CZ2000502A3 - Laundry detergent compositions containing enzymes that break down carbohydrate gums - Google Patents

Abstract

The present invention relates to laundry detergent compositions, comprising a saccharide gum degrading enzyme providing excellent cleaning performance, especially food stain/soil removal, dingy cleaning and whiteness benefits.

Description

Technical field

The present invention relates to laundry detergent compositions containing enzymes that degrade carbohydrate gums.

State of the art

Detergent compositions today include complex combinations of active ingredients that meet certain specific needs. In particular, contemporary detergent compositions generally include detergent enzymes that provide benefits in cleaning and caring for fabrics, and more specifically cellulase and amylase enzymes.

The effectiveness of cellulolytic enzymes, i.e. cellulases, in the field of textile cleaning and fabric roughness reducing agents has been known for some time. Cellulase activity consists in the hydrolysis of cellulose fibers or substrates by cellulase and, depending on the specific function of the cellulase, which is endo- or exocellulase and possibly hemicellulase. Cellulose structures are depolymerized or broken down into smaller and therefore more soluble or dispersible parts. In particular, this activity causes fabrics to be cleaned, revived, softened and generally their tactile characteristics are improved.

amylases are known in the art to cause stain removal from naturally occurring or artificially added starches contained in food stains or soils, or amylases are added as a finishing agent.

Food stains or soils constitute the majority of stains or soils that bother consumers, and they often contain food additives. Neutralizing agents, acidulants, antioxidants, preservatives, sweeteners, enzymes, stabilizers, and thickeners such as hydrocolloids and emulsifiers are commonly used food additives. In particular, consumers are demanding increasing amounts of texturizing agents as fat substitutes to reduce fat and calorie content. The market for hydrocolloid texturizing and stabilizing agents, also known as food gums, is growing at a rate of 4% per year, with xanthan gum growing at a rate of 6% to 8% per year and carrageenan gum growing at a rate of 3% per year (Chemical Week, June 19, 1996, pp. 32-34).

The term gum refers to a group of industrially used polysaccharides (long-chain polymers) or their derivatives that hydrate with hot or cold water to form viscous solutions, dispersions, or gels. Gums are classified as natural and modified natural gums, including seaweed extracts, plant extrudates, seed or root gums, and gums obtained by microbial fermentation. Modified (semisynthetic) gums include cellulose and starch derivatives and some synthetic gums such as low molecular weight methoxylated pectin, propylene glycol alginate, and carboxymethyl and hydroxypropyl guar gum (Gums in Encyclopedia of Chemical Technology, 4th ed., vol. 12, pp. 842-862, J. Baird, Kelco Division of Merck). See also Carbohydrate Chemistry for Food Scientists (Eagan Press - 1997) R.L. Whistler and J.N. BeMiller, Chapter 4, pages 63 to 89 and Direct Food Additives in Fruit Processing, P. Laslo, Bioprinciples and Applications, Volume 1, Chapter II, pages 313 to 325 (1996) Technomie publishing.

Some of these gums, such as xanthan gum (E415, CEE number), gellan gum (E416), guar gum (E412) and tragacanth (E413), are widely used alone or in combination in many foods (Gums in the ECT book, 4th edition, volume 12, pages 842 to 862, J. Baird, Kelco Division of Merck). Guar gum in particular is often used in foods as a thickener and binder of free water in sauces and salad dressings.

• · Other examples include algin gums known for their use in fruit juices, canned and preserved foods, tragacanth gums used in salad dressings, xanthan gums for dairy products and beverages, gellans found in toppings, frozen and dairy products, and agar in ice creams.

The specificity of these food gums is that upon hydration with water they form a highly to very highly viscous solution. Some of these gums, such as guar gum, algin gum, gum arabic and karaya gum, are also used in the paper industry and are chosen for their high affinity for cellulose fibers (Industrial gums, R.L. Whistler and J.N. BeMiller (Academie Press - 1973)). Their ability to flocculate clays and other inorganic materials such as calcium salts is also used in other applications such as water treatment. The high viscosity of these food gums is desirable for all of the above foods and other uses.

However, it has been surprisingly found that these food gums strongly adsorb to the cotton fibers of fabrics, thereby adhering stains/dirt to the fabric. This phenomenon occurs even when the gums are present in the food compositions in very small amounts, such as 0.01% to 5%, more typically 0.01% to 0.8%.

• · • ·

It has also been surprisingly found that the ability of these food gums to flocculate clays results in the fabrics appearing dull and yellow. This is very important because the overall performance of detergents is judged not only by its ability to remove stains and dirt, but also by its ability to prevent the redeposition of dirt or breakdown products of dirt or any insoluble salt on the washed fabric. The redeposition effect results in the washed fabric being covered with an unsightly film which results in visible streaks or spots which remain intact until the end of the washing process. This residue left on the fabric results in the fabric appearing dull and yellow.

As can be seen from the above, there is a continuing need for laundry detergent compositions which provide excellent overall cleaning performance. It is an object of the present invention to provide a laundry detergent composition which provides excellent cleaning and whitening performance, in particular excellent stain/soil removal, dullness removal and whitening performance.

These requirements are met by the development of laundry detergent compositions containing enzymes that degrade carbohydrate gums.

Surprisingly, it has been found that the laundry detergent composition of the present invention containing enzymes that degrade carbohydrate gums provides excellent stain/soil removal, dullness removal and bleaching performance resulting from the hydrolysis of food grade carbohydrate gums binding stains/soils from fabrics or clays to cotton fabrics. It has further been found that the performance of the laundry detergent compositions of the present invention is enhanced by the addition of selected surfactants, an additional enzyme, a builder and/or a bleaching system.

GB2-169-393 describes a method for removing cellulose impurities and other vegetable impurities from fabrics in dyeing and finishing mills by treating • · · · · · · • · · · · · > ♦ · · 4 4 4 4 » 44····· ·« · with an enzymatic agent containing cellulolytic and pectolytic enzymes, which allow the concentration of H ₂ SO ₄ to be reduced to below 2% during carbonization of the fabric.

WO96/06532 relates to compositions capable of killing or arresting the growth of microbial cells using basic proteins or peptides of biological origin, e.g. protamine or protamine sulfate. For some bacteria or fungi, these compositions further comprise oxidoreductase or cell wall degrading enzymes, such as type II endoglycosidase, lysozyme and/or chitinase.

WO95/35362 describes dishwashing detergents, including laundry detergents, and in particular household cleaning agents, containing cell wall degrading enzymes such as pectinases and/or hemicellulases and optionally cellulases. These agents are particularly suitable for removing vegetable stains and dirt and grime of a similar structure. These cell wall degrading enzymes degrade the structural components of plant cell walls, such as structured polysaccharides (cellulose, hemicelluloses, pectins) and include cellulolytic, pectin degrading and hemicellulose degrading enzymes. There are a large number of plant cell wall degrading enzymes. Cellulolytic enzymes are divided into three groups: endoglucanases, exoglucanases or cellobiohydrolases and β-glucosidases. A large number of pectin degrading enzymes are known: examples are pectinesterase, pectin lyase, pectate lyase and endo- or exopolygalacturonases. In addition to these enzymes that degrade smooth regions, enzymes that degrade branched regions have also been found. A large number of enzymes are known to degrade hemicellulose structures, such as xylanase, galactanase, arabinases, lichenases, and mannanases.

However, the use of enzymes that degrade carbohydrate gums for excellent cleaning performance on cotton fabrics in laundry detergent compositions has not been previously discovered.

The essence of the invention

Summary of the invention

The present invention relates to laundry detergent compositions containing enzymes that degrade carbohydrate gums, providing excellent cleaning performance on cotton fabrics, particularly in stain/soil removal, dullness removal and bleaching effects.

Detailed description of the invention

An essential ingredient of the laundry detergent compositions of the present invention is an enzyme that degrades carbohydrate gums. These enzymes are capable of hydrolyzing non-starch non-cellulosic food polysaccharides that have a viscosity of greater than 800 cPs in a 1% solution, expressed as a weight percent, (measured in water at 25°C, Brookfield Synchro-Lectic viscometer at 20 rpm).

Surprisingly, it has been found that the laundry detergent compositions of the present invention provide excellent cleaning and bleaching effects, particularly in removing food stains/soils and cleaning stains/soils caused by mildew.

Without wishing to be bound by any particular theory, it is believed that the enzyme that degrades the carbohydrate gum hydrolyzes the gum-based food additives present in food stains/soils binding the stains/soils to the cotton fibers. These non-starch non-cellulosic food polysaccharides have been found to have a high affinity for cotton fibers and thus bind the stains/soils to the fabric. Therefore, hydrolysis of these non-starch non-cellulosic food polysaccharides will release the stains/soils from the cotton fabric.

In addition, it has been surprisingly found that the laundry detergent composition of the present invention achieves appreciable cleaning of soiled fabrics and good bleaching efficiency. Without being bound by any particular theory, it is believed that carbohydrates, after being broken down into components, during partial cleaning of food stains/soils by currently used detergent compositions, are redeposited on the fabric and react with particulate contaminants such as clay-like compounds, resulting in soiling of cotton fabrics. The carbohydrate gum degrading enzymes of the present invention hydrolyze the carbohydrate film deposited on the fabric and therefore prevent flocculation of these compounds with particulate contaminants.

Without wishing to be bound by any particular theory, it is also believed that the enzymatic action of the gum-degrading enzymes of the present invention renders food stains/soils and fabric stains/soils more accessible to other detergent components of the laundry detergent composition, and in particular, it has been found that the effectiveness of the laundry detergent composition of the present invention is enhanced by combination with a selected surfactant, additional enzyme, builder and/or bleaching system.

The enzymes of the present invention have primary or secondary activity on non-starch non-cellulosic food polysaccharides that have a viscosity of greater than 800 cPs in a 1% solution, expressed as a weight percent, such as agar, algin, karawa, tragacanth, guar gum, xanthan gum and/or mixtures thereof.

Examples of industrial gums used individually or in combination as food additives are:

- seed gums such as guar gum, quince seed gum, linseed gum and ochre gums, tamarind, larch arabinogalactan;

- plant exudates such as gum arabic, ghatti, karaya, tragacanth;

- seaweed extracts such as algin, agar, carrageenan, fucoidan, furcellaran, and

- biosynthetic gums such as xanthan.

• ♦ 9 · « 9 9 9 9

9 9 9 9 9 · * · Μ • · » 4 4 · 9 9 · 4 4 • 4444 4 4 4 44 44 44 44 4 · ·«· 4··· • 44 4 9 4 9 9 9 9 9

Suitable enzymes for the purposes of the present invention have the following major or minor enzymatic activity:

arabinases, endoarabanases (E.C. 3.2.1.99) such as endo-α-Ι,5-arabinosidase, exoarabanase (E.C. 3.2.1.55), exo A (α-1,2; α-1,3) arabinofuranosidase, exo B (α-1,3; α-1,5) arabinofuranosidase;

(α-1,2; α-1,3) fucosidase, α-1,6-fucosidase (E.C.

3.2.1.127);

β-l,2-galactanase, β-l,3-galactanase (E.C. 3.2.1.90), β-l,4-galactanase, β-l,6-galactanase, galactanase is also called arabino galactan galactosidase (E.C. 3.2.1.89), a and β galactosidase (E.C. 3.2.1.22 & 23), (E.C. 3.2.1.102) (E.C.

3.2.1.103) β-mannosidase (3.2.1.25), α-mannosidase (3.2.1.24), β-1,2-mannosidase, α-1,2-mannosidase, (E.C. 3.2.1.113) (E.C.

3.2.1.130), α-1,2-1,6-mannosidase (3.2.1.137), β-1,3-mannosidase (E.C. 3.2.1.77), β-1,4-mannosidase (E.C. 3.2.1.137), β-1,6-mannosidase (E.C. 3.2.1.101), α-1,3-1,6-mannosidase (E.C.

3.2.1.114), β-1, 4-mannobiosidase (E.C. 3.2.1.100);

glucuronosidase (E.C. 3.2.1.131), glucuronidase (E.C.

3.2.1.31), exo 1,2 or 1,4 glucuronidase;

- agarase (E.C. 3.2.1.81), carrageenase (E.C. 3.2.1.83), α-1,2-xanthan lyase, poly(α-L-guluronate) lyase, also called alginase II (E.C. 4.2.2.11).

Preferred enzymes that degrade carbohydrate gums are: mannosidases, β-mannosidase, endo-1,4-β-D-mannosidase, endo-1,2-β-D-mannosidase and exo-1,3-β-D-mannosidase;

galactosidases, exo-1,β-β-D-galactosidase and

1,3-β-D-galactosidase;

glucuronidases, glucuronosidases and exo-1,2-or

1.4- glucuronidase ♦ 9 • 9 9 9 9 9

- arabinases, endo-α-Ι,5-arabinosidase, exoarabanase, exo A (α-1,2, α-1,3) arabinofuranosidase, exo B (α-1,3, α-1,5) arabinofuranosidase;

xanthan lyase, poly(α-L-guluronate) lyase, agarase and carrageenase.

Preferred enzymes that degrade carbohydrate gums, intended for specific non-starch non-cellulosic food polysaccharides that have a viscosity of greater than 800 cPs in a 1% solution, expressed as a percentage by weight, are in particular the following: Guar gum (guar powder, jaguar gum) hydrolyzing enzymes, *************************** (known as food additives E 410, E 412 and 21CFR 184, 1339 and 13423) are mannosidase, galactomannosidase, preferably endo mannosidase and galactomannosidase, such as Gamanase®, which is a galactomannanase from Aspergillus niger. Preferred enzymes for degrading xanthan gums are mannosidase, glucuronosidase and glucosidase. For degrading Karaya gum, galactosidase and rhamnogalacturonase are preferred enzymes. For the degradation of tragacanth gum, the preferred enzymes are galacturonase, galactosidase, fucosidase and arabanase. The preferred enzymes for the degradation of gellan, agar and carrageenan gums are glucosidase, rhamnosidase and glucuronidase, agarase and carrageenase. The preferred enzymes that degrade the mannopyranosyluron and gulopyranosyluron units contained in alginate are mannuronase and guluronase.

The following three mannan-degrading enzymes are included in the present invention: EC 3.2.1.25 β-mannosidase, EC 3.2.1.78 endo-1,4-β-mannosidase, hereinafter referred to as mannanase, and EC 3.2.1.100 1,4-β-mannobiosidase (IUPAC Classification, Enzyme Nomenclature, 1992, ISBN 0-1-227165-3 Academie Press).

Even more preferably, the laundry detergent compositions of the present invention comprise β-1,4-mannosidase (EC 3.2.1.78) referred to as mannanase. The term mannanase is further or

9*9 · · 99 · 9 9 9. · ♦ • 999 · 99 9

9999999 99 9

9 999 9999

999 9 99 9 99 99 galactomannanase refers to a mannanase enzyme defined in the art as the one officially called mannan endo-1,4-beta-mannosidase and alternatively referred to as beta-mannanase and endo-1,4-mannanase and catalyzes the random hydrolysis reactions of 1,4-beta-D-mannosidic linkages in mannans, galactomannans, glucomannans and galactoglucomannans.

Specifically, mannasases (EC 3.2.1.78) form a group of polysaccharides that degrade mannans and designate enzymes that are capable of cleaving polysaccharide chains containing mannose units, i.e. they are capable of cleaving glycosidic bonds in mannans, glucomannans and galactoglucomannans. Mannans are polysaccharides that have a backbone composed of β-1,4-linked mannos, glucomannans are polysaccharides that have a backbone composed of more or less regularly alternating β-1,4-linked mannos and glucose, and galactomannans and galactoglucomannans are mannans and glucomannans with β-1,6-linked galactose branches. These compounds are optionally acetylated.

The degradation of galactomannans and galactoglucomannans is facilitated by complete or partial removal of the galactose side branches. Furthermore, the degradation of acetylated mannans, glucomannans, galactomannans and galactoglucomannans is facilitated by complete or partial deacetylation. The acetyl groups are removed alkalinely or by mannan acetylesterases. The oligomers that are released by mannanases or by a combination of mannanases and α-galactosidases and/or mannan acetylesterases are further degraded by β-mannosidase and/or β-glucosidase to release maltose.

Mannanases have been identified in several organisms of the genus Bacillus. For example, Talbot et al., Appl. Environ. Microbiol., vol. 56, no. 11, pp. 3505-3510 (1990) describes a β-mannanase obtained from Bacillus stearothermopilus in the form of a dimer with a molecular weight of 162 kDa and a pH optimum of 5.5-7.5. Mendoza et al., World J. Microbiol. Biotech., vol. 10, no. 5, pp. 551- • · · · · · · · ♦ · · · · · · 9 9

9999 9 9 9 9999 9 φ · ♦ · ·· ··· · · « · ··· · 99 9 99 99

555 (1994), describes a β-mannanase obtained from Bacillus subtilis with a molecular weight of 38 kDa with an activity optimum at pH 5.5 and

55C and pI 4.8. JP-0304706 describes β-mannanase obtained from

Bacillus sp. with a molecular weight of 373 kDa, measured by gel filtration, pH optimum 8 to 10 and pI 5.3 to 5.4. JP-63056289 describes the preparation of an alkaline, heat-stable β-mannanase which hydrolyzes β-1,4-D-mannopyranoside bonds of, for example, mannans and produces manno-oligosaccharides. JP-63036774 describes the microorganism Bacillus FERM P-8856 which produces β-mannanase and β-mannosidase at alkaline pH. JP-08051975 describes alkaline β-mannanases from the alkalophilic Bacillus sp. AM-001. Purified mannanase from Bacillus amyloliquefaciens useful in paper bleaching and a method for its preparation is described in WO 97/11164. WO 91/18974 describes hemicellulases such as glucanase, xylanase or mannanase active at extreme pH and temperature. WO 94/25576 describes an enzyme from Aspergillus aculeatus. CBS 101.43 has mannanase activity which is useful for degrading and modifying material from plant or algal cell walls. WO 93/24622 describes mannanases isolated from Trichoderma reseei useful for bleaching lignocellulosic pulps. Hemicellulases capable of degrading mannan containing hemicelluloses are described in WO 91/18974 and purified mannanases from Bacillus amyloliquefaciens are described in WO 97/11164.

In particular, these mannanase enzymes will be alkaline mannanases as defined below, most preferably mannanases originating from bacterial sources. The detergent composition according to the present invention comprises in particular an alkaline mannanase selected from mannanases from Bacillus agaradherens strain and/or Bacillus subtilis strain 168, gene yght.

The term alkaline mannanase enzyme includes enzymes that have an enzymatic activity of at least 10%, preferably at least 25%, more preferably at least 40%, of maximum activity at a given pH in the range of 7 to 12, preferably

7.5 to 10.5.

· 9 9 9 9 9 « 9 · 9 9 9

9 9 · 9 9 9

999999* 99 9

9 9·9 9999

999 9 99 9 99 99

Most preferably, the laundry detergent composition of the present invention will comprise an alkaline mannanase from Bacillus agaradherens. This mannanase is:

i) a polypeptide produced by Bacillus agaradherens, NCIMB 40482 or ii) a polypeptide comprising the amino acid sequence set out in positions 32 to 343 of SEQ ID NO: 2 or iii) an analogue of a polypeptide defined in points i) or ii) which is at least 70% homologous to that polypeptide or is derived from that polypeptide by the substitution, deletion or addition of one or more amino acids or is immunologically reactive with a polyclonal antibody raised against that peptide in its pure form.

The present invention also includes an isolated polypeptide having mannanase activity selected from the group consisting of (a) polynucleotide molecules encoding a polypeptide having mannanase activity and comprising the oligonucleotide sequence set forth in SEQ ID NO: 1 from nucleotide 97 to nucleotide 1029;

(b) homologs of (a) ;

(c) polynucleotide molecules that encode a polypeptide having mannanase activity that is at least 70% identical to the amino acid sequence of SEQ ID NO: 2, from amino acid 32 to amino acid 343;

(d) molecules complementary to (a), (b) or (c); and (e) degenerate nucleotide sequences of (a), (b), (c) or (d).

Plasmid pSJ1678 containing a polynucleotide molecule (DNA sequence) encoding mannanase according to the present invention was transferred into an Escherichia coli strain, which was deposited by the inventors under the Budapest Agreement on the International Recognition of Microorganisms Deposited for the Purposes of Patent Procedure at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, • 9 9 · · · · · • · ···· « 9 · 9

9999 9 9 · ···« 9 · · * · • 9 9 9 9 9 9 9 9

99 9 99 99

Mascherorder Weg lb, D-38124 Brausnchweig, Federal Republic of Germany, May 18, 1998 under number DSM 12180.

The second most preferred enzyme is mannanase from Bacillus subtilisis strain 168 which is a mannanase that:

i) is encoded by the coding portion of the DNA sequence identification number 5 or an analogous sequence and/or ii) is a polypeptide comprising the amino acid sequence set forth in sequence identification number 6 or iii) is an analogous polypeptide as defined in point ii) which is at least 70% homologous to this polypeptide or is derived from this polypeptide by the substitution, deletion or addition of one or more amino acids or is immunologically reactive with a polyclonal antibody raised against this peptide in its pure form.

The present invention also includes an isolated polypeptide having mannanase activity selected from the group consisting of (a) polynucleotide molecules encoding a polypeptide having mannanase activity and comprising the nucleotide sequence set forth in SEQ ID NO: 5;

(b) homologs of (a);

(c) polynucleotide molecules that encode a polypeptide having mannanase activity that is at least 70% identical to the amino acid sequence of SEQ ID NO: 6;

(d) molecules complementary to (a), (b) or (c); and (e) degenerate nucleotide sequences of (a), (b), (c) or (d).

DEFINITION

Before discussing the present invention in detail, the following terms will first be defined:

The term ortholog (or homologous species) refers to a polypeptide or protein obtained from one species that is homologous to an analogous peptide or protein from another species.

* * *· * • ♦ · * · · · • * · · · · · · • ······ ·

The term paralog refers to a polypeptide or protein obtained from a given species that is homologous to a different peptide or protein from the same species.

The term expression vector refers to a DNA molecule, linear or circular, which contains a segment encoding a polypeptide of interest, operably linked to other segments that ensure its transcription. Such other segments include a promoter and a termination sequence and optionally include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal and the like. Expression vectors are generally derived from plasmid or viral DNA or contain elements of both. An expression vector according to the present invention is any expression vector that is routinely subjected to recombinant DNA procedures and the choice of vector will often depend on the host cell into which the vector is inserted. Thus, the vector may be an autonomously replicating vector, i.e. a vector that exists as an extrachromosomal entity whose replication is independent of chromosome replication, e.g. a plasmid. Alternatively, a vector is one that, upon introduction into a host cell, integrates into the genome of the host cell and replicates together with the chromosomes into which it has been integrated.

The term recombinantly expressed, as used herein in connection with the expression of polypeptides or proteins, is defined as is common in the art. Recombinant expression of a protein is generally accomplished using an expression vector as defined above.

The term isolated, as applied to a polynucleotide molecule, indicates that the polynucleotide has been removed from its natural genetic environment and is therefore free of other inappropriate or unwanted coding sequences and is in a form suitable for use in production systems for genetically engineered proteins. Such isolated molecules are those that are obtained from their natural environment and include cDNA and genomic clones.

9

9

9« 9 the term cloned protein/polypeptide, the protein is found behind

The isolated DNA molecules of the present invention do not contain other genes with which they are normally associated, but may contain naturally occurring 5' and 3' untranscribed regions, such as promoters and terminators. Identification of the linked regions is obvious to those skilled in the art (see, for example, Dynan and Tijan, Nature 316: 774-778, 1985).

The term isolated polynucleotide can alternatively be replaced by polynucleotide. When used for then the term isolated indicates that it is under conditions other than those in the natural environment. In a preferred form, the isolated protein does not contain other proteins, in particular other homologous proteins (i.e. homologous impurities (see below)). It is preferred to provide the protein in greater than 40% purity, even more preferably in greater than 60% purity. It is even more preferred to provide the protein in highly pure form, i.e. in greater than 80% purity, even more preferably in greater than 95% purity and most preferably in greater than 99% purity, as determined by SDS-PAGE.

The term isolated protein/polypeptide can alternatively be replaced by the term purified protein/polypeptide.

The term homologous impurity refers to any impurity (e.g., a polypeptide other than a polypeptide of the present invention) that originates from a homologous cell from which the polypeptide of the present invention was originally obtained.

The term "derived from", as used in the present invention in connection with a particular microbial source, refers to a polynucleotide and/or polypeptide produced from a particular source or from a cell into which the source gene has been inserted.

The term operably linked, in relation to parts of DNA, indicates that the parts are arranged such that their function is consistent for their intended purposes, e.g., transcription is initiated at a promoter and proceeds through the coding region to the terminator.

4 • «·· 44 ·4

444 4444

4444 4444

4444 4 4 4 4444 4 4 4 4 4

44 4 4444

44 · 44 44

The term polynucleotide refers to a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end. Polynucleotides include RNA and DNA and can be isolated from natural sources, synthesized in vitro, or prepared by combining natural or synthetic molecules.

The term complements of polynucleotide molecules refers to polynucleotide molecules that have a complementary base sequence and a reversed orientation compared to a reference sequence. For example, the sequence 5' ATGCACGGG 3' is complementary to the sequence 5' CCCGTGCAT 3'.

The term degenerate nucleotide sequence refers to a sequence of nucleotides that includes one or more degenerate codons (compared to a reference polynucleotide molecule that encodes a polypeptide). Degenerate codons contain a different triplet of nucleotides, but encode the same amino acid residue (i.e., triplets GAU and GAC both encode Asp).

The term promoter refers to the portion of a gene containing DNA sequences that allow RNA polymerase to bind and initiate transcription. Promoter sequences are generally, but not always, found in the 5' non-coding regions of genes.

The term secretory signal sequence refers to a DNA sequence that encodes a polypeptide (secretory peptide) that, as a component of a larger polypeptide, guides the larger polypeptide through the secretory pathway of the cell in which it is synthesized. The larger peptide is generally cleaved to remove the secretory peptide during passage through the secretory pathway.

How to use the sequences of the present invention to obtain other related sequences.

The sequence information disclosed in the present invention, which relates to the polynucleotide sequence encoding the mannanase of the present invention, can be used as a tool for identifying other homologous mannanases. For example, polymerase chain reaction (PCR) can be used to amplify i w*.— • 9

9999

9 «» * · • · · 9 9 9 9

9 9 9 9 9 9 9

9 9999 9 9 99 9

9 9 9 9 9 9 9

99 9 9 9 9 9 sequences encoding other homologous mannanases from various bacterial sources, particularly other Bacillus strains.

Activity test procedure

A polypeptide of the present invention having mannanase activity can be assayed for mannanase activity according to standard assay procedures known in the art, such as plating the test solution into 4 mm diameter holes cut in an agar plate containing 0.2% AZCL galactomannan (carob), i.e., a substrate for the endo-1,4-beta-D-mannanase assay available as Cat. No. I-AZGMA from Megazyme at a cost of $110 for 3 grams.

(The Internet address of Megazyme is: http://www.megazyme.com/Purchase/index.html).

Polynucleotides

An isolated polynucleotide of the present invention will hybridize to comparably large regions of SEQ ID NO: 1 or a sequence that is complementary to that sequence, at least under moderately stringent conditions.

Specifically, the polynucleotides of the present invention will hybridize to a denatured double-stranded DNA probe that either contains the entire sequence set forth at positions 97 to 1029 of SEQ ID NO: 1, or any probe that contains a subsequence of SEQ ID NO: 1 of at least 100 base pairs in length, at least under moderate conditions, but preferably under conditions that are severe, which are described in more detail below. Suitable experimental conditions for determining moderate or severe hybridization between a nucleotide probe and a homologous DNA or RNA sequence include soaking a filter containing DNA fragments or RNA to be hybridized in 5 x SSC (sodium chloride/sodium citrate, Sambrook et al., 1989) for 10 minutes and prehybridizing the filter in a solution of 5 x SSC, x Denhardt's solution (Sambrook et al., 1989), 0.5% SDS, and

100 pg/ml denatured sonicated salmon sperm DNA • · ···· «· · · · *· • · · · · · ♦ • · · · * · · · • · · *··* * · « · · • « · · · · · « * ··· · ·* · ♦· 0 (Sambrook et al., 1989) followed by hybridization in the same solution containing 10 ng/ml of random primer (Feinberg, A.P. and Vogelstein, B. (1993) Anal. Biochem. 132: 6 to 13), 32P-dCTP labeled (specific activity greater than 1 x 109 cpm/pg) probe for 12 hours at 45 °C. The filter was then washed twice for 30 minutes in 2 x SSC, 0.5% SDS at at least 60°C (moderately severe conditions), more preferably at least 65°C (moderately severe/severe conditions), even more preferably at least 70°C (severe conditions), and most preferably at least 75°C (very severe conditions).

