JPH0227985A - Phenol oxidase gene (1) - 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 (1), and more specifically to a white rot fungus that has the ability to produce and secrete phenol oxidase [particularly Coriolus hirsutus]. IFO49
17) ) derived phenol oxidase gene (I)
Regarding.

Phenol oxidase gene (1) can be introduced into various organisms to produce phenol oxidase that can be used for biological pulping, biobleaching, decolorization of industrial wastewater, pretreatment for wood saccharification, and as a reagent for clinical trials. can do.

[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 produce pulp. Attempts have been made to develop biological pulping to
No. 03), Shikashi, and white rot fungi have the ability to not only decompose lignin but also cellulose and hemicellulose, which are the raw materials for pulp, and have the problem of reducing pulp yield. There is. 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 ability of white-rot fungi to decompose lignin 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. Laccase, which has similar activity to phenol oxidase of white-rot fungi, is known from Neurospora crassa (Neurospora crassa).
Cloning of the laccase gene of A. urosporacrassa (U, A.

Garmann, K., Lerch; (1988) J
, Biol, Chem, 263.885-896), but its amino acid sequence is different from that of the phenol oxidase of the present invention,
Furthermore, lignin degradation by Neurospora crassa laccase has not yet been 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, as a new organism that efficiently expresses only the ability to decompose lignin, an organism that is endowed with the ability to produce phenol oxidase of a white rot fungus can be considered. This provides an oxidase gene (1).

(Means for Solving the Problems) The present invention provides the following formula: ^1a I 1eGlyProThrAlaAspLe
This invention relates to a phenoloxidase gene (I) in which a DNA encoding the amino acid sequence of uThrI 1eSerAsnAlaIJluVa1 is separated by an intron.

Furthermore, the present invention provides the above-mentioned phenol oxidase gene (
I) DNA that hybridizes to natural, synthetic,
or is obtained by semi-synthesis, and is related to the DNA encoding the amino acid sequence by nucleotide substitution, nucleotide insertion, nucleotide sequence inversion, or other mutation, and
This invention relates to a phenol oxidase gene (1), which is a DNA encoding a polypeptide having phenol oxidase activity, or the gene is separated by an intron.

Furthermore, the present invention provides phenol oxidase gene (1)
It relates to recombinant DNA ligated to vector DNA.

Furthermore, the present invention provides phenol oxidase gene (1)
relates to a phenol oxidase gene (1) having the DNA sequence shown below.

GTATCGTGAGTCTACATTCGGTCTG
ATATGATCAATTACTTACGACAACC
CCATCTTCCG to GA 6'l'l;[iTl;
ALiL, ALL+ljL, L, ACGCCTGCGG
CCGGTGACAACGTCACCATCCGCTT
CCGCAC A detailed description of the present invention follows.

<Synthesis of DNA probe> Phenoloxidase gene (I) was extracted from the cosmid library using colony hybridization method.
The DNA probe required for cloning is synthesized based on the partial amino acid sequence of phenol oxidase. The partial amino acid sequence of phenol oxidase is disclosed in JP-A-61285989 and JP-A-62-2201.
No. 89 and the amino acid sequence from the N-terminus of phenol oxidase produced and purified by the method of JP-A-62-220190 and purified phenol oxidase with BrC.
N-decomposition (Cole, R.

D: Methods Enzymol, Dan, 315
-317 (1967)) or tryptic digestion (Li
n, L, -N, & Brandts, J. F.: Bi
.

chemistry 22.553 (1983))
L, Amino acid sequence from the N-terminus of the isolated polypeptide was analyzed using the Edman decomposition method (Edman, P, & Hen5
chen, eight, protein sequence
termination, 2'nd de, +Spri
nger-Verlag, Berlin+pp232~
279 (1975)].

The DNA probe can be synthesized by any of the phosphothiester method, the phosphotriester method, the phosphite method, and the amidite method, which is an improved method thereof.

<Preparation of chromosomal DNA> Any organism can be used in the present invention as long as it has phenol oxidase, but white rot fungi that produce and secrete phenol oxidase with particularly high enzymatic activity [e.g. Kawaratake (IFO49)
17), Kawaratake (IFO30340), and Scale (IFO5714)) are good.

