WO2005054475A1 - エンドグルカナーゼstceおよびそれを含むセルラーゼ調製物 - Google Patents
エンドグルカナーゼstceおよびそれを含むセルラーゼ調製物 Download PDFInfo
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- WO2005054475A1 WO2005054475A1 PCT/JP2004/015733 JP2004015733W WO2005054475A1 WO 2005054475 A1 WO2005054475 A1 WO 2005054475A1 JP 2004015733 W JP2004015733 W JP 2004015733W WO 2005054475 A1 WO2005054475 A1 WO 2005054475A1
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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)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/189—Enzymes
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
- C11D3/16—Organic compounds
- C11D3/38—Products with no well-defined composition, e.g. natural products
- C11D3/386—Preparations containing enzymes, e.g. protease or amylase
- C11D3/38636—Preparations containing enzymes, e.g. protease or amylase containing enzymes other than protease, amylase, lipase, cellulase, oxidase or reductase
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
- C11D3/16—Organic compounds
- C11D3/38—Products with no well-defined composition, e.g. natural products
- C11D3/386—Preparations containing enzymes, e.g. protease or amylase
- C11D3/38645—Preparations containing enzymes, e.g. protease or amylase containing cellulase
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01004—Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
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- D—TEXTILES; PAPER
- D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
- D06M—TREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS OR FIBROUS GOODS MADE FROM SUCH MATERIALS
- D06M16/00—Biochemical treatment of fibres, threads, yarns, fabrics, or fibrous goods made from such materials, e.g. enzymatic
- D06M16/003—Biochemical treatment of fibres, threads, yarns, fabrics, or fibrous goods made from such materials, e.g. enzymatic with enzymes or microorganisms
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- D—TEXTILES; PAPER
- D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
- D21H—PULP COMPOSITIONS; PREPARATION THEREOF NOT COVERED BY SUBCLASSES D21C OR D21D; IMPREGNATING OR COATING OF PAPER; TREATMENT OF FINISHED PAPER NOT COVERED BY CLASS B31 OR SUBCLASS D21G; PAPER NOT OTHERWISE PROVIDED FOR
- D21H11/00—Pulp or paper, comprising cellulose or lignocellulose fibres of natural origin only
- D21H11/16—Pulp or paper, comprising cellulose or lignocellulose fibres of natural origin only modified by a particular after-treatment
- D21H11/20—Chemically or biochemically modified fibres
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- D—TEXTILES; PAPER
- D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
- D21H—PULP COMPOSITIONS; PREPARATION THEREOF NOT COVERED BY SUBCLASSES D21C OR D21D; IMPREGNATING OR COATING OF PAPER; TREATMENT OF FINISHED PAPER NOT COVERED BY CLASS B31 OR SUBCLASS D21G; PAPER NOT OTHERWISE PROVIDED FOR
- D21H25/00—After-treatment of paper not provided for in groups D21H17/00 - D21H23/00
- D21H25/18—After-treatment of paper not provided for in groups D21H17/00 - D21H23/00 of old paper as in books, documents, e.g. restoring
-
- D—TEXTILES; PAPER
- D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
- D06M—TREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS OR FIBROUS GOODS MADE FROM SUCH MATERIALS
- D06M2101/00—Chemical constitution of the fibres, threads, yarns, fabrics or fibrous goods made from such materials, to be treated
- D06M2101/02—Natural fibres, other than mineral fibres
- D06M2101/04—Vegetal fibres
- D06M2101/06—Vegetal fibres cellulosic
-
- D—TEXTILES; PAPER
- D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
- D06M—TREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS OR FIBROUS GOODS MADE FROM SUCH MATERIALS
- D06M2200/00—Functionality of the treatment composition and/or properties imparted to the textile material
- D06M2200/35—Abrasion, pilling or fibrillation resistance
-
- D—TEXTILES; PAPER
- D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
- D06M—TREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS OR FIBROUS GOODS MADE FROM SUCH MATERIALS
- D06M2200/00—Functionality of the treatment composition and/or properties imparted to the textile material
- D06M2200/50—Modified hand or grip properties; Softening compositions
Definitions
- the present invention relates to an endoglucanase STCE (Endoglucanases STCE), a cellulase preparation containing the same, and a method for treating a cellulose-containing fiber using the same.
- endoglucanase STCE Endoglucanases STCE
- cellulose-containing fibers have been treated with cellulose in order to impart desired properties to the fibers.
- desired properties for example, in the textile industry, to improve the feel and appearance of cellulose-containing fibers, or to give colored cellulose-containing fibers the appearance of a "stone wash" that provides a localized change in color.
- Treatment with cellulase is performed [European Patent No. 307,564 (Patent Document 1)].
- a purified 43 kD endorcanase component (EGV) derived from the genus Humicola in a detergent is compared with a conventional cellulase preparation which is a mixture of a plurality of conventional cellulase components. It is known that there is about 30 times the clarifying activity (the activity of removing fluff generated in the dough and increasing the color of the dough) [WO 91/17243 (Patent Document 6)]. In addition, it is known that the reaction of endoglucanase NCE5 derived from the genus Humicola (hereinafter sometimes abbreviated as “NCE5”) in a detergent causes clarification of colored dough [International Publication No.
- tap water Western used for washing is generally hardness divalent cations such as Kogu Ca 2T and Mg 2+ are included in a large amount. Since the divalent cation significantly reduces the cleaning ability of the detergent in the detergent, the detergent contains a builder to adsorb the divalent cation ["Fragrance Journal", 1995, Vol. 11, p. 33-55 (Non-Patent Document 1)].
- the type and amount of builders to be added to detergents are devised by region.
- cellulase activity which is not limited to detergent cleaning ability, is also affected by these divalent cations [Mansfield, SD et al., "Enzyme Microb.
- Non-Patent Document 2 Non-Patent Document 2
- Jenkins CC AND Suberkropp, K., "Freshwater Biology", Vol. 33, No. 2, 1995, p. 245-253 (Non-Patent Document 3)]. Therefore, depending on the hardness of the water, the cellulase activity is inhibited and a satisfactory clarifying effect cannot be obtained, and conversely, the cellulase activity is enhanced and the strength of the dough decreases.
- filamentous fungi belonging to the genus Aspergillus for example, filamentous fungi belonging to the genus Aspergillus, Humicola, or Trichoderma.
- Aspergillus which produces neutral cellulases rather than Trichoderma filamentous fungi which produces acidic cellulases.
- filamentous fungi of the genus Humicola as a host.
- bacteria of the genus Humicola with high productivity are extremely excellent hosts [International Publication No. 01Z90375 (Patent Document 7) and International Publication No. 98Z03640. (Patent Document 11)].
- Patent Document 1 European Patent No. 307,564
- Patent Document 2 European Patent No. 220,016
- Patent Document 3 International Publication No. 95Z02675 pamphlet
- Patent Document 4 WO 97Z30143 pamphlet
- Patent Document 5 International Publication No. 98Z08926 pamphlet
- Patent Document 6 International Publication No. 91Z17243 pamphlet
- Patent Document 7 International Publication No. 01Z90375 pamphlet
- Patent Document 8 International Publication No. 00Z24879 pamphlet
- Patent Document 9 International Publication No. 98Z03667 pamphlet
- Patent Document 10 International Publication No.98Z11239 pamphlet
- Patent Document 11 International Publication No. 98Z03640 pamphlet
- Non-Patent Document 1 "Fragrance Journal”, 1995, vol. 11, p. 33-55
- Non-Patent Document 2 Mansfield, S.D. et al., "Enzyme Microb. Technol.” 23, 1
- Non-Patent Document 3 Jenkins, C.C.C. AND Suberkropp, K., "Freshwater Biology,., Vol. 33, No. 2, 1995, p. 245-253
- the present inventors have not reported any isolation of a cellulase enzyme or a cellulase gene.
- a novel protein having high V and endalcanase activity and a gene thereof have been found from microorganisms belonging to the genus Staphvlotrichum, which has not been provided, and provided.
- the present inventor has developed a novel protein having a high and endalcanase activity isolated from a microorganism belonging to the genus StaDhylotrichum of the present invention. It has been found that the pigmented material has extremely strong clarifying activity with respect to the contained colored fabric.
- the endoglucanase of the present invention derived from Staphylotrichum cocosporum (Staphylotrichum coccosporum), particularly the endoglucanase STCE1 (hereinafter sometimes abbreviated as “STCE1”), which is one example thereof, is used in typical European detergents.
- NCE5 endalcanase N CE5
- WO 01Z90375 pamphlet endalcanase N CE5
- Endoglucanase had a clarifying activity that was about 80 times or more higher than that of RCEI (WO 00Z24879 pamphlet).
- the surprising finding that the endalcanase STCE1 has a stable clarifying activity regardless of the hardness of tap water has been obtained.
- the endalcanases NCE4 (International Publication No. 98Z03640) and NCE5 (International Publication No. 01/90375), which are known to have high fuzz removal activity on cellulose-containing fibers are used in tap water. While the clarification activity increases with increasing hardness, Endalcanase R CEI (WO 00Z24879 pamphlet) decreases with increasing hardness in tap water.
