Classifications

• • C—CHEMISTRY; METALLURGY
  • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
  • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
  • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
  • C11D3/16—Organic compounds
  • C11D3/38—Products with no well-defined composition, e.g. natural products
  • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
  • C11D3/38618—Protease or amylase in liquid compositions only
• • C—CHEMISTRY; METALLURGY
  • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
  • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
  • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
  • C11D3/16—Organic compounds
  • C11D3/38—Products with no well-defined composition, e.g. natural products
  • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
  • C11D3/38645—Preparations containing enzymes, e.g. protease or amylase containing cellulase
• • C—CHEMISTRY; METALLURGY
  • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
  • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
  • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
  • C11D3/16—Organic compounds
  • C11D3/38—Products with no well-defined composition, e.g. natural products
  • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
  • C11D3/38654—Preparations containing enzymes, e.g. protease or amylase containing oxidase or reductase
• • C—CHEMISTRY; METALLURGY
  • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
  • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
  • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
  • C11D3/16—Organic compounds
  • C11D3/38—Products with no well-defined composition, e.g. natural products
  • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
  • C11D3/38663—Stabilised liquid enzyme compositions

Definitions

• the present invention relates, inter alia, to liquid enzyme compositions containing (i) one or more enzymes in from relatively high to very high concentration, and (ii) one or more appropriate enzyme inhibitors, more particularly reversible inhibitors, which exhibit strong enzyme-inhibitory properties and/or which are present at a concentration such as to cause strong enzyme inhibition.
• the invention further relates to:
• an enzyme-containing, multi-component composition such as a liquid composition, e.g. a cleaning composition such as a detergent composition, prepared using an inhibitor-stabilized liquid enzyme composition of the invention as the source of enzyme;
• an enzyme-containing, multi-component composition such as a cleaning composition, e.g. a detergent composition
• a cleaning composition e.g. a detergent composition
• the enz ⁇ me(s) in question is/are introduced in the form of an inhibitor-stabilized liquid enzyme composition of the invention, i.e. the enzymes(s) is/are in the form of an inhibitor-stabilized composition when incorporated into the multi-component composition;
• an enzyme-containing, multi-component composition such as a cleaning composition, e.g. a detergent composition, prepared by a process of the latter type.
• the invention moreover relates to the use of a liquid enzyme composition according to the invention in the preparation or manufacture of an enzyme- containing, multi-component composition, such as a cleaning composition, e.g. a detergent composition.
• a cleaning composition e.g. a detergent composition.
• boric acid and boronic acids are known to reversibly inhibit proteolytic enzymes.
• a discussion of the inhibition of a serine protease, subtilisin, by boronic acid is given in Molecular & Cellular Biochemistry 51 . 1983, pp. 5-32.
• 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 being the poorest inhibitor. However, boronic acids bearing aromatic groups such as phenyl, 4-methoxyphenyl or 3,5- dichlorophenyl are described as being very good inhibitors, with 3,5- dichlorophenylboronic acid as a particularly effective one (see Keller et al, Biochem. Biophvs. Res. Comm. 176. 1991 , pp. 401 -405).
• aryl boronic acids which are substituted at the 3-position of the aryl group relative to boron are unexpectedly good reversible protease inhibitors.
• acetamidobenzene boronic acid is described in the latter document as being a particularly effective inhibitor of proteolytic enzymes.
• IC inhibitor activity
• a pre-prepared enzyme composition containing enzyme(s) and appropriate (reversible) enzyme inhibitor(s). It will be particularly convenient and advantageous that the pre- prepared enzyme composition contains a very high concentration of at least one enzyme, and possibly of several enzymes of the type(s) in question, since this will, among other things, enable the preparation of large volumes or quantities of an enzyme-containing, multi-component composition using relatively small volumes or quantities of pre-prepared enzyme/inhibitor composition.
• the stability of the enzyme(s) for which an inhibitor is present will, in the nature of things, be expected to be high.
• numerous pre-prepared compositions of this type will be expected to be capable of storage for relatively prolonged periods of time after manufacture without significant - or at least without unacceptable - loss of enzymatic activity.
• the present invention thus relates to a liquid composition
• a liquid composition comprising: (i) an enzyme in an amount exceeding 40 ⁇ M; and (ii) a reversible enzyme inhibitor in an amount effective to enhance the storage stability of the enzyme in a multi- component composition (e.g. a detergent composition, such as a liquid detergent composition) into which the liquid composition is subsequently incorporated.
• a multi- component composition e.g. a detergent composition, such as a liquid detergent composition
• a liquid composition of the invention may, for example, be a predominantly aqueous composition, or a predominantly non-aqueous composition (e.g. a composition comprising as solvent a high proportion of one or more non-aqueous - but normally water-miscible - liquids, generally organic liquids (such as alcohols, glycols or the like). Liquid compositions of the invention will themselves normally be non-detergent compositions.
