Process for preparation of 2, 5-furandicarboxylic acid

Abstract

The invention also relates to a method for preparing 2, 5-furandicarboxylic acid. The invention relates to a method for the in vitro oxidation of 5-formyl-2-furancarboxylic acid present in an aqueous solution to 2, 5-furandicarboxylic acid by treatment with an NAD (P) +-dependent oxidoreductase, in which NAD (P) H generated during the oxidation is enzymatically oxidized back to NAD (P) + by means of a dehydrogenase; and then removing the enzyme.

Description

TECHNICAL FIELD

[0001] The present invention relates to a process for the production of 2,5-furan dicarboxylic acid (FDCA) from 5-formyl-2-furancarboxylic acid (FFA). BACKGROUND

[0002] In 2018, about 90% of the globally produced plastics (400 million tons) were based on fossil feedstocks, the remaining fractions were from recycled plastics (9%), biobased plastics (1%) and carbon dioxide-based plastics (<1%) (Carus et al. 2020). At the same time, the global demand for plastics continues to grow. In 2019, the annual CO2 emissions of the entire life cycle of plastics amounted to 0.86 Gt, which corresponds to the CO2 emissions of 189 coal-fired power plants operating at full capacity (500 MW). It is predicted that by 2050, the emissions will increase to 2.8 Gt (corresponding to 615 coal-fired power plants) (Hamilton et al. 2019).

[0003] Polyethylene terephthalate (PET) is one of the plastics based on fossil feedstocks and is mainly used in the form of beverage packaging. It is a polycondensation polymer which is produced from terephthalic acid (1,4-benzenedicarboxylic acid) and ethylene glycol (ethane-1,2-diol) by dehydration.

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

[0005] Ethylene glycol is produced by hydrolysis of ethylene oxide (at 200 °C), which in turn is obtained from the oxidation of ethylene (derived from fossil feedstocks) (Berger 2016). Due to the fossil origin of the starting materials (p-xylene and ethylene), PET cannot usually be described as sustainable.

[0006] To achieve the 1.5 °C target of the Paris Climate Agreement and thus limit the negative effects of climate change, sustainable alternatives must be found for petroleum-chemical-based plastics. For example, for ethylene glycol, processes based on renewable resources exist. By way of example, ethylene can also be produced by dehydration of bioethanol obtained in a fermentative manner from glucose or starch (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 make from sustainable feedstocks and therefore needs to be replaced. 2,5-furan dicarboxylic acid (FDCA) is an excellent replacement for terephthalic acid, which is mainly obtained from catalytic upgrading of biomass. Polymerization of FDCA with ethylene glycol yields PEF (polyethylene furanoate), which has a heteroaromatic furan ring instead of a benzene ring in its structure. PEF is a thermoplastic like PET, but features significantly higher biodegradability, as well as superior thermal (higher glass transition temperature, lower melting temperature) and mechanical properties (higher stiffness) compared to PET. The most important property of PEF is its reduced permeability for gases such as O2 and CO2. This is particularly important for beverages, as it enables longer shelf life (prevents de-gassing of carbonated beverages; prevents oxidation processes induced by diffusing oxygen) (de Jong et al., 2022).

[0008] A review of various chemical syntheses can be found in the article of 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 prepared via enzymatic or chemical hydrolysis to D-glucose, which is subsequently isomerized (enzymatically or chemically) to D-fructose. D-fructose is converted to HMF by dehydration (loss of three water molecules in total). Common systems for fructose dehydration include inorganic acids (such as sulfuric acid or hydrochloric acid) on the one hand and (Bronsted or Lewis) acidic solid catalysts on the other hand (Cong et al., 2021; US 9617234 B1).

[0010] HMF has both alcohol and aldehyde functional groups, which must be oxidized to carboxylic acid groups to obtain FDCA. The three oxidation steps that are necessary can be performed in various ways: chemical ways using heterogeneous or homogeneous catalysts, electrochemical ways, and biocatalytic (enzymatic ways or with whole cells) ways.

[0011] The direct precursor of FDCA is FFA (5-formyl-2-furancarboxylic acid), which can be prepared, for example, by acid-catalyzed dehydration from 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 acetic acid / water mixtures at 80 °C in a flow reactor. In the last step, FFA can be oxidized to FDCA with oxygen as oxidant and a metal (e.g. Pt-Ru / C) or metal salt (Co & Mn-salts) as catalyst, or with hydrogen peroxide (US 10087161 B2).

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

[0013] In general, the aforementioned chemical preparation processes of FDCA starting with FFA as intermediate are characterized by unfavorable reaction conditions (high temperature and pressure), the formation of by-products, the use of expensive and partly toxic catalysts (such as cobalt salts) and thus cannot be described as sustainable.

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

[0015] FFA can be prepared by enzymatic oxidation of HMF, mainly with O2 as oxidant.

[0016] One group of enzymes for the preparation of FFA from HMF are aryl-alcohol oxidases (AAO, EC 1.1.3.7). Carro et al. (2015) used an AAO from the fungus Pleurotus eryngii to oxidize 3 mM HMF to FFA (98 mol%) and a small amount of FDCA within 4 hours.

