JP2001522784A - Antibacterial activity of laccase - Google Patents

Abstract

(57) Abstract: A microorganism and / or antimicrobial treatment method of the virus, in the presence of oxygen O _2, comprising the step of treating with an effective amount fungi laccase and one or more enhancers, enhancer The method wherein the agent has formula (I).

Description

DETAILED DESCRIPTION OF THE INVENTION

FIELD OF THE INVENTION The present invention uses a combination of a laccase enzyme and an enhancer that can kill or inhibit microorganisms, especially in laundry, on hard surfaces, on skin, teeth or mucous membranes. The present invention relates to a method of antimicrobial treatment and to a composition for preserving foods, cosmetics, paints, coatings, etc., comprising a laccase enzyme, an enhancer acting as an electron donor.

BACKGROUND OF THE INVENTION Various enzymatic antimicrobial compositions are known in the art. For example, WO 94/04127 discloses a stabilized dentifrice composition capable of producing an effective antibacterial concentration of hypothianite ions. The composition contains an oxidase capable of producing hydrogen peroxide and a peroxidase enzyme capable of oxidizing thiocyanate ions normally present in saliva to antibacterial hypothiocyanite ions. Suitable peroxidases include lactoperoxidase, myeloperoxidase, salivary peroxidase and chloro-peroxidase.

[0003] European Patent Application No. 0 500 387 discloses an enzymatic antimicrobial composition comprising a haloperoxidase such as myeloperoxidase, eosinophil oxidase, lactoperoxidase and chloroperoxidase, in the presence of peroxide and halide. A composition that selectively binds to a target microorganism and suppresses its growth is disclosed. WO 95/27046 discloses an antimicrobial composition comprising vanadium chloroperoxidase, halide ions, and hydrogen peroxide or a hydrogen peroxide generator.

[0004] Laccase has the general formula:

Embedded image Is an enzyme that catalyzes the oxidation of a substrate by

[0005] Enzymes have been used in the paper and pulp industry (WO 94/29510) for bleaching in laundry washing (WO 91/05839, EP 91610032, DE4008894, JP-A-64-60693). However, use of the enzyme for antimicrobial purposes was not suggested.

[0006] WO 96/10079 discloses a general formula for bleaching dyes in solution:

Embedded image Discloses a method of oxidizing a compound with a phenol oxidase in the presence of an enhancer.

[0007] WO 97/28257 and WO 97/00038 disclose methods for inactivation of microorganisms or viruses by polyphenol oxidase obtained from bacteria of the genus Bacillus in the presence of oxygen and enhancers. . An object of the present invention is a method for the antimicrobial treatment of microbial cells or microorganisms, ie for disinfection or storage, a method which is easy to use and is an effective alternative to known disinfection and storage methods. It is to provide. SUMMARY OF THE INVENTION In the presence of oxygen (O ₂ ), an effective amount of a fungal laccase enzyme and an effective amount of one or more of the following formulas:

[0008]

Embedded image

[0009] The treatment of microorganisms and / or viruses comprising treating the microorganisms and / or viruses with an enhancer represented by the formula, surprisingly, exhibits a previously unknown synergistic antibacterial action. Was.

[0010] Therefore, based on these findings, the present invention provides, in a first aspect, oxygen (O _Two ) Or oxygen (O_Two) An effective amount of laccase in the presence of a source and said increase.
An enzymatic antimicrobial method comprising treating microorganisms and / or viruses with a strong agent
provide. The present invention, for example, food, beverages, cosmetics, contact lens products, food ingredients,
For antimicrobial treatment for preservation of paint or enzyme compositions; human or animal
For antibacterial treatment on skin, hair, mouth, mucous membranes, wounds, bruises or on eyes;
For antibacterial treatment of laundry; and related to hard surface cleaning or disinfection
Useful when antimicrobial treatment is required for antimicrobial treatment.

Thus, in a further aspect, the invention relates to the antimicrobial activity of microorganisms and / or viruses present in, for example, laundry in a washing machine and / or in a liquid used to soak, wash or rinse the laundry. Method of treatment, antimicrobial treatment of microorganisms and / or viruses on or in human or animal skin, hair, oral cavity, mucous membranes, teeth, eyes, wounds, bruises, antimicrobial of microorganisms and / or viruses on cosmetics or in cosmetics Provided are a method of treatment, a method of antimicrobial treatment of microorganisms and / or viruses on or in contact lenses, and a method of antimicrobial treatment of microorganisms and / or viruses present on or in hard surfaces.

DETAILED DESCRIPTION OF THE INVENTION Definitions In the context of the present invention, the term antimicrobial refers to bactericidal,
It should be understood as bacteriostatic, fungicidal or fungistatic. In the context of the present invention, it should be understood that the term "bactericidal" can kill bacterial cells. In the context of the present invention, the term "bacteriostatic" should be understood to be capable of inhibiting bacterial growth, ie inhibiting bacterial cell growth.

In the context of the present invention, it should be understood that the term “bactericidal” can kill fungal cells. In the context of the present invention, the term "fungal growth inhibiting" is to be understood as being able to inhibit fungal growth, ie to inhibit fungal cell growth. The term "microorganism" means a virus, bacterium or cell thereof, fungus or cell thereof. The term "hard surface" as used herein relates to any surface that is essentially impermeable to microorganisms. Examples of hard surfaces are metals, such as stainless steel alloys, plastic / synthetic polymers, rubber, boards, glass, timber,
Paper, textile, concrete, rock, marble, gypsum and ceramic materials,
These can be optionally coated with, for example, paint, enamel, polymer and the like.

Antimicrobial Activity Without being bound by this theory, it is believed that the major reaction in the antimicrobial activity of the combination laccase / enhancer system of the present invention is the oxidation of essential proteins and enzymes sulfhydryl groups or other cellular sites. Can be Laccase enzymes can catalyze the O ₂ -dependent oxidation of electron donors, such as enhancers. Oxidized enhancers perform an electrophilic attack on microbial components, causing chemical modification of essential enzymes, transport systems and other functional components.

Sulfhydryl groups are particularly susceptible to electrophilic attack and are usually present in higher amounts than other easily oxidized groups. Most aspects of antimicrobial activity can correlate with chemical modification of these components. Antimicrobial activity is encouraged by effects that increase the stability of the oxidized material, provided that these effects do not interfere with their electrophilic properties or their ability to penetrate antimicrobial membranes. O ₂ itself is an oxidizing agent, but the O ₂ molecule is stabilized and only reacts slowly with biological material. Laccase oxidation catalyzed by the enhancer preserves the oxidizing power O ₂ to form more readily react.

The reaction catalyzed by laccase is shown as follows: n · O ₂ + m · AH _p → 2 · n · H ₂ O + m · A, mp = 4 (where AH _p and A Are the oxidized and reduced forms of a suitable electron donor);
Alternatively, the following equation:

[0017]

Embedded image

In the case of the enhancers / agents of the invention, the oxygen can be supplied from the air by diffusion or by aeration or can be generated by oxygen-releasing compounds. The antimicrobial action can be obtained against various microorganisms such as bacteria, fungi and / or viruses. In certain embodiments of the invention, the antimicrobial effect is on one strain over another strain, e.g., on bacteria over fungi or vice versa, or on positive bacteria over gram-negative bacteria or vice versa. Greater than.

Enzymes The following enzymes, especially recombinant enzymes and / or substantially purified enzymes, are preferred. In the context of the present invention, "laccase" includes enzymes that fall under the enzyme category EC 1.10.3.2.

Preferably, the laccase used is like a species of the genus Polyporus, in particular like Polyporus pinisitus or Polyporus versicolor, or Mycelioptera
hthera) species, such as M. thermophira, or Rhizoctonia species, especially Rhizoctonia praticola or Rhizoctonia solani
) Or species of the genus Rhus, especially Rhus vernifera (Rhus verni)
cifera).

In a particular embodiment of the invention, the oxidoreductase is a laccase, for example a laccase of the genus Polyporus, in particular the Polyporus pinisitus laccase [Trametez laccase described in WO 96/00290.
Virosa (also called Trametes villosa) laccase) (Novo Nordisk Biote
laccase of the genus Myseliophtera, especially WO 95/33836
Myceliophthera thermophila as described in
) Laccase (Novo Nordisk Biotech., Inc.).

Further, the laccase is a laccase of the genus Scytalidium,
For example, Citadium thermophilium described in WO 95/33837 (S. the
laccase (Novo Nordisk Biotech., Inc.) or a laccase of the species of the genus Pyricularia, for example, SI under the trade name SIGMA No. L5510.
Pyricularia oryzae laccase available from GMA, or laccase of the species of the genus Corprinus, such as C. cinereus laccase, especially C. cinereus.
su) IFO 30116 laccase, or a laccase of the species Rhizoctonia, such as Rh. solani laccase, in particular WO 95/07
Neutral rhizoctonia having an optimum pH in the range of 6.0 to 8.5 described in 988
Solani (Rh. Solani) laccase (Novo Nordisk A / S).

Laccase is a fungus such as Collybia, Fomes, Lentinus, Pleurotus, Aspergill.
illus), Neurospora, Podospora, Phlebia, such as P. radiata (WO 92/01046)
), Species of the genus Coriolus, for example, C. hirsi.
tus) or Botrytis.

[0024] Various enhancers are known to act as an electron donor to laccase for enhancer various purposes (e.g., WO 94/12620, WO 94/12621, WO 95/01626 and
WO 96/00179). The following formula:

[0025]

Embedded image The one or more enhancers that have, however, are, however, in the context of the present invention:
It has been shown to be surprisingly effective.

In a preferred embodiment of the present invention, the letter A in the above formula is -D, -CH = CH
-D, -CH = CH-CH = CH-D, -CH = ND, -N = ND or-
N = CH-D [where D is -CO-E, -SO_Two-E, -N-XY and -N ⁺ -XYZ wherein E is -H, -OH, -R or -OR, and X and
And Y and Z may be the same or different and are selected from -H and -R
, R is C₁~ C₁₆Alkyl, preferably C₁~ C₈Of this
An alkyl is a saturated or unsaturated branched or unbranched chain, and optionally
Which may be substituted with a xy, sulfo or amino group).
And B and C may be the same or different;
And C_mH_{2m + 1}(Where m = 1, 2, 3, 4 or 5).

In the above formula, A may be located at the meta position, as shown, instead of at the para position relative to the hydroxy group. In certain embodiments of the invention, the enhancer has the formula:

[0028]

Embedded image

Wherein A is a group such as —H, —OH, —CH ₃ , —OCH ₃ , —O (CH ₂ ) nCH ₃ , and n = 1, 2, 3, 4, 5, 6, 7, or 8]. Said combinations of enhancers are suitably applied sequentially or simultaneously and may have further synergistic effects if different microorganisms can exhibit a particular sensitivity to a particular enhancer. Thus, a particular embodiment of the invention comprises a laccase enzyme (EC 1.10.3.2) and the formula:

[0030]

Embedded image

An antimicrobial composition comprising at least two different enhancers, wherein A, B and C may be substituents as described above, wherein the laccase and the enhancer are in an antimicrobial effective amount With respect to the composition present in the composition. The composition can be a solid particulate or powder formulation, or it can be a liquid. Solid particulate compositions are disclosed in U.S. Pat.No. 4,106,991, U.S. Pat.No. 4,661,452, WO 97/31088 or WO 95/33.
039 and optionally coated by methods known in the art.

