CN110564746A

CN110564746A - Acid-resistant tannase, and gene and application thereof

Info

Publication number: CN110564746A
Application number: CN201910716884.3A
Authority: CN
Inventors: 肖安风; 邵嫄; 张永辉; 翁惠芬; 杨秋明; 茹毅; 肖琼
Original assignee: Jimei University
Current assignee: Jimei University
Priority date: 2019-08-05
Filing date: 2019-08-05
Publication date: 2019-12-13

Abstract

The invention relates to the field of bioengineering, and provides a gene for coding acid-resistant tannase, wherein the amino acid sequence of the enzyme is shown as SEQ ID NO: 1, the nucleic acid sequence of the gene is shown as SEQ ID NO: 2, respectively. The invention also discloses an expression vector and a recombinant strain containing the gene; a method for preparing the tannase and application of the tannase. The recombinant Aspergillus niger tannase provided by the embodiment of the invention has acid resistance which is obviously higher than that of the existing tannase, and is stable in a pH range of 2-7, so that the recombinant Aspergillus niger tannase has obvious advantages in food industry, particularly in acid fruit juice beverage treatment; the preparation method provided by the embodiment of the invention has higher tannase yield; in addition, the tannase provided by the embodiment of the invention can be used for industrial mass production of gallic acid under the condition of not controlling pH, so that the industrial cost is greatly reduced.

Description

Acid-resistant tannase, and gene and application thereof

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to acid-resistant tannase, and a gene and application thereof.

Background

The tannase has wide application, is not only applied to clarification of beverages and prevention of ester deterioration in the beverages in the food industry, but also can be used for stabilizing malt polysaccharide in some beverages, improving flavor and being used as a sensitive analysis probe for determining the structure of natural gallic acid esters; during the feed processing process, tannase treatment is adopted, so that the utilization rate of the plant protein of the livestock can be greatly improved, and the cost of animal husbandry is reduced; gallic Acid (GA) is a main product produced in the hydrolysis of a substrate by tannase, is an important precursor substance necessary for the synthesis of trimethoprim (trimethoprim) compounds in the pharmaceutical industry and Propyl Gallate (PG) as an antioxidant in foods, and has recently been used as a raw material for semiconductor photosensitive resins. Therefore, the effective utilization and deep development of tannase become the research hot spot at home and abroad at present.

In the 20 th century, Freudenberg, when cultured in aspergillus niger, found tannase contained in its hyphae; yosuken et al (2004) isolated tannase-producing lactobacilli from human feces and fermented foods. Bartaa et al (2005) have conducted analytical studies on tannase-producing ability of 35 Aspergillus and 25 Penicillium strains to screen out new tannase-producing varieties. However, the activity of tannase produced by strains separated and screened from the nature is not very high, so that the construction of the high-yield engineering bacteria of the tannase by adopting the modern biotechnology is a main way for improving the activity of the tannase. Hatameto et al (1996) cloned the tannase gene of Aspergillus oryzae and transformed the plasmid with tannase gene into Aspergillus oryzae Zeanol strain with low tannase yield, increasing its tannase yield; this lays a foundation for constructing a recombinant system to express tannase efficiently.

The pH value of the prior tannase is suitable in a partial acid and neutral range, and the optimal temperature is between 20 and 60 ℃. This makes the enzyme monoponinase limited during food production, especially the processing of acidic juice beverages. Thus, the existing tannase production technology still needs to be improved.

Disclosure of Invention

The present invention is directed to solving, at least to some extent, one of the technical problems in the art described above. Therefore, the first objective of the invention is to propose a gene encoding acid-resistant tannase. The acid-resistant tannase has the characteristic of high activity under an acidic condition, and meets the industrial requirements of food industry, particularly the treatment of acidic fruit juice beverages.

The second purpose of the invention is to provide an expression vector.

the third purpose of the invention is to propose a recombinant strain.

the fourth purpose of the invention is to provide a method for preparing the acid-resistant tannase.

The fifth purpose of the invention is to propose the application of the acid-resistant tannase.

In a first aspect of the present invention, there is provided a gene encoding an acid-resistant tannase enzyme having an amino acid sequence as set forth in SEQ ID NO: 1, the nucleic acid sequence of the gene is shown as SEQ ID NO: 2, respectively.

the invention separates and clones the nucleic acid sequence of the acid-resistant tannase Tan1 based on a thermal asymmetric PCR method, and sequence analysis results show that the full length of the acid-resistant tannase structural gene is 1713bp, the first 51 bits are signal peptide coding sequences and no intron sequences. The tannase gene obtained by the invention is different from the sequence disclosed by the prior person through Blast comparison, and is a new sequence.

The amino acid sequence of the acid-resistant tannase Tan1 is shown as SEQ ID NO: 1 is shown. Wherein the enzyme comprises 570 amino acids and a stop codon, the 17 th amino acid before the N-terminal analysis is a signal peptide sequence, and the theoretical molecular weight of the mature acid-resistant tannase is 61.14kDa after the signal peptide is removed. The tannase obtained by the invention has biological activity, the enzyme activity is 49.75U/mL, and the tannase has better acid resistance.