• compositions for cleaning dentures, contact lenses or hard surfaces e.g. in slaughterhouses, or in the food processing industry
• compositions for use in the leather industry e.g. for de-hairing and/or de-fatting of animal hides
• compositions for desizing textiles e.g. for de-hairing and/or de-fatting of animal hides
• compositions for washing denim textiles to achieve a "stone-washed" appearance e.g. for laundry washing, dishwashing and the like
• the use of such compositions for such purposes is within the scope of the invention.
• Enzyme classification numbers (EC numbers) referred to in the present specification with claims are in accordance with the Recommendations (1992) of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology. Academic Press Inc., 1992.
• a liquid composition of the invention contains at least one enzyme.
• Suitable enzymes include any commercially available enzyme. Particularly relevant enzymes include enzymes selected from the group consisting of proteases (i.e. peptidases, EC 3.4), amylases (classified under EC 3.2.1 ), lipases (including those classified under EC 3.1.1.3), cellulases (including EC 3.2.1.4) and oxidoreductases (EC 1 ), e.g. peroxidases (EC 1.1 1 ) and oxidases [including enzymes classified under EC 1 .10.3, such as laccases (EC 1 .10.3.2)], and any mixture thereof. Mixtures of enzymes from the same class (e.g. mixtures of different proteases, different lipases, etc.) are also included.
• proteases i.e. peptidases, EC 3.4
• amylases classified under EC 3.2.1
• lipases including those classified under EC 3.1.1.3
• cellulases including
• the amount(s) of enzyme(s) in the liquid composition will vary according to the type of enzyme(s) and the intended use of the liquid composition.
• any enzyme present will preferably be present in an amount in the range of 0.2-50% by weight (w/w) of the liquid composition (calculated on the basis of pure enzyme protein), often 0.5-25% w/w, such as 1-10% w/w, e.g. 2-8% w/w of the liquid composition.
• a preferred concentration range for an enzyme present in a liquid composition of the invention will be from about 50 ⁇ W ⁇ to about 20 mM, often from 100 ⁇ W ⁇ to 10 mM, such as from 500 ⁇ M to 5 mM, e.g.
• protease proteolytic enzyme
• Suitable proteases include those of animal, vegetable or microbial origin, especially microbial origin, as well as chemically produced or protein engineered (genetically engineered) mutants (variants) in which one or more amino acids have been substituted, inserted and/or deleted relative to the amino acid sequence of an enzyme of one of the latter types, and which exhibit proteolytic activity.
• the protease may, e.g., be a serine peptidase, preferably an alkaline microbial protease or a trypsin-like protease.
• alkaline proteases are subtilisins, especially those derived from Bacillus, e.g. subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168 (described in WO 89/06279).
• Examples of commercial Bacillus subtilisins are AlcalaseTM, SavinaseTM, EsperaseTM and DurazymTM products, all available from Novo Nordisk A/S.
• protease products such as AlcalaseTM, EsperaseTM and SavinaseTM
• AlcalaseTM EsperaseTM
• SavinaseTM are available as liquids (e.g. SavinaseTM 16.0 L, Type DX and Type EX) which are well suited Wide infra) to the preparation of protease-containing liquid compositions according to the present invention.
• trypsin-like proteases examples include trypsin (e.g. of porcine or bovine origin) and the Fusarium protease described in WO 89/06270.
• Amylases Any amylase (amylolytic enzyme) suitable for use in a liquid composition can be used. Suitable amylases include those of bacterial and fungal origin, as well as chemically produced or protein engineered (genetically engineered) mutants (variants) in which one or more amino acids have been substituted, inserted and/or deleted relative to the amino acid sequence of an enzyme of one of the latter types, and which exhibit amylolytic activity. Amylases include, for example, ⁇ -amylases (EC 3.2.1 .1 ), e.g. obtained from a particular strain of B. licheniformis. described in more detail in British Patent Specification No. 1 ,296,839.
• a very suitable ⁇ -amylase is TermamylTM, which is available (inter alia as a liquid product) from Novo Nordisk A/S.
• Lipases Any lipase (lipolytic enzyme) suitable for use in a liquid composition can be used. Suitable lipases include those of bacterial and fungal origin, as well as chemically produced or protein engineered (genetically engineered) mutants (variants) in which one or more amino acids have been substituted, inserted and/or deleted relative to the amino acid sequence of an enzyme of one of the latter types, and which exhibit lipolytic activity.
• a very suitable lipase is that obtained by cloning the gene from Humicola lanu ⁇ inosa and expressing the gene in Asper ⁇ illus orvzae. as described in EP 0 258 068, which is available (inter alia as a liquid product) from Novo Nordisk A/S under the tradename LipolaseTM.
