CN102719413A

CN102719413A - Novel tannase and application thereof

Info

Publication number: CN102719413A
Application number: CN2012101322770A
Authority: CN
Inventors: 刘玉焕; 姚健; 范新炯
Original assignee: National Sun Yat Sen University
Current assignee: Sun Yat Sen University; National Sun Yat Sen University
Priority date: 2012-04-28
Filing date: 2012-04-28
Publication date: 2012-10-10

Abstract

The invention discloses a novel tannase. An amino acid sequence of the tannase is shown as SEQ ID NO.3. The invention further discloses a cDNA of the novel tannase, and a nucleotide sequence of the cDNA is shown as SEQ ID NO.1. The invention further discloses an expression vector containing the novel tannase cDNA, recombinant tannase, a preparation method of the recombinant tannase and degradation application of the recombinant tannase by taking propyl gallate, tannic acid, epicatechin gallate, epigallocatechin-3-gallate, chlorogenic acid, rosmarinic acid and ethyl ferulate as substrates. The novel tannase and recombinant tannase have soluble expression in an escherichia coli expression system with high efficiency, and the recombinant tannase has efficient degradation effect on tannic acid, epicatechin gallate, epigallocatechin-3-gallate, chlorogenic acid, rosmarinic acid and ethyl ferulate.

Description

A kind of novel tannase and application thereof

Technical field

The invention belongs to the genetically engineered field, be specifically related to a kind of from the grand genomic library of bottom silt separation screening to novel tannase gene, heterogenous expression and an application thereof.

Background technology

In the production process of tea drink, can become muddy after the high temperature extraction liquid cooling but of tealeaves, and produce flocks (tea cream), this has had a strong impact on the mouthfeel and the fragrance of tea drink.The forming process of tea cream is: when temperature is higher; Materials such as tea-polyphenol in the tea extract and oxide compound thereof, trimethyl-xanthine, protein and lipid all exist with free state; Reduction along with temperature; Polyphenols in the millet paste (especially theoflavin, thearubigins and gallic acid ester etc.) forms complex compound with amino, the proteinic peptidyl hydrogen bonded of the ketone of trimethyl-xanthine respectively, and generation is condensed and precipitated gradually.In the production of tea drinks process, the method that need take physics, chemistry or enzyme to handle is eliminated tea cream or is made it to form solvend.Method with removing " tea cream " has physics method, chemical method and enzyme process.Wherein, because enzyme process has economy, efficiently and advantage such as non-secondary pollution than physics method and chemical method, become current main research focus.

In the last few years; Utilize pure culture technigne from environment, to screen the mikrobe that can produce tannase; But kind is very limited, mainly concentrates on Aspergillus, penicillium and the Rhizopus of Mycophyta, and these microbial enzyme amounts that screen are few; Enzymic activity is not high yet, causes the increase of production cost.So, investigator's (like aspergillus oryzae and lactobacillus plantarum) clone tannase gene from these mikrobes that screened is arranged, then it is expressed in engineering bacteria.But the less stable of these reorganization degrading enzymes has certain limitation in practical application.Up to the present, but all research about tannase all is aimed in the environment culturing micro-organisms comes unfolded.We know; Containing a large amount of Microbial resources in the environment; And the mikrobe above 99% in these mikrobes is difficult to study through the mode of traditional mikrobe separation and Culture; So traditional mikrobe separation and Culture technology has limited people's utilization and exploitation voluntary to the mikrobe that can produce tannase greatly.The appearance of grand genomics; Then solved the problem of this respect,, directly from environmental sample, extracted genome DNA because it has avoided the difficult problem of environmental microorganism separation and Culture; Set up grand genomic library, and then therefrom screen new gene or biologically active substance.Microorganism hereditary information that grand genomic library has comprised is all in the environment (educable with can not cultivate); Therefore; Excavate and utilize in the environment those can not the culturing micro-organisms resource aspect, great potential is arranged, become the field, forward position and the focus of current microbiological research in the world.So far Chinese scholars has been through this technology having screened like new gene such as glycase, zytase, proteolytic enzyme, cellulase, esterase/lypase of success from environment, and these enzymes have some new enzymatic properties and industrial application potentiality.Up to the present, do not utilize grand genome-based technologies from environment, to screen the research report of tannase gene as yet.

