Process for preparing 2,5-diformylfuran

Abstract

The invention relates to a process for preparing 2,5-diformylfuran by oxidising 5-(hydroxymethyl)furfural, which is present dissolved in a single-phase aqueous solution, with an oxidase in vitro to form 2,5-diformylfuran, which is separated from the aqueous solution, characterised by the combination of the measures whereby: i) the process is carried out at a temperature between 20°C and 40°C; ii) at the beginning of the oxidation, the single-phase aqueous solution contains at least 35 g / l of dissolved 5-(hydroxymethyl)furfural; and iii) the oxidase is a 5-(hydroxymethyl)furfural oxidase.

Description

[0001] Method for the production of 2,5-diformylfuran

[0002] The invention relates to a process for the production of 2,5-diformylfuran by oxidizing 5-(hydroxymethyl)furfural to 2,5-diformylfuran with an oxidase.

[0003] Background of the invention

[0004] 2,5-Diformylfuran (DFF; 2,5-furandicarbaldehyde) is a heteroaromatic dialdehyde that, due to its reactive aldehyde functions, can be used, for example, as a bifunctional cross-linking reagent for proteins (as a bio-based alternative to glutaraldehyde) (Danielli et al., 2022).

[0005] DFF can also be used to produce polymers, surfactants, fluorescent materials, pharmaceuticals and aerogels (Dai, 2021; Acosta et al., 2021; Derflinger et al., 2021).

[0006] The starting material for the production of DFF is 5-(hydroxymethyl)furfural (HMF), which can be obtained, for example, from cellulose (and thus from renewable resources). Enzymatic or chemical hydrolysis of cellulose yields D-glucose, which is subsequently isomerized to D-fructose enzymatically or chemically. Dehydration (removal of three H₂O molecules) yields HMF from D-fructose. Common systems for the dehydration of fructose include mineral acids such as H₂SO₄ or HCl, and solid (Brpnsted or Lewis) acid catalysts (Cong et al., 2021; US ​​9617234 Bl).

[0007] The oxidation step (conversion of the hydroxymethyl group into a formyl group) can be accomplished either chemically-catalytically or biocatalytically.

[0008] For the heterogeneous catalytic oxidation of HMF to DFF with oxygen, various transition metal catalysts (e.g., vanadium, manganese, molybdenum, ruthenium oxides) can be used (Dai, 2021). As an alternative to the generally unsustainable and sometimes expensive metal catalysts, a process exists in which HMF is first produced from D-fructose by dehydration in the presence of DMSO (Amarasekara et al., 2008), which is then oxidized to DFF by adding catalytic amounts of NaBr or HBr to the reaction mixture. The disadvantages of this process are the high reaction temperatures (150 °C) and the formation of byproducts (from DMSO) that are difficult to remove (Laugel et al., 2014).

[0009] Halliday et al. (2003) also describe a two-step process for the production of DFF from D-fructose. Dehydration was carried out using a cation exchange resin (AG 50W-X8) in DMSO (25.5 h at 80 °C). The resin was then filtered off, and the HMF in solution was oxidized to DFF with oxygen in the presence of 5 mol% V₂O₅ (17 h at 150 °C). For work-up, the reaction solution was diluted with dichloromethane, filtered, washed with water, and filtered through silica gel. The crude product obtained after evaporation of the solvent still contained impurities such as dimethyl sulfone (MejSOj) and dimethyl sulfide (IVIezS), which were formed from DMSO. Therefore, the crude product was first purified by vacuum sublimation and then by Soxhlet extraction (using cyclohexane as the extraction solvent) with simultaneous filtration through silica gel. In this way, DFF could be obtained with a purity of >99% and a yield of 42%.

[0010] A detailed overview of the chemical-catalytic production of DFF from HMF or directly from D-fructose and other carbohydrates can be found in the article by Dai (2021).

