JPH0525492A - Detergent composition - Google Patents

Abstract

PURPOSE:To obtain a detergent excellent in detergency against protein dirt, particularly water-insoluble protein dirt at collars and sleeves by incorporating a specific alkaline protease in the detergent. CONSTITUTION:One 1kg of a detergent composition (e.g. weakly alkaline powdery detergent for clothes) is blended with 0.1-6,000 P.U. of an alkaline protease produced by Bacillus sp. KSM-K16 or K14, which has an optimum pH of 10.0-12.5 (casein substrate; reacted at 40 deg.C for 10min), an optimum temperature of 50-60 deg.C (casein substrate; reacted at pH10.0 in the absence of Ca<2+> ions) and decomposition activity of 40KFU/APU or more for human keratinus fiber.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a detergent composition, and more particularly to a detergent composition having excellent detergency against protein stains.

[0002]

2. Description of the Prior Art Various proteases have been conventionally incorporated in detergents for clothes, detergents for automatic dishwashers and the like for the purpose of removing protein stains. As such a protease, an alkaline protease that acts in the alkaline region is used, and typical examples thereof include alcalase and sabinase (Novo Nordisk) API-2.
1 (manufactured by Showa Denko KK), Maxacal (manufactured by Gist Brocades), etc., but these alkaline proteases have sufficient activity against water-insoluble protein stains on the collar, sleeve and mouth, which are problems in home washing. There is a problem that the cleaning agent containing it does not have a sufficient effect of removing protein stains.

On the other hand, Ya enzyme (JP-A-61-280278) and the like have been reported as stable surfactants, but this is generally around room temperature because the active region is on the high temperature side. However, there is a problem that it is not suitable for cleaning with.

[0004]

Therefore, there is a demand for a detergent composition excellent in detergency of protein stains, which contains an alkaline protease having an activity in a wide temperature range and a high activity for water-insoluble proteins. Was there.

[0005]

[Means for Solving the Problems] Therefore, the inventors of the present invention have made extensive studies to solve the above problems and found that if a water-insoluble protein, particularly an alkaline protease having a high degrading activity on human keratin keratin fibers is added. It was found that a detergent having excellent detergency for protein stains, particularly water-insoluble protein stains such as collars and sleeves, can be obtained, and the present invention has been completed.

That is, the present invention provides a detergent composition comprising an alkaline protease having a degrading activity on human keratin keratin fibers of 40 KFU / APU or more.

In the present invention, human keratin keratin fiber degrading activity is human keratin keratin fiber degrading activity (KFU).
Is expressed as a ratio to the urea-modified hemoglobin degrading activity (APU), which is a water-soluble protein, and is specifically performed by the method described below.

In the present invention, the alkaline protease means that the enzyme is stable in the alkaline region and exhibits protease activity. In the present invention, the one having the following optimum pH and optimum temperature is particularly used. preferable. (1) Optimum pH (casein substrate, reaction at 40 ° C for 10 minutes) 10.0 to 12.5 (2) Optimum temperature (casein substrate, Ca ²⁺ -free pH 10.0
Reaction at 50-60 ° C

The alkaline protease used in the present invention is not particularly limited as long as it satisfies the above conditions, but specifically, alkaline protease K-16.
And alkaline protease K-14.

The alkaline proteases K-16 and K-14 used in the present invention are Bacillus (Bacillus) .
The microorganism belonging to the genus s ) can be cultivated and collected from the culture. Examples of the microorganism include those having the following mycological properties.

Alkaline protease K-16 producing bacterium (A) Morphological characteristics (a) Cell shape and size: bacillus 0.8-1.0 μm x
2.2-25 μm (b) Polymorphism: None. (C) Motility: Periflagellates are present and motility is present. (D) Spores [size, shape, position]: 1.0 to 1.2 μm
× 1.4 to 2.2 μm, oval, central quasi-end, slightly sporangial swelling. (E) Gram stain: Positive (f) Anti-acidity: Negative (g) Growth morphology on broth agar plate: Round, foliate, smooth surface, pale yellow, translucent colonies. (H) Growth on gravy agar slope: irregular foliate, slightly rough surface, pale yellow, translucent colonies. (I) Broth liquid culture: Good growth, turbidity and no pellicle. (J) Meat broth gelatin stab culture: Good growth and liquefaction. (K) Litmus milk: Peptone, but no solidification of milk and no change in litmus.

(B) Physiological properties (a) Reduction of nitrate: Positive (b) Denitrification reaction: Negative (c) MR test: Negative (d) VP test: Positive (e) Indole formation: Negative (f) Sulfide Hydrogen production: Negative (g) Starch hydrolysis: Positive (h) Utilization of citric acid: Positive (i) Utilization of inorganic nitrogen source: Utilizing nitrates, but not ammonium salts. (J) Pigment formation: Negative (k) Urease: Negative (l) Oxidase: Positive (m) Catalase: Positive (n) Growth temperature range: 55 ° C or lower (o) Growth pH range: pH 6.6 to 10 It can grow at 3. (P) Attitude toward oxygen: Aerobic (q) OF test: Oxidized type (O type) (r) Tolerance to sodium chloride: Grow in the presence of 10% sodium chloride. (S) Acid production and gas production from sugar

[0013]

[Table 1]

Regarding the above-mentioned mycological properties, "Burger's Manual of Systematic Bacteriology (Williams & Wilkins, 19
1984) "(Bergey's Manual of
Systematic Bacteriology)
As a result of the examination in accordance with the above description, it is appropriate that this strain belong to Bacillus subtilis . However, while Bacillus subtilis does not grow at all in pH 10 medium, this strain
It grows well even at pH 10. Also, while Bacillus subtilis cannot grow at 55 ° C, this strain is 55 ° C.
There are differences such as the fact that they can grow even in Japan.

