DE102009007272A1 - New nucleic acid sequence, which encodes a polypeptide with alcohol dehydrogenase activity, comprising e.g. nucleic acid sequence that hybridizes under stringent conditions with specific nucleic acid sequence, useful to produce polypeptide - Google Patents

Abstract

Nucleic acid sequence (I), which encodes a polypeptide with an alcohol dehydrogenase activity, comprising (a) a nucleic acid sequence with the sequence represented in a sequence of glycolate oxidase-alcohol dehydrogenase (GO-ADH), (b) a nucleic acid sequence that hybridizes under stringent conditions with the nucleic acid sequence according to sequence of GO-ADH or the complementary sequence to it, or (c) a nucleic acid sequence, which has a homology of at least 50%, preferably 90% to the sequence of GO-ADH or to the sequence of GO-ADH complementary sequence, is new. Independent claims are included for: (1) a polypeptide comprising a polypeptide encoded by (I), or a polypeptide with a homology of at least 75%, preferably 90% to the polypeptide of the polypeptide encoded by the sequence of GO-ADH; (2) a recombinant expression system or recombinant vector comprising (I); (3) primers or probes for producing (I); (4) producing an improved recombinant polypeptide with an alcohol dehydrogenase activity based on (I), comprising cloning (I) in a suitable vector and transferring it into a suitable expression system, and detecting the formed polypeptide with improved activity, selectivity and/or stability and isolating; and (5) a whole-cell catalyst comprising a cloned gene coding for a polypeptide.

Description

The The present invention is directed to nucleic acids and by they are directed to encoded polypeptides, wherein the polypeptides are a Have alcohol dehydrogenase activity. The polypeptides or nucleic acids originate from Gluconobacter oxydans or are derived from them. Furthermore, the present is directed Invention on expression systems, primers, methods of preparation of improved nucleic acids or encoded by them Polypeptides and their use for the preparation of enantiomerically enriched Alcohols, hydroxyketones or hydroxyaldehydes. Also the invention is directed to whole cell catalysts containing the corresponding Alcohol dehydrogenase (ADH) and coupled reaction systems directed to the preparation of the enantiomerically enriched products.

A variety of biological reactions is catalyzed by alcohol dehydrogenases, whereby alcohol substrates are oxidized to the corresponding ketones or aldehydes, as well as the opposite reduction of aldehyde or ketone to the alcohol is catalyzed. The described reactions are usually reversible and proceed in the presence of nicotinamide adenine dinucleotide (NAD ⁺ / NADH) or nicotinamide adenine dinucleotide phosphate (NADP ⁺ / NADPH) as coenzyme (cofactor).

In addition to the outstanding importance of alcohol dehydrogenases in biological processes, these enzymes are also considered to be interesting catalysts for organic-chemical synthesis for the production of alcohols, ketones or aldehydes. The recovery of optically active organic compounds, eg. As alcohols, hydroxy-aldehydes or hydroxy ketones, biocatalytic way is becoming increasingly important. Of particular interest are biocatalysts that allow regioselective reactions. As a way to large-scale synthesis of these compounds, the coupled use of two dehydrogenases under Cofaktorregenerierung or the substrate-coupled Coenzymregenerierung has proven (as a current, comprehensive overview of the prior art, see: W. Hummel, Adv. Biochem. Engineering / Biotechnology 1997, 58, 145-184 ).

For A technical use of ADHs is limited by the substrate spectra the enzymes described. Each enzyme has only a limited spectrum compounds, and the enzymes have characteristic regions and stereoselectivities, which the use also limit. In addition, there is generally only a minor Proportion of alcohol dehydrogenases in the for technical applications required recombinant form.

Therefore, there is a great interest in the discovery of other alcohol dehydrogenases with new enzymatic properties, especially for the regio- and stereoselective reaction of polyol compounds. It is well known that microorganisms are capable of oxidizing hydroxyl groups of a polyol, but usually secondary alcohol groups are oxidized. For example, glycerol is oxidized by organisms of the genus Gluconobacter or Acetobacter to dihydroxyacetone ( Prescott, SC and Dunn, CG (1959) Industrial Microbiology, McGraw Hill Book Co., Inc., New York, "The Acetic Acid Bacteria and Sone of Their Biochemical Activities", p. 428-469 ).

From Of particular interest as products are glyceraldehyde (formula scheme 1) or homologs by oxidation of the corresponding polyols (Scheme 2, R = aromatic or aliphatic side chain) can be formed.

Glyceraldehyde is an industrially important compound that is chemically produced, for example, by osmium tetraoxide catalyzed reaction of acrolein; However, osmium is toxic and recovery from the product solution poses a major technological problem. An enzyme catalyzed process leading to glyceraldehyde has been reported by Upjohn Co. (US Pat. US 4,353,987 ) in which a methanol dehydrogenase is used which oxidizes glycerol to glyceraldehyde. The enzyme is a so-called quinoprotein enzyme, coenzyme of the reaction is an unnatural compound, the phenazine methosulfate. However, the reactions described in the patent are not complete. With whole cells of Methylobacterium organophilum producing methanol dehydrogenase, 35% glyceraldehyde is obtained; If enzyme (as crude extract) is used, only 1 g / L glyceraldehyde is obtained from a substrate solution of 100 g / L.

task Therefore, it is the object of the present invention to provide a nucleic acid sequence which codes for alcohol dehydrogenases, the to overcome the disadvantages of the enzymes present in the prior art capital. In particular, such alcohol dehydrogenases should also oxidize regioselectively polyols or hydroxyketones or

hydroxyaldehydes reduce stereoselectively. Particularly preferred are those Enzymes that are NAD- or NADP-dependent, because of These coenzyme regeneration methods are well known. Farther should the alcohol dehydrogenases the known enzymes in terms on economic aspects, in particular with regard to Superior stability, activity and / or selectivity be.

Farther It is the object of the invention to provide a method for the expression of Indicate alcohol dehydrogenases, which have the disadvantages of the stand the technology to overcome existing enzymes.

• a) einer Nukleinsäuresequenz mit der in Sequenz GO-ADH dargestellten Sequenz,
• b) einer Nukleinsäuresequenz, die unter stringenten Bedingungen mit der Nukleinsäuresequenz gemäß Sequenz GO-ADH oder der dazu komplementären Sequenz hybridisiert,
• c) einer Nukleinsäuresequenz, die eine Homologie von mindestens 50%, bevorzugt mindestens mindestens 75%, noch bevorzugter von mindestens 80% und besonders bevorzugt von über 90% zur Sequenz GO-ADH oder zu der zur Sequenz GO-ADH komplementären Sequenz aufweist.

This object is achieved with regard to the nucleic acid sequence according to the invention by a:
Nucleic acid sequence coding for a polypeptide having alcohol dehydrogenase activity selected from the group:

• a) a nucleic acid sequence having the sequence shown in sequence GO-ADH,
• b) a nucleic acid sequence which hybridizes under stringent conditions with the nucleic acid sequence according to sequence GO-ADH or the sequence complementary thereto,
• c) a nucleic acid sequence which has a homology of at least 50%, preferably at least 75%, more preferably at least 80%, and most preferably more than 90% to the sequence GO-ADH or to the sequence complementary to the sequence GO-ADH.

• a) einer Nukleinsäuresequenz mit der in Sequenz GO-ADH dargestellten Sequenz,
• b) einer Nukleinsäuresequenz, die unter stringenten Bedingungen mit der Nukleinsäuresequenz gemäß Sequenz GO-ADH oder der dazu komplementären Sequenz hybridisiert,
• c) einer Nukleinsäuresequenz, die eine Homologie von mindestens 50%, bevorzugt mindestens mindestens 75%, noch bevorzugter von mindestens 80% und besonders bevorzugt von über 90% zur Sequenz GO-ADH oder zu der zur Sequenz GO-ADH komplementären Sequenz aufweist, dadurch gekennzeichnet, dass man
• a) eine Nukleinsäuresequenzen gemäß Anspruch 1 einer Mutagenese unterwirft,
• b) die aus a) erhältliche Nukleinsäuresequenz in einen geeigneten Vektor kloniert und dieses in ein geeignetes Expressionsystem transferiert und
• c) die gebildeten Polypeptide mit verbesserter Aktivität und/oder Selektivität und/oder Stabilität detektiert und isoliert.

With regard to the method, the problem is solved by:
A method for producing an improved recombinant polypeptide having alcohol dehydrogenase activity starting from nucleic acid sequences selected from the group:

• a) a nucleic acid sequence having the sequence shown in sequence GO-ADH,
• b) a nucleic acid sequence which hybridizes under stringent conditions with the nucleic acid sequence according to sequence GO-ADH or the sequence complementary thereto,
• c) a nucleic acid sequence which has a homology of at least 50%, preferably at least 75%, more preferably at least 80% and particularly preferably more than 90% to the sequence GO-ADH or to the sequence complementary to the sequence GO-ADH marked that one
• a) subjecting a nucleic acid sequence according to claim 1 to a mutagenesis,
• b) the nucleic acid sequence obtainable from a) is cloned into a suitable vector and this is transferred into a suitable expression system and
• c) detecting and isolating the polypeptides formed with improved activity and / or selectivity and / or stability.