[0011] Biocatalytic processes have also been described for the production of DFF, which are characterized by milder reaction conditions (less cost- and energy-intensive), the generation of less waste (such as solvents or by-products) and biodegradable catalysts (cells or enzymes).

[0012] Acosta et al. (2021) describe the fungus Fusarium culmorum EAN 51 for the production of DFF from HMF. 92% of the starting material (50 mM HMF) was oxidized to DFF in 31 h. The DFF formed was separated from the reaction medium by extraction with ethyl acetate.

[0013] Li et al. (2023) used Escherichia co / / cells expressing galactose oxidase (GOase), a copper-containing enzyme, to oxidize HMF produced from bread residues or D-fructose (dehydration in the strongly eutectic solvent betaine-lactic acid) to DFF, yielding 0.631 g DFF / g D-fructose and 0.323 g DFF / g bread.

[0014] Enzymatic (in vitro) methods for the oxidation of HMF to DFF are also known. Enzymes that catalyze the oxidation of HMF to DFF include alcohol oxidases (AO; e.g., from Candida boidinii, Hansenula sp., or Pichia pastoris), aryl alcohol oxidases, and the commercially available pyranose-2-oxidase from Cariolus sp. (US 8183020 B2; Qin et al., 2015).

[0015] Another enzyme for the oxidation of HMF to DFF is HMF oxidase (HMFO; EC 1.1.3.47), which is found in bacteria, fungi, and also in the honeybee (Apis mellifera). Tjallinks et al. (2023) used “beeHMFO” to oxidize 50 mM HMF to DFF (91% in 29 h), with the product of overoxidation (5-formyl-2-furan carboxylic acid = FFA) being found as a byproduct (7%).

[0016] For example, the GOase from Dactylium dendroides can oxidize 30 mM HMF to DFF with 92% efficiency within 96 h in the presence of horseradish peroxidase (HRP; for GOase activation) and catalase (for decomposition of the resulting hydrogen peroxide) (Qin et al., 2015). Cajnko et al. (2020) tested a range of commercially available enzymes (AO from Pichia pastoris; GOase from Dactylium dendroides; catalase from Aspergillus niger; laccase from Trametes versicolor; a fungal lignin peroxidase; and HRP) on 10 mM HMF and observed DFF formation only for AO and GOase (conversion: 25.6% for AO and 5.1% for GOase). FFA (3.1% conversion) was also found in the case of AO. By combining AO and catalase, the turnover was increased from 25.6% to 97.5% (in 72 h).The conversion of GOase to oxidation could be increased by the addition of catalase and HRP; however, the authors used sodium phosphate buffer (pH 7) for the enzymatic reactions (Cajnko et al., 2020). As Qin et al. (2015) observed, the conversions of (copper-containing) GOase in phosphate buffers are lower than in other media (such as sodium acetate buffer or deionized water), which could be due to the formation of sparingly soluble Cu3(PO4h).

[0017] McKenna et al. (2017) used a variant of galactose oxidase (GOase M3-5) to oxidize HMF to DFF. Using a system consisting of GOase M3-5, HRP, and catalase, 100 mM HMF was oxidized to DFF with 80% efficiency in 1 h. The authors observed the highest conversions in phosphate buffers, although the wild-type enzyme yields lower conversions in phosphate buffers (see above). The enzyme system is also described in US 10344307 B2.

[0018] Milic et al. (2024) used wild-type GOase immobilized on an epoxy carrier to oxidize 50 mM HMF to DFF (maximum yield 11.3% in 72 h; with biocatalyst exchange every 24 h) in a 50 / 50 (v / v) mixture of ethyl acetate (EtOAc) and 0.1 M sodium phosphate buffer (pH 7.4). The use of an organic phase (EtOAc) serves two purposes: 1. to increase the solubility of HMF and DFF, and 2. to prevent the adsorption of DFF and HMF to the carrier surface. No oxidation of HMF was observed in pure EtOAc when using free GOase.

