KR20120014766A - Tannic acid-degradable laccase 3 of cryphonectria parasitica and usage thereof - Google Patents

Abstract

PURPOSE: A novel laccase protein and a method for preparing the same are provided to be used in pulp, food, and fiber industries. CONSTITUTION: A laccase 3 which decomposes tannic acid is isolated from Cryphonectria parasitica. The laccase 3 has an amino acid of sequence number 7. A recombinant expression vector contains a region encoding laccase 3. The region is a region encoding mature laccase 3 and is regulated by inducible promoter. The inducible promoter is GAL1(galactose kinase) promoter. The recombinant expression vector is pYEGLAC3 or pYEGALLAC3. A yest host cell transformed by the recombinant expression vector is Saccharomyces sp.

Description

Tannic acid-degradable laccase 3 of Cryphonectria parasitica and usage thereof}

The present invention relates to a novel laccase protein and a use thereof, and more particularly, a recombinant expression vector comprising a laccase 3 derived from Cryphonectria parasitica that degrades tannic acid, and a coding region thereof. The present invention relates to a yeast host cell transformed with the vector, a method for producing LaCase 3 by culturing the host cell, and the use of LaCase 3.

Lacase is multi-copper-binding phenoloxidases (EC 1.10.3.2), first detected in the Japanese lac tree Toxicodendron verniciflua ; Lacaze was found not only in many other plants, but also in many insects and various fungi. Lacase is particularly widely present in ligninolytic basidiomycetes, and more than 125 basophilic laccase genes are known. In fungi, the biological functions of laccases vary, so that laccases are involved in the processes of various cells, including lignin degradation, spore formation, pigment production, fruiting body formation, and onset.

Lacases use oxygen as the final electron acceptor to catalyze the oxidation of many aromatic compounds such as diphenols, methoxy-substituted monophenols and aromatic amines. Many laccases are characterized by one type-1, one type-2 and two type-3 copper ions. One electron at a time is removed from the substrate by Type 1 copper ions, transferred to Type 2 / Type 3 copper sites, and oxygen molecules are reduced to water. Due to the low substrate specificity of laccases, the industrial use of laccases has led to the degradation of lignin, the discoloration of wastewater, the discoloration of fiber dyes, the treatment of drinking water and food, the sulfidation and solubilization of coal through enzyme-based biosensors, and the harmful environmental pollution. Deformation and inactivation of materials. In addition, recent studies have shown that the substrate specificity of this enzyme can be further extended by the presence of redox mediators. If diversification and widening of substrate specificity are possible, laccase may be the most important biocatalyst in mold biotechnology.

At least three laccases are present in the chestnut blight fungus Cryphonectria parasitica . Of these, Lacaze 3 is interesting. This is because the protein is specifically induced by tannic acid, which is structurally not related to inducers of ferulic acid and other commonly known fungal laccases such as 2,5-xyldine.

Phenolic metabolism in plants is complex and produces compounds ranging from pigments in flowers to complex phenols in plant cell wall lignin. However, phenol, known as tannic acid, is clearly distinguished from phenols of other plants in terms of the reactivity and biological activity of the compounds. Tannic acid is a water-soluble phenolic compound having a molecular weight of 500 to 3,000 and has the characteristic characteristic of precipitating alkaloids, gelatin and other proteins. What distinguishes tannic acid from other phenols is their ability to precipitate proteins. Thus, enzymes that can degrade tannic acid and are not sensitive to the presence of tannic acid can be useful for various applications of plant tissue materials.

It is quite difficult to express laccase in a non-fungal system, which includes Saccharomyces cerevisiae, Trichoderma ressei, Aspergillus aurise, Pichia pastoris, Kluyberomyces lactis, A. and A. Niger. Several fungi have been used for heterologous expression of laccases. Although P. pastoris laccase has been used as a host for heterologous expression, Saccharomyces cerevisiae is known to be available. However, techniques for mass production of laccases in active form are not yet known.

The inventors of the present invention first identified that laccase 3 derived from Cryponectria parasitea has a function of decomposing tannic acid, while studying a method for mass production of an enzyme capable of decomposing tannic acid in an active form. By culturing Saccharomyces cerevisiae transformed in a medium lacking uracil, it was confirmed that the mass production of laccase 3 possessing enzymatic activity was completed, and the present invention was completed.

One aspect of the invention relates to laccase 3 from Cryphonectria parasitica , which degrades tannic acid.

Another aspect of the invention relates to a recombinant expression vector comprising the coding region of Lacase 3.

Another aspect of the invention relates to a yeast host cell transformed with the recombinant expression vector.

Another aspect of the invention relates to a method for producing Lacase 3 by culturing the yeast host cell.

Another aspect of the invention relates to a composition for wood decoloration and degradation comprising the laccase 3, the yeast host cell or a culture thereof.

Another aspect of the present invention relates to a dye discoloration composition comprising the laccase 3, the yeast host cell or a culture thereof.

