JPH0762153B2 - Enzyme-containing detergent composition - Google Patents

Description

Detailed Description of the Invention

TECHNICAL FIELD The present invention relates to a detergent composition containing an enzyme having excellent detergency. 2. Description of the Related Art Conventionally, it is well known that an enzyme such as amylase, protease, and cellulase is blended in order to obtain a detergent composition having improved cleaning power against dirt on clothes (Japanese Patent Publication No. 47-45802). 48-30646, JP-A-47-373.
No. 3, No. 52-128904, No. 53-56204, No. 56
-129298). Among them, especially Bacillus
Alkaline protease produced from s) is generally used, and "Alcalase" (Alcalase), "Esperase" (Esperase) from Novo Industry Co.,
Commercially available under the trade names of "Savinase", "Maxatase" from Gist Brokers, and "Bioprase" from Nagase Seikagaku Corporation. However, the detergent composition containing these enzymes,
Although the effect of improving the detergency by the enzyme is to some extent, the effect is still insufficient, and the development of an enzyme-containing detergent composition that exerts a further effect of improving the detergency has been struggled. OBJECT OF THE INVENTION The present invention provides an enzyme-containing detergent composition having an excellent effect of improving detergency. Composition of the Invention The enzyme-containing detergent composition of the present invention comprises an alkaline protease produced from Bacillus sp. Y strain (Mikoken Kenjoyori No. 1029) and other kinds of alkaline proteases. And an enzyme. Hereinafter, the present invention will be described in more detail. The alkaline protease used in the present invention is a novel enzyme, and one strain belonging to the genus Bacillus, Bacillus sp. Y, found as a result of searching for alkaline protease-producing bacteria widely in nature.
It was produced from a strain. Bacillus SP Y stock is
BP-1029) has been deposited with the Institute of Microbial Technology, Institute of Industrial Technology. The Bacillus sp. Y strain is described in the application specification filed as Japanese Patent Application No. 60-123021. The details of the science will be described below. In addition, the bergey's manual
of Determinative Bacteriology 8th Edition (1974), RE
It carried out according to the search table of Gorden (1972). A medium of pH 10 was prepared by adding 1% of sodium carbonate. Temperature and
The optimum growth range for pH was measured with a temperature gradient biophoto recorder. A. Morphological properties The following morphological characteristics are observed when culturing on broth agar at 35 ° C for 2 days. 1) Cell shape and size: Bacillus, 0.4-0.5 μm × 1.7-1.9 μm. 2) Polymorphism: None. 3) Motility: Periflagellated and motile. 4) Spores: Spores are formed, and the cells swell near the tip during formation. The spores that have formed heat are of the lemon type, and the size is 0.7-0.9 μm × 1.0-1.2 μm. 5) Gram stainability: positive. 6) Anti-acid: Negative. B. Culture properties 1) Meat broth agar plate culture: Grows at pH 7.0 to form round, flat, and full-edge colonies. The surface of the colony was smooth and glossy, the peripheral part was light brown, and the center part was translucent light brown. It grows at pH 10.0 and forms round, flat, and full-edge colonies. The surface of the colony is smooth, shiny and cream colored. 2) Meat broth agar slant culture: It grows in a wide band at pH 7.0 and pH 10.0 and forms shiny cream or light brown colonies. A slight reddish brown pigment is formed. 3) Liquid culture of broth: Grows at pH 7.0 but does not form pellicle. It grows well at pH 10.0 and does not form pellicle. 4) Meat broth gelatin stab culture: slightly liquefies at pH 7.0. It liquefies at pH 10.0. 5) Growth is very poor with litmus milk pH 7.0. It grows at pH 10.0. No coagulation of milk is seen. Because the medium is alkaline,
Discoloration of litmus is unknown. C. Physiological properties 1) Reduction of nitrate: Positive. 2) Denitrification reaction: negative. 3) MR test: negative. 4) VR test: negative. 5) Indole formation: negative. 6) Generation of hydrogen sulfide: negative. 7) Hydrolysis of starch: positive. 8) Utilization of citric acid: Not used in Koser's medium. Chri
Slightly used in stensen medium. 9) Use of inorganic nitrogen source: Nitrate is not used. No ammonium salt is used. 10) Pigment formation: A water-soluble reddish brown pigment is produced together with the bacterial cell composition. 11) Urease: Positive. 12) Oxidase: Positive. 13) Catalase: positive. 14) Growth temperature range: around 33 to 35 ℃ (20 to 47
Good). 15) Growth pH range: around 10.0 (60 to 12.0) is good. 16) Attitude toward oxygen: Grows under aerobic and anaerobic conditions. 17) OF test: negative. 18) Acid and gas production from sugars: D-glycose, D-
It produces acid from mannose, D-fructose, maltose, sucrose, trehalose, D-mannite and starch, but does not produce gas. No acid or gas is produced from L-arabinose, D-xylose, D-galactose, lactose, inosit or glycerin. D. Other properties 1) Tolerance to sodium chloride: Grow in 10% NaCl. To summarize the above properties, first, this strain is a bacterium belonging to the genus Bacillus because it is a calatase-positive, facultatively aerobic, gram-positive bacillus having thermospores. Since this strain seems to belong to Bacillus alcalophilus, RIKEN and Horikoshi et al.