- the endalcanase STCE1 of the present invention had a stable clarifying activity regardless of the hardness of tap water. As described above, there is no knowledge about a cellulase having a high clarifying activity and a stable activity in detergents in Europe and the United States regardless of the hardness of tap water.
- staphylotricum 'cocosporum-derived endoglucanase STCE1 can be used for transformation using Humicola insolens (Humicola insolens), which is a heterologous strain from staphylotrichum cocosporum, as a host. It has been found that endoglucanase STC E1 gene is expressed in large amounts without any modification operation.
- Humicola insolens Humicola insolens
- the present invention relates to a microorganism derived from a microorganism belonging to the genus Staphylotrichum.
- An object of the present invention is to provide a novel protein having undulcanase activity, a gene thereof, and a cellulase preparation containing the protein and having good properties.
- the present invention also provides a host cell transformed with the gene encoding the protein, and a method for culturing the host cell to collect a target protein. Further, the present invention provides a method for treating a cellulose-containing fiber with the protein of the present invention or the cellulase preparation of the present invention.
- the present invention includes the following inventions.
- a protein derived from a microorganism belonging to the genus Staphvlotrichum and having endoglucanase activity is derived from a microorganism belonging to the genus Staphvlotrichum and having endoglucanase activity.
- N-terminal amino acid sequence has the sequence represented by SEQ ID NO: 1,
- N-terminal amino acid sequence has the sequence represented by SEQ ID NO: 1,
- Staphylotrichum * The protein according to any one of (1) to (4), which is derived from cocosporum (Staphylotrichum coccosporum).
- a homologous protein comprising an amino acid sequence having at least 85% homology with a protein comprising the amino acid sequence represented by SEQ ID NO: 3, and having an endalcanase activity;
- (iii) encodes a protein that hybridizes under stringent conditions with a polynucleotide consisting of the 64th to 948th bases in the base sequence represented by SEQ ID NO: 2 and that has a protein having an endalcanase activity under stringent conditions.
- Polynucleotide consisting of the 64th to 948th bases in the base sequence represented by SEQ ID NO: 2 and that has a protein having an endalcanase activity under stringent conditions.
- a filamentous fungus is a microorganism belonging to the genus Humicola, the genus Trichoderma (Trichoderma), the genus Staphylotrichum, the genus Aspergillus (Aspergillus), the genus Fusarium (Fusarium), or the genus Acremonium.
- the filamentous fungus is Humicola insolens (Humicola insolens) or Trichoderma 'viride rrichoderma
- a method for producing a protein comprising a step of collecting the protein according to any one of (1) to (6) from a host cell obtained by culture or a culture thereof.
- a cellulase preparation comprising the protein according to any one of (6) and (16).
- a detergent composition comprising the protein according to any one of (6) and (16) or the cellulase preparation according to (17).
- a method comprising the step of contacting the protein according to item, the cellulase preparation according to item (17), or the detergent composition according to item (18).
- (21) A method for reducing the weight of cellulose-containing fibers for the purpose of improving the feel and appearance of the cellulose-containing fibers, wherein the cellulose-containing fibers are treated with the protein according to any one of (1)-(6) and (16). Contacting with the cellulase preparation according to (17), or the detergent composition according to (18).
- a method comprising contacting the protein with the cellulase preparation according to (17) or the detergent composition according to (18).
- a method for reducing the rate at which the cellulose-containing fiber starts to stiffen or reducing the stiffness of the cellulose-containing fiber comprising: A method comprising contacting the protein according to any one of 6), the cellulase preparation according to (17), or the detergent composition according to (18).
- a method for improving the drainage of paper pulp which comprises preparing the protein according to any one of (1)-(6) and (16) or the cellulase preparation according to (17).
- a method comprising the step of treating with an object.
- a method for improving the digestibility of animal feed comprising the steps of: (1) the protein according to any one of (1) to (6) and (16), or the cellulase according to (17); A method comprising the step of treating with a preparation.
- the protein of the present invention is used for the purpose of clarifying the color of cellulose-containing fiber, reducing fluff, improving feel and appearance, local change in color, and reducing stiffness. It is useful for detergents as well as for textile processing.
- a protein having an endoglucanase activity having an endoglucanase activity
- endalcanase refers to an enzyme having an endalcanase activity, that is, endo 1,4
- endalcanase activity in the present specification means CMCase activity.
- CMCase activity means the activity of hydrolyzing carboxymethylcellulose (CMC, manufactured by Tokyo Chemical Industry Co., Ltd.), and the amount of reducing sugar released after incubating the test protein and CMC solution for a certain period of time. The amount of enzyme that produces 1 ⁇ mol of glucose-equivalent reducing sugar per minute is defined as 1 unit.
- Endalcanase activity can be measured, for example, by the following procedure.
- Ma First add 0.5 mL of the solution containing the test protein to 0.5 mL of 50 mmol ZL sodium acetate buffer (pH 6.0) containing 2% CMC, and incubate at 50 ° C for 30 minutes. .
- the generated reducing sugar concentration of the obtained reaction solution is determined by the 3,5-dinitrosalicylic acid method (DNS method). That is, add 3.OmL of DNS reagent to 1.OmL of the reaction solution 30 minutes after the reaction, incubate in a boiling water bath for 5 minutes, dilute with 8.OmL of distilled water, and measure the absorbance at 540 nm.
- DNS method 3,5-dinitrosalicylic acid method
- a calibration curve is prepared using the glucose solution that has been diluted stepwise, and the amount of reducing sugars produced in the enzyme reaction solution is determined in terms of glucose. The activity is calculated based on the amount of enzyme that produces 1 ⁇ mol of reducing sugar equivalent to glucose per minute as one unit.
- the DNS reagent can be prepared according to the description in the literature (for example, "Biochemical Experiment Method 1 Quantitative Determination of Reducing Sugars", p. 19-120, Sakuzo Fukui, Gakkai Shuppan Center). It can be prepared by such a procedure.
- the protein according to the present invention is a filamentous fungus, specifically, staphylotricum (
- a cow preferably belonging to the genus Staphvlotrichum), preferably Staphylotrichum 'Cocosporum, more preferably Staphylotrichum' Cocosporum.
- coccosporum can be obtained from IFO 31817 strain. These may be mutants thereof.
- the protein according to the present invention typically has the amino acid sequence at the N-terminal thereof represented by SEQ ID NO: 1.
- the N-terminal amino acid sequence can be determined, for example, by the method described in Example 2 below.
- a protein derived from the genus Stadylotrichum having the following properties:
- proteins having the following properties are proteins having the following properties:
- N-terminal amino acid sequence has the sequence represented by SEQ ID NO: 1,
- the average molecular weight by SDS-PAGE can be determined by the method described in Example 1.
- a protein comprising the amino acid sequence represented by SEQ ID NO: 3, and a modified protein thereof or a homologous protein thereof.
- the "protein containing the amino acid sequence represented by SEQ ID NO: 3" includes, for example, a protein having the amino acid sequence represented by SEQ ID NO: 3, an amino acid sequence obtained by adding a signal peptide to the amino acid sequence represented by SEQ ID NO: 3 And a protein consisting of an amino acid sequence having an appropriate marker sequence added to the N-terminus and Z- or C-terminus of the amino acid sequence represented by SEQ ID NO: 3.
- the signal peptide is, for example, an amino acid sequence consisting of a 21-amino acid residue encoded from a nucleotide sequence beginning with ATG at position 13 and ending at positions 61-63 in the nucleotide sequence represented by SEQ ID NO: 2 (SEQ ID NO: 2). 33).
- the marker sequence for example, a sequence for facilitating confirmation of polypeptide expression, confirmation of intracellular localization, or purification, and the like can be used.
- a histidine 'tag, a hemadaltune' tag, or a myc epitope can be used.
- the "modified protein” in the present invention refers to the amino acid sequence represented by SEQ ID NO: 3, in which one or more (preferably one or several) amino acids are deleted, substituted, inserted, or It is a protein containing an added amino acid sequence and having an endalcanase activity.
- the number of amino acids involved in modification such as “deletion, substitution, insertion, or addition” is preferably 110, more preferably 110, and still more preferably 116.
- the amino acid to be modified is not particularly limited as long as it can maintain or improve the activity of the endoclucanase, for example, a catalytic domain, a linker domain, or a cellulose-binding domain. Amino acids contained in the region can be modified.
- the modified protein includes a protein having an amino acid sequence in which one or more amino acid residues are conservatively substituted in the amino acid sequence represented by SEQ ID NO: 3 and which has an endalcanase activity. Include.
- conservative substitution means replacing one or more amino acid residues with another chemically similar amino acid so as not to substantially alter the activity of the protein. For example, a case where a certain hydrophobic residue is substituted by another hydrophobic residue, a case where a certain polar residue is substituted by another polar residue having the same charge, and the like can be mentioned.
- non-polar (hydrophobic) amino acids include alanine, norin, isoleucine, leucine, proline, tryptophan, phenylalanine, methionine and the like.
