Engineered redox enzymes containing rosmmann folds

Abstract

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

Description

Technical Field

[0001] This invention relates to the field of enzyme engineering, and in particular to enzymes capable of using the Rothman folded common motif GX. n GX m G / A enzymes that bind NAD(H) and / or NADP(H). Background Technology

[0002] Biocatalytic redox processes are widely used to replace expensive chemical synthesis. These processes can be carried out in a shorter time using milder reaction conditions (ambient pressure and temperature) and do not generate large amounts of waste.

[0003] Redox reactions, such as the stereoselective reduction of ketone groups or the regioselective oxidation of hydroxyl groups, are of particular interest to the pharmaceutical and food industries and are primarily carried out by redox enzymes, accompanied by the nicotinamide cofactor NAD(P). + The interconversion of NAD(P) (oxidized form) and NAD(P)H (reduced form). These redox reactions require stoichiometric amounts of NAD(P). + The conversion can be accomplished using NAD(P)H; however, adding such cofactors in industrial processes is very expensive and not cost-effective. Since cofactors and enzymes are costly components in biocatalysis, enzyme engineering is crucial for improving cost-effectiveness.

[0004] NAD(P)(H)-binding is often achieved through Rothmann folding. The structural fingerprint of Rothmann folding is a fragment of 30 to 35 residues from the first two β chains and the phosphate-binding helix (Bellamicini, 1996). When the βαβ sequences constituting the functional 3D motifs in various NAD(P)(H)-binding proteins are compared, the initial Rothmann concordant sequence GX is found. 1−2 -GXXG was identified (Rosman et al., 1975; Dym and Eisenberg, 2001). This is due to the presence of a substitute cofactor-binding concordance sequence (GX) in glutathione reductase from *E. coli*. 1−2 The shared sequence GXXXGIG (SEQ ID No. 30) was recently reported by Brakoulias and Jackson (2004), and therefore needed to be modified, ultimately resulting in the more general Rothman shared sequence GX. n GX m G / A (n ≤ 3 and m ≤ 2).

[0005] Since the Rosmann fold is present in a large number of oxidoreductases, engineering this universal cofactor-binding moiety would represent a promising and universally effective strategy for developing oxidoreductases with improved activity.

[0006] Therefore, one object of the present invention is to provide an oxidoreductase variant that exhibits enhanced activity compared to the parental oxidoreductase. Summary of the Invention

[0007] This invention relates to a variant of a parental NAD(P)H-dependent oxidoreductase comprising (a) a gene having a shared motif GX n GX m G / A, GX preferred n GX m The Rothman fold of G, wherein n is 1, 2 or 3 and m is 1 or 2, and (b) a glycine residue located 8 to 14 amino acid residues downstream of the common motif, wherein the glycine residue is mutated in this variant.

[0008] Surprisingly, it was found that, compared with the parental NAD(P)H-dependent oxidoreductase, mutation of glycine residues located 8 to 14 amino acid residues downstream of the Rothman fold common motif of the NAD(P)H-dependent oxidoreductase showed a significant increase in enzyme activity of more than 10%, preferably 20%, more preferably 30%, and even more preferably 40% or more.

[0009] like Figure 1 The comparison of typical enzymes containing Rothmann folds reveals a more complex Rothmann shared motif, GX. n GX m G / A (n ≤ 3 and m ≤ 2), the motif containing an additional conserved glycine (G) residue as a secondary prototype feature. The additional conserved G located 8 to 14 amino acid residues downstream of the Rothmann common motif will be referred to throughout this document as “secondary G”, or, when mutated, as “mutated secondary G”. Therefore, this invention relates to a mutated NAD(P)(H)-dependent oxidoreductase comprising a common motif GX. n -GX m The variant consists of a Rothman fold of -G / A (n ≤ 3 and m ≤ 2) and a secondary glycine (G) located at the C-terminus of the common motif, which is mutated at the secondary glycine (G).

[0010] Another aspect of the present invention relates to a method for obtaining a parental NAD(P)H-dependent oxidoreductase variant with increased enzymatic activity compared to the parental oxidoreductase, comprising the following steps:

[0011] - Provides a parental oxidoreductase containing (a) a shared motif GX n GX m The Rothman fold of G / A, where n is 1, 2, or 3 and m is 1 or 2, and (b) the glycine residue located 8 to 14 amino acid residues downstream of the common motif, and

[0012] - Mutate the glycine residue.

[0013] Another aspect of the invention relates to proteins having NADH and / or NADPH oxidase activity, preferably stable proteins, comprising an amino acid sequence selected from the group consisting of:

[0014] i) An amino acid sequence that has at least 80% sequence identity with SEQ ID No. 1,

[0015] ii) An amino acid sequence encoded by a nucleic acid sequence having at least 80% identity with SEQ ID No. 2, and

[0016] iii) An amino acid sequence encoded by a nucleic acid that binds to a nucleic acid molecule complementary to the nucleic acid sequence SEQ ID No. 2 under stringent conditions, wherein the stringent conditions preferably include washing at 65°C and at a salt concentration of 0.1 to 2 × SSC.

[0017] Compared to other known NADH and / or NADPH oxidases, proteins as defined above exhibiting NADH and / or NADPH oxidase activity demonstrate significantly higher stability. The proteins of this invention maintain stable activity over a longer period compared to other NADH and / or NADPH oxidases. This allows for increased NAD... + and / or NADP + The yield is important, for example, if the protein of the present invention is used in a cofactor recycling system or as a cofactor recycling system. Furthermore, the presence of an organic cosolvent has a much lower effect on the enzymatic activity of the protein of the present invention compared to other NADH and / or NADPH oxidases. Therefore, the protein of the present invention can be considered a stable or organic solvent-stable enzyme, i.e., an NADH and / or NADPH oxidase.

[0018] SEQ ID No. 1:

[0019] MKVVVGCTHAGTAAVKTILNEHPDASVSVYERNDNVSFLSCGIALYVGGVVKDPAGLFYSSPEELASMGAKINMEHNVKNIDNENKVVVIENLKTGETFEESYDKLVMTTGSWPIIPPIDGINSENILLCKNYNQANEIIKESKNAKKIVIVGGGYIGIELVEAFAESGKQVTLVDGLDRILNKYLDAEFTSVLEHDLQERGVTLALNQTVEKFVANESGAVTAVKTPVGEYEADLVILCVGFKPNTDLLKDKVEMLPNGAIVVDEYMRTSDEAIFAAGDSCAVHYNPTGGSAYIPLATNAVRMGALVGKNIVSPTVKYRGTQATSGLYLFGFNIGSTGLTENSAPHFGVEVRSVVEDNYRPEFMPTEKVTMKLVYEVGTNRIVGGQIMSKYDVTQSANTLSLCVQNKMTIEDLAYVDFFFQPHFDRPWNYLNILAQAAVEQERKLAK

[0020] SEQ ID No. 2:

[0021]

[0022] The protein of the present invention, comprising or consisting of SEQ ID No. 1, is a novel NADH oxidase isolated from Gram-positive bacteria of the genus *Carnobacteria* (e.g., *Carnobacterium divergens*), and can be used in industrial processes such as cofactor regeneration. This NADH oxidase, and the protein of the present invention, is characterized by a high total turnover number (TTN) and stability even during conversion with key substances that can cause enzyme denaturation, degradation, and / or inhibition. The protein of the present invention can also act as an NADPH oxidase, or even as an NAD(P)H oxidase, as described in more detail below.

[0023] Another aspect of the invention relates to nucleic acid molecules encoding proteins according to the invention, particularly variants of the parental NAD(P)H-dependent oxidoreductase as described above.

