Process for preparation of 2, 5-furandicarboxylic acid

Abstract

The invention relates to a method for producing 2, 5-furandicarboxylic acid by the in vitro oxidation of 5-formyl-2-furancarboxylic acid 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 oxidized back to NAD (P) + by means of an NAD (P) H oxidase via an enzymatic reaction, the enzyme is then removed.

Description

[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

[0003] In 2018, about 90% (4 Mt) of the globally produced plastics were based on fossil feedstocks, the remaining part was based on 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 is also growing. In 2019, the annual carbon dioxide emissions of the whole life cycle of plastics were 0.86 Gt, which is equivalent to the carbon dioxide emissions of 189 coal-fired power plants operating at full capacity (500 MW). It is estimated that this figure will increase to 2.8 Gt by 2050 (equivalent to 615 coal-fired power plants) (Hamilton et al., 2019).

[0004] One of the plastics based on fossil feedstocks is polyethylene terephthalate (PET), which is mainly used for beverage packaging. It is a polycondensation polymer produced from terephthalic acid (1,4-benzenedicarboxylic acid) and ethylene glycol (ethane-1,2-diol) by a dehydration reaction.

[0005] Terephthalic acid is produced on an industrial scale by a process called AMOCO: by oxidizing p-xylene (1,4-dimethylbenzene) in the presence of cobalt acetate, manganese acetate and HBr in acetic acid at about 200 °C in atmospheric oxygen (Tomás et al., 2013).

[0006] Ethylene glycol is produced by the hydrolysis of ethylene oxide (at 200 °C), which in turn is obtained by the oxidation of ethylene (obtained from fossil feedstocks) (Berger, 2016). Due to the fossil origin of the starting materials (p-xylene and ethylene), PET cannot usually be called a sustainable product.

[0007] In order to achieve the 1.5 °C target of the Paris Climate Agreement, thus limiting the negative effects of climate change, sustainable alternatives must be found for plastics based on oil chemistry. For example, for ethylene glycol, there are methods for its production based on renewable resources. For example, ethylene can also be produced by the dehydration of bioethanol, which is obtained from the fermentation of glucose or starch (e.g. Fan et al., 2013). Methods for the fermentation of xylose and / or glucose to ethylene glycol by means of metabolically engineered microorganisms (Escherichia coli or Saccharomyces cerevisiae) are also known (Salusjärvi et al., 2019).

[0008] The second building block of PET is the aromatic compound terephthalic acid, which is difficult to produce from sustainable raw materials and therefore needs to be replaced. 2,5-Furfurandicarboxylic acid (FDCA) is an excellent alternative to terephthalic acid, primarily obtained through the catalytic modification of biomass. FDCA is polymerized with ethylene glycol to produce polyethylene furanate (PEF), in which the benzene ring is replaced by a heteroaromatic furan ring. Similar to PET, PEF is a thermoplastic, but compared to PET, PEF has significantly higher biodegradability and better thermal properties (higher glass transition temperature, lower melting temperature) and mechanical properties (higher rigidity). However, the most important property of PEF is its reduced permeability to gases such as oxygen and carbon dioxide, which is particularly important for beverages as it can extend shelf life (preventing carbonated beverages from leaking and preventing the spread of oxygen from initiating oxidation processes) (de Jong et al., 2022).

[0009] Cong et al. (2021) outlined a variety of chemical synthesis methods in their article.

[0010] For FDCA, the most important starting material is undoubtedly 5-(hydroxymethyl)furfural (HMF), which can be obtained from, for example, cellulose, and thus from renewable sources. Cellulose is hydrolyzed enzymatically or chemically to produce D-glucose, which is then isomerized enzymatically or chemically to produce D-fructose. D-fructose undergoes a dehydration reaction (removing a total of three water molecules) to yield HMF. Common systems for fructose dehydration are primarily inorganic acids, such as H₂SO₄ or HCl, and secondarily (Brønsted or Lewis) acidic solid catalysts (Cong et al., 2021; US ​​9617234 B1).

[0011] HMF contains alcohol and aldehyde functional groups, which need to be oxidized to carboxyl groups to obtain FDCA. The three-step oxidation reaction required for this process can be carried out in a variety of ways: chemical methods using heterogeneous or homogeneous catalysts, electrochemical methods, and biocatalytic methods (enzymatic or whole-cell).

[0012] The direct precursor of FDCA is FFA (5-formyl-2-furanoic acid), which can be prepared, for example, by acid-catalyzed dehydration of the sugar acid derivatives 2-keto-D-gluconate (2KGA) or 5-keto-D-gluconate (5KGA). In a flow reactor at 80 °C, 2KGA can be dehydrated to FFA by treatment with HBr in an acetic acid / water mixture. In the final step, FFA can be oxidized to FDCA using oxygen as an oxidant, a metal (e.g., Pt-Ru / C) or metal salts (cobalt and manganese salts) as catalysts, or hydrogen peroxide (US 10087161 B2).

[0013] The acid-catalyzed dehydration of 5KGA to produce FFA is also described in EP 3265450 B1. The final oxidation step is carried out in acetic acid at 180 °C under oxygen overpressure in the presence of catalytic amounts of cobalt acetate, manganese acetate, and NaBr (similar to the Amoco process).

[0014] Generally, the above-mentioned chemical methods for preparing FDCA starting from FFA have disadvantages such as harsh reaction conditions (high temperature and high pressure), generation of by-products, and use of expensive and partially toxic catalysts (cobalt salts), and therefore cannot be called sustainable methods.

[0015] Alternatively, biocatalytic processes can be employed, which exhibit high selectivity under mild reaction conditions and utilize biodegradable catalysts such as cells or enzymes (Cong et al., 2021).

[0016] FFA can be prepared by enzymatic oxidation of HMF (mainly using oxygen as the oxidant).

[0017] One class of enzymes used to prepare FFA from HMF is aromatic alcohol oxidase (AAO, EC 1.1.3.7). Carro et al. (2015) used AAO from the fungus Pleurotus eryngii to oxidize 3 mM HMF to FFA (98 mol%) in 4 hours, generating a small amount of FDCA.

[0018] AAO-catalyzed aldehyde oxidation occurs via the corresponding geminal diol (aldehyde hydrate), where the aldehyde hydration in FFA is lower than that in the precursor 2,5-dicarboxyfuran (DFF) (DFF: 53%, FFA: 8%), explaining why FFA is preferentially generated over FDCA. The byproduct H₂O₂ from AAO-catalyzed oxidation chemically oxidizes FFA to FDCA (Carro et al., 2015). In a follow-up study by Serrano et al. (2019) on AAO in *Pleurotus eryngii*, it was found that when H₂O₂ was removed using catalase, HMF could be completely oxidized to FDCA because H₂O₂ inhibits the final oxidation step from FFA to FDCA. In this way, using the AAO mutant, 1.5 mM HMF could be oxidized to 1.6 mM FDCA within 6 days.

