CZ2000506A3 - Laundry detergent and/or fabric care composition - 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

A laundry detergent and / or fabric care composition and method for cleaning fabrics

Technical field

The present invention relates to laundry detergent and / or fabric care compositions containing mannanase and clay. The present invention also relates to a method of cleaning fabrics which uses said laundry detergent and / or fabric care composition.

BACKGROUND OF THE INVENTION

The performance of the detergent product is assessed by a number of factors, including the ability to remove soils and the ability to prevent the re-depositing of soiled or disintegrated soiling products on the items being washed. Therefore, detergent compositions today comprise 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 care. Moreover, the need for such detergent compositions which not only have good cleaning properties but also have fabric softening and other fabric care properties is well known in the art.

In today's detergent and fabric care compositions, fabric softeners are generally used to provide a soft feeling. European Patent Application A177 165 discloses the use of fabric softeners together with cellulase in detergent compositions. European Patent Application A 495 258 describes detergent compositions comprising a surfactant, a builder system and fabric softener clay and cellulase.

Food and cosmetic stains / stains are a major part of customer-related stains / stains and often contain food ingredients such as thickeners / stabilizers. Hycolloid gums and emulsifying agents are often used as food ingredients. The term gum means a group of industrially useful polysaccharides (long chain polymer) or derivatives thereof that hydrate in hot or cold water to form viscous solutions, dispersions or gels. Erasers are classified as natural and modified. Natural gums include sea seed extracts, plant secreted products, seed or root resins and microbial fermentation resins. Modified (semisynthetic) resins include cellulose and starch derivatives and some synthetic gums such as low methoxy pectin, propylene glycol alginate, and carboxymethyl and hydroxypropyl guar resins (Gums in Encyclopedia Chemical Technology, 4th edition, 12, pages 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), breadfruit (E410), are widely used alone or in combination in many food applications (Gums in Encyclopedia Chemical Technology, 4th Edition, 12, pages 842-862, J. Baird , Kelco Division of Merck).

The guar gum used in these food and cosmetic dyes is obtained from the seeds of the butterfly's endosperma Cyamopsis tetragonoloba. Guar gum (also called guaran) extracted from dicotyledon seed consists of a 1-4, bD-mannopyranosyl backbone unit and is used as a thickening agent in flavoring and in frozen products and in cosmetics (H.-D. Belitz Food Chemistry, p. 243, English version of the second edition, Springer-verlag, 1987, ISBN 0-387-15043-9 (US) {Carbohydrate Chemistry for Food Scientists, RL Wistler, Eagan Press, 1997, ISBN 0-913250-92-9) and {Industriai Gum, second edition, R. L. Whistler, p. 308, Academic Press, 1973, ISBN, 0-12-74-6252-x). Breadfruit gum (also called carob gum or St. John's bread) is also used in the food industry and is extracted from the seeds of evergreen plants grown in the Mediterranean region. Breadfruit gum probably differs from the structure of guar gum only by a small number of D-galactosyl side • 9 • 9 9 9

9 9 9 9 9 9 and has the same 1-4, b-D-mannopyranosyl backbone. For legume seeds, the water-soluble galactomannan is the main storage saccharide, which in some cases represents up to 20% by weight. of the total dry weight. Galactomannan has α-galactose attached to 0-6 mannose residues and can also be acetylated to varying degrees on 0-2 and 0-3 mannose residues.

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

It is therefore an object of the present invention to provide laundry detergent and / or fabric care compositions which exhibit optimum fabric softener performance and provide optimum stain removal and cleaning performance, particularly cosmetic and food stains.

The above objects are suited to a composition of laundry detergent and / or fabric care compositions which comprises mannanase enzyme and clay as a fabric softener.

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

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

having a molecular weight of 38,000, an optimum activity at pH 5.0 / 55 ° C and a pI of 4.8. Japanese Patent 0304706 describes β-mannanase derived from Bacillus sp. with a molecular weight of 37,000 ± 3000 measured by gel filtration, with an optimum pH of 8 to 10 and a pI of 5.3 to 3.0

5.4. Japanese patent 63056289 describes the production of an alkaline, thermostable β-mannanase that hydrolyzes β-1,4-D-mannopyranoside bonds of eg mannan and provides manno: oligo: carbohydrates. Japanese patent 63036774 relates to Bacillus microorganism FERM P-8856, which produces β-mannanase and β-mannosidase at alkaline pH. Purified Bacillus amyloliguefaciens mannanase and a process for its preparation, which is useful in pulp and paper bleaching, are described in WO 97/11164. WO 91/18974 discloses hemicellulase, such as glucanase, xylanase or mannanase, active at extreme pH and temperature, and the manufacture thereof. WO 94/25576 discloses an enzyme exhibiting mannanase activity derived from Aspergillus aculeatus CBS 101.43, which can be used for a variety of purposes where degradation or modification of plant cell wall or algae wall material is desired. WO 93/24622 describes mannanase isolated from Trichoderma reesia for bleaching lignocellulose pulp.

However, until now, a synergistic combination of mannanase and clay has not been described for optimum fabric softener performance and for optimum stain removal and cleaning performance, especially cosmetic and food stains in laundry detergent and / or fabric care compositions.

SUMMARY OF THE INVENTION

The present invention relates to laundry detergent and / or manganese and clay containing care compositions for optimum fabric softener performance and stain removal and cleaning performance, particularly cosmetic and food stains.

It is known in the art that clay is not compatible with some hydrocolloid gums contained, for example, 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, Second Edition, RL 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 fabric softening effect. In addition, it has been observed that the clay and the soiling from the laundry detergent are re-deposited on the guar gum residues and cause staining. Surprisingly, it has been found that the mannanase enzyme hydrolyzes hydrocolloid gums, especially guar gums. This enzymatic hydrolysis results in the absence of guar gum during washing. There is therefore no flocculation of clay and the clay retains its full fabric care potential. In addition, there are no guar gum residues left on the fabric, and there is therefore no repositioning of the clay and soiling at the subsequent wash load and thus no staining.

Mannnas enzyme: An essential element of the laundry detergent 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-β-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, Academic Press).

The laundry detergent 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 of mannan endo-1,4-beta-mannosidase and with alternate names beta-mannanase and endo-1,4-mannanase and catalyses the following reaction: random hydrolysis of 1,4-beta -D-mannoside bonds in mannan, galactomannan, glucomannan and galactoglucamannans.

In particular, mannanases (EC 3.2.1.78) represent a group of polysaccharases that degrade mannan and mean enzymes that are capable of cleaving polyosic chains containing mannose units, i.e., they are capable of cleaving glycosidic bonds in mannan, glucomannan, galakotmannan, and galactoglucomannan. Mannanes are polysaccharides having a backbone consisting of β-1,4-linked mannose, glucomannans are polysaccharides having a backbone consisting of more or less regularly repeating β-1,4-linked mannose, and glucose, galactomannan and galactoglucomannan are mannan and glucomannan with α-1,6-linked galactose side branches. These compounds may be acetylated.

The degradation of galactomannan and galactoglucomannan is facilitated by the full or partial removal of galactose side chains. Further, the degradation of acetylated mannan, glucomannan, galactomannan and galactoglucomannan is facilitated by full or partial deacetylation. Acetyl groups can be removed by alkaline or mannan acetylesterases. Oligomers that are released from mannanases or a combination of mannanases and α-galactosidase and / or mannan-acetyl esterases 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 β-mannanase derived from Bacillus steaiOthermophilus in a dimer with a molecular weight of 162,000 and an optimum pH of 5.5 to 7.5. Mendoza et al., World J. Microbiol. Biotech. 1994, 10 (5), 551-555, discloses Bacillus subtilis-derived β-mannanase 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 β-mannanase derived from Bacillus sp. with a molecular weight of 370,000 as measured by gel filtration, with 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 β-1,4-D-mannopyranoside bonds of eg mannan and provides manno-oligo-saccharides. Japanese patent 63036774 relates to Bacillus tt 9, 9, 9, 9, 9, 9, 9, 9, 9 99 99 of the microorganism FERM P-8856, which produces β-mannanase and β-mannosidase at alkaline pH. Japanese Patent 08051975 discloses alkaline beta-mannanases from alkalophilic Bacillus sp. AM-001. Purified Bacillus amyloliguefaciens mannanase useful in the bleaching of pulp and paper and a process for its preparation is described in WO 97/11164. WO 91/18974 discloses a hemicellulase, such as glucanase, xylanase or mannanase, active at extreme pH and temperature. WO 94/25576 discloses an enzyme from Aspergillus aculeatus CBS 101.43 which exhibits mannanase activity and which can be used to degrade or modify the cell wall material of plants or algae. WO 93/24622 describes mannanase isolated from Trichoderma reseei, useful for bleaching lignocellulose pulp. A hemicellulase capable of degrading mannan-containing hemicellulose is described in WO 91/18974. Purified mannanase from Bacillus amyloliquefaciens is described in WO97 / 11164.

In particular, the mannanase enzyme will be alkaline mannanase as defined below, most preferably 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 length.

The term alkali mannanase enzyme is intended to include an enzyme having an enzymatic activity of at least 10, preferably at least 25, more preferably at least 40% of its maximum aquarium at a given pH in the range of from 7 to 12, preferably from 7.5 to 10, 5.

Most preferably, the laundry detergent 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 shown at positions up to 343 of SEQ ID NO: 2. id. No 2

. 9 4 4 4 4

4 4 4

4 4 ·

4 4 4 4

9 9 9 4 5

9 99 99

9 · · 4 4 4

4 4 4 4 4

MKKKLSQIYHLIICTLIISVGIMGITTSPSAASTGFYVDGNTLYDANGQPFVMRGIN

HGHAWYKDTASTAIPAIAEQGANTIRIVLSDGGQWEKDDIDTIREVIELAEQNKM

VAWEVHDATGRDSRSDLNRAVDYWÍEMKDALIGKEDTVIINIANEWYGSWDGS

AlVADGYlDVIPKLRDAGLTHTLMVDAAGWGQYPQSIHDYGQDVFNADPLKNTM

FSIHMYEYAGGDANTVRSNIDRVIDQDLALVIGEFGHRHTDGDVDEDTILSYSEE

T3TGWLAWSWKGNSTEWDYLDLSE9WAGQHLTDWGNRIVHGADGLQETSKP

3TVFTDDNGGHPEPPTATTLYDFEGSTQGWHGSNVTGGPWSVTEWGASGNY

8L.KADVNLTSNSSHELYSEQSRNLHGYSQLNATVRHANWGNPGNGMNARLYV

KVGSDYTWHSGPFTRINSSNSGTTLSFDLNNIENSHHVREIGVQFSAADNSSGQ

TALYVDNVTLR (SEQ ID NO: 2) or iii) a polypeptide analogue as defined in vi) or 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 a 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

A YGGCGGTCAATGGGAAAAAGACGACATTGACACCATTCGTGAAGTCATTG

AGCTTGCGGAGCAAAATAAAATGGTGGCTGTCGTTGAAGTTCATGATGCCA

CGGGTCGCGATTCGCGCAGTGATT! 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

GCCCTTGGTCCGTAACAGAATGGGGTGCTTCAGGTAACTACTCTTTAAAAGC

C GATGTAAATTTAACCTC AAATTCTi CACATGAACTGTATAGTGAACAAAGTC gtaatctacacggatactctcagctcaacgcaaccgttcGccatgccaattg

GGGAAATCCCGGTAATGGCATGAATGCAAGACTTTACGTGAAAACGGGCTC

TGATTATACATGGCATAGCGGTCCTTTTACACGTATCAATAGCTCCAACTCA

GGAACAACGTTATCTTTTGATTTAAACAACATCGAAAATAGTCATCATGTTAG

GGAAATAGGCGTGCAATTTTCAGCGGCAGATAATAGCAGTGGTCAAACTGC

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

b) molecules homologous to a),

c) polynucleotide molecules that encode a polypeptide having mannanase activity and which is at least 70% identical to the amino acid sequence of SEQ. id. 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 a), b), c) or d).

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

9 9 9 9 9 9 9 9 9 9

99 99 99 9 · 99

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

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

i) is encoded by the coding part of the DNA sequence shown 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 GTT ACA GCC ATG ACA CAT TTT CTA TGG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG GAA TTC GCC AAA GCC TCA GCC GCC TCA GCC GCC TCA GCC GCC TCA AAA GCC TCA GCC AAA GCC TCA GCC AAA ACG GAC TTC AAG AGT AAC AGA AAC AGA TAC AAC AGA TAC AAC AGA TAC ATG ATT ATA ATG ATG ATG ATG ATT ATG ATG ATG ATT ATG ATG ATT ATG ATT ATG ATT ATG ATT ATG ATT ATG ATT ATG ATT ATG ATT ATG ATT ATG ATT ATG ATT ATG ATT ATG ATT ATG ATT ATG ATT ATG ATT ATG ATT TCA 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 ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG SEQ. id. No. 5) ··················· 9 · 9 · · 9 Or an analogue of said sequence and / or ii) means a polypeptide comprising an amino acid sequence as set forth in SEQ. id. No 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 (ii) that 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; 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 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 a),

c) polynucleotide molecules that encode a polypeptide having mannanase activity and which 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 a), b), c) or d).

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

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

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

The term expression vector refers to a DNA molecule, linear or circular, comprising a segment encoding a polypeptide of interest, operably linked to other segments that provide for its transcription. These additional segments may include a promoter and 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. The expression vector of the invention may be any expression vector that is suitably subjected to recombinant DNA techniques. The choice of vector will often depend on the host cell into which the vector is to be introduced. Thus, the vector may be an autonomously replicating vector, i.e., a vector that exists as an extra-chromosomal particle whose replication is independent of chromosome replication, e.g., a plasmid. Vector can also mean

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

99 99 • 9 9 * 99

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

The term recombinant expressed or recombinantly expressed as used herein in connection with expression of a polypeptide or protein is defined according to standard definition in the art. Recombinant expression of the protein is usually performed using the expression vector immediately described 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 devoid of other external or unwanted coding sequences and is in a form suitable for use in genetically engineered protein production systems. These isolated molecules are those that are separated from their natural environment and include cDNAs and genomic clones. The isolated DNA molecules of the present invention do not contain other genes with which they are usually associated, but may contain naturally occurring 5 'and 3' untranslated regions, such as promoters and terminators. The identification of associated regions will be apparent to the skilled artisan (see, for example, Dynan and Tijan: Nature 1985, 376, 774-778).