Another aspect of the present invention relates to a composition for decomposing tannin acid of a food comprising the laccase 3, the yeast host cell or a culture thereof.

Hereinafter, the present invention will be described in detail.

Lacase 3 of the present invention is a protein induced expression by tannic acid derived from Cryphonectria parasitica , and is further characterized by decomposing tannic acid.

Lacquer scope of claim 3 according to the invention is SEQ ID NO: refers to a functional equivalent of a protein and the protein consisting of the amino acid sequence shown in Fig. 7 (MALRAFLSLLPALSLVTAAPSVEPYQLSKRCVNSADDRSCWGDYDLSTNYYDEAPFTNVTREYWFNIVNTTASPDGVEKIVLAVNGSIPGPTIVADWGDTVVVHVTNSMQNNGSSIHFHGIRQNFTNQNDGVASITQCPTAPGDTTTYTWRALQYGTSWWHSHFYVQAWDGVYGGIQINGPATANYDEDLGVVSMMDWDHDTADNLVLQSAVTGPPTMANGLINGMNVYTLEDNSTVGSRYEMEFTSGQRYRLRLVNTAADTHFKFTIDNHTMEVIASDFVPIQPYTTDVVNIGIGQRYDVIVTADAPSDNYWMRAIAQLACSDNDNPDNIKAIIRYSDAANSTADPTSTATADASVDECVDEPAASLVPYLAIPAGGSADVTDNFAVAVEQLAGRVDWAMGNTTFINEWGYPSIQQVYDGNNTWGESQNVIQLPQANEWVYWIVQTTMGVTHPMHMHGHDFWVLGAGTGTFDSATASLSTVNVPRRDVTMLPASGWTVIAFITDNPGVWLTHCHIAWHTSEGLAVQMVERESEIVDLIDADLMNSTCGAWNDYANKVALVQDDSGI). A "functional equivalent" means at least 70%, preferably 80% or more, more preferably 90% or more sequence homology with the amino acid sequence shown in SEQ ID NO: 7 as a result of the addition, substitution or deletion of amino acids. It refers to a protein that exhibits substantially the same physiological activity as the protein represented by SEQ ID NO: 7. "Substantially homogeneous physiological activity" means that it has activity to decompose tannic acid.

For the expression of Lacase 3 of the present invention, it is preferred to prepare a recombinant expression vector by first operably linking the coding region of Lacase 3 to an appropriate expression vector. Here, it is preferable that the coding region of laccase 3 is the coding region of the laccase 3 matured (secreted sequence removed).

In addition to the coding region of Lacase 3, the recombinant expression vector of the present invention may include a selective marker for selection of transformants and a replication origin for propagation in host cells. In addition, the recombinant expression vector of the present invention may comprise a transcriptional or translational regulatory sequence capable of acting in a host cell as operably linked to the coding region of Lacase 3 of the present invention as desired.

In the present invention, 'regulatory sequence' refers to nucleic acid sequences essential or beneficial for expression of Lacase 3 of the present invention. Each regulatory sequence can be natural or foreign to the nucleic acid sequence encoding the polypeptide (ie, laccase 3). Such regulatory sequences include, but are not limited to, secretory sequences, polyadenylation sequences, propeptide sequences, promoters, enhancer or upstream activation sequences, and transcription terminators, and the like. Regulatory sequences in the present invention preferably include a promoter and a secretory sequence. Other regulatory sequences may optionally be included to further increase the expression of Lacase 3 of the present invention.

In the present invention, 'promoter' refers to a nucleic acid sequence essential for initiating the laccase 3 transcription of the present invention. Promoters of the invention can be constitutive or inducible. The constitutive promoter expresses less amount of Lacase 3 than the inducible promoter, but is more preferred when using the transformant itself. Inducible promoters can express higher amounts of Lacasease 3 than the constitutive promoter, and are preferred forms of the constitutive promoter when utilizing isolated and purified Lacasease 3.

Examples of promoters that act in the host cell of the present invention include a promoter of the yeast alpha-mating system, a promoter of the yeast triose phosphatase isomerase (TPI), and a glycolytic process of yeast. Promoters of glycolytic or alcohol dehydrogenase (ADH) genes and enzymes involved in yeast galactose metabolism (e.g., galactose kinase (GAL1) and uridine diphosphoglucose 4-epimerase (GAL10) ), GPD (glyceraldehyde-3-phosphate dehydrogenase), MOX (methano oxidase), AOX (alcohol oxidase) promoter and the like.

In the present invention, the 'secretory sequence (signal peptide)' refers to an amino acid sequence that induces the expressed protein to be transported out of the cell membrane. In general, surface proteins or secreted proteins that are transported out of the cell membrane have an N-terminal sequence that is cut by the motive peptidase in the cell membrane. In the present invention, but not limited to the secretion sequence, alpha-factor (α-factor) secretion sequence, killer toxin leader secretion sequence, invertase secretion sequence, alpha-amylase (α-amylase) Secretion sequences and the like can be used. In the present invention, rice peptide amylase 1A ( Ramy 1A) signal peptide (ASP) is particularly preferred.

