Protein with tannase activity and related biological material and application thereof

Abstract

The invention provides a protein with tannase activity as well as a related biological material and application thereof. The protein provided by the invention is the protein at the 19th-584th site of the amino acid sequence of SEQ ID NO: 1, has tannase activity, is excellent in enzymatic property, can rapidly catalyze and degrade tannin of different types, and has the characteristic of wide substrate specificity; in addition, the protein provided by the invention also has the characteristics of good cold adaptability and strong acid resistance, and can stably play a catalytic degradation role in a wide temperature and pH range. The protein with tannase activity provided by the invention has great application potential in the industries of food, feed and the like.

Description

Technical Field

[0001] The invention relates to the technical field of enzyme engineering, and in particular to a protein with tannase activity and related biological materials and applications thereof. Background Art

[0002] Tannins are a class of natural polyphenol compounds widely found in the plant kingdom, which give foods such as tea, fruit and wine their unique flavor and astringency. High levels of tannins can form insoluble complexes with proteins and other nutrients, affecting the nutritional value and digestibility of foods, and can also increase the astringency of foods, affecting taste and commercial applications. Tannase (EC 3.1.1.20) can effectively hydrolyze ester bonds and phenol bonds in tannins to generate gallic acid and glucose or other phenolic compounds, thereby improving the quality of tannin-containing products. Therefore, the research and application of tannase has become a hot topic in the fields of enzyme engineering and food technology.

[0003] In order to achieve large-scale application of tannase, various microbial hosts have been reported to express tannase heterologously, including Escherichia coli, Saccharomyces cerevisiae, and some fungal expression systems (Lekshmi et al., Environmental Research, 2021, 201, 111625). These microbial hosts are widely used because of their rapid reproduction, easy genetic manipulation, and high-density fermentation. In recent years, Aspergillus niger has gradually been used for heterologous expression of tannase due to its excellent performance in exogenous protein secretion and industrial fermentation, and has shown the potential to increase yield and enzyme activity stability. For example, Lekshmi et al. introduced the tannase encoding gene into Aspergillus niger, which effectively increased the yield of tannase and showed good enzyme activity and secretion efficiency. In addition, Aspergillus niger not only has a high exogenous protein secretion ability, but also has stability in high-density fermentation, which is suitable for large-scale industrial production. These studies show that Aspergillus niger, as a heterologous expression host of tannase, can show advantages in improving enzyme production and secretion efficiency. Especially in industrial applications, its stable fermentation characteristics provide feasibility for large-scale production of tannase.

[0004] Tannase has shown broad application prospects in many fields. In the food industry, tannase is used to treat tannins in wine to improve taste and clarity; in tea processing, tannase helps reduce the tannin content in tea, thereby reducing astringency and improving tea quality; in feed processing, tannase can degrade tannins in feed and improve feed utilization; in leather manufacturing, tannase helps soften leather. Chinese invention patent with publication number CN101481714A discloses a method for preparing ellagic acid by enzymatic method using pomegranate peel as raw material, wherein pomegranate peel is used as substrate, and crude ellagic acid is obtained through enzymatic reaction of tannase and ellagic tannin, and then pure ellagic acid is obtained; Chinese invention patent with publication number CN101701234A discloses a method for extracting ellagic acid from pomegranate peel residue by using biological enzyme, wherein the pomegranate peel residue is treated with biological composite enzyme (cellulose A, cellulase B and hydrolase), and the ellagic acid extraction rate reaches 12.69-24%; Chinese invention patent with publication number CN116121080A discloses an Aspergillus aculeatus and its application in the production of tannase and gallic acid, wherein Aspergillus aculeatus is used and pomegranate peel is used as fermentation medium to efficiently produce tannase (2.14U / mL) and gallic acid (yield 1.7mg / mL). However, tannases still have problems such as low enzyme activity, poor substrate specificity and single product in the application process. Therefore, it is very important to discover tannases with high catalytic efficiency and wide application range. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a tannase with high catalytic efficiency and wide application range.

[0006] In a first aspect, the present invention provides a protein, wherein the protein is any one of the following A1)-A5):

[0007] A1) a protein whose amino acid sequence includes positions 19-584 of SEQ ID NO: 1;

[0008] A2) a fusion protein obtained by fusing a signal peptide to the amino terminus of the protein shown in A1);

[0009] A3) a protein having tannase activity obtained by substituting and / or deleting and / or adding amino acid residues of any of the proteins shown in A1) to A2);

[0010] A4) a protein having 80% or more identity with any of the proteins shown in A1) to A2) and having tannase activity;

[0011] A5) A fusion protein obtained by fusing a protein tag to the carboxyl terminal and / or amino terminal of any of the proteins shown in A1) to A2).

[0012] Among the above proteins, the amino acid sequence of the protein described in A1) includes positions 19 to 584 of SEQ ID NO: 1. SEQ ID NO: 1 includes 584 amino acids in total. The amino acid sequence of SEQ ID NO: 1 is specifically as follows:

[0013] .

[0014] Furthermore, the amino acid sequence of the protein described in A1) is positions 19-584 of SEQ ID NO:1.

[0015] In the above protein, the signal peptide described in A2) can be selected from one of the following: saccharifying enzyme pglaa signal peptide, glucose oxidase gox signal peptide, amylase amya signal peptide, phytase phya signal peptide, and acid phosphatase phyb signal peptide. Specifically, the amino acid sequence of the saccharifying enzyme pglaa signal peptide is SEQ ID NO: 1, positions 1 to 18; the amino acid sequence of the glucose oxidase gox signal peptide is SEQ ID NO: 2; the amino acid sequence of the amylase amya signal peptide is SEQ ID NO: 3; the amino acid sequence of the phytase phya signal peptide is SEQ ID NO: 4; and the amino acid sequence of the acid phosphatase phyb signal peptide is SEQ ID NO: 5.

[0016] SEQ ID NO:2:MQTLLVSSLVVSLAAALPHYIR;

[0017] SEQ ID NO:3: MMVAWWSLFLYGLQVAAPALA;

[0018] SEQ ID NO:4: MGVSAVLLPLYLLSGVTSGLA;

[0019] SEQ ID NO: 5: MPRTSLLTLACALATGASA.

