WO2022268194A1 - Application of tannase in preparation of gallic acid - Google Patents

Abstract

An application of tannase in the preparation of gallic acid, belonging to the technical field of enzyme engineering. The application comprises the following steps: 1) preparing a tannic acid solution with a concentration of 25%-35% (w/v), and adjusting the pH of the tannic acid solution to 4.0-5.0; 2) adding the tannic acid solution obtained in step 1) to a reaction kettle, adding tannase according to a substrate addition amount of 36-54 U/g, and hydrolyzing to generate gallic acid. The tannase used is derived from Aspergillus niger ATCC 13496, the amino acid sequence thereof has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology in sequence with SEQ ID NO: 13 or a mature polypeptide thereof. Also disclosed is a method for preparing tannase by using the amino acid sequence of tannase expressed by an Aspergillus niger CICC 2462 strain.

Description

Application of a kind of tannase in preparation of gallic acid technical field

The invention belongs to the technical field of enzyme engineering and relates to an application of tannase in preparing gallic acid.

Background technique

Tannase (Tannase, E.C.3.1.1.20), also known as tannase, is a tannin-based hydrolase, which has a hydrolysis effect on acids with two phenol groups, such as tannic acid, and can hydrolyze galloyl Tannins and alkyl gallates produce small molecular substances such as gallic acid, glucose and alkyl alcohols. Based on the efficient hydrolysis of tannins, tannase has been widely used in the fields of food, beverage, wine making, medicine, and chemical industry.

The sources of microorganisms capable of producing tannase are very rich, mainly fungal Aspergillus, Penicillium and Rhizopus, especially Aspergillus niger, Aspergillus oryzae and Aspergillus flavus (Sunny D, Gunjan M, Kumar SA .Recent trends and advancements in microbial tannase-catalyzed biotransformation of tannins: a review. International Microbiology, 2018, 21.). Prior art focuses on Aspergillus and Penicillium mostly to the emphasis of tannase fermentative production research, often improves fermentative enzyme production activity (LV Rodríguez-Durán by way of mutagenesis breeding selection and improvement production strain and by optimizing fermentation conditions etc. , Valdivia-Urdiales B, Contreras-Esquivel JC, et al. Novel strategies for upstream and downstream processing of tannin acyl hydrolase. Enzyme research, 2011, 2011:823619.). However, the tannase produced by this method generally has the problems of low production efficiency and high production cost, and factors such as the mixing of enzyme components obviously limit the large-scale industrial application of tannase. Due to the wide variety of tannase and the different properties of enzymes from different sources, it is very difficult to find a suitable tannase for a specific field. There are literatures and patents reporting the use of genetic engineering to construct recombinant bacteria to produce tannase, but the hydrolysis effect of these enzymes on tara tannin is not obvious, and the use of tannase to hydrolyze tara tannin to efficiently produce gallic acid Industrial applications have not been reported yet.

As an important fine chemical, gallic acid is widely used in food, chemical, pharmaceutical and other fields. Although the current industrialized preparation of gallic acid methods (acid method and alkali method) has the advantages of mature technology, the preparation process will also produce a large amount of waste water and waste salt, which will cause serious environmental pollution (Chinese patent documents CN106242966 B and CN108003012A). With the strengthening of environmental protection supervision, the process of preparing gallic acid by chemical method is no longer suitable for the needs of development. Therefore, in recent years, studies on tannin enzymatic methods have been carried out at home and abroad. There are reports in the literature that gallic acid is prepared by microbial fermentation, but this method has a long production cycle and incomplete hydrolysis. Chinese patent document CN1083532A has been improved on this basis, by first cultivating microorganisms, and then adding raw materials to prepare gallic acid, but this method also has the problems of long production cycle, low product yield, and complicated operation, which is not suitable for large-scale production , and cannot compete with chemical methods (Lokeswari N, Jaya RK. Optimization of Gallic Acid Production from Terminalia Chebula by Aspergillus niger. Journal of Chemistry, 2007, 4(2): 287-293.). So far, there is no industrial application report of directly using tannase to prepare gallic acid. The main reasons are three aspects: 1) there is no suitable enzyme and corresponding sequence that can efficiently hydrolyze tara tannin; 2) there is no Develop its corresponding production strains and can effectively reduce production costs; 3) No corresponding gallic acid preparation process has been developed.

Contents of the invention

In order to solve the deficiencies in the prior art, the object of the present invention is to provide the application of a novel tannase in the high-efficiency preparation of gallic acid.

The amino acid sequence of the tannase has at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with SEQ ID NO: 13 or its mature polypeptide.

In a preferred embodiment, the amino acid sequence of the tannase is SEQ ID NO: 13 or the mature polypeptide of the polypeptide shown in SEQ ID NO: 13, and the tannase is derived from Aspergillus niger ATCC 13496, the optimum reaction temperature The temperature is 65-75°C, and the optimum reaction pH is 4.0-5.0.