[0024] For example, the nucleic acid molecule of the present invention can be part of a carrier. Therefore, another aspect of the present invention relates to a carrier comprising a nucleic acid molecule according to the present invention.

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

[0026] The nucleic acid molecule and / or vector of the present invention can be part of a host cell. Particularly preferred is that the host cell of the present invention is capable of expressing the protein of the present invention from the nucleic acid molecule and / or the vector. The protein can be secreted from the host cell or expressed intracellularly.

[0027] Another aspect of the invention relates to the use of proteins according to the invention or host cells according to the invention, or their lysates or homogenates, for the oxidation of NADH to NAD. + and / or oxidize NADPH to NADP + Its uses.

[0028] The protein of this invention can be used to oxidize NADH to NAD. + and / or oxidize NADPH to NADP + Lysates or homogenates from host cells expressing the proteins of this invention can also be used to oxidize NADH to NAD. + and / or oxidize NADPH to NADP + .

[0029] Another aspect of the present invention relates to oxidizing NADH to NAD. + and / or oxidize NADPH to NADP +The method includes the step of incubating a protein or host cell lysate according to the invention with NADH and / or NADPH.

[0030] Another aspect of the invention relates to proteins or host cells or their lysates or homogenates in a cofactor recycling system or as a cofactor recycling system, according to the invention.

[0031] One aspect of the present invention relates to a method for the enzymatic oxidation of a compound, wherein NADH is produced by NAD+ oxidation. + The formation and / or NADPH is caused by NADP + The method involves oxidizing NADH to NAD by adding a protein or host cell lysate according to the invention. + and / or oxidize NADPH to NADP + The steps. Attached Figure Description

[0032] Figure 1 This shows a comparison with a typical Rothman folding enzyme; this reveals the location of GX n GX m The presence of a conserved secondary G at the C-terminus of the G / A motif.

[0033] Figure 2 The comparison and relative activities of the reference and G-mutant oxidoreductases are shown.

[0034] Figure 3 The sequence alignment and activity measurement of the G170 CdivNox mutant are shown. Detailed Implementation

[0035] Compared to the parental NAD(P)H-dependent oxidoreductase, the “variants” of the parental NAD(P)H-dependent oxidoreductase contain at least one variation in the amino acid sequence, i.e., at least one mutated amino acid residue. Further mutations at other positions in the amino acid sequence of the parental NAD(P)H-dependent oxidoreductase are possible, in addition to the claimed mutated glycine residue.

[0036] The “parental” NAD(P)H-dependent oxidoreductase can be any oxidoreductase that contains a glycine residue at a specific position in the Rosmann fold.

[0037] "NAD(P)H-dependent oxidoreductase" is a type of enzyme that can bind NAD(P)H and catalyze the oxidation of NADH and / or NADPH and / or NAD. + and / or NADP +These are oxidoreductases that reduce NAD(P)H. These oxidoreductases typically contain a Rothmann fold, characterized by a continuous arrangement of β-chains linked by α-helices, and capable of accommodating NAD(P)H and / or NAD(P). + .

[0038] "Oxidoreductases" are enzymes that catalyze redox reactions. These include enzymes that act on the CH-OH group of a donor, enzymes that act on the aldehyde or bridging group of a donor, enzymes that act on the CH-CH group of a donor, enzymes that act on the CH-NH2 group of a donor, enzymes that act on the CH-NH group of a donor, enzymes that act on NADH or NADPH as donors, enzymes that act on hydrogen as donors, and enzymes that act on reduced flavin-redoxin as a donor. Enzymes catalyzing these redox reactions include enzymes that use NAD or NADP as acceptors, or enzymes that use cytochromes as acceptors, or enzymes that use oxygen as acceptors, or enzymes that use disulfides as acceptors, or enzymes that use quinones or similar compounds as acceptors.

[0039] Rossmann folds from different oxidoreductases contain the conserved motif GX. n GX m G / A, GX preferred n GX m G, where n is 1, 2, or 3 and 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, with glycine being the most preferred. In particularly preferred motifs, n is 1 and m is 2.

[0040] The shared motif “G / A” means “G or A” or “glycine or alanine”.

[0041] The glycine residue to be mutated is located 8 to 14 amino acid residues downstream of the conserved Rossmann fold motif. In this paper, "downstream" refers to the glycine residue located 8 to 14 amino acid residues downstream of the GX motif. n GX m The G / A motif is located at the C-terminus of 8 to 14 amino acid residues. The general formula / common sequence can be GX. n GX m G / AX o G, where o is an integer between 7 and 13, preferably 8 to 13, more preferably 9 to 13, even more preferably 9 to 12, even more preferably 9 to 11, and especially 10. X can be any amino acid residue.

[0042] As used in this article, amino acids are classified as follows:

[0043] Polar amino acids are serine (Ser, S), threonine (Thr, T), cysteine ​​(Cys, C), glutamine (Gln, Q), and asparagine (Asn, N). Positively charged amino acids are lysine (Lys, K), arginine (Arg, R), and histidine (His, H). Negatively charged amino acids are aspartic acid (Asp, D) and glutamic acid (Glu, E). Nonpolar amino acids are glycine (Gly, G), alanine (Ala, A), valine (Val, V), leucine (Leu, L), isoleucine (Ile, I), methionine (Met, M), proline (Pro, P), phenylalanine (Phe, F), tyrosine (Tyr, Y), and tryptophan (Trp, W).

[0044] As used in this paper, the terms "sequence identity" and "identity" refer to the percentage of identical nucleotide or amino acid matches between at least two nucleotide or amino acid sequences aligned using a normalization algorithm. This type of algorithm can insert gaps in the compared sequences in a normalized and reproducible manner to optimize the alignment between the two sequences, thereby enabling a more meaningful comparison.

[0045] The percentage of identity between sequences can be determined using one or more computer algorithms or programs known in the art or described herein. According to the invention, the Basic Local Alignment Search Tool (BLAST) provided by the National Center for Biotechnology Information (NCBI) (Altschul et al., 1990) is preferred. The BLAST software family contains several programs, including a tool called “BLAST 2 Sequence” for direct pairwise comparisons of two nucleotide or amino acid sequences. “BLAST 2 Sequence” is also interactively accessible and usable via the NCBI World Wide Web page on the Internet. The blastn program (for nucleotide sequences) defaults to a word length (W) of 28, an expectation value (E) of 0.05, M = 1, N = -2, and double-stranded comparisons. For amino acid sequences, the blastp program defaults to a word length of 3, an expectation value (E) of 0.05, and a BLOSUM62 scoring matrix (Henikoff & Henikoff, 1989), an alignment (B) of 50, an expectation value (E) of 0.05, M = 1, N = -2.

[0046] According to a preferred embodiment of the present invention, the glycine residue to be mutated may be replaced or deleted.

[0047] To achieve the desired effect of the variants of the invention, the glycine residue may be replaced or omitted by another amino acid residue.

[0048] According to a particularly preferred embodiment of the invention, the glycine residue may be replaced by an amino acid residue selected from the group consisting of asparagine, arginine, cysteine, tyrosine, threonine, glutamine, leucine, alanine, aspartic acid, and proline.

[0049] Includes GX n GX m G / A or GX n GX m The variables n and m of the common motif in the Rothman fold composed of G / A can be different. In a preferred embodiment of the 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.

[0050] The oxidoreductase variants of the present invention can be present in whole cells and / or suspensions, in homogenates and / or lysates of the corresponding cells from which they are formed, with lysates being particularly preferred.