According to the acid-resistant tannase Tan1 disclosed by the embodiment of the invention, the optimal pH value is 5.0, and the acid-resistant tannase Tan1 is stable within the range of pH 2-7; the optimum temperature is 50 ℃, and the enzyme activity is higher within the range of 30-50 ℃. Compared with tannase Tan2(Tan2, Yu X W, Li Y Q, et al 2008) derived from the literature, the tannase Tan1 has the characteristic of high activity under acidic conditions, and meets the industrial requirements of the food industry, particularly the treatment of acidic fruit juice and beverages. In addition, the wide temperature activity range of the compound has potential application value in the fields of feed, medicine, wastewater treatment and the like.

In a second aspect of the present invention, according to an embodiment of the present invention, there is provided an expression vector comprising the above-described polynucleotide encoding acid-resistant tannase. Wherein the polynucleotide is a DNA sequence.

in a third aspect of the present invention, according to an embodiment of the present invention, there is provided a recombinant strain obtained by transforming a host cell with the aforementioned expression vector. Wherein, the host cell adopts pichia GS 115.

In a fourth aspect of the present invention, according to an embodiment of the present invention, there is provided a method for preparing acid-resistant tannase, comprising the steps of:

(1) Taking Aspergillus niger genome DNA as a template, and amplifying a tannase gene sequence by designing two pairs of specific primers and utilizing a thermal asymmetric PCR method; wherein the preservation number of the Aspergillus niger is CCTCC NO: m2019358, deposited under the name Aspergillus niger FJ 0118;

(2) Cloning the tannase gene into a plasmid to obtain the expression vector;

(3) Transforming the expression vector into a host cell to obtain the recombinant strain;

(4) And performing induction expression on the recombinant strain on a fermentation tank to obtain the acid-resistant tannase.

According to a further embodiment of the invention, in step (1), the specific primers are:

An upstream specific primer: the sequence of QTan-F1 is shown as SEQ ID NO: 3, the sequence of QTan-F2 is shown as SEQ ID NO: 4;

A downstream specific primer: the sequence of QTan-R1 is shown as SEQ ID NO: 5, the sequence of QTan-R2 is shown as SEQ ID NO: 6.

According to a further embodiment of the present invention, the inducing the expression of the acid-resistant tannase in the step (4) is performed by using a methanol fermenter, and the inducing the expression by the methanol fermenter comprises: a glycerol batch fermentation stage, a starvation stage and a methanol fed-batch stage.

According to a further embodiment of the present invention, the method further comprises separating and purifying the acid-resistant tannase into: purifying protein with 1.6 × 20cm anion exchange column, and balancing column with citric acid buffer solution at flow rate of 1 mL/min; adding acid-resistant crude tannase enzyme solution, incubating for 20min at a flow rate of 1 mL/min; eluting with citric acid buffer solution containing NaCl at flow rate of 1mL/min, collecting and concentrating eluates of each step, and analyzing protein purification by SDS-PAGE.

According to the embodiment of the invention, two pairs of specific primers are designed, a nucleic acid sequence for coding aspergillus niger tannase mature protein is amplified from an aspergillus niger genome by a thermal asymmetric PCR method, the amplified nucleic acid sequence is cloned to a vector, after a certain copy number is obtained, a restriction enzyme is connected to a pichia pastoris expression vector pPIC9K, a recombinant expression vector pPIC9K-Tan1 is constructed, an electric shock method is adopted to transform pichia pastoris GS115, methanol is adopted to perform induced expression, and tannase in centrifugal supernatant is purified to obtain the acid-resistant tannase.

Therefore, the expression vector pPIC9K-Tan1 can efficiently express the target enzyme protein under the induction condition of methanol; the pichia pastoris expression system has less self-secreted background protein and is easy to purify, and DEAE anion exchange chromatography is adopted to purify the protein in one step, so that the loss of enzyme protein is reduced; the pichia pastoris expression system has low glycosylation degree and low nutritional requirement, can adopt a cheap culture medium, and can realize high-density fermentation; the recombinant tannase obtained by the method can be used for treating a juice sample with low pH value, and has the effects of removing astringency and clarifying; and almost completely degrading tannic acid to produce gallic acid under the condition of not controlling pH, thereby greatly simplifying the process operation.

in a fifth aspect of the invention, the invention also provides the application of the acid-resistant tannase in the food industry according to the embodiment of the invention, wherein the acid-resistant tannase is used in the treatment process of acid fruit juice beverages to reduce the content of ester catechin, and the acid-resistant tannase is indicated to be applicable to acid fruit juice treatment.