Liquid compositions can be prepared as stabilized liquid compositions by the addition of conventional stabilizers, as slurry compositions, or as compositions having enzymes in a protected form. Protected enzymes can be prepared by the methods disclosed in European Patent Application No. 238,216 or WO 97/41215. Such enhancers are commercially available or can be prepared by methods known in the art.

Effective amount of laccase Laccase is a medium used in antimicrobial treatment, ie, a washing liquid, cosmetic, food or beverage, or a medium used for antimicrobial treatment of other items, ie, washing liquid, disinfectant. 0.00001 to 100 mg / L, preferably 0.001 to 10
mg / L, for example from 0.1 to 5 mg / L.

Effective amount of enhancer The enhancer is a medium used in antimicrobial treatment, ie, a laundry liquor, cosmetic, food or beverage, or a medium used for antimicrobial treatment of other items, ie, a wash liquor, It may be present in disinfecting solutions, preservatives, footbaths and the like at 0.00001 to 500 mM, preferably 0.0001 to 5 mM, for example 0.001 to 0.050 mM.

The medium in which the catalytic reaction between the laccase and the enhancer is intended to take place is preferably aqueous and can be a liquid, aerosol, gel, paste or slurry depending on the intended use of the invention. . The laccase and enhancer may be included separately or together in a solid formulation intended for the preparation of the aforementioned media. The antimicrobial efficacy of the present invention depends, inter alia, on the medium, which can be adapted to the intended use of the present invention. The properties to be adapted are, inter alia, the solubility or mobility of the laccase, enhancer and / or O ₂ , the rate of the catalytic reaction, which also depends, for example, on the pH, the temperature and the buffer of the medium.

[0036] The medium can also affect the half-life of the radicals generated by the enhancer upon catalysis with laccase and O ₂ . Without being limited to any theory, it is anticipated by the present invention that a positive correlation is observed between the half-life of a radical in a medium and its potency. The half-life of the radical depends, inter alia, on the pH of the medium, the temperature and the buffer. The medium may contain auxiliaries such as wetting agents, thickening agents, buffers, stabilizers, fragrances, coloring agents, fillers, chelating agents and the like (eg, from detergent compositions). Useful wetting agents are surfactants, ie, nonionic, anionic, amphoteric or zwitterionic surfactants.

As mentioned above, laccases are well suited in the context of the present invention because, among other reasons, they catalyze the oxidation by molecular oxygen. Thus, the reaction takes place in a vessel in which the atmosphere is free (or, in other reactions, in which air, or another oxygen-containing gas is introduced), and in which the reaction using oxidase as an enzyme is carried out as an oxidant. Gaseous oxygen is available, however, it is desirable to forcibly aerate the liquid medium during the reaction to ensure proper supply of oxygen.

[0038] by pH among others laccase and properties of enhancing agents used in the medium, pH of the medium used is usually 5 to 11, often 6 to 10, should be in the range of for example 6.5 to 8.5 preferably . Temperature in the Medium In many embodiments of the present invention, a temperature in the range of 10-65 ° C, more preferably 30-50 ° C, should be used.

Processing Time The processing time depends, inter alia, on the type of treatment, the type of item being treated, the characteristics of the medium, such as temperature and pH, and the type and amount of enzymes and enhancers used. For storage purposes, processing time can range with the expected life of the item being stored. For disinfection purposes, treatment times ranging from 1 to 120 minutes may be used. In many cases, processing times in the range of 5 to 20 minutes are appropriate.

Processing of Laundry As mentioned above, the present invention relates to microorganisms or viruses present on the laundry, for example in a washing machine, and / or in the liquid used to soak, wash or rinse the laundry. Provide a method for antimicrobial treatment of Laccases and enhancers can be incorporated into the detergent composition.

Surfactant System of the Detergent Composition The detergent composition of the present invention contains a surfactant system, wherein the surfactant is non-ionic and / or anionic and / or cationic and / or cationic. And / or zwitterionic and / or zwitterionic and / or semipolar surfactants. Surfactants are typically present at a level of 0.1% to 60% by weight.

[0042] Surfactants are preferably formulated to be compatible with the enzyme components present in the composition. In liquid or gel compositions, the surfactant is most preferably formulated such that it promotes, or at least does not reduce, the stability of any enzymes in these compositions. Preferred systems for use according to the present invention include one or more nonionic and / or anionic surfactants described herein as surfactants.

Polyethylene, polypropylene and polybutylene oxide condensates of alkylphenols are suitable for use as the nonionic surfactant of the surfactant system of the present invention, with polyethylene oxide condensates being preferred. These compounds include the condensation products of alkyl phenols with alkyl phenols having an alkyl group of about 6 to about 14, preferably about 8 to about 14, carbon atoms in straight or branched chain form. In a preferred embodiment, the ethylene oxide is present in an amount equal to about 2 to about 25 moles, more preferably about 3 to about 15 moles of ethylene oxide per mole of alkylphenol.

Commercially available nonionic surfactants of this type include Igepal ™ CO-630 (
GAF Corporation), Triton ™ X-45, X-114, X-100 and X-102 (all Rohm & Haas Company). These surfactants are commonly referred to as alkylphenol alkoxylates (eg, alkylphenol ethoxylates).

The condensation products of primary and secondary aliphatic alcohols with about 1 to about 25 moles of ethylene oxide are useful for use as nonionic surfactants in the nonionic surfactant systems of the present invention. Are suitable. The alkyl chain of the aliphatic alcohol can be straight or branched, primary or secondary, and generally has from about 8 to about 22 carbon atoms. Preferred are condensation products of alcohols having alkyl groups having about 8 to about 20, more preferably about 10 to about 18, carbon atoms and having about 2 to about 10 moles of ethylene oxide per mole of alcohol. About 2 to about 7 moles, most preferably 2 to 5 moles, of ethylene oxide are present in the condensation product per mole of alcohol.

Examples of commercially available nonionic surfactants of this type include Tergitol ™
15-S-9 (condensation product of a C ₁₁ -C ₁₅ linear alcohol with 9 moles of ethylene oxide), Tergitol ™ 24-L-6 (6 moles of a C ₁₂ -C ₁₄ primary alcohol with Is a condensation product with ethylene oxide and has a narrow molecular weight distribution.
Neodol ™ 45-9 (condensation product of a C ₁₄ -C ₁₅ linear alcohol with 9 moles of ethylene oxide), Neodol ™ 23-3 (C ₁₂ -C ₁₃ linear alcohol) With 3.0 moles of ethylene oxide),

Neodol ™ 45-7 (condensation product of a C ₁₄ -C ₁₅ linear alcohol with 7 moles of ethylene oxide), Neodol ™ 45-5 (a C ₁₄ -C ₁₅ linear alcohol with Condensation product with 5 moles of ethylene oxide) (Shell Chemical Company),
Km (TM) EOB (the condensation product of alcohol with 9 moles ethylene oxide _{C 13 ~C 15) (Procter &} Gamble Company), and Genapol LA 050 (C ₁₂ ~
Condensation products of alcohols with 5 moles of ethylene oxide C ₁₄₎ (Hoechst) and the like. The preferred range of HLB for these products is 8-11, most preferred
8-10.

Further useful as nonionic surfactants in the surfactant system of the present invention are those disclosed in US Pat. No. 4,565,540, having about 6 to about 30, preferably about 10 to about 16, carbon atoms and having hydrophobic groups.
No. 647, as well as from about 1.3 to about 10, preferably from about 1.3 to about 10.
Polysaccharides having a hydrophilic group containing about 3, most preferably about 1.3 to about 2.7 saccharide units, such as polyglycosides. Any reducing sugar having 5 or 6 carbon atoms can be used.

For example, the glucose, galactose and galactosyl moieties can be replaced in place of the glucosyl moieties (optionally, the hydrophobic group is attached at 2-, 3-, 4-etc. Yields glucose or galactose). The intersugar linkage may be, for example, between a position of the additional saccharide unit and the 2-, 3-, 4- and / or 6-position on said saccharide unit.

Preferred alkyl polyglycosides have the following formula: R ² O (C _n H _2n O) _t (glycosyl) _x , wherein R ² is alkyl, alkylphenyl, hydroxyalkyl, hydroxyalkylphenyl and mixtures thereof. Selected from the group consisting of:
The alkyl group has about 10 to about 18, preferably about 12 to about 14, carbons and n is 2 or 3
, Preferably 2, t is from 0 to about 10, preferably 0, and x is about 1.3.
From about 1.3 to about 3, preferably from about 1.3 to about 3, and most preferably from about 1.3 to about 2.7).

Glycosyl is preferably obtained from glucose. To prepare these compounds, an alcohol or alkyl polyethoxy alcohol is first formed and then reacted with glucose or a source of glucose to form a glucoside (attached at the 1-position). The additional glucosyl units are then in their 1-position and the aforementioned glycosyl units 2-, 3-, 4- and / or 6-position, preferably preferentially 2
-Can be coupled to a position.

[0052] Condensation products of ethylene oxide with a hydrophobic base formed by the condensation of propylene oxide with propylene glycol are also suitable for use as the additional nonionic surfactant system of the present invention. The hydrophobic portion of these compounds preferably has a molecular weight of about 1500 to about 1800 and is water-insoluble. The addition of a polyoxyethylene moiety to this hydrophobic moiety increases the water solubility of the molecule as a whole, maintaining the liquid character of the product to the point that the polyethylene content is about 50% of the total weight of the condensation product This corresponds to a condensation with up to about 40 moles of ethylene oxide. Examples of these types of compounds include some commercially available Pluronic ™ surfactants (
BASF).

Also suitable for use as the nonionic surfactant of the nonionic surfactant system of the present invention are the condensation products of ethylene oxide with substances resulting from the reaction of propylene oxide and ethylenediamine. is there. The hydrophobic portion of these products consists of the reaction product of ethylenediamine and an excess of propylene oxide and generally has a molecular weight of about 2500 to about 3000. The hydrophobic moiety contains from about 40% to about 80% polyoxyethylene by weight of the condensation product, and from about 5,000 to
Condensed with ethylene oxide to have a molecular weight of about 11,000. Examples of this type of nonionic surfactant include some commercially available Tetronic ™ compounds (BASF).