As is clear from the above results, it is appropriate that this strain belong to Bacillus subtilis.
At the differ in some respects, and because also differs from other known strains, the present strain Bacillus sp (Ba
cillus sp. ) Named KSM-K16, and deposited at the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology as Micromachine Research Institute No. 11418.

Alkaline protease K-14 producing bacterium (A) Morphological characteristics (a) Cell shape and size: bacillus 0.8-1.0 μm x
2.5-25.5 μm (b) Polymorphism: None. (C) Motility: Periflagellates are present and motility is present. (D) Spores [size, shape, position]: 1.0 to 1.2 μm
× 1.5 to 2.2 μm, elliptical shape, central quasi-edge, swelling of sporangia a little. (E) Gram stain: Positive (f) Anti-acidity: Negative

(B) Physiological properties (a) Reduction of nitrate: Positive (b) Denitrification reaction: Negative (c) MR test: Negative (d) VP test: Positive (e) Indole formation: Negative (f) Sulfide Hydrogen production: Negative (g) Starch hydrolysis: Positive (h) Utilization of citric acid: Positive (i) Utilization of inorganic nitrogen source: Utilizing nitrates, but not ammonium salts. (J) Pigment formation: Negative (k) Urease: Negative (l) Oxidase: Positive (m) Catalase: Positive (n) Growth temperature range: 55 ° C or lower (o) Growth pH range: pH 6.6 to 10 It can grow at 3. (P) Attitude toward oxygen: Aerobic (q) OF test: Oxidized type (O type) (r) Tolerance to sodium chloride: Grow in the presence of 10% sodium chloride. (S) Acid production and gas production from sugar

[0018]

[Table 2]

Regarding the above-mentioned mycological properties, "Burger's Manual of Systematic Bacteriology (Williams & Wilkins, 19
1984) "(Bergey's Manual of
Systematic Bacteriology)
As a result of the examination in accordance with the above description, it is appropriate that this strain belong to Bacillus subtilis . However, while Bacillus subtilis does not grow at all in pH 10 medium, this strain
It grows well even at pH 10. Also, while Bacillus subtilis cannot grow at 55 ° C, this strain is 55 ° C.
There are differences such as the fact that they can grow even in Japan.

As is clear from the above results, it is appropriate that this strain belong to Bacillus subtilis,
At the differ in some respects, and because also differs from other known strains, the present strain Bacillus sp (Ba
cillus sp. ) Named KSM-K14, and deposited at the Institute of Microbial Science and Technology of the Institute of Industrial Technology as Microorganism Research Institute No. 12587.

In order to produce alkaline proteases K-16 and K-14 using these strains, the cells may be inoculated into a suitable medium and cultured according to a conventional method.

As the medium to be used, any medium can be used as long as it can be used for culturing ordinary microorganisms and can be used for this strain. It is preferable to include an appropriate amount of the source.

The carbon source and the nitrogen source are not particularly limited, but examples thereof include corn gluten meal, soybean flour, corn steep liquor, casamino acid, yeast extract, pharmamedia, sardines and meat. Examples include extract, peptone, hypro, adipower, corn meal, soybean meal, coffee meal, cottonseed oil meal, cultivator, amiflex and adipron, zest, azix and the like. Further, as the carbon source, assimilable carbon source, for example, arabinose, xylose, glucose, mannose, fructose, galactose, sucrose, maltose, lactose, sorbitol, mannitol, inositol, glycerin, soluble starch or inexpensive molasses, invert sugar Etc., and organic acids that can be assimilated, such as acetic acid. In addition, phosphoric acid, Mg ²⁺ , Ca ²⁺ , Mn ²⁺ , Z
Inorganic salts such as n ²⁺ , Co ²⁺ , Na ⁺ , and K ⁺ , and if necessary, inorganic and organic micronutrient sources can be appropriately added to the medium.

Collection and purification of the objective substances, alkaline proteases K-16 and K-14, from the thus obtained culture can be carried out in accordance with a general enzyme collection and purification means.

That is, the cells are separated by centrifuging or filtering the culture, and the cells and the culture filtrate are separated by a conventional means such as a salting out method or an isoelectric focusing method.
Proteins may be precipitated by a solvent precipitation method (methanol, ethanol, isopropyl alcohol, acetone, etc.), or ultrafiltration (for example, Diaflow membrane Y).
C, manufactured by Amicon) to obtain the desired alkaline protease. In the salting-out method, for example, ammonium sulfate (30
˜70% saturated fraction), for solvent precipitation, for example, after enzyme precipitation in 75% ethanol, filtration or centrifugation,
It is also possible to make it into a freeze-dried powder by desalting. Here, as the desalting method, a general method such as dialysis or a gel filtration method using Sephadex G-25 or the like is used.