In a sixth aspect of the present invention, the present invention also proposes the use of the above acid-resistant tannase for the production of gallic acid, according to the examples of the present invention.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Drawings

FIG. 1 shows the PCR amplification electrophoresis result of the Aspergillus niger tannase Tan1 gene; m: DL5000marker, 1-3: gradient amplification products;

FIG. 2 shows the PCR identification of pMD19T-Tan1 cloning vector from bacterial liquid; wherein M: DL5000marker, 1-8: results of amplification with the universal primers, 9-16: specific primer amplification result;

FIG. 3 shows the result of electrophoresis of the linearized expression vector pPIC9K-Tan 1; m: DL15000marker, 1: non-linearized plasmid, 2: linearizing the plasmid;

FIG. 4 shows electrophoresis results of wall-breaking PCR for identifying positive yeast transformants; m: DL5000marker, 1-5: specific primer verification, 6-10: verifying the universal primer;

FIG. 5 shows SDS-PAGE results of expression products expressed by positive transformants induced by methanol; 1: protein marker, 2-16: samples are taken every 8 hours within 8-120 hours;

FIG. 6 is a graph showing the activity change of tannase Tan1 expressed on the pot of methanol-induced positive transformant;

FIG. 7 is SDS-PAGE electrophoretic analysis of the purification result of Tan 1; m: protein marker 1: unpurified protein, 2: purifying the protein;

FIG. 8 is a graph showing the optimum reaction temperature of recombinant tannase; a: tan1, b: tan 2;

FIG. 9 is a thermal stability curve of recombinant tannase; a: tan1, b: tan 2;

FIG. 10 is a pH curve for optimal reaction of recombinant tannase; a: tan1, b: tan 2;

FIG. 11 is a pH stability curve of recombinant tannase; a: tan1, b: tan 2;

FIG. 12 is a liquid chromatogram of a blueberry fruit powder extract treated by recombinant tannase. 1: and (3) GA, 2: GC, 3: EGC, 4: EGCG, 5: EC, 6: GCG (1, 2, 3, 5 are non-ester catechin, 4, 6 are ester catechin);

FIG. 13 is a graph showing how tannic acid is hydrolyzed by the recombinant tannase enzyme method to produce gallic acid;

FIG. 14 is a reaction thin layer chromatography of gallic acid produced by tannin hydrolysis by recombinant tannase; a: tan1 reaction liquid thin layer chromatography, b: tan2 reaction liquid thin layer chromatography, 1: tannic acid standard, 2: gallic acid standard, 3: tannic acid and gallic acid mixed standard, 4: reaction for 0min sample, 5: reaction for 20min sample, 6: reaction for 40min sample, 7: reaction 80min sample, 8: the reaction time was 120 min.

Detailed Description

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the drawings are exemplary and intended to be illustrative of the invention and are not to be construed as limiting the invention.

The following disclosure provides many different embodiments, or examples, for implementing different embodiments of the invention. To simplify the disclosure, specific embodiments or examples are described below. Of course, they are merely examples and are not intended to limit the present 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 of chemistry, molecular biology, and the like, which are within the capabilities of persons skilled in the art. In addition, unless otherwise indicated, nucleic acids are written from left to right in the 5 'to 3' direction and amino acid sequences are written from left to right in the amino terminus to carboxy terminus direction.

the invention is described below by way of illustrative specific examples, which do not limit the scope of the invention in any way. Specifically, the following are mentioned: the reagents used in the present invention are commercially available unless otherwise specified.

Test materials and reagents

1. Bacterial strain and carrier: the Aspergillus niger (Aspergillus niger sp.FJ0118) used by the invention is preserved in the China center for type culture collection in 2019, 05 and 16 months, the preservation number is CCTCC NO: M2019358, the Aspergillus niger sp.FJ0118 Aspergillus niger FJ0118 is classified and named as Aspergillus niger sp.FJ0118, the preservation address is as follows: wuhan, Wuhan university. Cloning vector pMD-19T was purchased from TaKaRa, Pichia expression vector pPIC9k and strain GS115 were purchased from Invitrogen.

2. Enzymes and other biochemical reagents: the endonuclease and ligase were purchased from TaKaRa, rhodanine was purchased from aladddin, and others were made available from general Biochemical Agents.

3. Solution and medium:

(1) PDA medium (for preservation of aspergillus niger): peeling potato 200g, boiling with distilled water until it is soft but not rotten, filtering with eight layers of gauze to obtain juice, adding glucose 20g and agar 20g, metering to volume 1L, subpackaging to test tubes, sterilizing with high pressure steam at 121 deg.C for 20min, and cooling on inclined plane for use.

(2) LB culture medium: 10% of peptone, 5% of yeast powder, 10% of NaCl and 2% of agar, and is used for culturing escherichia coli.

(3) YPD medium: 10% of yeast powder, 20% of peptone, 20% of glucose and pH6.0, and is used for culturing pichia pastoris.

(4) MD culture medium; 20g of glucose and 20g of agar powder are dissolved in 0.9L of distilled water, autoclaved at 121 ℃ for 20min, and sterile 10 XYNB 100mL and 0.02% biotin 2mL are respectively added for use in the plate culture of Pichia pastoris.

(5) BMGY medium: 10g of yeast powder and 20g of peptone are dissolved in 0.7L of distilled water, sterilized at 121 ℃ for 20min under high pressure, and after cooling, 100mL (1M, pH 6.0) of sterile phosphate buffer solution, 10 XYNB 100mL, 100mL of 10 Xglycerol solution and 2mL of 0.02% biotin solution are respectively added into a super-clean workbench for culturing the pichia pastoris.

(6) BMMY medium: the carbon source in the BMGY medium was replaced with 100mL of 0.5% final methanol solution for Pichia pastoris induction culture.

