Chemical enzymatic route for synthesis of 2,5-furan dicarboxylic acid

Abstract

The application uses cheap and easily available hexose acid as a starting material, first obtains two precursors of 2,5-furan dicarboxylic acid synthesis, namely hexahydroxy acid and 4-deoxy-5-dehydro-hexahydroxy acid through enzyme catalytic reaction, and then 4-deoxy-5-dehydro-hexahydroxy acid is dehydrated under acidic conditions to obtain 2,5-furan dicarboxylic acid.

Description

Technical Field

[0001] This invention relates to the chemical enzymatic synthesis of 2,5-furandicarboxylic acid, and particularly to the application of engineered galactose oxidase, aldehyde dehydrogenase and gluconic acid dehydratase in the synthesis of 2,5-furandicarboxylic acid. Background Technology

[0002] 2,5-Furandicarboxylic acid (FDCA), a monomer with unique properties used in building polymers such as resins, is considered a green alternative to terephthalic acid (currently the main compound in polymer and resin manufacturing). Although significant progress has been made in FDCA synthesis technology over the past two decades, its industrial-scale production still faces numerous challenges, resulting in high prices and hindering large-scale application. Currently, the mainstream route for FDCA production involves the dehydration of fructose to prepare HMF, followed by the catalytic oxidation of HMF to form FDCA. However, this route suffers from poor HMF stability, unsatisfactory catalyst selectivity and stability, and the need for a neutralization step, leading to low overall yield, high impurity levels, and a complex purification process.

[0003] In addition to the route of synthesizing FDCA from HMF and its derivatives prepared from fructose, FDCA can also be prepared by dehydration cyclization of hexanoic acids (such as galactosic acid, glucosic acid, mannosic acid, etc.).

[0004] Gluconic acid exhibits good stability and can be dehydrated to form FDCA under acid catalysis (J. Kim et al., Biomass and Bioenergy, 181(2024), 107049). However, current solvents, acid catalysts, and process conditions still present challenges, such as equipment corrosion and environmental issues. Furthermore, 4-deoxy-5-dehydroglucaric acid (DDG), formed by the dehydration of gluconic acid, is one of the precursors for FDCA synthesis. Compared to gluconic acid, the reaction pathway for FDCA synthesis using DDG is shorter and contains fewer impurities, making it more advantageous for FDCA synthesis.

[0005] The preparation of gluconic acid still faces many challenges. Current methods for synthesizing gluconic acid mainly involve microbial fermentation and chemical oxidation. Chemical oxidation typically requires very harsh reaction conditions, such as strong oxidants like nitric acid at high temperatures and pressures. It also exhibits low selectivity, produces numerous byproducts, and poses significant challenges to the separation and purification of the compounds. When using fermentation to prepare gluconic acid, the acidity of the fermentation product leads to a decrease in the pH of the fermentation system, limiting the yield. Summary of the Invention

[0006] This invention aims to obtain two precursors for FDCA synthesis—hexonia acid and 4-deoxy-5-dehydro-hexonia acid—through an enzymatic reaction using inexpensive and readily available hexonia acid as a starting material. The synthetic route for hexonia acid is as follows: Figure 1 As shown.

[0007] The hexonic acid raw material can be gluconic acid, galactonic acid, mannitol, allonic acid, iduconic acid, etc.

