JPH08308581A - Manganese peroxidase gene - Google Patents

Abstract

PURPOSE: To obtain the subject gene useful for depolymerizing and decomposing lignin in production of paper and pulp, and mass production of manganese peroxidase useful for lignin decomposition as pretreatment for saccharifying a cellulose. CONSTITUTION: This manganese peroxidase gene encodes an amino acid sequence of the formula (including a case in which the amino acid sequence is not deficient in or not substituted with Asn-103, Arg-44, His-48 and His-177 but is deficient in and substituted with one or plural amino acids and still has manganese peroxidase activity). The manganese peroxidase gene is collected in primary selection by colony hybridization from a cDNA library prepared from a cell not producing a lignin peroxidase during production of manganese peroxidase from Coriolus hirsutun and in secondary selection using a synthetic DNA probe prepared from sequences AsnAlaAlaCysCysAla and LysMetThrValLeuGly.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a manganese peroxidase gene, a recombinant plasmid containing the gene, and a transformant containing the recombinant plasmid. Manganese peroxidase acts on lignin and has a function of lowering or degrading the molecular weight of lignin, so that it can be used in various steps in the production of paper pulp from a lignocellulosic material such as wood. That is, pulping process, pulp bleaching process,
It can be used to reduce the molecular weight of lignin or decompose it in the wastewater treatment process. Furthermore, in the field of so-called cellulosic biomass utilization, it can be used for the purpose of enhancing saccharification efficiency by decomposing lignin as a pretreatment for saccharification of cellulose.

[0002]

BACKGROUND ART Manganese peroxidase is an enzyme that decomposes lignin and is first isolated and purified from Phanerochaete chrysosporium [Arch. Biochem. Biophys., 242, 329-341 (19).
85)], the molecular weight is 46,000 or less, the iron-containing enzyme is presumed, hydrogen peroxide is required for the enzymatic action, and the enzymatic reaction is Mn (II) -dependent, It does not act on compounds in which the 4-position phenolic hydroxyl group of the lignin model compound has become a methoxyl group,
Its main feature is that it has been confirmed to act on a phenolic lignin model.

As for manganese peroxidase, Pleurotus ostreatus (Pleurotus ostreatus) is used for microorganisms other than Phanelocete chrysosporium [JP-A-3-132295, B
See IOTECHNOL. LETT., Volume 15, No.3, 289-294 (1993)], Trametes versicolor [Acta Chem
ica Scandinavica, Volume B41, 762-765 (1987), Arch. Bioc
hem. Biophys., Vol. 300, No. 1, 57-62 (1993)], Physia radiata [Arch. Bioc.
hem. Biophys., 279, 25-31 (1990)], Lentinus edodes [Appl. Microbiol. Biotechnol.,
Volume 33, 359-365 (1990)], Kakegawatake (Panus tigr
inus) [Biotec. Appl. Biochem., 13, 291-302 (1991)
See], Phanerochaete
sordida) [Appl. Environ. Microbiol., Volume 60, No. 2,
599-605 (1994)] and the like, but no purified example other than the above strains is known.

[0004] Each of the above-mentioned bacteria can find some manganese peroxidase isozymes in its culture, and it can be considered that various enzymes are involved in lignin degradation. FaneloKete chrysosporium, oyster mushroom, Kawaratake mushroom, Kogaresurokotake mushroom, Shiitake mushroom, Physcomitrella patens and Fanerokate
In any strain of Soldida, when producing manganese peroxidase using a wild strain, it is necessary to keep the nitrogen source concentration in the medium to a low concentration, and the enzyme is grown while the cells are grown in a nutrient-rich medium. Since it could not be produced, mass production of manganese peroxidase was difficult. Similarly to the strains described above, manganese peroxidase can be produced by stationary culture also in Pleurotus cornucopiae, but its production is small and further improvement of the production method is desired for practical use.

Attempts have been made to date to obtain a mutant strain that produces manganese peroxidase under a high nitrogen source concentration [Appl. Environ. Microbiol., Volume 57, No. 9, 2591-259.
6 (1991)], but it has not yet been put to practical use. In addition, it is considered that a large amount of enzyme is produced by gene recombination, but the manganese peroxidase gene has not been isolated from this bacterium.

Among the above-mentioned manganese peroxidase-producing bacteria, the gene thereof is isolated from funerocete.
Chrysosporium [J. Biol. Chem., 264, 5036-5040
(1989), J. Biol. Chem., 264, 13531-13535 (1989)], and industrial production using the manganese peroxidase gene of this bacterium has not been performed yet. The reason why it is difficult to obtain the manganese peroxidase gene is that the base sequence of this gene has high homology with the base sequence of lignin peroxidase, and there are many isozymes in lignin peroxidase, and there are many genes. It may be difficult to distinguish peroxidase. For example, in the case of Phanelocete chrysosporium, lignin peroxidase gene 6
Species [Nature, Volume 326, 520-523 (1987) (Corrigendum, 328
Volume, 742), Gene, Volume 60, 93-102 (1987) (Corrigendum, 69
Volume, 369 (1988)), Gene, Volume 97, 191-198 (1991), Bioche
m. Biophys. Res. Commun., Volume 162, 673-680 (1989), Bi
ochem. Biophys. Res. Commun., 73, 994-1000 (1990)
Reference], two manganese peroxidase genes have been reported, but the homology between these genes is very high at 57% to 86%.

In general, in order to obtain a purified protein gene, a synthetic DNA probe having a base sequence deduced from the amino acid sequence of the purified protein is prepared,
Although the gene to which this binds is selected and obtained, since the manganese peroxidase gene has high homology with the lignin peroxidase gene, it is difficult to make a synthetic DNA probe that selectively binds only to the manganese peroxidase gene. Even in Arakawakawatake, funerocete
Similar to Chrysosporium, there are many genes for lignin peroxidase isozymes [see JP-A-5-252958, JP-A-5-252959, JP-A-5-260978], and synthesis using slight differences. With a DNA probe,
It was difficult to selectively obtain only the manganese peroxidase gene.

Further, unlike a chromosomal DNA library containing all genes, in the case of a cDNA library in which only the genes of the protein being produced are collected, it is possible to produce only manganese peroxidase without producing lignin peroxidase. If possible, a library containing no lignin peroxidase gene can be prepared, and the manganese peroxidase gene can be selectively obtained. However, when cultured by the above-mentioned conventional culture method, Agarella agaricus also produces lignin peroxidase at the same time as it produces manganese peroxidase, and it is difficult to produce a cDNA library containing no lignin peroxidase gene. It was

Further, when mass-producing manganese peroxidase, it may be possible to utilize the gene of Phanelocete chrysosporium, but since this bacterium is limited in use from the standpoint of plant protection, it is not available in Japan. Production by seed was necessary. Further, the manganese peroxidase of Pleurotus cornucopiae has an optimum pH higher than that of any of the other strains known so far, and is advantageous for industrial use. Therefore, mass production has been desired.

[0010]

DISCLOSURE OF THE INVENTION The present invention provides a gene coding for manganese peroxidase derived from Aracia moss, which is necessary for more efficient industrial production of manganese peroxidase by utilizing gene recombination technology. It is intended to be provided.

[0011]

The present invention provides the amino acid sequence of SEQ ID NO: 1 which provides manganese peroxidase, or one or more amino acids added, deleted or substituted in the amino acid sequence of SEQ ID NO: 1 (provided that , Asn-103, Arg-44, His-48 and His-177 are the target amino acids for addition, deletion or substitution) and manganese peroxidase-encoding amino acid sequence having enzymatic activity It is in the oxidase gene.