(7) Fermentation medium on tank: 80.1mL of 85% phosphoric acid, 0.093% CaSO4, 1.82% K2SO4, 0.41% KOH, 1.5% MgSO4 & 7H2O, 4% glycerol, 0.5% yeast extract, 0.5% peptone and 0.1% defoaming oil (v/v), dissolving, then, determining the volume to 3L, injecting into a fermentation tank, sterilizing at 121 ℃ for 20min under high pressure, cooling and then preparing for use in the culture of Pichia pastoris.

It should be noted that: the molecular biological experiments, which are not specifically described in the following examples, were performed according to the methods listed in molecular cloning, a laboratory manual (third edition) j. sambrook, or according to the kit and product instructions.

Example 1 Aspergillus niger genome extraction

inoculating Aspergillus niger sp.FJ0118 strain on PDA slant, culturing at 30 deg.C for 4 days, preparing spore suspension with physiological saline, and adjusting to OD₆₀₀2.0, the spore suspension was inoculated into 50mL of PDA liquid medium with glass beads, cultured with shaking at 30 ℃ and 180rpm for 24 hours, centrifuged at 12000rpm to collect the cells, the cells were rapidly ground in liquid nitrogen until they were ground into fine white powder, and then Genomic DNA was extracted using the Plant Genomic DNA Extraction Kit according to the instructions.

Example 2 PCR amplification of tannase Gene

primers were designed using Primer Premier 5 of Premier company according to the sequence information of tannase gene (XM _001390374.1) published on NCBI, and the Primer sequences were as follows:

Tan-F：5’-ATGTCCAAGTTCCTCCTCTGGACC-3’(SEQ ID NO：3)；

Tan-R：5’-CTAGTAGATCGGAATGTCATACGCATCCA-3’(SEQ ID NO：4)；

PCR amplification was performed using the A.niger genomic DNA of example 1 as a template. The reaction parameters are as follows: denaturation at 95 ℃ for 5min, denaturation at 94 ℃ for 30sec, gradient annealing at 58-68 ℃ for 30sec, extension at 72 ℃ for 4min, heat preservation at 72 ℃ for 10min after 28 cycles to obtain a segment of about 2000bp, performing agarose gel electrophoresis verification after amplification, wherein the electrophoresis result is shown in figure 1, and the segment is recovered and sent to Berry biotechnology, Inc. for sequencing.

According to the nucleotide sequence obtained by sequencing, two sets of Tail-PCR specific primers at the upstream and the downstream are respectively designed: the design direction is the unknown region direction needing amplification, and the position of QTan-F2/R2(SEQ ID NO: 5 and SEQ ID NO: 7) is designed at the inner side of QTan-F1/R1(SEQ ID NO: 6 and SEQ ID NO: 8). The distance between every two primers is 60-100 bp, the length of the primers is generally 22-30nt, and the annealing temperature is 60-65 ℃. They were named QTan-F1, QTan-F2 (upstream specific primers), QTan-R1, and QTan-R2 (downstream specific primers) respectively as shown in Table 1.

TABLE 1 tannase Tail-PCR specific primers

Obtaining a flanking sequence of a known gene sequence by reverse Tail-PCR, recovering the amplified product and then sending the product to Berry biotechnology, Inc. for sequencing. The total length of the tannase Tan1 gene after splicing is 1713bp, 570 amino acids are coded, and a stop codon is coded.

Analysis using SignalP (http:// www.cbs.dtu.dk/services/SignalP) showed that the first 17 amino acids of the N-terminus of tannase after splicing were signal peptides. The theoretical molecular weight of the mature protein encoded by the gene is predicted to be 61.14 kDa.

EXAMPLE 3 construction of recombinant cloning plasmid pMD19T-Tan1

1) The tannase gene amplification product is connected with a cloning vector

The primer for redesigning the complete tannase gene obtained in example 2 was amplified, and the obtained product was recovered and purified and ligated with pMD19-T Simple vector (purchased from Takara Co.) to construct a recombinant cloning plasmid.

The primers used for the cloning of the spliced tannase gene were as follows:

WTan-F：5’-CCTCCTTCAGTCTCTCCTTTACCTTTG-3’(SEQ ID NO：9)；

WTan-R：5’-GATGATTCCAGTTGTGGCTGGTATTA-3’(SEQ ID NO：10)；

Ligation was performed according to the instructions provided in the pMD19-T Simple vector ligation kit. The following ingredients were added sequentially to a 0.2mL PCR tube:

after mixing, the mixture is centrifuged instantly and connected for 4 hours at 16 ℃ to obtain a connection product.

2) preparation of competent cells for chemical transformation of E.coli DH5 alpha

Firstly, using LB plate culture medium, using inoculating loop to pick out colibacillus (-20 deg.C glycerol preservation strain), grading and marking on the plate, and inversely culturing for 14-16h at 37 deg.C.

② an activated E.coli DH5 alpha single colony is picked from an LB plate, inoculated in 5mL of LB liquid culture medium and cultured for 12h under shaking at 37 ℃.

③ mixing the culture in a ratio of 1: 100 in 100mL LB liquid medium, and shake-culturing at 37 ℃ to OD₆₀₀When the culture medium was changed to about 0.5, the medium was placed on ice to stop the culture.

and fourthly, transferring 1mL of the bacterial liquid into a 1.5mL centrifuge tube, centrifuging for 10min at 4000rpm and 4 ℃, and removing the supernatant.

