Process for the preparation of 2,5-furandicarboxylic acid

Abstract

A method for preparing 5-hydroxymethyl-2-furanoic acid (HMFA), wherein NAD(P) is used... + Treatment with a β-dependent aldehyde dehydrogenase was used to oxidize 5-hydroxymethylfurfural (HMF) present in aqueous solution in vitro to 5-hydroxymethyl-2-furanoic acid (HMFA) to form NAD(P)H; subsequently, NAD(P)H oxidase was used to enzymatically regenerate the NAD(P)H formed during oxidation back into NAD(P). + The NAD(P)H oxidase is characterized by comprising an amino acid sequence selected from the group consisting of: i) an amino acid sequence having at least 80% identity with SEQ ID NO:14, SEQ ID NO:16 or SEQ ID NO:18; ii) an amino acid sequence encoded by a nucleic acid having at least 80% identity with SEQ ID NO:13, SEQ ID NO:15 or SEQ ID NO:17; and iii) an amino acid sequence encoded by a nucleic acid bound under stringent conditions to a nucleic acid molecule having a nucleic acid sequence having SEQ ID NO:13, SEQ ID NO:15 or SEQ ID NO:17.

Description

Technical Field

[0001] This invention relates to a method for preparing 2,5-furandicarboxylic acid (FDCA) from 5-(hydroxymethyl)furfural (HMF). Furthermore, this invention relates to a method for preparing 5-hydroxymethyl-2-furancarboxylic acid (HMFA) from 5-(hydroxymethyl)furfural (HMF). Background Technology

[0002] In 2018, approximately 90% (400 million tons) of plastics produced globally were based on fossil fuels, with the remaining share attributed to recycled plastics (9%), bio-based plastics (1%), and CO2-based plastics (<1%) (Carus et al., 2020). Meanwhile, global demand for plastics continues to grow. In 2019, annual CO2 emissions from plastics over their entire lifecycle amounted to 860 million tons, equivalent to the CO2 emissions from 189 fully operational (500 MW) coal-fired power plants. This is projected to increase to 2.8 billion tons (equivalent to 615 coal-fired power plants) by 2050 (Hamilton et al., 2019).

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

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

[0005] 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 feedstocks) (Berger, 2016). Due to the fossil origin of its starting materials (paraxylene and ethylene), PET cannot generally be described as sustainable.

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

[0007] The second component of PET (Baustein, an aromatic compound of terephthalic acid) is difficult to obtain from sustainable raw materials and therefore must be replaced. 2,5-Furfurandicarboxylic acid (FDCA) is an excellent alternative to terephthalic acid, primarily obtained through the catalytic upgrading of biomass. FDCA is polymerized with ethylene glycol to produce PEF (polyethylene furanate), in which the benzene ring is replaced by a heteroaromatic furan ring in its structure. Like PET, PEF is a thermoplastic, but compared to PET, PEF is characterized by significantly higher biodegradability, better thermal properties (higher glass transition temperature, lower melting temperature), and mechanical properties (higher stiffness). However, the most important property of PEF is its reduced permeability to gases such as O2 and CO2. This is particularly important for beverages, as it can extend their shelf life (preventing carbonated beverages from becoming carbonated; preventing oxidation processes through diffused oxygen) (de Jong et al., 2022).

[0008] Currently, the most important starting material for FDCA is 5-(hydroxymethyl)furfural (HMF), which can be obtained, for example, from cellulose (and thus from renewable feedstocks). Enzymatic or chemical hydrolysis of cellulose produces D-glucose, which is subsequently isomerized (enzymatically or chemically) to D-fructose. D-fructose is then dehydrated (eliminating three water molecules) to convert it to HMF. Common systems used for fructose dehydration include inorganic acids such as H₂SO₄ or HCl and (Brønsted or Lewis) acidic solid catalysts (Cong et al., 2021; US ​​9617234 B1).

[0009] HMF possesses both alcohol and aldehyde functional groups, and needs to be oxidized to carboxylic acid groups to obtain FDCA. The three necessary oxidation steps can be carried out in various ways: chemically using heterogeneous or homogeneous catalysts, electrochemically, and biocatalystically (enzymatically or using whole cells).

[0010] A review of various chemical synthesis methods can be found in the article by Cong et al. (2021).

[0011] Biocatalytic processes offer several advantages over traditional chemical processes. For example, the use of enzymes or cells allows for highly selective chemical reactions under mild reaction conditions (aqueous media, room temperature, ambient pressure), and the catalysts used are also biodegradable (Cong et al., 2021).

[0012] The intermediate for the oxidation of HMF to FDCA is 5-hydroxymethyl-2-furanoic acid (HMFA), which can be used, for example, as a component of low polyesters with polymers such as ε-caprolactone as a comonomer (Todea et al., 2019). Furthermore, HMFA possesses cytotoxic and antitumor properties (Munekata and Tamura, 1981) and can be used as a nematicide against nematodes such as *Bursaphelenchus xylophilus* and *Caenorhabditis elegans* (Kimura et al., 2007).

[0013] The oxidation of HMF to HMFA can be achieved via biocatalysis. For example, resting cells of the bacterium *Deinococcus wulumuqiensis* R12 were able to produce 511 mM HMFA in 20 hours with an 85% yield and a productivity of 44 g / (l·d) in a fed-batch process (0.75 mmol HMF added every 5 hours). 2,5-Di(hydroxymethyl)furan (BHMF) was formed only as a trace byproduct (<1%) (Cang et al., 2019).

[0014] Aldehyde dehydrogenase (ALDH) is an enzyme that catalyzes the oxidation of aldehyde groups to carboxylic acid groups. For example, the ALDH of the organism Raoultella ornithinolytica BF60 has been described to oxidize HMF to HMFA (Hossain et al., 2017).