The present invention further provides a manganese peroxidase gene represented by the base sequence of SEQ ID NO: 2.
The present invention also resides in a manganese peroxidase gene containing a promoter region contained in the base sequence of SEQ ID NO: 3. The present invention also has one or more amino acids added, deleted or substituted in the DNA encoding the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 1 (provided that Asn-103, Arg-44, His -48 and His-1
(Except when 77 is an amino acid to be added, deleted or substituted)) and a recombinant plasmid containing a DNA encoding an amino acid sequence having manganese peroxidase enzymatic activity.

Further, the present invention is a recombinant plasmid containing the manganese peroxidase gene represented by the nucleotide sequences of SEQ ID NOs: 2 and 3. Furthermore, the present invention resides in a transformant containing the above recombinant plasmid. Hereinafter, the present invention will be described in detail. The manganese peroxidase structural gene of the present invention has the A at position 44 in SEQ ID NO: 2.
Base sequence of 1138th TCG (Ser) from TG (Met), or 93rd ATG in SEQ ID NO: 3
It consists of the nucleotide sequence of the 1504th TCG (Ser) from (Met). This base sequence encodes 365 amino acids, and encodes a leader sequence of 26 amino acids from the N-terminus and manganese peroxidase of 339 amino acids, respectively. The leader sequence consisting of 26 amino acids is a typical secretory protein signal sequence that retains a basic amino acid lysine (Lys) residue near the N-terminus and is followed by a region containing many hydrophobic amino acids. It was

The chromosomal DNA (SEQ ID NO: 3) is composed of 5 introns (154 to 2) according to the GT-AG rule in the structural gene.
11th, 555 to 615th, 817 to 880th, 1123 to 1193th, 1432 to 1494th) are included. The 103rd asparagine counted from the N-terminal amino acid valine of the mature protein is considered to be an N-glycosylated amino acid residue. In addition, heme-iron coordinated proximal histidine, which is characteristic of peroxidase, is 177
The fourth histidine, the distalarginine involved in the decomposition of hydrogen peroxide is the 44th arginine, and the distalhistidine also involved in the decomposition of hydrogen peroxide is 4
It is considered to be the 8th histidine. These positions have high homology with other peroxidases, but the sequences before and after them are completely different (Table 1).
reference).

[0015]

[Table 1] Amino acid sequence around proximal and distal histidine Enzyme ────────────────────────────────── A: Book enzyme of the invention B: Coliorus hirstus LPO4 C: P.chrysosporium MPO H4 D: P.chrysosporium LPO H8 E: P.chrysosporium LPO H2 Distal histidine around a: 39 ValHisGluSerLeuArgLeuThrPheHisAspAlaIle 51 B: 39 GlyHisAspSerLeuArgLeuThrPheHisAspGlyIle 51 C: 37 AlaHisGluValIleArgLeuThrPheHisAspAlaThr 49 D: 38 AlaHisGluSerIleArgLeuValPheHisAspSerIle 50 E: 39 AlaHisGluAlaLeuArgMetValPheHisASpSerIle 51 Proximal histidine around A: 168 AspGluValValAlaLeuLeuAlaSerHisThrIleAla 180 B: 170 IleLeuThrValTrpLeuLeuThrAlaHisThrValAla 182 C: 164 PheGluValValSerLeuLeuAlaSerHIsThrValAla 176 D: 167 LeuGluLeuValTrpMetLeuSerAlaHisSerValAla 179 E: 168 IleGluThrValTrpLeuLeuSerAlaHisSerIleAla 180 ────────────── ──── ───────────────

The manganese peroxidase gene of the present invention differs from the manganese peroxidase H4 gene of Phanelocete chrysosporium in various points as shown in Table 2, and its homology is 59% in the nucleotide sequence,
The amino acid sequence was 52%, which is completely different from the manganese peroxidase H4 gene of Phanelocete chrysosporium.

[0017]

[Table 2] Comparison between the gene of the present invention and the manganese peroxidase H4 gene of Phanelocete chrysosporium ───────────────────────────── ────── Enzyme of the present invention Phanelocete H4 ────────────────────────────────── Intron number 5 7 places Leader sequence amino acid number 26 residues 24 residues Structural gene 365 amino acids 358 amino acids Molecular weight 39985 38152 N-glycosylation site Asn-103 Asn-100 Asn-155 Asn-184 Asn-241 Distalarginine Arg-44 Arg- 42 Distal Histidine His-48 His-46 Proximal Histidine His-177 His-173 ────────── ───────────────────────

The manganese peroxidase gene of Phanelocete chrysosporium has three characteristic amino acid substitutions, such as substitution of acidic amino acids and basic amino acids, as compared with the lignin peroxidase gene.
Although there are some places, the gene of the present invention also showed the same amino acid substitution as compared with the lignin peroxidase gene of Pleurotus cornucopiae. However, in Phanelocete chrysosporium, manganese peroxidase has a characteristic 7-residue amino acid insertion not found in lignin peroxidase near the 230th position from the N-terminal amino acid of the mature protein. No such insertion was found in the oxidase gene. (See Table 3)

[0019]

[Table 3] Insertion of amino acids characteristic of the manganese peroxidase of Fanelocate chrysosporium ─────────────────────────────── Enzyme A: Enzyme of the present invention B: Coliorus hirstus LPO4 C: Coliorus hirstus LPO6 E: P.chrysosporium MPO H3 F: P.chrysosporium MPO H4 G: P.chrysosporium LPO H8 H: P.chrysosporium LPO H2 Insertion of amino acid sequence A: 227 GluSerProLeu --------------------- HisGlyGluLeu 234 B: 229 GluSerProLeu -------------------- ------ GluGlyGluLeu 236 C: 228 GluSerProLeu --------------------- AlaGlyGluLeu 235 E: 226 MetSerProLeuProLeuGlySerGlySerAspThrGlyGluLeu 240 F: 227 AlaSerProLeuProLeuGlySerGlySerAspThrGluGerLluGerSluAlu -------------------- ProGlyGluLeu 236 H: 229 ThrSerProLeu --------------------- ProGlyGluLeu 236 ─ ──────────────────────────────────

From these facts, the manganese peroxidase gene of the present invention is a novel manganese peroxidase gene having a different amino acid sequence from known peroxidases. In the present invention, in addition to the manganese peroxidase gene encoding the amino acid sequence of SEQ ID NO: 1, one or more amino acids are added, deleted or substituted in the amino acid sequence of SEQ ID NO: 1 (provided that Asn- (Except when 103, Arg-44, His-48 and His-177 are amino acids targeted for addition, deletion or substitution) and a manganese peroxidase gene encoding an amino acid sequence having manganese peroxidase enzymatic activity It is included in the manganese peroxidase gene of the present invention. Then, here, addition, deletion or substitution of amino acids is a known technique for site-directed mutagenesis (for example, Nuclei
c Acid Reseach, Vol.10, No.20, 6487-6500, (1982)), and regarding addition, deletion or substitution of amino acids, one or more amino acids means site-specific mutagenesis. It means a number of amino acids that can be added, deleted or substituted by the method.

[0021] The manganese peroxidase gene of the present invention is a cDNA synthesized from the mRNA of Pleurotus cornucopiae.
It can be obtained from a library or a chromosomal DNA library as follows.

<Preparation of cDNA library> mR containing the manganese peroxidase gene of Pleurotus cornucopiae
The cDNA library is constructed from NA as follows. As long as Aragekawaratake can be used as long as it has the ability to produce manganese peroxidase, the mutant strain NTG III-55 derived from the Arakawakawatake IFO 4917 strain [Ministry of Industrial Science (currently, Life Research Institute) deposit No. FPRMP-14046] is particularly suitable. Bacteria suitable for preparing a cDNA library of Pleurotus cornucopiae are any ones as long as they are producing manganese peroxidase and not producing lignin peroxidase.