Fifthly, the operation is carried out according to the instruction of the conventional Cell Preparation Kit (Takara company prepares the Kit for large intestine competence).

Sixthly, the competent cells are divided into 50 mu L/tube on ice and preserved at the temperature of 80 ℃ to obtain the competent cells DH5 alpha.

3) Transformation of cloning vector and identification of positive transformant

The above competent cells DH 5. alpha. were removed from the freezer at-80 ℃ and thawed in a quick ice bath. Adding the ligation product into a colibacillus infected cell DH5 alpha, gently mixing, carrying out ice bath for 30min, carrying out water bath heat shock for 90s at 42 ℃, immediately carrying out ice bath for 2min, adding 1mL of LB liquid culture medium, and recovering for 2h at 37 ℃. Then, 200. mu.L of the bacterial suspension was applied to an LB plate containing Amp resistance (final concentration: 100. mu.g/mL) and cultured in an inverted state at 37 ℃ for 12 to 16 hours.

positive single colonies were picked and inoculated into 5mL of LB liquid medium containing Amp resistance (final concentration: 100. mu.g/mL), and cultured overnight at 37 ℃ and 180 rpm. Then, PCR verification of positive single colonies and sequencing analysis of the bacterial liquid show that the cloning vector is successfully transformed.

The total volume of the PCR verified reaction was 15. mu.L, and the following ingredients were added sequentially to a 0.2mL PCR tube:

and (3) after uniform mixing, performing instantaneous centrifugation, wherein the reaction parameters are as follows: denaturation at 94 deg.C for 2 min; denaturation at 94 ℃ for 30sec, gradient annealing at 60 ℃ for 30sec, extension at 72 ℃ for 1min for 30sec, and heat preservation at 72 ℃ for 2min after 28 cycles.

The PCR result of the bacterial liquid is shown in FIG. 2, and the comprehensive sequencing result shows that the Aspergillus niger tannase gene is successfully connected to the pMD19-T vector.

EXAMPLE 4 construction of recombinant expression vector pPIC9K-Tan1

1) preparation of tannase gene fragment to be inserted:

Considering that the signal peptide sequence carried by the gene itself can interfere the heterologous expression of pichia pastoris, the predicted signal peptide is removed when designing the primer, and the enzyme cutting site is added, the primer sequence is as follows:

MTan-F：5’-GAGACCTAGGGCGACTCTCGAGCAGGTCTGTA-3 '(SEQ ID NO: 11), 5' incorporates a protective base and an Avr II cleavage site (underlined), respectively

MTan-R：5’-ATTGCGGCCGCCTAGTAGATCGGAATGTCATACGCATC-3 '(SEQ ID NO: 12), 5' incorporates a protecting base and a Not I cleavage site (underlined)

pMD19T-Tan1 recombinant plasmid is used as a template, the total reaction volume is 50 mu L, and the following components are sequentially added into a 0.2mL PCR tube:

And (3) after uniform mixing, performing instantaneous centrifugation, wherein the reaction parameters are as follows: denaturation at 95 deg.C for 2 min; denaturation at 95 ℃ for 20sec, gradient annealing at 60 ℃ for 20sec, extension at 72 ℃ for 1min, and heat preservation at 72 ℃ for 5min after 28 cycles.

The Aspergillus niger tannase gene from which the signal peptide was removed was recovered using a small DNA fragment rapid gel recovery kit (purchased from Takara) according to the procedures provided in the product instructions.

According to the enzyme cutting sites carried by the designed upstream and downstream primers, the recovered and purified tannase gene PCR product is subjected to double enzyme cutting by using Avr II enzyme and Not I enzyme, and a 20 mu L enzyme cutting system is as follows:

mixing, centrifuging instantaneously, and digesting at 37 deg.C for 12 h. The cleavage products were electrophoresed on 1.0% agarose gel, and the results were observed. The band of interest was cut and placed into a 1.5mL centrifuge tube. Using a small amount of DNA fragment flash gel recovery kit (purchased from Takara Co.), purification and recovery were carried out according to the procedures provided in the product instructions.

2) Pichia pastoris expression vector pPIC9K (from Invitrogen) and recovery thereof

The double digestion was carried out using a 20. mu.L system as follows:

Mixing, centrifuging instantaneously, and digesting at 37 deg.C for 12 h. The cleavage products were electrophoresed on 1.0% agarose gel, and the results were observed. The bands of interest were cut and placed into 1.5mL centrifuge tubes. Purification and recovery were carried out using a small DNA fragment flash gel recovery kit (purchased from Takara) according to the procedures provided in the product instructions.

3) ligation of the enzyme-cleaved fragment to an expression vector

The Aspergillus niger tannase gene obtained in the step 1) is enzymatically linked with the expression vector pPIC9K obtained in the step 2) to construct an expression vector pPIC9K-Tan 1. The linking system is as follows:

Mixing, centrifuging instantly, and connecting at 16 deg.C for 20min to obtain the final product.

