Method for synthesizing furanic acid compound through chemoenzyme cascade catalysis of 5-hydroxymethylfurfural

Abstract

The invention discloses a method for synthesizing a furan acid compound through chemoenzyme cascade catalysis of 5-hydroxymethylfurfural, and the furan acid compound is any one of 5-hydroxymethyl-2-furancarboxylic acid, 5-formyl furan-2-carboxylic acid and 2, 5-furandicarboxylic acid; 5-hydroxymethylfurfural is used as a substrate, free or immobilized alcohol dehydrogenase, peroxygenase and galactose oxidase are used as catalysts, and the furanic acid compound is selectively generated under mild conditions. According to the synthesis method disclosed by the invention, in-situ regeneration and utilization of the cofactor and H2O2 can be realized at the same time, and the usage amount of the cofactor and H2O2 is reduced; the product synthesized by the synthesis method is good in selectivity and relatively high in yield, and has a relatively good industrial application prospect in preparation of furanic acid compounds through biological catalysis.

Description

Technical Field

[0001] The present invention relates to the technical field of biosynthesis, and particularly relates to a method for chemically enzymatic cascade catalysis of 5-hydroxymethylfurfural to synthesize furanoic acid compounds. Background Art

[0002] 5-Hydroxymethylfurfural (HMF) is an important bio-based chemical, which is mainly formed by dehydration of hexose sugars produced by degradation of renewable resource lignocellulose. It has broad application prospects and is listed as one of the "top 10 + 4" bio-based chemicals by the US Department of Energy, with a large market scale globally. Due to the presence of multiple functional groups (furan ring, alcohol hydroxyl group, and aldehyde group) in the structure of HMF, it has high reactivity and can be catalytically converted into various derivatives with higher added value, such as FDCA (2,5-furandicarboxylic acid), HMFCA (5-hydroxymethyl-2-furoic acid), DFF (2,5-furandialdehyde), etc. Among them, FDCA is considered to be the most valuable chemical obtained from the current biomass platform and has wide applications in multiple fields such as plastics, pharmaceuticals, spices, and textile industries. HMFCA is also a chemical with broad application prospects. It can not only be directly used to manufacture polyesters, but is also a key starting material for synthesizing renewable terephthalic acid (TPA), and also has important applications in the pharmaceutical industry. However, the synthetic route for it has rarely been reported.

[0003] Currently, the methods for catalytically converting HMF into high-value derivatives in industry still rely on traditional chemical catalysis, but it has disadvantages such as harsh reaction conditions (high temperature, strong acid, strong base, etc.) and dependence on noble metal catalysts. Especially, high temperature conditions will accelerate the degradation of HMF and generate unnecessary by-products. Biocatalysis usually occurs under mild reaction conditions, and biocatalysts have high selectivity, biodegradability, and environmental friendliness. Therefore, for the unstable characteristics of HMF, biocatalytic conversion is a good alternative. However, due to the poor selectivity of enzyme-catalyzed HMF, by-products are often generated while producing the target product. Therefore, there has been no reported route for highly selectively synthesizing HMFCA, FFCA (5-formylfuran-2-carboxylic acid), and FDCA in the same system. In addition, in the systems reported above, enzyme-catalyzed oxidation of HMF often requires the participation of cofactors and oxidant H 2 O 2 However, the low efficiency of the cofactor regeneration system and the inhibition problem of H 2 O 2 on enzymes reduce the overall catalytic efficiency of the reaction, and the TON (turnover number) values of enzymes are relatively low. Summary of the Invention

[0004] The object of the present invention is to provide a method for chemically enzymatic cascade catalysis of 5-hydroxymethylfurfural to synthesize furanoic acid compounds in view of the problem that the existing biocatalytic conversion technology cannot efficiently synthesize furanoic acid compounds such as HMFCA, FFCA, and FDCA. Based on the above background technology, the present invention constructs an F1-mediated multi-enzyme cascade catalytic system by using an artificial bridged flavin F1 with excellent catalytic oxygen ability, and efficiently and highly selectively oxidizes HMF to generate various high-value chemicals such as HMFCA, FFCA, and FDCA. Using HMF as a substrate and an artificial flavin as a catalyst to in-situ regenerate NAD + and H 2 O 2 , through the catalytic reactions of an organic small molecule catalyst - artificial flavin, free or immobilized alcohol dehydrogenase, free or immobilized galactose oxidase, and free or immobilized peroxygenase, furanoic acid compounds such as HMFCA, FFCA, and FDCA can be selectively synthesized.