In the present invention, a known yeast expression vector can be used for the introduction and expression of the Lacase 3 gene. In the embodiment of the present invention, pYEGPD-TER and pYEGAL-TER were used. The vector has a GPD promoter and galactose-1-phosphate uridinyl transferase ( GAL7 ) terminator, and a GAL1 promoter and GAL7 terminator, respectively.

As for the recombinant expression vector of this invention, pYEGLAC3 which has a cleavage map shown in FIG. 1A, or "pYEGALLAC3" which has a cleavage map shown in FIG. 1B is preferable. More preferred is 'pYEGALLAC3' comprising the GAL1 promoter, which is an inducible promoter.

Yeasts that can be used in the present invention include, but are not limited to, Saccharomyces genus, Trichoderma genus, Aspergillus genus, Pichia genus, Cluiveromyces genus and the like. Preferably it is yeast derived from the genus Saccharomyces, More preferably, it is Saccharomyces cerevisiae, Saccharomyces pastorinus, Saccharomyces bayanus, Saccharomyces berry. In an embodiment of the present invention, S. cerevisiae 2805 ( MATα pep4 :: HIS3 prb1-δ can1 GAL2 his3 -200 ura3 -52 ).

As a method of introducing the recombinant expression vector of the present invention into a host cell, chemical treatment such as, but not limited to, calcium phosphate or calcium chloride / rubidium chloride method, lithium acetate method, electroporation method, electroinjection method, PEG, etc. The method by using, the method using a gene gun, etc. are mentioned. Specifically, yeast is described by (1) Simon, MI et al., Editors, Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, vol.194, p.182-7, Academic press, Inc. New York; (2) Ito, et al. Journal of Bacteriology, 1983, vol. 153, p. 163; (3) Hinnen et al. Proceedings of the National Academy of Sciences USA, 1978, vol. 75, p.1920 and (4) Clontech Laboratories, Inc, Palo Alto. Transformation can be made using techniques known from Calif., USA (Yeastmaker ^™ Yeast Transformation System Kit).

In the present invention, the laccase 3 is prepared by culturing yeast host cells transformed with a recombinant expression vector comprising a coding region of laccase 3 derived from C. parasitica in a medium lacking uracil.

In the present invention, "ura ^- deficient medium" means a medium in which host cells are viable and laccase 3 can be expressed without uracil being included. The uracil deficient medium of the present invention preferably comprises a yeast nitrogen base without amino acids, adenine, tryptophan, casamino acid, dextrose, more preferably 5 Yeast nitrogen base, 0.01-0.05 g / l adenine, 0.01-0.05 g / l tryptophan, 3-9 g / l casamino acid and 15-25 g / l dextrose .

In one embodiment, the uracil deficient medium of the present invention comprises a yeast nitrogen base that does not include 6.7 g / l amino acid, 0.03 g / l adenine, 0.03 g / l tryptophan, 5 g / l casamino acid, 20 g / l dextrose, 20 g / l agar.

In one embodiment of the present invention, when the transformed host cell is cultured in a medium containing uracil, it is preferable to add an aspartyl protease inhibitor to the medium. This is because the inhibitor can inhibit the loss of activity of laccase 3. Representative examples of the inhibitor include pepstatin.

On the other hand, Lacasease 3 may have a detrimental effect on the growth of host cells. In the present invention, it was confirmed that the activity of laccase 3 decreases as the culture progresses. Therefore, in the present invention, in the state in which Lacasease 3 is expressed, short-term culturing is appropriate. Specifically, the culturing period is 1 to 5 days, preferably 2 to 4 days. Herein, the state in which laccase 3 is expressed is a state in which the expression of laccase 3 is induced by including a corresponding inducer in the medium when laccase 3 is under the control of an inducible promoter, and laccase 3 is a constitutive promoter. If under the control of the host cell is in a live state, because the expression of Lacase 3 occurs, it means that the host cell is alive, that is, the state in culture.

Lacase 3 produced according to the present invention is characterized by maintaining enzyme activity. For example, laccase 3 can degrade phenolic compounds such as tannic acid.

Lacase 3 of the present invention can be isolated from culture by methods known in the art. For example, the polypeptide can be separated from the medium by conventional methods including, but not limited to, centrifugation, filtration, extraction, spray drying, evaporation or precipitation. Furthermore, polypeptides can be purified through a variety of methods known in the art, including chromatography, electrophoresis, fractional solubility, SDS-PAGE or extraction.

Lacaze 3 of the present invention characterized by decomposing tannic acid can be applied to pulp industry, textile industry, food industry and the like. Here, the yeast host cell transformed with the recombinant expression vector including the coding region of Lacase 3 itself, its culture or its purified and isolated LaCase 3 can be used.

