WO2007029630A1 - Novel ceramidase and use thereof - Google Patents
Novel ceramidase and use thereof Download PDFInfo
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- WO2007029630A1 WO2007029630A1 PCT/JP2006/317422 JP2006317422W WO2007029630A1 WO 2007029630 A1 WO2007029630 A1 WO 2007029630A1 JP 2006317422 W JP2006317422 W JP 2006317422W WO 2007029630 A1 WO2007029630 A1 WO 2007029630A1
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- ceramidase
- dna
- amino acid
- protein
- transformant
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
- C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G18/00—Polymeric products of isocyanates or isothiocyanates
- C08G18/06—Polymeric products of isocyanates or isothiocyanates with compounds having active hydrogen
- C08G18/83—Chemically modified polymers
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J11/00—Recovery or working-up of waste materials
- C08J11/04—Recovery or working-up of waste materials of polymers
- C08J11/10—Recovery or working-up of waste materials of polymers by chemically breaking down the molecular chains of polymers or breaking of crosslinks, e.g. devulcanisation
- C08J11/105—Recovery or working-up of waste materials of polymers by chemically breaking down the molecular chains of polymers or breaking of crosslinks, e.g. devulcanisation by treatment with enzymes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
- C12Y305/01—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amides (3.5.1)
- C12Y305/01023—Ceramidase (3.5.1.23)
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J2375/00—Characterised by the use of polyureas or polyurethanes; Derivatives of such polymers
- C08J2375/04—Polyurethanes
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W30/00—Technologies for solid waste management
- Y02W30/50—Reuse, recycling or recovery technologies
- Y02W30/62—Plastics recycling; Rubber recycling
Definitions
- the present invention relates to a novel ceramidase, a DNA (gene) encoding the enzyme, a method for decomposing plastics using the enzyme, and the like.
- plastic fiber which is insoluble and hydrophobic because of its insolubility and is recovered as a monomer or oligomer. They often contain urethane bonds or amide bonds in various proportions, and it is necessary to efficiently cleave the urethane bonds or amide bonds for recovery and reuse. Enzymes that catalyze are anxious.
- Non-patent Document 1 or 2 There have been reports of microorganisms that decompose plastics and fibers containing urethane bonds and amide bonds.
- Non-patent Document 1 or 2 the enzymes derived from microorganisms described in them do not degrade the urethane-binding moiety but degrade the polyester moiety or the polyester moiety.
- Patent Documents 1 to 5 There is a report on a microorganism having a urethane-binding ability belonging to the genus Rhodococcus (Patent Document 6), but it is a low molecular weight urethane compound that is actually degraded by the microorganism.
- NB 1 Microbial degradation of polyurethane, polyester polyurethanes and polyester polyurethanes.
- Non-Patent Document 2 Biodegradation of polyurethane: a review, G. T. Howard. Int. Biodet. Biodeg., 49, 245-252 (2002)
- Patent Document 1 JP-A-01-240179
- Patent Document 2 JP-A-01-300892
- Patent Document 3 Japanese Patent Laid-Open No. 03-175985
- Patent Document 4 JP 04-325079
- Patent Document 5 JP-A 09-192633
- Patent Document 6 JP-A-2004-261103
- the present inventor has developed a urethane bond contained in a polymer substance derived from a mold having a high ability to grow on a hydrophobic solid surface among microorganisms, and a plastic containing a urethane bond that solves the above problems.
- a new enzyme that degrades Z or amide bonds is obtained, and a method for decomposing plastics using such an enzyme is provided.
- the present invention relates to the following aspects.
- a protein having ceramidase activity which also has an amino acid sequence ability in which one or several amino acids have been deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1.
- a protein having ceramidase activity which also has an amino acid sequence ability in which one or several amino acids have been deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1.
- a DNA for recombination comprising the DNA according to any one of embodiments 2 to 4!
- ⁇ 10 '' eukaryotic microorganism is a yeast, or eukaryotic filamentous fungus selected from the group of Aspergillus, Penicillium, Trichoderma, Rhypus, Metalithium, Acremonium, and Mucor
- Aspergillus Penicillium
- Trichoderma Trichoderma
- Rhypus Trichoderma
- Metalithium Trilithium
- Acremonium and Mucor
- a method for producing ceramidase comprising culturing the transformant according to any one of embodiments 7 to 13 in a medium and collecting ceramidase from the culture.
- a method for decomposing a polymer material comprising contacting the transformant according to any one of Embodiments 7 to 13 with the polymer material.
- the embodiment is characterized in that the polymeric material is selected from the group consisting of polyurethane, polyester containing urethane bonds in any proportion, polypropylene, polyvinyl chloride, nylon, polystyrene, starch, and mixtures thereof, Embodiment 15 The method as described in any one of -19. [21] The method according to any one of aspects 15 to 19, wherein the polymer substance is a biodegradable plastic.
- biodegradable plastic is polylactic acid, polybutylene succinic acid, polybutylene succinic acid 'adipic acid, aliphatic polyester, polystrength prolatatone, or polyhydroxybutyric acid.
- a novel ceramidase capable of decomposing a urethane bond and a Z or amide bond in a polymer substance having a molecular weight of several tens of thousands has been obtained as the koji mold, and its amino acid sequence and the DNA base sequence encoding it. And a method for decomposing plastics using the enzyme is provided.
- FIG. 1 is a SDS-PAGE photograph showing the state of separation of Aspergillus oryzae-derived proteins that strongly interact with hydrophobic columns.
- FIG. 2 shows an outline of the construction of a ceramidase high expression system.
- FIG. 3 is a photograph of a gel obtained by electrophoresis of the culture supernatant of a strain with a high expression of ceramidase.
- FIG. 4 is a graph showing the effect of ceramidase on the degradation of PBS.
- FIG. 5 is a graph showing a decrease in PBS intramolecular urethane bonds by ceramidase.
- FIG. 7 is a graph showing a state of degradation of a ceramide fluorescent substrate of ceramidase.
- ceramidase means an enzyme that degrades urethane bonds and Z or amide bonds. Therefore, in this specification, “ceramidase activity” means ceramide, plastic, It means activity capable of specifically decomposing urethane bonds and / or amide bonds contained in substances such as chemical fibers. Specifically, urethane bonds described in Example (7) of this specification It can be measured by decomposition or decomposition reaction of a ceramide fluorescent substrate (C12-NBD-ceramide) described in Example (8).
- the amino acid to be deleted, substituted or added is preferably a homologous amino acid (polar / nonpolar amino acid, sparse Aqueous 'hydrophilic amino acids, positive' negatively charged amino acids, aromatic amino acids, etc.), or the loss or attachment of amino acids can greatly affect the three-dimensional structure and / or local charge state of proteins. Those that do not change or are substantially unaffected are preferred.
- the ceramidase of the present invention having such a deleted, substituted or added amino acid includes, for example, site-specific mutagenesis (point mutagenesis, cassette mutagenesis, etc.), gene homologous recombination, primers It can be easily prepared by appropriately combining methods known to those skilled in the art such as extension method and PCR method.
- under stringent conditions means the degree of homology between each base sequence, for example, about 80% or more, preferably about 90% or more, more preferably on the average on the whole. It means a condition in which a hybrid is specifically formed only between nucleotide sequences having high homology, such as about 95% or more.
- the conditions include a sodium concentration of 150 to 900 mM, preferably 600 to 900 mM, and a pH of 6 to 8 at a temperature of 60 ° C to 68 ° C.
- Hybridization is, for example, a method described in Current Protocol in Molecular Biology (.edited by Frederick M. Ausubel et al, 1987). In the case of using a commercially available library, it can be carried out according to the method described in the attached instruction manual.
- the DNA of the present invention can be prepared by methods known to those skilled in the art.
- the Aspergillus oryzae RIB40 strain described in the Examples independent administrative corporation Liquor Research Institute: 3-1, No. 7 Higashihiroshima Kagamiyama, can be stored under the same number and can be distributed
- other commercially available filamentous fungi such as Neisseria gonorrhoeae can be easily cloned by the method described in the Examples.
- it is prepared by chemical synthesis well known to those skilled in the art or by amplification using PCR using the primer of the present invention. Things also come up. Therefore, the DNA of the present invention can be obtained by those skilled in the art such as genomic DNA, cDNA and synthetic DNA. It can be of any known type.
- the DNA encoding the ceramidase of the present invention was inserted into an appropriate DNA for recombination such as a plasmid vector, a phage vector, and various hybrid vectors, and thus obtained.
- an appropriate DNA for recombination such as a plasmid vector, a phage vector, and various hybrid vectors, and thus obtained.
- Various cells can be transformed with the expression vector.
- This recombination DNA is any vector that can be handled by recombinant DNA techniques. These vectors can be appropriately selected depending on the host cell to be introduced. When the vector is introduced into a host cell, the whole or a part of the vector can be inserted into one or more places in the genome of the host cell. Examples of such vectors include pET-12Bb and pAURlOl for E. coli hosts and PNEN142 for gonococcal hosts.
- the expression vector of the present invention typically includes various regulatory sequences known to those skilled in the art, such as various regulatory sequences such as various promoters, enhancers and silencers, ribosome binding sites, signal sequences, and translation initiation sequences. It can optionally contain elements and other genes encoding exogenous or endogenous proteins, various drug resistance genes, genes that complement auxotrophy, and the like.
- the promoter required to express the ceramidase of the present invention depends on the host cell obtained by transformation, but has transcription activity in the selected host cell and is homologous to the host cell or It can be any DNA sequence that can be derived from a gene encoding a protein that is heterologous. In these transformants, it is desirable that the suppression of production induction of each substance is released.
- the gene encoding the ceramidase of the present invention can be expressed under the control of a constitutive expression promoter or various inducible expression promoters. As a result, ceramidase is highly expressed, and is produced in a large amount on the cell surface or outside the cells, thereby promoting plastic degradation and further promoting production of useful substances.
- a prokaryotic microorganism in the present invention, a prokaryotic microorganism, a eukaryotic microorganism, a plant cell, an insect cell, an avian cell containing an egg, a mammalian cell, or the like may be used as a host cell transformed with a vector containing a DNA encoding ceramidase.
- prokaryotic microorganisms include Escherichia, Bacillus, or Streptomyces griseus or Streptococcus.
- the genus Streptomyces such as Sericolor can be used as a host.
- eukaryotes examples include yeasts such as Saccharomyces and Pichia, Aspergillus oryzae and Aspergillus sau, etc.
- filamentous fungi and basidiomycetes such as Trichoderma can also be selected.
- insect cells examples include Drosophila melanogaster and silkworm.
- a suitable promoter that regulates the transcription of the gene encoding the cutinase mutant of the present invention hosted by a prokaryotic microorganism the lac promoter of Escherichia coli Escherichia coli, the ⁇ -amylase of Bacillus licheniformis
- the promoter of the gene (amyL), the promoter of the ⁇ -amylase gene (amyQ) of Bacillus' Amiguchi liqufaciens The promoter of the gene (amyL), the promoter of the ⁇ -amylase gene (amyQ) of Bacillus' Amiguchi liqufaciens.
- promoters examples include Saccharomyces cerevisiae galactosidase gene, Aspergillus oryzae takaamylase gene, enolase gene, xylanase gene, phosphodarcokinase gene Examples include promoters such as the Dalcoamylase gene, Rhizomucor. Myehai aspartic protease gene, Aspergillus'-Gar's darcoamylase gene, and Rhizomucor's myehai lipase gene.
- the expression vector (DNA for recombination) containing the DNA of the present invention includes, for example, calcium chloride method, protoplast-PEG method, electopore position method, Ti plasmid method, particle gun method, baculovirus method, etc. It can be introduced into a host cell by any known method, and a transformant can be produced. Furthermore, the co-transformation method using multiple types of recombinant DNA is also possible.
- the transformant useful in the present invention is obtained by another one or more recombination DNAs containing a gene encoding any exogenous or endogenous protein.
- a gene encoding any exogenous or endogenous protein.
- Such genes include various esterases represented by cutinase and lipase as described in International Publication No. WO2004 / 038016A1 pamphlet.
- the ceramidase of the present invention obtained by PCR amplification or the like instead of the above expression vector It is also possible to obtain the transformant of the present invention by using an appropriate DNA fragment itself containing a gene encoding. In such a case, it can be used for transformation as a composition such as a solution containing an appropriate buffer solution and other auxiliary agents in addition to the DNA fragment to be obtained.
- the ceramidase of the present invention is preferably used for the production of ceramidase in a transformed host cell (transformant) having DNA encoding the enzyme, and cultured under U ⁇ conditions to express the mutant.
- a transformed host cell transformant
- it can be produced by secreting the expressed mutant extracellularly and recovering it from its host cell and Z or medium.
- the medium used for culturing the host cells is appropriately selected from any medium known to those skilled in the art, which is suitable for growing the transformed host cells of the present invention and expressing the ceramidase of the present invention. can do.
- the secreted ceramidase is produced by a suitable combination of any means known to those skilled in the art, for example, separation of media and cells by centrifugation or filtration, and salts such as ammonium sulfate. It can be recovered from the medium by precipitation of the protein components of the medium, followed by the use of hydrophobic chromatography, ion exchange chromatography, affinity chromatography, or other chromatographic techniques.
- the ceramidase of the present invention has an activity of specifically decomposing a urethane bond and a Z or amide bond, polyurethane and polyester, polypropylene, polyvinyl chloride, polyurethane containing an urethane bond in an arbitrary ratio, It can be advantageously used to decompose high molecular weight materials such as nylon, polystyrene, and starch.
- biodegradable plastics means “a simpler molecular level that retains the sufficient functions required for its intended use in the state of use, and when it is disposed of, by the action of microorganisms in the soil or water. It is a substance that should be called “plastic that can be broken down to Based on the degree of degradation! / Furthermore, it can be divided into “completely degradable biodegradable plastics” and “partially degradable (collapsed) biodegradable plastics”. It can be broadly divided into “production system”, “natural polymer system”, and “chemical synthesis system”. Any of these types of biodegradable plastics can be used in the method of the present invention.
- the plastic decomposition reaction may be carried out by any reaction system known to those skilled in the art (for example, aqueous and solid phase systems) and reaction conditions (solvent, medium, temperature and pH etc.) can be selected as appropriate.
- reaction system known to those skilled in the art
- reaction conditions solvent, medium, temperature and pH etc.
- Examples of the solvent used when hydrolyzing a polymer substance such as a biodegradable plastic by the method of the present invention include water, a buffer solution, an organic solvent and water, or a mixture of an organic solvent and a buffer solution. It is done. A surfactant, an organic substance, an inorganic substance, or the like can be added to these solvents as necessary. It is preferable to add a buffering agent to the solvent in order to stabilize the pH and improve the hydrolysis efficiency.
- the pH of the solvent is preferably maintained in the range 6-10, more preferably 79.
- the plastic in the decomposition method of the present invention, can be decomposed by allowing the ceramidase of the present invention or the esterase to coexist in the culture system.
- These substances may be those produced by the microorganisms themselves, and in addition to them, they can be added from outside the culture system.
- the amount and ratio of addition and various conditions such as the timing of addition can be appropriately selected by those skilled in the art. These substances need not be added at the same time, but may be added sequentially at each stage of the process.
- the above-described transformant was decomposed into a culture system! / ⁇ coexisted with a high molecular weight substance, and the transformant was brought into contact with the substance. It is possible to disassemble the plastic.
- This method can be carried out in any culture system known to those skilled in the art such as a liquid culture system containing a plastic emulsion and a solid culture system using a plastic solid pellet or plastic powder.
- the genes encoding ceramidase and esterase can be introduced into different microorganisms, and the reaction can be carried out using the plural types of transformants thus obtained.
- genes encoding these enzymes introduced into the transformant of the present invention are not limited to one type, and each can be recombined with a plurality of types of enzyme genes.
- each can be recombined with a plurality of types of enzyme genes.
- it is carried out in a co-culture system in which these multiple types of microorganisms are cultured at the same time, or the method of the present invention is composed of a plurality of continuous culture stages.
