Engineered rossman fold-containing oxidoreductases

Abstract

The present invention relates to a variant of a parent NAD(P)H dependent oxidoreductase comprising (a) a Rossman fold with a consensus motif GXnGXmG / A, wherein n is 1, 2 or 3 and m is 1 or 2, and (b) a glycine residue being located 8 to 14 amino acid residues downstream of said consensus motif, wherein in the variant the glycine residue is mutated.

Description

[0001] ENGINEERED ROSSMAN FOLD -CONTAINING OXIDOREDUCTASES

[0002] TECHNICAL FIELD

[0003] The present invention relates to the field of enzyme enginering, in particular enzymes capable of binding NAD(H) and / or NADP (H) using the Rossman fold consensus motif GXnGXmG / A.

[0004] BACKGROUND ART

[0005] Biocatalytic redox processes are widely used to replace expensive chemical synthesis, the processes can be performed in shorter time using milder reaction conditions (ambient pressure and temperature) and, furthermore, no extensive amounts of waste are formed .

[0006] Redox reactions like the stereoselective reduction of keto groups or the regioselective oxidation of hydroxy groups are of special interest for pharma and food industries and are mostly performed by oxidoreductases , accompanied by the interconversion of the nicotinamide cofactors NAD(P)+ (oxidized form) and NAD(P)H (reduced form) . These redox reactions require a stoichiometric amount of NAD(P)+ or NAD(P)H for the conversion to be completed, however, the addition of such cofactors in industrial processes is very expensive and not cost-efficient. As cofactors and enzymes are expensive components of biocatalytic processes, enzyme engineering is essential for enhancing cost-efficiency.

[0007] NAD(P) (H) -binding is achieved in many cases by the Rossman fold. The structural fingerprint of the Rossman fold is a stretch of thirty to thirty-five residues from the first two p strands and the connecting phosphate-binding helix (Bellamicini , 1996) . When the pap sequences that make up the functional 3D motif in various NAD(P) (H) -binding proteins were aligned, the initial Rossman consensus sequences G-X1-2-G-X-X-G was identified (Rossman et al., 1975; Dym and Eisenberg, 2001) . This consensus required modifications due to an alternative cofactor binding consensus (G- X1-2-G-X-X-A) in the glutathione reductase from E. coli (Scrutton et al., 1987) , and the more recently reported consensus G-X-X-X-G-I-G (SEQ ID No. 30) by Brakoulias and Jackson (2004) that ended up with the more general Rossman consensus GXnGXmG / A (n < 3, m < 2) . As the Rossman fold is found in a plethora of oxidoreductases , engineering this general cofactor-binding moiety would represent a promising and universally valid strategy to develop oxidoreductases with improved activity.

[0008] Therefore, it is an object of the present invention to provide oxidoreductase variants showing an improved activity compared to the parent oxidoreductases.

[0009] SUMMARY OF THE INVENTION

[0010] The present invention relates to a variant of a parent NAD(P)H dependent oxidoreductase comprising (a) a Rossman fold with a consensus motif GXnGXmG / A, preferably GXnGXmG, wherein n is 1, 2 or 3 and m is 1 or 2, and (b) a glycine residue being located 8 to 14 amino acid residues downstream of said consensus motif, wherein in the variant the glycine residue is mutated.

[0011] It was surprisingly found that mutating a glycine residue located 8 to 14 amino acid residues downstream of the Rossman fold consensus motif of a NAD(P)H dependent oxidoreductase show significantly increased enzymatic activity by more than 10%, preferably 20%, more preferably 30%, even more preferably 40% or more as compared to the parent NAD(P)H dependent oxidoreductase.

[0012] As shown in the alignment of Fig. 1 typical Rossman fold comprising enzymes, revealed the more complex Rossman consensus motif, GXnGXmG / A (n < 3, m < 2) that harbours an additionally conserved glycine (G) residue as a secondary prototypical feature. The additionally conserved G being located 8 to 14 amino acid residues downstream of the Rossman consensus motif will be termed "secondary G" throughout the text or, when mutated, "mutated secondary G" . Thus, the present invention relates to a mutated NAD(P) (H) -dependent oxidoreductase, comprising a Rossman fold with the consensus motif G-Xn-G-Xm-G / A (n < 3, m < 2) and a secondary glycine (G) located 8 to 14 amino acids C-terminal of said consensus motif, wherein the variant is mutated at the secondary glycine (G) .

[0013] A further aspect of the present invention relates to a method for obtaining a variant of a parent NAD(P)H dependent oxidoreductase having an increased enzymatic activity compared to the parent oxidoreductase comprising the steps of providing a parent oxidoreductase comprising (a) a Rossman fold with a consensus motif GXnGXmG / A, wherein n is 1 , 2 or 3 and m is 1 or 2 , and (b ) a glycine residue being located 8 to 14 amino acid residues downstream of said consensus moti f , and mutating said glycine residue .

[0014] Another aspect of the present invention relates to a protein, preferably stable protein, having NADH and / or NADPH oxidase activity comprising an amino acid sequence selected from the group consisting of i ) an amino acid sequence having at least 80% sequence identity with SEQ ID No . 1 , ii ) an amino acid sequence encoded by a nucleic acid sequence having an identity to SEQ ID No . 2 of at least 80% , and iii ) an amino acid sequence encoded by a nucleic acid which binds under stringent conditions to a nucleic acid molecule complementary to the nucleic acid sequence SEQ ID No . 2 , wherein the stringent conditions preferably comprise washing at 65 ° C, and at a salt concentration of 0 . 1 to 2x SSC .

[0015] A protein as defined above exhibiting an NADH and / or NADPH oxidase activity shows a signi ficant higher stability compared to other known NADH and / or NADPH oxidases . The activity of the protein of the present invention remains stable for a much longer period of time compared to other NADH and / or NADPH oxidases . This allows to increase the yield of NAD+ and / or NADP+, which is important i f the protein of the present invention is used in a cofactor recycling system or as a cofactor recycling system, for instance . Also the presence of organic co-solvents has a much lower ef fect on the enzymatic activity of the protein of the present invention compared to other NADH and / or NADPH oxidases . Hence , the protein of the present invention can be considered as a stable or organic solvent stable enzyme , i . e . NADH and / or NADPH oxidase .

[0016] SEQ ID No . 1 :

[0017] MKWWGCTHAGTAAVKTILNEHPDASVSVYERNDNVSFLSCGIALYVGGWKDPAGLFYSSPEE

[0018] LASMGAKINMEHNVKNIDNENKVWIENLKTGETFEESYDKLVMTTGSWPI IPPIDGINSENILL CKNYNQANEI IKESKNAKKIVIVGGGYIGIELVEAFAESGKQVTLVDGLDRILNKYLDAEFTSVL EHDLQERGVTLALNQTVEKFVANESGAVTAVKTPVGEYEADLVILCVGFKPNTDLLKDKVEMLPN GAIWDEYMRTSDEAI FAAGDSCAVHYNPTGGSAYIPLATNAVRMGALVGKNIVSPTVKYRGTQA TSGLYLFGFNIGSTGLTENSAPHFGVEVRSVWEDNYRPEFMPTTEKVTMKLVYEVGTNRIVGGQ IMSKYDVTQSANTLSLCVQNKMTIEDLAYVDFFFQPHFDRPWNYLNILAQAAVEQERKLAK

[0019] SEQ ID No . 2 :

[0020] ATGAAAGTAGTAGTAGTAGGCTGTACACATGCAGGAACAGCGGCAGTTAAGACG

[0021] AT T T T AAAT GAAC AT C C AGAT G C AT GAG TAT GAG TAT AT GAG C G T AAT GAG AAT G T C T C A TTTCTATCTTGTGGGATTGCGTTGTATGTTGGTGGAGTTGTGAAAGATCCTGCAGGTTTG T T T TAT T CAAGT CCAGAAGAAC T T GCAT CAAT GGGCGCGAAAAT TAACAT GGAACACAAT G T GAAAAAT AT AGAT AAT GAGAAT AAG GTCGTAGTAATT GAGAAT T T AAAAAC AG G C GAA AGAT T T GAAGAAAGC TAT GATAAGT T GGTAAT GACAAC T GGAT CAT GGCCAAT TAT T CC T C CAAT T GAT G GAAT CAAT AG T GAAAAT AT TCTTTTGTG T AAAAAC T AT AAC C AAG C AAAT GAAAT TAT TAAAGAAT CAAAAAAT GC TAAAAAGAT T GT CAT T GT T GGT GGT GGC TATAT T GGAATTGAATTAGTTGAGGCATTTGCAGAATCTGGCAAGCAAGTGACGCTAGTTGATGGA T T AGAT C G T AT T T T AAAC AAAT AT T T AGAT G C T GAAT T C AC TTCTGTTT T AGAG CAT GAT TTACAAGAAAGAGGCGTTACGCTAGCTTTAAACCAAACCGTCGAGAAATTTGTTGCCAAT GAATCAGGTGCTGTGACAGCTGTGAAAACACCAGTTGGAGAATATGAGGCTGATTTAGTT ATTTTATGTGTTGGATT T AAAC C AAAT AC TGATTTGTT GAAG GAT AAAG T AGAGAT G T T G CCAAATGGTGCCATCGTAGTGGATGAATATATGAGAACAAGCGATGAAGCGATTTTTGCT GCTGGCGATAGTTGCGCGGTTCATTATAATCCAACTGGAGGCTCTGCGTATATTCCGTTA GCTACAAATGCAGTTAGAATGGGAGCTTTAGTTGGGAAAAATATTGTTTCTCCAACAGTT AAATATCGTGGCACGCAAGCAACTTCTGGTTTATATTTATTTGGTTTTAATATAGGTTCA ACCGGATTGACTGAAAATAGCGCTCCTCATTTTGGCGTAGAGGTTCGTTCAGTAGTTGTA GAAGAT AAT T AT C G T C C AGAG TTTATGCC GACAAC AGAGAAAG T AAC GAT GAAAT T AG T T TAT GAAG TAG GAAC GAAT CGGATTGTTG GAG G T C AAAT C AT G T C AAAAT AT GAT G T GAC A C AAT C T G C C AAT AC G T T AT C T T T AT G T G T T C AAAAT AAAAT GAC GAT T GAG GAT T T G G C T TATGTAGATTTCTTCTTCCAACCTCACTTTGATCGTCCTTGGAACTATTTAAATATTTTA GCGCAAGCAGCTGTTGAGCAAGAGCGTAAACTAGCAAAATAA

[0022] The protein of the present invention comprising or consisting of SEQ ID No . 1 is a new NADH oxidase isolated from gram-positive bacteria of the genus Carnobacteria, e . g . Carnobacterium divergens , and can be used, for instance , in industrial processes for cofactor regeneration . This NADH oxidase as well as the proteins of the present invention are characteri zed by high total turnover numbers ( TTN) and are stable even in processes , where critical substances , which can lead to denaturation, degradation and / or inhibition of the enzyme , are converted . The protein of the present invention is also able to act as NADPH oxidase or even as NAD ( P ) H oxidase as described in more detail below .

