A method for synthesizing gallic acid using electrochemical driving

Abstract

The present application relates to a method for synthesizing gallic acid by electrochemical driving, wherein p-hydroxybenzoic acid hydroxylase is loaded on the working electrode of a three-electrode system to construct an electrochemical synthesis system with p-hydroxybenzoic acid as the substrate, and electrochemical technology is used to replace NADPH as the driving force and electron source of the reaction, so that the synthesis of gallic acid by directly using p-hydroxybenzoic acid without expensive coenzyme NADPH and by electrochemical driving of p-hydroxybenzoic acid hydroxylase can be realized. The synthesis method is simple, green and efficient, and is suitable for in vitro large-scale synthesis of gallic acid, thereby reducing the reaction cost. In addition, the enzyme catalytic efficiency can be regulated by artificial technical means through electrochemical technology, and the electrochemical signal can be used as a monitoring means to realize the visualization of the biological enzyme catalytic reaction process.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of electrochemical synthesis, and particularly relates to a method for synthesizing gallic acid by electrochemical driving. BACKGROUND

[0002] Gallic acid, as an important natural polyphenol compound, has important market applications in the fields of food, medicine, cosmetics and agriculture due to its wide biological activity and potential health benefits. It is not only widely used as a preservative and antioxidant in the food industry, but also used as a precursor for drug synthesis in the pharmaceutical industry, and as a key ingredient for improving skin quality and anti-aging ingredients in cosmetics. At the same time, it can be used as a synthetic dye, ink, developer, synthetic oil antioxidant, and extraction of rare metals. Gallic acid is an important antioxidant in the chip industry, which protects the metal surface, prevents pollution and improves the performance of photoresist. Gallic acid also has important applications in the field of national defense, such as corrosion-resistant coatings, bulletproof materials, stealth materials, propellant stabilizers, and electronic devices.

[0003] Current synthesis methods of gallic acid mainly include chemical synthesis and extraction from plants. The chemical synthesis method involves complex steps, and the use of large amounts of organic solvents has environmental impact and high cost. Although the plant extraction method is relatively simple, it is limited by the source of raw materials such as the quality of plant growth, foreign trade policy, international relations, etc., and the production cost is uncontrollable. Especially the production process is seriously polluted (such as the use and discharge of strong acid and strong base), and with the change and strict requirements of environmental protection policy, the cost of environmental evaluation and pollution treatment is increasing, resulting in increased cost. Therefore, it is urgent to develop a green, controllable and economic new synthesis route and process to improve its economic and environmental friendliness, so as to better meet the growing market demand. SUMMARY

[0004] To solve the above technical problems, the application provides a method for synthesizing gallic acid by electrochemical driving.

[0005] The technical scheme adopted by the application is: a method for synthesizing gallic acid by electrochemical driving, an electrochemical reaction three-electrode system is constructed, the three electrodes include a counter electrode, a reference electrode and a working electrode, and p-hydroxybenzoic acid hydroxylase is loaded on the working electrode; p-hydroxybenzoic acid is used as a substrate, and a voltage is applied to the three-electrode system to synthesize gallic acid.

[0006] Preferably, an expression vector capable of expressing p-hydroxybenzoic acid hydroxylase is constructed, which is transformed into a host bacterium, and after fermentation, p-hydroxybenzoic acid hydroxylase is separated and purified.

[0007] Preferably, pET-21a is used as an expression vector, and E. coli-BL21(DE3) is used as an expression host, and the p-hydroxybenzoic acid hydroxylase amino acid sequence is shown in SEQ ID No. 1.

[0008] Preferably, the p-hydroxybenzoic acid hydroxylase is drop-coated on a carbon-based working electrode to achieve the loading of the p-hydroxybenzoic acid hydroxylase.

[0009] Preferably, the working electrode is an electrode based on carbon paper, carbon cloth, thermal cracking graphite or carbon nanotube substrate, or a carbon material modified electrode.

