Method for synthesizing gallic acid by electrochemical driving

Abstract

The invention relates to a method for synthesizing gallic acid by electrochemical driving, which comprises the following steps of: loading p-hydroxybenzoic acid hydroxylase on a heavy working electrode of a three-electrode system to construct an electrochemical synthesis system taking p-hydroxybenzoic acid as a substrate, and replacing NADPH (Nicotinamide Adenine Dinucleotide Phosphate) by using an electrochemical technology to serve as a driving force and an electron source of reaction, so that the purpose of synthesizing gallic acid without expensive coenzyme NADPH can be realized. The gallic acid is synthesized by electrically driving p-hydroxybenzoic acid hydroxylase and directly utilizing p-hydroxybenzoic acid. The synthesis method is easy to operate, green, efficient and suitable for in-vitro large-scale synthesis of gallic acid, the reaction cost is reduced, in addition, the enzyme catalysis efficiency can be regulated and controlled through an electrochemical technology by means of an artificial technological means, and visualization of the biological enzyme catalysis reaction process is achieved by taking an electric signal as a monitoring means.

Description

Technical Field

[0001] The invention belongs to the technical field of electrochemical synthesis, and in particular relates to a method for synthesizing gallic acid by utilizing electrochemical drive. Background Art

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

[0003] The current synthesis methods of gallic acid mainly include chemical synthesis and extraction from plants. The chemical synthesis method involves cumbersome and complicated steps, and the large amount of organic solvents used causes environmental impact and has high costs. Although the plant extraction method is relatively simple, it is limited by the source of raw materials such as the good or bad plant growth year, foreign trade policies, international relations, etc., and the production cost is uncontrollable; especially the production process is seriously polluting (such as the use and discharge of strong acids and alkalis). With the changes and strict requirements of environmental protection policies, environmental assessment and pollution treatment investment are also increasing, resulting in increased costs. Based on this, it is urgent to develop new green, controllable and economical synthesis pathways and processes to improve their economy and environmental friendliness, so as to better adapt to the growing market demand. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a method for synthesizing gallic acid using electrochemical drive.

[0005] The technical solution adopted by the present invention is: a method for synthesizing gallic acid using electrochemical drive, constructing an electrochemical reaction three-electrode system, the three electrodes including a counter electrode, a reference electrode and a working electrode, and the working electrode is loaded with p-hydroxybenzoate hydroxylase; using p-hydroxybenzoic acid as a substrate, applying voltage to the three-electrode system to convert and synthesize gallic acid.

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

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

[0008] Preferably, the p-hydroxybenzoate hydroxylase is drop-coated onto a carbon-based working electrode to achieve complexation of the p-hydroxybenzoate hydroxylase.

[0009] Preferably, the working electrode is an electrode based on carbon paper, carbon cloth, pyrolyzed graphite or carbon nanotubes, or an electrode modified with a carbon material.

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

[0011] Preferably, the source of oxygen 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 100 rpm, -0.67V-0V (vs SHE), a scan rate of 20 mV / s, and 37°C to observe the non-enzymatic catalysis and to determine the enzyme loading. After the reaction curve is stable, p-hydroxybenzoic acid is added to the solution as a reaction substrate to observe the changes in the CV curve. At the same time, the chronoamperometry is performed at -0.35V (vs SHE) to monitor the current changes.

[0013] Preferably, the specific steps are:

[0014] Step 1: In vitro synthesis of p-hydroxybenzoate hydroxylase;

[0015] Step 2: drop-coating p-hydroxybenzoate hydroxylase onto the carbon-based working electrode, and incubating to achieve loading of p-hydroxybenzoate hydroxylase;

[0016] Step 3: Add 50 mM TAPS, pH 6.0 buffer to the reaction cell of the three-electrode system, and assemble the loaded working electrode into the three-electrode system;

[0017] Step 4: Set the scanning test current range to -0.67V-0V (vs SHE) and the scanning rate to 20mv / s to perform a scanning test of the cyclic voltammetry curve to observe the non-enzyme catalytic signal characteristics of the enzyme on the electrode;

[0018] Step 5: Add p-hydroxybenzoic acid as a substrate to the reaction, and observe the enzyme-catalyzed signal changes under the conditions of -0.67V-0V (vs SHE) and a scan rate of 20mv / s.

[0019] Preferably, when the current change is monitored by chronoamperometry at -0.35 V (vs SHE), the current signal increases after the substrate is added, and the current signal gradually decreases after the substrate is converted into gallic acid, and the reaction ends when the current signal returns to the starting current signal.

