CZ2000506A3 - Laundry detergent composition and / or fabric care composition and fabric cleaning method - 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

Laundry detergent and/or fabric care composition and method for cleaning fabrics

Technical field

The present invention relates to a laundry detergent composition and/or fabric care composition comprising mannanase and clay. The present invention also relates to a method of cleaning fabrics using said laundry detergent composition and/or fabric care composition.

State of the art

The performance of a detergent product is judged by numerous factors, including the ability to remove soil and the ability to prevent the redeposition of soil or soil breakdown products on the articles being washed. Therefore, detergent compositions today contain a complex combination of active ingredients that meet certain specific needs. Current detergent compositions typically include surfactants and detergent enzymes that provide fabric cleaning and fabric care. In addition, the need for detergent compositions that not only exhibit good cleaning properties but also have fabric softening and other fabric care properties is well recognized in the art.

In current detergent and fabric care compositions, softening clays are commonly used to provide a soft feel. European patent application A177 165 describes the use of softening clays together with cellulase in detergent compositions. European patent application A 495 258 describes detergent compositions which comprise a surfactant, a builder system and softening clays and cellulase.

Food and cosmetic stains/stains represent a major part of customer-related stains/stains and often contain food additives such as thickeners/stabilizers. Hydrocolloid gums and emulsifiers are often used as food additives. The term gum refers to a group of industrially useful polysaccharides (long-chain polymers) or their derivatives that hydrate in hot or cold water to form viscous solutions, dispersions or gels. Gums are classified as natural and modified. Natural gums include extracts from marine seeds, products secreted by plants, resins from seeds or roots, and resins obtained by microbial fermentation. Modified (semisynthetic) gums include cellulose and starch derivatives and some synthetic gums such as low-methoxylated pectin, propylene glycol alginate, and carboxymethyl and hydroxypropyl guar gum (Gums in Encyclopedia of Chemical Technology, 4th ed., 12, pp. 842-862, J. Baird, Kelco division of Merck). See also Carbohydrate Chemistry for Food Scientists (Eagan Press, 1997) by R.L. Whistler and J.N. BeMiller, chap. 4, pp. 63-89, and Direct Food Additives in Fruit Processing by P. Laszlo, Bioprinciples and Applications 1, chap. II, pp. 313-325 (1996), Technomie publishing. Some of these gums, such as guar gum (E412), baobab (E410), are widely used alone or in combinations in many food applications (Gums in Encyclopedia of Chemical Technology, 4th edition, 12, pages 842 to 862, J. Baird, Kelco division of Merck).

Guar gum, used in these food and cosmetic colorants, is obtained from the endosperm seeds of the legume plant Cyamopsis tetragonoloba. Guar gum (also called guaran) extracted from the dicotyledonous seed consists of a 1-4,b-D-mannopyranosyl backbone unit and is used as a thickening agent in flavorings and in frozen products and cosmetics (H.-D. Belitz Food Chemistry, p. 243, English version second edition, Springer-verlag, 1987, ISBN 0-387-15043-9 (USA)) {Carbohydrate Chemistry for Food Scientists, R.L. Wistler, Eagan Press, 1997, ISBN 0-913250-92-9) and {Industrial Gum, second edition, R. L. Whistler, p. 308, Academie Press, 1973, ISBN, 0-12-74-6252-x). Locust bean gum (also called locust bean gum or St. John's wort) is also used in the food industry and is extracted from the seeds of an evergreen plant grown in the Mediterranean region. Locust bean gum probably differs in structure from guar gum only by a small number of D-galactosyl side chains • 9 • 9 9 9

9 9 9 9 9 9 • · 999 ·· 9 chains and has the same 1-4,b-D-mannopyranosyl backbone. In legume seeds, water-soluble galactomannan is the main storage carbohydrate, sometimes accounting for up to 20% by weight of the total dry weight. Galactomannan has α-galactose attached to 0-6 mannose residues and may also be acetylated to varying degrees at 0-2 and 0-3 mannose residues.

However, clay is not compatible with some of the hydrocolloid gums contained in these food and cosmetic colorants. It is reported in the art that guar gum induces clay precipitation due to its potential to coagulate sludge or clay particles (see Industrial Gum, 2nd ed., R.L. Whistler, p. 307, Academic Press, 1973, ISBN, 0-12-74-6252-x).

The object of the present invention is therefore to provide laundry detergent compositions and/or fabric care compositions which exhibit optimal softening performance and provide optimal stain removal and cleaning performance, in particular of cosmetic and food stains.

The above objects are met by a formulation of laundry detergent compositions and/or fabric care compositions which comprises a mannanase enzyme and clay as a fabric softening agent.

It has further been found that the performance of the detergent compositions of the present invention is enhanced by the addition of laundry detergent compositions and/or fabric care compositions selected from a builder, a cellulase and/or a cationic surfactant.

Mannanases have been identified in several Bacillus organisms. For example, Talbot et al.: Appl. Environ. Microbiol. 1990, 56(11), 3505-3510, describe a β-mannanase derived from Bacillus stearothermophilus in a dimer with a molecular weight of 162,000 and a pH optimum of 5.5-7.5. Mendoza et al.: World J. Microbiol. Biotech. 1994, 10(5), 551-555, describes a β-mannanase derived from Bacillus Subtilis with

with a molecular weight of 38,000, an activity optimum of pH 5.0/55 °C and a pl of 4.8. Japanese patent 0304706 describes a β-mannanase derived from Bacillus sp. with a molecular weight of 37,000 ± 3,000 measured by gel filtration, an activity optimum of pH 8 to 10 and a pl of 5.3 to

5.4. Japanese Patent 63056289 describes the production of an alkaline, thermostable β-mannanase which hydrolyzes β-1,4-D-mannopyranoside bonds of e.g. mannans to yield manno:oligo:saccharides. Japanese Patent 63036774 relates to the Bacillus microorganism FERM P-8856 which produces β-mannanase and β-mannosidase at alkaline pH. A purified mannanase from Bacillus amyloliguefaciens and a process for its production which is useful in the bleaching of pulp and paper is described in WO 97/11164. WO 91/18974 describes a hemicellulase such as a glucanase, xylanase or mannanase active at extreme pH and temperature and the production thereof. WO 94/25576 describes an enzyme having mannanase activity derived from Aspergillus aculeatus CBS 101.43 which can be used for various purposes where degradation or modification of plant or algal cell wall material is desired. WO 93/24622 describes a mannanase isolated from Trichoderma reesia for bleaching lignocellulosic pulp.

However, until now, a synergistic combination of mannanase and clay for optimal fabric softening performance and for optimal stain removal and cleaning performance, particularly of cosmetic and food stains, in laundry detergent compositions and/or fabric care compositions has never been described before.

The essence of the invention

The present invention relates to laundry detergent compositions and/or fabric care compositions containing mannanase and clay to achieve optimal softening performance and to achieve optimal stain removal and cleaning performance, particularly of cosmetic and food stains.

It is known in the art that clay is incompatible with some hydrocolloid gums, such as those found in food and cosmetic stains. Guar gum has been reported to induce clay precipitation due to its potential to coagulate sludge or clay particles (see Industrial Gum, 2nd ed., R.L. Whistler, p. 307, Academic Press, 1973, ISBN, 0-12-74-6252-x).

Without wishing to be bound by theory, it has been found that the flocculated clay has a reduced softening effect. In addition, it has been observed that clay and soil from the wash load redeposit on the guar gum residues and cause soiling by stains. Surprisingly, it has been found that the mannanase enzyme hydrolyzes hydrocolloid gums, in particular guar gum. This enzymatic hydrolysis results in the absence of guar gum in the wash. Thus, no flocculation of the clay occurs and the clay retains its full fabric care potential. In addition, no guar gum residues remain on the fabric and thus no redeposition of clay and soil occurs in the next wash load and thus no staining by stains.

Mannanase enzyme: An essential element of the laundry detergent compositions and/or fabric care compositions of the present invention is the mannanase enzyme.

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-B-mannosidase, hereinafter referred to as mannanase, and EC 3.2.1.100: 1,4B-mannobiosidase (IUPAC Classification - Enzyme Nomenclature, 1992 ISBN 0-12-227165-3, Academie Press).

The laundry detergents and/or fabric care compositions of the present invention more preferably comprise β-1,4-mannosidase (E.C. 3.2.1.78) referred to as mannanase. The term mannanase or galactomannanase refers to a mannanase enzyme defined in the art with the official name mannan endo-1,4-beta-mannosidase and with the alternative names beta-mannanase and endo-1,4-mannanase and catalyzes the following reaction: the random hydrolysis of 1,4-beta-D-mannosidic bonds in mannans, galactomannans, glucomannans and galactoglucomannans.

Specifically, mannanases (EC 3.2.1.78) represent a group of polysaccharides that degrade mannans and are enzymes that are able to cleave polysaccharide chains containing mannose units, i.e. they are able to cleave glycosidic bonds in mannans, glucomannans, galactomannans and galactoglucomannans. Mannans are polysaccharides that have a basic skeleton that is composed of β-1,4-linked mannose, glucomannans are polysaccharides that have a basic skeleton that is composed of more or less regularly repeating β-1,4-linked mannose and glucose, galactomannans and galactoglucomannans are mannans and glucomannans with α-1,6-linked galactose side branches. These compounds can be acetylated.

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

Mannanases have been identified in several Bacillus organisms. For example, Talbot et al.: Appl. Environ. Microbiol. 1990, 56(77), 3505-3510, describes a β-mannanase derived from Bacillus steaiOthermophilus in a dimer with a molecular weight of 162,000 and a pH optimum of 5.5-7.5. Mendoza et al.: World J. Microbiol. Biotech. 1994, 10(5), 551-555, describes a β-mannanase derived from Bacillus subtilis with a molecular weight of 38,000, an activity optimum at pH 5.0/55°C and a pI of 4.8. Japanese Patent 0304706 describes a β-mannanase derived from Bacillus sp. with a molecular weight of 370,000 measured by gel filtration, an optimum pH of 8 to 10 and a pI of 5.3 to 5.4. Japanese patent 63056289 describes the production of an alkaline, thermostable β-mannanase which hydrolyzes the β-1,4-D-mannopyranoside bonds of e.g. mannans to yield manno-oligosaccharides. Japanese patent 63036774 relates to Bacillus tt ·· · · · · · · · · · · · · · · ·· Japanese Patent 08051975 describes alkaline beta-mannanases from alkalophilic Bacillus sp. AM-001. A purified mannanase from Bacillus amyloliguefaciens useful in the bleaching of pulp and paper and a process for its production are described in WO 97/11164. WO 91/18974 describes a hemicellulase such as a glucanase, xylanase or mannanase active at extreme pH and temperature. WO 94/25576 describes an enzyme from Aspergillus aculeatus CBS 101.43 which exhibits mannanase activity and which can be used for the degradation or modification of plant or algal cell wall material. WO 93/24622 describes a mannanase isolated from Trichoderma reseei useful for the bleaching of lignocellulosic pulp. A hemicellulase which is capable of degrading hemicellulose containing mannan is described in WO 91/18974. Purified mannanase from Bacillus amyloliquefaciens is described in WO97/11164.

The mannanase enzyme will in particular be an alkaline mannanase as defined below, most preferably a mannanase derived from a bacterial source. In particular, the laundry detergent composition of the present invention will comprise an alkaline mannanase selected from mannanases from Bacillus agaradherens strain and/or Bacillus subtilisis strain 168, gene yght.

The term alkaline mannanase enzyme is intended to include an enzyme that has an enzymatic activity of at least 10, preferably at least 25, more preferably at least 40% of its maximum activity at a given pH in the range of from 7 to 12, preferably from 7.5 to 10.5.

Most preferably, the laundry detergent compositions and/or fabric care compositions of the present invention will comprise an alkaline mannanase from Bacillus agaradherens. Said mannanase means

i) a polypeptide produced by Bacillus agaradherens, NCIMB 40482, or ii) a polypeptide comprising the amino acid sequence as set forth in positions up to 343 of SEQ ID NO: 2

9 4 4 4 4

4 4 4

4 4 ·

4 4 4 4

9 9 9 4 4

9 99 99

9 « · 4 4 4

4 4 4 4 4

MKKKLSQIYHLIICTLIISVGIMGITTSPSAASTGFYVDGNTLYDANGQPFVMRGIN

HGHAWYKDTASTAIPAIAEQGANTIRIVLSDGGQWEKDDIDTIREVIELAEQNKM

VAWEVHDATGRDSRSDLNRAVDYWÍEMKDALIGKEDTVIINIANEWYGSWDGS

AlVADGYlDVIPKLRDAGLTHTLMVDAAGWGQYPQSIHDYGQDVFNADPLKNTM

FSIMHYEYAGGDANTVRSNIDRVIDQDLALVIGEFGHRHTDGDVDEDTILSYSEE

T3TGWLAWSWKGNSTEWDYLDLSE9WAGQHLTDWGNRIVHGADGLQETSKP

3TVFTDDNGGHPEPPTATTLYDFEGSTQGWHGSNVTGGPWSVTEWGASGNY

8L.KADVNLTSNSSHELYSEQSRNLHGYSQLNATVRHANWGNPGNGMNARLYV

KVGSDYTWHSGPFTRINSSNGTTLSFDLNNIENSHHVREIGVQFSAADNSSGQ

TALYVDNVTLR (SEQ ID NO: 2) or iii) an analogue of the polypeptide defined in i) or ii), which is at least 70% homologous to said polypeptide or which is derived from said polypeptide by the substitution, deletion or addition of one or more amino acids or which is immunologically reactive with a polyclonal antibody against said polypeptide in purified 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 a nucleotide sequence as set forth in SEQ ID NO: 1

ATGAAAAAAAAGTTATCACAGATTTATCATTTAATTATTTGCACACTTATAATA

AGTGTGGGAATAATGGGGATTACAACGTCCCCATCAGCAGCAAGTACAGGC

TTTTATGTTGATGGCAATACGTTATATGACGCAAATGGGCAGCCATTTGTCAT

GAGAGGTATTAACCATGGACATGCTTGGTATAAAGACACCGCTTCAACAGCT

ATTCCTGCCATTGCAGAGCAAGGCGCCAACACGATTCGTATTGTTTTATCAG

AND YGGCGGTCAATGGAAAAAGACGACATTGACACCATTCGTGAAGTCATTG

AGCTTGCGGAGCAAAATAAAATGGTGGCTGTCGTTGAAGTTCATGATGCCA

CGGGTCGCGATTCGGCAGTGATT! AAATCGAGCCGTTGATTATTGGATAG

A/áATGAAAGATGCGCTTATCGGTAAaGAAGATACGGTTATTATTAACATTGCA • 9 · · 9 9 · · · · · • · ·· ·· · 9 9 9 *

99 999 9 999 99 9

9999 9 9 · 9 9999

99 99 99 99 99

AACGAGTGGTATGGGAGTTGGGATGGCTCAGCTTGGGCCGATGGCTATATT

GATGTCATTCCGAAGCTTCGCGATGCCGGCTTAACACACACCTTAATGGTTG

ArGCAGCAGGATGGGGGCAATATCCGCAATCTATTCATGATTACGGACAAG

ATGTGTTTAATGCAGATCCGTTAAAAAATACGATGTTCTCCATCCATATGTAT

GAGTATGCTGGTGGTGATGCTAACACTGTTAGAICAAATATTGATAGAGTCA

TčGATCAAGACCTTGCTCTCGTAATAGGTGAATTCGGTCATAGACATACTGA

TGGTGATGTTGATGAAGATACAATCCTTAGTTATTCTGAAGAAACTGGCACA

GGGTGGCTCGCTTGGTCTTGGAAAGGCAACAGTACCGAATGGGACTATTTA

GACCTTTCAGAAGACTGGGCTGGTCAACATTTAACTGATTGGGGGAATAGAA

TTGTCCACGGGGCCGATGGCTTACAGGAAACCTCCAAACCATCCACCGTAT rrACAGATGATAACGGTGGTCACCCTGAACCGCCAACTGCTACTACCTTGTA

TGACTTTGAAGGAAGCACACAAGGGTGGCATGGAAGCAACGTGACCGGTG

GCCCTTGGTCCGTAACAGAATGGGGTGCTTCAGGTAACTACTCTTTAAAGC

C GATGTAAATTTAACCTC AAATTCTí CACATGAACTGTATAGTGAACAAAGTC gtaatctacacggatactctcagctcaacgcaaccgttcGccatgccaattg

GGGAAATCCCGGTAATGGCATGAATGCAAGACTTTACGTGAAAACGGGCTC

TGATTATACATGGCATAGCGGTCCTTTTACACGTATCAATAGCTCCAACTCA

GGAACAACGTTATCTTTTGATTTAAACAACATCGAAAATAGTCATCATGTTAG

GGAAATAGGCGTGCAATTTTCAGCGGCAGATAATAGCAGTGGTCAAACTGC

TOTATACGTTGATAACGTTACTTTAAGATAG (SEQ ID NO: 1) from nucleotide 97 to nucleotide 1029,

b) molecules homologous to ad a),

c) polynucleotide molecules that encode a polypeptide that has mannanase activity and that is at least 70% identical to the amino acid sequence of SEQ ID NO: 6.2 from amino acid residue 32 to amino acid residue 343,

d) molecules complementary to a), b) or c) and

e) degenerate nucleotide sequences of a), b), c) or d).

Plasmid pSJ1678, which contains a polynucleotide molecule (DNA sequence) encoding a mannanase according to the present invention, was transformed into strain ·· · · · · · · · · · · 9 9

9 9 9 9 9 9 9 9 9 9

9 99 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 9 9 9 9

99 99 99 9· 99

Escherichia coli, which was deposited by the inventor under the Budapest Agreement on the International Recognition of Deposited Microorganisms for the Purposes of Patent Procedure at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig, Germany, on May 18, 1998, under the deposit number DSM 12180.

The second most preferred enzyme is mannanase from Bacillus subtilisis strain 168, which

i) is encoded by the coding portion of the DNA sequence set forth in SEQ ID NO: 5

AAT TGG CGC ATA CTG TGT CGC CTG TGA ATC CTA ATG CCC AGC AGA CAA CAA AAA CAG TGA TGA ACT GGC TTG CGC ACC TGC CGA ACC GAA CGG AAA ACA GAG TCC TTT CCG GAG CGT TCG GAG GTT ACA GCC ATG 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 AAA 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 GíAC 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 G'!T 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' (seq. id. no. 5) ·· ·· • · « · · · · ···· • · ·· * · « • ·· ··· · ··· ·· 9 • · · · · · · 9 · ·· · ·* ·· »« ·· ** ·· or an analog of the indicated sequence and/or ii) means a polypeptide containing the amino acid sequence as indicated in the seq. id. C. 6 ______ ____________ ydhT 1

Ll ^: KKHTISLLIIFLLASAVLAKPIEAHTVSPVNPNAQQTTKTVMNWLAHL 50 ydhT 51

PNRTENRVLSGAFGGYSHDTFSMAEADRIRSATGQSPAIYGCDYARGWLE 100 ydhT 101

TANIEDSIDVSCNGDLMSYWKNGGIPQISLHLANPAFQSGHFKTPITNDQ 150 ydhT 151

YKNILDSATAEGKRLNAMLSKIADGLQELENQGVPVLFRPLHEMNGEWFW 200 ydhT 201

WGLTSYNQKDNERISLYKQLYKKIYHYMTDTRGLDHLIWVYSPDANRDFK250 ydhT 251

TDFYPGASYVDIVGLDAYFQDAYSINGYDQLTALNKPFAFTEVGPQTANG 300 ydhT 301

SFDYSLFINAIKQKYPKTIYFLAWNDEV7SAAVNKGASALYHDSWTLNKGE 350 ydhT 351

IWNGDSLTPIVE*. 363 (SEQ ID NO: 6) or iii) means an analogue of a polypeptide as defined in ad ii) which is at least 70% homologous to said polypeptide or which is derived from said polypeptide by substitution, deletion or addition of one or more amino acids, or which is immunologically reactive with a polyclonal antibody against said polypeptide in purified form.

The present invention also includes an isolated polypeptide having mannanase activity selected from the group consisting of ·* ·· ·* ··«· 99 99 • » · * 9 9 9 9 9 9 9

9 99 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 9 9 9 9

99 99 99 99 99

a) polynucleotide molecules encoding a polypeptide having mannanase activity and comprising a nucleotide sequence as set forth in SEQ ID NO: 5,

b) molecules homologous to ad a),

c) polynucleotide molecules that encode a polypeptide that has mannanase activity and 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).

In the following section, definitions of certain terms will be provided before further discussion of the present invention.

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

The term paralog refers to a polypeptide or protein derived from a given species that is homologous to another polypeptide or protein from the same species.