No.221 (ATCC 21522), No.58 (Japanese Patent Publication No. 48-2792, 50-1)
6435), and No. D-6 (see Japanese Examined Patent Publication No. 56-4236)
And mycological properties were compared. This strain and Bacillus alcalophilus No. 21, No. 58, and No. D-6 are identical in that they grow well in an alkaline medium (pH 10), but with respect to the use of inorganic nitrogen source, this strain is , Nitrates and ammonium salts cannot be used, whereas the above-mentioned known strains can be used. In addition, in the range of growth pH, this strain had a pH of 6
To pH12, No.221 is pH7 to pH11, No.58, No.D-6 is pH7.5 to pH11, the above-mentioned known strain, pH7.0 or less They differ in that they cannot grow. Even in the range of growth temperature, this strain is 20 ℃ to 47 ℃
The optimum temperature range is from 33 ℃ to 35 ℃, while No.221 can grow up to 55 ℃ and No.58 up to 45 ℃, and the optimum temperature is from 37 ℃ to 40 ℃. -6 also has a high optimum temperature of 35 ° C to 40 ° C, which is different from this strain. In addition, when this strain and acid production from sugars are compared with No. D-6, this strain shows L-
No acid is produced from arabinose, D-xylose, D-galactose and glycerin, whereas No. D-6 is produced. Furthermore, this strain grows even under 10% sodium chloride, and is distinguished from the above-mentioned known strains. As described above, this strain is different from the known strain of Bacillus alcalophilus, but from the above-mentioned mycological properties, Bacillus
It is appropriate to judge that it is an alicophilus green bacterium. Alkaline protease obtained by culturing Bacillus sp. Y strain is a combination of Ya enzyme and Yb enzyme.
There are two types, Japanese Patent Application No. 60-123022 and Japanese Patent Application No. 6123, respectively.
No. 0-286944. The Bacillus sp. Y strain can be cultured, for example, as follows. First, a liquid medium containing 2% soluble starch and 0.02% magnesium sulfate, and 1 dry yeast
%, And a liquid medium containing 0.1% potassium hydrogen phosphate were sterilized separately at 121 ° C. for 20 minutes, and 20 ml of each was 500
Dispense into a 1 ml Sakaguchi flask, and add sterile sodium carbonate to the flask to a final concentration of 1%, and add 50 ml.