It is known that when a wild strain is cultivated using a medium having a small nitrogen source used for producing manganese peroxidase, it produces manganin peroxidase and lignin peroxidase at the same time. However, the mutant strain of the present invention produces about 10 times more manganese peroxidase than the wild strain even when it is cultured in a medium having a small nitrogen source, and lignin peroxidase produces an extremely small amount, It is possible to obtain cells that are particularly suitable for preparing a cDNA library for obtaining the manganese peroxidase gene.

The culturing method is not particularly limited, and as the nutrient source of the medium, those generally used for culturing microorganisms can be widely used. The carbon source may be any assimilable one, for example glucose,
Maltose, pulp, wood flour, etc. are used. Any available nitrogen compound may be used as the nitrogen source, and for example, peptone, soytone, ammonium tartrate, soybean lectin and the like are used. Other salts such as phosphates, sulfates, magnesium, calcium, potassium, sodium, copper, manganese, zinc, etc. are used as necessary. The culturing temperature may be appropriately changed within a range in which the producing bacterium can grow.
C. to 40.degree. C. are preferred. .

The obtained bacterial cells are immediately frozen in liquid nitrogen, crushed using, for example, a mortar, Waring blender, etc., and RNA is extracted from plant cells [Molecular Cl
oningA Laboratory Manual / 2nd Edition (1989)]. The mRNA in the obtained RNA is concentrated and purified through an affinity column such as oligo dT cellulose, and then a portion of this is used to synthesize cDNA, which is then cloned into an E. coli cloning vector such as λgt10 via an appropriate linker or adapter. (Manufactured by Amersham) to prepare a cDNA library.

These operations are carried out by a generally known method,
For example, the above-mentioned "Molecular Cloning A Laboratory Manual"
Can be performed according to the method of. Also commercially available mRN
It can be easily carried out by appropriately using kits such as A extraction kit, cDNA synthesis kit, cDNA cloning kit, ligation kit and the like.

<Preparation of Chromosome DNA Library> A medium capable of proliferating Pleurotus cornucopiae, for example, 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%) cultured cells are collected by filtration and frozen in liquid nitrogen.

Frozen cells were ground in a mortar and the like and the method of Yelton et al. [Proc. Natl. Acad. Sci. USA, 81, 1470 (198)
4)] and the like to prepare chromosomal DNA. The chromosomal DNA is cleaved with an appropriate restriction enzyme that can be introduced into a vector, ligated to the vector, introduced into E. coli, and transformed to prepare a chromosomal DNA library. In this case, the host vector system used is not particularly limited, but preferably Escherichia coli is used as a host and a phage vector such as EMBL3 (manufactured by Toyobo Co., Ltd.) is used. Depending on the type of vector used, it may be desirable to cut the chromosomal DNA with an appropriate restriction enzyme and then fractionate by a conventional method such as agarose gel electrophoresis or sucrose density gradient centrifugation, and arrange the lengths appropriately. There is also.

<Preparation of Synthetic DNA Probe> The enzyme protein purified according to the method described above is digested with lysyl endopeptidase (manufactured by Wako Pure Chemical Industries, Ltd.), and each peptide is subjected to HPLC using a reverse phase column such as ODS. Fractionate. After separation and purification, the amino acid sequence from the N terminus is analyzed by the Edman method. A synthetic DNA probe can be prepared by comparing these sequences with the known lignin peroxidase of Pleurotus cornucopiae and searching for a portion having a small homology and a small combination of base sequences deduced from those amino acids.

<Isolation of Manganese Peroxidase Gene from Library and Determination of Its Nucleotide Sequence> The manganese peroxidase cDNA gene is prepared from cells producing manganese peroxidase but not producing lignin peroxidase. The selected cDNA library can be selected by colony hybridization or plaque hybridization. The manganese peroxidase gene utilizes the high degree of homology between the lignin peroxidase gene and the manganese peroxidase gene to identify the lignin peroxidase gene [α-
^The probe labeled with ³² P] dCTP can be used as a probe to selectively obtain it. At this time, it is also possible to use the above-mentioned synthetic DNA probe end-labeled with [γ- ³² P] ATP as a probe. Next, the genes selected for the primary selection were subjected to the secondary selection by Southern hybridization using a synthetic DNA probe, and the target manganese peroxidase c was determined from the strength of the binding.
A DNA gene can be selectively obtained.

The manganese peroxidase chromosomal gene was obtained by colony hybridization or plaque hybridization from the above chromosomal library using the obtained manganese peroxidase cDNA gene labeled with [α- ³² P] dCTP as a probe. You can select. At this time, in order to eliminate a large number of mixed lignin peroxidase genes and obtain a chromosomal gene corresponding to the obtained manganese peroxidase cDNA gene, it is necessary to perform washing under more severe conditions than the usual method.

Further, the nucleotide sequence of the manganese peroxidase gene isolated by the above method can be determined by the method of Sanger et al. [Pro
c. Natl. Acad. Sci. USA, Vol. 74, 5463 (1977)]. cDNA synthesis from mRNA, λgt10
The clone selected from the cDNA library prepared in step 1 is used as an E. coli vector pBluescriptIISK + (Stratagene)
Strain obtained by subcloning into Escherichia coli JM109 / pBSMPOC1
Deposited with the Institute of Biotechnology, Institute of Industrial Science and Technology, deposit number FERM P
It has been deposited as -14932. In addition, clones selected from the chromosomal DNA library were cloned into E. coli vector pBlu.
The strain E. coli JM109 / pBSMPOG1 obtained by subcloning into the EcoRI site of escriptIISK + has been deposited at the Biotechnology Institute of Biotechnology under the deposit number FERM P-14933.

<Recombinant DNA, Transformant> The manganese peroxidase gene of the present invention represented by the nucleotide sequence of SEQ ID NO: 2 or 3 determined by the above method is incorporated into a plasmid vector to obtain an expression recombinant plasmid. You can In this case, the plasmid vector is not particularly limited, and λ phage, cosmid vector, pUC, pBluescript, etc. are appropriately used. Furthermore, yeast and Bacillus subtilis vectors are also used. In addition, a transformant can be obtained by introducing the above recombinant plasmid into a desired host cell. Escherichia coli, Bacillus subtilis and yeast are used as host cells.

[0034]

[Example 1]

<Preparation of cDNA library> Arakawakawatake IF
Manganese peroxidase CH- from O-4917 strain
1 High-producing mutant strain NTG III-55 was cultivated at 28 ° C. using an air-lift type 2 l culture tank containing 1.5 l of low concentration nitrogen source medium (medium of Bonnarme et al.) Shown in Table 4.

[0035]

[Table 4] Bonnarme's medium ───────────────────────────── glucose 10 g D-diammonium tartrate 0.2 g sodium tartrate (PH 4.5) 20 mM KH ₂ PO ₄ 2 g MgSO ₄ .7H ₂ O 0.5 g CaCl ₂ .2H ₂ O 0.1 g thiamine hydrochloride 1 mg Tween 20 0.5 g veratryl alcohol 2.5 mM trace amount Elemental solution ^* 70 ml ───────────────────────────── Total volume 1000 ml * Trace elemental solution ───────── _{──────────────────── MgSO 4 3 g MnSO 4 0.5} g NaCl 1.0 g FeSO 4 · 7H 2 O 0.1 g CoCl 2 0.1 _{g ZnSO 4 · 7H 2 O 0.1} g CuSO 4 0.1 g AlK (SO 4 ) ₂・ 12H ₂ O 10 mg H ₃ BO ₄ 10 mg Na ₂ MoO ₄・ 2H ₂ O 10 mg Nitrile acetate 1.5 g ─────────────────────── ───────

On the second day of culture, soybean lectin (0.1 g /
l) and veratryl alcohol (2.5 mM) were added,
Bacterial cells on the 3rd day of culture in which manganese peroxidase production was active and lignin peroxidase was not produced were collected, washed, frozen in liquid nitrogen, and ground in a mortar. The equipment and reagents used below are [Molecu
lar Cloning A Laboratory Manual / 2nd "Edition (198
9)] The one treated with diethylpyrocarbonate was used.
Total RNA was extracted from about 5 g of the crushed cells using an RNA extraction kit manufactured by Amersham, according to its manual.

