Method for producing 2,5-furandicarboxylic acid

Abstract

The invention also relates to a method for producing 2,5-furandicarboxylic acid. According to said method, 5-formyl-2-furancarboxylic acid, which is present in an aqueous solution, is oxidized by treatment with an NAD(P)+-dependent oxidoreductase in vitro to 2,5-furandicarboxylic acid, wherein the NAD(P)H formed during the oxidation is enzymatically oxidized back to NAD(P)+ by means of a dehydrogenase, after which the enzymes are removed.

Description

[0001] Process for the preparation of 2,5-furandicarboxylic acid

[0002] The invention relates to a process for the preparation of 2,5-furandicarboxylic acid (FDCA) from 5-formyl-2-furancarboxylic acid (FFA).

[0003] Background of the invention

[0004] In 2018, approximately 90% of the plastics produced worldwide (400 Mt) were based on fossil raw materials, with the remaining shares being recycled (9%), bio-based (1%), and CO2-based plastics (<1%) (Carus et al., 2020). At the same time, the global demand for plastics is also growing. In 2019, the annual CO2 emissions from the entire life cycle of plastics were 0.86 Gt, equivalent to the CO2 emissions of 189 coal-fired power plants operating at full capacity (500 MW). This figure is projected to rise to 2.8 Gt (equivalent to 615 coal-fired power plants) by 2050 (Hamilton et al., 2019).

[0005] One of the plastics based on fossil raw materials is polyethylene terephthalate (PET), which is primarily used in beverage packaging. It is a condensation polymer produced from terephthalic acid (1,4-benzenedicarboxylic acid) and ethylene glycol (ethane-1,2-diol) with the elimination of water.

[0006] Terephthalic acid is produced industrially by oxidation of p-xylene (1,4-dimethylbenzene) with atmospheric oxygen at approximately 200 °C in the presence of cobalt acetate, manganese acetate and HBr in acetic acid in the so-called AMOCO process (Tomäs et al., 2013).

[0007] Ethylene glycol is produced by the hydrolysis of ethylene oxide (at 200 °C), which in turn is obtained by the oxidation of ethylene (derived from fossil raw materials) (Berger, 2016). Due to the fossil sources of the raw materials (p-xylene and ethylene), PET cannot generally be considered sustainable.

[0008] To achieve the 1.5-degree target of the Paris Climate Agreement and thus limit the negative impacts of climate change, sustainable alternatives for petrochemical-based plastics must be found. For ethylene glycol, for example, there are processes based on renewable resources. For example, ethylene can also be produced by dehydrating bioethanol obtained by fermentation from glucose or starch (e.g., Fan et al., 2013). Fermentative conversions of xylose and / or glucose to ethylene glycol using metabolically engineered microorganisms (Escherichia coli or Saccharomyces cerevisiae) are also known (Salusjärvi et al., 2019). The second building block of PET, the aromatic compound terephthalic acid, is difficult to produce from sustainable raw materials and therefore must be replaced.An excellent substitute for terephthalic acid is 2,5-furandicarboxylic acid (FDCA), which is predominantly obtained from the catalytic upgrading of biomass. The polymerization of FDCA with ethylene glycol produces PEF (polyethylene furanoate), which contains a heteroaromatic furan ring instead of the benzene ring. Like PET, PEF is a thermoplastic, but compared to PET, it is characterized by significantly higher biodegradability and better thermal (higher glass transition temperature, lower melting temperature) and mechanical properties (higher stiffness). The most important property of PEF, however, is its reduced permeability to gases such as O2 and CO2. This is particularly important for beverages, as it can extend the shelf life of beverages (preventing off-gassing of carbonated beverages; preventing oxidation processes caused by diffused oxygen) (de Jong et al., 2022).

[0009] An overview of various chemical syntheses can be found in the article by Cong et al. (2021).

[0010] By far the most important starting material for FDCA is 5-(hydroxymethyl)furfural (HMF), which can be obtained, for example, from cellulose (and thus from renewable raw materials). Enzymatic or chemical hydrolysis of cellulose produces D-glucose, which is subsequently isomerized (enzymatically or chemically) to D-fructose. Dehydration (removal of a total of three H2O molecules) converts D-fructose to HMF. Common systems for the dehydration of fructose include mineral acids such as H2SO4 or HCl, and solid catalysts (Bronsted or Lewis) acid (Cong et al., 2021; US ​​9617234 B1).

[0011] HMF contains an alcohol and an aldehyde group, which must be oxidized to carboxylic acid groups to obtain FDCA. The three necessary oxidation steps can be carried out in various ways: chemically with heterogeneous or homogeneous catalysts, electrochemically, and biocatalytically (enzymatically or with whole cells).

[0012] The direct precursor of FDCA is FFA (5-formyl-2-furancarboxylic acid), which can be produced, 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 HBr in an acetic acid / water mixture in a flow reactor at 80 °C. In a final step, FFA can be oxidized to FDCA using either oxygen as the oxidant and metals (e.g., Pt-Ru / C) or metal salts (Co and Mn salts) as catalysts, or hydrogen peroxide (US 10087161 B2). The acid-catalyzed dehydration of 5KGA to FFA is also described in EP 3265450 B1. The final oxidation is carried out in acetic acid in the presence of catalytic amounts of Co acetate, Mn acetate and NaBr with an overpressure of oxygen at 180 °C (analogous to the AMOCO process).

[0013] In general, the chemical production processes of FDCA starting from FFA as an intermediate are characterized by unfavorable reaction conditions (high temperatures and pressures), the formation of by-products and the use of expensive and sometimes toxic (co-salts) catalysts and therefore cannot be described as sustainable.

[0014] An alternative is biocatalytic processes that are highly selective under mild reaction conditions and use biodegradable catalysts such as cells or enzymes (Cong et al., 2021).

[0015] FFA can be produced by the enzymatic oxidation of HMF (mainly with O2 as the oxidant).

[0016] One group of enzymes used to produce FFA from HMF are the 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, in small amounts, to FDCA in 4 h.

[0017] The AAO-catalyzed oxidation of the aldehyde groups proceeds via the corresponding geminal diols (aldehyde hydrates). The aldehyde groups in FFA exhibit a lower degree of hydration (DFF: 53%, FFA: 8%) compared to the precursor 2,5-diformylfuran (DFF), which explains the preferential formation of FFA over FDCA. H2O2, which is produced as a byproduct of the AAO-catalyzed oxidations, chemically oxidizes FFA to FDCA (Carro et al., 2015). In a later study by Serrano et al. (2019) on the AAO of P. eryngii, it was shown that HMF can also be completely oxidized to FDCA when a catalase is used to remove H2O2, since the final oxidation step of FFA to FDCA is inhibited by H2O2. In this way, 1.5 mM HMF could be oxidized to 1.6 mM FDCA in 6 d using an AAO mutant.

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

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

[0020] Zhang et al. (2019) immobilized a laccase (CotA-TJ102) from Bacillus subtilis TJ-102 on magnetic nanoparticles, which oxidized 83.3% of the starting material HMF to FFA (after 10 recycling 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 substrate HMF (100 mM) to FFA in 48 h. In addition to activating GOase M3.5, HRP also serves to regenerate NAD(P). + , which is required as a cofactor for SADH.

[0022] US 10344307 B2 describes further enzyme (systems) for the oxidation of HMF to FFA: 1) NAD(P)-dependent ketoreductase & NAD(P)H oxidase for cofactor regeneration, 2) NAD(P)-dependent aldehyde dehydrogenase & NAD(P)H oxidase and 3) xanthine oxidoreductase (such as the periplasmic aldehyde oxidoreductase (PaoABC) from E. coli; oxidizes HMF to 5-hydroxymethyl-2-furancarboxylic acid (HMFA) and DFF to FFA), galactose oxidase variant M3-5 (GOase M3-5; oxidizes HMF to DFF and HMFA to FFA) & horseradish peroxidase (HRP; for activation of GOase M3.5). For example, 50 mM DFF can be completely oxidized to FFA in 2 h using PaoABC at pH 6. At pH 7 and 8, however, the oxidation proceeds completely to FDCA. The enzyme system mentioned under 3) has also been 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 of FFA to FDCA.

[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; a fungal lignin peroxidase (LPO), and HRP) on 10 mM FFA and observed the formation of significant amounts of FDCA (11.6% for AO, 1.1% for laccase, and 3.2% for LPO) after 72 h only for AO, laccase, and LPO. 5-Hydroxymethyl-2-furancarboxylic acid (HMFA) was found as a byproduct (up to 18.2% for AO).

[0025] Another enzyme for the oxidation of FFA to FDCA is the nonspecific peroxygenase (UPO, EC 1.11.2.1; requires H2O2 as an oxidant) from Agrocybe aegerita, which can be used in combination with AAO to completely oxidize HMF to FDCA. Thus, 90% of the FFA (3 mM) can be oxidized to FDCA in 120 h (Carro et al., 2015). In addition to the oxidation of FFA to FDCA, the oxidation of HMF to FFA via DFF is also catalyzed by UPO (Lappe et al., 2021). Jia et al. (2017) used an enzyme system consisting of horse liver alcohol dehydrogenase (HLADH) and human hemoglobin (oxidized by H2O2 NADH to NAD + ) to oxidize 96% of the substrate (10 mM FFA) to FDCA in 60 h.

[0026] US 8183020 B2 describes the enzymatic oxidation of FFA to FDCA using a commercially available chloroperoxidase from Caldariomyces fumago (EC 1.11.1.10) with H2O2 as oxidant.

[0027] Aldehyde dehydrogenases (ALDHs) are enzymes that catalyze the oxidation of aldehyde groups to carboxylic acid groups. An ALDH has been described for the organism Raoultella ornithinolytica BF60 that can oxidize FFA to FDCA and HMF to HMFA (Hossain et al., 2017).

[0028] An aldehyde dehydrogenase (from Thermus thermophilus) was also used by Shortall et al. (2023) to oxidize one aliphatic (hexanal) and three aromatic aldehydes (p-tolualdehyde, benzaldehyde, and terephthalaldehyde) to the corresponding carboxylic acids in a coupled bienzymatic flow reactor. For this purpose, ALDH was immobilized directly from an E. coli lysate in one reactor module. L-lactate dehydrogenase was immobilized in a second reactor module for cofactor regeneration.

[0029] 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, benzylic, heteroaromatic, and bicyclic aldehydes. In the oxidation of 20 mM HMF, 90% yield of HMFA was achieved with ALDH from bovine lens and 91% yield was achieved with ALDH from E. coli in 24 h. The substrate FFA was not tested.