[0005] In order to achieve the above object, the technical solution provided by the present invention is as follows:

[0006] A method for chemically enzymatic cascade catalysis of 5-hydroxymethylfurfural (HMF) to synthesize furanoic acid compounds, wherein the furanoic acid compounds are any one of 5-hydroxymethyl-2-furoic acid (HMFCA), 5-formylfuran-2-carboxylic acid (FFCA), and 2,5-furandicarboxylic acid (FDCA);

[0007] The first enzyme cascade system is subjected to a first catalytic reaction to generate 5-hydroxymethyl-2-furoic acid (HMFCA), and a reaction solution containing 5-hydroxymethyl-2-furoic acid is obtained; the first enzyme cascade system includes: 5-hydroxymethylfurfural, alcohol dehydrogenase, peroxygenase, cofactor, artificial flavin, and solvent;

[0008] Inactivate or remove the alcohol dehydrogenase and the peroxygenase in the reaction solution containing 5-hydroxymethyl-2-furoic acid, and add galactose oxidase thereto for a second catalytic reaction to generate 5-formylfuran-2-carboxylic acid (FFCA);

[0009] Add galactose oxidase to the reaction solution containing 5-hydroxymethyl-2-furoic acid for a third catalytic reaction to generate 2,5-furandicarboxylic acid (FDCA).

[0010] Wherein, the alcohol dehydrogenase is any one of the following a1 to a5:

[0011] a1, alcohol dehydrogenase HLADH derived from Horse liver, and its UniProtKB of the amino acid sequence is P00327;

[0012] a2, alcohol dehydrogenase ADH-A derived from Rhodococcus ruber DSM 44541, with its UniProtKB of the amino acid sequence being Q8KLT9;

[0013] a3, alcohol dehydrogenase AaADH derived from Aromatoleum aromaticum bacterium strain EbN1

[0014] with its UniProtKB of the amino acid sequence being Q5P1J5;

[0015] a4, alcohol dehydrogenase PpADH derived from Paracoccus pantotrophus, with its UniProtKB of the amino acid sequence being A0A1I5GPJ0;

[0016] a5, alcohol dehydrogenase APDH derived from RMM Microcompartment of Mycobacterium smegmatis, with its UniProtKB of the amino acid sequence being A0QP46.

[0017] Among them, the peroxygenase is a peroxygenase variant AaeUPO derived from Agrocybe aegerita, with its UniProtKB of the amino acid sequence being B9W4V6.

[0018] Among them, the galactose oxidase is galactose oxidase Goase derived from Dactylium dendroides, with its UniProtKB of the amino acid sequence being P0CS93.

[0019] Among them, the alcohol dehydrogenase, peroxygenase and galactose oxidase are immobilized enzymes or free enzymes.

[0020] Free alcohol dehydrogenase, free peroxygenase and free galactose oxidase can exist in the form of recombinant cells, crude enzyme solution, crude enzyme powder or pure enzyme for catalysis.

[0021] Free alcohol dehydrogenase and free galactose oxidase can be obtained by purchasing from the market, and free peroxygenase can be prepared by the method in the literature (Molina-Espeja P, Garcia-Ruiz E, Gonzalez-Perez D, et al. Directed evolution of unspecific peroxygenase from Agrocybe aegerita. [J]. Applied & Environmental Microbiology, 2014, 80(11): 3496 - 3507).

[0022] The preparation methods of immobilized alcohol dehydrogenase, peroxidase and galactose oxidase are as follows:

[0023] Add methacrylic anhydride to an aqueous sodium alginate solution with a concentration of 20 - 40 g / L (the mass - volume ratio of sodium alginate to methacrylic anhydride is 1 g:5 - 10 mL), mix evenly to obtain a mixed solution. Adjust the pH of the mixed solution to 8 - 9 with an alkali solution, then stir - react at 0 - 5 °C for 24 - 36 h. After the reaction, wash the generated solid polymer product with absolute ethanol 3 - 4 times, and then place it in a vacuum drying oven at 35 - 45 °C for 6 - 8 h to finally obtain solid powder of sodium alginate with double bonds (SA - MA). Prepare an aqueous sodium alginate solution with double bonds with a concentration of 50 - 100 g / L, add a photo - initiator lithium phenyl phosphate (the mass ratio of sodium alginate with double bonds to lithium phenyl phosphate is 1:0.1 - 2), mix evenly, and irradiate with 400 - 420 nm blue light for 3 - 5 min to obtain a porous sodium alginate hydrogel immobilization material. Immerse the porous sodium alginate hydrogel immobilization material in a pure enzyme solution with a concentration of 2 - 5 g / L, the mass ratio of the immobilization material to the pure enzyme is 1:20 - 50. After standing at room temperature for 2 - 4 h, wash the immobilization material with pure water to obtain immobilized enzyme.