Preferred for use as nonionic surfactants in the surfactant system of the present invention are polyethylene oxide condensates of alkyl phenols, primary and secondary aliphatic alcohols with about 1 to about 25 moles. Condensation products with ethylene oxide, alkyl polysaccharides and mixtures thereof. Most preferred are three to five alkylphenol ethoxylates C ₈ -C ₁₄ having ethoxy group, and two to ten of alcohol ethoxylates of C ₈ -C ₁₈ having ethoxy group (preferably, an average C ₁₀ ), As well as mixtures thereof. Highly preferred nonionic surfactants have the formula:

[0055]

Embedded image

(Wherein R¹Is H or R¹Is C_1-4Hydrocarbyl, 2-hydr
Roxyethyl, 2-hydroxypropyl or a mixture thereof;^TwoIs C _5-31 And Z is at least three hydroxyl groups.
Having a linear hydrocarbyl chain directly attached to the chain
Ruby or its alkoxylated derivatives)
It is a surfactant. Preferably, R¹Is methyl and R^TwoIs linear C_11-15No
Lucil or C_16-18Alkyl or alkenyl chain of, for example, coconut
Or a mixture thereof, and Z is reduced in a reductive amination reaction.
Derived from source sugars such as glucose, fructose, maltose or lactose
It is.

Highly preferred anionic surfactants include alkyl alkoxylated sulfate surfactants. This example has the formula RO (A) mSO ₃ M, where
R is a hydroxyalkyl group having an alkyl component of the alkyl or C ₁₀ -C ₂₄ unsubstituted C ₁₀ -C _24, preferably an alkyl or hydroxyalkyl of C ₁₂ -C _20, more preferably alkyl of C ₁₂ -C ₁₈ Or hydroxyalkyl, A is an ethoxy or propoxy unit, m is greater than zero, typically from about 0.5 to about 6, preferably from about 0.5 to about 3, and M is H or, for example, a metal cation. A water-soluble salt or acid of an ion (eg, a cation that can be an ammonium or substituted ammonium cation) such as sodium, potassium, lithium, calcium, magnesium, and the like.

Alkyl ethoxylated sulfates, as well as alkyl propoxylated sulfates, are contemplated herein. Particular examples of substituted ammonium cations include methyl-, dimethyl, trimethyl-ammonium cations, and quaternary ammonium cations, such as tetramethyl-ammonium and dimethylpiperazinium cations, and alkylamines, such as ethylamine, Examples thereof include those obtained from diethylamine, triethylamine, a mixture thereof, and the like.

Exemplary surfactants include C ₁₂ -C ₁₈ alkyl polyethoxylate (1.0) sulfate (C ₁₂ -C ₁₈ E (1.0) M), C ₁₂ -C ₁₈ alkyl polyethoxylate (2.25 ) sulfate (of _{C 12 ~C 18 (2.25) M} ), and alkyl polyethoxylate of C ₁₂ -C ₁₈ (3.0) sulfate (E of _{C 12 ~C 18 (3.0) M} )
, And C ₁₂ alkyl polyethoxylate (4.0) of the -C ₁₈ sulfate (C ₁₂ -C ₁₈ of E (4.0) M) is (here, M is is conveniently selected from sodium and potassium) .

Suitable anionic surfactants used are alkyl ester sulfonate surfactants, such as “The Journal of the American Oil Chemists Society”, 52
(1975), a linear ester pp. 323-329 in accordance with carboxylic acids of C ₈ -C ₂₀ which are sulfonated with gaseous SO ₃ (i.e., fatty acids). Suitable starting materials include
Natural fat substances obtained from tallow, coconut oil and the like are included.

Preferred alkyl ester sulfonate surfactants, especially for laundry applications, include:
The following structural formula:

Embedded image

Wherein R ³ is a C ₈ -C ₂₀ hydrocarbyl, preferably alkyl or a combination thereof, and R ⁴ is a C ₁ -C ₆ hydrocarbyl, preferably alkyl,
Or a combination thereof, and M is a cation that forms a water-soluble salt with the alkyl ester sulfonate). Suitable salt forming cations include metals such as sodium, potassium and lithium, and substituted or unsubstituted ammonium cations such as monoethanolamine, diethanolamine and triethanolamine. Preferably, R ³ is C ₁₀ -C ₁₆ alkyl and R ⁴ is methyl, ethyl or isopropyl. Particularly preferred are methyl ester sulfonates wherein R ³ is C ₁₀ -C ₁₆ alkyl.

Other suitable cationic surfactants include those having the formula ROSO ₃ M, wherein R is preferably a C ₁₀ -C ₂₄ hydrocarbyl, preferably a C ₁₀ -C ₂₀ alkyl component. Alkyl or hydroxyalkyl, more preferably C ₁₂ -C ₁₈ alkyl or hydroxyalkyl, and M is H or a cation, such as an alkali metal cation (eg, sodium, potassium, lithium), or ammonium or substituted ammonium (Eg, methyl-, dimethyl- and trimethylammonium) cations, and

Aqueous solution of quaternary ammonium cations such as tetramethyl-ammonium and dimethylpiperidinium cations, and quaternary ammonium cations obtained from alkylamines such as ethylamine, diethylamine, triethylamine and mixtures thereof. And alkyl sulfate surfactants that are acidic salts or acids. Typically, C ₁₂ -C ₁₆ alkyl chains are preferred for low wash temperatures (eg, below about 50 ° C.), and C ₁₆ -C ₁₈ alkyl chains for high wash temperatures (eg, above about 50 ° C.). Is preferred.

Other anionic surfactants useful for cleaning purposes may also be included in the laundry detergent compositions of the present invention. Examples of these include salts (e.g., sodium, potassium, ammonium and substituted ammonium salts such as mono-, - di - and triethanolamine salts), primary or secondary C ₈ -C ₂₂ alkane sulfonates, olefin sulfonates of C ₈ -C _24, for example, British Patent specification No. 1,082,
_C8-24 alkyl polyglycol ether sulfonates (up to 10 moles of ethylene oxide) prepared by sulfonation of the pyrolysis products of alkaline earth metal citrates as described in Containing),
Alkyl glycerol sulfonate, fatty acyl glycerol sulfonate,

Fatty oleyl glycerol sulfate, alkyl phenol ethylene oxide ether sulfate, paraffin sulfonate, alkyl phosphate, isethionates such as acyl isethionate, N-acyl taurate, alkyl succinates and sulfosuccinates, sulfosuccinates monoester (particularly saturated and unsaturated monoesters of C ₁₂ -C ₁₈₎ and sulfosuccinates diesters of succinate (especially diesters of saturated and unsaturated C ₆ -C _12), acyl sarcosinates, sulfates of alkylpolysaccharides,

For example, sulfates, branched primary alkylsulfates, alkylpolyethoxycarboxylates of alkyl polyglycosides (the following non-ionic non-sulfated compounds), such as the formula RO (CH ₂ CH ₂ O) _k -CH ₂ COO ^- M ⁺ (wherein, R is an alkyl C ₈ -C _22, k is an integer of 0 - 10, and M is a soluble salt-forming cation) include those having a. Resin acids and hydrogenated resin acids are also suitable, examples include, for example, rosin, hydrogenated rosin and resin acids and hydrogenated resin acids present in or obtained from tall oil. Can be

[0068] Alkylbenzenesulfonates are highly preferred. Particularly preferred is a linear (linear) alkyl benzene sulfonate (LAS) in which the alkyl group preferably has 10 to 18 carbon atoms. A further example is "Surface Active Agents and Detergents" (Vol. I and II by
Schwartz, Perry and Berch). A variety of such surfactants are generally described in U.S. Pat. No. 3,929,678 (Laughlin et al., Column 23, line 58 to column 29, line 23; 1975).
Issued on December 30, 2012). When included therein, the laundry detergent compositions of the present invention typically contain from about 1% by weight to
About 40%, preferably about 3% to about 20%, by weight of such anionic surfactants are included.

The laundry detergent compositions of the present invention may include cationic, amphoteric, amphoteric and semi-polar surfactants, and non-ionic and / or anionic other than those described herein. Surfactants may also be included. Cationic surfactants suitable for use in the laundry detergent compositions of the present invention are those having long chain hydrocarbyl groups. Examples of such cationic surfactants include ammonium surfactants, such as alkyltrimethylammonium halides, and surfactants having the formula:

[R ² (OR ³ ) _y ] [R ⁴ (OR ³ ) _y ] ₂ R ⁵ N ⁺ X ⁻ (wherein, R ² is alkyl having about 8 to about 18 carbon atoms in the alkyl chain or An alkylbenzene group, and R ³ is —CH ₂ CH ₂ —, —CH ₂ CH (CH ₃ ) —
_{, -CH 2 CH (CH 2 OH} ) -, - CH 2 CH 2 CH 2 - and is selected from the group consisting of mixtures thereof, each of R ^4, the C ₁ -C ₄ alkyl, a C ₁ -C ₄ Hydroxyalkyl, a benzyl ring structure formed by linking two R ⁴ groups, —CH ₂ CHOHCHOHCOR ⁶ CHOHCH ₂ OH, where R ⁶ is any hexose or hexose polymer having a molecular weight of less than about 1000. And when y is not 0, it is hydrogen, and R ⁵ is the same as R ⁴ or is an alkyl chain, wherein the total number of carbon atoms of R ² + R ⁵ is 18 or less, and y is Each is 0-10, the sum of the y values is 0-15, and X is any compatible cation).

[0071] Highly preferred cationic surfactants are useful water-soluble quaternary ammonium compound in the compositions of the present invention having the _{formula: R 1 R 2 R 3 R} 4 N + X - ( i) wherein R ₁ is C ₈ -C ₁₆ alkyl and R ₂ , R ₃ and R ₄ are independently C ₁ -C ₄ alkyl, C ₁ -C ₄ hydroxyalkyl, benzyl and −
_{(C 2 H 40) x H} ( wherein, x has a value of 2-5, and X is an anion). No more than one of R ₂ , R ₃ or R ₄ should be benzyl.

Preferred alkyl chain lengths for R ₁ are C ₁₂ -C ₁₅ , especially where the alkyl group is a chain length obtained from coconut or palm kernel fat, or synthetically by olefin amplification or OXO alcohol synthesis. The resulting mixture of chain lengths. Preferred groups for R ₂ , R ₃ and R ₄ are methyl and hydroxyethyl groups, and the anion X may be selected from halide, methosulfate, acetate and phosphate ions.

Examples of suitable quaternary ammonium compounds of formula (i) for use herein are: coconut trimethylammonium chloride or bromide; coconut methyldihydroxyethylammonium chloride or bromide; decyltriethylammonium chloride _{Decyldimethylhydroxyethylammonium} chloride or bromide; C12-15 dimethylhydroxyethylammonium chloride or bromide; coconut dimethylhydroxyethylammonium chloride or bromide; myristyltrimethylammonium methyl sulfate; lauryldimethylbenzylammonium chloride or bromide; lauryldimethyl ( Ethenoxy) ₄ ammonium chloride or bromide;

The choline ester (R ¹ is:

Embedded image And R ₂ R ₃ R ₄ is methyl. Compound of formula (i). Di-alkylimidazoline [compound of formula (i)].