The polynucleotide sequence of the tannase comprises: 1) the polynucleotide sequence of SEQ ID NO:5; and/or, 2) the cDNA sequence of SEQ ID NO:5; and/or, 3) under very stringent conditions A polynucleotide sequence that hybridizes to the polynucleotide sequence of 1) or 2) or its full-length complementary strand. Those skilled in the art will appreciate that, because of the well-known degeneracy in the genetic code, different polynucleotides encoding the same amino acid sequence can be designed and prepared using routine skills, and that codon optimization for a particular host cell is also within the skill of the art well-known in.

Tens of thousands of tannase coding sequences have been disclosed in the prior art. For example, NCBI has published more than 18,000 tannase coding genes derived from different strains, and only the tannins from Aspergillus niger ATCC 13496 There are more than a dozen kinds of enzymes, but so far no research has found a tannase variety with excellent performance and suitable for large-scale industrial production of gallic acid. It has been reported in the literature that Aspergillus niger ATCC 13496 can produce tannase (Sharma S, Bhat TK, Dawra RK. Isolations, purification and properties of tannase from Aspergillus niger van Tieghem. World Journal of Microbiology & Biotechnology, 1999), but this document also discloses The tannase derived from Aspergillus niger ATCC 13496 is an intracellular enzyme. Due to the low yield of intracellular enzyme, it cannot be applied industrially. Therefore, this document gives reverse instructions to those skilled in the art. So far, there is no source of Aspergillus niger ATCC 13496 Further research on tannase. The inventor provides a kind of Aspergillus niger ATCC 13496 source of tannase through a large number of screening verification experiments, it is the tannase of extracellular secretion (it is numbered as Tan3 in the present invention), the aminoacid sequence of this enzyme is as SEQ ID NO : Shown in 13, the property of this tannase is significantly better than the tannase in the prior art.

The tannase Tan3 has the following chemical properties:

1) Function: act on the depsipyl acid bond to hydrolyze;

2) Molecular weight: about 800,000 Da (determined by SDS-PAGE);

3) Temperature stability: Stability remains above 90% until 65°C (pH4.0, 30 minutes heat treatment);

4) Optimum temperature: about 70°C;

5) Optimum pH: about 4.0;

6) Acid resistance: under the condition of pH 2.5, the loss of enzyme activity is within 2% (30°C) for 5 hours;

7) Substrate specificity: It acts well on tannin and gallate.

In a specific embodiment of the present invention, the present invention utilizes Aspergillus niger CICC 2462 and above-mentioned sequence to prepare high enzymatic activity (enzymatic activity can reach 1560.0U/mL), good thermostable tannase, this tannase is at higher It can still maintain enzyme activity for a long time in a certain temperature range, and the tannase can continuously hydrolyze tara tannins with a concentration of up to 30%, showing good substrate tolerance.

The present invention also proposes a method for efficiently preparing gallic acid by using the aforementioned tannase, which specifically includes the following steps:

1) Prepare a tannic acid solution with a concentration of 25%-35% (w/v), and adjust the pH of the tannic acid solution to 4.0-5.0;

2) Add the tannic acid solution obtained in step 1) into the reaction kettle, add tannase according to the substrate addition amount of 36-54U/g, and hydrolyze to generate gallic acid.

In a specific embodiment, the preparation method of the gallic acid is as follows:

1) Weigh 1-2 parts by mass of Tara powder raw material and place it in a raw material extraction tank, add 3-5 parts by mass of pure water to fully stir and dissolve;

2) Stir and extract at 30-35°C for 6-8 hours, and centrifuge to obtain a tannic acid extract;

3) The tannic acid extract is heated and concentrated until the concentration of tannic acid reaches 25%-35% (w/v), and the pH of the concentrated solution is adjusted to 4.0-5.0;

4) Take the tannic acid extract solution with a concentration of 25%-35% (w/v) in step 3) and add it to the reactor, and add the tannic acid extract of the present invention according to the substrate addition amount of 36U/g-54U/g. Tannase, hydrolyze to generate gallic acid, and control the reaction temperature at 35°C-40°C throughout the hydrolysis process.

In another specific embodiment, the preparation method of the gallic acid is as follows:

1) Weigh 2 kg of tara powder raw material and place it in a raw material extraction tank, add 10 kg of pure water;

2) At room temperature of 35°C, stirring and extracting for 6 hours, centrifuging and filtering to obtain about 9L of tannic acid extract;

3) 9L of tannic acid extract is concentrated until the concentration of tannic acid reaches 30%;

4) Take 500ml of concentrated tannic acid extract solution with a substrate concentration of about 30% (w/v) in the reaction kettle, adjust the pH to 4.0, add tannase enzyme solution, the amount of enzyme solution added per gram of substrate It is 36U, and the water bath controls the reaction temperature to 40°C. After reacting for 8 hours, detect the reduction of substrate tannic acid and the production of product gallic acid, and calculate the conversion rate.

The tannase described in the present invention can continuously hydrolyze tannin to generate gallic acid under a tannin substrate with a concentration of up to 30% (w/v), and the hydrolysis period is short, only 8 hours are required for complete hydrolysis, and the conversion rate is as high as 99.5% , the purity of the prepared gallic acid is 99%, so that the use of the tannase can be better applied to industrial mass production of gallic acid, and has obvious advantages compared with the preparation of gallic acid by chemical methods.