Molecules to which the oligonucleotide probe hybridizes under these conditions are detected using X-ray film.

As previously mentioned, the isolated polynucleotides of the present invention include DNA and RNA. Methods for isolating DNA and RNA are well known in the art. DNA and RNA encoding genes of interest can be cloned in Gene Bank or DNA libraries using methods known in the art.

Polynucleotides encoding polypeptides having mannanase activity of the present invention are then identified and isolated, for example, by hybridization or PCR.

The present invention further provides polypeptides that are counterparts and polynucleotides from other bacterial strains (orthologs or paralogs). Of particular interest are mannanase polypeptides from gram-positive alkalophilic strains, particularly Bacillus species.

Substances homologous to polypeptides having mannanase activity of the present invention can be cloned using the information and means provided by the present invention in combination with conventional cloning techniques. For example, a DNA sequence of the present invention can be cloned using chromosomal DNA obtained from a cell type that expresses the protein. Suitable sources of DNA can be identified by Northern blotting with probes designed from the sequences described in the present invention. A library is then prepared from the chromosomal DNA of the positive cell line. The DNA sequence of the present invention encoding a polypeptide having mannanase activity can then be isolated by various methods, such as testing with probes designed from the sequences described in the present application and claims or with one or more sets of degenerate probes based on the described sequences. The DNA sequence of the present invention can also be cloned by polymerase chain reaction or PCR (Mullis, U.S. patent 4,683,202) using primers designed from the sequences described in the present invention. Among other methods, the DNA library can be used to transform or inoculate host cells and the expression of the DNA of interest can be detected by an antibody (monoclonal or polyclonal) raised against mannanase cloned from B. agaradherens, NCIMB 40482, expressed and purified as described in the Materials and Methods section and in Example 1, or by an activity assay that involves a polypeptide having mannanase activity.

The mannanase encoding part of the DNA sequence cloned into the plasmid pSJ1678 present in Escherichia coli DSM 12180 and/or an analogous DNA sequence according to the present invention can be cloned from a strain of Bacillus agaradherens bacteria, preferably from the strain NCIMB 40482, producing an enzyme having mannan degrading activity or from another or related organism as described in the present invention.

Alternatively, an analogous sequence can be constructed based on a DNA sequence obtainable from a plasmid present in Escherichia coli DSM 12180 (which is believed to be identical to the attached SEQ ID NO: 1), e.g., which is a subsequence thereof and/or by introducing nucleotide substitutions that do not result in a different amino acid sequence of the mannanase encoded by the DNA sequence, but which corresponds to the codon usage of the host organism intended for production of the enzyme or by introducing nucleotide substitutions that result in a different amino acid sequence (i.e., a variant of the mannan degrading enzyme of the present invention).

Polypeptides

The amino acid sequence of amino acids 32 to 343 in SEQ ID NO: 2 is the mature mannanase sequence.

The present invention also provides mannanase polypeptides that are substantially homologous to the polypeptide of SEQ ID NO: 2 and substances that are homologous thereto (paralogs or orthologs). The term substantially homologous is used in the present invention to refer to polypeptides that have 70%, preferably at least 80%, more preferably at least 85% and even more preferably at least 90% sequence identity with the sequence set forth at amino acids 32 to 343 of SEQ ID NO: 2 or orthologs or paralogs thereof. Such polypeptides will preferably be at least 95% identical and most preferably 98% or more identical to the sequence set forth at amino acids 32 to 343 of SEQ ID NO: 2 or orthologs or paralogs thereof. The percent sequence is determined by conventional means, using programs known in the art, such as GAP provided in the GCG program package (Program Manual for the Wisconsin Package, version 8, August 1994, Genetics Computer Group, 575, Science Drive, Madison, Wisconsin, USA 53711), which is described in Needleman, S.B., and Wunsch, C.D. (1970), Journal of

Molecular Biology, 48, 443-453, which is incorporated herein by reference. GAP is used to compare polypeptide sequences with the following settings: GAP creation penalty is set to 3.0 and GAP extension penalty is set to 0.1.

The sequence identity of a polynucleotide molecule is determined by similar procedures using GAP with the following settings for DNA sequence comparison: GAP creation penalty has a value of 5.0 and GAP extension penalty has a value of 0.3.

identities with computer • · · · · · · ······ ·· · · · · · · · · · · · · « · · ·

The preparation of enzymes according to the present invention preferably starts from microorganisms, preferably from bacteria, algae or fungi, in particular from bacteria such as bacteria belonging to the Bacillus group, preferably to an alkalophilic strain of Bacillus, which can be selected from the group consisting of Bacillus agadherens and highly related Bacillus bacteria, which are preferably at least 95% and more preferably 98% homologous to Bacillus agadherens when comparing 16S rDNA sequences.

Essentially homologous proteins and polypeptides are characterized by having one or more amino acids exchanged, omitted, or added. These changes are preferably small, i.e., conservative amino acid substitutions (see Table 2) and other substitutions that do not significantly affect the shape or activity of the protein or polypeptide, small deletions, typically from one to 30 amino acids, and small extensions at the amino or carboxyl terminus, such as a methionine residue at the amino terminus, a small linker peptide of up to 20 to 25 residues, or small extensions that facilitate purification (affinity tag), such as the polyhistidine portion of protein A (Nilsson et al., EMBO J., 4: 1075, 1985; Nilsson et al., Methods Enzymol., 198: 3, 1991; see generally Ford et al., Protein Expression and Purification, 2: 95-107, 1991, which are incorporated herein by reference). DNA encoding affinity tags are available from commercial suppliers (e.g., Pharmacia Biotech, Piscataway, NJ; New England Biolabs, Beverly, MA).

However, even though the described changes are preferably small, they are sometimes of a larger nature, such as the joining of larger polypeptides of 300 amino acids or more, both at the amino terminus and at the carboxyl terminus of the mannanase polypeptides of the present invention.

• · • · · · · • · · ·

Table 1

Conservative amino acid substitutions

Basic: arginine, lysine, histidine

Acidic: glutamic acid, aspartic acid

Polar: glutamine, asparagine

Hydrophobic: leucine, isoleucine, valine

Aromatic: phenylalanine, tryptophan, tyrosine

Small: glycine, alanine, serine, threonine, methionine

In addition to the 20 standard amino acids, the amino acid residues of the polypeptide of the present invention may be replaced by non-standard amino acids (such as 4-hydroxyproline, 6-N-methyllysine, 2-aminoisobutanoic acid, isovaline, and α-methylserine). A limited number of non-conservative amino acids, amino acids not encoded by the genetic code, and unnatural amino acids may be used as replacement amino acid residues. Unnatural amino acids have been modified after protein synthesis and/or have a chemical structure in their side chain that is different from the chemical structure of standard amino acids. Unnatural amino acids may be chemically synthesized or are, preferably, commercially available and include pipecolic acid, thiazolidinecarboxylic acid, dehydroproline, 3- and 4-methylproline, and 3,3-dimethylproline.

Essential amino acids in the mannanase polypeptides of the present invention are identified by techniques known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, Science 244: 1081-1085, 1989). In alanine scanning mutagenesis, a single alanine mutation is made at each site in the molecule and the resulting mutated molecules are assayed for biological activity (i.e., mannanase activity) to identify amino acid residues that are essential for the activity of the molecule. See also Hilton et al., J. Biol. Chem., 271:

• φ

φ· φ φφ φ * φ · φφφφ φ φ φφφφφφφ φφ φ

4699-4708, 1996. The active site of an enzyme or other biological interaction can also be determined by physical analysis of the structure, which is determined by techniques such as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling in conjunction with mutation of amino acids from the putative constant site. See, for example, de Vos et al., Science, 255: 306-312, 1992, Smith et al., J, Mol. Biol., 224: 899-904, 1992, Wlodaver et al., FEBS Lett., 309: 59-64, 1992. The fact that amino acids are essential can also be deduced from homology analysis with polypeptides that are related to the polypeptides of the present invention.

Multiple amino acid substitutions can be made and tested by known methods of mutagenesis and/or recombination and then by appropriate testing procedures such as those described in Reidhaar-Olson and Sauer, Science,. 241: 53-57, 1988, Bowie and Sauer, Proc. Natl. Acad. Sci. USA 86: 2152-2156, 1989, WO 95/17413 or WO 95/22625. In short, these authors describe procedures for simultaneous changes at two or more sites in a polypeptide or recombination of different mutations (WO 95/17413, WO 95/22625) and subsequent selection of functional polypeptides, sequencing of the mutagenized peptides to determine the number of possible substitutions at each site. Other methods can be used, including phage display (e.g., Lowman et al., Biochem, 30: 10832-10837, 1991; Ladner et al., U.S. Patent No. 5,223,409; Huse, WIPO Publication WO 92/06204) and site-directed mutagenesis (Derbyshire et al., Gene 46: 145, 1986; Ner et al., DNA 7: 127, 1988).

The described mutagenesis methods can be combined with high-throughput automated screening methods to determine the activity of cloned mutagenized polypeptides in host cells. Mutagenized DNA molecules that encode active polypeptides can be recovered from host cells and rapidly sequenced using modern equipment. These procedures allow rapid determination of the importance of individual amino acid sequences • 9

9999

• 9 9

9999 9 9

9 9 9

9 · residues in polypeptides of interest and can be used for polypeptides of unknown structure.

Using the methods described, one of skill in the art can identify and/or prepare various polypeptides that are substantially homologous to residues 32 to 343 in SEQ ID NO: 2 and retain the mannanase activity of the wild-type protein.

Protein production

The proteins and polypeptides of the present invention, including full-length polypeptides, fragments thereof, and fusion proteins, can be produced in genetically engineered host cells using conventional techniques. Suitable host cells are those that can be transformed or transfected with exogenous DNA and grown in culture, and include bacteria, fungal cells, and higher eukaryotic cells grown in culture. Bacterial cells, especially cells grown in culture that are Gram-positive, are preferred. Gram-positive cells from the genus Bacillus are particularly preferred, namely cells from the group of Bacillus subtilis, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacillus lautus, Bacillus thuringiensis, Bacillus licheniformis, and Bacillus agaradherens, and especially Bacillus agaradherens.

Techniques for manipulating cloned DNA molecules and introducing exogenous DNA into various host cells are described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY, 1987, and Bacillus subtilis and other Gram-positive Bacteria, Sonensheim et al., 1993, American Society for Microbiology, Washington D.C., which are incorporated herein by reference.

In general, the DNA sequences encoding the mannanase of the present invention are operably linked to other genetic elements necessary for their expression, and generally include a transcription promoter and a terminator in an expression vector. The vector will also generally contain one or more selectable markers and one or more origins of replication, although one skilled in the art will recognize that in some systems the selectable markers are on separate vectors and replication of the exogenous DNA is accomplished by integration into the genome of the host cell. The selection of promoters, terminators, selectable markers, vectors, and other elements is a routine task within the skill of one skilled in the art. Many such elements are described in the literature and are available from commercial suppliers.

In order to direct the polypeptide into the secretory pathway of the host cell, a secretion signal sequence (also known as a leader sequence, preprosequence, or presequence) is placed in the expression vector. The secretion signal sequence may be native to the polypeptide in question, or may be derived from another secreted protein, or may be synthesized de novo. Many suitable secretion sequences are known in the art, and reference may be made to Bacillus subtilis and other Gram-positive bacteria, Sonensheim et al., 1993, American Society for Microbiology, Washington D.C. and Cutting, S.M. (editor) Molecular biological methods for bacillus, John Wiley and Sons, 1990, for further descriptions of suitable secretion signal sequences, particularly for the Bacillus host cell. The secretion signal sequence is linked to the DNA sequence in the correct reading frame. Secretory signal sequences are generally located 5' of the DNA sequence encoding the polypeptide of interest, although some signal sequences may be located anywhere in the DNA sequence of interest (see, Welch et al., U.S. Patent No. 5,037,746 , Holland et al., U.S. Patent No. 5,143,830 ).

Transformed or transfected host cells are grown in culture according to conventional procedures in a culture medium containing nutrients and other components required for the growth of the selected host cells. A variety of suitable media, including defined media and complex media, are known in the art and generally include a carbon source, a nitrogen source, essential amino acids, vitamins and minerals. The media also optionally contain such components as growth factors or serum, as appropriate. The growth medium will generally be selected for cells containing exogenously added DNA by, for example, selecting drugs or by deficiency of essential nutrients, which are supplemented with a selectable marker carried on an expression vector or cotransfected into the host cell.

Protein isolation

When the expressed recombinant polypeptide is secreted, the polypeptide can be purified outside the growth medium. Preferably, the host cells are removed from the medium (e.g., by centrifugation) prior to purification of the polypeptides.

If the expressed recombinant polypeptide is not secreted from the host cell, then the cell is preferably disrupted and the polypeptide is released into an aqueous extract, which is the first step in these purification techniques. Preferably, the host cells are removed from the medium (e.g., by centrifugation) prior to purification of the polypeptides.

Cell disruption is accomplished by conventional methods such as lysozyme treatment or high pressure treatment. For further descriptions of such cell disruption techniques, see Robert K. Scobes, Protein Purification, 2nd ed., Springer-Verlag).

Whether or not the expressed recombinant polypeptides (or chimeric polypeptides) are secreted, they can be purified using fractionation and/or conventional purification procedures and media.

Ammonium sulfate precipitation and acidic or chaotropic extraction can be used for sample fractionation. Examples of clean-up steps include hydroxyapatite, size exclusion, FPLC, and reversed-phase HPLC. Also suitable ion exchange media include derivatized dextrans, agarose, cellulose, polyacrylamide, specialty silica gels, and the like. Preferred are PEI, DEAE,

9 9 9 9 9 9

9 9 9 9 ·· 9

9999999 99 · • 9 9 9 9 9 9 9 9

9 9 9 9 9 99 99

QAE and Q derivatives, with DEAE Sepharose High Flow (Pharmacia, Piscataway, NJ) being most preferred. Examples of chromatographic media include phenyl, butyl or octyl derivatized media such as Phenyl Sepharose FF (Pharmacia), Toypearl butyl 650 (Toso Haas, Montgomeryville, PA), Octyl Sepharose (Pharmacia) and the like, or polyacrylic resins such as Amberchrom CG 71 (Toso Haas) and the like. Suitable solid supports include glass beads, silica gel resins, cellulose resins, agarose beads, cross-linked agarose beads, polystyrene beads, cross-linked polyacrylamide resins and the like, which are insoluble under the conditions under which they are used. These solid supports can be modified with reactive groups that allow attachment of proteins via amino groups, carboxyl groups, thiol groups, hydroxyl groups and/or sugar groups. Examples of chemistries for such attachment include cyanogen bromide activation, N-hydroxysuccinimide activation, epoxide activation, thiol activation, hydrazide activation, and coupling of carboxyl and amine derivatives via the carbodiimide method. These and other solid supports are well known, widely used in the art and are available from commercial suppliers.

The choice of a particular method is based on routine design and is determined in part by the properties of the selected support. See, for example, the book Affinity Chromatography: Principles & Methods, Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988.

The polypeptides of the present invention or fragments thereof can also be prepared by chemical synthesis. The polypeptides of the present invention are monomers or multimers, glycosylated or non-glycosylated, pegylated or non-pegylated, and may or may not include a starting methionine amino acid residue.

Based on the sequence information described in the present invention, a full-length DNA sequence encoding

« · · • φ φ φ φφφφ φ • ····· · · mananasu according to the present invention and comprising the DNA sequence shown in sequence identification No. 1 at least as the DNA sequence from position 97 to position 1029.

Cloning is carried out by standard procedures known in the art, such as preparation of a gene library from a Bacillus strain, particularly from the species B. agaradherens, NCIMB 40482;

plating such library onto suitable substrate plates;

identification of a clone comprising a polynucleotide sequence of the present invention by standard hybridization techniques using a probe based on sequence identification No. 1, or identification of a clone from the Bacillus agaradherens NCIMB 40482 gene library by an inverse PCR procedure using primers based on sequence information from sequence identification No. 1. For further details regarding inverse PCR, reference is made to the book M.J. McPherson et al., PCR A Practical approach, Information Press Ltd., Oxford, England).

Based on the sequence information described in the present invention (SEQ ID NO: 1, SEQ ID NO: 2), the isolation of homologous polynucleotide sequences encoding homologous mannanases of the present invention using a similar strategy using gene libraries from related microbial organisms, in particular gene libraries from other strains of the Bacillus species, such as an alkalophilic Bacillus species, is routine work for those skilled in the art.

Alternatively, DNA encoding the mannan or galactomannan degrading enzymes of the present invention can be conventionally cloned by well-known procedures from suitable sources, such as any of the above-mentioned organisms, using synthetic oligonucleotide probes prepared based on a DNA sequence obtainable from a plasmid present in Escherichia coli DSM 12180.

• · ····

9 9

9 9

9 9 · · · · 9 9 9 9

9 9 *· 9 9 9 99

The polynucleotide molecule of the present invention can be isolated from Escherichia coli DSM 12180, in which the plasmid obtained by cloning is stored. The present invention also relates to an isolated substantially pure biological culture of the Escherichia coli strain, DSM 12180.

In the present context, the term enzyme preparation means a means for obtaining either a conventional enzymatic fermentation product, optionally isolated and purified from a single species of microorganism, where this preparation usually comprises a number of different enzymatic activities; or a mixture of monocomponent enzymes, preferably enzymes derived from bacteria or fungi, using conventional recombinant techniques, where these enzymes are fermented and optionally isolated and purified separately and come from different sources, preferably fungal or bacterial sources; or a fermentation product of a microorganism which acts as a host cell for the expression of a recombinant mannanase, but which simultaneously produces other enzymes, e.g. pectin-degrading enzymes, proteases or cellulases, and which is a natural fermentation product from the microorganism, i.e. an enzyme complex normally produced by the corresponding natural microorganism.

The method of preparing an enzyme composition according to the present invention comprises cultivating a microorganism, e.g., a wild type, capable of producing mannanase under conditions permitting the formation of the enzyme and recovering the enzyme from the culture. The cultivation is carried out using conventional fermentation techniques, e.g., cultivation in shake flasks or shake fermenters, thereby ensuring sufficient aeration of the growth medium and inducing the production of the mannanase enzyme. The growth medium optionally contains a conventional nitrogen source, such as peptone, yeast extract or casamic acid, a limited amount of a carbon source, such as dextrose or sucrose, and an inducer, such as guar gum or locust bean gum. Recovery of the enzyme is carried out using conventional techniques, e.g., by separating the biomass and the supernatant • 0 • 0·· 0000 • 0 0000 0000

000000 0000000 00 0

by centrifugation or filtration, obtaining a solution above the disrupted cells if the enzyme is intracellular, optionally by subsequent purification, as described in EP 0 406 314 or by crystallization, as described in WO 97/15660.

Immunological cross-reactivity

Polyclonal antibodies used for immunological cross-reactivity determination are prepared using purified mannanase enzymes. More specifically, the anti-mannanase antiserum of the present invention is obtained from immunized rabbits (or other rodents) by the procedures described in N. Axelsen et al., A Manual of Quantitative Immunoelectrophoresis, Blackwell Scientific Publications, 1973, Chapter 23, or A. Johnston and R. Thorpe, Immunochemistry in Practice, Blackwell Scientific Publications, 1982 (specifically pages 27 to 31). Purified immunoglobulins can be obtained from the antiserum, for example, by precipitation with salts ((NH ₄ ) ₂ SO ₄ ) followed by dialysis and ion exchange chromatography, e.g. on DAEA-Sephadex. Immunochemical characterization of proteins is performed by Outcherlony double diffusion analysis (O. Outcherlony in Handbook of Experimental Immunology (D. M. Weir, editor), Blackwell Scientific Publications, 1967, pages 655 to 706), cross-linked immunoelectrophoresis (N. Axelsen et al., A Manual of Quantitative Immunoelectrophoresis, Blackwell Scientific Publications, 1973, chapters 3 and 4) or rocket immunoelectrophoresis (N. Axelsen et al., A Manual of Quantitative Immunoelectrophoresis, Blackwell Scientific Publications, 1973, chapter 2).

Examples of useful bacteria producing enzymes or enzyme compositions according to the present invention are Gram-positive bacteria, preferably from the division Bacillus/Lactobacillus, preferably a strain from the genus Bacillus, more preferably a strain of Bacillus agaradherens, especially a strain of Bacillus agaradherens, NCIMB 40482.

• · 99 9 ·» ·· · · · » · · 9 · • 9* 9 999 9 99 9

9999 · 9 9 9999 99 99 9

9 999 9999

999 9 99 9 99 99

The present invention includes an isolated mannanase having the properties described above and free from homologous impurities, produced using conventional recombinant techniques.

Determination of the catalytic activity (ManU - mannanase unit) of mannanase

Colorimetric test: 0.2% was used as substrate

AZCL-Galactomannan (Megazyme, Australia) in 0.1 M glycine buffer pH 10.0. The assay is performed in 1.5 ml Eppendorf microtubes on a thermal shaker with agitation at 40°C. Incubation of 0.750 ml substrate with 0.05 ml enzyme for 20 minutes, centrifugation for 4 minutes at 15,000 rpm. The color of the solution is measured at 600 nm in a 1 cm cuvette. One mannanase unit (ManU)' corresponds to an absorbance of 0.24 in 1 cm.

Obtaining mannanase from Bacillus agaradherens NCIMB 40482

Tribes

Bacillus agaradherens NCIMB 40482 containing a mannanase enzyme encoding DNA sequence.

E. coli strain: E. coli SJ2 cells (Diderichsen, B., Wedsted, U., Hedegaard, L., Jensen, B.R., Sjoholm, C. (1990) Cloning of aldB, which encodes alpha-acetolactate decarboxylase, an exoenzyme from Bacillus brevis, J. Bacteriol., 172, 4315-4321), were prepared and transformed by electroporation using a Gene Pul ser electroporator from BIO-RAD according to the procedure described by the manufacturer.

B. subtilis PL2306. This strain is B. subtilis DN1885 with the apr and npr genes disrupted (Diderichsen, B., Wedsted, U., Hedegaard, L., Jensen, B.R., Sjoholm, C. (1990) Cloning of aldB, which encodes alpha-acetolactate decarboxylase, an exoenzyme from Bacillus brevis, J. Bacteriol., 172, 4315-4321) in the transcription unit of the known Bacillus subtilis cellulase gene, resulting in cellulase-negative cells. The disruption is performed as described in the book Bacillus • 9

9*9* ·

9 99 9* • 9 9 9 9 *9 * 99* 9 9*9

9 9999 9 9 9 9 9

9 999 *99* *99 9 9* 9 99 99 subtilis and other Gram-Positive Bacteria, A.L. Sonenshein, J.A. Hoch and Richard Losick (editors), American Society for Microbiology, p. 618 (1993).

Competent cells were prepared and transformed according to the procedure described in R.E. Yasbin, G.A. Wilson and F.E. Young (1975) Transformation and transfection in Lysogenic strains of Bacillus subtilis'. evidence for selective induction of prophage in competent cells, J. Bacteriol., 121: 296-304.

Plasmids pSL1678 (described in more detail in WO 94/19454, which is incorporated herein by reference).

pMOL944. This plasmid is a pUBUO derivative containing elements that allow propagation of the plasmid in Bacillus subtilis, a kanamycin resistance gene, and has a strong promoter - and a signal peptide cloned from the amyL gene of B. licheniformis ATCC14580. The signal peptide containing a SacII site allows for the routine cloning of DNA encoding the mature portion of the protein by infusion with the signal peptide. This results in the expression of a pre-protein that is directed to the outside of the cell.

The plasmid was constructed using conventional genetic engineering techniques, which are briefly described below.

Construction of pMOL944

Plasmid pUBUO (T. McKenzie et al., 1986, Plasmid 15:93-103) was digested with the unique restriction enzyme NciII. A PCR fragment amplified from the amyL promoter encoded on plasmid pDN1981 (P.L. Jorgensen et al., 1990, Gene, 96, pp. 37-41) was digested with NciI and inserted into NciI digested pUBUO to yield plasmid pSJ2624.

The two PCR primers used have the following sequences:

LWN5494 5'-GTCGCCGGGGCGGCGCCTATCAATTGGTAACTGTATCTCAGC-3'

LWN54 95 5'-GTCGCCCGGGAGCTCTGATCAGGTACCAAGCTTGTCGACCTGCAGAATG

AGGCAGCAAGAAGAT-3’

Primer LWN5494 inserts a NotI center into the plasmid.

99 99

9 9 9 9 9 9 9

9 9 9 9 9 9 9 9 9

9999 99 9 999· 99 99 9

9 9 9 9 9 9 9

99 9 99 99

Plasmid pSJ2624 was then digested with SacI and NotI, and a new PCR fragment amplified from the amyL promoter encoded on pDN1981 was digested with SacI and NotI, and this DNA fragment was inserted into SacI-NotI digested pSJ2624, thereby obtaining plasmid pSJ2670.

This cloning replaces the original amyL promoter by cloning the same promoter but in the opposite direction. The two primers used for PCR amplification have the following sequences:

LWN5938 5'-GTCGGCGGCCGCTGATCACGTACCAAGCTTGTCGACCTGCAGAATGAGG

CAGCAAGAAGAT-3'

LWN5939 5'-GTCGGAGTCCTATCAATTGGTAACTGTATCTCAGC-3'

Plasmid pSJ2670 was digested with restriction enzymes PstI and BclI, and a PCR fragment amplified from the cloned DNA sequence encoding alkaline amylase SP722 (described in International Patent Application published as WO95/26397, which is incorporated herein by reference) was digested with PstI and BclI and inserted, yielding plasmid pMOL944. The two primers used for amplification have the following sequence.

LWN78 64 5'-AACAGCTGATCACGACTGATCTTTAGCTTGGCAC-3'

LWN7 901 5'-AACTGCAGCCGCGGCACATCATAATGGGACAAATGGG-3'

Primer LWN7901 inserts a SacII center into the plasmid.

Cloning of the mannanase gene from Bacilius agaradherens

Genomic DNA preparation

The Bacillus agaradherens strain NCIMB 40482 was propagated in liquid medium according to the procedure described in WO94/01532. After 16 hours of incubation at 30°C and 300 rpm, the cells were harvested and genomic DNA was isolated according to the procedure described in Pitcher, D.G., Saunders, N.A., Owen, R.J. (1989), Rapid extraction of bacterial genomic DNA with guanidinium thiocyanate. Lett. Appl. Microbiol., 8, 151-156.

Gene library preparation

Genomic DNA was partially digested with the restriction enzyme Sau3A and size-separated by electrophoresis on a 0.7% agarose gel. Fragments of 2 kb to 7 kb were isolated by electrophoresis on DEAE cellulose paper • · «· · ·· ·» ·· · · · · » · · · * · · · · · · e · « · • ·♦»» · · · »··· · · · · «

7>Λ · · · · · ···

-D «·· 9 99 9 99 99 (Dretzen, G., Bellard, M., Sassone-Corsi, P., Chambon, P. (1981), A reliable method for the recovery of DNa fragments from agarose and acrylamide gels. Anal. Biochem., 112, 295-298).

The isolated DNA fragments were ligated into BamHI digested pSJ1678 plasmid DNA and the ligation mixture was used to transform E. coli SJ2.

Identification of positive clones

The DNA library in E. coli constructed as described was screened on LB agar plates containing 0.2% AZCL-galactomannan (Megazyme) and 9 μΐ/ml chloramphenicol by incubation overnight at 37 °C. Clones showing mannanase activity were indicated by a light blue diffuse environment. Plasmid DNA from one of these clones was isolated by Qiagen plasmid spin preparation on 1 ml of nutrient medium overnight (cells incubated at 37 °C in TY with 9 μΐ/ml chloramphenicol and shaking at 250 rpm.

This clone (MB525) was further characterized by DNA sequencing of the cloned Sau3A DNA fragment, where this DNA sequencing was performed upstream of the primers using a Taq deoxy end-cycle sequencing kit (Perkin-Elmer, USA), fluorescently labeled ends and appropriate oligonucleotides as primers.

