WO2019038060A1 - Detergent composition comprising xanthan lyase variants ii - Google Patents

Detergent composition comprising xanthan lyase variants ii Download PDF

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Publication number
WO2019038060A1
WO2019038060A1 PCT/EP2018/071108 EP2018071108W WO2019038060A1 WO 2019038060 A1 WO2019038060 A1 WO 2019038060A1 EP 2018071108 W EP2018071108 W EP 2018071108W WO 2019038060 A1 WO2019038060 A1 WO 2019038060A1
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Prior art keywords
seq
variant
xanthan lyase
amino acids
region
Prior art date
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PCT/EP2018/071108
Other languages
French (fr)
Inventor
Nina Mussmann
Susanne Wieland
Christian DEGERING
Rune Nygaard MONRAD
Rajendra Kulothungan Sainathan
Sohel DALAL
Geetha Hiremath MENDEZ
Shilpi Agarwal
Allan Svendsen
Mette Louise Dissing OVERGAARD
Vasudeva P. RAO
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Henkel Ag & Co. Kgaa
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Application filed by Henkel Ag & Co. Kgaa filed Critical Henkel Ag & Co. Kgaa
Priority to EP18755157.7A priority Critical patent/EP3673060A1/en
Priority to US16/638,800 priority patent/US20210130744A1/en
Publication of WO2019038060A1 publication Critical patent/WO2019038060A1/en

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    • 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
    • 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
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y402/00—Carbon-oxygen lyases (4.2)
    • C12Y402/02—Carbon-oxygen lyases (4.2) acting on polysaccharides (4.2.2)
    • C12Y402/02012—Xanthan lyase (4.2.2.12)
    • C11D2111/12—
    • C11D2111/14—

Definitions

  • the present invention relates to detergent compositions, such as laundry compositions and dish wash compositions, including hand wash and automatic dish wash compositions, comprising novel xanthan lyase variants exhibiting alterations relative to the parent xanthan lyase in one or more properties such as: detergent stability (e.g. improved stability in a detergent composition) and/or storage stability (e.g. improved storage stability in a detergent composition).
  • detergent stability e.g. improved stability in a detergent composition
  • storage stability e.g. improved storage stability in a detergent composition
  • the present invention further relates to detergent compositions comprising novel xanthan lyase variants having activity on xanthan gum.
  • the invention also relates to methods for producing and using the compositions of the invention. Variants described herein are particularly suitable for use in cleaning processes and detergent compositions.
  • Xanthan gum is a polysaccharide derived from the bacterial coat of Xanthomonas campestris. It is produced by the fermentation of glucose, sucrose, or lactose by the Xanthomonas campestris bacterium. After a fermentation period, the polysaccharide is precipitated from a growth medium with isopropyl alcohol, dried, and ground into a fine powder. Later, it is added to a liquid medium to form the gum.
  • Xanthan gum is a natural polysaccharide consisting of different sugars which are connected by several different bonds, such as ⁇ -D-mannosyl- ⁇ -D-1 ,4-glucuronosyl bonds and ⁇ -D-glucosyl- ⁇ -D-1 ,4-glucosyl bonds.
  • Xanthan gum is at least partly soluble in water and forms highly viscous solutions or gels. Complete enzymatic degradation of xanthan gum requires several enzymatic activities including xanthan lyase activity and endo- ⁇ -1 ,4-glu- canase activity.
  • Xanthan lyases are enzymes that cleave the ⁇ -D-mannosyl- ⁇ -D-1 ,4-glucuronosyl bond of xanthan and have been described in the literature.
  • Xanthan lyases are known in the art, e.g. two xanthan lyases have been isolated from Paenibacillus alginolyticus XL-1 (e.g. Ruijssenaars ef al. (1999) 'A pyruvated mannose-specific xanthan lyase involved in xanthan degradation by Paenibacillus alginolyticus XL-1 ', Appl. Environ. Microbiol.
  • Glycoside hydrolases are enzymes that catalyse the hydrolysis of the glycosyl bond to release smaller sugars. There are over 100 classes of glycoside hydrolases which have been classified, see Henrissat ei al. (1991 ) 'A classification of glycosyl hydrolases based on amino-acid sequence similarities', J. Biochem.
  • glycoside hydrolase family 9 consists of over 70 different enzymes that are mostly endo-glucanases (EC 3.2.1 .4), cellobiohydrolases (EC 3.2.1.91 ), ⁇ -glucosidases (EC 3.2.1 .21 ) and exo ⁇ -glucosaminidase (EC 3.2.1.165).
  • xanthan gum has been used as an ingredient in many consumer products including foods (e.g. as thickening agent in salad dressings and dairy products) and cosmetics (e.g. as stabilizer and thickener in toothpaste and make-up, creams and lotions to prevent ingredients from separating and to provide the right texture of the product).
  • xanthan gum has found use in the oil industry as an additive to regulate the viscosity of drilling fluids etc.
  • the widespread use of xanthan gum has led to a desire to degrade solutions, gels or mixtures containing xanthan gum thereby allowing easier removal of the by-products.
  • Xanthan lyases and endoglucanases for the degradation of xanthan gum and the use of such enzymes for cleaning purposes, such as the removal of xanthan gum containing stains, and in the drilling and oil industries are known in the art, e.g. WO2013167581 A1.
  • the known xanthan lyase having SEQ ID NO: 2 was found to be sensitive to the presence of deter- gents. To improve applicability and/or cost and/or the performance of such enzymes there is an ongoing search for variants with altered properties, such as increased stability, e.g. improved stability in a detergent composition. However, mutagenesis of large enzymes followed by purification and functional analysis of mutant libraries can be very expensive and laborious.
  • xanthan lyase having SEQ ID NO: 2 is a large enzyme (>1000 residues), it is difficult and expensive to randomly target its properties for improvement of, e.g., stability in a detergent composition.
  • the present invention identifies regions in the protein sequence/structure of the known xanthan lyase having SEQ ID NO: 2 that are relevant for e.g. storage stability, and therefore provides an important guidance on where to mutate a xanthan lyase in order to stabilize the molecule in a detergent.
  • the present invention relates to detergent compositions comprising a xanthan lyase variant, comprising an alteration (e.g., a substitution, deletion or insertion) at one or more positions in a region selected from the group consisting of: region 7 corresponding to amino acids 1 to 153 of SEQ ID NO: 2, region 8 corresponding to amino acids 177 to 613 of SEQ ID NO: 2, region 9 corresponding to amino acids 659 to 730 of SEQ ID NO: 2, region 10 corresponding to amino acids 804 to 806 of SEQ ID NO: 2, region 1 1 corresponding to amino acids 847 to 871 of SEQ ID NO: 2, region 12 corresponding to amino acids 886 to 902 of SEQ ID NO: 2, and region 13 corresponding to amino acids 1005 to 1037 of SEQ ID NO: 2.
  • an alteration e.g., a substitution, deletion or insertion
  • the present invention relates to detergent compositions comprising a xanthan lyase variant, comprising an alteration (e.g., a substitution, deletion or insertion) at one or more positions in a region selected from the group consisting of: region 7 corresponding to amino acids 1 to 153 of SEQ ID NO: 2, region 8 corresponding to amino acids 177 to 613 of SEQ ID NO: 2, region 9 corresponding to amino acids 659 to 730 of SEQ ID NO: 2, region 10 corresponding to amino acids 804 to 806 of SEQ ID NO: 2, region 1 1 corresponding to amino acids 847 to 871 of SEQ ID NO: 2, region 12 corresponding to amino acids 886 to 902 of SEQ ID NO: 2, and region 13 corresponding to amino acids 1005 to 1037 of SEQ ID NO: 2, wherein said variant has at least 60% and less than 100% sequence identity to SEQ ID NO: 2, preferably said xanthan lyase variant having an activity on xanthan gum.
  • an alteration e.g., a substitution,
  • the present invention relates to detergent compositions comprising a xanthan lyase variant having at least 61 %, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 2.
  • the present invention relates to detergent compositions comprising a xanthan lyase variant comprising an alteration (e.g., a substitution, deletion or insertion) at one or more positions in a region selected from the group consisting of:
  • region 7 corresponding to amino acids 1 to 153 of SEQ ID NO: 2, e.g., said alteration at one or more positions selected from the group consisting of positions: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64,
  • region 8 corresponding to amino acids 177 to 613 of SEQ ID NO: 2, e.g., said alteration at one or more positions selected from the group consisting of positions: 177, 178, 179, 180, 181 , 182, 183, 184, 185, 186, 187, 188, 189, 190, 191 , 192, 193, 194, 195, 196, 197, 198, 199, 200, 201 , 202, 203, 204, 205, 206, 207, 208, 209, 210, 211 , 212, 213, 214, 215, 216, 217, 218, 219, 220, 221 ,
  • region 9 corresponding to amino acids 659 to 730 of SEQ ID NO: 2, e.g., said alteration at one or more positions selected from the group consisting of positions: 659, 660, 661 , 662, 663, 664, 665, 666, 667, 668, 669, 670, 671 , 672, 673, 674, 675, 676, 677, 678, 679, 680, 681 , 682, 683, 684, 685, 686, 687, 688, 689, 690, 691 , 692, 693, 694, 695, 696, 697, 698, 699, 700, 701 , 702, 703,
  • region 10 corresponding to amino acids 804 to 806 of SEQ ID NO: 2, e.g., said alteration at one or more positions selected from the group consisting of positions: 804, 805 and 806, wherein said positions correspond to amino acid positions of SEQ ID NO: 2,
  • v) region 11 corresponding to amino acids 847 to 871 of SEQ ID NO: 2, e.g., said alteration at one or more positions selected from the group consisting of positions: 847, 848, 849, 850, 851 , 852, 853, 854, 855, 856, 857, 858, 859, 860, 861 , 862, 863, 864, 865, 866, 867, 868, 869, 870 and 871 , wherein said positions correspond to amino acid positions of SEQ ID NO: 2,
  • region 12 corresponding to amino acids 886 to 902 of SEQ ID NO: 2, e.g., said alteration at one or more positions selected from the group consisting of positions: 886, 887, 888, 889, 890, 891 , 892, 893, 894, 895, 896, 897, 898, 899, 900, 901 and 902, wherein said positions correspond to amino acid positions of SEQ ID NO: 2,
  • region 13 corresponding to amino acids 1005 to 1037 of SEQ ID NO: 2, e.g., said alteration at one or more positions selected from the group consisting of positions: 1005, 1006, 1007, 1008, 1009, 1010, 101 1 , 1012, 1013, 1014, 1015, 1016, 1017, 1018, 1019, 1020, 1021 , 1022, 1023, 1024, 1025, 1026, 1027, 1028, 1029, 1030, 1031 , 1032, 1033, 1034, 1035, 1036 and 1037, wherein said positions correspond to amino acid positions of SEQ ID NO: 2.
  • the present invention relates to detergent compositions comprising a xanthan lyase variant comprising an alteration (e.g., a substitution, deletion or insertion) at one or more positions in two or more regions selected from the group consisting of:
  • region 13 corresponding to amino acids 1005 to 1037 of SEQ ID NO: 2.
  • the present invention relates to detergent compositions comprising a xanthan lyase variant having an alteration (e.g., a substitution, deletion or insertion) at one or more positions selected from the group consisting of positions: 9, 15, 46, 58, 66, 89, 95, 100, 106, 109, 183, 188, 190, 203, 204, 221 , 229, 234, 238, 240, 242, 243, 257, 258, 291 , 293, 316, 320, 324, 329, 333, 339, 341 , 352, 354, 360, 377, 399, 400, 419, 440, 450, 451 , 454, 458, 481 , 492, 567, 568, 578, 579, 582, 664, 672, 703, 728, 843, 855, 887, 892, 1008 and 1016 of SEQ ID NO: 2.
  • an alteration e.g., a substitution, deletion or insertion
  • the present invention relates to detergent compositions comprising a xanthan lyase variant of the invention further having an alteration at one or more positions selected from the group consisting of positions 624, 631 , 635, 649, 656, 752, 752, 754, 757, 769, 775, 777, 800, 801 , 875, 911 , and 915 wherein numbering is according to SEQ ID NO: 2.
  • the present invention relates to detergent compositions comprising a xanthan lyase variant having one or more substitutions selected from the group consisting of: K9R, N15T, L46D, A58L, S66H, Q89Y, K95E, S100D, N106Y, Q109R, Q109D, Q109F, Q109K, Q109A, K183Q, K183R, V188I, A190Q, A203P, K204R, A221 P, E229N, E229S, I234V, I238W, I238L, I238M, I240W, N242S, G243V, Y257W, R258E, K291 R, A293G, A293P, K316R, K320R, L324Q, K329R, K333R, L339M, 1341 P, V352I, S354P, K360R, K360G, F377Y, N399K, K
  • the xanthan lyase variant comprised in the detergent compositions of the invention comprises one of the following set of substitutions:
  • the xanthan lyase variant comprised in the detergent compositions of the invention comprises one of the following set of substitutions:
  • the xanthan lyase variant as described herein is one that does not comprise any amino acid alteration at a position outside of regions 7, 8, 9, 10, 1 1 , 12 and 13.
  • the xanthan lyase variant thus does not comprise any alteration (e.g., a substitution, deletion or insertion) in a region selected from the group consisting of: region 1 corresponding to amino acids 154 to 176 of SEQ ID NO: 2, region 2 corresponding to amino acids 614 to 658 of SEQ ID NO: 2, region 3 corresponding to amino acids 731 to 803 of SEQ ID NO: 2, region 4 corresponding to amino acids 807 to 846 of SEQ ID NO: 2, region 5 corresponding to amino acids 872 to 885 of SEQ ID NO: 2, and region 6 corresponding to amino acids 903 to 1004 of SEQ ID NO: 2.
  • the present invention relates to detergent compositions comprising a xanthan lyase variant having activity on xanthan gum; preferably said activity comprises xanthan lyase EC 4.2.2.12 activity, further preferably said activity is xanthan lyase EC 4.2.2.12 activity.
  • the present invention relates to detergent compositions comprising a xanthan lyase variant having an improved stability in a detergent composition compared to a parent xanthan lyase (e.g., with SEQ ID NO: 2).
  • the present invention relates to detergent compositions comprising a xanthan lyase variant having a half-life improvement factor (HIF) of >1 .0 relative to a parent xanthan lyase.
  • HIF half-life improvement factor
  • the present invention relates to detergent compositions comprising at least one xanthan lyase variant as described herein.
  • the invention relates to detergent compositions comprising a composition comprising an isolated xanthan lyase variant having activity on xanthan gum as described herein.
  • the composition further comprises an isolated polypeptide having GH9 endoglucanase activity.
  • the composition further comprises an isolated polypeptide having xanthan endoglucanase activity.
  • the invention relates to a composition comprising a xanthan lyase variant of the invention and an isolated polypeptide having xanthan endoglucanase activity.
  • a detergent composition of the invention comprises one or more detergent components for degrading xanthan gum.
  • the present invention relates to use of a detergent composition of the present invention, wherein said use is selected from the group consisting of: use for degrading xanthan gum, and use in a cleaning process, such as laundry or hard surface cleaning such as dish wash.
  • the present invention further relates to the use of a detergent composition of the invention for degrading xanthan gum or for washing or cleaning textiles and/or hard surfaces, such as dish wash, wherein the composition has an enzyme detergency benefit. ln some aspects, the present invention also relates to methods of degrading xanthan gum using detergent compositions of the invention, wherein xanthan gum is on the surface of a hard surface or textile.
  • SEQ ID NO: 1 is the DNA sequence of the parent mature xanthan lyase from a strain of a Paenibacillus sp.
  • SEQ ID NO: 2 is the amino acid sequence of the mature polypeptide encoded by SEQ ID NO: 1.
  • cDNA means a DNA molecule that can be prepared by reverse transcription from a mature, spliced, mRNA molecule obtained from a eukaryotic or prokaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA.
  • the initial, primary RNA transcript is a precursor to mRNA that is processed through a series of steps, including splicing, before appearing as mature spliced mRNA.
  • cleaning or detergent application means applying the xanthan lyase of the application in any composition for the purpose of cleaning or washing, by hand, machine or automated, a hard surface or a textile.
  • cleaning composition refers to compositions that find use in the removal of undesired compounds from items to be cleaned, such as textiles, dishes, and hard surfaces.
  • the terms encompass any materials/compounds selected for the particular type of cleaning composition desired and the form of the product (e.g., liquid, gel, powder, granulate, paste, or spray compositions) and includes, but is not limited to, detergent compositions (e.g., liquid and/or solid laundry detergents and fine fabric detergents; hard surface cleaning formulations, such as for glass, wood, ceramic and metal counter tops and windows; carpet cleaners; oven cleaners; fabric fresheners; fabric softeners; and textile and laundry pre- spotters, as well as dish wash detergents).
  • detergent compositions e.g., liquid and/or solid laundry detergents and fine fabric detergents
  • hard surface cleaning formulations such as for glass, wood, ceramic and metal counter tops and windows
  • carpet cleaners oven cleaners
  • fabric fresheners fabric softeners
  • textile and laundry pre- spotters as well as dish wash detergents
  • the detergent formulation may contain one or more additional enzymes (such as xanthan lyases, proteases, amylases, lipases, cutinases, cellulases, xanthan lyases, xyloglucanases, pectinases, pectin lyases, xanthanases, peroxidases, haloperoxygenases, catalases and mannanases, or any mixture thereof), and/or components such as surfactants, builders, chelators or chelating agents, bleach system or bleach components, polymers, fabric conditioners, foam boosters, suds suppressors, dyes, perfume, tannish inhibitors, optical brighteners, bactericides, fungicides, soil suspending agents, anti-corrosion agents, enzyme inhibitors or stabilizers, enzyme activators, transferase(s), hydrolytic enzymes, oxido reductases, bluing agents and fluorescent dyes,
  • additional enzymes such as xanthan ly
  • Coding sequence means a polynucleotide, which directly specifies the amino acid sequence of a polypeptide.
  • the boundaries of the coding sequence are generally determined by an open reading frame, which begins with a start codon such as ATG, GTG, or TTG and ends with a stop codon such as TAA, TAG, or TGA.
  • the coding sequence may be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.
  • Colour clarification During washing and wearing loose or broken fibers can accumulate on the surface of the fabrics. One consequence can be that the colours of the fabric appear less bright or less intense because of the surface contaminations. Removal of the loose or broken fibers from the textile will partly restore the original colours and looks of the textile.
  • colour clarification is meant the partial restoration of the initial colours of textile.
  • control sequences means nucleic acid sequences necessary for expression of a polynucleotide encoding a mature polypeptide of the present invention.
  • Each control sequence may be native (i.e., from the same gene) or foreign (i.e., from a different gene) to the polynucleotide encoding the polypeptide or native or foreign to each other.
  • control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator.
  • the control sequences include a promoter, and transcriptional and trans- lational stop signals.
  • the control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the polynucleotide encoding a polypeptide.
  • corresponding to refers to a way of determining the specific amino acid of a sequence wherein reference is made to a specific amino acid sequence.
  • reference is made to a specific amino acid sequence.
  • the skilled person would be able to align another amino acid sequence to said amino acid sequence that reference has been made to, in order to determine which specific amino acid may be of interest in said another amino acid sequence. Alignment of another amino acid sequence with e.g. the sequence as set forth in SEQ ID NO: 2, or any other sequence listed herein, has been described elsewhere herein. Alternative alignment methods may be used, and are well-known for the skilled person.
  • Degrading xanthan gum and xanthan gum degrading activity are used interchangeably and are defined as the depolymerisation, degradation or breaking down of xanthan gum into smaller components.
  • the degradation of xanthan gum can either be the removal of one or more side chain saccharides, the cutting of the backbone of xanthan gum into smaller components or the removal of one or more side chain saccharides and the cutting of the backbone of xanthan gum into smaller components.
  • a preferred assay for measuring degradation of xanthan gum is described in Example 3 herein.
  • Non-limiting examples of the xanthan gum degrading activity include xanthan lyase EC 4.2.2.12 activity.
  • Detergent component the term "detergent component” is defined herein to mean the types of chemicals which can be used in detergent compositions.
  • detergent components are surfactants, hydrotropes, builders, co-builders, chelators or chelating agents, bleaching system or bleach components, polymers, fabric hueing agents, fabric conditioners, foam boosters, suds suppressors, dispersants, dye transfer inhibitors, fluorescent whitening agents, perfume, optical brighteners, bactericides, fungicides, soil suspending agents, soil release polymers, anti-redeposition agents, enzyme inhibitors or stabilizers, enzyme activators, antioxidants, and solubilizers.
  • the detergent composition may comprise of one or more of any type of detergent component.
  • Detergent composition refers to compositions that find use in the removal of undesired compounds from items to be cleaned, such as textiles, dishes, and hard surfaces.
  • the detergent composition may be used to e.g. clean textiles, dishes and hard surfaces for both household cleaning and industrial cleaning.
  • the terms encompass any materials/compounds selected for the particular type of cleaning composition desired and the form of the product (e.g., liquid, gel, powder, granulate, paste, or spray compositions) and includes, but is not limited to, detergent compositions (e.g., liquid and/or solid laundry detergents and fine fabric detergents; hard surface cleaning formulations, such as for glass, wood, ceramic and metal counter tops and windows; carpet cleaners; oven cleaners; fabric fresheners; fabric softeners; and textile and laundry pre-spotters, as well as dish wash detergents).
  • detergent compositions e.g., liquid and/or solid laundry detergents and fine fabric detergents
  • hard surface cleaning formulations such as for glass, wood, ceramic and metal counter tops and windows
  • carpet cleaners oven cleaners
  • fabric fresheners fabric softeners
  • textile and laundry pre-spotters as well as dish wash detergents
  • the detergent formulation may contain one or more additional enzymes (such as endoglucanases, xanthan lyases, proteases, amylases, lichenases, lipases, cutinases, cellulases, xanthan lyases, xyloglucanases, pectinases, pectin lyases, xanthanases, peroxidases, haloperoxygenases, catalases and mannanases, or any mixture thereof), and/or components such as surfactants, builders, chelators or chelating agents, bleach system or bleach components, polymers, fabric conditioners, foam boosters, suds suppressors, dyes, perfume, tannish inhibitors, optical bright- eners, bactericides, fungicides, soil suspending agents, anti-corrosion agents, enzyme inhibitors or stabilizer
  • additional enzymes such as endoglucanases, xanthan lyases, proteases, amylases, lichen
  • Dish wash refers to all forms of washing dishes, e.g. by hand or automatic dish wash. Washing dishes includes, but is not limited to, the cleaning of all forms of crockery such as plates, cups, glasses, bowls, all forms of cutlery such as spoons, knives, forks and serving utensils as well as ceramics, plastics, metals, china, glass and acrylics.
  • Dish washing composition refers to all forms of compositions for cleaning hard surfaces.
  • the present invention is not restricted to any particular type of dish wash composition or any particular detergent.
  • Endoglucanase means an endo-1 ,4- or endo-1 ,3;1 ,4-beta-D-glu- can 4-glucanohydrolase (e.g., EC 3.2.1.4) that catalyses endohydrolysis of 1 ,4-beta-D-glycosidic linkages in cellulose, cellulose derivatives (such as carboxymethyl cellulose and hydroxyethyl cellulose), lichenin, beta-1 ,4 bonds in mixed beta-1 ,3/beta-1 ,4 glucans such as cereal beta-D-glucans, xyloglucans, xanthans and other plant material containing cellulosic components.
  • EG endo-1 ,4- or endo-1 ,3;1 ,4-beta-D-glu- can 4-glucanohydrolase (e.g., EC 3.2.1.4) that catalyses endohydrolysis of 1 ,4-beta-D-g
  • Endoglucanase activity can be determined by measuring reduction in substrate viscosity or increase in reducing ends determined by a reducing sugar assay (Zhang et al., 2006, Biotechnology Advances 24: 452-481 ).
  • Enzyme detergency benefit The term "enzyme detergency benefit" is defined herein as the advantageous effect an enzyme may add to a detergent compared to the same detergent without the enzyme.
  • Important detergency benefits which can be provided by enzymes are stain removal with no or very little visible soils after washing and or cleaning, prevention or reduction of redeposition of soils released in the washing process an effect that also is termed anti-redeposition, restoring fully or partly the whiteness of textiles, which originally were white but after repeated use and wash have obtained a greyish or yellowish appearance an effect that also is termed whitening.
  • Textile care benefits which are not directly related to catalytic stain removal or prevention of redeposition of soils are also important for enzyme detergency benefits.
  • Examples of such textile care benefits are prevention or reduction of dye transfer from one fabric to another fabric or another part of the same fabric an effect that is also termed dye transfer inhibition or anti- backstaining, removal of protruding or broken fibers from a fabric surface to decrease pilling tendencies or remove already existing pills or fuzz an effect that also is termed anti-pilling, improvement of the fabric- softness, colour clarification of the fabric and removal of particulate soils which are trapped in the fibers of the fabric or garment.
  • Enzymatic bleaching is a further enzyme detergency benefit where the catalytic ac- tivity generally is used to catalyze the formation of bleaching component such as hydrogen peroxide or other peroxides.
  • expression includes any step involved in the production of a polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.
  • Expression vector means a linear or circular DNA molecule that comprises a polynucleotide encoding a polypeptide and is operably linked to control sequences that provide for its expression.
  • fragment means a polypeptide having one or more amino acids absent from the amino and/or carboxyl terminus of a mature polypeptide; wherein the fragment has xanthan lyase activity.
  • a fragment contains at least 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94% or 95% of the number of amino acids of the mature polypeptide.
  • Endoglucanase variant having activity on xanthan gum pre-treated with xanthan lyase The term "Endoglucanase variant having activity on xanthan gum pre-treated with xanthan lyase" or an "endoglucanase having activity on xanthan gum pre-treated with xanthan lyase and belonging to the GH9 class of glycosyl hydrolases" is defined as a polypeptide comprising a domain belonging to the GH9 class of glycosyl hydrolases, and having activity (e.g., enzymatic activity, xanthan degrading activity, endoglucanase EC 3.2.1 .4 activity) on xanthan gum pre-treated with xanthan lyase.
  • activity e.g., enzymatic activity, xanthan degrading activity, endoglucanase EC 3.2.1 .4 activity
  • Xanthan lyase variant having activity on xanthan gum is defined as a polypeptide that cleaves the ⁇ -D-mannosyl- ⁇ -D-1 ,4-glucuronosyl bond of xanthan (e.g., xanthan lyase EC 4.2.2.12 activity).
  • a preferred assay for measuring activity on xan- than gum is disclosed in Example 3 herein.
  • Examples of the xanthan lyase variants having activity on xan- than gum are xanthan lyase polypeptides as such.
  • polypeptides that that cleaves the ⁇ -D-mannosyl- ⁇ -D-l ,4-glucuronosyl bond of xanthan are examples of the xanthan lyase polypeptides as such.
  • Half-life refers to the time it takes for an enzyme to lose half of its enzymatic activity under a given set of conditions. It is denoted as T1 ⁇ 2 and is measured in hours (hrs). Half-lifes can be calculated at a given detergent concentration and storage temperature for parent (e.g. wild-type) and/or variants, as the degradation follows an exponential decay and the incubation time (hours) is known, i.e. according to the following formulas:
  • T112 (wild-type) (Ln (0.5)/Ln (RA-wild-type/100))*Time
  • 'RA' is the residual activity in percent and 'Time' is the incubation time in hours.
  • Half-life improvement factor is the improvement of half-life of a variant compared to the parent polypeptide, such as the parent xanthan lyase.
  • a half-life improvement factor (HIF) under a given set of storage conditions can be calculated as
  • the wild-type (wt) is incubated under the same storage conditions (detergent concentration and incubation temperature) as the variant.
  • the incubation time for wild-type and variant is different e.g. 1 hr for wild-type and 168 hrs for the most stable variants.
  • the half-life improvement factor may also be calculated based on the half-life of a parent xanthan lyase (see the definition of "parent” below) that is not necessarily a wild-type.
  • Hard surface cleaning is defined herein as cleaning of hard surfaces wherein hard surfaces may include floors, tables, walls, roofs etc. as well as surfaces of hard objects such as cars (car wash) and dishes (dish wash). Dish washing includes but are not limited to cleaning of plates, cups, glasses, bowls, and cutlery such as spoons, knives, forks, serving utensils, ceramics, plastics, metals, china, glass and acrylics.
  • host cell means any cell type that is susceptible to transformation, transfection, transduction, or the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention.
  • host cell encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.
  • Improved property means a characteristic associated with a variant that is improved compared to the parent. Such improved properties include, but are not limited to, catalytic efficiency, catalytic rate, chemical stability, oxidation stability, pH activity, pH stability, specific activity, stability under storage conditions, chelator stability, substrate binding, substrate cleavage, substrate specificity, substrate stability, surface properties, thermal activity, and thermostability.
  • Improved wash performance is defined herein as a (variant) enzyme (also a blend of enzymes, not necessarily only variants but also backbones, and in combination with certain cleaning composition etc.) displaying an alteration of the wash performance of a protease variant relative to the wash performance of the parent protease variant e.g. by increased stain removal.
  • wash performance includes wash performance in laundry but also e.g. in dish wash.
  • Isolated means a substance in a form or environment that does not occur in nature.
  • isolated substances include (1 ) any non-naturally occurring substance, (2) any substance including, but not limited to, any enzyme, variant, nucleic acid , protein, peptide or cofactor, that is at least partially removed from one or more or all of the naturally occurring constituents with which it is associated in nature; (3) any substance modified by the hand of man relative to that substance found in nature; or (4) any substance modified by increasing the amount of the substance relative to other components with which it is naturally associated (e.g. , multiple copies of a gene encoding the substance; use of a stronger promoter than the promoter naturally associated with the gene encoding the substance).
  • An isolated substance may be present in a fermentation broth sample.
  • Laundering relates to both household laundering and industrial laundering and means the process of treating textiles with a solution containing a cleaning or detergent composition of the present invention.
  • the laundering process can for example be carried out using e.g. a household or an industrial washing machine or can be carried out by hand .
  • Mature polypeptide means a polypeptide in its final form following translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc.
  • the mature polypeptide is amino acids 1 to 1037 of SEQ ID NO: 2.
  • a host cell may produce a mixture of two of more different mature polypeptides (i.e. , with a different C-terminal and/or N-terminal amino acid) expressed by the same polynucleotide. It is also known in the art that different host cells process polypeptides differently, and thus, one host cell expressing a polynucleotide may produce a different mature polypeptide (e.g. , having a different C-terminal and/or N-terminal amino acid) as compared to another host cell expressing the same polynucleotide.
  • Mature polypeptide coding sequence means a polynucleotide that encodes a mature polypeptide having enzymatic activity such as activity on xanthan gum pre-treated with xanthan lyase or xanthan lyase activity.
  • the mature polypeptide coding sequence is nucleotides 1 to 31 1 1 of SEQ ID NO: 1 .
  • nucleic acid construct means a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or which is synthetic, which comprises one or more control sequences.
  • operably linked means a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide such that the control sequence directs expression of the coding sequence.
  • parent means any polypeptide with xanthan lyase activity to which an alteration is made to produce the enzyme variants as described herein.
  • the parent is a xanthan lyase having the identical amino acid sequence of the variant, but not having the alterations at one or more of the specified positions. It will be understood that the expression “having identical amino acid sequence” relates to 100% sequence identity.
  • Non-limiting examples of parent xanthan lyases include the mature parent xanthan lyase having SEQ ID NO: 2.
  • Sequence identity The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity".
  • sequence identity is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later.
  • the parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.
  • the output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:
  • the sequence identity between two deoxyribonucleotide se- quences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 5.0.0 or later.
  • the parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix.
  • the output of Needle labelled "longest identity" is used as the percent identity and is calculated as follows:
  • Stringency conditions The different stringency conditions are defined as follows.
  • very low stringency conditions means for probes of at least 100 nucleotides in length, pre- hybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 25% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 45°C.
  • low stringency conditions means for probes of at least 100 nucleotides in length, prehybrid- ization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 25% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 50°C.
  • medium stringency conditions means for probes of at least 100 nucleotides in length, pre- hybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 35% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 55°C.
  • medium-high stringency conditions means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 35% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 60°C.
  • high stringency conditions means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 65°C.
  • very high stringency conditions means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 70°C.
  • Subsequence means a polynucleotide having one or more nucleotides absent from the 5' and/or 3' end of a mature polypeptide coding sequence; wherein the subsequence encodes a fragment having enzymatic activity, such as activity on xanthan gum pre-treated with xanthan lyase or xanthan lyase activity.
  • Textile means any textile material including yarns, yarn intermediates, fibers, non- woven materials, natural materials, synthetic materials, and any other textile material, fabrics made of these materials and products made from fabrics (e.g., garments and other articles).
  • the textile or fabric may be in the form of knits, wovens, denims, non-wovens, felts, yarns, and towelling.
  • the textile may be cellulose based such as natural cellulosics, including cotton, flax/linen, jute, ramie, sisal or coir or manmade cellulo- sics (e.g.
  • the textile or fabric may also be non-cellulose based such as natural polyamides including wool, camel, cashmere, mohair, rabbit and silk or synthetic polymer such as nylon, aramid, polyester, acrylic, polypropylene and spandex elastane, or blends thereof as well as blend of cellulose based and non- cellulose based fibers.
  • non-cellulose based such as natural polyamides including wool, camel, cashmere, mohair, rabbit and silk or synthetic polymer such as nylon, aramid, polyester, acrylic, polypropylene and spandex elastane, or blends thereof as well as blend of cellulose based and non- cellulose based fibers.
  • blends are blends of cotton and/or rayon/viscose with one or more companion material such as wool, synthetic fibers (e.g.
  • Fabric may be conventional washable laundry, for example stained household laundry.
  • fabric or garment it is intended to include the broader term textiles as well.
  • Textile care benefits which are not directly related to catalytic stain removal or prevention of redeposition of soils, are also important for enzyme detergency benefits.
  • textile care benefits are prevention or reduction of dye transfer from one textile to another textile or another part of the same textile an effect that is also termed dye transfer inhibition or anti-backstaining, removal of protruding or broken fibers from a textile surface to decrease pilling tendencies or remove already existing pills or fuzz an effect that also is termed anti-pilling, improvement of the textile-softness, colour clarification of the textile and removal of particulate soils which are trapped in the fibers of the textile.
  • Enzymatic bleaching is a further enzyme detergency benefit where the catalytic activity generally is used to catalyse the formation of bleaching component such as hydrogen peroxide or other peroxides or other bleaching species.
  • variant means a polypeptide (e.g., a xanthan lyase polypeptide) comprising an alteration, i.e., a substitution, insertion, and/or deletion, at one or more positions.
  • a substitution means replacement of the amino acid occupying a position with a different amino acid;
  • a deletion means removal of the amino acid occupying a position;
  • an insertion means adding one or more amino acids, e.g., 1 -5 amino acids adjacent to and immediately following the amino acid occupying a position.
  • xanthan lyase variants as described herein include xanthan lyase variants having an activity on xanthan gum.
  • Non-limiting examples of variants as described herein further include variants having at least 20%, e.g., at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% xanthan lyase activity of the mature parent xanthan lyase of SEQ ID NO: 2.
  • a preferred assay for measuring activity on xanthan gum is disclosed in Example 3 herein.
  • Stability means resistance or the degree of resistance to change, unfolding, dis- integration, denaturation or activity loss.
  • Non-limiting examples of stability include conformational stability, storage stability and stability during use, e.g. during a wash process and reflects the stability of a polypeptide (e.g. a xanthan lyase variant according to the invention) as a function of time, e.g. how much activity is retained when said polypeptide (e.g. said xanthan lyase variant) is kept in solution, in particular in a detergent solution.
  • the stability is influenced by many factors, e.g. presence of chelator(s), pH, temperature, detergent composition, e.g.
  • the xanthan lyase stability may be measured using a half-life improvement factor (HIF) as described in Example 3 herein, e.g. determined relative to the xanthan lyase having SEQ ID NO: 2.
  • HIF half-life improvement factor
  • Improved stability is defined herein as increased stability in a detergent composition (e.g. , in solutions), relative to the stability of the parent xanthan lyase, relative to a xanthan lyase having the identical amino acid sequence of the variant, but not having the alterations at one or more of the specified positions, or relative to SEQ ID NO: 2.
  • improved stability and “increased stability” include “improved chemical stability”, “detergent stability” and "improved detergent stability”.
  • Improved chemical stability is defined herein as a variant enzyme displaying retention of enzymatic activity after a period of incubation in the presence of a chemical or chemicals, either naturally occurring or synthetic, which reduces the enzymatic activity of the parent enzyme. Improved chemical stability may also result in variants being more able (e.g., better that the parent) to catalyze a reaction in the presence of such chemicals.
  • the improved chemical stability is an improved stability in a detergent, in particular in a liquid detergent.
  • detergent stability or “improved detergent stability is in particular an improved stability of the xanthan lyase compared to the parent xanthan lyase, when a xanthan lyase variant of the present invention is mixed into a liquid detergent formulation.
  • Conformational stability means a resistance or a degree of resistance to conformational change, unfolding or disintegration. Accordingly, the term “less conformationally stable” means less resistant or having lesser degree of resistance to conformational change, unfolding or disintegration.
  • instability means lack of stability.
  • instability include conformational instability, unfolding, denaturation, disintegration, activity loss.
  • Wash performance is used as an enzyme's ability to remove stains present on the object to be cleaned during e.g. wash or hard surface cleaning.
  • the improvement in the wash performance may be quantified by calculating the so-called intensity value (Int) in 'Automatic Mechanical Stress Assay (AMSA) for laundry' or the remission value (Rem).
  • the mature polypeptide disclosed in SEQ ID NO: 2 is used to determine the corresponding amino acid residue in another xanthan lyase.
  • the amino acid sequence of another xanthan lyase is aligned with the mature polypeptide disclosed in SEQ ID NO: 2, and based on the alignment, the amino acid position number corresponding to any amino acid residue in the mature polypeptide disclosed in SEQ ID NO: 2 is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice , 2000, e Trteanl.ds Genet. 16: 276-277), preferably version 5.0.0 or later.
  • the parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.
  • Identification of the corresponding amino acid residue in another xanthan lyase can be determined by an alignment of multiple polypeptide sequences using several computer programs including, but not limited to, MUSCLE (multiple sequence comparison by log-expectation; version 3.5 or later; Edgar, 2004, Nucleic
  • proteins of known structure For proteins of known structure, several tools and resources are available for retrieving and generating structural alignments. For example, the SCOP superfamilies of proteins have been structurally aligned, and those alignments are accessible and downloadable.
  • Two or more protein structures can be aligned using a variety of algorithms such as the distance alignment matrix (Holm and Sander, 1998, Proteins 33: 88-96) or combinatorial extension (Shindyalov and Bourne, 1998, Protein Engineering 1 1 : 739-747), and implementation of these algorithms can additionally be utilized to query structure databases with a structure of interest in order to discover possible structural homologs (e.g., Holm and Park, 2000, Bioinformatics 16: 566-567).
  • Insertions For an amino acid insertion, the following nomenclature is used: Original amino acid, position, original amino acid, inserted amino acid. Accordingly, the insertion of lysine after glycine at position 195 is designated “Gly195GlyLys" or “G195GK”. An insertion of multiple amino acids is designated [Original amino acid, position, original amino acid, inserted amino acid #1 , inserted amino acid #2; etc.]. For example, the insertion of lysine and alanine after glycine at position 195 is indicated as "Gly195GlyLysAla" or "G195GKA”. An indication of an insertion at a particular position is understood as being an insertion after the original amino acid residue. For example, an "insertion at position 195" is understood to be an insertion after the original residue in position 195.
  • the inserted amino acid residue(s) are numbered by the addition of lower case letters to the position number of the amino acid residue preceding the inserted amino acid residue(s).
  • the sequence would thus be:
  • Variants comprising multiple alterations are separated by addition marks ("+"), e.g., "Arg170Tyr+Gly195Glu” or “R170Y+G195E” representing a substitution of arginine and glycine at positions 170 and 195 with tyrosine and glutamic acid, respectively.
  • Variants comprising multiple alterations are separated by a comma (",”), e.g., "R170Y,G195E”.
  • brackets e.g., Arg170[Tyr,Gly] or in one- letter code R170 [Y,G].
  • the known xanthan lyase having SEQ ID NO: 2 is a large enzyme (>1000 residues), it is therefore extremely laborious and expensive to target its properties for improvement of, e.g., stability in a detergent composition.
  • the present invention narrows down the number of residues to target when trying to stabilize xanthan lyase molecules using protein engineering to a region selected from the group consisting of: region 7 corresponding to amino acids 1 to 153 of SEQ ID NO: 2, region 8 corresponding to amino acids 177 to 613 of SEQ ID NO: 2, region 9 corresponding to amino acids 659 to 730 of SEQ ID NO: 2, region 10 corresponding to amino acids 804 to 806 of SEQ ID NO: 2, region 1 1 corresponding to amino acids 847 to 871 of SEQ ID NO: 2, region 12 corresponding to amino acids 886 to 902 of SEQ ID NO: 2, and region 13 corresponding to amino acids 1005 to 1037 of SEQ ID NO: 2.
  • the present invention dramatically narrows down the number of residues to target when trying to stabilize xanthan lyase molecules using protein engineering.
  • the regions in the protein sequence of the known xanthan lyase having SEQ ID NO: 2 that have an impact on stability of the molecule, e.g. during storage in a liquid detergent composition are the following: region 7 corresponding to amino acids 1 to 153 of SEQ ID NO: 2, region 8 corresponding to amino acids 177 to 613 of SEQ ID NO: 2, region 9 corresponding to amino acids 659 to 730 of SEQ ID NO: 2, region 10 corresponding to amino acids 804 to 806 of SEQ ID NO: 2, region 11 corresponding to amino acids 847 to 871 of SEQ ID NO: 2, region 12 corresponding to amino acids 886 to 902 of SEQ ID NO: 2, and region 13 corresponding to amino acids 1005 to 1037 of SEQ ID NO: 2.
  • This embodiment relates to an important guidance on where to mutate a xanthan lyase in order to stabilize the molecule in a deter- gent.
  • the present invention relates to detergent compositions comprising a xanthan lyase variant, comprising an alteration (e.g., a substitution, deletion or insertion) at one or more positions in a region selected from the group consisting of: region 7 corresponding to amino acids 1 to 153 of SEQ ID NO: 2, region 8 corresponding to amino acids 177 to 613 of SEQ ID NO: 2, region 9 corresponding to amino acids 659 to 730 of SEQ ID NO: 2, region 10 corresponding to amino acids 804 to 806 of SEQ ID NO: 2, region 1 1 corresponding to amino acids 847 to 871 of SEQ ID NO: 2, region 12 corresponding to amino acids 886 to 902 of SEQ ID NO: 2, and region 13 corresponding to amino acids 1005 to 1037 of SEQ ID NO: 2, wherein said variant has at least 60% and less than 100% sequence identity to SEQ ID NO: 2; preferably said xanthan lyase variant has activity on xanthan gum, further preferably said activity is a xan-
  • the present invention relates to detergent compositions comprising a xanthan lyase variant, comprising an alteration (e.g., a substitution, deletion or insertion) at one or more positions in a region selected from the group consisting of:
  • region 9 corresponding to amino acids 659 to 730 of SEQ ID NO: 2
  • region 10 corresponding to amino acids 804 to 806 of SEQ ID NO: 2
  • region 13 corresponding to amino acids 1005 to 1037 of SEQ ID NO: 2.
