Oxidase mutant and application thereof in green synthesis of aryl carboxylic acid

Abstract

The invention discloses an oxidase mutant, a coding gene thereof, a recombinant vector constructed by the coding gene, a recombinant genetically engineered bacterium obtained by transforming the recombinant vector, and application of the oxidase mutant in green synthesis of aryl carboxylic acid. Wild type oxidase AAO is modified through directed evolution, the broad-spectrum oxidase mutant AAO-T170A / F432Y / F79A / S385A / Y123L is obtained, and the broad-spectrum oxidase mutant AAO-T170A / F432Y / F79A / S385A / Y123L can efficiently catalyze one-step oxidation of various aryl alcohols or aldehydes into corresponding aryl carboxylic acid, is applied to green synthesis of aryl carboxylic acid, and has the advantages of high efficiency, wide application prospect and the like. The catalytic product yield, the substrate compatibility and the stability are remarkably improved compared with those of wild type oxidase OAA, and the method is suitable for the fields of bio-based materials, medicines and fine chemical engineering.

Description

Technical Field

[0001] The present invention relates to the technical field of biocatalysis, and in particular to an oxidase mutant and application thereof in the green synthesis of aromatic carboxylic acids. Background Art

[0002] Aromatic carboxylic acids (such as 2,5-furandicarboxylic acid (FDCA) and benzoic acid) are core raw materials for the synthesis of polyesters, pharmaceuticals (such as aspirin), and fragrances. Traditional chemical methods for preparing aromatic carboxylic acids rely on the catalytic oxidation of aromatic aldehydes using precious metals (Au / Pt), which suffer from the following drawbacks:

[0003] 1. Substrate limitations: only applicable to specific structures (e.g. 5-hydroxymethylfurfural HMF must contain hydroxyl groups);

[0004] 2. Harsh conditions: high temperature (>120℃), high pressure oxygen and strong acidic medium;

[0005] 3. Environmental burden: Precious metals are difficult to recycle and produce toxic by-products.

[0006] Although the electrochemical method for preparing aromatic carboxylic acids has mild conditions, the electrodes are easily deactivated, the cost is high, there are additional requirements for the conductive properties of the reaction solvent, and it is difficult to scale up.

[0007] Existing biological methods (such as multi-enzyme cascade systems) for preparing aromatic carboxylic acids, although mild, require coordinating the suitable reaction conditions of multiple enzymes, and will stay in intermediate products (such as 2,5-furandicarboxaldehyde DFF, 5-formylfuran-2-carboxylic acid FFCA) for a long time, causing intermediate accumulation, thereby affecting the enzyme conversion efficiency. Summary of the Invention

[0008] The purpose of the present invention is to provide an oxidase mutant and its application in the green synthesis of aromatic carboxylic acids, so as to solve the deficiencies of the prior art.

[0009] The present invention adopts the following technical solutions:

[0010] In a first aspect, the present invention provides an oxidase mutant, the amino acid sequence of which is shown in SEQ ID NO: 2.

[0011] In a second aspect, the present invention provides a gene encoding the above-mentioned oxidase mutant, the nucleotide sequence of which is shown in SEQ ID NO:4.

[0012] The third aspect of the present invention provides a recombinant vector constructed with the gene encoding the oxidase mutant.

[0013] The fourth aspect of the present invention provides a recombinant genetically engineered bacterium obtained by transformation of the above-mentioned recombinant vector.

[0014] The fifth aspect of the present invention provides the use of the above-mentioned oxidase mutant in the green synthesis of aromatic carboxylic acids.

[0015] Furthermore, when used, the catalyst can catalyze the synthesis of aromatic carboxylic acids from substrates, wherein the substrates include carbocyclic aldehydes, carbocyclic alcohols, heterocyclic aldehydes, and heterocyclic alcohols;

[0016] The carbocyclic aldehydes include aromatic carbocyclic aldehydes, and the aromatic carbocyclic aldehydes include benzene ring aldehydes;

[0017] The carbocyclic alcohols include aromatic carbocyclic alcohols, and the aromatic carbocyclic alcohols include benzene alcohols;

[0018] The heterocyclic aldehydes include aromatic heterocyclic aldehydes, and the aromatic heterocyclic aldehydes include furan ring aldehydes;

[0019] The heterocyclic alcohols include aromatic heterocyclic alcohols, and the aromatic heterocyclic alcohols include furan ring alcohols.