[0010] Preferably, the reaction system includes 5mM TAPS buffer with a pH of 6.0, and the concentration of p-hydroxybenzoic acid is 0.04-100mM; and oxygen is introduced as the reaction substrate.

[0011] Preferably, the oxygen source is pure oxygen or air.

[0012] Preferably, after the three-electrode device is assembled, the cyclic voltammetry (CV) curve of the reaction is tested at 100rpm, -0.67V-0V (vs SHE), a scan rate of 20mV / s, and 37℃, which is used to observe the non-enzymatic catalysis and determine the loading of the enzyme; after the reaction curve is stable, p-hydroxybenzoic acid is added to the solution as the reaction substrate to observe the change of the CV curve. Meanwhile, the chronoamperometry method is used to monitor the current change at -0.35V (vs SHE) in the later stage.

[0013] Preferably, the specific steps are as follows:

[0014] Step one: in vitro synthesis of p-hydroxybenzoic acid hydroxylase;

[0015] Step two: drop-coating the p-hydroxybenzoic acid hydroxylase on a carbon-based working electrode, and incubating to achieve the loading of the p-hydroxybenzoic acid hydroxylase;

[0016] Step three: adding 50mM TAPS buffer with a pH of 6.0 to the reaction pool of the three-electrode system, and assembling the loaded working electrode into the three-electrode system;

[0017] Step four: setting the scan test current range to -0.67V-0V (vs SHE) and the scan rate to 20mv / s to perform cyclic voltammetry curve scanning test, and observing the non-enzymatic catalysis signal characteristics of the enzyme on the electrode;

[0018] Step five: adding p-hydroxybenzoic acid as the substrate to the reaction, and observing the signal change of the enzyme catalysis under the condition of -0.67V-0V (vs SHE) and a scan rate of 20mv / s.

[0019] Preferably, when the chronoamperometry method is used to monitor the current change, the current signal increases after the substrate is added, and the current signal gradually decreases when the substrate is converted into gallic acid, and returns to the initial current signal, which is the end of the reaction.

[0020] The present application has the advantages and positive effects that the synthetic method is simple, green and efficient, and is suitable for in vitro scale synthesis of gallic acid, and no NADPH is needed in the process, the reaction electrons and driving force come from the electrode, and protons come from the solution, so the reaction cost is significantly reduced; by changing the size and direction of the applied current, the electron transfer rate between the enzyme and the electrode is regulated, and then the enzyme reaction rate is regulated, so as to realize the regulation of enzyme catalytic efficiency by artificial technical means; in addition, the reaction process can be monitored by using the electrical signal as a monitoring means, so as to realize the visualization of the biological enzyme catalytic reaction process. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 Schematic diagram of FAD conversion in the pobA enzyme on the electrode;

[0022] Figure 2 12% SDS-PAGE electrophoresis map of the purified pobA enzyme;

[0023] Figure 3 Reaction diagram of monitoring the activity of the pobA enzyme by using NADPH;

[0024] Figure 4 CV diagram of the pobA enzyme on the PGE electrode with the change of the scanning speed;

[0025] Figure 5 CV diagram of the pobA enzyme on the PGE electrode with the change of the scanning speed;

[0026] Figure 6 CV response change diagram of the pobA enzyme on the PGE electrode to the substrate;

[0027] Figure 7 HPLC detection result diagram of the conversion product of the electrochemical enzyme catalysis. DETAILED DESCRIPTION

[0028] The embodiments of the present application will be described below in combination with the drawings.

[0029] The present application relates to a method for synthesizing gallic acid by electrochemical driving. First, the p-hydroxybenzoic acid hydroxylase (pobA) is heterologously expressed, and the p-hydroxybenzoic acid hydroxylase with good performance can be obtained in large quantities; an electrochemical three-electrode reaction system is constructed, the p-hydroxybenzoic acid hydroxylase is placed in the electrochemical three-electrode system, and the electrochemical technology is used to replace NADPH as the driving force and electron source of the reaction, so that the expensive coenzyme NADPH is not needed to drive the p-hydroxybenzoic acid carboxylase to catalyze the p-hydroxybenzoic acid (4-HBA) to be converted into 3.4-dihydroxybenzoic acid and gallic acid (GA) in turn through two consecutive hydroxylation reactions.