Prepare a culture solution of. A Bacillus sp. Y strain is inoculated into the culture solution, and the culture solution is cultured at 30 ° C. for 15 hours to prepare a seed culture solution. 100 ml of the seed culture is added to an enzyme tank containing 3.5 liters of a medium having the same composition, and the mixture is cultivated by aeration and stirring for 70 hours while supplying 3.5 liters of air at 30 ° C. to the microbial fluid. The method for separating and producing the Ya enzyme and the Yb enzyme is as shown in Fig. 1. First, the microorganism culture solution was centrifuged at 10,000 rpm for 5 minutes to obtain a supernatant. The supernatant was then subjected to 70% saturation ammonium sulfate salting out. Further, the obtained precipitate was dissolved in 20 mM Tris-hydrochloric acid buffer solution (Ca ion 2 mM added, pH 7.2) and dialyzed against the same buffer solution to obtain Y crude enzyme (hereinafter, simply referred to as Y enzyme). It was Subsequently, the Y enzyme solution was added to diethylaminoethyl (DEA
E) -53 Cellulose was subjected to anion exchange chromatography and eluted with 10 mM borate buffer (pH 9.3) to obtain a non-adsorbed fraction. Subsequently, the non-adsorbed fraction was again subjected to ammonium sulfate salting out at 70% saturation. The obtained precipitate was again dissolved in 20 mM Tris-hydrochloric acid buffer solution (Ca ion 2 mM added, pH 7.2),
It was dialyzed against the same buffer. Furthermore, the solution was subjected to gel perchromatography on Tropar HW-55 to give 20 mM
Elution was performed with a Tris-hydrochloric acid buffer solution (Ca ion added 2 mM, pH 7.2), and active fractions were collected. Further, the fraction was subjected to salting out with 70% saturated ammonium sulfate, and the obtained precipitate was dialyzed against 20 mM Tris-hydrochloric acid buffer solution (Ca ion 2 mM added, pH 7.2). Finally, in order to remove the denatured protein, the active fraction after dialysis was passed through Millipore fistar to obtain purified Ya enzyme (hereinafter, simply referred to as Ya enzyme). On the other hand, the obtained Y enzyme solution was added to diethylaminoethyl (DE
AE) -53 Cellulose by anion exchange chromatography 20mM Tris-hydrochloric acid buffer (Ca ion 2mM addition, pH
After eluting the Ya enzyme as a non-adsorbed fraction in 7.2),
The same buffer containing 0.5 M sodium chloride was used for elution with a linear concentration gradient to obtain a crude fraction of Yb enzyme. The elution curve of the chromatography is shown in FIG. Then, the Yb crude fraction was again subjected to salting out with ammonium sulfate at 70% saturation. The obtained precipitate was dissolved in 50 mM phosphate buffer (pH 7.0) and dialyzed against the same buffer. Further, the solution was subjected to hemoglobin-agarose and affinity column chromatography and eluted with 50 mM phosphate buffer (pH 7.0) to collect active fractions. Further, the fraction was subjected to salting out with ammonium sulfate at 70% saturation, and the resulting precipitate was dissolved in 20 mM Tris-salt solution buffer (addition of Ca ion 2 mM, pH 7.2) and dialyzed against the same buffer. Furthermore, the solution was subjected to gel perchromatography of Tropar HW-55 and eluted with 20 mM Tris-hydrochloric acid buffer solution (Ca ion added 2 mM, pH 7.2) to collect active fractions. Further, the fraction was subjected to 70% saturation ammonium sulfate salting out, the resulting precipitate was dissolved in 20 mM Tris-hydrochloric acid buffer solution (Ca ion 2 mM added, pH 7.2), dialyzed against the same buffer solution, and purified. Y
b enzyme (hereinafter, simply referred to as Yb enzyme) was obtained. Elution curves of gel perchromatography using purified Ya enzyme and Yb enzyme as samples are shown in FIG. 12 and FIG. 13, respectively. Toyopearl HW-55 was used as the resin, and 20 mM Tris-HCl buffer (pH 7.2, Ca ion 2 mM) was used as the eluent.