- Polar (neutral) amino acids include glycine, serine, threonine, tyrosine, glutamine, asparagine, cysteine, and the like.
- positively charged (basic) amino acids include arginine, histidine, and lysine.
- negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
- the “homologous protein” in the present invention has at least 85%, preferably 90%, and most preferably 95% or more homology (sequence identity) with the protein containing the amino acid sequence represented by SEQ ID NO: 3.
- a protein having an amino acid sequence having the same and having an endalcanase activity are based on the homology search program FASTA3 [Science, 227, 1435-1441 (1985); Proc. Natl. Acad. ScI. USA, 85, 2444- 2448 (1988); http: //www.ddbj.nig.ac.jp/E-mail/homology—j.html], numerical values calculated using default (initial setting) parameters (identity) Is shown.
- endoglucanase STCE1 or endoglucanase STCE3.
- endalcanase STCEJ these are collectively referred to as “endalcanase STCEJ”.
- the endalcanase STCE of the present invention is an endalcanase belonging to family 45 as shown in Example 2 described later.
- Known endarcana belonging to family 45 Examples of the enzyme include, for example, enduclucanase NCE4 (WO98Z03640) or NCE5 (WO01Z90375) derived from the genus Humicola, or RCEI (Endotalucanase) derived from the genus Rhizopus (Eta). International Publication No. 00Z24879 pamphlet).
- Figs. 1 and 2 show the amino acid sequence [signal peptide (SEQ ID NO: 33) and mature protein (SEQ ID NO: 3)] of the endoglucanase STCE1 of the present invention, and a known endoglucanase belonging to family 45. Results of comparing amino acid sequences of NCE4 [signal peptide (SEQ ID NO: 34) and mature protein (SEQ ID NO: 35)] and NCE5 [signal peptide (SEQ ID NO: 36) and mature protein (SEQ ID NO: 37)] Is shown.
- Figure 1 shows the results for the first half (N-terminal side), and Figure 2 shows the results for the second half (C-terminal side).
- the symbol “*” indicates that the amino acid is common to STCE1.
- the endalcanase belonging to family 45 has a catalytic domain (No. 1 to No. 207) as a common domain, and optionally further comprises a linker. It may have one region (Linker) (No. 208-258) and Z or cellulose binding domain (CBD) (No. 259-295).
- the numbers shown in parentheses after each domain are the amino acid numbers in the amino acid sequence of the endalcanase STCE1 (SEQ ID NO: 3).
- a region containing a large number of conserved amino acids may affect the enzymatic activity of the endalcanase of the present invention (for example, STCE1).
- the amino acid modification for example, deletion, substitution, insertion, and Z or addition, particularly conservative substitution
- other regions or amino acids can increase its enzymatic activity. It is possible to obtain a modified protein or a homologous protein maintaining the above at a high V and probability without requiring excessive experimentation.
- an amino acid that is not common between the respective endalcanases may be modified to another amino acid (preferably, a conservatively replaceable similar amino acid)
- another amino acid preferably, a conservatively replaceable similar amino acid
- the modified protein or homologous protein of the present invention may have any region, for example, a catalytic region, a linker, or the like, as long as it has an endalcanase activity (that is, the endalcanase activity before modification is maintained or improved). Includes proteins in which amino acids in the region or in the cellulose binding region have been modified.
- the protein of the present invention can be obtained, for example, by isolating and purifying microbial activity as described in Example 1.
- a polynucleotide encoding the protein of the present invention is expressed in an appropriate host by gene recombination technology, and the produced protein is isolated and purified. Can be.
- a protein comprising the amino acid sequence represented by SEQ ID NO: 3, a modified protein thereof, and a polynucleotide encoding a homologous protein are provided.
- the amino acid sequence of a protein the nucleotide sequence encoding it can be easily determined, and various nucleotide sequences encoding the protein of the present invention can be selected.
- the term “polynucleotide” includes both DNA and RNA, and DNA is preferable.
- polynucleotide of the present invention is typically selected from the group consisting of:
- the number of bases which may be deleted, substituted, inserted or added is preferably 1 to 30 , More preferably 118, more preferably 119.
- the "stringent conditions" in the above (iii) means that a probe containing a sequence having the 64th to 948th basicity in the nucleotide sequence represented by SEQ ID NO: 2 and a homologous protein are encoded. While the probe hybridizes with the polynucleotide, the probe is controlled to such an extent that the probe does not hybridize with the endalcanase NCE5 gene (WO 01Z90375).
- an ECL direct DNA ZRNA labeling detection system manufactured by Amersham
- a probe a full-length nucleotide sequence encoding the amino acid sequence of labeled endalcanase STCE1
- the probe was added, and hybridization at 42 ° C for 15 hours was performed, followed by 0.4% according to the attached operation method.
- the polynucleotide according to the present invention may be a naturally occurring polynucleotide, a fully synthesized polynucleotide, or a polynucleotide synthesized using a part of a naturally occurring polynucleotide. Good.
- a typical method for obtaining the polynucleotide according to the present invention a method commonly used in the field of genetic engineering, for example, based on partial amino acid sequence information, from a genomic library of Staphylotrichum 'coco sporum is used. A method of performing screening using the prepared appropriate DNA probe is exemplified.
- a typical nucleotide sequence encoding the amino acid sequence of the endalcanase STCE1 according to the present invention has a sequence of the 64th to 948th nucleotides in the nucleotide sequence represented by SEQ ID NO: 2. Things.
- the nucleotide sequence represented by SEQ ID NO: 2 is It has an open reading frame that starts with ATG and ends with TAA at 949-951.
- the nucleotide sequence of 64-66 corresponds to the N-terminal amino acid of the mature protein of endoglucanase STCE1 consisting of 295 amino acid residues.
- a nucleotide sequence encoding a protein comprising the amino acid sequence represented by SEQ ID NO: 3, a modified protein thereof and a homologous protein (hereinafter referred to as "polynucleotide according to the present invention") can be replicated in a host microorganism, and And an expression vector containing the protein encoded by the nucleotide sequence in an expressible state.
- This expression vector can be constructed on the basis of a self-replicating vector, that is, an extrachromosomal independent body whose replication does not depend on chromosome replication, for example, a plasmid.
- the expression vector when introduced into a host microorganism, it may be inserted into the genome of the host microorganism and replicated together with the chromosome in which it has been integrated.
- the procedure and method for constructing a vector according to the present invention can use those commonly used in the field of genetic engineering.
- the expression vector according to the present invention has a nucleotide sequence controlling its expression, in addition to the polynucleotide according to the present invention, in order to actually introduce the vector into a host microorganism and express a protein having a desired activity. And a gene marker for selecting a transformant.
- Nucleotide sequences that control expression include those encoding promoters, terminators, and signal peptides.
- the promoter is not particularly limited as long as it exhibits transcriptional activity in the host microorganism, and can be obtained as a nucleotide sequence that controls the expression of a gene encoding a protein of the same or different type as the host microorganism.
- the signal peptide is not particularly limited as long as it contributes to secretion of the protein in the host microorganism, and is derived from a gene encoding the same or different type of protein as the host microorganism. It can be obtained from the nucleotide sequence.
- the gene marker in the present invention may be appropriately selected depending on the method for selecting a transformant. For example, a gene encoding drug resistance and a gene complementing auxotrophy can be used.
- a microorganism transformed with the expression vector there is provided a microorganism transformed with the expression vector.
- the host vector system is not particularly limited, and for example, a system using Escherichia coli, actinomycetes, yeast, filamentous fungi and the like, and a fusion protein expression system with other proteins using them can be used. Further, transformation of a microorganism with the expression vector can be carried out according to the method commonly used in this field.
- the transformant can be obtained by culturing the transformant in an appropriate medium and isolating the protein according to the present invention from the host cell or culture. Therefore, according to another aspect of the present invention, there is provided a method for producing the above-mentioned novel protein according to the present invention.
- the culture of the transformant and its conditions may be essentially equivalent to those of the microorganism used. After culturing the transformant, the method of recovering the target protein may be any of those commonly used in this field.
- yeast cell capable of expressing an endalcanase, which is encoded by the nucleotide sequence of the polynucleotide according to the present invention.
- the yeast cells in the present invention include, for example, microorganisms belonging to the genus Saccharomyces (Saccharomvces), the genus Nosenzenula (Hansenula), or the genus Pichia (£ khk), such as Saccharomyces cerevisiae.
- a method for expression in a filamentous fungus belonging to the incomplete fungi is provided.
- Preferred host filamentous fungi in the present invention include the genus Humicola, the genus Trichoderma, the genus Staphvlotrichum, the genus Aspergillus, the genus Fusarium or the genus Acremonium. And more preferably those belonging to the genus Humicola or Trichoderma. More specifically, Humicola insolens, Humicola thermoidea, and the like. thermoidea), Trichotenorema. Billia (Trichoderma viride), Trichodenorema. Lacey (nchoaerma
- Trichoderno longibrachiatum Trichoderma longibrachiatum
- Staphylo tricum 'Cocosporum Staphylo tricum 'Cocosporum
- Trichoderma viride Trichoderma
- the present invention includes the protein of the present invention (for example, a protein comprising the amino acid sequence represented by SEQ ID NO: 3, or a modified or homologous protein thereof, or a protein obtained by culturing the host cell of the present invention). It also relates to cellulase preparations.