[0020] Among the above proteins, the fusion protein described in A2) is at least one selected from potanA-pglaaS, potanA-goxS, potanA-amyaS, potanA-phyaS or potanA-phybS;

[0021] The potanA-pglaaS is a protein whose amino acid sequence includes SEQ ID NO: 1;

[0022] The potanA-goxS is a protein obtained by replacing the amino acid residues at positions 1-18 of SEQ ID NO: 1 with SEQ ID NO: 2, while keeping the other amino acid residues of SEQ ID NO: 1 unchanged; that is, in potanA-goxS, the amino acid residue (R) at position 22 of SEQ ID NO: 2 and the amino acid residue (S) at position 19 of SEQ ID NO: 1 are connected, and the glucose oxidase gox signal peptide is located at the N-terminus of the protein with an amino acid sequence of positions 19-584 of SEQ ID NO: 1.

[0023] The potanA-amyaS is a protein obtained by replacing the amino acid residues at positions 1-18 of SEQ ID NO:1 with SEQ ID NO:3, while keeping the other amino acid residues of SEQ ID NO:1 unchanged; that is, in potanA-amyaS, the amino acid residue (A) at position 21 of SEQ ID NO:3 and the amino acid residue (S) at position 19 of SEQ ID NO:1 are connected, and the amylase amya signal peptide is located at the N-terminus of the protein whose amino acid sequence is positions 19-584 of SEQ ID NO:1.

[0024] The potanA-phyaS is a protein obtained by replacing the amino acid residues at positions 1 to 18 of SEQ ID NO: 1 with SEQ ID NO: 4, while keeping the other amino acid residues of SEQ ID NO: 1 unchanged; that is, in potanA-phyaS, the amino acid residue (A) at position 21 of SEQ ID NO: 4 and the amino acid residue (S) at position 19 of SEQ ID NO: 1 are connected, and the phytase phya signal peptide is located at the N-terminus of the protein whose amino acid sequence is positions 19 to 584 of SEQ ID NO: 1.

[0025] The potanA-phybS is a protein obtained by replacing the amino acid residues at positions 1-18 of SEQ ID NO:1 with SEQ ID NO:5, while keeping the other amino acid residues of SEQ ID NO:1 unchanged; that is, in potanA-phyb, the amino acid residue (A) at position 19 of SEQ ID NO:5 and the amino acid residue (S) at position 19 of SEQ ID NO:1 are connected, and the acid phosphatase phyb signal peptide is located at the N-terminus of the protein with the amino acid sequence of positions 19-584 of SEQ ID NO:1.

[0026] In the above-mentioned proteins, identity refers to the identity of the amino acid sequence. The identity of the amino acid sequence can be determined using a homology search site on the Internet, such as the BLAST page on the NCBI homepage website. For example, in Advanced BLAST2.1, by using blastp as a program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as a Matrix, setting the Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively, and searching for the identity of a pair of amino acid sequences, the identity value (%) can be obtained.

[0027] In the above proteins, the above 80% identity can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.

[0028] Further, in A4), the protein has at least 90% identity with the protein shown in any one of A1)-A2). Further, in A4), the protein has at least 95% identity with the protein shown in any one of A1)-A2). Further, in A4), the protein has at least 99% identity with the protein shown in any one of A1)-A2).

[0029] In the above proteins, the protein tag refers to a polypeptide or protein that is fused and expressed with the target protein using DNA in vitro recombination technology to facilitate the expression, detection, tracing and / or purification of the target protein. The protein tag can be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag and / or a SUMO tag, etc.

[0030] Experiments have shown that the protein provided by the invention has tannase activity. The temperature of the tannase enzymatic reaction of the protein is 10-60° C. and the pH is 2-8.

[0031] Furthermore, the protein is potanA-goxS, and potanA-goxS has the highest enzyme activity of tannase at 45° C., that is, the optimum temperature of the tannase enzyme reaction of potanA-goxS is 45° C. potanA-goxS has the highest enzyme activity of tannase at pH 6.0, that is, the optimum pH of the tannase enzyme reaction of potanA-goxS is 6.0.

[0032] In a second aspect, the present invention provides a biomaterial related to any of the above-mentioned proteins, wherein the biomaterial is any one of the following B1)-B8):

[0033] B1) a nucleic acid molecule encoding the protein provided by the first aspect of the present invention;

[0034] B2) an expression cassette containing the nucleic acid molecule described in B1);

[0035] B3) a recombinant vector containing the nucleic acid molecule described in B1);

[0036] B4) a recombinant vector containing the expression cassette described in B2);

[0037] B5) a recombinant microorganism containing the nucleic acid molecule described in B1);

[0038] B6) a recombinant microorganism containing the expression cassette described in B2);

[0039] B7) a recombinant microorganism containing the recombinant vector described in B3);

[0040] B8) A recombinant microorganism containing the recombinant vector described in B4).

[0041] In the above biological material, the nucleic acid molecule described in B1) is a linear DNA molecule, specifically any one of the DNAs shown in B11)-B17) below:

[0042] B11) a DNA molecule whose coding sequence is SEQ ID NO: 6;

[0043] B12) the coding sequence is a DNA molecule formed by connecting the 54th nucleotide of SEQ ID NO:7 and the 1st nucleotide of SEQ ID NO:6;

[0044] B13) the coding sequence is a DNA molecule formed by connecting the 66th nucleotide of SEQ ID NO:8 and the 1st nucleotide of SEQ ID NO:6;

[0045] B14) the coding sequence is a DNA molecule formed by connecting the 63rd nucleotide of SEQ ID NO:9 and the 1st nucleotide of SEQ ID NO:6;

[0046] B15) the coding sequence is a DNA molecule formed by connecting the 63rd nucleotide of SEQ ID NO:10 and the 1st nucleotide of SEQ ID NO:6;

[0047] B16) the coding sequence is a DNA molecule formed by connecting the 57th nucleotide of SEQ ID NO:11 and the 1st nucleotide of SEQ ID NO:6;

[0048] B17) A DNA molecule that has more than 80% identity with the DNA molecule described in any one of B11 to B16) and encodes the same protein.