From 2 mg of the total RNA, manufactured by Pharmacia
Using the mRNA Purification Kit, a poly (A) RNA fraction was prepared according to its manual. Then 5 μg of poly (A) R
Double-stranded cDNA was synthesized from NA using a cDNA synthesis kit manufactured by Amersham according to its manual. Amersham cD from the synthesized double-stranded cDNA
Using the NA cloning kit, according to the manual, a cDNA library at λgt10 was prepared as follows. The EcoRI adapter was ligated to the cDNA, and the unreacted adapter was removed by a gel filtration column.
A fraction containing p or more cDNA fragments was taken out. Furthermore, after the above-mentioned cDNA fragment fraction was treated with kinase, phenol,
It was recovered by chloroform treatment and ethanol precipitation, air-dried and then dissolved in 10 μl of TE solution.

1 μg of λgt10 arm and 25 ng of cDNA were mixed and ligated in an attached ligation buffer containing 2.5 units of T4 DNA ligase at 16 ° C. for 16 hours. Then, in vitro packaging was performed according to the manual to prepare a cDNA library. Above cD
Recombinant λ phage from E. coli library NM
514 were infected and allowed to form plaques on LB agar plates. As a result, 1.6 × 10 ⁸ plaques / μg
It was possible to create a cDNA library with the titer of the insert cDNA.

<Preparation of Synthetic DNA Probe> Manganese peroxidase CH-derived from Arakawakawatake IFO4917 strain
1 A method of culturing a high-producing strain NIGIII-55 strain and treating the culture solution on the 7th day of culture using an ion exchange column, a gel filtration column, an HPLC column, etc. (described in Example 2 of Japanese Patent Application No. 6-66941). The enzyme protein purified by the method described in 1.) was digested with lysyl endopeptidase, and each peptide was digested with Vydac2
HPLC using 18TP54 column (solvent A: 0.1% TFA / DWB:
Fractionation was performed with 0.1% TFA / Acetonitrile, A: 5 → 55% / 50 min). The four peptide fragments were separated and purified, and the amino acid sequence was analyzed from the N terminus according to the Edman method. The results are shown in Fig. 1. These sequences were compared with the amino acid sequence of the known lignin peroxidase from Pleurotus cornucopiae (Fig. 2).
Searching for a portion (shown by an underline in FIG. 2) having a small homology and a small combination of base sequences deduced from those amino acids, four kinds of synthetic DNA probes were prepared from the amino acid sequences at two positions. Synthetic DNA produced
The probe is shown in FIG.

<Isolation of the manganese peroxidase cDNA gene from the cDNA library> The manganese peroxidase gene is isolated from the above-mentioned cDNA library by a conventional method [Molecular Cloning A Laboratory Manua.
l / 2nd "Edition (1989)] plaque hybridization.

E. coli NM514 was infected with the phage so that 4,000 to 6,000 plaques appeared on a petri dish having a diameter of 90 mm, and a nylon membrane was placed on the plate for 2 minutes when the plaque was about 0.5 mm to 1 mm. The plaque was transferred to the membrane. Then, according to the manual of the Amersham blotting membrane, the denaturing operation with the denaturing solution was performed once, and the neutralization operation with the neutralizing solution was performed twice.
Furthermore, 2 x SSC (1 x SSC: 150 mM NaC
1, 15 mM trisodium citrate) and air-dried, and then the recombinant DNA was immobilized on the membrane by UV irradiation to obtain a plaque hybridization membrane. After pre-hybridization, add 2 ml of hybridization solution (5
X SSC, 1 x Denhardt's solution, 1% SDS, 100 μg /
ml salmon-denatured DNA), and further 1 × 10 ⁶ cpm
Add a probe that has been radiolabeled to give
Hybridization was performed at 24 ° C. for 24 hours. As a probe, lignin peroxidase gene
No. 260978] was labeled with [α- ³² P] dCTP by a giga labeling kit manufactured by Amersham,
The manganese peroxidase gene was selected by utilizing the high homology between the lignin peroxidase gene and the manganese peroxidase gene. After hybridization, the membrane was washed in 2 × SSC containing 1% SDS at room temperature for 30 minutes with shaking according to a conventional method, and further washed in 0.1% SSC containing 1% SDS at room temperature for 30 minutes. After drying, autoradiography using an intensifying screen was performed to detect about 20 positive plaques. Phage DNA was prepared from these positive plaques by liquid culture by liquid culture, cleaved with restriction enzyme EcoRI, and subjected to 1% agarose gel electrophoresis with the amount of DNA in each sample kept constant. The DNA in the gel was transferred to a membrane by performing denaturation operation, neutralization operation, blotting operation, and fixation operation by UV according to the manual of a blotting membrane manufactured by Amersham Co., and used as a membrane for Southern hybridization. After completion of the prehybridization, 10 ml of a hybridization solution and a synthetic DNA probe were added so that the concentration was 1 × 10 ⁶ cpm / ml, and hybridization was carried out at 40 ° C. for 24 hours. Synthetic DNA probe is T4DNA as usual.
It was end-labeled with [γ- ³² P] ATP by the kinase. For selection of the manganese peroxidase cDNA gene, four types of probes 1a, 1b, 2a and 2b shown in FIG. 1 were used. The membrane after hybridization was washed twice by shaking in 2 × SSC at room temperature for 30 minutes, dried, and then autoradiographed. As a result of autoradiography, a positive DNA fragment of 1.4 kbp was detected in which the two kinds of probe 1a and probe 2b commonly hybridized more strongly than others. The fragment was pBluescriptIISK + manufactured by Stratagene, according to a conventional method.
Subcloned into the EcoRI site of
MPOC1) is shown in FIG. Escherichia coli was transformed with the plasmid, prepared in large quantities, and the nucleotide sequence was determined. As a result, manganese peroxidase C derived from Arakawakawatake mushroom IFO4917 strain was obtained.
A method of culturing H-1 high-producing strain NIGIII-55 strain and treating the culture solution on the 7th day of culture using an ion exchange column, gel filtration column, HPLC column, etc. (Example of Japanese Patent Application No. 6-66941) Two
According to the described method), the amino acid sequence of the enzyme was purified (according to 4 positions, 70 amino acids in total, see FIG. 1). The obtained manganese peroxidase cDNA gene (MP
E. coli JM109 / pBSMPOC transformant containing OC1)
1 has been deposited at the Institute of Biotechnology, Institute of Industrial Science and Technology, with the deposit number FERM P-14932 of the Institute for Life Science.

The determination of the nucleotide sequence is carried out by the Takara Shuzo Deletion Kit and Toyobo's Sequence Kit Sequen.
This was done using cing PRO. The results are as shown in SEQ ID NO: 2. The amino acid sequence deduced from the DNA sequence of SEQ ID NO: 2 is shown in SEQ ID NO: 1.