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

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

[0019] Qin et al. (2015) tested various laccases with 20 mol% TEMPO ((2,2,6,6-tetramethylpiperidin-1-yl)oxy) as mediator on 30 mM HMF. With laccase from the fungus Panus conchatus, 82% of the starting material could be oxidized to FFA within 96 h (with 4% of 2,5-diformylfuran (DFF) and 10% of FDCA as by-products).

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

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

[0022] US 10344307 B2 describes other enzyme systems for the oxidation of HMF to FFA: 1) NAD(P)-dependent ketone reductase and NAD(P)H oxidase (for cofactor regeneration); 2) NAD(P)-dependent aldehyde dehydrogenase and NAD(P)H oxidase; 3) xanthine oxidoreductase (such as periplasmic aldehyde oxidoreductase (PaoABC) from E. coli, which oxidizes HMF to 5-hydroxymethyl-2-furoic acid (HMFA) and DFF to FFA), galactose oxidase variant M 3-5 (GOase M 3-5Horseradish peroxidase (HRP, for activation of GOase M 3-5 For example, using PaoABC at pH 6, 50 mM DFF can be completely oxidized to FFA within 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 for the last oxidation step from FFA to FDCA are also known.

[0024] Cajnko et al. (2020) tested a series of commercially available enzymes (alcohol oxidase (AO) from Pichia pastoris, galactose oxidase from Dactylium dendroides, catalase from Aspergillus niger, laccase from Trametes versicolor, fungal lignin peroxidase (LPO), and HRP) on 10 mM FFA, only AO, laccase, and LPO could be observed to produce significant amounts of FDCA after 72 hours (AO, 11.6%; laccase, 1.1%; LPO, 3.2%). 5-Hydroxymethyl-2-furoic acid (HMFA) was found as a by-product (up to 18.2% for AO).

[0025] Another enzyme for the oxidation of FFA to FDCA is the non-specific peroxidase (UPO, EC 1.11.2.1, requires H2O2 as oxidant) from Agrocybe aegerita, which can be used in combination 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 the oxidation of FFA to FDCA, UPO also catalyzes the oxidation of HMF via DFF to FFA (Lappe et al., 2021).

[0026] Jia et al. (2017) used an enzyme system consisting of horse liver alcohol dehydrogenase (HLADH) and human hemoglobin (using H2O2 to oxidize NADH to NAD + ) to oxidize 96% of the substrate (10 mM FFA) to FDCA within 60 hours.

[0027] US 8183020 B2 describes the enzymatic oxidation of FFAs to FDCA using H2O2 as oxidant with a commercially available chloroperoxidase from Caldariomyces fumago (EC 1.11.1.10).

[0028] Aldehyde dehydrogenases (ALDHs) are enzymes that catalyze the oxidation of aldehyde groups to carboxylic acid groups. For the Raoultella ornithinolytica BF60 organism, an ALDH was described that can oxidize FFAs to FDCA and HMF to HMFA (Hossain et al. 2017).

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

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

[0031] US 10344307 B2 describes a method for the oxidation of DFF with a commercially available ALDH (in combination with an NAD(P)H oxidase) that produces FFAs, FDCA, or a mixture of both depending on the case (DFF concentration of 10 to 100 mM). With increasing DFF concentration, more FFA than FDCA is obtained (10 mM substrate: 100% FDCA; 50 mM substrate: 80% FDCA, 20% FFA; 100 mM substrate: 20% FDCA, 80% FFA (conversion after 3 hours each)). 10 mol% to 50 mol% of cofactor (based on substrate amount) was used for the oxidation in US 10344307 B2.

[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, 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 using NAD(P) + Treatment with a oxidoreductase-dependent enzyme oxidizes 5-formyl-2-furanoic acid (FFA) in aqueous solution to 2,5-furandicarboxylic acid (FDCA) in vitro; a dehydrogenase enzymatically oxidizes the NAD(P)H generated during oxidation back to NAD(P). + The enzyme is then removed. 2,5-Furandicarboxylic acid can then be separated from the solution by precipitation.

[0036] "In vitro" means that this method is carried out outside the organism, that is, not in intact cells, nor in fermentation.

[0037] Sugars (especially D-fructose) or aldehydes or ketones (especially acetone) are used as the oxidizing agents for NAD(P)H to NAD(P). + The substrate of dehydrogenase.

[0038] Appendix Figure 1 A preferred embodiment of the method according to the present invention is illustrated schematically.

[0039] NAD(P) used to oxidize FFA to FDCA + The preferred oxidoreductase for the dependent enzyme is an aldehyde dehydrogenase.

[0040] NAD(P) used to oxidize FFA to FDCA + The oxidoreductase-dependent enzyme preferably contains or consists of an amino acid sequence selected from or composed of the following:

[0041] i) Having an amino acid sequence that is at least 80% identical to SEQ ID No. 2, SEQ ID No. 8, SEQ ID No. 4, or SEQ ID No. 6;

[0042] ii) an amino acid sequence encoded by a nucleic acid having at least 80% identity with SEQ ID No. 1, SEQ ID No. 7, SEQ ID No. 3 or SEQ ID No. 5; and

[0043] iii) an amino acid sequence encoded by a nucleic acid which binds under stringent conditions to the complementary strand of a nucleic acid molecule having the nucleic acid sequence of SEQ ID No. 1, SEQ ID No. 7, SEQ ID No. 3 or SEQ ID No. 5.