- a cellulase preparation includes, in addition to the cellulase enzyme, for example, excipients (eg, lactose, sodium salt, sorbitol, etc.), preservatives, and Z or nonionic surfactants Can be contained.
- excipients eg, lactose, sodium salt, sorbitol, etc.
- preservatives e.g., sorbitol, etc.
- Z or nonionic surfactants can be contained.
- the form of the cellulase preparation may be either solid or liquid, and specific examples include powders, granules, granules, non-dusty granules, and liquid preparations.
- the cellulase preparations of the present invention include other senorylase enzymes, such as cellobiohydrolase, j8-dalcosidase, and Z, or other endalcanases other than the endalcanase of the present invention, in addition to the protein of the present invention.
- Non-dusty granules (preferably, non-dispersible granules), which are a kind of cellulase preparation, can be produced by a usual dry granulation method. That is, the protein of the present invention in a powdered state is neutral and does not affect the activity of the endalcanase, but does not affect the activity of inorganic salts (eg, sodium sulfate and sodium salt) and the activity of the endalcanase.
- inorganic salts eg, sodium sulfate and sodium salt
- one or more nonionic surfactants After mixing with one or more selected minerals (eg, bentonite, montmorillonite) and neutral organics (eg, starch or granular cellulose), one or more nonionic surfactants or Add multiple powders or finely suspended suspensions and mix or knead well.
- a synthetic polymer for example, polyethylene glycol or the like
- a natural polymer for example, starch or the like
- mold the product It is possible to produce non-dusty granules by molding into a spherical shape and drying.
- the amount of the one or more nonionic surfactants to be added is not particularly limited, but is preferably 0.1 to 50% by weight, more preferably 0.1 to 30% by weight, based on the whole cellulase preparation of the present invention. %, More preferably 1 to 10% by weight. It is also possible to control oxygen permeation and moisture permeation by coating the granule surface with a polymer, etc.
- a liquid preparation (preferably, a stabilized liquid form), which is one of the cellulase preparations, is added to a solution containing the protein of the present invention with an endalcanase stabilizing agent (eg, a synthetic agent).
- an endalcanase stabilizing agent eg, a synthetic agent.
- Polymer, natural polymer, etc.), and if necessary, inorganic salts and Z or a synthetic preservative can be added to prepare the composition.
- one or more nonionic surfactants can be blended.
- the amount of one or more nonionic surfactants added is not particularly limited, but is preferably 0.1 to 50% by weight, more preferably 0.1 to 50% by weight, based on the whole cellulase preparation of the present invention.
- the content is 30% by weight, more preferably 110% by weight.
- the present invention provides a detergent composition comprising the protein of the present invention or the cellulase preparation of the present invention.
- the detergent composition of the present invention may also contain a surfactant (which may be aionic, nonionic, cationic, amphoteric or zwitterionic or a mixture thereof).
- the detergent composition of the present invention may also include other detergent components known in the art, such as builders, bleach, bleach activators, corrosion inhibitors, sequestrants, soil release polymers, fragrances, other Enzymes (eg, proteases, lipases, amylases, etc.), enzyme stabilizers, formulation aids, optical brighteners, and Z or foam enhancers may also be included.
- Typical amphoteric surfactants are linear alkylbenzene sulfonates (LAS), alkyl sulfates (AS), alpha olefin sulfonates (AOS), and polyoxyethylene alkyl ether sulfates. (AES), a sulfo fatty acid ester salt (a-SFMe), and alkali metal salts of natural fatty acids.
- nonionic surfactants include polyoxyethylene alkyl ethers (AE), alkyl polyethylene glycol ethers, nonyl phenol polyethylene glycol ethers, fatty acid methyl ester ethoxylates, sucrose, and fatty acid esters of glucose, and alkyl darcosides. Polyethoxyl And esters of alkyl dalcosides.
- the method for treating the cellulose-containing fiber of the present invention is performed by bringing the protein of the present invention, the cellulase preparation of the present invention, or the detergent composition of the present invention into contact with the cellulose-containing fiber.
- Properties of cellulose-containing fibers that can be improved by the fiber treatment method of the present invention include the following.
- the fiber treatment method of the present invention comprises adding the protein of the present invention, the cellulase preparation of the present invention, or the detergent composition of the present invention to water in which the fibers are immersed or can be immersed.
- it can be performed in a fiber immersion step, a washing step, or a rinsing step.
- Conditions such as the contact temperature and the amount of the protein, cellulase preparation or detergent composition of the present invention to be added may be appropriately determined in consideration of other various conditions.
- the protein, cellulase preparation, or proteinase preparation of the invention at a protein concentration of 0.01-20 mgZL at a temperature of about 10-60 ° C. It is preferred to use a detergent composition.
- the protein and the cellulase of the present invention having a protein concentration of 0.1 to 50 mg ZL at a temperature of about 10 to 60 ° C. It is preferable to use an object or a detergent composition.
- the protein of the present invention having a protein concentration of 0.01 to 20 mgZL at a temperature of about 10 to 60 ° C and a cellulase preparation can be prepared. It is preferred to use an object or a detergent composition.
- the protein of the present invention having a protein concentration of 0.1 lOOmgZL at a temperature of about 20-60 ° C. It is preferred to use a cellulase preparation, or a detergent composition.
- the method described above can reduce the rate at which the cellulose-containing fiber starts to stiffen, or reduce the stiffness of the cellulose-containing fiber, at a temperature of about 10 to 60 ° C, at 0.01 to 20 mg / L. It is preferred to use the protein, cellulase preparation or detergent composition of the invention at a protein concentration of.
- the present invention relates to a method for deinking used paper, wherein the protein of the present invention or the cellulase preparation of the present invention is used in the step of treating the used paper with a deinking chemical to deink.
- the protein or cellulase preparation of the present invention improves the efficiency of deinking when applied to waste paper, waste paper strength is also useful in the process of producing recycled paper. According to the above-described deinking method, the whiteness of the used paper can be improved because the residual ink fiber is significantly reduced.
- the deinking chemical is not particularly limited as long as it is generally used for deinking used paper.
- Examples of the deinking chemical include alkalis such as sodium hydroxide and sodium carbonate, sodium silicate, hydrogen peroxide, and the like.
- Examples include phosphates, aron-based surfactants, non-on-based surfactants, and collecting materials such as oleic acid, and as auxiliary agents, pH stabilizers, chelating agents, or dispersants. And the like.
- the waste paper to which the above-described deinking method can be applied is not particularly limited as long as it is generally called waste paper.
- the waste paper include newspaper paper, magazine waste paper, and lower grades containing mechanical pulp and i-Danigaku pulp.
- Printed waste paper such as intermediate-grade printed waste paper, high-quality waste paper made of chemical pulp, and coated papers of these.
- the above-described deinking method can be applied to any paper other than what is generally called used paper, as long as the paper has ink attached thereto.
- the present invention relates to a method for improving the drainage of paper pulp, the method comprising a step of treating paper pulp with the protein of the present invention or the cellulase preparation of the present invention.
- the norp to which the method can be applied is not particularly limited, For example, waste paper pulp, recycled paperboard pulp, kraft pulp, sulfite pulp, processing heat treatment, and other high-yield pulp may be mentioned.
- the present invention relates to a method for improving digestibility of animal feed, which comprises a step of treating the animal feed with the protein of the present invention or the cellulase preparation of the present invention.
- dalkan in animal feed is appropriately reduced in molecular weight, so that digestibility of animal feed can be improved.
- digestibility of dalcan in feed can be improved.
- a method for improving the digestibility of an animal feed comprising the step of treating the animal feed with the protein or cellulase preparation of the present invention.
- the staphylotricum 'cocosporum (Staphvlotrichum coccosporum) IFO 31817 strain derived from the endalcanase STCE according to the present invention was deposited in Japan in 1985 at the Yeast Research Institute, and has a domestic accession number. Is IFO 31817. Also, on September 28, 2004, the Patent Organism Depositary Center for Patent Organisms, National Institute of Advanced Industrial Science and Technology (address: 305-8566, Tsukuba, Higashi, Ibaraki, Japan) ), And the international deposit number is FERM BP-10135.
- IFO 31817 strain is currently commissioned as NBRC 3 1817 by the Biotechnology Division of the National Institute of Technology and Evaluation (2-5-8 Kazusa Kadami Park, Kisarazu, Chiba 292-0818, Japan). The fact that the strain is open to the public and available for sale has been clarified by a certificate issued by the headquarters [November 19, 2003].
- Escherichia coli DH5a ZpUCl 18 of the present invention transformed with plasmid pUCl 18 -STCEex in which STCE1 gene was inserted into the ⁇ mHI site of plasmid pUCl 18-STCEex strain was obtained in 2003 (Heisei 15). It was deposited on December 1 at the National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary Center (original deposit) and transferred to the International Depositary on September 15, 2004 (Heisei 16)! You.
