JP2001340074A - Cellulase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a cellulase without being inhibited with a cellobiose, to provide a method for producing the cellulase and to obtain a detergent composition comprising the cellulase. SOLUTION: This cellulase is a new enzyme having the optical reaction pH of about 10 without being inhibited by the cellobiose which is one of reactional products, resistant to surfactants and chelating agents and useful as an enzyme for detergents for clothes, etc., biomass and treating fibers.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a cellulase useful as an enzyme for detergents and the like.

[0002]

2. Description of the Related Art Cellulose is a main component of plant cell walls, and is a typical biomass effectively used for clothing, paper, building materials and the like. Since the structure of cellulose is a macromolecule in which glucose is linearly linked to β-1,4, it can be converted into a fuel substance or a metabolite of higher added value by decomposition. For this reason, research on cellulase as an enzyme for decomposing cellulose and effective utilization of the reaction product have been widely performed by research institutions around the world. In general, the cellulases to be studied are enzymes derived from fungi and anaerobic bacteria which have an optimum reaction pH for medium acidity and can satisfactorily degrade crystalline cellulose.

It has been found that alkalophilic Bacillus bacteria produce alkaline cellulases (Japanese Patent Publication No. 50-2).
No. 8515), the application of cellulase to heavy detergents for clothing has become possible, and alkaline cellulase (Japanese Patent Publication No.
No. 23158, JP-B-6-30578, etc.) have been incorporated into clothing detergents, and cellulase has established itself as a detergent enzyme alongside protease, lipase and amylase.

[0004]

However, most of the conventional reaction cellulases for detergents have an optimum reaction pH of around 8 to 9,
When the pH of the heavy detergent for clothing is 10 or more, it often does not work effectively. One of the general characteristics of cellulases is that the enzyme activity is inhibited by cellobiose, one of the reaction products (for example, Creuzet et al., Biochimie, 65 , 149-156 (198
3)). Cellulase activity is inhibited by the reaction product, cellobiose, which does not lead to complete decomposition of the cellulose into oligosaccharides for effective utilization as biomass, which hinders effective utilization and increases the time required for fiber scouring. However, there are problems such as the following.

Accordingly, an object of the present invention is to find a cellulase which does not inhibit the enzyme activity by cellobiose and has an optimum reaction pH in a highly alkaline region.

[0006]

Means for Solving the Problems The present inventors screened cellulase-producing bacteria from nature,
The present inventors have found a microorganism that does not inhibit the enzyme activity by cellobiose and produces cellulase having an optimum reaction pH in a highly alkaline region.

That is, the present invention provides a cellulase which is not inhibited by cellobiose and a method for producing the same. The cellulase of the present invention is not inhibited by cellobiose at pH 10;
Cellulase activated by cellobiose in
Particularly, an alkaline cellulase having an optimum reaction pH in an alkaline region is preferable. As the type of hydrolysis of the cellulase of the present invention, an endo-type which randomly acts on an amorphous region of cellulose to produce various oligosaccharides is preferable.

[0008] Alkaline cellulases having the following enzymatic properties are preferred. 1. Action: Decomposes carboxymethyl cellulose in a liquefied form. It is not inhibited by the reaction product cellobiose and is activated by cellobiose at pH 10. 2. Substrate specificity: Decomposes carboxymethylcellulose, lichenan, crystalline cellulose, and cellooligosaccharides of cellotriose or higher to generate reducing sugars. 3. Optimal reaction pH: around pH 10 (glycine-sodium hydroxide buffer)

Further, in addition to the properties 1-3, the following enzymes are particularly preferably alkaline cellulases having properties. 4. Optimal reaction temperature: around 55 ° C (pH 10, glycine-
4. Sodium hydroxide buffer) 5. Stable pH range: pH 6-11.5 (30 ° C, 60 minutes) Heat resistance: stable up to around 55 ° C (pH 7.5 Tris-
(HCl buffer, 15 minutes) 7. Molecular weight: about 50 kDa (SDS electrophoresis) 8. Isoelectric point: about pH 4.2 (isoelectric focusing method) Amino terminal sequence: Asp-Asn-Ser-Val
-Val-Gly-Gln-Asn-Gly-Gln-
Leu-Ser-Ile

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The cellulase of the present invention is produced by culturing a producing bacterium and collecting from the culture. Such cellulase-producing bacteria include bacteria belonging to the genus Bacillus, for example, Bacillus having the following mycological properties.
SP KSM-N252 strain.

(Bacillus sp. KSM-N252)
(Bacteriological properties of strain) A Morphological properties (a) Shape and size of cells: bacilli (0.4 to 0.6 ×
(B) polymorphism: no (c) motility: yes (d) spore shape, size, position, swelling: oval,
0.6-1.0 × 0.8-1.4 μm, subordinate position, swelling (e) Gram staining: indefinite, but containing 0.1% by weight of sodium carbonate (hereinafter simply referred to as%) It does not grow on violet (CVT) agar. (F) Acid resistance: negative

B. Cultural properties (a) Liquid medium for general bacteria (pH 5.7, medium 1): does not grow (b) Liquid medium for general bacteria (pH 6.8, medium 1): does not grow (c) ) Agar medium for general bacteria (pH 6.5, medium 2): does not grow. (D) Agar medium for general bacteria (pH 8.5, medium 2): grows. Medium 1 is a nutrient broth (Difco) medium 2. Is a nutrient agar (Difco), the indicated amount was added, and the pH of the culture medium 1 was adjusted to a predetermined pH with dilute hydrochloric acid and the culture medium 2 with sodium carbonate.

