WO2007029630A1

WO2007029630A1 - Novel ceramidase and use thereof

Info

Publication number: WO2007029630A1
Application number: PCT/JP2006/317422
Authority: WO
Inventors: Shinsaku Ohtaki; Toru Takahashi; Takahiro Tanaka; Hitoshi Fujita; Yohei Yamagata; Keietsu Abe; Fumihiko Hasegawa; Katsuya Gomi
Original assignee: Japan Science And Technology Agency; Tohoku University
Priority date: 2005-09-07
Filing date: 2006-09-04
Publication date: 2007-03-15
Also published as: JP4953317B2; JPWO2007029630A1

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

Specification

Novel ceramidase and its use

Technical field

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.

Background art

[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] 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] 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.

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)

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

Disclosure of the invention

Problems to be solved by the invention

[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.

Means for solving the problem

[0006] That is, the present invention relates to the following aspects.

[1] The following ceramidase (a) or (b) protein:

(a) a protein comprising the amino acid sequence represented by SEQ ID NO: 1,

(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] DNA encoding the following protein (a) or (b):

(a) a protein comprising the amino acid sequence represented by SEQ ID NO: 1,

[3] DNA of (a) or (b) below:

(a) DNA consisting of the base sequence shown in SEQ ID NO: 1,

(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] The DNA according to embodiment 2 or 3, which is cDNA.

[5] A DNA for recombination comprising the DNA according to any one of embodiments 2 to 4!

[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] A mode in which the host of the transformant is a prokaryotic microorganism or a eukaryotic microorganism

7. The transformant according to 7.

[9] The transformant according to embodiment 8, wherein the prokaryotic microorganism is of the genus Escherichia, Bacillus, or Streptomyces.

`` 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] The transformant according to embodiment 10, wherein the host is Aspergillus oryzae or Aspergillus soe.

[12] The transformant according to any one of embodiments 7 to 11, further comprising a recombination DNA containing a DNA encoding an esterase.

[13] The transformant according to embodiment 12, wherein the esterase is lipase or cutinase

[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] A method for decomposing a polymer substance using the ceramidase according to embodiment 1.

[16] A urethane bond contained in the polymer substance using the ceramidase according to embodiment 1, and

A method of breaking the Z or amide bond.

[17] A method for degrading a polymer substance using the combination of ceramidase and esterase according to embodiment 1.

[18] The method according to embodiment 16, wherein the esterase is lipase or cutinase.

[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] 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] 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.

The invention's effect

[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.

Brief Description of Drawings

[0008] 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.

[Figure 6] Shows the structure of BHB.

FIG. 7 is a graph showing a state of degradation of a ceramide fluorescent substrate of ceramidase.

BEST MODE FOR CARRYING OUT THE INVENTION

[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] 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] 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.

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] 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] 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] 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] 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] 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.

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] 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] 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] 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] 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] 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] 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] 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] 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] 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] 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] 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] 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.

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] 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] 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] 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] 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] 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.

Example

[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) Separation of hydrophobic carrier-adsorbed protein from PBSA degradation products by Aspergillus oryzae

Spore 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

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] 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) Construction of vector for high expression by Aspergillus oryzae

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] 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] 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

), 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] 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) Preparation of gonococci with high expression of ceramidase

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] 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

twenty four

.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

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

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

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] 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) 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) Purification of Neisseria gonorrhoeae ceramidase

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

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] (Condition 1) 20 μ 1 of 50 mM Tris-HCl buffer (pH 8.5)

(Condition 2) Add 10 μl of 50 mM Tris-HCl buffer (pH 8.5) and 101 of 100 μg / ml ceramidase solution.

(Condition 3) Mix 10 μl of 50 mM Tris-HCl buffer (pH 8.5) with 100 μg of 100 μg / ml cutinase solution.

(Condition 4) Mix 10 μl of a 100 μg / ml cutinase solution with 10 μl of a 100 μg / ml ceramidase solution.

[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) Urethane bond degradation of ceramidase

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), 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) Degradation of ceramide fluorescent substrate of Neisseria gonorrhoeae 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. 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.

Industrial applicability

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

The scope of the claims

[I] Ceramidase, a protein of the following (a) or (b):

(a) a protein comprising the amino acid sequence represented by SEQ ID NO: 1,

[2] DNA encoding the following protein (a) or (b):

(a) a protein comprising the amino acid sequence represented by SEQ ID NO: 1,

[3] DNA of (a) or (b) below:

(a) DNA consisting of the base sequence shown in SEQ ID NO: 1,

[4] The DNA according to claim 2 or 3, which is cDNA.

[5] A DNA for recombination comprising the DNA according to any one of!

6. The DNA for recombination according to claim 5, wherein the DNA for recombination is a plasmid vector.

[7] A transformant having the DNA for recombination according to claim 5 or 6.

8. The transformant according to claim 7, wherein the host of the transformant is a prokaryotic microorganism or a eukaryotic microorganism.

[9] The transformant according to claim 8, wherein the prokaryotic microorganism is Escherichia, Bacillus, or Streptomyces.

[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] The transformant according to claim 10, wherein the host is Aspergillus oryzae or Aspergillus soe.

[12] The recombinant DNA further comprising a DNA encoding an esterase. The transformant according to any one of No.!

[13] The transformant according to claim 12, wherein the esterase is lipase or cutinase.

[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. A method for decomposing a polymer substance using the ceramidase according to claim 1.

[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] A method for decomposing a polymer substance using the combination of ceramidase and esterase according to claim 1.

18. The method according to claim 16, wherein the esterase is lipase or cutinase.

[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] 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] The method according to any one of claims 15 to 19, wherein the polymer substance is a biodegradable plastic.

[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.