The term expression vector means a DNA molecule, linear or circular, which contains a segment encoding the polypeptide of interest, operably linked to other segments which ensure its transcription. These other segments may include a promoter and a termination sequence and may optionally contain 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 may contain elements of both. An expression vector according to the invention may mean any expression vector which is suitably subjected to DNA recombination procedures. The choice of vector will often depend on the host cell into which the vector is to be introduced. Thus, a vector may mean an autonomously replicating vector, i.e. a vector which exists as an extrachromosomal particle whose replication is independent of chromosome replication, e.g. a plasmid. A vector may also mean

9« 9 9 «99« · 9 9 9

9 9 9 9 9 • 9 »9 • 9*9 99 · «9 9 9 • * * 9 9 9 9

9 9 9 9 *

99 99 •9 9*99

9 9 9 99 such a vector that, when introduced into a host cell, integrates into the host cell genome and replicates together with the chromosome(s) into which it is integrated.

The term recombinantly expressed or recombinantly expressed, as used herein in connection with the expression of a polypeptide or protein, is defined according to standard definitions in the art. Recombinant expression of a protein is typically accomplished using an expression vector as described immediately above.

The term isolated, when applied to a polynucleotide molecule, indicates that the polynucleotide has been removed from its natural genetic environment and is therefore free of other extraneous or unwanted coding sequences and is in a form suitable for use in genetically engineered protein production systems. These isolated molecules are those molecules that are separated from their natural environment and include cDNA and genomic clones. 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' untranslated regions, such as promoters and terminators. Identification of associated regions will be apparent to one skilled in the art (see, for example, Dynan and Tijan: Nature 1985, 376, 774-778).

The term isolated polynucleotide may also be referred to as a cloned polynucleotide. When applied to a protein/polypeptide, the term isolated means that the protein is in a state other than its natural environment. In a preferred form, the isolated protein exists essentially free of other proteins, particularly free of other homologous proteins (i.e., homologous impurities (see below)). It is preferred to obtain the protein in greater than 40% pure form, more preferably greater than 60% pure form. Even more preferred is to obtain the protein which is in a highly pure form, i.e. greater than 80%, more preferably greater than 95%, and even more preferably greater than 99% (w/w) pure form, as determined by SDS-PAGE.

• · · ·· · 4 4 4 4

44 4 4 · 4 4 4 4

4 4 4 4 4 444 44 4 •4 4 4 44 4 44 4 44 4

44 44 44 44

The term isolated protein/polypeptide may alternatively also be referred to as purified protein/polypeptide.

The term homologous impurity refers to any impurity (e.g., a polypeptide other than a polypeptide of the invention) that originates from a homologous cell from which the polypeptide of the invention was originally obtained. The term obtained from, as used herein in connection with a specific microbial source, refers to a polynucleotide and/or polypeptide produced by a specific source or cell into which a gene from that source has been inserted.

The term operably linked, when applied to DNA segments, refers to the fact that the segments are arranged so that they function in accordance with their intended purposes, e.g., transcription begins at a promoter and proceeds through the coding segment to a terminator.

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

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

• · ·· 99 99 99 99

The term promoter refers to the part of a gene containing DNA sequences that ensure the binding of RNA polymerase and the initiation of transcription. Promoter sequences are usually, 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, directs the larger polypeptide through the secretory pathway of the cell, below which it is synthesized. The larger polypeptide is usually cleaved to remove the secretory peptide during transport through the secretory pathway.

How to use the sequence of the invention to obtain other related sequences: Information about the disclosed sequences of the invention, which relate to the polynucleotide sequence encoding the mannanase of the invention, can be used as a tool to identify other homologous mannanases. For example, polymerase chain reaction (PCR) can be used to amplify sequences encoding other homologous mannanases from various microbial sources, particularly various Bacillus species.

Activity Assay: A polypeptide of the invention having mannanase activity can be assayed for mannanase activity according to standard assay procedures known in the art, such as applying the solution to be assayed to 4 mm diameter holes punched into agar plates containing 0.2% (w/w) AZCL galactomannan (carob), i.e., an endo-1,4-beta-D-mannanase assay substrate available as Cat. No. I-AZGMA from Megazyme at a price of US$110.00 for 3 grams (Megazyme website address: http://www.megazyme.com/Purchase/index.html).

Polynucleotides: An isolated polynucleotide of the invention will hybridize to regions of SEQ ID NO: 1 of similar size or to a sequence complementary thereto under at least mild selective conditions.

• ·

99 9

The polynucleotides of the invention will in particular hybridize to a denatured double-stranded DNA probe comprising either the full sequence set forth between positions 97 and 1029 of SEQ ID NO:1 above or any probe comprising subsequences of SEQ ID NO:1 of at least 100 nucleotide pairs in length under at least mildly selective conditions, but preferably under highly selective conditions, as described in detail below. Suitable experimental conditions for determining hybridization with moderate or high selectivity between a nucleotide probe and a homologous DNA or RNA sequence include pre-soaking the filter containing the DNA fragments or RNA to be hybridized in 5 x SSC (sodium chloride/sodium citrate, Sambrook et al. 1989) for 10 minutes and pre-hybridizing the filter in a solution of 5 x SSC, 5 x Denhardt's solution (Sambrook et al. 1989), 0.5% (w/w) SDS and 100 pg/ml denatured sonicated salmon sperm DNA (Sambrook et al. 1989), followed by hybridization in the same solution containing a concentration of 10 ng/ml of random primer (Freiberg A.P. and Vogelstein B.: Anal. Biochem. 1983, 132, 6-13.), 32P-dCTP-labeled (specific activity greater than 1.109 counts per minute/pg) probe for 10 min. 12 hours at 45°C. The filter is then washed twice for 30 minutes in 2 x SSC, 0.5% SDS at at least 60°C (moderate selectivity), more preferably at least 65°C (moderate/high selectivity), more preferably at least 70°C (high selectivity), and more preferably at least 75°C (very high selectivity).

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

As mentioned above, 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 the genes in question can be cloned in gene banks or DNA libraries by methods known in the art.

Polynucleotides encoding polypeptides having mannanase activity of the invention are then identified and isolated, for example by hybridization or PCR. The present invention further provides counterpart polypeptides and polynucleotides from various bacterial

strains (orthologies or paralogs). Of particular interest are mannanase polypeptides from gram-positive alkalophilic strains, including Bacillus species.

Species homologs of the polypeptide having mannanase activity of the invention can be cloned using the information and means provided by the present invention in combination with conventional cloning techniques. For example, DNA sequences of the present invention can be cloned using chromosomal DNA obtained from a cell type that expresses the protein. Suitable DNA sources can be identified by probing Northern blots with probes that are designed according to the sequences described in this invention. A library is then prepared from the chromosomal DNA of the positive cell line. DNA sequences of the invention encoding the polypeptide having mannanase activity can then be isolated by a variety of methods, such as probing with probes designed according to the sequences described in the present specification and claims or with one or more sets of degenerate probes based on the sequences described. DNA sequences of the invention can also be cloned by polymerase chain reaction or PCR (Mullis: U.S. Patent 4,683,202) using primers designed according to the sequences described in this invention. Among other methods, a DNA library can be used to transform or transfect host cells, and expression of the DNA in question can be detected by an antibody (monoclonal or polyclonal) against a 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 involving a polypeptide having mannanase activity.

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

···· ·· ·· ♦ · ··

An analogous sequence can also 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 linked SEQ ID NO: 1), e.g. as a subsequence thereof, and/or by introducing nucleotide substitutions that do not counteract another amino acid sequence of the mannanase encoded by the DNA sequence, but which corresponds to a codon of the host organism that is considered for the production of the enzyme, or by introducing nucleotide substitutions that may provide a different amino acid sequence (i.e. a variant of the mannan-degrading enzyme according to the invention).

Polypeptides; Amino acid sequence No. 32 to 343 of SEQ ID NO: 2 is a mature mannanase sequence.

The present invention also provides mannanase polypeptides that are substantially homologous to the polypeptide of SEQ ID NO:2 and its species homologs (paralogs or orthologs). The term substantially homologous is used herein 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 in amino acids 32 to 343 of SEQ ID NO:2 or orthologs or paralogs thereof. These polypeptides will more preferably be at least 95% identical, and most preferably 98% or more identical, to the sequence set forth in amino acids 32 to 343 of SEQ ID NO:2 or orthologs or paralogs thereof. Percent sequence identity is determined by conventional methods using computer programs known in the art as GAP, which are available 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) as described by Needleman S.B. and Wunsch C.D.: Journal of Molecular Biology 1970, 48, 443-453, which is incorporated herein by reference in its entirety. GAP is used with the following settings for polypeptide sequence comparison: GAP creation penalty 3.0 and GAP extension penalty 0.1.

> · · ( ► · · 4 • · ··

The sequence identity of polynucleotide molecules is determined in similar ways using GAP with the following settings for DNA sequence comparison: GAP creation penalty 5.0 and GAP extension penalty 0.3.

The enzyme preparation according to the invention is preferably derived from a microorganism, preferably a bacterium or a fungus, in particular a bacterium such as a bacterium belonging to the genus Bacillus, preferably an alkalophilic Bacillus strain, which may be selected from the group consisting of the species Bacillus agaradherens and closely related Bacillus species, all species being preferably at least 95%, even more preferably at least 98% homologous to Bacillus agaradherens according to aligned 16S rDNA sequences.

Substantially homologous proteins and polypeptides are characterized by having one or more amino acid substitutions, deletions, or additions. These changes are preferably minor in nature, i.e. conservative amino acid substitutions (see Table 2) and other substitutions that do not significantly affect the folding or activity of the protein or polypeptide, small deletions, typically one to about 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 about 20 to 25 residues, or small extensions that facilitate purification (affinity tag), such as a polyhistidine stretch, protein A (Nilsson et al.: EMBO J. 1985, 4, 1075; Nilsson et al.: Methods Enzymol. 1991, 198, 3. See generally Ford et al.: Protein Expression and Purification 1991, 2, 95-107, which are incorporated herein by reference. DNAs encoding affinity tags are available from commercial suppliers (e.g. Pharmacia Biotech, Piscataway, NJ.; New England Biolabs, Beverly, Ma.). While the changes described above are preferably of a minor nature, such changes can also be of a larger nature, such as the attachment of larger polypeptides of up to 300 amino acids or more as both amino- and carboxyl-terminal extensions to the mannanase polypeptide of the invention.

• 4 4444 • 4 44

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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, non-standard amino acids (such as 4-hydroxyproline, 6-N-methyllysine, 2-aminoisobutyric acid, isovaline, and α-methylserine) may be substituted for the amino acid residues of the polypeptide of the present invention. A limited number of non-conservative amino acids, amino acids not encoded by the genetic code, and unnatural amino acids may be substituted for the amino acid residues. Unnatural amino acids have also been modified after protein synthesis and/or have a chemical structure of their side chain(s) different from that 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 can be identified by techniques known in the art, such as site-directed mutagenesis or alanine-guided mutagenesis (Cunningham and Wells: Science 1989, 244, 1081-1085). In the latter method, individual alanine mutations are introduced at each residue in the molecule and the resulting mutated molecules are tested for biological activity (i.e., mannanase activity) to identify amino acid residues that are critical for the activity of the molecule. See also Hilton et al.: J. Biol. Chem. 1996, 271, 4699-4708.

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The active site of an enzyme or other biological interaction can also be determined by physical analysis of the structure, such as by methods such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with mutation of the putative amino acid contact site. See, for example, de Vos et al.: Science, 1992, 255, 306-312; Smith et al.: J. Mol. Biol. 1992, 224, 899-904; Wlodaver et al.: FEBS Lett. 1999, 309, 59-64. The identities of essential amino acids can also be inferred from homology analysis with polypeptides that are related to the polypeptides of the present invention.

Multiple amino acid substitutions can also be made and can be tested by known methods of mutagenesis, recombination and/or shuffling with subsequent evaluation procedures, such as those described by Reidhaar-Olson and Sauer (Science 1988, 241, 53-57.), Bowie and Sauer (Proc. Nat. Acad. Sci. USA 1989, 86, 2152-2156.), in WO 95/17413 or WO 95/22625. Briefly, these authors describe methods of simultaneously randomly changing two or more positions in a polypeptide or or recombining/shuffling different mutations (WO 95/17413, WO 95/22625), followed by selection of a functional polypeptide and then sequencing the mutagenized polypeptides to determine the spectrum of substitutions available at each position. Other methods that can be used include phage display (e.g., Lowman et al.: Biochem. 1991, 30,10832-10837; Ladner et al.: U.S. Patent No. 5,223,409; Huse: WIPO Publication WO 92/06204) and site-directed mutagenesis (Derbyshire et al.: Gene 1986, 46,145; Ner et al.: DNA 1988, 7,127).

Mutagenesis/shuffling methods as described above can be combined with high-throughput, automated assay methods for detecting the activity of cloned, mutagenized polypeptides in host cells. Mutagenized DNA molecules that encode active polypeptides can be isolated from host cells and rapidly sequenced using modern equipment. These methods allow rapid determination of the importance of individual amino acid residues in the polypeptide of interest and can be applied to polypeptides • · · · • ·· 9 ·· 99

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9 9 9 of unknown structure. Using the above methods, one skilled in the art can identify and/or prepare a variety of polypeptides that are substantially homologous to residues 32 to 343 of SEQ ID NO:2 and that retain the mannanase activity of the native protein.

Protein Production: The proteins and polypeptides of the present invention, including full-length proteins, fragments thereof, and fusion proteins, can be produced by genetically engineering constructed host cells according to conventional methods. Suitable host cells are those cell types that can be transformed or transfected with exogenous DNA and can be grown in culture. They include bacteria, fungal cells, and cultured higher eukaryotic cells. Bacterial cells, especially cultured cells of gram-positive organisms, are preferred. Gram-positive cells of the genus Bacillus are particularly preferred, such as cells from the group consisting 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, especially Bacillus agaradherens.

Techniques for manipulating cloned DNA molecules and introducing exogenous DNA into a variety of host cells are described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 1989, Ausubel et al. (eds.), in Current Protocols in Molecular Biology, John Wiley and Sons, Inc., N.Y., 1987, and in Bacillus subtilis and Other Gram-Positive Bacteria, Sonensheim et al., 1993, American Society for Microbiology, Washington D.C., which are incorporated herein by reference.

Generally, the DNA sequence encoding the mannanase of the present invention is operably linked to other genetic elements required for its expression, generally including a promoter and a transcription terminator in an expression vector. This vector will typically also contain one or more selectable markers and one or more origins of replication, although those skilled in the art will recognize that some ·♦»· • 9 9 9 9

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9f selectable marker systems can be obtained on separate vectors and replication of the exogenous DNA can be achieved by integration into the genome of the host cell. The selection of promoters, terminators, selectable markers, vectors and other elements is within the skill of one skilled in the art. Many of these elements are described in the literature and are available from commercial suppliers.

To direct polypeptides into the secretory pathway of the host cell, a secretory signal sequence, also known as a leader sequence, prepro sequence, or pre sequence, is provided in the expression vector. The secretory signal sequence may be a polypeptide or may be derived from another secreted protein or synthesized de novo. Numerous suitable secretory signal sequences are known in the art. For a further description of suitable secretory signal sequences, particularly for secretion in a Bacillus host cell, reference may be made to Bacillus subtilis and Other Gram-Positive Bacteria, Sonnensheim et al., 1993, American Society for Microbiology, Washington D.C., and Cutting S.M. (eds.): Molecular Biological Methods for Bacillus, John Wiley and Sons, 1990. The secretory signal sequence is linked to the DNA sequence in the correct reading frame. The secretory signal sequence is usually located 5' to the DNA sequence encoding the polypeptide of interest, although some signal sequences may be located elsewhere in the DNA sequence of interest (see, e.g., Welch et al.: U.S. Patent No. 5,037,743, Holland et al.: U.S. Patent No. 5,143,830).

Transformed or transfected cells are cultured by conventional methods in a culture medium that contains nutrients and other components required for the growth of the selected host cells. A variety of suitable media are known in the art, including defined media and complex media that generally contain a carbon source, a nitrogen source, essential amino acids, vitamins and minerals. The media may also contain components such as growth factors or serum, as desired. The growth medium will generally be selected from cells containing exogenously added DNA, for example, by drug selection or deficiency in an essential nutrient, which is supplemented with a selectable marker carried on an expression vector or co-transfected into the host cell.

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Protein Isolation: When the expressed recombinant polypeptide is secreted, it can be purified from the growth medium. The expression host cells are preferably removed from the medium before the polypeptide is purified (e.g., by centrifugation).

If the expressed recombinant polypeptide is not secreted from the host cell, the host cell is preferably disrupted and the polypeptide is released into an aqueous extract as the first step of this purification method. Preferably, the expressing host cells are isolated from the medium before the cells are disrupted (e.g., by centrifugation).

Cell disruption can be accomplished by conventional methods such as lysozyme cleavage or by subjecting the cells to high pressure. For further descriptions of cell disruption methods, see Robert K. Scobes, Protein Purification, Second Edition, Springer-Verlag.

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

Ammonium sulfate precipitation and acidic or chaotropic extraction can be used to fractionate samples. Examples of purification steps include hydroxyapatite, size exclusion chromatography, FPLC, and reversed-phase high-performance liquid chromatography. Suitable anion exchange media include derivatized dextrans, agarose, cellulose, polyacrylamide, silicates, and the like. PEI, DEAE, QAE, and Q derivatives are preferred, with DEAE fast-flowing sepharose (Pharmacia, Piscataway, NJ.) being particularly preferred. Examples of chromatographic media include media derivatized with a phenyl, butyl, or octyl group, such as Phenyl-Sepharose FF (Pharmacia), Toyopearl 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 carriers include • 9 99 99 9999 99 99

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99 999 9 999 99 9 •999 9999 9999 ·· ·· 99 ·· 99 99 glass beads, silica 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 supports may be modified with reactive groups that allow attachment of proteins via amino groups, carboxyl groups, mercaptan groups, hydroxyl groups, and/or carbohydrate groups. Examples of coupling chemistries include cyanogen bromide activation, N-hydroxysuccinimide activation, epoxide activation, mercaptan activation, hydrazide activation, and carboxyl and amine derivatives for carbodiimide coupling chemistries. These and other media are well known, widely used in the art, and available from commercial suppliers.

The selection of the appropriate method is a matter of routine and is partly determined by the properties of the chosen support. See, for example, Affinity ChrOmatography: Principles and Methods, Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988.

The polypeptides of the invention or fragments thereof may also be prepared by chemical synthesis. The polypeptides of the invention may be monomers or multimers, may be glycosylated or non-glycosylated, pegylated or non-pegylated, and may or may not contain an initial methionine amino acid residue.

Based on the sequence information described herein, a full-length DNA sequence encoding a mannanase according to the invention and comprising the DNA sequence set forth in SEQ ID NO: 1, at least the DNA sequence from position 97 to position 1029, can be cloned.

Cloning is performed by standard procedures known in the art, such as:

- preparation of a genomic library from a Bacillus strain, especially the B. agaradherens strain, NCIMB 40482,

44 • 4 4 • · 44 • 4 4 • 4 4

44

44' 4444 44 44 ·* 4 4 · 4 4 • · 4 4 4 4 4

4 444 44 4

44 4 4 4 4 4 «· ·· 44 44

- seeding this library onto suitable plates with substrate,

- identifying a clone containing the polynucleotide sequence of the invention by standard hybridization methods using a probe based on SEQ ID NO: 1 or

- identifying the colony from said Bacillus agaradherens NCIMB 40482 genomic library by an inverse PCR strategy using primers based on sequence information from SEQ ID NO: 1. Reference is made to M.J. McPherson et al. (PCR A practical approach, Information ress Ltd., Oxford, England) for further details regarding inverse PCR.

Based on the sequence information described herein (SEQ ID NO: 1 and SEQ ID NO: 2), it is a routine task for one skilled in the art to isolate homologous polynucleotide sequences encoding homologous mannanases of the invention by a similar strategy using genomic libraries from related microbial organisms, particularly genomic libraries from other strains of the genus Bacillus, such as alkalophilic Bacillus species.

DNA encoding a mannan or galactomannan-degrading enzyme of the invention may also be prepared by well-known methods, conveniently by cloning it from a suitable source, 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.

The polynucleotide molecule of the present invention can therefore be isolated from Escherichia coli, DSM 12180, in which the plasmid obtained by cloning as described above is inserted. 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 is intended to mean either a conventional enzymatic fermentation product, possibly isolated and purified, from a single species of microorganism, this preparation usually containing numerous different ·* ··♦· ·· ·*

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99 enzymatic activity, or means a mixture of single-component enzymes, preferably enzymes derived from bacterial or fungal species using conventional recombinant techniques, wherein these enzymes have been fermented and possibly isolated and purified separately and may originate from different species, preferably from fungal or bacterial species, or means a fermentation product of a microorganism which acts as a host cell for the expression of a recombinant mannanase, but in which the microorganism simultaneously produces other enzymes, e.g. pectin degrading enzymes, proteases or cellulases, and is a naturally occurring fermentation product of the microorganism, i.e. an enzyme complex conventionally produced by the corresponding naturally occurring microorganism.

