JPH078993B2 - Cleaning composition - Google Patents

Description

TECHNICAL FIELD The present invention relates to a detergent composition containing alkaline pullulanase.

[Conventional technology]

Blending an enzyme with a detergent has been practiced for a long time. The enzyme in the cleaning agent acts as a cleaning auxiliary agent, for example, in a detergent for clothes, various stains and stains attached to clothes, and in a detergent for a tableware, oils and fats remaining on the tableware, proteins, It has the function of decomposing or degrading starch etc. to facilitate removal.

In particular, α-amylase has been conventionally used to remove starchy stains, and it is possible to improve the detergency against starchy stains by immersing the item to be washed in the α-amylase-containing cleaning solution for a long time. it can.

Further, the present inventors have found that a specific pullulanase effectively acts on starchy stains strongly adhered to dishes, fibers, etc.,
It was found that the detergency can be significantly improved (Japanese Patent Application No.
63-285424).

[Problems to be Solved by the Invention]

However, most of the pullulanase conventionally found in nature are classified as so-called neutral or acid pullulanase, which shows the maximum and stable enzyme activity in the neutral neutral acidic region, and therefore is a dishwashing detergent. Alternatively, the presence of pullulanase which is suitable for a detergent composition for clothes, that is, which exhibits maximum activity in an alkaline region or has alkali resistance, that is, an alkali pullulanase and an alkali resistant pullulanase is actually very small.

Incidentally, here, the alkaline pullulanase refers to those having an optimum pH in the alkaline region, and the alkali-resistant pullulanase has an optimum pH in the neutral to acidic region, but even in the alkaline region It has a sufficient activity as compared with the activity in the above and retains stability. Further, the neutral means a pH range of 6 to 8, and the alkaline means a pH range higher than that.

Conventionally, as an alkaline pullulanase, Japanese Patent Publication Sho 53-27786
Only those described in the publication are known. However, this is an enzyme that has an optimum pH in the alkaline region and has a broader substrate specificity than conventionally known pullulanase, but the optimum pH is 8-9 and weak alkaline. Since it is in the region, there is a problem that it is not suitable for use as a detergent component. It also has the disadvantage that the enzyme is unstable and the productivity of the enzyme is poor at the same time.
It was not suitable for industrial fermentative production.

[Means for Solving the Problems]

Under such circumstances, the present inventors have conducted earnest research on alkaline pullulanase suitable as a detergent component, having an optimum pH on the higher pH side, and being stable and having good productivity. We have found that alkaline pullulanase meets these requirements and completed the present invention.

That is, the present invention provides a detergent composition containing an alkaline pullulanase having the following enzymatic properties.

1) Action Decomposes α-1,6 glucoside bond of pullulan to produce maltotriose. It also hydrolyzes the α-1,6 glucosidic bond of starch, amylopectin, glycogen or their partial degradation products.

2) Substrate specificity Among sugars having a branched structure branched by an α-1,6 glucoside bond, a branched structure having a degree of polymerization of maltose or higher is hydrolyzed.

3) Working pH and optimum pH The working pH is in the range of pH 5 to 11, and the optimum pH is in the range of 9.5 to 11.

4) pH stability It is extremely stable in the range of pH 8 to 10, and has a relative activity of 50% or more even in the range of pH 7 to 10.5 (by treatment at 45 ° C for 10 minutes).

5) Working temperature range and optimum temperature It works in the range of 10 to 60 ° C, and the optimum working temperature is about 50 ° C.

6) Temperature stability It is extremely stable up to 40 ℃ (10mM glycine-pH9.5).
Treatment with salt-sodium hydroxide buffer for 30 minutes).

7) Molecular weight The molecular weight by sodium dodecyl sulfate electrophoresis is 120,00.
It is 0 ± 5,000.

8) Effect of metal ions It is inhibited by Hg ²⁺ , Cd ²⁺ , Mn ²⁺ and Pb ²⁺ .

9) Effects of surfactants Linear alkylbenzene sulfonate sodium, alkyl sulfate sodium salt, polyoxyethylene alkyl sulfate sodium salt, sodium α-olefin sulfonate, sodium α-sulfonated fatty acid ester, sodium alkyl sulfonate, sodium It is hardly inhibited by surfactants such as didodecylsulfate, soap and softanol.

10) Effect of chelating agent EDTA, EGTA, citric acid and zeolite are hardly affected by the activity.

11) Protease resistance It has strong resistance to alkaline protease.

The alkaline pullulanase used in the present invention is produced by, for example, Bacillus sp. KSM-AP1876 (FERM P-10887), which is a kind of alkalophilic microorganism.

This microorganism has the following mycological properties. In addition, the medium used for classification of the strains below is one of the following medium 1 to 21.
There are 21 types, each of which contains 0.5% by weight (hereinafter simply referred to as%) of separately sterilized sodium carbonate (Na ₂ CO ₃ ).