Summary of the invention

First purpose of the present invention provides the cDNA of a kind of novel tannase and said novel tannase.

Second purpose of the present invention provides a kind of cloning process of grand genomics of above-mentioned novel tannase.

The 3rd purpose of the present invention provides a kind of expression vector that contains above-mentioned tannase cDNA.

The 4th purpose of the present invention provides a kind of reorganization tannase that utilizes above-mentioned expression vector establishment and preparation method thereof, and the application of said reorganization tannase.

For realizing first purpose of the present invention, the technical scheme of taking is: a kind of novel tannase, the aminoacid sequence of said tannase is as shown in SEQ ID NO.3.

A kind of cDNA of above-mentioned novel tannase, the nucleotide sequence of said cDNA is shown in SEQ ID NO.1.

For realizing second purpose of the present invention, the technical scheme of taking is: a kind of grand genomics cloning process of above-mentioned novel tannase comprises following steps: extract total DNA of ocean, South Sea mud and purifying; With the total DNA behind the purifying after the BamHI enzyme is cut processing; Be connected on the pUC118 carrier, electric shock transforms to import in the bacillus coli DH 5 alpha sets up grand genomic library, obtains positive colony through high flux screening; Through order-checking and BLAST relatively and design primer, thus be cloned into the purpose fragment.

For realizing the 3rd purpose of the present invention, the technical scheme of taking is: a kind of expression vector that comprises the cDNA of aforesaid novel tannase.

For realizing the 4th purpose of the present invention, the technical scheme of taking is: a kind of preparation method of the tannase of recombinating may further comprise the steps: transform host cell with the described expression vector of claim 4, the culture transformation body obtains the reorganization tannase from culture.

Preferably, among the preparation method of above-mentioned said reorganization tannase, said host cell is intestinal bacteria.

Preferably; The preparation method of said reorganization tannase specifically may further comprise the steps:, be connected with pET-28a (+) carrier through BamHI and HindIII double digestion with the described purpose fragment of claim 4, be converted into e. coli bl21; Induce through IPTG, obtain highly-soluble and express.

Preferably, among the preparation method of above-mentioned said reorganization tannase, described IPTG final concentration is 0.4mM, and inducing temperature is 25 ℃.

A kind of employing is the reorganization tannase for preparing of method as stated.

The application of a kind of above-mentioned described reorganization tannase in degraded Tenox PG, Weibull, plain catechin and gallate, NVP-XAA 723, chlorogenicacid, rosmarinic acid and Ferulic acid ethylester.Said reorganization tannase has Degradation to catechin and other substrate.

Beneficial effect of the present invention is:

(1) the present invention screens a tannase gene from the grand genomic library of bottom silt; Through genetic engineering technique to its functional study; Find this gene efficient soluble-expression in intestinal bacteria,, obtain a single protein band through protein purification and SDS-PAGE electrophoresis; Deduct fusion rotein, confirm that tentatively the molecular weight of this tannase is about 54kDa.

(2) the present invention is cloned into the dna sequence dna shown in the SEQ ID NO.1 on the prokaryotic expression carrier, and the transformed into escherichia coli competent cell obtains recombinant protein through the abduction delivering to positive colony, studies its zymologic property, and the result is following:

1. in coli expression system, this recombinant protein has highly-soluble expresses.

