The present Invention relates to a liquid composition, comprising a subtilisin and a subtilisin stabilizer.
Storage stability problems are well known with liquids containing enzyme(s). Especially in enzyme-containing liquid detergents a major problem, in particular If the detergent contains protease, is that of ensuring enzyme activity over time
The prior art has dealt extensively with improving the storage stability, for example by adding a protease inhibitor.
Bone acid and boronic acids are known to reversibly inhibit proteolytic enzymes. A discussion of the inhibition of one serine protease, subtilisin, by boronic acid is provided in
Boronic acids have very different capacities as subtilisin inhibitors. Boronic acids containing only alkyl groups such as methyl, butyl or 2-cyclohexylethyl are poor inhibitors with methylboronic acid as the poorest inhibitor, whereas boronic acids bearing aromatic groups such as phenyl, 4-methoxyphenyl or 3,5-dichlorophenyl are good inhibitors with 3,5-dichlorophenylboronic acid as a particularly effective one (see
It is also claimed that aryl boronic acids which have a substitution at the 3-position relative to boron are unexpectedly good reversible protease Inhibitors. Especially, acetamidophenyl boronic acid is claimed to be a superior inhibitor of proteolytic enzymes (see WHO 92/19707)
In
It has been found that borates are reprotoxic in animals and therefore there is a demand to find alternatives which can be used to stabilize the enzymes in liquid compositions. In a particular embodiment of the present invention the liquid composition does not comprise borate or boric acid
In
One object of the present invention is to provide a stable liquid composition.
It has surprisingly been found that certain aryl carboxylic acids have extraordinary good capacities as subtilisin stabilizers in liquids, especially the stabilizers indicated below.
The present invention provides thus a liquid composition comprising a subtilisin and a subtilisin stabilizer as described below.
The present invention relates to stabilization of subtilisins in liquid compositions. The present invention provides a liquid composition comprising a subtilisin and a subtilisin stabilizer which is:
3-chlorobenzoic acid, 4-chlorobenzoic acid, 3,5-dichlorobenzoic acid, 3-chlorophenylacetic acid 3-chlorophehyl-3-propionic acid, and their corresponding salts
In a particular embodiment of the present invention the liquid composition comprises 0.001-20% w/w of the stabilizer. In a more particular embodiment of the present invention the liquid composition comprises 0.01-15% of the stabilizer. In an even more particular embodiment of the present invention the liquid composition comprises 0.1-10% w/w of the stabilizer. In a most particular embodiment of the present invention the liquid composition comprises 0.5-5 % of the stabilizer. In a further particular embodiment of the present invention the liquid composition comprises 1.5 to 5% w/w of the stabilizer.
In a particular embodiment of the present invention the liquid composition comprises 0.05-1000 mM of the stabilizer. In a more particular embodiment of the present invention the liquid composition comprises 0.5-750 mM of the stabilizer. In an even more particular embodiment of the present invention the liquid composition comprises 5-500 mM of the stabilizer. In a most particular embodiment of the present invention the liquid composition, comprises 25-250 mM of the stabilizer
In a particular embodiment the present invention provides a liquid detergent composition comprising a surfactant, a subtilisin and a subtilisin stabilizer of any of the formulas disclosed above.
It has surprisingly been found that the stabilizer also have a stabilizing effect at pH above 8 such as above 9.
According to the invention the liquid composition contains at least one subtilisin Mixtures of subtilisins are also included.
In a particular embodiment the liquid composition comprises more than 2 different enzymes. In a particular embodiment a liquid composition comprising two or more enzymes in which the first enzyme is a subtilisin and the second enzyme is selected from the group consisting of amylases, lipases, cellulases, lyases and oxidoreductases is preferred. In a more particular embodiment the second enzyme is a lipase.
It Is to be understood that subtilisin variants (produced, for example, by recombinant techniques) are included within the meaning of the term subtilisin. Examples of such subtilisin variants are disclosed, e.g. in
Subtilisins are well-characterized physically and chemically. In addition to knowledge of the primary structure (amino add sequence) of these enzymes, over 50 high resolution X-ray structures of subtilisins have been determined which delineate the binding of substrate, transition state, products, at least three different protease inhibitors, and,define the structural consequences for natural variation (
One subgroup of the subtilases, I-SI, comprises the "classical" subtilisins, such as subtilisin 168, subtilisin BPN', subtilisin Carlsberg (ALCALASE®, Novozymes A/S), and subtilisin DY.
A further subgroup of the subtilases I-S2, is recognised by Siezen et al. (supra). Sub-group IS2 proteases are described as highly alkaline subtilisins and comprise enzymes such as subtilisin PB92 (MAXACAL®, Gist-Brocades NV), subtilisin 309 (SAVINASE®, Novozymes A/S), subtilisin 147 (ESPERASE®, Novozymes A/S), and alkaline elastase YaB.