[0023] A further aspect of the present invention relates to a nucleic acid molecule encoding a protein according to the present invention, in particular the variant of a parent NAD ( P ) H dependent oxidoreductase as mentioned above .

[0024] The nucleic acid molecule of the present invention can be part of a vector, for instance . Hence , a further aspect of the present invention relates to a vector comprising a nucleic acid molecule according to the present invention .

[0025] A further aspect of the present invention relates to a host cell comprising a nucleic acid molecule or a vector according to the present invention .

[0026] The nucleic acid molecule and / or the vector of the present invention can be part of a host cell . It is particularly preferred that the host cell of the present invention is able to express a protein of the present invention from said nucleic acid molecule and / or said vector . The protein can be secreted from the host cell or expressed intracellularly .

[0027] Another aspect of the present invention relates to the use of a protein according to present invention or a host cell according to the present invention or a lysate or homogenate thereof for oxidi zing NADH to NAD+ and / or NADPH to NADP+ .

[0028] The protein of the present invention can be used to oxidi ze NADH to NAD+ and / or NADPH to NADP+ . A lysate or a homogenate from a host cell expressing the protein of the present invention can be used to oxidi ze NADH to NAD+ and / or NADPH to NADP+ as well .

[0029] A further aspect of the present invention relates to a method for oxidi zing NADH to NAD+ and / or NADPH to NADP+ comprising the step of incubating a protein or a lysate of a host cell according to the present invention with NADH and / or NADPH .

[0030] Another aspect of the present invention relates to a protein or a host cell according to the present invention or a lysate or a homogenate thereof in a cofactor recycling system or as a cofactor recycling system . An aspect of the present invention relates to a method for the enzymatic oxidation of a compound, where NADH is formed from NAD+ and / or NADPH is formed from NADP+, comprising the step of oxidizing NADH to NAD+ and / or NADPH to NADP+ by adding a protein or a lysate of a host cell according to the present invention.

[0031] SHORT DESCRIPTION OF THE FIGURES

[0032] Fig. 1 shows an alignment of typical Rossman fold enzymes; this reveals the existence of a conserved secondary G located C-terminal of the GXnGXmG / A motif.

[0033] Fig. 2 shows an alignment and relative activities of reference as well as G-mutated oxidoreductases .

[0034] Fig. 3 shows a sequence alignment and activity measurements of G170 CdivNox mutants.

[0035] DESCRIPTION OF EMBODIMENTS

[0036] A "variant" of a parent NAD(P)H dependent oxidoreductase comprises at least one variation in the amino acid sequence, i.e. at least one mutated amino acid residue, compared to the parent NAD(P)H dependent oxidoreductase. Further mutations at other positions of the amino acid sequence of the parent NAD(P)H dependent oxidoreductase apart from the claimed mutated glycine residue are possible .

[0037] A "parent" NAD(P)H dependent oxidoreductase can be any oxidoreductase comprising a glycine residue at the defined position in the Rossman fold.

[0038] A "NAD(P)H dependent oxidoreductase" is an oxidoreductase capable of binding NAD(P)H and catalyzing the oxidation of NADH and / or NADPH and / or the reduction of NAD+ and / or NADP+ . Such oxidoreductase comprise typically a Rossman fold which is characterized by succession of p-strands connected by a-helices and that can accommodate NAD(P)H and / or NAD(P)+.

[0039] "Oxidoreductases" are enzymes, which catalyse oxidoreduction reactions, comprising the enzymes acting on the CH-OH group of donors, acting on the aldehyde or oxo group of donors, acting on the CH-CH group of donors, acting on the CH-NH2 group of donors, acting on the CH-NH group of donors, acting on NADH or NADPH as donors, acting on hydrogen as donor, acting on reduced flavodoxin as donor. Enzymes, which catalyse the oxidoreduction reactions of above, comprising the enzymes using NAD or NADP as acceptor, or a cytochrome as acceptor, or oxygen as acceptor, or a disulfide as acceptor, or a quinone or similar compound as acceptor.

[0040] Rossman folds from different oxidoreductases comprise the conserved motif GXnGXmG / A, preferably GXnGXmG, wherein n is 1, 2 or 3, m is 1 or 2 and X can be any amino acid residue. The C-terminal amino acid residue of the motif can be glycine or alanine, whereby glycine is most preferred. In a particularly preferred motif n is 1 and m is 2.

[0041] "G / A" of the consensus motif means "G or A", "glycine or alanine" .

[0042] The glycine residue to be mutated is located 8 to 14 amino acid residues downstream of the conserved Rossman fold motif. "Downstream", as used herein, means that the glycine residue is located 8 to 14 amino acid residues from the C-terminal end of the GXnGXmG / A motif. A general f ormula / consensus sequence may be GXnGXmG / AXoG, wherein o is an integer between 7 to 13, preferably 8 to 13, preferably 9 to 13, more preferably 9 to 12, more preferably 9 to 11, in particular 10. X can be any amino acid residue.

[0043] Amino acid classification, as used herein, is as follows:

[0044] Polar amino acids are Serine (Ser, S) , Threonine (Thr, T) , Cysteine (Cys, C) , Glutamine (Gin, Q) and Asparagine (Asn, N) . Positively charged amino acids are Lysine (Lys, K) and Arginine (Arg, R) as well as Histidine (His, H) . Negatively charged amino acids are Aspartate (Asp, D) and Glutamate (Glu, E) . Non-polar amino acids are Glycine (Gly, G) , Alanine (Ala, A) , Valine (Vai, V) , Leucine (Leu, L) , Isoleucine (lie, I) , Methionine (Met, M) , Proline (Pro, P) , Phenylalanine (Phe, F) , Tyrosine (Tyr, Y) and Tryptophan ( Trp, W) .

[0045] The terms "sequence identity" and "identity", as used herein, refer to the percentage of identical nucleotide or amino acid matches between at least two nucleotide or amino acid sequences aligned using a standardized algorithm. Such an algorithm can, in a standardized and reproducible manner, insert gaps in the compared sequences to optimize the alignment between two sequences, thus achieving a more meaningful comparison of the two sequences.

[0046] Percent identity between sequences may be determined using one or more computer algorithms or programs known in the prior art or described herein. According to the present invention, the Basic Local Alignment Search Tool (BLAST) provided by the National Center for Biotechnology Information (NCBI) (Altschul et al., 1990) , is preferably used. The BLAST software series contains several programs, including a tool referred to as "BLAST 2 Sequences", which is used for direct pairwise comparison of two nucleotide or amino acid sequences. "BLAST 2 Sequences" can also be accessed and used interactively via the NCBI World Wide Web page on the Internet. The blastn program (for nucleotide sequences) uses as defaults a word length (W) of 28, an expectation (E) of 0.05, M = 1, N = -2, and a comparison of both strands. For amino acid sequences, the blastp program uses as defaults a word length of 3 and an expectation (E) of 0.05 and the BLOSUM62 scoring matrix (Henikoff & Henikoff, 1989) , alignments (B) of 50, expectation (E) of 0.05, M = 1, N = -2.

[0047] According to a preferred embodiment of the present invention the glycine residue to be mutated can be substituted or deleted.

[0048] In order to achieve the claimed effect of the variant of the present invention, the glycine residue may be substituted with another amino acid residue or deleted.

[0049] According to a particularly preferred embodiment of the present invention the glycine residue can be substituted with an amino acid residue selected from the group consisting of asparagine, arginine, cysteine, tyrosine, threonine, glutamine, leucine, alanine, aspartic acid and proline.

[0050] The consensus motif of the Rossman fold comprising or consisting of GXnGXmG / A may vary in regard to the variables n and m. In a preferred embodiment of the present invention n is 1 and m is 2, or n and m are 2, or n is 2 and m is 1, or n is 3 and m is 1.

[0051] The oxidoreductase variants of the present invention may be present in whole cells and / or in a suspension, in the homogenate and / or in the lysate of the corresponding cells forming them, lysates being particularly preferred.

[0052] Suspension in this context means a suspension of resting cells. These are harvested (separated from the culture medium) after cultivation and used as a paste or suspended in a suitable buffer system. In contrast to fermentative processes, which also work with whole cells, the resting cells can no longer grow due to the absence of carbon sources and nutrients, but only serve to convert substrates. Homogenate in this context stands for a physically and / or chemically treated suspension (e.g., treated by pressure, lysozyme, or ultrasound) , releasing cellular components from the cells. A lysate is obtained when the insoluble cellular components of the homogenate are removed by, for example, filtration or centrifugation .

