Modified Rossmannfold-containing oxidoreductase

Abstract

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

Description

[Technical Field]

[0001] This invention relates to the field of enzyme engineering, particularly the Rossmann fold consensus motif GX. n GX m This relates to enzymes capable of binding to NAD(H) and / or NADP(H) using G / A.

[0002] Biocatalytic oxidation-reduction processes are widely used as an alternative to expensive chemical synthesis, allowing for shorter processing times using milder reaction conditions (atmospheric pressure and temperature), and furthermore, without generating excessive waste.

[0003] Redox reactions such as stereoselective reduction of keto groups and region-selective oxidation of hydroxyl groups are of particular interest in the pharmaceutical and food industries. These reactions are primarily carried out by oxidoreductases and involve the interconversion of nicotinamide cofactors NAD(P)+ (oxidized form) and NAD(P)H (reduced form). While these redox reactions require stoichiometric amounts of NAD(P)+ or NAD(P)H to complete the conversion, the addition of cofactors in industrial processes is extremely costly and inefficient. Since cofactors and enzymes are expensive components in biocatalytic processes, enzyme engineering is essential for improving cost efficiency.

[0004] NAD(P)(H) binding is often achieved by the Rossman fold. The structural fingerprint of the Rossman fold is a series of 30-35 residues from the first two β-chains and the phosphate-binding helix (Bellamicini, 1996). The βαβ sequences that constitute the functional 3D motif of various NAD(P)(H)-binding proteins are aligned with the early Rossman consensus sequence GX 1-2 -GXXG was identified (Rossman et al., 1975; Dym and Eisenberg, 2001). This consensus is the alternative cofactor binding consensus (GX) in glutathione reductase in E. coli. 1-2-G-X-X-A) (Scrutton et al., 1987), or the consensus G-X-X-X-G-I-G (SEQ ID NO: 30) more recently reported by Brakoulias and Jackson (2004), and finally to the more general Rossmann consensus GX n GX m reached G / A (n ≤ 3, m ≤ 2).

[0005] Since the Rossmann fold is present in many oxidoreductases, modifying this common cofactor-binding region would be a promising and universally effective strategy for developing oxidoreductases with improved activity.

[0006] Therefore, an object of the present invention is to provide an oxidoreductase variant that exhibits improved activity compared to the parent oxidoreductase. SUMMARY OF THE INVENTION MEANS FOR SOLVING THE PROBLEM

[0007] The present invention relates to a variant of a parent NAD(P)H-dependent oxidoreductase comprising (a) a Rossmann fold having a consensus motif GX n GX m G / A, preferably GX n GX m G (where 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 consensus motif (the glycine residue is mutated in this variant).

[0008] Surprisingly, when the glycine residue located 8 to 14 amino acid residues downstream of the Rossmann fold consensus motif of the NAD(P)H-dependent oxidoreductase was mutated, the enzyme activity was significantly increased by 10% or more, preferably 20%, more preferably 30%, and even more preferably 40% or more compared to the parent NAD(P)H-dependent oxidoreductase.

[0009] As shown in the alignment in Figure 1, a typical Rossman fold containing an enzyme is a more complex Rossman consensus motif, GX n GX m The G / A (n≦3, m≦2) relationship is revealed, and the conserved glycine (G) residue is retained as a secondary prototype feature. The additionally conserved G located 8-14 amino acid residues downstream of the Rothman consensus motif is referred to as "secondary G" throughout the text, or as "mutant secondary G" if mutated. Therefore, the present invention relates to the consensus motif GX n -GX m The mutant NAD(P)(H)-dependent oxidoreductase comprises a Rossmann fold having -G / A (n≦3, m≦2) and a secondary glycine (G) located at the 8-14 amino acid C-terminus of the consensus motif, wherein the mutant is mutated in the secondary glycine (G).

[0010] A further aspect of the present invention is a method for obtaining a variant of a parental NAD(P)H-dependent oxidoreductase having increased enzymatic activity compared to the parental oxidoreductase, comprising the following steps. (a) Consensus Motif GX n GX m A step to provide a parent oxidoreductase comprising (b) a Rossmann fold having G / A, where n is 1, 2, or 3 and m is 1 or 2, and (b) a glycine residue located downstream of 8 to 14 amino acid residues of the consensus motif, and A step of mutating the glycine residue.

[0011] Another aspect of the present invention relates to a protein having NADH and / or NADPH oxidase activity and comprising an amino acid sequence selected from the group consisting of the following, preferably a stable protein. i) An amino acid sequence having at least 80% sequence identity with Sequence 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 under stringent conditions to a nucleic acid molecule complementary to the nucleic acid sequence of SEQ ID NO: 2, wherein the stringent conditions preferably include washing at 65° C. and a salt concentration of 0.1-2×SSC.