The enzyme solution thus obtained may be used as it is, or may be further purified and crystallized by a known method before use. For further purification of the enzyme, for example, adsorption chromatography such as hydroxyapatite chromatography, DEAE-Sephadex, DEAE-cellulose, CM-cellulose or CM-.
Ion exchange chromatography such as biogel and molecular sieve gel chromatography such as Sephadex or biogel may be appropriately combined and fractionated and purified.

The alkaline proteases K-16 and K-14 of the present invention thus obtained have the following enzymatic properties. In the following, the enzyme activity was measured as follows.

Casein-degrading activity 50 mM boric acid-NaOH buffer (pH 1 containing 1% of casein)
0.0) 1 ml is mixed with 0.1 ml of enzyme solution, 40 ° C,
After reacting for 10 minutes, a reaction stop solution (0.123M trichloroacetic acid-0.246M sodium acetate-0.369
2 ml of M acetic acid) was added and the mixture was allowed to stand at 30 ° C. for 20 minutes. Then, it was filtered through a filter paper (Whatman, No. 2), and the protein degradation product in the filtrate was subjected to the Foreign Lowry method (Lowry, O.I.
H. et al. J. Biol. Chem. , Vol
193, p265 (1951)). In addition, 1P. U. Is 1 under the above reaction conditions.
The amount of enzyme that liberates 1 mmol of tyrosine per minute was used.

Human keratin fibril degrading activity (1) Degrading activity against urea-modified hemoglobin Anson-hemoglobin method (Anson, ML J. et al.
Gen. Physiol . , Vol22, p79 (19
38)) modified method. That is, the final concentration of urea-modified hemoglobin used as a substrate was 14.7 mg /
in a solution prepared to have a temperature of 25 ° C and pH of 1
After reacting at 0.5 for 10 minutes, trichloroacetic acid is added to the reaction solution to a final concentration of 31.25 mg / ml. The trichloroacetic acid-soluble component is colored with a phenol reagent. This color is 1 mmol of tyrosine and the color is 1
The activity per 10 minutes of reaction was determined from the calibration curve for APU, and the activity was measured per minute. That is, 1 APU means the amount of protease that gives a trichloroacetic acid-soluble content having the same coloration as 1 mmol of tyrosine is colored by the phenol reagent in 1 minute. (2) Degradation activity against human keratin fibers (2-1) Preparation of keratin fibers Human calcaneum (keratin) is scraped off with a scalpel, finely cut with scissors, and then washed with ion-exchanged water. 1M of keratin 8M
It is suspended in 20 to 50 ml of 50 mM Tris-hydrochloric acid buffer solution (pH 8.0) containing urea and 25 mM β-mercaptoethanol, and stirred overnight with a stirrer. The swollen keratin is ground with a Teflon homogenizer and then centrifuged (30,000 × g, 30 min). After filtering the supernatant using filter paper, the filtrate is dialyzed against ion-exchanged water, freeze-dried and powdered to be used as keratin fibers. (2-2) Measurement of Degradation Activity 1 mg / ml of prepared keratin fibers and 2 × 10 ⁻³ APU / protease of prepared keratin fibers in 0.1 M carbonate buffer (pH 10.5)
Add to make up to ml. Rotation speed 100rpm with magnetic stirrer at temperature of 30 ℃ (stirrer piece 35m
m) for a certain period of time, add phenylmethylsulfonylfluoride to the reaction mixture (final concentration 2 mM),
Filter through a 0.5 μm membrane filter. The solubilized protein in the filtrate is measured as Tyrosine by the Lowry method. (2-3) Calculation of Human Keratin Keratin Fiber Degrading Activity The amount of protease that produces a solubilized protein corresponding to 1 mg of tyrosine as a degradation product from human keratin keratin fibers in 1 minute is defined as 1KFU.

Enzymology of alkaline protease K-16
Property (1) Action It acts on various proteins under highly alkaline conditions. In particular, it has an activity of 48 KFU / APU against human keratin.

(2) Substrate specificity The specificity of alkaline protease K-16 for various substrates was compared with other commercially available proteases. The substrate used was casein, urea-modified hemoglobin, animal hair keratin,
Elastin was used to measure the degradation activity against them. Fifty
1% of each substrate in mM boric acid-NaOH buffer (pH 10.0)
(Urea-modified hemoglobin is 2.2%), and each enzyme solution 0.5 × 10 ⁻⁴ P. U. (Elastin is 3.5 × 10 ^-4
P. U. ) Was added and the reaction was carried out at 40 ° C. for 10 minutes.
Table 3 shows the activity of each enzyme when the activity of alkaline protease K-16 in each substrate is 100.

[0032]

[Table 3]

As is clear from this result, alkaline protease K-16 decomposes water-soluble and water-insoluble proteins well, and is compared with commercially available enzyme A and commercially available enzyme B which are often used as detergent enzymes at present. In particular, it exhibits an excellent action on elastin.

