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1. WO1999001544 - VARIANTS D'ENDO-1,4-$g(b)-GLUCANASE DE FAMILLE 6 ET COMPOSITIONS NETTOYANTES CONTENANT DE TELS COMPOSES

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[ EN ]

FAMILY 6 ENDO-1,4-β-GLUCANASE VARIANTS AND CLEANING COMPOSITIONS CONTAINING THEM

The present invention relates to cleaning compositions, including laundry detergent compositions and fabric softener or fabric conditioning compositions, containing an endo-1,4-β-glucanase of the glycosyl hydrolase family 6, preferably an improved variant of a parent Humicola endoglucanase or Humicola-like cellulase; the improved variants; and a method of constructing the variants.

BACKGROUND OF THE INVENTION
Performance of a cleaning composition, for use in a washing or cleaning method, such as a laundry, dishwashing or surface cleaning method, is judged by a number of factors, including the ability to remove soils, the ability to prevent the re-deposition of the soils, or, in case of laundry, the ability to maintain the original colours of the washed garment and the ability to maintain fabric or garment durability. The anti-harshening or softening effect of cellulase on fabrics and the fabric care (colour care/colour clarification) effect is known, e.g. from GB 1 368 599 and EP 269 168, along with other very beneficial cellulolytic effects such as particulate soil removal and de-pilling.
Fabric conditioning or fabric softener compositions, in particular compositions to be used in the rinse cycle of laundry washing processes, are also well known. Typically, such compositions contain a water-insoluble quaternary-ammonium fabric softening agent, the most commonly used having been di-long alkyl chain ammonium chloride. Fabric conditioning compositions comprising cellulase have also been suggested, e.g. in US
5,445,747, in particular compositions using a specific ~43kD cellulase obtained from the fungus Humicola insolens.
Cellulose is a polymer of glucose linked by β-1,4-glucosidic bonds. Cellulose chains form numerous intra- and intermolecular hydrogen bonds, which result in the formation of insoluble cellulose microfibrils. Microbial hydrolysis of cellulose to glucose involves the following three major classes of cellulases: endo-1,4-β-glucanases (EC 3.2.1.4), which cleave β-1,4-glucosidic links randomly throughout cellulose molecules; cellobiohydrolases (EC 3.2.1.91) (exoglucanases), which digest cellulose from the nonreducing end; and β-glucosidases (EC
3.2.1.21), which hydrolyse cellobiose and low-molecular-mass cellodextrins to release glucose. Most cellulases consist of a cellulose-binding domain (CBD) and a catalytic core or catalytic domain (CAD = catalytically active domain) separated by a linker rich in proline and hydroxy amino acid residues. All cellulases hydrolyse by either a "retaining" or "inverting" mechanism.
Cellulases are produced by many microorganisms and are often present in multiple forms. Recognition of the economic significance of the enzymatic degradation of cellulose has promoted an extensive search for industrially useful microbial cellulases. As a result, the enzymatic properties and the primary structures of a large number of cellulases have been investigated. On the basis of the results of a hydrophobic cluster analysis of the amino acid sequence of the catalytically active domain (CAD), these cellulases have been placed into 11 different families of glycosyl hydrolases (Henrissat, 1991; Henrissat et al., 1993). One of these families is known as the cellulase family B or as the glycosyl hydrolase family 6. Up till now, the following enzymes have been identified as belonging to this family: Agaricus bisporus exoglucanase 3 (cel3), Cellulomonas fimi endoglucanase A (cenA), Cellulomonas fimi exoglucanase A (cbhA), Microspora bispora endoglucanase A (celA), Streptomyces halstedii endoglucanase A (celA), Streptomyces strain KSM-9 endoglucanase 1 (casA), Thermomonospora fusca endoglucanase E-2 (celB), Trichoderma reesei exoglucanase II (cbh2), and probably Neocallimastix patriciarum exoglucanase (celA) (Denman et al., 1996) and Orpinomyces sp. (celA). The following two conserved regions have been used as signature patterns (PROSITE: PDOC00563. February 1997): V-x-Y-x(2)-P-x-R-D-C-[GSAF]-x(2)-[GSA](2)-x-G; and [LIVMYA]-[LIVA]-[LIVT]-[LIV]-E-P-D-[SAL]-[LI]-[PSAG]. The first conserved region contains a conserved aspartic acid residue which is potentially involved in the catalytic mechanism; the aspartate is followed by a cysteine which is involved in a di sulfide bond. The second conserved region contains an aspartate which seems to be the proton donor in the catalytic mechanism.
WO 97/20025 and WO 97/20026 discloses detergent compositions comprising an endoglucanase from Thermomonospora fusca.
An interesting feature of family 6 is that it contains both endoglucanases and exoglucanases which show definite differences in amino acid sequence, the exoglucanases having extra amino acid insertions. Without being bound to this theory it is presently believed that cellulolytic enzymes belonging to family 6 are inverting type enzymes, i.e. hydrolyse the β-1,4-glucosidic bond with inversion of anomeric configuration. The inverting mechanism involves protonation of the glycosidic oxygen of the scissile bond by an acidic amino acid residue
(general acid catalyst) with concerted attack of a water molecule at the anomeric carbon. The nucleophilicity of this water molecule is greatly increased through deprotonation by a basic amino acid residue (general basic catalyst). The partial positive charge formed at the anomeric carbon in the transition state is stabilised through resonance with the ring oxygen. This gives the transition state significant oxocarbonium ion character which is stabilised by electrostatic interactions with the nearby carboxylate side chains and by specific binding interactions with the sugar in its half-chair conformation. In general, only glutamate and aspartate residues act directly as general acid or base catalysts in glycosidases (Damude et. al. 1996).
Detergent compositions with cellulases, either monocomponent endoglucanases or cellulase enzyme systems, i.e. a mixture of cellulases, have successfully been used commercially for some years. However, these compositions are neither recommendable for use in a presoaking bath nor for use in case of prolonged storage of the washed and rinsed wet laundry, e.g. in the washing machine prior to line or tumbler drying, since such prolonged enzymatic impact may result in a weakening of the fabric or garment presumably due to the actual (but unknown) mechanisms by which the cellulase types hitherto used in cleaning compositions have acted on the cellulose-containing or cellulosic fabric.
Thus, it is an object of the present invention to provide cleaning compositions containing enzymes with cellulolytic ac tivity which enzymes provides colour clarification (colour care benefits) and possibly also soil removal of the laundry without any substantial weakening thereof when the laundry is subjected to pre-soaking or wet storage.

SUMMARY OF THE INVENTION
We have now found that endo-1,4-β-glucanases of the glycosyl hydrolase family 6 may valuably be incorporated into cleaning compositions at such a level that at least about 25% of the total weight of cellulolytic active enzyme protein present in the composition derives from the family 6 endoglucanase. The inclusion of such enzymes provides colour care benefits, i.e. colour clarification of laundry containing cotton or other cellu-losic fabrics. It is known that such colour care benefit is also provided by endo-1, 4-β-glucanases of the glycosyl hydrolase families 5, 7, 45, and 12. However, we now have surprisingly found that, in contrast to endoglucanases belonging to other families, application of family 6 endoglucanases in cleaning compositions delivers an important improvement in the degree of fabric durability, i.e. a considerable reduction in fabric weakening due to subjecting the laundry to a pre-soaking bath or prolonged wet storage of the washed or rinsed laundry within the washing machine.
Accordingly, in a first aspect the present invention relates to cleaning compositions comprising one or more enzymes having cellulolytic activity wherein at least 25% of the total weight of cellulolytic active enzyme protein derives from the presence of a Humicola endo-1,4-β-glucanase or Humicola-like cellulase (endo-type (Cel6B) or exo-type (Cel6A))of the glycosyl hydrolase family 6, the Humicola-like cellulase being an enzyme comprising a catalytically core domain having an amino acid sequence being at least 35% homologous to the appended SEQ ID
NO: 4.
By using the present invention, it is now possible to use , high performance cleaning compositions in any cleaning or laundering method without a substantial, negative impact on fabric durability.

In second and third aspects, the invention provides a method of constructing a variant of a parent Humicola family 6 endo-beta-1,4-glucanase or a Humicola-like family 6 cellulase which variant has endo-beta-1,4-glucanase activity and improved detergent compability as compared to the parent endo-beta-1,4-glucanase or cellulase; and variants provided by the method.
By using the protein engineering method of the invention it is now possible to provide well-performing endoglucanases from enzymatic starting material originally having different activities and/or different properties, eg can a cellobiohydrolase enzyme with poor detergent compability be engineered into a well-performing endoglucanase enzyme based on the findings disclosed herein.

THE DRAWINGS
In the accompanying drawings.
Fig. 1 shows ClustalW multiple sequence alignment of Family 6 cellulases. The ! SS_HI_CEL6B row shows the definition of α-helical (H) and β-strand (S) regions.
Fig. 2 shows the nucleotide sequence of pCA6H from BamHI-XbaI; the translational initiation codon is underlined (see example 3).
Fig. 3 shows the nucleotide sequence of pC6H from BamHI-XbaI the translational initiation codon is underlined (see example 3).
Figure 4: Secondary structure elements (strand and helix only) of catalytic core domain of Humicola insolens Cel6B as determined by DSSP for the two independent molecules in the asymmetric unit. (H) α-helix, (3) 3-10-helix, (S) β-strand.
Figure 5: The loop regions encompassing the binding cleft in the catalytic core region of Humicola insolens Cel6B. (L) indicate the defined loop regions encompassing the binding cleft,

(H) α-helical structure in both molecules, (S) β-strand regions in both molecules.
Fig. 6 shows the loop regions encompassing the binding cleft in Humicola insolens Cel6A as determined from sequence alignment to Humicola insolens EGIV (Cel6B). The numbering refers to the mature full length protein.

Fig. 7 shows the loop regions encompassing the binding cleft in Humicola insolens Cel6A as determined from analysis of X-ray structure.
Fig. 8: Residues on the surface of Humicola insolens Cel6B catalytic core domain and Neocallimastix patriciarum catalytic core domain (Q12646) are shown in bold and underline (see example 6).
Figure 9: Residues on the surface of Humicola insolens Cel6B catalytic core domain and Orpinomyces sp. CelA catalytic core domain (P78720) are shown in bold and underline (see example 6).
Figure 10: Residues on the surface of Humicola insolens Cel6B catalytic core domain and Orpinomyces sp. CelC catalytic core domain (P78721) are shown in bold and underline (see example 6).
In addition to the drawings, the present specification contains two appendices:
Appendix 1 shows the structural coordinates of Humicola insolens EG VI (Cel6B) endo-beta-1,4-glucanase.
Appendix 2 shows the structural coordinates of Humicola insolens Cel6A cellulase.

DETAILED DESCRIPTION OF THE INVENTION
Cellulase Numbering
In the context of this invention a specific numbering of amino acid residue positions in cellulolytic enzymes is
employed. By aligning the amino acid sequences of known
cellulases, as in figure 1 below, it is possible to
unambiguously allot an amino acid position number to any amino acid residue in any cellulolytic enzyme, if its amino acid sequence is known.
In figure 1 a number of selected amino acid sequences of cellulases of different microbial origin are aligned.
Using the numbering system originating from the amino acid sequence of the cellulase (endo-β-1,4-glucanase EG VI) obtained from the strain of Humicola insolens, DSM 1800, disclosed in e.g. Fig.1, aligned with the amino acid sequence of a number of other cellulases, it is possible to indicate the position of an amino acid residue in a cellulolytic enzyme unambiguously.
In describing the various cellulase variants produced or contemplated according to the invention, the following nomenclatures are adapted for ease of reference:
[Original amino acid; Position; Substituted amino acid]
Accordingly, the substitution of glutamine with histidine in position 119 is designated as Q119H.
Amino acid residues which represent insertions in relation to the amino acid sequence of the cellulase from Humicola insolens, are numbered by the addition of letters in
alphabetical order to the preceding cellulase number, such as e.g. position *21aV for the "inserted" valine (V), where no amino acid residue is present, between lysine at position 21 and alanine at position 22 of the amino acid sequence of the
cellulase from Humicola insolens, cf. Table 1.
Deletion of a proline (P) at position 49 in the amino acid sequence of the cellulase from Humicola insolens is
indicated as P49*.
Multiple mutations are separated by slash marks ("/"), e.g. Q119H/Q146R, representing mutations in positions 119 and 146 substituting glutamine (Q) with histidine (H), and glutamine (Q) acid with arginine (R), respectively.
If a substitution is made by mutation in e.g. a cellulase derived from a strain of Humicola insolens, the product is designated e.g. "Humicola insolens/*49P".
All positions referred to in this application by cellulase numbering refer, unless otherwise stated, to the cellulase numbers described above, and are determined relative to the amino acid sequence of the cellulase derived from Humicola insolens Cel6B.
Definitions
In the specification and claims, the term "endoglucanase" is intended to denote enzymes with cellulolytic activity, espe-cially endo-1,4-β-glucanase activity, which are classified in EC 3.2.1.4 according to the Enzyme Nomenclature (1992) and are capable of catalysing (endo) hydrolysis of 1,4-β-D-glucosidic link- ages in cellulose, lichenin and cereal β-D-glucans including

1,4-linkages in β-D-glucans also containing 1,3-linkages.
In the present context, the term "inverting type endoglucanase" means an endo-β-1,4-glucanase which hydrolyses the glycosidic bond with net inversion of anomeric configuration, i.e. which operate via a direct displacement of the leaving group by water: one residue acts as a general acid and the other as a general base.
In the present context, the term "retaining type endoglucanase" means an endo-β-1,4-glucanase" which hydrolyses the glycosidic bond with net retention of anomeric configuration, i.e. which utilizes a double-displacement mechanism involving a glycosyl-enzyme intermediate: one residue functions as general acid and general base while the other acts as a nucleophile and leaving group (McCarter et al., 1994).

