JP2007054050A - Alkaline xylanase - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an enzyme useful as a detergent enzyme acting in an alkaline region, having xyloglycan decomposition activity and activity to decompose crystalline cellulose and provide a gene for the enzyme. <P>SOLUTION: The alkaline xylanase has the following enzymatic properties. (1) Action: It hydrolyzes β-1,4-glycoside bond of xylan, (2) substrate specificity: it decomposes xylan and crystalline cellulose, especially xylan, (3) optimum reaction pH: the optimum reaction pH is about 8.5 in the case of using xylan as a substrate and reacting at 40°C, (4) pH stability: the enzyme treated at 5°C for 20 hr using xylan as a substrate has residual activity corresponding to ≥80% of the activity of untreated enzyme at pH 4.0-10.5 revealing high stability of the enzyme, (5) optimum reaction temperature: the optimum reaction temperature is about 50°C in the case of reacting at pH 9.0 for 20 min using xylan as a substrate, (6) thermal stability: the enzyme treated at pH 7.0 for 20 min using xylan as a substrate has residual activity corresponding to ≥90% of the activity of untreated enzyme at ≤50°C revealing high stability of the enzyme and (7) molecular weight: the molecular weight is about 40 kDa determined by SDS-polyacrylamide gel electrophoresis. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a xylanase useful as a cleaning enzyme and a gene encoding the xylanase.

  Various detergents including clothing detergents are blended with various hydrolases such as protease, amylase and lipase in order to improve the detergency. Cellulase is a general term for enzymes that hydrolyze the β-1,4 glycosidic bond of cellulose, but it has little effect on cellulose molecules in the crystalline region, which is the basis of the fiber, and is a gel structure formed by hydrated cellulose in the amorphous region. It is useful as a detergent enzyme for clothing because it softens the spillage and causes the dirt contained therein to flow out. Recently, Alkali cellulase derived from alkalophilic Bacillus genus bacteria has been found by Minegoshi (Patent Document 1, Non-Patent Document 1), and various alkali cellulases that can be blended in heavy laundry detergents have been developed (Patent Document 2). ~ 5).

By the way, among stains and stains adhering to clothing, one that is difficult to remove by washing is a stain derived from vegetables and fruits. In the stains derived from vegetables and fruits attached to clothing, the pigments are bound to constituents in the cell walls of vegetables and fruits by covalent bonds or physical bonds. The components of cell walls of vegetables and fruits to which pigments are bound adhere to cotton fibers of clothing, which is difficult to remove by washing. Since the cell wall components are crystalline cellulose and hemicellulose such as xyloglican (xylan), a detergent containing multiple hemicellulases such as cellulase and xylanase has been reported for the purpose of removing these stains. (Patent Document 6).
If there is an enzyme that acts on the crystalline region of cellulose to the extent that it does not damage cotton fibers and also decomposes xylan, stains derived from vegetables and fruits can be removed effectively, and washing for clothing etc. also from the viewpoint of cost and productivity However, cellulases currently incorporated in detergents are not known to have xyloglican-degrading activity (xylanase activity) and to act on the crystalline portion of cellulose.

  In addition, clothing manufactured from fabrics using cellulosic synthetic fibers (cotton rayon), hemp, cannabis, jute, ramie, etc., are repeatedly washed and the fibers are mechanically broken and crushed. As a result, fading occurs in the colored fabric. Accordingly, there is a need for a cleaning agent that is less prone to fluffing and fading.

On the other hand, in recent years, with the development of genetic engineering, production of detergent enzymes has also been mass-produced by genetic recombination. Alkaline cellulase is no exception, and many genes have already been cloned and base sequences determined and used in actual production.
Japanese Patent Publication No. 50-28515 Japanese Patent Publication No. 60-23158 Japanese Patent Publication No. 6-030578 US Pat. No. 4,945,053 Japanese Patent Laid-Open No. 10-313859 Japanese National Patent Publication No. 11-500464 Horikoshi & Akiba, Alkalophilic Microorganisms, Springer, Berlin, (1982)

  The present invention relates to providing an enzyme useful as a detergent enzyme that acts in an alkaline region, has xyloglycan degrading activity, and has crystalline cellulose degrading activity, and a gene thereof.

  The present inventors screened alkali xylanase-producing bacteria from nature, found a microorganism that produces an enzyme that meets the above-mentioned purpose, and succeeded in cloning an alkaline xylanase gene from the microorganism. completed.

That is, the present invention relates to an alkaline xylanase having the following enzymological properties.
1. Action:
Hydrolyzes β1,4-glycosidic bond of xylan.
2. Substrate specificity:
Decomposes xylan and crystalline cellulose, especially xylan.
3. Optimum reaction pH:
When xylan is reacted as a substrate at 40 ° C., the optimum reaction pH is about 8.5.
4). pH stability:
When treated with xylan as a substrate at 5 ° C. for 20 hours, it exhibits an untreated residual activity of 80% or more at pH 4.0 to 10.5.
5. Optimal reaction temperature:
When the reaction is carried out for 20 minutes at pH 9.0 using xylan as a substrate, the optimum reaction temperature is about 50 ° C.
6). Thermal stability:
When treated with xylan as a substrate at pH 7.0 for 20 minutes, it exhibits a residual activity of 90% or more of untreated up to 50 ° C.
7). Molecular weight:
The molecular weight by SDS-polyacrylamide gel electrophoresis is about 40 kDa.

In addition, the present invention relates to the protein described in any of (a) to (c) below and a gene encoding the protein.
(A) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 (b) an amino acid sequence shown in SEQ ID NO: 2 consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added, and xylanase activity (C) a protein comprising an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 2 and having xylanase activity

The present invention also relates to a gene comprising the polynucleotide described in any of (a) to (d) below.
(A) a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1 (b) a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1, wherein one or several bases are deleted, substituted or added A polynucleotide encoding a protein having xylanase activity (c) a polynucleotide which hybridizes with a polynucleotide comprising the base sequence represented by SEQ ID NO: 1 under stringent conditions and encodes a protein having xylanase activity (D) a polynucleotide comprising a base sequence having 80% or more identity with the base sequence represented by SEQ ID NO: 1 and encoding a protein having xylanase activity

  The present invention also relates to a recombinant vector containing the gene and a transformant containing the recombinant vector.

  The present invention also relates to a xylanase production method for culturing the above transformant and collecting xylanase.

  The present invention also relates to a detergent composition comprising the above alkaline xylanase or a protein having xylanase activity.

  The novel xylanase of the present invention is a novel enzyme having xylanase activity and crystalline cellulose hydrolyzing activity, and is useful as an enzyme for detergents such as clothing detergents. Moreover, if the alkaline xylanase gene of the present invention is used, the enzyme can be produced in a single and a large amount.

The alkaline xylanase of the present invention is a novel enzyme and has the following enzymological properties.
(1) Action Acts on xylan and hydrolyzes it.

(2) Substrate specificity Hydrolyzes xylan and crystalline cellulose (CP).
That is, when each substrate is allowed to act at 40 ° C. in a pH 9.0 sodium glycine hydroxide buffer, xylan and CP are hydrolyzed, and xylan is particularly well decomposed.
Carboxymethylcellulose (CMC), AZCL-avicel, AZCL-curdlan, AZCL-mannan and colloidal chitin are not hydrolyzed.

(3) Optimum reaction pH and pH dependency When enzyme activity is measured under the following conditions, the optimum reaction pH when xylan is used as a substrate is about pH 8.5, pH 8.0 to 9.0. It exhibits 90% or more of the maximum activity, and 60% or more of the maximum activity at pH 7 to 10 (FIG. 1A). That is, when the reaction is performed at 40 ° C. using xylan as a substrate, the optimum reaction pH is about 8.5.
Further, when CP is used as a substrate, the optimum reaction pH is about pH 5.5, showing 90% or more of the maximum activity at pH 4.5 to 9.0, and 60% or more of the maximum activity at pH 4 to 9.5. Shown (FIG. 1B). Thus, the alkali xylanase of the present invention exhibits a very unusual property that the optimum reaction pH differs depending on the substrate.