[0019] Other known AAOs are derived from the mycobacterium MS1601, which completely oxidizes 4 g / l (31.7 mM) HMF to FFA within 120 hours (Sayed et al., 2022), and from the fungus Moesziomyces antarcticus, which oxidizes 2 mM HMF to 99.6 mol% FFA and 0.4 mol% FDCA within 24 hours, and oxidizes 40% of FFA (2 mM) to FDCA within 144 hours (Lappe et al., 2021).

[0020] Qin et al. (2015) tested various laccases in 30 mM HMF using 20 mol% TEMPO ((2,2,6,6-tetramethylpiperidin-1-yl)-oxy) as the medium. Using laccase from the fungus *Panus conchatus*, 82% of the starting material was oxidized to FFA within 96 hours, with byproducts of 4% 2,5-dicarboxyfuran (DFF) and 10% FDCA.

[0021] Zhang et al. (2019) immobilized laccase (CotA-TJ102) from Bacillus subtilis TJ-102 onto magnetic nanoparticles, and used it to oxidize 83.3% of the starting material HMF to FFA (after 10 cycles) with a selectivity greater than 96%.

[0022] Jia et al. (2019) used an enzyme system consisting of galactose oxidase (GOase), alcohol dehydrogenase (SADH) from Synechocystis sp., and HRP to oxidize 97% of the substrate HMF (100 mM) to FFA within 48 hours. Besides activating GOase M… 3-5 In addition, HRP is also used for NAD(P) + The regeneration of NAD(P), and NAD(P) + It is a cofactor required by SADH.

[0023] US 10344307 B2 discloses other enzymes (systems) for oxidizing HMF to FFA: 1) NAD(P)-dependent ketone reductases and NAD(P)H oxidases (for cofactor regeneration); 2) NAD(P)-dependent aldehyde dehydrogenases and NAD(P)H oxidases; and 3) xanthine oxidoreductases, such as periplasmic aldehyde oxidoreductase (PaoABC) from Escherichia coli, which oxidizes HMF to 5-hydroxymethyl-2-furanoic acid (HMFA) and DFF to FFA; and galactose oxidase variant M.3-5 (GOase M) 3-5 It oxidizes HMF to DFF and HMFA to FFA; and horseradish peroxidase (HRP) is used to activate GOase M. 3-5 For example, at pH 6, PaoABC can completely oxidize 50 mM DFF to FFA within 2 hours, while at pH 7 and 8, the oxidation can proceed completely to FDCA. The enzyme system in 3) above is also described in detail in journal articles (McKenna et al., 2015; McKenna et al., 2017).

[0024] Besides PaoABC and AAO, other enzymes that can catalyze the final step of FFA oxidation to FDCA are also known.

[0025] Cajnko et al. (2020) tested several 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. For 10 mM FFA, only AO, laccase, and LPO showed significant FDCA production after 72 hours (11.6% for AO, 1.1% for laccase, and 3.2% for LPO). A byproduct was found to be 5-hydroxymethyl-2-furanoic acid (HMFA) (AO byproduct content was as high as 18.2%).

[0026] Another enzyme used to oxidize FFA to FDCA is a nonspecific peroxygenase from *Agrocybe aegerita* (UPO, EC 1.11.2.1; requires H2O2 as an oxidant), which can be used in conjunction with AAO to completely oxidize HMF to FDCA. Thus, 90% of FFA (3 mM) can be oxidized to FDCA within 120 hours (Carro et al., 2015). In addition to catalyzing the oxidation of FFA to FDCA, UPO also catalyzes the oxidation of HMF to FFA via DFF (Lappe et al., 2021).

[0027] Jia et al. (2017) used horse liver alcohol dehydrogenase (HLADH) and human hemoglobin (NADH to NAD through H2O2 oxidation of NADH to NAD) + An enzyme system composed of 10 mM FFA oxidizes 96% of the substrate (10 mM FFA) to FDCA within 60 hours.

[0028] US 8183020 B2 describes a method for enzymatically oxidizing FFA to FDCA using commercially available chloroperoxidase (EC1.11.1.10) from Caldariomyces fumago, wherein H2O2 is used as the oxidant.

[0029] Aldehyde dehydrogenases (ALDHs) are a class of enzymes that catalyze the oxidation of aldehyde groups to carboxylic acid groups. A specific ALDH from *Raoultella ornithinolytica* BF60 has been described, which can oxidize FFA to FDCA and HMF to HMFA (Hossain et al., 2017).

[0030] US 10344307 B2 describes a method for oxidizing DFF using commercially available ALDH (in combination with NAD(P)H oxidase), which, depending on the reaction conditions (DFF concentration from 10 to 100 mM), produces FFA, FDCA, or a mixture thereof. As the DFF concentration increases, 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, all conversions after 3 hours). For the oxidation reaction, US 10344307 B2 uses 10 to 50 mol% of cofactors (based on the amount of substrate).

[0031] The methods described in this section often have drawbacks such as low substrate concentration or uneconomical addition of large amounts of cofactors.

[0032] The present invention aims to provide a method for preparing FDCA that improves upon the method described in US 10344307 B2, particularly by achieving higher conversion rates at higher substrate concentrations while reducing the amount of cofactors required. Summary of the Invention

[0033] The objective of this invention is achieved through the following technical solution: by using NAD(P) + Treatment with a formyl-2-furanoic acid (FFA) in aqueous solution was performed in vitro to 2,5-furandicarboxylic acid. During the oxidation process, NAD(P)H generated was oxidized back to NAD(P) by an enzymatic reaction catalyzed by NAD(P)H oxidase. + The enzyme is then removed, characterized by the use of an H2O-forming NAD(P)H oxidase having an amino acid sequence selected from the group consisting of:

[0034] i) Having an amino acid sequence that is at least 80% identical to SEQ ID No. 8, SEQ ID No. 14, SEQ ID No. 12 or SEQ ID No. 10;

[0035] ii) An amino acid sequence encoded by a nucleic acid having at least 80% identity with SEQ ID No. 7, SEQ ID No. 13, SEQ ID No. 11, or SEQ ID No. 9; and

[0036] 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. 7, SEQ ID No. 13, SEQ ID No. 11 or SEQ ID No. 9 under stringent conditions.

[0037] FDCA can then be separated from solution by precipitation and / or crystallization.

[0038] A preferred embodiment of the method of the present invention is as follows: Figure 1 As shown.

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

[0040] NAD(P) used to oxidize FFA to FDCA + Type-dependent oxidoreductases contain, or preferably select, amino acid sequences from the group consisting of:

[0041] i) Having an amino acid sequence that is at least 80% identical to SEQ ID No. 16, SEQ ID No. 2, 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. 15, SEQ ID No. 1, SEQ ID No. 3, or SEQ ID No. 5; and

[0043] 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. 15, SEQ ID No. 1, SEQ ID No. 3 or SEQ ID No. 5 under stringent conditions.