By the nucleic acid sequence according to the invention as well as the inventive method succeeds Solution of the described tasks in surprising simple, but not less advantageous in nature and Wise. With the indicated nucleic acid sequences can recombinant alcohol dehydrogenases of the type from Gluconobacter oxydans are obtained in high yields in host organisms, such as z. B. Escherichia coli, express and which in a high Activity also sterically demanding polyols with high Selectivity to the corresponding enantiomerically enriched Can implement connections. Also the stability the thus obtained alcohol dehydrogenases leaves a Use on an industrial scale under economic Aspects appear to be particularly advantageous. So can with the polypeptides encoded by the nucleic acids Alcohol dehydrogenase activity from racemic glyceraldehyde stereoselectively reduced the D-enantiomers and thus enantiomerically enriched L-glyceraldehyde are recovered.

The enzyme according to the invention preferably catalyses the following reaction (formula scheme 1):

Besides Ketones are also reduced or secondary alcohols are oxidized.

A detailed analysis of the dehydrogenase genes of the bacterium Gluconobacter oxydans has shown that this organism is NAD (P) -dependent Contains enzymes that surprisingly glycerol not to dehydroxyacetone, but by oxidation of the terminal React hydroxyl group to glyceraldehyde. Similar gene sequences are also present in Pseudomonas aeruginosa or Hypocrea jecorina. Because of the good activity of the recombinant enzyme G. oxydans will be mainly this enzyme in the following described. In addition, the characterization of this enzyme has demonstrated that the reduction of glyceraldehyde with this enzyme is stereoselective runs. If the racemate of glyceraldehyde is used, only the D-enantiomer is reduced, with complete Sequence of this reaction enantiomerically pure L-glyceraldehyde won can be (equation 4). Technologically, this reaction has the Advantage that the regeneration of the Conzyms NADPH in an advantageous For example, done with glucose and glucose dehydrogenase can.

In The present invention, in addition to the nucleic acid sequences the sequence GO-ADH also shows those that are under stringent Conditions with the nucleic acid sequence according to the invention or their complementary hybridize or others, by suitable mutagenesis methods have been improved. In particular are includes such nucleic acids, which are alleles or functional Variants of the nucleic acid sequences according to the invention represent. Functional variants preferably have a homology of at least 50%, preferably at least 66%, more preferably from at least 70% more preferably at least 80% and especially preferably more than 90% to the sequence GO-ADH.

So has, for example, the sequence PA-ADH from Pseudomonas aeruginosa a match with the sequence GO-ADH of 66% and also encodes a peptide with corresponding Alcohol dehydrogenase activity.

The procedure for improving the nucleic acid sequences according to the invention by mutagenesis methods is well known to the person skilled in the art. Suitable mutagenesis methods are all methods available to the person skilled in the art for this purpose. In particular, these are saturation mutagenesis, random mutagenesis, in vitro recombination methods and site-directed mutagenesis ( Eigen, M. and Gardiner, W., Evolutionary molecular engineering based on RNA replication, Pure Appl. Chem. 1984, 56, 967-978 ; Chen, K. and Arnold, F., Enzymes engineering for nonaqueous solvents: random mutagenesis to enhance the activity of subtilisin E in polar organic media. Bio / Technology 1991, 9, 1073-1077 ; Horwitz, M. and Loeb, L., Promoters Selected From Random DNA Sequences, Proc Natl Acad Sci USA 83, 1986, 7405-7409 ; Dube, D. and L. Loeb, Mutants Generated By The Insertion Of Random Oligonucleotides Into The Active Site Of The Beta-Lactamase Gene, Biochemistry 1989, 28, 5703-5707 ; Stemmer, PC, Rapid evolution of a protein in vitro by DNA shuffling, Nature 1994, 370, 389-391 and Stemmer, PC, DNA shuffling by random fragmentation and reassembly: In vitro recombination for molecular evolution. Proc Natl Acad Sci USA 91, 1994, 10747-10751 ).

The obtained new nucleic acid sequences are according to the following methods are cloned into a host organism and the polypeptides thus expressed detected by suitable screening methods and then isolated.

The Information from the sequence GO-ADH can be used to generate primers be used to directly allelic forms by PCR, z. In others To identify and identify Gluconobacter species strains clone. In addition, due to the sequence information Probes to find other naturally occurring functional Variants of the nucleic acids according to the invention and thus the corresponding coded enzyme variants are used. Starting from the sequence GO-ADH or from alleles or natural occurring functional variants may, for. B. via PCR using a defective DNA polymerase Bank of artificially generated functional enzyme variants obtained become.

The term "under stringent conditions" is used herein as in Sambrook et al. ( Sambrook, J .; Fritsch, EF and Maniatis, T. (1989), Molecular cloning: a laboratory manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York ), understood. Preferably, a stringent hybridization according to the of the present invention when washed after washing for one hour with 1 x SSC (150 mM sodium chloride, 15 mM sodium citrate, pH 7.0) and 0.1% SDS (sodium dodecyl sulfate) at 50 ° C, preferably 55 ° C, more preferably 62 ° C and most preferably at 68 ° C and more preferably for 1 hour with 0.2 x SSC and 0.1% SDS at 50 ° C, more preferably 55 ° C, more preferably 62 ° C and most preferably at 68 ° C still a positive hybridization signal is observed.

The polypeptides of the invention are in technical Processes due to the already indicated regio- and stereoselectivity and the extended substrate spectrum.

With This includes allelic or functional variants the polypeptides of the invention. Under one functional variant in the context of this invention is an alcohol dehydrogenase with an amino acid sequence homology to the sequence GO-ADH of at least 75%, preferably at least 77%, more preferably from above 85% very preferred of over 90% understood.

So shows, for example, the polypeptide encoded by the sequence PA-ADH an amino acid-based homology to that through the sequence GO-ADH coded polypeptide of 77%. The polypeptide encoded by PA-ADH shows a substantially comparable alcohol dehydrogenase activity, such as the polypeptides encoded by the sequence GO-ADH. By mutagenesis (see above) can amino acid substitutions in the inventive Polypeptides are added, but the activity and / or selectivity and / or stability of the polypeptide may not be significantly reduced.

So For example, amino acids that are not are located in the active center and are not expected to the replacement by an amino acid of the same group to a significantly changed three-dimensional structure leads, by an amino acid of the same group be replaced. For example, it can be expected that certain Amino acids from the group with non-polar side chains (eg alanine by valine) can be interchanged, without this having a (significant) impact on a catalytic or biochemical function of the enzyme in the context of the invention would have. On Based on his expertise, the expert can make appropriate conclusions also for replacing other amino acid types, for example, the replacement of acidic amino acids by others create acidic amino acids.

The polypeptides of the invention having alcohol dehydrogenase activity can also posttranslational modifications, such as As glycosylations or phosphorylations have.

In another preferred embodiment, the polypeptides of the invention further contain at least one heterologous amino acid segment which labels these polypeptides as fusion proteins. Heterologous constituents of the fusion protein according to the invention can be, for example, tags (eg his tag, flag tag, maltose-binding protein) which can be used in the purification of the fusion proteins according to the invention. In other embodiments, the heterologous components may have their own enzymatic activity. In such a case, the two enzymatic components are preferably linked by a linker such as a flexible 6-10 amino acid glycine or glycine-serine linker to ensure functionality of the components. As used herein, the term "heterologous" may mean, on the one hand, that the components of the fusion protein naturally do not co-covalently bond together, and, on the other hand, that the components are of different species Fusion proteins are usually produced by recombinant DNA technology (see Sambrook et al. , supra).

In In another embodiment, the present invention relates on recombinant expression systems or recombinant plasmids / vectors comprising one or more of the invention Nucleic acids. Under an expression system is a system for recombinant expression of the invention Nucleic acids and thus for the recombinant production of Understand polypeptides of the invention.

This preparation may preferably be carried out in microorganisms or other hosts transformed or transfected with appropriate nucleic acid sequences or vectors (see below) (the terms "transformation" and "transfection" are used identically according to this invention). Suitable host cells include cells of unicellular microorganisms such as bacterial cells. As microorganisms in this regard are prokaryotes, such as E. coli, Bacillus subtilis or Pseudomonas sp. Furthermore, bacteria of the genera / species Lactobacillus, Bacillus, Rhodococcus, Campylobacter, Caulobacter, Mycobacterium, Streptomyces, Neisseria, Ralstonia, as well as Agrobacterium for the expression of the nucleic acid according to the invention be used. Corresponding strains are available in the art and can be obtained, at least in part, from international depositaries such as ATCC or DMSZ. Likewise, eukaryotes, such as mammalian cells, insect cells or plant cells or organisms such. B. yeasts such as Hansenula polymorpha, Pichia sp., Saccharomyces cerevisiae, are used for the recombinant production of the polypeptides.