[0020] Furthermore, the benzene ring aldehydes include benzaldehyde and 4-carboxybenzaldehyde, benzaldehyde is synthesized into benzoic acid, and 4-carboxybenzaldehyde is synthesized into terephthalic acid;

[0021] The benzene ring alcohols include benzyl alcohol and 4-hydroxymethylbenzoic acid. Benzyl alcohol is used to synthesize benzoic acid, and 4-hydroxymethylbenzoic acid is used to synthesize terephthalic acid.

[0022] The furan ring aldehydes include 5-hydroxymethylfurfural, 2-furaldehyde, and 5-formyl-2-furancarboxylic acid. 5-hydroxymethylfurfural is synthesized into 2,5-furandicarboxylic acid, 2-furaldehyde is synthesized into 2-furancarboxylic acid, and 5-formyl-2-furancarboxylic acid is synthesized into 2,5-furandicarboxylic acid.

[0023] The furan ring alcohols include 2-hydroxymethylfuran and 2,5-dihydroxymethylfuran. 2-hydroxymethylfuran is used to synthesize 2-furancarboxylic acid, and 2,5-dihydroxymethylfuran is used to synthesize 2,5-furandicarboxylic acid.

[0024] Furthermore, the addition amount of the oxidase mutant is 10-500 mg / L, and the substrate concentration is 9-100 mM.

[0025] Furthermore, the reaction conditions are: the solvent is PBS buffer with a pH of 7.5-8.5, the oxidant is oxygen or air, stirring, the reaction temperature is room temperature-40°C, the reaction pressure is normal pressure, and the reaction time is 8-24 hours.

[0026] Furthermore, after the reaction is completed, centrifugation or filtration is performed, and the supernatant or filtrate is purified to obtain the target carboxylic acid, wherein the purification includes acidification crystallization or membrane separation.

[0027] Beneficial effects of the present invention:

[0028] The present invention modified the wild-type oxidase AAO through directed evolution to obtain a broad-spectrum oxidase mutant AAO-T170A / F432Y / F79A / S385A / Y123L. This oxidase can efficiently catalyze the one-step oxidation of various aromatic alcohols or aldehydes to the corresponding aromatic carboxylic acids. It is applicable to the green synthesis of aromatic carboxylic acids. Compared with the wild-type oxidase OAA, it has significantly improved catalytic product yield, substrate compatibility, and stability. It is suitable for the fields of bio-based materials, pharmaceuticals, and fine chemicals. Its technical advantages include:

[0029] 1. Substrate universality: It can catalyze the oxidation of various aromatic alcohols or aldehydes containing carbon rings (such as benzene rings) and heterocyclic rings (such as furan rings) to carboxylic acids;

[0030] 2. One-step conversion: The oxidation reaction can be completed in one step with a single enzyme, avoiding the inhibition of intermediate products in the multi-enzyme cascade and shortening the reaction path;

[0031] 3. Industrial compatibility: High substrate tolerance, with a substrate concentration of up to 100 mM. Mild reaction conditions allow for a product yield of over 99% at room temperature -40°C, normal pressure, and near-neutral to weakly alkaline conditions. DETAILED DESCRIPTION

[0032] The present invention will be further explained below in conjunction with the examples. The following examples are only used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0033] Wild-type oxidase AAO is derived from Mycena metata, and its amino acid sequence is shown in SEQ ID NO:1, and its nucleotide sequence is shown in SEQ ID NO:3. Directed evolution of wild-type oxidase AAO yielded a broad-spectrum oxidase mutant, AAO-T170A / F432Y / F79A / S385A / Y123L, whose amino acid sequence is shown in SEQ ID NO:2, and its nucleotide sequence is shown in SEQ ID NO:4. The oxidase mutant AAO-T170A / F432Y / F79A / S385A / Y123L has the following mutations relative to wild-type oxidase AAO:

[0034] The threonine (T) at position 170 mutated to alanine (A), the phenylalanine (F) at position 432 mutated to tyrosine (Y), the threonine (F) at position 79 mutated to alanine (A), the serine (S) at position 385 mutated to alanine (A), and the tyrosine (Y) at position 123 mutated to leucine (L).