〔Measurement condition〕
Using 1% (w / v) xylan and 1% (w / v) CP as substrates, each 0.1 M buffer (glycine-hydrochloric acid buffer pH 2.5, pH 3, pH 3.5, citrate- Sodium citrate buffer pH 3, pH 4, pH 5, acetate buffer pH 3.5, pH 4.5, pH 5.5, phosphate buffer pH 6.0, pH 6.5, pH 7.0, Tris-HCl buffer pH 7.0, pH 8.0, pH 8.5, pH 9.0, sodium glycine hydroxide pH 8.5, pH 9.0, pH 9.5, pH 10, pH 10.5, sodium borate sodium hydroxide buffer pH 9.5, pH 10.5, Reaction is carried out at 40 ° C. for 20 minutes in pH 10.8 and sodium phosphate buffer (pH 11, pH 12), and xylanase activity and CP degradation activity are measured (at the pH value indicating the maximum activity). It is indicated by a relative activity of 100%). For measurement of xylanase activity, 0.1 unit / mL of xylan decomposition activity was used, and for measurement of CP decomposition activity, 0.1 mL of 0.25 unit / mL enzyme solution of CP decomposition activity was used.

(4) pH stability When enzyme activity is measured under the following conditions, both xylanase activity and CP degradation activity are 80% or more in the range of pH 4 to pH 10.5, and are stable in a wide pH range. (FIG. 2). That is, when treated with xylan as a substrate at 5 ° C. for 20 hours, it exhibits an untreated residual activity of 80% or more at pH 4.0-10.5.

〔Measurement condition〕
Each 0.1 M buffer (glycine-hydrochloric acid buffer pH 2.5, pH 3, pH 3.5, citrate-sodium citrate buffer pH 3, pH 4, pH 5, acetate buffer pH 3.5, pH 4.5, pH 5. 5 acetate buffer pH 4.5, pH 5.5, phosphate buffer pH 6.0, pH 6.5, pH 7.0, Tris-HCl buffer pH 7.0, pH 8.0, pH 8.5, pH 9.0, glycine water Sodium oxide pH 8.5, pH 9.0, pH 9.5, pH 10, pH 10.5, sodium borate sodium hydroxide buffer pH 9.5, pH 10.5, pH 10.8 and sodium phosphate hydroxide buffer pH 11, pH 12 ) After treatment at 5 ° C. for 20 hours, the activity is measured by the xylanase standard activity measurement method and the CPase standard activity measurement method described in Reference Examples (without pH buffer solution treatment). Sex indicated by residual activity was 100%). In the measurement of xylanase activity, an enzyme solution of 0.1 unit / mL for xylanase activity and 0.25 unit / mL for CP degradation activity was used for measurement of CP degradation activity.

(5) Optimum reaction temperature When enzyme activity is measured under the conditions shown below, the optimum reaction temperature for xylanase activity is about 50 ° C, and shows 60% or more of the maximum activity at 40 ° C to 55 ° C. (FIG. 3A). That is, when the reaction is carried out for 20 minutes at pH 9.0 using xylan as a substrate, the optimum reaction temperature is about 50 ° C.
Moreover, the optimal reaction temperature of CP decomposition activity is about 55 degreeC, and shows 60% or more of maximum activity at 40 to 60 degreeC (FIG. 3B).

〔Measurement condition〕
In accordance with the xylanase standard activity measurement method and CPase standard activity measurement method described in the Reference Examples, the reaction temperature is 20 ° C., 30 ° C., 40 ° C., 50 ° C., 55 ° C., 60 ° C., 70 ° C. and 80 ° C. (maximum The relative activity is shown with the value at the temperature at which the activity was indicated as 100%). For measurement of xylanase activity, 0.05 unit / mL of xylan decomposition activity was used, and for measurement of CP decomposition activity, 0.1 mL of 0.125 unit / mL enzyme solution of CP decomposition activity was used.

(6) Temperature stability When enzyme activity was measured under the following conditions, both xylanase activity and CP degradation activity had a residual activity of 90% or more after heat treatment at 50 ° C for 20 minutes, up to 50 ° C. Is stable. In addition, 2 mM calcium chloride slightly improves the stability of the enzyme against heat (FIG. 4). That is, when treated with xylan as a substrate at pH 7.0 for 20 minutes, untreated 90% or more residual activity is exhibited up to 50 ° C.

〔Measurement condition〕
Xylanase activity was measured at 0.1 unit / mL for xylan degradation activity, and CP degradation activity was measured at 0.25 unit / mL for CP degradation activity at 30 ° C, 40 ° C, 50 ° C, 60 ° C, 70 ° C. After heat treatment for 20 minutes in 0.1M phosphate buffer (pH 7.0) at each temperature of 80 ° C., 80 ° C., and 90 ° C., the xylanase standard activity measurement method and the CPase standard activity measurement method described in Reference Examples were used. The residual activity is then measured (indicated by the residual activity with 100% of the untreated activity relative to heat). It was performed in a system with and without 2 mM calcium chloride.

(7) Molecular weight SDS-PAGE standard LOW (Bio-Rad) is used as a molecular weight marker, gel is run at 30 mA for about 40 minutes using a gel with a gel concentration of 12.5%, and the molecular weight is about 40 kDa. . Since SDS-electrophoresis produces a fluctuation of about ± 5000 Da, the molecular weight is allowed to vary by about 5000 Da.

The protein comprising the amino acid sequence shown in (a) SEQ ID NO: 2 of the present invention acts on xyloglican and acts on crystalline cellulose and activity to break down its β1,4-glycosidic bond, and its β1,4-glycosidic bond It is a novel xylanase having an activity of degrading.
The protein of the present invention includes (a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 and a protein equivalent to the protein.

  Here, examples of the equivalent protein include (b) a protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2 and having xylanase activity. It is done.

  Here, the amino acid sequence in which one or several amino acids are deleted, substituted or added is preferably an amino acid sequence in which 1 to 10 amino acids are deleted, substituted or added. The above addition includes addition of one to several amino acids at both ends.

Comparing the identity of the amino acid sequence of the protein having the amino acid sequence shown in SEQ ID NO: 2 of the present invention (alkaline xylanase; hereinafter also referred to as N546b xylanase) and the known enzyme, the xylanase produced by the Bacillus sp. Strain YA355 strain (Ju-Hyun, Y et J.Microbiol.Biotechnol., 3,139-145,1993) the highest identity with a 76%, Bacillus sp .41M-1 strain derived xylanase (Nakai et al. Nucleic Acids Symp. Ser., 31, 235-236, 1994)) and 76%, and xylanase derived from the Dictyoglomus thermophilum strain was 51%. From the results of substrate specificity and homology search, the enzyme of the present invention was considered to be classified as xylanase.
However, the alkali xylanase of the present invention has the highest identity with the amino acid sequences of other enzymes at 76%, and the optimum pH of xylan decomposition activity is in the alkaline region as shown in the examples below. The alkali xylanase of the present invention is a novel enzyme because the optimum pH of the crystalline cellulose-degrading activity with the substrate in the acidic region is different from the optimum pH depending on the type of substrate. I can judge.

  That is, when the protein equivalent to the alkaline xylanase provided in the present invention is appropriately aligned with the corresponding sequence in the amino acid sequence shown in SEQ ID NO: 2, (c) 80% or more with the amino acid sequence shown in SEQ ID NO: 1, More preferably 85% or more, still more preferably 90% or more, and particularly preferably 95% or more of a protein having an xylanase activity.

  The identity of the above amino acid sequence is the amino acid sequence homology search method using BLAST2 that exists in the homology search tool in the genome net of Kyoto University Chemical Research Institute Bioinformatics Center. The program uses BLASTP and the option parameter is the default setting. The values (Scoring matrix is BLOSUM62, no filter, Alignment view is pairwise).