[0015] Zhang et al. tested whole-cell Escherichia coli biocatalysts expressing ALDH (coniferaldehyde DH, vanillin DH 1, vanillin DH 2, or succinylpyridine DH) from *Comamonas testosteroni* SC1588 for the oxidation of aromatic and heteroaromatic aldehydes to their respective acids. Vanillin DH 1 was best suited for the oxidation of HMF to HMFA, and additional expression of NADH oxidase (from *Lactobacillus brevis*) improved the yield and reduced the formation of the byproduct BHMF. In this manner, 250 mM HMF could be oxidized to HMFA within 9 hours with a yield of 95 ± 2% (Zhang et al., 2020).

[0016] In another study by Knaus et al., three different purified aldehyde dehydrogenases (from the bovine lens, *Escherichia coli*, and *Pseudomonas putida*) were tested in combination with NADH oxidase from *Streptococcus mutans* for cofactor regeneration in 61 different aliphatic aldehydes, aryl aliphatic aldehydes, benzyl aldehydes, heteroaryl aldehydes, and bicyclic aldehydes. In oxidation with 20 mM HMF, ALDH from the bovine lens achieved a 90% HMFA yield over 4 hours, while ALDH from *E. coli* achieved a 91% yield over 24 hours. Scale-up the reaction to 2 g HMF, with the addition of *E. coli* ALDH in the form of lyophilized *E. coli* cells (NOX-free), yielded 1.37 g HMFA (61% yield) (Knaus et al., 2018).

[0017] The combination of ALDH with NAD(P)H oxidase (commercial NOX-009 from Prozomix, Ltd.) is also described in US 10344307 B2, with 20 mol% of cofactor (NAD) + or NADP + Together, they completely oxidize 10 mM HMF to HMFA within 30 minutes.

[0018] Qin et al. (2015) used xanthine oxidase (XO) (a molybdenum-dependent enzyme) from Escherichia coli to oxidize 26 mM HMF to HMFA with oxygen (94% yield over 7 hours).

[0019] Another member of the xanthine oxidase family that catalyzes the oxidation of HMF to HMFA is periplasmic aldehyde oxidase (PaoABC) from Escherichia coli (McKenna et al., 2015; McKenna et al., 2017; US 10344307 B2).

[0020] Carro et al. (2015) described a nonspecific peroxidase (UPO, EC 1.11.2.1; requiring H2O2 as an oxidant) from the fungus Agrocybe aegerita that oxidized 3 mM HMF to 97% HMFA within 24 hours.

[0021] HMFA itself can be enzymatically oxidized to 5-formyl-2-furanoic acid (FFA) or FDCA. Typically, each oxidation step (HMFA → FFA and FFA → FDCA) requires two different enzymes.

[0022] Mathieu et al. (2020) described the oxidation of 10 mM HMFA to FFA with aryl alcohol oxidase (AAO) from the fungus Colletotrichum graminicola in the presence of catalase and HRP (horseradish peroxidase) (46% conversion after 16 hours).

[0023] In a study by Cleveland et al. (2021), two aryl alcohol oxidases from the fungi *Fusarium graminearum* (FgrAAO) and *Fusarium oxysporum* (FoxAAO) were characterized. They were able to convert 10 mM HMFA (also in the presence of catalase and HRP) to FFA within 16.5 hours (FgrAAO conversion: 84%, FoxAAO conversion: 96%). Neither aryl alcohol oxidase showed activity against FFA.

[0024] Cajnko et al. (2020) tested the effects of a series of commercially available enzymes (alcohol oxidase (AO) from Pichia pastoris; galactose oxidase from Dactylium dendroides; catalase from Aspergillus niger; laccase from Trametes versicolor; fungal lignin peroxidase (LPO); and HRP) on 10 mM FFA. Significant amounts of FDCA (11.6% from AO, 1.1% from laccase, and 3.2% from LPO) were observed only after 72 hours. HMFA was found as a byproduct (AO at a maximum of 18.2%). The maximum conversion of HMFA (10 mM) to FDCA via HRP or LPO within 72 hours was 4.0% (the maximum conversion to FFA was 0.6%).

[0025] Lappe et al. (2021) described a UPO from the Antarctic bacterium *Moesziomyces antarcticus* that completely oxidized 2 mM HMFA to FFA (99.2%) and FDCA (0.8%) within 144 hours. The 2 mM FFA could then be further converted to 40% FDCA within 144 hours.

[0026] Carro et al. (2015) used UPO from A. aegerita to catalyze the oxidation of FFA (3 mM) to FDCA (90% conversion after 120 hours).

[0027] Jia et al. (2017) used horse liverol dehydrogenase (HLADH) and human hemoglobin (using H2O2 to oxidize 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 the enzymatic oxidation of FFA to FDCA using H2O2 as an oxidant with commercially available chloroperoxidase (EC 1.11.1.10) from Caldariomyces fumago.

[0029] McKenna et al. (2017) used PaoABC and galactose oxidase M 3-5 (GOase M 3-5 (In combination with catalase and HRP, it is used for the oxidation of HMF to FDCA. Via GOase M) 3-5 The oxidation of HMF to 2,5-dicarboxyfuran (DFF) and the oxidation of HMFA to FFA were catalyzed, with the remaining steps catalyzed by PaoABC. HRP was added as GOase M. 3-5 An activator is essential; otherwise, the oxidation of HMFA to FFA will proceed very slowly, thus representing the bottleneck of the cascade reaction.

