Process for preparation of 2, 5-furandicarboxylic acid

Abstract

The invention relates to a method for producing 2, 5-furandicarboxylic acid by the in vitro oxidation of 5-formyl-2-furancarboxylic acid, which is present in an aqueous solution, by treatment with an aldehyde dehydrogenase, to 2, 5-furandicarboxylic acid, in which NAD (P) H produced during the oxidation is enzymatically oxidized back to NAD (P) + by means of a dehydrogenase and D-fructose or acetone; and then removing the enzyme.

Description

Technical Field

[0001] This invention relates to a method for preparing 2,5-furandicarboxylic acid (FDCA) from 5-formyl-2-furancarboxylic acid (FFA). Background Technology

[0002] In 2018, approximately 90% (400 million tons) of plastics produced globally were based on fossil fuels, with the remaining share coming from recycled plastics (9%), bio-based plastics (1%), and CO2-based plastics (<1%) (Carus et al., 2020). Meanwhile, global demand for plastics continues to grow. In 2019, the annual CO2 emissions over the entire lifecycle of plastics reached 0.86 Gt, equivalent to the CO2 emissions of 189 fully operational (500 MW) coal-fired power plants. Emissions are projected to increase to 2.8 Gt (equivalent to 615 coal-fired power plants) by 2050 (Hamilton et al., 2019).

[0003] Polyethylene terephthalate (PET) is a fossil-based plastic primarily used in beverage packaging. It is a condensation polymer prepared by dehydrating terephthalic acid (1,4-phthalic acid) and ethylene glycol (ethane-1,2-diol).

[0004] Terephthalic acid was industrially prepared by oxidizing p-xylene (1,4-dimethylbenzene) with atmospheric oxygen at about 200 °C in the presence of HBr in cobalt acetate, manganese acetate and acetic acid in the so-called AMOCO process (Tomás et al., 2013).

[0005] Ethylene oxide is prepared by hydrolysis of ethylene oxide (at 200°C), while ethylene oxide is in turn obtained by oxidation of ethylene (derived from fossil feedstocks) (Berger, 2016). Due to the fossil origin of the starting materials (paraxylene and ethylene), PET cannot generally be described as sustainable.

[0006] To achieve the 1.5°C target set by the Paris Climate Agreement and thereby limit the negative impacts of climate change, sustainable alternatives to petrochemical-based plastics must be found. For example, for ethylene glycol, there are processes based on renewable resources. Ethylene can also be produced, for instance, by dehydrating bioethanol obtained from glucose or starch through fermentation (e.g., Fan et al., 2013). The fermentation of xylose and / or glucose into ethylene glycol using metabolically engineered microorganisms (Escherichia coli or Saccharomyces cerevisiae) is also known (Salusjärvi et al., 2019).

[0007] The second building block of PET, the aromatic compound terephthalic acid, is difficult to obtain from sustainable raw materials, thus requiring alternatives. 2,5-Furfurandicarboxylic acid (FDCA) is an excellent alternative to terephthalic acid, primarily obtained through the catalytic upgrading of biomass. The polymerization of FDCA with ethylene glycol produces PEF (polyethylene furanate), which has a heteroaromatic furan ring in its structure instead of a benzene ring. PEF, like PET, is a thermoplastic, but compared to PET, it is characterized by significantly higher biodegradability, as well as superior thermal properties (higher glass transition temperature, lower melting temperature) and mechanical properties (higher stiffness). The most important property of PEF is its reduced permeability to gases such as O2 and CO2. This is particularly important for beverages, as it can extend shelf life (preventing degassing of carbonated beverages; preventing oxidation processes initiated by diffused oxygen) (de Jong et al., 2022).

[0008] A review of various chemical synthesis methods can be found in the article by Cong et al. (2021).

[0009] Currently, the most important starting material for FDCA is 5-hydroxymethylfurfural (HMF), which can be obtained, for example, from cellulose (and thus from renewable feedstocks). Cellulose is hydrolyzed enzymatically or chemically to produce D-glucose, which is then isomerized (enzymatically or chemically) to D-fructose. D-fructose is then converted to HMF by dehydration (removal of three water molecules). Common systems for fructose dehydration include, on the one hand, inorganic acids (such as sulfuric or hydrochloric acid) and on the other hand, acidic solid catalysts (Brenstein or Lewis) (Cong et al., 2021; US ​​9617234 B1).

[0010] HMF contains both alcohol and aldehyde functional groups and must be oxidized to carboxylic acid groups to obtain FDCA. The three necessary oxidation steps can be carried out in various ways: chemically using heterogeneous or homogeneous catalysts, electrochemically, and biocatalystically (enzymatically or with whole-cell catalysis).

[0011] The direct precursor of FDCA is FFA (5-formyl-2-furanoic acid), which can be prepared, for example, by acid-catalyzed dehydration of the sugar acid derivatives 2-keto-D-gluconate (2KGA) or 5-keto-D-gluconate (5KGA). 2KGA can be dehydrated to FFA by treatment with hydrogen bromide in an acetic acid / water mixture at 80 °C in a flow reactor. In a final step, FFA can be oxidized to FDCA using oxygen as an oxidant and a metal (e.g., Pt-Ru / C) or metal salt (Co & Mn-salts) as a catalyst, or oxidized by hydrogen peroxide (US 10087161 B2).

[0012] EP 3265450 B1 also describes the acid-catalyzed dehydration of 5KGA to FFA. The final oxidation is carried out at 180°C under oxygen overpressure in the presence of catalytic amounts of cobalt acetate, manganese acetate, and NaBr in acetic acid (similar to the AMOCO process).

[0013] Overall, the aforementioned chemical preparation process for FDCA, which starts with FFA as an intermediate, is characterized by unfavorable reaction conditions (high temperature and high pressure), the generation of byproducts, and the use of expensive and partially toxic catalysts (such as cobalt salts), and therefore cannot be described as sustainable.

[0014] Biocatalytic processes offer the following advantages as an alternative: they proceed with high selectivity under mild reaction conditions and use biodegradable catalysts (such as cells or enzymes) (Cong et al., 2021).

[0015] FFA can be prepared by enzymatic oxidation of HMF (mainly using O2 as an oxidant).

[0016] One group of enzymes used to prepare FFA from HMF is aryl alcohol oxidase (AAO, EC 1.1.3.7). Carro et al. (2015) used AAO from the fungus Pleurotus eryngii to oxidize 3 mM HMF to FFA (98 mol%) and a small amount of FDCA in 4 hours.

