JPH0227986A - Phenol oxidase gene (ii) - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a phenol oxidase gene (II), and more specifically to a white rot fungus having the ability to produce and secrete phenol oxidase [particularly Coriolus hirsuLus]. IFO4
917) ] metsenji 1--RNA below, mR
The phenol oxidase gene (abbreviated as NA) derived from
) regarding.

This phenol oxidase gene (II) does not contain introns and can be recombined into various organisms to be used in biological pulping, biobleaching, decolorization of industrial wastewater, pretreatment for wood saccharification, and as a reagent for clinical trials. can produce phenol oxidase.

[Prior art]

Phenol oxidase is an enzyme that oxidizes phenols in the presence of molecular oxygen to produce 0-quinone or p-quinone, and is known to contain copper as a prosthetic group. Phenol oxidase is widely distributed in the animal and plant kingdom, but phenol oxidase produced by some fungi called white rot fungi is considered to be particularly useful industrially.

White rot fungi are known to have a high ability to decompose lignin in lignocellulosic materials such as wood, and these white rot fungi are inoculated into lignocellulosic materials, cultured, and a portion of the lignin is decomposed to manufacture valves. Trials of biological pulping have been carried out (Japanese Patent Laid-Open No. 50-4690)
No. 3). However, white rot fungi have the ability to not only decompose lignin but also cellulose and hemicellulose, which are the raw materials for pulp, and suffer from the problem of reduced bulb yield. Another problem was that the decomposition of lignin by white-rot fungi occurs late in growth due to secondary metabolism, so it takes time.

The lignin decomposition ability of white-rot fungi is thought to be largely due to the phenol oxidase produced and secreted by white-rot fungi, and attempts have been made to clone the gene, but no success has been achieved so far. In addition, regarding roughcase, which has an activity similar to the phenol oxidase of white-rot fungi, Neurospora °crassa (Neurospora crassa)
Cloning of the laccase gene of S. urosporacrassa (U, S.

Germann, Lerch; (1988) J.
Biol, Chem. Not reported.

[Problem to be solved by the invention]

The present invention aims to solve the above-mentioned conventional problems and to make it possible to create various new organisms that only produce phenol oxidase.

It is believed that several types of enzymes are involved in the biodegradation of lignin in nature, and phenol oxidase produced by white rot fungi plays a central role, and is essential for research on lignin decomposition. It is an enzyme.

Therefore, an organism endowed with the ability to produce phenol oxidase can be considered as a new organism that efficiently expresses only the ability to decompose lignin, and the present invention aims to develop the mRNA of white rot fungi necessary for imparting the ability to produce phenol oxidase.
The present invention provides the phenol oxidase gene (If) derived from the phenol oxidase gene (If).

[Means to solve the problem]

The present invention has the following formula: ValAsnAspGln PheSer II eAspG I yH1sAsp
LeuThr I lel l aG 1uVa l
The present invention relates to a phenol oxidase gene (n) consisting of a DNA encoding the amino acid sequence of Asp:ier (wherein X is Ala or Pro).

In addition, the one where X in the above formula is Ala is defined as phenol oxidase gene (n) (OJ-POM 5), and
is Pro's phenol oxidase gene (n)
(Set as OJ-POO20.

Furthermore, the present invention provides a DNA that hybridizes to the above DNA.
A sequence which is obtained naturally, synthetically or semi-synthetically and is related to the DNA sequence of claim 1 by nucleotide substitutions, nucleotide insertions, nucleotide sequence inversions and other mutations. The present invention relates to a phenol oxidase gene (n) consisting of a DNA encoding a polypeptide having phenol oxidase activity.

Furthermore, the present invention provides the above-mentioned phenol oxidase gene (
n) is linked to vector DNA.

Furthermore, the present invention provides that the phenol oxidase gene (n) is expressed by the following formula % TCGGTAACTACTGGATTCGCGCCAA
CCCCAAACTTCGGCAA or the following formula (OJ-POM TTTGTGAACCAGTGCCCTATTTCCT
CTGGGCACTCGTTCCTCGGTAACTA
CTGGATTCGCGCCAACCCCAACTTC
The present invention relates to a phenol oxidase gene (II) having the DNA sequence GGCA^TCGATGTCAAACGACCAG.

Next, the present invention will be explained in detail.

