Recombinant strain for preparing gallic acid by enzyme method and construction method of recombinant strain

Abstract

The invention relates to the technical field of tannase, in particular to a recombinant strain for preparing gallic acid through an enzyme method and a construction method of the recombinant strain, the amino acid sequence of tannase is SEQ ID NO.1, tannase of Aspergillus niger CBS 513.88 is selected, and the nucleotide sequence of the tannase is SEQ ID NO.2. According to the invention, tannase genes in aspergillus niger are optimized, the performance of catalytic hydrolysis of tara tannin is improved, and a gene integrated recombinant aspergillus niger strain is constructed and is applied to production of tannase.

Description

Technical Field

[0001] The present invention relates to the technical field of tannase, and specifically provides a recombinant strain for the enzymatic preparation of gallic acid and a method for constructing the same. Background Art

[0002] Tannase is a serine esterase that catalyzes the hydrolysis of galloyl ester bonds in gallotannins to release gallic acid, which is an important intermediate in the chemical and pharmaceutical industries. It can also be used to treat tannins and proteins in beer, making it clear and transparent. It can also be used to remove the astringency of persimmons and other products, and for manufacturing instant tea to prevent the turbidity of fermented tea. By referring to the first crystal structure of fungal tannase from Aspergillus niger, this enzyme has a typical α / β-hydrolase fold domain with a large inserted cap domain, which together form a bowl-shaped hemisphere with the surface depression surrounded by N-linked glycans. Gallic acid binds at the junction of the two domains in the concave through forming two hydrogen bond networks with adjacent residues. One is formed around the carboxyl group of gallic acid, involving residues from the hydrolase fold domain, including residues from the catalytic triad, which consists of Ser206, His485, and Asp439. The other is formed around the three hydroxyl groups of the compound, mainly involving residues from the cap domain, including Gln238, Gln239, His242, and Ser441. Gallic acid binds in a sandwich-like mode through hydrophobic contact with Ile442. It is found that all these residues are highly conserved in fungal and yeast tannases.

[0003] Currently, tannase is mainly applied to treat tannins and proteins in glucose, beer, etc., making them clear and transparent. It can also be used to remove the astringency of persimmons and other products, and for manufacturing instant tea to prevent the turbidity of fermented tea. Its application in the industrial production of gallic acid from Chinese gallnut and tara gallic acid is very limited, especially for the latter.

[0004] There is little research on tannase applied to the hydrolysis of tara tannin. Even in the few studies, the catalytic efficiency of the enzyme is low, the fermentation enzyme activity is low, and the production performance of the constructed strains is not ideal. Summary of the Invention

[0005] The purpose of the present invention is to provide a recombinant strain for the enzymatic preparation of gallic acid and a method for constructing the same, so as to solve the problems in the above background art that there is little research on tannase applied to the hydrolysis of tara tannin. Even in the few studies, the catalytic efficiency of the enzyme is low, the fermentation enzyme activity is low, and the production performance of the constructed strains is not ideal.

[0006] To achieve the above object, the present invention provides the following technical solutions. A recombinant strain for the enzymatic preparation of gallic acid, wherein the amino acid sequence of the tannase is SEQ ID NO.1, the tannase gene is derived from Aspergillus niger CBS 513.88, and the nucleotide sequence is SEQ ID NO.2. Based on the SEQ ID NO.2 sequence, the sequence was optimized according to the codon preference of Aspergillus niger and the restriction enzyme cleavage sites at both ends, and the optimized nucleotide sequence is SEQ ID NO.3.

[0007] Preferably, the host cell of the tannase is a recombinant strain of Aspergillus niger SHBCC D10064 CBS 513.88. SEQ ID NO.3 was digested with restriction enzymes Bsu36I and SmaI, and the plasmid Pan7-Pxyn1-cre was also digested with restriction enzymes Bsu36I and SmaI, and then the two were ligated to construct an expression vector containing the target gene.

[0008] Preferably, the optimized tannase gene was integrated into the Aspergillus niger genome through the Pan7-Pxyn1-cre plasmid to construct a recombinant strain. This expression vector was transformed into the host cell [Aspergillus niger WFCCSW-JZ-001] through protoplasts, and the target recombinant strain (preservation number: CCTCC NO: M2024696) was constructed through screening markers. This culture was received by the China Center for Type Culture Collection on April 15, 2024, and registered. The preservation address is Wuhan University, Wuhan, China, or [Aspergillus niger WFCCSW-JZ-002]. Through screening markers, the target recombinant strain (preservation number: CCTCC NO: M 2024697) was constructed. This culture was received by the China Center for Type Culture Collection on April 15, 2024, and registered. The preservation address is Wuhan University, Wuhan, China.