[0049] In the above biological materials, the DNA molecule shown in B11) encodes an amino acid sequence of positions 19-584 of SEQ ID NO:1, named potanA; the DNA molecule shown in B12) encodes a protein with an amino acid sequence of SEQ ID NO:1, namely potanA-pglaaS; the DNA molecule shown in B13) encodes a protein with an amino acid sequence of SEQ ID NO:1 with amino acid residues 1-18 replaced by SEQ ID NO:2, namely potanA-goxS; the DNA molecule shown in B14) encodes a protein with amino acid sequence of SEQ ID NO:1 with amino acid residues 1-18 replaced by SEQ ID NO:3, namely potanA-amyaS; the DNA molecule shown in B15) encodes a protein with amino acid sequence of SEQ ID NO:1 with amino acid residues 1-18 replaced by SEQ ID NO:4, namely potanA-phyaS; the DNA molecule shown in B16) encodes a protein with amino acid sequence of SEQ ID NO:1 with amino acid residues 1-18 replaced by SEQ ID NO:5, namely potanA-phybS.

[0050] In the above biological material, the expression cassette containing the nucleic acid molecule described in B1) described in B2) refers to a DNA capable of expressing the tannase in a host cell. The expression cassette may also include a regulatory sequence necessary for expressing the tannase coding sequence. The regulatory sequence at least includes a promoter and termination signals for transcription and translation.

[0051] Furthermore, the promoter is a strong glaA promoter to increase the expression level of tannase.

[0052] Among the above biological materials, the recombinant vector described in B3)-B4) is obtained by connecting the saccharifying enzyme promoter PglaA, the signal peptide encoding gene, the tannase encoding gene, the tryptophan synthase gene terminator TtrpC, the PgpdA promoter, the pyrG screening marker gene, and the saccharifying enzyme terminator TglaA gene in sequence, replacing the fragment between the 3' end of the M13R site (AGCTCAGAATTAACCCT) and the M13F site (ACTGGCCGTCGTTTTAC) of the pEASY-Blunt plasmid, and introducing a restriction site at the 5' end of the saccharifying enzyme promoter PglaA, while keeping the other parts of the sequence of the pEASY-Blunt plasmid unchanged.

[0053] In the above biological materials, the recombinant microorganisms described in B5)-B8) can be obtained by introducing the coding gene of the protein into a receptor microorganism to obtain a recombinant microorganism expressing the protein. Further, the receptor microorganism is Aspergillus niger, and the saccharifying enzyme gene site of the strain is a transcriptionally active site, which helps to increase the expression of tannase.

[0054] In the above biological materials, the recombinant microorganisms described in B5) to B8) can specifically be recombinant Aspergillus niger expressing the protein provided by the first aspect of the present invention obtained by introducing the recombinant vector into Aspergillus niger.

[0055] In a third aspect, the present invention provides a method for preparing tannase, comprising: expressing the coding gene of the protein provided in the first aspect of the present invention in an organism to obtain tannase; the organism is a microorganism, a plant or a non-human animal.

[0056] In the above method, the microorganism may be Aspergillus niger.

[0057] In the above method, expressing the protein encoding gene in a microorganism comprises introducing the protein encoding gene into a recipient microorganism to obtain a recombinant microorganism expressing the protein, and culturing the recombinant microorganism to express the protein.

[0058] In the above method, the cultivation of the recombinant microorganism specifically includes seed liquid cultivation and fermentation cultivation. The culture medium used in the seed liquid cultivation stage can be DPY culture medium, the culture temperature is 30-36°C, the rotation speed is 200-220rpm, and the culture time is 110-130h to obtain seed liquid; the cultured seed liquid is inoculated into the fermentation medium according to a certain volume ratio for scale fermentation, and the fermentation medium includes nutrients required for the growth of recombinant microorganisms, such as corn starch hydrolysate, defatted soybean powder, corn steep liquor and ammonium sulfate, etc. The fermentation temperature is controlled at 30-36°C, the rotation speed is 200-300rpm, and the pH of the fermentation medium is controlled to be 5.0-5.5; after the starch hydrolysate in the fermentation medium is completely consumed, the corn starch hydrolysate can be continuously supplemented for the growth of the recombinant microorganism. During the entire fermentation process, the dissolved oxygen level is maintained at a level of ≥10%.

[0059] In a fourth aspect, the present invention provides use of the protein provided in the first aspect as tannase.

[0060] In a fifth aspect, the present invention provides use of the protein provided in the first aspect or the biomaterial provided in the second aspect in the preparation of a tannase preparation.

[0061] In a sixth aspect, the present invention provides a method for enzymatically hydrolyzing tannins in a product, comprising: mixing a product containing tannins with the protein described in the first aspect, and performing an enzymatic reaction so that the protein hydrolyzes the tannins in the product.

[0062] In the above method, the product is selected from at least one of food and feed. The food may be persimmon, tea and other foods rich in tannin.

[0063] In the above method, the pH of the enzymatic hydrolysis reaction system may be 2-8, specifically 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8 or a range between any two of the above point values.

[0064] In the above method, the temperature of the enzymatic hydrolysis reaction system is 10-60°C, specifically 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C or a range between any two of the above point values.

[0065] In a seventh aspect, the present invention provides a method for preparing garnet forest by an enzymatic method using pomegranate peel as raw material, wherein pomegranate peel or a pomegranate peel extract is mixed with the protein described in the first aspect above, and an enzymatic hydrolysis reaction is performed to obtain the garnet forest.

[0066] In the above method, the pomegranate peel extract can be an acid extract of pomegranate peel, for example, obtained by crushing the pomegranate peel into powder and mixing the powder with a citrate buffer solution.

[0067] In the above method, the pH of the enzymatic hydrolysis reaction system may be 2-8, specifically 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8 or a range between any two of the above point values.

[0068] In the above method, the temperature of the enzymatic hydrolysis reaction system is 10-60°C, specifically 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C or a range between any two of the above point values.