Composition of the used medium (% indicated): Medium 1. Nutrient broth, 0.8; Agar powder (Wako Pure Chemical Industries, Ltd.), 1.5 Medium 2. Nutrient broth, 0.8 Medium 3. Nutrient broth, 0.8; Gelatin, 20.0 ;
Agar powder (Wako Pure Chemical Industries, Ltd.), 1.5 medium 4. Bactrytomas milk, 10.5 medium 5. Nutrient broth, 0.8; KNO ₃ , 0.1 medium 6. Bactopeptone, 0.7; NaCl, 0.5; Glucose, 0.5 medium 7. SIM Agar medium (manufactured by Eiken Chemical), indicated amount medium 8. TSI agar medium (manufactured by Eiken Chemical), indicated amount medium 9. Yeast extract, 0.5; Bactopeptone, 1.5; K ₂ HPO ₄ ,
_{0.1; MgSO 4 · 7H 2 O} , 0.02; soluble starch, 2.0; agar powder (manufactured by Wako Pure Chemical), 1.5 Medium 10. causer medium (manufactured by Eiken Chemical), indicated amounts Medium 11. Christensen medium (manufactured by Eiken Chemical Co., ), Indicated amount medium 12. yeast extract, 0.05; Na ₂ SO ₄ , 0.1; KH ₂ PO ₄ , 0.1; glucose, 1.0 yeast extract, 0.05; Na ₂ SO ₄ , 0.1; KH ₂ PO ₄ , 0.1; glucose,
1.0; CaCl ₂・ 2H ₂ O, 0.05; MnSO ₄・_{4 to} 6H ₂ O, 0.01; FeSO ₄・ 7
_{H 2 O, 0.001; MgSO 4} · 7H 2 O, as the 0.02 nitrogen source, sodium nitrate, sodium nitrite, 0.25% ammonium chloride and ammonium phosphate, respectively, 0.2025%, 0.158
% And 0.195% were added to the above medium and used.

Medium 13. King A medium "Eiken" (manufactured by Eiken Chemical), indicated amount medium 14. King B medium "Eiken" (manufactured by Eiken Chemical), indicated amount medium 15. Urea medium "Eiken" (Eiken) Chemical), indicated amount medium 16. Cytochrome oxidase test paper (Nippon Pharmaceutical) medium 17. 3% hydrogen peroxide water medium 18. Bactopeptone, 0.5; Yeast extract, 0.5; K ₂ HPO ₄ ,
0.1; _{glucose, 1.0; MgSO 4 · 7H 2} O, 0.02 medium 19. Bacto peptone, 2.7; NaCl, 5.5; K 2 HPO 4, 0.3; glucose, 0.5; bromothymol blue, 0.06; agar powder (manufactured by Wako Pure Chemical Industries, Ltd. ), 1.5 medium _{20. (NH 4) 2 HPO 4} , 0.1; KCl, 0.02; MgSO 4 · 7H 2 O, 0.0
2; Yeast extract, 0.05; Sugar, 1.0 Medium 21. Casein, 0.5; Yeast extract, 0.5; Glucose, 1.0; K ₂
HPO ₄ , 0.1; MgSO ₄ / 7H ₂ O, 0.02; agar powder (manufactured by Wako Pure Chemical Industries, Ltd.), 1.5 [mycological properties] (a) Microscopic observation result The size of the bacterial cell is 1.0 to 2.2 μm × 2.2. It is a rod-shaped bacterium of ~ 4.4 μm and has an oval endospore (0.8 ~ 1.0 μm x
1.0-1.8 μm). It has periflagellates and is motile. Gram stain is indeterminate. There is no acid resistance.

(B) Growth state in various media Meat agar plate culture (medium 1) Growth condition is good. The shape of the settlement is circular, the surface is smooth, and the peripheral edge is smooth or wavy.

The color tone of the village is translucent, milky white and glossy.

Meat agar slope culture (medium 1) Grow. The state is spread, glossy and milky white translucent.

 Broth liquid culture (medium 2) Grows.

 Meat broth gelatin stab culture (medium 3) Good growth condition. Liquefaction of gelatin is observed.

Litmus milk medium (medium 4) No milk coagulation or peptone formation is observed. Discoloration of litmus cannot be determined because the medium is alkaline.

(C) Physiological properties Nitrate reduction and denitrification (Medium 5) Nitrate reduction is positive. Denitrification reaction is negative.

MR test (medium 6) It cannot be judged because the medium is alkaline.

 VP test (medium 6) negative.

 Formation of indole (medium 7) Negative.

 Production of hydrogen sulfide (medium 8) Negative.

 Starch hydrolysis (medium 9) positive.

Use of citric acid Negative in Coser medium (medium 10). It is not possible to determine whether it is positive or negative in Kristensen medium (medium 11).

 Use of inorganic nitrogen source (medium 12) Used with nitrates, ammonium salts, and nitrites.

 Pigment formation (medium 13,14) Negative.

 Urease (medium 15) negative.