Analysis of the sequencing results was performed according to Devereux et al. (1984) Nucleic Acids Res., 12, 387-395. The mannanase coding sequence is given as SEQ ID NO: 1. The deduced protein sequence is given as SEQ ID NO: 2.

Subcloning and expression of mannanase in B. subtilis

The mannanase encoding DNA sequence of the present invention was PCR amplified using a PCR primer set consisting of two oligonucleotides:

0 000« ♦ t 0 ·» 00

0 * 0 0 0 0

000 0 00 0

0 00·0 00 00 0

0 0 0 0 0 0

0 « 0 0 0 0

Mananasa-upper-SacII

5' -CAT TCT GCA GCC GCG GCA GCA AGT ACA GGC TTT TAT GTT GAT

GG-3'

Mananasa-lower-Notl

5'-GAC GAC GTA CAA GCG GCC GCG CTA TTT CCC TAA CAT GAT GAT ATT TTC G-3'

The Scall and Notl restriction sites are underlined.

Chromosomal DNA isolated from B. agaradherens NCIMB 40482 by the procedure previously described was used as a template in a PCR reaction performed with Amplitaq DNA Polymerase (Perkin Elmer) according to the manufacturer's instructions. The PCR reaction was performed in PCR buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl ₂ , 0.01% (w/v) gelatin) containing 200 μΜ dNTPs, 2.5 units AmpliTaq polymerase (Perkin-elmer, Cetus, USA) and 100 pmol of each primer.

The PCR reaction was performed using a thermal DNA cycler (Landgraf, Germany). One incubation at 94°C for 1 minute was followed by thirty PCR cycles consisting of denaturation at 94°C for 30 seconds, cooling to 60°C for 1 minute, and extension at 72°C for 2 minutes. Five μΐ aliquots of the amplification product were analyzed by electrophoresis on a 0.7% agarose gel (NuSieve, EMC). The size of the DNA fragments was 1.4 kb, demonstrating the correct amplification of the gene segment.

Subcloning of PCR fragment

Forty-five μΐ aliquots of PCR products produced by the procedure previously described were purified using the QIAquick PCR purification kit (Qiagen, USA) according to the manufacturer's instructions. The purified DNA was eluted in 50 μΐ 10 mM Tris-HCl, pH 8.5.

pMOL944 (5 μρ) and twenty-five μΐ of the purified PCR fragment were digested with SacII and NotI, electrophoresed in a 0.8% low-temperature gelling agarose gel (SeaPlaque GTG, EMC), and the corresponding fragments were excised from the gel and purified.

»« ·· ·> · · · • · * · • · · 4» • 9 9 9 • 9 99 using the QIAquick Gel extraction kit (Qaigen, USA) according to the manufacturer's instructions. The isolated PCR DNA fragments were then ligated into pMOL944 digested with SacII-NotI and purified. Ligation was performed overnight at 16 °C using 0.5 µg of DNA fragment, one unit of T4 DNA ligase and T4 ligase buffer (Boehringer Mannheim, Germany).

The ligation mixture was used to transform competent B. subtilis PL23006. Transformed cells were plated on plates containing LBPG-10 µg/ml Kanamycin. After 18 hours of incubation at 37°C, colonies were observed on the plates. Some clones were analyzed by isolating plasmid DNA from the overnight culture medium.

One such positive clone was plated several times on the agar plate used above, this clone was named MB594. Clone MB594 was grown overnight in TY-10 ug/ml kanamycin at 37°C and the next day 1 ml of cells were used to isolate plasmid from cells using Qiaprep Spin Plasmid Miniprep Kit No. 27106 based on the manufacturer's recommendations for B. subtilis plasmid preparation. This DNA was then sequenced and the DNA sequence corresponding to the mature part of the mannanase was determined, i.e. positions 94 to 1404 of the attached sequence identification no. 3. The derived mature protein is shown as sequence identification no. 4. It is apparent that the 3' end of the mannanase encoded by sequence identification no. 1 was changed to the end shown in sequence identification no. 3 due to the design of the lower primer used in the PCR. The resulting amino acid sequence is shown in sequence identification no. 4 and it is apparent that the C terminus of sequence identification no. 2 (SHHVREIGVQFSAADNSSGQTALYVDNVTLR) was changed to the C terminus of sequence identification no. 4 (IIMLGK).

Media

TY (described in Ausubel, F.M. et al. (eds.) Current Protocols in molecular Biology, John Wiley and Sons, 1995).

• 9

999 9 9 9 9 9 99 9 Q-7 9 9999 99 9 9999 99 99 9

O / 99999 9999

999 9 99 9 99 99

LB agar (described in Ausubel, F.M. et al. (eds.) Current Protocols in molecular Biology”, John Wiley and Sons,

1995).

LBPG is LB agar (see above) supplemented with 0.5% glucose and 0.05 M potassium phosphate at pH 7.0.

BPX medium is described in EP 0 506 780 (WO 91/091129)

Expression, purification and characterization of mannanase from Bacillus agaradherens

Clone MB 594 obtained by the procedure described in the Material and Methods section was grown in 25 x 200 ml BPX medium with 10 µg/ml kanamycin in 500 ml shake flasks for 5 days at 37°C at 300 rpm.

Then, 6,500 ml of the culture fluid of clone MB 594 (batch no. 9813) from the shake flasks was combined and the pH was adjusted to 5.5.

Then, 146 ml of cationic reagent (C521) and 292 ml of anionic reagent (A130) were added with stirring, which caused precipitation. The precipitated material was separated by centrifugation using a Sorval RC 3B centrifuge at 9,000 rpm at 6 °C for 20 minutes. The resulting solution was further purified using a Whatman GF/D and C glass filter and finally concentrated on a 10 kDa filter.

The pH of this concentrate was adjusted to 7.5 with sodium hydroxide. The clear solution was applied to an anion exchange chromatography column containing 900 ml of Q-Sepharose equilibrated with 50 mmol Tris at pH 7.5. The mannanase-active band was eluted with a sodium chloride gradient.

The pure enzyme gave a single band on SDS-PAGE with a molecular weight of 38 kDa. The amino acid sequence of the mannanase enzyme, i.e. the transcribed DNA sequence, is given as sequence identification number 2.

Determination of kinetic constants

Substrate: Locust bean gum and reducing sugar analysis (PHBAH); Sigma Locust bean gum (G-0753).

A kinetic assay using different concentrations of locust bean gum and incubation for 20 minutes at 40°C at pH 10 gave:

K _cat : 467 per second

Κ„,: 0.08 grams per I

Molecular weight: 38 kDa pi (isoelectric point): 4.2

The determined temperature optimum for mannanase was 60 °C.

The activity profile versus pH showed a maximum between pH 8 and 10.

DSC differential scanning calorimetry gave a melting point of 77°C at pH 7.5 in tris buffer, indicating that the enzyme is very thermally stable.

Detergent compatibility using 0.2% AZCL-Galactomannan as substrate and incubation at 40°C as previously described demonstrated excellent compatibility with common liquid detergents and good compatibility with common powder detergents.

Obtaining mannanase 168 from Bacillus subtilis β-mannanase from Bacillus subtilis was characterized and purified by the following procedure.

The Bacillus subtilis gene was examined for homology to the known Bacillus sp. β-mannanase gene sequence (Mendoza et al., Biochemica et Biophysica Acta, 1243: 552-554, 1995).

The ydhT coding region, the product of which was unknown, showed 58% similarity to the known Bacillus β-mannanase. The following oligonucleotides were designed to amplify the sequences encoding the mature part of the putative β-mannanase: 5'-GCT CAA TTG GCG CAT ACT GTG TCG CCT GTG-3' and 5'-GAC GGA TCC CGG ATT CAC TCA ACG ATT GGC G-3'. Total genomic DNA from Bacillus subtilis strain 1A95 was used as a template to amplify the ydhT mature region using the above primers. PCR was performed using the GENE-AMP PCR kit with AMPLITAQ DNA polymerase (Perkin Elmer, Applied Biosystems, Foster City, CA). An initial melting time of 5 min at 95 °C was followed by 25 cycles with the following program: melting at 95°C for 1 minute, cooling at 55°C for 2 minutes, and extension at 72°C for 2 minutes. After the last cycle, the reaction was held at 72°C for 10 minutes to ensure complete extension. PCR products were purified using the QIAquick PCR Purification Kit (Qiagen, Chatsworth, CA).

The ydhT mature region amplified from Bacillus subtilis strain 1A95 was inserted into the expression vector pPG1524 (previously described) as follows. The amplified 1028 bp fragment was digested with Mfe I and BamH I. The expression vector pPG 1527 was digested with EcdR 1 and BamH I. The restriction products were purified using the QIAquick PCR purification kit (Qaigen, Chatsworth, CA). The two fragments were ligated using DNA ligase (13 hours, 16°C) and used to transform the competent E. coli strain DH5-a. Ampicillin-resistant colonies were grown for DNA preparation. The DNA was then characterized by restriction analysis. Plasmid pPG3200 containing the mature region of the ydhT gene. Plasmid pPG3200 was then used to transform the competent Bacillus subtilis strain PG 632 (Saunders et al., 1992).

Seven kanamycin-resistant Bacillus subtilis clones and one control clone PG 632 were grown in 20 ml of 20/20/5 medium (20 g/l tryptone, 20 g/l yeast extract, 5 g/l NaCl) supplemented with 1 ml of 25% maltrin, 120 μΐ 10 mM MnCl ₂ and 20 μΐ 50 mg/ml kanamycin. The clones were grown overnight in 250 ml shake flasks at 37°C at 250 rpm to express the protein. The cells were centrifuged for 15 min at 14,000 rpm. One μΐ of the centrifuged solution was diluted with 99 μΐ of 50 mM sodium acetate (pH 6.0). One μΐ of this diluted solution was then assayed using endo-1,4-β-Mananase Beta-Mannazyme Tabs (Megazyme, Ireland) according to the manufacturer's instructions. The absorbance was read at 590 nm on a Beckman DU640 spectrophotometer. Clone 7 showed the highest absorbance at 1.67. The PG632 control showed no absorbance at 590 nm.

The centrifuged solution was analyzed by SDS-PAGE on a 10% to 20% Tris-glycine gel (Novex, San Diego, CA) to confirm the expected protein size of 38 kDa. Samples were prepared as follows. A 500 μΐ sample of ydhT clone 7 and PG 632 centrifuged solutions were precipitated with 55.5 μΐ of 100% trichloroacetic acid (Sigma), washed with 100 μΐ of 5% trichloroacetic acid, resuspended in 50 μΐ of Tris-glycine SDS sample buffer (Novex), and boiled for 5 min. One μΐ of each sample was subjected to gel electrophoresis at 30 mA for 90 min. A broad protein band was observed for clone 7 ydhT at 38 kDa.

Fermentation of Bacillus subtilis ydhT clone 7 in a volume of 10 L was carried out in a B-Braun Biostat C fermentor. The fermentation conditions were as follows. The cells were grown for 18 hours in 20/20/5 medium at 37°C. At the end of the fermentation, the cells were removed and the solution was concentrated to a volume of 1 liter using a tangential flow filtration system. The final yield of β-mannanase in the concentrated solution was determined to be 3 g/L.

Purification of β-mannanase from the fermentation broth was carried out as follows: 500 ml of the fermentation broth was centrifuged for 10 minutes at 10,000 rpm at 4 °C. The centrifuged solution was then dialyzed overnight at 4 °C in two 4-l batches in 10 mM potassium phosphate (pH 7.2) through a Spectrapor membrane with a molecular weight cutoff of 12,000 to 14,000 (Spectrum). The dialyzed solution was centrifuged for 10 minutes at 10,000 rpm at 4 °C. An anion exchange column packed with 300 ml of high-flow Q-Sepharose (Pharmacia) was equilibrated with 1 liter of 10 mM potassium phosphonate (pH 7.2) at 20°C, and then 300 ml of the centrifuged solution was poured onto the column.

Two fractions of 210 ml (sample A) and 175 ml (sample B) were obtained. These two fractions were assayed as previously described except that the solutions were diluted with 199 μΐ of 50 mM sodium acetate (pH 6.0) and showed absorbances of 0.38 and 0.52, respectively. Two μΐ of each sample were added to 8 μΐ of Tris-glycine SDS sample buffer (Novex, CA) and boiled for 5 minutes. The resulting samples were electrophoresed on a 10% to 20% Tris-Glycine gel (Novex, CA) for 90 minutes at 30 mA. A major band corresponding to 38 kDa was present in both samples and accounted for more than 95% of the total protein. BCA protein assay (Pierce) was performed on both samples according to the manufacturer's instructions, using bovine serum albumin as a standard, and sample A and sample B contained 1.3 mg/ml and 1.6 mg/ml β-mannanase, respectively. The identity of the protein was confirmed by ion spray mass spectrometry and amino acid sequence analysis from the amino terminus.

The determination of the enzymatic activity of purified β-mannanase samples was performed as follows. All assays used endo-1,4-β-Mananase Beta-Mannazyme Tabs (Megazyme, Ireland), which has been previously described. The activity in the pH range 3.0 to 9.0 was determined in 50 mM citrate phosphate buffer, 50 mM CAPSO (Sigma) was used for the determination of activity at pH 9.5, and 20 mM CAPS buffer was used for the pH range 10.0 to 11.0. The pH optimum for Bacillus subtilis β-mannanase was determined to be 6.0 to 6.5. The temperature activity profile was determined in 50 mM citrate phosphate buffer (pH 6.5). The enzyme showed optimal activity at 40 °C to 45 °C. Bacillus subtilis β-mannanase retained • ·· · 9 9

9 9 9 · • · · · · · · · · · · · · · · · · · · · · · · · · · · · · significant activity at temperatures below 15 °C and above 80 ’C. The specific activity against β-1,4-galactomannan was determined to be 160,000 gmol/min*mg β-mannanase using endo-1,4-h-Mananase Beta-Mannazyme Tabs (Megazyme, Ireland) according to the manufacturer’s instructions. The nucleotide and amino acid sequences of Bacillus subtilis β-mannanase are given as SEQ ID NOs 5 and 6.

The carbohydrate gum degrading enzyme is included in the laundry detergent compositions of the present invention generally in an amount of 0.0001% to 2% by weight, more preferably 0.0001% to 0.1% by weight, most preferably 0.0006% to 0.02% by weight of pure enzyme per weight of the composition.

The carbohydrate gum degrading enzyme of the present invention has, in addition to an enzymatic core comprising a catalytic domain, a cellulose binding domain (CBD), wherein the cellulose binding domain and the enzymatic core (catalytically active domain) of the enzyme are operably linked. The cellulose binding domain (CBD) exists as an integral part of the encoded enzyme or the CBD from another source is inserted into the enzyme, thereby forming an enzyme hybrid. In this context, the term cellulose binding domain should be understood as defined in Peter Tomme et al., Cellulose-Binding Domains Classification and Properties in the book Enzymatic Degradation of Insoluble Carbohydrates, John N. Saddler and Michael H. Penner (eds.), ACS Symposium Series No. 618, 1996. This definition classifies more than 120 cellulose-binding domains into 10 divisions (I to X) and shows that CBDs are found in various enzymes such as cellulases, xylanases, mannanases, arabinofuranosidases, acetylesterases and chitinases. CBDs have also been found in algae, e.g. in the red alga Porphyra purpurea, in the form of a non-hydrolytic polysaccharide-binding protein, see Peter Tomme et al., Cellulose-Binding Domains Classification and Properties in Enzymatic Degradation of Insoluble Carbohydrates, John N. Saddler and Michael H. Penner (eds.), ACS Symposium Series No. 618, 1996. However, most CBDs are from cellulases and xylanases. CBDs are found at the N and C termini of proteins or are internal. Enzymatic hybrids are known in the art, see e.g. WO 90/00609 and WO 95/16782 and are prepared by transferring a DNA construct comprising at least a DNA fragment encoding a cellulose binding domain ligated, with or without a linker, to a DNA sequence encoding a carbohydrate gum degrading enzyme into a host cell and growing the host cell so that it expresses the linked gene. Enzyme hybrids can be described as CBD-MR-X, where CBD is the N-terminal or C-terminal region of the amino acid sequence corresponding to at least the cellulose binding domain; MR is the middle part (linker) and optionally just a bond or short linking group consisting preferably of 2 to 100 carbon atoms, even more preferably of 2 to 40 carbon atoms; or preferably from 2 to 100 amino acids, even more preferably from 2 to 40 amino acids and X is the N-terminal or C-terminal region of the enzyme of the present invention.

The enzymes are of any suitable origin, such as plant, animal, bacterial, fungal and yeast. The origin is further mesophilic or extremophilic (psychrophilic, psychrotropic, thermophilic, barophilic, alkalophilic, acidophilic, halophilic, etc.). Purified or non-purified forms of these enzymes can be used. It is now common practice to modify the original natural types of enzymes by protein or genetic engineering techniques in order to optimize their effectiveness in the cleaning compositions of the present invention. For example, variants can be designed in which the compatibility of the enzyme with the common components of such compositions is increased. Alternatively, a variant can be designed in which the optimum pH, bleaching and chelating stability, catalytic activity and the like of the enzyme are precisely adjusted to the needs of a particular cleaning application.

• ·

In particular, attention should be paid to oxidation-sensitive amino acids for bleach stability and surface tension for detergent compatibility. The isoelectric point of such enzymes can be modified by replacing some charged amino acids, e.g. increasing the isoelectric point will help improve compatibility with anionic surfactants. The stability of enzymes can be further increased by e.g. creating additional salt bridges and strengthening metal-binding centers to increase stability under chelation.

Detergent ingredients

The laundry detergent compositions of the present invention also optionally contain other detergent ingredients. The precise nature of these other ingredients and the amount in which they are used will depend on the physical form of the composition and the nature of the cleaning operation for which they are used.

The laundry detergent compositions of the present invention preferably further comprise detergent components selected from certain surfactants, other enzymes, a builder and/or a bleaching system.

The laundry detergent compositions of the present invention are liquid, in the form of pastes, gels, sticks, tablets, sprays, foams, powders or in the form of granules. Granular compositions are sometimes also in compact form, liquid compositions are sometimes also in concentrated form.

A preferred type of gel detergent is a high-performance laundry gel composition comprising 15% by weight to 40% by weight of an anionic surfactant component consisting of: (i) 15% by weight to 25% by weight of alkyl polyethoxylated sulfates in which the alkyl consists of 10 to 22 carbon atoms and the polyethoxylated chain contains 0.5 to 15, preferably 0.5 to 5, and more preferably 0.5 to 4 ethylene oxide units; and (ii) 15% by weight to 20% by weight of fatty acids.

The gel compositions of the present invention further comprise one or more additional detersive ingredients selected from the group consisting of non-citrate builders, optical brighteners, soil release polymers, dye transfer inhibitors, polymeric dispersants, enzymes, suds suppressors, fragrances, dyes, salt builders, hydrotropes, anti-redeposition agents, anti-fouling agents, color fixatives, anti-fragrance agents, and mixtures thereof.

The gel compositions of the present invention have a viscosity of 100 cP to 4,000 cP, preferably 300 cP to 3,000 cP, more preferably 500 cP to 2,000 cP, at a rotation speed of 20 s ^-1 and are stable during storage.

Without being bound by any particular theory, it is believed that the presence of electrolytes affects the viscosity of the gel compositions. The gel-like nature of the compositions of the present invention is influenced by the choice of surfactants and the amount of electrolytes present. In preferred embodiments of the present invention, the compositions will further comprise 0% by weight to 10% by weight, preferably 2% by weight to 6% by weight, more preferably 3% by weight to 5% by weight of a suitable electrolyte or its acid equivalent. Sodium citrate is a highly preferred electrolyte for use in the present invention.

The compositions of the present invention optionally contain 0% by weight to 10% by weight solvents and hydrotropes. Without being bound by any particular theory, it is believed that the presence of solvents and hydrotropes can affect the equilibrium of the structured arrangement of the compositions versus the isotropic arrangement. Solvent refers to solvents generally used in the detergent industry, including alkyl mono-, di- and trialcohols, ethylene glycol, propylene glycol, propanediol, ethanediol, glycerin, etc. Hydrotrope refers to hydrotropes generally used in the detergent industry, including short chain surfactants that help solubilize other surfactants. Other examples of hydrotropes include cumene, xylene or toluene sulfonate, urea, alkyl (C ₈

4 44 4 44 44 • · · ··· 4 4 4 4

4 4 4 4 4 · 4 44 4 λ _r 4 4444 · 4 44444 4 4 44 4

4θ 444444444

444 4 44 4 44 44 or shorter chain) carboxylates, alkyl (C ₈ or shorter chain) sulfates and ethoxylated sulfates.

The fatty acids used in the present invention include saturated and/or unsaturated fatty acids obtained from natural sources or prepared synthetically. Examples of fatty acids include caproic, lauric, myristic, palmitic, stearic, arachidonic and behenic acid. Other fatty acids include palmitoleic, oleic, linoleic, linolenic and ricinoleic acid.

The compositions of the present invention can be formulated into compositions suitable for hand washing or machine washing, including laundry additives and compositions suitable for use in soaking and/or pre-washing heavily soiled fabrics, softening compositions added to the rinse.

When the compositions of the present invention are formulated as compositions suitable for use in automatic washing machines, the compositions of the present invention preferably comprise a surfactant and a builder and one or more other detergent components, preferably selected from organic polymeric compounds, bleaching agents, other enzymes, suds suppressors, dispersants, soap-based dispersants, soil suspending agents and soil re-deposition preventing agents and corrosion inhibitors. The detergent compositions also optionally comprise softening agents, such as other detergent components. Such compositions containing enzymes hydrolyzing carbohydrate gums provide fabric cleaning, stain removal, bleaching, softening, color enhancement and color transfer prevention.

The compositions of the present invention can also be used as additive detergent products. Such additive products are intended to supplement or enhance the effect of conventional detergent compositions.

• · · · • φ φ φ • φ φ φ φ φ · φ φφ φφ

If desired, the density of the detergent compositions according to the present invention at 20°C is 400 g/l to 1200 g/l, preferably 500 g/l to 950 g/l.

The compact form of the compositions of the present invention is best characterized by its density and, from the composition point of view, the amount of inorganic builder salts, which are common ingredients of detergent compositions in powder form; in conventional detergent compositions, builder salts are present in a substantial amount, typically 17% to 35% by weight of the total composition. In compact compositions, builder salts are present in an amount not exceeding 15% by weight of the total composition, preferably not exceeding 10% by weight, and most preferably not exceeding 5% by weight of the composition. The inorganic builder salts as used in the compositions of the present invention are selected from salts of alkali and alkaline earth metal sulfates and chlorides. The preferred builder salt is sodium sulfate.

The liquid detergent compositions of the present invention are optionally also in concentrated form, which means that the liquid detergent compositions of the present invention contain a lower amount of water compared to conventional liquid detergents. Typically, the water content of a concentrated liquid detergent is preferably less than 40% by weight, more preferably less than 30% by weight, and most preferably less than 20% by weight of the detergent composition.

Surfactant system

The laundry detergent composition of the present invention generally comprises a surfactant system in which the surfactant is selected from the group consisting of nonionic and/or anionic and/or cationic and/or ampholytic and/or zwitterionic and/or semipolar surfactants. Preferably, the laundry detergent compositions of the present invention comprise a nonionic, anionic and/or cationic surfactant.

9 ·· 99 • · · 9 9 · 9 9 9 9

999 9 999 9 99 · · ···· · 9 99999 9 9 ·· 9

4θ 9 9 999 9999

999 9 99 9 99 99

Surprisingly, it has been found that laundry detergent compositions according to the present invention further comprising a nonionic, anionic and/or cationic surfactant have improved stain and food soil removal, better clean faded fabrics and have improved bleaching effect.

Without being bound by any particular theory, it is believed that enzymatic hydrolysis results in small particles that are more easily removed by nonionic surfactants regardless of the specific contaminant. Preferred nonionic surfactants are alkyl ethoxylates AE3 to AE7. It is also believed that the combination of cationic surfactants with enzymatic hydrolysis of enzyme-degradable carbohydrate gums provides an improved overall effect.

The surfactant is typically present in an amount of from 0.1% to 60% by weight. More preferred amounts are from 1% to 35% by weight and most preferably from 1% to 30% by weight of the laundry detergent composition of the present invention.

The surfactant is preferably used so that it is compatible with the enzymatic components present in the composition. In liquid or gel compositions, the surfactant is used so that it enhances or at least does not impair the stability of the enzyme in these compositions.

Nonionic surfactants

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

9 9 9

9 9 9 9 9 9 • 9 9 9

9 9 9

9 9 9

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

Condensation products of primary and secondary aliphatic alcohols with 1 mole to 25 moles of ethylene oxide are suitable for use as a nonionic surfactant in the nonionic surfactant system of the present invention. The alkyl chain of the aliphatic alcohol is straight or branched, primary or secondary and generally consists of 8 to 22 carbon atoms. Preferred are condensation products of alcohols having an alkyl group consisting of 8 to 20 carbon atoms, more preferably 10 to 18 carbon atoms, with 2 moles to 10 moles of ethylene oxide per mole of alcohol. In said condensation products, 2 moles to 7 moles of ethylene oxide and most preferably 2 moles to 5 moles of ethylene oxide per mole of alcohol are present. Examples of commercially available nonionic surfactants of this type include Tergitol” 15-S-9 (a condensation product of a C _n to C ₁₅ linear alcohol with 9 moles of ethylene oxide), Tergitol” 24-L-6 NMW (a condensation product of a C ₁₂ to C ₁₄ primary alcohol with 6 moles of ethylene oxide with a narrow molecular weight distribution), both of which are sold by Union Carbide Corporation; Neodol™ 45-9 (condensation product of a C ₁₄ to C _x5 linear alcohol with 9 moles of ethylene oxide), Neodol” 23-3 (condensation product of a C ₁₂ to C ₁₃ linear alcohol with 3.0 moles of ethylene oxide), Neodol” 45-7 (condensation product of a C ₁₄ to C ₁₅ linear alcohol with 7 moles of ethylene oxide), Neodol” 45-5 (condensation product of a C ₁₄ to C ₁₅ linear alcohol with 5 moles of ethylene oxide) sold by Shell Chemical Company; Kyro” EOB (condensation product of a C ₁₃ to C ₁₅ alcohol with 9 moles of ethylene oxide), sold by The Procter & Gamble Company; and Genapol LA 030 or 050 (condensation product of C ₁₂ to C ₁₄ alcohol with 3 moles or with 5 moles • * 99 9 99 99

999 999 9999

999 9 999 9 «9 «

9999 99 9 9999 99 99 9

9 999 9999

999 9 99 9 99 99 ethylene oxide), sold by Hoechst. The preferred HLB range in these products is 8 to 11 and most preferably 8 to 10.

Also useful as a nonionic surfactant in the surfactant system of the present invention are the alkyl polysaccharides described in U.S. Patent 4,565,647, Llenado, issued January 21, 1986, which contain hydrophobic groups consisting of 6 to 30 carbon atoms, preferably 10 to 16 carbon atoms, and a polysaccharide, e.g., a polyglycoside, the hydrophilic group containing 1.3 to 10, preferably 1.3 to 3, most preferably 1.3 to 2.7 sugar units. Any reducing saccharide consisting of 5 or 6 carbon atoms may be used, e.g., glucose, galactose, and galactosyl groups may be substituted for glycosyl groups (optionally with hydrophobic groups attached at the 2-,

3-, 4-, etc., forming glucosides or galactosides). The bond between sugars is, for example, between the one-position of one sugar and the 2-, 3-, 4- and/or 6-position of the preceding sugar.

Preferred alkyl polyglycosides are compounds of general formula I:

R ² O (C _n H _2n O) _t (glycosyl) _x (I) where R ² is selected from the group consisting of alkyl, alkylphenyl, hydroxyalkyl, hydroxyalkylphenyl and mixtures thereof, in which the alkyls consist of 10 to 18 carbon atoms, preferably 12 to 14 carbon atoms, n is 2 or 3, preferably 2, t is 0 to 10, preferably 0, and x is 1.3 to 10, preferably 1.3 to 3, most preferably 1.3 to 2.7. Glycosyl is preferably derived from glucose. In the preparation of these compounds, an alcohol or alkylpolyethoxy alcohol is first produced and then reacted with glucose or a glucose source to form a glucoside (attachment at the 1-position). Further glycosyl units can then be attached via their 1-position to the 2-, 3-, 4- and/or 6-position of the preceding glycosyl unit, preferably in particular to the 2-position.

