CN1289665C - Recombined aspergillus oryzae tannase and its expression and purification - Google Patents

Abstract

The present invention relates to a method for expressing and purifying a recombinat aspergillus oryzae tannase. The present invention amplifies a nucleotide sequence coding an aspergillus oryzae tannase mature protein from an aspergillus oryzae gene by a PCR method, clones the nucleotide sequence to a methanol yeast expression vector pPIC9K, constructs an expression vector pPIC9K-TAN, uses an electric shock method to convert pichia KM71 to obtain a recombinat strain and uses a high density fermentation method for the induction expression of the recombinant tannase. Expression products are distributed in fermentation supernatant liquid, and the expression amount is 200 mg/L. The present invention establishes a method for purifying the recombinant aspergillus oryzae tannase, and DEAE anion-exchange column chromatography for one-step purification. The present invention also comprises a step of constructing constitutive expression vectors of pGAPZalpha A-TAN and pGAPZalpha A-ETAN of the recombinant tannase in the pichia, obtains an engineering strain with tannase activity by converting the pichia strain KM71H and is capable of expressing the recombinant tannase in a secretion mode. The recombinant aspergillus oryzae tannase has bioactivity.

Description

Recombinant Aspergillus oryzae tannase and its expression and purification method

technical field

The invention relates to a recombinant aspergillus oryzae tannase and its secretion, expression and purification in methanolic yeast. The invention also relates to the application of the recombinant Aspergillus oryzae tannase in industrial production.

Background technique

Tannin, also known as tannin, is widely distributed in higher plants, including monocots, dicots and ferns. Howes listed more than 600 species of 87 families of tannin-containing plants in the world. Tannin widely exists in food and feed, and is one of the main antinutritional factors in food, especially in feed. The anti-nutritional effect of tannin mainly has three aspects: (1) Tannin interacts with salivary proteins and glycoproteins in the oral cavity, causing tissue astringency and causing a series of unpleasant reactions. Tannins in feed can cause animals to reduce feed intake. When the tannin content in pasture exceeds 20mg/g dry matter, poultry refuses to eat. (2) There are a large number of phenolic hydroxyl groups and aromatic ring structures in tannin molecules, which can form complexes with proteins, causing protein coagulation and precipitation, and affecting protein digestion and absorption. Tannin and protein complex (Tannin Protein Complex) are difficult to be decomposed by proteases in the intestinal tract of mammals. If the tannin content of leguminous pasture is too high, the nutritional value of the feed will be reduced. (3) Tannins can reduce the activity of digestive enzymes in the mammalian intestine and have adverse effects on the gastrointestinal mucosa. Tannins form complexes with digestive enzymes and interfere with their biological activity. In vitro experiments showed that tannin extracts (such as broad bean shells) in the feed reduced the activity of trypsin, chymotrypsin and α-amylase. Tannic acid or chlorinated tannic acid caused changes in the gastric and duodenal mucosa of rats, causing excessive gastric mucus secretion, gastric mucosal necrosis, gallbladder atrophy, and decreased oxygen consumption of small intestinal epithelial cells and other adverse reactions. In addition, tannins are very toxic to ruminants, and cattle and sheep will be acutely poisoned when eating tannin-containing oak trees and some tropical leguminous trees, stems and leaves. In the production of aqueous herbal tea beverages, the rich tannins in the tea cause the precipitation of tea milk. Tannins precipitate with proteins in beer and juice, causing cloudiness in the product. Unnecessary tannins must be removed in the production of food, beverages, and poultry feed ^[9] . Using tannase to degrade redundant tannins is a technique with broad application prospects.

Tannin is the antagonist of most microorganisms and has toxic effects on microorganisms, but some microorganisms are resistant to tannins. These microorganisms degrade tannins through various mechanisms and methods. In 1913, Knudson isolated Aspergillus niger and Penicillum sp. to degrade tannin, and for the first time studied the fermentation degradation of tannin. In the past decade, many gut bacteria have been found to have the ability to degrade tannins. Osawa successively isolated Streptococcus bovis and Enterobacteriaceae degrading tannin-protein complexes from the feces and cecum of sloths. Nenuetr et al. reported the isolation of tannin-degrading bacteria in the gut of horses. In 1994, Makkai found that Sportrichum Preluerulentum could effectively degrade condensed tannins in oak. White rot (Ceriporiopsis subvermispora) chemically modifies the condensed tannins that are abundant in the leaves, thereby increasing the digestibility of the leaves threefold. Tanninyl hydrolase (EC3.1.1.20) is commonly called Tannase (Tannase). Tannase hydrolyzes the ester bond of tannin to produce gallic acid and glucose. Tannase is the key enzyme in the biodegradation of tannins. Progressive research on tannase began in the 1960s. Tannase was isolated and purified from Aspergillus oryzae, Aspergillus niger and Candida successively. Tannase is also isolated from certain bacterial cultures. Adachi's research on the physical and chemical properties of Aspergillus oryzae tannase proved that tannase is a typical serine esterase, which is consistent with the experimental results of Barthomeuf et al. in 1994.