Was used), and the expansion was performed by the ascending method. Also,
Figure 14 shows the elution curve of high performance liquid chromatography using purified Ya enzyme as a sample. The model is Waters WI
Using SP-710B, two I-125 columns were connected in series and eluted with 50 mM phosphate buffer. As is clear from the above, the Ya and Yb enzymes were completely purified. That is, Y enzyme, which is an alkaline protease obtained by culturing Bacillus sp. Y strain, is a mixture of Ya enzyme and Yb enzyme, and the ratio thereof is Ya enzyme / Yb enzyme = 90 / 10 to 50/50. Next, the Ya enzyme, Yb enzyme, and Y enzyme obtained according to the above-mentioned method will be described. [1] Substrate specificity The action of Ya and Yb enzymes is protein hydrolysis. The substrate specificity of the enzyme is shown in Table 1. In addition, the substrate specificity of the mixture with the Ya enzyme and the Yb enzyme simultaneously produced from the Bacillus sp. Y strain was also evaluated and compared. A enzyme [Bacillus Liche
The relative decomposition rate when the activity of an alkaline protease (trade name: Alcalase, Novo Co.) isolated from Niformis) was set to 100 was measured under the following conditions. Conditions: Temperature 35 ℃ pH 10.5 (50mM borate buffer) Reaction time 60 minutes Substrate temperature 1%, but 0.4% for hemoglobin Enzyme usage 100APU / ml However, egg white is 500APU / ml Proteolysis rate or activity is measured by Anson- He followed Hagiwara's modified method. The absorbance of the reaction solution passed after the reaction was 275n.
It was measured at m. The enzyme activity for releasing 1 μg of tyrosine in 1 minute was defined as 1 alkaline protease unit (APU). From this table, the Ya enzyme has a strong specificity for keratin,
It can be seen that the Yb enzyme has a high specificity for egg white and is weak for the insoluble protein keratin. In addition, Y enzyme, which is a mixture of Ya enzyme and Yb enzyme, has a characteristic of acting strongly on a wide range of substrates than known A enzyme. [2] Optimum pH and stable pH range Figs. 3 and 4 show graphs of the optimum pH and stable pH range of Ya enzyme and Yb enzyme. The buffer solutions used are as follows. pH range Buffer 3.5- 5.5 Acetic acid 4.5- 7.0 Citric acid 6.0- 8.0 Phosphoric acid 7.5- 9.0 Tris-HCl 8.0- 9.0 Boric acid-HCl 9.0-10.5 Glycine-NaOH 9.5-11.0 Boric acid-NaOH 11.0-12.0 Phosphoric acid- NaOH 12.0-13.0 KCl-NaOH 20 mM containing 0.6% casein to determine the optimum pH.
Add each enzyme to each buffer at about 400 APU / ml, and
The reaction was performed at 5 ° C for 10 minutes and the activity was measured. The relative activity at each pH when the activity at the optimum pH was set to 100 was determined. Stable pH
When examining the area, each enzyme was added to each buffer of 20 mM.
The activity was measured after adding 400 APU / ml and incubating at 25 ° C. for 24 hours. The relative activity at each pH was determined by setting the activity before the incubator as 100. As can be seen from FIG. 3, the optimum pH of the Ya enzyme is 10.0 to 12.5 and the stable pH range is 6.5 to 13.0. As can be seen from FIG. 4, the optimum pH of Yb enzyme is 9.0 to 10.0,
The stable pH range is 6.5 to 12.0. [3] Optimum temperature and thermostability The optimal temperature and thermostability of Ya and Yb enzymes are shown in Figs. As a substrate for investigating the optimum temperature
Each enzyme was added to a buffer solution containing 0.6% casein at pH 10.5 and reacted at each temperature for 10 minutes. The relative activity at each temperature was determined by setting the activity at 35 ° C as 100. The heat resistance was examined as follows. 50 mM boric acid-NaOH buffer (pH 10.5 at 35 ° C)
About 400 APU / ml of enzyme was added to the mixture, heat-treated at each temperature for 10 minutes, ice-cooled, and the activity was measured. As can be seen from Fig. 5, the optimum temperature of the Ya enzyme is 70 ° C, and the activity is maintained up to the temperature of 55 ° C. As can be seen from FIG. 6, the optimum temperature of Yb enzyme is 65 or
It is in the range of 70 ° C and maintains 100% activity up to a temperature of 50 ° C. [4] Ultraviolet absorption spectrum The ultraviolet absorption spectra of Ya and Yb enzymes are shown in Figs. 7 and 8. The sample was treated with 50 mM Tris-HCl buffer (pH