[0020] The advantages and positive effects of the present invention are as follows: the synthetic method is simple to operate, green and efficient, and is suitable for in vitro large-scale synthesis of gallic acid. No NADPH is required in the process, and the electrons and driving force of the reaction come from the electrode, and the protons come from the solution, so the reaction cost is significantly reduced; by changing the magnitude and direction of the applied current, the electron transfer rate between the enzyme and the electrode is regulated, and then the enzymatic reaction rate is regulated, so that the enzyme catalytic efficiency can be regulated by artificial technical means; in addition, the reaction progress can be monitored by means of electrical signals, so that the visualization of the biological enzyme catalytic reaction process can be realized. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0022] Figure 2 12% SDS-PAGE electrophoresis of pobA enzyme purification;

[0023] Figure 3 Reaction diagram of pobA enzyme activity monitored using NADPH;

[0024] Figure 4 CV diagram of pobA on PGE electrode with changing scan rate;

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

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

[0027] Figure 7 HPLC detection of electrochemical enzyme-catalyzed conversion products. DETAILED DESCRIPTION

[0028] The embodiments of the present invention are described below with reference to the accompanying drawings.

[0029] The present invention relates to a method for synthesizing gallic acid by electrochemical drive. First, p-hydroxybenzoate hydroxylase (pobA) is heterologously expressed, and p-hydroxybenzoate hydroxylase with good performance can be efficiently and massively obtained; an electrochemical three-electrode reaction system is constructed, and p-hydroxybenzoate hydroxylase is placed in the electrochemical three-electrode system, and electrochemical technology is used to replace NADPH as the driving force and electron source of the reaction, so that p-hydroxybenzoate carboxylase can be driven without expensive coenzyme NADPH, and p-hydroxybenzoic acid (4-HBA) is catalyzed to be converted into 3.4-dihydroxybenzoic acid and gallic acid (GA) in sequence through two consecutive hydroxyl addition reactions.

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

[0031] First, a plasmid capable of heterologously expressing p-hydroxybenzoate hydroxylase is constructed, which is transformed into a vector strain, and a large amount of p-hydroxybenzoate hydroxylase is obtained by heterologous expression. The p-hydroxybenzoate hydroxylase is separated and purified for use in constructing a reaction system. When used, the p-hydroxybenzoate hydroxylase is added dropwise to the working electrode, and the amount of p-hydroxybenzoate hydrogenase used each time is 0.56-5.6 μM, and the incubation time on the working electrode is 5-60 minutes; after incubation, the p-hydroxybenzoate 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; a platinum wire is used as a counter electrode, a silver / silver chloride electrode is used as a reference electrode, and the working electrode material can be an electrode based on carbon paper, carbon cloth, pyrolytic graphite, carbon nanotubes, etc. or an electrode modified with carbon materials, and the p-hydroxybenzoate hydroxylase is loaded on the working electrode when used.

[0032] Using electrochemical techniques as a substitute for NADPH and driving force in the traditional p-hydroxybenzoate hydroxylase reaction, such as Figure 1As shown in the figure, in this process, FAD in pobA directly obtains electrons from the electrode and is converted into FADH, and then FADH reacts with O 2 The reaction forms FADH-O-OH, and then the hydroxyl group on FADH-O-OH is transferred to p-hydroxybenzoic acid to react with gallic acid, and FADH-OH is generated at the same time. FADH-OH is automatically dehydrated to generate FAD, and the next round of reaction is carried out again. In this process, NADPH is not required. The electrons and driving force of the reaction come from the electrode, and the protons come from the solution. In this process, the electrochemical technology can be used to achieve adjustable and visual monitoring of the reaction.

[0033] Add 50mM TAPS pH 6.0 buffer to the reaction cell of the electrochemical reaction system, assemble the loaded working electrode into the three-electrode system; set the scanning test current range to -0.67V-0V (vs SHE), and the scanning rate to 20mv / s to perform scanning test of cyclic voltammetry curve, and observe the signal characteristics of non-enzyme catalysis of enzyme on the electrode; add p-hydroxybenzoic acid as substrate to the reaction, and observe the signal changes of enzyme catalysis under the conditions of -0.67V-0V (vs SHE) and scanning rate of 20mv / s. At the beginning after adding the substrate, apply -0.67V—0V vs. silver / silver chloride voltage to the working electrode, and use cyclic voltammetry to monitor the reaction process. Later, apply -0.35V vs. silver / silver chloride voltage to the working electrode, and use chronoamperometry to monitor the reaction process.

[0034] When the chronoamperometry was used to monitor the current changes at -0.35 V (vs SHE), the current signal increased after the substrate was added. After the substrate was converted into gallic acid, the current signal gradually decreased and the reaction ended when it returned to the starting current signal.