• Cellulases Any cellulase (cellulolytic enzyme) suitable for use in a liquid composition can be used. Suitable cellulases include those of bacterial and fungal origin, as well as chemically produced or protein engineered (genetically engineered) mutants (variants) in which one or more amino acids have been substituted, inserted and/or deleted relative to the amino acid sequence of an enzyme of one of the latter types, and which exhibit cellulolytic activity. Suitable cellulases are disclosed, for example, in US 4,435,307. A very suitable cellulase is that produced by a strain of Humicola insolens. available from Novo Nordisk A/S under the tradename CelluzymeTM.
• Peroxidases Any peroxidase suitable for use in a liquid composition, e.g. liquid detergent composition, can be used. Suitable peroxidases herein include those of plant, bacterial and fungal origin, as well as chemically produced or protein engineered (genetically engineered) mutants (variants) in which one or more amino acids have been substituted, inserted and/or deleted relative to the amino acid sequence of an enzyme of one of the latter types, and which exhibit peroxidase activity. 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 a peroxidase according to PCT/DK90/00260.
• Coprinus e.g. C. cinerius or C. macrorhizus
• Bacillus e.g. B. pumilus
• Oxidases Any oxidase suitable for use in a liquid composition, e.g. liquid detergent composition, can be used.
• Suitable oxidases herein include those of bacterial and fungal origin, as well as chemically produced or protein engineered (genetically engineered) mutants (variants) in which one or more amino acids have been substituted, inserted and/or deleted relative to the amino acid sequence of an enzyme of one of the latter types, and which exhibit oxidase activity.
• suitable oxidases are laccases derived from strains of Aspergillus, Neurospora (e.g. N. crassa). Trametes (e.g. T. villosa) or Myceliophthora (e.g. M. thermophila).
• Enzyme inhibitors (enzvme stabilizers)
• a liquid composition according to the invention contains at least one reversible enzyme inhibitor, normally one which is an inhibitor for at least one enzyme present in the liquid composition.
• a liquid composition of the invention may, in addition to containing a first type of enzyme and a reversible inhibitor for that type of enzyme, contain a second, third... etc. type of enzyme (e.g. an enzyme of one of the types mentioned above) one or more of which is not accompanied by a reversible inhibitor therefor.
• liquid enzyme composition which contains one or more enzymes, in amounts as disclosed herein, but which instead of containing an inhibitor for one or more of these enzymes contains an inhibitor for another type of enzyme with which the liquid composition may subsequently be contacted.
• One example hereof would be a liquid composition containing, as the only enzyme, a lipase together with a protease inhibitor; the protease inhibitor would then protect the lipase with respect to degradation by a protease which - deliberately or inadvertently - may subsequently be brought into contact with the lipase.
• Protease inhibitors One particularly interesting class of reversible protease inhibitors is constituted by the boronic acids [R'B(OH) 2 ] and borinic acids [R'R"B(OH)] (where R' and R" are organic substituents, e.g. optionally substituted aryl or heterocyclic substituents), some examples of which have been mentioned above. Further examples of relevant compounds of this type may be selected among those mentioned in WO 92/19707, in EP 0 478 050 A1 and in EP O 51 1 456 A1.
• R- is an optionally substituted fused aromatic ring structure containing 14 or 18 carbon atoms in the ring, or an optionally substituted monocyclic or fused aromatic heterocyclic ring structure containing up to 17 carbon atoms in the ring, or an optionally substituted monocyclic or fused quinonoid ring structure containing up to 18 carbon atoms in the ring;
• R has the formula:
• X is the same or different and selected from hydrogen, C,-C e alkyl, substituted C,-C ⁇ alkyl, aryl, substituted aryl, hydroxy, hydroxyl derivative, halogen, amine, alkylated amine, amine derivative, nitro, thiol, thiol derivative, aldehyde, acid, acid salt, ester, sulfonate or phosphonate, and o, p and q may be the same or different and may each be 0, 1 or 2; m and n may be the same or different and may each be 0 or 1 ;
• R 3 is the same or different as R-, and selected from R, or R 3 is a hydroxyl group, or R, and R 3 are both optionally substituted monocyclic or dicyclic aromatic ring structures.
• optionally substituted ring structures are such that the substituents on the ring structure are freely chosen, but they are preferably selected from hydrogen, C,-C ⁇ alkyl, substituted alkyl, aryl, substituted aryl, hydroxy, hydroxyl derivative, halogen, amine, alkylated amine, amine derivative, nitro, thiol, thiol derivative, aldehyde, acid, acid salt, ester, sulfonate and phosphonate.
• Boronic and borinic acid derivatives may be prepared using methods well known to those skilled in the art, for example by using one of the following methods:
• Borinic acid derivatives may be prepared according to method b, above. However, the ratio of Grignard reagent to borate adopted is then 2: 1 .