[0012] The protein defined as above and exhibiting NADH and / or NADPH oxidase activity exhibits significantly higher stability compared to other known NADH oxidases and / or NADPH oxidases. The activity of the protein of the present invention maintains stability for a much longer period compared to other NADH oxidases and / or NADPH oxidases. Thereby, + and / or NADP + the yield of can be increased, which is important when the protein of the present invention is used in a cofactor regeneration system or as a cofactor regeneration system. Also, the presence of an organic cosolvent has a much lower impact on the enzyme activity of the protein of the present invention compared to other NADH oxidases and / or NADPH oxidases. Therefore, the protein of the present invention can be regarded as a stable or organic solvent-stable enzyme, that is, an NADH oxidase and / or NADPH oxidase.

[0013] SEQ ID NO: 1:

Number

[0014] SEQ ID NO: 2:

Number

[0015] The protein of the present invention, including or comprising SEQ ID NO: 1, is a novel NADH oxidase isolated from Gram-positive bacteria of the genus Carnobacterium (e.g., Carnobacterium divergens) and can be used, for example, in cofactor regeneration in industrial processes. This NADH oxidase and the protein of the present invention are characterized by a high total turnover number (TTN) and are stable even in processes in which important substances that may cause enzyme denaturation, degradation, and / or inhibition are converted. The protein of the present invention can also act as an NADPH oxidase and, furthermore, as an NAD(P)H oxidase, as will be described in more detail below.

[0016] Further aspects of the present invention relate to nucleic acid molecules encoding proteins according to the present invention, in particular to mutants of the parental NAD(P)H-dependent oxidoreductase described above.

[0017] The nucleic acid molecule of the present invention can, for example, be part of a vector. Therefore, another aspect of the present invention relates to a vector comprising the nucleic acid molecule according to the present invention.

[0018] A further aspect of the present invention relates to a host cell comprising a nucleic acid molecule or vector according to the present invention.

[0019] The nucleic acid molecule and / or vector of the present invention can be part of a host cell. In particular, it is preferable that the host cell of the present invention can express the protein of the present invention from the nucleic acid molecule and / or vector. This protein can be secreted from the host cell or expressed within the cell.

[0020] Another aspect of the present invention relates to the use of the protein according to the present invention, or a host cell according to the present invention, or its lysate or homogenate, for oxidizing NADH to NAD+ and / or NADPH to NADP+.

[0021] The protein of the present invention can be used to oxidize NADH to NAD+ and / or NADPH to NADP+. Lysates or homogenates derived from host cells expressing the protein of the present invention can also be used to oxidize NADH to NAD+ and / or NADPH to NADP+.

[0022] A further 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 host cell lysate according to the present invention with NADH and / or NADPH.

[0023] Another aspect of the present invention relates to a protein, host cell, lysate or homogenate thereof, in or as a cofactor regeneration system.

[0024] Another aspect of the present invention relates to a method for the enzymatic oxidation of a compound, comprising 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, wherein NADH is formed from NAD+ and / or NADPH is formed from NADP+. [Brief explanation of the drawing]

[0025] [Figure 1] Figure 1 shows a typical Rossmannfold enzyme alignment; this reveals the presence of a conserved secondary G located at the C-terminus of the GXnGXmG / A motif. [Figure 2] Figure 2 shows the alignment and relative activity of the reference, as well as the G mutant oxidoreductase. [Figure 3] Figure 3 shows the sequence alignment and activity measurement of the G170 CdivNox mutation. [Modes for carrying out the invention]

[0026] A “mutant” of the parent NAD(P)H-dependent oxidoreductase is defined as having at least one mutation in its amino acid sequence compared to the parent NAD(P)H-dependent oxidoreductase, i.e., at least one mutant amino acid residue. In addition to the claimed mutant glycine residue, further mutations may exist at other positions in the amino acid sequence of the parent NAD(P)H-dependent oxidoreductase.

[0027] The "parent" NAD(P)H-dependent oxidoreductase can be any oxidoreductase that contains a glycine residue at the defined position in the Rossmann fold.

[0028] An "NAD(P)H-dependent oxidoreductase" is an oxidoreductase that binds to NAD(P)H and can catalyze the oxidation of NADH and / or NADPH, or the reduction of NAD+ and / or NADP+. Such oxidoreductases typically contain a Rossmann fold characterized by a series of β-chains linked by α-helices, and can accommodate NAD(P)H and / or NAD(P)+.