- the culture conditions (medium, temperature, pH, etc.) of the transformant in the above reaction can be appropriately selected depending on the condition of the host known to those skilled in the art.
- any surfactant or biosurfactant known to those skilled in the art such as disclosed in the above-mentioned international publication, is added to the reaction system by, for example, adding it from the outside, It is also possible to further accelerate the decomposition of molecular substances.
- the decomposition method of the present invention it is possible to recover a constituent monomer that has been dissolved and solubilized by a polymer substance, an oligomer in which a plurality of them are polymerized, or a salt thereof.
- the constituent monomer is synthesized by polycondensation, copolymerization, ring-opening polymerization, etc. of polylactic acid, polybutylene succinate, polybutylene succinate-coadipate, polyhydroxybutyrate, and Z or poly force prolacton.
- the above high molecular weight substance is a decomposed soluble monomer component or a material such as an oligomer obtained by polymerizing a plurality of them, and is the only nutrient source (carbon source) of the transformant.
- carbon source a nutrient source
- Nitrogen source Alternatively, other carbon sources and nitrogen sources can be added separately to the culture system.
- various carbons such as glucose as a carbon source, organic nitrogen sources as a nitrogen source, such as peptone, meat extract, yeast extract, corn 'strip' liquor, etc., inorganic nitrogen sources such as ammonium sulfate, salt ⁇ Ammumum can be contained.
- the medium may contain cations such as sodium ion, potassium ion, calcium ion, magnesium ion, sulfate ion, chlorine ion, phosphorus ion.
- cations such as sodium ion, potassium ion, calcium ion, magnesium ion, sulfate ion, chlorine ion, phosphorus ion.
- Inorganic salts composed of anions such as acid ions may be included.
- trace elements such as vitamins and nucleic acids can be contained.
- the polymer substance to be decomposed may be contained as a constituent element of a so-called "composite material".
- “composite material” is a material that is generally composed of two or more different material forces, for example, a material composed of plastic, various metals, and other inorganic material forces. Such composite materials are used for various purposes in various fields as various industrial materials.
- the plastic portion is selectively decomposed to recover the plastic as a monomer or oligomer, while being substantially free of plastic.
- Other parts such as metal can be recovered.
- the supernatant was subjected to Octy ⁇ cellulofine type-S (Seikagaku Corporation) saturated with 10 mM Tris-HCl buffer, pH 8.0, 20% saturated ammonium sulfate, and then 1 liter of purified water.
- the column was washed with.
- the washed column was eluted with a linear gradient of 0-70% ethanol.
- 0.2 ml of 100% (w / v) triclonal acetic acid was added to 0.8 ml of each elution fraction, mixed well, and then allowed to stand in ice for 20 minutes. These samples are 15,000 Xg, 4.
- the protein without gel was transferred to a PVDF membrane, the target protein band was cut out from the PVDF membrane, and the amino acid sequence of the amino terminal region was analyzed.
- the amino acid sequence of the amino acid terminal region of the target protein was “ASDDSVFLLG” (corresponding to the 50th to 59th amino acids in the amino acid sequence of SEQ ID NO: 1).
- Drosophila melanogaster neutral ceramidase (BAC77635.1) and 36%
- cellular slime mold Dictyostelium discoideum neutral ceramidase ( AAB69633.1) and 36%
- zebrafish Danio rerio neutral ceramidase (BAD69590.1) and 36% homology, indicating that this protein is a neutral ceramidase. It was suggested.
- a set of oligonucleotides was designed with reference to the base sequence around the cloned target gene (SEQ ID NO: 5 '-GTTGCGCACGtgTTCTAATGTCG-3, SEQ ID NO: 3: 5). GCGATGTCTAgATCCCCGAGTCC-3,).
- PCR reaction was performed using the Aspergillus oryzae RIB40 genomic DNA as a template. The amplification reaction was carried out after 30 cycles of denaturation of vertical DNA at 95 ° C for 3 minutes and holding at 95 ° C for 1 minute, 55 ° C for 1 minute, 72 ° C for 3 minutes. Fully extended at 5 ° C for 5 minutes and kept at 4 ° C. When the amplified fragment was confirmed by agarose electrophoresis, amplification of about 3,269 base pairs was observed.
- This amplified fragment was digested with PmaC I (Takara Shuzo) and Xba I (Takara Shuzo), and the digested fragment was subjected to agarose gel electrophoresis, and DNA was extracted using prep A gene (BioRad). This was used as an inserted DNA fragment.
- the plasmid pNEN142 DNA 5 ⁇ g having the Dalcore mirase promoter sequence (P-enoA142) in the base sequence was digested with PmaC I and XbaI, and subjected to phenol extraction and ethanol precipitation treatment in the usual manner. Al force Liphosphatase (Takara Shuzo) removed the 5'-terminal phosphate.
- This reaction solution was subjected to phenol extraction and ethanol precipitation by a conventional method, and then dissolved in TE to obtain a vector-DNA solution.
- 1 ⁇ g of vector DNA and 1.5 g of the inserted DNA fragment were ligated with T4 DNA ligase (Takara Shuzo) to obtain a ligated DNA solution.
- a single colony of E. coli transformed with the desired plasmid DNA was inoculated into 3 ml of LB liquid medium supplemented with 100 ug / ml ampicillin and shaken at 37 ° C. Cultured. Transfer 1.5 ml of the culture to a 2 ml Eppendorf tube, centrifuge at 15,000 X g for 1 min, and precipitate with 100 ⁇ l ice-cold TEG (25 mM Tris-HC1, 10 mM EDTA, 50 mM) Glucose, pH 8.0) was suspended, 200 ⁇ l of 0.2 ⁇ NaOH-1% SDS was added and gently stirred, and then 150 1 of 3 M NaOAc pH 5.2 was added and mixed.
- plasmid DNA to be transformed pNEN-Cer and pNEN142 prepared above were used. 10 g of these plasmid DNAs were completely digested with BamHI, phenol extraction and ethanol precipitation were performed by conventional methods, and then dissolved in 101 TE to obtain a DNA solution for transformation.
- Aspergillus oryzae niaD300 strain (Nitrate reductase gene (niaD) deficient mutant derived from RIB40 strain: Independent Administrative Institution Liquor Research Institute: Higashihiroshima Kagamiyama 3-chome No. 7-1, stored under the same number
- the spore suspension was added to YPD liquid medium and cultured with shaking at 30 ° C for 12 hours. Bacteria were collected from the culture using a glass filter. Transfer the cells to a 50 ml centrifuge tube and add 10 ml protoplasting solution (0.6 M KC1, 0.2 M NaH PO, pH 5
- the protoplast was washed three times at pH 7.5, and centrifuged as 3000 xg at 4 ° C for 5 minutes to obtain a precipitate.
- This protoplast was suspended in 1.2 M sorbitol, 50 mM CaCl, Tris-HC1 buffer, pH 7.5 so as to be 1 ⁇ 10 9 protoplasts / ml.
- Protoplast suspension Add 100 1 and 12.5 1 Soli (50 (w / v%) PEG # 4000, 50 mM CaCl, 10 mM Tris-HCl buffer, pH 7.5, each of the aforementioned plasmid DNA solutions for transformation to the suspension 100 1.
- PH 7.5 was mixed well.
- the protoplast suspension was mixed with Czapek-Dox soft agar medium that had been warmed to 55 ° C. and overlaid on Czapek-Dox agar medium. Thereafter, the cells were cultured at 30 ° C. until spores were formed.
- the conidia pattern is vigorously removed with a platinum needle and suspended in 0.01% (v / v%) Tween 80. This suspension is diluted and spread on Czapek-Dox agar medium at 30 ° C. The single spore was isolated by repeatedly culturing in the same manner. Single spore separation was confirmed by modifying the Hondel method (spore PCR method). Conidia was added to a 1.5 ml microtube containing 200 1 YPD medium with a platinum needle and cultured at 30 ° C for 40 hours.
- PCR Thermal Cycler PERSONAL (Takara Shuzo) was used as the PCR device.
- the plasmid DNA used for transformation was in the form of a cage. Amplification reaction was performed at 95 ° C for 3 minutes, denaturing the cage DNA, followed by 30 cycles of 94 ° C for 1 minute, 55 ° C for 1 minute, 7 2 ° C for 3 minutes, then 72 ° C for 5 minutes.
- ceramidase gene (sequence) was located downstream of the P-enoA142 promoter sequence on the chromosomal DNA of Aspergillus oryzae. The presence of the inserted sequence No. 1) was confirmed, and it was confirmed that PNEN142 was also transformed.
- the amino acid sequence of the amino terminal region is “NTEFA”, and the deduced amino acid sequence of ceramidase (the amino acid sequence in the amino acid sequence of SEQ ID NO: 1) A high expression of ceramidase was confirmed (Fig. 3).
- Ceramidase high-expressing strain spores were inoculated into a 3 liter volumetric flask containing 1 liter of YPM medium at 1 ⁇ 10 6 spores / ml and cultured at 30 ° C. for 24 hours.
- the culture solution was filtered with MIRACLOTH (CALBIOCHEM) to obtain a culture supernatant.
- Ammonium sulfate was added to the culture supernatant so as to be 40% saturation, and then centrifuged at 10,000 ⁇ g, 4 ° C. for 40 minutes to obtain a supernatant fraction.
- the supernatant fraction was subjected to Octy ⁇ cellulofine type-S (Seikagaku Corporation) equilibrated with 10 mM Tris-HCl buffer, pH 8.0, 40% saturated ammonium sulfate, and the column was washed with 1 liter of purified water. did. The washed column was eluted with a linear gradient of 0-70% ethanol. All eluted fractions were subjected to SDS-PAGE, and fractions containing 100 kDa ceramidase were collected.
- the collected fraction is dialyzed against 10 mM Tris-HCl buffer (pH 8.0) and applied to DEAE-cellulofine A-500 equilibrated with the same buffer, and the adsorbed fraction is linear gradient of NaCl, 0-0.5M. And eluted. All the collected fractions were subjected to SDS-PAGE, and a single band of ceramidase of 100 kDa was confirmed by silver staining. This was used as a purified preparation of ceramidase. The purified ceramidase was dialyzed against milliQ water and lyophilized to obtain a dry powder of ceramidase. [0051] (6) Effect of Aspergillus ceramidase on the degradation of PBSA emulsion
- PBSA degradation of purified ceramidase preparation was examined. Ceramidase freeze-dried powder was dissolved in 50 mM Tris-HCl buffer (pH 8.5) and adjusted to 100 ⁇ g / ml. In addition, PBSA-degrading enzyme kojinase-derived cutinase was also dissolved in 50 mM Tris-HCl buffer (pH 8.5) to a concentration of 100 ⁇ g / ml. Add 100 ⁇ 1 of 0.15 (w / v) PBSA emulsion suspended in 50 mM Tris-HCl buffer (pH 8.5) to the wells of a 96-well microtiter plate, and add enzymes to each well under the following conditions. .
- a microtiter plate supplemented with a solution of each condition was kept at 45 ° C, and the degree of decomposition of the PBSA (molecular weight: 60,000) emulsion was detected at an absorbance of 630 nm every 10 minutes.
- the turbidity of each well before incubation was 100%, and the degradation degree of PBSA at each time was calculated (Fig. 4).
- condition 1 where no enzyme was added and condition 2 where only ceramidase was added, there was almost no degradation of the PBSA emulsion.
- PBSA degradation was observed under condition 3 where PBSA degrading enzyme cutinase was added.
- ceramidase a decrease in the peak appearing at the retention time of 22.7 minutes was observed with the addition of ceramidase (Fig. 5).
- the peak appearing at 22.7 minutes is an ester compound consisting of two molecules of 1,4 butanediol and one molecule of 1,6-hexamethylene diisocyanate (hereinafter referred to as “BHB” (FIG. 6) for convenience).
- BHB 1,6-hexamethylene diisocyanate
- Met. BHB contains two urethane bonds in its molecule. Since ceramidase has the effect of cleaving the amide bond in the ceramide molecule, ceramidase cleaves the structurally similar urethane bond of BHB.
- ceramidase The activity of ceramidase was performed by partially modifying the method described in Journal 'Ob' Biological 'Chemistry, Vol. 275, pp. 3462-3468 (2000). That is, 550 pmol of C12-NBD-ceramide (Avanti Polar Lipids, In) and an appropriate amount of ceramidase in a 25 mM Tris-HCl buffer (pH 7.5) of 20 1 at 37 ° C. Incubated for 30 minutes. The reaction was stopped by incubating the reaction in a boiling water bath for 5 minutes.
- ceramidase of the present invention it is possible to efficiently and economically use high molecular weight materials such as polyurethane and polyester, polypropylene, polyvinyl chloride, nylon, polystyrene, and polyurethane containing urethane bonds in any proportion, particularly biodegradable plastics. And it becomes possible to disassemble easily.
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Abstract
[PROBLEMS] To obtain a novel enzyme which is derived from a microorganism, particularly a mold showing a high growth capability on a plastic surface having an urethane bond and can break an urethane bond and/or an amide bond in a polymeric substance, and to provide a method for degradation of a plastic material having an urethane bond by using the enzyme. [MEANS FOR SOLVING PROBLEMS] Disclosed is a ceramidase comprising either of the proteins (a) and (b): (a) a protein comprising the amino acid sequence depicted in SEQ ID NO:1; and (b) a protein comprising an amino acid sequence having the deletion, substitution or addition of one or several amino acid residues in the amino acid sequence depicted in SEQ ID NO:1 and having a ceramidase activity.
Description
明 細 書 Specification
新規なセラミダーゼ及びその利用 Novel ceramidase and its use
技術分野 Technical field
[0001] 本発明は、新規なセラミダーゼ、該酵素をコードする DNA (遺伝子)、及び該酵素 を利用するプラスチック分解方法等に関する。 The present invention relates to a novel ceramidase, a DNA (gene) encoding the enzyme, a method for decomposing plastics using the enzyme, and the like.
背景技術 Background art
[0002] 資源の有効利用の為には、不溶性かつ疎水性のために難分解性であるプラスチッ クゃィ匕学繊維を分解してモノマーやオリゴマーとして回収再利用することが望まれる 。それらにはウレタン結合またはアミド結合が種々の割合で含まれている場合が多く 、回収再利用のためには効率良くウレタン結合またはアミド結合を切断することが必 要であり、そのような反応を触媒する酵素が切望されている。 [0002] For effective use of resources, it is desired to decompose and recycle the plastic fiber which is insoluble and hydrophobic because of its insolubility and is recovered as a monomer or oligomer. They often contain urethane bonds or amide bonds in various proportions, and it is necessary to efficiently cleave the urethane bonds or amide bonds for recovery and reuse. Enzymes that catalyze are anxious.
[0003] これまでにウレタン結合やアミド結合を含むプラスチックやィ匕学繊維を分解する微 生物の報告はある(非特許文献 1又は 2)。し力しながら、それらに記載された微生物 由来の酵素はウレタン結合部分を分解するものではなぐポリエステル部位ゃポリエ 一テル部位を分解するものである。 [0003] There have been reports of microorganisms that decompose plastics and fibers containing urethane bonds and amide bonds (Non-patent Document 1 or 2). However, the enzymes derived from microorganisms described in them do not degrade the urethane-binding moiety but degrade the polyester moiety or the polyester moiety.
[0004] プラスチック等の固体に直接作用してウレタン結合部位を直接切断する酵素は殆ど 知られていない。ウレタン結合を分解する細菌も極めて稀に報告されている力 その 殆どが低分子ウレタンの分解菌であり(特許文献 1〜5)。また、ロドコッカス属に属す るウレタン結合分解能を有する微生物に関する報告もあるが (特許文献 6)、該微生 物により実際に分解されているのは低分子量のウレタンィ匕合物である。 [0004] Few enzymes are known that act directly on solids such as plastics to directly cleave urethane binding sites. Bacteria that break down urethane bonds are reported very rarely, most of them are low-molecular-weight urethane-degrading bacteria (Patent Documents 1 to 5). In addition, there is a report on a microorganism having a urethane-binding ability belonging to the genus Rhodococcus (Patent Document 6), but it is a low molecular weight urethane compound that is actually degraded by the microorganism.