• φ ΦΦΦ· »· » φφ φφ • Φ · ο φ · φ φφφφ φφφφ φ · φ φφφφ · φ φ φ φ φ φ φφφ φφφφ φφφ φ φφ φ ·Φ φφ

Also suitable for use as an additional nonionic surfactant system according to the present invention are condensation products of ethylene oxide with a hydrophobic base formed by the condensation of propylene oxide with propylene glycol. The hydrophobic part of these compounds preferably has a molecular weight of 1500 to 1800 and is not soluble in water. The addition of polyoxyethylene units to this hydrophobic part leads to an increase in the solubility of the molecule as a whole in water and the fluid character of the product is maintained up to the point where the polyethylene content is 50% by weight of the total weight of the condensation product, which corresponds to condensation with up to 40 moles of ethylene oxide. Examples of compounds of this type include some commercially available surfactants such as Plurafac™ LF404 and Pluronic”, sold by BASF.

Also suitable for use as nonionic surfactants in the nonionic surfactant system of the present invention are condensation products of ethylene oxide with products formed by the reaction of propylene oxide and ethylenediamine. The hydrophobic group of these products consists of the reaction product of ethylenediamine and excess propylene oxide and generally has a molecular weight of 2500 to 3000. This hydrophobic group is condensed with ethylene oxide to such an extent that the resulting condensation product contains 40% to 80% by weight of polyoxyethylene and has a molecular weight of 5000 to 11000. Examples of nonionic surfactants of this type include some of the commercially available Tetronic™ compounds sold by BASF.

For use as a nonionic surfactant in the surfactant system of the present invention, polyethylene oxide condensates of alkylphenols, condensation products of primary and secondary aliphatic alcohols with 1 mole to 25 moles of ethylene oxide, alkyl polysaccharides and mixtures thereof are preferred. Most preferred are _C8 to _C14 alkylphenol ethoxylates containing 3 to 15 ethoxy groups and _C8 to _C18 alcohol ethoxylates (preferably _C10 on average) containing 2 to 10 ethoxy groups and mixtures thereof.

• · ·0 0 00 00

000 00« 0000

000 0 000 0 00 ·

0000 0 0 0 0000 0 0 0 0 0 0 0 *00 0000 *00 · 0« 0 00 0·

Highly preferred nonionic surfactants are polyhydroxy fatty acid amide surfactants of the general formula II:

R ² - C - N - Z

II I

R ¹ (II) where R ¹ is H or R ¹ is a C3 to C4 hydrocarbon, 2-hydroxyethyl, 2-hydroxypropyl or a mixture thereof, R ² is a C5 to _C31 hydrocarbon and Z is a polyhydroxy hydrocarbon having a linear hydrocarbon chain with at least 3 hydroxyl groups directly attached to the chain or an alkoxylated derivative thereof. Preferably R ¹ is methyl, R ² is a straight C _n to C ₁₅ alkyl or C ₁₆ to ,C ₁₈ alkyl or alkenyl such as alkyl derived from coconut oil or a mixture thereof, and Z is obtained from a reducing sugar such as glucose, fructose, maltose, lactose by reductive amination.

Anionic surfactants

Preferred anionic surfactants for use in the present invention are alkyl ester sulfates and linear alkylbenzene surfactants. Suitable anionic surfactants used are linear alkylbenzene sulfonate and alkyl ester sulfonate surfactants including linear C ₈ to C ₂₀ carboxylic acid esters (i.e. fatty acids) which are sulfonated by the gaseous S0 ₃ process according to The Journal of the American Oil Chemists Society, 52 (1975), pp. 323-329. Suitable starting materials include natural fatty substances which are derived from tallow, palm oil, etc.

Preferred alkyl ester sulfonate surfactant, especially for

use when washing, include alkyl ester sulfonate surfactants general formula III: 0 II R ³ - CH - I C - OR ⁴ 1 Sat ₃ M

(III)

where R ³ is a C8 to C20 hydrocarbon, preferably alkyl or a combination thereof, R ⁴ is a Cx to _C6 hydrocarbon, preferably alkyl or a combination thereof, and M is a cation which forms a water-soluble salt with the alkyl ester sulfonate. Suitable salt-forming cations include metals such as sodium, potassium and lithium and substituted or unsubstituted ammonium cations such as monoethanolamine, diethanolamine and triethanolamine. Preferably R ³ is a C10 to C16 alkyl and R ⁴ is methyl, ethyl or isopropyl. Especially preferred are methyl ester sulfonates in which R ³ is a C10 to _C10 alkyl.

Other suitable anionic surfactants include alkyl sulfate surfactants, which are water-soluble salts or acids of the general formula IV:

ROSO3M (IV) where R is preferably a C ₁₀ to C ₂₄ hydrocarbon, preferably an alkyl or hydroxyalkyl that includes a C ₁₀ to C ₂₀ alkyl moiety, more preferably a C ₁₂ to C ₁₈ alkyl or hydroxyalkyl and M is H or a cation, e.g. an alkali metal cation (e.g. sodium, potassium, lithium) or an ammonium or substituted ammonium cation (e.g. methyl-, dimethyl- and trimethylammonium cation and quaternary ammonium cations such as tetramethylammonium cation and dimethylpiperidinium cation and quaternary ammonium cations derived from alkylamines such as ethylamine, diethylamine, triethylamine and mixtures thereof and the like). Typically, C ₁₂ to C ₁₆ alkyls are preferred for lower wash temperatures (e.g. less than 50 °C) and C ₁₆ to C ₁₈ alkyls are preferred for higher wash temperatures (e.g. greater than 50 °C).

Other anionic surfactants useful for cleaning purposes may be used in the detergent compositions of the present invention. These include salts (including, for example, sodium, potassium, ammonium and substituted ammonium salts such as mono-, diatriethanolamine salts) of soap, C ₈ to C ₂₂ primary or secondary alkane sulfonates, C ₈ to C ₂₄ olefin sulfonates, • φ • · Φ · e 9 φφφφ ···.· · 1,082, 179, C ₈ to C ₂₄ alkyl polyglycol ether sulfates (containing up to 10 moles of ethylene oxide), alkyl glycerol sulfonates, fatty acid acyl glycerol sulfonates, glycerol sulfates derived from fatty oils, alkyl phenol ethylene oxide ether sulfates, paraffin sulfonates, alkyl phosphates, isethionates such as acyl isethionates, N-acyl taurates, alkyl succinamates and sulfosuccinates, monoesters of sulfosuccinates (especially saturated and unsaturated C ₁₂ to C ₁₈ monoesters) and diesters of sulfosuccinates (especially saturated and unsaturated C ₆ to C ₁₂ diesters), acyl sarcosinates, alkyl polysaccharide sulfates such as alkyl polyglucoside sulfates (nonionic unsulfonated compounds, which are described below), branched primary alkyl sulfates and alkyl polyethoxycarboxylates such as compounds of the general formula In:

RO (CH ₂ CH ₂ O) _k -CH ₂ COO-M ⁺ (V) where R is C ₈ to C ₂₂ alkyl, k is an integer from 1 to 10 and M is a cation forming a soluble salt. Also suitable are resin acids and hydrogenated resin acids such as rosin, hydrogenated rosin and resin acids and hydrogenated resin acids present in or derived from tall oil.

Other examples are given in the book Surface Active Agents and Detergents (Vols. I and II, Schwartz, Perry and Berch). Various such surfactants are also generally described in U.S. Patent 3,929,678, issued December 30, 1975, Laughlin et al., column 23, line 58 to column 29, line 23, which are incorporated herein by reference).

When used, the laundry detergent compositions of the present invention typically contain from 1% by weight to 40% by weight, preferably from 3% by weight to 20% by weight, of such anionic surfactants.

• ···· * ·

Highly preferred anionic surfactants, including alkyl alkoxylated sulfate surfactants, according to the present invention are water-soluble salts or acids of the general formula VI:

RO (A) _m SOjM (VI) where R is unsubstituted C ₁₀ to C ₂₄ alkyl or hydroxyalkyl, containing a C ₁₀ to C ₂₄ alkyl moiety, preferably C ₁₂ to C ₂₀ alkyl or hydroxyalkyl, even more preferably C ₁₂ to C ₁₈ alkyl or hydroxyalkyl, A is ethoxyl or propoxyl, m is greater than zero, typically 0.5 to 6, more preferably 0.5 to 3 and M is H or a cation, which is, for example, a metal cation (e.g. sodium, potassium, lithium, calcium, magnesium, etc.), ammonium or substituted ammonium cation. Alkyl ethoxylated sulfates as well as alkyl propoxylated sulfates are also suitable for the present invention. Specific examples of substituted ammonium cations include methyl, dimethyl, trimethylammonium cations and quaternary ammonium cations such as tetramethylammonium and dimethylpiperidinium cations and those derived from alkylamines such as ethylamine, diethylamine, triethylamine,

their mixtures 'and the like. Examples of surfactants are C ₁₂ until ^18 alkyl polyethoxylate (1.0) sulfate (C ₁₂ -C ₁₆ E (1, 0)M), 0-12 until whose alkyl polyethoxylate (2,25) sulfate (C ₁₂ -C ₁₈ E (2.25)M), c ₁₂ until C ₁₈ alkyl polyethoxylate (3.0) sulfate (C ₁₂ -C ₁₈ E (3, 0)M) a c ₁₂ until C ₁₈ alkyl polyethoxylate (4.0) sulfate (C ₁₂ -C ₁₈ E (4, 0)M), where M is

selected from sodium and potassium.

Cationic surfactants

Cationic detergent surfactants suitable for use in laundry detergent compositions of the present invention are those having a single long chain hydrocarbon group. Examples of such cationic surfactants include ammonium surfactants such as alkyltrimethylammonium halides and surfactants of general formula VII:

• · • · · · · · · · · · · · · · · · · · · · · · · · · · · · 9 · · · * ···· «·· « ·· 9 99 99 [R ² (OR ³ )y] [R ⁴ (OR ³ )y] ₂ R ⁵ N ⁺ X' (VII) where R ² is alkyl or alkylbenzyl, consisting of 8 to 18 carbon atoms in the alkyl chain; each R ³ is selected from the group consisting of -CH2CH2~, -CH2CH (CH3)-, CH2CH (CH2OH)-, -CH2CH2CH2- and mixtures thereof; each R ⁴ is selected from the group consisting of Cx to C ₄ alkyl, _Cx to C ₄ hydroxyalkyl, a benzylic ring structure formed by the connection of two R ⁴ groups,

-CH ₂ -CHOH-CHOHCOR ⁶ CHOHCH ₂ OH, where R ⁶ is any hexose or hexose polymer with a molecular weight of less than 1000 and where Y is not 0; R ⁵ is the same as R ⁴ or is an alkyl in which the total number of carbon atoms of R ² plus R ⁵ is not greater than 18; each y is 0 to 10 and the total sum of y is 0 to 15; and X is any compatible anion.

A quaternary ammonium surfactant suitable for use according to the present invention is a compound of general formula VIII:

(VIII) where R1 is a short chain alkyl (C6 to C10) or alkylamidoalkyl of the general formula IX:

_{c6 -c10} ·

(IX) y is 2 to 4, preferably 3,

R2 is H or C1 to C3 alkyl, x is 0 to 4, preferably 0 to 2, most preferably 0,

R3, R4 and R5 are the same or different and are either short-chain alkyl (C1 to C3) or alkoxylated alkyl of the general formula X:

• · · · · · · · · · • · » · · * · · · « ··· · · · · · · 9 • ···· · · · ··· « Φ · · · • · ··· · · · · · · • · · · « · · · · · · where R6 is C _x to C ₄ alkyl and az is 1 or 2

X' is an anion, preferably a halide, e.g. methyl sulfate.

Preferred quaternary ammonium surfactants are those corresponding to the compound of general formula VIII, and

R _x is C ₈ , C ₁₀ or a mixture thereof, x=o,

R ₃ , R ₄ = CH ₃ and R ₅ = CH ₂ CH ₂ OH.

Highly preferred cationic surfactants are water-soluble quaternary ammonium compounds useful in the compositions of the present invention of general formula XI:

chloride or

R ₁ R ₂ R ₃ R ₄ N ⁺ X (XI) where R _x is C ₈ to C ₁₆ alkyl, each of R ₂ , R ₃ and R ₄ is independently C _x to C ₄ alkyl, C _x to C ₄ hydroxyalkyl, benzyl and ~(C ₂ H ₄₀ ) _x H, where x is 2 to 5 and X is an anion. No more than one of R ₂ , R ₃ or R ₄ should be benzyl. The preferred alkyl chain length R _x is C ₁₂ to C ₁₅ especially when the alkyl is a mixture of chains of different lengths derived from coconut oil or palm oil or is derived synthetically by olefin construction or OXO alcohol synthesis. Preferred groups for R ₂ R ₃ and R ₄ are methyl and hydroxyethyl and the anion X is selected from the group consisting of halide, methylsulfonate, acetate and phosphate.

Examples of suitable quaternary ammonium compounds of general formula XI for use in the present invention are:

(coco alkyl)trimethylammonium chloride or bromide;

(coco alkyl)methyldihydroxyethylammonium chloride or bromide;

decyltriethylammonium chloride;

decyldimethylhydroxyethylammonium chloride or bromide;

«9 * 9 9 · · • 9 · · · · · • · 9 9 9 9 9 9

9999999 99 · • 9 · 9 9 9 9 9 • · 9 9» 99

C ₁₂ to C ₁₅ dimethylhydroxyethylammonium chloride or bromide;

(coco alkyl)dimethylhydroxyethylammonium chloride or bromide;

myristyltrimethylammonium methylsulfate; lauryldimethylbenzylammonium chloride or bromide; lauryldimethyl(ethenoxy)ammonium chloride or bromide; choline esters (compounds of general formula XI, where R _x is

CH ₂ -CH ₂ -OCC ₁₂ to C ₁₄ alkyl and R 1 , R 2 and R ₄ are methyl).

II dialkylimidazoles (compounds of general formula XI).

Other cationic surfactants useful in the present invention are also described in U.S. Patent 4,228,044, Cambre, issued October 14, 1980 and in European Patent Application EP 000,224.

Typical cationic fabric softening ingredients include water-insoluble quaternary ammonium fabric softening actives or their corresponding amine precursors, the most commonly used being di-(long-chain alkyl)ammonium chloride or methyl sulfate.

Of these, the preferred cationic softeners are as follows:

1) di(tallow alkyl)dimethylammonium chloride (DTDMAC);

2) di(hydrogenated tallow alkyl)dimethylammonium chloride;

3) di(hydrogenated tallow alkyl)dimethylammonium methyl sulfate;

4) distearyldimethylammonium chloride;

5) dioleyldimethylammonium chloride;

6) dipalmitylhydroxyethylmethylammonium chloride;

7) stearylbenzyldimethylammonium chloride;

8) di(tallow alkyl)trimethylammonium chloride;

9) di(hydrogenated tallow alkyl)trimethylammonium chloride;

10) C ₁₂ to C ₁₄ alkyl hydroxyethyldimethylammonium chloride;

11) C ₁₂ to C ₁₈ alkyl dihydroxyethylmethylammonium chloride;

12) di(stearoyloxyethyl)dimethylammonium chloride (DSOEDMAC);

• 9 99 9 99 9 9 • · · ♦ · · 9 9 9 9

9 9 9 9 9 9 9 9 9 9

9999 9 9 9 9999 φ * φ Φ

Φ Φ Φ · Φ φφφφ «ΦΦ 4 ΦΦ Φ ΦΦ ··

13) di(tallow alkyl-oxyethyl)dimethylammonium chloride;

14) di(tallow alkyl)imidazolinium methyl sulfate;

15) 1-[2-(tallow alkyl)ylamidoethyl]-2-(tallow alkyl)ylimidazolinium methyl sulfate.

Biodegradable quaternary ammonium compounds have been introduced as alternatives to the traditionally used di(long-chain alkyl)ammonium chlorides and methyl sulfates. Such quaternary ammonium compounds contain long-chain alk(en)yl groups interrupted by functional groups such as carboxyl groups. Such materials and fabric softening compositions containing them are described in many publications such as EP-A-0,040,562 and EP-A-0,239,910.

The quaternary ammonium compounds and amine precursors of the present invention are compounds of general formula XII:

R3 _/R2

N—(CH ₂ ) _n -Q-Tl Rl

X ^- (xii) or compounds of general formula XIII:

R2 /

—(CH ₂ ) _n —CH-CH ₂

A. 6 i

I

XT (xiii) where Q is selected from the group consisting of -OC(O)-, -C(O)-O-, -OC(O)-O-, -NR ⁴ -C(O)~, -C(O)-NR ⁴ -,

R ¹ is (CH ₂ ) _n -QT ² or T ³ ,

R ² is (CH ₂ ) _m -QT ⁴ or T ⁵ or R ³ ,

R ³ is C _x to C ₄ alkyl or C _x to C ₄ hydroxyalkyl or H,

R ⁴ is H or C _x to C ₄ alkyl or C _x to C ₄ hydroxyalkyl,

T ¹ , T ² , T ³ , T ⁴ , T ⁵ are independently C _n to C ₂₂ alkyl or alkenyl,

000 0 00 0 • ···· 0 · 0 00 00 0 0 0 0 0 • · 000 0000

0 0 0 00 0 00 0 0 n and m are integers 1 to 4 and

X' is an anion compatible with the plasticizer. Non-limiting examples of anions compatible with the plasticizer include chloride or methyl sulfate.

Alkyl or alkenyl, chains T ¹ , T ² , T ³ , T ⁴ , T ⁵ must consist of at least 11 carbon atoms. The chain is straight or branched. Tallow is a common and inexpensive source of long chain alkyls and alkenyls. Particularly preferred are compounds in which T ¹ , T ² , T ³ , T ⁴ , T ⁵ represent a mixture of long chain materials, typically from tallow.

Specific examples of quaternary ammonium compounds suitable for use in aqueous fabric softening compositions of the present invention include:

1) N,N-di(tallow alkyl - oxyethyl)-N,N-dimethylammonium chloride;

2) N,N-di(tallow alkyl-oxyethyl)-N-methyl-N-(2-hydroxyethyl)ammonium methyl sulfate;

3) N,N-di[2-(tallow alkyl)oxy-2-oxoethyl]-N,N-dimethylammonium chloride;

4) N,N-di[2-(tallow alkyl)oxyethylcarbonyloxyethyl]-N,N-dimethylammonium chloride;

5) N-[2-(tallow alkyloxy-2-ethyl]-N-[2-(tallow alkyl-2-oxoethyl]-N,N-dimethylammonium chloride;

6) N,N,N-tri[(tallow alkyl)yloxyethyl]-N-methylammonium chloride;

7) N-[2-(tallow alkyl)yloxy-2-oxoethyl]-N-(tallow alkyl)-N,N-dimethylammonium chloride; and

8) 1,2-di(tallow alkyl)yloxy-3-trimethylammoniumpropane chloride;

and mixtures of these materials.

When using such cationic surfactants in the laundry detergent compositions of the present invention, the cationic surfactants will typically comprise from 0.2% by weight to 25% by weight, preferably from 1% by weight to 8% by weight.

• · * · · 4 • 4 4 4 4 4

444444 44

4 4 ·

4 4 4 4 4

reductase, pullulan,

Conventional detergent enzymes

Laundry detergent compositions preferably contain, in addition to enzymes that degrade carbohydrate gums, one or more enzymes that have a cleaning effect, care for fabrics and/or finish the fabric, preferably cellulases and/or amylases.

Surprisingly, it has been found that laundry detergent compositions of the present invention containing an additional enzyme, in particular cellulase and/or amylase, have improved efficacy in removing food stains and soils, cleaning laundry and bleaching. In particular, it has been found that cellulolytic enzymes are particularly useful in breaking down food additives on cellulosic polysaccharides and are therefore useful in aiding in the removal of stains/soils from cotton fabrics.

Without being bound by any particular theory, it is believed that this improved cleaning effect is the result of the combined action of the enzymatically hydrolyzing cellulase enzyme on the cotton fabric and the carbohydrate gum degrading enzyme on the stain-binding polysaccharide on the cotton fabric. Similarly, improved cleaning effect occurs when amylase is applied to the finishing agent covering the surface of the cotton fabric and the carbohydrate gum degrading enzyme is applied to the stain-binding polysaccharide on the cotton fabric.

Such enzymes include enzymes selected from celluloses, hemicelluloses, peroxidases, proteases, glucoamylases, amylases, xylanases, lipases, phospholipases, esters, cutins, pectins, keratins, oxidases, phenoloxidases, lipoxygenases, lignins, tannes, pentosans, β-glucans, arabinosides, hyaluronides, chondritins, laccases or mixtures thereof.

Cellulases useful in the present invention include both bacterial and fungal cellulases. Preferably, they have a pH optimum of 5 to 12 and a specific activity of greater than 50 CEVU/mg (CEVU cellulose viscosity unit). Suitable cellulases are described in U.S. Patent 4,435,307, Barbesgoard et al.,

WO96/02653, which describe fungal cellulases

J61078384 and produced • · · · ♦ · · «· • · » · * 9 9 9 9 9

9 9 9 9 9 9 9 9 9 9

99999· 9999999 99 9 • · 9 9 9 9 9 9 9

9 * ·· · 99 99

Humicola insolens, Trichoderma, Thielavia and Sporotrichum.

EP 739 982 describes cellulases isolated from new species

Bacillus. Suitable cellulases are also described in GB-A-2,075,028; GB-A-2,095,275; DE-OS-2,247,832 and WO95/26398.

Examples of such cellulases are cellulases produced by the strain Humicola insolens (Humicola grisea var. thermoidea), in particular the strain Humicola DSM 1800.

Other suitable cellulases are cellulases derived from Humicola insolens with a molecular weight of 50 kDa, an isoelectric point of 5.5 and containing 415 amino acids, and a 43 kDa endoglucanase derived from Humicola insolens, DSM 1800, exhibiting cellulase activity, the preferred endoglucanase component having the amino acid sequence described in PCT patent application No. WO 91/17243. Also suitable are cellulases such as the EGIII cellulases from Trichoderma longibrachiatum described in WO94/21801, Genencor, published September 29, 1994. Particularly suitable cellulases are cellulases that protect colors. Examples of such cellulases are the cellulases described in European patent application No. 91202879.2, filed November 6, 1991 (Novo), and Carezyme and Celluzyme (Novo Nordisk A/S) are particularly useful. See also WO91/17244 and WO91/21801. Other cellulases suitable for fabric care and/or their cleaning properties are described in WO96/34092, WO96/17996 and WO95/24471.

These cellulases are normally used in detergent compositions in amounts of 0.0001% by weight to 2% by weight of pure enzyme based on the weight of the detergent composition.

Amylases (α and/or β) can be used to remove sugar-based stains. WO94/02597, Novo Nordisk A/S, published February 3, 1994, describes cleaning compositions containing mutant amylases.

See also WO95/10603, Novo Nordisk A/S, published April 20, 1995.

Other amylases known for use in cleaning compositions include α- and β-amylases. α-amylases are known in the art.

4 4 · subject matter and include those described in U. No. 5,003,257; EP 252,666; WO91/00353; FR

EP 285,123·; EP 525,610; EP 368,341 and British ♦ · 4 4*4«

4 4 4 · 44 ·

4444444 44 4 • · · 4 4 4 4

4 44 44

S. Patent 2,676,456; Patent Application No. 1,296,839 (Novo). Other suitable amylases are the enhanced stability amylases described in WO94/18314, published August 18, 1994 and WO96/05295, Genencor, published February 22, 1996 and amylase variants having further modifications available from Novo Nordisk A/S, described in WO 95/10603, published April 1995. Also suitable are the amylases described in EP 277 216,

WO95/26397 and WO96/23873 (all from Novo Nordisk).

Examples of commercial α-amylase products are Purafect Ox Am® from Genencor and Termamyl®, Ban®, Fungamyl® and Duramyl®, all available from Novo Nordisk A/S Denmark. WO95/26397 describes other suitable amylases: α-amylases which have a specific activity at least 25% higher than Termamyl® in the temperature range of 25°C to 55°C and at pH values of 8 to 10, measured using the Phadebas® α-amylase activity assay. Variants of said enzymes are described in WO96/23873 (Novo Nordisk). Other amylolytic enzymes with improved properties in relation to the level of activity and a combination of thermal stability and higher activity are described in WO95/35382.

The amylolytic enzymes are present in the detergent compositions of the present invention in amounts of 0.0001% to 2% by weight, preferably 0.00018% to 0.06% by weight, more preferably 0.00024% to 0.048% by weight of pure enzyme based on the weight of the composition.

A preferred combination is a laundry detergent composition which is a mixture of commonly used enzymes such as protease, amylase, lipase, cutinase and/or cellulase in conjunction with one or more plant cell wall degrading enzymes.

Peroxidase enzymes are used in combination with oxygen sources, e.g. percarbonate, perborate, persulfate, hydrogen peroxide, etc. and with phenolic

9 · • · · ♦ 9 * 9 • · substrate, such as bleaching enhancing molecules. They are used for bleaching the solution, i.e. . they prevent the transfer of dyes or pigments from substrates during washing to other substrates in the washing solution. Peroxidase enzymes are known in the art, for example, horseradish peroxidase, ligninase and haloperoxidase, such as chlor bromoperoxidase. Detergent compositions containing peroxidase are described, for example, in PCT International Application WO 89/099813, WO89/09813 and in European Patent Application EP No. 91202882.6, filed November 6, 1991 and EP No. 96870013.8, filed February 20, 1996. Laccase enzyme is also suitable.

The builders are used in an amount of 0.1% by weight to 5% by weight based on the total weight of the composition. Preferred builders are substituted phenothiazines and phenoxazines, 10-phenothiazinepropionic acid (PPT), 10-ethylphenothiazine-4-carboxylic acid (EPC), 10-phenoxazinepropionic acid (POP) and 10-methylphenoxazine (described in WO 94/12621) and substituted syringates (C3 to C5 substituted alkyl syringates) and phenols. Preferred sources of hydrogen peroxide are sodium percarbonate or sodium perborate.

These peroxidases are normally used in a detergent composition in an amount of 0.0001% by weight to 2% by weight of pure enzyme based on the total weight of the composition.

Other preferred enzymes for use in detergent compositions of the present invention include lipases. Suitable lipase enzymes for use in detergents include those produced by microorganisms of the Pseudomonas group, such as Pseudomonas stutzeri ATCC 19.154, as described in British Patent 1,372,034. Suitable lipases include those which show a positive immunological cross-reaction with an antibody to lipase produced by the microorganism Pseudomonas fluorescent IAM 1057. This lipase is available from Amano Pharmaceutical Co. Ltd., Nagoya, Japan, under the trade name Lipase P Amano, hereinafter referred to as

4» 44

4 4 4

4 4 4

4 4 4

4 4 «

4 4 4

Amano-P. Other suitable commercially available lipases include Amano-CES, lipases from Chromobacter viscosum, e.g. Chromobacter viscosum var. lipolyticum NRRLB 3673 from Toyo Jozo Co., Tagata, Japan, Chromobacter viscosum lipases from U.S. Biochemical Corp., USA and Disoynth Co., Netherlands, and lipases from Pseudomonas gladioli. Particularly suitable lipases are lipases such as M1 Lipasa® and Lipomax® (Gist-Brocades) and Lipolase® and Lipolase Ultra® (Novo), which have been found to be very effective when used in combination with the compositions of the present invention. Also suitable are the lipolytic enzymes described in EP 258 068, WO 92/05249 and WO 95/22615 of Novo Nordisk and in WO 94/03578, WO 95/35381 and WO 96/00292 of Unilever.

Also suitable are cutinases [EC 3.1.1.50], which can be considered a special type of lipase, namely lipases, which do not require surface activation. The addition of cutinas to detergent compositions has been described, for example, in WO-A-88/09367 (Genencor); WO 90/09446 (Plant Genetic System) and WO 94/14963 and WO 94/14964 (Unilever).

Lipases and/or cutinases are normally used in a detergent composition in an amount of 0.0001% by weight to 2% by weight of pure enzyme based on the total weight of the composition.

