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EP1261698A1 - Family 5 xyloglucanases - Google Patents

Family 5 xyloglucanases

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Publication number
EP1261698A1
EP1261698A1 EP01909563A EP01909563A EP1261698A1 EP 1261698 A1 EP1261698 A1 EP 1261698A1 EP 01909563 A EP01909563 A EP 01909563A EP 01909563 A EP01909563 A EP 01909563A EP 1261698 A1 EP1261698 A1 EP 1261698A1
Authority
EP
European Patent Office
Prior art keywords
enzyme
xyloglucanase
seq
dna
sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP01909563A
Other languages
German (de)
French (fr)
Inventor
Reinhard Wilting
Mads Eskelund Bjoernvad
Markus Sakari Kauppinen
Martin Schülein
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Novozymes AS
Original Assignee
Novozymes AS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Novozymes AS filed Critical Novozymes AS
Publication of EP1261698A1 publication Critical patent/EP1261698A1/en
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/38—Products with no well-defined composition, e.g. natural products
    • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
    • C11D3/38636—Preparations containing enzymes, e.g. protease or amylase containing enzymes other than protease, amylase, lipase, cellulase, oxidase or reductase
    • D—TEXTILES; PAPER
    • D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
    • D06L—DRY-CLEANING, WASHING OR BLEACHING FIBRES, FILAMENTS, THREADS, YARNS, FABRICS, FEATHERS OR MADE-UP FIBROUS GOODS; BLEACHING LEATHER OR FURS
    • D06L1/00—Dry-cleaning or washing fibres, filaments, threads, yarns, fabrics, feathers or made-up fibrous goods
    • D06L1/12—Dry-cleaning or washing fibres, filaments, threads, yarns, fabrics, feathers or made-up fibrous goods using aqueous solvents
    • D06L1/14—De-sizing
    • D—TEXTILES; PAPER
    • D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
    • D06M—TREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS OR FIBROUS GOODS MADE FROM SUCH MATERIALS
    • D06M16/00—Biochemical treatment of fibres, threads, yarns, fabrics, or fibrous goods made from such materials, e.g. enzymatic
    • D06M16/003—Biochemical treatment of fibres, threads, yarns, fabrics, or fibrous goods made from such materials, e.g. enzymatic with enzymes or microorganisms
    • D—TEXTILES; PAPER
    • D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
    • D06M—TREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS OR FIBROUS GOODS MADE FROM SUCH MATERIALS
    • D06M2101/00—Chemical constitution of the fibres, threads, yarns, fabrics or fibrous goods made from such materials, to be treated
    • D06M2101/02—Natural fibres, other than mineral fibres
    • D06M2101/04—Vegetal fibres
    • D06M2101/06—Vegetal fibres cellulosic

Definitions

  • the present invention relates to xyloglucanases belonging to family 5 of glycosyl hydrolases, preferably to enzymes exhibiting xyloglucanase activity as their major enzymatic activity m the neutral and alkaline pH ranges; to a method of producing such enzymes; and to methods for using such enzymes m the textile, detergent and cellulose fiber processing industries .
  • Xyloglucan is a major structural polysaccha ⁇ de m the primary (growing) cell wall of plants. Structurally, xyloglucans consists of a cellulose-like beta-1 , 4-lmked glucose backbone, which is frequently substituted with various side chains. The xyloglucans of most dicotyledonous plants, some monocotyledons and gymnosperms are highly branched polysaccharides m which approx. 75% of the glucose residues m the backbone bear a glycosyl side chain at O- ⁇ . The glycosyl residue that is directly attached to the branched glucose residue is invariably alfa-D-xylose .
  • the xyloglucans Up to 50% of the side chains m the xyloglucans contain more than one residue due to the presence of beta-D-galactose or alfa-L-fucose- (1-2) -beta-D- galactose moieties at 0-2 of the xylose residues (C. Ohsumi and T. Hayashi (1994) Plant and Cell Physiology 35:963-967; G. J. McDougall and S. C. Fry (1994) Journal of Plant Physiology 143:591-595; J. L. Acebes et al . (1993) Phytochemistry 33:1343- 1345) .
  • the xyloglucan extracted from cotton fibers yielded glucose, xylose, galactose and fucose m the ratio of 50:29:12:7 (Hayashi et al . , 1988).
  • Xyloglucans produced by solanaceous plants are unusual m that typical only 40% of the beta-1 , 4-lmked glucose residues bear a glycosyl side chain at 0-6. Furthermore, up to 60% of the xylose residues are substituted at 0-2 with alfa-L- arabinose residues and some solanaceous plants, such as potato, also have xyloglucans with beta-D-galactose substituents at 0-2 of some of the xylose residues (York et al (1996)) .
  • Xyloglucan is believed to function m the primary wall of plants by cross-linking cellulose-micro fibrils, forming a cellulose-xyloglucan network. This network is considered necessary for the structural integrity of primary cell -walls (Carpita et al . , 1993). Another important function of xyloglucan is to act as a repository for xyloglucan subun t oligosaccharides that are physiologically active regulators of plant cell growth. Xyloglucan subunits may also modulate the action of a xyloglucan endotransglycosylase (XET) , a cell wall associated enzyme that has been hypothesized to play a role m the elongation of plant cell walls. Therefore xyloglucan might play an important role m wall loosening and consequently cell expansion (Fry et al . , 1992) .
  • XET xyloglucan endotransglycosylase
  • xyloglucan As the major polysaccha ⁇ de storage reserve.
  • This type of xyloglucan which is localized m massive thickenings on the inside of the seed cotyledon cell wall, is composed mainly of glucose, xylose and galactose (Rose et al . , 1996) .
  • Xyloglucanase activity is not included m the classification of enzymes provided by the Enzyme Nomenclature (1992) . Hitherto, this enzymatic activity has simply been classified as glucanase activity and has often been believed to be identical to cellulolytic activity (EC 3.2.1.4), i.e. activity against ⁇ -1 , 4-glycos ⁇ d ⁇ c linkages m cellulose or cellulose derivative substrates, or at least to be a side activity m enzymes having cellulolytic activity.
  • a true xyloglucanase is a true xyloglucan specific enzyme capable of catalyzing the solubilisation of xyloglucan to xyloglucan oligosaccharides but which does not exhibit substantial cellulolytic activity, e.g. activity against the conventionally used cellulose-like substrates CMC (carboxymethylcellulose) , HE cellulose and Avicel (macrocrystalline cellulose) .
  • CMC carboxymethylcellulose
  • HE cellulose HE cellulose
  • Avicel macrocrystalline cellulose
  • Xyloglucanase activity is described by Vincken et al . (1997) who characterizes three different endoglucanases from Trichoderma viride (similar to T. reesei ) which all have high activity against cellulose or CMC and show that the Endol (belonging to family 5 of glycosyl hydrolases, see Henrissat, B. et al . (1991, 1993)) has essentially no (i.e.
  • the inventors have now found enzymes having substantial xyloglucanase activity, which enzymes belong to family 5 of glycosyl hydrolases and exhibit excellent performance in conventional detergent compositions, especially liquid detergent compositions. All the found xyloglucanases are endogenous to a strain belonging to Paenibacillus pabuli or Paenibacillus sp .
  • the present invention relates to a xyloglucanase enzyme belonging to family 5 of glycosyl hydrolases, which enzyme is endogenous to a strain of Paenibacillus .
  • the strain of Paenibacillus belongs to the group consisting of the species Paenibacillus pabuli , the strain Paenibacillus sp . , DSM 13330, and strains of Paenibacillus sp . having a higher degree of identity with the Paenibacillus pabuli type strain ATCC 43899 than the strain Paenibacillus sp . , DSM 13330, when subjected to 16S RNA analysis .
  • the invention relates in further aspects to a family 5 xyloglucanase which is (a) a polypeptide encoded by the DNA sequence of positions 840-1931 of SEQ ID NO : 1, (b) a polypeptide produced by culturing a cell comprising the sequence of SEQ ID NO: 1 under conditions wherein the DNA sequence is expressed;
  • a xyloglucanase enzyme having a sequence of at least 85% identity to positions 33-395 of SEQ ID NO: 2 when identity is determined by GAP provided in the GCG program package using a GAP creation penalty of 3.0 and GAP extension penalty of 0.1; or
  • the invention provides an expression vector comprising a DNA segment which is e.g. a polynucleotide molecule of the invention; a cell comprising the DNA segment or the expression vector; and a method of producing a exhibiting xyloglucanase enzyme, which method comprises culturing the cell under conditions permitting the production of the enzyme, and recovering the enzyme from the culture.
  • a DNA segment which is e.g. a polynucleotide molecule of the invention
  • a cell comprising the DNA segment or the expression vector
  • a method of producing a exhibiting xyloglucanase enzyme comprises culturing the cell under conditions permitting the production of the enzyme, and recovering the enzyme from the culture.
  • the invention provides an isolated family 5 xyloglucanase enzyme characterized m (1) being free from homologous impurities and (11) being produced by the method described above.
  • the novel enzyme of the present invention is useful for the treatment of cellulosic material, especially cellulose- containing fiber, yarn, woven or non-woven fabric.
  • the treatment can be carried out during the processing of cellulosic material into a material ready for garment manufacture or fabric manufacture, e.g. m the desizmg or scouring step; or during industrial or household laundering of such fabric or garment.
  • the present invention relates to a detergent composition comprising a xyloglucanase enzyme having substantial xyloglucanase activity m the neutral or alkaline range; and to use of the enzyme of the invention for the treatment of cellulose-containing fibers, yarn, woven or non-woven fabric.
  • the present invention has now made it possible to use a xyloglucanase m detergent compositions for removing or bleaching certain soils or stains present on laundry, especially soils and spots resulting from xyloglucan-containmg food, plants, and the like. Further, it is contemplated that treatment with detergent compositions comprising the novel enzyme can prevent binding of certain soils to the xyloglucan left on the cellulosic material.
  • “obtained from” or “obtainable from” as used herein m connection with a specific source means that the enzyme is produced or can be produced by the specific source, or by a cell m which a gene from the source have been inserted.
  • the xyloglucanase of the invention may be obtained from a gram-positive bacterium belonging to a strain of the genus Bacillus, in particular a strain of Paenibacillus .
  • the xyloglucanase of the invention is obtained from the species Paenibacillus pabuli that is represented by the type strain ATCC 43899, this type strain being publicly available from American Type Culture Collection (ATCC) . It is at present contemplated that a DNA sequence encoding an enzyme with an amino acid sequence identity of at least 85% to the enzyme of the invention may be obtained from other strains belonging to the species Paenibacillus pabuli and such strains belonging to the species Paenibacillus sp . which, when subjected to a conventional 16S RNA analysis, have a higher degree of identity with the Paenibacillus pabuli type strain ATCC 43899 than the strain Paenibacillus sp .
  • ATCC American Type Culture Collection
  • the strain Paenibacillus sp . , DSM 13330 has an identity, compared with the strain Paenibacillus pabuli ATCC 43899, of at least 95%, more specifically of at least 97%. Further, the strain Paenibacillus sp . was deposited by the inventors according to the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg lb, D- 38124 Braunschweig, Federal Republic of Germany, on 17 February 2000 under the deposition number DSM 13330. The deposit was made by Novo Nordisk A/S and was later assigned to Novozymes A/S.
  • a plasmid comprising a DNA sequence encoding a xyloglucanase of the invention has been transformed into a strain of the Escherichia coli which was deposited by the inventors according to the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg lb, D- 38124 Braunschweig, Federal Republic of Germany, on 6 December 1999 under the deposition number DSM 13183. The deposit was made by Novo Nordisk A/S and was later assigned to Novozymes A/S. It is contemplated that the DNA sequence of this plasmid comprises the DNA sequence of SEQ ID NO: 1.
  • the term "enzyme preparation” is intended to mean either be a conventional enzymatic fermentation product, possibly isolated and purified, from a single species of a microorganism, such preparation usually comprising a number of different enzymatic activities; or a mixture of monocomponent enzymes, preferably enzymes derived from bacterial or fungal species by using conventional recombmant techniques, which enzymes have been fermented and possibly isolated and purified separately and which may originate from different species, preferably fungal or bacterial species; or the fermentation product of a microorganism which acts as a host cell for expression of a recombmant xyloglucanase, but which microorganism simultaneously produces other enzymes, e.g. xyloglucanases, proteases, or cellulases, being naturally occurring fermentation products of the microorganism, i.e. the enzyme complex conventionally produced by the corresponding naturally occurring microorganism.
  • expression vector denotes a DNA molecule, linear or circular, that comprises a segment encoding a polypeptide of interest operably linked to additional segments that provide for its transcription.
  • additional segments may include promoter and terminator sequences, and may optionally include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, and the like.
  • Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both.
  • the expression vector of the invention may be any expression vector that is conveniently subjected to recombmant DNA procedures, and the choice of vector will often depend on the host cell into which the vector is to be introduced.
  • the vector may be an autonomously replicating vector, i.e.
  • a vector that exists as an extra chromosomal entity, the replication of which is independent of chromosomal replication e.g. a plasmid.
  • 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.
  • the term "recombmant expressed” or “recombmantly expressed” used herein connection with expression of a polypeptide or protein is defined according to the standard definition the art. Recombmant expression of a protein is generally performed by using an expression vector as described immediately above.
  • isolated when applied to a polynucleotide molecule, denotes that the polynucleotide has been removed from its natural genetic milieu and is thus free of other extraneous or unwanted coding sequences, and is m a form suitable for use within genetically engineered protein production systems.
  • isolated molecules are those that are separated from their natural environment and include cDNA and genomic clones.
  • Isolated DNA molecules of the present invention are free of other genes with which they are ordinarily associated, but may include naturally occurring 5 ' and 3 ' untranslated regions such as promoters and terminators. The identification of associated regions will be evident to one of ordinary skill m the art (see for example, Dynan and Tijan, Nature 316 : 774-78, 1985).
  • the term "an isolated polynucleotide” may alternatively be termed "a cloned polynucleotide” .
  • the term "isolated” indicates that the protein is found m a condition other than its native environment.
  • the isolated protein is substantially free of other proteins, particularly other homologous proteins (i.e. "homologous impurities” (see below) ) .
  • homologous impurities i.e. "homologous impurities” (see below)
  • isolated protem/polypeptide may alternatively be termed "purified protem/polypeptide” .
  • homologous impurities means any impurity (e.g. another polypeptide than the polypeptide of the invention) , which originate from the homologous cell where the polypeptide of the invention is originally obtained.
  • obtained from as used herein m connection with a specific microbial source, means that the polynucleotide and/or polypeptide produced by the specific source, or by a cell m which a gene from the source have been inserted.
  • operably linked when referring to DNA segments, denotes that the segments are arranged so that they function m concert for their intended purposes, e.g. transcription initiates m the promoter and proceeds through the coding segment to the terminator
  • polynucleotide denotes a single- or double- stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end.
  • Polynucleotides include RNA and DNA, and may be isolated from natural sources, synthesized in vi tro, or prepared from a combination of natural and synthetic molecules .
  • complements of polynucleotide molecules denotes polynucleotide molecules having a complementary base sequence and reverse orientation as compared to a reference sequence. For example, the sequence 5' ATGCACGGG 3' is complementary to 5 ' CCCGTGCAT 3 ' .
  • degenerate nucleotide sequence denotes a sequence of nucleotides that includes one or more degenerate codons (as compared to a reference polynucleotide molecule that encodes a polypeptide) .
  • Degenerate codons contain different triplets of nucleotides, but encode the same ammo acid residue (i.e., GAU and GAC triplets each encode Asp) .
  • promoter denotes a portion of a gene containing DNA sequences that provide for the binding of RNA polymerase and initiation of transcription. Promoter sequences are commonly, but not always, found m the 5' non-codmg regions of genes.
  • secretory signal sequence denotes a DNA sequence that encodes a polypeptide (a "secretory peptide") that, as a component of a larger polypeptide, directs the larger polypeptide through a secretory pathway of a cell m which it is synthesized.
  • secretory peptide a polypeptide that, as a component of a larger polypeptide, directs the larger polypeptide through a secretory pathway of a cell m which it is synthesized.
  • the larger peptide is commonly cleaved to remove the secretory peptide during transit through the secretory pathway.
  • an isolated polynucleotide of the invention will hybridize to similar sized regions of SEQ ID NO: 1 or SEQ ID NO: 3, or a sequence complementary thereto, under at least medium stringency conditions .
  • polynucleotides of the invention will hybridize to a denatured double- stranded DNA probe comprising either the full sequence shown m SEQ ID NO: 1 or the sequence shown m positions 840-1931 of SEQ ID NO: 1 or the full sequence shown SEQ ID NO: 3 or the sequence shown m positions 693-1896 of SEQ ID NO : 3 or any probe comprising a subsequence of SEQ ID NO: 5 or SEQ ID NO: 3 having a length of at least about 100 base pairs under at least medium stringency conditions, but preferably at high stringency conditions as described m detail below.
  • Suitable experimental conditions for determining hybridization at medium or high stringency between a nucleotide probe and a homologous DNA or RNA sequence involve pre -soaking of the filter containing the DNA fragments or RNA to hybridize m 5 x SSC (Sodium chloride/Sodium citrate,
  • the isolated polynucleotides of the present invention include DNA and RNA.
  • Methods for isolating DNA and RNA are well known m the art.
  • DNA and RNA encoding genes of interest can be cloned m Gene Banks or DNA libraries by means of methods known m the art .
  • Polynucleotides encoding polypeptides having endoglucanase activity of the invention are then identified and isolated by, for example, hybridization or PCR.
  • the present invention further provides counterpart polypeptides and polynucleotides from different bacterial strains (orthologs or paralogs) .
  • xyloglucanase polypeptides from gram-positive alkalophilic strains, including species of Bacillus .
  • xyloglucanase peptides from strains of Paenibacillus, which are very closely related to the species Paenibacillus pabuli , exemplified by the strain ATCC 43899 that is the type strain of Paenibacillus pabuli .
  • Species homologues of a polypeptide with xyloglucanase activity of the invention can be cloned using information and compositions provided by the present invention m combination with conventional cloning techniques.
  • a DNA sequence of the present invention can be cloned using chromosomal DNA obtained from a cell type that expresses the protein. Suitable sources of DNA can be identified by probing Northern blots with probes designed from the sequences disclosed herein. A library is then prepared from chromosomal DNA of a positive cell line.
  • a DNA sequence of the invention encoding an polypeptide having xyloglucanase activity can then be isolated by a variety of methods, such as by probing with probes designed from the sequences disclosed m the present specification and claims or with one or more sets of degenerate probes based on the disclosed sequences.
  • a DNA sequence of the invention can also be cloned using the polymerase chain reaction, or PCR (Mullis, U.S.
  • the DNA library can be used to transform or transfect host cells, and expression of the DNA of interest can be detected with an antibody (monoclonal or polyclonal) raised against the xyloglucanase cloned from Paenibacillus pabuli , e.g. from the type strain deposited as ATCC 43899, or from Paenibacillus sp . , DSM 13330, expressed and purified as described in Materials and Methods and the examples, or by an activity test relating to a polypeptide having xyloglucanase activity.
  • an antibody monoclonal or polyclonal
  • sequence of amino acids in positions 33-395 of SEQ ID NO: 2 and positions 33-400 of SEQ ID NO : 4, respectively, is a mature xyloglucanase sequence comprising the catalytic active domain.
  • sequence of am o acids of SEQ ID NOS: 5-9 represents N-terminal or C-terminal sequences of mature xyloglucanase sequences, cf. example 2.
  • the present invention also provides xyloglucanase polypeptides that are substantially homologous to the polypeptide of amino acids in position 33-395 of SEQ ID NO: 2 and species homologs (paralogs or orthologs) thereof.
  • substantially homologous is used herein to denote polypeptides having 85%, preferably at least 88%, more preferably at least 90%, and even more preferably at least 95%, sequence identity to the sequence shown in amino acids nos. 33- 395 of SEQ ID NO: 2 or its orthologs or paralogs.
  • Such polypeptides will more preferably be at least 98% identical to the sequence shown in amino acids in positions 33-395 of SEQ ID NC : 2 or its orthologs or paralogs.
  • 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 m its entirety.
  • GAP is used with the following settings for polypeptide sequence comparison: GAP creation penalty of 3.0 and GAP extension penalty of 0.1.
  • the following sequence identity was found for the appended SEQ ID NOS: 2 and 4:
  • Sequence identity of polynucleotide molecules is determined by similar methods using GAP with the following settings for DNA sequence comparison: GAP creation penalty of 5.0 and GAP extension penalty of 0.3.
  • Substantially homologous proteins and polypeptides are characterized as having one or more ammo acid substitutions, deletions or additions. These changes are preferably of a minor nature, that is conservative ammo acid substitutions (see Taole 2) and other substitutions that do not significantly affect the folding or activity of the protein or polypeptide; small deletions, typically of one to about 30 ammo acids; and small ammo- or carboxyl -terminal extensions, such as an am o- termmal methionme residue, a small linker peptide of up to about 20-25 residues, or a small extension that facilitates purification (an affinity tag), such as a poly-histidme tract, protein A (Nilsson et al . , EMBO J.
  • an affinity tag such as a poly-histidme tract, protein A (Nilsson et al . , EMBO J.
  • non-standard ammo acids such as 4 -hydroxyprolme, 6-iV-methyl lysme, 2- am oisobuty ⁇ c acid, isovaline and a-methyl serme
  • non-standard ammo acids such as 4 -hydroxyprolme, 6-iV-methyl lysme, 2- am oisobuty ⁇ c acid, isovaline and a-methyl serme
  • Unnatural ammo acids can be chemically synthesized, or preferably, are commercially available, and include pipecolic acid, thiazolidme carboxylic acid, dehydroprolme, 3- and 4-
  • Essential ammo acids m the xyloglucanase polypeptides of the present invention can be identified according to procedures known m the art, such as site-directed mutagenesis or alan e- scannmg mutagenesis (Cunningham and Wells, Science 244 : 1081-
  • Mutagenesis/shuffling methods as disclosed above can be combined with high-throughput , automated screening methods to detect activity of cloned, mutagenized polypeptides m host cells.
  • 35 polypeptides can be recovered from the host cells and rapidly sequenced using modern equipment . These methods allow the rapid determination of the importance of individual ammo acid residues m a polypeptide of interest, and can be applied to polypeptides of unknown structure.
  • the xyloglucanase enzyme of the invention may, m addition to the enzyme core comprising the catal tically domain, also comprise a cellulose binding domain (CBD) , the cellulose binding domain and enzyme core (the catalytically active domain) of the enzyme being operably linked.
  • the cellulose binding domain (CBD) may exist as an integral part the encoded enzyme, or a CBD from another origin may be introduced into the xyloglucanase thus creating an enzyme hybrid.
  • the term "cellulose-bmdmg domain” is intended to be understood as defined by Peter Tomme et al . "Cellulose-Bmdmg Domains: Classification and Properties" "Enzymatic Degradation of Insoluble Carbohydrates", John N.
  • CBDs are found m various enzymes such as cellulases, xylanases, mannanases, arabinofuranosidases , acetyl esterases and chitmases.
  • CBDs have also been found m algae, e.g. the red alga Porphyra purpurea as a non-hydrolytic polysaccharide- bmdmg protein, see Tomme et al . , op . ci t .
  • Enzyme hybrids are known m the art, see e.g. WO 90/00609 and WO 95/16782, and may be prepared by transforming into a host cell a DNA construct comprising at least a fragment of DNA encoding the cellulose- bmdmg domain ligated, with or without a linker, to a DNA sequence encoding the xyloglucanase and growing the host cell to express the fused gene. Enzyme hybrids may be described by the following formula:
  • CBD is the N-termmal or the C-termmal region of an ammo acid sequence corresponding to at least the cellulose- bmdmg 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 about 100 ammo acids, more preferably of from 2 to 40 ammo acids
  • X is an N-termmal or C-termmal region of a polypeptide encoded by the polynucleotide molecule of the invention.
  • Polyclonal antibodies, especially monospecific polyclonal antibodies, to be used determining immunological cross- reactivity may be prepared by use of a purified xyloglucanolytic enzyme. More specifically, antiserum against the xyloglucanase of the invention may be raised by immunizing rabbits (or other rodents) according to the procedure described by N. Axelsen et al . m: A Manual of Quantitative Immunoelectrophoresis , Blackwell Scientific Publications, 1973, Chapter 23, or A. Johnstone and R. Thorpe, Immunochemistry m Practice, Blackwell Scientific Publications, 1982 (more specifically p. 27-31) .
  • Purified lmmunoglobulms may be obtained from the antisera, for example by salt precipitation ((NH 4 ) 2 S0 4 ) , followed by dialysis and ion exchange chromatography, e . g. on DEAE-Sephadex.
  • Immunochemical characterization of proteins may be done either by Outcherlony double-diffusion analysis (0. Ouchterlony m: Handbook of Experimental Immunology (D.M. Weir, Ed.), Blackwell Scientific Publications, 1967, pp. 655-706), by crossed immunoelectrophoresis (N. Axelsen et al . , supra, Chapters 3 and 4), or by rocket immunoelectrophoresis (N. Axelsen et al . , Chapter 2) .
  • a recombmant vector comprising a DNA construct encoding the enzyme of the invention may be any vector, which may con- veniently be subjected to recombmant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced.
  • the vector may be an autonomously replicating vector, i.e. a vector that exists as an extra chromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid.
  • the vector may be one which, when introduced into a host cell, is integrated into the host cell genome m part or m its entirety and replicated together with the chromosome (s) into which it has been integrated.
  • the vector is preferably an expression vector which the DNA sequence encoding the enzyme of the invention is operably linked to additional segments required for transcription of the DNA.
  • the expression vector is derived from plasmid or viral DNA, or may contain elements of both.
  • operably linked indicates that the segments are arranged so that they function in concert for their intended purposes, e.g. transcription initiates m a promoter and proceeds through the DNA sequence coding for the enzyme .
  • the promoter may be any DNA sequence, which shows transc ⁇ ptional activity m the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell .
  • 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 amyloliquefaci ens alpha-amylase gene, the Bacillus subtilis alkaline protease gene, or the Bacillus pumilus xylosidase gene, or the phage Lambda P R or P L promoters or the E. coll lac, trp or tac promoters.
  • the DNA sequence encoding the enzyme of the invention may also, if necessary, be operably connected to a suitable terminator.
  • the recombmant vector of the invention may further comprise a DNA sequence enabling the vector to replicate m the host cell m question.
  • the vector may also comprise a selectable marker, e.g. a gene the product of which complements a defect m the host cell, or a gene encoding resistance to e.g. antibiotics like kanamyc , chloramphenicol , erythromycm, tetracyclme, spectmomycme, or the like, or resistance to heavy metals or herbicides .
  • a secretory signal sequence also known as a leader sequence, prepro sequence or pre sequence
  • the secretory signal sequence is joined to the DNA sequence encoding the enzyme m the correct reading frame .
  • Secretory signal sequences are commonly positioned 5 ' to the DNA sequence encoding the enzyme.
  • the secretory signal sequence may be that normally associated with the enzyme or may be from a gene encoding another secreted protein.
  • the cloned DNA molecule introduced into the host cell may be either homologous or heterologous to the host m question. If homologous to the host cell, i.e. produced by the host cell m nature, it will typically be operably connected to another promoter sequence or, if applicable, another secretory signal sequence and/or terminator sequence than m its natural environment.
  • the term "homologous” is intended to include a DNA sequence encoding an enzyme native to the host organism m question.
  • heterologous is intended to include a DNA sequence not expressed by the host cell m nature. Thus, the DNA sequence may be from another organism, or it may be a synthetic sequence.