The cloning is carried out according to methods which are well known to the person skilled in the art ( Sambrook, J .; Fritsch, EF and Maniatis, T. (1989), Molecular cloning: a laboratory manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York ). Preferably, E. coli strains are used for this purpose, with very particular preference being given to E. coli XL1 Blue, NM 522, JM101, JM109, JM105, RR1, DH5α, TOP10, HB101, BL21 codon plus, BL21 (DE3 ) codon plus, BL21, BL21 (DE3), MM294, W3110, DSM14459 ( EP1444367 ).

Further can the recombinant production of the inventive Polypeptides are made in a non-human host. suitable eukaryotic cells include CHO cells, HeLa cells and others.

The transformation or transfection described above can be carried out by known methods, for. By calcium phosphate co-precipitation, lipofection, electroporation, PEG / DMSO method, particle bombardment or viral / bacteriophage infection. The cell according to the invention may contain the recombinant nucleic acid in extrachromosomal or chromosomally integrated form. Transfection and transformation protocols are known to the person skilled in the art ( Chan and Cohen. 1979. High Frequency Transformation of Bacillus subtilis Protoplasts by Palsmid DNA. Mol Gen Genet. 168 (1): 111-5 ; Kieser et al. 2000. Practical Streptomyces Genetics. The John Innes Foundation Norwich. ; Sambrook et al. 1989. Molecular Cloning. A Laboratory Manual. In: second ed. Cold Spring Harbor Laboratory Press. Cold Spring Harbor. NY. ; Irani and Rowe. 1997. Enhancement of transformation in Pseudomonas aeruginosa PAO1 by Mg2 + and heat. Biotechniques 22: 54-56 ).

In Another preferred embodiment is the host a transgenic non-human animal. Transgenic non-human Animals may be by methods known in the art getting produced. The transgenic invention non-human animal may prefer different genetic constitutions exhibit. It can be (i) the gene of a Overexpress nucleic acid sequence constitutively or inducibly, (ii) the endogenous gene of a gene of the invention Containing nucleic acid sequence in inactivated form, (iii) the endogenous gene of a nucleic acid sequence according to the invention completely or partially by a mutated gene of a Nucleic acid sequence of the invention (iv) conditional and tissue-specific overexpression or underexpression of the gene of a gene according to the invention Have nucleic acid sequence or (v) a conditional and tissue-specific knock-out of the gene of an inventive Having nucleic acid sequence. Preferably contains the transgenic animal additionally an exogenous gene of an inventive Nucleic acid sequence under control of overexpression permitting promoter. Alternatively, the endogenous gene of a Nucleic acid sequence by activation and / or replacement of the own promoter are overexpressed. Preferably the endogenous promoter of the gene of a Nucleic acid sequence a genetic change which leads to an increased expression of the gene. The genetic modification of the endogenous promoter comprises thereby both a mutation of individual bases as well as deletion and Insertion mutations.

In a particularly preferred embodiment of the host according to the invention, this is a transgenic rodent, preferably a transgenic mouse, a transgenic rabbit, a transgenic rat, or a transgenic sheep, a transgenic cow, a transgenic goat or a transgenic pig. Mice have many advantages over other animals. They are easy to maintain and their physiology is considered a model system for humans. The production of such gene-manipulated animals is well known to the person skilled in the art and is carried out by customary methods (cf. Hogan, B., Beddington, R., Costantini, F. and Lacy, E. (1994), Manipulating the Mouse Embryo ; A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY ; WO91 / 08216 ). Alternatively or additionally, cell culture systems, in particular human cell culture systems, can also be used for the applications described for the non-human transgenic animal according to the invention.

The coding nucleic acid sequences can be cloned into conventional plasmids / vectors and expressed after transfection of microorganisms or other host cells with such vectors in cell culture. In principle, all embodiments which are available to the person skilled in the art for this purpose are suitable as plasmids or vectors. Such plasmids and vectors may, for. B. Studier and Mitar worker ( Studier, WF; Rosenberg AH; Dunn JJ; Dubendroff JW; Use of the T7 RNA polymerase to direct expression of cloned genes, Methods Enzymol. 1990, 185, 61-89 ) or the brochures of Novagen, Promega, New England Biolabs, Clontech or Gibco BRL. Further preferred plasmids and vectors can be found in: Glover, DM (1985), DNA cloning: a practical approach, Vol. I-III, IRL Press Ltd., Oxford ; Rodriguez, RL and Denhardt, D.T. (eds) (1988), Vectors: a survey of molecular cloning vectors and their uses, 179-204, Butterworth, Stoneham ; Goeddel, DV, Systems for heterologous gene expression, Methods Enzymol. 1990, 185, 3-7 ; Sambrook, J .; Fritsch, EF and Maniatis, T. (1989), Molecular cloning: a laboratory manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York ,

Plasmids with which the gene construct described in this invention can preferably be cloned into the host organism are: pUC18 (Roche Biochemicals), pKK-177-3H (Roche Biochemicals), pBTac2 (Roche Biochemicals), pKK223-3 (Amersham Pharmacia Biotech), pKK-233-3 (Stratagene) or pET (Novagen). Suitable vectors are for. B. also pET-21a (+) for E. coli, but also other expression vectors for prokaryotic cell monoclonal and vectors for eukaryotes, such. Yeasts and insect or mammalian cells can be used. As suitable vectors for yeasts have z. As the pREP vector or the pINT vector proved. For expression in insect cells z. B. Baculovirus vectors as in EP127839 or EP549721 disclosed, and for expression in mammalian cells are z. B. SV40 vectors, which are commonly available. In a particularly preferred embodiment, the nucleic acid sequence of the invention introduced into the vector is additionally fused to a histidine tag provided by the vector. Preferably, the introduced nucleic acid sequence is cloned in pET vectors, for example pET-21a (+) or pET22b, such that the transcription is under the control of the IPTG-regulatable promoter present in the vector. Alternatively, rhamnose-regulatable promoters are preferably used.

The vectors can in addition to the usual markers such. B. antibiotic resistance genes, other functional nucleotide sequences for regulation, in particular for the repression or induction of the expression of the ADH gene and / or a reporter gene. As promoters are preferably regulatable weak promoters such. The rha promoter or the nmt1 promoter, or regulatable strong promoters, such as. As the lac, ara, lambda, pL, T7 or T3 promoter used. The coding DNA fragments must be transcribable in the vectors from a promoter. Other proven promoters are z. B. the baculovirus polyhedrin promoter for expression in insect cells (see, eg. EP127839 ) or the SV40 early promoter or LTR promoters z. From MMTV ( Mouse Mammary Tumor Virus; Lee et al. (1981) Nature, 294 (5838), 228-232 ).

The Expression vectors according to the invention can additional functional sequence regions, such. B. an origin of replication, Operators, or contain termination signals.

In In another embodiment, the present invention relates to partial sequences of the nucleic acid sequences according to the invention, these subsequences, preferably from at least 5 or 10, are preferred 50, more preferably 100, most preferably 150 consecutive Nucleotides, more preferably from at least 300 consecutive Nucleotides of the nucleic acids according to the invention consist. In particular, these are primers for the preparation of the invention Nucleic acid sequences by PCR or LCR or probes for obtaining the nucleic acid sequences according to the invention. The primers are derived from the 3'- or 5'-end of the invention Nucleic acid sequences. Preferably, they additionally have common Interfaces, such. B. NdeI at the 5 'end and at the 3' end one BamHI interface, on.

• a) die Nukleinsäuresequenzen einer Mutagenese unterwirft,
• b) die aus a) erhältlichen Nukleinsäuresequenzen in einen geeigneten Vektor kloniert und diesen in ein geeignetes Expressionsystem transferiert und
• c) die gebildeten Polypeptide mit verbesserter Aktivität und/oder Selektivität und/oder Stabilität detektiert und isoliert.

In a further embodiment, the present invention relates to a process for the preparation of improved rec-polypeptides (recombinant polypeptides) having alcohol dehydrogenase activity starting from nucleic acid sequences according to the invention, wherein

• a) subjecting the nucleic acid sequences to mutagenesis,
• b) cloning the nucleic acid sequences obtainable from a) into a suitable vector and transferring it into a suitable expression system, and
• c) detecting and isolating the polypeptides formed with improved activity and / or selectivity and / or stability.

Also an object of the invention are rec polypeptides or these encoding nucleic acid sequences, which according to a just described methods are available.