[0009] Preferably, the optimal reaction temperature of the tannase is 30 to 45 °C, and the optimal reaction pH is 4.0 to 5.5. The recombinant strain was fermented to obtain tannase, and relevant biochemical characteristics and application technologies were studied.

[0010] A method for constructing a recombinant strain for the enzymatic preparation of gallic acid, wherein the preparation of gallic acid using tannase specifically includes the following steps:

[0011] 1) Prepare a turbid solution of tara powder with a tara tannic acid conversion mass concentration of 1%-35% (w / w), and adjust the pH of the reaction solution to 4.0-6.0;

[0012] 2) Add the reaction solution obtained in step 1) to a reaction kettle, add tannase according to the substrate addition amount of 20 - 60 U / g, and hydrolyze to produce gallic acid.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0014] 1. Optimize the tannase gene in Aspergillus niger, improve the performance of catalytic hydrolysis of tara tannin, and construct a gene-integrated recombinant Aspergillus niger strain for the production of tannase. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is a graph of the optimal reaction temperature of the biochemical characteristics of the tannase of the present invention;

[0016] Figure 2 It is a graph of the optimal reaction pH of the biochemical characteristics of the tannase of the present invention;

[0017] Figure 3 It is a graph of the temperature tolerance of the biochemical characteristics of the tannase of the present invention;

[0018] Figure 4 It is a graph of the pH tolerance of the biochemical characteristics of the tannase of the present invention;

[0019] Figure 5 It is a schematic diagram of the plasmid information of Pan7 - Pxyn1 - cre of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0020] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0021] Please refer to Figures 1-5 , an embodiment provided by the present invention:

[0022] Example 1:

[0023] A method for constructing a recombinant strain for the enzymatic preparation of gallic acid:

[0024] Artificial synthesis of the Aspergillus niger tannase gene:

[0025] According to the gene sequence encoding tannase publicly available on NCBI (https: / / www.ncbi.nlm.nih.gov / genome / ) (NCBI Reference Sequence: XM_001401772.1, GeneID: 4990850), with the restriction enzyme cleavage sites Bsu36I and SmaI designed at both ends, Sangon Biotech (Shanghai) Co., Ltd. was commissioned for gene synthesis, and finally SEQ ID NO.2 was obtained.

[0026] Example 2:

[0027] Optimization of the Aspergillus niger tannase gene sequence:

[0028] According to the codon preference of Aspergillus niger, the corresponding nucleotides were optimized and changed, and finally SEQ ID NO.3 was obtained. The alignment with the NO:2 sequence is as follows:

[0029] Example 3 Construction of the tannase expression vector

[0030] The NO.3 sequence was ligated to the Pan7-Pxyn1-cre plasmid to construct a tannase expression vector. The specific process is as follows:

[0031] Enzyme digestion and gel recovery

[0032] The enzyme digestion reaction system is as follows:

[0033]

[0034]

[0035] Electrophoresis detection

[0036] Detection was performed using 1% agarose gel electrophoresis. Add 4 μl of 6X loading Dye to the 50 μl enzyme digestion system, mix well, load 25 μl of the sample into each gel well, and load 2 μl of SM0331 Marker into the adjacent gel well. After electrophoresis for 20 min, place it in an ultraviolet gel imager and compare it with the Marker to confirm the position of the target band size.

[0037] Gel recovery

[0038] 3.1 Gel cutting: Cut the required target fragment gel block with a scalpel in the ultraviolet gel imager and put it into a 2 ml EP tube.

[0039] 3.2 Gel solubilization: Add Buffer B2 at 3 - 6 times the weight of the gel block and solubilize it in a 55°C water bath for 10 min.