[0053] In a further embodiment, the oxidoreductase variants of the present invention may also be immobilized on a solid support material .

[0054] In a further embodiment of the invention, oxidoreductase variants are performed on enzymes, which catalyze oxido-reduction reactions, comprising enzymes acting on the CH-OH group of donors, comprising enzymes acting on the aldehyde or oxo group of donors, comprising enzymes acting on the CH-CH group of donors, comprising enzymes acting on the CH-NH2 group of donors, comprising enzymes acting on the CH-NH group of donors, comprising enzymes acting on NADH or NADPH as donors, comprising enzymes acting on hydrogen as donor or comprising enzymes acting on reduced flavodoxin as donor.

[0055] Enzymes, which catalyze the oxido-reduction reactions stated above, comprise enzymes using NAD or NADP as acceptor, or a cytochrome as acceptor, or oxygen as acceptor, or a disulfide as acceptor, or a quinone or similar compound as acceptor.

[0056] According to a preferred embodiment of the present invention the oxidoreductase is an oxidoreductase selected from the enzyme class EC 1, preferably selected from the group consisting of EC 1.1 acting on the CH-OH group of donors, EC 1.2 acting on the aldehyde or oxo group of donors, EC 1.3 acting on the CH-CH group of donors, EC 1.4 acting on the CH-NH2 group of donors, EC 1.5 acting on the CH-NH group of donors, EC 1.6 acting on NADH or NADPH, EC 1.7 Acting on other nitrogenous compounds as donors, EC 1.12 acting on hydrogen as donor and EC 1.19 acting on reduced flavodoxin as donor.

[0057] The EC number as implemented by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMB) , is a numerical classification scheme for enzymes, based on the chemical reactions they catalyze (Webb, 1992) with frequent updates listed by comprehensive enzyme information systems, e.g. BRENDA. According to a further preferred embodiment of the present invention the NAD(P)H dependent oxidoreductase is selected from the group consisting of NAD(P)H oxidase, xylitol dehydrogenase, glucose 1-dehydrogenase, aldehyde dehydrogenase, hydroxysteroid dehydrogenase, lactate dehydrogenase, glucitol (sorbitol) dehydrogenase and SDR family oxidoreductase.

[0058] According to a preferred embodiment of the present invention the NAD(P)H dependent oxidoreductase is an NAD(P)H oxidase comprising an amino acid sequence selected from the group consisting of i) an amino acid sequence having at least 80% sequence identity with SEQ ID No. 1, ii) an amino acid sequence encoded by a nucleic acid sequence having an identity to SEQ ID No. 2 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid which binds under stringent conditions to a nucleic acid molecule complementary to the nucleic acid sequence SEQ ID No . 2.

[0059] According to another preferred embodiment of the present invention the variant of the NAD(P)H dependent oxidoreductase, when aligned to the sequence of SEQ ID No. 1, comprises a mutation at position 170 of SEQ ID No. 1.

[0060] According to another preferred embodiment of the present invention the NAD(P)H dependent oxidoreductase is a xylitol dehydrogenase comprising an amino acid sequence selected from the group consisting of i) an amino acid sequence having at least 80% sequence identity with SEQ ID No. 16, ii) an amino acid sequence encoded by a nucleic acid sequence having an identity to SEQ ID No. 17 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid which binds under stringent conditions to a nucleic acid molecule complementary to the nucleic acid sequence SEQ ID No . 17. According to another preferred embodiment of the present invention the variant of the xylitol dehydrogenase , when aligned to the sequence of SEQ ID No . 16 , comprises a mutation at position 194 of SEQ ID No . 16 .

[0061] SEQ ID No . 16 :

[0062] MSTPENLSFVLQKPFDVKFEDRPIPKLSDPYSVKIQVKKTGICGSDVHYFTHGAIGDFWKAPMV LGHESSGWLEVGSEVKSLKVGDRVAMEPGVPSRHSDEYKSGRYNLCPHMAFAATPPYDGTLCKY YILPEDFCVKLPEHVSLEEGALVEPLSVAVHSSKLGNIKPGSHVAI YGAGPVGLLVAAVASAFGA E S VT 11 DL VE S RLNLAKE L GAT AT VQVD FKD T PRE S AAKWAANNG I AP D W I DAS GAE AS INSA INAIRPGGTYVQVGMGKPDVSFPIATLIGKELTVKGSFRYGYGDYPLAVSLLASGKVNVKKLITH EVKFEDAAEAFQLVRDGKAIKCI INGPE

[0063] SEQ ID No . 17 :

[0064] ATGTCTACTCCTGAAAACTTATCTTTTGTTTTACAAAAGCCTTTTGACGTCAAGTTCGAGGATAG ACCCATCCCCAAGTTGTCTGATCCTTACTCTGTCAAGATCCAAGTCAAGAAGACTGGTATCTGTG GTAGTGATGTTCATTACTTCACCCATGGAGCTATTGGTGACTTTGTCGTCAAGGCCCCAATGGTC CTTGGTCACGAATCCAGTGGTGTTGTCTTGGAAGTCGGTAGCGAGGTCAAGTCACTCAAGGTTGG TGACAGAGTCGCTATGGAGCCTGGTGTTCCCAGCAGACACTCTGATGAATACAAGTCCGGTAGAT ACAACTTGTGTCCTCACATGGCATTTGCTGCTACTCCTCCTTATGATGGTACTCTTTGTAAATAC TATATTCTTCCTGAAGACTTCTGTGTCAAGTTACCTGAGCACGTTTCTTTGGAAGAGGGTGCTTT AGTTGAACCTTTAAGTGTTGCGGTTCACTCCTCTAAGTTGGGTAACATTAAGCCCGGTAGCCATG TTGCCATTTACGGTGCAGGACCTGTTGGTTTGTTAGTTGCCGCAGTCGCCAGTGCTTTTGGTGCT GAATCTGTTACTATTATTGATCTTGTTGAATCTAGACTTAACCTTGCCAAGGAGTTAGGTGCTAC TGCCACTGTTCAAGTTGATTTCAAGGATACTCCTAAGGAGTCTGCTGCTAAGGTTGTTGCCGCTA ACAACGGAATTGCCCCTGATGTTGTCATTGATGCTTCTGGTGCTGAGGCTTCCATTAATAGTGCT ATCAATGCCATCAGACCTGGTGGCACTTACGTTCAAGTCGGTATGGGTAAGCCTGATGTCTCTTT CCCCATTGCTACTTTAATTGGTAAGGAGCTTACTGTTAAGGGATCTTTCAGATATGGTTACGGTG ACTACCCTCTTGCTGTCAGCTTGCTTGCTAGTGGAAAAGTCAATGTTAAAAAGTTGATTACCCAT GAAGTCAAGTTTGAGGATGCTGCTGAAGCTTTCCAATTGGTTAGAGATGGAAAGGCTATCAAGTG CATCATTAACGGCCCTGAGTAA

[0065] According to a preferred embodiment of the present invention the NAD ( P ) H dependent oxidoreductase is a glucose 1-dehydrogenase comprising an amino acid sequence selected from the group consisting of i ) an amino acid sequence having at least 80 % sequence identity with SEQ ID No . 18 , i i ) an amino acid sequence encoded by a nucleic acid sequence having an identity to SEQ ID No . 19 o f at least 80 % , and i i i ) an amino acid sequence encoded by a nucleic acid which binds under stringent conditions to a nucleic acid mol ecule complementary to the nucleic acid sequence SEQ ID No . 19 .

[0066] SEQ ID No . 18 :

[0067] MYPDLKGKWAI TGAASGLGKAMAIRFGKEQAKWINYYSNKQDPNEVKEEVIKAGGEAVWQGD VTKEEDVKNIVQTAINEFGTLDIMINNAGLENPVPSHEMPLKDWDKVI STNLTGAFLGSREAIKY FVENDIKGNVINMSSVHEVI PWPLFVHYAASKGGIKLMTETLALEYAPKGIRVNNIGPGAINTPI NAEKFADPKQRADVESMI PMGYIGEPEE IAAVAAWLASKEASYVTGI TLFADGGMTQYPS FQAGR G

[0068] SEQ ID No . 19 :

[0069] ATGTATCCAGATTTAAAAGGAAAAGTTGTCGCTATTACAGGAGCTGCTTCAGGATTAGGGAAGGC AATGGCCATTCGCTTCGGCAAGGAGCAGGCAAAAGTGGTTATCAACTACTACAGCAATAAGCAGG ATCCGAACGAGGTAAAGGAAGAGGTCATCAAGGCGGGCGGTGAAGCTGTTGTCGTCCAAGGAGAC G T AAC AAAAGAG GAAGAT G T AAAAAAC AT C G T C C AAAC AG C GAT T AAC GAG T T C G G TAG AC T C GA TATTATGATTAATAATGCCGGTCTTGAAAATCCCGTTCCTTCTCATGAAATGCCGCTGAAGGATT GGGATAAAGTAATCAGCACGAACTTAACGGGCGCCTTTTTAGGAAGCCGTGAAGCGATTAAATAT T T T G T T GAAAAC GAT AT AAAAG GAAAT G T C AT T AAT AT G T C GAG C G TAG AT GAAG TGATTCCGTG GCCATTATTTGTTCACTATGCGGCAAGTAAAGGCGGAATCAAGCTGATGACGGAAACATTGGCGC TGGAATATGCGCCGAAAGGCATTCGTGTCAACAATATCGGGCCAGGCGCGATCAACACGCCAATC AAT GC T GAAAAAT T T GC T GAT CC TAAGCAGAGAGCAGAT GTAGAAAGCAT GAT T CCGAT GGGATA TATCGGTGAACCGGAGGAAATTGCGGCAGTAGCAGCCTGGCTTGCTTCGAAGGAAGCCAGCTACG TCACAGGCATCACGTTATTCGCGGACGGCGGTATGACCCAATATCCTTCCTTCCAGGCAGGACGC GGATAA

[0070] According to a pre ferred embodiment o f the present invention the NAD ( P ) H dependent oxidoreductase i s a aldehyde dehydrogenase compri s ing an amino acid sequence selected from the group cons i sting o f i ) an amino acid sequence having at least 80 % sequence identity with SEQ ID No . 20 , i i ) an amino acid sequence encoded by a nucleic acid sequence having an identity to SEQ ID No . 21 o f at least 80 % , and i i i ) an amino acid sequence encoded by a nucleic acid which binds under stringent conditions to a nucleic acid mol ecule complementary to the nucleic acid sequence SEQ ID No . 21 .