[0069] The protein provided by the present invention has tannase activity and excellent enzymatic properties, can rapidly catalyze and degrade different types of tannins, and has the characteristics of wide substrate specificity; in addition, the protein provided by the present invention also has the characteristics of good cold adaptability and strong acid resistance, and can stably exert catalytic degradation in a wide range of temperature and pH. The protein with tannase activity provided by the present invention has great application potential in the food, feed and other industries. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1This is a diagram showing the process of tannase production by high-density fermentation of recombinant Aspergillus niger;

[0071] Figure 2 This is the electrophoresis diagram of tannase purification, where M is the protein standard, lane 1 is the crude enzyme solution, and lane 2 is the pure enzyme;

[0072] Figure 3 The optimum pH determination curve of tannase; wherein, ● represents glycine phosphate buffer with a pH of 2.0-4.0, ▲ represents acetate buffer with a pH of 3.0-6.5, ■ represents MES buffer with a pH of 6.5-7.5, □ represents citric acid buffer with a pH of 3.0-6.5, and ○ represents Tris hydrochloric acid buffer with a pH of 7.0-8.0;

[0073] Figure 4 The pH stability determination curve of tannase; wherein, ● represents glycine phosphate buffer with a pH of 2.0-4.0, ▲ represents acetate buffer with a pH of 3.0-6.5, ■ represents MES buffer with a pH of 6.5-7.5, □ represents citric acid buffer with a pH of 3.0-6.5, and ○ represents Tris hydrochloric acid buffer with a pH of 7.0-8.0;

[0074] Figure 5 This is the optimum temperature determination curve of tannase;

[0075] Figure 6 This is a curve diagram for the temperature stability determination of tannase. DETAILED DESCRIPTION

[0076] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings in the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments, and they should not be understood as limitations on the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. In the description of the present invention, it should be understood that the terms used are only for descriptive purposes and cannot be understood as indicating or implying relative importance.

[0077] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0078] The method for determining tannase activity in the following examples is as follows:

[0079] 250 μL of 100 mM citrate buffer (pH 6.0) prepared 10 mM propyl gallate as substrate, add 250 μL of diluted enzyme solution, and react at 45°C for 5 min. After the reaction, add 300 μL of methanol solution of rhodanine (the mass volume concentration of rhodanine is 0.667 m / V%) to terminate the reaction, shake evenly and stabilize in a 45°C water bath for 5 min, then add 200 μL of KOH (700 mmol / L) solution for color reaction, shake evenly and stabilize in a 45°C water bath for 5 min, finally add 4 mL of deionized water, and stabilize in a 40°C water bath for 10 min, then measure the OD520 value, and calculate the amount of gallic acid generated.

[0080] Definition of enzyme activity unit: Under the conditions of pH 6.0 and temperature 45°C, the amount of enzyme required to hydrolyze propyl gallate to produce 1 μmol of gallic acid per minute is 1 enzyme activity unit (U).

[0081] Example 1 Cloning and expression of tannase encoding gene

[0082] 1. Cloning of the gene encoding tannase

[0083] The cDNA of Penicillium oxalicum was extracted, and the cDNA of Penicillium oxalicum was used as a template to perform PCR amplification using an upstream primer, a downstream primer, and Fast Pfu DNA polymerase (purchased from Takara). The nucleotide sequence of the upstream primer was SEQ ID NO: 12, and the nucleotide sequence of the downstream primer was SEQ ID NO: 13. The PCR reaction procedure was as follows: 95°C pre-denaturation for 3 min; 95°C denaturation for 20 s, 55°C annealing for 20 s, 72°C extension for 30 s, and 34 cycles; 72°C extension for 5 min; 12°C incubation for 10 min. The PCR product was purified and recovered using a 1% agarose gel recovery kit.

[0084] Upstream primer (SEQ ID NO: 12): 5′-AGCATCATTACACCTCAGCAAGCGTCACTTCTGCAACGTCCCT-3′;

[0085] Downstream primer (SEQ ID NO: 13): 5′-TTTCAGTAACGTTAAGTTTAGTATACTGGGATTGGCACACCG-3′.

[0086] The nucleotide sequence of the amplified tannase encoding gene is shown in SEQ ID NO: 6 and is named PoTanA.

[0087] 2. Construction of recombinant plasmid containing tannase encoding gene

[0088] 2.1. Using the Gibson assembly method, the glucoamylase promoter PglaA, the glucoamylase pglaa signal peptide coding sequence, the tannase coding gene PoTanA, the tryptophan synthase gene terminator TtrpC, the PgpdA promoter, the pyrG selection marker gene, the glucoamylase terminator TglaA gene and the pEASY-Blunt plasmid ( -Blunt cloning kit, Beijing Quanshijin Biotechnology Co., Ltd.) backbone was assembled and connected, and a restriction site was introduced at the 5' end of the saccharifying enzyme promoter PglaA to construct a recombinant plasmid Blunt-pyrG-pgpdA-potanA-pglaaS. The nucleotide sequence of the recombinant plasmid Blunt-pyrG-pgpdA-potanA-pglaaS is shown in SEQ ID As shown in NO:14, wherein positions 205-221 are M13R sites, positions 235-251 are exogenously introduced exogenous sequences containing NOTI restriction endonuclease recognition sites, positions 252-1251 are saccharifying enzyme promoter PglaA, positions 1252-1305 are saccharifying enzyme signal peptide pglaaS encoding gene, positions 1306-2986 are tannase encoding gene PoTanA, positions 2987-3757 are tryptophan synthase gene terminator TtrpC, positions 3758-4837 are PgpdA promoter, positions 4838-5749 are pyrG screening marker gene, positions 5750-6836 are saccharifying enzyme terminator TglaA, positions 6837-6853 are M13F recognition sites, and the rest are sequences of the pEASY-Blunt plasmid backbone.

[0089] The recombinant plasmid Blunt-pyrG-pgpdA-potanA-pglaaS was transformed into E. coli Trans5α competent cells. Positive transformants were screened on LB plates containing 50 μg / mL kanamycin. After overnight culture, monoclonal colonies were picked, and colony PCR amplification was performed using primers YZ-Pgla-F and YZ-TtrpC-R, and sequencing was performed. The nucleotide sequence of YZ-Pgla-F is shown in SEQ ID NO:15, and the nucleotide sequence of YZ-TtrpC-R is shown in SEQ ID NO:16. The plasmid was extracted from the transformant with the correct sequencing result and named Blunt-pyrG-pgpdA-potanA-pglaaS.

[0090] YZ-Pgla-F: 5′-CTGAAGTGCTTCCTCCCTTTTAGACGC-3′;

[0091] YZ-TtrpC-R: 5′-TGGACAAATGAACGTATCTTATCGAGATCCT-3′.