[0029] "Oxidoreductase" refers to enzymes that catalyze redox reactions, including enzymes that act on the CH-OH group of a donor, the aldehyde or oxo group of a donor, the CH-CH group of a donor, the CH-NH2 group of a donor, the CH-NH group of a donor, NADH or NADPH as a donor, hydrogen as a donor, and reduced flavodoxin as a donor. It also includes enzymes that catalyze the above redox reactions and use NAD or NADP as an acceptor, or cytochrome as an acceptor, or oxygen as an acceptor, or disulfide as an acceptor, or quinone or a similar compound as an acceptor.

[0030] Rossmanfolds derived from different oxidoreductases are a preserved motif of GX n GX m G / A, preferably GX n GXm The motif includes G, where 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, with glycine being the most preferred. In the particularly preferred motif, n is 1 and m is 2.

[0031] The consensus motif "G / A" means "G or A" or "glycine or alanine."

[0032] The mutated glycine residue is located 8 to 14 amino acid residues downstream of the conserved Rossmanfold motif. Here, "downstream" refers to the glycine residue being GX n GX m This means that it is located 8 to 14 amino acid residues from the C-terminus of the G / A motif. The general formula and consensus sequence is GX n GX m G / AX o G may be an integer between 7 and 13, preferably 8 and 13, preferably 9 and 13, more preferably 9 and 12, more preferably 9 and 11, and particularly preferably 10. X can be any amino acid residue.

[0033] The amino acids used in this invention are classified as follows: 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).

[0034] In this invention, 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 standardized algorithm. Such an algorithm can optimize the alignment between two sequences by inserting gaps into the compared sequences in a standardized and reproducible manner, thereby enabling a more meaningful comparison of the two sequences.

[0035] The percentage identity between sequences may be determined using one or more computer algorithms or programs known in the prior art or described in the present invention. According to the present invention, the Basic Local Alignment Search Tool (BLAST) provided by the National Center for Biotechnology Information (NCBI) is preferably used (Altschul et al., 1990). The BLAST software series includes several programs, including a tool called "BLAST 2 Sequences," used for direct comparison of two nucleotide or amino acid sequences. "BLAST 2 Sequences" can also be accessed and used from each other through the NCBI World Wide Webpage on the Internet. The blastn program (for nucleotide sequences) uses a default word length (W) of 28, an expected value (E) of 0.05, M=1, N=-2, and compares both strands. For amino acid sequences, the blastp program uses the following default settings: word length 3, expected value (E) 0.05, BLOSUM62 scoring matrix (Henikoff & Henikoff, 1989), alignment (B) 50, expected value (E) 0.05, M=1, N=-2.

[0036] According to a preferred embodiment of the present invention, the mutated glycine residue can be substituted or deleted.

[0037] To obtain the claimed effects of the mutants of the present invention, the glycine residue may be substituted or deleted with another amino acid residue.

[0038] 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.

[0039] GX n GX m Includes G / A or GX n GX m The Rothmanfold consensus motif consisting of G / A may vary with respect 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.

[0040] The oxidoreductase variants of the present invention may be present in whole cells and / or in suspensions, in homogenates, and / or in the lysates of the corresponding cells forming them, particularly preferably in lysates.

[0041] In this context, suspension refers to suspensions of quiescent cells. These are recovered after culturing (separated from the culture medium) and used as a paste or suspended in a suitable buffer system. While the fermentation process deals with whole cells, quiescent cells cannot grow due to a lack of carbon sources and nutrients and are only useful for substrate conversion. In this context, homogenate refers to a suspension that has been physically and / or chemically treated (e.g., by pressure, lysozyme, or sonication) to release cellular components from the cells. Lysate is obtained by removing the insoluble cellular components of the homogenate, for example, by filtration or centrifugation.

[0042] In a further embodiment, the oxidoreductase variant of the present invention may be immobilized on a solid support material.

[0043] In a further embodiment of the present invention, the oxidoreductase mutation is performed on an enzyme that catalyzes redox reactions, comprising an enzyme that acts on the CH-OH group of a donor, an enzyme that acts on the aldehyde or oxo group of a donor, an enzyme that acts on the CH-CH group of a donor, an enzyme that acts on the CH-NH2 group of a donor, an enzyme that acts on the CH-NH group of a donor, an enzyme that acts on NADH or NADPH as a donor, an enzyme that acts on hydrogen as a donor, or an enzyme that acts on reduced flavodoxin as a donor.

[0044] The enzymes that catalyze the above-mentioned redox reactions include enzymes that use NAD or NADP as an acceptor, or cytochrome as an acceptor, or oxygen as an acceptor, or disulfide as an acceptor, or quinones or similar compounds as acceptors.

