CA2049084A1 - Detergent compositions containing subtilisin mutants - Google Patents

Abstract

C 6127 (R) ABSTRACT OF THE DISCLOSURE

The subject invention relates to mutant subtilisin proteases having substitution in at least 1 amino acid residue and to their use in structured detergent compositions in view of the enhanced stability they provide. In one embodiment of the invention, the enhanced enzyme stability in structured liquids comprising a decoupling polymer is shown and in a second embodiment, the enhanced stability of the mutant enzyme in a structured liquid comprising perborate is shown.
None of these effects would have been predicted in advance.

Description

C 6127 (R) STRUCTURED LIOUID DETERGENT COMPOSITIONS
CONTAINING SUBTILISIN MUTANTS

BACKGROUND AND PRIOR ART
This invention relates to structured liquid detergent com-positions containing mutant protease enzymes which provide enhanced stability.

The modification of subtilisin proteases by substitution at an amino acid site is known in the art. US-A-4 760 025 (Genencor), for example, claims subtilisin mutants with amino acid substitutions at amino acid sites 32, 155, 104, 222, 166, 64, 33, 169, 217 or 157 which are different from subtilisins naturally produced by B. amyloliquefaciens. These amino acid substitutions are said to lead to increased oxidation stability of the protease.

WO 87/04461, assigned to Amgen, discloses the substitution in Bacillus subtilisins of alternative amino acids (i.e. serine, valine, threonine, cysteine, glutamine and isoleucine) for ASN, GLY or ASN-GLY sequences (specifically at position 218).
These mutations are said to increase the stability of the enzyme at high temperatures or over a broader pH range than the wild type. WO 88/08033, also to Amgen, claims mutations which modify calcium-binding capacity (to replace an amino acid with a negatively charged residue such as ASP or GLU) and optionally a deletion and/or replacement of either residue of ASN-GLY sequences which results in better pH and thermal stability and higher specific activities. The reference claims that sites 41, 75, 76, 77, 78, 79, 80, 81, 208, and 214 may be replaced by a negatively charged amino acid and ASN may be replaced by SER, VAL, THR, CYS, GLU, or ILE in ASN-GLY sequences.

EP-A-342 177 (Procter & Gamble) discloses compositions comprising a protease with a speci~ic mutation. The liquid 2 C ~127 (R) compositions do not appear to be structured liquids as contemplated by the subject invention.

These references do not disclose structured liquid detergent compositions comprising the mutants of the subject invention or the advantages provided by the use of these mutants in these detergent compositions.

US-A-4 980 288 (Genex) discloses the subtilisin mutants which are used in the liquid detergent compositions of the invention. Although the use of mutants in some washing preparations is disclosed (Claims 6 and 7), there is no teaching of the use of these mutants in structured detergent compositions and no recognition of the advantages provided by the use of the mutants in such compositions Specifically, US-A-~ 980 288 discloses the use of a specific mutant (i.e. GX7150) in heavy duty liquid WISK (trade mark).
There is no teaching of what specific composition is used and no teaching that mutant enzymes may be used in structured liquid compositions. Further, there is no teaching or suggestion that mutant enæymes are effective in structured detergent compositions containing bleaches.

Accordingly, it is an object of the invention to show that mutant enzymes as described herein can be effectively used in structured liquids generally, in structured liquids comprising decoupling polymers and in structured liquids comprising active bleach compounds.
SUMMARY OF THE INVENTION
The subject invention provides liquid detergent compositions which are structured with detergent active material and, optionally, dissolved electrolyte, which comprise a detergent active structure existing as a separate phase within a predominantly aqueous phase wherein said composition comprises:

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3 C 6127 ~R) (1) from about 5~ to about 65% by weight detergent-active;
(2) from 0% to 50% by weight builder;
(3) from 0 to 30% by weight electrolyte;

(4) a mutant subtilisin protease added in sufficient quantity to have an activity level of 0.01 to 100,000 GU/g having substitutions in 1 or more amino acid residues compared to the wild type or commercially available subtilisin; and (5) the remainder water and minor ingredients, wherein the composition contains a sufficient amount of detergent active material and, optionally, sufficient dissolved electrolyte to result in a structure of a lamellar droplets dispersed in a continuous aqueous phase.
The pH of the detergent composition may range from about 7 to about 13, preferably 8 to about 11.

According to the invention, when certain modified mutant subtilisin proteases are used in the structured liquid detergent compositions of the invention, enhanced stability is observed. The effectiveness of such mutants in structured liquids could not have been predicted in advance.

In one embodiment of the invention, the structured liquid comprises a deflocculating polymer which can be used to allow incorporation of greater amounts of actives and electrolytes while keeping viscosity down and not affecting stability.
Once again, it could not have been predicted in advance that mutant enzymes would perform effectively in structured compositions comprising a deflocculating polymer.

~n a second embodiment of the invention, the structured liquid comprises a bleach active. The enhanced activity of a mutant enzyme in a bleach containing structured liquid could not have been predicted in advance.

4 C 6127 (R) BRIEF DESCRIPTION OF THE FIGURES
Figure 1 shows a plot of viscosity against lamellar phase volume fraction for a typical structured composition described herein below.
S

DETAILED DESCRIPTION OF THE INVENTION
The compositions which comprise the mutant proteases of the invention are compositions in which particles of solid material can be suspended by a structure formed from detergent-active material, the detergent-active existing as a separate phase dispersed within predominately aqueous phase.
This aqueous phase contains dissolved electrolyte.

Three common product forms of this type are liquids for heavy duty fabrics washing, liquid abrasive and general purpose cleaners. In the first class, the suspen~ed solid can be substantially the same as the dissolved electrolyte, being an excess of same beyond the solubility limit. This solid is usually present as a detergency builder, i.e. to counteract the effects of calcium ion water hardness in the wash. In addition, it may be desirable to suspend substantially insoluble particles of bleach, for example diperoxydodecanoic acid (DPDA). In the second class, the suspended solid is usually a particulate abrasive, insoluble in the system. In that case the electrolyte is a different, water-soluble material, present to contribute to structuring of the active material in the dispersed phase. In certain cases, the abrasive can, however, comprise partially soluble salts which dissolve when the product is diluted. In the third class, the structure is usually used for thickening products to give consumer-preferred flow properties, and sometimes to suspend pigment particles. Compositions of the first kind are described, for example, in our patent specification EP-A-38 101, whilst examples of those in the second category are described in our specification EP-A-140 452. Those in the third category are, for example, described in US-A-4 244 840.

C 6127 (R) More specifically, the present invention is concerned with aqueous liquid detergent compositions which contain sufficient detergent-active material and, optionally, sufficiently dissolved electrolyte to result in a structure of lamellar droplets dispersed in a continuous aqueous phase.

Lamellar droplets are a particular class of surfactant structures which, inter alia, are already known from a variety of references, e.g. H.A. Barnes, "Detergents", Ch.2.
in K. Walters (Ed), "Rheometry: Industrial Applications'l, J.
Wiley & Sons, Letchworth 1980.