[0008] Specifically, this invention uses gluconic acid as a starting material to synthesize glucocortic acid, and the specific process includes two steps, such as... Figure 2 Reaction A and reaction B are shown in the figure. Reaction A is an enzymatic reaction in which gluconic acid is converted into L-guluronic acid by an oxidase in the presence of oxygen. The oxidases involved in catalyzing reaction A are diverse, including L-lactic acid oxidase (EC 1.1.3.2), glucose oxidase (EC 1.1.3.4), hexose oxidase (EC 1.1.3.5), L-gulonolactone oxidase (EC 1.1.3.8), galactose oxidase (EC 1.1.3.9), pyranose oxidase (EC 1.1.3.10), L-sorbose oxidase (EC 1.1.3.11), alcohol oxidase (EC 1.1.3.13), N-acylhexosamine oxidase (EC 1.1.3.29), polyvinyl alcohol oxidase (EC 1.1.3.30), D-arabinose-1,4-lactone oxidase (EC 1.1.3.37), D-mannitol oxidase (EC 1.1.3.40), and alditol. The oxidases involved include 5-hydroxymethylfurfural oxidase (EC1.1.3.41), 3-deoxy-α-D-manno-octulose oxidase (EC1.1.3.47), and 3-deoxy-α-D-manno-octulose oxidase-8-oxidase (EC1.1.3.48). More specifically, the oxidase catalyzing reaction A is a galactose oxidase variant, modified to possess the oxidase activity of oxidizing the 6-hydroxyl group of gluconic acid, thereby obtaining the precursor L-guluronic acid for the synthesis of gluconic acid. The active site of galactose oxidase consists of a single copper ion coordinated to two histidine residues, one tyrosine residue, and a cross-linked cysteine-tyrosine unit. Therefore, the reaction requires copper ions to participate in the formation of catalytic activity. During the reaction, some copper ions at the enzyme's active site exist in a semi-reduced state, leading to the loss of enzyme activity. This application uses horseradish peroxidase (HRP, EC 1.11.1.7) as an activator of galactose oxidase, via 1e -The oxidative half-reduction form of galactose oxidase gives it catalytic activity, and the hydrogen peroxide produced in the reaction is decomposed by catalase (EC 1.11.1.6) to avoid the inhibition of the enzyme by hydrogen peroxide.

[0009] Reaction B is also an oxidation reaction, where L-guluronic acid is oxidized to D-gluconic acid. The catalyst for reaction B can be a metal catalyst (such as Au, Pt, Pd, Ru, Rh, Ir, etc.) or an enzyme. Enzymes that can catalyze reaction B include alcohol dehydrogenases (EC 1.1.1.1, EC 1.1.1.2), ketone reductases, and uronate dehydrogenases (EC 1.1.1.203). More specifically, the enzyme that can catalyze reaction B is uronate dehydrogenase (UDH); UDH requires the coenzyme NAD. + or NADP + This process completes the oxidation of the substrate, generating NADH or NADPH. In the presence of oxygen, NADH or NADPH is catalyzed by NAD(P)H oxidases (NOx, EC 1.6.3.1, EC 1.6.3.4) to oxidize to NAD. + or NADP + This enables the cyclic regeneration of coenzymes.

[0010] Figure 2 Reaction A shown can use catalase purchased from Sigma, catalog number 02071-5ML-F; or horseradish peroxidase purchased from Sigma, catalog number P8125-50KU.

[0011] pass Figure 1 After the reaction yields hexonic acid, it can be directly dehydrated and cyclized under acidic conditions via chemical methods to generate FDCA. The specific process is as follows: Figure 3 As shown. However, hexonic acid readily forms 1,5-hexonic acid lactone during the reaction, thus easily forming [a specific type of lactone] in the reaction. Figure 4 The 4-hydroxy-6-carboxy-2-pyranone (HOPC) impurity shown affects the yield of the product FDCA.

[0012] To reduce reaction byproducts and improve reaction efficiency, this invention utilizes Figure 5 The reaction shown further catalyzes hexonic acid with a dehydrating enzyme to generate 4-deoxy-5-dehydro-hexonic acid. Using this intermediate for the synthesis of FDCA results in a shorter and more efficient reaction pathway, and avoids the formation of the impurity HOPC.

[0013] The hexonic acid mentioned in the above route can be D / L-gluconic acid, D / L-galactonic acid, D / L-mannonic acid, D / L-idusonic acid, D / L-aloacetic acid, D / L-altraric acid, (2S,3R,4R,5S)-2,3,4,5-tetrahydroxyadipic acid, etc.