A method for producing an enzyme preparation according to the invention, the method comprising culturing a microorganism, e.g. a wild-type strain, capable of producing mannanase under conditions permitting the production of the enzyme, and isolating the enzyme from the culture. The culturing may be carried out using conventional fermentation techniques, e.g. by culturing in a culture flask or in stirred fermentors to ensure sufficient aeration of the growth medium, which induces the production of the mannanase enzyme. The growth medium may contain a conventional nitrogen source, such as peptone, yeast extract or casamic acids, a reduced amount of a conventional carbon source, such as dextrose or sucrose, and an inducing agent, such as guar gum or acacia gum. The isolation may be carried out by conventional methods, e.g. by separating the biomass and supernatant by centrifugation or filtration, by isolating the supernatant or broken cells if the enzyme in question is intracellular, with possible subsequent further purification as described in European patent 0406 314, or by crystallization as described in WO 97/15660.

Immunological cross-reactivity: Polyclonal antibodies used for immunological cross-reactivity determination can be prepared using purified mannanase enzyme. In more detail, the antiserum against mannanase according to the invention can be obtained by immunizing rabbits (or other rodents) by the procedure φφ φ ΦΦΦ φ · • · φ φ φ φ φφ φ · φ φ ♦ φ · φφ φφ φφ φφ • · Φ V φ · φ φ • φ φ φ φ • · · 9

ΦΦΦ according to N. Axelsen et al. in: A Manual of Quantitative Immunoelectrophoresis, Blackwell Scientific Publications, 1973, chapter 23, or A. Johnston and R. Thorpe: Immunochemistry in Practice, Blackwell Scientific Publications, 1982 (for details, pp. 27 to 31). Purified immunoglobulins can be obtained from antisera, for example by precipitation with salts (ammonium sulfate) followed by dialysis and chromatography on an ion exchange, e.g. on DEAE-sephadex. Immunochemical characterization of proteins can be performed either by Ouchterlony double diffusion analysis (O. Ouchterlony in: Handbook of Experimental Immunology (D.M. Weir, ed.), Blackwell Scientific Publications, 1967, pp. 655-706) crossed immunoelectrophoresis (N. Axelsen et al.: see above, chapters 3 and 4) or rocket immunoelectrophoresis (N. Axelsen et al.: chapter 2).

Examples of useful bacteria producing the enzyme or enzyme preparation according to the invention are gram-positive bacteria, preferably from the Bacillus/Lactobacillus subdivision, preferably a strain of the genus Bacillus, more preferably a strain of Bacillus agaradherens, especially a strain of Bacillus agaradherens, NCIMB 40482.

The present invention includes an isolated mannanase that has the properties described above, is free from homologous impurities, and is produced by conventional recombinant methods.

Determination of catalytic activity (ManU) of mannanase: Colorimetric assay: Substrate: 0.2% (w/w) AZCL-galactomannan (Megazyme, Australia) from carob in 0.1M glycine buffer, pH 10.0. The assay is performed in a 1.5 ml Eppendorf microtube on a thermomixer with stirring and temperature control at 40 °C. Incubation, 20 minutes 0.750 ml substrate with 0.05 ml enzyme, was stopped by centrifugation at 15,000 rpm for 4 minutes. The color of the supernatant is measured at 600 nm in a 1 cm cuvette. One ManU (mannanase unit) provides 0.24 abs in 1 cm.

Obtaining Bacillus agaradherens mannanase NCIMB 40482: Strains: Bacillus agaradherens NCIMB 40482 contains a mannanase enzyme encoding DNA sequence.

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

6. 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.: Cloning of aldB, which encodes alpha-acetolactate-deccarboxylase, an exoenzyme from Bacillus brevis. J. Bacteriol. 1990, 172, 4315 to 4321.) disrupted in the transcription unit of the known Bacillus subtilis cellulase gene, resulting in cellulase-negative cells. The disruption is carried out essentially as described in the literature (A.L. Sonenshein, J.A. Hoch and Richard Losick (eds.): Bacillus subtilis and other Gram-Positive Bacteria, American Society for Microbiology, p. 618.).

The appropriate cells were prepared and transformed as described by Yasbin R.E., Wilson G.A. and Young F.E.: Transformation and transfection in lysogenic strains of Bacillus subtilis: evidence for selective induction of prophage in competent cells. J. Bacteriol. 1975,121,296 to 304.

Plasmids: Plasmid pSJ1678 (as described in detail in WO 94/19454, which is incorporated herein by reference in its entirety).

pMOL.944: This plasmid is a pUB110 derivative that essentially contains elements that make the plasmid replicable in Bacillus subtilis, a kanamycin resistance gene and a strong promoter and a signal peptide cloned from the amylL gene of B. licheniformis ATCC 14580. The signal peptide contains a SacII site, making it convenient to clone the DNA encoding the mature portion of the protein in a junction with the signal peptide. This results in the expression of a pre-protein that is directed outside the cell.

•Φ «ΦΦΦ • · φφφφ φφ φ φφφφ • ΦΦΦ φφ φ φφφφ φ φφ ΦΦΦ φ ΦΦΦ φφ φ

ΦΦΦ· · φ φ · φφφφ

ΦΦΦ· φφ φφ φφφφ

The plasmid was constructed by conventional genetic engineering methods, which are briefly described as follows.

Construction of pMOL944: Plasmid pUB110 (McKenzie T. et al.: Plasmid, 1968,

15, 93-103.) was digested with the unique restriction enzyme NciI. A PCR fragment amplified from the amyL promoter encoded on plasmid pDN1981 (P.J. Jorgensen et al.: Gene 1990, 96, 37-41.) was digested with NciI and inserted into pI1B110 digested with NciI to form plasmid pSJ2624.

The two primers used had the following sequences:

#LWN5494

5'-GTCGCCGGGGCGGCCGCTATCAATTGGTAACTGTATCTCAGC-3'a #LWN5495

5'-GTCGCCCGGGAGCTCTGATCAGGTACCAAGCTTGTCGACCTGCAGAATGAGG

CAGCAAGAAGAT-3'.

Primer# LWN5494 inserts the NotI site into the plasmid.

Plasmid pSJ2624 was then digested with SacI and NotI, and a new PCR fragment amplified at the amyL protomorph encoded on pDN1981 was digested with SacI and NotI. This DNA fragment was inserted into SacI-NotI digested pSJ2624 to form plasmid pSJ2670.

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

#LWN5938

5'-GTCGGCGGCCGCTGATCACGTACCAAGCTTGTCGACCTGCAGAATGAGGCA GCAAGAAGAT-3' and

4 4 4*4 *· • 4 · · · · • · 44 4 4 4

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44 44 44

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4 « *4 4 # LWN5939

5'-GTCGGAGCTCTATCAATTGGTAACTGTATCTCAGC-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 WO 95/26397, which is incorporated herein by reference in its entirety) was digested with PstI and BclI and inserted, yielding plasmid pMOL944. The two primers used for PCR amplification had the following sequences:

#LWN7864

5'-AACAGCTGATCACGACTGATCTmAGCTTGGCAC-3'a #LWN7901

5'-AACTGCAGCCGCGGCACATCATAATGGGACATGGG-3'.

Primer# LWN7901 inserts the SacII site into the plasmid.

Cloning of the mannanase gene from Bacillus agaradherens'. Preparation of genomic DNA: The Bacillus agaradherens strain NCIMB 40482 was propagated in liquid medium as described in WO 94/01532. After 16 hours of incubation at 30°C and 300 rpm, the cells were isolated. Genomic DNA was isolated according to the method described by Pitcher et al. (Pitcher D.G., Saunders N.A., Owen R.J.: Rapid extraction of bacterial genomic DNA with guanidium thiocyanate. Letí. Appl. Microbiol. 1989, 8,151-156.).

Genomic library construction: Genomic DNA was partially digested with the restriction enzyme Sau3A and size-separated by electrophoresis on a 0.7% (w/w) agarose gel. Fragments with molecular weights between 2000 and 7000 were isolated by electrophoresis on DEAE-cellulose paper (Dretzen G., Bellard M., Sassone-Corsi P., Chambon P.: A reliable method for the recovery of DNA fragments from agarose and arylamide gels. Anal. Biochem. 1981, 112, 295 to 298.).

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

Identification of positive clones: The DNA library in E. coli, constructed as described above, was tested on LB agar plates containing 0.2% (w/w) AZCL-galactomannan (Megazyme) and 9 µg/ml chloramphenicol and incubated overnight at 37°C. Clones expressing mannanase activity appeared with a blue diffuse halo. Plasmid DNA from one of these clones was isolated by Qiagen plasmid spin preparation on 1 ml overnight in nutrient culture (cells were incubated at 37°C in TY with 9 µg/ml chloramphenicol and shaken at 250 rpm).

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

Analysis of the sequencing data was performed according to Devereux et al.: Nucleic Acids Res. 1984, 12, 387-395. The sequence encoding mannanase is set forth in SEQ ID NO: 1, supra. The deduced protein sequence is set forth in SEQ ID NO: 2, supra.

Subcloning and expression of mannanase in B. subtilis: The mannanase encoding DNA sequence of the invention was PCR amplified using a PCR primer set consisting of two oligonucleotides:

mannanasa. hom í. Sucking I

5-CAT TCT GCA GCC GCG GCA GCA AGT ACA GGC TTT TAT GTT GAT GG-3' and mannanase.lower.Notl

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

• · 0 ·

0 00 • · s t • · 0 0 ··

0 0

0·

0 0 ·

0 C 0

00 ·

I 0 0 » 0 0 » 0 0

00

SacII and NotII restriction sites are underlined.

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

The PCR reaction was performed using a DNA thermal cycler (Landgraf, Germany). One incubation at 94°C for 1 minute was followed by thirty PCR cycles using a cycling profile of denaturation for 30 seconds at 94°C, annealing for 1 minute at 60°C, and extension for 2 minutes at 72°C. Aliquots of the amplified product of 5 microliters were analyzed by electrophoresis in 0.7% (w/w) agarose gels (NuSieve, FMC). The appearance of a DNA fragment with a molecular weight of 1400 indicates amplification of the relevant gene segment.

Subcloning of PCR fragment: Aliquots (45 μΙ each) of the PCR products obtained as described above were purified using the QIAquick PCR purification kit (Qiagen, USA) according to the manufacturer's instructions. The purified DNA was eluted in 50 μΙ of 10 mM Tris-HCl, pH 8.5.

pg of pMOL944 and 25 μΙ of the purified PCR fragment were digested with SacII and NotI, electrophoresed in 0.8% (w/w) low-temperature gelling agarose gels (SeaPlaque GTG, FMC), the relevant fragments were excised from the gel and purified with the QIAquick Gel extraction kit (Qiagen, USA) according to the manufacturer's instructions. The isolated PCR DNA fragment was then ligated with the purified pMOL944, which had been previously digested with SacII-NotI. Ligation was performed overnight at 16°C using 0.5 pg of each DNA fragment, 1 unit of T4 DNA ligase, and T4 ligase buffer (Boehringer Mannheim, Germany).

The ligation mixture was used to transform the respective B. subtilis PL2306. Transformed cells were plated on LBPG-10 μg/ml kanamycin plates. After 18 hours of incubation at 37 °C, colonies were visible on the plates. Several colonies were analyzed by isolating plasmid DNA from the overnight culture medium.

One of these positive clones was streaked several times on agar plates as above. This clone was designated MB594. Clone MB594 was grown overnight in TY-10 μg/ml kanamycin at 37°C. The next day, 1 ml of cells was used to isolate plasmid from the cells using the Qiaprep Spin Plasmid Miniprep Kit # 27106 according to the manufacturer's recommendations for the preparation of B. subtilis plasmid preparations. This DNA was sequenced. The DNA sequence corresponding to the mature part of the mannanase was found to be the portion from position 94 to 1404 of the attached sequence ID No. 3.

ATGAAAAAAAAGTTATCACAGATTTATCATTTAATTATTTGCACACTTATAATA

AGTGTGGGAATAATGGGGATTACAACGTCCCCATCAGCAGCAAGTACAGGC tttatgttgatggcAatacgttat/\tgacgcaaatgggcagccatttgtcat

GAGÁGGTATTAACCATGGACATGCTTGGTATAAAGACACCGCTTCAACAGCT

ATTCCTGCCATTGCAGAGCAAGGCGCCAACACGATTCGTATTGTTTTATCAG

ATGGCGGTCAATGGGAAAAGACGACATTGACACCATTCGTGAAGTCATTG

AGČŤŤGCGGAGCAAAAIA^ÁATrGGŤGGCTGTCGTTGAAGTTCATCAŤGCCA''

CGGGTCGCGATTCGCGCAGTGATTTAAATCGAGCCGTTGATTATTGGATAG

AftATGAAAGATGCGCTTATCGGTAAAGAAGATACGGTTATTArrAACATTGCA /\ACGAGTGGTATGGGAGTTGGGATGGCTCAGCTTGGGCCGATGGCTATATT

GATGTCATTCCGAAGCTTCGCGATGCCGGCTTAACACACACCTTAATGGTTG

ATGCAGCAGGATGGGGGCAATATCCGCAATCTATTCATGATTACGGACAAG

ATGTGTTTAATGCAGATCCGTTAAA/u\ATACGATGTTCTCCATCCATATGTAT

GÁGTATGCTGGTGGTGATGCTAACAGTGTTTAGATCAAATATTGATAGAGTCA

TAGATCAAGACCTTGCTCTCGTAATAGGTGAATTCGGTCATAGACATACTGA

TGGTGATGTTGATGAAGATACAATCCTTAGTTATTCTGAAGAAACTGGCACA

GGGTGGCTCGCTTGGTCTTGGAAAGGCAACAGTACCGAATGGGACTATTTA

GACCTTTCAGAAGACTGGGCTGGTCAACATTTAACTGATTGGGGGAATAGAA

7GTCCACGGGGCCGATGGCTTACAGGAAACCTCCAAACCATCCACCGTAT r/ACAGATGATAACGGTGGTCACCCTGAACCGCCAACTGCTACTACCTTGTA • ·

7GACTTTGAAGGAAGCACACAAGGGTGGCATGGAAGCAACGTGACCGGTG

GCCCTTGGTCCGTAACAGAATGGGGTGCTTCAGGTAACTACTCTTTAAAGC

CGATGTAAATTTAACCTCAAATTCTTCACATGAACTGTATAGTGAACAAAGTC

GTAATCTACACGGATACTCTCAGCTCAACGCAACCGTTCGCCATGCCAATTG

GGGAAATCCCGGTAATGGCATGAATGCAAGACTTTACGTGAAAACGGGCTC

TGATTATACATGGCATAGCGGTCCTjTTACACGTATCAATAGCTCCAACTCA

GGAACAACGTTATCTTTTGATTTAAAv:AACATCGAAAATATCATCATGTTAGG

G;\AATAG (SEQ ID NO: 3)

The derived mature protein is shown in SEQ ID NO: 4

MHKKLSQlYHLIICTLlISVGIMGITrŠPSAASTGFYVDGNTLYDANGQPFVMRGIN

HGHAWYKDTASTAIPAIAEQGANTIRIVLSDGGGWEKDDIDTIREVIELAEQNKM , vawevhdatgrdsrsdlnravdywiemkdáíjgkedtviíniaňýgsvvďgs AWADGYIDVIPKLRDAGLTHTLMVDAAGWGQYPQSIHDYGQDVFNADPLKNTM FSIHMYEYAGGDANTVRSNIDRVIDCiDLALVIGEFGHRHTDGDVDEDTILSYSEE TSTGWLAWSWKGNSTEWDYLDLSEÚWAGQHLTDWGNRIVHGADGLQETSKP STVFTDDNGGHPEPPTATTLYDFEGSTQGWHGSNVTGGPWSVTEWGASGNY SLKADVNLTSNSSHELYSEQSRNLHGYSQLNATVRHANWGNPGNGMNARLYV KTGSDYTWHSGPFTRINSSNGTTLSFDLNNIENIIMLGK (seq. id. no. 4)

It was found that the 3' end of the mannanase encoded by the sequence SEQ ID NO: 1 was changed to the end shown in SEQ ID NO: 3 due to the design of the lower primer used in the PCR. The resulting amino acid sequence is shown in SEQ ID NO: 4. It is clear that the carbon terminus of SEQ ID NO: 2 (SHHVREIGVQFSAADNSSGQTALYVDNVTLR) is changed to the C terminus of SEQ ID NO: 4 (IIMLGK).

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

• · • · · · • ·· ·

LB agar (as 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% (w/w) glucose and 0.05 M potassium phosphate, pH 7.0.

BPX medium is described in European patent 0 506 780 (document WO 91/09129).

Expression, purification and characterization of mannanase from Bacillus agaradherens: Clone MB 594, obtained as described above, was cultured in 25 x 200 ml BPX medium with 10 µg/ml kanamycin in two 500 ml shake flasks for 5 days at 37 °C and 300 rpm.

6500 ml of culture fluid of clone MB 594 (batch # 9813) was isolated from shake flasks and the pH was adjusted to 5.5. 146 ml of cation exchange resin (C521) and 292 ml of anion exchange resin (A1390) were added for flocculation while stirring. The flocculated material was separated by centrifugation using a Sorval RC 3B centrifuge at 9000 rpm for 20 minutes at 6°C. The supernatant was clarified with Whatman GF/D and C glass filters and finally concentrated on a filter with a cut-off size of 10,000 wt. units.

The pH of 750 ml of this concentrate was adjusted with sodium hydroxide to

7.5. The clear solution was applied to anion exchange chromatography with a 900 ml Q-Sepharose column equilibrated with 50 mM Tris, pH 7.5. The bound mannanase activity was eluted using a sodium chloride gradient.

The pure enzyme gave a single band in SDS-PAGE with a molecular weight of 38,000. The amino acid sequence of the mannanase enzyme, i.e. the translated DNA sequence, is given in the above-described SEQ ID NO: 2.

• ·

9

Determination of kinetic constants: Substrate: Locust bean gum and reducing sugar analysis (PHBAH). Locust bean gum from Sigma (G-0753).

Kinetic determinations using various concentrations of locust bean gum and incubation for 20 minutes at 40°C and pH 10 gave Kkat: 467 per sec.

Kn: 0.08 grams per liter molecular weight: 38,000 pl (isoelectric point): 4.2.

The optimum temperature for mannanase was found to be 60°C.

The pH activity profile showed maximum activity 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 this enzyme is very thermally stable.

Detergent compatibility using 0.2% (w/w) AZCL-galactomannan from carob as substrate and incubation as above at 40°C shows excellent compatibility with conventional liquid detergents and good compatibility with conventional powder detergents.

Obtaining Bacillus subtilisis mannanase 168: Bacillus subtilisis β-mannanase was characterized and purified as follows. The Bacillus subtilis genome was examined for homology with the known Bacillus sp. β-mannanase gene sequence (Mendoza et al.: Biochemica et Biophysica Acta 1995, 1243, 552-554.). The coding region of ydhT, the product of which is 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 for amplifying 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, Ka., USA). An initial annealing time of 5 minutes at 95 °C was followed by 25 cycles with the following program: annealing at 95 °C for 1 minute, annealing at 55 °C for 2 minutes, and extension at 72 °C for two minutes. After the last cycle, the reaction was held at 72 °C for 10 minutes to complete the extension. PCR products were purified using the QIAquick PCR purification kit (Qiagen, Chatsworth, Ka., USA).

The ydhT mature region amplified from Bacillus subtilis strain 1A95 was inserted into the expression vector pPG1524 (described above) as follows. The amplified fragment of 1028 nucleotide pairs was digested with MfeI and BamHI. The expression vector pPG1527 was digested with EcoRI and BamHI. The restriction products were purified using the QIAquick PCR purification kit (Qiagen, Chatsworth, Ka., USA). The two fragments were ligated using T4 DNA ligase (13 hours, 16°C) and used to transform the corresponding E. coli strain DH5-a. Ampicillin-resistant colonies were cultured for DNA preparation. The DNA was characterized by restriction analysis. Plasmid pPG3200 contains the mature region of the ydhT gene. Plasmid pPG3200 was then used to transform the corresponding Bacillus subtilis strain PG 632 (Saunders et al. 1992).

Seven kanamycin-resistant Bacillus subtilis clones and one PG 632 control clone were picked and 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% maltin, 120 μΙ 10 mM MnCl ₂ and 20 μΙ 50 mg/ml kanamycin. The clones were grown overnight in 250 ml flasks with shaking at 250 rpm at 37 °C for protein expression. The cells were then centrifuged for 15 min at 14,000 rpm. One microliter of each supernatant was diluted in 99 μΙ 50 mM sodium acetate (pH 6.0). One μΙ of this dilution was assayed using endo-1,4-B-mannanase Beta-Mannazyme Tabs (Megazyme, Ireland) according to the manufacturer's instructions. Absorbance was measured at 590 nm • · ··4 ·

on a Beckman DU640 spectrophotometer. Clone 7 showed the highest absorbance of 1.67. Control PG632 showed no absorbance at 590 nm.