Other cationic surfactants useful herein are described in US Pat. No. 4,228,044
And EP 000 224. When included therein, the laundry detergent compositions of the present invention typically contain from 0.2% by weight to
About 25%, preferably from about 1% to about 8%, by weight of such cationic surfactants are included.

[0076] Amphoteric surfactants are also suitable for use in the laundry detergent compositions of the present invention. These surfactants can be broadly described as aliphatic derivatives of secondary or tertiary amines or as aliphatic derivatives of heterocyclic secondary and tertiary amines, where the aliphatic groups are linear. Or it may be branched. One of the aliphatic substituents has at least 8 carbons, typically from about 8 to about 18 carbons, and at least one contains an anionic water solubilizing group, e.g., carboxy, sulfonate, sulfate. I do. For examples of amphoteric surfactants, see, for example, U.S. Patent No. 3,929,678 (col. 19, lines 18-35).

When included therein, the laundry detergent compositions of the present invention typically contain from 0.2% to about
15% by weight, preferably from about 1% to about 10% by weight of such amphoteric surfactants are included. Zwitterionic surfactants are also suitable for use in laundry detergent compositions. These surfactants can be used as derivatives of secondary or tertiary amines, or as derivatives of heterocyclic secondary and tertiary amines, or as derivatives of quaternary ammonium, quaternary phosphonium or tertiary sulfonium compounds. Can be described extensively.
For examples of zwitterionic surfactants, see, for example, US Pat. No. 3,929,678 (column 19).
Line 38 to column 22, line 48). When included therein, the laundry detergent compositions of the present invention typically comprise from 0.2% by weight to about
15% by weight, preferably from about 1% to about 10% by weight of such zwitterionic surfactants are included.

Semipolar nonionic surfactants are a special category of nonionic surfactants, such as one alkyl moiety having about 10 to about 18 carbons and about 1 to about carbon atoms. A water-soluble amine oxide containing two moieties selected from the group consisting of an alkyl group of 3 and a hydroxyalkyl group; one alkyl moiety having about 10 to about 18 carbon atoms; and an alkyl moiety having about 1 to about 3 carbon atoms. A water-soluble phosphine oxide containing two moieties selected from the group consisting of
Water-soluble sulfoxides containing one alkyl moiety and one moiety selected from the group consisting of alkyl and hydroxyalkyl groups having about 1 to about 3 carbon atoms. The semipolar nonionic detersive surfactant includes the following formula:

[0079]

Embedded image

(Wherein R^ThreeIs an alkyl, hydroxyalkyl, or alkyl having about 8 to about 22 carbon atoms.
A phenyl group or a mixture thereof;^FourIs a carbon having about 2 to about 3 carbon atoms.
A alkylene or hydroxyalkylene group, or a mixture thereof, wherein x is
0 to about 3 and R^FiveEach represents an alkyl or hydro-hydrogen having from about 1 to about 3 carbon atoms.
Polyesters containing xyalkyl groups or about 1 to about 3 ethylene oxide groups
Amine oxide surfactants having a tylene oxide group). R ^Five The groups are linked together, for example, via an oxygen or nitrogen atom to form a ring structure
obtain.

These amine oxide surfactants include, in particular, C ₁₀ -C ₁₈ alkyldimethylamine oxides and C ₈ -C ₁₂ alkoxyethyldihydroxyethylamine oxides. When included therein, the laundry detergent compositions of the present invention typically comprise from 0.2% by weight to about
15% by weight, preferably from about 1% to about 10% by weight of such semipolar nonionic surfactants.

Builder System The composition of the present invention may further contain a builder system. Any conventional builder system is suitable for use herein, examples of which include aluminosilicate materials, silicates, polycarboxylates and fatty acids, materials such as ethylenediaminetetraacetic acid, sequestering agents such as Aminopolyphosphonates, especially ethylenediaminetetramethylenephosphonic acid and diethylenetriaminepentamethylenephosphonic acid. Although less preferred for obvious environmental reasons, phosphate builders may also be used herein.

Suitable builders are inorganic ion exchange materials, generally inorganic aluminosilicate hydrates, especially synthetic zeolite hydrates, such as zeolites A, X, B, HS or M
It may be AP hydrate. Another suitable inorganic builder material is a layered silicate, such as SKS-6 (Hoechst)
It is. SKS-6 is a crystalline layered silicate composed of sodium silicate (Na ₂ Si ₂ O ₅ ).

Suitable polycarboxylates containing one carboxy group include lactic acid, glycolic acid and their ether derivatives as disclosed in Belgian Patent Nos. 831,368, 821,369 and 821,370. No. Polycarboxylates containing two carboxy groups include succinic acid, malonic acid, (ethylenedioxy) diacetate, maleic acid, diglycolic acid, tartaric acid, tartronic acid and water-soluble salts of fumaric acid, and German Offenle -enschrift 2,446,686 and 2,446,487, ether carboxylates as described in U.S. Pat. No. 3,935,257, and sulfinyl carboxylates as described in Belgian Patent 840,623.

[0085] Polycarboxylates containing three carboxy groups include, in particular, water-soluble citrates, aconitrates and citraconates, as well as GB 1,379,
Succinate derivatives such as carboxymethyloxysuccinate described in US Pat. No. 241; lactoxysuccinate described in Dutch Patent Application No. 7205873; and 2-lactide described in British Patent No. 1,387,447. Oxa-1,
Oxypolycarboxylate materials such as 1,3-propanetricarboxylate are mentioned.

[0086] Polycarboxylates containing four carboxy groups include those disclosed in British Patent No. 1,
Oxydisuccinates described in U.S. Pat. No. 261,829 include 1,1,2,2-ethanetetracarboxylate and 1,1,3,3-propanetetracarboxylate containing a sulfo substituent, Nos. 1,398,421 and 1,398,422, and the sulfosuccinate derivatives disclosed in U.S. Pat.No. 3,936,448, and the sulfonated pyrolytic citrates described in British Patent 1,082,179, while Polycarboxylates containing phosphon substituents are
It is disclosed in British Patent 1,439,000.

The alicyclic and heterocyclic polycarboxylates include cyclopentane-cis, cis-tetracarboxylate, cyclopentadienidepentacarboxylate, 2,3,4,5-tetrahydrofuran-cis, cis, cis Tetracarboxylate, 2,5-tetrahydrofuran-cis, discarboxylate, 2,2,5,5-tetrahydrofuran-tetracarboxylate, 1,2,3,4,5,6-hexane-hexacarboxylate and Mention may be made of the carboxymethyl derivatives of polyhydric alcohols such as sorbitol, mannitol and xylitol. Aromatic polycarboxylates include the derivatives of melitic acid, pyromellitic acid and phthalic acid disclosed in GB 1,425,343.

Among the above, preferred polycarboxylates are hydroxycarboxylates containing up to 3 carboxy groups per molecule, especially citrates. Preferred builder systems for use in the compositions of the present invention include water-insoluble aluminosilicate builders, such as zeolite A, or layered silicates (SKS-
6) and water-soluble carboxylate chelating agents, such as mixtures of citric acid.

[0090] Chelating agents suitable for inclusion in the detergent compositions of the present invention include ethylenediamine.
-N, N‘-disuccinic acid (EDDS) or their alkali metals and alkalis
Earth metal, ammonium or substituted ammonium salts, or mixtures thereof
is there. Preferred EDDS compounds are the free acid forms and their sodium or mag
Nesium salt. Examples of such preferred sodium salts of EDDS include Na _Two EDDS and Na_FourEDDS. Such preferred magnesium in EDDS
Examples of salts include MgEDDS and Mg_TwoEDDS. Magnesium salts
Most preferred for inclusion in the compositions of the present invention.

Preferred builder systems include water-insoluble aluminosilicate builders, such as a mixture of zeolite A and a water-soluble carboxylate chelating agent, such as citric acid. Other builder substances that may form part of the builder system for use in the particulate composition include alkali metal carbonates, bicarbonates, inorganic substances such as silicates,
And organic substances such as organic phosphates, aminopolyalkylene phosphates and aminopolycarboxylates. Other suitable water-soluble organic salts are homo- or copolymer acids or salts thereof, wherein the polycarboxylic acid contains at least two carboxyl groups separated from each other by no more than two carbon atoms. I do.

A polymer of this type is disclosed in GB-A-1,596,756. Examples of such salts are polyacrylates having a molecular weight of 2000 to 5000, as well as copolymers thereof with maleic anhydride, such substances having a molecular weight of 20,000 to 70,000, in particular about 40,000. The detergency builder salt is usually included in an amount of 5% to 80% by weight of the composition. Preferred levels of builders for liquid detergents are between 5% and 30%.

Enzymes Preferred detergent compositions include, in addition to the enzyme preparations of the present invention, other enzyme (s) that provide cleaning performance and / or fabric protection. Such enzymes include proteases, lipases, cutinases, amylases, cellulases, peroxidases and other oxidases (eg laccase)
Is mentioned.

Proteases : Any protease suitable for use in alkaline solutions can be used. Suitable proteases include those of animal, plant or microbial origin. Bacterial origin is preferred. Includes chemically or genetically modified mutants. The protease may be a serine protease, preferably an alkaline microbial protease or a trypsin-like protease. Examples of alkaline proteases are subtilisins, especially subtilisins from the genus Bacillus, such as subtilisin novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168 (as described in WO 89/06279). Examples of trypsin-like proteases are trypsin (eg, of porcine or bovine origin), and WO
89/06279.

Preferred commercially available protease enzymes include Novo Nordisk A / S (Denmar) under the trade names Alcalase, Sabinase, Primase, Durazyme and Esperase.
k), sold by Genencor International under the trade name Maxatase, Maxacal, Maxapen, Properase, Perfect and Perfect OXP
al. and those sold by Solvay Enzymes under the trade names Opticlean and Optimase. Protease enzymes are present at a level of 0.00001% to 2% enzyme protein by weight of the composition, preferably at 0.
It may be incorporated into the composition of the present invention at a level of 0001% to 1%, more preferably at a level of 0.001% to 0.5%, more preferably at a level of 0.01% to 0.2% enzyme protein.

Lipase : Any lipase suitable for use in alkaline solutions can be used. Suitable lipases include those of bacterial or fungal origin. Chemically or genetically modified mutants are included. Examples of useful lipases include, for example, Humicola lanuginosa lipase of the genus Humicola as described in EP 258 068 and EP 305 216, EP 230 068 Rhizomucor of the genus Rhizomucor as described in
iehei) lipase,

Candida lipases of the genus Candida as described in EP 214 761, for example C. antarctica lipase, such as C. antarctica lipase A or B Pseudomonas lipases as described in EP 218 272, for example Pseudomonas alkaligenes (P. alcal
igenes) and Pseudomonas pseudoalcaligenes (P. pseudoalcalig)
enes) lipases, such as P. cepacia lipase as described in EP 331 376;

Pseudomonas stutzeri lipase and P. fluores as described, for example, in GB 1,372,034
cens) lipase, Bacillus lipase such as Bacillus subtilis (
B. subtilis) lipase (Dartois et al., (1993), Biochemica et Biophysics)
ca acta 1131, 253-260), B. stearotherophilus (B. stearothe)
rmophilus) lipase (JP 64/744992) and B. pumilus
) Lipase (WO 91/164221).