2. be substrate with the Tenox PG, the optimal reactive temperature of the recombinant protein of survey is 30 ℃, and is comparatively stable when temperature is lower than 45 ℃, and 45 ℃ of insulation 12h still possess 75% relative activity; Optimal reaction pH is 6.4, under neutrality or solutions of weak acidity, preserves stable; The NaCl of 4M shows higher salt tolerance to the not influence of its enzymic activity; Measure when metals ion and biochemical reagents influence enzyme activity and find 1mMCa ²⁺, Cd ²⁺And Mg ²⁺Its enzymic activity there is activation, 1mM Mn ²⁺, Cu ²⁺, Zn ²⁺, Co ²⁺, A1 ³⁺, EDTA, urea and Triton X-100 be to the not significantly influence of its enzymic activity, and 1mM Ag ⁺, Cr ²⁺, beta-mercaptoethanol, Tween 80 and PMSF have had strong inhibitory effects to its enzymic activity.

(3) the present invention is when research reorganization tannase is the Degradation of substrate with 0.5mg/mL Tenox PG, Weibull, L-Epicatechin gallate, NVP-XAA 723, chlorogenicacid, rosmarinic acid and Ferulic acid ethylester; Analyze discovery through HPLC; Under 30 ℃ of conditions; Through behind the 40min; Weibull, L-Epicatechin gallate, NVP-XAA 723, chlorogenicacid and Ferulic acid ethylester be complete hydrolysis, and nearly 88% rosmarinic acid is hydrolyzed.

Description of drawings

Fig. 1 is the SDS-PAGE electrophorogram in the embodiment of the invention 1.

Among Fig. 1, M is standard protein molecular weight maker, and 1 is the recombinant protein crude extract, and 2 is purified recombinant albumen.

Fig. 2 is the influence figure of temperature to reorganization tannase enzymic activity.

Fig. 3 is that (◇ represents 25 ℃ to temperature to the stable figure that influences of reorganization tannase; ● represent 35 ℃; Zero represents 45 ℃; ■ represents 50 ℃; represents 55 ℃).

Fig. 4 is the influence figure of pH to reorganization tannase enzymic activity.

(it is 5.0 that ■ represents pH to Fig. 5 to the stable figure that influences of reorganization tannase for pH; It is 6.0 that △ represents pH; It is 7.0 that represents pH; Zero to represent pH be 8.0; It is 9.0 that ◇ represents pH; ● representing pH is 10.0; ◆ representing pH is 11.0).

Fig. 6 is the influence figure of NaCl to the Tan410 enzymic activity.

Fig. 7 is the HPLC analysis of Tenox PG, Weibull, L-Epicatechin gallate (ECG), NVP-XAA 723 (EGCG), chlorogenicacid, rosmarinic acid and Ferulic acid ethylester degraded.

Embodiment

The present invention is further described the object of the invention, technical scheme and advantage for better explaining below in conjunction with accompanying drawing and specific embodiment.

Acquisition, the gene cloning and expression of the foundation of embodiment 1 grand genomic library and positive colony

1, the extraction of genomic dna

(1) gets bottom silt sample 4g, put into the aseptic centrifuge tube of 50ml;

(2) add 13.5ml DNA extraction liquid buffer, 37 ℃, 220r/min shaking table concussion 30min;

(3) add 1.5ml 20%SDS;

(4) 65 ℃ of water-bath 2h, during every put upside down centrifuge tube gently several times up and down, mixing at a distance from 20min;

(5) 6000 * g, 4 ℃ of centrifugal 10min;

(6) get supernatant, add isopyknic chloroform, put upside down gently several times up and down;

(7) 16000 * g, 4 ℃ of centrifugal 10min;

(8) repeat (6), (7) step twice;

(9) get supernatant, add the Virahol of 0.6v, behind the mixing, room temperature is placed 1-2h gently;

(10) 16000 * g, 4 ℃ of centrifugal 20min;

Twice of (11) 75% washing with alcohol;

(12) air-dry back ddH ₂O is resuspended.

2, test kit method purify DNA: reclaim the test kit specification sheets according to glue and carry out.