[0030] US 10344307 B2 describes a method for the oxidation of DFF using a commercially available ALDH (in combination with an NAD(P)H oxidase), which, depending on the conditions (DFF concentrations from 10 to 100 mM), leads to FFA, FDCA, or mixtures. With increasing DFF concentration, more FFA is obtained than FDCA (10 mM substrate: 100% FDCA; 50 mM substrate: 80% FDCA, 20% FFA; 100 mM substrate: 20% FDCA, 80% FFA (conversion after 3 h in each case)). Between 10 and 50 mol% cofactor (based on the amount of substrate) is used for the oxidations in US 10344307 B2.

[0031] Yuan et al. (2018) used engineered whole cells (resting cells) of Raoultella ornithinolytica BF60, in which the two genes adhPS and alkR were deleted to suppress the reduction of HMF to the corresponding diol, to oxidize HMF to FDCA (265 mM; 96% yield) in a fed-batch process in 144 h. Furthermore, an in vitro conversion of FFA to FDCA is described, which leads to lower conversions compared to the whole-cell process.

[0032] The methods known in the prior art generally have disadvantages such as low substrate concentrations or long reaction times.

[0033] This is where the object of the present invention comes in and aims to provide an improved process for the production of FDCA.

[0034] Detailed description of the invention

[0035] The object of the invention is achieved by treating 5-formyl-2-furancarboxylic acid (FFA), which is present in an aqueous solution, with an NAD(P) + -dependent oxidoreductase in vitro to 2,5-furandicarboxylic acid (FDCA), whereby the NAD(P)H produced during the oxidation is enzymatically converted back to NAD(P) by means of a dehydrogenase + oxidized, after which the enzymes are removed. The 2,5-furandicarboxylic acid can then be separated from the solution by precipitation.

[0036] In vitro means that the process in question is carried out outside an organism, i.e. not in a whole cell and not fermentatively.

[0037] As a substrate for dehydrogenase to oxidize NAD(P)H to NAD(P) + Sugars, of which D-fructose is particularly preferred, or aldehyde or keto compounds, of which acetone is particularly preferred, are used.

[0038] A preferred embodiment of the method according to the invention is shown schematically in the attached Figure 1.

[0039] At the NAD(P) + -dependent oxidoreductase for the oxidation of FFA to FDCA is preferably an aldehyde dehydrogenase.

[0040] A NAD(P) +-dependent oxidoreductase for the oxidation of FFA to FDCA preferably comprises or consists of an amino acid sequence which is selected from the group consisting of: i) an amino acid sequence which has an identity to SEQ ID No. 2, SEQ ID No. 8, SEQ ID No. 4 or SEQ ID No. 6 of at least 80%, ii) an amino acid sequence which is encoded by a nucleic acid which has an identity to SEQ ID No. 1, SEQ ID No. 7, SEQ ID No. 3 or SEQ ID No. 5 of at least 80%, and iii) an amino acid sequence which is encoded by a nucleic acid which binds under stringent conditions to a complementary strand of the nucleic acid molecule having the nucleic acid sequence SEQ ID No. 1, SEQ ID No. 7, SEQ ID No. 3 or SEQ ID No. 5.

[0041] SEQ ID No. 1:

[0042] ATGTCACGCTATGAACTGCTGATCGATGGTCGCCTGCAGGCGGCCGAGCACTACGACCGGGTGATCGA

[0043] CCCGGCCAGCGAAGAAATCGTTGGCGAAGCCGCCCGCGCCAGCCTGGAGCAGGTCGACGCGGCGGTG

[0044] GATGCCGCACACCGCGCCTTCCCGGCCTGGGCCACCGATCTCGACGTCCGCCGCCAGAGCCTGGCCCG

[0045] AGCCGCCGAACGAGTACGCGAGAATGCCCAGGCGCTGGCCGAGCTGATCACCCGCGAACAGGGTCGC

[0046] CCACTGCGCTCGACCCTGGAGGAAGTGGCTGGCGTCGCCGCCACTTTCGAGCACCACGCGCAGCTGGA

[0047] GCTACCCGCCGACACCCAGTTGCGCGACGACGGCGAGCGCCTGGTGCGCATCACCCGCAAACCGCTGG

[0048] GGGTGGTCGCCGCGATCACCCCGTGGAACGTCCCGCTGATCCTGCTGGTATTGAAGATCGCGCCTGCC

[0049] CTGCACGCCGGCAACACCGTGGTGGCCAAGCCCTCGGAGCACACGCCGCTGTCTACCCTGCTGCTGGC

[0050] GCGACTGCTGGGCGATGTGTTCCCCGCTGGCGTGTTCAACGTGGTTGCCGGTGCGGGCGAGGTTGGC

[0051] GAACACCTGGTACGTCATCCGCGGGTGCGCCACGTGACCTTCACCGGCAGCGTCGCCACCGGCAAGCG

[0052] CCTGTATGCCGGCGCGGGGGACGACCTCAAGCGTCTTACCCTGGAACTGGGCGGCAACGACGCCGCG

[0053] CTGGTACTGGAAGACGCCGACCTCGACGCCATCGTCGAACCGCTGTTCTGGGGCGCCTTCTGGAACAG

[0054] CGGCCAGGTGTGCTTTGCGATCAAGCGCCTGTACGTGCATGACAGCCTGTTCGAACCGTTACTGGCGA

[0055] AACTCGCCGAGCGCGCCCAGCGCACCCGCCTCGGTCATGGGCTCGACCCGCAGACGGAACTGGGGCC

[0056] GCTGACCAACGCACAGCAACTGGAACGGGTCATCGCCCTGGTGGAAGACGCCAAGGCTCACGGAGCA

[0057] CGCATCCGCAGCGGCGGCGTACGGCCCGACGGTCCCGGCTACTTCTACCCGCCGACCCTGGTCAGCGG

[0058] CGTGGCGGCCGGCGTCGCGCTGGTGGACGAGGAACAGTTCGGCCCGGTGTTGCCGGTGATCTCCTTCC

[0059] GCAACGAAGAGGACGCCATTACCCAGGCCAACGCCAGCCACTACGGCCTCGGCGCCTCGGTGTGGACC

[0060] CGCGACCTGGCGCGCGGCGAAGCCATCGCCAGGCGGCTGGAGGCGGGCCTGGCCTGGGTCAACCAGC

[0061] ACGGCCACATCCAGCCCGGCGCGCCCAAGGGCGGGCACAAGTGGAGCGGGCTCGGCTACGAAGGCG

[0062] GGCAGCGCGGCTATGAGGCCTTCAGCGAGCTGCAGGTGCTGAACATTTCGCGGCGCTAA

[0063] SEQ ID Nr. 2:

[0064] MSRYELLIDGRLQAAEHYDRVIDPASEEIVGEAARASLEQVDAAVDAAHRAFPAWATDLDVRRQSLARAAE

[0065] RVRENAQALAELITREQGRPLRSTLEEVAGVAATFEHHAQLELPADTQLRDDGERLVRITRKPLGVVAAITP

[0066] WNVPLILLVLKIAPALHAGNTVVAKPSEHTPLSTLLLARLLGDVFPAGVFNVVAGAGEVGEHLVRHPRVRHV

[0067] TFTGSVATGKRLYAGAGDDLKRLTLELGGNDAALVLEDADLDAIVEPLFWGAFWNSGQVCFAIKRLYVHDS

[0068] LFEPLLAKLAERAQRTRLGHGLDPQTELGPLTNAQQLERVIALVEDAKAHGARIRSGGVRPDGPGYFYPPTL VSGVAAGVALVDEEQFGPVLPVISFRNEEDAITQANASHYGLGASVWTRDLARGEAIARRLEAGLAWVNQ

[0069] HGHIQPGAPKGGHKWSGLGYEGGQRGYEAFSELQVLNISRR

[0070] SEQ ID Nr. 3:

[0071] ATGAAATCGTATCAGGGATTGGCTGACAAGTGGATTAAGGGCAGTGGGGAAGAATACCTTGATATTAA

[0072] TCCGGCTGATAAGGATCACGTATTAGCTAAGATAAGATTATATACAAAAGATGACGTTAAAGAAGCTAT

[0073] AAACAAGGCTGTAGCCAAATTCGACGAATGGTCAAGGACTCCAGCACCTAAAAGAGGCTCAATATTAC

[0074] TTAAGGCAGGGGAATTAATGGAACAAGAAGCCCAAGAGTTTGCGCTATTGATGACATTAGAGGAGGG

[0075] TAAGACTCTCAAGGATAGTATGTTTGAAGTGACAAGAAGTTATAATTTACTGAAATTTTATGGAGCATT

[0076] AGCATTTAAGATATCTGGGAAAACGCTTCCTTCAGCAGATCCTAATACTAGGATATTTACAGTAAAGGA

[0077] ACCCTTAGGCGTAGTAGCTTTAATTACGCCGTGGAATTTCCCATTATCAATACCAGTATGGAAATTGGCT

[0078] CCAGCCTTGGCTGCGGGTAACACTGCAGTAATAAAACCAGGCGACGAAAACACCGTTAATGGTAGCCAA

[0079] ATTGGTAGAAGTGTTGTCTAAAGCTGGATTGCCAGAGGGTGTCGTGAATTTAGTAGTTGGTAAGGGAA

[0080] GTGAAGTCGGAGATACCATAGTAAGTGATGATAATATAGCTGCAGTATCATTTACTGGATCAACCGAG

[0081] GTAGGTAAGAGAATTTACAAACTCGTAGGAAATAAAAATAGAATGACAAGAATTCAACTAGAGCTAGG

[0082] AGGTAAAAACGCGTTATATGTGGATAAGAGCGCTGACTTAACGTTAGCTGCTGAATTAGCCGTAAGAG

[0083] GAGGATTTGGACTAACCGGTCAATCATGTACTGCAACTAGTAGGTTAATAATTAACAAGGATGTATATA

[0084] CTCAATTTAAACAAAGACTACTAGAAAGAGTTAAGAAGTGGAGAGTAGGACCGGGTACTGAAGATGTT

[0085] GATATGGGTCCAGTTGTAGATGAAGGTCAATTTAAGAAAGACTTAGAATATATAGAATACGGAAAGAA

[0086] TGTGGGAGCAAAACTAATTTATGGTGGAAATAATACCAGGGAAGGGATATTTCCTAGAACCTACAA

[0087] TTTTCGAAGGAGTCACATCTGATATGAGGCTATTTAAAGAAGAGATTTCGGTCCAGTACTTAGTGTCA

[0088] CTGAGGCAAAAGATTTAGATGAGGCTATAAGGCTAGTTAACGCTGTAGACTATGGACATACAGCTGGA

[0089] ATAGTCGCAAGCGATATCAAGGCGATTAACGAGTTCGTTAGTAGGGTAGAGGCAGGAGTTATAAAGG

[0090] TTAATAAGCCAACAGTCGGACTGGAATTGCAAGCACCATTTGGTGGTTTTAAGAATTCTGGAGCCACTA

[0091] CGTGGAAAGAGATGGGAGAAGATGCTTTAGAGTTCTACCTTAAGGAGAAGACAGTATACGAAGGCTG GTAA

[0092] SEQ ID No. 4:

