JPH0746995B2 - Phenol oxidase gene (1) - Google Patents

Description

TECHNICAL FIELD The present invention relates to a phenol oxidase gene (I), and more specifically, it is a white-rot fungus capable of producing and secreting phenol oxidase [particularly Coriolus coriorum]. hirsutus IFO4917)]-derived phenol oxidase gene (I).

By introducing the phenol oxidase gene (I) into various organisms, it produces phenol oxidase that can be used as a reagent for biological pulping, biobleaching, decolorization of industrial wastewater, pretreatment of wood saccharification, and clinical trials. can do.

[Prior art]

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

It is known that white-rot fungi have a high ability to decompose lignin in lignocellulosic materials such as wood. This white-rot fungus is inoculated into a lignocellulosic material, cultured, and a part of the lignin is decomposed to produce pulp. Attempts have been made to achieve biological pulping (Japanese Patent Application Laid-Open No. 50-46903).
issue). However, the white-rot fungus not only decomposes lignin, but also has an ability to decompose cellulose and hemicellulose, which are raw materials of pulp, and thus has a problem of lowering pulp yield. Further, there is a problem that it takes time because the lignin decomposition of the white rot fungus occurs in the latter stage of growth by secondary metabolism.

It is considered that the lignin degrading power of white rot fungi is largely due to the phenol oxidase produced and secreted by the white rot fungus, and attempts to clone the gene have been made, but they have not been successful yet. For laccase, which has a similar activity to the phenol oxidase of white-rot fungi, Neurospora crassa (Neur
ospora crassa) laccase gene cloning (UAGarmann, K. Lerch; (1988) J. Biol. Chem. 263,885-
896), but its amino acid sequence is different from the amino acid sequence of the phenol oxidase of the present invention, and the lignin degradation by laccase of Neurospora crassa has not been reported yet.

[Problems to be Solved by the Invention]

An object of the present invention is to solve the above-mentioned conventional problems and to make it possible to produce various novel organisms that produce only phenol oxidase.

Several types of enzymes are considered to be involved in the biodegradation of lignin in the natural world, but the phenol oxidase produced by white-rot fungi plays a central role in it, and is essential for research on lignin degradation. It is an enzyme.

Therefore, as a novel organism that efficiently expresses only the lignin degrading ability, an organism to which the phenol oxidase-producing ability of the white rot fungus has been imparted is considered, and the present invention is a phenol oxidase gene necessary for imparting the phenol oxidase-producing ability ( I) is provided.

[Means for Solving the Problems]

The present invention has the following formula: Relates to a phenol oxidase gene (I) in which the DNA encoding the amino acid sequence of is isolated by an intron.

Furthermore, the present invention provides a DNA hybridizing to the above-mentioned phenol oxidase gene (I), which is natural, synthetic,
Alternatively, it is obtained by semi-synthesis and is associated with the DNA encoding the amino acid sequence by substitution of nucleotides, insertion of nucleotides and inversion of nucleotide sequence or other mutations, and the phenol oxidase activity is The present invention relates to a phenol oxidase gene (I), which is a DNA encoding a polypeptide having the gene, or in which the gene is divided by an intron.

Furthermore, the present invention provides a phenol oxidase gene (I)
Relating to recombinant DNA.

Furthermore, the present invention provides a phenol oxidase gene (I)
Relates to a phenol oxidase gene (I) having a DNA of the following sequence.

The present invention will be described in detail below.

<Synthesis of DNA probe> Phenol oxidase gene (I) from cosmid library using colony hybridization method
The DNA probe required to clone
It is synthesized based on the partial amino acid sequence of phenol oxidase. The partial amino acid sequence of phenol oxidase is described in JP-A-61-285989, JP-A-62-220189, and
BrCN decomposition [Cole, RD: Methods] of the amino acid sequence from the N-terminus of the phenol oxidase produced and purified by the method disclosed in JP-A-62-220190 and the purified phenol oxidase.
Enzymol. 11 , 315-317 (1967)] or trypsin decomposition [Lin, L.-N. & Brandts. JF: Bio-chemistry 22 , 553 (19
83)], and the amino acid sequence from the N-terminus of the isolated polypeptide is analyzed by the Edman degradation method [Edman, P. & Henschen, A. Prot.
ein sequence determination, 2'nd de., Springer-Verla
g, Berlin, pp232-279 (1975)].

The synthesis of the DNA probe can be carried out by any of the phosphodiester method, the phosphophotoester method, the phosphite method and the amidite method which is an improved method thereof.