Lacase 3 of the present invention can be used in the pulp industry to decolorize and decompose wood. In addition, laccase 3 can be used for the bleaching of dyes in the textile industry. For example, wastewater from dyes acts as an environmental pollutant, so its discoloration is essential. As shown in Example 3, Lacase 3 of the present invention can degrade malachite green G. In addition, Lacasease 3 of the present invention can be used to decompose tannic acid in food. Tannic acid can be desirable to remove as needed because it has a bitter taste.

According to the present invention, it is possible to mass-produce a laccase capable of decomposing tannic acid in a state in which enzyme activity is maintained.

Lacase 3 of the present invention can be used in various fields such as the pulp, food, and textile industries.

1A shows a cleavage map of the recombinant expression vector pYEGLAC3 of the present invention.
Figure 1b shows a cleavage map of the recombinant expression vector pYEGALLAC3 of the present invention.
Figure 2a shows the expression level of laccase 3 by the culture days of the transformant TYEGLAC3-1 of the present invention incubated for 5 days in ura ^- selection medium.
Figure 2b is cultured for 5 days in the ura ^- selection medium of the transformant TYEGLAC3-1 of the present invention, it shows the activity and cell growth of laccase 3 by the number of days culture.
FIG. 2C shows that the transformant TYEGALLAC3-6 of the present invention is incubated for 5 days in YEPD medium (non-induction, NI) or ura ^- selective medium (induction, I) supplemented with galactose, and activity of laccase 3 according to the number of days of culture. And cell growth.
Figure 3 is a result of staining with a DMOP substrate solution after electrophoresis of the culture supernatant of the transformant TYEGLAC3-1 according to the present invention (A), Kusami blue staining (B), analyzed by Western blot Results (C) are shown respectively (Lanes 1 and 2: lamease 1 and 5 mU of Trametes versicolor, lane 3: culture supernatant of mimic transformants, lane 4: culture supernatant lane of TYEGLAC3-1 lane 5: 40-fold concentrate of culture supernatant of TYEGLAC3-1).
Figure 4 is a transformant TYEGLAC3-1 according to the present invention is cultured in a medium supplemented with tannic acid, and shows the degree of color development around colonies (1: mimetic transformant, 2: TYEGLAC3-1, 3: TYEGLAC3-5 , 4: TYEGLAC3-7).
Figure 5 shows the results confirmed by staining with DMOP substrate solution for the protection of laccase 3 activity by various proteases (1: mimetic transformant, 2: TYEGLAC3-1, 3: TYEGLAC3-5, YEPD: fresh YEPD , SCB: culture supernatant of TYEGLAC3-1 incubated in YEPD)
Figure 6 shows the dye bleaching effect of Lacase 3 according to the present invention (1: negative control (ura ^- medium), culture of lac3 expressing transformant-about 10-20mU, 3: the same for 2 repeats 2 times Equivalent amount of culture was added, 4: Commercial laccase (Lacase isolated from the Trametes vericolor strain), 5: 3-10 mU of the same laccase as No. 4.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention as defined by the appended claims. It will be obvious to you.

Example 1: Materials and Methods

1-1. Strains and Culture Conditions

Plasmids were stored and amplified in Escherichia coli HB010 or DH5α. CDNA encoding a third lacquer of Cryphonectria parasitica was derived using the fact that a prior (Chung HJ et al Mol Plant- Microbe Interact 2008, 21:. 1582-1590), the production of the heterologous S. lac3 celebrity bicyclic Ke 2805 (MATα pep4 :: HIS3 prb1-δ can1 GAL2 his3-200 ura3-52 ) was used.

S. cerevisiae was maintained in YEPD medium (10 g / l yeast extract; 20 g / l peptone; 20 g / l dextrose). Uracil ^- deficient medium (ura-) yeast nitrogen base without 6.7 g / l amino acid, 0.03 g / l adenine and tryptophan, 5 g / l casamino acid, 20 g / l dextrose, 20 g / l Agar) was used to screen for transformants at 30 ° C. Prepare a primary inoculum from 5 ml of uracil selective culture incubated for 24 hours, and 1 x 10 ⁷ cells in a 300-ml Erlenmeyer flask containing 40 ml of YEPD broth or ura ^- selective broth. Moved to. The expression culture was incubated at 30 ° C. for 2-3 days with continuous stirring (200 rpm), centrifuged at 3,000 g, and the culture supernatant was recovered to analyze the activity of laccase.