<Synthesis of DNA probe) The DNA probe required to confirm that mRNA derived from the phenol oxidase gene (II) is included in the mRNA 0 of white rot fungi and to clone the phenol oxidase gene from the cDNA library is as follows. Synthesized based on the partial amino acid sequence of phenol oxidase.

The partial amino acid sequence of phenol oxidase is the amino acid sequence from the N-terminus of phenol oxidase produced and purified by the method of JP-A-61-285989, JP-A-62-220189, and JP-A-62-220190. BrCN decomposition (Co
le, R, D,: Methods Enzywol,
11, 315-317 (1967)) or tryptic digestion (Lin, L, -N, & Brandts.

J, F.: Bio-chemistry 22.553
(1983)) L, the amino acid sequence from the N-terminus of the isolated polyseptide was analyzed using the Edman degradation method (Edn+an, P
, & Hen5chen, A. Protein-5eq
uence deter+++1nation,
2'nd de,, Springer-Verla
G, Berlin, pp 232-279 (1975
).

The DNA probe can be synthesized by any one of the phosphothiester method, phosphotriester method, phosphite method, and the modified amidite method.

(Preparation of mRNA) Any organism that can be used in the present invention can be any organism that has phenol oxidase, but especially white rot fungi that produce and secrete phenol oxidase with high enzymatic activity [e.g. (IFO49
17), C. versicolor (IFO30340), and S. scale (IPO8714)) are good.

The composition of the medium for growing and propagating the white rot fungi may be any medium as long as it is a medium in which the white rot fungi produce phenol oxidase.

Glucose is used as the main carbon source, but other carbon sources that can be assimilated by white rot fungi may also be used. Yeast extract and polypeptone are used as the main nitrogen sources, but they can be assimilated by white rot fungi. Inorganic nitrogen compounds such as ammonium salts and nitrates, and organic nitrogen-containing substances such as urea and casein can also be used. In addition, inorganic salts such as calcium salts, magnesium salts, potassium salts, phosphates, manganese salts, zinc salts, iron salts, cornstarch liquor, vitamins,
It is also possible to add nutritional substances and growth-promoting substances such as amino acids and nucleic acids.

Inoculate white rot fungi into the medium from the first stage and culture.

When the phenol oxidase activity in the culture reaches its maximum, collect the bacteria and freeze in liquid nitrogen.

Total mRNA corresponding to proteins such as phenol oxidase can be extracted from white rot fungi by a conventional method. For example, white rot fungi are mixed with 2 to 5 volumes of NP-40,
A surfactant such as SO5 or Triton

After solubilization and centrifugation, add cold ethanol to the supernatant and R
There is a method of precipitating NA. In addition, the tissue was disrupted in a guanidine thiocyanate solution, and after ethanol precipitation,
There is also the GTC method, in which total mRNA is extracted by repeatedly dissolving the precipitate with guanidine hydrochloride. In addition, the method of Broda et al. (J, Microbiol, Methods, 4
．． (1985) 155-1623 and commercially available RNA extraction kit [RPN, 1264 manufactured by Amajam Japan ■.
) can also be used to extract total mRNA.

Alternatively, if necessary, a method can be carried out in which polysomes in the process of synthesizing phenol oxidase are precipitated using an antibody corresponding to phenol oxidase, and mRNA is extracted from this using a surfactant or the like.

The poly(A)m RNA of the present invention can be purified by a purification method using an adsorption column such as oligo(dT) cellulose or poly(U) cellulose, fractionation using a sig sugar density gradient centrifugation method, etc. .

In order to confirm the presence of mRNA corresponding to the target phenol oxidase in the total mRNA obtained as described above, the mRNA is translated into a protein, and the protein is identified using an antibody or the like. You can do the following method. For example, Re ticu 1, a system commonly used to translate mRNA into protein.
It is possible to confirm that the mRNA corresponding to phenol oxidase has activity by translating it into a protein using a cell-free system such as oxyte-lysate (reticulocyte lysate) or heat germ (wheat germ).

In addition, dot hybridization using a phenol oxidase DNA probe or Northern
Confirmation can also be performed by plot hybridization.

The mRNA obtained as described above was in vitro
.

Synthesize the cDNA and insert it into an appropriate vector etc.
It can be used to construct a cDNA library for cloning the phenol oxidase gene (n).

