JP2001309781A - New alkaline cellulase - Google Patents

Abstract

(57) An alkaline cellulase having an isoelectric point of about 9.3 as measured by isoelectric focusing, a method for producing the same,
A detergent composition containing the alkaline cellulase and a microorganism producing the alkaline cellulase. [Effect] It has a high isoelectric point, is resistant to surfactants and chelating agents, and is useful as an enzyme for detergents such as clothing.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an alkaline cellulase useful as a detergent enzyme, a method for producing the same, and a microorganism producing the alkaline cellulase.

[0002]

BACKGROUND OF THE INVENTION Clothing,
Cellulose used in paper, building materials, and the like can be converted into fuel substances and higher value-added metabolites by decomposition. Therefore, cellulase, an enzyme that degrades the cellulose, has been studied in a wide variety of research institutions around the world. Numerous cellulases from species and anaerobic bacteria have been found.

On the other hand, digging in 1982 (Japanese Patent Publication No. 50-2)
No. 8515, Horikoshi & Akiba, Alkalophilic Mi
croorganisms, Springer, Berlin, 1982) found alkaline cellulase derived from an alkalophilic Bacillus bacterium, suggesting the application of cellulase, which had been considered difficult, to heavy detergents for clothing. It has been established as a detergent enzyme along with protease, lipase and amylase when incorporated into detergents and the like (JP-B-60-23158, JP-B-6-3).
0578, US Pat. No. 4,945,053).

In general, it has been empirically recognized that proteases, lipases, amylases, and the like, which exhibit excellent effects as detergent enzymes, all have high isoelectric points. Higher enzymes are also considered to have higher detergency. Therefore, cellulase as a detergent enzyme is also required to have a high isoelectric point, but alkaline cellulase having a high isoelectric point has only been found to be derived from some molds (Fusarium oxysporum). (WO95 / 24471), its isoelectric point is 9, and its detergency is not always sufficient.

It is an object of the present invention to provide a novel alkaline cellulase having a high isoelectric point and excellent detergency.

[0006]

The present inventors have screened various cellulase-producing bacteria to obtain alkaline cellulase having a high isoelectric point from the natural world. The present inventors have found a novel alkaline cellulase useful as an enzyme and completed the present invention.

That is, the present invention provides an alkaline cellulase having an isoelectric point of about 9.3 as measured by isoelectric focusing, a method for producing the same, and a detergent composition containing the alkaline cellulase. It is.

[0008] The present invention also provides a Bacillus sp.
M-N257 strain and FERM P-17473
The microorganisms deposited as the above are provided.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION The alkaline cellulase of the present invention comprises:
It is characterized by its isoelectric point of about 9.3, but it has resistance to oxidizing agents, does not easily affect its activity, etc. due to the easy formation of disulfide bonds in enzyme proteins. In view of the above, it is preferable that cysteine is not contained as a constituent amino acid of the enzyme protein. Further, when the reaction is carried out with a glycine-sodium hydroxide buffer solution (pH 8.5), the optimum reaction temperature is about 55 ° C. Is preferred.

Further, the alkaline cellulase of the present invention preferably further has the following enzymatic properties. 1. Action: Good decomposition of carboxymethyl cellulose in liquefied form. 2. Substrate specificity: Decomposes carboxymethylcellulose, lichenan, crystalline cellulose and cellooligosaccharides higher than cellotriose to produce reducing sugars. 3. Optimal reaction pH: works at least pH 5-10,
The optimum pH is around 8.5. 4. Optimum reaction temperature: When the reaction is performed with a glycine-sodium hydroxide buffer (pH 8.5), the optimum reaction temperature is about 55 ° C. 5. Stable pH range: When treated at 30 ° C for 60 minutes,
It is stable in the pH range of 5-11. 6. Heat resistance: 1 in Tris-HCl buffer (pH 7.0)
It is stable up to 55 ° C in the treatment for 5 minutes. 7. Molecular weight: The estimated molecular weight by SDS polyacrylamide gel electrophoresis is about 43 kDa.

[0011] The alkaline cellulase of the present invention is produced by culturing the producing bacterium and collecting from the culture. Examples of such an alkaline cellulase-producing bacterium include a bacterium belonging to the genus Bacillus, for example, a Bacillus sp. KSM-N257 strain having the following mycological properties.