The chemical preparation of gallic acid reported in the patent document CN108003012A is complex in operation, and the reaction process requires high temperature and high pressure and a large amount of acid and alkali. Compared with the above-mentioned method, the method for efficiently preparing gallic acid proposed by the present invention by using the aforementioned tannase is simple in operation, does not require high temperature and high pressure for the reaction, and does not use a large amount of acid and alkali, resulting in a large amount of waste water and waste salt , seriously pollute the environment. The preparation of gallic acid by the microbial method reported in the patent document CN1083532A is complex in operation, the reaction cycle is as long as more than 35 hours, the conversion rate is only about 73%, and the concentration of the hydrolyzed substrate is only about 8%, which is not suitable for large-scale production. The present invention proposes The method for preparing gallic acid is more simple and controllable, the concentration of the hydrolyzed substrate can reach 30%, the conversion rate is as high as 99.5%, and the cycle is only about 8 hours. Based on the above, the method of using the aforementioned tannase to efficiently prepare gallic acid proposed by the present invention has obvious advantages, and can be better applied to industrial mass production of gallic acid.

The present invention also proposes a method for preparing tannase, the method utilizes Aspergillus niger CICC 2462 bacterial strain to express the amino acid sequence of tannase, and the amino acid sequence of said tannase has SEQ ID NO: 13 or its mature polypeptide At least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity; preferably, the amino acid sequence of the tannase is shown in SEQ ID NO:13 or SEQ ID NO:13 The mature polypeptide of the polypeptide. Aspergillus niger CICC 2462 strain is used to express the tannase of the present invention, the product has high enzyme activity, low production cost, and is beneficial to industrialized production.

Tannase (Tannase, E.C.3.1.1.20): also known as tannase. A tanninyl hydrolase that hydrolyzes acids with two phenolic groups, such as tannin. The enzyme can be produced by molds such as Aspergillus niger and Aspergillus oryzae. Preferably, the tannase is from the genus Aspergillus, such as Aspergillus niger; more preferably, the amino acid sequence of the tannase is shown in SEQ ID NO:13.

The term "amino acid sequence" is synonymous with and used interchangeably with the terms "polypeptide", "protein" and "peptide". Where such amino acid sequences exhibit activity, they are referred to as "enzymes". The conventional one-letter or three-letter codes for amino acid residues are used, with the amino acid sequence presented in standard amino to carboxy-terminal orientation (ie, N→C).

Mature polypeptide: The term "mature polypeptide" means a polypeptide in its final form after translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, and the like. It is known in the art that host cells can produce a mixture of two or more different mature polypeptides (ie, having different C-terminal and/or N-terminal amino acids) expressed from the same polynucleotide. In a specific example of the present invention, the protein encoded by SEQ ID NO:5 is predicted to be 583 amino acids, and a signal peptide of 19 residues is predicted using SignalP program version 3.0 (Nielsen et al., 1997, Protein Engineering 10:1-6) , the predicted mature protein contains 564 amino acids with a predicted molecular weight of 61.8 kDa and an isoelectric point of 4.64.

cDNA: The term "cDNA" means a DNA molecule capable of being prepared by reverse transcription from a mature, spliced mRNA molecule obtained from a eukaryotic cell. cDNA lacks intronic sequences normally present in the corresponding genomic DNA. The initial, primary RNA transcript is a precursor to mRNA that is processed through a series of steps including splicing and then emerges as mature spliced mRNA.

Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity". A particular sequence has at least a certain percentage of amino acid residues identical to those of a given reference sequence when aligned using the CLUSTALW algorithm with preset parameters. The default parameters of the CLUSTALW algorithm are: Deletions are counted as residues that are not identical compared to the reference sequence. Deletions occurring at any terminus are included. For example, a variant 500 amino acid residue polypeptide that lacks five amino acid residues at the C-terminus has a percent sequence identity of 99% (495/500 identical residues x 100) relative to the parental polypeptide, such variants consist of The language "variants having at least 99% sequence identity to the parent" encompasses.

Very Stringent Conditions: means sheared and denatured salmon sperm DNA at 42°C in 5X SSPE, 0.3% SDS, 200 μg/ml for probes of at least 100 nucleotides in length following standard Southern blotting procedures Prehybridize and hybridize with 50% formamide for 12 to 24 hours. The carrier material was finally washed three times with 2X SSC, 0.2% SDS at 65°C for 15 minutes each.

The term "about" refers to ±10% of the stated value.