[0044] SEQ ID No. 1:

[0045]

[0046] SEQ ID No. 2:

[0047] MSRYELLIDGRLQAAEHYDRVIDPASEEIVGEAARASLEQVDAAVDAAHRAFPAWATDLDVRRQSLARAAERVRENAQALAELITREQGRPLRSTLEEVAGVAATFEHHAQLELPADTQLRDDGERLVRITRKPLGVVAAITPWNVPLILLVLKIAPALHAGNTVVAKPSEHTPLSTLLLARLLGDVFPAGVFNVVAGAGEVGEHLVRHPRVRHVTFTGSVATGKRLYAGAGDDLKRLTLELGGNDAALVLEDADLDAIVEPLFWGAFWNSGQVCFAIKRLYVHDSLFEPLLAKLAERAQRTRLGHGLDPQTELGPLTNAQQLERVIALVEDAKAHGARIRSGGVRPDGPGYFYPPTLVSGVAAGVALVDEEQFGPVLPVISFRNEEDAITQANASHYGLGASVWTRDLARGEAIARRLEAGLAWVNQHGHIQPGAPKGGHKWSGLGYEGGQRGYEAFSELQVLNISRR

[0048] SEQ ID No. 3:

[0049]

[0050] SEQ ID No. 4:

[0051] MKSYQGLADKWIKGSGEEYLDINPADKDHVLAKIRLYTKDDVKEAINKAVAKFDEWSRTPAPKRGSILLKAGELMEQEAQEFALLMTLEEGKTLKDSMFEVTRSYNLLKFYGALAFKISGKTLPSADPNTRIFTVKEPLGVVALITPWNFPLSIPVWKLAPALAAGNTAVIKPATKTPLMVAKLVEVLSKAGLPEGVVNLVVGKGSEVGDTIVSDDNIAAVSFTGSTEVGKRIIKLVGNKNRMTRIQLELGGKNALYVDKSADLTLAAELAVRGGFGLTGQSCTATSRLIINKDVYTQFKQRLLERVKKWRVGPGTEDVDMGPVVDEGQFKKDLEYIEYGKNVGAKLIYGGNIIPGKGYFLEPTIFEGVTSDMRLFKEEIFGPVLSVTEAKDLDEAIRLVNAVDYGHTAGIVASDIKAINEFVSRVEAGVIKVNKPTVGLELQAPFGGFKNSGATTWKEMGEDALEFYLKEKTVYEGW

[0052] SEQ ID No. 5:

[0053]

[0054] SEQ ID No. 6:

[0055] MSTFHLLIDGHLQASDQSDVVINPATELEVGRAPRASATQVDQAVEAAHQAFHRWASQPEVRQQALLGAAAAIRQHADALARLITQEQGRPLHFTQGEVAGAAATFEHYAGFAAPSDVVLQQDEQKRVSIERRPFGVVAAITPWNVPIILLVLKIAPALKAGNTVVAKPSEYTPLSTLYLGEILKDVFPPGVLNVIAGDGQVGARLASHPLVQKVTFTGSVATGKKLYASAAQDVKRLTLELGGNDAAIVLDDANVDAIAEKIFWGAFWNSGQVCFAIKRLYVHERVFQPLLDALVKRAQKTRVGDGQLPGTELGPLTNKAQFERVISLVEDARRHGATIHSGGAALPGPGYFYPPTLVTGIGAGVALVDEEQFGPVLPLIPFRDEQEAVRQANDSPFGLGASVWTANPERGLALVRQLQAGLAWVNQHGDIHPGAPKGGYKSSGLGYEGGLRGYDEFSELQVVNAALV

[0056] SEQ ID No. 15:

[0057]

[0058] SEQ ID No. 16:

[0059] MSDSRYTDLGLQPLAGEWRHGRAGRRLKVSNPFDGSLLLEIEQADRDDLDAAYAKAAEVQPAWAALGPSARAAVLYKAVEVFDRRHEEIVDWIIRESGSTRLKAEIEWGAARAITLESASFP ARVHGRIVESDVPGKESRVYRSAIGVVGVISPWNFPLHLTQRSIAPALALGNAVVVKPASDTPVCGGLLLARIFEEAGLPAGLFSVVVGPGSEIGDAFVEHPVPGLVTFTGSTPVGRNIGRI ASGGAHLKHVALELGGNSPFVVLGDADLEQAVNAAVFGKFLHQGQICMAINRIIVEDSLYDAFAARFVERVKGLRVGDPQRADTAVGPIVNARQLEGLLEKIRLARQEGAKPLYEGGVDGQL LAPHVFGEVTATMEIARDEIFGPLVGLLRARDEAHALELANASEYGLSSAVFSRDLERAVRFARQLRAGMTHVNDIPVNDEANAPFGGEKNSGLGRFNGDWAIEEFTTDHWISVQHAPRQYPF

[0060] The oxidoreductases listed herein for oxidizing FFA to FDCA preferably contain an amino acid sequence having at least 80%, more preferably 85%, more preferably 90%, more preferably 95%, more preferably 98%, more preferably 99%, and especially 100% identity with SEQ ID No. 2, SEQ ID No. 4, or SEQ ID No. 6.

[0061] Alternatively, the oxidoreductase for oxidizing FFA to FDCA preferably comprises an amino acid sequence encoded by a nucleic acid having at least 80%, more preferably 85%, more preferably 90%, more preferably 95%, more preferably 98%, more preferably 99%, and particularly 100% identity with SEQ ID No. 1, SEQ ID No. 3, or SEQ ID No. 5. The nucleic acid encoding the oxidoreductase of the present invention for oxidizing FFA to FDCA particularly preferably comprises the nucleic acid sequence SEQ ID No. 1, SEQ ID No. 3, or SEQ ID No. 5, or a combination thereof.

[0062] As used in this article, “identity” refers to the percentage of identical nucleotides or amino acids matched between at least two nucleotide or amino acid sequences aligned using a normalization algorithm (alignment). This algorithm can insert gaps in the aligned sequences in a normalized and reproducible manner to optimize the alignment between the two sequences, thereby enabling a more meaningful comparison.

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

[0064] Alternatively, the oxidoreductase used to oxidize FFA to FDCA preferably comprises an amino acid sequence encoded by a nucleic acid that binds to the complementary strand of a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 1, SEQ ID No. 3, or SEQ ID No. 5 under stringent conditions. As used herein, “stringent conditions” refers to conditions based on which so-called specific hybridization occurs without non-specific hybridization. For example, stringent conditions may include hybridization at 45 °C in 6 × SSC (sodium chloride / sodium citrate), followed by washing at 50 to 65 °C with 0.2 to 1 × SSC, 0.1% SDS; or such conditions may include hybridization at 65 to 70 °C in 1 × SSC, followed by washing at 65 to 70 °C with 0.3 × SSC. Hybridization can be performed by methods known in the art, such as those described by J. Sambrook et al. in Molecular Cloning: A Laboratory Manual, Second Edition (1989, Cold Spring Harbor Laboratory).

