JP3027844B2 - Lignin peroxidase gene - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a lignin peroxidase (hereinafter abbreviated as LPO) cDNA, a recombinant plasmid containing the same, and a transformant containing the recombinant plasmid. LPO has a function of acting on lignin to lower the molecular weight or decompose it, so that it can be used in various processes in the production of paper pulp from lignocellulose materials such as wood. That is, it can be used for depolymerizing or decomposing lignin in a pulping step, a pulp bleaching step, a wastewater treatment step and the like. Furthermore, in the field of so-called cellulosic biomass utilization, lignin can be decomposed as a pretreatment for saccharification of cellulose, so that it can be applied for the purpose of increasing saccharification efficiency.

[0002]

2. Description of the Related Art LPO is a lignin-degrading enzyme and is used as an enzyme for decomposing lignin.
chrysosporium), which contains heme as a prosthetic group, has a molecular weight of about 42,000, requires hydrogen peroxide for enzymatic action, and has a phenolic hydroxyl group at position 4 of the lignin model compound. Has been found to act on compounds having a methoxyl group (FEBS Lett., Vol. 169, pp. 247-250 (1984)).

[0003] In recent years, LPO is a white rot fungus other than Fanerochaete chrysosporium, for example, Arakawakawatake (see Japanese Patent Application Laid-Open No. 64-27468), Yakeirotake (Japanese Patent Application Laid-Open No. 2-30348).
3), Kawaratake (Acta Chem. Scandinavica B41 Volume 766-
P.769 (1987)], and Scarlet mushroom (Phlebia r.
adiata) (see Biochem. J. Vol. 254, p. 877 (1988)) and the like. The purified example is not known other than the above strains.

[0004] In any of these bacteria, several LPO isozymes can be found in the culture thereof, and it can be considered that various enzymes are involved in lignin degradation. Fanerochaete chrysosporium, Kawatake mushroom, Arakawakawatake mushroom and Koganeshawaurotake mushroom, if LPO is to be produced using a wild strain, the nitrogen source concentration in the medium should be kept low, Due to restrictions such as increasing the oxygen partial pressure, mass production of LPO was difficult.

[0005] To date, attempts have been made to obtain mutants that produce LPO under a high nitrogen source concentration and under the same oxygen partial pressure as the atmosphere [Japanese Patent Laid-Open Nos. 64-27469 and 2-728.
75]. However, Fanerocaete, the most studied,
Chrysosporium is a phytopathogenic bacterium, and its import into the country is prohibited. Therefore, industrial production using F. chrysosporium as a host is difficult in Japan.

[0006] Therefore, there is a demand for the development of a technology for production using a safe strain grown in Japan.
No O-producing bacterium has its gene isolated other than Koganes agaricus, and no industrial production utilizing this LPO gene has been performed yet. Yakeirotake is LPO not only by stationary culture but also by shaking culture.
But the amount is small (see JP-A-2-303483),
Further improvement of the production method is desired for practical use.

As another solution, mass production of LPO by applying gene recombination technology is considered, but there has been no successful case so far. This is considered to be because the structure of the LPO gene successfully cloned so far was difficult to produce in large quantities by a microorganism serving as a host of a recombinant DNA such as Escherichia coli or yeast.

[0008]

DISCLOSURE OF THE INVENTION The present inventors have developed a novel novel method suitable for large-scale production of LPO from a library of cDNA and chromosomal DNA prepared based on mRNA isolated from LPO-producing Bacillus edulis cells. Successful cloning of the LPO gene has led to the completion of the present invention.

[0009]

That is, the present invention provides a lignin peroxidase cDNA represented by the nucleotide sequence of SEQ ID NO: 2. The present invention is also a recombinant plasmid containing the lignin peroxidase cDNA represented by the nucleotide sequence of SEQ ID NO: 2.

[0010] Further, the present invention is a transformant containing the recombinant plasmid. Hereinafter, the present invention will be described in detail. The structural gene of LPO of the present invention comprises the nucleotide sequence of TCT from 461st ATG to 1804th in SEQ ID NO: 1 and the nucleotide sequence of 1136th TCT (Ser) from 21st ATG to SEQ ID NO: 2. This base sequence
It encodes 372 amino acids and is 23
It encodes an amino acid leader sequence and a 349 amino acid lignin peroxidase, respectively.

[0011] The chromosomal DNA (SEQ ID NO: 1) has four introns (721 to 721) having the GT-AG rule in the structural gene.
775th, 991-1048th, 1594-1652th, 1718-1774
), And TATATAA near 380 is TA
TAbox, CGTCAATTG from the 258th or GCCCATGCA from the 339th is the so-called CAA
Considered a Tbox.

The asparagine at position 103 counted from the N-terminal amino acid valine of the mature protein is considered to be an N-glycosylated amino acid residue. In addition, it is considered that proxymal histidine, to which heme metal found in peroxidase binds, is histidine at position 177, and distal histidine, which is involved in the decomposition of hydrogen peroxide, is histidine at position 48. (See Table 1).