Random and site-directed mutations of the subtilase gene have both arisen from knowledge of the physical and chemical properties of the enzyme and contributed information relating to subtilase's catalytic activity, substrate specificity, tertiary structure, etc (
More recent publications covering this area are
Examples of commercially available subtilisins include Kannase™, Evertase™, Esperase™, Alcalase™, Durazym™, Savinase™, Ovozyme™, Pyrase™, and Clear-Lens™ Pro (all available from Novozymes A/S, Bagsvaerd, Denmark). Other preferred include those described in
Other commercially available subtilisins include Maxatase™, Maxacal™, Maxapem™, Opticlean™, Propease™, Purafect™ and Purafect Ox™ (available from Genencor International Inc., Gist-Brocades, BASF, or DSM Nutritional Products).
Lipases: Suitable lipases include those of bacterial or fungal origin Chemically or genetically modified mutants are included.
Examples of useful lipases include a Humicola lanugi-nosa lipase, e.g., as described in
Furthermore, a number of cloned lipases may be useful, including the Penicillium camenbertii lipase described by
Other types of lipolytic enzymes such as culinases may also be useful, e g., a cutinase derived from Pseudomonas mendocina as described in WHO 88/09367, or a culinase derived from Fusarium solani pisi (e.g. described in
Examples of commercially available lipases include Lipex™, Lipoprime™, Lipopan™, Lipolase™, Lipolase™ Ultra, Lipozyme™, Palatase™, Resinase™, Novozym™ 435 and Lecitase™ (all available from Novozymes A/S).
Other commercially available lipases include Lumafast™ (Pseudomonas mendocina lipase from Genencor International Inc.), Lipomax™ (Ps pseudoalcaligenes lipase from Gist Brocades/Genencor Int. Inc ; and Bacillus sp. lipase from Solvay enzymes. Further lipases are available from other suppliers such as Lipase P "Amano" (Amano Pharmaceutical Co. Ltd.).
Amylases. Suitable amylases (α and/or ß) include those of bacterial or fungal origin. Chemically or genetically modified mutants are included. Amylases include, for example, a-amylases obtained from a special strain of B. licheniformis, described in more detail in British Patent Specification No.
Cellulases: Suitable cellulases include those of bacterial or fungal origin Chemically or genetically modified mu-tants are included. Suitable cellulases are disclosed in
Oxidoreductases: Any oxidoreductase suitable for use in a liquid composition, e.g., peroxidases or oxidases such as laccases, can be used herein. Suitable peroxidases herein include those of plant, bacterial or fungal origin. Chemically or genetically modified mutants are included. Examples of suitable peroxidases are those derived from a strain of Coprinus, e.g., C. cinerius or C. macrorhizus, or from a strain of Bacillus, e.g., B. pumilus, particularly peroxidase according to
Preferred oxidoreductases in the context of the invention are peroxidases (EC 1.11.1), laccases (EC 1.10.3.2) and glucose oxidases (EC 1.1.3.4)]. An Example of a commercially available, oxidoreductase is Gluzyme™ (enzyme available from Novozymes A/S). Further oxidoreductases are available from other suppliers. Preferred transferases are transferases in any of the following sub-classes:
The composition may be any composition, but particularly suitable composition are cleaning compositions, personal care compositions, textile processing compositions e.g. bleaching, pharmaceutical compositions, leather processing compositions, pulp or paper processing compositions, food and beverage compositions and animal feed compositions.
In a particular embodiment of the present invention the liquid composition is a liquid detergent, eg, laundry detergent or dishwashing detergent. In a more particular embodiment of the present invention the composition comprises a surfactant
The invention is further directed to the use of the liquid enzyme additive in liquid detergent composition.
The liquid composition may be a concentrated product to be added to liquid detergents. The amount of enzyme used in the liquid composition is thus very high. In a particular embodiment of the present invention the amount of enzyme present in the liquid composition is at least 1.5 g/L In a more particular embodiment of the present invention the amount of enzyme is at least 5 g/L. In an even more particular embodiment of the present invention the amount of enzyme present is at least 10 g/L. In a most particular embodiment of the present invention the amount of enzyme present is at least 20 g/L such as even above 25 g/L. In a particular embodiment the amount of enzyme does not exceed 200 g/L. In a more particular embodiment of the present invention the amount of enzyme does not exceed 150 g/L. In a most particular embodiment of the present invention the amount of enzyme present in the liquid composition is less than 100 g/L.
In a particular embodiment of the present invention the liquid composition has a pH of more than 7.5 In a more particular embodiment the pH of the liquid composition is at least 7 7. ln a most particular embodiment the pH of the liquid composition is at least 8.0.
In a most particular embodiment of the present invention the liquid composition contain one or more detergent builders. In a particular embodiment of the present invention the liquid composition comprises at least 1% w/w of detergent builders. In a more particular embodiment of the present invention the liquid composition comprises at least 2% w/w of detergent builders. In a most particular embodiment of the present invention the liquid composition comprises at least 5% w/w of detergent builders.
The present Invention is further described by the following examples which should not be construed as limiting the scope of the invention.
In a particular embodiment of the present invention the liquid composition is a liquid detergent composition. In a more particular embodiment of the present invention the liquid composition is a liquid detergent composition for dishwashing or for laundry.