[0035] Example 1 Construction of recombinant Escherichia coli expressing wild-type oxidase AAO

[0036] 1. Obtaining the recombinant plasmid pET28a-AAO

[0037] The wild-type oxidase AAO sequence (amino acid sequence as shown in SEQ ID NO: 1, nucleotide sequence as shown in SEQ ID NO: 3) was sent to a gene synthesis company (Suzhou Jinweizhi Biotechnology Co., Ltd.) to prepare the wild-type oxidase plasmid pET28a-AAO based on the pET-28a plasmid, i.e., the recombinant plasmid pET28a-AAO.

[0038] 2. Transformation of recombinant plasmid pET28a-AAO into E. coli BL21 (DE3)

[0039] To each tube, add 10 μL of recombinant plasmid pET28a-AAO solution (approximately 8 ng / μL) to 100 μL of E. coli BL21(DE3) competent cell suspension (OD600 = 0.4-0.6). Gently mix and incubate on ice for 30 min. Transfer to a 42°C water bath and heat shock for 90 s. Quickly transfer to an ice bath and cool for 3 min. Add 700 μL of LB liquid medium (LB liquid medium formula: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, pH 7.0; same below) to each tube and incubate at 37°C, shaking at 200 rpm for 1 h. After incubation, the bacterial solution was centrifuged at 3000 rpm for 2 min, 600 μL of the supernatant was discarded, and the remaining bacterial solution was mixed and spread onto LB solid medium plates (LB solid medium formula is: LB liquid medium formula plus 1.5 m / v% agar powder; the same below) containing 50 μg / mL kanamycin sulfate, and incubated inverted at 37°C overnight.

[0040] Preserve the genetically engineered E. coli BL21(DE3) / pET28a-AAO: On the next day, pick a single colony and place it in 20 ml of LB liquid medium. Cultivate at 37°C, 200 rpm until OD600 = 0.8-1.0. Collect the bacterial suspension and store it in a glycerol tube at -80°C.

[0041] Example 2 Construction of recombinant Escherichia coli expressing the oxidase mutant AAO-T170A / F432Y / F79A / S385A / Y123L

[0042] 1. Obtaining the recombinant plasmid pET28a-AAO-T170A / F432Y / F79A / S385A / Y123L

[0043] The oxidase mutant AAO-T170A / F432Y / F79A / S385A / Y123L sequence (amino acid sequence as shown in SEQ ID NO: 2, nucleotide sequence as shown in SEQ ID NO: 4) was sent to a gene synthesis company (Suzhou Jinweizhi Biotechnology Co., Ltd.) to prepare the oxidase mutant plasmid pET28a-AAO-T170A / F432Y / F79A / S385A / Y123L based on the pET-28a plasmid, i.e., the recombinant plasmid pET28a-AAO-T170A / F432Y / F79A / S385A / Y123L.

[0044] 2. Transformation of recombinant plasmid pET28a-AAO-T170A / F432Y / F79A / S385A / Y123L into E. coli BL21 (DE3)

[0045] To each tube of 100 μL of E. coli BL21(DE3) competent cell suspension (OD600 = 0.4-0.6), add 10 μL of the recombinant plasmid pET28a-AAO-T170A / F432Y / F79A / S385A / Y123L solution (approximately 8 ng / μL). Gently mix and incubate on ice for 30 minutes. Transfer to a 42°C water bath and heat shock for 90 seconds. Quickly transfer to an ice bath and cool for 3 minutes. Add 700 μL of LB liquid medium to each tube and incubate at 37°C at 200 rpm for 1 hour. After incubation, centrifuge the culture at 3000 rpm for 2 minutes, discard 600 μL of the supernatant, mix the remaining culture, and plate onto a LB solid medium plate containing 50 μg / mL kanamycin sulfate. Incubate inverted at 37°C overnight.