Suitable proteases are subtilisins, which are obtained from specific strains of B. subtilis and B. licheniformis (subtilisin BPN and BPN'). One suitable protease is obtained from a Bacillus strain which has maximum activity in the pH range of 8 to 12, developed and sold as ESPERASE® by Novo Industries A/S of Denmark, hereinafter referred to as Novo. The preparation of this enzyme and analogous enzymes is described in GB 1,243,784 by Novo. Other suitable proteases include ALCALASE®, DURAZYM® and SAVINASE® from Novo and MAXATASE®, MAXACAL®, PROPERASE® and MAXAPEM® (a protein developed as Maxacal) from Gist-Brocades. Proteolytic enzymes also include modified bacterial serine proteases, such as those

9 99 9 99 99

99» 999 9999

9 9 9 999 9 99 · • 9999 99 9 9999 99 99 9

9 999 9999

999 9 99 9 99 99 described in European Patent Application Serial No. 87 303761.8, filed April 28, 1987 (specifically pages 17, 24 and 98) and referred to herein as Protease B and in European Patent Application 199,404, Venegas, published October 29, 1986, which relates to a modified bacterial serine proteolytic enzyme referred to herein as Protease A. Also suitable is the protease referred to herein as Protease C, which is a variant of the alkaline serine protease from Bacillus in which lysine at position 27 is replaced by arginine, tyrosine at position 104 is replaced by valine, serine at position 123 is replaced by asparagine and alanine at position 274 is replaced by threonine. Protease C is described in EP 90915958:4, corresponding to WO 91/06637, published May 16, 1991. Genetically modified variants, in particular Protease C, are also encompassed by the present invention.

The preferred protease is designated Protease D and is a hydrolase variant having an amino acid sequence not found in nature that is derived from a carbonyl hydrolase precursor by substituting different amino acids for different amino acid residues at positions that are equivalent to position +76 in the carbonyl hydrolase, preferably also in combination with one or more amino acid residues at positions equivalent to a position selected from the group consisting of +99, +101, +103, +104, +107, +123, +127, +105, +109, +126, +128, +135, +156, +166, +195, +197, +204, +206, +210, +216, +217, +218, +222, +260, +265 and/or +274 according to the numbering of Bacillus amyloliquefaciens subtilisin as described in WO95/10591 and in the patent application of C. Ghosh et al., Bleaching Compositions Comprising Protease Enzymes, which has U.S. Serial No. 08/322,677, filed October 13, 1994. Also suitable is a carbonyl hydrolase variant of the protease described in WO95/10591, which has an amino acid sequence that is derived from the precursor carbonyl hydrolase by substituting various amino acids at positions that are equivalent in the precursor enzyme.

9999 9

9 99 99

9 9 9 9 9 9

999 9 99 9

9 9999 99 99 9

9 9 9 9 9 9

9 9* 99 position +210 in combination with one or more of the following residues +33, +62, +67, +76, +100, +101, +103, +104, +107, +128, +129, +130, +132, +135, +156, +158, +164, +166, +167, +170, +209, +215, +217, +218 and +222, where the position numbering corresponds to the native subtilisin from Bacillus amyloliquefaciens or the corresponding amino acid residues in other carbonyl hydrolases or subtilisins, such as Bacillus lentus subtilisin (co-pending patent application US Serial No. 60/048,550, filed June 4, 1997).

Also suitable for the present invention are the proteases described in patent applications EP 251 446 and WO 91/06637, the BLAP® protease described in WO91/02792 and its variants described in WO 95/23221.

See also the high pH protease from Bacillus sp. NCIMB 40338 described in WO 93/18140 A, Novo. Enzymatic detergents comprising the protease, one or more other enzymes and a reverse protease inhibitor are described in WO 92/03529 A, Novo. If desired, a protease having reduced adsorptive properties and increased hydrolysis efficiency is available, described in WO 95/07791, Procter & Gamble. A recombinant trypsin-like protease suitable for use in detergents of the present invention is described in WO 94/25583, Novo. Other suitable proteases are described in EP 516 200, Unilever.

Proteolytic enzymes are used in the detergent compositions of the present invention in an amount of 0.0001% by weight to 2% by weight, preferably 0.001% by weight to 0.2% by weight, more preferably 0.005% by weight to 0.1% by weight of pure enzyme based on the total weight of the composition.

The enzymes are of any suitable origin, such as plant, animal, bacterial, fungal and yeast. The origin is further mesophilic or extremophilic (psychrophilic, psychrotropic, thermophilic, barophilic, alkalophilic, acidophilic, halophilic, etc.). Purified or non-purified forms of these enzymes can be used.

Nowadays, it is common practice to modify the original natural types

9

9 9 9 9 9 9 9

9 9 9 9 9 9 9 9 9

9999 99 9 9999 99 99 9 • · 9 9 9 9 9 9

9 99 9 99 99 enzymes by protein or genetic engineering techniques to optimize their effectiveness in the cleaning compositions of the present invention. For example, variants can be designed in which the compatibility of the enzyme with the conventional components of such compositions is increased. Alternatively, a variant can be designed in which the optimum pH, bleaching and chelating stability, catalytic activity, etc. of the enzyme is precisely tailored to the needs of a particular cleaning application.

In particular, attention should be paid to oxidation-sensitive amino acids for bleach stability and surface tension for detergent compatibility. The isoelectric point of such enzymes can be modified by replacing some charged amino acids, e.g. increasing the isoelectric point will help improve compatibility with anionic surfactants. The stability of enzymes can be further enhanced by e.g. creating additional salt bridges and strengthening metal binding centers to increase stability against chelation. Special attention should be paid to cellulases, as most cellulases have separate binding domains (CBDs). The properties of such enzymes can be further modified by modifications in these domains.

These enzymes are normally used in detergent compositions in amounts of 0.0001% by weight to 2% by weight of pure enzyme based on the total weight of the composition. The enzymes can be added as separate components (tablets, granules, stabilized liquids, etc.) containing a single enzyme or as a mixture of two or more enzymes (e.g. co-granulates).

Other ingredients that can be used in detergents are enzyme oxidation inhibitors, which are described in co-pending European Patent Application 92870018.6, filed

January 31, 1992. Examples of such enzyme oxidation inhibitors are ethoxylated tetraethylene polyamines.

Many enzymatic materials and compositions for their use in synthetic detergent compositions are also described in WO 9307263 A and WO 9307260 A, Genencor International, WO 8908694 A, Novo and US 3,553,139, · 00 0 00 00 »0 0 0 0 0 0**0 ··· · 0 0 0 · 0 0 · • 0000 0 0 0 0000 0 0 * 0 ·

0 000 00*0

0*0 · 00 * *0 0*

January 5, 1971, McCarty et al. Enzymes are further described in U.S. 4,101,457, Plače et al., July 18, 1978, and in U.S. 4,507,219, Hughes, March 26, 1985. Enzyme materials useful in liquid detergent compositions and their inclusion in such compositions are described in U.S. 4,261,868, Hora et al.,

April 14, 1981. Enzymes intended for use in detergents are stabilized in various ways. Techniques for stabilizing enzymes are described and exemplified in US 3,600,319, 17.

August 1971, Gedge et al., EP 199,405 and EP 200,586,

October 29, 1986, Venegas. Stabilization of the enzyme system is also described, for example, in US 3,519,570. A useful strain of Bacillus sp. AC 13 providing proteases, xylanases and cellulases is described in WO 9401532 A, Novo.

Bleaching agent

Surprisingly, it has been found that laundry detergent compositions of the present invention further comprising a bleaching agent, particularly a bleaching system with a bleach activator, are more effective in removing food stains/soils, removing soiling and bleaching. Without being bound by any particular theory, it is believed that bleaching systems with a bleach activator are more effective in treating smaller chromophoric particles resulting from enzymatic hydrolysis of carbohydrate gums, particularly at lower temperatures.

Other optional detergent ingredients that can be used in the laundry detergent compositions of the present invention include bleaching agents such as hydrogen peroxide, PB 1, PB 4 and percarbonate with a particle size of 400 microns to 800 microns.

These bleaching agents include one or more oxygen bleaching agents and, depending on the bleaching agent selected, one or more bleach activators. When used, the oxygen bleaching compounds are typically present in an amount of from 1% by weight to 25% by weight.

• 9 9 9 » 9 9 1

9 9 <

> 9 9 1

9 9 4

99 include methachloroperoxybenzoate hexahydrate

The bleaching agent for use in the present invention is any bleaching agent used in laundry detergent compositions including oxygen bleaches as well as others known in the art. The bleaching agent suitable for the present invention is an activated or non-activated bleaching agent.

One group of oxygen bleaching agents that can be used are bleaching agents based on peroxycarboxylic acids and their salts. Suitable examples of this group of agents include magnesium monoperoxyphthalate, magnesium, 4-nonylamino-4-oxoperoxybutanoic acid and diperoxydodecanedioic acid. Such bleaching agents are described in U.S. Patent 4,483,781, U.S. Patent Application 740,446, European Patent Application 0,133,354 and U.S. Patent 4,412,934. Highly preferred bleaching agents include 6-nonylamino-6-oxoperoxycaproic acid, which is described in U.S. Patent 4,634,551.

Another group of bleaching agents that can also be used are halogen bleaching agents. Examples of halogen bleaching agents include, for example, trichloroisocyanuric acid and sodium and potassium dichloroisocyanurates and N-chloro and N-bromo alkane sulfonamides. Such materials are normally added in an amount of 0.5% to 10% by weight of the final product, preferably in an amount of 1% to 5% by weight.

Hydrogen peroxide releasing agents can be used in combination with bleach activators such as tetraacetylethylenediamine (TAED), nonanoyloxybenzenesulfonate (NOBS, described in US Patent 4,412,934), 3,5-trimethylhexanoloxybenzenesulfonate (ISONOBS, described in EP 120,591) or pentaacetylglucose (PAG) or N-nonanoyl-6-aminocaproic acid phenol sulfate ester (NACA-OBS, described in WO94/28106), which is perhydrolyzed to the peroxyacid form, which is the active bleaching species, resulting in improved bleaching performance. Acylated citrate esters, such as those described in

described in co-pending European Patent Application No. 91870207.7 and an unsymmetrical acyclic imide bleach activator of the general formula XIV:

About About

which is described by Procter & Gamble in copending U.S. patent applications Serial No. 60/022,786 (filed July 30, 1996) and Serial No. 60/028,122 (filed October 15, 1996), wherein R _T is a C ₇ to C ₁₃ saturated or unsaturated linear or branched alkyl, R ₂ is a C ₂ to C ₈ saturated or unsaturated linear or branched alkyl, and R ₃ is a C _x to C ₄ saturated or unsaturated linear or branched alkyl.

Useful bleaching agents, including peroxyacids and bleaching systems, include bleach activators and peroxy bleach compounds for use in detergent compositions of the present invention are described in our co-pending applications USSN 08/136,626, PCT/US95/07823,

WO95/27772, WO95/27773, WO95/27774 and WO95/27775.

Hydrogen peroxide may also be present by adding an enzymatic system (i.e. an enzyme and its substrate) capable of producing hydrogen peroxide at the beginning or during washing and/or rinsing. Such enzyme systems are described in EP patent application 91202655.6 filed on October 9, 1991.

Metal-containing catalysts for use in bleaching compositions include cobalt-containing catalysts such as cobalt pentaamine acetate and manganese-containing catalysts such as those described in EPA 549,271, EPA 549,272, EPA 458,397, US 5,246,621, EPA 458,398, US 5,194,416 and US 5,114,611. Bleaching compositions containing peroxide compounds, a manganese-containing bleaching catalyst and a chelating agent are described in patent application No. 94870206.3.

·* · • · ·«·* ·

Bleaching agents other than oxygen bleaching agents are known in the art and may also be used. One type of non-oxygen bleaching agent that is of particular interest is photoactivated bleaching agents, such as sulfonated zinc and/or aluminum phthalocyanines. These materials are deposited on the laundry during the wash cycle. Upon exposure to light in the presence of oxygen, such as on hanging clothes left to dry in daylight, the sulfonated zinc phthalocyanine is activated and, in turn, bleaches the substrate. A preferred zinc phthalocyanine and photoactivated bleaching process are described in U.S. Patent 4,033,718. Typically, detergent compositions contain from 0.025% by weight to 1.25% by weight of sulfonated zinc phthalocyanine.

Filler system

The laundry detergent compositions of the present invention preferably comprise a builder, more preferably an inorganic builder, most preferably Zeolite A, layered silicate and/or sodium tripolyphosphate. Surprisingly, it has been found that the laundry detergent composition of the present invention further comprising a builder has improved performance in removing food stains/soils, removing soiling and bleaching. Without being bound to any particular theory, it is believed that the carbohydrate gums sequester calcium and thus reduce enzymatic hydrolysis. Therefore, the use of a builder is expected to remove the sequestered calcium and improve the action of the enzymes degrading the carbohydrate gums.

Any conventional filler system is suitable for use in the present invention, including aluminosilicate materials, silicates, polycarboxylates, alkyl or alkenyl succinic acids and fatty acids, materials such as ethylenediaminetetraacetate, diethylenetriaminepentamenethylene acetate, metal ion scavengers such as aminopolyphosphonates, particularly ethylenediaminetetramethylenephosphonic acid, and

99 • · φφφφ • φ φφ φ ·* 9

9 9

9 9 9

9 9 9999

9 9 9

99 9

9 9

9 «

Φ Φ Φ

Φ Φ Φ

ΦΦ 99 diethylenetriaminepentamethylenephosphonic acid. Phosphate fillers can also be used.

Suitable fillers are inorganic ion exchange materials, commonly inorganic hydrated silicate materials, more particularly hydrated synthetic zeolites such as hydrated zeolite A, X, B, HS or MAP.

Another suitable inorganic filler material is a layered silicate, e.g. SKS-6 (Hoechst). SKS-6 is a crystalline layered silicate consisting of sodium silicate (Na ₂ Si ₂ O 2 ).

Suitable polycarboxylates containing one carboxyl group include lactic acid, glycolic acid and their ether derivatives, which are described in Belgian Patent Nos. 831,368, 821,369 and 821,370. Polycarboxylates containing two carboxyl groups include water-soluble salts of succinic acid, malonic acid, ethylenedioxydiacetic acid, maleic acid, diglycolic acid, tartaric acid, tartronic acid and fumaric acid as well as the ether carboxylates described in German Offenlegenschrift 2,446,686 and 2,446,687 and U.S. Patent No. 3,935,257 and the sulfinyl carboxylates described in Belgian Patent No. 840,623. Polycarboxylates containing three carboxyl groups include, in particular, water-soluble citrates, aconitrates and citraconates as well as succinic acid derivatives such as carboxymethyloxysuccinates described in British Patent No. 1,379,241, lactoxysuccinates described in Dutch Application 7205873 and oxypolycarboxylate materials such as 2-oxa-1,1,3-propanetricarboxylates described in British Patent No. 1,387,447.

Polycarboxylates containing four carboxyl groups include the oxydisuccinates described in British Patent No. 1,261,829, 1,1,2,2-ethanetetracarboxylates, 1,1,3,3-propanetetracarboxylates and 1,1,2,3-propanetetracarboxylates. Polycarboxylates containing sulfo substituents include • ·· · ·· ·· • ··· 4 4 * · ·· 4 · · 4 4 4 4

4444 9 9 9 9999 9 9 99 9

9 9 9 9 9 9 9

99 9 99 99 sulfosuccinate derivatives described in British Patents Nos. 1,398,421 and 1,398,422 and in US Patent No. 3,936,448 and sulfonated pyrolyzed citrates described in British Patent No. 1,082,179, while polycarboxylates containing phosphonic substituents are described in British Patent No. 1,439,000.

Alicyclic and heterocyclic polycarboxylates include cyclopentane-cis,cis,cis-tetracarboxylates, cyclopentadienide pentacarboxylates, 2,3,4,5-tetrahydrofuran-cis,cis,cis-tetracarboxylates, 2,5-tetrahydrofuran-cis-dicarboxylates, 2,2,5,5-tetrahydrofurantetracarboxylates, 1,2,3,4,5,6-hexanehexacarboxylates and carboxymethyl derivatives of polyhydroxy alcohols such as sorbitol, mannitol and xylitol. Aromatic polycarboxylates include derivatives of mellitic acid, pyromellitic acid and phthalic acid described in British Patent No. 1,425,343.

are hydroxycarboxylates of agents, fillers

Preferred polycarboxylates containing up to three carboxyl groups per molecule, more particularly citrates.

Preferred builder systems for use in the present invention include a mixture of a water-insoluble aluminosilicate builder, such as zeolite A or layered silicate (SKS-6), and a water-soluble carboxylate chelating agent, such as citric acid. Other preferred builder systems include a mixture of water-insoluble aluminosilicate builders, such as zeolite A, and a water-soluble carboxylate chelating agent, such as citric acid. Preferred systems for use in the liquid detergent compositions of the present invention are soaps and polycarboxylates.

Other filler materials that form part of the filler system intended for use in granular compositions include inorganic materials such as alkali metal carbonates, bicarbonates and silicates and organic materials such as organic phosphonates, aminopolyalkylenephosphonates and aminopolycarboxylates.

Other suitable water-soluble organic salts are homo- or copolymeric acids or their salts, in which the polycarboxylic acid consists of at least two carboxyl residues separated from each other by not more than two carbon atoms. Polymers of this type are described in GB-A-1,596,756. Examples of such salts are polyacrylates with a molecular weight of 2000 to 5000 and their copolymers with maleic anhydride, which have a molecular weight of 20,000 to 70,000 and in particular 40,000.

Builder salts are normally used in detergents in amounts of from 5% to 80% by weight based on the weight of the composition, preferably in amounts of from 10% to 70% by weight and most preferably in amounts of from 30% to 60% by weight.

Another surfactant system

The laundry detergent compositions of the present invention also contain other cationic, ampholytic, zwitterionic and semipolar surfactants, as well as nonionic and/or anionic surfactants than those already described in the present invention.

Ampholytic surfactants are also suitable for use in the laundry detergent compositions of the present invention. These surfactants can be generally described as aliphatic derivatives of secondary or tertiary amines or aliphatic derivatives of heterocyclic secondary and tertiary amines in which the aliphatic moiety is straight or branched chain. One of the aliphatic substituents contains at least eight carbon atoms, typically 8 to 18 carbon atoms, and at least one contains an anionic water-solubilizing group, e.g., carboxyl, sulfonate, sulfate. See U.S. Patent No. 3,929,678, Laughlin et al., issued Dec. 30, 1975, at col. 19, lines 18 to 35, for examples of ampholytic surfactants.

e · tertiary quaternary group soluble

When used, the laundry detergent compositions of the present invention typically contain from 0.2% by weight to 15% by weight, preferably from 1% by weight to 10% by weight, of such ampholytic surfactants.

Zwitterionic surfactants are also suitable for use in the detergent compositions of the present invention. These surfactants can be generally described as derivatives of secondary and tertiary amines, derivatives of heterocyclic secondary and amines, or derivatives of quaternary ammonium, phosphonium, or tertiary sulfonium compounds. See U.S. Patent No. 3,929,678, Laughlin et al., issued December 30, 1975, at column 19, line 38 to column 22, line 48, for examples of zwitterionic surfactants.

When used, the laundry detergent compositions of the present invention typically contain from 0.2% by weight to 15% by weight, preferably from 1% by weight to 10% by weight, of such zwitterionic surfactants.

Semipolar nonionic surfactants are special nonionic surfactants that include water-soluble amine oxides containing one alkyl consisting of 10 to 18 carbon atoms and 2 groups selected from the group consisting of alkyls and hydroxyalkyls consisting of 1 to 3 carbon atoms, water-soluble phosphine oxides containing one alkyl consisting of 10 to 18 carbon atoms and 2 groups selected from the group consisting of alkyls and hydroxyalkyls consisting of 1 to 3 carbon atoms, and water-soluble sulfoxides containing one alkyl consisting of 10 to 18 carbon atoms and 2 groups selected from the group consisting of alkyls and hydroxyalkyls consisting of 1 to 3 carbon atoms.

Semipolar nonionic surfactants include amine oxide surfactants of the general formula XV:

O

AND

R3(OR4)xN(R5)2 (XV) • 9 9 • · 9 9 9

9999 99999«

99 9 99 99 where R ³ is alkyl, hydroxyalkyl or alkylphenyl or a mixture thereof consisting of 8 to 22 carbon atoms, R ⁴ is alkylene or hydroxyalkylene consisting of 2 to 3 carbon atoms or a mixture thereof, x is 0 to 3 and each R ⁵ is alkyl or hydroxyalkyl consisting of 1 to 3 carbon atoms or polyethylene oxide consisting of 1 to 3 ethylene oxide groups. The R ⁵ are optionally attached to each other, e.g. via an oxygen atom or a nitrogen atom, thereby forming a cyclic structure.

These amine oxide surfactants include in particular C ₁₀ to C ₁₈ alkyldimethylamine oxides and C ₈ to C ₁₂ alkoxyethyldihydroxyethylamine oxides.

When used, the laundry detergent compositions of the present invention typically contain from 0.2% by weight to 15% by weight, preferably from 1% by weight to 10% by weight, of such semipolar surfactants.

The laundry detergent composition according to the present invention further comprises a cosurfactant selected from the group of primary or tertiary amines. Primary amines suitable for use according to the present invention are of the type R _X NH ₂ , where R _x is C ₆ to C ₁₂ , preferably C ₆ to C ₁₀ alkyl or R ₄ X(CH ₂ ) _n , where X is -O-, -C(O)NH or -NH-, R ₄ is C ₆ to C ₁₂ alkyl and n is 1 to 5, preferably 3. The R _x alkyl is straight or branched and is optionally interrupted by up to 12, preferably less than 5 ethylene oxide units.

Preferred amines according to the present invention are n-alkylamines. Suitable amines for use according to the present invention are selected from the group consisting of 1-hexylamine, 1-octylamine, 1-decylamine and laurylamine. Other preferred primary amines include C8 to C10 oxypropylamine, octyloxypropylamine, 2-ethylhexyloxypropylamine, lauryl amidopropylamine and amidopropylamine.

Suitable tertiary amines for use in the present invention include tertiary amines of the type R ₁ R ₂ R ₃ N, where R _x and R ₂ are C _x to C ₈ alkyls or a group of the general formula

XVI:

• 4 • · • · · · · · · alkyl or R ₃ is

R ₄ is C ₄ to C ₁₂ , n

CH,

Rs.

-CH—o) / ·

H x (xvi)

R ₃ is either C ₆ to C

12' preferably C ₆ to C

10'

NH-,

R ₄ X(CH ₂ ) _n , where X is -O-, -C(O)NH- or is 1 to 5, preferably 2 to 3. R ₅ is H or C _x to C ₂ alkyl and x is 1 to 6.

R ₃ and R ₄ are linear or branched, R ₃ is alkyl which is optionally interrupted by up to 12, preferably less than 5, ethylene oxide groups.

Preferred tertiary amines are amines of the type R ₁ R ₂ R ₃ N, where R _x is C 8 to C ₁₂ alkyl, R ₂ and R ₃ are C _r to C ₃ or a group of general formula XVI, where R ₅ is H or CH ₃ and x = 1 to 2.

Also preferred are amidoamines of the general formula XVII:

O

R1—C—NH—( _CH2 ) _n —N-( _R2 ) ₂ (XVII) where _R1 is _C6 to _C12 alkyl, n is 2 to 4, preferably n is 3, _R2 and _R3 are C1 to _C4 .

The most preferred amines according to the present invention include 1-octylamine, 1-hexylamine, 1-decylamine, 1-dodecylamine, C8 to C10oxypropylamine, N-coco-1,3-diaminopropane, (coconut alkyl)dimethylamine, lauryldimethylamine, laurylbis(hydroxyethyl)amine, cocobis(hydroxyethyl)amine, laurylamine propoxylated 2 moles, octylamine propoxylated 2 moles, laurylamidopropyldimethylamine, C8 to C10 amidopropyldimethylamine and C10 amidopropyldimethylamine.

The most preferred amines for use in the compositions of the present invention are 1-hexylamine, 1-octylamine, 1-decylamine, 1-dodecylamine. Particularly desirable are n-dodecyldimethylamine and bishydroxyethyl(coconut alkyl)amine and heptaethoxylated oleylamine, lauryl amidopropylamine and cocoamidopropylamine.

·· φ φφ φφ φ · φ ΦΦΦ· • φφφ · φφ φ φφ φφφφφφφ φφ φ φ φ φφφφ φ φ φ φ φφ

Color Care and Fabric Care Benefits Technologies that provide a type of color care may also be included in the present invention. Examples of such technologies are metal catalysts for color maintenance. Such metal catalysts are described in co-pending European Patent Application No. 92870181.2. Color fixing agents, polyolefin dispersions for anti-crease and improved water absorption, fragrance and amino-functional polymer for color care, and fragrance are other examples of color care/fabric care technologies and are described in co-pending patent application No. 968701140.9, filed November 9, 1996.

Fabric softening agents may also be included in the laundry detergent compositions of the present invention, ^these agents being of the inorganic type or organic type. Examples of inorganic softening agents are the smectite clays disclosed in GB-A-1 400 898 and in USP 5,019,292. Organic softening agents include water-soluble tertiary amines, which are described in GB-A-1 514 276 and EP-B 0 011 340 and their combinations with mono C12 to C14 quaternary ammonium salts, which are described in EP-B-0 026 527 and EP-B-0 026 528 and di(long chain)amides, which are described in EP-B-0 242 919. Other useful organic components in a fabric softening system are high molecular weight polyethylene oxide materials, which are described in EP-A-0 299 575 and 0 313 146.

The amount of smectite clay is normally 2% to 20% by weight, preferably 5% to 15% by weight, with the material being added to the balance of the composition as a dry blended component. Organic fabric softeners such as water-insoluble tertiary amines or amides with two long chains are used in amounts of 0.5% to 5% by weight, normally 1% to 3% by weight, while high molecular weight polyethylene oxides and water-soluble cationic • * « 9 9 9 9 9 9 9 9 QO · ···· » · ····· · 9 99 9 OU 9 9 999 9999

9 9 9 9 9 9 9 9 99 materials are added in amounts of 0.1% by weight to 2% by weight, normally 0.15% by weight to 1.5% by weight. These materials are normally added to that part of the composition which is to be spray dried, although in some cases it is better to add them as a dry mixture of particles or to spray them as a molten mixture onto or into the solid components of the composition.

Chelating agents

The laundry detergent compositions of the present invention optionally contain one or more iron and/or manganese chelating agents. Such chelating agents may be selected from the group consisting of aminocarboxylates, aminophosphonates, polyfunctionally substituted aromatic chelating agents, and mixtures thereof, all of which are defined below! Without being bound by any particular theory, it is believed that the beneficial effects of these materials are due in part to their exceptional ability to remove iron and manganese ions from laundry solutions by forming chelates.

Aminocarboxylates useful as optional chelating agents include ethylenediaminetetraacetates, N-hydroxyethylenediaminetetraacetates, nitrile triacetates, ethylenediamine tetrapropionates, triethylenetetraminehexacetates, diethylenetriaminepentaacetates and ethanoldiglycines, their alkali metal, ammonia and substituted ammonia salts, and mixtures thereof.

Aminophosphonates including ethylenediaminetetrakis(methylenephosphonates) such as DEQUEST are also suitable for use in the compositions of the present invention, where at least small total amounts of phosphorus are permitted in the detergent composition. Preferably, these aminophosphonates do not contain alkyls or alkenyls longer than 6 carbon atoms.

Polyfunctionally substituted aromatic chelating agents are also useful in the compositions of the present invention. See U.S. Patent 3,812,044, issued May 21, 1974,

0*0 0 0* 9

0 ******* 0* * • · · * * * * • · 0 0 0 00

Connor et al. Preferred compounds of this type in acid form are dihydroxydisulfobenzenes, such as 1,2-dihydroxy-3,5-disulfobenzene.

A preferred biodegradable chelating agent for use in the present invention is ethylenediamine disuccinate (EDDS), particularly its [S,S] isomer, which is described in U.S. Patent 4,704,233, November 3, 1987, to Hartman and Perkins.

The compositions of the present invention optionally also contain water-soluble salts of methylglycinediacetic acid (MGDA) or its acid form as chelating agents or binders, for example, usable with insoluble fillers such as zeolites, layered silicates and the like.

When used, these chelating agents generally comprise from 0.1% by weight to 15% by weight of the detergent compositions of the present invention. More preferably, when used, the chelating agents comprise from 0.1% by weight to 3.0% by weight of such compositions.