• naphthalene boronic acids which are described in WO 95/29223 (which was unpublished at the priority date of the present application) and which comprise compounds having the following general formulas:
• R R 2 , R 3 , R 4 , R 5 , R 6 and R 7 are the same or different and are selected from hydrogen, C r C e alkyl, substituted C C 6 alkyl, aryl, substituted aryl, hydroxy, hydroxyl derivative, halogen, amine, alkylated amine, amine derivative, nitro, thiol, thiol derivative, aldehyde, acid, acid salt, ester, sulfonate and phos- phonate.
• Naphthalene boronic acid derivatives may be likewise be prepared using methods well known to those skilled in the art, for example by using a Grignard preparation as follows:
• the Grignard reagent is prepared by the slow dropwise addition of the appropriate bromonaphthalene starting material in sodium dried ether to magnesium turning in sodium dried ether. The reaction is promoted by the addition of a small iodine crystal.
• Trimethylborate or tri-n-butylborate in sodium dried ether is cooled to -70°C, and the Grignard reagent is added dropwise over a period of 2 hours while keeping the organoborate solution at -70°C and continuously agitating.
• the reaction mixture is allowed to warm to room temperature overnight, whereupon it is hydrolysed by the dropwise addition of cold dilute sulphuric acid.
• the ether layer is separated and the aqueous layer extracted with ether.
• the ether-containing fractions are combined and the solvent removed.
• the residue is made strongly alkaline, and any methanol or butanol so formed is removed.
• the alkaline solution is cooled and the resulting crystals of the desired boronic acid are removed by filtration. All products are preferably recrystallized from distilled water.
• naphthalene boronic acids may also be prepared using either direct lithiation of the naphthalene and/or lithiation of the bromide.
• boronic acids of interest in the context of the invention include the following: benzofuran-2- boronic acid; phenyl-boronic acid; 4-bromophenyl-boronic acid; 4-formylphenyl- boronic acid; 3-acetamidophenyl-boronic acid; 3,5-dichlorophenyl-boronic acid; 5-chlorothiophene-2-boronic acid; naphthalene-1 -boronic acid; naphthalene-2- boronic acid; and 6-hydroxynaphthalene-2-boronic acid.
• Further specific compounds of this type which are of interest in the context of the invention are mentioned in the table given in Example 2 herein (vide infra).
• Inhibitors of the boronic or borinic acid type may be introduced or incorporated into liquid compositions of the invention in the form of the acids themselves (e.g. dissolved in an appropriate water-miscible organic solvent, such as mono- propylene glycol or the like), or, if appropriate (e.g. for solubility reasons), as salts (e.g. alkali metal salts, such as sodium or potassium salts).
• an appropriate water-miscible organic solvent such as mono- propylene glycol or the like
• salts e.g. alkali metal salts, such as sodium or potassium salts.
• Reversible amylase inhibitors of possible relevance in the context of the invention may be found, e.g., among compounds of the so- called "acarbose" type (i.e. pseudo-oligosaccharides containing an acarviosine moiety and one or more maltose units).
• acarbose i.e. pseudo-oligosaccharides containing an acarviosine moiety and one or more maltose units.
• Other compounds of relevance as inhibitors for, in particular, ⁇ -amylases include maltose and maltotriose.
• Compounds such as methyl- ⁇ r-glucoside, as well as cycloamyloses, e.g. cyclohexa- and/or cycloheptaamylose may be of relevance as reversible inhibitors for ⁇ -amylases.
• Reversible cellulase inhibitors include 4-thiol-cellooligosaccharides [see, e.g., C. Schou et al., Journal of Carbohydrate Chemistry 12 (1993) pp. 743-752].
• Reversible lipase inhibitors include boronic and borinic acids selected among those described above and/or mentioned below (such as, e.g., optionally substituted phenyl boronic acids). Degree of enzyme inhibition
• the desired degree (strength, extent) of inhibition of an enzyme present in a liquid composition according to the invention will depend, among other things, on the purpose for which the composition is intended.
• the molar ratio of reversible inhibitor to enzyme will be chosen to be such that at least about one inhibitor molecule is present per active site of the enzyme in question, which will of course require a molar ratio of inhibitor (I) to enzyme (E) of at least 1 : 1 .
• a lower l:E molar ratio e.g. about 0.8: 1 , about 0.7: 1 , about 0.6:1 or even about 0.5:1 , may, however, be appropriate.
• a molar ratio of inhibitor to enzyme of at least 5, such as at least 1 5.
• l:E molar ratios of at least 50 or at least 100, or even higher ratios, may be appropriate.
• the molar ratio of inhibitor to enzyme will be chosen, inter alia, on the basis of considerations relating to the percentage of free (uninhibited) enzyme which it is desired to have present during the actual use of a multi-component, enzyme- containing composition based on a liquid composition of the invention.