[0024] Preferably, the preparation method of the porous sodium alginate hydrogel immobilization material is as follows: Add methacrylic anhydride to an aqueous sodium alginate solution with a concentration of 20 g / L (the mass - volume ratio of sodium alginate to methacrylic anhydride is 1 g:5 - 10 mL), mix evenly to obtain a mixed solution. Adjust the pH of the mixed solution to 8 with 5 mol / L NaOH aqueous solution, then stir - react at 0 °C at 600 rpm for 24 h. After the reaction, wash the generated solid polymer product with absolute ethanol 3 - 4 times, and then place it in a vacuum drying oven at 40 °C for 6 - 8 h to finally obtain solid powder of sodium alginate with double bonds (SA - MA). Prepare an aqueous sodium alginate solution with double bonds with a concentration of 50 g / L, add a photo - initiator lithium phenyl phosphate (the mass ratio of sodium alginate with double bonds to lithium phenyl phosphate is 1:0.1 - 2), mix evenly, and irradiate with 405 nm blue light for 3 - 5 min to obtain a porous sodium alginate hydrogel immobilization material.

[0025] Among them, the cofactor is NAD + (nicotinamide adenine dinucleotide) and / or NADP + (nicotinamide adenine dinucleotide phosphate), preferably NAD + .

[0026] Among them, the artificial flavin is 7 - trifluoromethyl - 1,10 - ethylene - isoalloxazine chloride (F1), and its chemical structural formula is shown in Formula 1:

[0027]

[0028] Among them, the solvent is a phosphate buffer solution with a concentration of 50 - 100 mM and a pH value of 6.0 - 8.0, preferably a phosphate buffer solution with a concentration of 50 mM and a pH value of 7.0.

[0029] Among them, in the first enzyme cascade system, the initial concentration of the 5 - hydroxymethylfurfural is 10 - 50 mM; the initial concentration of the alcohol dehydrogenase dependent on NAD + is 0.5 - 10 U / mL; the initial concentration of the peroxygenase dependent on H 2 O 2 is 0.1 - 10 U / mL; the initial concentration of the cofactor is 0.1 - 2.0 mM, preferably 0.1 mM; the initial concentration of the artificial flavin is 0.1 - 1 mM, preferably 0.1 mM; the initial concentration of the galactose oxidase in the reaction solution containing 5 - hydroxymethyl - 2 - furancarboxylic acid is 3 - 20 U / mL.

[0030] Among them, the reaction conditions for the first catalytic reaction, the second catalytic reaction, and the third catalytic reaction are: the rotation speed is 100 - 500 rpm, the reaction temperature is 25 - 35 °C, the reaction time is 2 - 144 h, and the reaction is carried out in an air atmosphere or an oxygen atmosphere. Preferably, the reaction temperature for the first catalytic reaction, the second catalytic reaction, and the third catalytic reaction is 30 °C, and the reaction time is 12 h.

[0031] Preferably, the inactivation is to inactivate the reaction solution containing 5 - hydroxymethyl - 2 - furancarboxylic acid at 80 - 95 °C for 5 - 10 min.

[0032] Figure 1 This is the total reaction flow chart for the preparation of three kinds of furanoic acids by the catalytic reaction of alcohol dehydrogenase coupled with peroxygenase and galactose oxidase in the present invention.

[0033] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0034] The present invention provides a brand - new green biosynthesis route. Using HMF as a raw material, it can selectively synthesize a variety of furanoic acid compounds in the same system, and both the yield and selectivity reach more than 99%; the use of artificial flavin can simultaneously regenerate NAD + and H 2 O 2 , realizing the internal circulation of coenzymes, reducing the usage amounts of cofactors and H 2 O 2 ; the synthesis method of the present invention is easy to operate, and the product yield is relatively high, having good industrial application prospects in the selective biocatalytic preparation of different furanoic acids. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The following further specifically describes the present invention in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become clearer.

[0036] Figure 1 This is the overall reaction flow chart for the preparation of three furanoic acids by the coupling of alcohol dehydrogenase, peroxygenase, and galactose oxidase for the present invention.