[0030] Electrochemistry is a scientific technology that uses electrical energy to drive chemical reactions and electrochemical processes. It plays an important role in energy storage, material synthesis, environmental protection, and analytical chemistry. This technology can be combined with biological enzyme catalysis to achieve efficient in vitro biosynthesis. The core of this technology can utilize the high selectivity and efficiency of enzymes, and the controllability and reconfigurability of electrochemical reactions to achieve precise catalysis and efficient synthesis of various chemical reactions. At the same time, electrochemistry uses electrical energy to replace chemical energy as the driving force, especially reducing the use of traditional expensive coenzymes such as NADH / NADPH, further reducing production costs. Moreover, during the electrochemical catalytic reaction process, electrical signals can be used as a monitoring means to monitor the reaction process in real time, achieving visualization of the biological enzyme catalytic reaction process. Finally, electrochemistry can regulate the rate of electron transfer between the enzyme and the electrode by changing the size and direction of the applied current, thereby regulating the enzyme reaction rate and achieving artificial technical regulation of enzyme catalytic efficiency.

[0031] First, a plasmid capable of heterologously expressing p-hydroxybenzoic acid hydroxylase is constructed, which is transformed into a carrier strain to heterologously express p-hydroxybenzoic acid hydroxylase in large quantities, and the p-hydroxybenzoic acid hydroxylase is separated and purified for use in constructing the reaction system. When used, the p-hydroxybenzoic acid hydroxylase is added dropwise to the working electrode, and the amount of p-hydroxybenzoic acid hydroxylase used each time is 0.56-5.6 μM, and the incubation time of p-hydroxybenzoic acid hydroxylase on the working electrode is 5-60 minutes; after incubation, the p-hydroxybenzoic acid hydroxylase is loaded on the working electrode to provide enzyme catalysis in the electrochemical reaction system. A three-electrode system is constructed, including a counter electrode, a reference electrode, and a working electrode; platinum wire is used as the counter electrode, silver / silver chloride electrode is used as the reference electrode, and the working electrode material can be carbon paper, carbon cloth, thermal cracking graphite, carbon nanotube electrode or carbon material modified electrode, and the p-hydroxybenzoic acid hydroxylase is loaded on the working electrode when used.

[0032] The electrochemical technology is used as a substitute for NADPH and a driving force in the traditional p-hydroxybenzoic acid hydroxylase reaction, such as Figure 1As shown, in the process, FAD in pobA is directly converted into FADH by receiving electrons from the electrode, and then FADH reacts with O2 to form FADH-O-OH, and then the hydroxyl group on FADH-O-OH is transferred to p-hydroxybenzoic acid to generate gallic acid, and FADH-OH is automatically dehydrated to generate FAD, and the next round of reaction is performed again. In this process, NADPH is not required, and the electrons and driving force of the reaction come from the electrode, and protons come from the solution, and in this process, the reaction can be adjusted and visualized by electrochemical technology.

[0033] Add 50 mM TAPS pH 6.0 buffer to the reaction cell of the electrochemical reaction system, and assemble the loaded working electrode into a three-electrode system; set the scanning test current range to -0.67V-0V (vs SHE), and the scanning speed to 20mv / s to perform cyclic voltammetry scanning test, and observe the non-enzymatic catalytic signal characteristics of the enzyme on the electrode; add p-hydroxybenzoic acid as a substrate to the reaction, and also under the condition of -0.67V-0V (vs SHE) and a scanning speed of 20mv / s, observe the signal change of the enzyme catalysis. After adding the substrate, initially apply -0.67V-0V vs. silver / silver chloride voltage to the working electrode, and use cyclic voltammetry to monitor the reaction process, and later apply -0.35V vs. silver / silver chloride voltage to the working electrode, and use chronoamperometry to monitor the reaction process.