When it was dissolved in 8.0) and the ultraviolet absorption spectrum was measured, the Ya enzyme showed maximum absorption at a wavelength of 276 nm, and the extinction coefficient E ^1% _{1 cm} at that wavelength was calculated to be 7.4. On the other hand, Yb enzyme showed the maximum absorption at the wavelength of 278 nm, and the extinction coefficient at that wavelength of ^-1% _{1 cm} was calculated to be 9.5. [5] Effect of metal ion The effect of metal ion on the activity of Ya enzyme and Yb enzyme was investigated. The results are shown in Tables 2 and 3. About 20 mM boric acid-NaOH buffer (pH 10.5) with Ya or Yb enzyme
400 APU / ml was added, various metal salts were further added at a concentration of 1 mM, and the residual activity was measured after the treatment under each predetermined condition. The numerical value is expressed as the relative activity with the activity at 0 minute being 100. From this Table 2, the activity of the Ya enzyme is inhibited by the addition of copper sulfate, silver nitrate, mercuric chloride and cadmium chloride,
It can be seen that the addition of calcium chloride increases the stability of the activity against heat. In addition, from Table 3, Yb enzyme is copper sulfate, silver nitrate, second chloride
It can be seen that the activity is blocked by the addition of mercury. Alkaline protease produced by bacteria belonging to the genus Bacillus, since increasing the thermal stability typically by Ca ^2+, Ca ²⁺
To see the effect of 50 mM boric acid-NaOH containing 5 mM Ca ²⁺
Add about 400 APU / ml of enzyme to the buffer (pH 10.5 at 35 ° C),
After heat treatment at each temperature for 10 minutes and ice-cooling, the activity was measured to determine the residual activity. For comparison, the evaluation was also performed under the condition that Ca ²⁺ was not added. The results are shown in Table 4. The numerical value is expressed as the relative activity with the activity at 0 minute being 100. It was found that the addition of Ca ions improves the stability against heat by about 5 ° C for the Ya enzyme and about 10 ° C for the Yb enzyme. [6] Effects of inhibitors The effects of various inhibitors on the Ya and Yb enzymes were investigated. The conditions and method are as follows. 50 mM Tris-
800 APU / ml of Ya or Yb enzyme in hydrochloric acid buffer (pH 7.2)
Was prepared. After adding each inhibitor and incubating at 35 ° C. for 30 minutes, the residual activity was measured. The value is shown as a relative activity when the value without addition of an inhibitor is 100. The results are shown in Table 5. As can be seen from this table, when the casein is used as a substrate, the Ya enzyme and the Yb enzyme have EDTA (ethylenediaminetetraacetic acid) and PCBM (p-chloromercury benzoic acid), antipain (Antipain), chymostatin (Chymostati).
The activity is not inhibited by n), but the activity is inhibited by DFP (diisopropylfluorophosphate) and PMSF (phenylmethanesulfinyl fluoride), and thus it is a protease having serine at the active center. [7] Molecular Weight The molecular weights of Ya enzyme and Yb enzyme were examined by gel permeation chromatography. Toyopearl HW-55 was used as the packing material, and 20 mM Tris-hydrochloric acid buffer solution (Ca ion 2 mM added, pH 7.
2) was used as the eluent. A standard curve was prepared using the following proteins (molecular weights in parentheses) as standard proteins. Protein albumin (43,000), thermolysin (37,500), subtilisin (27,600), chymotrypsinogen (25,700), myoglobin (17,200), and cytochrome C (11,700) were used. The calibration curve is shown in FIG. By this method, the molecular weight of the Ya enzyme was determined to be 21,000 and the molecular weight of the Yb enzyme was determined to be 40,000. [8] Isoelectric point The isoelectric points of Ya enzyme and Yb enzyme were examined by isoelectric focusing method. Pharmalite 3-10 was used as the column carrier. The isoelectric focusing patterns of Ya enzyme and Yb enzyme are shown in Fig. 10.
Shown in Figures and 11. By this method, the isoelectric point of Ya enzyme is
10.1, the isoelectric point of Yb enzyme was determined to be 5.1.