[0019] Due to its low boiling point (77 °C), EtOAc is inevitably blown out of the reaction vessel by the oxygen required for oxidation. Birmingham et al. therefore used a higher-boiling solvent (diethyl carbonate, bp = 126 °C) as a co-solvant in their process. Using the GOase M7-2A, HMF (100 g / L; 793 mM), which still contains some impurities from the production process, can be oxidized to DFF (96% conversion) within 6 h in the presence of HRP, catalase, and diethyl carbonate. At a substrate concentration of 150 g / L (corresponding to 1.19 M), only 62% conversion can be achieved within 6 h. In a further experiment (see Supplementary Table 7 in Birmingham et al.), the influence of different solvent mixtures (single-phase or two-phase) on the reaction of 250 mM HMF was tested in comparison to buffer (sodium phosphate buffer, pH 7.4). The highest conversions (75% and 71%) were achieved with 40% EtOAc (two-phase) and 71% EtOAc (higher pH 7.4), respectively.10% DMSO (single-phase) was obtained as a cosolvant; in pure buffer, at least 50% of the HM Fs could be converted to DFF. To reduce process costs, the authors suggest replacing the HRP with a suitable chemical or electrochemical activator (Birmingham et al., 2021). The two-phase process was also described in EP 3444355 Al.

[0020] EP 3444354 A1 describes a process for separating DFF from an aqueous solution by cooling. This previously known process is very complex: In a first reaction vessel (vessel A), 250 mM (31.5 g / l) of pre-purified HMF was oxidized in 800 ml of potassium phosphate buffer at 20 °C using GOase, HRP, and catalase. In a second reaction vessel (vessel B), 200 ml of potassium phosphate buffer (pH 7) was cooled to 2 °C. The reaction mixture from vessel A, containing dissolved HMF and DFF, was continuously pumped into vessel B at a rate of 37 ml / min, where the DFF precipitated due to cooling. The aqueous supernatant was pumped back into vessel A for further reaction. After a reaction time of 6 h, DFF precipitated as a solid. Additional DFF could be obtained by evaporating the aqueous reaction solution, but the DFF obtained in this way still contained impurities of the starting material.

[0021] US 8183020 B2 describes the use of an alcohol oxidase from Hansenula sp. for the oxidation of 35 g / l HMF and the use of a pyranose oxidase from Cariolus sp. for the oxidation of 37.8 g / l HMF. The DFF formed was detected by thin-layer chromatography but not quantified.

[0022] Wu et al. (2019) describe the oxidation of HMF to DFF using an enzyme system consisting of GOase, HRP, and catalase immobilized on copper phosphate nanoflowers. The immobilized system resulted in higher DFF yields than the corresponding free enzymes. At a concentration of 200 mM (25.2 g / L) HMF, the immobilized enzyme system exhibited a sharp decline in DFF yield, from initially over 80% to approximately 20% at 400 mM (50.4 g / L) HMF. With the free enzymes, DFF formation ceased altogether at concentrations as low as 300 mM.

[0023] This is where the object of the present invention comes in, and it aims to provide an improved biocatalytic process for the production of 2,5-diformylfuran (DFF).

[0024] Detailed description of the invention

[0025] This problem is solved according to the invention by oxidizing 5-(hydroxymethyl)furfural, which is dissolved in a single-phase aqueous solution, in vitro with an oxidase to 2,5-diformylfuran, which is separated from the aqueous solution, and is characterized by the combination of the measures, i) that the process is carried out at a temperature between 20 °C and 40 °C, ii) that the single-phase aqueous solution contains at least 35 g / l of dissolved 5-(hydroxymethyl)furfural at the beginning of the oxidation, and iii) that the oxidase is a 5-(hydroxymethyl)furfural oxidase.

[0026] The oxidase for the oxidation of HMF to DFF is a 5-(hydroxymethyl)furfural oxidase (HMFO; EC 1.1.3.47).

[0027] It has been shown that the 2,5-diformylfuran formed during oxidation precipitates directly as a solid.