特干文献 1: Microbial degradation of polyurethane, polyester polyurethanes and p olyether polyurethanes. T. Nakajima— Kambe, Y. Shigeno— Akutsu, N. Nomura, F. On uma, T. Nakahara. Appl. Microbiol. Biotechnol. , 51, 134 - 140 (1995) NB 1: Microbial degradation of polyurethane, polyester polyurethanes and polyester polyurethanes. T. Nakajima— Kambe, Y. Shigeno— Akutsu, N. Nomura, F. On uma, T. Nakahara. Appl. Microbiol. Biotechnol. , 134-140 (1995)
非特許文献 2 : Biodegradation of polyurethane: a review, G. T. Howard. Int. Biodet. Biodeg., 49, 245-252 (2002) Non-Patent Document 2: Biodegradation of polyurethane: a review, G. T. Howard. Int. Biodet. Biodeg., 49, 245-252 (2002)
特許文献 1:特開平 01-240179 Patent Document 1: JP-A-01-240179
特許文献 2:特開平 01-300892
特許文献 3:特開平 03-175985 Patent Document 2: JP-A-01-300892 Patent Document 3: Japanese Patent Laid-Open No. 03-175985
特許文献 4:特開平 04-325079 Patent Document 4: JP 04-325079
特許文献 5:特開平 09-192633 Patent Document 5: JP-A 09-192633
特許文献 6:特開 2004-261103 Patent Document 6: JP-A-2004-261103
発明の開示 Disclosure of the invention
発明が解決しょうとする課題 Problems to be solved by the invention
[0005] そこで本発明者は、上記課題を解決すベぐウレタン結合を含有するプラスチックに 微生物のうち疎水性固体表面での生育能力の高いカビに由来する、高分子物質に 含まれるウレタン結合及び Z又はアミド結合を分解する新規な酵素を取得し、そのよ うな酵素を利用したプラスチックの分解方法を提供するものである。 [0005] Therefore, the present inventor has developed a urethane bond contained in a polymer substance derived from a mold having a high ability to grow on a hydrophobic solid surface among microorganisms, and a plastic containing a urethane bond that solves the above problems. A new enzyme that degrades Z or amide bonds is obtained, and a method for decomposing plastics using such an enzyme is provided.
課題を解決するための手段 Means for solving the problem
[0006] 即ち、本発明は以下の各態様に係るものである。 [0006] That is, the present invention relates to the following aspects.
[1] 以下の (a)または (b)の蛋白質であるセラミダーゼ: [1] The following ceramidase (a) or (b) protein:
(a)配列番号 1に示されるアミノ酸配列からなる蛋白質、 (a) a protein comprising the amino acid sequence represented by SEQ ID NO: 1,
(b)配列番号 1に示されるアミノ酸配列において 1もしくは数個のアミノ酸が欠失、置 換もしくは付加されたアミノ酸配列力もなり、かつセラミダーゼ活性を有する蛋白質。 (b) a protein having ceramidase activity, which also has an amino acid sequence ability in which one or several amino acids have been deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1.
[2] 以下の (a)または (b)の蛋白質をコードする DNA: [2] DNA encoding the following protein (a) or (b):
(a)配列番号 1に示されるアミノ酸配列からなる蛋白質、 (a) a protein comprising the amino acid sequence represented by SEQ ID NO: 1,
(b)配列番号 1に示されるアミノ酸配列において 1もしくは数個のアミノ酸が欠失、置 換もしくは付加されたアミノ酸配列力もなり、かつセラミダーゼ活性を有する蛋白質。 (b) a protein having ceramidase activity, which also has an amino acid sequence ability in which one or several amino acids have been deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1.
[3] 以下の (a)または (b)の DNA: [3] DNA of (a) or (b) below:
(a)配列番号 1に示される塩基配列からなる DNA、 (a) DNA consisting of the base sequence shown in SEQ ID NO: 1,
(b)配列番号 1に示される塩基配列力 なる DNAと相補的な塩基配列とストリンジ ン トな条件でハイブリダィズし、かつセラミダーゼ活性を有する蛋白質をコードする DNA (b) DNA that encodes a protein that hybridizes under stringent conditions with a complementary nucleotide sequence to the DNA having the nucleotide sequence shown in SEQ ID NO: 1 and that has ceramidase activity
[4] cDNAである態様 2または 3記載の DNA。 [4] The DNA according to embodiment 2 or 3, which is cDNA.
[5] 態様 2〜4の!、ずれか一項に記載の DNAを含む組換え用 DNA。 [5] A DNA for recombination comprising the DNA according to any one of embodiments 2 to 4!
[6] 組換え用 DNAがプラスミドベクターである、態様 5記載の組換え用 DNA。
[7] 態様 5叉は 6記載の組換え用 DNAを有する形質転換体。 [6] The recombination DNA according to embodiment 5, wherein the recombination DNA is a plasmid vector. [7] A transformant having the DNA for recombination according to embodiment 5 or 6.
[8]形質転換体の宿主が原核微生物叉は真核微生物であることを特徴とする、態様 [8] A mode in which the host of the transformant is a prokaryotic microorganism or a eukaryotic microorganism
7記載の形質転換体。 7. The transformant according to 7.
[9]原核微生物がェシェリシァ属、バチルス属、叉はストレプトマイセス属であることを 特徴とする、態様 8記載の形質転換体。 [9] The transformant according to embodiment 8, wherein the prokaryotic microorganism is of the genus Escherichia, Bacillus, or Streptomyces.
「10」真核微生物が、酵母、叉は、ァスペルギルス属、ぺニシリウム属、トリコデルマ属 、リズプス属、メタリチウム属、アクレモニゥム属、及びムコール属カもなる群力 選択 される真核糸状菌であることを特徴とする、態様 8記載の形質転換体 `` 10 '' eukaryotic microorganism is a yeast, or eukaryotic filamentous fungus selected from the group of Aspergillus, Penicillium, Trichoderma, Rhypus, Metalithium, Acremonium, and Mucor The transformant according to aspect 8, characterized in that
[11] 宿主がァスペルギルス ·オリゼ叉はァスペルギルス ·ソーェであることを特徴と する、態様 10記載の形質転換体。 [11] The transformant according to embodiment 10, wherein the host is Aspergillus oryzae or Aspergillus soe.
[12] 更にエステラーゼをコードする DNAを含む組換え用 DNAを有する、態様 7〜 11の 、ずれか一項に記載の形質転換体。 [12] The transformant according to any one of embodiments 7 to 11, further comprising a recombination DNA containing a DNA encoding an esterase.
[13] エステラーゼがリパーゼまたはクチナーゼである、態様 12記載の形質転換体 [13] The transformant according to embodiment 12, wherein the esterase is lipase or cutinase
[14] 態様 7〜13のいずれか一項に記載の形質転換体を培地に培養し、培養物か らセラミダーゼを採取することを含む、セラミダーゼの製造法。 [14] A method for producing ceramidase, comprising culturing the transformant according to any one of embodiments 7 to 13 in a medium and collecting ceramidase from the culture.
[15] 態様 1記載のセラミダーゼを用いて高分子物質を分解する方法。 [15] A method for decomposing a polymer substance using the ceramidase according to embodiment 1.
[16] 態様 1記載のセラミダーゼを用いて高分子物質に含まれるウレタン結合及び [16] A urethane bond contained in the polymer substance using the ceramidase according to embodiment 1, and
Z又はアミド結合を分解する方法。 A method of breaking the Z or amide bond.
[17] 態様 1記載のセラミダーゼとエステラーゼとの組み合わせを用いて高分子物 質を分解する方法。 [17] A method for degrading a polymer substance using the combination of ceramidase and esterase according to embodiment 1.
[18] エステラーゼがリパーゼまたはクチナーゼである、態様 16記載の方法。 [18] The method according to embodiment 16, wherein the esterase is lipase or cutinase.
[19] 態様 7〜13のいずれか一項に記載の形質転換体を高分子物質に接触させる ことを含む、高分子物質を分解する方法。 [19] A method for decomposing a polymer material, comprising contacting the transformant according to any one of Embodiments 7 to 13 with the polymer material.
[20] 高分子物質がポリウレタン、任意の割合でウレタン結合を含むポリエステル、 ポリプロピレン、ポリ塩化ビニール、ナイロン、ポリスチレン、デンプン、及びそれらの 混合物から成る群から選択されることを特徴と刷る、態様 15〜 19のいずれか一項に 記載の方法。
[21] 高分子物質が生分解性プラスチックであることを特徴とする、態様 15〜19の V、ずれか一項に記載の方法。 [20] The embodiment is characterized in that the polymeric material is selected from the group consisting of polyurethane, polyester containing urethane bonds in any proportion, polypropylene, polyvinyl chloride, nylon, polystyrene, starch, and mixtures thereof, Embodiment 15 The method as described in any one of -19. [21] The method according to any one of aspects 15 to 19, wherein the polymer substance is a biodegradable plastic.
[22] 生分解性プラスチックがポリ乳酸、ポリブチレンコハク酸、ポリブチレンコハク 酸'アジピン酸、脂肪族ポリエステル、ポリ力プロラタトン、叉はポリハイドロキシ酪酸で ある態様 21記載の方法。 [22] The method according to embodiment 21, wherein the biodegradable plastic is polylactic acid, polybutylene succinic acid, polybutylene succinic acid 'adipic acid, aliphatic polyester, polystrength prolatatone, or polyhydroxybutyric acid.
発明の効果 The invention's effect
[0007] 本発明により、数万もの分子量を有する高分子物質中のウレタン結合及び Z又は アミド結合を分解する新規なセラミダーゼが麹菌力 取得され、そのアミノ酸配列及 びそれをコードする DNAの塩基配列が特定され、該酵素を利用したプラスチックの 分解方法等が提供される。 [0007] According to the present invention, a novel ceramidase capable of decomposing a urethane bond and a Z or amide bond in a polymer substance having a molecular weight of several tens of thousands has been obtained as the koji mold, and its amino acid sequence and the DNA base sequence encoding it. And a method for decomposing plastics using the enzyme is provided.
図面の簡単な説明 Brief Description of Drawings
[0008] [図 1]疎水性カラムに対して強 、相互作用を示す麹菌由来のタンパク質の分離の様 子を示す SDS-PAGEの写真である。 [0008] FIG. 1 is a SDS-PAGE photograph showing the state of separation of Aspergillus oryzae-derived proteins that strongly interact with hydrophobic columns.
[図 2]セラミダーゼ高発現系の構築の概略を示す。 FIG. 2 shows an outline of the construction of a ceramidase high expression system.
[図 3]セラミダーゼ高発現麹菌株の培養上清を電気泳動したゲルの写真である。 FIG. 3 is a photograph of a gel obtained by electrophoresis of the culture supernatant of a strain with a high expression of ceramidase.
[図 4]セラミダーゼによる PBSの分解促進効果を示すグラフである。 FIG. 4 is a graph showing the effect of ceramidase on the degradation of PBS.
[図 5]セラミダーゼによる PBS分子内ウレタン結合の減少を示すグラフである。 FIG. 5 is a graph showing a decrease in PBS intramolecular urethane bonds by ceramidase.
[図 6]BHBの構造を示す。 [Figure 6] Shows the structure of BHB.
[図 7]セラミダーゼのセラミド蛍光基質の分解の様子を示すグラフである。 FIG. 7 is a graph showing a state of degradation of a ceramide fluorescent substrate of ceramidase.
発明を実施するための最良の形態 BEST MODE FOR CARRYING OUT THE INVENTION
[0009] 本発明にお ヽて、「セラミダーゼ」とは、ウレタン結合及び Z又はアミド結合を分解す る酵素を意味し、従って、本明細書において「セラミダーゼ活性」とは、セラミド、ブラ スチックや化学繊維等の物質に含まれるウレタン結合及び/又はアミド結合を特異 的に分解することができる活性を意味し、具体的には、本明細書中の実施例(7)に 記載されたウレタン結合分解、又は、実施例(8)に記載されたセラミド蛍光基質 (C12 -NBD-セラミド)の分解反応によって測定することが出来る。 [0009] In the present invention, "ceramidase" means an enzyme that degrades urethane bonds and Z or amide bonds. Therefore, in this specification, "ceramidase activity" means ceramide, plastic, It means activity capable of specifically decomposing urethane bonds and / or amide bonds contained in substances such as chemical fibers. Specifically, urethane bonds described in Example (7) of this specification It can be measured by decomposition or decomposition reaction of a ceramide fluorescent substrate (C12-NBD-ceramide) described in Example (8).
[0010] 本発明のセラミダーゼに関して、配列番号 1に示されるアミノ酸配列において、欠失 、置換又は付加されるアミノ酸は、好ましくは、同族アミノ酸 (極性 ·非極性アミノ酸、疎
水性'親水性アミノ酸、陽性'陰性荷電アミノ酸、芳香族アミノ酸など)同士が置換され る力、又は、アミノ酸の欠失若しくは付カ卩によって、蛋白質の三次元構造及び/又は 局所的電荷状態に大きな変化が生じない、又は、実質的にそれらが影響を受けない ようなものが好ましい。又、本発明蛋白質のセラミダーゼ活性が維持されるために、配 列番号 1に示されるアミノ酸配列からなるセラミダーゼに含まれる機能ドメイン内のアミ ノ酸は保持されることが望ましい。このような欠失、置換又は付加されるアミノ酸を有 する本発明のセラミダーゼは、例えば、部位特異的変異導入法 (点突然変異導入及 びカセット式変異導入等)、遺伝子相同組換え法、プライマー伸長法、及び PCR法 等の当業者に周知の方法を適宜組み合わせて、容易に作成することが可能である。 [0010] Regarding the ceramidase of the present invention, in the amino acid sequence shown in SEQ ID NO: 1, the amino acid to be deleted, substituted or added is preferably a homologous amino acid (polar / nonpolar amino acid, sparse Aqueous 'hydrophilic amino acids, positive' negatively charged amino acids, aromatic amino acids, etc.), or the loss or attachment of amino acids can greatly affect the three-dimensional structure and / or local charge state of proteins. Those that do not change or are substantially unaffected are preferred. In order to maintain the ceramidase activity of the protein of the present invention, it is desirable to retain the amino acid in the functional domain contained in the ceramidase consisting of the amino acid sequence represented by SEQ ID NO: 1. The ceramidase of the present invention having such a deleted, substituted or added amino acid includes, for example, site-specific mutagenesis (point mutagenesis, cassette mutagenesis, etc.), gene homologous recombination, primers It can be easily prepared by appropriately combining methods known to those skilled in the art such as extension method and PCR method.
[0011] 本明細書において、「ストリンジ ントな条件下」とは、各塩基配列間の相同性の程 度力 例えば、全体の平均で約 80%以上、好ましくは約 90%以上、より好ましくは約 95%以上であるような、高い相同性を有する塩基配列間のみで、特異的にハイプリ ッドが形成されるような条件を意味する。具体的には、例えば、温度 60°C〜68°Cに おいて、ナトリウム濃度 150〜900mM、好ましくは 600〜900mM、 pH 6〜8であるような 条件を挙げることが出来る。 [0011] In the present specification, "under stringent conditions" means the degree of homology between each base sequence, for example, about 80% or more, preferably about 90% or more, more preferably on the average on the whole. It means a condition in which a hybrid is specifically formed only between nucleotide sequences having high homology, such as about 95% or more. Specifically, for example, the conditions include a sodium concentration of 150 to 900 mM, preferably 600 to 900 mM, and a pH of 6 to 8 at a temperature of 60 ° C to 68 ° C.
ハイブリダィゼーシヨンは、例えば、カレント 'プロトコ一ノレズ'イン'モレキュラ^ ~ ·バイ ォロン■ ~" (Current protocols in molecular biology (.edited by Frederick M. Ausubel et al, 1987) )に記載の方法等、当業界で公知の方法あるいはそれに準じる方法に従 つて行なうことができる。また、市販のライブラリーを使用する場合、添付の使用説明 書に記載の方法に従って行なうことができる。 Hybridization is, for example, a method described in Current Protocol in Molecular Biology (.edited by Frederick M. Ausubel et al, 1987). In the case of using a commercially available library, it can be carried out according to the method described in the attached instruction manual.
