JPH03290498A - Cleanser composition - Google Patents

Abstract

PURPOSE:To provide a cleanser composition effectively acting on the starch stains strongly adhered to table wares, fibers, etc., and having a remarkably improved cleanability by compounding an alkali pullulanase or alkali-resistant pullulanase having an alpha-amylase activity. CONSTITUTION:The objective composition comprises an alkali pullulanase or alkali-resistant pullulanase having an alpha-amylase activity in an amount of usually 0.1-10wt.%, the pullulanase being produced by Bacillus sp. KSM-AP1378 (FERM 10886). The purified enzyme may be used as the alkali or alkali-resistant pullulanase and a culture solution may be used as such as the crude enzyme.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a detergent composition containing an alkali or alkali-resistant pullulanase.

[Conventional technology]

Adding enzymes to cleaning agents has been practiced for a long time. Enzymes in detergents act as cleaning aids; for example, in laundry detergents, they remove various dirt and stains on clothing, and in dishwashing detergents, they remove oils and fats, proteins, etc. that remain on the surface of dishes. It functions to decompose or alter starch, etc., making it easier to remove.

Conventionally, α-amylase has been used to remove particularly starchy stains, and by soaking the items to be washed for a long time in a cleaning solution containing α-amylase, the cleaning power against starchy stains can be improved. Can be done.

The present inventors have discovered that the combined use of α-amylase and pullulanase effectively acts on starchy stains firmly attached to tableware, fibers, etc., and can significantly improve the cleaning power. Filed (Japanese Patent Application No. 63-285424)
.

[Problem to be solved by the invention]

However, most of the pullulanases conventionally found in nature are classified as so-called neutral or acidic pullulanases, which show maximum and stable enzymatic activity in the neutral to acidic region. The reality is that there are extremely few pullulanases that are suitable for laundry detergent compositions, that is, have maximum activity in the alkaline region or are alkaline tolerant, so-called alkaline pullulanases and alkali-tolerant pullulanases.

Here, alkaline pullulanase refers to one that has an optimal pH in the alkaline region, and alkaline-resistant pullulanase has an optimal pH in the neutral to acidic region, but it also has an optimal pH in the alkaline region. It refers to something that has sufficient activity and maintains stability compared to the activity in the field. Further, neutral refers to a pH range of 6 to 8, and alkaline refers to a pH range higher than that.

Conventionally, as alkaline pullulanase, the
Only the one described in Publication No. 786 is known. However, this enzyme has an optimum pH in the alkaline region, and although it has characteristics such as broader substrate specificity than conventionally known pullulanase, its optimum pH is 8 to 9.
Since it is in the weak alkaline range, there was a problem that it was not suitable for use as a cleaning agent component. In addition, it also has the drawbacks of unstable enzymes and poor enzyme productivity, making it unsuitable for industrial fermentation production.

[Means to solve the problem]

Under these circumstances, the present inventors have conducted intensive research on pullulanase that is more suitable as a cleaning agent component, and have found that alkali or alkali-resistant pullulanase having α-amylase activity is effective against tableware, even when α-amylase is not used in combination. The present invention was completed based on the discovery that it effectively acts on starch stains firmly attached to fibers, etc., significantly improves cleaning power, and is stable and has good productivity.

That is, the present invention provides a cleaning composition characterized by containing an alkali or alkali-resistant pullulanase having α-amylase activity.

Examples of alkali or alkali-resistant pullulanase that can be used in the present invention include Bacillus sp., which is a type of alkaliphilic microorganism shown below.
, ) KSM-APL37g (FBRM P-10886
) is produced by alkaline pullulanase.

This microorganism exhibits the following mycological properties.

In addition, the following medium 1 was used for classifying bacterial strains in the following.
~21 types, all of which contain 0.5% by weight of separately sterilized sodium carbonate (Na2CD-) (hereinafter referred to as
(simply referred to as %).

Composition of the medium used (displayed as %): Medium 1. Nutriendo broth, 0.8; powdered agar (
Wako Tsumugi Pharmaceutical Co., Ltd.), 1.5 culture jt2. Nicot trient broth, 0.8 medium 3
．． Nutriendo broth, 0.8: Gelatin, 2
0.0; agar powder (manufactured by Wako Tsumugi Pharmaceutical Co., Ltd.), 1.5 medium 4. Bata Tolitoma Milk, 10.5 Medium 5. Nutriendo broth, 0.8: KNO3,0,1 medium 6. Batatopebutone, 0.7; NaCl, 0,5;
Glucose, 0.5 medium7. 31M agar medium (Suzaku Kagaku), indicated amount medium 8. TSI agar medium (Suzaku Chemical), indicated amount medium 9
．． Yeast extract, 0.5; Bactopeptone, 1.5;
2) IP[14,0,1; Mg5D4-7H20,
0.02; Soluble starch, 2.0; Agar powder (manufactured by Wako Tsumugi Pharmaceutical Co., Ltd.), 1.5 Medium 10. Courser medium (Nusaku Kagaku)
, indicated amount medium 11. Christensen medium (Nusaku Kagaku)
, indicated amount medium 12. ■Yeast extract, 0.05; NazSO
4,0,1; KH, PO,, 0°1; Glucose, 1.
0■ Yeast extract, 0.05; NaaSO-, 0
, 1; Kt12P[1,, 0, 1; Glucose, 1.
0; CaC1,-2H20,0,05; Mn5
Oa"4~6FIJ, 0.01; Fe5Oa'7H2[
1,0,001; Mg5044HJ, 0.02 As nitrogen sources, sodium nitrate, sodium nitrite, ammonium chloride and ammonium phosphate were each used at 0.2
5%, 0, 2025%, 0.158% and 0.195%
It was used in addition to the medium of (1) and (2) above so that the following results were obtained.