[0017] The oxidation of the aldehyde group catalyzed by AAO proceeds via the corresponding geminal diol (aldehyde hydrate). The aldehyde group in FFA has a lower degree of hydration compared to the precursor 2,5-diformylfuran (DFF) (DFF: 53%, FFA: 8%), which explains the preferential formation of FFA over FDCA. The byproduct H₂O₂ generated by AAO-catalyzed oxidation chemically oxidizes FFA to FDCA (Carro et al., 2015). In a subsequent study on AAO from Pleurotus eryngii (2019), Serrano et al. (2019) showed that HMF could be completely oxidized to FDCA if catalase was used to remove H₂O₂, as H₂O₂ inhibits the final oxidation step from FFA to FDCA. In this way, using the AAO mutant, 1.5 mM HMF could be oxidized to 1.6 mM FDCA within 6 days.

[0018] Other known AAOs are from Mycobacterium sp. strain MS1601 (which completely oxidizes 4 g / L (31.7 mM) HMF to FFA within 120 hours) (Sayed et al., 2022) and from the fungus Moesziomyces antarcticus (which oxidizes 2 mM HMF to 99.6 mol% FFA and 0.4 mol% FDCA within 24 hours; and oxidizes 40% of FFA (2 mM) to FDCA within 144 hours) (Lappe et al., 2021).

[0019] Qin et al. (2015) tested various laccases against 30 mM HMF using 20 mol% TEMPO ((2,2,6,6-tetramethylpiperidin-1-yl)oxy) as a mediator. Using laccase from the fungus Panus conchatus, 82% of the starting material was oxidized to FFA (with 4% 2,5-dicarboxyfuran (DFF) and 10% FDCA as byproducts) within 96 hours.

[0020] Zhang et al. (2019) immobilized laccase (CotA-TJ102) from Bacillus subtilis TJ-102 onto magnetic nanoparticles, which could oxidize 83.3% of the starting material HMF to FFA (after 10 cycles) with selectivity >96%.

[0021] Jia et al. (2019) used an enzyme system consisting of GOase, an alcohol dehydrogenase (SADH) from the genus Synechocystis sp., and HRP to oxidize 97% of the HMF substrate (100 mM) to FFA within 48 hours. HRP activated GOase M... 3-5 In addition, it helps regenerate NAD(P). + It is a cofactor required by SADH.

[0022] US 10344307 B2 describes other enzyme systems for oxidizing HMF to FFA: 1) NAD(P)-dependent ketone reductases and NAD(P)H oxidases (for cofactor regeneration); 2) NAD(P)-dependent aldehyde dehydrogenases and NAD(P)H oxidases; 3) xanthine oxidoreductases (such as periplasmic aldehyde oxidoreductase (PaoABC) from Escherichia coli, which oxidizes HMF to 5-hydroxymethyl-2-furanoic acid (HMFA) and DFF to FFA), galactose oxidase variant M 3-5 (GOase M) 3-5(oxidizing HMF to DFF and HMFA to FFA) and horseradish peroxidase (HRP, used to activate GOase M) 3-5 For example, using PaoABC at pH 6, 50 mM DFF can be completely oxidized to FFA in 2 hours; while at pH 7 and 8, oxidation can proceed completely to FDCA. The enzyme system described in 3) is also described in detail in journal articles (McKenna et al., 2015; McKenna et al., 2017).

[0023] In addition to PaoABC and AAO, other enzymes are known for the final oxidation step from FFA to FDCA.

[0024] Cajnko et al. (2020) tested 10 mM FFA with a range of commercially available enzymes (alcohol oxidase (AO) from *Pichia pastoris*, galactose oxidase from *Dactylium dendroides*, catalase from *Aspergillus niger*, laccase from *Trametesversicolor*, fungal lignin peroxidase (LPO), and HRP). Only AO, laccase, and LPO were observed to generate significant amounts of FDCA after 72 hours (AO, 11.6%; laccase, 1.1%; LPO, 3.2%). 5-Hydroxymethyl-2-furanoic acid (HMFA) (up to 18.2% for AO) was found as a byproduct.

[0025] Another enzyme used to oxidize FFA to FDCA is a nonspecific peroxidase from *Agrocybe aegerita* (UPO, EC 1.11.2.1, requiring H2O2 as an oxidant), which can be used in conjunction with AAO to completely oxidize HMF to FDCA. In this way, 90% of FFA (3 mM) can be oxidized to FDCA within 120 hours (Carro et al., 2015). In addition to oxidizing FFA to FDCA, UPO also catalyzes the oxidation of HMF to FFA via DFF (Lappe et al., 2021).

[0026] Jia et al. (2017) used an enzyme system composed of horse liver alcohol dehydrogenase (HLADH) and human hemoglobin (using H2O2 to oxidize NADH to NAD). + The substrate (10 mM FFA) was oxidized to FDCA within 60 hours.

[0027] US 8183020 B2 describes the enzymatic oxidation of FFA to FDCA using H2O2 as an oxidant with commercially available chloroperoxidase from Caldariomyces fumago (EC1.11.1.10).

[0028] Aldehyde dehydrogenase (ALDH) is an enzyme that catalyzes the oxidation of aldehyde groups to carboxylic acid groups. For the ornithine-soluble Raoultella ornithinolytica BF60 organism, an ALDH that can oxidize FFA to FDCA and HMF to HMFA has been described (Hossain et al., 2017).

[0029] Shortall et al. (2023) used an aldehyde dehydrogenase (from *Thermus thermophilus*) to oxidize an aliphatic aldehyde (hexanal) and three aromatic aldehydes (p-tolualdehyde, benzaldehyde, and terephthalaldehyde) to their corresponding carboxylic acids in a coupled two-enzyme flow reactor. For this purpose, the ALDH was immobilized directly from *E. coli* cell lysates in one reactor module. L-lactate dehydrogenase, used for cofactor regeneration, was immobilized in a second reactor module.

[0030] In another study by Knaus et al. (2018), three different purified aldehyde dehydrogenases (derived from the bovine lens, Escherichia coli, and Pseudomonas putida) were tested in combination with NADH oxidase from Streptococcus mutans for cofactor regeneration for 61 different aliphatic aldehydes, aryl aliphatic aldehydes, benzyl aldehydes, heteroaromatic aldehydes, and bicyclic aldehydes. In oxidation with 20 mM HMF, ALDH from the bovine lens achieved a 90% yield of HMFA within 4 hours, while ALDH from E. coli achieved a 91% yield within 24 hours. FFA substrates were not tested.