The preparation of the required for the production of improved rec-polypeptides nucleic acid sequences and their expression in hosts will be addressed in the further course of the description and applies here ent speaking.

Also The invention relates to mutants of alcohol dehydrogenase with altered coenzyme specificity, in particular such mutants accepting NAD instead of NADP.

In In a next embodiment, the present invention is directed Invention on the use of the invention (rec) polypeptides for the preparation of enantiomerically enriched Alcohols, hydroxyketones or hydroxyaldehydes. The implementation of appropriate Substrates to enantiomerically enriched products with the help of Alcohol dehydrogenases is known in principle to the person skilled in the art (see above references).

in der R₁ und R₂ verschieden voneinander sind und folgende Strukturen umfassen können:
(C₁-C₂₀)-Alkyl, (C₁-C₈)-Alkoxy, HO-(C₁-C₈)-Alkyl, (C₂-C₈)-Alkoxyalkyl,
(C₁-C₈)-Alkoxycarbonyl, (C₆-C₁₈)-Aryl, (C₇-C₁₉)-Aralkyl, (C₃-C₁₈)-Heteroaryl,
(C₄-C₁₉)-Heteroaralkyl, (C₁-C₈)-Alkyl-(C₆-C₁₈)-Aryl, (C₁-C₈)-Alkyl-(C₃-C₁₈)-Heteroaryl,
(C₃-C₈)-Cycloalkyl, (C₁-C₈)-Alkyl-(C₃-C₈)-Cycloalkyl, (C₃-C₈)-Cycloalkyl-(C₁-C₈)-Alkyl, oder R₁ und R₂ bilden eine (C₃-C₅)-Alkylenbrücke.Examples of enantiomerically enriched compounds which can be prepared from the corresponding precursors are also familiar to the person skilled in the art. They can be explained by the following general equation:

in which R ₁ and R _{2 are} different from one another and may comprise the following structures:
(C ₁ -C ₂₀ ) -alkyl, (C ₁ -C ₈ ) -alkoxy, HO- (C ₁ -C ₈ ) -alkyl, (C ₂ -C ₈ ) -alkoxyalkyl,
(C ₁ -C ₈ ) -alkoxycarbonyl, (C ₆ -C ₁₈ ) -aryl, (C ₇ -C ₁₉ ) -aralkyl, (C ₃ -C ₁₈ ) -heteroaryl,
(C ₄ -C ₁₉ ) heteroaralkyl, (C ₁ -C ₈ ) -alkyl (C ₆ -C ₁₈ ) -aryl, (C ₁ -C ₈ ) -alkyl (C ₃ -C ₁₈ ) -heteroaryl,
(C ₃ -C ₈ ) -cycloalkyl, (C ₁ -C ₈ ) -alkyl (C ₃ -C ₈ ) -cycloalkyl, (C ₃ -C ₈ ) -cycloalkyl (C ₁ -C ₈ ) -alkyl, or R ₁ and R ₂ form a (C ₃ -C ₅ ) -alkylene bridge.

Particularly preferred substrates for the alcohol dehydrogenases according to the invention are R ₂ = OH, NH ₂ , SH or other heteroatom-containing groups. Glyceraldehyde is a preferred compound. Further substrates are also ketoester compounds, in particular α-, β-, γ-keto ester compounds.

A Another embodiment of the invention relates to the use of Nucleic acid sequences of the invention for the preparation of whole-cell catalysts or the use of the Nucleic acid sequences of the invention for mutagenesis.

The invention likewise provides a whole-cell catalyst comprising a cloned gene coding for a polypeptide having alcohol dehydrogenase activity according to the invention and a cloned gene for an enzyme which is suitable for regenerating NADPH or, in the presence of NAD-dependent mutants, NADH, in particular one Formate dehydrogenase, a glucose dehydrogenase or an NADP or NAD regenerating enzyme such as NAD (P) H-oxidase, lactate dehydrogenase or glutamate dehydrogenase. The advantage of a whole-cell catalyst is the simultaneous expression of both polypeptide systems (alcohol dehydrogenase and an enzyme for coenzyme regeneration), with which only one rec organism must be attracted to the reaction. In order to tailor the expression of the polypeptides in terms of their conversion rates, the corresponding coding nucleic acid sequences can be accommodated on different plasmids with different copy numbers and / or different strength promoters can be used for different levels of expression of the nucleic acid sequences. In such tuned enzyme systems advantageously accumulation of an optionally inhibiting acting intermediate compound does not occur and the considered reaction can proceed in an optimal overall speed. However, this is well known to the person skilled in the art ( Gellissen, G .; Piontek, M .; Dahlems, U .; Jenzelewski, V .; Gavagan, JW; DiCosimo, R .; Anton, DL; Janowicz, ZA (1996), Recombinant Hansenula polymorpha as a biocatalyst. Coexpression of the spinach glycolate oxidase (GO) and the S. cerevisiae catalase T (CTT1) gene, Appl. Microbiol. Biotechnol. 46, 46-54; Farwick, M .; London, M .; Dohmen, J .; Dahlems, U .; Gellissen, G .; Strasser, AW ; DE19920712 ).

The nucleic acid sequences according to the invention can therefore be used preferably for the production of rec-polypeptides. Recombinant techniques well known to those skilled in the art provide organisms which are capable of providing the subject polypeptide in sufficient quantity for a technical process. The rec polypeptides according to the invention are prepared by genetic engineering methods known to the person skilled in the art ( Sambrook, J .; Fritsch, EF and Maniatis, T. (1989), Molecular cloning: a laboratory manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York ; Balbas, P. and Bolivar, F. (1990), Design and construction of expression plasmid vectors in E.coli, Methods Enzymol. 185, 14-37 ; Rodriguez, RL and Denhardt, D.T (eds) (1988), Vectors: a survey of molecular cloning vectors and their uses, 205-225, Butterworth, Stoneham ). With regard to the general procedure (PCR, cloning, expression, etc.), reference is also made to the following literature and the text cited therein: Universal Genome Walker ^™ Kit User Manual, Clontech, 3/2000 and literature cited therein; Triglia T .; Peterson, MG and Kemp, DJ (1988), A procedure for in vitro amplification of DNA segments that lie outside the boundaries of known sequences, Nucleic Acids Res. 16, 8186 ; Sambrook, J .; Fritsch, EF and Maniatis, T. (1989), Molecular cloning: a laboratory manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York ; Rodriguez, RL and Denhardt, D.T (eds) (1988), Vectors: a survey of molecular cloning vectors and their uses, Butterworth, Stoneham ,

About that In addition, the present invention relates in a next Aspect having a coupled enzymatic reaction system a cofactor dependent enzymatic transformation of a Precursor (substrate) with an inventive Polypeptide and an enzymatic regeneration of the cofactor (NAD (P) H). The enzymatic regeneration of the cofactor should advantageously with those discussed above in the context of the whole cell catalyst Enzymes, but it can also be electrochemical or performed by chemical oxidation without the use of enzymes become. As a reaction system, each vessel can be understood be performed in which the reaction of the invention reactors of any kind (loop reactor, stirred tank, Enzyme membrane reactor, etc.), or diagnostic kits in any form. at Use of a formate dehydrogenase is the cofactor regeneration using formic acid or salts thereof as reducing agent. Alternatively, however, other enzymatic or substrate-based cofactor-generating systems (eg NADH oxidase, glucose dehydrogenase) be used.

A final use of the invention (rec) polypeptides with alcohol dehydrogenase activity or the whole-cell catalyst according to the invention their use in a process for stereoselective reduction of aldehydes. For the implementation of aldehydes are suitable aqueous solvents buffered accordingly are.

Preferably, the reduction is carried out at a temperature between 10 and 80 ° C, more preferably between 20 and 50 ° C (see 2 ). The preferred pH for the enzymatically catalyzed reduction of carbonyl compounds with the alcohol dehydrogenase according to the invention is between pH 4 and pH 8, more preferably between pH 4.5 and pH 6.5 (see 1a ). For the oxidation of hydroxy groups, the preferred pH is between 7.5 and 11, more preferably between 8 and 10 (see 1b ).