[0065] The present invention also discloses the use of an oxidoreductase in the oxidation of FFA to FDCA, wherein the oxidoreductase comprises an amino acid sequence selected from the group consisting of:

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

[0067] ii) An amino acid sequence encoded by a nucleic acid having at least 80% identity with SEQ ID No. 1, SEQ ID No. 3, or SEQ ID No. 5; and

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

[0069] The NAD(P)H oxidase used for cofactor regeneration may be derived from one of the following groups: EC 1.6.3.1 (H2O2-forming NAD(P)H oxidase), EC 1.6.3.2 (H2O-forming NAD(P)H oxidase), EC 1.6.3.3 (H2O2-forming NADH oxidase), and EC 1.6.3.4 (H2O-forming NADH oxidase), with H2O-forming oxidases being particularly preferred.

[0070] The H2O-forming NAD(P)H oxidases that are particularly preferred to use contain or preferably have an amino acid sequence selected from the group consisting of:

[0071] i) Having an amino acid sequence that is at least 80% identical to SEQ ID No. 8, SEQ ID No. 14, SEQ ID No. 12, or SEQ ID No. 10;

[0072] ii) An amino acid sequence encoded by a nucleic acid having at least 80% identity with SEQ ID No. 7, SEQ ID No. 13, SEQ ID No. 11, or SEQ ID No. 9; and

[0073] 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. 7, SEQ ID No. 13, SEQ ID No. 11 or SEQ ID No. 9 under stringent conditions.

[0074] SEQ ID No. 7:

[0075]

[0076] SEQ ID No. 8:

[0077] MKVVVGCTHAGTAAVKTILNEHPDASVSVYERNDNVSFLSCGIALYVGGVVKDPAGLFYSSPEELASMGAKINMEHNVKNIDNENKVVVIENLKTGETFEESYDKLVMTTGSWPIIPPIDGINSENILLCKNYNQANEIIKESKNAKKIVIVGGGYIGIELVEAFAESGKQVTLVDGLDRILNKYLDAEFTSVLEHDLQERGVTLALNQTVEKFVANESGAVTAVKTPVGEYEADLVILCVGFKPNTDLLKDKVEMLPNGAIVVDEYMRTSDEAIFAAGDSCAVHYNPTGGSAYIPLATNAVRMGALVGKNIVSPTVKYRGTQATSGLYLFGFNIGSTGLTENSAPHFGVEVRSVVEDNYRPEFMPTEKVTMKLVYEVGTNRIVGGQIMSKYDVTQSANTLSLCVQNKMTIEDLAYVDFFFQPHFDRPWNYLNILAQAAVEQERKLAK

[0078] SEQ ID No. 9:

[0079]

[0080] SEQ ID No. 10:

[0081] MSKIVIVGANGHAGTAINDNYGSENEVVVFDQNSNISFLGCGMALWIGKQISGPQGLFYADKESLEAKGAKIYMESPVTAIDYDAKRVTALVNGQEHVESYEKLILATGSTPILPPIKGAAIKEGSRDFEATLKNLQFVKLYQNAEDVINKLQDKSQNLNRIAVVGAGYIGVELAEAFKRLGKEVILIDVVDTCLAGYYDQDLSEMMRQNLEDHGIELAFGETVKAIEGDGKVERIVTDKASHDVDMVILAVGFRPNTALGNAKLKTFRNGAFLVDKKQETSIPDVYAIGDCATVYDNAINDTNYIALASNALRSGIVAGHNAAGHKLESLGVQGSNGISIFGLNMVSTGLTQEKAKRFGYNPEVTAFTDFQKASFIEHDNYPVTLKIVYDKDSRLVLGAQMASKEDMSMGIHMFSLAIQEKVTIERLALLDYFFLPHFNQPYNYMTKAALKAK

[0082] SEQ ID No. 11:

[0083]

[0084] SEQ ID No. 12:

[0085] MKVAVIGCTHAGTAAVKTILTENDDVEVVVFERNDNISFLSCGIALYVGGVVKDVNGLFYSDPSELESLGATVYMKHNVLSFDENTKVIQVENMETGEHFQESYDKLVIATGSWPIIPDLPGLDLENVMLCKNFKHAQELIQTKQDKKRVAVIGAGYIGIELVEAFAEDGKEVVLIDGADRVLPKYLDQEMTDLLEASLVDHGVQMQLGEFVESFLADDEGKVRAVKTSKGEYECDMAVLCVGFQPNTELYKGKLETMPNGAIIVDDYMHTSHPDIYACGDSCAVNYNPNDGHAYIPLATNAVRMGSLVGKNIKADRVKYRGTQSTSGLKLFGWNIGSTGVTDNSASSFNLETRSVYVEDNYRPEFMPTTEKVYMKLVYEVGTNRVVGGQLMSKYDITQSANTLSLAIQTKQTIEDLAYVDFFFQPHFDRPWNYLNILAQAALSQEEELAQN

[0086] SEQ ID No. 13:

[0087]

[0088] SEQ ID No. 14:

[0089] MKVVVGCTHAGTSAVKTILNEHPNTEVTVFERNDNVSFLSCGIALYVGGVVKDPAGLFYSNPEELTEMGATVHMEHNVTNIDTVAKKVTVENMQTGEVFEESYDKLVNTTGSWPIVPPISGIESKNILLCKNYNQANEIIRQAKDKQKVVIVGGGYIGIELVEAFAESGKDVTLIDGLDRILNKYLDPEFDTDILEHDLQERGIKLALNQTVNGFEANENGEVTKVVTSENSFETEMVIMCVGFRPNNELLKDKVDMLPNGAIIVDEYMRTSDKDIYAAGDSCAVHYNPNGGSAYIPLATNAVRMGTLVGKNIVEPSVKYRGTQSTSGLYLFGFNIGSTGVNVNSASHFGLDVRSVVVEDYYRPEFMPTNEKVLMKLVYEVGTNRIVGGQVMSKYDITQSANTLSLAVQNKMTIEDLAYVDFFFQPVFDRPWNYLNLLAQAAVEQERKIATGTEVTV

[0090] SEQ ID No. 17:

[0091]

[0092] SEQ ID No. 18:

[0093] MKVVVVGCTHAGTAAVKTILNEHPDASVSVYERNDNVSFLSCGIALYVGGVVKDPAGLFYSSPEELASMGAKINMEHNVKNIDNENKVVVIENLKTGETFEESYDKLVMTTG SWPIIPPIDGINSENILLCKNYNQANEIIKESKNAKKIVIVGGGYIAIELVEAFAESGKQVTLVARSDRILRKYLDAEFTSVLEHDLQERGVTLALNQTVEKFVANESGAVTA VKTPVGEYEADLVILCVGFKPNTDLLKDKVEMLPNGAIVVDEYMRTSDEAIFAAGDSCAVHYNPTGGSAYIPLATNAVRMGALVGKNIVSPTVKYRGTQATSGLYLFGFNIGS TGLTENSAPHFGVEVRSVVVEDNYRPEFMPTTEKVTMKLVYEVGTNRIVGGQIMSKYDVTQSANTLSLCVQNKMTIEDLAYVDFFFQPHFDRPWNYLNILAQAAVEQERKLAK

[0094] The preferred H2O-forming NAD(P)H oxidase comprises or is preferably composed of an amino acid sequence having at least 80%, more preferably 85%, more preferably 90%, more preferably 95%, more preferably 98%, more preferably 99%, and particularly 100% identity with SEQ ID No. 8, SEQ ID No. 14, SEQ ID No. 12, or SEQ ID No. 10. More preferably, the H2O-forming NAD(P)H oxidase comprises or is composed of the amino acid sequence of SEQ ID No. 8, SEQ ID No. 14, SEQ ID No. 12, or SEQ ID No. 10.

[0095] Alternatively, the H2O-forming NAD(P)H oxidase preferably comprises an amino acid sequence encoded by a nucleic acid having at least 80%, more preferably 85%, more preferably 90%, more preferably 95%, more preferably 98%, more preferably 99%, and particularly 100% identity with SEQ ID No. 7, SEQ ID No. 13, SEQ ID No. 11, or SEQ ID No. 9. More preferably, the nucleic acid encoding the H2O-forming NAD(P)H oxidase comprises or consists of the nucleic acid sequence SEQ ID No. 7, SEQ ID No. 13, SEQ ID No. 11, or SEQ ID No. 9.

[0096] The method of this invention particularly preferably uses NAD(P)H oxidase, which is capable of reducing NADP or oxidizing NADPH. In this document, a mutant of SEQ ID No. 8 is particularly preferred, which comprises or consists of the amino acid sequence SEQ ID No. 18. This mutant is preferably encoded by the nucleic acid sequence SEQ ID No. 17.

[0097] Another aspect of the invention relates to the use of H2O-forming NAD(P)H oxidase, which comprises or consists of an amino acid sequence selected from the group consisting of:

[0098] i) Having an amino acid sequence that is at least 80% identical to SEQ ID No. 8, SEQ ID No. 14, SEQ ID No. 12 or SEQ ID No. 8;

[0099] ii) An amino acid sequence encoded by a nucleic acid having at least 80% identity with SEQ ID No. 7, SEQ ID No. 13, SEQ ID No. 11, or SEQ ID No. 9; and

[0100] 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. 7, SEQ ID No. 13, SEQ ID No. 11 or SEQ ID No. 9 under stringent conditions.

[0101] In another preferred embodiment of the method of the present invention, the concentration of FFA in the aqueous solution is 5-200 g / l, particularly preferably 5-100 g / l.

[0102] The preferred temperature range is 15 to 50 °C, and the particularly preferred temperature range is 15 to 40 °C.

[0103] The optimal pH range for the reaction is 5 to 9.

[0104] In another preferred variant of the method of the present invention, the enzyme is present in a suspension, homogenate and / or lysate of the corresponding enzyme-producing cells, wherein the lysate is particularly preferred.

[0105] The suspensions used in this article refer to suspensions of resting cells. These resting cells are harvested (separated from the culture medium) after culture and suspended in a suitable buffer system. Unlike fermentation methods that also use whole cells, these resting cells cannot be regrowed due to the removal of carbon sources and nutrients, and are used only for substrate transformation (Lin & Tao, 2017). The homogenates used in this article refer to suspensions that have undergone physical and / or chemical treatment (e.g., by pressure, lysozyme, or sonication) in which cellular components are released from the cells. Lysates are obtained by removing insoluble cellular components from the homogenate, for example by filtration or centrifugation (see “Enzyme Production and Lysate Preparation”).

[0106] In another variant, the enzyme may also be modified at the N-terminus with a water-soluble polymer (such as polyethylene glycol), immobilized in or on a solid matrix, or as part of a fusion protein.

[0107] In another variant, the enzyme may exist in powder form, lyophilized form, or spray-dried form.

[0108] Preferred embodiments of the present invention will be described in more detail in the following examples.

[0109] Material

[0110] 5-Formyl-2-furanoic acid (FFA) and 2,5-furandicarboxylic acid (FDCA) were purchased from TCI; potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and sodium dodecyl sulfate (SDS) were purchased from Carl Roth; NAD + Disodium NADH, NADP + Disodium salt, tetrasodium NADPH and acetonitrile were purchased from PanReac AppliChem (ITW Reagents); and triethanolamine was purchased from Chem-Lab NV.

[0111] Enzyme production and preparation of lysates

[0112] Summary of expression of recombinant enzymes in E. coli

[0113] To produce recombinases in *E. coli* strains, genomic DNA or a synthetic genomic DNA equivalent matching the *E. coli* codons was first used as a template. The gene to be expressed was amplified by PCR using additional specific oligonucleotides carrying restriction endonuclease recognition sequences, and the gene was isolated from the reaction mixture. After digestion with restriction enzymes SphI and HindIII, the gene fragment encoding the target enzyme was ligated into the backbone of the expression vector pQE70-Kan, which had been digested with SphI and HindIII. The ligation product was transformed into chemically competent *E. coli* Top10F cells, and the resulting colonies were used for plasmid isolation and restriction enzyme digestion analysis.

[0114] The cloning results were verified by restriction enzyme digestion and DNA sequencing. The resulting construct carried the target gene, which was controlled by the IPTG-inducible T5 promoter. 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 clone was inoculated into LB medium for expression testing.

[0115] The next day, inoculation with optical density OD 550 The expression culture was prepared at 0.02 mg / L and shaken at 37°C until OD200 increased. 550 It reached 0.3. The temperature was then lowered to 25 °C, when the OD... 550 When the concentration reached 0.5, the culture was induced with 0.1 mM IPTG. After 22 hours, the culture was harvested (separated from the culture medium as a cell pellet by centrifugation), and the expression of the recombinase was analyzed by SDS-polyacrylamide gel electrophoresis and activity assay (using Use-Test or optical enzymatic assay).

[0116] Preparation of cell lysates by sonication

[0117] To prepare the cell suspension, the cell pellet prepared by the above method is weighed into a suitable container, and a buffer solution (e.g., triethanolamine (TEA)-hydrochloric acid) and lysozyme (final concentration 0.5 mg / ml) are added and stirred to dissolve. The biomass mass fraction is typically 20%, with the remainder being buffer solution.

[0118] Cell disruption was performed using a Branson Sonifier 450. The suspension was sonicated three times, with 15 ultrasonic pulses each time. Instrument settings: timer = 15; duty cycle = 50; output control = 3-5.