The term isolated polynucleotide may also be termed 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 substantially free of other proteins, particularly free of other homologous proteins (i.e., homologous impurities (see below)). It is preferred to obtain the protein in more than 40% pure form, more preferably in greater than 60% pure form. It is even more preferred to obtain a protein that is in highly pure form, i.e. greater than 80%, more preferably greater than 95% and even more preferably greater than 99% (w / w) of the pure form, as determined by SDS-PAGE.

4 4 4 4

44 4 4 4

4 4 4 4 4 444 44 4 • 4 4 4 44 4 4 44

44

Alternatively, the term isolated protein / polypeptide may also be referred to as purified protein / polypeptide.

The term homologous impurities means any impurity (eg, 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. As used herein, a term derived from a specific microbial source means 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 referring to DNA segments refers to the fact that the segments are arranged to function in accordance with their intended purposes, e.g., transcription begins at the promoter and proceeds to the coding segment at the 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 can be isolated from natural sources, synthesized in vitro, or can be prepared by combining natural and synthetic molecules.

The term polynucleotide molecule complements refers to polynucleotide molecules having an additional polynucleotide sequence and an inverted orientation as 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 (as compared to a reference polynucleotide molecule that encodes a polypeptide). Degenerate codons contain different nucleotide triplets but encode the same amino acid residues (i.e., both GAU and GAC triplets encode Asp).

• 99 99 99 99

The term promoter refers to a portion of a gene containing DNA sequences that provide for binding of RNA polymerase and initiation of transcription. Promoter sequences are usually, but not always, found in the 5 'non-coding regions of the genes.

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

How to Use the Sequence of the Invention to Obtain Other Related Sequences: Information on the disclosed sequences of the invention relating to a polynucleotide sequence encoding a mannanase of the invention can be used as a tool to identify other homologous mannanases. For example, a polymerase chain reaction (PCR) can be used to amplify sequences encoding other homologous mannanases from different microbial sources, particularly different Bacillus species.

Activity Test: A polypeptide of the invention having mannanase activity can be tested for mannanase activity according to standard test procedures known in the art, such as applying the solution to be tested to 4 mm holes punched in agar plates containing 0, 2% (w / w) AZCL galactomannan (carob), ie substrate for endo-1,4-beta-D-mannanase testing available as Cat # I-AZGMA from Megazyme at US $ 110.00 for 3 grams ( Megazyme's web address: http: /www.megazyme.com/Purchase/index.html).

Polynucleotides: The isolated polynucleotide of the invention will hybridize to the regions of SEQ. id. No. 1 of similar sizes or with a sequence complementary thereto under at least mild selective conditions.

• ·

99 9

In particular, the polynucleotides of the invention will hybridize to a denatured double-stranded DNA probe comprising either the full sequence shown between positions 97 and 1029 of the above sequence. id. 1 or any probe containing the subsequences of SEQ. id. No. 1 having a length of at least 100 nucleotide pairs under at least mild selective conditions, but preferably under high selectivity conditions, as described in detail below. Suitable experimental conditions for determining hybridization under mild or high selectivity between a nucleotide probe and a homologous DNA or RNA sequence include pre-soaking a filter containing DNA fragments or RNA for hybridization in 5 x SSC (sodium chloride / sodium citrate, Sambrook et al. 1989) for 10 minutes and filter pre-hybridization in 5 x SSC, 5 x Denhardt's solution (Sambrook et al. 1989), 0.5% SDS and 100 µg / ml denatured sonicated salmon sperm DNA (Sambrook et al 1989), followed by hybridization in the same solution containing a concentration of 10 ng / ml random primer (Freiberg AP and Vogelstein B .: Anal. Biochem. 1983, 132, 6-13), 32P-dCTP-labeled (specific activity greater than 1.109 counts per minute) pg) probes for 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 (mild selectivity), more preferably at least 65 ° C (mum / high selectivity), even more preferably at least 70 ° C (high) selectivity) and even more preferably at least 75 ° C (very high selectivity).

The molecules with 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 of interest can be cloned in gene banks or DNA libraries by methods known in the art.

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

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

Species homologues of a mannanase activity polypeptide of the invention can be cloned using the information and means obtained according to the invention in combination with conventional cloning techniques. For example, the DNA sequences of the present invention can be cloned using chromosomal DNA obtained from a type of cell that expresses a protein. Suitable DNA sources can be identified by probing Northem blots with probes that are designed according to the sequences described herein. A library is then prepared from the chromosomal DNA of the positive cell line. The DNA sequences of the invention encoding a polypeptide having mannanase activity can then be isolated in a variety of ways, such as probing with probes designed according to the sequences described in the present specification and claims, or one or more rows of degenerate probes based on the described sequences. The DNA sequence of the invention may also be cloned by polymerase chain reaction or PCR (Mullis: U.S. Patent 4,683,202) using primers designed according to the sequences described herein. Among other methods, a DNA library can be used to transform or transfect host cells, and the expression of the DNA of interest can be detected by an antibody (monoclonal or polyclonal) against mannanase cloned from B. agaradherens, NCIMB 40482, expressed and purified as described in the Materials and Methods section, and in Example 1, or an activity assay for 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 of the invention may be cloned from a strain of the bacterial Bacillus agaradherens species, preferably strain NCIMB 40482, producing an enzyme with mannan degrading activity or other or related organism as described herein.

····································

An analogous sequence may also be constructed based on the DNA sequence obtainable from the 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 the introduction of nucleotide sequences. substitutions that do not counteract another amino acid sequence of the mannanase encoded by the DNA sequence, but which correspond to the codon of the host organism that is contemplated for enzyme production, or by introducing a nucleotide substitution that can provide a different amino acid sequence (i.e., a mannan degrading enzyme variant of the invention).

Polypeptides; Amino acid sequences # 32-343 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 homologues (paralogs or orthologs). The term substantially homologous is used herein to refer to polypeptides having 70%, preferably at least 80%, more preferably at least 85%, and even more preferably at least 90% sequence identity to the sequence set forth in amino acids 32-343 of Seq. id. or their orthologs or paralogs. More preferably, the polypeptides will be at least 95% identical and most preferably at least 98% or more identical to the sequences set forth in amino acids 32 to 343 of SEQ. id. or its orthologs or paralogs. Percent sequence identity is determined by conventional methods, using computer programs known in the art as GAP, which are obtained in the GCG (Program Manual for the Wisconsin Package, version 8, August 1994, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin). , USA 53711) as described by Needleman SB 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 to compare polypeptide sequence: GAP creation penalty 3.0 and GAP extension penalty 0.1.

> · · (► 4 · 4)

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

The enzyme preparation of the invention is preferably derived from a microorganism, preferably a bacterium or fungi, 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 Bacillus agaradherens and very related Bacillus species. wherein all species are preferably at least 95%, even more preferably at least 98% homologous to Bacillus agaradherens according to the aligned 16S rDNA sequences.

Substantially homologous proteins and polypeptides are characterized in that they have one or more amino acid substitutions, deletions, or additions. These changes are preferably of a minor nature, i.e. conservative amino acid substitutions (see Table 2) and other substitutions that do not significantly affect protein or polypeptide folding or activity, small deletions, typically one to about 30 amino acids, and small elongations at the amino or amino acid. a carboxyl terminus, such as an amino terminus methionine residue, a small binding peptide of up to about 20 to 25 residues, or small elongations that facilitate purification (affinity tag), such as a polyhistidine region, protein A (Nilsson et al.: EMBO J. 1985 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. are available from commercial suppliers (e.g., Pharmacia Biotech, Piscataway, N.J .; New England Biolabs, Beverly, Ma.). and, such changes may also be of a greater nature, such as linking larger polypeptides with up to 300 amino acids or more as both an amino and carboxyl terminus extension to the mannanase polypeptide of the invention.

• 4,444 • 4,444

4 · 4 4 4

4 4 4 4 4

4 4 4 4

4 44

Table 1

Conservative amino acid substitutions

basic: arginine, lysine, histidine sour: 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-methylysine, 2-aminoisobutyric acid, isovaline, and α-methylserine) can be substituted for the amino acid residues of the polypeptide of the invention. A limited number of non-conservative amino acids, non-genetically encoded amino acids, and unnatural amino acids may be substituted for 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. The unnatural amino acids may be chemically synthesized or preferably are 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 according to techniques known in the art, such as site-directed mutagenesis or alanine-monitored mutagenesis (Cunningham and Wells: Science 1989, 244, 1081-1085). According to the latter method, individual alanine mutations are introduced at each residue in the molecule, and the resulting mutated molecules are tested for biological activity (ie, mannanase activity) to identify amino acid residues that are critical to the activity of the molecule. See also Hilton et al., J. Biol. Chem. 1996, 271, 4699-4708.

9

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9 • ·

The active site of the enzyme or other biological interaction can also be determined by physical structure analysis, such as by methods such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, along 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. Essential amino acid identities can also be inferred from homology analysis with polypeptides that are related to the polypeptides of the invention.

Multiple amino acid substitutions may also be made and may be tested by known mutagenesis, recombination, and / or rearrangement methods with the following evaluation procedure, such as those described by Reidhaar-Olson and Sauer (Science 1988, 241, 53-57). Bowie and Sauer (Proc. Natl. Acad. Sci. USA 1989, 86, 2152-2156), WO 95/17413 or WO 95/22625. Briefly, these authors describe methods for simultaneous random change of two or more positions in a polypeptide, or recombination / shuffling of various mutations (WO 95/17413, WO 95/22625) followed by selection of a functional polypeptide and then sequencing of mutagenized polypeptides to determine the spectrum of available substitutions in every position. Other methods that can be used include phage display (eg, Lowman et al., Biochem. 1991, 30, 103232-10837; Ladner et al., U.S. Patent No. 5,223,409; Huse, WIPO WO 92/9). And region-directed mutagenesis (Derbyshire et al., Gene 1986, 46, 145; Ner et al., DNA 1988, 7,127).

The mutagenesis / transposition methods as described above may be combined with high throughput, automated evaluation methods to detect 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 9 9

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9 9 9 unknown structures. Using the above methods, one of skill in the art can identify and / or prepare a variety of polypeptides that are substantially homologous to residues 32-343 of sequence. id. No. 2 and which retain the natural protein mannanase activity.

Protein Production: The proteins and polypeptides of the present invention, including full-length proteins, fragments thereof, and fusion proteins, can be produced by genetically engineered host cells according to conventional methods. Suitable host cells are those cell types that can be transformed or transfected with exogenous DNA and can grow in culture. They include bacteria, fungal cells and cultured higher eukaryotic cells. Preferred are bacterial cells, particularly cultured cells of gram-positive organisms. Particularly preferred are gram-positive cells of the genus Bacillus, such as cells from the group consisting of Bacillus subtilis, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillus amaguoliquefaciens, Bacillus coagulans, Bacillus thiusus, Bacillus lautus, Bacillus lautus, Bacillus lautus, agaradherens, especially Bacillus agaradherens.

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

In general, the DNA sequence encoding the mannanase of the present invention is operably linked to other genetic elements required for its expression, generally comprising a promoter and a transcription terminator in the expression vector. This vector will usually also contain one or more selectable markers and one or more origins of replication, although those skilled in the art will appreciate that some of the markers will be known.

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

To direct the polypeptides into the secretory pathway of the host cell, an expression signal sequence, also known as leader sequence, prepro sequence or pre sequence, is obtained 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 secretion signal sequences are known in the art. For 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. (ed.): Molecular Biological Methods for Bacillus, John Wiley & Sons, 1990. The secretory signal sequence is fused to the DNA sequence at the correct reading phase. 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). No. 037,743, Holland et al., U.S. Patent No. 5,143,830).

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

• 444

4 ·· • · 4 4 • 44 • 4 4 4

4 4 4

44 • 4 4 4 4 • 4 φ

4 4

4 4

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

If the expressed recombinant polypeptide is not secreted from the host cell, the host cell is preferably broken and released into the aqueous extract, which is the first step of this purification method. Preferably, the expression host cells are isolated from the medium before they are broken (eg by centrifugation).

Cell disruption can be accomplished by conventional methods such as lysozyme digestion 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 expressed recombinant polypeptides (or chimeric polypeptides) are secreted can be purified by fractionation and / or conventional purification methods.

Ammonium sulfate precipitation and acid or chaotropic extraction may be used for sample fractionation. Examples of purification steps include hydroxyapatite, size chromatography, FPLC, and reverse-phase high performance liquid chromatography. Suitable anion exchange media include derivatized dextrans, agarose, cellulose, polyacrylamide, silicates and the like. Preferred are PEI, DEAE, QAE and Q derivatives, with DEAE rapid flow sepharose (Pharmacia, Piscataway, NJ.) Being particularly preferred. Examples of chromatographic media include those derivatized with phenyl, butyl or octyl groups such as Phenyl-Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso Haas, Mongomeryville, Pa.), Octyl-Sepharose (Pharmacia) and the like, or polyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the like. Suitable solid supports include: 9 99 99 9999 99 99

9 9 9 9 9 9 9 9 9

9 9 9 9 9 9 9 9 9

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 not soluble under the conditions under which they are used. These carriers can be modified with reactive groups which allow the attachment of proteins by amino groups, carboxyl groups, mercaptan groups, hydroxyl groups and / or carbohydrate groups. Examples of condensation chemistry include cyanogen bromide activation, N-hydroxysuccinimide activation, epoxide activation, mercaptan activation, hydrazide activation, and carboxylic and amine derivatives for carbodiimide condensation chemistry. These and other media are well known, widely used in the art, and are available from commercial suppliers.

The choice of the method is a matter of routine and is in part determined by the properties of the carrier selected. See, for example, Affinity Chromatography: Principles and Methods, Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988.

Polypeptides of the invention or fragments thereof may also be prepared by chemical syntheses. 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 methionic amino acid residue.

Based on the sequence information described herein, the full length DNA sequence of the mannanase coding sequence of the invention and containing the DNA sequence shown in SEQ. id. 1, at least a DNA sequence from position 97 to position 1029.

Cloning is accomplished by standard techniques known in the art, such as:

- preparation of a genomic library from Bacillus strain, in particular 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 «· ··

- sowing this library onto suitable substrate plates,

identifying a clone containing the polynucleotide sequence of the invention by standard hybridization methods using the probe based on SEQ. id. 1 or

identifying a column from said Bacillus agaradherens NCIMB 40482 genomic library by inverse PCR strategies using primers based on sequence information. id. No. 1. Reference to M.J. McPherson et al. (PCR A Practical Approach, Information Research Ltd., Oxford, England) is provided herein for further details regarding inverse PCR.