[0046] Preservation of genetically engineered E. coli BL21(DE3) / pET28a-AAO-T170A / F432Y / F79A / S385A / Y123L:

[0047] On the next day, a single colony was picked and placed in 20 ml LB liquid medium, cultured at 37°C and 200 rpm until OD600 = 0.8-1.0, and the bacterial solution was collected and stored in a glycerol tube at -80°C.

[0048] Example 3 Induced expression culture of recombinant Escherichia coli

[0049] The obtained E. coli BL21(DE3) / pET28a-AAO, E. coli BL21(DE3) / pET28a-AAO-T170A / F432Y / F79A / S385A / Y123L, and E. coli BL21(DE3) (i.e., pET-28a empty vector transformants, serving as a negative control group) were inoculated into 20 mL of LB liquid medium containing 50 μg / mL kanamycin sulfate and cultured with shaking at 37°C, 200 rpm overnight. 10 mL of the culture was transferred to 1 L of LB liquid medium containing 50 μg / mL kanamycin sulfate and cultured with shaking at 37°C, 200 rpm until the OD600 reached approximately 0.6-0.8. The inducer, isopropyl-β-D-thiogalactopyranoside, was added to the culture at a final concentration of 0.5 mM and induced at 25°C, 200 rpm, for 12 h. After induction, centrifuge at 12,000 rpm for 10 minutes, discard the supernatant, and collect the bacterial pellet. Dissolve the collected bacterial pellet in PBS buffer (20 mM, pH 7.0) (wet bacterial cells: PBS volume ratio = approximately 1:10) and disrupt with ultrasound (power 150 W, frequency 20 kHz) for 30 minutes (working time 2 seconds, rest time 3 seconds). Centrifuge the disrupted cells at 12,000 rpm for 40 minutes, collect the supernatant and pellet, and analyze protein expression by SDS-PAGE. Clear bands at the corresponding molecular weight indicate correct protein expression.

[0050] Example 4 Purification of Recombinant Wild-Type Oxidase Protein (AAO) and Recombinant Oxidase Mutant Protein (AAO-T170A / F432Y / F79A / S385A / Y123L) (Expression in Recombinant E. coli BL21 (DE3) System)

[0051] Recombinant wild-type oxidase protein (AAO) and recombinant oxidase mutant protein (AAO-T170A / F432Y / F79A / S385A / Y123L) were purified using a His-Trap HP affinity column.

[0052] Pick a single colony of a positive clone (from Example 1 and Example 2, respectively) and inoculate it into 20 mL of LB liquid medium containing 50 μg / mL kanamycin sulfate, and culture it at 37°C and 200 rpm overnight. Take 10 mL of the culture solution and transfer it to 1 L of LB liquid medium containing 50 μg / mL kanamycin sulfate, and culture it at 37°C and 200 rpm until the OD600 is approximately 0.6–0.8. Add the inducer isopropyl-β-D-thiogalactoside at a final concentration of 0.5 mM to the culture, and culture it at 25°C and 200 rpm for 12 hours. After the induction culture is completed, centrifuge it at 12000 rpm for 10 minutes, discard the supernatant, and collect the bacterial precipitate. Wash the precipitate three times with pre-cooled (4°C) physiological saline (0.9wt% NaCl), centrifuge it at 12000 rpm for 5 minutes each time, and discard the supernatant. Finally, collect the bacterial precipitate (i.e., wet bacteria).