C Physiological properties (a) Nitrate reduction: positive (b) Denitrification reaction: negative (c) VP test: negative (d) Indole formation: negative (e) Hydrogen sulfide formation: negative (f) Starch hydrolysis: positive (g) Casein hydrolysis: negative (h) Gelatin liquefaction: positive (i) Utilization of citric acid: positive (j) Catalase: positive (k) Oxidase: positive (l) Growth temperature Range: 11 to 44 ° C (m) pH range for growth: pH 7.6 to 10.5 (n) Effect of enzymes on growth: Grow under anaerobic conditions. (O) Gas production from glucose: negative (p) Sodium chloride tolerance: grows in the presence of 7% sodium chloride. (Q) Hydrolysis of hippuric acid: negative (r) Hydrolysis of 4-methylumbelliferyl-β-D-glucuronide (MUG): negative (s) Utilization of sugar: glucose, arabinose, xylose, mannitol, galactose , Sucrose,
Mannose, maltose, lactose, trehalose,
Growth is observed using fructose, melibiose, ribose, salicin and the like as carbon sources. Glycerol, rhamnose, inositol and sorbitol cannot be used as carbon sources.

As described above, Bacillus SP KSM-N2
52 strains were alkalophilic bacteria that did not grow on a neutral medium, and were catalase-positive spore-forming bacilli, so they were determined to be alkalophilic Bacillus bacteria. Therefore, Nielsen et al. Newly described the alkalophilic Bacillus bacterium in terms of the morphology and physiological properties of this strain (Micr
obiology, 141 , 1745-1761, 1995), and as a result, the strain was considered to be a strain closely related to Bacillus suldo alcalophilus. However, its properties do not completely match those of the known Bacillus sudo alcalophilus, and do not match the properties of other Bacillus species. Bacillus SP KSM-N252
(FERMP-17474).

To produce the cellulase of the present invention using a cellulase-producing bacterium such as Bacillus sp. KSM-N252, the strain is inoculated into a medium containing an assimilable carbon source, a nitrogen source and other essential nutrients. Shaking culture or aeration-agitation culture may be performed according to the method. The pH of the medium is
What is necessary is just to adjust to pH suitable for cellulase production of the present invention using sodium carbonate or the like.

The collection and purification of the cellulase from the culture thus obtained can be carried out according to a general method. That is, the bacterial cells are removed from the culture by centrifugation or filtration, and the target enzyme can be concentrated from the resulting culture supernatant by conventional methods, for example, salting out, solvent precipitation, ultraconcentration, and the like. it can. The enzyme is precipitated under conditions such as ammonium sulfate (30-90% saturated fraction) as an example of the salting-out method and cold acetone (50% or more) as an example of the solvent precipitation, followed by centrifugation, desalting, and freezing. A dry powder or a spray-dried powder can be obtained. Dialysis as a desalting method,
Gel filtration or ultrafiltration using Sephadex G-10 or the like is used. The enzyme solution or the dry powder thus obtained can be used as it is, but can be further crystallized or granulated by a known method.

The alkaline cellulase derived from Bacillus sp. KSM-N252, which is an example of the cellulase of the present invention, has the following properties. In addition, the measurement of the enzyme activity was performed as follows.

(Method for measuring enzyme activity) 0.1 mL of 0.5 M glycine-sodium hydroxide buffer solution (pH 1) was placed in a test tube.
0.0), 0.4 mL of 2.5% (w / v) carboxymethylcellulose (A01MC; Nippon Paper), 0.4 mL of deionized water, 0.1 mL of an appropriately diluted enzyme solution (10 mM Tris Hydrochloric acid buffer, pH 7.5) and allowed to react for 20 minutes, followed by 1 mL of dinitrosalicylic acid reagent (0.5% dinitrosalicylic acid, 30% Rochelle salt,
(A 1.6% aqueous sodium hydroxide solution), and the reducing sugar was colored in a boiling water bath for 5 minutes. Quench in an ice-water bath, 4
mL of deionized water was added and the absorbance at 535 nm was measured to determine the amount of reducing sugar produced. A blank was prepared by adding a dinitrosalicylic acid reagent to a reaction solution treated without adding an enzyme solution, and then adding an enzyme solution to develop a color in the same manner. One unit (1 U) of the enzyme is 1 unit under the above reaction conditions.
The amount was such that 1 μmol of reducing sugar equivalent to glucose was produced per minute.

(1) Molecular weight (SDS electrophoresis) The enzyme solution treated with SDS (100 ° C., 3 minutes)
SDS electrophoresis was performed using a 2.5% acrylamide gel. Phosphorylase b (97.
4kDa), serum albumin (67kDa), ovalbumin (45kDa), carbonic anhydrolase (31kD)
a), using a trypsin inhibitor (21.5 kDa) and α-lactalbumin (14.4 kDa), a calibration curve was prepared from the respective mobilities and molecular weights, and the molecular weight of the enzyme was estimated to be about 50 kDa. .

(2) Isoelectric point The isoelectric focusing of the enzyme was performed on a PAG plate (Pharmacia) having a pH of 3.5 to 9.5 using a Multi Four II electrophoresis system (Pharmacia). After the electrophoresed gel was stained with Coomassie brilliant blue G-250, the isoelectric point of the enzyme was determined from the calibration curve obtained from the isoelectric point of the standard protein and the mobility, and was determined to be around pH 4.2. Was done.