[0031] US 10344307 B2 describes a method for oxidizing DFF with commercially available ALDH (in combination with NAD(P)H oxidase), which produces FFA, FDCA, or a mixture of both, depending on the DFF concentration (10 to 100 mM). More FFA is produced than FDCA as the DFF concentration increases (10 mM substrate: 100% FDCA; 50 mM substrate: 80% FDCA, 20% FFA; 100 mM substrate: 20% FDCA, 80% FFA (conversion rates after 3 hours)). US 10344307 B2 uses 10 mol% to 50 mol% cofactors (based on substrate amount) for oxidation.

[0032] Yuan et al. (2018) used engineered whole-cell (resting cells) of *Rauvolfia ornithine-lysinic* BF60, in which two genes, adhP3 and alkR, were knocked out to inhibit the reduction of HMF to the corresponding diol, in order to oxidize HMF to FDCA (265 mM, 96% yield) in a fed-batch process over 144 hours. Furthermore, an in vitro conversion from FFA to FDCA was described, but it resulted in a lower conversion rate compared to the whole-cell process.

[0033] Existing technologies generally have drawbacks such as low substrate concentration or long reaction time.

[0034] This is the purpose of the present invention, which is to provide an improved method for preparing FDCA. Detailed Implementation

[0035] The object of this invention is achieved by: in vitro oxidizing 5-formyl-2-furancarboxylic acid present in aqueous solution to 2,5-furandicarboxylic acid by treatment with an aldehyde dehydrogenase, wherein the dehydrogenase enzymatically oxidizes NAD(P)H generated during oxidation back to NAD(P) using D-fructose or acetone as a substrate. + The enzyme is then removed, characterized by a combination of the following measures:

[0036] NAD(P) used to oxidize 5-formyl-2-furancarboxylic acid to 2,5-furandicarboxylic acid. + Aldehyde-dependent dehydrogenases have amino acid sequences selected from the following groups:

[0037] i) Has an amino acid sequence that is at least 80% identical to SEQ ID No. 2, SEQ ID No. 4, or SEQ ID No. 6;

[0038] ii) An amino acid sequence encoded by a nucleic acid having at least 80% identity with SEQ ID No. 1, SEQ ID No. 3, or SEQ ID No. 5; and

[0039] iii) An amino acid sequence encoded by a nucleic acid that binds to the complementary strand of a nucleic acid molecule having a nucleic acid sequence of SEQ ID No. 1, SEQ ID No. 3, or SEQ ID No. 5 under stringent conditions.

[0040] Furthermore, it involves oxidizing NAD(P)H to NAD(P). + The dehydrogenase used is:

[0041] a) Xylitol dehydrogenases having an amino acid sequence selected from the following groups:

[0042] i) Has an amino acid sequence that is at least 80% identical to SEQ ID No. 8;

[0043] ii) An amino acid sequence encoded by a nucleic acid having at least 80% identity with SEQ ID No. 7; and

[0044] iii) An amino acid sequence encoded by a nucleic acid that binds to the complementary strand of a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 7 under stringent conditions.

[0045] D-fructose was used as a substrate.

[0046] or

[0047] b) NAD(P)H-dependent alcohol dehydrogenases having an amino acid sequence selected from the following groups:

[0048] i) Has an amino acid sequence that is at least 80% identical to SEQ ID No. 10;

[0049] ii) An amino acid sequence encoded by a nucleic acid having at least 80% identity with SEQ ID No. 9; and

[0050] iii) An amino acid sequence encoded by a nucleic acid that binds to the complementary strand of a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 9 under stringent conditions.

[0051] Acetone is used as the substrate.

[0052] "In vitro" means that this method is carried out outside the organism, that is, not in intact cells, nor in fermentation.

[0053] Within the meaning of this patent specification and claims, NAD(P) + NAD(P)H are cofactors of the corresponding enzymes.

[0054] Appendix Figure 1 A preferred embodiment of the method according to the present invention is illustrated schematically.

[0055] The sequences described above are described in the following paragraphs.

[0056] SEQ ID No. 1:

[0057]

[0058] SEQ ID No. 2:

[0059]

[0060] SEQ ID No. 3:

[0061]

[0062] SEQ ID No. 4:

[0063]

[0064] SEQ ID No. 5:

[0065]

[0066] SEQ ID No. 6:

[0067]

[0068] The aldehyde dehydrogenase described herein comprises an amino acid sequence having at least 80%, more preferably 85%, more preferably 90%, more preferably 95%, more preferably 98%, more preferably 99%, and particularly 100% identity with SEQ ID No. 2, SEQ ID No. 4, or SEQ ID No. 6.

[0069] Alternatively, the aldehyde dehydrogenase comprises an amino acid sequence encoded by a nucleic acid having at least 80%, more preferably 85%, more preferably 90%, more preferably 95%, more preferably 98%, more preferably 99%, and particularly 100% identity with SEQ ID No. 1, SEQ ID No. 3, or SEQ ID No. 5. Particularly preferably, the nucleic acid encoding the aldehyde dehydrogenase according to the invention comprises or is composed of the nucleic acid sequence SEQ ID No. 1, SEQ ID No. 3, or SEQ ID No. 5.

[0070] As used herein, the term "identity" refers to the percentage of identical nucleotides or amino acids matched between at least two nucleotide or amino acid sequences aligned to each other using a normalization algorithm ("alignment"). This algorithm can insert gaps ("Gaps") in aligned sequences in a normalized and reproducible pattern to optimize the alignment between two sequences, thereby enabling a more meaningful comparison between the two sequences.

[0071] 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 present invention, identity is determined using the Basic Local Alignment Search Tool (BLAST) (Altschul et al., 1990), provided by the National Center for Biotechnology Information (NCBI). The BLAST software suite includes several programs, including a tool called “BLAST 2 Sequences,” which can be used for direct pairwise comparisons of two nucleotide or amino acid sequences. “BLAST 2 Sequences” is interactively accessible and used online via the NCBI World Wide Web. The blastn program (for nucleotide sequences) uses the following default parameters: word length (W) 11, expected value (E) 10, M=5, N=-4, and aligns two strands. For amino acid sequences, the blastp program uses the following default parameters: word length 3 and expected value (E) 10, BLOSUM62 score matrix (Henikoff & Henikoff, 1989), alignment (B) 50, expected value (E) 10, M=5, N=-4.