The composition of the medium for growing and propagating white-rot fungi uses glucose as the main carbon source, but other carbon sources that can be assimilated by white-rot fungi may be used, and yeast extract and polypeptone are used as the main nitrogen sources. Inorganic nitrogen compounds such as ammonium salts and nitrates that are used but can be assimilated by white rot fungi, urea,
Organic nitrogen-containing substances such as casein can also be used. In addition, inorganic salts such as calcium salts, magnesium salts, potassium salts, phosphates, manganese salts, zinc salts, and iron salts, nutritional substances such as corn staple liquor, vitamins, amino acids, and nucleic acids, and growth-promoting substances. It is also possible to add.

White rot fungi are inoculated into the above medium and cultured.

After culturing, the bacteria are collected, frozen in liquid nitrogen, crushed in a mortar, and then chromosomal DNA is extracted and purified by the phenol extraction method to obtain chromosomal DNA used for cosmid library construction.

If chromosomal DNA is treated with proteinase before phenol extraction, chromosomal DNA can be extracted with high efficiency.

<Construction of cosmid library of chromosomal DNA> Cosmid vector pHc79 (Hohn, B, and C
o11ins.

J. (1980) Gene 11, 291) to construct a cosmid library of chromosomal DNA. Cosmid vector pHC79 is a commercially available product [e.g., Bethesda Research Laboratory
5earch Lab.

5358SA (manufactured by Ratories) and 567795 (manufactured by Boehringer Mannheim Yamanouchi) can be used.

The above chromosomal DNA was digested with restriction enzyme 5au3Ar (Takara Shuzo ■
Partially disassembled with 32-46Kb (1082A)
A chromosomal DNA fragment with a size of 1 kilobase pair is obtained. On the other hand, cosmid vector pHC79 was injected with restriction enzyme BamHI (
1010S (manufactured by Takara Shuzo), dephosphorylated, added the above partially degraded chromosomal DNA fragment, and
A DNA strand binding reaction is performed using 4DNA ligase [Takara Shuzo Co., Ltd. 20118].

The resulting binding reaction product was packaged using a commercially available in vitro packaging kit [for example, AmaJam Japan ■ N,
334Y, Promega Biotic (Pr.

P3151 (manufactured by mega Biotec) and inserted into mature phage particles, and E. coli DH1 (ATCC) was inserted into mature phage particles.
33849), and per 1 μg of chromosomal DNA,
Approximately 50,000 Ap' (ampicillin resistant) strains are obtained and used as a cosmid library of chromosomal DNA.

<Cloning of phenol oxidase gene (1)> Approximately 10,000 recombinant E. coli strains from the cosmid library were cultured in LB medium containing ampicillin (Batato tryptone L).
og/e, Bite Ease] Extract 5 g/l
, Sodium chloride 10g/1, Agar 15g/I!
,) to form colonies on top.

The colonies were filtered using a commercially available nitrocellulose filter or nylon filter [for example, RPN, 82C manufactured by Amajam Japan, AO45BO82C manufactured by Toyo Roshi ■.
) and the conventional method (Grunstein + M, &
D, S, Hogness: Proc, N
att, Acad, Sci, USA 72.39
61 (1975)) to immobilize the DNA on the filter.

Label (Ri
chardson, C.C. (1965) Proc.
Natl Acad, Sci.

Mouth, S, A, 54.158-161. E. coli containing the phenol oxidase gene (I) is selected by hybridizing with the synthetic NA probe prepared in [Reference], and the cosmid is extracted and purified by a conventional method.

In order to limit the part containing the phenol oxidase gene (1) in the chromosomal DNA fragment integrated into the cosmid and perform subcloning, we used antienzyme old nam (1060S manufactured by Zenshuzo Co., Ltd.) or EcoRl (Zen Shuzo Co., Ltd.). 10405) or SmaI (1085A, manufactured by Zenshuzo Co., Ltd.), separated into molecular weights by agarose gel electrophoresis, and immobilized on a filter.
The A fragment is hybridized with a synthetic NA probe labeled with 2P. The DNA fragment that hybridizes with the synthetic NA probe was transferred to the plasmid vector pUc19 (Y
anisch-Perron+C, Vieira, J.
and Messing, J. (1985) Gene
, 33.103+Messing, J. (1983).
Method in Enzymology, 101
．． 20-78° Zenshuzo 3219) and create a restriction enzyme map.

D containing the obtained phenol oxidase gene (1)
Integration of the NA fragment into vector DNA is carried out as follows. A vector DNA fragment is prepared by cutting the vector DNA with an appropriate restriction enzyme. Next, a DNA fragment containing the phenol oxidase gene (I) and vector DNA
Treat the mixture of fragments with T4 DNA ligase. Vector DNAs used include pBR322 and pUc1.
8, pUc19, [Manufactured by Zenshuzo ■ 3050, 3218
, 3219:1 etc. In addition, restriction enzymes include old ndnl, EcoRI, PstI [Zen Shuzo ■
1073S), Bawl I, etc.