[0071] SEQ ID No . 20 :

[0072] MSVPVQHPMYIDGQFVTWRGDAWIDWNPATEAVI SRI PDGQAEDARKAIDAAERAQPEWEALPA lERASWLRKI SAGIRERASE I SALIVEEGGKIQQLAEVEVAFTADYIDYMAEWARRYEGE I IQSD RPGENILLFKRALGVTTGILPWNFPFFLIARKMAPALLTGNT IVIKPSEFTPNNAIAFAKIVDE I GLPRGVFNLVLGRGETVGQELAGNPKVAMVSMTGSVSAGEKIMATAAKNI TKVCLELGGKAPAIV MDDADLELAVKAIVDSRVINSGQVCNCAERVYVQKGI YDQFVNRLGEAMQAVQFGNPAERNDIAM GPLINAAALERVEQKVARAVEEGARVAFGGKAVEGKGYYYPPTLLLDVRQEMS IMHEETFGPVLP WAFDTLEDAI SMANDSDYGLTSS IYTQNLNVAMKAIKGLKFGETYINRENFEAMQGFHAGWRKS GIGGADGKHGLHEYLQTQWYLQS

[0073] SEQ ID No . 21 :

[0074] ATGTCAGTACCCGTTCAACATCCTATGTATATCGATGGACAGTTTGTTACCTGGCGTGGAGACGC ATGGATTGATGTGGTAAACCCTGCTACAGAGGCTGTCATTTCCCGCATACCCGATGGTCAGGCCG AGGATGCCCGTAAGGCAATCGATGCAGCAGAACGTGCACAACCAGAATGGGAAGCGTTGCCTGCT ATTGAACGCGCCAGTTGGTTGCGCAAAATCTCCGCCGGGATCCGCGAACGCGCCAGTGAAATCAG TGCGCTGATTGTTGAAGAAGGGGGCAAGATCCAGCAGCTGGCTGAAGTCGAAGTGGCTTTTACTG CCGACTATATCGATTACATGGCGGAGTGGGCACGGCGTTACGAGGGCGAGATTATTCAAAGCGAT CGTCCAGGAGAAAATATTCTTTTGTTTAAACGTGCGCTTGGTGTGACTACCGGCATTCTGCCGTG GAACTTCCCGTTCTTCCTCATTGCCCGCAAAATGGCTCCCGCTCTTTTGACCGGTAATACCATCG T C AT T AAAC C T AG T GAAT T T AC G C C AAAC AAT GCGATTGCATTCGC C AAAAT C G T C GAT GAAAT A GGCCTTCCGCGCGGCGTGTTTAACCTTGTACTGGGGCGTGGTGAAACCGTTGGGCAAGAACTGGC GGGTAACCCAAAGGTCGCAATGGTCAGTATGACAGGCAGCGTCTCTGCAGGTGAGAAGATCATGG CGACTGCGGCGAAAAACATCACCAAAGTGTGTCTGGAATTGGGGGGTAAAGCACCAGCTATCGTA ATGGACGATGCCGATCTTGAACTGGCAGTCAAAGCCATCGTTGATTCACGCGTCATTAATAGTGG G CAAG T G T G TAAC T G T G CAGAAC G T G T T TAT G TAG AGAAAG G CAT T TAT GAT GAG T T C G T GAAT C GGCTGGGTGAAGCGATGCAGGCGGTTCAATTTGGTAACCCCGCTGAACGCAACGACATTGCGATG GGGCCGTTGATTAACGCCGCGGCGCTGGAAAGGGTCGAGCAAAAAGTGGCGCGCGCAGTAGAAGA

[0075] AGGGGCGAGAGTGGCGTTCGGTGGCAAAGCGGTAGAGGGGAAAGGATATTATTATCCGCCGACAT

[0076] TGCTGCTGGATGTTCGCCAGGAAATGTCGATTATGCATGAGGAAACCTTTGGCCCGGTGCTGCCA

[0077] GTTGTCGCATTTGACACGCTGGAAGATGCTATCTCAATGGCTAATGACAGTGATTACGGCCTGAC

[0078] CTCATCAATCTATACCCAAAATCTGAACGTCGCGATGAAAGCCATTAAAGGGCTGAAGTTTGGTG

[0079] AAACTTACATCAACCGTGAAAACTTCGAAGCTATGCAAGGCTTCCACGCCGGATGGCGTAAATCC

[0080] GGTATTGGCGGCGCAGATGGTAAACATGGCTTGCATGAATATCTGCAGACCCAGGTGGTTTATTT

[0081] ACAGTCTTAA

[0082] According to a pre ferred embodiment o f the present invention the NAD ( P ) H dependent oxidoreductase i s a hydroxysteroid dehydrogenase compri s ing an amino acid sequence selected from the group cons i sting o f i ) an amino acid sequence having at least 80 % sequence identity with SEQ ID No . 22 , i i ) an amino acid sequence encoded by a nucleic acid sequence having an identity to SEQ ID No . 23 o f at least 80 % , and i i i ) an amino acid sequence encoded by a nucleic acid which binds under stringent conditions to a nucleic acid mol ecule complementary to the nucleic acid sequence SEQ ID No . 23 .

[0083] SEQ ID No . 22 :

[0084] MDMGLKDKWLI TGGGGGIARGIERAFATEGAKFILTDLFPGGLEAAKEELERDFGSEVFT ILAN

[0085] GSVEEEVRAAVEAGAEHFGGRIDVLINNAQASASGLTLVQHSEEDFDLAVRSGLYATFFYMKHAY

[0086] PYLKETAGSVINFASGAGIGGNPGQSSYAAAKEGIRGMSRVAASEWGPDNINVNIVCPIVMTKAL

[0087] EEWREREPEMYEKNVKAI PLGRFGDAEKDVGRVCVFLASPDAS FVTGDT IMVQGGSGMKP

[0088] SEQ ID No . 23 :

[0089] ATGGACATGGGCTTGAAGGACAAGGTGGTGCTGATCACCGGCGGTGGCGGCGGTATCGCTCGGGG

[0090] TATCGAGCGCGCATTCGCCACCGAGGGTGCGAAGTTCATCCTCACCGACCTGTTCCCCGGCGGTC

[0091] TGGAAGCGGCGAAGGAGGAGCTTGAGCGCGATTTCGGCTCCGAAGTGTTCACGATTCTCGCGAAC

[0092] GGATCGGTGGAAGAGGAGGTGCGTGCGGCGGTCGAAGCGGGCGCCGAGCACTTCGGCGGGCGCAT

[0093] CGACGTCCTGATCAACAACGCGCAGGCGTCGGCGTCGGGATTGACGCTGGTGCAGCATTCGGAGG

[0094] AGGACTTCGACCTGGCGGTTCGATCCGGCCTGTACGCGACGTTCTTCTACATGAAGCATGCCTAC CCTTACCTGAAGGAGACGGCGGGATCCGTCATCAACTTCGCTTCCGGCGCGGGCATCGGCGGCAA

[0095] TCCAGGACAGAGCTCCTACGCTGCGGCGAAAGAGGGCATCCGAGGCATGAGCCGCGTGGCCGCAT

[0096] CGGAGTGGGGCCCCGACAACATCAACGTGAACATCGTATGCCCTATCGTCATGACGAAGGCGCTC

[0097] GAGGAATGGCGCGAGCGCGAGCCCGAGATGTACGAGAAGAACGTGAAGGCGATCCCGCTCGGACG

[0098] CTTCGGCGACGCGGAGAAGGACGTAGGACGCGTGTGCGTGTTCCTGGCCAGTCCCGATGCCTCGT

[0099] TCGTAACGGGCGACACCATCATGGTGCAGGGCGGTTCCGGCATGAAGCCGTAA

[0100] According to a pre ferred embodiment o f the present invention the NAD ( P ) H dependent oxidoreductase i s a lactate dehydrogenase compri s ing an amino acid sequence selected from the group cons i sting o f i ) an amino acid sequence having at least 80 % sequence identity with SEQ ID No . 24 , i i ) an amino acid sequence encoded by a nucleic acid sequence having an identity to SEQ ID No . 25 o f at least 80 % , and i i i ) an amino acid sequence encoded by a nucleic acid which binds under stringent conditions to a nucleic acid mol ecule complementary to the nucleic acid sequence SEQ ID No . 25 .