  • the present invention relates to detergent compositions comprising a xanthan lyase variant, comprising an alteration (e.g. , a substitution, deletion or insertion) at one or more positions in two or more regions selected from the group consisting of: region 7 corresponding to amino acids 1 to 153 of SEQ ID NO: 2, region 8 corresponding to amino acids 177 to 613 of SEQ ID NO: 2, region 9 corresponding to amino acids 659 to 730 of SEQ ID NO: 2, region 10 corresponding to amino acids 804 to 806 of SEQ ID NO: 2, region 1 1 corresponding to amino acids 847 to 871 of SEQ ID NO: 2, region 12 corresponding to amino acids 886 to 902 of SEQ ID NO: 2, and region 13 corresponding to amino acids 1005 to 1037 of SEQ ID NO: 2, wherein said variant has at least 60% and less than 100% sequence identity to SEQ ID NO: 2; preferably said xanthan lyase variant has activity on xanthan gum, further preferably said activity is a x
  • the present invention relates to detergent compositions comprising a xanthan lyase variant as described herein having multiple alterations (such as 2, 3, 4, 5, 6, 7, 8, 9 or 10) in one region (e.g. , of SEQ ID NO: 2 or another parent xanthan lyase) selected from the group consisting of: region 7 corresponding to amino acids 1 to 153 of SEQ ID NO: 2, region 8 corresponding to amino acids 177 to 613 of SEQ ID NO: 2, region 9 corresponding to amino acids 659 to 730 of SEQ ID NO: 2, region 10 correspond- ing to amino acids 804 to 806 of SEQ ID NO: 2, region 1 1 corresponding to amino acids 847 to 871 of SEQ ID NO: 2, region 12 corresponding to amino acids 886 to 902 of SEQ ID NO: 2, and region 13 corresponding to amino acids 1005 to 1037 of SEQ ID NO: 2, wherein said variant has at least 60% and less than 100% sequence identity to SEQ ID NO: 2, preferably said variant has activity on xanthan
  • the present invention relates to detergent compositions comprising a xanthan lyase variant as described herein having multiple alterations (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10) in multiple regions (e.g., 2, 3, 4, 5, 6 or 7) (e.g., of SEQ ID NO: 2 or another parent xanthan lyase) selected from the group consisting of: region 7 corresponding to amino acids 1 to 153 of SEQ ID NO: 2, region 8 corresponding to amino acids 177 to 613 of SEQ ID NO: 2, region 9 corresponding to amino acids 659 to 730 of SEQ ID NO: 2, region 10 corresponding to amino acids 804 to 806 of SEQ ID NO: 2, region 1 1 corresponding to amino acids 847 to 871 of SEQ ID NO: 2, region 12 corresponding to amino acids 886 to 902 of SEQ ID NO: 2, and region 13 corresponding to amino acids 1005 to 1037 of SEQ ID NO: 2, wherein said variant has at least 60% and less than 100% sequence identity to SEQ ID NO: 2
  • the present invention relates to detergent compositions comprising xanthan lyase variants, comprising an alteration (e.g., a substitution, deletion or insertion) at one or more positions of the mature parent polypeptide (e.g., SEQ ID NO: 2), wherein each alteration is independently a substitution, insertion or deletion, wherein the variant has xanthan lyase activity.
  • an alteration e.g., a substitution, deletion or insertion
  • the variant has sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, to the amino acid sequence of the parent xanthan lyase.
  • the variant has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to the mature polypeptide of SEQ ID NO: 2.
  • the present invention relates to detergent compositions comprising a xanthan lyase variant as described herein, having at least 61 %, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 2.
  • a variant comprises an alteration at one or more positions corresponding to positions 9, 15, 46, 58, 66, 89, 95, 100, 106, 109, 183, 188, 190, 203, 204, 221 , 229, 234, 238, 240, 242, 243, 257, 258, 291 , 293, 316, 320, 324, 329, 333, 339, 341 , 352, 354, 360, 377, 399, 400, 419, 440, 450, 451 , 454, 458, 481 , 492, 567, 568, 578, 579, 582, 664, 672, 703, 728, 843, 855, 887, 892, 1008 and 1016.
  • a variant comprises an alteration at two positions corresponding to any of positions 9, 15, 46, 58, 66, 89, 95, 100, 106, 109, 183, 188, 190, 203, 204, 221 , 229, 234, 238, 240, 242, 243, 257, 258, 291 , 293, 316, 320, 324, 329, 333, 339, 341 , 352, 354, 360, 377, 399, 400, 419, 440, 450, 451 , 454, 458, 481 , 492, 567, 568, 578, 579, 582, 664, 672, 703, 728, 843, 855, 887, 892, 1008 and 1016.
  • a variant comprises an alteration at three positions corresponding to any of positions 9, 15, 46, 58, 66, 89, 95, 100, 106, 109, 183, 188, 190, 203, 204, 221 , 229, 234, 238, 240, 242, 243, 257, 258, 291 , 293, 316, 320, 324, 329, 333, 339, 341 , 352, 354, 360, 377, 399, 400, 419, 440, 450, 451 , 454, 458, 481 , 492, 567, 568, 578, 579, 582, 664, 672, 703, 728, 843, 855, 887, 892, 1008 and 1016.
  • a variant com- prises an alteration at four or more positions, e.g. five, six, seven, eight, nine, ten or more positions, corresponding to positions 9, 15, 46, 58, 66, 89, 95, 100, 106, 109, 183, 188, 190, 203, 204, 221 , 229, 234, 238, 240, 242, 243, 257, 258, 291 , 293, 316, 320, 324, 329, 333, 339, 341 , 352, 354, 360, 377, 399, 400, 419, 440, 450, 451 , 454, 458, 481 , 492, 567, 568, 578, 579, 582, 664, 672, 703, 728, 843, 855, 887, 892, 1008 and 1016.
  • the variant comprises or consists of an alteration at a position corresponding to position
  • the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.
  • the variant comprises or consists of the substitution K9R of the mature polypeptide of SEQ ID NO: 2.
  • the variant comprises or consists of an alteration at a position corresponding to position 15.
  • the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.
  • the variant comprises or consists of the substitution N15T of the mature polypeptide of SEQ ID NO: 2.
  • the variant comprises or consists of an alteration at a position corresponding to position 46.
  • the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.
  • the variant comprises or consists of the substitution L46D of the mature polypeptide of SEQ ID NO: 2.
  • the variant comprises or consists of an alteration at a position corresponding to position 58.
  • the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.
  • the variant comprises or consists of the substitution A58L of the mature polypeptide of SEQ ID NO: 2.
  • the variant comprises or consists of an alteration at a position corresponding to position
  • the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.
  • the variant comprises or consists of the substitution S66H of the mature polypeptide of SEQ ID NO: 2.
  • the variant comprises or consists of an alteration at a position corresponding to position 89.
  • the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.
  • the variant comprises or consists of the substitution Q89Y of the mature polypeptide of SEQ ID NO: 2.
  • the variant comprises or consists of an alteration at a position corresponding to position 95.
  • the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.
  • the variant comprises or consists of the substitution K95E of the mature polypeptide of SEQ ID NO: 2.
  • the variant comprises or consists of an alteration at a position corresponding to position 100.
  • the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.
  • the variant comprises or consists of the substitution S100D of the mature polypeptide of SEQ ID NO: 2.
  • the variant comprises or consists of an alteration at a position corresponding to position 106.
  • the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.
  • the variant comprises or consists of the substitution N106Y of the mature polypeptide of SEQ ID NO: 2.
  • the variant comprises or consists of an alteration at a position corresponding to position
  • the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.
  • the variant comprises or consists of the substitution Q109R, Q109D, Q109F, Q109K or Q109A of the mature polypeptide of SEQ ID NO: 2.
  • a preferred substitution at a position corresponding to position 109 is Q109R.
  • the variant comprises or consists of an alteration at a position corresponding to position 183.
  • the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.
  • the variant comprises or consists of the substitution K183Q or K183R of the mature polypeptide of SEQ ID NO: 2.
  • the variant comprises or consists of an alteration at a position corresponding to position 188.
  • the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.
  • the variant comprises or consists of the substitution V188I of the mature polypeptide of SEQ ID NO: 2.
  • the variant comprises or consists of an alteration at a position corresponding to position 190.
  • the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.
  • the variant comprises or consists of the substitution A190Q of the mature polypeptide of SEQ ID NO: 2.
  • the variant comprises or consists of an alteration at a position corresponding to position
  • the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.
  • the variant comprises or consists of the substitution A203P of the mature polypeptide of SEQ ID NO: 2.
  • the variant comprises or consists of an alteration at a position corresponding to position
  • the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.
  • the variant comprises or consists of the substitution K204R of the mature polypeptide of SEQ ID NO: 2.
  • the variant comprises or consists of an alteration at a position corresponding to position 221.
  • the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.
  • the variant comprises or consists of the substitution A221 P of the mature polypeptide of SEQ ID NO: 2.
  • the variant comprises or consists of an alteration at a position corresponding to position 229.
  • the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.
  • the variant comprises or consists of the substitution E229N or E229S of the mature polypeptide of SEQ ID NO: 2.
  • the variant comprises or consists of an alteration at a position corresponding to position 234.
  • the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.
  • the variant comprises or consists of the substitution I234V of the mature polypeptide of SEQ ID NO: 2.
  • the variant comprises or consists of an alteration at a position corresponding to position 238.
  • the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.
  • the variant comprises or consists of the substitution I238W, I238L or I238M of the mature polypeptide of SEQ ID NO: 2.
  • Preferred substitutions at a position corresponding to position 238 are I238W and I238L.
  • the variant comprises or consists of an alteration at a position corresponding to position
  • the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.
  • the variant comprises or consists of the substitution I240W of the mature polypeptide of SEQ ID NO: 2.
  • the variant comprises or consists of an alteration at a position corresponding to position 242.
  • the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.
  • the variant comprises or consists of the substitution N242S of the mature polypeptide of SEQ ID NO: 2.
  • the variant comprises or consists of an alteration at a position corresponding to position 243.
  • the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.
  • the variant comprises or consists of the substitution G243V of the mature polypeptide of SEQ ID NO: 2.
  • the variant comprises or consists of an alteration at a position corresponding to position
  • the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.
  • the variant comprises or consists of the substitution Y257W of the mature polypeptide of SEQ ID NO: 2.
  • the variant comprises or consists of an alteration at a position corresponding to position
  • the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.
  • the variant comprises or consists of the substitution R258E of the mature polypeptide of SEQ ID NO: 2.
  • the variant comprises or consists of an alteration at a position corresponding to position
  • the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.
  • the variant comprises or consists of the substitution K291 R of the mature polypeptide of SEQ ID NO: 2.
  • the variant comprises or consists of an alteration at a position corresponding to position 293.
  • the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.
  • the variant comprises or consists of the substitution A293G or A293P of the mature polypeptide of SEQ ID NO: 2.
  • the variant comprises or consists of an alteration at a position corresponding to position 316.
  • the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.
  • the variant comprises or consists of the substitution K316R of the mature polypeptide of SEQ ID NO: 2. ln one aspect, the variant comprises or consists of an alteration at a position corresponding to position 320.
  • the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.
  • the variant comprises or consists of the substitution K320R of the mature polypeptide of SEQ ID NO: 2.
  • the variant comprises or consists of an alteration at a position corresponding to position
  • the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.
  • the variant comprises or consists of the substitution L324Q of the mature polypeptide of SEQ ID NO: 2.
  • the variant comprises or consists of an alteration at a position corresponding to position 329.
  • the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.
  • the variant comprises or consists of the substitution K329R of the mature polypeptide of SEQ ID NO: 2.
  • the variant comprises or consists of an alteration at a position corresponding to position 333.
  • the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.
  • the variant comprises or consists of the substitution K333R of the mature polypeptide of SEQ ID NO: 2.
  • the variant comprises or consists of an alteration at a position corresponding to position 339.
  • the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.
  • the variant comprises or consists of the substitution L339M of the mature polypeptide of SEQ ID NO: 2.
  • the variant comprises or consists of an alteration at a position corresponding to position 341.
  • the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.
  • the variant comprises or consists of the substitution 1341 P of the mature polypeptide of SEQ ID NO: 2.
  • the variant comprises or consists of an alteration at a position corresponding to position
  • the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.
  • the variant comprises or consists of the substitution V352I of the mature polypeptide of SEQ ID NO: 2.
  • the variant comprises or consists of an alteration at a position corresponding to position 354.
  • the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.
  • the variant comprises or consists of the substitution S354P of the mature polypeptide of SEQ ID NO: 2.
  • the variant comprises or consists of an alteration at a position corresponding to position 360.
  • the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.
  • the variant comprises or consists of the substitution K360G or K360R of the mature polypeptide of SEQ ID NO: 2.
  • the variant comprises or consists of an alteration at a position corresponding to position 377.
  • the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.
  • the variant comprises or consists of the substitution F377Y of the mature polypeptide of SEQ ID NO: 2.
  • the variant comprises or consists of an alteration at a position corresponding to position
  • the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.
  • the variant comprises or consists of the substitution N399K of the mature polypeptide of SEQ ID NO: 2.
  • the variant comprises or consists of an alteration at a position corresponding to position 400.
  • the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.
  • the variant comprises or consists of the substitution K400R of the mature polypeptide of SEQ ID NO: 2.
  • the variant comprises or consists of an alteration at a position corresponding to position 419.
  • the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.
  • the variant comprises or consists of the substitution F419Y of the mature polypeptide of SEQ ID NO: 2.
  • the variant comprises or consists of an alteration at a position corresponding to position 440.
  • the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.
  • the variant comprises or consists of the substitution N440K of the mature polypeptide of SEQ ID NO: 2.
  • the variant comprises or consists of an alteration at a position corresponding to position
  • the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.
  • the variant comprises or consists of the substitution D450P of the mature polypeptide of SEQ ID NO: 2.
  • the variant comprises or consists of an alteration at a position corresponding to position
  • the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.
  • the variant comprises or consists of the substitution K451 E or K451 R of the mature polypeptide of SEQ ID NO: 2.
  • the variant comprises or consists of an alteration at a position corresponding to position 454.
  • the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.
  • the variant comprises or consists of the substitution A454V of the mature polypeptide of SEQ ID NO: 2.
  • the variant comprises or consists of an alteration at a position corresponding to position 458.
  • the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.
  • the variant comprises or consists of the substitution D458S of the mature polypeptide of SEQ ID NO: 2. ln one aspect, the variant comprises or consists of an alteration at a position corresponding to position 481.
  • the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.
  • the variant comprises or consists of the substitution K481 R of the mature polypeptide of SEQ ID NO: 2.
  • the variant comprises or consists of an alteration at a position corresponding to position
  • the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.
  • the variant comprises or consists of the substitution A492L of the mature polypeptide of SEQ ID NO: 2.
  • the variant comprises or consists of an alteration at a position corresponding to position 567.
  • the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.
  • the variant comprises or consists of the substitution K567R of the mature polypeptide of SEQ ID NO: 2.
  • the variant comprises or consists of an alteration at a position corresponding to position 568.
  • the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.
  • the variant comprises or consists of the substitution G568A of the mature polypeptide of SEQ ID NO: 2.
  • the variant comprises or consists of an alteration at a position corresponding to position
  • the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.
  • the variant comprises or consists of the substitution S578K or S578R of the mature polypeptide of SEQ ID NO: 2.
  • the variant comprises or consists of an alteration at a position corresponding to position
  • the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.
  • the variant comprises or consists of the substitution S579R or S579K of the mature polypeptide of SEQ ID NO: 2.
  • a preferred substitution at a position corresponding to position 579 is S579R.
  • the variant comprises or consists of an alteration at a position corresponding to position 582.
  • the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.
  • the variant comprises or consists of the substitution S582K of the mature polypeptide of SEQ ID NO: 2.
  • the variant comprises or consists of an alteration at a position corresponding to position
  • the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.
  • the variant comprises or consists of the substitution T664K of the mature polypeptide of SEQ ID NO: 2.
  • the variant comprises or consists of an alteration at a position corresponding to position 672.
  • the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.
  • the variant comprises or consists of the substitution N672D of the mature polypeptide of SEQ ID NO: 2.
  • the variant comprises or consists of an alteration at a position corresponding to position 703.
  • the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.
  • the variant comprises or consists of the substitution I703L of the mature polypeptide of SEQ ID NO: 2.
  • the variant comprises or consists of an alteration at a position corresponding to position 728.
  • the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.
  • the variant comprises or consists of the substitution M728V of the mature polypeptide of SEQ ID NO: 2.
  • the variant comprises or consists of an alteration at a position corresponding to position
  • the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.
  • the variant comprises or consists of the substitution A843P of the mature polypeptide of SEQ ID NO: 2.
  • the variant comprises or consists of an alteration at a position corresponding to position 885.
  • the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.
  • the variant comprises or consists of the substitution K855R of the mature polypeptide of SEQ ID NO: 2.
  • the variant comprises or consists of an alteration at a position corresponding to position 887.
  • the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.
  • the variant comprises or consists of the substitution K887R of the mature polypeptide of SEQ ID NO: 2.
  • the variant comprises or consists of an alteration at a position corresponding to position 892.
  • the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.
  • the variant comprises or consists of the substitution N892Y, N892W or N892F of the mature polypeptide of SEQ ID NO: 2.
  • a preferred substitution at a position corresponding to position 892 is N892Y.
  • the variant comprises or consists of an alteration at a position corresponding to position 1008.
  • the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.
  • the variant comprises or consists of the substitution N1008D of the mature polypeptide of SEQ ID NO: 2.
  • the variant comprises or consists of an alteration at a position corresponding to position 1016.
  • the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.
  • the variant comprises or consists of the substitution K1016T of the mature polypeptide of SEQ ID NO: 2.
  • the present invention relates to detergent compositions comprising a xanthan lyase variant as described herein, having an alteration at one or more positions selected from the group consisting of positions: 9, 15, 46, 58, 66, 89, 95, 100, 106, 109, 183, 188, 190, 203, 204, 221 , 229, 234, 238, 240, 242, 243, 257, 258, 291 , 293, 316, 320, 324, 329, 333, 339, 341 , 352, 354, 360, 377, 399, 400, 419, 440, 450, 451 , 454, 458, 481 , 492, 567, 568, 578, 579, 582, 664, 672, 703, 728, 843, 855, 887, 892, 1008 and 1016 of SEQ ID NO: 2, wherein each position corresponds to the positions of SEQ ID NO: 2.
  • the xanthan lyase variant comprised in the detergent compositions of the invention further comprises an alteration at one or more positions selected from the group consisting of: 624, 631 , 635, 649, 656, 752, 752, 754, 757, 769, 775, 777, 800, 801 , 875, 911 , and 915 wherein numbering is according to SEQ ID NO: 2.
  • the present invention relates to detergent compositions comprising a xanthan ly- ase variant as described herein having one or more substitutions selected from the group consisting of: K9R, N15T, L46D, A58L, S66H, Q89Y, K95E, S100D, N106Y, Q109R, Q109D, Q109F, Q109K, Q109A, K183Q,K183R, V188I, A190Q, A203P, K204R, A221 P, E229N, E229S, I234V, I238W, I238L, I238M, I240W, N242S, G243V, Y257W, R258E, K291 R, A293G, A293P, K316R, K320R, L324Q, K329R, K333R, L339M, 1341 P, V352I, S354P, K360G, K360R, F377Y, N3
  • the invention relates to detergent compositions comprising a xanthan lyase variant selected from the group consisting of the xanthan lyase variants set forth in Table 1 herein.
  • the invention relates to detergent compositions comprising a xanthan lyase variant selected from the group consisting of the xanthan lyase variants set forth in Table 2 herein.
  • the invention relates to detergent compositions comprising a xanthan lyase variant selected from the group consisting of the xanthan lyase variants set forth in Table 3 herein.
  • the invention relates to detergent compositions comprising a xanthan lyase variant selected from the group consisting of the xanthan lyase variants set forth in Table 4 herein.
  • the variants may further comprise one or more additional alterations at one or more other positions in regions 7, 8, 9, 10, 1 1 , 12 and/or 13.
  • amino acid changes may be of a minor nature, that is conservative amino acid substitutions or insertions that do not significantly affect the folding and/or activity of the protein; small deletions, typically of 1-30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to 20-25 residues; or a small extension that facilitates purification by changing net charge or another function, such as a poly-histidine tract, an antigenic epitope or a binding domain.
  • conservative substitutions are within the groups of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and aspara- gine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, threonine and methionine).
  • Amino acid substitutions that do not generally alter specific activity are known in the art and are described, for example, by H. Neurath and R.L. Hill, 1979, In, The Proteins, Academic Press, New York.
  • amino acid changes are of such a nature that the physico-chemical properties of the polypeptides are altered.
  • amino acid changes may improve the thermal stability of the poly- peptide, alter the substrate specificity, change the pH optimum, and the like.
  • Essential amino acids in a polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081 -1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for xanthan lyase activity to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271 : 4699-4708.
  • the active site of the enzyme or other biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al. , 1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver ef a/., 1992, FEBS Lett. 309: 59-64.
  • the identity of essential amino acids can also be inferred from an alignment with a related polypeptide.
  • the present invention relates to detergent compositions comprising a xanthan lyase variant as described herein, having a total number of alterations compared to SEQ ID NO: 2 between 1 and 20, e.g., between 1 and 10 or between 1 and 5, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 alterations.
  • the present invention relates to detergent compositions comprising a xanthan lyase variant as described herein, having an activity on xanthan gum, preferably said activity on xanthan gum is a xanthan gum degrading activity, further preferably said xanthan gum degrading activity is EC 4.2.2.12 activity.
  • the variant has an improved stability in a detergent composition compared to a parent enzyme (e.g., SEQ ID NO: 2).
  • the present invention relates to detergent compositions comprising a xanthan lyase variant as described herein, having an improved stability in a detergent composition compared to the parent xanthan lyase (e.g., with SEQ ID NO: 2).
  • the present invention relates to detergent compositions comprising a xanthan ly- ase variant as described herein, having a half-life improvement factor (HIF) of >1 .0; preferably having a half- life improvement factor (HIF) of >1.0, preferably at least 1.2, such as at least 1 .5, e.g. at least 2.0, relative to a parent xanthan lyase.
  • HIF half-life improvement factor
  • HIF half-life improvement factor
  • the present invention relates to detergent compositions comprising a xanthan lyase variant as described herein, wherein a half-life improvement factor (HIF) is determined after incubation of said xanthan lyase variant in a detergent composition at 25°C or 30°C for a time period from about 30 min to about 20 hours.
  • HIF half-life improvement factor
  • the parent xanthan lyase may be (a) a polypeptide having at least 60% sequence identity to the mature polypeptide of SEQ ID NO: 2; (b) a polypeptide encoded by a polynucleotide that hybridizes under low stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 1 , or (ii) the full-length complement of (i); or (c) a polypeptide encoded by a polynucleotide having at least 60% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 1.
  • the parent has a sequence identity to the mature polypeptide of SEQ ID NO: 2 of at least 60%, e.g. , at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, which have xanthan lyase activity.
  • the amino acid sequence of the parent differs by up to 10 amino acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the mature polypeptide of SEQ ID NO: 2.
  • the parent comprises or consists of the amino acid sequence of SEQ ID NO: 2. In another aspect, the parent comprises or consists of the mature polypeptide of SEQ ID NO: 2. In another aspect, the parent is a fragment of the mature polypeptide of SEQ ID NO: 2 containing at least 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94% or 95% of the number of amino acids of SEQ ID NO: 2. In another embodiment, the parent is an allelic variant of the mature polypeptide of SEQ ID NO: 2.
  • the parent is encoded by a polynucleotide that hybridizes under very low stringency conditions, low stringency conditions, medium stringency conditions, medium-high stringency conditions, high stringency conditions, or very high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 1 , or (ii) the full-length complement of (i) (Sambrook , 1989, Moleceutlaalr. Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, New York).
  • polypeptide of SEQ ID NO: 1 The polynucleotide of SEQ ID NO: 1 or a subsequence thereof, as well as the polypeptide of SEQ ID NO:
  • nucleic acid probes may be used to design nucleic acid probes to identify and clone DNA encoding a parent from strains of different genera or species according to methods well known in the art.
  • probes can be used for hybridization with the genomic DNA or cDNA of a cell of interest, following standard Southern blotting procedures, in order to identify and isolate the corresponding gene therein.
  • Such probes can be considerably shorter than the entire sequence, but should be at least 15, e.g., at least 25, at least 35, or at least 70 nucleotides in length.
  • the nucleic acid probe is at least 100 nucleotides in length, e.g.
  • Both DNA and RNA probes can be used.
  • the probes are typically labeled for detecting the corresponding gene (for example, with 32 P, 3 H, 35 S, biotin, or avidin). Such probes are encompassed by the present invention.
  • a genomic DNA or cDNA library prepared from such other strains may be screened for DNA that hybridizes with the probes described above and encodes a parent.
  • Genomic or other DNA from such other strains may be separated by agarose or polyacrylamide gel electrophoresis, or other separation techniques.
  • DNA from the libraries or the separated DNA may be transferred to and immobilized on nitrocellulose or other suitable carrier material.
  • the carrier material is used in a Southern blot.
  • hybridization indicates that the polynucleotide hybridizes to a labeled nucleic acid probe corresponding to (i) SEQ ID NO: 1 ; (ii) the mature polypeptide coding sequence of SEQ ID NO: 1 ; (iii) the full-length complement thereof; or (iv) a subsequence thereof; under very low to very high stringency conditions.
  • Molecules to which the nucleic acid probe hybridizes under these conditions can be detected using, for example, X-ray film or any other detection means known in the art.
  • the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 1.
  • the nucleic acid probe is a polynucleotide that encodes the polypeptide of SEQ ID NO: 2; the mature polypeptide thereof; or a fragment thereof.
  • the nucleic acid probe is SEQ ID NO: 1.
  • the parent is encoded by a polynucleotide having a sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 1 of at least 60%, e.g. , at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%.
  • the polypeptide may be a hybrid polypeptide in which a region of one polypeptide is fused at the N-terminus or the C-terminus of a region of another polypeptide.
  • the parent may be a fusion polypeptide or cleavable fusion polypeptide in which another polypeptide is fused at the N-terminus or the C-terminus of the polypeptide of the present invention.
  • a fusion polypeptide is produced by fusing a polynucleotide encoding another polypeptide to a polynucleotide of the present invention.
  • Techniques for producing fusion polypeptides are known in the art, and include ligating the coding sequences encoding the polypeptides so that they are in frame and that expression of the fusion polypeptide is under control of the same promoter(s) and terminator.
  • Fusion polypeptides may also be constructed using intein technology in which fusion polypeptides are created post-translationally (Cooper , 1993, EMBeOt al. J. 12: 2575-2583; Dawson et al. , 1994, Science 266: 776-779).
  • a fusion polypeptide can further comprise a cleavage site between the two polypeptides. Upon secretion of the fusion protein, the site is cleaved releasing the two polypeptides.
  • cleavage sites include, but are not limited to, the sites disclosed in Martinet al. , 2003, J. Ind. Microbiol. Biotechnol. 3: 568- 576; Svetina et al., 2000, J. Biotechnol. 76: 245-251 ; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol.
  • the parent may be obtained from microorganisms of any genus.
  • the term "obtained from” as used herein in connection with a given source shall mean that the parent encoded by a polynucleotide is produced by the source or by a strain in which the polynucleotide from the source has been inserted.
  • the parent is secreted extracellularly.
  • the parent may be a bacterial enzyme.
  • the parent may be a Gram-positive bacterial polypeptide such as a Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, or Streptomyces enzyme, or a Gram-negative bacterial polypeptide such as a Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, llyobacter, Neisseria, Pseudomonas, Salmonella, or Ureaplasma enzyme.
  • the parent is a Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus li- cheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillus thuringiensis enzyme.
  • the parent is a Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equi subsp. zooepidemicus enzyme.
  • the parent is a Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, or Streptomyces lividans enzyme.
  • the parent may be a fungal enzyme.
  • the parent may be a yeast enzyme such as a Candida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia enzyme; or a filamentous fungal enzyme such as an Acremonium, Agaricus, Alternaria, Aspergillus, Aureobasidium, Botry- ospaeria, Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Cop- totermes, Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium, Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex, Lentinula, Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mu- cor, Myceliophthora, Neocallimastix,
  • the parent is a Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccha- romyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, or Saccharomyces oviformis enzyme.
  • the parent is an Acremonium cellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium luck- nowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chryso- sporium tropicum, Chrysosporium zonatum, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwel- lense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum,
  • the parent is a Paenibacillus sp. xanthan lyase, e.g. , the xanthan lyase of SEQ ID NO: 2.
  • the invention encompasses both the perfect and imperfect states, and other taxonomic equivalents, e.g., anamorphs, regardless of the species name by which they are known. Those skilled in the art will readily recognize the identity of appropriate equivalents.
  • ATCC American Type Culture Collection
  • DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH
  • CBS Centraalbureau Voor Schimmelcultures
  • NRRL Northern Regional Research Center
  • the parent may be identified and obtained from other sources including microorganisms isolated from nature (e.g., soil, composts, water, etc.) or DNA samples obtained directly from natural materials (e.g. , soil, composts, water, etc.) using the above-mentioned probes. Techniques for isolating microorganisms and DNA directly from natural habitats are well known in the art. A polynucleotide encoding a parent may then be obtained by similarly screening a genomic DNA or cDNA library of another microorganism or mixed DNA sample.
  • the polynucleotide can be isolated or cloned by utilizing techniques that are known to those of ordinary skill in the art (see, e.g., Sambrook et al, 1989, supra). Preparation of variants
  • Methods for obtaining a variant having xanthan lyase activity may comprise: (a) introducing into a parent xanthan lyase an alteration at one or more positions corresponding to positions 9, 15, 46, 58, 66, 89, 95, 100, 106, 109, 183, 188, 190, 203, 204, 221 , 229, 234, 238, 240, 242, 243, 257, 258, 291 , 293, 316, 320, 324, 329, 333, 339, 341 , 352, 354, 360, 377, 399, 400, 419, 440, 450, 451 , 454, 458, 481 , 492, 567, 568, 578, 579, 582, 664, 672, 703, 728, 843, 855, 887, 892, 1008 and 1016 of the mature polypeptide of SEQ ID NO: 2, wherein the variant has xanthan lyase activity; and (b
  • Methods for obtaining a variant having xanthan lyase activity may further comprise introducing an alteration (e.g., a substitution, deletion or insertion) at one or more positions corresponding to positions 624, 631 , 635, 649, 656, 752, 752, 754, 757, 769, 775, 777, 800, 801 , 875, 91 1 , and 915 wherein numbering is according to SEQ ID NO: 2.
  • an alteration e.g., a substitution, deletion or insertion
  • the variants can be prepared using any mutagenesis procedure known in the art, such as site-directed mutagenesis, synthetic gene construction, semi-synthetic gene construction, random mutagenesis, shuffling, etc.
  • Site-directed mutagenesis is a technique in which one or more mutations are introduced at one or more defined sites in a polynucleotide encoding the parent.
  • Site-directed mutagenesis can be accomplished in vitro by PCR involving the use of oligonucleotide primers containing the desired mutation. Site-directed mutagenesis can also be performed in vitro by cassette mutagenesis involving the cleavage by a restriction enzyme at a site in the plasmid comprising a polynucleotide encoding the parent and subsequent ligation of an oligonucleotide containing the mutation in the polynucleotide. Usually the restriction enzyme that digests the plasmid and the oligonucleotide is the same, permitting sticky ends of the plasmid and the insert to ligate to one another. See, e.g. , Scherer and Davis, 1979, Proc. Natl. Acad. Sci. USA 76: 4949-4955; and Barton ei al., 1990, Nucleic Acids Res. 18: 7349-4966.
  • Site-directed mutagenesis can also be accomplished in vivo by methods known in the art. See, e.g., U.S. Patent Application Publication No. 2004/0171154; Storici ei al., 2001 , Nature Biotechnol. 19: 773-776; Kren et al., 1998, Nat. Med. 4: 285-290; and Calissano and Macino, 1996, Fungal Genet. Newslett. 43: 15- 16.
  • Any site-directed mutagenesis procedure can be used in the present invention.
  • Synthetic gene construction entails in vitro synthesis of a designed polynucleotide molecule to encode a polypeptide of interest. Gene synthesis can be performed utilizing a number of techniques, such as the multiplex microchip-based technology described by Tian et al. (2004, Nature 432: 1050-1054) and similar technologies wherein oligonucleotides are synthesized and assembled upon photo-programmable microflu- idic chips.
  • Single or multiple amino acid substitutions, deletions, and/or insertions can be made and tested using known methods of mutagenesis, recombination, and/or shuffling, followed by a relevant screening procedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241 : 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413; or WO 95/22625.
  • Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al. , 1991 , Biochemistry 30: 10832-10837; U.S. Patent No. 5,223,409; WO 92/06204) and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner et al. , 1988, DNA 7: 127).
  • Mutagenesis/shuffling methods can be combined with high-throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides expressed by host cells (Ness et al. , 1999, Nature Biotechnology 17: 893-896). Mutagenized DNA molecules that encode active polypeptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide.
  • Semi-synthetic gene construction is accomplished by combining aspects of synthetic gene construction, and/or site-directed mutagenesis, and/or random mutagenesis, and/or shuffling.
  • Semi-synthetic construction is typified by a process utilizing polynucleotide fragments that are synthesized, in combination with PCR techniques. Defined regions of genes may thus be synthesized de novo, while other regions may be amplified using site-specific mutagenic primers, while yet other regions may be subjected to error-prone PCR or non-error prone PCR amplification. Polynucleotide subsequences may then be shuffled.
  • the present invention relates to detergent compositions comprising at least one (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10) xanthan lyase variant as described herein.
  • the present invention relates to detergent compositions comprising at least one
  • composition further comprises one or more detergent components.
  • the present invention relates to detergent compositions comprising at least one (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10) xanthan lyase variant as described herein, further comprising one or more additional enzymes selected from the group comprising or consisting of: endoglucanases, proteases, amylases, lipases, cutinases, cellulases, xanthan lyases, xyloglucanases, pectinases, pectin lyases, xantha- nases, peroxidases, haloperoxygenases, catalases and mannanases, or any mixture thereof.
  • additional enzymes selected from the group comprising or consisting of: endoglucanases, proteases, amylases, lipases, cutinases, cellulases, xanthan lyases, xyloglucanases, pectinases, pectin lyases, x
  • the present invention relates to detergent compositions comprising at least one (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10) xanthan lyase variant as described herein, further comprising one or more detergent components, wherein said detergent composition is in form of a bar, a homogenous tablet, a tablet having two or more layers, a pouch having one or more compartments, a regular or compact powder, a granule, a paste, a gel, or a regular, compact or concentrated liquid.
  • the present invention relates to use of a detergent composition of the invention, wherein said use is selected from the group comprising or consisting of: use for degrading xanthan gum and use in a cleaning process, such as laundry or hard surface cleaning such as dish wash.
  • the present invention relates to use of a detergent composition of the invention, wherein said composition has an enzyme detergency benefit.
  • the present invention relates to a method for degrading xanthan gum comprising: applying a detergent composition of the invention to a xanthan gum.
  • the present invention relates to a method for degrading xanthan gum comprising: applying a detergent composition of the invention to a xanthan gum, wherein said xanthan gum is on the surface of a textile or hard surface, such as dish wash.
  • Said polynucleotides encoding a xanthan lyase variant comprise an alteration at one or more positions selected from the group consisting of: i) region 7 corresponding to amino acids 1 to 153 of SEQ ID NO: 2, ii) region 8 corresponding to amino acids 177 to 613 of SEQ ID NO: 2, iii) region 9 corresponding to amino acids 659 to 730 of SEQ ID NO: 2, iv) region 10 corresponding to amino acids 804 to 806 of SEQ ID NO: 2, v) region 11 corresponding to amino acids 847 to 871 of SEQ ID NO: 2, vi) region 12 corresponding to amino acids 886 to 902 of SEQ ID NO: 2, and vii) region 13 corresponding to amino acids 1005 to 1037 of SEQ ID NO: 2, wherein said variant has at least 60% and less than 100% sequence identity to SEQ ID NO: 2.
  • the techniques used to isolate or clone a polynucleotide include isolation from genomic DNA or cDNA, or a combination thereof.
  • the cloning of the polynucleotides from genomic DNA can be effected, e.g., by using the well-known polymerase chain reaction (PCR) or antibody screening of expression libraries to detect cloned DNA fragments with shared structural features. See, e.g. , Innis ef a/., 1990, PCR: A Guide to Methods and Application, Academic Press, New York.
  • PCR polymerase chain reaction
  • nucleic acid amplification procedures such as ligase chain reaction (LCR), ligation activated transcription (LAT) and polynucleo- tide-based amplification (NASBA) may be used.
  • LCR ligase chain reaction
  • LAT ligation activated transcription
  • NASBA polynucleo- tide-based amplification
  • the polynucleotides may be cloned in a strain of Bacillus subtilis or E. coli, or a related organism and thus, for example, may be an allelic or species variant of the polypeptide encoding region of the polynucleotide.
  • Modification of a polynucleotide encoding a polypeptide may be necessary for synthesizing polypeptides substantially similar to the polypeptide.
  • the term "substantially similar" to the polypeptide refers to non- naturally occurring forms of the polypeptide. These polypeptides may differ in some engineered way from the polypeptide isolated from its native source, e.g. , variants that differ in specific activity, thermostability, pH optimum, or the like.
  • the variants may be constructed on the basis of the polynucleotide presented as the mature polypeptide coding sequence of SEQ ID NO: 1 , e.g., a subsequence thereof, and/or by introduction of nucleotide substitutions that do not result in a change in the amino acid sequence of the polypeptide, but which correspond to the codon usage of the host organism intended for production of the enzyme, or by introduction of nucleotide substitutions that may give rise to a different amino acid sequence.
  • nucleotide substitution see, Ford (1991)l., 'Protein Expression and Purification', 2: 95-
  • nucleic acid constructs comprising a polynucleotide encoding a variant as described herein operably linked to one or more control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.
  • nucleic acid constructs comprising a polynucleotide encoding a xanthan lyase variant, comprising an alteration at one or more positions selected from the group consisting of: i) region 7 corresponding to amino acids 1 to 153 of SEQ ID NO: 2, ii) region 8 corresponding to amino acids 177 to 613 of SEQ ID NO: 2, iii) region 9 corresponding to amino acids 659 to 730 of SEQ ID NO: 2, iv) region 10 corresponding to amino acids 804 to 806 of SEQ ID NO: 2, v) region 11 corresponding to amino acids 847 to 871 of SEQ ID NO: 2, vi) region 12 corresponding to amino acids 886 to 902 of SEQ ID NO: 2, and vii) region 13 corresponding to amino acids 1005 to 1037 of SEQ ID NO: 2, wherein said variant has at least 60% and less than 100% sequence identity to SEQ ID NO: 2 operably linked to one or more control sequences that direct the expression of the coding sequence
  • a polynucleotide may be manipulated in a variety of ways to provide for expression of the polypeptide.
  • Manipulation of the polynucleotide prior to its insertion into a vector may be desirable or necessary depending on the expression vector.
  • the techniques for modifying polynucleotides utilizing recombinant DNA methods are well known in the art.
  • the control sequence may be a promoter, a polynucleotide that is recognized by a host cell for expres- sion of a polynucleotide encoding a polypeptide of the present invention.
  • the promoter contains transcriptional control sequences that mediate the expression of the polypeptide.
  • the promoter may be any polynucleotide that shows transcriptional activity in the host cell including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.
  • suitable promoters for directing transcription of the nucleic acid constructs of the present invention in a bacterial host cell are the promoters obtained from the Bacillus amyloliquefaciens alpha- amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus licheniformis penicillinase gene (penP), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus subtilis levansu- crase gene (sacB), Bacillus subtilis xylA and xylB genes, Bacillus thuringiensis crylllA gene (Agaisse and Lereclus, 1994, Molecular Microbiology 13: 97-107), E.
  • E. coli lac operon E. coli trc promoter (Egon ei al. , 1988, Gene 69: 301-315), Streptomyces coelicolor agarase gene (dagA), and prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731 ), as well as the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80: 21 -25). Further promoters are described in "Useful proteins from recombinant bacteria" in Gilbert et al. , 1980, Scientific American 242: 74-94; and in Sambrook et al., 1989, supra. Examples of tandem promoters are disclosed in WO 99/43835.
  • promoters for directing transcription of the nucleic acid constructs of the present invention in a filamentous fungal host cell are promoters obtained from the genes for Aspergillus nidulans acetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Fusarium oxysporum trypsin-like protease (WO 96/00787), Fusarium venenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Daria (WO 00/56900), Fusarium venenatum Quinn (
  • useful promoters are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1 ), Saccharomyces cerevisiae galactokinase (GAL1 ), Saccharomyces cerevisiae alcohol dehydro- genase/glyceraldehyde-3-phosphate dehydrogenase (ADH1 , ADH2/GAP), Saccharomyces cerevisiae tri- ose phosphate isomerase (TPI), Saccharomyces cerevisiae metallothionein (CUP1 ), and Saccharomyces cerevisiae 3-phosphoglycerate kinase.
  • ENO-1 Saccharomyces cerevisiae enolase
  • GAL1 Saccharomyces cerevisiae galactokinase
  • ADH1 glyceraldehyde-3-phosphate dehydrogenase
  • TPI Saccharomyces cerevisiae
  • the control sequence may also be a transcription terminator, which is recognized by a host cell to terminate transcription.
  • the terminator is operably linked to the 3'-terminus of the polynucleotide encoding the polypeptide. Any terminator that is functional in the host cell may be used in the present invention.
  • Preferred terminators for bacterial host cells are obtained from the genes for Bacillus clausii alkaline protease (aprH), Bacillus licheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA (rrnB).
  • Preferred terminators for filamentous fungal host cells are obtained from the genes for Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase, Aspergil- lus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.
  • Preferred terminators for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1 ), and Saccharomyces cerevisiae glyceralde- hyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are described by Romanos et al., 1992, supra.
  • the control sequence may also be an mRNA stabilizer region downstream of a promoter and upstream of the coding sequence of a gene which increases expression of the gene.
  • suitable mRNA stabilizer regions are obtained from a Bacillus thuringiensis crylllA gene (WO 94/25612) and a Bacillus subtilis SP82 gene (Hue et al. , 1995, Journal of Bacteriology 177: 3465- 3471 ).
  • the control sequence may also be a leader, a nontranslated region of an mRNA that is important for translation by the host cell.
  • the leader is operably linked to the 5'-terminus of the polynucleotide encoding the polypeptide. Any leader that is functional in the host cell may be used.
  • Preferred leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.