The supernatant was analyzed by SDS-PAGE on a 10 to 20% (wt) Tris-Glycine gel (Novex, San Diego, Ka., USA) to confirm the expected protein size of 38,000 wt units. Samples were prepared as follows. A 500μΙ sample of ydhT clone 7 and PG 632 supernatants were precipitated with 55.5 μΙ of 100% (wt) trichloroacetic acid (Sigma), washed with 100 μΙ of 5% (wt) trichloroacetic acid, resuspended in 50 μΙ of Tris-Glycine SDS sample buffer (Novex), and boiled for five minutes. One μΙ of each sample was subjected to gel electrophoresis at 30 mA for 90 minutes. A large protein band with a molecular weight of 38,000 corresponding to ydhT clone 7 was observed.

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

Purification of β-mannanase from the fermentation supernatant was carried out as follows: 500 ml of the supernatant was centrifuged for 10 minutes at 10,000 rpm and 4 °C. The centrifuged supernatant was then dialyzed at 4 °C in two 4-L exchanges of 10 mM potassium phosphate (pH 7.2) through a Spectrapor membrane with a molecular weight cutoff of 12,000 to 14,000 (Speetrum). The dialyzed supernatant was centrifuged for 10 minutes at 10,000 rpm at 4 °C. A 200 ml column of fast-flowing Q sepharose anion exchange (Pharmacia) was equilibrated with 1 liter of 10 mM potassium phosphate (pH 7.2) at 20 °C. 300 ml of the supernatant was loaded onto the column. Two flow-through fractions of 210 ml (sample A) and 175 ml (sample B) were isolated. These two fractions were analyzed as above except that the samples were diluted with 199 μΙ of 50 mM sodium acetate (pH 6.0) and showed absorbances of 0.38, • * · · · · • · The resulting samples were electrophoresed on a 10 to 20% Tris-glycine gel (Novex, Ka., USA) for 90 minutes at 30 mA. A major band corresponding to a molecular weight of 38,000 was present in each sample. This band contained more than 95% by weight 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. Samples A and B contained 1.3 and 1.6 mg/ml of B-mannanase, respectively. The identity of the protein was confirmed by ion spray mass spectrometry and amino-terminal amino acid sequencing.

Purified β-mannanase samples were used to characterize enzyme activity as follows. All assays used endo-1,4-B-mannanase Beta-Mannazyme Tabs (Megazyme, Ireland) as described above. Activity in the pH range 3.0 to 9.0 was determined in 50 mM citrate phosphate buffer, activity at pH 9.5 was determined in 50 mM CAPSO (Sigma) and activity at pH 10.0 to 11.0 in 50 mM CAPS buffer. The optimum pH for Bacillus subtilis B-mannanase was found to be 6.0 to

6.5. The thermal activity profile was determined in 50 mM citrate phosphate buffer (pH 6.5). The enzyme showed optimal activity at 40 to 45 °C. Bacillus subtilis B-mannanase retained significant activity at less than 15 °C and more than 80 °C. The specific activity for B-1,4-galactomannan was determined as 160,000 pmol/min.mg B-mannanase using endo-1,4-B-mannanase Beta-Mannazyme Tabs (Megazyme, Ireland) according to the manufacturer's instructions. The nucleotide and amino acid sequences of Bacillus subtilisis B-mannanase are given in SEQ ID NO: 5 and SEQ ID NO: 6.

Mannanase is included in the compositions according to the invention preferably in an amount of from 0.0001 to 2, more preferably from 0.0005 to 0.1, most preferably from 0.001 to 0.02% by weight of pure enzyme based on the weight of the composition.

The enzyme according to the invention, in addition to the enzyme core containing the catalytic domain, also contains a cellulose binding domain (CBD), both of which are together

4 4 4 4 4 • · • · • 44 4 4 • 4 ·· ··

4 4 4

4 4 4

4 4 4

44 cleavably linked. The cellulose binding domain (CBD) may exist as an integral part of the encoded enzyme or a CBD of another origin may be introduced into the enzyme, thus forming an enzyme hybrid. In this context, the term cellulose binding domain is intended as defined by 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. This definition classifies more than 120 cellulose binding domains into 10 groups (I to X) and shows that CBDs are found in various enzymes such as cellulases, xylanases, mannanases, arabinofuranosidases, acetylesterases and chitinases. CBDs are also found in algae, e.g. in the red alga Porhyra purpurea, as a non-hydrolytic polysaccharide binding protein, see Tomme et al. (as cited above). However, most CBDs are cellulases and xylanases. CBDs have been found to be at the N or C termini of proteins or to be internal. Enzyme hybrids are known in the art, see e.g. WO 90/00609 and WO 95/16782, and can be prepared by transforming into a host cell a DNA construct comprising at least a DNA fragment encoding a cellulose binding domain linked, with or without a linker, to a DNA sequence encoding a mannanase enzyme and a growing host cell, such that the linked gene is expressed. Enzyme hybrids can be described by the following general formula

CBD-MR-X, in which CBD represents the N-terminal or C-terminal region of the amino acid sequence corresponding to at least the cellulose binding domain, MR represents the middle region (linker) and may represent a bond or a short linking group preferably with 2 to 100 carbon atoms, more preferably with 2 to 40 carbon atoms, or preferably with 2 to 100, more preferably with 2 to 40 amino acids, and X represents the N-terminal or C-terminal region of the enzyme according to the present invention.

The above enzymes may be of any suitable origin, such as plant, animal, bacterial, fungal and yeast origin. The origin may further be

4 44 44 4444 44 44 • 444 4 · 4 4444

44 44 4 4444

44 444 4 444 44 4

44 4 4 44 4 44 4

4· 44 44 44 44 44 mesophilic or extremophilic (psychrophilic, psychrotropic, thermophilic, barophilic, alkalophilic, acidophilic, halophilic, etc.). Purified or non-purified forms of these enzymes may be used. It is now common practice to modify natural enzymes by protein/genetic engineering techniques to optimize their performance in the cleaning compositions of the invention. For example, variants may be designed to increase the compatibility of the enzyme with ingredients commonly encountered in such compositions. Other variants may also be designed to adjust the optimum pH, stability to bleaches and chelating agents, catalytic activity and similar properties of the enzyme variant to suit the particular cleaning application.

Particular attention should be paid to oxidation-sensitive amino acids for bleach stability and surface charges for surfactant compatibility. The isoelectric point of such enzymes can be modified by substitution of some charged amino acids, e.g. increasing the isoelectric point can help improve compatibility with anionic surfactants. The stability of these enzymes can be further enhanced by e.g. formation of salt addition bridges and strengthening of metal binding sites to increase stability towards chelating agents.

Softening clay: A second essential component of the present laundry detergent compositions and/or fabric care compositions is softening clay. Any clay known in the art or mixtures thereof may be used in the present invention. Preferred examples are described in British Patent Specification 1,400,898 or U.S. Patent 5,019,292.

Among these clays are included various heat-treated kaolins and various multilayer smectites or bentonites, also called montmorillonite. As is known in the art, preferred smectite clays exhibit a cation exchange capacity of at least 50 millieq. per 100 grams of clay, corresponding to a layer charge of 0.2 to 0.6. Other preferred clays are those having particle sizes in the range of 5 to 50 microns.

• · ·· · * ·»·· · · ·· ···· ·· · · · · • φ ·Φ · · · «··· φ ·· · · φ · φ · φ φ · φ «••Φ · · · · · · · φ ·· ·· φ · ·· · φ ··

Other preferred smectite clays are hectorite clays of the general formula [(Mg ₃ . _x Li _x )Si ₄ . ^{yMe lll} yOw(OH ₂ . _z F _z )r ^(x+y <(x+^IVI ^{n+ )} , where y is 0 or if y is 0, Me ¹¹¹ is an aluminum, iron or boron atom, M ⁿ⁺ is a monovalent (n=1) or divalent (n=2) metal ion, for example selected from sodium, potassium, magnesium, calcium and strontium. The value (x+y) is the charge of the hectorite clay layer. Hectorite clays suitable for detergent compositions according to the present invention have a layer charge distribution such that at least 50% is in the range of 0.23 to 0.31.

Preferred are hectorite clays of natural origin which have a layer charge distribution such that at least 65% is in the range of 0.23 to 0.31.

Specific non-limiting examples of smectite clay minerals for fabric softening are:

sodium montmorillonite: Borck ^(R) , Volclay BC ^(R) , Gelwhite GP ^(R> , Thixo-Jel ^(R) ,

Ben-A-Gel ^(R) , Sodium Hectorite: Veegum F ^(R) and Laponite SP ^(R) , sodium saponite: Barasym NAS 100 ^(R) , calcium montmorillonite; Soft Clark ^(R) , Gelwhite L ^w , Imvite K ^(R) , CSM-Clay ^(R) from

Kimolous and lithium hectorite: Barasym LIH 200 ^(R) .

The amount of softening clay used in the present invention depends on the form of the laundry detergent and/or fabric care compositions. In general, it may range from a lower limit of 0.1, 3 or 4 to an upper limit of 50, 25 or 15% by weight.

»0 ·«··

AND*

0 0 0 0 « » 00 · • 0 0 · 0 · • · · 0 · ·· • · 0 « · 0 • · · · · · · • · · 0 0 0 « · 0 · · · 0 •0 00 00

Detergent Ingredients: The laundry detergent and/or fabric care compositions of the invention must contain at least one additional detergent ingredient. The precise nature of this additional ingredient and the amounts included therein will depend on the physical form of the composition and the nature of the cleaning operation for which it is used.

The laundry detergent and/or fabric care compositions of the present invention preferably further comprise a laundry detergent and/or fabric care component selected from cellulase, a builder selected from zeolite, sodium triphosphate and/or layered silicate, a cationic surfactant and/or mixtures thereof.

The laundry detergent compositions and/or fabric care compositions according to the invention may be in the form of a liquid, paste, gel, stick, tablet, spray, foam, powder or in the form of granules. Granular compositions may also be in compact form and liquid compositions may also be in concentrated form.

The compositions of the invention can be formulated, for example, as hand and machine wash detergent compositions including laundry additive compositions and compositions suitable for use in soaking and/or pretreating stained fabrics and as rinse softeners.

When formulated as compositions suitable for use in an automatic washing machine, the compositions of the invention preferably comprise both a surfactant system, which may comprise an anionic, cationic, nonionic or zwitterionic surfactant or a mixture thereof, and a builder system, which may comprise a phosphate builder, a non-phosphate inorganic zeolite, a layered silicate, an organic builder such as citrate and one or more detergent components, which are preferably selected from organic polymeric compounds, bleaching agents, other enzymes, suds suppressors, dispersants, lime soap type dispersants, soil suspending agents, anti-redeposition agents and corrosion inhibitors. Detergents ♦ φ ···* <ι· <· < · v * · ·· • · · · * • * · ·

94

4 9

9 4

9 9

9 9 9

94

99 • 4 9 9

9 4 · • · Φ 4 • » t ·

9 4 may also contain softening agents other than the inorganic clay claimed in the present invention as additional detergent ingredients. Compositions containing mannanase and clay may provide fabric cleaning, stain removal, softening action and color retention when formulated as laundry detergent compositions.

The compositions of the invention may also be used as detergent additive products in solid or liquid form. These additive products are intended to supplement or as an agent that improves the performance of conventional detergent compositions and may be added at any stage of the cleaning process.

If desired, the laundry detergent compositions of the invention have a density in the range of from 400 to 1200 g/liter, preferably from 500 to 950 g/liter of composition, measured at 20°C.

The compact form of the compositions of the invention is best expressed by the density and, in terms of the composition, the amount of inorganic filler salt. Inorganic filler salts are conventional additives to detergent compositions in powdered form. In conventional detergent compositions, filler salts are present in substantial amounts, typically in an amount of 17 to 35% by weight of the total composition. In compact compositions, the filler salt is present in amounts not exceeding 15% by weight of the total composition, preferably not exceeding 10% and most preferably not exceeding 5% by weight of the composition. Inorganic filler salts, as intended in the present compositions, are selected from alkali metal and alkaline earth metal sulfates and chlorides. A preferred filler salt is sodium sulfate.

The liquid detergent compositions of the present invention may also be in concentrated form. In such a case, the liquid detergent compositions of the present invention will contain a lower amount of water compared to conventional liquid detergents. The water content of the concentrated liquid detergent is typically preferably less than 40, more preferably less than 30, and most preferably less than 20% by weight of the detergent composition.

Suitable detergent compounds for use in the invention are selected from the group consisting of the compounds described below.

Surfactant System: The laundry detergent compositions and/or fabric care compositions of the present invention may further comprise a surfactant system in which the surfactant is selected from nonionic and/or anionic and/or cationic and/or ampholytic and/or zwitterionic and/or semipolar surfactants. The laundry detergent compositions and/or fabric care compositions of the present invention will preferably further comprise a cationic surfactant. Surprisingly, it has been found that said compositions further comprising a cationic surfactant provide improved cleaning performance and improved softening.

The additional surfactant is typically present in an amount of from 0.1 to 60% by weight. A more preferred amount is 1 to 35%, most preferred 1 to 30% by weight, by weight of the laundry detergent compositions and/or fabric care compositions of the invention.

The surfactant is preferably formulated to be compatible with the enzyme components present in the composition. In liquid or gel compositions, the surfactant is most preferably formulated to promote or at least not degrade the stability of any enzyme in the composition.

Polyethylene, polypropylene and polybutylene oxide condensates of alkylphenols are suitable for use as the nonionic surfactant of the surfactant systems of the present invention, with polyethylene oxide condensates being preferred. These compounds include condensation products of alkylphenols having an alkyl group of 6 to 14 carbon atoms, preferably 8 to 14 carbon atoms, in either a straight or branched chain configuration, and alkylene oxide. In a preferred embodiment, the ethylene oxide is present in an amount of 2 to 25 moles, more preferably 3 to 15 moles of ethylene oxide per mole of alkylphenol. Commercially available nonionic surfactants of this type include Igepal™ CO-630, manufactured by GAF Corporation, and Triton™ X-45, X-114, X-100 and X-102, all manufactured by Rohm & Haas Company. These surfactants are usually referred to as alkylphenol alkoxylates (e.g., alkylphenol ethoxylates).

Condensation products of primary and secondary aliphatic alcohols with 1 to 25 moles of ethylene oxide are suitable for use as the nonionic surfactant of the nonionic surfactant system of the present invention. The alkyl chain of the aliphatic alcohol may be either straight or branched, primary or secondary, and usually contains 8 to 22 carbon atoms. Condensation products of alcohols having an alkyl group of 8 to 20 carbon atoms, more preferably from 10 to 18 carbon atoms, with 2 to 10 moles of ethylene oxide per mole of alcohol are preferred. In said condensation products, 2 to 7 moles of ethylene oxide and most preferably 2 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 (condensation product of a linear alcohol having 11 to 15 carbon atoms with 9 moles of ethylene oxide), Tergitol™ 24-L-6 NMW (condensation product of a primary alcohol having 12 to 14 carbon atoms with 6 moles of ethylene oxide with a narrow molecular weight distribution), both sold by Union Carbide Corporation, Neodol™ 45-9 (condensation product of a linear alcohol having 14 to 15 carbon atoms with 9 moles of ethylene oxide), Neodol™ 23-3 (condensation product of a linear alcohol having 12 to 13 carbon atoms with 3 moles of ethylene oxide), NEODOL™ 45-7 (condensation product of a linear alcohol having 14 to 15 carbon atoms with 7 moles of ethylene oxide), Neodol™ 45-5 (condensation product of a linear alcohol having 14 to 15 carbon atoms with 7 moles of ethylene oxide), product of a linear alcohol with 14 to 15 carbon atoms with 5 moles of ethylene oxide), manufactured by Shell Chemical Company, Kyro™ EOB (condensation product of an alcohol with 13 to 15 carbon atoms with 9 moles of ethylene oxide), manufactured by The Procter and Gamble Company, and Genapol LA 030 or 050 (condensation product of an alcohol with 12 to 14 carbon atoms φ φ with 3 or 5 moles of ethylene oxide), manufactured by Hoechst. The preferred HLB range in these products is 8 to 11 and most preferably from 8 to 10.

In the compositions of the invention, alkyl polysaccharides, such as those described in U.S. Patent 4,565,647 to Llenada, issued January 21, 1986, which have a hydrophobic group of 6 to 30, preferably 10 to 16 carbon atoms, and a polysaccharide, e.g., a polyglycoside, the hydrophilic group containing from 1.3 to 10, preferably 1.3 to 3, most preferably 1.3 to 2.7 saccharide units, are also useful nonionic surfactants of the surfactant system of the present invention. Any reducing saccharide having 5 or 6 carbon atoms may be used, e.g. glucose, galactose and galactosyl groups may be substituted by glucosyl groups (optionally a hydrophobic group is attached at the 2-, 3-, 4-, etc. position, thus giving glucose or galactose as opposed to glucoside or galactoside). The bonds between sugars may be, for example, between one position of the next carbohydrate units and the 2-, 3-, 4- and/or 6- positions of the preceding carbohydrate units.

Preferred alkyl polyglycosides are compounds of the general formula

R ² O(C _n H _2n O)t(glycosyl) _x , wherein R ₂ is selected from the group consisting of alkyl, alkylphenyl, hydroxyalkyl, hydroxyalkylphenyl and mixtures thereof, in which the alkyl groups contain from 10 to 18, preferably from 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 from 1.3 to 3, most preferably from 1.3 to 2.7. Glycosyl is preferably derived from glucose. In the preparation of these compounds, an alcohol or alkylpolyethoxy alcohol is first formed and then reacted with glucose or a glucose source to form a glucoside (attached at the 1-position). Further glycosyl units may then be attached between their position 1 and positions 2, 3, 4 and/or 6 of the preceding glycosyl units, preferably predominantly at position 2.

«

Condensation products of ethylene oxide with a hydrophobic base formed by the condensation of propylene oxide with propylene glycol are also suitable for use as further nonionic surfactant systems according to the present invention. The hydrophobic portion of these compounds will preferably have a molecular weight of from 1500 to 1800 and will be insoluble in water. The addition of polyoxyethylene groups to this hydrophobic portion tends to increase the solubility of the molecule as a whole in water and this liquid character of the product is maintained up to the point where the polyoxyethylene 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 of the commercially available surfactants Plurafac™ LF404 and Pluronic™ manufactured by BASF.

Condensation products of ethylene oxide with the reaction products of propylene oxide with ethylenediamine are also suitable for use as the nonionic surfactant of the nonionic surfactant system of the present invention. The hydrophobic portion of these products consists of the reaction products of ethylenediamine with an excess of propylene oxide and generally has a molecular weight of from 2500 to 3000. This hydrophobic portion is condensed with ethylene oxide to such an extent that the condensation product contains from 40 to 80% by weight of polyoxyethylene and has a molecular weight of from 5000 to 11,000. Examples of this type of nonionic surfactant include some of the commercially available Tetronic™ compounds manufactured by BASF.

For use as nonionic surfactants in the surfactant systems of the present invention, polyethylene oxide condensates of alkylphenols, condensation products of primary and secondary aliphatic alcohols with 1 to 25 moles of ethylene oxide, alkyl polysaccharides, and mixtures thereof are preferred. Most preferred are alkyl (C8-C14) phenol ethoxylates having 3 to 15 ethoxy groups and alcohol (C8-C18) ethoxylates (preferably having an average of 10 carbon atoms) having 2 to 10 ethoxy groups, and mixtures thereof.

Highly preferred nonionic surfactants are surfactants of the polyhydroxy fatty acid amide type of the general formula • · » « » « • · r ² -cnz ¹¹ >1 OR ¹ in which R ¹ represents a hydrogen atom or a hydrocarbon group with 1 to 4 carbon atoms, 2-hydroxyethyl, 2-hydroxypropyl or mixtures thereof, R ² represents a hydrocarbon group with 5 to 31 carbon atoms and Z represents a polyhydroxy hydrocarbon group with a linear hydrocarbon chain with at least three hydroxyl groups directly attached to the chain or an alkoxylated derivative thereof. Preferably, R ¹ is methyl, R ² is a straight alkyl of 11 to 15 carbon atoms or an alkyl or alkenyl chain of 16 to 18 carbon atoms, such as coco alkyl or mixtures thereof, and Z is preferably derived from a reducing sugar, such as glucose, fructose, maltose and lactose, in a reductive amination reaction.

Suitable anionic surfactants used herein include linear alkylbenzene sulfonate, alkyl ester sulfonate surfactants comprising linear esters of carboxylic acids having 8 to 20 carbon atoms (i.e. fatty acids) which are sulfonated with sulfur dioxide gas according to The Journal of the American Oil Chemists Society 1975, 52, 323-329. Suitable starting materials would include natural fatty substances such as those derived from tallow, palm oil, etc.