[0012] 本発明 DNAは当業者に公知の方法で調製することが出来る。例えば、実施例に 記載されたァスペルギルス ·ォリゼ (Aspergillus oryzae) RIB40株(独立行政法人酒類 総合研究所:東広島巿鏡山三丁目 7番 1号、において同番号にて保存され、分譲可 能)、又は、市販されているその他の麹菌等の糸状菌の適当な株力 実施例で記載 した方法によって容易にクローユングすることが出来る。或は、本明細書に記載され た本発明 DNAの塩基配列又はアミノ酸産配列の情報に基づき、当業者に周知の化 学合成、又は、本発明のプライマーを使用した PCRにより増幅して調製することも出 来る。従って、本発明の DNAは、ゲノム DNA, cDNA及び合成 DNA等の当業者に
公知の任意の種類であり得る。 [0012] The DNA of the present invention can be prepared by methods known to those skilled in the art. For example, the Aspergillus oryzae RIB40 strain described in the Examples (independent administrative corporation Liquor Research Institute: 3-1, No. 7 Higashihiroshima Kagamiyama, can be stored under the same number and can be distributed) Alternatively, other commercially available filamentous fungi such as Neisseria gonorrhoeae can be easily cloned by the method described in the Examples. Alternatively, based on the information on the base sequence or amino acid production sequence of the DNA of the present invention described in the present specification, it is prepared by chemical synthesis well known to those skilled in the art or by amplification using PCR using the primer of the present invention. Things also come up. Therefore, the DNA of the present invention can be obtained by those skilled in the art such as genomic DNA, cDNA and synthetic DNA. It can be of any known type.
[0013] 当業者に周知の任意の方法に従い、本発明のセラミダーゼをコードする DNAを、 プラスミドベクター、ファージベクター、及び各種の混成ベクター等の適当な組換え用 DNAに挿入し、こうして得られた発現ベクターを用いて各種の細胞を形質転換する ことができる。この組換え用 DNAは、組換え DNA手法によって取り扱うことが可能な 任意のベクターである。これらのベクターは、その導入すべき宿主細胞に依存して適 当に選択することが出来る。該ベクターは、宿主細胞の中に導入する際に、宿主細 胞のゲノムの中にその全体あるいはその一部がゲノム中の 1箇所以上に糸且込まれる ことができる。このようなベクターの一例として大腸菌宿主用の pET- 12Bbや pAURlOl 、及び、麹菌宿主用の PNEN142等を挙げることができる。 [0013] According to an arbitrary method known to those skilled in the art, the DNA encoding the ceramidase of the present invention was inserted into an appropriate DNA for recombination such as a plasmid vector, a phage vector, and various hybrid vectors, and thus obtained. Various cells can be transformed with the expression vector. This recombination DNA is any vector that can be handled by recombinant DNA techniques. These vectors can be appropriately selected depending on the host cell to be introduced. When the vector is introduced into a host cell, the whole or a part of the vector can be inserted into one or more places in the genome of the host cell. Examples of such vectors include pET-12Bb and pAURlOl for E. coli hosts and PNEN142 for gonococcal hosts.
[0014] 本発明の発現ベクターには、典型的には、当業者に公知の、各種プロモーター、ェ ンハンサー及びサイレンサー等の各種調節配列、リボソーム結合部位、シグナル配 列、および翻訳開始配列等の各種要素ならびにその他の外来性あるいは内在性タ ンパク質をコードする遺伝子、各種薬剤耐性遺伝子、栄養要求性を相補する遺伝子 等を任意に含むことができる。 [0014] The expression vector of the present invention typically includes various regulatory sequences known to those skilled in the art, such as various regulatory sequences such as various promoters, enhancers and silencers, ribosome binding sites, signal sequences, and translation initiation sequences. It can optionally contain elements and other genes encoding exogenous or endogenous proteins, various drug resistance genes, genes that complement auxotrophy, and the like.
[0015] 本発明のセラミダーゼを発現させるに必要なプロモーターは、形質転換によって得 られた宿主細胞に依存するが、選択した宿主細胞において、転写活性を有し、かつ 宿主細胞に対して相同的または異種的であるタンパク質をコードする遺伝子に由来 することができる任意の DNA配列であることができる。尚、これら形質転換体におい ては各物質の生産誘導の抑制が解除されていることが望ましい。その為には、例え ば、本発明のセラミダーゼをコードする遺伝子を構成的発現プロモーター又は各種 の誘導型発現プロ一ター等の制御下で発現させることができる。その結果、セラミダ 一ゼが高発現され、細胞表面又は菌体外に多量に生産されプラスチック分解が促進 され、更に、有用物質の生産が促進される。 [0015] The promoter required to express the ceramidase of the present invention depends on the host cell obtained by transformation, but has transcription activity in the selected host cell and is homologous to the host cell or It can be any DNA sequence that can be derived from a gene encoding a protein that is heterologous. In these transformants, it is desirable that the suppression of production induction of each substance is released. For this purpose, for example, the gene encoding the ceramidase of the present invention can be expressed under the control of a constitutive expression promoter or various inducible expression promoters. As a result, ceramidase is highly expressed, and is produced in a large amount on the cell surface or outside the cells, thereby promoting plastic degradation and further promoting production of useful substances.
[0016] 本発明においてセラミダーゼをコードする DNAを含むベクターによって形質転換さ れる宿主細胞として、原核微生物、真核微生物、植物細胞、昆虫細胞、卵を含む鳥 類細胞、哺乳類細胞等を用いることができる。たとえば、原核微生物の例としてはェ シエリヒア属、バチルス属、又は、ストレプトマイセス'グリセウス若しくはストレプトコッカ
ス ·セリカラー等のストレブトマイセス属を宿主とすることができる。真核生物としては、 サッカロミセス属及びピヒア属等の酵母、ァスペルギルス ·ォリゼ及びァスペルギルス 'ソーェ等のァスペルギルス属、ぺニシリウム属、リゾプス属、メタリチウム属、モナスカ ス属、アクレモニゥム属、及びムコール属等の糸状菌、並びに、トリコデルマ属等の担 子菌など力も選択することができる。昆虫細胞としてはキイ口ショウジヨウバエ、カイコ 等の細胞を用いることができる。 [0016] In the present invention, a prokaryotic microorganism, a eukaryotic microorganism, a plant cell, an insect cell, an avian cell containing an egg, a mammalian cell, or the like may be used as a host cell transformed with a vector containing a DNA encoding ceramidase. it can. For example, examples of prokaryotic microorganisms include Escherichia, Bacillus, or Streptomyces griseus or Streptococcus. The genus Streptomyces such as Sericolor can be used as a host. Examples of eukaryotes include yeasts such as Saccharomyces and Pichia, Aspergillus oryzae and Aspergillus sau, etc. The ability of filamentous fungi and basidiomycetes such as Trichoderma can also be selected. Examples of insect cells that can be used include Drosophila melanogaster and silkworm.
[0017] 従って、例えば、原核微生物を宿主とする本発明のクチナーゼ変異体をコードする 遺伝子の転写を調節する適当なプロモーターの例として、大腸菌ェシエリシァ'コリの lacプロモータ、バシルス'リケニフォルミスの α -アミラーゼ遺伝子(amyL)のプロモー タ、バシルス 'アミ口リクファシエンスの α -アミラーゼ遺伝子(amyQ)のプロモータを挙 げることができる。 Therefore, for example, as an example of a suitable promoter that regulates the transcription of the gene encoding the cutinase mutant of the present invention hosted by a prokaryotic microorganism, the lac promoter of Escherichia coli Escherichia coli, the α-amylase of Bacillus licheniformis The promoter of the gene (amyL), the promoter of the α-amylase gene (amyQ) of Bacillus' Amiguchi liqufaciens.
[0018] また、真核微生物を宿主とした際に用いることのできるプロモーターの例として、サ ッカロミセス ·セリピシェのガラクトシダーゼ遺伝子、ァスペルギルス ·ォリゼのタカアミ ラーゼ遺伝子、エノラーゼ遺伝子、キシラナーゼ遺伝子、ホスホダルコキナーゼ遺伝 子、ダルコアミラーゼ遺伝子、リゾムコール.ミエハイのァスパラギン酸プロテアーゼ遺 伝子、ァスペルギルス' -ガーのダルコアミラーゼ遺伝子、リゾムコール'ミエハイのリ パーゼ遺伝子などのプロモーターを挙げることができる。 [0018] Examples of promoters that can be used when a eukaryotic microorganism is used as a host include Saccharomyces cerevisiae galactosidase gene, Aspergillus oryzae takaamylase gene, enolase gene, xylanase gene, phosphodarcokinase gene Examples include promoters such as the Dalcoamylase gene, Rhizomucor. Myehai aspartic protease gene, Aspergillus'-Gar's darcoamylase gene, and Rhizomucor's myehai lipase gene.
[0019] 本発明の DN Aを含む発現ベクター(組換え用 DNA)は、例えば、塩化カルシウム 法、プロトプラスト- PEG法、エレクト口ポレーシヨン法、 Tiプラスミド法、パーテイクルガ ン法、バキュロウィルス法などの公知の任意の方法によって宿主細胞へと導入でき、 形質転換体を作成することができる。更に、複数種の組換え DNAを用いるコトランス フエクシヨン法によっても可能である。 [0019] The expression vector (DNA for recombination) containing the DNA of the present invention includes, for example, calcium chloride method, protoplast-PEG method, electopore position method, Ti plasmid method, particle gun method, baculovirus method, etc. It can be introduced into a host cell by any known method, and a transformant can be produced. Furthermore, the co-transformation method using multiple types of recombinant DNA is also possible.
[0020] 尚、本発明に力かる形質転換体は、上記ベクターのほかに、任意の外来性あるい は内在性タンパク質をコードする遺伝子を含む別の 1つまたはそれ以上の組換え用 DNAによって形質転換されることができる。このような遺伝子の例として、国際公開 番号 WO2004/038016A1パンフレットに記載されているようなクチナーゼ及びリパーゼ に代表される各種エステラーゼを挙げることが出来る。 [0020] In addition to the above vector, the transformant useful in the present invention is obtained by another one or more recombination DNAs containing a gene encoding any exogenous or endogenous protein. Can be transformed. Examples of such genes include various esterases represented by cutinase and lipase as described in International Publication No. WO2004 / 038016A1 pamphlet.
[0021] 上記発現ベクターの代わりに、 PCR増幅等により取得される本発明のセラミダーゼ
をコードする遺伝子を含む適当な DNA断片自体を用いて本発明の形質転換体を得 ることも可能である。そのような場合には、カゝかる DNA断片にカロえてさらに適当な緩 衝液及びその他の助剤を任意に含む溶液等の組成物として形質転換に使用するこ とがでさる。 [0021] The ceramidase of the present invention obtained by PCR amplification or the like instead of the above expression vector It is also possible to obtain the transformant of the present invention by using an appropriate DNA fragment itself containing a gene encoding. In such a case, it can be used for transformation as a composition such as a solution containing an appropriate buffer solution and other auxiliary agents in addition to the DNA fragment to be obtained.
[0022] 本発明のセラミダーゼは、該酵素をコードする DNAを有する形質転換された宿主 細胞 (形質得転換体)をセラミダーゼの生産に好ま Uヽ条件で培養し、該変異体を発 現させ、好ましくは、発現させた変異体を細胞外に分泌させ、その宿主細胞および Z または培地から回収することにより製造することができる。宿主細胞の培養に用いる 培地は、当業者に公知である任意の培地の中から、本発明の形質転換された宿主 細胞を増殖させ、本発明のセラミダーゼを発現させるために適当なものを適宜選択 することができる。 [0022] The ceramidase of the present invention is preferably used for the production of ceramidase in a transformed host cell (transformant) having DNA encoding the enzyme, and cultured under U ヽ conditions to express the mutant. Preferably, it can be produced by secreting the expressed mutant extracellularly and recovering it from its host cell and Z or medium. The medium used for culturing the host cells is appropriately selected from any medium known to those skilled in the art, which is suitable for growing the transformed host cells of the present invention and expressing the ceramidase of the present invention. can do.
[0023] 宿主細胞力 分泌されたセラミダーゼは、当業者に公知の任意の手段の適当な組 み合わせ、例えば、遠心または濾過による培地と細胞の分離、および硫酸アンモ-ゥ ムの様な塩による培地のタンパク質成分の沈殿、及びこれに続く疎水クロマトグラフィ 一、イオン交換クロマトグラフィー、ァフィ-ティークロマトグラフィー、又はその他のク 口マトグラフィ一の使用により培地から回収することができる。 [0023] Host cell force The secreted ceramidase is produced by a suitable combination of any means known to those skilled in the art, for example, separation of media and cells by centrifugation or filtration, and salts such as ammonium sulfate. It can be recovered from the medium by precipitation of the protein components of the medium, followed by the use of hydrophobic chromatography, ion exchange chromatography, affinity chromatography, or other chromatographic techniques.
[0024] 本発明のセラミダーゼは、ウレタン結合及び Z又はアミド結合を特異的に分解する 活性を有して ヽるので、ポリウレタン及び任意の割合でウレタン結合を含むポリエステ ル、ポリプロピレン、ポリ塩化ビニール、ナイロン、ポリスチレン、及びデンプン等の高 分子物質の分解に有利に使用することができる。 [0024] Since the ceramidase of the present invention has an activity of specifically decomposing a urethane bond and a Z or amide bond, polyurethane and polyester, polypropylene, polyvinyl chloride, polyurethane containing an urethane bond in an arbitrary ratio, It can be advantageously used to decompose high molecular weight materials such as nylon, polystyrene, and starch.
[0025] 高分子物質の好適例として、生分解性プラスチックを挙げることが出来る。一般に、 「生分解性プラスチック」とは、「使用状態ではその使用目的において必要とされる充 分な機能を保ち、廃棄されたときには土中又は水中の微生物の働きによって、より単 純な分子レベルにまで分解されるプラスチック」ともいうべき物質である。分解の程度 に基づ!/ヽて、「完全分解型生分解性プラスチック」と「部分分解 (崩壊)型生分解性プ ラスチック」とに分けられ、更に、材料及び製造方法力もは、「微生物生産系」、「天然 高分子系」及び「ィ匕学合成系」に大きく分けることが出来る。本発明方法では、これら のいずれの種類の生分解性プラスチックも使用することが出来る。
[0026] 本発明方法で分解される生分解性プラスチックの代表的例として、ポリ乳酸、ポリブ チレンコハク酸(PBS)、ポリブチレンコハク酸'アジピン酸(PBSA)、脂肪属ポリエステ ル、ポリ力プロラタトン、叉はポリハイドロキシ酪酸を挙げることが出来る。これらの物質 は通常、数万の分子量を有するものである。 [0025] Preferable examples of the polymer substance include biodegradable plastics. In general, “biodegradable plastics” means “a simpler molecular level that retains the sufficient functions required for its intended use in the state of use, and when it is disposed of, by the action of microorganisms in the soil or water. It is a substance that should be called “plastic that can be broken down to Based on the degree of degradation! / Furthermore, it can be divided into “completely degradable biodegradable plastics” and “partially degradable (collapsed) biodegradable plastics”. It can be broadly divided into “production system”, “natural polymer system”, and “chemical synthesis system”. Any of these types of biodegradable plastics can be used in the method of the present invention. [0026] Representative examples of biodegradable plastics that are decomposed by the method of the present invention include polylactic acid, polybutylene succinic acid (PBS), polybutylene succinic acid 'adipic acid (PBSA), fatty polyester, polystrength prolatathone, For example, polyhydroxybutyric acid can be mentioned. These materials usually have a molecular weight of tens of thousands.
[0027] 更に、国際公開番号 WO2004/038016A1パンフレットに記載されているような、クチ ナーゼ及びリパーゼに代表される各種エステラーゼを本発明のセラミダーゼと組み 合わせて生分解性プラスチック等の高分子物質をより効率的、高収率で分解すること が可能となる。 [0027] Furthermore, various esterases typified by cutinase and lipase as described in the pamphlet of International Publication No. WO2004 / 038016A1 are combined with the ceramidase of the present invention to obtain a polymer substance such as a biodegradable plastic. It is possible to decompose efficiently and with high yield.