- the international accession number (the domestic accession number following the international accession number in []) is FERM BP-10127 [FERM P-19602].
- the Humicola insolens MN200-1 strain which can serve as a host for the expression vector of the present invention, was deposited on July 15, 1996 (Heisei 8), Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (AIST). Was deposited in Japan (original deposit) and transferred to an international deposit on June 13, 1997 (Heisei 9)!
- the international accession number (the number in parentheses following the international accession number is domestic accession number) is FERM BP-5977 [FERM P-15736].
- Trichoderma viride M C300-1 strain which can serve as a host for the expression vector of the present invention, was deposited on September 9, 1996 (April, 1996) with the National Institute of Advanced Industrial Science and Technology (Patented Organism). It was deposited at the center in Japan (original deposit), and on August 11, 1997, power was transferred to the international deposit.
- the international accession number (the number in [] following the international accession number is the domestic accession number) is FERM BP-6047 [FERM P-15842].
- Example 1 Isolation and Purification of Staphylotricum 'Cocosporum Powerful Component Having Absorbent Cotton Fibril Release Activity'
- This crude cellulase preparation was prepared to a final concentration of 1.5 mol ZL ammonium sulfate solution, and then equilibrated with 1.5 mol ZL ammonium sulfate solution.
- a HiTrap TM PhenylHP column (Amersham Biotech) (Manufactured by Science Inc.) and the stepwise elution method of ammonium sulfate concentration in deionized water is 1.5 molZL, 0.9 molZL, 0.75 mol / L, 0.6 mol / L, 0.15 molZL, OmolZL And fractionated. Among them, the fraction eluted when the concentration of ammonium sulfate was 0.75 mol ZL and Omol ZL showed strong activity to release cotton wool fibrils.
- the eluted fraction of the ammonium sulfate OmolZL was desalted using UltraFree ZBiomax-5K (manufactured by Millipore) and adjusted to 50 mmol ZL acetate buffer (pH 4.0).
- the mixture was applied to a MonoS 5Z5HR column (manufactured by Amersham Bioscience) equilibrated with a 50 mmol / L acetate buffer (pH 4.0).
- the fraction was eluted from 50 mmol / L acetate buffer (pH 4.0) to ImolZL sodium chloride in 50 mmol ZL acetate buffer (pH 5.0) by linear gradient elution.
- the fraction obtained when the sodium salt concentration was about 0.05 mol ZL had a strong activity of releasing cotton wool fibrils. This fraction was isolated as STCE1.
- the fraction eluted with 0.75 mol ZL of ammonium sulfate is desalted similarly using UltraFree ZBiomax-5K (Millipore), and then becomes 50 mmol ZL acetate buffer (pH 4.0). It was adjusted and applied to a MonoS 5Z5HR column (manufactured by Amersham Biosciences) equilibrated with 50 mmol ZL acetate buffer (pH 4.0). Then, a 50 mmol ZL acetic acid buffer (pH 4.0) was eluted with a linear gradient elution method from a 50 mmol ZL acetic acid buffer (pH 5.0) to ImolZL sodium chloride, and fractionated.
- the STCE1 fraction and the STCE3 fraction showed single bands on SDS-PAGE, respectively, and their average molecular weights (MW) were about 49 kD and about 45 kD, respectively.
- SDS-PAGE was carried out using a safety cell mini STC-808 electrophoresis tank (made of Tefcon® earth) and precast migel 10% —SDS-PAGEmini, 1.0 mm gel thickness (manufactured by Tefco). Dyeing was performed according to the method described in the product instruction manual.
- a molecular weight marker a precision protein standard (manufactured by Bio-Rad Laboratories) was used.
- the STCE1 fraction and the STCE3 fraction also had CMCase activity in the difference!
- Absorbent cotton fibril releasing activity was carried out by a method improved from the method of Neena Din et al. (Neena Din et al., "Biotechnology", 9 (1991), p. 1096-1099). That is, the amount of fluff released from absorbent cotton when the reaction was carried out under the following conditions using a washing fastness tester was measured at an absorbance of 600 nm.
- Example 1 the obtained STCE1 and STCE3 fractions were subjected to SDS-PAGE, and then electrically transferred to PVDF membrane Proplot TM (manufactured by Applied Biosystems). Then, the cells were stained with a CBB staining solution (0.1% Kumasi Blue G250, 30% methanol, 10% acetic acid), decolorized, and air-dried.
- CBB staining solution (0.1% Kumasi Blue G250, 30% methanol, 10% acetic acid
- Example 1 STCE1 and STCE3 obtained in Ultra Free ZBiomax-5K Desalted using Lipoa) and freeze-dried.
- the freeze-dried STCE1 and STCE3 were placed in a microtube having a capacity of about 4 O ⁇ g ⁇ l and 5 mL, and 500 L of a reducing buffer solution was added thereto and dissolved. Thereto was added 1.4 mg of dithiothreitol, sealed with nitrogen, sealed, and allowed to stand for 5 hours. Further, 2.7 mg of monoiodine acetic acid was added, and the mixture was allowed to stand still for 30 minutes in the absence of light to carry out carboxymethylation of cysteine residues of the protein.
- LE-3 Asn-Ala-Asp-Asn-Pro-Thr-Phe-Thr-Phe-Arg (10 residues) (SEQ ID NO: 6)
- LE-4 Ala- Ser-Vatro Asn-Gin-Pro-Va Phe-Ala-Cys (10 residues) (SEQ ID NO: 7)
- LE-5 Pro-Gly-Cys-Tyr-Trp-Arg-Phe (7 residues) (SEQ ID NO: 8)
- LE-10 Asn-Ala-Asp-Asn-Pro-Thr-Phe-Thr-Phe-Arg (10 residues) (SEQ ID NO: 13)
- LE-11 Ala— Ser— Va Asn— Gin— Pro— Va Phe — Ala— Cys— Ser— Ala— Asn— Phe— Gin— Arg (1 6 residues) (SEQ ID NO: 14)
- STCE3 was also considered to be an endalcanase belonging to family 45. However, since the corresponding internal amino acid sequences of STCE1 and STCE3 were completely identical, it was suggested that STCE3 might be a partial degradation product derived from STCE1.
- Example 2 Using the crudely purified cellulase preparation obtained in Example 1 and the purified endalcanase STCE1, the activity of removing fluff on cotton knitted fabric was evaluated as follows.
- the pre-dyed blue cotton knit fabric was fluffed in a large dish with a surfactant and rubber balls. Thereafter, the fluffed blue cotton knit fabric is subjected to a fuzz removal treatment under the following conditions, whereby a crudely purified cellulase preparation solution required to remove approximately 50% of the formed fuzz by visual evaluation and a purification end.
- the protein concentration of dalcanase STCE1 was calculated.
- washing fastness tester L-12 (made by Daiei Kagaku Seiki Seisakusho Co., Ltd.)
- Protein concentration was determined using a protein assay kit (Bio-Rad Laboratories). Serum albumin was quantified as a standard. The results are shown in Table 1.
- the protein concentration was determined using a protein assay kit (manufactured by Bio-Rad Laboratories) using bovine serum albumin as a standard. The results are shown in Table 2.
- Purified endodalcanase NCE5 International Publication No. 01Z90375 pamphlet
- RCEI RCEI [RCEI-H4 lacking the cellulose binding domain] (25 kD)
- International Publication WO02 / 42474 pamphlet International Publication WO02 / 42474 pamphlet
- the pre-dyed blue cotton knit fabric was fluffed in a large dish with a surfactant and rubber balls. Then, by treating the fluffed blue cotton knit fabric in a detergent under the following conditions, the purified endoglucanases STCE1, NCE4, NCE5, and NCE5 required for about 50% of the formed fluff to be visually removed. And RCEI protein concentrations were calculated.
- washing fastness tester L-12 (made by Daiei Kagaku Seiki Seisakusho Co., Ltd.)
- Treatment solution Artificial hardness water (25FH: Deionized water 80mmol ZL calcium chloride, 20m Manufactured by diluting 1000FH artificial hardness water containing molZL magnesium chloride with deionized water. )
- the protein concentration was determined using a protein assay kit (manufactured by Bio-Rad Laboratories) using bovine serum albumin as a standard. Table 3 shows the results.
- Example 6 Hardness in tap water gives the fluff removing activity of various purified endalcanases.
- Purified endorcanase STCE1 obtained in Example 1, culture of Humicola 'insolens.
- Endocalcanase NCE5 International Publication No. 01Z90375 pamphlet
- Purified Endorcanase NCE4 International Publication No.
- the fluffed brown cotton knit fabric was treated in artificial hardness water of various hardnesses (OFH, 5FH, 10FH, 20FH, 40FH) using four types of purified endalcanase solutions under the following conditions.
- the protein concentrations of the purified endalcanases STCE1, NCE5, NCE4, and RCEI required to visually remove about 50% of the formed fuzz are calculated, and the reciprocal value of the protein concentration is calculated.
- the removal activity value was used.