  • Suitable leaders for yeast host cells are obtained from the genes for Saccharomyces cerevisiae eno- lase (ENO-1 ), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha- factor, and Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).
  • the control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3'-terminus of the polynucleotide and, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence that is functional in the host cell may be used.
  • Preferred polyadenylation sequences for filamentous fungal host cells are obtained from the genes for Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger alpha-gluco- sidase Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.
  • the control sequence may also be a signal peptide coding region that encodes a signal peptide linked to the N-terminus of a polypeptide and directs the polypeptide into the cell's secretory pathway.
  • the 5'-end of the coding sequence of the polynucleotide may inherently contain a signal peptide coding sequence naturally linked in translation reading frame with the segment of the coding sequence that encodes the polypeptide.
  • the 5'-end of the coding sequence may contain a signal peptide coding sequence that is foreign to the coding sequence.
  • a foreign signal peptide coding sequence may be required where the coding sequence does not naturally contain a signal peptide coding sequence.
  • a foreign signal peptide coding sequence may simply replace the natural signal peptide coding sequence in order to enhance secretion of the polypeptide.
  • any signal peptide coding sequence that directs the expressed polypeptide into the secretory pathway of a host cell may be used.
  • Effective signal peptide coding sequences for bacterial host cells are the signal peptide coding sequences obtained from the genes for Bacillus NCIB 1 1837 maltogenic amylase, Bacillus licheniformis sub- tilisin, Bacillus licheniformis beta-lactamase, Bacillus stearothermophilus alpha-amylase, Bacillus stea- rothermophilus neutral proteases (nprT, nprS, nprM), and Bacillus subtilis prsA. Further signal peptides are described by Simonen and Palva, 1993, Microbiological Reviews 57: 109-137.
  • Effective signal peptide coding sequences for filamentous fungal host cells are the signal peptide coding sequences obtained from the genes for Aspergillus niger neutral amylase, Aspergillus n/ger glucoamyl- ase, Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicola insolens xanthan lyase V, Humicola lanuginosa lipase, and Rhizomucor miehei aspartic proteinase.
  • Useful signal peptides for yeast host cells are obtained from the genes for Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase. Other useful signal peptide coding sequences are described by Romanos et al., 1992, supra.
  • the control sequence may also be a propeptide coding sequence that encodes a propeptide positioned at the N-terminus of a polypeptide.
  • the resultant polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases).
  • a propolypeptide is generally inactive and can be converted to an active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide.
  • the propeptide coding sequence may be obtained from the genes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor miehei aspartic proteinase, and Saccharomyces cerevisiae alpha-factor.
  • the propeptide sequence is positioned next to the N-terminus of a polypeptide and the signal peptide sequence is positioned next to the N-terminus of the propeptide sequence.
  • regulatory sequences that regulate expression of the polypeptide relative to the growth of the host cell.
  • regulatory systems are those that cause expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound.
  • Regulatory systems in prokaryotic systems include the lac, tac, and trp operator systems.
  • yeast the ADH2 system or GAL1 system may be used.
  • filamentous fungi the Aspergillus niger glucoamylase promoter, Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzae glu- coamylase promoter may be used.
  • Other examples of regulatory sequences are those that allow for gene amplification.
  • these regulatory sequences include the dihydrofolate reductase gene that is amplified in the presence of methotrexate, and the metallothionein genes that are amplified with heavy metals.
  • the polynucleotide encoding the polypeptide would be operably linked with the regulatory sequence.
  • recombinant expression vectors comprising a polynucleotide encoding a variant as described herein, a promoter, and transcriptional and translational stop signals. Accordingly, also disclosed are recombinant expression vectors comprising a polynucleotide encoding a xanthan lyase variant, comprising an alteration at one or more positions selected from the group consisting of: i) region 7 corresponding to amino acids 1 to 153 of SEQ ID NO: 2, ii) region 8 corresponding to amino acids 177 to 613 of SEQ ID NO: 2, iii) region 9 corresponding to amino acids 659 to 730 of SEQ ID NO: 2, iv) region 10 corresponding to amino acids 804 to 806 of SEQ ID NO: 2, v) region 1 1 corresponding to amino acids 847 to 871 of SEQ ID NO: 2, vi) region 12 corresponding to amino acids 886 to 902 of SEQ ID NO: 2, and vii) region 13 corresponding to amino acids 100
  • the various nucleotide and control sequences may be joined together to produce a recombinant expression vector that may include one or more convenient restriction sites to allow for insertion or substitution of the polynucleotide encoding the polypeptide at such sites.
  • the polynucleotide may be ex- pressed by inserting the polynucleotide or a nucleic acid construct comprising the polynucleotide into an appropriate vector for expression.
  • the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.
  • the recombinant expression vector may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can bring about expression of the polynucleotide.
  • the choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced.
  • the vector may be a linear or closed circular plasmid.
  • the vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromoso- mal entity, the replication of which is independent of chromosomal replication, e.g. , a plasmid, an extrachro- mosomal element, a minichromosome, or an artificial chromosome.
  • the vector may contain any means for assuring self-replication.
  • the vector may be one that, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated.
  • a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, or a transposon may be used.
  • the vector preferably contains one or more selectable markers that permit easy selection of trans- formed, transfected, transduced, or the like cells.
  • a selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like.
  • bacterial selectable markers are Bacillus licheniformis or Bacillus subtilis dal genes, or markers that confer antibiotic resistance such as ampicillin, chloramphenicol, kanamycin, neomycin, spec- tinomycin, or tetracycline resistance.
  • Suitable markers for yeast host cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1 , and URA3.
  • Selectable markers for use in a filamentous fungal host cell include, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents thereof.
  • Preferred for use in an Aspergillus cell are Aspergillus nidulans or Aspergillus oryzae amdS and pyrG genes and a Streptomyces hygroscopicus bar gene.
  • the vector preferably contains an element(s) that permits integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.
  • the vector may rely on the polynucleotide's sequence encoding the polypeptide or any other element of the vector for integration into the genome by homologous or non-homologous recombination.
  • the vector may contain additional polynucleotides for direct- ing integration by homologous recombination into the genome of the host cell at a precise location(s) in the chromosome(s).
  • the integrational elements should contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000 base pairs, and 800 to 10,000 base pairs, which have a high degree of sequence identity to the corresponding target sequence to enhance the probability of homologous recombination.
  • the integrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell.
  • the integrational elements may be non-encoding or encoding polynucleotides.
  • the vector may be integrated into the genome of the host cell by non-homologous recombination.
  • the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question.
  • the origin of replication may be any plasmid replicator mediating autonomous replication that functions in a cell.
  • the term "origin of replication" or "plasmid replicator” means a polynucleotide that enables a plasmid or vector to replicate in vivo.
  • bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permitting replication in E. coli, and pUB1 10, pE194, pTA1060, and ⁇ permitting replication in Bacillus.
  • origins of replication for use in a yeast host cell are the 2 micron origin of replication,
  • ARS1 ARS4
  • CEN3 the combination of ARS1 and CEN3
  • ARS4 and CEN6 the combination of ARS4 and CEN6
  • AMA1 and ANSI examples of origins of replication useful in a filamentous fungal cell are AMA1 and ANSI (Gems ef a/., 1991 , Gene 98: 61-67; Cullen et al., 1987, Nucleic Acids Res. 15: 9163-9175; WO 00/24883). Isolation of the AMA1 gene and construction of plasmids or vectors comprising the gene can be accomplished accord- ing to the methods disclosed in WO 00/24883.
  • More than one copy of a polynucleotide of the present invention may be inserted into a host cell to increase production of a polypeptide.
  • An increase in the copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the polynucleotide where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the polynucleotide, can be selected for by cultivating the cells in the presence of the appropriate selectable agent.
  • the procedures used to ligate the elements described above to construct the recombinant expression vectors of the present invention are well known to one skilled in the art (see, e.g., Sambrook , 1989, et al. supra). Host cells
  • recombinant host cells comprising a polynucleotide encoding a variant as described herein operably linked to one or more control sequences that direct the production of a variant of the present invention. Accordingly, disclosed herein are recombinant host cells, comprising a polynucleotide encoding a xanthan lyase variant, comprising an alteration at one or more positions selected from the group consisting of: i) region 7 corresponding to amino acids 1 to 153 of SEQ ID NO: 2, ii) region 8 corresponding to amino acids 177 to 613 of SEQ ID NO: 2, iii) region 9 corresponding to amino acids 659 to 730 of SEQ
  • region 10 corresponding to amino acids 804 to 806 of SEQ ID NO: 2
  • region 1 1 corresponding to amino acids 847 to 871 of SEQ ID NO: 2
  • region 12 corresponding to amino acids 886 to 902 of SEQ
  • region 13 corresponding to amino acids 1005 to 1037 of SEQ ID NO: 2, wherein said variant has at least 60% and less than 100% sequence identity to SEQ ID NO: 2 operably linked to one or more control sequences that direct the production of a xanthan lyase variant, comprising an alteration at one or more positions selected from the group consisting of: i) region 7 corresponding to amino acids 1 to
  • region 8 corresponding to amino acids 177 to 613 of SEQ ID NO: 2
  • region 9 corresponding to amino acids 659 to 730 of SEQ ID NO: 2
  • region 10 corresponding to amino acids 804 to 806 of SEQ ID NO: 2
  • region 11 corresponding to amino acids 847 to 871 of SEQ ID NO: 2
  • region 13 corresponding to amino acids 1005 to 1037 of SEQ ID NO: 2, wherein said variant has at least 60% and less than 100% sequence identity to SEQ ID NO: 2.
  • a construct or vector comprising a polynucleotide is introduced into a host cell so that the construct or vector is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector as described earlier.
  • the term "host cell” encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication. The choice of a host cell will to a large extent depend upon the gene encoding the polypeptide and its source.
  • the host cell may be any cell useful in the recombinant production of a polypeptide of the present invention, e.g., a prokaryote or a eukaryote.
  • the prokaryotic host cell may be any Gram-positive or Gram-negative bacterium.
  • Gram-positive bacteria include, but are not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lacto- coccus, Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces.
  • Gram-negative bacteria include, but are not limited to, Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, llyobac- ter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.
  • the bacterial host cell may be any Bacillus cell including, but not limited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus fir- mus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.
  • Bacillus alkalophilus Bacillus amyloliquefaciens
  • Bacillus brevis Bacillus circulans
  • Bacillus clausii Bacillus coagulans
  • Bacillus fir- mus Bacillus lautus
  • Bacillus lentus Bacillus licheniformis
  • Bacillus megaterium Bacillus pumilus
  • Bacillus stearothermophilus Bacillus
  • the bacterial host cell may also be any Streptococcus cell including, but not limited to, Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equi subsp. zooepidemicus cells.
  • the bacterial host cell may also be any Streptomyces cell including, but not limited to, Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, and Streptomy- ces lividans cells.
  • the introduction of DNA into a Bacillus cell may be effected by protoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen. Genet. 168: 11 1-1 15), competent cell transformation (see, e.g., Young and Spizizen, 1961 , J. Bacteriol. 81 : 823-829, or Dubnau and Davidoff-Abelson, 1971 , J. Mol. Biol. 56: 209- 221 ), electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751 ), or conjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169: 5271-5278).
  • protoplast transformation see, e.g., Chang and Cohen, 1979, Mol. Gen. Genet. 168: 11 1-1 15
  • competent cell transformation see, e.g., Young and Spizizen, 1961 , J. Bacteriol. 81
  • the introduction of DNA into an E. coli cell may be effected by protoplast transformation (see, e.g. , Hanahan, 1983, J. Mol. Biol. 166: 557-580) or electroporation (see, e.g., Dower ei ai, 1988, Nucleic Acids Res. 16: 6127-6145).
  • the introduction of DNA into a Streptomyces cell may be effected by protoplast transformation, electroporation (see, e.g., Gong ei a/., 2004, Folia Microbiol. (Praha) 49: 399-405), conjugation (see, e.g. , Mazodier et al , 1989, J. Bacteriol.
  • DNA into a Pseudomonas cell may be effected by electroporation (see, e.g., Choi et al, 2006, J. Microbiol. Methods 64: 391 -397) or conjugation (see, e.g., Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71 : 51 -57).
  • the introduction of DNA into a Streptococcus cell may be effected by natural competence (see, e.g., Perry and Kuramitsu, 1981 , Infect. Immun. 32: 1295-1297), protoplast transformation (see, e.g., Catt and Jollick, 1991 , Microbios 68: 189-207), electroporation (see, e.g., Buckley et al , 1999, Appl. Environ. Microbiol. 65: 3800-3804), or conjugation (see, e.g., Clewell, 1981 , Microbiol. Rev. 45: 409-436).
  • any method known in the art for introducing DNA into a host cell can be used.
  • the host cell may also be a eukaryote, such as a mammalian, insect, plant, or fungal cell.
  • the host cell may be a fungal cell.
  • "Fungi” as used herein includes the phyla Ascomycota, Basidiomy- cota, Chytridiomycota, and Zygomycota as well as the Oomycota and all mitosporic fungi (as defined by Hawksworth et al, In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK).
  • the fungal host cell may be a yeast cell.
  • yeast as used herein includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes). Since the classification of yeast may change in the future, for the purposes of this invention, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, Passmore, and Davenport, editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).
  • the yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schiz- osaccharomyces, or Yarrowia cell, such as a Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytica cell.
  • the fungal host cell may be a filamentous fungal cell.
  • "Filamentous fungi” include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra).
  • the filamentous fungi are generally characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic. In contrast, vegetative growth by yeasts such as Saccharomyces cerevisiae is by budding of a unicellular thallus and carbon catabolism may be fermentative.
  • the filamentous fungal host cell may be an Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Mag- naporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cell.
  • the filamentous fungal host cell may be an Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium in- ops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium
  • Fungal cells may be transformed by a process involving protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se. Suitable procedures for transformation of Aspergillus and Trichoderma host cells are described in EP 238023, Yelton , 1984, Proetc.al. Natl. Acad. Sci. USA 81 : 1470-1474, and Christensen et al., 1988, Bio/Technology 6: 1419-1422. Suitable methods for transforming Fusarium species are described by Malardier , 1989, Geentea 7l.8: 147-156, and WO 96/00787. Yeast may be transformed using the procedures described by Becker and Guarente, In Abelson, J.N.
  • Also disclosed are methods of producing (e.g., in vitro or ex vivo methods) a variant comprising: (a) cultivating a host cell of the present invention under conditions suitable for expression of the variant; and (b) recovering the variant.
  • methods of producing (e.g., in vitro or ex vivo methods) a variant comprising: (a) cultivating a host cell comprising a polynucleotide encoding a xan- than lyase variant, comprising an alteration at one or more positions selected from the group consisting of: i) region 7 corresponding to amino acids 1 to 153 of SEQ ID NO: 2, ii) region 8 corresponding to amino acids 177 to 613 of SEQ ID NO: 2, iii) region 9 corresponding to amino acids 659 to 730 of SEQ ID NO: 2, iv) region 10 corresponding to amino acids 804 to 806 of SEQ ID NO: 2, v) region 1 1 corresponding to amino acids 847 to 871
  • a variant of the present invention comprising (a) cultivating a cell, which in its wild-type form produces the polypeptide, under con- ditions conducive for production of the polypeptide; and (b) recovering the polypeptide.
  • the cell is a Paenibacillus cell, or a Microbacterium cell.
  • the host cells are cultivated in a nutrient medium suitable for production of the polypeptide using methods known in the art.
  • the cell may be cultivated by shake flask cultivation, or small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermenters performed in a suitable medium and under conditions allowing the polypeptide to be expressed and/or isolated.
  • the cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g. , in catalogues of the American Type Culture Collection). If the polypeptide is secreted into the nutrient medium, the polypeptide can be recovered directly from the medium. If the polypeptide is not secreted, it can be recovered from cell lysates.
  • the variant polypeptide may be detected using methods known in the art that are specific for the polypeptides such as methods for determining cellulose or xanthan lyase activity. These detection methods include, but are not limited to, use of specific antibodies, formation of an enzyme product, or disappearance of an enzyme substrate. For example, an enzyme assay may be used to determine the activity of the polypeptide.
  • the variant polypeptide may be recovered using methods known in the art.
  • the polypep- tide may be recovered from the nutrient medium by conventional procedures including, but not limited to, collection, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation.
  • the variant polypeptide may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g. , ammo- nium sulfate precipitation), SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson and Ryden, editors, VCH Publishers, New York, 1989) to obtain substantially pure polypeptides.
  • chromatography e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion
  • electrophoretic procedures e.g., preparative isoelectric focusing
  • differential solubility e.g. , ammo- nium sulfate precipitation
  • SDS-PAGE SDS-PAGE
  • extraction see, e.g., Protein Purification, Janson
  • the variant polypeptide is not recovered, but rather a host cell expressing the polypeptide is used as a source of the variant polypeptide.
  • the variants according to the invention have improved stability in detergent compositions compared to a parent enzyme or compared to a xanthan lyase having the identical amino acid sequence of the variant, but not having an alteration (e.g., a substitution, deletion or insertion) at one or more of the specified positions or compared to the xanthan lyase with SEQ ID NO: 2, wherein activity and/or stability in detergent is measured as disclosed in Example 3 herein.
  • an alteration e.g., a substitution, deletion or insertion
  • the detergent compositions may comprise additional components.
  • additional components is within the skill of the artisan and includes conventional ingredients, including the exemplary non-limiting components set forth below.
  • the choice of components may include, for fabric care, the consideration of the type of fabric to be cleaned, the type and/or degree of soiling, the temperature at which cleaning is to take place, and the formulation of the detergent product.
  • components mentioned below are categorized by general header according to a particular functionality, this is not to be construed as a limitation, as a component may comprise additional functionalities as will be appreciated by the skilled artisan.
  • the detergent composition may be suitable for the laundering of textiles such as e.g. fabrics, cloths or linen, or for cleaning hard surfaces such as e.g. floors, tables, or dish wash.
  • a variant as described herein may be added to a detergent composition in an amount corresponding to 0.0001-200 mg of enzyme protein, such as 0.0005-100 mg of enzyme protein, preferably 0.001-30 mg of enzyme protein, more preferably 0.005-8 mg of enzyme protein, even more pref- erably 0.01 -2 mg of enzyme protein per litre of wash liquor.
  • a composition for use in automatic dishwash (ADW), for example, may include 0.0001 %-50%, such as 0.001 %-20%, such as 0.01 %-10%, such as 0.05-5% of enzyme protein by weight of the composition.
  • a composition for use in laundry granulation or a solid/granular laundry composition in general may include 0.0001 %-50%, such as 0.001 %-20%, such as 0.01 %-10%, such as 0.05%-5% of enzyme protein by weight of the composition.
  • a composition for use in laundry liquid may include 0.0001 %-10%, such as 0.001-7%, such as 0.1 %-5% of enzyme protein by weight of the composition.
  • the enzyme(s) of the detergent composition of the invention may be stabilized using conventional stabilizing agents, e.g., a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid, and the composition may be formulated as described in, for example, WO92/19709 and WO92/19708.
  • a polyol such as propylene glycol or glycerol
  • a sugar or sugar alcohol lactic acid, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid
  • a low detergent concentration system includes detergents where less than about 800 ppm of detergent components are present in the wash water.
  • Japanese detergents are typically considered low detergent concentration system as they have approximately 667 ppm of detergent components present in the wash water.
  • a medium detergent concentration includes detergents where between about 800 ppm and about
  • North American detergents are generally considered to be medium detergent concentration systems as they have approximately 975 ppm of detergent components present in the wash water.
  • a high detergent concentration system includes detergents where greater than about 2000 ppm of detergent components are present in the wash water.
  • European detergents are generally considered to be high detergent concentration systems as they have approximately 4500-5000 ppm of detergent components in the wash water.
  • Latin American detergents are generally high suds phosphate builder detergents and the range of detergents used in Latin America can fall in both the medium and high detergent concentrations as they range from 1500 ppm to 6000 ppm of detergent components in the wash water. Such detergent compositions are all embodiments of the invention.
  • a polypeptide of the present invention may also be incorporated in the detergent formulations disclosed in WO97/07202, which is hereby incorporated by reference.
  • the detergent composition may comprise one or more surfactants, which may be anionic and/or cationic and/or non-ionic and/or semi-polar and/or zwitterionic, or a mixture thereof.
  • the detergent composition includes a mixture of one or more non-ionic surfactants and one or more anionic surfactants.
  • the surfactant(s) is typically present at a level of from about 0.1 % to 60% by weight, such as about 1 % to about 40%, or about 3% to about 20%, or about 3% to about 10%.
  • the surfactant(s) is chosen based on the desired cleaning application, and includes any conventional surfactant(s) known in the art. Any surfactant known in the art for use in detergents may be utilized.
  • the detergent When included therein the detergent will usually comprise from about 1 % to about 40% by weight, such as from about 5% to about 30%, including from about 5% to about 15%, or from about 20% to about 25% of an anionic surfactant.
  • anionic surfactants include sulfates and sulfonates, in particular, linear alkylbenzenesulfonates (LAS), isomers of LAS, branched alkylbenzenesulfonates (BABS), phenylalkanesulfonates, alpha-olefinsulfonates (AOS), olefin sulfonates, alkene sulfonates, alkane-2,3- diylbis(sulfates), hydroxyalkanesulfonates and disulfonates, alkyl sulfates (AS) such as sodium dodecyl sul- fate (SDS), fatty alcohol sulfates (FAS), primary alcohol sulfates (PAS), alcohol
  • the detergent When included therein the detergent will usually comprise from about 0% to about 10% by weight of a cationic surfactant.
  • cationic surfactants include alklydimethylethanolamine quat (ADMEAQ), cetyltrimethylammonium bromide (CTAB), dimethyldistearylammonium chloride (DSDMAC), and alkylbenzyldimethylammonium, alkyl quaternary ammonium compounds, alkoxylated quaternary am- monium (AQA) compounds, and combinations thereof.
  • the detergent When included therein the detergent will usually comprise from about 0.2% to about 40% by weight of a non-ionic surfactant, for example from about 0.5% to about 30%, in particular from about 1 % to about 20%, from about 3% to about 10%, such as from about 3% to about 5%, or from about 8% to about 12%.
  • a non-ionic surfactant for example from about 0.5% to about 30%, in particular from about 1 % to about 20%, from about 3% to about 10%, such as from about 3% to about 5%, or from about 8% to about 12%.
  • Non-limiting examples of non-ionic surfactants include alcohol ethoxylates (AE or AEO), alcohol propox- ylates, propoxylated fatty alcohols (PFA), alkoxylated fatty acid alkyl esters, such as ethoxylated and/or propoxylated fatty acid alkyl esters, alkylphenol ethoxylates (APE), nonylphenol ethoxylates (NPE), al- kylpolyglycosides (APG), alkoxylated amines, fatty acid monoethanolamides (FAM), fatty acid diethanola- mides (FADA), ethoxylated fatty acid monoethanolamides (EFAM), propoxylated fatty acid monoethanolamides (PFAM), polyhydroxy alkyl fatty acid amides, or N-acyl N-alkyl derivatives of glucosamine (glu- camides, GA, or fatty acid glucamide, FAGA), as well as products available under the trade
  • the detergent When included therein the detergent will usually comprise from about 0% to about 10% by weight of a semipolar surfactant.
  • semipolar surfactants include amine oxides (AO) such as alkyldimethylamineoxide, N-(coco alkyl)-N,N-dimethylamine oxide and N-(tallow-alkyl)-N,N-bis(2-hydroxy- ethyl)amine oxide, fatty acid alkanolamides and ethoxylated fatty acid alkanolamides, and combinations thereof.
  • AO amine oxides
  • the detergent When included therein the detergent will usually comprise from about 0% to about 10% by weight of a zwitterionic surfactant.
  • zwitterionic surfactants include betaine, alkyldimethylbeta- ine, sulfobetaine, and combinations thereof. Hvdrotropes
  • a hydrotrope is a compound that solubilises hydrophobic compounds in aqueous solutions (or oppositely, polar substances in a non-polar environment).
  • hydrotropes typically have both hydrophilic and a hydrophobic character (so-called amphiphilic properties as known from surfactants); however, the molecular structure of hydrotropes generally do not favor spontaneous self-aggregation, see e.g. review by Hodgdon and Kaler (2007), Current Opinion in Colloid & Interface Science 12: 121-128. Hydrotropes do not display a critical concentration above which self-aggregation occurs as found for surfactants and lipids forming micel- lar, lamellar or other well defined meso-phases.
  • hydrotropes show a continuous-type aggregation process where the sizes of aggregates grow as concentration increases.
  • many hydrotropes alter the phase behaviour, stability, and colloidal properties of systems containing substances of polar and non-polar character, including mixtures of water, oil, surfactants, and polymers.
  • Hydrotropes are classically used across industries from pharma, personal care, food, to technical applications.
  • Use of hydrotropes in detergent compositions allow for example more concentrated formulations of surfactants (as in the process of compacting liquid detergents by removing water) without inducing undesired phenomena such as phase separation or high viscosity.
  • the detergent may comprise 0-5% by weight, such as about 0.5 to about 5%, or about 3% to about
  • hydrotrope Any hydrotrope known in the art for use in detergents may be utilized.
  • hydrotropes include sodium benzene sulfonate, sodium p-toluene sulfonate (STS), sodium xylene sulfonate (SXS), sodium cumene sulfonate (SCS), sodium cymene sulfonate, amine oxides, alcohols and polyglycolethers, sodium hydroxynaphthoate, sodium hydroxynaphthalene sulfonate, sodium ethylhexyl sulfate, and combinations thereof.
  • the detergent composition may comprise about 0-65% by weight, such as about 5% to about 45% of a detergent builder or co-builder, or a mixture thereof.
  • the level of builder is typi- cally 40-65%, particularly 50-65%.
  • the builder and/or co-builder may particularly be a chelating agent that forms water-soluble complexes with Ca and Mg. Any builder and/or co-builder known in the art for use in laundry detergents may be utilized.
  • Non-limiting examples of builders include zeolites, diphosphates (pyrophosphates), triphosphates such as sodium triphosphate (STP or STPP), carbonates such as sodium carbonate, soluble silicates such as sodium metasilicate, layered silicates (e.g. , SKS-6 from Hoechst), ethan- olamines such as 2-aminoethan-1 -ol (MEA), diethanolamine (DEA, also known as iminodiethanol), trieth- anolamine (TEA, also known as 2,2',2"-nitrilotriethanol), and carboxymethyl inulin (CMI), and combinations thereof.
  • zeolites diphosphates (pyrophosphates), triphosphates such as sodium triphosphate (STP or STPP), carbonates such as sodium carbonate, soluble silicates such as sodium metasilicate, layered silicates (e.g. , SKS-6 from Hoechst), ethan- olamines
  • the detergent composition may also comprise 0-20% by weight, such as about 5% to about 10%, of a detergent co-builder, or a mixture thereof.
  • the detergent composition may include a co-builder alone, or in combination with a builder, for example a zeolite builder.
  • co-builders include ho- mopolymers of polyacrylates or copolymers thereof, such as poly(acrylic acid) (PAA) or copoly(acrylic acid/maleic acid) (PAA PMA).
  • Further non-limiting examples include citrate, chelators such as aminocar- boxylates, aminopolycarboxylates and phosphonates, and alkyl- or alkenylsuccinic acid .
  • NTA 2,2',2"-nitrilotriacetic acid
  • EDTA ethylenediaminetetraacetic acid
  • DTPA diethylenetri- aminepentaacetic acid
  • IDS iminodisuccinic acid
  • EDDS ethylenediamine-N, N'-disuccinic acid
  • MGDA methylglycinediacetic acid
  • GLDA glutamic acid-N ,N-diacetic acid
  • HEDP 1 -hydroxyethane-1 ,1 -diphos- phonic acid
  • EDTMPA ethylenediaminetetra-(methylenephosphonic acid)
  • DTPMPA or DTMPA diethylenetriamine- pentakis(methylenephosphonic acid)
  • EDG N-(2-hydroxyethyl)iminodiacetic acid
  • ASMA aspartic acid-N-monoacetic acid
  • ASDA aspartic acid-N,N-diacetic acid
  • ASDA aspart
  • the detergent may comprise 0-50% by weight, such as about 0.1 % to about 25%, of a bleaching system.
  • a bleaching system Any bleaching system known in the art for use in laundry detergents may be utilized .
  • Suitable bleaching system components include bleaching catalysts, photobleaches, bleach activators, sources of hydrogen peroxide such as sodium percarbonate and sodium perborates, preformed peracids and mixtures thereof.
  • Suitable preformed peracids include, but are not limited to, peroxycarboxylic acids and salts, percarbonic acids and salts, perimidic acids and salts, peroxymonosulfuric acids and salts, for example, Oxone (R), and mixtures thereof.
  • Non-limiting examples of bleaching systems include peroxide-based bleaching systems, which may comprise, for example, an inorganic salt, including alkali metal salts such as sodium salts of perborate (usually mono- or tetra-hydrate), percarbonate, persulfate, perphosphate, persilicate salts, in combination with a peracid-forming bleach activator.
  • the term bleach activator is meant herein as a compound which reacts with peroxygen bleach like hydrogen peroxide to form a peracid. The peracid thus formed constitutes the activated bleach.
  • Suitable bleach activators to be used herein include those belonging to the class of esters amides, imides or anhydrides.
  • Suitable examples are tetracetylethylene diamine (TAED), sodium 4-[(3,5,5-trimethylhexanoyl)oxy]benzene sulfonate (ISONOBS), diperoxy dodecanoic acid, 4-(dodecanoyloxy)benzenesulfonate (LOBS), 4-(decanoyloxy)benzenesulfonate, 4-(decanoyloxy)benzoate (DOBS), 4-(nonanoyloxy)-benzenesulfonate (NOBS), and/or those disclosed in W098/17767.
  • TAED tetracetylethylene diamine
  • ISONOBS sodium 4-[(3,5,5-trimethylhexanoyl)oxy]benzene sulfonate
  • DOBS 4-(decanoyloxy)benzenesulfonate
  • NOBS 4-(nonanoyloxy)-benzenesulfonate
  • ATC acetyl triethyl citrate
  • ATC or a short chain triglyceride like triacetin has the advantage that it is environmental friendly as it eventually degrades into citric acid and alcohol.
  • acetyl triethyl citrate and triacetin has a good hydrolytical stability in the product upon storage and it is an efficient bleach activator.
  • ATC provides a good building capacity to the laundry additive.
  • the bleaching system may comprise peroxyacids of, for example, the amide, imide, or sulfone type.
  • the bleaching system may also comprise peracids such as 6-(phthalimido)peroxyhexanoic acid (PAP).
  • PAP 6-(phthalimido)peroxyhexanoic acid
  • the bleaching system may also include a bleach catalyst.
  • the bleach component may be an organic catalyst selected from the group consisting of organic catalysts having the following formulae:
  • each R is independently a branched alkyl group containing from 9 to 18 carbons or linear alkyl group containing from 1 1 to 18 carbons, more preferably each R is independently selected from the group consisting of 2-propylheptyl, 2-butyloctyl, 2-pentylnonyl, 2-hexyldecyl, n-dodecyl, n-tetradecyl, n-hexadecyl, n-octadecyl, iso-nonyl, iso-decyl, iso- tridecyl and iso-pentadecyl.
  • Suitable bleaching systems are described, e.g. in WO2007/087258, WO2007/087244, WO2007/087259 and WO2007/087242.
  • Suitable photobleaches may for example be sulfonated zinc phthalocyanine.
  • the detergent may comprise 0-10% by weight, such as 0.5-5%, 2-5%, 0.5-2% or 0.2-1 % of a polymer. Any polymer known in the art for use in detergents may be utilized.
  • the polymer may function as a co- builder as mentioned above, or may provide anti-redeposition, fiber protection, soil release, dye transfer inhibition, grease cleaning and/or anti-foaming properties. Some polymers may have more than one of the above-mentioned properties and/or more than one of the below-mentioned motifs.
  • Exemplary polymers include (carboxymethyl)cellulose (CMC), polyvinyl alcohol) (PVA), poly(vinylpyrrolidone) (PVP), poly(eth- yleneglycol) or poly(ethylene oxide) (PEG), ethoxylated poly(ethyleneimine), carboxymethyl inulin (CMI), and polycarboxylates such as PAA, PAA PMA, poly-aspartic acid, and lauryl methacrylate/acrylic acid copolymers, hydrophobically modified CMC (HM-CMC) and silicones, copolymers of terephthalic acid and oligomeric glycols, copolymers of poly(ethylene terephthalate) and poly(oxyethene terephthalate) (PET- POET), PVP, poly(vinylimidazole) (PVI), poly(vinylpyridine-N-oxide) (PVPO or PVPNO) and polyvinylpyrrol- idone-vinylim
  • Further exemplary polymers include sulfonated polycarboxylates, polyeth- ylene oxide and polypropylene oxide (PEO-PPO) and diquaternium ethoxy sulfate.
  • Other exemplary polymers are disclosed in, e.g., WO 2006/130575. Salts of the above-mentioned polymers are also contemplated .
  • the detergent compositions of the present invention may also include fabric hueing agents such as dyes or pigments, which when formulated in detergent compositions can deposit onto a fabric when said fabric is contacted with a wash liquor comprising said detergent compositions and thus altering the tint of said fabric through absorption/reflection of visible light.
  • fabric hueing agents alter the tint of a surface as they absorb at least a portion of the visible light spectrum.
  • Suitable fabric hueing agents include dyes and dye-clay conjugates, and may also include pigments.
  • Suitable dyes include small molecule dyes and polymeric dyes.
  • Suitable small molecule dyes include small molecule dyes selected from the group consisting of dyes falling into the Colour Index (C.I.) classifications of Direct Blue, Direct Red, Direct Violet, Acid Blue, Acid Red, Acid Violet, Basic Blue, Basic Violet and Basic Red, or mixtures thereof, for example as described in WO2005/03274, WO2005/03275, WO2005/03276 and EP1876226 (hereby incorporated by reference).
  • the detergent composition preferably comprises from about 0.00003 wt% to about 0.2 wt%, from about 0.00008 wt% to about 0.05 wt%, or even from about 0.0001 wt% to about 0.04 wt% fabric hueing agent.
  • the composition may comprise from 0.0001 wt% to 0.2 wt% fabric hueing agent, this may be especially preferred when the composition is in the form of a unit dose pouch.
  • Suitable hueing agents are also disclosed in, e.g. WO 2007/087257 and WO2007/087243.
  • the detergent additive as well as the detergent composition may comprise one or more additional enzymes such as a xanthan lyase, protease, lipase, cutinase, an amylase, lichenase, carbohydrase, cellu- lase, pectinase, mannanase, arabinase, galactanase, xylanase, oxidase, e.g., a laccase, and/or peroxidase.
  • additional enzymes such as a xanthan lyase, protease, lipase, cutinase, an amylase, lichenase, carbohydrase, cellu- lase, pectinase, mannanase, arabinase, galactanase, xylanase, oxidase, e.g., a laccase, and/or peroxidas
  • the properties of the selected enzyme(s) should be compatible with the selected detergent, (; ' .e., pH-optimum, compatibility with other enzymatic and non-enzymatic ingredients, etc.), and the en- zyme(s) should be present in effective amounts.
  • Suitable cellulases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Suitable cellulases include cellulases from the genera Bacillus, Pseudomo- nas, Humicola, Fusarium, Thielavia, Acremonium, e.g., the fungal cellulases produced from Humicola in- solens, Myceliophthora thermophila and Fusarium oxysporum disclosed in US 4,435,307, US 5,648,263, US 5,691 ,178, US 5,776,757 and WO 89/09259.
  • cellulases are the alkaline or neutral cellulases having color care benefits.
  • Examples of such cellulases are cellulases described in EP 0 495 257, EP 0 531 372, WO 96/11262, WO 96/29397, WO 98/08940.
  • Other examples are cellulase variants such as those described in WO 94/07998, EP 0 531 315, US 5,457,046, US 5,686,593, US 5,763,254, WO 95/24471 , WO 98/12307 and PCT/DK98/00299.
  • Example of cellulases exhibiting endo-beta-1 ,4-glucanase activity are those having de- scribed in WO02/099091 .
  • cellulases include the family 45 cellulases described in W096/29397, and especially variants thereof having substitution, insertion and/or deletion at one or more of the positions corresponding to the following positions in SEQ ID NO: 8 of WO 02/099091 : 2, 4, 7, 8, 10, 13, 15, 19, 20, 21 , 25, 26, 29, 32, 33, 34, 35, 37, 40, 42, 42a, 43, 44, 48, 53, 54, 55, 58, 59, 63, 64, 65, 66, 67, 70, 72, 76, 79, 80, 82, 84, 86, 88, 90, 91 , 93, 95, 95d, 95h, 95j, 97, 100, 101 , 102, 103, 113, 114, 1 17, 119, 121 , 133, 136, 137, 138, 139, 140a, 141 , 143a, 145, 146, 147, 150e, 150j, 151 , 152, 153,
  • cellulases include CelluzymeTM, and CarezymeTM (Novozymes A/S), Clazi- naseTM, and Puradax HATM (Genencor International Inc.), and KAC-500(B)TM (Kao Corporation).
  • the additional enzyme may be another protease or protease variant.
  • the protease may be of animal, vegetable or microbial origin, including chemically or genetically modified mutants. Microbial origin is pre- ferred. It may be an alkaline protease, such as a serine protease or a metalloprotease.
  • a serine protease may for example be of the S1 family, such as trypsin, or the S8 family such as subtilisin.
  • a metalloproteases protease may for example be a thermolysin from e.g. family M4, M5, M7 or M8.
  • subtilases refers to a sub-group of serine protease according to Siezen et al., Protein Engng. 4 (1991 ) 719-737 and Siezen et al. Protein Science 6 (1997) 501 -523.
  • Serine proteases are a subgroup of proteases characterized by having a serine in the active site, which forms a covalent adduct with the substrate.
  • the subtilases may be divided into 6 sub-divisions, i.e. the Subtilisin family, the Thermitase family, the Proteinase K family, the Lantibiotic peptidase family, the Kexin family and the Pyrolysin family.
  • the protease may be a subtilase, such as a subtilisin or a variant hereof.
  • subtilisins are those derived from Bacillus such as subtilisin lentus, Bacillus lentus, subtilisin Novo, subtilisin Carlsberg, Bacillus licheniformis, subtilisin BPN', subtilisin 309, subtilisin 147 and subtilisin 168 described in WO 89/06279 and protease PD138 (WO 93/18140). Additional serine protease examples are described in WO 98/0201 15, WO 01/44452, WO 01/58275, WO 01/58276, WO 03/006602 and WO 04/099401.
  • subtilase variants may be those having mutations in any of the positions: 3, 4, 9, 15, 27, 36, 68, 76, 87, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104, 106, 118, 120, 123, 128, 129, 130, 160, 167, 170, 194, 195, 199, 205, 217, 218, 222, 232, 235, 236, 245, 248, 252 and 274 using the BPN' numbering.
  • subtilase variants may comprise the mutations: S3T, V4I, S9R, A15T, K27R, *36D, V68A, N76D, N87S.R, *97E, A98S, S99G,D,A, S99AD, S101 G,M,R S103A, V104I,Y,N, S106A, G118V.R, H120D.N, N123S, S128L, P129Q, S130A, G160D, Y167A, R170S, A194P, G195E, V199M, V205I, L217D, N218D, M222S, A232V, K235L, Q236H, Q245R, N252K, T274A (using BPN' numbering).
  • a further preferred protease is the alkaline protease from Bacillus lentus DSM 5483, as described for example in WO 95/23221 , and variants thereof which are described in WO 92/21760, WO 95/23221 , EP 1921147 and EP 1921 148.
  • trypsin-like proteases are trypsin (e.g. of porcine or bovine origin) and the Fusarium pro- tease described in WO 89/06270 and WO 94/25583.
  • useful proteases are the variants described in WO 92/19729, WO 98/201 15, WO 98/201 16, and WO 98/34946, especially the variants with substitutions in one or more of the following positions: 27, 36, 57, 76, 87, 97, 101 , 104, 120, 123, 167, 170, 194, 206, 218, 222, 224, 235, and 274.
  • metalloproteases are the neutral metalloprotease as described in WO 07/044993.
  • Preferred commercially available protease enzymes include AlcalaseTM, CoronaseTM, DuralaseTM, Du- razymTM, EsperaseTM, EverlaseTM, KannaseTM, LiquanaseTM, Liquanase UltraTM, OvozymeTM, PolarzymeTM, PrimaseTM, RelaseTM, SavinaseTM and Savinase UltraTM, (Novozymes A/S), AxapemTM (Gist-Brocases N.V.), BLAP and BLAP X (Henkel AG & Co.
  • Suitable lipases and cutinases include those of bacterial or fungal origin. Chemically modified or protein engineered mutant enzymes are included. Examples include lipase from Thermomyces, e.g. from T. lanu- ginosus (previously named Humicola lanuginosa) as described in EP258068 and EP305216, cutinase from Humicola, e.g. H. insolens (WO96/13580), lipase from strains of Pseudomonas (some of these now renamed to Burkholdeha), e.g. P. alcaligenes or P. pseudoalcaligenes (EP218272), P. cepacia (EP331376), P.
  • Thermomyces e.g. from T. lanu- ginosus (previously named Humicola lanuginosa) as described in EP258068 and EP305216
  • cutinase from Humicola e
  • sp. strain SD705 (WO95/06720 & WO96/27002), P. wisconsinensis (WO96/12012), GDSL-type Strepto- myces lipases (W010/065455), cutinase from Magnaporthe grisea (W010/107560), cutinase from Pseudo- monas mendocina (US5,389,536), lipase from Thermobifida fusca (W01 1 /084412), Geobacillus stearother- mophilus lipase (W01 1/084417), lipase from Bacillus subtilis (W011/084599), and lipase from Streptomy- ces griseus (W01 1/150157) and S. pristinaespiralis (W012/137147).
  • lipases sometimes referred to as acyltransferases or perhydrolases, e.g. acyl- transferases with homology to Candida antarctica lipase A (W010/1 11 143), acyltransferase from Mycobac- terium smegmatis (WO05/56782), perhydrolases from the CE 7 family (WO09/67279), and variants of the M. smegmatis perhydrolase in particular the S54V variant used in the commercial product Gentle Power Bleach from Huntsman Textile Effects Pte Ltd (W010/100028).
  • acyltransferases or perhydrolases e.g. acyl- transferases with homology to Candida antarctica lipase A (W010/1 11 143), acyltransferase from Mycobac- terium smegmatis (WO05/56782), perhydrolases from the CE 7 family (WO09/67279), and variant
  • lipase variants such as those described in EP407225, WO92/05249, WO94/01541 , W094/25578, W095/14783, WO95/30744, W095/35381 , W095/22615, WO96/00292, WO97/04079, WO97/07202, WO00/34450, WO00/60063, WO01/92502, WO07/87508 and WO09/109500.
  • Preferred commercial lipase products include LipolaseTM, LipexTM; LipolexTM and LipocleanTM (Novo- zymes A/S), Lumafast (originally from Genencor) and Lipomax (originally from Gist-Brocades).