For use, the subject polypeptide may be used in native form as homogeneously purified compounds or as a recombinantly produced enzyme. Furthermore, the (rec) polypeptide can also be used as part of an intact host organism or in conjunction with the disrupted and arbitrarily highly purified cell mass of the host organism. Also possible is the use of the enzymes in immobilized form ( Sharma BP; Bailey LF and Messing RA (1982), Immobilized Biomaterials - Techniques and Applications, Angew. Chem. 94, 836-852 ). Advantageously, the immobilization is carried out by lyophilization ( Paradkar, VM; Dordick, JS (1994), Aqueous-Like Activity of Chymotrypsin Dissolved in Nearly Anhydrous Organic Solvents, J. Am. Chem. Soc. 116, 5009-5010; Mori, T. ; Okahata, Y. (1997), A variety of lipidated glycoside hydrolases as effective glycosyl transfer catalysts in homogeneous organic solvents, Tetrahedron Lett. 38, 1971-1974 ; Otamiri, M .; Adlercreutz, P .; Matthiasson, B. (1992), Complex formation between chymotrypsin and ethyl cellulose as a means to solubilize the enzyme in active form in toluene, Biocatalysis 6, 291-305 ). Very particular preference is given to lyophilization in the presence of surface-active substances, such as aerosol OT or polyvinylpyrrolidone or polyethylene glycol (PEG) or Brij 52 (diethylene glycol mono-cetyl ether) ( Kamiya, N .; Okazaki, S.-Y .; Goto, M. (1997), surfactant horseradish peroxidase complex catalytically active in anhydrous benzene, Biotechnol. Tech. 11, 375-378 ). See Most preferably, the immobilization on Eupergit ^®, in particular Eupergit C and Eupergit 250L ^{® ®} (Röhm) (for review: E. Katchalski-Katzir, DM Kraemer, J. Mol. Catal. B: enzyme. (2000), 10, 157 ). Likewise preferred is immobilization on Ni-NTA in combination with the polypeptide modified by attachment of a His-tag (hexa-histidine) ( Petty, KJ (1996), metal-chelate affinity chromatography In: Ausubel, FM et al. eds. Current Protocols in Molecular Biology, Vol. 2, New York: John Wiley and Sons ). The use as CLECs is also conceivable ( St. Clair, N .; Wang, Y.-F .; Margolin, AL (2000), Cofactor-bound cross-linked enzyme crystals (CLEC) of alcohol dehydrogenase, Angew. Chem. Int. Ed. 39, 380-383 ). These measures make it possible to generate from (rec) polypeptides, which become unstable by organic solvents, those which can work in mixtures of aqueous and organic solvents or entirely in organic form.

The reaction of ketones with the polypeptides according to the invention is preferably carried out as follows. The polypeptides are added in the desired form (free, immobilized, in host organisms or as a whole cell catalyst) to the aqueous solution. While maintaining the optimum temperature or the optimum pH range, the ketone, if appropriate the cofactor and, if appropriate, the cofactor regenerating agent are added to this mixture. After the reaction has taken place, the alcohol obtained can be isolated from the reaction mixture by methods known to those skilled in the art (crystallization, extraction, chromatography).

following Scheme 5 shows the reaction equation of the reaction of racemic Glyceraldehyde to L-glyceraldehyde by ADH-catalyzed reduction.

It could surprisingly be shown that the inventive expressible proteins have an enzymatic alcohol dehydrogenase activity have. In particular, aliphatic hydroxyaldehydes or aldehydes with polyol structure are of the inventive Alcohol dehydrogenase reduced (see experimental part).

Farther the alcohol dehydrogenase according to the invention is characterized by their excellent regioselectivity. So will Glyceraldehyde reduced with good activity while the structurally analogous compound dihydroxyacetone practically unreacted becomes. Furthermore, the claimed alcohol dehydrogenase is distinguished by their excellent stereoselectivity. So will D-glyceraldehyde reacted with very good activity, the L enantiomers are not. This stereoselectivity remains also obtained when used as substrate the racemic mixture becomes. The enzyme reduces only the D component from this mixture. This implementation can be completed, so that as Product of complete reduction highly enantiomer-enriched L-glyceraldehyde remains in the reaction solution, it can thus ee values> 99% be achieved.

Under optically enriched (enantiomerically enriched, enantiomerically enriched) Compounds in the context of the invention, the presence of an optical Antipode in mixture with the other in> 50 mol% understood.

Under The term nucleic acid sequences are used to describe all types of single-stranded or double-stranded DNA as also subsumes RNA or mixtures thereof. The inventive Nucleic acid sequence can therefore be a DNA or an RNA molecule be. It is preferred that the nucleic acid molecule is a cDNA or an mRNA molecule. According to the invention the DNA molecule furthermore a genomic DNA molecule be.

Of the Term "complementary" means according to the invention, that complementarity over the whole Area of the nucleic acid molecule according to the invention extends without gaps. In other words, it is preferred according to the invention that the complementarity is 100% above the entire region of the sequence according to the invention, d. H. extends from the illustrated 5 'end to the illustrated 3' end.

The Improvement of activity and / or selectivity and / or stability means according to the invention that the polypeptides are more active and / or more selective or among those used Reaction conditions are more stable. While the activity and the stability of the enzymes for the technical Naturally, the application should be as high as possible should be in terms of selectivity then one Improve speech if either the substrate selectivity decreases, but the enantioselectivity of the enzymes increased is.

Of the Expression "homology" (or identity) As used herein, by the equation H (%) = [1-V / X] × 100 where H is homology, X is the total number Nucleobases / amino acids of the comparison sequence is and V the number of different nucleobases / amino acids the sequence to be considered is based on the comparison sequence. In any case, with the term nucleic acid sequences, which encode polypeptides comprising all sequences, according to the degeneration of the genetic code appear possible.

Suitable (C ₁ -C ₈ ) -alkyl radicals are methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, heptyl or octyl together with all their bonding isomers. A (C ₁ -C ₂₀ ) -alkyl radical is in the Rah men of the definition according to the invention a corresponding radical with 1 to max. 20 C atoms. A (C ₃ -C ₂₀ ) -alkyl radical is within the scope of the definition according to the invention a corresponding radical having 3 to max. 20 C atoms. The radical (C ₁ -C ₈ ) -alkoxy corresponds to the radical (C ₁ -C ₈ ) -alkyl with the proviso that it is bonded via an oxygen atom. By (C ₂ -C ₈ ) alkoxyalkyl are meant residues in which the alkyl chain is interrupted by at least one oxygen function, whereby two oxygen atoms can not be linked together. The number of carbon atoms indicates the total number of carbon atoms contained in the radical. A (C ₃ -C ₅ ) -alkylene bridge is a carbon chain with three to five carbon atoms, which chain is bound to the molecule under consideration via two different carbon atoms.

The radicals just described may be monosubstituted or polysubstituted by halogens and / or (C ₁ -C ₈ ) alkoxycarbonyl and / or N, O, P, S, Si atom-containing radicals. These are in particular alkyl radicals of the abovementioned type which have one or more of these heteroatoms in their chain or which are bonded to the molecule via one of these heteroatoms.

By (C ₃ -C ₈ ) -cycloalkyl is meant cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl radicals, etc. These may be with one or more halogens and / or N, O, P, S, Si atom-containing radicals be substituted and / or have N, O, P, S atoms in the ring, such as. 1-, 2-, 3-, 4-piperidyl, 1-, 2-, 3-pyrrolidinyl, 2-, 3-tetrahydrofuryl, 2-, 3-, 4-morpholinyl.

A (C ₃ -C ₈ ) -cycloalkyl (C ₁ -C ₈ ) -alkyl radical denotes a cycloalkyl radical as described above which is bonded to the molecule via an alkyl radical as indicated above.

(C ₁ -C ₈ ) -alkoxycarbonyl in the context of the invention means an alkyl radical as defined above with max. 8 C atoms, which is bound via an O (C = O) function.

(C ₁ -C ₈ ) -Acyloxy in the context of the invention means an alkyl radical as defined above with max. 8 C atoms, which is bound via a (C = O) O function.

(C ₁ -C ₈ ) acyl means in the context of the invention an alkyl radical as defined above with max. 8 C atoms, which is bound via a (C = O) function.

By a (C ₆ -C ₁₈ ) -aryl radical is meant an aromatic radical having 6 to 18 C atoms. In particular, these include compounds such as phenyl, naphthyl, anthryl, phenanthryl, biphenyl or to the molecule in question annelierte systems of the type described, such as. Indenyl systems which optionally have (C ₁ -C ₈ ) -alkyl, (C ₁ -C ₈ ) -alkoxy, (C ₂ -C ₈ ) -alkoxyalkyl, NH (C ₁ -C ₈ ) -alkyl, N ((C ₁ -C ₈ ) -alkyl) ₂ , OH, O (C ₁ -C ₈ ) -alkyl, NO ₂ , NH (C ₁ -C ₈ ) -acyl, N ((C ₁ -C ₈ ) Acyl) ₂ , F, Cl, CF ₃ , (C ₁ -C ₈ ) acyl, (C ₁ -C ₈ ) acyloxy, (C ₇ -C ₁₉ ) aralkyl, (C ₄ -C ₁₉ ) heteroaralkyl may be substituted.

A (C ₇ -C ₁₉ ) -aralkyl radical is a (C ₆ -C ₁₈ ) -aryl radical bonded to the molecule via a (C ₁ -C ₈ ) -alkyl radical.