The enzyme
In a preferred embodiment of the present invention, the cleaning composition comprises a Humicola endo-1,4-β-glucanase or Humicola-like cellulase of the glycosyl hydrolase family 6 in an amount corresponding to at least 25%, preferably at least 30%, more preferably at least 40%, even more preferably at least 90%, especially at least 98%, of the total weight of enzyme protein having cellulolytic activity.
In the present context, the term "Humicola-like cellulase" denotes an endoglucanase or an exoglucanase (cellobiohydrolase) comprising a catalytically core domain which has an amino acid sequence being at least 35% homologous to SEQ ID NO: 4. This is explained in further detail below.
It is believed that no naturally occurring microorganism is capable of producing a cellulase complex comprising a family 6 endoglucanase in an amount of at least 25% by weight of the total amount of enzyme protein having cellulolytic activity. Accordingly, family 6 endoglucanase will usually be present in a mixture of other enzymes having cellulolytic activity. This mixture may either be a conventional fermentation product, possibly isolated and purified, from a single species of a microorganism.

Besides family 6 endoglucanase, examples of other cellulolytic enzymes usually present in a fungal cellulolytic mixture, i.e. a cellulase complex produced by a fungal species, are endo-1,4-β-glucanases of the glycosyl hydrolase families 5, 7, 12, or 45; and examples of other cellulolytic enzymes usually present in a bacterial cellulolytic mixture, i.e. a cellulase complex produced by a bacterial species, are endo-1,4-β-glucanases of the glycosyl hydrolase families 5, 8, 9, 12, 41, 45 or 48. The mixture may also be 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. The mixture may also be the fermentation product of a microorganism which acts as a host cell for expression of a recombinant endoglucanase, e.g. a family 6 endoglucanase, but which microorganism simultaneously produces other cellulases being naturally occurring fermentation products of the microorganism, i.e. the cellulase complex conventionally produced by the corresponding naturally occurring microorganism. Examples of useful recombinantly producible endo-1,4-β-glucanases of the glycosyl hydrolase family 45 are disclosed e.g. in WO91/ 17243, WO94/07998, and WO96/29397 which are hereby incorporated by reference. Examples of other useful endo-1,4-β-glucanases of the glycosyl hydrolase families 5, 7, 8, 9, 12, 41 and 48 are disclosed e.g. in Henrissat, 1991, and in Henrissat et al, 1993, which are hereby incorporated by reference.
In another preferred embodiment, essentially all cellulolytic activity present in the composition of the invention resuits from one single enzyme component, i.e. a monocomponent endo-1,4-β-glucanase of the glycosyl hydrolase family 6. Examples of endo-1,4-β-glucanases of the glycosyl hydrolase family 6 are those derived from the species Humicola insolens (eg EG VI also denoted Cel6B), Neocallimastix patriciarum, Orpinomyces sp. Further, it is comtemplated that the species Trichoderma reesei and Fusarium oxysporum produces enzymes which are suitable as starting material for the protein engineering method by which well-performing family 6 endoglucanase variants can be constructed.
In general, the family 6 endo-1,4-β-glucanase may be present in the cleaning composition of the present invention in an amount corresponding to from about 1 ECU to about 100000 ECU per liter washing or rinsing solution.
Preferably, the family 6 endo-1,4-β-glucanase, either native or variant, comprises one or two cellulose-binding domains (CBD) operably linked to the catalytic domain.
A cellulose binding domain (CBD) is a polypeptide which has high affinity for or binds to water-insoluble forms of cellulose and chitin, including crystalline forms.
CBDs are found as integral parts of large protein
complexes consisting of two or more different polypeptides, for example in hydrolytic enzymes (hydrolases) which typically are composed of a catalytic domain containing the active site for substrate hydrolysis, and a carbohydrate-binding domain or cellulose-binding domain (CBD) for binding to the insoluble matrix. Such enzymes can comprise more than one catalytic domain and one, two or three CBDs and optionally one or more
polypeptide regions linking the CBD(s) with the catalytic domain(s), the latter regions usually being denoted a "linker". Examples of hydrolytic enzymes comprising a CBD are 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 polysaccharidebinding protein, see Peter Tomme et al. "Cellulose-Binding
Domains: Classification and Properties" in "Enzymatic
Degradation of Insoluble Carbohydrates", John N. Saddler and Michael H. Penner (Eds.), ACS Symposium Series, No. 618, 1996. However, most of the known CBDs are from cellulases and
xylanases.
In this context, the term "cellulose-binding domain" is intended to be understood as defined by Tomme et al., op. cit. This definition classifies more than 120 cellulose-binding domains into 10 families (I-X) which may have different
functions or roles in connection with the mechanism of substrate binding. However, it is anticipated that new family representatives and additional CBD families will appear in the future.
In the protein complex, typically a hydrolytic enzyme, a CBD is located at the N or C termini or is internal.
A monomeric CBD typically consists of more than about 30 and less than about 250 amino acid residues. For example, a CBD classified in Family I consists of 33-37 amino acid residues; a CBD classified in Family IIa consists of 95-108 amino acid residues; and a CBD classified in Family VI consists of 85-92 amino acid residues. Accordingly, the molecular weight of a monomeric CBD will typically be in the range of from about 4kD to about 40kD, and usually below about 35kD.
CBDs may be useful as a single domain polypeptide or as a dimer, a trimer, or a polymer; or as a part of a protein hybrid. Chimeric protein hybrids are known in the art, see e.g. WO 90/00609, WO 94/24158 and WO 95/16782, and comprise a cellulose binding domain (CBD) from another origin, preferably from another microbial origin, than the chimeric protein as such, which CBD exists as an integral part of the protein. Typically, the chimeric protein hybrids are enzyme hybrids, i.e. contain a catalytic domain together with the binding domain.
Chimaric protein hybrids and enzyme hybrids can be
prepared by transforming into a host cell a DNA construct comprising at least a fragment of DNA encoding the cellulosebinding domain (CBD) ligated, with or without a linker, to a DNA sequence encoding the protein or enzyme and growing the host cell to express the fused gene. The recombinant fusion protein or enzyme hybrids may be described by the following formula:

CBD - MR - X

wherein CBD is the N-terminal or the C-terminal region of an amino acid sequence corresponding to at least the cellulose-binding domain; MR is the middle region (the linker), and may be a bond, or a short linking group preferably of from about 2 to about 100 carbon atoms, more preferably of from 2 to 40 carbon atoms; or is preferably from about 2 to to about 100 amino acids, more preferably of from 2 to 40 amino acids; and X is an N-terminal or C-terminal region of a polypeptide encoded by the DNA sequence encoding the protein or enzyme.
However, recombinant fusion protein or enzyme hybrids having an internal CBD are also contemplated.

The method of constructing enzyme variants and the variants
In a preferred embodiment, the invention provides a method of constructing a variant of a parent Humicola family 6 endo-beta-1,4-glucanase, which variant has endo-beta-1,4-glucanase activity and improved detergent compability as compared to the parent endobeta-1,4-glucanase, which method comprises i) analysing the structure of the parent Humicola family 6 endo-beta-1,4-glucanase to identify at least one amino acid residue or at least one structural part of the Humicola family 6 endo-beta-1,4-glucanase catalytically core domain structure, which amino acid residue or structural part is believed to be of relevance for altering the detergent compatibility of the parent Humicola family 6 endo-beta-1,4-glucanase as evaluated on the basis of structural or
functional considerations, ii) constructing a Humicola family 6 endo-beta-1,4-glucanase variant, which as compared to the parent Humicola family 6 endo-beta-1,4-glucanase has been modified in the amino acid residue or structural part identified in i) so as to alter the detergent compatibility, and, optionally, iii) testing the resulting Humicola family 6 endo-beta-1,4-glucanase variant with respect to detergent compatibility.
Preferably, the structural part to be modified is the binding cleft, the loop region encompassing the binding cleft, or the side chain of the catalytic acid Aspl39.
In another preferred embodiment, the invention provides a method of constructing a variant of a parent Humicola-like family 6 cellulase, which variant has endo-beta-1,4-glucanase activity and improved detergent compatibility as compared to the parent cellulase, which method comprises i) comparing the three-dimensional structure of the Humicola endo-beta-1,4-glucanase with the structure of a Humicola-like cellulase, ii) identifying a part of the Humicola-like cellulase structure which is different from the Humicola endo-beta-1,4-glucanase structure and which from structural or functional considerations is contemplated to be responsible for differences in the detergent compatibility of the Humicola endo-beta-1,4-glucanase and Humicola-like cellulase, iii) modifying the part of the Humicola-like cellulase identified in ii) whereby a Humicola-like endo-beta-1,4-glucanase variant is obtained, which has an improved detergent compatibility compared to the parent Humicola-like cellulase, and optionally, iv) testing the resulting Humicola-like endo-beta-1,4-glucanase variant with respect to detergent compatibility.
Preferably, the part of the Humicola-like cellulase is modified so as to resemble the corresponding part of the Humicola family 6 endo-beta-1,4-glucanase.
The modification is, in step iii) of the method, accomplished by deleting one or more amino acid residues of the part of the Humicola-like cellulase to be modified; or the modification is accomplished by replacing one or more amino acid residues of the part of the Humicola-like cellulase to be modified with the amino acid residues occupying corresponding positions in the Humicola endo-beta-1,4-glucanase; or the modification is accomplished by insertion of one or more amino acid residues present in the
Humicola endo-beta-1,4-glucanase into a corresponding position in the Humicola-like cellulase.
In a preferred embodiment, the parent Humicola endo-beta-1,4-glucanase is derived from a strain of Humicola insolens, more preferably from the strain Humicola insolens, DSM 1800.
By the term "improved detergent compability" as used herein is meant improved properties of the enzyme with respect to
enzymatic activity and stability in commercial detergent
compositions. More specifically, these improved properties are improved enzymatic performance or enzymatic activity at a high pH, preferably at a pH above 8, more preferably above 9, especially at a pH about or above 10; improved stability towards conventional commercial detergent composition ingredients such as anionic or non-ionic surfactants, cf. examples 4-7; improved thermal
stability; and improved resistance to oxidation (ie improved compatibility towards conventional detergent composition
ingredients such as bleaching agents).

The three-dimensional structure of Humicola insolens Cel6B (EG VI) catalytic core domain The three-dimensional structure of the catalytic core domain of the Humicola insolens Cel6B fungal cellulase was solved by X-ray crystallographic methods. The extent of the catalytic core domain used for the experiment was the 347 amino acid resi-dues starting from position 27 of SEQ ID NO: 4 (and including position 373 of SEQ ID NO:4).
The obtained three-dimensional structure is believed to be representative for the structure of the any fungal endoglucanase catalytic core domain belonging to family 6 of glycosyl hydrola-ses (Henrissat B. "A classification of glycosyl hydrolases based on amino-acid sequence similarities." Biochem. J. 280 309-316 (1991). Henrissat B., Bairoch A. "New families in the classification of glycosyl hydrolases based on amino-acid sequence similarities. Biochem. J. 293 781-788 (1993). Henrissat B., Bairoch A. "Updating the sequence-based classification of glycosyl hydrolases." Biochem. J. 316 695-696 (1996) .Davies G., Henrissat B. "Structures and mechanisms of glycosyl hydrolases." Structure 3 853-859 (1995)).
The structure was solved in accordance with the principles for X-ray crystallographic methods given in "X-Ray Structure Determination", Stout, G.K. and Jensen, L.H., John Wiley and Sons, Inc. N.Y. 1989. The structural coordinates of the catalytic core domain of the Humicola insolens Cel6B fungal cellulase solved at 1.6Å resolution are given in Appendix 1 in a conventional Brook-haven Protein Data Bank (PDB) format (E. E. Abola, F. C. Bernstein, S. H. Bryant, T. F. Koetzle, and J. Weng, Protein Data Bank, in Crystallographic Databases-Information Content, Software Systems, Scientific Applications, F. H. Allen, G. Berger-hoff, and R. Sievers, eds., Data Commission of the International Union of Crystallography, Bonn/Cambridge/Chester (1987) pp. 107-132. ; F. C. Bernstein, T. F. Koetzle, G. J. B. Williams, E. F. Meyer, Jr., M. D. Brice, J. R. Rodgers, O. Kennard, T. Shimanouchi, and M. Tasumi, The Protein Data Bank: a Computer-based Archival File for Macromolecular Structures, J. Mol. Biol. 112, 535-542 (1977); http://www.pdb.bnl.gov/).
The structure contains two independent molecules in the asymmetric unit identified by the letters A and B respectively. Only the part from residue G3 to A347 are detectable in the X-ray structure. It is thought that the remaining residues are disordered under these crystallization and data collection conditions and therefore not detectable in the X-ray structure. It is to be understood that Appendix 1 forms part of the present application.
The structure of the catalytic core domain of the Humicola insolens Cel6B fungal cellulase exhibits the distorted barrel topology first described for a family 6 glycoside hydrolase by the Trichoderma reesei CBHII structure (J.Rouvinen et.al.
"Three-dimensional structure of cellobiohydrolase from Thrichoderma reesei" Science 249, p380-386 (1990)). The catalytic Brønsted acid (D139) and the catalytic base (D316) are located on each side of a cleft at a distance of 9.12Å and 9.64Å for the two independent molecules respectively consistent with the catalytic mechanism occurring with inversion of the anomeric configuration. A third acidic residue (D180) is located close to the Brønsted acid having the effect of stabilizing the protonated form of the D139 thereby making the enzyme active even at alkaline conditions.
The secondary structure of the core domain of the Humicola insolens Cel6B fungal cellulase as determined by the DSSP program (W.Kabsch & C.Sander, Dictionary of protein secondary structure: pattern recognition of hydrogen bond and geometrical features. Biopolymers 22, 2577-2637 (1983)) is shown in figure 4.
The three-dimensional structure of the catalytic core domain of the Humicola insolens Cel6A cellulase was solved by X-ray crystallographic methods as described above and is shown in Appendix 2.