Such lamellar dispersions are used to endow properties such as consumer-preferred flow behaviour and/or turbid appearance. Many are also capable of suspending particulate solids such as detergency builders or abrasive particles.
Examples of such structured liquids without suspended solids are given in US-A-4 244 840, whilst examples where solid particles are suspended are disclosed in specifications EP-A-160 342; EP-A-38 101; EP-A-104 452 and also in the aforementioned US-A-4 244 840. Others are disclosed in EP-A-151 ~84, where the lamellar droplet are called 'spherulites'.

The presence of lamellar droplets in a liquid detergent product may be detected by means known to those skilled in the art, for examp:Le optical techniques, various rheometrical measurements. X-ray or neutron diffraction, and electron microscopy.
The lamellar droplets consist of an onion like configuration of concentric hi-layers of surfactant molecules, between which is trapped water or electrolyte solution (aqueous phase). Systems in which such droplets are close-packed provide a very desirable combination of physical stability and solid-suspending properties with useful flow properties.

6 C 6127 (R) In general, the viscosity and stability of the product depend on the volume fraction of the liquid which is occupied by the droplets. Generally speaking, the higher the volume fraction of the dispersed lamellar phase (droplets), the better the stability. However, higher volume fractions also lead to increased viscosity which in the limit can result in an unpourable product. This results in a compromise being reached. When the volume fraction is around 0.6 or higher, the droplets are just touching (space filling). This allows reasonable stability with an acceptable viscosity ( so no more than 2.5 Pas, preferably no more than 1 Pas at a shear rate of 21 s-1). This volume fraction also endows useful solid-suspending properties. Conductivity measurements are known to provide a useful way of measuring the volume fraction, when compared with the conductivity of the continuous phase.

Figure 1 shows a plot of viscosity against lamellar phase volume fraction for a typical composition of known kind:
Surfactants* 20 Sodium formate 5 or 7.5 Sodium citrate. 2 aq 10 Bora~ 3-5 25 Tinopal CBS-X 0.1 Perfume 0.15 Water Balance * (1) Sodium linear alkylbenzene sulfonate;
(2) Lauryl ether sulphate; and (3) C12-C13 ethoxylated alcohol, 6EO

It will be seen that there is a window bounded by lower volume fraction of 6.7 corresponding to the onset of instability and an upper volume fraction of 0.83 or 0.9 corresponding to a viscosity of 1 Pas or 2 Pas, respectively.

7 C 6127 (R) This is only one such plot and in many cases the lower volume fraction can be 0.6 or slightly lower.

A complication factor in the relationship between stability and viscosity on the one hand and volume fraction of the lamellar droplets is the degree of flocculation of the droplets. When flocculation occurs between the lamellar droplets at a given volume fraction, the viscosity of the corresponding product will increase owing to the formation of a network throughout the liquid. Flocculation may also lead to instability because deformation of the lamellar droplets, owing to flocculation, will make their packing more efficient. Consequently, more lamellar droplets will be required for stabilization by the space- filling mechanism, which will again lead to a further increase of the viscosity.

The volume fraction of droplets is increased by increasing the surfactant concentration and flocculation between the lamellar droplets occurs when a certain threshold value of the electrolyte concentration is crossed at a given level of surfactant (and fixed ratio between any different surfactant components). Thus, in practice, the effects referred to above mean that there is a limit to the amount of surfactant and electrolyte which can be incorporated whilst still having an acceptable product. In principle, higher surfactant levels are required for increased detergency (cleaning performance).
Increased electrolyte levels can also be used for better detergency, or are sometimes sought for secondary benefits such as building.
The dependency of stability and/or viscosity upon volume fraction can be favourably influenced by incorporating a deflocculating polymer comprising a hydrophilic backbone and one or more hydrophobic side-chains.

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8 C 6127 (R) As indicat~d above, one embodiment of the invention is the use of the mutant enzymes in structured liquids containing such a decoupling polymer.

The deflocculating polymer allows, if desired, the incorporation of greater amounts of surfactants and/or electrolytes than would otherwise be compatible with the need for a stable, low-viscosity product. It also allows (if desired) incorporation of greater amounts of certain other ingredients to which, in general, lamellar dispersions have been highly stability-sensitive. Further details of these are given hereinbelow.

The use of the decoupling polymer thus allows formulation of stable, pourable products wherein the volume fraction of the lamellar phase is 0.5, 0.6 or higher, but with combinations or concentrations of ingredients not possible hitherto.

The volume fraction of the lamellar droplet phase may be determined by the following method. The composition is centrifuged, say at 40,000 G for 12 hours, to separa~e the composition into a clear (continuous aqueous) layer, a turbid active-rich (lamellar) layer and (if solids are suspended) a solid particle layer. The conductivity of the continuous aqueous phase, the lamellar phase and of the total composition before centrifugation are measured. From these, the volume fraction of the lamellar phase is calculated, using the Bruggeman equation, as disclosed in American Physics, 2~, 636 (1935). When applying the equation, the conductivity of the total composition must be corrected for the conductivity inhibition owing to any suspended solids present. The degree of correction necessary can be determined by measuring the conductivity of a model system. This has the formulation of the total composition but without any surfactant. The difference in conductivity of the model system, when continuously stirred (to disperse the solids) and at rest (so the solids set11e), indicates the effect of g C 6127 (R) suspended solids in the real composition. Alternatively, the real composition may be subjected to mild centrifugation (say 2,000 G for 1 hour) to just remove the solids. The conductivity of the upper layer is that of the suspending base (aqueous continuous phase with dispersed lamellar phase, minus solids).

It should be noted that, if the centrifugation at 40,000 G
fails to yield a separate continuous phase, the conductivity of the aforementioned model system at rest can serve as the conductivity of the continuous aqueous phase. For the conductivity of the lamellar phase, a value of 0.8 can be used, which is typical for most systems. In any event, the contribution of this term in the equation is often negligible.

Preferably, the viscosity of the aqueous continuous phase is less than 25 mPas, most preferably less than 15 mPas, especially less than 10 mPas, these viscosities being measured using a capillary viscometer, for example an Ostwald viscometer.

The deflocculating polymer used in the structured composition of the invention is fully described in U.S. Serial No.
365,080, filed June 12, 1989 (assigned to the same assignee as the present invention) and this application is hereby incorporated by reference.

The various ingredients within the structured compositions of the invention is set forth below:

Deterqent-Active The compositions of the invention comprise a detergent-active material usually incorporated in liquid detergent formulations.

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C 6127 (R) The active detergent material may be an alkali metal or alkanolamine soap or a 10 to 24 carbon atom fatty acid, including polymerized fatty acids, or an anionic, nonionic, cationic, zwitterionic or amphoteric synthetic detergent material, or mixtures of any of these.