In addition, a number of cloned lipases are useful, examples of which include Yamaha
(1991, Gene 103, 61-67), Penicillium (Penicill)
genus Penicillium camembertii lipase, Geotrichom genus Geotrichum candidum (Geotrich)
um candidum) lipase (Schimada, Y. et al., (1989), J. Biochem., 106,
383-388) and various lipases of the genus Rhizopus, such as Rhizopus sp.
R. delemar lipase (Hass, MJ et al., (1991), Gene 109, 11)
7-113), Rhizopus niveus (R. niveus) lipase (Kugimiya et al., (
1992), Biosci. Biotech. Biochem. 56, 716-719) and Rhizopus oryzae lipase.

Other types of lipolytic enzymes such as cutinase are also useful, for example, Pseudomonas as described in WO 88/09367, for example.
) Of the genus Pseudomonas mendocina, or cutinase from the genus Fusarium Fusarium solanii
rium solani pisi) (for example, described in WO 90/09446).

Particularly suitable lipases are M1 Lipase ™, Rumafast ™ and Lipomax ™ (Genencor), Lipolase ™ and Lipolase Ultra ™ (Novo Nordisk A / S), and lipase P "Amano" (Amano Phar
lipase such as maceutical Co. Ltd.). Lipases are usually present at levels of 0.00001% to 2% enzyme protein by weight of the detergent composition, preferably 0.0001% to 1%, more preferably 0.001% to 0.5%,
More preferably, it is incorporated into the detergent composition at a level of 0.01% to 0.2% enzyme protein.

Amylase : Any amylase (α and / or β) suitable for use in an alkaline solution can be used. Suitable amylases include those of bacterial or fungal origin. Chemically or genetically modified mutants are included. Amylases include, for example, α-alpha from a particular strain of B. licheniformis described in more detail in GB 1,296,839.
-Amylase. Commercially available amylase is available from Duramil ™, Termamyl ™, Fungamyl ™ and Van ™ (Novo Nordisk A / S),
And Lapidase ™ and Maxamil ™ (Genencor).

Amylase is usually present at a level of 0.00001% to 2% enzyme protein by weight of the detergent composition, preferably 0.0001% to 1%, more preferably 0.001% to 0.5%.
, More preferably at a level of 0.01% to 0.2% enzyme protein in the detergent composition.

Cellulase : Any cellulase suitable for use in an alkaline solution can be used. Suitable cellulases include those of bacterial or fungal origin. Chemically or genetically modified mutants are included. Suitable cellulases are disclosed in U.S. Pat. No. 4,435,307 which discloses a fungal cellulase produced from Humicola insolens of the genus Humicola. Particularly suitable cellulases are those having a color-protecting effect. Examples of such cellulases are the cellulases described in EP-A-0 495 257.

Examples of commercially available cellulases include Humicola insolens
) Produced by the strains of Cellzyme (Novo Nordisk A / S), and KA
C-500 (B) (trademark) (Kao Corporation). Cellulases are usually present at levels of enzyme protein between 0.00001% and 2% of the weight of the detergent composition, preferably between 0.0001% and 1%, more preferably between 0.001% and 0.5%.
, More preferably at a level of 0.01% to 0.2% enzyme protein in the detergent composition.

Peroxidase / Oxidase : The peroxidase enzyme is used in combination with hydrogen peroxide or a source thereof (eg, percarbonate, perborate or persulfate). Oxidase enzymes are used in combination with oxygen. Both types of enzymes are preferably used for "solution bleaching", i.e. in order to prevent the transfer of textile dyes from one dyed fabric to another in the washing liquor during washing, e.g. WO 94/12621
And with enhancers as described in WO 95/01426. Suitable peroxidases / oxidases include those of plant, bacterial or fungal origin. Chemically or genetically modified mutants are included.

The peroxidase and / or oxidase enzymes are usually present at a level of 0.00001% to 2% of the enzyme protein by weight of the detergent composition, preferably 0.0001% to 1%.
%, More preferably from 0.001% to 0.5%, more preferably from 0.01% to 0.2%, of the enzyme protein is incorporated into the detergent composition. Mixtures of the aforementioned enzymes, in particular mixtures of proteases, amylases, lipases and / or cellulases, are encompassed herein.

The enzyme of the present invention, or any other enzyme incorporated into the detergent composition, is usually at a level of 0.00001% to 2% enzyme protein by weight of the detergent composition, preferably from 0.0001% to 1%, more preferably 0.001% to 0.5%, more preferably 0.1%.
It is incorporated into detergent compositions at levels of 01% to 0.2% enzyme protein.

Bleaching agents : Additional optional detergent components that may be included in the detergent compositions of the present invention include bleaching agents having a particle size of 400 to 800 microns, such as PB1, PB4 and percarbonates. Can be These bleach components may include one or more oxygen bleaches and, depending on the bleach selected, one or more bleach activators. When present, the oxygen bleaching compound is typically present at a level of about 1% to about 25%. Generally, the bleaching compound is an optional additional component in a non-liquid formulation, such as a granular detergent.

The bleach components for use herein can be any bleach useful for detergent compositions, including oxygen bleaches, as well as others known in the art. Bleaches suitable for the present invention can be activated or deactivated bleaches.

One class of oxygen bleaches that can be used include percarboxylic acid bleaches and salts thereof. Suitable examples of this type of bleach include magnesium monoperoxyphthalate hexahydrate, magnesium salt of metachloroperbenzoic acid, 4-nonylamino-
4-oxoperoxybutyric acid and diperoxidedecanedioic acid.
Such bleaches are disclosed in U.S. Pat. No. 4,483,781, U.S. Pat. No. 740,446, EP 0 133 354 and U.S. Pat. No. 4,412,934. Highly preferred bleaches also include 6-nonylamino-6-oxoperoxycaproic acid as described in U.S. Pat. No. 4,634,551.

Another type of bleach that may be used includes halogen bleaches. Examples of hypohalite bleaches include, for example, trichloroisocyanuric acid, and sodium and potassium dichloroisocyanurate, and N-chloro and N-bromoalkanesulfonamides. Such materials are usually added at 0.5 to 10% by weight, preferably 1 to 5% by weight of the final product.

Hydrogen peroxide releasing agents include bleach activators such as tetra-acetylethylenediamine (TAED), nonanoyloxybenzenesulfonate (NOBS, US Pat. No. 4,412,93).
4), 3,5-trimethyl-hexanoloxybenzenesulfonate (described in
ISONOBS, described in EP 120 591) or pentaacetylglucose (PA
Although they can be used in combination with G), they are perhydrolyzed to produce peracid as an active bleaching species, resulting in improved bleaching action. Also very suitable are the bleach activators C8 (6-octanamido-caproyl) oxybenzenesulfonate,
C9 (6-nonanamidocaproyl) oxybenzenesulfonate and C10 (
6-decaneamidocaproyl) oxybenzenesulfonate or a mixture thereof. Further suitable activators are acylated citrate esters as described in European Patent Application No. 91870207.7.

Useful bleaching agents, including peroxyacids and bleaching activators and bleaching systems, including peroxygen bleaching compounds for use in the cleaning compositions of the present invention, are described in US Pat.
It is described in SN 08 / 136,626. Hydrogen peroxide may also be present by adding an enzyme system (ie, an enzyme, and thus a substrate) that allows for the generation of hydrogen peroxide at or during the beginning and during the washing and / or rinsing steps. Such an enzyme system is disclosed in European Patent Publication EP 0 537 381.

[0114] Bleaches other than oxygen bleaches are known in the art and may be utilized herein. One class of non-oxygen bleaches of particular interest includes light activated bleach, such as zinc sulfonated and / or aluminum phthalocyanine. These substances can be deposited on the support during the washing step. Upon irradiation with light, the sulfonated zinc phthalocyanine is activated in the presence of oxygen, for example by hanging the garment and drying with sunlight, so that the support is bleached.

A preferred zinc phthalocyanine and light activated bleaching process is described in US Pat. No. 4,033,71
It is described in No. 8. Typically, the detergent composition contains from about 0.025% to about 1.25% by weight of the sulfonated zinc phthalocyanine. The bleach may also include a manganese catalyst. Manganese catalysts are, for example, "Efficient
manganese catalysts for low-temperature bleaching ", Nature 369, 1994, p
It may be one of the compounds described on pages 637-639.

Foam Inhibitor : Another optional ingredient is a foam inhibitor, examples of which include silicone and silica-silicone mixtures. Silicones are generally represented by alkalized polysiloxane materials, while silica is commonly used in finely divided form,
Examples include silica airgels and xerogels, as well as various types of hydrophobic silica. These substances can be mixed in as particles, in which case
The foam control agent is advantageously releasably incorporated into a water-soluble or water-dispersible, substantially non-surfactant detergent-impermeable carrier. Alternatively, the foam control agent may be applied by being dissolved or dispersed in a liquid carrier and sprayed on one or more other components.

[0117] Preferred silicone foam control agents are disclosed in US Patent No. 3,933,672. Other particularly useful foam inhibitors are the self-emulsifying silicone foam inhibitors described in German patent application DTOS 2,646,126. Examples of such compounds are Dow Corning
DC-544, which is a siloxane glycol copolymer.
Particularly preferred foam control agents are foam suppressor systems, including mixtures of silicone oils and 2-alkyl-alkanols. Suitable 2-alkyl-alkanols are 2-
Butyl-octanol, which is commercially available under the trade name Isophor 12R.

[0118] Such foam suppressant systems are described in European Patent Publication EP 0 593 841. Particularly preferred silicone foam control agents are described in European Patent Application No. 92201649.8. The composition may include a silicone / silica mixture in combination with a fumed non-porous silica, such as Aerosil (r). Said foam inhibitors are usually used at a level of from 0.001% to 2%, preferably from 0.01% to 1% by weight of the composition.

Other Constituents : Other constituents used in the detergent composition, such as soil sedimentation inhibitors, soil release agents, optical brighteners, abrasives, bactericides, haze inhibitors, colorants and / or Or an encapsulated or unencapsulated fragrance may be used. A particularly suitable encapsulating material is a water-soluble capsule consisting of a matrix of a polysaccharide and a polyhydroxy compound as described in GB 1,464,616.

Other suitable water-soluble encapsulating materials include dextrins derived from non-gelling starch esters of substituted dicarboxylic acids as described in US Pat. No. 3,455,838. These acid-ester dextrins are preferably prepared from starches such as waxy corn (maize), waxy sorghum (sorghum), sago, tapioca and potatoes. Suitable examples of such encapsulating materials include N-
N-Lok manufactured by National Starch. The N-Lok encapsulation material consists of modified corn starch and glucose. Starch can be modified by the addition of monofunctional substituents, such as octenyl succinic anhydride.

