Patent Description:
There is a permanent desire to improve the performance of automatic dishwashing compositions and their environmental profile.

Due to environmental concerns phosphate is increasingly being replaced by biodegradable complexing agents. These complexing agents can have a strong binding capacity for metals and/or are used in high levels and can negatively affect the performance of enzymes, in particular complexing agents can negatively affect proteases by extracting the structural calcium metal ions of the protease. The proteases can be affected in product and/or in-use. They can be more affected under fully built or overbuilt conditions, i.e., when a composition comprises high level of complexing agent and the composition is used in soft water because there will be more free builder to complex with the structural calcium metal ions of the protease. For the toughest items, consumers would usually select hot, long automatic dishwashing cycles. These cycles create a lot of stress on enzymes.

Automatic dishwashing compositions can be designed to have optimum performance under certain in-use conditions, for example a composition can be designed to have optimum performance in a soft water cycle, however a composition that has optimum performance in soft water might not have optimum performance in a hard water cycle and vice versa.

The object of the present invention is to provide a phosphate-free automatic dishwashing composition that provides better cleaning when used in soft water and preferably under different water hardness's. It is also desirable that the composition provides improved performance even under stressed conditions such as heavily soiled load washed in hot, long cycles.

<CIT> relates to variants of Bacillus gibsonii.

<CIT> relates to a phosphate-free automatic dishwashing cleaning composition comprising an organic complexing agent and a protease.

<CIT> relates to an automatic dishwashing detergent composition comprising an amylase and a protease.

<CIT> relates to LG12-clade protease enzymes.

According to the first aspect of the present invention, there is provided a phosphate-free automatic dishwashing cleaning composition. The composition comprises a complexing agent system and a novel protease. The composition presents improved cleaning performance on egg and sugary stains such as crème brule. The composition performs really well even when soft water is used in the automatic dishwashing process and even when a hot long cycle is used.

According to the second aspect of the invention there is provided a method of automatic dishwashing using soft water.

The elements of the composition of the invention described in connexion with the first aspect of the invention apply mutatis mutandis to the other aspects of the invention.

The present invention encompasses an automatic dishwashing cleaning composition. The composition is phosphate-free and comprises a complexing agent system and a protease. The composition delivers improved cleaning versus cleaning compositions comprising conventional proteases under a plurality of conditions. The composition provides good proteinaceous cleaning, in particular on egg and crème brulee soils. The invention also encompasses methods of automatic dishwashing with soft water and also methods of automatic dishwashing with soft water using hot, long cycles.

By "soft" water is herein meant water having a hardness of less than about <NUM> gpg (<NUM> ppm). Grain per gallon (gpg) is a unit of water hardness defined as <NUM> grain (<NUM> milligrams) of calcium carbonate dissolved in <NUM> gallon of water (<NUM>). It translates into <NUM> parts per million (ppm).

By "hot" cycle is herein understood a dishwashing program in which the main cycle is performed at a temperature above <NUM>, preferably above <NUM>.

By "long" cycle is herein understood a dishwashing program in which the main cycle has a duration of at least <NUM>, preferably at least <NUM> and more preferably at least <NUM> minutes and especially at least <NUM> minutes.

The composition of the invention comprises a variant protease, the variant proteases have a defined percentage of identity with respect to a reference protease identified by SEQ ID NO <NUM>).

The protease of the composition of the invention is herein sometimes referred to as "the protease of the invention". The proteases having any of sequences ID NO:<NUM> to <NUM> are herein sometimes referred to as "the reference protease".

The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity".

The term "variant" means a protease comprising a mutation, i.e., a substitution, insertion, and/or deletion, at one or more (e.g., several) positions relative to the reference protease. A substitution means replacement of the amino acid occupying a position with a different amino acid; a deletion means removal of the amino acid occupying a position; and an insertion means adding an amino acid adjacent to and immediately following the amino acid occupying a position. The variants of the present invention have at least <NUM>% identity with the reference protease.

The variant presents at least <NUM>%, more preferably at least <NUM>% identity with the protease of SEQ ID NO: <NUM>. SEQ ID NO: <NUM> corresponds to B. gibsonii-clade subtilisin Bgi02446. Also described herein, not part of the invention are variants with at least <NUM>%, more preferably at least <NUM>% identity with the protease of SEQ ID NO: <NUM>. SEQ ID NO: <NUM> corresponds to B. lentus subtilisin. Further described, not part of the invention are variants with at least <NUM>%, more preferably at least <NUM>% identity with one of the proteases of sequences SEQ ID NO: <NUM>-<NUM>.

The term "wild-type" protease means a protease expressed by a naturally occurring microorganism, such as a bacterium, yeast, or filamentous fungus found in nature.

In describing enzyme variants herein, the following nomenclature is used for ease of reference: Original amino acid(s): position(s): substituted amino acid(s).

According to this nomenclature, for instance the substitution of glutamic acid for glycine in position <NUM> is shown as G195E. A deletion of glycine in the same position is shown as G195*, and insertion of an additional amino acid residue such as lysine is shown as G195GK. Where a specific enzyme contains a "deletion" in comparison with other enzyme and an insertion is made in such a position this is indicated as *36D for insertion of an aspartic acid in position <NUM>. Multiple mutations are separated by pluses, i.e.: S99G+V102N, representing mutations in positions <NUM> and <NUM> substituting serine and valine for glycine and asparagine, respectively. Where the amino acid in a position (e.g. <NUM>) may be substituted by another amino acid selected from a group of amino acids, e.g. the group consisting of N and I, this will be indicated by V102N, I.