[0092] 2.2. The saccharifying enzyme pglaa signal peptide encoding gene in the recombinant plasmid Blunt-pyrG-pgpdA-potanA-pglaaS was replaced with the glucose oxidase gox signal peptide encoding gene (SEQ ID NO: 8), the amylase amya signal peptide encoding gene (SEQ ID NO: 9), the phytase phya signal peptide encoding gene (SEQ ID NO: 10) or the acid phosphatase phyb signal peptide encoding gene (SEQ ID NO: 11), and the assembly method was the same as the above steps. The recombinant plasmids were named Blunt-pyrG-pgpdA-potanA-goxS, Blunt-pyrG-pgpdA-potanA-amyaS, Blunt-pyrG-pgpdA-potanA-phyaS and Blunt-pyrG-pgpdA-potanA-phybS, respectively.

[0093] Blunt-pyrG-pgpdA-potanA-goxS is obtained by replacing the signal peptide pglaA coding gene of the recombinant plasmid Blunt-pyrG-pgpdA-potanA-pglaas with the DNA molecule shown in SEQ ID NO: 8, while keeping other sequences on the recombinant plasmid Blunt-pyrG-pgpdA-potanA-pglaas unchanged. Blunt-pyrG-pgpdA-potanA-amyaS is obtained by replacing the signal peptide pglaA coding sequence of the recombinant plasmid Blunt-pyrG-pgpdA-potanA-pglaas with the DNA molecule shown in SEQ ID NO: 9, while keeping other sequences on the recombinant plasmid Blunt-pyrG-pgpdA-potanA-pglaas unchanged. Blunt-pyrG-pgpdA-potanA-phyaS is obtained by replacing the signal peptide pglaA coding sequence of the recombinant plasmid Blunt-pyrG-pgpdA-potanA-pglaas with the DNA molecule shown in SEQ ID NO: 10, while keeping other sequences on the recombinant plasmid Blunt-pyrG-pgpdA-potanA-pglaas unchanged. Blunt-pyrG-pgpdA-potanA-phybS is obtained by replacing the signal peptide pglaA coding sequence of the recombinant plasmid Blunt-pyrG-pgpdA-potanA-pglaas with the DNA molecule shown in SEQ ID NO: 11, while keeping other sequences on the recombinant plasmid Blunt-pyrG-pgpdA-potanA-pglaas unchanged.

[0094] 3. Aspergillus niger protoplast transformation and recombinant bacteria screening

[0095] The above five recombinant plasmids were transformed into Aspergillus niger protoplasts. Aspergillus niger is described in the literature: Wang, S., Zhang, P., Xue, Y., Yan, Q., Li, X., & Jiang, Z. (2021). Characterization of a novel aspartic protease from Rhizomucor miehei expressed in Aspergillus niger and its application in production of ACE-inhibitory peptides. Foods, 10 (12), 2949. The colonies on the transformation plate were picked up and streaked on potato agar medium for subculture. The single colonies obtained by isolation and culture were picked up with a toothpick and 1 cm 2 , inoculated into 30 mL shake flask fermentation medium, fermented at 34 ° C, 220 rpm for 120 h. After the fermentation, the bacteria were collected and the fermentation supernatant was determined to determine the tannase activity. The results are shown in Table 1.

[0096] Table 1 Comparison of enzyme activities of different recombinant strains

[0097] Name of plasmid in recombinant bacteria Features and applications Enzyme activity (U / mL) Blunt-pyrG-pgpdA-potanA-pglaaS Glucoamylase pglaA signal peptide 30.1 Blunt-pyrG-pgpdA-potanA-goxS Glucose oxidase gox signal peptide 90.6 Blunt-pyrG-pgpdA-potanA-amyaS Amylase amya signal peptide 60.3 Blunt-pyrG-pgpdA-potanA-phyaS Phytase phya signal peptide 26.3 Blunt-pyrG-pgpdA-potanA-phybS Acid phosphatase phyB signal peptide 66.5

[0098] Example 2 Tannase production by high-density fermentation of Aspergillus niger

[0099] According to the enzyme activity determination results shown in Table 1, recombinant bacteria with higher tannase activity were selected for fermentation in a fermenter. Specifically, the recombinant strain (Blunt-pyrG-pgpdA-potanA-goxS) was inoculated into 20 mL DPY medium and cultured at 35°C and 220 rpm for 3 days to obtain a primary seed solution. The primary seed solution was inoculated into 300 mL DPY medium at an inoculation amount of 3-5%, and cultured at 34°C and 220 rpm for 5 days to obtain a secondary seed solution. 300 mL of the secondary seed was inoculated into a 5L fermenter containing 2.7L fermentation medium (composed of (w / v) 5% corn starch hydrolysate, 4% defatted soy flour, 5% corn steep liquor and 1% ammonium sulfate) for fermentation. During the fermentation process, the tank pressure was controlled to be 0.05-0.06 MPa, and the initial air volume was 1m 3 / h, temperature was 35°C, pH was 5.5, and stirring speed was 300rpm. After fermentation for 60h and the corn starch hydrolysate in the culture medium was completely consumed, the feeding batch phase began. An intermittent feeding strategy was adopted, and 100mL of 50% (w / v) corn starch hydrolysate was added every 24h. During the entire fermentation process, the dissolved oxygen level was maintained at ≥10%. After 24h of fermentation, the fermentation broth was collected and the enzyme activity of tannase in the supernatant was tested. The test results are as follows Figure 1 As shown, according to Figure 1 It can be seen that with the extension of fermentation time, the enzyme activity of tannase in the fermentation broth gradually increased. After 168h of fermentation, the enzyme activity of tannase in the fermentation broth reached 1000U / mL.