[0044] SEQ ID No. 1 :

[0045]

[0046] SEQ ID No. 2:

[0047]

[0048] SEQ ID No. 3:

[0049]

[0050] SEQ ID No. 4:

[0051]

[0052] SEQ ID No. 5:

[0053]

[0054] SEQ ID No. 6:

[0055]

[0056] SEQ ID No. 7:

[0057]

[0058] SEQ ID No. 8:

[0059]

[0060] The oxidoreductase for oxidizing FFA to FDCA described herein preferably comprises an amino acid sequence which is at least 80%, more preferably 85%, more preferably 90%, more preferably 95%, more preferably 98%, more preferably 99%, in particular 100% identical to SEQ ID No. 2, SEQ ID No. 8, SEQ ID No. 4 or SEQ ID No. 6.

[0061] Alternatively, the oxidoreductase for oxidizing FFA to FDCA preferably comprises an amino acid sequence which is encoded by a nucleic acid which is at least 80%, more preferably 85%, more preferably 90%, more preferably 95%, more preferably 98%, more preferably 99%, in particular 100% identical to SEQ ID No. 1, SEQ ID No. 7, SEQ ID No. 3 or SEQ ID No. 5. Particularly preferably, the nucleic acid encoding the oxidoreductase for oxidizing FFA to FDCA according to the present application comprises or consists of the nucleic acid sequence SEQ ID No. 1, SEQ ID No. 7, SEQ ID No. 3 or SEQ ID No. 5.

[0062] As used herein, the term "identity" refers to the percentage of matching identical nucleotides or amino acids between at least two nucleotide sequences or amino acid sequences aligned with each other using a standardized algorithm ("alignment"). The algorithm can insert gaps ("Gaps") in the alignment sequence in a standardized and reproducible pattern to optimize the alignment between the two sequences, thereby enabling a more meaningful comparison of the two sequences.

[0063] 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 application, identity is determined using the Basic Local Alignment Search Tool (BLAST) provided by the National Center for Biotechnology Information (NCBI) (Altschul et al., 1990). The BLAST software suite comprises a variety of programs, including a tool called "BLAST 2 Sequences", which can be used for a direct pairwise comparison of two nucleotide sequences or amino acid sequences. "BLAST 2 Sequences" is accessible and usable online interactively via the NCBI World Wide Web site. The blastn program (for nucleotide sequences) uses the following default parameters: wordlength (W) 11, expectation (E) 10, M=5, N=-4, and aligns both strands. For amino acid sequences, the blastp program uses the following default parameters: wordlength 3 and expectation (E) 10, BLOSUM62 scoring matrix (Henikoff & Henikoff, 1989), alignment (B) 50, expectation (E) 10, M=5, N=-4.

[0064] Alternatively, the oxidoreductase for oxidizing FFA to FDCA preferably comprises an amino acid sequence encoded by a nucleic acid which binds under stringent conditions to the complementary strand of a nucleic acid molecule having the nucleic acid sequence of SEQ ID No. 1, SEQ ID No. 7, SEQ ID No. 3 or SEQ ID No. 5. As used herein, "stringent conditions" refer to conditions under which only so-called specific hybrids, but not non-specific hybrids, are formed. For example, stringent conditions include hybridization in 6x SSC (sodium chloride / sodium citrate) at 45°C, followed by washing with 0.2 to 1 x SSC, 0.1 % SDS at 50-65°C; or such conditions can include hybridization in 1 x SSC at 65-70°C, followed by washing with 0.3 x SSC at 65-70°C. Hybridization can be carried out by conventionally known methods, such as those described in J. Sambrook et al. in Molecular Cloning: A Laboratory Manual (2ndEdition, Cold Spring Harbor Laboratory, 1989).

[0065] The present application also relates to the use of an oxidoreductase for oxidizing FFA to FDCA, wherein the oxidoreductase comprises an amino acid sequence selected from the group consisting of:

[0066] i) having an amino acid sequence which is at least 80% identical to SEQ ID No. 2, SEQ ID No. 8, SEQ ID No. 4 or SEQ ID No. 6;

[0067] ii) an amino acid sequence encoded by a nucleic acid which is at least 80% identical to SEQ ID No. 1, SEQ ID No. 7, SEQ ID No. 3 or SEQ ID No. 5; and

[0068] iii) an amino acid sequence encoded by a nucleic acid which binds under stringent conditions to the complementary strand of a nucleic acid molecule having the nucleic acid sequence of SEQ ID No. 1, SEQ ID No. 7, SEQ ID No. 3 or SEQ ID No. 5.

[0069] According to a preferred embodiment, a sugar or an aldehyde or a ketone compound is used as a substrate for the dehydrogenase enzyme for the enzymatic oxidation of NAD(P)H.

[0070] Preferably, D-fructose is used as a sugar and acetone is used as a ketone compound.

[0071] In a further embodiment, D-glucose is used as a sugar and acetone is used as a ketone compound.