Definition of the binding cleft of a three-dimensional structure of an enzyme belonging to Family 6 of glycosyl hydrolases:
A binding cleft is defined as consisting of the largest cave (pocket) on the surface of an enzyme and can extend beyond this pocket.
Using WHAT IF (G.Vriend, WHAT IF: a molecular modelling and drug design program. J.Mol. Graph. 8, 52-56, (1990) version
19980317-1938) applying the AACAVI command of the MAP menu with a PROBE RADIUS of 1.4 Å the residues on the surface of the largest cave (pocket) can be detected.

The binding cleft in contact with the substrate can consist of more residues than those in the concave cleft detected above. Those can be detected by visual inspection of the threedimensional structure e.g. using the program InsightII (© 1996, Molecular Simulations Incorporated) finding surface exposed residues extending from the concave cleft defined above. Surface exposure are detected either by the DSSP program (se below) or by the Surface Molecule command of InsightII.

Definition of the binding cleft of the catalytic core domain of

Humicola insolens Cel6B.
Applying the method to the three-dimensional structure of the catalytic core domain of Humicola insolens Cel6B the concave part of the binding cleft as detected by WHAT IF is defined as comprising of the following residues: W52, S54, Y86, D92, P138,

D139, D180, A182, N183, W186, N219, V220, S221, N222, W282,

K310, P311, E314, S315, D316, A327 and G328.
By visual inspection using InsightII the complete binding cleft is defined as comprising of the following residues: N14,

The loop regions encompassing the binding cleft:
Given the binding cleft as described above, the loop regions encompassing the binding cleft is defined as the regions of contiguous sequence not belonging to a α-helical region or a β- strand region in any of the determined structures. In this definition the 3-10 helices are included in the loop definition as they are not seen as an integral part of the inner core structure. Using this definition together with the secondary structure information in figure 4 the binding cleft encompassing loops are


This can be seen graphically in figure 5.

Residues in proximity:
To detect residues in proximity of each other the Subset zone command of the Insightll program is applied. The command detects residues or individual atoms within a defined distance from a predefined subset, groups of residues or groups of atoms. The Subset list command can be used to investigate the result.

Residues within 5 Å of residues in the binding cleft:
Based on the above definition of the binding cleft the following residues are within 5 Å of the residues in the binding cleft (including the residues in the binding cleft): L12, V13,

Residues within 2.5 Å of residues in the binding cleft:
Based on the above definition of the binding cleft the following residues are within 2.5 Å of the residues in the binding cleft (including the residues in the binding cleft): V13,


Residues within 15.0 Å of D139 side chain in the three-dimensional structure of Humicola insolens Cel6B catalytic core domain:
The following residues are found to be within 15.0 Å of an atom in the side chain of the catalytic acid (D139) defined as any of the atoms CB, CG, OD1 or OD2 in one of the two independent structures in the three-dimensional structure of Humicola insolens Cel6B catalytic core domain: F50, W52, 153, S54 , L83,
C93 , S94, A95, G96,

316, G317, Q318,

Residues within 10.0 Å of D139 side chain in the three- dimensional structure of Humicola insolens Cel6B catalytic core domain:
The following residues are found to be within 10.0 Å of an atom in the side chain of the catalytic acid (D139) defined as any of the atoms CB, CG, OD1 or OD2 in one of the two independent structures in the three-dimensional structure of Humicola insolens Cel6B catalytic core domain: W52, V84, L85, Y86, L88,

Residues within 5.0 Å of D139 side chain in the three-dimensional structure of Humicola insolens Cel6B catalytic core domain:
The following residues are found to be within 5.0 Å of an atom in the side chain of the catalytic acid (D139) defined as any of the atoms CB, CG, OD1 or OD2 in one of the two independent structures in the three-dimensional structure of Humicola insolens Cel6B catalytic core domain: D92, P138, D139, A140, N143, D180, A182, W186.

Residues on the surface of the molecule:
Residues on the surface of the three-dimensional structure of a molecule is defined as those having any part of their surface exposed to the solvent as calculated by the DSSP program (W.Kabsch & C.Sander, Dictionary of protein secondary structure: pattern recognition of hydrogen bond and geometrical features. Biopolymers 22, 2577-2637 (1983)).
For three-dimensional structure of the catalytic core domain of the Humicola insolens Cel6B fungal cellulase the application of the DSSP program to both of the molecules reveiled that the following residues were defined as being on the surface of the molecule:

Residues within 15.0 Å of D139 side chain in the three- dimensional structure of Humicola insolens Cel6B catalytic core domain and defined as being on the surface:
The following residues are found to be within 15.0 Å of an atom in the side chain of the catalytic acid (D139) defined as any of the atoms CB, CG, OD1 or OD2 in one of the two independent structures in the three-dimensional structure of Humicola insolens Cel6B catalytic core domain and are defined as being on the surface of the molecule: W52, S54, Y86, L88, D90, R91, D92,

Residues within 10.0 Å of D139 side chain in the three- dimensional structure of Humicola insolens Cel6B catalytic core domain and defined as being on the surface:
The following residues are found to be within 10.0 Å of an atom in the side chain of the catalytic acid (D139) defined as any of the atoms CB, CG, OD1 or OD2 in one of the two independent structures in the three-dimensional structure of Humicola insolens Cel6B catalytic core domain and are defined as being on the surface of the molecule: W52, Y86, L88, D90, R91, D92, C93,

Residues within 5.0 Å of D139 side chain in the three- dimensional structure of Humicola insolens Cel6B catalytic core domain and defined as being on the surface:
The following residues are found to be within 5.0 Å of an atom in the side chain of the catalytic acid (D139) defined as any of the atoms CB, CG, OD1 or OD2 in one of the two independent structures in the three-dimensional structure of Humicola insolens Cel6B catalytic core domain and are defined as being on the surface of the molecule: D92, P138, D139, A140, N143, D180, A182, W186.

Improved Stability Towards Anionic Surfactants
In order so stabilize an enzyme against denaturation by anionic tensides mutations/deletions of surface exposed residues are performed. The mutation is towards a more negatively charged residue, and preferably from a potentially positively charged residue (His, Lys or Arg, more preferably Arg). These points are thought to be anchor points for the anionic tensides especially the potentially positively charged residues and more preferably the Argini- ne residues.
The mutations from neutrally charged surface residues towards potentially negatively charged residues (Asp or Glu) should preferably be performed at points where the sequence holds the equivalent amide (Asn or Gln).
Mutating surface exposed residues towards more negatively charged residues for the core domain of Humicola insolens Cel6B comprises of the following mutations:
Neutral residues to be mutated to Asp or Glu (excluding His):


Preferably at the points where the sequence already contains Asn or Gin, which in the core domain of Humicola insolens Cel6B comprises residues: N4 , N14, Q23, Q26, Q34, N36, Q44, N55,


Preferably mutations should be performed at surface exposed positions containing a potentially positively charged residue (His, Lys or Arg) mutating to a residue not belonging to this group. In the core domain of Humicola insolens Cel6B this comprises residues: R9, K20, R25, R31, K39, K41, K46, R60, R70, K73,

Improved thermal stability
A enzyme can be stabilized towards thermal denaturation can by substitution of a naturally occurring amino acid residue other than proline with a proline residue at positions in the structure where the backbone dihedral angle φ (phi) are in the interval [-90° < φ < -40°] and where the back bone amide proton of the residue to be substituted does not participate as donor in a hydrogen bond. Preferably the residue should be outside α-helical regions as well as β-strand regions. More preferably the back bone ψ (psi) dihedral angle should be in the intervals: [- 180° < ψ < -150°] or [-80° < ψ < 10°] or [100° < ψ < 180°]. The dihedral angles as well as the potential hydrogen bonds involving the back bone amide proton can be investigated using the program DSSP. A hydrogen bond involving the back bone amide proton is defined as those with an energy determined by DSSP smaller than or equal to -1.4 kcal/mole.
Applying this method to the three-dimensional structure of the catalyic core domain of Humicola insolens Cel6B rsults in the following positions as targets for Xxx -> Pro mutations: N4,

Capping of alpha-helices
Due to the helix dipole created due to alignment of the many polar atoms in the backbone an alpha-helix exhibits a dipo le with a partial positive charge at the N-terminus and a partial negative charge at the C-terminus. This dipole can be further stabilized by introduction of opposite charges or partial charges at the ends or removal of equal charges or partial char-ges. The most well know example is the N-capping of the N-terminal of the alpha Helix with a Asn residue which can satisfy a hydrogen bond donor in the back bone which would else be unpaired. Alternatively an Asp residue located at the N-terminal can counteract the partial positive charge and stabilize the en-zyme structure. From a structural analysis other substitutions can be found which will place a residue close to a helix terminal with a stabilizing charge or partial charge.
Examination of the tree-dimensional structure of Humicola insolens Cel6B catalytic core domain results in the following potentially stabilizing mutations in the N-terminal regions of the alpha helices: N14D ; N55D ; S149N,D ; N183D ; K192Q ;
F331N,D.
And the following potentially stabilizing mutations in the C-terminal regions of the alpha-helices: V13K,R ; E77Q ;
S128N,Q,K,R ; T148N,Q,K,R ; S168N,Q,K,R ; L193Q ; D344N,Q,K,R.

Satisfaction of internal hydrogen bonds and salt bridges
"Unsatisfied" hydrogen bond donors and/or acceptors as well as unpaired buried charged groups from potentially charged residues can destabilize an enzyme structure. Removing the unsatisfied partner by mutagenesis to a residue without these properties or mutation of neighboring residues to fulfill the unsatisfied hydrogen bond or salt bridge can most often stabilize the enzyme structure. These unsatisfied hydrogen bond/salt bridge partners can be found using the WHAT CHECK routine which is an integral part of the WHAT IF program.
Applying the WHAT CHECK routine on the complete three dimensional structure of Humicola insolens Cel6B catalytic core domain followed by subsequent visual analysis using Insightll results in the following mutations to satisfy unsatisfied hydrogen bonds/salt bridges: L11Y ; T49A,S,N,Q,V,M,G ; N55G,A,S,T ;

Residues on the surface of internal cavities
This defines residues which are found to be on the surface of internal cavities in the enzyme structure. To detect these the options CAVITY and AACAVI in the WHAT IF program is used. A probe radius of 1.4 Å is typically used to detect internal cavities where mutations could be performed. The mutations are preferentially mutating to a residue with a larger side chain, thereby decreasing the volume of the cavity, or mutating to a residue with a smaller side chain, thereby increasing the volume of the cavity making it possible for a water molecule to be accommodated in the cavity. Both methods can increase the thermal stability of the enzyme structure. Residues having their side chain exposed to the cavity as determined by the AACAVI command in WHAT IF or by visual inspection using e.g. the Insightll program are prefered targets for mutagenesis.
In Humicola insolens Cel6B this results to: V13, N14, Y17,



Preferably the following residues having their side chains exposed to the cavity in a favorable position for mutagenesis as judged visually using InsightII: V13, N14 , Y17, S18, L21, V40, V43, L88, V117, F120, L136, E137, A140, 1141, Q159, 1163, A166, L170, L179, S217, V220, N284, F290, V309, S315, F331, Y334.
Preferably the following mutations to decrease the volume of said cavities (in one letter code): V13L, I,F, Y,W;

Improved Stability Towards Oxidation
Some amino acid residues are sensitive towards oxidation by oxidative detergents and will in their oxidized form have altered properties e.g. catalytic properties, stability, pH optimum. Surface exposed residues of the type Met are most labile towards oxidation. Tyr or Trp are also known to be labile towards oxidation. Mutation of surface exposed residues of the above mentioned type will remove the sensitivity towards oxidation. This comprises the residues: Y17, Y42, Y51, W52, Y86, Y112, M144, Y165, W186, W189, Y226, Y234, W279, W282, M321, M329, W330, Y334, M337, more preferably those which are also present in the binding cleft: Y51, W52, Y86, W186, W189, W279, W282.

Altered pH profile
The pH profile of an enzyme can be altered by changing the electrostatic environment of the active site. Especially the electrostatic field at the position of the catalytic proton donor is a determinant of the alkalinisity of the enzyme. A change in the electrostatic field at the point of the catalytic proton donor towards a more negative electrostatic field can increase the apparent pKa of the catalytic proton donor, and thereby increase the activity at more alkaline conditions. This change in the electrostatic field can be obtained by mutations/deletions or insertions of residues in the vicinity of the catalytic proton donor as follows:
1) Deletion of potentially positively charged residues.
2) Mutation of potentially positively charged residues to neutral or potentially negatively charged residues.
3) Mutation of neutral residues to potentially negatively charged residues.
4) Insertion of potentially negatively charged residues.
The mutations should preferably be made to surface exposed residues and preferably not more than 15Å from the catalytic proton donor, more preferably not more than loÅ from the cataly tic proton donor and most preferably nor more than δÅ from the catalytic proton donor.
Insertions/deletions should only be made in loop/turn regions and preferably not more than 15Å from the catalytic proton donor.
This results in the following positions:


Preferably on the surface within 10Å of the catalytic proton donor (D139) : W52, Y86, L88, C93, S94, A95 , S99, L136, P138,

Altering the pH profile of an enzyme (2)
Another method to alter the pH profile of an enzyme is to mutate the residues in or close to the binding cleft. This will create a variant enzyme where the electrostatics of the active site will be changed either directly due to altered charges or partial charges in the binding cleft, or due to altered geometry around the active site changing the degree of burial of the active site residues. These changes should be made not more than 5Å from a residue in the binding cleft, and preferably not more than 2.5Å from a residue in the binding cleft most preferably mutating residues in the binding cleft.