[0072] Alternatively, aldehyde dehydrogenase comprises an amino acid sequence encoded by a nucleic acid that binds to the complementary strand of a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 1, SEQ ID No. 3, or SEQ ID No. 5 under stringent conditions. As used herein, “stringent conditions” refers to conditions under which only so-called specific hybrids are formed and no non-specific hybrids are formed. For example, stringent conditions include hybridization at 45°C and 6×SSC (sodium chloride / sodium citrate), followed by washing at 50–65°C with 0.2–1×SSC and 0.1% SDS; or such conditions may include hybridization at 65–70°C and 1×SSC, followed by washing at 65–70°C with 0.3×SSC. Hybridization can be performed by conventional known methods, such as those described by J. Sambrook et al. in *Molecular Cloning: A Laboratory Manual* (2nd edition, Cold Spring Harbor Laboratory, 1989).

[0073] This invention also relates to the application of aldehyde dehydrogenase, wherein the amino acid sequence comprising the aldehyde dehydrogenase is selected from the group consisting of:

[0074] i) Has an amino acid sequence that is at least 80% identical to SEQ ID No. 2, SEQ ID No. 4, or SEQ ID No. 6;

[0075] ii) An amino acid sequence encoded by a nucleic acid having at least 80% identity with SEQ ID No. 1, SEQ ID No. 3, or SEQ ID No. 5; and

[0076] iii) An amino acid sequence encoded by a nucleic acid that binds to the complementary strand of a nucleic acid molecule having a nucleic acid sequence SEQ ID No. 1, SEQ ID No. 3 or SEQ ID No. 5 under strict conditions.

[0077] SEQ ID No. 7:

[0078]

[0079] SEQ ID No. 8:

[0080]

[0081] The xylitol dehydrogenase described herein comprises an amino acid sequence having at least 80%, more preferably 85%, more preferably 90%, more preferably 95%, more preferably 98%, more preferably 99%, and particularly 100% identity with SEQ ID No. 8.

[0082] Alternatively, the xylitol dehydrogenase comprises an amino acid sequence encoded by a nucleic acid having at least 80%, more preferably 85%, more preferably 90%, more preferably 95%, more preferably 98%, more preferably 99%, and particularly 100% identity with SEQ ID No. 7. Particularly preferably, the nucleic acid encoding the xylitol dehydrogenase according to the invention comprises or is composed of the nucleic acid sequence SEQ ID No. 7.

[0083] Alternatively, xylitol dehydrogenase comprises an amino acid sequence encoded by a nucleic acid bound to the complementary strand of a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 7 under stringent conditions. As used herein, “stringent conditions” refers to conditions under which only so-called specific hybrids are formed and no non-specific hybrids are formed. For example, stringent conditions include hybridization at 45°C and 6×SSC (sodium chloride / sodium citrate), followed by washing at 50–65°C with 0.2–1×SSC and 0.1% SDS; or such conditions may include hybridization at 65–70°C and 1×SSC, followed by washing at 65–70°C with 0.3×SSC. Hybridization can be performed by conventional known methods, such as those described by J. Sambrook et al. in *Molecular Cloning: A Laboratory Manual* (2nd edition, Cold Spring Harbor Laboratory, 1989).

[0084] This invention also relates to the use of xylitol dehydrogenase for the enzymatic oxidation of NAD(P)H by the production of D-sorbitol from D-fructose, wherein the xylitol dehydrogenase comprises an amino acid sequence selected from the group consisting of:

[0085] i) Has an amino acid sequence that is at least 80% identical to SEQ ID No. 8;

[0086] ii) An amino acid sequence encoded by a nucleic acid having at least 80% identity with SEQ ID No. 7; and

[0087] iii) An amino acid sequence encoded by a nucleic acid that binds to the complementary strand of a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 7 under stringent conditions.

[0088] D-fructose was used to oxidize NAD(P)H to NAD(P). + The substrate of xylitol dehydrogenase, an enzyme of dehydrogenase.

[0089] SEQ ID No. 9:

[0090]

[0091] SEQ ID No. 10:

[0092]

[0093] The NAD(P)H-dependent alcohol dehydrogenase described herein comprises an amino acid sequence having at least 80%, more preferably 85%, more preferably 90%, more preferably 95%, more preferably 98%, more preferably 99%, and particularly 100% identity with SEQ ID No. 10.

[0094] Alternatively, the NAD(P)H-dependent alcohol dehydrogenase comprises an amino acid sequence encoded by a nucleic acid having at least 80%, more preferably 85%, more preferably 90%, more preferably 95%, more preferably 98%, more preferably 99%, and particularly 100% identity with SEQ ID No. 9. Particularly preferably, the nucleic acid encoding the NAD(P)H-dependent alcohol dehydrogenase according to the invention comprises or is composed of the nucleic acid sequence SEQ ID No. 9.

[0095] Alternatively, the NAD(P)H-dependent alcohol dehydrogenase comprises an amino acid sequence encoded by a nucleic acid bound to the complementary strand of a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 9 under stringent conditions. As used herein, “stringent conditions” refers to conditions under which only so-called specific hybrids are formed and no non-specific hybrids are formed. For example, stringent conditions include hybridization at 45°C and 6×SSC (sodium chloride / sodium citrate), followed by washing at 50–65°C with 0.2–1×SSC and 0.1% SDS; or such conditions may include hybridization at 65–70°C and 1×SSC, followed by washing at 65–70°C with 0.3×SSC. Hybridization can be performed by conventional known methods, such as those described by J. Sambrook et al. in *Molecular Cloning: A Laboratory Manual* (2nd edition, Cold Spring Harbor Laboratory, 1989).

[0096] This invention also relates to the use of an NAD(P)H-dependent alcohol dehydrogenase for the enzymatic oxidation of NAD(P)H by the conversion of ketones or aldehydes into alcohols, wherein the alcohol dehydrogenase comprises an amino acid sequence selected from the group consisting of:

[0097] i) Has an amino acid sequence that is at least 80% identical to SEQ ID No. 10;

[0098] ii) An amino acid sequence encoded by a nucleic acid having at least 80% identity with SEQ ID No. 9; and

[0099] iii) An amino acid sequence encoded by a nucleic acid that binds to the complementary strand of a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 9 under stringent conditions.

[0100] Acetone was used to oxidize NAD(P)H to NAD(P). + The substrate of NAD(P)H-dependent alcohol dehydrogenase.

[0101] SEQ ID No. 11:

[0102]

[0103] SEQ ID No. 12:

[0104]

[0105] SEQ ID No. 13:

[0106]

[0107] SEQ ID No. 14:

[0108]

[0109] The xylose reductase described herein comprises an amino acid sequence having at least 80%, more preferably 85%, more preferably 90%, more preferably 95%, more preferably 98%, more preferably 99%, and particularly 100% identity with SEQ ID No. 12 or SEQ ID No. 14.