[0072] In a variant of the process according to the application, the enzymatic regeneration of NAD(P)H is accomplished using a xylitol dehydrogenase (XDH; EC 1.1.1.9) or a sorbitol dehydrogenase (EC 1.1.1.14, EC 1.1.1.15), the former being particularly preferred.

[0073] The xylitol dehydrogenase used for the enzymatic oxidation of NAD(P)H by generating D-sorbitol from D-fructose preferably comprises or consists of an amino acid sequence selected from the group consisting of:

[0074] i) an amino acid sequence having at least 80% identity to SEQ ID No. 20;

[0075] ii) an amino acid sequence encoded by a nucleic acid having at least 80% identity to SEQ ID No. 19; and

[0076] iii) an amino acid sequence encoded by a nucleic acid which binds under stringent conditions to the complementary strand of a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 19.

[0077] SEQ ID No. 19:

[0078]

[0079] SEQ ID No. 20:

[0080]

[0081] The xylitol 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%, in particular 100% identity to SEQ ID No. 20.

[0082] Alternatively, the xylitol 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%, in particular 100% identity to SEQ ID No. 19. Particularly preferably, the nucleic acid encoding the xylitol dehydrogenase according to the application comprises or consists of the nucleic acid sequence SEQ ID No. 19.

[0083] Alternatively, the xylitol dehydrogenase preferably comprises an amino acid sequence encoded by a nucleic acid which binds under stringent conditions to the complementary strand of a nucleic acid molecule having the nucleic acid sequence of SEQ ID No. 19. As used herein, "stringent conditions" refer to conditions under which only so-called specific hybrids, but not non-specific hybrids, are formed. For example, stringent conditions include hybridization in 6x SSC (sodium chloride / sodium citrate) at 45°C, followed by washing with 0.2 to 1 x SSC, 0.1% SDS at 50-65°C; or such conditions can include hybridization in 1 x SSC at 65-70°C, followed by washing with 0.3 x SSC at 65-70°C. Hybridization can be carried out by conventionally known methods, such as those described in J. Sambrook et al. in "Molecular Cloning: A Laboratory Manual" (2nded., Cold Spring Harbor Laboratory, 1989).

[0084] The present application also relates to the use of a xylitol dehydrogenase for the enzymatic oxidation of NAD(P)H by generating 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 which is at least 80% identical to SEQ ID No. 20;

[0086] ii) is encoded by a nucleic acid which has at least 80% identity to SEQ ID No. 19; and

[0087] iii) is encoded by a nucleic acid which binds under stringent conditions to the complementary strand of a nucleic acid molecule having the nucleic acid sequence of SEQ ID No. 19.

[0088] In other variants of the process according to the application, the enzymatic oxidation of NAD(P)H is accomplished using an alcohol dehydrogenase (EC 1.1.1.1 or EC 1.1.1.2).

[0089] The NAD(P)H-dependent alcohol dehydrogenase used for the enzymatic oxidation of NAD(P)H by generating 2-propanol from acetone preferably comprises or consists of an amino acid sequence selected from the group consisting of:

[0090] i) has an amino acid sequence which is at least 80% identical to SEQ ID No. 10 or SEQ ID No. 12;

[0091] ii) is encoded by a nucleic acid which has at least 80% identity to SEQ ID No. 9 or SEQ ID No. 11 ; and

[0092] iii) an amino acid sequence encoded by a nucleic acid which binds under stringent conditions to the complementary strand of a nucleic acid molecule having the nucleic acid sequence of SEQ ID No. 9 or SEQ ID No. 11.

[0093] SEQ ID No. 9:

[0094]

[0095] SEQ ID No. 10:

[0096]

[0097] SEQ ID No. 11:

[0098] SEQ ID No. 12:

[0099]

[0100] The alcohol dehydrogenase described herein preferably comprises an amino acid sequence which is at least 80%, more preferably 85%, more preferably 90%, more preferably 95%, more preferably 98%, more preferably 99%, in particular 100% identical to SEQ ID No. 10 or SEQ ID No. 12.

[0101] Alternatively, the alcohol dehydrogenase preferably comprises an amino acid sequence encoded by a nucleic acid which is at least 80%, more preferably 85%, more preferably 90%, more preferably 95%, more preferably 98%, more preferably 99%, in particular 100% identical to SEQ ID No. 9 or SEQ ID No. 11. Particularly preferably, the nucleic acid encoding the alcohol dehydrogenase according to the application comprises or consists of the nucleic acid sequence of SEQ ID No. 9 or SEQ ID No. 11.

[0102] Alternatively, the alcohol dehydrogenase preferably comprises an amino acid sequence encoded by a nucleic acid which binds under stringent conditions to the complementary strand of a nucleic acid molecule having the nucleic acid sequence of SEQ ID No. 9 or SEQ ID No. 11. As used herein, "stringent conditions" refer to conditions under which only so-called specific hybrids, but not non-specific hybrids, are formed. For example, stringent conditions include hybridization in 6x SSC (sodium chloride / sodium citrate) at 45°C, followed by washing with 0.2 to 1 x SSC, 0.1% SDS at 50-65°C; or such conditions can include hybridization in 1 x SSC at 65-70°C, followed by washing with 0.3 x SSC at 65-70°C. Hybridization can 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).