[0101] SEQ ID No . 24 :

[0102] MMNKHVNKVAL I GAGFVGS S YAFAL INQG I TDELWI DVNKEKAMGDVMDLNHGKAFAPQPVKT S YGTYEDCKDADIVCICAGANQKPGETRLELVEKNLKI FKGIVSEVMASGFDGI FLVATNPVDILT YATWKFSGLPKERVIGSGTTLDSARFRFMLSEYFGAAPQNVHAHI IGEHGDTELPVWSHANVGGV PVSELVEKNDAYKQEELDQIVDDVKNAAYHI IEKKGATYYGVAMSLARI TKAILHNENS ILTVST YLDGQYGADDVYIGVPAWNRGGIAGI TELNLNEKEKEQFLHSAGVLKNILKPHFAEQKVN

[0103] SEQ ID No . 25 :

[0104] ATGATGAACAAACATGTAAATAAAGTAGCTTTAATCGGAGCGGGTTTTGTTGGAAGCAGTTATGC ATTTGCGTTAAT T AAC C AAG GAAT C AC AGAT GAG CTTGTGGTCATTGATG T AAAT AAAGAAAAAG CAATGGGCGATGTGATGGATTTAAACCACGGAAAGGCGTTTGCGCCACAACCGGTCAAAACATCT TACGGAACATATGAAGACTGCAAGGATGCTGATATTGTCTGCATTTGCGCCGGAGCAAACCAAAA AC C T G G T GAGAC AC G C C T T GAAT TAG T AGAAAAGAAC T T GAAGAT T T T C AAAG GCATCGTTAGTG AAGTCATGGCGAGCGGATTTGACGGCATTTTCTTAGTCGCGACAAATCCGGTTGATATCCTGACT TACGCAACATGGAAATTCAGCGGCCTGCCAAAAGAGCGGGTGATTGGAAGCGGCACAACACTTGA TTCTGCGAGATTCCGTTTCATGCTGAGCGAATACTTTGGCGCAGCGCCTCAAAACGTACACGCGC ATATTATCGGAGAGCACGGCGACACAGAGCTTCCTGTTTGGAGCCACGCGAATGTCGGCGGTGTG CCGGTCAGTGAACTCGTTGAGAAAAACGATGCGTACAAACAAGAGGAGCTGGACCAAATTGTAGA TGATGTGAAAAACGCAGCTTACCATATCATTGAGAAAAAAGGCGCGACTTATTATGGGGTTGCGA T GAGT C T T GC T CGCAT TACAAAAGCCAT T C T T CATAAT GAAAACAGCATAT TAAC T GT CAGCACA

[0105] TATTTGGACGGGCAATACGGTGCAGATGACGTGTACATCGGTGTGCCGGCTGTCGTGAATCGCGG AGGGATCGCAGGTATCACTGAGCTGAACTTAAATGAGAAAGAAAAAGAACAGTTCCTTCACAGCG CCGGCGTCCT T AAAAAC AT T T T AAAAC CTCATTTTG C AGAAC AAAAAG T C AAC T AA

[0106] According to a pre ferred embodiment o f the present invention the NAD ( P ) H dependent oxidoreductase i s a glucitol ( sorbitol ) dehydrogenase SDR fami ly oxidoreductase compri s ing an amino acid sequence selected from the group cons i sting o f i ) an amino acid sequence having at least 80 % sequence identity with SEQ ID No . 2 6 , i i ) an amino acid sequence encoded by a nucleic acid sequence having an identity to SEQ ID No . 27 o f at least 80 % , and i i i ) an amino acid sequence encoded by a nucleic acid which binds under stringent conditions to a nucleic acid mol ecule complementary to the nucleic acid sequence SEQ ID No . 27 .

[0107] SEQ ID No . 2 6 :

[0108] MGAVTEKTKMYALTFYFLKKGTCQMTHTVPQNMKAAVMHNTRE IKIETLPVPDINHDEVLIKVMA VGICGSDLHYYTNGRIGNYWEKPFILGHECAGE IAAVGSSVDQFKVGDRVAVEPGVTCGRCEAC KEGRYNLCPDVQFLATPPVDGAFVQYIKMRQDFVFLI PDSLS YEEAALIEPFSVGIHAAARTKLQ PGST IAIMGMGPVGLMAVAAAKAFGAGT I IVTDLEPLRLEAAKKMGATHI INIREQDALEE IKT I TNDRGVDVAWE TAGNPAALQS ALAS VRRGGKLAI VGL P S QNE I PLNVP F I ADNE I D I YG I FRYAN TYPKGIEFLASGIVDTKHLVTDQYSLEQTQDAMERALQFKNECLKVMVYPNR

[0109] SEQ ID No . 27 :

[0110] AT GGGGGCAGTAACAGAGAAAACAAAAAT GTATGCAC T TACAT T T TAT T T T C TAAAGAAAGGAAC

[0111] TTGCCAAATGACTCACACAGTACCTCAAAACATGAAAGCGGCTGTTATGCACAACACAAGAGAGA TCAAAATTGAAACATTGCCTGTGCCTGATATCAATCATGATGAAGTGTTGATTAAGGTGATGGCT GTCGGAATTTGCGGATCTGATCTGCATTACTATACAAATGGCCGAATAGGCAACTATGTTGTGGA AAAACCATTTATCCTTGGGCATGAATGTGCGGGTGAAATTGCCGCTGTCGGATCATCTGTCGATC AATTCAAGGTGGGAGACCGCGTCGCTGTAGAGCCGGGTGTTACGTGCGGACGCTGTGAGGCGTGC AAAGAAGGACGCTATAATCTTTGCCCGGATGTACAGTTTTTGGCTACACCGCCGGTAGACGGTGC GTTTGTCCAATATATTAAAATGCGTCAGGACTTTGTTTTTTTAATCCCAGACTCACTTTCTTATG AAGAAGCTGCTTTGATCGAGCCGTTTTCTGTCGGTATCCATGCGGCGGCCAGAACGAAGCTACAG CCCGGATCAACGATTGCGATTATGGGGATGGGCCCTGTTGGGTTAATGGCTGTTGCCGCAGCTAA AGCATTTGGGGCAGGCACAATCATTGTCACCGACTTAGAGCCGCTGCGGTTAGAAGCTGCGAAAA AAAT GGGAGCGAC T CACAT TAT TAATATACGT GAACAGGAT GCAC T T GAAGAGAT TAAAACGAT C ACGAATGATAGAGGCGTTGATGTTGCTTGGGAAACAGCAGGGAATCCAGCGGCATTGCAATCCGC ACTGGCTTCTGTGCGCCGGGGCGGAAAATTGGCGATTGTCGGTTTGCCTTCACAGAACGAGATTC CGCTCAACGTGCCGTTTATTGCGGATAATGAGATTGATATTTACGGGATCTTCCGTTATGCCAAT

[0112] ACGTATCCAAAGGGAATCGAATTTCTTGCTTCAGGCATTGTGGACACGAAGCATCTAGTAACGGA CCAATATTCGCTGGAGCAGACGCAAGATGCGATGGAGCGGGCGCTTCAATTTAAGAATGAATGTT TAAAAGTGATGGTGTATCCAAATCGCTGA

[0113] According to a pre ferred embodiment o f the present invention the NAD ( P ) H dependent oxidoreductase i s a SDR fami ly oxidoreductase compri s ing an amino acid sequence selected from the group cons i sting o f i ) an amino acid sequence having at least 80 % sequence identity with SEQ ID No . 28 , i i ) an amino acid sequence encoded by a nucleic acid sequence having an identity to SEQ ID No . 29 o f at least 80 % , and i i i ) an amino acid sequence encoded by a nucleic acid which binds under stringent conditions to a nucleic acid mol ecule complementary to the nucleic acid sequence SEQ ID No . 29 .

[0114] SEQ ID No . 28 :

[0115] MTDRLKGKVAIVTGGTLGIGLAIADKFVEEGAKWI TGRHADVGEKAAKS IGGTDVIRFVQHDAS

[0116] DEAGWTKLFDTTEEAFGPVTTWNNAGIAVSKSVEDTTTEEWRKLLSVNLDGVFFGTRLGIQRMK

[0117] NKGLGAS I INMSS IEGFVGDPTLGAYNASKGAVRIMSKSAALDCALKDYDVRVNTVHPGYIKTPL

[0118] VDDLE GAEEMMS QRTKT PMGH I GE PND I AW I CVYLAS DE S KFAT GAE FWDGG YTAQ

[0119] SEQ ID No . 29 : ATGACTGATCGTTTAAAAGGCAAAGTAGCAATTGTAACTGGCGGTACCTTGGGAATTGGCTTGGC

[0120] AATCGCTGATAAGTTTGTTGAAGAAGGCGCAAAGGTTGTTATTACCGGCCGTCACGCTGATGTAG

[0121] GTGAAAAAGCTGCCAAATCAATCGGCGGCACAGACGTTATCCGTTTTGTCCAACACGATGCTTCT

[0122] GATGAAGCCGGCTGGACTAAGTTGTTTGATACGACTGAAGAAGCATTTGGCCCAGTTACCACGGT

[0123] TGTCAACAATGCCGGAATTGCGGTCAGCAAGAGTGTTGAAGATACCACAACTGAAGAATGGCGCA

[0124] AGCTGCTCTCAGTTAACTTGGATGGTGTCTTCTTCGGTACCCGTCTTGGAATCCAACGTATGAAG

[0125] AATAAAGGACTCGGAGCATCAATCATCAATATGTCATCTATCGAAGGTTTTGTTGGTGATCCAAC

[0126] TCTGGGTGCATACAACGCTTCAAAAGGTGCTGTCAGAATTATGTCTAAATCAGCTGCCTTGGATT

[0127] GCGCTTTGAAGGACTACGATGTTCGGGTTAACACTGTTCATCCAGGTTATATCAAGACACCATTG

[0128] GTTGACGATCTTGAAGGGGCAGAAGAAATGATGTCACAGCGGACCAAGACACCAATGGGTCATAT

[0129] CGGTGAACCTAACGATATCGCTTGGATCTGTGTTTACCTGGCATCTGACGAATCTAAATTTGCCA

[0130] CTGGTGCAGAATTCGTTGTCGATGGTGGATACACTGCTCAATAA

[0131] As used herein, stringent conditions refer to conditions under which so-called speci fic hybrids , but not non-speci fic hybrids , are formed .