Medium 13. King A medium “Nusaku” (manufactured by Nousaku Kagaku).

Instruction amount Medium 14. King B medium “Nusaku” (manufactured by Nousaku Kagaku).

Indicated amount medium 15. Urea medium “Suzaku” (Suzaku Kagaku), indicated amount medium 16. Filter paper for cytochrome oxidase test (Nippon Pharmaceutical) Medium 17.3% hydrogen peroxide aqueous medium 18. Batatopebutone, 0.5; Yeast extract,
0.5°K, HPO, 0,1; Glucose, 1.
0; Mg5O+・7H20,0,02 culture jl! ! 19. Batatopebuton, 2.7; Nai
, 5,5; HPD, 0.3; Glucose, 0.
5; Bromothymol blue, 0.06, agar powder (manufactured by Wako Tsumugi Pharmaceutical), 1.5 medium 20. (NH=)JPDi, 0.1; KCj
! , 0.02; Mg5Ov7H20,0,02;
Yeast extract, 0.05; sugar, 1°0 medium 21. Casein, 0.5; Yeast extract, 0.5; Glucose,
1.0; K2HPO4,0,1; Mg5Oa・
7H2D, 0.02; agar powder (manufactured by Wako Tsumugi Pharmaceutical),
1.5 [Mycological properties] (a) Microscopic observation results Large scale of bacterial cells, 0.8-2.4umX1.8-4.0
It is a microbacillus with an oval endospore (1 μm) at one end of the bacterial body.
, 0 to 1.2 μm x 1.2 to 1.4 μm>. It also has periflagella and is motile. Durham staining is indeterminate.

It has no anti-acid properties.

6) Growth status in various media■ Broth agar plate culture (medium 1.) Growth status is good. The shape of the village is circular, the surface is smooth, and the periphery is smooth. The color of the village is yellow, translucent and shiny.

■ Broth agar slant culture (medium 1.) Grows. Its condition is spread-like, glossy, and yellow and translucent.

■ Meat juice liquid culture (medium 2.) Grow.

■ Meat juice gelatin puncture culture (medium 3.) Growth condition is good. Liquefaction of gelatin is observed.

■ Ridmus milk medium (medium 4.) No coagulation or peptonization of milk was observed.

Discoloration of lidmus cannot be determined because the medium is alkaline.

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

■ MR test (medium 6.) Because the medium is alkaline, it is not possible to determine whether it is negative or positive.

■ VP test (medium 6.) negative.

■ Raw tL of indole<medium 7. ) negative.

■ Production of hydrogen sulfide (medium 8.) negative.

■ Starch hydrolysis (medium 9.) Positive.

■ Utilization of citric acid Negative in Cosar medium (medium 10.). Christensen medium (medium 11.) cannot determine whether it is positive or negative.

■ Use of inorganic nitrogen sources (medium 12.) Both nitrates, ammonium salts, and nitrites are used.

■ Pigment live tL (medium 13..14,) negative.

[Phase] Urease (medium 15.) negative.

0 Oxidase (medium 16.) negative.

■ Catalase (medium 17.) Positive.

0 Growth range (medium 18.) The temperature range for growth is 20-40℃, the optimal temperature range for growth is 3
The temperature is 0 to 35°C.

The pH range for growth is pH 7 to 10.5, and the optimum pH for growth is p.
It is H1O.

■ Attitude towards oxygen Aerobic.

■ 0-F test (medium 19.) Due to its alkaline nature, discoloration cannot be determined.

Grows only under aerobic conditions.

[Phase] Sugar utilization (medium 20.) L-arabinose, D-xylose, D-glucose,
D-mannose, D-7 lactose, D-galactose, maltose, sucrose, lactose, trehalose, D-sorbitol, D-mannite, inosit, glycerin, starch, raffinose, salicin, D-ribose and dextrin are utilized.

■ Growth in a salt-containing medium (modification of medium 1) It grows at a salt concentration of 7%, but cannot grow at a salt concentration of 10%.

[Phase] Casein degradation (medium 21.) Positive.

Based on the above examination of mycological properties,
Bergey's Manual of Det
erminative Bacteriology) 8th Edition and The Genus Bacillus ("'The Gen
us Bacillus”Ruth.