[0053] Weigh 2g of wet cells, add 20mL of a buffer solution containing 20mM Tris and 150mM NaCl (pH 8.0) to suspend the cells, and perform ultrasonication (power 150W, frequency 20KHz) in an ice bath for 30min (working time 2s, rest time 3s). The broken cells were centrifuged at 4°C and 12000rpm for 30min, and the supernatant was collected as a crude enzyme solution. The crude enzyme solution was purified using a His-Trap HP affinity column produced by GE Healthcare. After ultrafiltration and desalination, the pure enzyme solution can be directly used for the green synthesis of aromatic carboxylic acids or freeze-dried with a freeze dryer and stored for later use (the following examples use the freeze-dried product for the green synthesis of aromatic carboxylic acids).

[0054] Example 5: Synthesis of terephthalic acid from 4-carboxybenzaldehyde catalyzed by wild-type oxidase AAO

[0055] A 50 mL Erlenmeyer flask was charged with 10 mL of 50 mM PBS buffer (pH 8.0), followed by the addition of 13.6 mg of 4-carboxybenzaldehyde and 0.5 mg of wild-type AAO oxidase (prepared in Example 4). The pH of the reaction system was adjusted with 0.1 M sodium hydroxide solution to maintain a pH of 8.0 ± 0.1. The reaction was continued at 30°C and 180 rpm for 8 h, with the reaction system open to the atmosphere. After completion of the reaction, the system was heated to 100°C and boiled for 5 min to terminate the reaction. The product, terephthalic acid, was then analyzed by high-performance liquid chromatography (HPLC).

[0056] Example 6 HPLC detection of oxidation products

[0057] The samples were detected using an Agilent 1260 high performance liquid chromatograph with a UV detector.

[0058] Liquid phase conditions: Sepax HP-C18 column (4.6 x 250 mm, 3 μm), mobile phase: 0.2% (v / v) aqueous phosphoric acid:methanol (volume ratio = 80:20), flow rate: 0.8 ml / min, run time: 25 min, column temperature: 35°C, detection wavelength: 262 nm. The reaction solution from Example 5 was tested, and the conversion rate of terephthalic acid, the product of the synthesis of 4-carboxybenzaldehyde, was 1.9%.

[0059] Example 7 Wild-type oxidase AAO catalyzes the synthesis of 2,5-furandicarboxylic acid from 5-formyl-2-furancarboxylic acid

[0060] A 50 mL Erlenmeyer flask was charged with 10 mL of 50 mM PBS buffer (pH 8.0), followed by the addition of 14 mg of 5-formyl-2-furancarboxylic acid and 0.5 mg of wild-type AAO oxidase (prepared in Example 4). The pH of the reaction system was adjusted with 0.1 M sodium hydroxide solution to maintain a pH of 8.0 ± 0.1, and the reaction was continued at 30° C. and 180 rpm for 8 h, with the reaction system open to the atmosphere. After completion of the reaction, the system was heated to 100° C. and boiled for 5 min to terminate the reaction. The product was then analyzed by high-performance liquid chromatography (HPLC) (the same liquid chromatography conditions as in Example 6) to determine the conversion of 2,5-furandicarboxylic acid from 5-formyl-2-furancarboxylic acid, which was 0.4%.

[0061] Example 8: Synthesis of terephthalic acid from 4-carboxybenzaldehyde catalyzed by oxidase mutant AAO-T170A / F432Y / F79A / S385A / Y123L

[0062] A 50 mL conical flask was charged with 10 ml of 50 mM PBS buffer (pH 8.0), followed by the addition of 13.6 mg of 4-carboxybenzaldehyde and 0.1 mg of the oxidase mutant AAO-T170A / F432Y / F79A / S385A / Y123L (prepared in Example 4). The pH of the reaction system was adjusted to 8.0 ± 0.1 using 0.1 M sodium hydroxide solution. The reaction was continued at 30° C., 180 rpm, for 8 h, with the reaction system open to the atmosphere. After completion of the reaction, the system was heated to 100° C. and boiled for 5 min to terminate the reaction. The reaction was then subjected to HPLC analysis (the same liquid phase conditions as in Example 6) to determine the conversion of terephthalic acid, the product of 4-carboxybenzaldehyde synthesis, which was 99.2%.