3, grand genome electrophoresis detection: the purity and the quality that detect total DNA with 1% agarose gel electrophoresis

4, enzyme is cut genome DNA: with the partially digested total DNA of restriction enzyme BamHI, reclaim the endonuclease bamhi of 3-10kb, method is with test kit method purify DNA.

5, the electrophoresis detection of endonuclease bamhi: method is with grand genome electrophoresis detection.

6, the structure of cloning vector and conversion: the endonuclease bamhi that recovery is obtained is connected with pUC118/BamHI (BAP) carrier and spends the night, and connects product and carries out behind the purifying and electric transformed competence colibacillus cytomixis with PCR product purification test kit, places 5min on ice; In the 2mm electroporation container (still) of transfer DNA/cell mixture to precooling, to the electroporation container carry out pulse (2,500V) (testing time constant; Should be more than 4), the LB that adds 900 μ L immediately to the electroporation container, 37 ℃; 200r/min; 45min is cultivated in concussion, and converted product is applied to and contains 100 μ g/mL penbritins (Amp), on the flat board of 0.5mM sec.-propyl-β-D-thio-galactose pyran-glucoside (IPTG) and 100 μ M 5-bromo-4-chloro-3-indoles-β-D-galactosides (X-Gal); Flat board is inverted in 37 ℃ of incubators, cultivates 16-20h.Make up a storage capacity thus and reached 96000 transformants, grand genomic library that variety is good.

7, the evaluation of library screening and positive colony

(1) preliminary screening of tannase gene

Institute's adularescent transformant in the library transferred to contain on substrate (X-caprylate) screening culture medium; Behind 37 ℃ of cultivation 48h, picking produces clone's of blue hydrolysis circle on screening culture medium.

(2) the multiple sieve of tannase gene

Picking produces clone's of blue hydrolysis circle on screening culture medium, be inoculated into 10ml and contain in the LB substratum of 100 μ g/mLAmp, 0.5mM IPTG 37 ℃; The 220r/min incubated overnight, 12000 * g, centrifugal 1min collects thalline; Phosphate buffered saline buffer (100mM, pH 6.8) washs thalline once, centrifugal collection thalline; The phosphate buffered saline buffer thalline that suspends again, the ultrasonic disruption thalline, centrifugal collection supernatant (crude enzyme liquid) is as the active detection of tannase.Get 200 μ L crude enzyme liquids; The Tenox PG that adds 200 μ L 15mM, 37 ℃, water-bath 5min; Then add 200 μ L methyl alcohol rhodanines (0.667%); Add 200 μ L KOH (0.5M) behind 37 ℃ of placement 5min, place 5min, can make Tenox PG solution become mauve positive clone for 37 ℃ by safran.Obtain positive colony (pUC118-13B) through screening.

The plasmid of positive colony is sent to order-checking company to check order; Sequencing result shows that the nucleotide sequence of tannase gene is by 1476 based compositions, and its nucleotide sequence is shown in SEQ ID NO.1; The polypeptide of this dna encoding; Contain 491 amino acid, its aminoacid sequence is shown in SEQ ID NO.3, with its called after Tan410.Wherein SEQ ID NO.2 is the map of SEQ ID NO.1 and SEQ ID NO.3.

8, the clone of gene fragment: according to sequencing result design primer: F1 and F2, the HindIII and the BamHI restriction enzyme site that can insert pET-28a (+) carrier introduced at the primer two ends, and primer sequence is following:

P1:5 '-CGC GGATCCATGCTGCCCGCCTTCTGCCGC-3 ', underscore are BamHI restriction enzyme site sequence;

P2:5 '-CCC AAGCTTATAAGTCTTGGGTTCGTAGG-3 ', underscore are HindIII restriction enzyme site sequence.