The beneficial effects of the present invention include:

1) The present invention provides a tannase that can efficiently hydrolyze tara tannin. The enzymatic properties of the tannase are superior. Under the condition of pH 2.5, the loss of enzyme activity is within 2% for 5 hours (30° C.) , can continuously hydrolyze tannin to generate gallic acid under a tannin substrate with a concentration of up to 30% (w/v), and the conversion rate is greater than 99.0%, so that it can be applied to large-scale industrial enzymatic preparation of gallic acid;

2) The present invention proposes a production method of tannase, the Aspergillus niger CICC 2462 of the embodiment of the present invention can utilize the tannase sequence of the present invention to prepare high enzyme activity (enzyme activity can reach 1560.0U/mL), heat Stable tannase can still maintain enzyme activity for a long time in a relatively high temperature range, and the tannase has good substrate tolerance. Compared with the direct use of natural production strains for fermentation, the preparation process of this method Simple, stable, high production efficiency, high production cost, and a single enzyme component. There are patent reports using Pichia pastoris to express tannase, but in this process, a large amount of methanol needs to be continuously added. Methanol is toxic and difficult to separate , apparently, the method for preparing tannase of the present invention can avoid such problem;

3) Although the current industrialized preparation of gallic acid (acid method and alkali method) has the advantages of mature technology, the preparation process will also produce a large amount of waste water and waste salt, which will cause serious environmental pollution. There are literature and patent reports using microbial methods to prepare gallic acid, but this method has the problems of long production cycle, low product yield, and complicated operation, which is not suitable for large-scale production and cannot compete with chemical methods. There is no technical report on the direct use of tannase to prepare gallic acid. The present invention discloses a method for preparing gallic acid by enzymatic hydrolysis, which can continuously hydrolyze tannin to generate gallic acid under the tannin substrate with a concentration of up to 30% (w/v) Acid is equivalent to the chemical method in terms of production cycle and conversion rate, and it is easy to operate, mild in conditions, friendly to the environment, and very suitable for large-scale production applications.

Description of drawings

Fig. 1 is the pGla-amds plasmid map of the present invention.

Figure 2 is the SDS-PAGE of the shake flask culture broth of the tannase recombinant Aspergillus niger expression strain of the present invention.

Fig. 3 is the determination of the optimum temperature of tannase from different sources of the present invention.

Fig. 4 is the determination of the optimum pH of tannase from different sources of the present invention.

Fig. 5 is the temperature stability of Tannase Tan3 of the present invention.

Fig. 6 is the acid resistance test of the tannase Tan3 of the present invention.

Fig. 7 is the curve of gallic acid produced by enzymatic hydrolysis of tannic acid by recombinant tannase.

detailed description

The present invention will be further described in detail in conjunction with the following specific embodiments and accompanying drawings. The process, conditions, experimental methods, etc. for implementing the present invention, except for the content specifically mentioned below, are common knowledge and common knowledge in this field, and the present invention has no special limitation content.

The disclosure of the invention provides many different embodiments or examples for implementing different implementations of the invention. In order to simplify the disclosure of the present invention, specific embodiments or examples are described below. Of course, they are only examples and are not intended to limit the invention. In addition, the present invention provides examples of various specific processes and materials, and one of ordinary skill in the art will recognize the applicability of other processes and/or the use of other materials. The practice of the present invention will employ, unless otherwise indicated, conventional techniques in chemistry, molecular biology and the like, which are within the capability of a person skilled in the art. Also, herein, unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation and amino acid sequences are written left to right in amino to carboxy orientation.

The construction of embodiment 1 tannase expression plasmid

The construction method of the tannase expression plasmid comprises the following parts:

1) Linearize the pUC57 plasmid with vector-F and vector-R primers;

2) selection marker amdS expression cassette, synthesized by GenScript Company, the sequence of the amdS expression cassette is shown in SEQ ID NO:1;

3) A DNA fragment containing the gla promoter and terminator of the Aspergillus niger glucoamylase gene, synthesized by GenScript, the sequence is shown in SEQ ID NO:2;

4) Tannase genes were derived from Aspergillus niger CBS 513.88, Aspergillus niger An76, Aspergillus niger ATCC 13496, Aspergillus kawachii IFO 4308, Aspergillus niger FJ0118, Aspergillus oryzae 40, Aspergillus fumigatus RIB Aspergillus fumigatus Af293 and Penicillium rubens Wisconsin 54-1255 (the nucleotide sequence is SEQ ID NO: 3-10, the amino acid sequence is SEQ ID NO: 11-18), respectively named these genes as tan1, tan2, tan3, tan4, tan5, tan6, tan7, tan8, and these genes were synthesized by GenScript company.

First, primers amdS-F and amdS-R, gla-F and gla-R were used to amplify the amdS expression cassette with recombination arms and the DNA fragment containing the gla promoter and terminator by PCR respectively, and the Gibson Master Mix Kit (E2611 , New England Biolabs) recombined the above linearized pUC57 vector, amdS expression cassette, and gla promoter and terminator DNA fragments to obtain the pGla-amdS plasmid, and the sequence was confirmed to be correct by sequencing. This plasmid can be used for insertion of tannase gene after linearization by AflII and XhoI sites.