(3) Amino terminal sequence After adsorbing this enzyme to a prosolve filter, the amino acid sequence from the amino terminal to the 13th amino acid sequence was examined by a protein sequencer (476A; Applied Biosystem). Asp-Asn-Ser -Val-V
al-Gly-Gln-Asn-Gly-Gln-Le
u-Ser-Ile.

(4) Optimum reaction pH acetate buffer (pH 4-6), phosphate buffer (pH 6-
8), Tris-HCl buffer (pH 7-9), Glycine-
The optimum reaction pH was examined using each buffer (50 mM) of a sodium hydroxide buffer (pH 8 to 11), a carbonate buffer (pH 9 to 11), and a phosphate-sodium hydroxide buffer (pH 11 to 12.5). As a result, this enzyme showed the highest reaction rate in a glycine-sodium hydroxide buffer solution at pH 10. Further, it had an activity of 80% or more of the maximum activity between pH 8 and 11 (FIG. 1).

(5) Optimal reaction temperature 50 mM glycine-sodium hydroxide buffer (pH 10)
The enzyme reaction was performed at each temperature of 30 ° C to 80 ° C, and the optimum reaction temperature was examined. As a result, the enzyme showed an optimum reaction temperature around 55 ° C., and was inactivated at a temperature higher than that (FIG. 2).

(6) Stable pH range Acetate buffer (pH 4-6), MOPS buffer (pH 6-6)
8), phosphate buffer (pH 6-8), Tris-HCl buffer (pH 7-9), glycine-sodium hydroxide buffer (pH 8-11), carbonate buffer (pH 9-11), borate buffer The enzyme was added to each buffer solution (50 mM) of (pH 9 to 11) and a phosphate-sodium hydroxide buffer solution (pH 11 to 12.5), and the temperature was kept at 30 ° C. for 30 minutes. As a result, the activity of this enzyme before treatment was 100
%, It was extremely stable in the pH range of 6 to 11.5 (FIG. 3).

(7) Heat resistance The enzyme was added to a 50 mM Tris-hydrochloric acid buffer (pH 7.5), and the remaining activity was measured after being kept at 30 ° C. to 70 ° C. for 15 minutes. The enzyme is 5
It was stable up to 5 ° C. (FIG. 4).

(8) Substrate Specificity When the decomposition rate for carboxymethylcellulose is 100% under the standard activity measurement conditions, the decomposition rate is high at 368% for lichenan, 91% for phosphate-swollen Avicel, and xylan Was 20%. However, curdlan and laminarin did not decompose at all. The decomposition reaction of glucoside of cellotetraoside from the paranitrophenyl group derivative was performed in a 50 mM glycine-sodium hydroxide buffer solution (pH 8.0). As a result, the enzyme can decompose substrates equal to or higher than paranitrophenyl cellobioside, and 44% for paranitrophenyl cellobioside, 117% for paranitrophenyl cellotrioside, and 110% for paranitrophenyl tetraoside. % Relative rate (decomposition rate for carboxymethylcellulose at pH 8.0 is 10
(Assuming 0%). In addition, sigmacell 101 (Sigma) or Avicel SF which is insoluble cellulose is used.
(Funakoshi) as a substrate, 50 mM phosphate buffer (pH
6.0) for 1 hour, 2% and 0.58
% (When the decomposition rate for carboxymethylcellulose at pH 6.0 was 100%).

(9) Decomposition mode of substrate 50 mM glycine-sodium hydroxide buffer (pH 1)
0), 1.0% carboxymethylcellulose (A10M
C: Nippon Paper Industries Co., Ltd.) was placed in an Ostwald viscometer (No. 3; Shibata Kagaku Kogyo Co., Ltd.), kept at 30 ° C. for 10 minutes, and then the enzyme was added to make a total volume of 10 mL. After 5 to 30 minutes, the viscosity was measured, and the reaction solution was withdrawn at each time, the amount of reducing sugar produced was examined by the dinitrosalicylic acid method, and the amount of cleavage was calculated. As a result, as shown in FIG. 5, the present enzyme degraded carboxymethyl cellulose in an endo format. Next, using cellooligosaccharides (G1-G6) as substrates, 50
The decomposition reaction was performed at 30 ° C. for 2 hours in a mM glycine-sodium hydroxide buffer (pH 8.0). Thereafter, a fixed amount was spotted on a thin layer chromatography plate, and developed with a solvent system of butanol / ethanol / deionized water = 5/3/1 (v / v). An anisaldehyde-sulfuric acid solution was used to detect sugar spots. As a result, cellotriose (G
From G.3, G2 and G1 spots from cellotetraoside (G4)
G3 and G2 spots were detected from cellopentaoside (G5), and G5, G4, G3, G2 and G1 spots were detected from cellohexaoxide. Therefore, it was found that this enzyme can degrade G3 or more cellooligosaccharides.

(10) Effect of cellobiose 50 mM glycine-sodium hydroxide buffer (pH 1)
0), 1.0% carboxymethylcellulose (A10M
C; Nippon Paper Industries Co., Ltd.) and a reaction solution composed of 50-100 mM cellobiose in an Ostwald viscometer (No. 3).
After incubating at 10 ℃ for 10 minutes, add this enzyme and add 10mL
And As a result of measuring the viscosity after 5 to 30 minutes, as shown in FIG. 6, the enzyme was not inhibited at all in the presence of a high concentration of cellobiose, but rather the enzyme activity was promoted. On the other hand, when commercially available cellulases A and B were added to a reaction solution containing 50 mM glycine-sodium hydroxide buffer (pH 9), 1.0% carboxymethylcellulose, and 100 mM cellobiose, the enzyme activities of both cellulases were inhibited ( (FIG. 7).