[0045] According to a preferred embodiment of the present invention, the oxidoreductase is an oxidoreductase selected from enzyme class EC1, and is preferably selected from the group consisting of EC1.1 acting on the CH-OH group of the donor, EC1.2 acting on the oxo group of the donor, EC1.3 acting on the CH-CH group of the donor, EC1.4 acting on the CH-NH2 group of the donor, EC1.5 acting on the CH-NH group of the donor, EC1.6 acting on NADH or NADPH, EC1.7 acting on other nitrogen compounds as donors, EC1.19 acting on hydrogen as a donor, and EC1.19 acting on reduced flavodoxin as a donor.

[0046] The EC number, implemented by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMB), is a numerical classification system based on the chemical reactions catalyzed by enzymes (Webb, 1992), and is frequently updated by comprehensive enzyme information systems, such as BRENDA.

[0047] According to a more 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 oxidoreductases.

[0048] 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 the following: i) An amino acid sequence having at least 80% sequence identity with Sequence ID No. 1, ii) An amino acid sequence encoded by a nucleic acid sequence having at least 80% identity with Sequence ID No. 2, iii) An amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a nucleic acid molecule complementary to the nucleic acid sequence of SEQ ID NO: 2.

[0049] According to another preferred embodiment of the present invention, the variant of NAD(P)H-dependent oxidoreductase includes a mutation at position 170 of SEQ ID NO: 1 when aligned to the sequence of SEQ ID NO: 1.

[0050] 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 the following: i) An amino acid sequence having at least 80% sequence identity with sequence number 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 under stringent conditions to a nucleic acid molecule complementary to the nucleic acid sequence of SEQ ID NO: 17.

[0051] According to another preferred embodiment of the present invention, the variant of xylitol dehydrogenase includes a mutation at position 194 of SEQ ID NO: 16 when aligned to the sequence of SEQ ID NO: 16.

[0052] Sequence ID 16:

number

[0053] Sequence ID 17:

number

[0054] 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 the following: i) An amino acid sequence having at least 80% sequence identity with Sequence 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 under stringent conditions to a nucleic acid molecule complementary to the nucleic acid sequence of SEQ ID NO: 19.

[0055] Sequence ID 18:

number

[0056] Sequence ID 19:

number

[0057] 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 the following: 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 at least 80% identity with SEQ ID NO: 21, and iii) An amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a nucleic acid molecule complementary to the nucleic acid sequence of SEQ ID NO: 21.

[0058] Sequence ID 20:

number

[0059] Sequence ID 21:

number

[0060] 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 the following: 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 at least 80% identity with SEQ ID NO: 23, and iii) An amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a nucleic acid molecule complementary to the nucleic acid sequence of SEQ ID NO: 23.

[0061] Sequence ID 22:

number

[0062] Sequence ID 23:

number

[0063] 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 the following: i) An amino acid sequence having at least 80% sequence identity with sequence number 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 under stringent conditions to a nucleic acid molecule complementary to the nucleic acid sequence of SEQ ID NO: 25.

[0064] Sequence ID 24:

number

[0065] Sequence ID 25:

number

[0066] 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 the following: 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 at least 80% identity with Sequence ID No. 27, and iii) An amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a nucleic acid molecule complementary to the nucleic acid sequence of SEQ ID NO: 27.

[0067] Sequence ID 26:

number

[0068] Sequence ID 27:

number

[0069] 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 the following: i) An amino acid sequence having at least 80% sequence identity with sequence number 28, ii) an amino acid sequence encoded by a nucleic acid sequence having at least 80% identity with sequence number 29, and iii) An amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a nucleic acid molecule complementary to the nucleic acid sequence of SEQ ID NO: 29.

[0070] Sequence ID 28:

number

[0071] Sequence ID 29:

number

[0072] The stringent conditions used here refer to conditions under which so-called specific hybrids are formed, but nonspecific hybrids are not.

[0073] Hybridization may be carried out by conventionally known methods, such as those described in J. Sambrook et al, Molecular Cloning, A Laboratory Manual, 2nd Ed, Cold Spring Harbor Laboratory (1989). Stringent conditions are defined as washing at 65°C with a salt concentration of 0.1–2 × SSC (where 1 × SSC is understood as a mixture of 0.15 M sodium chloride / 0.015 M citrate).