Further, the tannase expression cassette was constructed as follows: use primer pairs tan1F/tan1R, tan1F/tan1R, tan1F/tan1R, tan1F/tan1R, tan1F/tan1R, tan1F/tan1R, tan1F/tan1R, tan1F/tan1R, PCR to expand the band The tan1~tan8 genes with recombination arms were then recombined with the linearized pGla-amdS plasmid using the Gibson Master Mix Kit (E2611, New England Biolabs) to obtain ptan1-amdS, ptan2-amdS, ptan3- Eight plasmids including amdS, ptan4-amdS, ptan5-amdS, ptan6-amdS, ptan7-amdS, ptan8-amdS were sequenced and confirmed. These plasmids can be used for protoplast transformation after linearization with the HindIII site.

The relevant primer sequences are as follows:

The primers used in the embodiment of the present invention 1 in table 1

Transformation integration of embodiment 2 tannase expression cassette

The tannase expression cassette in the embodiment of the present invention 1 is introduced into the Aspergillus niger CICC 2462 bacterial strain (purchased from China Industrial Microorganism Culture Collection Management Center CICC) by protoplast transformation method, and the specific operation steps are as follows:

(1) Preparation of protoplasts: Inoculate Aspergillus niger mycelia in nutrient-rich TZ liquid medium (0.8% beef extract powder, 0.2% yeast extract, 0.5% peptone, 0.2% NaCl, 3% sucrose, pH 5.8) After culturing for 48 hours, the mycelium was collected by filtration through Mira-cloth (Calbiochem Company) and washed with 0.7M NaCl (pH 5.8); after the mycelium was dried, it was transferred to a medium containing 1% cellulase (Sigma) and helicase ( Sigma) 1% and lysozyme (Sigma) 0.2% enzymolysis solution (pH 5.8), at 30 ℃, 65rpm enzymolysis 3h; then put the enzymolysis solution containing protoplasts on ice and wipe the mirror with four layers Paper filtration, the obtained filtrate was centrifuged gently at 3000rpm, 4°C for 10min, the supernatant was discarded, and the protoplasts attached to the tube wall were washed once with STC solution (1M D-Sorbitol, 50mM CaCl ₂ , 10mM Tris, pH 7.5) , and finally resuspend the protoplasts in an appropriate amount of STC solution.

(2) Protoplast transformation: Add 10 μl (concentration: 1000ng/μl) of the DNA fragment containing the tannase expression cassette obtained by linearization with HindIII into 100 μl of protoplast suspension, mix well, place it at room temperature for 25 minutes, and divide it into 3 Add a total of 900 μl PEG solution each time, mix well and place at room temperature for 25 minutes, then centrifuge at room temperature for 10 minutes at 3000 rpm, discard the supernatant, resuspend the protoplasts attached to the tube wall in 1ml STC solution, and mix with pre-cooled to about 45°C Acetamide medium (3% sucrose, 0.05% KCl, 0.1% K ₂ HPO ₄ 3H ₂ O 0.1%, 0.001% FeSO ₄ , 0.0244% MgSO ₄ , 0.06% acetamide, 0.34% CsCl) was mixed and plated, and the plates were After coagulation, put it in a 34°C incubator and cultivate it for 4-5 days. Pick the transformants to a new acetamide medium plate and place them in a 34°C incubator for another 4-5 days. The grown transformants are called positive. Turn.

Shake flask culture and enzyme activity assay of embodiment 3 tannase recombinant expression strain

1. Determination of Tannase Enzyme Activity

1) Definition of enzyme activity unit

Under the conditions of 30°C and pH 5.0, the amount of enzyme required to degrade propyl gallate (PG) solution and release 1 μmol of gallic acid per minute is defined as one enzyme activity unit (U).

2) Reagent

All reagents refer to analytical grade reagents and secondary water in accordance with GB/T 6682, unless other requirements are specified.

2.1 0.1mol/L disodium hydrogen phosphate-0.05mol/L citric acid buffer

Accurately weigh 73.56g of disodium hydrogen phosphate dodecahydrate, accurately weigh 20.45g of citric acid monohydrate, add 800ml of water to dissolve, adjust the volume to 2000ml after dissolving, adjust the pH to 5.0, and store in refrigerator at 4°C for one month.

2.2 0.5mol/l KOH solution

Accurately weigh 28.055g of KOH, add 800ml of water to dissolve, dilute to 1000ml after dissolution, and store at room temperature.

2.3 0.667% (W/V) rhodanine solution (methanol rhodanine)

Accurately weigh 0.667g of rhodanine, add 80ml of methanol to dissolve, dilute to 100ml with methanol after dissolution, and store at room temperature for 15 days.

2.4 Gallic acid standard solution, the concentration is 10mmol/l

Weigh 0.1701 g of anhydrous gallic acid, add phosphoric acid-citric acid buffer solution to dissolve, dilute to 100 ml, and store in a refrigerator at 4°C for 15 days.

2.5 Propyl gallate (PG) solution, the concentration is 10mmol/L

Weigh 0.2122 g of propyl gallate, add 80 ml of disodium hydrogen phosphate-0.05 mol/L citric acid buffer, stir and heat with magnetic force until it is completely dissolved, cool, and dilute to 100 ml with buffer. Shake well before use and watch out for crystals. If it is necessary to heat and dissolve before use, it is effective for 10 days when stored in a refrigerator at 4°C.