(11) Influence of Surfactant In a reaction system in which various surfactants were added to a concentration of 0.05% (w / v), the activity of the enzyme was measured. As a result, it was found that the present enzyme can express the activity extremely stably in the presence of each surfactant (Table 1).

[0030]

[Table 1]

(12) Influence of chelating agent The enzyme of the present invention was incubated at 50 ° C. in a 50 mM MOPS buffer (pH 7.0) containing 5 mM EDTA, EGTA and orthophenanthrin at 30 ° C. for 30 minutes. Activity was measured. Thereafter, the remaining activity was examined by a standard activity measurement method. As a result, this enzyme was extremely stable to any chelating agent.

(13) Influence of Metal Salt Enzyme reaction was carried out by adding 0.5 mM of each metal salt to the standard activity measurement conditions. As a result, it was found that the present enzyme was activated by about 10% by cobalt chloride and ferric chloride. It was inhibited by about 20% by mercury chloride and about 10% by copper chloride and cadmium chloride. Other metal ions such as manganese, magnesium, zinc, nickel, aluminum, copper, strontium, barium, lead, barium, beryllium, and palladium had almost no effect.

As described above, the alkaline cellulase of the present invention is a novel enzyme which is not inhibited by cellobiose, one of the reactants, has an optimum reaction pH of around 10, and is resistant to surfactants and chelating agents. It is useful as an enzyme for cleaning agents, biomass, and fiber treatment.

The amount of the cellulase to be added to the detergent composition of the present invention is not particularly limited as long as the cellulase exhibits an activity. In consideration of the above, it is preferably 100,000 U or less, and more preferably 10,000 U or less.

The thus obtained detergent composition containing the cellulase of the present invention has an excellent detergency as shown in Example 2 below.

When the cellulase of the present invention is used in a powder detergent composition, the granules produced according to the method described in JP-A No. 62-257990 are used after the production of the detergent particles in order to prevent inactivation or decomposition by heat. Mixing is preferred.

The detergent composition of the present invention can use various enzymes in addition to the cellulase of the present invention. For example,
Hydrolases, oxidases, reductases, transferases, lyases, isomerases, ligases, synthetases and the like. Among them, proteases, cellulases other than the present invention, keratinase, esterase, cutinase, amylase, lipase, pullulanase, pectinase, mannanase, glucosidase, glucanase, cholesterol oxidase, peroxidase, laccase, and the like are particularly preferable, and protease, cellulase, amylase, and lipase are particularly preferable. preferable. Examples of the protease include commercially available alcalase, esperase, savinase, evalase (above, Novozyme), properase, Plafect (Genencor), and KAP (Kao). As cellulase, cellzyme, carezyme (above, Novozyme), KAC (Kao), JP-A-10-3138
Alkaline cellulase (Kao) produced by Bacillus sp. Examples of amylase include Termamyl, Duramil (above, Novozyme), Plaster (Genencor), and KAM (Kao). These enzymes are from 0.001 to 1
0%, preferably 0.03 to 5% is blended.

The detergent composition of the present invention may contain known detergent components. Examples of the known detergent components include the following.

(1) Surfactant The surfactant is incorporated in the detergent composition in an amount of 0.5 to 60% by weight (hereinafter simply referred to as "%"), particularly 10 to 45% for a powdery detergent composition, and a liquid detergent composition. It is preferable to mix 20 to 50% of the product. When the detergent composition of the present invention is a bleach detergent or an automatic dishwasher detergent, the surfactant is generally blended in an amount of 1 to 10%, preferably 1 to 5%.

As the surfactant used in the detergent composition of the present invention, one or a combination of an anionic surfactant, a nonionic surfactant, an amphoteric surfactant and a cationic surfactant can be exemplified. And preferably an anionic surfactant and a nonionic surfactant.

As anionic surfactants, those having 10 to 10 carbon atoms
A sulfate of an alcohol having 18 carbon atoms, a sulfate of an alkoxylated alcohol having 8 to 20 carbon atoms, an alkylbenzene sulfonate, a paraffin sulfonate, α-
Olefin sulfonates, α-sulfofatty acid salts, α-sulfofatty acid alkyl ester salts or fatty acid salts are preferred. In the present invention, particularly, the alkyl chain has 10 to 14 carbon atoms,
More preferably, a linear alkylbenzene sulfonate of 12 to 14 is preferable, and as a counter ion, alkali metal salts and amines are preferable, and sodium and / or potassium, monoethanolamine, and diethanolamine are particularly preferable.

Examples of the nonionic surfactant include polyoxyalkylene alkyl (C 8-20) ether, alkyl polyglycoside, polyoxyalkylene alkyl (C 8-20) phenyl ether, polyoxyalkylene sorbitan fatty acid (C 8 Preferred are 8-22) esters, polyoxyalkylene glycol fatty acid (8-22 carbon atoms) esters, and polyoxyethylene polyoxypropylene block polymers. In particular, as a nonionic surfactant, 4 to 20 moles of an alkylene oxide such as ethylene oxide or propylene oxide was added to an alcohol having 10 to 18 carbon atoms [HLB value (calculated by the Griffin method) was 10.5 to 15.
0, preferably 11.0 to 14.5].