[0037] Figure 2 This is the reaction flow chart for the preparation of HMFCA by the coupling of alcohol dehydrogenase and peroxygenase for Example 1.

[0038] Figure 3 This is the reaction flow chart for the preparation of FFCA by the coupling of alcohol dehydrogenase, peroxygenase, and galactose oxidase for Example 2.

[0039] Figure 4 This is the reaction flow chart for the preparation of FDCA by the coupling of alcohol dehydrogenase, peroxygenase, and galactose oxidase for Example 3. Specific Embodiments

[0040] The following further illustrates the present invention according to the following embodiments. It should be understood that the following embodiments are only used to illustrate the present invention and not to limit the present invention.

[0041] For the specific technologies or conditions not specified in the embodiments, they shall be in accordance with the technologies or conditions described in the literature in this field or in accordance with the product specifications. For the reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through regular channels.

[0042] For the quantitative tests in the following embodiments, three repeated tests are set, and the results are averaged.

[0043] The specific enzyme activities of the HLADH enzyme, PpADH enzyme, ADH-A enzyme, AaADH enzyme, APADH enzyme, GOase enzyme, and AaeUPO enzyme described in the following embodiments are 40 U / mg, 32 mU / mg, 28 mU / mg, 15 mU / mg, 20 U / mg, 40 U / mg, and 23 U / mg respectively.

[0044] One unit of activity (U) of an alcohol dehydrogenase (HLADH enzyme, PpADH enzyme, ADH-A enzyme, AaADH enzyme, and APADH enzyme) is defined as the amount of enzyme required to catalyze the formation of 1 μmol of HMFCA from 1 μmol of HMF per minute.

[0045] One unit of activity (U) of a GOase enzyme is defined as the amount of enzyme required to catalyze the formation of 1 μmol of FFCA from 1 μmol of HMFCA per minute.

[0046] One unit (U) of AaeUPO enzyme activity is defined as the amount of enzyme required to catalyze the formation of 1 μmol of HMFCA from 1 μmol of HMF per minute.

[0047] In the following examples, the substrate conversion rate is calculated as follows: Substrate conversion rate = (Initial substrate concentration - Substrate concentration in the reaction solution) / Initial substrate concentration × 100%; The product yield is calculated as follows: Product yield = (Actual product concentration / Theoretical product concentration) × 100%.

[0048] Example 1: Catalysis of HMF to HMFCA by free alcohol dehydrogenase and free peroxygenase

[0049] 1. To a PBS buffer solution with a concentration of 50 mM and a pH value of 7.0, add 10 mM of HMF, 0.1 mM of NAD + , 0.1 mM of F1, 3 U / mL of free AaeUPO pure enzyme, and 1.5 U / mL of free alcohol dehydrogenase (HLADH, PpADH, ADH - A, AaADH, or APADH) pure enzyme in sequence to form an enzyme cascade reaction system. After reacting the reaction system at 30 °C for 24 h, a reaction solution is obtained.

[0050] 2. Take 200 μL of the reaction solution, dilute it to 1 mL with the above PBS buffer solution, shake it well, inactivate it at 95 °C for 5 minutes, and filter it with a filter membrane to obtain a detection sample. Perform liquid chromatography (HPLC) detection on the sample. The HMF conversion rate and HMFCA yield are shown in Table 1. Figure 2 This is the reaction flow chart for the preparation of HMFCA by coupling alcohol dehydrogenase with peroxygenase to catalyze HMF in this example.

[0051] Table 1 HMF conversion rate and HMFCA yield

[0052]

[0053] Example 2: Catalysis of HMF to FFCA by free alcohol dehydrogenase, free galactose oxidase, and free peroxygenase

[0054] 1. To a PBS buffer solution with a concentration of 50 mM and a pH value of 7.0, add 10 mM of HMF, 0.1 mM of NAD +, 0.1 mM of F1, 3 U / mL of free AaeUPO pure enzyme, and 1.5 U / mL of free alcohol dehydrogenase (HLADH, PpADH, ADH-A, AaADH, or APADH) pure enzyme were added to form an enzyme cascade reaction system. After reacting the reaction system at 30 °C for 12 h, the reaction solution was inactivated at 95 °C for 5 min, and then free GOase pure enzyme with an initial concentration of 10 U / mL was added to the reaction solution. After continuing the reaction for 6 h, the reaction solution was obtained.