[0028] 上記方法において、プラスチックの分解反応は、目的 ·規模などに応じて、当業者 に公知の任意の反応系(例えば、水溶系及び固相系)及び反応条件 (溶媒、培地、 温度及び pHなど)を適宜選択して実施することが出来る。 [0028] In the above method, the plastic decomposition reaction may be carried out by any reaction system known to those skilled in the art (for example, aqueous and solid phase systems) and reaction conditions (solvent, medium, temperature and pH etc.) can be selected as appropriate.
[0029] 生分解性プラスチック等の高分子物質を本発明方法で加水分解する際に用いられ る溶媒としては、水、緩衝液、有機溶媒と水、あるいは、有機溶媒と緩衝液の混合物 が挙げられる。これらの溶媒には、必要に応じ、界面活性剤、有機物、無機物等を添 加することができる。溶媒には、 pHを安定させ、加水分解効率を向上させるために、 緩衝剤を添加することが好ましい。溶媒の pHは、好ましくは 6— 10、より好ましくは 7 9の範囲に維持する。 [0029] Examples of the solvent used when hydrolyzing a polymer substance such as a biodegradable plastic by the method of the present invention include water, a buffer solution, an organic solvent and water, or a mixture of an organic solvent and a buffer solution. It is done. A surfactant, an organic substance, an inorganic substance, or the like can be added to these solvents as necessary. It is preferable to add a buffering agent to the solvent in order to stabilize the pH and improve the hydrolysis efficiency. The pH of the solvent is preferably maintained in the range 6-10, more preferably 79.
[0030] 本発明の分解方法において、本発明のセラミダーゼ、又は、それと上記エステラー ゼを培養系に共存させることにより、プラスチックを分解することが出来る。これらの物 質は微生物自体により産生されたものでも良いし、それらに加えて、更に培養系の外 部より添加することが可能である。添加の量及び割合'添加の時期等の諸条件は当 業者が適宜選択することが出来る。これらの物質は必ずしも同時に添加する必要は なぐ方法の各段階で逐次的に添加することも可能である。 [0030] In the decomposition method of the present invention, the plastic can be decomposed by allowing the ceramidase of the present invention or the esterase to coexist in the culture system. These substances may be those produced by the microorganisms themselves, and in addition to them, they can be added from outside the culture system. The amount and ratio of addition and various conditions such as the timing of addition can be appropriately selected by those skilled in the art. These substances need not be added at the same time, but may be added sequentially at each stage of the process.
[0031] 或 、は、本発明の分解方法にお!、て、上記の形質転換体の培養系に分解した!/ヽ 高分子物質に共存させ、形質転換体を該物質に接触させることによってプラスチック を分解することが可能である。この方法は、例えば、プラスチック乳化液を含む液体 培養系及びプラスチック固型ペレット又はプラスチック粉体等を使用する固体培養系 等の当業者に公知の任意の培養系にお 、て行うことが出来る。
[0032] 本発明方法にぉ 、て、セラミダーゼ及びエステラーゼをコードする遺伝子を夫々別 の微生物に導入して、こうして得られた複数種類の形質転換体を用いて反応させるこ ともできる。尚、本発明の形質転換体において導入される、これら酵素をコードする遺 伝子は一種類とは限らず、夫々、複数種類の酵素遺伝子で組換えることも可能であ る。複数種類の微生物を使用する場合には、これら複数種類の微生物を同時に培養 する共培養系で実施したり、又は、本発明方法が複数の連続的な培養段階から構成 されており、その各段階において、夫々前の段階で得られた培養液に更に異なる微 生物を逐次的に作用させて処理することも可能である。 Alternatively, in the degradation method of the present invention, the above-described transformant was decomposed into a culture system! / ヽ coexisted with a high molecular weight substance, and the transformant was brought into contact with the substance. It is possible to disassemble the plastic. This method can be carried out in any culture system known to those skilled in the art such as a liquid culture system containing a plastic emulsion and a solid culture system using a plastic solid pellet or plastic powder. [0032] According to the method of the present invention, the genes encoding ceramidase and esterase can be introduced into different microorganisms, and the reaction can be carried out using the plural types of transformants thus obtained. It should be noted that the genes encoding these enzymes introduced into the transformant of the present invention are not limited to one type, and each can be recombined with a plurality of types of enzyme genes. When multiple types of microorganisms are used, it is carried out in a co-culture system in which these multiple types of microorganisms are cultured at the same time, or the method of the present invention is composed of a plurality of continuous culture stages. However, it is also possible to sequentially treat different microorganisms on the culture solution obtained in the previous step.
[0033] 上記反応における、形質転換体の培養条件 (培地、温度及び pHなど)は、当業者 に公知の条件力 宿主の種類等に応じて適宜選択することが出来る。更に、上記国 際公開に開示されているような、当業者に公知の任意の界面活性物質若しくはバイ ォサーファクタントを、例えば外部より添加することによって反応系に共存させ、それ らの存在下で高分子物質の分解を更に促進することも可能である。 [0033] The culture conditions (medium, temperature, pH, etc.) of the transformant in the above reaction can be appropriately selected depending on the condition of the host known to those skilled in the art. Furthermore, any surfactant or biosurfactant known to those skilled in the art, such as disclosed in the above-mentioned international publication, is added to the reaction system by, for example, adding it from the outside, It is also possible to further accelerate the decomposition of molecular substances.
[0034] 本発明の分解方法によって、高分子物質が分解され、可溶化した構成成分モノマ 一若しくはそれらが複数個重合したオリゴマー、又は、それらの塩を回収することがで きる。ここで、構成成分モノマーとは、ポリ乳酸、ポリブチレンサクシネート、ポリブチレ ンサクシネートーコーアジペート、ポリヒドロキシブチレート、及び Z又は、ポリ力プロラ タトンを重縮合、共重合、開環重合等により合成する際の原料となるモノマーのことで あり、脂肪族カルボン酸、脂肪族ヒドロキシカルボン酸、脂肪族ジオール、脂肪族ジ カルボン酸、環状エステル等が含まれる。 [0034] According to the decomposition method of the present invention, it is possible to recover a constituent monomer that has been dissolved and solubilized by a polymer substance, an oligomer in which a plurality of them are polymerized, or a salt thereof. Here, the constituent monomer is synthesized by polycondensation, copolymerization, ring-opening polymerization, etc. of polylactic acid, polybutylene succinate, polybutylene succinate-coadipate, polyhydroxybutyrate, and Z or poly force prolacton. It is a monomer used as a raw material for this, and includes aliphatic carboxylic acids, aliphatic hydroxycarboxylic acids, aliphatic diols, aliphatic dicarboxylic acids, cyclic esters and the like.
[0035] 上記培養系において、上記の高分子物質は分解された可溶ィ匕した構成成分モノマ 一若しくはそれらが複数個重合したオリゴマー等の物質自体が形質転換体の唯一の 栄養源 (炭素源,窒素源)として消費されることもある。或いは、他の炭素源、窒素源 を別途、培養系に添加することも出来る。例えば、炭素源としてはグルコース等の各 種糖類、窒素源としては有機窒素源、例えばペプトン、肉エキス、酵母エキス、コーン 'スチープ'リカー等、無機窒素源、例えば硫酸アンモ-ゥム、塩ィ匕アンモ-ゥム等を 含有することができる。更に所望により、培地中には、ナトリウムイオン、カリウムイオン 、カルシウムイオン、マグネシウムイオン等の陽イオンと硫酸イオン、塩素イオン、リン
酸イオン等の陰イオンとからなる無機塩類を含まれていてもよい。さらに、ビタミン類、 核酸類等の微量要素を含有されることもできる。 [0035] In the above culture system, the above high molecular weight substance is a decomposed soluble monomer component or a material such as an oligomer obtained by polymerizing a plurality of them, and is the only nutrient source (carbon source) of the transformant. , Nitrogen source). Alternatively, other carbon sources and nitrogen sources can be added separately to the culture system. For example, various carbons such as glucose as a carbon source, organic nitrogen sources as a nitrogen source, such as peptone, meat extract, yeast extract, corn 'strip' liquor, etc., inorganic nitrogen sources such as ammonium sulfate, salt匕 Ammumum can be contained. Further, if desired, the medium may contain cations such as sodium ion, potassium ion, calcium ion, magnesium ion, sulfate ion, chlorine ion, phosphorus ion. Inorganic salts composed of anions such as acid ions may be included. Furthermore, trace elements such as vitamins and nucleic acids can be contained.
[0036] 更に、本発明のセラミダーゼを用いる全ての方法において、分解の対象となる高分 子物質は、所謂「複合材料」の一構成要素として含まれているものでもよい。ここで、 「 複合材料」とは一般的に 2種類以上の異なる物質力も構成されている材料であり、例 えば、プラスチックと各種金属及びその他の無機物質力 構成されて 、るものがある 。このような複合材料は各種産業素材として多方面で各種の目的に利用されている。 [0036] Furthermore, in all methods using the ceramidase of the present invention, the polymer substance to be decomposed may be contained as a constituent element of a so-called "composite material". Here, “composite material” is a material that is generally composed of two or more different material forces, for example, a material composed of plastic, various metals, and other inorganic material forces. Such composite materials are used for various purposes in various fields as various industrial materials.
[0037] 従って、上記の方法によって、例えば、プラスチックを含む複合材料から、プラスチ ック部分を選択的に分解して、プラスチックをモノマー又はオリゴマーとして回収し、 一方で、プラスチックを実質的に含まない金属等のその他の部分を回収することが出 来る。 [0037] Therefore, by the above-described method, for example, from a composite material containing plastic, the plastic portion is selectively decomposed to recover the plastic as a monomer or oligomer, while being substantially free of plastic. Other parts such as metal can be recovered.
実施例 Example
[0038] 以下、実施例に即して本発明を具体的に説明するが、本発明の技術的範囲はこれ らの記載によって何等制限されるものではない。尚、実施例における各種遺伝子操 作【ま、 Current protocols in molecular biology ^edited by Fredrick M. Aausubel et al. , 1987)に記載されている方法に従った。尚、以下の実施例で用いた麹菌ァスペルギ ルス 'ォリゼ (Aspergillus oryzae) RIB40株は、独立行政法人酒類総合研究所 (東広 島巿鏡山三丁目 7番 1号)より分与可能である。 [0038] Hereinafter, the present invention will be described in detail with reference to examples. However, the technical scope of the present invention is not limited by these descriptions. It should be noted that the methods described in various gene manipulations in the Examples (Current protocols in molecular biology ^ edited by Fredrick M. Aausubel et al., 1987) were followed. In addition, the Aspergillus oryzae RIB40 strain used in the following examples can be distributed from the Incorporated Administrative Agency, Liquor Research Institute (No. 7-1, Kagamiyama 3-chome, Higashihiroshima).
[0039] (1)麹菌による PBSA分解産物から疎水性担体吸着タンパク質の分離 [0039] (1) Separation of hydrophobic carrier-adsorbed protein from PBSA degradation products by Aspergillus oryzae
1 X 106個胞子 I mlとなるように麹菌ァスペルギルス'ォリゼ(Aspergillus oryzae) RIB 40株の胞子懸濁液を 3リットル容坂ロフラスコ内の 1 (w/v %) PBSA乳化液を唯一の 炭素源とした Czapek-Dox培地 1リットルに接種した。 30°Cにて 7日間振盪培養し、 その培養液を MIRACROTH (CALBIOCHEM (商品登録) )で濾過し菌体を除 、た。 その濾液を 20 %の硫酸アンモ-ゥムで飽和させ 1時間氷冷し、 10,000 X g、 4°Cにて 3 0分間遠心分離による沈殿を除去した。その上清を 10 mM Tris-HCl緩衝液、 pH 8. 0、 20 %飽和硫酸アンモ-ゥムで飽和させた Octy卜 cellulofine type- S (生化学工業) に供した後、 1リットルの精製水でカラムを洗浄した。洗浄後のカラムを 0-70 %ェタノ ールの直線勾配で溶出した。
[0040] 各溶出フラクション 0.8 mlに 0.2 mlの 100 % (w/v) トリクロ口酢酸をカ卩え、よく混合 した後、氷中に 20分間静置した。これらのサンプルを 15,000 X g、 4。C、 20分間遠 心分離し、上清を完全に除いた後、 15 1の SDS化溶液 { 5 % (w/v) SDS、 5 % (v/v) 2-メルカプトエタノール、 0.12 M Tris-塩酸緩衝液(pH 8.8)、 0.05 % (w/v)ブロモフ ェノールブルー、 10 % (w/v)グリセロール }に溶解させた。これらのサンプルを沸騰湯 浴中で 5分間煮沸し、 SDS-PAGEに供した。 SDS- PAGE後のゲルはシルベストステ イン、 PAGE蛋白質用(ナカライテスタ)を用いて行い、方法は説明書に記載されてい る方法に基づいて行った。溶出画分中に約 100 kDaのタンパク質が検出された(図 1)。以後の SDS-PAGEと PVDF膜へのブロッテイングの方法は Schaggerら (Schagge r, H., et al. (1987) Anal. Biochem. , 166, 368- 379)の方法にしたがった。泳動板は、 1 60mm X 160mm, lmm厚のものを使用し、電流 10mA—定で泳動を開始しサンプル のグリセロール前線が泳動板の先端まで泳動したところで電気泳動を止めた。 SDS-PSpore suspension of Aspergillus oryzae RIB 40 strain to 1 X 10 6 spores I ml of 1 (w / v%) PBSA emulsion in a 3 liter volumetric flask 1 liter of Czapek-Dox medium as a source was inoculated. The culture was shaken at 30 ° C for 7 days, and the culture was filtered with MIRACROTH (CALBIOCHEM (registered trademark)) to remove the cells. The filtrate was saturated with 20% ammonium sulfate and ice-cooled for 1 hour, and the precipitate was removed by centrifugation at 10,000 × g and 4 ° C. for 30 minutes. The supernatant was subjected to Octy 卜 cellulofine type-S (Seikagaku Corporation) saturated with 10 mM Tris-HCl buffer, pH 8.0, 20% saturated ammonium sulfate, and then 1 liter of purified water. The column was washed with. The washed column was eluted with a linear gradient of 0-70% ethanol. [0040] 0.2 ml of 100% (w / v) triclonal acetic acid was added to 0.8 ml of each elution fraction, mixed well, and then allowed to stand in ice for 20 minutes. These samples are 15,000 Xg, 4. C, centrifuge for 20 minutes, remove the supernatant completely, and then remove the 15 1 SDS solution {5% (w / v) SDS, 5% (v / v) 2-mercaptoethanol, 0.12 M Tris- It was dissolved in a hydrochloric acid buffer (pH 8.8), 0.05% (w / v) bromophenol blue, 10% (w / v) glycerol}. These samples were boiled in boiling water bath for 5 minutes and subjected to SDS-PAGE. The gel after SDS-PAGE was performed using sylvest stain and PAGE protein (Nacalai Tester), and the method was performed based on the method described in the manual. A protein of about 100 kDa was detected in the eluted fraction (Fig. 1). Subsequent SDS-PAGE and blotting methods on PVDF membranes followed the method of Schagger et al. (Schagger, H., et al. (1987) Anal. Biochem., 166, 368-379). The electrophoresis plate used was 160 mm x 160 mm, lmm thick, and the electrophoresis was started at a current of 10 mA constant, and the electrophoresis was stopped when the glycerol front of the sample migrated to the tip of the electrophoresis plate. SDS-P
AGE泳動後ゲルないタンパク質を PVDF膜に転写し、 PVDF膜より目的タンパク質 のバンドを切り抜き、ァミノ末端領域のアミノ酸配列の解析を行った。その結果、 目的 タンパク質のァミノ末端領域のアミノ酸配列は「 ASDDSVFLLG」(配列番号 1のァミノ 酸配列における第 50〜59番目のアミノ酸に相当)であった。 After AGE migration, the protein without gel was transferred to a PVDF membrane, the target protein band was cut out from the PVDF membrane, and the amino acid sequence of the amino terminal region was analyzed. As a result, the amino acid sequence of the amino acid terminal region of the target protein was “ASDDSVFLLG” (corresponding to the 50th to 59th amino acids in the amino acid sequence of SEQ ID NO: 1).