Tannase has a wide range of applications in industrial production and has attracted increasing attention. In the production of water-soluble herbal tea beverages, tannase is used to degrade the tannins rich in tea, so as to avoid the formation of tea milk caused by tannins. Tannins precipitate with proteins in beer and fruit juices, causing turbidity, and tannase can be used as a clarifying agent in the production of such beverages. In feed processing, adding tannin enzyme can reduce the adverse effects of tannin and improve the utilization rate of feed. When purifying proteins by taking advantage of the protein-precipitating properties of tannins, tannase can be used to remove tannins from the precipitate. In addition, tannase is also used in the production of gallic acid and gallic acid esters. Gallic acid is a kind of widely used fine chemical raw material. It is used in medicine to make triclopyrim (for sulfonamide synergist), bifendate (for liver disease), and keguancao (for coronary heart disease) and other drugs. Propyl gallate, glycerol gallate, etc. are used as antioxidants and preservatives in food. Gallic acid is also used in the synthesis of dyes and reagents for chemical analysis.

Tannase has traditionally been extracted from the fermentation broth of tannase-producing strains. In 1997, Lane et al prepared a tannase preparation from Aspergillus oryzae. However, the amount of enzyme produced is relatively small, and it is difficult to purify, so the cost of extracting tannase from the fermentation broth of tannase-producing strains is high and the efficiency is low, so it is difficult to popularize and apply. Hatameto cloned the tannase gene of Aspergillus oryzae in 1996. The plasmid PT1 with the tannase gene was transformed into the strain Aspergillus orygze Aol with low tannase production to increase the tannase production of the host bacteria. This lays the foundation for the production of recombinant tannase using a high-efficiency expression system.

As a new generation of yeast expression system, methanol yeast expression system has many advantages compared with the existing major recombinant protein production systems. As a eukaryote, the methanol yeast expression system has the advantages of eukaryotic expression systems such as Saccharomyces cerevisiae expression system, has the post-translational processing function of eukaryotic proteins, and has the intracellular environment and sugar suitable for the correct folding of eukaryotic gene expression products. The chain processing system can also secrete exogenous proteins into the medium for purification. This is not found in prokaryotic expression systems such as Escherichia coli (E.coli). Compared with animal cell expression systems, the methanol yeast system has the characteristics of rapid reproduction, easy cultivation, and low cost, and it is also easy to carry out molecular genetic manipulation. Methanol yeast has advantages over Saccharomyces cerevisiae in the following aspects: stable passage of recombinant strains, high expression level and easy industrial scale-up of production scale. The expression level of exogenous protein in methanolic yeast system is 10-100 times higher than that in Saccharomyces cerevisiae system. This is because methanolic yeast possesses the AOX promoter. The AOX promoter is one of the strongest regulatory eukaryotic promoters among the promoters that have been studied so far, so the expression of foreign genes driven by the AOX promoter has a very large and efficient expression ability. Since Gzegy et al first expressed hepatitis B surface antigen (HbsAg) in Pichia pastoris (P. pastoris) in 1987, at least 40 foreign proteins have been expressed in P. pastoris in just a few years. In 1997, Waterham isolated and cloned the promoter of glyceraldehyde-3-phosphate dehydrogenase (GAP) in Pichia pastoris, which is a constitutive efficient promoter. The GAP promoter can promote the high-efficiency expression of foreign genes during the growth of cells without induction. Due to its high efficiency and more convenient operation, the GAP promoter has great application potential and is an effective substitute for the AOX promoter. In addition, the secretory expression of P. pastoris has a very good advantage that the level of secreted protein of P. pastoris is very low. This means that when growing on P. pastoris minimal medium, most of the protein in the culture is the target protein, which facilitates the purification of the target protein. In the glycosylation of secreted proteins, Pichia pastoris does not hyperglycosylate like Saccharomyces cerevisiae, which is another advantage of Pichia pastoris.