• a satisfactorily high level of free enzyme e.g. a protease or a lipase
• the washing medium which is typically water to which the the detergent composition in question has been added.
• a parameter of importance is the inhibition constant (defined above) for reversible inhibition of an enzyme.
• K expressed in the conventional manner in mol/l (M)
• M mol/l
• a more desirable "window of inhibition" in connection with detergent enzymes will, however, often be such that an inhibitor(s) associated with a particular enzyme exerts an inhibition corresponding to about 3 • 10 7 M ⁇ K ⁇ ⁇ 1 • 10 3 M, such as 4.3 • 10 '7 M ⁇ K, ⁇ 4.5 * 10 "4 M.
• preferred enzyme-containing liquid compositions of the invention are compositions in which the ratio between: (a) the total molar concentration (which may be denoted [l] t ) of a reversible enzyme inhibitor, I, present in the composition; and
• the inhibition constant (K-; expressed in mol/l) for inhibition of an enzyme which is present in the composition and which is reversibly inhibited by the inhibitor in question; is at least 50 (i.e. [I] t /K; > 50), such as at least 100 or, frequently, at least 250.
• a practical upper limit for the ratio [l] t /Kj in liquid compositions of the invention will normally be about 10000 (i.e. 10 4 ). For many purposes, a value for the ratio [l] t /Kj in the range of 250-5000, often in the range of 250-2500, will be appropriate.
• the value of the ratio [l] t /Kj may be regarded as a measure of the "inhibitory capacity" of a liquid composition of the invention.
• a high value - and thereby a high “inhibitory capacity" - may be achieved, for example, by incorporating, in a liquid composition, a relatively modest to low concentration of a strongly inhibiting inhibitor, or by incorporating a relatively high concentration of a more weakly inhibiting inhibitor.
• liquid compositions according to the invention comprising enzyme(s) and reversible enzyme inhibitor(s) may be dried by appropriate methods (e.g. by lyophilization or by spray-drying.
• the dried enzyme/inhibitor product may be then be comminuted (e.g. by milling) and suspended or slurried at an appropriate concentration in a non-aqueous liquid vehicle, e.g. a non-ionic surfactant (such as SoftanolTM from BP), to form a slurry product.
• a non-aqueous liquid vehicle e.g. a non-ionic surfactant (such as SoftanolTM from BP)
• a typical goal will be attainment of at least 50% inhibition of a chosen enzyme (e.g. a protease) in the detergent composition per s_e, and an amount of free enzyme in the washing medium corresponding to at least 50% of the total amount of that enzyme.
• a chosen enzyme e.g. a protease
• a detergent composition incorporating a liquid composition of the invention will, apart from enzyme(s) and inhibitor(s), comprise a surfactant, and will normally be a liquid detergent composition.
• the detergent composition may, e.g., be a laundry detergent composition or a dishwashing detergent composition.
• a liquid detergent composition may be aqueous, e.g. typically containing up to 70% of water and 0-30% of organic solvent, or substantially non-aqueous.
• the detergent composition will comprise one or more surfactants, each of which may be anionic, nonionic, cationic, or amphoteric (zwitterionic).
• the detergent will usually contain 0-50% of anionic surfactant such as linear alkylbenzene- sulfonate (LAS), alpha-olefinsulfonate (AOS), alkyl sulfate (fatty alcohol sulfate) (AS), alcohol ethoxysulfate (AEOS or AES), secondary alkanesulfonates (SAS), alpha-sulfo fatty acid methyl esters, alkyl- or alkenylsuccinic acid, or soap.
• anionic surfactant such as linear alkylbenzene- sulfonate (LAS), alpha-olefinsulfonate (AOS), alkyl sulfate (fatty alcohol sulfate) (AS), alcohol ethoxysulfate (AEOS or AES), secondary alkanesulf
• nonionic surfactant such as alcohol ethoxylate (AEO or AE), alcohol propoxylate, carboxylated alcohol ethoxylates, nonylphenol ethoxylate, alkylpolyglycoside, alkyldimethylamine oxide, ethoxylated fatty acid monoethanolamide, fatty acid monoethanolamide, or polyhydroxy alkyl fatty acid amide (e.g. as described in WO 92/06154).
• the detergent contains 1 -65% of a detergent builder, but some dishwashing detergents may contain even up to 90% of a detergent builder, or complexing agent such as zeolite, diphosphate, triphosphate, phosphonate, citrate, nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTMPA), alkyl- or alkenylsuccinic acid, soluble silicates or layered silicates (e.g. SKS-6 from Hoechst).
• zeolite diphosphate, triphosphate, phosphonate, citrate, nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTMPA), alkyl- or alkenylsuccinic acid, soluble silicates or layered silicates (e.g. SKS-6 from Hoechst).
• the detergent builders may be subdivided into phosphorus-containing and non- phosphorous-containing types.