[0051] The suspension in this context refers to a suspension of resting cells. These cells are harvested after culture (isolated from the culture medium) and used as a paste or suspended in a suitable buffer system. Unlike fermentation processes that also use whole cells, resting cells cease growth due to the lack of carbon and nutrients and are used solely for substrate transformation. The homogenate in this context represents a suspension that has undergone physical and / or chemical treatment (e.g., by pressure, lysozyme, or sonication) to release cellular components from the cells. Lysates are obtained when insoluble cellular components of the homogenate are removed, for example, by filtration or centrifugation.

[0052] In another embodiment, the oxidoreductase variant of the present invention can also be immobilized on a solid support material.

[0053] In another embodiment of the invention, the oxidoreductase variant is carried out on an enzyme that catalyzes a redox reaction, the enzyme including enzymes that act on the CH-OH group of the donor, enzymes that act on the aldehyde or bridging group of the donor, enzymes that act on the CH-CH group of the donor, enzymes that act on the CH-NH2 group of the donor, enzymes that act on the CH-NH group of the donor, enzymes that act on NADH or NADPH as a donor, enzymes that act on hydrogen as a donor, or enzymes that act on reduced flavin reductase as a donor.

[0054] Enzymes that catalyze the above-mentioned redox reactions include enzymes that use NAD or NADP as acceptors, or enzymes that use cytochromes as acceptors, or enzymes that use oxygen as acceptors, or enzymes that use disulfides as acceptors, or enzymes that use quinones or similar compounds as acceptors.

[0055] According to a preferred embodiment of the present invention, the oxidoreductase is selected from enzyme EC 1, preferably selected from the group consisting of EC 1.1 which acts on the CH-OH group of the donor, EC 1.2 which acts on the aldehyde or bridging group of the donor, EC 1.3 which acts on the CH-CH group of the donor, EC 1.4 which acts on the CH-NH2 group of the donor, EC 1.5 which acts on the CH-NH group of the donor, EC 1.6 which acts on NADH or NADPH, EC 1.7 which acts on other nitrogen-containing compounds as donors, EC 1.12 which acts on hydrogen as a donor, and EC 1.19 which acts on reduced flavin redox proteins as donors.

[0056] The EC numbering system, implemented by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMB), is a scheme for numerically classifying enzymes based on the chemical reactions they catalyze (Webb, 1992), and it is frequently updated by comprehensive enzyme information systems (such as BRENDA).

[0057] According to another 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:

[0059] i) An amino acid sequence that has at least 80% sequence identity with SEQ ID No. 1,

[0060] ii) An amino acid sequence encoded by a nucleic acid sequence having at least 80% identity with SEQ ID No. 2, and

[0061] iii) An amino acid sequence encoded by a nucleic acid that binds to a nucleic acid molecule complementary to the nucleic acid sequence SEQ ID No. 2 under strict conditions.

[0062] According to another preferred embodiment of the invention, the variant of the NAD(P)H-dependent oxidoreductase contains a mutation at position 170 of SEQ ID No. 1 when compared with the sequence of SEQ ID No. 1.

[0063] According to another preferred embodiment of the invention, the NAD(P)H-dependent oxidoreductase is a xylitol dehydrogenase comprising an amino acid sequence selected from the group consisting of:

[0064] i) An amino acid sequence that has at least 80% sequence identity with SEQ ID No. 16.

[0065] ii) An amino acid sequence encoded by a nucleic acid sequence having at least 80% identity with SEQ ID No. 17, and

[0066] iii) An amino acid sequence encoded by a nucleic acid that binds to a nucleic acid molecule complementary to the nucleic acid sequence SEQ ID No. 17 under strict conditions.

[0067] According to another preferred embodiment of the invention, when compared with the sequence of SEQ ID No. 16, the variant of xylitol dehydrogenase contains a mutation at position 194 of SEQ ID No. 16.

[0068] SEQ ID No. 16:

[0069] MSTPENLSFVLQKPFDVKFEDRPIPKLSDPYSVKIQVKKTGICGSDVHYFTHGAIGDFVVKAPMVLGHESSGVVLEVGSEVKSLKVGDRVAMEPGVPSRHSDEYKSGRYNLCPHMAFAATPPYDGTLCKYYILPEDFCVKLPEHVSLEEGALVEPLSVAVHSSKLGNIKPGSHVAI YGAGPVGLLVAAVASAFGAESVTIIDLVESRLNLAKELGATATVQVDFKDTPKESAAKVVAANNGIAPDVVIDASGAEASINSAINAIRPGGTYVQVGMGKPDVSFPIATLIGKELTVKGSFRYGYGDYPLAVSLLASGKVNVKKLITHEVKFEDAAEAFQLVRDGKAIKCIINGPE

[0070] SEQ ID No. 17:

[0071]

[0072] 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:

[0073] i) An amino acid sequence that has at least 80% sequence identity with SEQ ID No. 18.

[0074] ii) An amino acid sequence encoded by a nucleic acid sequence having at least 80% identity with SEQ ID No. 19, and

[0075] iii) An amino acid sequence encoded by a nucleic acid that binds to a nucleic acid molecule complementary to the nucleic acid sequence SEQ ID No. 19 under strict conditions.

[0076] SEQ ID No. 18:

[0077] MYPDLKGKVVAITGAASGLGKAMAIRFGKEQAKVVINYYSNKQDPNEVKEEVIKAGGEAVVVQGDVTKEEDVKNIVQTAINEFGTLDIMINNAGLENPVPSHEMPLKDWDKVISTNLTGAFLGSREAIKY FVENDIKGNVINMSSVHEVIPWPLFVHYAASKGGIKLMTETLALEYAPKGIRVNNIGPGAINTPINAEKFADPKQRADVESMIPMGYIGEPEEIAAVAAWLASKEASYVTGITLFADGGMTQYPSFQAGRG

[0078] SEQ ID No. 19:

[0079] ATGTATCCAGATTTAAAAGGAAAAGTTGTCGCTATTACAGGAGCTGCTTCAGGATTAGGGAAGGCAATGGCCATTCGCTTCGGCAAGGAGCAGGCAAAAGTGGTTATCAACTACAGCAATAAGCAGGATCCGAACGAGGTAAAGGAAGAGGTCATCAAGGCGGGCGGTGAAGCTGTTGTCGTCCAAGGAGACG TAACAAAAGAGGAAGATGTAAAAAACATCGTCCAAACAGCGATTAACGAGTTCGGTACACTCGATATTATGATTAATAATGCCGGTCTTGAAAATCCCGTCCTTCTCCATGAAATGCCGCTGAAGGATTGGGATAAAGTAATCAGCACGAACTTAACGGGCGCCTTTTTAGGAAGCCGTGAAGCGATTAAATATTTT GTTGAAAACGATATAAAAGGAAATGTCATTAATATGTCGAGCGTACATGAAGTGATTCCGTGGCCATTATTTGTTCACTATGCGGCAAGTAAAGGCGGAATCAAGCTGATGACGGAAACATTGGCGCTGGAATATGCGCCGAAAGGCATTCGTGTCAACAAATATCGGGCCAGGCGCGATCAACACGCCAATCAATG CTGAAAAATTTGCTGATCCTAAGCAGAGAGCAGATGTAGAAAGCATGATTCCGATGGGATATATCGGTGAACCGGAGGAAATTGCGGCAGTAGCAGCCTGGCTTGCTTCGAAGGAAGCCAGCTACGTCACAGGCATCACGTTATTCGCGGACGGCGGTATGACCCAATATCCTTCCTTCCAGGCAGGACGCGGATAA

[0080] According to a preferred embodiment of the present invention, the NAD(P)H-dependent oxidoreductase is an aldehyde dehydrogenase comprising an amino acid sequence selected from the group consisting of:

[0081] i) An amino acid sequence that has at least 80% sequence identity with SEQ ID No. 20.