 Oxidase (medium 16) Neither positive nor negative can be determined.

 Catalase (medium 17) positive.

Growth range (medium 18) Growth temperature range is 20-40 ℃, optimum growth temperature range is 30-35
It was ℃.

The growth pH range was pH 7 to 10.5, and the optimum growth pH was pH 10.

 Attitude toward oxygen Aerobic.

 OF test (medium 19) Discoloration cannot be determined because it is alkaline.

Only grows in aerobic conditions.

Utilization of sugar (medium 20) L-arabinose, D-xylose, D-glucose,
D-mannose, D-fructose, D-galactose, maltose, sucrose, lactose, trehalose, D-sorbit, D-mannitol, glycerin, starch, salicin, D-ribose and dextrin are used.

Growth in a salt-containing medium (medium 1 is modified) It grows at a salt concentration of 5%, but cannot grow at 7%.

 Casein degradation (medium 21) positive.

Based on the above studies on mycological properties,
Bergey's Mannual of Determinative Bacteriolg
y) 8th Edition and The Genus B.
acillus "Ruth, E.Gordon, Agriculture Handbook No.427,
Agricultural Research Service, USDepartment of Ag
riculture Washington DC, (1973)), and as a result of comparative search, this strain was found to be Bacillus (Ba) which is a spore bacillus.
cillus) is recognized as a kind of genus. However, since this strain cannot grow in the neutral region and shows good growth exclusively in the high pH region, recently, Horikoshi and Akiba (“Alkaloph
ilic Microorganism ", Japan Scientific Society Press
(Tokyo), published in 1982) and belongs to the so-called alkalophilic microorganisms, which is tentatively distinguished from conventional neutral-growing Bacillus bacteria.

Furthermore, since the mycological properties of this strain do not match with any of the known alkalophilic Bacillus, it was determined as a new strain and named Bacillus sp. KSM-AP1876. -10887) was deposited at the Institute of Microbial Technology, Ministry of International Trade and Industry.

In order to obtain the alkaline pullulanase used in the present invention using the above-mentioned microorganism, the microorganism may be ingested in the medium and cultured according to a conventional method. Further, it is preferable that the medium contains an appropriate amount of assimilable carbon source and nitrogen source. The carbon source and the nitrogen source are not particularly limited,
For example, as a nitrogen source, corn gluten meal, soy flour, corn steep liquor, casamino acid, yeast extract,
Pharmamedia, meat extract, tryptone, soyton,
High Pro, Aji Power, Soy Bean Meal, Cottonseed Oil Cake,
Examples thereof include organic nitrogen sources such as cultivators, adiprons and zests, and inorganic nitrogen sources such as ammonium sulfate, ammonium nitrate, ammonium phosphate, ammonium carbonate, sodium nitrate and ammonium acetate. Further, as the carbon source, soluble starch, insoluble starch, amylopectin, glycogen, in addition to branched oligosaccharides generated by partial decomposition of pullulan, carbon sources that can be assimilated, for example, glucose, maltose, arabinose, xylose, ribose, mannose, Examples thereof include fructose, galactose, maltose, sucrose, lactose, trehalose, mannitol, sorbitol, glycerin, and assimilable organic acids such as acetic acid. In addition, phosphate, magnesium salt, calcium salt, manganese salt, zinc salt, cobalt salt,
Inorganic salts such as sodium salts and potassium salts, and if necessary, inorganic and organic micronutrient sources can be appropriately added to the medium.

Collection and purification of the target substance, alkaline pullulanase, from the thus obtained culture can be carried out in accordance with a general enzyme collection and purification means. That is, a crude enzyme solution can be obtained by removing the bacterial cells from the culture solution by a usual solid-liquid separation means such as centrifugation or filtration. This crude enzyme solution can be used as it is, but if necessary, a crude enzyme is obtained by a separation means such as a salting-out method, a precipitation method, an ultrafiltration method, and further purified and crystallized by a known method. It is also possible to use it as a purified enzyme.

Hereinafter, an example of a method for purifying the alkaline pullulanase used in the present invention will be described in more detail.