Examples of anionic synthetic detergents are salts (including sodium, potassium, ammonium and substituted ammonium salts) such as mono-, di- and triethanolamine salts of 9 to 20 carbon alkylbenzene sulphonates, 8 to 22 carbon primary or secondary alkane sulphonates, 8 to 24 carbon olefin sulphonates, sulphonated polycarboxylic acids prepared by sulphonation of the pyrolyzed product of alkaline earth metal citrates, e.g., as described in GB-A-l 082 179, 8 to 22 carbon alkylsulphates, 8 to 24 carbon alkylpolyglycol-ether-sulphates, -carboxylates and -phosphates (containing up to 10 moles of ethylene oxide); further examples are described in "Surface Active Agents and Detergents" (Vol. I and II) by Schwartz, Perry and Berch. Any suitable anionic may be used and the examples are not intended to be limiting in any way.

Examples of nonionic synthetic detergents are the condensation products of ethylene oxide, propylene oxide and/or butylene oxide with 8 to 18 carbon alkylphenols, 8 to 18 carbon primary or secondary aliphatic alcohols, 8 to 18 carbon fatty acid amides; further examples of nonionics include tertiary amine oxides with ~ to 18 carbon alkyl chain and two l to 3 carbon alkyl chains. The above reference also describes further examples of nonionics.
The average number of moles of ethylene oxide or propylene oxide present in the above nonionics varies from 1-30;
mixtures of various nonionics, including mixtures of nonionics with a lower and a higher degree of alkoxylation, may also be used.

11 C 6127 (R) Examples of cationic detergents are the quaternary ammonium compounds such as alkyldimethylammonium halogenides.

Examples of amphoteric or zwitterionic detergents are N-alkylamino acids, sulphobetaines, condensation products of fatty acids with protein hydrolysates; but owing to their relatively high costs they are usually used in combination with an anionic or a nonionic detergent. Mixtures of the various types of active detergents may also be used, and preference is given to mixtures of an anionic and a nonionic detergent-active. Soaps (in the form of their sodium, potassium and substituted ammonium salts) of fatty acids may also be used, preferably in conjunction with an anionic and/or nonionic synthetic detergent.
Builders Builders which can be used according to this invention include conventional alkaline detergency builders, inorganic or organic, which can be used at levels from 0% to about 50%
by weight of the composition, preferably from 1% to about 30%
by weight, most preferably from about 20% to about 30%.

Examples of suitable inorganic alkaline detergency builders are water-soluble alkalimetal phosphates, polyphosphates, borates, silicates and also carbonates. Specific examples of such salts are sodium and potassium triphosphates, pyrophosphates, orthophosphates, hexametaphosphates, tetraborates, silicates and carbonates.

Examples of suitable organic alkaline detergency builder salts are: (1) water-soluble amino polycarboxylates, e.g.
sodium and potassium ethylenediaminetetraacetates, nitrilotriacetates and N-(2-hydroxyethyl)-nitrilo-diacetates; (2) water-soluble salts of phytic acid, e.g., sodium and potassium phytates (see US-A-2 379 942); (3) water-soluble polyphosphonates, including specifically, sodium, potassium and lithium salts of ethane-l-hydroxy-2~90~
12 C 6127 (R) l,l-diphosphonic acid; sodium, potassium and lithium salts of methylene diphosphonic acid; sodium, potassium and lithium salts of ethylene diphosphonic acid; and sodium, potassium and lithium salts of ethane-1,1,2-triphosphonic acid. Other examples include the alkali metal salts of ethane-2-carboxy-l,ldiphosphonic acid hydroxymethanediphosphonic acid, carboxyldiphosphonic acid, ethane-l-hydroxy-1,1,2-tri-phosphonic acid, ethane-2-hydroxy-1,1,2-triphosphonic acid, propane-1,1,3,3-tetraphosphonic acid, propane-1,1,2,3-tetraphosphonic acid, and propane- 1,2,2,3-tetraphosphonic acid; (4) water-soluble salts of polycarboxylate polymers and co-polymers as described in US-A-3 308 067.

In addition, polycarboxylate builders can be used satisfactorily, including water-soluble salts of mellitic acid, citric acid, and carboxymethyloxysuccinic acid and salts of polymers of itaconic acid and maleic acid. Certain zeolites or aluminosilicates can be used. One such aluminosilicate which is useful in the compositions of the invention is an amorphous water-insoluble hydrated compound of the formula Nax(yAlO2.SiO2), wherein x is a number from 1.0 to 1.2 and y is l, said amorphous material being further characterized by an Mg++ exchange capacity of from about 50 mg eq. CaC03/g and a particle diameter of from ahout 0.01 micron to about 5 microns. This ion-exchange builder is more fully described in GB-A-1 470 250.

A second water-insoluble synthetic aluminosilicate ion-exchange material useful herein is crystalline in nature and has the formula Naz[(AlO2)y.(SiO2)]xH2O, wherein z and y are integers of a least 6; the molar ratio of z to y is in the range from 1.0 to about 0.5, and x is an integer from about 15 to about 264; said aluminosilicate ion-exchange material having a particle size diameter from about 0.1 micron to about 100 microns; a calcium ion-exchange capacity on an anhydrous basis of at least about 200 milligrams equivalent of CaCO3 hardness per gram; and a calcium exchange rate on an 13 C 6127 (R) anhydrous basis of at least about 2 grams/gallon/minute/gram.
These synthetic aluminosilicates are more fully described in GB-A-l 429 143.

Electrolyte The compositions of the present invention require sufficient electrolyte to cause the formation of a lamellar phase by the soap/surfactant to endow solid suspending capability. The selection of the particular type(s) and amount of electrolyte to bring this into being for a given choice of soap/
surfactant is effected using methodology very well known to those skilled in the art. It utilizes the particular techniques described in a wide variety of references. One such technique entails conductivity measurements. The detection of the presence of such a lamellar phase is also very well known and may be effected by, for example, optical and electron microscopy or x-ray diffraction, supported by conductivity measurement.

As used herein, the term electrolyte means any water- soluble salt. The amount of electrolyte should be sufficient to cause formation of a lamellar phase by the soap/surfactant to endow solid suspending capability. Preferably, the composition comprises at least 1.0% by weight, more preferably at least 5.0% by weight, most preferably at least 17.0% by weight of electrolyte. The electrolyte may also be a detergency builder, such as the inorganic builder sodium tripolyphosphate, or it may be a non-functional electrolyte such as sodium sulphate or chloride. Preferably, the inorganic builder comprises all or part of the electrolyte.

The compositions of the invention are capable of suspending particulate solids, although particularly preferred are those systems where such solids are actually in suspension. The solids may be undissolved electrolyte, the same as or different from the electrolyte in solution, the latter being saturated in electrolyte. Additionally or alternatively, they ~J~ 3~
14 C 6127 (R) may be materials which are substantially insoluble in water alone. Examples of such substantially insoluble materials are aluminosilicate builders and particles of calcite abrasive.