A. Morphological properties (a) Cell shape and size: Bacillus (0.6-0.8 ×
(B) polymorphism: no (c) motility: yes (d) spore shape, size, position, swelling: oval,
0.8 to 1.0 × 1.0 to 1.6 μm, subordinate position, swelling. (E) Gram staining: undefined, but does not grow on crystal violet (CVT) agar medium. (F) Acid resistance: negative

B. Physiological properties (a) Reduction of nitrate (medium 1): positive (b) Denitrification reaction (medium 1): negative (c) MR test (medium 2): positive (pH 5-5.5) (d) VP test (Medium 2): negative (e) Indole formation (medium 3): negative (f) Hydrogen sulfide formation (medium 4): negative (g) Starch hydrolysis (medium 5): positive (h) Casein hydrolysis Degradation (medium 6): negative (i) Liquefaction of gelatin (medium 7): negative (j) Utilization of citric acid (medium 8): negative (k) catalase: positive (l) oxidase (medium 9): positive (m ) Urease (medium 10): negative (n) Temperature range of growth (medium 11): 13-14 ° C. (o) pH range of growth (medium 12): pH 6-10 (p) Effect of oxygen on growth (medium 13) ): Growing under anaerobic conditions. (Q) Sodium chloride resistance (medium 14): grows in the presence of 10% sodium chloride. (R) Gas production from glucose (medium 15): negative (s) Acid production from sugar (medium 16): glucose, arabinose, xylose, mannitol, galactose,
Acid production is observed from sucrose, mannose, maltose, lactose, trehalose, fructose, melibiose, ribose, salicin, glycerol, rhamnose and the like, and no acid production is observed from sorbitol and inositol.

Here, each of the culture media 1 to 16 has the following composition. Medium 1: Nutrient broth (Difco) 0.8% by weight, potassium nitrate 0.1% by weight Medium 2: Bacto peptone (Difco) 0.7% by weight, sodium chloride 0.5% by weight, glucose (separately sterilized)
5% by weight Medium 3: SIM medium, filter paper for indole production test (Nissui Pharmaceutical) Medium 4: TSI agar medium (Eiken Chemical) Medium 5: 1.5% by weight of bactopeptone (Difco), 0.5% of yeast extract %, Soluble starch 2.0% by weight,
Potassium monohydrogen phosphate 0.1% by weight, magnesium sulfate heptahydrate 0.02% by weight, agar 1.5% by weight Medium 6: Yeast extract 0.5% by weight, glucose 2.0% by weight, casein 0.5 % By weight, potassium monohydrogen phosphate 0.1% by weight, magnesium sulfate heptahydrate 0.02% by weight, agar 1.5% by weight Medium 7: Nutrient broth (Difco) 0.8% by weight, gelatin 1.2 Medium 8: Simmons medium (Eiken Chemical) Medium 9: Cytochrome oxidase test filter paper (Nissui Pharmaceutical) Medium 10: Urea medium (Eiken Chemical) Medium 11: Nutrient broth Medium 12 : Sodium carbonate in nutrient broth
Add hydrochloric acid after sterilization to adjust pH. Medium 13: Anaerobic agar (Difco) Medium 14: Add sodium chloride to nutrient agar (Difco). Medium 15: adjusted to 1.0% by weight of bactopeptone, 0.5% by weight of sodium chloride, 1.0% by weight of glucose, 0.002% by weight of phenol red, and pH 7.2 to 7.4. Medium 16: ammonium monohydrogen phosphate 0.1% by weight, potassium chloride 0.02% by weight, magnesium sulfate heptahydrate 0.02% by weight, yeast extract 0.02% by weight, agar 1.
5% by weight, bromocresol purple 0.0006% by weight, saccharide (sterilized by filtration) 1.0% by weight

As described above, the morphology and physiological properties of the KSM-N257 strain are described in Bergey's Manual of Systematic Ba.
cteriology "(Williams & Wilkins, 1984), the strain was considered to be a strain closely related to Bacillus circulans. However, its properties do not match known Bacillus circulans,
Since this strain does not match the properties of other Bacillus bacteria, this strain was sent to the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology as a new Bacillus bacterium. Bacillus sp. KSM-N257 strain (FER
MP-17473).

