WO2002077242A2 - Family 74 xyloglucanases - Google Patents

Abstract

Xyloglucanases belonging to family 74 of glycosyl hydrolases are derived from the genus Jonesia, especially from the strain Jonesia sp, DSM 14140. It is contemplated that the xyloglucanases exhibit high performance in conventional detergent compositions.

Description

FAMILY 74 XYLOGLUCANASES

The present invention relates to xyloglucanases belonging to family 74 of glycosyl hydrolases, preferably to enzymes exhibiting xyloglucanase activity as their major enzymatic activity in the neutral and alkaline pH ranges; to an enzyme preparation comprising the enzyme of the invention; to a method of producing such enzymes; and to methods for using such enzymes in the textile, detergent and cellulose fiber processing industries .

BACKGROUND OF THE INVENTION

Xyloglucanase activity has not yet been included in the classification of enzymes provided by the Enzyme Nomenclature (1992) . Hitherto, this enzymatic activity has simply been classified as glucanase activity and has often been believed to be identical to cellulolytic activity (EC 3.2.1.4), i.e. activity against β-1, 4-glycosidic linkages in cellulose or cellulose derivative substrates, or at least to be a side activity in enzymes having cellulolytic activity. However, a true xyloglucanase is a true xyloglucan specific enzyme capable of catalyzing the solubilisation of xyloglucan to xyloglucan oligosaccharides but which does not exhibit substantial cellulolytic activity, e.g. activity against the conventionally used cellulose-like substrates CMC (carboxymethylcellulose) , HE cellulose and Avicel (microcrystalline cellulose) . A xyloglucanase cleaves the beta-¹- _f 4-glycosidic linkages in the backbone of xyloglucan.

Xyloglucanase activity is disclosed in Nincken, J.P., Beldman, G. , and Voragen, A.G.J. Substrate-specificity of endoglucanases - what determines xyloglucanase activity. Carbohydrate Research 298 (4) :299-310, 1997, wherein three different endoglucanases Endol, EndoV and EndoVI from Trichoderma viride (similar to T. reesei) are characterized. Endol, EndoV and EndoVI belongs to family 5, 7 and 12 of glycosyl hydrolases, respectively, see Henrissat, B. (1991) , A classification of glycosyl hydrolases based on amino acid sequence similaritites . Biochem. J. , 280:309-316, and Henrissat, B. and A. Bairoch (1993), New families in the classification of glycosyl hydrolases base^'d on amino acid sequence similaritites . Biochem. J. , 293:781-788.

International Patent Publication WO 94/14953 discloses a family 12 xyloglucanase (EG II) cloned from the fungus

Aspergillus aculeatus and expressed in the fungus Aspergillus oryzae .

International Patent Publication WO 99/02663 discloses xyloglucanases cloned from Bacillus licheniformis (family 12) and Bacillus agaradhaerens (family 5) and expressed in Bacillus subtilis .

International Patent Application PCT/DKOl/00132 discloses xyloglucanases cloned from Paenibacillus pabuli (family 5) and expressed in Bacillus subtilis . International Patent Application PCT/DKOl/00116 discloses xyloglucanases cloned from Paenibacillus polymyxa (family 44) and expressed in Bacillus subtilis .

It is an object of the present invention to provide an enzyme with a high xyloglucanase activity at an alkaline pH and exhibiting excellent performance in conventional detergent compositions .

SUMMARY OF THE INVENTION

The inventors have now found enzymes having substantial xyloglucanase activity, which enzymes belong to family 74 of glycosyl hydrolases . It is contemplated that these xyloglucanase enzymes perform excellent in conventional detergent compositions, especially in liquid detergent compositions.

The inventors have succeeded in cloning and expressing a family 74 xyloglucanase derived from the marine organism Jonesia.

Accordingly, in a first aspect the invention relates to a polypeptide exhibiting xyloglucanase activity as its major enzymatic antivity, which enzyme belongs to family 74 of glycosyl hydrolases and is endogenous to a bacterium, particularly an alkalophilic bacterium, and more particularly a bacterial strain belonging to the genus Jonesia .

In a further aspect, the invention relates- to a family 74 xyloglucanase selected from one of (a) a polypeptide encoded by the DNA sequence of positions 72-2820 of SEQ ID NO: 1; (b) a polypeptide produced by culturing a cell comprising the sequence of positions 72-2820 of SEQ ID NO : 1 under conditions wherein the DNA sequence is expressed; (c) a xyloglucanase enzyme having a sequence of at least 60% identity to positions 24-940 of SEQ ID NO: 2 when identity is determined by the Clustal method with a PAM250 residue table and the default settings of the Megalign program in the Lasergene package using a GAP creation penalty of 3 and Ktuple of 1; and (d) a polypeptide encoded by a DNA sequence that hybridizes to the DNA sequence of SEQ ID NO : 1 under medium stringency conditions, wherein the medium stringency conditions comprise hybridization in 5xSSC at 45°C and washing in 2xSSC at 60°C; and to an isolated polynucleotide molecule encoding a polypeptide having xyloglucanase activity which polynucleotide molecule hybridizes to a denatured double- stranded DNA probe under medium stringency conditions, wherein ^■ the probe is selected from the group consisting of DNA probes comprising the sequence shown in positions 72-2820 of SEQ ID N0:1, and DNA probes comprising a subsequence of positions 72- 2820 of SEQ ID NO-.l, the subsequence having a length of at least about 100 base pairs.

In further aspects, the invention provides an expression vector comprising a DNA segment which is e.g. a polynucleotide molecule of the invention; a cell comprising the DNA segment or the expression vector; and a method of producing a exhibiting xyloglucanase enzyme, which method comprises culturing the cell under conditions permitting the production of the enzyme, and recovering the enzyme from the culture . In yet another aspect the invention provides an isolated xyloglucanase enzyme being free from homologous impurities .

The novel enzyme of the present invention is useful for the treatment of cellulosic material, especially cellulose- containing fiber, yarn, woven or non-woven fabric. The treatment can be carried out during the processing of cellulosic material into a material ready for garment manufacture or fabric manufacture, e.g. in the desizing or scouring step; or during industrial or household laundering of such fabric or garment . Accordingly, in further aspects the present invention relates to an enzyme preparation comprising a xyloglucanase enzyme of the invention and conventional fillers or stabilizers; a detergent composition comprising a xyloglucanase enzyme having substantial xyloglucanase activity in the neutral or alkaline range; and to use of the enzyme of the invention for the treatment of cellulose-containing fibers, yarn, woven or non- woven fabric .

The present invention has now made it possible to use a xyloglucanase in detergent compositions, preferably liquid laundry detergent compositions, for removing or bleaching certain soils or stains present on laundry, especially soils and spots resulting from xyloglucan-containing food, plants, and the like. Further, it is contemplated that treatment with detergent compositions comprising the novel enzyme can prevent binding of . certain soils to the xyloglucan left on the cellulosic material.

DETAILED DESCRIPTION OF THE INVENTION

MICROBIAL SOURCES

The xyloglucanase of the invention may be obtained from a bacterium belonging to a strain of the genus Jonesia, in particular a strain of Jonesia sp.

In a preferred embodiment, the xyloglucanase of this invention is obtained from the strain Jonesia sp . , DSM 14140. It is at present contemplated that a DNA sequence encoding an enzyme having similar functional properties as the xyloglucanase from with Jonesia sp . , DSM 14140 and having an amino acid sequence identity of at least 60% to this xyloglucanase may be obtained from other strains belonging to the genus Jonesia . The strain Jonesia sp . was deposited by the inventors according to the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg lb, D-38124 Braunschweig, Federal Republic of Germany, on 23 February 2001 under the deposition number DSM 14140. DEFINITIONS

For the purpose of the present invention the term "obtained from", "derived from", "obtainable from" or "derivable from", as used herein in connection with a specific source, means that the enzyme is produced or can be produced by the specific source, or by a cell in which a gene from the source have been inserted.

In the present context, the term "enzyme preparation" is intended to mean either be a conventional enzymatic fermentation product, possibly isolated and purified, from a single species of a microorganism, such preparation usually comprising a number of different enzymatic activities; or a mixture of monocomponent enzymes, preferably enzymes derived from bacterial or fungal species by using conventional recombinant techniques, which enzymes have been fermented and possibly isolated and purified separately and which may originate from different species, preferably fungal or bacterial species; or the fermentation product of a microorganism which acts as a host cell for expression of a recombinant xyloglucanase, but which microorganism simultaneously produces other enzymes, e.g. other xyloglucanases, proteases, or cellulases, being naturally occurring fermentation products of the microorganism, i.e. the enzyme complex conventionally produced by the corresponding naturally occurring microorganism. In the present context the term "expression vector" denotes a DNA molecule, linear or circular, that comprises a segment encoding a polypeptide of interest operably linked to additional segments that provide for its transcription. Such additional segments may include promoter and terminator sequences, and may optionally include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, and the like. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both. The expression vector of the invention may be any expression vector that is conveniently subjected to recombinant DNA procedures, and 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

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FAMILY 74 The xyloglucanase according to the invention has been found to show a 49% homology with a glycoside hydrolase from Streptomyces (GenBank/GenPept/SwissProt/AL031515/CAA20642.1/086727) belonging to the glycoside hydrolase family 74 presently containing 6 members (http://afmb.cnrs-mrs.fr/~cazy/CAZY/index.html) . For further reference regarding classification of glycosyl hydrolases see also Coutinho, P.M. and Henrissat, B. (1999) In "Recent Advances in Carbohydrate Bioengineering" , H.J. Gilbert, G. Davies, B. Henrissat and B. Svensson eds., The Royal Society of Chemistry, Cambridge, pp. 3-12; and Coutinho, P.M. and Henrissat, B. (1999) In "Genetics, Biochemestry and Ecology of Cellulose Degradation", K. Ohmiya, K. Hayashi, K. Sakka, Y. Kobayashi, S. Karita and T. Kimura eds., Uni Publishers Co., Tokyo, pp. 15-23; Henrissat B., Bairoch A., Biochem. J. 316:695- 696(1996) "Updating the sequence-based classification of glycosyl hydrolases"; Davies G., Henrissat B., Structure ^■ 3 : 853- 859(1995) "Structures and mechanisms of glycosyl hydrolases".

POLYNUCLEOTIDES

Within preferred embodiments of the invention an isolated polynucleotide of the invention will hybridize to similar sized regions of SEQ ID NO: 1 or a sequence complementary thereto, under at least medium stringency conditions.

In particular polynucleotides of the invention will hybridize to a denatured double-stranded DNA probe comprising either the full sequence shown in SEQ ID NO: 1 or the sequence shown in positions 72-2820 of SEQ ID NO : 1 or any probe comprising a subsequence of SEQ ID NO:l having a length of at least about 100 base pairs under at least medium stringency conditions, but preferably at high stringency conditions as described in detail below. Suitable experimental conditions for determining hybridization at medium or high stringency between a nucleotide probe and a homologous DNA or RNA sequence involve pre-soaking of the filter containing the DNA fragments or RNA to w w t t μ μ

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0 ii μQ rt 3 rt Ω ti H ti rt 0 φ Cb Φ ØJ 0 0 ø Φ Φ 00 tr rt 0 0 tr J μ- ø 0 -~ 0 ø s μ- 3 Φ 03 Φ H O o O rt 0 Ω Cb ii U_ 0 ø ii

Φ 3 Φ 0 < CQ rt CQ 0 CQ μ- Hi Φ < 13 Φ CO 0 φ ϋ S ^< rt tr ø- 0 ra 3 ^• — ' rt H μ- μ- O ø Ω CQ μ- TJ φ Cb ø φ tr X μ- 0 ø ø 03 0 . — . . — , Φ μ> Φ 3 0 μ- • Cb ø 03 Ω 0 μ- rt Φ Cb 0 μ- 0 03 TJ φ ø rt ø 0 Cb rt rt μ-¹ tr tr 3 OΛ ø 3 1 3

TJ α 0 μ-

0 Φ ø rt ^ Ω Ω ø Ω rt 3 ^• ! ^■<: 03 Hi tr φ μ- μ- φ o X TJ φ a o ø H N

3 <! μ- μ- rt 0 CQ 0 . — . μ- TJ TJ ø tϋ μ- ti Φ Ω C ιQ . o S H • O φ

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03 rt φ Ω Cb < CQ ø μ- 0 O < Cb 03 03 Ω μ- 0 μ- tr ø 0 ω Ω 10 - ø Ω 0 n o CQ

0 0 CQ φ Φ ø μ- Cb Hi φ 0 H φ μ- φ ø ø 0 0 μ- Ω o rt O φ —* tr 0 b Ω

0 μ- φ 0 Cb ø Φ ø Φ TJ ii ; ø N ø φ f ø Ω rt ø Ω ^■-S ^ H rt ω Φ 0 Φ 3 ii ø ø 0 rt CQ D rt ø rt 0 rt ø 0 ^ Φ 03 tr — μ- H Hi TJ t ii rt rt μ> in μ- , — .

Ω Φ rt H- tr μ- o¹ μ- TJ 0 μ- rt μ- Ω 0 tr 0 0 Cb • -. ø μ- 0 H ^►ϋ CQ ø o ø CQ φ Cb 0 ^■<; 0 0 & Hi 0 Cb ø Ω O μ- 0 0 $ Φ 0 Cb - 0 rt vQ ø ti O 1 - μ- 0 o X 0

CQ μ- CQ ø Hi 0 μ, CD Φ ii ø 0 0 0 $ φ ti tr ø Φ Q tr Cb ø Cb

Hi ø rt CD 0 0 03 ø Φ ø μ- Ω 0 rt Φ Φ 0 0 Φ ω Φ Ω > μ- • ^"0 α ø μ-

0 ii <! rt t Ω 0 O 3 μ. 0 Hi 0 ø μ- I-¹ α tr α Ω 0 o Hi ø ^ø φ ø ø

Hi 0 Φ Φ φ 0 . 0 Hi 0 rt 0 O ti CQ ø 0 μ- a Φ Cb 0 ^*≤. Ω Hi 1 CQ μ> 0 Cb 3

3 ø Ω 0 TJ 0 Co H CQ μ- ti ti Φ 0 ø > φ μ-¹ Φ "< 3 o 1 ) u. 3 tr α rt tr TJ 0 rt 0 03 Cb rt TJ rt Φ μ- rt μ- <J _^ — ' μ- H . 0 co μ^j 0 TJ Ω a ø μ- 0 ti tr 03 H O 3 *-" φ - V rt O tr ς^ rt 0 03 Φ CQ Φ ø 0 V£> ii ii tr

0 μ- φ Φ S TJ ø Hi . 03 Φ O tr μ-¹ Φ tr ø 0 Ω 0 0 Φ 0 μ* tr ø Ω '--' o Cb Φ

Ω ø μQ CQ μ- ø ii fo Φ μ- φ Cb rt ø <! ω rt K. Φ ø 0 - Hi rt tr 0

Ω Φ ø Φ Ω Φ *. ø 03 M O Hi ø ^ ø ø ø Φ ø 3 Φ rt Φ H Cb 0 - a ti

0 M Ω φ ø H> TJ co TJ w Hi ii TJ Φ ti 0 ja rt Cb Ω 0 0 μ- 03 tr Φ Ω Hi 03 tr μ-

0 0 03 rt 0 rt α Φ 0 O ii μ- TJ rt Ω ti s Φ H H o Cb < Φ 0 Φ H Cb

0 • 0 μ- Q s; Ω H TJ 3 0 Cb rt • φ rt Cb ø Φ Φ 3 μ- 0 0 0 03 μ- φ tr rt μ- φ Cb s tr μ- 0 0 < φ μ- ø • 03 0 o 0 H _^^ CQ rt PJ 0 \_>

Φ tr 1 ø Cb Φ μ- ø TJ H Cb μ- ø Cb φ TJ μ- rt TJ h 3 O 03 Φ ti 0 rt M μ- CQ

T) Φ 0 < H Ω H tr rt μ- Cb rt Φ α 0 ø μ- ϋ Φ o ISO J μ^j ø ≤ 0 0 N 0 μ- Φ ii φ 0 s; *> tr μ- μ- Hi φ μ- 03 ω a s H CQ Cb Φ ii ØJ φ 03 rt Φ ϋ rt ø Cb

Cb Ω ø 03 μ- H μ- Ω Hi 03 Hi > φ Φ Hi TJ φ X rt Ω rt μ- Cb Φ μ- rt μ-

Φ rt Φ rt μ- rt 0J ø μ- ø Φ μ- tr ø rt ø ø Φ ii μ- φ 0 Cb 0 μ- 0 ø tr 0 X μ- ø tr O ti rt Ω μ-¹ ii Ω φ 0 ?r ø t 0 0 J H φ TJ CQ Ω Hi μ- ø tr 0 0 3 rt 0 ø ø 0 CQ . Φ Φ ø φ 0 Cb < O ø 0 Ω ti 0J Hi ti CQ 0J μ- ø ^<< ω ø μ- rt Φ 3 ø >_ H tr ii 0 0 μ- Cb Cb X 0 tr Φ Φ Ω • Ω » O O . — . Ω

Hi Cb TJ μ- * <! Φ 0 rt 0 0 ø 0 03 Φ 1 tr μ- H Hi - Hi ø CQ 0 μ- μ- Φ H μ- ø φ 03 Ω μ- rt ø CQ ti td 0 ti Φ •< øJ φ ø t ^ μ- 0 Hi rt φ X ø Φ ø Hi 0 H rt rt ø tr φ Cb a Hi rt ø tr i o tπ Ω • μ» tr Ω 3 ti

Cb TJ 03 - o Q "< φ rt 0 H X α 0 μ- ^ tr ØJ H' ø • o rt o H 0 tr rt ø ti μ- Ω H ø ti Φ Ω TJ μ- *<;

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^ Q CQ 3 0 Ω H Φ ø Φ Φ ii 0 0 μ- 03 μ- ti H ØJ ^ o\° μ- KΩ μ- a. 0 -

03 α tr rt 0 0 μ-¹ 03 ii rt CQ Ω 03 μ- φ rt μ^ rt CO 3 N X Hi

TJ Φ !-. μ- μ- ø 03 rt μ- 0 M μ- 0 0 0 3 Cb ØJ ØJ CQ tr ^■ ! ω ø ω μ- CQ ii 03 ø 0 ø Φ 03 ø rt ø tr Cb Hi μ- ω μ-¹ rt Φ *— ' rt ØJ Φ 0

0 ø ø 03 M rt ø μ-< Φ Ω ti μ- 0 N rt Φ CQ tr 0 μ- rt rt 3 tr rt rt Φ ^ ti H Cb 0 ø ø rt rt φ 0J μ^j Hi μ- > Hi 0 3 φ tr μ- tr μ- ø ø Φ 0 H. CQ μ- tr 03 ^1 03 φ 0 μ- CQ 0 ø o 0 ti ti ø Φ 0 ø μ- tr 0 μ- ø Φ in rt ØJ rt μ^j tr ø 0 ø H 0

CQ ø Cb ø CQ φ cQ o ω rt φ H μ- μ- 0 D ^ Φ 03 O rt Φ ii ø ø ti

Northern blots with probes designed from the sequences disclosed herein. A library is then prepared from chromosomal DNA of a positive cell line. A DNA sequence of the invention encoding an polypeptide having xyloglucanase activity can then be isolated by a variety of methods, such as by probing with probes designed from the sequences disclosed in the present specification and claims or with one or more sets of degenerate probes based on the disclosed sequences. A DNA sequence of the invention can also be cloned using the polymerase chain reaction, or PCR (Mullis, U.S. Patent 4,683,202), using primers designed from the sequences disclosed herein. Within an additional method, the DNA library can be used to transform or transfect host cells, and expression of the DNA of interest can be detected with an antibody (monoclonal or polyclonal) raised against the xyloglucanase cloned from Jonesia sp. , e.g. from Jonesia sp . , DSM 14140, expressed and purified as described in Materials and Methods and the Examples, or by an activity test relating to a polypeptide having xyloglucanase activity.