  • the amylase may be an alpha-amylase, a beta-amylase or a glucoamylase and may be of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Amylases include, for example, alpha-amylases obtained from Bacillus, e.g. , a special strain of Bacillus licheniformis, described in more detail in GB 1 ,296,839.
  • amylases are those having SEQ ID NO: 3 in WO 95/10603 or variants having 90% se- quence identity to SEQ ID NO: 3 thereof.
  • Preferred variants are described in WO 94/02597, WO 94/18314, WO 97/43424 and SEQ ID NO: 4 of WO 99/019467, such as variants with substitutions in one or more of the following positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 178, 179, 181 , 188, 190, 197, 201 , 202, 207, 208, 209, 21 1 , 243, 264, 304, 305, 391 , 408, and 444 of SEQ ID NO: 3 in WO 95/10603.
  • amylases are variants of SEQ ID NO: 1 of WO 2016/203064 having at least 75% sequence identity to SEQ ID NO: 1 thereof.
  • Preferred variants are variants comprising a modification in one or more positions corresponding to positions 1 , 54, 56, 72, 109, 113, 1 16, 134, 140, 159, 167, 169, 172, 173, 174, 181 , 182, 183, 184, 189, 194, 195, 206, 255, 260, 262, 265, 284, 289, 304, 305, 347, 391 , 395, 439, 469, 444, 473, 476, or 477 of SEQ ID NO: 1 , wherein said alpha-amylase variant has a sequence identity of at least 75% but less than 100% to SEQ ID NO: 1 .
  • amylases which can be used are amylases having SEQ ID NO: 6 in WO 02/010355 or variants thereof having 90% sequence identity to SEQ ID NO: 6.
  • Preferred variants of SEQ ID NO: 6 are those having a deletion in positions 181 and 182 and a substitution in position 193.
  • amylase examples are hybrid alpha-amylase comprising residues 1-33 of the alpha-amylase derived from B. amyloliquefaciens shown in SEQ ID NO: 6 of WO 2006/066594 and residues 36-483 of the B. licheniformis alpha-amylase shown in SEQ ID NO: 4 of WO 2006/066594 or variants having 90% se- quence identity thereof.
  • Preferred variants of this hybrid alpha-amylase are those having a substitution, a deletion or an insertion in one of more of the following positions: G48, T49, G107, H156, A181 , N190, M197, 1201 , A209 and Q264.
  • hybrid alpha-amylase comprising residues 1-33 of the alpha-amylase derived from B. amyloliquefaciens shown in SEQ ID NO: 6 of WO 2006/066594 and residues 36-483 of SEQ ID NO: 4 are those having the substitutions:
  • amylase examples are amylases having SEQ ID NO: 6 in WO 99/019467 or variants thereof having 90% sequence identity to SEQ ID NO: 6.
  • Preferred variants of SEQ ID NO: 6 are those having a substitution, a deletion or an insertion in one or more of the following positions: R181 , G182, H183, G184, N195, I206, E212, E216 and K269.
  • Particularly preferred amylases are those having deletion in positions G182 and H183 or positions H183 and G184.
  • Additional amylases are those having SEQ ID NO: 1 , SEQ ID NO: 2 or SEQ ID NO: 7 of WO 96/023873 or variants thereof having 90% sequence identity to SEQ ID NO: 1 , SEQ ID NO: 2 or SEQ ID NO: 7.
  • Pre- ferred variants of SEQ ID NO: 1 , SEQ ID NO: 2 or SEQ ID NO: 7 are those having a substitution, a deletion or an insertion in one or more of the following positions: 140, 181 , 182, 183, 184, 195, 206, 212, 243, 260, 269, 304 and 476. More preferred variants are those having a deletion in positions 182 and 183 or positions 183 and 184.
  • Most preferred amylase variants of SEQ ID NO: 1 , SEQ ID NO: 2 or SEQ ID NO: 7 are those having a deletion in positions 183 and 184 and a substitution in positions 140, 195, 206, 243, 260, 304 and 476.
  • amylases which can be used are amylases having SEQ ID NO: 2 of WO 08/153815, SEQ ID NO: 10 in WO 01/66712 or variants thereof having 90% sequence identity to SEQ ID NO: 2 of WO 08/153815 or 90% sequence identity to SEQ ID NO: 10 in WO 01/66712.
  • Preferred variants of SEQ ID NO: 10 in WO 01/66712 are those having a substitution, a deletion or an insertion in one of more of the following positions: 176, 177, 178, 179, 190, 201 , 207, 211 and 264.
  • amylases which can be used are amylases having SEQ ID NO: 2 of WO 09/061380 or variants thereof having 90% sequence identity to SEQ ID NO: 2.
  • Preferred variants of SEQ ID NO: 2 are those having a substitution, a deletion or an insertion in one of more of the following positions: Q87, Q98, S125, N128, T131 , T165, K178, R180, S181 , T182, G183, M201 , F202, N225, S243, N272, N282, Y305, R309, D319, Q320, Q359, K444 and G475.
  • More preferred variants of SEQ ID NO: 2 are those having the substitution in one of more of the following positions: Q87E.R, Q98R, S125A, N128C, T131 I, T165I, K178L, T182G, M201 L, F202Y, N225E.R, N272E.R, S243Q,A,E,D, Y305R, R309A, Q320R, Q359E, K444E and G475K and/or deletion in position R180 and/or S181.
  • Most preferred amylase variants of SEQ ID NO: 2 are those having the substitutions:
  • variant optionally further comprises a substitution at position 243 and/or a deletion at position 180 and/or position 181.
  • amylases are the alpha-amylase having SEQ ID NO: 12 in WO01/66712 or a variant having at least 90%, such as at least 95%, sequence identity to SEQ ID NO: 12.
  • Preferred amylase variants are those having a substitution, a deletion or an insertion in one of more of the following positions of SEQ ID NO: 12 in WO01/66712: R28, R118, N174; R181 , G182, D183, G184, G186, W189, N195, M202, Y298, N299, K302, S303, N306, R310, N314; R320, H324, E345, Y396, R400, W439, R444, N445, K446, Q449, R458, N471 , N484.
  • Particular preferred amylases include variants having a deletion of D183 and G184 and having the substitutions R1 18K, N195F, R320K and R458K, and a variant additionally having substitutions in one or more position selected from the group: M9, G149, G182, G186, M202, T257, Y295, N299, M323, E345 and A339, most preferred a variant that additionally has substitutions in all these positions.
  • amylases are DuramylTM, TermamylTM, FungamylTM, StainzymeTM, Stainzyme PlusTM, NatalaseTM and BANTM (Novozymes A S), RapidaseTM and PurastarTM (from Genencor International Inc.).
  • Suitable peroxidases/oxidases include those of plant, bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful peroxidases include peroxidases from Coprinus, e.g. , from C. cinereus, and variants thereof as those described in WO 93/24618, WO 95/10602, and WO 98/15257.
  • peroxidases include GuardzymeTM (Novozymes A/S).
  • the detergent enzyme(s) may be included in a detergent composition by adding separate additives containing one or more enzymes, or by adding a combined additive comprising all of these enzymes.
  • a detergent additive of the invention i.e., a separate additive or a combined additive, can be formulated, for example, as a granulate, liquid, slurry, etc.
  • Preferred detergent additive formulations are granulates, in particular non-dusting granulates, liquids, in particular stabilized liquids, or slurries.
  • Non-dusting granulates may be produced, e.g., as disclosed in US 4,106,991 and 4,661 ,452 and may optionally be coated by methods known in the art.
  • waxy coating materials are poly(ethylene oxide) products (polyethyleneglycol, PEG) with mean molar weights of 1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon atoms and in which there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- and triglycerides of fatty acids.
  • Liquid enzyme preparations may, for instance, be stabilized by adding a polyol such as propylene glycol, a sugar or sugar alcohol, lactic acid or boric acid according to established methods.
  • Protected enzymes may be prepared according to the method disclosed in EP 238,216.
  • any detergent components known in the art for use in laundry detergents may also be utilized.
  • Other optional detergent components include anti-corrosion agents, anti-shrink agents, anti-soil redeposition agents, anti-wrinkling agents, bactericides, binders, corrosion inhibitors, disintegrants/disintegration agents, dyes, enzyme stabilizers (including boric acid, borates, CMC, and/or polyols such as propylene glycol), fabric conditioners including clays, fillers/processing aids, fluorescent whitening agents/optical brighteners, foam boosters, foam (suds) regulators, perfumes, soil-suspending agents, softeners, suds suppressors, tarnish inhibitors, and wicking agents, either alone or in combination.
  • Any ingredient known in the art for use in laundry detergents may be utilized. The choice of such ingredients is well within the skill of the artisan.
  • the detergent compositions of the present invention can also contain dispersants.
  • powdered detergents may comprise dispersants.
  • Suitable water-soluble organic materials include the homo- or co-polymeric acids or their salts, in which the polycarboxylic acid comprises at least two car- boxyl radicals separated from each other by not more than two carbon atoms.
  • Suitable dispersants are for example described in Powdered Detergents, Surfactant science series volume 71 , Marcel Dekker, Inc.
  • the detergent compositions of the present invention may also include one or more dye transfer inhibiting agents.
  • Suitable polymeric dye transfer inhibiting agents include, but are not limited to, polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidones and polyvinylimidazoles or mixtures thereof.
  • the dye transfer inhibiting agents may be present at levels from about 0.0001 % to about 10%, from about 0.01 % to about 5% or even from about 0.1 % to about 3% by weight of the composition.
  • Fluorescent whitening agent The detergent compositions of the present invention will preferably also contain additional components that may tint articles being cleaned, such as fluorescent whitening agent or optical brighteners. Where present the brightener is preferably at a level of about 0,01 % to about 0,5%. Any fluorescent whitening agent suitable for use in a laundry detergent composition may be used in the composition of the present invention. The most commonly used fluorescent whitening agents are those belonging to the classes of diaminostilbene-sulphonic acid derivatives, diarylpyrazoline derivatives and bisphenyl-dis- tyryl derivatives.
  • diaminostilbene-sulphonic acid derivative type of fluorescent whitening agents include the sodium salts of: 4,4'-bis-(2-diethanolamino-4-anilino-s-triazin-6-ylamino) stilbene-2,2'-di- sulphonate; 4,4'-bis-(2,4-dianilino-s-triazin-6-ylamino) stilbene-2.2'-disulphonate; 4,4'-bis-(2-anilino-4(N- methyl-N-2-hydroxy-ethylamino)-s-triazin-6-ylamino) stilbene-2,2'-disulphonate, 4,4'-bis-(4-phenyl-2,1 ,3-tri- azol-2-yl)stilbene-2,2'-disulphonate; 4,4'-bis-(2-anilino-4(1 -methyl-2-hydroxy-ethylamino)-s-triazin-6-yla- min
  • Preferred fluorescent whitening agents are Tinopal DMS and Tinopal CBS available from Ciba-Geigy AG, Basel, Switzerland.
  • Tinopal DMS is the disodium salt of 4,4'-bis-(2-morpholino-4 anilino-s-triazin-6-ylamino) stilbene disulphonate.
  • Tinopal CBS is the disodium salt of 2,2'-bis-(phenyl-styryl) disulphonate.
  • fluorescent whitening agents is the commercially available Parawhite KX, supplied by Paramount Minerals and Chemicals, Mumbai, India.
  • Other fluorescers suitable for use in the invention include the 1 -3-diaryl pyrazolines and the 7-alkylaminocoumarins.
  • Suitable fluorescent brightener levels include lower levels of from about 0.01 , from 0.05, from about 0.1 or even from about 0.2 wt % to upper levels of 0.5 or even 0.75 wt%.
  • the detergent compositions of the present invention may also include one or more soil release polymers which aid the removal of soils from fabrics such as cotton and polyester based fabrics, in particular the removal of hydrophobic soils from polyester based fabrics.
  • the soil release polymers may for example be non-ionic or anionic terephthalate based polymers, polyvinyl caprolactam and related copolymers, vinyl graft copolymers, polyester polyamides see for example Chapter 7 in Powdered Detergents, Surfactant science series volume 71 , Marcel Dekker, Inc.
  • Another type of soil release polymers are amphiphilic alkoxylated grease cleaning polymers comprising a core structure and a plurality of alkox- ylate groups attached to that core structure.
  • the core structure may comprise a polyalkylenimine structure or a polyalkanolamine structure as described in detail in WO 2009/087523 (hereby incorporated by reference).
  • random graft co-polymers are suitable soil release polymers Suitable graft co-polymers are described in more detail in WO 2007/138054, WO 2006/108856 and WO 2006/1 13314 (hereby incor- porated by reference).
  • Other soil release polymers are substituted polysaccharide structures especially substituted cellulosic structures such as modified cellulose derivatives such as those described in EP 1867808 or WO 2003/040279 (both are hereby incorporated by reference).
  • Suitable cellulosic polymers include cellulose, cellulose ethers, cellulose esters, cellulose amides and mixtures thereof. Suitable cellulosic polymers include anionically modified cellulose, nonionically modified cellulose, cationically modified cellulose, zwit- terionically modified cellulose, and mixtures thereof. Suitable cellulosic polymers include methyl cellulose, carboxy methyl cellulose, ethyl cellulose, hydroxyl ethyl cellulose, hydroxyl propyl methyl cellulose, ester carboxy methyl cellulose, and mixtures thereof.
  • the detergent compositions of the present invention may also include one or more anti-redeposition agents such as carboxymethylcellulose (CMC), polyvinyl alcohol (PVA), polyvinylpyr- rolidone (PVP), polyoxyethylene and/or polyethyleneglycol (PEG), homopolymers of acrylic acid, copolymers of acrylic acid and maleic acid, and ethoxylated polyethyleneimines.
  • CMC carboxymethylcellulose
  • PVA polyvinyl alcohol
  • PVP polyvinylpyr- rolidone
  • PEG polyethyleneglycol
  • homopolymers of acrylic acid copolymers of acrylic acid and maleic acid
  • the cellulose based polymers described under soil release polymers above may also function as anti-redeposition agents.
  • adjunct materials include, but are not limited to, anti-shrink agents, anti-wrinkling agents, bactericides, binders, carriers, dyes, enzyme stabilizers, fabric softeners, fillers, foam regulators, hy- drotropes, perfumes, pigments, sod suppressors, solvents, and structurants for liquid detergents and/or structure elasticizing agents.
  • the detergent composition may be in any convenient form, e.g., a bar, a homogenous tablet, a tablet having two or more layers, a pouch having one or more compartments, a regular or compact powder, a granule, a paste, a gel, or a regular, compact or concentrated liquid.
  • a bar e.g., a bar, a homogenous tablet, a tablet having two or more layers, a pouch having one or more compartments, a regular or compact powder, a granule, a paste, a gel, or a regular, compact or concentrated liquid.
  • a regular or compact powder e.g., a granule, a paste, a gel, or a regular, compact or concentrated liquid.
  • There are a number of detergent formulation forms such as layers (same or different phases), pouches, as well as forms for machine dosing unit.
  • Pouches can be configured as single or multi-compartments. It can be of any form, shape and material which is suitable for hold the composition, e.g. without allowing the release of the composition from the pouch prior to water contact.
  • the pouch is made from water soluble film which encloses an inner volume. Said inner volume can be divided into compartments of the pouch.
  • Preferred films are polymeric materials preferably polymers which are formed into a film or sheet.
  • Preferred polymers, copolymers or derivatives thereof are selected polyacrylates, and water soluble acrylate copolymers, methyl cellulose, carboxy methyl cellulose, sodium dextrin, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, malto dex- trin, poly methacrylates, most preferably polyvinyl alcohol copolymers and, hydroxypropyl methyl cellulose (HPMC).
  • the level of polymer in the film for example PVA is at least about 60%.
  • Preferred average molecular weight will typically be about 20,000 to about 150,000.
  • Films can also be of blend compositions comprising hydrolytically degradable and water soluble polymer blends such as polyactide and polyvinyl alcohol (known under the Trade reference M8630 as sold by Chris Craft In. Prod. Of Gary, Ind., US) plus plasticisers like glycerol, ethylene glycerol, Propylene glycol, sorbitol and mixtures thereof.
  • the pouches can comprise a solid laundry cleaning composition or part components and/or a liquid cleaning composition or part components separated by the water soluble film.
  • the compartment for liquid components can be different in composition than compartments containing solids. Ref: (US2009/0011970 A1 ).
  • Detergent ingredients can be separated physically from each other by compartments in water dissolv- able pouches or in different layers of tablets. Thereby negative storage interaction between components can be avoided. Different dissolution profiles of each of the compartments can also give rise to delayed dissolution of selected components in the wash solution.
  • a liquid or gel detergent which is not unit dosed, may be aqueous, typically containing at least 20% by weight and up to 95% water, such as up to about 70% water, up to about 65% water, up to about 55% water, up to about 45% water, up to about 35% water.
  • Other types of liquids including without limitation, alkanols, amines, diols, ethers and polyols may be included in an aqueous liquid or gel.
  • An aqueous liquid or gel detergent may contain from 0-30% organic solvent.
  • a liquid or gel detergent may be non-aqueous.
  • the enzymes of the invention may be added to laundry soap bars and used for hand washing laundry, fabrics and/or textiles.
  • laundry soap bar includes laundry bars, soap bars, combo bars, syndet bars and detergent bars.
  • the types of bar usually differ in the type of surfactant they contain, and the term laundry soap bar includes those containing soaps from fatty acids and/or synthetic soaps.
  • the laundry soap bar has a physical form which is solid and not a liquid, gel or a powder at room temperature.
  • the term solid is defined as a physical form which does not significantly change over time, i.e. if a solid object (e.g. laundry soap bar) is placed inside a container, the solid object does not change to fill the container it is placed in.
  • the bar is a solid typically in bar form but can be in other solid shapes such as round or oval.
  • the laundry soap bar may contain one or more additional enzymes, protease inhibitors such as peptide aldehydes (or hydrosulfite adduct or hemiacetal adduct), boric acid, borate, borax and/or phenylboronic acid derivatives such as 4-formylphenylboronic acid, one or more soaps or synthetic surfactants, polyols such as glycerine, pH controlling compounds such as fatty acids, citric acid, acetic acid and/or formic acid, and/or a salt of a monovalent cation and an organic anion wherein the monovalent cation may be for example Na + , K + or NH4 + and the organic anion may be for example formate, acetate, citrate or lactate such that the salt of a monovalent cation and an organic anion may be, for example, sodium formate.
  • protease inhibitors such as peptide aldehydes (or hydrosulfite adduct or hem
  • the laundry soap bar may also contain complexing agents like EDTA and HEDP, perfumes and/or different type of fillers, surfactants e.g. anionic synthetic surfactants, builders, polymeric soil release agents, detergent chelators, stabilizing agents, fillers, dyes, colorants, dye transfer inhibitors, alkoxylated polycarbonates, suds suppressers, structurants, binders, leaching agents, bleaching activators, clay soil removal agents, anti-redeposition agents, polymeric dispersing agents, brighteners, fabric softeners, perfumes and/or other compounds known in the art.
  • the laundry soap bar may be processed in conventional laundry soap bar making equipment such as but not limited to: mixers, plodders, e.g. a two stage vacuum plodder, extruders, cutters, logo-stampers, cooling tunnels and wrappers.
  • the invention is not limited to preparing the laundry soap bars by any single method.
  • the premix of the invention may be added to the soap at different stages of the process.
  • the premix containing a soap, an enzyme, optionally one or more additional enzymes, a protease inhibitor, and a salt of a monovalent cation and an organic anion may be prepared and the mixture is then plodded.
  • the enzyme and optional additional enzymes may be added at the same time as the protease inhibitor for example in liquid form.
  • the process may further comprise the steps of milling, extruding, cutting, stamping, cooling and/or wrapping.
  • the present invention also relates to methods of producing the composition.
  • the method may be relevant for the (storage) stability of the detergent composition: e.g. Soap bar premix method WO 2009/155557.
  • the present invention is also directed to methods for using the detergent compositions thereof.
  • the present invention may be used for example in any detergent application which requires the degradation of xanthan gum. Use to degrade xanthan gum
  • Xanthan gum has been used as an ingredient in many consumer products including foods and cosmetics and has found use in the oil industry. Therefore, the degradation of xanthan gum can result in improved cleaning processes, such as the easier removal of stains containing gums, such as xanthan gum.
  • the present invention is directed to the use of xanthan lyase variants as described herein or compo- sitions thereof to degrade xanthan gum.
  • the present invention is also directed to the use of detergent compositions comprising xanthan lyases as described herein to degrade xanthan gum. Degradation of xanthan gum can preferably be measured using the viscosity reduction assay (e.g., ViPr assay) or alternatively as described in Example 3 herein.
  • GH9 endoglucanase activity may alternatively be measured by assessment of reducing ends on xan- than gum pre-treated with xanthan lyase using the colorimetric assay developed by Lever (1972), Anal. Biochem. 47: 273-279, 1972.
  • a preferred embodiment is the use of 0.1 % xanthan gum pre-treated with xanthan lyase.
  • Degradation of xanthan gum pre-treated with xanthan lyase may be determined by calculating difference between blank and sample wherein a difference of more than 0.5 mAU, preferably more than 0.6 mAU, more preferably more than 0.7 mAU or even more preferably more than 0.8 mAU shows degra- dation of xanthan gum pre-treated with xanthan lyase.
  • Xanthan lyase activity may alternatively be measured by assessment of reducing ends on xanthan gum using the colorimetric assay developed by Lever (1972), Anal. Biochem. 47: 273-279, 1972.
  • a preferred embodiment is the use of 0.1 % xanthan gum.
  • Degradation of xanthan gum may be determined by calculating difference between blank and sample, wherein a difference of more than 0.1 mAU, preferably more than 0.15 mAU, more preferably more than 0.2 mAU or even more preferably more than 0.25 mAU, shows degradation of xanthan gum.
  • Xanthan lyase e.g. variants as described herein
  • endoglucanase activity may alternatively be measured by assessment of reducing ends on xanthan gum using the colorimetric assay developed by Lever (1972), Anal. Biochem. 47: 273-279, 1972.
  • a preferred embodiment is the use of 0.1 % xanthan gum.
  • Degradation of xanthan gum may be determined by calculating difference between blank and sample wherein a difference of more than 0.4 mAU, preferably more than 0.5 mAU, more preferably more than 0.6 mAU or even more preferably more than 0.8 mAU shows degradation of xanthan gum.
  • the invention also relates to methods for degrading xanthan gum comprising applying a detergent composition comprising one or more xanthan lyase variants as described herein to xanthan gum.
  • the invention further relates to methods for degrading xanthan gum comprising applying a detergent composition comprising one or more xanthan lyases to xanthan gum.
  • An embodiment is a method for degrading xanthan gum comprising applying a detergent composition comprising one or more xanthan lyase variants as described herein together with one or more endoglucanases to xanthan gum.
  • the present invention inter alia relates to the use of detergent compositions comprising xanthan lyase variants as described herein in cleaning processes such as the laundering of textiles and fabrics (e.g., household laundry washing and industrial laundry washing), as well as household and industrial hard surface cleaning, such as dish wash.
  • the xanthan lyase variants as described herein may be added to a detergent composition comprising of one or more detergent components.
  • xanthan lyase variants as described herein may be used together with an endoglu- canase(s) in detergent compositions for cleaning processes such as the laundering of textiles and fabrics (e.g. household laundry washing and industrial laundry washing), as well as household and industrial hard surface cleaning, such as dish wash.
  • the xanthan lyase variants as described herein together with an endoglucanase(s) may be added to a detergent composition comprising of one or more detergent com- ponents.
  • the polypeptides described herein may be added to and thus become a component of a detergent composition.
  • the detergent composition may be formulated, for example, as a hand or machine laundry detergent composition for both household and industrial laundry cleaning, including a laundry additive composition suitable for pre-treatment of stained fabrics and a rinse added fabric softener composition, or be formulated as a detergent composition for use in general household or industrial hard surface cleaning operations, or be formulated for hand or machine (both household and industrial) dishwashing operations.
  • the present invention relates to a detergent additive comprising a polypeptide as described herein.
  • the invention also relates to methods for degrading xanthan gum on the surface of a textile or hard surface, such as dish wash, comprising applying a detergent composition comprising one or more xanthan lyase variants as described herein to xanthan gum.
  • the invention relates to a method for degrading xanthan gum on the surface of a textile or hard surface, such as dish wash, comprising applying a detergent composition comprising one or more xanthan lyase variants as described herein together with one or more endoglucanases to xanthan gum.
  • the invention relates to a detergent com- position comprising one or more detergent components as described herein. It is contemplated that the use of a xanthan lyase variant as described herein alone may give an enzyme detergency benefit, preferably an enzyme detergency benefit on xanthan gum.
  • the invention relates to the use of a detergent composition comprising one or more detergent components and an isolated xanthan lyase variant as described herein together with a GH9 en- doglucanase. In some aspects, the invention relates to the use of a detergent composition comprising one or more detergent components and an isolated xanthan lyase variant as described herein together with a GH9 endoglucanase.
  • a detergent composition comprising a xanthan lyase variant, comprising an alteration (e.g., a substitution, deletion or insertion) at one or more positions in a region selected from the group consisting of: i) region 7 corresponding to amino acids 1 to 153 of SEQ ID NO: 2,
  • said variant has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, and less than 100% sequence identity to SEQ ID NO: 2; preferably said xanthan lyase variant has activity on xanthan gum, further preferably said activity is a xanthan gum degrading activity.
  • the detergent composition comprising a xanthan lyase variant of paragraph 1 , which is a variant of a parent xanthan lyase selected from the group consisting of:
  • the detergent composition comprising a xanthan lyase variant of paragraph 2, wherein the parent xanthan lyase has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the mature polypeptide of SEQ ID NO: 2.
  • the detergent composition comprising a xanthan lyase variant of any of paragraphs 2-3, wherein the parent xanthan lyase is encoded by a polynucleotide that hybridizes under low stringency conditions, medium stringency conditions, medium-high stringency conditions, high stringency conditions, or very high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 1 or (ii) the full-length complement of (i).
  • the detergent composition comprising a xanthan lyase variant of any of paragraphs 2-4, wherein the parent xanthan lyase is encoded by a polynucleotide having at least 60%, e.g., at least 65%, at least
  • the detergent composition comprising a xanthan lyase variant of any of paragraphs 2-5, wherein the parent xanthan lyase comprises or consists of the mature polypeptide of SEQ ID NO: 2.
  • the detergent composition comprising a xanthan lyase variant of any of paragraphs 2-6, wherein the parent xanthan lyase is a fragment of the mature polypeptide of SEQ ID NO: 2, wherein the fragment has xanthan lyase activity.
  • the detergent composition comprising a xanthan lyase variant of any of paragraphs 2-7, which has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to the amino acid sequence of the parent xanthan lyase.
  • the detergent composition comprising a xanthan lyase variant of any of paragraphs 1 -8, wherein said variant has at least 61 %, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 2.
  • the detergent composition comprising a xanthan lyase variant of any of paragraphs 1 -9, wherein said alteration (e.g., a substitution, deletion or insertion) at one or more positions is selected from the group consisting of alterations in positions: 9, 15, 46, 58, 66, 89, 95, 100, 106, 109, 183, 188, 190, 203, 204, 221 , 229, 234, 238, 240, 242, 243, 257, 258, 291 , 293, 316, 320, 324, 329, 333, 339, 341 , 352, 354, 360, 377, 399, 400, 419, 440, 450, 451 , 454, 458, 481 , 492, 567, 568, 578, 579, 582, 664, 672, 703, 728, 843, 855, 887, 892, 1008 and 1016 of SEQ ID NO: 2, wherein numbering is according to SEQ ID NO: 2,
  • the detergent composition comprising a xanthan lyase variant of any of claims 1-10, further comprising one or more alterations at one or more positions selected from the group consisting of position 624, 631 , 635, 649, 656, 752, 752, 754, 757, 769, 775, 777, 800, 801 , 875, 91 1 , and 915 wherein numbering is according to SEQ ID NO: 2.
  • the detergent composition comprising a xanthan lyase variant of any of paragraphs 1 -1 1 , having one or more substitutions selected from the group consisting of: K9R, N15T, L46D, A58L, S66H, Q89Y, K95E, S100D, N106Y, Q109R, Q109D, Q109F, Q109K, Q109A, K183Q.K183R, V188I, A190Q, A203P, K204R, A221 P, E229N, E229S, I234V, I238W, I238L, I238M, I240W, N242S, G243V, Y257W, R258E, K291 R, A293G, A293P, K316R, K320R, L324Q, K329R, K333R, L339M, 1341 P, V352I, S354P, K360R, K360G, F377Y, N399K
  • the detergent composition comprising a xanthan lyase variant of any of paragraphs 1 -13, comprising one of the following set of substitutions:
  • the detergent composition comprising a xanthan lyase variant of any of paragraphs 1 -14, wherein said variant does not comprise any amino acid alteration at a position outside of regions 7, 8, 9, 10, 11 , 12 and 13.
  • the detergent composition comprising a xanthan lyase variant of any of paragraphs 1 -15, wherein the total number of alterations compared to the parent xanthan lyase (e.g., SEQ ID NO: 2) is between 1 and 20, e.g. between 1 and 10 or between 1 and 5, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 alterations.
  • SEQ ID NO: 2 the total number of alterations compared to the parent xanthan lyase
  • the detergent composition comprising a xanthan lyase variant of any of paragraphs 1-16, wherein said activity on xanthan gum is a xanthan gum degrading activity, preferably said xanthan lyase variant has EC 4.2.2.12 activity.
  • the detergent composition comprising a xanthan lyase variant of any of paragraphs 1-17, wherein said variant has an improved stability in a detergent composition compared to a parent xanthan lyase (e.g., with SEQ ID NO: 2).
  • the detergent composition comprising a xanthan lyase variant of any of paragraphs 1-18, wherein said variant has a half-life improvement factor (HIF) of >1.0; preferably >1 .0, more preferably at least 1.2, such as at least 1 .5, e.g. at least 2.0, relative to a parent xanthan lyase, e.g. a xanthan lyase with SEQ
  • HAF half-life improvement factor
  • the detergent composition comprising a xanthan lyase variant of paragraph 19, wherein said half-life improvement factor (HIF) is determined after incubation of said xanthan lyase variant in a detergent composition at 25°C for a time period from about 30 min to about 20 hours.
  • HIF half-life improvement factor
  • the detergent composition comprising a xanthan lyase variant of any of paragraphs 1 -20, wherein said variant is selected from the group consisting of i) the xanthan lyase variants set forth in Table 1 herein, ii) the xanthan lyase variants set forth in Table 2 herein, iii) the xanthan lyase variants set forth in Table 3 herein, and iv) the xanthan lyase variants set forth in Table 4 herein.
  • additional en- zymes selected from the group consisting of: endoglucanases, proteases, amylases, lichenases, lipases, cutinases, cellulases, xanthan lyases, xyloglucanases, pectinases, pectin lyases, xanthanases, peroxidases, haloperoxygenases, catalases and mannan
  • a method for degrading xanthan gum comprising: applying a detergent composition of any of para- graphs 1-25 to a xanthan gum.
  • Example 1 Construction and expression of xanthan lyase variants
  • Xanthan lyase parent gene (i.e., SEQ ID NO: 1 ) was PCR assembled into a linear cassette containing the promoter system on the upstream and cat selection maker on the downstream.
  • SEQ ID NO: 1 Xanthan lyase parent gene was PCR assembled into a linear cassette containing the promoter system on the upstream and cat selection maker on the downstream.
  • >2 kb DNA sequence identical to the site of integration was included on both the sides of the cassette.
  • Genomic DNA prepared from the strain containing xanthan lyase parent gene (SEQ ID NO: 1 ) was used as template for generating the site-directed mutants. Mutagenic forward and reverse primers were used to generate an approximately 6 kb PCR fragment.
  • This fragment was used as a megaprimer along with another forward primer to amplify >8 kb DNA fragment.
  • This fragment contained the complete cassette (promoter system, xanthan lyase and cat gene along with homologous DNA sequence required for recombination at Pel locus) was used for transformation.
  • the triple promoter system used in the cassette has been described in WO 99/43835 and it consists of promoters from Bacillus licheniformis alpha-amylase gene (amyL), Bacillus amyloliquefaciens alpha-am- ylase gene (amyQ), and the Bacillus thuringiensis crylllA promoter including the stabilizing sequence.
  • Protease signal sequence from Bacillus clausii was included to export the protein out of the cells.
  • Generated variants of the mature parent xanthan lyase of SEQ ID NO: 2 are shown in Table 1 below. The presence of the alteration was confirmed by sequencing.
  • Bacillus organism containing a variant was inoculated in LB broth containing chloramphenicol (6 Mg/ml) and grown overnight at 37°C.
  • 2% of overnight culture was added to 300 ml of 10-R medium in 1000 ml baffled flask and grown at 30°C for 96 hrs. at 180 rpm.
  • 10-R medium contained 33 g/L Soluble starch, 6 g/L (NH ) 2 HP0 , 5 g/L Potato peptone, 1.2 g/L (MgS0 x 7H 2 0), 12 g/L KH2PO4, 5 g/L (Na 2 HP0 4 x 2H 2 0), 18 mL/L of Trace metal solution, 1.8 g/L K2SO4 and 0.1 g/L (CaCl2 x 2H2O) and 0.5 mL/L SB2121 (anti-foam agent).
  • Bacillus subtilis broth was clarified by centrifuging at 8000 x g for 30 minutes at 10°C followed by vacuum filtration using a combination of Seitz filter (K250) and WHATMAN glass filter GF/F grade in a Buchner funnel. Finally, the supernatant was filtered through 0.22 ⁇ Tangential flow filtration unit.
  • Xanthan lyase variants were purified using three-step automated tandem column chromatography.
  • Macro-Prep Methyl HIC column was pre-equilibrated with 50 mM Tris, pH 8.0 containing 1 M (NH4)2S04 and 1 mM CaCl2 buffer.
  • the clarified culture supernatant 250 ml_
  • 50 mM Tris, pH 8.0 containing 2 M (NH 4 ) 2 SO 4 andl mM CaCl2 buffer was diluted 1 :1 in-line with 50 mM Tris, pH 8.0 containing 2 M (NH 4 ) 2 SO 4 andl mM CaCl2 buffer to make the final concentration to 1 M.
  • the unbound or weakly bound protein was washed with the equilibration buffer until the Absorbance at 280 nm comes below 0.1 AU.
  • Elution was carried out using 50 mM Tris pH 8 containing 0.5 M (NH 4 ) 2 SO 4 and 1 mM CaCl2. Eluted protein peak was automatically loaded on MEP-Hy- percel column pre-equilibrated with 50 mM Tris, pH 8 containing 0.5 M (NH 4 ) 2 SO 4 and 1 mM CaCl2. The unbound or weakly bound protein was washed with the equilibration buffer until the Absorbance at 280 nm comes below 0.1 AU. The column was washed again with 50 mM Tris, pH 8 containing 1 mM CaCl2 to remove impurities.
  • the Purified protein was eluted with 50 mM Na-acetate, pH 5 containing 1 mM CaCl2. The eluted purified protein was automatically transferred to Sephadex G-25 column pre-equilibrated with 50 mM MOPS, pH 8 containingl mM CaCl2 for desalting.
  • Example 3 Detergent stability assay
  • Reagents for the detergent stability assay were prepared as follows:
  • a stock of 1.0 M MOPS buffer was prepared by dissolving 209.26 g of 3-Morpholinopropanesulfonic acid in Milli Q water. pH was adjusted to 7.5 using NaOH and the final volume of buffer was made up to 1000 ml. This buffer stock was stored at 4°C until use.
  • a 50 mM working solution of MOPS buffer was prepared by adding 50 ml of 1.0 M stock to 950 ml of Milli Q water.
  • a substrate solution of 0.4% w/v xanthan gum was freshly prepared by dissolving 400 mg of xanthan gum in 100 ml of Milli Q water.
  • a stock solution mix containing 1.0 M Na2C03, 0.17 M potassium sodium tartrate and 5 mM (Bi(N03)3 x 5H2O) was prepared by dissolving 106.99 g of Na2C03, 47.98 g of potassium sodium tartrate and 2.42 mg of (Bi(N03)3 x 5H2O) in Milli Q water for a final volume of 1000 ml. This stock solution mix was filtered and stored at room temperature.
  • PAHBAH p-hydroxybenzoic acid hydrazide
  • the in-detergent stability was determined by measuring the enzymatic activity present in culture supernatants of variants or wild-type controls after incubation with detergent (70% Persil Universal Gel deter- gent (PUG), final concentration) at 30°C for one hour.
  • detergent 50% Persil Universal Gel deter- gent (PUG), final concentration
  • Detergent stress was carried out by addition of 30 ⁇ of culture supernatant and 70 ⁇ of a Persil Universal Gel detergent (100%) into wells of 96-well microtitre plates which were shaken for 15 min at 1000 rpm. Two identical plates were produced whereof one plate was incubated at 4°C (unstressed plate) and the other plate was incubated at 30°C (stressed plate) for 1 hour. After incubation, samples from unstressed and stressed plates were diluted 50X with dilution buffer (50mM MOPS, 5 mM CaCh, pH 7.5).
  • dilution buffer 50mM MOPS, 5 mM CaCh, pH 7.5.
  • reaction mixtures were prepared in 96-well PCR plates. 50 ⁇ of diluted samples were mixed with 50 ⁇ of freshly prepared substrate solution and incubated at 40°C for 1 hour.
  • the residual activity (RA) for variants and wild-type controls were calculated as the percentage of enzymatic activity remaining after incubation at 30°C relative to the enzymatic activity remaining after incuba- tion at 4°C, i.e., according to the following formula:
  • Residual activity (RA) 100% * Abs405 (sample incubated at 30°C)/Abs405 (sample incubated at 4°C).
  • the detergent stability of purified variants was determined by measuring the enzyme activity of the purified protein after incubation with detergent (70% Persil Universal Gel detergent (PUG), final concentration for the variants of Table 3, and 90% for Table 4) at 30°C unless otherwise indicated (see Table 4) for the time indicated in Tables 3 and 4 below.
  • detergent 70% Persil Universal Gel detergent (PUG), final concentration for the variants of Table 3, and 90% for Table 4
  • Purified variants were diluted to a concentration of 200 ppm using 50 mM MOPS buffer.
  • 10 ⁇ of diluted purified samples were mixed with 90 ⁇ of Persil Universal Gel detergent (100%) into wells of 96-well microtitre plates which were shaken for 20 min at 1000 rpm. Two identical plates were produced whereof one plate was incubated at 4°C (unstressed plate) and the other plate was incubated at 30°C (stressed plate) for 1 hour. After incubation, samples from unstressed and stressed plates were diluted 50X with dilution buffer (50 mM MOPS, 1 mM CaCI 2 , pH 7.5). Enzymatic activity analysis of unstressed and stressed samples was done as described in section A. C. Calculating half-lives and half-life improvement factors (HIF)
  • T1/2 (variants) (Ln (0.5)/Ln (RA-variants/100))*Time
  • HIF half-life improvement factor
  • the incubation time for wild-type and variant is different, e.g. 1 h for wild-type and up to 168 h for the most stable variants.
  • the incubation temperature for some variants in Table 4 was increased by 2-5 degrees Celsius.
  • HIF half-life improvement factor
  • T1/2 (variants) (Ln (0.5)/Ln (RA-variants/100))*Time
  • the incubation time for wild-type and variant is different e.g. 1 h for wild- type and 840 h for the most stable variants, HIF values could not be calculated as the half-life of the wild- type could not be determined accurately. Stability of these variants are reported in terms of half-life (in hours).

Abstract

The present invention relates to detergent compositions comprising xanthan lyase variants and meth- ods for use of said compositions.

Description

DETERGENT COMPOSITION COMPRISING XANTHAN LYASE VARIANTS II
REFERENCE TO A SEQUENCE LISTING
This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference.
BACKGROUND OF THE INVENTION
Field of the Invention
The present invention relates to detergent compositions, such as laundry compositions and dish wash compositions, including hand wash and automatic dish wash compositions, comprising novel xanthan lyase variants exhibiting alterations relative to the parent xanthan lyase in one or more properties such as: detergent stability (e.g. improved stability in a detergent composition) and/or storage stability (e.g. improved storage stability in a detergent composition). The present invention further relates to detergent compositions comprising novel xanthan lyase variants having activity on xanthan gum. The invention also relates to methods for producing and using the compositions of the invention. Variants described herein are particularly suitable for use in cleaning processes and detergent compositions.
Description of the Related Art
Xanthan gum is a polysaccharide derived from the bacterial coat of Xanthomonas campestris. It is produced by the fermentation of glucose, sucrose, or lactose by the Xanthomonas campestris bacterium. After a fermentation period, the polysaccharide is precipitated from a growth medium with isopropyl alcohol, dried, and ground into a fine powder. Later, it is added to a liquid medium to form the gum. Xanthan gum is a natural polysaccharide consisting of different sugars which are connected by several different bonds, such as β-D-mannosyl- β-D-1 ,4-glucuronosyl bonds and β-D-glucosyl-β-D-1 ,4-glucosyl bonds. Xanthan gum is at least partly soluble in water and forms highly viscous solutions or gels. Complete enzymatic degradation of xanthan gum requires several enzymatic activities including xanthan lyase activity and endo- β-1 ,4-glu- canase activity. Xanthan lyases are enzymes that cleave the β-D-mannosyl-β-D-1 ,4-glucuronosyl bond of xanthan and have been described in the literature. Xanthan lyases are known in the art, e.g. two xanthan lyases have been isolated from Paenibacillus alginolyticus XL-1 (e.g. Ruijssenaars ef al. (1999) 'A pyruvated mannose-specific xanthan lyase involved in xanthan degradation by Paenibacillus alginolyticus XL-1 ', Appl. Environ. Microbiol. 65(6): 2446-2452, and Ruijssenaars et al. (2000), 'A novel gene encoding xanthan lyase of Paenibacillus alginolyticus strain XL-1 ', Appl. Environ. Microbiol. 66(9): 3945-3950). Glycoside hydrolases are enzymes that catalyse the hydrolysis of the glycosyl bond to release smaller sugars. There are over 100 classes of glycoside hydrolases which have been classified, see Henrissat ei al. (1991 ) 'A classification of glycosyl hydrolases based on amino-acid sequence similarities', J. Biochem. 280: 309-316 and the Uniprot website at www.cazy.org. The glycoside hydrolase family 9 (GH9) consists of over 70 different enzymes that are mostly endo-glucanases (EC 3.2.1 .4), cellobiohydrolases (EC 3.2.1.91 ), β-glucosidases (EC 3.2.1 .21 ) and exo^-glucosaminidase (EC 3.2.1.165). In recent years xanthan gum has been used as an ingredient in many consumer products including foods (e.g. as thickening agent in salad dressings and dairy products) and cosmetics (e.g. as stabilizer and thickener in toothpaste and make-up, creams and lotions to prevent ingredients from separating and to provide the right texture of the product). Further, xanthan gum has found use in the oil industry as an additive to regulate the viscosity of drilling fluids etc. The widespread use of xanthan gum has led to a desire to degrade solutions, gels or mixtures containing xanthan gum thereby allowing easier removal of the by-products. Xanthan lyases and endoglucanases for the degradation of xanthan gum and the use of such enzymes for cleaning purposes, such as the removal of xanthan gum containing stains, and in the drilling and oil industries are known in the art, e.g. WO2013167581 A1.