[0030] The methods presented in this article for the preparation of HMFA and FDCA typically have some drawbacks, such as low substrate concentrations, long reaction times, or uneconomical addition of large amounts of cofactors.

[0031] This is the object of the present invention, which aims to provide an improved method for preparing HMFA and FDCA, characterized by high substrate concentration and / or high conversion rate. Summary of the Invention

[0032] According to the present invention, the objective for preparing 5-hydroxymethyl-2-furanoic acid (HMFA) is achieved by: using NAD(P) + --dependent aldehyde dehydrogenase oxidizes 5-hydroxymethylfurfural (HMF) present in aqueous solution in vitro to form 5-hydroxymethyl-2-furanoic acid (HMFA), generating NAD(P)H. Subsequently, NAD(P)H oxidase enzymatically regenerates the NAD(P)H formed during oxidation back to NAD(P). + The NAD(P)H oxidase is characterized by comprising an amino acid sequence selected from the group consisting of:

[0033] i) Having an amino acid sequence that is at least 80% identical to SEQ ID NO: 14, SEQ ID NO: 16 or SEQ ID NO: 18;

[0034] ii) An amino acid sequence encoded by a nucleic acid having at least 80% identity with SEQ ID NO: 13, SEQ ID NO: 15, or SEQ ID NO: 17; and

[0035] iii) An amino acid sequence encoded by a nucleic acid that binds to a nucleic acid molecule having a nucleic acid sequence SEQ ID NO: 13, SEQ ID NO: 15 or SEQ ID NO: 17 under strict conditions.

[0036] Surprisingly, compared with NADH oxidases from Streptococcus mutans (Knaus et al., 2018) or NOX-009 (US 10344307 B2) described in the prior art, NADH oxidases with SEQ ID NO: 14, 16 and 18 were found to cause significantly higher conversion (see Examples 2 and 3).

[0037] According to the present invention, the purpose of preparing 2,5-furandicarboxylic acid (FDCA) is achieved by adding 5-hydroxymethyl-2-furancarboxylic acid (HMFA) to an aqueous solution after the formation of 5-hydroxymethyl-2-furancarboxylic acid (HMFA).

[0038] It has been shown that the addition of HMFO ensures that the oxidation of HMF does not stop at HMFA, but continues to the corresponding dicarboxylic acid (FDCA), because NAD(P) + --dependent aldehyde dehydrogenases also catalyze the final oxidation step from 5-formyl-2-furanoic acid (FFA) to FDCA.

[0039] In another variant of the method according to the invention, catalase is also added to the aqueous solution.

[0040] In another preferred variant of the method according to the invention, NAD(P)H formed during oxidation is at least partially enzymatically regenerated back to NAD(P) using an oxidoreductase. + Furthermore, ketone compounds are used as cosubstrates for oxidoreductases.

[0041] The preferred oxidoreductases used are alcohol dehydrogenase, xylitol dehydrogenase, sorbitol dehydrogenase, xylose reductase, or SDR family oxidoreductases.

[0042] The ketone compounds used are preferably ketoses, particularly D-fructose; aldoses, particularly D-glucose or D-xylose; or ketones, more preferably fatty ketones, particularly acetone.

[0043] A preferred embodiment of the method according to the invention is shown in the appendix. Figure 1The diagram is shown schematically. Here, name A represents 5-(hydroxymethyl)furfural (HMF), B represents 5-hydroxymethyl-2-furanoic acid (HMFA), C represents 5-formyl-2-furanoic acid (FFA), D represents 2,5-furandicarboxylic acid (FDCA), 1 represents aldehyde dehydrogenase, 2 represents NAD(P)H oxidase, and 3 represents HMF oxidase.

[0044] NAD(P) + - Aldehyde-dependent dehydrogenases preferably contain or consist of an amino acid sequence selected from or composed of the following:

[0045] i) Having an amino acid sequence that is at least 80% identical to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 or SEQ ID NO: 12;

[0046] ii) An amino acid sequence encoded by a nucleic acid having at least 80% identity with SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 11; and

[0047] iii) An amino acid sequence encoded by a nucleic acid that binds to a nucleic acid molecule having a nucleic acid sequence SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 11 under strict conditions.

[0048] SEQ ID NO: 1:

[0049]

[0050] SEQ ID NO: 2:

[0051]

[0052] SEQ ID NO: 3:

[0053]

[0054] SEQ ID NO: 4:

[0055]

[0056] SEQ ID NO: 5:

[0057]

[0058] SEQ ID NO: 6:

[0059]

[0060] SEQ ID NO: 7:

[0061]

[0062] SEQ ID NO: 8:

[0063]

[0064] SEQ ID NO: 9:

[0065]

[0066] SEQ ID NO: 10:

[0067]

[0068] SEQ ID NO: 11:

[0069]

[0070] SEQ ID NO: 12:

[0071]

[0072] The NAD(P) discussed in this article + --dependent aldehyde dehydrogenase preferably comprises an amino acid sequence having at least 80%, more preferably 85%, more preferably 90%, more preferably 95%, more preferably 98%, more preferably 99%, and particularly 100% identity with SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 or SEQ ID NO: 12.

[0073] Or, NAD(P) + --dependent aldehyde dehydrogenase preferably comprises an amino acid sequence encoded by a nucleic acid having at least 80%, more preferably 85%, more preferably 90%, more preferably 95%, more preferably 98%, more preferably 99%, and particularly 100% identity with SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 11. Particularly preferably, it encodes NAD(P) according to the invention. +The nucleic acid of the --dependent aldehyde dehydrogenase contains or is composed of the nucleic acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 11.