In all cases, the accepted IUPAC single letter or triple letter amino acid abbreviation is employed.

The numbering used in this patent is versus the sequences shown and not the BPN' numbering.

The relatedness between two amino acid sequences is described by the parameter "identity". For purposes of the present invention, the alignment of two amino acid sequences is determined by using the Needle program from the EMBOSS package (http://emboss. org) version <NUM>. The Needle program implements the global alignment algorithm described in <NPL>. The substitution matrix used is BLOSUM62, gap opening penalty is <NUM>, and gap extension penalty is <NUM>.

The degree of identity between an amino acid sequence of an enzyme used herein ("invention sequence") and a different amino acid sequence ("foreign sequence") is calculated as the number of exact matches in an alignment of the two sequences, divided by the length of the "invention sequence" or the length of the "foreign sequence", whichever is the shortest. The result is expressed in percent identity. An exact match occurs when the "invention sequence" and the "foreign sequence" have identical amino acid residues in the same positions of the overlap. The length of a sequence is the number of amino acid residues in the sequence.

The term "succinate based compound" and "succinic acid based compound" are used interchangeably herein.

The variant has at least <NUM>%, preferably at least <NUM>% identity with the amino acid sequence of SEQ ID NO:<NUM> and comprises the following substitutions: P54T, S126A, D127E and F128G.

The protease of the invention performs very well in phosphate-free compositions even when the compositions are used in soft water.

Preferred levels of protease in the composition of the invention include from about <NUM> to about <NUM>, more preferably from about <NUM> to about <NUM> of active protease per gram of the composition.

The automatic dishwashing cleaning composition can be in any physical form. It can be a loose powder, a gel or presented in unit dose form. Preferably it is in unit dose form, unit dose forms include pressed tablets and water-soluble packs. The automatic dishwashing cleaning composition of the invention is preferably presented in unit-dose form and it can be in any physical form including solid, liquid and gel form. The composition of the invention is very well suited to be presented in the form of a multi-compartment pack, more in particular a multi-compartment pack comprising compartments with compositions in different physical forms, for example a compartment comprising a composition in solid form and another compartment comprising a composition in liquid form. The composition is preferably enveloped by a water-soluble film such as polyvinyl alcohol. Especially preferred are compositions in unit dose form wrapped in a polyvinyl alcohol film having a thickness of less than <NUM>, preferably from <NUM> to <NUM>. The detergent composition of the invention weighs from about <NUM> to about <NUM> grams, preferably from about <NUM> to about <NUM> grams. This weight range fits comfortably in a dishwasher dispenser. Even though this range amounts to a low amount of detergent, the detergent has been formulated in a way that provides all the benefits mentioned herein above.

The composition is preferably phosphate free. By "phosphate-free" is herein understood that the composition comprises less than <NUM>%, preferably less than <NUM>% by weight of the composition of phosphate.

The composition of the invention is phosphate-free and comprises a complexing agent system.

For the purpose of this invention, a "complexing agent" is a compound capable of binding polyvalent ions such as calcium, magnesium, lead, copper, zinc, cadmium, mercury, manganese, iron, aluminium and other cationic polyvalent ions to form a water-soluble complex. The complexing agent has a logarithmic stability constant ([log K]) for Ca2+ of at least <NUM>. The stability constant, log K, is measured in a solution of ionic strength of <NUM>, at a temperature of <NUM>° C.

The composition of the invention comprises from <NUM>% to <NUM>% by weight of the composition of a complexing agent system. Preferably, the composition comprises a complexing agent selected from the group consisting of citric acid, methyl glycine diacetic acid (MGDA), glutamic-N,N-diacetic acid (GLDA), iminodisuccinic acid (IDS), carboxy methyl inulin, L-Aspartic acid N, N-diacetic acid tetrasodium salt (ASDA) and mixtures thereof. For the purpose of this invention, the term "acid", when referring to complexing agents, includes acid and salts thereof.

In a preferred embodiment, the composition comprises from <NUM>% to <NUM>% by weight of the invention of MGDA, more preferably the tri-sodium salt of MGDA. Compositions comprising this high level of MGDA perform well in soft water and also in long and/or hot cycles.

In a preferred embodiment, the composition comprises from <NUM>% to <NUM>% by weight of the invention of citric acid, more preferably sodium citrate. Compositions comprising citric acid perform well in soft water.

In a preferred embodiment, the complexing agent system comprises citric acid and MGDA preferably in a weight ratio of from about <NUM>:<NUM> to about <NUM>:<NUM>, more preferably from about <NUM>:<NUM> to about <NUM>:<NUM>.

Preferably, the composition of the invention comprises a dispersant polymer. A dispersant polymer can be used in any suitable amount from about <NUM> to about <NUM>%, preferably from <NUM> to about <NUM>%, more preferably from <NUM> to % by weight of the composition.

The dispersant polymer is capable to suspend calcium or calcium carbonate in an automatic dishwashing process.

The dispersant polymer has a calcium binding capacity within the range between <NUM> to <NUM> of Ca/g of dispersant polymer, preferably between <NUM> to <NUM> of Ca/g of dispersant polymer, more preferably <NUM> to <NUM> of Ca/g of dispersant polymer at <NUM>. In order to determine if a polymer is a dispersant polymer within the meaning of the invention, the following calcium binding-capacity determination is conducted in accordance with the following instructions:.