(Synthesis of cDNA) As a cDNA synthesis method, Gubler-Hoffm
Examples include the an method, the Rand method, the Okayama-Berg method, and variations thereof. For example, it can be carried out in a test tube by the following method. Using the above mRNA as a template and oligo (dT) as a primer, dNTP (=dAT
mRNA was extracted using reverse transcriptase (261OA manufactured by Zenshuzo Co., Ltd.) in the presence of P, dGTP, dCTP, dTTP).
Synthesize single-stranded cDNA complementary to RNAs.
Cut the mRNA with eH (2150A manufactured by Zenshuzo Co., Ltd.), use the mRNA as a primer, and add DNA polymerase I (2140A manufactured by Zenshuzo Co., Ltd.) in the presence of dNTPs.
Synthesize double-stranded cDNA using This synthesis method is cD
As an NA synthesis kit, Amajam Japan ■ (RP
N, 1256Y), Boehringer Mannheim Yamanouchi (1013882), and can be used.

<Construction of cDNA library> The above double-stranded cDNA is either ligated with synthetic linkers at both ends or treated with terminal transferase [Zen Shuzo
A cDNA library can be constructed by adding an appropriate foot part (for example, polyC) using 2230A (manufactured by Co., Ltd.) and ligating it to a plasmid vector or λ phage vector.

For example, double-stranded cDNA is ligated with EcoRI Linker using DNA ligase derived from T4 phage, then cut with restriction enzyme EcoRI (1040S manufactured by Zenshuzo Co., Ltd.) to obtain double-stranded cDNA with EcoRI sticky ends, and then used as a phage vector. Incorporated into the BcoR1 site of λgtll and in vitro packaging [Amajam
A cDNA library is constructed by performing the following steps: N, 334Y (manufactured by Japan ■, P3151 (manufactured by Promega Biotic)).

In addition, commercially available cDNAs of λgtll and λgtlo
A Library Kit [RPN manufactured by Amajam Japan ■, 1280. RPN, 1257. Promega Biotic P3010) can also be used.

<Cloning of phenol oxidase gene (11)> Using a phenol oxidase DNA probe labeled with a radioactive isotope from a cDNA library, phenol oxidase mRNA was isolated by Brak hybridization cidin or colony I hybridization.
Clone origin c origin A.

<Subcloning> Subcloning of the obtained phenol oxidase gene (n) is performed as follows. DNA containing the phenol oxidase gene (■) and vector DNA are cut with restriction enzymes to prepare cDNA fragments and vector DNA fragments. Next, the mixture of both is treated with T4 DNA ligase (2011A manufactured by Zenshuzo Co., Ltd.). Vector DNAs used include pBR322, pucte, p
Uc19, pUcllB (3050.32 manufactured by Zenshuzo Company)
18.3219.3318), etc. In addition, restriction enzymes include 1IindI[[, EcoRI, Pst
I, BamHI etc. [1060S manufactured by Zenshuzo Company, 1
040S.

10735, 10105).

(Determination of base sequence of phenol oxidase gene (n)) The cloned cDNA was transferred to the plasmid vector pU
c11B or pUc119, or pUc18. p
Subcloning into Uc19 or M13 phage.

In principle, the subcloned DNA fragment is Hen1
koff's method and Yanisch-Perron's method (Henikoff, S. (1984) Gene,
28.351-359 Yantsch-Perron
,C., ,Vieira, J., and Messing+
J. (1985) Gene+33.103-119)
To create a deletion mutant, you can also use a commercially available Delision Kit (Zen Shuzo 6030).

Deletion mutants are produced using the dideoxy method (Sang
er, P. (1981) 5science, 2'14.
1205-1210) to determine the base sequence. Commercially available sea fencing kit (6010A manufactured by Zenshuzo Co., Ltd.)
, 6015A, 317-01 manufactured by Nirapon Gene■
121) can also be used.

The cDNA derived from the mRNA of C. versicolor containing the phenol oxidase gene (II) is the plasmid pUc.
19 or subcloned into the HcoRI site within the multiple cloning site of pUcllB and injected into E. coli JM109 or MVl184 using a conventional method [for example, Led
erberg, E. M., & Cohen, S. N., Jo.
urnal of Bacteri-ology, -■
1.1072-1074 (1974)) and deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology.

This transformed E. coli OJ-POM-5, OJ-POM-
2 have been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology, and their respective deposit numbers are FER
M P-10055) and Microtechnical Research Institute No. 10061 (
FERM P-10061).