B. Gordon, Agriculture
Handbook N (L 427゜Agricu
ultural research 5service,
U,S.

mouth parts of agricultural
re Washington D, C,.

(1973)), and as a result of a comparative search, it was recognized that this strain is a type of genus Bacillus, which is a spore-bearing bacillus. However, since this strain cannot grow in a neutral region and only grows well in a highly alkaline region, Horikoshi and Akiba [Al
kalo, Pbilic Microorganism
, Japan Scientific 5ocie
ty Press (Tokyo), published in 1982], it is tentatively known as a so-called Alkalophilic microorganism.
It is distinguished from conventional Bacillus bacteria that grow in neutral conditions.

Furthermore, since the mycological properties of this strain do not match any of the known alkalophilic bacilli, we judged it to be a new strain and named it Bacillus sp. Deposited at the National Institute of Microbial Technology.

In order to obtain the alkaline pullulanase having α-amylase activity used in the present invention using the above-mentioned bacterial strain, the bacterial strain may be inoculated into a medium and cultured according to a conventional method. It is preferable that the medium used for culture contains appropriate amounts of assimilable carbon sources and nitrogen sources. The carbon source and nitrogen source are not particularly limited, but examples include corn gluten meal, soybean flour, corn steep liquor, casamino acid, yeast extract, Pharmamedia, meat extract, tryptone, soyton, Vibro, and Azipower. ,
Examples include organic nitrogen sources such as cottonseed oil cake, cultivator, aziprone, and zest, and inorganic nitrogen sources such as ammonium sulfate, ammonium nitrate, ammonium phosphate, ammonium carbonate, sodium nitrate, and ammonium acetate. Carbon sources include soluble starch, insoluble starch, amylopectin, glycogen, pullulan, and branched oligosaccharides produced by partial decomposition thereof, as well as utilizable carbon sources such as glucose, maltose, arabinose, xylose, ribose, mannose, Examples include flagose, galactose, maltose, sucrose, lactose, trehalose, mannitol, sorbitol, glycerin, and assimilated organic acids such as citric acid and acetic acid. In addition, inorganic salts such as phosphates, magnesium salts, calcium salts, manganese salts, zinc salts, cobalt salts, sodium salts, potassium salts, etc., and if necessary, inorganic and organic trace nutrients are added to the medium as appropriate. You can also.

In addition, the temperature during culturing is 20 to 40°C, especially 30 to 30°C.
The temperature is preferably 5°C, and the pH is preferably 8 to 1015, particularly 1O, and under these conditions, the culture is usually completed in 2 to 3 days.

The target substance, α-, from the culture thus obtained
Collection and purification of alkaline pullulanase having amylase activity can be carried out in accordance with general enzyme collection and purification methods. That is, after culturing, bacterial cells can be removed from the culture by conventional solid-liquid separation means such as centrifugation or filtration to obtain a crude enzyme solution. This crude enzyme solution can be used as it is, but if necessary, the crude enzyme can be obtained by separation methods such as salting-out method, precipitation method, ultrafiltration method, etc., and then purified and crystallized by a known method. It is also possible to use it as an enzyme.

An example of a preferred method for producing the alkaline pullulanase will be described below. For example, KSM-API378 strain, which belongs to the alkaline bacterium Bacillus genus, is mixed with 1% pullulan, 1% polypeptone, 0.5% yeast extract, and 0.1% 112PO.
,,0,25%Na*HPO4・12H20,0,02
%Mg5O<・7H10, 0.5% Sodium carbonate was cultured at 30° C. for 3 days with aerobic shaking, and the resulting culture was removed from the culture solution to obtain a supernatant. Next, ■D
It is purified by RAB cellulose adsorption, ■ α-cyclodextrin affinity chromatography, ■ DRAB) Yopal (manufactured by Toyo Soda) chromatography, and ■ Sephacryl (manufactured by Pharmacia) chromatography. The purified enzyme thus obtained is
Polyacrylamide gel electrophoresis (gel concentration 15%) and sodium dodecyl sulfate (SOS) electrophoresis gave a single band, and the yield of pullulanase activity was about 2%.

The alkaline pullulanase thus obtained can be suitably used as a component of the detergent composition of the present invention. The enzymatic chemical properties of this alkaline pullulanase will be explained below.

The enzyme activity was measured using the following buffer solutions (50 mM each) and according to the following method.

pH 4-6 Acetate buffer pH 6-8 Phosphate buffer (used for measuring pullulanase activity) pH (6-8 Trismaleide buffer (used for measuring α-amylase activity) pH 8-11 Glycine-salt-sodium hydroxide buffer pH 11 ~12 Potassium chloride-sodium hydroxide buffer enzyme activity measurement method: ■ Pullulanase activity In various buffer solutions, pullulan (the final concentration in the reaction system is
0.1- of the enzyme solution was added to the substrate solution (0.9-) in which 0.25%) was dissolved, and the mixture was reacted at 40°C for 300 minutes. After the reaction, 3.5-dinitrosalicylic acid (3°5-dinit
rosalicylic acid (DNS)')
The amount of reducing sugar was determined using the method. That is, the reaction solution 1.0-
Add NS reagent 1.0- to , heat at 100°C for 5 minutes to develop color, and after cooling, 4.07! of deionized water was added to dilute the solution, and colorimetry was carried out at a wavelength of 535 nm. The enzyme titer is
The amount of enzyme that produced reducing sugar equivalent to 1 μmol of glucose per minute was defined as 1 unit (IU).