The calcium binding capacity referred to herein is determined via titration using a pH/ion meter, such as the Mettler Toledo SevenMulti™ bench top meter and a PerfectION™ comb Ca combination electrode. To measure the binding capacity a heating and stirring device suitable for beakers or tergotometer pots is set to <NUM>, and the ion electrode with meter are calibrated according to the manufacturer's instructions. The standard concentrations for the electrode calibration should bracket the test concentration and should be measured at <NUM>. A stock solution of <NUM>/g of Ca is prepared by adding <NUM> of CaCl<NUM>-<NUM><NUM>O into <NUM> of deionised water, then dilutions are carried out to prepare three working solutions of <NUM> each, respectively comprising <NUM>/g, <NUM>/g, and <NUM>/g concentrations of Calcium. The <NUM> Ca/g working solution is used as the initial concentration during the titration, which is conducted at <NUM>. The ionic strength of each working solution is adjusted by adding <NUM>/L of NaCl to each. The <NUM> of <NUM> Ca/g working solution is heated and stirred until it reaches <NUM>. The initial reading of Calcium ion concentration is conducted at when the solution reaches <NUM> using the ion electrode. Then the test polymer is added incrementally to the calcium working solution (at <NUM>/L intervals) and measured after <NUM> minutes of agitation following each incremental addition. The titration is stopped when the solution reaches <NUM>/g of Calcium. The titration procedure is repeated using the remaining two calcium concentration working solutions. The binding capacity of the test polymer is calculated as the linear slope of the calcium concentrations measured against the grams/L of test polymer that was added.

The dispersant polymer preferably bears a negative net charge when dissolved in an aqueous solution with a pH greater than <NUM>.

The dispersant polymer can bear also sulfonated carboxylic esters or amides, in order to increase the negative charge at lower pH and improve their dispersing properties in hard water. The preferred dispersant polymers are sulfonated / carboxylated polymers, i.e., polymer comprising both sulfonated and carboxylated monomers.

Preferably, the dispersant polymers are sulfonated derivatives of polycarboxylic acids and may comprise two, three, four or more different monomer units. The preferred copolymers contain:.

At least one structural unit derived from a carboxylic acid monomer having the general formula (III):
<CHM>
wherein R<NUM> to R<NUM> are independently selected from hydrogen, methyl, linear or branched saturated alkyl groups having from <NUM> to <NUM> carbon atoms, linear or branched mono or polyunsaturated alkenyl groups having from <NUM> to <NUM> carbon atoms, alkyl or alkenyl groups as aforementioned substituted with -NH2 or -OH, or -COOH, or COOR<NUM>, where R<NUM> is selected from hydrogen, alkali metal, or a linear or branched, saturated or unsaturated alkyl or alkenyl group with <NUM> to <NUM> carbons;.

Preferred carboxylic acid monomers include one or more of the following: acrylic acid, maleic acid, maleic anhydride, itaconic acid, citraconic acid, <NUM>-phenylacrylic acid, cinnamic acid, crotonic acid, fumaric acid, methacrylic acid, <NUM>-ethylacrylic acid, methylenemalonic acid, or sorbic acid. Acrylic and methacrylic acids being more preferred.

Optionally, one or more structural units derived from at least one nonionic monomer having the general formula (IV):
<CHM>.

Wherein R<NUM> to R<NUM> are independently selected from hydrogen, methyl, phenyl or hydroxyalkyl groups containing <NUM> to <NUM> carbon atoms, and can be part of a cyclic structure, X is an optionally present spacer group which is selected from -CH<NUM>-, -COO-, -CONH- or -CONR<NUM>-, and R<NUM> is selected from linear or branched, saturated alkyl radicals having <NUM> to <NUM> carbon atoms or unsaturated, preferably aromatic, radicals having from <NUM> to <NUM> carbon atoms.

Preferred non-ionic monomers include one or more of the following: butene, isobutene, pentene, <NUM>-methylpent-<NUM>-ene, <NUM>-methylpent-<NUM>-ene, <NUM>,<NUM>,<NUM>-trimethylpent-<NUM>-ene, <NUM>,<NUM>,<NUM>-trimethylpent-<NUM>-ene, cyclopentene, methylcyclopentene, <NUM>-methyl-<NUM>-methyl-cyclopentene, hexene, <NUM>,<NUM>-dimethylhex-<NUM>-ene, <NUM>,<NUM>-dimethylhex-<NUM>-ene, <NUM>,<NUM>-dimethylhex-<NUM>-ene, <NUM>,<NUM>-dimethylhex-<NUM>-ene, <NUM>,<NUM>-dimethylhex-<NUM>-ene, cyclohexene, methylcyclohexene, cycloheptene, alpha olefins having <NUM> or more carbon atoms such as, dec-<NUM>-ene, dodec-<NUM>-ene, hexadec-<NUM>-ene, octadec-<NUM>-ene and docos-<NUM>-ene, preferred aromatic monomers are styrene, alpha methylstyrene, <NUM>-methylstyrene, <NUM>-dodecylstyrene, <NUM>-ethyl-<NUM>-bezylstyrene, <NUM>-cyclohexylstyrene, <NUM>-propylstyrol, <NUM>-vinylnaphtalene, <NUM>-vinylnaphtalene; preferred carboxylic ester monomers are methyl (meth)acrylate, ethyl (meth)acrylate, propyl (meth)acrylate, t-butyl (meth)acrylate, pentyl (meth)acrylate, hexyl (meth)acrylate, <NUM>-ethylhexyl (meth)acrylate, octyl (meth)acrylate, lauryl (meth)acrylate, stearyl (meth)acrylate and behenyl (meth)acrylate; preferred amides are N-methyl acrylamide, N-ethyl acrylamide, N-t-butyl acrylamide, N-<NUM>-ethylhexyl acrylamide, N-octyl acrylamide, N-lauryl acrylamide, N-stearyl acrylamide, N-behenyl acrylamide.