Preferred alkyl ester sulfonate surfactants, particularly for laundry applications, include alkyl ester sulfonate surfactants of the general formula

Oh, H"

^R3 -CH-C- ^ORr ,

I so ₃ m wherein R ³ represents a carbon group with 8 to 20 carbon atoms, preferably alkyl or a combination thereof, R ⁴ represents a hydrocarbon group with 1 to 6 carbon atoms, preferably alkyl or a combination thereof, and M represents a cation that forms a water-soluble salt with an 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. R ₃ preferably represents an alkyl with 10 to 16 carbon atoms and R ₄ represents methyl, ethyl or isopropyl. Methyl ester sulfonates in which R ³ represents an alkyl with 10 to 16 carbon atoms are particularly preferred.

Other suitable anionic surfactants include alkyl sulfate surfactants, which are water-soluble salts or acids of the general formula ROSO ₃ M, wherein R is preferably a hydrocarbon group of 10 to 24 carbon atoms, preferably alkyl or hydroxyalkyl with an alkyl component of 10 to 20 carbon atoms, more preferably alkyl or hydroxyalkyl of 12 to 18 carbon atoms, and M is hydrogen or a cation, e.g. an alkali metal cation (e.g. sodium, potassium, lithium) or ammonium or substituted ammonium (e.g. methyl, dimethyl and trimethyl ammonium cations and quaternary ammonium cations such as tetramethyl ammonium and dimethyl piperidinium cations, and quaternary ammonium cations derived from alkylamines such as ethylamine, diethylamine, triethylamine and mixtures thereof, and the like). Typically, alkyl chains with 12 to 16 carbon atoms are preferred for lower washing temperatures (e.g., for temperatures below 50°C) and alkyl chains with 16 to 18 carbon atoms are preferred for higher washing temperatures (e.g., above 50°C).

For cleaning purposes, other anionic surfactants may be included in the cleaning compositions of the present invention. These include salts (including, for example, sodium, potassium, ammonium and substituted ammonium salts such as mono-, di- and tri-ethanolamine salts) of soaps, primary or secondary alkane sulfonates having 8 to 22 carbon atoms, olefin sulfonates having 8 to 24 carbon atoms, sulfonated polycarboxylic acids prepared by sulfonating the pyrotized product of alkaline earth metal citrates, e.g. as described in British Patent Specification No. 1 082179, alkyl (with 8 to 24 carbon atoms) polyglycol ether sulfates (with up to 10 ethylene oxide moieties), alkyl glyceryl sulfonates, fatty acylglycerol sulfonates, fatty oleylglycerol sulfates, 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 monoesters with 12 to 18 carbon atoms) and diesters of sulfosuccinates (especially saturated and unsaturated diesters with 6 to 12 carbon atoms), acyl sarcosinates, sulfates alkyl polysaccharides, such as alkylpolyglucoside sulfates (nonionic non-sulfated compounds described below), branched-chain primary alkyl sulfates, and alkylpolyethoxycarboxylates, such as compounds of the general formula

RO(CH ₂ CH ₂ O) _k -CH ₂ COO'M ⁺ , where R is an alkyl of 8 to 22 carbon atoms, k is a number 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 described in Surface Active Agents and Detergents (Volumes I et tt, Schwartz, Perry and Berch). Such various surfactants are also generally described in U.S. Patent 3,929,678 to Laughlin et al., issued December 30, 1975, at column 23, line 58, through column 29, line 23 (incorporated herein by reference).

If included, the laundry detergent compositions and/or fabric care compositions of the present invention typically contain from 1 to 40, preferably from 3 to 20% by weight of these anionic surfactants.

Highly preferred anionic surfactants include alkyl alkoxylated sulfate surfactants. These surfactants are water-soluble salts or acids of the general formula RO(A) _m SO ₃ M, wherein R is an unsubstituted alkyl group of 10 to 24 carbon atoms or a hydroxyalkyl group with an alkyl moiety of 10 to 24 carbon atoms, preferably ·

• · • · an alkyl or hydroxylalkyl group with 12 to 20 carbon atoms, more preferably an alkyl or hydroxyalkyl group with 12 to 18 carbon atoms, A represents an ethoxy or propoxy unit, m represents a number greater than 0, typically between 0.5 and 6, more preferably between 0.5 and 3, and M represents a hydrogen atom or a cation, which may represent, for example, a metal cation (e.g. sodium, potassium, lithium, calcium, magnesium, etc.), an ammonium or substituted ammonium cation. This includes alkyl ethoxylated sulfates as well as alkyl propoxylated sulfates. Specific examples of substituted ammonium cations include methyl, dimethyl, and trimethyl ammonium cations, and quaternary ammonium cations such as tetramethyl ammonium and dimethyl piperidine cations, and cations derived from alkylamines such as ethylamine, diethylamine, triethylamine, and mixtures thereof, and the like. Examples of surfactants are alkyl(C12-C18)polyethoxylate (1.0) sulfate ( _C12 - _C18 E(1.0)M), alkyl(C12-C18)polyethoxylate (2.25) sulfate ( _C12 - _C18 E(2.25)M), alkyl(C12-C18)polyethoxylate (3.0) sulfate (C12- _C18 E(3.0)M) and alkyl(C12-C18)polyethoxylate (4.0) sulfate (C12- _C18 E(4.0)M), wherein M is suitably selected from sodium and potassium.

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

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

44 • · ♦ 4

4 4 4 • 4 4 4 • ·4 4 •4 44

If present, the laundry detergent compositions and/or fabric care compositions of the present invention typically contain from 0.2 to 15, preferably from 1 to 10% by weight of these ampholytic surfactants.

Zwitterionic surfactants are also suitable for use in laundry detergent compositions and/or fabric care compositions. These surfactants may be broadly described as derivatives of secondary and tertiary amines, derivatives of heterocyclic secondary and tertiary amines, or derivatives of quaternary ammonium, quaternary phosphonium, or tertiary sulfonium compounds. For examples of zwitterionic surfactants, see, e.g., U.S. Patent No. 3,929,678 to Laughlin et al., issued December 30, 1975, column 19, line 18, through column 22, line 48.

If present, the laundry detergent compositions and/or fabric care compositions of the present invention typically contain from 0.2 to 15, preferably from 1 to 10% by weight of these zwitterionic surfactants.

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

Semipolar nonionic detergent surfactants include amine oxide surfactants of the general formula

9* ·99 9

About

R ³ (OR ⁴ ) x N(R ⁵ ) ₂ , wherein R ³ represents an alkyl, hydroxyalkyl or alkylphenyl group or mixtures thereof with 8 to 22 carbon atoms, R ⁴ represents an alkylene or hydroxyalkylene group with 2 to 3 carbon atoms or mixtures thereof, x represents a number from 0 to 3, and each R ⁵ represents an alkyl or hydroxyalkyl group with 1 to 3 carbon atoms or a polyethylene oxide group with 1 to 3 ethylene oxide groups. The R ⁵ groups may be attached to each other, e.g., by an oxygen or nitrogen atom, so as to form a ring structure.

These amine oxide surfactants particularly include alkyl(C10-C18)dimethylamine oxides and alkoxy(C8-C12)ethyldihydroxyethylamine oxides.

If included, the cleaning compositions of the present invention typically contain from 0.2 to 15, preferably from 1 to 10% by weight of these semi-polar nonionic surfactants.

The laundry detergent compositions and/or fabric care compositions of the present invention may further comprise other surfactants which are selected from the group of primary or tertiary amines.

Suitable primary amines for use according to the invention include amines of the general formula RiNH ₂ , in which R-ι represents an alkyl chain of 6 to 12, preferably 6 to 10 carbon atoms or the group R4X(CH ₂ ) _n , X represents the group -O-, -C(O)NH- or -NH-, R ₄ represents an alkyl chain of 6 to 12 carbon atoms and n represents a number between 1 and 5, preferably 3. The alkyl chains Ri may be straight or branched and may be interrupted by up to 12, preferably less than 5 ethylene oxide groups. Preferred amines according to the above formula are alkylamines. Suitable amines for use according to the invention may be selected from 1-hexylamine, 1-octylamine, 1-decylamine and lau44 ··

4 · » • 4 4 4 • 4 4 4 4 ♦ · 4 4

Other preferred primary amines include oxypropylamine, octyloxypropylamine, 2-ethylhexyloxypropylamine, lurylamidopropylamine, and amidopropylamine having 8 to 10 carbon atoms.

Suitable tertiary amines for use in the invention include tertiary amines of the general formula R1R2R3H, in which R1 and _R2 represent alkyl chains of 1 to 8 carbon atoms or a group of the general formula

R ₅

-(CH ₂ -CH-O) _x H ,

R ₃ represents either an alkyl chain of 6 to 12 carbon atoms, preferably 6 to 10 carbon atoms, or R ₃ represents a group of the general formula R4X(CH ₂ ) _n , in which X represents the group -O-, -C(O)NH- or -NH-, R ₄ represents a group of 4 to 12 carbon atoms and n represents a number between 1 and 5, preferably 2 to 3. R ₅ represents a hydrogen atom or an alkyl of 1 to 2 carbon atoms and x represents a number between 1 to 6. R ₃ and R ₄ may be linear or branched and the alkyl chains of R ₃ may be interrupted by up to 12, preferably less than 5, ethylene oxide groups.

Preferred tertiary amines are amines of the general formula RiR ₂ R ₃ N, in which R 1 represents an alkyl chain with 6 to 12 carbon atoms, R ₂ and R ₃ represent an alkyl group with 1 to 3 carbon atoms or a group of the general formula

R ₅

-(CH ₂ -CH-O) _x H , in which R ₅ represents a hydrogen atom or a methyl group and x represents a number from 1 to 2.

Also preferred are amidoamines of the general formula

RrC(O)-NH-(CH ₂ )nN-(R ₂ ) ₂ • * φ ·· ·* φφ • φ φ · φ • · φφ φ • * · * · φ • · φ φ φ ·· ·♦ φφ in which Ri represents alkyl with 6 to 12 carbon atoms, n represents the number 2 to 4, preferably n represents the number 3, and R ₂ and R ₃ represent a group with 1 to 4 carbon atoms.

The most preferred amines according to the present invention include 1-octylamine, 1-hexylamine, 1-decylamine, 1-dodecylamine, oxypropylamine with 8 to 10 carbon atoms, N-coco 1,3-diaminopropane, coco alkyldimethylamine, lauryldimethylamine, laurylbis(hydroxyethyl)amine, coco bis(hydroxyethyl)amine, laurylamine propoxylated with 2 moles, octylamine propoxylated with 2 moles, laurylamidopropylmethylamine, amidopropyldimethylamine with 8 to 10 carbon atoms, and amidopropyldimethylamine with 10 carbon atoms.

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

Bleaching agents: Laundry detergent compositions and/or fabric care compositions may further comprise bleaching agents such as hydrogen peroxide, PB1, PB4 and percarbonate, which have a particle size of 400 to 800 pm. These bleaching agent components may comprise one or more oxygen bleaching agents and, depending on the bleaching agent selected, one or more bleach activators. If oxygen bleaching compounds are present, they are typically present in amounts of from 1 to 25% by weight.

The bleaching agent component for use in the present invention may be any bleaching agent useful in laundry detergent compositions and/or fabric care compositions containing oxygen bleaching agents as well as other agents known in the art. The bleaching agent suitable for the present invention may be an activated or non-activated bleaching agent.

• w · »· · • · · • · · • » · • · · · ·* ·» • * · » · · • ♦ · • ·» • * * · ··

One category of oxygen bleaching agents that can be used includes percarboxylic acid type bleaching agents and their salts. Suitable examples of this group of agents include magnesium monoperphthalate hexahydrate, magnesium salt of m-chloroperbenzoic acid, 4-nonylamino-4-oxoperbutyric acid and diperdodecanedioic acid. These 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 also include 6-nonylamino-6-oxopercapric acid as described in U.S. Patent 4,634,551.

Another category of bleaching agents that can be used includes halogenated bleaching agents. Examples of halogenated bleaching agents include, for example, trichloroisocyanuric acid, sodium and potassium dichloroisocyanurate, and N-chloro- and N-bromo-alkanesulfonamides. These 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 U.S. Patent 4,412,934), 3,5-trimethylhexanoloxybenzenesulfonate (ISONOBS, described in European Patent 120,591) or pentaacetylglucose (PAG) or N-nonanoyl-6-aminocapric acid phenolsulfonate ester (NACA-OBS, described in WO 94/28106), which perhydrolyze to form the peracid as active bleaching species, resulting in improved bleaching performance. Acylated citrate esters, such as the compounds described in the accompanying European Patent Application No. 91870207.7 and an unsymmetrical acyclic imide bleach activator of the following general formula described in copending U.S. Patent Application Serial Nos. 60/022,786 (Procter & Gamble, filed July 30, 1996) and 60/028,122 (filed October 15, 1996)

R ₃

Ο « φ φ ♦ e φ« • · · • Φ • ♦ Φ • · ·

ΦΦ • φ • Φ • ·

ΦΦ • · Φ • ♦ » • · Φ • * φ φ ·· ΦΦ * · · • φ φ « * · • φ φ φφ in which R1 represents a saturated or unsaturated linear or branched chain alkyl group with 7 to 13 carbon atoms, R ₂ represents a saturated or unsaturated linear or branched chain alkyl group with 1 to 8 carbon atoms and R ₃ represents a saturated or unsaturated linear or branched chain alkyl group with 1 to 4 carbon atoms.

Useful bleaching agents, including peracids and bleaching systems containing bleach activators and peroxygen bleaching compounds, for use in laundry detergent compositions and/or fabric care compositions of the invention are described in our accompanying U.S. Patent Nos. 08/136,626, PCT/US95/07823, WO 95/27772, WO 95/27773, WO 95/27774 and WO 95/27775.

Hydrogen peroxide may also be present by adding an enzymatic system (i.e. an enzyme and a substrate therefor) capable of generating hydrogen peroxide at the beginning or during the washing and/or rinsing process. Such enzymatic systems are described in European Patent Application 91202655.6, filed October 6, 1991.

Metal-containing catalysts for use in bleaching compositions include cobalt-containing catalysts such as pentaamine acetate cobalt salts and manganese-containing catalysts such as those described in European Patent Applications 549271, 549272, 458397, 458398 and in U.S. Patents 5,246,621, 5,194,416 and 5,114,611. A bleaching composition containing persulfuronin and a bleaching catalyst containing manganese and a chelating agent are described in Patent Application No. 94870206.3

Bleaching agents other than oxygen bleaching agents are also known in the art and may be used herein. One type of non-oxygen bleaching agent of particular interest is photoactivated bleaching agents, such as sulfonated zinc and/or aluminum phthalocyanines. These materials may be deposited on the substrate during the washing process. Upon exposure to light in the presence of oxygen, such as hanging clothes to dry in daylight, the sulfonated zinc phthalocyanine is activated and the substrate is thereby bleached. The 9 9 «

99 999 9 999 99 9 • · · 9 · · · · 9 99 9 worthy zinc phthalocyanine and photoactivated bleaching process are described in US Patent 4,033,718. Laundry detergent compositions and/or fabric care compositions will typically contain 0.025 to 1.25% by weight of sulfonated zinc phthalocyanine.

Builder System: The laundry detergent compositions and/or fabric care compositions of the present invention may further comprise a builder. The laundry detergent compositions and/or fabric care compositions of the present invention will preferably further comprise a builder selected from zeolite, sodium triphosphate and/or layered silicate. Surprisingly, it has been found that said compositions which further comprise a builder selected from zeolite, sodium triphosphate and/or layered silicate provide improved cleaning and softening performance.

Any conventional building system comprising aluminosilicate materials, silicates, polycarboxylates, alkyl or alkenyl succinic acid and fatty acids, materials such as ethylenediaminetetraacetate, diethylenetriaminepentamethyleneacetate, metal ion scavengers such as aminopolyphosphonates, especially ethylenediaminetetramethylenephosphonic acid and diethylenetriaminepentamethylenephosphonic acid, is suitable for use in the invention. Phosphate building components may also be used.

Suitable builders may be inorganic ion exchange materials, typically an inorganic hydrated aluminosilicate material, particularly a hydrated synthetic zeolite such as hydrated zeolite A, X, B, HS or MAP.

Another suitable inorganic building 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 ₅ ).

Suitable polycarboxylates containing one carboxyl group include lactic acid, glycolic acid and their ether derivatives as described in Belgian Patents Nos. 831,368, 821,369 and 821,370. Polycarboxylates containing two carboxyl groups include water-soluble salts of succinic acid, malonic acid, (ethylenedioxy)diacetic acid, maleic acid, diglycolic acid, tartaric acid, tartronic acid and fumaric acid, as well as ether carboxylates described in German Patent Nos. 2,446,686 and 2,446,687 and in U.S. Patent No. 3,935,257 and sulfinyl carboxylates described in Belgian Patent No. 640,623. Polycarboxylates containing carboxylic groups include in particular water-soluble citrates, aconitates and citraconates as well as succinate derivatives such as carboxymethyloxysuccinates described in British Patent Specification No. 1,379,241, lactoxysuccinates described in Dutch Patent Application 7,205,873 and oxypolycarboxylate materials such as 2-oxa-1,1,3-propanetricarboxylates described in British Patent Specification 1,387,447.

Polycarboxylates containing four carboxyl groups include 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 sulfosubstituents include sulfosuccinate derivatives described in British Patents Nos. 6,1398,421 and 6,1398,422 and in U.S. 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, cyclopentadiene 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-hexane hexacarboxylates and carboxymethyl derivatives of polyhydroxy alcohols such as sorbitol, mannitol and xylitol. Aromatic polycarboxylates include mellitic acid, pyromellitic acid and phthalic acid derivatives described in British Patent Specification 1,425,343.

9 • 9 • · • ••9 99 9 · · · ·

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

99 99 99 · · 9«

Of the above, preferred polycarboxylates are hydroxycarboxylates containing up to three carboxyl groups per molecule, especially citrates.

Preferred builder systems for use in the present compositions 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 system components include a mixture of a water-insoluble aluminosilicate builder, such as zeolite A, and a water-soluble carboxylate chelating agent, such as citric acid. Preferred builder systems for liquid laundry detergent compositions and/or fabric care compositions of the present invention are soaps and polycarboxylates.

Other builders which may form part of the builder system 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 co-polymeric acids or salts thereof in which the polycarboxylic acid contains at least two carboxyl groups separated by not more than two carbon atoms. Polymers of this type are described in British Patent Specification 1,596,756. Examples of such salts are polyacrylates having a molecular weight of 2000 to 5000 and their copolymers with maleic anhydride, such as copolymers having a molecular weight of from 20,000 to 70,000, especially around 40,000.

The salts of detergent builders are usually present in the composition in amounts of from 5 to 80, preferably from 10 to 70 and most usually from 30 to 60% by weight of the composition.

Conventional detergent enzymes: Laundry detergents and/or fabric care products may contain, in addition to the mannanase enzyme, one of the following:

or more enzymes which provide cleaning performance, provide fabric care and/or have disinfectant effects. The laundry detergent compositions and/or fabric care compositions of the present invention will preferably further comprise a cellulase. Surprisingly, it has been found that said compositions further providing a cellulase provide improved cleaning and improved fabric softening performance.

Said enzymes include enzymes selected from celluloses, hemicelluloses, peroxidases, proteases, glucoamylases, amylases, xylans, lipases, phospholipases, esters, cutins, pectins, keratins, reductases, oxidases, phenoloxidases, lipoxygenases, lignins, pullulans, tannins, pentosans, mallans, β-glucans, arabinosides, hyaluronides, chondroitins, laccases or mixtures thereof.

A suitable combination is laundry detergent compositions and/or fabric care compositions which contain a mixture of conventionally used enzymes, such as protease, amylase, lipase, cutinase and/or cellulase, together with one or more plant cell wall degrading enzymes.

Suitable proteases are the subtilisins obtained from the respective strains of B. subtilis and B. licheniformis (subtilisin BPN and BPN'). One suitable protease is obtained from a strain of Bacillus, has maximum activity in the pH range of 8 to 12, has been developed and is sold as Esperase ^(R) by Novo Industries A/S of Denmark, hereinafter referred to as Novo. The preparation of this enzyme and analogous enzymes is described in British Patent Specification 1,243,784 (Novo). Other suitable proteases include Alcalase ^(R) , Durazym ^(R) and Savinase ^(R) from Novo and Maxatase ^(R) , Maxacal ^(R) , Properase ^(R) and Maxapem ^(R) (protein engineered Maxacal) from Gist-Brocads. Proteolytic enzymes also include modified bacterial serine proteases such as those described in European Patent Application No. 87 303761.8, filed April 28, 1987 (especially pages 17, 24 and 98) and referred to herein as protease B, and in European Patent Application 199 404 Venegase, published October 29, 1986, which refers to a modified bacterial serine proteolytic enzyme, referred to herein as protease A. A suitable protease is referred to herein as protease C, which is · · · · · a variant of the alkaline serine protease from Bacillus in which lysine has replaced arginine at position 27, tyrosine has replaced valine at position 104, serine has replaced asparagine at position 123, and alanine has replaced threonine at position 274. Protease C is described in European Patent 90915958:4, which corresponds to WO 91/06637, published May 16, 1991. Genetically modified variants, in particular protease C, are also included.