  Here, having xylanase activity means an activity of hydrolyzing β1,4-glycoside bond in xyloglican, and the degree of the activity is the amino acid sequence represented by SEQ ID NO: 2 as long as the function is exhibited. It may be higher or lower than that of the protein consisting of the above.

  In addition, the protein equivalent to the alkaline xylanase has a hydrolytic activity of crystalline cellulose. The degrading activity of crystalline cellulose means the activity of hydrolyzing the β1,4-glycoside bond of crystalline cellulose, and the degree of the activity consists of the amino acid sequence represented by SEQ ID NO: 2 as long as it exhibits its function. It may be higher than or lower than that of protein.

  In addition, the protein equivalent to the alkaline xylanase may have additional properties. Such properties include, for example, properties that are superior in stability compared to the protein consisting of the amino acid sequence represented by SEQ ID NO: 2, properties that have different functions only at low temperatures and / or high temperatures, and properties that differ in optimum pH. Etc.

  The alkaline xylanase of the present invention may also be part of a larger protein, such as a fusion protein. Here, examples of the sequence added in the fusion protein include sequences useful for purification, such as multiple histidine residues, and additional sequences that ensure stability during recombinant production.

  The alkaline xylanase of the present invention had an optimum pH of about 8.5 when xylan was used as a substrate, and was about 5.5 depending on the substrate when cellulose powder (CP) was used as a substrate. Therefore, it is possible to adjust the balance between the xylanase activity and the decomposition activity of crystalline cellulose by adjusting the pH of the environment when used as a cleaning agent.

  The gene of the present invention encodes a protein consisting of the amino acid sequence shown by SEQ ID NO: 2, and preferably (a) a gene consisting of a polynucleotide consisting of the base sequence shown by SEQ ID NO: 1, and an equivalent thereof Gene. Here, the equivalent gene includes (b) a nucleotide sequence in which one or several bases are deleted, substituted or added in the polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1, and alkaline xylanase activity (C) a polynucleotide that encodes a protein that hybridizes with a polynucleotide comprising the base sequence represented by SEQ ID NO: 1 under stringent conditions and has an alkaline xylanase activity, (d) And a polynucleotide encoding a protein consisting of a base sequence having 80% or more identity with the base sequence shown in SEQ ID NO: 1 and having alkaline xylanase activity.

  Here, the stringent conditions include, for example, conditions described in Molecular cloning-a laboratory manual, 2nd edition (Sambrook et al., 1989). That is, 6XSSC (1XSSC composition: 0.15M sodium chloride, 0.015M sodium citrate, pH 7.0), 0.5% SDS, 5X Denhart and 100 mg / mL herring sperm DNA together with the probe at 65 ° C Examples include conditions of constant temperature for 8 to 16 hours and hybridization.

  In addition, the gene equivalent to the gene shown in (a) above has a higher level of mRNA expression and / or higher stability than the gene shown in (a), and the stability of the translated protein. It may have properties such as excellent properties.

  In addition, the search of the identity of the above-mentioned base sequence was also performed by a homology search method using BLAST2 present in the homology search tool in the genome net of Kyoto University Chemical Research Institute Bioinformatics Center. BLASTN was used as a program, and optional parameters were set at initial settings (Scoring matrix was BLOSUM62, No Filter, Alignment view was pairwise).

The alkaline xylanase gene of the present invention can be cloned from Bacillus Sp . KSM-N546 (FERM P-19729) or the like. As a cloning method, known means such as a shotgun method and a PCR method can be used. The method of using it is mentioned.

  In addition, the gene provided by the present invention can also be prepared by modifying a gene consisting of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 1 by a method such as planned or random mutagenesis. .

  Here, the design of the mutation when introducing the mutation systematically can be performed, for example, by taking into account the characteristic sequence on the gene sequence. Here, reference to a characteristic sequence can be performed, for example, by taking into account the three-dimensional structure prediction of the protein and homology with known proteins. Examples of the method for randomly introducing mutations include a PCR method and a mutagen treatment method. As a method of introducing mutation systematically, site-directed mutagenesis can be mentioned, and more specifically, for example, using Site-Directed Mutagenesis System Mutan-SuperExpress Km kit (Takara Bio). it can. In addition, a recombinant PCR (polymerase chain reaction) method (PCR protocols, Academic press, New York, 1990) can also be used.

  The gene of the present invention can also be obtained by obtaining a sequence that hybridizes to a polynucleotide containing a part or all of the gene of the present invention using an appropriate principle. The appropriate principle includes, for example, a PCR method using a polynucleotide containing a part of the gene of the present invention as a primer, and a method using a polynucleotide containing a part of the gene of the present invention as a probe. included.

The alkaline xylanase of the present invention can be obtained, for example, by inoculating the production microorganism into a natural or synthetic medium, culturing under normal culture conditions, and collecting from the culture. It is also possible to artificially express the above enzyme gene and collect it. Use of the gene is useful because the protein provided in the present invention can be produced in large quantities.
Here, as the alkaline xylanase producing microorganisms, for example Bacillus sp (Bacillus sp.) KSM-N546 (FERM P-19729) and the like.

  In order to produce an alkaline xylanase using the gene of the present invention, the alkaline xylanase gene is incorporated into any vector suitable for expressing the gene in the target host, and the host is prepared using the recombinant vector. What is necessary is just to culture | cultivate the transformant obtained by transformation, and extract | collect alkaline xylanase from the said culture solution. The culture may be carried out by inoculating a medium containing a carbon source, a nitrogen source and other essential nutrients that can assimilate microorganisms, and following a conventional method.

  In addition, in order to produce a recombinant vector containing the alkaline xylanase gene of the present invention, it is possible to maintain replication in a host cell, to stably express the enzyme, and to a vector that can stably hold the gene. An alkaline xylanase gene may be incorporated. Further, when Bacillus subtilis is used as a host, it is derived from Bacillus sp. KSM-64 (FERM BP-2886) described in JP-A No. 2000-287687 so that the alkaline xylanase can be highly expressed upstream of the alkaline xylanase gene. DNA fragments may be bound. Examples of such vectors include pUC18, pBR322, pHY300PLK and the like when Escherichia coli is used as a host, and pUB110, pHSP64 (Sumitomo et al., Biosci. Biotechnol, Biocem., 59, 2172-2175, when Bacillus subtilis is used as a host. 1995) or pHY300PLK.

In order to transform a host using the recombinant vector thus obtained, protoplast method, competent cell method, electroporation method and the like can be used. The host is not particularly limited, and any host can be used. For example, animals, animal-derived cells, plants, plant-derived cells, microorganisms and the like can be used, but microorganisms are preferable, and microorganisms include Bacillus Gram-positive bacteria such as genus (Bacillus subtilis), Gram-negative bacteria such as Escherichia coli (Escherichia coli), fungi such as Streptomyces genus (actinomycetes), Saccharomyces genus (yeast), Aspergillus genus (mold) are preferred, and Bacillus subtilis is preferred. More preferred are Bacillus subtilis 168 strain and Bacillus subtilis ISW1214 strain. As animal-derived cells and plant-derived cells, those established as cultured cells are preferred. Further, as the host, a strain lacking alkaline protease is preferable.

  The obtained transformant may be cultured under suitable conditions using a medium containing an assimilating carbon source, nitrogen source, metal salt, vitamin and the like. From the culture solution thus obtained, the enzyme can be collected and purified by a general method, and the necessary enzyme form can be obtained by freeze drying, spray drying, and crystallization.

  Collection and purification of the alkaline xylanase from the culture thus obtained can be performed according to a general method. That is, the cells can be removed from the culture by centrifugation or filtration, and the target enzyme can be concentrated from the obtained culture supernatant by conventional means. The enzyme solution or dry powder thus obtained can be used as it is, but can be further crystallized or granulated by a known method.

  The alkaline xylanase of the present invention has xylanase activity and crystalline cellulose hydrolysis activity, as shown in the Examples below. Therefore, it is useful as an enzyme for blending various detergent compositions.