POLYPEPTIDES

The sequence of amino acids nos. 24-940 of SEQ ID NO: 2 is a mature xyloglucanase sequence including the catalytic active domain. The present invention also provides xyloglucanase polypeptides that are substantially homologous to the polypeptide of amino acids nos. 24-940 of SEQ ID NO: 2 and species homologs (paralogs or orthologs) thereof. The term "substantially homologous" is used herein to denote polypeptides having 60% , preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 97%, and even more preferably at least 99%, sequence identity to the sequence shown in amino acids nos. 24- 940 of SEQ ID NO: 2 or their orthologs or paralogs. Such polypeptides will most preferably be 98% or more identical to the sequence shown in amino acids nos. 24-940 of SEQ ID NO : 2 or its orthologs or paralogs. Percent sequence identity is determined by conventional methods, by the Clustal method (Thompson, J.D., Higgins, D.G., and Gibson, T.J., (1994) , Nucleic Acids Research 22, 4673-4680) with the default settings of the Megalign program in the Lasergene package (DNAstart Inc., 1228 South Park Street, Madison, Wisconsin 53715) . The settings for multiple alignment are: GAP penalty of 10, and GAP length penalty 10; while the pair-wise alignment parameters are GAP penalty of 3 and Ktuple of 1.

Sequence identity of polynucleotide molecules is determined by the Clustal method (Thompson, J.D., Higgins, D.G., and Gibson, T.J., (1994) ,Nucleic Acids Research 22, 4673-4680) with the default settings of the Megalign program in the Lasergene package (DNAstart Inc., 1228 South Park Street, Madison, Wisconsin 53715) . The settings for multiple alignment are: GAP penalty of 10, and GAP length penalty 10; while the pair-wise alignment parameters are GAP penalty of 5 and Ktuple of 2. Substantially homologous proteins and polypeptides are characterized as having one or more amino acid substitutions, deletions or additions. These changes are preferably of a minor nature, that is conservative amino acid substitutions (see Table 1) and other substitutions that do not significantly affect the folding or activity of the protein or polypeptide; small deletions, typically of one to about 30 amino acids; and small amino- or carboxyl-terminal extensions, such as an amino- terminal methionine residue, a small linker peptide of up to about 20-25 residues, or a small extension that facilitates purification (an affinity tag), such as a poly-histidine tract, protein A (Nilsson et al . , EMBO J. 4:1075, 1985; Nilsson et al . , Methods Enzymol . 198 :3, 1991. See, in general Ford et al . , Protein Expression and Purification 2 : 95-107, 1991, which is incorporated herein by reference. DNAs encoding affinity tags are available from commercial suppliers (e.g., Pharmacia Biotech, Piscataway, NJ; New England Biolabs, Beverly, MA) .

However, even though the changes described above preferably are of a minor nature, such changes may also be of a larger nature such as fusion of larger polypeptides of up to 300 amino acids or more both as amino- or carboxyl-terminal extensions to a polypeptide of the invention having xyloglucanase activity.

Table 1 Conservative amino acid substitutions

Basic : arginine lysine histidine

Acidic glutamic acid aspartic acid

Polar: glutamine asparagine

Hydrophobic : leucine isoleucine valine

Aromatic phenylalanine tryptophan tyrosine

Small : glycine alanine serine threonine methionine

In addition to the 20 standard amino acids, non-standard amino acids (such as 4-hydroxyproline, 6-JV-methyl lysine, 2- aminoisobutyric acid, isovaline and a-methyl serine) may be substituted for amino acid residues of a polypeptide according to the invention. A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, and unnatural amino acids may be substituted for amino acid residues. "Unnatural amino acids" have been modified after protein synthesis, and/or have a chemical structure in their side chain(s) different from that of the standard amino acids. Unnatural amino acids can be chemically synthesized, or preferably, are commercially available, and include pipecolic acid, thiazolidine carboxylic acid, dehydroproline, 3- and 4- methylproline, and 3 , 3-dimethylproline . Essential amino acids in the xyloglucanase polypeptides of the present invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine- scanning mutagenesis (Cunningham and Wells, Science 244 : 1081- 1085, 1989). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for biological activity (i.e xyloglucanase activity) to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al . , J. Biol. Chem. 271:4699-4708, 1996. The active site of the enzyme or other biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction or photo affinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Nos et al . , Science 255 :306-312 , 1992; Smith et al . , J. Mol. Biol. 224:899-904, 1992; Wlodaver et al . , FEBS Lett . 309 : 59-64, 1992. The identities of essential amino acids can also be inferred from analysis of homologies with polypeptides, which are related to a polypeptide according to the invention.

Multiple amino acid substitutions can be made and tested using known methods of mutagenesis, recombination and/or shuffling followed by a relevant screening procedure, such as those disclosed by Reidhaar-Olson and Sauer (Science 243.: 53 -57, 1988) , Bowie and Sauer (Proc. Νatl . Acad. Sci. USA 86:2152-2156, 1989), W095/17413, or WO 95/22625. Briefly, these authors disclose methods for simultaneously randomizing two or more positions in a polypeptide, or recombination/shuffling of different mutations (W095/17413, W095/22625) , followed by selecting for functional a polypeptide, and then sequencing the mutagenized polypeptides to determine the spectrum of allowable substitutions at each position. Other methods that can be used include phage display (e.g., Lowman et al . , Biochem. 3_0: 10832- 10837, 1991; Ladner et al . , U.S. Patent No. 5,223,409; Huse, WIPO Publication WO 92/06204) and region-directed mutagenesis (Derbyshire et al . , Gene 46:145, 1986; Ner et al . , DNA 7:127, 1988) .

Mutagenesis/shuffling methods as disclosed above can be combined with high-throughput , automated screening methods to detect activity of cloned, mutagenized polypeptides in host cells. Mutagenized DNA molecules that encode active polypeptides can be recovered from the host cells and rapidly sequenced using modern equipment. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide of interest, and can be applied to polypeptides of unknown structure . Using the methods discussed above, one of ordinary skill in the art can identify and/or prepare a variety of polypeptides that are substantially homologous to residues 24 to 940 of SEQ ID NO: 2 and retain the xyloglucanase activity of the wild-type protein. The xyloglucanase enzyme of the invention may, in addition to the enzyme core comprising the catalytically domain, also comprise a cellulose binding domain (CBD) , the cellulose binding domain and enzyme core (the catalytically active domain) of the enzyme being operably linked. The cellulose-binding domain (CBD) may exist as an integral part the encoded enzyme, or a CBD from another origin may be introduced into the xyloglucanase thus creating an enzyme hybrid. In this context, the term "cellulose- binding domain" is intended to be understood as defined by Peter Tomme et al. "Cellulose-Binding Domains: Classification and Properties" in "Enzymatic Degradation of Insoluble

Carbohydrates", John N. Saddler and Michael H. Penner (Eds.), ACS Symposium Series, No. 618, 1996. This definition classifies more than 120 cellulose-binding domains into 10 families (I-X) , and demonstrates that CBDs are found in various enzymes such as cellulases, xylanases, mannanases, arabinofuranosidases, acetyl esterases and chitinases . CBDs have also been found in algae, e.g. the red alga Porphyra purpurea as a non-hydrolytic polysaccharide-binding protein, see Tomme et al . , op . ci t . However, most of the CBDs are from cellulases and xylanases, CBDs are found at the N and C termini of proteins or are internal. Enzyme hybrids are known in the art, see e.g. WO 90/00609 and WO 95/16782, and may be prepared by transforming into a host cell a DNA construct comprising at least a fragment of DNA encoding the cellulose-binding domain Iigated, with or without a linker, to a DNA sequence encoding the xyloglucanase and growing the host cell to express the fused gene. Enzyme hybrids may be described by the following formula:

CBD - MR - X ω ω w _o μ μ

Ui o Ul o un o Ul

replicating vector, i.e. a vector that exists as an extra chromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell genome in part or in its entirety and replicated together with the chromosome (s) into which it has been integrated.

The vector is preferably an expression vector in which the DNA sequence encoding the enzyme of the invention is operably linked to additional segments required for transcription of the DNA. In general, the expression vector is derived from plasmid or viral DNA, or may contain elements of both. The term, "operably linked" indicates that the segments are arranged so that they function in concert for their intended purposes, e.g. transcription initiates in a promoter and proceeds through the DNA sequence coding for the enzyme .

The promoter may be any DNA sequence, which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell .

Examples of suitable promoters for use in bacterial host cells include the promoter of the Bacillus stearothermophilus maltogenic amylase gene, the Bacillus licheniformis alpha- amylase gene, the Bacillus amyloliquefaciens alpha-amylase gene, the Bacillus subtilis alkaline protease gene, or the Bacillus pumilus xylosidase gene, or the phage Lambda P_R or P_L promoters or the E. coli lac, trp or tac promoters.

The DNA sequence encoding the enzyme of the invention may also, if necessary, be operably connected to a suitable terminator.

The recombinant vector of the invention may further comprise a DNA sequence enabling the vector to replicate in the host cell in question.

The vector may also comprise a selectable marker, e.g. a gene the product of which complements a defect in the host cell, or a gene encoding resistance to e.g. antibiotics like kanamycin, chloramphenicol, erythromycin, tetracycline, spectinomycine, or the like, or resistance to heavy metals or herbicides .

To direct an enzyme of the present invention into the secretory pathway of the host cells, a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) may be provided in the recombinant vector. The secretory signal sequence is joined to the DNA sequence encoding the enzyme in the correct reading frame. Secretory signal sequences are commonly positioned 5 ' to the DNA sequence encoding the enzyme . The secretory signal sequence may be that normally associated with the enzyme or may be from a gene encoding another secreted protein.

The procedures used to ligate the DNA sequences coding for the present enzyme, the promoter and optionally the terminator and/or secretory signal sequence, respectively, or to assemble ^' these sequences by suitable PCR amplification schemes, and to insert them into suitable vectors containing the information necessary for replication or integration, are well known to ^■ persons skilled in the art (cf., for instance, Sambrook et al . , op.cit . ) .

HOST CELLS

The cloned DNA molecule introduced into the host cell may be either homologous or heterologous to the host in question. If homologous to the host cell, i.e. produced by the host cell in nature, it will typically be operably connected to another promoter sequence or, if applicable, another secretory signal sequence and/or terminator sequence than in its natural environment. The term "homologous" is intended to include a DNA: sequence encoding an enzyme native to the host organism in question. The term "heterologous" is intended to include a DNA sequence not expressed by the host cell in nature. Thus, the DNA sequence may be from another organism, or it may be a synthetic sequence .

The host cell into which the cloned DNA molecule or the recombinant vector of the invention is introduced may be any cell, which is capable of producing the desired enzyme and includes bacteria, yeast, fungi and higher eukaryotic cells.

Examples of bacterial host cells which on cultivation are w w _J _o μ μ o in o o in i-a TJ 0 0 03 Ω φ 3 0 Φ μ Cb rt μ CQ 03 Cb μ- 0 Co 0 TJ tr CQ CQ 0¹ to Ω 0 to μ- Ω μ μ .μ 3ξ rt 0 X ø CO 0 φ μ- tr φ μ Φ μ- ø 0 ø 03 μ 0J rt π- 0 0 0 Φ

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H μ- tr 0 0. « . ø Hi 3 to Φ rt Ω tr ø Ω Φ H φ Φ > 03 Hi 0 μ ø ø μ- 0 b μ μ

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3 rj ø ø μ- μ. μ- rt H μ ø Φ 0 μ ø μ- 0 H μ 3 0 0 ø rt ø 0 μ- Ω μ-

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^ 3 0 0 3 tr Q » Ω Φ 3 TJ r CQ 03 ø rt 03 •> rt 0 Cb a tr 0 0 CQ

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0 *<: CQ ø μ-¹ N TJ ^ Hi 0 Φ -. 03 μ- N 0 H¹ rt Hi CQ rt 0 μ. μ a μ. H ø ø c 0 . tr tr φ

CQ 0 3 μ 3 rt Ω ø 03 ^a Φ μ rt ø TJ μ- ø ^ ø Φ tr tr rt μ W 0 ø μ μ. ø

Hi 0 μ. Φ rt 03 ct 3 μ ω 0 ø rt 0 ø φ g ø Ω Φ tq 0 μ _> a - ø o 0

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* tr 03 μ. TJ μ- φ ø ø Ω 0 Φ Φ Hi CD φ Ω 0 rt μ- fcr 0 μ. 0 Ω ? ø Ω

0 rt ø μ- Ω Ω *< CQ 3 ø μ ø rt tr tr rt μ- 0 μ. 3 0 0 Φ 0 W μ. ^ Ω μ-

0 a. μ ø μ ø tr μ rt CQ μ- ct Φ 0 0 0 Ω Φ TJ 0 0 ø - ø ø tr tr 0

Φ 0 0 rt < Φ tr μ TJ TJ o J • μ μ Ω Cb ^ μ φ 0 3 ^ CQ Ω μ. CQ tJ μ μ- μ- Φ 0 rt Φ 0 0 μ μ-¹ Φ rt μ- rt μ- μ 0 Q μ. ø μ. Q μ- ø μ- ø μ S ø 0 Ω ø ø μ- TJ 03 Ω ø H 3 φ Φ 0 ø ^ 0 tr . O rt μ to ø CQ rt

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CD CD H. Ct Φ Ω Ω Φ Cb rt φ Cb 0 3 tr Ω 0 Φ CQ 0 μ- CQ rt ø rt 0 rt ø tø μ &- μ- μ- μQ rt Φ μ- 03 rt 03 μ - Hi CQ Hi μ ø rt tr ø 0 Ω μ CS] μ- φ μ- k> ,0 03 <! ø Φ CO tr μ Ω TJ tr 0 φ 0 . 0 Hi 0 ø μ Φ - to μ. ø ø Ω 0 Φ 0 Ω 3 rt 03 rt Φ 3 ø *__ μ. TJ 0J Φ Cb _^ øJ Φ 0J 0 ø μ μ- μ- 3

<! tr 3 ti CD φ rt ø Ω Ω 03 Ω ct Cb μ 0J ^> Φ μ- Ω Cb Hi μ- a to α ø φ

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plant part such as seeds or leaves . Regulatory sequences are e.g. described by Tague et al, Plant, Phys., 86 , 506 , 1988 .

For constitutive expression the 35S-CaMV promoter may be used (Franck et al . , 1980. Cell 21: 285-294). Organ-specific promoters may e.g. be a promoter from storage sink tissues such as seeds, potato tubers, and fruits (Edwards & Coruzzi, 1990. Annu. Rev. Genet. 24: 275-303), or from metabolic sink tissues such as meristems (Ito et al . , 1994. Plant Mol. Biol. 24: 863- 878) , a seed specific promoter such as the glutelin, prolamin, globulin or albumin promoter from rice (Wu et al . , Plant and

Cell Physiology Vol. 39, No. 8 pp. 885-889 (1998)), a Vicia faba promoter from the legumin B4 and the unknown seed protein gene from Vicia faba described by Conrad U. et al, Journal of Plant Physiology Vol. 152, No. 6 pp. 708-711 (1998), a promoter from a seed oil body protein (Chen et al . , Plant and cell physiology vol. 39, No. 9 pp. 935-941 (1998), the storage protein napA promoter from Brassica napus, or any other seed specific promoter known in the art, e.g. as described in WO 91/14772. Furthermore, the promoter may be a leaf specific promoter such as the rbcs promoter from rice or tomato (Kyozuka et al . , Plant Physiology Vol. 102, No. 3 pp. 991-1000 (1993), the chlorella virus adenine methyltransferase gene promoter (Mitra, A. and Higgins, DW, Plant Molecular Biology Vol. 26, No. 1 pp. 85-93 (1994) , or the aldP gene promoter from rice (Kagaya et al . , Molecular and General Genetics Vol. 248, No. 6 pp. 668-674

(1995) , or a wound inducible promoter such as the potato pin2 promoter (Xu et al, Plant Molecular Biology Vol. 22, No. 4 pp. 573-588 (1993) .

A promoter enhancer element may be used to achieve higher expression of the enzyme in the plant. For instance, the promoter enhancer element may be an intron placed between the promoter and the nucleotide sequence encoding the enzyme. For instance, Xu et al . op ci t disclose the use of the first intron of the rice actin 1 gene to enhance expression. The selectable marker gene and any other parts of the expression construct may be chosen from those available in the art .

The DNA construct is incorporated into the plant genome according to conventional techniques known in the art, including Agrobacterium- ediated transformation, virus-mediated transformation, micro injection, particle bombardment, biolistic transformation, and electroporation (Gasser et al, Science, 244, 5 1293; Potrykus, Bio/Techn. 8, 535, 1990; Shimamoto et al, Nature, 338, 274, 1989) .

Presently, AsrroJacteri iΩ tumefaciens mediated gene transfer is the method of choice for generating transgenic dicots (for review Hooykas & Schilperoort, 1992. Plant Mol. 0 Biol. 19: 15-38), however it can also be used for transforming monocots, although other transformation methods are generally preferred for these plants. Presently, the method of choice for generating transgenic monocots is particle bombardment (microscopic gold or tungsten particles coated with the 5 transforming DNA) of embryonic calli or developing embryos (Christou, 1992. Plant J. 2: 275-281; Shimamoto, 1994. Curr. Opin. Biotechnol. 5: 158-162; Vasil et al . , 1992. Bio/Technology 10: 667-674) . An alternative method for transformation of monocots is based on protoplast transformation as described by 0 Omirulleh S, et al . , Plant Molecular biology Vol. 21, No. 3 pp. 415-428 (1993) .

Following transformation, the transformants having incorporated the expression construct are selected and regenerated into whole plants according to methods well known in 5 the art .

ENZYME COMPOSITIONS

In a still further aspect, the present invention relates to an enzyme composition comprising the xyloglucanase of the 0 invention.

The enzyme composition of the invention may, in addition to the xyloglucanase of the invention, comprise one or more other enzyme types, for instance hemicellulase such as xylanase and mannanase, cellulase or endo-β-1, -glucanase components, 5 chitinase, lipase, esterase, pectinase, cutinase, phytase, oxidoreductase (peroxidase, haloperoxidase, oxidase, laccase) , protease, amylase, reductase, phenoloxidase, ligninase, pullulanase, pectate lyase, pectin acetyl esterase, polygalacturonase, rhamnogalacturonase, pectin lyase, pectin methylesterase, cellobiohydrolase, transglutaminase; or mixtures thereof .