[0074] As used herein, the term "identity" refers to the percentage of identical nucleotide or amino acid matches between at least two nucleotide or amino acid sequences compared using a standardized algorithm ("alignment"). This algorithm can insert gaps in the compared sequences in a standardized and reproducible manner to optimize the alignment between the two sequences, thereby enabling a more meaningful comparison.

[0075] The percentage of identity between sequences can be determined using one or more computer algorithms or programs known in the prior 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 various programs, including a tool called “BLAST 2 Sequence”, for direct pairwise comparison of two nucleotide or amino acid sequences. “BLAST 2 Sequence” is also interactively accessible and usable via the NCBI World Wide Web address on the Internet. The blastn program (for nucleotide sequences) uses the following default parameters: word length (W) of 11, expectation value (E) of 10, M = 5, N = -4, and compares two strands. For amino acid sequences, the blastp program uses the following default parameters: word length 3, expected value (E) 10, BLOSUM62 score matrix (Henikoff and Henikoff, 1989), alignment (B) 50, expected value (E) 10, M = 5, N = -4.

[0076] Or, NAD(P) +--dependent aldehyde dehydrogenases preferably comprise an amino acid sequence encoded by a nucleic acid bound to a nucleic acid molecule having a nucleic acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 11 under stringent conditions. As used herein, stringent conditions refer to conditions under which a so-called specific hybrid is formed without the formation of a non-specific hybrid. For example, stringent conditions include hybridization at 45°C in 6×SSC (sodium chloride / sodium citrate), followed by washing with 0.2 to 1×SSC, 0.1% SDS at 50 to 65°C; or such conditions may include hybridization at 65 to 70°C in 1×SSC, followed by washing with 0.3×SSC at 65 to 70°C. 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, 2nd edition, Cold Spring Harbor Laboratory (1989).

[0077] NAD(P) was also disclosed. + The use of --dependent aldehyde dehydrogenases, in which NAD(P) + - Aldehyde-dependent dehydrogenases contain amino acid sequences selected from the following groups:

[0078] i) Having an amino acid sequence that is at least 80% identical to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 or SEQ ID NO: 12;

[0079] ii) An amino acid sequence encoded by a nucleic acid having at least 80% identity with SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 11; and

[0080] iii) An amino acid sequence encoded by a nucleic acid that binds to a nucleic acid molecule having a nucleic acid sequence SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 11 under strict conditions.

[0081] In a preferred variant of the method according to the invention, NAD(P)H oxidase is used to complete the NAD(P)H to NAD(P) conversion. + Enzymatic oxidation.

[0082] The NAD(P)H oxidase used for cofactor regeneration can be from one of the following groups: EC 1.6.3.1 (NAD(P)H oxidase (forming H2O2)), EC 1.6.3.2 (NAD(P)H oxidase (forming H2O)), EC 1.6.3.3 (NADH oxidase (forming H2O2)) and EC 1.6.3.4 (NADH oxidase (forming H2O)), with the H2O-forming type being particularly preferred.

[0083] The NAD(P)H oxidase used in particular for forming H2O preferably contains an amino acid sequence selected from or composed of the following:

[0084] i) Having an amino acid sequence that is at least 80% identical to SEQ ID NO: 14, SEQ ID NO: 16 or SEQ ID NO: 18;

[0085] ii) An amino acid sequence encoded by a nucleic acid having at least 80% identity with SEQ ID NO: 13, SEQ ID NO: 15, or SEQ ID NO: 17; and

[0086] iii) An amino acid sequence encoded by a nucleic acid that binds to a nucleic acid molecule having a nucleic acid sequence SEQ ID NO: 13, SEQ ID NO: 15 or SEQ ID NO: 17 under strict conditions.

[0087] SEQ ID NO: 13:

[0088]

[0089] SEQ ID NO: 14:

[0090]

[0091] SEQ ID NO: 15:

[0092]

[0093] SEQ ID NO: 16:

[0094]

[0095] SEQ ID NO: 17:

[0096]

[0097] SEQ ID NO: 18:

[0098]

[0099] The preferred H2O-forming NAD(P)H oxidase used preferably comprises or is composed of the following amino acid sequence: having an amino acid sequence that is at least 80%, more preferably 85%, even more preferably 90%, even more preferably 95%, even more preferably 98%, even more preferably 99%, and particularly 100% identical to SEQ ID NO: 14, SEQ ID NO: 16, or SEQ ID NO: 18. Particularly preferably, the nucleic acid encoding the H2O-forming NAD(P)H oxidase according to the invention comprises or is composed of the nucleic acid sequence SEQ ID NO: 14, SEQ ID NO: 16, or SEQ ID NO: 18.

[0100] Alternatively, the NAD(P)H oxidase that forms H2O preferably comprises an amino acid sequence encoded by a nucleic acid having at least 80%, more preferably 85%, even more preferably 90%, even more preferably 95%, even more preferably 98%, even more preferably 99%, and particularly 100% identity with SEQ ID NO: 13, SEQ ID NO: 15, or SEQ ID NO: 17. Particularly preferably, the nucleic acid encoding the NAD(P)H oxidase that forms H2O comprises or is composed of the nucleic acid sequence SEQ ID NO: 13, SEQ ID NO: 15, or SEQ ID NO: 17.

[0101] Alternatively, the NAD(P)H oxidase that forms H2O preferably comprises an amino acid sequence encoded by a nucleic acid that binds to a nucleic acid molecule having the nucleic acid sequence SEQ ID NO: 13, SEQ ID NO: 15, or SEQ ID NO: 17 under stringent conditions. As used herein, stringent conditions refer to conditions under which a so-called specific hybrid is formed without the formation of a non-specific hybrid. For example, stringent conditions include hybridization at 45°C in 6×SSC (sodium chloride / sodium citrate), followed by washing with 0.2 to 1×SSC, 0.1% SDS at 50 to 65°C; or such conditions may include hybridization at 65 to 70°C in 1×SSC, followed by washing with 0.3×SSC at 65 to 70°C. 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, 2nd edition, Cold Spring Harbor Laboratory (1989).