[0132] Hybridi zation may be performed by conventionally known procedures , such as those described in J . Sambrook et al , Molecular Cloning, A Laboratory Manual , 2nd Ed, Cold Spring Harbor Laboratory ( 1989 ) . The stringent condition is a condition in which washing is performed at 65 ° C, and at a salt concentration of 0 . 1 to 2x SSC (wherein IxSSC is understood to be a mixture of 0 , 15 M sodium chloride / 0 , 015 M sodium citrate ) .

[0133] Another aspect of the present invention relates to a protein, preferably stable protein, having NADH and / or NADPH oxidase activity comprising an amino acid sequence selected from the group consisting of i ) an amino acid sequence having at least 80% sequence identity with SEQ ID No . 1 , ii ) an amino acid sequence encoded by a nucleic acid sequence having an identity to SEQ ID No . 2 of at least 80% , and iii ) an amino acid sequence encoded by a nucleic acid which binds under stringent conditions to a nucleic acid molecule complementary to the nucleic acid sequence SEQ ID No . 2 , wherein the stringent conditions preferably comprise washing at 65 ° C, and at a salt concentration of 0 . 1 to 2x SSC . According to a preferred embodiment of the present invention the protein having NADH and / or NADPH oxidase activity of the present invention comprises a polypeptide consisting of the amino acid sequence GX1GX2X3X4, wherein XI is a non-polar amino acid residue, X2 is a non-polar amino acid residue, X3 is a non-polar amino acid residue and X4 is a non-polar amino acid residue.

[0134] According to another preferred embodiment of the present invention XI of GX1GX2X3X4 is glycine, and / or X2 of GXlGX2X3X4is tyrosine, and / or X3 of GXlGX2X3X4is isoleucine, and / or X4 of GXlGX2X3X4is glycine or alanine. Hence, according to a particularly preferred embodiment the protein of the present invention comprises the amino acid sequence GGGYIX4 (SEQ ID No. 3) , even more preferably GGGYIG (SEQ ID No. 4) or GGGYIA (SEQ ID No. 5) . Surprisingly, it turned out that a protein of the present invention comprising the amino acid sequence GGGYIG (SEQ ID No. 4) is able to oxidize NADH and / or NADPH, whereas a protein of the present invention comprising the amino acid sequence GGGYIA (SEQ ID No. 5) shows a significantly reduced or even no capability to oxidize NADH. The latter proteins are thus used preferably to oxidize NADPH.

[0135] According to a preferred embodiment of the present invention the protein of the present invention comprises a polypeptide consisting of the amino acid sequence GX1GX2X3X4X5X6X7X8X9X1 0X11X12 , preferably GGGYIX4X5X6X7X8X9X10X11X12 (SEQ ID No. 6) , GGGYIGX5X6X7X8X9X10X11X12 (SEQ ID No. 7) or GGGYIAX5X6X7X8X9X10X11X12 (SEQ ID No. 8) , wherein XI is a non-polar amino acid residue, X2 is a non-polar amino acid residue, X3 is a non-polar amino acid residue, X4 is a non-polar amino acid residue, X5 is a peptide consisting of 6 to 12, preferably 8 to 10, more preferably 10, amino acid residues, X6 is a non-polar amino acid residue, X7 is a peptide consisting of 4 to 8, preferably 4 to 6, more preferably 6, amino acid residues, X8 is an acidic amino acid residue or a non-polar amino acid residue, X9 is a polar amino acid residue or a basic amino acid residue, X10 is a non-polar amino acid residue or a polar amino acid residue, Xll is a peptide consisting of 2 to 6, preferably 2 to 4, more preferably 4, amino acid residues, and X12 is a polar amino acid residue or a basic amino acid residue, wherein XI is particularly preferred glycine, and / or X2 is particularly preferred tyrosine, and / or X3 is particularly preferred isoleucine, and / or X4 is particularly preferred glycine or alanine.

[0136] According to another preferred embodiment of the present invention X5 is a peptide consisting of amino acid sequence IELVEAFAES (SEQ ID No. 9) and / or X7 is a peptide consisting of amino acid sequence KQVTLV (SEQ ID No. 10) and / or Xll is a peptide consisting of amino acid sequence DRIL (SEQ ID No. 11) .

[0137] The protein of the present invention preferably comprises a polypeptide consisting of amino acid sequence GX1GX2X3X4 IELVEAFAESX6KQVTLVX8X9X10DRILX12 (SEQ ID No. 12) , more preferably GGGYIX4 IELVEAFAESX6KQVTLVX8X9X10DRILX12 (SEQ ID No. 13) , GGGYIGIELVEAFAESX6KQVTLVX8X9X1 0DRILX12 (SEQ ID No. 14) or GGGYIAIELVEAFAESX6KQVTLVX8X9X1 0DRILX12 (SEQ ID No. 15) , as a motif.

[0138] According to a particularly preferred embodiment of the present invention X6 is glycine, asparagine , arginine, cysteine, tyrosine, threonine, glutamine, leucine, alanine, aspartic acid and proline, and / or X8 is aspartic acid or alanine and / or X9 is glycine or arginine and / or X10 is leucine or serine and / or X12 is asparagine or arginine.

[0139] Surprisingly, it turned out that the substitution of some amino acid residues within the aforementioned motif allows to influence the substrate specificity of the protein of the present invention. The resulting protein variants may have a specificity for NAD(H) , NADP (H) or even both.

[0140] In a particularly preferred embodiment of the present invention X12 is asparagine. If the protein of the present invention comprises an asparagine residue at that position, the protein is able to oxidize NADH in a significantly higher degree as NADPH. Such a protein shows just a very reduced oxidation rate for NADPH. In another preferred embodiment of the present invention X12 is arginine. If the protein of the present invention comprises an arginine residue at that position, the protein is able to oxidize NADH as well as NADPH at significant levels. This enables the proteins of the present invention to be used in complex reaction systems, where two or more lead enzymes perform reaction cascades having different cofactor (NADH, NADPH) preferences.

[0141] According to a further preferred embodiment of the present invention X4 is non-polar, and X6 is a non-polar, polar or charged amino acid residue, preferably glycine, asparagine, arginine, cysteine, tyrosine, threonine, glutamine, leucine, alanine, aspartic acid and proline, X8 is a non-polar amino acid residue, preferably alanine, X10 is a polar amino acid residue, preferably serine, and X9 and X12 are a basic amino acid residue, preferably arginine. If the protein of the present invention comprises a motif with these amino acid residues, the protein is able to oxidize NADH to NAD+ and NADPH to NADP+ . Such a protein can be advantageously used in reaction mixtures where either NADH or NADPH or even both need to be oxidized to NAD+ and / or NADP+, respectively. For instance, enzymatic reactions involving the reduction of NAD+ to NADH and / or NADP+ to NADPH may comprise such a protein for cofactor regeneration .

[0142] According to another preferred embodiment of the present invention X4 is a non-polar amino acid residue, preferably alanine, X6 is a non-polar, polar or charged amino acid residue, preferably glycine, asparagine, arginine, cysteine, tyrosine, threonine, glutamine, leucine, alanine, aspartic acid and proline, X8 is a non-polar amino acid residue, preferably alanine, X10 is a polar amino acid residue, preferably serine, and X9 and X12 are a basic amino acid residue, preferably arginine. Proteins of the present invention comprising these amino acid residues within the aforementioned motif are able to oxidize NADPH, thus forming NADP+ .

[0143] According to a preferred embodiment of the present invention X4 and X6 are a non-polar amino acid residue, preferably glycine, X8 is an acidic amino acid residue, preferably aspartic acid, X9 is a polar amino acid residue, preferably glycine, X10 is a non-polar amino acid residue, preferably leucine, and X12 is a polar amino acid residue, preferably asparagine. If the aforementioned motif comprises these amino acid residues the protein of the present invention is able to oxidize NADH to NAD+ .

[0144] According to another preferred embodiment of the present invention the protein of the present invention comprises an amino acid sequence having at least 85%, preferably at least 90%, more preferably at least 95%, more preferably at least 98%, in particular 100%, sequence identity with SEQ ID No. 1, 16, 18, 20, 22, 24, 26 and / or 28. According to a preferred embodiment of the present invention the protein of the present invention comprises an amino acid sequence encoded by a nucleic acid sequence having an identity to SEQ ID No . 2 , 17 , 19 , 21 , 23 , 25 , 27 and / or 29 of at least 85% , preferably of at least 90% , more preferably of at least 95% , more preferably of at least 98 % , in particular of 100% .

[0145] The protein of the present invention can be modi fied once , twice , three , four or five times by a water-soluble polymer . A water- soluble polymer is polyethylene glycol , for example . The binding of the polyethylene glycol preferably takes place at the N-terminal end of the protein according to the present invention . The protein of the present invention can also be bound to a solid body such as polyethylene , polystyrene , polysaccharide , cellulose or cellulose derivatives .

[0146] According to a preferred embodiment of the present invention the protein of the present invention maybe truncated at the N- and / or C-terminus .

[0147] Furthermore , the protein of the present invention may be part of a fus ion protein . Such a fusion protein may comprise next to the protein of the present invention at least a further polypeptide at the N-terminal and / or C-terminal end . Fusion proteins can, for example , be separated more easily from other proteins or are expressed in the cells in larger amounts .

[0148] A further aspect of the present invention relates to a nucleic acid molecule encoding a protein according to the present invention . The nucleic acid molecule may be a DNA or an RNA molecule .

[0149] A further aspect of the present invention relates to a vector comprising a nucleic acid molecule according to the present invention .