[0028] The process according to the invention is therefore very easy to carry out: it does not require complex cooling of the reaction solution, nor is an organic co-solvant necessary. This offers a further advantage: the low solubility of the product in an aqueous medium allows for easy separation from the reaction solution.

[0029] Furthermore, it has surprisingly been shown that the 2,5-diformylfuran produced according to the inventive method precipitates from the aqueous solution in high purity.

[0030] The process according to the invention is shown schematically in the accompanying figure 1, where A stands for 5-(Hydroxymethyl)furfural (HMF), B for 2,5-Diformylfuran (DFF) and 1 for 5-(Hydroxymethyl)furfural oxidase.

[0031] In a preferred embodiment, the aqueous solution contains the 5-(hydroxymethyl)furfural dissolved at the beginning of the oxidation in concentrations between 50 g / l and 250 g / l, in particular between 150 g / l and 250 g / l.

[0032] It is further preferred if the aqueous solution contains catalase to destroy the hydrogen peroxide formed during oxidation.

[0033] The preferred temperature range is between 25 and 40 °C.

[0034] The particularly preferred pH range of the reaction is between pH 4 and pH 9.

[0035] The oxidizing agent for the reaction is oxygen, which is introduced into the reaction vessel either in the form of compressed air or in pure form. The oxygen concentration in the reaction mixture can also be increased by applying overpressure to the reaction vessel.

[0036] The oxidation is preferably carried out in vitro. The 5-(Hydroxymethyl)furfural oxidase can be present in a suspension, in the homogenate, or in the lysate of the corresponding enzyme-producing cells, with a lysate being preferred.

[0037] A preferred variant of the process according to the invention therefore consists in the fact that the 5-(hydroxymethyl)furfural oxidase is present in the single-phase aqueous solution in a dissolved state (lysate).

[0038] For the purposes of this description and the patent claims, "suspension" means a suspension of resting cells. These are harvested after cultivation (separated from the nutrient medium) and suspended in a suitable buffer system. In contrast to fermentative processes, which also utilize whole cells, the resting cells, due to the removal of carbon sources and nutrients, can no longer grow but serve only for the conversion of substrates (Lin & Tao, 2017). In this context, "homogenate" refers to a physically and / or chemically treated suspension (e.g., treated by pressure, lysozyme, or ultrasound) whereby the cell components are released from the cells. A lysate is obtained when the insoluble cell components of the homogenate are removed, for example, by filtration or centrifugation (see Enzyme Production & Lysate Preparation for details).

[0039] In another variant, 5-(hydroxymethyl)furfural oxidase can also be modified at the N-terminus with a water-soluble polymer such as polyethylene glycol, immobilized in or on a solid matrix, or be part of a fusion protein.

[0040] In another variant, 5-(Hydroxymethyl)furfural oxidase can be in powder form, in lyophilized or spray-dried form.

[0041] The process described here allows for the continuous production of DFF. The precipitated solid can be continuously separated from the aqueous reaction solution using a suitable separator, and the aqueous solution is returned to the reaction vessel. Additionally, fresh 5-(hydroxymethyl)furfural can be added to the reaction solution.

[0042] The following examples describe preferred embodiments of the invention in more detail.

[0043] materials

[0044] 5-(Hydroxymethyl)furfural (HMF) was supplied by Biosynth, 2,5-Diformylfuran (2,5-Furandicarbaldehyde; DFF) was supplied by Sigma-Aldrich, potassium dihydrogen phosphate, dipotassium hydrogen phosphate and sodium dodecyl sulfate (SDS) were supplied by Carl Roth, acetonitrile was supplied by PanReac AppliChem (ITW Reagents) and triethanolamine was supplied by Chem-Lab NV.