To produce the alkaline cellulase of the present invention using an alkaline cellulase-producing bacterium such as KSM-N257 strain, the bacterial strain is inoculated into a medium containing an assimilable carbon source, nitrogen source and other essential components. Shaking culture or aeration and stirring culture may be performed according to the method. There is no particular limitation on the carbon source and nitrogen source used, and carbon sources that can be assimilated, such as cellooligosaccharide, carboxymethylcellulose, hydroxyethylcellulose, lactose, galactose, arabinose,
Sucrose, maltose and the like can be mentioned. Examples of the nitrogen source include meat extract, fish meat extract, yeast extract, peptone, corn steep liquor and the like. In addition, phosphates, metal salts, and organic and inorganic trace nutrients can be appropriately added. The pH of the medium may be adjusted to a pH suitable for the production of the enzyme of the present invention using sodium carbonate or the like.

The collection and purification of alkaline cellulase from the obtained culture can be performed according to a general method. That is, the cells are removed by centrifugation or filtration from the culture solution, and the target enzyme can be concentrated from the obtained culture supernatant by a conventional method, for example, a salting-out method, a solvent precipitation method, or an ultraconcentration. 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. As a desalting method, dialysis, gel filtration using Sephadex G-10, ultrafiltration, or the like is used. The enzyme solution or the dried powder thus obtained can be used as it is, but can be further crystallized or granulated by a known method.

The alkaline cellulase of the present invention thus obtained has the following properties (1) to (11).
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 8.
5), 0.4 mL of 2.5% (w / v) carboxymethylcellulose (AO1MC; Nippon Paper), 0.4 mL of deionized water, 0.1 mL of appropriately diluted enzyme solution (diluted 10
mM Tris-HCl buffer, pH 7.5).
After reacting for 1 minute, 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 boiling water for 5 minutes. The mixture was quenched in ice water, 4 mL of deionized water was added, and the absorbance at 535 nm was measured to determine the amount of reducing sugar produced. In addition, after adding the dinitrosalicylic acid reagent to the reaction solution treated without adding the enzyme solution,
An enzyme solution was added thereto, and a color-developed product was prepared in the same manner. The enzyme unit (1 U) is 1 per minute under the above reaction conditions.
The amount was used to generate reducing sugar equivalent to μmol of glucose.

<Enzymatic properties> (1) Molecular weight (SDS electrophoresis) For the enzyme solution subjected to SDS treatment (100 ° C, 3 minutes),
SDS electrophoresis was performed using a 2.5% acrylamide gel. Phosphorylase b (97.
4 kDa), serum albumin (67 kDa), ovalbumin (45 kDa), carbonic anhydrolase (31 kDa), trypsin inhibitor (21.5 kDa)
Using Da) 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 determined. It was estimated to be about 43 kDa.

(2) Isoelectric point 2% (v / v) ampholine (Pharmalite pH 8)
-10.5; Pharmacia) added 5% (w / v)
The enzyme was subjected to isoelectric focusing using acrylamide gel (type 111; Vinerad). 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 migration point, and was determined to be about 9.3. Was done.

(3) Optimal reaction pH acetate buffer (pH 4-6), citrate buffer (pH 5.
5-7.5), MOPS buffer (pH 6-8), phosphate buffer (pH 6-8), Tris-HCl buffer (pH 7-
9), glycine-sodium hydroxide buffer (pH 8-1)
1), borate buffer (pH 9-11), carbonate buffer (p
H9-11), phosphate-sodium hydroxide buffer (pH
As a result of examining the optimal reaction pH using each buffer (50 mM) described in 11-12.5), this enzyme was found to be glycine-pH 8.5.
It showed the highest reaction rate in sodium hydroxide buffer. Further, the activity was 70% or more of the maximum activity between pH 7 and 9, and the activity was 10% or more of the maximum activity even in a wide range of pH 5 to 10 (FIG. 1).

(4) Optimal reaction temperature 50 mM glycine-sodium hydroxide buffer (pH 8.
In 5), an enzyme reaction was performed at each temperature of 30 ° C to 70 ° C, and the optimum reaction temperature was examined. As a result, the enzyme showed an optimum reaction temperature around 55 ° C. (FIG. 2).