[0041] 得られたアミノ酸配列をァスペルギルス.ォリゼゲノムデータベースの BLAST検索 を利用して相同検索を行ったところ完全一致した配列が取得できた。得られた配列 力も分子量が l OOkDaのタンパク質の全長アミノ酸配列を推定し、 日本 DNAデータバ ンク(http:〃 www.ddbj.nig.a jp/Welcome- j.html)にて相同性検索を行った結果、キ イロショウジヨウバエ、ドロソフイラ'メラノガスター(Drosophila melanogaster)の中性セ ラミダーゼ(BAC77635.1)と 36%、細胞性粘菌、デクテオステリゥム.デスコィディウム( Dictyostelium discoideum)の中性セラミダーゼ(AAB69633.1)と 36%、そしてゼブラフ イツシュ、ダニォ'レリオ(Danio rerio)の中性セラミダーゼ(BAD69590.1)と 36%の相 同性を示したことから、本タンパク質は中性セラミダーゼであることが示唆された。 [0041] When the obtained amino acid sequence was subjected to a homologous search using a BLAST search of the Aspergillus oryzae genome database, a completely matched sequence was obtained. The obtained sequence force was also estimated from the full-length amino acid sequence of a protein with a molecular weight of lOOkDa, and homology search was performed using the Japan DNA data bank (http: 〃 www.ddbj.nig.a jp / Welcome-e.html). As a result, Drosophila melanogaster neutral ceramidase (BAC77635.1) and 36%, cellular slime mold, Dictyostelium discoideum neutral ceramidase ( AAB69633.1) and 36%, and zebrafish, Danio rerio neutral ceramidase (BAD69590.1) and 36% homology, indicating that this protein is a neutral ceramidase. It was suggested.
[0042] (2)麹菌による高発現用ベクターの構築 [0042] (2) Construction of vector for high expression by Aspergillus oryzae
クロー-ングされた目的遺伝子の周辺塩基配列を参考に一組のオリゴヌクレオチド を設計した(配列番号 2: 5 ' - GTTGCGCACGtgTTCTAATGTCG- 3,、 配列番号 3: 5
GCGATGTCTAgATCCCCGAGTCC- 3,)。ァスペルギルス'ォリゼ RIB40株のゲノ ム DNAをテンプレートとして PCR反応を行った。増幅反応は、 95°C、 3分間铸型 DN Aを変性し、 95°C、 1分間、 55°C、 1分間、 72°C, 3分間保持するサイクルを 30サイ クル行った後、 72°C、 5分間で完全伸長させ、 4°Cで保持した。その PCRによる増幅 断片をァガロース電気泳動にて確認を行ったところ約 3,269塩基対の増幅が見られた A set of oligonucleotides was designed with reference to the base sequence around the cloned target gene (SEQ ID NO: 5 '-GTTGCGCACGtgTTCTAATGTCG-3, SEQ ID NO: 3: 5). GCGATGTCTAgATCCCCGAGTCC-3,). PCR reaction was performed using the Aspergillus oryzae RIB40 genomic DNA as a template. The amplification reaction was carried out after 30 cycles of denaturation of vertical DNA at 95 ° C for 3 minutes and holding at 95 ° C for 1 minute, 55 ° C for 1 minute, 72 ° C for 3 minutes. Fully extended at 5 ° C for 5 minutes and kept at 4 ° C. When the amplified fragment was confirmed by agarose electrophoresis, amplification of about 3,269 base pairs was observed.
[0043] この増幅断片を PmaC I (宝酒造)及び Xba I (宝酒造)で消化し、その消化断片をァ ガロースゲル電気泳動に供したゲル中より prep A gene (BioRad)を用いて DNAを 抽出し、これを挿入 DNA断片とした。次に塩基配列中にダルコアミラーゼのプロモ 一ター配列(P- enoA142)を有するプラスミド pNEN142 DNA 5 ^ gを PmaC I及び Xb a Iで消化し、定法によりフエノール抽出、エタノール沈殿処理をおこなった後、アル力 リフォスファターゼ(宝酒造)により 5 '末端のリン酸を除去した。この反応液を定法に よりフエノール抽出、エタノール沈殿処理をおこなった後 TEに溶解したものをべクタ 一 DNA溶液とした。次に、ベクター DNA 1 μ gと挿入 DNA断片 1.5 gを T4 D NAライゲース(宝酒造)により連結させ、連結 DNA溶液を得た。 [0043] This amplified fragment was digested with PmaC I (Takara Shuzo) and Xba I (Takara Shuzo), and the digested fragment was subjected to agarose gel electrophoresis, and DNA was extracted using prep A gene (BioRad). This was used as an inserted DNA fragment. Next, the plasmid pNEN142 DNA 5 ^ g having the Dalcore mirase promoter sequence (P-enoA142) in the base sequence was digested with PmaC I and XbaI, and subjected to phenol extraction and ethanol precipitation treatment in the usual manner. Al force Liphosphatase (Takara Shuzo) removed the 5'-terminal phosphate. This reaction solution was subjected to phenol extraction and ethanol precipitation by a conventional method, and then dissolved in TE to obtain a vector-DNA solution. Next, 1 μg of vector DNA and 1.5 g of the inserted DNA fragment were ligated with T4 DNA ligase (Takara Shuzo) to obtain a ligated DNA solution.
[0044] 連結 DNA溶液 10 μ 1に 10 μ 1の 10 X KCM (1 M KC1、 0.3 M CaCl [0044] Ligated DNA solution 10 μ 1 to 10 μ 1 of 10 X KCM (1 M KC1, 0.3 M CaCl
2、 0.5 M MgCl 2, 0.5 M MgCl
2 2
)、 7 1の 30%ポリエチレングリコール(# 6000)、 73 1の滅菌水を加え、よく撹拌し た。これを氷中でよく冷却した後、氷上解凍したコンビテントセル (大腸菌 DH5 a株: T !^1¾\)を100 1加え穏やかに撹拌し、氷中で 20分間、続いて室温で 10分間放置 した。これに、 200 μ ΐの LB液体培地をカ卩え、 100 μ § / mlのアンピシリンを添加し た LB平板培地にまき、 37°Cで一晩培養した。 ), 71 1 30% polyethylene glycol (# 6000), 73 1 sterilized water was added and stirred well. After cooling this well in ice, add 100 1 viable cells (E. coli DH5 a strain: T! ^ 1¾ \) thawed on ice, gently agitate, leave in ice for 20 minutes, and then stand at room temperature for 10 minutes. did. This, 200 μ ΐ of LB liquid medium Ka卩E, plated on LB plate medium supplemented with 100 μ § / ml ampicillin, and cultured overnight at 37 ° C.
[0045] こうして目的のプラスミド DNAを用いて形質転換した大腸菌の単一のコロニーを 3 mlの 100 u g / mlのアンピシリンを添カ卩した LB液体培地に植菌し、 37°Cでー晚振盪 培養した。 1.5 mlの培養液を 2 mlのエツペンドルフチューブに移して 15,000 X gで 1分 間遠心分離し、沈殿を 100 μ 1の氷冷した TEG (25 mM Tris- HC1、 10 mM EDTA、 50 mM Glucose, pH 8.0)で懸濁し、これに 200 μ 1の 0.2 Ν NaOH- 1 % SDSを加えて穏 やかに撹拌した後、 150 1の 3 M NaOAc pH 5.2を加えて混合した。これを 15,000 X g、 4 °Cで 5分間遠心分離し上清を回収し 450 μ 1のフエノール'クロ口ホルム'イソァ
ミルアルコール (25 : 24: 1)をカ卩えて激しく撹拌した後、 15,000 X g、室温で 5分間遠心 分離し上層を回収した。この溶液に- 20 °Cで氷冷した 900 1のエタノールを加えて- 20 °Cで 10分間放置後、 15,000 X gで 4 °Cで 5分間遠心分離した。沈殿を 500 1の 70 o/oエタノールでリンスしたのち、乾燥させ、最後に RNase (100 g / ml)を含む TE (10 mM Tris- HC1、 1 mM EDTA、 pH 8.0) 50 μ 1に溶解した。得られたプラスミドを PmaCI と Xbalで切断後、ァガロース電気泳動により挿入断片の存在を認め、麹菌形質転換 用プラスミドである pNEN-Cerの完成を確認した(図 2)。このプラスミド中の挿入 DNA 断片を ABI PRISM™ 377 DNA sequencer Long Read (PE Biosystem)のプロトコ一 ルに従い、 ABI PRISM™ 377 DNA sequencing system (PE Biosystem)にて解析した [0045] A single colony of E. coli transformed with the desired plasmid DNA was inoculated into 3 ml of LB liquid medium supplemented with 100 ug / ml ampicillin and shaken at 37 ° C. Cultured. Transfer 1.5 ml of the culture to a 2 ml Eppendorf tube, centrifuge at 15,000 X g for 1 min, and precipitate with 100 μl ice-cold TEG (25 mM Tris-HC1, 10 mM EDTA, 50 mM) Glucose, pH 8.0) was suspended, 200 μl of 0.2 μ NaOH-1% SDS was added and gently stirred, and then 150 1 of 3 M NaOAc pH 5.2 was added and mixed. This was centrifuged at 15,000 X g for 5 minutes at 4 ° C, and the supernatant was collected. 450 μ1 phenol 'cloform form' Mill alcohol (25: 24: 1) was added and stirred vigorously, and then centrifuged at 15,000 X g for 5 minutes at room temperature to recover the upper layer. To this solution, 900 1 ethanol cooled at −20 ° C. was added and left at −20 ° C. for 10 minutes, followed by centrifugation at 15,000 × g for 5 minutes at 4 ° C. Rinse the precipitate with 500 1 70 o / o ethanol, dry, and finally dissolve in 50 μ 1 of TE (10 mM Tris-HC1, 1 mM EDTA, pH 8.0) containing RNase (100 g / ml) . The resulting plasmid was cleaved with PmaCI and Xbal, and the presence of the inserted fragment was confirmed by agarose electrophoresis, confirming the completion of pNEN-Cer, a plasmid for gonococcal transformation (Fig. 2). The inserted DNA fragment in this plasmid was analyzed by ABI PRISM ™ 377 DNA sequencing system (PE Biosystem) according to the ABI PRISM ™ 377 DNA sequencer Long Read (PE Biosystem) protocol.
[0046] (3)セラミダーゼ高発現麹菌の作成 [0046] (3) Preparation of gonococci with high expression of ceramidase
ァスペルギルス 'ォリゼの形質転換はプロトプラスト- PEG法を改良した方法を用い、 形質転換するプラスミド DNAは上記で作製した pNEN- Cer及び pNEN142を用いた。 これらのプラスミド DNA10 gを BamHIで完全消化し、定法によりフエノール抽出、ェ タノール沈澱処理を行った後 10 1の TEに溶解し形質転換用 DNA溶液とした。 For the transformation of Aspergillus oryzae, a modified protoplast-PEG method was used, and for the plasmid DNA to be transformed, pNEN-Cer and pNEN142 prepared above were used. 10 g of these plasmid DNAs were completely digested with BamHI, phenol extraction and ethanol precipitation were performed by conventional methods, and then dissolved in 101 TE to obtain a DNA solution for transformation.
[0047] ァスペルギルス.ォリゼ niaD300株(RIB40株由来の硝酸還元酵素遺伝子(niaD)欠 損変異株:独立行政法人酒類総合研究所:東広島巿鏡山三丁目 7番 1号、において 同番号にて保存され、分譲可能)の胞子懸濁液を YPD液体培地に添加し、 30°C、 12 時間振盪培養した。培養液よりガラスフィルターを用いで集菌した。菌体を 50ml容の 遠心チューブに移し、 10mlのプロトプラスト化溶液 (0.6M KC1、 0.2M NaH PO、 pH 5 [0047] Aspergillus oryzae niaD300 strain (Nitrate reductase gene (niaD) deficient mutant derived from RIB40 strain: Independent Administrative Institution Liquor Research Institute: Higashihiroshima Kagamiyama 3-chome No. 7-1, stored under the same number The spore suspension was added to YPD liquid medium and cultured with shaking at 30 ° C for 12 hours. Bacteria were collected from the culture using a glass filter. Transfer the cells to a 50 ml centrifuge tube and add 10 ml protoplasting solution (0.6 M KC1, 0.2 M NaH PO, pH 5
2 4 twenty four
.5、 5mg/ ml Lysing enzyme (¾igma Chemical Co.)、 lOmg/ml Cellujase Onozuka R-l 0 (Yakult Pharmaceutical Ind.Co.,Ltd.)、 lOmg/ml Yatalase (TaKaRa) )を加え懸濁し 、 30°C90 rpm、 3時間振盪しプロトプラストィ匕反応を行った。滅菌した MIRACLOTH( CALBIOCHEM)にて濾過し、濾液中のプロトプラストを 3,000 x g , 4°C , 5分間遠心 分離することで沈澱として得た。 1.2Mソルビトール、 50 mM CaCl、 Tris- HC1 buffer .5, 5 mg / ml Lysing enzyme (¾igma Chemical Co.), lOmg / ml Cellujase Onozuka Rl 0 (Yakult Pharmaceutical Ind. Co., Ltd.), lOmg / ml Yatalase (TaKaRa)) and suspended, 30 ° C90 The protoplasty reaction was performed by shaking at rpm for 3 hours. The mixture was filtered through sterilized MIRACLOTH (CALBIOCHEM), and the protoplasts in the filtrate were centrifuged at 3,000 x g, 4 ° C for 5 minutes to obtain a precipitate. 1.2M sorbitol, 50 mM CaCl, Tris- HC1 buffer
2 2
、 pH7.5にて 3回プロトプラストを洗浄し、 3000 x g、 4°C , 5分間遠心分離すること で沈澱として得た。このプロトプラストを 1 X 109個プロトプラスト/ mlになるように 1.2M ソルビトール, 50mM CaCl、 Tris- HC1 buffer, pH7.5で懸濁した。プロトプラスト懸
濁液 100 1に前述の形質転換用プラスミド DNA溶液各 10 1と 12.5 1の Soli (50 ( w/v%) PEG # 4000、 50mM CaCl 、 10 mM Tris- HCl buffer、 pH7.5をカ卩えよく混合し The protoplast was washed three times at pH 7.5, and centrifuged as 3000 xg at 4 ° C for 5 minutes to obtain a precipitate. This protoplast was suspended in 1.2 M sorbitol, 50 mM CaCl, Tris-HC1 buffer, pH 7.5 so as to be 1 × 10 9 protoplasts / ml. Protoplast suspension Add 100 1 and 12.5 1 Soli (50 (w / v%) PEG # 4000, 50 mM CaCl, 10 mM Tris-HCl buffer, pH 7.5, each of the aforementioned plasmid DNA solutions for transformation to the suspension 100 1. Mix well
2 2
、氷中で 30分間放置した。次にこの混合液を 50 ml容の遠心チューブに移し、 lml の Soliをカ卩え混合した後、 2 mlの 1.2Mソルビトール、 50 mM CaCl 、 Tris- HCl buffer And left in ice for 30 minutes. Next, transfer this mixture to a 50 ml centrifuge tube, add lml of Soli, mix, and then add 2 ml of 1.2M sorbitol, 50 mM CaCl 2, Tris-HCl buffer.
2 2
、 pH7.5をカ卩えよく混合した。 55°Cに温めておいた Czapek-Dox軟寒天培地にプロト プラスト懸濁液をカ卩ぇ混合し、 Czapek-Dox寒天培地に重層した。その後、 30°Cで胞 子を形成するまで培養した。 PH 7.5 was mixed well. The protoplast suspension was mixed with Czapek-Dox soft agar medium that had been warmed to 55 ° C. and overlaid on Czapek-Dox agar medium. Thereafter, the cells were cultured at 30 ° C. until spores were formed.