  • the host cell into which the cloned DNA molecule or the recombmant vector of the invention is introduced may be any cell, which is capable of producing the desired enzyme and includes bacteria, yeast, fungi and higher eukaryotic cells.
  • Examples of bacterial host cells which on cultivation are capable of producing the enzyme of the invention may be a gram-positive bacteria such as a strain of Bacillus, m particular Bacillus alkalophilus , Bacillus amyloliquefaciens, Bacillus brevis, Bacillus lautus, Bacillus lentus , Bacillus licheniformis , Bacillus circulans , Bacillus coagulans , Bacillus mega theri um, Bacillus stearothermophilus , Bacillus subtilis and Bacillus thuringi ensis, a strain of Lactobacillus, a strain of Streptococcus, a strain of Streptomyces, m particular Strep to yces 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 m a manner known per se (cf . e.g. Sambrook et al . , supra) .
  • competent cells m a manner known per se (cf . e.g. Sambrook et al . , supra) .
  • enzyme bacteria such as
  • the enzyme may be retained m the cytoplasm, typically as insoluble granules (known as inclusion bodies) , or may be directed to the periplasmic space by a bacterial secretion sequence.
  • the cells are lysed and the granules are recovered and denatured after which the enzyme is refolded by diluting the denaturing agent.
  • 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 .
  • the enzyme When expressing the enzyme m gram-positive bacteria such as a strain of Bacillus or a strain of Streptomyces, the enzyme may be retained m the cytoplasm, or may be directed to the extra cellular medium by a bacterial secretion sequence.
  • 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 m particular Aspergillus awamori , Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, and Fusarium oxysporum, and a strain of Trichoderma , preferably Trichoderma harzianum, Trichoderma reesei and Tri choderma vi ri de .
  • Fungal cells may be transformed by a process involving protoplast formation and transformation of the protoplasts followed by regeneration of the cell wall m a manner known per se .
  • a strain of Aspergillus as a host cell is described m 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 . , m particular Kluyveromyces lacti s and Kluyveromyces marcianus, a strain of Pichia sp .
  • a strain of Saccharo yces a strain of Saccharo yces , m particular Saccharo yces carlsbergensis , Saccharomyces cerevisae, Saccharomyces kluyve ⁇ and Saccharomyces uvarum, a strain of Schizosaccharomyces sp . , m particular Schizosaccharomyces pombe, and a strain of Yarrowia sp . , m particular Yarrowia lipolyti ca .
  • 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 .
  • the present invention also relates to a method of producing the enzyme preparation of the invention, the method comprising culturing a microorganism capable of producing the xyloglucanase under conditions permitting the production of the enzyme, and recovering the enzyme from the culture.
  • Culturing may be carried out using conventional fermentation techniques, e.g. culturing m shake flasks or fermentors with agitation to ensure sufficient aeration on a growth medium inducing production of the xyloglucanase enzyme.
  • the growth medium may contain a conventional N-source such as peptone, yeast extract or casammo acids, a reduced amount of a conventional C-source such as dextrose or sucrose, and an mducer such as xyloglucan or composit plant substrates such as cereal brans (e.g. wheat bran or rice husk) .
  • the recovery may be carried out using conventional techniques, e.g. separation of bio-mass and supernatant by centrifugation or filtration, recovery of the supernatant or disruption of cells if the enzyme of interest is mtracellular, perhaps followed by further purification as described m EP 0 406 314 or by crystallization as described m WO 97/15660.
  • the present invention provides a method of producing an isolated enzyme according to the invention, wherein a suitable host cell, which has been transformed with a DNA sequence encoding the enzyme, is cultured under conditions permitting the production of the enzyme, and the resulting enzyme is recovered from the culture.
  • an isolated polypeptide e.g. an enzyme
  • isolated polypeptide may alternatively be termed "purified polypeptide" .
  • homologous impurities mean any impurities (e.g. other polypeptides than the enzyme of the invention), which originate from the homologous cell where the enzyme of the invention is originally obtained.
  • the homologous host cell may be a strain of Paenibacillus sp . or Paenibacillus pabuli .
  • the medium used to culture the transformed host cells may be any conventional medium suitable for growing the host cells m question.
  • the expressed xyloglucanolytic enzyme may conveniently be secreted into the culture medium and may be recovered there from by well-known procedures including separat- mg the cells from the medium by centrifugation or filtration, precipitating protemaceous components of the medium by means of a salt such as ammonium sulphate, followed by chromatographic procedures such as ion exchange chromatography, affinity chromatography, or the like.
  • the present invention also relates to a transgenic plant, plant part or plant cell which has been transformed with a DNA sequence encoding the xyloglucanase of the invention so as to express and produce this enzyme m recoverable quantities .
  • the enzyme may be recovered from the plant or plant part .
  • the transgenic plant can be dicotyledonous or monocotyledonous, for short a dicot or a monocot .
  • monocot plants are grasses, such as meadow grass (blue grass, Poa) , forage grass such as festuca, lolium, temperate grass, such as Agrostis, and cereals, e.g. wheat, oats, rye, barley, rice, sorghum and maize (corn) .
  • dicot plants are tobacco, legumes, such as lupins, potato, sugar beet, pea, bean and soybean, and cruciferous (family Brassicaceae) , such as cauliflower, oil seed rape and the closely related model organism Arabidopsis thaliana .
  • plant parts are stem, callus, leaves, root, fruits, seeds, and tubers.
  • plant tissues such as chloroplast, apoplast, mitochondria, vacuole, peroxisomes and cytoplasm are considered to be a plant part.
  • any plant cell, whatever the tissue origin, is considered to be a plant part.
  • the transgenic plant or plant cell expressing the enzyme of the invention may be constructed m accordance with methods known m the art.
  • the plant or plant cell is constructed by incorporating one or more expression constructs encoding the enzyme of the invention into the plant host genome and propagating the resulting modified plant or plant cell into a transgenic plant or plant cell .
  • the expression construct is a DNA construct which comprises a gene encoding the enzyme of the invention m operable association with appropriate regulatory sequences required for expression of the gene m the plant or plant part of choice.
  • the expression construct may comprise a selectable marker useful for identifying host cells into which the expression construct has been integrated and DNA sequences necessary for introduction of the construct into the plant question (the latter depends on the DNA introduction method to be used) .
  • the choice of regulatory sequences, such as promoter and terminator sequences and optionally signal or transit sequences is determined, e.g. based on when, where and how the enzyme is desired to be expressed.
  • the expression of the gene encoding the enzyme of the invention may be constitutive or inducible, or may be developmental, stage or tissue specific, and the gene product may be targeted to a specific tissue or plant part such as seeds or leaves .
  • Regulatory sequences are e.g. described by Tague et al , Plant, Phys . , 86, 506, 1988.
  • the 35S-CaMV promoter may be used (Franck et al . , 1980. Cell 21: 285-294) .
  • Organ-specific promoters may e.g. be a promoter from storage sink tissues such as seeds, potato tubers, and fruits (Edwards & Coruzzi, 1990. Annu. Rev. Genet.
  • the promoter may be a leaf specific promoter such as the rbcs promoter from rice or tomato (Kyozuka et al., Plant Physiology Vol . 102, No. 3 pp. 991-1000 (1993), the chlorella virus adenme methyltransferase gene promoter (Mitra, A.
  • a promoter enhancer element may be used to achieve higher expression of the enzyme in the plant.
  • the promoter enhancer element may be an intron placed between the promoter and the nucleotide sequence encoding the enzyme.
  • Xu et al . op ci t disclose the use of the first intron of the rice actin 1 gene to enhance expression.
  • the selectable marker gene and any other parts of the expression construct may be chosen from those available in the art .
  • the DNA construct is incorporated into the plant genome according to conventional techniques known in the art, including Agrobacterium-mediated transformation, virus-mediated transformation, micro injection, particle bombardment, biolistic transformation, and electroporation (Gasser et al, Science, 244, 1293; Potrykus, Bio/Techn. 8, 535, 1990; Shimamoto et al , Nature, 338, 274, 1989) .
  • transgenic dicots for review Hooykas & Schilperoort , 1992. Plant Mol. Biol. 19: 15-38
  • the method of choice for generating transgenic monocots is particle bombardment (microscopic gold or tungsten particles coated with the transforming DNA) of embryonic calli or developing embryos (Christou, 1992. Plant J. 2: 275-281; Shimamoto, 1994. Curr. Opin. Biotechnol . 5: 158-162; Vasil et al . , 1992.
  • the xyloglucanase has a relative activity at a temperature of 50°C, preferably of at least 60%, preferably at least 70%, compared 5 to the activity at the optimal temperature.
  • the relative xyloglucanase activity is at least 40%, preferably at least 50%; at a temperature of 70°C, the relative xyloglucanase activity is at least 40%, preferably at least 10 45%, especially at least 50%.
  • the present invention relates to an enzyme composition
  • an enzyme composition comprising an enzyme exhibiting is xyloglucanase activity as described above.
  • the enzyme composition of the invention may, in addition to the xyloglucanase of the invention, comprise one or more other enzyme types, for instance hemicellulase such as xylanase and mannanase, cellulase or endo- ⁇ -1 , 4-glucanase components,
  • chitinase lipase, esterase, pectinase, cutinase, phytase, oxidoreductase (peroxidase, haloperoxidase, oxidase, laccase) , protease, amylase, reductase, phenoloxidase, ligninase, pullulanase, pectate lyase, pectin acetyl esterase, polygalacturonase , rhamnogalacturonase, pectin lyase, pectin 5 methylesterase, cellobiohydrolase, transglutaminase; or mixtures thereof .
  • the enzyme composition may be prepared in accordance with methods known in the art and may be in the form of a liquid or a dry composition.
  • the enzyme composition may be 0 in the form of a granulate or a microgranulate.
  • the enzyme to be included in the composition may be stabilized in accordance with methods known in the art.
  • Xyloglucanases have potential uses in a lot of different industries and applications. Examples are given below of 5 preferred uses of the enzyme composition of the invention.
  • the dosage of the enzyme composition of the invention and other conditions under which the composition is used may be determined based on methods known in the art .
  • the xyloglucanase or xyloglucanase composition according to the invention may be useful for at least one of the following purposes.
  • colour clarification is meant the partly restoration of the initial colours of fabric or garment throughout multiple washing cycles.
  • de-pill g denotes removing of pills from the fabric surface.
  • soaking liquor denotes an aqueous liquor m 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 m a washing or laundering process .
  • washing liquor denotes an aqueous liquor m which laundry is subjected to a washing process, i.e. usually a combined chemical and mechanical action either manually or m a washing machine.
  • the washing liquor is an aqueous solution of a powder or liquid detergent composition.
  • rmsmg liquor denotes an aqueous liquor m which laundry is immersed and treated, conventionally immediately after being subjected to a washing process, m order to rinse the laundry, i.e. essentially remove the detergent solution from the laundry.
  • the rmsmg liquor may contain a fabric conditioning or softening composition.
  • the laundry subjected to the method of the present invention may be conventional washable laundry.
  • the major part of the laundry is sewn or unsown fabrics, including knits, wovens, denims, yarns, and towelling, made from cotton, cotton blends or natural or manmade cellulosics (e.g. originating from xylan-conta mg cellulose fibers such as from wood pulp) or blends thereof .
  • 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, polyvmyl alcohol fibers, polyvmyl chloride fibers, polyvmylidene chloride fibers, polyurethane fibers, polyurea fibers, aramid fibers), and cellulose-containing fibers (e.g. rayon/viscose, ramie, flax/linen, jute, cellulose acetate fibers, lyocell) .
  • cellulose-containing fibers e.g. rayon/viscose, ramie, flax/linen, jute, cellulose acetate fibers, lyocell
  • the detergent compositions according to the present invention comprise a surfactant system, wherein the surfactant can be selected from non-ionic and/or anionic and/or catiomc 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 m the composition.
  • the surfactant is most preferably formulated m such a way that it promotes, or at least does not degrade, the stability of any enzyme m these compositions.
  • Preferred systems to be used according to the present invention comprise as a surfactant one or more of the non-ionic and/or anionic surfactants described herein.
  • Polyethylene, polypropylene, and polybutylene oxide conden-sates of alkyl phenols are suitable for use as the non- lonic surfactant of the surfactant systems of the present invention, with the polyethylene oxide condensates being pre- ferred.
  • 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, m either a straight chain or branched-cham configuration with the alkylene oxide.
  • the ethylene oxide is present m 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.
  • non-ionic surfactants of this type include IgepalTM CO-630, marketed by the GAF Corporation; and TritonTM X-45, X- 114, X-100 and X-102, all marketed by the Rohm & Haas Company. These surfactants are commonly referred to as alkyl phenol alkoxylates (e.g., alkyl phenol ethoxylates) .
  • alkyl phenol alkoxylates e.g., alkyl phenol ethoxylates
  • the condensation products of primary and secondary aliphatic alcohols with about 1 to about 25 moles of ethylene oxide are suitable for use as the non-ionic surfactant of the non- ionic surfactant systems of the present invention.
  • the alkyl chain of the aliphatic alcohol can either be straight or branched, primary or secondary, and generally contains from about 8 to about 22 carbon atoms.
  • 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.
  • non- ionic surfactants of this type include TergitolTM 15-S-9 (The condensation product of C11-C15 linear alcohol with 9 moles ethylene oxide), TergitolTM 24-L-6 NMW (the condensation product of C ⁇ 2 -C ⁇ primary alcohol with 6 moles ethylene oxide with a narrow molecular weight distribution) , both marketed by Union Carbide Corporation; NeodolTM 45-9 (the condensation product of C ⁇ 4 -C ⁇ 5 linear alcohol with 9 moles of ethylene oxide), NeodolTM 23-3 (the condensation product of Ci 2 -Ci 3 linear alcohol with 3.0 moles of ethylene oxide) , NeodolTM 45-7 (the condensation product of C ⁇ 4 -C ⁇ 5 linear alcohol with 7 moles of ethylene oxide) , NeodolTM 45-5 (the condensation product of C14-C15 linear alcohol with 5 moles of ethylene oxide) marketed by Shell Chemical Company, Kyro TM EOB (the condensation product of C13-C15 alcohol with 9 moles ethylene
  • alkyl polysaccharides disclosed m 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 polysaccha ⁇ de, 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 substi- tuted 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
  • R 2 is selected from the group consisting of alkyl, alkyl phenyl, hydroxyalkyl , hydroxyaIky1phenyl , and mixtures thereof 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, preferably 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.
  • 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-pos ⁇ t ⁇ on) .
  • the additional glycosyl units can then be attached between their 1- position and the preceding glycosyl unit's 2-, 3-, 4-, and/or 6-pos ⁇ t ⁇ on, preferably predominantly the 2-pos ⁇ t ⁇ on.
  • the condensation products of ethylene oxide with a hydrophobic base formed by the condensation of propylene oxide with propylene glycol are also suitable for use as the additional non-ionic surfactant systems of the present invention.
  • the hydrophobic portion of these compounds will preferably have a molecular weight from about 1500 to about 1800 and will exhibit water insolubility.
  • the addition of polyoxyethylene moieties to this hydrophobic portion tends to increase the water solubility of the molecule as a whole, and the liquid character of the product is retained up to the point where the polyoxyethylene content is about 50% of the total weight of the condensation product, which corresponds to condensation with up to about 40 moles of ethylene oxide.
  • Examples of compounds of this type include certain of the commercially available PluronicTM surfactants, marketed by BASF.
  • non-ionic surfactant of the non-ionic surfactant system of the present invention are the condensation products of ethylene oxide with the product resulting from the reaction of propylene oxide and ethylenediamme .
  • the hydrophobic moiety of these products consists of the reaction product of ethylenediamme 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.
  • this type of non-ionic surfactant include certain of the commercially available TetronicTM compounds, marketed by BASF.
  • non-ionic surfactant of the surfactant systems of the present invention are polyethylene oxide condensates of alkyl phenols, condensation products of primary and secondary aliphatic alcohols with from about 1 to about 25 moles of ethyleneoxide, alkylpolysaccharides , and mixtures hereof. Most preferred is Cs-C ⁇ 4 alkyl phenol ethoxylates having from 3 to 15 ethoxy groups and Ca-C ⁇ 8 alcohol ethoxylates (preferably Cio av . ) having from 2 to 10 ethoxy groups, and mixtures thereof.
  • Highly preferred non- ionic surfactants are polyhydroxy fatty acid amide surfactants of the formula
  • R 1 wherein R 1 is H, or R 1 is C ⁇ - 4 hydrocarbyl , 2-hydroxyethyl , 2- hydroxypropyl or a mixture thereof, R 2 is C 5 31 hydrocarbyl, and Z is a polyhydroxy hydrocarbyl having a linear hydrocarbyl chain with at least 3 hydroxyls directly connected to the chain, or an alkoxylated derivative thereof.
  • R 1 is methyl
  • R 2 is straight C ⁇ -15 alkyl or Ci ⁇ -i ⁇ alkyl or alkenyl chain such as coconut alkyl or mixtures thereof
  • Z is derived from a reducing sugar such as glucose, fructose, maltose or lactose, m a reductive animation reaction.
  • Highly preferred anionic surfactants include alkyl alkoxylated sulfate surfactants.
  • Examples hereof are water soluble salts or acids of the formula R0(A) m S03M wherein R is an unsubstituted C ⁇ o-C- 24 alkyl or hydroxyalkyl group having a C10- C 24 alkyl component, preferably a C12-C20 alkyl or hydroxyalkyl, 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.
  • R is an unsubstituted C ⁇ o-C- 24 alkyl or hydroxyalkyl group having a C
  • 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 piperdimum cations and those derived from alkylammes such as ethylam e, diethylamme, t ⁇ ethylamme, mixtures thereof, and the like.
  • Exemplary surfactants are C ⁇ 2 -C ⁇ 8 alkyl polyethoxylate (1.0) sulfate (C ⁇ 2 -C ⁇ 8 E (1.0) M) , C ⁇ 2 -C ⁇ 8 alkyl polyethoxylate (2.25) sulfate (C ⁇ 2 -C ⁇ 8 (2.25) M, and C ⁇ 2 -C ⁇ 8 alkyl polyethoxylate (3.0) sulfate (C ⁇ 2 -C ⁇ 8 E (3.0) M) , and C ⁇ 2 -C ⁇ 8 alkyl polyethoxylate (4.0) sulfate (C ⁇ 2 -C ⁇ 8 E (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 C 8 -C 2 o 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: 0
  • R is a C 8 -C2o hydrocarbyl, preferably an alkyl, or combination thereof
  • R 4 is a C ⁇ -C 6 hydrocarbyl, preferably an alkyl, or combination thereof
  • 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 .
  • R 3 is C10-C16 alkyl
  • R 4 is methyl, ethyl or isopropyl .
  • the methyl ester sulfonates wherein R 3 is C ⁇ 0 -C ⁇ S alkyl.
  • alkyl sulfate surfactants which are water soluble salts or acids of the formula ROSO 3 M wherein R preferably is a C ⁇ 0 -C 24 hydrocarbyl, preferably an alkyl or hydroxyalkyl having a C10-C20 alkyl com- ponent, more preferably a C ⁇ 2 -C ⁇ 8 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.
  • R preferably is a C ⁇ 0 -C 24 hydrocarbyl, preferably an alkyl or hydroxyalkyl having a C10-C20 alkyl com- ponent, more preferably a C ⁇ 2 -C ⁇ 8 alkyl or hydroxyalkyl
  • M is H or a cation, e.g., an alkali metal cation (e.g.
  • alkylamines such as ethylamine, diethylamine, triethylamine, and mixtures thereof, and the like.
  • alkyl chains of C ⁇ 2 -C ⁇ s are preferred for lower wash temperatures (e.g. below about 50 °C) and Ci6-C ⁇ 8 alkyl chains are preferred for higher wash temperatures (e.g. above about 50 °C) .
  • anionic surfactants useful for detersive purposes can also be included in the laundry detergent compositions of the present invention. Theses can include salts (including, for example, sodium, potassium, ammonium, and substituted ammonium salts such as mono- di- and t ⁇ ethanolamme salts) of soap, C 8 - C 2 2 primary or secondary alkanesulfonates , C 8 -C4 olef sulfonates , sulfonated polycarboxylic acids prepared by sulfonation of the pyrolyzed product of alkaline earth metal citrates, e.g., as described m British patent specification No.
  • salts including, for example, sodium, potassium, ammonium, and substituted ammonium salts such as mono- di- and t ⁇ ethanolamme salts
  • C 8 - C 2 2 primary or secondary alkanesulfonates C 8 -C4 olef sulfonates
  • 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, lsethionates such as the acyl lsethionates , N-acyl taurates, alkyl succmamates and sulfosuccmates , monoesters of sulfosuccmates (especially saturated and unsaturated C ⁇ 2 -C ⁇ 8 monoesters) and diesters of sulfosuccmates (especially saturated and unsaturated C6-C12 diesters), acyl sarcosmates , sulfates of alkyl polysaccharides such
  • 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 m “Surface Active Agents and Detergents” (Vol. I and II by Schwartz, Perry and Berch) . A variety of such surfactants are also generally disclosed m US 3,929,678, (Column 23, line 58 through Column 29, line 23, herein incorporated by reference) . When included therein, the laundry detergent compositions of the present invention typically comprise from about 1% to about 40%, preferably from about 3% to about 20% by weight of such anionic surfactants .
  • the laundry detergent compositions of the present invention may also contain cationic, ampholytic, zwitterionic , and semi-polar surfactants, as well as the non-ionic 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:
  • R 2 is an alkyl or alkyl benzyl group having from about 8 to about 18 carbon atoms in the alkyl chain
  • each R 3 is selected form the group consisting of -CH 2 CH 2 -, -CH 2 CH (CH 3 ) - , -
  • each R 4 is selected from the group consisting of C1-C4 alkyl, C1-C4 hydroxyalkyl, benzyl ring structures formed by joining the two R 4 groups, -CH 2 CHOHCHOHCOR 6 CHOHCH 2 OH, wherein R 6 is any hexose or hexose polymer having a molecular weight less than about 1000, and hydrogen when y is not 0; R 5 is the same as R 4 or is an alkyl chain, wherein the total number of carbon atoms or R plus R 5 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:
  • Ri is C 8 -C ⁇ 6 alkyl
  • each of R 2 , R 3 and R 4 is independently C 1 -C 4 alkyl, C 1 -C 4 hydroxy alkyl, benzyl, and -(C2H 40 ) X H where x has a value from 2 to 5, and X is an anion.
  • R2 , R3 or R 4 should be benzyl.
  • the preferred alkyl chain length for Ri 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.
  • R 2 R 3 and R 4 are methyl and hydroxyethyl groups and the anion X may be selected from halide, methosulphate, acetate and phosphate ions.
  • suitable quaternary ammonium compounds of formulae (I) for use herein are: coconut t ⁇ methyl ammonium chloride or bromide; coconut methyl dihydroxyethyl ammonium chloride or bromide; decyl t ⁇ ethyl ammonium chloride; decyl dimethyl hydroxyethyl ammonium chloride or bromide; C12 is dimethyl hydroxyethyl ammonium chloride or bromide; coconut dimethyl hydroxyethyl ammonium chloride or bromide; my ⁇ styl trimethyl ammonium methyl sulphate; lauryl dimethyl benzyl ammonium chloride or bromide; lauryl dimethyl (ethenoxy) 4 ammonium chloride or bromide; cholme esters (compound
  • CH2-CH2-O-C-C1214 alkyl and R2R3R4 are methyl
  • di-alkyl lmidazolmes [compounds of formula (1)] .
  • 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 m 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 m which the aliphatic radical can be straight- or branched-cham.
  • 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-solubilizmg group, e.g. carboxy, sulfonate, sulfate. See US 3,929,678 (column 19, lines 18-35) for examples of ampholytic surfactants.
  • the laundry detergent compositions of the present invention typically comprise from 0.2% to about 15%, preferably from about 1% to about 10% by weight of such ampholytic surfactants.
  • Zwitterionic surfactants are also suitable for use in laundry detergent 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.
  • the laundry detergent compositions of the present invention typically comprise from 0.2% to about 15%, preferably from about 1% to about 10% by weight of such 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:
  • R 3 is an alkyl, hydroxyalkyl, or alkyl phenyl group or mixtures thereof containing from about 8 to about 22 carbon atoms
  • R 4 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
  • each R 5 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 R 5 groups can be attached to each other, e.g., through an oxygen or nitrogen atom, to form a ring structure .
  • These amme oxide surfactants m particular include C ⁇ 0 -C ⁇ 8 alkyl dimethyl amme oxides and C 8 -C ⁇ 2 alkoxy ethyl dihydroxy ethyl amme oxides .
  • the laundry detergent compositions of the present invention typically comprise from 0.2% to about 15%, preferably from about 1% to about 10% by weight of such semi-polar nonionic surfactants.
  • compositions according to the present invention may further comprise a builder system.
  • a builder system Any conventional builder system is suitable for use herein including alummosilicate materials, silicates, polycarboxylates and fatty acids, materials such as ethylenediamme tetraacetate, metal ion sequestrants such as ammopolyphosphonates, particularly ethylenediamme tetramethylene phosphonic acid and diethylene t ⁇ amme pentamethylenephosphomc acid.
  • phosphate builders can also be used herein.
  • Suitable builders can be an inorganic ion exchange material, commonly an inorganic hydrated alummosilicate material, more particularly a hydrated synthetic zeolite such as hydrated zeolite A, X, B, HS or MAP.
  • SKS-6 is a crystalline layered silicate consisting of sodium silicate (Na 2 Si2 ⁇ 5 ) .
  • Suitable polycarboxylates containing one carboxy group include lactic acid, glycolic acid and ether derivatives thereof as disclosed m Belgian Patent Nos. 831,368, 821,369 and 821,370.
  • Polycarboxylates containing two carboxy groups include the water-soluble salts of succmic acid, malonic acid, (ethylenedioxy) diacetic acid, maleic acid, diglycollic acid, tartaric acid, tartronic acid and fumaric acid, as well as the ether carboxylates described m German Offenle-ensch ⁇ ft 2,446,686, and 2,446,487, US 3,935,257 and the sulfmyl carboxylates described m Belgian Patent No. 840,623.
  • Polycarboxylates containing three carboxy groups include, m particular, water-soluble citrates, aconitrates and citraconates as well as succ ate derivatives such as the carboxymethyloxysucc ates described m British Patent No. 1,379,241, lactoxysuccmates described Netherlands Application 7205873, and the oxypolycarboxylate materials such as 2 -oxa-1 , 1 , 3 -propane t ⁇ carboxylates described m British Patent No. 1,387,447.
  • Polycarboxylates containing four carboxy groups include oxydisuccmates disclosed m British Patent No. 1,261,829, 1 , 1 , 2 , 2 , -ethane tetracarboxylates, 1 , 1 , 3 , 3 -propane tetrac7arboxylates containing sulfo substituents include the sulfosuccmate derivatives disclosed m British Patent Nos.
  • 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-c ⁇ s, discarboxylates , 2 , 2 , 5 , 5 , -tetrahydrofuran - tetracarboxylates, 1,2,3,4,5,6- hexane - hexacarboxylates and carboxymethyl derivatives of polyhyd ⁇ c alcohols such as sorbitol , mannitol and xylitol.
  • Aromatic polycarboxylates include mellitic acid, pyromellitic acid and the phthalic acid derivatives disclosed m British Patent No. 1,425, 343.
  • the preferred polycarboxylates are hydroxy- carboxylates containing up to three carboxy groups per molecule, more particularly citrates.
  • Preferred builder systems for use the present compositions include a mixture of a water- insoluble alummosilicate builder such as zeolite A or of a layered silicate (SKS-6) , and a water-soluble carboxylate chelatmg agent such as citric acid.