A preferred protease, designated protease D, is a carbonyl hydrolase variant with an amino acid sequence that does not occur in nature, which is derived from a precursor carbonyl hydrolase by substituting with different amino acids a plurality of amino acid residues at a position of said carbonyl hydrolase that is equivalent to position +76, preferably also in combination with one or more amino acid residue positions equivalent to those selected from the group consisting of +99, +101, +103, +104, +107, +123, +27, +105, +109, +126, +128, +135, +156, +166, +195, +197, +204, +206, +210, +216, +217, +218, +222, +260, +265 and/or +274 according to the Bacillus amyloliquefaciens numbering. subtilisin, as described in WO 95/10591 and in the patent application of C. Ghose et al.: Bleaching Compositions Comprising Protease Enzymes, U.S. Patent Application No. 08/322,677, filed October 13, 1994. Also suitable is a variant of the carbonyl hydrolase protease described in WO 95/10591, which has an amino acid sequence derived by replacing a plurality of amino acid residues in the enzyme precursor corresponding to position +210 in combination with one or more of the following residues: +33, +62, +67, +76, +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 naturally occurring Bacillus amyloliquefaciens subtilisin or equivalent amino acid residues in other carbonyl hydrolases. hydrolases or subtilisins, such as Bacillus lentus subtilisin (co-pending U.S. patent application 60/048,550, filed June 4, 1997).

Also suitable according to the present invention are the proteases described in European Patent Application 251,446 and WO 91/06637, the BLAP ^(R) protease described in WO 91/02792 and their 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 a protease, one or more other enzymes and a reversible protease inhibitor are described in WO 92/03529 A (Novo). If desired, a protease with reduced adsorption and increased hydrolysis is available as described in WO 95/07791 (Procter & Gamble). A recombinant, trypsin-like protease for detergents suitable for use in the present invention is described in WO 94/25583 (Novo). Other suitable proteases are described in European Patent 516,200 (Unilever).

The proteolytic enzymes are present in the laundry detergent compositions and/or fabric care compositions of the present invention in an amount of from 0.0001 to 2, preferably from 0.001 to 0.2, more preferably from 0.005 to 0.1% by weight of pure enzyme based on the weight of the composition.

Cellulases useful in the present invention include both bacterial and fungal cellulases, preferably those having a pH optimum between 5 and 12 and a specific activity of about 50 CEVU/mg (CEVU stands for cellulose viscosity unit). Suitable cellulases are described in U.S. Patent 4,435,307 to Barbesgoard et al., Japanese Patent 61078384 and WO 96/02653, which describes fungal cellulases from Humicola insolens, Trichoderma, Thielavia and Sporotrichum. European Patent 739,682 describes cellulases isolated from novel Bacillus species. Suitable cellulases are described in British Patent Applications 2,075,028 and 2,095,275, German Patent Application No. OS 2,247,832 and WO 95/26398.

Examples of these cellulases are cellulases produced by the Humicola insolens strain (Humicola grisea var. thermoidea), especially Humicola strain DSM 1800.

Other suitable cellulases are cellulases derived from Humicola insolens with a molecular weight of 50,000, an isoelectric point of 5.5 and containing 415 amino acids, and an endoglucanase with a molecular weight of about 43,000 derived from Humicola insolens, DSM 1800, exhibiting cellulase activity; a preferred endoglucanase component has the amino acid sequence described in PCT patent application no. WO 91/17243. Also suitable cellulases are the EDIII cellulases from Trichoderma longibrachiatum, described in WO 94/21801, Genencor, published September 29, 1994. Particularly suitable cellulases are cellulases which have color care properties. Examples of such cellulases are the cellulases described in European patent application no. 9120287.9.2, filed November 6, 1991 (Novo). Carezyme and Celluzyme (Novo Nordisk A/AS) are particularly useful. See also WO 91/17244 and WO 91/21801. Other suitable cellulases with fabric care and/or cleaning properties are described in WO 96/34092, WO 96/17994 and WO 95/24471.

Said cellulases are usually contained in laundry detergent compositions and/or fabric care compositions in amounts of from 0.0001 to 2% by weight of pure enzyme based on the weight of the laundry detergent compositions and/or fabric care compositions.

Peroxidase enzymes are used in combination with oxygen sources, e.g. percarbonate, perborate, persulfate, hydrogen peroxide, etc. with a phenolic substrate as a molecular bleaching enhancer. They are used for bleaching solutions, i.e. to prevent the transfer of dyes or pigments removed from substrates during washing to other substrates present in the wash solution. Peroxidase enzymes known in the art include, for example, horseradish peroxidase, ligninase and haloperoxidases such as chloro- and bromo-peroxidase. Laundry detergent compositions and/or fabric care compositions containing peroxidase are described, for example, in PCT International Application WO 89/099813, WO 89/09813, in European Patent Application No. 91202882.6, filed November 6, 1991, and in European Patent 96870013.8, filed February 20, 1996. Laccase enzyme is also suitable.

Enhancers are usually present in an amount of from 0.1 to 5% by weight of the total composition. Preferred enhancers are substituted phenothiazine and phenoxazine 10-phenothiazinepropionic acid (PPT), 10-ethylphenothioazine-4-carbo4 44 44

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44 xylic acid (EPC), 10-phenoxazinepropionic acid (POP) and 10-methylphenoxazine (described in WO 94/12621) and substituted syringates (C3-C5 alkyl substituted syringates) and phenols. Preferred sources of hydrogen peroxide are sodium percarbonate or sodium perborate.

The peroxidases mentioned are usually contained in laundry detergent compositions and/or fabric care compositions in amounts of from 0.0001 to 2% by weight of pure enzyme based on the weight of the laundry detergent compositions and/or fabric care compositions.

Other suitable enzymes which may be included in the laundry detergent and/or fabric care compositions of the present invention are lipases. Suitable lipase enzymes for use in detergents include those produced by microorganisms of the Pseudomonas group, such as Pseudomonas stutzeri ATCC 19154, as described in British Patent Specification 1,372,304. Suitable lipases include those lipases which show a positive immunological cross-reaction with the lipase antibody produced by the Pseudomonas fluorescent IAM 1057 microorganism. This lipase is available from Amano Pharmaceutical Co. Ltd., Nagoya, Japan, under the trade name Lipase P Amano, hereinafter also referred to as Amano-P. Other commercial 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 US Biochemical Corp., USA, and Diosynth Co., Netherlands, and lipases from Pseudomonas gladioli. Particularly suitable lipases are lipases such as M1 Lipase ^(R) and Lipomax ^(R) (Gist-Brocades) and Lipolase ^(R) and Lipolase Ultra ^(R) (Novo), which have been found to be very effective when combined with the compositions of the present invention. Also suitable are the lipolytic enzymes described in European Patent 258,068, WO 92/05249 and WO 95/22615 (Novo Nordisk) and WO 94/03578, WO 95/35381 and WO 96/00292 (Unilever).

• · 99··

Also suitable are cutinases [EC 3.1.1.50], which can be considered as a special type of lipase, namely lipases, which do not require interfacial 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 included in laundry detergent compositions and/or fabric care compositions in an amount of from 0.0001 to 2% by weight of pure enzyme based on the weight of the laundry detergent compositions and/or fabric care compositions.

Amylases (α and/or β) may be included in the detergents for the removal of carbohydrate-based stains. WO 94/02597, Novo Nordisk A/S, published February 3, 1994, describes detergent compositions that include mutated amylases. See also WO 95/10603, Novo Nordisk A/S, published April 20, 1995. Other amylases known for use in detergent compositions include both α- and β-amylases. α-Amylases are known in the art and include those amylases described in U.S. Pat. No. 5,003,257, European Patent 252,666, WO 91/00353, French Patent 2,676,456, European Patent 285123, European Patent 525,610, European Patent 368,341 and British Patent No. 1,296,839 (Novo). Other suitable amylases are the enhanced stability amylases described in WO 94/18314, published August 18, 1994, and WO 96/05295, Genencor, published February 22, 1996, and amylase variants with further modification in the immediate precursor available from Novo Nordisk A/S, described in WO 95/10603, published April 1995. Suitable amylases are also described in European Patent 277,216 and in WO 95/26397 and WO 96/23873 (all Novo Nordisk).

Examples of commercial α-amylase products are Purafect Ox Am ^(R> from Genencor and Termamyl ^CR) , Ban ^(R) , Fungamyl ^(R) and Duramyl ^(R) , all available from Novo Nordisk A/S, Denmark. WO 95/26397 describes other suitable amylases: α-amylases which are characterized by a specific activity at least 25% higher than that of

99

9 9 9 9

9 9 9

9 9 9 9 9

9 9 9 9

99 ♦ » 9 99 9

9 9 9 9 9 ♦ 9 99 9 ·

9 9 9 9 9 9

9 9 9 9 9

99 99 specific activity of Termamyl ^(R) at a temperature in the range of 25 to 55 °C and a pH in the range of 8 to 10, measured by the Phadebas ^(R) α-amylase activity test. Variants of the above enzymes are suitable, as described in WO 96/23873 (Novo Nordisk). Other amylolytic enzymes with improved properties in terms of activity level and a combination of thermostability and higher activity levels are described in WO 95/35382.

The amylolytic enzymes are included in the laundry detergent compositions and/or fabric care compositions of the present invention in an amount of from 0.0001 to 2, preferably from 0.00018 to 0.06, more preferably from 0.00024 to 0.08% by weight of pure enzyme based on the weight of the composition.

The above enzymes may be of suitable origin, such as plant, animal, bacterial, fungal and yeast origin. The origin may further be mesophilic or extremophilic (psychrophilic, psychrotropic, thermophilic, barophilic, alkalophilic, acidophilic, halophilic, etc.). Purified or non-purified forms of these enzymes may be used. It is now common practice to modify natural enzymes by protein/genetic engineering techniques in order to optimize their performance in the detergent compositions of the invention. For example, variants may be designed such that the compatibility of the enzyme with the ingredients typically encountered in these compositions is increased. Other variants may also be designed so that the optimum pH, stability towards bleaching or chelating agents, catalytic activity and similar properties of the enzyme variant are adjusted to suit the particular cleaning application.

Particular attention should be paid to oxidation-sensitive amino acids for bleach stability and to surface charges for surfactant compatibility. The isoelectric point of such enzymes can be modified by substitution of some charged amino acids, e.g. increasing the isoelectric point can help improve compatibility with anionic surfactants. The stability of these enzymes can be further enhanced by e.g. formation of salt addition bridges and strengthening of calcium binding sites to increase stability towards chelating agents. Special attention

99··

99

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9 9 9 9

99 • 9 99 • 999 9 ·

9 99 9 9

9 9 9 · 9 9 • · · · 9 9

99 99 must be devoted to cellulases, because most cellulases have separate binding domains (CBDs). The properties of such enzymes can be altered by modifications in these domains.

The enzymes are normally present in laundry detergent compositions and/or fabric care compositions in amounts of from 0.0001 to 2% by weight of pure enzyme based on the weight of the laundry detergent compositions and/or fabric care compositions. The enzymes may be added as separate individual components (pieces, granules, stabilized liquids, etc. containing one enzyme) or as mixtures of two or more enzymes (e.g. cogranulates).

Other suitable detergent additives which may be added are enzyme oxidation scavengers which are described in the accompanying European Patent Application 92870018.6, filed January 31, 1992. Examples of these are ethoxylated tetraethylene polyamines.

Many enzyme materials and means for incorporating them into synthetic detergent compositions are also described in WO 93/07263 A and WO 93/07260 A (Genencor International), WO 89/08694 A (Novo), and U.S. Patent 3,553,139, January 5, 1971, to McCarty et al. Enzymes are further described in U.S. Patent No. 4,101,457 to Place et al., July 18, 1978, and U.S. Patent No. 4,507,219 to Hughes,

March 26, 1985. Enzyme materials useful for liquid detergent compositions and their inclusion in such compositions are described in U.S. Patent No. 4,261,868 to Hory et al., April 14, 1981. Enzymes for use in detergent compositions can be stabilized in various ways. Methods for stabilizing enzymes are described and exemplified in U.S. Patent 3,600,319 to Gedge et al., August 17, 1971, in European Patent 199405 and in European Patent 200,586 to Venegas, October 29, 1986. Enzyme stabilization systems are also described, for example, in U.S. Patent 3,519,570. A useful Bacillus, sp. AC13, providing proteases, xylanases and cellulases, is described in WO 94/01532 A (Novo).

·»··

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Color care and fabric care effects: This also includes technologies that provide color retention. Examples of these technologies are metal catalysts for color retention. These metal catalysts are described in the accompanying European Patent Application No. 92870181.2. Dye fixing agents, polyolefin dispersion for anti-wrinkle and improved water absorbency, perfume and amine-functional polymer (PCT/US 97/16546) for color treatment and for perfume impression are other examples of color/fabric care technologies and are described in the accompanying European Patent Application No. 96870140.9, filed November 7, 1996.

Fabric softening agents may also be included in the laundry detergent and/or fabric care compositions of the present invention. These agents may be inorganic or organic. Organic softening agents include water-insoluble tertiary amines as described in British Patent Specification A 1 514 276 and European Patent Application B 0 011 340. Their combinations with mono(C 12-14) quaternary ammonium salts are described in European Patent Applications B 0 026 527 and B 0 026 528 and amides with two long chains are described in European Patent Application B 0 242 919. Other useful organic components of fabric softening systems include high molecular weight polyethylene oxide materials as described in European Patent Applications A 0 299 575 and 0 313146.

Organic fabric softening agents such as water-insoluble tertiary amines or two long chain amide materials are included in the compositions in amounts of from 0.5 to 5, normally from 1 to 3% by weight, while high molecular weight polyethylene oxide materials and water-soluble cationic materials are added in amounts of from 0.1 to 2, normally from 0.15 to 1.5% by weight. These materials are normally added to the spray dried portion of the composition, although in some cases it may be more convenient to add them as a dry bulk mixture or to spray them as a molten liquid onto other solid components of the composition.

Chelating agents: The laundry detergent compositions and/or fabric care compositions according to the invention may optionally also contain one or more chelating agents »♦ 999· ·· ·· ♦ · 1 • · « » · · 1

I 9 9 « ·« ·♦ iron and/or manganese agents. These chelating agents may be selected from the group consisting of aminocarboxylates, aminophosphonates, polyfunctionally substituted aromatic chelating agents, and mixtures thereof, all as described below. Without wishing to be bound by theory, it is believed that the beneficial effects of these materials reside in part in their exceptional ability to remove iron and manganese ions from laundry solutions by forming soluble chelates.

Aminocarboxylates useful as optional chelating agents include ethylenediaminetetraacetates, N-hydroxyethylethylenediaminetriacetates, nitriletriacetates, ethylenediaminetetrapropionates, triethylenetetraminehexaacetates, diethylenetriaminepentaacetates, and ethanoldiglycines, their alkali metal salts, ammonia, and their substituted ammonium salts, and mixtures thereof.

Aminophosphonates are also suitable for use in the compositions of the invention as chelating agents, provided that at least low levels of total phosphorus are permitted in the detergent compositions. These agents include ethyldiaminetetrakis(methylenephosphonates), such as Dequest. These aminophosphonates preferably do not contain an alkyl or alkenyl group of more than 6 carbon atoms.

Polyfunctionally substituted aromatic chelating agents are also useful in the compositions of the invention. See U.S. Patent 3,812,044 to Connor et al., issued May 21, 1974. Preferred compounds of this type in the acid form are dihydroxydisulfobenzenes, such as 1,2-dihydroxy-3,5-disulfobenzene.

A preferred biodegradable chelating agent used herein is ethylenediamine disuccinate (EDDS), particularly the [S,S]-isomer, as described in U.S. Patent 4,704,233 to Hartman and Perkins, November 3, 1987.

The compositions of the invention may also contain water-soluble salts (or the acid form) of methylglycinediacetic acid (MGDA) as a chelating agent ♦0 0000

00

0 0 0 t 0 0 *

0 0 0

0 0 0

00 or a co-used building component useful for example with insoluble building components such as zeolites, layered silicates and the like.

If used, these chelating agents will typically be present in the laundry detergent and/or fabric care compositions of the invention in amounts of from 0.1 to 15% by weight of the compositions of the invention. More preferably, if used, these chelating agents will be present in the compositions in amounts of from 0.1 to 3.0% by weight of the compositions.

Foam Suppressors: Another optional ingredient is a foam suppressor, exemplified by silicones and silica-silicone blends. Silicones can generally be represented by alkylated polysiloxane materials, while silica is normally used in finely powdered form, exemplified by silica aerogels and xerogels and hydrophobic silicas of various types. These materials can be incorporated into the compositions as particles in which the foam suppressor is preferably releasably incorporated in a water-soluble or water-dispersible carrier that is substantially impermeable to non-surfactant detergent. The foam suppressor can also be dissolved or dispersed in a liquid carrier and applied by spraying onto one or more other ingredients. A preferred silicone suds control agent is described by Bartollot et al.: U.S. Patent 3,933,672. Other particularly useful suds suppressors are the self-emulsifying silicone suds suppressors described in German Patent Application DTOS 2,646,126, published April 28, 1977. An example of such a compound is DC-544, commercially available from Dow Corning, which is a siloxane/glycol copolymer. A particularly preferred suds control agent is a suds suppressor system 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.

This foam suppressor system is described in the accompanying European Patent Application No. 92870174.7, filed November 10, 1992.

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44 4·

Particularly preferred silicone suds control agents are described in the accompanying European Patent Application No. 92201649.8. Said compositions may comprise a silicone/silica blend in combination with a powdered non-porous silica such as Aerosil ^(R) .

Foam suppressors, as described above, are typically used in amounts of from 0.001 to 2, preferably from 0.01 to 1% by weight of the composition.

Other Ingredients: Other ingredients such as soil suspending agents, soil release agents, optical brighteners, abrasives, bactericides, fade inhibitors, coloring agents and/or encapsulated or unencapsulated perfumes may be used in laundry detergent compositions and/or fabric care compositions.

Particularly suitable encapsulation materials are water-soluble capsules consisting of a matrix of polysaccharide and polyhydroxy compounds, as described in British Patent Specification 1,464,616. Other suitable water-soluble encapsulation materials include dextrins derived from non-gelatinizing starch esters of substituted dicarboxylic acids, as described in U.S. Patent No. 3,455,838. These dextrin acid esters are preferably prepared from starches derived from waxy maize, waxy sorghum, sago, tapioca and potato. Suitable examples of encapsulation materials include N-Lok manufactured by National Starch. The N-Lok encapsulation material consists of modified corn starch and glucose. This starch is modified by the addition of monofunctional substituted groups, such as octenyl succinic anhydride.

Anti-soil re-deposition and soil suspension agents suitable for use in the present invention include cellulose derivatives such as methylcellulose, carboxymethylcellulose and hydroxyethylcellulose, and homo- or co-polymers of polycarboxylic acids or their salts. Polymers of this type include polyacrylates and copolymers of maleic anhydride and acrylic acid, as mentioned above as building blocks, as well as copolymers of maleic anhydride with ethylene, methylvinylether or methacrylic acid, the maleic anhydride constituting at least 20 mole percent of the copolymer. These materials are normally used in amounts of from 0.5 to 10 wt.%, more preferably from 0.75 to 8, most preferably from 1 to 6 wt.%.

Preferred optical brighteners are anionic in nature. Examples thereof are disodium 4,4'-bis-(2-diethanolamino-4-anilino-1,3,5-triazin-6-ylamino)stilbene-2:2'-disulfonate, disodium 4,4'-bis-(2-morpholino-4-anilino-1,3,5-triazin-6-ylamino)stilbene-2:2'-disulfonate, disodium 4,4'-bis-(2,4-dianilino-1,3,5-triazin-6-ylamino)stilbene-2:2'-disulfonate, monosodium 4',4-bis-(2,4-dianilino-1,3,5-triazin-6-ylamino)stilbene-2-sulfonate, disodium 4,4'-bis-(2-anilino-4-(N-methyl-N-2-hydroxyethylamino)-1,3,5-triazin-6-ylamino)stilbene-2:2'-disulfonate, disodium salt of 4,4'-bis-(4-phenyl-1,1,5-triazol-1,3,5-ylamino)stilbene-2:2'-disulfonate, disodium salt of 4,4'-bis-(2-anilino-4-(1-methyl-2-hydroxyethylamino)-1,3,5-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 brightening agents are the specific brightening agents described in European patent 753 567.