[0040] 3.3 Column Chromatography Recovery: Transfer all the completely dissolved gel mixture into a DNA adsorption column, centrifuge at 6000 rpm for 30 sec at high speed, and discard the liquid in the collection tube. Add 600 μl of Wash Solution to the above adsorption column, centrifuge at 7500 rpm for 30 sec, and discard the liquid in the collection tube. (Repeat once) Centrifuge the washed adsorption column and collection tube at 12000 rpm at high speed for 1 min, and replace the collection tube with a clean 1.5 ml EP tube. Open the column lid and place it in a 55°C drying oven for 10 minutes to remove residual ethanol. Add 60 μl of sterilized ddH2O (eluent) to the adsorption membrane, let it stand at room temperature for 2 min, and centrifuge at 12000 rpm at high speed for 1 min. The obtained DNA solution is stored at -20°C or used for subsequent experiments.

[0041] 3.4 Quantification and Preservation: Quantify the obtained product using a spectrophotometer and record the relevant data.

[0042] Ligation and Transformation

[0043] Ligation

[0044] Digest the Pan7-Pxyn1-cre plasmid with SmaI and Bsu36I to generate two different sticky ends, enabling the target gene to be inserted into the vector molecule directionally.

[0045] Ligation Reaction System:

[0046]

[0047]

[0048] Operation Steps:

[0049] First, mix it evenly and centrifuge at low speed for 15 sec, then incubate the sticky ends at 16°C for more than 0.5 h, and finally perform a transformation experiment using competent cells DH5α.

[0050] Transformation

[0051] 2.1 Preparation: Thaw the competent cells DH5α and the ligation product in an ice bath.

[0052] 2.2 Transfer the ligation product: Pipette 10 μl of the ligation product and add it to the dissolved competent cell suspension (gently mix), and place it in an ice bath for 15 min.

[0053] 2.3 Heat Activation: Place the competent cell suspension in a 42°C constant temperature water bath, incubate for 45 sec, and then place it in an ice bath for 5 min.

[0054] 2.4 Bacterial liquid culture: Add 500 μl of LB medium (antibiotic-free) to the competent cells after ice bath, and then place them in a constant temperature shaker at 37°C at 180 rmp for 45 min.

[0055] 2.5 Plate coating: Use a pipette to directly coat 0.1 ml of the transformed bacterial liquid on an LB solid culture plate containing Amp, and invert it in a constant temperature incubator at 37°C overnight.

[0056] 2.6 Check the results: Take out the culture plate the next day and observe the colonies on the culture plate. Pick a single colony and transfer it to an LB liquid medium containing Amp, place it in a constant temperature shaker at 37°C at 180 rmp. After 12 - 16 h, coat it on an LB solid culture plate containing Amp, and invert it in a constant temperature incubator at 37°C overnight (repeat 3 times to extract the plasmid).

[0057] Example 4:

[0058] Construction of recombinant strain containing the target tannase:

[0059] ① Aspergillus niger strain, enzymes and reagents

[0060] Ⅰ Aspergillus niger strain:

[0061] We constructed an Aspergillus niger strain with high expression of tannase using Aspergillus niger SHBCC D10064 CBS 513.88.

[0062] Ⅱ Enzymes and reagents:

[0063] Enzyme solution: 0.5% snail enzyme, 0.5% cellulase

[0064] SMC solution: 5 mmol / L K2HPO4, 5 mmol / L KH2PO4, 0.8 mol / L Mg SO4, pH 5.5

[0065] STC solution: 10 mmol / L Tris-HCl, 50 mmol / L Ca Cl2, 1.2 mol / L sorbitol, pH 7.5

[0066] Plate screening medium (g / L): (1) Sucrose 30, dipotassium hydrogen phosphate 1, magnesium sulfate 0.5, agar 15; (2) Tannic acid 20, bromophenol blue 0.04. Sterilize at 115°C for 20 min, sterilize (1) and (2) separately, and mix them after cooling to 40°C and then pour the plates.

[0067] PEG buffer: 50% (W / V) PEG - 4000, 1 mmol / L Ca Cl2, 10 mmol / L Tris-HCl, pH 7.5.

[0068] YAG medium: 5 g of yeast powder, 20 g of glucose, 1 mL of 1000X trace elements

[0069] 1000X trace elements: 22 g of ZnSO4·7H2O, 11 g of H3BO3, 5 g of MnCl2·4H2O, 5 g of FeSO4·7H2O, 1.6 g of CoCl2·5H2O, 1.6 g of CuSO4, 1.1 g of (NH4)6Mo7O24·4H2O, 50 g of Na-EDTA

[0070] ② Screening steps for tannase highly expressed Aspergillus niger strains

[0071] Ⅰ Obtaining of Aspergillus niger mycelia

[0072] The optimal conditions for the growth of Aspergillus niger mycelia are inoculating 1×108 spores of Aspergillus niger with an inoculation age of 5 d into 100 mL of liquid medium and culturing them in a shaker at 37 °C and 180 r / min.