[9] Amino acid composition Amino acid composition of Ya enzyme and Yb enzyme [Amino acid analyzer JL
C-200A (JEOL) used] was investigated. In addition, tryptophan was measured by an alkali decomposition method, and cystine was measured by a performance acid oxidation method. Its composition is shown in Table 6 in comparison with that of known proteases. As a result, compared to other enzymes, Ya enzyme is tryptophan,
Yb enzymes such as serine and valine show remarkable differences in amino acid composition such as tryptophan, histidine, arginine, aspartic acid, glycine and alanine. [10] Elemental analysis values Table 7 shows the elemental analysis values for Ya and Yb enzymes. Finally, as a summary, various properties of the Ya enzyme and the Yb enzyme are shown in Table 8 below by comparing with the A enzyme and a known alkaline protease produced by an alkaliphilic bacterium of the genus Bacillus. Other similar known alkaline proteases (E-1, E-
2, API-21, No, 221, see the note in Table 8), the optimum pH is A enzyme, E-1, E-2 and API-21 is 1
0-11, No.221 is 11-12, Ya enzyme is 10-12.5 and the region is wide on the high pH side, and the optimum pH of Yb enzyme is 9-10.
Low compared to the enzyme and other known alkaline proteases. Next, the optimum temperature is around 70 ° C for Ya enzyme and Yb enzyme, whereas 60 ° C for A enzyme, No.221 and 45-50 ° C for API-21.
In E-1 and E-2, it is higher than that of Ya enzyme and Yb enzyme at 75 ° C., which is also different. In addition, Ya enzyme and Yb enzyme in the presence of 5mM Ca ²⁺ ion,
Similar to enzymes A, No. 221, and API-21, it has a heat resistance of about 5
Although improved by 10 ° C, E-1 and E-2 produced by Bacillus No. D-6 strain (see note in Table 8) are different in that thermal stability due to Ca ²⁺ ion is not observed. . Furthermore, since the molecular weight of Yb enzyme is 40,000, which is larger than that of known alkaline proteases, and the isoelectric point is as low as 5.1,
Obviously another kind. From the above, this enzyme is different from any of the conventionally known alkaline proteases. Therefore, it is appropriate to judge that this enzyme is a novel enzyme.
And Yb. The amount of the alkaline protease (Y enzyme) of the present invention is not particularly limited, but preferably 50 per 1 kg of the detergent composition.
~ 10000 APU, more preferably 1000-5000 APU. Examples of the enzyme other than the alkaline protease used in the present invention include amylase, lipase, protease, cellulase, and the like. Among them, alkaline protease produced from the genus Bacillus is preferable, and novo industry "Alcalase" of the company, "Esperase" (Esperase),
Savinase, Gist Brokers' Maxatase, Maxacar
l), and Nagase Seikagaku's "Pioplase" and the like. One or more of these enzymes are blended in the detergent composition. The blending amount is not particularly limited, but is preferably 0.05 to 10 wt%, more preferably 0.1 to 5 wt% in the detergent composition. If desired, the cleaning composition of the present invention may further contain optional components. As an optional component, a surfactant, a builder, which is generally used in a detergent composition,
Anti-recontamination agents, bleaching agents, enzymes, fluorescent whitening agents, hydrotropes, inorganic salts, fragrances and the like can be mentioned. As the surface active agent, an anionic surface active agent, a nonionic surface active agent, a cationic surface active agent, an amphoteric surface active agent or a zwitterionic surface active agent is used. As the anionic surfactant, usual sulfonate-based, sulfate-based, phosphate-based anionic surfactants and soaps are used. Examples of the sulfonate anionic surfactant include a C _8-22 linear or branched alkylbenzene sulfonate, a C _8-22 linear alkyl sulfonate, and a C _8-22 long-chain olefin sulfonate. And so on. Further, as the sulfate type anionic surfactant, a C _8-22 straight chain or branched chain alkyl or alkenyl sulfate ester salt, a C _8-22 polyoxyethylene (EO = 1 to 7 mol) straight chain or _Examples thereof include branched alkyl or alkenyl ether sulfuric acid ester salts, C _8-22 polyoxyethylene (EO = 1 to 7 mol) linear or branched alkyl phenyl ether sulfuric acid ester salts, and the like. Here, EO indicates the average number of moles of ethylene oxide added. As the phosphate-based anionic surfactant, monoalkyl (or alkenyl) of C _{8 to 22,} a dialkyl (or alkenyl) or Sesukirin salts, polyoxyethylene (EO = 1 to 7 of the C _{8 to 22}