■ α-Amylase activity Add 0.1- of enzyme solution to 0.9- of substrate solution prepared by dissolving soluble starch (final concentration in reaction system: 0.25%) in various MIr solutions, and add 0.1- of enzyme solution to 5D' C for 15 minutes.

After the reaction, reducing sugars were quantified by the ([1NS) method.

That is, 1.0 wl of reaction solution! Niguchi NS reagent 1.0rr11
was added, heated at 100℃ for 5 minutes to develop color, and after cooling,
．． 0ml! diluted by adding deionized water to a wavelength of 535n.
Colorimetry was carried out at m. The enzyme titer is 1 μl per minute.
0Ji! The amount of enzyme that produces reducing sugar equivalent to glucose was defined as 1 unit (1U).

[Enzyme chemical properties]

(2) Action It acts on pullulan and soluble starch, producing mainly maltotriose from the former, and mainly maltotetraose and maltopentaose from the latter. It also acts on glycogen to produce maltotetraose and maltopentaose (Figure 1).

■ Substrate specificity Acts on pullulan, soluble starch and glycogen (Table 1).

Table 1 below (margin) Action pH and optimum action pi (The action pH of this enzyme on pullulan is in the range of 5 to 12, and the optimum action p+ is found to be in the range of 8.5 to 10.

The pullulanase activity at each pH was measured using 0.25% pullulan, 10mM acetate buffer (pt14-5), phosphate buffer (pH6-8), and glycine-salt-sodium hydroxide buffer (pH9-10.5). FIG. 2(a) shows the results of a reaction at 40° C. for 300 minutes using a reaction system of potassium chloride-sodium hydroxide buffer (pt(11-12)).

Further, the effective pH for soluble starch is in the range of 4 to 12, and the optimum action pal is found in the range of pH 7 to 9.5.

The α-amylase activity at each pH was determined using 0.25% soluble starch, 10mM acetic acid M buffer (p) 14-5, trismaleide buffer (pH 6-8), glycine-salt-hydroxide) IJum buffer. (pH 9-10.5) and carbonate buffer (pflll-12) at 50°C.
The results of measurement after reacting for 15 minutes are shown in FIG. 2 (b).

■ p) I stability The pH stability of this enzyme against pullulan is between pH 6 and 10.
．． Approved within the range of 5.

In addition, the pullulanase activity at each pH (0.25% pullulan, 10-acetate buffer (pH 4-5), phosphate buffer (pH 6-8), glycine-salt-sodium hydroxide buffer (pH 9-10) .5) and a reaction system of potassium chloride-sodium hydroxide buffer (pH 11 to 12),
The results of the reaction at 45°C for 10 minutes are shown in Figure 3 (a).
).

In addition, p)I stability against soluble starch was determined at pH 4 to 1.
It is recognized within the range of 2.

The α-amylase activity at each pH was measured using 0.25% pullulan, 10d acetate buffer (pH 4-5), trismaleide buffer (pH 6-8), glycine-salt-sodium hydroxide buffer (pH 11-12.5). ) and a carbonate buffer (pH 11 to 12), the reaction was carried out at 50° C. for 15 minutes, and the measurement results are shown in FIG. 3 (b).

■ Action temperature range and optimum action temperature The activity of this enzyme against pullulan and soluble starch is 10
It is observed in the range of ~65°C, and the optimum temperature is found to be about 50°C (Fig. 4(a) and (b)).

■ Temperature stability Regarding this enzyme, the temperature was changed under the conditions of pf (9.5).
When we investigated the conditions for inactivation by treating at each temperature for 30 minutes, we found that it is extremely stable up to 45°C (Fig. 5 (a)
) and (b)).

(2) Molecular Weight The molecular weight determined by SO3 electrophoresis (gel concentration 7.5%) is approximately 200.000±5.000.

■ Influence of metal ions on pullulanase activity: Hg2+, Mn2+, Pb2+
is inhibited by In addition, α-amylase activity is Hg”,
Inhibited by Mn2+Pb'', Cd'', Zn'+ (
Table 2).

As is clear from Table 2 below, the pullulanase activity and α-amylase activity of this enzyme are inhibited by different types of metal ions.

■ Directly affected by surfactants Sodium alkylbenzene sulfonate, polyoxyethylene alkyl sulfate sodium salt, α-
Treatment at 40°C for 15 minutes with a 0.05% solution of various surfactants such as sodium olefin sulfonate, sodium α-sulfonated fatty acid ester, sodium alkylsulfonate, SO3, Stone M, and Softanol (registered trademark). However, the activity is hardly inhibited.