[0073] Ⅱ Enzymolysis of Aspergillus niger mycelia

[0074] 1) Preparation of enzyme lysate: The complex enzyme system is 0.50% snail enzyme + 0.50% cellulase

[0075] 2) Preparation of Aspergillus niger mycelia: Place filter cloth and fine needle filter paper in a glass funnel, (collect mycelial balls by filtering with eight layers of gauze), then wash the mycelial balls with 200 mL of sterile distilled water, filter to collect fresh Aspergillus niger mycelia, after washing the mycelia with sterile water, rinse them with SMC solution to remove the residual liquid medium.

[0076] 3) Enzymolysis of Aspergillus niger mycelia: Take 1.00 g of fresh mycelia and place them in 10 mL of enzyme lysate, and enzymolyze them at 37 °C and 100 r / min for 3 h.

[0077] Ⅲ Collection of Aspergillus niger protoplasts

[0078] Add 10 mL of an equal volume of STC solution to the protoplast forward lysate, gently pipette up and down 5 times with a large pipette tip to release the protoplasts attached to the mycelia into the solution. Collect the protoplasts with a sterilized double-layer fine needle filter paper into a new sterile centrifuge tube, centrifuge at 10 °C and 3000 r / min for 10 min, and discard the supernatant; resuspend the protoplasts gently with STC, centrifuge and wash 3 times at 10 °C and 3000 r / min, and finally resuspend the precipitate with 200 μL of STC to obtain an Aspergillus niger protoplast suspension. Calculate the protoplast concentration and yield through a hemocytometer for protoplast regeneration and transformation.

[0079] Ⅳ High expression of Aspergillus niger tannase gene mediated by protoplast transformation method and screening of transformants

[0080] Add the following transformation system into a 50 mL centrifuge tube in sequence and flick gently to mix: 100 μL of Aspergillus niger 513.88 protoplast suspension (the number of protoplast cells should reach 1×107 cells / mL); 10 μL of tannase expression plasmid (or the expression plasmid of the optimized tannase DNA sequence) (concentration 10 μg / μL); 25 μL of PEG buffer. After ice-bathing for 30 min, slowly add 1 mL of PEG buffer, mix well, accurately time for 5 min, then add 2 mL of STC solution in sequence. Add the above system onto the tannase screening medium and spread evenly. Incubate upright for 1 h and then incubate upside down in a 37 °C incubator for 3 d to 4 d.

[0081] Transformants are obtained through hygromycin resistance screening. Those with genomic DNA extracted for polyclonal amplification of tannase are positive transformants. At the same time, use the genomic DNA of the original strain as a negative control to detect whether the tannase gene is amplified.

[0082] Example 5:

[0083] Fermentation of the target recombinant strain:

[0084] Fermentation broth formula: (1 liter) 5 g of yeast powder, 20 g of glucose, 20 g of xylose, 1 mL of 1000x trace elements, 1 L of ddH2O.

[0085] Prepare liquid seeds from the target recombinant strain, inoculate them into a fermenter, and ferment according to the above fermentation formula at 37 °C, pH 5.0, and 500 rpm. Start sampling every 24 h to detect enzyme activity at 48 h. The specific fermentation enzyme activity levels are as follows:

[0086]

[0087] Enzyme activity detection method:

[0088] Tannase activity detection kit (ultraviolet spectrophotometry)

[0089] Product trademark: solarbio

[0090] Catalog number: BC4070

[0091] Specification: 50T / 24S

[0092] Kit composition

[0093]

[0094] Solution preparation:

[0095] 1. Reagent 1: Take 1 vial before use, add 1.5 mL of absolute ethanol and mix well to dissolve. The unused reagent can be stored at 2 - 8 °C for one week.

[0096] 2. Standard: 5 mg of propyl gallate. Before use, add 1.178 mL of absolute ethanol and mix well to dissolve, preparing a standard solution of 20 μmol / mL. Store at 2 - 8 °C for two weeks.