In this way, recombinant DNA in which the phenol oxidase gene (1) is linked to vector DNA can be obtained.

Determination of the nucleotide sequence of the phenol oxidase gene (1)> The DNA fragment subcloned into the plasmid vector pUc19 was, in principle, prepared using the Hen1koff method and the Yanisch-Perron method (Henikoff's method).
f, S, (1984) Gene+ 28+ 351~
359 Yanisch-Perron, C., Vie.
ira.

J. and Messing, J. (1985) Ge
ne, 33, 103-119), but a commercially available Delicidine Kit (6030, manufactured by Zenshuzo Co., Ltd.) can also be used.

Deletion mutants are produced using the dideoxy method (Sang
er, F. (1981) 5science, Yu 14.1
205-1210) to determine the base sequence. Commercially available sea fencing kit [6010A manufactured by Zenshuzo Co., Ltd.
6015A, manufactured by Nirapon Gene ■ 317-0112
11 can also be used.

The EcoRI fragment of the chromosomal DNA of C. versicolor containing the phenol oxidase gene (1) is plasmid p.
EcoR1 within the multiple cloning site of tl(:19
E. coli JM109 in the form subcloned to
(ATCC53323) using conventional methods [for example, Lederb
erg, E.

M, & Cohen+S, N, Journal of
Bacteriology+ 119+1072~10
74 (1974)). This transformed E. coli 0J-POG-E 1 has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology, and its deposit number is FERM P-10048.

(Recombinant DNA) The phenol oxidase gene (1) thus obtained is used by incorporating it into the vector DNA of microorganisms, plants, and animals, and introducing it into microorganisms, plants, and animals to produce phenol oxidase or this improved protein. The goal is to make it possible to create new organisms that produce large quantities of.

Integration of the phenol oxidase gene (I) into a vector can usually be carried out in vitro as follows.

Both ends of the DNA of the phenol oxidase gene (1) are treated with exonuclease if necessary, and NAs are connected to each end as necessary, or a plurality of bases in combinations that can be annealed are polymerized.

This is then incorporated into the desired vector. The method of integration involves cleaving the vector with an appropriate restriction enzyme and, if necessary, polymerizing an appropriate linker or a plurality of bases in an annealing-enabled combination. Double-stranded DNA processed in this way
and vector DNA and ligate using T4 ligase.

The obtained recombinant DNA is introduced into vector hosts such as microorganisms, plant cells, and animal cells.

Examples of hosts that can be used here include yeasts of the genus Satucharomyces such as Satucharomyces cerevisiae, cells of plants of the Solanaceae family such as tobacco and petunia, and Ba.
Animal cultured cells such as 1bIC3T3 are preferred. Vectors used for these hosts are illustrated below. pJDB219. as a yeast vector. YEρ13゜YRp7.
Ylpl, pYC, pTC2, Ti as a plant vector
Various vectors derived from plasmids, various vectors derived from cauliflower mosaic virus (including binary vectors), and nopSV derived from SV40 as animal vectors.
K, p+ r-nβ-, psVlf, + to 11+
, pβ2X, pSXβF, etc. However, Ti
In the case of plasmid-derived plants and lids, the obtained recombinant DNA is once introduced into Agrobacterium tumefaciens 737, etc., and the wood recombinant microorganism is infected by GO-culture into plant cells. This allows the recombinant DNA to be introduced into the host plant.

In addition, in the present invention, amino acids and polypeptides are I
It shall be abbreviated according to the method adopted by the UPAC-IUB Committee on Biochemistry (CBN), for example, the following abbreviations are used.

la ＾rg sn 8sp ys in lu ly lis 1e ea ys et he Pr.

er hr rp yr al L-Alanine-Arginite-Asparagine L-Aspartic acid L-Cysteine-Glutamine-Glutamic acid-Glycine-Histidine-Isoleucine L-Leucine L-Lysine-Methionine-Phenylalanine-Proline -Serine -Threonine L-1-Liptophan -Tyrosine L-Valine Furthermore, the DNA sequence is abbreviated by the type of base contained in each deoxyribonucleotide constituting it, and 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 (1) derived from the chromosome 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 In).

Three types of white rot fungi [Arage kawaratake (IFO4917)
).