  • a suitable chelant for inclusion m the detergent composi-ions m accordance with the invention is ethylened ⁇ amme-N,N' -disuccimc 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 Na 2 EDDS and Na 4 EDDS .
  • Examples of such preferred magnesium salts of EDDS include MgEDDS and Mg 2 EDDS .
  • the magnesium salts are the most preferred for inclusion m compositions m accordance with the invention.
  • Preferred builder systems include a mixture of a water- insoluble alummosilicate builder such as zeolite A, and a water soluble carboxylate chelatmg agent such as citric acid.
  • a water- insoluble alummosilicate builder such as zeolite A
  • a water soluble carboxylate chelatmg agent such as citric acid.
  • Other builder materials that can form part of the builder system for use m granular compositions include inorganic materials such as alkali metal carbonates, bicarbonates, silicates, and organic materials such as the organic phosphonates, ammo polyalkylene phosphonates and ammo polycarboxylates .
  • Other suitable water-soluble organic salts are the homo- or co-polymeric acids or their salts, m 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 m GB-A-1 , 596 , 756.
  • 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 m amounts of from 5% to 80% by weight of the composition. Preferred levels of builder for liquid detergents are from 5% to 30%.
  • Enzymes Preferred detergent compositions, m addition to the enzyme preparation of the invention, comprise other enzyme (s) which provides cleaning performance and/or fabric care benefits .
  • Such enzymes include proteases, lipases, cutmases, amylases, cellulases, peroxidases, oxidases (e.g. laccases) .
  • protease suitable for use m 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 serme protease, preferably an alkaline microbial protease or a trypsm-like protease.
  • alkaline proteases are subtilisms, especially those derived from Bacillus, e.g., subtilism Novo, subtilism Carlsberg, subtilism 309, subtilism 147 and subtilism 168 (described WO 89/06279) .
  • trypsm-like proteases are tryps (e.g. of porcine or bovine origin) and the Fusarium protease described m WO 89/06270.
  • Preferred commercially available protease enzymes include those sold under the trade names Alcalase, Sav ase, 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 m 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 m alkaline solutions can be used. Suitable lipases include those of bac- te ⁇ al or fungal origin. Chemically or genetically modified mutants are included.
  • useful lipases include a Humicola lanugmosa lipase, e.g., as described m EP 258 068 and EP 305 216, a Rhizomucor miehei lipase, e.g., as described m EP 238 023, a Candida lipase, such as a C. antarctica lipase, e.g., the C . antarctica lipase A or B described EP 214 761, a Pseudomonas lipase such as a P. alcaligenes and P. pseudoalcaligenes lipase, e.g., as described m EP 218 272, a P.
  • a Humicola lanugmosa lipase e.g., as described m EP 258 068 and EP 305 216
  • a Rhizomucor miehei lipase e.g., as described m EP 238 023
  • cepac a lipase e.g., as described m EP 331 376, a P. stutze ⁇ lipase, e.g., as disclosed m 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) .
  • cloned lipases may be useful, including the Penicillium camemberti lipase described by Yamaguchi et al . , (1991), Gene 103, 61-67), the Geot ⁇ cum 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.
  • lipolytic enzymes such as cutmases may also be useful, e.g., a cutmase derived from Pseudomonas mendocma as described m WO 88/09367, or a cutmase derived from Fusarium solani pisi (e.g. described m WO 90/09446) .
  • lipases such as Ml LipaseTM, Luma fastTM and LipomaxTM (Genencor) , Lipolase and
  • Lipolase UltraTM Novartis A/S
  • Lipase P "Amano” Novartis P "Amano Pharmaceutical Co. Ltd.
  • the lipases are normally incorporated m 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 alkaline solutions can be used. Suitable amylases include those of bacterial or fungal origin. Chemically or genetically mod- if ⁇ ed mutants are included. Amylases include, for example, a- amylases obtained from a special strain of B. licheniformis, described m more detail m GB 1,296,839. Commercially T I TM TM available amylases are Duramyl , Termamyl , Fungamyl and BAN
  • the amylases are normally incorporated m 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 m 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 m US 4,435,307 which discloses fungal cellulases produced from Humi - cola msolens, WO 96/34108 and WO 96/34092 which disclose bacterial alkalophilic cellulases (BCE 103) from Bacillus, and m 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 m European patent application No. 0 495 257.
  • Cellulases include CelluzymeTM and CarezymeTM produced by a strain of Humicola insolens (Novo Nordisk A/S), KAC-500(B)TM (Kao Corporation), and PuradaxTM (Genencor International) .
  • Cellulases are normally incorporated 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 combination with hydrogen peroxide or a source thereof (e.g. a percarbonate, perborate or persulfate) . Oxidase enzymes are used m 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 m a wash liquor, preferably together with an enhancing agent as described m 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 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, m particular a mixture of a protease, an amylase, a lipase and/or a cellulase.
  • the enzyme of the invention is normally incorporated 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 such as PBl, 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%.
  • bleaching compounds are optional added components m 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 m the art.
  • the bleaching agent suitable for the present invention can be an activated or non-activated bleaching agent.
  • 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-nonylammo-4-oxoperoxybutyr ⁇ c acid and diperoxydodecanedioic acid. Such bleaching agents are disclosed m US 4,483,781, US 740,446, EP 0 133 354 and US 4,412,934. Highly preferred bleaching agents also include 6- nonylammo-6-oxoperoxycapro ⁇ c acid as described m US 4,634,551. Another category of bleaching agents that can be used encompasses the halogen bleaching agents.
  • hypohalite bleaching agents examples include t ⁇ chloro lsocyanu ⁇ c 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 m combination with bleach activators such as tetra- acetylethylenediamme (TAED) , nonanoyloxybenzenesulfonate (NOBS, described m US 4,412,934), 3 , 5-t ⁇ methyl- hexsanoloxybenzenesulfonate (ISONOBS, described m EP 120 591) or pentaacetylglucose (PAG) , which are perhydrolyzed to form a peracid as the active bleaching species, leading to improved bleaching effect.
  • bleach activators such as tetra- acetylethylenediamme (TAED) , nonanoyloxybenzenesulfonate (NOBS, described m US 4,412,934), 3 , 5-t ⁇ methyl- hexsanoloxybenzenesulfonate (ISONOBS, described m EP 120 591) or pentaacet
  • bleach activators C8 (6-octanam ⁇ do-caproyl) oxybenzene-sulfonate, C9(6- nonanamido caproyl) oxybenzenesulfonate and CIO (6-decanam ⁇ do caproyl) oxybenzenesulfonate or mixtures thereof.
  • acylated citrate esters such as disclosed European Patent Application No. 91870207.7.
  • Useful bleaching agents including peroxyacids and bleaching systems comprising bleach activators and peroxygen bleaching compounds for use m cleaning compositions according to the invention are described m 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 rmsmg process.
  • an enzymatic system i.e. an enzyme and a substrate therefore
  • Such enzymatic systems are disclosed m European Patent Application EP 0 537 381.
  • Bleaching agents other than oxygen bleaching agents are also known m 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 zmc and/or aluminium phthalocyanmes . These materials can be deposited upon the substrate during the washing process. Upon irradiation with light, m the presence of oxygen, such as by hanging clothes out to dry m the daylight, the sulfonated z c phthalocyanme is activated and, consequently, the substrate is bleached.
  • Preferred zmc phthalocyanme and a photoactivated bleaching process are described m US 4,033,718.
  • detergent composition will contain about 0.025% to about 1.25%, by weight, of sulfonated zmc phthalocyanme.
  • Bleaching agents may also comprise a manganese catalyst.
  • the manganese catalyst may, e.g., be one of the compounds described m "Efficient manganese catalysts for low-temperature bleaching", Nature 369, 1994, pp. 637-639.
  • a suds suppressor exemplified by silicones, and silica-silicone mixtures.
  • Silicones can generally be represented by alkylated polysiloxane materials, while silica is normally used m finely divided forms exemplified by silica aerogels and xerogels and hydrophobic silicas of various types. Theses materials can be incorporated as particulates , m which the suds suppressor is advantageously releasably incorporated m a water-soluble or waterdispersible, substantially non surface-active detergent impermeable carrier. Alternatively the suds suppressor can be dissolved or dispersed a liquid carrier and applied by spraying on to one or more of the other components.
  • a preferred silicone suds controlling agent is disclosed m US 3,933,672.
  • Other particularly useful suds suppressors are the self-emulsifying silicone suds suppressors, described m German Patent Application DTOS 2,646,126.
  • An example of such a compound is DC- 544, commercially available form Dow Corning, which is a siloxane-glycol copolymer.
  • Especially preferred suds controlling agent are the suds suppressor system comprising a mixture of silicone oils and 2-alkyl-alkanols .
  • Suitable 2- alkyl-alkanols are 2-butyl-octanol which are commercially available under the trade name Isofol 12 R.
  • Such suds suppressor system are described m European Patent Application EP 0 593 841.
  • compositions can comprise a silicone/ silica mixture in combination with fumed nonporous silica such as Aeros ⁇ l R .
  • the suds suppressors described above are normally employed at levels of from 0.001% to 2% by weight of the composition, preferably from 0.01% to 1% by weight.
  • m detergent compositions may be employed such as soil-suspending agents, soil-releasing agents, optical b ⁇ ghteners, abrasives, bacte ⁇ cides , tarnish inhibitors, coloring agents, and/or encapsulated or nonencapsulated perfumes.
  • encapsulating materials are water soluble capsules which consist of a matrix of polysaccha ⁇ de and polyhydroxy compounds such as described m GB 1,464,616.
  • Suitable water soluble encapsulating materials comprise dextrms derived from ungelatmized starch acid esters of substituted dicarboxylic acids such as described m US 3,455,838. These acid-ester dextrms 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 succmic 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, methylvmyl 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 m character, examples of which are disodium 4 , 4 ' -bis- (2-d ⁇ ethanolammo-4- anilmo -s- tr ⁇ azm-6-ylammo) st ⁇ lbene-2 : 2 ' disulphonate, disodium 4, - 4 ' -bis- (2 -morpholmo-4-an ⁇ lmo-s-tr ⁇ azm-6- ylammo-st ⁇ lbene-2 : 2 ' - disulphonate, disodium 4,4' - bis- (2,4- d ⁇ an ⁇ lmo- ⁇ -tr ⁇ azm-6-ylammo) st ⁇ lbene-2 : 2 ' - disulphonate, monosodium 4 ',4'' - bis- (2 , 4-d ⁇ an ⁇ lmo-s-tr ⁇ -azm-6 ylamino) st ⁇ lbene-2 -sulphon
  • 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.
  • Soil release agents useful m compositions of the present invention are conventionally copolymers or terpolymers of terephthalic acid with ethylene glycol and/or propylene glycol units various arrangements. Examples of such polymers are disclosed m US 4,116,885 and 4,711,730 and EP 0 272 033.
  • a particular preferred polymer m accordance with EP 0 272 033 has the formula:
  • 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", m the present context most of said copolymers herein will be endcapped by sulphobenzoate groups.
  • 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 m detail m EP 311 342.
  • Fabric softening agents can also be incorporated into laundry detergent compositions m accordance with the present invention. These agents may be inorganic or organic type. Inorganic softening agents are exemplified by the smectite clays disclosed m GB-A-1 400898 and m US 5,019,292. Organic fabric softening agents include the water insoluble tertiary ammes as disclosed m GB-A1 514 276 and EP 0 011 340 and their combination with mono C ⁇ 2 -C ⁇ 4 quaternary ammonium salts are disclosed EP-B-0 026 528 and di-long-cham amides as disclosed EP 0 242 919. Other useful organic ingredients of fabric softening systems include high molecular weight polyethylene oxide materials as disclosed m EP 0 299 575 and 0 313 146.
  • Levels of smectite clay are normally m 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 ammes 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 m 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.
  • the detergent compositions according to the present invention may also comprise from 0.001% to 10%, preferably from 0.01% to 2%, more preferably form 0.05% to 1% by weight of polymeric dye- transfer inhibiting agents.
  • Said polymeric dye- transfer inhibiting agents are normally incorporated into detergent compositions order to inhibit the transfer of dyes from colored fabrics onto fabrics washed therewith. These polymers have the ability of complexmg or adsorbing the fugitive dyes washed out of dyed fabrics before the dyes have the opportunity to become attached to other articles m the wash.
  • Especially suitable polymeric dye-transfer inhibiting agents are polyam e N-oxide polymers, copolymers of N-vmyl- pyrrolidone and N-vmylimidazole, polyvmylpyrrolidone polymers, polyvmyloxazolidones and polyvmylimidazoles or mixtures thereof .
  • the detergent composition according to the invention can be m liquid, paste, gels, bars or granular forms.
  • Non-dustmg granulates may be produced, e.g., as disclosed m US 4,106,991 and 4,661,452 (both to Novo Indust ⁇ A/S) and may optionally be coated by methods known m the art.
  • waxy coating materials are pol (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 m which the alcohol contains from 12 to 20 carbon atoms and which there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- and triglyce ⁇ des of fatty acids.
  • pol (ethylene oxide) products polyethyleneglycol , PEG
  • ethoxylated nonylphenols having from 16 to 50 ethylene oxide units
  • ethoxylated fatty alcohols m which the alcohol contains from 12 to 20 carbon atoms and which there are 15 to 80 ethylene oxide units
  • fatty alcohols fatty acids
  • mono- and di- and triglyce ⁇ des of fatty acids are given m GB 1483591.
  • Granular compositions according to the present invention can also be m "compact form", i.e. they may have a relatively higher density than conventional granular detergents, i.e. form 550 to 950 g/1; m 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, typi- cally sodium sulphate; "Compact" detergent typically comprise not more than 10% filler salt.
  • the liquid compositions according to the present invention can also be "concentrated form", m such case, the liquid detergent compositions according to the present invention will contain a lower amount of water, compared to conventional liquid detergents. Typically, the water content of the concentrated liquid detergent is less than 30%, more preferably less than 20%, most preferably less than 10% by weight of the detergent compositions.
  • the compositions of the invention may for example, be formulated as hand and machine laundry detergent compositions including laundry additive compositions and compositions suitable for use m the pretreatment of stained fabrics, rinse added fabric softener compositions, and compositions for use m general household hard surface cleaning operations and dishwashing operations.
  • LAS Sodium linear C12 alkyl benzene sulphonate
  • TAS Sodium tallow alkyl sulphate
  • XYEZS C ⁇ - C ⁇ 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
  • CFAA C12 - C14 alkyl N-methyl glucamide
  • TFAA Ci6 - Cis alkyl N-methyl glucamide
  • NaSKS-6 Crystalline layered silicate of formula d-NaS ⁇ 2 05
  • Phosphate Sodium t ⁇ polyphosphate 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 Alummosilicate of formula
  • Perborate Anhydrous sodium perborate monohydrate bleach, empirical formula NaB0 2 .H0 2
  • PB4 Anhydrous sodium perborate tetrahydrate Percarbonate: Anhydrous sodium percarbonate bleach of empirical formula 2Na 2 C0 3 .3H 2 0 2
  • CMC Sodium carboxymethyl cellulose
  • DETPMP Diethylene t ⁇ amme penta (methylene phosphonic acid) , marketed by Monsanto under the Tradename Dequest 2060
  • Granular Suds suppressor 12% Silicone/silica, 18% stearyl alcohol, 70% starch granular form
  • HMWPEO High molecular weight polyethylene oxide
  • TAE 25 Tallow alcohol ethoxylate (25)
  • a granular fabric cleaning composition m accordance with the invention may be prepared as follows:
  • Enzyme of the invention 0.1
  • a compact granular fabric cleaning composition (density 800 g/1) in accord with the invention may be prepared as follows:
  • Enzyme of the invention 0.1
  • Granular suds suppressor 3.5 water/minors Up to 100%
  • 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:
  • Enzyme of the invention 0.10 0.05
  • Granular fabric cleaning compositions m accordance with the invention which provide "Softening through the wash” capability may be prepared as follows:
  • Zeolite A 15.0 15.0 MA/AA 4.0 4.0 DETPMP 0.4 0.4 Perborate 15.0 Percarbonate - 15.0
  • Heavy duty liquid fabric cleaning compositions m accordance with the invention may be prepared as follows:
  • Enzyme of the invention 0.10 0.05
  • xyloglucan from tamarind seeds supplied by Megazyme, Ireland has a complex branched structure with glucose, xylose, galactose and arabinose m the ratio of 45:36:16:3. Accordingly, it is strongly believed that an enzyme showing catalytic activity on this xyloglucan also has catalytic activity on other xyloglucan structures from different sources (angiosperms or gy nosperms) .
  • Cotton suspension culture xyloglucan MW 100,000 kDa was obtained from Professor A. Mort of Oklahoma State University. IH NMR (D20, 80°C) of xyloglucans was used to compare the monosaccharide composition of samples of different origin. The integrals of the anome ⁇ c signals from the commercial sample fully agree with the composition given by Megazyme. However, the cotton xyloglucan seems to have a different structure. There appears to be much less galactose and about half of galactose residues are fucosylated. Furthermore, the molar ratio between xylose and glucose is smaller (0.63 compared to 0.77 for the tamarind) , which suggest a more open structure of cotton xyloglucan. These findings agree with results obtained with xyloglucan from cotton cells (Buchala et al, Acta Bot . Neerl. 42, 1993, 213-219).
  • Paenibacillus pabuli e.g. the type strain ATCC 43899, and Paenibacillus sp . , DSM 13330, comprises a DNA sequence encoding a family 5 xyloglucanase of the invention.
  • E. coli DSM 13183, comprises the plasmid containing the DNA encoding the xyloglucanase of the invention (SEQ ID NO: 1) .
  • Other strains SEQ ID NO: 1.
  • E. coli hosts XLl-Blue MRF " and XLOLR E. coli strains were provided by Stratagene Inc. (USA) and used according to the manufacturer's instructions.
  • This strain is the B . subtilis DN1885 with disrupted apr and npr genes Diderichsen et al . (1990) disrupted in the transcriptional unit of the known Bacillus subtilis cellulase gene, resulting in cellulase negative cells. The disruption was performed essentially as described in Sonenshein et al . (1993) .
  • Bacillus subtili s MB1053-1 This strain is PL 2306 in which the pectate lyase gene Pel has been disrupted resulting in a pectate lyase negative strain. The disruption was performed essentially as described in Sonenshein et al . (1993) .
  • Competent cells were prepared and transformed as described by Yasbin et al . (1975) .
  • Plasmids pBK-CAMV Stratagene inc. La Jolla CA., USA.
  • Bacteriophage ZAP Express Stratagene inc. La Jolla CA. , USA. pMOL944.
  • This plasmid is a pUBHO derivative essentially containing elements making the plasmid propagatable in Bacillus subtilis, kanamycin resistance gene and having a strong promoter and signal peptide cloned from the amyL gene of B . licheniformis ATCC 14580.
  • the signal peptide contains a SacII site making it convenient to clone the DNA encoding the mature part of a protein in-fusion with the signal peptide. This results in the expression of a Pre-protein, which is directed towards the exterior of the cell .
  • the plasmid was constructed by means of ordinary genetic engineering and is briefly described in the following. Construction of pMOL944 :
  • the pUBHO plasmid (McKenzie, T. et al . , 1986,) was digested with the unique restriction enzyme Neil .
  • the two PCR primers used have the following sequences:
  • the primer #LWN5494 inserts a Notl site m the plasmid.
  • the plasmid pSJ2624 was then digested with Sad and Notl and a new PCR fragment amplified on amyL promoter encoded on the pDN1981 was digested with Sad and Notl and this DNA fragment was inserted m the Sacl-Notl digested pSJ2624 to give the plasmid pSJ2670.
  • This cloning replaces the first amyL promoter cloning with the same promoter but m the opposite direction.
  • the two primers used for PCR amplification have the following sequences : #LWN5938 5 " -GTCGGCGGCCGCTGATCACGTACCAAGCTTGTCGACCTGCAGAATG AGGCAGCAAGAAGAT - 3 "
  • the plasmid pSJ2670 was digested with the restriction enzymes Pstl and Bell and a PCR fragment amplified from a cloned DNA sequence encoding the alkaline amylase SP722 (International Patent Application published as W095/26397 which is hereby incorporated by reference) was digested with Pstl and Bell and inserted to give the plasmid pMOL944.
  • the two primers used for PCR amplification have the following sequence: #LWN7864 5 ⁇ -AACAGCTGATCACGACTGATCTTTTAGCTTGGCAC-3 ' #LWN7901 5 ⁇ -AACTGCAGCCGCGGCACATCATAATGGGACAAATGGG -3'
  • the primer #LWN7901 inserts a SacII site m the plasmid.
  • LB agar (as described m Ausubel, F. M. et al , 1995) .
  • LBPG is LB agar supplemented with 0.5% Glucose and 0.05 M potassium phosphate, pH 7.0.
  • AZCL-Xyloglucan is added to LBPG-agar to 0.5 % AZCL- Xyloglucan is from Megazyme, Australia.
  • BPX media is described m EP 0 506 780 (WO 91/09129) .
  • NZY agar per liter 5 g of NaCl, 2 g of MgS04 , 5 g of yeast extract, 10 g of NZ amme (casein hydrolysate) , 15 g of agar; add deionized water to 1 litre, adjust pH with NaOH to pH 7.5 and autoclave
  • NZY broth (per litre) 5 g of NaCl, 2 g of MgS04 , 5 g of yeast extract, 10 g of NZ amme (casein hydrolysate) ; add deionized water to 1 litre, adjust pH with NaOH to pH 7.5 and autoclave NZY Top Agar (per litre) 5 g of NaCl, 2 g of MgS04 , 5 g of yeast extract, 10 g of NZ amme (casein hydrolysate), 0.7 % (w/v) agarose; add deionized water to 1 litre, adjust pH with NaOH to pH 7.5 and autoclave .
  • the xyloglucanase activity is measured using AZCL- xyloglucan from Megazyme, Ireland, (htt : //www .megazyme . com/purchase/index . tml) as substrate.
  • a solution of 0.2 % of the blue substrate is suspended m a 0.1 M phosphate buffer pH 7.5 under stirring.
  • the solution is distributed under stirring to 1.5 ml Eppendorf tubes (0.75 ml to each) , 50 ⁇ l enzyme solution is added and they are incubated m an Eppendorp Thermomixer model 5436 for 20 minutes at 40°C with a mixing of 1200 rpm. After incubation the colored solution is separated from the solid by 4 minutes centrifugation at 14,000 rpm and the absorbance of the supernatant is measured at 600 nm.
  • One XyloU unit is defined as the amount of enzyme resulting m an absorbance of 0.24 m a 1 cm cuvette at 600 nm.
  • CMC method CMC unit
  • CMC units is measured using 0.1 M Mops buffer pH 7.5 at 40°C. 20 mm incubation and determination of the formation of reducing sugars using PHAB : One CMC unit corresponds to the formation of 1 micromole glucose equivalent per mm.
  • a strain of Paenibacillus pabuli was grown m TY with pH adjusted pH 7. After 24 hours incubation at 30°C and 300 rpm, the cells were harvested and genomic DNA was isolated by the method described below.
  • the Paenibacillus pabuli strain was propagated m liquid media as described above.
  • the cells were harvested, and genomic DNA was isolated by the method described by Pi tcher et al . 1989.
  • Genomic DNA of Paenibacillus pabuli was partially digested with restriction enzyme Sau3A, and size-fractionated by elec- trophoresis on a 0.7 % agarose gel (SeaKem agarose, FMC, USA) . Fragments between 4 and 10 kb m size were isolated and concentrated to a DNA band by running the DNA fragments backwards on a 1.5 % agarose gel followed by extraction of the fragments from the agarose gel slice using the GFX gel extraction kit according to the manufacturer's instructions (Amersham Pharmacia Biotech, USA) . To construct a genomic library, ca .
  • plaque-forming units from the genomic library were plated on NZY-agar plates containing 0.1 % AZCL-xyloglucan (MegaZyme, Australia) using E. coli XLl-Blue MRF' (Stratagene, USA) as a host, followed by incubation of the plates at 37°C for 24 hours.
  • E. coli XLl-Blue MRF' (Stratagene, USA) as a host, followed by incubation of the plates at 37°C for 24 hours.
  • a single xyloglucanase-positive lambda clone was identified by the formation of blue hydrolysis halo around the positive phage clone.
  • the clone was recovered from the screening plate by coring the TOP-agar slice containing the plaque of interest into 500 ⁇ l of SM buffer and
  • the xyloglucanase-positive lambdaZAPExpress clone was plaque-purifled by plating an aliquot of the cored phage stock on NZY plates containing 0.1 % AZCL-xyloglucan as above.
  • a single, xyloglucanase-positive lambda clone was cored into 500 ⁇ l of SM buffer and 20 ⁇ l of chloroform, and purified by one more plating round as described above .
  • E. coli XLl-Blue cells (Stratagene, La Jolla Ca . ) were 5 prepared and resuspended lOmM MgS04 as recommended by
  • the xyloglucanase-positive phagemid clones were characterized by the formation of blue hydrolysis halos around the positive colonies. These were further analyzed by restriction enzyme digests of the isolated phagemid DNA (QiaSpm kit, Qiagen, USA) with EcoRI, Pstl, EcoRI-Pstl, and
  • 80 ng of target DNA from the genomic xyloglucanase clone pXYG1009 was transposon-tagged using the pGPS-2 donor plasmid 35 and the GPS-1 Genome Priming System from New England
  • E. coli DH10B cells Gibco-BRL, USA
  • the transformed E. coli cells were plated on LB agar plates containing kanamycm (20 ⁇ g/ml) , and chloramphemcol (15 ⁇ g/ml) .
  • Primers based on the DNA sequence of XYG1009 were designed for PCR amplification (polymerase chain reaction) and are given below as Primer 1 and Primer 2. PCR was carried out using the following protocol :
  • Primer 1 (10M) 1 . . 0 1
  • Primer 2 (10M) 1 . . 0 1
  • PCR Master mix (Advanced Biotechnologies Ltd. Surrey KT22 7Ba, UK, catalogue number AB-0575) containing Taq DNA polymerase, dNTPs, MgCl 2 and reaction buffer.
  • the PCR amplification conditions were Step 1 94 °C 2min Step 2 94°C 30sec
  • Step 3 55°C 30sec Step 4 72°C 2mm Steps 2-4 were repeated for 30 cycles Step 5 72°C 2mm Step 6 4°C hold temperature
  • Primer 1 CAT TCT GCA GCC GCG GCA GCG GAC GCT TCG CAA ATA GTG TC
  • Primer 2 GCG TTG AGA CGC GCG GCC GCT TAT TGC ATA CCT TGC ATG ATC GC
  • SEQ ID NO: 5 PCR product denoted XYG 1035 sequenced from
  • SEQ ID NO: 6 PCR product denoted XYG 1035 sequenced from C-termmal end
  • SEQ ID NO: 7 PCR product denoted XYG 1036 sequenced and overlapping from both ends
  • SEQ ID NO: 8 PCR product denoted XYG 1037 sequenced from N-terminal end
  • SEQ ID NO: 9 PCR product denoted XYG 1037 sequenced from
  • ammo acid sequences of the PCR amplified fragments were compared to that of SEQ ID NO: 2 and the number of ammo acid substitutions are given below.
  • the nomenclature is such that the numbering is based on the ammo acid sequence position in SEQ ID NO: 2.
  • the ammo acids are represented by single letters where the letter preceding the number is the ammo acid SEQ ID NO: 2 and the letter following the number is the changed ammo acid in the PCR amplified sequence.
  • five ammo acid substitutions could be detected (N94S, T197A, T241A, F286S, N379K) where the numbering is based on SEQ ID NO: 2.