  The cleaning composition of the present invention can be used in combination with various enzymes other than the alkaline xylanase of the present invention. Examples of such enzymes include hydrolase, oxidase, reductase, transferase, lyase, isomerase, ligase, synthetase, etc., among which protease, cellulase, keratinase, esterase, cutinase, amylase, lipase, pullulanase , Pectinase, mannanase, glucosidase, glucanase, cholesterol oxidase, peroxidase, laccase, etc. are preferred, protease, cellulase, amylase, and lipase are more preferred, and cellulase having crystalline cellulose resolution is particularly preferred.

    As shown in Examples described later, by combining the alkaline xylanase of the present invention and cellulase having a crystalline cellulose resolving power, fluff of clothing made of cotton fibers can be efficiently removed. Therefore, according to this, it is also possible to prevent the fading caused by the generation of fluff in the colored fabric. Examples of cellulases having such a crystalline cellulose resolving power include, for example, KSM-S237 cellulase (JP-A-10-313859), KSM-N252 cellulase (JP-A-2001-340074), KSM-N145 cellulase (JP-A-2005-287441). Gazette), Carezyme (registered trademark, Novozymes) and the like.

  The cleaning composition of the present invention can contain a known cleaning component. Such components include, for example, surfactants, divalent metal ion scavengers, alkali agents, recontamination inhibitors, bleaches, builders, softeners, reducing agents (sulfites, etc.) known in the field of clothing detergents. , Foam suppressants (such as silicone), fragrances, and other additives.

The surfactant is preferably added in an amount of 0.5 to 60% by mass in the detergent composition, particularly 10 to 45% by mass when the powdery detergent composition is used, and the liquid detergent composition. Is preferably 20 to 50% by mass.
Examples of the surfactant include one or a combination of an anionic surfactant, a nonionic surfactant, an amphoteric surfactant, and a cationic surfactant, and preferably an anionic surfactant. Agent, nonionic surfactant.

  Examples of the anionic surfactant include a sulfate ester salt of an alcohol having 10 to 18 carbon atoms, a sulfate ester salt of an alkoxylate of an alcohol having 8 to 20 carbon atoms, an alkylbenzene sulfonate, a paraffin sulfonate, and an α-olefin sulfone. Acid salts, α-sulfo fatty acid salts, α-sulfo fatty acid alkyl ester salts or fatty acid salts are preferred. In the present invention, a linear alkylbenzene sulfonate having an alkyl chain having 10 to 14 carbon atoms, more preferably 12 to 14 carbon atoms is preferable, and an alkali metal salt or an amine is preferable as the counter ion. Or potassium, monoethanolamine, and diethanolamine are preferable.

  Nonionic surfactants include polyoxyalkylene alkyl (carbon number 8-20) ether, alkyl polyglycoside, polyoxyalkylene alkyl (carbon number 8-20) phenyl ether, polyoxyalkylene sorbitan fatty acid (carbon number 8-8). 22) Ester, polyoxyalkylene glycol fatty acid (carbon number 8 to 22) ester, and polyoxyethylene polyoxypropylene block polymer are preferable. In particular, as a nonionic surfactant, 4 to 20 moles of alkylene oxide such as ethylene oxide or propylene oxide is added to an alcohol having 10 to 18 carbon atoms [HLB value (calculated by Griffin method) is 10.5 to 15. 0, preferably from 11.0 to 14.5] polyoxyalkylene alkyl ethers are preferred.

  The divalent metal ion scavenger is added in an amount of 0.01 to 50% by mass, preferably 5 to 40% by mass. Examples of the divalent metal ion scavenger include condensed phosphates such as tripolyphosphate, pyrophosphate and orthophosphate, aluminosilicates such as zeolite, synthetic layered crystalline silicate, nitrilotriacetate, Examples include ethylenediaminetetraacetate, citrate, isocitrate, polyacetal carboxylate and the like. Of these, crystalline aluminosilicate (synthetic zeolite) is particularly preferable, and among A-type, X-type, and P-type zeolite, A-type is particularly preferable. As the synthetic zeolite, those having an average primary particle size of 0.1 to 10 μm, particularly 0.1 to 5 μm are preferably used.

  The alkali agent is blended in an amount of 0.01 to 80% by mass, preferably 1 to 40% by mass. In the case of a powdery detergent composition, examples include alkali metal carbonates such as sodium carbonate collectively called dense ash and light ash, and amorphous alkali metal silicates such as JIS No. 1, No. 2, No. 3, etc. . These inorganic alkaline agents are effective in forming the skeleton of the particles when the detergent is dried, and can obtain a detergent that is relatively hard and excellent in fluidity. Examples of other alkali agents include sodium sesquicarbonate and sodium hydrogen carbonate, and phosphates such as tripolyphosphate also have an action as an alkali agent. Moreover, as an alkaline agent used when setting it as a liquid detergent composition, in addition to the said alkaline agent, sodium hydroxide and mono, di, or triethanolamine can be used, and it is as a counter ion of an activator. Can also be used

  The recontamination inhibitor is preferably blended in an amount of 0.001 to 10% by mass, preferably 1 to 5% by mass. Examples of the recontamination preventive agent include polyethylene glycol, carboxylic acid polymer, polyvinyl alcohol, polyvinyl pyrrolidone and the like. Among these, the carboxylic acid-based polymer has a function of capturing metal ions and a function of dispersing solid particle dirt from clothing into a washing bath, in addition to the ability to prevent recontamination. The carboxylic acid polymer is a homopolymer or copolymer such as acrylic acid, methacrylic acid, and itaconic acid. The copolymer is preferably a copolymer of the above monomer and maleic acid, and has a molecular weight of several thousand to 100,000. preferable. In addition to the above carboxylic acid polymers, polymers such as polyglycidyl salts, cellulose derivatives such as carboxymethyl cellulose, and aminocarboxylic acid polymers such as polyaspartic acid also have metal ion scavengers, dispersants, and recontamination preventing ability. Therefore, it is preferable.

  Bleaching agents such as hydrogen peroxide and percarbonate are preferably blended in an amount of 1 to 10% by mass. When using a bleaching agent, 0.01-10 mass% of bleach activators (activators), such as tetraacetylethylenediamine (TAED) and Unexamined-Japanese-Patent No. 6-316700 description, can be mix | blended.

  Examples of the fluorescent agent include biphenyl type fluorescent agents (for example, Tinopearl CBS-X) and stilbene type fluorescent agents (for example, DM type fluorescent dye). It is preferable to blend 0.001 to 2 mass% of the fluorescent agent.

  The detergent composition of the present invention can be produced according to a conventional method by combining the alkali xylanase of the present invention and the above-mentioned known cleaning components. The form of the detergent can be selected depending on the application, and for example, it can be liquid, powder, granule, paste, solid and the like.

  The detergent composition thus obtained can be used as a powder detergent for clothing, a liquid detergent for clothing, a detergent for automatic dishwashers, a detergent for modifying cotton fibers, and the like.

Example 1 Screening for Alkaline Xylanase-Producing Bacteria Suspension of soil in various parts of Japan was heat-treated at 80 ° C. for 30 minutes and applied to an agar plate medium having the composition shown in Table 1 below. The cells were statically cultured in a 30 ° C. incubator for 3 days, and after the growth of the bacteria, those in which dissolution spots accompanying the decomposition of xylan were detected were selected, and single colonization was repeated. As a result, Bacillus sp. KSM-N546 strain was obtained.

Example 2 Production and Purification of Alkaline Xylanase (N546b Xylanase) from Bacillus sp. KSM-N546 Strain
The Bacillus sp. KSM-N546 strain was subjected to shaking culture at 30 ° C. for 3 days using a liquid medium having the composition shown in Table 2.