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

C. versicolor Califolia Ala-Arg-Gln-Ala-ValAla-Ar
g-Gln-Ala-Val 17th row Pr of the above arrangement
The following DNA corresponds to the 25th Val from o.
The probe was synthesized. However, ■ is deoxyinosine.

In addition, B
Decomposition with rCN and reverse phase high performance liquid chromatography [
(Elution conditions, column: Phenyl-5PW RP (
(manufactured by Toyo Soda Co., Ltd.), eluent 20% acetonitrile 10
．． 1% TFA to 75% Acetritrile 10.1% TF
The results of analysis of the amino acid sequence of the polypeptide separated using the Edman degradation method (concentration gradient elution into L, room temperature) are shown below.

7 lakeka 7 latake Met-Ala-Phe-^
5n-Phe Kawaratake Met-Ala
-Phe-^5n-Phe scale M
et-Ala-Phe-5n-Phe The following DNA probe was synthesized to correspond to the above amino acid sequence.

The above results show that the amino acid sequences of phenol oxidase produced and secreted by white rot fungi have very high homology, and by using the DNA probe used in the present invention, it is possible to clone the phenol oxidase of any white rot fungus. can. Therefore, in the following examples, the phenol oxidase gene (
1) The cloning method will be explained.

Example 2 <Preparation of chromosomal DNA> A. nigra (IFO4917) was grown in 11 YPD medium (yeast extract 10 g/A, polypeptone 20 g/
f, glucose 20 g/l) was inoculated into a 52-volume Erlenmeyer flask and cultured with shaking at 27°C for 7 days. After culturing, the cells were harvested and frozen in liquid nitrogen, resulting in approximately 20 g of frozen bacterial cells.

10 g of frozen bacterial cells were crushed using a mortar under liquid nitrogen for about 15 minutes. Buffered and (pre-warmed at 42°C)
0,1M NaC1,0,111Tris-ICI,
Proteinase K (final concentration 100 μg/mf Heringer Mannheim Yamanouchi 161519) was added to 10 mZ (0.1 M EDTA, pH 8), and 5 g of crushed bacterial cells were placed therein and reacted for 2 hours with gentle stirring. Equal amount of TE (10mM Tris-HCI
, 1mM EDTA, pH 8) After extracting chromosomal DNA with saturated phenol, perform ethanol precipitation, dissolve again in 5d TH, and incubate with RNaseA at 37°C for 30 minutes.
(Final concentration: 10 Qug/ml, Takara Shuzo 11) to remove RNA. Equilibrium density gradient centrifugation using CsCl (Beckman, νTi800-tar, 15°C
, 50 krpm, 16 hours) to obtain 1.5 tags of purified chromosomal DNA.

Example 3 <Construction of cosmid library of chromosomal DNA> Restriction enzyme 5au3 was added to the purified chromosomal DNA 2501Ig as described above.
Add AI, partially decompose at 37°C, and remove 5% from the partially decomposed product.
~25% C:F density gradient centrifugation (Beckman 5W
40 Ti rotor, 15°C, 22,5krpm,
About 4 μg of chromosomal DNA fragments of 32 to 46 Kb were obtained.

After adding the restriction enzyme BamHI and reacting at 37°C for 12 hours to completely decompose, dephosphorylating with alkaline phosphatase (Takara Shuzo 2250A) at 37°C for 30 minutes, and phenol-extracting 10 μg of cosmid vector and 32 Mix 1 μg of ~46 Kb chromosomal DNA fragment, and
4 Add DNA ligase and react at 15°C overnight.
A binding reaction of NA chains was performed.

The resulting binding reaction product was pancaged using an in vitro packaging kit manufactured by Amajam Japan, and as a result of infecting indicator bacteria DI11, lu
Approximately 50,000 strains of Ap' (
An ampicillin-resistant) strain was obtained and used as a chromosomal DNA cosmid library.

Example 4 (Cloning of phenol oxidase gene (I)) Approximately 5,000 strains of recombinant E. coli from a cosmid library were inoculated with 20% of recombinant E. coli containing ampicillin (final concentration 50 μg/n/).
Colonies were formed on two sheets of LB agar medium.

Colonies were transferred to two nitrocellulose filters (manufactured by Amajam Japan ■).