[0082] ii) An amino acid sequence encoded by a nucleic acid sequence having at least 80% identity with SEQ ID No. 21, and

[0083] iii) An amino acid sequence encoded by a nucleic acid that binds to a nucleic acid molecule complementary to the nucleic acid sequence SEQ ID No. 21 under strict conditions.

[0084] SEQ ID No. 20:

[0085] MSVPVQHPMYIDGQFVTWRGDAWIDVVNPATEAVISRIPDGQAEDARKAIDAAERAQPEWEALPAIERASWLRKISAGIRERASEISALIVEEGGKIQQLAEVEVAFTADYIDYMAEWA RRYEGEIIQSDRPGENILLFKRALGVTTGILPWNFPFFLIARKMAPALLTGNTIVIKPSEFTPNNAIAFAKIVDEIGLPRGVFNLVLGRGETVGQELAGNPKVAMVSMTGVSAGEKIMA TAAKNITKVCLELGGKAPAIVMDDADLELAVKAIVDSRVINSGQVCNCAERVYVQKGIYDQFVNRLGEAMQAVQFGNPAERNDIAMGPLINAAALERVEQKVARAVEEGARVAFGGKAVE GKGYYYPPPTLLLDVRQEMSIMHEETFGPVLPVVAFDTLEDAISMANDSDYGLTSSIYTQNLNVAMKAIKGLKFGETYINRENFEAMQGFHAGWRKSGIGGADGKHGLHEYLQTQVVYLQS

[0086] SEQ ID No. 21:

[0087]

[0088] According to a preferred embodiment of the present invention, the NAD(P)H-dependent oxidoreductase is a hydroxysteroid dehydrogenase comprising an amino acid sequence selected from the group consisting of:

[0089] i) An amino acid sequence that has at least 80% sequence identity with SEQ ID No. 22.

[0090] ii) An amino acid sequence encoded by a nucleic acid sequence having at least 80% identity with SEQ ID No. 23, and

[0091] iii) An amino acid sequence encoded by a nucleic acid that binds to a nucleic acid molecule complementary to the nucleic acid sequence SEQ ID No. 23 under strict conditions.

[0092] SEQ ID No. 22:

[0093] MDMGLKDKVVLITGGGGGIARGIERAFATEGAKFILTDLFPGGLEAAKEELERDFGSEVFTILANGSVEEEVRAAVEAGAEHFGGRIDVLINNAQASASGLTLVQHSEEDFDLAVRSGLYATFFYMK HAYPYLKETAGSVINFASGAGIGGNPGQSSYAAAKEGIRGMSRVAASEWGPDNINVNIVCPIVMTKALEEWREREPEMYEKNVKAIPLGRFGDAEKDVGRVCVFLASPDASFVTGDTIMVQGGSGMKP

[0094] SEQ ID No. 23:

[0095] ATGGACATGGGCTTGAAGGACAAGGTGGTGCTGATCACCGGCGGTGGCGGCGGTATCGCTCGGGGTATCGAGCGCGCATTCGCCACCGAGGGTGCGAAGTTCATCCTCACCGACCTGTTCCCCGGCGGTCTGGAAGCGGCGAAGGAGGAGCTTGAGCGCGATTTCGGCTCCGAAGTGTTCACGATTCTCGCG AACGGATCGGTGGAAGAGGAGGTGCGTGCGGCGGTCGAAGCGGGCGCCGAGCACTTCGGCGGGCGCATCGACGTCCTGATCAACAACGCGCAGGCGTCGGCGTCGGGATTGACGCTGGTGCAGCATTCGGAGGAGGACTTCGACCTGGCGGTTCGATCCGGCCTGTACGCGACGTTCTTCTACATGAAGCAT GCCTACCCTTACCTGAAGGAGACGGCGGGATCCGTCATCAACTTCGCTTCCGGCGCGGGCATCGGCGGCAATCCAGGACAGAGCTCCTACGCTGCGGCGAAAGAGGGCATCCGAGGCATGAGCCGCGTGGCCGCATCGGAGTGGGGCCCCGACAACATCAACGTGAACATCGTATGCCCTATCGTCATGACG AAGGCGCTCGAGGAATGGCGCGAGCGCGAGCCCGAGATGTACGAGAAGAACGTGAAGGCGATCCCGCTCGGACGCTTCGGCGACGCGGAGAAGGACGTAGGACGCGTGTGCGTGTTCCTGGCCAGTCCCGATGCCTCGTTCGTAACGGGCGACACCATCATGGTGCAGGGCGGTTCCGGCATGAAGCCGTAA

[0096] According to a preferred embodiment of the present invention, the NAD(P)H-dependent oxidoreductase is a lactate dehydrogenase comprising an amino acid sequence selected from the group consisting of:

[0097] i) An amino acid sequence that has at least 80% sequence identity with SEQ ID No. 24.

[0098] ii) An amino acid sequence encoded by a nucleic acid sequence having at least 80% identity with SEQ ID No. 25, and

[0099] iii) An amino acid sequence encoded by a nucleic acid that binds to a nucleic acid molecule complementary to the nucleic acid sequence SEQ ID No. 25 under strict conditions.

[0100] SEQ ID No. 24:

[0101] MMNKHVNKVALIGAGFVGSSYAFALINQGITDELVVIDVNKEKAMGDVMDLNHGKAFAPQPVKTSYGTYEDCKDADIVCICAGANQKPGETRLELVEKNLKIFKGIVSEVMASGFDGIFLVATNPVDILTYATWKFSGLPKERVIGSGTTLDSAFRFML SEYFGAAPQNVHAHIIGEHGDTELPVWSHANVGGVPVSELVEKNDAYKQEELDQIVDDVKNAAYHIIEKKGATYYGVAMSLARITKAILHNENSILTVSTYLDGQYGADDVYIGVPAVVNRGGIAGITELNLNEKEKEQFLHSAGVLKNILKPHFAEQKVN

[0102] SEQ ID No. 25:

[0103] ATGATGAACAAACATGTAAATAAAGTAGCTTTAATCGGAGCGGGTTTTGTTGGAAGCAGTTATGCATTTGCGTTAATTAACCAAGGAATCACAGATGAGCTTGTGGTCATTGATGTAAATAAAGAAAAAGCAATGGGCGATGTGATGGATTTAAACCACGGAAAGGCGTTTGCGCCACAACCGGTCAAAACATCTTACGGAACATATGAAGACTGCAAGGATGCTGATATTGTCTGCATTTGCGCCGGAGCAAACCAAAAACCTGGTGAGACACGCCTTGAATTAGTAGAAAAGAACTTGAAGATTTTCAAAGGCATCGTTAGTGAAGTCATGGCGAGCGGATTTGACGGCATTTTCTTAGTCGCGACAAATCCGGTTGATATCCTGACTTACGCAACATGGAAATTCAGCGGCCTGCCAAAAGAGCGGGTGATTGGAAGCGGCACAACACTTGATTCTGCGAGATTCCGTTTCATGCTGAGCGAATACTTTGGCGCAGCGCCTCAAAACGTACACGCGCATATTATCGGAGAGCACGGCGACACAGAGCTTCCTGTTTGGAGCCACGCGAATGTCGGCGGTGTGCCGGTCAGTGAACTCGTTGAGAAAAACGATGCGTACAAACAAGAGGAGCTGGACCAAATTGTAGATGATGTGAAAAACGCAGCTTACCATATCATTGAGAAAAAAGGCGCGACTTATTATGGGGTTGCGATGAGTCTTGCTCGCATTACAAAAGCCATTCTTCATAATGAAAACAGCATATTAACTGTCAGCACATATTTGGACGGGCAATACGGTGCAGATGACGTGTACATCGGTGTGCCGGCTGTCGTGAATCGCGGAGGGATCGCAGGTATCACTGAGCTGAACTTAAATGAGAAAGAAAAAGAACAGTTCCTTCACAGCGCCGGCGTCCTTAAAAACATTTTAAAACCTCATTTTGCAGAACAAAAAGTCAACTAA

[0104] According to a preferred embodiment of the present invention, the NAD(P)H-dependent oxidoreductase is a glucitol (sorbitol) dehydrogenase SDR family oxidoreductase comprising an amino acid sequence selected from the group consisting of:

[0105] i) An amino acid sequence that has at least 80% sequence identity with SEQ ID No. 26.