Mol) monoalkyl (or alkenyl), dialkyl (or alkenyl) or sesquiphosphate. Soaps include _C8-24 saturated or unsaturated fatty acid salts. The cations as counterions of these anionic surfactants are, for example, alkali metal or alkaline earth metal ions such as sodium, potassium and magnesium, alkanolamines such as monoethanolamine, diethanolamine and triethanolamine, and ammonium. is there. As the nonionic surfactant, C _8-22 polyoxyethylene (EO = 1 to 25 mol) linear or branched alkyl or alkenyl ether, C _8-18 polyoxyethylene (EO = 1-25 Mol) alkyl or alkenyl phenyl ether, oxyalkylene adduct such as polyoxyethylene polyoxypropylene block copolymer, C _8-24 saturated or unsaturated fatty acid alkanolamide or its alkylene oxide adduct, C _12-14
And a tertiary amine oxide of Examples of the amphoteric surfactant include dimethyldialkyl ( _C8-18 ) alkylcarboxybetaine, dialkyl ( _C8-18 ) aminoalkylenecarboxylate, and 2-alkyl-1-carboxy-1-hydroxyethylimidazolium betaine. To be As the cationic surfactant, the following general formula (I),
Examples include those represented by (II) and (III). (In the formula, at least one of R ₁ , R ₂ , R ₃ , and R ₄ is a C _12-24 alkyl or alkenyl group, and the other is a C _1-4 alkyl group, a hydroxyalkyl group, or a benzyl group. , X represents halogen.) (In the formula, R ₅ and R ₆ are C _12-24 alkyl or alkenyl groups, R ₇ is C _1-4 alkyl group, hydroxyalkyl group or benzyl group, R ₆ is H or CH ₃ , and n is 1 ~
An integer of 5 and X represents halogen. ) (In the formula, R ₉ and R ₁₀ are C _12-24 alkyl or alkenyl groups, l and m are integers of 1-20, and X represents halogen.) These surfactants may be used alone. Good,
Two or more kinds may be combined, and the blending amount is suitably 10 to 50% by weight, preferably 20 to 40% by weight. Examples of the builder include phosphates such as orthophosphate, pyrophosphate, tripolyphosphate, metaphosphate and hexametaphosphate; amino such as nitrilotriacetic acid, ethylenediaminetetraacetic acid, hydroxyethylethylenediaminetriacetic acid and diethylenetriaminepentaacetic acid. Carboxylic acids or salts thereof; polyvalent carboxylic acids such as citric acid, diglycolic acid, malic acid, oxalic acid, tartaric acid, succinic acid or salts thereof; polyacrylic acid, polymethacrylic acid, acrylic acid copolymers, polymaleic acid Acid, polyfumaric acid, polyelectrolytes such as maleic anhydride copolymers, or salts thereof;
Non-electrolytic polymers such as polyvinyl alcohol, polyethylene glycol and carboxymethyl cellulose; crystalline or amorphous aluminosilicates represented by the general formula (IV), inorganic salts such as carbonates; triethanolamine, diethanolamine, monoethanolamine, etc. Alkanolamines of. x (M ₂ O or M′O) ・ Al ₂ O ₃・ y (SiO ₂ ) ・ w (H ₂ O)
(IV) (In the formula, M is an alkali metal, M'is an alkaline earth metal that can be exchanged with calcium, x, y, and w are the number of moles of each component, and generally x is 0.7-1.5y is 1
3 and w are arbitrary numbers. ) These builders are used singly or in combination of two or more, and are preferably mixed in the detergent composition in an amount of 1 to 50% by weight, more preferably 5 to 30% by weight. Anti-recontamination agents include carboxymethyl cellulose, polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, etc., and fluorescent whitening agents include 4,4'-bis (2-sulfostyryl) -biphenyl salt, 4,4'-bis (4-chloro-3-sulfostyryl) -biphenyl salt, 2- (stilphenyl) naphthothiazole derivative, 4,4'-bis (triazol-2-yl) stilbene derivative, bis (triazinylamino) stilbene disulfonic acid Examples of the hydrotrope include lower alcohols, polyhydric alcohols, lower aryl sulfonic acids or salts thereof, and each of these components may be used alone or in combination of two or more. EFFECTS OF THE INVENTION According to the present invention, by using the novel alkaline protease produced from Bacillus sp. Y strain in combination with other kinds of enzymes, excellent detergency improving effect is exhibited, and An extremely highly practical detergent composition having significantly improved detergency as compared with the known enzyme-containing detergent composition can be realized. Next, the present invention will be described in more detail by way of examples.