Definition of Humicola-like cellulases and their sequences
The present invention includes variants of sequences having at least 35% identity to the catalytic core domain of Humicola insolens Cel6B. Percent sequence identity is determined by conventional methods, by means of computer programs known in the art such as GAP provided in the GCG program package (Program Manual for the Wisconsin Package, Version 8, August 1994, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA
53711) as disclosed in Needleman, S.B. and Wunsch, CD., (1970), Journal of Molecular Biology, 48, 443-453, which is hereby incorporated by reference in its entirety, ie using the GAP algo- rithm of the GCG package version 8 using a gap creation penalty of 3.00 and a gap extension penalty of 0.10 and all other parameters are kept at their default value. The catalytic core domain of Humicola insolens Cel6B is defined as the 347 residues used for the X-ray structure determination (positions 27-374 of SEQ ID NO: 4). Only the part of the sequence extending from the start of the alignment to the catalytic core domain of Humicola insolens Cel6B to the last residue aligning with to the catalytic core domain of Humicola insolens Cel6B are included (as seen in Figure 1 (1A+1B)). Following known sequences are within the definition: Orpinomyces sp. SPTREMBL entry p78720 [residues 128- 459], Orpinomyces sp. SPTREMBL entry p78721 [residues 127-449], Humicola insolens Cel6B (endocellulase CMC 38K in patent
WO9311249-A) [residues 27-379], Trichoderma reesei (Trichoderma longibrachiatum) SwissProt entry p07987 [residues 112-471], Fusarium oxysporum SwissProt entry p46236 [residues 103-462], Humicola insolens Cel6A and patent number JP 1996126492-A/ 1) [residues 112-473], Acremonium cellulolyticus (patent number

WO9733982-A1) [residues 86-437], Penicillium purpurogenum
(Presented at The Annual Meeting of Japan Society for Bioscience, Biotechnology, and Agrochemistry, April 1-2, 1998, Nagoya, Japan. The sequence was recorded on videotape) [residues 96-457], Agaricus bisporus SwissProt entry p49075 [residues 87-438], Phanerochaete chrysosporium SPTREMBL entry q02321
[residues 103-460], Neocallimastix patriciarum SPTREMBL entry q12646 [residues 98-428], Humicola insolens EMBL entry E11341

[residues 115-476].

Multiple alignment of sequences of the invention
Sequences having more than 35.0% identity to the catalytic core domain of Humicola insolens Cel6B as defined above can be aligned using the multiple alignment program Clustal W ver. 1.7 (Thompson et.al. Nucleic Acids Research Vol. 22 , No. 22 pp. 4673-4680 (1994)) which is able to include secondary structure information in the alignment. The secondary structure of the catalytic core domain of Humicola insolens Cel6B as defined previously in fig. 4 for the α-helix and β-strand regions can be included in the input for a profile/structure alignment. Only positions belonging to α-helical region or a β-strand region in both og the independant molecules are considered as being in a to α-helical region or a β-strand region respectively (see also fig. 1A/B). Using this information as the 1st profile and using the remaining sequences as the 2nd profile. The option Align sequences to 1st profile is used to align sequences to the sequence of the catalytic core domain of Humicola insolens Cel6B taking the structural elements into account. No alterations is made to the default parameters. The result of the alignment are seen in figure 1A/B. This alignment is used to identify the positions equivalent to positions in the catalytic core domain of Humicola insolens Cel6B.

Alignment of new seguence to known alignment
To align a new sequence with more than 35.0% sequence identity as determined by the GAP program to the known alignment in Fig. 4 the Profile/Structure alignment option of ClustalW is applied. Only the part of the sequence extending from the start of the GAP alignment to the catalytic core domain of Humicola insolens Cel6B to the last residue aligning with to the catalytic core domain of Humicola insolens Cel6B are included. The alignment in Fig. 4 is read as 1st profile and the new sequence is read as 2nd profile. The option Align sequences to 1st profile is used to align a new sequence to the sequence alignment in Fig. 4. No alterations is made to the default parameters. From such an alignment residues in a new sequence at positions equivalent to positions in the catalytic core domain of Humicola insolens Cel6B can be identified.

Structure based seguence alignment
An more preferred way of identifying equivalent residues between a "new" sequence ant the catalytic core domain of Humicola insolens Cel6B is to determine the three-dimensional X-ray structure fold of the "new" sequence and apply a structure based sequence alignment as implemented in the Modeler 97.0 program included in the Homology 97.0 package from MSI INC. using the MALIGN3D command with the GAP_PENALTIES_3D parameters set to 0.0 and 1.75 and the FIT_ATOMS set to CA. This alignment will find residues at structurally equivalent positions, i.e. having their CA atoms not more than 3.5Å apart in a structural superposition. From this alignment equivalent residues in a "new" sequence can be identified.

Increased color care activity by trimming of binding cleft loops.
The Humicola insolens Cel6B is able to perform color clarification as seen in examples 1 and 2 and has activity towards CMC. Neocallimastix patriciarum SPTREMBL entry q12646 have been shown to have activity towards CMC, the same is believed to be the case for Orpinomyces sp. SPTREMBL entry p78720 and Orpinomyces sp. SPTREMBL entry p78721. The origin of this is thought to be a more open binding cleft caused by one or more of the binding cleft encompassing loops being shorter that in the other fungal family 6 cellulases, preferably one of the four longer loops: Y86-N107, N219-D242, L272-P287 or W308-F331 (Humicola insolens Cel6B numbering) or equivalent regions as determined by the multiple sequence alignment, more preferably the regions N219-D242 or W308-F331 which are seen in the multiple sequence alignment to be different in length. The extent of the loop regions can be trimmed (ie made shorter) by deletion of individual residues which together with mutation of neighboring residues can optimize the color care effect. The loop manipulations can be performed using site directed mutagenesis, region specific random mutagenesis using spiked oligonucleotides, protein family shuffling or by other methods.

Methods of preparing endoglucanase variants
Several methods for introducing mutations into genes are known in the art. Cloning of cellulase-encoding DNA sequences and methods for generating mutations at specific sites within the cellulase-encoding sequence are mentioned in the following. Cloning a DNA sequence encoding a cellulase
The DNA sequence encoding a parent cellulase may be isolated from any cell or microorganism producing the cellulase in question, using various methods well known in the art. First, a genomic DNA and/or cDNA library should be constructed using chromosomal DNA or messenger RNA from the organism that produces the cellulase to be studied. Then, if the amino acid sequence of the cellulase is known, homologous, labelled oligonucleotide probes may be synthesized and used to identify cellulase-encoding clones from a genomic library prepared from the organism in question. Alternatively, a labelled oligonucleotide probe containing sequences homologous to a known cellulase gene could be used as a probe to identify cellulase-encoding clones, using hybridization and washing conditions of lower stringency.
A method for identifying cellulase-encoding clones involves inserting cDNA into an expression vector, such as a plasmid, transforming cellulase-negative fungi with the resulting cDNA library, and then plating the transformed fungi onto agar
containing a substrate for cellulase, thereby allowing clones expressing the cellulase to be identified.
Alternatively, the DNA sequence encoding the enzyme may be prepared synthetically by established standard methods, e.g. the phosphoroamidite method. In the phosphoroamidite method, oligonucleotides are synthesized, e.g. in an automatic DNA synthesizer, purified, annealed, ligated and cloned in appropriate vectors.
Finally, the DNA sequence may be of mixed genomic and synthetic origin, mixed synthetic and cDNA origin or mixed genomic and cDNA origin, prepared by ligating fragments of synthetic, genomic or cDNA origin (as appropriate, the fragments corresponding to various parts of the entire DNA sequence), in accordance with standard techniques. The DNA sequence may also be prepared by polymerase chain reaction (PCR) using specific primers.
Site-directed mutagenesis
Once a cellulase-encoding DNA sequence has been isolated, and desirable sites for mutation identified, mutations may be introduced using synthetic oligonucleotides. These oligonucleotides contain nucleotide sequences flanking the desired mutation sites; mutant nucleotides are inserted during oligonucleotide synthesis. In a specific method, a single-stranded gap of DNA, bridging the cellulase-encoding sequence, is created in a vector carrying the cellulase gene. Then the synthetic nucleotide, bearing the desired mutation, is annealed to a homologous portion of the single-stranded DNA. The remaining gap is then filled in with T7 DNA polymerase and the construct is ligated using T4 ligase. A
specific example of this method is described in Morinaga et al. (1984). US 4,760,025 discloses the introduction of oligonucleotides encoding multiple mutations by performing minor alterations of the cassette. However, an even greater variety of mutations can be introduced at any one time by the Morinaga method, because a multitude of oligonucleotides, of various lengths, can be
introduced.
Another method of introducing mutations into cellulase-encoding DNA sequences is described in Nelson and Long (1989). It involves the 3-step generation of a PCR fragment containing the desired mutation introduced by using a chemically synthesized DNA strand as one of the primers in the PCR reactions. From the PCR-generated fragment, a DNA fragment carrying the mutation may be isolated by cleavage with restriction endonucleases and reinserted into an expression plasmid.
Random mutagenesis
The random mutagenesis of a DNA sequence encoding a parent cellulase may conveniently be performed by use of any method known in the art.
For instance, the random mutagenesis may be performed by use of a suitable physical or chemical mutagenizing agent, by use of a suitable oligonucleotide, or by subjecting the DNA sequence to PCR generated mutagenesis. Furthermore, the random mutagenesis may be performed by use of any combination of these mutagenizing agents. The mutagenizing agent may, e.g., be one which induces transitions, transversions, inversions, scrambling, deletions, and/or insertions.
Examples of a physical or chemical mutagenizing agent suitable for the present purpose include ultraviolet (UV) irradiation, hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), O-methyl hydroxylamine, nitrous acid, ethyl methane sulphonate
(EMS), sodium bisulphite, formic acid, and nucleotide analogues. When such agents are used, the mutagenesis is typically performed by incubating the DNA sequence encoding the parent enzyme to be mutagenized in the presence of the mutagenizing agent of choice under suitable conditions for the mutagenesis to take place, and selecting for mutated DNA having the desired
properties.
When the mutagenesis is performed by the use of an oligonucleotide, the oligonucleotide may be doped or spiked with the three non-parent nucleotides during the synthesis of the oligonucleotide at the positions which are to be changed. The doping or spiking may be done so that codons for unwanted amino acids are avoided. The doped or spiked oligonucleotide can be incorporated into the DNA encoding the cellulase enzyme by any published technique, using e.g. PCR, LCR or any DNA polymerase and ligase.
When PCR-generated mutagenesis is used, either a chemically treated or non-treated gene encoding a parent cellulase enzyme is subjected to PCR under conditions that increase the misincorporation of nucleotides (Deshler 1992; Leung et al.,
Technique, Vol.1, 1989, pp. 11-15).
A mutator strain of E. coli (Fowler et al., Molec. Gen. Genet., 133, 1974, pp. 179-191), S. cereviseae or any other microbial organism may be used for the random mutagenesis of the DNA
encoding the cellulase enzyme by e.g. transforming a plasmid containing the parent enzyme into the mutator strain, growing the mutator strain with the plasmid and isolating the mutated plasmid from the mutator strain. The mutated plasmid may subsequently be transformed into the expression organism.
The DNA sequence to be mutagenized may conveniently be present in a genomic or cDNA library prepared from an organism expressing the parent cellulase enzyme. Alternatively, the DNA sequence may be present on a suitable vector such as a plasmid or a
bacteriophage, which as such may be incubated with or otherwise exposed to the mutagenizing agent. The DNA to be mutagenized may also be present in a host cell either by being integrated in the genome of said cell or by being present on a vector harboured in the cell. Finally, the DNA to be mutagenized may be in isolated form. It will be understood that the DNA sequence to be subjected to random mutagenesis is preferably a cDNA or a genomic DNA sequence.
In some cases it may be convenient to amplify the mutated DNA sequence prior to the expression step or the screening step being performed. Such amplification may be performed in accordance with methods known in the art, the presently preferred method being PCR-generated amplification using oligonucleotide primers prepared on the basis of the DNA or amino acid sequence of the parent enzyme.
Subsequent to the incubation with or exposure to the mutagenizing agent, the mutated DNA is expressed by culturing a suitable host cell carrying the DNA sequence under conditions allowing expression to take place. The host cell used for this purpose may be one which has been transformed with the mutated DNA sequence, optionally present on a vector, or one which was carried the DNA sequence encoding the parent enzyme during the mutagenesis treatment. Examples of suitable host cells are fungal hosts such as Aspergillus niger or Aspergillus oryzae.
The mutated DNA sequence may further comprise a DNA sequence encoding functions permitting expression of the mutated DNA sequence.
Localized random mutagenesis
The random mutagenesis may advantageously be localized to a part of the parent cellulase in question. This may, e.g., be advantageous when certain regions of the enzyme have been
identified to be of particular importance for a given property of the enzyme, and when modified are expected to result in a variant having improved properties. Such regions may normally be
identified when the tertiary structure of the parent enzyme has been elucidated and related to the function of the enzyme.
The localized random mutagenesis is conveniently performed by use of PCR-generated mutagenesis techniques as described above or any other suitable technique known in the art.
Alternatively, the DNA sequence encoding the part of the DNA sequence to be modified may be isolated, e.g. by being inserted into a suitable vector, and said part may subsequently be subjected to mutagenesis by use of any of the mutagenesis methods discussed above.
With respect to the screening step in the above-mentioned method of the invention, this may conveniently be performed by use of aa filter assay based on the following principle:
A microorganism capable of expressing the mutated cellulase enzyme of interest is incubated on a suitable medium and under suitable conditions for the enzyme to be secreted, the medium being provided with a double filter comprising a first protein-binding filter and on top of that a second filter exhibiting a low protein binding capability. The microorganism is located on the second filter. Subsequent to the incubation, the first filter comprising enzymes secreted from the microorganisms is separated from the second filter comprising the microorganisms. The first filter is subjected to screening for the desired enzymatic activity and the corresponding microbial colonies present on the second filter are identified.