• phosphorus-containing inorganic alkaline detergent builders include the water-soluble salts, especially alkali metal pyrophosphates, orthophosphates, polyphosphates and phosphonates.
• non-phosphorus-containinginorganic builders include water-soluble alkali metal carbonates, borates and silicates as well as layered disilicates and the various types of water-insoluble crystalline or amorphous alumino silicates of which zeolites is the best known representative.
• suitable organic builders include alkali metal, ammonium or substituted ammonium salts of succinates, malonates, fatty acid malonates, fatty acid sulphonates, carboxymethoxy succinates, polyacetates, carboxylates, polycarboxylates, aminopolycarboxylates and polyacetyl carboxylates.
• the detergent may also be unbuilt, i.e. essentially free of detergent builder.
• the detergent may comprise one or more polymers.
• examples are carboxymethylcellulose (CMC), poly(vinylpyrrolidone) (PVP), polyethyleneglycol (PEG), poly. vinyl alcohol) (PVA), polycarboxylates such as polyacrylates, polymaleates, maleic/acrylic acid copolymers and lauryl methacrylate/acrylic acid copolymers.
• the detergent composition may contain bleaching agents of the chlorine/bromine- type or the oxygen-type.
• the bleaching agents may be coated or encapsulated.
• examples of inorganic chlorine/bromine-type bleaches are lithium, sodium or calcium hypochlorite or hypobromite as well as chlorinated trisodium phosphate.
• the bleaching system may also comprise a H 2 O 2 source such as perborate or percarbonate which may be combined with a peracid-forming bleach activator such as tetraacetylethylenediamine (TAED) or nonanoyloxybenzenesulfonate (NOBS).
• TAED tetraacetylethylenediamine
• NOBS nonanoyloxybenzenesulfonate
• organic chlorine/bromine-type bleaches are heterocyclic N-bromo and N-chloro imides such as trichloroisocyanuric, tribromoisocyanuric, dibromoisocyanuric and dichloroisocyanuric acids, and salts thereof with water solubilizing cations such as potassium and sodium.
• Hydantoin compounds are also suitable.
• the bleaching system may also comprise peroxyacids of, e.g., the amide, i ide, or sulfone type.
• oxygen bleaches are preferred, for example in the form of an inorganic persalt, preferably with a bleach precursor or as a peroxy acid compound.
• suitable peroxy bleach compounds are alkali metal perborates, both tetrahydrates and monohydrates, alkali metal percarbonates, persilicates and perphosphates.
• Preferred activator materials are TAED or NOBS.
• the enzymes of the detergent composition of the invention are incorporated in the detergent composition in the form of a liquid enzyme/inhibitor composition according to the invention.
• enzyme-stabilizing substances may be incorporated, e.g. a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid, boric acid, or a boric acid derivative such as an aromatic borate ester (see, e.g., WO 92/19709 and WO 92/19708).
• the detergent may also contain other conventional detergent ingredients such as, e.g., fabric conditioners including clays, deflocculant material, foam boosters/foam depressors (in dishwashing detergents foam depressors), suds suppressors, anti-corrosion agents, soil-suspending agents, anti-soil-redeposition agents, dyes, dehydrating agents, bactericides, optical brighteners, or perfume.
• fabric conditioners including clays, deflocculant material, foam boosters/foam depressors (in dishwashing detergents foam depressors), suds suppressors, anti-corrosion agents, soil-suspending agents, anti-soil-redeposition agents, dyes, dehydrating agents, bactericides, optical brighteners, or perfume.
• the pH (measured in aqueous solution at use concentration) will usually be neutral or alkaline, e.g. in the range of 7-1 1 .
• laundry detergent compositions within the scope of the invention include:
• An aqueous liquid detergent composition comprising
• Alcohol ethoxylate e.g. C 12 . 15 alcohol, 7 EO or C 12 . 15 alcohol, 5 EO
• Polymers e.g. PVP, PEG 0 - 3%
• Enzymes (calculated as pure enzyme 0.0001 - 0.2% protein)
• An aqueous structured liquid detergent composition comprising
• Linear alkylbenzenesulfonate (calculated as acid) 1 5 - 21 %
• Alcohol ethoxylate e.g. C 12 . 15 alcohol, 7
• Polymers e.g. PEG, PVP 0 3%
• Anchoring polymers such as, e.g., lauryl methacrylate/acrylic acid copolymer; molar 0 3% ratio 25: 1 ; MW 3800
• Enzymes (calculated as pure enzyme pro ⁇ 0.0001 - 0.2% tein)
• An aqueous liquid detergent composition comprising
• Alcohol ethoxysulfate e.g. C 12 . 15 alcohol, 2-3 EO 8 15%
• Alcohol ethoxylate e.g. C 12 15 alcohol, 7
• Soap as fatty acid e.g. lauric acid 0 3%
• Hydrotrope e.g. sodium toluensulfonate 2 6%
• Enzymes (calculated as pure enzyme pro ⁇ 0.0001 - 0.2% tein)
• An aqueous liquid detergent composition comprising
• Alcohol ethoxylate e.g. C 12 . 15 alcohol, 7 EO, or C 12 , 15 alcohol, 5 EO
• Polymer e.g. maleic/acrylic acid copolymer, anchoring polymer such as, e.g., lauryl methacrylate/acrylic acid copolymer 0 3%
• Enzymes (calculated as pure enzyme 0.0001 - 0.2% protein)
• a detergent composition formulated as a non-aqueous detergent liquid comprising a liquid nonionic surfactant such as, e.g., linear alkoxylated primary alcohol, a builder system (e.g. phosphate), enzyme and alkali.