In the context of the invention, a (C ₃ -C ₁₈ ) -heteroaryl radical denotes a five-, six- or seven-membered aromatic ring system comprising 3 to 18 C atoms, which heteroatoms such. B. nitrogen, oxygen or sulfur in the ring. As such heteroaromatics, especially radicals are considered, such as 1-, 2-, 3-furyl, such as 1-, 2-, 3-pyrrolyl, 1-, 2-, 3-thienyl, 2-, 3-, 4-pyridyl, 2-, 3-, 4-, 5-, 6-, 7-indolyl, 3-, 4-, 5-pyrazolyl, 2-, 4-, 5-imidazolyl, acridinyl, quinolinyl, phenanthridinyl, 2-, 4- , 5-, 6-pyrimidinyl. The heteroaromatics may be substituted in the same way as the abovementioned (C ₆ -C ₁₈ ) aryl radicals.

By (C ₄ -C ₁₉ ) heteroaralkyl is meant a heteroaromatic system corresponding to the (C ₇ -C ₁₉ ) aralkyl radical.

When Halogens (Hal) are fluorine, chlorine, bromine and iodine in question.

Under The term aqueous solvent becomes water or a solvent mixture consisting mainly of water with water-soluble organic solvents such as As alcohols, especially methanol or ethanol, or ethers, like THF or dioxane, understood

The Obtaining and biochemical characterization of an enzyme with which optically active compounds such as glyceraldehyde can be obtained, is the example of glycerol dehydrogenase from Gluconobacter oxydans described below.

Examples:

Example 1: Detection, Sequencing and cloning the glyceraldehyde-converting ADH gene from Gluconobacter oxydans

outgoing from the protein sequence of GlyDH from Hypocrea jecorina was one in silico screening for alcohol dehydrogenase (ADH) genes with the Genome performed by G. oxydans. After cloning and Expression of various ADH genes were photometric activity tests carried out with enzyme-containing crude extract. An important Criterion for the evaluation of the ADHs was the substrate selectivity, d. H. According to the invention suitable enzymes should Glyceraldehyde (GA) much better than the structurally analogous compound Dihydroxyacetone (DHA). Therefore, all recombinant enzymes were both tested with GA as well as with DHA containing test approaches per 1 ml: 100 mM TEA buffer pH 7.0; 0, 24 mM NADH or NADPH; 10 mM DHA or GA and 10 μl enzyme (crude cell extract). Surprisingly In these tests, a protein could be found, the glyceraldehyde with good activity, while dihydroxyacetone practically not accepted. The photometric test found: Glyceraldehyde reduction with NADPH: 67 U / ml; Glyceraldehyde-reduction with NADH: 0.5 U / ml; Dihydroxyacetone reduction with NADPH: 1.5 U / ml; Dihydroxyacetone reduction with NADH: 0 U / ml. The results show moreover, that it is an NADPH-dependent enzyme, NADH shows only a negligible activity. Since in this reaction an aldehyde is reduced to an alcohol, it must be the catalysing enzyme to an alcohol dehydrogenase (ADH) act. Derived from the name of her originally it was called GO-ADH.

Example 2: Heterologous expression of GO-ADH

to Expression of GO-ADH were E. coli BL21 (DE3) cells with the vector pGOADH transformed and cultured in ampicillin-containing LB medium. The cells were grown to an optical density (at 600 nm) of 0.5 incubated at 37 ° C and the expression of GO-ADH at Achieve the stated optical density with 0.3 mM IPTG solution induced. Thereupon the cell suspension became for 18 hours incubated at 25 ° C to give the optimal yield of GO-ADH to achieve.

Example 3: Investigation of the substrate spectrum recombinant alcohol dehydrogenase

In a first step is the preparation of crude extract containing the recombinant alcohol dehydrogenase from Gluconobacter oxydans, by cell disruption of the strain E. coli BL21 (DE3) pGOADH. To do this 52 g of the cell pellet with 120 ml Tris-HCl buffer (pH 7.5, 100 mM) and resuspended therein, leaving a biomass concentration of about 433 g / L is present. Subsequently, 1 ml of this Suspension under cooling for 2 × 2 min unlocked by ultrasound. After centrifugation (14,000 rpm; 10 min), a supernatant is obtained, which is subsequently as enzyme solution (crude extract) for activity determination and preparative experiments. The photometric Activity determination can be more oxidative and reductive Direction, for the reduction are used: 100 mM TEA buffer pH 7; 0, 24 mM NADPH; 10 mM substrate (glyceraldehyde or other keto compounds); 10 μl of enzyme (crude extract). For the oxidation reaction are used: 100 mM potassium phosphate pH 9.7; 2mM NADP; 100 mM substrate (glycerol or other alcohols); 30 mM ammonium sulfate; 10 μl of enzyme (crude extract). The reaction is performed in a 1 cm cuvette, to the components in the cuvette are mixed, the cuvette placed in the photometer and the data recording started. The enzyme solution is added just before the measurement begins. The activity of the enzyme is determined by changing the photometrically Concentration of NADPH determined in a given time interval. The measurement is carried out at a temperature of 30 ° C for the reduction and a temperature of 37 ° C for the oxidation, a wavelength of 340 nm and with a Measuring time of 1 min.

The Reactions can be made with both crude extract protein as well with partially or completely purified enzyme preparation be performed. For purification, the crude extract be partially purified with anion exchange chromatography (purification on Q-Sepharose FF material equilibrated with 0.1 M TEA buffer (pH 8.0), eluting with a gradient of 0-0.6 M NaCl). A simple method for almost complete cleaning is based on an enzyme that has a so-called His-tag (hexahistidine) at the N-terminus wearing. Such a modified enzyme can be simpler Manner bound to a nickel-NTA column (Qiagen) (equilibrated with 50 mM TEA buffer pH 7) and then with an imidazole gradient (from 0 to 500 mM). His-tag and the following Elution allow a very efficient cleaning of the Enzyme, you get a practical with this purification step homogeneous enzyme preparation.

The results are in U / mg enzyme protein attached in Table 1 (oxidation) and Tab. 2 (reduction) posed. In addition to the aldehydes and ketones listed in Tab. 2, the protected glyceraldehyde, the isopropylidene-glyceraldehyde, is also accepted with good activity.

Tab. 1: Activity of GO-ADH in oxidation of various substrates. An enzyme preparation with N-terminal His-tag was used, which was highly purified by chromatography on Ni-NTA. The determination of the Crude extract protein content in the enzyme solution according to the Bradford's method yielded a protein content of 4.3 mg per ml of enzyme solution.

Tab. 2: Activity of GO-ADH in the reduction of various substrates An enzyme preparation with N-terminal His-tag was used, which was highly purified by chromatography on Ni-NTA. The determination of the Crude extract protein content in the enzyme solution according to the Bradford's method yielded a protein content of 1.2 mg per ml of enzyme solution.

Example 4: Dependency of Activity of recombinant alcohol dehydrogenase of pH

In order to determine the dependence of the activity of the recombinant GO-ADH on the pH, the enzyme activities were measured photometrically using the oxidative or reductive assay described in Example 3, in which case only buffers at different pH values were used. The evaluations revealed ( 1a and 1b ) showed that the enzyme for the reductive direction (reduction of glyceraldehyde with NADPH) had a pH optimum at pH 5.5, whereas in the oxidative direction it showed the highest activity at pH 10.0.

Example 5: Dependence of Activity of the recombinant alcohol dehydrogenase of the temperature

To determine the dependence of the activity of the recombinant GO-ADH on the temperature, the enzyme activities were measured photometrically with the reductive test described in Example 3, only the temperature was varied in the range from 5 to 60 ° C. 2 shows that maximum activity is achieved at 55 ° C.

Example 6: Temperature stability recombinant alcohol dehydrogenase

To determine the temperature stability of the recombinant GO-ADH, the enzyme was preincubated at different temperatures between 5 and 58 ° C. for 10 min and then the residual activity was determined using the reductive standard test (see Example 3) with glyceraldehyde and NADPH. 3 shows that activity decreases significantly at 42 ° C due to this preincubation.

Example 7: Storage stability of recombinant alcohol dehydrogenase

Crude extract of the recombinant alcohol dehydrogenase was stored at 22 ° C., 4 ° C. and -20 ° C., and the residual activity was determined after certain time intervals using the standard test (glyceraldehyde and NADPH) described in Example 3. 4 shows the course of the residual activity under the different storage conditions.