Preferred sulfonated monomers include one or more of the following: <NUM>-acrylamido-<NUM>-propanesulfonic acid, <NUM>-acrylamido-<NUM>-propanesulfonic acid, <NUM>-acrylamido-<NUM>-methyl-<NUM>-propanesulfonic acid, <NUM>-methacrylamido-<NUM>-methyl-<NUM>-propanesulfonic acid, <NUM>- methacrylamido-<NUM>-hydroxy-propanesulfonic acid, allylsulfonic acid, methallylsulfonic acid, allyloxybenzenesulfonic acid, methallyloxybenzenesulfonic acid, <NUM>-hydroxy-<NUM>- (<NUM>-propenyloxy) propanesulfonic acid, <NUM>-methyl-<NUM>-propen-<NUM>-sulfonic acid, styrenesulfonic acid, vinylsulfonic acid, <NUM>-sulfopropyl, <NUM>-sulfo-propylmethacrylate, sulfomethacrylamide, sulfomethylmethacrylamide and mixtures of said acids or their water-soluble salts.

Preferably, the polymer comprises the following levels of monomers: from about <NUM> to about <NUM>%, preferably from about <NUM> to about <NUM>% by weight of the polymer of one or more carboxylic acid monomer; from about <NUM> to about <NUM>%, preferably from about <NUM> to about <NUM>% by weight of the polymer of one or more sulfonic acid monomer; and optionally from about <NUM>% to about <NUM>%, preferably from about <NUM> to about <NUM>% by weight of the polymer of one or more non-ionic monomer. An especially preferred polymer comprises about <NUM>% to about <NUM>% by weight of the polymer of at least one carboxylic acid monomer and from about <NUM>% to about <NUM>% by weight of the polymer of at least one sulfonic acid monomer.

Preferred commercial available polymers include: Alcosperse <NUM>, Aquatreat AR <NUM> and Aquatreat MPS supplied by Alco Chemical; Acumer <NUM>, Acumer <NUM>, Acusol <NUM> and Acusol <NUM> supplied by Rohm & Haas; Goodrich K-<NUM>, K-<NUM> and K-<NUM> supplied by BF Goodrich; and ACP <NUM> supplied by ISP technologies Inc. Particularly preferred polymers are Acusol <NUM> and Acusol <NUM> supplied by Rohm & Haas.

Suitable dispersant polymers include anionic carboxylic polymer of low molecular weight. They can be homopolymers or copolymers with a weight average molecular weight of less than or equal to about <NUM>,<NUM>/mol, or less than or equal to about <NUM>,<NUM>/mol, or less than or equal to about <NUM>,<NUM>/mol, or from about <NUM>,<NUM> to about <NUM>,<NUM>/mol, preferably from about <NUM>,<NUM> to about <NUM>,<NUM>/mol. The dispersant polymer may be a low molecular weight homopolymer of polyacrylate, with an average molecular weight of from <NUM>,<NUM> to <NUM>,<NUM>, particularly from <NUM>,<NUM> to <NUM>,<NUM>, and particularly preferably from <NUM>,<NUM> to <NUM>,<NUM>.

The dispersant polymer may be a copolymer of acrylic with methacrylic acid, acrylic and/or methacrylic with maleic acid, and acrylic and/or methacrylic with fumaric acid, with a molecular weight of less than <NUM>,<NUM>. Their molecular weight ranges from <NUM>,<NUM> to <NUM>,<NUM> and more preferably from <NUM>,<NUM> to <NUM>,<NUM> and in particular <NUM>,<NUM> to <NUM>,<NUM>/mol. and a ratio of (meth)acrylate to maleate or fumarate segments of from <NUM>:<NUM> to <NUM>:<NUM>.

The dispersant polymer may be a copolymer of acrylamide and acrylate having a molecular weight of from <NUM>,<NUM> to <NUM>,<NUM>, alternatively from <NUM>,<NUM> to <NUM>,<NUM>, and an acrylamide content of less than <NUM>%, alternatively less than <NUM>%, by weight of the dispersant polymer can also be used. Alternatively, such dispersant polymer may have a molecular weight of from <NUM>,<NUM> to <NUM>,<NUM> and an acrylamide content of from <NUM>% to <NUM>%, by weight of the polymer.

Dispersant polymers suitable herein also include itaconic acid homopolymers and copolymers. Alternatively, the dispersant polymer can be selected from the group consisting of alkoxylated polyalkyleneimines, alkoxylated polycarboxylates, polyethylene glycols, styrene co-polymers, cellulose sulfate esters, carboxylated polysaccharides, amphiphilic graft copolymers and mixtures thereof.

The composition of the invention preferably comprises from about <NUM> to about <NUM>%, more preferably from about <NUM> to about <NUM>% of bleach, preferably percarbonate, by weight of the composition.

Inorganic and organic bleaches are suitable for use herein. Inorganic bleaches include perhydrate salts such as perborate, percarbonate, perphosphate, persulfate and persilicate salts. The inorganic perhydrate salts are normally the alkali metal salts. The inorganic perhydrate salt may be included as the crystalline solid without additional protection. Alternatively, the salt can be coated. Suitable coatings include sodium sulphate, sodium carbonate, sodium silicate and mixtures thereof. Said coatings can be applied as a mixture applied to the surface or sequentially in layers.