■ Effect of chelating agents Chelating agents EDTA (10cnM) and EGT
A (10mM>) pullulanase activity is hardly inhibited, but α-amylase activity is significantly inhibited.Also, α-amylase activity inhibited by the chelating agent is restored when Ca2+ is added again (Table 2). ).

■ Alkaline proteases such as protease-resistant maxatase (manufactured by CIBIS) and savinase (manufactured by Novo) coexist during activity measurement (0
, 2AU/I1), it has strong resistance to all proteases.

As is clear from the above enzyme chemical properties, this enzyme is
It is a new enzyme with different physical and chemical properties from conventional pullulanase having α-amylase activity.

Furthermore, in order to further clarify the novel points of this enzyme, the results of a comparison of its physicochemical properties with a conventionally reported pullulanase having α-amylase activity are shown in Table 3 below.

As is clear from Table 3 in the margin below, this alkaline pullulanase is
Pullulanase-amylase complex enzyme derived from Bacillus subtilis TO and Bacillus circulans F-2
Its physicochemical properties are clearly different from the amylase having pullulanase activity derived from the origin.

The above-mentioned alkali or alkali-resistant pullulanase having α-amylase activity is usually contained in the detergent composition of the present invention at 0.0%.
It is blended in an amount of 1 to 10% by weight. As the alkali or alkali-resistant pullulanase, a purified enzyme may be used, or the culture solution may be used as it is as a crude enzyme.

Other commonly used detergent ingredients to be added to the cleaning composition of the present invention are not particularly limited and may be added as desired depending on the use and purpose. The ingredients will be described below.

(1) The amount of the surfactant to be blended is not particularly limited, but it is preferably blended in an amount of 0.5 to 60% by weight.As the surfactant that can be used in the cleaning composition of the present invention, anionic surfactants can be used. Examples of the agent include alkylbenzene sulfonates, alkyl or alkenyl ether sulfates, alkyl or alkenyl sulfates, olefin sulfonates, alkanesulfonates, saturated or unsaturated fatty acid salts, alkyl or alkenyl ether carboxylates, α-
Sulfo fatty acid salts or esters, amino acid type surfactants,
Examples of amphoteric surfactants include N-acyl amino acid type surfactants, alkyl or alkenyl acid phosphate esters, alkyl or alkenyl phosphate esters or salts thereof, and examples of amphoteric surfactants include carboxy or sulfobetaine type surfactants. Examples include polyoxyalkylene alkyl or alkenyl ether, polyoxyethylene alkylphenyl ether, higher fatty acid alkanolamide or its alkylene oxide adduct, sucrose fatty acid ester, fatty acid glycerin monoester, alkylamine oxide, alkyl glycoside, etc. Examples of the surfactant include quaternary ammonium salts.

When the cleaning composition of the present invention is used as a detergent for automatic dishwashers, the surfactant is preferably a low-foaming or non-foaming nonionic surfactant.

Examples of surfactants of this type include alkoxylated nonionic surfactants, where the alkoxy moiety is selected from the group consisting of ethylene oxide, propylene oxide, and mixtures thereof. A specific example of such a surfactant is BASF Japan's “Pl
urafac (registered trademark) LF403”, Plura
fac LF1300" and Nippon Shokubai Kagaku Kogyo ■'s "Softanol BP7045" (registered trademark). When the detergent composition of the present invention is used as an automatic dishwasher detergent, no surfactant is added to the composition. It is preferable that it is blended in an amount of .5 to 30% by weight.

(2) Alkaline agents such as carbonates, bicarbonates, silicates, borates, alkanolamines, or inorganic electrolytes such as sulfates are usually mixed in an amount of 0 to 90% by weight.(3) Tripolyphosphate, pyrroline acid salts, phosphates such as orthophosphates, salts of phosphonic acids such as ethane-1,1-diphosphonate, salts of phosphonocarboxylic acids such as 2-phosphonobutane-1,2-dicarboxylic acid, aspartic acid,
Salts of amino acids such as glutamic acid, aminopolyacetates such as nitrilotriacetate and ethylenediaminetetraacetate, polymer chelating agents such as polyacrylic acid and polyaconitic acid, salts of organic acids such as oxalic acid and citric acid, aluminosilicate The divalent metal ion scavenger, such as a salt, is usually incorporated into the composition in an amount of 0 to 50% by weight.

(4) Bleaching agents such as sodium percarbonate, sodium percarbonate, sodium hypochlorite, dichloroisosialic acid, etc. are 0 to 85% by weight.
It is blended.

(5) Other minor components include polyethylene glycol, recontamination inhibitors such as carboxymethyl cellulose, enzymes such as protease, lipase, α-amylase, and cellulase, enzyme deactivation inhibitors such as sulfites, fluorescent dyes, and blue tints. Additives, dyes, anti-caking agents, solubilizers, enzyme or bleach activators, metal corrosion inhibitors, and the like can be added as necessary.