[0150] The term "vector" , as used herein, refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked . Useful vectors are those capable of autonomous replication and / or expression of the nucleic acid linked to it . Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as "expression vectors" . Expres sion vectors used in recombinant DNA technology often have the form of a "plasmid" , which usually refers to a circular double-stranded DNA loop that does not bind to a chromosome when in the form of a vector . As used herein, "plasmid" and "vector" are used interchangeably because plasmids are the most commonly used form of vector . However, other forms of expression vectors that perform equivalent functions and are subsequently known in the art are also included .

[0151] Suitable cloning vectors for overexpression and production of the proteins of the present invention include , for example , pKK223- 3 , pTrc99a, pUC, pTZ , pSK, pBluescript , pGEM, pQE , pET , PHUB, pPLc, pKC30 , pRMl / pRM9 , pTrxFus , pAS l , pGEx, pMAL or pTrx .

[0152] The nucleic acid molecule of the present invention encoding the protein of the present invention may be operably linked to a promoter . The term "operably linked" , as used herein, means that a selected nucleotide sequence ( such as encoding a protein described herein) is in close proximity to the promoter to al low the promoter to regulate the expression of the selected nucleic acid molecule . In addition, the promoter is located upstream of the selected nucleotide sequence in the direction of transcription and translation . By "operably linked" is meant that when an appropriate molecule ( such as a transcriptional activator protein) is bound to a regulatory sequence , the nucleotide sequence and the regulatory sequence are linked in such a way that the nucleic acid molecule / gene is expressed .

[0153] Suitable expression promoters include , for example , trp-lac ( tac ) -promotor, trp-lac ( trc ) promotor, lac-promotor , T7-promotor and XpL-promotor .

[0154] Another aspect of the present invention relates to a host cell comprising a nucleic acid molecule or a vector according to the present invention .

[0155] Host cells comprising a nucleic acid molecule or a vector according to the present invention can be used for producing the protein of the present invention . Suitable host cells include bacteria like E . coli and yeast cells like Komagataella phaf fii .

[0156] A further aspect of the present invention relates to the use of a protein or a host cell according to the present invention or a lysate or homogenate thereof for oxidi zing NADH to NAD+ and / or NADPH to NADP+ .

[0157] The protein of the present invention can be used in any method for oxidi zing NADH to NAD+ and / or NADPH to NADP+ . The protein may be an isolated protein (e.g. from a host cells as mentioned above) or an isolated immobilized protein or may be part of whole cell or an immobilized whole cell and a cell homogenate or a cell lysate. The use of cell lysates is particularly preferred because lysates comprise cofactors NADH / NAD+ and NADPH / NADP+ so that in complex reaction mixtures involving oxidoreductases , for instance, it is not necessary to add further cofactors.

[0158] Another aspect of the present invention relates to a method for oxidizing NADH to NAD+ and / or NADPH to NADP+ comprising the step of incubating a protein or a host cell according to the present invention or a lysate or homogenate thereof with NADH and / or NADPH.

[0159] A further aspect of the present invention relates to the use of a protein or a host cell according to the present invention or a lysate or homogenate thereof in a cofactor recycling system or as a cofactor recycling system.

[0160] The protein of the present invention can be used in cofactor recycling systems where NADH is recycled to NAD+ and / or NADPH is recycled to NADP+ . Such cofactor recycling systems are well-known to the person skilled in the art. The protein of the present invention can also be used as a cofactor recycling system in reactions where NADH has to be recycled to NAD+ and / or NADPH has to be recycled to NADP+ .

[0161] The temperature optimum of the NAD(P)H oxidase of the present invention lies between 20°C and 45°C and the optimum pH between 6.0 and 8.5. The NAD(P)H oxidase shows good temperature and pH stabilities and is stable for more than 80 hours when incubated at pH 7.0 and 30 °C. Hence, the NAD(P)H oxidase of the present invention is much more stable compared to other NAD(P)H oxidases known in the art. Furthermore, it surprisingly exhibits high stability in aqueous solutions comprising organic co-solvents, such as acetone and isopropanol. It could be shown that the NAD(P)H oxidase of the present invention is stable and enzymatically active in aqueous solutions comprising up to 25% (v / v) , preferably up to 20% (v / v) , more preferably up to 15% (v / v) , of an organic solvent like an alcohol, such as isopropanol, or acetone. Hence, it is particularly preferred to use the NAD(P)H oxidase of the present invention in aqueous reaction mixtures comprising the aforementioned amounts of organic solvents or in aqueous reaction mixtures comprising 0.5 to 25% (v / v) , preferably 1 to 20% (v / v) , more preferably 1 to 15% (v / v) , of at least one organic cosolvent. Under such conditions the NAD(P)H oxidase of the present invention is enzymatically active for a minimum of 20 h at 30°C.

[0162] The protein of the present invention can be used in the method of the present invention either as whole cells, cell homogenate, cell lysate, or in a completely (at least 95%, preferably at least 98%) or partially purified state. The method of the present invention is carried out with the protein according to the invention or with cells containing the protein according to the invention. In doing so, the cells used can be provided in a native, permeabilized or lysed state.

[0163] EXAMPLE

[0164] Material and. Methods : Production of the enzymes & production of the lysa tes

[0165] General information on the expression of recombinant enzymes in E. coli .

[0166] For recombinant enzyme production in an Escherichia coii strain, the gene to be expressed was first amplified in a PCR using the genomic DNA or its equivalent synthetically adapted to the codon usage of E. coii as a template together with specific oligonucleotides additionally carrying recognition sequences for restriction endonucleases and isolated from the reaction mixture. After nucleic acid digestion with restriction enzymes SphI and Hindlll, the gene fragment encoding the target enzyme was ligated into the backbone of the expression vector pQE70-Kan cut with SphI and Hindlll. The ligation product was transformed into chemically competent E. coii cells ToplOF and the resulting colonies are used for plasmid isolation and restriction analysis.

[0167] The result of the cloning step was verified by restriction enzyme digestion and DNA sequencing. The resulting construct carries the target gene under the IPTG-inducible T5 promoter.

[0168] For overexpression of the enzyme in E. coii, the resulting expression plasmid was transformed into the competent expression cells RB791. After 24 h incubation at 37 °C, resulting colonies were inoculated into LB medium for expression assays. The next day, expression cultures were inoculated and shaken at 37 °C until an OD550 of 0.3 was reached. The temperature was then lowered to 25 °C and the cultures were induced with 0.5 mM IPTG when an OD550 of 0.5 was reached. After 22 h, the cultures were harvested (separated from the medium by centrifugation in the form of a cell pellet) and analyzed for expression of the recombinant enzyme by SDS gel electrophoresis and an activity assay (use in use assay or optical enzymatic assay) .

[0169] Production of cell lysates by Retsch disruption

[0170] To prepare a cell suspension (volume 0.5 - 1.0 mL) , the cell pellet prepared according to the above procedure was weighed into a suitable vessel and dissolved in buffer (100 mM triethanolamine (TEA-HC1) pH 7) . The mass fraction of biomass is usually 10%, with the remainder accounted for by the buffer. A Retsch MM 400 Homogenizer was used for cell disruption.

[0171] The homogenates were centrifuged for 1 min at 4 °C and 16000 g to separate the insoluble cell fragments and obtain the lysate. Kinetic measurements

[0172] Activity measurements with enzyme preparations were performed as follows :

[0173] A defined volume (10 pl) of enzyme dilution was mixed in a cuvette of a total volume of one mL consisting of: a) 0.2 mM NAD+ and 100 mM TEA-HC1 pH8.0 plus 200 mM D-sorbitol or b) 0.2 mM NAD(P)H and 100 mM TEA-HC1 pH7.0.

[0174] Measurement a) was performed with GmXylDH or mutant.

[0175] Measurement b) was performed with NAD oxidases (Nox) .

[0176] The change of absorbance (AA, delta A) measured at 340 nm is a measure of enzyme activity according to Lambert-Beer's law (AA = s-Ac-d; where s is 6.22 1 / (mmol -cm) for NAD(P)H; Ac (delta c) is the change in concentration and d is the optical path length of the cuvette (1 cm) . One enzyme unit U corresponds to the amount of enzyme that catalyzes the conversion of one pmol substrate per minute under defined conditions. The activity given below are expressed as percent of reference (unmodified enzyme) [calculated as activity (mutant) divided by activity (reference) ] . PCR-mediated Mutagenesis Mutations were generated using standard overlap extension PCR (for example described by Hilgarth and Lanigan (2019) ) .

[0177] Example 1

[0178] Activity measurements of mutated, secondary G oxdioreductases

[0179] A sequence alignment of wild-type oxidoreductases used for mutation experiments is shown in Fig. 1. The database accession numbers (GenBank, Uniprot) of the enzymes analyzed are shown in the following table. Sequence alignment of reference (bold) and mutated oxidoreductases as well as their relative activities (expressed as percent of reference) are shown in Fig. 2. All variants mutated at the secondary G showed increased activity. Note that the WjogaNox reference already harbours a mutated secondary G (D170) . Reverse mutation of the WjogaNox back to G (W ogaNox_D170G) showed reduced activity as expected.

[0180] Example 2

[0181] Activity screening of mutated secondary G oxidoreductases

[0182] For screening an oxidoreductase library with variants mutated at the secondary G, CdivNOX served as reference. CdivNox G170 was mutated to hydrophobic (A,C,L,P,Y) , hydrophilic (N,Q,T) or charged (D,R) amino acids. Sequence alignment of reference (bold) and mutated Nox as well as their relative activities (expressed as percent of reference) are shown in Fig. 3. All secondary G variants showed increased activity up to 211%.

[0183] LITERATURE

[0184] Altschul, S. F., Gish, W., Miller, W., Myers, E. W., & Lipman, D. J. (1990) . Basic local alignment search tool. Journal of Molecular Biology, 215(3) , 403-410. https: / / doi.org / 10.1016 / S0022- 2836 (05) 80360-2

[0185] Bellamacina CR. The nicotinamide dinucleotide binding motif: a comparison of nucleotide binding proteins. FASEB J. 1996 Sep; 10 (11) : 1257-69.