The evaluation in each example was performed according to the following test methods.・ Washing power US.Testing's Terg-O-Tometer was used as a washing device, and 10 pieces of protein-containing wet artificial dirt cloth, sebum cloth, and washing knitted cloth were added to the bath ratio 30 times, and 120r.pm
Wash at 25 ℃ for 10 minutes. After washing, 900 ml of a predetermined washing concentration is used, and rinsing is performed with 900 ml of water for 3 minutes. The water used was 3 ° DH. The detergency is calculated by the following formula. The detergency test method is based on Oil Chemistry 30,432 (1981) "Research on New Artificial Soils (1st Report)". Example 1 Polyoxyethylene alkyl ether sulfuric acid ester sodium salt (C13, linearity 50%, EO = 3) 20 wt%,
Polyoxyethylene alkyl ether (C12-13,
A linear composition of 80%, EO = 15) 15 wt% was used as a standard composition, and various liquid detergent compositions containing alkanolamine, paratoluenesulfonic acid, and enzyme were prepared as shown in Table 9 below. The cleaning power of each composition was evaluated, and the results are shown in Table 9. Example 2 Linear alkylbenzene sulfonate sodium 10 wt%, α
-Sodium olefin sulfonate 10wt%, sodium silicate (SiO / Na ₂ O molar ratio 2) 10wt%, sodium carbonate
Granular detergent composition containing 10 wt%, polyethylene glycol (# 6000) 1 wt%, tallow soap 1 wt%, zeolite (4A type) 15 wt%, water 5 wt% as standard, and containing enzyme as shown in Table 10. The composition was prepared and the detergency of each composition was evaluated. The results are shown in Table 10.

[Brief description of drawings]

FIG. 1 is a flow sheet showing the purification steps of Ya enzyme and Yb enzyme. FIG. 2 is a graph showing the elution curve of YAE DEAE-53 cellulose subjected to anion exchange chromatography. Fig. 3 shows the optimum pH and stable pH range of Ya enzyme, and Fig. 4 shows
3 is a graph showing the optimum pH and stable pH range of Yb enzyme. Fig. 5 shows the optimum temperature and thermostability of Ya enzyme, and Fig. 6 shows Yb.
It is a graph which shows the optimal temperature and heat resistance of an enzyme. FIG. 7 is a graph showing the ultraviolet absorption spectrum curve of the Ya enzyme, and FIG. 8 is a graph showing the ultraviolet absorption spectrum curve of the Yb enzyme. FIG. 9 is a graph showing a calibration curve for determining the molecular weights of the Ya enzyme and the Yb enzyme. FIG. 10 is a graph showing the isoelectric focusing pattern of the Ya enzyme, and FIG. 11 is a graph showing the isoelectric focusing pattern of the Yb enzyme. FIG. 12 is a graph showing a gel perchromatography elution curve of Ya enzyme and FIG. 13 is a graph showing Yb enzyme, respectively. FIG. 14 is a graph showing a high performance liquid chromatography elution curve of Ya enzyme.

Claims (1)

[Claims] 1. An enzyme-containing detergent composition characterized by containing an alkaline protease produced from Bacillus sp. Y strain (Mikko Kenjoyori No. 1029) and an enzyme of another species. object.