(Recombinant DNA) The phenol oxidase gene (n) obtained in this way is used by incorporating it into the vector DNA of microorganisms (especially prokaryotes), plants, and animals, and introducing it into microorganisms, plants, and animals. The aim is to make it possible to create new organisms that produce oxidase or this improved protein in significant amounts. The method for integrating the phenol oxidase gene (n) into a vector is to cleave the vector with an appropriate restriction enzyme, and if necessary, polymerize an appropriate linker or a plurality of bases in an annealing possible combination. Double-stranded DNA and vector DNA processed in this way
Mix A and connect using ligase.

The obtained recombinant DNA is introduced into a vector host microorganism. Suitable host microorganisms include microorganisms of the genus Escherichia such as Escherichia coli, microorganisms of the genus Bacillus such as Bacillus subtilis, and microorganisms of the genus Satucharomyces such as Satucharomyces cerevisiae. Vectors used for these microorganisms are illustrated below. (See Protein Nucleic Acid Enzyme, Vol. 28, No. 4 (1981)).

EK-based plasmid vector (stringent type) p
scroll, pRK353. pRK646. pR
K248. pDF41, etc., EK-based plasmid vectors (relaxed type) Ca1E1゜pVH51, pAc
105. R5F2124. pcRl, pMB9.
BR313, pBR322, pBR324, pBR3
25, pBR327, pBR328, pKY2289゜pKY2700. pKN80. pKc? , pKB
15B, pMX2004. pAcYcl.

pAcYc184. λgt/λC, λgt/λB, λ-ES/λ of λgt-based phage vectors such as λdu1
B, λZJvir・λB, λ8 LO to λB, λWES
-Ta205. λDam, λgtl1, etc., Sharon vector Sharon 4A, Sharon 3A, Sharon 16
A, Sharon 13A, Sharon 148, Sharon 15A
, Sharon 8, Sharon 10. Sharon 17. Sharon 20 etc., Tiolais group vector L512. λZEQS.

λZYV5φ, JZUVφ2. , JZUVφ3
．． JYEQSφ1. JYEQSφ.

λYEQSφ31λ3all11λS51, etc., Bacillus subtilis plasmid vector pTA1015. pLs15.
pT^1020. pLS28. pLs13゜pTA
1050. pTA1060. pTA1030. p
Staphylococcus-derived plasmid vector pT127, such as TA1031. pc194, pc221.
pC223, pUB112. pUBllo, psA
O501, psA.

501, psA2100. pE194. ρTP4.
pips et al., yeast vector pJDB219. YEp1
3. YRp7. Ylpl, pYC, pTC2.

Microbial vectors, e.g. Pstl such as pBR322
Alternatively, insert promoter regions derived from E. coli, such as lac and tr, into EcoRI 5ite or other sites depending on the purpose.
It is possible to integrate the phenol oxidase by connecting it in-frame to p and tac, transform it into a suitable host, and express the phenol oxidase in the host.

In addition, in plants, T-DN derived from Ti-plasmid
The gene is introduced into plants using a similar method such as the pAL1050 vector containing the twrjil region of A.

When inserting recombinant DNA, it is connected in-frame to the leader sequence of tmr, and the stop codon of tmr is also used.

In the present invention, amino acids and polypeptides are rupAc
-tue shall be abbreviated according to the method adopted by the Committee on Biochemistry (CBN), for example, the following abbreviations are used.

^1a L-alanine Arg L-arginine^sn L-asparagine Asp L-aspartic acid Cys L-cystine Gln L-glutamine Glu L-glutamic acid cty Glycine 11is L-histidine 11e L-isoleucine Leu L-leucine Lys L-lysine Met L-methionine Phe L-phenylalanine Pro L-proline Ser L-serine Thr, L-threonine Trp L-tributophane Tyr L-tyrosine Val L-valine Also, the sequence of DNA is determined by the bases contained in each deoxyribonucleotide that makes up the DNA. shall be abbreviated by type,
The following abbreviations are used.

A Adenine (represents deoxyadenylic acid) C Cytosine (represents deoxycytidylic acid) G Guanine (represents deoxyguanylic acid) T Thymine (represents deoxythymidylic acid) [Example] The following examples are as follows: The cloning and nucleotide sequence determination of the phenol oxidase gene (II) derived from the mRNA of white rot fungi will be described in detail.