[0097] II. Principle of Detection Method

[0098] Use propyl gallate (PG) as the substrate for the enzymatic reaction of tannase. It has a characteristic absorption peak at 270 nm. Calculate the tannase activity based on the change in absorbance before and after the reaction. The amount of enzyme that reduces 1 nmol of PG per minute in the reaction system is defined as one enzyme activity unit (U).

[0099] III. Detection Instruments and Equipment

[0100] UV - visible spectrophotometer, low - temperature centrifuge, water bath, adjustable pipette, micro - quartz cuvette.

[0101] IV. Determination of Enzyme Activity in Fermentation Broth

[0102] I) Sample Preparation:

[0103] Enzyme solution preparation: Take 1 mL of fermentation broth and place it in a 1.5 mL Eppendorf tube. Centrifuge at 10000 rpm for 10 min at 4 °C. Take the supernatant and place it on ice for further measurement.

[0104] II) Determination Steps:

[0105] (1) Pre - heat the UV - visible spectrophotometer for 30 min and adjust the wavelength to 270 nm. Zero with distilled water.

[0106] (2) Dilution of the standard solution: Take 10 μL of the 20 μmol / mL standard solution, add 3990 μL of the extraction solution, mix well, and prepare a standard solution of 0.05 μmol / mL for use. Prepare it freshly before use.

[0107] (3) Treatment of the control tube sample: Pipette 0.1 mL of the fermentation broth supernatant into a 1.5 mL Eppendorf tube, place it in a boiling water bath for 5 min, cool to room temperature, and measure it as a control.

[0108] (4) Sampling table: Add the following reagents into 1.5 mL Eppendorf tubes respectively

[0109]

[0110]

[0111] After reacting in a water bath at 40 °C for 10 min, immediately place it in a boiling water bath for 5 min, cool to room temperature, centrifuge at 10000 rpm for 10 min at room temperature, and take the supernatant for measurement.

[0112] Add the following reagents into the micro - quartz cuvette respectively

[0113]

[0114] After thorough mixing, measure the absorbance at 270 nm, denoted as A of the test tube and A of the control tube respectively, and calculate ΔA = A of the control tube - A of the test tube.

[0115] (III) Calculation of tannase activity

[0116] Tannase (U / mL) = ΔA ÷ (A of the standard ÷ C of the standard) × F × 1000 × V of the enzymatic reaction ÷ T ÷ V of the sample = (A of the control tube - A of the test tube) ÷ A of the standard × 166.67

[0117] ΔA: = A of the control tube - A of the test tube

[0118] A of the standard: Absorbance of the 0.05 μmol / mL standard solution

[0119] C of the standard: Concentration of the standard solution, 0.05 μmol / mL

[0120] F: Dilution factor of the supernatant, and the dilution factor is 20

[0121] 1000: 1 μmol = 1000 nmol

[0122] V of the enzymatic reaction: Total volume of the enzymatic reaction, 1 mL

[0123] T: Reaction time, 10 min

[0124] V of the sample: Volume of the sample added, 0.1 mL

[0125] Example 6:

[0126] Study on the enzymatic properties of the target tannase:

[0127] Experimental method

[0128] (1) Optimum temperature

[0129] Define the tannase activity measured at 35 °C as 100% enzyme activity. By adjusting the temperature of the water bath to 30 °C, 35 °C, 40 °C, 50 °C, 60 °C, 70 °C, 80 °C to change the temperature during the enzyme reaction, investigate the effect of measurement under different temperature conditions on the enzyme activity. Set two parallel samples for each group.

[0130] (2) Optimum pH value

[0131] The tannase activity measured under the condition of reaction pH value of 5 was defined as 100% enzyme activity. The crude enzyme was diluted with citrate-phosphate buffer solutions with pH values of 3.5, 4.5, 5.5, 6.5, and 7.5 respectively to change the enzyme reaction pH value. The preparation methods of each pH buffer solution are shown in Appendix 2 of this article. The effects of different pH conditions on the enzyme activity were investigated. Two parallel samples were set for each group.

[0132] (3) Temperature tolerance

[0133] The initial enzyme activity of the fermentation broth was defined as 100% enzyme activity. The fermentation broth was stored in a water bath at 30 °C, 40 °C, 50 °C, 60 °C, 70 °C, and 80 °C for 30 min respectively, and then the enzyme activity was detected to investigate the temperature tolerance of tannase. Two parallel samples were set for each group.