[0063] Example 9 Oxidase mutant AAO-T170A / F432Y / F79A / S385A / Y123L catalyzes the synthesis of 2,5-furandicarboxylic acid from 5-formyl-2-furancarboxylic acid

[0064] A 50 mL Erlenmeyer flask was charged with 10 mL of 50 mM PBS buffer (pH 8.0), followed by the addition of 14 mg of 5-formyl-2-furancarboxylic acid and 0.2 mg of the oxidase mutant AAO-T170A / F432Y / F79A / S385A / Y123L (prepared in Example 4). The pH of the reaction system was adjusted with 0.1 M sodium hydroxide solution to maintain a pH of 8.0 ± 0.1. The reaction was stirred at 30° C. and 180 rpm for 8 h, with the reaction system open to the atmosphere. After completion of the reaction, the system was heated to 100° C. and boiled for 5 min to terminate the reaction. The product was then analyzed by HPLC (the liquid phase conditions were the same as in Example 6) to determine the conversion rate of 2,5-furandicarboxylic acid from 5-formyl-2-furancarboxylic acid, which was 99.0%.

[0065] Example 10: Synthesis of terephthalic acid from 4-hydroxymethylbenzoic acid catalyzed by oxidase mutant AAO-T170A / F432Y / F79A / S385A / Y123L

[0066] A 50 mL conical flask was charged with 10 mL of 50 mM PBS buffer (pH 8.0), followed by the addition of 15 mg of 4-hydroxymethylbenzoic acid and 0.2 mg of the oxidase mutant AAO-T170A / F432Y / F79A / S385A / Y123L (prepared in Example 4). The pH of the reaction system was adjusted with 0.1 M sodium hydroxide solution to maintain a pH of 8.0 ± 0.1. The reaction was stirred at 30° C. and 180 rpm for 8 h, with the reaction system open to the atmosphere. After completion of the reaction, the system was heated to 100° C. and boiled for 5 min to terminate the reaction. The product was then analyzed by high-performance liquid chromatography (HPLC) (liquid phase conditions were the same as in Example 6) to determine the conversion of terephthalic acid to 4-hydroxymethylbenzoic acid, which was 99.2%.

[0067] Example 11 Oxidase mutant AAO-T170A / F432Y / F79A / S385A / Y123L catalyzes the synthesis of 2,5-furandicarboxylic acid from 5-hydroxymethylfurfural

[0068] A 50 mL conical flask was charged with 10 mL of 50 mM PBS buffer (pH 8.0), followed by the addition of 12.6 mg of 5-hydroxymethylfurfural and 0.5 mg of the oxidase mutant AAO-T170A / F432Y / F79A / S385A / Y123L (prepared in Example 4). The pH of the reaction system was adjusted with 0.1 M sodium hydroxide solution to maintain a pH of 8.0 ± 0.1. The reaction was stirred at 30° C. and 180 rpm for 8 h, with the reaction system open to the atmosphere. After completion of the reaction, the system was heated to 100° C. and boiled for 5 min to terminate the reaction. The product was then analyzed by high-performance liquid chromatography (HPLC) (the same liquid phase conditions as in Example 6) to determine the conversion of 5-hydroxymethylfurfural to 2,5-furandicarboxylic acid, which was 32.2%.

[0069] Example 12 Oxidase mutant AAO-T170A / F432Y / F79A / S385A / Y123L catalyzes the synthesis of 2,5-furandicarboxylic acid from 5-hydroxymethylfurfural

[0070] A 50 mL conical flask was charged with 10 mL of 50 mM PBS buffer (pH 8.0), followed by the addition of 12.6 mg of 5-hydroxymethylfurfural and 0.5 mg of the oxidase mutant AAO-T170A / F432Y / F79A / S385A / Y123L (prepared in Example 4). The pH of the reaction system was adjusted with 0.1 M sodium hydroxide solution to maintain a pH of 8.0 ± 0.1. The reaction was continued at 30° C., 180 rpm, and the reaction system was open to the atmosphere for 16 h. After completion of the reaction, the system was heated to 100° C. and boiled for 5 min to terminate the reaction. The product was then analyzed by high-performance liquid chromatography (HPLC) (the same liquid phase conditions as in Example 6) to determine the conversion of 5-hydroxymethylfurfural to 2,5-furandicarboxylic acid, which was 97.1%.