<Preparation of chromosomal DNA> The organisms that can be used in the present invention are all white rot fungi that produce and secrete phenol oxidase having a particularly high enzymatic activity as long as it has phenol oxidase (e.g., algae). Kawatake (IFO491
7), Kawaratake (IFO30340), Kaigaratake (IFO871)
4)] is 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 modified by the white-rot fungus may be used, and yeast extract, polypeptone as the main nitrogen source. Uses ammonium salts that can be assimilated by white-rot fungi, inorganic nitrogen compounds such as nitrates, urea,
Organic nitrogen-containing materials such as casein can also be used. In addition, inorganic salts such as calcium salt, magnesium salt, potassium salt, phosphate, manganese salt, zinc salt, iron salt, corn steep liquor, vitamins, amino acids,
It is also possible to add nutritional substances such as nucleic acids and growth promoting substances.

The above-mentioned medium is inoculated with a white rot fungus and cultured. After culturing,
After collecting the cells, freezing in liquid nitrogen and crushing in a mortar, the chromosomal DNA is extracted and purified by the phenol extraction method to obtain the chromosomal DNA used for cosmid library construction.

Chromosomal DNA can be extracted with high efficiency if proteinase treatment is performed in advance before extracting the chromosomal DNA with phenol.

<Construction of cosmid library of chromosomal DNA> Cosmid vector pHC79 [Hohn, B. and Collins, J. (198
0) Gene 11 , 291] to construct a cosmid library of chromosomal DNA. The cosmid vector pHC79 is commercially available [eg Bethesda Research Laboratory (Bethes
5358SA manufactured by da Research Labo-ratories) and 567795 manufactured by Boehringer Mannheim Yamanouchi Co., Ltd.] can be used.

Restriction enzyme Sau3AI [Takara Shuzo Co., Ltd. 108
2A] is partially decomposed to obtain a chromosomal DNA fragment having a size of 32 to 46 Kb (kilobase pairs). On the other hand, the cosmid vector pHC79 was completely digested with the restriction enzyme BamHI (Takara Shuzo Co., Ltd., 1010S), dephosphorylated, and the above partially digested chromosomal DNA fragment was added, and T4 DNA ligase (Takara Shuzo Co., Ltd.) was added. Made 2011A)
The binding reaction of the DNA chains is carried out by.

A commercially available in vitro packaging kit (for example, manufactured by Amersham Japan Co., Ltd.)
N.334Y, Promega Biotec P3151] was used to insert into mature phage particles, and Escherichia coli DH1 (ATCC33849) was infected with about 50,000 strains of Ap ^r (ampicillin) per 1 μg of chromosomal DNA. Resistant strain)
Use as a chromosomal DNA cosmid library.

<Cloning of phenol oxidase gene (I)> About 10,000 strains of recombinant Escherichia coli in a cosmid library were treated with LB medium containing ampicillin (Bactryptone 10 g / l, bait yeast extract 5 g / l, sodium chloride 10 g / l, agar 15 g). /
l) Allow colonies to form on top.

The colony is replicated on a commercially available nitrocellulose filter or nylon filter [eg, Amersham Japan KK RPN.82C, Toyo Roshi Kaisha A045B082C], and a conventional method [Grunstein, M. & D.S. Hogness : Proc.N
atl.Acad.Sci.USA 72 , 3961 (1975)] to fix the DNA on the filter.

DNA on the filter and radioisotope ³² P [Amersham
Japan PB10168] label [Richardson, C.
C (1965) Proc. Natl Acad. Sci. USA 54 , 158-161, hybridized with a synthetic DNA probe to select Escherichia coli having a phenol oxidase gene (I) incorporated therein, and extract a cosmid by a conventional method. Purify.

The restriction enzyme Hind III [Takara Shuzo Co., Ltd. 1060S] or EcoR I [Takara Shuzo (Takara Shuzo ( Co., Ltd. 1040S] or Sma I [Takara Shuzo Co., Ltd. 1085A], and separated by molecular weight by agarose gel electrophoresis.
Hybridize the chromosomal DNA fragment immobilized on the filter with the ³² P-labeled synthetic DNA probe. A DNA fragment that hybridizes with the synthetic DNA probe was added to the plasmid vector pUC19 [Yanisch-Perron, C. Vieira, J. and Messing,
J. (1985) Gene, 33 , 103, Messing, J (1983) Method in E
nzymology, 101 , 20-78, 3219 manufactured by Takara Shuzo Co., Ltd.] to create a restriction enzyme map.