[0110] Alternatively, the xylose reductase comprises an amino acid sequence encoded by a nucleic acid having at least 80%, more preferably 85%, more preferably 90%, more preferably 95%, more preferably 98%, more preferably 99%, and particularly 100% identity with SEQ ID No. 11 or SEQ ID No. 13. Particularly preferably, the nucleic acid encoding the xylose reductase according to the invention comprises or is composed of the nucleic acid sequence SEQ ID No. 11 or SEQ ID No. 13.

[0111] Alternatively, xylose reductase comprises an amino acid sequence encoded by a nucleic acid that binds to the complementary strand of a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 11 or SEQ ID No. 13 under stringent conditions. As used herein, “stringent conditions” refers to conditions under which only so-called specific hybrids are formed and no non-specific hybrids are formed. For example, stringent conditions include hybridization at 45°C and 6×SSC (sodium chloride / sodium citrate), followed by washing at 50–65°C with 0.2–1×SSC and 0.1% SDS; or such conditions may include hybridization at 65–70°C and 1×SSC, followed by washing at 65–70°C with 0.3×SSC. Hybridization can be performed by conventional known methods, such as those described by J. Sambrook et al. in *Molecular Cloning: A Laboratory Manual* (2nd edition, Cold Spring Harbor Laboratory, 1989).

[0112] This invention also relates to the application of xylose reductase, wherein the xylose reductase comprises an amino acid sequence selected from the group consisting of:

[0113] i) Has an amino acid sequence that is at least 80% identical to SEQ ID No. 12 or SEQ ID No. 14;

[0114] ii) An amino acid sequence encoded by a nucleic acid having at least 80% identity with SEQ ID No. 11 or SEQ ID No. 13; and

[0115] iii) An amino acid sequence encoded by a nucleic acid that binds to the complementary strand of a nucleic acid molecule having a nucleic acid sequence SEQ ID No. 11 or SEQ ID No. 13 under stringent conditions.

[0116] D-glucose or L-arabinose are used as the oxidant for NAD(P)H to NAD(P). + The substrate of xylose reductase, a dehydrogenase.

[0117] SEQ ID No. 15:

[0118]

[0119] SEQ ID No. 16:

[0120]

[0121] The alcohol dehydrogenase described herein comprises an amino acid sequence having at least 80%, more preferably 85%, more preferably 90%, more preferably 95%, more preferably 98%, more preferably 99%, and particularly 100% identity with SEQ ID No. 16.

[0122] Alternatively, the alcohol dehydrogenase comprises an amino acid sequence encoded by a nucleic acid having at least 80%, more preferably 85%, more preferably 90%, more preferably 95%, more preferably 98%, more preferably 99%, and particularly 100% identity with SEQ ID No. 15. Particularly preferably, the nucleic acid encoding the alcohol dehydrogenase according to the invention comprises or is composed of the nucleic acid sequence SEQ ID No. 15.

[0123] Alternatively, the alcohol dehydrogenase comprises an amino acid sequence encoded by a nucleic acid bound to the complementary strand of a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 15 under stringent conditions. As used herein, “stringent conditions” refers to conditions under which only so-called specific hybrids are formed and no non-specific hybrids are formed. For example, stringent conditions include hybridization at 45°C and 6×SSC (sodium chloride / sodium citrate), followed by washing at 50–65°C with 0.2–1×SSC and 0.1% SDS; or such conditions may include hybridization at 65–70°C and 1×SSC, followed by washing at 65–70°C with 0.3×SSC. Hybridization can be performed by conventional known methods, such as those described by J. Sambrook et al. in *Molecular Cloning: A Laboratory Manual* (2nd edition, Cold Spring Harbor Laboratory, 1989).

[0124] This invention also relates to the use of alcohol dehydrogenases, wherein the alcohol dehydrogenase comprises an amino acid sequence selected from the group consisting of:

[0125] i) Has an amino acid sequence that is at least 80% identical to SEQ ID No. 16;

[0126] ii) An amino acid sequence encoded by a nucleic acid having at least 80% identity with SEQ ID No. 15; and

[0127] iii) An amino acid sequence encoded by a nucleic acid that binds to the complementary strand of a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 15 under strict conditions.

[0128] Dihydroxyacetone was used as the agent for oxidizing NAD(P)H to NAD(P). + The substrate of alcohol dehydrogenase.

[0129] Mannitol dehydrogenases used for the enzymatic oxidation of NAD(P)H preferably contain an amino acid sequence selected from or composed of the following:

[0130] i) Has an amino acid sequence that is at least 80% identical to SEQ ID No. 18;

[0131] ii) An amino acid sequence encoded by a nucleic acid having at least 80% identity with SEQ ID No. 17; and

[0132] iii) An amino acid sequence encoded by a nucleic acid that binds to the complementary strand of a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 17 under strict conditions.

[0133] SEQ ID No. 17:

[0134]

[0135] SEQ ID No. 18:

[0136]

[0137] The mannitol dehydrogenase described herein preferably comprises an amino acid sequence having at least 80%, more preferably 85%, more preferably 90%, more preferably 95%, more preferably 98%, more preferably 99%, and particularly 100% identity with SEQ ID No. 18.

[0138] Alternatively, the mannitol dehydrogenase preferably comprises an amino acid sequence encoded by a nucleic acid having at least 80%, more preferably 85%, more preferably 90%, more preferably 95%, more preferably 98%, more preferably 99%, and particularly 100% identity with SEQ ID No. 17. Particularly preferably, the nucleic acid encoding the mannitol dehydrogenase according to the invention comprises or is composed of the nucleic acid sequence SEQ ID No. 17.

[0139] Alternatively, the mannitol dehydrogenase preferably comprises an amino acid sequence encoded by a nucleic acid that binds to the complementary strand of a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 17 under stringent conditions. As used herein, “stringent conditions” refers to conditions under which only so-called specific hybrids are formed and no non-specific hybrids are formed. For example, stringent conditions include hybridization at 45°C and 6×SSC (sodium chloride / sodium citrate), followed by washing at 50–65°C with 0.2–1×SSC and 0.1% SDS; or such conditions may include hybridization at 65–70°C and 1×SSC, followed by washing at 65–70°C with 0.3×SSC. Hybridization can be performed by conventional known methods, such as those described by J. Sambrook et al. in *Molecular Cloning: A Laboratory Manual* (2nd edition, Cold Spring Harbor Laboratory, 1989).