[0106] ii) An amino acid sequence encoded by a nucleic acid sequence having at least 80% identity with SEQ ID No. 27, and

[0107] iii) An amino acid sequence encoded by a nucleic acid that binds to a nucleic acid molecule complementary to the nucleic acid sequence SEQ ID No. 27 under strict conditions.

[0108] SEQ ID No. 26:

[0109] MGAVTEKTKMYALTFYFLKKGTCQMTHTVPQNMKAAVMHNTREIKIETLPVPDINHDEVLIKVMAVGICGSDLHYYTNGRIGNYVVEKPFILGHECAGEIAAVGSSVDQFKVGDRVAVEPGVTCGRCEACKEGRYNLCPDVQFLATPPVDGAFVQYIKMRQDFVFLIPDSLSYEEAALIEPFSVGIHA AARTKLQPGSTIAIMGMGPVGLMAVAAAKAFGAGTIIVTDLEPLRLEAAKKMGATHIINIREQDALEEIKTITNDRGVDVAWETAGNPAALQSALASVRRGGKLAIVGLPSQNEIPLNVPFIADNEIDIYGIFRYANTYPKGIEFLASGIVDTKHLVTDQYSLEQTQDAMERALQFKNECLKVMVYPNR

[0110] SEQ ID No. 27:

[0111]

[0112] According to a preferred embodiment of the present invention, the NAD(P)H-dependent oxidoreductase is an SDR family oxidoreductase comprising an amino acid sequence selected from the group consisting of:

[0113] i) An amino acid sequence that has at least 80% sequence identity with SEQ ID No. 28.

[0114] ii) An amino acid sequence encoded by a nucleic acid sequence having at least 80% identity with SEQ ID No. 29, and

[0115] iii) An amino acid sequence encoded by a nucleic acid that binds to a nucleic acid molecule complementary to the nucleic acid sequence SEQ ID No. 29 under strict conditions.

[0116] SEQ ID No. 28:

[0117] MTDRLKGKVAIVTGGTLGLAIADKFVEEGAKVVITGRHADVGEKAAKSIGGTDVIRFVQHDASDEAGWTKLFDTTEEAFGPVTTVVNNAGIAVSKSVEDTTTEEWRKLLSVNLDGVFFGTRLGI QRMKNKGLGASIINMSSIEGFVGDPTLGAYNASKGAVRIMSKSAALDCALKDYDVRVNTVHPGYIKTPLVDDLEGAEEMMSQRTKTPMGHIGEPNDIAWICVYLASDESKFATGAEFVVDGGYTAQ

[0118] SEQ ID No. 29:

[0119] ATGACTGATCGTTTAAAAGGCAAAGTAGCAATTGTAACTGGCGGTACCTTGGGAATTGGCTTGGCAATCGCTGATAAGTTTGTTGAAGAAGGCGCAAAGGTTGTTATTACCGGCCGTCACGCTGATGTAGGTGAAAAAGCTGCCAAATCAATCGGCGGCACAGACGTTATCCGTTTTGTCCAACACGAT GCTTCTGATGAAGCCGGCTGGACTAAGTTGTTTGATACGACTGAAGAAGCATTTGGCCCAGTTACCACGGTTGTCAACAATGCCGGAATTGCGGTCAGCAAGAGTGTTGAAGATACCACAACTGAAGAATGGCGCAAGCTGCTCTCAGTTAACTTGGATGGTGTCTTCTTCGGTACCCGTCTTGGAATCC AACGTATGAAGAATAAAGGACTCGGAGCATCAATCATCAATATGTCATCTATCGAAGGTTTTGTTGGTGATCCAACTCTGGGTGCATACAACGCTTCAAAAGGTGCTGTCAGAATTATGTCTAAATCAGCTGCCTTGGATTGCGCTTTGAAGGACTACGATGTTCGGGTTAACACTGTTCATCCAGGTTA TATCAAGACACCATTGGTTGACGATCTTGAAGGGGCAGAAGAAATGATGTCACAGCGGACCAAGACACCAATGGGTCATATCGGTGAACCTAACGATATCGCTTGGATCTGTGTTTACCTGGCATCTGACGAATCTAAATTTGCCACTGGTGCAGAATTCGTTGTCGATGGTGGATACACTGTCAATAA

[0120] As used in this article, strict conditions refer to the conditions for the formation of so-called specific hybrids rather than non-specific hybrids.

[0121] Hybridization can be performed according to conventional and 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 conditions are washing at 65°C and with a salt concentration of 0.1 to 2 × SSC (where 1 × SSC is understood as a mixture of 0.15 M sodium chloride and 0.015 M sodium citrate).

[0122] Another aspect of the invention relates to a protein having NADH and / or NADPH oxidase activity, preferably a stable protein, comprising an amino acid sequence selected from the group consisting of:

[0123] i) An amino acid sequence that has at least 80% sequence identity with SEQ ID No. 1,

[0124] ii) An amino acid sequence encoded by a nucleic acid sequence having at least 80% identity with SEQ ID No. 2, and

[0125] iii) An amino acid sequence encoded by a nucleic acid that binds to a nucleic acid molecule complementary to the nucleic acid sequence SEQ ID No. 2 under stringent conditions, wherein the stringent conditions preferably include washing at 65°C and at a salt concentration of 0.1 to 2 × SSC.

[0126] According to a preferred embodiment of the present invention, the protein of the present invention having NADH and / or NADPH oxidase activity comprises a polypeptide consisting of the amino acid sequence GX1GX2X3X4, wherein X1 is a nonpolar amino acid residue, X2 is a nonpolar amino acid residue, X3 is a nonpolar amino acid residue, and X4 is a nonpolar amino acid residue.

[0127] According to another preferred embodiment of the invention, X1 in GX1GX2X3X4 is glycine, and / or X2 in GX1GX2X3X4 is tyrosine, and / or X3 in GX1GX2X3X4 is isoleucine, and / or X4 in GX1GX2X3X4 is glycine or alanine. Therefore, according to a particularly preferred embodiment, the protein of the 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, results show that the protein of the invention comprising the amino acid sequence GGGYIG (SEQ ID No. 4) is capable of oxidizing NADH and / or NADPH, while the protein of the invention comprising the amino acid sequence GGGYIA (SEQ ID No. 5) shows a significantly reduced or even absent ability to oxidize NADH. Therefore, the latter protein is preferred for oxidizing NADPH.

