culture ), centrifuged at 5000 * g / 10 min, washed once with 10 mM TRIS * HCl, pH 7.0 and resuspended in 2 mL of the same buffer.
  • the consumption of reduced cosubstrates during the reduction of ketones can be monitored at 340 nm.
  • 1 unit (1 U) corresponds to the amount of enzyme which reduces 1 ⁇ of ketone in 1 min.
  • a batch procedure instead of a dosage for the pyridin-4-yl-ethanone was tested since it simplifies the handling in the pilot plant. Furthermore, it was tested as a solvent instead of 2-butanol / 'so-propanol. In another experiment, the amount of / 'so-propanol was reduced from 40% to 20%, in order to shorten the subsequent distillation time.
  • the Reduktionsausträge are first filtered through Celite ®, respectively.
  • a first extraction is then carried out with twice the amount (w / w) of ethyl acetate optionally at room temperature or at 50 ° C to obtain a faster phase separation.
  • a Mulm Mrs between aqueous and organic phase This can be separated with the organic (a) or with the aqueous phase (b).
  • a further filtration is required.
  • 78% of the desired product are obtained as a light-sand solid.
  • Further extraction of the aqueous phase with 150% (w / w) ethyl acetate and work up according to (a) gives a further 10%, d. H. a total of 88% yield of (S) -1-pyridin-4-yl-ethanol in the 4L laboratory scale (ee> 99%, formerly purity> 98%).
  • a common nuclear hydrogenation catalyst was tested for the intended reaction. It was a supported metal catalyst consisting of 5% Ru on Al 2 0 3 . These contacts are intended for the fixed bed and were therefore ground for a batch procedure.

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Abstract

L'invention concerne un procédé de fabrication d'alcools optiquement actifs N-hétérocycliques de la formule (I), dans laquelle R1 représente des groupes alkyle, lesquels, de nouveau, peuvent être substitués une ou plusieurs fois par alkyle, halogène, SH.SR3, OH, OR3, NO2, CN, CO, COOR3, NR3R4 ou NR3R3R5+X-, où R3, R4 et R5 représentent indépendamment les uns des autres H ou un reste alkyle inférieur ou alcoxy inférieur et X- représente un contre-ion, R2 représente des groupes hétéroaryle à teneur en N, lesquels, de nouveau, peuvent être substitués une ou plusieurs fois par alkyle, halogène, SH.SR3, OH, OR3, NO2, CN, CO, COOR3, NR3R4 ou NR3R3R5+X-, où R3, R4 et R5 représentent indépendamment les uns des autres H ou un reste alkyle inférieur ou alcoxy inférieur et X- représente un contre-ion, par réduction de la cétone correspondante, la réduction étant réalisée avec une déshydrogénase ayant la séquence polypeptidique SEQ ID NO:2 ou NO:4, ou ayant une séquence polypeptidique dans laquelle jusqu'à 25 % des restes acide aminé sont modifiés par rapport à SEQ ID NO:2 ou NO:4 par délétion, insertion, substitution ou une combinaison de celles-ci.
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