[0121] Anti-redeposition and anti-fouling agents suitable for use herein include cellulose derivatives such as methylcellulose, carboxymethylcellulose and hydroxyethylcellulose, and homo- or copolymer polycarboxylic acids or salts thereof. Can be Polymers of this type include the polyacrylates and maleic anhydride-acrylic acid copolymers described above as builders, and copolymers of maleic anhydride with ethylene, methyl vinyl ether or methacrylic acid, where maleic anhydride comprises at least 20 mole percent of the copolymer. Constitute.
These materials are normally used at a level of from 0.5% to 10%, more preferably from 0.75% to 8%, most preferably from 1% to 6% by weight of the composition.

Preferred optical brighteners are anionic, examples of which are 4,4′-bis- (
2-diethanolamino-4-anilino-s-triazin-6-ylamino) stilbene-2: 2 'disodium disulfonate, disodium 4,4'-bis- (2
-Morpholino-4 -anilino-s-triazine-6-ylamino-stilbene-2:
Disodium 2'-disulfonate, 4,4'-bis- (2,4-dianilino-s-triazin-6-ylamino) stilbene-2: 2 'disodium disulfonate, 4
', 4 "-bis- (2,4-dianilino-s-triazin-6-ylamino) stilbene-2-sulfonic acid monosodium salt,

4,4′-bis- (2-anilino-4- (N-methyl-N-2-hydroxyethylamine) -s-triazin-6-ylamino) stilbene-2,2′-disulfonate disodium, 4,4'-bis- (4-phenyl-2,1,3-triazol-2-yl) stilbene-2,2 'disodium disulfonate, 4,4'-bis- (2-anilino-4- ( 1-methyl-2-hydroxyethylamino) -s-triazin-6-ylamino) stilbene-2,2 ′ disodium disulfonate, 2
(Stilbyl-4 "-(naphth-1 ', 2': 4,5) -triazole-2" -sodium sulfonate and 4,4'-bis- (2-sulfostyryl) biphenyl.

[0124] Other useful polymeric substances are polyethylene glycols, especially those having a molecular weight of 1000-1000.
It is 10,000, especially 2000-8000, most preferably about 4000. They are,
It is used at a level of from 0.20% to 5% by weight, more preferably from 0.25% to 2.5% by weight. These polymers and the aforementioned homo- or copolymer polycarboxylate salts are used to improve whiteness retention, textile ash deposition, and cleaning performance on clay, proteinaceous and oxidizable soils in the presence of transition metal impurities. It is beneficial.

Soil release agents useful in the compositions of the present invention are conventionally copolymers or terpolymers of terephthalic acid and ethylene glycol and / or propylene glycol units in various formulations. Examples of such polymers are described in U.S. Pat.
Nos. 6,885 and 4,711,730 and EP 0 272 033. A particularly preferred polymer according to EP 0 272 033 has the formula: (CH ₃ (PEG) ₄₃ ) _0.75 (POH) _0.25 [T- (PO) _2.8 (T-PEG) _0.4 ] T (PO
H) _0.25 ((PEG) ₄₃ CH ₃ ) _{0.75 where} PEG is-(OC ₂ H ₄ ) O-, PO is (OC ₃ H ₆ O), and T is (pOOC ₆ H ₄ CO)).

Even more preferred are modified polymers such as dimethyl terephthalate,
A random copolymer of dimethylsulfoisophthalate, ethylene glycol and 1,2-propanediol, the terminal groups mainly consisting of sulfobenzoate and supplementary monoesters of ethylene glycol and / or 1,2-propanediol . The goal is to obtain a polymer flanked on both ends by sulfobenzoate groups, and "primarily", in the context of the present invention, most of the copolymers mentioned herein are flanked by sulfobenzoate groups. . However, some copolymers are not completely crowned, and thus their end groups consist of monoesters of ethylene glycol and / or 1,2-propanediol, and , May consist of such species.

[0127] The polyesters selected herein are about 46% by weight dimethyl terephthalic acid, about 16% by weight 1,2-propanediol, about 10% by weight ethylene glycol, about 13% by weight dimethyl It contains sulfobenzoic acid and about 15% by weight sulfoisophthalic acid and has a molecular weight of about 3000. Polyesters and their production methods
It is described in detail in EP 311 342.

Softeners : Fabric softeners can also be incorporated into the laundry detergent compositions of the present invention. These agents can be of the inorganic or organic type. Inorganic softeners are exemplified by the green clay disclosed in British Patent Application No. 1,400,898 and US Patent No. 5,019,292. Organic fabric softeners include water-insoluble tertiary amines such as those disclosed in UK Patent Application No. 1,514,276 and EP 0,011,340, EP-B-0
With a mono-C ₁₂ -C ₁₄ quaternary ammonium salt as disclosed in
And combinations with double long chain amides as disclosed in EP 0 242 919. Other useful organic components of the fabric softening system include high molecular weight polyethylene oxide materials as disclosed in EP 0 299 575 and EP 0 313 146.

The level of green clay is usually between 5% and 15% by weight, more preferably 8% by weight.
-12% by weight, the material being added to the remaining formulation as a dry-mix component. Organic fabric softeners, such as water-insoluble tertiary amines or bilong amide materials, are incorporated at levels of 0.5% to 5% by weight, usually 1% to 3% by weight, while high molecular weight polyethylene oxide Substances and water-soluble cationic substances are 0.
It is added at a level of 1% to 2% by weight, usually 0.15% to 1.5% by weight. These substances are usually added to the spray-dried portion of the composition, but in some cases, they are added to other solid components of the composition as dry mixed particles, or It may be more convenient to spray them as a melt.

Polymer transfer inhibitor : The detergent composition of the present invention comprises 0.001 to 10% by weight, preferably 0.01 to 2% by weight, more preferably 0.05 to 1% by weight of a polymer transfer agent. Dye inhibitors may also be included. The polymeric transfer dyes are usually incorporated into detergent compositions to prevent the transfer of dyes from the colored fabric onto the fabric being laundered together. These polymers have the ability to complex or adsorb discolorable dyes washed out of the dyed fabric before the dyes have a chance to bind to other articles in the wash liquor.

Particularly suitable polymeric transfer dyes are polyamine N-oxide polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole, polyvinylpyrrolidone polymers, polyvinyloxazolididone and polyvinylimidazole or combinations thereof. . The addition of such a polymer also enhances the performance of the enzyme of the present invention. The detergent composition of the present invention may be in liquid, paste, gel, stick or granular form.

For example, dust-free granules such as those disclosed in US Pat. Nos. And No. (both Novo Industri A / S) can be prepared and optionally coated by methods known in the art. Examples of waxy coating materials are poly (ethylene oxide) products (polyethylene glycol, PEG) having an average molecular weight of 1000 to 20,000; ethoxylated nonylphenols having 16 to 50 ethylene oxide units;
Ethoxylated fatty alcohols having from 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and the mono- and di- and triglycerides of fatty acids. Film-forming coating materials suitable for application by fluidized bed technology are shown in GB 1483593.

The particulate compositions of the present invention can also be in “compressed form”. That is, they have a relatively higher density than conventional granular detergents, i.e., 550-950 g / l, and in such cases, the granular detergent compositions of the present invention have lower levels than conventional granular detergents. It contains an amount of “inorganic filler salt”. Typical filler salts are the alkaline earth metal salts of sulfates and chlorides, typically sodium sulfate. "Compressed" detergents typically include up to 10% filler salt. The liquid composition of the present invention may also be in “concentrated form”. In such a case, the liquid detergent composition of the present invention contains a lower amount of water than a conventional liquid detergent. Typically, the water content of the concentrated liquid detergent is 30% by weight of the detergent composition.
Less than 20% by weight, more preferably less than 10% by weight.

The compositions of the present invention include, for example, laundry detergent compositions for hand and machine washing, such as laundry additive compositions, and compositions suitable for use in the pretreatment of soiled fabrics, rinse aid fabric softeners. The composition can be formulated as a composition for use in home hard surface cleaning and dishwashing operations. The following examples are intended to illustrate compositions according to the present invention, but do not necessarily limit or otherwise define the scope of the present invention.

In the detergent composition, the abbreviations of the constituents have the following meanings: LAS: linear C ₁₂ sodium alkylbenzenesulfonate TAS: tallow sodium alkyl sulfate XYAS: C _{1X to} C _1Y sodium alkyl sulfate SS: 2 - secondary soap surfactants 25EY butyl octanoate: average Y moles of ethylene oxide condensed with C ₁₂ -C ₁₅ predominantly linear primary alcohol 45EY: was engaged average Y moles of ethylene oxide condensed C predominantly linear primary alcohol ₁₄ -C ₁₅

XYEZS: C _1X -C _1Y sodium alkylsulfate condensed with an average of Z moles of ethylene oxide per mole Nonionic: C ₁₃ -C ₁₅ mixed ethoxylated / propoxylated fatty alcohol. Average degree of ethoxylation 3.8 and average degree of propoxylation 4.5. Pull la fax
Sold by BASF GmBH under the trade name LF404. CFAA: alkyl N of C ₁₂ -C ₁₄ - methyl glucamide TFAA: - methyl glucamide alkyl N of C ₁₆ -C ₁₈

Silicate: amorphous sodium silicate (SiO ₂ : Na ₂ O ratio = 2.0) NaSKS-6: crystallized silicate of formula δ-Na ₂ Si ₂ O ₅ Carbonate: anhydrous sodium carbonate phosphorus Acid salt: sodium tripolyphosphate MA / AA: 1: 4 maleic / acrylic acid copolymer. Polyacrylate having an average molecular weight of about 80,000: A polyacrylate homopolymer having an average molecular weight of 8,000. PA30
Sold by BASF GmBH.