[0074] Another aspect of the present invention relates to a protein having NADH and / or NADPH oxidase activity and comprising an amino acid sequence selected from the group consisting of the following, preferably a stable protein. i) An amino acid sequence having at least 80% sequence identity with Sequence ID No. 1, ii) An amino acid sequence encoded by a nucleic acid sequence having at least 80% identity with SEQ ID NO: 2, iii) an amino acid sequence encoded by a nucleic acid that binds to a nucleic acid molecule complementary to the nucleic acid sequence of SEQ ID NO: 2 under stringent conditions, wherein stringent conditions preferably include washing at 65°C and a salt concentration of 0.1 to 2 × SSC.

[0075] According to a preferred embodiment of the present invention, the protein having NADH and / or NADPH oxidase activity comprises a polypeptide consisting of the amino acid sequence GX1GX2X3X4, where 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.

[0076] According to another preferred embodiment of the present 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 present invention comprises the amino acid sequence GGGYIX4 (SEQ ID NO: 3), more preferably GGGYIG (SEQ ID NO: 4) or GGGYIA (SEQ ID NO: 5). Surprisingly, the protein of the present invention comprising the amino acid sequence GGGYIG (SEQ ID NO: 4) can oxidize NADH and / or NADPH, but the protein of the present invention comprising the amino acid sequence GGGYIA (SEQ ID NO: 5) was found to have significantly reduced or no ability to oxidize NADH. Therefore, the latter protein is preferably used to oxidize NADPH.

[0077] 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: 8), where 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 consisting of 6 to 12, preferably 8 to 10, more preferably 10 amino acid residues, and X6 is a nonpolar amino acid X7 is a peptide consisting of 4 to 8 amino acid residues, preferably 4 to 6, more preferably 6; 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 consisting of 2 to 6 amino acid residues, preferably 2 to 4, more preferably 4; X12 is a polar amino acid residue or a basic amino acid residue, where X1 is particularly preferably glycine, and / or X2 is particularly preferably tyrosine, and / or X3 is particularly preferably isoleucine, and / or X4 is particularly preferably glycine or alanine.

[0078] According to another preferred embodiment of the present invention, X5 is a peptide consisting of the amino acid sequence IELVEAFAES (SEQ ID NO: 9), and / or X7 is a peptide consisting of the amino acid sequence KQVTLV (SEQ ID NO: 10), and / or X11 is a peptide consisting of the amino acid sequence DRIL (SEQ ID NO: 11).

[0079] The protein of the present invention preferably comprises a polypeptide consisting of the amino acid sequence GX1GX2X3X4IELVEAFAESX6KQVTLVX8X9X10DRILX12 (SEQ ID NO: 12) as a motif, more preferably GGGYIX4IELVEAFAESX6KQVTLVX8X9X10DRILX12 (SEQ ID NO: 13), GGGYIGIELVEAFAESX6KQVTLVX8X9X10DRILX12 (SEQ ID NO: 14), or GGGYIAIELVEAFAESX6KQVTLVX8X9X10DRILX12 (SEQ ID NO: 15).

[0080] 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.

[0081] Surprisingly, it was found that substitutions of several amino acid residues within the aforementioned motif affected the substrate specificity of the protein of the present invention. The resulting protein variants may exhibit specificity for NAD(H), NADP(H), or both.

[0082] In a particularly preferred embodiment of the present invention, X12 is asparagine. When the protein of the present invention contains an asparagine residue at that position, the protein can oxidize NADH to NADPH at a remarkably high level. Such proteins are shown to have a very low oxidation rate to NADPH. In another preferred embodiment of the present invention, X12 is arginine. When the protein of the present invention contains an arginine residue at that position, the protein can oxidize NADH and NADPH at remarkably high levels. This allows the protein of the present invention to be used in complex reaction systems, and two or more lead enzymes can carry out a reaction cascade with different cofactor selectivity (NADH, NADPH).

[0083] According to a more preferred embodiment of the present invention, X4 is nonpolar, and 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 can oxidize NADH to NAD+ and NADPH to NADP+. Such proteins can be advantageously used in reaction mixtures where it is necessary to oxidize NADH or NADPH or both to NAD+ and / or NADP+, respectively. For example, an enzymatic reaction related to the reduction from NAD+ to NADH and / or from NADP+ to NADPH may contain a protein for cofactor regeneration.

[0084] According to another preferred embodiment of the present 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 present invention containing these amino acid residues within the aforementioned motif can oxidize NADPH and thus form NADP+.

[0085] According to a preferred embodiment of the present 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. When the aforementioned motif includes these amino acid residues, the protein of the present invention can oxidize NADH to NAD+.

[0086] 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 90%, more preferably 98%, and particularly preferably 100% sequence identity with SEQ ID NOs: 1, 16, 18, 20, 22, 24, 26, and / or 28.