  • the xyloglucanase encoding DNA sequence of the invention (SEQ ID NO: 1) was PCR amplified using the PCR primer set consisting of these two oligo nucleotides:
  • the oligonucleotides were used in a PCR reaction in HiFidelityTM PCR buffer (Boehringer Mannheim, Germany) supplemented with 200 ⁇ M of each dNTP, 2.6 units of HiFidelityTM Expand enzyme mix and 200 pmol of each primer. Chromosomal DNA isolated from Paenibacillus pabuli as described above was used as template.
  • the PCR reaction was performed using a DNA thermal cycler (Landgraf , Germany) .
  • One incubation at 94°C for 1 min followed by ten cycles of PCR performed using a cycle profile of denaturation at 94°C for 15 sec, annealing at 60°C for 60 sec, and extension at 72°C for 120sec, followed by twenty cycles of denaturation at 94°C for 15 sec, 60°C for 60 sec and 72°C for 120 sec (at this elongation step 20 sec are added every cycle) .
  • Five- ⁇ l aliquots of the amplification product was analysed by electrophoresis in 0.7 % agarose gels (NuSieve, FMC) . The appearance of a DNA fragment size 1.2 kb indicated proper amplification of the gene segment.
  • PCR fragment Forty-five- ⁇ l aliquots of the PCR products generated as described above were purified using QIAquick PCR purification kit (Qiagen, USA) according to the manufacturer's instructions. The purified DNA was eluted in 50 ⁇ l of lOmM Tris-HCl, pH 8.5. 5 ⁇ g of pMOL944 and twenty- five- ⁇ l of the purified PCR fragment was digested with SacII and Notl, electrophoresed m 0.7 % agarose gels (NuSieve, FMC) , the relevant fragments were excised from the gels, and purified using QIAquick Gel extraction Kit (Qiagen, USA) according to the manufacturer's instructions.
  • QIAquick Gel extraction Kit Qiagen, USA
  • the isolated PCR DNA fragment was then ligated to the SacII-Notl digested and purified pMOL944. The ligation was performed overnight at 16°C using 0.5 ⁇ g of each DNA fragment, 1 U of T4 DNA ligase and T4 ligase buffer (Boehrmger Mannheim, Germany) .
  • the ligation mixture was used to transform competent B. subtilis PL2306.
  • the transformed cells were plated onto LBPG- 10 ⁇ g/ml of Kanamycm-agar plates. After 18 hours incubation at 37°C colonies were seen on plates. Several clones were analyzed by isolating plasmid DNA from overnight culture broth.
  • MB1040 One such positive clone was restreaked several times on agar plates as used above; this clone was called MB1040.
  • the clone MB1040 was grown overnight m TY-lO ⁇ g/ml Kanamycm at 37°C, and next day 1 ml of cells were used to isolate plasmid from the cells using the Qiaprep Spin Plasmid Mmiprep Kit #27106 according to the manufacturers recommendations for B . subtilis plasmid preparations.
  • This DNA was sequenced and revealed a DNA sequence identical to the part of the xyloglucanase gene m SEQ ID NO: 1 encoding the mature xyloglucanase.
  • Example 3 The clone MB1040 obtained as described above m Example 3 was grown m BPX media with 10 ⁇ g/ml of Kanamycm m 500ml two baffled shake flasks for 5 days at 37°C at 300 rpm, whereby 4000 ml of culture broth was obtained with a pH of 5.8. Then 180 ml of cationic agent (C521 10%) and 360 ml of anionic agent (A130 0.1%) was added during agitation for flocculation.
  • the flocculated material was separated by centrifugation using a Sorval RC 3B centrifuge at 10000 rpm for 30 mm at 6°C.
  • the total volume of the resulting supernatant was 4200 ml.
  • the supernatant was clarified using Whatman glass filters GF/D and C and finally concentrated on a filtron UF membrane with a cut off of 10 kDa .
  • the total volume of 1450 ml was adjusted to pH 8.0.
  • Phenyl -Sepharose hydrophobic chromatography was carried out. To the solution was added ammonium sulphate to a final concentration of 1.2 M.
  • the column was adjusted with the same solution and the enzyme solution was added.
  • the xyloglucanase bound to the column and the pure xyloglucanase was eluted using water.
  • the xyloglucanase containing a 95% pure band m SDS -PAGE at 40 kDa was concentrated and formulated with 30% MPG for trials .
  • the pure enzyme gave a single band m SDS -PAGE of 40 kDa and an isoelectric point of around 8.9.
  • ADASQIVS The following sequence was found by N-terminal determination of the pure enzyme: ADASQIVS; the theoretical MW estimated from the found N-termmus ADASQIVS is 40535.06 Da.
  • the protein concentration was determined using a molar extinction coefficient of 94590 (based on the ammo acid composition deducted from the sequence) .
  • the pH activity profiles showed more than 50% relative activity between pH 6.0 and 8.0 at 40°C.
  • the temperature optimum was 50° at pH 7.5.
  • the ammo acid sequence SEQ ID NO: 2 deducted from the DNA sequence (SEQ ID NO: 1) shows that the coding region code for: positions 1-32 signal peptide, and positions 33-395 catalytic domain belonging to glycosyl hydrolase family 5.
  • SEQ ID NO: 2 is 83% homologous (Blast) with: CelA EMBL entry: Y12512 (Blanco A., Diaz P., Martinez J., Vidal T., Torres A.L., Pastor F.I.J.; "Cloning of a new endoglucanase gene from Bacillus sp . BP-23 and characterisation of the enzyme. Performance paper manufacture from cereal straw”; Appl . Microbiol. Biotechnol . 50:48-54(1998)).
  • SEQ ID NO: 2 is 30% homologous (Blast) with Bacillus agaradhaerens xyloglucanase disclosed m WO99/02663.
  • the substrate is xyloglucan (amyloid) from tamarind seeds (the substrate is commercially available from Megazyme) . Buffer 0.1 M sodium phosphate, pH 7.5.
  • the substrate was prepared as a stock solution containing 5 gram per 1 m buffer. After mixing it was heated using a magnetic stirrer until a clear solution was obtained. The solution was then cooled to 40 °C and kept m a temperature controlled water bath at 40°C. The diluted enzyme solution of 0.5 ml was preheated for 10 mm. and mixed with 1.0 ml substrate and incubated for 20 mm.
  • reducing sugars The formation of reducing sugars is determined by using p- hydroxy-benzoic-acid-hydrazide (PHBAH) modified from Lever (1972) using 5 gram of potassium sodium tartrate m addition to 1.5 gram of PHBAH. Glucose is used as reference for determination of the reducing groups.
  • PHBAH p- hydroxy-benzoic-acid-hydrazide
  • the xyloglucanase enzyme also had an activity of 0.2 CMC units per mg protein.
  • the xyloglucanase of the invention is characterised by having a high specific activity on xyloglucan (tamarind gum from Megazyme) and very low cellulase activity on CMC (endoglucanase activity) .
  • the xyloglucanase encoding DNA sequence of the invention was PCR amplified using the PCR primer set consisting of these two oligo nucleotides:
  • the oligonucleotides were used in a PCR reaction in HiFidelityTM PCR buffer (Boehringer Mannheim, Germany) supplemented with 200 ⁇ M of each dNTP, 2.6 units of HiFidelityTM Expand enzyme mix and 200 pmol of each primer. Chromosomal DNA isolated from Paenibacillus sp . , DSM 13330, was used as template.
  • the PCR reaction was performed using a DNA thermal cycler (Landgraf , Germany) .
  • One incubation at 94°C for 1 min followed by ten cycles of PCR performed using a cycle profile of denaturation at 94°C for 15 sec, annealing at 60°C for 60 sec, and extension at 72°C for 120sec, followed by twenty cycles of denaturation at 94°C for 15 sec, 60°C for 60 sec and 72°C for 120 sec (at this elongation step 20 sec are added every cycle) .
  • Five- ⁇ l aliquots of the amplification product was analysed by electrophoresis in 0.7 % agarose gels (NuSieve, FMC) .
  • the appearance of a DNA fragment size 1.3 kb indicated proper amplification of the gene segment.
  • the isolated PCR DNA fragment was then ligated to the Pstl-Notl digested and purified pMOL944.
  • the ligation was performed overnight at 16°C using 0.5 ⁇ g of each DNA fragment, 1 U of T4 DNA ligase and T4 ligase buffer (Boehrmger Mannheim, is Germany) .
  • the ligation mixture was used to transform competent B . subtili s MB1053-1.
  • the transformed cells were plated onto LBPG-10 ⁇ g/ml of Kanamycm-agar plates. After 18 hours incubation at 37°C colonies were seen on plates.
  • PL3381 One such positive clone was restreaked several times on agar plates as used above; this clone was called PL3381.
  • the clone PL3381 was grown overnight m TY-lO ⁇ g/ml Kanamycm at
  • the clone PL3381 obtained as described example 5 was incubated 4000 ml of BPX containing 10 ⁇ g/ml of Kanamycm and grown for 5 days at 37°C at 300 rpm, final pH was 5.64.
  • the fermentation medium was flocculated using cationic flocculation agent C521 (10% solution) and 0.1% solution of anionic agent A130: To 4000 ml of broth was added 180 ml of C521 (10%) simultaneously with 360 ml of A130 under stirring at room temperature.
  • the flocculated material was separated by centrifugation using a Sorval RC 3B centrifuge at 4,500 rpm for 30 minutes.
  • the supernatant was clarified using Whatman glass filter number F. In total was obtained 4000 ml of clear solution.
  • the liquid was concentrated into 400 ml, using filtron ultraflltration with a MW cut off of 10 kDa.
  • the concentrate was batch treated with 200 gram Q- Sepharose equilibrated with 25 mM T ⁇ s pH 7.5.
  • the unbound material was stabilized with 30% MPG and the xyloglucanase was used for detergent cleaning.
  • the enzyme is very active m the commercial liquid detergents sold by The Procter & Gamble Company under the brand names Ariel and Tide.
  • ADFRSLNASQIVSEMG ammo acid sequence shown m SEQ ID NO: 4 deduced from the DNA sequence shown SEQ ID NO: 3 with a 32 ammo acid pro sequence.
  • the calculated MW from the deduced sequence was 40 kDa and the calculated pi was 8.89.
  • the molar extinction coefficient at 280 nm was 93390.
  • DSC Disc Scanning Calorimetry
  • m sodium acetate buffer at pH 6.0 showed a melting temperature around 68.6°C.
  • the oligonucleotides were used m a PCR reaction m
  • HiFidelityTM PCR buffer (Boehrmger Mannheim, Germany) supplemented with 200 ⁇ M of each dNTP, 2.6 units of
  • the PCR reaction was performed using a DNA thermal cycler (Landgraf , Germany) .
  • One incubation at 94°C for 1 mm followed by ten cycles of PCR performed using a cycle profile of denaturation at 94°C for 15 sec, annealing at 60°C for 60 sec, and extension at 72°C for 120sec, followed by twenty cycles of denaturation at 94°C for 15 sec, 60°C for 60 sec and 72°C for 120 sec (at this elongation step 20 sec are added every cycle) .
  • Five- ⁇ l aliquots of the amplification product was analysed by electrophoresis m 0.7 % agarose gels (NuSieve, FMC) . The appearance of a DNA fragment size 1.2 kb indicated proper amplification of the gene segment.
  • PCR fragment Forty-flve- ⁇ l aliquots of the PCR products generated as described above were purified using QIAquick PCR purification kit (Qiagen, USA) according to the manufacturer's instructions. The purified DNA was eluted 50 ⁇ l of lOmM T ⁇ s-HCl, pH 8.5.
  • the ligation mixture was used to transform competent B. subtilis PL2306.
  • the transformed cells were plated onto LBPG- 10 ⁇ g/ml of Kanamycm-agar plates. After 18 hours incubation at 37°C colonies were seen on plates. Several clones were analyzed by isolating plasmid DNA from overnight culture broth.
  • MB1067 One such positive clone was restreaked several times on agar plates as used above, this clone was called MB1067.
  • the clone MB1067 was grown overnight TY-lO ⁇ g/ml Kanamycm at 37°C, and next day 1 ml of cells were used to isolate plasmid from the cells using the Qiaprep Spin Plasmid Mmiprep Kit #27106 according to the manufacturers recommendations for B . subtilis plasmid preparations.
  • This DNA was sequenced and revealed a DNA sequence identical to the part of the xyloglucanase gene m SEQ ID NO: 10 encoding the mature xyloglucanase represented by the derived protein sequence m SEQ ID NO: 11.
  • the clone MB1067 obtained as described m example 7 was incubated m 4200 ml of BPX containing mg/ml kanamycm from shake flasks with a final pH of 7.5.
  • the fermentation medium was flocculated using 42 ml 50% W/W CaCl 2 , 42 ml 11% Na-alummate and 20% formic acid followed by adding 105 ml of C521 (10%) simultaneously with 315 ml of A130 under stirring at room temperature.
  • the flocculated material was separated by centrifugation using a Sorval RC 3B centrifuge at 4,500 rpm for 30 minutes.
  • the supernatant was clarified using Whatman glass filter number F. In total was obtained 3900 ml of clear solution.
  • the liquid was concentrated into 400 ml, using filtron ultraflltration with a MW cut off of 10 kDa.
  • the concentrate was batch treated with 200 gram Q- Sepharose equilibrated with 25 mM Tris pH 7.5.
  • the unbound material was stabilized with 30% MPG and the xyloglucanase was used for detergent cleaning.
  • the enzyme is very active in liquid Ariel and Tide.

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Abstract

Xyloglucanases belonging to family 5 of glycosyl hydrolases are derived from strains of Paenibacillus, especially from strains of Paenibacillus pabuli. The xyloglucanases show high performance in conventional liquid detergent compositions.

Description

FAMILY 5 XYLOGLUCANASES
The present invention relates to xyloglucanases belonging to family 5 of glycosyl hydrolases, preferably to enzymes exhibiting xyloglucanase activity as their major enzymatic activity m the neutral and alkaline pH ranges; to a method of producing such enzymes; and to methods for using such enzymes m the textile, detergent and cellulose fiber processing industries .
BACKGROUND OF THE INVENTION
Xyloglucan is a major structural polysacchaπde m the primary (growing) cell wall of plants. Structurally, xyloglucans consists of a cellulose-like beta-1 , 4-lmked glucose backbone, which is frequently substituted with various side chains. The xyloglucans of most dicotyledonous plants, some monocotyledons and gymnosperms are highly branched polysaccharides m which approx. 75% of the glucose residues m the backbone bear a glycosyl side chain at O-β. The glycosyl residue that is directly attached to the branched glucose residue is invariably alfa-D-xylose . Up to 50% of the side chains m the xyloglucans contain more than one residue due to the presence of beta-D-galactose or alfa-L-fucose- (1-2) -beta-D- galactose moieties at 0-2 of the xylose residues (C. Ohsumi and T. Hayashi (1994) Plant and Cell Physiology 35:963-967; G. J. McDougall and S. C. Fry (1994) Journal of Plant Physiology 143:591-595; J. L. Acebes et al . (1993) Phytochemistry 33:1343- 1345) . On acid hydrolysis, the xyloglucan extracted from cotton fibers yielded glucose, xylose, galactose and fucose m the ratio of 50:29:12:7 (Hayashi et al . , 1988).
Xyloglucans produced by solanaceous plants are unusual m that typical only 40% of the beta-1 , 4-lmked glucose residues bear a glycosyl side chain at 0-6. Furthermore, up to 60% of the xylose residues are substituted at 0-2 with alfa-L- arabinose residues and some solanaceous plants, such as potato, also have xyloglucans with beta-D-galactose substituents at 0-2 of some of the xylose residues (York et al (1996)) .
Xyloglucan is believed to function m the primary wall of plants by cross-linking cellulose-micro fibrils, forming a cellulose-xyloglucan network. This network is considered necessary for the structural integrity of primary cell -walls (Carpita et al . , 1993). Another important function of xyloglucan is to act as a repository for xyloglucan subun t oligosaccharides that are physiologically active regulators of plant cell growth. Xyloglucan subunits may also modulate the action of a xyloglucan endotransglycosylase (XET) , a cell wall associated enzyme that has been hypothesized to play a role m the elongation of plant cell walls. Therefore xyloglucan might play an important role m wall loosening and consequently cell expansion (Fry et al . , 1992) .
The seeds of many dicotyledonous species contain xyloglucan as the major polysacchaπde storage reserve. This type of xyloglucan, which is localized m massive thickenings on the inside of the seed cotyledon cell wall, is composed mainly of glucose, xylose and galactose (Rose et al . , 1996) .
Seeds of the tamarind tree Tamaπndus mdica became a commercial source of gum m 1943 when the gum was found useful as a paper and textile size. Sizing of ute and cotton with tamarind xyloglucan has been extensively practiced m Asia owing to the low cost of the gum and to its excellent properties. Food applications of tamarind xyloglucan include use m confections, ams and jellies and as a stabilizer m ice cream and mayonnaise (Whistler et al . , 1993) .
Xyloglucanase activity is not included m the classification of enzymes provided by the Enzyme Nomenclature (1992) . Hitherto, this enzymatic activity has simply been classified as glucanase activity and has often been believed to be identical to cellulolytic activity (EC 3.2.1.4), i.e. activity against β-1 , 4-glycosιdιc linkages m cellulose or cellulose derivative substrates, or at least to be a side activity m enzymes having cellulolytic activity. However, a true xyloglucanase is a true xyloglucan specific enzyme capable of catalyzing the solubilisation of xyloglucan to xyloglucan oligosaccharides but which does not exhibit substantial cellulolytic activity, e.g. activity against the conventionally used cellulose-like substrates CMC (carboxymethylcellulose) , HE cellulose and Avicel (macrocrystalline cellulose) . A xyloglucanase cleaves the beta-1 , 4-glycosidic linkages in the backbone of xyloglucan.
Xyloglucanase activity is described by Vincken et al . (1997) who characterizes three different endoglucanases from Trichoderma viride (similar to T. reesei ) which all have high activity against cellulose or CMC and show that the Endol (belonging to family 5 of glycosyl hydrolases, see Henrissat, B. et al . (1991, 1993)) has essentially no (i.e. very little) activity against xyloglucan, and that EndoV (belonging to the family 7 of glycosyl hydrolases) and EndoIV (belonging to the family 12 of glycosyl hydrolases) both have activity against xyloglucan and CMC, respectively, of the same order of magnitude . International Patent Publication WO 94/14953 discloses a family 12 xyloglucanase (EG II) cloned from the fungus Aspergillus aculeatus and expressed in the fungus Aspergillus oryzae .
International Patent Publication WO 99/02663 discloses xyloglucanases cloned from Bacillus licheniformis (family 12) and Bacillus agaradhaerens (family 5) and expressed in Bacillus subtilis .
It is an object of the present invention to provide an enzyme with a high xyloglucanase activity, which have an excellent performance in conventional detergent compositions, especially liquid detergents for household laundering.
SUMMARY OF THE INVENTION
The inventors have now found enzymes having substantial xyloglucanase activity, which enzymes belong to family 5 of glycosyl hydrolases and exhibit excellent performance in conventional detergent compositions, especially liquid detergent compositions. All the found xyloglucanases are endogenous to a strain belonging to Paenibacillus pabuli or Paenibacillus sp .
Accordingly, the present invention relates to a xyloglucanase enzyme belonging to family 5 of glycosyl hydrolases, which enzyme is endogenous to a strain of Paenibacillus . Preferably, the strain of Paenibacillus belongs to the group consisting of the species Paenibacillus pabuli , the strain Paenibacillus sp . , DSM 13330, and strains of Paenibacillus sp . having a higher degree of identity with the Paenibacillus pabuli type strain ATCC 43899 than the strain Paenibacillus sp . , DSM 13330, when subjected to 16S RNA analysis .
The inventors have also succeeded in cloning and expressing a family 5 xyloglucanase from the above species and strains, i.e. the invention relates in further aspects to a family 5 xyloglucanase which is (a) a polypeptide encoded by the DNA sequence of positions 840-1931 of SEQ ID NO : 1, (b) a polypeptide produced by culturing a cell comprising the sequence of SEQ ID NO: 1 under conditions wherein the DNA sequence is expressed;
(c) a xyloglucanase enzyme having a sequence of at least 85% identity to positions 33-395 of SEQ ID NO: 2 when identity is determined by GAP provided in the GCG program package using a GAP creation penalty of 3.0 and GAP extension penalty of 0.1; or
(d) a polypeptide encoded by the xyloglucanase encoding part of the DNA sequence obtainable from the plasmid in Escheri chia coli DSM 13183; and to an isolated polynucleotide molecule encoding a polypeptide having xyloglucanase activity which polynucleotide molecule hybridizes to a denatured double- stranded DNA probe under medium stringency conditions, wherein the probe is selected from the group consisting of DNA probes comprising the sequence shown in positions 840-1931 of SEQ ID NO: 1, positions 693-1896 of SEQ ID NO : 3 , and DNA probes comprising a subsequence of positions 840-1931 of SEQ ID NO : 1 or positions 693-1896 of SEQ ID NO : 3 , the subsequence having a length of at least about 100 base pairs.
In further aspects, the invention provides an expression vector comprising a DNA segment which is e.g. a polynucleotide molecule of the invention; a cell comprising the DNA segment or the expression vector; and a method of producing a exhibiting xyloglucanase enzyme, which method comprises culturing the cell under conditions permitting the production of the enzyme, and recovering the enzyme from the culture.
In yet another aspect the invention provides an isolated family 5 xyloglucanase enzyme characterized m (1) being free from homologous impurities and (11) being produced by the method described above.
The novel enzyme of the present invention is useful for the treatment of cellulosic material, especially cellulose- containing fiber, yarn, woven or non-woven fabric. The treatment can be carried out during the processing of cellulosic material into a material ready for garment manufacture or fabric manufacture, e.g. m the desizmg or scouring step; or during industrial or household laundering of such fabric or garment.
Accordingly, m further aspects the present invention relates to a detergent composition comprising a xyloglucanase enzyme having substantial xyloglucanase activity m the neutral or alkaline range; and to use of the enzyme of the invention for the treatment of cellulose-containing fibers, yarn, woven or non-woven fabric. The present invention has now made it possible to use a xyloglucanase m detergent compositions for removing or bleaching certain soils or stains present on laundry, especially soils and spots resulting from xyloglucan-containmg food, plants, and the like. Further, it is contemplated that treatment with detergent compositions comprising the novel enzyme can prevent binding of certain soils to the xyloglucan left on the cellulosic material.
DETAILED DESCRIPTION OF THE INVENTION
MICROBIAL SOURCES
For the purpose of the present invention the term
"obtained from" or "obtainable from" as used herein m connection with a specific source, means that the enzyme is produced or can be produced by the specific source, or by a cell m which a gene from the source have been inserted.
It is at present contemplated that the xyloglucanase of the invention may be obtained from a gram-positive bacterium belonging to a strain of the genus Bacillus, in particular a strain of Paenibacillus .
In a preferred embodiment, the xyloglucanase of the invention is obtained from the species Paenibacillus pabuli that is represented by the type strain ATCC 43899, this type strain being publicly available from American Type Culture Collection (ATCC) . It is at present contemplated that a DNA sequence encoding an enzyme with an amino acid sequence identity of at least 85% to the enzyme of the invention may be obtained from other strains belonging to the species Paenibacillus pabuli and such strains belonging to the species Paenibacillus sp . which, when subjected to a conventional 16S RNA analysis, have a higher degree of identity with the Paenibacillus pabuli type strain ATCC 43899 than the strain Paenibacillus sp . , DSM 13330. It is contemplated that the strain Paenibacillus sp . , DSM 13330, has an identity, compared with the strain Paenibacillus pabuli ATCC 43899, of at least 95%, more specifically of at least 97%. Further, the strain Paenibacillus sp . was deposited by the inventors according to the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg lb, D- 38124 Braunschweig, Federal Republic of Germany, on 17 February 2000 under the deposition number DSM 13330. The deposit was made by Novo Nordisk A/S and was later assigned to Novozymes A/S.
A plasmid comprising a DNA sequence encoding a xyloglucanase of the invention has been transformed into a strain of the Escherichia coli which was deposited by the inventors according to the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg lb, D- 38124 Braunschweig, Federal Republic of Germany, on 6 December 1999 under the deposition number DSM 13183. The deposit was made by Novo Nordisk A/S and was later assigned to Novozymes A/S. It is contemplated that the DNA sequence of this plasmid comprises the DNA sequence of SEQ ID NO: 1.
DEFINITIONS In the present context, the term "enzyme preparation" is intended to mean either be a conventional enzymatic fermentation product, possibly isolated and purified, from a single species of a microorganism, such preparation usually comprising a number of different enzymatic activities; or a mixture of monocomponent enzymes, preferably enzymes derived from bacterial or fungal species by using conventional recombmant techniques, which enzymes have been fermented and possibly isolated and purified separately and which may originate from different species, preferably fungal or bacterial species; or the fermentation product of a microorganism which acts as a host cell for expression of a recombmant xyloglucanase, but which microorganism simultaneously produces other enzymes, e.g. xyloglucanases, proteases, or cellulases, being naturally occurring fermentation products of the microorganism, i.e. the enzyme complex conventionally produced by the corresponding naturally occurring microorganism.
In the present context the term "expression vector" denotes a DNA molecule, linear or circular, that comprises a segment encoding a polypeptide of interest operably linked to additional segments that provide for its transcription. Such additional segments may include promoter and terminator sequences, and may optionally include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, and the like. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both. The expression vector of the invention may be any expression vector that is conveniently subjected to recombmant DNA procedures, and the choice of vector will often depend on the host cell into which the vector is to be introduced. Thus, the vector may be an autonomously replicating vector, i.e. a vector that exists as an extra chromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome (s) into which it has been integrated. The term "recombmant expressed" or "recombmantly expressed" used herein connection with expression of a polypeptide or protein is defined according to the standard definition the art. Recombmant expression of a protein is generally performed by using an expression vector as described immediately above.
The term "isolated", when applied to a polynucleotide molecule, denotes that the polynucleotide has been removed from its natural genetic milieu and is thus free of other extraneous or unwanted coding sequences, and is m a form suitable for use within genetically engineered protein production systems. Such isolated molecules are those that are separated from their natural environment and include cDNA and genomic clones. Isolated DNA molecules of the present invention are free of other genes with which they are ordinarily associated, but may include naturally occurring 5 ' and 3 ' untranslated regions such as promoters and terminators. The identification of associated regions will be evident to one of ordinary skill m the art (see for example, Dynan and Tijan, Nature 316 : 774-78, 1985). The term "an isolated polynucleotide" may alternatively be termed "a cloned polynucleotide" .
When applied to a protem/polypeptide , the term "isolated" indicates that the protein is found m a condition other than its native environment. In a preferred form, the isolated protein is substantially free of other proteins, particularly other homologous proteins (i.e. "homologous impurities" (see below) ) . It is preferred to provide the protein m a greater than 40% pure form, more preferably greater than 60% pure form. Even more preferably it is preferred to provide the protein m a highly purified form, i.e., greater than 80% pure, more preferably greater than 95% pure, and even more preferably greater than 99% pure, as determined by SDS-PAGE.
The term "isolated protem/polypeptide may alternatively be termed "purified protem/polypeptide" . The term "homologous impurities" means any impurity (e.g. another polypeptide than the polypeptide of the invention) , which originate from the homologous cell where the polypeptide of the invention is originally obtained. The term "obtained from" as used herein m connection with a specific microbial source, means that the polynucleotide and/or polypeptide produced by the specific source, or by a cell m which a gene from the source have been inserted.