Contents of the invention

The invention provides a new Aspergillus oryzae tannase and its coding gene; the invention also provides the expression of the above-mentioned new gene and the purification method and application of the expression product.

The present invention has designed a pair of specific primers to encode the nucleotide sequence of Aspergillus oryzae tannase mature protein (its encoded mature peptide sequence is shown in <400>2 sequence in the sequence listing) (as <400 in the sequence listing) >1 sequence) was amplified from the Aspergillus oryzae gene by PCR method, cloned into Pichia pastoris expression vector pPIC9K (purchased from Invitrogen Company), constructed expression vector pPIC9K-TAN, and transformed Pichia pastoris KM71 ( purchased from Invitrogen Company) to obtain recombinant strains. The type of host bacteria, shake flask culture conditions, and fermentation culture conditions were explored and optimized, and the expression level of the recombinant protein reached 200mg/L.

In the present invention, the tannase gene is also cloned into the Pichia pastoris expression vector pGAPZαA (purchased from Invitrogen Company), and the expression framework of the gene is transformed by the PCR method, and the constitutive expression vectors pGAPZαA-TAN and pGAPZαA-TAN and pGAPZαA- etan. The Pichia pastoris strain KM71H (purchased from Invitrogen) was transformed by electroporation to obtain the recombinant strain, which was expressed in a secreted form under shake flask culture conditions.

The present invention also explores and optimizes the purification conditions of the recombinant tannase protein, and the expression product is purified by one-step DEAE Sepharose (purchased from Amersham Biosciences) anion exchange chromatography, and the recombinant tannase protein with a purity of more than 95% can be obtained.

The recombinant tannase obtained by the invention has biological activity.

The present invention constructs the expression plasmid pPIC9k-TAN containing the mature peptide coding sequence of Aspergillus oryzae tannase protein (see Figure 13 for the construction process) and the expression plasmids pGAPZαA-TAN and pGAPZαA-ETAN (see Figure 14 for the construction process)

The present invention constructs the expression plasmid pPIC9K-TAN of the Aspergillus oryzae tannase mature protein coding sequence, and the expression plasmid vector can be digested by EcoRI/NotI to obtain a 1.7kbp fragment, which is the Aspergillus oryzae tannase mature peptide encoding sequence. The tannase genes in the expression plasmids pGAPZαA-TAN and pGAPZαA-ETAN were cloned by XhoI and NotI. Since the tannase mature protein coding sequence contains an XhoI restriction site at 1405 bp, the expression plasmids can be obtained by XhoI/NotI double restriction digestion Two fragments of about 300bp and 1.4kb.

The replication method of the expression plasmid vector of the present invention: with reference to the Sambrook (Sambrook, et al.1989, Molecularcloing.Cold Spring Harbor Laboratory Press.USA) method, transform the plasmid in the E.Coli.DH5α bacterial strain by the _CaCl method, and transform pPIC9K -TAN bacteria were cultured with LB medium containing 100 μg/ml ampicillin, bacteria transformed with pGAPZαA-TAN and pGAPZαA-ETAN were cultured with 25 μg/ml low-salt LB medium, and the plasmid was extracted by alkaline method.

Description of drawings

Fig. 1 is the electrophoresis result of the tannase gene amplified from the Aspergillus oryzae gene by PCR. 1: PCR-amplified tannase gene; 2: negative control; M: 1kb DNA marker.

Fig. 2 is the electrophoresis result of enzyme digestion and identification of tannase methanol yeast expression vector vector pPIC9k-TAN. 1: pPIC9k-TAN(EcoR I+Not I); 2: pPIC9k(EcoR I+Not I); M1: 1kb DNAmarker.

Fig. 3 is the electrophoresis result of PCR identification of the recombinant methanolic yeast strain integrated with vector pPIC9k-TAN. 1: Positive clone; 2: Negative control; M1: 1kb DNA marker.

Fig. 4 is the result of SDS-PAGE electrophoresis of the expression product of recombinant tannase induced by expression vector pPIC9k-TAN with methanol. 1: fermentation supernatant before induction; 2-5: fermentation supernatant 1-4 days after methanol induction; 6: protein molecular weight standard.

Figure 5 is the SDS-PAGE electrophoresis analysis of the purification results of recombinant tannase. 1: Fermentation supernatant; 2: DEAE anion exchange purified tannase (non-reducing); 3: Purified tannase deglycosylated (non-reducing); 4: Protein molecular weight standard; 5: DEAE anion exchange purification tannase (reduction); 6: deglycosylation (reduction) of purified tannase.

Fig. 6 is the optimal reaction temperature curve of recombinant tannase.