[0128] According to a preferred embodiment of the present invention, the protein of the present invention comprises a polypeptide consisting of the amino acid sequence GX1GX2X3X4X5X6X7X8X9X10X11X12, preferably GGGYIX4X5X6X7X8X9X10X11X12 (SEQ ID No. 6), GGGYIGX5X6X7X8X9X10X11X12 (SEQ ID No. 7) or GGGYIAX5X6X7X8X9X10X11X12 (SEQ ID No. 7). 8), wherein X1 is a nonpolar amino acid residue, X2 is a nonpolar amino acid residue, X3 is a nonpolar amino acid residue, X4 is a nonpolar amino acid residue, X5 is a peptide composed of 6 to 12, preferably 8 to 10, more preferably 10 amino acid residues, X6 is a nonpolar amino acid residue, X7 is a peptide composed of 4 to 8, preferably 4 to 6, more preferably 6 amino acid residues, X8 is an acidic amino acid residue or a nonpolar amino acid residue, X9 is a polar amino acid residue or a basic amino acid residue, X10 is a nonpolar amino acid residue or a polar amino acid residue, X11 is a peptide composed 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 X1 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.

[0129] According to another preferred embodiment of the present invention, X5 is a peptide composed of the amino acid sequence IELVEFAES (SEQ ID No. 9), and / or X7 is a peptide composed of the amino acid sequence KQVTLV (SEQ ID No. 10), and / or X11 is a peptide composed of the amino acid sequence DRIL (SEQ ID No. 11).

[0130] The protein of the present invention preferably comprises a polypeptide as a motif consisting of the amino acid sequence GX1GX2X3X4IELVEAFAESX6KQVTLVX8X9X10DRILX12 (SEQ ID No. 12), more preferably GGGYIX4IELVEAFAESX6KQVTLVX8X9X10DRILX12 (SEQ ID No. 13), GGGYIGIELVEAFAESX6KQVTLVX8X9X10DRILX12 (SEQ ID No. 14) or GGGYIAIELVEAFAESX6KQVTLVX8X9X10DRILX12 (SEQ ID No. 15).

[0131] 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.

[0132] Surprisingly, the results show that substitution of certain amino acid residues in the above motif can affect the substrate specificity of the proteins of the present invention. The resulting protein variants can be specific for NAD(H), NADP(H), or even both.

[0133] In a particularly preferred embodiment of the invention, X12 is asparagine. If the protein of the present invention contains an asparagine residue at this position, the protein is capable of oxidizing NADH to a significantly higher degree than NADPH. Such a protein has a very low oxidation rate of NADPH. In another preferred embodiment of the invention, X12 is arginine. If the protein of the present invention contains an arginine residue at this position, the protein is capable of oxidizing both NADH and NADPH at a significant level. This allows the protein of the present invention to be used in complex reaction systems in which two or more lead enzymes perform reaction cascades with different cofactor (NADH, NADPH) preferences.

[0134] According to a further preferred embodiment of the present invention, X4 is nonpolar, X6 is a nonpolar, polar, or charged amino acid residue, preferably glycine, asparagine, arginine, cysteine, tyrosine, threonine, glutamine, leucine, alanine, aspartic acid, and proline, X8 is a nonpolar amino acid residue, preferably alanine, X10 is a polar amino acid residue, preferably serine, and X9 and X12 are basic amino acid residues, preferably arginine. If the protein of the present invention contains a motif having these amino acid residues, the protein is capable of oxidizing NADH to NAD. + And it oxidizes NADPH to NADP. + Such proteins can be advantageously used in situations where NADH or NADPH, or even both, need to be oxidized to NAD. + and / or NADP + In the reaction mixture. For example, involving NAD. + Restore to NADH and / or NADP + The enzymatic reaction that reduces NADPH may involve proteins that are used for cofactor regeneration.

[0135] According to another preferred embodiment of the invention, X4 is a nonpolar amino acid residue, preferably alanine; X6 is a nonpolar, polar, or charged amino acid residue, preferably glycine, asparagine, arginine, cysteine, tyrosine, threonine, glutamine, leucine, alanine, aspartic acid, and proline; X8 is a nonpolar amino acid residue, preferably alanine; X10 is a polar amino acid residue, preferably serine; and X9 and X12 are basic amino acid residues, preferably arginine. Proteins of the invention containing these amino acid residues in the above motif are capable of oxidizing NADPH to form NADP. + .

[0136] According to a preferred embodiment of the invention, X4 and X6 are nonpolar amino acid residues, preferably glycine; X8 is an acidic amino acid residue, preferably aspartic acid; X9 is a polar amino acid residue, preferably glycine; X10 is a nonpolar amino acid residue, preferably leucine; and X12 is a polar amino acid residue, preferably asparagine. If the aforementioned motif contains these amino acid residues, the protein of the present invention is capable of oxidizing NADH to NAD. + .

[0137] 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%, and particularly 100% sequence identity with SEQ ID No. 1, SEQ ID No. 16, SEQ ID No. 18, SEQ ID No. 20, SEQ ID No. 22, SEQ ID No. 24, SEQ ID No. 26 and / or SEQ ID No. 28.

[0138] 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 at least 85%, preferably at least 90%, more preferably at least 95%, more preferably at least 98%, and particularly 100% identity with the nucleic acid sequences of SEQ ID No. 2, SEQ ID No. 17, SEQ ID No. 19, SEQ ID No. 21, SEQ ID No. 23, SEQ ID No. 25, SEQ ID No. 27 and / or SEQ ID No. 29.

[0139] The proteins of this invention can be modified once, twice, three times, four times, or five times with a water-soluble polymer. For example, the water-soluble polymer is polyethylene glycol. According to the invention, the binding of polyethylene glycol preferably occurs at the N-terminus of the protein. The proteins of this invention can also be bound to solids, such as polyethylene, polystyrene, polysaccharides, cellulose, or cellulose derivatives.

[0140] According to a preferred embodiment of the present invention, the protein of the present invention may be truncated at the N-terminus and / or C-terminus.

[0141] Furthermore, the protein of the present invention can be part of a fusion protein. In addition to the protein of the present invention, such a fusion protein may also contain at least one additional polypeptide at its N-terminus and / or C-terminus. For example, the fusion protein can be more easily isolated from other proteins or expressed in larger quantities in cells.

[0142] Another aspect of the invention relates to a nucleic acid molecule encoding a protein according to the invention. The nucleic acid molecule may be a DNA or RNA molecule.

[0143] Another aspect of the present invention relates to a carrier comprising a nucleic acid molecule according to the present invention.

[0144] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. Useful vectors are those capable of autonomously replicating and / or expressing the nucleic acid to which they are linked. Vectors capable of directing the expression of genes to which they are effectively linked are referred to herein as "expression vectors." Expression vectors used in recombinant DNA technology are typically in the form of "plasmids," which generally refer to a circular double-stranded DNA loop that does not bind to a chromosome when present as 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.

[0145] Suitable cloning vectors for overexpressing and producing the proteins of the present invention include, for example, pKK223-3, pTrc99a, pUC, pTZ, pSK, pBluescript, pGEM, pQE, pET, PHUB, pPLc, pKC30, pRM1 / pRM9, pTrxFus, pAS1, pGEx, pMAL, or pTrx.

[0146] The nucleic acid molecules of the present invention encoding the proteins of the present invention can be operatively linked to a promoter. As used herein, the term "operatively linked" means that a selected nucleotide sequence (e.g., encoding a protein described herein) is adjacent to a promoter to allow the promoter to regulate the expression of the selected nucleic acid molecule. Furthermore, the promoter is located upstream of the selected nucleotide sequence in both the transcriptional and translational directions. "Operationally linked" also refers to the manner in which the nucleotide and regulatory sequences are linked when a suitable molecule (e.g., a transcriptional activator protein) binds to the regulatory sequence, enabling the expression of the nucleic acid molecule / gene.