[0100] Example 3 Purification of tannase and determination of its enzymatic properties

[0101] 1. Purification of tannase

[0102] The fusion protein was purified by SP (SepharoseTM) ion exchange chromatography column. The specific steps are as follows: the fermentation broth after the fermentation of Example 2 was centrifuged at 10000rpm for 5min, and the supernatant was collected. 20mL of the supernatant was dialyzed overnight in buffer A (20mM pH 4.0 glycine hydrochloride buffer) to obtain a crude enzyme solution at a temperature of 4°C. The crude enzyme solution was loaded on the SP ion exchange chromatography column balanced with buffer A at 0.5mL / min. After washing the unbound protein with 5mL of buffer A, eluted with 40% buffer B (500mM NaCl, 20mM pH 4.0 glycine-HCl buffer) for 15min, then eluted with a gradient of 40%-55% buffer B for 15min, then eluted with 55% buffer B for 15min, and finally eluted with a gradient of 55%-100% buffer B for 30min. Detect the OD 280nm of the eluate and collect the elution peak. The fractions with high enzyme activity were collected and dialyzed overnight in a citrate-hydrochloric acid buffer (20 mM, pH 6.0). The fusion protein PoTanA-goxS was obtained after ultrafiltration and concentration, and the protein purity was analyzed by SDS-PAGE.

[0103] The SDS-PAGE purification of the fusion protein is shown in Figure 2 As shown. Figure 2 It can be seen that the size of the fusion protein PoTanA-goxS is 64 kDa, which is consistent with the expected size. The amino acid sequence is obtained by replacing the amino acid residues 1-18 of SEQ ID NO: 1 with SEQ ID NO: 2, while keeping the other amino acid residues of SEQ ID NO: 1 unchanged. The specific protein is as follows:

[0104] .

[0105] The enzyme activity and protein concentration of the crude enzyme solution and the pure enzyme solution were measured, and the purification table is shown in Table 2. After one-step purification by SP ion exchange chromatography, the specific enzyme activity increased from 133.01 U / mg to 192.07 U / mg, the purification factor was 1.4, and the recovery rate was 67.6%.

[0106] Table 2 Purification of tannase PoTanA-goxS

[0107] Purification steps Total enzyme activity (U) Total protein (mg) Specific enzyme activity (U / mg) Purification multiple Recovery rate (%) Crude enzyme solution 7964.25 59.88 133.01 1 100 SP chromatography column 5384 28.03 192.07 1.44 67.6

[0108] 2. Determination of the optimum pH

[0109] The enzyme activity of the tannase prepared above was measured at 45°C in different 50mM buffer systems, and the relative enzyme activity was calculated with the highest enzyme activity as 100%. The various buffers are as follows:

[0110] 1) Glycine-HCl buffer (pH 2.0-4.0);

[0111] 2) Acetate buffer (pH 3.0-6.5);

[0112] 3) Citric acid buffer (pH 3.0-6.5);

[0113] 4) MES (morpholineethanesulfonic acid) buffer (pH 6.5-7.5);

[0114] 5) Tris-HCl (tris(hydroxymethyl)aminomethane-hydrochloric acid) buffer (pH 7.0-8.0);

[0115] Test results such as Figure 3 As shown, according to Figure 3 It was found that the optimum pH for the enzyme reaction of tannase PoTanA-goxS was 6.0.

[0116] 3. pH stability determination

[0117] The tannase PoTanA-goxS was diluted with the five pH buffers mentioned above, placed in a 30°C water bath for 30 minutes, quickly placed in ice water for 30 minutes, and then the residual enzyme activity was measured. The enzyme activity of the untreated tannase was taken as 100%, and the relative enzyme activity of PoTanA-goxS after different pH treatments was calculated.

[0118] The results are as follows Figure 4 As shown, the tannase PoTanA-goxS still retained more than 80% of the enzyme activity after being incubated in the pH range of 2.5-6.5 for 30 minutes.

[0119] 4. Determination of the optimum temperature

[0120] After the tannase PoTanA-goxS was appropriately diluted with 50 mM citric acid buffer (pH 6.0), the enzyme activity was measured at different temperatures (10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60°C). The highest enzyme activity was taken as 100%, and the relative enzyme activity was calculated.

[0121] The results of the test are as follows Figure 5 As shown, it can be seen that the optimum temperature for the enzyme reaction of tannase PoTanA-goxS is 45°C.

[0122] 5. Determination of temperature stability

[0123] After the tannase PoTanA-goxS was appropriately diluted with 50mM citric acid buffer (pH 6.0), it was kept at different temperatures (10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60°C) for 30 minutes, and then quickly placed in ice water for 30 minutes, and then the enzyme activity was measured. The enzyme activity of the untreated tannase was taken as 100%, and the relative enzyme activity of PoTanA-goxS after treatment at different temperatures was calculated.

[0124] The results of the test are as follows Figure 6 As shown, it can be seen that after the tannase PoTanA-goxS was treated below 45°C for 30 minutes, more than 80% of the enzyme activity remained, and it had good temperature stability.

[0125] Example 4 Substrate specificity of tannase

[0126] 1 mg / mL solution containing different substrates was prepared in 50 mM citric acid buffer (pH 6.0), including methyl gallate (MG), ethyl gallate (EG), propyl gallate (PG), gallocatechin gallate (GCG), epicatechin gallate (ECG), epigallocatechin gallate (EGCG), catechin gallate (CG) and tannic acid (TA). 900 μL substrate solution was mixed with 100 μL diluted enzyme solution, kept at 45°C for 20 min, inactivated at 100°C for 10 min, the amount of gallic acid generated was determined, and the enzyme activity was calculated. The calculation results are shown in Table 3.

[0127] Table 3 Substrate specificity of tannase PoTanA-goxS

[0128] Substrate Specific enzyme activity (U / mg) Relative enzyme activity (%) Methyl Gallate 213 100 Ethyl Gallate 160 75 Propyl Gallate 192 90 Epigallocatechin gallate 17 8.2 Epicatechin gallate 4 2 Epigallocatechin gallate 3 1.3 Epigallocatechin gallate 16 7.5 Tannic acid 10 4.5

[0129] According to Table 3, the tanninase PoTanA-goxS showed enzyme activity on different substrates, among which the specific enzyme activity on methyl gallate was the highest, which was 213U / mg, followed by propyl gallate (192U / mg) and ethyl gallate (160U / mg). PoTanA-goxS also had good catalytic activity on natural tannins, with a specific enzyme activity of 17U / mg on catechin gallate and 16U / mg on catechin gallate.

[0130] Example 5 Application of tannase in persimmon juice

[0131] 1. Optimization of enzyme dosage

[0132] Persimmon juice was obtained by processing persimmons with a juicer. 20 mL of persimmon juice was added with different enzyme amounts of 0, 0.1, 0.5, 1, and 2 U / mL of tanninase PoTanA-goxS, treated at 45°C for 90 min, and then boiled at 100°C for 5 min to inactivate. The tannin content in the treated persimmon juice was determined. The results are shown in Table 4.