(2) Divalent metal ion scavenger The divalent metal ion scavenger is incorporated in an amount of 0.01 to 50%, preferably 5 to 40%. Examples of the divalent metal ion scavenger used in the cleaning composition of the present invention include condensed phosphates such as tripolyphosphate, pyrophosphate and orthophosphate, aluminosilicates such as zeolite, and synthetic layered crystalline silicic acid. Salts, nitrilotriacetate, ethylenediaminetetraacetate, citrate, isocitrate, polyacetalcarboxylate, and the like. Of these, crystalline aluminosilicates (synthetic zeolites) are particularly preferred, and A-type, X-type
Of the zeolite type and P type, A type is particularly preferred. As the synthetic zeolite, those having an average primary particle size of 0.1 to 10 μm, particularly 0.1 to 5 μm are suitably used.

(3) Alkali agent The alkaline agent is 0.01 to 80%, preferably 1 to 40%.
Be blended. In the case of powder detergents, alkali metal carbonates such as sodium carbonate, which are collectively referred to as dense ash and light ash, and amorphous alkali metal silicates such as JIS Nos. 1, 2, and 3 are exemplified. These inorganic alkaline agents are effective in forming the skeleton of particles when the detergent is dried, and a relatively hard detergent having excellent fluidity can be obtained. Other alkalis include sodium sesquicarbonate and sodium hydrogen carbonate, and phosphates such as tripolyphosphate also have an action as an alkali agent. In addition, as the alkaline agent used in the liquid detergent, sodium hydroxide, as well as the above alkaline agent, and mono, di, or triethanolamine can be used, and can also be used as a counter ion of the activator.

(4) Anti-soil redeposition agent The anti-soil redeposition agent is 0.001 to 10%, preferably 1 to 5%.
%. Examples of the anti-redeposition agent used in the cleaning composition of the present invention include polyethylene glycol, carboxylic acid-based polymers, polyvinyl alcohol, and polyvinylpyrrolidone. Among these, the carboxylic acid-based polymer has a function of trapping metal ions and a function of dispersing solid particle stains from clothes into a washing bath, in addition to the ability to prevent recontamination.
The carboxylic acid-based polymer is a homopolymer or copolymer of acrylic acid, methacrylic acid, itaconic acid, etc.
The copolymer is preferably a copolymer of the above monomer and maleic acid, and preferably has a molecular weight of several thousand to 100,000. In addition to the carboxylic acid-based polymers, polymers such as polyglycidylate, cellulose derivatives such as carboxymethylcellulose, and aminocarboxylic acid-based polymers such as polyaspartic acid also have a metal ion scavenger, a dispersant, and a re-contamination preventing ability. It is preferred.

(5) Bleaching agents For example, bleaching agents such as hydrogen peroxide and percarbonate are 1 to 10%.
It is preferable to mix them. When using a bleaching agent, use tetraacetyl ethylenediamine (TAED) or
A bleaching activator (activator) as described in JP-A-316700 can be blended in an amount of 0.01 to 10%.

(6) Fluorescent Agent Examples of the fluorescent agent used in the cleaning composition of the present invention include a biphenyl type fluorescent agent (eg, Tinopearl CBS-X) and a stilbene type fluorescent agent (eg, a DM type fluorescent dye). . It is preferable that the fluorescent agent is added in an amount of 0.001 to 2%.

(7) Other Ingredients The detergent composition of the present invention contains a builder, a softening agent, a reducing agent (such as a sulfite), a foam inhibitor (such as a silicone), a fragrance known in the field of clothing detergents. , And other additives.

The detergent composition of the present invention can be produced according to a conventional method by combining the cellulase obtained by the above method and the above-mentioned known cleaning components. The form of the detergent can be selected according to the use, and for example, can be liquid, powder, granules and the like. The detergent composition thus obtained can be used as a clothing detergent, a bleach detergent, a detergent for automatic dishwashers, and the like.

When the detergent composition of the present invention is a powder detergent, it is particularly preferred that the detergent composition is as follows. 10 to 50% by weight of the surfactant (preferably 15 to
45% by weight), 10 to 50% by weight (preferably 20 to 40% by weight) of a water-soluble inorganic substance, and 5 to 5% of a water-insoluble inorganic substance.
0% by weight (preferably 10 to 40% by weight), wherein the total of the water-soluble inorganic substance and the water-insoluble inorganic substance is 4%.
A cleaning composition comprising particles such as 0-70% by weight (preferably 45-65% by weight). Further, a detergent composition which is a particle capable of releasing bubbles having a diameter of 1/10 or more of the particle diameter from the inside of the particle in the process of dissolving the particle in water.

As the surfactant used for such particles, various surfactants as described in (1) can be used. Further, as the water-soluble inorganic substance, carbonates, hydrogencarbonates, sulfates, sulfites, hydrogensulfates, hydrochlorides, or alkali metal salts such as phosphates, water-soluble inorganic salts such as ammonium salts or amine salts, and And low molecular weight water-soluble organic acid salts such as citrate and fumarate. As the water-insoluble inorganic substance, those having an average primary particle size of 0.1 to 20 μm are preferable. For example, clays such as crystalline or amorphous aminosilicates, silicon dioxide, hydrated silicate compounds, pearlite, bentonite, etc. There are compounds and the like, and crystalline or amorphous aluminosilicates, silicon dioxide, and hydrated silicate compounds are preferred. Among them, crystalline aluminosilicates are preferable in terms of sequestering ability and supporting ability of a surfactant. Is preferred.

