WO2007029630A1 - Nouvelle céramidase et son utilisation - Google Patents

Nouvelle céramidase et son utilisation Download PDF

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Publication number
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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WIPO (PCT)
Prior art keywords
ceramidase
dna
amino acid
protein
transformant
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PCT/JP2006/317422
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English (en)
Japanese (ja)
Inventor
Shinsaku Ohtaki
Toru Takahashi
Takahiro Tanaka
Hitoshi Fujita
Yohei Yamagata
Keietsu Abe
Fumihiko Hasegawa
Katsuya Gomi
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Japan Science And Technology Agency
Tohoku University
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Priority to JP2007534383A priority Critical patent/JP4953317B2/ja
Publication of WO2007029630A1 publication Critical patent/WO2007029630A1/fr

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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)
    • 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
    • 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
    • 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)
    • 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
    • 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

Le problème à résoudre dans le cadre de l’invention consiste à obtenir une nouvelle enzyme dérivée d’un micro-organisme, en particulier d’une moisissure à potentiel de croissance élevé sur une surface plastique présentant une liaison uréthane, et pouvant briser une liaison uréthane et/ou une liaison amide dans une substance polymère, ainsi qu’à trouver un procédé de dégradation d’un matériau plastique présentant une liaison uréthane à l’aide de l’enzyme. La solution proposée est une céramidase comprenant l’une des protéines (a) et (b) : (a) est une protéine comprenant la séquence amino-acide décrite dans le document SEQ ID NO:1 et (b) une protéine comprenant une séquence amino-acide après délétion, substitution ou ajout d’un ou de plusieurs résidus amino-acides dans la séquence amino-acide décrite en SEQ ID NO:1 et possédant une activité céramidase.
PCT/JP2006/317422 2005-09-07 2006-09-04 Nouvelle céramidase et son utilisation WO2007029630A1 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
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WO2010050482A1 (fr) * 2008-10-27 2010-05-06 東洋製罐株式会社 Méthode de production d'un oligomère et/ou d'un monomère par dégradation d'une résine biodégradable
WO2013134801A2 (fr) * 2012-03-12 2013-09-19 Eurofoam Gmbh Procédé de valorisation matière de polyuréthanes
CN111254135A (zh) * 2020-03-03 2020-06-09 江南大学 一种应用性能提高的脲酶突变体
JP2020132781A (ja) * 2019-02-21 2020-08-31 トヨタ紡織株式会社 繊維強化プラスチックの分解方法
CN113115588A (zh) * 2018-06-21 2021-07-13 科思创知识产权两合公司 用于聚氨酯酶促分解的新型氨基甲酸酯水解酶
CN114286837A (zh) * 2019-08-16 2022-04-05 科思创知识产权两合公司 分解聚醚聚氨酯的方法

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WO2004038016A1 (fr) * 2002-10-23 2004-05-06 Tohoku Techno Arch Co., Ltd Procede de degradation du plastique et procede de production de substance utile dans lequel ledit procede est utilise

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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 *

Cited By (14)

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CN102264912B (zh) * 2008-10-27 2016-01-06 东洋制罐株式会社 降解生物降解性树脂而生成低聚物和/或单体的方法
CN102264912A (zh) * 2008-10-27 2011-11-30 东洋制罐株式会社 降解生物降解性树脂而生成低聚物和/或单体的方法
US8501445B2 (en) 2008-10-27 2013-08-06 Toyo Seikan Kaisha, Ltd. Method for producing oligomer and/or monomer by degrading biodegradable resin
WO2010050482A1 (fr) * 2008-10-27 2010-05-06 東洋製罐株式会社 Méthode de production d'un oligomère et/ou d'un monomère par dégradation d'une résine biodégradable
US9284432B2 (en) 2008-10-27 2016-03-15 Toyo Seikan Kaisha, Ltd. Method for degrading a readily-degradable resin composition
US8846355B2 (en) 2008-10-27 2014-09-30 Toyo Seikan Kaisha, Ltd. Method for degrading biodegradable resin
WO2013134801A2 (fr) * 2012-03-12 2013-09-19 Eurofoam Gmbh Procédé de valorisation matière de polyuréthanes
WO2013134801A3 (fr) * 2012-03-12 2014-01-23 Eurofoam Gmbh Procédé de valorisation matière de polyuréthanes
CN113115588A (zh) * 2018-06-21 2021-07-13 科思创知识产权两合公司 用于聚氨酯酶促分解的新型氨基甲酸酯水解酶
JP2021531036A (ja) * 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 ポリウレタンの酵素的分解のための新規なウレタナーゼ
JP2020132781A (ja) * 2019-02-21 2020-08-31 トヨタ紡織株式会社 繊維強化プラスチックの分解方法
CN114286837A (zh) * 2019-08-16 2022-04-05 科思创知识产权两合公司 分解聚醚聚氨酯的方法
CN111254135A (zh) * 2020-03-03 2020-06-09 江南大学 一种应用性能提高的脲酶突变体
CN111254135B (zh) * 2020-03-03 2021-09-28 江南大学 一种应用性能提高的脲酶突变体

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