Figure 7 is the thermostability curve of recombinant tannase.

Fig. 8 is the optimal reaction pH curve of recombinant tannase.

Figure 9 is the acid-base stability curve of recombinant tannase.

Fig. 10 is the SDS-PAGE electrophoresis result of the cell culture supernatant expressed by the recombinant tannase through the expression vector pGAPZαA-TAN. 1: protein molecular weight standard; 2-9: culture supernatants cultured for 2-9 days.

Fig. 11 is the screening result of the tannin plate of the Pichia pastoris strain KM71H transformed with the expression vectors pGAPZαA-TAN and pGAPZαA-ETAN. A: Pichia strain KM71H transformed with pGAPZαA-TAN; B: Pichia strain KM71H transformed with pGAPZαA-ETAN.

Fig. 12 is a schematic diagram of the expression framework of tannase in the expression vectors pGAPZαA-TAN and pGAPZαA-ETAN. A: the expression framework of the tannase gene in the expression vector pGAPZαA-TAN; B: the expression framework of the tannase gene in the expression vector pGAPZαA-ETAN.

Figure 13 is a schematic diagram of the construction of the yeast expression vector pPIC9k-TAN for tannase methanol.

Figure 14 is a schematic diagram of the construction of expression vectors pGAPZαA-TAN and pGAPZαA-ETAN.

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings and embodiments, which will help those of ordinary skill in the art understand the present invention, but the present invention is not limited in any form.

Embodiment one, the PCR amplification of Aspergillus oryzae tannase gene

The extraction of the Aspergillus oryzae genome refers to the experimental scheme of "Modern Molecular Biology Experimental Technology", according to the two-terminal sequence of the mature protein encoded by the tannase gene and the multi-enzyme cutting site of the Pichia pastoris expression vector pPIC9K (purchased from Invitrogen Company), Synthesize a pair of primers with the following sequence:

Upstream primer, 5'G GAATTC GCTTCTTTTTACCGATGTGTGCAC3' (single underlined part is EcoRI restriction sequence);

downstream primers, (The single underlined part is the NotI restriction sequence, and the double underlined part is the stop codon).

PCR amplification and gene cloning were carried out according to conventional methods. The PCR product is about 1700bp, as shown in Figure 1, encoding 570 amino acid residues.

Example 2. Construction of Tannase Methanol Yeast Expression Vector pPIC9k-TAN

The Aspergillus oryzae tannase gene amplified by PCR was cloned into the corresponding restriction restriction window of the methanolic yeast expression vector pPIC9K with EcoRI and NotI double digestion, and the expression vector pPIC9K-TAN was obtained. The construction process is shown in Figure 13 and identified by restriction digestion (Fig. ) and sequencing analysis showed that the cloned gene was the target gene.

Example 3. Transformation of yeast strains and screening of positive clones

The expression plasmid pPIC9K-TAN was single-cut and linearized with PmeI, and Pichia pastoris KM71 (purchased from Invitrogen) was transformed by electroporation, while the same linearized empty vector pPIC9K was transformed as a control. Positive clones were screened on MD solid culture plates, and positive clones were further screened with G418-resistant YPD plates. The transformants were further identified by PCR. The PCR primers were α-factor sequencing primer (5'-TACTATTGCCAGCATTGCTGC-3') and 3'AOX1 sequencing primer (5'-GCAAATGGCATTCTGACATCC-3'), and Taq DNA polymerase was used for The PCR reaction uses the extracted positive clone genome as a template, and the reaction cycle conditions are 94°C for 5 minutes, 94°C for 45 seconds, 52°C for 30 seconds, 72°C for 2 minutes, 32 cycles, 72°C for 10 minutes, and 4°C storage. Agarose gel electrophoresis detection shows that in Figure 3, a specific DNA band of about 1.8 kb was obtained by PCR amplification, which was consistent with the expected size.

Embodiment four, the high-density fermentation induction expression of recombinant Aspergillus oryzae tannase

Single clones were selected from the YPD plate, inoculated in 20ml BMGY medium, and cultured at 30°C and 240rpm for 20h. Inoculate into 300ml BMGY medium at a ratio of 1:50, cultivate at 30°C and 240rpm until OD600=2-6, and inoculate the fermenter.

Add 3L fermentation base medium to a 7L fermenter, sterilize at 121°C for 20 minutes, adjust the temperature to 30°C, adjust the pH to 5.5 with ammonia water, add PTM1 (4.35ml L ^-1 ), and inoculate seed bacteria (1:10). During the fermentation process, the temperature is controlled at 30°C, the ventilation rate is maintained at 2vvm, and the rotation speed is controlled at 300-800rpm to maintain more than 20% of dissolved oxygen.