Alkaline Bacillus bacterium KSM-AP1876 strain was prepared by using 1% pullulan, 0.2% tryptone, 0.1% yeast extract, 0.03% KH ₂ P.
O ₄ , 0.02% CaCl ₂ · 2H ₂ O, 0.1% (NH ₄ ) ₂ SO ₄ , 0.001% Fe
_{SO 4 · 7H 2 O, 0.0001} % MnCl 2 · 4H 2 O, 0.02% MgSO 4 · 7H 2 O
And 0.5% sodium carbonate in a culture medium at 30 ° C. for 3 days under aerobic shaking culture to remove bacterial cells from the resulting culture solution to obtain a supernatant. Next, DEAE-cellulose powder was added to the supernatant to completely remove pullulanase in the supernatant.
Adsorb on cellulose. Next, the resin is washed with 10 mM Tris-HCl buffer (pH 8), and then the enzyme is eluted with the same buffer containing 0.6 M sodium chloride. Furthermore, after dialysis and concentration against 10 mM Tris-hydrochloric acid buffer solution (pH 8), D equilibrated with the same buffer solution was used.
EAE-cellulose DE52 is adsorbed and the same buffer is used to
Elute with a ~ 1 M sodium chloride concentration gradient, collect the active fractions, concentrate using an ultrafiltration membrane with an average molecular weight cut-off of 10,000, and dialyze overnight against the same buffer containing 0.1 M sodium chloride.
This was concentrated, then dialyzed, and after adsorption on a Sephacryl S-200 column equilibrated with the same buffer containing 0.1 M sodium chloride, 0.1
Elute with the same buffer containing M sodium chloride, collect the active powder,
Further, it is adsorbed on the DEAE Toyopearl 650S column. The adsorbed enzyme is eluted with a 0.1-1 M sodium chloride concentration gradient in 10 mM Tris-hydrochloric acid buffer (pH 8), and the active fraction is collected.
The collected active fractions were concentrated using an ultrafiltration membrane and then adsorbed on a Butyl Toyopearl 650S column equilibrated with the same buffer containing 2M ammonium sulfate, and 10 mM Tris-
The active fraction is collected by elution with a concentration gradient of 2 to 0 M ammonium sulfate using a hydrochloric acid buffer solution (pH 8). The collected active fractions are concentrated using an ultrafiltration membrane and then dialyzed against the same buffer overnight. The purified enzyme thus obtained was subjected to polyacrylamide gel electrophoresis (gel concentration 15%).
And sodium dodecyl sulfate (SDS) electrophoresis gave a single band with an activity yield of about 4%.

The enzymatic chemical properties of the alkaline pullulanase thus obtained are described below.

The enzyme activity was measured according to the following method using the following buffer solutions (each addressed to 10 mM).

pH4-6 Acetate buffer pH6-8 Phosphate buffer pH8-11 Glycine-saline-sodium hydroxide buffer pH11-12 Potassium chloride-sodium hydroxide buffer Enzyme activity measurement method: Pullulan (reaction system in various buffers The final concentration in is 0.
0.1 ml of the enzyme solution was added to 0.9 ml of the substrate solution in which 25%) was dissolved and reacted at 40 ° C. for 30 minutes. After the reaction, 3,5-dinitrosalicylic acid (DNS)
The reducing sugar was quantified by the method. That is, D to 1.0 ml of reaction solution
NS reagent (1.0 ml) was added, color was developed by heating at 100 ° C. for 5 minutes, and after cooling, 4.0 ml of deionized water was added to dilute, and colorimetric determination was performed at a wavelength of 535 nm. The titer of the enzyme is 1 unit (1 unit of the amount of enzyme that produces a reducing sugar equivalent to 1 μmol glucose per minute.
U).

(Enzymatic properties) Action It decomposes the α-1,6 glucoside bond of pullulan to form maltotriose. It also hydrolyzes α-1,6 glucosidic bonds of starch, amylopectin, glycogen or their partial degradation products.

Substrate specificity Among branched sugars branched by α-1,6 glucoside bond,
Hydrolyzes a branched structure having a degree of polymerization equal to or higher than maltose (Table 1).

Working pH and optimum pH The working pH is in the range of pH 5 to 11, and the optimum pH is in the range of pH 9.5 to 11.

In addition, pullulanase activity at each pH, 0.25% pullulan, 10 mM acetate buffer (pH 4 ~ 6), phosphate buffer (pH 6 ~
8.5), glycine-saline-sodium hydroxide buffer (pH
8.5 to 11) and a potassium chloride-sodium hydroxide buffer (pH 11 to 12) reaction system were used to react at 40 ° C for 30 minutes, and the results are shown in Fig. 1.

pH stability It is extremely stable at pH 8 to 10, and even at pH 7 to 10.5
Maintain activity above 50%.

In addition, pullulanase activity at each pH, 0.25% pullulan, 10 mM acetate buffer (pH 4 ~ 6), phosphate buffer (pH 6 ~
8.5), glycine-saline-sodium hydroxide buffer (pH
The results measured by using the reaction system of 8.5 to 11) and a potassium chloride-sodium hydroxide buffer solution (pH 11 to 12) at 45 ° C. for 10 minutes are shown in FIG.

Operating temperature range and optimum temperature Operates in a wide range of 10 ℃ to 60 ℃, and the optimum working temperature is about 50 ℃.
It is observed at ° C (Fig. 3).

Temperature stability The temperature of this enzyme was changed under conditions of pH 9.5, and the inactivation condition was examined by treating it for 30 minutes at each temperature.
It is extremely stable up to ℃, and even at 50 ℃ about 50
It has a residual activity of not less than% (Fig. 4).

Molecular weight The molecular weight by SDS electrophoresis (gel concentration 7.5%) is about 120,
It is 000 ± 5,000.