[0093] M KSYQGLADKWIKGSGEEYLDINPADKDHVLAKIRLYTKDDVKEAINKAVAKFDEWSRTPAPKRGSILLKAG

[0094] ELMEQEAQEFALLMTLEEGKTLKDSM FEVTRSYNLLKFYGALAFKISGKTLPSADPNTRIFTVKEPLGVVALIT

[0095] PWNFPLSIPVWKLAPALAAGNTAVIKPATKTPLMVAKLVEVLSKAGLPEGVVNLVVGKGSEVGDTIVSDNI

[0096] AAVSFTGSTEVGCRYKLVGNKNRMTRIQLGGKNALYVDKSADLLAELAVRGGFGLTGQSCTATSRLII

[0097] NKDVYTQFKQRLLERVKKWRVGPGTEDVDMGPVVDEGQFKKDLEYIEYGKNVGAKLIYGNIIPGKGYFLE PTIFEGVTSDM RLFKEEIFGPVLSVTEAKDLDEAIRLVNAVDYGHTAGIVASDIKAINEFVSVKVKVKVPT

[0098] VGLELQAPFGGFKNSGATTWKEMGEDALEFYLKKTVYEGW

[0099] SE ID Nr. 5:

[0100] ATGTCGACATTTCATTTACTTATTGATGGCCATCTGCAAGCCAGTGACCAATCCGATGTGGTGATCAACC

[0101] CCGCGACCGAGCTGGAAGTCGGGCGTGCCCCACGTGCCAGCGCCACCCAGGTCGACCAGGCAGTGGA

[0102] AGCCGCACATCAGGCATTTCATCGCTGGGCATCACAGCCTGAGGTGCGCCAGCAGGCACTGCTGGGCG

[0103] CGGCCGCCGCCATTCGCCAGCATGCCGATGCCCTGGCACGCCTGATCACGCAAGAGCAGGGGCGGCCA

[0104] TTGCACTTTACCCAGGGCGAGGTGGCCGGGGCTGCTGCTACCTTTGAACACTATGCCGGGTTTGCCGCG

[0105] CCATCGGATGTGGTGCTGCAGCAGGACGAACAAAAGCGGGTCAGCATTGAGCGCAGGCCGTTTGGCG

[0106] TAGTGGCTGCCATTACGCCCTGGAATGTGCCCATCATCCTGCTGGTACTGAAAATCGCCCCGGCCTTGA

[0107] AGGCTGGCAATACCGTGGTCGCCAAGCCATCGGAATACACCCCGCTTTCTACCTTGTACCTGGGTGAAA

[0108] TTCTGAAAGATGTATTTCCGCCCGGCGTGCTGAACGTGATAGCCGGTGACGGTCAGGTGGGGGCGCGC

[0109] CTGGCATCGCATCCGCTGGTGCAGAAGGTGACGTTCACCGGCAGCGTGGCAACCGGAAAAAAACTCTA

[0110] TGCCAGCGCCGCGCAGGATGTAAAACGCCTGACGCTGGAACTGGGCGGTAACGATGCGGCCATCGTG

[0111] CTGGACGATGCCAATGTCGATGCCATTGCCGAGAAGATTTTCTGGGGCGCCTTCTGGAATAGCGGTCA

[0112] GGTGTGCTTTGCCATCAAGCGCCTCTATGTGCACGAGCGTGTTTTCCAGCCCTTGCTCGATGCCCTGGT

[0113] GAAACGCGCGCAAAAAACCCGCGTGGGCGATGGCCAGCTGCCGGGTACCGAGCTGGGGCCGCTTACC

[0114] AACAAAGCCCAGTTTGAGCGCGTGATATCGCTGGTGGAAGACGCCAGACGCCATGGCGCCACCATTCA

[0115] TTCAGGCGGTGCTGCATTGCCTGGCCCCGGCTATTTCTATCCGCCCACCCTGGTCACCGGCATAGGCGC

[0116] AGGTGTCGCGCTGGTGGATGAAGAGCAGTTCGGACCAGTATTGCCGCTGATTCCTTTCCGTGATGAGC

[0117] AAGAGGCGGTGCGTCAGGCCAACGACAGTCCGTTTGGTCTGGGCGCTTCGGTTTGGACCGCCAATCCCC

[0118] GAACGCGGCCTGGCGCTGGTACGCCAGCTCCAGGCCGGGCTCGCCTGGGTCAACCAGCATGGCGATAT

[0119] CCATCCCGGTGCGCCCAAGGGCGGCTACAAATCCAGTGGCCTTGGCTACGAGGGCGGGCTGCGTGGCT

[0120] ATGACGAGTTCAGCGAGCTGCAAGTCGTCAATGCGGCGCTGGTTTAA

[0121] SEQ ID No. 6:

[0122] MSTFHLLIDGHLQASDQSDWINPATELEVGRAPRASATQVDQAVEAAHQAFHRWASQPEVRQQALLGA

[0123] AAAIRQHADALARLITQEQGRPLHFTQGEVAGAAATFEHYAGFAAPSDVVLQQDEQKRVSIERRPFGVVAA

[0124] ITPWNVPIILLVLKIAPALKAGNTTVVAKPSEYTPLSTLYLGEILKDVFPPGVLNVIAGDGQVGARLASHPLVQK

[0125] VTFTGSVATGKKLYAAQDVKRLTLELGGNDAAIVLDDANVDAIAEKIFWGAFWNSGQVCFAIKRLYVHE

[0126] RVFQPLLDALVKRAQKTRVGDGQLPGTELGPLTNKAQFERVISLVEDARRHGATIHSGGAALPGPGYFYPPT LVTGIGAGVALVDEEQFGPPVLPLIPFRDEQEAVRQANDSPFGLGASVWTANPERGLALVRQLQAGLAWVN

[0127] QHGDIHPGAPKGGYKSSGLGYEGGLRGYDEFSELQVVNAALV

[0128] SE ID Nr. 7:

[0129] ATGAGCGATTCCCGTTATACCGACCTCGGTCTCCAGCCCCCTGGCCGGCGAGTGGCGCCACGGCCGGGC

[0130] CGGCCGCCGGCTGAAGGTGAGCAACCCGTTCGACGGCAGCCTGCTGCTGGAGATCGAGCAGGCCGAC

[0131] CGCGACGACCTCGATGCCGCCTACGCCAAGGCCGCCGAGGTCCAGCCGGCATGGGCCGCGCTCGGGC

[0132] CCTCGGCACGCGCGGCGGTACTGTACAAGGCGGTGGAGGTGTTCGACCGCCGCCACGAGGAGATCGT

[0133] CGACTGGATCATCCGCGAGTCCGGCAGCACCCGCCTGAAGGCCGAGATCGAATGGGGCGCGCGCGC

[0134] GCGATCACCCTGGAGTCGGCGTCGTTCCCGGCACGGGTGCACGGGCGCATCGTCGAGTCCGACGGTGCC

[0135] GGGCAAGGAAAGCCGGGTCTACCGCAGCGCCATCGGCGTGGTCGGGGTGATCAGCCCCGTGGAACTTC

[0136] CCGCTGCACCTGACCCAGCGTTCCATCGCCCCGGCCCTGGCGCTGGGCAACGCGGTGGTGGTCAAGCC

[0137] GGCCAGCGACACGCCGGTCTGCGGCGGACTGCTGCTGGCGCGGATCTTCGAAGAGGCCGGGCTGCCG

[0138] GCCGGGCTGTTCAGCGTGGTGGTCGGCCCCGGCAGCGAGATCGGCGACGCCTTCGTCGAGCACCCGG

[0139] TGCCGGGCCTGGTGACCTTCACCGGATCGACCCCGGTGGGCCGCAACATCGGCCGCATCGCCAGCGGC

[0140] GGCGCGCACCTCAAGCACGTGGCGCTGGAGCTGGGCGGCAACAGTCCGTTCGTGGTGCTCGGCGACG

[0141] CCGATCTGGAGCAGGCGGTGAATGCCGCGGTGTTCGGCAAGTTCCTCCACCAGGGGCAGATCTGCATG

[0142] GCGATCAACCGCATCATCGTCGAGGACAGCCTCTACGACGCTTTCGCCGCGCGCTTCGTCGAGCGGGTC

[0143] AAGGGTCTCCGGGTCGGCGATCCGCAGCGCGCCGATACCGCGGTCGGGCCGATCGTCAACGCGCGCC

[0144] AGCTCGAAGGCCTGCTGGAAAAGATCCGCCTGGCCCGCCAGGAAGGCGCCAAGCCGCTGTACGAGGG

[0145] CGGCGTCGATGGGCAGTTGCTGGCTCCGCACGTATTCGGCGAGGTCACCGCGACGATGGAGATCGCCC

[0146] GCGATGAAATCTTCGGCCCGCTGGTCGGCCTGCTCCGCGCGCGCGACGAGGCGCATGCGCTGGAGTTG

[0147] GCCAACGCCAGCGAATACGGGCTGTCCAGCGCGGTGTTCAGCCGCGACCTGGAACGCGCGGTGCGCTT

[0148] TGCCCGCCAGCTTCGCGCGGGGATGACCCACGTCAACGACATTCCGGTGAACGACGAGGCCAACGCGC

[0149] CCTTCGGCGGCGAGAAGAACTCCGGACTTGGCCGCTTCAACGGCGACTGGGCCATCGAGGAATTCACC ACCGACCACTGGATCAGCGTGCAGCACGCGCCGCGCCAGTACCCGTTCTAA

[0150] SEQ ID Nr. 8:

[0151] MSDSRYTDLGLQPLAGEWRHGRAGRRLKVSNPFDGSLLLEIEQADRDDLDAAYAKAAEVQPAWAALGPSA

[0152] RAAVLYKAVEVFDRRHEEIVDWIIRESGSTRLKAEIEWGAARAITLESASFPARVHGRIVESDVPGKESRVYRS

[0153] AIGVVGVISPWNFPLHLTQRSIAPALALGNAVVVKPASDTPVCGGLLLARIFEEAGLPAGLFSWVGPGSEIG

[0154] DAFVEHPVPGLVTFTGSTPVGRNIGRIASGGAHLKHVALELGGNSPFVVLGDADLEQAVNAAVFGKFLHQG

[0155] QICMAINRIIVEDSLYDAFAARFVERVKGLRVGDPQRADTAVGPIVNARQLEGLLEKIRLARQEGAKPLYEGG VDGQLLAPHVFGEVTATMEIARDEIFGPLVGLLRARDEAHALELANASEYGLSSAVFSRDLERAVRFARQLR AGMTHVNDIPVNDEANAPFGGEKNSGLGRFNGDWAIEEFTTDHWISVQHAPRQYPF

[0156] The oxidoreductases mentioned here for the oxidation of FFA to FDCA preferably comprise an amino acid sequence which has an identity to SEQ ID No. 2, SEQ ID No. 8, SEQ ID No. 4 or SEQ ID No. 6 of at least 80%, more preferably 85%, more preferably 90%, more preferably 95%, more preferably 98%, more preferably 99%, in particular 100%.