Alkali metal percarbonates, particularly sodium percarbonate is the preferred bleach for use herein. The percarbonate is most preferably incorporated into the products in a coated form which provides in-product stability.

Potassium peroxymonopersulfate is another inorganic perhydrate salt of utility herein.

Typical organic bleaches are organic peroxyacids, especially dodecanediperoxoic acid, tetradecanediperoxoic acid, and hexadecanediperoxoic acid. Mono- and diperazelaic acid, mono- and diperbrassylic acid are also suitable herein. Diacyl and Tetraacylperoxides, for instance dibenzoyl peroxide and dilauroyl peroxide, are other organic peroxides that can be used in the context of this invention.

Further typical organic bleaches include the peroxyacids, particular examples being the alkylperoxy acids and the arylperoxy acids. Preferred representatives are (a) peroxybenzoic acid and its ring-substituted derivatives, such as alkylperoxybenzoic acids, but also peroxy-α-naphthoic acid and magnesium monoperphthalate, (b) the aliphatic or substituted aliphatic peroxy acids, such as peroxylauric acid, peroxystearic acid, ε-phthalimidoperoxycaproic acid[phthaloiminoperoxyhexanoic acid (PAP)], o-carboxybenzamidoperoxycaproic acid, N-nonenylamidoperadipic acid and N-nonenylamidopersuccinates, and (c) aliphatic and araliphatic peroxydicarboxylic acids, such as <NUM>,<NUM>-diperoxycarboxylic acid, <NUM>,<NUM>-diperoxyazelaic acid, diperoxysebacic acid, diperoxybrassylic acid, the diperoxyphthalic acids, <NUM>-decyldiperoxybutane-<NUM>,<NUM>-dioic acid, N,N-terephthaloyldi(<NUM>-aminopercaproic acid).

Bleach activators are typically organic peracid precursors that enhance the bleaching action in the course of cleaning at temperatures of <NUM>° C and below. Bleach activators suitable for use herein include compounds which, under perhydrolysis conditions, give aliphatic peroxoycarboxylic acids having preferably from <NUM> to <NUM> carbon atoms, in particular from <NUM> to <NUM> carbon atoms, and/or optionally substituted perbenzoic acid. Suitable substances bear O-acyl and/or N-acyl groups of the number of carbon atoms specified and/or optionally substituted benzoyl groups. Preference is given to polyacylated alkylenediamines, in particular tetraacetylethylenediamine (TAED), acylated triazine derivatives, in particular <NUM>,<NUM>-diacetyl-<NUM>,<NUM>-dioxohexahydro-<NUM>,<NUM>,<NUM>-triazine (DADHT), acylated glycolurils, in particular tetraacetylglycoluril (TAGU), N-acylimides, in particular N-nonanoylsuccinimide (NOSI), acylated phenolsulfonates, in particular n-nonanoyl- or isononanoyloxybenzenesulfonate (n- or iso-NOBS), decanoyloxybenzoic acid (DOBA), carboxylic anhydrides, in particular phthalic anhydride, acylated polyhydric alcohols, in particular triacetin, ethylene glycol diacetate and <NUM>,<NUM>-diacetoxy-<NUM>,<NUM>-dihydrofuran and also triethylacetyl citrate (TEAC). If present the composition of the invention comprises from <NUM> to <NUM>, preferably from <NUM> to <NUM>% by weight of the composition of bleach activator, preferably TAED.

The composition herein preferably contains a bleach catalyst, preferably a metal containing bleach catalyst. More preferably the metal containing bleach catalyst is a transition metal containing bleach catalyst, especially a manganese or cobalt-containing bleach catalyst.

Bleach catalysts preferred for use herein include manganese triazacyclononane and related complexes; Co, Cu, Mn and Fe bispyridylamine and related complexes; and pentamine acetate cobalt(III) and related complexes. Especially preferred bleach catalyst for use herein are <NUM>,<NUM>,<NUM>-trimethyl-<NUM>,<NUM>,<NUM>-triazacyclononane (Me-TACN) and <NUM>,<NUM>, <NUM>,<NUM>- tetramethyl-<NUM>,<NUM>,<NUM>-triazacyclononane (Me/Me-TACN).

Preferably the composition of the invention comprises from <NUM> to <NUM>, more preferably from <NUM> to <NUM>% of bleach catalyst by weight of the composition. Preferably the bleach catalyst is a manganese bleach catalyst.

The composition of the invention preferably comprises an inorganic builder. Suitable inorganic builders are selected from the group consisting of carbonate, silicate and mixtures thereof. Especially preferred for use herein is sodium carbonate. Preferably the composition of the invention comprises from <NUM> to <NUM>%, more preferably from <NUM> to <NUM>% and especially from <NUM> to <NUM>% of sodium carbonate by weight of the composition.

Surfactants suitable for use herein include non-ionic surfactants, preferably the compositions are free of any other surfactants. Traditionally, non-ionic surfactants have been used in automatic dishwashing for surface modification purposes in particular for sheeting to avoid filming and spotting and to improve shine. It has been found that non-ionic surfactants can also contribute to prevent redeposition of soils.