(3) Optimum pH Casein was added to various pH buffer solutions (50 mM) to a final concentration of 0.91%, and alkaline protease K-16 was added.
5.2 × 10 ⁻⁵ P. U. In addition, the reaction was carried out at 40 ° C. for 10 minutes to measure the activity. The activity at the optimum pH is set to 100, and each
The activity at pH was expressed relatively. The results are shown in Fig. 1. Figure 1
As is clear from the above, the optimum pH of alkaline protease K-16 is 11.0 to 12.3. The various buffers used and their pH ranges are as follows. Acetate buffer pH 3.9 to 5.7 Phosphate buffer pH 6.6 to 8.3 Carbonate buffer pH 9.2 to 10.9 Phosphate-NaOH buffer pH 10.9 to 12.7 KCl-NaOH buffer pH 10 .9 to 12.6

(4) pH stability 7.9 × in the same buffer (20 mM) as used in (3).
10 ⁻³ P. U. Alkaline protease K-16 was added and the mixture was allowed to stand at 25 ° C. for 48 hours. The treatment solution was diluted 40-fold with 50 mM boric acid-NaOH buffer (pH 10.0), and the activity was measured. Enzyme activity before treatment is 100% and each pH
Relative activity was determined. The results are shown in Figure 2. As is clear from FIG. 2, alkaline protease K-16 has a stable region of pH 6.0 to 12.0 in the absence of Ca ²⁺ ,
In the presence of 2 mM Ca ²⁺ , its stability range is pH 5.0-12.0.
Met.

(5) 50 mM boric acid containing 0.91% casein as an optimal temperature substrate
A NaOH buffer solution (pH 10.0) was added with 3.1 × 10 ⁻⁵ P. U. Alkaline protease K-16 was added and reacted at each temperature for 10 minutes. The relative activity at each temperature was determined by setting the activity at 40 ° C as 100%. The results are shown in Figure 3. As is clear from FIG. 3, the optimum temperature of alkaline protease K-16 was 55 ° C. in the absence of Ca ²⁺ , and was 5 mM Ca ^2+.
It was 70 ° C. in the presence.

(6) 1.6 × 10 in a heat-resistant 20 mM boric acid-NaOH buffer (pH 9.5)
^-3 P. U. Alkaline Protease K-16 was added, heat-treated at each temperature for 10 minutes, cooled with ice, then 50 mM boric acid-NaOH
It was diluted 5-fold with a buffer solution (pH 10.0). And 0.91
The activity was measured using% casein as a substrate. Relative activity at each treatment temperature was determined with the activity of the untreated as 100%.
The results are shown in Fig. 4. As is clear from FIG. 4, alkaline protease K-16 was 5 mM at 50 ° C. in the absence of Ca ^2+.
In the presence of Ca ^2+, under the above heat treatment conditions up to 60 ° C, 90
% Activity was maintained.

(7) Molecular Weight The molecular weight of alkaline protease K-16 was examined by sodium dodecyl sulfate (SDS) -polyacrylamide gel electrophoresis. For the molecular weight marker, a marker kit for low molecular weight (Bio-Rad), that is, phosphorylase b (molecular weight: 97,400), bovine serum albumin (molecular weight: 66,200), ovalbumin (molecular weight: 42,
700), carbonic anhydrase (molecular weight: 3
1,000), soybean trypsin inhibitor (molecular weight:
21,500), lysozyme (molecular weight: 14,400)
Was used. By this method, alkaline protease K-1
The molecular weight of 6 was determined to be 28,000 ± 1,000.

(8) Isoelectric point The isoelectric point of alkaline protease K-16 was examined by the isoelectric focusing method. Servelite 9-11 was used as the amphoteric carrier for the column. By this method, the isoelectric point of alkaline protease K-16 was found to be 10.5 or higher.

(9) Effect of metal ion Alkaline protease K-1 for various metal ions
The effect on 6 was investigated. First, 20 mM boric acid-
Various metal salts were added to a NaOH buffer solution (pH 9.5) at a concentration of 1 mM, and 3.9 × 10 ⁻³ P. U. Add the enzyme of 3
It was left at 0 ° C. for 20 minutes. After that, 50 mM boric acid-NaOH
The residual activity was measured by diluting 5 times with a buffer solution (pH 10.0). The residual activity was expressed as a relative value to the enzyme activity treated in the same manner without addition of a metal salt. The results are shown in Table 4. As is clear from this result, the alkaline protease K-
It can be seen that the activity of 16 is inhibited by Hg ²⁺ and Cu ²⁺ . Further, it is understood from the results of the above (5) and (6) that the thermal stability is improved by Ca ²⁺ .

[0041]

[Table 4]

(10) Effects of inhibitors Alkaline protease K is used for general enzyme inhibitors.
The effect on -16 was investigated. Various inhibitors were added to 10 mM phosphate buffer (pH 7.0) to a predetermined concentration, and alkaline protease K-16 7.9 x 1 was added thereto.
0 ^-3 P. U. Was added and the mixture was allowed to stand at 30 ° C. for 20 minutes. Then, the treatment liquid was diluted 20 times with ion-exchanged water, and the residual activity was measured. The residual activity was expressed as a relative value to the enzyme activity treated in the same manner without the addition of an inhibitor. The results are shown in Table 5. As is clear from these results, the activity of alkaline protease K-16 was inhibited by diisopropylfluorophosphate (DFP), phenylmethanesulfonyl fluoride (PMSF), and chymostatin, which are serine protease inhibitors. It can be seen that the protease has a serine residue in the center.