[0045] Production of enzymes & production of lysates

[0046] General information on the expression of recombinant enzymes in E. coli

[0047] For recombinant enzyme production in an Escherichia co / / strain, the gene to be expressed was first amplified in PCR using genomic DNA or its synthetically adapted equivalent (adapted to the codon usage of E. coli) as a template, along with specific oligonucleotides that additionally carry recognition sequences for restriction endonucleases. The resulting DNA was then isolated from the reaction mixture. After nucleic acid digestion with the restriction enzymes Sphl and Hind II I, the gene fragment encoding the target enzyme was ligated into the Sphl- and Hind II I-receptor backbone of the expression vector pQE70-Kan. The ligation product was transformed into chemically competent E. co / / cells (ToplOF), and the resulting colonies were used for plasmid isolation and restriction analysis.

[0048] The result of the cloning step was verified by restriction enzyme digestion and DNA sequencing. The resulting construct carries the target gene under the IPTG-inducible T5 promoter.

[0049] For the overexpression of the enzyme in E. coli, the resulting expression plasmid was transformed into competent expression cells RB791. After 24 h incubation at 37 °C, the resulting colonies were inoculated into LB medium for expression assays.

[0050] The following day, expression cultures with an optical density (OD550) of 0.02 were inoculated and shaken at 37 °C until an OD550 of 0.3 was reached. The temperature was then lowered to 25 °C, and the cultures were induced with 0.1 mM IPTG when an OD550 of 0.5 was reached. After 22 h, the cultures were harvested (separated from the medium by centrifugation into a cell pellet) and analyzed for the expression of the recombinant enzyme using SDS-gel electrophoresis and activity determination (for use in a use test).

[0051] Production of cell lysates using Sonifier digestion

[0052] To prepare a cell suspension, the cell pellet produced according to the above procedure was weighed into a suitable container, mixed with buffer (e.g., triethanolamine (TEA) - HCl), and dissolved with stirring. The biomass fraction is typically 20% by mass, with the remainder consisting of the buffer. A Branson Sonifier 450 was used for cell disruption. The suspension was treated three times with 15 ultrasonic pulses each (device settings: Timer = 15; Duty Cycle = 50; Output Control = 3-5).

[0053] The resulting homogenate was centrifuged for 10 min at 4 °C and 16000 rpm (Eppendorf centrifuge 5417R) to separate the insoluble cell fragments and obtain the lysate.

[0054] Table 1. Enzyme types and donor organisms for the enzyme used.

[0055] Note *: In the NCBI Protein Database, the HMF oxidase from P. nitroreducens is classified as glucose-methanol-choline (GMC) oxidoreductase, which as a superfamily also includes the HMF oxidases (Vinambres et al., 2020).

[0056] Note **: The HMF oxidase used also catalyzes the reaction of DFF to the “overoxidation product” 5-formylfuran carboxylic acid (FFA).

[0057] Analytical methods

[0058] High Performance Liquid Chromatography (HPLC)

[0059] High-performance liquid chromatography (HPLC) was used to quantify HMF and DFF. Detection was performed using a UV detector. A Phenomenex Rezex ROA organic acid H+ (8%) column with a suitable guard column was used for the measurement and isocratically eluted with 1 mM sulfuric acid.

[0060] The following examples describe preferred variants of the process according to the invention in more detail. The lysate used in these examples was produced according to the process described above. Example 1

[0061] Oxidation of HMF to DFF

[0062] The reaction was carried out in a Labfors 5 benchtop bioreactor (Infors AG). A glass reactor with an attached stirrer, pH electrode, and C2 sensor was used. pH control was achieved by adding 5M NaOH or 5M H2SO4.

[0063] Initially, 62 g of HMF, 257.2 ml of deionized water, and 100 ml of a 500 mM potassium phosphate buffer (pH 7) were placed in the reactor and heated to 20 °C with stirring. A clear brown solution was obtained (final HMF concentration 125 g / l; 993 mM).

[0064] To start the reaction, 3.6 kL of HMFO lysate (corresponding to 58 U / g substrate) was added. The oxygen supply (via a sparger) was set to 0.05 l / min.

[0065] After some time, a deposit of DFF formed.