[0034] When monitoring the current change by chronoamperometry under the condition of -0.35V (vs SHE), after the addition of the substrate, the current signal increases, and when the substrate is converted into gallic acid, the current signal gradually decreases and returns to the initial current signal, which is the end of the reaction.

[0035] The above method can realize electrochemical-driven synthesis of gallic acid, and by changing the size and direction of the applied current, the electron transfer rate between the enzyme and the electrode can be regulated, and thus the enzyme reaction rate can be regulated, and the enzyme catalytic efficiency can be regulated by artificial technical means. In addition, the reaction process can be monitored by electrical signals to realize the visualization of the biological enzyme catalytic reaction process.

[0036] The embodiments of the present application will be described below with reference to the accompanying drawings, wherein the experimental methods not specifically described in the operation steps are performed according to the corresponding product instructions. The instruments, reagents and consumables used in the examples can be purchased from commercial companies unless otherwise specified.

[0037] Example 1: Preparation of p-hydroxybenzoic acid hydroxylase

[0038] 1.1 Expression and purification of pobA protein

[0039] The recombinant plasmid (pobA-pET21a) was constructed according to the information of p-hydroxybenzoic acid hydroxylase amino acid sequence (SEQ ID No. 1) and nucleotide sequence (SEQ ID No. 2) obtained from NCBI accession number NC_002516.2, and His tag was added at the C terminal.

[0040] SEQ ID No. 1:

[0041] MKTQVAIIGAGPSGLLLGQLLHKAGIDNVILERQTPDYVLGRIRAGVLEQGMVDLLREAGVDRRMARDGLVHEGVEIAFAGQRRRIDLKRLSGGKTVTVYGQTEVTRDLMEAREACGATTVYQAAEVRLHDLQGERPYVTFERDGERLRLDCDYIAGCDGFHGISRQSIPAERLKVFERVYPFGWLGLLADTPPVSHELIYANHPRGFALCSQRSATRSRYYVQVPLSEKVEDWSDERFWTELKARLPSEVAEKLVTGPSLEKSIAPLRSFVVEPMQHGRLFLAGDAAHIVPPTGAKGLNLAASDVSTLYRLLLKAYREGRGELLERYSAICLRRIWKAERFSWWMTSVLHRFPDTDAFSQRIQQTELEYYLGSEAGLATIAENYVGLPYEEIEHHHHHH

[0042] SEQ ID No. 2:

[0043]

[0044] The constructed plasmid was transformed into competent E. coli BL21(DE3) cells, which were cultured in LB medium containing 0.1 mg / mL ampicillin resistance (AMP + ), at 37°C, 200 rpm for 6 hours, then 1 mM IPTG was added when the OD 600 value was between 0.6-0.8, and expression and induction were carried out at 16°C, 160 rpm for 16 hours. After the end of the culture, the culture solution was centrifuged at 4000 rpm for 40 min, the supernatant was discarded, and the bacterial cells were collected, then resuspended and diluted with 50 mM Tris, pH 6.0 buffer, and stored in a -80°C ultra-low temperature refrigerator for use.

[0045] The collected bacterial cells were crushed under a pressure of 1200 kPa for 3 minutes. Then, Ni-NTA affinity resin (5 mL, Cytiva) was used for affinity purification, followed by gradient elution, where Buffer 1: 50 mM Tris, 150 mM NaCl, 25 mM imidazole, pH 7.4; Buffer 2: 50 mM Tris, 150 mM NaCl, 300 mM imidazole, pH 7.4; Buffer 3: 50 mM Tris, 150 mM NaCl, 500 mM imidazole, pH 7.4; using Buffer 1 containing 25 mM imidazole is used to wash off non-specifically bound impurities bound to the nickel column; using Buffer 2 containing 300 mM imidazole is used to elute the target protein bound to the nickel column; using Buffer 3 containing 500 mM imidazole is used to completely elute the target protein from the nickel column. The samples eluted by different buffers were detected respectively. The purity of the target protein was verified by 12% SDS-PAGE, and the protein concentration was detected by the Bradford method, and 10% glycerol was added for aliquot storage at -80°C. The prepared pobA protein was detected by electrophoresis for purity, as shown in Figure 2 , where the stream is the effluent after the crushed bacterial solution flows through the Ni-NTA affinity resin. From Figure 2 The results show that the pobA protein was successfully prepared and purified.