[0035] The above method can realize electrochemically driven synthesis of gallic acid, and by changing the magnitude and direction of the applied current, the electron transfer rate between the enzyme and the electrode can be regulated, thereby regulating the rate of the enzymatic reaction, thereby achieving artificial technical means to control the enzyme catalytic efficiency; in addition, the reaction progress can be monitored by electrical signals to achieve visualization of the enzyme catalytic reaction process.

[0036] The scheme of the present invention is described below in conjunction with the accompanying drawings, wherein the experimental methods without specific operating steps are all carried out in accordance with the corresponding product instructions, and the instruments, reagents, and consumables used in the examples can all be purchased from commercial companies unless otherwise specified.

[0037] Example 1: Preparation of p-hydroxybenzoate hydroxylase

[0038] 1.1 Expression and purification of pobA protein

[0039] The amino acid sequence (SEQ ID No. 1) and nucleotide sequence (SEQ ID No. 2) of p-hydroxybenzoate hydroxylase were prepared according to NCBI accession number NC_002516.2 to construct a recombinant plasmid (pobA-pET21a), and a His tag was added at the C-terminus.

[0040] SEQ ID No.1:

[0041] MKTQVAIIGAGPSGLLLGQLLHKAGIDNVILERQTPDYVLGRIRAGVLEQGMVDLLREAGVDRRMARDGLVHEGVEIAFAGQRRRIDLKRLSGGKTVYGQTEVTRDLMEAREACGATTVYQAAEVRLHDLQGERPYVTFERDGERLRLDCDYIAAGCDGFHGISRQSIPAERLKVFERVYPFGWLGLLADTPPVSHELI YANHPRGFALCSQRSATRSRYYVQVPLSEKVEDWSDERFWTELKARLPSEVAEKLVTGPSLEKSIAPLRSFVVEPMQHGRLFLAGDAAHIVPPTGAKGLNLAASDVSTLYRLLLKAYREGRGELLERYSAICLRRIWKAERFSWWMTSVLHRFPDTDAFSQRIQQTELEYYLGSEAGLATIAENYVGLPYEEIEHHHHHH

[0042] SEQ ID No.2:

[0043]

[0044] The constructed plasmid was transformed into competent Escherichia coli BL21 (DE3) cells and cultured in an inoculum containing 0.1 mg / mL ampicillin resistance (AMP + ), cultured at 37°C, 200 rpm for 6 h, and then at OD 600 When the value is between 0.6 and 0.8, add 1 mM IPTG, and express and induce for 16 hours at 16°C and 160 rpm. After the culture is completed, centrifuge the culture solution at 4000 rpm for 40 minutes, discard the supernatant, collect the bacteria, and then resuspend and dilute with 50 mM Tris, pH 6.0 buffer, and store in a -80°C ultra-low temperature refrigerator for later use.

[0045] The collected bacteria were crushed under 1200 kPa pressure for 3 minutes. Ni-NTA affinity resin (5 mL, Cytiva) was then 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 mMTris, 150 mM NaCl, 500 mM imidazole, pH 7.4; Buffer 1 containing 25 mM imidazole was used to wash away non-specifically bound impurities bound to the nickel column; Buffer 2 containing 300 mM imidazole was used to elute the target protein bound to the nickel column; Buffer 3 containing 500 mM imidazole was used to completely elute the target protein on the nickel column. The samples eluted with different buffers were tested respectively. The purity of the target protein was verified by 12% SDS-PAGE, and the protein concentration was detected by Bradford method. After adding 10% glycerol, the protein was aliquoted and stored at -80°C for future use. The purity of the prepared pobA protein was detected by electrophoresis. Figure 2 As shown in FIG. 1 , the flow stream is the outflow liquid after the broken bacterial liquid flows through the Ni-NTA affinity resin. Figure 2 The results show that pobA protein 1 was successfully prepared and purified. 2 Detection of pobA enzyme activity

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

[0047] Example 2: Construction of electrochemical reaction system

[0048] 2.1 Preparation of thermal graphite electrode

[0049] Select the appropriate electrode original protected by polytetrafluoroethylene (PTFE), scrape and polish it with a knife. Ultrasonicate the graphite rod with anhydrous ethanol for 15 minutes; put silver glue in the sleeve, insert the graphite rod of appropriate size into the polytetrafluoroethylene shell, and the size of the graphite rod is 2mm×2mm×20mm (length×width×height). After drying overnight in a 45℃ oven, scrape off the excess silver glue on the edge of the graphite. Wrap 1.5 circles of transparent tape around the shell (make sure the level is slightly higher than the graphite) to form a mold, then stir the mixed epoxy resin AB glue evenly, gently pour it into the top of the sleeve, cool to room temperature, and polish it with coarse sandpaper until the graphite head is exposed, and then polish it with P400 sandpaper to make it smooth. Finally, use the ohm function on the multimeter to detect the electrode resistance of about 4 or 5Ω, and prepare a thermal cracking graphite electrode.