The term "operably linked", when referring to DNA segments, denotes that the segments are arranged so that they function m concert for their intended purposes, e.g. transcription initiates m the promoter and proceeds through the coding segment to the terminator
The term "polynucleotide" denotes a single- or double- stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end. Polynucleotides include RNA and DNA, and may be isolated from natural sources, synthesized in vi tro, or prepared from a combination of natural and synthetic molecules . The term "complements of polynucleotide molecules" denotes polynucleotide molecules having a complementary base sequence and reverse orientation as compared to a reference sequence. For example, the sequence 5' ATGCACGGG 3' is complementary to 5 ' CCCGTGCAT 3 ' . The term "degenerate nucleotide sequence" denotes a sequence of nucleotides that includes one or more degenerate codons (as compared to a reference polynucleotide molecule that encodes a polypeptide) . Degenerate codons contain different triplets of nucleotides, but encode the same ammo acid residue (i.e., GAU and GAC triplets each encode Asp) .
The term "promoter" denotes a portion of a gene containing DNA sequences that provide for the binding of RNA polymerase and initiation of transcription. Promoter sequences are commonly, but not always, found m the 5' non-codmg regions of genes.
The term "secretory signal sequence" denotes a DNA sequence that encodes a polypeptide (a "secretory peptide") that, as a component of a larger polypeptide, directs the larger polypeptide through a secretory pathway of a cell m which it is synthesized. The larger peptide is commonly cleaved to remove the secretory peptide during transit through the secretory pathway.
POLYNUCLEOTIDES
Within preferred embodiments of the invention an isolated polynucleotide of the invention will hybridize to similar sized regions of SEQ ID NO: 1 or SEQ ID NO: 3, or a sequence complementary thereto, under at least medium stringency conditions .
In particular polynucleotides of the invention will hybridize to a denatured double- stranded DNA probe comprising either the full sequence shown m SEQ ID NO: 1 or the sequence shown m positions 840-1931 of SEQ ID NO: 1 or the full sequence shown SEQ ID NO: 3 or the sequence shown m positions 693-1896 of SEQ ID NO : 3 or any probe comprising a subsequence of SEQ ID NO: 5 or SEQ ID NO: 3 having a length of at least about 100 base pairs under at least medium stringency conditions, but preferably at high stringency conditions as described m detail below. Suitable experimental conditions for determining hybridization at medium or high stringency between a nucleotide probe and a homologous DNA or RNA sequence involve pre -soaking of the filter containing the DNA fragments or RNA to hybridize m 5 x SSC (Sodium chloride/Sodium citrate,
Sambrook et al . 1989) for 10 mm, and prehybπdization of the filter m a solution of 5 x SSC, 5 x Denhardt ' s solution (Sambrook et al . 1989), 0.5 % SDS and 100 μg/ml of denatured sonicated salmon sperm DNA (Sambrook et al . 1989), followed by hybridization in the same solution containing a concentration of lOng/ml of a random-primed (Femberg, A. P. and Vogelstem, B (1983) Anal. Biochem. 132:6-13), 32P-dCTP-labeled (specific activity higher than 1 x 109 cpm/μg) probe for 12 hours at ca . 45°C. The filter is then washed twice for 30 minutes m 2 x SSC, 0.5 % SDS at least 60°C (medium stringency), still more preferably at least 65 °C (medium/high stringency) , even more preferably at least 70 °C (high stringency) , and even more preferably at least 75°C (very high stringency) . Molecules to which the oligonucleotide probe hybridizes under these conditions are detected using an x-ray film.
As previously noted, the isolated polynucleotides of the present invention include DNA and RNA. Methods for isolating DNA and RNA are well known m the art. DNA and RNA encoding genes of interest can be cloned m Gene Banks or DNA libraries by means of methods known m the art .
Polynucleotides encoding polypeptides having endoglucanase activity of the invention are then identified and isolated by, for example, hybridization or PCR.
The present invention further provides counterpart polypeptides and polynucleotides from different bacterial strains (orthologs or paralogs) . Of particular interest are xyloglucanase polypeptides from gram-positive alkalophilic strains, including species of Bacillus . Of special interest are xyloglucanase peptides from strains of Paenibacillus, which are very closely related to the species Paenibacillus pabuli , exemplified by the strain ATCC 43899 that is the type strain of Paenibacillus pabuli . Species homologues of a polypeptide with xyloglucanase activity of the invention can be cloned using information and compositions provided by the present invention m combination with conventional cloning techniques. For example, a DNA sequence of the present invention can be cloned using chromosomal DNA obtained from a cell type that expresses the protein. Suitable sources of DNA can be identified by probing Northern blots with probes designed from the sequences disclosed herein. A library is then prepared from chromosomal DNA of a positive cell line. A DNA sequence of the invention encoding an polypeptide having xyloglucanase activity can then be isolated by a variety of methods, such as by probing with probes designed from the sequences disclosed m the present specification and claims or with one or more sets of degenerate probes based on the disclosed sequences. A DNA sequence of the invention can also be cloned using the polymerase chain reaction, or PCR (Mullis, U.S. Patent 4,683,202), using primers designed from the sequences disclosed herein Within an additional method, the DNA library can be used to transform or transfect host cells, and expression of the DNA of interest can be detected with an antibody (monoclonal or polyclonal) raised against the xyloglucanase cloned from Paenibacillus pabuli , e.g. from the type strain deposited as ATCC 43899, or from Paenibacillus sp . , DSM 13330, expressed and purified as described in Materials and Methods and the examples, or by an activity test relating to a polypeptide having xyloglucanase activity.
POLYPEPTIDES :
The sequence of amino acids in positions 33-395 of SEQ ID NO: 2 and positions 33-400 of SEQ ID NO : 4, respectively, is a mature xyloglucanase sequence comprising the catalytic active domain. The sequence of am o acids of SEQ ID NOS: 5-9 represents N-terminal or C-terminal sequences of mature xyloglucanase sequences, cf. example 2.
The present invention also provides xyloglucanase polypeptides that are substantially homologous to the polypeptide of amino acids in position 33-395 of SEQ ID NO: 2 and species homologs (paralogs or orthologs) thereof. The term " substantially homologous" is used herein to denote polypeptides having 85%, preferably at least 88%, more preferably at least 90%, and even more preferably at least 95%, sequence identity to the sequence shown in amino acids nos. 33- 395 of SEQ ID NO: 2 or its orthologs or paralogs. Such polypeptides will more preferably be at least 98% identical to the sequence shown in amino acids in positions 33-395 of SEQ ID NC : 2 or its orthologs or paralogs. 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 m its entirety. GAP is used with the following settings for polypeptide sequence comparison: GAP creation penalty of 3.0 and GAP extension penalty of 0.1. The following sequence identity was found for the appended SEQ ID NOS: 2 and 4:
SEQ ID #2 SEQ ID #4 SEQ ID #2 100% 88%
Sequence identity of polynucleotide molecules is determined by similar methods using GAP with the following settings for DNA sequence comparison: GAP creation penalty of 5.0 and GAP extension penalty of 0.3.
Substantially homologous proteins and polypeptides are characterized as having one or more ammo acid substitutions, deletions or additions. These changes are preferably of a minor nature, that is conservative ammo acid substitutions (see Taole 2) and other substitutions that do not significantly affect the folding or activity of the protein or polypeptide; small deletions, typically of one to about 30 ammo acids; and small ammo- or carboxyl -terminal extensions, such as an am o- termmal methionme residue, a small linker peptide of up to about 20-25 residues, or a small extension that facilitates purification (an affinity tag), such as a poly-histidme tract, protein A (Nilsson et al . , EMBO J. 4:1075, 1985; Nilsson et al , Methods Enzymol . 198 : 3 , 1991. See, m general Ford et al . , Protein Expression and Purification 2: 95-107, 1991, which is incorporated herein by reference. DNAs encoding affinity tags are available from commercial suppliers (e.g., Pharmacia Biotech, Piscataway, NJ; New England Biolabs, Beverly, MA) .
However, even though the changes described above preferably are of a minor nature, such changes may also be of a larger nature such as fusion of larger polypeptides of up to 300 ammo acids or more both as ammo- or carboxyl -terminal extensions to a polypeptide of the invention having xyloglucanase activity.
Taole 1 Conservative ammo acid substitutions Basic: argmme lysme histidme Acidic ; glutamic acid aspartic acid
Polar: glutamme asparag e
Hydrophobic leucine lsoleucme valme
Aromatic phenylalanme tryptophan
10 tyrosme
Small : glycme alanme serme threonme
15 methionme
In addition to the 20 standard ammo acids, non-standard ammo acids (such as 4 -hydroxyprolme, 6-iV-methyl lysme, 2- am oisobutyπc acid, isovaline and a-methyl serme) may be
20 substituted for ammo acid residues of a polypeptide according to the invention. A limited number of non-conservative ammo acids, ammo acids that are not encoded by the genetic code, and unnatural ammo acids may be substituted for ammo acid residues. "Unnatural ammo acids" have been modified after
25 protein synthesis, and/or have a chemical structure m their side chain (s) different from that of the standard ammo acids.
Unnatural ammo acids can be chemically synthesized, or preferably, are commercially available, and include pipecolic acid, thiazolidme carboxylic acid, dehydroprolme, 3- and 4-
30 methylprolme, and 3 , 3 -dimethylprol e .
Essential ammo acids m the xyloglucanase polypeptides of the present invention can be identified according to procedures known m the art, such as site-directed mutagenesis or alan e- scannmg mutagenesis (Cunningham and Wells, Science 244 : 1081-
35 1085, 1989) . In the latter technique, single alanme mutations are introduced at every residue m the molecule, and the resultant mutant molecules are tested for biological activity (i.e. xyloglucanase activity) to identify ammo acid residues that are critical to the activity of the molecule. See also, Hilton et al . , J. Biol. Chem. 271 =4699-4708, 1996. The active site of the enzyme or other biological interaction can also be determined by physical analysis of structure, as determined by 5 such techniques as nuclear magnetic resonance, crystallography, electron diffraction or photo affinity labeling, conjunction with mutation of putative contact site ammo acids. See, for example, de Vos et al . , Science 25_5: 306-312 , 1992; Smith et al., J. Mol. Biol. 224:899-904, 1992; Wlodaver et al . , FEBS lo Lett . 309 : 59-64 , 1992. The identities of essential ammo acids can also be inferred from analysis of homologies with polypeptides, which are related to a polypeptide according to the invention.
Multiple ammo acid substitutions can be made and tested is using known methods of mutagenesis, recombination and/or shuffling followed by a relevant screening procedure, such as those disclosed by Reidhaar-Olson and Sauer (Science 241:53-57, 1988), Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152- 2156, 1989), W095/17413, or WO 95/22625. Briefly, these authors
20 disclose methods for simultaneously randomizing two or more positions m a polypeptide, or recombmation/shuffling of different mutations (W095/17413, W095/22625) , followed by selecting for functional a polypeptide, and then sequencing the mutagenized polypeptides to determine the spectrum of allowable
25 substitutions at each position. Other methods that can be used include phage display (e.g., Lowman et al . , Biochem. 30 : 10832- 10837, 1991; Ladner et al . , U.S. Patent No. 5,223,409; Huse, WIPO Publication WO 92/06204) and region-directed mutagenesis (Derbyshire et al . , Gene 46:145, 1986; Ner et al . , DNA 7:127,
30 1988) .
Mutagenesis/shuffling methods as disclosed above can be combined with high-throughput , automated screening methods to detect activity of cloned, mutagenized polypeptides m host cells. Mutagenized DNA molecules that encode active
35 polypeptides can be recovered from the host cells and rapidly sequenced using modern equipment . These methods allow the rapid determination of the importance of individual ammo acid residues m a polypeptide of interest, and can be applied to polypeptides of unknown structure.
Using the methods discussed above, one of ordinary skill m the art can identify and/or prepare a variety of polypeptides that are substantially homologous or identical to residues 33 to 395 of SEQ ID NO : 2 and retain the xyloglucanase activity of the wild-type protein.
The xyloglucanase enzyme of the invention may, m addition to the enzyme core comprising the catal tically domain, also comprise a cellulose binding domain (CBD) , the cellulose binding domain and enzyme core (the catalytically active domain) of the enzyme being operably linked. The cellulose binding domain (CBD) may exist as an integral part the encoded enzyme, or a CBD from another origin may be introduced into the xyloglucanase thus creating an enzyme hybrid. In this context, the term "cellulose-bmdmg domain" is intended to be understood as defined by Peter Tomme et al . "Cellulose-Bmdmg Domains: Classification and Properties" "Enzymatic Degradation of Insoluble Carbohydrates", John N. Saddler and Michael H. Penner (Eds.), ACS Symposium Series, No. 618, 1996. This definition classifies more than 120 cellulose-bmdmg domains into 10 families (I-X), and demonstrates that CBDs are found m various enzymes such as cellulases, xylanases, mannanases, arabinofuranosidases , acetyl esterases and chitmases. CBDs have also been found m algae, e.g. the red alga Porphyra purpurea as a non-hydrolytic polysaccharide- bmdmg protein, see Tomme et al . , op . ci t . However, most of the CBDs are from cellulases and xylanases, CBDs are found at the N and C termini of proteins or are internal . Enzyme hybrids are known m the art, see e.g. WO 90/00609 and WO 95/16782, and may be prepared by transforming into a host cell a DNA construct comprising at least a fragment of DNA encoding the cellulose- bmdmg domain ligated, with or without a linker, to a DNA sequence encoding the xyloglucanase and growing the host cell to express the fused gene. Enzyme hybrids may be described by the following formula:
CBD - MR - X wherein CBD is the N-termmal or the C-termmal region of an ammo acid sequence corresponding to at least the cellulose- bmdmg 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 about 100 ammo acids, more preferably of from 2 to 40 ammo acids; and X is an N-termmal or C-termmal region of a polypeptide encoded by the polynucleotide molecule of the invention.
IMMUNOLOGICAL CROSS-REACTIVITY
Polyclonal antibodies, especially monospecific polyclonal antibodies, to be used determining immunological cross- reactivity may be prepared by use of a purified xyloglucanolytic enzyme. More specifically, antiserum against the xyloglucanase of the invention may be raised by immunizing rabbits (or other rodents) according to the procedure described by N. Axelsen et al . m: A Manual of Quantitative Immunoelectrophoresis , Blackwell Scientific Publications, 1973, Chapter 23, or A. Johnstone and R. Thorpe, Immunochemistry m Practice, Blackwell Scientific Publications, 1982 (more specifically p. 27-31) . Purified lmmunoglobulms may be obtained from the antisera, for example by salt precipitation ((NH4)2 S04) , followed by dialysis and ion exchange chromatography, e . g. on DEAE-Sephadex. Immunochemical characterization of proteins may be done either by Outcherlony double-diffusion analysis (0. Ouchterlony m: Handbook of Experimental Immunology (D.M. Weir, Ed.), Blackwell Scientific Publications, 1967, pp. 655-706), by crossed immunoelectrophoresis (N. Axelsen et al . , supra, Chapters 3 and 4), or by rocket immunoelectrophoresis (N. Axelsen et al . , Chapter 2) .
RECOMBINANT EXPRESSION VECTORS
A recombmant vector comprising a DNA construct encoding the enzyme of the invention may be any vector, which may con- veniently be subjected to recombmant 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 that exists as an extra chromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell genome m part or m its entirety and replicated together with the chromosome (s) into which it has been integrated.
The vector is preferably an expression vector which the DNA sequence encoding the enzyme of the invention is operably linked to additional segments required for transcription of the DNA. In general, the expression vector is derived from plasmid or viral DNA, or may contain elements of both. The term, "operably linked" indicates that the segments are arranged so that they function in concert for their intended purposes, e.g. transcription initiates m a promoter and proceeds through the DNA sequence coding for the enzyme .
The promoter may be any DNA sequence, which shows transcπptional activity m the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell . Examples of suitable promoters for use in bacterial host cells include the promoter of the Bacillus stearothermophilus maltogenic amylase gene, the Bacillus licheniformis alpha- amylase gene, the Bacillus amyloliquefaci ens alpha-amylase gene, the Bacillus subtilis alkaline protease gene, or the Bacillus pumilus xylosidase gene, or the phage Lambda PR or PL promoters or the E. coll lac, trp or tac promoters.
The DNA sequence encoding the enzyme of the invention may also, if necessary, be operably connected to a suitable terminator. The recombmant vector of the invention may further comprise a DNA sequence enabling the vector to replicate m the host cell m question.
The vector may also comprise a selectable marker, e.g. a gene the product of which complements a defect m the host cell, or a gene encoding resistance to e.g. antibiotics like kanamyc , chloramphenicol , erythromycm, tetracyclme, spectmomycme, or the like, or resistance to heavy metals or herbicides . To direct an enzyme of the present invention into the secretory pathway of the host cells, a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) may be provided m the recombmant vector. The secretory signal sequence is joined to the DNA sequence encoding the enzyme m the correct reading frame . Secretory signal sequences are commonly positioned 5 ' to the DNA sequence encoding the enzyme. The secretory signal sequence may be that normally associated with the enzyme or may be from a gene encoding another secreted protein.
The procedures used to ligate the DNA sequences coding for the present enzyme, the promoter and optionally the terminator and/or secretory signal sequence, respectively, or to assemble these sequences by suitable PCR amplification schemes, and to insert them into suitable vectors containing the information necessary for replication or integration, are well known to persons skilled m the art (cf., for instance, Sambrook et al . ,
HOST CELLS
The cloned DNA molecule introduced into the host cell may be either homologous or heterologous to the host m question. If homologous to the host cell, i.e. produced by the host cell m nature, it will typically be operably connected to another promoter sequence or, if applicable, another secretory signal sequence and/or terminator sequence than m its natural environment. The term "homologous" is intended to include a DNA sequence encoding an enzyme native to the host organism m question. The term "heterologous" is intended to include a DNA sequence not expressed by the host cell m nature. Thus, the DNA sequence may be from another organism, or it may be a synthetic sequence.
The host cell into which the cloned DNA molecule or the recombmant vector of the invention is introduced may be any cell, which is capable of producing the desired enzyme and includes bacteria, yeast, fungi and higher eukaryotic cells.
Examples of bacterial host cells which on cultivation are capable of producing the enzyme of the invention may be a gram-positive bacteria such as a strain of Bacillus, m particular Bacillus alkalophilus , Bacillus amyloliquefaciens, Bacillus brevis, Bacillus lautus, Bacillus lentus , Bacillus licheniformis , Bacillus circulans , Bacillus coagulans , Bacillus mega theri um, Bacillus stearothermophilus , Bacillus subtilis and Bacillus thuringi ensis, a strain of Lactobacillus, a strain of Streptococcus, a strain of Streptomyces, m particular Strep to yces 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 m a manner known per se (cf . e.g. Sambrook et al . , supra) . When expressing the enzyme bacteria such as
Escherichia coli , the enzyme may be retained m 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 m gram-positive bacteria such as a strain of Bacillus or a strain of Streptomyces, the enzyme may be retained m the cytoplasm, or may be directed to the extra cellular 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, m particular Aspergillus awamori , Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, and Fusarium oxysporum, and a strain of Trichoderma , preferably Trichoderma harzianum, Trichoderma reesei and Tri choderma vi ri de .
Fungal cells may be transformed by a process involving protoplast formation and transformation of the protoplasts followed by regeneration of the cell wall m a manner known per se . The use of a strain of Aspergillus as a host cell is described m 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 . , m particular Kluyveromyces lacti s and Kluyveromyces marcianus, a strain of Pichia sp . , a strain of Saccharo yces , m particular Saccharo yces carlsbergensis , Saccharomyces cerevisae, Saccharomyces kluyveπ and Saccharomyces uvarum, a strain of Schizosaccharomyces sp . , m particular Schizosaccharomyces pombe, and a strain of Yarrowia sp . , m particular Yarrowia lipolyti ca . 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 .
METHOD OF PRODUCING A XYLOGLUCANOLYTIC ENZYME
In another aspect, the present invention also relates to a method of producing the enzyme preparation of the invention, the method comprising culturing a microorganism capable of producing the xyloglucanase under conditions permitting the production of the enzyme, and recovering the enzyme from the culture. Culturing may be carried out using conventional fermentation techniques, e.g. culturing m shake flasks or fermentors with agitation to ensure sufficient aeration on a growth medium inducing production of the xyloglucanase enzyme. The growth medium may contain a conventional N-source such as peptone, yeast extract or casammo acids, a reduced amount of a conventional C-source such as dextrose or sucrose, and an mducer such as xyloglucan or composit plant substrates such as cereal brans (e.g. wheat bran or rice husk) . The recovery may be carried out using conventional techniques, e.g. separation of bio-mass and supernatant by centrifugation or filtration, recovery of the supernatant or disruption of cells if the enzyme of interest is mtracellular, perhaps followed by further purification as described m EP 0 406 314 or by crystallization as described m WO 97/15660.
Further, the present invention provides a method of producing an isolated enzyme according to the invention, wherein a suitable host cell, which has been transformed with a DNA sequence encoding the enzyme, is cultured under conditions permitting the production of the enzyme, and the resulting enzyme is recovered from the culture.
As defined herein, an isolated polypeptide (e.g. an enzyme) is a polypeptide which is essentially free of other polypeptides, e.g., at least about 20% pure, preferably at least about 40% pure, more preferably about 60% pure, even more preferably about 80% pure, most preferably about 90% pure, and even most preferably about 95% pure, as determined by SDS -PAGE. The term "isolated polypeptide" may alternatively be termed "purified polypeptide" .
When an expression vector comprising a DNA sequence encoding the enzyme is transformed into a heterologous host cell it is possible to enable heterologous recombmant production of the enzyme of the invention.
Thereby it is possible to make a highly purified or monocomponent xyloglucanolytic composition, characterized m being free from homologous impurities.
In this context, homologous impurities mean any impurities (e.g. other polypeptides than the enzyme of the invention), which originate from the homologous cell where the enzyme of the invention is originally obtained.
In the present invention the homologous host cell may be a strain of Paenibacillus sp . or Paenibacillus pabuli . The medium used to culture the transformed host cells may be any conventional medium suitable for growing the host cells m question. The expressed xyloglucanolytic enzyme may conveniently be secreted into the culture medium and may be recovered there from by well-known procedures including separat- mg the cells from the medium by centrifugation or filtration, precipitating protemaceous components of the medium by means of a salt such as ammonium sulphate, followed by chromatographic procedures such as ion exchange chromatography, affinity chromatography, or the like.
The present invention also relates to a transgenic plant, plant part or plant cell which has been transformed with a DNA sequence encoding the xyloglucanase of the invention so as to express and produce this enzyme m recoverable quantities . The enzyme may be recovered from the plant or plant part .
The transgenic plant can be dicotyledonous or monocotyledonous, for short a dicot or a monocot . Examples of monocot plants are grasses, such as meadow grass (blue grass, Poa) , forage grass such as festuca, lolium, temperate grass, such as Agrostis, and cereals, e.g. wheat, oats, rye, barley, rice, sorghum and maize (corn) .
Examples of dicot plants are tobacco, legumes, such as lupins, potato, sugar beet, pea, bean and soybean, and cruciferous (family Brassicaceae) , such as cauliflower, oil seed rape and the closely related model organism Arabidopsis thaliana .
Examples of plant parts are stem, callus, leaves, root, fruits, seeds, and tubers. In the present context, also specific plant tissues, such as chloroplast, apoplast, mitochondria, vacuole, peroxisomes and cytoplasm are considered to be a plant part. Furthermore, any plant cell, whatever the tissue origin, is considered to be a plant part.
Also included withm the scope of the invention are the progeny of such plants, plant parts and plant cells.
The transgenic plant or plant cell expressing the enzyme of the invention may be constructed m accordance with methods known m the art. In short the plant or plant cell is constructed by incorporating one or more expression constructs encoding the enzyme of the invention into the plant host genome and propagating the resulting modified plant or plant cell into a transgenic plant or plant cell .
Conveniently, the expression construct is a DNA construct which comprises a gene encoding the enzyme of the invention m operable association with appropriate regulatory sequences required for expression of the gene m the plant or plant part of choice. Furthermore, the expression construct may comprise a selectable marker useful for identifying host cells into which the expression construct has been integrated and DNA sequences necessary for introduction of the construct into the plant question (the latter depends on the DNA introduction method to be used) . The choice of regulatory sequences, such as promoter and terminator sequences and optionally signal or transit sequences is determined, e.g. based on when, where and how the enzyme is desired to be expressed. For instance, the expression of the gene encoding the enzyme of the invention may be constitutive or inducible, or may be developmental, stage or tissue specific, and the gene product may be targeted to a specific tissue or plant part such as seeds or leaves . Regulatory sequences are e.g. described by Tague et al , Plant, Phys . , 86, 506, 1988. For constitutive expression the 35S-CaMV promoter may be used (Franck et al . , 1980. Cell 21: 285-294) . Organ-specific promoters may e.g. be a promoter from storage sink tissues such as seeds, potato tubers, and fruits (Edwards & Coruzzi, 1990. Annu. Rev. Genet. 24: 275-303), or from metabolic sink tissues such as meristems (Ito et al . , 1994. Plant Mol. Biol. 24: 863- 878), a seed specific promoter such as the glutelm, prolamm, globulin or albumin promoter from rice (Wu et al . , Plant and Cell Physiology Vol. 39, No. 8 pp. 885-889 (1998)), a Vicia faba promoter from the legumm B4 and the unknown seed protein gene from Vi cia faba described by Conrad U. et al , Journal of Plant Physiology Vol. 152, No. 6 pp. 708-711 (1998), a promoter from a seed oil body protein (Chen et al . , Plant and cell physiology vol. 39, No. 9 pp. 935-941 (1998), the storage protein napA promoter from Brassica napus , or any other seed specific promoter known m the art, e.g. as described m WO 91/14772. Furthermore, the promoter may be a leaf specific promoter such as the rbcs promoter from rice or tomato (Kyozuka et al., Plant Physiology Vol . 102, No. 3 pp. 991-1000 (1993), the chlorella virus adenme methyltransferase gene promoter (Mitra, A. and Higgms, DW, Plant Molecular Biology Vol. 26, No. 1 pp . 85-93 (1994) , or the aldP gene promoter from rice (Kagaya et al . , Molecular and General Genetics Vol. 248, No. 6 pp. 668-674 (1995), or a wound inducible promoter such as the potato pin2 promoter (Xu et al , Plant Molecular Biology Vol. 22, No. 4 pp. 573-588 (1993) .
A promoter enhancer element may be used to achieve higher expression of the enzyme in the plant. For instance, the promoter enhancer element may be an intron placed between the promoter and the nucleotide sequence encoding the enzyme. For instance, Xu et al . op ci t disclose the use of the first intron of the rice actin 1 gene to enhance expression.
The selectable marker gene and any other parts of the expression construct may be chosen from those available in the art .
The DNA construct is incorporated into the plant genome according to conventional techniques known in the art, including Agrobacterium-mediated transformation, virus-mediated transformation, micro injection, particle bombardment, biolistic transformation, and electroporation (Gasser et al, Science, 244, 1293; Potrykus, Bio/Techn. 8, 535, 1990; Shimamoto et al , Nature, 338, 274, 1989) .
Presently, Agrobacterium tumefaciens mediated gene transfer is the method of choice for generating transgenic dicots (for review Hooykas & Schilperoort , 1992. Plant Mol. Biol. 19: 15-38) , however it can also be used for transforming monocots, although other transformation methods are generally preferred for these plants. Presently, the method of choice for generating transgenic monocots is particle bombardment (microscopic gold or tungsten particles coated with the transforming DNA) of embryonic calli or developing embryos (Christou, 1992. Plant J. 2: 275-281; Shimamoto, 1994. Curr. Opin. Biotechnol . 5: 158-162; Vasil et al . , 1992. Bio/Technology 10: 667-674). An alternative method for transformation of monocots is based on protoplast transformation as described by Omirulleh S, et al . , Plant Molecular biology Vol. 21, No. 3 pp. 415-428 (1993).