Scum-suppressing agent

Another optional ingredient is a suds suppressor, exemplified by silicones and silicone-silica blends. Silicones can be generally described as alkylated polysiloxane materials, while silica is normally used in finely divided form, exemplified by silica aerogels and hydrophobic silicas of various types. These materials can be used as particles in which the suds suppressor is preferably releasably incorporated into a water-soluble or water-dispersible, substantially surfactant-free, detergent-impermeable carrier. Alternatively, the suds suppressor can be dissolved or dispersed in a liquid carrier and applied by spraying onto one or more other ingredients.

9· 9 9999 • 99 9 · 9 999· 9 9 9 9 999 9 99 9

QQ 9 9999 9 9 ······· 99 ·

About Z. ········ • 99 9 99 * 99 99

Preferred silicone suds suppressors are described in U.S. Patent 3,933,672, Bartollota et al. Other particularly useful suds suppressors are the self-eluting silicone suds suppressors described in German Patent Application DTOS 2,646,126, issued April 28, 1977. An example of such a compound is DC-544, commercially available from Dow Corning, which is a siloxane-glycol copolymer. Particularly preferred suds suppressor systems are suds suppressor systems comprising a mixture of silicone oils and 2-alkyl alkanols. A suitable 2-alkyl alkanol is 2-butyl octanol, which is commercially available under the trade name Isofol 12 R.

Such a suds suppression system is described in copending European Patent Application No. 92870174.7 filed November 10, 1992.

Particularly preferred silicone suds suppressing agents are described in co-pending European Patent Application No. 92201649.8. Such compositions comprise silicone/silica blends in combination with non-porous silica such as Aerosil®.

Susceptors are normally used in amounts of 0.001% to 2% by weight of the composition, preferably 0.01% to 1% by weight.

Other folders

Other ingredients used in laundry detergent compositions, such as soil suspending agents, soil release agents, optical brighteners, abrasives, bactericides, color inhibitors, coloring agents, and/or encapsulated or unencapsulated fragrances, may also be used.

Particularly suitable encapsulating materials are water-soluble capsules consisting of a matrix of polysaccharide and polyhydroxy compounds, which are described in GB 1,464,616. Other suitable water-soluble encapsulating materials are dextrins derived from non-gelatinized acid esters of starch with substituted dicarboxylic acids, which are described in US 3,455,838. These dextrin acid esters are preferably prepared from starches such as corn, sago, tapioca and potato. Suitable examples of such encapsulating materials include N-Lok manufactured by National Starch. N-Lok encapsulating material consists of modified corn starch and glucose. The starch is modified by the addition of monofunctionally substituted groups such as octenyl succinic anhydride.

The soil release agents and soil suspending agents of the present invention include cellulose derivatives such as methylcellulose, carboxymethylcellulose and hydroxyethylcellulose and homo- or copolymeric polycarboxylic acids or salts thereof. Polymers of this type include polyacrylates and copolymers of maleic anhydride and acrylic acid, which have already been mentioned as fillers, as well as copolymers of maleic anhydride with ethylene, methylvinylether or methacrylic acid, wherein the maleic anhydride constitutes at least 20 mole percent of the copolymer. These materials are normally used in amounts of 0.5% to 10% by weight, more preferably 0.75% to 8% by weight, most preferably 1% to 6% by weight of the composition.

Preferred optical brighteners are of anionic nature and examples thereof are 4,4'-bis-(2-diethanolamino-4-anilino-s-triazin-6-ylamino)stilbene-2:2'-disulfonate disodium salt, 4,4'-bis-(2-morpholino-4-anilino-s-triazin-6-ylamino)stilbene-2:2'-disulfonate disodium salt, 4,4 ¹ -bis-(2,4-dianilino-s-triazin-6-ylamino)stilbene-2:2'-disulfonate disodium salt, 4',4-(2,4-dianilino-s-triazin-6-ylamino)stilbene-2-sulfonate monosodium salt, disodium salt 4,4'-bis-(2-anilino-4-(Nmethyl-N-2-hydroxyethylamino)-s-triazin-6-ylamino)stilbene-2,2'-disulfonate, disodium

4,4'-bis-(4-phenyl-2,1,3-salt • · ····

-triazol-2-yl)stilbene-2,2'-disulfonate, disodium salt of 4,4'-bis-(2-anilino-4-(1-methyl-2-hydroxyethylamino)-s-triazin-6-ylamino)stilbene-2,2'-disulfonate, sodium salt of 2-(stilbyl-4-(naphtho-1', 2':4,5)-1,2,3-triazole-2-sulfonate and 4,4'-bis-(2-sulfostyryl)biphenyl. Highly preferred brighteners are in particular the brighteners of co-pending European Patent Application No. 95201943.8.

Other useful polymeric materials are polyethylene glycols, having a molecular weight of 2000 to 8000 and polyethylene glycols, having a molecular weight of 1000 to 10,000, more preferably 4000. These are used in amounts of 0.20% to 5% by weight, more preferably 0.25% by weight. These polymers and copolymer polycarboxylates are valuable for improving bleaching and cleaning effects on clay, protonable and oxidizable soils in the presence of transition metal impurities.

Soil release agents useful in the compositions of the present invention are commonly used copolymers or terpolymers of terephthalic acid with ethylene glycol and/or propylene glycol units in various configurations. Examples of such polymers are described in U.S. Patent Nos. 4,116,885 and 4,711,730 and European Published Patent Application No. 0 272 033. Particular preferred polymers according to EP-A-0 272 033 are (CH ₃ (PEG) ₄₃ ) _{0 .75} (POH) ₀₍₂₅ [(T-PO) _{2 .8} (TPEG) ₀₍₄ ]T(PO-H) _0<25 ( (PEG) ₄₃ CH ₃ ) _o<75 where PEG is -(OC ₂ H ₄ )O-, PO is (OC ₃ H ₆ O) and T is (pcOC ₆ H ₄ CO).

Also very useful are modified polyesters such as random copolymers of dimethyl terephthalate, dimethyl sulfoisophthalate, ethylene glycol and 1,2-propanediol, where the end groups consist primarily of sulfobenzoate and secondarily of monoesters of ethylene glycol and/or propanediol. The aim is to obtain a polymer terminated at both ends with sulfobenzoate groups, primarily in the present context meaning that • 44 4 ·♦ ·4 • · · 4 9 · 4 4 • · · · 4 ♦ 4 4 4 4 •44« «4 4 4444 44 44 4 • 4 4 4 4444

44 4 44 44 most of the copolymers of the present invention are terminated with sulfobenzoate groups. However, some copolymers are less than completely terminated and therefore their end groups consist of monoesters of ethylene glycols and/or propane-1,2-diol, hence the secondary designation of such compounds.

Selected polyesters of the present invention contain 46% by weight of dimethylterephthalic acid, 16% by weight of propane-1,2-diol, 10% by weight of ethylene glycols, 13% by weight of dimethylsulfobenzoic acid and 15% by weight of sulfoisophthalic acid and have a molecular weight of 3000. Polyesters and their methods of preparation are described in more detail in EPA 311 342.

It is well known in the art that free chlorine in tap water rapidly deactivates enzymes contained in detergent compositions. Therefore, the use of chlorine scavengers such as perborate, ammonium sulfate, sodium sulfite or polyethyleneimine in an amount of 0.1% by weight of the total composition will improve the stability of the detergent enzymes. Compositions containing chlorine scavengers are described in patent application 92870018.6, filed

January 31, 1992.

Alkoxylated polycarboxylates, such as those prepared from polyacrylates, are useful in the present invention for improved grease removal performance, such materials are described in WO 91/08281 and PCT 90/01815 at page 4 et seq., which are incorporated herein by reference. Chemically, these materials include polyacrylates having one ethoxyl side chain for every 7 to 8 acrylate units. The side chains are

- (CH ₂ CH ₂ O) _{r a} (CH ₂ ) _n CH ₃ , where m is 2 to 3 and n is 6 to 12. The side chains are ester-linked to the polyacrylate backbone, forming a comb-type polymer structure. The molecular weight varies but is typically in the range of 2000 to 50,000. Such alkoxylated polycarboxylates comprise from 0.05% by weight to 10% by weight of the compositions of the present invention.

Dispersants

The laundry detergent compositions of the present invention also contain dispersants. Suitable water-soluble organic salts are homo- or copolymers of acids or their salts in which the polycarboxylic acid comprises at least two carboxyl radicals separated from each other by not more than two carbon atoms. Polymers of this type are described in GB-A-1,596,756. Examples of such salts are polyacrylates having a molecular weight of 2000 to 5000 and their copolymers with maleic anhydride having a molecular weight of 1000 to 100,000.

In particular, an acrylate-methacrylate copolymer such as 480N with a molecular weight of 4000 can be added to the laundry detergent compositions of the present invention in an amount of 0.5% by weight to 20% by weight of the composition.

The compositions of the present invention optionally comprise a core soap peptizer compound which preferably has a core soap dispersion power (LSDP) as defined in the present invention of not more than 8, preferably not more than 7, most preferably not more than 6. The core soap peptizer compound is preferably used in an amount of 0% by weight to 20% by weight.

A numerical measure of the effectiveness of a core soap peptizer is the core soap dispersion power (LSDP), which is determined by the core soap dispersion test described in H.C. Boghetty and C.A. Bergman, J. Am. Oil. Chem. Soc., vol. 27, pp. 88-90, (1950). This core soap dispersion test is widely used by those skilled in the art, see, for example, the following review articles, W.N. Linfield, Surfactant Science Series, vol. 7, p. 3, W.N. Linfield, Tenside surf. det., vol. 27, pp. 159-163 (1990), and M.K. Nagarajan, W.F. Masler, Cosmetics and Toiletries, vol.

• 9

9 • · 9 9 9 · 9 · ·· 9999 9999

9999 99 9 9999 · 9 99 9

99 9 9999

99 9 99 99

104, pages 71-73 (1989). LSDP is the weight percent ratio of dispersant to sodium oleate required to disperse a core soap deposit formed by 0.025 g of sodium oleate in 30 ml of water having a hardness of 333 ppm CaCO ₃ (Ca:Mg = 3:2) equivalent.

Surfactants that have good core soap peptization ability include some amine oxides, betaines, sulfobetaines, alkyl ethoxy sulfates, and ethoxylated alcohols.

Examples of surfactants having an LSDP of not more than 8 for use in the present invention include _C16 to _C18 dimethylamine oxides, _C12 to _C18 alkyl ethoxy sulfates with a medium degree of ethoxylation of 1 to 5, especially _C12 to _C15 alkyl ethoxy sulfate surfactants with a degree of ethoxylation of 3 (LSDP = 4), and _C14 to _C15 ethoxylated alcohols with a medium degree of ethoxylation of either 12 (LSDP = 6) or 30, sold under the trade names Lutensol A012 and Lutensol A030 by BASF GmbH.

Polymeric core soap peptizers suitable for use in the present invention are described in M.K. Nagarajan, W.F. Masler, Cosmetics and Toiletries, Vol. 104, pp. 71-73 (1989).

Hydrophobic bleaches such as 4-[N-octanoyl-6-aminohexanoyl]benzenesulfonate, 4-[N-nonanoyl-6-aminohexanoyl]benzenesulfonate, 4-[N-decanoyl-6-aminohexanoyl]benzenesulfonate and mixtures thereof and nonanoyloxybenzenesulfonate together with hydrophilic/hydrophobic bleaches can also be used as core soap peptizing compounds.

Color transfer attenuation

The laundry detergent compositions of the present invention also contain compounds that inhibit the transfer of color from one fabric to another resulting from the solubilization and suspension of dyes during the washing of colored fabrics.

Polymeric dye transfer dampening agents

The laundry detergent compositions of the present invention also contain 0.001% by weight to 10% by weight, preferably 0.01% by weight to 2% by weight, more preferably 0.05% by weight to 1% by weight of polymeric dye transfer inhibiting agents. These polymeric dye transfer inhibiting agents are normally used in laundry detergent compositions to prevent the transfer of dyes from colored fabrics to other fabrics washed with them. These polymers have the ability to complex or adsorb unstable dyes that are washed out of the dyed fabrics before other fabrics in the wash solution are dyed.

Particularly suitable polymeric dye transfer suppressants are polyamine-N-oxide polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole, polyvinylpyrrolidone polymers, polyvinyloxazolidones and polyvinylimidazoles, or mixtures thereof.

The addition of such polymers will also improve the effectiveness of the enzymes of the present invention.

a) polyamine-N-oxide polymers

Polyamine-N-oxide polymers suitable for use in the present invention contain units of the general formula XVIII:

P

AND

A _x

AND

R (XVIII) where P is a polymerizable unit to which an R-N-O group is attached or where the R-N-O group forms part of a polymerizable unit or a combination of both,

0 0

II II II

A is NC, CO, C, -O-, -S-, -N-, x is 0 or 1,

R are aliphatic, ethoxylated aliphatic, aromatic, heterocyclic or alicyclic groups or any combination thereof to which the nitrogen of the N-0 group is attached or in which the nitrogen of the N-0 group is part of these groups.

Group N-0 is a group of general formula XIX:

• 9

9···

9 99 99 • 9 9 9 9 9 ·

999 9 99 9

9999999 99 ·

9 9 9 9999

99 9 99 99 (Rl) _χ -Ν-(R2) _y I (R3) _from (XIX) or XX:

AND

-N-(Rl) _x (XX) where Rl, R2 and R3 are aliphatic groups, aromatic, heterocyclic or alicyclic groups or combinations thereof, x and/or y and/or z is 0 or 1 and the nitrogen of the N-0 group is attached to them or the nitrogen of the N-0 group forms part of these groups.

The N-0 group is part of the polymerizable unit (P) or is attached to the polymer backbone, or a combination of both.

Suitable polyamine N-oxides in which the N-O group forms part of the polymerizable unit include polyamine N-oxides in which R is selected from aliphatic, aromatic, alicyclic or heterocyclic groups.

One group of these polyamine N-oxides includes the group of polyamine N-oxides in which the nitrogen of the N-O group forms part of the R group. Preferred polyamine N-oxides are those in which R is a heterocyclic group such as pyridine, pyrrole, imidazole, pyrrolidine, piperidine, quinoline, acridine and derivatives thereof.

Another group of polyamine N-oxides includes a group of polyamine N-oxides in which the nitrogen of the N-0 group is attached to the R group.

Other suitable polyamine N-oxides are polyamine oxides in which the N-O group is attached to a polymerizable unit.

A preferred group of these polyamine N-oxides are polyamine N-oxides of general formula XVIII, where R are aromatic, t · ·· · ·· ·# ·· ··· · · # « · ♦ ···· » · · · • ♦··· * · · ··· · · · · · · · • · · · · · · ♦

........* ·* heterocyclic or alicyclic groups in which the nitrogen of the N-0 functional group is part of the R groups.

Examples of these groups are polyamine oxides in which R is a heterocyclic compound such as pyridine, pyrrole, imidazole and their derivatives.

Another preferred group of polyamine N-oxides are polyamine oxides of the general formula XVIII, in which R are aromatic, heterocyclic or alicyclic groups, in which the nitrogen of the N-O functional group is attached to the R groups.

Examples of this group are polyamine oxides in which the R groups are aromatic, such as phenyl.

Any polymer backbone can be used as long as the resulting amine oxide polymer is water soluble and resists dye transfer. Examples of suitable polymer backbones are polyvinyls, polyalkylenes, polyesters, polyethers, polyamides, polyimides, polyacrylates, and mixtures thereof.

The amine-N-oxide polymers of the present invention typically have an amine to amine-N-oxide ratio of 10:1 to 1:1,000,000.

However, the amount of amine oxide groups present in the polyamine oxide polymer varies according to the particular copolymerization or the particular degree of N-oxidation. Preferably, the ratio of amine to amine N-oxide is 2:3 to 1:1,000,000. More preferably, it is 1:4 to 1:1,000,000, most preferably, it is 1:7 to 1:1,000,000. The polymers of the present invention include random or block copolymers in which one type of monomer is an amine N-oxide and the other type of monomer is or is not an amine N-oxide. The amine oxide unit of the polyamine N-oxides has a pKa <10, preferably a pKa <7, most preferably a pKa <6.

Polyamine oxides can be obtained in most cases at any degree of polymerization. The degree of polymerization is not critical for the material as long as it has the desired solubility in water and the appropriate power to suspend dyes.

• 4 • 44«

4 44 44

4 4 4 4 4 4 » 444 4 44 4

4444444 44 4

4 44 4 444 4

444 4 4» 4 44 44

Typically, the average molecular weight is 500 to 1,000,000, preferably 1,000 to 50,000, even more preferably 2,000 to 30,000, and most preferably 3,000 to 20,000.

b) copolymers of N-vinylpyrrolidone and N-vinylimidazole

The N-vinylimidazole/N-vinylpyrrolidone polymers used in the present invention have an average molecular weight of 5,000 to 1,000,000, preferably 5,000 to 200,000.

Highly preferred polymers for use in detergent compositions of the present invention include a polymer selected from N-vinylimidazole/N-vinylpyrrolidone copolymers, wherein the polymer has an average molecular weight of 5,000 to 50,000, more preferably 8,000 to 30,000, most preferably 10,000 to 20,000.

The average molecular weight is determined by light scattering, which is described in Barth, H.G. and Mays J.W., Chemical Analysis Vol. 113, Modern Methods of Polymer Characterization.

Highly preferred N-vinylimidazole/N-vinylpyrrolidone copolymers have an average molecular weight of 5,000 to 50,000, more preferably 8,000 to 30,000, and most preferably 10,000 to 20,000.

N-vinylimidazole/N-vinylpyrrolidone copolymers of the stated average molecular weight provide excellent results in inhibiting dye transfer and do not negatively affect the cleaning performance of detergent compositions in which they are contained.

The N-vinylimidazole/N-vinylpyrrolidone copolymers of the present invention have a molar ratio of N-vinylimidazole to N-vinylpyrrolidone of 1 to 0.2, more preferably 0.8 to 0.3, and most preferably 0.6 to 0.4.

c) Polyvinylpyrrolidone

The detergent compositions of the present invention also utilize polyvinylpyrrolidone (PVP) having an average molecular weight of 2500 to 400,000, preferably 5000 to 200,000, more preferably 5000 to 50,000, and most preferably 5000 to 15,000. Suitable polyvinylpyrrolidones are commercially available from ISP.

4 4 44 99

4 4 4 4*4

9 9 9 · 4 · 4

9 9999999 9 9 ·

4 444 4444

444 4 44 4 44 44 • » »•44

Corporation, New York, NY and Montreal, Canada under the trade names PVP K-15 (viscosity molecular weight 10,000), PVP

K-30 (average molecular weight 40,000), PVP K-60 (average molecular weight 160,000) and PVP K-90 (average molecular weight 360,000). Other suitable polyvinylpyrrolidones, which are commercially available from BASF Corporation, include Sokalan HP 165 and Sokalan HP 12, as well as polyvinylpyrrolidones known to those skilled in the detergent art (see, for example,

EP-A-262,897 and EP-A-256,696).

d) Polyvinyloxazolidone

The detergent compositions of the present invention also utilize polyvinyloxazolidone.

dye transfer inhibitor Polyvinyloxazolidones have an average molecular weight of 2500 to 400,000, preferably 5000 to

200,000, more preferably 5,000 to 50,000, and most preferably 5,000 to 15,000.

e) Polyvinylimidazole

The detergent compositions of the present invention also utilize polyvinylimidazole as a dye transfer inhibitor. These polyvinylimidazoles have an average molecular weight of 2500 to 400,000, preferably 5000 to 200,000, more preferably 5000 to 50,000, and most preferably 5000 to 15,000.

f) Cross-linked polymers

Crosslinked polymers are polymers whose backbones are interconnected to some extent, these connections being chemical or physical in nature, perhaps with active groups on the backbone or on the branches, crosslinked polymers were described in the Journal of Polymer Science, volume 22, pages 1035 to 1039.

In one embodiment, the crosslinked polymers were made to form a three-dimensional solid structure that traps dyes in pores formed by this three-dimensional structure. In another embodiment, the crosslinked polymers capture dyes by swelling.

Such crosslinked polymers are described in copending patent application 94870213.9.

• 4 ·· · *· 04 · ··· 4 4 · 4 • 4 4 4 4 4 4 » 4 44 4

444444 4444444 44 4

4 4 44 4404 • 44 4 4* 4 44 44 together

Examples of embodiments of the invention

Washing method

The compositions of the present invention can be used in principle in any washing or cleaning process, including soaking, prewashing and processes that involve rinsing, in which a special rinse aid component is added.

The process described in the present invention involves contacting the fabric with a washing solution in a conventional manner and is described in more detail in the following examples.

The process of the present invention is commonly carried out as a cleaning process. The cleaning is preferably carried out at a temperature of 5°C to 95°C, in particular 10°C to 60°C. The pH of the resulting solution is preferably 7 to 11. Preferably, the laundry detergent component of the present invention, in the form of a 1% solution in water, expressed as a percentage by weight, will have a pH value of 7 to 9.5 (hence it is referred to as an alkaline detergent).

The following examples are intended to illustrate the means of the present invention, but do not constitute any limitation or other definition of the scope of the present invention.

Enzyme quantities in detergent compositions are expressed as the weight of pure enzyme per weight of the total composition and, unless otherwise stated, detergent ingredients are expressed as the weight of the total ingredients. The abbreviations for ingredients have the following meanings:

LAS: sodium linear _Cn to _C13 alkylbenzene sulfonate

TAS: sodium tallow alkyl sulfate

CxyAS: C _lx to C _ly sodium alkyl sulfate

CxySAS: C _lx to C _ly secondary (2, 3) sodium sulfate

CxyEz : C _lx to C _ly mainly linear primary alcohol condensed on average with moles of ethylene oxide • · ·· 99 *· · 99 ··

9 9 9 9 9 9

9 9 · 9 9 9 9 • · 9 *999 9 · · · * • 9 9 Λ · 9 9 9 9

999 9 9« * 99 99

CxyEzS: C _lx to C _ly sodium alkyl sulfate condensed with an average of 10 moles of ethylene oxide

QAS : R ₂ 'N+(CH ₃ ) ₂ (C ₂ H ₄ OH) with R ₂ = C ₁₂ to C ₁₄

QAS 1 : R ₂ *N+(CH ₃ ) ₂ (C ₂ H ₄ OH) with R ₂ = C ₈ to C _n

APA: _C8 to _C10 amidopropyldimethylamine

Soap: Sodium linear alkyl carboxylate derived from a mixture

80/20 fatty acids from tallow and coconut oil

Nonionic: C ₁₃ to C ₁₅ mixed ethoxylated/propoxylated fatty alcohol with an average degree of ethoxylation of 3.8 and an average degree of propoxylation of 4.5

Neodol 45-13: C14-C15 linear primary alcohol ethoxylate, sold by Shell Chemical Co.

Quat: a quaternary surfactant selected from one or more of the following: lauryltrimethylammonium chloride, myristyltrimethylammonium chloride, palmityltrimethylammonium chloride, (coconut alkyl)trimethylammonium chloride, (coconut alkyl)trimethylammonium methylsulfate, (coconut alkyl)dimethylmonohydroxyethylammonium chloride, (coconut alkyl)dimethylmonohydroxyethylammonium methylsulfate, stearyldimethylmonohydroxyethylammonium chloride, stearyldimethylmonohydroxyethylammonium methylsulfate, di- _C12 to _C14 alkyldimethylammonium chloride

STS: sodium toluenesulfonate

CFAA: C ₁₂ to C ₁₄ alkyl N-methylglucamine

TFAA: C ₁₆ to C ₁₈ alkyl N-methylglucamine

TPKFA: C ₁₂ to C ₁₄ terminated, fully cleaved fatty acids

DEQA: di(tallow alkyl - oxyethyl)dimethylammonium chloride

DEQA(2) : di(tallow alkyl - oxyethyl)hydroxyethyldimethylammonium methyl sulfate

DTDMAMS : di(tallow alkyl)dimethylammonium• · · · ♦ ···· ··* · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·♦· · · · · « · • · · · · · ' · · · ·

...... * *· ··

SDASA methyl sulfate : stearyldimethylamine : triple-pressed stearic acid in a ratio of 1 : 2

Silicate: amorphous sodium silicate (ratio SiO ₂ : NaO ₂ =1.6

up to 3.2) Zeolite A : hydrated sodium aluminosilicate Na ₁₂ (AlO ₂ SiO ₂ ) ₁₂ ·27Η ₂ Ο where the particle size is 0.1 micrometer to 10 micrometers (weight is expressed on an anhydrous basis) Na-SKS-6 : crystalline layered silicate 6-Na ₂ Si ₂ O ₅ Citrate : trisodium citrate dihydrate with an activity of 86.4% and a particle size of 425 microns to 850 microns Borate : sodium borate

Carbonate: anhydrous sodium carbonate with a particle size of 200 micrometers to 900 micrometers

Bicarbonate: anhydrous sodium bicarbonate with particle size

400 micrometers to 1200 micrometers Sulphate : anhydrous sodium sulfate Mg sulfate : anhydrous magnesium sulfate STPP : trisodium polyphosphate TSPP : tetrasodium pyrophosphate MA/AA : random copolymer of acrylate with maleate in the ratio 4:1 with an average molecular weight of 70,000 to MA/AA 1 80,000 : random copolymer of acrylate with maleate in the ratio 6;4 with an average molecular weight of 10,000 AA : sodium polyacrylate polymer with an average molecular weight of 4,500 PB1 : anhydrous sodium peroxyborate monohydrate NaBO ₂ »H ₂ O ₂ PB4 : anhydrous sodium peroxyborate tetrahydrate NaBO · 311.0 · HO

Peroxides* 99 999 9999

9 9 9 9 9 9 9 9 9 9 « 9 9··· 9 99 ·

9999 99 9 9999 99 99 9

9 999 9999

...........

carbonate: anhydrous sodium percarbonate 2Na ₂ CO ₃ · 3H ₂ O ₂

At DCC : sodium dichloroisocyanurate TAED : tetraacetylethylenediamine NOBS : nonanoyloxybenzenesulfonate in the form of sodium salt

NACA-OBS : (6-nonamidocaproyl)oxybenzenesulfonate

DTPA : diethylenetriaminepentaacetic acid HEDP : 1,1-hydroxyethanediphosphonic acid DETPMP : diethyltriaminepenta(methylene)phosphonate sold by Monsanto under the trade name Dequest 2060 EDDS : ethylenediamine-N,Ν'-disuccinic acid, (S,S)isomer in the form of the sodium salt

Photoactivated bleach: sulfonated zinc phthalocyanine encapsulated in a dextrin soluble polymer

Photoactivated bleach 1: sulfonated aluminum phthalocyanine encapsulated in

dextrin soluble polymer PAAC : cobalt(III) pentaamine acetate Mananasa : mannanase sold under the trade name Gamanasa® by Novo Nordisk A/S and/or galactomanase extracted from the enzyme product sold under the trade name Rohapec® BIL by Rohm

Alkaline

mananasa : mananase from Bacillus agaradherens, NCIMB 40482 Protease : proteolytic enzyme sold under the trade names Savinasa, Alcalasa, Durazym by Novo Nordisk A/S, Maxacal, Maxapem sold by Gist-Brocades and proteases covered by patents WO91/06637 and/or WO95/10591 and/or EP 251 446. Amylase : amylolytic enzyme sold under the trade name Purafact OX Am® described in WO94/18314, WO96/05265 sold by Genencor, Termamyl®, Fungamyl® and Duramyl®, all available from Novo Nordisk A/S and those described in

• · • · · • · · ·

WO95/26397

Lipase: a lipolytic enzyme sold under the trade names Lipolase, Lipolase Ultra by Novo Nordisk A/S, and Lipomax sold by Gist-Brocades.