Hg ²⁺ of influence 1mM of metal ion, Cd ^2+, and strongly inhibited by Mn ^2+, it has been slightly marginalized in Pb ^2+.

Effects of surfactants Linear alkylbenzene sulfonate sodium, alkyl sulfate sodium salt, polyoxyethylene alkyl sulfate sodium salt, sodium α-olefin sulfonate, sodium α-sulfonate fatty acid ester, sodium alkyl sulfonate, SDS, soap , Various kinds of surfactants such as Softanol (registered trademark).
Almost no inhibition of activity even after treatment with a 05% solution at 40 ° C for 15 minutes.

Effects of chelating agents The chelating agents EDTA (10 mM), EGTA (10 Mm), citric acid (0.05%) and zeolite (0.05%) hardly inhibit the activity.

Protease-resistant API-21 (manufactured by Showa Denko KK), Maxatase (manufactured by IBIS), savinase, alcalase, Esperase (manufactured by Novo) and other alkaline proteases coexist at the time of activity measurement (0.2 AU /), any protease Also has a strong resistance to. The above alkaline pullulanase is usually added in the detergent composition of the present invention in an amount of 0.1 to 10% by weight.

As the alkali or alkali-resistant pullulanase, a purified enzyme may be used, or the culture solution may be directly used as a crude enzyme.

There is no particular limitation on the other components used in the detergent composition of the present invention, which are commonly used in detergents, and they may be optionally incorporated according to the intended use and purpose. Hereinafter, those compounding ingredients are described.

(1) The blending amount of the surfactant is not particularly limited, but preferably 0.5 to 60% by weight. As the surfactant that can be used in the detergent composition of the present invention, as the anionic surfactant, alkylbenzene sulfonate, alkyl or alkenyl ether sulfate, alkyl or alkenyl sulfate, olefin sulfonate, Alkane sulfonate, saturated or unsaturated fatty acid salt, alkyl or alkenyl ether carboxylate, α-sulfo fatty acid salt or ester, amino acid type surfactant, N-acyl amino acid type surfactant, alkyl or alkenyl acid phosphate ester, Alkyl or alkenyl phosphates or salts thereof, amphoteric surfactants such as carboxy or sulfobetaine type surfactants, and nonionic surfactants such as polyoxyalkylene alkyl or alkenyl ethers and polyoxyethylene alkyl surfactants. Vinyl ether, higher fatty acid alkanolamides or alkylene oxide adducts thereof, sucrose fatty acid esters,
Examples include fatty acid glycerin monoester, alkylamine oxide, and alkyl glycoside, and examples of the cationic surfactant include quaternary ammonium salt.

When the detergent composition of the present invention is used as a detergent for an automatic dishwasher, the surfactant is preferably a low-foaming or non-foaming nonionic surfactant. Examples of this type of surfactant include alkoxylated nonionic surfactants (wherein the alkoxy portion is selected from the group consisting of ethylene oxide, propylene oxide and mixtures thereof). Specific examples of such a surfactant include:
BASF Japan "Plurafac (registered trademark) LF403", "Pl
Examples include urafac LF1300 "and" Softanol EP7045 "(registered trademark) manufactured by Nippon Shokubai Kagaku Kogyo Co., Ltd. When the detergent composition of the present invention is used as a detergent for an automatic dishwasher, the surfactant is contained in the composition. It is preferable that 0.5 to 30% by weight is blended.

(2) Inorganic electrolytes such as carbonates, bicarbonates, silicates, borates, alkanolamines or other alkaline agents, or sulfates are usually added in an amount of 0 to 90% by weight.

(3) Phosphates such as tripolyphosphate, pyrophosphate, orthophosphate, phosphonic acid salts such as ethane-1,1-diphosphonate, phosphonos such as 2-phosphonobutane-1,2-dicarboxylic acid Carboxylic acid salts, amino acid salts such as aspartic acid and glutamic acid, amino polyacetates such as nitrilotriacetate and ethylenediamine tetraacetate, polymer chelating agents such as polyacrylic acid and polyaconitic acid, oxalic acid, citric acid, etc. The organic acid salt, divalent metal ion scavenger such as aluminosilicate, etc. is usually 0 to 50% by weight in the composition.
Be compounded.

(4) 0 to 85% by weight of bleaching agents such as sodium percarbonate, sodium perborate, sodium hypochlorite, and dichloroisoaric acid.
Be compounded.

(5) As other minor components, anti-redeposition agents such as polyethylene glycol and carboxymethyl cellulose,
Enzymes such as proteases, lipases, α-amylases, cellulases, enzyme deactivation inhibitors such as sulfites, fluorescent dyes, bluing agents, pigments, anti-caking agents, solubilizers, enzymes or bleach activators A metal corrosion inhibitor or the like can be added as needed.