(5) Stable pH range Acetate buffer (pH 4-6), citrate buffer (pH 4-6)
7), MOPS buffer (pH 6-8), phosphate buffer (pH 6-8), Tris-HCl buffer (pH 7-9),
Glycine-sodium hydroxide buffer (pH 8-11),
Each buffer (20 mM) of carbonate buffer (pH 9-11) and phosphate-sodium hydroxide buffer (pH 11-12.5)
The enzyme was added thereto, and the temperature was kept at 30 ° C. for 60 minutes, and then the residual activity was measured. As a result, the activity of this enzyme was 100
%, It was extremely stable in the pH range of 5-11 (FIG. 3).

(6) Heat resistance The enzyme was added to a 50 mM Tris-hydrochloric acid buffer (pH 7.0), 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). Similar results were obtained when 1 mM calcium chloride was added.

(7) Substrate Specificity When the decomposition rate for carboxymethylcellulose was 100% under the standard activity measurement conditions, the decomposition rate for lichenan was as high as 287%, but for xylan, curdlan and laminarin, Did not decompose at all. It was decomposed at a reaction rate of 0.12% with respect to sigma cell 101 (Sigma), which is an insoluble cellulose, and with a reaction rate of 0.013% with respect to Avicel SF (Funakoshi). However, the glycoside (p) which is a paranitrophenyl group (pNP) derivative
Glycine-sodium hydroxide buffer (pH 8.5) for cellotetraoside (pNPG4) from NPG1)
Did not decompose at all.

(8) Decomposition mode of substrate 50 mM glycine-sodium hydroxide buffer (pH 8.
5), 1.0% carboxymethylcellulose (A1OM
C: Nippon Paper Industries Co., Ltd.) was placed in an Ostwald viscometer, and the temperature was kept constant 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 amount of reducing sugar produced in the reaction solution at each time was examined by the dinitrosalicylic acid method to calculate the amount of cleavage. As a result, FIG.
As shown in the figure, this enzyme was found to degrade carboxymethylcellulose in an endo format. Next, degradation was performed at 30 ° C. for 2 hours in a 50 mM glycine-sodium hydroxide buffer (pH 8.5) using cellooligosaccharide (G1-G6) as a substrate. Thereafter, a fixed amount was spotted on a thin-layer chromatography plate (silica gel 60, no. 5567; Merck), butanol / ethanol / deionized water = 5 /
The development was performed in a 3/1 (v / v) solvent system. An anisaldehyde-sulfuric acid solution was used to detect sugar spots. As a result, this enzyme converts cellopentaoside (G5) into G2 and G
Decomposed into 3. In addition, cellohexaoxide (G6)
Or G2 and G4. However, it did not act on oligosaccharides below cellotetraoside (G4).

(9) 0.05% or 0.0% of surfactant
In a reaction system added to 1% (w / v),
The activity of the enzyme was measured. As a result, it was found that in the presence of each surfactant, the present enzyme can express 70% or more of the activity as compared with the control (Table 1).

[0029]

[Table 1]

(10) Influence of a chelating agent The enzyme was incubated at 50 ° C. in a 50 mM Tris-HCl buffer (pH 7.5) containing 5 mM EDTA, EGTA and orthophenanthrin at 30 ° C. for 30 minutes and then diluted 10-fold. Then, the remaining activity was examined by a standard activity measuring method. As a result, this enzyme was extremely stable to any chelating agent.

(11) Influence of Metal Salt An enzyme reaction was carried out by adding 0.5 mM of each metal salt to standard activity conditions. As a result, this enzyme was 17%
It was found to be activated to some degree. Other metal ions such as manganese, magnesium, zinc, nickel, cadmium, aluminum, copper, iron, strontium, barium, lead, barium, beryllium, and baradium had little effect.

In addition, the alkaline cellulase of the present invention was hydrolyzed with hydrochloric acid, and the amino acid composition was examined using an amino acid analyzer (L-8500, manufactured by Hitachi, Ltd.). As a result, no cysteine was contained in the constituent amino acids.

As described above, the cellulase of the present invention is a novel enzyme having an isoelectric point of about 9.3 and an optimum reaction temperature of 55 ° C., and is resistant to surfactants and chelating agents. It is useful as an enzyme for detergents, biomass, and fiber treatment.

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

The thus obtained detergent composition containing the alkaline cellulase of the present invention has excellent detergency as shown in Example 4 below.