The enzyme composition may be prepared in accordance with methods known in the art and may be in the form of a liquid or a dry composition. For instance, the enzyme composition may be in granulate or microgranulate form. The enzyme to be included in the composition may be stabilized in accordance with methods known in the art . Xyloglucanases have potential uses in a lot of different industries and applications. Examples are given below of preferred uses of the enzyme composition of the invention. The dosage of the enzyme composition of the invention and other conditions under which the composition is used may be determined based on methods known in the art .

The xyloglucanase or xyloglucanase composition according to the invention may be useful for at least one of the following purposes .

USES

Use in the detergent industry

During washing and wearing, dyestuff from dyed fabrics or garment will conventionally bleed from the fabric, which then looks faded and worn. Removal of surface fibers from the fabric will partly restore the original colours and looks of the fabric. By the term "colour clarification", as used herein, is meant the partly restoration of the initial colours of fabric or garment throughout multiple washing cycles.

The term "de-pilling" denotes removing of pills from the fabric surface.

The term "soaking liquor" denotes aqueous liquor in which laundry may be immersed prior to being subjected to a conventional washing process. The soaking liquor may contain one or more ingredients conventionally used in a washing or laundering process.

The term "washing liquor" denotes aqueous liquor in which laundry is subjected to a washing process, i.e. usually a combined chemical and mechanical action either manually or in a w ω t t μ μ in o Ul o O Ul

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invention comprise as a surfactant one or more of the non-ionic and/or anionic surfactants described herein.

Polyethylene, polypropylene, and polybutylene oxide conden-sates of alkyl phenols are suitable for use as the non- ionic surfactant of the surfactant systems of the present invention, with the polyethylene oxide condensates being preferred. These compounds include the condensation products of alkyl phenols having an alkyl group containing from about 6 to about 14 carbon atoms, preferably from about 8 to about 14 carbon atoms, in either a straight chain or branched-chain configuration with the alkylene oxide. In a preferred embodiment, the ethylene oxide is present in an amount equal to from about 2 to about 25 moles, more preferably from about 3 to about 15 moles, of ethylene oxide per mole of alkyl phenol. Commercially available non-ionic surfactants of this type include Igepal™ CO- 630, marketed by the GAF Corporation; and Triton™ X-45, X-114, X-100 and X-102, all marketed by the Rohm & Haas Company. These surfactants are commonly referred to as alkyl phenol alkoxylates (e.g., alkyl phenol ethoxylates) . The condensation products of primary and secondary aliphatic alcohols with about 1 to about 25 moles of ethylene oxide are suitable for use as the non-ionic surfactant of the non-ionic surfactant systems of the present invention. The alkyl chain of the aliphatic alcohol can either be straight or branched, primary or secondary, and generally contains from about 8 to about 22 carbon atoms. Preferred are the condensation products of alcohols having an alkyl group containing from about 8 to about 20 carbon atoms, more preferably from about 10 to about 18 carbon atoms, with from about 2 to about 10 moles of ethylene oxide per mole of alcohol . About 2 to about 7 moles of ethylene oxide and most preferably from 2 to 5 moles of ethylene oxide per mole of alcohol are present in said condensation products. Examples of commercially available non-ionic surfactants of this type include Tergitol™ 15-S-9 (The condensation product of C_ι:1.-C₁₅ linear alcohol with 9 moles ethylene oxide) , Tergitol™ 24-L-6 NMW (the condensation product of C₁₂-C₁₄ primary alcohol with 6 moles ethylene oxide with a narrow molecular weight distribution) , both marketed by Union Carbide Corporation; ω to t μ H

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3 , most preferably from about 1.3 to about 2.7. The glycosyl is preferably derived from glucose. To prepare these compounds, the alcohol or alkylpolyethoxy alcohol is formed first and then reacted with glucose, or a source of glucose, to form the glucoside (attachment at the 1-position) . The additional glycosyl units can then be attached between their 1-position and the preceding glycosyl units 2-, 3-, 4-, and/or 6-position, preferably predominantly the 2-position.

The condensation products of ethylene oxide with a hydrophobic base formed by the condensation of propylene oxide with propylene glycol are also suitable for use as the additional non-ionic surfactant systems of the present invention. The hydrophobic portion of these compounds will preferably have a molecular weight from about 1500 to about 1800 and will exhibit water insolubility. The addition of polyoxyethylene moieties to this hydrophobic portion tends to increase the water solubility of the molecule as a whole, and the liquid character of the product is retained up to the point where the polyoxyethylene content is about 50% of the total weight of the condensation product, which corresponds to condensation with up to about 40 moles of ethylene oxide. Examples of compounds of this type include certain of the commercially available Pluronic™ surfactants, marketed by BASF. Also suitable for use as the non-ionic surfactant of the non-ionic surfactant system of the present invention, are the condensation products of ethylene oxide with the product resulting from the reaction of propylene oxide and ethylenediamine. The hydrophobic moiety of these products consists of the reaction product of ethylenediamine and excess propylene oxide, and generally has a molecular weight of from about 2500 to about 3000. This hydrophobic moiety is condensed with ethylene oxide to the extent that the condensation product contains from about 40% to about 80% by weight of polyoxyethylene and has a molecular weight of from about 5,000 to about 11,000. Examples of this type of non-ionic surfactant include certain of the commercially available Tetronic™ compounds, marketed by BASF.

Preferred for use as the non-ionic surfactant of the surfactant systems of the present invention are polyethylene oxide condensates of alkyl phenols, condensation products of primary and secondary aliphatic alcohols with from about 1 to about 25 moles of ethylene oxide, alkyl polysaccharides, and mixtures hereof. Most preferred are C₈-C₁₄ alkyl phenol ethoxylates having from 3 to 15 ethoxy groups and C_β-C_x8 alcohol ethoxylates (preferably C₁₀ avg.) having from 2 to 10 ethoxy groups, and mixtures thereof.

Highly preferred non-ionic surfactants are polyhydroxy fatty acid amide surfactants of the formula

R² - C - N - Z,

I I I I

0 R¹ wherein R¹ is H, or R¹ is Cχ.₄ hydrocarbyl, 2-hydroxyethyl, 2- hydroxypropyl or a mixture thereof, R² is C₅.₃₁ hydrocarbyl, and Z is a polyhydroxy hydrocarbyl having a linear hydrocarbyl chain with at least 3 hydroxyls directly connected to the chain, or an alkoxylated derivative thereof. Preferably, R¹ is methyl, R² is straight C_:1.₁₅ alkyl or C₁₆.₁₈ alkyl or alkenyl chain such as coconut alkyl or mixtures thereof, and Z is derived from a reducing sugar such as glucose, fructose, maltose or lactose, in a reductive amination reaction.

Highly preferred anionic surfactants include alkyl alkoxylated sulfate surfactants. Examples hereof are water soluble salts or acids of the formula RO(A)_mS03M wherein R is an unsubstituted C_1Q-C-₂₄ alkyl or hydroxyalkyl group having a C_1Q-C₂₄ alkyl component, preferably a C_ι2-C₂₀ alkyl or hydro-xyalkyl, more preferably C₁₂-C₁₈ alkyl or hydroxyalkyl, A is an ethoxy or propoxy unit, m is greater than zero, typically between about 0.5 and about 6, more preferably between about 0.5 and about 3, and M is H or a cation which can be, for example, a metal cation (e.g., sodium, potassium, lithium, calcium, magnesium, etc.), ammonium or substituted-ammonium cation. Alkyl ethoxylated sulfates as well as alkyl propoxylated sulfates are contemplated herein. Specific examples of substituted ammonium cations include methyl-, dimethyl, trimethyl-ammonium cations and quaternary ammonium cations such as tetramethyl-ammonium and dimethyl piperdinium cations and those derived from alkylamines such as ethylamine, diethyl amine, triethylamine, mixtures thereof, and the like. Exemplary surfactants are C₁₂-C₁₈ alkyl polyethoxylate (1.0) sulfate (C₁₂-C_1SE (1.0)M) , C₁₂-C₁₈ alkyl polyethoxylate (2.25) sulfate (C₁₂-C₁₈ (2.25) M, and C₁₂-C₁₈ alkyl polyethoxylate (3.0) sulfate (C₁₂-C_1SE (3.0) M) , and C₁₂-C₁₈ alkyl polyethoxylate (4.0) sulfate (C₁₂-C₁₈E (4.0) M) , wherein M is conveniently selected from sodium and potassium. Suitable anionic surfactants to be used are alkyl ester sulfonate surfactants including linear esters of C₈-C₂₀ carboxylic acids (i.e., fatty acids), which are, sulfonated with gaseous S0₃ according to "The Journal of the American Oil Chemists Society", 52 (1975), pp. 323-329. Suitable starting materials would include natural fatty substances as derived from tallow, palm oil, etc. The preferred alkyl ester sulfonate surfactant, especially for laundry applications, comprise alkyl ester sulfonate surfactants of the structural formula:

0

I I R³ - CH - C - OR⁴

SO,M

wherein R³ is a C₈-C₂₀ hydrocarbyl, preferably an alkyl, or combination thereof, R⁴ is a

hydrocarbyl, preferably an alkyl, or combination thereof, and M is a cation, which forms a water-soluble salt with the alkyl ester sulfonate. Suitable salt-forming cations include metals such as sodium, potassium, and lithium, and substituted or unsubstituted ammonium cations, such as monoethanolamine, diethanolamine, and triethanolamine. Preferably, R³ is C₁₀-C_ιε alkyl, and R⁴ is methyl, ethyl or isopropyl . Especially preferred are the methyl ester sulfonates wherein R³ is C₁₀-C_X6 alkyl.

Other suitable anionic surfactants include the alkyl sulfate surfactants which are water soluble salts or acids of , the formula ROS0₃M wherein R preferably is a C₁₀-C₂₄ hydrocarbyl, preferably an alkyl or hydroxyalkyl having a C₁₀-C₂₀ alkyl component, more preferably a C₁₂-C₁₈ alkyl or hydroxyalkyl, and M is H or a cation, e.g., an alkali metal cation (e.g. sodium, potassium, lithium), or ammonium or substituted ammonium (e.g. methyl-, dimethyl-, and trimethyl ammonium cations and quaternary ammonium cations such as tetramethyl-ammonium and dimethyl piperdinium cations and quaternary ammonium cations derived from alkylamines such as ethylamine, diethylamine, triethylamine, and mixtures thereof, and the like) . Typically, alkyl chains of C₁₂-C₁₆ are preferred for lower wash temperatures (e.g. below about 50 °C) and C₁₆-C₁₈ alkyl chains are preferred for higher wash temperatures (e.g. above about 50°C) .

Other anionic surfactants useful for detersive purposes can also be included in the laundry detergent compositions of the present invention. Theses can include salts (including, for example, sodium, potassium, ammonium, and substituted ammonium salts such as mono- di- and triethanolamine salts) of soap, C₈- C₂₂ primary or secondary alkanesulfonates, C₈-C₂₄ olefinsulfonates, sulfonated polycarboxylic acids prepared by sulfonation of the pyrolyzed product of alkaline earth metal citrates, e.g., as described in British patent specification No. 1,082,179, C₈-C₂₄ alkylpolyglycolethersulfates (containing up to 10 moles of ethylene oxide) ; alkyl glycerol sulfonates, fatty acyl glycerol sulfonates, fatty oleyl glycerol sulfates, alkyl phenol ethylene oxide ether sulfates, paraffin sulfonates, alkyl phosphates, isethionates such as the acyl isethionates, N-acyl taurates, alkyl succinamates and sulfosuccinates, monoesters of sulfosuccinates (especially saturated and unsaturated C₁₂-C₁₈ monoesters) and diesters of sulfosuccinates (especially saturated and unsaturated C_β-C₁₂ diesters), acyl sarcosinates, sulfates of alkylpolysaccharides such as the sulfates of alkylpolyglucoside (the nonionic nonsulfated compounds being described below) , branched primary alkyl sulfates, and alkyl polyethoxy carboxylates such as those of the formula RO(CH₂CH₂O)_k-CH₂C00-M+ wherein R is a C₈-C₂₂ alkyl, k is an integer from 1 to 10, and M is a soluble salt forming cation. Resin acids and hydrogenated resin acids are also suitable, such as rosin, hydrogenated rosin, and resin acids and hydrogenated resin acids present in or derived from tall oil.

Alkylbenzene sulfonates are highly preferred. Especially preferred are linear (straight-chain) alkyl benzene sulfonates (LAS) wherein the alkyl group preferably contains from 10 to 18 carbon atoms .

Further examples are described in "Surface Active Agents and Detergents" (Vol. I and II by Schwartz, Perrry and Berch) . A variety of such surfactants are also generally disclosed in US 3,929,678, (Column 23, line 58 through Column 29, line 23, herein incorporated by reference) .

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

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

Cationic detersive surfactants suitable for use in the laundry detergent compositions, of the present invention are those having one long-chain hydrocarbyl group. Examples of such cationic surfactants include the ammonium surfactants such as alkyltrimethylammonium halogenides, and those surfactants having the formula :

[R² (OR³ ) _y] [R⁴ (OR³ ) _y] ₂R⁵N+X-

wherein R² is an alkyl or alkyl benzyl group having from about 8 to about 18 carbon atoms in the alkyl chain, each R³ is selected form the group consisting of -CH₂CH₂-, -CH₂CH (CH₃) - , - CH₂CH(CH₂0H) -, -CH₂CH₂CH₂-, and mixtures thereof; each R⁴ is selected from the group consisting of C_3.-C₄ alkyl, C1_.-C₄ hydroxyalkyl, benzyl ring structures formed by joining the two R⁴ groups, -CH₂CHOHCHOHCOR⁶CHOHCH₂OH, wherein R⁶ is any hexose or hexose polymer having a molecular weight less than about 1000, and hydrogen when y is not 0; R⁵ is the same as R⁴ or is an alkyl chain, wherein the total number of carbon atoms or R² plus R⁵ is not more than about 18; each y is from 0 to about 10, and the sum of the y values is from 0 to about 15; and X is any compatible anion.

Highly preferred cationic surfactants are the water soluble quaternary ammonium compounds useful in the present composition having the formula:

R₁R₂R₃R₄N⁺X- (i)

wherein R_x is C₈-Cι₆ alkyl, each of R₂, R₃ and R₄ is independently C_!-C₄ alkyl, C-Ci hydroxy alkyl, benzyl, and -(C₂H₄₀)_XH where x has a value from 2 to 5, and X is an anion. Not more than one of R₂, R₃ or R₄ should be benzyl. The preferred alkyl chain length for R_x is C₁₂-C₁₅, particularly where the alkyl group is a mixture of chain lengths derived from coconut or palm kernel fat or is derived synthetically by olefin build up or 0X0 alcohols synthesis. Preferred groups for R₂R₃ and R₄ are methyl and hydroxyethyl groups and the anion X may be selected from halide, methosulphate, acetate and phosphate ions.

Examples of suitable quaternary ammonium compounds of formulae (i) for use herein are: coconut trimethyl ammonium chloride or bromide ; coconut methyl dihydroxyethyl ammonium chloride or bromide; decyl triethyl ammonium chloride; decyl dimethyl hydroxyethyl ammonium chloride or bromide; C₁₂_₁₅ dimethyl hydroxyethyl ammonium chloride or bromide; coconut dimethyl hydroxyethyl ammonium chloride or bromide; myristyl trimethyl ammonium methyl sulphate; lauryl dimethyl benzyl ammonium chloride or bromide ; lauryl dimethyl (ethenoxy)₄ ammonium chloride or bromide; choline esters (compounds of formula (i) wherein R_x is

CH₂-CH₂-0-C-C₁₂.₁₄ alkyl and R₂R₃R₄ are methyl) .

0

di-alkyl imidazolines [compounds of formula (i)] . Other cationic surfactants useful herein are also described in US 4,228,044 and in EP 000 224.

When included therein, the laundry detergent compositions of the present invention typically comprise from 0.2% to about ω ω to tυ μ μ

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Ω 0 μ >c μ- ø rt rt - 0 μ 0 μ- 3 0 rt rt rt o C^Q ø rt 0 μ μ. μ 3 Hi •

0 3 0 CD μ. 03 3 0 0 μ- tr 3 0 Ω <J TJ μ 03 0 μ- tr Φ μ- co CO : 0 J ø 0

0 tr 0 H φ 0 tr ø rt tr 0 φ TJ ø μ; Φ 0 Hi . tr 0 Φ μ; Φ Ω tr 0 ø 0 Ω tr rt rt 0 H μ- μ- μ- 03 0 ø μ μ- 0 μ Ω CD 03 0 0 ø μ rt rt Ω Φ 0 3 Cb CO rt 0 ø tr 0 0 CQ Φ Φ Ω Ω . 0 Φ ø ø μ; μ- Ω ø Φ μ- α S3 0 0 ø μ- H μ- 0 ø μ- Φ rt tr μ Ω rt tr rt rt μ- Φ 0 μ- 0 rt rt rt rt μ- Ω O ø ø 03 Cb ø μ. rt 0 rt ø 0 rt μ; co μ; ø Cb 0 Ω Hi μ- ø ø rt ø rt rt Φ μ- rt μ- CQ H Φ 0 φ μ- 0 H TJ - |J 03 3 0 ø μ> TJ - 03 10 φ tr ø μ rt 03 Cb 0 03 H ø μ σ TJ Cb 0 ø μ o\ μ- ∞ 3 03 03 ø rt o\° μ- ø μ 0 μ- 0 Φ - Φ ø o\°

CQ 0 TJ 03 Hi Ω Hi Ω rt — - 0 φ Φ 03 03 Ω rt μ ID 1 0 μ- μ D 03 ø tr Hi to 03 rt ø

0 rt tr Hi 0 rt 0 tr en ø Ω Ω • rt 0 03 cQ rt 0 Ω μ rt

Hi TJ 0 0 Ω μ Hi 0 Ω 0 Φ Hi φ μ- 0 0 ø 0 H φ 0 D 0 tr μ- μ μ 0 Φ 0 μ 03 0 0 μ Cb rt 0 φ ø 0 0 μ_ μ H Ω - μ-¹ μ> ø rt ø μ- Hi

0 Ω ø TJ 0 3 0 φ 0 ø μ. μ 3 Cb Cb tr Φ ø μ; rt σi 0 oo rt μ ø tr ø ø

3 0 tr tr rt 3 0 tr 0 α 0 0 φ tr 0 ø -J. tr μ; μ-¹ Φ rt tr ø 0 μ- ø rt S3 rt 0 Ω ø Φ CD μ μ 03 ø 0 Ω 0 ø oo μ- Ω 0 μ- Cb tr 03 0 ø 03 ø ø μ- tr ø ø 03 ø 0 0 X μQ μ; μ. φ ø 0 ø rt 0 Φ μ TJ φ 0 0 tr μ- rt Φ ø φ tr rt rt 3 Cb 0 ω ø 03 rt 3 Cb CO _^-_^ μ- μ ø 3 tr rt

0 03 μ- o φ ø TJ μ 3 -. ø 0 0 O 0 J μ • Ω N tr 03 tr μ- 0 03 TJ CD

0 rt μ> 0 ø CQ 0 μ μ H μ μ; TJ ID rt - ø ø 0 H μ μ; 0 μ- 0 rt μ ø rt μ 0 00 rr μ- co X CQ μ rt 1 φ o μ- to Φ Cb Cb μ 03 o μ- ø ø 0 φ μ- 0 Φ μ- o\° ø μ- 0 03 o\° 03 Cb Φ CD μ Hi ø o\o 03 b 0 CQ ø ø 03 Ω 03 rt

H CQ Ω Cb Hi 0 μ» 0 0 Φ Φ 03 -. ø rt rt 0 μ- Φ Φ 3 0 tr Ω μ- Φ ø tr ø Φ μ TJ o tr rt CTi ø Φ Φ Ω rt tr rt 0 CQ rt O tr μ- Cb TJ ø tr μ. rt 0 μ 03 0 ø 03 μ; Hi Φ 0 -J μ μ μ rt ø μ; Hi Φ μ 0 ø Φ ø φ tr rt H

0 Hi tr 3 Ω rt tr TJ μ μ Hi oo μ. rt rt 0 tr μ μ M 0 3 rt Cb μ 0 φ S3

0 Ω 0 0 H φ S3 0 CQ μ- μ- 0 S3 0 CQ Ώ ø 03 1 S3 μ- rt μ- φ ø 0 ø 0 0 ø φ Ω Φ 3 Φ N _^-, TJ 0 ø rt φ Φ 3 φ - TJ - oo Ω tr <J μ- ø Hi μ- tr ø tr CQ ø μ- μ- ø 5^> Ω tr μ μ 03 μ- 0 - tr μ- ø Ω <! 0 CQ

0 PC 0 rt 0 μ- tr ø ø CQ o rt μ- 0 0 μ; μj Hi CQ o rt " 0 Ω 0 Ω rt φ μ tr ø μ; ct ø 0 CQ 0 3 μ-¹ tr . rt M D Ω 0 tr • μ- φ 0 ø μ- tr μ- Cb ø rt rt o μ- rt rt 0 μ- rt to Ω rt ø TJ ø ø ø μ rt to Ω ø . Cb μ ø < φ rt ø

3 ø μ- rt ø Ω o\° 0 Φ 3 tr 3 3

10 ø o\° 0 φ CQ σ rt φ μ μ- CD 0

CQ 03 μ- μ> ø φ ø 0 μ 0 0 μ- μ- 0 0 03 • ø 0 tr 03 μ- 0 Φ Mi μ ø CQ H rt Hi rt TJ μ- ø ø ø tr 03 Hi rt TJ rt ø O Φ <!