[0013]

[Table 1]

The LPO gene of the present invention is
It differs from the LPO H8 gene of Chrysosporium in various points as shown in Table 2, and has a homology of 70% in the nucleotide sequence and 61% in the amino acid sequence. It is completely different from a gene. Further, as can be seen from Table 1, the amino acid sequence was different from that of known peroxidase, and it was a novel LPO gene.

[0015]

[Table 2]

The LPO gene of the present invention can be obtained as follows from a library of cDNA synthesized based on chromosomal DNA or mRNA of Pleurotus ostreatus. <Preparation of chromosomal DNA library> A medium capable of growing P. aeruginosa, for example, Belcos peptone medium (glucose 2%, polypeptone 0.5%, yeast extract 0.2%,
The cells grown by culturing with (KH ₂ PO ₄ 0.1%, MgSO ₄ .7H ₂ O 0.05%) are collected by filtration and frozen with liquid nitrogen.

The cells are ground in a mortar or the like, and chromosome D is extracted by a conventional method for extracting chromosomal DNA, such as the method of Yelton et al. [Proc. Natl. Acad. Sci. USA Vol. 81, 1470 (1984)].
Prepare NA. The chromosomal DNA is cut with an appropriate restriction enzyme capable of being introduced into a vector, ligated to a vector, introduced into Escherichia coli, and transformed to prepare a chromosomal DNA library. In this case, the host vector system to be used is not particularly limited. Preferably, Escherichia coli is used as a host, and a cosmid vector (such as pHC79) is used. Depending on the type of vector used, after chromosomal DNA is cut with an appropriate restriction enzyme,
In some cases, it is better to fractionate by a conventional method such as agarose gel or sucrose density gradient centrifugation and adjust the length to an appropriate range. <Preparation of cDNA library>
Preparation of a cDNA library from mRNA containing a gene is performed as follows.

Although any strain of Yakeirotake may be used, for example, IFO5307 strain can be used. Cells that are suitable for preparing a cDNA library of the strain may be any cells during LPO production.
It can be obtained by culturing according to the method shown in 83. The cells thus obtained are immediately frozen with liquid nitrogen, crushed using, for example, a mortar, a Waring blender or the like, to extract RNA from plant cells.
ular Cloning A Laboratory Manual / 2nd Edition (198
Extract RNA according to 9). M in the obtained RNA
The RNA is concentrated and purified through an affinity column such as oligo dT cellulose, and a portion of this is
NA is synthesized and ligated to an E. coli cloning vector, for example, λgt10, via an appropriate linker or adapter to prepare a cDNA library. These operations are performed by generally known methods, for example, the aforementioned “Molecular Cloth”.
ningA Laboratory Manual ". It can be easily carried out by appropriately using commercially available kits such as an mRNA extraction kit, a cDNA synthesis kit, a cDNA cloning kit, and a ligation kit. [LPO gene from library] Isolation and determination of its nucleotide sequence) The lignin peroxidase gene of F. chrysosporium was obtained by the method of Asada et al. (Appl.
Microbiol. Biotechnol. Vol. 29, pp. 469-473 (1988)]
Can be prepared. Escherichia coli HB101 containing the gene
/ pBR322LPO has been deposited with the National Institute of Microbial Technology under the deposit number FERM P-10714.

Restriction enzymes XhoI and SmaI for lignin peroxidase gene of F. chrysosporium
And the resulting 0.59 kb fragment was digested with a conventional nick translation method [Sambrook et al., "Molecular C
loning A Laboratory Manual. The 2nd Edition "] ³²
P-labelling can be used as a probe to select from the chromosomal DNA library or cDNA library by hybridization.

The chromosomal DNA was partially digested with San3AI, ligated with cosmid pHC79, transduced into Escherichia coli, selected by colony hybridization, and subcloned into pUC118. E. coli JM109 / pUCLPOBj was obtained from Deposited with the National Institute of Microbial Technology under the deposit number FERM P-12007. In addition, a strain E. coli XL-1 obtained by synthesizing cDNA from mRNA, selecting from a library of E. coli NM514 strain prepared with λgt10 by plaque hybridization, preparing λDNA from the strain, and subcloning it into pBluescript. / pBSLPO-2 has been deposited with the National Institute of Microbial Industry and Technology under the deposit number FERM P-12008.

The nucleotide sequence of the LPO gene isolated by the above method is determined by the method of Sanger et al., Proc. Natl. Acad. Sci. USA
Vol. 74, No. 5463 (1977). <Recombinant DNA, transformant> The LPO gene represented by the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2 can be inserted into a plasmid vector to obtain an expression recombinant plasmid. In this case, the plasmid vector is not particularly limited, and λ phage, cosmid vector, pUC, pBluescript and the like are appropriately used. further,
Yeast and Bacillus subtilis vectors can also be used.

In addition, a transformant can be obtained by introducing the above-mentioned recombinant plasmid into a desired host cell. Escherichia coli and yeast are used as host cells.