Mutant Subtilisin Protease Proteins exist in a dynamic equilibrium between a folded, ordered state and an unfolded, disordered state. This equilibrium in part reflects the short range interactions between the different segments of the polypeptide chain which tend to stabilize the protein's structure, and, on the other hand, those thermodynamic forces which tend to promote the randomization of the molecule.

The largest class of naturally occurring proteins is made up of enzymes. Each enzyme generally catalyses a different kind of chemical reaction, and is usually highly specific in its function. Enzymes have been studied to determine correlations between the three-dimensional structure of the enzyme and its activity or stability.
The amino acid sequence of an enzyme determines the characteristics of the enzyme, and the enzyme's amino acid sequence is specified by the nucleotide sequence of a gene coding for the enzyme. A change of the amino acid sequence of an enzyme may alter the enzyme's properties to varying degrees, or may even inactivate the enzyme, depending on the location, nature and/or magnitude of the change in the amino acid sequence.
. .
Although there may be slight variations in a distinct type of naturally occurring enzyme within a given species or organism, enzymes of a specific type produced by organisms of the same species generally are substantially identical with respect to substrate specificity, thermal stability, activity levels under various conditions (e.g. temperature and pH), oxidation stability, and the like. Such characteristics of a naturally occurring or "wild-type~' enzyme are not necessarily C 6127 (R) optimized for utilization outside of the natural environment of the enzyme. It may thus be desirable to alter a natural characteristic of an enzyme to optimize a certain property of the enzyme for a specific use, or for use in a specific environment.

Amino acids are naturally occurring compounds that are the building blocks of proteins. The natural amino acids are usually abbreviated to either three letters or one letter.
The most common amino acids, and their symbols, are given in Table 1. The amino acids are joined head to tail to form a long main chain. Each kind of amino acid has a different side group.

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16 C 6127 (R) Table l. Amino acid names and abbreviations ________________________________________________________ Amino acid Three letter code Single letter code _____________________________________________ __________ S Alanine Ala A
Arginine Arg R
Aspartic acid Asp D
Asparagine Asn N
Cysteine Cys C
10 Glutamic acid Glu E
Glutamine Gln Q
Glycine Gly G
Histidine His H
Isoleucine Ile 15 Leucine Leu L
Lysine Lys K
Methionine Met M
Phenylalanine Phe F
Proline Pro P
20 Serine Ser S
Threonine Thr T
Tryptophan Trp W
Tyrosine Tyr Y
Valine Val V

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17 C 6127 (R) All amino acids have the same atoms in the main chain and differ only in the side chains. The main-chain atoms are a nitrogen, two carbons, and one oxygen. The first atom is the nitrogen, called N. The next atom is a carbon and is called the alpha-carbon. Side groups are attached to this alpha-carbon. The alpha-carbon is connected to the carbonyl carbon which is called C. C is connected to the carbonyl oxygen (called O) and to the N of the next residue. The side group atoms are given names composed of the symbol for the element (C, O, N, S), a Greek letter (alpha, beta, gamma, delta, epsilon, zeta and eta), and perhaps an Arabic numeral if the side group is forked.

The subtilisin enzymes used in the detergent compositions of this invention have been modified by m~tating the various nucleotide sequences that code for the enzymes. Use of the modified subtilisin enzymes provides enhanced stability in the compositions.

The subtilisin enzymes of this invention belong to a class of enzymes known as proteases. A protease is a catalyst for the cleavage of peptide bonds. An example of this cleavage is given below:

/ \ / \ /
H N H
H H H
+H20 prote~se R I ; 1 2 , c~{ fQ c H OH H-N N
H H H

18 C 6127 (R) one type of protease is a serine protease. A serine protease will catalyze the hydrolysis of peptide bonds in which there is an essential serine residue at the active site. Serine proteases can be inhibited by phenylmethyl sulphonylflu.oride and by diisopropylfluoro-phosphate.

A subtilisin is a serine protease produced by Gram positive bacteria or by fungi. The amino acid sequences of seven subtilisins are known. These include five subtilisins from Bacillus strains (subtilisin BPN', subtilisin Carlsberg, subtilisin DY, subtilisin amylosacchariticus~ and mesenticopeptidase). (Vasantha et al., "Gene for alkaline protease and neutral protease from Bacillus amyloliguefaciens contain a large open-reading frame between the regions coding for signal sequence and mature protein", J. Bacteriol.
159_811-819 (1984); Jacobs et al., "Cloning sequencing and expression of subtilisin Carlsberg form Bacillus licheniformis", Nucleic Acids Res. 13:8013-8926 (1985);
Nedkov et al., "Determination of the comple~e amino acid sequence of subtilisin DY and its comparison with the primary structures of the subtilisin BPN', Carlsberg and amylosacchariticus", Biol. Chem. Hoppe-Seyler 366:421-430 (1985); Kurihara et al., "Subtilisin amylosacchariticus", J. Biol. Chem. 247:5619-5631 (1972); and Svendsen et al., "Complete amino acid sequence of alkaline mesenteri.co-peptidase", FEBS Let. 196:228-232 (1986)).

The amino acid sequence of the subtilisin thermitase from Thermoactinomyces vulqaris is also known. (Meloun et al., "Complete primary structure of thermitase from Thermoactinomyces vulgarls and its structural features related to the subtilisin-type proteases", FEBS Lett.
183:195-200 (1985). The amino acid sequences from two fungal proteinases are known: Proteinase K from Tritirachium album (Jany et al., "proteinase K from Tritirachium album Limber", Biol. Chem. Hoppe-Seyler 366:485-492 (1985)) and thermomycolase from the thermophilic fungus, Malbranchea 1~ C 6127 (R) pulchella (Gaucher et al., "Endopeptidases: Thermomycolin", Methods Enzymol. 45:415~433 (1976)).

These enzymes have been shown to be related to subtilisin BPN', not only through their primary sequences and enzymological properties, but also by comparison of ~-ray crystallographic data. (McPhalen et al., "Crystal and molecular structure of the inhibitor eglin from leeches in complex with subtilisin Carlsberg", ~EBS Lett. 188:55 58 (1985) and Pahler et al., "Three-dimensional structure of fungal proteinase K reveals similarity to bacterial subtilisin", EMB0 J. 3:1311-1314 (1984).) The mutated enzymes used in the compositions of the invention may be introduced into any serine protease which has at least 50% and preferably 80% amino acid sequence homology with the sequence referenced above for subtilisin BPN', subtilisin Carlsberg, subtilisin DY, subtilisin amylosacchariticus, mesenticopeptidase, thermitase, proteinase K, or thermomycolase, and therefore may be considered homologous.

Thus, the mutated subtilisin enzymes used in the detergent composition of this invention have at least one of the specific amino acid position substitutions shown in Table 2.
In Table 2, the naturally occurring amino acid and position number is given first with the arrow to the right indicating the amino acid substitution. The mutations were made using subtilisin BPN'. However, as explained herein, these mutations can be introduced at analogous positions in other serine proteases using oligonucleotide-directed mutagenesis.