Ω oo ø

0 0 CQ rt 0 Φ 0 o 0 H μ- Φ φ Φ Φ 0 0 H Ω ø 0 0 μ- 03 ø ø 0 0 0 X CQ 03 CO ø CO ø 03 03 CD 03 00 0 ø Φ Hi rt ø 0 μ TJ Cb 0 Hi Ω μ- 0 0 ø μ- μ- -. 3 -. -. tr μ- 0 ø μ- 1 μ Φ rt μ- m Ω tr 03 0 ø ø Cb μ Ω tr rt Ω μ ø Ω tr rt ω tr ø 0 0 < tr rt tr

0 to Φ tr 0 μ Φ μ . tr 0 μ- 0 0 0 tr 0 μ- in 0 03 3 Hi Φ Φ tr

0 0 0 μ^j tr 03 ø 0 μ- μ μ 0 ø 0 X rt 03 03 CD Φ 0 ø P? 0 0 rt ø Cb rt ø μ. rt Φ

Cb rt μ. ø Hi 03 ø φ 03 tr 0 μ; Φ Hi

atoms; and water-soluble sulfoxides containing one alkyl moiety from about 10 to about 18 carbon atoms and a moiety selected from the group consisting of alkyl and hydroxyalkyl moieties of from about 1 to about 3 carbon atoms .

Semi-polar nonionic detergent surfactants include the amine oxide surfactants having the formula :

O

R³(0R⁴)xN(R⁵)2

wherein R³ is an alkyl, hydroxyalkyl, or alkyl phenyl group or mixtures thereof containing from about 8 to about 22 carbon atoms; R⁴ is an alkylene or hydroxyalkylene group containing from about 2 to about 3 carbon atoms or mixtures thereof; x is from 0 to about 3 : and each R⁵ is an alkyl or hydroxyalkyl group containing from about 1 to about 3 carbon atoms or a polyethylene oxide group containing from about 1 to about 3 ethylene oxide groups . The R⁵ groups can be attached to each other, e.g., through an oxygen or nitrogen atom, to form a ring structure .

These amine oxide surfactants in particular include C₁₀-C₁₈ alkyl dimethyl amine oxides and C₈-C₁₂ alkoxy ethyl dihydroxy ethyl amine oxides . When included therein, the laundry detergent compositions of the present invention typically comprise from 0.2% to about 15%, preferably from about 1% to about 10% by weight of such semi-polar nonionic surfactants.

Builder system

The compositions according to the present invention may further comprise a builder system. Any conventional builder system is suitable for use herein including aluminosilicate materials, silicates, polycarboxylates and fatty acids, materials such as ethylenediamine tetraacetate, metal ion sequestrants such as aminopolyphosphonates, particularly ethylenediamine tetramethylene phosphonic acid and diethylene triamine pentamethylenephosphonic acid. Though less preferred w w t to μ μ in o Ul O Ul o in

Ω Cb S3 TJ H 03 rt μ> 0 tr 3 3 tr Hi μ. μ- tr μ. - 0 Φ X μ. 0 0 Φ 0

Ω 03 μ- μ ιo rt H μ. Cb rt t μ

Ω 0 KΩ Hi μ - Cb μ Φ Φ ø

0 μ^j φ 00 0 ø to μ- 0 μ μ 03 0

TJ 0 03 Ω •* 03 rt μ- μ- Φ tr φ 03 TJ CD μP* 0 -j to 0 13 Φ ø ø O Cb <!

0 φ 0 Φ to Ω - Ω 0 Cb μ^j 0 μ- rt μ- Cb Cb μ» Ω μ 1 Ω 0 -. -. μ- tr 0 ø Ω μ. μ- tr φ μ- μ. rt N rt Φ ø

0 μ. μ- Ω Ω 0 ø 0 rt ø Ω tr φ 3 Ω ø μ 03

Φ Ω ø 0 μ- ø 0 X tr 0 0 φ 0 0 0 tr φ

1 H μ rt Cb rt μ. ø ct μ μ μ^j μ 3 μ^j μ- Φ

Ω μ- U3 tr μ φ μ^j ø φ tr μ- Φ 3 φ ø 0 μ- Ω μ 0 0 μ> ø Φ CO 0 03 rt 0 <!

03 μ- X rt ^•- Cb rt ø φ TJ ø tr μ-

- 0 rt μ. Φ ω φ Φ rt Cb μ. μ- 0 H 0 μ

Ω 0 μ- 03 D μ 03 Φ μ- rt μ μ; μ- 0 μ- Cb 03 ø oo μ- rt 03 0 ø rt ø

03 tr rt Cb < Ω μ Ω rt tr μ- 0 Cb 3

1 tr Φ Φ μp> 0 0 ø H Φ ^j > Ω φ φ

Ω Φ 13 03 03 to rt ø Ω 0 03 Φ t ø μ ø μ- r 0 Ω to μ- rt ø 03 μ- CD rt

03 Φ rt Ω μ < ø μ Φ Ω μ- 03 ø 0 ø

1 μ φ 0 μ- 0 φ μ- tr Cb 0 ø ** μ 0 Ω rt 0 0 ø tr 0 D ø 0 0 0 μ^j μ 0

Φ Ω rt rt Φ Cb μ- X μ- rt μ K μ: CQ ø μ rt μ. ø Cb Cb ø μ; ø ø CQ CO 0 φ μ Ω a μ- μ- μ- CQ μ- 0 tr (II ø 0 ø μ- ø 03 0 ϋd ø ø 0 μ- Φ co

Ω μ- • μ- ø Ω 03 rt μ μ- μ- μ tr Ω O ø Ω ø ω α ø Φ μ- ø Ω μ. 0 ø μ μ> CQ CO 0 μ^j 03 rt CQ 2 Cb tr ø CO tr TJ ^ μ 03 Hi μ- tr ^ μ μ; -.

0 0 TJ μ- ιo φ 0 CD Hi ø 13 0 Cb μ-

H CO

% tr rt - Cb μ> tr 0 μ- . rt μ ø TJ μ; μ; KO 0 μ- κo 03 - ø μ^j Φ 0 tr

M Ω CQ 03 ω μ- 0 μ> TJ μ Cb Cb rt μ 0 ø ø o TJ tr σι ø tr 0 Φ Φ CQ 03 rt 2, o tr - 03 ω rt Ω μ 03 Cb 0 TJ

Φ tr o 0 13 ω rt - φ ø j 0 tr

03 0 • ø ø μ μ- lo ø μ 3 ø 0 μ- 0

X Φ rt 00 μ- rt 1 rt tr 0J rt Ω rt μ. φ - ct ø TJ 0 rt tr ø φ

Ω CO ø μ- Φ μ Φ

0 0 rt 0 03 ø 0 a X Φ 3 μ- μ. 0 μ. μ rt μ- 0 tr

Ω rt tr 0 tr rt TJ • μ- μ- ø ø 0

H Φ CD a Cb 03 0 CQ 0 Ω 0 μ-

0 03 rt 0 13 ø μ> μ μ^j CO Φ M

TJ μ- • rt ø μ- Φ *• 0 N μ- X Cb

Φ μ- rt tr rt ø to 0 μ- Φ Ω φ ø 0 μ> φ Φ Ω en TJ D O μ- tr μ rt Ω φ -• ø H μ> 03 Ω ØJ 03 ø 0 o CD rt ø μ- ø ø

Cb 0 rt oo 0 Cb oo μ- ø ct t CQ Ω μ- Cb 03 to a Φ to 0 μ; Φ φ φ 0 φ Φ - Ml 0 CO Ω Φ ø ø ø μ> O 03 rt μ CO μ- μ -j 0 • tr 0 Φ 0 ø

Cb φ κo 0 Φ Cb Cb Ω μ^j

Φ ct φ tr 03 Φ 0 Cb ø

D

TJ 0 TJ TJ O 3 03 03 μ- Hi s td TJ Hi 3 3 Cb μ- 0 03 ø Ω 3 Ω H rt TJ 0 1 t rt TJ

0 μ 0 tr μ- ø μ. O ø O CQ X μ 0 μ- Φ μ- O CQ μ- 0 O 0 tr O Φ Φ

H H 0 rt 03 H 03 μ td ø Φ μ X rt 03 ø Φ ø 3 μ μ tf φ Ω tr t rt 0 μ. Ω μ. 03 μ- φ rt 0 O D 3 Hi 3 rt ø 0 CO ø μ- 3 TJ Φ tr to μ; O Φ μ rt

Ω 0 Ω TJ Ω μ Φ r μ- ϋ TJ Φ ø Ω rt Ω μ- 0 O in TJ Ω tr X in ø ø

0 1 0 tr ø μ- 3 H 0 0 CO μ ø μ - Ω μ- ø ø 03 TJ X tr 0 0 0 - Ω Ω μ TJ O μ 0 rt ø Ω φ tr TJ Ω Φ μ ø Φ μ- ø > 03 rt O μ- 13 0 μ; Ω ω rt μ Ω in 0 ø tr 0 rt tr ø φ \-> Hi rt μ ø 03 Φ frCD ø ø ø φ CD rt μ μ Hi ιf_. tr tr 03 0 μ μ

0 H tr 0 0 D CO 0 tr Ω Φ Φ ø ø Cb 3 μ- ø l Ω μ- μ- Φ rt ø CO ØJ 0 μ 1 tr tr

X φ X rt » μ Φ 0 Hi 03 Cb O et rt 3 Ω Ω ø tr _^-_^ H 0 Hi μ- rt rt . H X CD tr rt 0 0 . _ μ μ; Φ CO μ μ 0 Φ μ- Hi 03 tr tr O Ω μ- O μ- 0 Φ Ω Φ tr μ- μ. 0 0 Φ X X

Φ 03 ø 0 0 tr H μ 0 S 0 Φ φ ø 0 O rt 0 « Ω 03 μ 0 CD φ Ω Ω X rt μ. ; μ- μ D 0 - ø Ω CD tr 0 0 μ ø CQ CQ Cb μ μ- Ω μ ø 03 CO ø μ 0 tr μ; μ

Ω μ- 0 rt Cb tr Φ 0 X 3 Φ 0 μ- Ul rt td Φ ø μ- Cb tr 1 μ- Φ ø Ω 0 0 rt h-¹ ø 0 0

Ω μ- Φ 0 μ- μj μ- Cb μ- D Ω 0 0 0 3 Cb ø H Ω CTi φ ø Cb μ O tr Ω Φ 0 0 tr rt rt ø rt 03 3 0 0 μ- ø ø D tr 3 Cb Hi ø Φ μ- Ω ø 0 μ- 03 CO rt μ . Φ Φ

Ω 0 ø . μ- μ CD ø Cb ø 0 tr μ- . _^-_^ O Ω rt tr tr μ. ct < Cb Φ Cb D 03 μ- Ω tr ø CQ φ rt 03 0 Ω TJ 03 0 O td φ Ω μ 0 0 ø ø Φ μ- 03 03 μ >• -.

C μ- H 0 ø 0 CQ μ Φ μ- μ- O ' μ 0 3 TJ μ O tr μ- 0 μ- Cb μ- Ω μ- - Cb ø 0 0

Cb Φ ø H μ H H 3 μ-¹ S3 Φ Ω 0 Φ μ- ø φ Ω μ ø Hi to to

Ω 03 TJ μ- Pc ι-3 Φ μ α

0 3 Ω μ- Cb TJ ' Hi rt O Φ 03 CO μ- H Cb Cb Cb rt μ- rt μ μ-¹ tr 0 0 _". -

0 S3 0 Ω 0 ø 0 tr Ω φ O — • rt Φ φ CD μ Hi 0 ø ø φ ø φ μ ø tr μ- 0 μ- Cb μ in 10

3 0 ø H 0 rt φ 0 μ 03 μ φ tr tr ø Ω ø μ μ ø iQ Φ <J Cb rt ø 1 -

TJ μ rt μ; 3 μ- μ^j Φ H rt μ- μ

3 μ O 3 CQ 0 rt μ- 3 rt ø Φ O Ω ø rt μ Φ 0 0 ø μ 0 Φ 03 rt ø Φ Ml ø μ rt μ ct Cb S3 03 μ- CD Φ ø TJ rt ø Φ - μ- rt μ H rt 3 μ μ- rt μ. μ- CQ Cb CQ Φ μ- tr Hi ø 0 X μ; CO TJ μ μ- 3 - μ 1 rt in

03 tr 1 Pc φ φ 0 μ- tr 03 O ø td ø Cb rt rt Φ O rt Ω rt CD . Φ < Φ tr μ I

Φ Φ 03 μ. μ rt Ω M ø ø rt ø t

03 μ- 0 H μ- ø 0 03 CQ μ- Φ 03 Φ 3 a Φ ø tr μ Φ tr ø rt rt Hi φ H 3 0 rt ø r

CQ ø α 03 td rt Φ μ- μ μ Φ O Φ 03 H ø X Φ tr Φ μ H Φ 0 3 -> 3 μ- CQ CO μ- α Φ ø μ- 1 0 Φ 3 μ μ- ø rt μ. rt ø 0 TJ ct 0 Cb 03 μ- ø 0 0 α Cb 0 < 0 03 03 CD rt μ Cb rt ø g μ Cb μ rt 03 tr Φ O Ω o tr CQ Φ ø 3 Φ μ- 3 CO Φ Ω 0 0 tr Φ μ- μ- μ- Φ ø μ ø

0 ø CD 0 Φ μ μ- 03 ø ø Pc ø N Hi Hi μ Cb 03 Ω rt rt Ω 0 tr

Φ TJ CQ μ μ- ct 0 ø ø rt

03 μ- Ω 03 Ω 3 0 ø ø φ O Φ Ω O tr ø 1 μ;

Φ rt tr 0 tr rt rt 03 Ω Ω 0 0 H 0 0 3 μ- 03 tr O ø μ Φ TJ H ø j μ Hi Cb ø CD 0 0 Ω 0 μ- Ω 0 Ω μ^j 3 ø 3 O μ- O μ- 0 O Ω M tr 0 μ

CO - μ 03 tr ø 0 ø 03 Ω 0 O rt Cb Ct TJ ø 0 O φ μ- S3 ø Ω 03 μ- ø 0 μ 0 rt CQ TJ 0 ø ø 0 tr Cb μ 03

03 φ 0 μ- 3 ø rt ø 03 0 μ. Φ Cb ø Cb X 0 1 μ- 0 tr 0 rt CD Ω Φ Cb 0 Ct 0 ø φ μ- Ω Φ rt Φ μ Ω Cb Cb φ μ. ø Hi rt ø ø o CD φ Hi tr 0 ø

S3 μ- ø CD μ- O CO 0 ø ø μ* 0 3 rt rt a tr CQ μ- ø Φ tr 0 μ μ^j 1 ø μ Φ Cb 0 ø μ μ μ- 0 μ μ- TJ X μ- ø Ω μ

0 S3 Ω ø rt ø μ 0 Ω 03 1 ø X tr ø μ. μ; < t tr Ω 3 03 t i 3 φ Φ μ 03 CO Φ tr rt μ- ø tr r D φ Φ tr H φ μ- α Φ 0 •» ct rt 0 O μ- μ. 0 μ 0 φ 03 ø

Ω μ- C tr tr X μ ø rt X t o μ- rt CD

0 Ω ø CQ μ- 0 TJ Ω O X rt O

S3 tr Hi d 0 0

Hi

00 μ rt ø . φ μ. 03 tr CQ μ; μ 3 rt μ- - 1 μ tr 0 Ω Cb 0 μ- -> rt μ- Φ . Φ 03 l_ι 0 0 φ μ -^> μ- Φ O < Cb tr rt 0 μ 0 φ μ rt μ- 0 rt td PC Φ Cb 0 Hi o 0 rt φ μ> μ- Ω

0 rt CO 0 Q μ rt μ rt μ tr t

3 α ø td rt rt 0 0 Φ r 0 TJ 0 rt μ- . Ul - D μ-

X tr Cb ø tr μ- μ- Φ Φ Φ CQ μ- t O α Ω 03

Cb X tr tr H Φ rt Φ ø tr μ TJ μ. Φ ø ø 0 ø O Ω rt

CO CO ø Φ Φ μ- Cb Φ μ^j Φ 03 03 tr rt μ- > O _^ ø ~

H" μ 0 ø o Hi > O tr rt a 3 μ ø μ- μ Ω φ 03 μ- μ Hi 10 μ φ 3 Ω 0J μ 0 Hi Φ μ- TJ Hi Φ Φ 0 CQ

0J tr 0 Φ TJ ø 13 Ω O -. tr Ω μ μ- rt CQ rt Ω TJ ø μ 0 3 φ φ μ; ø Φ μ 0J 3 TJ 0 μ-

0 rt ø φ ø tr μ- ø ø μ- Φ rt μ φ rt 0 0 «. μ Ω d φ Φ Hi φ μ- ø ø X 03

Cb tr 0 03 ø φ Cb ø ø φ H α 03 Φ - 0 ø rt ø μ Φ Ω rt H in μ. ~ μ- φ - μ- • Cb S3 <! φ Hi 0 α μ φ rt Φ 3 ø ø μ- μ- μ; -.