The fermentation is divided into three stages: the growth stage, from adding seed bacteria, culturing for about 16 to 24 hours, until the glycerol in the fermenter is exhausted, showing a sudden rise in dissolved oxygen; then entering the glycerol growth-promoting stage, adding 50% glycerol ( Contains PTM1, 12ml L ^-1 ), feeding rate is 18ml L ^-1 h ^-1 , lasts 4~6h; finally enters the induction period, adjusts the pH to 5.0 with ammonia water, feeds 100% methanol (contains PTM1, 12ml L ^{-1 1} ), the flow rate increased linearly from 1ml L ^-1 h ^-1 to 3ml L ^-1 h ^-1 over 10h, and lasted for 120h. The protein expression of the fermentation supernatant was detected by SDS-PAGE, as shown in Figure 4.

Embodiment five, the purification of recombinant Aspergillus oryzae tannase

The fermentation broth was centrifuged at 8000rpm at 4°C for 30min, the supernatant was desalted with Sephendex G-25, replaced with 10mmol/L citric acid pH5.5 buffer, and then purified by DEAE Sepharose Fast Flow ion exchange chromatography column, the elution system was 10mmol/L L citric acid pH5.5 buffer, NaCl linear gradient (0-0.5mol/L), pH5.5. The eluted samples were analyzed and identified by SDS-PAGE (Fig. 5). The target protein fractions were combined and dialyzed in double distilled water. After dialysis, the samples were freeze-dried and stored at -20°C.

Embodiment six, the activity identification of recombinant Aspergillus oryzae tannase

The activity identification of recombinant Aspergillus oryzae tannase refers to the method of Beverini (1990). 1 IU of enzyme activity defines the amount of enzyme required to hydrolyze 1 μmol of tannic acid in 1 minute. Enzyme activity detection The enzyme activity of the recombinant Aspergillus oryzae tannase we prepared reached 50,000IU/g.

Embodiment seven, recombinant Aspergillus oryzae tannase optimal reaction temperature and thermostability determination

Enzyme activity was measured at different temperatures (10°C-70°C). When measuring thermal stability, the enzyme solution was incubated at different temperatures for 10 minutes, and then its residual enzyme activity was measured. The optimum reaction temperature of the recombinant tannase was measured to be 40° C., and the thermal stability range was 10-40° C. ( FIG. 6 , FIG. 7 ).

Embodiment eight, recombinant Aspergillus oryzae tannase optimum reaction pH and acid-base stability determination

Na ₂ HPO ₄ -citric acid buffer solutions (pH 2.2-8.0) with different pH values were prepared, with 0.5 pH units as a gradient. When determining the optimum reaction pH value, the tannase was dissolved in deionized water and diluted 5 times with Na ₂ HPO ₄ -citric acid buffer solution with different pH values; then the enzyme activity was measured. When measuring acid-base stability, tannase was dissolved in buffer solutions with different pH values, and after incubation at 30° C. for one hour, 20 times the volume of 50 mM citric acid buffer solution with pH 5.5 was added, and then the enzyme activity was measured. As a result of the measurement, the optimum reaction pH value of the recombinant tannase was pH6.0, and the acid-base stability range was pH4-6 (Fig. 8, Fig. 9).

Example 9. Construction of recombinant tannase Pichia pastoris constitutive expression vectors pGAPZαA-TAN and pGAPZαA-ETAN

Synthetic primers were designed according to the partial signal peptide sequence and tannase coding sequence of the expression vector pGAPZαA (purchased from Invitrogen):

Upstream primers:

(1): 5'CCG CTCGAG AAAAGA GCTTCTTTTTACCGATGTGTGCAC3' (the underlined part is the XhoI restriction site in the vector signal peptide sequence),

(单下划线部分为载体信号肽序列中的XhoI酶切位点，双下划线部分为载体信号肽序列末端的Ste13蛋白酶切割序列)；(2):

(The single underlined part is the XhoI restriction site in the carrier signal peptide sequence, and the double underlined part is the Ste13 protease cleavage sequence at the end of the carrier signal peptide sequence);

Downstream primer: 5'TCGCTGGACTGGCCATAAAAGC3' (the downstream primer is a sequence at about 480bp in the tannase coding sequence).