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

DNA encoding the mannan or galactomannan-degrading enzyme of the invention can also be prepared by well known methods, suitably cloned from a suitable source, such as any of the above organisms, using synthetic oligonucleotide probes prepared based on the DNA sequence obtainable from the plasmid present in Escherichia coli DSM 12180.

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

In the present context, the term enzyme preparation is meant to mean either a conventional enzymatic fermentation product, possibly isolated and purified, from a single species of microorganism, this preparation usually containing a number of different enzymes.

9 9

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99

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99 • 9 9 9

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99 enzymatic activity, or means a mixture of one-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 fungal or bacterial species , or means a fermentation product of a microorganism that acts as a host cell for the expression of recombinant mannanase but at the same time it produces other enzymes, eg pectin, protease or cellulase degrading enzymes, and are naturally occurring fermentation products of the microorganism, ie enzyme a complex conventionally produced by the corresponding naturally occurring microorganism.

A method for producing an enzyme preparation of the invention, the method comprising culturing a microorganism, eg, a wild strain capable of producing mannanase under conditions permitting the production of the enzyme, and isolating the enzyme from the culture. Cultivation can be carried out using conventional fermentation techniques, for example, by cultivation in a culture flask or in fermenters with agitation to ensure sufficient aeration of the growth medium, which induces mannanasin enzyme production. The growth medium may comprise a conventional nitrogen source such as peptone, yeast extract or casaminic acids, a reduced amount of a conventional carbon source such as dextrose or sucrose, and an inducing agent such as guar gum or breadfruit gum. Isolation may be accomplished by conventional means, e.g., by separating the biomass and supernatant by centrifugation or filtration, isolating the supernatant or broken cells if the enzyme of interest is intracellular, with possible subsequent purification as described in European patent 0406 314, or by crystallization as described in WO 97/15660.

Immunological cross-reactivity: Polyclonal antibodies that are used to determine immunological cross-reactivity can be prepared using purified mannanase enzyme. In particular, the anti-mannanase antiserum of the invention can be obtained by immunizing rabbits (or other rodents) by the procedure of the procedure of φφ postupem postupem postupem postupem postupem postupem postupem postupem postupem postupem postupem postupem postupemφφφφφφφφφφφφφφφφΦΦΦΦΦΦΦΦΦΦΦΦΦΦΦΦ • φ φ φ · · · 9

ΦΦ ΦΦ according to Ν. 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, pages 27-31). Purified immunoglobulins can be obtained from antisera, for example by precipitation of salts (ammonium sulfate) followed by dialysis and ion exchange chromatography, e.g. DEAE-sephadex. Immunochemical characterization of proteins can be performed either by Ouchterlony's two-diffusion analysis (O. Ouchterlony in: Handbook of Experimental Immunology (DM Weir, eds.), Blackwell Scientific Publications, 1967, pp. 655-706) by cross-immunoelectrophoresis (N. Axelsen et al .: see above, chapters 3 and 4) or by rocket immunoelectrophoresis (N. Axelsen et al .: chapter 2).

Examples of useful bacteria producing the enzyme or enzyme preparation of the invention are Gram positive bacteria, preferably from the Baciilus / 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 encompasses isolated mannanase having the properties described above, free of homologous impurities and produced by conventional recombinant methods.

Determination of Mannanase Catalytic Activity (ManU): Colorimetric Assay: Substrate: 0.2% (w / w) AZCL-galactomannan (Megazyme, Australia) from carob in 0.1 M glycine buffer, pH 10.0. The test is carried out in a 1.5 ml Eppendorf microtube on a thermomixer with stirring and temperature control at 40 ° C. Incubation, for 20 minutes, 0.750 ml of substrate with 0.05 ml of 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 at 1 cm.

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

99

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99

£. coli strain: E. coli SJ2 cells (Diderichsen B., Wedsted U., Hedegaard L., Jensen BR, Sjoholm C .: Cloning of aldB, which encodes alpha-acetolactatecarboxylase, an exoenzyme from Bacillus brevis, J. Bacteriol. 172, 4315-4321) and were 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 interrupted apr and npr genes (Diderichsen B., Wedsted U., Hedegaard L., Jensen BR, Sjoholm C .: Cloning of aldB, which encodes alpha-acetolactat-deckarboxylase, an exoenzyme from Bacillus brevis (J. Bacteriol. 1990, 172, 4315-43).) broken in the transcription unit of the known Bacillus subtilis cellulase gene, resulting in cellulase negative cells. Disruption is performed 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.).

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 detailed 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 causing the plasmid to be reproducible in Bacillus subtilis, a kanamycin resistance gene, and a strong promoter and a signal peptide cloned from the amylL B. licheniformis gene ATCC 14580. The signal peptide contains a SacII site, making it convenient to clone the DNA encoding the mature portion of the protein in conjunction with the signal peptide. This leads to the expression of the pre-protein which is directed out of the cell.

• Φ ΦΦΦ · · · • • • • • φ φ φ φ φ φ φ φ

ΦΦΦ · · φ φ · φφφφ

ΦΦΦ · φφ φφ φφφφ

The plasmid was constructed by conventional genetic engineering techniques, 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 Ncil. The PCR fragment amplified from the amyL promoter encoded on plasmid pDN1981 (P.J.Jorgensen et al., Gene 1990, 96, 37-41) was digested with Ncil and inserted into pI1110 digested with Ncil to give plasmid pSJ2624.

The two primers used had the following sequences:

# LWN5494

5'-GTCGCCGGGGCGGCCGCTATCAATTGGTAACTGTATCTCAGC-3'a # LWN5495

5'-GTCGCCCGGGAGCTCTGATCAGGTACCAAGCTTGTCGACCTGCAGAATGAGG

CAGCAAGAAGAT-3 '.

Primer # LWN5494 insertes into the plasmid instead of NotI.

Plasmid pSJ2624 was then digested with SacI and NotI, and a new PCR fragment amplified for the amyL protomer encoded by pDN1981 was digested with SacI and NotI. This DNA fragment was inserted into SacI-NotI digested pSJ2624 to give plasmid pSJ2670.

This cloning replaces the first amyL space 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 * 44 4 4 4

4 4 4 4 4 5

4 4 4 4 4 5

44 44 44

4 4

4 4

4 LWN5939

5'-GTCGGAGCTCTATCAATTGGTAACTGTATCTCAGC-3 '.

Plasmid pSJ2670 was digested with the restriction enzymes PstI and Bell, and the PCR fragment amplified from the cloned DNA sequence coding for the 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 Bell and inserted. to obtain plasmid pMOL944. The two primers used for PCR amplification had the following sequences:

# LWN7864

5'-AACAGCTGATCACGACTGATCTmAGCTTGGCAC-3'a # LWN7901

5'-AACTGCAGCCGCGGCACATCATAATGGGACATGGG-3 '.

Primer # LWN7901 inserted into the plasmid instead of SacII.

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

Genomic library construction: Genomic DNA was partially digested with the restriction enzyme Sau3A and resolved by electrophoresis on a 0.7% 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 recovering DNA fragments from agarose and aerylamide gels. Anal. Biochem., 1981, 112, 295-198).

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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 E. coli DNA library, constructed as described above, was tested on LB agar plates containing 0.2% (w / w) AZCL-galactomannan (Megazyme) and 9 g / ml chloramphenicol and incubated overnight at 37 ° C. Clones expressing mannanase activity appeared with blue diffuse aureole. Plasmid DNA from one of these clones was isolated with a Qiagen plasmid spin preparation per ml overnight in 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 a Taq deoxy-termination cycle sequencing kit (Perkin-Elmer, USA), fluorescently labeled terminators, and appropriate oligonucleotides as primers.

Sequencing data analysis was performed according to Devereux et al., Nucleic Acids Res. 1984, 12, 387-395. The sequence encoding the mannanase is set forth in SEQ. id. The deduced protein sequence is set forth in SEQ. id. No 2.

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

mannanasa. homí. Suction I

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

5 '-GAC GAC GTA CAA GCG GCC GCG CTA TTT CCC TAA 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 0

00

SacII and Notll restriction sites are underlined.

The chromosomal DNA isolated from B. agaradherens NCIMB 40482 as described above was used as a template in the 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% (w / v) gelatin) containing 200 μΜ of each dNTP, 2.5 AmpliTaq polymerase units (Perkin-Elmer, Cetus, USA) and 100 pmoles 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 performed using a denaturation cycle profile of 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% agarose gels (NuSieve, FMC). The appearance of a 1400 molecular weight DNA fragment indicates appropriate amplification of the gene segment.

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

pg pMOL944 and 25 μΙ of the purified PCR fragment were digested with SacII and NotI, electrophoresed in 0.8% low temperature agarose gels (SeaPlaque GTG, FMC), the respective fragments were excised from the gel and purified by QIAquick Gel extraction kit (Qiagen, USA) according to the manufacturer's instructions. The isolated PCR DNA fragment was then ligated with purified pMOL944, which had been previously digested with SacII-NotI. Ligation was performed overnight at 16 ° C using 0.5 µg 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. The transformed cells were plated on LBPG-10 µg / ml kanamycin plate. After 18 hours incubation at 37 ° C, colonies were visible on the plates. Several columns were analyzed by isolating the plasmid DNA from the culture medium overnight.

One of these positive clones was streaked several times on agar plates as described above. This clone was called MB594. The MB594 clone was grown overnight in TY-10 µg / ml kanamycin at 37 ° C. The next day, 1 ml cells were used to isolate the 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 part from position 94 to 1404 of the linked sequence id. No 3.

ATGAAAAAAAAGTTATCACAGATTTATCATTTAATTATTTGCACACTTATAATA

AGTGTGGGAATAATGGGGATTACAACGTCCCCATCAGCAGCAAGTACAGGC ttttatgttgatggcAatacgttat / \ tgacgcaaatgggcagccatttgtcat

GAGÁGGTATTAACCATGGACATGCTTGGTATAAAGACACCGCTTCAACAGCT

ATTCCTGCCATTGCAGAGCAAGGCGCCAACACGATTCGTATTGTTTTATCAG

ATGGCGGTCAATGGGAAAAAGACGACATTGACACCATTCGTGAAGTCATTG

AGČŤGCGGAGCAAAAIA ^ ÁATrGGŤGGCTGTCGTTGAAGTTCATCAŤGCCA ''

CGGGTCGCGATTCGCGCAGTGATTTAAATCGAGCCGTTGATTATTGGATAG

AftATGAAAGATGCGCTTATCGGTAAAGAAGATACGGTTATTArrAACATTGCA / \ ACGAGTGGTATGGGAGTTGGGATGGCTCAGCTTGGGCCGATGGCTATATT

GATGTCATTCCGAAGCTTCGCGATGCCGGCTTAACACACACCTTAATGGTTG

ATGCAGCAGGATGGGGGCAATATCCGCAATCTATTCATGATTACGGACAAG

ATGTGTTTAATGCAGATCCGTTAAA / u \ ATACGATGTTCTCCATCCATATGTAT

GÁGTATGCTGGTGGTGATGCTAACAGTGTTAGATCAAATATTGATAGAGTCA

TAGATCAAGACCTTGCTCTCGTAATAGGTGAATTCGGTCATAGACATACTGA

TGGTGATGTTGATGAAGATACAATCCTTAGTTATTCTGAAGAAACTGGCACA

GGGTGGCTCGCTTGGTCTTGGAAAGGCAACAGTACCGAATGGGACTATTTA

GACCTTTCAGAAGACTGGGCTGGTCAACATTTAACTGATTGGGGGAATAGAA

7GTCCACGGGGCCGATGGCTTACAGGAAACCTCCAAACCATCCACCGTAT r / ACAGATGATAACGGTGGTCACCCTGAACCGCCAACTGCTACTACCTTGTA • ·

7GACTTTGAAGGAAGCACACAAGGGTGGCATGGAAGCAACGTGACCGGTG

GCCCTTGGTCCGTAACAGAATGGGGTGCTTCAGGTAACTACTCTTTAAAAGC

CGATGTAAATTTAACCTCAAATTCTTCACATGAACTGTATAGTGAACAAAGTC

GTAATCTACACGGATACTCTCAGCTCAACGCAACCGTTCGCCATGCCAATTG

GGGAAATCCCGGTAATGGCATGAATGCAAGACTTTACGTGAAAACGGGCTC

TGATTATACATGGCATAGCGGTCCTjTTACACGTATCAATAGCTCCAACTCA

GGAACAACGTTATCTTTTGATTTAAAv: AACATCGAAAATATCATCATGTTAGG

G; \ AATAG (SEQ ID NO: 3)

The derived mature protein is shown in SEQ. id. No. 4

MHKKLSQlYHLIICTLlISVGIMGITrŠPSAASTGFYVDGNTLYDANGQPFVMRGIN

HGHAWYKDTASTAIPAIAEQGANTIRIVLSDGGQWEKDDIDTIREVIELAEQNKM, vawevhdatgrdsrsdlnravdywiemkdáíjgkedtviíňíaňěwýgsvvďgs AWADGYIDVIPKLRDAGLTHTLMVDAAGWGQYPQSIHDYGQDVFNADPLKNTM FSIHMYEYAGGDANTVRSNIDRVIDCiDLALVIGEFGHRHTDGDVDEDTILSYSEE TSTGWLAWSWKGNSTEWDYLDLSEÚWAGQHLTDWGNRIVHGADGLQETSKP STVFTDDNGGHPEPPTATTLYDFEGSTQGWHGSNVTGGPWSVTEWGASGNY SLKADVNLTSNSSHELYSEQSRNLHGYSQLNATVRHANWGNPGNGMNARLYV KTGSDYTWHSGPFTRINSSNSGTTLSFDLNNIENIIMLGK (Seq. Id. No. 4)

It was found that the 3 'end of the mannanase encoded by the sequence of 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 PCR. The resulting amino acid sequence is shown in SEQ. id. It is apparent that the carbon end of SEQ. id. No. 2 (SHHVREIGVQFSAADNSSGQTALYVDNVTLR) is changed to the C terminus of SEQ. id. No. 4 (IIMLGK).

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

· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

LB agar (as described by Ausubel F. M. et al. (Ed.) 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 (WO 91/09129).

Mannanase expression, purification and characterization from Bacillus agaradherens: The MB 594 clone 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. minute.

6500 ml of MB 594 clone culture liquid (batch # 9813) was collected from the shake flasks and the pH was adjusted to 5.5. During stirring, 146 ml of cation exchange resin (C521) and 292 ml of anion exchange resin (A1390) were added for flocculation. The flocculated material was separated by centrifugation using a Sorval RC 3B centrifuge at 9,000 rpm for 20 minutes at 6 ° C. The supernatant was clarified with Whatman GF / D and C glass filters and finally concentrated on a 10,000 wt. units.