[0046] The purified p-hydroxybenzoic acid hydroxylase was diluted to a concentration of 1 mg / mL and 2 mg / mL using 50 mM Tris, pH 6.0 buffer, and then added to a 50 mM Tris, pH 6.0 buffer containing 5 mM 4-HBA, 0.1 mM NADPH for reaction, and the change in ultraviolet absorption value at 340 nm was detected using a multi-well enzyme marker to analyze the utilization of NADPH by pobA, and the results are as shown in Figure 3The results show that the enzyme activity of the pobA can be confirmed in the presence of 4-HBA and NADPH.

[0047] Example 2: Construction of electrochemical reaction system

[0048] 2.1 Preparation of thermal cracking graphite electrode

[0049] The electrode element protected by polytetrafluoroethylene (PTFE) was selected and scraped with a knife. The graphite rod was ultrasonically treated with anhydrous ethanol for 15 min; silver glue was loaded into the sleeve, and a graphite rod of appropriate size was inserted into the polytetrafluoroethylene shell. The size of the graphite rod was 2 mm x 2 mm x 20 mm (length x width x height). After drying in a 45°C oven overnight, the excess silver glue on the edges of the graphite was scraped off. Wrap 1.5 turns of transparent tape around the shell (make sure it is slightly higher than the graphite) to form a mold. Then mix the epoxy resin AB glue evenly, pour it gently into the top of the sleeve, and after cooling to room temperature, polish it with coarse sandpaper until the graphite head is exposed. Then polish it flat with P400 sandpaper. Finally, use the ohm function of a multimeter to detect the electrode resistance, which is about 4 or 5 Ω. Thus, the thermal cracking graphite electrode is prepared.

[0050] 2.2 Construction of three-electrode system

[0051] A platinum wire was used as the counter electrode, a silver / silver chloride electrode was used as the reference electrode, and a carbon electrode was used as the working electrode to construct a three-electrode system. The working electrode materials mainly included carbon paper, carbon cloth, thermal cracking graphite, carbon nanotube-based electrodes, or carbon material-modified electrodes. The p-hydroxybenzoic acid hydroxylase prepared in Example 1 was directly added to the working electrode, and combined at 4°C for 10 minutes.

[0052] The three-electrode device was assembled for protein film voltammetry (PFV) experiments. The counter electrode was a platinum wire, the reference electrode was an Ag / AgCl electrode, the reference pool was a 0.1M NaCl buffer, and the reference pool was connected to the electrochemical reaction pool through a Luggin capillary. The tip of the Luggin capillary was located near the working electrode. 20 mL of 50 mM TAPS pH 6.0 buffer was added to the reaction pool, and the PGE electrode incubated with p-hydroxybenzoic acid hydroxylase was assembled on a Wanhong rotary motor to control the rotation rate of the PGE electrode in the reaction pool. The bottom end was placed about 1 cm away from the liquid surface.

[0053] 2.3 Electrochemical reaction system

[0054] The concentration of the substrate p-hydroxybenzoic acid was 0.04-100 mM. The reaction system used oxygen as the reaction substrate, and the oxygen source could be pure oxygen or air.

[0055] The cyclic voltammetry (CV) curve of the reaction was tested at 100 rpm, -0.67 V-0 V (vs SHE), a scan rate of 20 mV / s, and 37°C. The non-enzymatic catalysis was observed to determine the loading of the enzyme. After the reaction curve was stable, p-hydroxybenzoic acid was added to the solution as a reaction substrate, and the CV curve change was observed. In the later stage, the chronoamperometry method was used to monitor the current change at -0.35 V (vs SHE) under the condition of -0.35 V (vs SHE) and a temperature of 37°C at a rotation speed of 100 rpm.