• a liquid nonionic surfactant such as, e.g., linear alkoxylated primary alcohol, a builder system (e.g. phosphate), enzyme and alkali.
• the detergent may also comprise anionic surfactant and/or a bleach system.
• Dishwashing and other multi-component, enzyme-containing compositions examples include the following: 1 ) LIQUID DISHWASHING COMPOSITION WITH CLEANING SURFACTANT SYSTEM, COMPRISING:
• TED Tetraacetylethylenediamine
• Liquid nonionic surfactant e.g. alcohol ethoxylates 2.0 - 10.0%
• Liquid carrier selected from higher glycols, polyglycols, polyoxides, glycolethers 25.0 - 45.0%
• Stabilizer e.g. a partial ester of phosphoric acid and a C 1 ⁇ -C 18 alkanol
• Foam suppressor e.g. silicone 0 1 .5%
• Liquid nonionic surfactant e.g. alcohol ethoxylates 2.0 - 10.0%
• Stabilizing system e.g. mixtures of finely divided silicone and low molecular weight dialkyl polyglycol ethers
• Clay gel thickener e.g. bentonite 0.0 - 10.0%
• Liquid carrier selected from higher lycols, polyglycols, polyoxides and glycol ethers Balance 4) THIXOTROPIC LIQUID AUTOMATIC DISHWASHING COMPOSITION COMPRISING:
• Aluminium tristearate 0 0.1 %
• TED Tetraacetylethylenediamine
• the manganese catalyst may, e.g., be one of the compounds described in "Efficient manganese catalysts for low-temperature bleaching", Nature 369. 1994, pp. 637-639.
• the "enzymes" content of the above formulations may include the content of enzyme inhibitor(s) present.
• a particular formulation encompassed within one of the types recited above may contain an organic solvent of a type which is not specifically mentioned above in connection with that type of formulation, but which has functioned, e.g., as a solubilizing solvent for an inhibitor present in a liquid enzyme/inhibitor composition of the invention which has been incorporated in the particular formulation in question.
• a liquid enzyme/inhibitor composition of the invention of the invention may be incorporated in a detergent composition so as to give an enzyme concentration which is conventionally employed in detergents. It is at present contemplated that, for a detergent composition of the invention, a liquid enzyme/inhibitor composition of the invention may, e.g., be incorporated in an amount which - for a given enzyme present in the enzyme/inhibitor composition - corresponds to an amount of enzyme in the range 0.00001 -1 mg (calculated as pure enzyme protein) of enzyme per liter of wash liquor. Tests of Stabilizers
• inhibitory effectiveness of an inhibitor may be tested in various ways, illustrated here by the following two tests for the case of a protease/protease inhibitor:
• Liquid enzyme/inhibitor composition is added to a liquid detergent formulation which is stored under well-defined conditions.
• the enzyme activity of each enzyme is determined as a function of time (e.g. after 1 , 3, and 7 days).
• D P and D L are denatured (i.e. non-active) protease and lipase.
• reaction rate constants are derived from storage stability data by the use of a parameter estimation method (Gauss- Newton with the Levenberg modification).
• the storage stability data give the concentration of (P + PI) and L as a function of time.
• Reaction III is much faster than the other reactions and equilibrium is assumed in the calculations.
• Reaction IV is excluded from the system to reduce the number of parameters thereby describing the stability of the inhibited enzyme by only one reaction rate constant (from equation V).
• the concentration of inhibitor may be assumed, as a reasonable approximation, to be constant.
• reaction rate constants are somewhat sensitive to small variations in the data, but the sensitivity is reduced significantly by giving the results relative to the value for boric acid.
• boric acid as well as closely related or equivalent substances such as alkali metal borates (e.g.
• borax and boric oxide] for the purpose of inhibiting, in particular, proteases is well known (see, e.g., EP 0 451 924 A2), but the low inhibitory strength in combination with the generally rather poor solubility of such substances (which is further illustrated for boric acid in the working examples herein; vide infra) renders them generally unsuitable for use in the context of the present invention [in which the inhibitor(s) and/or amount(s) thereof present in a liquid composition of the invention should be such as to still give rise to good enzyme stabilization when the liquid composition is incorporated as a component of a multi- component composition].