[0133] Table 4 Effect of different tannin enzyme addition amounts on tannin content in persimmon juice

[0134]

[0135]

[0136] According to Table 4, tannase can effectively reduce the tannin content in persimmon juice. As the amount of tannase added increases, the tannin content gradually decreases. When the amount of enzyme added is 1U / mL, the tannin concentration is 0.37g / 100mL, and the content is 72.18% lower than before enzymolysis.

[0137] 2. Processing temperature optimization

[0138] Persimmon juice was obtained by processing persimmons with a juicer, 20 mL of persimmon juice was added with 1 U / mL of tannase, and the mixed system was treated at different temperatures (35, 40, 45, 50°C) for 90 min, and then boiled at 100°C for 5 min to inactivate. The content of tannin in persimmon juice before and after treatment was determined, and the results are shown in Table 5.

[0139] Table 5 Effect of different temperatures on tannin content in persimmon juice

[0140] Temperature(℃) Tannin concentration (g / 100mL) Tannin relative content (%) 30 0.53 39.85 35 0.42 31.58 40 0.33 24.81 45 0.37 27.82 50 0.56 42.11

[0141] According to Table 5, temperature has a great influence on the catalytic effect of tannase. As the temperature increases, the tannin content in persimmon juice decreases first and then increases. When the temperature is 40℃, the tannin concentration is 0.33g / 100mL, which is 75.19% lower than before enzymolysis.

[0142] 3. Processing time optimization

[0143] Persimmon juice was obtained by processing persimmons with a juicer. 20 mL of persimmon juice was added with 1 U / mL of tanninase PoTanA-goxS, treated at 40°C for a certain time (30, 60, 90, 120 min), and then boiled at 100°C for 5 min to inactivate it. The tannin content in the persimmon juice before and after treatment was determined. The results are shown in Table 6.

[0144] Table 6 Effect of different treatment times on tannin content in persimmon juice

[0145] Processing time (min) Tannin concentration (g / 100mL) Tannin relative content (%) 30 0.58 43.61 60 0.45 33.83 90 0.33 24.81 120 0.30 22.56

[0146] According to Table 6, the tannin content in persimmon juice decreases with the extension of tannase treatment time. When the treatment time reaches 120 min, the tannin content is 0.30 g / 100 mL, which is 77.44% lower than that before enzymolysis.

[0147] Example 6 Application of tannase in tea

[0148] 1. Optimization of enzyme dosage

[0149] Weigh 5g of green tea, mix it with water at a mass ratio of 1:50, extract it at 85℃ for 30min, stir it every 5min, and filter it to get tea soup. Take 20mL of tea soup and add tannin enzyme PoTanA-goxS, with enzyme dosage of 0, 0.1, 0.5, 1, 2U / mL, treat it at 45℃ for 90min, and then boil it at 100℃ for 5min to inactivate it. The contents of tannin and gallic acid in the tea soup before and after treatment were determined, and the results are shown in Table 7.

[0150] Table 7 Effects of different tannase addition amounts on tannin and gallic acid content in tea soup

[0151] Enzyme dosage (U / mL) Tannin concentration (mmol / L) Tannin relative content (%) Gallic acid concentration (mmol / L) 0 3.76 100 2.35 0.1 3.61 96.01 6.53 0.5 2.97 78.99 12.59 1 2.70 71.81 15.73 2 1.85 49.20 20.96

[0152] According to Table 7, as the amount of tannase added increased, the content of tannin in the tea soup decreased significantly, and the content of gallic acid increased significantly. When the amount of enzyme added was 2U / mL, the concentration of tannin in the tea soup was 1.85mmo / L, which was 50.80% lower than before enzymatic hydrolysis.

[0153] 2. Processing temperature optimization

[0154] Weigh 5g of green tea, mix at a tea-water mass ratio of 1:50, soak at 85℃ for 30min, stir every 5min, and filter to obtain tea soup. Take 20mL of tea soup and add tannin enzyme PoTanA-goxS, add 2U / mL of enzyme, treat at different temperatures (35, 40, 45, 50℃) for 90min, and then boil at 100℃ for 5min to inactivate. Determine the content of tannin and gallic acid in the tea soup before and after treatment. The results are shown in Table 8.

[0155] Table 8 Effect of different temperatures on the content of tannin and gallic acid in tea soup

[0156] Temperature(℃) Tannin concentration (mmol / L) Tannin relative content (%) Gallic acid concentration (mmol / L) 35 2.89 76.86 9.78 40 1.64 43.62 23.08 45 1.85 49.20 20.96 50 2.76 73.40 10.62

[0157] According to Table 8, as the temperature gradually increases, the tannin content in the tea soup shows a trend of first decreasing and then increasing, which is mainly caused by the denaturation of tannin enzymes caused by high temperature. When the treatment temperature is 40℃, the tannin concentration in the tea soup is 1.64mmo / L, which is 56.38% lower than before enzymatic hydrolysis.

[0158] 3. Processing time optimization

[0159] Weigh 5g of green tea, mix it with water at a mass ratio of 1:50, extract it at 85℃ for 30min, stir it every 5min, and filter it to get the tea soup. Take 20mL of tea soup and add tannin enzyme PoTanA-goxS at an enzyme amount of 2U / mL, treat it at 40℃ for a certain time (30, 60, 90, 120min), and then boil it at 100℃ for 5min to inactivate it. Determine the content of tannin and gallic acid in the tea soup before and after treatment.

[0160] Table 9 Effects of different treatment times on the contents of tannin and gallic acid in tea soup

[0161] Time (min) Tannin concentration (mmol / L) Tannin relative content (%) Gallic acid concentration (mmol / L) 30 2.68 71.28 13.14 60 2.15 57.18 17.79 90 1.64 43.62 23.08 120 1.07 28.46 31.45

[0162] According to Table 9, as the treatment time increases, the tannin content in the tea soup decreases significantly. When the treatment time is 120 min, the tannin concentration in the tea soup is 1.07 mmol / L, which is 71.54% lower than before enzymatic hydrolysis.