[0050] 2.2 Construction of three-electrode system

[0051] A three-electrode system was constructed with a platinum wire as a counter electrode, a silver / silver chloride electrode as a reference electrode, and a carbon electrode as a working electrode. The working electrode material was mainly an electrode based on carbon paper, carbon cloth, pyrolytic graphite, carbon nanotubes, etc., or an electrode modified with a carbon material. The p-hydroxybenzoate hydroxylase prepared in Example 1 was directly added dropwise to the working electrode and combined at 4°C for 10 minutes.

[0052] Assemble the three-electrode device for protein film voltammetry (PFV) experiment. The counter electrode is a platinum wire, the reference is an Ag / AgCl electrode, and the reference cell contains a 0.1M NaCl buffer. The reference cell is connected to the electrochemical reaction cell through a Luggin capillary, and the tip of the Luggin capillary is located near the working electrode. Add 20mL of 50mM TAPS pH 6.0 buffer to the reaction cell, and then assemble the PGE electrode incubated with p-hydroxybenzoate hydroxylase onto a Wantong rotary motor to control the rotation rate of the PGE electrode in the reaction cell. The bottom of the PGE electrode can be placed about 1cm from the liquid surface.

[0053] 2.3 Electrochemical reaction system

[0054] The concentration of the input substrate p-hydroxybenzoic acid is 0.04-100 mM. The reaction system uses oxygen as a reaction substrate, and the oxygen source can be pure oxygen or air.

[0055] The cyclic voltammetry (CV) curve of the reaction was tested at 100rpm, -0.67V-0V (vs SHE), a scan rate of 20mV / s, and 37°C. The non-enzymatic catalysis was observed and the enzyme loading was determined. After the reaction curve was stable, p-hydroxybenzoic acid was added to the solution as a reaction substrate to observe the changes in the CV curve. Later, the chronoamperometry was performed at -0.35V (vs SHE) to monitor the current changes. The conditions were -0.35V (vs SHE), 37°C, and a rotation speed of 100rpm.

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

[0057] The three-electrode system was assembled according to the method of Example 2, and 5 μL of 50 mg / mL pobA was added dropwise to the polished carbon electrode. The mixture was incubated at 4°C for 1 h to allow pobA to bind to the carbon electrode. ddH 2 O was rinsed to remove excess enzyme, and the treated working electrode was placed in the electrode system. The CV changes at scan rates of 5mv / s, 10mv / s, 15mv / s, 20mv / s, 25mv / s, 30mv / s, 35mv / s, 40mv / s, 45mv / s, and 50mv / s were tested at -0.76V to -0.1V (vs SHE) and 100rpm. The results are shown in the figure. Figure 4 As shown. The PGE electrode can effectively load pobA and can perform efficient electron transfer. When the scan rate is 20mv / 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 a carbon electrode as the speed changes

[0059] The three-electrode system was assembled according to the method of Example 2, and 5 μL of 50 mg / mL pobA was added dropwise to the polished carbon electrode. The mixture was incubated at 4°C for 1 h to allow pobA to bind to the carbon electrode. ddH 2 O was rinsed to remove excess enzyme, and the treated working electrode was placed in the electrode system. Under the conditions of -0.76V to -0.1V (vs SHE), 20mv / s, the CV changes of the electrode at 100rpm, 200rpm, 300rpm, 400rpm, 500rpm, 600rpm, 700rpm, and 800rpm were tested. The results are as follows: Figure 5 As shown, pobA can stably produce effects under various rotation speed conditions, which shows that pobA can be stably loaded on the PGE electrode.

[0060] Example 5: CV response changes of pobA to substrates on PGE electrode

[0061] The three-electrode system was assembled according to the method of Example 2, and 5 μL of 50 mg / mL pobA was added dropwise to the polished carbon electrode. The mixture was incubated at 4°C for 1 h to allow pobA to bind to the carbon electrode. ddH 2 O was used to rinse to remove excess enzymes, and the treated working electrode was placed in the electrode system. The CV changes of the electrode were tested under the conditions of -0.76V to -0.1V (vs SHE), 20mV / s, and 100rpm. After the reaction curve was stable, 40μM p-hydroxybenzoic acid (4-HBA) was added to the solution as a reaction substrate, and the changes in the CV curve were observed. The results are as follows: Figure 6 As shown, the reduction current signal was enhanced, which was significantly different from the electrochemical signal without substrate, mainly manifested in a reduction current signal close to -5 μA. It can be seen that the electrochemical technology can be used to drive p-hydroxybenzoate hydroxylase to convert and synthesize gallic acid.