[Example 2] <Preparation of chromosomal gene library> An agar piece having a diameter of 5 mm was punched out from a flat plate agar medium of Arakawakawatake (IFO 4917 strain) with a cork borer, and glucose / peptone medium (glucose 2%, polypeptone 0. 5%, 0.2% yeast _{extract, KH 2 PO 4 0.1%,} MgSO 4 · 7
H ₂ O 0.05%, adjusted to pH 4.5 with phosphoric acid) 200
The cells were inoculated in ml and cultured at 28 ° C. for 7 days with rotary shaking.
After collecting the bacterial cells, the bacterial cells were washed with 1 liter of sterilized water and frozen with liquid nitrogen. 5 g of this frozen microbial cell was crushed using a mortar. Transfer the crushed cells to a centrifuge tube, and use Lysis buffer (1
(00mM Tris (pH8.0), 100mM EDTA, 100mM NaCl and proteinase K were added to 100μg / ml)
10 ml was added and incubated at 55 ° C. for 3 hours.
After incubation, perform phenol extraction and chloroform extraction, and gradually add ethanol to the aqueous layer to add DNA.
When was precipitated, the chromosomal DNA was wound up and dissolved in a TE solution.

100 μg of the obtained chromosomal DNA was digested with restriction enzyme S
Partially decomposed with au3AI, fractionated by 10-40% sucrose density gradient ultracentrifugation (30,000 rpm, 18 hours), 30-
A 50 kbp DNA fragment fraction was collected. EMBL3BamH
The I-arm and the chromosomal DNA fragment of 30 to 50 kbp partially digested with Sau3AI were mixed at a molar ratio of 3: 1 to prepare T4D.
After performing a binding reaction with NA ligase at 16 ° C. overnight, the product was packaged with an in vitro packaging kit manufactured by Amersham Co. to prepare a rainbow trout chromosomal DNA library.

<Isolation of the manganese peroxidase gene from the chromosomal DNA library> The manganese peroxidase gene can be isolated from the chromosomal DNA library according to a conventional method [Sambrook et al., "Molecular Cloning AL".
aboratory Manual / 2nd "Edition (1989) by plaque hybridization. Escherichia coli P2392 (manufactured by Toyobo Co., Ltd.) was infected with the above chromosomal DNA library, and about 5,000 recombinant plaques were plated on an LB plate with a diameter of 90 mm. , The recombinant plaque was cultured at 37 ° C. until it grew to a diameter of about 1 mm, the nylon membrane was placed on the plate for 2 minutes, and the plaque was transferred to the membrane. Then, the filter was denatured, neutralized, washed, dried, and UV-treated to prepare a hybridization filter.

As a probe used for plaque hybridization, a manganese peroxidase cDNA gene isolated by the method shown in Example 1 was prepared as follows. Escherichia coli JM109 / pBSMPOC1 in which the gene was cloned was cultured, and 10 μg of the recombinant plasmid obtained by alkaline extraction from the cells was completely cleaved with restriction enzyme EcoRI.
A 1.4 kbp manganese peroxidase cDNA gene fragment was cut out by% agarose gel electrophoresis. 1 μg of the gene fragment was radiolabeled with [α- ³² P] dCTP according to the manual using a giga labeling kit manufactured by Amersham Co., and used as a probe.

Then, after the completion of the prehybridization, 10 ml of the hybridization solution and 1 × 10 ^{6 were} added.
The above probe was added so that the concentration would be cpm / ml.
Hybridization was performed at 24 ° C. for 24 hours. The membrane after hybridization was washed under a more severe condition than the usual method in order to eliminate the lignin peroxidase gene mixed in and to distinguish the chromosome fragment corresponding to the manganese peroxidase cDNA obtained in Example 1. 3% in 2 × SSC with 1% SDS at room temperature
It was washed by shaking for 0 minutes, washed in the same solution at 70 ° C. for 30 minutes, and further washed with 1% SDS at 70 ° C.
By washing in 1 × SSC for 30 minutes, many lignin peroxidase and manganese peroxidase isozyme genes that were present were detected as little as possible. After air-drying, autoradiography was performed to isolate 5 positive plaques to obtain 3 6.5 kbp EcoRI fragments and 2 4.3 kbp EcoRI fragments. These fragments were converted into Ec of pBluescript II SK + manufactured by Stratagene according to a conventional method.
It was subcloned into the oRI site and transformed into E. coli JM109. Subcloned DNA was prepared in large quantities and the nucleotide sequence was determined.
A manganese peroxidase chromosomal gene (MPOG1) corresponding to the gene could be obtained as a 6.5 kbp EcoRI fragment. The fragment was designated as the plasmid pB manufactured by Stratagene.
The recombinant plasmid ligated to the EcoRI site of luescriptIISK ⁺ is shown in FIG. E. coli with the combined plasmid
Strain E. coli JM109 / pBSMPOG1 obtained by transforming li JM109
Is deposit number of Life Research Institute at Institute of Biotechnology, Institute of Technology
Deposited as FERM P-14933. The nucleotide sequence of the gene is as shown in SEQ ID NO: 3.

<Structure of Manganese Peroxidase Gene> Comparison of the gene derived from the mRNA of SEQ ID NO: 2 with the manganese peroxidase gene derived from the chromosomal DNA of SEQ ID NO: 3 reveals the existence and secretion of an intron on the chromosomal DNA. The presence of the leader sequence involved was revealed. It is shown collectively in SEQ ID NO: 4. The introns on the chromosome are 154-121, 555-615, and 817 of SEQ ID NO: 4.
There were 5 positions of the ~ 880th, 1123 ~ 1193th, and 1432 ~ 1494th nucleotide sequences. The 26 amino acid sequence starting from the 145th methionine to arginine is the leader sequence, and as a mature protein, from the amino-terminal valine,
An open reading frame is found that encodes 339 amino acids up to the carboxyl-terminal serine.

The 103rd asparagine counted from the amino terminal amino acid valine of the mature protein is Asn.
It is considered to be N-glycosylated from the sequence of -X-Thr. In addition, proximal histidine coordinated by heme iron, which is common to many peroxidases, is 17
Seventh, distalarginine involved in the decomposition of hydrogen peroxide was found at the 44th position, and distalhistidine was found at the 48th position. These amino acids have high homology with other peroxidases. The sequence of was completely different.

In comparison with lignin peroxidase, there are three characteristic amino acid substitutions such as replacement of acidic amino acids with basic amino acids in the manganese peroxidase of Phanelocete chrysosporium.
The gene of the present invention also showed similar amino acid substitutions as compared with the lignin peroxidase gene of Pleurotus cornucopiae, and it was clearly considered to be a manganese peroxidase gene. However, in Phanelocete chrysosporium, manganese peroxidase has a characteristic 7-residue amino acid insertion not found in lignin peroxidase near the 230th position from the N-terminal amino acid of the mature protein. No such insertion was found in the oxidase gene. From these facts, the manganese peroxidase gene of the present invention has a different amino acid sequence from known peroxidases, and is a novel manganese peroxidase gene.

[0051]

INDUSTRIAL APPLICABILITY The present invention has made it possible to provide a novel manganese peroxidase chromosomal gene, a manganese peroxidase cDNA gene, recombinant plasmids and transformants thereof. As a result, efficient manganese peroxidase production using gene recombination technology can be performed.