[0147] Suitable expression promoters include, for example, the trp-lac (tac) promoter, the trp-lac (trc) promoter, the lac promoter, the T7 promoter, and the λpL promoter.

[0148] Another aspect of the invention relates to a host cell comprising a nucleic acid molecule or vector according to the invention.

[0149] Host cells containing nucleic acid molecules or vectors according to the present invention can be used to produce the proteins of the present invention. Suitable host cells include bacteria such as Escherichia coli and yeast cells such as Komagataella phaffii.

[0150] Another aspect of the invention relates to the use of proteins or host cells or their lysates or homogenates according to the invention for the oxidation of NADH to NAD. + and / or oxidize NADPH to NADP + Its uses.

[0151] The protein of this invention can be used to oxidize NADH to NAD. + and / or oxidize NADPH to NADP + In any of the methods. The protein can be an isolated protein (e.g., a protein from the host cell described above) or an isolated immobilized protein, or it can be a whole cell or a portion of an immobilized whole cell and a cell homogenate or cell lysate. The use of cell lysates is particularly preferred because the lysate contains the cofactor NADH / NAD. + and NADPH / NADP + Therefore, in complex reaction mixtures, such as those involving oxidoreductases, it is not necessary to add other cofactors.

[0152] Another aspect of the present invention relates to a method for oxidizing NADH to NAD. + and / or oxidize NADPH to NADP + The method includes the step of incubating the protein or host cell or its lysate or homogenate according to the invention with NADH and / or NADPH.

[0153] Another aspect of the invention relates to the use of the protein or host cell or its lysate or homogenate according to the invention in a cofactor recycling system or as a cofactor recycling system.

[0154] The protein of this invention can be used to recycle NADH into NAD. + And / or NADPH is recycled to NADP + The cofactor recycling system is well known to those skilled in the art. The protein of this invention can also be used as NADH must be recycled to NAD. + And / or NADPH must be recycled to NADP + The cofactor recycling system in the reaction.

[0155] The optimal temperature for the NAD(P)H oxidase of the present invention is between 20°C and 45°C, and the optimal pH is between 6.0 and 8.5. The NAD(P)H oxidase exhibits good temperature and pH stability, and remains stable for over 80 hours when incubated at pH 7.0 and 30°C. Therefore, the NAD(P)H oxidase of the present invention is significantly more stable than other NAD(P)H oxidases known in the prior art. Furthermore, it surprisingly exhibits high stability in aqueous solutions containing organic co-solvents such as acetone and isopropanol. It can be demonstrated that the NAD(P)H oxidase of the present invention is stable and possesses enzymatic activity in aqueous solutions containing up to 25% (v / v), preferably up to 20% (v / v), more preferably up to 15% (v / v) of an organic solvent (such as an alcohol (e.g., isopropanol) or acetone). Therefore, the NAD(P)H oxidase of the present invention is particularly preferred to be used in an aqueous reaction mixture containing the aforementioned amount of organic solvent, or in an aqueous reaction mixture containing 0.5 to 25% (v / v), preferably 1 to 20% (v / v), more preferably 1 to 15% (v / v) of at least one organic co-solvent. Under these conditions, the NAD(P)H oxidase of the present invention exhibits enzyme activity for at least 20 hours at 30°C.

[0156] The proteins of the present invention can be used in the methods of the present invention in whole-cell, cell homogenate, cell lysate, or completely (at least 95%, preferably at least 98%) or partially purified form. The methods of the present invention are carried out using the proteins according to the present invention or cells containing the proteins according to the present invention. In doing so, the cells used can be provided in a native, permeabilized, or lysed state.

[0157] Example

[0158] Materials and Methods: Production of Enzymes and Production of Lysates

[0159] General information about the expression of recombinases in Escherichia coli.

[0160] To produce recombinases in *E. coli* strains, the gene to be expressed was first amplified by PCR using genomic DNA or its synthetically adapted codons for *E. coli* as a template, along with specific oligonucleotides carrying restriction endonuclease recognition sequences, and then isolated from the reaction mixture. After digestion of the nucleic acid with restriction endonucleases SphI and HindIII, the gene fragment encoding the target enzyme was ligated to the backbone of the expression vector pQE70-Kan, which was digested with SphI and HindIII. The ligation product was transformed into chemically competent *E. coli* cells Top10F, and the resulting colonies were used for plasmid isolation and restriction analysis.

[0161] The results of the cloning process were verified by restriction enzyme digestion and DNA sequencing. The resulting construct carried the target gene under the IPTG-inducible T5 promoter.

[0162] To overexpress the enzyme in *E. coli*, the resulting expression plasmid was transformed into competent expression cells RB791. After incubation at 37°C for 24 hours, the resulting colonies were inoculated into LB medium for expression assay.

[0163] The next day, the expression culture was inoculated and shaken at 37°C until the OD550 reached 0.3. The temperature was then lowered to 25°C, and when the OD550 reached 0.5, the culture was induced with 0.5 mM IPTG. After 22 hours, the culture was harvested (separated from the medium as a cell pellet by centrifugation), and the expression of the recombinase was analyzed by SDS-PAGE and activity assays (in-use assay or optical enzyme assay).

[0164] Cell lysates were prepared using a Retsch lysing machine.

[0165] To prepare a cell suspension (0.5 mL to 1.0 mL), weigh the cell pellet prepared according to the above procedure into a suitable container and dissolve it in buffer (100 mM triethanolamine (TEA-HCl) pH 7). The biomass mass fraction is typically 10%, with the remainder being buffer. Cell disruption is performed using a Retsch MM 400 homogenizer.

[0166] The homogenate was centrifuged at 4°C and 16000g for 1 minute to separate insoluble cell debris and obtain lysates.

[0167] Dynamic measurement

[0168] The activity of enzyme preparations was measured as follows:

[0169] A certain volume (10 μl) of enzyme dilution solution was mixed with a total volume of 1 mL of solution in a cuvette. This solution consisted of the following components:

[0170] a) 0.2mM NAD + Add 200mM D-sorbitol to 100mM TEA-HCl pH 8.0, or

[0171] b) 0.2 mM NAD(P)H and 100 mM TEA-HCl pH 7.0.

[0172] Measurements were performed using GmXylDH or a mutant.

[0173] b) Measurements were performed using NAD oxidase (Nox).

[0174] The change in absorbance (ΔA, delta A) measured at 340 nm is a measure of enzyme activity according to the Lambert-Beer law (ΔA, delta A = ε∙Δc∙d; where, for NAD(P)H, ε is 6.22 l / (mmol·cm); Δc (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 1 μmol of substrate per minute under specified conditions. The activities given below are expressed as a percentage of the reference (unmodified enzyme) [calculated as activity (mutant) divided by activity (reference)].

[0175] PCR-mediated mutagenesis

[0176] Mutations were generated using standard overlap extension PCR (e.g., as described by Hilgarth and Lanigan (2019)).

[0177] Example 1

[0178] Activity measurement of mutant secondary G oxidoreductase

[0179] Sequence alignment of wild-type oxidoreductases used in mutation experiments, such as... Figure 1 As shown in the table below, the database access numbers (GenBank, Uniprot) of the analyzed enzymes are listed.

[0180]

[0181] Sequence alignments of the reference (bold) and mutant oxidoreductases, and their relative activities (expressed as a percentage of the reference), are shown in the figure. Figure 2 In the study, all variants with the mutation at secondary G showed increased activity. Note that WjogaNox references a variant already containing a mutated secondary G (D170). Reversing the mutation of WjogaNox back to G (WjogaNox_D170G) showed the expected decrease in activity.