The filter used for binding the enzymatic activity may be any protein binding filter e.g. nylon or nitrocellulose. The top filter carrying the colonies of the expression organism may be any filter that has no or low affinity for binding proteins e.g.
cellulose acetate or Durapore™. The filter may be pretreated with any of the conditions to be used for screening or may be treated during the detection of enzymatic activity.
The enzymatic activity may be detected by a dye, fluorescence, precipitation, pH indicator, IR-absorbance or any other known technique for detection of enzymatic activity.
The detecting compound may be immobilized by any immobilizing agent, e.g., agarose, agar, gelatine, polyacrylamide, starch, filter paper, cloth; or any combination of immobilizing agents. Expression of cellulase variants
According to the invention, a DNA sequence encoding the variant produced by methods described above, or by any alternative methods known in the art, can be expressed, in enzyme form, using an expression vector which typically includes control sequences encoding a promoter, operator, ribosome binding site, translation initiation signal, and, optionally, a repressor gene or various activator genes.
The recombinant expression vector carrying the DNA sequence encoding a cellulase variant of the invention may be any vector which may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced. Thus, the vector may be an
autonomously replicating vector, i.e. a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid, a bacteriophage or an extrachromosomal element, minichromosome or an artificial
chromosome. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.
In the vector, the DNA sequence should be operably connected to a suitable promoter sequence. 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 directing the transcription of the DNA sequence encoding a cellulase variant of the invention, especially in a fungal host, are those derived from the gene encoding A. oryzae TAKA amylase,

Rhizomucor miehei aspartic proteinase, A. niger neutral α-amylase, A. niger acid stable α-amylase, A. niger glucoamylase, Rhizomucor miehei lipase, A. oryzae alkaline protease, A. oryzae triose phosphate isomerase or A. nidulans acetamidase.
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 gen, or the Bacillus pumilus xylosidase gene, or the phage Lambda PR or PL promoters or the E. coli lac, trp or tac promoters.
The expression vector of the invention may also comprise a suitable transcription terminator and, in eukaryotes, polyadenylation sequences operably connected to the DNA sequence encoding the cellulase variant of the invention. Termination and polyadenylation sequences may suitably be derived from the same sources as the promoter.
The vector may further comprise a DNA sequence enabling the vector to replicate in the host cell in question. Examples of such sequences are the origins of replication of plasmids pUC19, pACYC177, pUB110, pE194, pAMB1 and pIJ702.
The vector may also comprise a selectable marker, e.g. a gene, the product of which complements a defect in the host cell, such as one which confers antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracyclin resistance. Furthermore, the vector may comprise Aspergillus selection markers such as amdS, argB, niaD and sC, a marker giving rise to hygromycin resistance, or the selection may be accomplished by co-transformation, e.g. as described in WO 91/17243.
The procedures used to ligate the DNA construct of the invention encoding a cellulase variant, the promoter, terminator and other elements, respectively, and to insert them into suitable vectors containing the information necessary for replication, are well known to persons skilled in the art (cf., for instance, Sambrook et al. (1989)).

The cell of the invention, either comprising a DNA construct or an expression vector of the invention as defined above, is
advantageously used as a host cell in the recombinant production of a cellulase variant of the invention. The cell may be
transformed with the DNA construct of the invention encoding the variant, conveniently by integrating the DNA construct (in one or more copies) in the host chromosome. This integration is generally considered to be an advantage as the DNA sequence is more likely to be stably maintained in the cell. Integration of the DNA constructs into the host chromosome may be performed according to conventional methods, e.g. by homologous or heterologous
recombination. Alternatively, the cell may be transformed with an expression vector as described above in connection with the different types of host cells.
The cell of the invention may be a cell of a higher organism such as a mammal or an insect, but is preferably a microbial cell, e.g. a bacterial or fungal cell.
Examples of bacterial host cells which on
cultivation are capable of producing the enzyme of the invention may be a gram-positive bacteria such as a strain of Bacillus, in particular Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus circulans, Bacillus coagulans, Bacillus megatherium, Bacillus stearothermophilus, Bacillus subtilis and Bacillus thuringiensis, a strain of Lactobacillus, a strain of Streptococcus, a strain of Streptomyces, in particular
Streptomyces lividans and Streptomyces murinus, or the host cell may be a gram-negative bacteria such as a strain of Escherichia coli.
The transformation of the bacteria may be effected by protoplast transformation, electroporation, conjugation, or by using competent cells in a manner known per se (cf. e.g.
Sambrook et al., supra).
When expressing the enzyme in a bacteria such as
Escherichia coli, the enzyme may be retained in the cytoplasm, typically as insoluble granules (known as inclusion bodies), or may be directed to the periplasmic space by a bacterial
secretion sequence. In the former case, the cells are lysed and the granules are recovered and denatured after which the enzyme is refolded by diluting the denaturing agent. In the latter case, the enzyme may be recovered from the periplasmic space by disrupting the cells, e.g. by sonication or osmotic shock, to release the contents of the periplasmic space and recovering the enzyme.
When expressing the enzyme in a gram-positive bacteria such as a strain of Bacillus or a strain of Streptomyces, the enzyme may be retained in the cytoplasm, or may be directed to the extracellular medium by a bacterial secretion sequence.
Examples of a fungal host cell which on cultivation are capable of producing the enzyme of the invention is e.g. a strain of Aspergillus or Fusarium, in particular Aspergillus awamori , Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, and Fusarium oxysporum, and a strain of Trichoderma, preferably Trichoderma harzianum, Trichoderma reesei and
Trichoderma viride.
Fungal cells may be transformed by a process involving protoplast formation and transformation of the protoplasts followed by regeneration of the cell wall in a manner known per se. The use of a strain of Aspergillus as a host cell is
described in EP 238 023 (Novo Nordisk A/S), the contents of which are hereby incorporated by reference.
Examples of a host cell of yeast origin which on
cultivation are capable of producing the enzyme of the invention is e.g. a strain of Hansenula sp., a strain of Kluyveromyces sp., in particular Kluyveromyces lactis and Kluyveromyces marcianus, a strain of Pichia sp., a strain of Saccharomyces, in particular Saccharomyces carlsbergensis, Saccharomyces
cerevisae, Saccharomyces kluyveri and Saccharomyces uvarum, a strain of Schizosaccharomyces sp., in particular
Schizosaccharomyces pombe, and a strain of Yarrowia sp., in particular Yarrowia lipolytica.
Examples of a host cell of plant origin which on
cultivation are capable of producing the enzyme of the invention is e.g. a plant cell of Solanum tuberosum or Nicotiana tabacum. In a yet further aspect, the present invention relates to a method of producing a cellulase variant of the invention, which method comprises cultivating a host cell as described above under conditions conducive to the production of the variant and
recovering the variant from the cells and/or culture medium.
The medium used to cultivate the cells may be any conventional medium suitable for growing the host cell in question and
obtaining expression of the cellulase variant of the invention. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g. as described in catalogues of the American Type Culture Collection).
The cellulase variant secreted from the host cells may conveniently be recovered from the culture medium by well-known procedures, including separating the cells from the medium by centrifugation or filtration, and precipitating proteinaceous components of the medium by means of a salt such as ammonium sulphate, followed by the use of chromatographic procedures such as ion exchange chromatography, affinity chromatography, or the like.

The cleaning or detergent or fabric conditioning compositions
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 an 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 an 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 washing machine. Conventionally, the washing liquor is an aqueous solution of a powder or liquid detergent composition.
The term "rinsing liquor" denotes an aqueous liquor in which laundry is immersed and treated, conventionally immediately after being subjected to a washing process, in order to rinse the laundry, i.e. essentially remove the detergent solution from the laundry. The rinsing liquor may contain a fabric conditioning or softening composition.
In another aspect, the present invention also relates to a process for machine treatment of fabrics which process comprises treating fabric during a rinse cycle of a machine washing process with a rinse solution containing the composition
according to the invention.
The laundry subjected to the composition or the method of the present invention may be conventional washable laundry.
Preferably, the major part of the laundry is sewn or unsewn fabrics, including knits, wovens, denims, yarns, and toweling, made from cotton, cotton blends or natural or manmade
cellulosics (e.g. originating from xylan-containing cellulose fibers such as from wood pulp) or blends thereof. Examples of blends are blends of cotton or rayon/viscose with one or more companion material such as wool, synthetic fibers (e.g.
polyamide fibers, acrylic fibers, polyester fibers, polyvinyl alcohol fibers, polyvinyl chloride fibers, polyvinylidene chloride fibers, polyurethane fibers, polyurea fibers, aramid fibers), and cellulose-containing fibers (e.g. rayon/viscose, ramie, flax/linen, jute, cellulose acetate fibers, lyocell). Cleaning composition according to claim 1 wherein the composition is a fabric softener or fabric conditioning composition for the treatment of fabrics.
The cleaning composition of the invention may be in the form of a fabric softener composition comprising from about 1% to about 90%, preferably from about 2% to about 50%, by weight of one or more cationic fabric softening agents, nonionic fabric softening agents, or mixtures thereof. In case of cationic fabric softening agents, such agents may advantageously comprise quaternary ammonium softening agents or amine precursors
thereof. A Specific example of a useful quaternary ammonium sof-tening agent is N,N-di(2-tallowoyl-oxy-ethyl)-N,N-dimethyl ammonium chloride.

DETERGENT AND FABRIC SOFTENER DISCLOSURE AND EXAMPLES surfactant system
The cleaning compositions according to the present
invention comprise a surfactant system, wherein the surfactant can be selected from nonionic and/or anionic and/or cationic and/or ampholytic and/or zwitterionic and/or semi-polar
surfactants.
The surfactant is typically present at a level from 0.1% to 60% by weight.
The surfactant is preferably formulated to be compatible with enzyme components present in the composition. In liquid or gel compositions the surfactant is most preferably formulated in such a way that it promotes, or at least does not degrade, the stability of any enzyme in these compositions.
Preferred systems to be used according to the present invention comprise as a surfactant one or more of the nonionic and/or anionic surfactants described herein.
Polyethylene, polypropylene, and polybutylene oxide condensates of alkyl phenols are suitable for use as the
nonionic 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 nonionic 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 alkylphenol 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 nonionic surfactant of the nonionic 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 nonionic surfactants of this type include Tergitol™ 15-S-9 (The condensation product of C11-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

Corporati •on; NeodolTM 45-9 (the condensation product of C14-C15 linear alcohol with 9 moles of ethylene oxide), Neodol™ 23-3 (the condensation product of C12-C13 linear alcohol with 3.0 moles of ethylene oxide), Neodol™ 45-7 (the condensation product of C14-Ci5 linear alcohol with 7 moles of ethylene oxide), Neodol™ 45-5 (the condensation product of C14-C15 linear alcohol with 5 moles of ethylene oxide) marketed by Shell
Chemical Company, Kyro™ EOB (the condensation product of C13-C15 alcohol with 9 moles ethylene oxide), marketed by The Procter & Gamble Company, and Genapol LA 050 (the condensation product of C12-C14 alcohol with 5 moles of ethylene oxide) marketed by Hoechst. Preferred range of HLB in these products is from 8-11 and most preferred from 8-10.
Also useful as the nonionic surfactant of the surfactant systems of the present invention are alkylpolysaccharides disclosed in US 4,565,647, having a hydrophobic group containing from about 6 to about 30 carbon atoms, preferably from about 10 to about 16 carbon atoms and a polysaccharide, e.g. a
polyglycoside, hydrophilic group containing from about 1.3 to about 10, preferably from about 1.3 to about 3, most preferably from about 1.3 to about 2.7 saccharide units. Any reducing saccharide containing 5 or 6 carbon atoms can be used, e.g., glucose, galactose and galactosyl moieties can be substituted for the glucosyl moieties (optionally the hydrophobic group is attached at the 2-, 3-, 4-, etc. positions thus giving a glucose or galactose as opposed to a glucoside or galactoside). The intersaccharide bonds can be, e.g., between the one position of the additional saccharide units and the 2-, 3-, 4-, and/or 6-positions on the preceding saccharide units.
The preferred alkylpolyglycosides have the formula

R2O(cnH2nO)t(glycosyl)x

wherein R2 is selected from the group consisting of alkyl, alkylphenyl, hydroxyalkyl, hydroxyalkylphenyl, and mixtures thereof in which the alkyl groups contain from about 10 to about 18, preferably from about 12 to about 14, carbon atoms; n is 2 or 3, preferably 2; t is from 0 to about 10, pre-ferably 0; and x is from about 1.3 to about 10, preferably from about 1.3 to about 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 nonionic 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 nonionic surfactant of the nonionic 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 nonionic surfactant i •nclude certai•n of the commerci.ally avai.lable Tetronic™
compounds, marketed by BASF.
Preferred for use as the nonionic 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 ethyleneoxide, alkylpolysaccharides, and mixtures hereof. Most preferred are C8-C14 alkyl phenol
ethoxylates having from 3 to 15 ethoxy groups and C8-C18 alcohol ethoxylates (preferably C10 avg.) having from 2 to 10 ethoxy groups, and mixtures thereof.
Highly preferred nonionic surfactants are polyhydroxy fatty acid amide surfactants of the formula



wherein R1 is H, or R is C1-4 hydrocarbyl, 2-hydroxyethyl, 2-hydroxypropyl or a mixture thereof, R2 is C5-31 hydrocarbyl, and Z is a polyhydroxyhydrocarbyl having a linear hydrocarbyl chain with at least 3 hydroxyIs directly connected to the chain, or an alkoxylated derivative thereof. Preferably, R1 is methyl, R2 is straight C11-15 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)mSO3M wherein R is an unsubstituted C10-C-24 alkyl or hydroxyalkyl group having a C10-C24 alkyl component, preferably a C12-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, diethylamine, triethylamine, mixtures thereof, and the like. Exemplary surfactants are C12-C18 alkyl polyethoxylate (1.0) sulfate (C12-C18E(1.0)M), C12-C18 alkyl polyethoxylate (2.25) sulfate (C12-C18(2.25)M, and C12-C18 alkyl polyethoxylate (3.0) sulfate (C12-C18E(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 SO3 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:


wherein R3 is a C8-C20 hydrocarbyl, preferably an alkyl, or combination thereof, R4 is a C1-C6 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, diethonolamine, and triethanolamine. Preferably, R3 is C10-C16 alkyl, and R4 is methyl, ethyl or isopropyl. Especially preferred are the methyl ester sulfonates wherein R3 is C10-C16 alkyl.
Other suitable anionic surfactants include the alkyl sulfate surfactants which are water soluble salts or acids of the formula ROSO3M 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 cleaning, especially 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 C6-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-CH2COO-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 cleaning, especially 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 cleaning 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(CH2OH)-, -CH2CH2CH2-, and mixtures thereof; each R4 is selected from the group consisting of C1-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 R1 is C8-C16 alkyl, each of R2, R3 and R4 is
independently C1-C4 alkyl, C1-C4 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 R1 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 OXO 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 R1 is


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 25%, preferably from about 1% to about 8% by weight of such cationic surfactants.
Ampholytic surfactants are also suitable for use in the laundry detergent compositions of the present invention. These surfactants can be broadly described as aliphatic derivatives of secondary or tertiary amines, or aliphatic derivatives of heterocyclic secondary and tertiary amines in which the
aliphatic radical can be straight- or branched-chain. One of the aliphatic substituents contains at least about 8 carbon atoms, typically from about 8 to about 18 carbon atoms, and at least one contains an anionic water-solubilizing group, e.g. carboxy, sulfonate, sulfate. See US 3,929,678 (column 19, lines 18-35) for examples of ampholytic surfactants.
When included therein, the cleaning, e.g. 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 ampholytic surfactants.
Zwitterionic surfactants are also suitable for use in cleaning compositions. These surfactants can be broadly
described as derivatives of secondary and tertiary amines, derivatives of heterocyclic secondary and tertiary amines, or derivatives of quaternary ammonium, quaternary phosphonium or tertiary sulfonium compounds. See US 3,929,678 (column 19, line 38 through column 22, line 48) for examples of zwitterionic surfactants.