Examples of proteases that can be used in the present invention include subtilisins obtained from specific bacterial strains such as Bacillus subtilis and Bacillus licheniformis. Examples of these include Maxatase® sold by Gist, Alcalase®, Esperase® and Sabinase® from Novo Industries.

Further, examples of α-amylase that can be used in the present invention include those obtained from Bacillus licheniformis, Bacillus subtilis, etc., and examples of commercially available products include “Termamyl” (registered trademark) manufactured by Novo Industries. )
, "Maxamil" (registered trademark a) manufactured by Gist Co., Ltd., and the like.

When the detergent composition of the present invention is used as a detergent for an automatic dishwasher, it is preferable to use an inorganic alkaline agent as a balance component other than the above-mentioned components in the case of a powder or granule composition. Examples of the inorganic alkaline agents include sodium pyrophosphate, sodium orthophosphate, sodium tripolyphosphate, sodium carbonate, sodium bicarbonate, sodium sesquicarbonate, borax, and sodium silicate. In particular, since IJium silicate has a metal corrosion inhibiting effect, it is desirable to use it together with other alkaline agents. In this case, 35 to 85% by weight of other alkaline agents and sodium silicate (SxOa/Na1O ratio of 1/1 to
4/1, preferably 271-2.5/1) 2-15
It is most preferable to use % by weight in combination. The inorganic alkaline agent has a detergent solution with a concentration of 0.05 to 1% by weight at a pH of 9.0 to 1.
Adjust the blending amount so that it becomes 1.0.

In the case of 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, etc. as a copper corrosion inhibitor to the automatic dishwasher detergent.

In addition, since phosphorus-free detergents are now mainstream among many cleaning agents, phosphate-containing detergents may become a social problem from an environmental standpoint. Therefore, it is also important to make it phosphorus-free without reducing its detergency against various stains.

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

(In the formula, X1t-H, -CHa, -CH,C00)I
or -C)l (DI()C00II, Y represents -H or -lobe) Among these, citric acid, malic acid, tartaric acid or water-soluble salts thereof are preferred. Examples of such salts include sodium salts, potassium salts, monoethanolamine salts, jetanolamine salts, triethanolamine salts, and the like.

Such components are contained in the cleaning composition of the present invention in an amount of 0.5 to 3
It is preferable to blend 0% by weight.

Furthermore, it is preferable to use 1 to 10% by weight of a polymer chelating agent as a divalent metal ion scavenger. Examples of polymer chelating agents include divalent metal ion-trapping polymer electrolytes as described in JP-A-57-145199, and polymers of acrylic acid or methacrylic acid,
Examples include acrylic acid methacrylic acid copolymers, their water-soluble bases, etc., and the average molecular weight is 1.500 to 100.00.
0, particularly preferably 3.000 to 20.000.

The cleaning composition of the present invention can be prepared by mixing the above-mentioned components according to a conventional method and used as a powder, granule or liquid cleaning agent for clothing, tableware, housing, etc.

〔Example〕

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

Production Example 1 (1) Soil from Tochigi City, Tochigi Prefecture was mixed into a medicine spoonful (approximately 0.5 g).
It was suspended in sterile physiological saline and heat-treated at 80°C for 15 minutes. The supernatant of this heat-treated liquid was diluted appropriately and applied to a separation agar medium (medium A). Next, this was heated at 30°C for 3
The cells were cultured for several days to form colonies. We selected those that formed a pellucid zone around the colony due to the dissolution of colored pullulan and colored starch, and obtained pullulanase-producing bacteria with α-amylase activity. Furthermore, the obtained bacteria were inoculated into a liquid medium B, and cultured with shaking at 30°C for 3 days. After culturing, the pullulanase activity and amylase activity of the centrifuged synovial fluid were measured at pH 10, and alkaline pullulanase-producing bacteria having α-amylase activity were screened.

By the above method, Bacillus sp. KSM, which is an alkaline pullulanase producing bacterium having α-amylase activity
- I was able to obtain 8 P1378 (FORM P-10886).

Medium A, pullulan 0.5% soluble starch 0.5% colored pullulan 0.2% colored starch 0.2% polypeptide 0.2% yeast extract
0.1% KH2PO40,03% (Nt14) 2SO,0,1% 11g5Oa”7HJ CaCj! *”2L[1 FeSOa・7t (J MnCj! a”4)1i0 Agar pH Medium B, pullulan soluble starch tryptone yeast extract HaPOi (N)Is)ssOa MgSL・7H20 CaC112L.

Fe50. '? )1.0 MnCJ 14Hz0 0,02% 0.02% 0.001% 0,0001% 1.5% 10.0 0.5% 0.5% 0.2% 0.1% 0.03% 0 .1% 0.02% 0.02% 0.001% 0.0001% pH 10.0 (2) Bacillus sp. KSM-^P, an alkaline pullulanase producing bacterium with α-amylase activity
The 1378 strain was inoculated into liquid medium B of Example 1, and cultured with shaking at 30°C for 3 days. After culturing, the bacterial cells were removed by centrifugation to obtain a crude enzyme solution. Furthermore, according to conventional methods, 100
% ethanol to form an ethanol dry powder, we were able to obtain an alkaline pullulanase enzyme preparation having the α-amylase activity shown in Table 4 below (the enzyme activity was determined by p
This is the measured value in H9).