[0157] Alternatively, the oxidoreductases for the oxidation of FFA to FDCA preferably comprise an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 1, SEQ ID No. 7, SEQ ID No. 3 or SEQ ID No. 5 of at least 80%, more preferably 85%, more preferably 90%, more preferably 95%, more preferably 98%, more preferably 99%, in particular 100%. Particularly preferably, the nucleic acid encoding the oxidoreductase according to the invention for the oxidation of FFA to FDCA 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.

[0158] The term "identity," as used herein, refers to the percentage of identical nucleotide or amino acid matches between at least two nucleotide or amino acid sequences aligned using a standardized algorithm. Such an algorithm can, in a standardized and reproducible manner, insert gaps into the compared sequences to optimize the alignment between two sequences, thus achieving a more meaningful comparison of the two sequences.

[0159] The percentage identity between sequences can be determined using one or more computer algorithms or programs known in the art or described herein. According to the invention, the Basic Local Alignment Search Tool (BLAST) (Altschul et al., 1990) provided by the National Center for Biotechnology Information (NCBI) is used to determine identity. The BLAST software suite includes various programs, including a tool called "BLAST 2 Sequences," which is used for the direct pairwise comparison of two nucleotide or amino acid sequences. "BLAST 2 Sequences" can also be accessed and used interactively via the NCBI World Wide Web. The blastn program (for nucleotide sequences) uses a word length (W) of 11, an expectation (E) of 10, M = 5, N = -4, and a comparison of both strands.For amino acid sequences, the blastp program uses a word length of 3 and an expectation (E) of 10 and the BLOSUM62.

[0160] Scoring matrix (Henikoff & Henikoff, 1989), alignments (B) of 50, expectation (E) of 10, M = 5, N = -4.

[0161] Alternatively, the oxidoreductases for oxidizing FFA to FDCA preferably comprise an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a complementary strand of the nucleic acid molecule having the nucleic acid sequence 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 so-called specific hybrids, but not non-specific hybrids, are formed. For example, the stringent conditions comprise hybridization in 6xSSC (sodium chloride / sodium citrate) at 45°C and then washing with 0.2 to 1xSSC, 0.1% SDS at 50 to 65°C; or such conditions may comprise hybridization in 1xSSC at 65 to 70°C and then washing with 0.3xSSC at 65 to 70°C. Hybridization can be performed by conventionally known methods, such as those described by J. Sambrook et al. in Molecular Cloning, A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory (1989).

[0162] The invention also relates to the use of an oxidoreductase for the oxidation of FFA to FDCA, wherein the oxidoreductase comprises an amino acid sequence selected from the group consisting of: i) an amino acid sequence having an identity to SEQ ID No. 2, SEQ ID No. 8, SEQ ID No. 4 or SEQ ID No. 6 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 1, SEQ ID No. 7, SEQ ID No. 3 or SEQ ID No. 5 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a complementary strand of the nucleic acid molecule having the nucleic acid sequence SEQ ID No. 1, SEQ ID No. 7, SEQ ID No. 3 or SEQ ID No. 5.

[0163] According to a preferred embodiment, a sugar or an aldehyde or a keto compound is used as a substrate of the dehydrogenase for the enzymatic oxidation of NAD(P)H.

[0164] D-fructose is preferably used as the sugar and acetone is preferably used as the keto compound.

[0165] In an alternative embodiment, D-glucose is used as the sugar and acetone as the keto compound. In a variant of the process according to the invention, 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), with the former being particularly preferred.

[0166] The xylitol dehydrogenase for the enzymatic oxidation of NAD(P)H by forming D-sorbitol from D-fructose preferably comprises or consists of an amino acid sequence selected from the group consisting of: i) an amino acid sequence having an identity to SEQ ID No. 20 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 19 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a complementary strand of the nucleic acid molecule having the nucleic acid sequence SEQ ID No. 19.

[0167] SEQ ID No. 19:

[0168] ATGAGCACACCGGAAAATCTGAGCTTTGTGCTGCAGAAACCGTTTGATGTGAAATTTGAAGATCGTCCGA TTCCGAAACTGAGCGATCCGTATAGCGTTAAAATTCAGGTGAAAAAAACCGGCATTTGCGGTAGTGATGT TCACTATTTCACCCATGGTGCAATTGGTGATTTTGTTGTTAAAGCACCGATGGTTCTGGGTCATGAAAGCA GCGGTGTTGTTCTGGAAGTTGGTAGCGAAGTTAAAAGCCTGAAAGTTGGTGATCGTGTTGCAATGGAAC CGGGTGTTCCGAGCCGTCATAGTGATGAGTATAAAAGCGGTCGTTATAATCTGTGTCCGCACATGGCATT TGCAGCAACCCCTCCGTATGATGGCACCCTGTGTAAATACTATATTCTGCCGGAAGATTTCTGCGTTAAAC TGCCGGAACATGTTAGCCTGGAAGAAGGTGCACTGGTTGAACCGCTGAGCGTTGCAGTTCATAGCAGCA AACTGGGTAACATTAAACCGGGTAGCCATGTTGCAATTTATGGTGCAGGTCCGGTTGGTCTGCTGGTTGC AGCAGTTGCAAGCGCATTTGGTGCAGAAAGCGTTACCATTATTGATCTGGTTGAAAGCCGTCTGAATCTG GCAAAAGAACTGGGTGCAACCGCAACCGTTCAGGTTGATTTTAAAGATACCCCGAAAGAAAGCGCAGCA AAAGTTGTTGCAGCAAATAATGGCATTGCACCGGATGTTGTTATTGATGCAAGCGGTGCAGAAGCAAGC ATTAATTCAGCCATTAATGCAATTCGTCCGGGTGGCACCTATGTTCAGGTGGGTATGGGTAAACCGGATG TGAGCTTTCCGATTGCAACCCTGATTGGTAAAGAACTGACCGTTAAAGGTAGCTTTCGTTATGGTTATGGT GATTATCCGCTGGCAGTTAGCCTGCTGGCAAGCGGTAAAGTTAATGTGAAAAAACTGATCACCCATGAAGTGAAATTCGAGGATGCAGCAGAAGCATTTCAGCTGGTTCGTGATGGTAAAGCCATTAAATGTATTATCA ACGGTCCGGAATAA

[0169] SEQ ID NO. 20: MSTPENLSFVLQKPFDVKFEDRPIPKLSDPYSVKIQVKKTGICGSDVHYFTHGAIGDFVVKAPMVLGHESSGVV LEVGSEVKSLKVGDRVAMEPGVPSRHSDEYKSGRYNLCPHMAFAATPPYDGTLCKYYILPEDFCVKLPEHVSLE EGALVEPLSVAVHSSKLGNIKPGSHVAIYGAGPVGLLVAAVASAFGAESVTIIDLVESRLNLAKELGATATVQVD FKDTPKESAAKVVAANNGIAPDVVIDASGAEASINSAINAIRPGGTYVQVGMGKPDVSFPIATLIGKELTVKGS FRYGYGDYPLAVSLLASGKVNVKKLITHEVKFEDAAEAFQLVRDGKAIKCIINGPE

[0170] The xylitol dehydrogenase mentioned here preferably comprises an amino acid sequence which has an identity to SEQ ID No. 20 of at least 80%, more preferably 85%, more preferably 90%, more preferably 95%, more preferably 98%, more preferably 99%, in particular 100%.

[0171] Alternatively, the xylitol dehydrogenase preferably comprises an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 19 of at least 80%, more preferably 85%, more preferably 90%, more preferably 95%, more preferably 98%, more preferably 99%, in particular 100%. Particularly preferably, the nucleic acid encoding the xylitol dehydrogenase according to the invention comprises or consists of the nucleic acid sequence SEQ ID No. 19.

[0172] Alternatively, the xylitol dehydrogenase preferably comprises an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a complementary strand of the nucleic acid molecule having the nucleic acid sequence SEQ ID NO: 19. As used herein, stringent conditions refer to conditions under which so-called specific hybrids, but not non-specific hybrids, are formed. For example, the stringent conditions comprise hybridization in 6xSSC (sodium chloride / sodium citrate) at 45°C and then washing with 0.2 to 1xSSC, 0.1% SDS at 50 to 65°C; or such conditions may comprise hybridization in 1xSSC at 65 to 70°C and then washing with 0.3xSSC at 65 to 70°C. The hybridization can be carried out by conventionally known methods, such as those described by J. Sambrook et al. in Molecular Cloning, A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory (1989).

[0173] The invention also relates to the use of a xylitol dehydrogenase for the enzymatic oxidation of NAD(P)H by forming D-sorbitol from D-fructose, wherein the xylitol dehydrogenase comprises an amino acid sequence selected from the group consisting of: i) an amino acid sequence having an identity to SEQ ID No. 20 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 19 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a complementary strand of the nucleic acid molecule having the nucleic acid sequence SEQ ID No. 19.

[0174] In a further variant of the process according to the invention, the enzymatic oxidation of NAD(P)H is accomplished by means of an alcohol dehydrogenase (EC 1.1.1.1 or EC 1.1.1.2).

[0175] The NAD(P)H-dependent alcohol dehydrogenase for the enzymatic oxidation of NAD(P)H by formation of 2-propanol from acetone or preferably consists of an amino acid sequence which is selected from the group consisting of: i) an amino acid sequence which has an identity to SEQ ID No. 10 or SEQ ID No. 12 of at least 80%, ii) an amino acid sequence which is encoded by a nucleic acid which has an identity to SEQ ID No. 9 or SEQ ID No. 11 of at least 80%, and iii) an amino acid sequence which is encoded by a nucleic acid which binds under stringent conditions to a complementary strand of the nucleic acid molecule having the nucleic acid sequence SEQ ID No. 9 or SEQ ID No. 11.