When the alkaline 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 to prevent deactivation or decomposition by heat. It is preferred to mix later.

The detergent composition of the present invention may use various enzymes in addition to the alkaline cellulase of the present invention. For example, hydrolase, oxidase, reductase, transferase, lyase, isomerase, ligase,
Synthetase and the like. Among them, protease, cellulase other than the present invention, keratinase, esterase, cutinase, amylase, lipase, pullulanase, pectinase, mannanase, glucosidase, glucanase, cholesterol oxidase, peroxidase,
Laccase and the like are preferred, and protease, cellulase, amylase and lipase are particularly preferred. Examples of the protease include commercially available alcalase, esperase, savinase, evalase (above, Novozyme), properase, Plafect (Genencor), and KAP (Kao). Examples of the cellulase include cellzyme, carezyme (Novozyme), KAC (Kao), alkaline cellulase (Kao) produced by Bacillus sp. Strain KSM-S237 described in JP-A-10-313859, and the like. Examples of amylase include Termamyl, Duramil (above, Novozyme), Plaster (Genencor), and KAM (Kao). These enzymes are 0.001-10%, preferably 0.03-5%
Be blended.

The cleaning composition of the present invention can contain known cleaning components. Examples of the known cleaning 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%. As the divalent metal ion scavenger used in the cleaning composition of the present invention, tripolyphosphate, pyrophosphate, condensed phosphates such as orthophosphate,
Aluminosilicates such as zeolites, synthetic layered crystalline silicates, nitrilotriacetate, ethylenediaminetetraacetate,
Citrate, isocitrate, polyacetal carboxylate and the like can be mentioned. Of these, crystalline aluminosilicates (synthetic zeolites) are particularly preferred, and A-type, X-type and P-type zeolites are particularly preferred. The synthetic zeolite has an average primary particle size of 0.1 to 10 μm, particularly 0.1 to 5 μm.
μm is preferably 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, collectively referred to as dense ash and light ash, and J
Amorphous alkali metal silicates such as IS No. 1, No. 2, and No. 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 a copolymer of acrylic acid, methacrylic acid, itaconic acid, or the like, and the copolymer is preferably a copolymer of the above monomer and maleic acid, and has a molecular weight of thousands to 100,000. preferable. In addition to the carboxylic acid-based polymers, polymers such as polyglycidylates, cellulose derivatives such as carboxymethylcellulose, and aminocarboxylic acids-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 agent A bleaching agent such as hydrogen peroxide or percarbonate is preferably blended in an amount of 1 to 10%. When a bleach is used, tetraacetyl ethylenediamine (TAED) or JP-A-6-3
A bleaching activator (activator) described in JP-A-16700 or the like can be incorporated 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 (for example, Tinopearl CBS-X) and a stilbene type fluorescent agent (for example, 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), and a fragrance known in the field of detergents for clothing. , 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 present 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. As the water-soluble inorganic substances, carbonates, hydrogencarbonates, sulfates, sulfites, hydrogensulfates, hydrochlorides, or alkali metal salts such as phosphates, water-soluble inorganic salts such as ammonium salts or amine salts, Low molecular weight water-soluble organic acid salts such as citrate and fumarate are exemplified. 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]

Next, the present invention will be described in detail with reference to examples.