[0140] This invention also relates to the use of mannitol dehydrogenase, wherein the mannitol dehydrogenase comprises an amino acid sequence selected from the group consisting of:

[0141] i) Has an amino acid sequence that is at least 80% identical to SEQ ID No. 18;

[0142] ii) An amino acid sequence encoded by a nucleic acid having at least 80% identity with SEQ ID No. 17; and

[0143] iii) An amino acid sequence encoded by a nucleic acid that binds to the complementary strand of a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 17 under strict conditions.

[0144] D-fructose was used to oxidize NAD(P)H to NAD(P). + The substrate of mannitol dehydrogenase.

[0145] In a preferred embodiment of the invention, in the method according to the invention, NAD(P) having an amino acid sequence having at least 80% identity with SEQ ID No. 2 is used. + The enzyme combines an aldehyde-dependent dehydrogenase with a xylitol dehydrogenase having an amino acid sequence that is at least 80% identical to that of SEQ ID No. 8.

[0146] In a preferred embodiment of the invention, in the method according to the invention, NAD(P) having an amino acid sequence having at least 80% identity with SEQ ID No. 4 is used. + The enzyme combines an aldehyde-dependent dehydrogenase with a xylitol dehydrogenase having an amino acid sequence that is at least 80% identical to that of SEQ ID No. 8.

[0147] In a preferred embodiment of the invention, in the method according to the invention, NAD(P) having an amino acid sequence having at least 80% identity with SEQ ID No. 6 is used. + The enzyme combines an aldehyde-dependent dehydrogenase with a xylitol dehydrogenase having an amino acid sequence that is at least 80% identical to that of SEQ ID No. 8.

[0148] In a preferred embodiment of the invention, in the method according to the invention, NAD(P) having an amino acid sequence having at least 80% identity with SEQ ID No. 2 is used. + The enzyme combines an aldehyde-dependent dehydrogenase and an NAD(P)H-dependent alcohol dehydrogenase having an amino acid sequence that is at least 80% identical to that of SEQ ID No. 10.

[0149] In a preferred embodiment of the invention, in the method according to the invention, NAD(P) having an amino acid sequence having at least 80% identity with SEQ ID No. 4 is used. + The enzyme combines an aldehyde-dependent dehydrogenase and an NAD(P)H-dependent alcohol dehydrogenase having an amino acid sequence that is at least 80% identical to that of SEQ ID No. 10.

[0150] In a preferred embodiment of the invention, in the method according to the invention, NAD(P) having an amino acid sequence having at least 80% identity with SEQ ID No. 6 is used. + The enzyme combines an aldehyde-dependent dehydrogenase and an NAD(P)H-dependent alcohol dehydrogenase having an amino acid sequence that is at least 80% identical to that of SEQ ID No. 10.

[0151] In other preferred embodiments of the method according to the invention, the concentration of FFA in the aqueous solution is 5-100 g / l.

[0152] The preferred temperature range is between 15 and 40°C.

[0153] The preferred reaction pH range is between pH 5 and pH 9.

[0154] In a preferred variant of the method according to the invention, the enzyme is present in an aqueous suspension, homogenate, and / or lysate of the corresponding cell that produces the enzyme, particularly preferably in the lysate.

[0155] In this context, "homogeneous material" refers to a suspension that has undergone physical and / or chemical treatment (e.g., by pressure, lysozyme, or ultrasound) in which cellular components are released from the cells. When insoluble cellular components are removed from the homogenate (e.g., by filtration or centrifugation), a "lysate" is obtained (see Enzyme Preparation and Lysate Preparation for details).

[0156] In other variants, the enzyme may be modified with a water-soluble polymer (such as polyethylene glycol) at its N-terminus, immobilized in or on a solid matrix, or as part of a fusion protein.

[0157] In other variants, the enzyme may be present in powder, lyophilized, or spray-dried form.

[0158] Preferred embodiments of the present invention are described in more detail below with reference to the examples.

[0159] Material

[0160] 5-Formyl-2-furanoic acid (FFA) and 2,5-furandicarboxylic acid (FDCA) were purchased from TCI; acetone, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium dodecyl sulfate (SDS), and D-sorbitol were purchased from Carl Roth; NAD + Disodium NADH, NADP + Disodium salt, tetrasodium salt of NADPH, acetonitrile and D-fructose were purchased from PanReac AppliChem (ITW Reagents); triethanolamine was purchased from Chem-Lab NV.

[0161] Enzyme preparation and preparation of lysates

[0162] Overview of recombinase expression in Escherichia coli

[0163] To prepare recombinant enzymes in *E. coli* strains, genomic DNA or its synthetic equivalent adapted to *E. coli* codons was first used as a template, along with specific oligonucleotides carrying restriction endonuclease recognition sequences, to amplify the gene to be expressed via PCR; the gene was then isolated from the reaction mixture. After digestion with restriction enzymes SphI and HindIII, the gene fragment encoding the target enzyme was ligated into the backbone of the SphI and HindIII-digested expression vector pQE70-Kan. The ligation product was transformed into chemically competent *E. coli* Top10F cells, and the resulting colonies were used for plasmid isolation and restriction analysis.

[0164] The cloning results were verified by restriction enzyme digestion and DNA sequencing. The resulting construct carried the target gene under the IPTG-inducible T5 promoter.

[0165] 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 testing.

[0166] The following day, inoculation with optical density OD 550 The expression culture was at 0.02 mg / L and shaken at 37°C until the OD value reached 0.02%. 550 Reaching 0.3. Then the temperature was lowered to 25°C, when OD... 550 When the concentration reached 0.5, the culture was induced with 0.1 mM IPTG. After 22 hours, the culture was harvested (separated from the culture medium as a cell pellet by centrifugation) and the expression of the recombinase was analyzed using SDS gel electrophoresis and activity assays (using the Use-Test or optical enzymatic assay).

[0167] Cell lysates were prepared by ultrasonic disruption.

[0168] To prepare the cell suspension, weigh the cell pellet prepared according to the above method and place it in a suitable container. Mix it with buffer and lysozyme (final concentration 0.5 mg / ml) (e.g., triethanolamine (TEA)-HCl) and stir to dissolve. The biomass mass fraction is usually 20%, with the remainder being buffer.

[0169] Cell disruption was performed using a Branson Sonifier 450. The suspension was sonicated three times, with 15 ultrasonic pulses each time (device settings: timer=15; duty cycle=50; output control=3-5).

[0170] The resulting homogenate was centrifuged at 4°C and 16,000 rpm for 10 minutes (Eppendorf Centrifuge 5417R) to separate insoluble cell debris and obtain lysate.