2 ~
C 6127 (R) Table 2 Mutations in subtilisln BPN' 1 Val8 -> Ile 2 Thr22 -~ Cys, Ser87 -> Cys 3 Thr22 -> Lys, Asn76 -> Asp 4 Met50 -> Phe Ser53 -> Thr 6 Ser63 -> Asp, Tyr217 -> Lys 7 Asn76 -> Asp 8 Ser78 -> Asp 9 TyrlO4 -> Val, Glyl28 -> Ser Alall6 -> Glu 11 Leul26 -> Ile 12 Glyl31 -> Asp 13 Glyl66 -> Ser 14 Glyl69 -> Ala Prol72 -> Asp 16 Prol72 -> Glu 17 Serl88 -> Pro 18 Gln206 -> Cys 19 Gln206 -> Tyr Ala216 -> Cys, Gln206 -> Cys 21 Tyr217 -> Lys 22 Tyr217 -> Leu 23 Asn218 -> Asp 24 Gln206 -> Tyr Ser248 -> Asp, Ser249 -> Arg 26 Thr254 -> Ala 27 Gln271 -~ Glu Stability of a mutated subtilisin in a given composition is expressed as the half life of the enzyme in hours at a given temperature, e.g., 37G.

Table 3 shows the strain designation of the host cell secreting the mutated subtilisin enzymes.

21 C 6127 (R) Table 3 Table 3 - Mutated Subtilisin BPN' Enzymes Strain Mutation 5 GX7130 Wild type GX7174 VAL8->ILE
GX7175 GLY169->ALA
GX7181 ASN218->ASP
THR22->CYS
SER87->CYS
GX7186 ASN218->SER
THR22->CYS
SER87->CYS
GLY169->ALA
15 GX7195 TYR217->LYS
GX7199 THR22->CYS
SER87->CYS
GLY169->ALA
PR0172->ASP
20 GX8303 MET50->PHE
GX8309 SER248->ASP
SER249->ARG
GX8314 GLN206->CYS
GX8321 THR22->CYS
SER87->CYS
GLY169->ALA
MET50->PHE
TYR217->LYS
ASN218->SER
30 GX8324 THR22->CYS
SER87->CYS
GLY169->ALA
MET50->PHE
TYR217->I,YS
ASN218 >SER
GLN206->CYS
GX8330 TYR217->LEU

22 C 6127 (R) GX8336 GLN206->TYR
GX8350 MET50->PHE
GLY169->ALA
GLN206->CYS
TYR217->LYS
ASN218->SER
ASN76->ASP
GX8352 SER63->ASP
TYR217->LYS

10 GX8354 GLN271->GLU
GX8363 THR22->LYS
ASN76->ASP
GX8372 MET50->PHE
GLY169->ALA
GLN206->CYS
TYR217->LYS
ASN76->ASP
SER78->ASP
ASN218->SER
20 GX8376 TYR104->VAL
GLY128->SER
GX7148 GLY131->ASP
GX7150 ASN218->SER
GX7164 ASN218->ASP
25 GX7178 SER188->PR0 GX7188 Al,A116->GLU
GX7189 LEU126->ILE
GX8301 ASN218->SER
GLY166->SER
30 GX8305 SER53->THR
GX8306 ASN218->SER
THR254->ALA
GX8315 ASN218->SER
GLY131->ASP
THR254->ALA
GX7159 THR22->CYS
SER87->CYS

2 ~
23 C 6127 (R) GX8307 GLN206->CYS
SER87->CYS
ALA216->CYS
GX7172 PRO172->ASP
GX8312 PRO172->GLU
GX8347 ASN76->ASP
GX8364 SER78~ASP
GX8373 ASN218->ASP
MET50->PHE
GLY169->ALA
GLN206->CYS
TYR217->LYS
ASN76->ASP
SER78~>ASP
15 GX8397 MET50->PHE
ASN76->ASP
GLY169->ALA
GLN206->CYS
ASN218->SER
20 GX8398 MET50->PHE
ASN76->ASP
GLN206->CYS
TYR217->LYS
ASN218->SER
25 GX8399 MET50->PHE
ASN76->ASP
ASN218->SER
GLN206->CYS

The subtilisin enzyme mutations shown in Tables 2 and 3, can be made on other proteases which are closely related, subtilisin Carlsberg for example. Closeness of relation is measured by comparison of amino acid sequences. There are many methods of aligning protein sequences, but the differences are only manifested when the degree of relatedness is quite small. The methods described in Atlas of Protein Sequence and Structure, Margaret O. Dayhoff editor, 24 C 6127 (R) Vol. 5, Supplement 2, 1976, National Biomedical Research Foundation, Georgetown University Medical Center, Washington, D.C., p. 3 ff., entitled SEARCH and ALIGN, define relatedness. As is well known in the art, related proteins 5 can differ in number of amino acids as well as identity of each amino acid along the chain. That is, there can be deletions or insertions when two structures are aligned for maximum identity. For example, subtilisin Carlsberg has only 274 amino acids, while subtilisin BPN' has 275 amino acids.
10 Aligning the two sequences shows that Carlsberg has no residue corresponding to ASN56 of subtilisin BPN'. Thus the amino acid sequence of Carlsberg would appear very different from BPN' unless a gap is recorded at location 56. Therefore an analogous substitution of position 218 of BPN' may be made 15 at location 218 of subtilisin Carlsberg, provided that the residues in Carlsberg are numbered by homology to BPN'.

In general, one should not transfer mutations if either subtilisin has a gap at, or immediately adjacent to, the site 20 of the mutation. Therefore, after aligning the amino acid sequences, those mutations at, or next to, gaps should be deleted from the list of desirable mutations and the mutation is not made.

25 One can use this reasoning to transfer all of the thermostable mutations described herein to other homologous serine proteases.

In brief, in order to introduce the mutation(s) for the 30 subtilisin, the gene coding for the desired subtilisin material generally is first isolated from its natural source and cloned in a cloning vector. Alternatively, mRNA which is transcribed from the gene of interest can be isolated from the source cell and converted into cDNA by reverse 35 transcription for insertion into a cloning vector. A cloning vector can be a phage or plasmid, and generally includes a replicon for autonomous replication of the vector in a micro-7" ~
C 6127 (R) organism independent of the genome of the micro-organism. A
cloning vector advantageously includes one or more phenotypic markers, such as DNA coding for antibiotic resistance, to aid in selection of ~icro-organisms transformed by the vector.

Procedures for insertion of DNA or cDNA into a vector for cloning purposes are well known in the art. These procedures generally include insertion of the gene coding for the subtilisin material into an opened restriction endonuclease site in the vector, and may involve addition of homopolymeric tails of deoxynucleotides to the ends of the gene and linking the gene to opened ends of a clonlng vector having complementary homopolymeric tails. A subtilisin gene can then be mutated by oligonucleotide-directed mutagenesis.
Oligonucleotide-directed mutagenesis, also called site-directed mutagenesis, is described in detail in Bryan et al., Proc. Natl. Acad. Sci. USA 83:3743-3745 (1986), incorporated herein by reference.