[0052]

[Sequence list]

 (1) SEQ ID NO: 1 Sequence length: 365 Sequence form: Amino acid Origin Biological name: Arakawakara (Coliorus hirstus) Strain name: IFO4917 Sequence characteristics 1-26 Signal peptide 27-365 Mature peptide Sequence: Met Ala Phe Lys Thr Leu Ala Ser Phe Val Ser Val Leu Ala Ala -25 -20 -15 Leu Gln Val Thr Asn Gly Ala Leu Thr Arg Arg Val Thr Cys Pro -10 -5 1 Asp Gly Val Asn Thr Ala Ser Asn Ala Ala Cys Cys Ala Leu Phe 5 10 15 Pro Val Leu Glu Asp Ile Gln Thr Asn Leu Phe Asp Gly Gly Glu 20 25 30 Cys Gly Glu Glu Val His Glu Ser Leu Arg Leu Thr Phe His Asp 35 40 45 Ala Ile Gly Ile Ser Pro Lys Ile Ala Ser Thr Gly Thr Phe Gly 50 55 60 Gly Gly Gly Ala Asp Gly Ser Ile Ala Ile Phe Ala Asp Ile Glu 65 70 75 Thr Asn Phe His Ala Asn Asn Gly Val Asp Glu Ile Ile Gly Glu 80 85 90 Gln Ala Pro Phe Ile Ala Arg His Asn Leu Thr Thr Ala Asp Phe 95 100 105 Ile Gln Phe Ala Gly Ala Ile Gly Val Ser Asn Cys Pro Gly Ala 110 115 120 Pro Arg Leu Asp Val Phe Val Gly Arg Lys Asp Ala Thr Gln Pro 125 130 135 Ala Pro Asp Leu Thr Val Pro Glu Pro Phe Asp Thr Val Asp Ser 140 145 150 Ile Leu Ala Arg Phe Ala Asp Ala Gly Gly Phe Ser Ser Asp Glu 155 160 165 Val Val Ala Leu Leu Ala Ser His Thr Ile Ala Ala Ala Asp His 170 175 180 Val Asp Pro Ser Ile Pro Gly Thr Pro Phe Asp Ser Thr Arg Ser 185 190 195 Leu Phe Asp Thr Gln Phe Phe Ile Glu Thr Gln Leu Arg Gly Thr 200 205 210 Leu Phe Pro Gly Thr Gly Gly Asn Gln Gly Glu Val Glu Ser Pro 215 220 225 Leu His Gly Glu Leu Arg Leu Gln Ser Asp Ser Glu Leu Ala Arg 230 235 240 Asp Ser Arg Thr Ala Cys Glu Trp Gln Ser Phe Val Asn Asn Gln 245 250 255 Ala Lys Leu Gln Ser Ala Phe Lys Ala Arg Phe Arg Lys Met Thr 260 265 270 Val Leu Gly His Ser Val Ser Asp Leu Val Asp Cys Ser Glu Val 275 280 285 Val Pro Thr Pro Pro Ala Pro Ala Ser Asn Ala His Phe Pro Ala 290 295 300 Gly Leu Gly Gln Ala Asp Val Glu Gln Ala Cys Ala Ser Thr Pro 305 310 315 Phe Pro Thr Leu Pro Thr Asp Pro Gly Pro Val Thr Ser Val Ala 320 325 330 Pro Val Pro Pro Ser *** 335

(2) SEQ ID NO: 2 Sequence length: 1323 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: cDNA Origin organism name: Coriorus hirstus strain name : IFO4917 Sequence features: 44-1138 CDS 44-121 Signal peptide 122-1138 Mature peptide 428-436 N-glycosylation site 251-253 Distalarginine 263-265 Distalhistidine 650-652 Proximal histidine Sequence: TTCTCCTCTT CTGAGCCATC TCTACTACCT CTCCTAGCAG ATCATGGCTT 50 TCAAGACACT CGCCTCCTTC GTCTCGGTTC TCGCCGCTCT CCAGGTCACC 100 AATGGTGCGC TCACTCGCCG GGTGACTTGC CCCGACGGTG TCAACACTGC 150 GTCCAACGCG GCGTGCTGCG CGCTCTTCCC CGTCCTCGAA GACATCCAGA 200 CAAACCTCTT CGACGGCGGC GAGTGCGGCG AGGAGGTGCA CGAGTCCCTC 250 CGTCTCACCT TCCACGACGC CATCGGCATC TCGCCCAAGA TCGCCTCCAC 300 CGGCACCTTT GGTGGCGGCG GCGCGGACGG CTCTATCGCC ATCTTCGCGG 350 ACATCGAGAC CAACTTCCAC GCGAACAACG GTGTCGACGA GATCATCGGC 400 GAGCAGGCGC CCTTCATCGC GCGCCACAAC CTCACCACTG CGGACTTCAT 450 CCAGTTCGCC GGGGCGATCG GCGTGAGCAA CTGCCCTGGT GCGCCGCGCC 500 TCGACGTCTT CGTCGGCCGC AAGGACGCGA CGCAGCCCGC GCCCGACTTG 550 ACCGTGCCCG AGCCGTTCGA CACCGTCGAC AGCATCCTCG CCCGCTTCGC 600 CGACGCTGGT GGCTTCAGCT CTGACGAGGT CGTCGCCCTC CTGGCCTCGC 650 ACACCATCGC TGCGGCTGAC CACGTCGACC CGAGCATCCC CGGCACGCCG 700 TTCGACTCCA CCCGGAGCCT GTTCGACACG CAGTTCTTCA TCGAGACCCA 750 GCTCCGCGGC ACGCTCTTCC CCGGCACCGG CGGCAACCAG GGCGAGGTCG 800 AGTCGCCCCT CCACGGCGAG CTCCGCCTCC AGTCCGACTC TGAGCTCGCG 850 CGTGACTCGC GCACCGCCTG CGAGTGGCAG TCCTTCGTCA ACAACCAGGC 900 GAAGCTCCAG TCCGCGTTCA AGGCGCGGTT CCGCAAGATG ACCGTGCTCG 950 GGCACAGCGT GAGCGACCTC GTCGACTGCT CCGAGGTCGT GCCCACGCCG 1000 CCCGCGCCCG CGAGCAACGC GCACTTCCCC GCCGGCCTCG GCCAGGGCGA 1050 CGTCGAGCAG GCCTGCGCCA GCACGCCCTT CCCGACCCTC CCCACCGACC 1100 CGGGCCCGGT CACCTCCGTG GCCCCTGTTC CCCCTTCGTA AACGACACAG 1150 GAAGGATGAT GGTCTTGGTT GGCACGGCCG AGGGCTATTA GAACATCTGG 1200 TCTTGGCTTC ACACACACAT TCTTGAGGGT TCACATGATT CT TGGATAGA 1250 GCGCATCTGC ACATGTTTTC GGCTTAATAT ATTGCTGTTG TTATGTTCAG 1300 CTTTACCTTT AAAAAAAAAA AAA 1323