Examples of proteases that can be used in the present invention include subtilisins obtained from specific strains such as Bacillus subtilis and Bacillus licheniformis. Examples of these are "Maxatase" (registered trademark) sold by Gist, "Alcalase" (registered trademark) and "Esperase" (registered trademark) manufactured by Novo Industry.
And “Sabinase” (registered trademark) and the like.

Examples of α-amylase that can be used in the present invention include those obtained from Bacillus licheniformis, Bacillus subtilis, and the like. As an example of a commercially available product, "Termamyl" (registered trademark) manufactured by Novo Industry Co., Ltd. ,
"Maxamil" (registered trademark) of Gist, Inc. and the like can be mentioned.

When the detergent composition of the present invention is used as a detergent for an automatic dishwasher, an inorganic alkaline agent is preferably used as a balance component other than the above components in the case of a powder composition. Examples of the inorganic alkaline agent include sodium pyrophosphate, sodium orthophosphate, sodium tripolyphosphate, sodium carbonate, sodium bicarbonate, sodium sesquicarbonate, borax, and sodium silicate. In particular, since sodium silicate has a metal corrosion inhibiting action, it is desirable to use it together with other alkaline agents. In this case, other alkaline agents 35 to 85 wt% and sodium silicate (SiO ₂ / Na ₂ O ratio of 1 / 1-4 / 1, preferably 2 / 1-2.
5/1) It is most preferable to use 2 to 15% by weight. As for the inorganic alkaline agent, the pH of the detergent solution having a concentration of 0.05 to 1% by weight is 9.0
Adjust the blending amount to be ~ 11.0. For liquids,
The balance component is water.

It is also effective to add a fatty acid having a hydrocarbon chain length of about 8 to 18, benzotriazole, or the like as a copper corrosion inhibitor to the detergent for an automatic dishwashing machine.

In addition, since most phosphorus-free detergents are the mainstream of detergents, phosphate-containing detergents may become a social problem from the viewpoint of environmental problems. Therefore, with the spread of automatic dishwashers, it is also important to eliminate phosphorus without deteriorating the cleaning power against various stains.

When dephosphorizing, it is preferable to use a hydroxy polyvalent carboxylic acid represented by the following general formula (I) or a water-soluble salt thereof as a divalent metal ion scavenger.

(In the formula, X is —H, —CH ₃ , —CH ₂ COOH or —CH (OH) CO
OH, Y represents -H or -OH) Of these, citric acid, malic acid, tartaric acid or their water-soluble salts are preferable. Examples of such salts include sodium salts, potassium salts, monoethanolamine salts, diethanolamine salts, triethanolamine salts and the like.

Such a component is preferably added to the detergent composition of the present invention in an amount of 0.5 to 30% by weight.

Further, it is preferable to additionally use 1 to 10% by weight of a polymer chelating agent as a divalent metal ion scavenger. Examples of the polymer chelating agent include divalent metal ion-trapping polymer electrolytes as described in JP-A-57-145199, but polymers of acrylic acid or methacrylic acid, acrylic acid methacrylic acid copolymer Coalesce, water-soluble salts thereof, etc., the average molecular weight is 1,500 to 100,000, particularly 3,000 to 20,00.
0 is preferable.

The detergent composition of the present invention can be used as a powder, granular or liquid detergent for clothes, tableware, homes, etc. by mixing the above components according to a conventional method.

〔Example〕

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

Production Example 1 (1) 1 scoop of soil (approximately 0.5 g) of soil in Yokohama City, Kanagawa Prefecture,
It was suspended in sterile physiological saline and heat-treated at 80 ° C for 15 minutes. The supernatant of this heat treatment solution was appropriately diluted and applied to a separation agar medium (medium A). Then, this was cultured at 30 ° C. for 3 days to form a colony. Those that form a transparent zone based on the dissolution of pullulan around the settlement were selected and pullulanase-producing bacteria were obtained. Further, the obtained bacterium was inoculated into the liquid medium of the medium B, and cultivated with shaking at 30 ° C. for 3 days. After culturing, the supernatant liquid centrifuged was measured for pullulanase activity at pH 10 to screen for alkaline pullulanase-producing bacteria.

By the above-mentioned method, the alkaline pullulanase-producing bacterium Bacillus sp. KSM-AP1876 (FERM P-10887) could be obtained.

Medium A. 0.1% pullulan 0.8% colored pullulan 0.2% polypeptone 0.2% yeast extract _{KH 2 PO 4 0.03% (NH} 4) 2 SO 4 0.1% MgSO 4 · 7H 2 O 0.02% CaCl 2 · 2H 2 O 0.02% FeSO 4・ 7H ₂ O 0.001% MnCl ₂・ 4H ₂ O 0.0001% Agar 1.5% Na ₂ CO ₃ 0.5% pH 10.0 Medium B. Pullulan 1% Tryptone 0.2% Yeast extract 0.1% KH ₂ PO ₄ 0.03% (NH ₄ ) ₂ SO ₄ 0.1% MgSO ₄ / 7H ₂ O 0.02% CaCl ₂ / 2H ₂ O 0.02% FeSO ₄ / 7H ₂ O 0.001% MnCl ₂ / 4H ₂ O 0.0001% Na ₂ CO ₃ 0.5% pH 10.0 (2) Alkaline pullulanase The Bacillus sp. KSM-AP1876 strain, which is a producing bacterium, was inoculated into the liquid medium B of Reference Example 1 and cultivated with shaking at 30 ° C for 3 days. After culturing, the bacterial cells were removed by centrifugation to obtain a crude pullulanase enzyme solution. Further, according to a usual method, ethanol dry powder was prepared, and Table 2 below was used.
The crude enzyme preparation shown in was obtained. The enzyme activity in Table 2 is pH 9
It is the measured value in).