[0171] Table 1. Enzyme types and donor organisms used in the examples (ALDH = aldehyde dehydrogenase)

[0172]

[0173] This ALDH is classified as 2,5-dioxovalerate dehydrogenase (catalyzing the oxidation of 2,5-dioxovalerate to α-ketoglutarate) in the NCBI protein database (accession number WP_009990943.1).

[0174] Analytical methods

[0175] High-performance liquid chromatography (HPLC)

[0176] Quantification of FFA and FDCA was performed using HPLC (High Performance Liquid Chromatography). Detection was performed using a UV detector. Measurements were performed using a Phenomenex Rezex ROA-Organic Acid H+ (8%) column with a corresponding guard column, eluted isocratically with 1 mM sulfuric acid.

[0177] D-fructose and D-sorbitol were quantitatively analyzed using high-performance liquid chromatography (HPLC). Detection was performed using a refractive index detector. Measurements were taken using a Phenomenex Rezex RCM-monosaccharide Ca2+ column with a corresponding guard column, eluted isocratically with 3.5% isopropanol.

[0178] Enzyme activity determination (optical enzymatic assay)

[0179] Enzyme activity in the lysates was determined using a Shimadzu UV-1900 spectrophotometer. For this purpose, the generation or consumption of NAD(P)H was tracked by absorbance changes at 340 nm. Measurements were performed using 0.2 mM cofactor (NAD(P)H). + The measurement was performed using either NADH / NADPH. For this purpose, 20 μl of 10 mM cofactor stock solution was added to a cuvette (Greiner bio-one polystyrene semi-micro cuvette), and the pH was adjusted to the desired value with 870 μl of 100 mM TEA-HCl buffer. 10 μl of lysate (diluted or undiluted) and 100 μl of substrate solution were added to the cuvette, and measurements were started immediately thereafter. Measurements were performed at a standard temperature of 25 °C. The extinction coefficient (ε = 6220 L·mol⁻¹) of NADH / NADPH at 340 nm was used as the criterion. -1 ·cm -1The enzyme activity of the lysate can be determined, expressed in U / ml (based on lysate volume) or U / g (based on the biomass used in preparation). 1 U represents the conversion of 1 μmol of substrate per minute (1 U = 1 μmol / min = 1.67 × 10⁻⁶). -8 kat).

[0180] Preferred variations of the method according to the invention are described in more detail below through examples. The pyrolytes used in these examples are all prepared by the method described above.

[0181] Example 1

[0182] Oxidation of 5-formyl-2-furanoic acid to 2,5-furandicarboxylic acid—using XDH and D-fructose as cofactor regeneration.

[0183] The reaction was carried out in a multi-module BioXplorer benchtop bioreactor (HEL). A stainless steel reactor (maximum volume 400 ml) equipped with a stirrer and pH electrode was used as the vessel. The pH was controlled by adding 5M NaOH or 1M H2SO4.

[0184] Initially, 5.5 g FFA and 16 g D-fructose (final concentration 600 mM) were added to 100 ml of 100 mM potassium phosphate buffer (pH 7), and the mixture was heated to 20°C with stirring. Then, 20 ml of ALDH I lysate, 13 ml of XDH lysate, and 3 ml of 10 mM NAD were added. + Solution (final concentration 0.2 mM). Adjust the total volume of the reaction mixture to 150 ml by adding deionized water.

[0185] For analysis, 50 μl of the mixture was combined with 200 μl of acetonitrile and incubated in an Eppendorf thermostatic mixer at 85 °C and 1200 rpm for 15 min. The sample was briefly centrifuged, mixed with 750 μl of deionized water and vortexed, then centrifuged at maximum centrifugation force (max. g.) for 5 min. 250 μl of the supernatant was diluted in an HPLC vial with 750 μl of acetonitrile / water mixture (1 / 4 v / v) and measured by HPLC (UV detection).

[0186] Four hours later, FFA was completely oxidized to FDCA.

[0187] For post-processing, the reactor contents were heated to 70°C and stirred at this temperature for 1 hour. After centrifugation to remove denatured proteins, the supernatant was filtered through a pleated filter. This yielded a clear solution, which was acidified to pH < 2 with 10 ml of 12 M H₂SO₄ solution. After cooling to 4°C, a precipitate formed, which was collected by filtration. In this way, 5.1 g of FDCA could be separated as a solid.

[0188] Example 2

[0189] Oxidation of 5-formyl-2-furancarboxylic acid to 2,5-furandicarboxylic acid—cofactor regeneration using ADH and acetone.

[0190] The reaction was carried out in a Labfors benchtop bioreactor (Infors AG). A glass reactor (3.4 L volume) equipped with a stirrer and pH electrode was used as the vessel. The pH was controlled by adding 5 M NaOH or 1 M H2SO4.

[0191] Initially, 27.7 g of FFA was placed in 550 ml of 100 mM potassium phosphate buffer (pH 7), and the solution was heated to 20°C with stirring. Then, 67 ml of ALDH I lysate, 30 ml of ADH lysate, and 10 ml of 10 mM NAD were added. + Solution (final concentration 0.2 mM) and 15 ml of acetone. In addition, an overpressure of 320 mbar was applied.

[0192] For analysis, 50 μl of the mixture was combined with 200 μl of acetonitrile and incubated in an Eppendorf thermostatic mixer at 85 °C and 1200 rpm for 15 min. The sample was briefly centrifuged, mixed with 750 μl of deionized water, vortexed, and then centrifuged at maximum centrifugation for 5 min. 250 μl of the supernatant was diluted in an HPLC vial with a 750 μl acetonitrile / water mixture (1 / 4 v / v) and measured by HPLC (UV detection).

[0193] After 4.5 hours, FFA was completely oxidized to FDCA. Similar to Example 1, FDCA could be separated as a solid.

[0194] Example 3

[0195] 5-Formyl-2-furanoic acid was oxidized to 2,5-furandicarboxylic acid using aldehyde dehydrogenase II (ALDH II), and cofactor regeneration was performed using XDH and D-fructose.

[0196] Mix the following components in a glass vial: 300 μl FFA solution (11.9 g / l), 10 μl deionized water, 50 μl ALDH II lysate, 50 μl 1 M potassium phosphate buffer (pH 8), 50 μl 1.5 M D-fructose solution, and 40 μl XDH lysate. Incubate the mixture with continuous shaking (Eppendorf thermostatic mixer, 20 °C, 800 rpm) for a total of 20 hours.

[0197] For analysis, 50 μl of the mixture was combined with 200 μl of acetonitrile and incubated in an Eppendorf thermostatic mixer at 85 °C and 1200 rpm for 15 min. The sample was briefly centrifuged, mixed with 750 μl of deionized water, vortexed, and then centrifuged at maximum centrifugation for 5 min. 200 μl of the supernatant was transferred to an HPLC vial with an inner tube for measurement by HPLC (UV detection).