Example 1 Screening of Alkaline Cellulase Producing Bacteria Suspensions of soils from various parts of Japan in sterilized water were treated at 80.degree.
And then applied to an agar plate medium having the following composition [2.0% carboxymethylcellulose (A10M
C), 1.0% meat extract (Oxoid), 1.0% bactopeptone (Difco), sodium chloride 1.0
%, Potassium dihydrogen phosphate 0.1%, sodium carbonate 0.5% (separate sterilization)]. After static cultivation in a 30 ° C. incubator for 3 days, the cells were grown, and then 0.1% (w / v) Congo Red solution was poured and stained. Thereafter, the cells were decolorized with a 1 M sodium chloride solution, and those in which lysis spots associated with the decomposition of carboxymethyl cellulose were detected around the grown bacteria were selected, and single colonization was repeated. Polypeptone S (Nihon Pharmaceutical) 2.0%, fish meat extract (Wako Pure Chemical Industries) 1.0%, potassium monohydrogen phosphate 0.15
%, Yeast extract (Difco) 0.1%, magnesium sulfate heptahydrate 0.07%, carboxymethylcellulose 0.1%, sodium carbonate 0.5% (separate sterilization) at 30 ° C. Shaking culture was performed for 3 days. Ammonium sulfate was added to the culture supernatant to 90% saturation, and the precipitate was dissolved in a small amount of 10 mM Tris-HCl buffer (pH 7.5) containing 2 mM calcium chloride, and dialyzed against the buffer overnight. . Take a part of the dialysis solution and use Phase-sy
stem (Pharmacia; IEF gel, pH 3.0-
9.0) to perform isoelectric focusing. After the electrophoresed gel was immersed in 10 mM phosphate buffer (pH 7.0), the agar plate containing carboxymethylcellulose [1.0
% Carboxymethylcellulose, 50 mM glycine-sodium hydroxide buffer (pH 9.0), 1.5% agar]. After leaving at 30 ° C for 3 hours, remove the gel,
The 0.1% Congo Red solution was poured. 1 after about 10 minutes
M sodium chloride solution, and those having a high isoelectric point where discoloration spots associated with the decomposition of carboxymethylcellulose were detected were selected. Bacillus sp. KS
M-N257 strain was obtained.

Example 2 Bacillus sp. KSM-
Production of Alkaline Cellulase by N257 Strain The Bacillus sp. KSM-N257 strain obtained by the above screening was cultured aerobically at 30 ° C. for 3 days by adding 50 mL of medium to a 500 mL Sakaguchi flask. The medium composition was Polyppetone 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 0.07%, carboxymethylcellulose 0.1%, sodium carbonate 0.5% (separate sterilization). As a result, a productivity of about 330 U / L was obtained.

Example 3 Purification of Alkaline Cellulase A culture of Bacillus sp. KSM-N257 was centrifuged (8000 × g, 15 minutes, 4 ° C.) to obtain a supernatant (4.5 L). 90% ammonium sulfate
Add to saturation and centrifuge (10,000 ×
g, 15 minutes, 4 ° C.). Small one
After dissolving in 0 mM Tris-HCl buffer (pH 7.2),
The buffer was dialyzed overnight. Before using the dialysis solution
DEAE Toyopearl 650M column (2.5 × 2) equilibrated with 0 mM Tris-HCl buffer (pH 7.2)
0 cm; Tosoh). When about 300 mL of the equilibration buffer was used to wash and elute the non-adsorbed protein, cellulase was present in the non-adsorbed fraction, and this fraction was collected (63%).
0 mL) and ammonium sulfate. The precipitate was collected by centrifugation (10,000 × g, 15 minutes, 4 ° C.), dissolved in a small amount of 10 mM phosphate buffer (pH 6.0), and dialyzed against the same buffer overnight. Then, the column was attached to a CM Toyopearl 650M column (2.5 × 20 cm; Tosoh) equilibrated with 10 mM phosphate buffer (pH 6.0), and washed and eluted with about 300 mL of the same buffer. Further, using a buffer solution (300 mL each) containing 0 to 0.15 M sodium chloride, the adsorbed protein was eluted by a concentration gradient elution method. As a result, about 0.03M
The alkaline cellulase activity was eluted near the sodium chloride concentration of This fraction was collected and concentrated by ultrafiltration (YM3 membrane; Amicon) (33 mL). This was again applied to a CM Toyopearl 650 M column (2.5 × 20 cm) equilibrated in the same manner as described above, and adsorbed by a concentration gradient elution method using a buffer solution (200 mL each) containing 0 to 0.15 M sodium chloride. The protein was eluted.
Alkaline cellulase activity was eluted near the sodium chloride concentration of about 0.03 M. This fraction was collected and subjected to ultrafiltration (YM
(3 membranes). The resulting enzyme solution was previously equilibrated with a 10 mM Tris-hydrochloric acid buffer (pH 8.5). A DEAE Toyopearl 650S column (1.
5 × 13 cm) and washed and eluted with about 80 mL of the equilibration buffer. The cellulase fraction is eluted in the non-adsorbed part, and this fraction is collected and subjected to ultrafiltration (YM10 membrane).
(1.1 mL, 165 U, 9.3 mg)
protein). By the above purification operation, this enzyme is about 100
Purified 2-fold, the activity yield was 18%. The purified alkaline cellulase preparation obtained here exhibited the above-mentioned enzymatic properties.