[0056] Example 3: CV of pobA on PGE electrode with scan rate change

[0057] The three-electrode system was assembled according to the method of Example 2, 5 μL of 50 mg / mL pobA was added to the carbon electrode which had been polished, and incubated at 4°C for 1 h to allow the combination of pobA and carbon electrode. The treated working electrode was placed in the electrode system. The CV change was tested at -0.76 V to -0.1 V (vs SHE) at a scan rate of 20 mV / s, 100 rpm, 200 rpm, 300 rpm, 400 rpm, 500 rpm, 600 rpm, 700 rpm, and 800 rpm, respectively. The results are shown in Figure 4 The PGE electrode can effectively load pobA and can efficiently transfer electrons. At a scan rate of 20 mV / s, the characteristics of non-enzymatic catalysis can be clearly observed, and a stable signal can be generated.

[0058] Example 4: CV of pobA on carbon electrode with rotation speed change

[0059] The three-electrode system was assembled according to the method of Example 2, 5 μL of 50 mg / mL pobA was added to the carbon electrode which had been polished, and incubated at 4°C for 1 h to allow the combination of pobA and carbon electrode. The treated working electrode was placed in the electrode system. The CV change was tested at -0.76 V to -0.1 V (vs SHE) at a scan rate of 20 mV / s, 100 rpm, 200 rpm, 300 rpm, 400 rpm, 500 rpm, 600 rpm, 700 rpm, and 800 rpm, respectively. The results are shown in Figure 5 The PGE electrode can effectively load pobA and can efficiently transfer electrons. At a scan rate of 20 mV / s, the characteristics of non-enzymatic catalysis can be clearly observed, and a stable signal can be generated.

[0060] Example 5: CV response change of pobA on PGE electrode to substrate

[0061] The three-electrode system was assembled according to the method of Example 2, 5 μL of 50 mg / mL pobA was added dropwise to the carbon electrode which had been polished, and incubated at 4°C for 1 h to allow the binding of pobA to the carbon electrode. The excess enzyme was removed by washing with ddH2O, and the treated working electrode was placed in the electrode system. The CV of the electrode was tested at -0.76 V to -0.1 V (vs SHE), 20 mV / s, 100 rpm, and after the reaction curve was stable, 40 μM p-hydroxybenzoic acid (4-HBA) was added to the solution as the reaction substrate, and the CV curve was observed. The results are shown in Figure 6 The signal of the reduction current was enhanced, and there was a clear contrast with the electrochemical signal without the substrate, mainly showing a reduction current signal of about -5 μA. It can be seen that the electrochemical technology can drive the p-hydroxybenzoic acid hydroxylase to convert to synthesize gallic acid.

[0062] Example 6: Detection of the electrochemical reaction product by high performance liquid chromatography (HPLC);

[0063] The electrochemical catalysis of p-hydroxybenzoic acid to gallic acid by pobA was carried out according to the method of Example 5, and after the reaction was completed, the reaction solution was detected by HPLC. The liquid chromatography detection conditions were as follows: a Kromasil ODS-C18 column (250 mm x 4.6 mm, 5 μm) was used, 0.1% phosphoric acid (mobile phase A) - acetonitrile (mobile phase B) was used as the mobile phase for gradient elution (0-5 min, 0% B→10% B; 5-8 min, 10% B→20% B; 8-12 min, 20% B→40% B; 12-15 min, 40% B→100% B; 15-18 min, 100% B; 18-20 min, 100% B→0% B; 20-25 min, 0% B), the flow rate was 1 mL / min, the detection wavelength was 254 nm, the column temperature was 25°C, and the detection time was 25 min. The detection results are shown in Figure 7 The product and the substrate were eluted at different time periods, wherein the No. 1 peak at about 9 min was the final product gallic acid (GA), the No. 2 peak at 11 min was the intermediate product 3,4-dihydroxybenzoic acid (3,4-DHA), and the No. 3 peak at 13 min was the substrate p-hydroxybenzoic acid (4-HBA). Example 5 successfully prepared gallic acid (GA), and in addition, the electrochemical enzyme catalytic preparation of gallic acid can be monitored and separated by HPLC.