[0102] Another aspect of the invention relates to the use of NAD(P)H oxidases that form H2O, comprising or consisting of an amino acid sequence selected from or composed of the group consisting of:

[0103] i) Having an amino acid sequence that is at least 80% identical to SEQ ID NO: 14, SEQ ID NO: 16 or SEQ ID NO: 18;

[0104] ii) An amino acid sequence encoded by a nucleic acid having at least 80% identity with SEQ ID NO: 13, SEQ ID NO: 15, or SEQ ID NO: 17; and

[0105] iii) An amino acid sequence encoded by a nucleic acid that binds to a nucleic acid molecule having SEQ ID NO: 13, SEQ ID NO: 15 or SEQ ID NO: 17 under strict conditions.

[0106] In another preferred embodiment of the method according to the invention, the concentration of HMF in the aqueous solution is 15-130 g / l.

[0107] The preferred temperature range is 18 to 40°C.

[0108] Particularly preferred is a reaction pH range of pH 6 to pH 9.

[0109] In another preferred variant of the method according to the invention, the enzyme is present in the suspension, homogenate and / or lysate of the corresponding cells that produced it, particularly preferably the lysate.

[0110] In this context, suspension refers to a suspension of resting cells. Resting cells are harvested after culture (separated from the nutrient medium) and suspended in a suitable buffer system. Unlike fermentation processes that also use whole cells, resting cells cease growth due to the removal of carbon sources and nutrients but are used solely for substrate transformation (Lin and Tao, 2017). In this context, homogenate refers to a suspension that has undergone physical and / or chemical treatment (e.g., with pressure, lysozyme, or sonication), in which cellular components are released from the cells. Lysates are obtained when insoluble cellular components in the homogenate are removed (e.g., by filtration or centrifugation) (see Enzyme Generation and Lysate Preparation).

[0111] In another variant, the enzyme may also be modified at its N-terminus with a portion of a water-soluble polymer, such as polyethylene glycol, or a fusion protein, immobilized in or on a solid matrix.

[0112] In another variant, the enzyme can be in powder form, lyophilized form, or spray-dried form.

[0113] Preferred embodiments of the present invention are described in more detail with reference to the following examples.

[0114] Material

[0115] 5-(hydroxymethyl)furfural (HMF) was purchased from Biosynth; 5-formyl-2-furanic acid (FFA) and 2,5-furandicarboxylic acid (FDCA) were purchased from TCI; 2,5-diformylfuran (DFF) and 5-hydroxymethyl-2-furanic acid (HMFA) were purchased from Sigma-Aldrich; acetone, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and sodium dodecyl sulfate (SDS) were purchased from Carl Roth; NAD + Disodium NADH, NADP + Disodium salt, tetrasodium salt of NADPH, and acetonitrile were purchased from PanReac AppliChem (ITW Reagent); triethanolamine was purchased from Chem-Lab NV. Commercial NADH oxidase NOX-009 was purchased from Prozomix Limited, UK.

[0116] Enzyme production and lysate preparation

[0117] A universal procedure for expressing recombinases in Escherichia coli

[0118] For the production of recombinases in *E. coli* strains, the gene to be expressed is first amplified by PCR using genomic DNA or its synthetic equivalent optimized for *E. coli* codons as a template, along with specific oligonucleotides carrying additional restriction endonuclease recognition sequences. The amplified gene fragment is then isolated from the reaction mixture. After digestion with restriction enzymes SphI and HindIII, the gene fragment encoding the target enzyme is ligated into the backbone of the expression vector pQE70-Kan, which has been digested with SphI and HindIII. The ligation product is transformed into chemically competent *E. coli* Top10F' cells, and the resulting colonies are used for plasmid isolation and restriction analysis.

[0119] The cloning results were verified by restriction enzyme digestion and DNA sequencing. The resulting construct contained the target gene under the control of the IPTG-inducible T5 promoter.

[0120] To overexpress the enzyme in *E. coli*, the resulting expression plasmid was transformed into competent expression strain RB791 cells. After incubation at 37°C for 24 hours, the resulting colonies were inoculated into LB medium for expression screening.

[0121] The next day, the expression culture was inoculated to 550 nm (OD). 550 The optical density at point (0.02) is 0.02. After shaking at 37℃ until the OD value is reached... 550 Reaching 0.3. Then the temperature was lowered to 25°C, and at OD... 550When the concentration reached 0.5, the culture was induced with 0.1 mM IPTG. After 22 hours, the culture was harvested (by centrifugation to form a cell pellet) and the recombinase expression in the culture was analyzed using SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and activity assays (using a test or optical enzymatic assay).

[0122] Cell lysates prepared by ultrasonic treatment

[0123] To prepare the cell suspension, the cell pellet obtained using the above procedure is weighed into a suitable container, resuspended in a buffer solution supplemented with lysozyme (final concentration 0.5 mg / mL, e.g., potassium phosphate buffer), and dissolved by stirring. Biomass typically comprises 20% by mass, with the remainder consisting of the buffer solution.

[0124] Cell disruption was performed using a Branson Sonifier 450. The suspension was subjected to three rounds of sonication, each round consisting of 15 sonic pulses (instrument settings: timer = 15; duty cycle = 50; output control = 3-5).