1-2. Plasmid Construction

The cDNA clone of the lac3 gene was used conventionally (Chung HJ et al. Mol Plant-Microbe Interact 2008, 21: 1582-1590). At the 5 'and 3' ends of the mature peptides of the laccase 3 protein, forward 5'-GGTCTAGAATGCAGGTGCTGAACACC-3 '(SEQ ID NO: 1) and reverse 5'-GGTCTAGATTATATGCCC GAATC GTCC-3' (SEQ ID NO: 2) as primers and To generate an XbaI restriction enzyme site via overlap extension PCR using overlap forward 5'-AACTTGA CAGCCGGGGCTCCCAGTGTCGAG-3 '(SEQ ID NO: 3) and overlap reverse 5'-CTCGAC ACTGGGA GCCCCGG CTGTCAAGTT-3' (SEQ ID NO: 4) It was then fused with a rice peptide-derived amylase 1A ( Ramy 1A) signal peptide (ASP). The amplified gene was cloned into pGEM T-Easy vector (Promega), analyzed via restriction enzyme digestion, and confirmed by DNA sequencing. To construct yeast expression vectors, pGEM T-Easy vectors were digested with XbaI to cut out ASP / LAC3 fusion fragments and inserted into pYEGPD-TER and pYEGAL-TER. The vector has a glyceraldehyde-3-phosphate dehydrogenase ( GPD ) promoter and a galactose-1-phosphate uridilyl transferase ( GAL7 ) terminator, and a galactokinase 1 ( GAL1 ) promoter and a GAL7 terminator, respectively. have. The plasmids thus formed were named pYEGLAC3 (FIG. 1A) and pYEGALLAC3 (FIG. 1B).

1-3. Transformation and Selection of Saccharomyces cerevisiae

The constructed recombinant vector was introduced into S. cerevisiae 2805 using the lithium acetate method, and the transformants thus formed were screened in ura ⁻ medium. Colony PCR and transformation with E. coli were performed using plasmids sampled from the transformants to confirm the presence of recombinant plasmids in yeast. Yeast introduced with pYEGLAC3 was named TYEGLAC3 and yeast introduced with pYEGALLAC3 was named TYEGALLAC3.

Stability of the plasmid introduced into the yeast was measured as follows. Samples incubated in non-selective YEPD medium were serially diluted with sterile H ₂ O to 200 colony-forming units (CFU) and then plated on ura ^- selective and non-selective plates. And the relative value of CFU was confirmed.

1-3. Analysis of laccase

Lacase activity in the filtrate of the culture was confirmed by spectrophotometric analysis using 10 mM 2,6-dimethophenol (DMOP) dissolved in sodium tartrate buffer of pH 3.4 as a substrate. The laccase unit was defined as a 1.0 / min A ₄₆₈ increase at 25 ° C.

For active staining of native gels, native polyacrylamide gel electrophoresis (PAGE) was performed under non-denaturing conditions. Lacase activity was visualized in the gel using DMOP substrate solution.

A medium supplemented with tannic acid was prepared by adding 0.5% (w / v) tannic acid to the selection medium lacking uracil. The transformants were then cultured in a medium supplemented with tannic acid to visualize the activity of laccase through the color development of the medium.

1-4. Protease Inhibition Assay

To determine whether the inhibition of laccase activity is due to the presence of proteases in the culture medium, several protease inhibitors such as aprotinin, EDTA, E-64 protease inhibitors, lupetin, pepstatin and phenylmethylsulphonyl fluoride (PMSF) may be used. Pre-incubation with the culture medium was carried out for 10 hours at ℃. This precultured culture medium was mixed with the culture medium containing the laccase activity and left to culture for an additional 24 hours, and then the residual laccase activity was measured.

1-5. Western blot analysis of expressed laccase 3

Constructs of some genes comprising domains from residues 231 to 567 were expressed in E. coli as hexahistidine fusion proteins and purified by nickel affinity chromatography according to the manufacturer's instructions (Takara). CDNA encoding some laccase 3 was PCR by using primers 5'-GCAGCTCGAG CTCGAGGACAACAGC-3 '(SEQ ID NO: 5) and 5'-CCCAAGCTT TTATATGCCCGA-3' (Reverse) (SEQ ID NO: 6) Amplified. Primers were modified to incorporate restriction enzymes XhoI and HindIII, respectively. Cleaved lac3 (1014 bp) was inserted into the XhoI and HindIII sites of the expression vector pColdII DNA (Takara). The recombinant plasmid thus formed was transformed with E. coli strain BL21. Induction, purification and identification of recombinant Lacasease 3 using an anti-hexahistidine antibody was performed according to the manufacturer's instructions.

Anti-CpLac3 antibodies were obtained by injecting some 100 μg of purified Lacasease 3 into 6-week old BALB / c mice and boosting them with the same amount of laccase emulsified in incomplete Freund's adjuvant two weeks later. . On day 5 after booster injection multiple serum was obtained and western blot analysis was performed.

Example 2. Results

2-1. Analysis of Transformed S. cerevisiae

10-20 S. cerevisiae transformants representing recombinant plasmids were selected in ura ^- medium. Colony PCR was performed on the DNA samples obtained from the transformants. The 1.8 kb DNA fragment was amplified and the recombinant plasmid was delivered to the yeast. In addition, E. coli transformation with DNA samples from all selected transformants confirmed that the recombinant plasmid was present in yeast.