[0103] The present application also relates to the use of an NAD(P)H-dependent alcohol dehydrogenase for the enzymatic oxidation of NAD(P)H by generating an alcohol from a ketone or aldehyde, wherein the alcohol dehydrogenase comprises an amino acid sequence selected from the group consisting of:

[0104] i) an amino acid sequence having at least 80% identity to SEQ ID No. 10 or SEQ ID No. 12;

[0105] ii) an amino acid sequence encoded by a nucleic acid having at least 80% identity to SEQ ID No. 9 or SEQ ID No. 11 ; and

[0106] iii) an amino acid sequence encoded by a nucleic acid which binds under stringent conditions to the complementary strand of a nucleic acid molecule having the nucleic acid sequence of SEQ ID No. 9 or SEQ ID No. 11.

[0107] In other variants of the process according to the application, the enzymatic oxidation of NAD(P)H is accomplished using a xylose reductase (EC 1.1.1.307, EC 1.1.1.430, EC 1.1.1.431).

[0108] The xylose reductase used for the enzymatic oxidation of NAD(P)H by generating D-sorbitol from D-glucose or L-arabitol from L-arabinose preferably comprises or consists of an amino acid sequence selected from the group consisting of:

[0109] i) an amino acid sequence having at least 80% identity to SEQ ID No. 14 or SEQ ID No. 16;

[0110] ii) an amino acid sequence encoded by a nucleic acid having at least 80% identity to SEQ ID No. 13 or SEQ ID No. 15; and

[0111] iii) an amino acid sequence encoded by a nucleic acid which binds under stringent conditions to the complementary strand of a nucleic acid molecule having the nucleic acid sequence of SEQ ID No. 13 or SEQ ID No. 15.

[0112] SEQ ID No. 13:

[0113]

[0114] SEQ ID No. 14:

[0115]

[0116] SEQ ID No. 15:

[0117]

[0118] SEQ ID No. 16:

[0119]

[0120] The xylose reductase described herein preferably comprises an amino acid sequence which is at least 80%, more preferably 85%, more preferably 90%, more preferably 95%, more preferably 98%, more preferably 99%, in particular 100% identical to SEQ ID No. 14 or SEQ ID No. 16.

[0121] Alternatively, the xylose reductase preferably comprises an amino acid sequence which is encoded by a nucleic acid which is at least 80%, more preferably 85%, more preferably 90%, more preferably 95%, more preferably 98%, more preferably 99%, in particular 100% identical to SEQ ID No. 13 or SEQ ID No. 15. Particularly preferably, the nucleic acid encoding the xylose reductase according to the application comprises or consists of the nucleic acid sequence SEQ ID No. 13 or SEQ ID No. 15.

[0122] Alternatively, the xylose reductase preferably comprises an amino acid sequence which is encoded by a nucleic acid which binds under stringent conditions to the complementary strand of a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 13 or SEQ ID No. 15. As used herein, "stringent conditions" refer to conditions under which only so-called specific hybrids, but not non-specific hybrids, are formed. For example, stringent conditions include hybridization in 6x SSC (sodium chloride / sodium citrate) at 45°C, followed by washing with 0.2 to 1 x SSC, 0.1% SDS at 50-65°C; or such conditions can include hybridization in 1 x SSC at 65-70°C, followed by washing with 0.3 x SSC at 65-70°C. The hybridization can 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).

[0123] The present application also relates to the use of a xylose reductase for the enzymatic oxidation of NAD(P)H, wherein the xylose reductase comprises an amino acid sequence selected from the group consisting of:

[0124] i) an amino acid sequence which is at least 80% identical to SEQ ID No. 14 or SEQ ID No. 16;

[0125] ii) an amino acid sequence which is encoded by a nucleic acid which is at least 80% identical to SEQ ID No. 13 or SEQ ID No. 15; and

[0126] iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to the complementary strand of a nucleic acid molecule having the nucleic acid sequence of SEQ ID No. 13 or SEQ ID No. 15.

[0127] In other variants of the process according to the application, the enzymatic oxidation of NAD(P)H is accomplished using a mannitol dehydrogenase (EC 1.1.1.67; EC 1.1.1.138; EC 1.1.1.255).

[0128] The mannitol dehydrogenase used for the enzymatic oxidation of NAD(P)H by generating D-mannitol from D-fructose preferably comprises or consists of an amino acid sequence selected from the group consisting of:

[0129] i) an amino acid sequence having at least 80% identity to SEQ ID No. 18;

[0130] ii) an amino acid sequence encoded by a nucleic acid having at least 80% identity to SEQ ID No. 17; and

[0131] iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to the complementary strand of a nucleic acid molecule having the nucleic acid sequence of SEQ ID No. 17.

[0132] SEQ ID No. 17:

[0133]

[0134] SEQ ID No. 18:

[0135]

[0136] 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%, in particular 100% identity to SEQ ID No. 18.

[0137] 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%, in particular 100% identity to SEQ ID No. 17. Particularly preferably, the nucleic acid encoding the mannitol dehydrogenase according to the application comprises or consists of the nucleic acid sequence of SEQ ID No. 17.