Using pPIC9K-TAN as a template, PCR amplification was carried out with two sets of primers respectively, and the size of the amplified fragment was about 500bp, cut into two fragments after double enzyme digestion with XhoI and SphI, and the fragment of about 100bp was recovered; the plasmid pPIC9K-TAN was double-digested with SphI and NotI, and the tannase gene fragment with a size of 1.4kb was recovered; these two fragments were connected to the corresponding XhoI/NotI digestion window of the expression vector pGAPZαA, and the expression vector pGAPZαA-TAN was constructed and pGAPZαA-ETAN, the construction process is shown in Figure 14; the expression frame of the tannase gene in the expression vector is shown in Figure 12, the tannase in pGAPZαA-TAN is directly connected to the end of the carrier signal peptide, and the tannase gene in pGAPZαA-ETAN It is connected to the end of the protease cleavage site of Pichia pastoris protein Ste13, that is, there are 4 amino acid residues EAEA between the signal peptide sequence and tannase; the vector is successfully constructed after enzyme digestion and DNA sequencing.

Embodiment 10. Transformation of Pichia pastoris strains and screening of positive clones

The expression vectors pGAPZαA-TAN and pGAPZαA-ETAN were linearized with AvrII respectively, and Pichia pastoris KM71H was transformed by electroporation, while the same linearized empty vector pGAPZαA was used as a control. The transformed cells were screened with zeocine resistance plate to select resistant clones, and then screened with 0.2% tannic acid YNB + 0.5% glucose plate to screen out tannase active strains. Among them, the recombinant strain with tannase activity can form a transparent tannin degradation circle around the colony ( FIG. 11 ).

Example 11. Constitutive expression of recombinant tannase through the expression vector pGAPZαA-TAN

Pick a single colony from the YPD plate, inoculate it into 50ml YPD medium, cultivate it at 30°C and 240rpm, add a glucose solution with a final concentration of 0.5% every 24 hours, and take the culture supernatant for SDS-PAGE analysis. There is a characteristic protein band at 100kDa, which is the recombinant tannase product (Figure 10).

Sequence Listing

<110> Sun Yat-Sen University

<120> Recombinant Aspergillus oryzae tannase and its expression and purification method

<160>2

<210>1

<211>1710

<212>DNA

<213> Aspergillus oryzae

<220>

<221>mat peptide

<222>(1)...(570)

<400>1

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Ala Ser Phe Thr Asp Val Cys Thr Val Ser Asn Val Lys Ala Ala Leu

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Pro Ala Asn Gly Thr Leu Leu Gly Ile Ser Met Leu Pro Ser Ala Val

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Thr Tyr Asp Tyr Cys Asn Val Thr Val Ala Tyr Thr His Thr Gly Lys

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Gly Asp Lys Val Val Ile Lys Tyr Ala Phe Pro Lys Pro Ser Asp Tyr

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Glu Asn Arg Phe Tyr Val Ala Gly Gly Gly Gly Gly Phe Ser Leu Ser Ser

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Asp Ala Thr Gly Gly Leu Ala Tyr Gly Ala Val Gly Gly Ala Thr Asp

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Ala Gly Tyr Asp Ala Phe Asp Asn Ser Tyr Asp Glu Val Val Leu Tyr

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Gly Asn Gly Thr Ile Asn Trp Asp Ala Thr Tyr Met Phe Ala Tyr Gln

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Ala Leu Gly Glu Met Thr Arg Ile Gly Lys Tyr Ile Thr Lys Gly Phe

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Tyr Gly Gln Ser Ser Asp Ser Lys Val Tyr Thr Tyr Tyr Glu Gly Cys

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Ser Asp Gly Gly Arg Glu Gly Met Ser Gln Val Gln Arg Trp Gly Glu

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Glu Tyr Asp Gly Ala Ile Thr Gly Ala Pro Ala Phe Arg Phe Ala Gln

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Gln Gln Val His His Val Phe Ser Ser Glu Val Glu Gln Thr Leu Asp

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Tyr Tyr Pro Pro Pro Cys Glu Ser Lys Lys Ile Val Asn Ala Thr Ile

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Ala Ala Cys Asp Pro Leu Asp Gly Arg Thr Asp Gly Val Val Ser Arg

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Thr Asp Leu Cys Lys Leu Asn Phe Asn Leu Thr Ser Ile Ile Gly Glu

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Pro Tyr Tyr Cys Ala Ala Gly Thr Ser Thr Ser Leu Gly Phe Gly Phe

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Ser Asn Gly Lys Arg Ser Asn Val Lys Arg Gln Ala Glu Gly Ser Thr