Utilizing two primers, is that template is carried out pcr amplification reaction with plasmid pUC118-13B, and the PCR system is following:

The PCR reaction conditions is: 94 ℃ of preparatory sex change 4min, and cycling condition is 94 ℃, 1min; 60 ℃, 1min; 72 ℃, 1.5min, totally 5 circulations, 94 ℃, 1min; 52.5 ℃, 1min; 72 ℃, 1.5min, totally 24 circulations, last 72 ℃ are extended 10min.The PCR product with PCR product purification test kit purifying after with BamHI, HindIII double digestion 24h; With through the same pET-28a (+) that handles (Invitrogen) expression vector be connected, Transformed E scherichia coli BL21 (DE3), conversion fluid are coated on the LB solid medium that contains kantlex (50 μ g/mL); 37 ℃ of overnight cultures; The single colony inoculation of picking 10 strains extracts DNA at random, after the double digestion checking, delivers order-checking.

10, the acquisition and the molecular weight detection of reorganization tannase Tan410 crude enzyme liquid

To contain through the bacterial strain of sequence verification correct plasmid and rule to the LB solid medium that contains kantlex (50 μ g/mL); 37 ℃ of overnight cultures; Picking reorganization bacterium is seeded in the LB liquid nutrient medium that contains kantlex (50 μ g/mL) at random; 37 ℃, 220r/min shaking table overnight cultures are forwarded to by 1: 100 inoculum size in the LB liquid nutrient medium that contains kantlex (50 μ g/mL) of 50mL, when growing to OD ₆₀₀Added IPTG at=0.7 o'clock to final concentration 0.4mM, 25 ℃, 200r/min are cultivated 12 hours (OD ₆₀₀=3), the centrifugal 5min of 14000 * g abandons supernatant; Thalline is resuspended in 50mL, in the phosphate buffered saline buffer of 0.1M (pH=6.4), with ultrasonic disruption appearance (Sonics company) smudge cells; 4 ℃, the centrifugal 20min of 14000 * g collects supernatant; Obtain big and heavy histone, with Ni-NTAAgerose (QIAGEN) affinity column purification of recombinant proteins, affinity column concrete operations step is undertaken by QIAGEN Company products specification sheets with big and heavy histone.

The crude enzyme liquid and the liquid of protease behind the purifying that obtain are carried out the SDS-PAGE gel electrophoresis analysis; The SDS-PAGE electrophoresis result shows; The said nucleotide sequence encoded polypeptide of SEQ ID NO.1 is efficiently expressed in Escherichia coli BL21 (DE3); And all recombinant proteins all are soluble, and no inclusion body forms, and the molecular weight size of recombinant protein Tan410 is about 54kDa according to a preliminary estimate.(shown in accompanying drawing 1).

The zymologic property research of embodiment 2 reorganization tannase Tan410

Get 200 μ L crude enzyme liquids, add the Tenox PG of 200 μ L 15mM, 37 ℃; Water-bath 5min then adds 200 μ L methyl alcohol rhodanines (0.667%), adds 200 μ L KOH (0.5M) behind 37 ℃ of placement 5min; Place 5min for 37 ℃; Simultaneously do contrast with the crude enzyme liquid of deactivation, 520nm measures absorbance value, and absorption value shows that more greatly enzyme activity is high more.

1, reorganization tannase Tan410 optimal reactive temperature and thermostability

Be to measure its enzymic activity as stated above under 20-60 ℃ the condition in temperature of reaction, obtain its optimal reactive temperature when the highest (be designated as 100% with enzyme activity).Under 25 ℃, 35 ℃, 45 ℃, 50 ℃, 55 ℃ conditions, respectively enzyme is incubated 0.25h, 0.5h, 1h, 2h, 4h, 12h, measures residual enzyme for 30 ℃ and live, obtain the thermostability of enzyme.The result is shown in Fig. 2 and 3, and the optimal reactive temperature of Tan410 is 30 ℃; When temperature was lower than 45 ℃, Tan410 was stable, and 45 ℃ of insulation 12h still possess 75% relative activity, and along with the rising of temperature, the temperature stability of enzyme reduces rapidly, under 55 ℃ of conditions, handles 12h, and the relative activity of enzyme reduces to 15% rapidly.