[0014] Figure 5The dehydrating enzymes mentioned in the reaction may be gluconate dehydratase (EC 4.2.1.40), citrate dehydratase (EC 4.2.1.4), arabinose dehydratase (EC 4.2.1.5), galactose dehydratase (EC 4.2.1.6), alginate dehydratase (EC 4.2.1.7), mannolate dehydratase (EC 4.2.1.8), dihydroxy acid dehydratase (EC 4.2.1.9), 3-dehydroquinoline dehydratase (EC 4.2.1.10), phosphoglucose dehydratase (EC 4.2.1.12), L-arabinose dehydratase (EC 4.2.1.25), propylene glycol dehydratase (EC 4.2.1.28). ), glycerol dehydratase (EC4.2.1.30), L-tartrate dehydratase (EC4.2.1.32), isopropyl malonate dehydratase (EC4.2.1.33.3), (S)-2-methylmalonic acid dehydratase (EC4.2.1.34), (R)-2-methylmalonic acid dehydratase (EC4.2.1.35), galactogluconate dehydratase (EC4.2.1.39), gluconate dehydratase (EC4.2.1.39), glycoside dehydratase (EC4.2.1.30), glycerol dehydratase (EC4.2.1.30), L-tartrate dehydratase (EC4.2.1.32), isopropyl malonate dehydratase (EC4.2.1.33.3), glycoside dehydratase (EC4.2.1.34), glycoside dehydratase (EC4.2.1.35), galactogluconate dehydratase (EC4.2.1.39), glycoside dehydratase (EC4.2.1.30), glycoside dehydratase (EC4.2.1.32), glycoside dehydratase (EC4.2.1.33.3), glycoside dehydratase (EC4.2.1.34), glycoside dehydratase (EC4.2.1.35), glycoside dehydratase (EC4.2.1.35), glycoside dehydratase (EC4.2.1.39), glycoside dehydratase (EC4.2.1.30), glycoside dehydratase (EC4.2.1.32), glycoside dehydratase (EC4.2.1.33.3), glycoside dehydratase (EC4.2.1.34), glycoside dehydratase (EC4.2.1.35), 4.2.1.42), 2-dehydro-3-deoxy-L-arabinose dehydratase (EC 4.2.1.43), inosine-2 dehydratase (EC 4.2.1.44), CDP-glucose 4,6-dehydratase (4.2.1.45), TDP-glucose 4,6-dehydratase (EC 4.2.1.46), GDP-4,6-mannose dehydratase (EC 4.2.1.47), D-succinate L-Succinate dehydratase (EC4.2.1.67), L-Succinate dehydratase (EC4.2.1.68), UDP-glucose 4,6-dehydratase (EC4.2.1.76), 2-methylcitrate dehydratase (EC4.2.1.79), D-tartrate dehydratase (EC4.2.1.81), xylorate dehydratase (EC4.2.1.82), L-rhamnite dehydratase (EC4.2.1.82). 1.90), 2-methylisocitrate dehydratase (EC 4.2.1.99), 1,5-dehydro-D-fructose dehydratase (EC 4.2.1.111), 2-methylcitrate dehydratase (EC 4.2.1.117), linalool dehydratase (EC 4.2.1.127), D-lactic acid dehydratase (EC 4.2.1.130), gluconic acid / galactonate dehydratase (EC 4.2.1.14) 0), 2-dehydro-3-deoxy-D-arabinose dehydratase (EC4.2.1.141), L-galactose dehydratase (EC4.2.1.146), L-talarate dehydratase, EC (4.2.1.156), D-galactose dehydratase (EC4.2.1.158), 6-deoxy-6-sulfonic acid-D-glucose dehydratase (EC4.2.1.162), etc.

[0015] More specifically, this invention synthesizes FDCA from gluconic acid, which can be obtained by heating under acidic conditions, as shown in the specific route below. Figure 6 As shown in reaction C, the acid used is selected from one or more of sulfuric acid and hydrohalic acids (such as hydrofluoric acid, hydrochloric acid, hydrobromic acid, hydroiodic acid, etc.).

[0016] Besides the direct dehydration of gluconic acid to FDCA via acid catalysis, partial dehydration of gluconic acid can also be achieved via gluconic acid dehydratase (GlucD, EC4.2.1.40). Figure 7 Reaction D) as shown generates 4-deoxy-5-dehydroglucaric acid (DDG). Gluconate dehydratase is a magnesium-dependent enzyme. Besides magnesium ions, divalent metal ions such as manganese ions can also act as cofactors for gluconate dehydratase, participating in the enzyme's catalytic process. The magnesium ion concentration in the enzymatic dehydration reaction is 0.05-5 mM; the reaction temperature is 25-60℃, preferably 45℃; and the pH range is 7-10. Using DDG for FDCA synthesis results in a shorter and more efficient reaction pathway, avoiding the formation of the impurity HOPC.