[0055] 2. Take 200 μL of the reaction solution, dilute it to 1 mL with buffer, shake well, inactivate it at 95 °C for 5 minutes, and filter it through a membrane to obtain a test sample. The sample was detected by liquid chromatography (HPLC). The HMF conversion rate and FFCA yield are shown in Table 2. Figure 3 This is the reaction flow chart for the preparation of FFCA by coupling alcohol dehydrogenase, peroxygenase, and galactose oxidase to catalyze HMF in this example.

[0056] Table 2 HMF conversion rate and FFCA yield

[0057]

[0058] Example 3: Catalysis of HMF to produce FDCA by free alcohol dehydrogenase, free galactose oxidase, and free peroxygenase

[0059] 1. To a PBS buffer with a concentration of 50 mM and a pH value of 7.0, add HMF with an initial concentration of 10 mM, 0.1 mM of NAD + , 0.1 mM of F1, 3 U / mL of free AaeUPO pure enzyme, and 1.5 U / mL of free alcohol dehydrogenase (HLADH, PpADH, ADH-A, AaADH, or APADH) pure enzyme to form an enzyme cascade reaction system. After reacting the reaction system at 30 °C for 12 h, 10 U / mL of free GOase pure enzyme was added to the reaction solution. After continuing the reaction for 6 h, the reaction solution was obtained.

[0060] 2. Take 200 μL of the reaction solution, dilute it to 1 mL with buffer, shake well, inactivate it at 95 °C for 5 minutes, and filter it through a membrane to obtain a test sample. The sample was detected by liquid chromatography (HPLC). The HMF conversion rate and FDCA yield are shown in Table 3. Figure 4 This is the reaction flow chart for the preparation of FDCA by coupling alcohol dehydrogenase, peroxygenase, and galactose oxidase to catalyze HMF in this example.

[0061] Table 3 HMF conversion rate and FDCA yield

[0062]

[0063] Example 4 Synthesis of FFCA from HMF Catalyzed by Immobilized Alcohol Dehydrogenase, Immobilized Galactose Oxidase and Immobilized Peroxygenase

[0064] 1. To a PBS buffer solution with a concentration of 50 mM and a pH value of 7.0, add 10 mM of HMF, 0.1 mM of NAD + , 0.1 mM of F1, 3 U / mL of immobilized AaeUPO pure enzyme and 1.5 U / mL of immobilized alcohol dehydrogenase (HLADH, PpADH, ADH-A, AaADH or APADH) in sequence to form an enzyme cascade reaction system. After reacting the reaction system at 30 °C for 12 h, take out the immobilized alcohol dehydrogenase and AaeUPO, then add 10 U / mL of immobilized GOase to the reaction solution, and continue to react for 6 h to obtain a reaction solution. Take out the immobilized alcohol dehydrogenase, GOase and AaeUPO enzymes for the next batch of the reaction of synthesizing FFCA from HMF until the enzyme no longer catalyzes, and combine all the reaction solutions (the reaction solutions of the first 6 batches of catalytic reactions).

[0065] 2. Take 200 μL of the combined reaction solution, dilute it to 1 mL with buffer solution, shake it well and inactivate it at 95 °C for 5 minutes, and filter it with a filter membrane to obtain a detection sample. Detect the sample by liquid chromatography (HPLC). The HMF conversion rate and FFCA yield are shown in Table 4.

[0066] Table 4 HMF Conversion Rate and FFCA Yield of Multi-Batch Catalysis

[0067]

[0068] Example 5 Synthesis of FDCA from HMF Catalyzed by Immobilized Alcohol Dehydrogenase, Immobilized Galactose Oxidase and Immobilized Peroxygenase

[0069] 1. To a PBS buffer solution with a concentration of 50 mM and a pH value of 7.0, add 10 mM of HMF, 0.1 mM of NAD + , 0.1 mM of F1, 3 U / mL of immobilized AaeUPO pure enzyme and 1.5 U / mL of immobilized alcohol dehydrogenase (HLADH, PpADH, ADH-A, AaADH or APADH) in sequence to form an enzyme cascade reaction system. After reacting the reaction system at 30 °C for 12 h, add 10 U / mL of immobilized GOase to the reaction solution, and continue to react for 6 h to obtain a reaction solution. Take out the immobilized alcohol dehydrogenase, GOase and AaeUPO enzymes for the next batch of the reaction of synthesizing FDCA from HMF until the enzyme no longer catalyzes, and combine all the reaction solutions (the reaction solutions of the first 6 batches of catalytic reactions).