[0125] The resulting homogenate was centrifuged at 16,000 rpm for 10 minutes at 4°C (Eppendorf centrifuge 5417R) to remove insoluble cell debris and obtain lysate.

[0126] Table 1. Types of enzymes and donor organisms used in the examples (ALDH = aldehyde dehydrogenase)

[0127]

[0128] Note: In the NCBI protein database, HMF oxidase from *Pseudomonas nitroreductans* is classified as glucose-methanol-choline (GMC) oxidoreductase, which is also a superfamily that includes HMF oxidase (Viñambres et al., 2020).

[0129] Analytical methods

[0130] High-performance liquid chromatography (HPLC)

[0131] HPLC was used to quantify HMF, DFF, HMFA, FFA, and FDCA, and a UV detector was used for detection. Phenomenex Rezex ROA-organic acid H2O was then used with appropriate guard columns. + (8%) The column was eluted isocratically with 1 mM sulfuric acid for analysis.

[0132] Determination of enzyme activity (optical enzymatic assay)

[0133] Enzyme activity in the lysates was determined using a Shimadzu UV-1900 spectrophotometer. For this purpose, the formation or consumption of NAD(P)H was monitored by measuring changes in absorbance at 340 nm. 0.2 mM cofactor (NAD(P)H) was used. + Alternatively, NAD(P)H can be used for the assay. Briefly, place 20 μL of 10 mM cofactor stock solution in a cuvette (Greiner bio-one semi-micro polystyrene cuvette) and adjust the pH to the desired level with 870 μL of 100 mM TEA HCl buffer. Add 10 μL of lysate (diluted or undiluted) and 100 μL of substrate solution to the cuvette and begin the measurement immediately. All assays were performed at 25°C under standard conditions. The molar extinction coefficient of NADH / NADPH at 340 nm (ε = 6220 L·mol⁻¹) was used. -1 ·cm -1 Enzyme activity of the lysate was calculated in U / mL (relative to the volume of the lysate) or U / g (relative to the biomass used in the preparation). One unit (1 U) was defined as the conversion rate of 1 μmol of substrate per minute (1 U = 1 μmol / min = 1.67 × 10⁻⁶). -8 kat).

[0134] Preferred embodiments of the method according to the present invention are described in more detail through the following examples. The pyrolytes used in these examples were prepared using the above-described procedure.

[0135] Example 1

[0136] Oxidation of 5-hydroxymethylfurfural to 5-hydroxymethyl-2-furanic acid using various aldehyde dehydrogenases

[0137] The oxidation reaction was carried out in 2 mL glass vials containing 30 μL of NADH oxidase lysate and 50 μL of aldehyde dehydrogenase lysate in 250 mM potassium phosphate buffer (pH 7) (see Table 2 below).

[0138] The reaction was initiated by adding HMF or HMFA to a final concentration of 10 mM. The vials were incubated in an Eppendorf Thermomixer with continuous shaking (30°C, 800 rpm) for 20 hours.

[0139] For analysis, 50 μL of the reaction mixture was mixed with 200 μL of acetonitrile and incubated at 85 °C in an Eppendorf Thermomixer at 1200 rpm for 15 min. The sample was briefly centrifuged, mixed with 750 μL of ultrapure water, vortexed, and centrifuged at maximum relative centrifugal force (RCF) for 5 min. 200 μL of the supernatant was transferred to an HPLC vial and analyzed by HPLC with UV detection. The results are shown in Table 2 below.

[0140] Table 2

[0141]

[0142] The results in Table 2 show that when combined with NADH oxidase for cofactor regeneration, the aldehyde dehydrogenase tested in this paper only oxidizes HMF to HMFA without further oxidizing HMFA.

[0143] Example 2

[0144] Oxidation of 5-hydroxymethylfurfural to 5-hydroxymethyl-2-furanic acid - Comparison of different NADH oxidases

[0145] The oxidation reaction was performed in 2 mL glass vials containing 10 μL of ALDH I lysate, 5 U of NADH oxidase lysate (see Table 3 below), and 0.1 mM NAD+ in 250 mM potassium phosphate buffer (pH 7). + The solution.

[0146] The reaction was initiated by adding HMF to a final concentration of 20 g / L. The vials were incubated in an Eppendorf Thermomixer with continuous shaking (35°C, 800 rpm) for 20 hours.

[0147] For analysis, 50 μL of the reaction mixture was mixed with 200 μL of acetonitrile and incubated at 85 °C in an Eppendorf Thermomixer at 1200 rpm for 15 min. The sample was briefly centrifuged, mixed with 750 μL of ultrapure water, vortexed, and centrifuged at maximum relative centrifugal force for 5 min. 300 μL of the supernatant was transferred to an HPLC vial, diluted with 300 μL of 20% acetonitrile, and analyzed by HPLC with UV detection. The results are shown in Table 3 below.

[0148] Table 3

[0149]

[0150] Table 3 shows that the NADH oxidases used in this invention (SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18) produce significantly higher levels of HMFA compared to the NADH oxidases from Streptococcus mutans described by Knaus et al. (2018).

[0151] Example 3

[0152] Oxidation of 5-hydroxymethylfurfural to 5-hydroxymethyl-2-furanic acid - Comparison of different NADH oxidases

[0153] The oxidation reaction was performed in duplicate, each replicate in a Labfors benchtop bioreactor (Infors AG). The reaction vessel consisted of a 3.4L glass reactor equipped with a stirrer, O2 sensor, and pH electrode. The pH was controlled by adding 5 M NaOH or 1 M H2SO4.