[0017] 4-Deoxy-5-dehydro-gluconic acid (DDG) can be directly converted to FDCA by cyclization and dehydration under heating and acidic conditions via chemical methods. Figure 8 The acid used in reaction E is selected from one or more of sulfuric acid and hydrohalic acids (such as hydrofluoric acid, hydrochloric acid, hydrobromic acid, hydroiodic acid, etc.). Attached Figure Description

[0018] Figure 1 For the enzyme-catalyzed preparation of hexonic acid

[0019] Figure 2 To facilitate the enzyme-catalyzed synthesis of L-guluronic acid (reaction A) and D-gluconic acid (reaction B).

[0020] Figure 3 FDCA is formed by the direct dehydration and cyclization of hexanoic acid under heating and acidic conditions.

[0021] Figure 4 It is 4-hydroxy-6-carboxy-2-pyranone

[0022] Figure 5 Enzymatic preparation of 4-deoxy-5-dehydro-hexanoic acid and synthesis of FDCA from 4-deoxy-5-dehydro-hexanoic acid.

[0023] Figure 6 The reaction involves the dehydration of gluconic acid under acidic conditions to produce FDCA (reaction C).

[0024] Figure 7The gluconic acid undergoes partial dehydration under the catalysis of a dehydrating enzyme to produce 4-deoxy-5-dehydro-gluconic acid (reaction D).

[0025] Figure 8 The dehydration of 4-deoxy-5-dehydro-gluconic acid under acidic conditions yields FDCA (reaction E). Detailed Implementation Plan

[0026] The following examples further illustrate the present invention, providing a clear and complete description of its technical solutions. However, the present invention is not limited thereto; the described embodiments are only a part of the embodiments, not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Furthermore, the experimental methods involved in the following examples, but without specific conditions, are generally performed according to conventional or manufacturer-recommended conditions in the fields of biology and chemistry, and the reagents and equipment involved, but not specifically mentioned, are all commercially available.

[0027] Example 1

[0028] All reaction samples were analyzed using the following LC-MS method.

[0029]

[0030]

[0031] Example 2

[0032] In a 500 mL reaction flask, add 12.5 g sodium gluconate, 0.5 g SEQ ID No: 1 galactose oxidase mutant enzyme powder, 0.1 mL catalase enzyme solution, 25 mL copper sulfate stock solution (5 mM), 50 mM Tris-HCl (pH = 8), and 25 mL 1 g / L horseradish peroxidase stock solution. Start stirring, and the final reaction volume is 250 mL. React at room temperature for 24 h. LC-MS analysis showed that the yield of L-guluronic acid was 93.1%.

[0033] Example 3

[0034] The reaction solution obtained in Example 2 was heated at 60°C for 2 hours, filtered under reduced pressure, and the filtrate was collected. The filtrate was rotary evaporated at 50°C until 25% of its volume remained. The remaining rotary evaporated liquid was placed at 4°C overnight, filtered under reduced pressure, and the filter cake was washed twice with ice water to finally obtain 8g of L-guluronate sodium solid.

[0035] Example 4

[0036] In a 100 mL reaction flask, add 2.5 g of L-guluronate sodium solid prepared in Example 3, 0.5 g of wet cells expressing UDH as shown in SEQ ID No: 2, 0.5 g of wet cells expressing NOx as shown in SEQ ID No: 3, 50 mM Tris-HCl (pH = 9), and 5 mL of 1 g / L NAD. + The mother liquor and the final reaction volume were 50 mL. During the reaction, the pH was adjusted to pH 9 in real time with 5M sodium hydroxide. The reaction was carried out at 30℃ for 24 h. The sample was taken for LC-MS detection. 94% of the L-guluronate sodium in the reaction solution was converted to gluconic acid.

[0037] Example 5

[0038] In a 30 mL reaction flask, 1 g of potassium D-gluconic acid monophosphate, 0.005 g of wet cells expressing GlucD as shown in SEQ ID No: 4, 0.5 mL of 20 mM magnesium chloride stock solution, and 50 mM Tris-HCl (pH = 8) were added. The pH was adjusted to 8 with 5 M potassium hydroxide. Stirring was started, and the final reaction volume was 5 mL. The reaction was carried out at 30 °C for 5 h. LC-MS analysis showed that the conversion rate of potassium D-gluconic acid monophosphate to 4-deoxy-5-dehydro-gluconic acid was >99%.