Example 8: Use of the recombinant Alcohol dehydrogenase: recovery of enantiomeric-enriched Glyceraldehyde by racemate resolution with isolated enzymes

50 mM racemic glyceraldehyde, 100 U / ml GO-ADH, 100 mM glucose, 2 U / ml GDH and 0.5 mM NADP in 100 mM TEA pH 7 in a total volume of 1 ml were used. The reaction took place at 20 ° C. The reduction of GA was observed over 3 hours, at certain time intervals samples were taken and the concentration of the two glyceraldehyde enantiomers was determined by gas chromatography (GC determination method see Example 9). Cofactor regeneration took place with the help of the GDH. In 5 the result is shown, which indicates that GO-ADH reduces enantioselective glyceraldehyde, thereby degrading the D-enantiomer and enriching L-glyceraldehyde. An ee value of 95.2% could be determined after a reaction time of 4 h. Therefore, GO-ADH is a potential enzyme for the resolution of glyceraldehyde.

Example 9: Recovery of Enantiomer Enriched Glyceraldehyde with a whole cell catalyst

construction of a recombinant E. coli strain for whole-cell catalysis: As a whole-cell catalyst, a recombinant E coli strain was constructed, which is both the gene for the GO-ADH and a gene for a Contains enzyme whose gene product is the regeneration of NADPH can take over. As Regenerierungsenzym was the Glucose dehydrogenase (GDH) from Bacillus subtilis used. For the co-expression of GO-ADH and GDH became the expression vector pETDuet-1 (Novagen). This vector contains two multiple cloning sites besides an ampicillin resistance gene, each a T7 promoter and a ribosomal binding site precedes. The T7 terminator sits at the end of the second multiple Cloning site. The vector is for coexpression optimally suited for two genes.

For the gdh gene was constructed primers that restricted the gene EcoRI and NotI should be added at the ends. These primers were used in a PCR. The purified PCR product as well the vector pETDuet-1 was subsequently restricted subjected to the restriction enzymes EcoRI and NotI. The cut Vector was dephosphorylated with shrimp alkaline phosphatase. Both DNA fragments were ligated with the T4 DNA ligase after ligation competent expression strain E. coli XL1-Blue transformed. To Cultivation of some clones was followed by isolation of the plasmids with the following Restriction and gel electrophoretic analysis. The gdh sequence one of the obtained clones was confirmed by sequence analysis.

Restriction approaches of this plasmid and the goadh fragment, to which the ends NdeI and KpnI were added by PCR, were then carried out with the restriction enzymes NdeI and KpnI. After dephosphorylation of the vector with alkaline phosphatase from shrimp, ligation of the two DNA fragments with T4 DNA ligase and transformation with competent E. coli XL1-Blue cells were performed. From the colonies resulting after selection on LBamp plates, several clones were selected and grown in 5 ml LBamp medium overnight. From the cultures, the plasmid DNA was isolated, which was separated by restriction on an agarose gel electrophoresed. Sequencing of one of the resulting clones confirmed the correct gene sequence. 6 returns the plasmid map for the plasmid containing the genes for the two enzymes GO-ADH and GDH.

For the expression became the vector pAW-21 with competent E. coli BL21 (DE3) cells transformed. A single colony was inoculated into 5 ml of LBamp medium and shaken overnight at 37 ° C. Subsequently 1% was inoculated in 30 ml LBamp medium, shaken at 37 ° C and after reaching a OD600 of 0.5 with 1 mM IPTG induced gene expression. After 20 hours Shaking at 25 ° C, the cells were harvested.

Cell Culture: E. coli BL21 (DE3) pAW21: A single colony from an agar plate (LB / 100 mg / L ampicillin) is picked and grown in 5 ml LB medium (100 mg ampicillin / L) overnight at 37 ° C. An Erlenmeyer flask containing 30 mL LB medium (100 mg Amp./L) is inoculated with 300 μL of this culture and incubated over the weekend at room temperature. 500 mL LB medium (100 mg / L ampicillin) in a 2 L Erlenmey are inoculated with 5 ml of the culture and grown at 37 ° C to OD580 = 0.56, then induced with 1 mM IPTG and shaken at 25 ° C for 16 hours. After centrifugation at 4 ° C (30 min, 3200 G) 3.61 g of cells are obtained, which are stored at -20 ° C. Measurements of the Activity of the Enzymes GDH and GO-ADH in the Whole Cell Catalysts: 80 mg cells are suspended in 320 μL TEA buffer (50 mM, pH 7.0) by ultrasound (3 × 60 sec., 50% cycle, 50% intensity, 60 sec. each break) on ice. The activity of the soluble proteins is determined according to standard tests on the photometer, there were 151 U / ml for the GO-ADH (test with rac. Glyceraldehyd / NADPH) and 89 U / ml for the glucose dehydrogenase (test with 5 mM glucose, 0.25 mM NAD). The Bradford test showed a protein content of 15.6 mg / mL.

resolution using the whole-cell catalyst (recombinant E. coli cells): To obtain enantiomerically-enriched glyceraldehyde were used: 100 mM ammonium carbonate buffer, with conc. HCl to pH 8.0 set; 300 mM glyceraldehyde, 180 mM glucose, taking after Were re-dosed with 12.6 mM for 80 h; 56 g / L cells BL21 pAW21 (wet cell weight). Procedure: 300 mM racemic buffer in 30 ml Glyceraldehyde and 180 mM glucose presented, mixed with 1.7 g of whole-cell catalyst and stirred at room temperature (22 ° C) with exclusion of light. Regularly samples are taken, derivatized and measured by gas chromatography, at the same time became the pH regularly checked. The GC analysis shows at 71.5 hr reaction time, an ee value for the L-enantiomer of glyceraldehyde of 97.4%. After 75 hours of reaction were 10 ml of ammonium carbonate solution and 100 mg of glucose are added. A sample after 80 hrs reaction time showed on the GC that only the L-enantiomer was detectable. The reaction solution was centrifuged (10 min, 20,000 g), the clear supernatant (about 38 ml) is removed and stored at -20 ° C. For the GC analysis, the samples were each according to the following Method prepared: 120 μL samples were taken and centrifuged, 100 μL supernatant were derivatized with 150 μL ethyl acetate, 150 μL 1,4-dioxane, 100 μL Acetic anhydride and 10 μL of pyridine; the solution becomes directly injected. The concentration of the two glyceraldehyde enantiomers was determined under the following conditions: Column: CP-Chirasil-DEX CB (Varian), separation at 108 ° C (isothermal), carrier gas: Helium (200 ml / min), injection volume: 8 μl. Under these Conditions elute the various glycerides compounds following times: Peak Pair 1: D-Glyceraldehyde = 7.5 min, L-Glyceraldehyde = 7.6 min; Peak Pair 2: D-Glyceraldehyde = 10.2 min, L-Glyceraldehyde = 10.8 min; Peak Pair 3: D-Glyceraldehyde = 11.9 min, L-Glyceraldehyde = 14.10 min. These three product peaks are the variously derivatized glyceraldehydes (in the 1- or 2-position mono-derivatized or di-derivatized at 1- and 2-positions).

The 7 shows the evolution of the ee value for L-glyceraldehyde as a function of the reaction time.

Description of the figures

1 : Dependence of the activity of GO-ADH on the pH value;

1a ) Activity course for the reduction reaction. The activity of the reduction was measured photometrically with glyceraldehyde (racemic) and NADPH.

1b ) Activity profile for the oxidation reaction. The activity of the oxidation was measured photometrically with L-arabitol and NADP.

2 : Dependence of the activity of the recombinant GO-ADH on the temperature;

3 : Temperature stability of recombinant alcohol dehydrogenase;

4 : Storage stability of recombinant alcohol dehydrogenase;

5 : Recovery of enantiomerically-enriched glyceraldehyde by resolution with isolated enzymes;

6 : Plasmid map for the whole cell construct with the two genes coding for the two enzymes GO-ADH (GOX3) and glucose dehydrogenase (GDH). Given are the interfaces for cloning the two genes; Amp = ampicillin resistance;

7 : Development of the ee value for L-glyceraldehyde as a function of the reaction time for a conversion (reduction) of racemic glyceraldehyde with a recombinant whole-cell catalyst.

It follows Sequence listing according to WIPO St. 25. This can of the official publication platform of the DPMA become.

QUOTES INCLUDE IN THE DESCRIPTION

This list The documents listed by the applicant have been automated generated and is solely for better information recorded by the reader. The list is not part of the German Patent or utility model application. The DPMA takes over no liability for any errors or omissions.