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Thr Ser Tyr Gln Pro Ala Gln Asn Gly Thr Val Thr Ala Arg Gly Val

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Ala Val Ala Gln Ala Ile Tyr Asp Gly Leu His Asn Ser Arg Gly Glu

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Arg Ala Tyr Leu Ser Trp Gln Ile Ala Ser Glu Leu Ser Asp Ala Glu

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Thr Glu Tyr Asn Ser Asp Thr Gly Lys Trp Glu Leu Asn Ile Pro Ser

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Thr Gly Gly Glu Tyr Val Thr Lys Phe Ile Gln Leu Leu Asn Leu Asp

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Asn Leu Ser Asp Leu Asn Asn Val Thr Tyr Asp Thr Leu Val Asp Trp

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Met Asn Thr Gly Met Val Arg Tyr Met Asp Ser Leu Gln Thr Thr Leu

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Pro Asp Leu Thr Pro Phe Gln Ser Ser Ser Gly Gly Lys Leu Leu His Tyr

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His Gly Glu Ser Asp Pro Ser Ile Pro Ala Ala Ser Ser Val His Tyr

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Trp Gln Ala Val Arg Ser Val Met Tyr Gly Asp Lys Thr Glu Glu Glu

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Ala Leu Glu Ala Leu Glu Asp Trp Tyr Gln Phe Tyr Leu Ile Pro Gly

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Ala Ala His Cys Gly Thr Asn Ser Leu Gln Pro Gly Pro Tyr Pro Glu

485 490 495

aac aac atg gag att atg atc gac tgg gtc gag aac ggc aac aag ccg 1536

Asn Asn Met Glu Ile Met Ile Asp Trp Val Glu Asn Gly Asn Lys Pro

500 505 510

tcc cgt ctc aat gcc act gtt tct tcg ggt acc tac gcc ggc gag acc 1584

Ser Arg Leu Asn Ala Thr Val Ser Ser Ser Gly Thr Tyr Ala Gly Glu Thr

515 520 525

cag atg ctt tgc cag tgg ccc aag cgt cct ctc tgg cgc ggc aac tcc 1632

Gln Met Leu Cys Gln Trp Pro Lys Arg Pro Leu Trp Arg Gly Asn Ser

530 535 540

agc ttc gac tgt gtc aac gac gag aag tcg att gac agc tgg acc tac 1680

Ser Phe Asp Cys Val Asn Asp Glu Lys Ser Ile Asp Ser Trp Thr Tyr

545 550 555 560

gag ttc cca gcc ttc aag gtc cct gta tac 1710

Glu Phe Pro Ala Phe Lys Val Pro Val Tyr

565 570

<210>2

<211>570

<212>PRT

<213> Aspergillus oryzae

<220>

<221>mat peptide

<222>(1)...(570)