When included therein, the cleaning compositions of the present invention typically comprise from 0.2% to about 15%, preferably from about 1% to about 10% by weight of such
zwitterionic surfactants.
Semi-polar nonionic surfactants are a special category of nonionic surfactants which include water-soluble amine oxides containing one alkyl moiety of from about 10 to about 18 carbon atoms and 2 moieties selected from the group consisting of alkyl groups and hydroxyalkyl groups containing from about 1 to about 3 carbon atoms; watersoluble phosphine oxides containing one alkyl moiety of from about 10 to about 18 carbon atoms and 2 moieties selected from the group consisting of alkyl groups and hydroxyalkyl groups containing from about 1 to about 3 carbon 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:


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 cleaning 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 for obvious environmental reasons, phosphate builders can also be used herein.
Suitable builders can be an inorganic ion exchange
material, commonly an inorganic hydrated aluminosilicate
material, more particularly a hydrated synthetic zeolite such as hydrated zeolite A, X, B, HS or MAP.
Another suitable inorganic builder material is layered silicate, e.g. SKS-6 (Hoechst). SKS-6 is a crystalline layered silicate consisting of sodium silicate (Na2Si2O5).
Suitable polycarboxylates containing one carboxy group include lactic acid, glycolic acid and ether derivatives thereof as disclosed in Belgian Patent Nos. 831,368, 821,369 and
821,370. Polycarboxylates containing two carboxy groups include the water-soluble salts of succinic acid, malonic acid,
(ethylenedioxy) diacetic acid, maleic acid, diglycollic acid, tartaric acid, tartronic acid and fumaric acid, as well as the ether carboxylates described in German Offenle-enschrift
2,446,686, and 2,446,487, US 3,935,257 and the sulfinyl carboxylates described in Belgian Patent No. 840,623.
Polycarboxylates containing three carboxy groups include, in particular, water-soluble citrates, aconitrates and citraconates as well as succinate derivatives such as the
carboxymethyloxysuccinates described in British Patent No.
1,379,241, lactoxysuccinates described in Netherlands
Application 7205873, and the oxypolycarboxylate materials such as 2-oxa-1,1,3-propane tricarboxylates described in British Patent No. 1,387,447.

Polycarboxylates containing four carboxy groups include oxydisuccinates disclosed in British Patent No. 1,261,829,
1,1,2,2,-ethane tetracarboxylates, 1,1,3,3-propane
tetrac7arboxylates containing sulfo substituents include the sulfosuccinate derivatives disclosed in British Patent Nos.
1,398,421 and 1,398,422 and in US 3,936,448, and the sulfonated pyrolysed citrates described in British Patent No. 1,082,179, while polycarboxylates containing phosphone substituents are disclosed in British Patent No. 1,439,000.
Alicyclic and heterocyclic polycarboxylates include cyclopentane-cis,cis-cis-tetracarboxylates, cyclopentadienide pentacarboxylates, 2,3,4,5-tetrahydro-furan - cis, cis, cis-tetracarboxylates, 2,5-tetrahydro-furan-cis, discarboxylates, 2,2,5,5,-tetrahydrofuran - tetracarboxylates, 1,2,3,4,5,6-hexane - hexacarboxylates and carboxymethyl derivatives of polyhydric alcohols such as sorbitol, mannitol and xylitol. Aromatic polycarboxylates include mellitic acid, pyromellitic acid and the phthalic acid derivatives disclosed in British Patent No. 1,425,343.
Of the above, the preferred polycarboxylates are hydroxycarboxylates containing up to three carboxy groups per molecule, more particularly citrates.
Preferred builder systems for use in the present
compositions include a mixture of a water-insoluble
aluminosilicate builder such as zeolite A or of a layered silicate (SKS-6), and a water-soluble carboxylate chelating agent such as citric acid.
A suitable chelant for inclusion in the cleaning compositions in accordance with the invention is ethylenediamine-N,N'-disuccinic acid (EDDS) or the alkali metal, alkaline earth metal, ammonium, or substituted ammonium salts thereof, or mixtures thereof. Preferred EDDS compounds are the free acid form and the sodium or magnesium salt thereof. Examples of such preferred sodium salts of EDDS include Na2EDDS and Na4EDDS.
Examples of such preferred magnesium salts of EDDS include
MgEDDS and Mg2EDDS. The magnesium salts are the most preferred for inclusion in compositions in accordance with the invention.

Preferred builder systems include a mixture of a water-insoluble aluminosilicate builder such as zeolite A, and a water soluble carboxylate chelating agent such as citric acid.
Other builder materials that can form part of the builder system for use in granular compositions include inorganic materials such as alkali metal carbonates, bicarbonates, silicates, and organic materials such as the organic
phosphonates, amino polyalkylene phosphonates and amino
polycarboxylates.
Other suitable water-soluble organic salts are the homoor co-polymeric acids or their salts, in which the
polycarboxylic acid comprises at least two carboxyl radicals 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 cleaning compositions, in addition to the family 6 endo-β-1,4-glucanase, comprise other enzyme(s) which provides cleaning performance and/or fabric care benefits.
Such enzymes include proteases, lipases, cutinases, amylases, other 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 Novo Nordisk A/S (Denmark), 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 M1
Lipase™, Luma fast™ and Lipomax™ (Genencor), Lipolase™ and Lipolase Ultra™ (Novo Nordisk A/S), and Lipase P "Amano" (Amano Pharmaceutical Co. Ltd.).
The lipases 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.
Amylases: Any amylase (a and/or b) suitable for use in alkaline solutions can be used. Suitable amylases include those of bacterial or fungal origin. Chemically or genetically modified mutants are included. Amylases include, for example, aamylases obtained from a special strain of B. licheniformis. described in more detail in GB 1,296,839. Commercially available amylases are Duramyl TM, TermamylTM, FungamylTM and BANTM
(available from Novo Nordisk A/S) and Rapidase™ and Maxamyl P™ (available from Genencor).
The amylases 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.
Cellulases: Any cellulase suitable for use in alkaline solutions can be used. Suitable cellulases include those of bacterial or fungal origin. Chemically or genetically modified mutants are included. Suitable cellulases are disclosed in US 4,435,307 which discloses fungal cellulases produced from Humicola insolens, in WO 96/34108 and WO 96/34092 which disclose bacterial alkalophilic cellulases (BCE 103) from Bacillus, and in WO 94/21801, US 5,475,101 and US 5,419,778 which disclose EG III cellulases from Trichoderma. Especially suitable cellulases are the cellulases having colour care benefits. Examples of such cellulases are cellulases described in European patent
application No. 0 495 257 and the endoglucanase of the present i InventiIon. CommerciIally avaiIlable cellulases i.nclude CelluzymeTM and Carezyme™ produced by a strain of Humicola insolens (Novo Nordisk A/S), KAC-500(B)™ (Kao Corporation), and Puradax™
(Genencor International).
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 protein by weight of the composition.

Bleaching agents
Additional optional detergent ingredients that can be included in the cleaning or detergent compositions of the present invention include bleaching agents such as PB1, PB4 and percarbonate with a particle size of 400-800 microns. These bleaching agent components can include one or more oxygen bleaching agents and, depending upon the bleaching agent chosen, one or more bleach activators. When present oxygen bleaching compounds will typically be present at levels of from about 1% to about 25%. In general, bleaching compounds are optional added components in non-liquid formulations, e.g. granular detergents.

The bleaching agent component for use herein can be any of the bleaching agents useful for detergent compositions including oxygen bleaches as well as others known in the art.
The bleaching agent suitable for the present invention can be an activated or non-activated bleaching agent.
One category of oxygen bleaching agent that can be used encompasses percarboxylic acid bleaching agents and salts thereof. Suitable examples of this class of agents include magnesium monoperoxyphthalate hexahydrate, the magnesium salt of meta-chloro perbenzoic acid, 4-nonylamino-4-oxoperoxybutyric acid and diperoxydodecanedioic acid. Such bleaching agents are disclosed in US 4,483,781, US 740,446, EP 0 133 354 and US 4,412,934. Highly preferred bleaching agents also include 6-nonylamino-6-oxoperoxycaproic acid as described in US 4,634,551.

Another category of bleaching agents that can be used encompasses the halogen bleaching agents. Examples of hypohalite bleaching agents, for example, include trichloro isocyanuric acid and the sodium and potassium dichloroisocyanurates and N-chloro and N-bromo alkane sulphonamides. Such materials are normally added at 0.5-10% by weight of the finished product, preferably 1-5% by weight.
The hydrogen peroxide releasing agents can be used in combination with bleach activators such as tetraacetylethylenediamine (TAED), nonanoyloxybenzenesulfonate (NOBS, described in US 4,412,934), 3,5-trimethylhexsanoloxybenzenesulfonate (ISONOBS, described in EP 120 591) or pentaacetylglucose (PAG), which are perhydrolyzed to form a peracid as the active bleaching species, leading to improved bleaching effect. In addition, very suitable are the bleach activators C8 (6-octanamido-caproyl) oxybenzene-sulfonate, C9 (6-nonanamido caproyl) oxybenzenesulfonate and CIO (6-decanamido caproyl) oxybenzenesulfonate or mixtures thereof. Also suitable activators are acylated citrate esters such as disclosed in European Patent Application No. 91870207.7.
Useful bleaching agents, including peroxyacids and
bleaching systems comprising bleach activators and peroxygen bleaching compounds for use in cleaning compositions according to the invention are described in application USSN 08/136,626.
The hydrogen peroxide may also be present by adding an enzymatic system (i.e. an enzyme and a substrate therefore) which is capable of generation of hydrogen peroxide at the beginning or during the washing and/or rinsing process. Such enzymatic systems are disclosed in European Patent Application EP 0 537 381.
Bleaching agents other than oxygen bleaching agents are also known in the art and can be utilized herein. One type of non-oxygen bleaching agent of particular interest includes photoactivated bleaching agents such as the sulfonated zinc and-/or aluminium phthalocyanines. These materials can be deposited upon the substrate during the washing process. Upon irradiation with light, in the presence of oxygen, such as by hanging clothes out to dry in the daylight, the sulfonated zinc
phthalocyanine is activated and, consequently, the substrate is bleached. Preferred zinc phthalocyanine and a photoactivated bleaching process are described in US 4,033,718. Typically, detergent composition will contain about 0.025% to about 1.25%, by weight, of sulfonated zinc phthalocyanine.
Bleaching agents may also comprise a manganese catalyst. The manganese catalyst may, e.g., be one of the compounds described in "Efficient manganese catalysts for low-temperature bleaching", Nature 369, 1994, pp. 637-639.

Suds suppressors
Another optional ingredient is a suds suppressor,
exemplified by silicones, and silica-silicone mixtures.
Silicones can generally be represented by alkylated polysiloxane materials, while silica is normally used in finely 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-alkylalkanols 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 AerosilR.
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 conventionally used in cleaning or 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-(2,4-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-ylamino) stilbene-2,2'disulphonate, sodium 2(stilbyl-4''-(naphtho-1',2':4,5)-1,2,3, - triazole-2''-sulphonate and 4,4'-bis(2-sulphostyry1)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)43)0.75(POH)0.25[T-PO)2.8(T-PEG)0.4]T(POH)0.25((PEG)43CH3)0.75

where PEG is -(OC2H4)O-, PO is (OC3H6O) 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 cleaning 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 cleaning, especially laundry 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-transfer inhibiting agents are normally incorporated into detergent compositions in order to inhibit the transfer of dyes from colored fabrics onto fabrics washed therewith. These polymers have the ability of complexing or adsorbing the fugitive dyes washed out of dyed fabrics before the dyes have the opportunity to become attached to other articles in the wash.
Especially suitable polymeric dye-transfer inhibiting agents are polyamine N-oxide polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole, polyvinylpyrrolidone polymers, polyvinyloxazolidones and polyvinylimidazoles or mixtures thereof .
Addition of such polymers also enhances the performance of the enzymes according the invention.
The cleaning composition according to the invention can be in liquid, paste, gels, bars or granular forms.
Non-dusting granulates may be produced, e.g., as disclosed in US 4,106,991 and 4,661,452 (both to Novo Industri A/S) and may optionally be coated by methods known in the art. Examples of waxy coating materials are poly (ethylene oxide) products (polyethyleneglycol, PEG) with mean molecular weights of 1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon atoms and in which there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono-and di- and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application by fluid bed techniques are given in GB 1483591.
Granular compositions according to the present invention can also be in "compact form", i.e. they may have a relatively higher density than conventional granular detergents or cleaning compositions, i.e. form 550 to 950 g/l; in such case, the granular detergent compositions according to the present
invention will contain a lower amount of "Inorganic filler salt", compared to conventional granular detergents; typical filler salts are alkaline earth metal salts of sulphates and chlorides, typically sodium sulphate; "Compact" detergent typically comprise not more than 10% filler salt. The liquid compositions according to the 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 .
In another preferred embodiment, the cleaning composition is a granular detergent composition containing no more than 40%, preferably no more than 15%, by weight of inorganic filler salt. 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 C1x - C1Y 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: C1X - C1Y 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 (SiO2:Na2O ratio = 2.0)

NaSKS-6: Crystalline layered silicate of formula d-Na2Si2O5

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 (AlO2SiO2)12· 27H2O 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 NaBO2·H2O2
PB4 : Anhydrous sodium perborate tetrahydrate
Percarbonate: Anhydrous sodium percarbonate bleach of empirical formula 2Na2CO3·3H2O2
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, N' -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:


Detergent Example II

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

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:

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

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

Cellulolytic Activity
The cellulolytic activity may be measured in endo-cellulase units (ECU), determined at pH 7.5, with carboxymethyl cellulose (CMC) as substrate.
The ECU assay quantifies the amount of catalytic activity present in the sample by measuring the ability of the sample to reduce the viscosity of a solution of carboxy-methylcellulose (CMC). The assay is carried out at 40°C; pH 7.5; 0.1M phosphate buffer; time 30 min; using a relative enzyme standard for reducing the viscosity of the CMC Hercules 7 LFD substrate; enzyme concentration approx. 0.15 ECU/ml. The arch standard is defined to 8200 ECU/g.