The known xanthan lyase having SEQ ID NO: 2 was found to be sensitive to the presence of deter- gents. To improve applicability and/or cost and/or the performance of such enzymes there is an ongoing search for variants with altered properties, such as increased stability, e.g. improved stability in a detergent composition. However, mutagenesis of large enzymes followed by purification and functional analysis of mutant libraries can be very expensive and laborious. SUMMARY OF THE INVENTION
Since the known xanthan lyase having SEQ ID NO: 2 is a large enzyme (>1000 residues), it is difficult and expensive to randomly target its properties for improvement of, e.g., stability in a detergent composition.
In some aspects, the present invention identifies regions in the protein sequence/structure of the known xanthan lyase having SEQ ID NO: 2 that are relevant for e.g. storage stability, and therefore provides an important guidance on where to mutate a xanthan lyase in order to stabilize the molecule in a detergent.
In some aspects, the present invention relates to detergent compositions comprising a xanthan lyase variant, comprising an alteration (e.g., a substitution, deletion or insertion) at one or more positions in a region selected from the group consisting of: region 7 corresponding to amino acids 1 to 153 of SEQ ID NO: 2, region 8 corresponding to amino acids 177 to 613 of SEQ ID NO: 2, region 9 corresponding to amino acids 659 to 730 of SEQ ID NO: 2, region 10 corresponding to amino acids 804 to 806 of SEQ ID NO: 2, region 1 1 corresponding to amino acids 847 to 871 of SEQ ID NO: 2, region 12 corresponding to amino acids 886 to 902 of SEQ ID NO: 2, and region 13 corresponding to amino acids 1005 to 1037 of SEQ ID NO: 2.
In some aspects, the present invention relates to detergent compositions comprising a xanthan lyase variant, comprising an alteration (e.g., a substitution, deletion or insertion) at one or more positions in a region selected from the group consisting of: region 7 corresponding to amino acids 1 to 153 of SEQ ID NO: 2, region 8 corresponding to amino acids 177 to 613 of SEQ ID NO: 2, region 9 corresponding to amino acids 659 to 730 of SEQ ID NO: 2, region 10 corresponding to amino acids 804 to 806 of SEQ ID NO: 2, region 1 1 corresponding to amino acids 847 to 871 of SEQ ID NO: 2, region 12 corresponding to amino acids 886 to 902 of SEQ ID NO: 2, and region 13 corresponding to amino acids 1005 to 1037 of SEQ ID NO: 2, wherein said variant has at least 60% and less than 100% sequence identity to SEQ ID NO: 2, preferably said xanthan lyase variant having an activity on xanthan gum.
In some aspects, the present invention relates to detergent compositions comprising a xanthan lyase variant having at least 61 %, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 2.
In some aspects, the present invention relates to detergent compositions comprising a xanthan lyase variant comprising an alteration (e.g., a substitution, deletion or insertion) at one or more positions in a region selected from the group consisting of:
i) region 7 corresponding to amino acids 1 to 153 of SEQ ID NO: 2, e.g., said alteration at one or more positions selected from the group consisting of positions: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64,
65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104, 105, 106, 107, 108, 109, 1 10, 1 1 1 , 112, 113, 114, 1 15, 1 16, 1 17, 118, 119, 120, 121 , 122, 123, 124, 125, 126, 127, 128, 129, 130, 131 , 132, 133, 134, 135, 136, 137, 138, 139, 140, 141 , 142, 143, 144, 145, 146, 147, 148, 149, 150, 151 , 152 and 153, wherein said positions correspond to amino acid positions of SEQ ID NO:
2,
ii) region 8 corresponding to amino acids 177 to 613 of SEQ ID NO: 2, e.g., said alteration at one or more positions selected from the group consisting of positions: 177, 178, 179, 180, 181 , 182, 183, 184, 185, 186, 187, 188, 189, 190, 191 , 192, 193, 194, 195, 196, 197, 198, 199, 200, 201 , 202, 203, 204, 205, 206, 207, 208, 209, 210, 211 , 212, 213, 214, 215, 216, 217, 218, 219, 220, 221 ,
222, 223, 224, 225, 226, 227, 228, 229, 230, 231 , 232, 233, 234, 235, 236, 237, 238, 239, 240, 241 , 242, 243, 244, 245, 246, 247, 248, 249, 250, 251 , 252, 253, 254, 255, 256, 257, 258, 259, 260, 261 , 262, 263, 264, 265, 266, 267, 268, 269, 270, 271 , 272, 273, 274, 275, 276, 277, 278, 279, 280, 281 , 282, 283, 284, 285, 286, 287, 288, 289, 290, 291 , 292, 293, 294, 295, 296, 297, 298, 299, 300, 301 , 302, 303, 304, 305, 306, 307, 308, 309, 310, 31 1 , 312, 313, 314, 315, 316,
317, 318, 319, 320, 321 , 322, 323, 324, 325, 326, 327, 328, 329, 330, 331 , 332, 333, 334, 335, 336 337, 338, 339, 340, 341 , 342, 343, 344, 345, 346, 347, 348, 349, 350, 351 , 352, 353, 354, 355 356, 357, 358, 359, 360, 361 , 362, 363, 364, 365, 366, 367, 368, 369, 370, 371 , 372, 373, 374 375, 376, 377, 378, 379, 380, 381 , 382, 383, 384, 385, 386, 387, 388, 389, 390, 391 , 392, 393 394, 395, 396, 397, 398, 399, 400, 401 , 402, 403, 404, 405, 406, 407, 408, 409, 410, 41 1 , 412 413, 414, 415, 416, 417, 418, 419, 420, 421 , 422, 423, 424, 425, 426, 427, 428, 429, 430, 431 432, 433, 434, 435, 436, 437, 438, 439, 440, 441 , 442, 443, 444, 445, 446, 447, 448, 449, 450 451 , 452, 453, 454, 455, 456, 457, 458, 459, 460, 461 , 462, 463, 464, 465, 466, 467, 468, 469 470, 471 , 472, 473, 474, 475, 476, 477, 478, 479, 480, 481 , 482, 483, 484, 485, 486, 487, 488 489, 490, 491 , 492, 493, 494, 495, 496, 497, 498, 499, 500, 501 , 502, 503, 504, 505, 506, 507 508, 509, 510, 511 , 512, 513, 514, 515, 516, 517, 518, 519, 520, 521 , 522, 523, 524, 525, 526 527, 528, 529, 530, 531 , 532, 533, 534, 535, 536, 537, 538, 539, 540, 541 , 542, 543, 544, 545 546, 547, 548, 549, 550, 551 , 552, 553, 554, 555, 556, 557, 558, 559, 560, 561 , 562, 563, 564 565, 566, 567, 568, 569, 570, 571 , 572, 573, 574, 575, 576, 577, 578, 579, 580, 581 , 582, 583 584, 585, 586, 587, 588, 589, 590, 591 , 592, 593, 594, 595, 596, 597, 598, 599, 600, 601 , 602, 603, 604, 605, 606, 607, 608, 609, 610, 611 , 612 and 613, wherein said positions correspond to amino acid positions of SEQ ID NO: 2,
iii) region 9 corresponding to amino acids 659 to 730 of SEQ ID NO: 2, e.g., said alteration at one or more positions selected from the group consisting of positions: 659, 660, 661 , 662, 663, 664, 665, 666, 667, 668, 669, 670, 671 , 672, 673, 674, 675, 676, 677, 678, 679, 680, 681 , 682, 683, 684, 685, 686, 687, 688, 689, 690, 691 , 692, 693, 694, 695, 696, 697, 698, 699, 700, 701 , 702, 703,
704, 705, 706, 707, 708, 709, 710, 71 1 , 712, 713, 714, 715, 716, 717, 718, 719, 720, 721 , 722, 723, 724, 725, 726, 727, 728, 729 and 730, wherein said positions correspond to amino acid positions of SEQ ID NO: 2,
iv) region 10 corresponding to amino acids 804 to 806 of SEQ ID NO: 2, e.g., said alteration at one or more positions selected from the group consisting of positions: 804, 805 and 806, wherein said positions correspond to amino acid positions of SEQ ID NO: 2,
v) region 11 corresponding to amino acids 847 to 871 of SEQ ID NO: 2, e.g., said alteration at one or more positions selected from the group consisting of positions: 847, 848, 849, 850, 851 , 852, 853, 854, 855, 856, 857, 858, 859, 860, 861 , 862, 863, 864, 865, 866, 867, 868, 869, 870 and 871 , wherein said positions correspond to amino acid positions of SEQ ID NO: 2,
vi) region 12 corresponding to amino acids 886 to 902 of SEQ ID NO: 2, e.g., said alteration at one or more positions selected from the group consisting of positions: 886, 887, 888, 889, 890, 891 , 892, 893, 894, 895, 896, 897, 898, 899, 900, 901 and 902, wherein said positions correspond to amino acid positions of SEQ ID NO: 2,
vii) region 13 corresponding to amino acids 1005 to 1037 of SEQ ID NO: 2, e.g., said alteration at one or more positions selected from the group consisting of positions: 1005, 1006, 1007, 1008, 1009, 1010, 101 1 , 1012, 1013, 1014, 1015, 1016, 1017, 1018, 1019, 1020, 1021 , 1022, 1023, 1024, 1025, 1026, 1027, 1028, 1029, 1030, 1031 , 1032, 1033, 1034, 1035, 1036 and 1037, wherein said positions correspond to amino acid positions of SEQ ID NO: 2.
In some aspects, the present invention relates to detergent compositions comprising a xanthan lyase variant comprising an alteration (e.g., a substitution, deletion or insertion) at one or more positions in two or more regions selected from the group consisting of:
i) region 7 corresponding to amino acids 1 to 153 of SEQ ID NO: 2,
ii) region 8 corresponding to amino acids 177 to 613 of SEQ ID NO: 2,
iii) region 9 corresponding to amino acids 659 to 730 of SEQ ID NO: 2,
iv) region 10 corresponding to amino acids 804 to 806 of SEQ ID NO: 2, e
v) region 11 corresponding to amino acids 847 to 871 of SEQ ID NO: 2,
vi) region 12 corresponding to amino acids 886 to 902 of SEQ ID NO: 2, and
vii) region 13 corresponding to amino acids 1005 to 1037 of SEQ ID NO: 2.
In some aspects, the present invention relates to detergent compositions comprising a xanthan lyase variant having an alteration (e.g., a substitution, deletion or insertion) at one or more positions selected from the group consisting of positions: 9, 15, 46, 58, 66, 89, 95, 100, 106, 109, 183, 188, 190, 203, 204, 221 , 229, 234, 238, 240, 242, 243, 257, 258, 291 , 293, 316, 320, 324, 329, 333, 339, 341 , 352, 354, 360, 377, 399, 400, 419, 440, 450, 451 , 454, 458, 481 , 492, 567, 568, 578, 579, 582, 664, 672, 703, 728, 843, 855, 887, 892, 1008 and 1016 of SEQ ID NO: 2.
In one aspect, the present invention relates to detergent compositions comprising a xanthan lyase variant of the invention further having an alteration at one or more positions selected from the group consisting of positions 624, 631 , 635, 649, 656, 752, 752, 754, 757, 769, 775, 777, 800, 801 , 875, 911 , and 915 wherein numbering is according to SEQ ID NO: 2.
In some aspects, the present invention relates to detergent compositions comprising a xanthan lyase variant having one or more substitutions selected from the group consisting of: K9R, N15T, L46D, A58L, S66H, Q89Y, K95E, S100D, N106Y, Q109R, Q109D, Q109F, Q109K, Q109A, K183Q, K183R, V188I, A190Q, A203P, K204R, A221 P, E229N, E229S, I234V, I238W, I238L, I238M, I240W, N242S, G243V, Y257W, R258E, K291 R, A293G, A293P, K316R, K320R, L324Q, K329R, K333R, L339M, 1341 P, V352I, S354P, K360R, K360G, F377Y, N399K, K400R, F419Y, N440K, D450P, K451 E, K451 R, A454V, D458S, K481 R, A492L, A492H, K567R, G568A, S578K, S578R, S579R, S579K, S582K, A624E, T631 N, S635E, T649K, I656V, T664K, N672D, I703L, M728V, G738L, P752K, P752R, G753E, S754E, S754R, S757D, A769D, L775A, D777R, V800P, D801 G, A843P, K855R, K875T, K887R, N892Y, N892W, N892F, A91 1V, T915A, N1008D and K1016T.
In a preferred embodiment, the xanthan lyase variant comprised in the detergent compositions of the invention comprises one of the following set of substitutions:
Figure imgf000007_0001
Figure imgf000008_0001
Figure imgf000009_0001
Figure imgf000010_0001
In a more preferred embodiment, the xanthan lyase variant comprised in the detergent compositions of the invention comprises one of the following set of substitutions:
Figure imgf000010_0002
Figure imgf000011_0001
Figure imgf000012_0001
In a particular aspect, the xanthan lyase variant as described herein is one that does not comprise any amino acid alteration at a position outside of regions 7, 8, 9, 10, 1 1 , 12 and 13. In this aspect, the xanthan lyase variant thus does not comprise any alteration (e.g., a substitution, deletion or insertion) in a region selected from the group consisting of: region 1 corresponding to amino acids 154 to 176 of SEQ ID NO: 2, region 2 corresponding to amino acids 614 to 658 of SEQ ID NO: 2, region 3 corresponding to amino acids 731 to 803 of SEQ ID NO: 2, region 4 corresponding to amino acids 807 to 846 of SEQ ID NO: 2, region 5 corresponding to amino acids 872 to 885 of SEQ ID NO: 2, and region 6 corresponding to amino acids 903 to 1004 of SEQ ID NO: 2.
In some aspects, the present invention relates to detergent compositions comprising a xanthan lyase variant having activity on xanthan gum; preferably said activity comprises xanthan lyase EC 4.2.2.12 activity, further preferably said activity is xanthan lyase EC 4.2.2.12 activity.
In some aspects, the present invention relates to detergent compositions comprising a xanthan lyase variant having an improved stability in a detergent composition compared to a parent xanthan lyase (e.g., with SEQ ID NO: 2).
In some aspects, the present invention relates to detergent compositions comprising a xanthan lyase variant having a half-life improvement factor (HIF) of >1 .0 relative to a parent xanthan lyase.
In some aspects, the present invention relates to detergent compositions comprising at least one xanthan lyase variant as described herein. In another aspect, the invention relates to detergent compositions comprising a composition comprising an isolated xanthan lyase variant having activity on xanthan gum as described herein. In a further aspect, the composition further comprises an isolated polypeptide having GH9 endoglucanase activity. In a further aspect, the composition further comprises an isolated polypeptide having xanthan endoglucanase activity. In a preferred embodiment, the invention relates to a composition comprising a xanthan lyase variant of the invention and an isolated polypeptide having xanthan endoglucanase activity.
In another aspect, a detergent composition of the invention comprises one or more detergent components for degrading xanthan gum.
In some aspects, the present invention relates to use of a detergent composition of the present invention, wherein said use is selected from the group consisting of: use for degrading xanthan gum, and use in a cleaning process, such as laundry or hard surface cleaning such as dish wash.
In some aspects, the present invention further relates to the use of a detergent composition of the invention for degrading xanthan gum or for washing or cleaning textiles and/or hard surfaces, such as dish wash, wherein the composition has an enzyme detergency benefit. ln some aspects, the present invention also relates to methods of degrading xanthan gum using detergent compositions of the invention, wherein xanthan gum is on the surface of a hard surface or textile.
OVERVIEW OF SEQUENCE LISTING SEQ ID NO: 1 is the DNA sequence of the parent mature xanthan lyase from a strain of a Paenibacillus sp.
SEQ ID NO: 2 is the amino acid sequence of the mature polypeptide encoded by SEQ ID NO: 1. DEFINITIONS
cDNA: The term "cDNA" means a DNA molecule that can be prepared by reverse transcription from a mature, spliced, mRNA molecule obtained from a eukaryotic or prokaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA. The initial, primary RNA transcript is a precursor to mRNA that is processed through a series of steps, including splicing, before appearing as mature spliced mRNA.
Cleaning or Detergent Application: the term "cleaning or detergent application" means applying the xanthan lyase of the application in any composition for the purpose of cleaning or washing, by hand, machine or automated, a hard surface or a textile.
Cleaning Composition: the term "cleaning composition" refers to compositions that find use in the removal of undesired compounds from items to be cleaned, such as textiles, dishes, and hard surfaces. The terms encompass any materials/compounds selected for the particular type of cleaning composition desired and the form of the product (e.g., liquid, gel, powder, granulate, paste, or spray compositions) and includes, but is not limited to, detergent compositions (e.g., liquid and/or solid laundry detergents and fine fabric detergents; hard surface cleaning formulations, such as for glass, wood, ceramic and metal counter tops and windows; carpet cleaners; oven cleaners; fabric fresheners; fabric softeners; and textile and laundry pre- spotters, as well as dish wash detergents). In addition to the xanthan lyase, the detergent formulation may contain one or more additional enzymes (such as xanthan lyases, proteases, amylases, lipases, cutinases, cellulases, xanthan lyases, xyloglucanases, pectinases, pectin lyases, xanthanases, peroxidases, haloperoxygenases, catalases and mannanases, or any mixture thereof), and/or components such as surfactants, builders, chelators or chelating agents, bleach system or bleach components, polymers, fabric conditioners, foam boosters, suds suppressors, dyes, perfume, tannish inhibitors, optical brighteners, bactericides, fungicides, soil suspending agents, anti-corrosion agents, enzyme inhibitors or stabilizers, enzyme activators, transferase(s), hydrolytic enzymes, oxido reductases, bluing agents and fluorescent dyes, antioxidants, and solubilizers.
Coding sequence: The term "coding sequence" means a polynucleotide, which directly specifies the amino acid sequence of a polypeptide. The boundaries of the coding sequence are generally determined by an open reading frame, which begins with a start codon such as ATG, GTG, or TTG and ends with a stop codon such as TAA, TAG, or TGA. The coding sequence may be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.
Colour clarification: During washing and wearing loose or broken fibers can accumulate on the surface of the fabrics. One consequence can be that the colours of the fabric appear less bright or less intense because of the surface contaminations. Removal of the loose or broken fibers from the textile will partly restore the original colours and looks of the textile. By the term "colour clarification", as used herein, is meant the partial restoration of the initial colours of textile.
Control sequences: The term "control sequences" means nucleic acid sequences necessary for expression of a polynucleotide encoding a mature polypeptide of the present invention. Each control sequence may be native (i.e., from the same gene) or foreign (i.e., from a different gene) to the polynucleotide encoding the polypeptide or native or foreign to each other. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and trans- lational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the polynucleotide encoding a polypeptide.
Corresponding to: The term "corresponding to" as used herein, refers to a way of determining the specific amino acid of a sequence wherein reference is made to a specific amino acid sequence. E.g. for the purposes of the present invention, when references are made to specific amino acid positions, the skilled person would be able to align another amino acid sequence to said amino acid sequence that reference has been made to, in order to determine which specific amino acid may be of interest in said another amino acid sequence. Alignment of another amino acid sequence with e.g. the sequence as set forth in SEQ ID NO: 2, or any other sequence listed herein, has been described elsewhere herein. Alternative alignment methods may be used, and are well-known for the skilled person.
Degrading xanthan gum and xanthan gum degrading activity: The terms "degrading xanthan gum" and "xanthan gum degrading activity" are used interchangeably and are defined as the depolymerisation, degradation or breaking down of xanthan gum into smaller components. The degradation of xanthan gum can either be the removal of one or more side chain saccharides, the cutting of the backbone of xanthan gum into smaller components or the removal of one or more side chain saccharides and the cutting of the backbone of xanthan gum into smaller components. A preferred assay for measuring degradation of xanthan gum is described in Example 3 herein. Non-limiting examples of the xanthan gum degrading activity include xanthan lyase EC 4.2.2.12 activity.
Detergent component: the term "detergent component" is defined herein to mean the types of chemicals which can be used in detergent compositions. Examples of detergent components are surfactants, hydrotropes, builders, co-builders, chelators or chelating agents, bleaching system or bleach components, polymers, fabric hueing agents, fabric conditioners, foam boosters, suds suppressors, dispersants, dye transfer inhibitors, fluorescent whitening agents, perfume, optical brighteners, bactericides, fungicides, soil suspending agents, soil release polymers, anti-redeposition agents, enzyme inhibitors or stabilizers, enzyme activators, antioxidants, and solubilizers. The detergent composition may comprise of one or more of any type of detergent component.
Detergent composition: the term "detergent composition" refers to compositions that find use in the removal of undesired compounds from items to be cleaned, such as textiles, dishes, and hard surfaces. The detergent composition may be used to e.g. clean textiles, dishes and hard surfaces for both household cleaning and industrial cleaning. The terms encompass any materials/compounds selected for the particular type of cleaning composition desired and the form of the product (e.g., liquid, gel, powder, granulate, paste, or spray compositions) and includes, but is not limited to, detergent compositions (e.g., liquid and/or solid laundry detergents and fine fabric detergents; hard surface cleaning formulations, such as for glass, wood, ceramic and metal counter tops and windows; carpet cleaners; oven cleaners; fabric fresheners; fabric softeners; and textile and laundry pre-spotters, as well as dish wash detergents). In addition to containing a xanthan lyase of the invention and/or a GH9 endoglucanase, the detergent formulation may contain one or more additional enzymes (such as endoglucanases, xanthan lyases, proteases, amylases, lichenases, lipases, cutinases, cellulases, xanthan lyases, xyloglucanases, pectinases, pectin lyases, xanthanases, peroxidases, haloperoxygenases, catalases and mannanases, or any mixture thereof), and/or components such as surfactants, builders, chelators or chelating agents, bleach system or bleach components, polymers, fabric conditioners, foam boosters, suds suppressors, dyes, perfume, tannish inhibitors, optical bright- eners, bactericides, fungicides, soil suspending agents, anti-corrosion agents, enzyme inhibitors or stabilizers, enzyme activators, transferase(s), hydrolytic enzymes, oxido reductases, bluing agents and fluorescent dyes, antioxidants, and solubilizers.
Dish wash: The term "dish wash" refers to all forms of washing dishes, e.g. by hand or automatic dish wash. Washing dishes includes, but is not limited to, the cleaning of all forms of crockery such as plates, cups, glasses, bowls, all forms of cutlery such as spoons, knives, forks and serving utensils as well as ceramics, plastics, metals, china, glass and acrylics.
Dish washing composition: The term "dish washing composition" refers to all forms of compositions for cleaning hard surfaces. The present invention is not restricted to any particular type of dish wash composition or any particular detergent.
Endoglucanase: The term "endoglucanase" or "EG" means an endo-1 ,4- or endo-1 ,3;1 ,4-beta-D-glu- can 4-glucanohydrolase (e.g., EC 3.2.1.4) that catalyses endohydrolysis of 1 ,4-beta-D-glycosidic linkages in cellulose, cellulose derivatives (such as carboxymethyl cellulose and hydroxyethyl cellulose), lichenin, beta-1 ,4 bonds in mixed beta-1 ,3/beta-1 ,4 glucans such as cereal beta-D-glucans, xyloglucans, xanthans and other plant material containing cellulosic components. Endoglucanase activity can be determined by measuring reduction in substrate viscosity or increase in reducing ends determined by a reducing sugar assay (Zhang et al., 2006, Biotechnology Advances 24: 452-481 ). Enzyme detergency benefit: The term "enzyme detergency benefit" is defined herein as the advantageous effect an enzyme may add to a detergent compared to the same detergent without the enzyme. Important detergency benefits which can be provided by enzymes are stain removal with no or very little visible soils after washing and or cleaning, prevention or reduction of redeposition of soils released in the washing process an effect that also is termed anti-redeposition, restoring fully or partly the whiteness of textiles, which originally were white but after repeated use and wash have obtained a greyish or yellowish appearance an effect that also is termed whitening. Textile care benefits, which are not directly related to catalytic stain removal or prevention of redeposition of soils are also important for enzyme detergency benefits. Examples of such textile care benefits are prevention or reduction of dye transfer from one fabric to another fabric or another part of the same fabric an effect that is also termed dye transfer inhibition or anti- backstaining, removal of protruding or broken fibers from a fabric surface to decrease pilling tendencies or remove already existing pills or fuzz an effect that also is termed anti-pilling, improvement of the fabric- softness, colour clarification of the fabric and removal of particulate soils which are trapped in the fibers of the fabric or garment. Enzymatic bleaching is a further enzyme detergency benefit where the catalytic ac- tivity generally is used to catalyze the formation of bleaching component such as hydrogen peroxide or other peroxides.
Expression: The term "expression" includes any step involved in the production of a polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.
Expression vector: The term "expression vector" means a linear or circular DNA molecule that comprises a polynucleotide encoding a polypeptide and is operably linked to control sequences that provide for its expression.
Fragment: The term "fragment" means a polypeptide having one or more amino acids absent from the amino and/or carboxyl terminus of a mature polypeptide; wherein the fragment has xanthan lyase activity. In one aspect, a fragment contains at least 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94% or 95% of the number of amino acids of the mature polypeptide.
Endoglucanase variant having activity on xanthan gum pre-treated with xanthan lyase: The term "Endoglucanase variant having activity on xanthan gum pre-treated with xanthan lyase" or an "endoglucanase having activity on xanthan gum pre-treated with xanthan lyase and belonging to the GH9 class of glycosyl hydrolases" is defined as a polypeptide comprising a domain belonging to the GH9 class of glycosyl hydrolases, and having activity (e.g., enzymatic activity, xanthan degrading activity, endoglucanase EC 3.2.1 .4 activity) on xanthan gum pre-treated with xanthan lyase.
Xanthan lyase variant having activity on xanthan gum: The term "Xanthan lyase variant having activity on xanthan gum" is defined as a polypeptide that cleaves the β-D-mannosyl-β-D-1 ,4-glucuronosyl bond of xanthan (e.g., xanthan lyase EC 4.2.2.12 activity). A preferred assay for measuring activity on xan- than gum is disclosed in Example 3 herein. Examples of the xanthan lyase variants having activity on xan- than gum, are xanthan lyase polypeptides as such. Thus, polypeptides that that cleaves the β-D-mannosyl- β-D-l ,4-glucuronosyl bond of xanthan.
Half-life: The term "half-life" refers to the time it takes for an enzyme to lose half of its enzymatic activity under a given set of conditions. It is denoted as T½ and is measured in hours (hrs). Half-lifes can be calculated at a given detergent concentration and storage temperature for parent (e.g. wild-type) and/or variants, as the degradation follows an exponential decay and the incubation time (hours) is known, i.e. according to the following formulas:
T1 /2 (variant) = (Ln (0.5)/Ln (RA-variant/100))*Time
T112 (wild-type) = (Ln (0.5)/Ln (RA-wild-type/100))*Time
Where 'RA' is the residual activity in percent and 'Time' is the incubation time in hours.
Half-life improvement factor: The term "Half-life improvement factor" or "H IF" is the improvement of half-life of a variant compared to the parent polypeptide, such as the parent xanthan lyase. A half-life improvement factor (HIF) under a given set of storage conditions (detergent concentration and temperature) can be calculated as
Figure imgf000017_0001
Where the wild-type (wt) is incubated under the same storage conditions (detergent concentration and incubation temperature) as the variant. In the cases, where the difference in stability between wild-type and variant is too big to accurately assess half-life for both wild-type and variant using the same incubation time, the incubation time for wild-type and variant is different e.g. 1 hr for wild-type and 168 hrs for the most stable variants.
A preferred way of calculating HIF is also described in Example 3 herein. The half-life improvement factor may also be calculated based on the half-life of a parent xanthan lyase (see the definition of "parent" below) that is not necessarily a wild-type.
Hard surface cleaning: The term "Hard surface cleaning" is defined herein as cleaning of hard surfaces wherein hard surfaces may include floors, tables, walls, roofs etc. as well as surfaces of hard objects such as cars (car wash) and dishes (dish wash). Dish washing includes but are not limited to cleaning of plates, cups, glasses, bowls, and cutlery such as spoons, knives, forks, serving utensils, ceramics, plastics, metals, china, glass and acrylics.
Host cell: The term "host cell" means any cell type that is susceptible to transformation, transfection, transduction, or the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.
Improved property: The term "improved property" means a characteristic associated with a variant that is improved compared to the parent. Such improved properties include, but are not limited to, catalytic efficiency, catalytic rate, chemical stability, oxidation stability, pH activity, pH stability, specific activity, stability under storage conditions, chelator stability, substrate binding, substrate cleavage, substrate specificity, substrate stability, surface properties, thermal activity, and thermostability.
Improved wash performance: The term "improved wash performance" is defined herein as a (variant) enzyme (also a blend of enzymes, not necessarily only variants but also backbones, and in combination with certain cleaning composition etc.) displaying an alteration of the wash performance of a protease variant relative to the wash performance of the parent protease variant e.g. by increased stain removal. The term "wash performance" includes wash performance in laundry but also e.g. in dish wash.
Isolated: The term "isolated" means a substance in a form or environment that does not occur in nature. Non-limiting examples of isolated substances include (1 ) any non-naturally occurring substance, (2) any substance including, but not limited to, any enzyme, variant, nucleic acid , protein, peptide or cofactor, that is at least partially removed from one or more or all of the naturally occurring constituents with which it is associated in nature; (3) any substance modified by the hand of man relative to that substance found in nature; or (4) any substance modified by increasing the amount of the substance relative to other components with which it is naturally associated (e.g. , multiple copies of a gene encoding the substance; use of a stronger promoter than the promoter naturally associated with the gene encoding the substance). An isolated substance may be present in a fermentation broth sample.
Laundering: The term "laundering" relates to both household laundering and industrial laundering and means the process of treating textiles with a solution containing a cleaning or detergent composition of the present invention. The laundering process can for example be carried out using e.g. a household or an industrial washing machine or can be carried out by hand .
Mature polypeptide: The term "mature polypeptide" means a polypeptide in its final form following translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc. In one aspect, the mature polypeptide is amino acids 1 to 1037 of SEQ ID NO: 2.
It is known in the art that a host cell may produce a mixture of two of more different mature polypeptides (i.e. , with a different C-terminal and/or N-terminal amino acid) expressed by the same polynucleotide. It is also known in the art that different host cells process polypeptides differently, and thus, one host cell expressing a polynucleotide may produce a different mature polypeptide (e.g. , having a different C-terminal and/or N-terminal amino acid) as compared to another host cell expressing the same polynucleotide.
Mature polypeptide coding sequence: The term "mature polypeptide coding sequence" means a polynucleotide that encodes a mature polypeptide having enzymatic activity such as activity on xanthan gum pre-treated with xanthan lyase or xanthan lyase activity. In one aspect, the mature polypeptide coding sequence is nucleotides 1 to 31 1 1 of SEQ ID NO: 1 .
Mutant: The term "mutant" means a polynucleotide encoding a variant. Nucleic acid construct: The term "nucleic acid construct" means a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or which is synthetic, which comprises one or more control sequences.
Operably linked: The term "operably linked" means a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide such that the control sequence directs expression of the coding sequence.
Parent: The term "parent" or "parent xanthan lyase" means any polypeptide with xanthan lyase activity to which an alteration is made to produce the enzyme variants as described herein. In one aspect, the parent is a xanthan lyase having the identical amino acid sequence of the variant, but not having the alterations at one or more of the specified positions. It will be understood that the expression "having identical amino acid sequence" relates to 100% sequence identity. Non-limiting examples of parent xanthan lyases include the mature parent xanthan lyase having SEQ ID NO: 2.
Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity". For purposes of the present invention, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:
(Identical Residues x 100)/(Length of Alignment - Total Number of Gaps in Alignment)
For purposes of the present invention, the sequence identity between two deoxyribonucleotide se- quences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labelled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:
(Identical Deoxyribonucleotides x 100)/(Length of Alignment - Total Number of Gaps in Alignment)
Stringency conditions: The different stringency conditions are defined as follows.
The term "very low stringency conditions" means for probes of at least 100 nucleotides in length, pre- hybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 25% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 45°C.
The term "low stringency conditions" means for probes of at least 100 nucleotides in length, prehybrid- ization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 25% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 50°C.
The term "medium stringency conditions" means for probes of at least 100 nucleotides in length, pre- hybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 35% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 55°C.
The term "medium-high stringency conditions" means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 35% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 60°C.
The term "high stringency conditions" means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 65°C.
The term "very high stringency conditions" means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 70°C.
Subsequence: The term "subsequence" means a polynucleotide having one or more nucleotides absent from the 5' and/or 3' end of a mature polypeptide coding sequence; wherein the subsequence encodes a fragment having enzymatic activity, such as activity on xanthan gum pre-treated with xanthan lyase or xanthan lyase activity.
Textile: The term "textile" means any textile material including yarns, yarn intermediates, fibers, non- woven materials, natural materials, synthetic materials, and any other textile material, fabrics made of these materials and products made from fabrics (e.g., garments and other articles). The textile or fabric may be in the form of knits, wovens, denims, non-wovens, felts, yarns, and towelling. The textile may be cellulose based such as natural cellulosics, including cotton, flax/linen, jute, ramie, sisal or coir or manmade cellulo- sics (e.g. originating from wood pulp) including viscose/rayon, ramie, cellulose acetate fibers (tricell), lyocell or blends thereof. The textile or fabric may also be non-cellulose based such as natural polyamides including wool, camel, cashmere, mohair, rabbit and silk or synthetic polymer such as nylon, aramid, polyester, acrylic, polypropylene and spandex elastane, or blends thereof as well as blend of cellulose based and non- cellulose based fibers. Examples of blends are blends of cotton and/or rayon/viscose with one or more companion material such as wool, synthetic fibers (e.g. polyamide fibers, acrylic fibers, polyester fibers, polyvinyl alcohol fibers, polyvinyl chloride fibers, polyurethane fibers, polyurea fibers, aramid fibers), and cellulose-containing fibers (e.g. rayon/viscose, ramie, flax/linen, jute, cellulose acetate fibers, lyocell). Fabric may be conventional washable laundry, for example stained household laundry. When the term fabric or garment is used it is intended to include the broader term textiles as well.
Textile care benefit: "Textile care benefits", which are not directly related to catalytic stain removal or prevention of redeposition of soils, are also important for enzyme detergency benefits. Examples of such textile care benefits are prevention or reduction of dye transfer from one textile to another textile or another part of the same textile an effect that is also termed dye transfer inhibition or anti-backstaining, removal of protruding or broken fibers from a textile surface to decrease pilling tendencies or remove already existing pills or fuzz an effect that also is termed anti-pilling, improvement of the textile-softness, colour clarification of the textile and removal of particulate soils which are trapped in the fibers of the textile. Enzymatic bleaching is a further enzyme detergency benefit where the catalytic activity generally is used to catalyse the formation of bleaching component such as hydrogen peroxide or other peroxides or other bleaching species.
Variant: The term "variant" means a polypeptide (e.g., a xanthan lyase polypeptide) comprising an alteration, i.e., a substitution, insertion, and/or deletion, at one or more positions. A substitution means replacement of the amino acid occupying a position with a different amino acid; a deletion means removal of the amino acid occupying a position; and an insertion means adding one or more amino acids, e.g., 1 -5 amino acids adjacent to and immediately following the amino acid occupying a position. Non-limiting examples of xanthan lyase variants as described herein include xanthan lyase variants having an activity on xanthan gum. Non-limiting examples of variants as described herein further include variants having at least 20%, e.g., at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% xanthan lyase activity of the mature parent xanthan lyase of SEQ ID NO: 2. A preferred assay for measuring activity on xanthan gum is disclosed in Example 3 herein.
Stability: The term "stability" means resistance or the degree of resistance to change, unfolding, dis- integration, denaturation or activity loss. Non-limiting examples of stability include conformational stability, storage stability and stability during use, e.g. during a wash process and reflects the stability of a polypeptide (e.g. a xanthan lyase variant according to the invention) as a function of time, e.g. how much activity is retained when said polypeptide (e.g. said xanthan lyase variant) is kept in solution, in particular in a detergent solution. The stability is influenced by many factors, e.g. presence of chelator(s), pH, temperature, detergent composition, e.g. amount of builder(s), surfactant(s), chelator(s) etc. The xanthan lyase stability may be measured using a half-life improvement factor (HIF) as described in Example 3 herein, e.g. determined relative to the xanthan lyase having SEQ ID NO: 2.
Improved stability: The term "improved stability" or "increased stability" is defined herein as increased stability in a detergent composition (e.g. , in solutions), relative to the stability of the parent xanthan lyase, relative to a xanthan lyase having the identical amino acid sequence of the variant, but not having the alterations at one or more of the specified positions, or relative to SEQ ID NO: 2. The terms "improved stability" and "increased stability" include "improved chemical stability", "detergent stability" and "improved detergent stability".
Improved chemical stability: The term "improved chemical stability" is defined herein as a variant enzyme displaying retention of enzymatic activity after a period of incubation in the presence of a chemical or chemicals, either naturally occurring or synthetic, which reduces the enzymatic activity of the parent enzyme. Improved chemical stability may also result in variants being more able (e.g., better that the parent) to catalyze a reaction in the presence of such chemicals. In a particular aspect of the invention the improved chemical stability is an improved stability in a detergent, in particular in a liquid detergent. The term "deter- gent stability" or "improved detergent stability is in particular an improved stability of the xanthan lyase compared to the parent xanthan lyase, when a xanthan lyase variant of the present invention is mixed into a liquid detergent formulation.
Conformational stability: The term "conformational stability" means a resistance or a degree of resistance to conformational change, unfolding or disintegration. Accordingly, the term "less conformationally stable" means less resistant or having lesser degree of resistance to conformational change, unfolding or disintegration.
Instability: The term "instability" means lack of stability. Non-limiting examples of instability include conformational instability, unfolding, denaturation, disintegration, activity loss.
Wash performance: The term "wash performance" is used as an enzyme's ability to remove stains present on the object to be cleaned during e.g. wash or hard surface cleaning. The improvement in the wash performance may be quantified by calculating the so-called intensity value (Int) in 'Automatic Mechanical Stress Assay (AMSA) for laundry' or the remission value (Rem).
Conventions for Designation of Variants
For purposes of the present invention, the mature polypeptide disclosed in SEQ ID NO: 2 is used to determine the corresponding amino acid residue in another xanthan lyase. The amino acid sequence of another xanthan lyase is aligned with the mature polypeptide disclosed in SEQ ID NO: 2, and based on the alignment, the amino acid position number corresponding to any amino acid residue in the mature polypeptide disclosed in SEQ ID NO: 2 is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice , 2000, e Trteanl.ds Genet. 16: 276-277), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.
Identification of the corresponding amino acid residue in another xanthan lyase can be determined by an alignment of multiple polypeptide sequences using several computer programs including, but not limited to, MUSCLE (multiple sequence comparison by log-expectation; version 3.5 or later; Edgar, 2004, Nucleic
Acids Research 32: 1792-1797), MAFFT (version 6.857 or later; Katoh and Kuma, 2002, Nucleic Acids Research 30: 3059-3066; Katoh ei a/. , 2005, Nucleic Acids Research 33: 511 -518; Katoh and Toh, 2007, Bioinformatics 23: 372-374; Katoh ei a/., 2009, Methods in Molecular Biology 537:_39-64; Katoh and Toh, 2010, Bioinformatics 26:_1899-1900), and EMBOSS EMMA employing ClustalW (1 .83 or later; Thompson ei a/. , 1994, Nucleic Acids Research 22: 4673-4680), using their respective default parameters.
When the other enzyme has diverged from the mature polypeptide of SEQ ID NO: 2 such that traditional sequence-based comparison fails to detect their relationship (Lindahl and Elofsson, 2000, J. Mol. Biol. 295: 613-615), other pairwise sequence comparison algorithms can be used. Greater sensitivity in sequence- based searching can be attained using search programs that utilize probabilistic representations of poly- peptide families (profiles) to search databases. For example, the PSI-BLAST program generates profiles through an iterative database search process and is capable of detecting remote homologs (Altschul et ai , 1997, Nucleic Acids Res. 25: 3389-3402). Even greater sensitivity can be achieved if the family or super- family for the polypeptide has one or more representatives in the protein structure databases. Programs such as GenTHREADER (Jones, 1999, J. Mol. Biol. 287: 797-815; McGuffin and Jones, 2003, Bioinformat- ics 19: 874-881 ) utilize information from a variety of sources (PSI-BLAST, secondary structure prediction, structural alignment profiles, and solvation potentials) as input to a neural network that predicts the structural fold for a query sequence. Similarly, the method of Gough et al , 2000, J. Mol. Biol. 313: 903-919, can be used to align a sequence of unknown structure with the superfamily models present in the SCOP database. These alignments can in turn be used to generate homology models for the polypeptide, and such models can be assessed for accuracy using a variety of tools developed for that purpose.
For proteins of known structure, several tools and resources are available for retrieving and generating structural alignments. For example, the SCOP superfamilies of proteins have been structurally aligned, and those alignments are accessible and downloadable. Two or more protein structures can be aligned using a variety of algorithms such as the distance alignment matrix (Holm and Sander, 1998, Proteins 33: 88-96) or combinatorial extension (Shindyalov and Bourne, 1998, Protein Engineering 1 1 : 739-747), and implementation of these algorithms can additionally be utilized to query structure databases with a structure of interest in order to discover possible structural homologs (e.g., Holm and Park, 2000, Bioinformatics 16: 566-567).
In describing the variants, the nomenclature described below is adapted for ease of reference. The accepted lUPAC single letter or three letter amino acid abbreviation is employed.
Substitutions. For an amino acid substitution, the following nomenclature is used: Original amino acid, position, substituted amino acid. Accordingly, the substitution of threonine at position 226 with alanine is designated as "Thr226Ala" or "T226A". Multiple mutations are separated by addition marks ("+"), e.g., "Gly205Arg + Ser41 1 Phe" or "G205R + S411 F", representing substitutions at positions 205 and 41 1 of glycine (G) with arginine (R) and serine (S) with phenylalanine (F), respectively.
Deletions. For an amino acid deletion, the following nomenclature is used: Original amino acid, position, *. Accordingly, the deletion of glycine at position 195 is designated as "Gly195*" or "G195*". Multiple deletions are separated by addition marks ("+"), e.g., "Gly195* + Ser411 *" or "G195* + S41 1 *".
Insertions. For an amino acid insertion, the following nomenclature is used: Original amino acid, position, original amino acid, inserted amino acid. Accordingly, the insertion of lysine after glycine at position 195 is designated "Gly195GlyLys" or "G195GK". An insertion of multiple amino acids is designated [Original amino acid, position, original amino acid, inserted amino acid #1 , inserted amino acid #2; etc.]. For example, the insertion of lysine and alanine after glycine at position 195 is indicated as "Gly195GlyLysAla" or "G195GKA". An indication of an insertion at a particular position is understood as being an insertion after the original amino acid residue. For example, an "insertion at position 195" is understood to be an insertion after the original residue in position 195.