(3) SEQ ID NO: 3 Sequence length: 2294 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Chromosomal DNA Origin organism name: Coriorus hirstus strain Name: IFO4917 Sequence features: 93-1507 CDS 93-228 Signal peptide 229-1507 Mature peptide 154-211 Intron 555-615 Intron 817-881 Intron 1123-1193 Intron 1430-1494 Intron 535-543 N-glycosylation site 358 -360 Distal arginine 370-372 Distal histidine 882-884 Proximal histidine sequence: GACTTAGTAA AGAGGGTAGG TCGACGTCTG GTGTCCTCAG AGCCTCGGTT 50 TCTCCTCTTC TGAGCCATCT CTACTACCTC TCCTAGCAGA TCATGGCTTT 100 CAAGACACTC GCCTCCTTCG TCTCGGTTCT CGCCGCTCTC CAGGTGACCA 150 ATGGTGCGTG TCTACTCACA TTCCTAGTCC AAGGCCTCGG GCTGACCAGA 200 TCTGCCCACA GGTGCGCTCA CTCGCCGGGT GACTTGCCCC GACGGTGTCA 250 ACACTGCGTC CAACGCGGCG TGCTGCGCGC TCTTCCCGG T CCTCGAAGAC 300 ATCCAGACAA ACCTCTTCGA CGGCGGCGAG TGCGGCGAGG AGGTGCACGA 350 GTCCCTCCGT CTCACCTTCC ACGACGCCAT CGGCATCTCG CCCAAGATCG 400 CCTCCACTGG CACGTTCGGT GGTGGCGGCG CGGACGGCTC TATCGCCATC 450 TTCGCGGACA TCGAGACCAA CTTCCACGCG AACAACGGTG TCGACGAGAT 500 CATCGGCGAG CAGGCTCCCT TCATCGCGCG CCACAACCTC ACCACTGCGG 550 ACTTGTACGT CGATCTGCGC CCGTAAACCC CGCCTGTCAC AACGCTGATC 600 ATCCGCGAGC GCGAGCATCC AGTTCGCCGG GGCGATCGGC GTGAGCAACT 650 GCCCTGGGGC GCCGCGCCTC GACGTCTTCG TCGGCCGCAA GGACGCGACG 700 CAGCCCGCGC CCGACTTGAC CGTGCCCGAG CCGTTCGACA CCGTCGACAG 750 CATCCTCGCC CGCTTCGCCG ACGCTGGTGG CTTCAGCTCT GACGAGGTCG 800 TCGCCCTCCT GGCCTCGTAC GTTATCTCCA GCACCTCTAC TTTCTACTGT 850 GCGGCGACTA ACGTGTTTGT TTGTGCTCAG GCACACCATC GCTGCGGCTG 900 ACCACGTCGA CCCGAGCATC CCCGGCACGC CGTTCGACTC CACCCGGAGC 950 CTGTTCGACA CGCAGTTCTT CATCGAGACC CAGCTCCGCG GCACGCTCTT 1000 CCCGGGCACT GGGGGAAACC AGGGCGAGGT CGAGTCGCCC CTCCACGGCG 1050 AGCTCCGCCT CCAGTCCGAC TCTGAGCTCG CGCGCGACTC GCGCACCGCC 1100 TGCGAGTGGC AGTCCTTCGT CAGTGCGTAC CCC TCTCTCC TCCTGCCCAC 1150 CTCTTACTCC CCTGGCTGAC GCGCCGTCCG CGCAACCCTA CAGACAACCA 1200 GGCGAAGCTC CAGTCCGCGT TCAAGGCGCG GTTCCGCAAG ATGACCGTGC 1250 TCGGGCACAG CGTGAGCGAC CTCGTCGACT GCTCCGAGGT CGTGCCCACG 1300 CCGCCCGCGC CCGCGAGCAA CGCGCACTTC CCCGCCGGCC TCGGCCAGGC 1350 CGACGTCGAG CAGGCCTGCG CCAGCACGCC CTTCCCGACC CTTCCCACCG 1400 ACCCGGGCCC GGTCACCTCC GTCGCTCCTG TGTAAGTCCC GTTCCAGTGA 1450 GGTCCTTTTG AGGAAACATC GATGCTGATT TGGCGCTCTT GCAGCCCCCC 1500 TTCGTAAACG TACACAGGGA AGATGATGGT CTTGGTTGGC ACGGCCGAGG 1550 GCTATTAGAA CATCTGGTCT TGGCTTCCAC ACACATTCTT GAGGGTTCGC 1600 ATGATTTTTG GATAGAGCAA TTCTGCACAT GTTTTCGGCT TAATATATTG 1650 CTTGCTGCTG TTATGTTCAG CTTTACCTTT TGAACTCGAA TGAACTGCAG 1700 GTGAAGTGCG GATTGTAGAC GCATTCCGAT GTCCAAGCGA AGATCTACGG 1750 ATACAAGCCA ATCCCGTTCC CTTGATCTAC AAGTCCGTAC AGTCCTTCCG 1800 AGACGTCTAG CGTTGTCGCA AGGGCCCAAT GGCCATATTT TCTATGAGGA 1850 TTTGAGGAGA GTGCGAATGT CGTGGGCCAT CTGTGTAGCT CTGCCTCATC 1900 GTCGAAGTCG TATGTGTATT GTTTCTCGGC CTGCAATTGG AGTGAGCGAA 1950 ACTTCCCGAG AGCAC AGAAT GGATGCTGTA TGGCTCACCG TTCGCAACAC 2000 GAGTTGGTGA GTCTCTTCAA TCGTCCGACA CTCTATTATA ACGTCCTTAT 2050 TAGTTGTTCC GTCGGTCCTA ACGTCCAATT TCTCTCCAGC ACGAGGGAAC 2100 AGGAGTGTAT GCTTGACATC CGACGCCCTC GGCCGCCCCT CCCTCTCCTC 2150 CACAAGCACG CACAGTACCC GCGGCGCAGA CGTCAAGAGG AGCACGCACG 2200 GCCGGTTCTT CTTCAAACAC CGCTTGCGCA CGTGCGACCT CACTGCCGCG 2250 ACGCGGGACG GTCTCGCAAG CGCACAGACC GCGGACCTAC ATGC 2294