[0071] Example 13 Oxidase mutant AAO-T170A / F432Y / F79A / S385A / Y123L catalyzes the synthesis of 2,5-furandicarboxylic acid from 5-hydroxymethylfurfural

[0072] A 50 mL conical flask was charged with 10 mL of 50 mM PBS buffer (pH 7.5), followed by the addition of 12.6 mg of 5-hydroxymethylfurfural and 0.5 mg of the oxidase mutant AAO-T170A / F432Y / F79A / S385A / Y123L (prepared in Example 4). The pH of the reaction system was adjusted with 0.1 M sodium hydroxide solution to maintain a pH of 7.5 ± 0.1. The reaction was continued at 30° C., 180 rpm, and the reaction system was open to the atmosphere for 18 h. After completion of the reaction, the system was heated to 100° C. and boiled for 5 min to terminate the reaction. The product was then analyzed by high-performance liquid chromatography (HPLC) (the same liquid phase conditions as in Example 6) to determine the conversion of 5-hydroxymethylfurfural to 2,5-furandicarboxylic acid, which was 95.3%.

[0073] Example 14 Oxidase mutant AAO-T170A / F432Y / F79A / S385A / Y123L catalyzes the synthesis of 2,5-furandicarboxylic acid from 5-hydroxymethylfurfural

[0074] A 50 mL conical flask was charged with 10 mL of 50 mM PBS buffer (pH 8.5), followed by the addition of 12.6 mg of 5-hydroxymethylfurfural and 0.5 mg of the oxidase mutant AAO-T170A / F432Y / F79A / S385A / Y123L (prepared in Example 4). The pH of the reaction system was adjusted with 0.1 M sodium hydroxide solution to maintain a pH of 8.5 ± 0.1. The reaction was continued at 30° C., 180 rpm, and the reaction system was open to the atmosphere for 16 h. After completion of the reaction, the system was heated to 100° C. and boiled for 5 min to terminate the reaction. The product was then analyzed by high-performance liquid chromatography (HPLC) (the same liquid phase conditions as in Example 6) to determine the conversion of 5-hydroxymethylfurfural to 2,5-furandicarboxylic acid, which was 97.4%.

[0075] Example 15 Oxidase mutant AAO-T170A / F432Y / F79A / S385A / Y123L catalyzes the synthesis of 2,5-furandicarboxylic acid from 5-hydroxymethylfurfural

[0076] A 50 mL conical flask was charged with 10 mL of 80 mM PBS buffer (pH 8.5), followed by the addition of 63 mg of 5-hydroxymethylfurfural and 2.5 mg of the oxidase mutant AAO-T170A / F432Y / F79A / S385A / Y123L (prepared in Example 4). The pH of the reaction system was adjusted with 0.1 M sodium hydroxide solution to maintain a pH of 8.5 ± 0.1. The reaction was continued at 30° C., 180 rpm, and the reaction system was open to the atmosphere for 16 h. After completion of the reaction, the system was heated to 100° C. and boiled for 5 min to terminate the reaction. The product was then analyzed by high-performance liquid chromatography (HPLC) (the same liquid phase conditions as in Example 6) to determine the conversion of 5-hydroxymethylfurfural to 2,5-furandicarboxylic acid, which was 96.7%.