In such cases the inserted amino acid residue(s) are numbered by the addition of lower case letters to the position number of the amino acid residue preceding the inserted amino acid residue(s). In the above example, the sequence would thus be:
Figure imgf000024_0001
Multiple alterations. Variants comprising multiple alterations are separated by addition marks ("+"), e.g., "Arg170Tyr+Gly195Glu" or "R170Y+G195E" representing a substitution of arginine and glycine at positions 170 and 195 with tyrosine and glutamic acid, respectively. Variants comprising multiple alterations are separated by a comma (","), e.g., "R170Y,G195E".
Different alterations. Where different alterations can be introduced at a position, the different alterations are separated by a comma, e.g., "Arg170Tyr,Glu" represents a substitution of arginine at position 170 with tyrosine or glutamic acid. Thus, "Tyr167Gly,Ala+Arg170Gly,Ala" designates the following variants:
"Tyr167Gly+Arg170Gly", "Tyr167Gly+Arg170Ala", "Tyr167Ala+Arg170Gly", and
"Tyr167Ala+Arg170Ala".
Alternatively, different alterations or may be indicated using brackets, e.g., Arg170[Tyr,Gly] or in one- letter code R170 [Y,G].
DETAILED DESCRIPTION OF THE INVENTION
The known xanthan lyase having SEQ ID NO: 2 is a large enzyme (>1000 residues), it is therefore extremely laborious and expensive to target its properties for improvement of, e.g., stability in a detergent composition. In some aspects, the present invention narrows down the number of residues to target when trying to stabilize xanthan lyase molecules using protein engineering to a region selected from the group consisting of: region 7 corresponding to amino acids 1 to 153 of SEQ ID NO: 2, region 8 corresponding to amino acids 177 to 613 of SEQ ID NO: 2, region 9 corresponding to amino acids 659 to 730 of SEQ ID NO: 2, region 10 corresponding to amino acids 804 to 806 of SEQ ID NO: 2, region 1 1 corresponding to amino acids 847 to 871 of SEQ ID NO: 2, region 12 corresponding to amino acids 886 to 902 of SEQ ID NO: 2, and region 13 corresponding to amino acids 1005 to 1037 of SEQ ID NO: 2.
In one embodiment, the present invention dramatically narrows down the number of residues to target when trying to stabilize xanthan lyase molecules using protein engineering.
Variants
In one embodiment, the regions in the protein sequence of the known xanthan lyase having SEQ ID NO: 2 that have an impact on stability of the molecule, e.g. during storage in a liquid detergent composition, are the following: region 7 corresponding to amino acids 1 to 153 of SEQ ID NO: 2, region 8 corresponding to amino acids 177 to 613 of SEQ ID NO: 2, region 9 corresponding to amino acids 659 to 730 of SEQ ID NO: 2, region 10 corresponding to amino acids 804 to 806 of SEQ ID NO: 2, region 11 corresponding to amino acids 847 to 871 of SEQ ID NO: 2, region 12 corresponding to amino acids 886 to 902 of SEQ ID NO: 2, and region 13 corresponding to amino acids 1005 to 1037 of SEQ ID NO: 2. This embodiment relates to an important guidance on where to mutate a xanthan lyase in order to stabilize the molecule in a deter- gent.
In one embodiment, the present invention relates to detergent compositions comprising a xanthan lyase variant, comprising an alteration (e.g., a substitution, deletion or insertion) at one or more positions in a region selected from the group consisting of: region 7 corresponding to amino acids 1 to 153 of SEQ ID NO: 2, region 8 corresponding to amino acids 177 to 613 of SEQ ID NO: 2, region 9 corresponding to amino acids 659 to 730 of SEQ ID NO: 2, region 10 corresponding to amino acids 804 to 806 of SEQ ID NO: 2, region 1 1 corresponding to amino acids 847 to 871 of SEQ ID NO: 2, region 12 corresponding to amino acids 886 to 902 of SEQ ID NO: 2, and region 13 corresponding to amino acids 1005 to 1037 of SEQ ID NO: 2, wherein said variant has at least 60% and less than 100% sequence identity to SEQ ID NO: 2; preferably said xanthan lyase variant has activity on xanthan gum, further preferably said activity is a xan- than gum degrading activity.
In one embodiment, the present invention relates to detergent compositions comprising a xanthan lyase variant, comprising an alteration (e.g., a substitution, deletion or insertion) at one or more positions in a region selected from the group consisting of:
i) region 7 corresponding to amino acids 1 to 153 of SEQ ID NO: 2,
ii) region 8 corresponding to amino acids 177 to 613 of SEQ ID NO: 2,
iii) region 9 corresponding to amino acids 659 to 730 of SEQ ID NO: 2, iv) region 10 corresponding to amino acids 804 to 806 of SEQ ID NO: 2,
v) region 1 1 corresponding to amino acids 847 to 871 of SEQ ID NO: 2,
vi) region 12 corresponding to amino acids 886 to 902 of SEQ ID NO: 2,
vii) region 13 corresponding to amino acids 1005 to 1037 of SEQ ID NO: 2.
In one embodiment, the present invention relates to detergent compositions comprising a xanthan lyase variant, comprising an alteration (e.g. , a substitution, deletion or insertion) at one or more positions in two or more regions selected from the group consisting of: region 7 corresponding to amino acids 1 to 153 of SEQ ID NO: 2, region 8 corresponding to amino acids 177 to 613 of SEQ ID NO: 2, region 9 corresponding to amino acids 659 to 730 of SEQ ID NO: 2, region 10 corresponding to amino acids 804 to 806 of SEQ ID NO: 2, region 1 1 corresponding to amino acids 847 to 871 of SEQ ID NO: 2, region 12 corresponding to amino acids 886 to 902 of SEQ ID NO: 2, and region 13 corresponding to amino acids 1005 to 1037 of SEQ ID NO: 2, wherein said variant has at least 60% and less than 100% sequence identity to SEQ ID NO: 2; preferably said xanthan lyase variant has activity on xanthan gum, further preferably said activity is a xanthan gum degrading activity.
In one embodiment, the present invention relates to detergent compositions comprising a xanthan lyase variant as described herein having multiple alterations (such as 2, 3, 4, 5, 6, 7, 8, 9 or 10) in one region (e.g. , of SEQ ID NO: 2 or another parent xanthan lyase) selected from the group consisting of: region 7 corresponding to amino acids 1 to 153 of SEQ ID NO: 2, region 8 corresponding to amino acids 177 to 613 of SEQ ID NO: 2, region 9 corresponding to amino acids 659 to 730 of SEQ ID NO: 2, region 10 correspond- ing to amino acids 804 to 806 of SEQ ID NO: 2, region 1 1 corresponding to amino acids 847 to 871 of SEQ ID NO: 2, region 12 corresponding to amino acids 886 to 902 of SEQ ID NO: 2, and region 13 corresponding to amino acids 1005 to 1037 of SEQ ID NO: 2, wherein said variant has at least 60% and less than 100% sequence identity to SEQ ID NO: 2, preferably said variant has activity on xanthan gum, further preferably said activity is a xanthan gum degrading activity.
In one embodiment, the present invention relates to detergent compositions comprising a xanthan lyase variant as described herein having multiple alterations (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10) in multiple regions (e.g., 2, 3, 4, 5, 6 or 7) (e.g., of SEQ ID NO: 2 or another parent xanthan lyase) selected from the group consisting of: region 7 corresponding to amino acids 1 to 153 of SEQ ID NO: 2, region 8 corresponding to amino acids 177 to 613 of SEQ ID NO: 2, region 9 corresponding to amino acids 659 to 730 of SEQ ID NO: 2, region 10 corresponding to amino acids 804 to 806 of SEQ ID NO: 2, region 1 1 corresponding to amino acids 847 to 871 of SEQ ID NO: 2, region 12 corresponding to amino acids 886 to 902 of SEQ ID NO: 2, and region 13 corresponding to amino acids 1005 to 1037 of SEQ ID NO: 2, wherein said variant has at least 60% and less than 100% sequence identity to SEQ ID NO: 2, preferably said variant has activity on xanthan gum, further preferably said activity is a xanthan gum degrading activity.
In one embodiment, the present invention relates to detergent compositions comprising xanthan lyase variants, comprising an alteration (e.g., a substitution, deletion or insertion) at one or more positions of the mature parent polypeptide (e.g., SEQ ID NO: 2), wherein each alteration is independently a substitution, insertion or deletion, wherein the variant has xanthan lyase activity.
In an embodiment, the variant has sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, to the amino acid sequence of the parent xanthan lyase.
In one embodiment, the variant has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to the mature polypeptide of SEQ ID NO: 2.
In one embodiment, the present invention relates to detergent compositions comprising a xanthan lyase variant as described herein, having at least 61 %, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 2.
In another aspect, a variant comprises an alteration at one or more positions corresponding to positions 9, 15, 46, 58, 66, 89, 95, 100, 106, 109, 183, 188, 190, 203, 204, 221 , 229, 234, 238, 240, 242, 243, 257, 258, 291 , 293, 316, 320, 324, 329, 333, 339, 341 , 352, 354, 360, 377, 399, 400, 419, 440, 450, 451 , 454, 458, 481 , 492, 567, 568, 578, 579, 582, 664, 672, 703, 728, 843, 855, 887, 892, 1008 and 1016. In another aspect, a variant comprises an alteration at two positions corresponding to any of positions 9, 15, 46, 58, 66, 89, 95, 100, 106, 109, 183, 188, 190, 203, 204, 221 , 229, 234, 238, 240, 242, 243, 257, 258, 291 , 293, 316, 320, 324, 329, 333, 339, 341 , 352, 354, 360, 377, 399, 400, 419, 440, 450, 451 , 454, 458, 481 , 492, 567, 568, 578, 579, 582, 664, 672, 703, 728, 843, 855, 887, 892, 1008 and 1016. In another aspect, a variant comprises an alteration at three positions corresponding to any of positions 9, 15, 46, 58, 66, 89, 95, 100, 106, 109, 183, 188, 190, 203, 204, 221 , 229, 234, 238, 240, 242, 243, 257, 258, 291 , 293, 316, 320, 324, 329, 333, 339, 341 , 352, 354, 360, 377, 399, 400, 419, 440, 450, 451 , 454, 458, 481 , 492, 567, 568, 578, 579, 582, 664, 672, 703, 728, 843, 855, 887, 892, 1008 and 1016. In another aspect, a variant com- prises an alteration at four or more positions, e.g. five, six, seven, eight, nine, ten or more positions, corresponding to positions 9, 15, 46, 58, 66, 89, 95, 100, 106, 109, 183, 188, 190, 203, 204, 221 , 229, 234, 238, 240, 242, 243, 257, 258, 291 , 293, 316, 320, 324, 329, 333, 339, 341 , 352, 354, 360, 377, 399, 400, 419, 440, 450, 451 , 454, 458, 481 , 492, 567, 568, 578, 579, 582, 664, 672, 703, 728, 843, 855, 887, 892, 1008 and 1016.
In one aspect, the variant comprises or consists of an alteration at a position corresponding to position
9. In one embodiment, the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In a particular embodiment, the variant comprises or consists of the substitution K9R of the mature polypeptide of SEQ ID NO: 2.
In one aspect, the variant comprises or consists of an alteration at a position corresponding to position 15. In one embodiment, the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In a particular embodiment, the variant comprises or consists of the substitution N15T of the mature polypeptide of SEQ ID NO: 2.
In one aspect, the variant comprises or consists of an alteration at a position corresponding to position 46. In one embodiment, the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In a particular embodiment, the variant comprises or consists of the substitution L46D of the mature polypeptide of SEQ ID NO: 2.
In one aspect, the variant comprises or consists of an alteration at a position corresponding to position 58. In one embodiment, the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In a particular embodiment, the variant comprises or consists of the substitution A58L of the mature polypeptide of SEQ ID NO: 2.
In one aspect, the variant comprises or consists of an alteration at a position corresponding to position
66. In one embodiment, the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In a particular embodiment, the variant comprises or consists of the substitution S66H of the mature polypeptide of SEQ ID NO: 2.
In one aspect, the variant comprises or consists of an alteration at a position corresponding to position 89. In one embodiment, the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In a particular embodiment, the variant comprises or consists of the substitution Q89Y of the mature polypeptide of SEQ ID NO: 2.
In one aspect, the variant comprises or consists of an alteration at a position corresponding to position 95. In one embodiment, the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In a particular embodiment, the variant comprises or consists of the substitution K95E of the mature polypeptide of SEQ ID NO: 2.
In one aspect, the variant comprises or consists of an alteration at a position corresponding to position 100. In one embodiment, the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In a particular embodiment, the variant comprises or consists of the substitution S100D of the mature polypeptide of SEQ ID NO: 2.
In one aspect, the variant comprises or consists of an alteration at a position corresponding to position 106. In one embodiment, the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In a particular embodiment, the variant comprises or consists of the substitution N106Y of the mature polypeptide of SEQ ID NO: 2.
In one aspect, the variant comprises or consists of an alteration at a position corresponding to position
109. In one embodiment, the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In a particular embodiment, the variant comprises or consists of the substitution Q109R, Q109D, Q109F, Q109K or Q109A of the mature polypeptide of SEQ ID NO: 2. A preferred substitution at a position corresponding to position 109 is Q109R.
In one aspect, the variant comprises or consists of an alteration at a position corresponding to position 183. In one embodiment, the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In a particular embodiment, the variant comprises or consists of the substitution K183Q or K183R of the mature polypeptide of SEQ ID NO: 2.
In one aspect, the variant comprises or consists of an alteration at a position corresponding to position 188. In one embodiment, the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In a particular embodiment, the variant comprises or consists of the substitution V188I of the mature polypeptide of SEQ ID NO: 2.
In one aspect, the variant comprises or consists of an alteration at a position corresponding to position 190. In one embodiment, the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In a particular embodiment, the variant comprises or consists of the substitution A190Q of the mature polypeptide of SEQ ID NO: 2.
In one aspect, the variant comprises or consists of an alteration at a position corresponding to position
203. In one embodiment, the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In a particular embodiment, the variant comprises or consists of the substitution A203P of the mature polypeptide of SEQ ID NO: 2.
In one aspect, the variant comprises or consists of an alteration at a position corresponding to position
204. In one embodiment, the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In a particular embodiment, the variant comprises or consists of the substitution K204R of the mature polypeptide of SEQ ID NO: 2.
In one aspect, the variant comprises or consists of an alteration at a position corresponding to position 221. In one embodiment, the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In a particular embodiment, the variant comprises or consists of the substitution A221 P of the mature polypeptide of SEQ ID NO: 2.
In one aspect, the variant comprises or consists of an alteration at a position corresponding to position 229. In one embodiment, the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In a particular embodiment, the variant comprises or consists of the substitution E229N or E229S of the mature polypeptide of SEQ ID NO: 2.
In one aspect, the variant comprises or consists of an alteration at a position corresponding to position 234. In one embodiment, the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In a particular embodiment, the variant comprises or consists of the substitution I234V of the mature polypeptide of SEQ ID NO: 2.
In one aspect, the variant comprises or consists of an alteration at a position corresponding to position 238. In one embodiment, the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In a particular embodiment, the variant comprises or consists of the substitution I238W, I238L or I238M of the mature polypeptide of SEQ ID NO: 2. Preferred substitutions at a position corresponding to position 238 are I238W and I238L.
In one aspect, the variant comprises or consists of an alteration at a position corresponding to position
240. In one embodiment, the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In a particular embodiment, the variant comprises or consists of the substitution I240W of the mature polypeptide of SEQ ID NO: 2.
In one aspect, the variant comprises or consists of an alteration at a position corresponding to position 242. In one embodiment, the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In a particular embodiment, the variant comprises or consists of the substitution N242S of the mature polypeptide of SEQ ID NO: 2.
In one aspect, the variant comprises or consists of an alteration at a position corresponding to position 243. In one embodiment, the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In a particular embodiment, the variant comprises or consists of the substitution G243V of the mature polypeptide of SEQ ID NO: 2.
In one aspect, the variant comprises or consists of an alteration at a position corresponding to position
257. In one embodiment, the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In a particular embodiment, the variant comprises or consists of the substitution Y257W of the mature polypeptide of SEQ ID NO: 2.
In one aspect, the variant comprises or consists of an alteration at a position corresponding to position
258. In one embodiment, the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In a particular embodiment, the variant comprises or consists of the substitution R258E of the mature polypeptide of SEQ ID NO: 2.
In one aspect, the variant comprises or consists of an alteration at a position corresponding to position
291. In one embodiment, the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In a particular embodiment, the variant comprises or consists of the substitution K291 R of the mature polypeptide of SEQ ID NO: 2.
In one aspect, the variant comprises or consists of an alteration at a position corresponding to position 293. In one embodiment, the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In a particular embodiment, the variant comprises or consists of the substitution A293G or A293P of the mature polypeptide of SEQ ID NO: 2.
In one aspect, the variant comprises or consists of an alteration at a position corresponding to position 316. In one embodiment, the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In a particular embodiment, the variant comprises or consists of the substitution K316R of the mature polypeptide of SEQ ID NO: 2. ln one aspect, the variant comprises or consists of an alteration at a position corresponding to position 320. In one embodiment, the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In a particular embodiment, the variant comprises or consists of the substitution K320R of the mature polypeptide of SEQ ID NO: 2.
In one aspect, the variant comprises or consists of an alteration at a position corresponding to position
324. In one embodiment, the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In a particular embodiment, the variant comprises or consists of the substitution L324Q of the mature polypeptide of SEQ ID NO: 2.
In one aspect, the variant comprises or consists of an alteration at a position corresponding to position 329. In one embodiment, the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In a particular embodiment, the variant comprises or consists of the substitution K329R of the mature polypeptide of SEQ ID NO: 2.
In one aspect, the variant comprises or consists of an alteration at a position corresponding to position 333. In one embodiment, the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In a particular embodiment, the variant comprises or consists of the substitution K333R of the mature polypeptide of SEQ ID NO: 2.
In one aspect, the variant comprises or consists of an alteration at a position corresponding to position 339. In one embodiment, the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In a particular embodiment, the variant comprises or consists of the substitution L339M of the mature polypeptide of SEQ ID NO: 2.
In one aspect, the variant comprises or consists of an alteration at a position corresponding to position 341. In one embodiment, the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In a particular embodiment, the variant comprises or consists of the substitution 1341 P of the mature polypeptide of SEQ ID NO: 2.
In one aspect, the variant comprises or consists of an alteration at a position corresponding to position
352. In one embodiment, the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In a particular embodiment, the variant comprises or consists of the substitution V352I of the mature polypeptide of SEQ ID NO: 2.
In one aspect, the variant comprises or consists of an alteration at a position corresponding to position 354. In one embodiment, the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In a particular embodiment, the variant comprises or consists of the substitution S354P of the mature polypeptide of SEQ ID NO: 2.
In one aspect, the variant comprises or consists of an alteration at a position corresponding to position 360. In one embodiment, the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In a particular embodiment, the variant comprises or consists of the substitution K360G or K360R of the mature polypeptide of SEQ ID NO: 2. In one aspect, the variant comprises or consists of an alteration at a position corresponding to position 377. In one embodiment, the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In a particular embodiment, the variant comprises or consists of the substitution F377Y of the mature polypeptide of SEQ ID NO: 2.
In one aspect, the variant comprises or consists of an alteration at a position corresponding to position
399. In one embodiment, the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In a particular embodiment, the variant comprises or consists of the substitution N399K of the mature polypeptide of SEQ ID NO: 2.
In one aspect, the variant comprises or consists of an alteration at a position corresponding to position 400. In one embodiment, the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In a particular embodiment, the variant comprises or consists of the substitution K400R of the mature polypeptide of SEQ ID NO: 2.
In one aspect, the variant comprises or consists of an alteration at a position corresponding to position 419. In one embodiment, the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In a particular embodiment, the variant comprises or consists of the substitution F419Y of the mature polypeptide of SEQ ID NO: 2.
In one aspect, the variant comprises or consists of an alteration at a position corresponding to position 440. In one embodiment, the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In a particular embodiment, the variant comprises or consists of the substitution N440K of the mature polypeptide of SEQ ID NO: 2.
In one aspect, the variant comprises or consists of an alteration at a position corresponding to position
450. In one embodiment, the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In a particular embodiment, the variant comprises or consists of the substitution D450P of the mature polypeptide of SEQ ID NO: 2.
In one aspect, the variant comprises or consists of an alteration at a position corresponding to position
451. In one embodiment, the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In a particular embodiment, the variant comprises or consists of the substitution K451 E or K451 R of the mature polypeptide of SEQ ID NO: 2.
In one aspect, the variant comprises or consists of an alteration at a position corresponding to position 454. In one embodiment, the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In a particular embodiment, the variant comprises or consists of the substitution A454V of the mature polypeptide of SEQ ID NO: 2.
In one aspect, the variant comprises or consists of an alteration at a position corresponding to position 458. In one embodiment, the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In a particular embodiment, the variant comprises or consists of the substitution D458S of the mature polypeptide of SEQ ID NO: 2. ln one aspect, the variant comprises or consists of an alteration at a position corresponding to position 481. In one embodiment, the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In a particular embodiment, the variant comprises or consists of the substitution K481 R of the mature polypeptide of SEQ ID NO: 2.
In one aspect, the variant comprises or consists of an alteration at a position corresponding to position
492. In one embodiment, the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In a particular embodiment, the variant comprises or consists of the substitution A492L of the mature polypeptide of SEQ ID NO: 2.
In one aspect, the variant comprises or consists of an alteration at a position corresponding to position 567. In one embodiment, the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In a particular embodiment, the variant comprises or consists of the substitution K567R of the mature polypeptide of SEQ ID NO: 2.
In one aspect, the variant comprises or consists of an alteration at a position corresponding to position 568. In one embodiment, the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In a particular embodiment, the variant comprises or consists of the substitution G568A of the mature polypeptide of SEQ ID NO: 2.
In one aspect, the variant comprises or consists of an alteration at a position corresponding to position
578. In one embodiment, the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In a particular embodiment, the variant comprises or consists of the substitution S578K or S578R of the mature polypeptide of SEQ ID NO: 2.
In one aspect, the variant comprises or consists of an alteration at a position corresponding to position
579. In one embodiment, the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In a particular embodiment, the variant comprises or consists of the substitution S579R or S579K of the mature polypeptide of SEQ ID NO: 2. A preferred substitution at a position corresponding to position 579 is S579R.
In one aspect, the variant comprises or consists of an alteration at a position corresponding to position 582. In one embodiment, the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In a particular embodiment, the variant comprises or consists of the substitution S582K of the mature polypeptide of SEQ ID NO: 2.
In one aspect, the variant comprises or consists of an alteration at a position corresponding to position
664. In one embodiment, the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In a particular embodiment, the variant comprises or consists of the substitution T664K of the mature polypeptide of SEQ ID NO: 2.
In one aspect, the variant comprises or consists of an alteration at a position corresponding to position 672. In one embodiment, the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In a particular embodiment, the variant comprises or consists of the substitution N672D of the mature polypeptide of SEQ ID NO: 2.
In one aspect, the variant comprises or consists of an alteration at a position corresponding to position 703. In one embodiment, the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In a particular embodiment, the variant comprises or consists of the substitution I703L of the mature polypeptide of SEQ ID NO: 2.
In one aspect, the variant comprises or consists of an alteration at a position corresponding to position 728. In one embodiment, the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In a particular embodiment, the variant comprises or consists of the substitution M728V of the mature polypeptide of SEQ ID NO: 2.
In one aspect, the variant comprises or consists of an alteration at a position corresponding to position
843. In one embodiment, the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In a particular embodiment, the variant comprises or consists of the substitution A843P of the mature polypeptide of SEQ ID NO: 2.
In one aspect, the variant comprises or consists of an alteration at a position corresponding to position 885. In one embodiment, the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In a particular embodiment, the variant comprises or consists of the substitution K855R of the mature polypeptide of SEQ ID NO: 2.
In one aspect, the variant comprises or consists of an alteration at a position corresponding to position 887. In one embodiment, the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In a particular embodiment, the variant comprises or consists of the substitution K887R of the mature polypeptide of SEQ ID NO: 2.
In one aspect, the variant comprises or consists of an alteration at a position corresponding to position 892. In one embodiment, the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In a particular embodiment, the variant comprises or consists of the substitution N892Y, N892W or N892F of the mature polypeptide of SEQ ID NO: 2. A preferred substitution at a position corresponding to position 892 is N892Y.
In one aspect, the variant comprises or consists of an alteration at a position corresponding to position 1008. In one embodiment, the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In a particular embodiment, the variant comprises or consists of the substitution N1008D of the mature polypeptide of SEQ ID NO: 2.
In one aspect, the variant comprises or consists of an alteration at a position corresponding to position 1016. In one embodiment, the amino acid at this position may substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In a particular embodiment, the variant comprises or consists of the substitution K1016T of the mature polypeptide of SEQ ID NO: 2. In one embod- iment, the present invention relates to detergent compositions comprising a xanthan lyase variant as described herein, having an alteration at one or more positions selected from the group consisting of positions: 9, 15, 46, 58, 66, 89, 95, 100, 106, 109, 183, 188, 190, 203, 204, 221 , 229, 234, 238, 240, 242, 243, 257, 258, 291 , 293, 316, 320, 324, 329, 333, 339, 341 , 352, 354, 360, 377, 399, 400, 419, 440, 450, 451 , 454, 458, 481 , 492, 567, 568, 578, 579, 582, 664, 672, 703, 728, 843, 855, 887, 892, 1008 and 1016 of SEQ ID NO: 2, wherein each position corresponds to the positions of SEQ ID NO: 2.
In one embodiment the xanthan lyase variant comprised in the detergent compositions of the invention, further comprises an alteration at one or more positions selected from the group consisting of: 624, 631 , 635, 649, 656, 752, 752, 754, 757, 769, 775, 777, 800, 801 , 875, 911 , and 915 wherein numbering is according to SEQ ID NO: 2.
In one embodiment, the present invention relates to detergent compositions comprising a xanthan ly- ase variant as described herein having one or more substitutions selected from the group consisting of: K9R, N15T, L46D, A58L, S66H, Q89Y, K95E, S100D, N106Y, Q109R, Q109D, Q109F, Q109K, Q109A, K183Q,K183R, V188I, A190Q, A203P, K204R, A221 P, E229N, E229S, I234V, I238W, I238L, I238M, I240W, N242S, G243V, Y257W, R258E, K291 R, A293G, A293P, K316R, K320R, L324Q, K329R, K333R, L339M, 1341 P, V352I, S354P, K360G, K360R, F377Y, N399K, K400R, F419Y, D450P, K451 E, K451 R, A454V, D458S, K481 R, A492L, K567R, G568A, S578K, S578R, S579R, S579K, S582K, A624E, T631 N, S635E, T649K, I656V, T664K, N672D, I703L, M728V, G738L, P752K, P752R, G753E, S754E, S754R, S757D, A769D, L775A, D777R, V800P, D801 G, A843P, K855R, K875T, K887R, N892Y, N892W, N892F, A911V, T915A, N1008D and K1016T, wherein numbering is according to SEQ ID NO: 2.
Figure imgf000035_0001
Figure imgf000036_0001
Figure imgf000037_0001
In a particular embodiment, the invention relates to detergent compositions comprising a xanthan lyase variant selected from the group consisting of the xanthan lyase variants set forth in Table 1 herein.
In a particular embodiment, the invention relates to detergent compositions comprising a xanthan lyase variant selected from the group consisting of the xanthan lyase variants set forth in Table 2 herein.
In a particular embodiment, the invention relates to detergent compositions comprising a xanthan lyase variant selected from the group consisting of the xanthan lyase variants set forth in Table 3 herein.
In a particular embodiment, the invention relates to detergent compositions comprising a xanthan lyase variant selected from the group consisting of the xanthan lyase variants set forth in Table 4 herein.
The variants may further comprise one or more additional alterations at one or more other positions in regions 7, 8, 9, 10, 1 1 , 12 and/or 13.
The amino acid changes may be of a minor nature, that is conservative amino acid substitutions or insertions that do not significantly affect the folding and/or activity of the protein; small deletions, typically of 1-30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to 20-25 residues; or a small extension that facilitates purification by changing net charge or another function, such as a poly-histidine tract, an antigenic epitope or a binding domain.
Examples of conservative substitutions are within the groups of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and aspara- gine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, threonine and methionine). Amino acid substitutions that do not generally alter specific activity are known in the art and are described, for example, by H. Neurath and R.L. Hill, 1979, In, The Proteins, Academic Press, New York. Common substitutions are Ala/Ser, Val/lle, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/lle, Leu Val, Ala/Glu, and Asp/Gly.
Alternatively, the amino acid changes are of such a nature that the physico-chemical properties of the polypeptides are altered. For example, amino acid changes may improve the thermal stability of the poly- peptide, alter the substrate specificity, change the pH optimum, and the like.
Essential amino acids in a polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081 -1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for xanthan lyase activity to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271 : 4699-4708. The active site of the enzyme or other biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al. , 1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver ef a/., 1992, FEBS Lett. 309: 59-64. The identity of essential amino acids can also be inferred from an alignment with a related polypeptide.
In one embodiment, the present invention relates to detergent compositions comprising a xanthan lyase variant as described herein, having a total number of alterations compared to SEQ ID NO: 2 between 1 and 20, e.g., between 1 and 10 or between 1 and 5, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 alterations.
In one embodiment, the present invention relates to detergent compositions comprising a xanthan lyase variant as described herein, having an activity on xanthan gum, preferably said activity on xanthan gum is a xanthan gum degrading activity, further preferably said xanthan gum degrading activity is EC 4.2.2.12 activity.
In an embodiment, the variant has an improved stability in a detergent composition compared to a parent enzyme (e.g., SEQ ID NO: 2).
In one embodiment the present invention relates to detergent compositions comprising a xanthan lyase variant as described herein, having an improved stability in a detergent composition compared to the parent xanthan lyase (e.g., with SEQ ID NO: 2).
In one embodiment, the present invention relates to detergent compositions comprising a xanthan ly- ase variant as described herein, having a half-life improvement factor (HIF) of >1 .0; preferably having a half- life improvement factor (HIF) of >1.0, preferably at least 1.2, such as at least 1 .5, e.g. at least 2.0, relative to a parent xanthan lyase. A preferred way of calculating a half-life improvement factor (HIF) is described in Example 3 herein.
In one embodiment, the present invention relates to detergent compositions comprising a xanthan lyase variant as described herein, wherein a half-life improvement factor (HIF) is determined after incubation of said xanthan lyase variant in a detergent composition at 25°C or 30°C for a time period from about 30 min to about 20 hours.
Parent
The parent xanthan lyase may be (a) a polypeptide having at least 60% sequence identity to the mature polypeptide of SEQ ID NO: 2; (b) a polypeptide encoded by a polynucleotide that hybridizes under low stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 1 , or (ii) the full-length complement of (i); or (c) a polypeptide encoded by a polynucleotide having at least 60% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 1.
In an aspect, the parent has a sequence identity to the mature polypeptide of SEQ ID NO: 2 of at least 60%, e.g. , at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, which have xanthan lyase activity. In one aspect, the amino acid sequence of the parent differs by up to 10 amino acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the mature polypeptide of SEQ ID NO: 2.
In another aspect, the parent comprises or consists of the amino acid sequence of SEQ ID NO: 2. In another aspect, the parent comprises or consists of the mature polypeptide of SEQ ID NO: 2. In another aspect, the parent is a fragment of the mature polypeptide of SEQ ID NO: 2 containing at least 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94% or 95% of the number of amino acids of SEQ ID NO: 2. In another embodiment, the parent is an allelic variant of the mature polypeptide of SEQ ID NO: 2.
In another aspect, the parent is encoded by a polynucleotide that hybridizes under very low stringency conditions, low stringency conditions, medium stringency conditions, medium-high stringency conditions, high stringency conditions, or very high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 1 , or (ii) the full-length complement of (i) (Sambrook , 1989, Moleceutlaalr. Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, New York).
The polynucleotide of SEQ ID NO: 1 or a subsequence thereof, as well as the polypeptide of SEQ ID
NO: 2 or a fragment thereof, may be used to design nucleic acid probes to identify and clone DNA encoding a parent from strains of different genera or species according to methods well known in the art. In particular, such probes can be used for hybridization with the genomic DNA or cDNA of a cell of interest, following standard Southern blotting procedures, in order to identify and isolate the corresponding gene therein. Such probes can be considerably shorter than the entire sequence, but should be at least 15, e.g., at least 25, at least 35, or at least 70 nucleotides in length. Preferably, the nucleic acid probe is at least 100 nucleotides in length, e.g. , at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides, at least 600 nucleotides, at least 700 nucleotides, at least 800 nucleotides, or at least 900 nucleotides in length. Both DNA and RNA probes can be used. The probes are typically labeled for detecting the corresponding gene (for example, with 32P, 3H, 35S, biotin, or avidin). Such probes are encompassed by the present invention.
A genomic DNA or cDNA library prepared from such other strains may be screened for DNA that hybridizes with the probes described above and encodes a parent. Genomic or other DNA from such other strains may be separated by agarose or polyacrylamide gel electrophoresis, or other separation techniques. DNA from the libraries or the separated DNA may be transferred to and immobilized on nitrocellulose or other suitable carrier material. In order to identify a clone or DNA that hybridizes with SEQ ID NO: 1 or a subsequence thereof, the carrier material is used in a Southern blot.
For purposes of the present invention, hybridization indicates that the polynucleotide hybridizes to a labeled nucleic acid probe corresponding to (i) SEQ ID NO: 1 ; (ii) the mature polypeptide coding sequence of SEQ ID NO: 1 ; (iii) the full-length complement thereof; or (iv) a subsequence thereof; under very low to very high stringency conditions. Molecules to which the nucleic acid probe hybridizes under these conditions can be detected using, for example, X-ray film or any other detection means known in the art.
In one aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 1. In another aspect, the nucleic acid probe is a polynucleotide that encodes the polypeptide of SEQ ID NO: 2; the mature polypeptide thereof; or a fragment thereof. In another aspect, the nucleic acid probe is SEQ ID NO: 1.
In another embodiment, the parent is encoded by a polynucleotide having a sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 1 of at least 60%, e.g. , at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%.
The polypeptide may be a hybrid polypeptide in which a region of one polypeptide is fused at the N-terminus or the C-terminus of a region of another polypeptide.
The parent may be a fusion polypeptide or cleavable fusion polypeptide in which another polypeptide is fused at the N-terminus or the C-terminus of the polypeptide of the present invention. A fusion polypeptide is produced by fusing a polynucleotide encoding another polypeptide to a polynucleotide of the present invention. Techniques for producing fusion polypeptides are known in the art, and include ligating the coding sequences encoding the polypeptides so that they are in frame and that expression of the fusion polypeptide is under control of the same promoter(s) and terminator. Fusion polypeptides may also be constructed using intein technology in which fusion polypeptides are created post-translationally (Cooper , 1993, EMBeOt al. J. 12: 2575-2583; Dawson et al. , 1994, Science 266: 776-779).
A fusion polypeptide can further comprise a cleavage site between the two polypeptides. Upon secretion of the fusion protein, the site is cleaved releasing the two polypeptides. Examples of cleavage sites include, but are not limited to, the sites disclosed in Martinet al. , 2003, J. Ind. Microbiol. Biotechnol. 3: 568- 576; Svetina et al., 2000, J. Biotechnol. 76: 245-251 ; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13: 498-503; and Contreras et al., 1991 , Biotechnology 9: 378-381 ; Eaton et al., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995, Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure, Function, and Genetics 6: 240-248; and Stevens, 2003, Drug Discovery World 4: 35-48.
The parent may be obtained from microorganisms of any genus. For purposes of the present invention, the term "obtained from" as used herein in connection with a given source shall mean that the parent encoded by a polynucleotide is produced by the source or by a strain in which the polynucleotide from the source has been inserted. In one aspect, the parent is secreted extracellularly.
The parent may be a bacterial enzyme. For example, the parent may be a Gram-positive bacterial polypeptide such as a Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, or Streptomyces enzyme, or a Gram-negative bacterial polypeptide such as a Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, llyobacter, Neisseria, Pseudomonas, Salmonella, or Ureaplasma enzyme.
In one aspect, the parent is a Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus li- cheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillus thuringiensis enzyme.
In another aspect, the parent is a Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equi subsp. zooepidemicus enzyme.
In another aspect, the parent is a Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, or Streptomyces lividans enzyme.
The parent may be a fungal enzyme. For example, the parent may be a yeast enzyme such as a Candida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia enzyme; or a filamentous fungal enzyme such as an Acremonium, Agaricus, Alternaria, Aspergillus, Aureobasidium, Botry- ospaeria, Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Cop- totermes, Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium, Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex, Lentinula, Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mu- cor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Piromyces, Poitrasia, Pseudoplectania, Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea, Verticillium, Volvariella, or Xylaria enzyme.
In another aspect, the parent is a Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccha- romyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, or Saccharomyces oviformis enzyme. In another aspect, the parent is an Acremonium cellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium luck- nowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chryso- sporium tropicum, Chrysosporium zonatum, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwel- lense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa, Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium funiculosum, Penicil- lium purpurogenum, Phanerochaete chrysosporium, Thielavia achromatica, Thielavia albomyces, Thielavia albopilosa, Thielavia australeinsis, Thielavia fimeti, Thielavia microspora, Thielavia ovispora, Thielavia peruviana, Thielavia setosa, Thielavia spededonium, Thielavia subthermophila, Thielavia terrestris, Tnchoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride enzyme.
In another aspect, the parent is a Paenibacillus sp. xanthan lyase, e.g. , the xanthan lyase of SEQ ID NO: 2.
It will be understood that for the aforementioned species, the invention encompasses both the perfect and imperfect states, and other taxonomic equivalents, e.g., anamorphs, regardless of the species name by which they are known. Those skilled in the art will readily recognize the identity of appropriate equivalents.
Strains of these species are readily accessible to the public in a number of culture collections, such as the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).
The parent may be identified and obtained from other sources including microorganisms isolated from nature (e.g., soil, composts, water, etc.) or DNA samples obtained directly from natural materials (e.g. , soil, composts, water, etc.) using the above-mentioned probes. Techniques for isolating microorganisms and DNA directly from natural habitats are well known in the art. A polynucleotide encoding a parent may then be obtained by similarly screening a genomic DNA or cDNA library of another microorganism or mixed DNA sample. Once a polynucleotide encoding a parent has been detected with the probe(s), the polynucleotide can be isolated or cloned by utilizing techniques that are known to those of ordinary skill in the art (see, e.g., Sambrook et al, 1989, supra). Preparation of variants
Methods for obtaining a variant having xanthan lyase activity, may comprise: (a) introducing into a parent xanthan lyase an alteration at one or more positions corresponding to positions 9, 15, 46, 58, 66, 89, 95, 100, 106, 109, 183, 188, 190, 203, 204, 221 , 229, 234, 238, 240, 242, 243, 257, 258, 291 , 293, 316, 320, 324, 329, 333, 339, 341 , 352, 354, 360, 377, 399, 400, 419, 440, 450, 451 , 454, 458, 481 , 492, 567, 568, 578, 579, 582, 664, 672, 703, 728, 843, 855, 887, 892, 1008 and 1016 of the mature polypeptide of SEQ ID NO: 2, wherein the variant has xanthan lyase activity; and (b) recovering the variant.
Methods for obtaining a variant having xanthan lyase activity may further comprise introducing an alteration (e.g., a substitution, deletion or insertion) at one or more positions corresponding to positions 624, 631 , 635, 649, 656, 752, 752, 754, 757, 769, 775, 777, 800, 801 , 875, 91 1 , and 915 wherein numbering is according to SEQ ID NO: 2.
The variants can be prepared using any mutagenesis procedure known in the art, such as site-directed mutagenesis, synthetic gene construction, semi-synthetic gene construction, random mutagenesis, shuffling, etc.
Site-directed mutagenesis is a technique in which one or more mutations are introduced at one or more defined sites in a polynucleotide encoding the parent.
Site-directed mutagenesis can be accomplished in vitro by PCR involving the use of oligonucleotide primers containing the desired mutation. Site-directed mutagenesis can also be performed in vitro by cassette mutagenesis involving the cleavage by a restriction enzyme at a site in the plasmid comprising a polynucleotide encoding the parent and subsequent ligation of an oligonucleotide containing the mutation in the polynucleotide. Usually the restriction enzyme that digests the plasmid and the oligonucleotide is the same, permitting sticky ends of the plasmid and the insert to ligate to one another. See, e.g. , Scherer and Davis, 1979, Proc. Natl. Acad. Sci. USA 76: 4949-4955; and Barton ei al., 1990, Nucleic Acids Res. 18: 7349-4966.
Site-directed mutagenesis can also be accomplished in vivo by methods known in the art. See, e.g., U.S. Patent Application Publication No. 2004/0171154; Storici ei al., 2001 , Nature Biotechnol. 19: 773-776; Kren et al., 1998, Nat. Med. 4: 285-290; and Calissano and Macino, 1996, Fungal Genet. Newslett. 43: 15- 16.
Any site-directed mutagenesis procedure can be used in the present invention. There are many commercial kits available that can be used to prepare variants.
Synthetic gene construction entails in vitro synthesis of a designed polynucleotide molecule to encode a polypeptide of interest. Gene synthesis can be performed utilizing a number of techniques, such as the multiplex microchip-based technology described by Tian et al. (2004, Nature 432: 1050-1054) and similar technologies wherein oligonucleotides are synthesized and assembled upon photo-programmable microflu- idic chips.
Single or multiple amino acid substitutions, deletions, and/or insertions can be made and tested using known methods of mutagenesis, recombination, and/or shuffling, followed by a relevant screening procedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241 : 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al. , 1991 , Biochemistry 30: 10832-10837; U.S. Patent No. 5,223,409; WO 92/06204) and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner et al. , 1988, DNA 7: 127).
Mutagenesis/shuffling methods can be combined with high-throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides expressed by host cells (Ness et al. , 1999, Nature Biotechnology 17: 893-896). Mutagenized DNA molecules that encode active polypeptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide.
Semi-synthetic gene construction is accomplished by combining aspects of synthetic gene construction, and/or site-directed mutagenesis, and/or random mutagenesis, and/or shuffling. Semi-synthetic construction is typified by a process utilizing polynucleotide fragments that are synthesized, in combination with PCR techniques. Defined regions of genes may thus be synthesized de novo, while other regions may be amplified using site-specific mutagenic primers, while yet other regions may be subjected to error-prone PCR or non-error prone PCR amplification. Polynucleotide subsequences may then be shuffled.
Embodiments
In one embodiment, the present invention relates to detergent compositions comprising at least one (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10) xanthan lyase variant as described herein.
In one embodiment, the present invention relates to detergent compositions comprising at least one
(e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10) xanthan lyase variant as described herein, wherein said composition further comprises one or more detergent components.