[0176] SEQ ID No. 9:

[0177] ATGAAAGCTGCAGTTGTGGAACAATTTAAAAAGCCGTTACAAGTGAAAGAAGTGGAAAAACCTAAGAT CTCAT ACGGGGAAGTATTAGTGCGCATCAAAGCGTGTGGGGTATGCCATACAGACTTGCATGCCGCAC ATGGCGACTGGCCTGTAAAGCCTAAACTGCCTCTCATTCCTGGCCATGAAGGCGTCGGTGTAATTGAAG AAGTAGGTCCTGGGGTAACACATTTAAAAGTTGGAGATCGCGTAGGTATCCCTTGGCTTTATTCGGCGT GCGGTCATTGTGACTATTGCTTAAGCGGACAAGAAACATTATGCGAACGTCAACAAAACGCTGGCTATT CCGTCGATGGTGGTTATGCTGAATATTGCCGTGCTGCAGCCGATTATGTCGTAAAAATTCCTGATAACTT ATCGTTTGAAGAAGCCGCTCCAATCTTTTGCGCTGGTGTAACAACATATAAAGCGCTCAAAGTAACAGG CGCAAAACCAGGTGAATGGGTAGCCATTTACGGTATCGGCGGGCTTGGACATGTCGCAGTCCAATACG CAAAGGCGATGGGGTTAAACGTCGTTGCTGTCGATTTAGGTGATGAAAAACTTGAGCTTGCTAAACAA CTTGGTGCAGATCTTGTCGTCAATCCGAAACATGATGATGCAGCACAATGGATAAAAGAAAAAGTGGG CGGTGTGCATGCGACTGTCGTCACAGCTGTTTCAAAAGCCGCGTTCGAATCAGCCTACAAATCCATTCG TCGCGGTGGTGCTTGCGTACTCGTCGGATTACCGCCGGAAGAAATACCTATTCCAATTTTCGATACAGT ATTAAATGGAGTAAAAATTATTGGTTCTATCGTTGGTACGCGCAAAGACTTACAAGAGGCACTTCAATT TGCAGCAGAAGGAAAAGTAAAAACAATTGTCGAAGTGCAACCGCTTGAAAACATTAACGACGTATTCGATCGTATGTTAAAAAGGGCAAATTAACGCCGCGTCGTGTTAAAAAGTAGATTAA

[0178] SEQ ID No. 10:

[0179] M KAAVVEQFKKPLQVKEVEKPKISYGEVLVRIKACGVCHTDLHAAHGDWPVKPKLPLIPGHEGVGVIEEVG

[0180] PGVTHLKVGDRVGIPWLYSACGHCDYCLSGQETLCERQQNAGYSVDGGYAEYCRAAADYWKIPDNLSFEE

[0181] AAPIFCAGVTTYKALKVTGAKPGEWVAIYGIGGLGHVAVQYACAMGLNVVAVDLGDEKLELAKQLGADLV

[0182] VNPKHDDAAQWIKEKVGGVHATVVTAVSKAAFESAYKSIRRGCACVLVGLPPEEIPIPIFDTVLNGVKIIGSIV

[0183] GTRKDLQEALQFAAEGKVKTIVEVQPLENINDVFDRMLKGQINGRVVLKVD

[0184] SEQ ID No. 11:

[0185] ATGAAAGGTTTTGCAATGCTCAGTATCGGTAAAGTTGGCTGGATTGAGAAGGAAAAGCCTGCTCCTGGC

[0186] CCATTTGATGCTATTGTAAGACCTCTAGCTGTGGCCCCTTGCACTTCGGACATTCATACCGTTTTTGAAGG

[0187] CGCCATTGGCGAAAGACATAACATGATACTCGGTCACGAAGCTGTAGGTGAAGTAGTTGAAGTAGGTAG

[0188] TGAGGTAAAAGATTTTAAACCTGGTGATCGCGTTGTTGTGCCAGCTATTACCCCTGATTGGCGGACCTCT

[0189] GAAGTACAAAGAGGATATCACCAGCACTCCGGTGGAATGCTGGCAGGCTGGAAATTTTCGAATGTAAAA

[0190] GATGGTGTTTTTGGTGAATTTTTTCATGTGAATGATGCTGATATGAATTTAGCACATCTGCCTAAAGAAAT

[0191] TCCATTGGAAGCTGCAGTTATGATTCCCGATATGATGACCACTGGTTTTCACGGAGCTGAACTGGCAGAT

[0192] ATAGAATTAGGTGCGACGGTAGCAGTTTTGGGTATTGGCCCAGTAGGTCTTATGGCAGTCGCTGGTGCC

[0193] AAATTGCGTGGAGCCGGAAGAATTATTGCCGTAGGCAGTAGACCAGTTTGTGTAGATGCTGCAAAATAC

[0194] TATGGAGCTACTGATATTGTAAACTATAAAGATGGTCCTATCGAAAGTCAGATTATGAATCTAACTGAAG

[0195] GCAAAGGTGTCGATGCTGCCATCATCGCTGGAGGAAATGCTGACATTATGGCTACAGCAGTTAAGATTG

[0196] TTAAACCTGGTGGCACCATCGCTAATGTAAATTATTTTGGCGAAGGAGAGGTTTTGCCTGTTCCTCGTCTT

[0197] GAATGGGGTTGCGGCATGGCTCATAAAACTATAAAAGGCGGGCTATGCCCCGGTGGACGTCTAAGAATG

[0198] GAAAGACTGATTGACCTTGTTTTTTATAAGCGTGTCGATCCTTCTAAGCTCGTCACTCACGTTTTCCGGGG

[0199] ATTTGACAATATTGAAAAAGCCTTTATGTTGATGAAAGACAAACCAAAAGACCTAATCAAACCTGTTGTA ATATTAGCATAA

[0200] SE ID No. 12:

[0201] MKGFAMLSIGKVGWIEKEKPAPGPFDAIVRPLAVAPCTSDIHTVFEGAIGERHNMILGHEAVGEVVEVGSEVK

[0202] DFKPGDRVVVPAITPDWRTSEVQRGYHQHSGGMLAGWKFSNVKDGVFGEFFHVNDADMNLAHLPKEIPLE

[0203] AAVMIPDMMTTGFHGAELADIELGATVAVLGIGPVGLMAVAGAKLRGAGRIIAVGSRPVCVDAAKYYGATDI VNYKDGPIESQIMNLTEGKGVDAAIIAGGNADIMATAVKIVKPGGTIANVNYFGEGEVLPVPRLEWGCGMA HKTIKGGLCPGGRLRMERLIDLVFYKRVDPSKLVTHVFRGFDNIEKAFMLMKDKPKDLIKPVVILA

[0204] The alcohol dehydrogenases mentioned here preferably comprise an amino acid sequence which has an identity to SEQ ID No. 10 or SEQ ID No. 12 of at least 80%, more preferably 85%, more preferably 90%, more preferably 95%, more preferably 98%, more preferably 99%, in particular 100%.

[0205] Alternatively, the alcohol dehydrogenases preferably comprise an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 9 or SEQ ID No. 11 of at least 80%, more preferably 85%, more preferably 90%, more preferably 95%, more preferably 98%, more preferably 99%, in particular 100%. Particularly preferably, the nucleic acid encoding the alcohol dehydrogenases according to the invention comprises or consists of the nucleic acid sequence SEQ ID No. 9 or SEQ ID No. 11.

[0206] Alternatively, the alcohol dehydrogenases preferably comprise an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a complementary strand of the nucleic acid molecule having the nucleic acid sequence SEQ ID NO: 9 or SEQ ID NO: 11. As used herein, stringent conditions refer to conditions under which so-called specific hybrids, but not non-specific hybrids, are formed. For example, the stringent conditions comprise hybridization in 6xSSC (sodium chloride / sodium citrate) at 45°C and then washing with 0.2 to 1xSSC, 0.1% SDS at 50 to 65°C; or such conditions may comprise hybridization in 1xSSC at 65 to 70°C and then washing with 0.3xSSC at 65 to 70°C. The hybridization can be carried out by conventionally known methods, such as those described by J. Sambrook et al. in Molecular Cloning, A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory (1989).

[0207] The invention also relates to the use of an NAD(P)H-dependent alcohol dehydrogenase for the enzymatic oxidation of NAD(P)H by forming an alcohol from a ketone or aldehyde, wherein the alcohol dehydrogenase comprises an amino acid sequence selected from the group consisting of: i) an amino acid sequence having an identity to SEQ ID No. 10 or SEQ ID No. 12 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 9 or SEQ ID No. 11 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a complementary strand of the nucleic acid molecule having the nucleic acid sequence SEQ ID No. 9 or SEQ ID No. 11.

[0208] In a further variant of the process according to the invention, the enzymatic oxidation of NAD(P)H is accomplished by means of a xylose reductase (EC 1.1.1.307, EC 1.1.1.430, EC 1.1.1.431).

[0209] The xylose reductase for the enzymatic oxidation of NAD(P)H by forming 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: i) an amino acid sequence having an identity to SEQ ID No. 14 or SEQ ID No. 16 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 13 or SEQ ID No. 15 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a complementary strand of the nucleic acid molecule having the nucleic acid sequence SEQ ID No. 13 or SEQ ID No. 15.

[0210] SEQ ID Nr. 13:

[0211] ATGGCCACGCCTACTATCAAGCTGAACAGCGGCTATGACATGCCCCTGGTGGGCTTTGGTCTGTGGAAG GTCAACAAGGAAACCTGCGCGGACCAGGTCTACGAGGCTATCAAGGCGGGCTACCGCTTGTTTGACGGT GCGTGCGACTATGGCAACGAAGTTGAGGCCGGCCAGGGTGTCGCTCGCGCCATCAAGGAAGGCATTGT GAAGCGTGAGGACCTCTTCATTGTGTCCAAGCTGTGGAACACGTTCCACGAGGCCGACAAGGTCGAGCC GATCGCGCGGAAGCAGCTGGCCGACTGGGGCCTCGACTACTTTGACCTGTACCTCATCCACTTCCCGATC GCGCTGAAGTACGTCGACCCGGCCGAGATCTACCCGCCGGGCTGGACGGGCACCAAGAAGGAGGTCGA GTTCAGCAACGCGACGATCCAGGAGACGTGGCAGGCCATGGAGACCCTGGTCGACAAGAAGCTGACGC GCAGCATCGGCATCAGCAACTTCAGCGCCCAGCTGATCATGGACCTGCTGCGGTACGCGCGCATCCGCCC CGCGACCTTGCAGATCGAGCACCACCCGTACCTGACGCAGCAGGCGCTGGTCGAGTACGTGCAGAAGGA GGGCATCGCCGTGACGGCGTACTCGTCCTTCGGCCCACTGAGCTTCCTGGAACTGGGCCACCAGGTCGCC AAGGACACGCCGCTGCTCTTCGAGCACTCGACCGTCAAGTCGATCGCCGAGAAGCACGGCAAGACGCCC GCCCAGGTGCTACTGCGCTGGGCCACCCAGCGCAACATCGCCGTCATCCCCAAGAGCAACAACCCGGGC CGCCTGGCGCAGAACCTGGACGTGACGGCGTGGGATCTGGAGCCCGCCGACATTGAGGCCTTGAGCGC GCTGAACAAGAACCTTCGATTCAACAACCCACCTAGCTACGGACTGTACATCCCGATCTTCGCTTAA