The particles preferably further contain 0.5 to 20% by weight of a water-soluble polymer from the viewpoint of washing performance.
1 to 15% by weight is more preferable, and 3 to 10% by weight is further preferable. Examples of the water-soluble polymer include polyalkylene glycol, carboxylic acid-based polymer, carboxymethylcellulose, soluble starch, saccharides and the like. Carboxylic acid polymers having a molecular weight of several thousands to 100,000 and polyethylene glycol are preferred. Particularly, salts of acrylic acid-maleic acid copolymer, polyacrylates and polyethylene glycol are preferred. Here, the salt includes a sodium salt, a potassium salt, and an ammonium salt.

The particles are preferably subjected to surface modification with a surface coating agent in view of fluidity and non-caking properties.
The surface coating agent is preferably 1 to 30% by weight of the particles,
25% by weight is more preferred, and 5 to 25% by weight is even more preferred. As the surface coating agent, for example, aluminosilicate, calcium silicate, silicon dioxide, bentonite,
Silicate compounds such as talc, clay, amorphous silica derivatives and crystalline silicate compounds, fine powders such as metal soaps and powdered surfactants, carboxymethyl cellulose, polyethylene glycol, sodium polyacrylate, copolymers of acrylic acid and maleic acid Or water-soluble polymers such as polycarboxylates such as salts thereof, and fatty acids. Among them, water-insoluble inorganic substances are preferred, and crystalline aluminosilicates, amorphous aluminosilicates, and crystalline silicate compounds are particularly preferred.

In terms of solubility, these particles are preferably at least 1/10, more preferably at least 1/5, further preferably at least 1/4, particularly preferably at least 1/3 of the particle diameter in the process of dissolving in water. Emit bubbles of the above diameter. It is preferable that bubbles of a predetermined size be generated within 120 seconds when dissolved in water in a state where the bubbles are released.
0 seconds or less are more preferable, and 45 seconds or less are still more preferable.
In order to release bubbles, it is only necessary to have pores (single or plural) capable of releasing bubbles of a predetermined size, and the form and structure of the particles are not particularly limited.

The method for producing the particles is not particularly limited, but from the viewpoint of solubility, 100 parts by weight of base granules obtained by spray-drying a slurry containing a water-soluble inorganic substance, a water-insoluble inorganic substance and, if necessary, a water-soluble polymer. It is preferable to use a method in which 5 to 80 parts by weight of a surface active agent is supported on each part, and then the surface is modified with a surface coating agent.

The particles are used in terms of convenience and waste reduction.
The bulk density measured by the method specified by JIS K 3362 is preferably at least 600 g / L, more preferably at least 700 g / L, even more preferably at least 800 g / L.
In terms of solubility, the bulk density is preferably 1600 g / L or less, more preferably 1300 g / L or less, and 1000 g / L or less.
g / L or less is more preferable.

The particles are JIS Z 8 in terms of solubility.
After oscillating for 5 minutes using a standard sieve of No. 801, the average particle size determined from the weight fraction depending on the size of the sieve is 150 to 70.
0 μm is preferable, and more preferably 150 to 600 μm
m, more preferably 180 to 400 μm. Also,
From the viewpoint of solubility, particles having a size of 177 to 350 μm are preferably 40% by weight or more, more preferably 50% by weight or more.

From the viewpoint of cleaning performance, these particles are preferably 50 to 99.5% by weight, more preferably 50 to 98% by weight, even more preferably 60 to 96% by weight, and more preferably 70 to 96% by weight in the detergent composition.
-94% by weight is particularly preferred.

[0059]

EXAMPLES Example 1 (1) Screening of Cellulase-Producing Bacteria Suspension of soil in various parts of Japan in sterilized water was carried out at 80 ° C and 30 ° C.
For 2 minutes, coated on an agar plate medium having the following composition [Carboxymethylcellulose (A10MC; Nippon Paper) 2.0%, meat extract (oxoid) 1.0%, bactopeptone (Difco) 1.0%, chloride Sodium 1.0%, potassium dihydrogen phosphate 0.1%, sodium carbonate 0.5% (separate sterilization), trypan blue 0.005%
(Separate sterilization)]. The culture was allowed to stand still for 3 days in an incubator at 30 ° C., and a single colony was repeatedly selected from those in which lysis spots associated with the decomposition of carboxymethyl cellulose were detected around the grown bacteria. Polypeptone S (Nippon Pharmaceutical) 2.0%, fish meat extract (Wako Pure Chemical) 1.
0%, potassium monohydrogen phosphate 0.15%, yeast extract (Difco) 0.1%, magnesium sulfate heptahydrate 0.0
3% using a liquid medium consisting of 7%, 0.1% carboxymethylcellulose, and 0.5% sodium carbonate (separately sterilized).
Shaking culture was performed at 0 ° C. for 3 days. Bacterial strains producing cellulase were selected, and Bacillus sp. KSM-N252 strain was obtained as a cellulase-producing bacterium that showed a strong activity particularly in a high alkaline region.

(2) Bacillus sp. KSM-N2
Production of cellulase by 52 strains The culture of Bacillus sp. KSM-N252 strain obtained by the above-mentioned screening was carried out in a 500-mL Sakaguchi flask with 100 mL of medium [polypeptone S 1.0%, fish meat extract 0.5%, enzyme extract 0.1%]. %, Potassium hydrogen phosphate 0.15%, magnesium sulfate heptahydrate 0.07%
%, Sodium glutamate 1.0%, lactose2.
0%, sodium carbonate 0.5% (separate sterilization)]
The reaction was performed aerobically at 0 ° C. for 4 days. The medium composition was the same as above. As a result, a productivity of about 1200 U / L was obtained.