In one embodiment, the present invention relates to detergent compositions comprising at least one (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10) xanthan lyase variant as described herein, further comprising one or more additional enzymes selected from the group comprising or consisting of: endoglucanases, proteases, amylases, lipases, cutinases, cellulases, xanthan lyases, xyloglucanases, pectinases, pectin lyases, xantha- nases, peroxidases, haloperoxygenases, catalases and mannanases, or any mixture thereof.
In one embodiment, the present invention relates to detergent compositions comprising at least one (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10) xanthan lyase variant as described herein, further comprising one or more detergent components, wherein said detergent composition is in form of a bar, a homogenous tablet, a tablet having two or more layers, a pouch having one or more compartments, a regular or compact powder, a granule, a paste, a gel, or a regular, compact or concentrated liquid.
In one embodiment, the present invention relates to use of a detergent composition of the invention, wherein said use is selected from the group comprising or consisting of: use for degrading xanthan gum and use in a cleaning process, such as laundry or hard surface cleaning such as dish wash.
In one embodiment, the present invention relates to use of a detergent composition of the invention, wherein said composition has an enzyme detergency benefit.
In one embodiment, the present invention relates to a method for degrading xanthan gum comprising: applying a detergent composition of the invention to a xanthan gum.
In one embodiment, the present invention relates to a method for degrading xanthan gum comprising: applying a detergent composition of the invention to a xanthan gum, wherein said xanthan gum is on the surface of a textile or hard surface, such as dish wash.
Polynucleotides
Disclosed herein are isolated polynucleotides encoding a variant as described herein. Said polynucle- otides encoding a xanthan lyase variant, comprise an alteration at one or more positions selected from the group consisting of: i) region 7 corresponding to amino acids 1 to 153 of SEQ ID NO: 2, ii) region 8 corresponding to amino acids 177 to 613 of SEQ ID NO: 2, iii) region 9 corresponding to amino acids 659 to 730 of SEQ ID NO: 2, iv) region 10 corresponding to amino acids 804 to 806 of SEQ ID NO: 2, v) region 11 corresponding to amino acids 847 to 871 of SEQ ID NO: 2, vi) region 12 corresponding to amino acids 886 to 902 of SEQ ID NO: 2, and vii) region 13 corresponding to amino acids 1005 to 1037 of SEQ ID NO: 2, wherein said variant has at least 60% and less than 100% sequence identity to SEQ ID NO: 2.
The techniques used to isolate or clone a polynucleotide are known in the art and include isolation from genomic DNA or cDNA, or a combination thereof. The cloning of the polynucleotides from genomic DNA can be effected, e.g., by using the well-known polymerase chain reaction (PCR) or antibody screening of expression libraries to detect cloned DNA fragments with shared structural features. See, e.g. , Innis ef a/., 1990, PCR: A Guide to Methods and Application, Academic Press, New York. Other nucleic acid amplification procedures such as ligase chain reaction (LCR), ligation activated transcription (LAT) and polynucleo- tide-based amplification (NASBA) may be used. The polynucleotides may be cloned in a strain of Bacillus subtilis or E. coli, or a related organism and thus, for example, may be an allelic or species variant of the polypeptide encoding region of the polynucleotide.
Modification of a polynucleotide encoding a polypeptide may be necessary for synthesizing polypeptides substantially similar to the polypeptide. The term "substantially similar" to the polypeptide refers to non- naturally occurring forms of the polypeptide. These polypeptides may differ in some engineered way from the polypeptide isolated from its native source, e.g. , variants that differ in specific activity, thermostability, pH optimum, or the like. The variants may be constructed on the basis of the polynucleotide presented as the mature polypeptide coding sequence of SEQ ID NO: 1 , e.g., a subsequence thereof, and/or by introduction of nucleotide substitutions that do not result in a change in the amino acid sequence of the polypeptide, but which correspond to the codon usage of the host organism intended for production of the enzyme, or by introduction of nucleotide substitutions that may give rise to a different amino acid sequence. For a general description of nucleotide substitution, see, Ford (1991)l., 'Protein Expression and Purification', 2: 95-
Figure imgf000045_0001
107. Nucleic acid constructs
Also disclosed herein are nucleic acid constructs comprising a polynucleotide encoding a variant as described herein operably linked to one or more control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences. Accordingly, dis- closed are nucleic acid constructs comprising a polynucleotide encoding a xanthan lyase variant, comprising an alteration at one or more positions selected from the group consisting of: i) region 7 corresponding to amino acids 1 to 153 of SEQ ID NO: 2, ii) region 8 corresponding to amino acids 177 to 613 of SEQ ID NO: 2, iii) region 9 corresponding to amino acids 659 to 730 of SEQ ID NO: 2, iv) region 10 corresponding to amino acids 804 to 806 of SEQ ID NO: 2, v) region 11 corresponding to amino acids 847 to 871 of SEQ ID NO: 2, vi) region 12 corresponding to amino acids 886 to 902 of SEQ ID NO: 2, and vii) region 13 corresponding to amino acids 1005 to 1037 of SEQ ID NO: 2, wherein said variant has at least 60% and less than 100% sequence identity to SEQ ID NO: 2 operably linked to one or more control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.
A polynucleotide may be manipulated in a variety of ways to provide for expression of the polypeptide.
Manipulation of the polynucleotide prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying polynucleotides utilizing recombinant DNA methods are well known in the art.
The control sequence may be a promoter, a polynucleotide that is recognized by a host cell for expres- sion of a polynucleotide encoding a polypeptide of the present invention. The promoter contains transcriptional control sequences that mediate the expression of the polypeptide. The promoter may be any polynucleotide that shows transcriptional activity in the host cell including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.
Examples of suitable promoters for directing transcription of the nucleic acid constructs of the present invention in a bacterial host cell are the promoters obtained from the Bacillus amyloliquefaciens alpha- amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus licheniformis penicillinase gene (penP), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus subtilis levansu- crase gene (sacB), Bacillus subtilis xylA and xylB genes, Bacillus thuringiensis crylllA gene (Agaisse and Lereclus, 1994, Molecular Microbiology 13: 97-107), E. coli lac operon, E. coli trc promoter (Egon ei al. , 1988, Gene 69: 301-315), Streptomyces coelicolor agarase gene (dagA), and prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731 ), as well as the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80: 21 -25). Further promoters are described in "Useful proteins from recombinant bacteria" in Gilbert et al. , 1980, Scientific American 242: 74-94; and in Sambrook et al., 1989, supra. Examples of tandem promoters are disclosed in WO 99/43835.
Examples of suitable promoters for directing transcription of the nucleic acid constructs of the present invention in a filamentous fungal host cell are promoters obtained from the genes for Aspergillus nidulans acetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Fusarium oxysporum trypsin-like protease (WO 96/00787), Fusarium venenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Daria (WO 00/56900), Fusarium venenatum Quinn (WO 00/56900), Rhizomucor miehei lipase, Rhizomucor miehei aspartic proteinase, Trichoderma reesei beta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, Trichoderma reesei xanthan lyase I, Trichoderma reesei xanthan lyase II, Trichoderma reesei xanthan lyase III, Trichoderma reesei xanthan lyase IV, Trichoderma reesei xanthan lyase V, Trichoderma reesei xylanase I, Trichoderma reesei xylanase II, Trichoderma reesei beta- xylosidase, as well as the NA2-tpi promoter (a modified promoter from an Aspergillus neutral alpha-amylase gene in which the untranslated leader has been replaced by an untranslated leader from an Aspergillus triose phosphate isomerase gene; non-limiting examples include modified promoters from an Aspergillus niger neutral alpha-amylase gene in which the untranslated leader has been replaced by an untranslated leader from an Aspergillus nidulans or Aspergillus oryzae triose phosphate isomerase gene); and mutant, truncated, and hybrid promoters thereof.
In a yeast host, useful promoters are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1 ), Saccharomyces cerevisiae galactokinase (GAL1 ), Saccharomyces cerevisiae alcohol dehydro- genase/glyceraldehyde-3-phosphate dehydrogenase (ADH1 , ADH2/GAP), Saccharomyces cerevisiae tri- ose phosphate isomerase (TPI), Saccharomyces cerevisiae metallothionein (CUP1 ), and Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other useful promoters for yeast host cells are described by Roma- nos et al., 1992, Yeast 8: 423-488.
The control sequence may also be a transcription terminator, which is recognized by a host cell to terminate transcription. The terminator is operably linked to the 3'-terminus of the polynucleotide encoding the polypeptide. Any terminator that is functional in the host cell may be used in the present invention.
Preferred terminators for bacterial host cells are obtained from the genes for Bacillus clausii alkaline protease (aprH), Bacillus licheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA (rrnB).
Preferred terminators for filamentous fungal host cells are obtained from the genes for Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase, Aspergil- lus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.
Preferred terminators for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1 ), and Saccharomyces cerevisiae glyceralde- hyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are described by Romanos et al., 1992, supra.
The control sequence may also be an mRNA stabilizer region downstream of a promoter and upstream of the coding sequence of a gene which increases expression of the gene. Examples of suitable mRNA stabilizer regions are obtained from a Bacillus thuringiensis crylllA gene (WO 94/25612) and a Bacillus subtilis SP82 gene (Hue et al. , 1995, Journal of Bacteriology 177: 3465- 3471 ).
The control sequence may also be a leader, a nontranslated region of an mRNA that is important for translation by the host cell. The leader is operably linked to the 5'-terminus of the polynucleotide encoding the polypeptide. Any leader that is functional in the host cell may be used.
Preferred leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.
Suitable leaders for yeast host cells are obtained from the genes for Saccharomyces cerevisiae eno- lase (ENO-1 ), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha- factor, and Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).
The control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3'-terminus of the polynucleotide and, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence that is functional in the host cell may be used.
Preferred polyadenylation sequences for filamentous fungal host cells are obtained from the genes for Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger alpha-gluco- sidase Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.
Useful polyadenylation sequences for yeast host cells are described by Guo and Sherman, 1995, Mol.
Cellular Biol. 15: 5983-5990.
The control sequence may also be a signal peptide coding region that encodes a signal peptide linked to the N-terminus of a polypeptide and directs the polypeptide into the cell's secretory pathway. The 5'-end of the coding sequence of the polynucleotide may inherently contain a signal peptide coding sequence naturally linked in translation reading frame with the segment of the coding sequence that encodes the polypeptide. Alternatively, the 5'-end of the coding sequence may contain a signal peptide coding sequence that is foreign to the coding sequence. A foreign signal peptide coding sequence may be required where the coding sequence does not naturally contain a signal peptide coding sequence. Alternatively, a foreign signal peptide coding sequence may simply replace the natural signal peptide coding sequence in order to enhance secretion of the polypeptide. However, any signal peptide coding sequence that directs the expressed polypeptide into the secretory pathway of a host cell may be used.
Effective signal peptide coding sequences for bacterial host cells are the signal peptide coding sequences obtained from the genes for Bacillus NCIB 1 1837 maltogenic amylase, Bacillus licheniformis sub- tilisin, Bacillus licheniformis beta-lactamase, Bacillus stearothermophilus alpha-amylase, Bacillus stea- rothermophilus neutral proteases (nprT, nprS, nprM), and Bacillus subtilis prsA. Further signal peptides are described by Simonen and Palva, 1993, Microbiological Reviews 57: 109-137. Effective signal peptide coding sequences for filamentous fungal host cells are the signal peptide coding sequences obtained from the genes for Aspergillus niger neutral amylase, Aspergillus n/ger glucoamyl- ase, Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicola insolens xanthan lyase V, Humicola lanuginosa lipase, and Rhizomucor miehei aspartic proteinase.
Useful signal peptides for yeast host cells are obtained from the genes for Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase. Other useful signal peptide coding sequences are described by Romanos et al., 1992, supra.
The control sequence may also be a propeptide coding sequence that encodes a propeptide positioned at the N-terminus of a polypeptide. The resultant polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases). A propolypeptide is generally inactive and can be converted to an active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding sequence may be obtained from the genes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor miehei aspartic proteinase, and Saccharomyces cerevisiae alpha-factor.
Where both signal peptide and propeptide sequences are present, the propeptide sequence is positioned next to the N-terminus of a polypeptide and the signal peptide sequence is positioned next to the N-terminus of the propeptide sequence.
It may also be desirable to add regulatory sequences that regulate expression of the polypeptide relative to the growth of the host cell. Examples of regulatory systems are those that cause expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Regulatory systems in prokaryotic systems include the lac, tac, and trp operator systems. In yeast, the ADH2 system or GAL1 system may be used. In filamentous fungi, the Aspergillus niger glucoamylase promoter, Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzae glu- coamylase promoter may be used. Other examples of regulatory sequences are those that allow for gene amplification. In eukaryotic systems, these regulatory sequences include the dihydrofolate reductase gene that is amplified in the presence of methotrexate, and the metallothionein genes that are amplified with heavy metals. In these cases, the polynucleotide encoding the polypeptide would be operably linked with the regulatory sequence. Expression vectors
Also disclosed herein are recombinant expression vectors comprising a polynucleotide encoding a variant as described herein, a promoter, and transcriptional and translational stop signals. Accordingly, also disclosed are recombinant expression vectors comprising a polynucleotide encoding a xanthan lyase variant, comprising an alteration at one or more positions selected from the group consisting of: i) region 7 corresponding to amino acids 1 to 153 of SEQ ID NO: 2, ii) region 8 corresponding to amino acids 177 to 613 of SEQ ID NO: 2, iii) region 9 corresponding to amino acids 659 to 730 of SEQ ID NO: 2, iv) region 10 corresponding to amino acids 804 to 806 of SEQ ID NO: 2, v) region 1 1 corresponding to amino acids 847 to 871 of SEQ ID NO: 2, vi) region 12 corresponding to amino acids 886 to 902 of SEQ ID NO: 2, and vii) region 13 corresponding to amino acids 1005 to 1037 of SEQ ID NO: 2, wherein said variant has at least 60% and less than 100% sequence identity to SEQ ID NO: 2 a xanthan lyase variant, comprising an alter- ation at one or more positions selected from the group consisting of: i) region 7 corresponding to amino acids 1 to 153 of SEQ ID NO: 2, ii) region 8 corresponding to amino acids 177 to 613 of SEQ ID NO: 2, iii) region 9 corresponding to amino acids 659 to 730 of SEQ ID NO: 2, iv) region 10 corresponding to amino acids 804 to 806 of SEQ ID NO: 2, v) region 11 corresponding to amino acids 847 to 871 of SEQ ID NO: 2, vi) region 12 corresponding to amino acids 886 to 902 of SEQ ID NO: 2, and vii) region 13 corresponding to amino acids 1005 to 1037 of SEQ ID NO: 2, wherein said variant has at least 60% and less than 100% sequence identity to SEQ ID NO: 2, a promoter, and transcriptional and translational stop codons.
The various nucleotide and control sequences may be joined together to produce a recombinant expression vector that may include one or more convenient restriction sites to allow for insertion or substitution of the polynucleotide encoding the polypeptide at such sites. Alternatively, the polynucleotide may be ex- pressed by inserting the polynucleotide or a nucleic acid construct comprising the polynucleotide into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.
The recombinant expression vector may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can bring about expression of the polynucleotide. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may be a linear or closed circular plasmid.
The vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromoso- mal entity, the replication of which is independent of chromosomal replication, e.g. , a plasmid, an extrachro- mosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one that, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, or a transposon, may be used.
The vector preferably contains one or more selectable markers that permit easy selection of trans- formed, transfected, transduced, or the like cells. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like.
Examples of bacterial selectable markers are Bacillus licheniformis or Bacillus subtilis dal genes, or markers that confer antibiotic resistance such as ampicillin, chloramphenicol, kanamycin, neomycin, spec- tinomycin, or tetracycline resistance. Suitable markers for yeast host cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1 , and URA3. Selectable markers for use in a filamentous fungal host cell include, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents thereof. Preferred for use in an Aspergillus cell are Aspergillus nidulans or Aspergillus oryzae amdS and pyrG genes and a Streptomyces hygroscopicus bar gene.
The vector preferably contains an element(s) that permits integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.
For integration into the host cell genome, the vector may rely on the polynucleotide's sequence encoding the polypeptide or any other element of the vector for integration into the genome by homologous or non-homologous recombination. Alternatively, the vector may contain additional polynucleotides for direct- ing integration by homologous recombination into the genome of the host cell at a precise location(s) in the chromosome(s). To increase the likelihood of integration at a precise location, the integrational elements should contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000 base pairs, and 800 to 10,000 base pairs, which have a high degree of sequence identity to the corresponding target sequence to enhance the probability of homologous recombination. The integrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell. Furthermore, the integrational elements may be non-encoding or encoding polynucleotides. On the other hand, the vector may be integrated into the genome of the host cell by non-homologous recombination.
For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question. The origin of replication may be any plasmid replicator mediating autonomous replication that functions in a cell. The term "origin of replication" or "plasmid replicator" means a polynucleotide that enables a plasmid or vector to replicate in vivo.
Examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permitting replication in E. coli, and pUB1 10, pE194, pTA1060, and ρΑΜβΙ permitting replication in Bacillus.
Examples of origins of replication for use in a yeast host cell are the 2 micron origin of replication,
ARS1 , ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6.
Examples of origins of replication useful in a filamentous fungal cell are AMA1 and ANSI (Gems ef a/., 1991 , Gene 98: 61-67; Cullen et al., 1987, Nucleic Acids Res. 15: 9163-9175; WO 00/24883). Isolation of the AMA1 gene and construction of plasmids or vectors comprising the gene can be accomplished accord- ing to the methods disclosed in WO 00/24883.
More than one copy of a polynucleotide of the present invention may be inserted into a host cell to increase production of a polypeptide. An increase in the copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the polynucleotide where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the polynucleotide, can be selected for by cultivating the cells in the presence of the appropriate selectable agent. The procedures used to ligate the elements described above to construct the recombinant expression vectors of the present invention are well known to one skilled in the art (see, e.g., Sambrook , 1989, et al. supra). Host cells
Also disclosed herein are recombinant host cells, comprising a polynucleotide encoding a variant as described herein operably linked to one or more control sequences that direct the production of a variant of the present invention. Accordingly, disclosed herein are recombinant host cells, comprising a polynucleotide encoding a xanthan lyase variant, comprising an alteration at one or more positions selected from the group consisting of: i) region 7 corresponding to amino acids 1 to 153 of SEQ ID NO: 2, ii) region 8 corresponding to amino acids 177 to 613 of SEQ ID NO: 2, iii) region 9 corresponding to amino acids 659 to 730 of SEQ
ID NO: 2, iv) region 10 corresponding to amino acids 804 to 806 of SEQ ID NO: 2, v) region 1 1 corresponding to amino acids 847 to 871 of SEQ ID NO: 2, vi) region 12 corresponding to amino acids 886 to 902 of SEQ
ID NO: 2, and vii) region 13 corresponding to amino acids 1005 to 1037 of SEQ ID NO: 2, wherein said variant has at least 60% and less than 100% sequence identity to SEQ ID NO: 2 operably linked to one or more control sequences that direct the production of a xanthan lyase variant, comprising an alteration at one or more positions selected from the group consisting of: i) region 7 corresponding to amino acids 1 to
153 of SEQ ID NO: 2, ii) region 8 corresponding to amino acids 177 to 613 of SEQ ID NO: 2, iii) region 9 corresponding to amino acids 659 to 730 of SEQ ID NO: 2, iv) region 10 corresponding to amino acids 804 to 806 of SEQ ID NO: 2, v) region 11 corresponding to amino acids 847 to 871 of SEQ ID NO: 2, vi) region
12 corresponding to amino acids 886 to 902 of SEQ ID NO: 2, and vii) region 13 corresponding to amino acids 1005 to 1037 of SEQ ID NO: 2, wherein said variant has at least 60% and less than 100% sequence identity to SEQ ID NO: 2.
A construct or vector comprising a polynucleotide is introduced into a host cell so that the construct or vector is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector as described earlier. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication. The choice of a host cell will to a large extent depend upon the gene encoding the polypeptide and its source.
The host cell may be any cell useful in the recombinant production of a polypeptide of the present invention, e.g., a prokaryote or a eukaryote.
The prokaryotic host cell may be any Gram-positive or Gram-negative bacterium. Gram-positive bacteria include, but are not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lacto- coccus, Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include, but are not limited to, Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, llyobac- ter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma. The bacterial host cell may be any Bacillus cell including, but not limited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus fir- mus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.
The bacterial host cell may also be any Streptococcus cell including, but not limited to, Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equi subsp. zooepidemicus cells.
The bacterial host cell may also be any Streptomyces cell including, but not limited to, Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, and Streptomy- ces lividans cells.
The introduction of DNA into a Bacillus cell may be effected by protoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen. Genet. 168: 11 1-1 15), competent cell transformation (see, e.g., Young and Spizizen, 1961 , J. Bacteriol. 81 : 823-829, or Dubnau and Davidoff-Abelson, 1971 , J. Mol. Biol. 56: 209- 221 ), electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751 ), or conjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169: 5271-5278). The introduction of DNA into an E. coli cell may be effected by protoplast transformation (see, e.g. , Hanahan, 1983, J. Mol. Biol. 166: 557-580) or electroporation (see, e.g., Dower ei ai, 1988, Nucleic Acids Res. 16: 6127-6145). The introduction of DNA into a Streptomyces cell may be effected by protoplast transformation, electroporation (see, e.g., Gong ei a/., 2004, Folia Microbiol. (Praha) 49: 399-405), conjugation (see, e.g. , Mazodier et al , 1989, J. Bacteriol. 171 : 3583-3585), or transduction (see, e.g., Burke et al, 2001 , Proc. Natl. Acad. Sci. USA 98: 6289-6294). The introduction of DNA into a Pseudomonas cell may be effected by electroporation (see, e.g., Choi et al, 2006, J. Microbiol. Methods 64: 391 -397) or conjugation (see, e.g., Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71 : 51 -57). The introduction of DNA into a Streptococcus cell may be effected by natural competence (see, e.g., Perry and Kuramitsu, 1981 , Infect. Immun. 32: 1295-1297), protoplast transformation (see, e.g., Catt and Jollick, 1991 , Microbios 68: 189-207), electroporation (see, e.g., Buckley et al , 1999, Appl. Environ. Microbiol. 65: 3800-3804), or conjugation (see, e.g., Clewell, 1981 , Microbiol. Rev. 45: 409-436). However, any method known in the art for introducing DNA into a host cell can be used.
The host cell may also be a eukaryote, such as a mammalian, insect, plant, or fungal cell.
The host cell may be a fungal cell. "Fungi" as used herein includes the phyla Ascomycota, Basidiomy- cota, Chytridiomycota, and Zygomycota as well as the Oomycota and all mitosporic fungi (as defined by Hawksworth et al, In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK).
The fungal host cell may be a yeast cell. "Yeast" as used herein includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes). Since the classification of yeast may change in the future, for the purposes of this invention, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, Passmore, and Davenport, editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).
The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schiz- osaccharomyces, or Yarrowia cell, such as a Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytica cell.
The fungal host cell may be a filamentous fungal cell. "Filamentous fungi" include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra). The filamentous fungi are generally characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic. In contrast, vegetative growth by yeasts such as Saccharomyces cerevisiae is by budding of a unicellular thallus and carbon catabolism may be fermentative.
The filamentous fungal host cell may be an Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Mag- naporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cell.
For example, the filamentous fungal host cell may be an Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium in- ops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothe- cioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radi- ata, Pleurotus eryngii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cell.
Fungal cells may be transformed by a process involving protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se. Suitable procedures for transformation of Aspergillus and Trichoderma host cells are described in EP 238023, Yelton , 1984, Proetc.al. Natl. Acad. Sci. USA 81 : 1470-1474, and Christensen et al., 1988, Bio/Technology 6: 1419-1422. Suitable methods for transforming Fusarium species are described by Malardier , 1989, Geentea 7l.8: 147-156, and WO 96/00787. Yeast may be transformed using the procedures described by Becker and Guarente, In Abelson, J.N. and Simon, M.I., editors, Guide to Yeast Genetics and Molecular Biology, Methods in Enzy- mology, Volume 194, pp 182-187, Academic Press, Inc., New York; Ito et al. , 1983, J. Bacteriol. 153: 163; and Hinnen et al. , 1978, Proc. Natl. Acad. Sci. USA 75: 1920. Methods of production
Also disclosed are methods of producing (e.g., in vitro or ex vivo methods) a variant, comprising: (a) cultivating a host cell of the present invention under conditions suitable for expression of the variant; and (b) recovering the variant. Accordingly, also disclosed herein are methods of producing (e.g., in vitro or ex vivo methods) a variant, comprising: (a) cultivating a host cell comprising a polynucleotide encoding a xan- than lyase variant, comprising an alteration at one or more positions selected from the group consisting of: i) region 7 corresponding to amino acids 1 to 153 of SEQ ID NO: 2, ii) region 8 corresponding to amino acids 177 to 613 of SEQ ID NO: 2, iii) region 9 corresponding to amino acids 659 to 730 of SEQ ID NO: 2, iv) region 10 corresponding to amino acids 804 to 806 of SEQ ID NO: 2, v) region 1 1 corresponding to amino acids 847 to 871 of SEQ ID NO: 2, vi) region 12 corresponding to amino acids 886 to 902 of SEQ ID NO: 2, and vii) region 13 corresponding to amino acids 1005 to 1037 of SEQ ID NO: 2, wherein said variant has at least 60% and less than 100% sequence identity to SEQ ID NO: 2, under conditions suitable for expression of the variant; and (b) optionally, recovering the variant.
Further disclosed are methods of producing (e.g., in vitro or ex vivo methods) a variant of the present invention, comprising (a) cultivating a cell, which in its wild-type form produces the polypeptide, under con- ditions conducive for production of the polypeptide; and (b) recovering the polypeptide. In a preferred aspect, the cell is a Paenibacillus cell, or a Microbacterium cell.
Also disclosed are methods of producing (e.g., in vitro or ex vivo methods) a variant of the present invention, comprising (a) cultivating a recombinant host cell of the present invention under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide.
The host cells are cultivated in a nutrient medium suitable for production of the polypeptide using methods known in the art. For example, the cell may be cultivated by shake flask cultivation, or small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermenters performed in a suitable medium and under conditions allowing the polypeptide to be expressed and/or isolated. The cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g. , in catalogues of the American Type Culture Collection). If the polypeptide is secreted into the nutrient medium, the polypeptide can be recovered directly from the medium. If the polypeptide is not secreted, it can be recovered from cell lysates.
The variant polypeptide may be detected using methods known in the art that are specific for the polypeptides such as methods for determining cellulose or xanthan lyase activity. These detection methods include, but are not limited to, use of specific antibodies, formation of an enzyme product, or disappearance of an enzyme substrate. For example, an enzyme assay may be used to determine the activity of the polypeptide.
The variant polypeptide may be recovered using methods known in the art. For example, the polypep- tide may be recovered from the nutrient medium by conventional procedures including, but not limited to, collection, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation.
The variant polypeptide may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g. , ammo- nium sulfate precipitation), SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson and Ryden, editors, VCH Publishers, New York, 1989) to obtain substantially pure polypeptides.
In an alternative aspect, the variant polypeptide is not recovered, but rather a host cell expressing the polypeptide is used as a source of the variant polypeptide. Compositions
In one certain aspect, the variants according to the invention have improved stability in detergent compositions compared to a parent enzyme or compared to a xanthan lyase having the identical amino acid sequence of the variant, but not having an alteration (e.g., a substitution, deletion or insertion) at one or more of the specified positions or compared to the xanthan lyase with SEQ ID NO: 2, wherein activity and/or stability in detergent is measured as disclosed in Example 3 herein.
Besides enzymes the detergent compositions may comprise additional components. The choice of additional components is within the skill of the artisan and includes conventional ingredients, including the exemplary non-limiting components set forth below. The choice of components may include, for fabric care, the consideration of the type of fabric to be cleaned, the type and/or degree of soiling, the temperature at which cleaning is to take place, and the formulation of the detergent product. Although components mentioned below are categorized by general header according to a particular functionality, this is not to be construed as a limitation, as a component may comprise additional functionalities as will be appreciated by the skilled artisan.
The detergent composition may be suitable for the laundering of textiles such as e.g. fabrics, cloths or linen, or for cleaning hard surfaces such as e.g. floors, tables, or dish wash.
Detergent compositions
In one embodiment, a variant as described herein may be added to a detergent composition in an amount corresponding to 0.0001-200 mg of enzyme protein, such as 0.0005-100 mg of enzyme protein, preferably 0.001-30 mg of enzyme protein, more preferably 0.005-8 mg of enzyme protein, even more pref- erably 0.01 -2 mg of enzyme protein per litre of wash liquor.
A composition for use in automatic dishwash (ADW), for example, may include 0.0001 %-50%, such as 0.001 %-20%, such as 0.01 %-10%, such as 0.05-5% of enzyme protein by weight of the composition.
A composition for use in laundry granulation or a solid/granular laundry composition in general, for example, may include 0.0001 %-50%, such as 0.001 %-20%, such as 0.01 %-10%, such as 0.05%-5% of enzyme protein by weight of the composition.
A composition for use in laundry liquid, for example, may include 0.0001 %-10%, such as 0.001-7%, such as 0.1 %-5% of enzyme protein by weight of the composition.
The enzyme(s) of the detergent composition of the invention may be stabilized using conventional stabilizing agents, e.g., a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid, and the composition may be formulated as described in, for example, WO92/19709 and WO92/19708.
In certain markets different wash conditions and, as such, different types of detergents are used. This is disclosed in e.g. EP 1 025 240. For example, In Asia (Japan) a low detergent concentration system is used, while the United States uses a medium detergent concentration system, and Europe uses a high detergent concentration system.
A low detergent concentration system includes detergents where less than about 800 ppm of detergent components are present in the wash water. Japanese detergents are typically considered low detergent concentration system as they have approximately 667 ppm of detergent components present in the wash water.
A medium detergent concentration includes detergents where between about 800 ppm and about
2000ppm of detergent components are present in the wash water. North American detergents are generally considered to be medium detergent concentration systems as they have approximately 975 ppm of detergent components present in the wash water.
A high detergent concentration system includes detergents where greater than about 2000 ppm of detergent components are present in the wash water. European detergents are generally considered to be high detergent concentration systems as they have approximately 4500-5000 ppm of detergent components in the wash water.
Latin American detergents are generally high suds phosphate builder detergents and the range of detergents used in Latin America can fall in both the medium and high detergent concentrations as they range from 1500 ppm to 6000 ppm of detergent components in the wash water. Such detergent compositions are all embodiments of the invention.
A polypeptide of the present invention may also be incorporated in the detergent formulations disclosed in WO97/07202, which is hereby incorporated by reference. Surfactants
The detergent composition may comprise one or more surfactants, which may be anionic and/or cationic and/or non-ionic and/or semi-polar and/or zwitterionic, or a mixture thereof. In a particular embodiment, the detergent composition includes a mixture of one or more non-ionic surfactants and one or more anionic surfactants. The surfactant(s) is typically present at a level of from about 0.1 % to 60% by weight, such as about 1 % to about 40%, or about 3% to about 20%, or about 3% to about 10%. The surfactant(s) is chosen based on the desired cleaning application, and includes any conventional surfactant(s) known in the art. Any surfactant known in the art for use in detergents may be utilized.
When included therein the detergent will usually comprise from about 1 % to about 40% by weight, such as from about 5% to about 30%, including from about 5% to about 15%, or from about 20% to about 25% of an anionic surfactant. Non-limiting examples of anionic surfactants include sulfates and sulfonates, in particular, linear alkylbenzenesulfonates (LAS), isomers of LAS, branched alkylbenzenesulfonates (BABS), phenylalkanesulfonates, alpha-olefinsulfonates (AOS), olefin sulfonates, alkene sulfonates, alkane-2,3- diylbis(sulfates), hydroxyalkanesulfonates and disulfonates, alkyl sulfates (AS) such as sodium dodecyl sul- fate (SDS), fatty alcohol sulfates (FAS), primary alcohol sulfates (PAS), alcohol ethersulfates (AES or AEOS or FES, also known as alcohol ethoxysulfates or fatty alcohol ether sulfates), secondary alkanesulfonates (SAS), paraffin sulfonates (PS), ester sulfonates, sulfonated fatty acid glycerol esters, alpha-sulfo fatty acid methyl esters (alpha-SFMe or SES) including methyl ester sulfonate (MES), alkyl- or alkenylsuccinic acid, dodecenyl/tetradecenyl succinic acid (DTSA), fatty acid derivatives of amino acids, diesters and monoesters of sulfo-succinic acid or soap, and combinations thereof.
When included therein the detergent will usually comprise from about 0% to about 10% by weight of a cationic surfactant. Non-limiting examples of cationic surfactants include alklydimethylethanolamine quat (ADMEAQ), cetyltrimethylammonium bromide (CTAB), dimethyldistearylammonium chloride (DSDMAC), and alkylbenzyldimethylammonium, alkyl quaternary ammonium compounds, alkoxylated quaternary am- monium (AQA) compounds, and combinations thereof.
When included therein the detergent will usually comprise from about 0.2% to about 40% by weight of a non-ionic surfactant, for example from about 0.5% to about 30%, in particular from about 1 % to about 20%, from about 3% to about 10%, such as from about 3% to about 5%, or from about 8% to about 12%. Non-limiting examples of non-ionic surfactants include alcohol ethoxylates (AE or AEO), alcohol propox- ylates, propoxylated fatty alcohols (PFA), alkoxylated fatty acid alkyl esters, such as ethoxylated and/or propoxylated fatty acid alkyl esters, alkylphenol ethoxylates (APE), nonylphenol ethoxylates (NPE), al- kylpolyglycosides (APG), alkoxylated amines, fatty acid monoethanolamides (FAM), fatty acid diethanola- mides (FADA), ethoxylated fatty acid monoethanolamides (EFAM), propoxylated fatty acid monoethanolamides (PFAM), polyhydroxy alkyl fatty acid amides, or N-acyl N-alkyl derivatives of glucosamine (glu- camides, GA, or fatty acid glucamide, FAGA), as well as products available under the trade names SPAN and TWEEN, and combinations thereof. When included therein the detergent will usually comprise from about 0% to about 10% by weight of a semipolar surfactant. Non-limiting examples of semipolar surfactants include amine oxides (AO) such as alkyldimethylamineoxide, N-(coco alkyl)-N,N-dimethylamine oxide and N-(tallow-alkyl)-N,N-bis(2-hydroxy- ethyl)amine oxide, fatty acid alkanolamides and ethoxylated fatty acid alkanolamides, and combinations thereof.
When included therein the detergent will usually comprise from about 0% to about 10% by weight of a zwitterionic surfactant. Non-limiting examples of zwitterionic surfactants include betaine, alkyldimethylbeta- ine, sulfobetaine, and combinations thereof. Hvdrotropes
A hydrotrope is a compound that solubilises hydrophobic compounds in aqueous solutions (or oppositely, polar substances in a non-polar environment). Typically, hydrotropes have both hydrophilic and a hydrophobic character (so-called amphiphilic properties as known from surfactants); however, the molecular structure of hydrotropes generally do not favor spontaneous self-aggregation, see e.g. review by Hodgdon and Kaler (2007), Current Opinion in Colloid & Interface Science 12: 121-128. Hydrotropes do not display a critical concentration above which self-aggregation occurs as found for surfactants and lipids forming micel- lar, lamellar or other well defined meso-phases. Instead, many hydrotropes show a continuous-type aggregation process where the sizes of aggregates grow as concentration increases. However, many hydrotropes alter the phase behaviour, stability, and colloidal properties of systems containing substances of polar and non-polar character, including mixtures of water, oil, surfactants, and polymers. Hydrotropes are classically used across industries from pharma, personal care, food, to technical applications. Use of hydrotropes in detergent compositions allow for example more concentrated formulations of surfactants (as in the process of compacting liquid detergents by removing water) without inducing undesired phenomena such as phase separation or high viscosity.
The detergent may comprise 0-5% by weight, such as about 0.5 to about 5%, or about 3% to about
5%, of a hydrotrope. Any hydrotrope known in the art for use in detergents may be utilized. Non-limiting examples of hydrotropes include sodium benzene sulfonate, sodium p-toluene sulfonate (STS), sodium xylene sulfonate (SXS), sodium cumene sulfonate (SCS), sodium cymene sulfonate, amine oxides, alcohols and polyglycolethers, sodium hydroxynaphthoate, sodium hydroxynaphthalene sulfonate, sodium ethylhexyl sulfate, and combinations thereof.
Builders and Co-Builders
The detergent composition may comprise about 0-65% by weight, such as about 5% to about 45% of a detergent builder or co-builder, or a mixture thereof. In a dish wash detergent, the level of builder is typi- cally 40-65%, particularly 50-65%. The builder and/or co-builder may particularly be a chelating agent that forms water-soluble complexes with Ca and Mg. Any builder and/or co-builder known in the art for use in laundry detergents may be utilized. Non-limiting examples of builders include zeolites, diphosphates (pyrophosphates), triphosphates such as sodium triphosphate (STP or STPP), carbonates such as sodium carbonate, soluble silicates such as sodium metasilicate, layered silicates (e.g. , SKS-6 from Hoechst), ethan- olamines such as 2-aminoethan-1 -ol (MEA), diethanolamine (DEA, also known as iminodiethanol), trieth- anolamine (TEA, also known as 2,2',2"-nitrilotriethanol), and carboxymethyl inulin (CMI), and combinations thereof.
The detergent composition may also comprise 0-20% by weight, such as about 5% to about 10%, of a detergent co-builder, or a mixture thereof. The detergent composition may include a co-builder alone, or in combination with a builder, for example a zeolite builder. Non-limiting examples of co-builders include ho- mopolymers of polyacrylates or copolymers thereof, such as poly(acrylic acid) (PAA) or copoly(acrylic acid/maleic acid) (PAA PMA). Further non-limiting examples include citrate, chelators such as aminocar- boxylates, aminopolycarboxylates and phosphonates, and alkyl- or alkenylsuccinic acid . Additional specific examples include 2,2',2"-nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA), diethylenetri- aminepentaacetic acid (DTPA), iminodisuccinic acid (IDS), ethylenediamine-N, N'-disuccinic acid (EDDS), methylglycinediacetic acid (MGDA), glutamic acid-N ,N-diacetic acid (GLDA), 1 -hydroxyethane-1 ,1 -diphos- phonic acid (HEDP), ethylenediaminetetra-(methylenephosphonic acid) (EDTMPA), diethylenetriamine- pentakis(methylenephosphonic acid) (DTPMPA or DTMPA), N-(2-hydroxyethyl)iminodiacetic acid (EDG), aspartic acid- N-monoacetic acid (ASMA), aspartic acid-N,N-diacetic acid (ASDA), aspartic acid-N-mono- propionic acid (ASMP), iminodisuccinic acid (IDA), N-(2-sulfomethyl)-aspartic acid (SMAS), N-(2-sulfoethyl)- aspartic acid (SEAS), N-(2-sulfomethyl)-glutamic acid (SMGL), N-(2-sulfoethyl)-glutamic acid (SEGL), N- methyliminodiacetic acid (MIDA), a-alanine-N,N-diacetic acid (a-ALDA), serine-N,N-diacetic acid (SEDA), isoserine-N,N-diacetic acid (ISDA), phenylalanine-N,N-diacetic acid (PHDA), anthranilic acid-N,N-diacetic acid (ANDA), sulfanilic acid-N,N-diacetic acid (SLDA), taurine-N,N-diacetic acid (TUDA) and sulfomethyl-N,N-diacetic acid (SMDA), N-(2-hydroxyethyl)-ethylidenediamine-N,N',N'-triacetate (HEDTA), diethanolgly- cine (DEG), diethylenetriamine penta(methylenephosphonic acid) (DTPMP), aminotris(meth- ylenephosphonic acid) (ATMP), and combinations and salts thereof. Further exemplary builders and/or co- builders are described in, e.g., WO 09/102854, US 5977053.
Bleaching Systems
The detergent may comprise 0-50% by weight, such as about 0.1 % to about 25%, of a bleaching system. Any bleaching system known in the art for use in laundry detergents may be utilized . Suitable bleaching system components include bleaching catalysts, photobleaches, bleach activators, sources of hydrogen peroxide such as sodium percarbonate and sodium perborates, preformed peracids and mixtures thereof. Suitable preformed peracids include, but are not limited to, peroxycarboxylic acids and salts, percarbonic acids and salts, perimidic acids and salts, peroxymonosulfuric acids and salts, for example, Oxone (R), and mixtures thereof. Non-limiting examples of bleaching systems include peroxide-based bleaching systems, which may comprise, for example, an inorganic salt, including alkali metal salts such as sodium salts of perborate (usually mono- or tetra-hydrate), percarbonate, persulfate, perphosphate, persilicate salts, in combination with a peracid-forming bleach activator. The term bleach activator is meant herein as a compound which reacts with peroxygen bleach like hydrogen peroxide to form a peracid. The peracid thus formed constitutes the activated bleach. Suitable bleach activators to be used herein include those belonging to the class of esters amides, imides or anhydrides. Suitable examples are tetracetylethylene diamine (TAED), sodium 4-[(3,5,5-trimethylhexanoyl)oxy]benzene sulfonate (ISONOBS), diperoxy dodecanoic acid, 4-(dodecanoyloxy)benzenesulfonate (LOBS), 4-(decanoyloxy)benzenesulfonate, 4-(decanoyloxy)benzoate (DOBS), 4-(nonanoyloxy)-benzenesulfonate (NOBS), and/or those disclosed in W098/17767. A particular family of bleach activators of interest was disclosed in EP624154 and particularly preferred in that family is acetyl triethyl citrate (ATC). ATC or a short chain triglyceride like triacetin has the advantage that it is environmental friendly as it eventually degrades into citric acid and alcohol. Furthermore, acetyl triethyl citrate and triacetin has a good hydrolytical stability in the product upon storage and it is an efficient bleach activator. Finally, ATC provides a good building capacity to the laundry additive. Alternatively, the bleaching system may comprise peroxyacids of, for example, the amide, imide, or sulfone type. The bleaching system may also comprise peracids such as 6-(phthalimido)peroxyhexanoic acid (PAP). The bleaching system may also include a bleach catalyst. In some embodiments the bleach component may be an organic catalyst selected from the group consisting of organic catalysts having the following formulae:
Figure imgf000061_0001
to 24 carbons or linear alkyl group containing from 11 to 24 carbons, preferably each R is independently a branched alkyl group containing from 9 to 18 carbons or linear alkyl group containing from 1 1 to 18 carbons, more preferably each R is independently selected from the group consisting of 2-propylheptyl, 2-butyloctyl, 2-pentylnonyl, 2-hexyldecyl, n-dodecyl, n-tetradecyl, n-hexadecyl, n-octadecyl, iso-nonyl, iso-decyl, iso- tridecyl and iso-pentadecyl. Other exemplary bleaching systems are described, e.g. in WO2007/087258, WO2007/087244, WO2007/087259 and WO2007/087242. Suitable photobleaches may for example be sulfonated zinc phthalocyanine.