[0048] 胞子形成後、白金針にて分生子柄を力きとり、 0.01 %(v/v%) Tween80に懸濁し、 この懸濁液を希釈し、 Czapek-Dox寒天培地に広げ 30°Cで培養することを繰り返し 単胞子分離した。単胞子分離の確認は Hondel法(胞子 PCR法)を改変しておこな つた。白金針にて 200 1の YPD培地が入った 1.5ml容マイクロチューブに分生子 を添カ卩し、 30°Cで 40時間培養した。培養菌体を新しい 1.5ml容マイクロチューブに 移し、 50 μ 1のプロトプラスト化溶液 0.8Μ KC1、 10mMクェン酸、 pH6.5、 2.5mg/ml Lysing enzyme (Signa Chemical し o.)、 2.5mg/ml Yatalase (TaKaRa) )をカロ;^て懸淘 し、 37°Cで一時間放置し、 5分間以上氷上に放置し菌体を沈澱させた。この上清 5 μ 1を铸型として PCRの反応系に用いた。 pNEN- Cerと ρΝΕΝ142の胞子 PCR用の プライマーとして配列番号 4 : 5,- CTTCCGTCCTCCAAGTTAGTCGA- 3, と配列番 号 5 : 5,- GTAGAATCACGAATGAGACCTTTGACGACC- 3,の 2種のオリゴヌタレ ォチドを合成した。 PCR用装置は PCR Thermal Cycler PERSONAL (宝酒造)を用 いた。ポジティブコントロールとして形質転換に使用にしたプラスミド DNAを铸型とし た。増幅反応は 95°C、 3分間铸型 DNAを変性し、 94°C 1分間、 55°C 1分間、 7 2°C 3分間保持するサイクルを 30サイクルおこなった後、 72°C、 5分間で完全伸長 させ 4 °Cで保持した。得られた PCR増幅断片をァガロース電気泳動に供したところ 、ポジティブコントロールと同じ位置に PCR断片の増幅が確認され、その結果より、 麹菌の染色体 DNA上に P-enoA142プロモーター配列下流にセラミダーゼ遺伝子( 配列番号 1)の挿入された配列の存在が確認され、 PNEN142もまた形質転換された ことが確認された。 [0048] After sporulation, the conidia pattern is vigorously removed with a platinum needle and suspended in 0.01% (v / v%) Tween 80. This suspension is diluted and spread on Czapek-Dox agar medium at 30 ° C. The single spore was isolated by repeatedly culturing in the same manner. Single spore separation was confirmed by modifying the Hondel method (spore PCR method). Conidia was added to a 1.5 ml microtube containing 200 1 YPD medium with a platinum needle and cultured at 30 ° C for 40 hours. Transfer the cultured cells to a new 1.5 ml microtube, 50 μl of protoplasting solution 0.8 Μ KC1, 10 mM citrate, pH 6.5, 2.5 mg / ml Lysing enzyme (Signa Chemical o.), 2.5 mg / ml Yatalase (TaKaRa)) was suspended and allowed to stand at 37 ° C for 1 hour and left on ice for 5 minutes to precipitate the cells. This supernatant (5 μl) was used in a PCR reaction system as a bowl. Two primers, SEQ ID NO: 4: 5, -CTTCCGTCCTCCAAGTTAGTCGA-3, and SEQ ID NO: 5: 5, -GTAGAATCACGAATGAGACCTTTGACGACC-3, were synthesized as primers for pNEN-Cer and ρΝΕΝ142 spore PCR. PCR Thermal Cycler PERSONAL (Takara Shuzo) was used as the PCR device. As a positive control, the plasmid DNA used for transformation was in the form of a cage. Amplification reaction was performed at 95 ° C for 3 minutes, denaturing the cage DNA, followed by 30 cycles of 94 ° C for 1 minute, 55 ° C for 1 minute, 7 2 ° C for 3 minutes, then 72 ° C for 5 minutes. Fully extended with and kept at 4 ° C. When the obtained PCR amplified fragment was subjected to agarose electrophoresis, amplification of the PCR fragment was confirmed at the same position as the positive control. From the results, the ceramidase gene (sequence) was located downstream of the P-enoA142 promoter sequence on the chromosomal DNA of Aspergillus oryzae. The presence of the inserted sequence No. 1) was confirmed, and it was confirmed that PNEN142 was also transformed.
[0049] (4)セラミダーゼ発現の確認
形質転換されたセラミダーゼ高発現麹菌とセラミダーゼ遺伝子を挿入して 、な ヽ麹 菌(pNEN142)を 3 mlの YPM培地( 1 (w/v%)酵母エキス , 2 (w/v%)ペプトン, 2 ( w/v%)マルトース)に 1 X 106胞子/ ml濃度で植菌し、 30°C , 24時間振盪培養した。 培養後、ガラスフィルターで菌体を濾過し、培養上清を得た。培養上清 0.2 mlに対 して 50 μ 1の 100 (w/v%)冷トリクロ口酢酸をカ卩え、よく混合した後、氷中に 20分間 放置した。サンプルを 15,000 X g , 4°C , 20分間遠心分離し上清を完全に除いた後 、沈澱を 15 1の SDS化溶液に溶解させたあと、沸騰湯浴中に 5分間放置し SDS化を 行い SDS-PAGEに供した。その後、電気泳動したゲルを PVDF膜に転写し、 PVDF 膜上より 100 kDaの断片を切り抜き、そのままァミノ末端アミノ酸配列の解析をおこな つた。セラミダーゼ高発現麹菌でのみ発現して!/、るタンパク質のペプチド配列の解析 の結果、ァミノ末端領域のアミノ酸配列が「NTEFA」であり、セラミダーゼの推定アミノ 酸配列(配列番号 1のアミノ酸配列における第 46〜50番目のアミノ酸)と完全一致し たためセラミダーゼの高発現が確認された(図 3)。 [0049] (4) Confirmation of ceramidase expression Transformed ceramidase high-expressing bacilli and ceramidase gene were inserted, and bacilli (pNEN142) was added to 3 ml of YPM medium (1 (w / v%) yeast extract, 2 (w / v%) peptone, 2 (w / v%) maltose) was inoculated at a concentration of 1 × 10 6 spores / ml and cultured with shaking at 30 ° C. for 24 hours. After culturing, the cells were filtered with a glass filter to obtain a culture supernatant. 50 μl of 100 (w / v%) cold triclonal acetic acid was added to 0.2 ml of the culture supernatant, mixed well, and then left on ice for 20 minutes. Centrifuge the sample at 15,000 Xg, 4 ° C for 20 minutes to completely remove the supernatant, dissolve the precipitate in 151 SDS solution, and leave it in a boiling water bath for 5 minutes to convert to SDS. Performed for SDS-PAGE. Subsequently, the electrophoresed gel was transferred to a PVDF membrane, a 100 kDa fragment was cut out from the PVDF membrane, and the amino terminal amino acid sequence was analyzed as it was. As a result of analysis of the peptide sequence of the protein that is expressed only in bacilli that highly express ceramidase! /, The amino acid sequence of the amino terminal region is “NTEFA”, and the deduced amino acid sequence of ceramidase (the amino acid sequence in the amino acid sequence of SEQ ID NO: 1) A high expression of ceramidase was confirmed (Fig. 3).
(5)麹菌セラミダーゼの精製 (5) Purification of Neisseria gonorrhoeae ceramidase
セラミダーゼ高発現株の胞子を 1 X 106個胞子/ mlになるように、 1リットルの YPM 培地を入れた 3リットル容坂ロフラスコに接種し 30°Cで 24時間培養した。培養液を MIRACLOTH (CALBIOCHEM)にて濾過し、培養上清を得た。培養上清に 40%飽和 となるように硫酸アンモ-ゥムをカ卩えた後、 10,000 X g , 4°C, 40分間遠心分離し、得 られた上清画分を得た。上清画分を 10 mMトリス-塩酸緩衝液, pH 8.0、 40 %飽和 硫安にて平衡化した Octy卜 cellulofine type-S (生化学工業)に供した後、 1リットルの 精製水でカラムを洗浄した。洗浄後のカラムを 0-70 %エタノールの直線勾配で溶出 した。溶出させた全画分を SDS-PAGEに供し、 100 kDaのセラミダーゼが含まれた画 分を回収した。回収した画分を 10 mM Tris-HCl緩衝液(pH 8.0)で透析し、同緩衝 液で平衡化した DEAE- cellulofine A- 500に供し、吸着画分を NaCl、 0- 0.5Mの直 線勾配で溶出させた。回収した全画分を SDS-PAGEに供し、銀染色により 100 kDa であるセラミダーゼの単一バンドが確認されたため、これをセラミダーゼの精製標品と した。精製されたセラミダーゼをミリ Q水に対して透析を行った後凍結乾燥を行い、セ ラミダーゼの乾燥粉末を得た。
[0051] (6)麹菌セラミダーゼの PBSA乳化液分解促進効果 Ceramidase high-expressing strain spores were inoculated into a 3 liter volumetric flask containing 1 liter of YPM medium at 1 × 10 6 spores / ml and cultured at 30 ° C. for 24 hours. The culture solution was filtered with MIRACLOTH (CALBIOCHEM) to obtain a culture supernatant. Ammonium sulfate was added to the culture supernatant so as to be 40% saturation, and then centrifuged at 10,000 × g, 4 ° C. for 40 minutes to obtain a supernatant fraction. The supernatant fraction was subjected to Octy 卜 cellulofine type-S (Seikagaku Corporation) equilibrated with 10 mM Tris-HCl buffer, pH 8.0, 40% saturated ammonium sulfate, and the column was washed with 1 liter of purified water. did. The washed column was eluted with a linear gradient of 0-70% ethanol. All eluted fractions were subjected to SDS-PAGE, and fractions containing 100 kDa ceramidase were collected. The collected fraction is dialyzed against 10 mM Tris-HCl buffer (pH 8.0) and applied to DEAE-cellulofine A-500 equilibrated with the same buffer, and the adsorbed fraction is linear gradient of NaCl, 0-0.5M. And eluted. All the collected fractions were subjected to SDS-PAGE, and a single band of ceramidase of 100 kDa was confirmed by silver staining. This was used as a purified preparation of ceramidase. The purified ceramidase was dialyzed against milliQ water and lyophilized to obtain a dry powder of ceramidase. [0051] (6) Effect of Aspergillus ceramidase on the degradation of PBSA emulsion
セラミダーゼ精製標品の PBSA分解について検討を行った。セラミダーゼの凍結乾 燥粉末を 50 mM Tris-HCl緩衝液 (pH 8.5)に溶解し、 100 μ g/mlとなるように調整 した。また、 PBSA分解酵素である麹菌由来のクチナーゼも 100 μ g/mlとなるように 5 0 mM Tris-HCl緩衝液(pH 8.5)に溶解した。 96穴マイクロタイタープレートのゥエル に 50 mM Tris-HCl緩衝液(pH 8.5)に懸濁してある 0.15 (w/v) PBSA乳化液 100 μ 1 を入れ、次の条件で各ゥエルに酵素を添加した。 PBSA degradation of purified ceramidase preparation was examined. Ceramidase freeze-dried powder was dissolved in 50 mM Tris-HCl buffer (pH 8.5) and adjusted to 100 μg / ml. In addition, PBSA-degrading enzyme kojinase-derived cutinase was also dissolved in 50 mM Tris-HCl buffer (pH 8.5) to a concentration of 100 μg / ml. Add 100 μ 1 of 0.15 (w / v) PBSA emulsion suspended in 50 mM Tris-HCl buffer (pH 8.5) to the wells of a 96-well microtiter plate, and add enzymes to each well under the following conditions. .
[0052] (条件 1) 20 μ 1の 50 mM Tris-HCl緩衝液(pH 8.5)を添カロ [0052] (Condition 1) 20 μ 1 of 50 mM Tris-HCl buffer (pH 8.5)
(条件 2) 10 μ 1の 50 mM Tris-HCl緩衝液(pH 8.5)と 10 1の 100 μ g/mlのセラミ ダーゼ溶液を混合して添加 (Condition 2) Add 10 μl of 50 mM Tris-HCl buffer (pH 8.5) and 101 of 100 μg / ml ceramidase solution.
(条件 3) 10 μ 1の 50 mM Tris-HCl緩衝液(pH 8.5)と 10 1の 100 μ g/mlのクチナ ーゼ溶液を混合して添カロ (Condition 3) Mix 10 μl of 50 mM Tris-HCl buffer (pH 8.5) with 100 μg of 100 μg / ml cutinase solution.
(条件 4) 10 μ 1の 100 μ g/mlのクチナーゼ溶液と 10 μ 1の 100 μ g/mlのセラミダ ーゼ溶液を混合して添カロ (Condition 4) Mix 10 μl of a 100 μg / ml cutinase solution with 10 μl of a 100 μg / ml ceramidase solution.
[0053] 各条件の溶液を添カ卩したマイクロタイタープレートを 45 °Cで保温し、 10分おきに PBSA (分子量: 6万)乳化液の分解度を吸光度 630 nmで検出した。保温前の各ゥ エルの濁度を 100 %とし、各時間における PBSAの分解度を算出した(図 4)。酵素を 加えなかった条件 1とセラミダーゼのみを加えた条件 2ではほとんど PBSA乳化液の 分解は見られなかった。また、 PBSA分解酵素であるクチナーゼを加えた条件 3にお いて PBSAの分解が見られた。そして、セラミダーゼとクチナーゼを混合した条件 4 において最大の PBSA分解活性が見られた。セラミダーゼとクチナーゼを混合した 場合に、クチナーゼを単独で作用させた場合に比べ、最大で 50 %の PBSA分解促 進効果が見られた。このことより、セラミダーゼはクチナーゼによる PBSA分解を促進 する効果があることが示された。 [0053] A microtiter plate supplemented with a solution of each condition was kept at 45 ° C, and the degree of decomposition of the PBSA (molecular weight: 60,000) emulsion was detected at an absorbance of 630 nm every 10 minutes. The turbidity of each well before incubation was 100%, and the degradation degree of PBSA at each time was calculated (Fig. 4). Under condition 1 where no enzyme was added and condition 2 where only ceramidase was added, there was almost no degradation of the PBSA emulsion. In addition, PBSA degradation was observed under condition 3 where PBSA degrading enzyme cutinase was added. The maximum PBSA degrading activity was observed under condition 4 where ceramidase and cutinase were mixed. When ceramidase and cutinase were mixed, up to 50% of PBSA degradation was promoted compared to when cutinase was used alone. This indicates that ceramidase has the effect of promoting PBSA degradation by cutinase.