- the fluff removal activity value at a hardness of 0FH is 100
- the relative value of the fluff removing activity at each hardness was determined.
- Treatment solution Artificial hardness water (OFH, 5FH, 10FH, 20FH, 40FH: Prepared by diluting 1000FH artificial hardness water with deionized water and 80mmolZL calcium chloride and 20mmolZL magnesium chloride mixed with deionized water. )
- Staphylotrichum cocosporum IFO 31817 strain in (T) medium (2.0% Abyssenolle, 2.0 % Yeast extract, 2.0% corn steep liquor, 1.0% glucose, 0.2% potassium phosphate) at 28 ° C for 72 hours, and the cells were collected by centrifugation.
- the obtained cells were freeze-dried, crushed with a blender, and dissolved in 8 mL of TE (10 mmol ZL Tris-HCl (pH 8.0), lmmol / L EDTA) buffer. To this was added 4 mL of TE buffer containing 10% SDS, and the mixture was kept at 60 ° C for 30 minutes.
- the precipitate is dissolved in 5 mL of TE buffer, 5 L of lOmgZmL of ribonuclease-18 (RNase A) solution is added, and the mixture is incubated at 37 ° C for 1 hour, and then 20 mgZmL of proteinase K (proteinase K). K) 50 L of the solution was added, and the mixture was kept at 37 ° C for 1 hour. Then, 3 mL of polyethylene glycol solution (20% PEG6000, 2.5 mol ZL sodium chloride) was added to precipitate DNA. The precipitate was dissolved in 500 L of TE buffer, and extracted twice with phenol-cloth form-isoamyl alcohol to precipitate ethanol. The precipitate was washed with 70% ethanol, dried, and dissolved in an appropriate amount of TE buffer to prepare a genomic DNA sample.
- Example 7 About 10 ⁇ g of the genomic DNA of staphylotricum 'cocosporum' obtained in (1) was digested with a plurality of restriction enzymes (EmRI, BamHI, Hindlll, XhoL ⁇ I, etc.). The sample was subjected to 8% agarose gel electrophoresis. This was transferred to a nylon membrane (Hybond N + nylon transfer membrane, manufactured by Amersham), DNA was fixed with 0.4 N sodium hydroxide, and washed with 5-fold concentration SSC (75 mmol ZL trisodium citrate, 750 mmol ZL sodium chloride). , Dried and the DNA fixed.
- a plurality of restriction enzymes EmRI, BamHI, Hindlll, XhoL ⁇ I, etc.
- the sample was subjected to 8% agarose gel electrophoresis. This was transferred to a nylon membrane (Hybond N + nylon transfer membrane, manufactured by Amersham), DNA was fixed with 0.4 N sodium hydrox
- the probe is to digest the plasmid pJND-c5 (International Patent Publication No. WO 01/90375) containing the cDNA of the NCE5 gene with ⁇ mHI, and then subject it to 0.8% agarose gel electrophoresis to recover a DNA fragment of about 700 bp. Obtained by This was labeled with an ECL direct DNAZ RNA labeling detection system (Amersham). Following pre-hybridization of the genomic DNA-immobilized membrane at 42 ° C for 1 hour according to the method described in the instructions attached to the kit, the labeled NCE5 probe was added thereto. Then 4 Hybridization was performed at 2 ° C for 15 hours.
- washing of the nylon membrane after the hybridization was performed according to the method described in the instruction manual attached to the kit. First, washing twice with 0.4% SDS and 6 mol ZL urea in 0.6 times concentration SSC (9 mmol ZL trisodium citrate, 90 mmol ZL sodium chloride) at 42 ° C for 20 minutes was repeated twice. Washing was performed twice with double concentration SSC at room temperature for 5 minutes. The washed nylon membrane was immersed in the attached detection solution for 1 minute, and then exposed to Fuji Medical X-ray film (Fuji Photo Film Co., Ltd.).
- the genomic DNA of Staphylotrichum cocosporum was digested with Es ⁇ RI and subjected to 0.8% agarose gel electrophoresis using SeaKemLE agarose (FMC).
- FMC SeaKemLE agarose
- a DNA fragment having a size of about 8 to 12 kbp was extracted and purified according to a standard method so as to contain around 10 kbp.
- This DNA fragment was ligated to a phage vector, Lambda DASH II vector (Stratagene). After ethanol precipitation, this was dissolved in TE buffer, and the entire amount was packaged in a lambda head using a Gigapack III Gold Packaging Kit (Stratagene), and the resulting phage was transformed into E. coli XL1-Blue MRA. The strain was infected. Cloning of the target gene was performed using a 5 ⁇ 10 4 phage library obtained by this method.
- the genomic DNA library (toRI library) obtained according to Example 7 (3) was transferred to a nylon membrane (Nybond N + Nylon transfer membrane, manufactured by Amersham), and 0.4N sodium hydroxide was added.
- the DNA was fixed with, washed with 5-fold concentration SSC and dried to fix the DNA.
- the probe of the NCE5 gene labeled in Example 7 (2) was added, and hybridization was performed at 42 ° C. for 15 hours.
- washing of the nylon membrane after the hybridization was performed in accordance with the method described in the instructions attached to the kit. First, washing with 0.4% SDS and 6 mol ZL urea was added to 0.6 times concentration SSC and washing at 42 ° C for 20 minutes twice, and then washing with 5 times concentration SSC at room temperature for 5 minutes. I went round. The washed nylon membrane was immersed in the attached detection solution for 1 minute, and then exposed to Fuji Medical X-ray film (Fuji Photo Film Co., Ltd.) to obtain four phage clones.
- E. coli XL1-Blue MRA strain was infected with phage, and phage particles were collected 18 hours later. These particles were treated with proteinase K and phenol according to the method of Grossberger (Grossberger, D., "Nucleic Acids Res.”, 15, 6737, 1987), and then phage DNA was separated by ethanol precipitation. .
- phage DNAs were digested with a plurality of restriction enzymes and subjected to 0.8% agarose gel electrophoresis.
- the DNA was transferred to a nylon membrane by the Southern method (Southern, EM, "J. Mol. Biol.”, 98, p. 503-517, 1975). went.
- the four types of phage DNAs showed a common hybridization pattern even when multiple restriction enzymes were cut.
- a signal strong in the band of about 4.4 kbp and a signal of about 2.5 kbp in common were obtained. This band showed a weak signal.
- this homologous gene exists over the DNA of about 4.4 kbp and about 2.5 kbp, and the DNAs of these two sizes are collected and sub- Crawling was done.
- the plasmid obtained by subcloning about 4.4 kbp of DNA was named pSTCE-Sal4.4, and the plasmid obtained by subcloning about 2.5 kbp of DNA was named pSTCE-Sal2.5.
- the nucleotide sequence analysis of the NCE5 homologous gene subcloned in Example 7 (5) proceeded as follows.
- An ALF DNA Sequencer II (Pharmacia Biotech) was used as a base sequence analyzer.
- As a sequencing gel an acrylamide carrier available as a No., Idoguchi Link Long Ranger (manufactured by FMC) was used.
- ALF grade reagent (Pharmacia Biotech) was used for various gel preparation reagents (N, N, ⁇ ', N'-tetramethylethylenediamine, urea, and ammonium persulfate).
- an auto-read sequencing kit (Pharmacia Biotech) was used for the base sequence decoding reaction. Gel preparation conditions, reaction conditions and electrophoresis conditions were set with reference to the respective instructions attached to the kit.
- nucleotide sequences of the approximately 4.4 kbp DNA fragment in the plasmid pSTCE-Sal4.4 and the approximately 2.5 kbp DNA fragment in the plasmid pSTCE-Sal2.5 were determined according to a conventional method. Furthermore, when the decoded base sequence was translated into an amino acid sequence, one open reading frame completely matched the N-terminal amino acid sequence of STCE1 and the internal amino acid sequence shown in Example 2. These results suggested that this NCE5 homologous gene is a gene encoding STCE1. Therefore, hereinafter, this NCE5 homologous gene is described as STCE1 gene.
- STCE1 is an endoglucanase belonging to family 45, which has a catalytic domain on the N-terminal side and a cellulose-binding domain on the C-terminal side. It turned out to be. However, since it was presumed that this DNA sequence contained a region considered to be an intron, the cDNA of the STCE1 gene was isolated by RT (reverse transcriptase) PCR.
- Staphylotrichum cocosporum IFO 31817 strain in 30 mL of (T) medium (2.0% Abycel, 2.0% yeast extract, 2.0% corn steep liquor, 1.0% glucose, 0.2% phosphorus (Monopotassium acid) at 28 ° C for 72 hours, and the cells were collected by centrifugation. The obtained cells were freeze-dried and finely crushed with a spatula. Total RNA was isolated using Isogen (Wako Pure Chemical Industries).
- mRNA was prepared using an mRNA isolation kit (Stratagene).
- 10 mL of the lysis buffer was added to 0.2 mL of the total RNA prepared above, and 5 mL of the oligo dT solution was further purified.