[0043]

[Table 5]

(11) Effect of surfactant 6.6 × 10 ^-2 P.I. U. Was added to 5 ml of 0.1 M Tris-hydrochloric acid buffer solution (pH 9.0, containing 10% ethanol) in which various kinds of surfactants of predetermined concentrations were dissolved, and the mixture was allowed to stand at 40 ° C. for 4 hours, and then 50 mM boric acid -NaOH buffer (pH 1
After 20-fold dilution with 0.0), the residual activity was measured. The activity at 0 minute treatment time was defined as 100%, and the residual activity was expressed as a relative value. The results are shown in Table 6. As is clear from these results, the alkaline protease K-16 exhibited high stability even when various surfactants were present at high concentrations (1-10%). From this, alkaline protease K-16
Is useful as a detergent component containing a surfactant.

[0045]

[Table 6]

Enzymology of alkaline protease K-14
Property (1) Action It acts on various proteins under highly alkaline conditions. In particular, it has an activity of 50 KFU / APU against human keratin.

(2) Optimum pH As a result of the same examination as in the case of the alkaline protease K-16, the optimum pH of the alkaline protease K-14 was 10.4-12.0, as is clear from FIG. there were. (3) pH stability As a result of the same examination as in the case of the alkaline protease K-16, as is clear from FIG. 6, the alkaline protease K-14 has a stable region of pH 7 in the absence of Ca ^2+.
.About.11.5, and its stable region was pH 5 to 12 in the presence of 2 mM Ca ²⁺ . (4) Optimum temperature As a result of studying in the same manner as in the case of the alkaline protease K-16, as is clear from FIG. 7, the optimal temperature of the alkaline protease K-14 is 55 ° C. in the absence of Ca ²⁺ . The temperature was 70 ° C. in the presence of 5 mM Ca ²⁺ .

(5) Heat resistance As a result of the same examination as in the case of the alkaline protease K-16, as is clear from FIG. 8, the alkaline protease K-14 was 5 ° C in the absence of Ca ²⁺ at 50 ° C and 5 mM Ca. ²⁺
In the presence, 90% or more of the activity was maintained up to 60 ° C. (6) Isoelectric point The isoelectric point of alkaline protease K-14 was measured by a rotophore isoelectric focusing method (manufactured by Bio-Rad).
As a result, the isoelectric point of alkaline protease K-14 was 1
It was 0.0 or more.

The amount of the above-mentioned alkaline protease compounded in the detergent composition of the present invention is not particularly limited as long as the alkaline protease is active, but the detergent composition 1
0.1 to 6,000 P./kg. U. , Especially 5 to 400
P. U. Is preferred.

Further, known detergent components can be blended in the detergent composition of the present invention. Examples of the known detergent components include the following.

(1) Surfactant: average carbon number 10 to 16
Linear alkylbenzenesulfonate having an alkyl group of 1 to 3, alkylethoxy having a linear or branched alkyl group having an average carbon number of 10 to 20 and adding an average of 0.5 to 8 mol of ethylene oxide in one molecule Sulfates, alkyl sulfates having an alkyl group with an average carbon number of 10 to 20, olefin sulfonates having an average of 10 to 20 carbon atoms in one molecule, alkanes having an average of 10 to 20 carbon atoms in one molecule Sulfonate, α-sulfofatty acid methyl or ethyl ester salt having an average of 10 to 20 carbon atoms in one molecule, higher fatty acid salt having an average of 8 to 20 carbons, straight or branched chain having an average of 10 to 20 carbons Anionic surface active agent such as an alkyl ether carboxylate having an average alkyl group of 0.5 to 8 moles in one molecule. Agents: Nonionic surfactants such as polyoxyethylene alkyl ethers having an alkyl group with an average carbon number of 10 to 20 and 1 to 20 moles of ethylene oxide added, higher fatty acid alkanolamides or alkylene oxide adducts thereof, etc. Betaine type amphoteric surfactants; sulfonic acid type amphoteric surfactants, phosphate ester type surfactants, amino acid type surfactants, cationic surfactants and the like. These surfactants are compounded in the detergent composition in an amount of 5 to 60% by weight (hereinafter simply referred to as%), particularly 10 to 45% for the powdery detergent composition and 20 to 20% for the liquid detergent composition. It is preferable to add 55%. When the detergent composition of the present invention is a bleaching agent, the surfactant is generally added in an amount of 1 to 10%, preferably 1 to 5%.

(2) Divalent metal ion scavenger: condensed polyphosphate such as tripolyphosphate, pyrophosphate, orthophosphate, aluminosilicate such as zeolite, synthetic layered crystalline silicate, nitrilotriacetate , Ethylenediamine tetraacetate, citrate, isocitrate, polyacetal carboxylate, etc. The divalent metal ion scavenger is 0 to
50%, preferably 5-40%. It is more preferable to use a divalent metal ion scavenger containing no phosphorus.

(3) Alkaline agent and inorganic salt: silicate,
Carbonates, sesquicarbonates, sulfates, alkanolamines, etc. These are blended from 0 to 80%.

(4) Anti-redeposition agent: polyethylene glycol, polyacrylic acid salt, polyacrylic acid copolymer,
Polyvinyl alcohol, polyvinyl pyrrolidone, carboxymethyl cellulose, etc. Anti-recontamination agent is 0-10
%, Preferably 1 to 5%.

(5) Enzymes: Enzymes such as cellulase, amylase, lipase, hemicellulase, β-glycosidase, glucose oxidase, cholesterol oxidase, and proteases other than the above alkaline proteases.
The above-mentioned alkaline protease has better compatibility with enzymes such as cellulase and lipase than conventionally known alkaline proteases, and if these enzymes are used in combination, the detergency can be further improved.