(3) Purification of Cellulase The culture of Bacillus sp. KSM-N252 was centrifuged (8000 × g, 15 minutes, 4 ° C.), and the supernatant (2
L) was obtained. This was concentrated to 115 mL using a module (AIP-1300; Asahi Kasei). Pre-concentrate 10
DEAE Toyopearl 650M column (5.5 × 18) equilibrated with mM Tris-HCl buffer (pH 7.5)
cm; Tosoh). After washing and eluting the non-adsorbed protein using about 1.5 L of the equilibration buffer, 0 to 0.
The adsorbed protein was eluted by the concentration gradient method of 4M sodium chloride. Fractions showing cellulase activity eluted near the concentration of 0.1 M sodium chloride were collected (325 mM).
L) and concentration by ultrafiltration (PM10 membrane; Amicon) to 25 mL. Next, DEAE-Bio equilibrated with 10 mM phosphate buffer (pH 6.0)
It was attached to a GelA column (2.5 × 24 cm; Bio-Rad) and washed and eluted with about 350 mL of the same buffer.
Further, a buffer solution containing 0 to 0.125 M potassium chloride (5
(00 mL each), and the adsorbed protein was eluted by the concentration gradient method. As a result, alkaline cellulase was eluted near the potassium chloride concentration of 0.1 to 0.125M. The fractions were collected and concentrated by ultrafiltration (PM10 membrane) (25 mL). This was previously equilibrated with 10 mM Tris-HCl buffer (pH 9.5).
The solution was applied to an AE-Bio-GelA column (2.5 × 11 cm) and contained a buffer (0 to 0.125 M potassium chloride (2
(50 mL each), and the adsorbed protein was eluted by the concentration gradient method, and further, the adsorbed protein was eluted with a buffer (250 mL each) containing 0.125 to 0.25 M potassium chloride. As a result, the cellulase was eluted near the potassium chloride concentration of 0.18 M, and this fraction was collected and concentrated by ultrafiltration (PM10 membrane) (1.
5 mL). The obtained concentrated solution was applied to a Bio-GelA05m column (1.5 × 75 cm; Bio-Rad) equilibrated with 10 mM Tris-HCl buffer (pH 7.5) containing 0.1 M potassium chloride, and subjected to gel filtration chromatography. . Fractions showing cellulase activity were collected (35 mL) and applied to a hydroxyapatite column (1.5 × 8 cm) which had been equilibrated with 10 mM phosphate buffer (pH 7.0) in advance. The cellulase passed without adsorbing to the column, but when this fraction was concentrated and subjected to SDS electrophoresis,
A uniform protein band with a molecular weight of about 50 kDa was detected. This enzyme was purified about 1000 times by the above purification operation, and the activity yield was 6%. The purified cellulase obtained here was an alkaline cellulase exhibiting the aforementioned enzymatic properties.

Example 2 Detergency Test (1) Preparation of Detergent A detergent test was carried out using the detergent described in Example 3 of WO 99/29830. That is, 1m ³ with stirring blades
After the water temperature reached 55 ° C., 48 kg of a 50% (w / v) aqueous sodium dodecylbenzenesulfonate solution and 135 kg of a 40% (w / v) aqueous sodium polyacrylate solution were added to the mixing tank. After stirring for 15 minutes, 120 kg of sodium carbonate, 60 kg of sodium sulfate, 9 kg of sodium sulfite, 3 k of fluorescent dye
g was added. After further stirring for 15 minutes, 300 kg of zeolite was added and stirred for 30 minutes to obtain a homogeneous slurry (water content in the slurry was 50% by weight). The base granules were obtained by spraying this slurry from a pressure spray nozzle installed near the top of the spray-drying tower. (The high-temperature gas supplied to the spray-drying tower was supplied at a temperature of 225 ° C. from the bottom of the tower, and from the top of the tower. Discharged at 105 ° C).

Next, 15 parts by weight of the nonionic surfactant and 50 parts by weight
A 15% by weight aqueous solution of sodium dodecylbenzenesulfonate and 1 part by weight of polyethylene glycol were heated at 70 ° C.
To obtain a mixed solution. 100 parts by weight of the base granules are charged into a Lodige mixer (manufactured by Matsuzaka Giken Co., Ltd., capacity 20 L, with jacket), and the main shaft (1
(50 rpm) and stirring of the chopper (4000 rpm) were started. In addition, warm water of 75 ° C was applied to the jacket at 10 L /
The mixture was poured in 3 minutes, and then stirred for 5 minutes. Further, the surface of the particles of the detergent particles was coated with 10 parts by weight of a crystalline aluminosilicate to obtain a final granular detergent.

<Raw Materials Used> Aqueous sodium dodecylbenzenesulfonate solution: Neoperex F65 (manufactured by Kao Corporation) Nonionic surfactant: Emulgen 108KM having an average ethylene oxide mole number of 8.5 (manufactured by Kao Corporation) ) Aqueous sodium polyacrylate solution: average molecular weight 1000
0 (manufactured according to the method described in Examples in Japanese Patent Publication No. 2-24283) Sodium carbonate: dense ash (manufactured by Central Glass Co., Ltd.) Zeolite: zeolite type 4A having an average particle size of 3.5 μm (Tosoh Corporation) )) Polyethylene glycol: K-PEG 6000 (average molecular weight 8500, manufactured by Kao Corporation) Fluorescent dye: Tinopearl CBS-X (manufactured by Ciba Geigy)

(2) Method of measuring detergency (a) Artificially contaminated oil and fat cloth An artificially contaminated oil and fat cloth described in JP-A-57-108199 was used.