[0087] 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 preferably 100% identity with SEQ ID NOs: 2, 17, 19, 21, 23, 25, 27, and / or 29.

[0088] The protein of the present invention can be modified once, twice, three times, four times, or five times with a water-soluble polymer. The water-soluble polymer is, for example, polyethylene glycol. Preferably, the polyethylene glycol is bonded at the N-terminus of the protein according to the present invention. The protein of the present invention can also be bonded to solid materials such as polyethylene, polystyrene, polysaccharides, cellulose, and cellulose derivatives.

[0089] According to a preferred embodiment of the present invention, the protein of the present invention may be cleaved at the N-terminus and / or C-terminus.

[0090] Furthermore, the protein of the present invention may be part of a fusion protein. Such a fusion protein may contain at least a further polypeptide at its N-terminus and / or C-terminus, adjacent to the protein of the present invention. For example, the fusion protein can be more easily isolated from other proteins or can be expressed in large quantities within cells.

[0091] Further aspects of the present invention relate to nucleic acid molecules encoding proteins according to the present invention. The nucleic acid molecule may be a DNA molecule or an RNA molecule.

[0092] A further aspect of the present invention relates to a vector comprising a nucleic acid molecule according to the present invention.

[0093] As used in this invention, the term "vector" refers to a nucleic acid molecule capable of transporting another linked nucleic acid. A useful vector is one that enables the autonomous replication and expression of the linked nucleic acid. A vector capable of dictating the expression of a functionally linked gene is referred to here as an "expression vector." Expression vectors used in recombinant DNA technology often take the form of a "plasmid," which, when in vector form, usually refers to a non-chromosome-bound double-stranded DNA loop. Plasmids are the most commonly used form of vector, and therefore, as used in this invention, "plasmid" and "vector" are used synonymously. However, other forms of expression vectors that perform equivalent functions and are later known in this art are also included.

[0094] Examples of cloning vectors suitable for the overexpression and production of the protein of the present invention include pKK223-3, pTrc99a, pUC, pTZ, pSK, pBluescript, pGEM, pQE, pET, PHUB, pPLc, pKC30, pRM1 / pRM9, pTrxFus, pAS1, pGEx, pMAL, and pTrx.

[0095] The nucleic acid molecule of the present invention encoding the protein of the present invention may be operably ligated to a promoter. As used herein, “operably ligated” means that the selected nucleotide sequence (e.g., encoding the protein described herein) is close to the promoter so that the promoter can regulate the expression of the selected nucleic acid molecule. Furthermore, the promoter is located upstream of the selected nucleotide sequence in the direction of transcription and translation. “Operatably ligated” means that when an appropriate molecule (e.g., a transcription-activating protein) binds to the regulatory sequence, the nucleotide sequence and the regulatory sequence are ligated so that the nucleic acid molecule / gene is expressed.

[0096] Suitable expression promoters include, for example, the trp-lac(tac)-promoter, trp-lac(trc)-promoter, lac-promoter, T7-promoter, and λpL-promoter.

[0097] Another aspect of the present invention relates to a host cell comprising a nucleic acid molecule or vector according to the present invention.

[0098] 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.

[0099] A further aspect of the present invention relates to the use of the protein, host cell, or lysate or homogenate thereof according to the present invention for oxidizing NADH to NAD+ and / or NADPH to NADP+.

[0100] The protein of the present invention can be used in any method for oxidizing NADH to NAD+ and / or NADPH to NADP+. The protein may be an isolated protein (e.g., derived from the aforementioned host cells), an isolated immobilized protein, a part of a whole cell, or a part of an immobilized whole cell and a cell homogenate or cell lysate. The use of cell lysates is particularly preferred because the lysates contain the cofactors NADH / NAD+ and NADPH / NADP+, and there is no need to add further cofactors in complex reaction mixtures, for example, in which oxidoreductase is involved.

[0101] 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 according to the present invention, or a host cell, or its lysate or homogenate, together with NADH and / or NADPH.

[0102] Further aspects of the present invention relate to the use of the protein, host cell, or lysate or homogenate thereof according to the present invention in or as a cofactor regeneration system.

[0103] The protein of the present invention can be used in cofactor regeneration systems in which NADH is regenerated to NAD+ and / or NADPH is recycled to NADP+. Such cofactor regeneration systems are well known to those skilled in the art. The protein of the present invention can also be used as a cofactor regeneration system in reactions in which it is necessary to regenerate NADH to NAD+ and / or NADPH to NADP+.