The detergent composition of the invention may for example be formulated as a hand or machine laundry detergent composition including a laundry additive composition suitable for pretreatment of stained fabrics and a rinse added fabric softener composition, or be formulated as a detergent composition for use in general household hard surface cleaning operations, or be formulated for hand or machine dishwashing operations.
Examples of subtilisins are, those derived from Bacillus, e.g., subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168 (described in
Examples of useful are the variants described in
Preferred commercially available subtilisins include Alcalase™, Savinase™, Primase™, Duralase™, Esperase™, and Kannase™ (Novozymes A/S), Maxatase™, Maxacal™, Maxapem™, Properase™, Purafect™, Purafect OxP™, FN2™, and FN3™ (Genencor International Inc.).
Lipases: Suitable lipases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Example of useful lipases include lipases from Humicola (synonym Thermomyces), e.g. from H. lanuginosa (T. lanuginasus) as described in
Other examples are lipase variants such as those described in
Preferred commercially available lipase enzymes include Lipolase™ and Lipolase Ultra™ (Novozymes A/S).
Amylases: Suitable amylases (α and/or β) include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Amylases include, for example, α-amylases obtained from Bacillus, e.g. a special strain of B. licheniformis, described in more detail in
Examples of useful amylases are the variants described in
Commercially available amylases are Duramyl™, Termamyl™, Fungamyl™ and BAN™ (Novozymes A/S). Rapidase™ and Purastar™ (from Genencor International Inc.).
Cellulases: Suitable cellulases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Suitable cellulases include cellulases from the genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, e.g. the fungal cellulases produced from Humicola lnsolens, Myceliophthora thermophila and Fusarium oxysporum disclosed in
Especially suitable cellulases are the alkaline or neutral cellulases having colour care benefits. Examples of such cellulases are cellulases described in
Commercially available cellulases include Celluzyme™, and Carezyme™ (Novozymes A/S), Clazinase™, and Puradax HA™ (Genencor International Inc.), and KAC-500(B)™ (Kao Corporation).
Peroxidases/Oxidases: Suitable peroxidases/oxidases include those of plant, bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful peroxidases include peroxidases from Coprinus, e.g from C. cinereus, and variants thereof as those described in
Commercially available peroxidases include Guardzyme™ (Novozymes A/S).
The detergent enzyme(s) may be included in a detergent composition by adding separate additives containing one or more enzymes, or by adding a combined additive comprising all of these enzymes. A liquid detergent may be aqueous, typically containing up to 70% water and 0-30 % organic solvent, or non-aqueous.
The detergent composition comprises one or more surfactants, which may be non-ionic including semi-polar and/or anionic and/or cationic and/or zwitterionic. The surfactants are typically present at a level of from 0.1% to 60% by weight.
When included therein the detergent will usually contain from about 1 % to about 40% of an anionic surfactant such as linear alkylbenzenesulfonate, alpha-olefinsulfonate, alkyl sulfate (fatty alcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate, alpha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid or soap.
When included therein the detergent will usually contain from about 0.2% to about 40% of a non-ionic surfactant such as alcohol ethoxylate, nonylphenol ethoxylate, alkylpolyglycoside, alkyldimethylamineoxide, ethoxylated fatty acid monoethanolamide, fatty acid monoethanolamide, polyhydroxy alkyl fatty add amide, or N-acyl N-alkyl derivatives of glucosamine ("glucamides"). The detergent may contain 0-65 % of a detergent builder or complexing agent such as zeolite, diphosphate, triphosphate, phosphonate, carbonate, citrate, nitrilotriacetic add, ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid, alkyl- or alkenylsuccinic add, soluble silicates or layered silicates (e.g. SKS-6 from Hoechst).
The detergent may comprise one or more polymers. Examples are carboxymethylcellulose, poly(vinylpyrrolidone), poly (ethylene glycol), poly(vinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazole), polycarboxylates such as polyacrylates, maleic/acrylic acid copolymers and lauryl methacrylate/acrylic add copolymers.
The detergent may contain a bleaching system which may comprise a H2O2 source such as perborate or percarbonate which may be combined with a peracid-forming bleach activator such as tetraacetylethylenediamine or nonanoyloxybenzenesulfonate. Alternatively, the bleaching system may comprise peroxyacids of e g the amide, imide, or sulfone type.
The detergent may also contain other conventional detergent ingredients such as e.g. fabric conditioners including clays, foam boosters, suds suppressors, anti-corrosion agents, soil-suspending agents, anti-soil redeposition agents, dyes, bactericides, optical brighteners, hydrotropes, tarnish inhibitors, or perfumes.
The inhibition constant Ki = [E][I]/[EI] for the inhibition of Savinase was determined using standard methods under the following conditions:
The inhibition constant Ki = [E][I]/[EI] for the inhibition of Savinase was determined using standard methods under the following conditions:
The inhibition constant Ki was determined using Sigma Plot 9.0, Enzyme Kinetics Module 1.1
The inhibition constant Ki = [E][I]/[EI] for the inhibition of Savinase was determined using standard methods under the following conditions:
This list of references cited by the applicant is for the reader's convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.