EXAMPLE 1
A. Wet storage test for cellulases
The following example is intended to describe the inven- tion that different inverting endoglucanases may differ significantly in respect to their ability to cause fabric weakening upon wet storage.
To illustrate that major differences can be observed between inverting endoglucanases from different cellulase families the following experiment can be made:
A new bleached wowen cotton (app. 350 g/m2) swatch
(25cmx25cm) is incubated at elevated dosage (100 kECU/1) for 7 days in Tris buffer pH 7 at 25°C and after this prolonged incubation the fabric is rinsed in MilliQ-water (25°C) for 10 min utes, line-dried and equilibrated in a constant climate room (60%RH, 20°C) for 48 hours. Finally the loss in tensile strength is measured on an Instron.
The relative tensile strength loss (%TSL) is quantified versus "enzyme blank", i.e. an experiment where the fabric is incubated in buffer without any enzyme present and the cellulase is then classified into one of the following 4 groups:

Class A (%TSL is in the range of [0%-25%])
Class B (%TSL is in the range of [25%-50%])
Class C (%TSL is in the range of [50%-75%])
Class D (%TSL is in the range of [75%-100%])

In the following example two inverting endoglucanases were tested:
a. `43 kD EGV from the fungal species Humicola insolens, DSM 1800, belonging to family 45 of glycosyl hydrolases and described in detail in WO 91/17243.
b. EGVI from the fungal species Humicola insolens, DSM 1800, belonging to the cellulase family 6 and having the amino acid sequence listed in SEQ ID NO: 4. The DNA sequence encoding for this enzyme is listed in SEQ ID NO: 3 (the coding region corresponding to positions 16-1356).

The following results were obtained from the evaluation: Enzyme Tensile strength class
EG V C

EG VI A
It is thus found that the cellulase belonging to family 6 is much less prone to cause tensile strength loss upon prolonged wet storage.

B. Colour Clarification in Terg-O-Meter
In this example the capability of family 6 endoglucanases to rejuvenate the colour of cotton textile is demonstrated using an assay for determining colour care benefits, i.e. "Color Clarification", of cotton cloth in a miniaturised washing machine, the 100 ml Terg-O-Meter.

250 ml beakers with 100 ml buffer (or detergent) eas positioned in a Terg-O-Meter and equilibrated to 35°C. Then two 7×7 cm swatches of black, woven cotton cloth was added to each beaker, the stirrers were put in motion, and finally enzyme was added: A) A blank, B) Three different dosages of a standard

(e.g. the commercial available enzyme preparation Celluzyme™), and C) Two different dosages of the family 6 endoglucanase. Incubation then proceeded for 30 minutes at 35°C
After the 30 minutes of incubation the swatches were rinsed in cold tap water for 10 minutes and dried in a tumble dryer.
The cycle of incubation and rinsing/drying was repeated once - or until the swatches clearly differed in respect to colour and/or fuzz in the swatches surface.
Finally the swatches were graded against the blank (no enzyme) and the standard (e.g. Celluzyme) swatches. Visual grading was performed by a panel of trained graders, and colour was measured with a remission spectrometer. Results are expressed in the table below as "Colour Clarification" (CC) of the swatches obtained per activity unit of enzyme.

EXAMPLE 2
"Colour Clarification" in Household Laundering
The capability of a family 6 endoglucanase to rejuvenate the colour of cotton textile was demonstrated using an assay for determining "Colour Clarification" of cotton cloth in a normal household washing machine.
Swatches of 7×7 cm swatches of black, woven cotton cloth were stapled to one common piece of cloth, and 7×7 cm swatches of blue knitted cotton cloth was stapled to another piece of cloth. This together with a standardised household load of laundry cloth was entered into a household washing machine.
Such loads were laundered in pH 7 buffer, and at the relevant step in the washing cycle enzyme was added: A) A blank, B) Three different dosages of a standard (e.g. the commercially available cellulase preparation Celluzyme™), and C) Two different dosages of the family 6 endoglucanase.
After laundering the loads were dried in a tumble dryer, and the cycle of incubation and rinsing/drying were repeated for a total of 14 times.
Finally the swatches were graded against the blank (no enzyme) and the standard (e.g. Celluzyme™) swatches. Visual grading was performed by a panel of trained graders, and colour was measured with a remission spectrometer. Results are expressed in the table below as "Colour Clarification" (CC) of the swatches obtained per activity unit of enzyme.

EXAMPLE 3
Cloning of Humicola insolens Cel6A and EG VI (Cel6B)
Cel6A and B cDNA clones were identified in a Humicola insolens cDNA expression library (disclosed in WO 91/17244 (Cel6A) and W093/11249(Cel6B)).
The expression plasmids for cel6A and B (pCA6H and pC6H) were constructed by PCR addition of adequate restrictions sites (BamHI-XbaI) to the individual CDS's, and introduction into XbaI- BamHI cut pCaHj418 vector. The resulting DNA sequences from BamHI- XbaI are given in Figures 2 and 3, respectively (the translational initiation codon is underlined in each sequence).
Cel6B (& cel6A) variants with the exeption of larger deletions/inserts (>9bp) were constructed by application of the Chameleon™ Double-stranded, site-directed Mutagenesis kit, from Stratagene. The following synthetic oligo-nucleotide were used as selection primer:
S/M GAATGACTTGGTTGACGCGTCACCAGTCAC, or
M/S GAATGACTTGGTTGAGTACTCACCAGTCAC.
S/M replaces the Seal site in the beta-lactamase gene of the plasmid with a Mlul site and M/S does the reverse. The later is used to introduce secondary mutations in variants generated by the first selection primer.
CA6H4 and 5 were made by SOE PCR utilizing the following primers:



With pCA6H as template (10 ng/100 ml) , Pwo (Boehringer) based PCR reactions were performed, under standard conditions, as recommended by the manufactor, with the following primer pairs:
1: TAKA-F/CA6H4-R
2: TAKA-R/CA6H4-F

3: TAKA-F/CA6H5-R
4: TAKA-R/CA6H5-F
96°C, 2' - 4X( 94 °C,30''- 50°C, 30''- 72°C, 45'') - 25x( 94 °C,30''- 57°C, 30"- 72°C, 45'') -72°C, 7'- 4°C, hold
The resulting products, 1&3 1398 bp, 2&4 153 bp were purified via agarose gel electrophoresis and applied in two new PCR's with templates as listed about 0.1 pmol/100 ml each:
5: PCRlprod.+ PCR2prod
6: PCR3prod.+ PCR4prod and TAKA-F/R as primers: 96°C, 2' - 4x( 94 °C, 30''-72°C, 30''-72°C, 45'') - 20x( 94 °C, 30''-57°C, 30''- 72°C, 45'') -72°C, 7'- 4°C, hold.
The resulting products of 1477 bp were purified via agarose gel elctrophoresis, subjected to BamH1-Xba1 restriction nuclease digestions and the resulting 1365 bp bands isolated as above and cloned into pCaHj418 Xba1 - BamH1 vector.

EXAMPLE 4
Trimming of binding cleft loops to increase activity
In order to alter Humicola insolens Cel6A to a Humicola endoglucanase type in order to create an enzyme having improved performance in colour clarification, mutations which reduce the length of one or more of the binding cleft encompassing loops was performed. The extent of the binding cleft encompassing loops can be determined either from the multiple sequence alignment or by solving the three dimensional X-ray structure of Humicola insolens Cel6A and perform the same analysis as described for Humicola insolens EGIV (Cel6B).
From the sequence alignment in fig. 1A/B the binding cleft loop regions of Humicola insolens Cel6A can be found as shown in Fig. 6.
The same analysis could be performed by solving the X-ray structure of Humicola insolens Cel6A catalytic core domain and performing the same analysis as described for the X-ray structure of Humicola insolens EG VI (Cel6B) catalytic core domain. In this case the result is a little different as shown in Fig. 7.
The four longer loops encompassing the binding cleft
(residues Y86-N107, N219-D242, L272-P287 or W308-F331 using Humicola insolens Cel6B numbering) are in the numbering scheme of Humicola insolens Cel6A V173-N195, N307-D330, K360-G376 and W397-F435 (using Humicola insolens Cel6A numbering) when the multiple sequence alignment method is used and it is Y174-N195, N307-D330, K360-Y391 and W397-F435 F435 (using Humicola insolens Cel6A numbering) when the X-ray structure method is used.
Constructions of loop trimming:
In one example (A) the loop W397-F435 (using Humicola insolens Cel6A numbering) which is equivalent to the W308-F331 in Humicola insolens Cel6B is mutagenized altering the sequence from
WVKPGGECDGTSDTTAARYDYHCGLEDALKPAPEAGOWF
to
WVKPGGECDGCGLEAGQF
(the underlined residues have been deleted)
thereby making the binding cleft more accessible and generating color care activity for the variant.
In another example (B) the same loop is shortened as in (A) and three extra mutations is introduced to alter the loop geometry (G420I+L421A+E422G in Humicola insolens Cel6A numbering):
WVKPGGECDGTSDTTAARYDYHCGLEDALKPAPEAGOWF
to
WVKPGGECDGCIAGAGQF
(A) and (B) were made by SOE PCR as described in example 3.
In a similar manner, the cel6A-type cellulases from the species Fusarium oxysporum, Trichoderma reesei, Agaricus bis-pora, Acremonium cellulyticus, Phanerochaete chrysosporium, Penicillium purpurogenum which are aligned in fig. 1A/B can be altered to a Humicola endoglucanase type enzyme (Cel6B-type).

EXAMPLE 5
Resistance to anionic surfactants in detergent.
As described it is possible to stabilize an enzyme against denaturation by anionic tensides by mutation/deletion of surface exposed residue(s) towards more negatively charged residue(s) i.e. removal of positively charged residue(s) and/or the introduction of negatively charged residue(s).
Resistance to anionic surfactants in detergent (A)
Variants of the present invention may show improved performance with respect to an altered sensitivity towards anionic surfactants (tensides). Anionic tensides are products frequently incorporated into detergent compositions. Unfolding of cellulases tested so far, is accompanied by a decay in the intrinsic fluorescence of the proteins. The intrinsic fluorescence derives from Trp side chains (and to a smaller extent Tyr side chains) and is sensitive to the hydrophobicity of the side chain environment. Unfolding leads to a more hydrophilic environment as the side-chains become more exposed to solvent, and this quenches fluorescence.
Fluorescence is followed on a Perkin/Elmer™ LS50 luminescence spectrometer. In practice, the greatest change in fluorescence on unfolding is obtained by excitation at 280 nm and emission at 340 nm. Slit widths (which regulate the magnitude of the signal) are usually 5 nm for both emission and excitation at a protein concentration of 5 μg/ml. Fluorescence is measured in 2-ml quartz cuvettes thermostatted with a circulating water bath and stirred with a small magnet. The magnet-stirrer is built into the spectrometer.
Unfolding can be followed in real time using the available software. Rapid unfolding (going to completion within less than 5-10 minutes) is monitored in the TimeDrive option, in which the fluorescence is measured every few (2-5) seconds. For slower unfolding, four cuvettes can be measured at a time in the cuvette-holder using the Wavelength Program option, in which the fluorescence of each cuvette is measured every 30 seconds. In all cases, unfolding is initiated by adding a small volume
(typically 50 μl) of concentrated enzyme solution to the thermostatted cuvette solution where mixing is complete within a few seconds due to the rapid rotation of the magnet.
Data are measured in the software program GraphPad
Prism. Unfolding fits in all cases to a single-exponential function from which a single half-time of unfolding (or unfolding rate constant) can be obtained. Typical unfolding conditions are 50 mM HEPES pH 7, 0-500 ppm LAS/250 ppm LAS, 25°C.
In both cases, the protein concentration is 5-10 μg/ml (the protein concentration is not crucial, as LAS is in excess).