[0212] SEQ ID No. 14: MATPTIKLNSGYDMPLVGFGLWKVNKETCADQVYEAIKAGYRLFDGACDYGNEVEAGQGVARAIKEGIVKRE DLFIVSKLWNTFHEADKVEPIARKQLADWGLDYFDLYLIHFPIALKYVDPAEIYPPGWTKEVKETQEFS WQAMETLVDKKLTRSIGISNFSAQLIMDLLRYARIRPATLQIEHHPYLTQQALVEYVQKEGIAVTAYSSFGPLSF LELGHQVAKDTPLLFEHSTVKSIAEKHGKTPAQLLRWATQRNIAVIPKSNNPGRLAQNLDVTAWDLEPADIEALNKALNFYFPYFYFGPLSF

[0213] SE ID Nr. 15:

[0214] ATGACATACCTCGCACCAACAGTTACCTTGAACAATGGATCCAAGATGCCGCTAGTCGGCTTGGGATGCT GGAAAATCCCAAACGAAGTGTGTGCCGAACAGGTGTACGAAGCCATCAAGTTGGGCTACCGCTTGTTCG ACGGCGCGCAGGACTACGCCAACGAAAAAGAGGTGGGCCAAGGTATTAACAGAGCCATCAAGGAAGGA ATCGTCAAGAGAGAAGACTTGGTCGTCGTTTCTAAGTTGTGGAACAGTTTCCACCACCCAGACAACGTGC GTACCGCAGTCGAAAGAACTTTGAACGACTTGCAATTGGACTACTTGGACTTGTTCTACATCCATTTCCCA TTGGCTTTCAAGTTCGTGCCACTAGACGAGAAGTACCCTCCAGGTTTCTACACAGGTAAGGACAATTTCG CCAAGGAAATCATCGAAGAGGAGCCTGTCCCAATCTTGGACACCTACAGAGCCCTTGAGAAGTTGGTCG ACGAAGGTTTGATCAAATCTTTGGGTATCTCAAACTTTTCGGGTGCATTGATCCAGGACTTGTTGCGTGGC GCCCGTATCAAGCCAGTCGCCTTGCAGATCGAACACCACCCATACTTGGTCCAGGACCGCTTGATCACGT ACGCCCAAAAGGTGGGCTTGCAAGTCGTCGCCTACTCCAGTTTCGGCCCACTATCCTTTGTCGAGTTGAA CAACGAAAAGGCCTTGCACACAAAGACTTTGTTCGAAAACGACACCATCAAGGCCATCGCTCAAAAACAC AACGTCACCCCATCCCACGTCTTGTTGAAGTGGTCCACCCAACGTGGTATCGCCGTCATTCCAAAGTCCTC CAAGAAGGAACGTCTCCTCGAGAACTTGAAGATCGAAGAGACCTTTACCTTGTCCGACGAAGAGATCAA GGAGATCAACGGCTTGGACCAGGGATTGAGATTTAACGACCCATGGGACTGGTTGGGCAACGAATTCCCAACCTTTATCTAA

[0215] SEQ ID No. 16:

[0216] MTYLAPTVTLNNGSKMPLVGLGCWKIPNEVCAEQVYEAIKLGYRLFDGAQDYANEKEVGQGINRAIKEGIVK REDLVVVSKLWNSFHHPDNVRTAVERTLNDLQLDYLDLFYIHFPLAFKFVPLDEKYPPGFYTGKDNFAKEIEEE PVPILDTYRALEKLVDEGLIKSLGISNFSGALIQDLLRGARIKPVALQIEHHPYLVQDRLITYAQKVGLQVVAYSSF GPLSFVELNNEKALHTKTLFENDTIKAIAQKHNVTPSHVLLKWSTQRGIAVIPKSSKKERLLENLKIEETFTLSDEE IKEINGLDQGLRFNDPWDWLGNEFPTFI

[0217] The xylose reductases mentioned here preferably comprise an amino acid sequence having an identity to SEQ ID No. 14 or SEQ ID No. 16 of at least 80%, even more preferably 85%, even more preferably 90%, even more preferably 95%, even more preferably 98%, even more preferably 99%, in particular 100%. Alternatively, the xylose reductases preferably comprise an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 13 or SEQ ID No. 15 of at least 80%, even more preferably 85%, even more preferably 90%, even more preferably 95%, even more preferably 98%, even more preferably 99%, in particular 100%. Particularly preferably, the nucleic acid encoding the xylose reductases according to the invention comprises or consists of the nucleic acid sequence SEQ ID No. 13 or SEQ ID No. 15.

[0218] Alternatively, the xylose reductases preferably comprise an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a complementary strand of the 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 so-called specific hybrids, but not non-specific hybrids, are formed. For example, the stringent conditions comprise hybridization in 6xSSC (sodium chloride / sodium citrate) at 45°C and then washing with 0.2 to 1xSSC, 0.1% SDS at 50 to 65°C; or such conditions may comprise hybridization in 1xSSC at 65 to 70°C and then washing with 0.3xSSC at 65 to 70°C. The hybridization can be carried out by conventionally known methods, such as those described by J. Sambrook et al. in Molecular Cloning, A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory (1989).

[0219] The invention 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: i) an amino acid sequence having an identity to SEQ ID No. 14 or SEQ ID No. 16 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 13 or SEQ ID No. 15 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a complementary strand of the nucleic acid molecule having the nucleic acid sequence SEQ ID No. 13 or SEQ ID No. 15.

[0220] In a further variant of the method according to the invention, the enzymatic oxidation of NAD(P)H is accomplished by means of a mannitol dehydrogenase (EC 1.1.1.67; EC 1.1.1.138; EC 1.1.1.255). The mannitol dehydrogenase for the enzymatic oxidation of NAD(P)H by forming D-mannitol from D-fructose preferably comprises or consists of an amino acid sequence selected from the group consisting of: i) an amino acid sequence having at least 80% identity to SEQ ID No. 18, ii) an amino acid sequence encoded by a nucleic acid having at least 80% identity to SEQ ID No. 17, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a complementary strand of the nucleic acid molecule having the nucleic acid sequence SEQ ID No. 17.

[0221] SEQ ID No. 17:

[0222] ATGGCTGCCAACGTCCCCAAGACCATGAAGGCTCTCAGATATGAGAAGCCTGAGACGTTCTCCATCGTCG ACATTCCCGTTCCCACTCTGCGTGAGAACGATGTCTTGATCAAGGTCAAGGCTTGCGGTGTCTGTGGTAC CGACCTGCACATTCACGAGGGAGAATTCCTTGCCAAGTTCCCTCTCGTTCCTGGCCACGAGACTGTCGGT GTTGTTGCCGCAGTTGGACCCAAGGTCAAGGGTTTCGAGATCGGTGACCGTGTTGTTGCCGACAACTCCG AGCTTTGCGGCCAATGCTTCTACTGCCGACGAGGAGAGGAGTTGCTCTGCGAGCACTTCGAAGCTCACG GTGTCACGATGAACGGCGGTTTCGCTGAGTACTGCGCCTACCCTGCCGGCCGTGTCTTCAAGATCAAGAA CCTCTCTGACGTGGACGCCACTCTGCTTGAGCCCGCGTCCTGCGCCGCTCACGGTCTGGACAAGATTGCC CCCAAGATGGGCTCGTCCGTCCTGGTGTTCGGCGCCGGTCCCACCGGTCTGGTCCTTGCTCAGATGCTCC GTCTGAACGGAGGATGCCGCGTCGTCGTCGCTGCGCCCGAGGGTCTGAAGATGGACCTGGCCCAGAAG CTCGGCGCTGGTGATGAATACGTTGCTCTTTCTCGCACGAACCCTCAGGCTCAGTTTGACAAGCTGAAGG CCGACAACCCGTACGGCTTCGACATTGTCGTCGAGGCTACCGGCAATGCCAAGATCCTGGAGGATGCCA TCAACTATGTCCGCCGTGGAGGCAAACTGGTCGTGTACGGTGTGTACGCGAACAAGGACCGCGTCTCGT GGCCCCCGAGCAAGATCTTCGGTGACGAAATCACCATTCTGGGTAGCTTCTCCGAGACCTACAAGTTCCC CGCCGCCATCGACTACCTGGACTCCGGCAAGGTGAAGGTCCAGGGCATCGTGAACAAGACCTTCCGGCTGGAGCAGTGGGAGGAGTGTCTGGCGTCGTTGAAGAACAAGAGCGCCATCAAGGCGGCGATCGTCTTTG ACTAA

[0223] SEQ ID No. 18:

[0224] MAANVPKTMKALRYEKPETFSIVDIPVPTLRENDVLIKVKACGVCGTDLHIHEGEFLAKFPLVPGHETVGVVAA VGPKVKGFEIGDRVVADNSELCGQCFYCRRGEELLCEHFEAHGVTM NGGFAEYCAYPAGRVFKIKNLSDVDA TLLEPASCAAHGLDKIAPKMGSSVLVFGAGPTGLVLAQMLRLNGGCRVVVAAPEGLKM DLAQKLGAGDEYV ALSRTNPQAQFDKLKADNPYGFDIVVEATGNAKILEDAINYVRRGGKLVVYGVYANKDRVSWPPSKIFGDEITI LGSFSETYKFPAAIDYLDSGKVKVQGIVNKTFRLEQWEECLASLKNKSAIKAAIVFD

[0225] The mannitol dehydrogenase mentioned here preferably comprises an amino acid sequence which has an identity to SEQ ID No. 18 of at least 80%, more preferably 85%, more preferably 90%, more preferably 95%, more preferably 98%, more preferably 99%, in particular 100%.

[0226] Alternatively, the mannitol dehydrogenase preferably comprises an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 17 of at least 80%, more preferably 85%, more preferably 90%, more preferably 95%, more preferably 98%, more preferably 99%, in particular 100%. Particularly preferably, the nucleic acid encoding the mannitol dehydrogenase according to the invention comprises or consists of the nucleic acid sequence SEQ ID No. 17.

[0227] Alternatively, the mannitol dehydrogenase preferably comprises an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a complementary strand of the nucleic acid molecule having the nucleic acid sequence SEQ ID NO: 17. As used herein, stringent conditions refer to conditions under which so-called specific hybrids, but not non-specific hybrids, are formed. For example, the stringent conditions comprise hybridization in 6xSSC (sodium chloride / sodium citrate) at 45°C and then washing with 0.2 to 1xSSC, 0.1% SDS at 50 to 65°C; or such conditions may comprise hybridization in 1xSSC at 65 to 70°C and then washing with 0.3xSSC at 65 to 70°C. The hybridization can be carried out by conventionally known methods, such as those described by J. Sambrook et al. in Molecular Cloning, A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory (1989).