[0138] Alternatively, the mannitol dehydrogenase preferably comprises an amino acid sequence which is encoded by a nucleic acid which binds under stringent conditions to the complementary strand of a nucleic acid molecule having the nucleic acid sequence of SEQ ID No. 17. As used herein, "stringent conditions" refer to conditions under which only so-called specific hybrids, but not non-specific hybrids, are formed. For example, stringent conditions include hybridization in 6x SSC (sodium chloride / sodium citrate) at 45°C, followed by washing with 0.2 to 1 x SSC, 0.1% SDS at 50-65°C; or such conditions can include hybridization in 1 x SSC at 65-70°C, followed by washing with 0.3 x SSC at 65-70°C. Hybridization can be carried out by conventionally known methods, such as those described in J. Sambrook et al. in "Molecular Cloning: A Laboratory Manual" (2nd ed., Cold Spring Harbor Laboratory, 1989).

[0139] The present application also relates to the use of a mannitol dehydrogenase, wherein the mannitol dehydrogenase comprises an amino acid sequence selected from the group consisting of:

[0140] i) an amino acid sequence which is at least 80% identical to SEQ ID No. 18;

[0141] ii) an amino acid sequence which is encoded by a nucleic acid which is at least 80% identical to SEQ ID No. 17; and

[0142] iii) an amino acid sequence which is encoded by a nucleic acid which binds under stringent conditions to the complementary strand of a nucleic acid molecule having the nucleic acid sequence of SEQ ID No. 17.

[0143] In other preferred embodiments of the process according to the application, the concentration of FFA in the aqueous solution is between 5 and 200 g / l, particularly preferably between 5 and 100 g / l.

[0144] The preferred temperature range is between 15 and 50°C, particularly preferably between 15 and 40°C.

[0145] The particularly preferred reaction pH range is between pH 5 and pH 9.

[0146] In preferred variants of the process according to the application, the enzyme is present in a homogenate and / or a lysate of the corresponding cell which produces the enzyme, wherein a lysate is particularly preferred. The enzyme can also be present in solid form in the aqueous reaction mixture.

[0147] In this context, a "homogenate" refers to a suspension which has been treated physically and / or chemically (for example by pressure, lysozyme or ultrasound), wherein the cellular components are released from the cells. A "lysate" is obtained when the insoluble cellular components in the homogenate are removed (for example by filtration or centrifugation) (see also the preparation of the enzyme and the preparation of the lysate).

[0148] In other variants, the enzyme can be modified at the N-terminus with a water-soluble polymer such as polyethylene glycol, immobilized in or on a solid matrix, or part of a fusion protein.

[0149] In other variants, the enzyme can be present in powder form, lyophilized form or spray-dried form.

[0150] The preferred embodiments of the present application are described in more detail with the following examples.

[0151] Materials

[0152] 5-Formyl-2-furancarboxylic acid (FFA) and 2,5-furan dicarboxylic acid (FDCA) were purchased from TCI; acetone, potassium dihydrogen phosphate, dibasic potassium phosphate, sodium dodecyl sulfate (SDS) and D-sorbitol were purchased from Carl Roth; NAD + , NADH disodium salt, NADP + disodium salt, NADPH tetrasodium salt, acetonitrile and D-fructose were purchased from PanReac AppliChem (ITW Reagents); triethanolamine was purchased from Chem-Lab NV.

[0153] Preparation of the enzyme and preparation of the lysate

[0154] Overview of recombinase expression in E. coli

[0155] To prepare the recombinant enzyme in E. coli strain, the gene to be expressed was first amplified by PCR using genomic DNA or its synthetic equivalent adapted to E. coli codon usage as template, together with specific oligonucleotides carrying additional restriction enzyme recognition sequences; and the gene was isolated from the reaction mixture. After nuclease cleavage with restriction enzymes Sphl and Hindlll, the gene fragment encoding the enzyme of interest was ligated into the backbone of the expression vector pQE70-Kan cut with Sphl and Hindlll. The ligation product was transformed into chemically competent E. coli Top 10F cells and the resulting colonies were used for plasmid isolation and restriction analysis.

[0156] The results of the cloning step were verified by restriction enzyme cleavage and DNA sequencing. The resulting construct carries the gene of interest under the IPTG inducible T5 promoter.

[0157] 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.

[0158] The next day, expression cultures with an optical density OD 550 of 0.02 were inoculated and shaken at 37°C until an OD550 When an OD of 0.3 was reached, the temperature was lowered to 25°C and, when an OD of 0.5 was reached, the culture was induced with 0.1 mM IPTG. After 22 hours, the culture was harvested (separation of the cell pellet from the culture medium by centrifugation) and analyzed for expression of the recombinant enzyme using SDS gel electrophoresis and activity assay (Use-Test or optical enzymatic assay). 550 When an OD of 0.3 was reached, the temperature was lowered to 25°C and, when an OD of 0.5 was reached, the culture was induced with 0.1 mM IPTG. After 22 hours, the culture was harvested (separation of the cell pellet from the culture medium by centrifugation) and analyzed for expression of the recombinant enzyme using SDS gel electrophoresis and activity assay (Use-Test or optical enzymatic assay).