(3) The crude enzyme solution obtained in (2) was purified according to the following procedure to obtain alkaline pullulanase having α-amylase activity. That is, DBAB cellulose powder was added to the supernatant of the crude enzyme solution, and the pullulanase in the supernatant was completely adsorbed onto the DRAB cellulose. Then 10rnM)! After washing the resin with J Soot-acid buffer (p) (8), the enzyme was eluted with the same buffer containing 0.6 M NaCl. Then 10mM)! J Soot-acid buffer (pH
After dialysis with 8), it was adsorbed onto an α-cyclodextrin affinity column equilibrated with the same buffer, eluted with the same buffer containing β-cyclodextrin, and the active fraction was collected. After dialysis, the collected active fractions were divided into 1
Adsorbed on DEAB Toyopearl 650S equilibrated with 10ff1 Tris-HCl buffer (pH 8).
I element was eluted in the same buffer using a concentration gradient of 0.1 to IM sodium chloride, and the active fractions were collected. The collected active fractions were dialyzed and then loaded onto a Sephacryl S-200 column equilibrated with the same buffer containing 0.1M NaCl.
It was eluted with the same tlNfr solution containing 1M sodium chloride, and the active fraction was collected. The collected active fractions were concentrated using an ultrafiltration membrane and then added to 1001M Tris-HCl buffer (pH 8).
was dialyzed overnight using Davis CDav for the alkaline pullulanase with α-amylase activity obtained.
is D, J,, ^nn, N, Y.

^cad, Sci, 121.404 (1964
) After performing electrophoresis according to the method of
It was confirmed that a single band was obtained by staining with brilliant blue (Figure 6).

(5) The alkaline pullulanase having α-amylase activity obtained in (4) was subjected to sodium dodecyl sulfate (SDS) electrophoresis according to a conventional method.

The results are shown in FIG. From this result, the molecular weight of this enzyme was 200.000±5.000.

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

(1) Washing conditions Washing machine used: Fully automatic dishwasher manufactured by Matsushita Electric (model NP)
-600) An aqueous cleaning solution is sprayed from a rotating nozzle to wash tableware placed on the upper surface of the spray trajectory.

Washing temperature: Gradually raise the temperature from 5°C to 55°C.

Washing water: Water with a hardness of 3.5° DH Washing concentration: 0.2% Washing time: 20 minutes of washing, 20 minutes of rinsing Amount of circulating water during washing: 2°51 (2) Evaluation of cleaning power Contaminated dishes with starch stains and Evaluation method (contaminated dish) Soft cooked rice was left at room temperature for 30 minutes, and 3g
The mixture was stretched and coated on a magnetic dish (diameter 25 cm), air-dried at room temperature overnight, and six plates each were washed.

(Starch stain cleaning power evaluation method) To determine the remaining starch, the blue area (P,) generated by the color reaction of iodine is measured by photojudgment, and the cleaning rate is determined from the initial contaminated area (So) using the formula below. Ta.

Cleaning rate = C (So P +) / S0] x 100 (
3) Cleaning composition Sodium citrate Sodium silicate No. 201 5 enzymes Table 5 or Table 6 Sodium carbonate Balance (Note) Numbers indicate weight %.

However, enzymes are indicated by their activity in detergents.

(4) Cleaning power test effect The test results are shown in Tables 5 and 6.

Table 5 Measurement was carried out by the Towako (CM-amylose/DBX method) method. IU is an enzyme unit in which an enzyme 100 decomposes exactly 10 μm of starch in 30 minutes at 37° C.

The enzyme activities in Table 5 are amylase, B-tesze The enzyme activities in Table 6 were measured by the following method.

Add 0.11nl of the enzyme solution to 0.9ml of a substrate solution containing pullulan (final concentration in the reaction system: 0.25%) dissolved in IJum buffer (pH 9, 0) (10mM glycine-salt-hydroxide). Add and react at 40°C for 30 minutes.

After the reaction, 3,5-dinitrosalicylic acid (3,5-din
itrosalicylic acid (DNS))
The amount of reducing sugar was determined using the method. That is, the reaction solution 1.0-
DNS reagent 1.0- was added to the solution, heated at 100° C. for 5 minutes to develop color, cooled, diluted with 4.0 Tnl of deionized water, and measured colorimetrically at a wavelength of 535 nm. The enzyme titer was defined as 1 unit (IU), which was the amount of enzyme that produced reducing sugar equivalent to 1 μl 10 1 of glucose per minute.

Example 2 (Clothing detergent) The cleaning conditions, detergency test, and results adopted in this example are as follows.

(1) Mix artificially contaminated cloth shiratama and cooked rice in a ratio of 9:1, dilute to 2 times with tap water, and add to a blender. This liquid is applied to a 10 cm x 10 cm test piece of cotton cloth in an amount of 2.5 to 5% of the weight of the cloth. It was dried at 20°C for 24 hours and used for experiments.