DN containing the resulting phenol oxidase gene (I)
Integration of the A fragment into the vector DNA is performed as follows. Vector D is obtained by cutting vector DNA with an appropriate restriction enzyme.
Prepare NA fragments. Then, a mixture of the DNA fragment containing the phenol oxidase gene (I) and the vector DNA fragment was added to T4.
Treat with DNA ligase. Vector DNAs used include pBR322, pUC18, pUC19, [3050,3 manufactured by Takara Shuzo Co., Ltd.].
218, 3219] and the like. As a restriction enzyme, Hind
III, EcoR I, Pst I [1073S manufactured by Takara Shuzo Co., Ltd.], BamH
I and so on.

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

<Determination of Nucleotide Sequence of Phenol Oxidase Gene (I)> The DNA fragment subcloned into the plasmid vector pUC19 can be obtained by the principle of Henikoff method and Yanisch-Perron method [Henikoff, S. (1984) Gene, 28 , 351-359 Yanisch -P
erron, C., Vieira, J. and Messing, J. (1985) Gene, 33 , 10
3 to 119] to create a deletion mutant, but a commercially available deletion kit [6030 manufactured by Takara Shuzo Co., Ltd.] can also be used.

The deletion mutant is based on the dideoxy method [Sanger,
F. (1981) Science, 214 , 1205-1210]. Commercially available sequencing kit [6010A, 6015A manufactured by Takara Shuzo Co., Ltd., 31 manufactured by Nippon Gene Co., Ltd.]
7-01121] can also be used.

The EcoR I fragment of the chromosomal DNA of Pleurotus cornucopiae containing the phenol oxidase gene (I) was subcloned into the EcoR I site within the multicloning site of the plasmid pUC19 in a form commonly used in E. coli JM109 (ATCC53323) [eg Lederberg.EM & Cohen, SNJournal]. of Bacteriol
ogy, 119 , 1072-1074 (1974)]. This transformed Escherichia coli 0J-P0G-E1 was deposited at the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology, and the deposit number is 1004
No. 8 (FERM P-10048).

<Recombinant DNA> Utilization of the phenol oxidase gene (I) thus obtained is carried out by using microorganism, plant and animal vectors.
It is intended to make it possible to create a novel organism which is incorporated into DNA or the like and introduced into microorganisms, plants and animals to produce a large amount of phenol oxidase or its improved protein.

Incorporation of the phenol oxidase gene (I) into a vector can usually be performed in vitro as follows.

Both ends of the phenol oxidase gene (I) DNA are treated with exonuclease as necessary, and
A plurality of bases in a combination capable of connecting DNA or being annealed is polymerized. Then, it is incorporated into the vector of interest. As a method for incorporating, a vector is cleaved with a suitable restriction enzyme, and if necessary, a plurality of suitable linkers or a combination of bases capable of annealing are polymerized. The double-stranded DNA processed in this way and the vector DNA are mixed and ligated using T4 ligase.

The resulting recombinant DNA is a microorganism that is the host of the vector,
Introduced into plants, plant cells and animal cells. There are various types of hosts that can be used here, and for example, yeast such as Saccharomyces genus such as Saccharomyces cerevisiae, solanaceous plant cells such as tobacco and petunia, and animal cultured cells such as BalbIC3T3 are preferable. The vector used for these hosts is illustrated below. PJDB219, YE as yeast vector
p13, YRp7, YIp1, pYC, pTC2, ( including binary type vectors) Various vectors are derived from various vectors and cauliflower mosaic virus-derived Ti plasmid as plant vectors, derived from SV ₄₀ as an animal vector pSVK _+, pI-11β
−, PSVH _in + K +, pβ2x, pSXβ +, etc. However,
In the case of a plant vector derived from the Ti plasmid, the obtained recombinant DNA was once transferred to Agrobacterium tumefaciens T.
Introduced into 37 etc., this recombinant microorganism is transformed into plant cells by CO-culture.
The recombinant DNA can be introduced into the host plant by infecting the host plant by infection.

In the present invention, amino acids and polypeptides are IUPA.
It is abbreviated according to the method adopted by the C-IUB Biochemistry Committee (CBN). For example, the following abbreviations are used.

Ala L-alanine Arg L-arginine Asn L-asparagine Asp L-aspartic acid Cys L-cysteine Gln L-glutamine Glu L-glutamic acid Gly glycine His L-histidine Ile 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-tryptophan Tyr L-tyrosine Val L-valine The DNA sequence is abbreviated by the type of base contained in each deoxyribonucleotide constituting it. The following abbreviations are used.