(4) SEQ ID NO: 4 Sequence length: 2294 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Chromosomal DNA Origin organism name: Coriorus hirstus strain Name: IFO4917 Sequence features: 93-1507 CDS 93-228 Signal peptide 229-1507 Mature peptide 154-211 Intron 555-615 Intron 817-881 Intron 1123-1193 Intron 1430-1494 Intron 535-543 N-glycosylation site 358 -360 Distal arginine 370-372 Distal histidine 882-884 Proximal histidine sequence: GACTTAGTAAAGAGGGTAGGTCGACGTCTGGTGTCCTCAGAGCCTCGGTT 50 TCTCCTCTTCTGAGCCATCTCTACTACCTCTCCTAGCAGATCATGGCTTT 100 MetAlaPhe -25 CAAGACACTCGCCTCCTTCGTCTCGGTTCTCGCCGCTCTCCAGGTGACCA 150 LysThrLeuAlaSerPheValSerValLeuAlaAlaLeuGlnValThrAsn -20 -15 -10 ATGGTGCGTGTCTACTCACATTCCTAGTCCAAGGCCTCGGGCTGACCAGA 200 TCTGCCCACAGGTGCGCTCACTCGCCGGGTGACTTGCCC CGACGGTGTCA 250 GlyAlaLeuThrArgArgValThrCysProAspGlyValAsn -5 -1 1 5 ACACTGCGTCCAACGCGGCGTGCTGCGCGCTCTTCCCGGTCCTCGAAGAC 300 ThrAlaSerAsnAlaAlaCysCysAlaLeuPheProValLeuGluAsp 10 15 20 ATCCAGACAAACCTCTTCGACGGCGGCGAGTGCGGCGAGGAGGTGCACGA 350 IleGlnThrAsnLeuPheAspGlyGlyGluCysGlyGluGluValHisGlu 25 30 35 40 GTCCCTCCGTCTCACCTTCCACGACGCCATCGGCATCTCGCCCAAGATCG 400 SerLeuArgLeuThrPheHisAspAlaIleGlyIleSerProLysIleAla 45 50 55 CCTCCACTGGCACGTTCGGTGGTGGCGGCGCGGACGGCTCTATCGCCATC 450 SerThrGlyThrPheGlyGlyGlyGlyAlaAspGlySerIleAlaIle 60 65 70 TTCGCGGACATCGAGACCAACTTCCACGCGAACAACGGTGTCGACGAGAT 500 PheAlaAspIleGluThrAsnPheHisAlaAsnAsnGlyValAspGluIle 75 80 85 90 CATCGGCGAGCAGGCTCCCTTCATCGCGCGCCACAACCTCACCACTGCGG 550 IleGlyGluGlnAlaProPheIleAlaArgHisAsnLeuThrThrAlaAsp 95 100 105 ACTTGTACGTCGATCTGCGCCCGTAAACCCCGCCTGTCACAACGCTGATC 600 Phe ATCCGCGAGCGCGAGCATCCAGTTCGCCGGGGCGATCGGCGTGAGCAACT 650 IleGlnPheAlaGlyAlaIleGlyValSerAsnCys 110 115 120 GCCCTGGGGCGCCGCGCCTCGACGTCTTCGTCGGCCGCAAGGACGCGACG 700 ProGlyAlaProArg LeuAspValPheValGlyArgLysAspAlaThr 125 130 135 CAGCCCGCGCCCGACTTGACCGTGCCCGAGCCGTTCGACACCGTCGACAG 750 GlnProAlaProAspLeuThrValProGluProPheAspThrValAspSer 140 145 150 CATCCTCGCCCGCTTCGCCGACGCTGGTGGCTTCAGCTCTGACGAGGTCG 800 IleLeuAlaArgPheAlaAspAlaGlyGlyPheSerSerAspGluValVal 155 160 165 170 TCGCCCTCCTGGCCTCGTACGTTATCTCCAGCACCTCTACTTTCTACTGT 850 AlaLeuLeuAlaSer 175 GCGGCGACTAACGTGTTTGTTTGTGCTCAGGCACACCATCGCTGCGGCTG 900 HisThrIleAlaAlaAlaAsp 180 ACCACGTCGACCCGAGCATCCCCGGCACGCCGTTCGACTCCACCCGGAGC 950 HisValAspProSerIleProGlyThrProPheAspSerThrArgSer 185 190 195 CTGTTCGACACGCAGTTCTTCATCGAGACCCAGCTCCGCGGCACGCTCTT 1000 LeuPheAspThrGlnPhePheIleGluThrGlnLeuArgGlyThrLeuPhe 200 205 210 215 CCCGGGCACTGGGGGAAACCAGGGCGAGGTCGAGTCGCCCCTCCACGGCG 1050 ProGlyThrGlyGlyAsnGlnGlyGluValGluSerProLeuHisGlyGlu 220 225 230 AGCTCCGCCTCCAGTCCGACTCTGAGCTCGCGCGCGACTCGCGCACCGCC 1100 LeuArgLeuGlnSerAspSerGluLeuAlaArgAspSerArgThrAla 235 240 245 TGCGAGTGGCAGTCCTTCGTCAGTGCGTACCCCTCTCTCCTCCTGCCCAC 1150 CysGluTrpGlnSerPheVal 2 50 255 CTCTTACTCCCCTGGCTGACGCGCCGTCCGCGCAACCCTACAGACAACCA 1200 AsnAsnGln GGCGAAGCTCCAGTCCGCGTTCAAGGCGCGGTTCCGCAAGATGACCGTGC 1250 AlaLysLeuGlnSerAlaPheLysAlaArgPheArgLysMetThrValLeu 260 265 270 275 TCGGGCACAGCGTGAGCGACCTCGTCGACTGCTCCGAGGTCGTGCCCACG 1300 GlyHisSerValSerAspLeuValAspCysSerGluValValProThr 280 285 290 CCGCCCGCGCCCGCGAGCAACGCGCACTTCCCCGCCGGCCTCGGCCAGGC 1350 ProProAlaProAlaSerAsnAlaHisPheProAlaGlyLeuGlyGlnAla 295 300 305 CGACGTCGAGCAGGCCTGCGCCAGCACGCCCTTCCCGACCCTTCCCACCG 1400 AspValGluGlnAlaCysAlaSerThrProPheProThrLeuProThrAsp 310 315 320 325 ACCCGGGCCCGGTCACCTCCGTCGCTCCTGTGTAAGTCCCGTTCCAGTGA 1450 ProGlyProValThrSerValAlaProVal 330 335 GGTCCTTTTGAGGAAACATCGATGCTGATTTGGCGCTCTTGCAGCCCCCC 1500 ProPro TTCGTAAACGTACACAGGGAAGATGATGGTCTTGGTTGGCACGGCCGAGG 1550 Ser *** GCTATTAGAACATCTGGTCTTGGCTTCCACACACATTCTTGAGGGTTCGC 1600 ATGATTTTTGGATAGAGCAATTCTGCACATGTTTTCGGCTTAATATATTG 1650 CTTGCTGCTGTTATGTTCAGCTTTACCTTTTGAACTCGAATGAACTGCAG 1700 GTGAAGTGCGGATTGTAGACGCATTCCGATGTCCAAGCGAAGATCTACGG 17 50 ATACAAGCCAATCCCGTTCCCTTGATCTACAAGTCCGTACAGTCCTTCCG 1800 AGACGTCTAGCGTTGTCGCAAGGGCCCAATGGCCATATTTTCTATGAGGA 1850 TTTGAGGAGAGTGCGAATGTCGTGGGCCATCTGTGTAGCTCTGCCTCATC 1900 GTCGAAGTCGTATGTGTATTGTTTCTCGGCCTGCAATTGGAGTGAGCGAA 1950 ACTTCCCGAGAGCACAGAATGGATGCTGTATGGCTCACCGTTCGCAACAC 2000 GAGTTGGTGAGTCTCTTCAATCGTCCGACACTCTATTATAACGTCCTTAT 2050 TAGTTGTTCCGTCGGTCCTAACGTCCAATTTCTCTCCAGCACGAGGGAAC 2100 AGGAGTGTATGCTTGACATCCGACGCCCTCGGCCGCCCCTCCCTCTCCTC 2150 CACAAGCACGCACAGTACCCGCGGCGCAGACGTCAAGAGGAGCACGCACG 2200 GCCGGTTCTTCTTCAAACACCGCTTGCGCACGTGCGACCTCACTGCCGCG 2250 ACGCGGGACGGTCTCGCAAGCGCACAGACCGCGGACCTACATGC 2294

[Brief description of drawings]

FIG. 1 is a diagram showing a partial amino acid sequence of manganese peroxidase purified from Pleurotus cornucopiae.

FIG. 2 is a diagram comparing a partial amino acid sequence of manganese peroxidase of Pleurotus cornucopiae and a lignin peroxidase of Pleurotus cornucopiae. Sequence portions suitable for making synthetic DNA probes are underlined.

FIG. 3 is a view showing the sequence of the synthetic DNA probe prepared.

FIG. 4 shows the manganese peroxidase cDNA gene (MPOC1) of Pleurotus cornucopiae as the Ec of pBluescriptIISK +.
The figure which shows the recombinant plasmid subcloned in oRI site.

FIG. 5 shows the EcoR of pBluescriptIISK + as the manganese peroxidase chromosomal gene (MPOG1) of Pleurotus cornucopiae.
The figure which shows the recombinant plasmid subcloned in I site.

Continuation of the front page (51) Int.Cl. ⁶ Identification code Office reference number FI technical display location (C12N 1/21 C12R 1:19) (C12N 9/08 C12R 1:19)

Claims (6)

[Claims] 1. An amino acid sequence of SEQ ID NO: 1 which produces manganese peroxidase, or one or more amino acids are added, deleted or substituted in the amino acid sequence of SEQ ID NO: 1 (provided Asn-103, Arg-44). , His
A manganese peroxidase gene encoding an amino acid sequence having -48 and His-177 as amino acids to be added, deleted or substituted) and having manganese peroxidase enzymatic activity. 2. A manganese peroxidase gene represented by the base sequence of SEQ ID NO: 2. 3. A manganese peroxidase gene containing a promoter region represented by the nucleotide sequence of SEQ ID NO: 3. 4. One or more amino acids are added, deleted or substituted in the DNA encoding the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 1 (provided Asn-103, Arg-44, His -48 and His-177 are excluded except when they are amino acids to be added, deleted or substituted) and a recombinant plasmid containing a DNA encoding an amino acid sequence having manganese peroxidase enzymatic activity. 5. A recombinant plasmid containing the manganese peroxidase gene represented by the nucleotide sequence of SEQ ID NO: 2 or 3. 6. A transformant containing the recombinant plasmid according to claim 4 or 5.