Preferably the composition of the invention comprises a non-ionic surfactant or a non-ionic surfactant system, more preferably the non-ionic surfactant or a non-ionic surfactant system has a phase inversion temperature, as measured at a concentration of <NUM>% in distilled water, between <NUM> and <NUM>, preferably between <NUM> and <NUM>. By a "non-ionic surfactant system" is meant herein a mixture of two or more non-ionic surfactants. Preferred for use herein are non-ionic surfactant systems. They seem to have improved cleaning and finishing properties and better stability in product than single non-ionic surfactants.

Phase inversion temperature is the temperature below which a surfactant, or a mixture thereof, partitions preferentially into the water phase as oil-swollen micelles and above which it partitions preferentially into the oil phase as water swollen inverted micelles. Phase inversion temperature can be determined visually by identifying at which temperature cloudiness occurs.

The phase inversion temperature of a non-ionic surfactant or system can be determined as follows: a solution containing <NUM>% of the corresponding surfactant or mixture by weight of the solution in distilled water is prepared. The solution is stirred gently before phase inversion temperature analysis to ensure that the process occurs in chemical equilibrium. The phase inversion temperature is taken in a thermostable bath by immersing the solutions in <NUM> sealed glass test tube. To ensure the absence of leakage, the test tube is weighed before and after phase inversion temperature measurement. The temperature is gradually increased at a rate of less than <NUM> per minute, until the temperature reaches a few degrees below the pre-estimated phase inversion temperature. Phase inversion temperature is determined visually at the first sign of turbidity.

Suitable nonionic surfactants include: i) ethoxylated non-ionic surfactants prepared by the reaction of a monohydroxy alkanol or alkyphenol with <NUM> to <NUM> carbon atoms with preferably at least <NUM> moles particularly preferred at least <NUM> moles, and still more preferred at least <NUM> moles of ethylene oxide per mole of alcohol or alkylphenol; ii) alcohol alkoxylated surfactants having a from <NUM> to <NUM> carbon atoms and at least one ethoxy and propoxy group. Preferred for use herein are mixtures of surfactants i) and ii).

Another suitable non-ionic surfactants are epoxy-capped poly(oxyalkylated) alcohols represented by the formula:.

R1O[CH2CH(CH3)O]x[CH2CH2O]y[CH2CH(OH)R2]     (I).

wherein R1 is a linear or branched, aliphatic hydrocarbon radical having from <NUM> to <NUM> carbon atoms; R2 is a linear or branched aliphatic hydrocarbon radical having from <NUM> to <NUM> carbon atoms; x is an integer having an average value of from <NUM> to <NUM>, more preferably about <NUM>; and y is an integer having a value of at least <NUM>, more preferably at least <NUM>.

Preferably, the surfactant of formula I, at least about <NUM> carbon atoms in the terminal epoxide unit [CH2CH(OH)R2]. Suitable surfactants of formula I, according to the present invention, are Olin Corporation's POLY-TERGENT® SLF-18B nonionic surfactants, as described, for example, in <CIT> by Olin Corporation.

The composition of the invention can comprise a protease in addition to the protease of the invention. A mixture of two or more proteases can contribute to an enhanced cleaning across a broader temperature, cycle duration, and/or substrate range, and provide superior shine benefits, especially when used in conjunction with an anti-redeposition agent and/or a sulfonated polymer.

Suitable proteases for use in combination with the variant proteases of the invention include metalloproteases and serine proteases, including neutral or alkaline microbial serine proteases, such as subtilisins (EC <NUM>. Suitable proteases include those of animal, vegetable or microbial origin. In one aspect, such suitable protease may be of microbial origin. The suitable proteases include chemically or genetically modified mutants of the aforementioned suitable proteases. In one aspect, the suitable protease may be a serine protease, such as an alkaline microbial protease or/and a trypsin-type protease. Examples of suitable neutral or alkaline proteases include:.

Especially preferred additional proteases for the detergent of the invention are polypeptides demonstrating at least <NUM>%, preferably at least <NUM>%, more preferably at least <NUM>%, even more preferably at least <NUM>% and especially <NUM>% identity with the wild-type enzyme from Bacillus lentus, comprising mutations in one or more, preferably two or more and more preferably three or more of the following positions, using the BPN' numbering system and amino acid abbreviations as illustrated in <CIT>:V68A, N76D, N87S, S99D, S99SD, S99A, S101G, S101M, S103A, V104N/I, G118V, G118R, S128L, P129Q, S130A, Y167A, R170S, A194P, V205I, Q206L/D/E, Y209W and/or M222S.

Most preferably the additional protease is selected from the group of proteases comprising the below mutations (BPN' numbering system) versus either the PB92 wild-type (SEQ ID NO:<NUM> in <CIT>) or the subtilisin <NUM> wild-type (sequence as per PB92 backbone, except comprising a natural variation of N87S).

Suitable commercially available additional protease enzymes include those sold under the trade names Alcalase®, Savinase®, Primase®, Durazym®, Polarzyme®, Kannase®, Liquanase®, Liquanase Ultra®, Savinase Ultra®, Ovozyme®, Neutrase®, Everlase®, Coronase®, Blaze®, Blaze Ultra® and Esperase® by Novozymes A/S (Denmark); those sold under the tradename Maxatase®, Maxacal®, Maxapem®, Properase®, Purafect®, Purafect Prime®, Purafect Ox®, FN3®, FN4®, Excellase®, Ultimase® and Purafect OXP® by Dupont; those sold under the tradename Opticlean® and Optimase® by Solvay Enzymes; and those available from Henkel/Kemira, namely BLAP (sequence shown in Figure29 of <CIT> with the following mutations S99D + S101 R + S103A + V104I + G159S, hereinafter referred to as BLAP), BLAP R (BLAP with S3T + V4I + V199M + V205I + L217D), BLAP X (BLAP with S3T + V4I + V205I) and BLAP F49 (BLAP with S3T + V4I + A194P + V199M + V205I + L217D); and KAP (Bacillus alkalophilus subtilisin with mutations A230V + S256G + S259N) from Kao.