[0023]

[Example] <Preparation of chromosomal DNA library> A oyster mushroom was cultured in a glucose / peptone medium (glucose 2%,
Polypeptone 0.5%, Yeast extract 0.2%, KH ₂ PO ₄ 0.1
%, MgSO ₄・ 7H ₂ O 0.05%) After shaking culture in 200 ml at 28 ° C for 7 days, the bacterial cells are collected by filtration, washed, water is removed as much as possible with filter paper, and frozen with liquid nitrogen. did. 10g of this cell
Was crushed with a mortar and pestle, and further crushed with a Waring blender. Transfer the crushed cells to a centrifuge tube, 2% SDS, 50 mM
Suspended in diethylpyrocarbonate-treated water containing EDTA,
Shake for 1 minute. Next, the mixture was kept at 68 ° C. for 15 minutes, left at room temperature, and centrifuged at 9000 rpm for 15 minutes to remove cell residue.
32 ml of 5M potassium acetate (pH 5.8) is added to 80 ml of the supernatant, stirred, left on ice for 1 hour, centrifuged at 4 ° C at 25,000 rpm for 15 minutes to remove proteins, and an equal volume of isopropanol is added to the supernatant The precipitate was collected by centrifugation at 25,000 × g for 30 minutes at room temperature, the supernatant was removed, and the precipitate was dried under reduced pressure. Precipitate T
It was dissolved in 3 ml of E buffer, treated with phenol / chloroform and treated with proteinase K at 37 ° C. for 1 hour according to a conventional method [Sambrook et al., Molecular Cloning A Laboratory.
Manual / 2nd Edition "(1989)].

Using 100 μg of the obtained chromosomal DNA as a restriction enzyme
It was partially digested with San3AI and fractionated by 10-40% sucrose density gradient ultracentrifugation (30,000 rpm, 18 hours) to obtain a 30 kb to 50 kb fraction. Cosmid vector pHC79 20 μg restriction enzyme
Digestion was sufficiently performed using 20 units of BamHI, treated with alkaline phosphatase, extracted with phenol, extracted with chloroform, precipitated with ethanol, and dissolved in TE buffer. 30-50 kb chromosome DN partially digested with the vector DNA and San3AI
The A fragment was mixed at a molar ratio of 3: 1 and subjected to a binding reaction overnight at 16 ° C. with T4 DNA ligase, and then packaged with an in vitro packaging kit of Amersham to infect Escherichia coli DH1 and ambicilin was lysed. Transduced strains were selected on LB plates containing the cells. The resulting colony was inoculated into LB medium containing ambicilin, and chromosome D
This was an NA library. <Isolation of LPO gene from chromosomal DNA library> Isolation of the LPO gene from the chromosomal DNA library is performed by colony hybridization [Sambrook].
"Molecular Cloning A Laboratory Manual / 2ndEdit
ion "(1989)].

A colony of the chromosomal DNA library grown on an agar medium was transferred to a membrane, and placed on an LB agar medium containing 200 μg / ml of chloramphenicol.
C. 24 hours. Subsequently, according to the manual of a colony / plaque screen (NEF-978X) manufactured by Du Pont, the cells were washed twice with 2 × SSC after denaturation twice and neutralization twice, and air-dried.

Subsequently, the plate was shaken overnight at 65 ° C. in a prehybridization solution (5 × SSC, 1 × Denhardt's solution, 1% SDS), and 50 μg of denatured salmon DNA, 1 × 10 ⁶ cpm per one membrane And hybridization was performed at 50 ° C. for 24 hours. After hybridization, the membrane contains 1% SDS according to a standard method.
The plate was washed several times with × SSC and 0.1 × SSC at different times, dried, and then positive colonies were detected by autoradiography using an intensifying screen.

The above probe was prepared as follows.
E. coli HB101 / pBR322LPO (FERM P-10714)
Propagated in LB medium containing 50μg / ml, and alkali-SDS method
pBR322LPO was prepared. pBR322LPO 10μg with restriction enzyme Xh
The fragment was cut with oI and SmaI, and a 0.59 Kb fragment was separated and extracted by 0.7% agarose gel electrophoresis.

The XhoI-SmaI fragment (0.59 Kb) 1 μm
g of [α- ³² P] CTP (15 TBq / mmol, Amersham PB1016
20 μl of nick translation reaction solution containing 2 μl of 5,370 MBq / ml (composition: dNTP containing 1 mM each of dATP, dTTP and dGTP)
Mixed solution '': 2 μl, 0.5 mM Tris-HCl buffer (pH 8.0) containing 50 mM MgCl _2, 10 mM β-mercaptoethanol: 2 μl, 0.
1ppm DNase: 1μl, DNA polymerase (Takara Shuzo 5
(Unit / μl): 1 μl, DNA solution: 10 μl, distilled water 2 μl)
Was incubated at 15 ° C. for 1 hour, the reaction was stopped by adding 30 μl of 0.5% SDS solution, and Sephadex G-50 equilibrated with TE buffer.
Of unreacted [α- ³² P]
Except for dCTP, a ³² P-labeled probe was prepared.