[0228] The invention also relates to the use of a mannitol dehydrogenase, wherein mannitol dehydrogenase comprises an amino acid sequence selected from the group consisting of: i) an amino acid sequence having an identity to SEQ ID No. 18 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 17 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a complementary strand of the nucleic acid molecule having the nucleic acid sequence SEQ ID No. 17.

[0229] In further preferred embodiments of the process according to the invention, the concentration of FFA in the aqueous solution is 5 - 200 g / l, particularly preferably 5 - 100 g / l.

[0230] The preferred temperature range is between 15 and 50 °C, the particularly preferred temperature range is between 15 and 40 °C.

[0231] The most preferred pH range of the reaction is between pH 5 and pH 9.

[0232] In a preferred variant of the method according to the invention, the enzymes are present in the homogenate and / or in the lysate of the corresponding cells producing them, with lysates being particularly preferred. The enzymes can also be present in solid form in the aqueous reaction mixture.

[0233] In this context, homogenate refers to a physically and / or chemically treated suspension (e.g., treated by pressure, lysozyme, or ultrasound), whereby the cellular components are released from the cells. A lysate is obtained when the insoluble cellular components of the homogenate are removed, for example, by filtration or centrifugation (see Enzyme Production & Lysate Preparation for details).

[0234] In another variant, the enzymes can also be modified at the N-terminus with a water-soluble polymer such as polyethylene glycol, immobilized in or on a solid matrix, or be part of a fusion protein.

[0235] In another variant, the enzymes can be in powder form, in lyophilized or spray-dried form.

[0236] The following examples describe preferred embodiments of the invention in more detail.

[0237] Materials

[0238] 5-Formyl-2-furancarboxylic acid (FFA) and 2,5-furandicarboxylic acid (FDCA) were obtained from TCI, acetone, potassium dihydrogen phosphate, di-potassium hydrogen phosphate, sodium dodecyl sulfate (SDS) and D-sorbitol were obtained from Carl Roth, NAD + , NADH disodium salt, NADP +Disodium salt, NADPH tetrasodium salt, acetonitrile, and D-fructose were purchased from PanReac AppliChem (ITW Reagents), and triethanolamine was purchased from Chem-Lab NV. Enzyme production and lysate preparation

[0239] General information on the expression of recombinant enzymes in E. coli

[0240] For recombinant enzyme production in an Escherichia coli strain, the gene to be expressed was first amplified by PCR using genomic DNA or its synthetic equivalent, synthetically adapted to the codon usage of E. coli, as a template, together with specific oligonucleotides additionally carrying recognition sequences for restriction endonucleases. The gene fragment encoding the target enzyme was isolated from the reaction mixture. After nucleic acid digestion with the restriction enzymes SphI and HindIII, the gene fragment encoding the target enzyme was ligated into the SphI and HindIII-cleaved backbone of the expression vector pQE70-Kan. The ligation product was transformed into chemically competent E. coli cells (ToplOF), and the resulting colonies were used for plasmid isolation and restriction analysis.

[0241] The result of the cloning step was verified by restriction enzyme digestion and DNA sequencing. The resulting construct carries the target gene under the IPTG-inducible T5 promoter.

[0242] For overexpression of the enzyme in E. coli, the resulting expression plasmid was transformed into the competent expression cells RB791. After 24 h of incubation at 37 °C, the resulting colonies were inoculated into LB medium for expression assays.

[0243] The next day, expression cultures with an optical density (OD550) of 0.02 were inoculated and shaken at 37°C until an OD550 of 0.3 was reached. The temperature was then lowered to 25°C, and the cultures were induced with 0.1 mM IPTG upon reaching an OD550 of 0.5. After 22 h, the cultures were harvested (separated from the medium by centrifugation in the form of a cell pellet) and analyzed for expression of the recombinant enzyme using SDS gel electrophoresis and an activity determination (use in a USE test or optical enzymatic assay).

[0244] Preparation of cell lysates using sonifier disruption

[0245] To prepare a cell suspension, the cell pellet prepared according to the above procedure was weighed into a suitable container and mixed with buffer and lysozyme (final concentration 0.5 mg / ml) (e.g., triethanolamine (TEA) - HCl) and dissolved with stirring. The mass fraction of biomass is typically 20%, with the remainder being the buffer. A Branson Sonifier 450 was used for cell disruption. The suspension was treated three times with 15 ultrasonic pulses each (device settings: Timer = 15; Duty Cycle = 50; Output Control = 3-5).

[0246] The resulting homogenate was centrifuged for 10 min at 4 °C and 16000 rpm (Eppendorf Centrifuge 5417R) to separate the insoluble cell fragments and obtain the lysate.

[0247] Table 1. Enzyme types and donor organisms for the enzymes used in the examples (ALDH = aldehyde dehydrogenase).

[0248] *This ALDH is classified in the NCBI Protein Database (entry WP_009990943.1) as 2,5-dioxopentanoate dehydrogenase (catalyzes the oxidation of 2,5-dioxopentanoate to a-ketoglutarate).

[0249] Analytical methods

[0250] High Performance Liquid Chromatography (HPLC)

[0251] HPLC (high-performance liquid chromatography) was used to quantify FFA and FDCA. Detection was performed using a UV detector. A Phenomenex Rezex ROA-Organic Acid H+ (8%) column with an appropriate precolumn was used for the measurement and eluted isocratically with 1 mM sulfuric acid.

[0252] High-performance liquid chromatography was used to quantify D-fructose and D-sorbitol. Detection is performed using a refractive index detector. A Phenomenex Rezex RCM-Monosaccharide Ca2+ column with an appropriate precolumn is used for the measurement and isocratically eluted with 3.5% isopropanol.

[0253] Determination of enzyme activities (optical-enzymatic assay)

[0254] Enzyme activities in the lysates were determined using a Shimadzu UV-1900 spectrophotometer. The formation or consumption of NAD(P)H was monitored at a wavelength of 340 nm via the change in absorbance. The measurements were performed with 0.2 mM cofactor (NAD(P) + or NAD(P)H). For this purpose, 20 μl of a 10 mM stock solution of the cofactor was placed in a cuvette (Greiner bio-one semi-micro cuvette made of polystyrene), and the desired pH was adjusted with 100 mM TEA-HCl buffer (870 μl). 10 μl of lysate (diluted or undiluted) and 100 μl of substrate solution were added to the cuvette, and the measurement was started immediately. The measurements were carried out at 25 °C as standard. The extinction coefficient of NADH / NADPH at 340 nm (E = 6220 L mol 1 cm 1), the enzyme activity of the lysate can be determined in U / ml (based on the volume of the lysate) or U / g (based on the biomass used for production). 1 U represents 1 pmol of substrate turnover per minute (1 U = 1 pmol / min = l.67-10 _8 kat). The following examples describe preferred variants of the method according to the invention in more detail. The lysates used in these examples were prepared according to the methods described above.

[0255] Example 1

[0256] Oxidation of 5-formyl-2-furancarboxylic acid to 2,5-furandicarboxylic acid - cofactor regeneration using XDH and D-fructose

[0257] The reaction was carried out in a BioXplorer benchtop bioreactor with a polyblock (HEL). The vessel used was a stainless steel reactor (max. volume 400 ml) with an attached stirrer and pH electrode. pH control was achieved by adding 5M NaOH or 1M H2SO4.

[0258] Initially, 5.5 g of FFA and 16 g of D-fructose (600 mM final concentration) were placed in 100 ml of a 100 mM potassium phosphate buffer (pH 7) and brought to 20 °C while stirring. Then, 20 ml of ALDH I lysate, 13 ml of XDH lysate, and 3 ml of a 10 mM NAD + -solution (final concentration 0.2 mM) was introduced. The total volume of the reaction mixture was brought to 150 ml by adding deionized water.

[0259] For analysis, 50 μl of the mixture was mixed with 200 μl of acetonitrile and incubated in an Eppendorf Thermomixer 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 for 5 min at max. 1 g. 250 μl of the supernatant was diluted in an HPLC vial with 750 μl of an acetonitrile / water mixture (1 / 4 v / v) and analyzed by HPLC (UV detection).

[0260] After 4 h, the FFA was completely oxidized to FDCA.

[0261] For processing, the reactor contents were heated to 70 °C and stirred at this temperature for 1 h. After centrifuging off denatured protein, the supernatant was filtered through a fluted filter. This yielded a clear solution, which was acidified to pH < 2 with 10 ml of a 12 M HzSCU solution. Cooling to 4 °C resulted in a precipitate, which was filtered off. In this way, 5.1 g of FDCA were isolated as a solid. Example 2

[0262] Oxidation of 5-formyl-2-furancarboxylic acid to 2,5-furandicarboxylic acid - cofactor regeneration using ADH and acetone

[0263] The reaction was carried out in a Labfors benchtop bioreactor (Infors AG). A glass reactor (volume 3.4 l) with a stirrer and pH electrode was used as the vessel. pH was controlled by adding 5M NaOH or 1M H2SO4.

[0264] Initially, 27.7 g of FFA were placed in 550 ml of a 100 mM potassium phosphate buffer (pH 7) and brought to 20 °C while stirring. Then, 67 ml of ALDH I lysate, 30 ml of ADH lysate, 10 ml of a 10 mM NAD + -solution (final concentration 0.2 mM) and 15 ml of acetone were added. Additionally, an overpressure of 320 mbar was applied.

[0265] For analysis, 50 μl of the mixture was mixed with 200 μl of acetonitrile and incubated in an Eppendorf Thermomixer 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 for 5 min at max. 1 g. 250 μl of the supernatant was diluted in an HPLC vial with 750 μl of an acetonitrile / water mixture (1 / 4 v / v) and analyzed by HPLC (UV detection).

[0266] After 4.5 h, the FFA was completely oxidized to FDCA. FDCA can be isolated as a solid analogously to Example 1.

[0267] Example 3

[0268] Oxidation of 5-formyl-2-furancarboxylic acid to 2,5-furandicarboxylic acid with aldehyde dehydrogenase II (ALDH II) and cofactor regeneration using XDH and D-fructose

[0269] The following components were mixed in a glass vial: 300 μl of an FFA solution (11.9 g / l), 10 μl of deionized water, 50 μl of ALDH II lysate, 50 μl of a 1 M potassium phosphate buffer (pH 8), 50 μl of a 1.5 M D-fructose solution, and 40 μl of XDH lysate. The mixture was incubated for a total of 20 h with continuous shaking (Eppendorf Thermomixer; 20 °C, 800 rpm).