Other useful polymeric materials are polyethylene glycols, particularly those having a molecular weight of 1000 to 10,000, particularly 2000 to 8000, and most preferably 4000. They are used in amounts of from 0.20 to 5, more preferably from 0.25 to 2.5 wt. %. These polymers and the previously mentioned homo- and co-polymeric polycarboxylate salts are valuable for improving whiteness retention, ash deposition on fabrics, and performing cleaning of clay, proteinaceous, and oxidizable soils in the presence of transition metal impurities.

The soil release agents useful in the compositions of the present invention are conventionally copolymers or terpolymers of terephthalic acid with ethylene glycol and/or propylene glycol units in various arrangements. Examples of such polymers are described in U.S. Patent Nos. 4,116,885 and 4,711,730 and in European Published Patent Application No. 0,272,033. A particularly preferred polymer according to European Patent Application No. 0,272,033 is a compound of the general formula *··»·· 9,999 99 99

9 9 9 9 9 9 9 9 9 9

9 99 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 · · · *

99 99 9 9 99 9 9 (CH ₃ (PEG) 4 3 ) o.7 ₅ (POH) _o.25 F-PO) 2. ₈ r-PEG) o.4]T(PO-H) _o.25 ((PEG) 4 ₃ CH ₃ ) o.7 ₅ , in which PEG represents the group -(OC ₂ H ₄ ) O-, PO represents the group (OC ₃ H ₆ O) and T represents the group (pcOC ₅ H ₄ CO).

Also very useful are modified polyesters, such as random copolymers of dimethyl terephthalate, dimethyl sulfoisophthalate, ethylene glycol and 1,2-propanediol, the end groups consisting 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 most of the copolymers of the invention will be end-capped with sulfobenzoate groups. However, some copolymers will be less than fully capped and their end groups may therefore consist of monoesters of ethylene glycol and/or propane-1,2-diol, which secondarily consist of these moieties.

Selected polyesters according to the invention contain 46 wt. % dimethylterephthalic acid, 16 wt. % propane-1,2-diol, 10 wt. % ethylene glycol, 13 wt. % dimethylsulfobenzoic acid and 15 wt. % sulfoisophthalic acid and have a molecular weight of around 3000. The polyesters and the method of their production are described in detail in European patent application 311 342.

It is well known in the art that free chlorine in tap water rapidly deactivates enzymes contained in detergent compositions. The use of a chlorine scavenger, such as perborate, ammonium sulfate, sodium sulfite or polyethyleneimine, in an amount greater than 0.1% by weight of the total composition, improves the stability of the detergent enzymes during washing. Compositions containing a chlorine scavenger are described in European Patent Application 92870018.6, filed January 31, 1992.

Alkoxylated polycarboxylates, such as those prepared from polyacrylates, are useful in the present invention to achieve further grease removal.

9,999

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M 99

9 · • ·· • · 9

These materials are described in WO 91/08281 and PCT 90/01815 at page 4 et seq., both of which are incorporated herein by reference. Chemically, these materials comprise polyacrylates having one ethoxy group for every 7 to 8 acrylate units in the side chain. The side chains have the general formula -(CH ₂ CH ₂ O) _m (CH ₂ ) n CH ₃ , where m is a number from 2 to 3 and n is a number from 6 to 12. The side chains are ester-linked to the polyacrylate backbone, thus providing a comb-type polymer structure. The molecular weight may vary, but is typically in the range of 2000 to 50,000. These alkoxylated polycarboxylates may be present in the compositions in an amount from 0.05 to 10% by weight.

Dispersing agents: The laundry detergent compositions and/or fabric care compositions of the present invention may also contain dispersing agents. Suitable water-soluble organic salts are homo- or co-polymeric acids or salts thereof, in which the polycarboxylic acid contains at least two carboxyl groups separated by not more than two carbon atoms. Polymers of this type are described in British Patent Specification No. A 1,596,756. Examples of these salts are polyacrylates of molecular weight 2000 to 5000 and their copolymers with maleic anhydride, such as copolymers of molecular weight from 1000 to 100,000.

In particular, copolymers of acrylates and methacrylates, such as 480N, which has a molecular weight of 4000, may be added to the laundry detergent and/or fabric care compositions of the present invention in an amount of from 0.5% to 20% by weight of the composition.

The compositions of the invention may comprise a lime soap peptizing compound having a lime soap dispersing power (LSDP), as defined herein below, of not more than 8, preferably not more than 7, most preferably not more than 6. The lime soap peptizing compound is preferably present in an amount of from 0% to 20% by weight.

V9 ···« ·· ··

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Φ· • · * « • · ·« • · · · · • · · · ·· *· • · · • · · • · 9 · • · · · ·· 99

A numerical measure of the effectiveness of a lime soap peptizing compound is given by the lime soap dispersion value (LSDP), which is measured using the lime soap dispersion test as described in the article by HC Borghetty and CA Bergman: J. Am. Oil. Chem. Soc. 1950, 27, 88-90. This milk of lime dispersion test is widely used in the practice of the art, and is referred to, for example, in the following review articles: WN Linfield: Surfactant Science Senes 7, 3, WN Linfield: Tenside Surf. Det. 1990, 27, 159-163, and Μ. K. Nagarajan, WF Masler Cosmetics and Toiletries 1989, 104, 71-73. LSDP means the ratio of % by weight of dispersant to sodium oleate required to disperse lime soap deposits formed by the action of 0.025 g of sodium oleate in 30 ml of water with a hardness equivalent of 333 ppm CaCO ₃ (Ca:Mg ratio = 3:2).

Surfactants that have good peptizing properties of lime soap compounds include certain 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 dimethylamine oxide having 16 to 18 carbon atoms, alkyl (12 to 18 carbon atoms) ethoxysulfates having an average degree of ethoxylation of from 1 to 5, especially alkyl (12 to 15 carbon atoms) ethoxysulfate surfactant having an ethoxylation degree of 3 (LSDP = 4), and ethoxylated alcohols having 14 to 15 carbon atoms with an average degree of ethoxylation of either 12 (LSDP = 6) or 30, sold under the trade names Lutensol A012 and Lutensol A030 by BASF GmbH.

Polymeric peptizing milks of lime suitable for use in the invention are described in the article by M. K. Nagarajan and W. F. Masler: Cosmetics and Toiletries 1989, 104, 71-73.

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

Hydrophobic bleaching agents such as 4-[N-octanoyl-6-aminohexanoyl]benzenesulfonate, 4-[N-nonanoyl-6-aminohexanoyl]benzenesulfonate, 4-[N-decanoyl-6-aminohexanoyl]benzenesulfonate and mixtures thereof and nonanoylbenzenesulfonate together with hydrophilic/hydrophobic bleaching agents can also be used as lime milk peptizing compounds.

Inhibition of Dye Transfer: The laundry detergent compositions and/or fabric care compositions of the present invention also contain compounds for inhibiting the transfer from one fabric to another of those dyes that are solubilized and suspended and that the fabric encounters during washing processes in which colored fabrics are washed.

Polymeric dye transfer inhibiting agents: The laundry detergent compositions and/or fabric care compositions of the present invention also contain from 0.001 to 10%, preferably from 0.01 to 2, more preferably from 0.05 to 1% by weight of polymeric dye transfer inhibiting agents. These polymeric dye transfer inhibiting agents are normally included in laundry detergent compositions and/or fabric care compositions to inhibit the transfer of dyes from colored fabrics to fabrics being washed with them. These polymers have the ability to complex or absorb volatile dyes washed out of colored fabrics before these dyes have a chance to adhere to other items during washing.

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

The addition of these polymers also increases the efficiency of the enzymes of the invention.

a) Polyamine-N-oxide polymers: Polyamine-N-oxide polymers preferred for use according to the invention contain units of the following general formula I • · · · • 4 ·· • ’ · · · · · 4 4 · ·

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

44 44 4 4 44 44

P

R wherein P represents a polymerizable unit to which the R-N-O group may be attached or where the R-N-O group may form part of the polymerizable unit or both.

A represents one of the following structures: -NC(O)-, -C(O)O-, -CO-, -S-, -0-, -N-, x represents the number 0 or 1 and R represents an aliphatic, ethoxylated aliphatic, aromatic, heterocyclic or alicyclic group or any combination thereof to which the nitrogen atom of the N-O group may be attached or part of which may form the nitrogen atom of the N-O group.

The group N-0 can mean a group of the following general formulas

About About

II (Ri)xN-(R ₂ ) _y or =N-(R ₁ ) _X ,

I (R ₃ ) _z in which Ri, R ₂ and R ₃ represent an aliphatic, aromatic, heterocyclic or alicyclic group or combinations thereof, x or/and or/and represent the number 0 or 1 and the nitrogen atom of the group N-0 may be attached to or may form part of these groups.

The N-0 group may be part of a polymerizable unit (P) or may be 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 said polyamine N-oxides includes the group of polyamine N-oxides in which the nitrogen atom of the N-O group forms part of the R group. Preferred polyamine N-oxides are those compounds in which R represents a heterocyclic group such as pyridine, pyrrole, imidazole, pyrrolidine, piperidine, quinoline, acridine and derivatives thereof.

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

Other suitable polyamine N-oxides are those 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 I, in which R represents an aromatic, heterocyclic or alicyclic group, in which the nitrogen atom of the functional group N-0 represents part of said group R.

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

Another preferred group of polyamine N-oxides are polyamine oxides of general formula I, in which R represents an aromatic, heterocyclic or alicyclic group, in which the nitrogen atom of the functional group N-0 is connected to said R groups.

Examples of these groups are polyamine oxides in which the R groups may represent an aromatic group such as a phenyl group.

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

• φ φ φ φφφφ • « φ φ «« φ φφφφ • ΦΦΦ « φ · · φ φ · φ φφ φφφ « φφφ φφ · φ φφ · φ · · φ φ φφ φ φφ φφ φφ «· ··

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 number of amine oxide groups present in the polyamine oxide polymer may vary depending on the particular copolymerization or the particular degree of N-oxidation. The amine to amine-N-oxide ratio is preferably from 2:3 to 1:1,000,000, more preferably from 1:4 to 1:1,000,000, most preferably from 1:7 to 1:1,000,000. Indeed, the polymers of the present invention include random or block copolymers where one type of monomer is an amine-N-oxide and the other type of monomer is either an amine-N-oxide or not. The amine oxide unit of the polyamine-N-oxides has a PKa < 10, preferably a PKa < 7, more preferably a PKa < 6.

Polyamine oxides can be obtained with almost any degree of polymerization. The degree of polymerization is not critical provided that the material has the desired water solubility and the desired dye suspending activity.

The average molecular weight is typically in the range of 500 to 1,000,000, preferably 1,000 to 50,000, more preferably 2,000 to 30,000, and most preferably 3,000 to 20,000.

b) Copolymers of N-vinylpyrrolidone and N-vinylimidazole: The polymers of N-vinylpyrrolidone and N-vinylimidazole used in the present invention have an average molecular weight in the range of from 5000 to 1,000,000, more preferably from 5000 to 200,000.

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

The average molecular weight range was determined by light scattering as described by Barth H.G. and Mays J.W.: Chemical Analyses 113, Modem Methods of Polymer Characterization.

• · • · · ·

Highly preferred N-vinylimidazole/N-vinylpyrrolidone copolymers have an average molecular weight in the range of from 5000 to 50,000, more preferably from 8000 to 30,000, and most preferably from 10,000 to 20,000.

The N-vinylimidazole/N-vinylpyrrolidone copolymers are characterized in that the above average molecular weight range provides excellent dye transfer inhibition properties without adversely affecting the cleaning performance of the detergent compositions prepared herein.

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

c) Polyvinylpyrrolidone: The laundry detergent and/or fabric care compositions of the present invention may also employ polyvinylpyrrolidone (PVP) having an average molecular weight of from 2500 to 400,000, preferably from 5000 to 200,000, more preferably from 5000 to 50,000, and most preferably from 5000 to 15,000. Suitable polyvinylpyrrolidones are commercially available from ISP Corporation, New York, N.Y., and Montreal, Canada, under the product names PVP K-15 (viscosity molecular weight 10,000), PVP K-30 (viscosity molecular weight 40,000), PVP K-60 (viscosity molecular weight 160,000), and PVP K-90 (viscosity molecular weight 360,000). Other suitable polyvinylpyrrolidones commercially available from BASF Cooperation include Sokalan HP 165 and Sokalan HP 12, polyvinylpyrrolidones known to those skilled in the detergent art (see, for example, European Patent Applications 262,897 and 256,696).

d) Polynyloxazolidone: The laundry detergent compositions and/or fabric care compositions of the present invention may also employ polyvinyloxazolidone as the polymeric dye transfer inhibiting agent. Said polyvinyloxazolidones have • ·

an average molecular weight of from 2500 to 400,000, preferably from 5000 to 200,000, more preferably from 5000 to 50,000 and most preferably from 5000 to 15,000.

e) Polyvinylimidazole: The laundry detergent compositions and/or fabric care compositions of the present invention may also employ polyvinylimidazole as the polymeric dye transfer inhibiting agent. Said polyvinylimidazoles have an average molecular weight of from 2500 to 400,000, preferably from 5000 to 200,000, more preferably from 5000 to 50,000, and most preferably from 5000 to 15,000.

f) Crosslinked polymers: Crosslinked polymers are polymers whose backbones are interconnected to some degree. These linkages may be chemical or physical in nature, possibly with active groups on the backbone or on the branches. Crosslinked polymers have been described in the Journal of Polymer Science 22, 1035-1039. In one embodiment, the crosslinked polymers are made to form a three-dimensional rigid structure that can entrap dyes in the pores formed by the three-dimensional structure. In another embodiment, the crosslinked polymers entrap these dyes by swelling. These crosslinked polymers are described in the accompanying patent application 94870213.9.

Washing Process: The compositions of the invention can be used in essentially any washing or cleaning process, including soaking processes, pretreatment processes, and processes with a rinsing step, in which a separate rinsing aid may be added.

The method described herein comprises contacting the materials with a washing solution in a conventional manner and as hereinafter exemplified. The method according to the invention is preferably carried out during the washing process. The cleaning method is preferably carried out at 5 to 95 °C, in particular between 10 and 60 °C. The pH value of the treatment solution is preferably from 7 to 12.

• 9 ··

9 9·9 ·

9

9

9 • 9 99 99 99 99 99

The following examples are intended to be illustrative of compositions of the present invention, but are not necessarily intended to limit or otherwise define the scope of the invention. In laundry detergent compositions and/or fabric care compositions, the amounts of enzyme are expressed as the weight of pure enzyme based on the weight of the total composition, and, unless otherwise indicated, detergent additives are expressed as the weight based on the total weight of the compositions. The abbreviated ingredient identifications in this specification have the following meanings:

LAS: sodium linear alkyl(C11-C13)benzenesulfonate

TAS: sodium tallow alkyl sulfate

CxyAS: alkyl(with 1 x to 1 y carbon atoms)sodium sulfate

CxySAS: secondary (2,3) alkyl (with 1 x to 1 y carbon atoms) sodium sulfate

CxyEz: predominantly linear primary alcohol with 1x to 1y carbon atoms condensed with an average of z moles of ethylene oxide CxyEzS: sodium alkyl sulfate with 1x to 1y carbon atoms condensed with an average of z moles of ethylene oxide

QAS: R ₂ .N ⁺ (CH ₃ ) 2 (C ₂ H4OH) with R ₂ representing a group with 12 to 14 carbon atoms

QAS 1: R ₂ .N ⁺ (CH ₃ ) 2 (C ₂ H ₄ OH) with R ₂ representing a group with 8 to 11 carbon atoms

APA: amido(C8-C10)propyldimethylamine soap: linear sodium alkyl carboxylate derived from a mixture (80/20) of tallow and coconut oil nonionic: mixed ethoxylated/propoxylated fatty alcohol (C13-C15) with an average degree of ethoxylation of 3.8 and an average degree of propoxylation of 4.5

Neodol 45-13: a linear primary alcohol (with 14 to 15 carbon atoms) ethoxylate sold by Shell Chemical Co.

STS: sodium toluenesulfonate

CFAA: alkyl(with 12 to 14 carbon atoms)N-methyl-glucamide

TFAA: alkyl(C16-C18)N-methyl-glucamide

TPKFA: fatty acids with 12 to 14 carbon atoms silicate: amorphous sodium silicate (SiO ₂ :Na ₂ O ratio is 1.6 to 3.2) ·· » «

9· •9 ·999 metasilicate: zeolite A:

NASKS-6:

citrate:

citric acid:

borate:

carbonate:

bicarbonate:

sulfate:

Magnesium sulfate: STPP:

TSPP:

MA/AA:

MA/AA 1:

AA:

PA30:

480N:

polygel/carbopol:

PB1:

PB4:

sodium metasilicate (SiO ₂ :Na ₂ O ratio = 1.0) hydrated sodium aluminosilicate of formula Na ₁₂ (AIO ₂ SiO ₂ )i ₂ .27 H ₂ O with a primary particle size ranging from 0.1 to 10 pm (weight expressed on an anhydrous basis) crystalline layered silicate of formula 6-Na ₂ Si ₂ O ₅ sodium citrate dihydrate with an activity of 86.4% with a particle size distribution between 425 pm and 850 pm anhydrous citric acid sodium borate anhydrous sodium carbonate with a particle size between 200 pm and 900 pm anhydrous sodium bicarbonate with a particle size between 400 pm and 1200 pm anhydrous sodium sulphate anhydrous magnesium sulphate anhydrous sodium triphosphate sodium diphosphate maleic/acrylic acid random copolymer in ratio 1:4 with an average molecular weight of 70,000 to 80,000 random maleic/acrylic acid copolymer in a ratio of 6:4 with an average molecular weight of 10,000 sodium polyacrylate polymer with an average molecular weight of 4,500 polyacrylic acid with an average molecular weight of 4,500 to 8,000 random acrylic/methacrylic acid copolymer in a ratio of 7:3, average molecular weight around 3,500 cross-linked polyacrylates with high molecular weight anhydrous sodium perborate monohydrate with the formula NaBO ₂ .H ₂ O ₂ anhydrous sodium perborate tetrahydrate with the formula NaBO ₂ .3 H ₂ OH ₂ O ₂

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

87 percarbonate: NaDCC: anhydrous sodium percarbonate formula 2 Na ₂ CO ₃ .3 H ₂ O ₂ 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: diethylenetriaminepenta(methylene)phosphonic acid sold by Monsanto under the trade name Dequest 2060 EDDS: ethylenediamine-N,N'-disuccinic acid, (S,S)-isomer in the form of its sodium salt MnTACN: Manganese 1,4,7-trimethyl-1,4,7-triazacyclononane

photoactivated bleaching agent: sulfonated zinc phthalocyanine encapsulated in a soluble dextrin polymer photoactivated bleaching agent 1: sulfonated aluminum phthalocyanine encapsulated

from the soluble polymer dextrin PAAC: cobalt(III) pentaamine acetate salt paraffin: paraffin oil sold under the trade name Winog 70 by Wintershall Regarding: sodium benzoate BzP: benzoyl peroxide mannanasa: mannanase from Bacillus agaradherens, NCIMB 40482 protease: a proteolytic enzyme sold under the trade names Savinase, Alcalase, and Durazym by Novo Nordisk A/S, Maxacal, and amylase: Maxapem, sold by Gist-Brocades, and proteases described in WO 91/06637 and/or WO 95/10591 and/or European Patent 251 446, amylolytic enzyme sold under the trade name Purafact Ox Am ^R , described in WO 94/18314 and WO 96/05295, sold by Genencor, Termamyl ^(R) , Funga-

44

4444 44 4 4 4 4 · «4 44 4 4444

94 444 4 444 44 4

4444 4444 4444

44 44 44 44 44

lipase: myl ^(R) and Duramyl ^(R) , all available from Novo Nordisk A/S, and those described in WO 95/26397 lipolytic enzyme sold under the trade name cellulase: Lipolase and Lipolase Ultra by Novo Nordisk A/S and Lipomax by Gist-Brocades a cellulite enzyme sold under the trade name Care- clay: zyme, Celluzyme and/or Endolase by Novo Nordisk A/S smectite clay or bentonite clay CMC: sodium carboxymethylcellulose PVP: polyvinyl polymer with an average molecular weight PVNO: 60,000 number average molecular weight poly(4-vinylpyridine)-N-oxide PVPVI: 50,000 copolymer of vinylimidazole and vinylpyrrolidone with an average clarifier 1: molecular weight 20,000 4,4'-bis(2-sulfostyryl)biphenyl disodium salt clarifier 2: 4,4'-bis(4-anilino-6-morpholino-1,3,5-triazin-2-yl)stil- disodium salt ben-2:2'-disulfonate silicone antifoam agent: polydimethylsiloxane foam regulator with siloxane- -oxyalkylene copolymer as dispersant with a ratio of the former to the latter from 10:1 to 100:1 granulated foam suppressor: 12% by weight silicone/silica, 18% opaquing agent: wt. stearyl alcohol, 70% wt. starch in granular form water-based monostyrene latex mixture, sold SRP1: BASF Aktiengesellschaft under the trade name Lytron 621 anionic end-capped polyesters SRP2: diethoxylated poly(1,2-propylene terephthalate) short block QEA: polymer bis((C ₂ H ₅ O)(C ₂ H4O) _n )(CH ₃ )-N ⁺ -C6Hi2-N ⁺ -(CH3)

44

4 4 4

4 4 4

4 4 4

4 4 4

44 ··♦· • · 4·

4*44 44

4 44 4 4 4

4 4 4 4 4 4

4 4 · 4 4 4

44 44 44

REI:

SCS:

HMWPEO:

PEGx:

PEO:

TEPAE:

bis(( _C2H5O )-( _C2H4O ))nwhere n is a number from 20 to 30 polyethyleneimine with an average molecular weight of 1800 and an average degree of ethoxylation of 7 ethyleneoxy groups per nitrogen atom sodium cumenesulfonate high molecular weight polyethylene oxide polyethylene glycol with a molecular weight of x polyethylene oxide with an average molecular weight of 5000 tetraethylenepentamine ethoxylate

Examples of embodiments of the invention

Example 1

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

I II III Blown powder zeolite A 13.0 13.0 15.0 sulfate - 3.0 - 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

9999 ·· • · · * • · ·· » · · · • · · 9

99

99

9 9 9 9

9 9 9 9

9 9 9 9 9

9 9 9 9

99 99

Agglomerate LAS 5.0 5.0 5.0 TAS 2.0 1.0 1.0 silicate 3.0 2.0 4.0 zeolite A 8.0 8.0 8.0 carbonate 7.0 4.0 4.0 Sprayer perfume 0.3 0.3 0.3 C45E7 2.0 2.0 2.0 C25E3 2.0 - - Dry ingredients citrate 3.0 - 2.0 bicarbonate - 3.0 - carbonate 8.0 15.0 10.0 TAED 6.0 2.0 5.0 PB1 9.0 7.0 10.0 PEO - - 0.2 bentonite clay 10.0 10.0 10.0 mannanasa 0.001 0.001 0.02 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 antifoaming agent 5.0 5.0 5.0 sulfate - 3.0 - density (g/liter) miscellaneous and minor components 850 850 top up to 100% 850

φφ φφ φ φ φ · φ φ φ φ φ φ · φ φ φ φ φφ φφ φφ φφ • φ φ · φ ΦΦΦ φ φ φ φ φ φ φ φφ φφ φφ φφφφ φ · φ

ΦΦΦ φ φ φ φ φ φ φφ φ·

Example 2

The following liquid detergent compositions were prepared according to the present invention (amounts are in parts by weight, enzyme means pure enzyme).