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

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. Bound mannanase activity was eluted using a sodium chloride gradient.

Pure enzyme yielded a single band in SDS-PAGE with a molecular weight of 38,000. The amino acid sequence of the mannanase enzyme, ie the translated DNA sequence, is shown in the above-described sequence. id. No 2.

• ·

9

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

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

: ": 0.08 grams per liter molecular weight: 38 000 µl (isoelectric point): 4.2.

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

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

DSC differential evaluation calorimetry gave 77 ° C as the melting point at pH 7.5 in Tris buffer, indicating that this enzyme is thermally very 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.

Recovery of 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 ydhT coding region, whose product is unknown, showed 58% similarity to the known Bacillus β-mannanase. The following oligonucleotides were designed to amplify sequences encoding the mature portion of the putative β-mannanase: 5-GCT CAA TTG GCG CAT ACT GTG TCG CCT GTG-3 'and 5'-GAC GGA TCG CGG ATT CAC TCA ACG ATT GGC G-3' . Total genomic DNA from Bacillus subtilis strain 1A95 was used

as a template to amplify the ydhT mature region using the above primers. PCR was performed using a GENE-AMP PCR kit with AMPLITAQ DNA polymerase (Perkin Elmer, Applied Biosystems, Foster City, CA). The initial melting time of 5 minutes at 95 ° C was followed by 25 cycles with the following program: melting 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 expansion. PCR products were purified using a 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 pairs of nucoeotides was digested with Mfel and BamH1. The expression vector pPG1527 was digested with EcoRI and BamH1. Restriction products were purified using a 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 respective DH5-α E coli strains. Ampicillin resistant colonies were cultured for DNA preparation. 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 Bacillus substilis kanamycin resistant clones and one PG 632 control clone were collected and grown in 20 ml 20/20/5 medium (20 g / l tryptone, 20 g / l yeast extract, 5 g / l NaCl) supplemented with 1 ml 25% mortar, 120 μΙ 10mM MnCl ₂ and 20 μΙ 50 mg / ml kanamycin. Clones were grown overnight in 250 ml flasks with shaking at 250 rpm at 37 ° C for protein expression. The cells were then centrifuged at 14,000 rpm for 15 minutes. One microliter of each supernatant was diluted in 99 μΙ 50mM sodium acetate (pH 6.0). One μΙ of this dilution was evaluated using endo-1,4-B-mannanase Beta-Mannazyme Tabs (Megazyme, Ireland) according to the manufacturer's instructions. Absorbance was evaluated at 590 nm • · ·· 4 ·

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

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

The fermentation of Bacillus subtilis ydhT clone 7 was also obtained in a B. Braun Biostat C fermenter. 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, 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 performed as follows: 500 ml of the supernatant was centrifuged at 10,000 rpm for 10 minutes at 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 separation of 12,000-14,000 (Speetrum). The dialyzed supernatant was centrifuged at 10,000 rpm for 10 minutes at 4 ° C. A 200 ml high-speed Q sepharose anion exchange column (Pharmacia) was equilibrated with 1 liter of 10 mM potassium phosphate (pH 7.2) at 20 ° C. 300 ml of the supernatant was applied to the column. Two effluent fractions of 210 ml (sample A) and 175 ml (sample B) were collected. The two fractions were analyzed as above except that the samples were diluted with 199 μΙ 50 mM sodium acetate (pH 6.0) and showed an absorbance of 0.38. · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 0.52. Two microliters of each sample was added to 8 μΙ of Tris-glycine SDS sample buffer (Novex, Ka., USA) and boiled for 5 minutes. The resulting samples were electrophoresed on a 10-20% 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. total protein. BCA protein analysis (Pierce) was performed on both samples according to the manufacturer's instructions using bovine serum albumin as standard. Samples A and B contained 1.3 and 1.6 mg / ml B-mannanase, respectively. The identity of the protein was confirmed by ion spray mass spectrometry and amino acid sequence analysis from the amino terminus.

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

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 β-mannanase retained significant activity at less than 15 ° C and more than 80 ° C. The specific activity for β-1,4-galactomannan was determined to be 160,000 pmol / min.mg β-mannanase using endo-1,4-β-mannanase Beta-Mannazyme Tabs (Megazyme, Ireland) according to the manufacturer's instructions. The nucleotide and amino acid sequences of Bacillus subtilisis β-mannanase are shown in SEQ. id. No. 5 and id. No. 6.

The mannanase is included in the compositions of 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. pure enzyme by weight of the composition.

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

4 4 4 4 4 • 4 • 4 ·· ··

4 4 4

4 4 4

4 4 4

44 are operably connected. The cellulose binding domain (CBD) may exist as an integral part of the encoded enzyme, or CBD of other origin may be introduced into the enzyme to form 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 the 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., the red algae of Porhyra purpurea, as a nonhydrolytic polysaccharide binding protein, see Tomme et al. (as above). However, most CBDs are cellulases and xylanases. CBDs have been found to be at the N or C termini of the proteins or are intrinsic. Enzyme hybrids are known in the art, see, for example, 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 bound, with or without a linker, to DNA a sequence encoding a mannanase enzyme and a growing host cell so that the fusion gene is expressed. Enzyme hybrids can be described by the following general formula

CBD-MR-X, wherein CBD is the N- or C-terminal region of the amino acid sequence corresponding to at least the cellulose binding domain, MR is the linker, and may be a bond or a short linking group preferably of 2 to 100 carbon atoms, more preferably 2 to 40 carbon atoms, or preferably 2 to 100, more preferably 2 to 40 amino acids, and X is the N-terminal or C-terminal region of the enzyme of the invention.

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

4 44 44 4444 44 44 444 4444

44 44 4444

44,444 4,444 44 4

43 4 4 43 4 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 the components usually encountered in such compositions. Other variants may also be designed such that optimal pH, stability to bleaching and chelating agents, catalytic activity, and similar properties of the enzyme variant are adjusted to suit a particular purification application.

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

Fabric softener: The second essential ingredient of the present detergent and / or fabric care compositions is fabric softener clay. According to the present invention, any clay used in the art, or mixtures thereof, can be used. Preferred examples are described in British Patent Specification 1,400,898 or in U.S. Pat. No. 5,019,292.

These clay include a variety of 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 milliequins. per 100 grams of clay, which corresponds to a layer charge of 0.2 to 0.6. Other preferred clays are those having particle sizes ranging from 5 to 50 microns.

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

Other preferred smectite clays are hectorite clays of the general formula [(Mg _{3. X} Li _x ) Si _4. YMe ^III 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 +} means a monovalent (n = 1) or divalent (n = 2) metal ion, for example selected from sodium, potassium, magnesium, calcium and strontium. The hectorite clays suitable for the detergent compositions of the present invention have a charge charge distribution of the layer such that at least 50% is in the range of 0.23 to 0.31.

Preference is given to naturally occurring hectorite clays having a charge distribution of the layer 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:

montmorillonite sodium: 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) , montmorillonite calcium; Soft Clark ^(R) , Gelwhite L ( ^w) , Imvite K ^(R) , CSM-Clay ^(R)

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

The amount of fabric softener to be used according to the present invention depends on the form of laundry detergent and / or fabric care compositions. In general, it can range from the lower limits of 0.1, 3 or 4 up to the upper limits of 50, 25 or 15% by weight.

»0 ·« ··

AND*

0 0 0 0 0 00 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 00

Detergent Ingredients: The laundry detergent and / or fabric care compositions of the invention must contain at least one additional detergent component. The exact nature of this additional component and the amounts included herein will depend upon 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 component selected from zeolite, sodium triphosphate and / or layered silicate, a cationic surfactant, and or mixtures thereof.

The laundry detergent and / or fabric care compositions of the invention may exist as a liquid, paste, gels, sticks, tablets, spray, foam, powder or granules. Granular compositions may also exist in compact form and liquid compositions may also exist in concentrated form.

The compositions of the invention may be formulated, for example, as hand and automatic laundry detergent compositions, including laundry additive compositions and compositions suitable for use in soaking and / or pretreatment of stained fabrics and as rinse fabric softeners.

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

94

4 9

9 4

9 9

9 9 9

94

100 • 4 9 9

9 4 · 4

Fabric softeners other than the inorganic clay claimed in the present invention may also be included as additional detergent ingredients. Compositions containing mannanase and clay may provide fabric cleansing, de-staining, fabric softeners, and color appearance 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 as a supplement or as an agent that improves the performance of conventional detergent compositions and can 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 the composition measured at 20 ° C.

The compact form of the compositions of the invention is best expressed in terms of density and in terms of composition by the amount of inorganic filler salt. Inorganic filler salts are conventional detergent compositions in powder form. In conventional detergent compositions, the filler salts are present in substantial amounts, typically 17-35% by weight. by weight of the whole composition. In compact compositions, the filler salt is present in amounts not exceeding 15% of the total composition, preferably not more than 10% and most preferably not more than 5% by weight. by weight of the composition. The inorganic filler salts, as meant in the present compositions, are selected from alkali metal and alkaline earth metal sulphates and chlorides. The preferred filler salt is sodium sulfate.

The liquid detergent compositions of the present invention may also exist in concentrated form. In such a case, the liquid detergent compositions of the present invention will contain less water as 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. 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 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 zwitterionic surfactants. and / or semi-polar surfactants. The laundry detergent 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 fabric softener.

The other surfactant is typically present in an amount of from 0.1 to 60 wt%. A more preferred amount is 1 to 35%, most preferably 1 to 30% by weight. by weight of the laundry detergent 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 such that it promotes or at least does not degrade the stability of any enzyme in such compositions.

The 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 the condensation products of alkylphenols having an alkyl group of 6 to 14 carbon atoms, preferably of 8 to 14 carbon atoms, in either a straight or branched chain configuration and an 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 alkyl phenol. 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 the Rohm & Haas Company. These surfactants are commonly referred to as alkylphenolalkoxylates (e.g., alkylphenol ethoxylates).

The 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. Preferred are the 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. 2 to 7 moles of ethylene oxide and most preferably 2 to 5 moles of ethylene oxide per mole of alcohol are present in said condensation products. Examples of commercially available nonionic surfactants of this type include Tergitol (TM) 15-S-9 (9-mol linear ethylene oxide condensation product), Tergitol (TM) 24-L-6 NMW (12-primary primary alcohol condensation product). up to 14 carbon atoms with 6 moles of narrow molecular weight ethylene oxide), both sold by Union Carbide Corporation, Neodol ™ 45-9 (a condensation product of a linear alcohol of 14 to 15 carbon atoms with 9 moles of ethylene oxide), Neodol ™ 23-3 ( the condensation product of a linear C 12 -C 13 carbon atom with 3 moles of ethylene oxide), NEODOL ™ 45-7 (the condensation product of a linear alcohol of 14 to 15 carbon atoms with 7 moles of ethylene oxide), Neodol ™ 45-5 up to 15 carbon atoms with 5 moles of ethylene oxide), manufactured by Shell Chemical Company, Kyro ™ EOB (condensation product of an alcohol of 13 to 15 carbon atoms with 9 moles of thylene oxide), manufactured by The Procter and Gamble Company, and Genapol LA 030 or 050 (a C 12 -C 14 alcohol condensation product with 3 or 5 moles of ethylene oxide), manufactured by Hoechst. The preferred HLB range in these products is from 8 to 11, and most preferably from 8 to 10.

Also useful in the compositions of the invention are useful nonionic surfactants of the surfactant system of the present invention as alkylpolysaccharides such as those described in U.S. Patent 4,565,647 Llenada, issued January 21, 1986, having a hydrophobic group of from 6 to 30; preferably having 10 to 16 carbon atoms, and a polysaccharide, eg, polyglycoside, the hydrophilic group contains from 1.3 to 10, preferably from 1.3 to 3, most preferably from 1.3 to 2.7 carbohydrate units. Any reducing carbohydrate of 5 or 6 carbon atoms may be used, e.g. glucose, galactose, and galactosyl groups may be substituted with glucosyl groups (optionally, the hydrophobic group is attached at the 2-, 3-, 4-, etc. position to provide glucose or galactose as opposed to glucoside or galactoside). Sugar linkages may be, for example, between one position of the other saccharide units and the 2-, 3-, 4- and / or 6- positions of the preceding saccharide units.

Preferred alkyl polyglycosides are compounds of formula

R ² O (C _n H 2 _n O) t (glycosyl) _x , wherein R ₂ is selected from the group consisting of alkyl, alkylphenyl, hydroxyalkyl, hydroxyalkylphenyl and mixtures thereof, wherein the alkyl groups contain from 10 to 18, preferably from 12 to 14 carbon atoms, n is 2 or 3, preferably 2, t is from 0 to 10, preferably 0, and x is from 1.3 to 10, preferably from 1.3 to 3, most preferably from 1.3 to 2.7. The glycosyl is preferably derived from glucose. In preparing these compounds, an alcohol or an alkylpolyethoxy alcohol is first formed and then reacted with glucose or a source of glucose to form the glucoside (attached at position 1). Additional glycosyl units may then be attached between their 1-position and the 2, 3, 4 and / or 6 positions of the preceding glycosyl units, preferably predominantly at the 2-position.

«

The hydrophobic base ethylene oxide condensation products formed by the condensation of propylene oxide with propylene glycol are also suitable for use as other nonionic surfactant systems of 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 moiety tends to increase the solubility of the molecule as a whole in water, and this liquid nature of the product is maintained to the point where the polyoxyethylene content is 50 wt%. based on the total weight of the condensation product corresponding to the condensation with up to 40 moles of ethylene oxide. Examples of compounds of this type include some of the commercially available Plurafac ™ LF404 and Pluronic ™ surfactants manufactured by BASF.

Also suitable for use as the nonionic surfactant of the nonionic surfactant system of the present invention are the condensation products of ethylene oxide with the products of the reaction of propylene oxide with ethylenediamine. The hydrophobic portion of these products consists of 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. polyoxyethylene and had a molecular weight of from 5,000 to 11,000. Examples of this type of nonionic surfactant include some commercially available Tetronic ™ compounds manufactured by BASF.