[0104] 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, remaining stable for more than 80 hours when incubated at pH 7.0 and 30°C. Therefore, the NAD(P)H oxidase of the present invention is far more stable than other NAD(P)H oxidases known in the art. Furthermore, it exhibits remarkably high stability even in aqueous solutions containing organic cosolvents such as acetone and isopropanol. The NAD(P)H oxidase of the present invention is shown to be stable and enzymatically active in aqueous solutions containing alcohols such as isopropanol or organic solvents such as acetone, with a maximum of 25% (v / v), preferably up to 20% (v / v), and more preferably up to 15% (v / v). Therefore, it is particularly preferable to use the NAD(P)H oxidase of the present invention in an aqueous reaction mixture containing the aforementioned amount of organic solvent, or in an aqueous reaction mixture containing at least one organic cosolvent in an amount of 0.5-25% (v / v), preferably 1-20% (v / v), and more preferably 1-15% (v / v). Under such conditions, the NAD(P)H oxidase of the present invention is enzymatically active for at least 20 hours at 30°C.

[0105] The proteins of the present invention can be used in the methods of the present invention as whole cells, cell homogenates, cell lysates, or in a completely (at least 95%, preferably at least 98%) or partially purified state. The methods of the present invention are carried out using the proteins of the present invention or using cells containing the proteins of the present invention. In doing so, the cells used can be provided in a native, membrane-permeable, or lysed state. [Examples]

[0106] Materials and methods: Enzyme production and lysate production General information on recombinant enzyme expression in Escherichia coli. For the purification of recombinant enzymes in E. coli strains, the gene to be expressed was first amplified by PCR using genomic DNA or an equivalent artificially adapted to the codon frequency of E. coli as a template, along with specific oligonucleotides to which the restriction enzyme recognition sequence was added. The resulting mixture was then isolated. After nucleic acid digestion with restriction enzymes SphI and HindIII, the gene fragment encoding the target enzyme was ligated to the backbone of the expression vector pQE70-Kan, which had been cleaved with SphI and HindIII. The ligation product was converted to chemically competent E. coli cells, Top10F, and the resulting colonies were used for plasmid isolation and restriction analysis.

[0107] The results of the cloning step were verified by restriction enzyme digestion and DNA sequencing. The resulting constructs transport the target gene under the IPTG-induced T5 promoter.

[0108] For enzyme overexpression in E. coli, the obtained expression plasmid was converted to competent expression cells RB791. After incubation at 37°C for 24 hours, the resulting colonies were inoculated into LB medium for expression assay.

[0109] The next day, the expression culture was inoculated, OD 550 The mixture was shaken at 37°C until it reached 0.3. After that, the temperature was lowered to 25°C, and the OD (Oxygen Demand) was adjusted. 550 When the value reached 0.5, culture was induced with 0.5 mM IPTG. After 22 hours, the culture was collected (separated from the medium by centrifugation in the form of a cell pellet), and recombinant enzyme expression was analyzed by SDS gel electrophoresis and activity assay (used in the assay or optical enzyme assay).

[0110] Cell lysate production by Letche disruption To prepare cell suspensions (volume 0.5–1.0 mL), the cell pellets prepared according to the above procedure were weighed into appropriate containers and dissolved in buffer (100 mM triethanolamine (TEA-HCl) pH 7). Biomass mass spectrometry typically yielded 10% of the sample, with the remainder being buffer. A Retsch MM 400 homogenizer was used for cell disruption.

[0111] The homogenate was centrifuged at 4°C and 16000g for 1 minute to separate insoluble cell fragments and obtain lysate.

[0112] Kinetic measurements Activity measurements using enzyme preparations were performed as follows: A defined amount (10 μl) of enzyme diluent was mixed into a 1 mL cuvette consisting of the following: a) 0.2mM NAD + and 100 mM TEA-HCl pH 8.0 and 200 mM D-sorbitol or b) 0.2 mM NAD(P)H and 100 mM TEA-HCl pH 7.0. Measurement a) was performed using GmXylDH or a mutant. Measurement b) was performed using NAD oxidase (Nox).

[0113] The change in absorbance (ΔA, delta A) measured at 340 nm is an indicator of enzyme activity based on the Lambert-Via law (ΔA = ε·Δc·d; where ε is 6.22 l / (mmol·cm) for NAD(P)H; 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 defined conditions. The activity shown below is expressed as a percentage of the reference (unmodified enzyme) [calculated as activity (mutant) divided by activity (reference)].

[0114] PCR-mediated mutagenesis Mutants were generated using standard overlap extension PCR (as described, e.g., by Hilgarth and Lanigan (2019)).

[0115] Example 1 Activity measurement of mutant secondary G oxidoreductase Figure 1 shows the sequence alignment of wild-type oxidoreductase used in the mutation experiment. The database accession numbers (GenBank, Uniprot) of the analyzed enzymes are shown in the table below.