• compounds such as boric acid, alkali metal borates (e.g. borax) and boric oxide will generally be excluded as inhibitors in the context of the present invention.
• An “improvement factor” may be defined as follows:
• IF defined in this manner thus provides a measure of the inhibition efficiency of a given inhibitor relative to boric acid.
• K The inhibition constant K
• the inhibition constant K may be determined by using standard methods; for reference see Keller et al, Biochem. Biophys. Res. Comm. 176. 1991 , pp.401 -405; J. Bieth in Baver-Svmposium "Proteinase Inhibitors”. pp. 463-469, Springer-Verlag, 1974, and Lone Kierstein Hansen in "Deter ⁇ mination of Specific Activities of Selected Detergent Proteases using Protease Activity. Molecular Weights. Kinetic Parameters and Inhibition Kinetics". PhD thesis, Novo Nordisk A/S and University of Copenhagen, 1991 . The determination may, if desired, be performed in the presence of non-enzyme components (surfactant(s), etc.) of a detergent composition.
• Conditions appropriate for determining K, values for inhibition of enzymes will typically be achieved by employing a buffer system which provides a suitable pH and which does not react or form complexes with the enzyme(s) or inhibitor(s) in question.
• a suitable buffer system for many enzyme/inhibitor interactions will, for example, be a glycyl-glycine buffer at a mildly alkaline pH, e.g. pH 8.6, and at ambient temperature (typically 25°C).
• K jf for the inhibition of the protease SavinaseTM were determined using standard methods under the following conditions:
• Succinyl-Alanine-Alanine-Proline-Phenylalanine-para-nitro-anilide SAAPFpNA (from Sigma, catalogue No. S-7388).
• the initial rate of substrate hydrolysis was determined at nine substrate concentrations in the range of 0.01 to 2 mM using a Cobas Fara automated spectrophotometer.
• the concentration of active enzyme [E was determined by active site titration using very strongly binding protein-type protease inhibitors.
• Inhibition constants K were calculated from plots of K m /k c ⁇ t as a function of the concentration of inhibitor. The inhibitors were assumed to be 100% pure, and the molar concentrations were determined from weighed amounts and molecular weights.
• the resulting values of the inhibition constants Kj for a series of boronic acid enzyme stabilizers (inhibitors) tested are listed below, together with values of the ratio [l] t /Ki for some of these inhibitors.
• KNPU Kero Novo Protease Units
• KLU Kelo Lipase Units
• One KNPU corresponds to 2.53 mg (ca. 9.4 * 10 '5 mmol) of pure enzyme protein.
• a pamphlet AF 220/1 - GB (available on request from Novo Nordisk A/S, Bagsvaerd, Denmark), concerning determination of SavinaseTM activity.
• One Lipase Unit is defined as the amount of enzyme which, under standard conditions (30.0°C, pH 7.0; with Gum Arabic as emulsifier and tributyrin as substrate) liberates 1 //mol of titrable butyric acid per minute.
• a pamphlet AF 95/5) describing this analytical method in more detail is available upon request from Novo Nordisk A/S, Bagsvaerd, Denmark.
• KLU corresponds to 0.2 mg (ca. 6.7 - 10 '6 mmol) of pure enzyme protein.
• Second test series For the first test series, equal parts by weight of inhibitor solution and of SavinaseTM 16.0 L, Type EX (liquid protease preparation containing 16 KNPU of protease per gram; Novo Nordisk A/S, Bagsvaerd, Denmark) were mixed, giving SavinaseTM 8 L EX preparations each containing 2% w/w of inhibitor.
• Second test series For the second test series, SavinaseTM 16.0 L EX, 4% w/w inhibitor in MPG, and pure MPG were mixed in a weight ratio of 50:37.5: 12.5, respectively, giving SavinaseTM 8 L EX preparations each containing 1 .5% w/w of inhibitor.
• composition of detergent base I (US tvoe):
• NansaTM 1 169/p 10.3 Linear Alkylbenzene Sulfonate, LAS
• BerolTM 452 3.5 Alkyl Ether Sulfate, AES
• composition of detergent base II is composition of detergent base II:
• composition of detergent base III is composition of detergent base III:
• OMOTM Micro was a retail product purchased in a Danish supermarket. The enzyme content therein was inactivated by treatment in a microwave oven (85°C, 5 minutes).
• liquid enzyme/inhibitor compositions according to the invention are as follows (all compositions were solutions of acceptable appearance):
• this composition has a molar inhibitor:enzyme (l:E) ratio of about 33.
• this composition has a molar l:E ratio of about 108.

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• 1996
  • 1996-01-09 EP EP96900086A patent/EP0802968A1/en not_active Withdrawn
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