4) ligation product transformed large intestine competent cell DH5 alpha and positive transformant verification

The competent cells DH 5. alpha. of example 3 were removed from the freezer at-80 ℃ and thawed in a rapid ice bath. Adding the ligation product obtained in the step 3) into an escherichia coli competent cell DH5 alpha, gently mixing, carrying out ice bath for 30min, carrying out water bath heat shock for 90s at 42 ℃, immediately carrying out ice bath for 2min, adding 1mL of LB liquid culture medium, and recovering for 2h at 37 ℃.200 mu L of the bacterial liquid was spread on an LB plate containing Amp resistance (final concentration: 100. mu.g/mL), and cultured in an inverted state at 37 ℃ for 12-16 hours.

and (3) selecting a positive single colony, inoculating the positive single colony into 5mL LB liquid culture medium containing Amp resistance (the final concentration is 100 mu g/mL), culturing the positive single colony at 37 ℃ and 180rpm overnight, verifying the positive single colony through bacterial liquid PCR (polymerase chain reaction) and indicating successful transformation of an expression vector through sequencing analysis.

the total reaction volume was 15. mu.L, and the following components were sequentially added to a 0.2mL PCR tube:

the comprehensive sequencing result shows that the Aspergillus niger tannase gene is successfully connected into a pPIC9K vector.

example 5 expression vector pPIC9K-Tan1 transformation of Pichia pastoris GS115

1) the expression vector pPIC9K-Tan1 constructed in example 4 was linearized by a single digestion with SalI, using a 20. mu.L system, and the following components were added:

mixing, instantaneous centrifugation, and reaction conditions: reacting at 37 ℃ for 12h, detecting whether the linearization is complete by agarose gel electrophoresis after the reaction is finished, and obtaining a result as shown in figure 3, wherein the linearization is a single band and the electrophoresis speed is slower than that of the non-linearization, and the PCR product is recovered and stored at-20 ℃ for later use.

2) Transformation of Yeast strains and screening for Positive clones

transforming the expression vector pPIC9K-Tan1 linearized in the step 1) into pichia pastoris GS115 competent cells through an electric shock transformation method, adding pre-cooled 1mol/L D-sorbitol solution, recovering for 2 hours at 30 ℃, then coating 200 mu L of bacterial liquid in an MD solid culture medium to primarily screen positive clones, performing inverted culture at 30 ℃ for 2-3 days, and observing the growth condition of single colonies.

Then, single colonies were picked from MD plates and inoculated into YPD plates containing G148 resistance (final concentration: 2.5mg/mL) for secondary screening of multiple copies, and cultured at 30 ℃ until colonies were grown. And selecting colonies, inoculating the colonies into a non-resistance liquid YPD culture medium, culturing for 16-18 h, and performing wall-breaking PCR identification on the bacteria liquid treated by TE/SDS to verify positive transformants.

The wall-broken bacteria liquid PCR system is as follows:

and (3) after uniform mixing, performing instantaneous centrifugation, wherein the reaction parameters are as follows: denaturation at 94 deg.C for 2 min; denaturation at 94 ℃ for 30sec, gradient annealing at 60 ℃ for 30sec, extension at 72 ℃ for 1min for 30sec, and heat preservation at 72 ℃ for 2min after 28 cycles. After the reaction is finished, agarose gel electrophoresis is carried out for analysis and identification, the result is shown in figure 4, five transformants are all shown as positive clones, and a transformant template is determined according to the sequencing result.

Example 6 Induction of high expression of recombinant A.niger tannase Tan1

1) Seed liquid preparation

The recombinant strain obtained by screening in example 5 was inoculated into YPD medium for activation, and cultured at 30 ℃ and 250rpm for 16-18 hours. Then, the activated strains are treated according to the proportion of 1: 100 percent of the seed culture medium is inoculated into YPD seed culture medium, and the seed culture medium is cultured for 16-18 h at 30 ℃ and 250rpm to obtain 150mL of seed solution.

2) Fermentation on tank

adding 3L of fermentation medium into a 5L fermentation tank, sterilizing at 121 ℃ for 20min, cooling, adding 13.2mL of trace element PTM, adjusting the pH value to 5.2 by ammonia water, and mixing according to the proportion of 1: inoculating seed liquid at a ratio of 20, controlling the temperature at 30 deg.C and the rotation speed at about 600rpm during fermentation. The fermentation is specifically divided into the following three stages:

Batch fermentation stage of glycerol: 16-24 h before fermentation culture (dissolved oxygen (DO) > 40%, ventilation volume: 4L), until the glycerol in the culture medium is exhausted (DO rapidly rises), supplementing the glycerol until the bacterial mass reaches 180 mg/mL.

② starvation stage: after the thalli reach the required density, glycerol is stopped being supplemented, Dissolved Oxygen (DO) rises rapidly, and no carbon source is added at the moment, so that the starvation state of the thalli is ensured for 30min, and the utilization of methanol by the thalli is prevented from being influenced.

③ methanol feeding stage: after starvation for 30min, the fermentation temperature was adjusted to 28 ℃ and the pH was maintained at about 5.0, and the methanol induction phase was started. Adding methanol (containing 1.2 percent of PTM) 12h before induction, wherein the feeding speed is 1.5 mL/h; after 12h induction, methanol was added at 3mL/h for 96 h.