2-2. Expression of LAC3 Protein Using a Constitutive Promoter

The expression of the trans gene encoding Lacase 3 by the GPD promoter was confirmed by enzyme assay using the supernatant of the culture and DMOP as the enzyme and substrate, respectively. As a result of measuring enzyme activity in the culture supernatant of the transformants cultured in YEPD medium for 3 days, no laccase activity was detected. However, for culture supernatants prepared from ura ^- selective broth, all transformants showed laccase activity. The transformant TYEGLAC3-1 showing the highest enzymatic activity was used for further analysis. Transformant TYEGLAC3-1 was incubated for 5 days in ura ^- selection medium and the samples were analyzed to obtain expression patterns over time of recombinant laccase 3 (FIG. 2A). The highest laccase activity was measured 2 days after culture, which was 20.0 mU / ml culture supernatant. As the culture proceeded, the enzyme activity rapidly decreased (FIG. 2B).

Natural gel electrophoresis of the culture supernatant and staining with DMOP substrate solution confirmed the presence of a band showing laccase activity (FIG. 3A). Kusami blue staining of the natural gel, it was confirmed the presence of a protein band at the corresponding position (Fig. 3 B). In addition, Western blot analysis confirmed the immune reactivity of the corresponding protein band (Fig. 3C). Culture supernatants of transformant TYEGLAC3-1 cultured in non-specific and nutrient rich YEPD media did not show any activity and immune responsive bands of laccase. These results show that ura ^- selection is essential for the transformant to exhibit laccase activity and is maintained in ura ^- selection culture medium. These results also show that the laccase activity in the culture supernatant is due to the protein product of the lac3 gene (FIG. 3).

Since laccase 3 derived from C. parasitica responds to tannic acid, the oxidation of tannic acid by the expressed laccase 3 was measured using a medium supplemented with tannic acid. Plates containing ura ^- selective medium supplemented with 0.5% tannic acid were prepared to culture the transformed cells. As shown in FIG. 4, dark brown color appeared around the colonies of the transformants. On the other hand, only the vector showed no such color response around the transformed cells (mimetic transformants). This means that the transformant can express and secrete laccase 3 into the medium, and the expressed recombinant laccase 3 possesses biochemical properties of oxidizing phenolic compounds such as tannic acid.

2-3. Plasmid Stability and Cell Growth of Recombinant Strains

CFU was measured in nonselective YEPD medium as well as selection medium, and plasmid stability and cell growth were confirmed by counting the number of cells using a hematocytometer. Plasmid stability was confirmed for transformants TYEGLAC3-1 and two randomly selected transformants TYEGLAC3-5 and -7. Plasmid stability of transformants grown on YEPD broth was measured by plating 200 μl of a suspension of cultured for 10 days diluted on ^{6 6} dilution in ura ^- selective plates and non-selective YEPD plates, resulting in at least 65% of cells being cultured for 3 days It was confirmed that the plasmid was lost after the incubation (Table 1). Even in cells cultured in ura ^- selection medium, although not as high as YEPD medium, high plasmid instability appeared, and 50% of the plated cells could not form separate colonies.

Cell growth of lac3 and mimic transformants was compared. Compared to mimic transformants showing 917 CFU on YEPD plates, lac3 transformants showed up to 40% growth. In addition, the cells are ura ^- was observed when the culturing in selection broth, more cells reduction (≥25%) of the growth (Table 1).

2-4. Effect of Cultured Broth on Expressed LAC3

While incubating TYEGLAC3-1 transformants in non-selective YEPD medium, the number of cells containing plasmids decreased significantly, but still a significant number of cells contained plasmids. Thus, we tried to determine why laccase activity was not detected in the culture supernatant of YEPD. To this end, the supernatant of ura ^- selective broth containing laccase activity was mixed with the culture supernatant of YEPD and the enzyme activity was measured. After stationary culture at 25 ° C. for 24 hours, no laccase activity was observed. However, mixing with fresh YEPD medium or reaction buffer showed laccase activity, indicating that the expressed laccase activity was sensitive to the cultured YEPD medium, while the laccase activity remained stable in ura ^- selective medium.

Instability of laccase activity expressed in cultured YEPD media was confirmed using various protease inhibitors. As shown in Figure 5, ura ^- lacquer activity in the supernatant of the selection broth was offset when mixed with the supernatant of the cultured medium it is YEPD broth. Residual laccase activity was observed when the cultured YEPD medium was mixed with supernatant of ura ^- selective broth containing laccase activity after prepolitical incubation with pepstatin, an aspartyl protease inhibitor. No protection was identified with aprotinin, EDTA, E-64 protease inhibitors, lupetin or PMSF.