Ω Ω tr 0 rt 1 Ω μ- Cb rt φ Cb co O 0 0 0 μ; Cb Ω tr CTl 0 Ω ø tr ø ø rt ø Φ Hi Ω Cb μ μ ' a O ø μ; 1 rt μ-

H 0 μ- Φ rt μ- μ ϋ CQ Φ μ a ø Cb tr Φ 03

03 3 S3 μ μ- 03 ø 1 O Φ Cb a TJ Ω 0 O ø μ Φ CD 1

0 Cb

Cb 0 O 0

03 μ. Cb μ- X _^

1 Φ rt ø Ω 1 . Ω ø μ Φ • tr μ- Φ 1 μ 1 0 Φ

separated form each other by not more than two carbon atoms .

Polymers of this type are disclosed in GB-A-1, 596 , 756. Examples of such salts are polyacrylates of MW 2000-5000 and their copolymers with maleic anhydride, such copolymers having a molecular weight of from 20,000 to 70,000, especially about 40,000.

Detergency builder salts are normally included in amounts of from 5% to 80% by weight of the composition. Preferred levels of builder for liquid detergents are from 5% to 30%.

Enzymes

Preferred detergent compositions, in addition to the enzyme preparation of the invention, comprise other enzyme (s) which provides cleaning performance and/or fabric care benefits . Such enzymes include proteases, lipases, cutinases, amylases, cellulases, peroxidases, oxidases (e.g. laccases) .

Proteases : Any protease suitable for use in alkaline solutions can be used. Suitable proteases include those of animal, vegetable or microbial origin. Microbial origin is preferred. Chemically or genetically modified mutants are included. The protease may be a serine protease, preferably an alkaline microbial protease or a trypsin-like protease. Examples of alkaline proteases are subtilisins, especially those derived from Bacillus, e.g., subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168 (described in WO 89/06279) . Examples of trypsin-like proteases are trypsin (e.g. of porcine or bovine origin) and the Fusarium protease described in WO 89/06270.

Preferred commercially available protease enzymes include those sold under the trade names Alcalase, Savinase, Primase, Durazym, and Esperase by Novozymes A/S, those sold under the tradename Maxatase, Maxacal, Maxapem, Properase, Purafect and Purafect OXP by Genencor International, and those sold under the tradename Opticlean and Optimase by Solvay Enzymes. Protease enzymes may be incorporated into the compositions in accordance with the invention at a level of from 0.00001% to 2% of enzyme protein by weight of the composition, preferably at a level of from 0.0001% to 1% of enzyme protein by weight of the composition, more preferably at a level of from 0.001% to 0.5% of enzyme protein by weight of the composition, even more preferably at a level of from 0.01% to 0.2% of enzyme protein by weight of the composition. Lipases : Any lipase suitable for use in alkaline solutions can be used. Suitable lipases include those of bacterial or fungal origin. Chemically or genetically modified mutants are included.

Examples of useful lipases include a Humicola lanuginosa lipase, e.g., as described in EP 258 068 and EP 305 216, a Rhizomucor miehei lipase, e.g., as described in EP 238 023, a Candida lipase, such as a C. antarctica lipase, e.g., the C. antarctica lipase A or B described in EP 214 761, a Pseudomonas lipase such as a P. alcaligenes and P. pseudoalcaligenes lipase, e.g., as described in EP 218 272, a P. cepacia lipase, e.g., as described in EP 331 376, a P. stutzeri lipase, e.g., as disclosed in GB 1,372,034, a P. fluorescens lipase, a Bacillus lipase, e.g., a B. subtilis lipase (Dartois et al . , (1993), Biochemica et Biophysica acta 1131, 253-260), a B. stearo- thermophilus lipase (JP 64/744992) and a B. pumilus lipase (WO 91/16422) .

Furthermore, a number of cloned lipases may be useful, including the Penicillium camembertii lipase described by Yamaguchi et al . , (1991), Gene 103, 61-67), the Geotricum candidum lipase (Schimada, Y. et al . , (1989), J. Biochem., 106, 383-388) , and various Rhizopus lipases such as a R. delemar lipase (Hass, M.J et al . , (1991), Gene 109, 117-113), a R. niveus lipase (Kugimiya et al . , (1992), Biosci . Biotech. Biochem. 56, 716-719) and a R. oryzae lipase. Other types of lipolytic enzymes such as cutinases may also be useful, e.g., a cutinase derived from Pseudomonas mendocina as described in WO 88/09367, or a cutinase derived from Fusarium solani pisi (e.g. described in WO 90/09446) . Especially suitable lipases are lipases such as Ml Lipase™, Luma fast™ and Lipomax™ (Genencor) , Lipolase™ and Lipolase Ultra™ (Novozymes A/S) , and Lipase P "Amano" (Amano Pharmaceutical Co. Ltd.) .

The lipases are normally incorporated in the detergent J to t μ μ

Ul o Ul o Ul o in

Ω & . μ- 0 Ω ø H μ- tr 0 μP> 3 tr 03 Ω TJ 3 o tr Ω > ø Cb 0 μ- 0 0 Ω TJ 3 o tr Ω

0 0 ø TJ Φ μ H ø 0 0 - ø 0 0 0 Φ μ 0 • μ; 0 3 Φ 3 Hi Hi H 0 Φ μ 0 . μ^ 0 μ a Ω TJ H φ H Ω μ_ μ* rt Ω 3 <! 0 μ o 3 CQ μ; 03 μ; μ- P? 3 < 0 μ o 3

TJ μ. H H H 3 rt 0 ω ø rt 0 TJ Φ rt Φ o S3 TJ •— - μ-¹ Ω H φ tr ø TJ Φ rt Φ o S3 TJ

0 Q 0 μ- 0 rt Ω Ω Φ in ø φ rt 0 φ o Φ 0 ø μ 0 Cb 0 o Φ o φ 0 μ 0 Cb Ω tr Φ μ μ. - rt μ μ- 03 μ- TJ H μ- CD ø CD μ- 03 Ω μ- CD μ- TJ μ> μ- 03 ø Φ 0 ø φ H KD μ- 0 0 03 μ- 0 Ω μ- 0 rt ø μ o\° CQ μ- ι-3 0 φ tr Φ 3 rt ø > μ- 0 ø μ o\° CQ μ- rt 0 03 H 0 0 o 0 ø Φ rt Hi Φ tr rt t Cb 03 Φ CQ 0 φ Φ 3 rt Hi Φ tr ΓΓ μ- Ω μ- φ Ω ø ~_\ 0 -J ø 03 μ- tr Mi rt rt μ- Φ Cb rt μ μ- tr Hi rt rt μ-

0 μ. Φ 0 03 φ to μ. μ M 0 Hi μ . Φ 0 0 to 0 0 ø μ- 03 0 Hi μ; φ 0 0 ø 0 ø 0 H 0 Φ S3 Φ 0 Ω 0 ø μ μ 0 ø 0 0 μ μ- tr ø ø 0 ø ø μ μ 0 ø

— ø H 0 H 03 CO H 0 tr μ 0 H • 0 S3 μ¹ Hi 3 TJ φ ø rt rt H 03 0 S3 ø H Hi

- 0 ø a μ 0 Φ O t 0 μ- μ- 0 0 3 Φ tr o\° 0 μ. μ- 0 03 0 Φ 3 Φ if o\° ø

N 0 Φ 03 H ø - Ω ø Hi 03 μ- rt rt Cb α 3 μ- 0 rt 03 μ- rt rt ø Φ • 0 - H tr Ω 0 tr Φ o cQ μ. 0 tr ø 0 0 0 ø ø μ μ- • • σ CQ μ. 0 tr ø ø Ω 03 Hi 0 μ- μ-¹ 0 Φ 03 _* tr Hi φ ø CD CD μ μ Φ μ 0 • tr Hi Φ ø

Cb ø φ o φ Φ μ TJ ø Cb 0 CQ •• o rt φ Φ 0 Φ Cb φ Hi ø

§ H 03 0 CO tr μ- Cb 0 g o rt 0

0 H rt φ Ω CO _: 3 0 CQ μ» rt Φ Ω H

13 . — . μP> H 3 μ- 2! 03 φ H CD s o\° 0 0 0 Φ μ. Cb Hi μ- ø μ. o\° 0 0 0 φ

0 a 0 ID 0 tr σi H O Ω Cb φ Hi ø N 3 <! ø 0 H Φ μ ø CQ Ω Hi ø N 3 <! μ 0 0 in H ø ι-3 ~ μ- • 0 Cb μ. rt μ. TJ φ μ 0 rt 0 Ω ø 0 0 rt TJ Φ

0 <! Cb 0 <! μ μp Ω 0 μ • 0 rt 3 0 Φ Cb 0 3 H¹ 0 3 0 rt 3 0

Cb 0 to 03 μ- μ. -J

CO O μ- Ω tr Φ φ 03 μ- ø μ. tr Φ Φ CO

0 N Ω in Φ ø o σi Ω Φ ø CQ CO Φ o Φ < μ- 0 ø s μ_ ø Cb 0 tr o Φ <! μ- 0 , 1 ø -J 03 CQ tr - φ 10 CD μ- μ- 0 . Φ TJ rt Mi 0 0 Φ φ μ Φ ø . Φ TJ rt Hi

3 μ • 0 H μP rt ø μ- H to Ω μ μ- μ X μ μ- CO Cb μ- 03 to Ω μ μ- φ Φ Cb Ω D_ o h-¹ H Hi ø • rt 0 o\° 0 0 0 Hi 3 0 3 ø TJ CQ 0 φ o\° 0 0 o . Hi

_^-, 03 N Ω Φ 0 φ μ¹ 0 O 0 tr ø 3 0 rt ø μ ø 3 ø Φ μ- 03 3 0 rt ø μ

Ω μ? 0 03 μ CO 0 μ^j Ω tr 0 0 TJ Ml Φ o H^{1 •} 3 Ω Ω > ø Φ . — . 0 TJ Hi Φ 0

Φ 3 3 Ω 0 3 ø 0 CQ φ tr 03 Hi 0 μ- 3 H \ μ. td μ- 3 Cb 0 Hi 0 μ-

0 " Φ 3 μ ø ø ø 03 0 0 Φ φ Φ 03 Hi 3 ø TJ μ. ø ; 03 Hi ø TJ

Φ CO 3 Φ μ- μ • Cb φ 0 Ω 3 Φ μ- μ μ o 13 3 μ> H M Ω 0 Φ μ- μ μ o

0 " — μ tr 03 Cb φ μ- Ω 03 0 rt 0 tr Φ . μ- 3; - 0 tr O 0 0 rt 0 tr Φ .

Ω - TJ Ω Φ Ω td α Ω Ω Φ 0 N μ- 3 μ; Hi o ø to CD 03 Φ 0 Cb IS! μ- Hi o

, — .. 3 μ .

0 μ μ- Cb ø 03 co 53 φ H 0 μ- 0 Φ o Ω _t i ID rt φ 3 μ- *-_\ ^ O Φ o μ c? 0 ø μ TJ rjd O 0 rt 3 ø o S3^' μ o 0 Ω 0 en μ 03 μ- rt o g ø o S3 μ o

[ Cb μ-¹ μ- Φ Φ in Ω H 0 ø φ - . Φ 0 σ μ Φ ø - Ω ø μ Φ • φ ø o

H Ω 0 ø Ω - td ID 0 0 μ. tr o μ- tr H TJ 0 CQ 00 μ- μ- ø tr o μ- tr H ø 1 Ω ; tr μ- μf en 03 0 TJ Φ o CQ o\° 0 Φ ø IO ø ø H tr TJ Φ o CQ o\° rt in Φ td Φ ø H H ^*\ ø Φ 0 CD φ μ < H tr μ^ μ ti 3 D Ω H¹ Φ μ <! μ> tr μ.

Φ o Cb 0 ø H D O ω CO CD μ φ 0 Φ o\° rt rt Ω • 0 μ-¹ μ . 0 Φ o\° ct rt μ o <j μ Φ - 10 μP> φ CO Hi rt ø 0 0 rt 0 Hi 0 0 CD rt 0 0 ø tr ø 0 Hi μ. -J ^• — ' O CQ 0 CQ 0 Φ rt 0 rt Φ μ Ω Cb 0 3 0 Φ rt 0 rt

0 ω μ. μ- TJ μ- -0 CO μ φ μ- μ μ- 3 0 Hi to Cb 0 03 Φ μ μ; μ- μ- 3 0 Hi to rt — Φ rt CD oo Hi to TJ Φ 0 ø ø 0 ø o\° , 0 3 . - rt ø 0 ø o\° μ- a 0 0 ø 03 0 μ μ φ Ω 0 μ o rt μ- ti 3 CQ ø ø μ o rt

0 tr ø • μ- S3 0 S3 0 Cb rt H 03 tr Φ . tr H 0 ø Cb Φ H Ml Φ 03 tr tr Φ . ' 0 ø . — . 03 M rt tr 3 tr Cb μ- μ- 0 φ μ. in Φ Φ Hi μ μ- 0 ø Φ H μj in φ Φ Hi

0 « rt Φ TJ td ø μ- μ- 0 03 Ω Cb TJ o\° <! rt rd Ω Ω μ Φ 03 Φ TJ o\° < ø μ 0 X tr Ω to Ω Ω Ω 0 Φ μ- S3 μ Ω Φ φ tr μ- tr rt S3 μ Ω φ Φ

*-' 0 ø Ω rt ø " tr 0 tr Φ H 0 Φ Φ 0 0 0 Φ ø φ Φ μ- μ- Hi Φ φ 0 0 0

. μ- Φ Φ φ 0 Cb o rt μ- Hi Hi 3 N § >. ø X Ω ø 0 μ- Hi H) 3 N ø ø TJ Cb μ. Cb CO μ. tr 0 CQ Φ TJ 0 Cb ³ μ^j μ- ø ø Ω μ CQ φ TJ 0 ; μ-¹ rt Ω μ- μ- Hi φ 0 H¹ tr μ Φ 0 Hi 3 Φ μ; Hi 3 μ-¹ tr • μ Φ O Hi 3

0 0 Φ φ 03 03 μ Cb 3 03 PC rt ø 0 03 Φ rt 0

Ω 0 Ω 0 0 Φ ø tr tsi μ- HI Φ o a TJ H 0 0 rt 0 ø CD Φ

Hi H CD μ-¹ 0 μ *< Cb 03 tr N μ- Hi

0 μ" H 0 3 μ- b H 0 H _ rt μ TJ μ 3 0 ø 0 - 0 ø μ- 0 μ- Hi < <! 3 Φ φ Φ 0 μ. rt μ TJ

0 μ. g μ- 0 μ CQ o 0 μ- - 3 Hi μ. 3 μ- 0 μ

Φ Hi H 03 03 &. Hl Hi ø Φ 0 3 o φ N μ- 03 0 rt μ- Φ o 3 0

CO 0 φ ø φ c? α μ- Φ rt ø rt 0 μ. μ-> - 0 Cb tr 0 rt 0 ø rt

CD O ø a CO Φ tr rt - φ rt 3 0 1 1 0 tr rt Φ

0 φ td Cb μ. Cb φ μ- Φ tr 03 φ μ-

Ω 03 Ω 1 ø ø 03 φ ø ø tr φ

Cellulases are normally incorporated in the detergent composition at a level of from 0.00001% to 2% of enzyme protein by weight of the composition, preferably at a level of from 0.0001% to 1% of enzyme protein by weight of the composition, more preferably at a level of from 0.001% to 0.5% of enzyme protein by weight of the composition, even more preferably at a level of from 0.01% to 0.2% of enzyme protein by weight of the composition.

Peroxidases/Oxidases : Peroxidase enzymes are used in combination with hydrogen peroxide or a source thereof (e.g. a percarbonate, perborate or persulfate) . Oxidase enzymes are used in combination with oxygen. Both types of enzymes are used for "solution bleaching", i.e. to prevent transfer of a textile dye from a dyed fabric to another fabric when said fabrics are washed together in a wash liquor, preferably together with an enhancing agent as described in e.g. WO 94/12621 and WO 95/01426. Suitable peroxidases/oxidases include those of plant, bacterial or fungal origin. Chemically or genetically modified mutants are included. Peroxidase and/or oxidase enzymes are normally incorporated in the detergent composition at a level of from 0.00001% to 2% of enzyme protein by weight of the composition, preferably at a level of from 0.0001% to 1% of enzyme protein by weight of the composition, more preferably at a level of from 0.001% to 0.5% of enzyme protein by weight of the composition, even more preferably at a level of from 0.01% to 0.2% of enzyme protein by weight of the composition.

Mixtures of the above mentioned enzymes are encompassed herein, in particular a mixture of a protease, an amylase, a lipase and/or a cellulase.