[0039] Example 6

[0040] In five 30 mL reaction flasks, 1 g of potassium D-gluconic acid monophosphate, 0.0025 g of wet cells expressing GlucD as shown in SEQ ID No: 4, 0.5 mL of 20 mM magnesium chloride stock solution, and 50 mM Tris-HCl (pH = 8) were added respectively. The pH was adjusted to 8 with 5 M potassium hydroxide. Stirring was started, and the final reaction volume was 5 mL. The reaction was carried out at 30℃, 35℃, 40℃, 45℃, 50℃, and 60℃ for 4 h respectively. Samples were taken for LC-MS detection. The conversion rate of potassium D-gluconic acid monophosphate to 4-deoxy-5-dehydro-gluconic acid is shown in the table below.

[0041] reaction temperature Conversion rate 30℃ 64％ 35℃ 94％ 40℃ 96％ 45℃ >99％ 50℃ 90％ 60℃ 75％

[0042] Example 7

[0043] In five 30 mL reaction flasks, 1 g of potassium D-gluconic acid monophosphate, 0.0025 g of wet cells expressing GlucD as shown in SEQ ID No: 4, 0.5 mL of 0.5 mM, 20 mM, and 50 mM magnesium chloride stock solutions, and 50 mM Tris-HCl (pH = 8) were added respectively. The pH was adjusted to 8 with 5 M potassium hydroxide. Stirring was started, and the final reaction volume was 5 mL. The reaction was carried out at 40 °C for 4 h. Samples were taken for LC-MS detection. The conversion rate of potassium D-gluconic acid monophosphate to 4-deoxy-5-dehydro-gluconic acid is shown in the table below.

[0044]

[0045]

[0046] Example 8

[0047] In five 30 mL reaction flasks, 1 g of potassium D-gluconic acid monophosphate, 0.0025 g of wet cells expressing GlucD as shown in SEQ ID No: 4, 0.5 mL of 20 mM magnesium chloride stock solution, and 50 mM Tris-HCl (pH = 8) were added respectively. The pH was adjusted to pH 7, pH 8, pH 9, and pH 10 respectively with 5 M potassium hydroxide. Stirring was started, and the final reaction volume was 5 mL. The reaction was carried out at 40 °C for 4 h. The conversion rate of potassium D-gluconic acid monophosphate to 4-deoxy-5-dehydro-gluconic acid was detected by LC-MS as shown in the table below.

[0048] reaction pH Conversion rate pH7 93％ pH8 96％ pH9 95％ pH10 89％

[0049] Example 9

[0050] In a 500 mL reaction flask, add 12.5 g sodium gluconate, 0.5 g of galactose oxidase mutant enzyme powder (SEQ ID No: 1), 2.5 g of wet cells expressing UDH (SEQ ID No: 2), 2.5 g of wet cells expressing NOx (SEQ ID No: 3), 0.125 g of wet cells expressing GlucD (SEQ ID No: 4), 0.1 mL of catalase solution, 25 mL of copper sulfate stock solution (5 mM), 50 mM Tris-HCl (pH = 9), 25 mL of 1 g / L horseradish peroxidase (HRP) stock solution, and 25 mL of 20 mM magnesium chloride stock solution. Start stirring, and the final reaction volume is 250 mL. During the reaction, adjust the pH to pH 8.5 in real time with 5 M sodium hydroxide. React at room temperature for 24 h. LC-MS analysis showed that the conversion rate of sodium gluconate to 4-deoxy-5-dehydro-gluconic acid was 99.2%.

[0051] Example 10

[0052] The reaction solution of Example 9 was heated at 60°C for 2 hours to denature and precipitate the protein. After filtration, the filtrate was freeze-dried to obtain 9.5 g of solid. 0.2 g, 0.5 g, and 1 g of the solid were added to n-butanol and concentrated sulfuric acid, with the concentration of concentrated sulfuric acid controlled at 5 M. The reaction was carried out at 60°C, 80°C, and 120°C for 5 hours. The final conversion rate of 4-deoxy-5-dehydro-gluconic acid to FDCA is shown in the table below.

[0053]

[0054] It should be understood that after reading the above description of the present invention, those skilled in the art can make various alterations or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A chemical enzymatic route for the synthesis of 2,5-furandicarboxylic acid, comprising the following enzymatic and chemical reactions: (i) Hexuronic acid was obtained by enzyme-catalyzed oxidation of hexuronic acid as the starting material; (ii) hexuronic acid was further oxidized by enzyme to obtain hexonic acid; (iii) hexonic acid was dehydrated by enzyme to obtain 4-deoxy-5-dehydro-hexonic acid; (iv) 4-deoxy-5-dehydro-hexonic acid was dehydrated under acidic conditions to obtain 2,5-furandicarboxylic acid.