The protease used in these compositions is used in an amount sufficient to have an activity of 0.01 to 100,000 GU/g based on the final composition. A GU is a glycine unit, which is the amount of proteolytic enzyme which under standard incubation conditions produces an amount of terminal NH2-groups equivalent to 1 microgramme/ml of glycine.

Water Finally, except for the stabilizer and optional components described below, water comprises the remainder of the compositions. Generally, the amount of water will vary from 30-80~ of the composition although this will depend on the amount of actives and the ingredients used.

Stabilizer Another component which may be optionally used in the compositions of the invention is a stabilizer or stabilizer system. The improvements in stability of the invention can be 8 ~
26 C 6127 (R) demonstrated in systems with or without enzyme stabilization systems although it is preferred that such systems be used.
When present, the stabilization system comprises from about 0.1 to ahout 15% of the composition.

The enzyme stabilization systems may comprise calcium ion, boric acid, propylene glycol and/or short chain carboxylic acids. The composition preferably contains from about 0.01 to about 50, preferably from about 0.1 to about 30, more preferably from about 1 to about 20 millimoles of calcium ion per liter. When calcium ion is used, the level of calcium ion should be selected so that there is always some minimum level available for the enzyme after allowing for complexation with builders, etc. in the composition. Any water-soluble calcium salt can be used as the source of calcium ion including calcium chloride, calcium formate, calcium acetate, and calcium propionate. A small amount of calcium ion, generally from 0.05 to about 2.5 millimoles per liter, is often also present in the composition due to calcium in the enzyme slurry and formula water.

Another enzyme stabilizer which may be used is propionic acid or a propionic acid salt capable of forming propionic acid.
When used, the stabilizer may be used in an amount from about 0.1% to about 15% by weight of the composition.

Another preferred enzyme stabilizer is polyols containing only carbon, hydrogen and oxygen atoms. They preferably contain from 2 to 6 carbon atoms and from 2 to 6 hydroxy groups. Examples include propylene glycol (especially 1,2 propanediol which is preferred), ethylene glycol, glycerol, sorbitol, mannitol and glucose. The polyol generally represents from about 0. 5% to about 15~o ~ preferably from about 1.0% to about 8 o- by weight of the composition.
The compositions herein may also optionally contain from about 0.25% to about 5%, most preferably from about 0.5% to 2~ 3~
27 C 6127 (R) about 3% by weight of boric acid. The boric acid may be, but is preferably not, formed by a compound capable of forming boric acid in the composition. Boric acid is preferred, although other compounds such as boric oxide, borax and other alkali metal borates (e.g. sodium ortho-, meta-, and pyroborate and sodium pentaborate) are suitable. Substituted boric acids (e.g. phenylboronic acid, butane boronic acid and p-bromo phenylboronic acid) can also be used instead of boric acid.

One especially preferred stabilization system is a polyol in combination with boric acid. Preferably, the weight ratio of polyol to boric acid added is at least 1, more preferably at least 1.3.
Optional Components In addition to the ingredients described hereinbefore, the preferred compositions herein frequently contain a series of optional ingredients which are used for the known functionality in conventional levels. While the inventive compositions are premised on aqueous enzyme-containing detergent compositions, it is frequently desirable to use a phase regulant. This component, together with water, then constitutes the solvent matrix for the claimed liquid compositions. Suitable phase regulants are well known in liquid detergent technology and, for example, can be represented by hydrotropes such as salts of alkylaryl sulfonates having up to 3 carbon atoms in the alkylgroup, e.g. sodium, potassium, ammonium and ethanolamine salts of xylene-, toluene-, ethyl benzene-, cumene-, and isopropyl benzene sulfonic acids. Alcohols may also be used as phase regulants. This phase regulant is frequently used in an amount from about 0.5% to about 20%, the sum of phase regulant and water is normally in the range from 35% to 65%.
The preferred compositions herein can contain a series of further optional ingredients which are mostly used in 2 ~ 8 ~
28 C 6127 (R) additive levels, usually below about 5%. Examples of the like additives include: polyacids, suds regulants, opacifiers, antioxidants, bactericides, dyes, perfumes, brighteners and the like.

The beneficial utilization of the claimed compositions under various usage conditions can require the utilization of a suds regulant. While generally all detergent suds regulants can be utilized, preferred for use herein are alkylated polysiloxanes such as dimethylpolysiloxane, also frequently termed silicones. The silicones are frequently used in a level not exceeding 0.5%, most preferably between 0.01% and 0.2%.

It can also be desirable to utilize opacifiers inasmuch as they contribute to create a uniform appearance of the concentrated liquid detergent compositions. Examples of suitable opacifiers include: polystyrene commercially known as LYTRON 621 manufactured by MONSANTO CHEMICAL CORPORATION.
The opacifiers are frequently used in an amount from 0.3% to 1.5%.

The compositions herein can also contain known antioxidants for their known utility, frequently radical scavengers, in the art established levels, i.e. 0.001% to 0.25% (by reference to total composition). 'rhese antioxidants are frequently introduced in conjunction with fatty acids.

The compositions of the invention may also contain other enzymes in addition to the proteases of the invention such as lipases, amylases and cellulases. When present, these enzymes may be used in an amount from about 0.01% to about 5% of the compositions.

In one embodiment of the invention, another optional ingredient which may be used in the structured liquids is a deflocculating polymer.

29 C 6127 (R) In general, a deflocculating polymer comprises a hydrophobic backbone and one or more hydrophobic side chains and allows, if desired, the incorporation of greater amounts of surfactants and/or electrolytes than would otherwise be.
compatible with the need for a stable, low-viscosity product as well as the incorporation, if desired, of greater amounts of other ingredients to which lamellar dispersions are highly stability-sensitive.

The hydrophilic backbone generally is a linear, branched or highly crosslinked molecular composition containing one or more types of relatively hydrophobic monomer units where monomers preferably are sufficiently soluble to form at least a 1% by weight solution when dissolved in water. The only limitations to the structure of the hydrophilic backbone are that they be suitable for incorporation in an active-structured aqueous liquid composition and that a polymer corresponding to the hydrophi]ic backbone made from the backbone monomeric constituents is relatively water sol~ble (solubility in water at ambient temperature and at pH of 3.0 to 12.5 is preferably more than 1 g/1). The hydrophilic backbone is also preferably predominantly linear, e.g., the main chain of backbone constitutes at least 50% by weight, preferably more than 75%, most preferably more than 90% by weight.