(B) Preparation of Cellulase Granules From the purified enzyme obtained in Example 1, JP-A-62-2579
No. 90 (600 U /
g).

(C) Washing conditions and method: The detergent was dissolved in 4 ° DH hard water at a constant temperature of 30 ° C.
Prepare 1 L of 667% detergent aqueous solution. Artificial fat stained cloth 5
A sheet (6 × 6 cm) was added to a detergent aqueous solution, left at 30 ° C. for 1 hour, and then the detergent solution and the artificially-stained cloth were transferred as they were to a stainless steel beaker for a tergotometer, and the tergotometer was used at 100 rpm at 30 ° C. Stir and wash for minutes.
After the artificially stained cloth was rinsed under running water, it was iron-pressed and subjected to reflectance measurement. The calculation of the cleaning rate was performed according to the following equation. The reflectance at 550 nm of the original cloth and the contaminated cloth before and after cleaning was measured with a self-recording color system (manufactured by Shimadzu Corporation), and the cleaning rate was calculated by the following equation. Cleaning rate = (Reflectance after cleaning−Reflectance before cleaning) / (Reflectance of original cloth−Reflectance before cleaning) × 100

The above-mentioned enzymes were added in various amounts to the granular detergent shown in (1) to conduct a detergency test. Table 2 shows the results. It can be seen that the cleaning composition of the present invention has excellent detergency.

[0069]

[Table 2]

[0070]

The cellulase of the present invention is not inhibited by cellobiose, one of the reaction products, and has an optimum reaction pH of pH
It has about 10 and is a novel enzyme resistant to surfactants and chelating agents, and is useful as a detergent for clothing, a biomass, and a fiber treatment enzyme.

[Brief description of the drawings]

FIG. 1 is a diagram showing the effect of pH on cellulase activity of the present invention.

FIG. 2 is a diagram showing the effect of temperature on the cellulase activity of the present invention.

FIG. 3 shows the effect of pH on cellulase stability of the present invention.

FIG. 4 is a diagram showing the effect of temperature on the cellulase stability of the present invention.

FIG. 5 is a graph showing the relationship between the decrease in viscosity due to the decomposition of carboxymethylcellulose by the cellulase of the present invention and the amount of reducing terminal generated.

FIG. 6 is a graph showing a decrease in viscosity associated with carboxymethylcellulose degradation in the presence of cellobiose at pH 10 by the cellulase of the present invention.

FIG. 7 is a graph showing a decrease in viscosity due to carboxymethylcellulose degradation in the presence of cellobiose at pH 9 by commercially available cellulases A and B.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Yoshihiro Hakamada 2606 Kabane-cho, Akaga-cho, Haga-gun, Tochigi Prefecture (72) Inventor Keiji Endo 2606, Kabashi-cho, Akabane-shi, Haga-gun, Tochigi Prefecture Kao Corporation ( 72) Inventor Toru Kobayashi 2606, Kabane-cho, Akaga-cho, Haga-gun, Tochigi Pref. In the Kao Research Institute (72) Inventor Shuji Kawai 2606, Akabane, Kaiga-cho, Haga-gun, Tochigi Pref. In Kao Research Institute (72) Inventor Yasuo Wada 1334 Minato, Wakayama-shi, Wakayama Pref. Kao Corporation Research Laboratory F-term (reference) 4B050 CC01 DD02 FF03E FF05E FF09E FF11E FF12E JJ03 KK02 KK03 KK05 KK10 LL04 LL05 4B065 AA15X AC12 AC15 BA23 BB03 BB12 BC17 BC16 BC17 BC16 AB19 AC08 BA09 DA01 EA12 EA16 EA28 EB30 EB36 EC03 ED02 FA47

Claims (7)

[Claims] 1. A cellulase that is not inhibited by cellobiose. 2. Cellulase activated by cellobiose at pH 10. 3. A cellulase having the following enzymatic properties: 1. Action: Decomposes carboxymethyl cellulose in a liquefied form. It is not inhibited by the reaction product cellobiose and is activated by cellobiose at pH 10. 2. Substrate specificity: Decomposes carboxymethylcellulose, lichenan, crystalline cellulose, and cellooligosaccharides of cellotriose or higher to generate reducing sugars. 3. 3. Optimal reaction pH: around pH 10 (glycine-sodium hydroxide buffer) Optimal reaction temperature: around 55 ° C (pH 10, glycine-
4. Sodium hydroxide buffer) 5. Stable pH range: pH 6-11.5 (30 ° C, 60 minutes) Heat resistance: stable up to around 55 ° C (pH 7.5 Tris-
(HCl buffer, 15 minutes) 7. Molecular weight: about 50 kDa (SDS electrophoresis) 8. Isoelectric point: about pH 4.2 (isoelectric focusing method) Amino terminal sequence: Asp-Asn-Ser-Val
-Val-Gly-Gln-Asn-Gly-Gln-
Leu-Ser-Ile 4. A method for producing cellulase, comprising culturing the cellulase-producing bacterium according to claim 1, and collecting the cellulase. 5. The method according to claim 4, wherein the cellulase-producing bacterium is a Bacillus bacterium. 6. The method for producing a cellulase according to claim 5, wherein the Bacillus bacterium is Bacillus sp. KSM-N252 (FERM P-17474). 7. A detergent composition containing the cellulase according to claim 1.