[0062] Example 6: Detecting electrochemical reaction products using high performance liquid chromatography (HPLC);

[0063] According to the method of Example 5, p-hydroxybenzoic acid was converted into gallic acid by electrochemical catalysis using pobA. After the reaction, the reaction solution was subjected to HPLC detection. The conditions for liquid phase detection were as follows: a Kro masil ODS-C18 chromatographic column (250 mm × 4.6 mm, 5 μm) was used, and 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), a flow rate of 1 mL / min, a detection wavelength of 254 nm, a column temperature of 25°C, and a detection time of 25 min. The detection results are shown in the figure. Figure 7 As shown, the product and the substrate peak in different time periods, wherein Peak 1 at about 9 minutes is the final product gallic acid (GA), Peak 2 at 11 minutes is the intermediate product 3,4-dihydroxybenzoic acid (3,4-DHA), and Peak 3 at the last 13 minutes is the input substrate p-hydroxybenzoic acid (4-HBA). Example 5 successfully prepared gallic acid (GA), and HPLC can also be used to monitor and separate the electrochemical enzyme-catalyzed preparation of gallic acid.

[0064] The embodiments of the present invention are described in detail above, but the contents described are only preferred embodiments of the present invention and cannot be considered to limit the scope of implementation of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the scope of the patent coverage of the present invention.

Claims

1. A method for synthesizing gallic acid using electrochemical drive, characterized in that: A three-electrode system for electrochemical reaction is constructed, wherein the three electrodes include a counter electrode, a reference electrode and a working electrode, and p-hydroxybenzoate hydroxylase is loaded on the working electrode. With p-hydroxybenzoic acid as a substrate, a voltage is applied to the three-electrode system to convert and synthesize gallic acid.

2. The method for synthesizing gallic acid by electrochemical drive according to claim 1, characterized in that: An expression vector capable of expressing p-hydroxybenzoate hydroxylase is constructed, transformed into a host bacterium, and the p-hydroxybenzoate hydroxylase is separated and purified after fermentation.

3. The method for synthesizing gallic acid by electrochemical drive according to claim 2, characterized in that: pET-21a was used as an expression vector, Escherichia coli-BL21 (DE3) was used as an expression host, and the amino acid sequence of p-hydroxybenzoate hydroxylase was shown in SEQ ID No.

1.

4. The method for synthesizing gallic acid by electrochemical drive according to claim 2, 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.

5. The method for synthesizing gallic acid by electrochemical drive according to claim 4, 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.

6. The method for synthesizing gallic acid by electrochemical drive according to any one of claims 1 to 5, characterized in that: The reaction system includes 50mM TAPS buffer, pH value is 6.0, and the concentration of p-hydroxybenzoic acid is 0.04-100mM; oxygen is introduced as a reaction substrate.

7. The method for synthesizing gallic acid by electrochemical drive according to claim 6, characterized in that: The oxygen source is pure oxygen or air.

8. The method for synthesizing gallic acid by electrochemical drive according to claim 6, 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 (vs SHE), a scan rate of 20 mV / s, and 37 ° C. After the reaction curve was stable, p-hydroxybenzoic acid was added to the solution as a reaction substrate, the changes in the CV curve were observed, and the chronoamperometry was used to monitor the current changes at -0.35 V (vs SHE).

9. The method for synthesizing gallic acid by electrochemical drive according to claim 1, characterized in that: The specific steps are: Step 1: In vitro synthesis of p-hydroxybenzoate hydroxylase; Step 2: drop-coating p-hydroxybenzoate hydroxylase onto the carbon-based working electrode, and incubating to achieve loading of p-hydroxybenzoate hydroxylase; Step 3: Add 50 mM TAPS pH to the reaction cell of the three-electrode system 6.0 buffer, assembling the loaded working electrode into a three-electrode system; Step 4: Set the scanning test current range to -0.67V-0V (vs SHE) and the scanning rate to 20mv / s to perform a scanning test of the cyclic voltammetry curve to observe the non-enzyme catalytic 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 -0.67V-0V (vs SHE) and a scan rate of 20mv / s.

10. The method for synthesizing gallic acid by electrochemical drive according to claim 9, characterized in that: When the chronoamperometry was used to monitor the current changes at -0.35 V (vs SHE), the current signal increased after the substrate was added. After 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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