[0182] Example 2

[0183] Activity screening of mutant secondary G oxidoreductase

[0184] To screen for oxidoreductase libraries containing variants with mutations at the secondary G position, CdivNox was used as a reference. CdivNox G170 mutations are performed to hydrophobic (A, C, L, P, Y), hydrophilic (N, Q, T), or charged (D, R) amino acids. Sequence alignments of the reference (bold) and mutated Nox, along with their relative activities (expressed as a percentage of the reference), are shown below. Figure 3 As shown, all secondary G variants exhibited an increase in activity of up to 211%.

[0185] References

[0186] 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

[0187] Bellamacina CR. The nicotinamide dinucleotide binding motif: a comparison of nucleotide binding proteins. FASEB J. 1996 Sep;10(11):1257-69.

[0188] 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).

[0189] Dym O, Eisenberg D. Sequence-structure analysis of FAD-containing proteins. Protein Sci. 2001 Sep;10(9):1712-28.

[0190] Henikoff, S., & Henikoff, J. G. (1992). Amino acid substitutionmatrices from protein blocks. Proceedings of the National Academy of Sciencesof the United States of America, 89(22), 10915–10919. https: / / doi.org / 10.1073 / pnas.89.22.10915

[0191] Hilgarth, R.S., & Lanigan T. M. (2019). Optimization of overlapextension PCR for efficient transgene construction. MethodsX, 7, 100759.https: / / doi.org / 10.1016 / j.mex.2019.12.001

[0192] Rossman, M. G., Liljas, A., Brändén, C. I., & Banaszak, L. J. (1975).2 Evolutionary and structural relationships among dehydrogenases. In Theenzymes (Vol. 11, pp. 61-102). Academic Press.

[0193] Sambrook, J., Fritsch, E. R., & Maniatis, T. (1989). MolecularCloning: A Laboratory Manual (2nd ed.). Cold Spring Harbor, NY: Cold SpringHarbor Laboratory Press.

[0194] Scrutton NS, Berry A, Perham RN. Purification and characterization ofglutathione reductase encoded by a cloned and over-expressed gene inEscherichia coli. Biochem J. 1987 Aug 1;245(3):875-80.

[0195] Webb, E. C. (1992). Enzyme nomenclature 1992. Recommendations of theNomenclature Committee of the International Union of Biochemistry andMolecular Biology on the Nomenclature and Classification of Enzymes。

Claims

1. A variant of a parental NAD(P)H-dependent oxidoreductase comprising (a) a shared motif GX n GX m The Rothman fold of G / A, wherein n is 1, 2 or 3 and m is 1 or 2, and (b) a glycine residue located 8 to 14 amino acid residues downstream of the common motif, wherein the glycine residue is mutated in the variant.

2. The variant according to 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, particularly 11 amino acid residues downstream of the common motif.

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

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

5. A variant according to 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 according to any one of claims 1 to 5, wherein the oxidoreductase is an oxidoreductase selected from enzyme class EC 1, preferably an oxidoreductase 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 according to 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 oxidoreductases.

8. The variant according to 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 that has at least 80% sequence identity with SEQ ID No. 1, ii) An amino acid sequence encoded by a nucleic acid sequence having at least 80% identity with SEQ ID No. 2, and iii) An amino acid sequence encoded by a nucleic acid that binds to a nucleic acid molecule complementary to the nucleic acid sequence SEQ ID No. 2 under strict conditions.

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

1.

10. A variant according to any one of claims 1 to 9, wherein 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 that has at least 80% sequence identity with SEQ ID No.

16. ii) An amino acid sequence encoded by a nucleic acid sequence having at least 80% identity with SEQ ID No. 17, and iii) An amino acid sequence encoded by a nucleic acid that binds to a nucleic acid molecule complementary to the nucleic acid sequence SEQ ID No. 17 under strict conditions.

11. The variant according to 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 that has at least 80% sequence identity with SEQ ID No.

18. ii) An amino acid sequence encoded by a nucleic acid sequence having at least 80% identity with SEQ ID No. 19, and iii) An amino acid sequence encoded by a nucleic acid that binds to a nucleic acid molecule complementary to the nucleic acid sequence SEQ ID No. 19 under strict conditions.

12. The variant according to any one of claims 1 to 9, wherein the NAD(P)H-dependent oxidoreductase is an aldehyde dehydrogenase comprising an amino acid sequence selected from the group consisting of: i) An amino acid sequence that has at least 80% sequence identity with SEQ ID No.

20. ii) An amino acid sequence encoded by a nucleic acid sequence having at least 80% identity with SEQ ID No. 21, and iii) An amino acid sequence encoded by a nucleic acid that binds to a nucleic acid molecule complementary to the nucleic acid sequence SEQ ID No. 21 under strict conditions.

13. The variant according to 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 that has at least 80% sequence identity with SEQ ID No.

22. ii) An amino acid sequence encoded by a nucleic acid sequence having at least 80% identity with SEQ ID No. 23, and iii) An amino acid sequence encoded by a nucleic acid that binds to a nucleic acid molecule complementary to the nucleic acid sequence SEQ ID No. 23 under strict conditions.

14. The variant according to 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 of: i) An amino acid sequence that has at least 80% sequence identity with SEQ ID No.

24. ii) An amino acid sequence encoded by a nucleic acid sequence having at least 80% identity with SEQ ID No. 25, and iii) An amino acid sequence encoded by a nucleic acid that binds to a nucleic acid molecule complementary to the nucleic acid sequence SEQ ID No. 25 under strict conditions.

15. The variant according to any one of claims 1 to 9, wherein the NAD(P)H-dependent oxidoreductase is a sorbitol dehydrogenase comprising an amino acid sequence selected from the group consisting of: i) An amino acid sequence that has at least 80% sequence identity with SEQ ID No.

26. ii) An amino acid sequence encoded by a nucleic acid sequence having at least 80% identity with SEQ ID No. 27, and iii) An amino acid sequence encoded by a nucleic acid that binds to a nucleic acid molecule complementary to the nucleic acid sequence SEQ ID No. 27 under strict conditions.

16. The variant according to any one of claims 1 to 9, wherein the NAD(P)H-dependent oxidoreductase is an SDR family oxidoreductase comprising an amino acid sequence selected from the group consisting of: i) An amino acid sequence that has at least 80% sequence identity with SEQ ID No.

28. ii) An amino acid sequence encoded by a nucleic acid sequence having at least 80% identity with SEQ ID No. 29, and iii) An amino acid sequence encoded by a nucleic acid that binds to a nucleic acid molecule complementary to the nucleic acid sequence SEQ ID No. 29 under strict conditions.

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

18. A vector comprising the nucleic acid molecule according to claim 17.

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

20. A method for obtaining a parental NAD(P)H-dependent oxidoreductase variant with increased enzymatic activity compared to the parental oxidoreductase, comprising the steps of: - Provides a parental oxidoreductase containing (a) a shared motif GX n GX m The Rothman fold of G / A, where n is 1, 2, or 3 and m is 1 or 2, and (b) the glycine residue located 8 to 14 amino acid residues downstream of the common motif, and - Mutate the 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, particularly 11 amino acid residues downstream of the common motif.

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

23. The method according to any one of claims 20 to 22, wherein the glycine residue is replaced by 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 according to 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 according to any one of claims 20 to 24, wherein the oxidoreductase is an oxidoreductase selected from enzyme class EC 1, preferably an oxidoreductase 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 according to 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 oxidoreductases.

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