The enzyme of the invention, or any other enzyme incorporated in the detergent composition, is normally incorporated in the detergent composition at a level from 0.00001% to 2% of enzyme protein by weight of the composition, preferably at a level from 0.0001% to 1% of enzyme protein by weight of the composition, more preferably at a level from 0.001% to 0.5% of enzyme protein by weight of the composition, even more preferably at a level from 0.01% to 0.2% of enzyme w w to to μ μ o Ul o in o Ul

Cb ø Ω TJ 3 Ω tr φ 0 μP Cb 0 3 3 rt φ tr 0 rt ø CQ rt 0 Cb Ω 0 μ- μ- ω TJ φ Ω 0 μ ø tr Ω H ø 0 - μ- Ω φ ø tr 0 Φ X tr 0 Φ ^μ<ι Ω φ 0 ø ø H¹ μ

CQ Φ 3 φ μ- Φ Ω 0 μ 03 μ- rt CQ φ Ω μ; φ ø ø TJ rt TJ 3 TJ Ω Ω Φ 0

Ω rt r Hi 0 Cb 0J 0 μ. μ> Ω Cb ø ø μ 0 ø CQ Φ μ- μ- Φ TJ ø 0 rt μ μ. μ- φ μ. μ Ω 3 H t H 1 Φ Φ 3 ø φ tr H μ Ω <! ø 0 μ ø 0 O Φ μ- μ-¹ ø μ 0 ø tr TJ 0 * 0 ø Ω 03 0 TJ 0 H μ- 0 ø 0 Cb ø rt Cb Cb t μ- tr Φ 0 m 0 0 ø μ- ø 3 U) 03 ø tr μ- Ml 0 O ø Hi φ μ. μQ μ^J h-> rt μ- Φ μ- Φ Φ H- ø

Φ rt rt tr tr Cb 0 Cb ø 03 ø μ- IO Φ Cb μ^j 0 . 03 ø Ω tr tr 0 r 0 •> 0 ø ø Ω Cb Cb 0

Cb tr μ- Φ H" Cb ø CQ 03 0 ø ifs. Cb 0 3 03 Φ rt φ Ω φ μ- μj μ Q rt tr Cb IQ tr

*< 0 • Φ Cb rt Φ rt 0 . Cb μ Φ μ- Φ tr C tr CQ ra φ μ- μ- μ. μ- H 0 ø; tr ø CQ tr 1 μ- μ- 0 3 0 03 Ω < tr ø μ- tr H tr • ø φ ø rt ø ø Φ μ^ μ> a Φ CQ Φ en K ø TJ 0 μ- 0 ø H Ω ø H Mi Φ Φ TJ Ω 03 ø μ- IQ S

0 S3 Cb 1 0 1 φ rt μ μ- φ TJ ø rt TJ rt rt Φ tr Q φ 0 0 ≤ o ø μ- Ω rt 0 φ q Φ μ- μ in rt tr l ø tr 0 CQ Cj μ Φ 0 ø Φ Φ Φ 0 Φ 0 μ Ω TJ tr 0 0 N tr tr ø ti μ- O Cb rt 0 o\° μ 0 rt φ Ω X tr O 0 μ TJ tr μ CQ Cb Ω l 0 Ω 3 tr μ φ Φ μ- φ 0 rt CQ μ- tr CQ o 0 Cb 03 0 0 H X tr Φ H Ω 0 tr CQ tr 0 μ- Φ ø rt μ- ø H CO tr μP* 0 Φ tr • 3 μ- - tr rt TJ : > μ. Φ μ Φ ø μ 0 μ- 0 Φ μ- ø 03 tr ø 0 CQ Cb rt

- 3 tr ø μ. in 0 0 ø φ Φ - Cb ø 0 μ μ. μ ø 03 0 ø 0 CQ Φ TJ Φ Ω Hi φ 0 μP μ- 1 3 Ml CQ μ TJ μP> o N X φ tr CQ rt CQ rt μ μ-¹ 0 rt TJ 0

H ø φ TJ S3 μ> 0 0 0 0 0 μ 00 Cb 0 X 0 0 ø S3 03 μ- Ω rt Φ tr 0 if Q φ rt Mi to Φ 0 Φ Φ o ø μ CQ μ X φ ω Φ μ- . ø X Hi 0 ø Φ ø 0 0 CD Cb O φ μ μ-

- Ω μ μ- o\° ?c ø Φ μ; μ; Hi - Ω Ω tr 3 μ. ø CQ 0 CQ ø 3 0 Φ Φ φ O ø CQ 0 rt

ID — tr o cq 0 Cb Φ ø Ω φ -J 0 ct TJ H 0 φ 03 Φ 03 TJ rt ø ø 1 rt φ 0 tr ω H_ X tr tr 0 X 0 0 μ 00 0 0 tr H μ- X 0 ø Φ ø - 0 rt Ω 0 oo CD ø 0 φ

0 μ- rt •< φ TJ tr Hi TJ μ H Φ Ω 0 Φ Ω μ. Ω rt 0 Hi rt ø M tr ø o rt μ-¹

^• — ^• td Ω Cb • 0 3 μ φ - Cb μ- 03 CQ rt 03 0 Φ 0 Φ 0 μ- Φ o CD Ω

- α rt Φ S3 03 rt TJ Φ tr 0 Cb μ- Cb 0 0 Φ μ- CD Ω • Cb <! X ø 0 Ω Cb 0

• — ' μ- φ 0 ø 0 μ- a o rt 0 Ω ø <! ø 0 0 CQ 03 Φ μi CQ 0 3 Ω 0 Φ 3 ω -• <! μ μ- 03 Φ Ω φ Ω tr CO μ- Φ Hi μ- ø μ- rt Hi 3 • μ-¹ CQ μ μ- tr 3 rt TJ

- ø Φ CQ TJ CQ » tr ø H Ω Cb t rt rt tr 0 TJ 0 CD Φ ø Ω TJ φ 0 in ø rt μ-¹ tr tr μ- μ- Ω ø Φ ! tr rt Φ ø φ μ 0 CQ μ ø CQ 3 μ ø 0 μ CQ

0 0 Φ ct 0 ø μ- ø tr Ω P> 0 0 Φ tr tr φ Cb tr μ ø μ Φ 0 φ 0 0 CO 03 CQ μ- rt ø μ ø ø 3 ø CQ μ- μ- Ω o Ω 0 X μ- -~ 0 03 Cb Φ ø Hi tr 0 μ ø μ- φ rt μ 0 CO 03 0 0 Ω ø Cb tr μ- 0 ø 03 φ Ω tr Φ Φ ø ø 0 μ-¹ rt Φ CD 13 rt 0 μ- μ- 0 μ- Hi 3 Cb ø CQ μ- μP> Cb μ . tr 0 tr H P? rt rt 0 TJ Hi φ • 03 μ- rt 0

3 0 03 ø μ- μ- 0 CQ ø ø μP H μ . Ω Ω μ- Φ Hi ø φ rt μ 0 Ω 0 μ> 0 ø φ μ; 0 CQ Ct Cb Ω Cb φ 0 D CQ CD 0 Cb tr ø ø 0 0 μ Hi 0 μ- 0 Ω tr X m - 0 μ- rt μ-¹ Ω tr Φ tr Φ ø CQ - CO 3 μ ø μ- cq Ω μ S3 CQ 0 μ O 3 tr o μ. tr 03 ø tr 0 tr ø Φ 03 μ^j rt φ Cb ø 0 μ- ø 03 ø ^{^} ø φ μ ø μ- 03 CQ Φ 13 CQ μ. X CQ • 0 rt 03 ø Φ CQ td Ω ø rt CD CQ ø μ- rt 0 Cb ø 0 ø Φ φ l 03 0 μ μ; 0 Φ Hi μ μ • rt CD φ 13 tr 0 φ CQ ø tr μ- rt 0 Φ H tr CQ ø 0 Φ μp> Hi Φ

1 tr 03 ø μ- co 0 μ- 03 Ω ø 1 *_"• 0 ø Φ CQ Φ ø CD rt 0 Cb

Φ rt ø 0 μ- Ω td μ rt o tr μ Hi CQ ø Ω φ φ ø 0 Ω tr tr 0 rt μ- ø rt 03 μ- Ω 03 tr X rt μ- CO μ> 1 rt Φ rt ø TJ rt 0 μ Cb rt 0 0 0 tr φ

N φ 03 tr 0 H ø tr tr H φ 0 tr 0 ø CQ μ tr 3 tr Q Cb 3 ø φ Φ Cb φ ø φ rt Ω tr Ω 0 3 0 Φ ø lo 0 X φ CQ rt rt Φ Φ φ TJ Φ Φ Φ μ» TJ Φ ø ø rt

0 μ ø Φ 3 μ; μ TJ rt Cb lo Ω 0 φ 03 tr ø 03 0 μ ø Cb \° O Ω Ω J TJ 03 φ 0 0 Cb 0 0 0 CD tr TJ 3 0 0 rt Φ ø 03 Φ rt ø 0 tr tr Φ μ

CQ 1 rt ø φ Ω μ- 0 ω μ- Φ ø rt ø rt . ø μ μ- μ- CQ Ω rt ø μ. μ- μ- μ Φ rt

0 tr TJ Φ ø μ- CD 0 ø in ø μ Q 03 0 t rt rr

Φ ø . 0 0 b ø ø Ω 03 tr

H μ μ μ 03 0 μ- μ CQ 0 ø Cb Ω . μ- 3 03 3 CQ CQ 0J Φ ø

Hi o μ- ø o 0 ø X Φ μ- ØJ μ- 0 Ω TJ ø 0 μ ø rt

0 0 C ø rt Ω Mi tr co Ω ø 0 μ. 03 ø 03 ø 0 ø 0 0 tr S3 μ 0 ø tr rt

0 03 0 Φ μ. φ H ø CQ tr μ- Ω 0 <! D 0 0 0 μ- Φ cQ CQ 0 Ω

0 φ Ω CD 03 0 tr 0 Cb φ 0 0 tr Φ Φ ø M φ φ ø μ- ø rt Cb rt ø τ1 0 _^ Cb ø rt 3 ø ct Φ ø μ- tr 0 rt tr ø ø 0 ø ø

Φ - 0 ø 0 03 en Φ rt μ; Cb l rt ø Φ rt rt rt rt < μ- μ ø μ 0 Φ lo CO 03 μ 03 Φ 0 μ- Ω 03 to φ CD Φ Φ tr

,—. ø φ Cb μ- tr Cb |J σ> μ- 0 O 0 0 in 0 ø φ a Ω 0 _^ ø Ω H" φ ø 0 ø μ- o\° Ω 0 S3 rt

Ω ø a i μ rt Cb Cb μ. ø • tr 0 μ- μ-

03 0 1 μ- in Φ Ω μ- Cb rt 0 O μ rt H 0 ø ø 0 H tr ø

- Φ • Ml ø CQ Hi 0

0J w t t μ μ

Ul o Ul o Ul O Ul w tr Cb μ] r Cb tr tr TJ Ω S3 0 \ TJ 0 0 td Φ tr S3 Φ rt tr tr td 0 Ω ø 0 tr TJ 0 tr

0 H Φ ø- μ. φ H H tr H μ- TJ 0 tr 0 H 13 0 Φ tr ø 0 ø Ω 0 0 Ω H Φ μ Φ o_ φ 03 Φ rt φ Φ rt 0 rt 0 μ 0 0 CD N CQ μ- N φ Φ μ rt TJ 0 rt φ μ X

CQ 0 Ω S3 φ 0 0 tr rt tr 0 rt 1 0 o μj μ- Ω μ; rt 0 ØJ 0 μ- μ 0 μ- 0 ø TJ CD

Ω μ 3 Φ μ Ω Ω 0 ^{^} 0 0 0 g ø tr 3 tr Ω o TJ <! o 0 <! Ω Ω Φ 0

03 tr μ- ø μ- CQ tr tr μ-¹ φ H rt H 0 X PC in ø) ø ø Φ tr tr Φ ø μ. 0 ø tr μ- ø ø

0 μ- tr ø 03 cQ φ μ- Φ 0 03 μ- tr 0 Ω μ. 0 03 ω rt μ- μ- rt Hi μ- μ- ø rt H 3 rt μ- Cb rt 0

TJ ø Φ CQ tr 0 ø Cb Ω CQ Φ 3 rt CQ O -J μ- ø 03 μ- tr μ- ø ø 03 ø 0 ^• — ' μ- 0 ø ø

•0 Q Cb ØJ φ t rt CQ μ; 0 tr μ- μ- Φ S3 Φ Ω CQ Ω Φ ø CQ CQ φ μ Cb μ CQ 0J ø o

H ø ø •. 0 0 rt CD ø < ø 0 0 ω Ω <! Hi 13 03 0 0 03 03 Ω X

Φ «. μ- Φ Ω Ω TJ 13 ø rt - 0 μ- ø Ω 00 03 0 ø 03 tr Φ Ω 03 0 0 X φ Φ ø μ; ø 0 "≤.

03 03 tr 0 0 μ μ μ- tr 0 rt tr μ- tr H μ; μ TJ ^ rt 0 μ; Ω Ω Hi rt rt tr

CQ a Φ μ- Hi 3 0 φ ø rt μ- 03 3 Φ ø μ- 03 0 03 Cb rt 3 ui Φ μ tr ø oo i-h tr μ. Φ

0 0 — ø TJ Ω Hi Φ 0 ø rt Cb Φ ø rt Cb tr rt μ μ- TJ ct tr ø Φ Φ TJ . — . φ Φ

H rt td Ω c 03 0 Φ Φ μ TJ 0 rt CQ φ ø H Φ 0 0 0 φ H¹ rt μ on Ω CQ N

03 0 Hi 0 0 03 CQ μ μ- Cb rt ø tr tr Ω tr 3 μ Φ 3 CQ 0 ø 3 φ 0 N 0 1 rt 0 H φ μ Hi rt ø H μ- CD μ 03 μ tr rt ct μ-¹ tr Φ 0 03 μ- φ 0 CQ 0 > Ω Φ μ. 0 • Ω ø 0

Φ μ- ø CQ Hi rt φ μ; φ Φ tr φ μ- CQ ø o _^-, 0 ø Cb Ω TJ μ. 0 μ-¹ Ω rt Ω φ

Ω Φ 0 μ- 0 Cb 0 ø ø ø ø φ 0 cQ Hi μ- μ 03 Ω tr TJ μ-¹ Φ ^• — rt H μ- 0 03 ω μ- μ. 0 0 0 μ Ω μ- TJ Cb H Ω CQ μ 0 μ • TJ Φ 0 μ- 0 03 ø 0 03 0 en Φ 03 rt 0 ø Φ N rt ø μ ø 0 tr rt rt Φ rt CQ φ Φ Hi 3 ø μ- rt 0 0 0 Φ Φ

<-_ ø rt ra rt μ- μ- Φ μ Ω μ- 0 CD tr Φ . μ Cb 0 TJ CQ Ω Φ H X ø 0 Hi

^ rt φ S3 Cb ø <! rt 03 μ- ; ø CQ ø Cb φ 0 0 Φ μ μ 0 Cb Hi 3 C 0

X 03 μ- ø rt 0 " b tr _^-_^

3 3 Cb μ- φ Ω ø tr Φ ø 0 CQ φ ø 0 μ- Φ ø tr μ- Cb 13 0 μ> 3 0 ø CQ rt φ ø CQ ø ø Cb rt 03 S3 μ ø μ- Ω 0 03 CQ μ- Ω 0 Φ Cb μ- Φ ! 0

KO ø j μ; N μ-¹ Ω TJ Φ Ω μ- ø rt tr Ω ø ø Cb μ 03 μ- Φ 0 μ- 0 ø o rt 0 Ω rt

ID ø μ- μ tr Cb Cb φ rt 0 CQ Ω φ μ" CQ rt Φ Φ μ- Φ ø ø 0 rt rt N 1 μ- Ω ^*—-' Φ

CQ ø Ω μ- rt ø tr φ Φ 0 0 μ 0 tr μ- ø tr CQ rt μ Φ Φ Ω 0 tr

- ø φ μ-¹ Ω 0 tr tr ø ; O Φ 03 ø Hi 0 03 μ- o N 3 Φ μ- 03 a 0 0 ø 0 μ- -— » ø • 03 ø φ ø ø Hi rt rt φ ø ø μ: ø Cb ø tr -• 0 rt 0 Φ TJ *- ø S3 H

TJ Φ CQ 0 TJ rt - Cb μ- S3 03 TJ tr tr Cb CQ 3 μ. • Φ μ 03 μ CQ tr o

TJ 03 tr ø 0 CQ 0 ø ι-3 ø Φ 0 0 Φ μ- Ω φ μ- 0 0 <! μ- Ω

Φ Ω rt μ- μ- Ω tr X CO tr 03 μ ø μ- ø Hi ø H" ø ø KD Φ 3 μ Φ 03 Ω a

0 tr ø ø μ; Ω rt μ. tr Φ ø rt 0 ø ø ø H Φ Ω Ω μ> CO μ- Hi μ-¹ μ TJ tr Ω en Ω tr M 0 0 0 - CQ μ- 03 Ω μ- rt 0 Cb tr 03 ø 0 tr oo rt X O — μ. φ bd

10 ø Φ TJ H 0 ø ø Φ ø Φ tr Ω μ- >_ td "\ μ. Cb 0 TJ 0 0 -j Φ rt 0 Ω 0 CO

-j rt μ 0 tr O μ- 03 rt ø CQ ø H μ; 0 0 Cb TJ μ- 0 Cb o μ ø 0 0 03 μ- μ -

1 ø 0 μ- Ω 0 ø φ tr 3 ø μ- Q μ μ μ 0 tr ø Ω μ- to CQ μ rt X 0 φ Φ en H ø 03 μ. 0 μ Φ μQ Φ TJ ø 03 0 N Φ 0 0 Φ μ- CQ rt ø o Φ Φ '≤ μ- CO Cb

10 μ. Φ Φ rt ~ ø 03 μ ft μ Φ TJ μ iQ 03 Ω μ- iQ O 03 tr rt - TJ Φ

ID CO ø O ø φ 03 0 0 Φ rt Cb Φ μ- Φ 0 TJ ø Ω <! • 0 ø Φ ø Φ 03

. rt 0 0 μ- o 10 ø 0 0 Ω Ω μ tr μ- tr ø ø tr μ rt 0 ø TJ l Ω rt ø ø tr H μ Ω

CO Hi ø • ω Cb rt tr φ μ- Φ ø tr ø 03 03 Φ μ- 3 rt Φ . tr tr Cb N H Φ tr μ

3 φ o - Hi CQ ø rt φ φ μ- TJ rt 03 0 TJ 0 μ φ Φ Φ 0 μ. μ-

Hi rt ø to -j 0 μ; O ø 03 μ^j 03 Φ μ ø 13 ø Φ μ Φ 0 0 μ 0 0 μ Ω 0 Cb Cb tr

0 tr ø m H 0 03 . 03 0 μ Φ Ω ø CQ μ ø ø CQ 03 03 Φ μ> Φ ø μ- μ Φ μ Φ CQ o\° CO TJ 0 -~ Φ μ- tr rt 0 rt rt α μ- μ; 0 o 1 μ ø 0 Cb

0 . tr rt rt tr Ω Hi 03 ø μ- φ TJ X Φ CO rt ø 0 Cb Hi 03 Φ CQ μ-¹

H Ω ø rt 0 tr Φ μ. TJ 0 0 rt ø ø μ μ- tr CO μ- ø Ω μ- • ^~. 0 μ; μ-

0 0 φ O m rt Φ Cb 0 ø ø CQ rt 0 Cb rt μ. a 0 Cb μ- 03 en H rt rt N ø

S3 3 CO 3 0 ø- ø ø μ- o Ω Φ tr ø Cb Ω 1 Hi tr O Φ

1 TJ φ ø 0J 03 N 0 tr rt ø ø ø > φ φ o 03 TJ 03 Cb 0 Φ Cb td rt 0 tr μ- Ω 0 μ- ø μ- Φ Φ Ω Φ CQ TJ CQ ø μ Cb oo φ 0 CO φ 0 μ- 13 φ 0 Ω 0 Ω rt tr ø CQ μ Cb Φ TJ CQ rt φ Cb ^ 0 μ 0 03 0 Ω 0 tr rt

3 ø 0 0 ø μ- 03 Ω μ- μ Cb ø rt ø • Hi μ- H Ω o 0 φ ø rt TJ 0 H

TJ Cb rt rt h-¹ <! rt ø 0 Φ N Cb ^ rt μ- rt O ø ω Ω X Cb Cb 03 ø Φ φ μ to

Φ 03 ø ø μ CQ Cb TJ μ- Φ TJ CQ Ω O tr μ CQ en 0 μ. 0 ø - ø 0 Hi o μ μ> μ. rt 0 μ- 0 ø 03 Φ 0 0 Φ Φ - μ CQ μ- μ- 3 Ω < 0 ø μ; . Φ rt ø 03 Ω ø rt Ω — ø en Cb Φ ø rt μ- Ω tr Φ μ σi rt CQ to Cb φ rt μ- 0 μ μ- tr ø to μ- ø ø Cb ID Cb 3 D ø rt iπ μ- rt 0 Hi Φ 0 en 0 tr 0 _^-_^ μ> μ • o\° μ- 0 φ 0 ø • iQ t-¹ cn 0

Φ - 03 ø Cb Cb φ 1

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 divided forms exemplified by silica aerogels and xerogels and hydrophobic silicas of various types. Theses materials can be incorporated as particulates, in which the suds suppressor is advantageously releasably incorporated in a water-soluble or waterdispersible, substantially non surface-active detergent impermeable carrier. Alternatively the suds suppressor can be dissolved or dispersed in a liquid carrier and applied by spraying on to one or more of the other components.