2. The method of claim 1, wherein the reactants in step (i) comprise gluconic acid, galactonic acid, mannitol, allonic acid, and idulaonic acid; and the enzymes used comprise L-lactate oxidase (EC1.1.3.2), glucose oxidase (EC1.1.3.4), hexose oxidase (EC1.1.3.5), L-gulonolactone oxidase (EC1.1.3.8), galactose oxidase (EC1.1.3.9), pyranose oxidase (EC1.1.3.10), L-sorbose oxidase (EC1.1.3.11), and alcohol oxidase (EC1.1.3.13). N-acylhexosamine oxidase (EC1.1.3.29), polyvinyl alcohol oxidase (EC1.1.3.30), D-arabinose-1,4-lactone oxidase (EC1.1.3.37), D-mannitol oxidase (EC1.1.3.40), alditol oxidase (EC1.1.3.41), 5-hydroxymethylfurfural oxidase (EC1.1.3.47), 3-deoxy-α-D-manno-octulose acid 8-oxidase (EC1.1.3.48); the hydrogen peroxide generated in step (i) is decomposed by catalase (EC 1.11.1.6).

3. The method of claim 1, wherein the enzymes used in step (ii) include alcohol dehydrogenase (EC 1.1.1.1, EC 1.1.1.2), ketone reductase, and aldehyde dehydrogenase (EC 1.1.1.203); this enzymatic reaction is simultaneously accompanied by the addition of coenzyme NAD+ or NADP+, and NAD(P)H oxidase (NOx, EC 1.6.3.1, EC 1.6.3.4) is used to achieve the cyclic regeneration of the coenzyme; the hexanoic acid obtained in step (ii) includes D / L-gluconic acid, D / L-galactonic acid, D / L-mannonic acid, D / L-iduronic acid, D / L-aloonic acid, D / L-altraric acid, and (2S,3R,4R,5S)-2,3,4,5-tetrahydroxyhexanoic acid.

4. The method of claim 1, wherein the enzymes used in step (iii) include gluconate dehydratase (EC 4.2.1.40), citrate dehydratase (EC 4.2.1.4), arabinose dehydratase (EC 4.2.1.5), galactobionic acid dehydratase (EC 4.2.1.6), alginate dehydratase (EC 4.2.1.7), mannolate dehydratase (EC 4.2.1.8), dihydroxy acid dehydratase (EC 4.2.1.9), 3-dehydroquinoline dehydratase (EC 4.2.1.10), phosphoglucose dehydratase (EC 4.2.1.12), L-arabinose dehydratase (EC 4.2.1.25), propylene glycol dehydratase, (EC 4.2.1.28) ), glycerol dehydratase (EC4.2.1.30), L-tartrate dehydratase (EC4.2.1.32), isopropyl malonate dehydratase (EC4.2.1.33.3), (S)-2-methylmalonic acid dehydratase (EC4.2.1.34), (R)-2-methylmalonic acid dehydratase (EC4.2.1.35), galactogluconate dehydratase (EC4.2.1.39), gluconate dehydratase (EC4.2.1.39), glycoside dehydratase (EC4.2.1.30), glycerol dehydratase (EC4.2.1.30), L-tartrate dehydratase (EC4.2.1.32), isopropyl malonate dehydratase (EC4.2.1.33.3), glycoside dehydratase (EC4.2.1.34), glycoside dehydratase (EC4.2.1.35), galactogluconate dehydratase (EC4.2.1.39), glycoside dehydratase (EC4.2.1.30), glycoside dehydratase (EC4.2.1.32), glycoside dehydratase (EC4.2.1.33.3), glycoside dehydratase (EC4.2.1.34), glycoside dehydratase (EC4.2.1.35), glycoside dehydratase (EC4.2.1.35), glycoside dehydratase (EC4.2.1.39), glycoside dehydratase (EC4.2.1.30), glycoside dehydratase (EC4.2.1.32), glycoside dehydratase (EC4.2.1.33.3), glycoside dehydratase (EC4.2.1.34), glycoside dehydratase (EC4.2.1.35), 4.2.1.42), 2-dehydro-3-deoxy-L-arabinose dehydratase (EC 4.2.1.43), inosine-2 dehydratase (EC 4.2.1.44), CDP-glucose 4,6-dehydratase (4.2.1.45), TDP-glucose 4,6-dehydratase (EC 4.2.1.46), GDP-4,6-mannose dehydratase (EC 4.2.1.47), D-succinate... Succinate dehydratase (EC 4.2.1.67), L-succinate dehydratase (EC 4.2.1.68), UDP-glucose 4,6-dehydratase (EC 4.2.1.76), 2-methylcitrate dehydratase (EC 4.2.1.79), D-tartrate dehydratase (EC 4.2.1.81), xylorate dehydratase (EC 4.2.1.82), L-rhamnolate dehydratase (EC 4.2.1.82). 1.90), 2-methyl isocitrate dehydratase (EC 4.2.1.99), 1,5-dehydro-D-fructose dehydratase (EC 4.2.1.111), 2-methylcitrate dehydratase (EC 4.2.1.117), linalool dehydratase (EC 4.2.1.127), D-lactic acid dehydratase (EC 4.2.1.130), gluconic acid / galactonate dehydratase (EC 4.2.1.1...). 40) 2-Dehydro-3-deoxy-D-arabinose dehydratase (EC4.2.1.141), L-galactose dehydratase (EC4.2.1.146), L-talarate dehydratase, EC4.2.1.156, D-galactose dehydratase (EC4.2.1.158), 6-deoxy-6-sulfonic acid-D-glucose dehydratase (EC4.2.1.162).