[0270] For analysis, 50 μl of the mixture was mixed with 200 μl of acetonitrile and incubated in an Eppendorf Thermomixer 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 for 5 min at max. 1 g. 200 μl of the supernatant was transferred to an HPLC vial with an insert and analyzed by HPLC (UV detection).

[0271] In this way, 40.8% of the FFA (7.1 g / l) was oxidized to FDCA.

[0272] Example 4

[0273] Oxidation of 5-formyl-2-furancarboxylic acid to 2,5-furandicarboxylic acid with aldehyde dehydrogenase III (ALDH III) and cofactor regeneration using ADH and acetone

[0274] The following components were mixed in a glass vial: 5.2 mg FFA, 325 μl deionized water, 35 μl ALDH 111 lysate, 100 μl of a 500 mM potassium phosphate buffer (pH 8), 15 μl acetone, 30 μl ADH lysate, and 5 μl of a 10 mM NAD +-solution. The mixture was incubated for a total of 24 h with continuous shaking (Eppendorf Thermomixer; 20 °C, 800 rpm).

[0275] For analysis, 50 μl of the mixture was mixed with 200 μl of acetonitrile and incubated in an Eppendorf Thermomixer 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 for 5 min at max. 1 g. 200 μl of the supernatant was transferred to an HPLC vial with an insert and analyzed by HPLC (UV detection).

[0276] In this way, 48.0% of the FFA was oxidized to FDCA.

[0277] Example 5

[0278] Oxidation of 5-formyl-2-furancarboxylic acid to 2,5-furandicarboxylic acid with aldehyde dehydrogenase II (ALDH II) and various dehydrogenases for cofactor regeneration

[0279] The following components were mixed in 3 glass vials (vials 1 - 3): 300 μl of an FFA solution (final concentration 7.1 g / l), 50 μl of a 1 M potassium phosphate buffer (pH 8), 50 μl of ALDH I I-lysate, 5 μl of a 10 mM NADP + solution, 50 μl of dehydrogenase lysate (see Table 2 below), 50 μl of a 1.5M substrate solution (see Table 2 below), and 5 μl of deionized water. The mixture was incubated with continuous shaking (Eppendorf Thermomixer; 20 °C, 800 rpm) for a total of 24 h.

[0280] For analysis, 50 μl of the mixture was mixed with 200 μl of acetonitrile and incubated in an Eppendorf Thermomixer 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 for 5 min at max. 1 g. 200 μl of the supernatant was transferred to an HPLC vial with an insert and analyzed by HPLC (UV detection).

[0281] The results are shown in Table 2 below.

[0282] Table 2.

[0283] The results in Table 2 show that different dehydrogenases react with different substrates for cofactor regeneration (in this case NADP + ) are suitable.

[0284] Example 6

[0285] Oxidation of 5-formyl-2-furancarboxylic acid to 2,5-furandicarboxylic acid with aldehyde dehydrogenase II (ALDH II) and cofactor regeneration using ADH and dihydroxyacetone

[0286] The following components were mixed in a glass vial: 143 μl of an FFA solution (final concentration 12 g / l), 125 μl of a 250 mM potassium phosphate buffer (pH 7), 10 μl of ALDH II lysate, 10 μl of a 5 mM NADP +solution, 10 μl of dehydrogenase lysate (see Table 2 below), 50 μl of a 300 g / l dihydroxyacetone solution, and 152 μl of deionized water. The mixture was incubated with continuous shaking (Eppendorf Thermomixer; 30 °C, 800 rpm) for a total of 20 h.

[0287] For analysis, 50 μl of the mixture was mixed with 200 μl of acetonitrile and incubated in an Eppendorf Thermomixer 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 for 5 min at max. 1 g. 200 μl of the supernatant was transferred to an HPLC vial with an insert and analyzed by HPLC (UV detection).

[0288] In this way, 66% of the FFA was oxidized to FDCA. Table 3.

[0289] Example 7

[0290] Oxidation of 5-formyl-2-furancarboxylic acid to 2,5-furandicarboxylic acid with aldehyde dehydrogenase III (ALDH III) and cofactor regeneration using ADH and acetone

[0291] The following components were mixed in a glass vial: 143 μl of an FFA solution (final concentration 12 g / l), 187 μl of deionized water, 10 μl of ALDH IV lysate, 125 μl of a 1 M potassium phosphate buffer (pH 7), 15 μl of acetone, 10 μl of ADH I I lysate, and 10 μl of a 10 mM NADP + -solution. The mixture was incubated with continuous shaking (Eppendorf Thermomixer; 30 °C, 800 rpm) for a total of 20 h.

[0292] For analysis, 50 μl of the mixture was mixed with 200 μl of acetonitrile and incubated in an Eppendorf Thermomixer 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 for 5 min at max. 1 g. 200 μl of the supernatant was transferred to an HPLC vial with an insert and analyzed by HPLC (UV detection).

[0293] In this way, 85.0% of the FFA was oxidized to FDCA.

[0294] Example 8

[0295] Oxidation of 5-formyl-2-furancarboxylic acid to 2,5-furandicarboxylic acid with aldehyde dehydrogenase

[0296] V (ALDH V) and cofactor regeneration using ADH and acetone The following components were mixed in a glass vial: 85 μl of a substrate solution (final concentration 6.5 g / l FFA), 255 μl deionized water, 25 μl ALDH V suspension, 100 μl of a 500 mM potassium phosphate buffer (pH 8), 15 μl acetone, 30 μl ADH I lysate, and 5 μl of a 10 mM NAD + -solution. The mixture was incubated with continuous shaking (Eppendorf Thermomixer; 20 °C, 800 rpm) for a total of 20 h.

[0297] For analysis, 50 μl of the mixture was mixed with 200 μl of acetonitrile and incubated in an Eppendorf Thermomixer 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 for 5 min at max. 1 g. 200 μl of the supernatant was transferred to an HPLC vial with an insert and analyzed by HPLC (UV detection).

[0298] In this way, no conversion of FFA to FDCA was observed.

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[0324] Henikoff, S., & Henikoff, J. G. (1992). Amino acid substitution matrices from protein blocks. Proceedings of the National Academy of Sciences of the United States of America, 89(22), 10915-10919. https: / / doi.org / 10.1073 / pnas.89.22.10915

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[0326] Protein [Internet], Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information. Accession No. WP_024766379.1, aldehyde dehydrogenase family protein [Pseudomonas nitroreducens]. Verfügbar unter: https: / / www.ncbi.nlm.nih.gOv / protein / WP_024766379.l / (Zugriff am 21.12.2023)

[0327] Protein [Internet], Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information. Accession No. WP_009990943.1, 2,5-dioxopentanoate dehydrogenase [Saccharolobus solfataricus]. Verfügbar unter: https: / / www.ncbi.nlm.nih.gOv / protein / WP_009990943.l / (Zugriff am 21.12.2023)

[0328] Protein [Internet], Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information. Accession No. WP_015829138.1, aldehyde dehydrogenase family protein [Methylovorus glucosotrophus]. Verfügbar unter: https: / / www.ncbi.nlm.nih.gOv / protein / WP_015829138.l / (Zugriff am 21.12.2023)

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Claims

Patent claims 1.Process for the preparation of 2,5-furandicarboxylic acid by reacting 5-formyl-2-furancarboxylic acid, which is present in an aqueous solution, with an NAD(P) + -dependent oxidoreductase in vitro to 2,5-furandicarboxylic acid, whereby the NAD(P)H produced during the oxidation is enzymatically converted back to NAD(P) by means of a dehydrogenase + is oxidized, after which the enzymes are removed. 2.Process according to claim 1, characterized in that the NAD(P) + -dependent oxidoreductase for the oxidation of 5-formyl-2-furancarboxylic acid to 2,5-furandicarboxylic acid is an aldehyde dehydrogenase. S.Process according to one of claims 1 or 2, characterized in that the NAD(P) +- dependent oxidoreductase for the oxidation of 5-formyl-2-furancarboxylic acid to 2,5-furandicarboxylic acid has an amino acid sequence which is selected from the group consisting of: i) an amino acid sequence which has an identity to SEQ ID No. 2, SEQ ID No. 8, SEQ ID No. 4 or SEQ ID No. 6 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 1, SEQ ID No. 7, SEQ ID No. 4 or SEQ ID No. 5 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid which binds under stringent conditions to a complementary strand of the nucleic acid molecule having the nucleic acid sequence SEQ ID No. 1, SEQ ID No. 7, SEQ ID No. 3 or SEQ ID No.

5.

4. Process according to one of claims 1 to 3, characterized in that a sugar or an aldehyde or a keto compound is used as a substrate of the dehydrogenase for the enzymatic oxidation of NAD(P)H.

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

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

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

19.

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

11.

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

15.

10. The method according to any one of claims 1 to 6, characterized in that a mannitol dehydrogenase is used as dehydrogenase for the enzymatic oxidation of NAD(P)H by forming D-mannitol from D-fructose, which has an amino acid sequence selected from the group consisting of: i) an amino acid sequence having an identity to SEQ ID No. 18 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 17 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a complementary strand of the nucleic acid molecule having the nucleic acid sequence SEQ ID No.

17.

11. Use of a xylitol dehydrogenase for the enzymatic oxidation of NAD(P)H by forming D-sorbitol from D-fructose, wherein the xylitol dehydrogenase comprises an amino acid sequence selected from the group consisting of: i) an amino acid sequence having an identity to SEQ ID No. 20 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 19 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a complementary strand of the nucleic acid molecule having the nucleic acid sequence SEQ ID No.

19.

12. Use of an NAD(P)H-dependent alcohol dehydrogenase for the enzymatic oxidation of NAD(P)H by forming an alcohol from a ketone or aldehyde, wherein the alcohol dehydrogenase comprises an amino acid sequence selected from the group consisting of: i) an amino acid sequence which has at least 80% identity to SEQ ID No. 10 or SEQ ID No. 12, ii) an amino acid sequence which is encoded by a nucleic acid which has at least 80% identity to SEQ ID No. 9 or SEQ ID No. 11, and iii) an amino acid sequence which is encoded by a nucleic acid which binds under stringent conditions to a complementary strand of the nucleic acid molecule having the nucleic acid sequence SEQ ID No. 9 or SEQ ID No.

11.

13. 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: i) an amino acid sequence having an identity to SEQ ID No. 14 or SEQ ID No. 16 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 13 or SEQ ID No. 15 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a complementary strand of the nucleic acid molecule having the nucleic acid sequence SEQ ID No. 13 or SEQ ID No.

15.

14. Use of a mannitol dehydrogenase for the enzymatic oxidation of NAD(P)H, wherein mannitol dehydrogenase comprises an amino acid sequence selected from the group consisting of: i) an amino acid sequence having an identity to SEQ ID No. 18 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 17 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a complementary strand of the nucleic acid molecule having the nucleic acid sequence SEQ ID No. 17.

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