Especially preferred for use herein in combination with the variant protease of the invention are commercial proteases selected from the group consisting of Properase®, Blaze®, Ultimase®, Everlase®, Savinase®, Excellase®, Blaze Ultra®, BLAP and BLAP variants.

Preferred levels of protease in the product of the invention include from about <NUM> to about <NUM>, more preferably from about <NUM> to about <NUM> and especially from about <NUM> to about <NUM> of active protease/g of composition.

Preferably the composition of the invention may comprise an amylase. Suitable alpha-amylases include those of bacterial or fungal origin. Chemically or genetically modified mutants (variants) are included. A preferred alkaline alpha-amylase is derived from a strain of Bacillus, such as Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus stearothermophilus, Bacillus subtilis, or other Bacillus sp. , such as Bacillus sp. NCBI <NUM>, NCBI <NUM>, NCBI <NUM>, DSM <NUM> (<CIT>) DSM <NUM>, DSMZ no. <NUM>, KSM AP1378 (<CIT>), KSM K36 or KSM K38 (<CIT>). Preferred amylases include:.

Preferably the amylase is an engineered enzyme, wherein one or more of the amino acids prone to bleach oxidation have been substituted by an amino acid less prone to oxidation. In particular it is preferred that methionine residues are substituted with any other amino acid. In particular it is preferred that the methionine most prone to oxidation is substituted. Preferably the methionine in a position equivalent to <NUM> in the AA560 enzyme listed as SEQ ID NO. <NUM> in <CIT> is substituted. Preferably, the methionine at this position is substituted with threonine or leucine, preferably leucine.

Suitable commercially available alpha-amylases include DURAMYL®, LIQUEZYME®, TERMAMYL®, TERMAMYL ULTRA®, NATALASE®, SUPRAMYL®, STAINZYME®, STAINZYME PLUS®, FUNGAMYL®, ATLANTIC®, INTENSA® and BAN® (Novozymes A/S, Bagsvaerd, Denmark), KEMZYM® AT <NUM> Biozym Biotech Trading GmbH Wehlistrasse 27b A-<NUM> Wien Austria, RAPIDASE® , PURASTAR9, ENZYSIZE®, OPTISIZE HT PLUS®, POWERASE®, PREFERENZ S® series (including PREFERENZ S1000® and PREFERENZ S2000® and PURASTAR OXAM® (DuPont. , Palo Alto, California) and KAM® (Kao, <NUM>-<NUM> Nihonbashi Kayabacho, <NUM>-chome, Chuo-ku Tokyo <NUM>-<NUM>, Japan). In one aspect, suitable amylases include ATLANTIC®, STAINZYME®, POWERASE®, INTENSA® and STAINZYME PLUS® and mixtures thereof.

Preferably, the product of the invention comprises at least <NUM>, preferably from about <NUM> to about <NUM>, more preferably from about <NUM> to about <NUM>, especially from about <NUM> to about <NUM> of active amylase/ g of composition.

Preferably, the protease and/or amylase of the composition of the invention are in the form of granulates, the granulates comprise more than <NUM>% of sodium sulfate by weight of the granulate and/or the sodium sulfate and the active enzyme (protease and/or amylase) are in a weight ratio of between <NUM>:<NUM> and <NUM>:<NUM> or preferably between <NUM>:<NUM> and <NUM>:<NUM> or more preferably between <NUM>:<NUM> and <NUM>:<NUM>.

Crystal growth inhibitors are materials that can bind to calcium carbonate crystals and prevent further growth of species such as aragonite and calcite.

Examples of effective crystal growth inhibitors include phosphonates, polyphosphonates, inulin derivatives, polyitaconic acid homopolymers and cyclic polycarboxylates.

Suitable crystal growth inhibitors may be selected from the group comprising HEDP (<NUM>-hydroxyethylidene <NUM>,<NUM>-diphosphonic acid), carboxymethylinulin (CMI), tricarballylic acid and cyclic carboxylates. For the purposes of this invention the term carboxylate covers both the anionic form and the protonated carboxylic acid form.

Cyclic carboxylates contain at least two, preferably three or preferably at least four carboxylate groups and the cyclic structure is based on either a mono- or bi-cyclic alkane or a heterocycle. Suitable cyclic structures include cyclopropane, cyclobutane, cyclohexane or cyclopentane or cycloheptane, bicyclo-heptane or bicyclo-octane and/or tetrhaydrofuran. One preferred crystal growth inhibitor is cyclopentane tetracarboxylate.

Cyclic carboxylates having at least <NUM>%, preferably <NUM>% of the carboxylate groups on the same side, or in the "cis" position of the 3D-structure of the cycle are preferred for use herein.

It is preferred that the two carboxylate groups, which are on the same side of the cycle are in directly neighbouring or "ortho" positions.

Preferred crystal growth inhibitors include HEDP, tricarballylic acid, tetrahydrofurantetracarboxylic acid (THFTCA) and cyclopentanetetracarboxylic acid (CPTCA). The THFTCA is preferably in the 2c,3t,4t,5c-configuration, and the CPTCA in the cis,cis,cis,cis-configuration. Especially preferred crystal growth inhibitor for use herein is HEDP.