2, reorganization tannase Tan410 optimal reaction pH and pH stability

In the pH4.0-9.0 damping fluid, measure its enzymic activity respectively as stated above, obtain its optimal reaction pH.30 ℃ of following conditions; Enzyme liquid is preserved 0.25h, 0.5h, 1h, 2h, 4h and 12h respectively in the damping fluid of pH 5.0,6.0,7.0,8.0,9.0,10.0 and 11.0; Measure residual enzymes for 30 ℃ and live, obtain pH stability when the highest (be designated as 100% with enzyme activity) of enzyme.The result is shown in Figure 4 and 5, and the optimal reaction pH of Tan410 is 6.4; Tan410 is stable in the scope of pH 6.0-7.0, is lower than pH 6.0 or is higher than pH7.0, and the enzyme activity of Tan410 descends rapidly along with the prolongation of child care time.

3, chemical reagent is to the influence of enzyme activity

In reaction system, add CaCl ₂, MgCl ₂, MnCl ₂, CuCl ₂, ZnSO ₄, AlCl ₃, CdCl ₂, CoCl ₂, AgNO ₃, Hg (NO ₃) ₂, Cr (NO ₃) ₂, EDTA, urea, beta-mercaptoethanol, PMSF, TritonX-100 and Tween 80, making its final concentration is 1mM, under standard conditions, surveys enzyme activity, is 100% with the enzyme liquid vigor that does not add chemical reagent.The result is as shown in table 1, Ca ²⁺, Cd ²⁺And Mg ²⁺Enzymic activity to Tan410 has activation, Mn ²⁺, Cu ²⁺, Zn ²⁺, Co ²⁺, Al ³⁺, EDTA, urea and Triton X-100 be to the not significantly influence of enzymic activity of Tan410, and Ag ⁺, Cr ²⁺, beta-mercaptoethanol, Tween 80 and PMSF have had strong inhibitory effects, Hg to the enzymic activity of Tan410 ²⁺The enzymic activity that then suppresses Tan410 fully.

Table 1 chemical reagent is to the influence of Tan410 enzymic activity

Chemical(1mM)	?Relative?activity(％)
		Control	100
CaCl ₂	126
		MgCl ₂	120
MnCl ₂	90
		CuCl ₂	108
ZnSO ₄	100
		AlCl ₃	97
CdCl ₂	121
		CoCl ₂	100
AgNO ₃	51
		Hg(NO ₃) ₂	0
Cr(NO ₃) ₂	52
		EDTA	86
urea	106
		β-mercaptoethanol	63
PMSF	32

TritonX-100	100
		Tween?80	47

4, NaCl is to the influence of Tan410 enzymic activity

In enzyme liquid, adding NaCl, to make its final concentration be 1M, 2M, 3M, 4M and 5M, and 30 ℃ of condition held 24h survey enzyme activity then under standard conditions, is 100% with the vigor of the enzyme liquid that do not add NaCl.The result is as shown in Figure 6, and NaCl with high concentration is to the not significantly influence of enzyme activity of Tan410, and when NaCl concentration arrived 5M, Tan410 still had 80% relative enzyme activity.

Embodiment 3 reorganization tannase Tan410 analyze the HPLC of Tenox PG, Weibull, L-Epicatechin gallate, hin acid esters, chlorogenicacid, rosmarinic acid and Ferulic acid ethylester degraded

Get 6 2ml centrifuge tubes; The enzyme liquid that adds 200 μ L respectively; The methyl gallate, Weibull, L-Epicatechin gallate, NVP-XAA 723, chlorogenicacid, rosmarinic acid and the Ferulic acid ethylester that then add the 200 μ L of 0.5mg/mL respectively, the machine of going up behind 30 ℃ of placement 40min carries out HPLC and analyzes.