Zeolite A: sodium aluminosilicate hydrate of the formula Na ₁₂ (AlO ₂ SiO ₂ ) .27H ₂ O. It has a primary particle size ranging from 1 to 10μ. Citrate: Trisodium citrate dihydrate Citric: Citric acid Perborate: Anhydrous sodium perborate monohydrate bleach. Empirical formula NaBO ₂ · H ₂ O ₂ PB4: anhydrous sodium perborate tetrahydrate Percarbonate: empirical formula 2Na ₂ CO ₂ · 3H ₂ O ₂ anhydrous sodium percarbonate bleach

TAED: tetraacetylethylenediamine CMC: sodium carboxymethylcellulose DETPMP: diethylenetriaminepenta (methylenephosphonic acid). Quest 20
Commercially available from Monsanto under 60 trade names. PVP: polyvinylpyrrolidone polymer EDDS: ethylenediamine-N, N'-succinic acid. [S, S] in the form of sodium salt
Isomer Foaming inhibitor: 25% paraffin wax, melting point 50 ° C, 17% hydrophobic silica, 58% paraffin oil

Granular foam inhibitor: 12% silicone / silica, 18% stearyl alcohol, 70%
Starch (particulate form) Sulfate: anhydrous sodium sulfate HMWPEO: high molecular weight polyethylene oxide TAE 25: tallow alcohol ethoxylate (25)

Detergent Example I A granular fabric cleaning composition of the present invention can be prepared as follows:

[0142]

[Table 1]

Detergent Example II The compressed granular fabric cleaning composition of the present invention (density 800 g / l) can be prepared as follows:

[0144]

[Table 2]

Detergent Example III A particulate fabric cleaning composition of the present invention that is particularly useful for washing colored fabrics can be prepared as follows:

[0146]

[Table 3]

Detergent Example IV A particulate fabric cleaning composition of the present invention that provides the ability to "soften the entire wash liquor" can be prepared as follows:

[0148]

[Table 4]

Detergent Example V A heavy duty liquid textile cleaning composition of the present invention can be prepared as follows:

[0150]

[Table 5]

Treatment of Human or Animal Parts The present invention provides a method for antimicrobial treatment of microorganisms or viruses present on or in human or animal skin, hair, oral cavity, mucous membranes, teeth, eyes, wounds or bruises. Thus, the present invention may be useful for disinfection, eg, treating acne or other skin infections, infections in the eyes or mouth, microbial growth on the feet, axilla, teeth (mouth care), wounds, bruises, and the like. The treatment may be applied by use of an aerosol, liquid, emulsion, gel, slurry, paste or solid containing the laccase and the enhancer.

Processing of Cosmetics, Food or Beverages or Other Products The present invention relates to foods, beverages, cosmetics, such as lotions, creams, gels, ointments,
It can be useful for the preservation of soaps, shampoos, conditioners, antiperspirants, deodorants, mouthwashes, contact lens products, footbath products, enzyme formulations or food ingredients. The present invention can be applied to non-preservative foods, beverages, cosmetics and food ingredients in an amount effective to achieve the desired antimicrobial effect. Contact Lens Treatment The present invention can be useful for cleaning and / or antimicrobial treatment of contact lenses.

Treatment of Hard Surfaces In general, the present invention is intended to provide a method that is useful for the antimicrobial treatment of any hard surface as described above. The treatment may be applied for general disinfection purposes, for example, disinfection of hospital wards, operating rooms, rooms for food treatment or other facilities requiring disinfection.

The hard surface can also be a cooling tower process equipment, a water treatment plant, a dairy, a processing plant for food or food additives, a chemical or pharmaceutical processing plant. The hard surface can also be a medical device or a sewage system. Thus, the present invention provides an antimicrobial method that is useful in conventional in place cleaning (CIP) systems. The laccase and enhancer used in the context of the present invention should be brought into contact with the surface in question through the medium and be present in an amount having an antibacterial action.

Storage of Paint / Storage of canned paint products has been accomplished in the art by adding non-enzymatic organic biocides to the paint. In the context of the present invention, the paint comprises a solid vehicle dissolved or dispersed in a liquid vehicle, such as water, an organic solvent and / or oil, and upon spreading on the surface, becomes a dry, lightly colored decorative and /
Or as a substance that leaves a protective coating. Typically, an isothiazolion, e.g., 5-chloro-2-methyl-4-thiazoli-3-one, is present at about 0.05 to 0.
It has been added to paint as a biocide in doses ranging from 5% to inhibit / prevent microbial growth in the paint.

However, the method of the present invention is suitably applied in this field, thereby replacing the use of toxic organic biocides with environmentally compatible enzymes, thereby reducing the use of previously existing toxic biocides. Solving the problem of environmental biohazard. Accordingly, the present invention provides a method of preserving a paint, comprising contacting said paint with a laccase and one or more enhancers of the present invention. further,
The present invention provides a paint composition comprising laccase and one or more enhancers of the present invention.

The paint is preferably a water-based paint, ie a solid paint is dispersed in an aqueous solution. The paint is 0-20%, preferably 0-10%, for example
It may contain 0-5% of organic solvents. 0.0001-100 mg, preferably 0.001-10 mg, of enzyme per liter of paint
/ L, e.g., 0.01-1 mg / L, is added to the paint while the enhancer is 10-50
It may be added to the paint composition in an amount of 0 μM, preferably 25-250 μM, for example 100 μM. The following examples illustrate the invention, but do not limit the invention.

Embodiment 1 Laccase antibacterial activity using different enhancers Methyl syringate (MeS), cinnamic acid, chlorogenic acid, syringaldehyde and PPT as pH enhancers
6 and the recombinant Myceliophthera laccase (rMtL, WO 95/33
836) and recombinant Polyporus laccase (rPpL, WO 96/0029)
0) was tested for antimicrobial activity (ie, bactericidal activity).

Air-flushed 0.05 M MES buffer (2- [
Bactericidal activity was evaluated in [N-morpholino] ethanesulfonic acid) (pH 6).
Pseudomonas aeruginosa (ATCC 10145) and Staphylococcus epidermidis (DS)
M20042), the bactericidal activity was determined. Tryptonized broth (Oxoid CM
129) 24 hours at 30 ° C. Among the cells were grown and diluted with MES buffer, a final cell concentration of about 10 ⁴ cfu / ml. The cell suspension was mixed with laccase (3 mg / L) and enhancer (0.1 mM) at 40 ° C for 20 minutes. Bactericidal activity was determined by incubation with Malthus Malthus.

The detection time measured by the Malthus instrument was converted to cfu / ml by a calibration curve.
When counting total viable cells, direct or indirect Malthus measurements were used (Malthus
Flexi M2060, Malthus Instrument Limited). Cell metabolism was determined by direct measurement and by conductance measurements on growth substrates. 3 ml by indirect measurement
Transfer the growth medium to the outer chamber of the indirect Malthus cell and add 0.5 ml of sterile KOH (0.1
M) was transferred to the inner compartment. The cell suspension was transferred to the outer compartment of the Malthus cell after enzyme treatment. Cells in the growth in the outer chamber to generate CO _2, which is soluble in KOH in the inner chamber, thereby changing the conductance of the KOH.

The amount of CO ₂ produced by the respiring cells that survived the enzymatic treatment was used to estimate the number of viable cells. If the conductance change can be measured in Malthus, record the detection time (dt). Using a calibration curve for cfu versus dt, dt was converted to colony numbers (Johansen et al., 1995. Journal of Applied Ba
cteriology 78: 297-303, Johansen et al. 1997, Applied and Environmental M
icrobiology 63: 3724-3728).

Using methylsyringate (MeS) as enhancer, rPpL and rMtL, pH =
6. Laccase = 3mg / L and enhancer 0.1mM, bactericidal activity (log (cfu / ml) =
Log of the number of cells killed). Under these experimental conditions, the other enhancers did not produce bactericidal activity (Figure 1; 1 = no enhancer included; 2 = methylsilingate; 3 = cinnamic acid; 4 = chlorogenic acid; 5 = PPT; 6 = Syringaldehyde)
However, the bactericidal activity of the laccase / enhancer system is dependent, inter alia, on pH and the like, so other enhancers may be beneficial, for example, at high pH.

The bactericidal activity of rMtL / MeS or rPpL / MeS is particularly pronounced against Staphylococcus epidermidis (FIG. 1), which is 100% killed by rPpL / MeS and rMtL / MeS
Only a few cells survived the MeS treatment. However, the bactericidal activity against Pseudomonas aeruginosa was observed as a cell reduction of 1-2 log units (FIG. 1).

Embodiment 2 FIG. In Miseri off Terra laccase and Methyl cylinder as described in bactericidal activity Example 1 of the gate in MES buffer 40 ° C. at pH6 using 3 2 multifactorial design, Miseri off Terra laccase (rMtL) and methyl cylindrical gate (MeS
) Was determined, however, higher cell concentrations (107-108 cfu / ml) were used. The rMtL levels tested were 0-2, 5-5 mg / L
And the MeS level was 0-5-10 mM.

The bactericidal activity against Pseudomonas aeruginosa ATCC 10145 is 5-10 mM
Of MeS. Increasing the rMtL concentration from 2.5 mg / L to 5 mg / L resulted in a decrease in bactericidal activity, and therefore the optimal concentration of the enzyme was 5 mg / L for bactericidal activity against Pseudomonas aeruginosa under these specific conditions. Lower. pH
The bactericidal activity obtained at high MeS concentration in 6 was very close to 100% bactericidal activity (
Figure 2 ). At pH 4, 100% bactericidal activity was obtained. The Gram-positive strain Staphylococcus epidermidis has a pH of 5-6 and a pH of 0.
At MeS concentrations higher than 5 mM, 100% kill was achieved by rMtL / MeS treatment. MeS was not bactericidal to the two test organisms at the concentrations used, unless combined with laccase.

Embodiment 3 FIG. The antimicrobial activity of rMtL and rPpL using MeS as an optimal pH enhancer for the antimicrobial activity of laccase / methylsyringate (ie, bactericidal activity)
pH 4-10, determined using the following buffer system: Homopipes (pH 4 and 5)
, MES (pH6), HEPES (pH7 and 8), HEPES / CAPS (pH9) and
CAPS (pH 10). Bactericidal activity was evaluated against Staphylococcus epidermidis at 40 ° C. as described in Example 1. rPpL and rMtL are both low p
H has bactericidal activity (FIG. 3); 1 = (rPpL = 2 mg / l and MeS = 25 mM); 2
= (RPpL = 2 mg / l and MeS = 50 mM); 3 = (rMrL = 2 mg / l and MeS = 25 mM)
2 = (rMtL = 2 mg / l and MeS = 50 mM).

The bactericidal activity of rPpL and rMtL increased with decreasing pH value, however, at the tested concentrations of MeS, the bactericidal activity was limited at pH values higher than 5. Significant bactericidal activity is obtained at higher pH values by increasing the MeS concentration, and thus, for example, using rPpL = 5 mg / L and MeS = 5 mM, at a bactericidal activity against Staphylococcus epidermidis at pH 9 was gotten. Bactericidal activity can be obtained at different conditions (pH, temperature, etc.) by choosing laccases and enhancers that are active under certain desired conditions.

Embodiment 4 FIG. Antimicrobial activity of Mycelioffera and Pleurotus mushroom laccase using different syringe gates against Staphylococcus epidermidis at pH 6 in MES buffer as described in Example 1 against Mycelioffera laccase (rMtL) and Polyporus laccase (rMtL) The antimicrobial activity (ie, bactericidal activity) of rPpL) was determined. Laccase
Used at a concentration of 3 mg / L. Acetosyringon, methylsyringate, ethylsyringate, butylsyringate and laurylsyringate were evaluated as electron donors at a concentration of 0.2 mM.

All the syringate compounds had bactericidal activity when combined with laccase. Lauryl silingate and acetosyringon also showed no bactericidal activity without laccase, however, the addition of laccase significantly increased the bactericidal activity (FIG. 4; 1 = enhancement) No agent; 2
= Methylsilingate; 3 = ethylsilingate; 4 = butylsilingate; 5
= Lauryl silingate; 6 = acetosyringone; dashed line = total kill).