Also preferred for use herein are partially decarboxylated polyitaconic acid homopolymers, preferably having a level of decarboxylation is in the range of <NUM> mole % to <NUM> mole %. Especially preferred polymer for use herein is Itaconix TSI® provided by Itaconix.

The crystal growth inhibitors are present preferably in a quantity from about <NUM> to about <NUM> %, particularly from about <NUM> to about <NUM> % and in particular, from <NUM> to <NUM> % by weight of the composition.

Metal care agents may prevent or reduce the tarnishing, corrosion or oxidation of metals, including aluminium, stainless steel and non-ferrous metals, such as silver and copper. Preferably the composition of the invention comprises from <NUM> to <NUM>%, more preferably from <NUM> to <NUM>% and especially from <NUM> to <NUM>% by weight of the product of a metal care agent, preferably the metal care agent is benzo triazole (BTA).

Glass care agents protect the appearance of glass items during the dishwashing process. Preferably the composition of the invention comprises from <NUM> to <NUM>%, more preferably from <NUM> to <NUM>% and specially from <NUM> to <NUM>% by weight of the composition of a metal care agent, preferably the glass care agent is a zinc containing material, specially hydrozincite. Other suitable glass care agents are polyethyleneimine (PEI). A particularly preferred PEI is Lupasol® FG, supplied by BASF.

The automatic dishwashing composition of the invention preferably has a pH as measured in <NUM>% weight/volume aqueous solution in distilled water at <NUM> of from about <NUM> to about <NUM>, more preferably from about <NUM> to less than about <NUM> and especially from about <NUM> to about <NUM>. The automatic dishwashing composition of the invention preferably has a reserve alkalinity of from about <NUM> to about <NUM>, more preferably from about <NUM> to about <NUM> at a pH of <NUM> as measured in NaOH with <NUM> grams of product at <NUM>.

A preferred automatic dishwashing composition of the invention comprises:.

Egg yolk removal by automatic dishwashing compositions comprising variant proteases were compared with the same compositions comprising the parent protease.

The compositions displayed in Table <NUM> were used. <NUM> of each composition were dissolved in a litre of deionized water to produce a cleaning solution having a pH of <NUM>. The corresponding protease was added to the cleaning solution at a level between <NUM>-<NUM>.

The cleaning performance of the proteases variants listed in Table <NUM> was tested relative to the parent using Automatic Dishwashing Compositions A, B and C (see Table <NUM>), as measured by the stain removal on egg yolk microswatches (PAS-<NUM>, Center for Testmaterials BV, Vlaardingen, Netherlands). The egg swatch stains were pre-sized to fit the microtiter plate (MTPs); standard <NUM> well plate. The stain removal of the PAS-<NUM> egg swatches was measured post wash versus a reference.

The MTPs were filled prior to protease addition with <NUM>/l of detergent and the detergent and deionized water.

After incubating the PAS-<NUM> swatches for <NUM> min at <NUM>, absorbance was read at <NUM> with a SpectraMax plate reader. Absorbance results were obtained by subtracting the value for a blank control (containing no protease) from each sample value (hereinafter "blank subtracted absorbance"). For each condition and variant, a performance index (PI) was calculated by dividing the blank subtracted absorbance by that of the parent protease at the same concentration. The value for the parent protease was determined from a standard curve of the parent protease which was included in the test and which was fitted to a Langmuir fit.

The protease activity of parent and subtilisin variants thereof was tested by measuring hydrolysis of N-suc-AAPF-pNA. The reagent solutions used for the AAPF hydrolysis assay were: <NUM> Tris/HCl pH <NUM>, containing <NUM>% TWEEN®-<NUM> (Tris dilution buffer); <NUM> Tris buffer pH <NUM>, containing <NUM> CaCl<NUM> and <NUM>% TWEEN®-<NUM> (Tris/Ca buffer); and <NUM> suc-AAPF-pNA in DMSO (suc-AAPF-pNA stock solution) (Sigma: S-<NUM>). A substrate working solution was prepared by adding <NUM> suc-AAPF-pNA stock solution to <NUM> Tris/Ca buffer and mixed well. An enzyme sample was added to a MTP (Greiner <NUM>) containing <NUM>/suc-AAPF-pNA working solution and assayed for activity at <NUM> over <NUM> with a SpectraMax plate reader in kinetic mode at room temperature (RT). The absorbance of a blank containing no protease was subtracted from each sample reading. The protease activity was expressed as mOD-min-<NUM>.

The stability of the variants described herein was measured by diluting the variants in stress buffer and measuring the proteolytic activity of the variants before and after a heat incubation step of <NUM> minutes at <NUM> using the AAPF assay described above. Stability was measured in Tris-EDTA (<NUM> Tris pH <NUM>; <NUM> EDTA; <NUM>% Tween <NUM>) buffered condition. % Residual activities were calculated by taking a ratio of the stressed to unstressed activity and multiplying by <NUM>.

Claim 1:
A phosphate-free automatic dishwashing cleaning composition comprising:
i) a protease wherein the protease is a variant having at least <NUM>% identity with the amino acid sequence of SEQ ID NO:<NUM> and comprising the following substitutions: P54T, S126A, D127E and F128G (using the SEQ ID NO: <NUM> numbering); and
ii) from <NUM> to <NUM>% by weight of the composition of a complexing agent system comprising from <NUM> to less than <NUM>% by weight of the composition of citric acid.