  The obtained culture supernatant was diluted about 10-fold with 50 mM Tris-HCl buffer (pH 7.5), added to SuperQ-Toyopearl 650M (Tosoh) equilibrated with the same buffer, and then columned with about 480 mL of the same buffer. The interior was washed to obtain a non-adsorbed fraction containing alkali xylanase. Subsequently, the non-adsorbed fraction was concentrated 20 times using a PM10 concentration membrane (Amicon) while adding 10 mM boric acid-sodium hydroxide buffer (pH 9.5). Next, it was applied to a DEAE-Toyopearl 650M column (1 cm × 10 cm) previously equilibrated with 10 mM boric acid-sodium hydroxide buffer (pH 9.5) to adsorb alkali xylanase. After washing the column with 200 mL of the same buffer, the adsorbed protein was eluted with 200 mL of 0-0.1 M KCl. Alkali xylanase active fractions were collected and desalted and concentrated with a PM10 concentration membrane. Further, it was attached to a CM-Toyopearl 650M column (1 cm × 10 cm) previously equilibrated with 10 mM phosphate buffer (pH 6.0). Alkaline xylanase was eluted in the non-adsorbed fraction and desalted and concentrated with a PM10 concentration membrane. Finally, it was attached to a Hydroxyapatite column (1 cm × 10 cm) previously equilibrated with 10 mM phosphate buffer (pH 7.0). Alkaline xylanase was eluted in the non-adsorbed fraction, and this was desalted and concentrated with a PM10 concentration membrane to obtain an enzyme-purified preparation purified to homogeneity by electrophoresis. The obtained purified sample was used for N-terminal amino acid sequence analysis.

Example 3 Acquisition of N546b Xylanase Gene Fragment (1) Restriction enzyme treatment of genomic DNA Acquisition of N546b xylanase gene was performed using LA PCR in vitro Cloning Kit (manufactured by Takara).
Genomic DNA was prepared from the cells of Bacillus sp. KSM-N546 obtained in Example 2 by the method of Saito and Miura (Biochim. Biophys. Acta, 72, 619-629, 1963). First, the genomic DNA of KSM-N546 strain was subjected to HindIII treatment. N546 strain genomic DNA 2 μL (1 μg), 10 × buffer 10 μL, deionized water 24 μL, and HindIII 2 μL (20 units) were mixed, treated at 37 ° C. for 3 hours, and after completion of the reaction, GFX PCR DNA and Gel Band Purification Kit ( (Manufactured by Pharmacia) and purified (25 μL).

(2) Ligation reaction To 20 μL of the purified DNA solution, 5 μL (100 ng DNA) of HindIII cassette from LA PCR in vitro Clonig Kit and 25 μL of Ligation High were added and allowed to stand overnight at 16 ° C., and then DNA was purified by GFX PCR DNA and Gel Band Purification Kit. Purified and recovered.

(3) Amplification of N546b xylanase gene fragment by PCR 26.5 μL of the ligation purification solution was added 5 μL of 10 × buffer in LA PCR in vitro Clonig Kit, 0.5 μL of LA Taq, 8 μL of dNTP mix, 5 μL of MgCl ₂ , primer C1 in the kit 1 μL (10 pmol) and 4 μL of composite primer I (SEQ ID NO: 3) estimated from the N-terminal sequence of N546b xylanase purified enzyme were mixed. PCR conditions were 94 ° C. for 2 minutes, followed by heat denaturation at 94 ° C. for 30 seconds, 55 ° C. for 2 minutes, and 72 ° C. for 2 minutes (30 cycles). A second PCR was performed using 1 μL of the obtained PCR solution as a template. 1 μL of template DNA solution was purified with 5 μL of 10 × buffer, LA Taq 0.5 μL, dNTP mix ₈ μL, MgCl ₂ 5 μL, cassette primer C2 1 μL (10 pmol), deionized water 26.5 μL, and N546b xylanase purified in LA PCR in vitro Clonig Kit 4 μL (10 pmol) of mixed primer II (SEQ ID NO: 4) estimated from the N-terminal sequence of the enzyme was mixed. PCR reaction conditions were 94 ° C for 1 minute after heat denaturation, 94 ° C for 30 seconds, 55 ° C for 2 minutes, 72 ° C for 1 minute (30 cycles). Using the thus obtained partial amplified fragment (about 1.2 kb) of the N546b xylanase gene, the partial base sequence was analyzed using the cassette primer C2 as a primer.

Example 4 Determination of the entire nucleotide sequence of the N546b xylanase gene by inverse PCR Inverse PCR primers III to VI (SEQ ID NOs: 5 to 8) based on the nucleotide sequence obtained from a partially amplified fragment (about 1.2 kb) of the 546b xylanase gene And inverse PCR was performed. KSM-N546b strain genomic DNA solution 5 μL (1 μg), 10 × buffer 10 μL, deionized water 82 μL, and SacI 3 μL (30 units) were mixed, followed by restriction enzyme treatment at 37 ° C. for 3 hours. The obtained genome degradation product was purified by GFX PCR DNA and Gel Band Purification Kit (20 μL) and then self-closed using Ligation High (16 ° C., 18 hours). The first PCR was performed using 1 μL of the obtained ligation mixture as template DNA. Inverse PCR primer III (SEQ ID NO: 5) and primer IV (SEQ ID NO: 6) were used. After mixing 1 μL of template DNA solution, 5 μL of each primer (1 μM), 10 μl buffer 5 μL, LA Taq 0.5 μL, dNTP mix ₈ μL, MgCl ₂ 5 μL, and deionized water 21.5 μL, Gene Amp PCR System 9700 (manufactured by Amersham Pharmacia) ). The reaction conditions were 94 ° C for 2 minutes after heat denaturation, 94 ° C for 1 minute, 55 ° C for 1 minute, 72 ° C for 1 minute (28 cycles), and 72 ° C for 1 minute. The obtained PCR product was purified by GFX PCR DNA and Gel Band Purification Kit. As a result, an amplified DNA fragment of about 5.5 kbp was obtained.
Next, a second inverse PCR was performed using the purified amplified DNA fragment as a template and inverse PCR primers V and VI. Specifically, 0.5 μL of template DNA solution, 5 μL of each primer (1 μM), 10 × buffer 5 μL, LA Taq 0.5 μL, dNTP mix ₈ μL, MgCl ₂ 5 μL, and deionized water 19 μL were mixed, and PCR was performed using Gene Amp PCR System 9700. Went. The reaction conditions were 94 ° C for 2 minutes after heat denaturation, 94 ° C for 30 seconds, 57 ° C for 1 minute, 72 ° C for 2 minutes 30 seconds (28 cycles), and 72 ° C for 2 minutes 30 seconds. The obtained PCR product was purified by GFX PCR DNA and Gel Band Purification Kit, then subjected to 1% (w / v) agarose gel electrophoresis, and the amplified DNA fragment of about 5.5 kb was recovered from the gel, purified, and primer The total base sequence (about 1.8 kbp) of the N546b xylanase gene was determined using V (SEQ ID NO: 7) and primer VI (SEQ ID NO: 8).

Example 5 Subcloning of N546b Xylanase Gene Primers were constructed based on the nucleotide sequence upstream of the decoded N546b xylanase gene promoter and downstream of the terminator (primer VII, primer VIII, SEQ ID NO: 9 and SEQ ID NO: 10, respectively), and the N546 strain genome was Genomic PCR was performed as a template. N546 strain genome 0.5 μL (1 μg), primer VII (BglII restriction enzyme recognition site addition, SEQ ID NO: 9), primer VIII (HindIII restriction enzyme recognition site addition, SEQ ID NO: 10) 5 μL, 10 × buffer 5 μL, LA Taq0 After mixing 5 μL, dNTP mix 8 μL, MgCl 25 μL, and deionized water 21 μL, PCR was performed using Gene Amp PCR System 9700. The reaction conditions were 94 ° C. for 1 minute heat denaturation, 98 ° C. for 10 seconds, 68 ° C. for 4 minutes 30 seconds (28 cycles). The obtained PCR product was purified with GFX PCR DNA and Gel Band Purification Kit (87 μL), 10 μL of 10 × buffer and 3 μL of HindIII (30 units) were added, and the mixture was treated with enzyme at 37 ° C. for 20 hours. The HindIII treatment solution was purified with GFX PCR DNA and Gel Band Purification Kit, and then similarly treated with BglII. After the restriction enzyme treatment solution was purified, it was ligated to the shuttle vector pHY300PLK previously treated with HindIII and BglII using Ligation High (16 ° C., 20 hours).
Using 3 μL of the resulting ligation mixture, 100 μL of E. coli JM109 (manufactured by Takara) was transformed. From the transformants obtained as tetracycline resistant strains, a strain carrying pHY300PLK (pHY546b) in which the target N546b xylanase gene was inserted was selected by colony PCR. Plasmids were recovered and purified from the selected transformants using Micro Prep Plasmid Purification Kit (manufactured by Amersham Pharmacia). Next, the obtained transformants by the protoplast method host bacterium Bacillus subtilis ISW1214 strain (manufactured by Yakult) with PHY546b. A DM3 regeneration agar medium (Table 3) containing 30 μg / mL of tetracycline hydrochloride (manufactured by Sigma) was used as the regeneration medium of the transformant.