The protein expression of the supernatant of the fermentation broth was examined by SDS-PAGE, and the results are shown in FIG. 5, in which the amount of the produced enzyme increased with the increase of the fermentation time.

the glycerol is supplemented every 8 hours to sample and determine biomass, the methanol induction stage every 8 hours to sample and determine enzyme activity and biomass, the result is shown in figure 6, the biomass can reach 330mg/mL at most, after 120 hours of fermentation induction on a tank, the enzyme activity reaches 49.75U/mL, and compared with the shake flask fermentation, the enzyme activity is improved by 53 times. ZHONG et al heterologously express the single tannase gene of aspergillus oryzae by using pichia pastoris, and the maximum enzyme activity is 7000 IU/L; selwal et al produced tannase by liquid fermentation of Penicillium, and the enzyme activity was 34.7U/mL. The recombinant Aspergillus niger tannase obtained by the method has enzyme activity higher than that of tannase obtained by Zhong, Selwal and the like.

Example 7 isolation and purification of recombinant A.niger tannase

the recombinant A.niger tannase obtained in example 6 above was purified using DEAE Sepharose Fast Flow on a column format of 1.6X 20 cm. The separation and purification method comprises the following steps:

The fermentation supernatant obtained in example 6 was filtered through a 0.22 μm filter to remove impurities, and then refrigerated at 4 ℃ for future use. Purifying with DEAE Sepharose Fast Flow ion exchange chromatography column, eluting with citric acid buffer solution (10mmol/L, pH5.0) and NaCl linear gradient (0.05-0.1mol/L) at Flow rate of 1mL/min, collecting 3mL per tube until OD is reached₂₈₀The value is zero.

Respectively determining protein content and tannase activity of each tube, mixing eluates with tannase activity, ultrafiltering and concentrating with 10kDa ultrafiltration membrane, and performing SDS-PAGE to verify and analyze purification conditions, wherein the result is shown in FIG. 7, and the purified product is a single band.

Example 8 determination of optimum reaction temperature and thermal stability of tannase

After a certain period of treatment at various temperatures (30, 40, 50, 60, 70 ℃), the enzyme activity was measured (in reference to the method of Sharma (2000), the amount of enzyme required to produce 1. mu. mol of gallic acid per minute at 30 ℃ is defined as one enzyme activity unit U). And (3) when measuring the thermal stability, keeping the enzyme solution at different temperatures for different times, measuring the residual enzyme activity, and taking the enzyme solution with the highest enzyme activity of 100% and the enzyme solution inactivated for 10min as a blank control. The results are shown in FIGS. 8 and 9. The optimal reaction temperature of the recombinant Aspergillus niger tannase Tan1 is 50 ℃, and the relative enzyme activity of more than 70% can be maintained at 65 ℃. After the recombinant tannase Tan1 is treated at the temperature of lower than 50 ℃ for 120min, the enzyme activity is basically stable and unchanged, but the enzyme activity tends to increase. Keeping the temperature at 60 ℃ for 120min and still keeping more than 50 percent of enzyme activity; the optimum reaction temperature of Tan2 is 70 deg.C, after 10min of treatment in 70 deg.C water bath environment, about 10% of tannase activity remains, and half-life periods at 60 deg.C and 50 deg.C are 8min and 20min respectively. The above results show that the two tannases have good thermal stability compared with the tannase from filamentous fungi reported in the literature, and Fuentes-Garibay et al report that the tannase from a strain of Aspergillus niger (Xerophilic Aspergillus niger) loses 50% of activity after being kept at 30 ℃ for 120min, which shows that the temperature stability difference of the recombinant tannase from different sources is significant. Wherein Tan1 has better heat resistance at high temperatures than Tan 2.

example 9 determination of optimum reaction pH and acid-base stability of tannase

Diluting the enzyme solution to a proper multiple with buffer solutions with different pH values (1 pH unit is a gradient), uniformly mixing 0.25mL of the enzyme solution with a substrate with the same volume and pH value, and measuring the activity of the tannase at 30 ℃. And (3) taking enzyme liquid with the highest enzyme activity of 100% and inactivation time of 10min under different pH conditions as a blank control, and researching the optimum pH of the recombinant tannase. When the acid-base stability is measured, enzyme solution is diluted by buffer solutions with different pH values, placed for 24 hours at 4 ℃, and the enzyme activity is measured under the standard condition. The measurement results are shown in fig. 10 and 11. The optimal reaction pH of the recombinant Aspergillus niger tannase Tan1 is 5.0. The research shows that the optimum pH of most of the tannase derived from the filamentous fungi is in an acidic range (4.3-6.5), and the research result is similar to the optimum pH of different tannases derived from the filamentous fungi. Particularly, Tan1 is stable in an acidic to neutral environment (pH is 2.0-7.0), and the enzyme activity is not basically lost (the residual relative enzyme activity is 97.65%) after the Tan1 is placed for 24 hours under the acidic condition; tan2 has an optimum reaction pH of 5.0 and a pH stability of 3 to 7. Tan1 shows better stability in a lower pH environment than Tan 2.

Example 10 comparison of recombinant Aspergillus niger tannase Tan1 with tannases from other sources

The literature reports that there are differences in the enzymatic properties of tannase from different sources. The pH value stability range of the fungus and plant tannase is generally between 4.0 and 6.0. The optimum pH value of the bacterium tannase is usually in a neutral range, and the stable range of the fungus tannase is 3.0-7.0. The data on the enzymatic properties of tannase are shown in Table 1, and compared with most of the tannase derived from fungi in Table 1, the enzyme Tan1 of the present invention has very good stability under acidic conditions, and can be used for the treatment of acidic foods.