A preferred silicone suds controlling agent is disclosed in US 3,933,672. Other particularly useful suds suppressors are the self-emulsifying silicone suds suppressors, described in German Patent Application DTOS 2,646,126. An example of such a compound is DC-544, commercially available form Dow Corning, which is a siloxane-glycol copolymer. Especially preferred suds controlling agent are the suds suppressor system comprising a mixture of silicone oils and 2-alkyl-alkanols . Suitable 2 -alkyl- alkanols are 2-butyl-octanol which are commercially available under the trade name Isofol 12 R.

Such suds suppressor system are described in European Patent Application EP 0 593 841. Especially preferred silicone suds controlling agents are described in European Patent Application No. 92201649.8. Said compositions can comprise a silicone/ silica mixture in combination with fumed nonporous silica such as Aerosil^R.

The suds suppressors described above are normally employed at levels of from 0.001% to 2% by weight of the composition, preferably from 0.01% to 1% by weight.

Other components

Other components used in detergent compositions may be employed such as soil-suspending agents, soil-releasing agents, optical brighteners, abrasives, bactericides, tarnish inhibitors, coloring agents, and/or encapsulated or nonencapsulated perfumes . Especially suitable encapsulating materials are water soluble capsules which consist of a matrix of polysaccharide and polyhydroxy compounds such as described in GB 1,464,616.

Other suitable water soluble encapsulating materials comprise dextrins derived from ungelatinized starch acid esters of substituted dicarboxylic acids such as described in US 3,455,838. These acid-ester ^■ dextrins are, preferably, prepared from such starches as waxy maize, waxy sorghum, sago, tapioca and potato. Suitable examples of said encapsulation materials include N-Lok manufactured by National Starch. The N-Lok encapsulating material consists of a modified maize starch and glucose. The starch is modified by adding monofunctional substituted groups such as octenyl succinic acid anhydride. Antiredeposition and soil suspension agents suitable herein include cellulose derivatives such as methylcellulose, carboxymethylcellulose and hydroxyethylcellulose, and homo- or co-polymeric polycarboxylic acids or their salts . Polymers of this type include the polyacrylates and maleic anhydride-acrylic acid copolymers previously mentioned as builders, as well as copolymers of maleic anhydride with ethylene, methylvinyl ether or methacrylic acid, the maleic anhydride constituting at least 20 mole percent of the copolymer. These materials are normally used at levels of from 0.5% to 10% by weight, more preferably form 0.75% to 8%, most preferably from 1% to 6% by weight of the composition.

Preferred optical brighteners are anionic in character, examples of which are disodium 4, 4 ' -bis- (2-diethanolamino-4- anilino -s- triazin-6-ylamino) stilbene-2 :2 ' disulphonate, disodium 4, - 4 ' -bis- (2-morpholino-4-anilino-s-triazin-6- ylamino-stilbene-2 : 2 ' - disulphonate, disodium 4,4' - bis- (2,4- dianilino-s-triazin-6-ylamino) stilbene-2 : 2 ' - disulphonate, monosodium 4 ',4'' - bis- (2 , -dianilino-s-tri-azin-6 ylamino) stilbene-2-sulphonate, disodium 4,4' -bis- (2-anilino-4- (N-methyl-N-2-hydroxyethylamino) -s-triazin-6-ylamino) stilbene- 2,2' - disulphonate, di-sodium 4,4' -bis- (4-phenyl-2, 1, 3- triazol-2-yl) -stilbene-2, 2 ' disulphonate, di-so-dium 4,4'bis(2- anilino-4- (1-methyl-2-hydroxyethylamino) -s-triazin-6-ylami- no) stilbene-2, 2 'disulphonate, sodium 2 (stilbyl-4 ' ' - (naphtho- 1' ,2 ' -.4,5) -1,2, 3, - triazole-2 ^•' -sulphonate and 4,4'-bis(2- sulphostyryl ) biphenyl .

Other useful polymeric materials are the polyethylene glycols, particularly those of molecular weight 1000-10000, more particularly 2000 to 8000 and most preferably about 4000. These are used at levels of from 0.20% to 5% more preferably from 0.25% to 2.5% by weight. These polymers and the previously mentioned homo- or co-polymeric poly-carboxylate salts are valuable for improving whiteness maintenance, fabric ash deposition, and cleaning performance on clay, proteinaceous and oxidizable soils in the presence of transition metal impurities.

Soil release agents useful in compositions of the present invention are conventionally copolymers or terpolymers of terephthalic acid with ethylene glycol and/or propylene glycol units in various arrangements. Examples of such polymers are disclosed in US 4,116,885 and 4,711,730 and EP 0 272 033. A particular preferred polymer in accordance with EP 0 272 033 has the formula :

(CH₃ (PEG) _«) ₀._7S (POH) ₀.₂₅ [T-PO) ₂.₈ (T-PEG) ₀.₄] T (POH) ₀.₂₅ ( (PEG) ₄₃CH₃) _..₇₅

where PEG is -(OC₂H₄)0-, PO is (OC₃H₆0) and T is (pOOC₆H₄CO) .

Also very useful are modified polyesters as random copolymers of dimethyl terephthalate, dimethyl sulfoisophthalate, ethylene glycol and 1, 2-propanediol, the end groups consisting primarily of sulphobenzoate and secondarily of mono esters of ethylene glycol and/or 1, 2-propanediol . The target is to obtain a polymer capped at both end by sulphobenzoate groups, "primarily", in the present context most of said copolymers herein will be endcapped by sulphobenzoate groups. However, some copolymers will be less than fully capped, and therefore their end groups may consist of monoester of ethylene glycol and/or 1, 2-propanediol, thereof consist "secondarily" of such species. The selected polyesters herein contain about 46% by weight of dimethyl terephthalic acid, about 16% by weight of 1,2- propanediol, about 10% by weight ethylene glycol, about 13% by weight of dimethyl sulfobenzoic acid and about 15% by weight of sulfoisophthalic acid, and have a molecular weight of about 3.000. The polyesters and their method of preparation are described in detail in EP 311 342.

Softening agents

Fabric softening agents can also be incorporated into laundry detergent compositions in accordance with the present invention. These agents may be inorganic or organic in type. Inorganic softening agents are exemplified by the smectite clays disclosed in GB-A-1 400898 and in US 5,019,292. Organic fabric softening agents include the water insoluble tertiary amines as disclosed in GB-A1 514 276 and EP 0 011 340 and their combination with mono C₁₂-C₁₄ quaternary ammonium salts are disclosed in EP-B-0 026 528 and di-long-chain amides as disclosed in EP 0 242 919. Other useful organic ingredients of fabric softening systems include high molecular weight polyethylene oxide materials as disclosed in EP 0 299 575 and 0 313 146.

Levels of smectite clay are normally in the range from 5% to 15%, more preferably from 8% to 12% by weight, with the material being added as a dry mixed component to the remainder of the formulation. Organic fabric softening agents such as the water-insoluble tertiary amines or dilong chain amide materials are incorporated at levels of from 0.5% to 5% by weight, normally from 1% to 3% by weight whilst the high molecular weight polyethylene oxide materials and the water soluble cationic materials are added at levels of from 0.1% to 2%, normally from 0.15% to 1.5% by weight. These materials are normally added to the spray dried portion of the composition, although in some instances it may be more convenient to add them as a dry mixed particulate, or spray them as molten liquid on to other solid components of the composition.

Polymeric dye-transfer inhibiting agents The detergent compositions according to the present invention may also comprise from 0.001% to 10%, preferably from 0.01% to 2%, more preferably form 0.05% to 1% by weight of polymeric dye- transfer inhibiting agents. Said polymeric dye- ω w t to μ μ

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3 ø Ω rt = Ω CQ CQ Cb to tr rt μ. tr ø ø CQ 0 0 μ- 0 μ^ 0 tr tr 03 rt

03 Φ ø = O μ- tr Ω 0 μ-

=: 3 φ 0 Φ Ω 3 Φ 0 iQ 03 Φ μ Hi Cb rt ø μ) μ ø 0 0 a 3 rt 03 Φ μ μ- μ-

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• 0 μ μ 0 ø Ω ø CQ μ μ μ- t 0 0 μ μp 0 Ω Q Ω 03 ø rt Φ μ- ø ø

TJ O CQ CQ Ω o Ω μ- μ- μ- μ- rt o rt ø id μ- rt _^ 0 CQ 0 tr μ.

a 0 Ω 0 0 03 CQ

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Φ rt rt rt μ- 0 Ω φ Hi 0 Ω 03 03 0 TJ 03 H 0 03 0 φ 0 Cb 3 X Φ φ ^ 0 ø rt

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- μ 9;* 0 μ μ- 03 ø H"

0 rt φ μ- rt ø ø tr μ μ- 0 CQ 0 tr 0 0 μ ø Cb Φ μ- μ- ø tr tr μ-¹

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• μ . Ω ø - — - μ- 0 3 03 0 μ- μ- 0 O

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present invention can also be in "concentrated form", in such case, the liquid detergent compositions according to the present invention will contain a lower amount of water, compared to conventional liquid detergents. Typically, the water content of the concentrated liquid detergent is less than 30%, more preferably less than 20%, most preferably less than 10% by weight of the detergent compositions.

The compositions of the invention may for example, be formulated as hand and machine laundry detergent compositions including laundry additive compositions and compositions suitable for use in the pretreatment of stained fabrics, rinse added fabric softener compositions, and compositions for use in general household hard surface cleaning operations and dishwashing operations . The following examples are meant to exemplify compositions for the present invention, but are not necessarily meant to limit or otherwise define the scope of the invention. In the detergent compositions, the abbreviated component identifications have the following meanings :

LAS: Sodium linear C₁₂ alkyl benzene sulphonate

TAS : Sodium tallow alkyl sulphate

XYAS : Sodium C_1X - C_1Y alkyl sulfate

SS: Secondary soap surfactant of formula 2-butyl octanoic acid

25EY: A C₁₂ - C₁₅ predominantly linear primary alcohol condensed with an average of Y moles of ethylene oxide 45EY: A C₁₄ - C₁₅ predominantly linear primary alcohol condensed with an average of Y moles of ethylene oxide XYEZS : C_1X - C_1Y sodium alkyl sulfate condensed with an average of Z moles of ethylene oxide per mole

Nonionic : C₁₃ - C₁₅ mixed ethoxylated/propoxylated fatty alcohol with an average degree of ethoxylation of 3.8 and an average degree of propoxylation of 4.5 sold under the tradename Plurafax LF404 by BASF Gmbh

CFAA: C₁₂ - C₁₄ alkyl N-methyl glucamide TFAA: C₁₆ - C₁₈ alkyl N-methyl glucamide Silicate: Amorphous Sodium Silicate (Si0₂:Na₂0 ratio = 2.0) NaSKS-6: Crystalline layered silicate of formula d-Na₂Si₂O_s

Carbonate : Anhydrous sodium carbonate

Phosphate : Sodium tripolyphosphate

MA/AA: Copolymer of 1:4 maleic/acrylic acid, average molecular weight about 80,000

Polyacrylate : Polyacrylate homopolymer with an average molecular weight of 8,000 sold under the tradename PA30 by BASF

GmbH

Zeolite A: Hydrated Sodium Aluminosilicate of formula Na₁₂ (Al0₂Si0₂) ₁₂. 27H₂0 having a primary particle size in the range from 1 to 10 micrometers

Citrate: Tri-sodium citrate dihydrate

Citric: Citric Acid

Perborate: Anhydrous sodium perborate monohydrate bleach, empirical formula NaB0₂.H₂0₂

PB4 : Anhydrous sodium perborate tetrahydrate

Percarbonate : Anhydrous sodium percarbonate bleach of empirical formula 2Na₂C0₃.3H₂0₂

TAED : Tetraacetyl ethylene diamine CMC: Sodium carboxymethyl cellulose

DETPMP : Diethylene triamine penta (methylene phosphonic acid) , marketed by Monsanto under the Tradename Dequest 2060

PVP: Polyvinylpyrrolidone polymer

EDDS: Ethylenediamine-N, N¹ -disuccinic acid, [S,S] isomer in the form of the sodium salt

Suds Suppressor: 25% paraffin wax Mpt 50 °C, 17% hydrophobic silica, 58% paraffin oil

Granular Suds suppressor: 12% Silicone/silica, 18% stearyl alcohol, 70% starch in granular form Sulphate: Anhydrous sodium sulphate

HMWPEO: High molecular weight polyethylene oxide

TAE 25: Tallow alcohol ethoxylate (25)

Detergent Example I

A granular fabric cleaning composition in accordance with the invention may be prepared as follows : Sodium linear C₁₂ alkyl 6.5 benzene sulfonate

Sodium sulfate 15.0

Zeolite A 26.0 Sodium nitrilotriacetate 5.0

Enzyme of the invention 0.1

PVP 0.5

TAED 3.0

Boric acid 4.0 Perborate 18.0

Phenol sulphonate 0.1

Minors Up to 100

Detergent Example II A compact granular fabric cleaning composition (density 800 g/1) in accord with the invention may be prepared as follows :

45AS 8.0

25E3S 2.0

25E5 3.0 25E3 3.0 TFAA 2.5

Zeolite A 17.0 NaSKS-6 12.0

Citric acid 3.0

Carbonate 7.0

MA/AA 5.0

CMC 0.4

Enzyme of the invention 0.1

TAED 6.0

Percarbonate 22.0

EDDS 0.3

Granular suds suppressor 3.5 water/minors Up to 100%

Detergent Example III

Granular fabric cleaning compositions in accordance with the invention which are especially useful in the laundering of coloured fabrics were prepared as follows

LAS 10.7 -

TAS 2.4 -

TFAA - 4.0

5 45AS 3.1 10.0

45E7 4.0 -

25E3S - 3.0

68E11 1.8 -

25E5 - 8.0

10 Citrate 15.0 7.0

Carbonate - 10

Citric acid 2.5 3.0

Zeolite A 32.1 25.0

Na-SKS-6 - 9.0

15 MA/AA 5.0 5.0

DETPMP 0.2 0.8

Enzyme of the invention 0.10 0.05

Silicate 2.5 -

Sulphate 5.2 3.0

20 PVP 0.5 -

Poly (4-vinylpyridine) -N- - 0.2

Oxide/copolymer of vinyl- imidazole and vinyl- pyrrolidone

25 Perborate 1.0 -

Phenol sulfonate 0.2 -

Water/Minors Up to 100%

Detergent Example IV

30 Granular fabric cleaning compositions in accordance with the invention which provide "Softening through the wash" capability may be prepared as follows :

45AS - 10.0

LAS 7.6

35 68AS 1.3

45E7 4.0

25E3 - 5.0

Coco-alkyl-dimethyl hydroxy- 1.4 1.0 ethyl ammonium chloride

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

Perborate 15.0 -

Percarbonate - 15.0

TAED 5.0 5.0 Smectite clay 10.0 10.0

HMWPEO - 0.1

Enzyme of the invention 0.10 0.05

Silicate 3.0 5.0

Carbonate 10.0 10.0 Granular suds suppressor 1.0 4.0

CMC 0.2 0.1

Water/Minors Up to 100%

Detergent Example V Heavy duty liquid fabric cleaning compositions in accordance with the invention may be prepared as follows

I II

LAS acid form - 25.0

Citric acid 5.0 2.0

25AS acid form 8.0 -

25AE2S acid form 3.0 -

25AE7 8.0 -

CFAA 5 -

DETPMP 1.0 1.0

Fatty acid 8 -

Oleic acid - 1.0

Ethanol 4.0 6.0

Propanediol 2.0 6 . 0

Enzyme of the invention 0.10 0.05

Coco-alkyl dimethyl - 3.0 hydroxy ethyl ammonium chloride Smectite clay - 5.0

PVP 2.0

Water / Minors Up to 100%

THE XYLOGLUCAN SUBSTRATE

In addition to the aforesaid about xyloglucan it should be noted that xyloglucan from tamarind seeds supplied by Megazyme, Ireland has a complex branched structure with glucose, xylose, galactose and arabinose in the ratio of 45:36:16:3. Accordingly, it is strongly believed that an enzyme showing catalytic activity on this xyloglucan also has catalytic activity on other xyloglucan structures from different sources (angiosperms or gymnosperms) .

MATERIALS AND METHODS Strains

Jonesia sp . , DSM 14140, producing the xyloglucanase of the invention.

Other strains

E. coli hosts: XLl-lue MRF- and XL0LR E. coli strains were provided by Stratagene Inc . (USA) and used according to the manufaturer' s instructions.

Plasmids

PBK-CAMV: Stratagene Inc., La Jolla, California, USA. Bacteriophage ZAP Express: Stratagene Inc., La Jolla, California, USA.

Media

TY (Ausubel, F. M. et al . (Eds.) "Current protocols in Molecular Biology". John Wiley and Sons, 1995) .

LB agar (Ausubel, F. M. et al . (Eds.) "Current protocols in Molecular Biology". John Wiley and Sons, 1995) . LBPG is LB agar supplemented with 0.5% Glucose and 0.05 M potassium phosphate, pH 7.0

AZC -xyloglucan is added to LBPG-agar to 0.5 %. AZCL- xyloglucan is from Megazyme, Ireland. BPX media is described in EP 0 506 780 (WO 91/09129) . NZY agar (per litre) 5 g of NaCl, 2 g of MgS04 , 5 g of yeast extract, 10 g of NZ amine (casein hydrolysate) , 15 g of agar; add de-ionized water to 1 litre, adjust pH with NaOH to pH 7.5 and autoclave

NZY broth (per litre) 5 g of NaCl, 2 g of MgS04, 5 g of yeast extract, 10 g of NZ amine (casein hydrolysate) ; add deionized water to 1 litre, adjust pH with NaOH to pH 7.5 and autoclave NZY Top Agar (per litre) 5 g of NaCl, 2 g of MgS04 , 5 g of yeast extract, 10 g of NZ amine (casein hydrolysate), 0.7 % (w/v) agarose; add de-ionized water to 1 litre, adjust pH with NaOH to pH 7.5 and autoclave .

Xyloglucanase assay (XyloU)

The xyloglucanase activity is measured using AZCL- xyloglucan from Megazyme, Ireland, (htt : //www. megazyme . com/purchase/index. html) as substrate .

A solution of 0.2 % of the blue substrate is suspended in a 0.1 M phosphate buffer pH 7.5 under stirring. The solution is distributed under stirring to 1.5 ml Eppendorf tubes (0.75 ml to each) , 50 μl enzyme solution is added and they are incubated in an Eppendorp Thermomixer model 5436 for 20 minutes at 40 °C with a mixing of 1200 rpm. After incubation the colored solution is separated from the solid by 4 minutes centrifugation at 14,000 rpm and the absorbance of the supernatant is measured at 600 nm.

One XyloU unit is defined as the amount of enzyme resulting in an absorbance of 0.24 in a 1 cm cuvette at 600 nm.

Isolation of Jonesia sp. , DSM 14140

An amount of marine sediment the size of a small pea was distributed on a minimal agar medium at pH 9 with 1% xyloglucan as C source and 0.025% AZCL-xyloglucan for xyloglucanase detection. The plate was incubated at 30°C for 4 days. A yellow colony with xyloglucanase activity was selected and identified as a Jonesia sp . and deposited as DSM 14140.

General molecular biology methods DNA manipulations and transformations were performed using standard methods of molecular biology (Sambrook et al . (1989) Molecular cloning: A laboratory manual, Cold Spring Harbor lab. Cold Spring Harbor, NY; Ausubel, F. M. et al . (eds.) "Current protocols in Molecular Biology". John Wiley and Sons, 1995; Harwood, C. R. , and Cutting, S. M. (eds.) "Molecular Biological Methods for Bacillus". John Wiley and Sons, 1990) .