Example 6 Production of Recombinant N546b Xylanase Transformants grown on DM3 regenerated agar medium were cultured using LB medium (1% bactotryptone, 0.5% yeast extract, 1% sodium chloride) (30 ° C., The culture supernatant (recombinant N546b xylanase crude enzyme solution) was obtained by centrifugation (8000 rpm, 20 minutes, 4 ° C.) for 72 hours and 125 rpm. The xylanase activity was 41 units and the CP degradation activity was 0.43 units.

Example 7 Various Properties of N546b Xylanase (1) Substrate Specificity According to the method shown in the Reference Example, the hydrolytic ability of this enzyme for CMC, CP, xylan, AZCL-avicel, AZCL-curdlan, AZCL-mannan and colloidal chitin As a result of the investigation, when the decomposition activity for xylan was 100, the decomposition activity for CP was 0.36. Moreover, it did not have hydrolytic ability with respect to CMC, AZCL-Avicel, AZCL-curdlan, AZCL-mannan and colloidal chitin.

(2) Optimum reaction pH
The substrate is 1% (w / v) xylan and 1% (w / v) CP, and each 0.1 M buffer (glycine-hydrochloric acid buffer pH 2.5, pH 3, pH 3.5, citrate- Sodium citrate buffer pH 3, pH 4, pH 5, acetate buffer pH 3.5, pH 4.5, pH 5.5, phosphate buffer pH 6.0, pH 6.5, pH 7.0, Tris-HCl buffer pH 7.0, pH 8.0, pH 8.5, pH 9.0, sodium glycine hydroxide pH 8.5, pH 9.0, pH 9.5, pH 10, pH 10.5, sodium borate sodium hydroxide buffer pH 9.5, pH 10.5, The measurement was carried out at 40 ° C. for 20 minutes in pH 10.8 and sodium phosphate buffer (pH 11, pH 12). For measurement of xylanase activity, 0.1 unit / mL of xylan degradation activity was used, and for measurement of CP degradation activity, 0.1 mL of 0.25 unit / mL enzyme solution of CP degradation activity was used. The relative activity was shown with the pH value indicating the maximum activity as 100%. The optimum reaction pH when xylan was used as a substrate showed 90% or more of the maximum activity at pH 8.0 to 9.0 around pH 8.5, and 60% or more of the maximum activity at pH 7-10. In addition, when CP was used as a substrate, the optimum reaction pH was about pH 5.5, pH 4.5 to 9.0 showed 90% or more of the maximum activity, and pH 4 to 9.5 showed 60% or more of the maximum activity. . It showed a very unusual property that the optimum reaction pH was different with different substrates (FIG. 1).

(3) pH stability Each 0.1 M buffer (glycine-hydrochloric acid buffer pH 2.5, pH 3, pH 3.5, citrate-sodium citrate buffer pH 3, pH 4, pH 5, acetate buffer pH 3.5, pH 4.5, pH 5.5 acetate buffer pH 4.5, pH 5.5, phosphate buffer pH 6.0, pH 6.5, pH 7.0, Tris-HCl buffer pH 7.0, pH 8.0, pH 8.5, pH 9.0, sodium glycine hydroxide pH 8.5, pH 9.0, pH 9.5, pH 10, pH 10.5, sodium borate sodium hydroxide buffer pH 9.5, pH 10.5, pH 10.8 and phosphoric acid hydroxylation After treatment at 5 ° C. for 20 hours in sodium buffer pH 11, pH 12), the activity was measured by the xylanase standard activity measurement method and the CPase standard activity measurement method described in Reference Examples. The activity was shown as the residual activity with the activity of the pH buffer solution untreated as 100%. In this experiment, 0.1 unit / mL (xylanase) and 0.25 unit / mL (CPase) enzyme solutions were used. As a result, both the xylanase activity and the CP degradation activity were found to be 80% or more in the range of pH 4 to pH 10.5 and stable over a wide pH range (FIG. 2).

(4) Optimum reaction temperature According to the xylanase standard activity measurement method and CPase standard activity measurement method described in the reference examples, the reaction temperature is 20 ° C, 30 ° C, 40 ° C, 50 ° C, 55 ° C, 60 ° C, 70 ° C and The reaction was carried out at ° C. The relative activity was shown with the value at the temperature showing the highest activity as 100%. For measurement of xylanase activity, 0.05 unit / mL of xylan degradation activity was used, and for measurement of CPase activity, 0.1 mL of 0.125 unit / mL enzyme solution of CPase degradation activity was used. As a result, the optimum reaction temperature of this enzyme for xylan and CP was examined. The optimum reaction temperature for xylanase activity was about 50 ° C., and the optimum reaction temperature for CP decomposition activity was about 55 ° C., both having an optimum reaction temperature around 50 ° C. (FIG. 3).

(5) Temperature stability The enzyme solution of 0.1 unit / mL (xylanase) and 0.25 unit / mL (CPase) is 30 degreeC, 40 degreeC, 50 degreeC, 60 degreeC, 70 degreeC, 80 degreeC, and 90 degreeC. After being heat-treated in 0.1 M phosphate buffer (pH 7.0) for 20 minutes, the residual activity was measured by the xylanase standard activity measurement method and the CPase standard activity measurement method described in Reference Examples. The residual activity was defined as 100% untreated activity against heat. It was performed in a system with and without 2 mM calcium chloride. As a result, both xylanase and CPase had a residual activity of 90% or more after heat treatment at 50 ° C. for 20 minutes, and were stable up to 50 ° C. With 2 mM calcium chloride, the stability of the enzyme to heat was slightly improved (FIG. 4).

(6) SDS-polyacrylamide gel electrophoresis SDS-polyacrylamide gel electrophoresis was performed according to the method of Laemmli et al. The molecular weight marker used was SDS-PAGE Standard Low (Bio-Rad). As standard proteins, phosphorose b (molecular weight; 97,400), serum albumin (molecular weight; 66,200), ovalbumin (molecular weight; 45, 000), carbonic anhydrase (molecular weight; 31,000), trypsin inhibitor (molecular weight; 21,500) and lysozyme (molecular weight; 14,400). When a gel having a gel concentration of 12.5% was subjected to electrophoresis at 30 mA / gel for about 40 minutes to determine the molecular weight, the molecular weight was about 40 k.

Reference Example Enzyme activity measurement method (1) CMCase activity measurement method [3.5-dinitrosalicylic acid (DNS) method]
Final concentration 1% (w / v) carboxymethylcellulose (CMC, manufactured by Nippon Paper Industries Co., Ltd.), 0.1 M enzyme solution diluted to an appropriate concentration in 0.1 mL substrate solution of 0.1 M glycine-sodium hydroxide (pH 9.0) After reacting for 20 minutes at 40 ° C., 1 mL of DNS solution was added and heat-treated in a boiling water bath for 5 minutes. After heat treatment, the mixture was cooled in ice water, 4 mL of deionized water was added and stirred, and the absorbance at 535 nm was measured using a U2000 spectrophotometer (Hitachi). One unit of enzyme was defined as an amount that liberates 1 μmol of glucose per minute.