Cited patent literature

• US 4353987 [0007]
• EP 1444367 [0031]
• WO 91/08216 [0035]
• EP 127839 [0037, 0038]
• - EP 549721 [0037]
• - DE 19920712 [0049]

Cited non-patent literature

• - W. Hummel, Adv. Biochem. Engineering / Biotechnology 1997, 58, 145-184 [0003]
• Prescott, SC and Dunn, CG (1959) Industrial Microbiology, McGraw Hill Book Co., Inc., New York, "The Acetic Acid Bacteria and Sone of Their Biochemical Activities", p. 428-469 [0005]
• Eigen, M. and Gardiner, W., Evolutionary molecular engineering based on RNA replication, Pure Appl. Chem. 1984, 56, 967-978 [0019]
• - Chen, K. and Arnold, F., Enzymes engineering for nonaqueous solvents: random mutagenesis to enhance the activity of subtilisin E in polar organic media. Bio / Technology 1991, 9, 1073-1077 [0019]
• Horwitz, M. and Loeb, L., Promoters Selected From Random DNA Sequences, Proc Natl Acad Sci USA 83, 1986, 7405-7409 [0019]
• - Dube, D. and L. Loeb, Mutants Generated By The Insertion Of Random Oligonucleotides Into The Active Site Of The Beta-Lactamase Gene, Biochemistry 1989, 28, 5703-5707 [0019]
• - Stemmer, PC, Rapid evolution of a protein in vitro by DNA shuffling, Nature 1994, 370, 389-391 and Stemmer, PC, DNA shuffling by random fragmentation and reassembly: In vitro recombination for molecular evolution. Proc Natl Acad Sci USA 91, 1994, 10747-10751 [0019]
• - Sambrook, J .; Fritsch, EF and Maniatis, T. (1989), Molecular cloning: a laboratory manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York [0022]
• - Sambrook et al. [0028]
• - Sambrook, J .; Fritsch, EF and Maniatis, T. (1989), Molecular cloning: a laboratory manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York [0031]
• - Chan and Cohen. 1979. High Frequency Transformation of Bacillus subtilis Protoplasts by Palsmid DNA. Mol Gen Genet. 168 (1): 111-5 [0033]
• - Kieser et al. 2000. Practical Streptomyces Genetics. The John Innes Foundation Norwich. [0033]
• - Sambrook et al. 1989. Molecular Cloning. A Laboratory Manual. In: second ed. Cold Spring Harbor Laboratory Press. Cold Spring Harbor. NY. [0033]
• - Irani and Rowe. 1997. Enhancement of transformation in Pseudomonas aeruginosa PAO1 by Mg2 + and heat. Biotechniques 22: 54-56 [0033]
• - Hogan, B., Beddington, R., Costantini, F. and Lacy, E. (1994), Manipulating the Mouse Embryo [0035]
• A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. [0035]
• - Studier, WF; Rosenberg AH; Dunn JJ; Dubendroff JW; Use of the T7 RNA polymerase to direct expression of cloned genes, Methods Enzymol. 1990, 185, 61-89 [0036]
• Glover, DM (1985), DNA cloning: a practical approach, Vol. I-III, IRL Press Ltd., Oxford [0036]
• Rodriguez, RL and Denhardt, D.T. (eds) (1988), Vectors: a survey of molecular cloning vectors and their uses, 179-204, Butterworth, Stoneham [0036]
• - Goeddel, DV, Systems for heterologous gene expression, Methods Enzymol. 1990, 185, 3-7 [0036]
• - Sambrook, J .; Fritsch, EF and Maniatis, T. (1989), Molecular cloning: a laboratory manual, 2nd Ed., Cold Spring Harbor Laboratory Press, New York [0036]
• - Mouse Mammary Tumor Virus; Lee et al. (1981) Nature, 294 (5838), 228-232 [0038]
• Gellissen, G .; Piontek, M .; Dahlems, U .; Jenzelewski, V .; Gavagan, JW; DiCosimo, R .; Anton, DL; Janowicz, ZA (1996), Recombinant Hansenula polymorpha as a biocatalyst. Coexpression of the spinach glycolate oxidase (GO) and the S. cerevisiae catalase T (CTT1) gene, Appl. Microbiol. Biotechnol. 46, 46-54; Farwick, M .; London, M .; Dohmen, J .; Dahlems, U .; Gellissen, G .; Strasser, AW [0049]
• - Sambrook, J .; Fritsch, EF and Maniatis, T. (1989), Molecular cloning: a laboratory manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York [0050]
• Balbas, P. and Bolivar, F. (1990), Design and construction of expression plasmid vectors in E.coli, Methods Enzymol. 185, 14-37 [0050]
• Rodriguez, RL and Denhardt, D.T. (eds) (1988), Vectors: a survey of molecular cloning vectors and their uses, 205-225, Butterworth, Stoneham [0050]
• - Triglia T .; Peterson, MG and Kemp, DJ (1988), A procedure for in vitro amplification of DNA segments that lie outside the boundaries of known sequences, Nucleic Acids Res. 16, 8186 [0050]
• - Sambrook, J .; Fritsch, EF and Maniatis, T. (1989), Molecular cloning: a laboratory manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York [0050]
• Rodriguez, RL and Denhardt, D.T. (eds) (1988), Vectors: a survey of molecular cloning vectors and their uses, Butterworth, Stoneham [0050]
• - Sharma BP; Bailey LF and Messing RA (1982), Immobilized Biomaterials - Techniques and Applications, Angew. Chem. 94, 836-852 [0054]
• - Paradkar, VM; Dordick, JS (1994), Aqueous-Like Activity of Chymotrypsin Dissolved in Nearly Anhydrous Organic Solvents, J. Am. Chem. Soc. 116, 5009-5010; Mori, T. [0054]
• Okahata, Y. (1997), A variety of lipidated glycoside hydrolases as effective glycosyl transfer catalysts in homogeneous organic solvents, Tetrahedron Lett. 38, 1971-1974 [0054]
• - Otamiri, M .; Adlercreutz, P .; Matthiasson, B. (1992), Complex formation between chymotrypsin and ethyl cellulose as a means to solubilize the enzyme in active form in toluene, Biocatalysis 6, 291-305 [0054]
• - Kamiya, N .; Okazaki, S.-Y .; Goto, M. (1997), surfactant horseradish peroxidase complex catalytically active in anhydrous benzene, Biotechnol. Tech. 11, 375-378 [0054]
• E. Katchalski-Katzir, DM Kraemer, J. Mol. Catal. B: enzyme. (2000), 10, 157 [0054]
• - Petty, KJ (1996), metal-chelate affinity chromatography In: Ausubel, FM et al. eds. Current Protocols in Molecular Biology, Vol. 2, New York: John Wiley and Sons [0054]
• - St. Clair, N .; Wang, Y.-F .; Margolin, AL (2000), Cofactor-bound cross-linked enzyme crystals (CLEC) of alcohol dehydrogenase, Angew. Chem. Int. Ed. 39, 380-383 [0054]

Claims (13)

Nucleic acid sequence responsible for encodes a polypeptide with alcohol dehydrogenase activity, selected from the group: a) a nucleic acid sequence with the sequence shown in sequence GO-ADH, b) a nucleic acid sequence, under stringent conditions with the nucleic acid sequence according to sequence GO-ADH or the complementary thereto Sequence hybridizes, c) a nucleic acid sequence, which has a homology of at least 50%, preferably at least 66%, more preferably at least 70%, more preferably at least 80% and more preferably more than 90% to the sequence GO-ADH or to the sequence complementary to the sequence GO-ADH. Nucleic acid sequence according to claim 1, these being one by mutagenesis of the sequence GO-ADH this altered sequence is. Nucleic acid sequence according to claim 1, which corresponds to the sequence PA-ADH. Nucleic acid sequence according to claim 3, these being one by mutagenesis of the sequence PA-ADH this altered sequence is. Polypeptide selected from the group: a) the polypeptides encoded by a nucleic acid sequence according to one of claims 1 to 4, b) polypeptide with a homology of at least 75%, preferably at least 77%, still more preferably at least 85%, and more preferably above 90% to the polypeptide of the polypeptide encoded by the sequence GO-ADH. Recombinant expression system or recombinant Vector comprising one or more nucleic acid sequences according to one of claims 1 to 4. Primers or probes for the preparation of a nucleic acid sequences according to one of claims 1 to 4. Process for producing an improved recombinant Polypeptides starting with alcohol dehydrogenase activity from a nucleic acid sequence according to a of claims 1 to 4, characterized in that you a) the nucleic acid sequence in a suitable Vector is cloned and transferred to a suitable expression system and b) the formed polypeptides with improved activity and / or selectivity and / or stability and isolated. Use of a polypeptide according to claim 5 for the preparation of enantiomerically enriched alcohols, ketones, Hydroxyketones, aldehydes or hydroxyaldehydes. Use of a polypeptide according to claim 9, wherein the preparation by means of Racemattrennverfahren and / or Oxidation of alcohols takes place. Whole-cell catalyst comprising a cloned gene coding for a polypeptide according to claim 5th The whole-cell catalyst according to claim 11, comprising a cloned gene coding for a polypeptide capable of converting NAD (P) ⁺ to NAD (P) H. The whole-cell catalyst according to claim 11, comprising a cloned gene coding for a polypeptide capable of converting NAD (P) H to NAD (P) ⁺ .