[0116] [Table 1]

[0117] Figure 2 shows the sequence alignment of the reference (bold) and mutant oxidoreductases, as well as their relative activity (expressed as a percentage of the reference). All mutants mutated in secondary G showed increased activity. Note that the reference of WjogaNox already retained the mutated secondary G (D170). The reverse mutation of WjogaNox to G (WjogaNox_D170G) showed decreased activity as expected.

[0118] Activity screening of mutant secondary G oxidoreductases For screening oxidoreductase libraries using mutants mutated via secondary G, CdivNOx was used as the reference. CdivNox G170 was mutated with hydrophobic (A, C, L, P, Y), hydrophilic (N, Q, T), or charged (D, R) amino acids. Figure 3 shows the sequence alignment of the reference (bold) and mutant Nox, as well as their relative activity (expressed as a percentage of the reference). All secondary G mutants showed up to a 211% increase in activity.

[0119] References JPEG2026523236000019.jpg221165

Claims

1. (a) Consensus Motif GX ｎ GX ｍ A variant of a parent NAD(P)H-dependent oxidoreductase comprising (b) a Rossman fold having G / A, where n is 1, 2, or 3 and m is 1 or 2, and (b) a glycine residue located 8 to 14 residues downstream of the consensus motif, wherein the variant has a mutation in the glycine residue.

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

3. A mutant according to claim 1 or 2, wherein a glycine residue is substituted or deleted.

4. A variant according to 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. 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. A variant according to any one of claims 1 to 5, wherein the oxidoreductase is selected from enzyme class EC1, preferably from the group consisting of EC1.1, EC1.2, EC1.3, EC1.4, EC1.5, EC1.6, EC1.7, EC1.12, and EC1.

19.

7. A 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. A 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 the following: i) An amino acid sequence having at least 80% sequence identity with Sequence 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 under stringent conditions to a nucleic acid molecule complementary to the nucleic acid sequence of Sequence ID No.

2.

9. The variant according to any one of claims 1 to 8, wherein when the variant of NAD(P)H-dependent oxidoreductase is aligned to the sequence of SEQ ID NO: 1, the variant includes a mutation at position 170 of SEQ ID NO:

1.

10. A mutant 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 the following, i) An amino acid sequence having at least 80% sequence identity with sequence number 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 under stringent conditions to a nucleic acid molecule complementary to the nucleic acid sequence of Sequence ID No.

17.

11. A mutant 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 the following, 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 at least 80% identity with SEQ ID NO: 19, and iii) An amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a nucleic acid molecule complementary to the nucleic acid sequence of SEQ ID NO:

19.

12. A mutant 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 the following, i) An amino acid sequence having at least 80% sequence identity with Sequence 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 under stringent conditions to a nucleic acid molecule complementary to the nucleic acid sequence of SEQ ID NO:

21.

13. A mutant 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 the following, i) An amino acid sequence having at least 80% sequence identity with Sequence 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 under stringent conditions to a nucleic acid molecule complementary to the nucleic acid sequence of SEQ ID NO:

23.

14. A 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 the following, i) An amino acid sequence having at least 80% sequence identity with Sequence 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 under stringent conditions to a nucleic acid molecule complementary to the nucleic acid sequence of SEQ ID NO:

25.

15. A variant according to 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 the following, 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 at least 80% identity with SEQ ID NO: 27, and iii) An amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a nucleic acid molecule complementary to the nucleic acid sequence of SEQ ID NO:

27.

16. A 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 the following, i) An amino acid sequence having at least 80% sequence identity with Sequence 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 under stringent conditions to a nucleic acid molecule complementary to the nucleic acid sequence of Sequence ID No.

29.

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 described in claim 17.

19. A host cell comprising the nucleic acid molecule described in claim 17, or the vector described in claim 18.

20. A method for obtaining a variant of parental NAD(P)H-dependent oxidoreductase having increased enzymatic activity compared to the parental oxidoreductase, comprising the following steps: (a) Consensus Motif GX ｎ GX ｍ To provide a parent oxidoreductase comprising (b) a Rossmann fold having G / A, where n is 1, 2, or 3 and m is 1 or 2, and (b) a glycine residue located downstream of 8 to 14 amino acid residues of the consensus motif, and To mutate the aforementioned glycine residue.

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

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

23. The method according to 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 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 selected from enzyme class EC1, preferably from the group consisting of EC1.1, EC1.2, EC1.3, EC1.4, EC1.5, EC1.6, EC1.7, EC1.12, and EC1.

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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