2-5. Expression of LAC3 Protein Using Inducible GAL1 Promoter

Expression of the trans gene encoding laccase 3 by the inducible GAL1 promoter was confirmed. The transformant containing the plasmid pYEGALLAC3 was incubated for 5 days in the induction medium containing 2% galactose, and the enzyme activity was confirmed in the culture supernatant of all transformants (FIG. 2C). Primary inoculum was prepared from 5 ml of medium containing 2% raffinose / 0.1% glucose as a carbon source while maintaining selectivity. Transformant TYEGALLAC3-6 showing 41.5 mU / ml culture supernatant with the highest enzymatic activity was used in subsequent examples. Transformants using the inducible GAL1 promoter showed higher laccase activity than the transformants TYEGALLAC3-1 expressing lac3 by the constitutive GPD promoter. Growth of TYEGALLAC3-6 transformants in YEPD medium was not reduced (Table 2), showing that growth did not decrease unless laccase expression occurred. The stability of the introduction of the plasmid (pYEGALLAC3-6) into the selected TYEGALLAC3-6 was also excellent so that at least 85% of the cells retained the plasmid after stationary culture for 3 days in non-selective medium. This result means that the stability of the plasmid is maintained as long as laccase expression is not induced.

Example 3: Validation of the dye bleaching effect of Lacase 3

Acid blue 147, Coomassie brilliant blue G250, Crystal violet, Methylene blue, Brilliant blue R, Malachite green on microplates After dispensing G (Malacite Green G), Remazol brilliant blue R, and Malachite Green base, cultures of ura ^- deficient medium (negative control) and lac3 expressing transformants (about 10 ~ 20mU), Lameze (Trametes vericolor) strain of Lacase (control 1mU or 10mU) was put in each of the stationary culture and observed for the bleaching effect of the dyes. As a result, it was confirmed that Lacase 3 of the present invention can discolor malachite green G.

<110> INDUSTRIAL COOPERATION FOUNDATION CHONBUK NATIONAL UNIVERSITY <120> Tannic acid-degradable laccase 3 of Cryphonectria parasitica and          usage <160> 7 <170> KopatentIn 1.71 <210> 1 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 ggtctagaat gcaggtgctg aacacc 26 <210> 2 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 ggtctagatt atatgcccga atcgtcc 27 <210> 3 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 aacttgacag ccggggctcc cagtgtcgag 30 <210> 4 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 ctcgacactg ggagccccgg ctgtcaagtt 30 <210> 5 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 gcagctcgag ctcgaggaca acagc 25 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 cccaagcttt tatatgcccg a 21 <210> 7 <211> 567 <212> PRT <213> Cryphonectria parasitica <400> 7 Met Ala Leu Arg Ala Phe Leu Ser Leu Leu Pro Ala Leu Ser Leu Val   1 5 10 15 Thr Ala Ala Pro Ser Val Glu Pro Tyr Gln Leu Ser Lys Arg Cys Val              20 25 30 Asn Ser Ala Asp Asp Arg Ser Cys Trp Gly Asp Tyr Asp Leu Ser Thr          35 40 45 Asn Tyr Tyr Asp Glu Ala Pro Phe Thr Asn Val Thr Arg Glu Tyr Trp      50 55 60 Phe Asn Ile Val Asn Thr Thr Ala Ser Pro Asp Gly Val Glu Lys Ile  65 70 75 80 Val Leu Ala Val Asn Gly Ser Ile Pro Gly Pro Thr Ile Val Ala Asp                  85 90 95 Trp Gly Asp Thr Val Val Val His Val Thr Asn Ser Met Gln Asn Asn             100 105 110 Gly Ser Ser Ile His Phe His Gly Ile Arg Gln Asn Phe Thr Asn Gln         115 120 125 Asn Asp Gly Val Ala Ser Ile Thr Gln Cys Pro Thr Ala Pro Gly Asp     130 135 140 Thr Thr Thr Tyr Thr Trp Arg Ala Leu Gln Tyr Gly Thr Ser Trp Trp 145 150 155 160 His Ser His Phe Tyr Val Gln Ala Trp Asp Gly Val Tyr Gly Gly Ile                 165 170 175 Gln Ile Asn Gly Pro Ala Thr Ala Asn Tyr Asp Glu Asp Leu Gly Val             180 185 190 Val Ser Met Met Asp Trp Asp His Asp Thr Ala Asp Asn Leu Val Leu         195 200 205 Gln Ser Ala Val Thr Gly Pro Pro Thr Met Ala Asn Gly Leu Ile Asn     210 215 220 Gly Met Asn Val Tyr Thr Leu Glu Asp Asn Ser Thr Val Gly Ser Arg 225 230 235 240 Tyr Glu Met Glu Phe Thr Ser Gly Gln Arg Tyr Arg Leu Arg Leu Val                 245 250 255 Asn Thr Ala Ala Asp Thr His Phe Lys Phe Thr Ile Asp Asn His Thr             260 265 270 Met Glu Val Ile Ala Ser Asp Phe Val Pro Ile Gln Pro Tyr Thr Thr         275 280 285 Asp Val Val Asn Ile Gly Ile Gly Gln Arg Tyr Asp Val Ile Val Thr     290 295 300 Ala Asp Ala Pro Ser Asp Asn Tyr Trp Met Arg Ala Ile Ala Gln Leu 305 310 315 320 Ala Cys Ser Asp Asn Asp Asn Pro Asp Asn Ile Lys Ala Ile Ile Arg                 325 330 335 Tyr Ser Asp Ala Ala Asn Ser Thr Ala Asp Pro Thr Ser Thr Ala Thr             340 345 350 Ala Asp Ala Ser Val Asp Glu Cys Val Asp Glu Pro Ala Ala Ser Leu         355 360 365 Val Pro Tyr Leu Ala Ile Pro Ala Gly Gly Ser Ala Asp Val Thr Asp     370 375 380 Asn Phe Ala Val Ala Val Glu Gln Leu Ala Gly Arg Val Asp Trp Ala 385 390 395 400 Met Gly Asn Thr Thr Phe Ile Asn Glu Trp Gly Tyr Pro Ser Ile Gln                 405 410 415 Gln Val Tyr Asp Gly Asn Asn Thr Trp Gly Glu Ser Gln Asn Val Ile             420 425 430 Gln Leu Pro Gln Ala Asn Glu Trp Val Tyr Trp Ile Val Gln Thr Thr         435 440 445 Met Gly Val Thr His Pro Met His Met His Gly His Asp Phe Trp Val     450 455 460 Leu Gly Ala Gly Thr Gly Thr Phe Asp Ser Ala Thr Ala Ser Leu Ser 465 470 475 480 Thr Val Asn Val Pro Arg Arg Asp Val Thr Met Leu Pro Ala Ser Gly                 485 490 495 Trp Thr Val Ile Ala Phe Ile Thr Asp Asn Pro Gly Val Trp Leu Thr             500 505 510 His Cys His Ile Ala Trp His Thr Ser Glu Gly Leu Ala Val Gln Met         515 520 525 Val Glu Arg Glu Ser Glu Ile Val Asp Leu Ile Asp Ala Asp Leu Met     530 535 540 Asn Ser Thr Cys Gly Ala Trp Asn Asp Tyr Ala Asn Lys Val Ala Leu 545 550 555 560 Val Gln Asp Asp Ser Gly Ile                 565