Example 1 <Synthesis of DNA probe> The DNA probe was synthesized using the amidite method.
This was carried out using a synthesizer (Nippon Zeon + GenetA-1 [[)].

Three types of white rot fungi
7).

Kawaratake (IFO30340), Kagaratake (IF
The results of analyzing the amino acid sequence from the N-terminus of phenol oxidase purified from O8714) ) up to the 25th stage using the Edman degradation method are shown below.

N-terminal 1st stage C. versicolor ^1a-8rg to Gln-^1a
-Vat scale mushroom ^Ia-Arg-Gln
-8Ia-Val-2 from the 17th stage Pro to the above
The following DNA probe was synthesized to correspond to Val in the 5th row.

However, ■ is deoxyinosine.

26mer-C (16*ix) 3'-CCI-GA
C-GGI-TTC-GCI-8G^-CA^-GCI
-GT-5゜T T GG In addition, the phenol oxidase of three types of white rot fungi was
Decomposition with rCN and reverse phase high performance liquid chromatography [
Elution conditions, column: Phenyl-5PW RP (manufactured by Toyo Soda), eluent 20% acetonitrile 10
．． 1% TFA to 75% Acetritrile 10.1% TF
The amino acid sequence of the polypeptide separated by concentration gradient elution to A (1 room temperature) was analyzed by Edman degradation, and the results are shown below.

Met-Ala-Phe-A
sn-Phe force 7 ratake Met-Ala
-Phe-Asn-Pheh Igaratake M
et-Ala-Phe-Asn-Phe The following DNA probe was synthesized to correspond to the above amino acid sequence.

From the above results, the homology of the amino acid sequences of phenol oxidase produced and secreted by white-rot fungi is extremely high, and by using the DNA probe used in the present invention, the phenol oxidase gene (n
) can also be cloned. Therefore, in the following example, a method for cloning the phenol oxidase gene (II) of C. versicolor will be described.

Example 2 <Preparation of mRNA> C. versicolor (IFO4917) was grown in 11 YPD medium (yeast extract 10 g/l, polypeptone 20 g/l).
I! , glucose 20g/β) was inoculated into a 51 volume Erlenmeyer flask, and cultured with shaking at 27°C for 6 days. After confirming that phenol oxidase was produced and secreted in the culture solution, the bacteria were collected and frozen in liquid nitrogen to obtain about 20 g of frozen bacterial cells.

11 m from 5 g of frozen bacterial cells by the method of Broda et al.
Total mRNA of g was extracted. Crush 5 g of frozen bacterial cells with a 100-liter Waring blender containing liquid nitrogen, and mix with 3 times the amount of TNS buffer [1xtri-iso-Propyl].
naphthalene 5ulphonic aci
d+ 200mM Tris-HC1+25mM EG
Dissolve in TA (pH 7,8), 250mM NaC+). Remove the pellet by centrifugation, add 0.5 g of phenol per 1 d of supernatant, and dissolve by keeping at 5-15°C. When all the phenol has dissolved, η amount of chloroform is added, and after centrifugation, the supernatant is collected, extracted twice with chloroform, and the total mRNA11 is extracted by ethanol precipitation.
mg was recovered.

In addition, when using a commercially available RNA extraction kit (manufactured by Amajam Japan), 6.7 μg was obtained from 3 g of frozen bacterial cells.
It was possible to extract the total mRNA of .

Total mRNA recovered by the above two methods was analyzed by Northern blot analysis using a phenoloxidase DNA probe.
Hybridization method (Tho-mas+P, S,
, Proc, Natl, Acad, Sci, USA 7
7, 5201 (1980)], it was confirmed that it contained mRNA derived from the phenol oxidase gene (II).

511 g of total mRNA was collected using the method of Maniatis et al. (Maniati et al.) using an oligo(dT) cellulose column.
s(thg, Mo1e-cular Cloning,
A Laboratory Manual+197~19
9.1982) to separate and purify poly(A) m RNA, and about 100 μg of poly(A) m
RNA was purified.

Example 3 (Synthesis of cDNA) Poly(A) mRNA derived from the white-rot fungus Aragekawarake
Synthesis of cDNA was performed by Gubler and Hoffman.
n method (U, Guvier & B, J, Hoffm
an; Gene, 25, 263-269 (1983
)) using a cDNA synthesis kit manufactured by Amajam Japan.