[0119] The resulting homogenate was centrifuged at 16,000 rpm for 10 minutes at 4°C (Eppendorf centrifuge 5417R) to separate insoluble cell debris and obtain lysates.

[0120] Table 1: Enzyme types and donor organisms used in the examples (ALDH = aldehyde dehydrogenase)

[0121]

[0122] This ALDH is classified as a 2,5-dioxovalerate dehydrogenase in the NCBI protein database (accession number WP_009990943.1), catalyzing the oxidation of 2,5-dioxovalerate to α-ketoglutarate. The amino acid sequence of NOX I shares 40.32% identity with NOX V and 35.93% with NOX VI. The amino acid sequence of NOX II shares 75.45% identity with NOX V and 47.22% with NOX VI. The amino acid sequence of NOX III shares 67.19% identity with NOX V and 45.33% with NOX VI. The amino acid sequence of NOX IV shares 79.02% identity with NOX V and 47.23% with NOX VI.

[0123] Analytical methods

[0124] High performance liquid chromatography (HPLC)

[0125] Quantitative analysis of FFA and FDCA was performed using high-performance liquid chromatography (HPLC). Detection was performed using a UV detector. Measurements were taken using a Phenomenex Rezex ROA-Organic Acid H+ (8%) column and a corresponding pre-column, with isocratic elution using 1 mM sulfuric acid.

[0126] Enzyme activity assay (optical enzymatic assay)

[0127] Enzyme activity in the lysates was determined using a Shimadzu UV-1900 spectrophotometer. To this end, the generation or consumption of NAD(P)H was tracked by changes in absorbance at 340 nm. Measurements were performed using 0.2 mM cofactor (NAD(P)H). + The assay was performed using either NADH / NADPH or NAD(P)H. For this purpose, 20 µl of 10 mM cofactor stock solution was added to a cuvette (polystyrene semi-micro cuvette, Greiner bio-one), and the pH was adjusted to the desired value (870 µl) with 100 mM TEA-HCl buffer. 10 µl of lysate (diluted or undiluted) and 100 µl of substrate solution were added to the cuvette, and the measurement was started immediately. Measurements were performed at 25 °C. The extinction coefficient of NADH / NADPH at 340 nm (ε = 6220 L mol) was used as a reference.-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 for preparation). 1 U represents the conversion of 1 µmol of substrate per minute (1 U = 1 µmol / min = 1.67 × 10⁻⁶). -8 kat).

[0128] The following examples will describe preferred variations of the method of the present invention in more detail. The pyrolytes used in these examples were prepared by the method described above.

[0129] Example 1: Oxidation of 5-formyl-2-furanoic acid to 2,5-furandicarboxylic acid using aldehyde dehydrogenase I (ALDH I).

[0130] 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.

[0131] Initially, 5.5 g of FFA was added to 150 ml of 100 mM potassium phosphate buffer (pH 7), and heated to 20 °C with stirring. Then, 20 ml of ALDH I lysate, 13 ml of NADH oxidase lysate, and 3 ml of 10 mM NAD were added. + Solution (final concentration 0.2 mM). Additionally, an overpressure of 320 mbar (O2 atmosphere) was applied.

[0132] For analysis, 200 µl of acetonitrile was mixed with 50 µl of the sample and incubated in an Eppendorf thermostatic mixer at 85 °C and 1200 rpm for 15 min. The sample was briefly centrifuged, 750 µl of deionized water was added, vortexed, and then centrifuged at maximum centrifugation for 5 min. 250 µl of the supernatant was diluted with 750 µl of acetonitrile / water mixture (1 / 4 v / v) in an HPLC vial and measured by HPLC (UV detection).

[0133] After 20 hours, FFA was completely oxidized to FDCA.

[0134] The reactor contents were then heated to 70°C and stirred at that temperature for 1 hour. After centrifugation to separate the denatured proteins, the supernatant was filtered through a pleated filter. A clear solution was obtained, and its pH was adjusted to <2 by adding 10 ml of 12M H₂SO₄ solution. After cooling to 4°C, FDCA precipitate formed, which was filtered and dried in a vacuum drying oven at 50°C.

[0135] Example 2: Oxidation of 5-formyl-2-furanoic acid to 2,5-furandicarboxylic acid using aldehyde dehydrogenase II (ALDH II).

[0136] Mix the following components in a glass sample vial: 300 µl FFA solution (11.9 g / l), 60 µl deionized water, 50 µl ALDH II lysate, 50 µl 1M potassium phosphate buffer (pH 8), and 40 µl NADH oxidase lysate. Incubate the sample for 20 hours with continuous shaking (Eppendorf thermostatic mixer; 20 °C, 800 rpm).

[0137] For analysis, 200 µl of acetonitrile was mixed with 50 µl of the sample and incubated in an Eppendorf thermostatic mixer at 85 °C and 1200 rpm for 15 minutes. The sample was briefly centrifuged, 750 µl of deionized water was added, and the mixture was vortexed, followed by centrifugation at maximum speed for 5 minutes. 200 µl of the supernatant was transferred to an HPLC vial with an insert sample tube, and the analysis was performed by HPLC (UV detection).

[0138] In this way, 34.5% of FFA (7.1 g / l) was oxidized to FDCA (measured concentration: 2.4 g / l).

[0139] Example 3: Oxidation of 5-formyl-2-furanoic acid to 2,5-furandicarboxylic acid using aldehyde dehydrogenase III (ALDH III).

[0140] Mix the following components in a glass sample vial: 5.1 mg FFA, 325 µl deionized water, 35 µl ALDH III lysate, 100 µl 500 mM potassium phosphate buffer (pH 8), 30 µl NADH oxidase lysate, and 5 µl 10 mM NAD. + Solution. Incubate the sample for 24 hours with continuous shaking (Eppendorf thermostatic mixer; 20 °C, 800 rpm).

[0141] For analysis, 200 µl of acetonitrile was mixed with 50 µl of the sample and incubated in an Eppendorf thermostatic mixer at 85 °C and 1200 rpm for 15 minutes. The sample was briefly centrifuged, 750 µl of deionized water was added, and the mixture was vortexed, followed by centrifugation at maximum speed for 5 minutes. 200 µl of the supernatant was transferred to an HPLC vial with an insert sample tube, and the analysis was performed by HPLC (UV detection).

[0142] In this way, 57.0% of FFA (10.2 g / l) was oxidized to FDCA (measured concentration: 5.9 g / l).

[0143] Example 4: Testing the oxidation of 5-formyl-2-furanoic acid to 2,5-furandicarboxylic acid using different NADH oxidases

[0144] The following components were mixed in seven glass sample vials: 143 µl FFA solution, 212 µl deionized water, 10 µl ALDH I lysate, 125 µl 1M potassium phosphate buffer (pH 7), and 10 µl NADH oxidase lysate (see Table 2 below). The samples were incubated for 20 hours with continuous shaking (Eppendorf thermostatic mixer; 30 °C, 800 rpm).