(6) Scavenger or reducing agent for available chlorine in tap water: As a scavenger for available chlorine, ammonium sulfate, urea, guanidine hydrochloride, guanidine carbonate, guanidine sulfamate, thiourea dioxide, monoethanolamine,
Examples include diethanolamine, triethanolamine, amino acids represented by glycine, sodium glutamate, and proteins such as bovine serum albumin and casein, as well as protein hydrolysis, meat extract, and fish meat extract. Examples of the reducing agent include alkali metal salts such as thiosulfates, sulfites, and nitrite salts, alkaline earth metal salts, and Rongalit C.

(7) Bleach: Percarbonate, perborate, sulfonated phthalocyanine zinc salt or aluminum salt, hydrogen peroxide, etc.

(8) Fluorescent dye: A fluorescent dye that is usually used in detergents.

(9) Solubilizing agent: In the case of a liquid detergent, the following solubilizing agents can be used. Lower alcohols such as ethanol, benzene sulfonates, lower alkyl benzene sulfonates such as p-toluene sulfonate, polyols such as propylene glycol.

(10) Others: In addition to the above, ingredients commonly used in detergents such as fragrances, anti-caking agents, enzyme activators, antioxidants, preservatives, pigments, bluing agents and bleach activators are required. Can be blended accordingly.

The detergent composition of the present invention can be produced according to a conventional method by combining the above-mentioned alkaline protease and the above-mentioned known detergent components. The form of the detergent can be selected according to the application, and can be liquid, powder, granules, or the like. Further, the cleaning composition of the present invention, a cleaning agent for clothes, a cleaning agent for an automatic dishwasher, a water pipe cleaning agent,
It can be used as a denture cleaning agent, a bleaching agent and the like.

[0062]

EFFECT OF THE INVENTION The detergent composition of the present invention has a high activity in the alkaline range, is resistant to alkali, and has a strong human keratin fibril-degrading activity.
For example, Bacillus S
p. ) KSM-K-16 (Ministry of Industrial Science, Microbiology No. 11418)
Live bacteria or, Bacillus sp. (Bacillus of
sp. ) KSM-K-14 (Microtechnology Research Institute, Microbiology Co., No. 12587
No.), the alkaline protease K-16 or the alkaline protease K-14 produced by live bacteria is contained, and it gives an excellent detergency to protein stains.

[0063]

EXAMPLES Next, the present invention will be specifically described with reference to Reference Examples and Examples, but the present invention is not limited to these Examples and the like. In the following,% means% by weight unless otherwise specified.

Reference Example 1 Isolation and collection of protease-producing strain: (1) About 1 g of a soil sample was sterilized in 10 ml of physiological saline.
And suspended at 80 ° C. for 20 minutes for heat treatment. Inoculate the keratin halo agar medium with 0.1 ml of the heat-treated supernatant,
Static culture was carried out at 30 ° C. for 48 hours. The composition of the keratin halo agar medium used is as shown below. Glucose 1% Yeast extract 0.2% Animal hair keratin 1% Carboxymethylcellulose 1% Potassium phosphate 0.1% Magnesium sulfate heptahydrate 0.02% Agar 1.5%

(2) Separately sterilized 10% sodium carbonate (1%) was added to the above medium composition to give a final pH of 10.5.
After the adjustment, the plate medium was prepared. After culturing, the halos around the grown colonies were selected and selected in the same medium.
Purification was performed 3 times to obtain a uniform protease-producing bacterium.

(3) These strains obtained in (2) above were inoculated into the following liquid medium and aerobically cultured at 30 ° C. for 48 hours with shaking. Glucose 2.0% Polypeptone S 1.0% Yeast extract 0.05% Potassium primary phosphate 0.1% Magnesium sulfate heptahydrate 0.02% Sodium carbonate (separate sterilization) 1.0% pH 10.5 After completion of the culture, the obtained culture was centrifuged (3000 rpm;
After 10 minutes), the bacteria were removed, and the obtained culture supernatant was used as an enzyme solution.

(4) A crude enzyme sample was prepared by freeze-drying from the enzyme solution obtained in (3) above, and evaluated for storage stability at 40 ° C. in a commercial liquid detergent. Among them, Bacillus sp. KSM-K16 strain and Bacillus sp. KSM-K14 strain were obtained as strains that produce the enzyme having the highest stability.

Reference Example 2 Cultivation of bacterial cells and purification of alkaline protease K-16: (1) Bacillus sp. KSM-K16, an alkalophilic bacterium obtained in Reference Example 1, was used in the following liquid medium (3.0
1) and aerobically cultivated at 30 ° C. for 48 hours with shaking to produce alkaline protease K-16. Glucose 2.0% Fish meat extract 1.0% Soybean powder 1.0% Magnesium sulfate 0.02% Potassium dihydrogen phosphate 0.1% pH 10.0