- the oligo dT was washed three times with a high salt buffer and twice with a low salt buffer, and then eluted with an lysis buffer heated to 68 ° C. This solution was precipitated with ethanol, and the precipitate was washed with 75% ethanol, dried, and dissolved in 15 / z L of water to obtain an mRNA fraction.
- STCE1-CN 5 GCGGATCCATGCGTTCCTCCCCCGTC-3 '(26mer) (SEQ ID NO: 18)
- the RT-PCR reaction was performed under the following conditions. First, add C-terminal primer and reverse After reacting with transcriptase, Taq polymerase (recombinant Taq, manufactured by Takara Shuzo), N-terminal primers, 94 ° C for 1 minute, 50 ° C for 2 minutes, 72 ° C for 2 minutes Amplification was performed by repeating the reaction conditions 30 times. The amplified fragment was one fragment of about lkbp as a result of agarose gel electrophoresis. This was subcloned into pUC18 (plasmid pUCSTCE1).
- the nucleotide sequence of the fragment was determined according to the method of Example 8. Furthermore, the intron was determined by comparing this nucleotide sequence with the nucleotide sequence of the genome. As a result of this analysis, the entire nucleotide sequence of the cDNA of STCE1 gene derived from Staphylotrichum 'cocosporum was determined (SEQ ID NO: 2). Based on the sequence information of the obtained cDNA of the STCE1 gene, a DNA fragment containing the STCE1 gene translation region containing introns was isolated. A mixture of the four kinds of phage DNAs used in Example 7 (5) was subjected to PCR using the primers STCE-HNBam and STCE-HCBam as a type III. The amplified fragment was separated and purified by agarose electrophoresis, cut with an EamHI trowel, and then separated and purified by agarose electrophoresis again.
- STCE-HNBam the primers STCE-HCBam
- the above fragment of about 1. lkbp was inserted into the ⁇ mHI site of plasmid PUC118 to obtain plasmid pUC118-STCEex (FERM P-19602). Furthermore, the nucleotide sequence of this inserted fragment was determined by the method described above, and the sequence of the intron and translation region of the STCE1 gene (SEQ ID NO: 22) was confirmed. In addition, the use of the primer STCE-HCBam at the time of PCR caused the termination codon sequence to change into a TAA force TGA. The amino acid sequence of the generated protein did not change.
- Expression vector in Humicola insolens MN200-1 strain (FERM BP-5977)
- One pJND—STCE1 was engineered such that the expressed protein fused the N-terminal 16 amino acid residues of NCE4 with the remaining amino acid residues of STCEl. That is, it was constructed using plasmid PJND-NCE4 in which the NCE4 gene (International Publication No. 98Z03640) was ligated to the ⁇ mHI site of plasmid pJDOl (International Publication No. 00Z24879).
- NCE4 match the N-terminal 16 amino acid residues of STCE1, even if the N-terminal 16 amino acid residues of NCE4 and the remaining amino acid residues of STCE1 are fused. This is because the expressed protein is identical to STCE1.
- the plasmid pJND-NCE4 was prepared as follows. First, the following primers were designed so that the NCE4 gene (WO98Z03640 pamphlet) could be ligated to the EamHI site of the plasmid pJDOl, with the sequence immediately upstream of the start codon and the EamHi site immediately downstream of the stop codon. According to the method described in Example 4 (1) 1) of WO 01Z90375 pamphlet, the plasmid pCNCE4 (WO 98Z03640 pamphlet) was subjected to mutation by PCR method and amplified by PCR.
- the plasmid subcloned at the ⁇ mHI site of the plasmid pJDOl (Example D1 (2) (b) of WO 00/24879) was designated as pJND-NCE4. .
- a fragment amplified by PCR using the plasmid pUC-STCE1 prepared in Example 9 (2) as a type I and STCE1-N-S9A4 and STCE1-C FokF as primers was separated and purified by agarose electrophoresis, and BamHI and BamHI were separated and purified. After cutting with Fokl, agarose electrophoresis again Separation and purification were performed.
- the aforementioned plasmid pJND-NCE4 was separated and purified by agarose gel electrophoresis using the PCR-amplified fragment with STCE1-N-FokR4 and STCE1-C-BamF primers, cut with BHI and £ 2kl, and re-against agarose gel. Separation and purification were performed by electrophoresis.
- STCE1-C-BamF 5'-GGGATCCTGGGACAAGATGC-3 '(20mer) (SEQ ID NO: 28)
- the above two fragments were inserted into the EamHI site of plasmid PJD01 to obtain plasmid pJND-STCEl. Furthermore, the nucleotide sequence of this inserted fragment was determined by the method described above, and it was confirmed that the fragment was the same as the STCE1 gene (SEQ ID NO: 2).
- the obtained cells were washed with 0.5 mol / L sucrose, and filtered through a 0.45 ⁇ m filter to obtain a protoplast-forming enzyme solution (3 mg ZmL ⁇ -glucuronidase, lmgZmL chitinase, lmgZmL zymoryase). (Zymolyas e), 0.5 mol / L sucrose) Suspended in 10 mL. The mixture was shaken at 30 ° C. for 60-90 minutes, and the mycelium was protoplasted.
- This protoplast was suspended in 1 mL of SUTC buffer solution, and 10 ⁇ g of a solution containing 10 ⁇ g of plasmid pJND-STCE1 was added to 100 ⁇ L of the suspension, and the mixture was allowed to stand on ice for 5 minutes. Then 4 A 100 / zL PEG solution (60% PEG4000, lOmmolZL calcium chloride, lOmmolZL tris-hydrochloride (pH 7.5)) was added to the mixture, and allowed to stand on ice for 20 minutes. Centrifuged at 2500 rpm for 10 minutes. The collected protoplasts were suspended in 1 mL of SUTC buffer, followed by centrifugation at 4000 rpm for 5 minutes, and finally suspended in 100 L of SUTC buffer.
- PEG solution 50% PEG4000, lOmmolZL calcium chloride, lOmmolZL tris-hydrochloride (pH 7.5)
- the protoplasts were placed on YMG medium (1% gnorecos, 0.4% yeast extract, 0.2% malt extract, 1% agar (pH 6.8)) supplemented with nodigromycin (200 ⁇ g / mL).
- the cells were overlaid with soft agar and cultured at 37 ° C for 5 days, and the formed colonies were used as transformants.
- Plasmid pJND-STCE1 was introduced into Humicola 'Insolens MN200-1 strain, and 40 strains showing hygromycin resistance were selected. These were cultured in (N) medium (5.0% Avicel, 2.0% yeast extract, 0.1% polypeptone, 0.03% magnesium sulfate, pH 6.8) at 37 ° C for 4 days, and obtained. The culture supernatant was analyzed by SDS-PAGE electrophoresis (precast migel 14% —SDS—PAGEmini, 1. Omm gel thickness (made of Tefcone earth)). As a result, it was confirmed that in 13 clones, a protein having a molecular weight of 45-49 kD, which is estimated to be STCE1, was significantly enhanced.
- the N-terminal amino acid sequence was determined to confirm that the protein overexpressed in (i) was derived from the STCE1 gene.
- the culture supernatant was separated by SDS-PAGE electrophoresis, the protein was electrically transferred to a PVDF membrane according to the method of Example 2, and the protein having a molecular weight of about 45 to 49 kD was subjected to a protein sequencer. As a result, it was consistent with the N-terminal amino acid sequence of the endalcanase STCE1 (SEQ ID NO: 1).
- Example 10 In the SDS-PAGE of (3), the culture supernatant of one of the 13 strains (4A-9 strain), of which expression was particularly remarkable, among 13 strains in which the expression of a protein having a molecular weight of 45-49 kD was confirmed was used.
- the fluff removal activity of the cotton knitted fabric was measured using the same.
- Non-transformant as control A culture supernatant derived from the parent strain was used.
- the method is as described in Example 3, and the fluff formed is visually observed by performing a fluff removal treatment with various culture supernatants on a blue cotton knitted fabric that has been previously fluffed under a treatment condition of pH 6, 40 ° C, and 1 hour.
- the protein concentration in the culture supernatant required for almost 100% removal in the evaluation was calculated.
- the protein concentration was determined using a protein assay kit (manufactured by Bio-Rad Laboratories) using bovine serum albumin as a standard. The results are shown in Table 5.
- Example 10 Using the culture supernatant of the STCE1 large expression strain (4A-9 strain) obtained in Example 10 and the parent strain (MN200-1 strain) which is a non-transformant, 12 oz. was subjected to a decolorizing treatment under the following conditions.
- a pre-dyed brown cotton knit fabric was fluffed in a large dish with a surfactant and rubber balls. Then, by treating the fluffed brown cotton knit fabric in a detergent under the following conditions, the protein concentration in the amount of the culture supernatant required for about 50% of the formed fluff to be visually removed is obtained. I asked.
- Treatment solution Artificial hardness water (25FH: prepared by diluting 1000FH artificial hardness water obtained by mixing 80 mmol ZL calcium chloride and 20 mmol ZL magnesium chloride in deionized water with deionized water.)
- washing fastness tester L-12 (made by Daiei Kagaku Seiki Seisakusho Co., Ltd.)