[0198] In this manner, 40.8% of FFA (7.1 g / l) was oxidized to FDCA.

[0199] Example 4

[0200] 5-Formyl-2-furanoic acid was oxidized to 2,5-furandicarboxylic acid using aldehyde dehydrogenase III (ALDH III), and cofactor regeneration was performed using ADH and acetone.

[0201] Mix the following components in a glass vial: 5.2 mg FFA, 325 μl deionized water, 35 μl ALDH III lysate, 100 μl 500 mM potassium phosphate buffer (pH 8), 15 μl acetone, 30 μl ADH I lysate, and 5 μl 10 mM NAD. + Solution. Incubate the mixture with continuous shaking (Eppendorf thermostatic mixer, 20°C, 800 rpm) for a total of 24 hours.

[0202] For analysis, 50 μl of the mixture was combined with 200 μl of acetonitrile and incubated in an Eppendorf thermostatic mixer at 85 °C and 1200 rpm for 15 min. The sample was briefly centrifuged, mixed with 750 μl of deionized water, vortexed, and then centrifuged at maximum centrifugation for 5 min. 200 μl of the supernatant was transferred to an HPLC vial with an inner tube for measurement by HPLC (UV detection).

[0203] In this manner, 48.0% of FFA (10.4 g / l) was oxidized to FDCA.

[0204] Example 5

[0205] 5-Formyl-2-furanoic acid was oxidized to 2,5-furandicarboxylic acid using aldehyde dehydrogenase II (ALDH II) and various dehydrogenases for cofactor regeneration.

[0206] Mix the following components in three glass vials (vials 1-3): 300 μl FFA solution (final concentration 7.1 g / L), 50 μl 1 M potassium phosphate buffer (pH 8), 50 μl ALDH II lysate, and 5 μl 10 mM NADP. + The mixture contained 50 μl of dehydrogenase lysate (see Table 2 below), 50 μl of 1.5 M substrate solution (see Table 2 below), and 5 μl of deionized water. The mixture was incubated with continuous shaking (Eppendorf thermostatic mixer, 20°C, 800 rpm) for a total of 24 hours.

[0207] For analysis, 50 μl of the mixture was combined with 200 μl of acetonitrile and incubated in an Eppendorf thermostatic mixer at 85 °C and 1200 rpm for 15 min. The sample was briefly centrifuged, mixed with 750 μl of deionized water, vortexed, and then centrifuged at maximum centrifugation for 5 min. 200 μl of the supernatant was transferred to an HPLC vial with an inner tube for measurement by HPLC (UV detection).

[0208] The results are shown in Table 2 below.

[0209] Table 2

[0210]

[0211] The results in Table 2 show that various dehydrogenases and different substrates are suitable for cofactor regeneration (NADP in this example). + ).

[0212] Example 6

[0213] 5-Formyl-2-furanoic acid was oxidized to 2,5-furandicarboxylic acid using aldehyde dehydrogenase II (ALDH II), and cofactor regeneration was performed using ADH and dihydroxyacetone.

[0214] Mix the following components in a glass vial: 143 μl FFA solution (final concentration 12 g / L), 125 μl 250 mM potassium phosphate buffer (pH 7), 10 μl ALDH II lysate, and 10 μl 5 mM NADP. +The mixture contained 10 μl of dehydrogenase lysate (see Table 2 below), 50 μl of 300 g / L dihydroxyacetone solution, and 152 μl of deionized water. The mixture was incubated with continuous shaking (Eppendorf thermostatic mixer, 30°C, 800 rpm) for a total of 20 hours.

[0215] For analysis, 50 μl of the mixture was combined with 200 μl of acetonitrile and incubated in an Eppendorf thermostatic mixer at 85 °C and 1200 rpm for 15 min. The sample was briefly centrifuged, mixed with 750 μl of deionized water, vortexed, and then centrifuged at maximum centrifugation for 5 min. 200 μl of the supernatant was transferred to an HPLC vial with an inner tube for measurement by HPLC (UV detection).

[0216] In this way, 66% of FFA is oxidized to FDCA.

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[0251] Protein [Internet]. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information. Accession No. CAA46053.1, alcohol dehydrogenase [Thermoanaerobacter brockii]. Available from: https: / / www.ncbi.nlm.nih.gov / protein / CAA46053.1

Claims

1. A method for preparing 2,5-furandicarboxylic acid, the method comprising in vitro oxidizing 5-formyl-2-furancarboxylic acid present in aqueous solution to 2,5-furandicarboxylic acid by treatment with an aldehyde dehydrogenase, wherein a dehydrogenase is used to enzymatically oxidize NAD(P)H generated during oxidation back to NAD(P) using D-fructose or acetone as a substrate. + ; then the enzyme is removed, characterized in that The following combination of measures: NAD(P) used to oxidize 5-formyl-2-furancarboxylic acid to 2,5-furandicarboxylic acid. + Aldehyde-dependent dehydrogenases have amino acid sequences selected from the following groups: i) Has an amino acid sequence that is at least 80% identical to SEQ ID No. 2, SEQ ID No. 4, or SEQ ID No. 6; ii) An amino acid sequence encoded by a nucleic acid having at least 80% identity with SEQ ID No. 1, SEQ ID No. 3, or SEQ ID No. 5; and iii) An amino acid sequence encoded by a nucleic acid that binds to the complementary strand of a nucleic acid molecule having a nucleic acid sequence of SEQ ID No. 1, SEQ ID No. 3, or SEQ ID No. 5 under stringent conditions. Furthermore, it involves oxidizing NAD(P)H to NAD(P). + The dehydrogenase used is: a) Xylitol dehydrogenases having an amino acid sequence selected from the following groups: i) Has an amino acid sequence that is at least 80% identical to SEQ ID No. 8; ii) An amino acid sequence encoded by a nucleic acid having at least 80% identity with SEQ ID No. 7; and iii) An amino acid sequence encoded by a nucleic acid that binds to the complementary strand of a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 7 under stringent conditions. D-fructose was used as the substrate. or b) NAD(P)H-dependent alcohol dehydrogenases having an amino acid sequence selected from the following groups: i) Has an amino acid sequence that is at least 80% identical to SEQ ID No. 10; ii) An amino acid sequence encoded by a nucleic acid having at least 80% identity with SEQ ID No. 9; and iii) An amino acid sequence encoded by a nucleic acid that binds to the complementary strand of a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 9 under stringent conditions. Acetone is used as the substrate.

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