(2) After completion of the culture, 3 l of the obtained culture was centrifuged (10,000 rpm; 5 minutes) to remove the bacteria,
The supernatant was freeze-dried. After dissolving 2 g of the freeze-dried powder in 10 ml of ion-exchanged water (crude enzyme solution), the solution was dialyzed against a 10 mM Tris-hydrochloric acid buffer solution (2 mM Ca ²⁺ added, pH 7.5) overnight using a dialysis membrane. 26 ml of dialysis solution (activity 3.15 PU / ml, specific activity 1.97P.
U. / Mg protein) was obtained. Next, CM-5 equilibrated with 10 mM Tris-hydrochloric acid buffer (adding 2 mM Ca ²⁺ , pH 7.5)
2 A column packed with a cellulose carrier was washed, the inside of the column was washed with the same buffer, and then alkaline protease K-16 was eluted with the same buffer containing 0 to 0.15 M sodium chloride. When this active fraction was collected, the total amount was 15 ml and the activity was 1.12 P. U. / Ml, specific activity 5.75 P. U.
/ Mg protein. Then, this solution was dialyzed overnight against 50 mM Tris-hydrochloric acid buffer solution (10 mM Ca ²⁺ , 0.2 M sodium chloride added, pH 8.0), and then subjected to ultrafiltration (Amicon, cut molecular weight 5, 000) and concentrate 5
Sephadex G-50 (Pharmacia) equilibrated with 0 mM Tris-hydrochloric acid buffer (10 mM Ca ²⁺ , 0.2 M sodium chloride added, pH 8.0) was subjected to gel filtration chromatography and developed in the same buffer. It was The total amount of the active fractions obtained here was 11.5 ml and the activity was 0.9 P.M. U. /
ml, specific activity 6.03 P. U. / Mg protein. The solution was dialyzed against ion-exchanged water overnight, and then the activity was 0.56.
P. U. / Ml, specific activity 5.60 P. U. A solution of / mg protein was obtained. This solution was freeze-dried to obtain an enzyme powder. The specific activity of the obtained enzyme was 1.87 P. U. / Mg, and human keratin keratin fibril-degrading activity was 48 KFU / APU.

Reference Example 3 Cultivation of bacterial cells and purification of alkaline protease K-14: (1) Bacillus sp. KSM-K14, an alkalophilic bacterium obtained in Reference Example 1, was used in the following liquid medium (3.0
1) and aerobically cultivated at 30 ° C. for 48 hours with shaking to produce alkaline protease K-14. Glucose 2.0% Fish meat extract 1.0% Soybean powder 1.0% Magnesium sulfate 0.02% Potassium potassium phosphate 0.1% pH 10.0 (2) From the culture obtained after the completion of culture, Purified enzyme powder of alkaline protease K-14 was obtained by the same method as in Reference Example 2. The human keratin keratin fibril-degrading activity of the obtained enzyme was 50 KFU / APU.

The cleaning test in the following examples was carried out under the following experimental conditions. 1) Natural collar cloth-contaminated cloth: A cotton gold cloth (# 2023 cloth) is sewn onto the collar of a shirt, and is worn by an adult boy for 3 days.
After wearing it, leave it at 25 ° C and 65% RH for 1 month, then divide the degree of dirt into three levels. Of these, the one with the most dirt is the one with the dirt symmetrical to the center point. The cloth was cut into half and used for the experiment.

2) Washing conditions and method: When washing a naturally contaminated cloth, a 9 cm × 30 cm naturally contaminated cloth is cut in half at symmetrical positions, and one of a pair of 9 cm × 15 cm contaminated cloths is used as an enzyme-free detergent. It was washed with an additive detergent, and one was washed with the detergent of the present invention as a comparative detergent. First, 15 pieces of naturally contaminated cloth
Sew each piece onto a cotton cloth of 50 cm x 50 cm, and in the case of powder detergent, put 1 kg of this contaminated cloth and cotton underwear together in 6 l of 0.417% detergent solution, soak it for 2 hours at 30 ° C, and then Toshiba. It was transferred to a washing machine "Ginga", and the total amount was adjusted to 30 liters. Then, it was washed by strong inversion for 10 minutes, and dried for judgment. In the case of liquid detergent, apply 20cc of detergent liquid evenly to the contaminated cloth. 1
After 0 minutes, 1 kg including cotton underwear was transferred to a Toshiba washing machine "Ginga", the total amount was 30 liters, washed by strong inversion for 10 minutes, and dried and used for judgment. The half-cut cloth washed with the standard detergent and the half-cut cloth washed with the detergent of the present invention were evaluated by a paired comparison by visual inspection. The cleaning cloths were ranked on the basis of standard stains, which were ranked in 10 stages, which represent the degree of stains. The detergency was represented by the detergency of the detergent of the present invention when the detergency of the standard detergent was 100. A difference in detergency index of 0.5 or more can be regarded as a significant difference.

3) Enzymes used: (a) Alkaline protease K-16 (the one obtained in Reference Example 2 was diluted 200 times with Glauber's salt and granulated) (b) Alkaline protease K-14 (reference Granules obtained by diluting the one obtained in Example 3 with Glauber's salt 200 times) (c) Protease (Novo Nordisk, Sabinase 4.0T) (d) Protease (Gist Brocades, Maxacar P- 400,000) (e) Protease (Showa Denko KK, API-21H) (f) Cellulase (Kao, Microtech Lab. No. 1138, 5)
00IU / g / granulated product) (g) Cellulase (Novo Nordisk, Cellzyme 1.0T) (h) Lipase (Novo Nordisk, Lipolase 1)
00T)

Example 1 A detergent for weakly alkaline powdered clothing having the composition shown in Table 7 was prepared.