[0154] In reactor I, 213.2 mL of deionized water and 15 mL of 10 mM NAD were added. + The solution, 200 mL of 500 mM potassium phosphate buffer (pH 7), 50 mL of ALDH I lysate, and 0.5 g of commercially available NADH oxidase powder (NOX-009) were mixed with stirring and heated to 35°C. The reaction was initiated by adding 20.5 g of HMF (approximately 80% purity).

[0155] In reactor II, 213.2 mL of deionized water and 15 mL of 10 mM NAD were added. + The solution, 200 mL of 500 mM potassium phosphate buffer (pH 7), 50 mL of ALDH I lysate, and 0.5 g of NADH oxidase powder (SEQ ID NO: 14) were mixed with stirring and heated to 35°C. The reaction was initiated by adding 20.5 g of HMF (approximately 80% purity).

[0156] In addition, an overpressure of 320 mbar was applied to all reactors, and the oxygen supply rate (via sprayer) was set to 0.05 L / min.

[0157] For analysis, 50 μL of the reaction mixture was mixed with 200 μL of acetonitrile and incubated at 85 °C in an Eppendorf Thermomixer at 1200 rpm for 15 min. The sample was briefly centrifuged, mixed with 750 μL of deionized water, vortexed, and centrifuged at maximum relative centrifugation for 5 min. 250 μL of the supernatant was transferred to an HPLC vial, diluted with 750 μL of acetonitrile / water mixture (1 / 4 v / v), and analyzed by HPLC with UV detection.

[0158] The results are presented as a concentration summary in Figure 2 The results show that cofactor regeneration using NADH oxidase (SEQ ID NO: 14) achieved a conversion rate of >99% within 4.5 hours, while only about 13% conversion was achieved using commercial NADH oxidase (NOX-009).

[0159] Example 4

[0160] Oxidation of 5-hydroxymethylfurfural via 5-hydroxymethyl-2-furancarboxylic acid to 2,5-furandicarboxylic acid (batch feeding process)

[0161] The reaction was carried out in a Labfors benchtop bioreactor (Infors AG). The reaction vessel consisted of a 3.4 L glass reactor equipped with a stirrer, an O2 sensor, and a pH electrode. The pH was controlled by adding 5 M NaOH or 1 M H2SO4.

[0162] Initially, 212.7 mL of deionized water, 200 mL of 500 mM potassium phosphate buffer (pH 7), and 15 mL of 10 mM NAD were added. + The solution, 50 mL of ALDH I lysate, and 21 kU of NADH oxidase lysate were mixed and heated to 30 °C with stirring. The reaction was initiated by adding 20.5 g of HMF (approximately 80% purity).

[0163] In addition, an overpressure of 320 mbar was applied and the oxygen supply rate (via the sprayer) was set to 0.05 L / min.

[0164] For analysis, 50 μL of the reaction mixture was mixed with 200 μL of acetonitrile and incubated at 85 °C in an Eppendorf Thermomixer at 1200 rpm for 15 min. The sample was briefly centrifuged, mixed with 750 μL of deionized water, vortexed, and centrifuged at maximum relative centrifugation for 5 min. 200 μL of the supernatant was transferred to an HPLC vial, diluted with 800 μL of acetonitrile / water mixture (1 / 4 v / v), and analyzed by HPLC with UV detection.

[0165] HMF was completely oxidized to HMFA within 2 hours. Therefore, 20.5 g HMF, 21 kU NADH oxidase lysate, and 30 mL ALDH I lysate were added at 2 hours and again at 4 hours.

[0166] At 7 hours, add 50 mL of HMFO lysate, 10 mL of catalase lysate, 30 mL of ALDH I lysate, and 21 kU of NADH oxidase lysate. At 25 hours, add 50 mL of HMFO lysate, 30 mL of ALDH I lysate, and 28 kU of NADH oxidase lysate.

[0167] Using this method, >99% of the HMF substrate was oxidized to FDCA within a total reaction time of 29 hours.

[0168] The reactor contents were then heated to 70°C and maintained for 60 minutes. The resulting precipitate was removed by centrifugation and decantation. The solution was treated with activated carbon (5% w / v) and stirred at 50°C for 30 minutes. The activated carbon was then removed by filtration through a P4 glass funnel filter using diatomaceous earth. The filtrate was acidified with concentrated H₂SO₄ to pH < 2 and cooled to 4°C. The resulting precipitate (FDCA) was collected by filtration.

[0169] In this way, 61 g of FDCA as a colorless solid was obtained.

[0170] Example 5

[0171] Oxidation of 5-hydroxymethylfurfural via 5-hydroxymethyl-2-furancarboxylic acid to 2,5-furandicarboxylic acid

[0172] The reaction was carried out in a Labfors benchtop bioreactor (Infors AG). The reaction vessel consisted of a 3.4 L glass reactor equipped with a stirrer, an O2 sensor, and a pH electrode. The pH was controlled by adding 5 M NaOH or 1 M H2SO4.

[0173] Initially, 304 mL of deionized water, 100 mL of 500 mM potassium phosphate buffer (pH 7), and 15 mL of 10 mM NAD were added. + The solution, 50 mL of ALDH I lysate, and 21 kU of NADH oxidase lysate were mixed and heated to 30°C with stirring. The reaction was initiated by adding 31 g of HMF (approximately 80% purity).

[0174] In addition, an overpressure of 320 mbar was applied and the oxygen supply rate (via the sprayer) was set to 0.05 L / min.

[0175] For analysis, 50 μL of the reaction mixture was mixed with 200 μL of acetonitrile and incubated at 85 °C in an Eppendorf Thermomixer at 1200 rpm for 15 min. The sample was briefly centrifuged, mixed with 750 μL of deionized water, vortexed, and centrifuged at maximum relative centrifugation for 5 min. 200 μL of the supernatant was transferred to an HPLC vial, diluted with 800 μL of acetonitrile / water mixture (1 / 4 v / v), and analyzed by HPLC with UV detection.