Following transformation, the transformants having incorporated the expression construct are selected and regenerated into whole plants according to methods well known in the art . THE ENZYME
In a preferred embodiment of the present invention, the xyloglucanase has a relative activity at a temperature of 50°C, preferably of at least 60%, preferably at least 70%, compared 5 to the activity at the optimal temperature.
In yet another preferred embodiment, at a temperature of 60°C, the relative xyloglucanase activity is at least 40%, preferably at least 50%; at a temperature of 70°C, the relative xyloglucanase activity is at least 40%, preferably at least 10 45%, especially at least 50%.
ENZYME COMPOSITIONS
In a still further aspect, the present invention relates to an enzyme composition comprising an enzyme exhibiting is xyloglucanase activity as described above.
The enzyme composition of the invention may, in addition to the xyloglucanase of the invention, comprise one or more other enzyme types, for instance hemicellulase such as xylanase and mannanase, cellulase or endo-β-1 , 4-glucanase components,
20 chitinase, lipase, esterase, pectinase, cutinase, phytase, oxidoreductase (peroxidase, haloperoxidase, oxidase, laccase) , protease, amylase, reductase, phenoloxidase, ligninase, pullulanase, pectate lyase, pectin acetyl esterase, polygalacturonase , rhamnogalacturonase, pectin lyase, pectin 5 methylesterase, cellobiohydrolase, transglutaminase; or mixtures thereof .
The enzyme composition may be prepared in accordance with methods known in the art and may be in the form of a liquid or a dry composition. For instance, the enzyme composition may be 0 in the form of a granulate or a microgranulate. The enzyme to be included in the composition may be stabilized in accordance with methods known in the art.
Xyloglucanases have potential uses in a lot of different industries and applications. Examples are given below of 5 preferred uses of the enzyme composition of the invention. The dosage of the enzyme composition of the invention and other conditions under which the composition is used may be determined based on methods known in the art . The xyloglucanase or xyloglucanase composition according to the invention may be useful for at least one of the following purposes.
USES
Use in the detergent industry
During washing and wearing, dyestuff from dyed fabrics or garment will conventionally bleed from the fabric, which then looks faded and worn. Removal of surface fibers from the fabric will partly restore the original colours and looks of the fabric. By the term "colour clarification", as used herein, is meant the partly restoration of the initial colours of fabric or garment throughout multiple washing cycles.
The term "de-pill g" denotes removing of pills from the fabric surface.
The term "soaking liquor" denotes an aqueous liquor m 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 m a washing or laundering process .
The term "washing liquor" denotes an aqueous liquor m which laundry is subjected to a washing process, i.e. usually a combined chemical and mechanical action either manually or m a washing machine. Conventionally, the washing liquor is an aqueous solution of a powder or liquid detergent composition. The term "rmsmg liquor" denotes an aqueous liquor m which laundry is immersed and treated, conventionally immediately after being subjected to a washing process, m order to rinse the laundry, i.e. essentially remove the detergent solution from the laundry. The rmsmg liquor may contain a fabric conditioning or softening composition.
The laundry subjected to the method of the present invention may be conventional washable laundry. Preferably, the major part of the laundry is sewn or unsown fabrics, including knits, wovens, denims, yarns, and towelling, made from cotton, cotton blends or natural or manmade cellulosics (e.g. originating from xylan-conta mg 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, polyvmyl alcohol fibers, polyvmyl chloride fibers, polyvmylidene chloride fibers, polyurethane fibers, polyurea fibers, aramid fibers), and cellulose-containing fibers (e.g. rayon/viscose, ramie, flax/linen, jute, cellulose acetate fibers, lyocell) .
DETERGENT DISCLOSURE AND EXAMPLES Surfactant system
The detergent compositions according to the present invention comprise a surfactant system, wherein the surfactant can be selected from non-ionic and/or anionic and/or catiomc 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 m the composition. In liquid or gel compositions the surfactant is most preferably formulated m such a way that it promotes, or at least does not degrade, the stability of any enzyme m these compositions.
Preferred systems to be used according to the present invention comprise as a surfactant one or more of the non-ionic and/or anionic surfactants described herein.
Polyethylene, polypropylene, and polybutylene oxide conden-sates of alkyl phenols are suitable for use as the non- lonic surfactant of the surfactant systems of the present invention, with the polyethylene oxide condensates being pre- ferred. 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, m either a straight chain or branched-cham configuration with the alkylene oxide. In a preferred embodiment, the ethylene oxide is present m an amount equal to from about 2 to about 25 moles, more preferably from about 3 to about 15 moles, of ethylene oxide per mole of alkyl phenol. Commercially available non-ionic surfactants of this type include Igepal™ CO-630, marketed by the GAF Corporation; and Triton™ X-45, X- 114, X-100 and X-102, all marketed by the Rohm & Haas Company. These surfactants are commonly referred to as alkyl phenol alkoxylates (e.g., alkyl phenol ethoxylates) . The condensation products of primary and secondary aliphatic alcohols with about 1 to about 25 moles of ethylene oxide are suitable for use as the non-ionic surfactant of the non- ionic surfactant systems of the present invention. The alkyl chain of the aliphatic alcohol can either be straight or branched, primary or secondary, and generally contains from about 8 to about 22 carbon atoms. Preferred are the condensation products of alcohols having an alkyl group containing from about 8 to about 20 carbon atoms, more preferably from about 10 to about 18 carbon atoms, with from about 2 to about 10 moles of ethylene oxide per mole of alcohol . About 2 to about 7 moles of ethylene oxide and most preferably from 2 to 5 moles of ethylene oxide per mole of alcohol are present in said condensation products. Examples of commercially available non- ionic surfactants of this type include Tergitol™ 15-S-9 (The condensation product of C11-C15 linear alcohol with 9 moles ethylene oxide), Tergitol™ 24-L-6 NMW (the condensation product of Cι2-Cι primary alcohol with 6 moles ethylene oxide with a narrow molecular weight distribution) , both marketed by Union Carbide Corporation; Neodol™ 45-9 (the condensation product of Cι4-Cι5 linear alcohol with 9 moles of ethylene oxide), Neodol™ 23-3 (the condensation product of Ci2-Ci3 linear alcohol with 3.0 moles of ethylene oxide) , Neodol™ 45-7 (the condensation product of Cι4-Cι5 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 TM 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 non-ionic surfactant of the surfactant systems of the present invention are alkyl polysaccharides disclosed m 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 polysacchaπde, 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 substi- tuted 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
R20 (CnH2n0) t (glycosyl ) x
wherein R2 is selected from the group consisting of alkyl, alkyl phenyl, hydroxyalkyl , hydroxyaIky1phenyl , and mixtures thereof 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, preferably 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-posιtιon) . The additional glycosyl units can then be attached between their 1- position and the preceding glycosyl unit's 2-, 3-, 4-, and/or 6-posιtιon, preferably predominantly the 2-posιtιon.
The condensation products of ethylene oxide with a hydrophobic base formed by the condensation of propylene oxide with propylene glycol are also suitable for use as the additional non-ionic surfactant systems of the present invention. The hydrophobic portion of these compounds will preferably have a molecular weight from about 1500 to about 1800 and will exhibit water insolubility. The addition of polyoxyethylene moieties to this hydrophobic portion tends to increase the water solubility of the molecule as a whole, and the liquid character of the product is retained up to the point where the polyoxyethylene content is about 50% of the total weight of the condensation product, which corresponds to condensation with up to about 40 moles of ethylene oxide. Examples of compounds of this type include certain of the commercially available Pluronic™ surfactants, marketed by BASF. Also suitable for use as the non-ionic surfactant of the non-ionic surfactant system of the present invention, are the condensation products of ethylene oxide with the product resulting from the reaction of propylene oxide and ethylenediamme . The hydrophobic moiety of these products consists of the reaction product of ethylenediamme and excess propylene oxide, and generally has a molecular weight of from about 2500 to about 3000. This hydrophobic moiety is condensed with ethylene oxide to the extent that the condensation product contains from about 40% to about 80% by weight of polyoxyethylene and has a molecular weight of from about 5,000 to about 11,000. Examples of this type of non-ionic surfactant include certain of the commercially available Tetronic™ compounds, marketed by BASF. Preferred for use as the non-ionic surfactant of the surfactant systems of the present invention are polyethylene oxide condensates of alkyl phenols, condensation products of primary and secondary aliphatic alcohols with from about 1 to about 25 moles of ethyleneoxide, alkylpolysaccharides , and mixtures hereof. Most preferred is Cs-Cι4 alkyl phenol ethoxylates having from 3 to 15 ethoxy groups and Ca-Cι8 alcohol ethoxylates (preferably Cio av . ) having from 2 to 10 ethoxy groups, and mixtures thereof. Highly preferred non- ionic surfactants are polyhydroxy fatty acid amide surfactants of the formula
R2 - C - N - Z,
0 R1 wherein R1 is H, or R1 is Cι-4 hydrocarbyl , 2-hydroxyethyl , 2- hydroxypropyl or a mixture thereof, R2 is C5 31 hydrocarbyl, and Z is a polyhydroxy hydrocarbyl having a linear hydrocarbyl chain with at least 3 hydroxyls directly connected to the chain, or an alkoxylated derivative thereof. Preferably, R1 is methyl, R2 is straight Cπ-15 alkyl or Ciβ-iβ 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, m a reductive animation reaction. Highly preferred anionic surfactants include alkyl alkoxylated sulfate surfactants. Examples hereof are water soluble salts or acids of the formula R0(A)mS03M wherein R is an unsubstituted Cιo-C-24 alkyl or hydroxyalkyl group having a C10- C24 alkyl component, preferably a C12-C20 alkyl or hydroxyalkyl, 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 piperdimum cations and those derived from alkylammes such as ethylam e, diethylamme, tπethylamme, mixtures thereof, and the like. Exemplary surfactants are Cι2-Cι8 alkyl polyethoxylate (1.0) sulfate (Cι2-Cι8E (1.0) M) , Cι2-Cι8 alkyl polyethoxylate (2.25) sulfate (Cι2-Cι8 (2.25) M, and Cι2-Cι8 alkyl polyethoxylate (3.0) sulfate (Cι2-Cι8E (3.0) M) , and Cι2-Cι8 alkyl polyethoxylate (4.0) sulfate (Cι2-Cι8E (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-C2o 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: 0
RJ CH C OR
S03M
wherein R is a C8-C2o hydrocarbyl, preferably an alkyl, or combination thereof, R4 is a Cι-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 Cι0-CιS 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 Cι0-C24 hydrocarbyl, preferably an alkyl or hydroxyalkyl having a C10-C20 alkyl com- ponent, more preferably a Cι2-Cι8 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 Cι2-Cιs are preferred for lower wash temperatures (e.g. below about 50 °C) and Ci6-Cι8 alkyl chains are preferred for higher wash temperatures (e.g. above about 50 °C) .
Other anionic surfactants useful for detersive purposes can also be included in the laundry detergent compositions of the present invention. Theses can include salts (including, for example, sodium, potassium, ammonium, and substituted ammonium salts such as mono- di- and tπethanolamme salts) of soap, C8- C22 primary or secondary alkanesulfonates , C8-C4 olef sulfonates , sulfonated polycarboxylic acids prepared by sulfonation of the pyrolyzed product of alkaline earth metal citrates, e.g., as described m 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, lsethionates such as the acyl lsethionates , N-acyl taurates, alkyl succmamates and sulfosuccmates , monoesters of sulfosuccmates (especially saturated and unsaturated Cι2-Cι8 monoesters) and diesters of sulfosuccmates (especially saturated and unsaturated C6-C12 diesters), acyl sarcosmates , sulfates of alkyl polysaccharides such as the sulfates of alkylpolyglucoside (the non-ionic nonsulfated compounds being described below) , branched primary alkyl sulfates, and alkyl polyethoxy carboxylates such as those of the formula RO (CH2CH20) k-CH2C00-M+ wherein R is a C8-C22 alkyl, k is an integer from 1 to 10, and M is a soluble salt forming cation. Resm acids and hydrogenated resm acids are also suitable, such as rosin, hydrogenated rosm, and resm acids and hydrogenated resm acids present m or derived from tall
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 m "Surface Active Agents and Detergents" (Vol. I and II by Schwartz, Perry and Berch) . A variety of such surfactants are also generally disclosed m US 3,929,678, (Column 23, line 58 through Column 29, line 23, herein incorporated by reference) . When included therein, the laundry detergent compositions of the present invention typically comprise from about 1% to about 40%, preferably from about 3% to about 20% by weight of such anionic surfactants . The laundry detergent compositions of the present invention may also contain cationic, ampholytic, zwitterionic , and semi-polar surfactants, as well as the non-ionic 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:
[R (OR3 [R4(OR3; ,] 2R N+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 R 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:
RιR2R3R4N+X" (i)
wherein Ri is C8-Cι6 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 Ri 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 tπmethyl ammonium chloride or bromide; coconut methyl dihydroxyethyl ammonium chloride or bromide; decyl tπethyl ammonium chloride; decyl dimethyl hydroxyethyl ammonium chloride or bromide; C12 is dimethyl hydroxyethyl ammonium chloride or bromide; coconut dimethyl hydroxyethyl ammonium chloride or bromide; myπstyl trimethyl ammonium methyl sulphate; lauryl dimethyl benzyl ammonium chloride or bromide; lauryl dimethyl (ethenoxy)4 ammonium chloride or bromide; cholme esters (compounds of formula (I) wherein R is
CH2-CH2-O-C-C1214 alkyl and R2R3R4 are methyl) .
II 0
di-alkyl lmidazolmes [compounds of formula (1)] .
Other cationic surfactants useful herein are also described m US 4,228,044 and m 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 m 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 m which the aliphatic radical can be straight- or branched-cham. 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-solubilizmg 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 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 laundry detergent 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 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 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:
O
R3(OR4)xN(R5)2
wherein R3 is an alkyl, hydroxyalkyl, or alkyl phenyl group or mixtures thereof containing from about 8 to about 22 carbon atoms; R4 is an alkylene or hydroxyalkylene group containing from about 2 to about 3 carbon atoms or mixtures thereof; x is from 0 to about 3 : and each R5 is an alkyl or hydroxyalkyl group containing from about 1 to about 3 carbon atoms or a polyethylene oxide group containing from about 1 to about 3 ethylene oxide groups . The R5 groups can be attached to each other, e.g., through an oxygen or nitrogen atom, to form a ring structure . These amme oxide surfactants m particular include Cι0-Cι8 alkyl dimethyl amme oxides and C8-Cι2 alkoxy ethyl dihydroxy ethyl amme oxides .
When included therein, the laundry detergent compositions of the present invention typically comprise from 0.2% to about 15%, preferably from about 1% to about 10% by weight of such semi-polar nonionic surfactants.
Builder system
The compositions according to the present invention may further comprise a builder system. Any conventional builder system is suitable for use herein including alummosilicate materials, silicates, polycarboxylates and fatty acids, materials such as ethylenediamme tetraacetate, metal ion sequestrants such as ammopolyphosphonates, particularly ethylenediamme tetramethylene phosphonic acid and diethylene tπamme pentamethylenephosphomc 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 alummosilicate 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 (Na2Si2θ5) .
Suitable polycarboxylates containing one carboxy group include lactic acid, glycolic acid and ether derivatives thereof as disclosed m Belgian Patent Nos. 831,368, 821,369 and 821,370. Polycarboxylates containing two carboxy groups include the water-soluble salts of succmic acid, malonic acid, (ethylenedioxy) diacetic acid, maleic acid, diglycollic acid, tartaric acid, tartronic acid and fumaric acid, as well as the ether carboxylates described m German Offenle-enschπft 2,446,686, and 2,446,487, US 3,935,257 and the sulfmyl carboxylates described m Belgian Patent No. 840,623. Polycarboxylates containing three carboxy groups include, m particular, water-soluble citrates, aconitrates and citraconates as well as succ ate derivatives such as the carboxymethyloxysucc ates described m British Patent No. 1,379,241, lactoxysuccmates described Netherlands Application 7205873, and the oxypolycarboxylate materials such as 2 -oxa-1 , 1 , 3 -propane tπcarboxylates described m British Patent No. 1,387,447.
Polycarboxylates containing four carboxy groups include oxydisuccmates disclosed m British Patent No. 1,261,829, 1 , 1 , 2 , 2 , -ethane tetracarboxylates, 1 , 1 , 3 , 3 -propane tetrac7arboxylates containing sulfo substituents include the sulfosuccmate derivatives disclosed m British Patent Nos.
1,398,421 and 1,398,422 and m US 3,936,448, and the sulfonated pyrolysed citrates described m British Patent No. 1,082,179, while polycarboxylates containing phosphone substituents are disclosed m 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-cιs, discarboxylates , 2 , 2 , 5 , 5 , -tetrahydrofuran - tetracarboxylates, 1,2,3,4,5,6- hexane - hexacarboxylates and carboxymethyl derivatives of polyhydπc alcohols such as sorbitol , mannitol and xylitol. Aromatic polycarboxylates include mellitic acid, pyromellitic acid and the phthalic acid derivatives disclosed m British Patent No. 1,425, 343. Of the above, the preferred polycarboxylates are hydroxy- carboxylates containing up to three carboxy groups per molecule, more particularly citrates.
Preferred builder systems for use the present compositions include a mixture of a water- insoluble alummosilicate builder such as zeolite A or of a layered silicate (SKS-6) , and a water-soluble carboxylate chelatmg agent such as citric acid. A suitable chelant for inclusion m the detergent composi-ions m accordance with the invention is ethylenedιamme-N,N' -disuccimc 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 m compositions m accordance with the invention.
Preferred builder systems include a mixture of a water- insoluble alummosilicate builder such as zeolite A, and a water soluble carboxylate chelatmg agent such as citric acid. Other builder materials that can form part of the builder system for use m granular compositions include inorganic materials such as alkali metal carbonates, bicarbonates, silicates, and organic materials such as the organic phosphonates, ammo polyalkylene phosphonates and ammo polycarboxylates . Other suitable water-soluble organic salts are the homo- or co-polymeric acids or their salts, m 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 m 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 m amounts of from 5% to 80% by weight of the composition. Preferred levels of builder for liquid detergents are from 5% to 30%.
Enzymes Preferred detergent compositions, m addition to the enzyme preparation of the invention, comprise other enzyme (s) which provides cleaning performance and/or fabric care benefits . Such enzymes include proteases, lipases, cutmases, amylases, cellulases, peroxidases, oxidases (e.g. laccases) .
Proteases : Any protease suitable for use m 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 serme protease, preferably an alkaline microbial protease or a trypsm-like protease. Examples of alkaline proteases are subtilisms, especially those derived from Bacillus, e.g., subtilism Novo, subtilism Carlsberg, subtilism 309, subtilism 147 and subtilism 168 (described WO 89/06279) . Examples of trypsm-like proteases are tryps (e.g. of porcine or bovine origin) and the Fusarium protease described m WO 89/06270.
Preferred commercially available protease enzymes include those sold under the trade names Alcalase, Sav ase, 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 m 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 m alkaline solutions can be used. Suitable lipases include those of bac- teπal or fungal origin. Chemically or genetically modified mutants are included.
Examples of useful lipases include a Humicola lanugmosa lipase, e.g., as described m EP 258 068 and EP 305 216, a Rhizomucor miehei lipase, e.g., as described m EP 238 023, a Candida lipase, such as a C. antarctica lipase, e.g., the C . antarctica lipase A or B described EP 214 761, a Pseudomonas lipase such as a P. alcaligenes and P. pseudoalcaligenes lipase, e.g., as described m EP 218 272, a P. cepac a lipase, e.g., as described m EP 331 376, a P. stutzeπ lipase, e.g., as disclosed m 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 camemberti lipase described by Yamaguchi et al . , (1991), Gene 103, 61-67), the Geotπcum 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 cutmases may also be useful, e.g., a cutmase derived from Pseudomonas mendocma as described m WO 88/09367, or a cutmase derived from Fusarium solani pisi (e.g. described m WO 90/09446) . Especially suitable lipases are lipases such as Ml 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 m 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 alkaline solutions can be used. Suitable amylases include those of bacterial or fungal origin. Chemically or genetically mod- ifιed mutants are included. Amylases include, for example, a- amylases obtained from a special strain of B. licheniformis, described m more detail m GB 1,296,839. Commercially T I TM TM available amylases are Duramyl , Termamyl , Fungamyl and BAN
TM TM (available from Novo Nordisk A/S) and Rapidase and Maxamyl P (available from Genencor) .
The amylases are normally incorporated m 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 m 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 m US 4,435,307 which discloses fungal cellulases produced from Humi - cola msolens, WO 96/34108 and WO 96/34092 which disclose bacterial alkalophilic cellulases (BCE 103) from Bacillus, and m 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 m European patent application No. 0 495 257. Commercially available cellulases include Celluzyme™ 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 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 combination with hydrogen peroxide or a source thereof (e.g. a percarbonate, perborate or persulfate) . Oxidase enzymes are used m 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 m a wash liquor, preferably together with an enhancing agent as described m 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 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, m particular a mixture of a protease, an amylase, a lipase and/or a cellulase.
The enzyme of the invention, or any other enzyme incorporated m the detergent composition, is normally incorporated 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 m the detergent compositions of the present invention include bleaching agents such as PBl, 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 m 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 m 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-nonylammo-4-oxoperoxybutyrιc acid and diperoxydodecanedioic acid. Such bleaching agents are disclosed m US 4,483,781, US 740,446, EP 0 133 354 and US 4,412,934. Highly preferred bleaching agents also include 6- nonylammo-6-oxoperoxycaproιc acid as described m 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 tπchloro lsocyanuπc 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 m combination with bleach activators such as tetra- acetylethylenediamme (TAED) , nonanoyloxybenzenesulfonate (NOBS, described m US 4,412,934), 3 , 5-tπmethyl- hexsanoloxybenzenesulfonate (ISONOBS, described m 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-octanamιdo-caproyl) oxybenzene-sulfonate, C9(6- nonanamido caproyl) oxybenzenesulfonate and CIO (6-decanamιdo caproyl) oxybenzenesulfonate or mixtures thereof. Also suitable activators are acylated citrate esters such as disclosed European Patent Application No. 91870207.7.
Useful bleaching agents, including peroxyacids and bleaching systems comprising bleach activators and peroxygen bleaching compounds for use m cleaning compositions according to the invention are described m 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 rmsmg process. Such enzymatic systems are disclosed m European Patent Application EP 0 537 381.
Bleaching agents other than oxygen bleaching agents are also known m 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 zmc and/or aluminium phthalocyanmes . These materials can be deposited upon the substrate during the washing process. Upon irradiation with light, m the presence of oxygen, such as by hanging clothes out to dry m the daylight, the sulfonated z c phthalocyanme is activated and, consequently, the substrate is bleached. Preferred zmc phthalocyanme and a photoactivated bleaching process are described m US 4,033,718. Typically, detergent composition will contain about 0.025% to about 1.25%, by weight, of sulfonated zmc phthalocyanme. Bleaching agents may also comprise a manganese catalyst. The manganese catalyst may, e.g., be one of the compounds described m "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 m finely divided forms exemplified by silica aerogels and xerogels and hydrophobic silicas of various types. Theses materials can be incorporated as particulates , m which the suds suppressor is advantageously releasably incorporated m a water-soluble or waterdispersible, substantially non surface-active detergent impermeable carrier. Alternatively the suds suppressor can be dissolved or dispersed a liquid carrier and applied by spraying on to one or more of the other components. A preferred silicone suds controlling agent is disclosed m US 3,933,672. Other particularly useful suds suppressors are the self-emulsifying silicone suds suppressors, described m German Patent Application DTOS 2,646,126. An example of such a compound is DC- 544, commercially available form Dow Corning, which is a siloxane-glycol copolymer. Especially preferred suds controlling agent are the suds suppressor system comprising a mixture of silicone oils and 2-alkyl-alkanols . Suitable 2- alkyl-alkanols are 2-butyl-octanol which are commercially available under the trade name Isofol 12 R. Such suds suppressor system are described m European Patent Application EP 0 593 841.
Especially preferred silicone suds controlling agents are described m European Patent Application No. 92201649.8. Said compositions can comprise a silicone/ silica mixture in combination with fumed nonporous silica such as AerosιlR. The suds suppressors described above are normally employed at levels of from 0.001% to 2% by weight of the composition, preferably from 0.01% to 1% by weight.
Other components
Other components used m detergent compositions may be employed such as soil-suspending agents, soil-releasing agents, optical bπghteners, abrasives, bacteπcides , tarnish inhibitors, coloring agents, and/or encapsulated or nonencapsulated perfumes.
Especially suitable encapsulating materials are water soluble capsules which consist of a matrix of polysacchaπde and polyhydroxy compounds such as described m GB 1,464,616.
Other suitable water soluble encapsulating materials comprise dextrms derived from ungelatmized starch acid esters of substituted dicarboxylic acids such as described m US 3,455,838. These acid-ester dextrms 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 succmic 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, methylvmyl 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 m character, examples of which are disodium 4 , 4 ' -bis- (2-dιethanolammo-4- anilmo -s- trιazm-6-ylammo) stιlbene-2 : 2 ' disulphonate, disodium 4, - 4 ' -bis- (2 -morpholmo-4-anιlmo-s-trιazm-6- ylammo-stιlbene-2 : 2 ' - disulphonate, disodium 4,4' - bis- (2,4- dιanιlmo-Ξ-trιazm-6-ylammo) stιlbene-2 : 2 ' - disulphonate, monosodium 4 ',4'' - bis- (2 , 4-dιanιlmo-s-trι-azm-6 ylamino) stιlbene-2 -sulphonate, disodium 4,4' -bis- (2-anιlmo-4- (N-methyl-N-2-hydroxyethylammo) -s-trιazm-6-ylammo) stilbene- 2,2' - disulphonate, di-sodium 4,4' -bis- ( -phenyl-2 , 1 , 3 - tπazol-2-yl) -stιlbene-2 , 2 ' disulphonate, di-so-dium 4,4 'bis (2- anιlmo-4- (1 -methyl -2 -hydroxyethylammo) -s-tπazm-6-ylamι- no) stιlbene-2 , 2 ' disulphonate, sodium 2 (stιlbyl-4 ' ' - (naphtho- 1 ' , 2 ' :4, 5) -1, 2, 3, - tπazole-2 '' -sulphonate and 4,4'-bιs(2- sulphostyryl) biphenyl .
Other useful polymeric materials are the polyethylene glycols, particularly those of molecular weight 1000-10000, more particularly 2000 to 8000 and most preferably about 4000. These are used at levels of from 0.20% to 5% more preferably from 0.25% to 2.5% by weight. These polymers and the previously mentioned homo- or co-polymeric poly-carboxylate salts are valuable for improving whiteness maintenance, fabric ash deposition, and cleaning performance on clay, protemaceous and oxidizable soils m the presence of transition metal impurities . Soil release agents useful m compositions of the present invention are conventionally copolymers or terpolymers of terephthalic acid with ethylene glycol and/or propylene glycol units various arrangements. Examples of such polymers are disclosed m US 4,116,885 and 4,711,730 and EP 0 272 033. A particular preferred polymer m accordance with EP 0 272 033 has the formula:
( CH3 ( PEG ) 43 ) o 75 ( POH ) 0 25 [ T - PO ) 2 8 ( T - PEG) 0 4 ] T ( POH ) 0 25 ( ( PEG) 43CH3 ) 0 75
where PEG is -(OC2H4)0-, PO is (OC3H60) and T is (pOOCsH4CO) . 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", m 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 m detail m EP 311 342.