[0066] During operation, samples were continuously taken from the reactor solution and analyzed as follows: 50 µl of the reactor solution were mixed with 200 µl of acetonitrile and incubated in an Eppendorf Thermomixer at 85 °C and 1200 rpm for 15 min. The sample was briefly centrifuged, mixed with 750 µl of deionized water, vortexed, and then centrifuged for 5 min at max. g. 200 µl of the supernatant were transferred to an HPLC vial with an insert and measured by HPLC (Rl detection).

[0067] After 30 hours of operation, 50 ml of the reaction mixture was taken off. The precipitated solid was filtered off directly, washed twice with 10 ml of water each time, and dried overnight in a vacuum drying oven at 40 °C.

[0068] In this way, 4.2 g of DFF could be isolated as a brownish solid (72% yield based on the extracted reaction mixture; analyte distribution: 98.8% DFF). No FFA could be detected in the solid. Example 2

[0069] Oxidation of HMF to DFF - product precipitation by cooling

[0070] The reaction was carried out in a Labfors 5 benchtop bioreactor (Infors AG). A glass reactor with an attached stirrer, pH electrode, and C2 sensor was used. pH control was achieved by adding 5M NaOH or 5M H2SO4.

[0071] Initially, 31 g of HMF, 288.2 ml of deionized water, and 100 ml of a 500 mM potassium phosphate buffer (pH 7) were placed in the reactor and heated to 20 °C while stirring. A clear brown solution was obtained (final HMF concentration 64 g / l; 508 mM).

[0072] To start the reaction, 3.6 kL of HMFO lysate (corresponding to 116 U / g substrate) was added. The oxygen supply (via a sparger) was set to 0.05 l / min.

[0073] After some time, a deposit of DFF formed.

[0074] During operation, samples were continuously taken from the reactor solution and analyzed as follows: 50 µl of the reactor solution were mixed with 200 µl of acetonitrile and incubated in an Eppendorf Thermomixer at 85 °C and 1200 rpm for 15 min. The sample was briefly centrifuged, mixed with 750 µl of deionized water, vortexed, and then centrifuged for 5 min at max. g. 200 µl of the supernatant were transferred to an HPLC vial with an insert and analyzed by HPLC (UV detection).

[0075] After 4 hours of operation, 50 ml of the reaction mixture was taken and incubated in a refrigerator at 2 °C. The subsequent work-up steps were carried out analogously to Example 1 (filtration and drying).

[0076] In this way, 1.6 g of DFF could be isolated as a brownish solid (65% yield based on the reaction mixture taken; 97.4% DFF).

[0077] Example 3

[0078] Oxidation of HMF to DFF

[0079] The reaction was carried out in a Labfors 5 benchtop bioreactor (Infors AG). A glass reactor with an attached stirrer, pH electrode, and C2 sensor was used. pH control was achieved by adding 5M NaOH or 5M H2SO4.

[0080] Initially, 25.2 g of HMF, 294 ml of deionized water, and 100 ml of a 500 mM potassium phosphate buffer (pH 7) were placed in the reactor and heated to 25 °C while stirring. A clear brown solution was obtained (final HMF concentration 50 g / l; 400 mM).

[0081] To start the reaction, 3.6 kL of HMFO lysate (corresponding to 143 U / g substrate) was added. The oxygen supply (via a sparger) was set to 0.05 l / min.

[0082] During operation, samples were continuously taken from the reactor solution and analyzed as follows: 50 µl of the reactor solution were mixed with 200 µl of acetonitrile and incubated in an Eppendorf Thermomixer at 85 °C and 1200 rpm for 15 min. The sample was briefly centrifuged, mixed with 750 µl of deionized water, vortexed, and then centrifuged for 5 min at max. g. 200 µl of the supernatant were transferred to an HPLC vial with an insert and analyzed by HPLC (UV detection).

[0083] Within one hour of starting the reaction, 80% of the HMF was oxidized. In addition, a precipitate of DFF formed. The DFF was filtered off analogously to Example 1.