(2) CPase (crystalline cellulose degradation activity) activity measurement method Final concentration 1% (w / v) cellulose powder (CP, SIGMACELL, type 101, manufactured by Sigma), 0.1 M glycine-sodium hydroxide (pH 9.0) After adding 0.1 mL of enzyme solution diluted to an appropriate concentration to 0.9 mL of the substrate solution and reacting at 40 ° C. for 1 to 5 hours, adding 1 mL of DNS solution and centrifuging (3000 rpm, 25 ° C., 15 minutes) 1 mL was collected, and 2 mL of deionized water was added and stirred. After heat treatment in a boiling water bath for 5 minutes, the heat treatment was followed by cooling in ice water, and the absorbance at 535 nm was measured using a U2000 spectrophotometer (Hitachi). The enzyme 1 unit was an amount that liberates 1 μmol of glucose per minute. This method was used as a standard activity measurement method for CPase (crystalline cellulose degradation activity).

(3) Xylanase activity measurement method Final concentration of 1% (w / v) xylan (Oat-spelt, manufactured by Fluka), 0.1M glycine-sodium hydroxide (pH 9.0) substrate solution to 0.9 mL to an appropriate concentration After adding 0.1 mL of diluted enzyme solution and reacting at 40 ° C. for 20 minutes, 1 mL of DNS solution was added and heat-treated in a boiling water bath for 5 minutes. After heat treatment, the mixture was cooled in ice water, centrifuged (3000 rpm, 25 ° C., 15 minutes), 1 mL was taken, and 4 mL of deionized water was added and stirred. Absorbance at 535 nm was measured using a U2000 spectrophotometer (Hitachi). The enzyme 1 unit was an amount that liberates 1 μmol of xylose per minute. This method was used as a xylanase standard activity measurement method.

(4) Avicellase activity measurement method Final concentration 1% (w / v) AZCL-Avicel (manufactured by Megazyme), 0.1 M glycine-sodium hydroxide (pH 9.0) substrate solution was diluted to an appropriate concentration to 0.9 mL After adding 0.1 mL of the enzyme solution and reacting at 40 ° C. for 4 hours, 3 mL of ethanol was added and stirred. After centrifugation (3000 rpm, 25 ° C., 15 minutes), the absorbance of the supernatant at 650 nm was measured using a U2000 spectrophotometer (Hitachi).

(5) Chitinase activity measurement method Using 1% (w / v) colloidal chitin substrate and 0.2 M glycine sodium hydroxide buffer (pH 9) so that the final concentrations are 0.4% and 0.1 M, respectively. To 0.9 mL of the prepared substrate solution, 0.1 mL of enzyme solution was added and reacted at 40 ° C. for 2 hours. After adding 1 mL of DNS solution to stop the reaction, it was boiled for 5 minutes in a boiling water bath. After cooling in ice water, the absorbance at 535 nm of the supernatant obtained by centrifugation (3000 rpm, 15 minutes, 25 ° C.) was measured with a microplate reader (VERSAmax, manufactured by Molecular Devices). In addition, after adding 1 mL of DNS solutions to 0.9 mL of substrate solutions, 0.1 mL of crude enzyme solutions were added and the same operation was performed, and it was set as the blank. One unit of enzyme was defined as the amount of enzyme that liberates 1 μmol of N-acetylglucosamine per minute.

(6) Mannanase activity measurement method 0.2 mL of 1% (w / v) AZCL-galactomannan (manufactured by Megazyme) solution, 0.2 mL of 0.5 M glycine sodium hydroxide buffer (pH 10), deionized water 0. After 5 mL and 0.1 mL of enzyme solution were mixed and reacted at 40 ° C. for 20 minutes, 3 mL of ethanol was added and centrifuged (3000 rpm, 15 minutes, 25 ° C.) to precipitate the residue. The absorbance at 600 nm of the supernatant was measured. 1ManU (mannanase unit) was an enzyme amount having an absorbance of 0.44 at 600 nm under the above reaction conditions (mananase 1 unit derived from Bacillus sp. AM001 strain gave an absorbance of 0.44 under the above reaction conditions. As a reference).

(7) β (1,3) -glucanase activity measurement method 1% (w / v) AZCL-curdlan (β (1,3) -glucan, manufactured by Megazyme) solution 0.2 mL, 0.5 M glycine sodium hydroxide Mix 0.2 mL of buffer solution (pH 10), 0.5 mL of deionized water and 0.1 mL of enzyme solution, react at 40 ° C. for 20 minutes, add 3 mL of ethanol, and centrifuge (3000 rpm, 15 minutes, 25 ° C.) And the residue was precipitated. The absorbance at 600 nm of the supernatant was measured.

Example 8 Fluff Removal Effect on Cotton Cloth Surface by Combination with Various Cellulases The fluff removal effect on the cotton fabric surface in the case of combining N546b xylanase of the present invention with the cellulase shown below was examined.
(1) Method of fluff removal treatment For fluff removal treatment, a test cloth (6cm x 6cm) is used that is obtained by stitching together a used blue knitted shirt fabric that has been fluffed by repeated washing using a commercially available detergent so that the surface of the fluff comes to the front. The test was performed using a Terg-o-meter using three pieces. The treatment was performed at least at 120 rpm for 2 hours using 1 L of the following cellulase or a treatment solution containing cellulase and xylanase of the present invention at a constant temperature of 40 ° C. After the treatment, it was rinsed well with tap water and finished with a dryer with several towels.
<Cellulase>
1) Renozyme (Novozyme)
2) Carezyme (Novozyme)
3) KSM-S237 cellulase (Japanese Patent Laid-Open No. 10-313859)
4) KSM-N252 cellulase (Japanese Patent Application 2001-043663)
5) KSM-N145 cellulase (JP 2005-287441)

(2) Measurement of the degree of fluff removal The degree of fluff removal was evaluated by the L value (brightness) of the Lab display system measured by the SCE (regular reflection light removal) method using a spectrocolorimeter (CM-3500d manufactured by Minolta). . Decrease in L value after treatment (decrease in lightness) = ΔL value relative to the L value of the sample before treatment was determined, and the degree of fluff removal was evaluated based on this ΔL value. In each test, ΔL values at 6 points were measured (N = 6), and the average value was calculated.

(3) Results When 0.02 U of CPSM activity measured with 100 mM glycine-sodium hydroxide buffer (pH 9.0) was used for KSM-N145 cellulase, N546b xylanase measured with the same buffer was used in a 0.3 U reaction system. When it was added to KSM-N145, it showed a higher degree of fluff removal compared to the use of KSM-N145 cellulase alone (FIG. 5).
Further, carezyme cellulase measured under the above conditions was used in a 0.01 U reaction system, and N546b xylanase measured under the same conditions was added as xylanase activity in 0.1 U, 0.5 U, and 1.0 U. As a result, as the addition amount increased, a higher degree of fluff removal was shown (FIG. 6).
Example 9

  Furthermore, 0.1 part by weight of N546 xylanase was blended with 100 parts by weight of the detergent having the composition shown in Table 4 to prepare the inventive detergent compositions (Prescription Examples 1 to 5). In the case of a granular cleaning agent, the detergent, which has been granulated with components excluding enzyme, PC, AC-1, and AC-2, and enzyme, PC, AC-1, and AC-2, respectively. By blending. The obtained cleaning agent has excellent cleaning power and is useful as a cleaning agent for clothing.