Softening agents
Fabric softening agents can also be incorporated into laundry detergent compositions m accordance with the present invention. These agents may be inorganic or organic type. Inorganic softening agents are exemplified by the smectite clays disclosed m GB-A-1 400898 and m US 5,019,292. Organic fabric softening agents include the water insoluble tertiary ammes as disclosed m GB-A1 514 276 and EP 0 011 340 and their combination with mono Cι2-Cι4 quaternary ammonium salts are disclosed EP-B-0 026 528 and di-long-cham amides as disclosed EP 0 242 919. Other useful organic ingredients of fabric softening systems include high molecular weight polyethylene oxide materials as disclosed m EP 0 299 575 and 0 313 146.
Levels of smectite clay are normally m 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 ammes 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 m some instances it may be more convenient to add them as a dry mixed particulate, or spray them as molten liquid on to other solid components of the composition.
Polymeric dye-transfer inhibiting agents
The detergent compositions according to the present invention may also comprise from 0.001% to 10%, preferably from 0.01% to 2%, more preferably form 0.05% to 1% by weight of polymeric dye- transfer inhibiting agents. Said polymeric dye- transfer inhibiting agents are normally incorporated into detergent compositions order to inhibit the transfer of dyes from colored fabrics onto fabrics washed therewith. These polymers have the ability of complexmg or adsorbing the fugitive dyes washed out of dyed fabrics before the dyes have the opportunity to become attached to other articles m the wash. Especially suitable polymeric dye-transfer inhibiting agents are polyam e N-oxide polymers, copolymers of N-vmyl- pyrrolidone and N-vmylimidazole, polyvmylpyrrolidone polymers, polyvmyloxazolidones and polyvmylimidazoles or mixtures thereof .
Addition of such polymers also enhances the performance of the enzymes according the invention.
The detergent composition according to the invention can be m liquid, paste, gels, bars or granular forms. Non-dustmg granulates may be produced, e.g., as disclosed m US 4,106,991 and 4,661,452 (both to Novo Industπ A/S) and may optionally be coated by methods known m the art. Examples of waxy coating materials are pol (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 m which the alcohol contains from 12 to 20 carbon atoms and which there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- and triglyceπdes of fatty acids. Examples of film-forming coating materials suitable for application by fluid bed techniques are given m GB 1483591.
Granular compositions according to the present invention can also be m "compact form", i.e. they may have a relatively higher density than conventional granular detergents, i.e. form 550 to 950 g/1; m 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, typi- cally sodium sulphate; "Compact" detergent typically comprise not more than 10% filler salt. The liquid compositions according to the present invention can also be "concentrated form", m such case, the liquid detergent compositions according to the present invention will contain a lower amount of water, compared to conventional liquid detergents. Typically, the water content of the concentrated liquid detergent is less than 30%, more preferably less than 20%, most preferably less than 10% by weight of the detergent compositions. The compositions of the invention may for example, be formulated as hand and machine laundry detergent compositions including laundry additive compositions and compositions suitable for use m the pretreatment of stained fabrics, rinse added fabric softener compositions, and compositions for use m 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 Cιχ - Cιγ alkyl sulfate
SS : Secondary soap surfactant of formula 2 -butyl octanoic acid
25EY: A Cχ2 - C15 predominantly linear primary alcohol condensed with an average of Y moles of ethylene oxide
45EY: A Cι4 - C15 predominantly linear primary alcohol condensed with an average of Y moles of ethylene oxide
XYEZS : Cιχ - Cιγ 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: Ci6 - Cis alkyl N-methyl glucamide
Silicate: Amorphous Sodium Silicate (Sι02:Na20 ratio = 2.0)
NaSKS-6: Crystalline layered silicate of formula d-NaSι205
Carbonate : Anhydrous sodium carbonate
Phosphate : Sodium tπpolyphosphate 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 Alummosilicate of formula
Nai2 (AIO2S1O2) 12 • 27H20 having a primary particle size m the range from 1 to 10 micrometers Citrate: Tπ-sodium citrate dihydrate
Citric: Citric Acid
Perborate: Anhydrous sodium perborate monohydrate bleach, empirical formula NaB02.H02
PB4 : Anhydrous sodium perborate tetrahydrate Percarbonate: Anhydrous sodium percarbonate bleach of empirical formula 2Na2C03.3H202
TAED : Tetraacetyl ethylene diamme
CMC: Sodium carboxymethyl cellulose
DETPMP : Diethylene tπamme penta (methylene phosphonic acid) , marketed by Monsanto under the Tradename Dequest 2060
PVP : Polyvmylpyrrolidone polymer
EDDS: Ethylenediamme-N, N ' -disuccmic acid, [S,S] isomer m 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 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 m accordance with the invention may be prepared as follows:
Sodium linear C12 alkyl 6.5 benzene sulfonate
Sodium sulfate 15.0 Zeolite A 26.0
Sodium nitrilotπacetate 5.0
Enzyme of the invention 0.1
PVP 0.5 TAED 3.0
Boric acid 4.0
Perborate 18.0
Phenol sulphonate 0.1
Minors Up to 100
Detergent Example II
A compact granular fabric cleaning composition (density 800 g/1) in accord with the invention may be prepared as follows:
45AS 8.0
25E3S 2.0
25E5 3.0
25E3 3.0
TFAA 2.5
Zeolite A 17.0
NaSKS-6 12.0
Citric acid 3.0
Carbonate 7.0
MA/AA 5.0
CMC 0.4
Enzyme of the invention 0.1
TAED 6.0
Percarbonate 22.0
EDDS 0.3
Granular suds suppressor 3.5 water/minors Up to 100%
Detergent Example III Granular fabric cleaning compositions in accordance with the invention which are especially useful in the laundering of coloured fabrics were prepared as follows:
LAS 10.7
TAS 2.4 TFAA - 4.0
45AS 3.1 10.0
45E7 4.0
25E3S - 3.0 68E11 1.8 -
25E5 - 8.0
Citrate 15.0 7.0
Carbonate - 10
Citric acid 2.5 3.0
Zeolite A 32.1 25.0
Na-SKS-6 - 9.0
MA/AA 5.0 5.0
DETPMP 0.2 0.8
Enzyme of the invention 0.10 0.05
Silicate 2.5 -
Sulphate 5.2 3.0
PVP 0.5 -
Poly (4 -vmylpyridme) -N- - 0.2
Oxide/copolymer of vinyl - lmidazole and vmyl- pyrrolidone
Perborate 1.0 -
Phenol sulfonate 0.2 -
Water/Minors Up to 100%
Detergent Example IV
Granular fabric cleaning compositions m accordance with the invention which provide "Softening through the wash" capability may be prepared as follows:
45AS - 10.0
LAS 7.6 -
68AS 1.3 -
45E7 4.0 -
25E3 - 5.0
Coco-alkyl- -dimethyl hydroxy- 1.4 1.0 ethyl ammonium chloride
Citrate 5.0 3.0
Na-SKS-6 - 11.0
Zeolite A 15.0 15.0 MA/AA 4.0 4.0 DETPMP 0.4 0.4 Perborate 15.0 Percarbonate - 15.0
TAED 5.0 5.0
Smectite clay 10.0 10.0
HMWPEO - 0.1
5 Enzyme of the invention 0.10 0.05
Silicate 3.0 5.0
Carbonate 10.0 10.0
Granular suds suppressor 1.0 4.0
CMC 0.2 0.1
10 Water/Minors Up to 100%
Detergent Example V
Heavy duty liquid fabric cleaning compositions m accordance with the invention may be prepared as follows:
15
I II
LAS acid form - 25.0
Citric acid 5.0 2.0
25AS acid forml 8.0 -
25AE7 8.0 -
CFAA 5 -
DETPMP 1.0 1.0
Fatty acid 8 -
25 Oleic acid - 1.0
Ethanol 4.0 6.0
Propanediol 2.0 6.0
Enzyme of the invention 0.10 0.05
Coco-alkyl dimethyl - 3.0
30 hydroxy ethyl ammonium chloride
Smectite clay - 5.0
PVP 2.0 -
Water / Minors Up to 100%
35
THE XYLOGLUCAN SUBSTRATE
In addition to the aforesaid about xyloglucan it should be noted that xyloglucan from tamarind seeds supplied by Megazyme, Ireland has a complex branched structure with glucose, xylose, galactose and arabinose m the ratio of 45:36:16:3. Accordingly, it is strongly believed that an enzyme showing catalytic activity on this xyloglucan also has catalytic activity on other xyloglucan structures from different sources (angiosperms or gy nosperms) .
Cotton suspension culture xyloglucan MW 100,000 kDa was obtained from Professor A. Mort of Oklahoma State University. IH NMR (D20, 80°C) of xyloglucans was used to compare the monosaccharide composition of samples of different origin. The integrals of the anomeπc signals from the commercial sample fully agree with the composition given by Megazyme. However, the cotton xyloglucan seems to have a different structure. There appears to be much less galactose and about half of galactose residues are fucosylated. Furthermore, the molar ratio between xylose and glucose is smaller (0.63 compared to 0.77 for the tamarind) , which suggest a more open structure of cotton xyloglucan. These findings agree with results obtained with xyloglucan from cotton cells (Buchala et al, Acta Bot . Neerl. 42, 1993, 213-219).
Xyloglucan (Megazyme) Cotton xyloglucan
Glucose 45 % 52 %
Xylose 35 % 33 %
Galactose 16 % 10 %
Fucose - 5 %
Arabinose <4 % a -
a Could not be detected m NMR
MATERIALS AND METHODS Strains
Paenibacillus pabuli , e.g. the type strain ATCC 43899, and Paenibacillus sp . , DSM 13330, comprises a DNA sequence encoding a family 5 xyloglucanase of the invention.
E. coli , DSM 13183, comprises the plasmid containing the DNA encoding the xyloglucanase of the invention (SEQ ID NO: 1) . Other strains
E. coli hosts: XLl-Blue MRF" and XLOLR E. coli strains were provided by Stratagene Inc. (USA) and used according to the manufacturer's instructions.
B . subtili s PL2306. This strain is the B . subtilis DN1885 with disrupted apr and npr genes Diderichsen et al . (1990) disrupted in the transcriptional unit of the known Bacillus subtilis cellulase gene, resulting in cellulase negative cells. The disruption was performed essentially as described in Sonenshein et al . (1993) .
Bacillus subtili s MB1053-1: This strain is PL 2306 in which the pectate lyase gene Pel has been disrupted resulting in a pectate lyase negative strain. The disruption was performed essentially as described in Sonenshein et al . (1993) .
Competent cells were prepared and transformed as described by Yasbin et al . (1975) .
Plasmids pBK-CAMV: Stratagene inc. La Jolla CA., USA.
Bacteriophage ZAP Express: Stratagene inc. La Jolla CA. , USA. pMOL944. This plasmid is a pUBHO derivative essentially containing elements making the plasmid propagatable in Bacillus subtilis, kanamycin resistance gene and having a strong promoter and signal peptide cloned from the amyL gene of B . licheniformis ATCC 14580. The signal peptide contains a SacII site making it convenient to clone the DNA encoding the mature part of a protein in-fusion with the signal peptide. This results in the expression of a Pre-protein, which is directed towards the exterior of the cell .
The plasmid was constructed by means of ordinary genetic engineering and is briefly described in the following. Construction of pMOL944 :
The pUBHO plasmid (McKenzie, T. et al . , 1986,) was digested with the unique restriction enzyme Neil . A PCR fragment amplified from the amyL promoter encoded on the plasmid pDN1981 (Jørgensen et al . , 1990) was digested with Neil and inserted the Neil digested pUBHO to give the plasmid pSJ2624. The two PCR primers used have the following sequences:
# LWN5494 5'-GTCGCCGGGGCGGCCGCTATCAATTGGTAACTGTATCTCAGC -3'
# LWN5495 5'-GTCGCCCGGGAGCTCTGATCAGGTACCAAGCTTGTCGACCTGCAGAA
TGAGGCAGCAAGAAGAT -3' The primer #LWN5494 inserts a Notl site m the plasmid. The plasmid pSJ2624 was then digested with Sad and Notl and a new PCR fragment amplified on amyL promoter encoded on the pDN1981 was digested with Sad and Notl and this DNA fragment was inserted m the Sacl-Notl digested pSJ2624 to give the plasmid pSJ2670. This cloning replaces the first amyL promoter cloning with the same promoter but m the opposite direction. The two primers used for PCR amplification have the following sequences : #LWN5938 5 " -GTCGGCGGCCGCTGATCACGTACCAAGCTTGTCGACCTGCAGAATG AGGCAGCAAGAAGAT - 3 "
#LWN5939 5 " -GTCGGAGCTCTATCAATTGGTAACTGTATCTCAGC - 3 "
The plasmid pSJ2670 was digested with the restriction enzymes Pstl and Bell and a PCR fragment amplified from a cloned DNA sequence encoding the alkaline amylase SP722 (International Patent Application published as W095/26397 which is hereby incorporated by reference) was digested with Pstl and Bell and inserted to give the plasmid pMOL944. The two primers used for PCR amplification have the following sequence: #LWN7864 5^ -AACAGCTGATCACGACTGATCTTTTAGCTTGGCAC-3 ' #LWN7901 5^ -AACTGCAGCCGCGGCACATCATAATGGGACAAATGGG -3'
The primer #LWN7901 inserts a SacII site m the plasmid.
Media
TY (as described m Ausubel, F. M. et al . 1995).
LB agar (as described m Ausubel, F. M. et al , 1995) . LBPG is LB agar supplemented with 0.5% Glucose and 0.05 M potassium phosphate, pH 7.0.
AZCL-Xyloglucan is added to LBPG-agar to 0.5 % AZCL- Xyloglucan is from Megazyme, Australia. BPX media is described m EP 0 506 780 (WO 91/09129) .
NZY agar (per liter) 5 g of NaCl, 2 g of MgS04 , 5 g of yeast extract, 10 g of NZ amme (casein hydrolysate) , 15 g of agar; add deionized water to 1 litre, adjust pH with NaOH to pH 7.5 and autoclave
NZY broth (per litre) 5 g of NaCl, 2 g of MgS04 , 5 g of yeast extract, 10 g of NZ amme (casein hydrolysate) ; add deionized water to 1 litre, adjust pH with NaOH to pH 7.5 and autoclave NZY Top Agar (per litre) 5 g of NaCl, 2 g of MgS04 , 5 g of yeast extract, 10 g of NZ amme (casein hydrolysate), 0.7 % (w/v) agarose; add deionized water to 1 litre, adjust pH with NaOH to pH 7.5 and autoclave .
Xyloglucanase assay (XyloU)
The xyloglucanase activity is measured using AZCL- xyloglucan from Megazyme, Ireland, (htt : //www .megazyme . com/purchase/index . tml) as substrate.
A solution of 0.2 % of the blue substrate is suspended m a 0.1 M phosphate buffer pH 7.5 under stirring. The solution is distributed under stirring to 1.5 ml Eppendorf tubes (0.75 ml to each) , 50 μl enzyme solution is added and they are incubated m an Eppendorp Thermomixer model 5436 for 20 minutes at 40°C with a mixing of 1200 rpm. After incubation the colored solution is separated from the solid by 4 minutes centrifugation at 14,000 rpm and the absorbance of the supernatant is measured at 600 nm.
One XyloU unit is defined as the amount of enzyme resulting m an absorbance of 0.24 m a 1 cm cuvette at 600 nm.
Cellulase assay (CMC method: CMC unit)
CMC units is measured using 0.1 M Mops buffer pH 7.5 at 40°C. 20 mm incubation and determination of the formation of reducing sugars using PHAB : One CMC unit corresponds to the formation of 1 micromole glucose equivalent per mm. The CMC
(Carboxy Methyl Cellulose 7L from Hercules) final concentration General molecular biology methods
Unless otherwise stated all DNA manipulations and transformations were performed using standard methods of molecular biology (Sambrook et al . (1989) Molecular cloning: A laboratory manual, Cold Spring Harbor lab., Cold Spring Harbor, NY; Ausubel, F. M. et al . (eds.) "Current protocols m Molecular Biology". John Wiley and Sons, 1995; Harwood, C. R. , and Cutting, S. M. (eds.) "Molecular Biological Methods for Bacillus". John Wiley and Sons, 1990). Enzymes for DNA manipulations were used according to the specifications of the suppliers, (e.g. restriction endonucleases, ligases etc. are obtainable from New England Biolabs , Inc . ) .
The following examples illustrate the invention.
EXAMPLE 1
Cloning of xyloglucanase encoding genes from Paenibacillus paJuli and Paenibacillus sp . , DSM 13330
Cultivation of donor strains
A strain of Paenibacillus pabuli was grown m TY with pH adjusted pH 7. After 24 hours incubation at 30°C and 300 rpm, the cells were harvested and genomic DNA was isolated by the method described below.
Isolation of genomic DNA
The Paenibacillus pabuli strain was propagated m liquid media as described above. The cells were harvested, and genomic DNA was isolated by the method described by Pi tcher et al . 1989.
Construction of a genomic library from Paenibacillus pabuli Genomic DNA of Paenibacillus pabuli was partially digested with restriction enzyme Sau3A, and size-fractionated by elec- trophoresis on a 0.7 % agarose gel (SeaKem agarose, FMC, USA) . Fragments between 4 and 10 kb m size were isolated and concentrated to a DNA band by running the DNA fragments backwards on a 1.5 % agarose gel followed by extraction of the fragments from the agarose gel slice using the GFX gel extraction kit according to the manufacturer's instructions (Amersham Pharmacia Biotech, USA) . To construct a genomic library, ca . 100 ng of purified, fractionated DNA from above was ligated with 1 μg of BamHI -cleaved, dephosphorylated la bdaZAPexpress vector arms (Stratagene, La Jolla CA, USA) for 24 hours at + 4 °C according to the manufacturer's instructions. A 3-μl aliquot of the ligation mixture was packaged directly using the GigaPacklll Gold packaging extract (Stratagene, USA) according to the manufacturers instructions (Stratagene) . The genomic lambdaZAPExpress phage library was titered using the E. coli XLl-Blue MRF-stram from Stratagene (La Jolla, USA) . The unamplified genomic library comprised of 5 x 106 plaque-forming units (pfu) with a vector background of less than 1 %.
Screening for xyloglucanase clones by functional expression in lambdaZAPExpress
Approximately 10 000 plaque-forming units (pfu) from the genomic library were plated on NZY-agar plates containing 0.1 % AZCL-xyloglucan (MegaZyme, Australia) using E. coli XLl-Blue MRF' (Stratagene, USA) as a host, followed by incubation of the plates at 37°C for 24 hours. A single xyloglucanase-positive lambda clone was identified by the formation of blue hydrolysis halo around the positive phage clone. The clone was recovered from the screening plate by coring the TOP-agar slice containing the plaque of interest into 500 μl of SM buffer and
20 μl of chloroform. The xyloglucanase-positive lambdaZAPExpress clone was plaque-purifled by plating an aliquot of the cored phage stock on NZY plates containing 0.1 % AZCL-xyloglucan as above. A single, xyloglucanase-positive lambda clone was cored into 500 μl of SM buffer and 20 μl of chloroform, and purified by one more plating round as described above . Single-clone in vivo excision of the phagemid from the xyloglucanase-positive lambdaZAPExpress clone
E. coli XLl-Blue cells (Stratagene, La Jolla Ca . ) were 5 prepared and resuspended lOmM MgS04 as recommended by
Stratagene (La Jolla, USA) . A 250-μl aliquot of the pure phage stock from the xyloglucanase-positive clone was combined m Falcon 2059 tube with 200 μl of XLl-Blue MRF' cells (OD600=1.0) and > 106 pfu/ml of the ExAssist M13 helper phage (Stratagene) ,
10 and the mixture was incubated at 37°C for 15 minutes. Three ml of NZY broth was added to the tube and the tube was incubated at 37°C for 2.5 hours. The tube was heated at 65°C for 20 minutes to kill the E . coli cells and bacteriophage lambda; the phagemid being resistant to heating. The tube was spun at 3000
15 rpm for 15 minutes to remove cellular debris and the supernatant was decanted into clean Falcon 2059 tubes. Aliquots of the supernatant containing the excised smgle-stranded phagemid were used to infect 200 μl of E. coli XLOLR cells (Stratagene, OD600=1.0 m lOmM MgS04) by incubation at 37°C for
20 15 minutes. 350μl of NZY broth was added to the cells and the tubes were incubated for 45 mm at 37°C. Aliquots of the cells were plated onto LB kanamycm agar plates and incubated for 24 hours at 37°C. Five excised single colonies were re-streaked onto LB kanamycm agar plates containing 0.1 % AZCL-xyloglucan
25 (MegaZyme, Australia) . The xyloglucanase-positive phagemid clones were characterized by the formation of blue hydrolysis halos around the positive colonies. These were further analyzed by restriction enzyme digests of the isolated phagemid DNA (QiaSpm kit, Qiagen, USA) with EcoRI, Pstl, EcoRI-Pstl, and
30 Hmdlll followed by agarose gel electrophoresis .
Nucleotide sequence analysis
80 ng of target DNA from the genomic xyloglucanase clone pXYG1009 was transposon-tagged using the pGPS-2 donor plasmid 35 and the GPS-1 Genome Priming System from New England
Biosystems, USA, according to the manufacturer's instructions. One μl of the transposition reaction mixture was electroporated into E. coli DH10B cells (Gibco-BRL, USA) according to the manufacturer's instructions, and the transformed E. coli cells were plated on LB agar plates containing kanamycm (20 μg/ml) , and chloramphemcol (15 μg/ml) . 100 colonies were re-tested on LB kanamycm and chloramphemcol plates containing 0.1 % AZCL- xyloglucan as substrate, and of these, 13 transposon-tagged pXYG1009 clones, showing no activity on the AZCL-xyloglucan substrate were, together with the pXYG1009 clone, chosen as plasmid templates for sequencing. The nucleotide sequence of the XYG1009 clone was determined from both strands by the dideoxy chain-termination method (Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 5463- 5467) using 500 ng of QiaQuick-purifled template (Qiagen, USA) , the Taq deoxy-termmal cycle sequencing kit (Perkm-Elmer, USA) , fluorescent labelled terminators and 5 pmol of either pBK-CMV polylmker primers (Stratagene, USA) or the primerS and primerN sequencing primers from the GPS-1 Genome Priming System (New England Biosystems, USA) . Analysis of the sequence data was performed according to Devereux et al . 1984. The sequence is shown m the appended SEQ ID NO: 1 and the deduced ammo acid sequence is shown the appended SEQ ID NO: 2.
In a similar manner, based on the strain Paenibacillus sp . , DSM 13330, the genomic xyloglucanase clone pXYG1022 was identified and analysed. The nucleotide sequence of the XYG1022 enzyme is shown m the appended SEQ ID NO: 3 and the deduced ammo acid sequence is shown in the appended SEQ ID NO: 4.
EXAMPLE 2 Identification of xyloglucanase genes by PCR amplification
Total DNA was isolated from different strains of Paenibacillus pabuli or Paenijbacillus sp . contemplated to belong to Paenibacillus pabuli using the commercially available kit, High Pure Template Preparation kit (Boehrmger Mannheim, catalogue number 1796828) according to the manufacturers instructions .
Primers based on the DNA sequence of XYG1009 (SEQ ID NO: 1) were designed for PCR amplification (polymerase chain reaction) and are given below as Primer 1 and Primer 2. PCR was carried out using the following protocol :
Reddy Mix* 22 . 5 1 Total DNA 0 . . 5 1
Primer 1 (10M) 1 . . 0 1 Primer 2 (10M) 1 . . 0 1
25.0 1
*Reddy Mix PCR Master mix (Advanced Biotechnologies Ltd. Surrey KT22 7Ba, UK, catalogue number AB-0575) containing Taq DNA polymerase, dNTPs, MgCl2 and reaction buffer. The PCR amplification conditions were Step 1 94 °C 2min Step 2 94°C 30sec
Step 3 55°C 30sec Step 4 72°C 2mm Steps 2-4 were repeated for 30 cycles Step 5 72°C 2mm Step 6 4°C hold temperature
5 litres of the reaction product was visualised following electrophoresis on a 1% agarose gel. A single band of approx. lkb was obtained for each of the samples. The PCR amplified band was sequenced using primers 1 and 2.
Primer 1 : CAT TCT GCA GCC GCG GCA GCG GAC GCT TCG CAA ATA GTG TC
Primer 2 : GCG TTG AGA CGC GCG GCC GCT TAT TGC ATA CCT TGC ATG ATC GC
One of the strains gave a sequence which was 100% identical on the DNA and ammo acid sequence listed m SEQ ID NOS: 1 and 2, respectively.
Three other five strains gave the (partial) ammo acid sequences listed m SEQ ID NOS: 5-9 as follows:
SEQ ID NO: 5: PCR product denoted XYG 1035 sequenced from
N-termmal end
SEQ ID NO: 6: PCR product denoted XYG 1035 sequenced from C-termmal end
SEQ ID NO: 7: PCR product denoted XYG 1036 sequenced and overlapping from both ends
SEQ ID NO: 8: PCR product denoted XYG 1037 sequenced from N-terminal end
SEQ ID NO: 9: PCR product denoted XYG 1037 sequenced from
C-termmal end
Comparison of the ammo acid sequences from the PCR amplified DNAs to the ammo acid sequence of XYG1009 from Paenibacillus pabuli (SEQ ID NO: 2) :
The ammo acid sequences of the PCR amplified fragments were compared to that of SEQ ID NO: 2 and the number of ammo acid substitutions are given below. The nomenclature is such that the numbering is based on the ammo acid sequence position in SEQ ID NO: 2. The ammo acids are represented by single letters where the letter preceding the number is the ammo acid SEQ ID NO: 2 and the letter following the number is the changed ammo acid in the PCR amplified sequence. In the PCR amplified fragment resulting XYG 1035 five ammo acid substitutions could be detected (N94S, T197A, T241A, F286S, N379K) where the numbering is based on SEQ ID NO: 2.
In the PCR amplified fragment resulting m XYG 1036 8 ammo acid substitutions could be detected (R174H, T197A, V224I, Y233F, T241A, Y247F, F286A, S335T) where the numbering is based on SEQ ID NO: 2.
In the PCR amplified fragment resulting m XYG 1037 9 ammo acid substitutions could be detected (T197A, A201P, V224I, Y233F, T241V, Y247F, F286A, A290S, A349E) where the numbering is based on SEQ ID NO : 2.
In the PCR amplified fragment resulting m XYG 1034 11 ammo acid substitutions could be detected (N61S, E73A, K76Q, V90I, I164V, V224I, Y233F, T241A, Y247F, F286A, S377Q) where the numbering is based on SEQ ID NO: 2.
EXAMPLE 3
Subcloning and expression in B . subtilis of the XYG1009 gene from Paenibacillus pabuli encoding for the xyloglucanase of the invention
Subcloning and expression of mature xyloglucanase in B. subtilis . The xyloglucanase encoding DNA sequence of the invention (SEQ ID NO: 1) was PCR amplified using the PCR primer set consisting of these two oligo nucleotides:
# 178765 5 '-CAT TCT GCA GCC GCG GCA GCG GAC GCT TCG CAA ATA GTG TC-3'
# 178766
5' -GCG TTG AGA CGC GCG GCC GCT TAT TGC ATA CCT TGC ATG ATC GC-
3' Restriction sites SacII and Notl are underlined
The oligonucleotides were used in a PCR reaction in HiFidelityTM PCR buffer (Boehringer Mannheim, Germany) supplemented with 200 μM of each dNTP, 2.6 units of HiFidelityTM Expand enzyme mix and 200 pmol of each primer. Chromosomal DNA isolated from Paenibacillus pabuli as described above was used as template.