Chromatographic condition is: Diamonsil C18 chromatographic column (250 * 4.6mm, 5 μ m), and 30 ℃ of column temperatures, the ultraviolet detection wavelength of gallic acid is 260nm, FLA and caffeinic ultraviolet detection wavelength are 320nm.Sample size is 0.5 μ L, and is quantitative with the peak area external standard method.

Mobile phase A: the PBS of acetonitrile B:pH 3.5, A: B=60: 40, flow velocity 0.8 μ L/min.The result sees Fig. 7; Under 30 ℃ of conditions, through behind the 40min, Weibull, L-Epicatechin gallate, NVP-XAA 723, chlorogenicacid and Ferulic acid ethylester be complete hydrolysis; Nearly 88% rosmarinic acid is hydrolyzed, and 46% methyl gallate is hydrolyzed.This explanation Tan410 has stronger avidity to natural substrate.

In sum; The described dna sequence dna that derives from bottom silt of SEQ ID NO.1 of the present invention can obtain efficient soluble expression in coli expression system; Its recombinant protein can Hydrolysable Tannins acid, L-Epicatechin gallate, NVP-XAA 723, rosmarinic acid, chlorogenicacid and Ferulic acid ethylester; Wherein to Rosmarinus officinalis, the hydrolytic action of chlorogenicacid and Ferulic acid ethylester is the peculiar character of Tan410; Mean except in tea drink, having the application potential important use potentiality are also arranged at aspects such as agriculture and industry waste processing.

Last institute should be noted that; Above embodiment is only in order to technical scheme of the present invention to be described but not to the restriction of protection domain of the present invention; Although the present invention has been done detailed description with reference to preferred embodiment; Those of ordinary skill in the art should be appreciated that and can make amendment or be equal to replacement technical scheme of the present invention, and do not break away from the essence and the scope of technical scheme of the present invention.

Claims

1. a novel tannase is characterized in that, the aminoacid sequence of said tannase is shown in SEQ ID NO.3.

2. the cDNA of novel tannase as claimed in claim 1 is characterized in that, the nucleotide sequence of said cDNA is shown in SEQ ID NO.1.

3. the grand genomics cloning process of novel tannase according to claim 1 is characterized in that, comprises following steps: extract total DNA of ocean mud and purifying; With the total DNA behind the purifying after the BamHI enzyme is cut processing; Be connected on the pUC118 carrier, electric shock transforms to import in the bacillus coli DH 5 alpha sets up grand genomic library, obtains positive colony through high flux screening; Through order-checking and BLAST relatively and design primer, thus be cloned into the purpose fragment.

4. expression vector that comprises the cDNA of novel tannase as claimed in claim 2.

5. the preparation method of the tannase of recombinating is characterized in that, may further comprise the steps: transform host cell with the described expression vector of claim 4, the culture transformation body obtains the reorganization tannase from culture.

6. the preparation method of reorganization tannase as claimed in claim 5 is characterized in that, said host cell is intestinal bacteria.

7. the preparation method of reorganization tannase as claimed in claim 6; It is characterized in that; Specifically may further comprise the steps:, be connected through BamHI and HindIII double digestion with the described purpose fragment of claim 4, be converted into e. coli bl21 with pET-28a (+) carrier; Induce through IPTG, obtain highly-soluble and express.

8. the preparation method of reorganization tannase as claimed in claim 7 is characterized in that, described IPTG final concentration is 0.4mM, and inducing temperature is 25 ℃.

9. the reorganization tannase for preparing of employing such as the said method of claim 5.

10. the application of reorganization tannase as claimed in claim 9 in degraded Tenox PG, Weibull, plain catechin and gallate, NVP-XAA 723, chlorogenicacid, rosmarinic acid and Ferulic acid ethylester.