[0170] Acetosyringone, methyl syringate, ethyl syringate, and butyl syringate provided 100% bactericidal activity against Staphylococcus epidermidis when combined with Tamatake mushroom laccase. In contrast, the bactericidal activity obtained with Mycelioffella laccase and acetosyringone was reduced compared to both the activity of Polyporus laccase and the activity obtained with other syringate compounds. . A different syringate compound was also evaluated against Pseudomonas aeruginosa, which is more resistant than Staphylococcus epidermidis. Acetosyringone was found to be the most bactericidal enhancer against Pseudomonas aeruginosa.

Embodiment 5 FIG. Synergy by different enhancer combinations The susceptibility of different microorganisms depends on the enhancer used, so that by combining the different enhancers and applying them simultaneously, a wide range of antibacterial activities can be obtained. Total antimicrobial activity against mixed cultures of different microorganisms is expected to be significantly increased when using the enhancer formulation. The enhancers are intended to be tested at sub-lethal concentrations (less than 100% bactericidal activity) and in different combinations against microorganisms having different physiological findings. Synergy can be determined by multifactor experiments using laccase and enhancers, such as acetosyringone, methylsyringate, ethylsyringate, butylsyringate and laurylsyringate.

Embodiment 6 FIG. Synergistic antibacterial activity of the combination of two enhancers The antibacterial activity of laccase using the two enhancers was assessed by Pseudomonas aeruginosa (ATCC 10145) and Staphylococcus epidermidis (DSM 20042)
At pH 6 and determined as described in Example 1. Methylsyringate and acetosyringone were used as enhancers, and laccase was rPpL (1 mg / L
) And using a 32 multi-factorial experimental design to determine antimicrobial activity. Staphylococcus epidermidis

Synergistic antimicrobial activity was found when the two enhancers were combined (FIG. 5; A = methylsyringate; B = acetosyringone). Acetosyringone caused about 1.5 log units of cell reduction in Pseudomonas aeruginosa, and methylsyringate caused about 3 log units of cell reduction, while the combination resulted in about 6-7 log units of cell reduction. A corresponding total kill of the test organisms resulted (FIG. 5).

Embodiment 7 FIG. Preservation of paint involves adding an amount of laccase and enhancer, for example, 1 mg of mycelioffera laccase and 10 μmol of methylsyringate (sterile filtered) to 100 mL of sterile water-based paint in a sterile jar. Can be accomplished by preparing a paint sample that includes the enzymatic sterilization system. To this paint sample,
Then, to a 100 mL sterile reference sample of the paint that does not contain the enzyme system, here, 200 μL of a 108 cfu / ml mixed culture sample of Pseudomonas species and Bacillus species is added.

Mix the culture in the paint, close the jar and leave at 30 or 40 ° C. After 3 or 4 days, samples are taken from each jar and mounted on agar plates using conventional methods to count the number of growing colonies from both samples. This procedure is repeated about 10 times (30 or 40 days) and the number of colonies from the paint sample containing the enzyme system is compared to the number from the reference sample.

[Brief description of the drawings]

FIG. 1 shows the antibacterial activity of different laccase systems against P. aeruginosa and S. epidermidis. The enzyme concentration used was 3 mg / L and the enhancer concentration used was 0.1 mM. The different enhancers tested were: 1 = no mediator; 2 = methyl syringate; 3 = cinnamic acid; 4 = chlorogenic acid; 5 = PPT; 6 = syringaldehyde. The bacterial activity of the different combinations is expressed as log cfu / ml, ie the log of the number of cfu killed per ml (cfu = colony forming units).

FIG. 2 shows Pseudomonas aeru at pH 6 and 40 ° C.
2 shows the reaction surface plots for the bactericidal activity of rMtL and MeS against C. ginosa) (expressed as the log against killed cfu / ml).

FIG. 3. FIG. 3 shows that Staphylococcus epidermidis by pH.
1 shows the bactericidal activity of laccase and methylsyringate against E. epidermidis (expressed as the log of killed cfu / ml). Laccase concentration = 2mg / L
Temperature = 40 ° C .; treatment time = 20 minutes. Different laccase and MeS concentrations were used:
1 = rPpL + 25 mM MeS; 2 = rPpL + 50 mM MeS; 3 = rMtL
+25 mM MeS; 4 == rMtL + 50 mM MeS.

FIG. 4 shows the bactericidal activity of different syringate compounds against Staphylococcus epidermidis in rMtL, rPpL or laccase-free combinations (expressed as log versus cfu killed / ml). The laccase concentration was 3 mg / L. The enhancer concentration was 0.2 mM. Temperature = 40 ° C .; pH = 6; treatment time = 20 minutes. The enhancers tested were: 1 = no enhancer; 2 = methylsyringate; 3 = ethylsyringate; 4 = butylsyringate; 5 = laurylsyringate; 6 = acetosyringone. The dashed line indicates the total kill of the tested microorganism.

FIG. 5 shows Pseudomonas aeru at pH 6 and 40 ° C.
2 shows the reaction surface plots for rPpL (1 mg / L) against B. ginosa) and the bactericidal activity of methylsilin gate and acetosyringone (expressed as log versus killed cfu / ml).

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat ゛ (Reference) C09D 5/14 C09D 5/14 4J038 201/00 201/00 C11D 3/386 C11D 3/386 3/48 3 / 48 G02C 13/00 G02C 13/00 (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW) , SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE, GH, GM, HR, HU, ID, IL, IS, JP, KE, KG, KP, KR , KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, UZ, VN, YU, ZW. DA09 4C058 AA03 AA09 AA12 AA21 AA22 AA23 AA28 BB07 JJ04 JJ23 4C083 AC471 AC472 AC481 AC482 AC551 AC691 AC791 AD471 CC05 CC33 CC38 DD22 DD31 DD41 EE10 EE18 EE34 4H003 AB19 AB27 AB31 AC08 AC13 BA09 BA12 DA01 EA18 DA01 EA18 DA05 EB16 EB22 EB 28 EB32 EB42 EC01 FA34 4H011 AA02 BA01 BA02 BB21 BC03 BC04 BC05 BC06 BC07 BC08 BC17 BC18 BC19 BC20 DA02 DA13 DD07 DH02 DH04 DH08 DH10 DH19 4J038 BA182 EA011 JA64 JC38 KA02 MA08 NA02 PC02 PC03 PC04 PC06 PC06

Claims (21)

[Claims] 1. A method for the antibacterial treatment of microorganisms and / or viruses, said method comprising the step of, in the presence of oxygen O ₂ , adding an effective amount of a fungal laccase (EC 1.10.3.2) enzyme and one or more of the same. Wherein the enhancer is of the following formula: Wherein A is -D, -CH = CH-D, -CH = CH-CH = CH-D,-
CH = ND, -N = ND or -N = CH-D wherein D is -CO-E,
-SO ₂ -E, -N-XY, and -N ⁺ -xyz (wherein, E is -H, -OH,
A -R or -OR, and X and Y and Z may be the same or different, are selected from -H and -R, R is alkyl of C ₁ -C _16, preferably C ₁ -C _{8 wherein} the alkyl is saturated or unsaturated, branched or unbranched, and is optionally substituted with a carboxy, sulfo, or amino group. And B and C
May be the same or different and may be selected from C _m H _{2m + 1} (1 ≦ m ≦ 5), represented by｝. 2. A laccase (EC 1.10.3.2) enzyme and the following formula: Wherein A is -D, -CH = CH-D, -CH = CH-CH = CH-D,-
CH = ND, -N = ND or -N = CH-D wherein D is -CO-E,
-SO ₂ -E, -N-XY, and -N ⁺ -xyz (wherein, E is -H, -OH,
A -R or -OR, and X and Y and Z may be the same or different, are selected from -H and -R, R is alkyl of C ₁ -C _16, preferably C ₁ -C _{8 wherein} the alkyl is saturated or unsaturated, branched or unbranched, and is optionally substituted with a carboxy, sulfo, or amino group. And B and C
May be the same or different and may be selected from C _m H _{2m + 1} (1 ≦ m ≦ 5). An antimicrobial composition comprising at least two different enhancers represented by: 3. The method of claim 2, wherein the enhancing agent has the following formula: Wherein A is -H, -OH, -CH_Three, -OCH_Three, -O (CH_Two) NCH _Three Wherein n = 1, 2, 3, 4, 5, 6, 7, 7 or 8. The method of claim 1, wherein the group is selected from the group consisting of: 4. The method according to claim 1, wherein said treating comprises the simultaneous use of two different enhancers. 5. The method of claim 1, wherein said treating comprises the simultaneous use of three different enhancers. 6. The laccase wherein the laccase is a species of Myceliophteera,
Seeds of Polyporus, seeds of Coprinus, Rhizoctonia (
Rhizoctonia seeds, Scytalidium seeds and pyriclaria (
The method according to claim 1, wherein the method is obtained from a fungus selected from the species of Pyricularia). 7. The method according to claim 7, wherein the laccase is a fungus Mycelioffera thermophila (My
celiophthera thermophira or Polyporus pinisis
tus). 8. An antibacterial treatment of microorganisms and / or viruses present in a liquid used for immersing, washing or rinsing the laundry on and / or rinsing the laundry, according to any of claims 1 and 3 to 7. Or the method of claim 1. 9. The method of claim 8, wherein the laundry is processed in a washing machine. 10. An antimicrobial treatment of microorganisms and / or viruses present in the skin, hair, oral cavity, mucous membranes, teeth, eyes, wounds or bruises of humans or animals.
The method according to any one of 1 and 3 to 7. 11. The method according to claim 1, for the antimicrobial treatment of microorganisms and / or viruses in cosmetics. 12. The method according to claim 1, for the antimicrobial treatment (storage or disinfection) of contact lenses. 13. A method according to claim 1 for the antimicrobial treatment of hard surfaces. 14. The method of claim 13, wherein said hard surface is a facility requiring disinfection. 15. The facility is a hospital room, a food or food additive treatment room, a water treatment room, a paper and / or pulp treatment room or a chemical or pharmaceutical treatment room.
14. The method according to 14. 16. The hard surface is a cooling tower process equipment, a water treatment plant, a dairy plant, a treatment plant for food or food additives, a paper and / or pulping plant, or a chemical or pharmaceutical processing plant. Item 14. The method according to Item 13. 17. The method of claim 13, wherein said hard surface is a surface of a sewage system. 18. The method of claim 13, wherein said hard surface is a surface of a medical device. 19. The method according to claim 1, for antimicrobial treatment in a clean-in-place (CIP) system. 20. A method for preserving an aqueous-based paint, as claimed in claim 1.
The method according to any one of claims 1 to 7, wherein the paint is laccase (EC 1.1).
0.3.2) and one or more enhancers according to claim 1. 21. A composition comprising a paint, a laccase (EC 1.10.3.2) and an enhancer.