The PCR reaction was performed using a DNA thermal cycler (Landgraf , Germany) . One incubation at 94°C for 1 min followed by ten cycles of PCR performed using a cycle profile of denaturation at 94°C for 15 sec, annealing at 60°C for 60 sec, and extension at 72°C for 120sec, followed by twenty cycles of denaturation at 94°C for 15 sec, 60°C for 60 sec and 72°C for 120 sec (at this elongation step 20 sec are added every cycle) . Five-μl aliquots of the amplification product was analysed by electrophoresis in 0.7 % agarose gels (NuSieve, FMC) . The appearance of a DNA fragment size 1.2 kb indicated proper amplification of the gene segment.
Subcloning of PCR fragment : Forty-five-μl aliquots of the PCR products generated as described above were purified using QIAquick PCR purification kit (Qiagen, USA) according to the manufacturer's instructions. The purified DNA was eluted in 50 μl of lOmM Tris-HCl, pH 8.5. 5 μg of pMOL944 and twenty- five-μl of the purified PCR fragment was digested with SacII and Notl, electrophoresed m 0.7 % agarose gels (NuSieve, FMC) , the relevant fragments were excised from the gels, and purified using QIAquick Gel extraction Kit (Qiagen, USA) according to the manufacturer's instructions. The isolated PCR DNA fragment was then ligated to the SacII-Notl digested and purified pMOL944. The ligation was performed overnight at 16°C using 0.5 μg of each DNA fragment, 1 U of T4 DNA ligase and T4 ligase buffer (Boehrmger Mannheim, Germany) .
The ligation mixture was used to transform competent B. subtilis PL2306. The transformed cells were plated onto LBPG- 10 μg/ml of Kanamycm-agar plates. After 18 hours incubation at 37°C colonies were seen on plates. Several clones were analyzed by isolating plasmid DNA from overnight culture broth.
One such positive clone was restreaked several times on agar plates as used above; this clone was called MB1040. The clone MB1040 was grown overnight m TY-lOμg/ml Kanamycm at 37°C, and next day 1 ml of cells were used to isolate plasmid from the cells using the Qiaprep Spin Plasmid Mmiprep Kit #27106 according to the manufacturers recommendations for B . subtilis plasmid preparations. This DNA was sequenced and revealed a DNA sequence identical to the part of the xyloglucanase gene m SEQ ID NO: 1 encoding the mature xyloglucanase.
EXAMPLE 4
Expression, purification and characterization of xyloglucanase from Paenibacillus pabuli The clone MB1040 obtained as described above m Example 3 was grown m BPX media with 10 μg/ml of Kanamycm m 500ml two baffled shake flasks for 5 days at 37°C at 300 rpm, whereby 4000 ml of culture broth was obtained with a pH of 5.8. Then 180 ml of cationic agent (C521 10%) and 360 ml of anionic agent (A130 0.1%) was added during agitation for flocculation. The flocculated material was separated by centrifugation using a Sorval RC 3B centrifuge at 10000 rpm for 30 mm at 6°C. The total volume of the resulting supernatant was 4200 ml. The supernatant was clarified using Whatman glass filters GF/D and C and finally concentrated on a filtron UF membrane with a cut off of 10 kDa . The total volume of 1450 ml was adjusted to pH 8.0. For obtaining a highly purified xyloglucanase a final step using Phenyl -Sepharose hydrophobic chromatography was carried out. To the solution was added ammonium sulphate to a final concentration of 1.2 M. The column was adjusted with the same solution and the enzyme solution was added. The xyloglucanase bound to the column and the pure xyloglucanase was eluted using water. The xyloglucanase containing a 95% pure band m SDS -PAGE at 40 kDa was concentrated and formulated with 30% MPG for trials .
Characterisation
The pure enzyme gave a single band m SDS -PAGE of 40 kDa and an isoelectric point of around 8.9.
The following sequence was found by N-terminal determination of the pure enzyme: ADASQIVS; the theoretical MW estimated from the found N-termmus ADASQIVS is 40535.06 Da.
The protein concentration was determined using a molar extinction coefficient of 94590 (based on the ammo acid composition deducted from the sequence) . The pH activity profiles showed more than 50% relative activity between pH 6.0 and 8.0 at 40°C.
The temperature optimum was 50° at pH 7.5.
The ammo acid sequence SEQ ID NO: 2 deducted from the DNA sequence (SEQ ID NO: 1) shows that the coding region code for: positions 1-32 signal peptide, and positions 33-395 catalytic domain belonging to glycosyl hydrolase family 5.
SEQ ID NO: 2 is 83% homologous (Blast) with: CelA EMBL entry: Y12512 (Blanco A., Diaz P., Martinez J., Vidal T., Torres A.L., Pastor F.I.J.; "Cloning of a new endoglucanase gene from Bacillus sp . BP-23 and characterisation of the enzyme. Performance paper manufacture from cereal straw"; Appl . Microbiol. Biotechnol . 50:48-54(1998)).
SEQ ID NO: 2 is 30% homologous (Blast) with Bacillus agaradhaerens xyloglucanase disclosed m WO99/02663.
Comparison example: Steady state kinetics on soluble xyloglucan and CMC (carboxymethylcellulose)
A method for determination of activity against xyloglucan has been developed.
The substrate is xyloglucan (amyloid) from tamarind seeds (the substrate is commercially available from Megazyme) . Buffer 0.1 M sodium phosphate, pH 7.5.
The substrate was prepared as a stock solution containing 5 gram per 1 m buffer. After mixing it was heated using a magnetic stirrer until a clear solution was obtained. The solution was then cooled to 40 °C and kept m a temperature controlled water bath at 40°C. The diluted enzyme solution of 0.5 ml was preheated for 10 mm. and mixed with 1.0 ml substrate and incubated for 20 mm.
The formation of reducing sugars is determined by using p- hydroxy-benzoic-acid-hydrazide (PHBAH) modified from Lever (1972) using 5 gram of potassium sodium tartrate m addition to 1.5 gram of PHBAH. Glucose is used as reference for determination of the reducing groups.
Results :
On xyloglucan a kCat of 1100 per sec was obtained with a kM of 0.2 gram per 1. On CMC could only be detected 0.7 kCat per sec with a very high KM above 50 gram per 1.
The xyloglucanase enzyme also had an activity of 0.2 CMC units per mg protein.
In conclusion, the xyloglucanase of the invention is characterised by having a high specific activity on xyloglucan (tamarind gum from Megazyme) and very low cellulase activity on CMC (endoglucanase activity) .
Immunological properties: At the Danish company DAKO, rabbit polyclonal monospecific serum was raised against the highly purified xyloglucanase using conventional techniques. The serum formed a nice single precipitate in agarose gels with the xyloglucanase of the invention.
EXAMPLE 5
Subcloning and expression in B . subtilis of the XYG1022 gene from Paenibacillus sp. , DSM 13330, encoding for the xyloglucanase of the invention
Subcloning and expression of mature xyloglucanase in B . subtilis .
The xyloglucanase encoding DNA sequence of the invention was PCR amplified using the PCR primer set consisting of these two oligo nucleotides:
188669:
5 " - CATTCTGCAGCCGCGGCCGCGGATTTCAGATCATTGAACGC -3'
189585 :
5'- GCGTTGAGACGCGCGGCCGCTTACTGTATACCCTGCATGATGGC -3' Restriction sites Pstl and Notl are underlined
The oligonucleotides were used in a PCR reaction in HiFidelityTM PCR buffer (Boehringer Mannheim, Germany) supplemented with 200 μM of each dNTP, 2.6 units of HiFidelityTM Expand enzyme mix and 200 pmol of each primer. Chromosomal DNA isolated from Paenibacillus sp . , DSM 13330, was used as template.
The PCR reaction was performed using a DNA thermal cycler (Landgraf , Germany) . One incubation at 94°C for 1 min followed by ten cycles of PCR performed using a cycle profile of denaturation at 94°C for 15 sec, annealing at 60°C for 60 sec, and extension at 72°C for 120sec, followed by twenty cycles of denaturation at 94°C for 15 sec, 60°C for 60 sec and 72°C for 120 sec (at this elongation step 20 sec are added every cycle) . Five-μl aliquots of the amplification product was analysed by electrophoresis in 0.7 % agarose gels (NuSieve, FMC) . The appearance of a DNA fragment size 1.3 kb indicated proper amplification of the gene segment. Subcloning of PCR ragment:
Forty- five-μl aliquots of the PCR products generated as described above were purified using QIAquick PCR purification kit (Qiagen, USA) according to the manufacturer's instructions. 5 The purified DNA was eluted m 50 μl of lOmM Tπs-HCl, pH 8.5. 5 μg of pMOL944 and twenty-flve-μl of the purified PCR fragment was digested with Pstl and Notl, electrophoresed m 0.7 % agarose gels (NuSieve, FMC) , the relevant fragments were excised from the gels, and purified using QIAquick Gel lo extraction Kit (Qiagen, USA) according to the manufacturer's instructions. The isolated PCR DNA fragment was then ligated to the Pstl-Notl digested and purified pMOL944. The ligation was performed overnight at 16°C using 0.5 μg of each DNA fragment, 1 U of T4 DNA ligase and T4 ligase buffer (Boehrmger Mannheim, is Germany) .
The ligation mixture was used to transform competent B . subtili s MB1053-1. The transformed cells were plated onto LBPG-10 μg/ml of Kanamycm-agar plates. After 18 hours incubation at 37°C colonies were seen on plates. Several clones
20 were analyzed by isolating plasmid DNA from overnight culture broth.
One such positive clone was restreaked several times on agar plates as used above; this clone was called PL3381. The clone PL3381 was grown overnight m TY-lOμg/ml Kanamycm at
25 37°C, and next day 1 ml of cells were used to isolate plasmid from the cells using the Qiaprep Spin Plasmid M iprep Kit #27106 according to the manufacturers recommendations for B . subtilis plasmid preparations. This DNA was sequenced and revealed a DNA sequence identical to the part of the
30 xyloglucanase gene m SEQ ID NO: 3 encoding the mature xyloglucanase .
EXAMPLE 6
Purification and characterization of xyloglucanase from 35 Paenibacillus sp . , DSM 13330
The clone PL3381 obtained as described example 5 was incubated 4000 ml of BPX containing 10 μg/ml of Kanamycm and grown for 5 days at 37°C at 300 rpm, final pH was 5.64. The fermentation medium was flocculated using cationic flocculation agent C521 (10% solution) and 0.1% solution of anionic agent A130: To 4000 ml of broth was added 180 ml of C521 (10%) simultaneously with 360 ml of A130 under stirring at room temperature. The flocculated material was separated by centrifugation using a Sorval RC 3B centrifuge at 4,500 rpm for 30 minutes. The supernatant was clarified using Whatman glass filter number F. In total was obtained 4000 ml of clear solution. The liquid was concentrated into 400 ml, using filtron ultraflltration with a MW cut off of 10 kDa.
The concentrate was batch treated with 200 gram Q- Sepharose equilibrated with 25 mM Tπs pH 7.5. The unbound material was stabilized with 30% MPG and the xyloglucanase was used for detergent cleaning. The enzyme is very active m the commercial liquid detergents sold by The Procter & Gamble Company under the brand names Ariel and Tide.
For obtaining a pure enzyme 2 ml of this partial pure enzyme was applied to a size chromatography (Superdex 75) column equilibrated with 0.1 M Sodium acetate pH 6.0. The xyloglucanase eluted as a single peak with a MW of 40 kDa m SDS-PAGE.
After electroblottmg of this band the N-termmal was determined as: ADFRSLNASQIVSEMG This is agreement with the ammo acid sequence shown m SEQ ID NO: 4 deduced from the DNA sequence shown SEQ ID NO: 3 with a 32 ammo acid pro sequence. The calculated MW from the deduced sequence was 40 kDa and the calculated pi was 8.89. The molar extinction coefficient at 280 nm was 93390. DSC (Disc Scanning Calorimetry) m sodium acetate buffer at pH 6.0 showed a melting temperature around 68.6°C.
EXAMPLE 7
Subcloning and expression in B . subtilis of the XYG1034 gene from Paenibacillus pabuli encoding for the xyloglucanase of the invention
Subcloning and expression of mature xyloglucanase m B . subtilis . The xyloglucanase encoding DNA sequence of the invention was PCR amplified using the PCR primer set consisting of these two oligo nucleotides:
# 178765
5 '-CAT TCT GCA GCC GCG GCA GCG GAC GCT TCG CAA ATA GTG TC-3'
# 178766
5' -GCG TTG AGA CGC GCG GCC GCT TAT TGC ATA CCT TGC ATG ATC GC-
3' Restriction sites SacII and Notl are underlined
The oligonucleotides were used m a PCR reaction m
HiFidelityTM PCR buffer (Boehrmger Mannheim, Germany) supplemented with 200 μM of each dNTP, 2.6 units of
HiFidelityTM Expand enzyme mix and 200 pmol of each primer, Chromosomal DNA isolated from a strain of Paenibacillus pabuli was used as template .
The PCR reaction was performed using a DNA thermal cycler (Landgraf , Germany) . One incubation at 94°C for 1 mm followed by ten cycles of PCR performed using a cycle profile of denaturation at 94°C for 15 sec, annealing at 60°C for 60 sec, and extension at 72°C for 120sec, followed by twenty cycles of denaturation at 94°C for 15 sec, 60°C for 60 sec and 72°C for 120 sec (at this elongation step 20 sec are added every cycle) . Five-μl aliquots of the amplification product was analysed by electrophoresis m 0.7 % agarose gels (NuSieve, FMC) . The appearance of a DNA fragment size 1.2 kb indicated proper amplification of the gene segment.
Subcloning of PCR fragment : Forty-flve-μl aliquots of the PCR products generated as described above were purified using QIAquick PCR purification kit (Qiagen, USA) according to the manufacturer's instructions. The purified DNA was eluted 50 μl of lOmM Tπs-HCl, pH 8.5. 5 μg of pMOL944 and twenty-flve-μl of the purified PCR fragment was digested with SacII and Notl, electrophoresed m 0.7 % agarose gels (NuSieve, FMC), the relevant fragments were excised from the gels, and purified using QIAquick Gel extraction Kit (Qiagen, USA) according to the manufacturer's instructions. The isolated PCR DNA fragment was then ligated to the SacII-Notl digested and purified pMOL944. The ligation was performed overnight at 16°C using 0.5 μg of each DNA fragment, 1 U of T4 DNA ligase and T4 ligase buffer (Boehrmger Mannheim, Germany) .
The ligation mixture was used to transform competent B. subtilis PL2306. The transformed cells were plated onto LBPG- 10 μg/ml of Kanamycm-agar plates. After 18 hours incubation at 37°C colonies were seen on plates. Several clones were analyzed by isolating plasmid DNA from overnight culture broth.
One such positive clone was restreaked several times on agar plates as used above, this clone was called MB1067. The clone MB1067 was grown overnight TY-lOμg/ml Kanamycm at 37°C, and next day 1 ml of cells were used to isolate plasmid from the cells using the Qiaprep Spin Plasmid Mmiprep Kit #27106 according to the manufacturers recommendations for B . subtilis plasmid preparations. This DNA was sequenced and revealed a DNA sequence identical to the part of the xyloglucanase gene m SEQ ID NO: 10 encoding the mature xyloglucanase represented by the derived protein sequence m SEQ ID NO: 11.
EXAMPLE 8
Purification and characterization of xyloglucanase XYG1034 cloned from Paenibacillus pabuli
The clone MB1067 obtained as described m example 7 was incubated m 4200 ml of BPX containing mg/ml kanamycm from shake flasks with a final pH of 7.5.
The fermentation medium was flocculated using 42 ml 50% W/W CaCl2, 42 ml 11% Na-alummate and 20% formic acid followed by adding 105 ml of C521 (10%) simultaneously with 315 ml of A130 under stirring at room temperature. The flocculated material was separated by centrifugation using a Sorval RC 3B centrifuge at 4,500 rpm for 30 minutes. The supernatant was clarified using Whatman glass filter number F. In total was obtained 3900 ml of clear solution.
The liquid was concentrated into 400 ml, using filtron ultraflltration with a MW cut off of 10 kDa. The concentrate was batch treated with 200 gram Q- Sepharose equilibrated with 25 mM Tris pH 7.5. The unbound material was stabilized with 30% MPG and the xyloglucanase was used for detergent cleaning. The enzyme is very active in liquid Ariel and Tide.
For obtaining a pure enzyme 2 ml of this partial pure enzyme was applied to a size chromatography (Superdex 75) column equilibrated with 0.1 M sodium acetate pH 6.0. The xyloglucanase eluted as a single peak with a MW of 40 kDa in SDS-PAGE.
After electroblotting of this band the N-terminal was determined as: ADASQIVSEMGAGWNLG
This is in agreement with the amino acid sequence shown in SEQ ID NO: 2 (XYG1009) deduced from the DNA sequence shown in SEQ ID NO: 1 with a 32 amino acid pro sequence. The calculated MW from the deduced sequence was 40 kDa and the calculated pi was 8.89. The molar extinction coefficient at 280 nm was 94590 based on the very homologues sequence of XYG1009.
DSC in sodium acetate buffer at pH 6.0 showed a melting temperature around 61.5°C.
LITERATURE
Ausubel, F. M. et al . (Eds.) "Current protocols m Molecular Biology". John Wiley and Sons, 1995.
N. C. Carpita and D. M. Gibeaut (1993) The Plant Journal 3:1- 30.
T. Christensen et al . Biotechnology vol 6 page 1419-1422, 1988.
Devereux et al . (1984) Nucleic Acids Res. 12, 387-395.
Dideπchsen, B., Wedsted, U. , Hedegaard, L., Jensen, B. R., Sjøholm, C. (1990) Cloning of aldB, which encodes alpha- acetolactate decarboxylase, an exoenzyme from Bacillus brevis . J. Bacteriol . 172:4315-4321.
Dretzen, G., Bellard, M. , Sassone-Corsi , P., Chambon, P. (1981) A reliable method for the recovery of DNA fragments from agarose and acrylamide gels. Anal. Biochem., 112, 295-298.
Eriksson, O.E. & Hawksworth, D.L.: Systema Ascomycetum vol 12 (1993) .
S. C. Fry et al (1992) Biochemical Journal 282:821-828
Hawksworth, D.L., Kirk, P.M., Sutton, B.C. and Pegler, D.N.: Dictionary of the fungi, International Mycological Institute, 616 pp (1995) ;
T. Hayashi and D. P. Delmer (1988) Carbohydrate Research 181:273-277.
Henπssat, B. 1991. A classification of glycosyl hydrolases based on ammo acid sequence similaπtites . Biochem. J., 280:309-316.
Henrissat, B., and A. Bairoch. 1993. New families the classification of glycosyl hydrolases based on ammo acid sequence similaritites . Biochem. J., 293:781-788.
Jύlich, W.: Higher Taxa of Basidiomycetes , Bibliotheca Mycologia 85, 485 pp (1981) .
Jørgensen, P.L. et al . , 1990, Gene, 96, p. 37-41.
McKenzie, T. et al . , 1986, Plasmid 15:93-103.
Leatherbarrow, R. J. (1992) Grafit version 3.0 Erithacus Software Ltd. Stames, U.K.
Lever, M. (1972) A new reaction for colormetric determination of carbohydrates. Anal. Biochem. 47, 273-279.
O'Donnell, K. : Zygomycetes m culture, University of Georgia, US, 257 pp (1979) .
Pitcher, D. G., Saunders, N. A., Owen, R. J. (1989). Rapid extraction of bacterial genomic DNA with guanidium thiocyanate. Lett. Appl . Microbiol . , 8, 151-156.
J. K. C. Rose et al (1996) Plant Physiology 110:493-499
Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 5463-5467
A.L. Sonenshein, J.A. Hoch and Richard Losick (Eds.) (1993) Bacillus subtilis and other Gram-Positive Bacteria, American Society for microbiology, p.618.
Vmcken, J.P., Beldman, G., and Voragen, A.G.J. Substrate- specificity of endoglucanases - what determines xyloglucanase activity. Carbohydrate Research 298 (4) : 299-310 , 1997.
Von Arx, J. A. : The genera of fungi sporulatmg m culture, 424 pp (1981) . R. L. Whistler and J. N. BeMiller (1993) Industrial gums: Polysaccharides and their derivatives, Academic Press Inc.
Yasbin, R.E., Wilson, G.A. and Young, F.E. (1975)
Transformation and transfection in lysogenic strains of Bacillus subtilis -. evidence for selective induction of prophage in competent cells. J. Bacteriol, 121:296-304.
W.S. York et al (1996) Carbohydrate Research 285:99-128.
Original (for SUBMISSION) - pnnted on 28.02.2001 11:14:44 AM -1 Form - PCT/RO/134 (EASY) Indications Relating to Deposited Microorganism(s) or Other Biological Material (PCT Rule 13bis) -1-1 Prepared using PCT-EASY Version 2 . 91 (updated 01 . 01 .2001) -2 International Application No.
-3 Applicant's or agent's file reference 6073 .204 - O
The indications made below relate to the deposited microorganism(s) or other biological material referred to in the description on: -1 page 6 -2 line 20 - 28 -3 Identification of Deposit -3-1 Name of depositary institution DSMZ -Deutsche Sammlung von
Mikroorganismen und Zellkulturen GmbH -3-2 Address of depositary institution Mascheroder Weg lb , D- 38124
Braunschweig, Germany -3-3 Date of deposit 17 February 2000 ( 17 . 02 . 2000 ) -3-4 Accession Number DSMZ 13330 -4 Additional Indications NONE -5 Designated States for Which all designated States Indications are Made -6 Separate Furnishing of Indications NONE
These indications will be submitted to the International Bureau later
The indications made below relate to the deposited microorganism(s) or other biological material referred to in the description on: -1 page 6 -7 -2 line 29 -2 -3 Identification of Deposit -3-1 Name of depositary institution DSMZ -Deutsche Sammlung von
Mikroorganismen und Zellkulturen GmbH -3-2 Address of depositary institution Mascheroder Weg lb, D- 38124
Braunschweig, Germany -3-3 Date of deposit 06 December 1999 ( 06 . 12 . 1999 ) -3-4 Accession Number DSMZ 13183 -4 Additional Indications NONE -5 Designated States for Which all designated States Indications are Made -6 Separate Furnishing of Indications NONE
These indications will be submitted to the International Bureau later Original (for SUBMISSION) - printed on 28.02.2001 11:14:44 AM FOR RECEIVING OFFICE USE ONLY
-5 This form was received by the international Bureau on: -5-1 Authorized officer

Claims

1. A xyloglucanase enzyme belonging to family 5 of glycosyl hydrolases, which enzyme is endogenous to a strain of Paenibacillus .
2 . The enzyme according to claim 1, wherein the strain of Paenibacillus belongs to the group consisting of the species Paenibacillus pabuli , Paenibacillus sp . , DSM 13330, and Paenibacillus sp . having a higher degree of identity with the Paenibacillus pabuli type strain ATCC 43899 than the strain Paenibacillus sp . , DSM 13330, when subjected to 16S RNA analysis .
3. A xyloglucanase belonging to family 5 of glycosyl hydrolases, which enzyme is selected from one of
(a) a polypeptide encoded by the DNA sequence of positions 840- 1931 of SEQ ID NO: 1 ;
(b) a polypeptide produced by culturing a cell comprising the sequence of SEQ ID NO: 1 under conditions wherein the DNA sequence is expressed;
(c) a xyloglucanase enzyme having a sequence of at least 85% identity to positions 33-395 of SEQ ID NO: 2 when identity is determined by GAP provided m the GCG program package using a GAP creation penalty of 3.0 and GAP extension penalty of 0.1;
(d) a polypeptide encoded by a DNA sequence that hybridizes to the DNA sequence of positions 840-1931 of SEQ ID NO : 1 under medium stringency conditions, wherein the medium stringency conditions comprise hybridization m 5xSSC at 45°C and washing m 2xSSC at 60°C; or
(e) a polypeptide encoded by the xyloglucanase encoding part of the DNA sequence obtainable from the plasmid m Escheri chia coli DSM 13183.
4. The xyloglucanase according to claim 3 which is obtained or obtainable from a bacterium, preferably from a Gram positive bacterium, preferably from the Bacillus/ Lactobacillus subdivision, preferably a species from the genus Paenibacillus , especially a Paenibacillus pabuli .
5. An isolated xyloglucanase enzyme, m which the enzyme is (I) free from homologous impurities, and (ii) produced by culturing a cell comprising the DNA sequence of positions 840-1931 of SEQ ID NO: 1 or of positions 693-1896 of SEQ ID NO: 3, wherein the enzyme is produced and isolated.
6. An enzyme preparation comprising the enzyme according to any of the claims 1, 3 or 5.
7. The preparation according to claim 6 which further comprises one or more enzymes selected from the group consisting of proteases, cellulases (endoglucanases) , β-glucanases , hemicellulases, lipases, peroxidases, laccases, _a γlases glucoamylases , cutmases, pectmases, reductases, oxidases, phenoloxidases, lignmases, pullulanases, pectate lyases, xyloglucanases, xylanases, pectin acetyl esterases, polygalacturonases , rhamnogalacturonases , pectin lyases, other mannanases, pectin methylesterases , cellobiohydrolases , transglutammases ; or mixtures thereof.
8. An isolated polynucleotide molecule encoding a polypeptide having xyloglucanase activity which polynucleotide molecule hybridizes to a denatured double-stranded DNA probe under medium stringency conditions, wherein the probe is selected from the group consisting of DNA probes comprising the sequence shown m positions 840-1931 of SEQ ID NO: 1, positions 693-1896 of SEQ ID NO: 3, and DNA probes comprising a subsequence of positions 840-1931 of SEQ ID NO: 1 or positions 693-1896 of SEQ ID NO: 3, the subsequence having a length of at least about 100 base pairs .
9. An expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment selected from the group consisting of (a) polynucleotide molecules encoding a polypeptide having xyloglucanase activity comprising a nucleotide sequence as shown m SEQ ID NO: 1 from nucleotide 840 to nucleotide 1931, (b) polynucleotide molecules encoding a polypeptide having xyloglucanase activity comprising a nucleotide sequence as shown in SEQ ID NO: 3 from nucleotide 693 to nucleotide 1896, (c) polynucleotide molecules encoding a 5 polypeptide having xyloglucanase activity that is at least 85% identical to the amino acid sequence of SEQ ID NO : 2 from amino acid residue 33 to amino acid residue 395, and (d) degenerate nucleotide sequences of (a) , (b) or (c) ; and a transcription terminator.
10
10. A cultured cell into which has been introduced an expression vector according to claim 9, wherein said cell expresses the polypeptide encoded by the DNA segment .
is
11. A method of producing a polypeptide having xyloglucanase activity comprising culturing a cell into which has been introduced an expression vector according to claim 9, whereby said cell expresses a polypeptide encoded by the DNA segment; and recovering the polypeptide.
20
12. A detergent composition comprising the enzyme preparation according to claim 7 or the enzyme according to any of the claims 1, 3 and 5.
25 13. A process for machine treatment of fabrics which process comprises treating fabric during a washing cycle of a machine washing process with a washing solution containing the enzyme preparation according to claim 7 or the enzyme according to any of the claims 1, 3 and 5.
30
14. Use of the enzyme preparation according to claim 7 or the enzyme according to any of the claims 1, 3 and 5 in the textile industry for improving the properties of cellulosic fibers, yarn, woven or non-woven fabric.
35
15. The use according to claim 14, wherein the enzyme preparation or the enzyme is used in a scouring process step.
16. Use of the enzyme preparation according to claim 7 or the enzyme according to any of the claims 1, 3 and 5 m the cellulose fiber processing industry for ratting of fibers selected from the group consisting of hemp, jute, flax and linen.
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