[0145] For analysis, 200 µl of acetonitrile was mixed with 50 µl of the sample and incubated in an Eppendorf thermostatic mixer at 85 °C and 1200 rpm for 15 minutes. The sample was briefly centrifuged, 750 µl of deionized water was added, and the mixture was vortexed, followed by centrifugation at maximum speed for 5 minutes. 200 µl of the supernatant was transferred to an HPLC vial with an insert sample tube, and the analysis was performed by HPLC (UV detection).

[0146] The results are shown in Table 2 below.

[0147]

[0148] Table 2 shows that using NADH oxidases I-IV resulted in FDCA yields >50%, while on the other hand, using NADH oxidases V and VI yields were only 13% to 21% in the same reaction time.

[0149] Example 5: Oxidation of 5-formyl-2-furanoic acid to 2,5-furandicarboxylic acid using NAD(P)H oxidase (NOX VII)

[0150] Mix the following components in a glass sample vial: 143 µl FFA solution (final concentration 12 g / L), 187 µl deionized water, 10 µl ALDH IV lysate, 125 µl 1M potassium phosphate buffer (pH 7), 10 µl NOX VII lysate, and 10 µl 10mM NADP. + Solution. The sample was incubated for 20 hours under continuous shaking (Eppendorf thermostatic mixer; 30°C, 800 rpm).

[0151] For analysis, 200 µl of acetonitrile was mixed with 50 µl of the sample and incubated in an Eppendorf thermostatic mixer at 85 °C and 1200 rpm for 15 minutes. The sample was briefly centrifuged, 750 µl of deionized water was added, and the mixture was vortexed, followed by centrifugation at maximum speed for 5 minutes. 200 µl of the supernatant was transferred to an HPLC vial with an insert sample tube, and the analysis was performed by HPLC (UV detection).

[0152] In this way, 65.0% of FFA is oxidized to FDCA.

[0153] literature

[0154] Carus, M., Dammer, L., Raschka, A., Skoczinski, P., & vom Berg, C. (2020). Renewable Carbon—Key to a Sustainable and Future-Oriented Chemical and Plastic Industry. https: / / renewable-carbon-initiative.com / wp-content / uploads / 2020 / 09 / 20-09-21_Paper_12-on-Renewable-Carbon.pdf (Access date: 17.10.2022)

[0155] Hamilton, LA, Feit, S., Muffett, C., Kelso, M., Rubright, SM, Bernhardt, C., Schaeffer, E., Moon, D., Morris, J., & Labbé-Bellas, R. (2019). Plastic & Climate: The Hidden Costs of a Plastic Planet. https: / / www.ciel.org / wp-content / uploads / 2019 / 05 / Plastic-and-Climate-FINAL-2019.pdf (Access date: 18.10.2022)

[0156] Tomás, R. A. F., Bordado, J. C. M., & Gomes, J. F. P. (2013). p-Xylene Oxidation to Terephthalic Acid: A Literature Review Oriented towardProcess Optimization and Development. Chemical Reviews, 113(10), 7421–7469.https: / / doi.org / 10.1021 / cr300298j

[0157] Berger, A. (2016). Ethylenglycol, RD-05-01999 in Böckler, F., Dill,B., Eisenbrand, G., Faupel, F., Fugmann, B., Gamse, T., Matissek, R.,Pohnert, G., Rühling, A., Schmidt, S., Sprenger, G. (Ed.), RÖMPP [Online],Stuttgart, Georg Thieme Verlag. https: / / roempp.thieme.de / lexicon / RD-05-01999(Retrieval date: 22.12.2023)

[0158] Fan, D., Dai, D.-J., Wu, H.-S. (2013). Ethylene Formation byCatalytic Dehydration of Ethanol with Industrial Considerations. Materials 6(1), 101-115. https: / / doi.org / 10.3390 / ma6010101 <unk>0000348<unk> It should be noted that the tag

[0159] in the original text seems to be incomplete or incorrect. I've translated it as <unk>0000348<unk> to maintain the integrity of the translation as much as possible while indicating the potential issue. If this is a specific format that needs to be preserved exactly, more context would be helpful for a more accurate translation.Salusjärvi, L., Havukainen, S., Koivistoinen, O., & Toivari, M.(2019). Biotechnological production of glycolic acid and ethylene glycol:current state and perspectives. Applied Microbiology and Biotechnology, 103,2525–2535. https: / / doi.org / 10.1007 / s00253-019-09640-2

[0160] de Jong, E., Visser, H. A., Dias, A. S., Harvey, C., Gruter, G.-J. M.(2022). The Road to Bring FDCA and PEF to the Market. Polymers, 14, 943.https: / / doi.org / 10.3390 / polym14050943

[0161] Cong, H., Yuan, H., Tao, Z., Bao, H., Zhang, Z., Jiang, Y., Huang,D., Liu, H., & Wang, T. (2021). Recent Advances in Catalytic Conversion ofBiomass to 2,5-Furandicarboxylic Acid. Catalysts, 11(9), 1113. https: / / doi.org / 10.3390 / catal11091113

[0162] Carro, J., Ferreira, P., Rodríguez, L., Prieto, A., Serrano, A.,Balcells, B., Ardá, A., Jiménez-Barbero, J., Gutiérrez, A., Ullrich, R.,Hofrichter, M., & Martínez, A. T. (2015). 5-hydroxymethylfurfural conversionby fungal aryl-alcohol oxidase and unspecific peroxygenase. FEBS Journal, 282(16), 3218–3229. https: / / doi.org / 10.1111 / febs.13177

[0163] Serrano, A., Calviño, E., Carro, J., Sánchez-Ruiz, M. I., Cañada, F.J., & Martínez, A. T. (2019). Complete oxidation of hydroxymethylfurfural tofurandicarboxylic acid by aryl-alcohol oxidase. Biotechnology for Biofuelsand Bioproducts, 12, 217. https: / / doi.org / 10.1186 / s13068-019-1555-z

[0164] Sayed, M., Gaber, Y., Junghus, F., Martín, E. V., Pyo, S.-H., &Hatti-Kaul, R. (2022). Oxidation of 5-hydroxymethylfurfural with a novel arylalcohol oxidase from Mycobacterium sp. MS1601. Microbial Biotechnology, 15(8), 2176–2190. https: / / doi.org / 10.1111 / 1751-7915.14052

[0165] Lappe, A., Jankowski, N., Albrecht, A., Koschorreck, K. (2021).Characterization of a thermotolerant aryl-alcohol oxidase from Moesziomycesantarcticus oxidizing 5-hydroxymethyl-2-furancarboxylic acid. AppliedMicrobiology and Biotechnology, 105, 8313–8327. https: / / doi.org / 10.1007 / s00253-021-11557-8