A positive colony was isolated and 3 ml of 50 μg / ml
Was cultured in an LB medium containing ampicillin, and a plasmid DNA was prepared by an alkali extraction method. <Detection of LPO Gene Fragment by Southern Hybridization> The above plasmid DNA was completely digested with the restriction enzyme EcoRI, subjected to agarose gel electrophoresis, and then subjected to Southern hybridization using an ordinary nylon membrane [the above-mentioned "Molecular Cloning A". Laboratory
Manual / 2nd Edition "] to detect the LPO gene.

A DNA fragment (about 3 kb) detected by Southern hybridization was excised from an agarose gel and ligated to the EcoRI site of pUC118 to transform Escherichia coli JM109. The obtained E. coli transformant E. coli JM109 / pUCLPOBj containing the LPO gene has been deposited with the National Institute of Microbial Industry and Technology as a microbial fungus deposit FERM P-12007.

The entire base sequence of the LPO gene was obtained from Takara Shuzo Co., Ltd.
Using a TAKARA kilosequence deletion kit and Toyobo Sequenase ^™ , the procedures were performed according to the respective manuals. The results are as shown in SEQ ID NO: 1. <Creation of cDNA library> Yake mushrooms were cultured in 10 ml of the above-described glucose-peptone medium for 5 days.
After collecting and washing the cells, the cells were frozen with liquid nitrogen and crushed with a Waring blender.

The instruments and reagents to be used below are prepared by a conventional method [Sambrook et al., Molecular Cloning A Laboratory Manua.
l / 2nd Edition (1989)] The product treated with diethyl pyrocarbonate was used. 50ml of 4M per 5g of ground cells
Add a guanidine thiocyanate solution and bring to 1-2 ° C at 37 ° C.
After centrifugation for 1 minute, the mixture was centrifuged at 12000 rpm for 15 minutes to remove the residue. 3 ml of the supernatant is placed on a centrifuge tube containing 9 ml of a 5.7 M CsCl, 0.01 M EDTA (pH 7.5) solution, and the mixture is placed at 20 ° C. with a swing rotor.
Centrifugation was performed at 31,000 rpm for 24 hours. The RNA precipitate was dissolved in 4M guanidine thiocyanate, centrifuged at 10,000 rpm for 10 minutes to remove insolubles, the RNA was precipitated by ethanol precipitation, dissolved in TE, and stored at -80 ° C until use.

From the total RNA fraction, 0.1 mg was obtained using Oligotex dT30 manufactured by Takara Shuzo Co., Ltd. according to the manual.
A poly (A) RNA fraction was prepared. Next, double-stranded cDNA was synthesized from 5 μg of poly (A) RNA using a cDNA synthesis kit manufactured by Pharmacia in accordance with the manual. From the synthesized double-stranded cDNA, a cDNA library using λgt10 was prepared as follows using a cDNA cloning kit manufactured by Amersham according to the manual. cDN
A was ligated with an EcoRI adapter, treated with kinase, and electrophoresed on a low-melting agarose gel to cut out a gel containing a DNA fragment of 0.5 kb or more. From gels, use conventional methods (Sambrook et al.)
Molecular Cloning A Laboratory Manual / 2nd Editio
The DNA was recovered according to n "), treated with phenol and chloroform, precipitated with ethanol, dried, and dissolved in 10 µl of TE.

1 μg of λgt10 arm, 100 ng of cDNA, T4
The arm and cDNA were ligated at 16 ° C. for 16 hours in a ligation buffer containing 2.5 units of DNA ligase, and packaged in vitro according to the manual.
The packaged λ phage infected E. coli NM514 and formed plaques on LB agar plates. As a result, 10 ⁷ plaques / μg of insert DNA could be obtained, and 80 to 90% of the insert DNA contained 0.6 to 2.0 kb of insert DNA.

E. coli NM514 was infected with phage so that 2000-3000 plaques appeared on a 90 mm diameter petri dish.
A DNA library was prepared. <Isolation of LPO gene from library> Isolation of the LPO gene from the library is performed by conventional plaque hybridization. Sambrook et al., Molecular Cloning A
Performed by Laboratory Manual / 2nd Edition.

That is, when the plaque is about 0.5 to 1 mm in size, a nylon membrane is placed on the plate for 2 minutes, the plaque is transferred to the membrane, denatured with 0.5N NaOH, neutralized, and washed in 2 × SSC solution. Dried. Subsequently, by the same method as the colony hybridization described above,
Positive plaques were detected.