[0084] literature

[0085] Danielli, C., van Langen, L., Boes, D., Asaro, F., Anselmi, S., Provenza, F., Renzi, M., & Gardossi, L. (2022).

[0086] 2.5-Furandicarboxaldehyde as a bio-based crosslinking agent replacing glutaraldehyde for covalent enzyme immobilization. RSC Advances, 12(55), 35676. https: / / doi.org / 10.1039 / D2RA07153C

[0087] Dai, J. (2021). Synthesis of 2,5-diformylfuran from renewable carbohydrates and its applications: A review. Green Energy & Environment, 6(1), 22-32. https: / / doi.Org / 10.1016 / j.gee.2020.06.013

[0088] Acosta, A. M., Turull, C. C., Cosovanu, D., Marti, N. S., & Canela-Garayoa, R. (2021). Novel and Efficient Biotechnological Approach to Produce 2,5-Diformylfuran from Biomass-Derived 5-Hydroxymethylfurfural. ACS Sustainable Chemistry & Engineering, 9(43), 14550-14558. https: / / doi.org / 10.1021 / acssuschemeng.lc05308

[0089] Derflinger, C., Kamm, B., & Paulik, C. (2021). Sustainable aerogels derived from bio-based 2,5- diformylfuran and depolymerization products of lignin. International Journal of Biobased Plastics, 3(1), 29-39. https: / / doi.org / 10.1080 / 24759651.2021.1877025

[0090] Cong, H., Yuan, H., Tao, Z., Bao, H., Zhang, Z., Jiang, Y., Huang, D., Liu, H., & Wang, T. (2021). Recent Advances in Catalytic Conversion of Biomass to 2,5-Furandicarboxylic Acid. Catalysts, 11(9), 1113. https: / / doi.org / 10.3390 / catalll091113

[0091] Amarasekara, A. S., Williams, L. D., & Ebede, C. C. (2008). Mechanism of the dehydration of D-fructose to 5-hydroxymethylfurfural in dimethyl sulfoxide at 150 °C: an NMR study. Carbohydrate Research, 343(18), 3021-3024. https: / / doi.Org / 10.1016 / j.carres.2008.09.008

[0092] Laugel, C., Estrine, B., Le Bras, J., Hoffmann, N., Marinkovic, S., & Muzart, J. (2014). NaBr / DMSO-lnduced Synthesis of 2,5-Diformylfuran from Fructose or 5-(Hydroxymethyl)furfural. ChemCatChem, 6(5), 1195- 1198. https: / / doi.org / 10.1002 / cctc.201400023

[0093] Halliday, G. A., Young, R. J., & Grushin, V. V. (2003). One-Pot, Two-Step, Practical Catalytic Synthesis of

[0094] 2.5-Diformylfuran from Fructose. Organic Letters, 5(11), 2003-2005. https: / / doi.org / 10.1021 / ol034572a

[0095] Li, Q„ Ma, C.-L., & He, Y.-C. (2023). Effective one-pot chemoenzymatic cascade catalysis of biobased feedstock for synthesizing 2,5-diformylfuran in a sustainable reaction system. Bioresource Technology, 378, 128965. https: / / doi.Org / 10.1016 / j.biortech.2023.128965 Qin, Y.-Z., Li, Y.-M., Zong, M.-H., Wu, H., & Li, N. (2015). Enzyme-catalyzed selective oxidation of 5-hydroxymethylfurfural (HMF) and separation of HMF and 2,5-diformylfuran using deep eutectic solvents. Green Chemistry, 17(7), 3718-3722. https: / / doi.org / 10.1039 / C5GC00788G

[0096] Tja II inks, G., Boverio, A., Jager, A. W., Kaya, S. G., Mattevi, A., & Fraaije, M. W. (2023). Efficient Oxidation of 5-Hydroxymethylfurfural Using a Flavoprotein Oxidase from the Honeybee Apis mellifera. ChemBioChem, 24(24), e202300588. https: / / doi.org / 10.1002 / cbic.202300588