5. The method of claim 1, wherein in step (iv), 4-deoxy-5-dehydro-hexanoic acid undergoes cyclization and dehydration under heating and in the presence of an inorganic acid to generate 2,5-furandicarboxylic acid, wherein the inorganic acid includes one or a combination of several selected from sulfuric acid and hydrohalic acids (such as hydrofluoric acid, hydrochloric acid, hydrobromic acid, hydroiodic acid, etc.).

6. A chemical enzymatic route for synthesizing 2,5-furandicarboxylic acid, comprising three or four enzymatic and chemical reactions: (a) starting from gluconic acid and catalytically oxidizing it to L-guluronic acid; (b) oxidizing L-guluronic acid by enzymatic or metal catalytic oxidation to D-gluconic acid; (c) dehydrating D-gluconic acid under heating and in the presence of an inorganic acid to generate 2,5-furandicarboxylic acid; or, (a) starting from gluconic acid and catalytically oxidizing it to L-guluronic acid; (b) oxidizing L-guluronic acid by enzymatic or metal catalytic oxidation to D-gluconic acid; (d) catalyzing D-gluconic acid with a dehydrating enzyme to generate 4-deoxy-5-dehydro-gluconic acid; (e) dehydrating 4-deoxy-5-dehydro-gluconic acid under acidic conditions to generate 2,5-furandicarboxylic acid.

7. The method of claim 6, wherein reaction (a) uses an oxidase and horseradish peroxidase is used as an activator of the oxidase, and the hydrogen peroxide produced in the reaction is decomposed by catalase.

8. The method of claim 7, wherein the oxidase used in reaction (a) comprises the sequence shown in SEQ ID No:

1.

9. The method of claim 6, wherein the enzyme that can be used in reaction (b) includes aldehyde dehydrogenase, and coenzyme NAD+ or NADP+ is added, and NAD(P)H oxidase is used to achieve the cyclic regeneration of the coenzyme.

10. The method of claim 9, wherein the aldehyde dehydrogenase used in reaction (b) comprises the sequence shown in SEQ ID No: 2, and the NAD(P)H oxidase used in reaction (b) comprises the sequence shown in SEQ ID No:

3.

11. The method of claim 6, wherein the dehydrating enzyme used in reaction (d) comprises the sequence shown in SEQ ID No:

4.

12. The method of claim 6, wherein in reaction (e), 4-deoxy-5-dehydro-gluconic acid is dehydrated under heating and in the presence of an inorganic acid to generate 2,5-furandicarboxylic acid, wherein the inorganic acid includes one or a combination of several selected from sulfuric acid and hydrohalic acids (such as hydrofluoric acid, hydrochloric acid, hydrobromic acid, hydroiodic acid, etc.).

13. An engineered oxidase polypeptide, the amino acid sequence of which is shown in SEQ ID No: 1.

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