From the positive plaque, phage DNA was prepared by a small volume liquid culture according to a conventional method, and the restriction enzyme NotI was prepared.
And cut with 1% low melting point agarose gel.
A kb fragment was obtained. The fragment was subcloned into the NtoI site of the phage script SK of Stratagene and transformed into Escherichia coli XL-1 Blue according to a conventional method. Subcloned DNA was prepared in large quantities and ultracentrifuged (32,000 rpm, 40 h
r, 20 ° C) and the nucleotide sequence was determined. The nucleotide sequence is determined by Takara Shuzo's Deletion Kit and United
The assay was performed using a kit of Sequenase manufactured by States Biochemical. The results are as shown in SEQ ID NO: 1.

The fragment digested with NotI was ligated with Stratagene.
A strain obtained by ligating to NotI site of plasmid pBluescript IISK ⁺ and transforming E. coli XL-1 Blue
E. coli XL-1 / pBSLPO-2 has been deposited at the Institute of Microbial Industry and Technology of the National Institute of Advanced Industrial Science and Technology as FERM P-12008, a microbial bacterium deposit. <Structure of LPO gene> Comparison between the LPO gene derived from the chromosomal DNA of SEQ ID NO: 1 and the gene derived from the mRNA of SEQ ID NO: 2 shows the presence of introns on the chromosomal DNA, and CAAT and TATAbox involved in controlling the expression
The presence of a region that is thought to be involved and a leader sequence involved in secretion were clarified. The sequence is collectively shown in SEQ ID NO: 3.

The intron on the chromosome is 721 of SEQ ID NO: 3.
775th, 991-1048th, 1594-1652th, 1718-17
It was 4 positions in the 74th nucleotide sequence. 380-386th TATATAA is TATAbox, 259-267th CGTCAATTG or 339-347th GC
CCATGCA is considered a CAATbox. The leader sequence is the 23 amino acid sequence from methionine at position 461 to arginine, and an open reading frame encoding 349 amino acids from amino-terminal valine to carboxyl-terminal serine is found as a mature protein.

The 103rd asparagine is Asn-XT
It is considered that N-glycosylation is performed based on the sequence of hr. Also, proxymal histidine, which binds to heme metal, which is commonly found in many peroxidases, appears at position 177, and distal histidine, which is involved in the decomposition of hydrogen peroxide, appears at position 48. It had a relatively high homology with peroxidase.

However, the sequence was completely different from that of the known LPO gene, and was a novel sequence.

[0042]

According to the present invention, a novel LPO cDNA,
The recombinant plasmid and the transformant could be provided. Thus, it has become possible to provide LPO having lignin-decomposing activity that is industrially useful at low cost.

[0043]