[0186] Brakoulias, A. & Jackson, R. M. Towards a structural classification of phosphate binding sites in protein-nucleotide complexes: An automated all-against-all structural comparison using geometric matching. Proteins. 56, 250-260 (2004) .

[0187] Dym 0, Eisenberg D. Sequence-structure analysis of FAD- containing proteins. Protein Sci. 2001 Sep; 10 ( 9) : 1712-28.

[0188] Henikoff, S., & Henikoff, J. G. (1992) . Amino acid substitution matrices from protein blocks. Proceedings of the National Academy of Sciences of the United States of America, 89(22) , 10915-10919. https: / / doi.org / 10.1073 / pnas .89.22.10915

[0189] Hilgarth, R.S., & Lanigan T. M. (2019) . Optimization of overlap extension PGR for efficient transgene construction. MethodsX, 7, 100759. https : / / doi . org / 10.1016 / j .mex.2019.12.001

[0190] Rossman, M. G., Liljas, A., Branden, C. I., & Banaszak, L. J. (1975) . 2 Evolutionary and structural relationships among dehydrogenases. In The enzymes (Vol. 11, pp . 61-102) . Academic Press .

[0191] Sambrook, J., Fritsch, E. R., & Maniatis, T. (1989) . Molecular Cloning: A Laboratory Manual (2nd ed.) . Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.

[0192] Scrutton NS, Berry A, Perham RN. Purification and characterization of glutathione reductase encoded by a cloned and over-expressed gene in Escherichia coli. Biochem J. 1987 Aug 1;245 (3) : 875-80.

[0193] Webb, E. C. (1992) . Enzyme nomenclature 1992. Recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology on the Nomenclature and Classification of Enzymes.

Claims

CLAIMS1. A variant of a parent NAD(P)H dependent oxidoreductase comprising (a) a Rossman fold with a consensus motif GXnGXmG / A, wherein n is 1, 2 or 3 and m is 1 or 2, and (b) a glycine residue being located 8 to 14 amino acid residues downstream of said consensus motif, wherein in the variant the glycine residue is mutated.

2. The variant of claim 1, wherein the glycine residue is located 9 to 14, preferably 10 to 14, more preferably 10 to 13, more preferably 10 to 12, in particular 11, amino acid residues downstream of said consensus motif.

3. The variant of claim 1 or 2, wherein the glycine residue is substituted or deleted.

4. The variant of any one of claims 1 to 3, wherein the glycine residue is substituted with an amino acid residue selected from the group consisting of asparagine, arginine, cysteine, tyrosine, threonine, glutamine, leucine, alanine, aspartic acid and proline.

5. The variant of any one of claims 1 to 4, wherein n is 1 and m is 2, or n and m are 2, or n is 2 and m is 1, or n is 3 and m is 1.

6. The variant of any one of claims 1 to 5, wherein the oxidoreductase is an oxidoreductase selected from enzyme class EC 1, preferably selected from the group consisting of EC 1.1, EC 1.2, EC 1.3, EC 1.4, EC 1.5, EC 1.6, EC 1.7, EC 1.12 and EC 1.19.

7. The variant of any one of claims 1 to 6, wherein the NAD(P)H dependent oxidoreductase is selected from the group consisting of NAD(P)H oxidase, xylitol dehydrogenase, glucose 1-dehydrogenase, aldehyde dehydrogenase, hydroxysteroid dehydrogenase, lactate dehydrogenase, Glucitol (sorbitol) dehydrogenase and SDR family oxidoreductase .

8. The variant of any one of claims 1 to 7, wherein the NAD(P)H dependent oxidoreductase is an NAD(P)H oxidase comprising an amino acid sequence selected from the group consisting of i) an amino acid sequence having at least 80% sequence identity with SEQ ID No. 1, ii) an amino acid sequence encoded by a nucleic acid sequence having an identity to SEQ ID No. 2 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid which binds under stringent conditions to a nucleic acid molecule complementary to the nucleic acid sequence SEQ ID No . 2.

9. The variant of any one of claims 1 to 8, wherein the variant of the NAD(P)H dependent oxidoreductase, when aligned to the sequence of SEQ ID No. 1, comprises a mutation at position 170 of SEQ ID No. 1.

10. The variant of any one of claims 1 to 9, wherein the NAD(P)H dependent oxidoreductase is an xylitol dehydrogenase comprising an amino acid sequence selected from the group consisting of i) an amino acid sequence having at least 80% sequence identity with SEQ ID No. 16, ii) an amino acid sequence encoded by a nucleic acid sequence having an identity to SEQ ID No. 17 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid which binds under stringent conditions to a nucleic acid molecule complementary to the nucleic acid sequence SEQ ID No . 17.

11. The variant of any one of claims 1 to 9, wherein the NAD(P)H dependent oxidoreductase is a glucose 1-dehydrogenase comprising an amino acid sequence selected from the group consisting of i) an amino acid sequence having at least 80% sequence identity with SEQ ID No. 18,ii) an amino acid sequence encoded by a nucleic acid sequence having an identity to SEQ ID No. 19 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid which binds under stringent conditions to a nucleic acid molecule complementary to the nucleic acid sequence SEQ ID No. 19.

12. The variant of any one of claims 1 to 9, wherein the NAD(P)H dependent oxidoreductase is a aldehyde dehydrogenase comprising an amino acid sequence selected from the group consisting of i) an amino acid sequence having at least 80% sequence identity with SEQ ID No. 20, ii) an amino acid sequence encoded by a nucleic acid sequence having an identity to SEQ ID No. 21 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid which binds under stringent conditions to a nucleic acid molecule complementary to the nucleic acid sequence SEQ ID No . 21.

13. The variant of any one of claims 1 to 9, wherein the NAD(P)H dependent oxidoreductase is a hydroxysteroid dehydrogenase comprising an amino acid sequence selected from the group consisting of i) an amino acid sequence having at least 80% sequence identity with SEQ ID No. 22, ii) an amino acid sequence encoded by a nucleic acid sequence having an identity to SEQ ID No. 23 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid which binds under stringent conditions to a nucleic acid molecule complementary to the nucleic acid sequence SEQ ID No. 23.

14. The variant of any one of claims 1 to 9, wherein the NAD(P)H dependent oxidoreductase is a lactate dehydrogenase comprising an amino acid sequence selected from the group consisting ofi) an amino acid sequence having at least 80% sequence identity with SEQ ID No. 24, ii) an amino acid sequence encoded by a nucleic acid sequence having an identity to SEQ ID No. 25 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid which binds under stringent conditions to a nucleic acid molecule complementary to the nucleic acid sequence SEQ ID No. 25.

15. The variant of any one of claims 1 to 9, wherein the NAD(P)H dependent oxidoreductase is a Glucitol (sorbitol) dehydrogenase comprising an amino acid sequence selected from the group consisting of i) an amino acid sequence having at least 80% sequence identity with SEQ ID No. 26, ii) an amino acid sequence encoded by a nucleic acid sequence having an identity to SEQ ID No. 27 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid which binds under stringent conditions to a nucleic acid molecule complementary to the nucleic acid sequence SEQ ID No . 27.

16. The variant of any one of claims 1 to 9, wherein the NAD(P)H dependent oxidoreductase is a SDR family oxidoreductase comprising an amino acid sequence selected from the group consisting of i) an amino acid sequence having at least 80% sequence identity with SEQ ID No. 28, ii) an amino acid sequence encoded by a nucleic acid sequence having an identity to SEQ ID No. 29 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid which binds under stringent conditions to a nucleic acid molecule complementary to the nucleic acid sequence SEQ ID No. 29.

17. A nucleic acid molecule encoding a protein according to any one of claims 1 to 16.

18. Vector comprising a nucleic acid molecule according to claim 17.

19. Host cell comprising a nucleic acid molecule according to claim 17 or a vector according to claim 18.

20. Method for obtaining a variant of a parent NAD(P)H dependent oxidoreductase having an increased enzymatic activity compared to the parent oxidoreductase comprising the steps of providing a parent oxidoreductase comprising (a) a Rossman fold with a consensus motif GXnGXmG / A, wherein n is 1, 2 or 3 and m is 1 or 2, and (b) a glycine residue being located 8 to 14 amino acid residues downstream of said consensus motif, and mutating said glycine residue.

21. The method of claim 20, wherein the glycine residue is located 9 to 14, preferably 10 to 14, more preferably 10 to 13, more preferably 10 to 12, in particular 11, amino acid residues downstream of said consensus motif.

22. The method of claim 20 or 21, wherein the glycine residue is substituted or deleted.

23. The method of any one of claims 20 to 22, wherein the glycine residue is substituted with an amino acid residue selected from the group consisting of asparagine, arginine, cysteine, tyrosine, threonine, glutamine, leucine, alanine, aspartic acid and proline.

24. The method of any one of claims 20 to 23, wherein n is 1 and m is 2, or n and m are 2, or n is 2 and m is 1, or n is 3 and m is 1.

25. The method of any one of claims 20 to 24, wherein the oxidoreductase is an oxidoreductase selected from the enzyme classEC 1, preferably selected from the group consisting of EC 1.1, EC 1.2, EC 1.3, EC 1.4, EC 1.5, EC 1.6, EC 1.7, EC 1.12 and EC 1.19.

26. The method of any one of claims 20 to 25, wherein the NAD(P)H dependent oxidoreductase is selected from the group consisting of NAD(P)H oxidase, xylitol dehydrogenase, glucose 1-dehydrogenase, aldehyde dehydrogenase, hydroxysteroid dehydrogenase, lactate dehydrogenase, Glucitol (sorbitol) dehydrogenase and SDR family oxidoreductase .

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