The hydrophilic backbone is composed of monomer units selected from a variety of units available for polymer preparation and linked by any chemical links including -0-, O o O
ll ll 11 - C-O, - C-C-, - C-O-, - C-N, - C-N, and - P -OH

Preferably the hydrophobic side chains are part of a monomer unit which is incorporated in the monomer by copolymerizing hydrophobic monomers and the hydrophilic monomer making set 2 ~ $ ~
C 6127 (R) the backbone. The hydrophobic side chains preferably include those which when isolated from their linkage are relatively water insoluble, i.e., preferably less than 1 g/1, more preferred less than 0.5 g/1, most preferred less than 0..1/
g/1 of the hydrophobic monomers, will dissolve in water at ambient temperature at pH of 3.0 to 12~5.

Preferably, the hydrophobic moieties are selected form siloxanes, saturated and unsaturated alkyl chains, e.g., having from 5 to 24 carbons, preferably 6 to 18, most preferred 8 to 16 carbons, and are optionally bonded to hydrophilic backbone via an alkoxylene or polyalkoxylene linkage, for example a polyethoxy, polypropoxy, or butyloxy (or mixtures of the same) linkage having from 1 to 50 alkoxylene groups. Alternatively, the hydrophobic side chain can be composed of relatively hydrophobic alkoxy groups, for example, butylene oxide and/or propylene oxide, in the absence of alkyl or alkenyl group.

Monomer units which may make up the hydrophilic backbone include:

tl) unsatureated, preferably mono-unsaturated, Cl_6 acids, ethers, alcohols, aldehydes, ketones or esters such as monomers of acrylic acid, methacrylic acid, maleic acid, vinyl-methyl ether, vinyl sulphonate or vinyl alcohol obtained by hydrolysis of vinyl acetate, acrolein;

(2) cyclic units, unsaturated or comprising other groups capable of forming inter-monomer linkages, such as saccharides and glucosides, alkoxy units and maleic anhydride;

(3) glycerol or other saturated polyalcohos.
Monomeric units comprising both the hydrophilic backbone and hydrophobic side chain may be substituted with groups such as g ~

31 C 6127 (R) amino, amine, amide, sulphonate, sulphate, phosphonate, phosphate, hydroxy, carboxyl and oxide groups.

The hydrophilic backbone is preferably composed of one Pr two monomer units but may contain three or more different types.
The backbone may also contain small amounts of relatively hydrophilic units such as those derived from polymers having a solubility of less than 1 g/1 in water provided the overall solubility of the polymer meets the requirements discussed above. Examples include polyvinyl acetate or polymethyl methacrylate.

The deflocculating polymer generally will comprise, when used, from about 0.1 to about 5% of the composition, preferably 0.1 to about 2% and most preferably, about 0.5 to about 1.5%.

Yet another optional component which may be used in a second embodiment of the invention are peroxygen bleaching compounds as well as bleach activator compounds.

The peroxygen bleaching compounds useful herein are those capable of yielding hydrogen peroxide in aqueous solution.
These compounds are well known in the art and include hydrogen peroxide and the alkali metal peroxides, organic peroxide bleaching compounds such as urea peroxide, and inorganic persalt bleaching compounds, such as the alkali metal perborates, percarbonates,perphosphates, and the like.
Mixtures of two or more such bleaching compounds can also be used, if desired.

Preferred peroxygen bleaching compounds include sodium perborate, commercially available in the form of mono- and tetra-hydrate, sodium carbonate peroxyhydrate, sodium pyrophosphate peroxyhydrate, urea peroxyhydrate, and sodium peroxide. Particularly preferred are sodium perborate tetrahydrate and, especially, sodium perborate monohydrate.

32 2~ 8~ (R) Sodium perborate monohydrate is especially preferred because it is very stable during storage and yet still dissolves very quickly in the bleaching solution. It is believed that such rapid dissolution results in the formation of higher le.vels of percarboxylic acid and, thus, enhanced surface bleaching performance.

The level of peroxygen bleach within compositions of the invention is form about 0.1% to about 95% and preferably from about 1% to about 60%. When the bleaching compositions within the invention are also detergent compositions it is preferred that the level of peroxygen bleach is from about 1% to about 20%.

Bleach activator compounds which may be used with the bleaching compounds have the general formula:

R - C - L
Wherein R is and alkyl group containing from about 5 to about 18 carbon atoms wherein the longest linear alkyl chain extending from and including the carbonyl carbon contains from about 6 to about 10 carbon atoms and L is a leaving group, the conjugate acid of which has a PKa in the range of from about 6 to about 13.

L can be essentially any suitable leaving group. A leaving group is any group that is displaced form the bleach activator as a consequence of the nucleophilic attack on the bleach activator by the perhydroxide anion. This, the perhydrolysis reaction, results in the formation of the percarboxylic acid. Generally, for a group to be a suitable leaving group is must exert an electron attracting effect.
This facilitates the nucleophilic attack by the perhydroxide anion. Leaving groups that exhibit such behaviour are those in which their conjugate acid has a PKa in the range of from 2 ~
33 C 6127 (R) about 6 to about 13, preferably from about 7 to about 11 and most preferably from about 8 to about 11.

Product pH
pH of the compositions of the invention is from about 7 to 13, preferably 8 to 11.

The compositions of the present invention may be prepared using the general techniques known in the art of the processing of liquid detergent products. However, the order of addition of components can be important. Thus, a preferred order of addition (with continuous mixing) is to add to the water the soluble electrolytes, then any insoluble material such as aluminosilicates, followed by the actives. The mixtures are then cooled below 30C, whereafter any minors and additional ingredients can be added. Finally, if necessary, the pH of the composition can be adjusted, e.g. by addition of a small quantity of caustic material. The enzyme is generally added after the mixture has been cooled but may be added while the mixture is still warm and the minors and additional ingredients are added.

In use, the compositions of the present invention will generally be diluted with water to form a wash liquor preferably comprising from 0.01 to 10%, more preferably from 0.1 to 3% by weight of said composition. The wash liquor is used for the washing of fabrics, for instance in an automatic washing machine.

The following examples are intended to illustrate the invention and facilitate its understanding and are not meant to limit the invention in any way.

2 ~
34 C 6127 (R) The stability of various wild-type subtilisins were compared to mutant subtilisin strain GX8397 (subtilisin with 5 amino acid mutations) in the following formulation: .

Structured Liquid Formulation A By Weiqht Primary alcohol ethoxylate 4.8 Linear alkylbenzene sulfonate 6.7 Zeolite 20.0 10 Citric acid 2.3 NAOH 1.8 Sodium xylene sulphonate 0.8 Calcium chloride 0.2 Water and minor ingredientsto 100%
15 pH 8.8 Enzyme Half-life at 37C ~hours) Improvement Alcalase (from Novo) 44 Wildtype BPN' 76 20 GX 8397 342 677*

* Relative to commercially available Alcalase; improvement was 350% relative to Wildtype BPN'. It could not have been predicted in advance that the mutant strain would function so effectively in a structured liquid such as that set forth above.