(3) About the crude enzyme solution obtained in (2), DEAE cellulose adsorption, DEAE cellulose (Whatman) chromatography, Sephacryl (Pharmacia) chromatography, DEAE Toyopearl (Toyo Soda) chromatography, butyl Toyopearl (manufactured by Toyo Soda Co., Ltd.) was purified to obtain alkaline pullulanase.

About the obtained alkaline pullulanase Davis (Davi
s DJ, Ann.NYAcad.Sci., 121,404 (1964)), followed by electrophoresis, followed by
It was confirmed to give a single band by staining with blue (Fig. 5).

(4) The alkaline pullulanase obtained in (3) was subjected to SDS electrophoresis according to a conventional method (Fig. 6). From this result, the molecular weight of this enzyme was 120,000 ± 5,000.

Example 1 (cleaning agent for automatic dishwasher) The cleaning conditions, the cleaning power test and the results adopted in this example are as follows.

(1) Washing conditions Washer used; fully automatic dishwasher manufactured by Matsushita Electric Industrial Co., Ltd. (model
NP-600) A type in which a cleaning agent aqueous solution is sprayed from a rotating nozzle to wash dishes placed on the upper surface of the spray track.

Washing temperature; Gradually raise from 5 ℃ to 55 ℃.

Washing water; water with hardness 3.5 ° DH Washing concentration; 0.2% Washing time; Washing 20 minutes, rinsing 20 minutes Circulating water volume during washing; 2.5 (2) Evaluation of cleaning power Contaminated dish of starch stain and evaluation method (contaminated dish) Mix white balls and cooked rice 9: 1, add equal amount of tap water to them, and mix with a mixer. 4 g of this dirt is evenly applied to a magnetic dish with a diameter of 22 cm, and air dried for 24 hours.

The above three contaminated dishes were subjected to the cleaning test.

(Evaluation method of detergency of starch stain) For the residual starch, the blue partial area (P ₁ ) generated by the color reaction of iodine was measured by photo determination, and the cleaning rate was calculated from the initial contaminated area (S ₀ ) by the following formula. I asked.

Cleaning rate = [(S ₀ −P ₁ ) / S ₀ ] × 100 (3) Cleaning composition Softanol EP7045 (manufactured by Nippon Shokubai Chemical Co., Ltd.) 2 Sodium citrate 201 Sodium silicate 5 Enzyme Table 3 Sodium carbonate balance (Note) Numbers indicate% by weight.

However, the enzyme is shown as the activity in the detergent.

(4) Detergency test results Table 3 shows the test results.

The enzyme activity in Table 3 was measured by the following method.

For 2 types of pullulanase, 0.9m of a substrate solution prepared by dissolving pullulan (final concentration in the reaction system is 0.25%) in 10mM glycine-salt-sodium hydroxide buffer (pH 9.0)
Add 0.1 ml of enzyme solution to l and incubate at 40 ℃ for 30 minutes.

For Termamyl 60T, a substrate solution prepared by dissolving soluble starch (final concentration in the reaction system is 0.25%) in 10 mM glycine-saline-sodium hydroxide buffer (pH 9).
0.1 ml of enzyme solution is added to 0.9 ml and reacted at 50 ° C for 15 minutes.

After the reaction, 3,5-dinitrosalicylic acid (3.5-dinitrosalic
The reducing sugar was quantified by the ylic acid (DNS) method. That is, add 1.0 ml of DNS reagent to 1.0 ml of reaction solution, and for 5 minutes at 100 ° C.
After heat-developed, the mixture was cooled, diluted with 4.0 ml of deionized water, and colorimetrically determined at a wavelength of 535 nm. Enzyme titer is 1
The amount of enzyme that produces a reducing sugar corresponding to 1 μmol of glucose per minute was defined as 1 unit (1 U).

Example 2 (Cleaning agent for clothing) The cleaning conditions, the cleaning power test and the results adopted in this example are as follows.

(1) Artificial polluted cloth White balls and cooked rice are mixed at a ratio of 9: 1, diluted twice with tap water, and mixed in a mixer. This solution is applied to a cotton cloth 10 cm × 10 cm test piece so that the weight of the cloth is 2.5 to 5%. It was dried at 20 ° C. for 24 hours and used for the experiment.