5 μg of 011go(dT) 12-18 (Pharmacia 27-7858-01) was added to 5 μg of poly(A) mRNA in the presence of 50 units of human fetal RNase inhibitor enzyme (HPRI) for 100 units of reversal. Approximately 30% of the enzyme was reacted at 42°C for 1.5 hours.
One 1icDNA was synthesized with a yield of . Add 4 to this reaction solution.
Add 1 unit of E. coli ribonuclease H and 115 units of E. coli DNA polymerase I and heat at 12°C for 1 hour.
After reacting at 22°C for 1 hour, the enzyme was inactivated by standing at 70°C for 10 minutes. After that, T4DN of IO unit
Add A polymerase and react at 37"C for 10 minutes.
Two-end strand cDNA was obtained with a yield of about 95%.

Example 4 (Construction of cDNA library) Commercially available λgtll cloning system [Amajam
A cDNA library was constructed using the following product (manufactured by Japan ■).

20 units of Eco
After reacting RI methylase at 37°C for 1 hour, Ec
oRI linker was attached to this, and 16 units of E
After adding coRI and reacting at 37°C for 2 hours, 5ep
It was purified by passing through a Harose CL4B column. λg
After ligation reaction with 1 μg of tll arm, in vitro
Packaging [^.

Becker & M, Gold; Proc, Natl.
, Acad, Sci, LIS^, 72.581 (19
75)] to obtain 10 recombinant λ phages, which were used as a cDNA library derived from the mRNA of Corsifolia versicolor.

Example 5 Cloning of phenol oxidase gene (II)> E. coli Y1090 strain was infected with the cDNA library derived from the mRNA of A. coli obtained in Example 4 to form plaques.

The clone containing the phenol oxidase gene (n) was labeled (
Richardson, C.C. (1965), P.
roc.

Natl, Acad, Sci, U, S, A,, 54.
The plaque hybridization method of Benton and Davis [-0D, Benton & R.
, W. Davis: 5science, 196 1
80 (1977)], two types of clones were selected.

7g i containing phenol oxidase gene (II)
Purification of DNA from phage was performed according to the method of Thomas and Davis (M, Thomas & R, W, D
avis; Journal of Molecula
r Biology, 91.315 (1974)).

Example 6 Determination of the base sequence of the phenol oxidase gene (II) Σ The phenol oxidase gene (I) obtained in Example 5
The 2 clone λgtll DNA containing I) was cut with the restriction enzyme EcoRI, the inserted phenol oxidase gene was excised, and the plasmid vector pUc
19 and into the EcoRr site of pUc118.

The clone subcloned into ρ[IC19 was OJ-
A clone subcloned into POM5 and puci 18 was designated as OJ-POM2.

A restriction enzyme cleavage map of the subcloned cDNA was prepared by a conventional method and is shown in FIG.

The restriction enzyme maps of OJ-POM5 and OJ-POM2 were the same.

Deletion mutants were created from the subcloned cDNA using a commercially available Precision Kit [manufactured by Takara Shuzo Co., Ltd.], and a commercially available M13 Sequencing Kit [manufactured by Takara Shuzo Co., Ltd.] was used to create deletion mutants.
The nucleotide sequence of the phenol oxidase gene (II) was determined by the dideoxy method using Takara Shuzo Co., Ltd.). At the same time, the entire amino acid sequence of phenol oxidase was determined. The base sequence and complete amino acid sequence of phenol oxidase are shown in Figure 2 (OJ-POM 5) and Figure 3 (OJ-P
Shown in OM 2).

OJ-POM5 and OJ-POM2 differed in 17 bases, but only differed in one amino acid.

〔Effect of the invention〕

The present invention has the following effects.

■ By providing the entire amino acid sequence of phenol oxidase, the role of phenol oxidase in lignin decomposition has been elucidated, which can be applied to biological pulping.

■ The DNA encoding phenol oxidase can be transferred to other organisms in various ways (for example, by ligating it to a vector such as a plasmid, cosmid, phage, or virus, and creating a recombinant by transformation or transduction; ,D
Recombination can be performed using a method in which a recombinant is created by directly introducing an NA fragment by electroporation, etc., and a new organism that produces a significant amount of phenol oxidase can be created.

■ Phenol oxidase produced in large quantities using a new organism that has recombined the DNA encoding phenol oxidase is free from contamination with cellulase and hemicellulase.
Can be used for biological pulping and does not reduce pulp yield.