3) Determination steps

3.1 Gallic acid standard curve drawing

Gallic acid standard solutions with different concentrations were prepared with disodium hydrogen phosphate-citric acid (0.1-0.05M) buffer at pH 5.0, and 9 different concentration gradients were prepared from 40-240 μmol/L. Take 0.5ml of gallic acid diluted standard solution and mix with 0.3ml of methanol rhodanine solution (0.667% W/V), add to all test tubes, bathe in water at 30°C for 5min, then add 4.2ml of KOH solution to all test tubes medium, 30°C for 10min. The buffer solution was used instead of the standard solution as a blank control, and the absorbance value (A520) was measured at 520nm. Draw a standard curve with concentration (mmol/L) as the abscissa and A520 as the ordinate.

3.2 Preparation of sample solution

The liquid sample can be directly diluted with buffer solution to the tannase activity of 0.03-0.15U/ml in the enzyme solution to be tested.

3.3 Sample determination

1) Take 4 clean test tubes, 1 blank tube and 3 test tubes. Add 0.25ml of PG solution to each tube in a 30°C water bath for 5-10 minutes, then add 0.25ml of the enzyme solution to be tested to the middle of the measuring tube at intervals of 15 seconds, and react in a 30°C water bath for 5 minutes;

2) Add 0.3ml of methanol rhodanine solution (0.667%, W/V) to all test tubes at intervals of 15s, and keep warm for 5min;

3) Add 4.2ml of KOH solution (0.5M) to all test tubes, add 0.25ml of enzyme solution to the blank tube, and place it at 30°C for 10 minutes (when the last sample is added to the KOH solution), reset to zero with the blank tube , Measure the absorbance of each tube solution at 520nm.

Calculation of sample enzyme activity:

XD=(△A520-b)×0.5×n/0.25/t/k

XD—the activity of tannase in sample diluent, U/ml;

△A520—Absorbance of enzyme reaction solution-Absorbance of enzyme blank sample;

K—the slope of the standard curve;

b—the intercept of the standard curve;

1/0.25—converted to 1ml enzyme solution;

t—enzymolysis reaction time, 5min;

0.5—0.5ml enzyme solution

n—the dilution factor of the sample.

Enzyme activity determination and protein electrophoresis analysis of tannase Aspergillus niger recombinant expression strain:

Inoculate positive transformants of tannase Aspergillus niger recombinant expression strain in Example 2 of the present invention into 50ml YPM medium (yeast extract 0.2%, peptone 0.2%, maltose 2%) shake flasks respectively, shake at 34°C and 220rpm The bed was cultured for 6 days, and the supernatant of the fermentation broth was collected by centrifugation, and the activity of the obtained sample was measured according to the above-mentioned tannase activity assay method, and the results are shown in Table 2 below. The results showed that the activity of tannase from different sources was significantly different. The samples after fermentation broth treatment were detected by protein electrophoresis (SDS-PAGE), and the results are shown in Figure 2. The molecular weights of tannases from different sources were all around 80kDa.

Table 2 Enzyme activity determination of recombinant expression strains of tannase from different sources

strain sequence source Enzyme activity U/ml Ptan1-amdS Aspergillus niger CBS 513.88 (SEQ ID NO: 3) 1342 Ptan2-amdS Aspergillus niger An76 (SEQ ID NO: 4) 50 Ptan3-amdS Aspergillus niger ATCC 13496 (SEQ ID NO: 5) 1560 Ptan4-amdS Aspergillus kawachii IFO 4308 (SEQ ID NO: 6) 1804 Ptan5-amdS Aspergillus niger FJ0118 (SEQ ID NO: 7) 239 Ptan6-amdS Aspergillus oryzae RIB40 (SEQ ID NO: 8) 3099 Ptan7-amdS Aspergillus fumigatus Af293 (SEQ ID NO: 9) 348 Ptan8-amdS Penicillium rubens Wisconsin 54-1255 (SEQ ID NO:10) 37 CICC2462 (control) the 0

Embodiment 4 Different sources of tannase enzymatic property determination

1) Optimum reaction temperature

According to the above-mentioned tannase activity assay method, the reaction was carried out at a reaction temperature of 20°C, 30°C, 35°C, 40°C, 50°C, 60°C, 70°C and 80°C. Each measurement result was expressed by using the value of the temperature showing the highest activity as 100% relative activity ( FIG. 3 ). The results analysis showed that the optimum reaction temperature of Tan1, Tan3, Tan4 and Tan6 was around 70°C, the optimum reaction temperature of Tan2, Tan5 and Tan7 was around 60°C, and the optimum reaction temperature of Tan8 was around 50°C.

2) Optimum reaction pH

According to the above-mentioned tannase activity assay method, using 10mM propyl gallate as a substrate, in each buffer solution (disodium hydrogen phosphate-citric acid buffer pH2.5, 3.0, 4.0, 5.0, phosphate buffer pH6.0, 7.0, 8.0), the measurement was carried out under the reaction conditions of 30°C and 5 minutes. The results of each measurement were expressed using the pH value showing the maximum activity value as 100% relative activity ( FIG. 4 ). The analysis of the results showed that the optimum reaction pH of Tan1 was around 7.0, the optimum reaction pH of Tan2 and Tan4 was around 6.0, the optimum reaction pH of Tan5, Tan6, Tan7 and Tan8 was around 5.0, and the optimum reaction pH of Tan3 was around 4.0.