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

Highly preferred nonionic surfactants are the surfactants are polyhydroxy fatty acid amides of the general formula • · »« »« • r ² · ¹¹ -cnz> 1 OR ¹ wherein R ¹ represents a hydrogen atom or a hydrocarbon group having 1 to 4 carbon atoms, 2-hydroxyethyl, 2-hydroxypropyl or a mixture thereof, R ² is a hydrocarbon group having 5-31 carbon atoms and Z is a polyhydroxyhydrocarbyl having a linear hydrocarbyl chain with at least three hydroxyl groups directly connected to the chain, or an alkoxylated derivative thereof. Preferably R ¹ is methyl, R ² is C 11 -C 15 straight alkyl or C 16 -C 18 alkyl or alkenyl, such as coconut 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 for use herein include linear alkylbenzene sulfonate, alkyl ester sulfonate surfactants comprising linear esters of C 8 -C 20 carboxylic acids (i.e. fatty acids) that are sulfonated with gaseous sulfur dioxide 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, especially for laundry applications, include alkyl ester sulfonate surfactants of the formula:

O, H "

R ³ -CH-C-OR ^r ,

Even with ₃ m in which R ³ represents uhlodíkovou group having 8-20 carbon atoms, preferably an alkyl, or combination thereof, R ⁴ is a hydrocarbon group having 1 to 6 carbon atoms, • · preferably an alkyl, or combination thereof, and M is a cation, which forms a water-soluble salt with an alkyl ester sulfonate. Suitable salt-forming cations include such metals as sodium, potassium and lithium, and substituted or unsubstituted ammonium cations such as monoethanolamine, diethanolamine and triethanolamine. Preferably R ₃ is C 10 -C 16 alkyl and R ₄ is methyl, ethyl or isopropyl. Particular preference is given to methyl ester sulfonates in which R ³ is C 10 -C 16 alkyl.

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

Other anionic surfactants may be included in the cleaning compositions of the present invention for cleaning purposes. These include salts (including, for example, sodium, potassium, ammonium and substituted ammonium salts such as mono-, di-, and triethanolamine salts) of soaps, primary or secondary C 8 -C 22 alkanesulfonates, C 8 -C 24 olefin sulfonates , sulfonated polycarboxylic acids prepared by sulfonation of the pyrotized alkaline earth citrate product, e.g., as described in British Patent Specification No. 1,082,179, alkyl (8 to 24 carbon atoms) polyglycol ether sulfates (with up to 10 ethylene oxide motifs), alkyl glycerol sulfonates, fatty acytglycerol sulfates fatty fatty acid glycerophsulfates, alkyl phenol ethylene oxide ether sulfates, paraffin sulfonates, alkyl phosphates, isethionates such as acyl isethionates , N-acyltaurates, alkylsuccinamates and sulfosuccinates, monoesters of sulfosuccinates (especially saturated and unsaturated monoesters (d) esters of sulfosuccinates (particularly saturated and unsaturated diesters of 6 to 12 carbon atoms), acyl sarcosinates, alkylpolysaccharide sulfates such as alkylpotyglucoside sulfates (nonionic unsulfated compounds described below), branched chain alkyl chain alkyl alkylsulfates and branched chain alkyl alkoxy sulfates; are compounds of formula

RO (CH ₂ CH ₂ O) _k -CH ₂ COO'M ⁺ , wherein R is an alkyl of 8 to 22 carbon atoms, k is an integer from 1 to 10, and M is a soluble salt cation. 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.

Additional examples are described in Surface Active Agents and Detergents (Volume I and tt, Schwartz, Perry and Berch). Such a variety of surfactants are also generally described in U.S. Pat. No. 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 present, the laundry detergent and / or fabric care compositions of the present invention typically comprise from 1 to 40%, preferably from 3 to 20% by weight. of these anionic surfactants.

Highly preferred anionic surfactants include alkylalkoxylated sulfate surfactants. These surfactants are water-soluble salts or acids of the formula RO (A) _m SO ₃ M, wherein R is unsubstituted C 10 -C 24 alkyl or C 10 -C 24 alkyl, preferably C 10 -C 24 alkyl. ·

An alkyl or hydroxyalkyl group having 12 to 20 carbon atoms, more preferably an alkyl or hydroxyalkyl group having 12 to 18 carbon atoms, A is an ethoxy or propoxy unit, m is 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 be, for example, a metal cation (e.g., sodium, potassium, lithium, calcium, magnesium, etc.), ammonium or substituted ammonium cation. These include 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 tetramethylammonium and dimethylpiperidine cations and alkylamine derived cations such as ethylamine, diethylamine, triethylamine, and mixtures thereof and the like. Examples of surfactants are the alkyl (C 12 -C 18) sulfate of the polyethoxylate (1,0) (C ₁₂ -C ₁₈ E (1.0) M), the alkyl (C 12 -C 18) sulfate of the polyethoxylate (2, C ₁ -C ₁₈ E). 25) (C ₁₂ -C ₁₈ E (2.25) M), alkyl (C 12 -C 18 alkyl) sulfate of polyethoxylate (3.0) (C 12 -C ₁₈ E (3.0) M) and alkyl sulfate (C 12-18) carbon atoms) of a polyethoxylate (4.0) (C 12 -C ₁₈ E (4.0) M), wherein M is suitably selected from sodium and potassium.

The laundry detergent 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 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 extensively described as aliphatic derivatives of secondary or tertiary amines or aliphatic derivatives of heterocyclic secondary and tertiary amines in which the aliphatic group may have a 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 December 30, 1975, at column 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 and / or fabric care compositions of the present invention typically comprise 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 and / or fabric care compositions. These surfactants can be extensively 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 Dec. 30, 1975, column 19, line 18, to column 22, line 48.

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

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

Semi-polar nonionic detergent surfactants include amine oxide surfactants of the formula

9 * · 98 9

O t

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

These amine oxide surfactants particularly include alkyl (C10-18) dimethylamine oxides and alkoxy (C8-12) ethyldihydroxyethylamine oxides.

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

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

Suitable primary amines for use herein include amines of the formula R ₁ NH ₂ , wherein R 6 represents an alkyl chain of 6 to 12, preferably 6 to 10 carbon atoms, or a group R 4 X (CH ₂ ) _n , X is -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 of R 1 may be straight or branched and may be interrupted up to 12, preferably with less than 5 ethylene oxide groups. Preferred amines of the above formula are alkylamines. Suitable amines for use in the invention may be selected from 1-hexylamine, 1-octylamine, 1-decylamine and lau44.

4 »· 4 4» · 4 4 ♦ · 4 4

0 · rylamine. Other preferred primary amines include oxypropylamine, octyloxypropylamine, 2-ethylhexyloxypropylamine, lurylamidopropylamine and C 8 -C 10 amidopropylamine.

Suitable tertiary amines for use herein include tertiary amines of formula R 1 R ₂ R ₃ H wherein R 1 and R ₂ are C 1 -C 8 alkyl chains or a group of the formula

R ₅

- (CH ₂ -CH-O) _x H,

R ₃ is either an alkyl chain of 6 to 12 carbon atoms, preferably of 6 to 10 carbon atoms, or R ₃ is a group of the formula R 4 X (CH ₂ ) _n wherein X is -O-, -C (O) NH- or -NH-, R ₄ is a group of 4 to 12 carbon atoms and n is a number between 1 and 5, preferably 2 to 3. R ₅ is a hydrogen atom or an alkyl of 1 to 2 carbon atoms and x is a number between 1 and 5 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 those of formula R ₁ R ₂ R ₃ N, wherein R 1 is an alkyl chain of 6 to 12 carbon atoms, R ₂ and R ₃ are an alkyl group of 1 to 3 carbon atoms, or a group of the formula

R ₅

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

Also preferred are amidoamines of the formula

RrC (O) -NH- (CH ₂ ) n N- (R ₂ ) ₂ • * φ ·· · * φφ • φ · φ · φ · φ · φ · · · · · φ · · φ φ · · · · wherein R 1 is C 6 -C 12 alkyl, n is 2 to 4, preferably n is 3, and R ₂ and R ₃ are C 1 -C 4 groups.

The most preferred amines of the present invention include 1-octylamine, 1-hexylamine, 1-decylamine, 1-dodecylamine, C 8 -C 10 oxypropylamine, N-coconut 1,3-diaminopropane, coconut alkyldimethylamine, lauryldimethylamine, laurylbis (hydroxyethyl) amine , coconut bis (hydroxyethyl) amine, 2-mol prophylated laurylamine, 2-mol prophylated octylamine, laurylamidopropylmethylamine, C 8 -C 10 amidopropyldimethylamine and C 10 amidopropyldimethylamine.

The most preferred amines for use in the compositions of the invention are 1-hexylamine, 1-octylamine, 1-decylamine and 1-dodecylamine. Particularly desirable are dodecyldimethylamine, bishydroxyethyl (coconut alkyl) amine, seven times ethoxylated oleylamine, laurylamidopropylamine and coconut amidopropylamine.

Bleaching Agents: The laundry detergent and / or fabric care compositions may further comprise bleaching agents such as hydrogen peroxide, PB1, PB4, and percarbonate having a particle size of 400-800 µm. These bleach 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 bleach component for use according to the invention may be any bleaching agent useful for laundry detergent and / or care agents containing oxygen bleaching agents as well as other agents known in the art. A bleaching agent suitable for the present invention may be an activated or non-activated bleaching agent.

• w · · · • * * * * * * * w w w w w w w

One category of oxygen bleaching agents that may be used includes percarboxylic acid bleaching agents and salts thereof. Suitable examples of this class of agents include magnesium monoperphthalate hexahydrate, magnesium salt of m-chloroperbenzoic acid, 4-nonylamino-4-oxo-butyric acid, and diperdodecanedioic acid. Such bleaching agents are described in U.S. Patent 4,483,781, U.S. Patent Application 740,446, European Patent Application 0 133,354, and U.S. Patent 4,412,934. Highly preferred bleaching agents also include 6-nonylamino-6-oxopercaproic acid as described U.S. Patent 4,634,551.

Another category of bleaching agents that may be used includes halogenated bleaching agents. Examples of hypohalite 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 wt. % by weight of the final product, preferably in an amount of 1 to 5 wt.

Hydrogen peroxide releasing agents can be used in combination with bleach activators such as tetraacetylethylenediamine (TAED), nonanoyloxybenzenesulfonate (NOBS, described in US patent 4,412,934), 3,5-trimethylhexanoloxybenzenesulfonate (ISONOBS, described in European patent 120,591g) or penta (PAG) or the phenol sulfonate ester of N-nonanoyl-6-aminocaproic acid (NACA-OBS, described in WO 94/28106), which is perhydrolyzed to produce peracid as active bleaching particles, resulting in improved bleaching performance. Suitable activators are also acylated citrate esters such as those disclosed in the accompanying European Patent Application No. 91870207.7 and the unsymmetrical acyclic imide bleach activator of the following general formula described in the accompanying U.S. Patent Application No. 60/022 786 (Procter & Gamble, filed July 30) 1996) and No. 60/028 122 (filed October 15, 1996)

R ₃

Ο ♦ • • • ·

ΦΦ • φ • ·

In which R1 represents a saturated or unsaturated alkyl group having a linear or branched chain of 7 to 13 carbon atoms R ₂ represents a saturated or unsaturated C 1 -C 8 linear or branched alkyl group and R ₃ represents a saturated or unsaturated C 1 -C 4 linear or branched alkyl group.

Useful bleaching agents, including peracids and bleach systems comprising bleach activators and peroxygen bleach compounds, for use in laundry detergent and / or fabric care compositions of the invention are described in our accompanying U.S. Application Serial Nos. 08/136 626, PCT / US95 / No. 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 (ie, an enzyme and a substrate thereof) that is capable of generating hydrogen peroxide at the beginning or during the washing and / or rinse process. Such enzyme systems are described in European Patent Application 91202655.6, filed October 6, 1991.

Metal-containing catalysts for use in bleaching agents include cobalt-containing catalysts such as pentaamine acetate-cobalt salts and manganese-containing catalysts such as those described in European Patent Applications 549271, 549 272, 458 397, 458 398 and US Patents 5,246 621, 5194416 and 5114611. A bleaching composition comprising perslouonene and a bleach catalyst comprising manganese and a chelating agent is disclosed in Patent Application No. 94870206.3.

Bleaching agents other than oxygen bleaching agents are also known in the art and can be used herein. One type of non-oxygen bleaching agent of particular interest includes photoactivated bleaching agents such as sulfonated zinc and / or aluminum phthalocyanines. These materials can be deposited on the substrate during the washing process. After irradiation with light in the presence of oxygen, such as hanging clothes for daylight drying, the sulfonated zinc phthalocyanine is activated and the substrate is bleached. Výro··· · · · · · · 9 9 · · ««

Zinc phthalocyanine and a photoactivated bleaching process are described in U.S. Patent 4,033,718. Laundry detergent and / or fabric care compositions will typically contain from 0.025 to 1%. 25 wt. sulfonated zinc phthalocyanine.

Building Component System: The laundry detergent and / or fabric care compositions of the present invention may further comprise a building component. The laundry detergent 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 weighing performance.

Any conventional builder system that includes aluminosilicate materials, silicates, polycarboxylates, alkyl or alkenylsuccinic acid and fatty acids, such materials as ethylenediaminetetraacetate, diethylenetriaminepentamethylene acetate, metal ion scavengers, such as aminopolyphenonephosphonates, especially aminopolyphenephosphine, especially aminopolyphosphine phosphonates diethylenetriaminepentamethylenephosphonic acid. Phosphate builders can also be used herein.

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

Another suitable inorganic building material is a layered silicate such as SKS-6 (Hoechst). SKS-6 is a crystalline layered silicate consisting of sodium silicate (Na ₂ Si ₂ O ₅ ).

Suitable polycarboxylates containing a single carboxyl group include lactic acid, glycolic acid, and ether derivatives thereof as described in Belgian patents Nos. 831,368, 821,369 and 821,370. Polycarboxylates containing two carboxyl groups include the 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 the ether carboxylates described in FR 2446686 and 2,446,687; U.S. Pat. No. 3,935,257 and the sulfinylcarboxylates described in Belgian Patent No. 640,623. Polycarboxylates containing three carboxyl groups in particular include water-soluble citrates, aconitates and citraconates as well as succinate derivatives such as the carboxymethyloxysuccinates described in British Patent Specification No. 5,902,249. 1,379,241, lacto-succinates disclosed in Dutch Patent Application 7,205,873 and oxypolycarboxylate materials such as 2-oxa-1,3,3-propane tricarboxylates described in British Patent 1,387,447.