Enzymes for DNA manipulations were used according to the specifications of the suppliers.

The following examples illustrate the invention.

EXAMPLE 1

Cloning of xyloglucanase encoding genes from Jonesia

Genomic DNA preparation

The strain Jonesia sp . , DSM 14140, was propagated in liquid TY medium. After 16 hours incubation at 30 °C and 300 rpm, the cells were harvested, and genomic DNA was isolated by the method describe by Duffner, F., Fitzsim ons, A., Brophy, G., O'Kiely, P, O'Connell, M. Journal Appl . Bact . 76: 583-591 (1994) "Dominance of Lactobacillus plantarum strains in grass silage as demonstrated by a novel competition assay".

Genomic library construction

A Lambda ZAP library was prepared from genomic DNA of the strain Jonesia sp . , DSM 14140. The ZAP Express cloning kit used was with BamHI digested and dephosphorylated arms from Stratagene. Isolated DNA was partially digested with Sau3A and size fractionated on a 1% agarose gel. DNA was excised from the agarose gel between 3 and 10 Kb and purified using Qiaspin DNA fragment purification procedure (Qiagen GmbH) . 100 ng of purified, fractionated DNA was Iigated with 1 μg of BamHI dephosphorylated ZAPexpress vector arms (4 degrees overnight) . Ligation reaction was packaged directly with GigaPacklll Gold according to the manufacturers instructions (Stratagene) . Phage libraries were titered with XLlblue mrf" (Stratagene) . Screening for xyloglucanase clones by functional expression in lambda ZAP Express

Approximately 10,000 plaque-forming units (pfu) from the genomic library were plated on NZY-agar plates containing 0.1 % AZCL-xyloglucan (MegaZyme, Ireland) , using E. coli XLl-Blue MRF' (Stratagene, USA) as a host, followed by incubation of the plates at 37 °C for 24 hours. Xyloglucanase-positive lambda clones were identified by the formation of blue hydrolysis halos around the positive phage clones. These were recovered from the screening plates by coring the TOP-agar slices containing the plaques of interest into 500 μl of SM buffer and 20 μl of chloroform. The xyloglucanase-positive lambda ZAP Express clones were plaque-purified by plating an aliquot of the cored phage stock on NZY plates containing 0.1 % AZCL-xyloglucan as above. Single xyloglucanase-positive lambda clones were cored into 500 μl of SM buffer and 20 μl of chloroform, and purified by one more plating round as described above.

Single-clone in vivo excision of the phagemids from the xyloglucanase-positive lambda ZAP Express clone

E. coli XLl-Blue cells (Stratagene, La Jolla Ca.) were prepared and resuspended in lOmM MgS04 as recommended by

Stratagene (La Jolla, USA) . 250μl aliquots of the pure phage stocks from the xyloglucanase-positive clone was combined in Falcon 2059 tubes with 200μl of XLl-Blue MRF' cells (OD600=1.0) and >10^ε pfu/ml of the ExAssist M13 helper phage (Stratagene) , and the mixture was incubated at 37 °C for 15 minutes. 3 ml of NZY broth was added to the tube and the tube was incubated at 37 °C for 2.5 hours. The tube was heated at 65 °C for 20 minutes to kill the E. coli cells and bacteriophage lambda, the phagemids being resistant to heating. The tube was spun at 3000 rpm for 15 minutes to remove cellular debris and the supernatant was decanted into a clean Falcon 2059 tube. Aliquots of the supernatant containing the excised single-stranded phagemids were used to infect 200μl of E. coli XLOLR cells (Stratagene, OD600=1.0 in lOmM MgS04) by incubation at 37 °C for 15 minutes. 350μl of NZY broth was added to the cells and the tubes were w t t μ μ in o in o Ul o Ul l- rt z a TJ α Ω rt ι-3 TJ ω 0 μ-

0 Φ 0 μ a O tr tr ^3 ø ø

3 μ 03 0 Φ > Φ φ 0 rt Ω μ- 3 H TJ 0 rt Ω 0 0 μ- Cb (D 0 z ø Hi X Φ tr ^μ. HI ø φ ι-3 0 μ ø μ- 0 μ . 03 tr¹ 0 ø¹ ø rt - rt 0 03 Φ μ O to rt

-o μ- rt Φ Φ H- rt 03 3 0 Ω μ- Φ μ- μ f μ- μ- . 0 iQ 0 ø C α

• 0 3 ø (D 0 03 rt ø iQ 0 ø ø μ- ø ø o > μ- 0 rt ø Hi

Φ ø Ω CQ H μ-¹ 0 Ω ø Φ ø o t 3 Φ H (D PC 0 0 0 μ- 3 μ

Φ Cb Φ μ- rt

^■a 0 ø Ω j

X rt 0 rt φ s; 0 0 μ- o Ω μ . - Hi rt α> Cb φ Hi 03 ø μ^j μ- in

H 0 μ μ- P _^-. μ Φ CQ o ø

0 Cb 0 Cb o lO Hi Φ tr ø 3

CQ 3 Φ Φ μ- μ TJ o μ- 0 μ-

H ^^ 0 0 μ- 0 0 > Φ CQ ø

0 CO tr 03 0 CQ 3 Cb Φ 03 μ> O ø

Ω 0 0 Φ 0 Φ φ μ- μ 0J

0 0 rt μQ 0 _^ td 0 ø- rt oV> s; rt

0 CQ tr 0 H rt μ; μ- Φ TJ

0 Φ φ μ. Ω μ- Cb < > μ ω

03 μ Ul 0 CQ ø 0 Ω μ φ N φ ø ι

Φ ** ct Ω H- ø ø 0 O rt o μ Φ m Cb μ. TJ tr¹ μ φ O tr Ά 0 μ . ø" Φ 03

Φ . ø 0 Cb X ø 03 0 X

H Cb Hi Φ Kj ø μ- CQ μ. 03 ø

0 CQ 03 Ω Cb 03 Φ rt ø

0 a rt μ- μ> 3 0 μ Cb μ-

CQ μ- tr ø- CQ o Ω tr μ- CQ φ μQ

03 Ω μ. Φ ø to ø 0 Cb H 0 μ- 0 C ø o 0 PC ø o rt rt CQ rt 0 Ω Ω φ Ω rt

0 φ ø' Φ Φ ø Φ 03 H 0 Cb 0 ω ø Φ ø Cb 03 Cb 0 0 tr rt - 0 H- 0 0 0 ø 0 tr Cb 3 TJ 0 td μ φ 0 rt Hi

Φ O μ- μ- X CQ 0 03 2 rt Φ

• Cb Ω KJ α ø Φ 0 Cb rt

CQ - Φ Ω lO 0 ø CQ ^{^}

H 0 X H μ- Cb φ ø f Hi Φ μ. 0 X μ; o 0 μ. μ N td 0

Ω 0 μ . t TJ rt φ *__ μ Ω

0 Cb 0 o 03 X tr 3 C Φ

03 Ω CQ . μ- K! Φ Ω Φ ø K3 H μ. o tr ø Ω tr •> ø

0 ø 0 H TJ ø ø CO

0 μ- Ω TJ o 0 μ t 3 ø- - H ø 0 H t 03 ø 0 μ. 0 s, μ. CQ 0 0 o μ- Ω 03 Ω 0 φ

Cb 0 0 03 • rt rt rt μ- μ μ μ 0 03 3 μ- Φ μ ø 03 φ

0 Φ μ- 13 < μ 0J

Cb Φ μ- M 0 0 TJ

0 > Ω 0 N μ- CQ rt

03 h-¹ 03 Φ 0 ø 0

Φ 0 3 Cb μ rt to 0 μ- Φ

Φ Cb tr α μ.

EXAMPLE 2

Expression of XYG1020 in E. coli

SEQ ID NO: 1 was amplified from Jonesia sp . , DSM 14140, genomic DNA by PCR and subcloned into an E. coli expression vector, pSE420 (Invitrogen) . Colonies were positive on LB agar plates containing ampicillin and AZCL-xyloglucan confirming that this sequence encodes the xyloglucanase.

EXAMPLE 3

Heterologous expression of XYG1020 in Aspergillus oryzae

Construction of the xyloglucanase expression cassette for Aspergillus expression SEQ ID NO:l was amplified from Jonesia sp . DSM 14140 genomic DNA by PCR using standard procedures using the primers: GACTAGCAAAGCTTCACCATGCGTTTACGAACCGTGGTGGCC (SEQ ID NO: 4) GCTAGTCCACCTCGAGGTATGACTAACCTCTAGTCAGCTAGCG (SEQ ID NO: 5) and a PTC-200 Peltier Thermal cycler (MJ Research, USA) and the Expand High Fidelity PCR System (Roche Molecular Biochemicals, Germany) according to the manufacturer's instructions. Thirty cycles of PCR was performed using a cycle profile of denaturation at 94 °C for 30 sec, annealing at 55 °C for 30 sec, and extension at 72 °C for 5 min. The amplification products were analyzed by electrophoresis in a 1 % agarose gel, and subsequently the ca. 2.8 kb XYG1020 PCR fragment was extracted from the gel using the GFX gel band purification kit (Amersham- Pharmacia Biotech, Uppsala, Sweden) according to the manufacturer's instructions. Both the purified PCR fragment and Aspergillus expression vector pMStr57 were double digested with HindiII and Xhol and the PCR fragment was Iigated to the vector using standard methods (Sambrook et al . 1989) . E. coli strain DH10B (supplied by Stratagene (La Jolla, Ca.)) was transformed with the ligation mixture, and transformants were screened for presence of the PCR insert in pMStr57 by restriction endonuclease analysis.

7An expression clone thus identified was designated pXYG1020-AO, and the sequence of the cloned PCR fragment was determined to ensure that no errors were introduced during amplification .

Transformation of Aspergillus oryzae

Transformation of Aspergillus oryzae is carried out as described by Christensen et al . , (1988), Biotechnology 6, 1419- 1422. A number of Aspergillus transformants are obtained which show a positive reaction on medium containing AZCL-xyloglucan confirming expression in the heterologous host.

Expression vector pMStr57

The Aspergillus oryzae expression plasmid pMStr57 contains an Aspergillus expression cassette based on the Aspergillus niger neutral amylase II promoter fused to the Aspergillus nidulans triose phosphate isomerase non-translated leader sequence (NA2TPI promoter) and the Aspergillus niger amyloglycosidase terminator (Tamg) . Also present on the plasmid is the Aspergillus selective marker amdS from Aspergillus nidulans enabling growth on acetamide as sole nitrogen source . These elements are cloned into the E . coli vector pUC19. pMStr57 is constructed from pCaHj483 (WO 98/00529) by switching out the EcoRI-XhoI promoter-bearing fragment for a modified version of the same promoter. The modifications were introduced by site directed mutagenesis to the sequence shown in SEQ ID NO: 3.

Table 2. List of main features in plasmid pMSTR57 (overall size in 6795bp)

EXAMPLE 4 Detergent stability Agar plugs were taken in triplicate from center of xyloglucanase positive colonies of Jonesia sp . , DSM 14140, applied to three sets of plates and embedded in 1% agarose in H strength tap water with 0.025% AZCL-xyloglucan as reference and either of the commercial laundry detergents Ariel Futur powder (dosage: 6.7 g/1) or Tide powder (dosage: 1 g/1) added in the test plates . The solidified plates were incubated overnight at 37 °C, and the detergent stability was evaluated by visual comparison of the blue zones around the agar plugs in the detergent plates with the reference. The activity following incubation in the presence of each of the commercial laundry detergents was similar to the activity of the reference, thus demonstrating full stability of the xyloglucanase of the invention in presence of conventional, commercial laundry detergent compositions.

EXAMPLE 5

Wash Evaluation of Xyloglucanases

Wash Example I The following provides general guidelines on how to perform a wash evaluation of detergent compositions containing xyloglucanases .

Preparation of tamarind gum swatches 5 1 ionized water is heated to 50 °C. 10 g Tamarind seed polysaccharide is added slowly during agitation. The suspension is agitated until the polymer is solubilized. Clean cotton swatches (style 400 from Testfabrics Inc.) are placed in the solution for 30 min, squeezed between two rolls and dried overnight. The swatches are rinsed an EU wash machine, dried overnight and cut into 5 cm x 5 cm pieces .

Preparation of dinginess swatches

Old white cotton fabrics are cut into 5 cm x 5 cm pieces .

Wash in Launder-o-meter Each beaker (500 ml) is added steel balls, 200 ml detergent solution and 6-12 dinginess or tamarind gum swatches. The wash cycle is conducted and the swatches are rinsed in tap water and air dried overnight at room temperature .

Iron oxide soiling

The following mixture is prepared and stirred for 30 min: 5,25g Fe₂0₃ (Merck 3924) 800 ml ionised water 42 ml Na₂C0₃ (5g/l) The swatches and iron oxide solution is placed in a Japanese washing machine (NA-F38A1M) using the programme "Large SPEEDY" (42 1 water, 3 min wash, 1 rinse, 3 min centrifuging) . The swatches are air dried overnight at room temperature .

Evaluation

Remission of the swatches is measured at 440 nm using a MacBeth ColorEye 7000 remission spectrophotometer . The results are expressed as Delta remission = R_enzyme, after soiling - R-control, after soi_li_ng where R is the remission at 440 nm .

Data

The effect on the tamarind gum swatches expresses the effect of the xyloglucanase during wash on tamarind gum containing stains e.g. food stains, whereas the effect on the dinginess swatches expresses the general cleaning effect of the xyloglucanase during wash. Original (for SUBMISSION) • printed on 26.03.2002 03:30:33 PM -1 Form - PCT/RO/134 (EASY) Indications Relating to Deposited Microorganlsm(s) or Other Biological Material (PCT Rule 13bis) -1-1 Prepared using PCT-EASY Version 2 . 92 (updated 01. 01.2002 ) -2 International Application No.

-3 Applicant's or agent's file reference 10153 .204-WO

The indications made below relate to the deposited microorganism(s) or other biological material referred to in the description on: -1 page 4 -2 line 31-37 -3 Identification of Deposit -3-1 Name of depositary institution DSMZ- Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH -3-2 Address of depositary institution Mascheroder Weg lb, D- 38124 Braunschweig, Germany -3-3 Date of deposit 23 February 2001 (23 . 02 .2001) -3-4 Accession Number DSMZ 14140 -4 Additional Indications NONE -5 Designated States for Which all designated States Indications are Made -6 Separate Furnishing of Indications NONE

These indications will be submitted to the International Bureau later

FOR RECEIVING OFFICE USE ONLY

-5 This form was received by the international Bureau on: -5-1 Authorized officer

Claims

1. A xyloglucanse enzyme belonging to family 74 of glycosyl hydrolases, which enzyme is endogenous to a bacterium.

2. The xyloglucanase enzyme of claim 1, wherein the bacterium comprises alkalophilic bacteria.

3. The xyloglucanase enzyme according to any of the preceding claims, wherein the bacterium belongs to the genus Jonesia .

4. The xyloglucanase enzyme according to any of the preceding claims, wherein the strain is Jonesia sp . , DSM 14140.

5. A xyloglucanase, which is selected from one of

(a) a polypeptide encoded by the DNA sequence of positions 72- 2820 of SEQ ID NO: 1;

(b) a polypeptide produced by culturing a cell comprising the sequence of positions 72-2820 of SEQ ID NO: 1 under conditions wherein the DNA sequence is expressed;

(c) a xyloglucanase enzyme having a sequence of at least 60% identity to positions 24-940 of SEQ ID NO: 2 when identity is determined by the Clustal method with a PAM250 residue table and the default settings of the Megalign program in the Lasergene package using a GAP creation penalty of 3 and Ktuple of 1; and

(d) a polypeptide encoded by a DNA sequence that hybridizes to the DNA sequence of SEQ ID NO: 1 under medium stringency conditions, wherein the medium stringency conditions comprise hybridization in 5xSSC at 45°C and washing in 2xSSC at 60°C.

6. The xyloglucanase according to claim 5 which is obtained or obtainable from a bacterium, preferably from a strain belonging to the genus Jonesia, preferably from Jonesia sp . , DSM 14140.

7. The xyloglucanase according to any of the claims 5 and 6, wherein the xyloglucanase is comprised in family 74 of glycosyl hydrolases .

8. The xyloglucanase of claim 5, which enzyme is isolated and free from homologous impurities.

9. An enzyme preparation comprising the enzyme according to any of the claims 1-8, and conventional fillers or stabilizers.

10. The preparation according to claim 9 which further . comprises one or more enzymes selected from the group consisting of proteases, cellulases (endoglucanases) , β-glucanases, hemicellulases, lipases, peroxidases, laccases, α-amylases, glucoamylases, cutinases, pectinases, reductases, oxidases, phenoloxidases, ligninases, pullulanases, pectate lyases, other xyloglucanases not belonging to family 74, xylanases, pectin acetyl esterases, polygalacturonases, rhamnogalacturonases, pectin lyases, other mannanases, pectin methylesterases, cellobiohydrolases, transglutaminases; or mixtures thereof.

11. An isolated polynucleotide molecule encoding a polypeptide having xyloglucanase activity which polynucleotide molecule hybridizes to a denatured double-stranded DNA probe under medium stringency conditions, wherein the probe is selected from the group consisting of DNA probes comprising the sequence shown in positions 72-2820 of SEQ ID NO: 1, and DNA probes comprising a subsequence of positions 72-2820 of SEQ ID NO: 1, the subsequence having a length of at least about 100 base pairs.

12. An expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment selected from the group consisting of (a) polynucleotide molecules encoding a polypeptide having xyloglucanase activity comprising a nucleotide sequence as shown in SEQ ID NO: 1 from nucleotide 72 to nucleotide 2820, (b) polynucleotide molecules encoding a polypeptide having xyloglucanase activity that is at least 60% identical to the amino acid sequence of SEQ ID NO: 2 from amino acid residue 24 to amino acid residue 940, and (c) degenerate nucleotide sequences of (a) or (b) ; and a transcription terminator.

13. A cultured cell into which has been introduced an expressionvector according to claim 12, wherein said cell expresses the polypeptide encoded by the DNA segment.

14. A method of producing a polypeptide having xyloglucanase activity, which method comprises culturing a cell into which has been introduced an expression vector according to claim 12, whereby said cell expresses a polypeptide encoded by the DNA segment; and recovering the polypeptide.

10

15. A detergent composition comprising the enzyme preparation according to claim 9 or the enzyme according to any of the claims 1-8.

15 16. A process for machine treatment of fabrics which process comprises treating fabric during a washing cycle of a machine washing process with a washing solution containing the enzyme preparation according to claim 9 or the enzyme according to any of the claims 1-8.

20

17. Use of the enzyme preparation according to claim 9 or the enzyme according to any of the claims 1-8 in the textile industry for improving the properties of cellulosic fibers, yarn, woven or non-woven fabric.

25

18. The use according to claim 17, wherein the enzyme preparation or the enzyme is used in a textile scouring process step.

30 19. Use of the enzyme preparation according to claim 9 or the enzyme according to any of the claims 1-8 in the cellulose fiber processing industry for ratting of fibers selected from the group consisting of hemp, jute, flax and linen.