Embodiment 5 Different source tannases are to the hydrolysis reaction of tannic acid

10% tannic acid was used as substrate. Take 500ml of 10% tannic acid solution and add them to the reactor, and add 900U of tannase Tan1, Tan2, Tan3, Tan4, Tan5, Tan6, Tan7 and Tan8 to the reactor respectively. All reactions only control the initial pH to 4.0, the pH is not controlled during the reaction, and the temperature is controlled at about 40°C during all the reactions. Sampling was performed every 2 hours to detect the reduction of tannic acid in the reaction solution, and the reaction was stopped after 18 hours of reaction. The conversion rate was calculated according to the reduction of tannic acid, and the degree of hydrolysis of tannic acid by tannase from different sources was judged according to the conversion rate. The results are shown in Table 3. According to the results, it can be seen that the hydrolysis activity of tanninase from different sources is significantly different. Tan3 has the highest hydrolysis activity to tannic acid, and the conversion rate can reach 98.5% without controlling the pH in the reaction process. According to the hydrolysis reaction of Tan3 to tannic acid, it can be seen that this reaction process does not use a large amount of acid and alkali, the reaction conditions are mild, and the conversion rate can reach more than 90%. Thus, Tan3 is very suitable for the preparation of gallic acid by enzymatic hydrolysis.

Table 3 Hydrolysis of tannic acid by tannase from different sources

enzyme Conversion rate Tan1 4.33% Tan2 3.82% Tan3 98.5% Tan4 8.57% Tan5 63.2% Tan6 5.66%

Tan7 61.54% Tan8 42.37%

In Example 3, the substrate used for the determination of enzyme activity is propyl gallate, which is a chemically synthesized simple small molecular compound; the substrate used in the hydrolysis reaction is tannic acid, which is a natural and complex macromolecular compound. Different enzymes have different affinities for different substrates, thus resulting in differences in enzyme activity. Part of the enzyme activity in Example 3 is higher than Tan3, which is aimed at the propyl gallate substrate, indicating that the affinity of these enzymes to propyl gallate is higher than Tan3; and in the present embodiment, the hydrolysis reaction adopts For tannic acid, the hydrolysis rate of Tan3 was significantly higher than that of other enzymes, indicating that the affinity of Tan3 for tannic acid was significantly higher than that of other enzymes.

The enzymatic property determination of embodiment 6 tannase Tan3

1) Temperature stability

50mM disodium hydrogen phosphate-citric acid buffer (pH4.0), at 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C and 80°C At each temperature, after 30 minutes of enzyme heat treatment, the residual activity was determined by the above-mentioned tannase activity assay. The results of each measurement were expressed using the activity not subjected to heat treatment as 100% residual activity ( FIG. 5 ). Under heat treatment at 65°C for 30 minutes, it has more than 90% residual activity and is stable until 65°C.

2) Acid resistance test

Under the condition of disodium hydrogen phosphate-citric acid buffer pH 2.5, after treatment at 30°C for 1, 2, 3, 4, 5 hours respectively, dilute with 0.2M disodium hydrogen phosphate-citric acid buffer (pH 4.0) 5-fold, and the activity was determined using the tannase activity assay described above. Each measurement result was expressed by using the pH value at which the maximum activity value appeared as 100% relative activity ( FIG. 6 ). According to the results, it can be seen that under the condition of pH 2.5, the loss of enzyme activity was within 2% (30° C.) for 5 hours, which indicated that the tannase Tan3 was very stable under acidic conditions. Use tannase to hydrolyze tannic acid to prepare gallic acid under acidic conditions, and with the generation of gallic acid, the reaction pH will gradually decrease and then stabilize (gallic acid is gradually saturated), the tannase Tan3 The acid resistance performance fully demonstrates that the enzyme Tan3 of the present invention is very suitable for the enzymatic preparation of gallic acid.

3) Effect of metal ions and inhibitors

20 mM of various metal ions and EDTA were added to 50 mM disodium hydrogen phosphate-citric acid buffer solution (pH 4.0), and after treatment at 30° C. for 30 minutes, the activity was measured by the above-mentioned tannase activity assay. The results are shown in Table 4. Each measurement result is shown with the activity when no addition was taken as 100% relative activity. According to the results, it can be seen that Tannase Tan3 is not inhibited by metal ions and EDTA.