Polycarboxylates containing four carboxyl groups include the oxydisuccinates described in British Patent Specification 1,261,829, 1,1,2,2-ethane tetracarboxylates, 1,1,3,3-propantetracarboxylates and 1,1,2, 3-propanetetracarboxylates. Sulfosubstituted-containing polycarboxylates include the sulfosuccinate derivatives disclosed in British Patent Specification 6. No. 1,398,421 and 1,398,422; and U.S. Patent No. 3,936,448 and the sulfonated pyrolyzed citrates described in British Patent No. 1,082,179, wherein polycarboxylates containing phosphone 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 meilitic acid, pyromilitic acid, and phthalic acid derivatives described in British Patent 1,425,343.

9 9 9 9 9 9 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, particularly citrates.

Preferred building 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 systems 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 building systems for liquid laundry detergent and / or fabric care compositions of the present invention are soaps and polycarboxylates.

Other building materials that may form part of the building system for use in granular compositions include inorganic materials such as alkali metal carbonates, bicarbonates and silicates, and organic materials such as organic phosphonates, aminopolyalkylene phosphonates, and aminopolycarboxylates. Other suitable water-soluble organic salts are homo- or co-polymeric acids or salts thereof, in which the polycarboxylic acid comprises at least two carboxyl groups separated by no more than two carbon atoms. Polymers of this type are described in British Patent No. 1,596,756. Examples of such salts are polyacrylates having a molecular weight of 2000-5000 and copolymers thereof with maleic anhydride, such as copolymers having a molecular weight of from 20,000 to 70,000, in particular about 40,000.

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

Conventional Detergent Enzymes: In addition to mannanase enzyme, laundry detergent and / or fabric care compositions may further contain one of the following:

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

Said enzymes include enzymes selected from cellulase, hemicellulase, peroxidase, protease, glucoamylase, amylase, xylanase, lipase, phospholipase, esterase, cutin, pectinase, keratanas, reductase, oxidase, phenoloxidase, lipoxygenase, ligninase, pullulanase, tannase, pentosanas, mallanase, β-glucanase, arabinosidase, hyaluronidase, chondroitinas, lackas or mixtures thereof.

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

Suitable proteases are subtilisins which are obtained from the respective strains of B. subtilis and B. licheniformis (subtilisin BPN and BPN '). One suitable protease is obtained from a Bacillus strain, has a maximum activity in the pH range of 8-12, 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 Maxap ^(R) (protein engineered Maxacal) from Gist- Proteolytic enzymes also include modified bacterial serine proteases, such as those described in European Patent Application No. 87 303761.8, filed April 28, 1987 (particularly pages 17, 24, and 98), 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 protease referred to herein as protease C, which is herein referred to as &quot; protease C &quot; A variant of the alkaline serine protease from Bacillus in which lysine replaced arginine at position 27, tyrosine replaced valine at position 104, serine replaced asparagine at position 123 and alanine replaced threonine at position 27 4. Protease C is described in European Patent 90915958: 4, which corresponds to WO 91/06637, published May 16, 1991. Also included are genetically modified variants, particularly protease C.

A preferred protease, referred to as protease D, is a variant of a non-naturally occurring carbonyl hydrolase that is derived from a precursor carbonyl hydrolase by substituting different amino acids for many amino acid residues at the position of said carbonyl hydrolase, which is equivalent to +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 numbering of Bacillus amyloliquefaciens subtilisin as described in WO 95/10591 and in Patent Application C. Ghose et al .: Bleaching Compositions Comprising Protease Enzymes, U.S. Patent Application No. 08 / 322,677, filed October 13, 1994. also a variant of carbonyl hydrolase prote as disclosed in WO 95/10591, which has an amino acid sequence derived by replacing multiple amino acid residues in an 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, wherein the position numbering corresponds to naturally occurring Bacillus amyloliquefaciens subtilisin or equivalent amino acid residues in other carbonyl hydrolase or subtilisins such as Bacillus lentus subtilisin (accompanying 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 variants thereof described in WO 95/23221.

• · · · · · · · · · · · · ·

See also 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 detergent compositions useful herein is described in WO 94/25583 (Novo). Other suitable proteases are described in European Patent 516 200 (Unilever).

Proteolytic enzymes are included in the laundry detergent 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. pure enzyme by weight of the composition.

Cellulases useful in the present invention include both bacterial and fungal type cellulases, preferably those having a pH optimum between 5 and 12 and a specific activity of about 50 CEVU / mg (CÉVU means 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 discloses fungal cellulase 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, in German patent OS 2,247,832 and in WO 95/26398.

Examples of such cellulases are cellulases produced by Humicola insolens (Humicola grisea var. Thermoidea), particularly Humicola by DSM 1800.

Other suitable cellulases are cellulases derived from Humicola insolens having a molecular weight of 50,000, an isolelectric point of 5.5 and containing 415 amino acids, and endoglucanase with a molecular weight of about 43,000 derived from Humicola insolens, DSM 1800, showing cellulase activity; a preferred endoglucanase component has the amino acid sequence described in PCT Patent Application No. WO 91/17243. Suitable cellulases are also EDIII cellulases from Trichoderma longibrachiatum, described in WO 94/21801, Genencor, published Sep. 29, 1994. Particularly suitable cellulases are cellulases having the color care property. Examples of such cellulases are those described in European Patent Application No. 9120287.9.2, filed Nov. 6, 1991. Particularly useful are Carezyme and Celluzyme (Novo Nordisk A / AS). 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 generally present in amounts of from 0.0001 to 2% by weight in detergent and / or fabric care compositions. pure enzyme by weight of laundry detergent and / or fabric care compositions.

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

The amplifiers are usually present in an amount of from 0.1 to 5% by weight. by weight of the whole composition. Preferred enhancers are substituted phenthiazine and phenoxazine 10-phenothiazinepropionic acid (PPT), 10-ethylphenothioazine-4-carbo.

4 4 4 4

4 4 4

4 4 4 4 4

4 4 4 4 •

44 xylic acid (EPC), 10-phenoxazinepropionic acid (POP) and 10-methylphenoxazine (described in WO 94/12621) and substituted syringates (C 3 -C 5 alkyl substituted syringates) and phenols. Preferred sources of hydrogen peroxide are sodium percarbonate or perborate.

Said peroxidases are usually present in amounts of from 0.0001 to 2% by weight in detergent and / or fabric care compositions. pure enzyme by weight of laundry detergent and / or fabric care compositions.

Other suitable enzymes that 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 family, such as Pseudomonas stutzeri ATCC 19154, as described in British Patent 1,372,304. Suitable lipases include those lipases that exhibit a positive immunological cross-reaction with a lipase antibody produced by Pseudomonas fluorescent IAM 1057. This lipase is available from Amano Pharmaceutical Co. Ltd., Nagoya, Japan, under the tradename Lipase P Amano, hereinafter also referred to as Amano-P. Other commercial lipases include Amano-CES, Chromobacter viscosum lipases, 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., The 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 compositions of the present invention. Also suitable are lipolytic enzymes described in European patent 258 068, in WO 92/05249 and WO 95/22615 (Novo Nordisk) and in WO 94/03578, WO 95/35381 and WO 96/00292 (Unilever).

• 99

Cutinases [EC 3.1.1.50] are also suitable, which can be considered as a special kind of lipase, namely lipases that do not require interfacial activation. Administration of cutin 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 an amount of from 0.0001 to 2% by weight of the detergent and / or fabric care compositions. pure enzyme by weight of laundry detergent and / or fabric care compositions.

Amylases (and and / or β) may be included in prostheses to remove carbohydrate-based stains. WO 94/02597, Novo Nordisk A / S, published February 3, 1994, discloses 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. Patent 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 Specification No. 1,296,839 (Novo). Other suitable amylases are the improved 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 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 discloses other suitable amylases: α-amylases, which have a specific activity at least 25% higher than

99

9

9 9 9

9

9

99 »9 99 9

9 9 9 9 9 ♦ 9 99 9 ·

9 9 9 9 9

9

The specific activity of Termamyl ^(R) at a temperature in the range of from 25 to 55 ° C and a pH in the range of from 8 to 10, as measured by the Phadebas ^(R) α-amylase activity assay. Variants of the above enzymes described in WO 96/23873 (Novo Nordisk) are suitable. 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.

Amylolytic enzymes are included in the laundry detergent 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. pure enzyme by weight of the composition.

The above enzymes may be of suitable origin, such as vegetable, animal, bacterial, fungal and yeast origin. Furthermore, the origin may 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 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 components usually encountered in such compositions is enhanced. Other variants may also be designed such that optimal pH, stability to bleaching or chelating agents, catalytic activity, and similar properties of the enzyme variant are adjusted to suit a particular purification application.

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

99 ··

99

9

9 9 9

9

9

99 • 9 99 • 999 9 ·

9 98 9 9

9 9 9 9 9 9 9

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

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

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

Many enzyme materials and compositions for their incorporation 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 3,555,339, Jan. 5, 2003. 1971, 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, U.S. Pat.

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

· »··

4 4 4

4 4 4

4 44

4 4 4

4 4 4

4 4

4 4

4 4

4 4 4

4 44

44

4 4 4

4 4 4

4 4 4 4

4 4 4

44

Effect of color care and fabric care: This also includes technologies that provide paint whiskers. Examples of such technologies are metal catalysts for color preservation. These metal catalysts are described in the accompanying European Patent Application No. 92870181.2. Dye fixing agents, anti-wrinkle polyolefin dispersion and improved water absorbability, perfume and amine function polymer (PCT / US 97/16546) for color treatment and perfume feel are other examples of dye / fabric care technologies and are described in the accompanying European No. 96870140.9, filed Nov. 7, 1996.

Fabric softeners 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 fabric softeners include water-insoluble tertiary amines as described in British Patent A 1 514 276 and European Patent B 0 011 340. Their combinations with mono (C 12 -C 14) quaternary ammonium salts are described in B 0 026 527 and B 0 026 528 and two long chain amides are described in European patent B 0 242 919. Other useful organic components of fabric softener systems include high molecular weight polyethylene oxide materials as described in the European patent application And 0 299 575 and 0 313146.

Organic fabric softeners such as water-insoluble tertiary amines or double 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 wt. These materials are normally added to the spray-dried portion of the composition, although in some cases it may be preferable to add them as a dry bulk composition or spray them as a molten liquid onto other solid components of the composition.

Chelating Agents: The laundry detergent and / or fabric care compositions of the present invention may also optionally contain one or more chelating agents.

Iron and / or manganese reagents. 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 lie in part in their exceptional ability to remove iron and manganese ions from washing solutions by forming soluble chelates.

Aminocarboxylates useful as optional chelating agents include ethylenediaminetetraacetates, N-hydroxyethylethylenediamine triacetates, nitrile triacetates, ethylenediaminetetrapropionates, triethylenetetramine hexaacetates, diethylenetriaminepentaacetates and ethanediglycines, and their salts with the alkali metal salts thereof.

Amino phosphonates are also suitable for use in the compositions of the invention as chelating agents when at least low amounts of total phosphorus are allowed in the detergent compositions. These agents include ethylenediaminetetrakis (methylene phosphonates) such as Dequest. These aminophosphonates preferably do not contain an alkyl or alkenyl group having 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 acid form are dihydroxydisulfobenzenes such as 1,2-dihydroxy-3,5-disulfobenzene.

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

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

00

0 0 0 t 0 0

0 0 0

0 0 0

Or a currently used builder component useful, for example, with insoluble builders such as zeolites, layered silicates and the like.

If used, these chelating agents will generally be present in amounts of from 0.1 to 15% by weight of the detergent and / or fabric care compositions of the present invention. 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 wt%. by weight of the compositions.

Foam Suppressors: Another optional ingredient is a suds suppressor, exemplified by silicones and silica-silicone mixtures. Silicones can generally be represented by alkylated polysiloxane materials, while silica is normally used in finely powdered form, examples of which are silica aerogels and xerogels and hydrophobic silicas of various types. These materials may be included in the compositions as particles in which the suds suppressor is preferably releasably included in a water-soluble or water-dispersible carrier that is substantially impermeable to the non-surfactant detergent. The suds suppressor may also be dissolved or dispersed in a liquid carrier and applied by spraying onto one or more of the other ingredients. A preferred silicone suds controlling agent is described by Bartollot et al., U.S. Pat. No. 3,933,672. Other particularly useful suds suppressors are the self-emulsifying silicone suds suppressors described in German Patent Application DTOS 2,664,126, 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 controlling agent is a suds suppressor system comprising a mixture of silicone oils and 2-alkyl alkanols. Suitable 2-alkyl alkanols are 2-butyloctanol, which is commercially available under the trade name Isofol 12 R.

This suds suppressor system is described in the accompanying European patent application N 92870174.7, filed November 10, 1992.

4 * 4444

44

4 4 4 4

4 4 4

4 4 4

44

4 4 4 4 4

44 44

4 4 4 4 4 5

4 4 4 4 4

44 4 ·

Particularly preferred silicone suds controlling agents are described in the accompanying European Patent Application No. 92201649.8. Said compositions can comprise a silicone / silica mixture in combination with a dust nonporous silica such as Aerosil ^(R).

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

Other Ingredients: Additional ingredients such as soil suspending agents, soil release agents, optical brighteners, abrasives, bactericides, gloss loss inhibitors, coloring agents and / or perfumes sealed may be used in laundry detergent and / or fabric care compositions. not encapsulated.

A particularly suitable capsule encapsulation material is a water-soluble capsule which consists of a matrix of polysaccharide and polyhydroxy compounds as described in British Patent 1,464,616. Other suitable water-soluble capsule encapsulation materials include dextrins derived from non-gelatinized starch esters substituted with These dextrin acid esters are preferably prepared from those starches derived from waxy maize, wax sorghum, sago, tapoic and potato. Suitable examples of encapsulating materials include N-Lok manufactured by National Starch. The N-Lok capsule material consists of modified corn starch and glucose. This starch is modified by the addition of monofunctional substituted groups such as octenylsuccinic anhydride.

Anti-redeposition agents and soil suspending agents useful in the present invention include cellulose derivatives such as methylcellulose, carboxymethylcellulose and hydroxyethylcellulose, and the homo- or co-polymeric polycarboxylic acids or salts thereof. Polymers of this type include polyacrylates and copolymers of maleic anhydride and acrylic acid, all mentioned above as builders, as well as copolymers of acid anhydride. Maleic acid with ethylene, methyl vinyl ether or methacrylic acid, maleic anhydride represents at least 20 mole percent of the copolymer. &lt; tb &gt; &lt; tb &gt; These materials are normally used in amounts of from 0.5 to 10 wt%, more preferably from 0.75 to 8 wt%, most preferably from 1 to 6 wt%.