1 II III IV LAS 25.0 - - - C25AS - 13.0 16.0 13.0 C25E3S - 2.0 2.0 4.0 C25E7 - - 4.0 4.0 TFAA - 6.0 6.0 6.0 APA 3.0 1.0 2.0 - TPKFA - 14.0 11.0 11.0 citric acid 1.0 1.0 1.0 1.0 dodecenyl/tetradecenyl- succinic acid 15.0 - - - rapeseed fatty acid 1.0 - 3.5 - ethanol 7.0 2.0 3.0 2.0 1,2-propanediol 6.0 8.0 10.0 13.0 monoethanolamine - - 9.0 9.0 TEPAE - - 0.4 0.3 DETPMP 2.0 1.2 1.0 - mannanasa 0.001 0.002 0.02 0.001 protease 0.08 0.02 0.01 0.02 lipase - - 0.003 0.003 amylase 0.004 0.01 0.01 0.01 cellulase - - 0.004 0.003 SEP 2 - - 0.2 0.1

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

boric acid 1.0 1.5 2.5 2.5 bentonite clay 4.0 5.0 4.0 4.0 clarifier 1 0.1 0.2 0.3 - foam suppressor 0.4 - - - opacifying agent 0.8 0.7 - - NaOH up to pH 8.0 7.5 8.0 8.2

various and minor components

Example 3

According to the present invention, the following granular detergent compositions have been prepared for fabrics having a fabric softening effect during washing.

II

C45AS LAS 7.6 10.0 C68AS 1.3 - C45E7 4.0 - C25E3 - 5.0 coco alkyl dimethyl hydroxyethyl ammonium chloride 1.4 1.0 citrate 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 PB1 15.0 - percarbonate - 15.0 TAED 5.0 5.0

44

4 4 4

4 4 4

4 4 4

4 4 4

44

4 4

4

4 4

4 4

4444

4

4 4

4

4 4

4· 44

smectite clay 10.0 10.0 HMWPEO - 0.1 mannanasa 0.001 0.02 protease 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 foam suppressor 1.0 4.0 CMC 0.2 0.1 miscellaneous and minority components up to 100%

Example 4

The following laundry bar detergent compositions were prepared according to the present invention (amounts are given in parts by weight, enzymes are expressed as pure enzyme).

I II III IV LAS - - 19.0 15.0 C28AS 26.0 13.5 - - sodium laurate 2.5 9.0 - - zeolite A 2.0 1.25 - - carbonate 20.0 3.0 13.0 8.0 calcium carbonate 27.5 39.0 31.0 - sulfate 5.0 5.0 3.0 5.0 TSPP 5.0 - - - STPP 5.0 15.0 10.0 -

99

9·· ··

999« · ·

9 9 9 9 9 * · 9 9 9 9 •9 99 99

9« 9999 • 9 99

9 9 9 9

9 9 ··

9 9 ·· 9 • 9 9 9 9

99 99

bentonite clay 4.0 10.0 4.0 4.0 DETPMP - 0.7 0.6 - CMC - 1.0 1.0 1.0 talc - - 10.0 11.0 silicate - - 4.0 5.0 PVNO 0.02 0.03 - 0.01 MA/AA 0.4 1.0 - - SEP 1 0.3 0.3 0.3 0.3 mannanasa 0.001 0.001 0.02 0.001 amylase - - 0.01 - protease - 0.004 - 0.003 lipase - 0.002 - 0.002 cellulase - 0.003 - - PEO - 0.2 - 0.2 perfume 1.0 0.5 0.3 0.2 magnesium sulfate - - 3.0 3.0 brightening agent 0.15 0.1 0.15 - photoactivated whitening reagent (ppm) - 15.0 15.0 15.0

In III VI In LAS 21.0 6.75 8.8 - C28AS - 15.75 11.2 22.5 sodium laurate - - - - zeolite A - 1.25 1.25 1.25 carbonate 10.0 15.0 13.0 8.0 calcium carbonate - 36.0 - 36.0 sulfate 3.0 - - 5.0

99

9 9 9

9 9 9

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

9V

9 9

9 9

9 9 9

99 ··*· *· 99

TSPP - 5.0 2.5 - STPP - 7.0 8.0 10.0 bentonite clay 5.0 4.0 4.0 4.0 DETPMP 0.6 0.7 0.7 0.7 CMC 1.0 - - 1.0 talc 10.0 - - - silicate 3.0 - - - PVNO - 0.02 - - MA/AA 0.2 0.4 0.5 0.4 SEP1 0.3 0.3 0.3 0.3 mannanasa 0.02 0.03 0.01 0.001 amylase - - 0.002 - protease 0.003 - - 0.003 lipase - - - - cellulase 0.0003 0.002 - - PEO 0.3 - - 0.3 perfume 0.4 - - 0.4 magnesium sulfate 3.0 - - - brightening agent - - - 0.1 photoactivated whitening reagent (ppm) 15.0 - - 15.0

99

9 9 9

9 9 9

9 9 9

9 9 9

99 • 9 • 9 9

9

9 9

9 9

9 •

9«r ···»

9 9

9 9

9 9 9

9 9 9

99

SEQUENCE LIST (1) GENERAL INFORMATION:

Applicant name: The Procter & Gamble Company Street: One Procter & Gamble Plaza City: Cincinnati, Ohio Country: USA Zip Code: 45202

Title of the invention: Laundry detergent and/or fabric care composition and method of cleaning fabrics

Number of sequences: 6

Computer reading form:

media type: floppy disk computer: IBM PC compatible operating system: PC-DOS/MS-DOS computer program (software): Patentln Release #1.0, version #1.25 (EPO)

Seq. ID. No. 1:

Sequence properties: length: 1407 nucleotide pairs type: nucleic acid strand type: single-stranded topology: linear

Molecule type: genomic DNA Original source:

Characters:

name/key: CDS location: 1 to 1482

Sequence description: sequence id. no. 1:

ATGAAAAAAAAGTTATCACAGATTTATCATTTAATTATTTGCACACTTATAATA

AGTGTGGGAATAATGGGGATTACAACGTCCCCATCAGCAGCAAGTACAGGC

ITTTATGTTGATGGCAATACGTTATATGACGCAAATGGGCAGCCATTTGTCAT

GAGAGGTATTAACCATGGACATGCTTGGTATAAAGACACCGCTTCAACAGCT

AÍTCCTGCCATTGCAGAGCAAGGCGCCAACACGATTCGTATTGTTTTATCAG

ATGGCGGTCAATGGGAAAAGACGACATTGACACCATTCGTGAAGTCATTG

AGCTTGCGGAGCAAAATAAAATGGTGGCTGTCGTTGAAGTTCATGATGCCA

CGGGTCGCGATTCGGCAGTGATTl AAATCGAGCCGTTGATTATTGGATAG

AAATGAAAGATGCGCTTATCGGTAAAGAAGTACGGTTATTATTAACATTGCA

AACGAGTGGTATGGGAGTTGGGATGGCTCAGCTTGGGCCGATGGCTATATT

GATGTCATTCCGAAGCTTCGCGATGCCGGCTTAACACACACCTTAATGGTTG

ATGCAGCAGGATGGGGGCAATATCCGCAATCTATTCATGATTACGGACAAG

ATGTGTTTAATGCAGATCCGTTAAAAAATACGATGTTCTCCATCCATATGTAT

GAGTATGCTGGTGGTGATGCTAACACTGTTAGATCAAATATTGATAGAGTCA

TAGATCAAGACCTTGCTCTCGTAATAGGTGAATTCGGTCATAGACATACTGA

TGGTGATGTTGATGAAGATACAATCCTTAGTTATTCTGAAGAAACTGGCACA

GGGTGGCTCGCTTGGTCTTGGAAAGGCAACAGTACCGAATGGGACTATTTA

GACCTTTCAGAAGACTGGGCTGGTCAACATTTAACTGATTGGGGGAATAGAA

TrGTCCACGGGGCCGATGGCTTACAGGAAACCTCCAAACCATCCACCGTAT

TTACAGATGATAACGGTGGTCACCC-TGAACCGCCAACTGCTACTACCTTGTA

TGACTTTGAAGGAAGCACACAAGGGTGGCATGGAAGCAACGTGACCGGTG

GCCCTTGGTCCGTAACAGAATGGGGTGCTTCAGGTAACTACTCTTTAAAGC

C GATGTAAATTTAACCTCAAATTCTl CACATGAACTGTATAGTGAACAAAGTC • · ·· · · · · · · · · gggaaatcccggtaatggcatgaatgcaagactttacgtgaaaacgggctc TGATTATACATGGCATAGCGGTCTÍTTACACGTATCAATAGCTCCAACTCA GGAACAACGTTATCTTTTGATTTAACAACATCGAAAATAGTCATCATGTTAG GGAAATAGGCGTGCAATTTTCAGCG GCAGATAATAGC AGTGGTCAAACTGC TGTATACGTTGATAACGTTACTTTAAGATAG

Seq. ID. No. 2:

Sequence properties:

length: 493 amino acids type: amino acid topology: linear

Molecule type: protein

Sequence description: sequence id. no. 2:

MKKKLSQIYHLIICTLIISVGIMGITTSPSAASTGFYVDGNTLYDANGQPFVMRGIN

HGHAWYKDTASTAIPAIAEQGANTIRIVLSDGGQWEKDDIDTIREVIELAEQNKM

VAWEVHDATGRDSRSDLNRAVDYWIEMKDALIGKEDTVIINIANEWYGSWDGS

AWADGYIDVIPKLRDAGLTHTLMVDAAGWGQYPQSIHDYGQDVFNADPLKNTM

FSIMHYEYAGGDANTVRSNIDRVIDQDLALVIGEFGHRHTDGDVDEDTILSYSEE

TGTGWLAWSWKGNSTEWDYLDLSEDWAGQHLTDWGNRIVHGADGLQETSKP

STVFTDDNGGHPEPPTATTLYDFEGSTQGWHGSNVTGGPWSVTEWGASGNY

SLKADVNLTSNSSHELYSEQSRNLHGYSQLNATVRHANWGNPGNGMNARLYV

KTGSDYTWHSGPFTRINSSNGTTLSFDLNNIENSHHVREIGVQFSAADNSSGQ

TALYVDNVTLR

Seq. ID. No. 3:

Sequence properties: length: 1407 nucleotide pairs type: nucleic acid ·♦·· ·· · · · · · · · • ·

Molecule type: genomic DNA

Sequence description: sequence id. no. 3:

ATGAAAAAAAAGTTATCACAGATTTATCATTTAATTATTTGCACACTTATAATA

AGTGTGGGAATAATGGGGATTACAACGTCCCCATCAGCAGCAAGTACAGGC

TTTTATGTTGATGGCAATACGTTATATGACGCAAATGGGCAGCCATTTGTCAT

GAGÁGGTATTAACCATGGACATGCTTGGTATAAAGACACCGCTTCAACAGCT

ATTCCTGCCATTGCAGAGCAAGGCGCCAACACGATTCGTATTGTTTTATCAG

ATGGCGGTCAATGGAAAAAGACGACATTGACACCATTCGTGAAGTCATTG

AGCTTGCGGAGCAAAATAAAATGGTGGCTGTCGTTGAAGTTCATGÁTGCCA

GGGGTCGCGATTCGGCAGTGATTl· AAATCGAGCCGTTGATTATTGGATAG

AAATGAAAGATGCGCTTATCGGTAAáGAAGATACGGTTATTATTAACATTGCA

AACGAGTGGTATGGGAGTTGGGATG GCTCAGCTTGGGCCGATGGCTATATT

GATGTCATTCCGAAGCTTCGCGATGCCGGCTTAACACACACCTTAATGGTTG

ÁTGCAGCAGGATGGGGGCAATATCCGCAATCTATTCATGATTACGGACAAG

ÁTGTGTTTAATGCAGATCCGTTAAAAAATACGATGTTCTCCATCCATATGTAT

GAGTATGCTGGTGGTGATGCTAACACTGTTAGATCAAATATTGATAGAGTCA

TAGATCAAGACCTTGCTCTCGTAATAGGTGAATTCGGTCATAGACATACTGA

TGGTGATGTTGATGAAGATACAATCCTTAGTTATTCTGAAGAAACTGGCACA

GGGTGGCTCGCTTGGTCTTGGAAAGGCAACAGTACCGAATGGGACTATTTA

GACCTTTCAGAAGACTGGGCTGGTCAACATTTAACTGATTGGGGGAATAGAA

7TGTCCACGGGGCGATGGCTTACAGGAAACCTCCAAACCATCCACCGTAT

TTACAGATGATAACGGTGGTCACCCTGAACCGCCAACTGCTACTACCTTGTA

TGACTTTGAAGGAAGCACACAAGGGTGGCATGGAAGCAACGTGACCGGTG

GGCCTTGGTCCGTAACAGAATGGGGTGCTTCAGGTAACTACTCTTTAAAGC

CGATGTAAATTTAACCTCAAATTCTTGACATGAACTGTATAGTGAACAAAGTC

GTAATCTACACGGATACTCTCAGCTCAACGCAACCGTTCGCCATGCCAATTG

GGGAAATCCCGGTAATGGCATGAATGCAAGACTTTACGTGAAAACGGGCTC

TGATTATACATGGCATAGCGGTCCTTTTACACGTATCAATAGCTCCAACTCA

GGAACAACGTTATCTTTTGATTTAAACAACATCGAAAATATCATCATGTTAGG

G)\AATAG • · • · · · • · · · • · ·· ·· · · · · • * · 9 · · · ·«· ·9

100

Seq. ID. No. 4:

Sequence properties: length: 468 amino acids type: amino acid topology: linear

Molecule type: protein

Sequence description: sequence id. no. 4:

vawevhdatgrdsrsdlnravdyviemkdalígkedtviinianewygswdgs AWADGYIDVIPKLRDAGLTHTLMVDAAGWGQYPQSIHDYGQDVFNADPLKNTM FSIHMYEYAGGDANTVRSNIDRVIDQDLALVIGEFGHRHTDGDVDEDTILSYSEE TGTGWLAWSWKGNSTEWDYLDSEDWAGQHLTDWGNRIVHGADGLQETSKP SWFTDDNGGHPEPPTATTLYDFEGSTQGWHGSNVTGGPWSVTEWGASGNY SLKADVNLTSNSSHELYSEQSRNLHGYSQLNATVRHANWGNPGNGMNARLYV KTGSDYTWHSGPFTRINSSNGTTL':f-DLNNIENHMLGK

MKKKLSQIYHLIICTLNSVGIMGITTSPSAASTGFYVDGNTLYDANGQPFVMRGIN

HGHAWYKDTASTAIPAIAEQGANTIRIVLSDGGQWEKDDIDTIREVIELAEQNKM

Seq. ID. No. 5:

Sequence properties: length: 1029 nucleotide pairs type: nucleic acid strand type: single-stranded topology: linear

Molecule type: genomic DNA * ·

101

Sequence description: sequence ID No. 5:

5' AAT TGG CGC ATA CTG TGT CGC CTG TGA ATC CTA ATG CCC AGC AGA CAA CAA AAA CAG TGA TGA ACT GGC TTG CGC ACC TGC CGA ACC

G.AA CGG AAA ACA GAG TCC TTT CCG GAG CGT TCG GAG GTT ACA GCC ATG 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 TAÁ ATG CCA TGC TCA GCA AAA TTG CTG ACG GAC TTC AAG AGT TGG AGA ACC AAG GTG TGC CTG TTC TGT TCA GGC QGC 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 ΑΠ TTA AAA CTG ÁTT TTTACC CGG GCG CGT CTT ACGTGGATATTG 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 CIT 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’

Seq. ID. No. 6:

Sequence properties: length: 363 amino acids type: amino acid topology: linear

102

Molecule type: protein

Sequence description: sequence ID No. 6:

yéhT 1

LFKKHTISLLIIFLLASAVLAKPIEAHTVSPVNPNAQQTTKTVMNWLAHL50 ydhT 51

PNRTENRVLSGAFGGYSHDTFSMAEADRIRSATGQSPAIYGCDYARGWLE 100 ydhT 101

TANIEDSIDVSCNGDLMSYWKNGGIPQISLHLANPAFQSGHFKTPITNDQ 150 ydhT 151

YKNILDSATAEGKRLNAMLSKIADGLOELENQGVPVLFRPLHEMNGEWFW 200 y:íhT 201

WGLTSYNQKDNERISLYKQLYKKlYFiYMTDTRGLDHLIWVYSPDANRDFK 250 ydhT 251

TDFYPGASYVDIVGLDAYFQDAYSINGYDQLTALNKPFAFTEVGPQTANG 300 ydhT 301

SFDYSLFINAIKQKYPKTIYFLAWNDEWSAAVNKGASALYHDSWTLNKGE 350 ydhT 351 tWNGDSLTPIVE*. 363

Claims (8)

A laundry detergent and / or fabric care composition, comprising a laundry detergent and / or fabric care ingredient, a mannanase enzyme and clay. A laundry detergent and / or fabric care composition according to claim 1, characterized in that mannanase is present in an amount of from 0.0001 to 2, preferably from 0.0005 to 0.5, more preferably from 0.001 to 0, 02 wt. pure enzyme based on the total composition. A laundry detergent and / or fabric care composition according to claims 1 to 3 2, characterized in that it contains clay in an amount of from 0.1 to 50, preferably from 3 to 25, more preferably from 4 to 15% by weight. of the total weight of the composition. A laundry detergent and / or fabric care composition according to claims 1 to 4 3, characterized in that smectite clay, preferably montmorillonite or hectorite clay, with a cation exchange capacity of at least 50 meq / 100 g is used as clay. A laundry detergent and / or fabric care composition according to claims 1 to 5 4, further comprising a building component selected from zeolite, sodium triphosphate, layered silicate and / or mixtures thereof. A laundry detergent and / or fabric care composition according to any one of the preceding claims, further comprising cellulase. A laundry detergent and / or fabric care composition according to any one of the preceding claims 1 to 6, further comprising: · 9 9908 99 9 · 9 9 · 99 99 99 9 9999 9,999,999,999,999 9,999 9,999 9,999 9 104 contains a cationic surfactant, preferably a surfactant comprising two long alkyl chains. A method for cleaning fabrics, characterized in that it uses a laundry detergent and / or fabric care composition according to any one of the preceding claims 1 to 7.