<400>2

Ala Ser Phe Thr Asp Val Cys Thr Val Ser Asn Val Lys Ala Ala Leu

1 5 10 15

Pro Ala Asn Gly Thr Leu Leu Gly Ile Ser Met Leu Pro Ser Ala Val

20 25 30

Thr Ala Asn Pro Leu Tyr Asn Gln Ser Ala Gly Met Gly Ser Thr Thr

35 40 45

Thr Tyr Asp Tyr Cys Asn Val Thr Val Ala Tyr Thr His Thr Gly Lys

50 55 60

Gly Asp Lys Val Val Ile Lys Tyr Ala Phe Pro Lys Pro Ser Asp Tyr

65 70 75 80

Glu Asn Arg Phe Tyr Val Ala Gly Gly Gly Gly Gly Phe Ser Leu Ser Ser

85 90 95

Asp Ala Thr Gly Gly Leu Ala Tyr Gly Ala Val Gly Gly Ala Thr Asp

100 105 110

Ala Gly Tyr Asp Ala Phe Asp Asn Ser Tyr Asp Glu Val Val Leu Tyr

115 120 125

Gly Asn Gly Thr Ile Asn Trp Asp Ala Thr Tyr Met Phe Ala Tyr Gln

130 135 140

Ala Leu Gly Glu Met Thr Arg Ile Gly Lys Tyr Ile Thr Lys Gly Phe

145 150 155 160

Tyr Gly Gln Ser Ser Asp Ser Lys Val Tyr Thr Tyr Tyr Glu Gly Cys

165 170 175

Ser Asp Gly Gly Arg Glu Gly Met Ser Gln Val Gln Arg Trp Gly Glu

180 185 190

Glu Tyr Asp Gly Ala Ile Thr Gly Ala Pro Ala Phe Arg Phe Ala Gln

195 200 205

Gln Gln Val His His Val Phe Ser Ser Glu Val Glu Gln Thr Leu Asp

210 215 220

Tyr Tyr Pro Pro Pro Cys Glu Ser Lys Lys Ile Val Asn Ala Thr Ile

225 230 235 240

Ala Ala Cys Asp Pro Leu Asp Gly Arg Thr Asp Gly Val Val Ser Arg

245 250 255

Thr Asp Leu Cys Lys Leu Asn Phe Asn Leu Thr Ser Ile Ile Gly Glu

260 265 270

Pro Tyr Tyr Cys Ala Ala Gly Thr Ser Thr Ser Leu Gly Phe Gly Phe

275 280 285

Ser Asn Gly Lys Arg Ser Asn Val Lys Arg Gln Ala Glu Gly Ser Thr

290 295 300

Thr Ser Tyr Gln Pro Ala Gln Asn Gly Thr Val Thr Ala Arg Gly Val

305 310 315 320

Ala Val Ala Gln Ala Ile Tyr Asp Gly Leu His Asn Ser Arg Gly Glu

325 330 335

Arg Ala Tyr Leu Ser Trp Gln Ile Ala Ser Glu Leu Ser Asp Ala Glu

340 345 350

Thr Glu Tyr Asn Ser Asp Thr Gly Lys Trp Glu Leu Asn Ile Pro Ser

355 360 365

Thr Gly Gly Glu Tyr Val Thr Lys Phe Ile Gln Leu Leu Asn Leu Asp

370 375 380

Asn Leu Ser Asp Leu Asn Asn Val Thr Tyr Asp Thr Leu Val Asp Trp

385 390 395 400

Met Asn Thr Gly Met Val Arg Tyr Met Asp Ser Leu Gln Thr Thr Leu

405 410 415

Pro Asp Leu Thr Pro Phe Gln Ser Ser Ser Gly Gly Lys Leu Leu His Tyr

420 425 430

His Gly Glu Ser Asp Pro Ser Ile Pro Ala Ala Ser Ser Val His Tyr

435 440 445

Trp Gln Ala Val Arg Ser Val Met Tyr Gly Asp Lys Thr Glu Glu Glu

450 455 460

Ala Leu Glu Ala Leu Glu Asp Trp Tyr Gln Phe Tyr Leu Ile Pro Gly

465 470 475 480

Ala Ala His Cys Gly Thr Asn Ser Leu Gln Pro Gly Pro Tyr Pro Glu

485 490 495

Asn Asn Met Glu Ile Met Ile Asp Trp Val Glu Asn Gly Asn Lys Pro

500 505 510

Ser Arg Leu Asn Ala Thr Val Ser Ser Ser Gly Thr Tyr Ala Gly Glu Thr

515 520 525

Gln Met Leu Cys Gln Trp Pro Lys Arg Pro Leu Trp Arg Gly Asn Ser

530 535 540

Ser Phe Asp Cys Val Asn Asp Glu Lys Ser Ile Asp Ser Trp Thr Tyr

545 550 555 560

Glu Phe Pro Ala Phe Lys Val Pro Val Tyr

565 570

Claims (3)

1. the recombination method of an aspergillus oryzae tannase gene is cut the site according to maturation protein two terminal sequences of tannase genes encoding and the multienzyme of yeast expression vector pPIC9K, synthetic a pair of primer, and sequence is as follows:

Upstream primer, 5 ' G GAATTCGCTTCTTTTACCGATGTGTGCAC3 ';

Downstream primer, 5 ' GCG GCGGCCGC GTATACAGGGACCTTGAAGGC3 '; Obtain aspergillus oryzae tannase gene shown in SEQ ID NO:1 through pcr amplification.

2. the aspergillus oryzae tannase expression of gene method of claim 1 gained, in Pichi strain KM71, adopt methyl alcohol to carry out abduction delivering by expression vector pPIC9K-TAN, perhaps in Pichi strain KM71H, carry out constitutive expression, need not induce by expression vector pGAPZ α A-TAN and pGAPZ α A-ETAN; The expression product of dual mode is all secreted outside born of the same parents, carries out purifying and obtains reorganization aspergillus oryzae tannase shown in SEQ ID NO:2 by obtaining culture supernatants.

3. aspergillus oryzae tannase expression of gene method as claimed in claim 2, the purifying that it is characterized in that described culture supernatants are with DEAE anion exchange chromatography single step purification.

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