A adenine (representing deoxyadenylic acid) C cytosine (representing deoxycytidylic acid) G guanine (representing deoxyguanylic acid) T thymine (representing deoxythymidylate) [Examples] [Examples] The cloning and nucleotide sequence determination of the phenol oxidase gene (I) derived from the chromosome of white rot fungus will be described in detail.

Example 1 <Synthesis of DNA probe> The DNA probe was synthesized by the amidite method using a DNA synthesizer (Zeon GenetA III, Japan).

The amino acid sequence from the N-terminal of phenol oxidase purified from three white-rot fungi [Arakawakaratake (IFO4917), Kawaratake (IFO30340), Kaigarake (IFO8714)] was analyzed by Edman degradation up to the 25th step. Show.

The following DNA probes were synthesized so as to correspond to Pro on the 17th row and Val on the 25th row in the above sequence. However, I is deoxyinosine.

In addition, the phenol oxidase of 3 kinds of white-rot fungi was added to Br.
Reversed phase high performance liquid chromatography [(elution conditions, column: Phenyl-5PW RP (Toyo Soda)), eluent 20% acetonitrile / 0.1% TFA to 75% acetolitril / 0.1% TFL The results obtained by analyzing the amino acid sequence of the polypeptide separated by gradient elution, greenhouse) by the Edman degradation method are shown below.

Arakawakaratake Met-Ala-Phe-Asn-Phe Kawaratake Met-Ala-Phe-Asn-Phe Kaigaraketake Met-Ala-Phe-Asn-Phe The following DNA probes were synthesized so as to correspond to the above amino acid sequences.

From the above results, the homology of the amino acid sequence of the phenol oxidase produced and secreted by the white rot fungus is very high, and by using the DNA probe used in the present invention, any white rot phenol oxidase can be cloned. it can. Therefore, in the following example,
A method for cloning the Phenol oxidase gene (I) of Pleurotus cornucopiae will be described.

Example 2 <Preparation of chromosomal DNA> Agarella charlotteum (IFO4917) was inoculated into a 5 l Erlenmeyer flask containing 1 YPD medium (yeast extract 10 g / l, polypeptone 20 g / l, glucose 20 g / l), and 27 ° C. The cells were cultivated with shaking for 7 days. After culturing, the cells were collected and frozen in liquid nitrogen.
Frozen cells of g were obtained.

Frozen cells of 10 g were crushed for about 15 minutes using a mortar under liquid nitrogen. A buffer solution (0.1M NaCl,
Proteinase K (final concentration 100 μg / ml Boehringer Mannheim Yamanouchi 161519) was added to 10 ml of 0.1 M Tris-HCl, 0.1 M EDTA, pH8, and 5 g of crushed cells was added to it and allowed to react for 2 hours with gentle stirring. It was Equivalent TE (10mM Tri
s-HC1,1mM EDTA, pH8) Chromosomal DNA was extracted with saturated phenol, ethanol precipitated, dissolved again in 5 ml TE, and treated with RNase A (final concentration 100 μg / ml Takara Shuzo Co., Ltd.) for 30 minutes at 37 ° C. RNA was removed. Equilibrium density gradient centrifugation using CsC1 (Beckman, VTi80 rotor, 15 ℃, 50krp
m, 16 hours) to obtain 1.5 mg of purified chromosomal DNA.

Example 3 <Construction of a cosmid library of chromosomal DNA> To 250 μg of the above-mentioned purified chromosomal DNA, the restriction enzyme Sau3AI was added and partially decomposed at 37 ° C., and the partially decomposed product was subjected to 5-25% sucrose density gradient centrifugation (Beckman. SW40Ti rotor, 15 ℃,
About 2 μg of chromosomal DNA fragment of 32 to 46 Kb was obtained at 22.5 krpm for 16 hours.

After adding the restriction enzyme BamHI and reacting at 37 ° C for 12 hours to completely decompose it, it was dephosphorized with alkaline phosphatase [2250A manufactured by Takara Shuzo Co., Ltd.] for 30 minutes at 37 ° C, and 10 µg of phenol-extracted cosmid vector was extracted. And 32-46 Kb chromosomal DNA
1 μg of the fragments were mixed, T4 DNA ligase was added, and the mixture was reacted overnight at 15 ° C. to carry out a DNA strand binding reaction.

The resulting binding reaction product was packaged using an in vitro packaging kit manufactured by Amersham Japan and infected with the indicator bacterium DH1, and as a result, about 50,000 strains of Ap ^r per 1 μg of chromosomal DNA. (Ampicillin resistance)
A strain was obtained and used as a chromosomal DNA cosmid library.