[0077] Example 16 Oxidase mutant AAO-T170A / F432Y / F79A / S385A / Y123L catalyzes the synthesis of 2,5-furandicarboxylic acid from 5-hydroxymethylfurfural

[0078] A 50 mL conical flask was charged with 10 mL of PBS buffer (120 mM, pH 8.5), followed by the addition of 126 mg of 5-hydroxymethylfurfural and 5 mg of the oxidase mutant AAO-T170A / F432Y / F79A / S385A / Y123L (prepared in Example 4). The pH of the reaction system was adjusted with 0.1 M sodium hydroxide solution to maintain a pH of 8.5 ± 0.1. The reaction was continued at 30° C., 180 rpm, and the reaction system was open to the atmosphere for 24 h. After completion of the reaction, the system was heated to 100° C. and boiled for 5 min to terminate the reaction. The product was then analyzed by high-performance liquid chromatography (HPLC) (the same liquid phase conditions as in Example 6) to determine the conversion of 5-hydroxymethylfurfural to 2,5-furandicarboxylic acid, which was 95.0%.

Claims

1. An oxidase mutant, characterized in that Its amino acid sequence is shown in SEQ ID NO:

2.

2. The gene encoding the oxidase mutant according to claim 1, characterized in that Its nucleotide sequence is shown in SEQ ID NO:

4.

3. A recombinant vector constructed with the gene encoding the oxidase mutant according to claim 2.

4. The recombinant genetically engineered bacteria obtained by transformation with the recombinant vector according to claim 3.

5. Use of the oxidase mutant according to claim 1 in the green synthesis of aromatic carboxylic acids.

6. The use according to claim 5, characterized in that When used, it catalyzes the synthesis of aromatic carboxylic acids from substrates, wherein the substrates include carbocyclic aldehydes, carbocyclic alcohols, heterocyclic aldehydes, and heterocyclic alcohols; The carbocyclic aldehydes include aromatic carbocyclic aldehydes, and the aromatic carbocyclic aldehydes include benzene ring aldehydes; The carbocyclic alcohols include aromatic carbocyclic alcohols, and the aromatic carbocyclic alcohols include benzene alcohols; The heterocyclic aldehydes include aromatic heterocyclic aldehydes, and the aromatic heterocyclic aldehydes include furan ring aldehydes; The heterocyclic alcohols include aromatic heterocyclic alcohols, and the aromatic heterocyclic alcohols include furan ring alcohols.

7. The use according to claim 6, characterized in that The benzene ring aldehydes include benzaldehyde and 4-carboxybenzaldehyde. Benzaldehyde is synthesized into benzoic acid, and 4-carboxybenzaldehyde is synthesized into terephthalic acid. The benzene ring alcohols include benzyl alcohol and 4-hydroxymethylbenzoic acid. Benzyl alcohol is used to synthesize benzoic acid, and 4-hydroxymethylbenzoic acid is used to synthesize terephthalic acid. The furan ring aldehydes include 5-hydroxymethylfurfural, 2-furaldehyde, and 5-formyl-2-furancarboxylic acid. 5-hydroxymethylfurfural is synthesized into 2,5-furandicarboxylic acid, 2-furaldehyde is synthesized into 2-furancarboxylic acid, and 5-formyl-2-furancarboxylic acid is synthesized into 2,5-furandicarboxylic acid. The furan ring alcohols include 2-hydroxymethylfuran and 2,5-dihydroxymethylfuran. 2-hydroxymethylfuran is used to synthesize 2-furancarboxylic acid, and 2,5-dihydroxymethylfuran is used to synthesize 2,5-furandicarboxylic acid.

8. The use according to any one of claims 5 to 7, characterized in that: The addition amount of the oxidase mutant is 10-500 mg / L, and the substrate concentration is 9-100 mM.

9. The use according to claim 8, characterized in that The reaction conditions are as follows: the solvent is PBS buffer with a pH of 7.5-8.5, the oxidant is oxygen or air, stirring, the reaction temperature is room temperature-40° C., the reaction pressure is normal pressure, and the reaction time is 8-24 hours.

10. The use according to claim 9, characterized in that After the reaction is completed, the mixture is centrifuged or filtered, and the supernatant or filtrate is purified to obtain the target carboxylic acid. The purification includes acidification crystallization or membrane separation.