[0064] The above embodiments of the present application are described in detail, but the content described is only the preferred embodiments of the present application, and cannot be considered as limiting the scope of the implementation of the present application. Any equivalent changes and improvements made within the scope of the present application should still belong to the patent coverage of the present application.

Claims

1. A method for synthesizing gallic acid using electrochemical drive, characterized in that: A three-electrode system for an electrochemical reaction is constructed. The three electrodes include a counter electrode, a reference electrode, and a working electrode. The working electrode is a carbon-based working electrode loaded with p-hydroxybenzoate hydroxylase. Using p-hydroxybenzoic acid as a substrate, a voltage is applied to the three-electrode system to convert and synthesize gallic acid. An expression vector capable of expressing p-hydroxybenzoate hydroxylase was constructed, transformed into a host bacterium, and the p-hydroxybenzoate hydroxylase was isolated and purified after fermentation. pET-21a was used as the expression vector and Escherichia coli-BL21 (DE3) was used as the expression host. The amino acid sequence of p-hydroxybenzoate hydroxylase is shown in SEQ ID No.

1.

2. The method for synthesizing gallic acid by electrochemical drive according to claim 1, characterized in that: The p-hydroxybenzoate hydroxylase was drop-coated onto the carbon-based working electrode to achieve the loading of the p-hydroxybenzoate hydroxylase.

3. The method for synthesizing gallic acid by electrochemical drive according to claim 2, characterized in that: The working electrode is an electrode based on carbon paper, carbon cloth, pyrolysis graphite or carbon nanotube, or an electrode modified with carbon material.

4. The method for synthesizing gallic acid by electrochemical drive according to any one of claims 1 to 3, characterized in that: The reaction system includes 50 mM TAPS buffer, pH 6.0, and p-hydroxybenzoic acid concentration of 0.04-100 mM; oxygen is introduced as a reaction substrate.

5. The method for synthesizing gallic acid by electrochemical drive according to claim 4, characterized in that: The oxygen source is pure oxygen or air.

6. The method for synthesizing gallic acid using electrochemical drive according to claim 4, characterized in that: After the three-electrode device was assembled, the cyclic voltammetry (CV) curve of the reaction was tested at 100 rpm, -0.67 V-0 V, a scan rate of 20 mV / s, and 37 °C. After the reaction curve stabilized, p-hydroxybenzoic acid was added to the solution as a reaction substrate, and the changes in the CV curve were observed. The current changes were monitored by chronoamperometry at -0.35 V.

7. The method for synthesizing gallic acid using electrochemical drive according to claim 1, characterized in that: The specific steps are: Step 1: Synthesis of p-hydroxybenzoate hydroxylase in vitro; Step 2: drop-coating p-hydroxybenzoate hydroxylase onto the carbon-based working electrode and incubating to achieve the loading of p-hydroxybenzoate hydroxylase; Step 3: Add 50 mM TAPS pH to the reaction cell of the three-electrode system 6.0 buffer, assemble the loaded working electrode into a three-electrode system; Step 4: Set the sweep test current range to -0.67 V-0 V and the sweep rate to 20 mV / s to perform a cyclic voltammetry curve sweep test to observe the non-enzymatic signal characteristics of the enzyme on the electrode; Step 5: Add p-hydroxybenzoic acid as a substrate to the reaction system and observe the enzyme-catalyzed signal changes under the conditions of -0.67 V to 0 V and a scan rate of 20 mv / s.

8. The method for synthesizing gallic acid using electrochemical drive according to claim 7, characterized in that: When the chronoamperometry was used to monitor the current changes at -0.35 V, the current signal increased after the substrate was added. When the substrate was converted into gallic acid, the current signal gradually decreased and the reaction ended when it returned to the starting current signal.

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