The impact of table 4 metal ions and inhibitors on Tan3

the Relative activity (%) Fe3+ 93 Cu2+ 83

Zn2+ 89 Ca2+ 108 Mg2+ 120 Mn2+ 95 EDTA 99 no added 100

Example 7 Utilize different sources of tannase to prepare gallic acid

1) Weigh 2 kg of tara powder raw material and place it in a raw material extraction tank, add 10L of pure water;

2) At room temperature of 35°C, stirring and extracting for 6 hours, centrifuging and filtering to obtain about 9L of tannic acid extract;

3) 9L of tannic acid extract was heated and concentrated until the concentration of tannic acid reached 30% (w/v), and the pH of the concentrated solution was adjusted to 4.0. Tannic acid content and purity are detected by HPLC method: chromatographic column: C18 (5 μm, 4.6mm × 250mm); column temperature: 25°C; mobile phase: acetonitrile/water=2:98 (containing 0.065% trifluoroacetic acid in acetonitrile containing 0.05% trifluoroacetic acid); flow rate: 1.0ml/min; injection volume: 10μl; detection wavelength: 278nm; gallic acid content and purity are detected by HPLC method: chromatographic column: C18 (5μm, 4.6mm×250mm); column Temperature: 25°C; mobile phase: acetonitrile/water = 2:98 (0.065% trifluoroacetic acid in water and 0.05% trifluoroacetic acid in acetonitrile); flow rate: 0.8ml/min; injection volume: 10μl; detection wavelength: 274nm ;

4) Take 500ml of the above-mentioned tannic acid extract concentrate with a substrate concentration of 30% (w/v) and add them to the reactor, and add 5400U of tannic acid to the reactor according to the amount of 36U/g Tannase Tan3, Tan5, Tan7, Tan8. The pH was not controlled during the reaction, and the temperature was controlled at about 40°C. Take a sample every 2 hours to detect the reduction of tannic acid in the reaction solution, calculate the conversion rate according to the reduction of tannic acid, and detect the content of gallic acid in the sample after the reaction is over, according to the amount of gallic acid generated and the reduction of tannic acid To determine the final conversion rate, the results are shown in Figure 7.

The results showed that tannase Tan5, Tan7 and Tan8 had very poor hydrolysis effect on tara tannin, and the highest conversion rate was only about 30%, while tannase Tan3 could be as high as 30% (w/v) concentration under the same conditions. The continuous hydrolysis of Tara tannin under the tannin substrate produces gallic acid, and the hydrolysis period is short. It only takes 8 hours for complete hydrolysis, and the conversion rate is as high as 99.5%. The sample after hydrolysis is decolorized by activated carbon and vacuum dried. 99%, so that the use of the tannase can be better applied to the industrial mass production of gallic acid, and has obvious advantages compared with the preparation of gallic acid by chemical methods.

In the description of this specification, descriptions referring to the terms "one embodiment", "some embodiments", "example", "specific examples", or "some examples" mean that specific features described in connection with the embodiment or example , structure, material or characteristic is included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms should not be understood as necessarily referring to the same embodiment or example. Furthermore, the specific features, structures, materials or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification.

The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the concept of the present invention, changes and advantages conceivable by those skilled in the art are all included in the present invention, and the appended claims are the protection scope.

Claims (11)

An application of tannase in the preparation of gallic acid, characterized in that the amino acid sequence of the tannase has at least 95% sequence identity with SEQ ID NO: 13 or its mature polypeptide. The application according to claim 1, wherein the amino acid sequence of the tannase has at least 96%, at least 97%, at least 98%, at least 99% or more of the sequence of SEQ ID NO: 13 or its mature polypeptide consistency. The application according to claim 1, wherein the amino acid sequence of the tannase is SEQ ID NO:13 or the mature polypeptide of the polypeptide shown in SEQ ID NO:13. application as claimed in claim 1, is characterized in that, described tannase is derived from aspergillus niger ATCC 13496. The application according to claim 1, characterized in that the optimum reaction temperature of the tannase is 65-75° C., and the optimum reaction pH is 4.0-5.0. Application as claimed in claim 1, is characterized in that, the polynucleotide sequence of described tannase comprises: 1) the polynucleotide sequence of SEQ ID NO:5; And/or, 2) the polynucleotide sequence of SEQ ID NO:5 cDNA sequence; and/or, 3) a polynucleotide sequence that hybridizes to the polynucleotide sequence of 1) or 2) or its full-length complementary strand under very stringent conditions. The application according to claim 1, characterized in that, utilizing tannase to prepare gallic acid specifically comprises the steps of: 1) Prepare a tannic acid solution with a concentration of 25%-35% (w/v), and adjust the pH of the tannic acid solution to 4.0-5.0; 2) Add the tannic acid solution obtained in step 1) into the reaction kettle, add tannase according to the substrate addition amount of 36-54U/g, and hydrolyze to generate gallic acid. The application according to claim 7, characterized in that the concentration of the tannic acid solution in step 1) is 30%. The application according to claim 7, characterized in that, in step 1), the pH of the tannic acid solution is adjusted to 4.0. The application according to claim 7, characterized in that, in step 2), the added amount of tannase is 36U/g substrate added amount. A method for preparing tannase, characterized in that, the method utilizes Aspergillus niger CICC 2462 bacterial strain to express the amino acid sequence of tannase, and the amino acid sequence of said tannase has at least More than 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity; Preferably, the amino acid sequence of the tannase is SEQ ID NO: 13 or the mature polypeptide of the polypeptide shown in SEQ ID NO: 13.

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