Claims (20)

Lacase derived from Cryphonectria parasitica which decomposes tannic acid 3. The method of claim 1,
Lacase 3, consisting of the amino acid sequence shown in SEQ ID NO: 7. A recombinant expression vector comprising the coding region of Lacase 3 according to claim 1. The method of claim 3,
The coding region of Lacase 3 is a recombinant expression vector, characterized in that the coding region of mature Lacase 3. The method of claim 3,
The coding region of Lacase 3 is recombinant expression vector, characterized in that under the control of the inducible promoter. 6. The method of claim 5,
The inducible promoter is a recombinant expression vector, characterized in that the GAL1 (galactose kinase) promoter. The method of claim 3,
The coding region of Lacase 3 is recombinant expression vector, characterized in that it is operatively connected with a signal peptide of amylase 1A ( Ramy 1A) derived from rice. The method of claim 3,
The recombinant expression vector is pYEGLAC3 having a cleavage map shown in Figure 1a or pYEGALLAC3 having a cleavage map shown in Figure 1b. Yeast host cell transformed with the recombinant expression vector according to claim 3. The method of claim 9,
The yeast is a host cell, characterized in that the genus Saccharomyces. The method of claim 10,
The genus Saccharomyces is a host cell, characterized in that Saccharomyces cerevisiae. A method for preparing laccase 3, comprising culturing the transformant according to claim 9 in a medium lacking uracil. The method of claim 12,
The uracil deficient medium comprises a yeast nitrogen base without amino acids, adenine, tryptophan, casamino acid, and dextrose. The method of claim 13,
The uracil deficient medium is a yeast nitrogen base not containing 5-8 g / l amino acid, 0.01-0.05 g / l adenine, 0.01-0.05 g / l tryptophan, 3-9 g / l casamino acid, 15-25 g / l l Production method characterized in that it comprises dextrose. The method of claim 12,
If Lacase 3 is expressed in the manufacturing method, characterized in that culture for 1 to 5 days. The method of claim 2,
Lacase 3 has an enzyme activity that decomposes tannic acid. A composition for decolorizing and degrading wood, comprising a yeast host cell or a culture thereof transformed with a recombinant expression vector comprising laccase 3 according to claim 1. A dye discoloration composition comprising a yeast host cell or a culture thereof transformed with a recombinant expression vector comprising a laccase 3 according to claim 1 and a coding region thereof. 19. The method of claim 18,
The dye is malachite green G, characterized in that the composition. Food composition for tannin acid degradation comprising a yeast host cell or a culture thereof transformed with a recombinant expression vector comprising a laccase 3, coding region thereof according to claim 1.

Images