(2) Washing conditions and method Dissolve the detergent in 4 ° DH hard water to prepare 0.665% detergent aqueous solution 1. Add 5 sheets of artificial cotton cloth to the detergent solution,
After standing at 40 ° C for 1 hour, the detergent solution and the artificially contaminated cloth are transferred as they are to a stainless beaker for a tergotometer, and washed by stirring with a tergotometer at 100 rpm, 20 ° C for 10 minutes. After rinsing under running water, it was dried at 20 ° C. for 24 hours and subjected to weight measurement.

(3) Evaluation of Detergency The weight of the original cloth before cleaning and the weight of the contaminated cloth before and after cleaning were measured, and the cleaning rate (%) was calculated by the following formula.

The value of each cleaning rate in Table 4 is an average value of 5 sheets.

(4) Detergent composition Linear sodium dodecyl benzene sulfonate 15 Alkyl ethoxy sulfate sodium (C _{14 to} C ₁₅ , ▲ ▼ ＝ 3mo
l) 5 4A-type zeolite 15 Sodium silicate 15 Sodium carbonate 15 Sodium polyacrylate (▲ ▼ = 8000) 1.5 Polyethylene glycol (▲ ▼ = 6000) 1.5 Fermentase Table 4 Fluorescent dye 0.5 Glauber's salt residual water 5 (Note) ) Numbers represent% by weight.

However, enzymes show activity in detergents.

(5) Detergency test results Table 4 shows the test results.

The enzyme activity in Table 4 was measured according to Table 3.

〔The invention's effect〕

As described above, the detergent composition of the present invention has an excellent detergency against starchy stains within a normal washing time.

[Brief description of drawings]

FIG. 1 is a drawing showing the relationship between the reaction pH and the relative activity of the alkaline pullulanase used in the present invention. FIG. 2 is a drawing showing the relationship between the treatment pH and residual activity of the alkaline pullulanase used in the present invention. FIG. 3 is a drawing showing the relationship between the reaction temperature (pH 9.5) and relative activity of the alkaline pullulanase used in the present invention. FIG. 4 is a drawing showing the relationship between the treatment temperature (pH 9.5) and residual activity of the alkaline pullulanase used in the present invention. FIG. 5 is a drawing showing the results of electrophoresis of alkaline pullulanase used in the present invention. FIG. 6 shows the alkaline pullulanase used in the present invention.
It is a figure which shows the result of SDS electrophoresis.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Internal reference number FI technical display location (C12N 9/44 C12R 1:07) (72) Inventor Katsuhiko Deuti 3413 Ishiicho, Utsunomiya City, Tochigi Prefecture 107 (56) Reference JP-A-3-87177 (JP, A)

Claims (3)

[Claims] 1. A detergent composition comprising an alkaline pullulanase having the following enzymatic properties. 1) Action Decomposes α-1,6 glucoside bond of pullulan to produce maltotriose. It also hydrolyzes the α-1,6 glucosidic bond of starch, amylopectin, glycogen or their partial degradation products. 2) Substrate specificity Among sugars having a branched structure branched by α-1,6 glucoside bond, a branched structure having a degree of polymerization of maltose or higher is hydrolyzed. 3) Working pH and optimum pH The working pH is in the range of pH 5 to 11, and the optimum pH is in the range of 9.5 to 11. 4) pH stability It is extremely stable in the range of pH 8 to 10, and has a relative activity of 50% or more even in the range of pH 7 to 10.5 (by treatment at 45 ° C for 10 minutes). 5) Working temperature range and optimum temperature It works in the range of 10 to 60 ° C, and the optimum working temperature is about 50 ° C. 6) Temperature stability It is extremely stable up to 40 ℃ (10mM glycine-pH9.5).
Treatment with salt-sodium hydroxide buffer for 30 minutes). 7) Molecular weight The molecular weight by sodium dodecyl sulfate electrophoresis is 120,00.
It is 0 ± 5,000. 8) Effect of metal ions It is inhibited by Hg ²⁺ , Cd ²⁺ , Mn ²⁺ and Pb ²⁺ . 9) Effects of surfactants Linear alkylbenzene sulfonate sodium, alkyl sulfate sodium salt, polyoxyethylene alkyl sulfate sodium salt, sodium α-olefin sulfonate, sodium α-sulfonated fatty acid ester, sodium alkyl sulfonate, sodium It is hardly inhibited by surfactants such as didodecylsulfate, soap and softanol. 10) Effect of chelating agent EDTA, EGTA, citric acid and zeolite are hardly affected by the activity. 11) Protease resistance It has strong resistance to alkaline protease. 2. The detergent composition according to claim 1, wherein the alkaline pullulanase is obtained by isolation from a culture of a microorganism belonging to the genus Bacillus. 3. The microorganism is Bacillus sp.
sp.) KSM-AP1876, and the cleaning composition according to claim 2, which has been deposited as Microorganism Research Institute No. 10887.