[Sequence list] (1) SEQ ID NO: 1 Sequence length: 1978 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Genomic DNA Origin Organism: Bjerkandera adusta strain Name: IFO5307 Sequence characteristics 259-267 E -35 signal 339-347 E -35 signal 380-386 E -10 signal 461-1804 P CDS 461-529 P sig peptide 530-1804 P mat peptide 721-774 intron 991- 1048 intron 1594-1652 intron 1717-1772 intron 891-893 P N-glycosylation site 671-673 P distal histidine 1171-1173 P proximal histidine CGCAGAAGCA CGCTCGGCAG 180 CGTCGCGACG CGATACTCTGGG GGCATGATTG AGCACAAGTT TCCCTGCGCC AATAGCACCC 240 TGCGCAACTC TATTCCGACG TCAATTGAAA GGCTACGCCA AAAATAACTT TCAACGCCAG 300 GATCTTGCAG TCGCTACGGC CGGGGGGCGCGGTCGATCGATCGCGCGGTCGGTC TA TCGAGAGGGT ATATAAGGTC GACCATCTAG CTCTTCCAAG TCTCCAAGCC 420 CAACCCGGTC TTCCGCAATT CCTCGAGCAA CTCAGCAGCA ATGGCCTTCA AGCAGCTCCT 480 CGCCACCCTC TCCGCCGCCC TTGTGGCGCA GGCAGCGATC ACCCGCCGCG TCGCCTGCCC 540 CGACGGCAAG AACACCGCGA TCAACGCGGC GTGCTGCAGC CTGTTCACGG CGCGCGACGA 600 CATCCAGAAG AACATGTTCG ACGGCGGGCA GTGCAACGAC ATTGCGCACC AGGCCATCCG 720 GCTGACCTTC CACGACGCCG TCGCGTTCTC GCCGACTCTG ACGGCGCAGG GCAAGTTTGG 780 GTGCGTACCA AAGTCCATCC CGAGGATTTG AACAGCATGC TGAGTTGGAC TACAGTGGTA 840 ACGGCGCCGA CGGGTCGATC ATGACCTTCG ACACCATCGA GACCAACTTC CACCCGAACA 900 TCGGCCTCGA CGAGATCGTG AACATCGAGA AGCCGTTCGC CCAGTTCCAC AACATGACCC 960 CCGGTGACTT CATCCACTTT GCGGGTGCCC TCGCCGTGAC GAACTGCCCC GGCGCACCCA 1020 CGCTCACCTT CTCCATCGGC CGTCCCCCAC GTACGCAGTT CCTCAAATGC GTCTTGCTTC 1080 GAATTTACTG ACGCAATGGC GTTTTTAGCG GTCGCCGCTG CCCCCGACGG CCTCGTCCCC 1140 GAGCCGTTCC ACACCGCGGA CCAGATCTTC GCGCGCATGC TCGACGCGCT CCAGTTCGAC 1200 GAGCTCGAGA CCGTGTGGGG CCTGATCGCG CACACCGTCG GCGCGTCCAA CGACGTCGAC 1260 CCGTCCATCC CCCGCACGCC C TTCGACAGC ACGCCCTCGA TCTTCGACGG CCAGTTCTTC 1320 ATCGACACGC AGCTGCGCGG CGAGCTCTTC CCTGGGCAGG GCGGGCTGCA AGGTGAAGTA 1380 GAGTCGCCGC TGAAGGGCCA GATCCGGCTG CAGTCCGACC ACATCATCGC GCGCGACTCG 1440 CGCTCGGCGT GCGAGTGGCA GTCGTTCGCG AACGACCACG ACAAGCTCAC GAACCGCTTC 1500 CAGTTCGTGA TCGAGACCCT GGCGATGGTC GGACAGGACC CGACGAACAT GATTGATTGC 1560 TCCGAGGTCA TCCCCATCCC CAAGGACCTC ACCGCGGCGC AGCTCACCCC CATGTTCCCC 1620 GCTGGAAAGA CCAACGCTGA TGTCGAGCAG GCTGTACGTG CCCTTCGCCT TCGCCATTTT 1680 CCGGACTAGA ACACTAACGA ATGATCTTAC AGTGCGCCGA CACGCCCTTC CCGTCCTTCG 1740 CCACTCGCCC GGGTCCCGCC ACCGCTGTTC CTCCCGTGTA CGTCTCCCCT CAGTCGATTA 1800 AAGCATCTAA CCTAACGATA TTCCTTTCCA CAGCCCCTCC GCCAAGACTG GAGCTCCCGG 1860 CTCTTAAGCG GATTTACTCC GAACCTCAAT CGGTATATGT CGTCGTCGGT GTATTAGAAT 1920 GAATGAAATG TATATAAGAT GTTTTGTATG CCACTAGATT TGTACTTCTA GCGAATTC // 1978 (2) SEQ ID NO: 2 sequence length: 1273 type of sequence : Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: cDNA Origin Organism Bjerkandera adusta Strain name: IFO5307 Sequence characteristics 259-267 E -35 signal 339-347 E -35 signal 380-386 E -10 signal 21-1136 P CDS 21-89 P sig peptide 90-1136 P mat peptide SEQ: CCTCGAGCAA CTCAGCAGCA ATGGCCTTCA AGCAGCTCCT CGCCACCCTC TCCGCCGCCC 60 TTGTGGCGCA GGCAGCGATC ACCCGCCGCG TCGCCTGCCC CGACGGCAAG AACACCGCGA 120 TCAACGCGGC GTGCTGCAGC CTGTTCACGG CGCGCGACGA CATCCAGAAG AACATGTTCG 180 ACGGCGGGCA GTGCAACGAC ATTGCGCACC AGGCCATCCG GCTGACCTTC CACGACGCCG 240 TCGCGTTCTC GCCGACTCTG ACGGCGCAGG