Preferred optical brighteners are anionic in nature. Examples thereof are 4,4'-bis- (2-diethanolamino-4-anilino-1,3,5-triazin-6-ylamino) stilbene-2: 2'-disulfonate disodium salt, 4,4'-bis disodium salt. - (2-morpholino-4-anilino-1,3,5-triazin-6-ylamino) -stilbene-2: 2'-disulfonate, 4,4'-bis- (2,4-dianilino-1, disodium salt, 3,5-triazin-6-ylamino) stilbene-2: 2'-disulfonate, 4 ', 4-bis- (2,4-dianilino-1,3,5-triazin-6-ylamino) -stilbene monosodium salt 2-sulfonate, 4,4'-bis- (2-anilino-4- (N-methyl-N-2-hydroxyethylamino) -1,3,5-triazin-6-ylamino) stilbene-2: 2 'disodium salt disulfonate, disodium salt of 4,4'-bis- (4-phenyl-6H-triazol-2-yl) stilbene-4-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 2 (stilbyl-4- (naphtho-T, 2 ': 4,5)) -1,2,3-triazole-2''-sulfonate and 4 ', 4-bis- (2-sulfostyryl) biphenyl Highly preferred brighteners are the specific brighteners described in European Patent 753,567.

Other useful polymeric materials are polyethylene glycols, especially those having a molecular weight of 1000 to 10,000, in particular 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% by weight. These polymers and the aforementioned homo- and co-polymeric polycarboxylate salts are valuable for improving whiteness preservation, ash deposition and clay purification, protein and oxidizable soiling in the presence of transit metal impurities.

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 configurations. Examples of these polymers are described in U.S. Patent Nos. 4,116,885 and 4,711,730 and European Published Patent Application No. 0 272 033. A particularly preferred polymer of European Patent Application 0 272 033 is a compound of the formula 9,999,999 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 9 9 9 9 9 · · ·

99 99 9 9 99 9 9 (CH ₃ (PEG) 43), 7 ₅ (POH), ₂₅ F-PO) second ₈ R-PEG) o 4] T (PO-H) o ₂₅ ((PEG) 4 _{3 CH3)} O.7 _5, wherein the PEG is - (OC ₂ H ₄₎ O-, PO is (OC ₃ H ₆ O) and T represents a group (pcOC ₅ H ₄ CO).

Modified polyesters are also very useful, such as random copolymers of dimethyl terephthalate, dimethyl sulfoisophthalate, ethylene glycol and 1,2-propanediol, the end groups consisting primarily of sulfobenzoate and secondly of monoesters of ethylene glycol and / or propanediol. The aim is to obtain a polymer at both ends terminated by sulfobenzoate groups, primarily in the present context most of the copolymers of the invention will be closed at the end by sulfobenzoate groups. However, some copolymers will be less than fully enclosed, and their end groups may therefore consist of an ethylene glycol monoester and / or propane-1,2-diol, which secondarily consists of these moieties.

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

It is well known in the art that free chlorine in tap water rapidly inactivates enzymes contained in detergent compositions. The use of a chlorine scavenger such as perborate, ammonium sulfate, sodium sulfite or polyethyleneimine in an amount above 0.1 wt. improves the stability of the detergent enzymes during washing by the weight of the entire composition. 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 invention to provide further grease removal.

9 999

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9 · · · · ·

9 9 9

Μ 99

9

These materials are described in WO 91/08281 and PCT 90/01815 on page 4 and on the following pages; both publications are incorporated herein by reference. Chemically, these materials contain 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 in which m is a number from 2 to 3 and n is a number from 6 to 12. The side chains are bonded via an ester bond to a polyacrylate backbone to provide a polymer crest type structure. The molecular weight may vary, but typically ranges from 2000 to 50,000. These alkoxylated polycarboxylates may be present in the compositions in an amount of from 0.05 to 10% by weight.

Dispersants: The laundry detergent and / or fabric care compositions of the present invention may also contain dispersants. Suitable water-soluble organic salts are homo- or co-polymeric acids or salts thereof, in which the polycarboxylic acid comprises at least two carboxyl groups separated by no more than two carbon atoms. Polymers of this type are described in British Patent No. 1,596,756. Examples of such salts are polyacrylates having a molecular weight of 2000 to 5000 and copolymers thereof with maleic anhydride, such as copolymers having a molecular weight of from 1000 to 100,000.

In particular, copolymers of acrylates and methacrylates such as 480N having 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 capability (LSDP) as defined herein no more than 8, preferably no more than 7, most preferably no more than 6. A lime soap peptizing compound is present with preferably in an amount of 0 to 20 wt.

V9 ··· «·· ··

«·« • • · • Μ

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

The numerical measure of the efficacy of a lime soap peptizing compound is given by the lime soap dispersing value (LSDP), which is measured using a 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 lime milk 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 by weight of dispersant to sodium oleate required to disperse lime soap deposits formed by 0.025 g sodium oleate in 30 ml of water having a hardness equivalent of 333 ppm CaCO ₃ (Ca: Mg ratio = 3: 2).

Surfactants having good lime soap peptizing compound properties include some amine oxides, betaines, sulfobetaines, alkyl ethoxy sulfates and ethoxylated alcohols.

Examples of surfactants having an LSDP of not more than 8 for use in the present invention include 16 to 18 carbon atoms dimethylamine oxide, alkyl (12 to 18 carbon atoms) ethoxy sulfates with an average degree of ethoxylation of from 1 to 5, especially alkyl ( (12 to 15 carbon atoms) ethoxylate sulfate surfactant with a degree of ethoxylation of 3 (LSDP = 4) and ethoxylated alcohols of 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; Lutensol A030 by BASF GmbH.

Polymeric peptizing lime milk suitable for use in the present invention is described in Article Μ. K. Nagarajan and W. F. Masler: Cosmetics and Toiletries 1989, 104, 71-73.

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.

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

Coloring dye transfer inhibiting agents: The laundry detergent 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 the polymeric transfer inhibiting agents. dyes. These polymeric dye transfer inhibiting agents are normally included in laundry detergent and / or fabric care compositions to inhibit the transfer of dyes from colored fabrics to the laundry agents. These polymers have the ability to form a complex or absorb volatile dyes washed out of the dyed fabrics before the dyes have the opportunity to adhere to other laundry articles.

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: The polyamine-N-oxide polymers preferred for use in the present invention comprise units of the following general formula (I): (4)

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

43 44 4 4 44 44

P

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

A is one of the following structures: -NC (O) -, -C (O) O-, -CO-, -S-, -O-, -N-, x is 0 or 1 and R is aliphatic, ethoxylated an aliphatic, aromatic, herocyclic or alicyclic group, or any combination thereof, to which a nitrogen atom of the N-O group may be attached or part of which may be a nitrogen atom of the NO group.

The group N-O may be a group of the following general formulas

O O

II (R 1) x N- (R ₂ ) _y or = N- (R ₁ ) _X ,

I (R _{3) z} in which R, R ₂ and R ₃ are aliphatic, aromatic, heterocyclic or alicyclic groups or combinations thereof, x or / and y or / and are 0 or 1; and the nitrogen of the N-0 can be attached or may form part of these groups.

The N-O group may be part of a polymerizable unit (P) or may be attached to a polymer backbone or a combination of both.

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

• · · · · · ·

One group of said polyamine N-oxides comprises a 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 wherein R is a heterocyclic group such as pyridine, pyrrole, imidazole, pyrrolidine, piperidine, quinoline, acridine and derivatives thereof.

Another group of said polyamine N-oxides comprises a group of polyamine N-oxides in which the nitrogen atom of the N-O group is attached to the group R

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 is the polyamine N-oxides of formula (I) wherein R is an aromatic, heterocyclic or alicyclic group wherein the nitrogen atom of the N-O functional group is part of said R group.

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

Another preferred group of polyamine N-oxides are those of formula I wherein R is an aromatic, heterocyclic or alicyclic group in which the nitrogen atom of the N-O function is attached to said R groups.

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

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

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

The amine-N-oxide polymers of the present invention typically have an amine to amine-N-oxide ratio of from 10: 1 to 1: 1,000,000. The ratio of amine to amine N-oxide 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. Polymers of the present invention indeed, they include random or block copolymers wherein one type of monomer is an amine N-oxide and the other type of monomer is either an amine N-oxide or does not. The amine oxide unit of the polyamine N-oxides has a PKa < 10, preferably PKa < 7, more preferably PKa < 6.

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

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

b) N-vinylpyrrolidone and N-vinylimidazole copolymers: The polymers of N-vinylpyrrolidone and N-vinylimidazole used in the present invention have an average molecular weight ranging from 5,000 to 1,000,000, more preferably from 5,000 to 200,000.

Highly preferred polymers for use in the detergent compositions of the present invention comprise a polymer selected from N-vinylimidazole / N-vinylpyrrolidone copolymers, said polymer having an average molecular weight ranging 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 Maysem J. W., Chemical Analyzes 113, Modem Methods of Polymer Characterization.

• • •

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

N-Vinylimidazole / N-vinylpyrrolidone copolymers are characterized in that said average molecular weight range provides excellent dye transfer inhibiting properties while not adversely affecting the cleaning performance of the detergent compositions 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 use polyvinylpyrrolidone (PVP) having an average molecular weight of from 2500 to 400,000, preferably from 5,000 to 200,000, more preferably from 5,000 to 50,000 and most preferably Suitable polyvinylpyrrolidones are commercially available from ISP Corporation, New York, NY, and Montreal, Canada, under the product names PVP K-15 (viscosity molecular weight 10,000), PVP K-30 (average molecular weight 40). PVP K-60 (average molecular weight 160,000) and PVP K-90 (average 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) Polinyloxazolidone: The laundry detergent and / or fabric care compositions of the present invention may also use polyvinyloxazolidone as a polymeric dye transfer inhibiting agent. The polyvinyloxazolidones have:

an average molecular weight of from 2500 to 400,000, preferably from 5,000 to 200,000, more preferably from 5,000 to 50,000, and most preferably from 5,000 to 15,000.

e) Polyvinylimidazole: The laundry detergent and / or fabric care compositions of the present invention may also use polyvinylimidazole as a polymeric dye transfer inhibiting agent. Said polyvinylimidazoles have an average molecular weight of from 2500 to 400,000, preferably from 5,000 to 200,000, more preferably from 5,000 to 50,000, and most preferably from 5,000 to 15,000.

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

Washing Method: The compositions of the invention may be used in virtually any washing or cleaning method, including soaking, pretreatment and rinse-stage methods in which a separate rinse aid may be added.

The method described herein comprises contacting the substances with the wash solution in a conventional manner and as exemplified hereinafter. The process according to the invention is preferably carried out during the washing process. The purification process is preferably carried out at 5 to 95 ° C, in particular between 10 and 60 ° C. The pH 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 exemplify the compositions of the present invention, but are not necessarily meant to limit or otherwise define the scope of the invention. In laundry detergent and / or fabric care compositions, the amounts of enzyme are expressed as the weight of pure enzyme by weight of the total composition and, unless otherwise indicated, detergent ingredients are expressed as weight of the total weight of the compositions. The abbreviated identifications of the components in this file have the following meanings:

LAS: linear alkyl (C 11 -C 13) sodium benzenesulfonate

TAS: sodium tallow alkyl sulphate

CxyAS: sodium (C 1 -C 1 alkyl) sodium sulfate

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

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

QAS: R ₂ .N ⁺ (CH ₃ ) 2 (C ₂ H ₄ OH) with R ₂ representing a group of 12 to 14 carbon atoms

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

APA: amido (C8-C10) propyldimethylamine soap: sodium linear alkylcarboxylate derived from a mixture of (80/20) tallow and coconut oil nonionic: mixed ethoxylated / propoxylated fatty alcohol (13 to 15 carbon atoms) with an average degree of ethoxylation of 3, 8 and an average degree of propoxylation of 4.5

Neodol45-13: a linear primary alcohol (C14-C15) ethoxylate sold by Shell Chemical Co.

STS: sodium toluenesulfonate

CFAA: alkyl (C 12 -C 14) N-methyl glucamide

TFAA: alkyl (16 to 18 carbon atoms) N-methyl glucamide

TPKFA: C12-C14 fatty acids 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 sulphate: STPP:

TSPP:

MA / AA:

MA / AA 1:

AA:

PA30:

480N:

polygel / carbopol:

PB1:

PB4:

Sodium metasilicate (SiO _2: Na ₂ O = 1.0) Hydrated Sodium Aluminosilicate of formula Na _{12 (AlO2 SiO2)} and ₂ .27 H ₂ O having a primary particle size in the range of 0.1 to 10 microns (weight expressed on anhydrous basis) crystalline layered silicate of formula 6-Na ₂ Si ₂ O ₅ sodium citrate dihydrate of 86.4% activity with particle size distribution between 425 µm and 850 µm anhydrous citric acid sodium borate anhydrous sodium carbonate with particles of between 200 µm 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 pyrophosphate 1: 4 random maleic acid / acrylic copolymer with an average molecular weight of 70,000 to 80,000 random maleic acid copolymer 6: 4 acrylic 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 4500 to 8000 random 7: 3 acrylic acid / methacrylic acid copolymer, average molecular weight of about 3500 high molecular weight crosslinked polyacrylates anhydrous sodium perborate monohydrate of formula NaBO ₂ .H ₂ O ₂ anhydrous sodium perborate tetrahydrate of formula OR 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 of the formula 2 Na ₂ CO ₃ .3 H ₂ O ₂ sodium dichloroisocyanurate TAED: tetraacetylethylenediamine NOBS: nonanoyloxybenzenesulfonate in the form of the sodium salt NACA-OBS: (6-nonamidocaproyl) oxybenzenesulfonate DTPA: diethylenetriaminepentaacetic acid HEDP: 1,1-hydroxyethane diphosphonic acid DETPMP: diethylenetriaminpenta (methylene) phosphonic acid sold by Monsanto under the trade name Dequest 2060 EDDS: ethylenediamine-N, N'-disuccinic acid, (S, S) -isomer in form its sodium salts MnTACN: Manganese 1,4,7-trimethyl-1,4,7-triazacyclononate