[0054] (7)セラミダーゼのウレタン結合分解 [0054] (7) Urethane bond degradation of ceramidase
ポリブチレンスクシネート(PBS、分子量: 6万)のペレット 4 mgを 2 mlの 50 mM Tri s塩酸緩衝液 (pH 8.5)に懸濁した。この PBSを含む溶液を二つに分け、片方に 100 1のセラミダーゼ溶液(100 mg/ml)をカ卩え、もう片方にはコントロールとして 100 μ
1の 50 mM Tris塩酸緩衝液 (pH 8.5)を加えた。この溶液を 37°Cで 12時間保温し た。それぞれの溶液に 500 μ 1のクチナーゼ溶液(185 μ g/ml)をカ卩ぇ 37 °Cにて 1 2時間保温し、 PBSのペレットを完全に分解した。この PBS分解物 400 μ 1を排除 分子量 1万のセントリカット超ミニ (クラボウ)に供し限外濾過を行った。この濾過物を 逆相クロマトグラフィーに供し、 PBS分解物の変化を観察した。尚、逆相クロマトグラフ ィ一の解析に用いた機種は L-4000 UV Detector (日立製作所)、 L-6200 Intelligent Pump (日立製作所)、 L-6000 Pump (日立製作所)であり、解析条件は以下の通りで ある。使用カラム: Shodex C18P-4E (昭和電工)、カラム温度:室温、流速: 1.0 ml/min 、溶離液 A:水 I酢酸 = 100 I 0.1 (v/v)、溶離液 B : ァセトニトリル I酢酸 = 100 /4 mg of a pellet of polybutylene succinate (PBS, molecular weight: 60,000) was suspended in 2 ml of 50 mM Tris hydrochloric acid buffer (pH 8.5). Divide this PBS-containing solution into two parts, and add 100 1 ceramidase solution (100 mg / ml) on one side and 100 μm on the other side as a control. 1 of 50 mM Tris hydrochloric acid buffer (pH 8.5) was added. This solution was incubated at 37 ° C for 12 hours. In each solution, 500 μl of cutinase solution (185 μg / ml) was incubated at 37 ° C. for 12 hours to completely decompose the PBS pellet. 400 μ1 of this PBS degradation product was excluded and subjected to ultrafiltration using a Centricut ultra-mini (Kurabo) with a molecular weight of 10,000. This filtrate was subjected to reverse phase chromatography, and changes in the PBS degradation product were observed. The models used for the analysis of reverse phase chromatography are L-4000 UV Detector (Hitachi), L-6200 Intelligent Pump (Hitachi), and L-6000 Pump (Hitachi). It is as follows. Column used: Shodex C18P-4E (Showa Denko), column temperature: room temperature, flow rate: 1.0 ml / min, eluent A: water I acetic acid = 100 I 0.1 (v / v), eluent B: acetonitrile I acetic acid = 100 /
0.1 (v/v)、溶出勾配条件 (Gradient B [%]) = 5 (0 min)→ 5 (5 min)→ 100 (50 min )→ 100 (60 min)→ 5 (61 min)→ 5 (75 min)。解析の結果、セラミダーゼの添カ卩によ つて保持時間 22.7分に出現するピークの減少が観察された(図 5)。この 22.7分に 出現するピークは 2分子の 1,4ブタンジオールと 1分子の 1,6-へキサメチレンジイソシ ァネートからなるエステルイ匕合物(以下、便宜上「 BHB」(図 6)とする)であった。 BHB はその分子内に二つのウレタン結合を含む。セラミダーゼはセラミド分子内のアミド 結合を分解する作用があるため、構造的に類似している BHBのウレタン結合をセラ ミダーゼが分解している。 0.1 (v / v), elution gradient condition (Gradient B [%]) = 5 (0 min) → 5 (5 min) → 100 (50 min) → 100 (60 min) → 5 (61 min) → 5 ( 75 min). As a result of the analysis, a decrease in the peak appearing at the retention time of 22.7 minutes was observed with the addition of ceramidase (Fig. 5). The peak appearing at 22.7 minutes is an ester compound consisting of two molecules of 1,4 butanediol and one molecule of 1,6-hexamethylene diisocyanate (hereinafter referred to as “BHB” (FIG. 6) for convenience). Met. BHB contains two urethane bonds in its molecule. Since ceramidase has the effect of cleaving the amide bond in the ceramide molecule, ceramidase cleaves the structurally similar urethane bond of BHB.
(8)麹菌セラミダーゼのセラミド蛍光基質の分解 (8) Degradation of ceramide fluorescent substrate of Neisseria gonorrhoeae ceramidase
セラミダーゼの活性はジャーナル'ォブ 'バイオロジカル 'ケミストリー、第 275卷、第 3 462-3468頁(2000)に記載されている方法を一部改変して行った。すなわち 20 1の 25 mMトリス塩酸緩衝液(pH 7.5)中に 550 pmolの、 C12- NBD-セラミド(Avanti Pola r Lipids, In )、及び適当量のセラミダーゼを溶解した反応混合液を 37°Cで 30分間ィ ンキュペートした。この反応液を沸騰水浴で 5分間インキュベートすることで反応を 停止した。得られた反応液を凍結乾燥し、水分を完全に除いた後、乾固物をクロロホ ルム Zメタノール =2Zl (V/V)に溶解し、シリカゲル薄層クロマトグラフィー(展開溶 媒:クロ口ホルム Zメタノール Z28%アンモニア水 = 90/20/0.5 (V/V/V) )で展開 した。その後、 Fla-2000 (フジフィルム)を用い、励起波長 473 nm、蛍光波長 520で 上記の反応によって生成した C12-NBD-脂肪酸の定量を行った。反応系にセラミダ
ーゼをカ卩えな力つた場合は C12-NBD-セラミドのスポットのみが検出された力 反応 系にセラミダーゼをカ卩えた場合において C12- NBD-セラミドの分解物である C12-NB D-脂肪酸のスポットが検出された(図 7)。このことより本麹菌セラミダーゼはセラミド分 解活性を有することが証明された。 The activity of ceramidase was performed by partially modifying the method described in Journal 'Ob' Biological 'Chemistry, Vol. 275, pp. 3462-3468 (2000). That is, 550 pmol of C12-NBD-ceramide (Avanti Polar Lipids, In) and an appropriate amount of ceramidase in a 25 mM Tris-HCl buffer (pH 7.5) of 20 1 at 37 ° C. Incubated for 30 minutes. The reaction was stopped by incubating the reaction in a boiling water bath for 5 minutes. The resulting reaction solution was freeze-dried to completely remove water, and the dried product was dissolved in chloroform Zmethanol = 2Zl (V / V) and silica gel thin layer chromatography (developing solvent: black mouth form). Z methanol Z28% aqueous ammonia = 90/20 / 0.5 (V / V / V)). Thereafter, Fla-2000 (Fuji Film) was used to quantify the C12-NBD-fatty acid produced by the above reaction at an excitation wavelength of 473 nm and a fluorescence wavelength of 520. Ceramida in the reaction system In the case of a strong reaction, only the C12-NBD-ceramide spot was detected. When a ceramidase was prepared in the reaction system, the degradation product of C12-NB D-fatty acid, which is a degradation product of C12-NBD-ceramide. A spot was detected (Figure 7). From these results, it was proved that the Bacillus subtilis ceramidase has ceramide-degrading activity.
産業上の利用可能性 Industrial applicability
本発明のセラミダーゼを用いて、ポリウレタン及び任意の割合でウレタン結合を含 むポリエステル、ポリプロピレン、ポリ塩化ビニール、ナイロン、ポリスチレン、及びデン プン等の高分子物質、特に生体分解プラスチックを効率的、経済的、かつ簡易に分 解することが可能となる。
By using the ceramidase of the present invention, it is possible to efficiently and economically use high molecular weight materials such as polyurethane and polyester, polypropylene, polyvinyl chloride, nylon, polystyrene, and polyurethane containing urethane bonds in any proportion, particularly biodegradable plastics. And it becomes possible to disassemble easily.
Claims
請求の範囲 The scope of the claims
[I] 以下の (a)または (b)の蛋白質であるセラミダーゼ: [I] Ceramidase, a protein of the following (a) or (b):
(a)配列番号 1に示されるアミノ酸配列からなる蛋白質、 (a) a protein comprising the amino acid sequence represented by SEQ ID NO: 1,
(b)配列番号 1に示されるアミノ酸配列において 1もしくは数個のアミノ酸が欠失、置 換もしくは付加されたアミノ酸配列力もなり、かつセラミダーゼ活性を有する蛋白質。 (b) a protein having ceramidase activity, which also has an amino acid sequence ability in which one or several amino acids have been deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1.
[2] 以下の (a)または (b)の蛋白質をコードする DNA: [2] DNA encoding the following protein (a) or (b):
(a)配列番号 1に示されるアミノ酸配列からなる蛋白質、 (a) a protein comprising the amino acid sequence represented by SEQ ID NO: 1,
(b)配列番号 1に示されるアミノ酸配列において 1もしくは数個のアミノ酸が欠失、置 換もしくは付加されたアミノ酸配列力もなり、かつセラミダーゼ活性を有する蛋白質。 (b) a protein having ceramidase activity, which also has an amino acid sequence ability in which one or several amino acids have been deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1.
[3] 以下の (a)または (b)の DNA: [3] DNA of (a) or (b) below:
(a)配列番号 1に示される塩基配列からなる DNA、 (a) DNA consisting of the base sequence shown in SEQ ID NO: 1,
(b)配列番号 1に示される塩基配列力 なる DNAと相補的な塩基配列とストリンジ ン トな条件でハイブリダィズし、かつセラミダーゼ活性を有する蛋白質をコードする DNA (b) DNA that encodes a protein that hybridizes under stringent conditions with a complementary nucleotide sequence to the DNA having the nucleotide sequence shown in SEQ ID NO: 1 and that has ceramidase activity
[4] cDNAである請求項 2または 3記載の DNA。 [4] The DNA according to claim 2 or 3, which is cDNA.
[5] 請求項 2〜4の!、ずれか一項に記載の DNAを含む組換え用 DNA。 [5] A DNA for recombination comprising the DNA according to any one of!
[6] 組換え用 DNAがプラスミドベクターである、請求項 5記載の組換え用 DNA。 6. The DNA for recombination according to claim 5, wherein the DNA for recombination is a plasmid vector.
[7] 請求項 5叉は 6記載の組換え用 DNAを有する形質転換体。 [7] A transformant having the DNA for recombination according to claim 5 or 6.
[8] 形質転換体の宿主が原核微生物叉は真核微生物であることを特徴とする、請求項 7記載の形質転換体。 8. The transformant according to claim 7, wherein the host of the transformant is a prokaryotic microorganism or a eukaryotic microorganism.
[9] 原核微生物がェシェリシァ属、バチルス属、叉はストレプトマイセス属であることを特 徴とする、請求項 8記載の形質転換体。 [9] The transformant according to claim 8, wherein the prokaryotic microorganism is Escherichia, Bacillus, or Streptomyces.
[10] 真核微生物が、酵母、叉は、ァスペルギルス属、ぺニシリウム属、トリコデルマ属、リ ズプス属、メタリチウム属、アクレモ-ゥム属、及びムコール属からなる群から選択され る真核糸状菌であることを特徴とする、請求項 8記載の形質転換体 [10] The eukaryotic microorganism is a yeast, or eukaryotic filamentous selected from the group consisting of Aspergillus, Penicillium, Trichoderma, Lipus, Metalithium, Acremum, and Mucor. The transformant according to claim 8, which is a fungus.
[I I] 宿主がァスペルギルス'オリゼ叉はァスペルギルス ·ソーェであることを特徴とする、 請求項 10記載の形質転換体。 [I I] The transformant according to claim 10, wherein the host is Aspergillus oryzae or Aspergillus soe.
[12] 更にエステラーゼをコードする DNAを含む組換え用 DNAを有する、請求項 7〜11
の!、ずれか一項に記載の形質転換体。 [12] The recombinant DNA further comprising a DNA encoding an esterase. The transformant according to any one of No.!
[13] エステラーゼがリパーゼまたはクチナーゼである、請求項 12記載の形質転換体。 [13] The transformant according to claim 12, wherein the esterase is lipase or cutinase.
[14] 請求項 7〜13のいずれか一項に記載の形質転換体を培地に培養し、培養物から セラミダーゼを採取することを含む、セラミダーゼの製造法。 [14] A method for producing ceramidase, comprising culturing the transformant according to any one of claims 7 to 13 in a medium and collecting ceramidase from the culture.
[15] 請求項 1記載のセラミダーゼを用いて高分子物質を分解する方法。 15. A method for decomposing a polymer substance using the ceramidase according to claim 1.
[16] 請求項 1記載のセラミダーゼを用いて高分子物質に含まれるウレタン結合及び Z又 はアミド結合を分解する方法。 [16] A method for decomposing a urethane bond and a Z or amide bond contained in a polymer substance using the ceramidase according to claim 1.
[17] 請求項 1記載のセラミダーゼとエステラーゼとの組み合わせを用いて高分子物質を 分解する方法。 [17] A method for decomposing a polymer substance using the combination of ceramidase and esterase according to claim 1.
[18] エステラーゼがリパーゼまたはクチナーゼである、請求項 16記載の方法。 18. The method according to claim 16, wherein the esterase is lipase or cutinase.
[19] 請求項 7〜13のいずれか一項に記載の形質転換体を高分子物質に接触させるこ とを含む、高分子物質を分解する方法。 [19] A method for decomposing a polymer substance, comprising contacting the transformant according to any one of claims 7 to 13 with the polymer substance.
[20] 高分子物質がポリウレタン、任意の割合でウレタン結合を含むポリエステル、ポリプ ロピレン、ポリ塩化ビニール、ナイロン、ポリスチレン、デンプン、及びそれらの混合物 から成る群から選択されることを特徴とする、請求項 15〜 19のいずれか一項に記載 の方法。 [20] The polymer material is selected from the group consisting of polyurethane, polyester containing urethane bonds in any proportion, polypropylene, polyvinyl chloride, nylon, polystyrene, starch, and mixtures thereof. Item 20. The method according to any one of Items 15 to 19.
[21] 高分子物質が生分解性プラスチックであることを特徴とする、請求項 15〜19のい ずれか一項に記載の方法。 [21] The method according to any one of claims 15 to 19, wherein the polymer substance is a biodegradable plastic.
[22] 生分解性プラスチックがポリ乳酸、ポリブチレンコハク酸、ポリブチレンコハク酸 'ァ ジピン酸、脂肪族ポリエステル、ポリ力プロラタトン、叉はポリハイドロキシ酪酸である 請求項 21記載の方法。
[22] The method according to claim 21, wherein the biodegradable plastic is polylactic acid, polybutylene succinic acid, polybutylene succinic acid adipic acid, aliphatic polyester, poly strength prolatatone, or polyhydroxybutyric acid.
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Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2010050482A1 (en) * | 2008-10-27 | 2010-05-06 | 東洋製罐株式会社 | Method for producing oligomer and/or monomer by degrading biodegradable resin |
WO2013134801A2 (en) * | 2012-03-12 | 2013-09-19 | Eurofoam Gmbh | Process for the material utilization of polyurethanes |
CN111254135A (en) * | 2020-03-03 | 2020-06-09 | 江南大学 | Urease mutant with improved application performance |
JP2020132781A (en) * | 2019-02-21 | 2020-08-31 | トヨタ紡織株式会社 | Method for decomposing fiber-reinforced plastic |
CN113115588A (en) * | 2018-06-21 | 2021-07-13 | 科思创知识产权两合公司 | Novel carbamate hydrolase for enzymatic decomposition of polyurethane |
CN114286837A (en) * | 2019-08-16 | 2022-04-05 | 科思创知识产权两合公司 | Method for decomposing polyether polyurethane |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2004038016A1 (en) * | 2002-10-23 | 2004-05-06 | Tohoku Techno Arch Co., Ltd | Method of degrading plastic and process for producing useful substance using the same |
-
2006
- 2006-09-04 WO PCT/JP2006/317422 patent/WO2007029630A1/en active Application Filing
- 2006-09-04 JP JP2007534383A patent/JP4953317B2/en active Active
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2004038016A1 (en) * | 2002-10-23 | 2004-05-06 | Tohoku Techno Arch Co., Ltd | Method of degrading plastic and process for producing useful substance using the same |
Non-Patent Citations (1)
Title |
---|
MAEDA H. ET AL.: "Purification and characterization of a biodegradable plastic-degrading enzyme from Aspergillus oryzae", APPL. MICROBIOL. BIOTECHNOL., vol. 67, no. 6, June 2005 (2005-06-01), pages 778 - 788, XP019331869 * |
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US8501445B2 (en) | 2008-10-27 | 2013-08-06 | Toyo Seikan Kaisha, Ltd. | Method for producing oligomer and/or monomer by degrading biodegradable resin |
WO2010050482A1 (en) * | 2008-10-27 | 2010-05-06 | 東洋製罐株式会社 | Method for producing oligomer and/or monomer by degrading biodegradable resin |
US9284432B2 (en) | 2008-10-27 | 2016-03-15 | Toyo Seikan Kaisha, Ltd. | Method for degrading a readily-degradable resin composition |
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JP2021531036A (en) * | 2018-06-21 | 2021-11-18 | コヴェストロ インテレクチュアル プロパティ ゲーエムベーハー ウント シーオー.カーゲー | A novel ureathanase for enzymatic degradation of polyurethane |
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