GCAAGTTTGG TGGTAACGGC GCCGACGGGT 300 CGATCATGAC CTTCGACACC ATCGAGACCA ACTTCCACCC GAACATCGGC CTCGACGAGA 360 TCGTGAACAT CGAGAAGCCG TTCGCCCAGT TCCACAACAT GACCCCCGGT GACTTCATCC 420 ACTTTGCGGG TGCCCTCGCC GTGACGAACT GCCCCGGCGC ACCCACGCTC ACCTTCTCCA 480 TCGGCCGTCC CCCACCGGTC GCCGCTGCCC CCGACGGCCT CGTCCCCGAG CCGTTCCACA 540 CCGCGGACCA GATCTTCGCG CGCATGCTCCG ACGCGCTCCGCC CGTCCGTCCGCCCGCGCGTCCGCCCGCGCGTCCGCCCGTCCGCGCGTCCGCCCGCGCGCGTCCGCCCGCGCGCGTCCGCCCGCGCGTCCGCC T CGACAGCACG CCCTCGATCT TCGACGGCCA GTTCTTCATC GACACGCAGC 720 TGCGCGGCGA GCTCTTCCCT GGGCAGGGCG GGCTGCAAGG TGAAGTAGAG TCGCCGCTGA 780 AGGGCCAGAT CCGGCTGCAG TCCGACCACA TCATCGCGCG CGACTCGCGC TCGGCGTGCG 840 AGTGGCAGTC GTTCGCGAAC GACCACGACA AGCTCACGAA CCGCTTCCAG TTCGTGATCG 900 AGACCCTGGC GATGGTCGGA CAGGACCCGA CGAACATGAT TGATTGCTCC GAGGTCATCC 960 CCATCCCCAA GGACCTCACC GCGGCGCAGC TCACCCCCAT GTTCCCCGCT GGAAAGACCA 1020 ACGCTGATGT CGAGCAGGCT TGCGCCGACA CGCCCTTCCC GTCCTTCGCC ACTCGCCCGG 1080 GTCCCGCCAC CGCTGTTCCT CCCGTCCCCT CCGCCAAGAC TGGAGCTCCC GGCTCTTAAG 1140 CGGATTTACT CCGAACCTCA ATCGGTATAT GTCGTCGTCG GTGTATTAGA ATGAATGAAA 1200 TGTATATAAG ATGTTTTGTA TGCCACTAGA TTTGTACTTC TAGCGAATTC TAGCTGATTC1 TGCGTC TGCCA TGCTCATC TAGCGAATTC Topology: linear Sequence type: genomic DNA Origin Organism: Bjerkandera adusta Strain: IFO5307 Sequence characteristics 259-267 E- 35 signal 339-347 E -35 signal 380-386 E -10 signal 461-1804 P CDS 461-529 P sig peptide 530-1804 P mat peptide 721-774 intron 991-1048 intron 1594-1652 intron 1717-1772 intron 891 -893 P N-glycosylation site 671-673 P distal histidine 1171-1173 P proximal histidine sequence: 10 20 30 40 50 60 CACGGTATGTCGCGTTCCTCCAGCGCCGCACAAAACTTACCAGTGACCGGTTTCGGCGCG 70 80 90 100 110 120 TGGAAGAGGAAGCGTCGTCGTCGTGAGTACGCCCTTCCCCGCCGCAATTTCTGCCACACT 130 140 150 160 170 180 TGGCGTGTATGTCCACAGGGTCGTTCCATGATGTGTGCAACGCAGAAGCACGCTCGGCAG 190 200 210 220 230 240 CGTCGCGACGCGATACTCTGGGCATGATTGAGCACAAGTTTCCCTGCGCCAATAGCACCC 250 260 270 280 290 300 TGCGCAACTCTATTCCGACGTCAATTGAAAGGCTACGCCAAAAATAACTTTCAACGCCAG 310 320 330 340 350 360 GATCTTGCAGTCGCTACGGCCGGGGGTGGCGATTGGCTGCCCATGCACGATTCGGCGCGA 370 380 390 400 410 420 TTACGGGGTATCGAGAGGGTATATAAGGTCGACCATCTAGCTCTTCCAAGTCTCCAAGCC 430 440 450 460 470 480 CAACCCGGTCTTCCGCAATTCCTCGAGCAACTCAGCAGCAATGGCCTTCAAGCAGCTCCT MetAlaPheLysGlnLeuLe 490 500 510 520 530 540 CGCCACCC TCTCCGCCGCCCTTGTGGCGCAGGCAGCGATCACCCGCCGCGTCGCCTGCCC uAlaThrLeuSerAlaAlaLeuValAlaGlnAlaAlaIleThrArgArgValAlaCysPr 550 560 570 580 590 600 CGACGGCAAGAACACCGCGATCAACGCGGCGTGCTGCAGCCTGTTCACGGCGCGCGACGA oAspGlyLysAsnThrAlaIleAsnAlaAlaCysCysSerLeuPheThrAlaArgAspAs 610 620 630 640 650 660 CATCCAGAAGAACATGTTCGACGGCGGGCAGTGCAACGACATTGCGCACCAGGCCATCCG pIleGlnLysAsnMetPheAspGlyGlyGlnCysAsnAspIleAlaHisGlnAlaIleAr 670 680 690 700 710 720 GCTGACCTTCCACGACGCCGTCGCGTTCTCGCCGACTCTGACGGCGCAGGGCAAGTTTGG gLeuThrPheHisAspAlaValAlaPheSerProThrLeuThrAlaGlnGlyLysPheGl 730 740 750 760 770 780 gtgcgtaccaaagtccatcccgaggatttgaacagcatgctgagttggactacagTGGTA y GlyA 790 800 810 820 830 840 ACGGCGCCGACGGGTCGATCATGACCTTCGACACCATCGAGACCAACTTCCACCCGAACA snGlyAlaAspGlySerIleMetThrPheAspThrIleGluThrAsnPheHisProAsnI 850 860 870 880 890 900 TCGGCCTCGACGAGATCGTGAACATCGAGAAGCCGTTCGCCCAGTTCCACAACATGACCC