[0097] Cajnko, M. M., Novak, U., Grilc, M., & Likozar, B. (2020). Enzymatic conversion 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 / sl3068-020-01705-z

[0098] McKenna, S. M., Mines, P., Law, P., Kovacs-Schreiner, K., Birmingham, W. R., Turner, N. J., Leimkühler, S., & Carnell, A. J. (2017). The continuous oxidation of HMF to FDCA and the immobilisation and stabilisation of periplasmic aldehyde oxidase (PaoABC). Green Chemistry, 19, 4660-4665. https: / / doi.org / 10.1039 / C7GC01696D

[0099] Milic, M., Byström, E., Dominguez de Maria, P., & Kara, S. (2024). Selective Oxidation of 5- Hydroxymethylfurfural to 2,5-Diformylfuran in Biphasic Media using Immobilized Galactose Oxidase: Proof of Concept and Limitations. ChemCatChem, 16(4), e202301384. https: / / doi.org / 10.1002 / cctc.202301384

[0100] Birmingham, W. R., Toftgaard Pedersen, A., Dias Gomes, M., Bpje Madsen, M., Breuer, M., Woodley, J. M., & Turner, N. J. (2021). Toward scalable biocatalytic conversion of 5-hydroxymethylfurfural by galactose oxidase using coordinated reaction and enzyme engineering. Nature Communications, 12, 4946. https: / / doi.org / 10.1038 / s41467-021-25034-3

[0101] Wu, Z., Shi, L., Yu, X., Zhang, S., & Chen, G. (2019). Co-lmmobilization of Tri-Enzymes for the Conversion of Hydroxymethylfurfural to 2,5-Diformylfuran. Molecules, 24(20), 3648. https: / / doi.org / 10.3390 / molecules24203648

[0102] Lin, B., & Tao, Y. (2017). Whole-cell biocatalysts by design. Microbial Cell Factories, 16, 106. https: / / doi.org / 10.1186 / sl2934-017-0724-7

[0103] Protein [Internet], Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information. Accession No. WP_024766380.1, GMC oxidoreductase [Pseudomonas nitroreducens]. Verfügbar unter: https: / / www.ncbi.nlm.nih.gOv / protein / WP_024766380.l (Zugriff am 29.04.2024) Vinambres, M., Espada, M., Martinez, A. T., & Serrano, A. (2020). Screening and Evaluation of New Hydroxymethylfurfural Oxidases for Furandicarboxylic Acid Production. Applied and Environmental Microbiology, 86(16), e00842-20. https: / / doi.org / 10.1128 / AEM.00842-20

Claims

Patent claims 1. A process for the production of 2,5-diformylfuran by oxidizing 5-(hydroxymethyl)furfural, which is dissolved in a single-phase aqueous solution, with an oxidase in vitro to 2,5-diformylfuran, which is separated from the aqueous solution, characterized by the combination of the measures, i) that the process is carried out at a temperature between 20 °C and 40 °C, ii) that the single-phase aqueous solution contains at least 35 g / l of dissolved 5-(hydroxymethyl)furfural at the beginning of the oxidation, and iii) that the oxidase is a 5-(hydroxymethyl)furfural oxidase.

2. The method according to claim 1, characterized in that the single-phase aqueous solution contains the 5-(hydroxymethyl)furfural dissolved in a concentration of between 50 g / l and 250 g / l at the beginning of the oxidation.

3. Method according to claim 2, characterized in that the single-phase aqueous solution contains the 5-(hydroxymethyl)furfural dissolved at a concentration of between 150 g / l and 250 g / l at the beginning of the oxidation.

4. Method according to one of claims 1 to 3, characterized in that it is carried out at a temperature between 25 °C and 40 °C.

5. Method according to one of claims 1 to 4, characterized in that the 5-(hydroxymethyl)furfural oxidase is present in the aqueous solution in a dissolved state.

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