With the colonies facing up, place the filter in denaturing solution (1.5M
Place on filter paper soaked in NaCl (1,0,5 M NaOH) and leave for 7 min, then filter in neutralizing solution (1,0,5 M NaOH).
It was placed on a filter paper soaked in 5M NaCl, 0,5M NaOH) and left for 3 minutes, then placed on a filter paper freshly soaked in a neutralizing solution and left for 3 minutes. Filter 2X5SC(0,
After washing with 3M NaCl (0.03M trisodium citrate) and air drying, the DNA was fixed on the filter by treatment at 80°C for 2 hours.

Synthetic NA probes 15mer-A and B, and 26m
er-C and D are radioactive isotopes (γ-: +zp) ATP
(manufactured by Amajam Japan) and T4 polynucleotide kinase (2021A, manufactured by Takara Shuzo) and hybridized with DNA immobilized on a filter. As a result, two types of synthetic NA probes, 15mer and 26mer, We were able to obtain a hybridizing clone, ie, an E. coli strain containing a cosmid into which the phenol oxidase gene (1) was integrated.

In order to limit the portion containing the phenoloxidase gene (I) in the chromosomal DNA fragment integrated into the cosmid and perform subcloning, the cosmid was cut with the restriction enzyme old ndI [or EcoRI or Srma T] and 1% The molecular weight was separated by agarose gel electrophoresis, and the Southern blotting method (Southern, E.
,M, ,J,Mo1. Biol, 98, 503-5
17+19.75 on the filter and tzp
Synthetic NA probe labeled with ° (26mer-C,
As a result of hybridization with D and 15+++er-A, B), the 26mar-CD probe
[I 5.3Kb, EcoRI 4.6Kb.

The 15mer-A and B probes hybridized to a 1.9 Kb DNA fragment.
3Kb, EcoRI 4.6, SmaI 2.
Hybridized to a 4 Kb DNA fragment.

After subcloning each DNA fragment into pUc19, a restriction enzyme physical map was created (Figure 1).

Example 5 Base sequence of phenol oxidase gene (1)> Plasmid I) Eco subcloned into UCl3
RI4.6Kb, Small 2.4Kb, Small
1.9Kb, l1indll [5,3Kb DNA
Using Takara Shuzo's Precision Kit, cut the fragments into 10 pieces.
Deletion mutants were created at intervals of 0 to 200 bp, and the nucleotide sequence of the phenol oxidase gene ([) was determined using a sequencing kit manufactured by Takara Shuzo Co., Ltd., and the amino acid sequence was also determined at the same time (Figure 2).

The phenol oxidase gene N) is EcoRI4.
It was found that it is encoded in chromosomal DNA in the form of a 6Kb DNA fragment divided into 10 introns.

〔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 Mi variant by transformation or transduction; ,
Recombination can be performed using a method in which DNA fragments are directly introduced by electroporation or the like to create recombinants, and a new organism that produces a significant amount of phenol oxidase can be created.

■ Phenol oxidase produced in large quantities by a new organism that has recombined the DNA encoding phenol oxidase is free from contamination with cellulases and hemicellulases, can be used for biological pulping, and does not reduce pulp yield.

■ Phenol oxidase produced by a new organism that has recombined the DNA encoding phenol oxidase.
The enzyme has high purity and can be used as an enzyme of excellent quality as a reagent for clinical trials, such as a reagent for measuring enzyme activity, a substrate, or a reagent for quantifying bilirubin.

[Brief explanation of the drawing]

FIG. 1 shows a restriction enzyme cleavage map near the phenol oxidase gene (1) on chromosomal DNA. Phenol oxidase is encoded in the shaded area. FIG. 2 shows the entire base sequence and amino acid sequence of the phenol oxidase gene (I). (The r11e heVaIVaITyrAspProAsnAspPr
oHi, sAlaSerLeuTyrAspValAs
pAsnAeAsnPhe AsnAspGln Diagram (Part 3 TGTGATTCTCCTCTTGGATATGCCCT
CTCACCTGHiSAlaPh eA1aVaIV
aIArgSerAlaG13/5er-1-hrVa
l-1-3/r IJkSn diagram (Part 4

Claims (4)

[Claims] (1) A phenol oxidase gene (I) consisting of DNA encoding the following amino acid sequence separated by an intron. [There is a gene sequence] (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 (I) which is a DNA encoding a polypeptide that has a phenol oxidase activity and has a phenol oxidase activity, or is obtained by dividing the gene with an intron. (3) A recombinant DNA in which the phenol oxidase gene (I) according to any one of claims 1 to 2 is linked to vector DNA. (4) The phenol oxidase gene (I) according to claim 1, wherein the phenol oxidase gene (I) has DNA having the following sequence. [There is a gene sequence]