[0163] Example 7 Application of tannase in feed

[0164] Bran, sorghum grains, soybean meal, and corn were crushed and passed through a 40-mesh sieve. The feed was prepared by mixing 30% sorghum grains, 30% corn, 20% soybean meal, 15% bran, and 5% premix in a mass ratio. Tanninase PoTanA-goxS was diluted and added to the feed in a feed-water mass ratio of 1:2. The enzyme addition amount was 10, 20, 40, 60, 80, and 100 U / g. The feed was treated at 40°C for 6 hours, and the tannin content in the feed before and after treatment was determined. The results are shown in Table 10.

[0165] Table 10 Effect of different enzyme addition amounts of tannase on tannin content in feed

[0166] Enzyme dosage (U / g) Tannin content (mg / g) Tannin relative content (%) 0 47.5 100 10 37.1 78.11 20 31.9 67.16 40 25.3 53.26 60 16.5 34.74 80 12.0 25.26 100 12.2 25.58

[0167] According to Table 10, after adding tannase, the tannin content in the feed was significantly reduced. When the addition amount was 80U / g, the tannin content in the feed was 12.0mg / g. Compared with the group without enzyme addition, the tannin in the feed was reduced by 74.74%.

[0168] Example 8 Application of tanninase in hydrolyzing pomegranate peel to extract pomegranate

[0169] The pomegranate peel was crushed and passed through a 40-mesh sieve to obtain pomegranate peel powder; 10 g of pomegranate peel powder was weighed into a 1L beaker, and 600 mL of 50 mmol / L citrate buffer (pH 6.0) was added to mix with it. The mixture was kept in an 80°C water bath for 2 hours, shaken every 30 minutes, and cooled to room temperature after being taken out to obtain a pomegranate peel extract. Tannase PoTanA-goxS was added to the pomegranate peel extract at enzyme dosages of 2, 4, 6, 8, and 10 U / mL, and treated at 40°C for 48 hours. The content of punica granatum in the pomegranate peel extract before and after treatment was determined, and the results are shown in Table 11.

[0170] Table 11 Effect of different enzyme addition amounts of tannase on the content of punica granatum in pomegranate peel

[0171] Enzyme dosage (U / mL) Concentration of garnet (mg / mL) Relative content of pomegranate forest (%) 0 2.23 100 2 2.61 117.04 4 2.90 130.04 6 3.84 172.20 8 3.81 170.85 10 3.62 162.33

[0172] According to Table 11, after adding tannase PoTanA-goxS, the concentration of puniculata in the pomegranate peel extract increased significantly. When the addition amount was 6 U / mL, the concentration of puniculata in the extract was 3.84 mg / mL, and the content increased by 72.20% compared with before hydrolysis.

[0173] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A protein, characterized in that The protein is any one of the following A1)-A5): A1) a protein whose amino acid sequence includes positions 19-584 of SEQ ID NO: 1; A2) a fusion protein obtained by fusing a signal peptide to the amino terminus of the protein shown in A1); A3) a protein having tannase activity obtained by replacing and / or deleting and / or adding amino acid residues of any of the proteins shown in A1) to A2); A4) a protein having 80% or more identity with any of the proteins shown in A1) to A2) and having tannase activity; A5) A fusion protein obtained by fusing a protein tag to the carboxyl terminal and / or amino terminal of any of the proteins shown in A1) to A2).

2. The protein according to claim 1, characterized in that The protein has tannase activity.

3. The protein according to claim 1 or 2, characterized in that A2), wherein the fusion protein is selected from at least one of potanA-pglaaS, potanA-goxS, potanA-amyaS, potanA-phyaS or potanA-phybS; The potanA-pglaaS is a protein whose amino acid sequence includes SEQ ID NO: 1; The potanA-goxS is a protein obtained by replacing amino acid residues 1 to 18 of SEQ ID NO: 1 with SEQ ID NO: 2, while keeping other amino acid residues of SEQ ID NO: 1 unchanged; The potanA-amyaS is a protein obtained by replacing the amino acid residues 1 to 18 of SEQ ID NO: 1 with SEQ ID NO: 3, while keeping the other amino acid residues of SEQ ID NO: 1 unchanged; The potanA-phyaS is a protein obtained by replacing the amino acid residues 1 to 18 of SEQ ID NO: 1 with SEQ ID NO: 4, while keeping the other amino acid residues of SEQ ID NO: 1 unchanged; The potanA-phybS is a protein obtained by replacing the amino acid residues 1 to 18 of SEQ ID NO: 1 with SEQ ID NO: 5, while keeping the other amino acid residues of SEQ ID NO: 1 unchanged.

4. A biomaterial related to the protein according to any one of claims 1 to 3, characterized in that: The biological material is any one of the following B1)-B8): B1) A nucleic acid molecule encoding the protein according to any one of claims 1 to 3; B2) an expression cassette containing the nucleic acid molecule described in B1); B3) a recombinant vector containing the nucleic acid molecule described in B1); B4) a recombinant vector containing the expression cassette described in B2); B5) a recombinant microorganism containing the nucleic acid molecule described in B1); B6) a recombinant microorganism containing the expression cassette described in B2); B7) a recombinant microorganism containing the recombinant vector described in B3); B8) A recombinant microorganism containing the recombinant vector described in B4).

5. A method for preparing tannase, characterized in that: include: The gene encoding the protein according to any one of claims 1 to 3 is expressed in an organism to obtain tannase; the organism is a microorganism, a plant or a non-human animal.

6. Use of the protein according to any one of claims 1 to 3 as tannase.

7. Use of the protein according to any one of claims 1 to 3 or the biomaterial according to claim 4 in the preparation of tannase preparations.

8. A method for enzymatically hydrolyzing tannin in a product, characterized in that: include: The product containing tannin is mixed with the protein according to any one of claims 1 to 3, and an enzymatic hydrolysis reaction is performed so that the protein can enzymatically hydrolyze the tannin in the product.

9. A method for preparing pomegranate peel by enzymatic method using pomegranate peel as raw material, characterized in that: The pomegranate peel or the extract of the pomegranate peel is mixed with the protein according to any one of claims 1 to 3, and subjected to enzymatic hydrolysis to obtain the pomegranate peel forest.

10. The method according to claim 8 or 9, characterized in that: The temperature of the enzymatic hydrolysis reaction is 10-60°C, and the pH is 2-8.

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