[0176] At 4.5 hours, 50 mL of ALDH I lysate and 21 kU of NADH oxidase lysate were added.

[0177] Within 7 hours, >99% of the HMF substrate was oxidized to HMFA, followed by the addition of 50 mL of HMFO lysate.

[0178] At 8 hours, 50 mL of ALDH I lysate and 21 kU of NADH oxidase lysate were added; at 11 hours, 80 mL of HMFO lysate was added.

[0179] Using this method, >99% of the HMF substrate was oxidized to FDCA within a total reaction time of 23 hours.

[0180] Example 6

[0181] Oxidation of 5-hydroxymethylfurfural from 5-hydroxymethyl-2-furanic acid to 2,5-furandicarboxylic acid – regeneration using cofactors of NADH oxidase and alcohol dehydrogenase.

[0182] The reaction was carried out in a Labfors benchtop bioreactor (Infors AG). The reaction vessel consisted of a 3.4 L glass reactor equipped with a stirrer, an O2 sensor, and a pH electrode. The pH was controlled by adding 5 M NaOH or 1 M H2SO4.

[0183] Initially, 212.7 mL of deionized water, 200 mL of 500 mM potassium phosphate buffer (pH 7), and 15 mL of 10 mM NAD were added. + The solution, 50 mL of ALDH I lysate, and 21 kU of NADH oxidase lysate were mixed and heated to 35 °C with stirring. The reaction was initiated by adding 20.5 g of HMF (approximately 80% purity).

[0184] In addition, an overpressure of 320 mbar was applied and the oxygen supply rate (via the sprayer) was set to 0.05 L / min.

[0185] For analysis, 50 μL of the reaction mixture was mixed with 200 μL of acetonitrile and incubated at 85 °C in an Eppendorf Thermomixer at 1200 rpm for 15 min. The sample was briefly centrifuged, mixed with 750 μL of deionized water, vortexed, and centrifuged at maximum relative centrifugation for 5 min. 200 μL of the supernatant was transferred to an HPLC vial, diluted with 800 μL of acetonitrile / water mixture (1 / 4 v / v), and analyzed by HPLC with UV detection.

[0186] Within 2.5 hours, >99% of the HMF substrate was oxidized to HMFA.

[0187] To further oxidize, 30 mL of HMFO lysate and 10 mL of catalase lysate were added. After 3.75 hours, 50 mL of ALDH I lysate, 30 mL of alcohol dehydrogenase lysate, and 15 mL of acetone were added.

[0188] Within 18 hours, >99% of HMFA was oxidized to FDCA.

[0189] Using this method, >99% of the HMF substrate was oxidized to FDCA within a total reaction time of 20.5 hours.

[0190] literature

[0191]

[0192]

[0193]

[0194]

[0195]

Claims

1. A method for preparing 5-hydroxymethyl-2-furanic acid (HMFA), wherein the preparation is carried out using NAD(P) + Treatment with a 2-dependent aldehyde dehydrogenase oxidizes 5-hydroxymethylfurfural (HMF) present in aqueous solution to 5-hydroxymethyl-2-furanoic acid (HMFA), forming NAD(P)H; subsequently, NAD(P)H oxidase enzymatically regenerates the NAD(P)H formed during oxidation back into NAD(P). + , Its features are, The NAD(P)H oxidase comprises an amino acid sequence selected from the group consisting of: i) Having an amino acid sequence that is at least 80% identical to SEQ ID NO: 14, SEQ ID NO: 16 or SEQ ID NO: 18; ii) An amino acid sequence encoded by a nucleic acid having at least 80% identity with SEQ ID NO: 13, SEQ ID NO: 15, or SEQ ID NO: 17; and iii) An amino acid sequence encoded by a nucleic acid that binds to a nucleic acid molecule having a nucleic acid sequence having SEQ ID NO: 13, SEQ ID NO: 15 or SEQ ID NO: 17 under strict conditions.

2. A method for preparing 2,5-furandicarboxylic acid, characterized in that, In the method according to claim 1, after the formation of 5-hydroxymethyl-2-furanoic acid (HMFA), 5-hydroxymethylfurfural oxidase is added to the aqueous solution.

3. The method according to claim 2, characterized in that, Catalase was also added to the aqueous solution.

4. The method according to any one of claims 1 to 3, characterized in that, Using oxidoreductases will at least partially enzymatically regenerate NAD(P)H formed during oxidation back into NAD(P). + Furthermore, ketone compounds are used as cosubstrates for the oxidoreductase.

5. The method according to claim 4, characterized in that, Ketosaccharides, aldoses, or ketones are used as the ketone compounds.

6. The method according to claim 5, characterized in that, D-fructose, D-glucose, or D-xylose are used as the ketose or aldose.

7. The method according to claim 5, characterized in that, Fatty ketones are used as the ketones in question.

8. The method according to claim 7, characterized in that, Acetone is used as the fatty ketone.

9. The method according to any one of claims 1 to 8, characterized in that, The NAD(P) + Aldehyde-dependent dehydrogenases have amino acid sequences selected from the following groups: i) Having an amino acid sequence that is at least 80% identical to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 or SEQ ID NO: 12; ii) An amino acid sequence encoded by a nucleic acid having at least 80% identity with SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 11; and iii) An amino acid sequence encoded by a nucleic acid molecule that binds to a nucleic acid molecule having a nucleic acid sequence having SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 11 under strict conditions.

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