[0070] 2. Take 200 μL of the combined reaction solution, dilute it to 1 mL with buffer, shake well, inactivate at 95 °C for 5 minutes, and filter with a filter membrane to obtain the test sample. The sample was detected by high performance liquid chromatography (HPLC). The HMF conversion rate and FDCA yield are shown in Table 5.

[0071] Table 5 HMF conversion rate and FDCA yield of multi-batch catalysis

[0072]

[0073] The present invention provides an idea and method for the chemoenzymatic cascade catalysis of 5-hydroxymethylfurfural to synthesize furanoic acid compounds. There are many methods and ways to specifically implement this technical solution. The above is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention. Each component not clearly defined in this embodiment can be realized by the prior art.

Claims

1. A method for synthesizing furanic acid compounds by 5-hydroxymethylfurfural catalyzed by chemical enzyme cascade, characterized in that: The furanic acid compound is any one of 5-hydroxymethyl-2-furancarboxylic acid, 5-formaldehyde furan-2-carboxylic acid and 2,5-furandicarboxylic acid; The first enzyme cascade system generates 5-hydroxymethyl-2-furancarboxylic acid through a first catalytic reaction to obtain a reaction solution containing 5-hydroxymethyl-2-furancarboxylic acid; the first enzyme cascade system comprises: 5-hydroxymethylfurfural, alcohol dehydrogenase, peroxygenase, cofactor, artificial flavin and solvent; The alcohol dehydrogenase and the peroxygenase in the reaction solution containing 5-hydroxymethyl-2-furancarboxylic acid are inactivated or removed, and galactose oxidase is added thereto to carry out a second catalytic reaction to generate 5-formaldehyde furan-2-carboxylic acid; The galactose oxidase is added to the reaction solution containing 5-hydroxymethyl-2-furancarboxylic acid to carry out a third catalytic reaction to generate 2,5-furandicarboxylic acid.

2. The method according to claim 1, characterized in that The alcohol dehydrogenase is any one of the following a1 to a5: a1, alcohol dehydrogenase HLADH from Horse liver, the amino acid sequence of which is P00327 in UniProtKB; a2, alcohol dehydrogenase ADH-A from Rhodococcus ruber DSM 44541, whose amino acid sequence UniProtKB is Q8KLT9; a3, alcohol dehydrogenase AaADH from Aromatoleum aromaticum bacterium strain EbN1, whose amino acid sequence UniProtKB is Q5P1J5; a4, alcohol dehydrogenase PpADH from Paracoccus pantotrophus, with the amino acid sequence of UniProtKB A0A1I5GPJ0; a5, alcohol dehydrogenase APADH from RMM Microcompartment of Mycobacterium smegmatis, whose amino acid sequence UniProtKB is A0QP46.

3. The method according to claim 1, characterized in that The peroxygenase is a peroxygenase variant AaeUPO derived from Agrocybeaegerita, and the UniProtKB of the amino acid sequence thereof is B9W4V6.

4. The method according to claim 1, characterized in that: The galactose oxidase is a galactose oxidase Goase derived from Dactylium dendroides, and the UniProtKB of the amino acid sequence thereof is P0CS93.

5. The method according to claim 1, characterized in that The alcohol dehydrogenase, peroxygenase and galactose oxidase are immobilized enzymes or free enzymes.

6. The method according to claim 1, characterized in that The cofactor is NAD + and / or NADP + .

7. The method according to claim 1, characterized in that The artificial flavin is 7-trifluoromethyl-1,10-ethylene isoalloxazine chloride.

8. The method according to claim 1, characterized in that The solvent is a phosphate buffer with a concentration of 50-100 mM and a pH value of 6.0-8.

0.

9. The method according to claim 1, characterized in that: In the first enzyme cascade system, the initial concentration of the 5-hydroxymethylfurfural is 10-50 mM, the initial concentration of the alcohol dehydrogenase is 0.5-10 U / mL, the initial concentration of the peroxygenase is 0.1-10 U / mL, the initial concentration of the cofactor is 0.1-2.0 mM, and the initial concentration of the artificial flavin is 0.1-1 mM; the initial concentration of the galactose oxidase in the reaction solution containing 5-hydroxymethyl-2-furancarboxylic acid is 3-20 U / mL.

10. The method according to claim 1, characterized in that The reaction conditions of the first catalytic reaction, the second catalytic reaction and the third catalytic reaction are: a rotation speed of 100 to 500 rpm, a reaction temperature of 25 to 35° C., a reaction time of 2 to 144 h, and the reaction is carried out in an air atmosphere or an oxygen atmosphere.

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