[0159] Preparation of cell lysate by sonication

[0160] To prepare the cell suspension, the cell pellet prepared according to the above method was weighed and placed in a suitable container and mixed with buffer and lysozyme (final concentration 0.5 mg / ml) (e.g. triethanolamine (TEA)-HCl) and stirred to dissolve. The mass fraction of the biomass was typically 20%, the remainder being buffer.

[0161] Cell disruption was performed using a Branson Sonifier 450. The suspension was subjected to three ultrasonic treatments, each with 15 ultrasonic pulses (device settings: timer = 15; duty cycle = 50; output control = 3-5).

[0162] The resulting homogenate was centrifuged at 16000 rpm for 10 minutes at 4°C (Eppendorf Centrifuge 5417R) to separate the insoluble cell debris and obtain the lysate.

[0163] Table 1 Enzyme types used in the examples and the donor organism of the enzymes (ALDH = aldehyde dehydrogenase)

[0164]

[0165] This ALDH is classified as 2,5-dioxopentanoate dehydrogenase (catalyzing the oxidation of 2,5-dioxopentanoate to alpha-ketoglutarate) in the NCBI protein database (accession number WP_009990943.1).

[0166] Analytical methods

[0167] High performance liquid chromatography (HPLC)

[0168] FFA and FDCA were quantified 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 and corresponding guard column, with isocratic elution using 1 mM sulfuric acid.

[0169] 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.

[0170] Determination of enzyme activity (optical enzymatic assay)

[0171] 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 -1 The 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).

[0172] 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.

[0173] Example 1

[0174] Oxidation of 5-formyl-2-furanoic acid to 2,5-furandicarboxylic acid—using XDH and D-fructose as cofactor regeneration.

[0175] 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.

[0176] 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.

[0177] 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 force (max. g.) 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).

[0178] Four hours later, FFA was completely oxidized to FDCA.

[0179] 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.

[0180] Example 2

[0181] Oxidation of 5-formyl-2-furancarboxylic acid to 2,5-furandicarboxylic acid—cofactor regeneration using ADH and acetone.

[0182] 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.

[0183] 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.

[0184] 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).

[0185] After 4.5 hours, FFA was completely oxidized to FDCA. Similar to Example 1, FDCA could be separated as a solid.

[0186] Example 3

[0187] 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.

[0188] 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.

[0189] 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).

[0190] In this manner, 40.8% of FFA (7.1 g / l) was oxidized to FDCA.

[0191] Example 4

[0192] 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.

[0193] 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 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.

[0194] 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).

[0195] In this way, 48.0% of FFA was oxidized to FDCA.

[0196] Example 5

[0197] 5-Formyl-2-furanoic acid was oxidized to 2,5-furandicarboxylic acid using aldehyde dehydrogenase II (ALDH II) and various dehydrogenases for cofactor regeneration.

[0198] 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.

[0199] 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).

[0200] The results are shown in Table 2 below.

[0201] Table 2

[0202]

[0203] The results in Table 2 show that various dehydrogenases and different substrates are suitable for cofactor regeneration (NADP in this example). + ).

[0204] Example 6

[0205] 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.

[0206] 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.

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

[0209] Table 3

[0210]

[0211] Example 7

[0212] 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.

[0213] Mix the following components in a glass vial: 143 μl FFA solution (final concentration 12 g / L), 187 μl deionized water, 10 μl ALDH IV lysate, 125 μl 1 M potassium phosphate buffer (pH 7), 15 μl acetone, 10 μl ADH II lysate, and 10 μl 10 mM NADP. + Solution. Incubate the mixture with continuous shaking (Eppendorf thermostatic mixer, 30°C, 800 rpm) for a total of 20 hours.

[0214] 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).

[0215] In this way, 85.0% of FFA is oxidized to FDCA.

[0216] Example 8

[0217] 5-Formyl-2-furanoic acid was oxidized to 2,5-furandicarboxylic acid using aldehyde dehydrogenase V (ALDH V), and cofactor regeneration was performed using ADH and acetone.

[0218] Mix the following components in a glass vial: 85 μl substrate solution (final concentration 6.5 g / l FFA), 255 μl deionized water, 25 μl ALDH V suspension, 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 20 hours.

[0219] 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).

[0220] In this manner, no conversion of FFA to FDCA was observed.

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[0252] Sakoda, H., & Imanaka, T. (1992). Cloning and sequencing of the gene coding for alcohol dehydrogenase of Bacillus stearothermophilus and rational shift of the optimum pH. Journal of Bacteriology, 174(4), 1397–1402. https: / / doi.org / 10.1128 / jb.174.4.1397-1402.1992

[0253] 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

[0254] Protein [Internet]. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information. Accession No. ACR78268.1, xylose reductase [Rasamsonia emersonii]. Available from: https: / / www.ncbi.nlm.nih.gov / protein / ACR78268.1

[0255] Protein [Internet]. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information. Accession No. XP_022674194.1, trifunctional aldehyde reductase / xylose reductase / glucose 1-dehydrogenase (NADP(+)) [Kluyveromyces marxianus DMKU3-1042]. Available from: https: / / www.ncbi.nlm.nih.gov / protein / XP_022674194.1

Claims

1. A method for preparing 2,5-furandicarboxylic acid, wherein the preparation is carried out by means of NAD(P) + Treatment with a oxidoreductase-dependent enzyme oxidizes 5-formyl-2-furancarboxylic acid, present in aqueous solution, to 2,5-furandicarboxylic acid in vitro, wherein NAD(P)H generated during oxidation is enzymatically oxidized back to NAD(P) by a dehydrogenase. + Then the enzyme is removed.