TABLE 1 enzymatic Properties of tannase from different sources

Ng data not given in the literature

Example 11 application of tannase in blueberry juice treatment

in view of the fact that the tannase obtained by the method is good in stability under an extremely acidic condition, the influence of adding the recombinant aspergillus niger tannase Tan1 in a blueberry powder leaching solution on the polyphenol content of the blueberry powder leaching solution is researched.

Extracting blueberry polyphenol:

Taking 0.3g of dried blueberry powder to a test tube with a plug, and mixing the dried blueberry powder and the test tube according to a volume ratio of 1: 30 adding an extraction solvent, adjusting the pH value to 2.0 by using 2M HCl, screwing a pipe plug, performing ultrasonic extraction for 1h (the frequency is 400Hz, the temperature is 45 ℃), centrifuging at 4000rpm for 5min, and separating supernatant from precipitate, wherein the supernatant is the polyphenol substances in the extracted blueberries.

Treating blueberry powder extract by recombinant aspergillus niger tannase:

And taking 4mL of the supernatant as a substrate, adding 1mL of recombinant Aspergillus niger tannase Tan1 enzyme solution, treating at 50 ℃ for 2h, and carrying out liquid phase analysis after the obtained sample passes through a membrane. As shown in fig. 12, the content of ester catechin decreased and the content of non-ester catechin increased after the enzyme treatment, indicating that the tannase Tan1 of the present application can be applied to the treatment of blueberry juice. Since ester catechin is a source of bitterness and astringency in fruit juice, reducing the proportion of ester catechin in blueberry polyphenol can reduce astringency in fruit juice and improve the storage quality of fruit juice.

Example 12 application of tannase to gallic acid production

Dissolving 2g tannic acid in 50 deg.C distilled water for 10min, adding 450U tannase Tan1 or Tan2, sampling every 20min to detect gallic acid generation, and maintaining the reaction temperature at 50 deg.C. And detecting the change of tannic acid and gallic acid in the leaching solution by using a thin layer, wherein the developing agent comprises: n-butanol: acetic acid: water (v/v/v) ═ 2:2:1, developer: 3% FeCl₃。

as shown in fig. 13, the initial pH of the reaction was 5.0, and as the reaction proceeded, the pH was maintained at substantially 3.0, under which the conversion rate of gallic acid of Tan1 reached substantially 100%, but the conversion rate of gallic acid of Tan2 reached only 34.5% at the maximum, corresponding to that shown in fig. 14, the thin layer analysis result showed that Tan1 was substantially free of tannic acid after hydrolysis reaction for 120min, and it was converted into gallic acid; however, a large amount of tannic acid still exists after Tan2 hydrolysis reaction for 120min, so Tan1 has more application potential under extremely acidic conditions compared with Tan 2.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not to be understood as necessarily referring to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples described in this specification can be combined and combined by those skilled in the art.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.

sequence listing

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Claims

1. a gene encoding acid-resistant tannase, wherein the amino acid sequence of the acid-resistant tannase is as shown in SEQ ID NO: 1, and the nucleic acid sequence of the gene is shown as SEQ ID NO: 2, respectively.

2. the gene encoding acid-resistant tannase according to claim 1, wherein the gene is obtained by PCR amplification from Aspergillus niger sp.FJ0118 deposited at the China center for type culture Collection at 2019, 05 and 16 days with the deposit number of CCTCC NO: M2019358.

3. an expression vector comprising the gene encoding acid-resistant tannase according to claim 1.

4. A recombinant strain obtained by transforming a host cell with the expression vector of claim 3.

5. The recombinant strain of claim 4, wherein the host cell is Pichia pastoris GS 115.

6. The preparation method for preparing the acid-resistant tannase is characterized by comprising the following steps of:

(2) Cloning the tannase gene into a plasmid to obtain the expression vector of claim 3;

(3) Transforming a host cell with the expression vector to obtain the recombinant strain of claim 4;

7. The method according to claim 6, wherein in the step (1), the specific primers are:

8. The method of claim 6, wherein the inducing the expression of the acid-resistant tannase in the step (4) is performed by using a methanol fermentor, and the inducing the expression by the methanol fermentor comprises: a glycerol batch fermentation stage, a starvation stage and a methanol fed-batch stage.

9. The method of claim 6, wherein: further comprising the step of separating and purifying the acid-resistant tannase, wherein the separation and purification are as follows: purifying protein with 1.6 × 20cm anion exchange column, and balancing column with citric acid buffer solution at flow rate of 1 mL/min; adding acid-resistant crude tannase enzyme solution at flow rate of 1mL/min, and incubating for 20 min; eluting with citric acid buffer solution containing NaCl at flow rate of 1mL/min, collecting and concentrating eluates of each step, and analyzing protein purification by SDS-PAGE.

10. Use of the acid-resistant tannase of claim 1 in the food industry.

11. Use of the acid-resistant tannase of claim 1 in the production of gallic acid.