Example 4 Detergency Test (1) Preparation of Detergent A detergency 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, the nonionic surfactant 15
1 part by weight of polyethylene glycol and 1 part by weight of polyethylene glycol were heated and mixed at 70 ° C. to prepare a mixed solution. Ladyge mixer (Matsuzaka Giken Co., Ltd., capacity 20
L, with a jacket), 100 parts by weight of the base granules described above, and a spindle (150 rpm) and a chopper (4000 rpm).
pm). In addition, warm water of 75 ° C. was flowed through the jacket at a rate of 10 L / min, and the above-mentioned mixed solution was charged therein for 3 minutes, and thereafter, stirring was performed 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 Granules of Alkaline Cellulase The purified enzyme obtained in Example 3 was
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 alkaline cellulase of the present invention is a novel enzyme having an isoelectric point of about 9.3 and an optimum reaction temperature of about 55 ° C., and is resistant to surfactants and chelating agents. It is useful as an enzyme for detergents. Further, according to the production method, the alkaline cellulase can be produced in large quantities and at low cost.

[Brief description of the drawings]

FIG. 1. Effect of p on alkaline cellulase activity of the present invention.
It is a figure showing the influence of H.

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

FIG. 3 is a graph showing the effect of pH on the stability of alkaline cellulase of the present invention.

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

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

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) (C12N 1/20 (C12N 1/20 C12R 1:07) C12R 1:07) (72) Inventor Yoshihiro Hakamada 2606 Kao Corporation, Research Laboratories, Kaigacho, Haga-gun, Tochigi Prefecture (72) Inventor Toru Kobayashi 2606 Akabane, Kaigamachi, Haga-gun, Tochigi Pref. 2606 Kao Corporation Research Laboratory (72) Inventor Yasuo Wada 1334 Minato, Wakayama-shi, Wakayama Prefecture Kao Research Laboratory F-term (reference) 4B050 CC01 CC07 DD02 FF04E FF05E FF11E KK01 KK02 KK03 KK05 KK20 LL04 4B065 AA15X AC14 AC14 CA31 CA57 4H003 AB19 AC08 DA01 EA16 EA28 EB22 EB30 EB36 EC03 FA07 FA28 FA47

Claims (9)

[Claims] 1. An alkaline cellulase having an isoelectric point of about 9.3 as measured by isoelectric focusing. 2. The alkaline cellulase according to claim 1, which does not contain cysteine as a constituent amino acid. 3. A glycine-sodium hydroxide buffer (p
H8.5), the optimum reaction temperature was about 5
3. The alkaline cellulase according to claim 1, which is at 5 ° C. 4. The alkaline cellulase according to claim 1, which has the following enzymatic properties. 1. Action: Good decomposition of carboxymethyl cellulose in liquefied form. 2. Substrate specificity: Decomposes carboxymethylcellulose, lichenan, crystalline cellulose and cellooligosaccharides higher than cellotriose to produce reducing sugars. 3. Optimal reaction pH: works at least pH 5-10,
The optimum pH is around 8.5. 4. Optimum reaction temperature: When the reaction is performed with a glycine-sodium hydroxide buffer (pH 8.5), the optimum reaction temperature is about 55 ° C. 5. Stable pH range: When treated at 30 ° C for 60 minutes,
It is stable in the pH range of 5-11. 6. Heat resistance: 1 in Tris-HCl buffer (pH 7.0)
It is stable up to 55 ° C in the treatment for 5 minutes. 7. Molecular weight: The estimated molecular weight by SDS polyacrylamide gel electrophoresis is about 43 kDa. 5. A method for producing an alkaline cellulase, comprising culturing the bacterium producing the alkaline cellulase according to claim 1 and collecting the alkaline cellulase from the culture. 6. The method for producing alkaline cellulase according to claim 5, wherein the alkaline cellulase-producing bacterium is a bacterium belonging to the genus Bacillus. 7. The bacterium belonging to the genus Bacillus is Bacillus sp.
The method for producing an alkaline cellulase according to claim 6, which is KSM-N257 strain (FERM P-17473). 8. Bacillus sp. KSM-N257
A microorganism named the strain and deposited as FERM P-17473. 9. A detergent composition comprising the alkaline cellulase according to claim 1.