Example 4 <Cloning of Phenol Oxidase Gene (I)> About 5,000 recombinant Escherichia coli of the cosmid library were formed into colonies on 20 LB agar medium containing ampicillin (final concentration 50 μg / ml). The colony was transferred to two nitrocellulose filters (manufactured by Amersham Japan KK). Place the filter, colony up, on filter paper soaked in denaturing solution (1.5M NaCl, 0.5M NaOH) and leave for 7 minutes, then filter the neutralizing solution (1.5M
It was placed on a filter paper dipped in NaCl, 0.5M NaOH) and left for 3 minutes, and then placed on a filter paper newly dipped in a neutralizing solution and left for 3 minutes. The filter was washed with 2 x SSC (0.3M NaCl, 0.03M trisodium citrate), air-dried, and then treated at 80 ° C for 2 hours, DN
A was fixed to the filter.

Synthetic DNA probes 15mer-A and B, and 26mer-C and D are radioactive isotopes [γ- ³² P] ATP (Amersham Japan KK) and T4 polynucleotide kinase (Takara Shuzo 2021A). Labeled with and hybridized with DNA immobilized on the filter, the results were 15mer and 26
A clone that hybridizes with two synthetic DNA probes of mer, that is, a phenol oxidase gene (I)
It was possible to obtain Escherichia coli having a cosmid in which P.

The cosmid restriction enzyme Hi is used for subcloning by limiting the portion containing the phenol oxidase gene (I) in the chromosomal DNA fragment integrated in the cosmid.
Cleavage with nd III, EcoR I or Sma I, and separation by molecular weight by 1% agarose gel electrophoresis, and Southern blotting (Southern, EM, J. Mol. Biol., 98 , 503-
Immobilized on the filter with 517,1975 and labeled with ³² P synthetic DNA probes (26mer-C, D and 15mer-A, B),
As a result of hybridization, the 26mer-C, D probe was Hin
d III5.3Kb, EcoR I4.6Kb, Sma I1.9Kb hybridized to the DNA fragment, 15mer-A, B probe, Hind III5.3Kb, Eco
It hybridized to DNA fragments of R 4.6 Kb and Sma I 2.4 Kb.

After subcloning each DNA fragment into pUC19,
A restriction enzyme physical map was prepared (Fig. 1).

Example 5 <Nucleotide sequence of phenol oxidase gene (I)> EcoR I4.6Kb, S subcloned into plasmid pUC19
ma I2.4Kb, Sma I1.9Kb, Hind III5.3Kb using a DNA fragment made by Takara Shuzo Co., Ltd., to create a deletion mutant every 100-200 bp, using a Takara Shuzo sequencing kit, The nucleotide sequence of the phenol oxidase gene (I) was determined, and at the same time, the amino acid sequence was also determined (Fig. 2).

Phenol oxidase gene (I) is EcoR I4.6KbD
Chromosome in the form of 10 introns in NA fragment
It was found to be encoded by DNA.

〔The invention's effect〕

 The present invention has the following effects.

Providing the complete amino acid sequence of phenol oxidase elucidates the role of phenol oxidase in lignin degradation and can be applied to biological pulping.

DNA encoding phenol oxidase can be used by other organisms in various ways (eg, plasmids, cosmids,
Recombinant can be carried out by ligating to a vector such as a phage or virus and preparing a recombinant by transformation or transduction, or a method of directly preparing a recombinant by introducing a DNA fragment by electroporation). It is possible to create a new organism that produces a large amount of phenol oxidase.

Phenol oxidase produced in a large amount in a novel organism in which DNA encoding phenol oxidase is recombined does not contain cellulase or hemicellulase, can be used for biological pulping, and does not reduce pulp yield.

Phenol oxidase produced by a novel organism that has recombined DNA encoding phenol oxidase is
It has a high degree of purity and can be used as an enzyme of excellent quality as a reagent for measuring clinical activity such as a reagent for measuring enzyme activity, a substrate, and a reagent for quantifying bilirubin.

[Brief description of drawings]

FIG. 1 shows a restriction enzyme cleavage map near the phenol oxidase gene (I) on chromosomal DNA. Phenol oxidase is coded in the shaded area. FIG. 2 shows the entire nucleotide sequence and amino acid sequence of the phenol oxidase gene (I).

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Claims (3)

[Claims] 1. A phenol oxidase gene (I) obtained by disrupting a DNA encoding the following amino acid sequence by an intron. 2. The phenol oxidase gene (I) according to claim 1, wherein the phenol oxidase gene (I) has a DNA sequence of the following sequence. 3. A recombinant DNA in which the phenol oxidase gene (I) according to any one of claims 1 and 2 is bound to vector DNA.