■ Phenol oxidase, which is produced by a new organism in which the DNA encoding phenol oxidase has been modified, has high purity and is of excellent quality as a reagent for clinical trials, such as reagents for enzyme activity measurement, substrates, and reagents for quantifying bilirubin. Can be used as an enzyme.

[Brief explanation of the drawing]

FIG. 1 shows a restriction enzyme cleavage map of the phenol oxidase gene (II) derived from the mRNA of Corsifolia versicolor. Phenol oxidase is encoded in the shaded area. Figure 2 shows the phenol oxidase gene (II) (O
The nucleotide sequence and entire amino acid sequence of J-POM 5) are shown,
Figure 3 shows the phenol oxidase gene (II) (OJ
-The base sequence and entire amino acid sequence of POM 2) are shown. Figure 2 (Part 1) A1aI1eG1yProThrA1aAspLeuT
hrI1eSerAsn8IaG1uVaISerPr
oAspG1yPheA1aArgG1nA1aVaI
VaIVaIAsnAsriValThrProG1y
ProLeuVa18]aG1yAsnLysG1yA
spArgPheG1nLeuAsnVaII1eAs
pAsnLeuThrAsnHisThrMetLeu
LysSerThrSerI1eHisTrpHisG
1yPhePheG1nLysG1yThrAsnTr
p'A1aAspG1yProA1aPheVaIAs
nG1nCysProI 1eSerSerG1yHi
sSerPheLeuTyrAspPheG1nVaJ
．． ProAspG1nAlaG1yThrPheTrp
TyrHisSerHisLeuSerThrG1nT
yrCysAspG1yLeuArgG1yProPh
eVaIVaITyrAspProAsnAs,pPr
oHisAIaSerLeuTyrAspVaIAsp
AsnAspAspThrVaII1eAsnLeuA
1aAspTrpTyrHisThrA1aA1aLy
sLeuG1yPr081aPheProLeuG1y
A1aAspA1aThrThrA1aA1aAspL
euA1aVaI I 1eAsnVaIThrLys
G1yLysArgTyrArgP}ieArgLeu
Figure 2 (Part 2) Va I SerLeuSerCys As pPro
As nH i s Thr-PheSer I 1
eAs pG 1 yH i s As pLeuTh
rI 1 eI 1eG 1 uVa IAspSer
I 1eAsnSerG1 nProLeuVa I
Va I AspSerI 1eG 1 n I 1e
PheA laA 1 aG 1 nArgTyrse
rPheVa I LeuAsnA 1 aAs pG
1 nAspVa I G 1 yAsnTyrTr
p I 1 eA rgA 1 aAs nProAs
nPheG 1 yAsnVa I G 1 yPhe
A 1 aG 1 yG 1 y I 1 eAsnS
erA 1 a I 1 eLeuArgTyrAs
pG lyA 1 aAs pProVa I G
1 uProThrThrThrG 1 nThrTh
rProThrLys ProLeuAsnG1uVa
IAspLeuHisProLeuA1aThrMet
81aVaIProG1ySerProVaIA1aG
1yG1yValAspTh-rA1aI 1eAsn
MetAIG 1 yA 1 aSerPheVa I
ProProThrVaAsnAlaG1nAspL
euLeuProserGIG 1 u I 1 eS
erPheProA 1 aThrA 1 aA 1Figure 3 (Part 3)

Claims (5)

[Claims] (1) Phenoloxidase gene (II) consisting of DNA encoding the following amino acid sequence. [There is a gene sequence] [X in the formula is Ala or Pro] (2) DNA that hybridizes to the DNA according to claim 1
A sequence which is obtained naturally, synthetically or semi-synthetically and is related to the DNA sequence of claim 1 by nucleotide substitutions, nucleotide insertions, nucleotide sequence inversions and other mutations. A phenol oxidase gene (II) consisting of DNA encoding a polypeptide having phenol oxidase activity. (3) A recombinant DNA in which the phenol oxidase gene (II) according to any one of claims 1 to 2 is linked to vector DNA. (4) The phenol oxidase gene (II) is expressed by the following formula (O
The phenol oxidase gene (II) according to claim 1, which has the DNA of J-POM5). [There is a gene sequence] (5) The phenol oxidase gene (II) is expressed by the following formula (O
The phenol oxidase gene (II) according to claim 1, which has the DNA of J-POM2). [There is a gene sequence]