[0134] (4) pH tolerance

[0135] The initial enzyme activity of the fermentation broth was defined as 100% enzyme activity. The fermentation broth was stored in buffer solutions with pH values of 3, 4, 5, 6, 7, 8, and 9 for 30 min respectively, and then the enzyme activity was detected. The preparation methods of each pH buffer solution are shown in Appendix 2 of this article. The pH tolerance of tannase was investigated. Two parallel samples were set for each group.

[0136] Example 7:

[0137] Hydrolysis application of tannase in tara tannin:

[0138] 1) Start stirring, add 600 L of process water and 400 kg of tara powder into a clean reaction kettle to prepare a turbid solution of tara powder with a mass concentration of 20% (w / w) of tara tannic acid (the tannic acid content in commercial tara powder is 50%), and adjust the pH of the reaction solution to 5.0;

[0139] 2) Start the heating / cooling system of the reaction kettle, adjust the temperature in the kettle to 35 °C, and add tannase according to the substrate addition amount of 36 U / g;

[0140] 3) After reacting at 35 °C for 8 h, raise the temperature to 80 °C - 90 °C, add 8 kg of activated carbon, and keep the temperature for decolorization for 1 h;

[0141] 4) After decolorization, pump the reaction solution into a plate and frame filter for pressure filtration, and transfer the filtrate into a freezing crystallization tank;

[0142] 5) After cooling the filtrate to 20 °C, keep the temperature for crystallization for 4 h, transfer the filtrate into a plate centrifuge to remove the mother liquor, and obtain wet gallic acid;

[0143] 6) Send the wet product into a vacuum double-cone dryer for drying;

[0144] 7) The obtained gallic acid product has a mass of 192.6 kg, a moisture content of 0.43%, a content of 99.8%, and a chromaticity < 80 Hazen;

[0145] 8) The yield (on dry basis) = mass of gallic acid × (1 - moisture content) ÷ mass of tara powder = 192.6 × (1 - 0.0043) ÷ 400 = 47.94%.

[0146] For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and can be implemented in other specific forms without departing from the spirit or basic characteristics of the present invention. Therefore, from any point of view, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be embraced by the present invention. Any reference signs in the claims should not be construed as limiting the claimed claim.

Claims

1. A recombinant strain for preparing gallic acid by enzymatic method, characterized in that: The invention comprises tannase: the amino acid sequence of the tannase is SEQ ID NO.1, the tannase gene is derived from Aspergillus niger CBS 513.88, the nucleotide sequence is SEQ ID NO.2, based on the sequence of SEQ ID NO.2, the sequence is optimized according to the codon preference of Aspergillus niger and the restriction enzyme sites at both ends, the optimized nucleotide sequence is SEQ ID NO.3, SEQ ID NO:3 is digested by Bsu36I and SmaI restriction enzymes, the plasmid Pan7-Pxyn1-cre is also digested by Bsu36I and SmaI restriction enzymes, the two are connected, an expression vector containing the target gene is constructed, and the expression vector is transformed into a host cell [Aspergillus niger CBS 513.88 (product name: Aspergillus niger SHBCC D10064 CBS 513.88), and the target recombinant strain (Accession No.: CCTCC M2024697) was constructed by screening markers. The recombinant strain was fermented to obtain tannase.

2. The recombinant strain for preparing gallic acid by enzymatic method according to claim 1, characterized in that: The host cell of the tannase is a recombinant strain of Aspergillus niger SHBCC D10064 CBS 513.

88.

3. The recombinant strain for preparing gallic acid by enzymatic method according to claim 2, characterized in that: The optimized tannase gene is integrated into the Aspergillus niger genome via the Pan7-Pxyn1-cre plasmid to construct a recombinant strain.

4. The recombinant strain for preparing gallic acid by enzymatic method according to claim 1, characterized in that: The optimal reaction temperature of the tannase is 30 to 45°C, and the optimal reaction pH is 4.0 to 5.

5.

5. A method for constructing a recombinant strain for preparing gallic acid by enzymatic method, characterized in that: The method for preparing gallic acid by utilizing tannase specifically comprises the following steps: 1) preparing a turbid solution of Tara powder having a Tara tannic acid conversion mass concentration of 1%-35% (w / w), and adjusting the pH of the reaction solution to 4.0-6.0; 2) The reaction solution obtained in step 1) is added to a reaction kettle, and tannase is added at a substrate addition amount of 20-60 U / g to hydrolyze and generate gallic acid.

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