Structured Liquid Formulation B By Weight Primary alcohol ethoxylate 16.0 30 Linear alkylbenzene sulphonate 9.7 Citric acid 7.0 NaOH 6.2 Fatty Acid 9-5 Calcium chloride 0.2 35 Water and minor ingredientsto 100%
pH 9.2 2~9Q~
C 6127 (R) Enzyme Half-life at 37C (hours) Improvement Savinase (from Novo) 90 As can be seen from the results above, the stability of the mutant strain GX8397, measured as the half-life of the enzyme at 37C, was significantly greater in the structured detergent compositions of the invention compared to either the commercially available enzyme or the wildtype tested in the same formulatiGns. Again, this stability enhancement could not have been predicted in advance.

The stability of wildtype BPN' was compared to mutant subtilisin with 6 or fewer amino acid mutations in the following Formulation:

Inqredient % by weiqht Primary alcohol ethoxylate 9.0 20 Linear alkylbenzene sulphonate 16.5 Zeolite 15.0 Citric acid 8.2 Potassium hydroxide 10.5 Fatty acid 4.5 25 Calcium chloride 0.2 Water & minors to 100%
P~ 9.1 The following results were obtained:
% Activity %
Enzyme No. Substitutions After 193 Hrs Increase Wildtype BPN' 0 33%
GX8397 5 85% 158 35 GX8347 1 57% 73 ~9~
36 C 6127 (R) The results show that the s~ability of the mutant enzymes was clearly superior to wildtype BPN' in the composition of the invention. The example also shows that the stability was significantly improved even when the enzyme was mutated,in as few as 1 amino acid site. (i.e., GX8347). These stability measures in structured liquids could not have been predicted in advance.

In this example, once again the stability of wildtype BPN' was tested against GX 8397. In this case, the structures composition also contained a deflocculating polymer incorporated to allow the formulation to contain a greater amount of active while maintaining stability.
Inqredient % by Weiqht LAS 28.0 Alcohol Ethoxylate 12.0 Sodium Citrate 10.0 20 Ethanolamine 2.0 Triethanolamine 2.0 Decoupling Polymer* 1.0 Fluorescer, Dye <1 Glycerol 5.0 25 Borax (5 Aq) 2.7 Water to 100 pH 10 Protease present at 12,000 GU/g.
* Decoupling polymer is an acrylic acid, lauryl methacrylate copolymer % Activity Remainina After Storaae Enzyme @ 37OC for 22 Days BPN' 27 ~9~

37 C 6127 (R) Again, an increase of almost 3-fold was observed. The effectiveness of a mutant enzyme in a structured liquid containing a decoupling polymer could not have been predicted in advance.

The following liquid detergent composition was prepared:
Ingredient % by Weight LAS 27.5 lO Alcohol Ethoxylate12.0 Citric Acid 6.7 Ethanolamine 2.0 Triethanolamine 2.0 Decoupling Polymer* 1.0 15 Fluorescer, Dye <1 Glycero] 5.0 Borax (5 Aq) 2.7 Potassium hydroxide 6.0 Sodium hydroxide 3.1 20 Water to lO0 pH 10 Protease present at 12,000 GU/g.

* Decoupling polymer is an acrylic acid, lauryl methacrylate copolymer ~ Activity Remaininq After Storage Enzyme @ 37C for 22 Days 30 BPN' 42 This example is similar to Example 3 and again shows the increased activity of the mutant enzyme in a structured liquid comprising a decoupling polymer (i.e. more than two-fold increase). The effectiveness of a mutant enzyme in suchcompositions is completely unpredictable.

2 ~ 8 4 38 C 6127 (R) Structured liquid formulation containing perborate.
Ingredient gO by Weight Linear alkylbenzene sulfonic acid 21.9 5 Alcohol Ethoxylate 9.0 Sodium perborate tetrahydrate 20.0 Sodium metaborate 2.6 Sodium hydroxide 3.5 Sodium citrate (dihydrate) 9.21 10 Citric acid anhydrous 0.78 Calcium chloride (dihydrate)0.15 Dequest 2060S* 0.40 Decoupling Polymer** 1.0 Sokalan PA50*** 0.20 15 Whitener 0.10 Water to 100 pH 9.0 Protease present at 12,000 GU/g.

20 * Bleaching stabilizing sequestrant ** Acrylic acid, lauryl methacrylate copolymer *** Polyacrylic acid compressing polymer Enz~me Half-lives at 37C (Hrs~
~X 8397 300 BPN' 30 once more the ten-fold increase in stability using a mutant enzyme in a perborate containing structured liquid simply was not and could not have been predicted in advance.
************

Claims (15)

1. A liquid detergent composition comprising a detergent active structure existing as a separate phase within a .
continuous aqueous phase wherein said composition comprises:
(1) from about 5% to about 65% by weight detergent-active;
(2) from 0% to 50% by weight builder;
(3) from 0 to 30% by weight electrolyte;
(4) a mutant subtilisin protease added in sufficient quantity to have an activity level of 0.01 to 100,000 GU/g having substitutions in l or more amino acid residues compared to the wild type or commercially available subtilisin; and (5) the remainder water and minor ingredients; and wherein the composition contains a sufficient amount of detergent active material to result in a structure of a lamellar droplets dispersed in said continuous aqueous phase.

2. A composition according to claim l, wherein the subtilisin is derived from Strain GX8350 and has the following substitutions:
MET50->PHE
GLY169->ALA
GLN206->CYS
TYR217->LYS
ASN218->SER
ASN76->ASP

3. A composition according to claim 1, wherein the subtilisin is derived from Strain GX8397 and has the following mutations:
MET50->PHE
ASN76->ASP
GLY169->ALA
GLN206->CYS
ASN218->SER

C 6127 (R)

4. A composition according to claim 1, wherein the subtilisin is derived from GX8398 and has the following mutations:
MET50->PHE
ASN76->ASP
GLN206->CYS
TYR217->LYS
ASN218->SER

5. A composition according to claim, 1 wherein the subtilisin is derived from Strain GX8399 and has the following mutations:
MET50->PHE
SN76->ASP
SN218->SER
GLN206->CYS

6. A composition according to claim 1, comprising 1-30%
builder.

7. A composition according to claim 1, comprising 5-30%
electrolyte.

8. A composition according to claim 1, additionally comprising 0.5 to about 15% by weight of an enzyme stabilizer or enzyme stabilization system.

9. A composition according to claim 8, wherein the enzyme stabilizer is propionic acid or a propionic acid salt capable of forming propionic acid.

10. A composition according to claim 8, wherein the enzyme stabilizer is an enzyme stabilization system comprising propylene glycol and horic acid.

11. A composition according to claim 1 wherein the structured liquid comprises a decoupling polymer.

41 C 6127 (R)

12. A composition according to claim 11 comprising the following:

13. A composition according to claim 1 wherein the structured liquid comprises a peroxygen bleaching compound.

14. A composition according to claim 13 comprising the following

15. A liquid detergent as claimed in claim 1 and substantially as described herein.