- Treatment solution Artificial hardness water (OFH, 5FH, 10FH, 20FH, 40FH: Prepared by diluting 1000FH artificial hardness water with deionized water and 80mmolZL calcium chloride and 20mmolZL magnesium chloride mixed with deionized water. )
- the protein concentration in the amount of the culture supernatant required to visually remove approximately 50% of the formed fluff was determined, and the reciprocal value of the protein concentration was defined as the fluff removal activity value. .
- the fuzz removal activity value at a hardness of 0FH was set to 100, relative values of the fuzz removal activity at various hardnesses were determined. The results are shown in Table 8.
- STCE1 For the STCE1 gene, the following mutation-introducing primers were designed so as to contain ⁇ mi in the sequence upstream of the start codon and 20mi downstream of the stop codon, and were amplified by PCR.
- STCEl-N-Smal For the STCE1 gene, the following mutation-introducing primers were designed so as to contain ⁇ mi in the sequence upstream of the start codon and 20mi downstream of the stop codon, and were amplified by PCR.
- PDH25 (Cullen, D., Leong, S.A., Wilson, LJ AND Henner, DJ, "Gene", 57, p. 21-26, 1987) originated in the plasmid STCE1-M2 site.
- the resistance cassette was inserted and the plasmid STCE1N-pCBl was constructed.
- the reaction conditions such as enzymes were in accordance with the instructions attached to the kit.
- Plasmid STCE1N-pCBl was constructed to express the STCE1 protein using its own initiation codon in the host Trichoderma.
- the STCE1 gene is designed with the following mutation-introducing primers containing 2 ⁇ 1 immediately upstream of the codon of the amino acid (Ala) encoding the N-terminus and 0 ⁇ 1 downstream of the stop codon, and is used for PCR. Amplified.
- TaKaRa LA Taq with GC buffer (Takara Shuzo) was used. Under reaction conditions! At last, the conditions of the instructions attached to the enzyme were followed. The sample after the reaction was separated by agarose gel electrophoresis, and further cut with restriction enzymes Sfilll and ⁇ to obtain a gene fragment STCE1-M of about 0.9 kbp.
- pCBl-M2 (WO98 / 11239) was digested with restriction enzymes and Xhol, and a 7.3 kbp fragment was recovered. The 0.9kbp gene fragment STCE1— M was ligated using TaKaRa DNA Ligation Kit Ver. 1 (Takara Shuzo) to produce plasmid STCE1-M2.
- Plasmid STCE1M-pCBl was constructed in host Trichoderma to express STCE1 protein in the form of a fusion protein with the pre-pro sequence of the CBHI protein, using the initiation codon from the vector.
- Trichoderma viride MC300-1 strain (FERM BP-6047) was added to an S medium (3.0% glucose, 1.0% yeast extract, 0.1% polypeptone, 0.14% ammonium sulfate, 0.1%).
- the cells were cultured in 2% potassium phosphate, 0.03% magnesium sulfate, pH 6.8) at 28 ° C for 24 hours, centrifuged at 3000 rpm for 10 minutes, and collected. The obtained cells were washed with 0.5 mol ZL sucrose, and filtered with a 0.45 m filter to obtain a protoplastidase enzyme solution (5 mg / mL Novozyme 234, 5 mg / mL Cellular Zeonodzuka R-10, 0.5 mol ZL).
- This protoplast is suspended in 1 mL of SUTC buffer, and 10 L of a DNA solution containing 10 ⁇ g of STCE1N-pCBl or STCE1M-pCBl is added to 100 ⁇ L of this, and the mixture is left on ice for 5 minutes. did.
- add 400 / ⁇ ⁇ ⁇ ⁇ 3 ⁇ 4 (60% ⁇ 4000, lOmmol ZL chloride, 10 mmol / L Tris-HCl (pH 7.5)), leave it on ice for 20 minutes, and add 10 mL of SUTC buffer. And centrifuged at 2500 rpm for 10 minutes.
- the collected protoplasts were suspended in 1 mL of SUTC buffer, then centrifuged at 4000 rpm for 5 minutes, and finally suspended in 100 ⁇ L of SUTC buffer.
- the protoplasts were prepared by adding igugromycin B (20 ⁇ g / mL) to potato potato dextrose (P D) Overlay on PD agar (1.3% potato dextrose agar, 17.1% sucrose) on agar medium (3.9% potato dextrose sagger, 17.1% sucrose) and at 28 ° C After culturing for 5 days, the formed colonies were used as transformants.
- Example 16 Identification of STCE1 in culture medium of Trichoderma's viride transformant of STCE1 gene and evaluation of fluff removing activity
- Plasmids STCE IN-pCBl and STCE1M-pCBl were introduced into Trichoderma 'viride MC300-1 strain, and 50 strains showing resistance to hygromycin B were selected. These were cultured in S medium at 37 ° C for 5 days, and the resulting culture supernatant was applied to a TSKgel TMS-250 column (4.6 mml. D.X 7.5 cm) (Tosoichi Co., Ltd.) HPLC eluted with a linear gradient from 0% to 80% acetonitrile in 0.05% TFA (trifluoroacetic acid) at a flow rate of 1. OmL / min and detected a peak at UV 280 nm.
- Example 16 the culture supernatant of STCE1 was expressed and the parent strain (MC300-1 strain), which was a non-transformant, was used. (5 mmol ZL sodium carbonate buffer) at 40 ° C for 1 hour, and the fluff is removed by performing a fluff removal treatment with various culture supernatants on a previously fluffed blue cotton knit fabric. Then, the protein concentration in the culture supernatant required to remove approximately 50% was calculated.
- the protein concentration was determined using a protein assay kit (manufactured by Bio-Rad Laboratories) using bovine serum albumin as a standard. Table 9 shows the results.
- Trichoderma viride (STCE lN-pCBl transformant) 2 7 Trichoderma 'viride (STCE lM-pCBl transformant) 2 8 Industrial applicability
- novel endalcanase STCE of the present invention can be applied to uses such as treatment of cellulose-containing fibers, deinking of waste paper, improvement of drainage of paper pulp, and improvement of digestibility of animal samples. it can.
- FIG. 1 The amino acid sequence [signal peptide (SEQ ID NO: 33) and mature protein (SEQ ID NO: 3)] of the endoglucanase STCE1 of the present invention, and NCE4 [signal peptide (known as endoglucanase belonging to family 45) SEQ ID NO: 34) and the mature protein (SEQ ID NO: 35)] and NCE5 [Signal peptide (SEQ ID NO: 36) and mature protein (SEQ ID NO: 37)]
- FIG. 9 is an explanatory diagram showing a result regarding an array.
- FIG. 2 is an explanatory diagram showing the result of the comparison result shown in FIG. 1 concerning the C-terminal sequence.
- the base sequences to be used are as follows: Primer NCE4—N-BamHI, Primer NCE4—C BamHI, Primer STCE1—N—S9A4, Primer Mer STCEl—C—FokF, Primer STCEl—N—FokR4, Primer STCEl—C—BamF, Primer STCEl—N—Smal, Primer STCEl—C—XhoI, Primer S TCEl—M—Sphl, and Primer STCEl—C—Xhol It is.
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Priority Applications (6)
Application Number | Priority Date | Filing Date | Title |
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ES04792875.9T ES2575526T3 (es) | 2003-12-03 | 2004-10-22 | Endoglucanasa STCE y preparación de celulasa que contiene la misma |
DK04792875.9T DK1700917T3 (en) | 2003-12-03 | 2004-10-22 | ENDOGLUCANASE STCE AND CELLULASE PREPARATION CONTAINING THE SAME |
JP2005515882A JP4547335B2 (ja) | 2003-12-03 | 2004-10-22 | エンドグルカナーゼstceおよびそれを含むセルラーゼ調製物 |
CN2004800361057A CN1902315B (zh) | 2003-12-03 | 2004-10-22 | 内切葡聚糖酶stce和含有内切葡聚糖酶的纤维素酶配制品 |
US10/581,717 US7595182B2 (en) | 2003-12-03 | 2004-10-22 | Endoglucanase STCE and cellulase preparation containing the same |
EP04792875.9A EP1700917B1 (en) | 2003-12-03 | 2004-10-22 | Endoglucanase stce and cellulase preparation containing the same |
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JP2003-404020 | 2003-12-03 | ||
JP2003404020 | 2003-12-03 |
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Also Published As
Publication number | Publication date |
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CN1902315A (zh) | 2007-01-24 |
DK1700917T3 (en) | 2016-07-25 |
ES2575526T3 (es) | 2016-06-29 |
JPWO2005054475A1 (ja) | 2007-12-06 |
US7595182B2 (en) | 2009-09-29 |
EP1700917A1 (en) | 2006-09-13 |
EP1700917B1 (en) | 2016-04-13 |
EP1700917A4 (en) | 2007-06-27 |
CN102766614A (zh) | 2012-11-07 |
JP4547335B2 (ja) | 2010-09-22 |
CN1902315B (zh) | 2012-05-02 |
US20070111278A1 (en) | 2007-05-17 |
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