Cellulase: a cellulolytic enzyme sold under the trade names Carezym, Celuzym, and/or Endolase by Novo Nordisk A/S

CMC: sodium carboxymethylcellulose

PVP: polyvinyl polymer with an average molecular weight of 60,000

PVNO: polyvinylpyridine-N-oxide with an average molecular weight of 50,000

PVPVI: copolymer of vinylimidazole and vinylpyrrolidone with an average molecular weight of 20,000

Clarifier 1: disodium 4,4'-bis(2-sulfostyryl)biphenyl Clarifier 2: disodium 4,4'-bis(4-aniline-6-morpholin-1,3,5-triazin-2-yl)stilbene-2,2'-disulfonate

Silicone antifoam agent: polydimethylsiloxane foam suppressant with siloxaneoxyalkylene copolymer as dispersant with a ratio of foam suppressant to dispersant of 10:1 to 100:1

Anti-soap agent: 12% silicone/silicon dioxide, 18% stearyl alcohol, 70% starch in granular form

Opacifier: water-based monostyrene latex mixture, sold by BASF Aktiengesellschaft under the trade name Lytron 621

SRP 1 : anionic terminated polyesters

SRP 2 : diethoxylated poly(1,2-propylene terephthalate) •ΦΦΦΦ · φ short block polymer

QEA: bis ( (C ₂ H ₅ O) (C ₂ H ₄ O) _n ) (CH ₃ )-N ⁺ -C 6 C 12-N ⁺ -(CH 3 ) bis ( (C ₂ H ₅ O) (C ₂ H ₄ O)) _n , where n = 20 to 30

PEI: medium molecular weight polyethyleneimine

1800 and a medium degree of ethoxylation of 7

Polymer A: modified polyamines PEI (Mr = 182) with an average degree of ethoxylation = 15

Polymer B: modified polyamines PEI (Mr = 600) with an average degree of ethoxylation =20

Polyamidepolyamine

SCS

HMWPEO

PEG _X

PEO: polyamide polyamines of the present invention are commercially sold under the trade names: Kymen®, Kymen 557H®, Kymen 557LX®, Reten® and Cartaretin®: sodium cumenesulfonate: high molecular weight polyethylene oxide: polyethylene glycol of molecular weight x: medium molecular weight polyethylene oxide

000

TEPAE : tetraethylenepentamine ethoxylate

Example 1

The following high density laundry detergent compositions were prepared according to the present invention:

I II III IV In VI LAS 8.0 8, 0 8, 0 2.0 6, 0 6, 0 TAS - 0.5 - 0.5 1.0 0.1 C46 (S)AS 2.0 2.5 - - - - C25AS - - - 7.0 4.5 5, 5 C68AS 2.0 5.0 7.0 - - - C25E5 - - 3.4 10, 0 4.6 4.6 C25E7 3.4 3.4 1.0 - - - C25E3S - - - 2, 0 5.0 4.5 QAS - 0.8 - - - - QAS 1 - - - 0.8 0.5 1.0 Zeolite A 18, 1 18.0 14.1 18, 1 20, 0 18.1 Lemon juice - - - 2.5 - 2.5

• · 4

4

Carbonate 13, 0 13, 0 27.0 10, 0 10, 0 13, 0 Na-SKS-6 - - - 10, 0 - 10, 0 Silicate 1.4 1.4 3, 0 0.3 0.5 0.3 Citrate - 1.0 - 3, 0 - - Sulphate 26, 1 26, 1 26, 1 6, 0 - - Mg sulfate 0.3 - - 0, 2 - 0.2 MA/AA 0.3 0.3 0.3 4.0 1.0 1.0 CMC 0, 2 0, 2 0.2 0.2 0.4 0.4 PB4 9.0 9.0 5.0 - - - Peroxy- carbonate — — — 18, 0 18.0 TAED 1.5 0.4 1.5 - 3, 9 4.2 NACA-OBS - 2, 0 1.0 - - - DETPMP 0.25 0.25 0.25 0.25 - - SEP 1 - - - 0.2 - 0.2 EDDS - 0.25 0.4 - ; 0.5 0.5 CFAA 1.0 - 2.0 - - HEDP 0.3 0.3 0.3 0.3 0.4 0.4 QEA - - - 0, 2 - 0.5 Mananasa 0.005 0.002 0.0008 0.001 0.002 0.001 Protease 0.009 0.009 0.01 0.04 0.05 0.03 Amylase 0.002 0.002 0.002 0.006 0.008 0.008 Cellulase 0.0007 - - 0.0007 0.0007 0.0007 Lipase 0.006 - - 0.01 0.01 0.01 Photoactivated bleach (ppm) 15 15 15 — 20 20 PVNO/PVPVI - - - 0.1 - - Brightener 1 0.09 0.09 0.09 - 0.09 0.09 Fragrance 0.3 0.3 0.3 0.4 0.4 0.4 Silicone anti- 0.5 0.5 0.5 — 0.3 0.3 foaming Density in g/1 850 850 850 850 850 850 Miscellaneous and small The rest until 100% leftovers

• · ·

100

9 9 9 9 9 9 9

9 9 9 9 9 9 9 9 9

9 9 9 9 9 9 9 9 9 9 · · · · · • · · · ···· • · Φ · f 9 9 9 9

Example 2

The following granular laundry detergents that

are usable especially when washing conditions in European washing machines, were prepared according to the present invention: I II III IV In VI LAS 5, 5 7.5 5.0 5.0 6, 0 7.0 TAS 1.25 1.9 - 0.8 0.4 0.3 C24AS/C25AS - 2.2 5.0 5.0 5.0 2.2 C25E3S - 0.8 1.0 1.5 3, 0 1.0 C45E7 3, 25 - - - - 3.0 TFAA - - 2, 0 - - - C25E5 - 5, 5 - - - - QAS 0.8 - - - - - QAS 1 - 0.7 1.0 0.5 1.0 0.7 STPP 19, 7 - - - - Zeolite A - 19, 5 25, 0 19, 5 20, 0 17, 0 NaSKS-6/Citric Acid - 10, 6 — 10, 6 — — (79:21) Na-SKS-6 - - 9.0 - 10, 0 10, 0 Carbonate 6.1 21.4 9.0 10, 0 10, 0 18, 0 Bicarbonate — 2, 0 7.0 5.0 2.0 Silicate 6, 8 - - 0.3 0.5 - Citrate - - 4.0 4.0 - - Sulphate 39, 8 - - 5.0 - 12, 0 Mg sulfate - - 0.1 0.2 0.2 - MA/AA 0.5 1.6 3, 0 4.0 1.0 1.0 CMC 0, 2 0.4 1.0 1.0 0.4 0.4 PB4 5.0 12, 7 - - - - Peroxy- carbonate — — — — 18.0 15.0 TAED 0.5 3.1 - - 5.0 - NACA-OBS 1.0 3, 5 - - - 2.5 DETPMP 0.25 0.2 0.3 0.4 - 0.2 HEDP - 0.3 - 0.3 0.3 0.3 QEA - - 1.0 1.0 1.0 - Mananasa 0.005 0.002 0.008 0.005 0.002 0.001 Protease 0.009 0.03 0.03 0.05 0.05 0.02

101 • 9 9 · · 9 « 9 9 9 9 9 9 9 · 9 9 9 9 9 9 9999 9 9 9 • 9 · 9 9 9 9 9999 9 9 9 9 9 « Lipase 0.003 0.003 0.006 0.006 0.006 0.004 Cellulase 0.0006 0.0006 0.0005 0.0005 0.0007 0.0007 Amylase 0.002 0.002 0.006 0.006 0.01 0.003 PVNO/PVPVI - - 0, 2 0, 2 - - PVP 0.9 1.3 - - - 0.9 SEP 1 - - 0.2 0.2 0.2 - Photoactivated 15 27 - - 20 20 bleach (ppm) Photoactivated bleach 1(ppm) 15 - - - - - Brightener 1 0.08 0.2 - - 0.09 0.15 Brightener 2 - 0.04 - - - - Fragrance 0.3 0.5 0.4 0.3 0.4 0.3 Silicone anti- 0.5 2.4 0.3 0.5 0.3 2.0 foaming Density in g/1 750 750 750 750 750 750 Miscellaneous and small The rest until 100%

leftovers

Example 3

The following detergents are usable:

especially under the conditions laundry in Europe washing machine, were prepared according to submitted I II invention: III IV In VI Blown powder LAS 6, 0 5.0 11, 0 11.0 6, 0 6, 0 TAS 2.0 - - - 2, 0 2, 0 Zeolite A 24.0 - - - 20, 0 20, 0 STPP - 27, 0 24.0 24.0 - - Sulphate 4.0 6, 0 13, 0 13, 0 - - MA/AA 1.0 4.0 6, 0 6, 0 2, 0 2.0 Silicate 1.0 7.0 3, 0 3, 0 3, 0 3, 0 CMC 1.0 1.0 0.5 0.5 0.6 0.6 Brightener 1 0.2 0.2 0.2 0.2 0.2 0.2 Silicone 1.0 1.0 1.0 1.0 0.3 0.3 antifoaming agent DETPMP 0.4 0.4 0.2 0.2 0.4 0.4

• φ ·φ · φφφφ φφφ φ φ · φφφφ φφφ φφφφ φφφφ • φ φ φ φ φ φ φ · φ φ φ φ φφ φ

102

Sprayed Brightener 0.02 - - - 0.02 0.02 C45E7 - - - - 5.0 5.0 C45E2 2.5 2.5 2, 0 2.0 - - C45E3 2, 6 2, 5 2, 0 2, 0 - - Fragrance 0.5 0.3 0.5 0.5 0.2 0.2 Silicone anti- 0.3 0.3 0.3 0.3 — — foaming Dry additives QEA - - - - 1.0 1.0 EDDS 0.3 - - - - - Sulphate 2.0 3, 0 5.0 5.0 10.0 IQ, 0 Carbonate 6, 0 13, 0 15, 0 15, 0 14.0 14.0 Lemon juice 2, 5 - - - 2.0 2.0 QAS 1 0.5 - - - . 0.5 0.5 Na-SKS-6 10, 0 - - - - - Peroxy- carbonate 18.5 — — — PB4 - 18.0 10, 0 10, 0 21.5 21, 5 TAED 2, 0 2.0 - - 2.0 2, 0 NACA-OBS 3, 0 2.0 4.0 4.0 - - Mananasa 0.005 0.002 0.0008 - 0.001 - · Alkaline - - - 0.001 - 0.002 mananasa Protease 0.03 0.03 0.03 0.03 0.03 0.03 Lipase 0.008 0.008 0.008 0.008 0.004 0.004 Amylase 0.003 0.003 0.003 0.003 0.003 0.003 Brightener 1 0.05 - - - 0.05 0.05 Miscellaneous and small The rest until 100% leftovers Example 4 Next granulated detergents were >prepared according to the present invention: I II III IV In VI Blown powder LAS 23.0 8, 0 7.0 9.0 7.0 7.0 TAS - - - - 1.0 -

C45AS 6, 0 6, 0 C45AES - 1.0 C45E35 - - Zeolite A 10, 0 18, 0 MA/AA - 0.5 MA/AA 1 7.0 - AA - 3, 0 Sulphate 5.0 6, 3 Silicate 10, 0 1, about Carbonate 15, 0 20, 0 PEG 4000 0.4 1.5 DTPA - 0.9 Brightener 2 0.3 0.2 Sprayed C45E7 - 2, 0 C25E9 3, 0 - C23E9 - - Fragrance 0.3 0.3 Agglomerates C4 5AS - 5.0 LAS - 2, 0 Zeolite A - 7.5 Carbonate - 4.0 PEG 4000 - 0.5 Miscellaneous (water, etc.) — 2, 0 Dry additives QAS - - Lemon juice - - PB4 - - PB1 4.0 1.0 Peroxy- carbonate — — Carbonate - 5, 3 NOBS 4.0 - Methylcellulose 0, 2 - Na-SKS-6 8.0 - STS - -

·· 9

103

• 9 4 4· 444444 4 • 4 4 4 4 94 9 4 4 9944 · · « 9 • 5.0 8, 0 - - 1.0 1.0 - - - 2.0 4, 0 14.0 12.0 10, 0 10, 0 - - - 2, 0 3, 0 2, 0 3, 0 3, 0 14.3 11.0 15, 0 19, 3 1.0 1.0 1.0 1.0 10, 0 20.7 8, 0 6, 0 1.5 1.0 1.0 1.0 0.5 - - 0.5 0.3 - 0.1 0.3 - - 2.0 2, 0 - - - - 1.5 2, 0 - 2, 0 0.3 2, 0 0.3 0.3 5.0 2, 0 - 5.0 2, 0 - - 2, 0 7.5 8.0 - 7.5 4.0 5.0 - 4.0 0.5 - - 0.5 2, 0 2.0 - 2.0

- - 1.0 — - - 2.0 - - - 12, 0 1.0 3, 0 2, 0 - - - - 2, 0 10.0 1.8 - 4.0 4.0 6, 0 - - 0.6 - - - - - - - - 2.0 - 1.0 -

«φ ·

104 • Φ Φ φφ · ΦΦ φ · φ φ · φ φ · · φ φ Φ φφφφ φ φ φ φ φ φ· · Φ • Φ • φ φ· Φ · φ • φ φ Culmene sulfonic acid - 1.0 - - 2, 0 Mananasa 0.005 0.002 0.001 0.008 0.001 0.001 Protease 0.02 0.02 0.02 0.01 0.02 0.02 Lipase 0.004 0.004 - 0.004 0.004 Amylase 0.003 0.002 - 0.003 - Cellulase 0.0005 0.0005 0.0005 0.0007 0.0005 0.0005 PVPVI - - - 0.5 0.1 PVP - - - 0.5 - PVNO 0.5 0.3 - - QEA - - - 1.0 - SEP 1 0.2 0.5 0.3 - 0.2 - Silicone antifoaming agent 0, 2 0.4 0.2 0.4 ο,ι Mg sulfate Miscellaneous and small residues 0.2 Remainder up to 100% 0.2 Example 5 Next detergents not containing none bleach that were prepared are particularly suitable for washing colored according to the present invention: fabrics, I II III IV In

Blown powder

Zeolite A 15, 0 15, 0 15.0 - Sulphate - - 5.0 - - LAS 3, 0 3, 0 3, 0 - - DETPMP 0.4 0.4 0.5 - - CMC 0.4 0.4 0.4 - - MA/AA 4.0 4.0 4.0 - Agglomerates C4 5AS - - - 11.0 11.0 LAS 6, 0 6, 0 5.0 - - TAS 3, 0 3, 0 2, 0 - - Silicate 4.0 4.0 4.0 - - Zeolite A 10, 0 10, 0 15.0 13.0 13.0 CMC - - - 0.5 0.5

4

105 ♦ 4 4 4 4 4 4

4«4 4 44 4

4444444 44 4 ♦ 4 4 4 4 4 4 • 4 4 4 4 <4

MA/AA - - 2, 0 2, 0 Carbonate 9.0 9.0 7.0 7.0 7.0 Sprayed Fragrance 0.3 0.3 0.3 0.5 0.5 C45E7 4.0 4.0 4.0 4.0 4.0 C25E3 2, 0 2, 0 2, 0 2, 0 2, 0 Dry additives MA/AA - - - 3, 0 3.0 Na-SKS-6 - - - 12.0 12.0 Citrate 10, 0 10, 0 - 8, 0 8, 0 Bicarbonate 7.0 7.0 3, 0 5.0 5.0 Carbonate 8, 0 8.0 5.0 7.0 7.0 PVPVI/PVNO 0.5 0.5 0.5 0.5 0.5 Mananasa 0.0008 - 0.0005 0.001 - Alkaline - 0.0008 - - 0.002 mananasa Protease 0.03 0.03 0.02 0.05 0.05 Lipase 0.008 0.008 0.008 0.008 0.008 Amylase 0.01 0.01 0.01 0.01 0.01 Cellulase 0.001 0.001 0.001 0.001 0.001 Silicone 5.0 5.0 5.0 5.0 5.0 anti-reagent foaming Sulphate - - 9.0 - - Density (g/1) 700 700 700 700 700 Miscellaneous and small Remainder to 100 %

leftovers

Example 6

The following detergent compositions were prepared according to the present invention:

Basic granules I II III IV Zeolite A 30, 0 22, 0 24, 0 10, 0 Sulphate 10, 0 5.0 10.0 7.0 MA/AA 3, 0 - - - AA - 1.6 2.0 - MA/AA 1 - 12, 0 - 6, 0 LAS 14, 0 10, 0 9, 0 20, 0

4

4 *

4*4

4 4*444 4

4

4 4 4

44 ► 4 4 4 » *4 4 > 4 4 4 » 4 4 4

4 4 4

106

C4 5AS 8, 0 7.0 9.0 7.0 C45AES - 1.0 1.0 - Silicate - 1.0 0.5 10, 0 Soap - 2.0 - - Brightener 1 0.2 0.2 0.2 0.2 Carbonate 6, 0 9.0 10, 0 10, 0 PEG 4000 - 1.0 1.5 - DTPA - 0.4 - - Sprayed C25E9 - - - 5.0 C45E7 1.0 1.0 - - C23E9 - 1.0 2, 5 - Fragrance 0.2 0.3 0.3 - Dry additives Carbonate 5.0 10, 0 18, 0 8, 0 PVPVI/PVNO 0.5 - 0.3 - Mananasa 0.005 0.002 0.0008 0.001 Protease 0.03 0.03 0.03 0.02 Lipase 0.008 - - 0.008 Amylase 0.002 - - 0.002 Cellulase 0.0002 0.0005 0.0005 0.0002 NOBS - 4.0 - 4.5 PB 1 1.0 5.0 1.5 6, 0 Sulphate 4.0 5.0 - 5.0 SEP 1 - 0.4 - - A dampening agent - 0.5 0.5 - suds formation Miscellaneous and small remnants The rest until 100%

Example 7

The following granular detergents are prepared according to the present invention:

I II means was

Blown Powder Zeolite A STPP Sulfate Carbonate

20, 0

20.0

III IV 15, 0 15, 0 - - 5.0 5.0 5.0 5.0

Φ Φ »· φ φφ Φ· • · · φφφ φφφφ φφφ φφφφ φφφφ φ φφφφ φ · φ φφφφ φ φ φ φ φ φ φφφ φφφφ φφφ * φφ φ φ · φφ

107

TAS - - 1.0 1.0 LAS 6, 0 6, 0 6, 0 6, 0 C68AS 2, 0 2.0 - - Silicate 3, 0 8.0 - - MA/AA 4.0 2.0 2.0 2, 0 CMC 0.6 0.6 0.2 0.2 Brightener 1 0, 2 0, 2 0.1 0.1 DETPMP 0.4 0.4 0.1 0.1 STS - - 1.0 1.0 Sprayed C45E7 5.0 5.0 4.0 4.0 Silicone agent 0.3 0.3 0.1 0.1 anti-foaming Fragrance 0.2 0.2 0.3 0.3 Dry additives QEA - - 1.0 ·' 1.0 Carbonate 14, 0 9.0 10.0 10.0 PB 1 1.5 2.0 - - PB 4 18.5 13, 0 13, 0 13.0 TAED 2.0 2, 0 2.0 2, 0 QAS - - 1.0 1.0 Photoactivated 15 ppm 15 ppm 15 ppm 15 ppm bleach Na-SKS-6 - - 3.0 3.0 Mananasa 0.005 0.002 0.0008 - Alkaline mannanase - - - 0.001 Protease 0.03 0.03 0.007 0.007 Lipase 0.004 0.004 0.004 0.004 Amylase 0.006 0.006 0.003 0.003 Cellulase 0.0002 0.0002 0.0005 0.0005 Sulphate 10, 0 20, 0 5.0 5.0 Density (g/1) 700 700 700 700 Miscellaneous and small remnants The rest up to 100%

108 • · ·* · 9· ·· • · · · · · · « · · ··· · ··· · ··· · ··· · ···················»* · · 9 9 9

9 9 9 9 9 9 9 9

999 9 99 9 9999

Example 8 The following detergent resources were prepared pc of the present invention: I II III Blown powder Zeolite A 15, 0 15, 0 15.0 Sulphate - 5.0 - LAS 3, 0 3, 0 3, 0 QAS - 1.5 1.5 DETPMP 0.4 0, 2 0.4 EDDS - 0.4 0.2 CMC 0.4 0.4 0.4 MA/AA 4.0 2.0 2, 0 Agglomerate LAS 5.0 5.0 5.0 TAS 2.0 2, 0 1.0 Silicate 3, 0 3.0 4.0 Zeolite A 8, 0 8.0 8, 0 Carbonate 8, 0 8.0 4.0 Sprayed Fragrance 0.3 0.3 0.3 C45E7 2.0 2, 0 2, 0 C25E3 2, 0 - - Dry additives Citrate 5.0 - 2.0 Bicarbonate - 3, 0 - Carbonate 8.0 15, 0 10, 0 TAED 6, 0 2.0 5.0 PB 1 14.0 7.0 10.0 PEO - - 0.2 Bentonite clay - - 10.0 Mananasa 0.005 0.002 0.0008 Protease 0.03 0.03 0.03 Lipase 0.008 0.008 0.008 Cellulase 0.001 0.001 0.001 Amylase 0.01 0.01 0.01 Silicone anti- 5.0 5.0 5.0

foaming

3.0

850 850

The rest up to 100%

109

0 4 4 4 4

4444 4 · 44441

4 * ·

4 44 0

Sulfate

Density (g/1) Miscellaneous and small residues

Example 9

The following detergents of the present invention:

850 funds were prepared according to

I II III IV LAS 18, 0 14, 0 24.0 20, 0 QAS 0.7 1.0 - 0.7 TFAA - 1.0 - - C25E56.5 - - 1.0 - C45E7 - 1.0 - - C45E3S 1.0 2, 5 1.0 - STPP 32.0 18, 0 30.0 ' 22.0 Silicate 9.0 5.0 9.0 8.0 Carbonate 11, 0 7.5 10, 0 5.0 Bicarbonate - 7.5 - PB 1 3, 0 1.0 - - PB 4 - 1.0 - - NOBS 2.0 1.0 - - DETPMP - 1.0 - - DTPA 0.5 - 0.2 0.3 SEP 1 0.3 0.2 - 0.1 MA/AA 1.0 1.5 2, 0 0.5 CMC 0.8 0.4 0.4 0.2 PEI - - 0.4 - Sulphate 20, 0 10, 0 20, 0 30, 0 Mg sulfate 0.2 - 0.4 0.9 Mananasa 0.005 0.002 0.005 0.001 Protease 0.03 0.03 0.02 0.02 Amylase 0.008 0.007 - 0.004 Lipase 0.004 - 0.002 - Cellulase 0.0003 - - 0.0001 Photoactivated 30 ppm 20 ppm - 10 ppm bleach Fragrance 0.3 0.3 0.1 0.2 Brightener 1/2 0.05 0.02 0.08 0.1

Miscellaneous and small residues Remainder up to 100%

110 • 0 00 0 00 00 •» 0 0 0 0 «000 • 0 · 0·0· 0 0 0 0 0 000000 0000000 00 0 0 0 000 00*0

000 0 00 0 00 00

Example 10

The following liquid detergent compositions were prepared according to the present invention (amounts are given as parts by weight, enzymes are given as pure enzyme):

I II III IV in LAS 11, 5 8, 8 - 3, 9 - C25E2.5S - 3, 0 18, 0 - 16.0 C45E2.25S 11.5 3, 0 - 15.7 - C23E9 - 2.7 1, 8 2.0 1.0 C23E7 3.2 - - - CFAA - - 5.2 - 3.1 TPKFA 1.6 - 2.0 0.5 2, 0 Lemon juice (50%) 6, 5 1.2 2, 5 4.4 2.5 Ca formate 0.1 0.06 0.1 - - Formate 0.5 0.06 0.1 0.05 0.05 SCS 4.0 1.0 3, 0 1.2 - Borate 0.6 - 3, 0 2.0 2.9 On hydroxide 5, 8 2, 0 3, 5 3.7 2.7 Ethanol 1.75 1.0 3, 6 4.2 2.9 1,2-propanediol 3, 3 2.0 8, 0 7.9 5, 3 Monoethanolamine 3, 0 1.5 1.3 2.5 0.8 TEPAE 1.6 - 1.3 1.2 1.2 Mananasa 0.005 0.001 0.002 0.0005 0.0002 Protease 0.03 0.01 0.03 0.02 0.02 Lipase - - 0.002 - - Amylase - - - 0.002 - Cellulase - - 0.0002 0.0005 0.0001 SEP 1 0.2 - 0.1 - - DTPA - - 0.3 - - PVNO - - 0.3 - 0.2 Brightener 1 0.2 0.07 0.1 - - Silicone 0.04 0.02 0.1 0.1 0.1

antifoaming agent

Miscellaneous and water

6»·· • · ···»

111 • 9 ·· * 9 9 · • · · · · • · · » • · · · « · »·

Example 11

The following liquid detergent compositions were prepared according to the present invention (amounts are given as parts

masses, enzymes are listed as clean enzyme): I II III IV LAS 10, 0 13, 0 9.0 - C25AS 4.0 1.0 2, 0 10, 0 C-25E3S 1.0 - - 3, 0 C25E7 6, 0 8.0 13, 0 2.5 TFAA - - - 4.5 APA - 1.4 - - TPKFA 2, 0 - 13, 0 7.0 Lemon juice 2, 0 3, 0 1.0 1.5 Dodecenyl/tetradecenyl amber 12, 0 10, 0 — - acid Fatty acid from 4.0 2.0 1.0 - rapeseed Ethanol 4.0 4.0 7.0 2.0 1,2-Propanediol 4.0 4.0 2, 0 7.0 Monoethanolamine - - - 5.0 Triethanolamine - - 8, 0 - TEPAE 0.5 - 0.5 0, 2 DETPMP 1.0 1.0 0.5 1.0 Mananasa 0.0002 0.0005 0.005 0.0005 Protease 0.02 0.02 0.01 0.008 Lipase - 0.002 - 0.002 Amylase 0.004 0.004 0.01 0.008 Cellulase - - - 0.002 SEP 2 0.3 - 0.3 0.1 Boric Acid 0.1 0.2 1.0 2, 0 Ca chloride - 0.02 - 0.01 Brightener 1 - 0.4 - - A buffering agent 0.1 0.3 - 0.1 suds formation Agent causing 0.5 0.4 - 0.3 turbidity NaOH so that 8.0 8, 0 7.6 7.7

the resulting pH was

Miscellaneous and water 112 « 9 »· · » · « «1 *··· 9 • · *·· » m · » • · · » • · · · . • · ···» · · • · · » ·· « .· Example 12 The following liquid detergents resources were ready according to the present invention (quantities are listed as parts masses, enzymes are listed as pure enzyme): I II III IV In LAS 25, 0 - - - - C25AS - 13, 0 18, 0 15, 0 18, 0 C25E3S - 2.0 2.0 4.0 2, 0 C25E7 - - 4.0 4.0 4.0 TFAA - 6, 0 8.0 8, 0 8, 0 APA 3, 0 1.0 2, 0 - 2.0 TPKFA - 15, 0 11.0 11.0 11.0 Lemon juice 1.0 1.0 1.0 ; 1.0 ¹ 1.0 Dodecenyl/tetradecenyl succinic acid 15, 0 Rapeseed fatty acid 1.0 — 3, 5 3, 5 Ethanol 7.0 2.0 3.0 2, 0 3, 0 1,2-Propanediol 6, 0 8, 0 10, 0 13, 0 10, 0 Monoethanolamine - - 9.0 9.0 9.0 TEPAE - - 0.4 0.3 0.4 DETPMP 2.0 1.2 1.0 - 1.0 Mananasa 0.0001 0 , 0002 0.005 0.0005 - Alkaline - - - - 0.005 mananasa Protease 0.08 0.02 0.01 0.02 0.01 Lipase - - 0.003 0.003 0.003 Amylase 0.004 0.01 0.01 0.01 0.01 Cellulase - - 0.004 0.003 0.004 SEP 2 - - 0.2 0.1 0.2 Boric Acid 1.0 1.5 2.5 2, 5 2.5 Bentonite clay 4.0 4.0 — Brightener 1 0.1 0.2 0.3 - 0.3 Soap Suppressant 0.4 — ~~ — —

• · ·· · ··

4 4 4 · · 4 • 44 4444 4 • 4444 4 4 · ···· 4 4 4 4 4

113 444 4 44 4 44 Agent causing turbidity 0.8 0.7 - - - NaOH so that 8, 0 7.5 8.0 8.2 8.0

The resulting pH was Various and water

Example 13

The following liquid detergents resources were ready according to the present invention (quantities are listed as parts mass, enzymes are listed as pure enzyme): I II III LAS 27, 6 18.9 27, 6 C4 5AS 13, 8 5.9 13, 8 C13E8 3, 0 3.1 3, 0 Oleic acid 3.4 2, 5 3.4 Citric acid 5.4 5.4 5.4 On hydroxide 0.4 3, 6 0.4 Ca formate 0.2 0.1 0.2 Formate - 0.5 Ethanol 7.0 - 7.0 Monoethanolamine 16, 5 8, 0 16, 5 1,2-propanediol 5, 9 5, 5 5.9 Xylenesulfonic acid - 2.4 - TEPAE 1.5 0.8 1.5 Protease 0.05 0.02 0.05 Mananasa 0.005 0.0002 - Alkaline mannanase - - 0.001 PEC - 0.7 - Brightener 2 0.4 0.1 0.4 Fragrance 0.5 0.3 0.5 Water and small debris Example 14 The following gel detergents resources were ready according to the present invention: I II III IV In C12-15E2.5S 21 20.2 22, 7 13, 6 20.2

• ·

C12LAS

C12-14 glucosamide

C12-14EO7

C12-15EO9

C8-10 amidopropylamine

CIO amidopropylamine Lemon k.