LAS-2: Alkyl benzene sulfonic acid “alkene L (carbon number of alkyl chain 10 to 14)” manufactured by Nisseki Detergent Co., Ltd. neutralized with 48% NaOH LAS-3: Alkyl benzene sulfone manufactured by Nisseki Detergent Co., Ltd. Acid "alkene L (alkyl chain having 10 to 14 carbon atoms)" neutralized with 50% KOH AS-2: Soda salt of Dobanol 25 sulfate (C ₁₂ -C ₁₅ sulfuric acid) manufactured by Mitsubishi Chemical Corporation SAS: Hoechst Japan Ltd. Hostapur SAS 93, C ₁₃ ~C ₁₈ alkanesulfonic sodium AOS: alpha-olefin sulfonate sodium SFE: palm oil-derived, alpha-sulfo fatty acid methyl ester sodium fatty acid salt: palmitic acid sodium AES-2: polyoxy Ethylene alkyl (C ₁₂ -C ₁₅ ) ether sulfate soda (EO average addition mole number 2)
AE-3: Nonideddo _{S-3 (C 12, C} 13 that the average 3 moles of EO alcohol, manufactured by Mitsubishi Chemical Corporation)
AE-4: Non-dead R-7 (average of 7.2 mol of EO added to C ₁₂ -C ₁₅ alcohol, manufactured by Mitsubishi Chemical Corporation)
AE-5: SOFTANOL 70 (C ₁₂ ~C ₁₅ that the average 7 moles of EO on the secondary alcohol, manufactured by Nippon Shokubai)
AG: Alkyl (derived from palm oil) glucoside (average polymerization degree 1.5)
Oil-absorbing carrier: TIXOLEX 25 (amorphous sodium aluminosilicate, manufactured by Cofran Chemical Co., Ltd., oil absorption capacity 235 ml / 100 g)
Crystalline silicate: SKS-6, δ-Na ₂ Si ₂ O ₅ , crystalline layered silicate, average particle size 20 μm, manufactured by Hoechst Tokuyama Corporation Amorphous silicate: JIS No. 1 sodium silicate STPP: tripolyphosphate soda NTA: nitrilotri Sodium acetate PAA: polyacrylic acid soda, average molecular weight 12000
AA-MA: Socaran CP5, acrylic acid-maleic acid copolymer CMC: Sunrose B1B, carboxymethylcellulose soda, manufactured by Sanyo Kokusaku Pulp Co., Ltd. (currently Nippon Paper Industries Co., Ltd.)
PEG: polyethylene glycol, average molecular weight 6000
PVP: polyvinylpyrrolidone, average molecular weight 40000, K value = 26-35
Fluorescent dye: Chinopearl CBS (Ciba Geigy) and Whitetex SA (Sumitomo Chemical) are blended at a weight ratio of 1: 1 (Note: In the case of liquid detergent, only Chinopearl CBS is blended)
Fragrance: The fragrance composition described in the examples of JP-A-8-239700 is used. Enzyme: Savinase 12.0TW (protease), lipolase 100T (lipase), Termamyl 60T (amylase), carezyme (cellulase) (the above enzymes are Novononor) Disk) and KAC500 (cellulase, manufactured by Kao) in a weight ratio of 2: 1: 1: 1 (note: liquid detergent is sabinase 16.0L (protease, manufactured by Novo Nordisk) only Combination)
PC: Sodium percarbonate, average particle diameter of 400 μm, coated with sodium metaborate AC-1: TAED, tetraacetylenediamine, manufactured by Hoechst AC-2: Lauroyloxybenzenesulfonic acid sodium

It is a graph which shows the optimal pH of the alkaline xylanase of this invention. A: Xylanase activity, B: CP (cellulose powder) decomposition activity. ×: Glycine-hydrochloric acid buffer pH 2.5, pH 3, pH 3.5, *: Citric acid-sodium citrate buffer pH 3, pH 4, pH 5, +: Acetic acid buffer pH 3.5, pH 4.5, pH 5.5, ●: Phosphate buffer pH 6.0, pH 6.5, pH 7.0, ○: Tris-HCl buffer pH 7.0, pH 8.0, pH 8.5, pH 9.0, ▲: Glycine sodium hydroxide pH 8.5, pH 9.0, pH 9.5, pH 10, pH 10.5, Δ: sodium borate sodium hydroxide buffer pH 9.5, pH 10.5, pH 10.8, □: sodium phosphate hydroxide buffer pH 11, pH 12. It is a graph which shows the pH stability of the alkaline xylanase of this invention. A: xylanase activity, B: CP degradation activity. ×: Glycine-hydrochloric acid buffer pH 2.5, pH 3, pH 3.5, *: Citric acid-sodium citrate buffer pH 3, pH 4, pH 5, +: Acetic acid buffer pH 3.5, pH 4.5, pH 5.5, ●: Phosphate buffer pH 6.0, pH 6.5, pH 7.0, ○: Tris-HCl buffer pH 7.0, pH 8.0, pH 8.5, pH 9.0, ▲: Glycine sodium hydroxide pH 8.5, pH 9.0, pH 9.5, pH 10, pH 10.5, Δ: sodium borate sodium hydroxide buffer pH 9.5, pH 10.5, pH 10.8, □: sodium phosphate hydroxide buffer pH 11, pH 12. It is a graph which shows the optimal reaction temperature of the alkaline xylanase of this invention. A: xylanase activity, B: CP degradation activity. It is a graph which shows the temperature stability of the alkali xylanase of this invention. A: xylanase activity, B: CP degradation activity. ●: No addition of 2 mM calcium chloride, ○ Addition of 2 mM calcium chloride It is a graph which shows the fluff removal effect on the surface of the cotton cloth by the combination of the alkali xylanase and cellulase (KSM-N145) of this invention. 546b: N546b xylanase, N145: KSM-N145 cellulase It is a graph which shows the fluff removal effect on the surface of the cotton cloth by the combination of the alkali xylanase and cellulase (carezyme) of this invention. care: Carezyme (Novozyme), 546b: N546b xylanase

Claims (11)

Alkaline xylanase having the following enzymological properties.
1. Action:
Hydrolyzes β1,4-glycosidic bond of xylan.
2. Substrate specificity:
Decomposes xylan and crystalline cellulose, especially xylan.
3. Optimum reaction pH:
When xylan is reacted as a substrate at 40 ° C., the optimum reaction pH is about 8.5.
4). pH stability:
When treated with xylan as a substrate at 5 ° C. for 20 hours, it exhibits an untreated residual activity of 80% or more at pH 4.0 to 10.5.
5. Optimal reaction temperature:
When the reaction is carried out for 20 minutes at pH 9.0 using xylan as a substrate, the optimum reaction temperature is about 50 ° C.
6). Thermal stability:
When treated with xylan as a substrate at pH 7.0 for 20 minutes, it exhibits a residual activity of 90% or more of untreated up to 50 ° C.
7). Molecular weight:
The molecular weight by SDS-polyacrylamide gel electrophoresis is about 40 kDa.   2. The alkaline xylanase according to claim 1, wherein the optimum reaction pH is about 5.5 when the cellulose powder is reacted at 40 ° C. as a substrate. The protein according to any one of (a) to (c) below.
(A) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 (b) an amino acid sequence shown in SEQ ID NO: 2 consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added, and xylanase activity (C) a protein comprising an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 2 and having xylanase activity   A gene encoding the protein according to claim 3. A gene comprising the polynucleotide according to any one of (a) to (d) below.
(A) a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1 (b) a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1, wherein one or several bases are deleted, substituted or added A polynucleotide encoding a protein having xylanase activity (c) a polynucleotide which hybridizes with a polynucleotide comprising the base sequence represented by SEQ ID NO: 1 under stringent conditions and encodes a protein having xylanase activity (D) a polynucleotide comprising a base sequence having 80% or more identity with the base sequence represented by SEQ ID NO: 1 and encoding a protein having xylanase activity   A recombinant vector containing the gene according to claim 4 or 5.   A transformant comprising the recombinant vector according to claim 6.   The transformant according to claim 7, wherein the host is a microorganism.   A method for producing xylanase, comprising culturing the transformant according to claim 7 or 8 and collecting xylanase.   A detergent composition comprising the alkali xylanase according to claim 1 or 2 or the protein according to claim 3.   Furthermore, the cleaning composition of Claim 10 formed by mix | blending the cellulase which has crystalline cellulose resolution | decomposability.

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