[0166] Qin, Y.-Z., Li, Y.-M., Zong, M.-H., Wu, H., & Li, N. (2015). Enzyme-catalyzed selective oxidation of 5-hydroxymethylfurfural (HMF) and separationof HMF and 2,5-diformylfuran using deep eutectic solvents. Green Chemistry,17(7), 3718–3722. https: / / doi.org / 10.1039 / C5GC00788G

[0167] Zhang, C., Chang, X., Zhu, L., Xing, Q., You, S., Qi, W., Su, R., &He, Z. (2019). Highly efficient and selective production of FFCA from CotA-TJ102 laccase-catalyzed oxidation of 5-HMF. International Journal ofBiological Macromolecules, 128, 132–139. https: / / doi.org / 10.1016 / j.ijbiomac.2019.01.104

[0168] Jia, H.-Y., Zong, M.-H., Zheng, G.-W., & Li, N. (2019). One-PotEnzyme Cascade for Controlled Synthesis of Furancarboxylic Acids from 5-Hydroxymethylfurfural by H2O2 Internal Recycling. ChemSusChem, 12(21), 4764–4768. https: / / doi.org / 10.1002 / cssc.201902199

[0169] McKenna, S. M., Leimkühler, S., Herter, S., Turner, N. J., & Carnell,A. J. (2015). Enzyme cascade reactions: Synthesis of furandicarboxylic acid(FDCA) and carboxylic acids using oxidases in tandem. Green Chemistry, 17,3271–3275. https: / / doi.org / 10.1039 / C5GC00707K

[0170] McKenna, S. M., Mines, P., Law, P., Kovacs-Schreiner, K., Birmingham,W. R., Turner, N. J., Leimkühler, S., & Carnell, A. J. (2017). The continuousoxidation of HMF to FDCA and the immobilisation and stabilisation ofperiplasmic aldehyde oxidase (PaoABC). Green Chemistry, 19, 4660–4665.https: / / doi.org / 10.1039 / C7GC01696D

[0171] Cajnko, M. M., Novak, U., Grilc, M., & Likozar, B. (2020). Enzymaticconversion reactions of 5-hydroxymethylfurfural (HMF) to bio-based 2,5-diformylfuran (DFF) and 2,5-furandicarboxylic acid (FDCA) with air:mechanisms, pathways and synthesis selectivity. Biotechnology for Biofuels,13, 66. https: / / doi.org / 10.1186 / s13068-020-01705-z

[0172] Jia, H.-Y., Zong, M.-H., Yu, H.-L., & Li, N. (2017). Dehydrogenase-Catalyzed Oxidation of Furanics: Exploitation of Hemoglobin CatalyticPromiscuity. ChemSusChem, 10(18), 3524–3528. https: / / doi.org / 10.1002 / cssc.201701288

[0173] Hossain, G. S., Yuan, H., Li, J., Shin, H., Wang, M., Du, G., Chen,J., & Liu, L. (2017). Metabolic Engineering of Raoultella ornithinolyticaBF60 for Production of 2,5-Furandicarboxylic Acid from 5-Hydroxymethylfurfural. Applied and Environmental Microbiology, 83(1), e02312-16. https: / / doi.org / 10.1128 / AEM.02312-16

[0174] Altschul, S. F., Gish, W., Miller, W., Myers, E. W., & Lipman, D. J.(1990). Basic local alignment search tool. Journal of Molecular Biology, 215(3), 403–410. https: / / doi.org / 10.1016 / S0022-2836(05)80360-2

[0175] Henikoff, S., & Henikoff, J. G. (1992). Amino acid substitutionmatrices from protein blocks. Proceedings of the National Academy of Sciencesof the United States of America, 89(22), 10915–10919. https: / / doi.org / 10.1073 / pnas.89.22.10915

[0176] Sambrook, J., Fritsch, E. R., & Maniatis, T. (1989). MolecularCloning: A Laboratory Manual (2nd ed.). Cold Spring Harbor, NY: Cold SpringHarbor Laboratory Press.

[0177] Lin, B., & Tao, Y. (2017). Whole-cell biocatalysts by design.Microbial Cell Factories, 16, 106. https: / / doi.org / 10.1186 / s12934-017-0724-7

[0178] 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]. Available at: https: / / www.ncbi.nlm.nih.gov / protein / WP_024766379.1 / (Accessed: 21.12.2023)

[0179] 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]. Available at: https: / / www.ncbi.nlm.nih.gov / protein / WP_009990943.1 / (Accessed: 21.12.2023)

[0180] 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]. Available at: https: / / www.ncbi.nlm.nih.gov / protein / WP_015829138.1 / (Accessed: 21.12.2023)

[0181] Matsumoto, J., Higuchi, M., Shimada, M., Yamamoto, Y., & Kamio, Y.(1996). Molecular Cloning and Sequence Analysis of the Gene Encoding the H2O-forming NADH Oxidase from Streptococcus mutans. Bioscience, Biotechnology,and Biochemistry, 60(1), 39–43. https: / / doi.org / 10.1271 / bbb.60.39。

Claims

1. A method for preparing 2,5-furandicarboxylic acid, said method being carried out by: using NAD(P) + 5-Formyl-2-furancarboxylic acid, present in aqueous solution, is oxidized in vitro to 2,5-furandicarboxylic acid by treatment with a oxidoreductase-dependent enzyme, wherein the NAD(P)H generated during the oxidation is oxidized back to NAD(P) by an enzymatic reaction catalyzed by NAD(P)H oxidase. + Subsequently, the enzyme is removed, characterized in that, The H2O-forming NAD(P)H oxidase is used, wherein the H2O-forming NAD(P)H oxidase 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. 8, SEQ ID No. 14, SEQ ID No. 12 or SEQ ID No. 10; ii) An amino acid sequence encoded by a nucleic acid having at least 80% identity with SEQ ID No. 7, SEQ ID No. 13, SEQ ID No. 11, or SEQ ID No. 9; and iii) An amino acid sequence encoded by a nucleic acid that binds to the complementary strand of a nucleic acid molecule having a nucleic acid sequence of SEQ ID No. 7, SEQ ID No. 13, SEQ ID No. 11 or SEQ ID No. 9 under stringent conditions.

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 aldehyde-dependent oxidoreductase is an 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. + Type-dependent oxidoreductases 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. 16, SEQ ID No. 2, SEQ ID No. 4, or SEQ ID No. 6; ii) An amino acid sequence encoded by a nucleic acid having at least 80% identity with SEQ ID No. 15, SEQ ID No. 1, SEQ ID No. 3, or SEQ ID No. 5; and iii) An amino acid sequence encoded by a nucleic acid that binds to the complementary strand of a nucleic acid molecule having a nucleic acid sequence of SEQ ID No. 15, SEQ ID No. 1, SEQ ID No. 3 or SEQ ID No. 5 under stringent conditions.

Images