From this table it is seen that mutation of residues resulting in the removal of positively charged residue(s) and/or the introduction of negatively charged residue (s) increase the resistance towards LAS.
Resistance to anionic surfactants in detergent (B)
The alteration of the surface electrostatics of an enzyme will influence the sensibility towards anionic tensides such as LAS (linear alkylbenzenesulfonate) . Especially variants where positive charged residues have been removed and/or negatively charged residues have been introduced will increase the resistance towards LAS, whereas the opposite, i.e. the introduction of positively charged residues and/or the removal of negatively charged residues will lower the resistance towards LAS. The residues Arg (R), Lys (K) and His (H) are viewed as positively or potentially positively charged residue and the residues Asp (D), Glu (E) and Cys (C) if not included in a disulphide bridge are viewed as negatively or potentially negatively charged residues. Positions already containing one of these residues are the primary target for mutagenesis, secondary targets are positions which has one of these residues on an equivalent position in another cellulase, and third target are any surface exposed re-sidue. In this experiment wild type Humicola insolens Cel6B cellulase are being compared to Humicola insolens Cel6B cellulase variants belonging to all three of the above groups, comparing the stability towards LAS in detergent.
Cellulase resistance to anionic surfactants was measured as activity on PASC (phosphoric acid swollen cellulose) in the presence of anionic surfactant vs. activity on PASC in the absence of anionic surfactant.
The reaction medium contained 5.0 g/l of a commercial regular powder detergent from the detergent manufacturer NOPA Den-mark. The detergent was formulated without surfactants for this experiment and pH adjusted to pH 7.0. Further the reaction medium included 0.5 g/l PASC and was with or without 1 g/l LAS
(linear alkylbenzenesulphonate), which is an anionic surfactant, and the reaction proceeded at the temperature 30°C for 30 minutes. Cellulase was dosed at 0.20 S-CEVU/1. After the 30 minutes of incubation the reaction was stopped with 2 N NaOH and the amount of reducing sugar ends determined through reduction of phydroxybenzoic acid hydrazide. The decrease in absorption of re duced p-hydroxybenzoic acid hydrazide relates to the cellulase activity.
The type of mutation and the resistance towards LAS for variants with increased LAS resistance is summarized in the following table:


From this table it is seen that mutation of residues resulting in the removal of positively charged residue (s) and/or the introduction of negatively charged residue (s) increase the resistance towards LAS.

EXAMPLE 6
Improving stability towards anionic surfactants of any Humicola- like family 6 cellulase
In order to stabilize any Humicola-like family 6 cellulase towards anionic surfactants, residues on the surface of the molecule should be mutated towards a more negatively charged surface as described in the text resulting in the removal of positively charged residue(s) and/or introduction of negatively charged residue(s). The residues on the surface of the molecule can be detected from the multiple sequence alignment in the following way: Residues at a position in the sequence equivalent to residues on the surface of the Humicola insolens Cel6A X-ray structure are thought to most likely be on the surface of a given family 6 cellulase. In the case of an insertion the inserted residue(s) are considered as being on the surface of the molecule if one of the flanking residues of the insertion is considered as being on the surface.
To achieve improved performance of the enzyme in color clarification a linker and a CBD have to bee attached to the catalytic core domain. In the cases where the wild type enzyme does not include the linker region and/or the CBD these segments can be included from another enzyme e.g. Humicola insolens Cel6B by standard techniques to achive a hybrid enzyme with the desired improved properties.
(A) Neocallimastix patriciarum
Taking the Neocallimastix patriciarum SPTREMBL entry q12646 as an example figure 8 shows the residues considered as being on the surface of the Neocallimastix patriciarum catalytic core domain.
Preferably potentially positively charged residues should be mutagenized to neutral or negatively charged residues. In the case of Neocallimastix patriciarum this results to (using the numbering scheme of Humicola insolens Cel6B): K4 , K16, R27, K43, K45, K67, K72, R91, K113, R122, R125, K131, R156, H159, K160, H183, K195, K201, K212, R214, K249, H262, R293, R295, K310, R318e, R323C, H332, R340, R343.
More preferably the positions which hold an Arg: R27, R91, R122, R125, R156, R214, R293, R295, R318e, R323c, R340 or R343.

Or preferably positions which in other Humicola-like family 6 cellulases have been shown to improve stability towards anionic tensides.
(B) Orpinomγces sp. CelA
Taking the Orpinomyces sp. CelA SPTREMBL entry p78720
(residues 128-459) as an example figure 9 shows the residues considered as being on the surface of the Orpinomyces sp. CelA catalytic core domain.
Preferably potentially positively charged residues should be mutagenized to neutral or negatively charged residues. In the case of Orpinomyces sp. CelA this results to (using the numbering scheme of Humicola insolens Cel6B): K16, K26, K27, K38, K40, K43, K45, K72, K111, K113, K128, K131, R153, R156, H159, K160, K169, H174, K176, H183, K201, K212, R214, K245, H247, R252, K257, R260, K262, R269, K286, R293, K295, K310, R318e, R321 or H332.
More preferably the positions which hold an Arg: R153, R156, R214, R252, R260, R269, R293, R318e or R321.
Or preferably positions which in other Humicola-like family 6 cellulases have been shown to improve stability towards anionic tensides (surfactants).

(C) Orpinomyces sp. CelC
Taking the Orpinomyces sp. CelC SPTREMBL entry p78721
(residues 127-449) as an example figure 10 shows the residues considered as being on the surface of the Orpinomyces sp. CelA catalytic core domain.
Preferably potentially positively charged residues should be mutagenized to neutral or negatively charged residues. In the case of Orpinomyces sp. CelC this results to (using the numbering scheme of Humicola insolens Cel6B) : K16, R27, K42, K43, R64, K72, R91, R131, H156, H159, K160, K169, K173, R176, H183, R195, R205, K212, R214, H247, R252, R257, R260, K262, R272, K286, R293, K310, R320 or H332.
More preferably the positions which hold an Arg: R27, R64, R91, R131, R176, R195, R205, R214, R252, R257, R260, R272, R293 or R320.
Or preferably positions which in other Humicola-like family 6 cellulases have been shown to improve stability towards anionic tensides (surfactants).

EXAMPLE 7
Alteration of pH activity profile
The pH activity profile of a cellulase is governed by the pH dependent behavior of specific titratable groups, typically the acidic residues in the active site. The pH profile can be altered by changing the electrostatic environment of these residues, either by substitution of residues involving charged or potentially charged groups such as Arg (R), Lys (K), Tyr (Y), His (H), Glu (E), Asp (D) or Cys (C) if not involved in a disulphide bridge or by changes in the surface accessibility of these specific titratable groups by mutation of these specific residues on the surface of the enzyme close to the proton donor as described above or by mutation of residues in the vicinity of the binding cleft as described herein, preferably by mutation (s) in the binding cleft within 5h, more preferably 2.5Å, of the substrate, or preferably by mutations within 10Å, more preferably δÅ, from the active site (D139).
In this example Humicola insolens Cel6B cellulase and variants of Humicola insolens Cel6B cellulase involving substi tution of charged or potentially charged residues have been tested for activity towards PASC at pH 7 and pH 10, respectively.

In order to determine the pH optimum for cellulases we have selected organic buffers because it is common known that e.g. borate forms covalent complexes with mono- and oligo-saccharides and phosphate can precipitate with Ca-ions. In DATA FOR
BIOCHEMICAL RESEARCH Third Edition OXFORD SCIENCE PUBLICATIONS page 223 to 241, suitable organic buffers has been found. In respect of their pKa values we decided to use Na-acetate in the range 4 - 5.5, MES at 6.0, MOPS in the range 6.5 - 7.5, Nabarbiturate 8.0 - 8.5 and glycine in the range 9.0 - 10.5.
Method:
The method is enzymatic degradation of carboxy-methylcellulose, at different pH's. Buffers are prepared in the range 4.0 to 10.5 with intervals of 0.5 pH unit. The analyze is based on formation of new reducing ends in carboxy-methyl-cellulose, these are visualized by reaction with PHBAH in strong alkaline environment, were they forms a yellow compound with absorption maximum at 410 nm.
Experimental Protocol:
Buffer preparation: 0.2 mol of each buffer substance is weighed out and dissolved in 1 liter of Milli Q water. 250 ml 0.2M buffer solution and 200 ml Milli Q water is mixed. The pH are measured using Radiometer PHM92 labmeter calibrated using standard buffer solutions from Radiometer. The pH of the buffers are adjusted to actual pH using 4M NaOH or 4M HCl and adjusted to total 500 ml with water. When adjusting Na-barbiturate to pH 8.0 there might be some precipitation, this can be re-dissolved by heating to 50°C.
Acetic acid 100% 0.2 mol = 12.01 g.
MES 0.2 mol = 39.04 g.
MOPS 0.2 mol = 41.86 g.
Na-barbiturate 0.2 mol = 41.24 g.
Glycine 0.2mol = 15.01 g.
Buffers: as disclosed in WO 98/12307, page 89.
The actual pH is measured in a series treated as the main values, but without stop reagent, pH is measured after 20 min. incubation at 40 °C.

Substrate Preparation:
2.0 g CMC , in 250 ml conic glass flask with a magnet rod, is moistened with 2.5 ml. 96% ethanol, 100 ml. Milli Q water is added and then boiled to transparency on a heating magnetic stirrer. Approximately 2 min. boiling. Cooled to room
temperature on magnetic stirrer.
Stop Reagent:
1.5 g PHBAH and 5 g K-Na-tartrate dissolved in 2% NaOH. Procedure:
There are made 3 main values and 2 blank value using 5 ml glass test tubes. (1 main value for pH determination )


Mixing on a Heidolph REAX 2000 mixer with permanent mix and maximum speed (9). No stirring during incubation on water bath with temperature control. Immediately after adding PHBAH-reagent and mixing the samples are boiled 10 min. Cooled in cold tap water for 5 min. Absorbance read at 410 nm.

Determination of Activity
The absorbance at 410 nm from the 2 Main values are added and divided by 2 and the 2 Blank values are added and divided by

2, the 2 mean values are subtracted. The percentages are
calculated by using the highest value as 100%.
The measured pH is plotted against the relative activity. Buffer reagents as disclosed in W098/ 12307, page 90.

Cellulase resistance to anionic surfactants was measured as activity on PASC (phosphoric acid swollen cellulose) at neutral pH (pH 7.0) vs. activity on PASC at alkaline pH (pH 10.0).
The reaction medium contained 5.0 g/l of a commercial regular powder detergent from the detergent manufacturer NOPA Denmark. The pH was adjusted to pH 7.0 and pH 10.0, respectively. Further the reaction medium included 0.5 g/l PASC, and the reaction proceeded at the temperature 30°C for 30 minutes. Cellulase was dosed at 0.20 S-CEVU/l. After the 30 minutes of incubation the reaction was stopped with 2 N NaOH and the amount of reducing sugar ends determined through reduction of p-hydroxybenzoic acid hydrazide. The decrease in absorption of reduced p- hydroxybenzoic acid hydrazide relates to the cellulase activity. The results are presented below, the activity at pH 10 relative to pH 7 is compared to that of wild type Humicola insolens Cel6B cellulase.


From the above table it is seen that the relative alkaline activity can be increased by creating variants involving potentially charged residues which are mutated towards a more negatively charged residue and/or by altering residues not more than 5Å from the residues in the binding cleft.

EXAMPLE 8
Variants with improved catalytic properties
The following site directed variants of Humicola insolens Cel6B (EG VI) endoglucanase were prepared as described above: K20E, K103Q, K103E, S94D, A95G.
The specific activity on CMC of the variants and the wild- type H. insolens endoglucanase were measured in the ECU (endo- cellulase unit) assay (cf. above under "Cellulolytic Activity") with the following results:

Wild-type 100%
K20E 109%
K103Q 120%
K103E 115%
S94D 180%
A95G 116%
All the tested variants have improved specific activity.

LITERATURE

Damude, H.G., V. Ferro, S.G. Withers, and R.A.J. Warren. 1996. Substrate specificity of endoglucanase A from Cellulomonas fimi: fundamental differences between endoglucanases and exoglucanases from family 6. Biochem. J., 315:467-472.

Denman, S., G-P. Xue, and B. Patel. 1996. Characterization of a Neocallimastix patriciarum cellulase cDNA (celA) homologous to Trichoderma reesei cellobiohydrolase II. Appl. Environ. Microbiol., 62 (6):1889-1896.

Henrissat, B. 1991. A classification of glycosyl hydrolases based on amino acid sequence similaritites. Biochem. J.,
280:309-316.

Henrissat, B., and A. Bairoch. 1993. New families in the classification of glycosyl hydrolases based on amino acid sequence similaritites. Biochem. J., 293:781-788.

McCarter, J.D., and S.G. Withers. 1994. Mechanisms of enzymatic glycoside hydrolysis. Current Opinion in Structural Biology, 4:885-892.

Irwin, D.C., Spezio, M., and Wilson, D.B. Activity studies of 8 purified cellulases - specificity, synergism, and binding domain effects. Biotechnology and Bioengineering. 42:1002-1013, 1993.

Shen, H., Meinke, A., Tomme, P. Damude, E. K., Kilburn, D. G., Miller, R. C. Warren, R. A. J. and Gilkes, N. R. Cellulomonas fimi Cellobiohydrolases. In Enzymatic Degradation of insoluble Carbohydrates, ed. J. N. Sadler and M. H. Penner ACS Symposium sreies 618, Washington 1995. Chapter 12, p. 174-196.

Sambrook et al. (1989) Molecular cloning: A laboratory manual, Cold Spring Harbor lab., Cold Spring Harbor, NY.

Schulein, M., Tikhomirov, D. F. and Schou, C. Humicola insolens Alkaline Cellulases. In Proceedings of the second TRICEL sympo sium on Trichoderma reesei cellulases and other hydrolases, Espoo 1993. Ed. by P. Souminen and T. Reinikainen. Foundation for Biotechnical and Industrial Fermentation Research 8, 1993, p. 109-116.

APPENDIX 1
The structural coordinated of the three-dimensional structure of the Humicola insolens Cel6B catalytic core domain.

The structural coordinates of the Humicola insolens Cel6B catalytic core domain as determined by X-ray crystallography. The format of the coordinates is the conventional Brookhaven Protein Data Bank (PDB) format:



APPENDIX 2

The structural coordinated of the three-dimensional structure of the Humicola insolens Cel6A catalytic core domain
The structural coordinates of the Humicola insolens Cel6A catalytic core domain as determined by X-ray crystallography. The format of the coordinates is the conventional Brookhaven Protein Data Bank (PDB) format. The residue numbering follows the sequence shown in appendix 2. Only the residues from G91 to F450 are detected in the X-ray structure.