(2) Washing conditions and method Dissolve detergent in 4° DH hard water and prepare 0.665% detergent aqueous solution 11 (enzyme activity in aqueous solution 1.98X 10"U, #
) Prepare it. Five artificially contaminated cotton cloths were added to an aqueous detergent solution, and left to stand at 40°C for 1 hour. The detergent solution and artificially contaminated cloth were then transferred to a stainless steel beaker for a turbotometer, and heated at 1100 rpm, 20°C, 1 hour using a turbotometer.
Wash with stirring for 0 minutes. After rinsing under running water, 20℃, 2
It was dried for 4 hours and subjected to weight measurement.

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

Cleaning rate (%) = The value of each cleaning rate in Table 2 is shown as the average value of 5 sheets.

(4) Detergent composition Type 4A zeolite Sodium silicate Sodium carbonate Sodium polyacrylate (MW = 8000) Polyethylene glycol (MW = 6000) Fluorescent dye Fluorescent sulfate 0.5 Remaining amount The enzyme activity in Table 7 is Measured by the enzyme activity assay method shown in the table.

Table 8 (5) Cleaning power test results The test results are shown in Tables 7 and 8.

Table 7 (Note) Alkaline pullulanase was obtained in Production Example 1. The enzyme activity in Table 8 was measured by the enzyme activity measurement method in Table 6.

〔Effect of the invention〕

As described above, the cleaning composition of the present invention has excellent cleaning power against starchy stains within a normal cleaning time.

[Brief explanation of drawings]

Figure 1 shows the enzymatic reaction when pullulan, amylopectin, amylose, and glycogen were used as substrates for an enzymatic reaction using alkaline pullulanase having α-amylase activity.
Paper chromatography showing the production of maltooligosaccharides. FIGS. 2(a) and 2(b) are drawings showing the relationship between the reaction pH and relative activity of alkaline pullulanase having α-amylase activity. FIGS. 3(a) and 3(b) are drawings showing the relationship between the treatment pH and the residual activity of alkaline pullulanase having α-amylase activity. FIGS. 4(a) and 4(b) are drawings showing the relationship between the reaction temperature (pH 9.5) and relative activity of alkaline pullulanase having α-amylase activity. FIGS. 5(a) and 5(b) are drawings showing the relationship between treatment temperature (pH 9.5) and residual activity of alkaline pullulanase having α-amylase activity. FIG. 6 is a drawing showing the results of electrophoresis of alkaline pullulanase having α-amylase activity according to the Davis method. FIG. 7 is a drawing showing the results of SO3 electrophoresis of alkaline pullulanase having α-amylase activity. that's all

Claims (5)

[Claims] (1) A cleaning composition characterized by containing an alkali or alkali-resistant pullulanase having α-amylase activity. (2) The detergent composition according to claim 1, wherein the alkaline pullulanase has an optimal action pH on pullulan in the range of 8.5 to 10 and an optimal action pH on soluble starch in the range of 7 to 9.5. thing. (3) The cleaning composition according to claim 2, wherein the alkaline pullulanase has the following enzymatic properties. 1) Action Acts on pullulan and soluble starch, producing mainly maltotriose from pullulan and producing mainly maltotetraose and maltopentaose from soluble starch. It also acts on glycogen, producing maltotetraose and maltopentaose. 2) Substrate specificity Acts on pullulan, soluble starch and glycogen. 3) Action pH and optimum action pH The action pH for pullulan is in the range of 5-12, and the optimum action pH is in the range of 8.5-10. Further, the effective pH for soluble starch is in the range of 4 to 12, and the optimum pH is in the range of 7 to 9.5. 4) pH stability Stable for pullulan in the pH range of 6 to 10.5, and stable for soluble starch in the pH range of 4 to 12 (by treatment at 45° C. for 10 minutes). 5) Working temperature range and optimum working temperature It acts on pullulan and soluble starch in the range of 10 to 65°C, and its optimum working temperature is about 50°C. 6) Temperature stability Extremely stable up to 45°C. (10 m of pH 9.5
M glycine-saline-sodium hydroxide buffer by treatment for 30 minutes). 7) Molecular weight The molecular weight determined by sodium dodecyl sulfate (SDS) electrophoresis is 200,000±5,000. 8) Influence of metal ions on pullulanase activity: Hg^2^+, Mn^2^+, Pb
It is inhibited by ^2^+. In addition, α-amylase activity is
g^2^+, Mn^2^+, Pb^2^+, Zn^2^
+, inhibited by Cd^2^+. (4) The cleaning composition according to any one of claims 1 to 3, wherein the alkali or alkali-resistant pullulanase is isolated and obtained from a culture of a microorganism belonging to the genus Bacillus. (5) Microorganisms are Bacillus sp.
s¥sp. 5. The detergent composition according to claim 4, which is designated as KSM-AP1378 and deposited as Fiber Science and Technology Research Institute No. 10886.