C12/14 fatty acid

Palm kernel fatty acid

Canola Fatty Acid Mananasa

Alkaline mannanase

Protease Lipase Amylase Cellulase Brightener 1 Polymer A Polymer B Polyamine-polyamide

Polyethoxylated polyamines

Dirt release agent

Ethanol

1,2-propanediol Monoethanolamine

NaOH

Boric acid Borax

Scum suppressant

114 • 9 9 9 99 9 9 99 9 9 9 9 9 - - - 9.1 - 4, 0 2, 5 - - 2, 5 4.5 - - - - - 0.6 0.6 0.6 0.6 1.3 - - - - - 1.3 1.3 1.3 1.3 1.0 5.0 1.0 1.0 5.0 - 10, 0 10, 0 10, 0 10, 0 8, 0 - - - - 8, 0 - - - - 0.0001 0.0002 0.005 0.0005 - - - - 0.0008 0.02 0.03 0.03 0.03 0.03 0.001 0.002 0.003 0.002 0.002 0.003 0.002 0.002 0.002 0.002 0.0007 0.0001 0.0001 0.0001 0.0001 0.15 0.15 0.15 0.15 0.15 0.7 0.6 0.6 0.6 0.6 - 1.2 1.2 1.2 1.2 2, 0 1.0 1.0 - 1.0 - 2, 0 - - 2.0 - 0.1 0.1 0.1 0.1 0.7 0.5 0.5 0.5 0.5 4.0 4.0 4.0 4.0 4.0 0.7 0.5 0.5 0.5 0.5 2, 8 7.0 7.0 7.0 7.0 2, 0 - - - - - 2.5 2, 5 2.5 2.5 - 0.1 0.1 0.1 0.1

• « ··· ···· ··· ··· ···· • ·

115 • · · · · · 4 · · Polydimethyl- siloxane 0.2 - - - Fragrance 0.5 0.75 0.75 0.75 0.75 Dye - 0.04 0.04 0.04 0.04 Miscellaneous and water The rest is up to 100%

Example 15

The following gel detergent compositions were prepared according to the present invention:

I II III IV C12-15E2.5S 18.2 22, 6 27, 6 22, 6 C12-15EO9 0.6 0.6 0.6 0.6 CIO amidopropylamine 1.3 1.3 1.3 1.3 Lemon juice 1.0 1.0 1.0 1.0 C12/14 fatty acid 10, 0 10, 0 .7.5 10, 0 Quat 1.0 5.0 - - Mananasa 0.005 0.001 0.002 0.0005 Protease 0.03 0.01 0.03 0.03 Lipase 0.002 0.002 0.002 0.002 Amylase 0.003 0.002 0.001 0.002 Cellulase 0.0001 0.0004 0.0001 0.0001 Brightener 1 0.15 0.15 0.15 0.15 Polymer A 0.6 0.3 0.6 0.6 Polymer B 1.2 0.6 1.2 1.2 Dirty release agent 0.1 0.1 0.1 0.1 Ethanol 0.5 0.5 0.5 0.5 1,2-propanediol 4.0 4.0 4.0 4.0 Monoethanolamine 0.5 0.5 0.5 0.5 NaOH 7.0 7.0 7.0 7.0 Boric acid - - - - Borax 2.5 2.5 2.5 - Soap Suppressant 0.1 0.1 0.1 0.1 Fragrance 0.75 0.75 0.75 0.75 Dye 0.04 0.04 0.04 0.04 Miscellaneous and water The rest until 100%

116

Example 16

The following granular laundry detergents

fabrics that have the ability to soften during washing were prepared according to the present invention: I II C45AS - 10.0 LAS 7.6 - C68AS 1.3 - C45E7 4.0 - C25E3 - 5.0 Coco-alkyldimethylhydroxy- ethylammonium chloride 1.4 1.0 Lemon juice 5.0 3, 0 Na-SKS-6 - 11.0 Zeolite A 15, 0 , 15, 0 MA/AA 4.0 4.0 DETPMP 0.4 0.4 PB 1 15, 0 - Percarbonate - 15, 0 TAED 5.0 5.0 Smectite clay 10, 0 10, 0 High molecular weight PEO — 0.1 Mananasa 0.01 0.001 Proetas 0.02 0.01 Lipase 0.02 0.01 Amylase 0.03 0.005 Cellulase 0.001 - Silicate 3, 0 5.0 Carbonate 10, 0 10, 0 Soap Suppressant 1.0 4.0 CMC 0, 2 0.1 Miscellaneous and water The rest until 100%

Example 17

The following rinse-addable softeners were prepared according to the present invention:

117

9 9 9 9 9 9 9 9 ··· 9 · 9 9*99 • 99 · 9 · · 9 · · *

999· 9 9 9 9999 99 99 9

9 999 9999

999 9 »9 9 «9 99

I II DEQA (2) 20, 0 20, 0 Alkaline mannanase - 0.002 Mananasa 0.0008 - Cellulase 0.001 0.001 HCL 0.03 0.03 Antifoaming agent 0.01 0.01 Blue dye 25 ppm 25 ppm CaCl ₂ 0.20 0.20 Fragrance 0.90 0.90 Miscellaneous and water The rest up to 100 Q, Ό Example 18 Following softening resources on fabric and resources added to fabrics before drying were prepared according to of the present invention: I II III IV In DEQA 2, 6 19.0 - - - DEQA (2) - - - 51, 8 DTMAMS - - 26, 0 - SDASA - 70, 0 42, 0 40.2 Stearic acid 0.3 - - - - from IV=0 Neodol 45-13 - 13, 0 - - Hydrochloric acid 0.02 0.02 - - - acid Ethanol - 1.0 - - Mananasa 0.0008 0.0002 0.0005 0.005 0.0002 Fragrance 1.0 1.0 0.75 1.0 1.5 Glycopersic S-20 - - - 15.4 Glycerol monostearate — 26.0 Digeranyl succinate - 0.38 - - Silicone 0.01 anti- 0.01 - foaming Electrolyte 0.1 - - - Clay - - 3, 0 - Dye 10 ppm 25 ppm 0.01 - -

·· ·· • · · • φ · φ · · · ·«····· φ φ · • · φ · · · · ·· · Φ· ·· ·· φ

118

Water and small debris

100% 100%

Example 19

The following laundry detergents in bar form

were prepared according to the submitted invention (quantity are listed as parts mass, enzymes I am listed like clean enzyme): I II III IV In III VI In LAS - - 19, 0 15, 0 21, 0 6.75 8, 8 - C2 8AS 30, 0 13, 5 - - - 15.75 11.2 22.5 For the laureate 2, 5 9, 0 - - - - - - Zeolite A 2.0 1.25 - - - 1.25 1.25 1.25 Carbonate 20, 0 3, 0 13, 0 8, 0 10, 0 15, 0 15, 0 10, 0 About 27.5 39, 0 35, 0 - - 40, 0 ' - 40, 0 Carbonate Sulphate 5.0 5.0 3, 0 5.0 3, 0 - - 5.0 TSPP 5.0 - - - - 5.0 2.5 - STPP 5.0 15, 0 10, 0 - - 7.0 8, 0 10.0 Bentonite - 10, 0 - - 5.0 - - - clay DETPMP - 0.7 0.6 - 0.6 0.7 0.7 0.7 CMC - 1.0 1.0 1.0 1.0 - - 1.0 Talc - - 10, 0 15, 0 10, 0 - - - Silicate - - 4.0 5.0 3, 0 - - - PVNO 0.02 0.03 - 0.01 - 0.02 - - MA/AA 0.4 1.0 - - 0.2 0.4 0.5 0.4 SEP 1 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 Mananasa 0.0005 0.0005 0.0008 0.0005 0.0002 0.0002 0.0001 0.0005 Amylase - - 0.01 - - 0.002 - Protease - 0.004 0.003 0.003 - - 0.003 Lipase - 0.002 - 0.002 - - - - Cellulase - 0.0003 - - 0.0003 0.0002 - - PEO - 0.2 - 0.2 0.3 - - 0.3 Fragrance 1.0 0.5 0.3 0.2 0.4 - - 0.4 Mg sulfate - - 3, 0 3, 0 3, 0 - - - Brightener 0.15 0.1 0.15 - - - - 0.1

· · 999 9999

999 9 999 9 99 9 « 9999 99 9 9999 99 99 99 9

9 999 9999

Photo-activated - 15.0 15.0 15.0 15.0 - - 15.0 bleach (ppm)

Example 20

The following detergent additive compositions were prepared according to the present invention:

I II III IV LAS - 5.0 5.0 5.0 STPP 30, 0 - 20, 0 20, 0 Zeolite A - 35, 0 20, 0 20, 0 PB 1 20, 0 15, 0 - - TAED 10, 0 8, 0 - - Mananasa 0.005 0.0002 0.001 - Alkaline mannanase - - - 0.005 Protease - 0.3 0.3 0.3 Amylase - 0.06 0.06 0.06 Small residues, water and The rest until 100%

various

Sequence list (1) General information

Submitter:

Name: : The Procter & Gamble Company Street: : One Procter & Gamble Plaza City: : Cincinnati, Ohio Country: USA

Postal code: 45202

Name of the invention: Laundry detergents with enzymes that break down carbohydrate gums containing

Number of sequences: 6

Computer-readable form: Media type: floppy disk

120 • · · · ♦ · φφφ • · · φφφ* φφφ • φ····· φφφφφφφ φφ • · φφφ φφφ • · φ φ φ* φ φφ

Computer: IBM PC compatible

Operating system: PC-DOS / MS-DOS Software: Patentln 1.0 version 1.25 (EPO)

Sequence identification number 1

Sequence characteristics:

Length: 1407 base pairs

Type: nucleic acid

Fiber content: single

Topology: linear

Molecule type: gene DNA

Original source

Features:

Name/Key: CDS

Location: 1-1428

Sequence description: sequence identification number 1

AT GAAAAAAAAG T TAT C AC AGAT T T AT CAT T T AAT TAT T T GCACAC T TAT AATA

AGTGTGGGAATAATGGGGATTACAACGTCCCCATCAGCAGCAAGTACAGGC

T T T TAT GT T GATGGCAATAC GT TATAT GAC GCAAATGGGCAGCCAT T TGTCAT

GAGAGGTATTAACCATGGACATGCTTGGTATAAAGACACCGCTTCAACAGCT

ATTCCTGCCATTGCAGAGCAAGGCGCCAACACGATTCGTATTGTTTTATCAG

ATGGCGGTCAATGGGAAAAGACGACATTGACACCATTCGTGAAGTCATTG

AGCTTGCGGAGCAAAATAAAATGGTGGCTGTCGTTGAAGTTCATGATGCCA

CGGGTCGCGATTCGCGCAGTGATTTAAATCGAGCCGTTGATTATTGGATAG

AAATGAAAGATGCGCTTATCGGTAAAGAAGTACGGTTATTATTAACATTGCA

AACGAGTGGTATGGGAGTTGGGATGGCTCAGCTTGGGCCGATGGCTATATT

GATGTCATTCCGAAGCTTCGCGATGCCGGCTTAACACACACCTTAATGGTTG

ATGCAGCAGGATGGGGGCAATATCCGCAATCTATTCATGATTACGGACAAG

ATGTGTTTAATGCAGATCCGTTAAAAAATACGATGTTCTCCATCCATATGTAT ♦ · · ·

121

9 99

GAGTATGCTGGTGGTGATGCTAACACTGTTAGATCAAATATTGATAGAGTCA

TAGATCAAGACCTTGCTCTCGTAATAGGTGAATTCGGTCATAGACATACTGA

TGGTGATGTTGATGAAGATACAATCCTTAGTTATTCTGAAGAAACTGGCACA

GGGTGGCTCGCTTGGTCTTGGAAAGGCAACAGTACCGAATGGGACTATTTA

GACCTTTCAGAAGACTGGGCTGGTCAACATTTAACTGATTGGGGGAATAGAA

TTGTCCACGGGGCCGATGGCTTACAGGAAACCTCCAAACCATCCACCGTAT

TTACAGATGATAACGGTGGTCACCCTGAACCGCCAACTGCTACTACCTTGTA

TGACTTTGAAGGAAGCACACAAGGGTGGCATGGAAGCAACGTGACCGGTG

GCCCTTGGTCCGTAACAGAATGGGGTGCTTCAGGTAACTACTCTTTAAAGC

CGATGTAAATTTAACCTCAAATTCTTCACATGAACTGTATAGTGAACAAAGTC

GTAATCTACACGGATACTCTCAGCTCAACGCAACCGTTCGCCATGCCAATTG

GGGAAATCCCGGTAATGGCATGAATGCAAGACTTTACGTGAAAACGGGCTC

TGATTATACATGGCATAGCGGTCCTTTTACACGTATCAATAGCTCCAACTCA

GGAACAACGTTATCTTTTGATTTAAACAACATCGAAAATAGTCATCATGTTÁG

GGAAATAGGCGTGCAATTTTCAGCGGCAGATAATAGCAGTGGTCAAACTGC

TCTATACGTTGATAACGTTACTTTAAGATAG

Sequence identification number 2

Sequence characteristics:

Length: 493 amino acids Type: amino acid Topology: linear

Molecule type: protein

Sequence description: sequence identification number 2

MKKKLSQIYHLIICTLIISVGIMGITTSPSAASTGFYVDGNTLYDANGQPFVMRGIN

HGHAWYKDTASTAIPAIAEQGANTIRIVLSDGGQWEKDDIDTIREVIELAEQNKM

VAWEVHDATGRDSRSDLNRAVDYWIEMKDALIGKEDTVIINIANEWYGSWDGS

AWADGYIDVIPKLRDAGLTHTLMVDAAGWGQYPQS1HDYGQDVFNADPLKNTM

FSIMHYEYAGGDANTVRSNIDRVIDQDLALVIGEFGHRHTDGDVDEDTILSYSEE

TGTGWLAWSWKGNSTEWDYLDLSEDWAGQHLTDWGNRIVHGADGLQETSKP • · • ·

122 .....

STVFTDDNGGHPEPPTATTLYDFEGSTQGWHGSNVTGGPWSVTEWGASGNY

SLKADVNLTSNSSHELYSEQSRNLHGYSQLNATVRHANWGNPGNGMNARLW

KTGSDYTWHSGPFTRINSSNGTTLSFDLNNIENSHHVREIGVQFSAADNSSGQ

TALYVDNVTLR

Sequence identification number 3

Sequence characteristics: Length: 1407 base pairs Type: nucleic acid Stranding: simple Topology: linear

Molecule type: gene DNA

Sequence description: sequence identification number 3

AT GAAAAAAAAG T T AT CAC AGAT T T AT C AT T TAAT TAT T T GCACAC T T ATAAT A AGTGTGGGAATAATGGGGATTACAACGTCCCCATCAGCAGCAAGTACAGGC

TTTTATGTTGATGGCAATACGTTATATGACGCAAATGGGCAGCCATTTGTCAT

GAGAGGTATTAACCATGGACATGCTTGGTATAAAGACACCGCTTCAACAGCT

ATTCCTGCCATTGCAGAGCAAGGCGCCAACACGATTCGTATTGTTTTATCAG

ATGGCGGTCAATGGGAAAAGACGACATTGACACCATTCGTGAAGTCATTG

AGCTTGCGGAGCAAAATAAAATGGTGGCTGTCGTTGAAGTTCATGATGCCA

CGGGTCGCGATTCGCGCAGTGATTTAAATCGAGCCGTTGATTATTGGATAG

AAATGAAAGATGCGCTTATCGGTAAAGAAGTACGGTTATTATTAACATTGCA

AACGAGTGGTATGGGAGTTGGGATGGCTCAGCTTGGGCCGATGGCTATATT

GATGTCATTCCGAAGCTTCGCGATGCCGGCTTAACACACACCTTAATGGTTG

ATGCAGCAGGATGGGGGCAATATCCGCAATCTATTCATGATTACGGACAAG

ATGTGTTTAATGCAGATCCGTTAAAAAAA.TACGATGTTCTCCATCCATATGTAT

GAGTATGCTGGTGGTGATGCTAACACTGTTAGATCAAATATTGATAGAGTCA

TAGATCAAGACCTTGCTCTCGTAATAGGTGAATTCGGTCATAGACATACTGA

TGGTGATGTTGATGAAGATACAATCCTTAGTTATTCTGAAGAAACTGGCACA

GGGTGGCTCGCTTGGTCTTGGAAAGGCAACAGTACCGAATGGGACTATTTA

123 • 9 · · • 9 9 9

9 9 9

99

GACCTTTCAGAAGACTGGGCTGGTCAACATTTAACTGATTGGGGGAATAGAA

TTGTCCACGGGGCCGATGGCTTACAGGAAACCTCCAAACCATCCACCGTAT

TTACAGATGATAACGGTGGTCACCCTGAACCGCCAACTGCTACTACCTTGTA

TGACTTTGAAGGAAGCACACAAGGGTGGCATGGAAGCAACGTGACCGGTG

GCCCTTGGTCCGTAACAGAATGGGGTGCTTCAGGTAACTACTCTTTAAAGC

CGATGTAAATTTAACCTCAAATTCTTCACATGAACTGTATAGTGAACAAAGTC

GTAATCTACACGGATACTCTCAGCTCAACGCAACCGTTCGCCATGCCAATTG

GGGAAATCCCGGTAATGGCATGAATGCAAGACTTTACGTGAAAACGGGCTC

TGATIATACATGGCATAGCGGTCCTTTTACACGTATCAATAGCTCCAACTCA

GGAACAACGTTATCTTTTGATTTAAACAACATCGAAAATATCATCATGTTAGG

GAAATAG

Sequence identification number 4

Sequence characteristics: Length: 468 amino acids Type: amino acid Topology: linear

Molecule type: protein

Sequence description: sequence identification number 4

MKKKLSQfYHLIICTLIISVGIMGITTSPSAASTGFYVDGNTLYDANGQPFVMRGIN

HGHAWYKDTASTAiPAIAEQGANTIRIVLSDGGQWEKDDIDTIREVIELAEQNKM

VAWEVHDATGRDSRSDLNRAVDYWIEMKDALIGKEDTVIINIANEWYGSWDGS

AWADGYIDVIPKLRDAGLTHTLMVDAAGWGQYPQSIHDYGQDVFNADPLKNTM

FSIHMYEYAGGDANTVRSNIDRVÍDQDLALVIGE FGHRHTDGDVDEDTILS YSEE

TGTGWLAWSWKGNSTEWDYLDLSEDWAGQHLTDWGNRIVHGADGLQETSKP

STVFTDDNGGHPEPPTATTLYDFEGSTQGWHGSNVTGGPWSVTEWGASGNY

SLKADVNLTSNSSHELYSEQSRNLHGYSQLNATVRHANWGNPGNGMNARLYV

KTGSDYTWHSGPFTRINSSNSGTTLSFDLNNIENIIMLGK ··

Μ · · 4 ♦ 9 9 9 9 • · · ···· · · · · • 4444 · 9 9 9999 9 9 9 9 9

9 9 9 9 9 9 9 9

124 ··· · ·· * ·· 44

Sequence identification number 5

Sequence characteristics:

Length: 1029 base pairs Type: nucleic acid Stranding: simple Topology: linear

Molecule type: gene DNA

Sequence description: sequence identification number 5

5' AAT TGG CGC ATA CTG TGT CGC CTG TGA ATC CTA ATG CCC AGC AGA

CAA CGG CAA AAA AAA ACA CAG GAG TGA TCC TGA ACT GGC GAG TTG CGT CGC TCG ACC GAG TGC GTT ČGA ACA ACC GCC GAA ATG TTT CCG ACA CAT TTT CTA TGG CTG AGG CTG ATA GAA TCC GAA GCG CCA CCG GGC AAT CGC CTG CTA TTT ATG GCT GCG ATT ATG CCA GAG GAT GGC TTG AAA CAG CAA ATA TTG AAG ATT CAA TAG ATG TAA GCT GCA ACG GCG ATT TAA TGT CGT ATT GGA AAA ATG GCG GAA TTC CGC AAA TCA GTT TGC ACC TGG CGA ACC CTG CTT TTC AGT CAG GGC ATT TTA AAA CAC CGA TTA CAA ATG ATC AGT ATA AAA ACA TAT TAG ATT CAG CAA CAG CGG AAG GGA AGC GGC TAA ATG CCA TGC TCA GCA TAA TTG CTG ACG GAC TTC AAG AGT TGG AGA ACC AAG GTG TGC CTG TTC TGT TCA GGC CGC TGC ATG AAA TGA ACG GCG AAT GGT TTT GGT GGG GAC TCA CAT CAT ATA ACC AAA AGG ATA ATG AAA GAA TCT CTC TAT ATA AAC AGC TCT ACA AGA AAA TCT ATC ATT ATA TGA CCG ACA CAA GAG GAC TTG ATC ATT TGA TTT GGG TTT ACT CTC CCG ACG CCA ACC GAG ATT TTA AAA CTG ATT TTT ACC CGG GCG CGT CTT ACG TGG ATA TTG TCG GAT TAG ATG CGT ATT TTC AAG ATG CCT ACT CGA TCA ATG GAT ACG ATC AGC TAA CAG CGC TTA ATA AAC CAT TTG CTT TTA CAG AAG TCG GCC CGC AAA CAG CAA ACG GCA GCT TCG ATT ACA GCC TGT TCA TCA ATG CAA TAA AAC AAA AAT ATC CTA AAA CCA TTT ACT TTC TGG CAT

GGA ATG ATG AAT GGA GCG CAG CAG TAA ACA AGG GTG CTT CAG CTT *

♦ · 9 *

9 9 ·

9 9 9

9 9 9

99

125

TAT ATC ATG ACA GCT GGA CAC TCA ACA AGG GAG AAA TAT GGA ATG GTG ATT CTT TAA CGC CAA TCG TTG AGT GAA TCC GGG ATC 3'

Sequence identification number 6

Sequence characteristics:

Length: 363 amino acids

Type: amino acid

Topology: linear

Molecule type: protein

Sequence description: sequence identification number 6 ydhT 1

LFKKHTISLLIIFLLASAVLAKPIEAHTVSPVNPNAQQTTKTVMNWLAHL 50 ydhT 51

PNRTENRVLSGAFGGYSHDTFSMAEADRIRSATGOSPAIYGCDYARGWLE 100 ydhT 101

TANIEDSIDVSCNGDLMSYWKNGGIPQISLHLANPAFOSGHFKTPITNDQ 150 ydhT 151

YKNILDSATAEGKRLNAMLSKIADGLQELENQGVPVLFRPLHEMNGEWFW 200 ydhT 201

WGLTSYNQKDNERISLYKOLYKKIYHYMTDTRGLDHLIWVYSPDANRDFK 250 ydhT 251

TDFYPGASYVDIVGLDAYFQDAYSINGYDQLTALNKPFAFTEVGPOTANG 300 ydhT 301

SFDYSLFINAIKQKYPKTIYFLAWNDEWSAAVNKGASALYHDSWTLNKGE 350 ydhT 351

IWNGDSLTPIVE*. 363

126 • · · · ···· * · · · · · 9 9 9 9

9999 9 9 9 9999 99 99 9

9 9 9 9 9 9 9 • · ·· · 99 99

Industrial applicability

The compositions of the present invention are industrially useful in the manufacture of detergents intended for cleaning fabrics and/or removing stains from fabrics and/or bleaching fabrics and/or softening fabrics and/or caring for the color appearance of fabrics and/or inhibiting the transfer of dyes from fabrics.

Claims (17)

PATENT CLAIMS A laundry detergent composition comprising a detergent component, a carbohydrate-degrading enzyme, which degrades non-starch, non-cellulosic food polysaccharides having a viscosity of more than 800 cPs in a 1% solution by weight. A detergent detergent composition according to claim 1, wherein the polysaccharide is selected from agar, algin, karawa, tragacanth, guar gum, locust beans, xanthan and / or mixtures thereof. Washing detergent composition according to claims 1 to 2, characterized in that the carbohydrate gum degrading enzyme is selected from mannosidase, in particular β-mannosidase, endo-1,4-β-D-mannosidase, endo-1,2-h-D-mannosidase; galactosidases, in particular exo-1,3-β-D-mannosidases, exo-1, δ-β-D-galactosidase and 1,3-β-D-galactosidase; glucuronidases, glucuronosidases, exo-1,2 or 1,4-glucuronidases; arabinases, in particular endo-a-1,5-arabinosidases, exo-arabanases, exo-A- (a-1,2; a-1,3) arabinofuranosidases, exo-B- (a-1,3; a-1 5) arabinofuranosidases; xanthanlyases, lyases (α-L-guluconate); agarases, carageenases and / or mixtures thereof. A detergent detergent composition according to claims 1 to 3 wherein the carbohydrate gum degrading enzyme is β-mannosidase (EC 3.2.1.78-mannanase). Washing detergent composition according to claims 1 to 4, characterized in that the carbohydrate-degrading enzyme is present in an amount of 0.0001% by weight to 2% by weight, preferably 0.0005% by weight to 0.1% by weight, even more preferably 0.0006% to 0.015% by weight of pure enzyme based on the total composition. r 128 • 44 4 4 4 4 4 4 4 4 4 4 4 4 4 4 44 44 44 44 «· ··· · ·· · ·· A detergent detergent composition according to claims 1 to 6, further comprising a surfactant selected from a nonionic and anionic surfactant, a cationic surfactant and / or mixtures thereof. A laundry detergent composition according to any one of the preceding claims, further comprising a further enzyme, preferably cellulase and / or amylase. A laundry detergent composition according to any one of the preceding claims, further comprising a builder, preferably an inorganic builder, more preferably a builder selected from zeolite A, layered silicates, sodium tripolyphosphate and / or mixtures thereof. A laundry detergent composition according to any preceding claim, further comprising an activated bleach system. A laundry detergent composition according to any one of the preceding claims, wherein the composition is in liquid form, in the form of a paste, gel, rod, tablet, spray, foam, powder or granules. A laundry detergent composition in gel form according to claim 10, comprising from 15% to 40% by weight of an anionic surfactant component comprising: (i) 5 to 25% by weight of alkyl polyethoxylate sulfates in which the alkyl consists of 10 to 22 carbon atoms and the polyethoxylate chain consists of 0.5 to 15, preferably 0.5 to 5 and even more preferably 0.5 to 4 , ethylene oxide units and (ii) from 5% to 20% by weight of fatty acids. 12. A detergent additive comprising a carbohydrate-degrading enzyme. 13. A fabric softening composition comprising a carbohydrate-degrading enzyme, 129 • · ··· · Τ] / <Όλ • · The enzyme degrades non-starch, non-cellulosic food polysaccharides with a viscosity of more than 800 cPs in a 1% solution by weight, and further contains a cationic surfactant containing two long chain lengths. Use of a carbohydrate-rubber degrading enzyme, wherein the enzyme degrades non-starch, non-cellulosic food polysaccharides with a viscosity of more than 800 cPs in a 1% by weight solution, in a laundry detergent for cleaning fabrics and / or removing stains from the fabric and / or bleaching fabrics and / or softening fabrics and / or caring for the color appearance of the fabric and / or dampening the transfer of dyes from the fabric. Use of a carbohydrate gum degrading enzyme according to claim 14 for removing non-starch, 1 non-cellulosic food polysaccharides having a viscosity of more than 800 cPs in a 1% solution, expressed as a percentage by weight. Use of a carbohydrate gum degrading enzyme according to claims 14 to 15, wherein the polysaccharide is selected from agar, algin, karawa, tragacanth, guar gum, locust beans, xanthan and / or mixtures thereof. Use of a carbohydrate gum degrading enzyme according to claims 14 to 16 and cellulase for the removal of non-starch food polysaccharides having a viscosity of more than 800 cPs in a 1% by weight solution.