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

of a soluble dextrin polymer PAAC: cobalt pentaamine acetate salt paraffin: paraffin oil sold under the trade name Winog 70 by Wintershall NaBz: sodium benzoate BzP: benzoyl peroxide mannanasa: mannanase from Bacillus agaradherens, NCIMB 40482 proteasa: a proteolytic enzyme sold under the tradename Savinase, Alcalase and Durazym by Novo Nordisk A / S, Maxacal and amylasa: Maxap, sold by Gist-Brocades, and proteases described in WO 91/06637 and / or WO 95/10591 and / or European Patent 251 446, an amylolytic enzyme sold under the trade name Purafact Ox Am ^R , described in WO 94/18314, and in 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

lipase: myl ^(R) and Duramyl ^(R) , all available to Novo Nordisk A / S, and those described in WO 95/26397, a lipolytic enzyme sold under the trade name celulasa: Lipolas and Lipolas Ultra by Novo Nordisk A / S and Lipomax Gist-Brocades 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: an average molecular weight polyvinyl polymer PVNO: 60 000 poly (4-vinylpyridine) -N-oxide having an average molecular weight PVPVI: 50 000 a copolymer of vinyl imidazole and vinyl pyrrolidone with an average brightener 1: molecular weight 20,000 4,4'-bis (2-sulfostyryl) biphenyl disodium salt brightener 2: Disodium salt of 4,4'-bis (4-anilino-6-morpholino-1,3,5-triazin-2-yl) stil- ben-2: 2'-disulfonate silicone antifoam agent: polydimethylsiloxane foaming regulator with siloxane- an oxyalkylene copolymer dispersant with a first to second ratio of from 10: 1 to 100: 1 granulated suds suppressor: 12 wt. Silicone / silica, 18% opacifying agent: wt. % stearyl alcohol, 70 wt. starch in granular form monostyrenlatex water-based blend, sold SRP1: BASF Aktiengesellschaft under the trade name Lytron 621 an anionic group at the ends of the closed polyesters SRP2: diethoxylated poly (1,2-propylene terephthalate) short block QEA: a polymer of bis ((C ₂ H ₅ O) (C ₂ H4O) _{n) (CH3)} -N ⁺ -C6Hi2-N ⁺ - (CH3)

44

4 4 4

4 4 4

4 4 4

4 4 4

44 ·· · 4 ·

4 * 44

4 4 4 4 4

4 4 4 4 4 5

4 4 4

44 44 44

ΡΕΙ:

CCT:

HMWPEO:

PEGx:

PEO:

TEPAE:

bis ((C 2 H ₅ O) - (C ₂ H4O)) n wherein n is 20 to 30 polyethylenimine with an average molecular weight of 1800 and an average degree of ethoxylation of 7 ethyleneoxy residues per nitrogen cumene sulphonate polyethylene with high molecular weight polyethylene glycol with a molecular weight x polyethylene oxide with an average molecular weight of 5000 tetraethylene pentamine ethoxylate

DETAILED DESCRIPTION OF THE INVENTION

Example 1

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

AND 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

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 Spraying agent 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 proteasa 0.03 0.03 0.03 lipase 0.008 0.008 0.008 celulalsa 0.001 0.001 0.001 amylasa 0.01 0.01 0.01 silicone antifoam 5.0 5.0 5.0 sulfate - 3.0 - density (g / liter) various and minor components 850 850 add up to 100% 850

φ φ · · φ φ φ φ φ φ · φ φ φ φ φ φ φ φ φ · · · · φ φ φ φ φ φ φ φ φ φ φ φ φ φ

ΦΦΦ φ φ φ φ φ φ φ φ ·

Example 2

According to the present invention, the following liquid detergent compositions have been prepared (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 proteasa 0.08 0.02 0.01 0.02 lipase - - 0.003 0.003 amylasa 0.004 0.01 0.01 0.01 celulasa - - 0.004 0.003 CFP 2 - - 0.2 0.1

φ · φ φ • · φ φ φ φ 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 brightener 1 0.1 0.2 0.3 - suds suppressor 0.4 - - - opacifying agent 0.8 0.7 - - NaOH until 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 fabric softeners.

II

C45AS LAS 7.6 10.0 C68AS 1.3 - C45E7 4.0 - C25E3 - 5.0 coconut alkyl-dimethyl-hydroxyethylammonium 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 proteasa 0.02 0.01 lipase 0.02 0.01 amylasa 0.03 0.005 celulasa 0.001 - silicate 3.0 5.0 carbonate 10.0 10.0 suds suppressor 1.0 4.0 CMC 0.2 0.1 various and minor components up to 100%

Example 4

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

AND 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 99 99

9 «9999 • 9 99

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 - - CFP 1 0.3 0.3 0.3 0.3 mannanasa 0.001 0.001 0.02 0.001 amylasa - - 0.01 - proteasa - 0.004 - 0.003 lipase - 0,002 - 0,002 celulasa - 0.003 - - PEO - 0.2 - 0.2 perfume 1.0 0.5 0.3 0.2 magnesium sulfate - - 3.0 3.0 a 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 99

9 V

9 9

9 9

9 9 9

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 SRP1 0.3 0.3 0.3 0.3 mannanasa 0.02 0.03 0.01 0.001 amylasa - - 0,002 - proteasa 0.003 - - 0.003 lipase - - - - celulasa 0.0003 0,002 - - PEO 0.3 - - 0.3 perfume 0.4 - - 0.4 magnesium sulfate 3.0 - - - a 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

98 • 9 • 9 9

9

9 9

9 9

9 •

9 «r ···»

9 9

9 9

9 9 9

9 9 9

99

LIST OF SEQUENCES (1) GENERAL INFORMATION:

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

Title of the Invention: A laundry detergent and / or fabric care composition and method for 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 features: length: 1407 nucleotide pairs type: nucleic acid fiber 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

ATGGCGGTCAATGGGAAAAAGACGACATTGACACCATTCGTGAAGTCATTG

AGCTTGCGGAGCAAAATAAAATGGTGGCTGTCGTTGAAGTTCATGATGCCA

CGGGTCGCGATTCGCGCAGTGATTl AAATCGAGCCGTTGATTATTGGATAG

AAATGAAAGATGCGCTTATCGGTAAAGAAGATACGGTTATTATTAACATTGCA

AACGAGTGGTATGGGAGTTGGGATGGCTCAGCTTGGGCCGATGGCTATATT

GATGTCATTCCGAAGCTTCGCGATGCCGGCTTAACACACACCTTAATGGTTG

ATGCAGCAGGATGGGGGCAATATCCGCAATCTATTCATGATTACGGACAAG

ATGTGTTTAATGCAGATCCGTTAAAAATACGATGTTCTCCATCCATATGTAT

GAGTATGCTGGTGGTGATGCTAACACTGTTAGATCAAATATTGATAGAGTCA

TAGATCAAGACCTTGCTCTCGTAATAGGTGAATTCGGTCATAGACATACTGA

TGGTGATGTTGATGAAGATACAATCCTTAGTTATTCTGAAGAAACTGGCACA

GGGTGGCTCGCTTGGTCTTGGAAAGGCAACAGTACCGAATGGGACTATTTA

GACCTTTCAGAAGACTGGGCTGGTCAACATTTAACTGATTGGGGGAATAGAA

TrGTCCACGGGGCCGATGGCTTACAGGAAACCTCCAAACCATCCACCGTAT

TTACAGATGATAACGGTGGTCACCC-TGAACCGCCAACTGCTACTACCTTGTA

TGACTTTGAAGGAAGCACACAAGGGTGGCATGGAAGCAACGTGACCGGTG

GCCCTTGGTCCGTAACAGAATGGGGTGCTTCAGGTAACTACTCTTTAAAAGC

C GATGTAAATTTAACCTCAAATTCTl CACATGAACTGTATAGTGAACAAAGTC • ·· · · · · · · · · · gggaaatcccggtaatggcatgaatgcaagactttacgtgaaaacgggctc TGATTATACATGGCATAGCGGTCCTÍTTACACGTATCAATAGCTCCAACTCA GGAACAACGTTATCTTTTGATTTAAACAACATCGAAAATAGTCATCATGTTAG 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

FSIHMYEYAGGDANTVRSNIDRVIDQDLALVIGEFGHRHTDGDVDEDTILSYSEE

TGTGWLAWSWKGNSTEWDYLDLSEDWAGQHLTDWGNRIVHGADGLQETSKP

STVFTDDNGGHPEPPTATTLYDFEGSTQGWHGSNVTGGPWSVTEWGASGNY

SLKADVNLTSNSSHELYSEQSRNLHGYSQLNATVRHANWGNPGNGMNARLYV

KTGSDYTWHSGPFTRINSSNSGTTLSFDLNNIENSHHVREIGVQFSAADNSSGQ

TALYVDNVTLR

Seq. id. No. 3:

Sequence properties: length: 1407 pairs of nucleotides type: nucleic acid type: nucleic acid single-stranded topology: linear

Molecule type: genomic DNA

Sequence description: sequence id. No. 3:

ATGAAAAAAAAGTTATCACAGATTTATCATTTAATTATTTGCACACTTATAATA

AGTGTGGGAATAATGGGGATTACAACGTCCCCATCAGCAGCAAGTACAGGC

TTTTATGTTGATGGCAATACGTTATATGACGCAAATGGGCAGCCATTTGTCAT

GAGÁGGTATTAACCATGGACATGCTTGGTATAAAGACACCGCTTCAACAGCT

ATTCCTGCCATTGCAGAGCAAGGCGCCAACACGATTCGTATTGTTTTATCAG

ATGGCGGTCAATGGGAAAAAGACGACATTGACACCATTCGTGAAGTCATTG

AGCTTGCGGAGCAAAATAAAATGGTGGCTGTCGTTGAAGTTCATGATGCCA

GGGGTCGCGATTCGCGCAGTGATTl · AAATCGAGCCGTTGATTATTGGATAG

AAATGAAAGATGCGCTTATCGGTAAáGAAGATACGGTTATTATTAACATTGCA

AACGAGTGGTATGGGAGTTGGGATG GCTCAGCTTGGGCCGATGGCTATATT

GATGTCATTCCGAAGCTTCGCGATGCCGGCTTAACACACACCTTAATGGTTG

ÁTGCAGCAGGATGGGGGCAATATCCGCAATCTATTCATGATTACGGACAAG

ÁTGTGTTTAATGCAGATCCGTTAAAAAATACGATGTTCTCCATCCATATGTAT

GAGTATGCTGGTGGTGATGCTAACACTGTTAGATCAAATATTGATAGAGTCA

TAGATCAAGACCTTGCTCTCGTAATAGGTGAATTCGGTCATAGACATACTGA

TGGTGATGTTGATGAAGATACAATCCTTAGTTATTCTGAAGAAACTGGCACA

GGGTGGCTCGCTTGGTCTTGGAAAGGCAACAGTACCGAATGGGACTATTTA

GACCTTTCAGAAGACTGGGCTGGTCAACATTTAACTGATTGGGGGAATAGAA

7TGTCCACGGGGCCGATGGCTTACAGGAAACCTCCAAACCATCCACCGTAT

TTACAGATGATAACGGTGGTCACCCTGAACCGCCAACTGCTACTACCTTGTA

TGACTTTGAAGGAAGCACACAAGGGTGGCATGGAAGCAACGTGACCGGTG

GGCCTTGGTCCGTAACAGAATGGGGTGCTTCAGGTAACTACTCTTTAAAAGC

CGATGTAAATTTAACCTCAAATTCTTGACATGAACTGTATAGTGAACAAAGTC

GTAATCTACACGGATACTCTCAGCTCAACGCAACCGTTCGCCATGCCAATTG

GGGAAATCCCGGTAATGGCATGAATGCAAGACTTTACGTGAAAACGGGCTC

TGATTATACATGGCATAGCGGTCCTTTTACACGTATCAATAGCTCCAACTCA

GGAACAACGTTATCTTTTGATTTAAACAACATCGAAAATATCATCATGTTAGG

G) \ AATAG • 9 · 9 · 9

100 ALIGN!

Seq. id. 4:

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

Molecule type: protein

Sequence description: sequence id. 4:

vawevhdatgrdsrsdlnravdyviemkdalígkedtviinianewygswdgs AWADGYIDVIPKLRDAGLTHTLMVDAAGWGQYPQSIHDYGQDVFNADPLKNTM FSIHMYEYAGGDANTVRSNIDRVIDQDLALVIGEFGHRHTDGDVDEDTILSYSEE TGTGWLAWSWKGNSTEWDYLDLSEDWAGQHLTDWGNRIVHGADGLQETSKP SWFTDDNGGHPEPPTATTLYDFEGSTQGWHGSNVTGGPWSVTEWGASGNY SLKADVNLTSNSSHELYSEQSRNLHGYSQLNATVRHANWGNPGNGMNARLYV KTGSDYTWHSGPFTRINSSNSGTTL ': f-DLNNIENHMLGK

MKKKLSQIYHLIICTLNSVGIMGITTSPSAASTGFYVDGNTLYDANGQPFVMRGIN

HGHAWYKDTASTAIPAIAEQGANTIRIVLSDGGQWEKDDIDTIREVIELAEQNKM

Seq. id. No 5:

Sequence features: length: 1029 nucleotide pairs type: nucleic acid fiber type: single-stranded topology: linear

Molecule type: genomic DNA * ·

101

Sequence description: sequence id. No 5:

5 'AAT TGG CGC ATA CTG TGG CGC CTG TGA ATC CTA ATG CCC AGC AGA CAA CAA AAA CAG TGA TGA ACT

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 CAG CAA ATA TTG AAG ATT CAA TAG ATG TAA GCT GCG ATT TAA TGT CGT ATT GGA AAA ATG GCG GAA TTC CGC AAA TCA GTT ACC TGG CGA ACC CTG CTT TTC AGT CAG GGC ATT TTA AAA CAC CGA ATG ATA AAA ACA TAT ATT CAG AAT GG 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 QGC TGC GGG GAC TCA CAT CAT ATA ATG ATA ATG ATA ATG ATA ATG ATA ATG ATA ATG ATA ATG ATA ATG ATA ATG ATA ATG ATA ATG ATA ATG ATA ATG ATA ATG ATA ATG ATA ATG ATA ATG ATA ATG ATA CTG ATT TTTACC CGG GCG CGT CTT ACGTGGATATTG TCG GAT TAG ATG CGT ATT TTC AAG ATG CCT ACT CGA TCA ATG GAT ACG ATC AGA TAA CAG CGC TTA ATA AAC CAT TTG CTT TTA CAG AAG ATT ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATT ATG ATT ATG ATT ATG ATT ATG ATT ATG ATT ATG ATT ATG ATT ATG ATT ATG ATT ATT ATT ATT ATT ATT ATT ATT ATT ATT ATT ATT ATT ATT ATT ATT ATT ATT ATG GTG ATT CTT TAA CGC ATA 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.