[0075]

[Table 7]

Table 8 shows the results of the washing test with the various detergents obtained. In the table, the detergent number is indicated by "Example number-symbol of enzyme used" and those not using enzyme are indicated by "Example number-⊚". (The same applies hereinafter) From the results shown in Table 8, it can be seen that the product of the present invention exhibits excellent detergency against natural collar stains as compared with conventional detergents containing an enzyme.

[0077]

[Table 8]

Example 2 A weak alkaline powder detergent was prepared by using various combinations of enzymes in the formulation of Example 1 as shown in Table 9. Table 9 shows the results of the cleaning test using the various detergents obtained. From the results in Table 9, it can be seen that, by using the alkaline protease K-16 in combination with cellulase and / or lipase, a synergistic effect of improving the detergency can be obtained with respect to natural collar stains.

[0079]

[Table 9]

Example 3 A detergent for weakly alkaline powdered clothing having the composition shown in Table 10 was prepared.

[0081]

[Table 10]

Table 11 shows the results of the washing test with the various detergents obtained. From the results in Table 11, it can be seen that the effects of the detergent of the present invention are effectively exhibited by using various builders.

[0083]

[Table 11]

Example 4 A detergent for weakly alkaline powdered clothing having the composition shown in Table 12 was prepared.

[0085]

[Table 12]

Table 13 shows the results of the washing test with the various detergents obtained. From the results shown in Table 13, it can be seen that the product of the present invention exhibits excellent detergency against natural collar stains as compared with conventional detergents containing an enzyme.

[0087]

[Table 13]

Example 5 A detergent for weakly alkaline powdered clothing having the composition shown in Table 14 was prepared.

[0089]

[Table 14]

Table 15 shows the results of the washing test with the various detergents obtained. From the results shown in Table 15, it can be seen that the product of the present invention exhibits excellent detergency against natural collar stains as compared with conventional detergents containing an enzyme.

[0091]

[Table 15]

Example 6 A detergent for weakly alkaline powdered clothing having the composition shown in Table 16 was prepared.

[0093]

[Table 16]

Table 17 shows the results of the washing test with the various detergents obtained. From the results in Table 17, it can be seen that the product of the present invention exhibits excellent detergency against natural collar stains as compared with conventional detergents containing an enzyme.

[0095]

[Table 17]

Example 7 A weak alkaline liquid detergent having the composition shown in Table 18 was prepared. The pH of the detergent stock solution was 9.6.

[0097]

[Table 18]

Table 19 shows the results of the enzyme stability test in the various liquid detergents obtained. 1) Enzyme used (i) Alkaline protease K-16 (one obtained in Reference Example 2 diluted with glycerol and water by 100 times and dissolved) (j) Alkaline protease K-14 (in Reference Example 3) The obtained product was diluted 100 times with glycerol and water and dissolved. (K) Protease (Novo Nordisk, Esperase 8.0L) (l) Protease (Novo Nordisk, Sabinase 8.0L) )

2) Stability test After mixing the liquid detergents of the respective compositions, the mixture was allowed to stand at 40 ° C. for 10 days and 20 days, and the activity of each enzyme was measured by the following measuring method. The residual rate was calculated.

[0100]

[Equation 1]

The liquid detergent of each composition was made into a 1% tap water solution (hardness 2.5 ° DH), and the enzyme activity was measured using an automatic analyzer Autoanalyzer (registered trademark, Technicon). The details of the measurement were according to the following documents. Reference: Analyst, 96 [2], p159-163.
(1971)

[0102]

[Table 19]

[0103]

[Brief description of drawings]

FIG. 1 Effects on the activity of alkaline protease K-16
It is a figure which shows the influence of pH.

FIG. 2 is a drawing showing the effect of pH on the stability of alkaline protease K-16.

FIG. 3 is a drawing showing the effect of temperature on the activity of alkaline protease K-16.

FIG. 4 is a drawing showing the effect of temperature on the stability of alkaline protease K-16.

FIG. 5: Effects on the activity of alkaline protease K-14
It is a figure which shows the influence of pH.

FIG. 6 is a drawing showing the effect of pH on the stability of alkaline protease K-14.

FIG. 7 is a drawing showing the effect of temperature on the activity of alkaline protease K-14.

FIG. 8 is a drawing showing the effect of temperature on the stability of alkaline protease K-14.

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Jun Hitomi             2321-61 C, Hoshakuji, Takanezawa-machi, Shioya-gun, Tochigi Prefecture             Its Wako 101

Claims (3)

[Claims] 1. A detergent composition comprising an alkaline protease having a degrading activity on human keratin keratin fibers of 40 KFU / APU or more. 2. The cleaning composition according to claim 1, wherein the alkaline protease has the following optimum pH and optimum temperature. (1) Optimum pH (casein substrate, reaction at 40 ° C for 10 minutes) 10.0 to 12.5 (2) Optimum temperature (casein substrate, Ca ²⁺ ion-free pH 1
Reaction at 0.0) 50-60 ° C 3. The alkaline protease is Bacillus sp. KSM-K16.
Alkaline protease K-16 or Bacillus sp. ( Bacil sp .
lus sp. ) KSM-K14 (Ministry of Microbiology Research Institute 125
87) which is an alkaline protease K-14 produced by the detergent composition according to claim 1 or 2.