leGlyLeuAspGluIleValAsnIleGluLysProPheAlaGlnPheHisAsnMetThrP 910 920 930 940 950 960 960 CCGGTGACTTCATCCACTTTGCGGGTGCCCTCGCCGTGAC GAACTGCCCCGGCGCACCCA roGlyAspPheIleHisPheAlaGlyAlaLeuAlaValThrAsnCysProGlyAlaProT 970 980 990 1000 1010 1020 CGCTCACCTTCTCCATCGGCCGTCCCCCACgtacgcagttcctcaaatgcgtcttgcttc hrLeuThrPheSerIleGlyArgProPro 1030 1040 1050 1060 1070 1080 gaatttactgacgcaatggcgtttttagCGGTCGCCGCTGCCCCCGACGGCCTCGTCCCC ProValAlaAlaAlaProAspGlyLeuValPro 1090 1100 1110 1120 1130 1140 GAGCCGTTCCACACCGCGGACCAGATCTTCGCGCGCATGCTCGACGCGCTCCAGTTCGAC GluProPheHisThrAlaAspGlnIlePheAlaArgMetLeuAspAlaLeuGlnPheAsp 1150 1160 1170 1180 1190 1200 GAGCTCGAGACCGTGTGGGGCCTGATCGCGCACACCGTCGGCGCGTCCAACGACGTCGAC GluLeuGluThrValTrpGlyLeuIleAlaHisThrValGlyAlaSerAsnAspValAsp 1210 1220 1230 1240 1250 1260 CCGTCCATCCCCCGCACGCCCTTCGACAGCACGCCCTCGATCTTCGACGGCCAGTTCTTC ProSerIleProArgThrProPheAspSerThrProSerIlePheAspGlyGlnPhePhe 1270 1280 1290 1300 1310 1320 ATCGACACGCAGCTGCGCGGCGAGCTCTTCCCTGGGCAGGGCGGGCTGCAAGGTGAAGTA IleAspThrGlnLeuArgGlyGluLeuPheProGlyGlnGlyGlyLeuGlnGlyGluVal 1330 1340 1350 1360 1370 1380 GAGTCGCCGCTGAAGGGCCAGATCCGGCTGCAGTCC GACCACATCATCGCGCGCGACTCG GluSerProLeuLysGlyGlnIleArgLeuGlnSerAspHisIleIleAlaArgAspSer 1390 1400 1410 1420 1430 1440 CGCTCGGCGTGCGAGTGGCAGTCGTTCGCGAACGACCACGACAAGCTCACGAACCGCTTC ArgSerAlaCysGluTrpGlnSerPheAlaAsnAspHisAspLysLeuThrAsnArgPhe 1450 1460 1470 1480 1490 1500 CAGTTCGTGATCGAGACCCTGGCGATGGTCGGACAGGACCCGACGAACATGATTGATTGC GlnPheValIleGluThrLeuAlaMetValGlyGlnAspProThrAsnMetIleAspCys 1510 1520 1530 1540 1550 1560 TCCGAGGTCATCCCCATCCCCAAGGACCTCACCGCGGCGCAGCTCACCCCCATGTTCCCC SerGluValIleProIleProLysAspLeuThrAlaAlaGlnLeuThrProMetPhePro 1570 1580 1590 1600 1610 1620 GCTGGAAAGACCAACGCTGATGTCGAGCAGGCTgtacgtgcccttcgccttcgccatttt AlaGlyLysThrAsnAlaAspValGluGlnAla 1630 1640 1650 1660 1670 1680 ccggactagaacactaacgaatgatcttacagTGCGCCGACACGCCCTTCCCGTCCTTCG CysAlaAspThrProPheProSerPheA 1690 1700 1710 1720 1730 1740 CCACTCGCCCGGGTCCCGCCACCGCTGTTCCTCCCGTgtacgtctcccctcagtcgatta laThrArgProGlyProAlaThrAlaValProProVal 1750 1760 1770 1780 1790 1800 aagcatctaacctaacgatattcctttccacagCCCCTCCGCCAAGACTGGAG CTCCCGG ProSerAlaLysThrGlyAlaProGl 1810 1820 1830 1840 1850 1860 CTCTTAAGCGGATTTACTCCGAACCTCAATCGGTATATGTCGTCGTCGGTGTATTAGAAT ySer // 1870 1880 1890 1900 1910 GAATGAAATGTATATAAGATGTTTTGTATGCCTCTAGATTG

──────────────────────────────────────────────────続 き Continuation of the front page (51) Int.Cl. ⁷ Identification code FI C12R 1: 645) There is an application for the application of Article 30, Paragraph 1 of the Patent Law. Microbial accession number FERM P-12008 Microbial accession number FERM P-12007 (56) Reference JP-A-2-303483 (JP, A) Gene, Vol. 89, (1990), p. 145-150 (58) Field surveyed (Int. Cl. ⁷ , DB name) C12N 15/00-15/90 C12N 1/00-1/38 BIOSIS (DIALOG) GenBank / EMBL / DDBJ (GENETYX) WPI ( DIALOG)

Claims (3)

(57) [Claims] 1. A lignin peroxidase cDNA represented by the nucleotide sequence of SEQ ID NO: 2. 2. A recombinant plasmid containing a lignin peroxidase cDNA represented by the nucleotide sequence of SEQ ID NO: 2. A transformant comprising the recombinant plasmid according to claim 2.