2. The method according to claim 1, characterized in that, The NAD(P) used for the oxidation of 5-formyl-2-furancarboxylic acid to 2,5-furandicarboxylic acid. + The oxidoreductase-dependent enzyme is aldehyde dehydrogenase.

3. The method according to claim 1 or 2, characterized in that, The NAD(P) used for the oxidation of 5-formyl-2-furancarboxylic acid to 2,5-furandicarboxylic acid. + Redox enzymes have amino acid sequences selected from the following groups: i) Having an amino acid sequence that is at least 80% identical to SEQ ID No. 2, SEQ ID No. 8, 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. 7, SEQ ID No. 4, 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. 7, SEQ ID No. 3 or SEQ ID No. 5 under stringent conditions.

4. The method according to any one of claims 1 to 3, characterized in that, Sugars, aldehydes, or ketones are used as substrates for dehydrogenases that enzymatically oxidize NAD(P)H.

5. The method according to claim 4, characterized in that, D-fructose is used as the sugar, and acetone is used as the ketone compound.

6. The method according to claim 4, characterized in that, D-glucose is used as the sugar, and acetone is used as the ketone compound.

7. The method according to any one of claims 1 to 6, characterized in that, Xylitol dehydrogenase is used as the dehydrogenase for the enzymatic oxidation of NAD(P)H, and the xylitol dehydrogenase has an amino acid sequence selected from the group consisting of: i) Has an amino acid sequence that is at least 80% identical to SEQ ID No. 20; ii) An amino acid sequence encoded by a nucleic acid having at least 80% identity with SEQ ID No. 19; 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. 19 under strict conditions.

8. The method according to any one of claims 1 to 6, characterized in that, A NAD(P)H-dependent alcohol dehydrogenase is used as the dehydrogenase for the enzymatic oxidation of NAD(P)H by the production of 2-propanol from acetone, wherein the NAD(P)H-dependent alcohol dehydrogenase has an amino acid sequence selected from the group consisting of: i) Having an amino acid sequence that is at least 80% identical to SEQ ID No. 10 or SEQ ID No. 12; ii) An amino acid sequence encoded by a nucleic acid having at least 80% identity with SEQ ID No. 9 or SEQ ID No. 11; 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 SEQ ID No. 9 or SEQ ID No. 11 under stringent conditions.

9. The method according to any one of claims 1 to 6, characterized in that, Xylose reductase is used as a dehydrogenase for the enzymatic oxidation of NAD(P)H by the production of D-sorbitol from D-glucose or L-arabinose from L-arabinose, wherein the xylose reductase has an amino acid sequence selected from the group consisting of: i) Has an amino acid sequence that is at least 80% identical to SEQ ID No. 14 or SEQ ID No. 16; ii) An amino acid sequence encoded by a nucleic acid having at least 80% identity with SEQ ID No. 13 or SEQ ID No. 15; 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 SEQ ID No. 13 or SEQ ID No. 15 under stringent conditions.

10. The method according to any one of claims 1 to 6, characterized in that, Mannitol dehydrogenase is used as the dehydrogenase for the enzymatic oxidation of NAD(P)H by the production of D-mannitol from D-fructose, said mannitol dehydrogenase having an amino acid sequence selected from the group consisting of: i) Has an amino acid sequence that is at least 80% identical to SEQ ID No. 18; ii) An amino acid sequence encoded by a nucleic acid having at least 80% identity with SEQ ID No. 17; 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. 17 under strict conditions.

11. An application 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: i) Has an amino acid sequence that is at least 80% identical to SEQ ID No. 20; ii) An amino acid sequence encoded by a nucleic acid having at least 80% identity with SEQ ID No. 19; 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. 19 under strict conditions.

12. 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: i) Having an amino acid sequence that is at least 80% identical to SEQ ID No. 10 or SEQ ID No. 12; ii) An amino acid sequence encoded by a nucleic acid having at least 80% identity with SEQ ID No. 9 or SEQ ID No. 11; 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 SEQ ID No. 9 or SEQ ID No. 11 under stringent conditions.

13. Application of xylose reductase in the enzymatic oxidation of NAD(P)H, wherein the xylose reductase comprises an amino acid sequence selected from the group consisting of: i) Has an amino acid sequence that is at least 80% identical to SEQ ID No. 14 or SEQ ID No. 16; ii) An amino acid sequence encoded by a nucleic acid having at least 80% identity with SEQ ID No. 13 or SEQ ID No. 15; 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 SEQ ID No. 13 or SEQ ID No. 15 under stringent conditions.

14. Application of mannitol dehydrogenase for the enzymatic oxidation of NAD(P)H, wherein the mannitol dehydrogenase comprises an amino acid sequence selected from the group consisting of: i) Has an amino acid sequence that is at least 80% identical to SEQ ID No. 18; ii) An amino acid sequence encoded by a nucleic acid having at least 80% identity with SEQ ID No. 17; 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. 17 under strict conditions.

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