Polymers
The detergent may comprise 0-10% by weight, such as 0.5-5%, 2-5%, 0.5-2% or 0.2-1 % of a polymer. Any polymer known in the art for use in detergents may be utilized. The polymer may function as a co- builder as mentioned above, or may provide anti-redeposition, fiber protection, soil release, dye transfer inhibition, grease cleaning and/or anti-foaming properties. Some polymers may have more than one of the above-mentioned properties and/or more than one of the below-mentioned motifs. Exemplary polymers include (carboxymethyl)cellulose (CMC), polyvinyl alcohol) (PVA), poly(vinylpyrrolidone) (PVP), poly(eth- yleneglycol) or poly(ethylene oxide) (PEG), ethoxylated poly(ethyleneimine), carboxymethyl inulin (CMI), and polycarboxylates such as PAA, PAA PMA, poly-aspartic acid, and lauryl methacrylate/acrylic acid copolymers, hydrophobically modified CMC (HM-CMC) and silicones, copolymers of terephthalic acid and oligomeric glycols, copolymers of poly(ethylene terephthalate) and poly(oxyethene terephthalate) (PET- POET), PVP, poly(vinylimidazole) (PVI), poly(vinylpyridine-N-oxide) (PVPO or PVPNO) and polyvinylpyrrol- idone-vinylimidazole (PVPVI). Further exemplary polymers include sulfonated polycarboxylates, polyeth- ylene oxide and polypropylene oxide (PEO-PPO) and diquaternium ethoxy sulfate. Other exemplary polymers are disclosed in, e.g., WO 2006/130575. Salts of the above-mentioned polymers are also contemplated .
Fabric hueing agents
The detergent compositions of the present invention may also include fabric hueing agents such as dyes or pigments, which when formulated in detergent compositions can deposit onto a fabric when said fabric is contacted with a wash liquor comprising said detergent compositions and thus altering the tint of said fabric through absorption/reflection of visible light. Fluorescent whitening agents emit at least some visible light. In contrast, fabric hueing agents alter the tint of a surface as they absorb at least a portion of the visible light spectrum. Suitable fabric hueing agents include dyes and dye-clay conjugates, and may also include pigments. Suitable dyes include small molecule dyes and polymeric dyes. Suitable small molecule dyes include small molecule dyes selected from the group consisting of dyes falling into the Colour Index (C.I.) classifications of Direct Blue, Direct Red, Direct Violet, Acid Blue, Acid Red, Acid Violet, Basic Blue, Basic Violet and Basic Red, or mixtures thereof, for example as described in WO2005/03274, WO2005/03275, WO2005/03276 and EP1876226 (hereby incorporated by reference). The detergent composition preferably comprises from about 0.00003 wt% to about 0.2 wt%, from about 0.00008 wt% to about 0.05 wt%, or even from about 0.0001 wt% to about 0.04 wt% fabric hueing agent. The composition may comprise from 0.0001 wt% to 0.2 wt% fabric hueing agent, this may be especially preferred when the composition is in the form of a unit dose pouch. Suitable hueing agents are also disclosed in, e.g. WO 2007/087257 and WO2007/087243.
Additional Enzymes
The detergent additive as well as the detergent composition may comprise one or more additional enzymes such as a xanthan lyase, protease, lipase, cutinase, an amylase, lichenase, carbohydrase, cellu- lase, pectinase, mannanase, arabinase, galactanase, xylanase, oxidase, e.g., a laccase, and/or peroxidase.
In general, the properties of the selected enzyme(s) should be compatible with the selected detergent, (;'.e., pH-optimum, compatibility with other enzymatic and non-enzymatic ingredients, etc.), and the en- zyme(s) should be present in effective amounts.
Cellulases
Suitable cellulases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Suitable cellulases include cellulases from the genera Bacillus, Pseudomo- nas, Humicola, Fusarium, Thielavia, Acremonium, e.g., the fungal cellulases produced from Humicola in- solens, Myceliophthora thermophila and Fusarium oxysporum disclosed in US 4,435,307, US 5,648,263, US 5,691 ,178, US 5,776,757 and WO 89/09259.
Especially suitable cellulases are the alkaline or neutral cellulases having color care benefits. Examples of such cellulases are cellulases described in EP 0 495 257, EP 0 531 372, WO 96/11262, WO 96/29397, WO 98/08940. Other examples are cellulase variants such as those described in WO 94/07998, EP 0 531 315, US 5,457,046, US 5,686,593, US 5,763,254, WO 95/24471 , WO 98/12307 and PCT/DK98/00299.
Example of cellulases exhibiting endo-beta-1 ,4-glucanase activity (EC 3.2.1.4) are those having de- scribed in WO02/099091 .
Other examples of cellulases include the family 45 cellulases described in W096/29397, and especially variants thereof having substitution, insertion and/or deletion at one or more of the positions corresponding to the following positions in SEQ ID NO: 8 of WO 02/099091 : 2, 4, 7, 8, 10, 13, 15, 19, 20, 21 , 25, 26, 29, 32, 33, 34, 35, 37, 40, 42, 42a, 43, 44, 48, 53, 54, 55, 58, 59, 63, 64, 65, 66, 67, 70, 72, 76, 79, 80, 82, 84, 86, 88, 90, 91 , 93, 95, 95d, 95h, 95j, 97, 100, 101 , 102, 103, 113, 114, 1 17, 119, 121 , 133, 136, 137, 138, 139, 140a, 141 , 143a, 145, 146, 147, 150e, 150j, 151 , 152, 153, 154, 155, 156, 157, 158, 159, 160c, 160e, 160k, 161 , 162, 164, 165, 168, 170, 171 , 172, 173, 175, 176, 178, 181 , 183, 184, 185, 186, 188, 191 , 192, 195, 196, 200, and/or 20, preferably selected among P19A, G20K, Q44K, N48E, Q1 19H or Q146 R.
Commercially available cellulases include Celluzyme™, and Carezyme™ (Novozymes A/S), Clazi- nase™, and Puradax HA™ (Genencor International Inc.), and KAC-500(B)™ (Kao Corporation).
Proteases
The additional enzyme may be another protease or protease variant. The protease may be of animal, vegetable or microbial origin, including chemically or genetically modified mutants. Microbial origin is pre- ferred. It may be an alkaline protease, such as a serine protease or a metalloprotease. A serine protease may for example be of the S1 family, such as trypsin, or the S8 family such as subtilisin. A metalloproteases protease may for example be a thermolysin from e.g. family M4, M5, M7 or M8.
The term "subtilases" refers to a sub-group of serine protease according to Siezen et al., Protein Engng. 4 (1991 ) 719-737 and Siezen et al. Protein Science 6 (1997) 501 -523. Serine proteases are a subgroup of proteases characterized by having a serine in the active site, which forms a covalent adduct with the substrate. The subtilases may be divided into 6 sub-divisions, i.e. the Subtilisin family, the Thermitase family, the Proteinase K family, the Lantibiotic peptidase family, the Kexin family and the Pyrolysin family. In one aspect of the invention the protease may be a subtilase, such as a subtilisin or a variant hereof. Further the subtilases (and the serine proteases) are characterised by having two active site amino acid residues apart from the serine, namely a histidine and an aspartic acid residue.
Examples of subtilisins are those derived from Bacillus such as subtilisin lentus, Bacillus lentus, subtilisin Novo, subtilisin Carlsberg, Bacillus licheniformis, subtilisin BPN', subtilisin 309, subtilisin 147 and subtilisin 168 described in WO 89/06279 and protease PD138 (WO 93/18140). Additional serine protease examples are described in WO 98/0201 15, WO 01/44452, WO 01/58275, WO 01/58276, WO 03/006602 and WO 04/099401. An example of a subtilase variants may be those having mutations in any of the positions: 3, 4, 9, 15, 27, 36, 68, 76, 87, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104, 106, 118, 120, 123, 128, 129, 130, 160, 167, 170, 194, 195, 199, 205, 217, 218, 222, 232, 235, 236, 245, 248, 252 and 274 using the BPN' numbering. More preferred the subtilase variants may comprise the mutations: S3T, V4I, S9R, A15T, K27R, *36D, V68A, N76D, N87S.R, *97E, A98S, S99G,D,A, S99AD, S101 G,M,R S103A, V104I,Y,N, S106A, G118V.R, H120D.N, N123S, S128L, P129Q, S130A, G160D, Y167A, R170S, A194P, G195E, V199M, V205I, L217D, N218D, M222S, A232V, K235L, Q236H, Q245R, N252K, T274A (using BPN' numbering). A further preferred protease is the alkaline protease from Bacillus lentus DSM 5483, as described for example in WO 95/23221 , and variants thereof which are described in WO 92/21760, WO 95/23221 , EP 1921147 and EP 1921 148.
Examples of trypsin-like proteases are trypsin (e.g. of porcine or bovine origin) and the Fusarium pro- tease described in WO 89/06270 and WO 94/25583. Examples of useful proteases are the variants described in WO 92/19729, WO 98/201 15, WO 98/201 16, and WO 98/34946, especially the variants with substitutions in one or more of the following positions: 27, 36, 57, 76, 87, 97, 101 , 104, 120, 123, 167, 170, 194, 206, 218, 222, 224, 235, and 274.
Examples of metalloproteases are the neutral metalloprotease as described in WO 07/044993.
Preferred commercially available protease enzymes include Alcalase™, Coronase™, Duralase™, Du- razym™, Esperase™, Everlase™, Kannase™, Liquanase™, Liquanase Ultra™, Ovozyme™, Polarzyme™, Primase™, Relase™, Savinase™ and Savinase Ultra™, (Novozymes A/S), Axapem™ (Gist-Brocases N.V.), BLAP and BLAP X (Henkel AG & Co. KGaA), Excellase™, FN2™, FN3™, FN4™, Maxaca™, Maxapem™, Maxatase™, Properase™, Purafast™, Purafect™, Purafect OxP™, Purafect Prime™ and Puramax™ (Genencor int.).
Lipases and Cutinases
Suitable lipases and cutinases include those of bacterial or fungal origin. Chemically modified or protein engineered mutant enzymes are included. Examples include lipase from Thermomyces, e.g. from T. lanu- ginosus (previously named Humicola lanuginosa) as described in EP258068 and EP305216, cutinase from Humicola, e.g. H. insolens (WO96/13580), lipase from strains of Pseudomonas (some of these now renamed to Burkholdeha), e.g. P. alcaligenes or P. pseudoalcaligenes (EP218272), P. cepacia (EP331376), P. sp. strain SD705 (WO95/06720 & WO96/27002), P. wisconsinensis (WO96/12012), GDSL-type Strepto- myces lipases (W010/065455), cutinase from Magnaporthe grisea (W010/107560), cutinase from Pseudo- monas mendocina (US5,389,536), lipase from Thermobifida fusca (W01 1 /084412), Geobacillus stearother- mophilus lipase (W01 1/084417), lipase from Bacillus subtilis (W011/084599), and lipase from Streptomy- ces griseus (W01 1/150157) and S. pristinaespiralis (W012/137147).
Further examples are lipases sometimes referred to as acyltransferases or perhydrolases, e.g. acyl- transferases with homology to Candida antarctica lipase A (W010/1 11 143), acyltransferase from Mycobac- terium smegmatis (WO05/56782), perhydrolases from the CE 7 family (WO09/67279), and variants of the M. smegmatis perhydrolase in particular the S54V variant used in the commercial product Gentle Power Bleach from Huntsman Textile Effects Pte Ltd (W010/100028).
Other examples are lipase variants such as those described in EP407225, WO92/05249, WO94/01541 , W094/25578, W095/14783, WO95/30744, W095/35381 , W095/22615, WO96/00292, WO97/04079, WO97/07202, WO00/34450, WO00/60063, WO01/92502, WO07/87508 and WO09/109500.
Preferred commercial lipase products include Lipolase™, Lipex™; Lipolex™ and Lipoclean™ (Novo- zymes A/S), Lumafast (originally from Genencor) and Lipomax (originally from Gist-Brocades).
Amylases
The amylase may be an alpha-amylase, a beta-amylase or a glucoamylase and may be of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Amylases include, for example, alpha-amylases obtained from Bacillus, e.g. , a special strain of Bacillus licheniformis, described in more detail in GB 1 ,296,839.
Examples of amylases are those having SEQ ID NO: 3 in WO 95/10603 or variants having 90% se- quence identity to SEQ ID NO: 3 thereof. Preferred variants are described in WO 94/02597, WO 94/18314, WO 97/43424 and SEQ ID NO: 4 of WO 99/019467, such as variants with substitutions in one or more of the following positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 178, 179, 181 , 188, 190, 197, 201 , 202, 207, 208, 209, 21 1 , 243, 264, 304, 305, 391 , 408, and 444 of SEQ ID NO: 3 in WO 95/10603.
Other amylases are variants of SEQ ID NO: 1 of WO 2016/203064 having at least 75% sequence identity to SEQ ID NO: 1 thereof. Preferred variants are variants comprising a modification in one or more positions corresponding to positions 1 , 54, 56, 72, 109, 113, 1 16, 134, 140, 159, 167, 169, 172, 173, 174, 181 , 182, 183, 184, 189, 194, 195, 206, 255, 260, 262, 265, 284, 289, 304, 305, 347, 391 , 395, 439, 469, 444, 473, 476, or 477 of SEQ ID NO: 1 , wherein said alpha-amylase variant has a sequence identity of at least 75% but less than 100% to SEQ ID NO: 1 .
Further amylases which can be used are amylases having SEQ ID NO: 6 in WO 02/010355 or variants thereof having 90% sequence identity to SEQ ID NO: 6. Preferred variants of SEQ ID NO: 6 are those having a deletion in positions 181 and 182 and a substitution in position 193.
Other amylase examples are hybrid alpha-amylase comprising residues 1-33 of the alpha-amylase derived from B. amyloliquefaciens shown in SEQ ID NO: 6 of WO 2006/066594 and residues 36-483 of the B. licheniformis alpha-amylase shown in SEQ ID NO: 4 of WO 2006/066594 or variants having 90% se- quence identity thereof. Preferred variants of this hybrid alpha-amylase are those having a substitution, a deletion or an insertion in one of more of the following positions: G48, T49, G107, H156, A181 , N190, M197, 1201 , A209 and Q264. Most preferred variants of the hybrid alpha-amylase comprising residues 1-33 of the alpha-amylase derived from B. amyloliquefaciens shown in SEQ ID NO: 6 of WO 2006/066594 and residues 36-483 of SEQ ID NO: 4 are those having the substitutions:
M197T;
H156Y+A181T+N190F+A209V+Q264S; or
G48+T49+G107+H156+A181 +N190+1201 +A209+Q264.
Further amylase examples are amylases having SEQ ID NO: 6 in WO 99/019467 or variants thereof having 90% sequence identity to SEQ ID NO: 6. Preferred variants of SEQ ID NO: 6 are those having a substitution, a deletion or an insertion in one or more of the following positions: R181 , G182, H183, G184, N195, I206, E212, E216 and K269. Particularly preferred amylases are those having deletion in positions G182 and H183 or positions H183 and G184.
Additional amylases are those having SEQ ID NO: 1 , SEQ ID NO: 2 or SEQ ID NO: 7 of WO 96/023873 or variants thereof having 90% sequence identity to SEQ ID NO: 1 , SEQ ID NO: 2 or SEQ ID NO: 7. Pre- ferred variants of SEQ ID NO: 1 , SEQ ID NO: 2 or SEQ ID NO: 7 are those having a substitution, a deletion or an insertion in one or more of the following positions: 140, 181 , 182, 183, 184, 195, 206, 212, 243, 260, 269, 304 and 476. More preferred variants are those having a deletion in positions 182 and 183 or positions 183 and 184. Most preferred amylase variants of SEQ ID NO: 1 , SEQ ID NO: 2 or SEQ ID NO: 7 are those having a deletion in positions 183 and 184 and a substitution in positions 140, 195, 206, 243, 260, 304 and 476.
Other amylases which can be used are amylases having SEQ ID NO: 2 of WO 08/153815, SEQ ID NO: 10 in WO 01/66712 or variants thereof having 90% sequence identity to SEQ ID NO: 2 of WO 08/153815 or 90% sequence identity to SEQ ID NO: 10 in WO 01/66712. Preferred variants of SEQ ID NO: 10 in WO 01/66712 are those having a substitution, a deletion or an insertion in one of more of the following positions: 176, 177, 178, 179, 190, 201 , 207, 211 and 264.
Further amylases which can be used are amylases having SEQ ID NO: 2 of WO 09/061380 or variants thereof having 90% sequence identity to SEQ ID NO: 2. Preferred variants of SEQ ID NO: 2 are those having a substitution, a deletion or an insertion in one of more of the following positions: Q87, Q98, S125, N128, T131 , T165, K178, R180, S181 , T182, G183, M201 , F202, N225, S243, N272, N282, Y305, R309, D319, Q320, Q359, K444 and G475. More preferred variants of SEQ ID NO: 2 are those having the substitution in one of more of the following positions: Q87E.R, Q98R, S125A, N128C, T131 I, T165I, K178L, T182G, M201 L, F202Y, N225E.R, N272E.R, S243Q,A,E,D, Y305R, R309A, Q320R, Q359E, K444E and G475K and/or deletion in position R180 and/or S181. Most preferred amylase variants of SEQ ID NO: 2 are those having the substitutions:
N128C+K178L+T182G+Y305R+G475K;
N128C+K178L+T182G+F202Y+Y305R+D319T+G475K;
S125A+N128C+K178L+T182G+Y305R+G475K; or
S125A+N128C+T131 I+T165I+K178L+T182G+Y305R+G475K wherein the variant optionally further comprises a substitution at position 243 and/or a deletion at position 180 and/or position 181.
Other examples of amylases are the alpha-amylase having SEQ ID NO: 12 in WO01/66712 or a variant having at least 90%, such as at least 95%, sequence identity to SEQ ID NO: 12. Preferred amylase variants are those having a substitution, a deletion or an insertion in one of more of the following positions of SEQ ID NO: 12 in WO01/66712: R28, R118, N174; R181 , G182, D183, G184, G186, W189, N195, M202, Y298, N299, K302, S303, N306, R310, N314; R320, H324, E345, Y396, R400, W439, R444, N445, K446, Q449, R458, N471 , N484. Particular preferred amylases include variants having a deletion of D183 and G184 and having the substitutions R1 18K, N195F, R320K and R458K, and a variant additionally having substitutions in one or more position selected from the group: M9, G149, G182, G186, M202, T257, Y295, N299, M323, E345 and A339, most preferred a variant that additionally has substitutions in all these positions.
Commercially available amylases are Duramyl™, Termamyl™, Fungamyl™, Stainzyme™, Stainzyme Plus™, Natalase™ and BAN™ (Novozymes A S), Rapidase™ and Purastar™ (from Genencor International Inc.).
Peroxidases/Oxidases
Suitable peroxidases/oxidases include those of plant, bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful peroxidases include peroxidases from Coprinus, e.g. , from C. cinereus, and variants thereof as those described in WO 93/24618, WO 95/10602, and WO 98/15257.
Commercially available peroxidases include Guardzyme™ (Novozymes A/S).
The detergent enzyme(s) may be included in a detergent composition by adding separate additives containing one or more enzymes, or by adding a combined additive comprising all of these enzymes. A detergent additive of the invention, i.e., a separate additive or a combined additive, can be formulated, for example, as a granulate, liquid, slurry, etc. Preferred detergent additive formulations are granulates, in particular non-dusting granulates, liquids, in particular stabilized liquids, or slurries.
Non-dusting granulates may be produced, e.g., as disclosed in US 4,106,991 and 4,661 ,452 and may optionally be coated by methods known in the art. Examples of waxy coating materials are poly(ethylene oxide) products (polyethyleneglycol, PEG) with mean molar weights of 1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon atoms and in which there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application by fluid bed techniques are given in GB 1483591. Liquid enzyme preparations may, for instance, be stabilized by adding a polyol such as propylene glycol, a sugar or sugar alcohol, lactic acid or boric acid according to established methods. Protected enzymes may be prepared according to the method disclosed in EP 238,216.
Adjunct materials
Any detergent components known in the art for use in laundry detergents may also be utilized. Other optional detergent components include anti-corrosion agents, anti-shrink agents, anti-soil redeposition agents, anti-wrinkling agents, bactericides, binders, corrosion inhibitors, disintegrants/disintegration agents, dyes, enzyme stabilizers (including boric acid, borates, CMC, and/or polyols such as propylene glycol), fabric conditioners including clays, fillers/processing aids, fluorescent whitening agents/optical brighteners, foam boosters, foam (suds) regulators, perfumes, soil-suspending agents, softeners, suds suppressors, tarnish inhibitors, and wicking agents, either alone or in combination. Any ingredient known in the art for use in laundry detergents may be utilized. The choice of such ingredients is well within the skill of the artisan.
Dispersants: The detergent compositions of the present invention can also contain dispersants. In particular powdered detergents may comprise dispersants. Suitable water-soluble organic materials include the homo- or co-polymeric acids or their salts, in which the polycarboxylic acid comprises at least two car- boxyl radicals separated from each other by not more than two carbon atoms. Suitable dispersants are for example described in Powdered Detergents, Surfactant science series volume 71 , Marcel Dekker, Inc.
Dye Transfer Inhibiting Agents: The detergent compositions of the present invention may also include one or more dye transfer inhibiting agents. Suitable polymeric dye transfer inhibiting agents include, but are not limited to, polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidones and polyvinylimidazoles or mixtures thereof. When present in a subject composition, the dye transfer inhibiting agents may be present at levels from about 0.0001 % to about 10%, from about 0.01 % to about 5% or even from about 0.1 % to about 3% by weight of the composition.
Fluorescent whitening agent: The detergent compositions of the present invention will preferably also contain additional components that may tint articles being cleaned, such as fluorescent whitening agent or optical brighteners. Where present the brightener is preferably at a level of about 0,01 % to about 0,5%. Any fluorescent whitening agent suitable for use in a laundry detergent composition may be used in the composition of the present invention. The most commonly used fluorescent whitening agents are those belonging to the classes of diaminostilbene-sulphonic acid derivatives, diarylpyrazoline derivatives and bisphenyl-dis- tyryl derivatives. Examples of the diaminostilbene-sulphonic acid derivative type of fluorescent whitening agents include the sodium salts of: 4,4'-bis-(2-diethanolamino-4-anilino-s-triazin-6-ylamino) stilbene-2,2'-di- sulphonate; 4,4'-bis-(2,4-dianilino-s-triazin-6-ylamino) stilbene-2.2'-disulphonate; 4,4'-bis-(2-anilino-4(N- methyl-N-2-hydroxy-ethylamino)-s-triazin-6-ylamino) stilbene-2,2'-disulphonate, 4,4'-bis-(4-phenyl-2,1 ,3-tri- azol-2-yl)stilbene-2,2'-disulphonate; 4,4'-bis-(2-anilino-4(1 -methyl-2-hydroxy-ethylamino)-s-triazin-6-yla- mino) stilbene-2,2'-disulphonate and 2-(stilbyl-4"-naptho-1 ,2':4,5)-1 ,2,3-trizole-2"-sulphonate. Preferred fluorescent whitening agents are Tinopal DMS and Tinopal CBS available from Ciba-Geigy AG, Basel, Switzerland. Tinopal DMS is the disodium salt of 4,4'-bis-(2-morpholino-4 anilino-s-triazin-6-ylamino) stilbene disulphonate. Tinopal CBS is the disodium salt of 2,2'-bis-(phenyl-styryl) disulphonate. Also preferred are fluorescent whitening agents is the commercially available Parawhite KX, supplied by Paramount Minerals and Chemicals, Mumbai, India. Other fluorescers suitable for use in the invention include the 1 -3-diaryl pyrazolines and the 7-alkylaminocoumarins. Suitable fluorescent brightener levels include lower levels of from about 0.01 , from 0.05, from about 0.1 or even from about 0.2 wt % to upper levels of 0.5 or even 0.75 wt%.
Soil release polymers: The detergent compositions of the present invention may also include one or more soil release polymers which aid the removal of soils from fabrics such as cotton and polyester based fabrics, in particular the removal of hydrophobic soils from polyester based fabrics. The soil release polymers may for example be non-ionic or anionic terephthalate based polymers, polyvinyl caprolactam and related copolymers, vinyl graft copolymers, polyester polyamides see for example Chapter 7 in Powdered Detergents, Surfactant science series volume 71 , Marcel Dekker, Inc. Another type of soil release polymers are amphiphilic alkoxylated grease cleaning polymers comprising a core structure and a plurality of alkox- ylate groups attached to that core structure. The core structure may comprise a polyalkylenimine structure or a polyalkanolamine structure as described in detail in WO 2009/087523 (hereby incorporated by reference). Furthermore, random graft co-polymers are suitable soil release polymers Suitable graft co-polymers are described in more detail in WO 2007/138054, WO 2006/108856 and WO 2006/1 13314 (hereby incor- porated by reference). Other soil release polymers are substituted polysaccharide structures especially substituted cellulosic structures such as modified cellulose derivatives such as those described in EP 1867808 or WO 2003/040279 (both are hereby incorporated by reference). Suitable cellulosic polymers include cellulose, cellulose ethers, cellulose esters, cellulose amides and mixtures thereof. Suitable cellulosic polymers include anionically modified cellulose, nonionically modified cellulose, cationically modified cellulose, zwit- terionically modified cellulose, and mixtures thereof. Suitable cellulosic polymers include methyl cellulose, carboxy methyl cellulose, ethyl cellulose, hydroxyl ethyl cellulose, hydroxyl propyl methyl cellulose, ester carboxy methyl cellulose, and mixtures thereof.
Anti-redeposition agents: The detergent compositions of the present invention may also include one or more anti-redeposition agents such as carboxymethylcellulose (CMC), polyvinyl alcohol (PVA), polyvinylpyr- rolidone (PVP), polyoxyethylene and/or polyethyleneglycol (PEG), homopolymers of acrylic acid, copolymers of acrylic acid and maleic acid, and ethoxylated polyethyleneimines. The cellulose based polymers described under soil release polymers above may also function as anti-redeposition agents.
Other suitable adjunct materials include, but are not limited to, anti-shrink agents, anti-wrinkling agents, bactericides, binders, carriers, dyes, enzyme stabilizers, fabric softeners, fillers, foam regulators, hy- drotropes, perfumes, pigments, sod suppressors, solvents, and structurants for liquid detergents and/or structure elasticizing agents.
Formulation of detergent products
The detergent composition may be in any convenient form, e.g., a bar, a homogenous tablet, a tablet having two or more layers, a pouch having one or more compartments, a regular or compact powder, a granule, a paste, a gel, or a regular, compact or concentrated liquid. There are a number of detergent formulation forms such as layers (same or different phases), pouches, as well as forms for machine dosing unit.
Pouches can be configured as single or multi-compartments. It can be of any form, shape and material which is suitable for hold the composition, e.g. without allowing the release of the composition from the pouch prior to water contact. The pouch is made from water soluble film which encloses an inner volume. Said inner volume can be divided into compartments of the pouch. Preferred films are polymeric materials preferably polymers which are formed into a film or sheet. Preferred polymers, copolymers or derivatives thereof are selected polyacrylates, and water soluble acrylate copolymers, methyl cellulose, carboxy methyl cellulose, sodium dextrin, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, malto dex- trin, poly methacrylates, most preferably polyvinyl alcohol copolymers and, hydroxypropyl methyl cellulose (HPMC). Preferably the level of polymer in the film for example PVA is at least about 60%. Preferred average molecular weight will typically be about 20,000 to about 150,000. Films can also be of blend compositions comprising hydrolytically degradable and water soluble polymer blends such as polyactide and polyvinyl alcohol (known under the Trade reference M8630 as sold by Chris Craft In. Prod. Of Gary, Ind., US) plus plasticisers like glycerol, ethylene glycerol, Propylene glycol, sorbitol and mixtures thereof. The pouches can comprise a solid laundry cleaning composition or part components and/or a liquid cleaning composition or part components separated by the water soluble film. The compartment for liquid components can be different in composition than compartments containing solids. Ref: (US2009/0011970 A1 ).
Detergent ingredients can be separated physically from each other by compartments in water dissolv- able pouches or in different layers of tablets. Thereby negative storage interaction between components can be avoided. Different dissolution profiles of each of the compartments can also give rise to delayed dissolution of selected components in the wash solution.
A liquid or gel detergent, which is not unit dosed, may be aqueous, typically containing at least 20% by weight and up to 95% water, such as up to about 70% water, up to about 65% water, up to about 55% water, up to about 45% water, up to about 35% water. Other types of liquids, including without limitation, alkanols, amines, diols, ethers and polyols may be included in an aqueous liquid or gel. An aqueous liquid or gel detergent may contain from 0-30% organic solvent. A liquid or gel detergent may be non-aqueous.
Laundry Soap Bars
The enzymes of the invention may be added to laundry soap bars and used for hand washing laundry, fabrics and/or textiles. The term laundry soap bar includes laundry bars, soap bars, combo bars, syndet bars and detergent bars. The types of bar usually differ in the type of surfactant they contain, and the term laundry soap bar includes those containing soaps from fatty acids and/or synthetic soaps. The laundry soap bar has a physical form which is solid and not a liquid, gel or a powder at room temperature. The term solid is defined as a physical form which does not significantly change over time, i.e. if a solid object (e.g. laundry soap bar) is placed inside a container, the solid object does not change to fill the container it is placed in. The bar is a solid typically in bar form but can be in other solid shapes such as round or oval.
The laundry soap bar may contain one or more additional enzymes, protease inhibitors such as peptide aldehydes (or hydrosulfite adduct or hemiacetal adduct), boric acid, borate, borax and/or phenylboronic acid derivatives such as 4-formylphenylboronic acid, one or more soaps or synthetic surfactants, polyols such as glycerine, pH controlling compounds such as fatty acids, citric acid, acetic acid and/or formic acid, and/or a salt of a monovalent cation and an organic anion wherein the monovalent cation may be for example Na+, K+ or NH4+ and the organic anion may be for example formate, acetate, citrate or lactate such that the salt of a monovalent cation and an organic anion may be, for example, sodium formate.
The laundry soap bar may also contain complexing agents like EDTA and HEDP, perfumes and/or different type of fillers, surfactants e.g. anionic synthetic surfactants, builders, polymeric soil release agents, detergent chelators, stabilizing agents, fillers, dyes, colorants, dye transfer inhibitors, alkoxylated polycarbonates, suds suppressers, structurants, binders, leaching agents, bleaching activators, clay soil removal agents, anti-redeposition agents, polymeric dispersing agents, brighteners, fabric softeners, perfumes and/or other compounds known in the art.
The laundry soap bar may be processed in conventional laundry soap bar making equipment such as but not limited to: mixers, plodders, e.g. a two stage vacuum plodder, extruders, cutters, logo-stampers, cooling tunnels and wrappers. The invention is not limited to preparing the laundry soap bars by any single method. The premix of the invention may be added to the soap at different stages of the process. For example, the premix containing a soap, an enzyme, optionally one or more additional enzymes, a protease inhibitor, and a salt of a monovalent cation and an organic anion may be prepared and the mixture is then plodded. The enzyme and optional additional enzymes may be added at the same time as the protease inhibitor for example in liquid form. Besides the mixing step and the plodding step, the process may further comprise the steps of milling, extruding, cutting, stamping, cooling and/or wrapping. Method of producing the composition
The present invention also relates to methods of producing the composition. The method may be relevant for the (storage) stability of the detergent composition: e.g. Soap bar premix method WO 2009/155557. Uses
The present invention is also directed to methods for using the detergent compositions thereof. The present invention may be used for example in any detergent application which requires the degradation of xanthan gum. Use to degrade xanthan gum
Xanthan gum has been used as an ingredient in many consumer products including foods and cosmetics and has found use in the oil industry. Therefore, the degradation of xanthan gum can result in improved cleaning processes, such as the easier removal of stains containing gums, such as xanthan gum. Thus, the present invention is directed to the use of xanthan lyase variants as described herein or compo- sitions thereof to degrade xanthan gum. The present invention is also directed to the use of detergent compositions comprising xanthan lyases as described herein to degrade xanthan gum. Degradation of xanthan gum can preferably be measured using the viscosity reduction assay (e.g., ViPr assay) or alternatively as described in Example 3 herein.
GH9 endoglucanase activity may alternatively be measured by assessment of reducing ends on xan- than gum pre-treated with xanthan lyase using the colorimetric assay developed by Lever (1972), Anal. Biochem. 47: 273-279, 1972. A preferred embodiment is the use of 0.1 % xanthan gum pre-treated with xanthan lyase. Degradation of xanthan gum pre-treated with xanthan lyase may be determined by calculating difference between blank and sample wherein a difference of more than 0.5 mAU, preferably more than 0.6 mAU, more preferably more than 0.7 mAU or even more preferably more than 0.8 mAU shows degra- dation of xanthan gum pre-treated with xanthan lyase.
Xanthan lyase activity may alternatively be measured by assessment of reducing ends on xanthan gum using the colorimetric assay developed by Lever (1972), Anal. Biochem. 47: 273-279, 1972. A preferred embodiment is the use of 0.1 % xanthan gum. Degradation of xanthan gum may be determined by calculating difference between blank and sample, wherein a difference of more than 0.1 mAU, preferably more than 0.15 mAU, more preferably more than 0.2 mAU or even more preferably more than 0.25 mAU, shows degradation of xanthan gum.
Xanthan lyase (e.g. variants as described herein) and endoglucanase activity may alternatively be measured by assessment of reducing ends on xanthan gum using the colorimetric assay developed by Lever (1972), Anal. Biochem. 47: 273-279, 1972. A preferred embodiment is the use of 0.1 % xanthan gum. Degradation of xanthan gum may be determined by calculating difference between blank and sample wherein a difference of more than 0.4 mAU, preferably more than 0.5 mAU, more preferably more than 0.6 mAU or even more preferably more than 0.8 mAU shows degradation of xanthan gum.
The invention also relates to methods for degrading xanthan gum comprising applying a detergent composition comprising one or more xanthan lyase variants as described herein to xanthan gum. The invention further relates to methods for degrading xanthan gum comprising applying a detergent composition comprising one or more xanthan lyases to xanthan gum. An embodiment is a method for degrading xanthan gum comprising applying a detergent composition comprising one or more xanthan lyase variants as described herein together with one or more endoglucanases to xanthan gum.
Use in detergents
The present invention inter alia relates to the use of detergent compositions comprising xanthan lyase variants as described herein in cleaning processes such as the laundering of textiles and fabrics (e.g., household laundry washing and industrial laundry washing), as well as household and industrial hard surface cleaning, such as dish wash. For this, the xanthan lyase variants as described herein may be added to a detergent composition comprising of one or more detergent components.
In some aspects, xanthan lyase variants as described herein may be used together with an endoglu- canase(s) in detergent compositions for cleaning processes such as the laundering of textiles and fabrics (e.g. household laundry washing and industrial laundry washing), as well as household and industrial hard surface cleaning, such as dish wash. For this, the xanthan lyase variants as described herein together with an endoglucanase(s) may be added to a detergent composition comprising of one or more detergent com- ponents.
The polypeptides described herein may be added to and thus become a component of a detergent composition. The detergent composition may be formulated, for example, as a hand or machine laundry detergent composition for both household and industrial laundry cleaning, including a laundry additive composition suitable for pre-treatment of stained fabrics and a rinse added fabric softener composition, or be formulated as a detergent composition for use in general household or industrial hard surface cleaning operations, or be formulated for hand or machine (both household and industrial) dishwashing operations. In a specific aspect, the present invention relates to a detergent additive comprising a polypeptide as described herein.
The invention also relates to methods for degrading xanthan gum on the surface of a textile or hard surface, such as dish wash, comprising applying a detergent composition comprising one or more xanthan lyase variants as described herein to xanthan gum. In some aspects, the invention relates to a method for degrading xanthan gum on the surface of a textile or hard surface, such as dish wash, comprising applying a detergent composition comprising one or more xanthan lyase variants as described herein together with one or more endoglucanases to xanthan gum. In some aspects, the invention relates to a detergent com- position comprising one or more detergent components as described herein. It is contemplated that the use of a xanthan lyase variant as described herein alone may give an enzyme detergency benefit, preferably an enzyme detergency benefit on xanthan gum.
In some aspects, the invention relates to the use of a detergent composition comprising one or more detergent components and an isolated xanthan lyase variant as described herein together with a GH9 en- doglucanase. In some aspects, the invention relates to the use of a detergent composition comprising one or more detergent components and an isolated xanthan lyase variant as described herein together with a GH9 endoglucanase.
The invention is further defined in the following paragraphs:
1. A detergent composition comprising a xanthan lyase variant, comprising an alteration (e.g., a substitution, deletion or insertion) at one or more positions in a region selected from the group consisting of: i) region 7 corresponding to amino acids 1 to 153 of SEQ ID NO: 2,
ii) region 8 corresponding to amino acids 177 to 613 of SEQ ID NO: 2,
iii) region 9 corresponding to amino acids 659 to 730 of SEQ ID NO: 2,
iv) region 10 corresponding to amino acids 804 to 806 of SEQ ID NO: 2,
v) region 11 corresponding to amino acids 847 to 871 of SEQ ID NO: 2,
vi) region 12 corresponding to amino acids 886 to 902 of SEQ ID NO: 2,
vii) region 13 corresponding to amino acids 1005 to 1037 of SEQ ID NO: 2;
wherein said variant has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, and less than 100% sequence identity to SEQ ID NO: 2; preferably said xanthan lyase variant has activity on xanthan gum, further preferably said activity is a xanthan gum degrading activity.
2. The detergent composition comprising a xanthan lyase variant of paragraph 1 , which is a variant of a parent xanthan lyase selected from the group consisting of:
a) a polypeptide having at least 60% sequence identity to the mature polypeptide of SEQ ID NO: 2; b) a polypeptide encoded by a polynucleotide that hybridizes under low stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 1 , or (ii) the full-length complement of (i); c) a polypeptide encoded by a polynucleotide having at least 60% identity to the mature polypeptide coding sequence of SEQ ID NO: 1 ; and
d) a fragment of the mature polypeptide of SEQ ID NO: 2, which has xanthan lyase activity.
3. The detergent composition comprising a xanthan lyase variant of paragraph 2, wherein the parent xanthan lyase has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the mature polypeptide of SEQ ID NO: 2.
4. The detergent composition comprising a xanthan lyase variant of any of paragraphs 2-3, wherein the parent xanthan lyase is encoded by a polynucleotide that hybridizes under low stringency conditions, medium stringency conditions, medium-high stringency conditions, high stringency conditions, or very high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 1 or (ii) the full-length complement of (i).
5. The detergent composition comprising a xanthan lyase variant of any of paragraphs 2-4, wherein the parent xanthan lyase is encoded by a polynucleotide having at least 60%, e.g., at least 65%, at least
70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 1.
6. The detergent composition comprising a xanthan lyase variant of any of paragraphs 2-5, wherein the parent xanthan lyase comprises or consists of the mature polypeptide of SEQ ID NO: 2.
7. The detergent composition comprising a xanthan lyase variant of any of paragraphs 2-6, wherein the parent xanthan lyase is a fragment of the mature polypeptide of SEQ ID NO: 2, wherein the fragment has xanthan lyase activity.
8. The detergent composition comprising a xanthan lyase variant of any of paragraphs 2-7, which has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to the amino acid sequence of the parent xanthan lyase.
9. The detergent composition comprising a xanthan lyase variant of any of paragraphs 1 -8, wherein said variant has at least 61 %, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 2.
10. The detergent composition comprising a xanthan lyase variant of any of paragraphs 1 -9, wherein said alteration (e.g., a substitution, deletion or insertion) at one or more positions is selected from the group consisting of alterations in positions: 9, 15, 46, 58, 66, 89, 95, 100, 106, 109, 183, 188, 190, 203, 204, 221 , 229, 234, 238, 240, 242, 243, 257, 258, 291 , 293, 316, 320, 324, 329, 333, 339, 341 , 352, 354, 360, 377, 399, 400, 419, 440, 450, 451 , 454, 458, 481 , 492, 567, 568, 578, 579, 582, 664, 672, 703, 728, 843, 855, 887, 892, 1008 and 1016 of SEQ ID NO: 2, wherein numbering is according to SEQ ID
NO: 2.
11 . The detergent composition comprising a xanthan lyase variant of any of claims 1-10, further comprising one or more alterations at one or more positions selected from the group consisting of position 624, 631 , 635, 649, 656, 752, 752, 754, 757, 769, 775, 777, 800, 801 , 875, 91 1 , and 915 wherein numbering is according to SEQ ID NO: 2.
12. The detergent composition comprising a xanthan lyase variant of any of paragraphs 1 -1 1 , having one or more substitutions selected from the group consisting of: K9R, N15T, L46D, A58L, S66H, Q89Y, K95E, S100D, N106Y, Q109R, Q109D, Q109F, Q109K, Q109A, K183Q.K183R, V188I, A190Q, A203P, K204R, A221 P, E229N, E229S, I234V, I238W, I238L, I238M, I240W, N242S, G243V, Y257W, R258E, K291 R, A293G, A293P, K316R, K320R, L324Q, K329R, K333R, L339M, 1341 P, V352I, S354P, K360R, K360G, F377Y, N399K, K400R, F419Y, N440K, D450P, K451 E, K451 R, A454V, D458S,
K481 R, A492L, A492H, K567R, G568A, S578K, S578R, S579R, S579K, S582K, A624E, T631 N, S635E, T649K, I656V, T664K, N672D, I703L, M728V, G738L, P752K, P752R, G753E, S754E, S754R, S757D, A769D, L775A, D777R, V800P, D801 G, A843P, K855R, K875T, K887R, N892Y, N892W, N892F, A911V, T915A, N1008D and K1016T wherein numbering is according to SEQ ID NO: 2. 13. The detergent composition comprising a xanthan lyase variant of any of paragraphs 1 -12, comprising one of the following set of substitutions:
Figure imgf000076_0001
Figure imgf000077_0001
Figure imgf000078_0001
Figure imgf000079_0001
The detergent composition comprising a xanthan lyase variant of any of paragraphs 1 -13, comprising one of the following set of substitutions:
Figure imgf000079_0002
Figure imgf000080_0001
Figure imgf000081_0001
15. The detergent composition comprising a xanthan lyase variant of any of paragraphs 1 -14, wherein said variant does not comprise any amino acid alteration at a position outside of regions 7, 8, 9, 10, 11 , 12 and 13.
16. The detergent composition comprising a xanthan lyase variant of any of paragraphs 1 -15, wherein the total number of alterations compared to the parent xanthan lyase (e.g., SEQ ID NO: 2) is between 1 and 20, e.g. between 1 and 10 or between 1 and 5, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 alterations.
17. The detergent composition comprising a xanthan lyase variant of any of paragraphs 1-16, wherein said activity on xanthan gum is a xanthan gum degrading activity, preferably said xanthan lyase variant has EC 4.2.2.12 activity.
18. The detergent composition comprising a xanthan lyase variant of any of paragraphs 1-17, wherein said variant has an improved stability in a detergent composition compared to a parent xanthan lyase (e.g., with SEQ ID NO: 2).
19. The detergent composition comprising a xanthan lyase variant of any of paragraphs 1-18, wherein said variant has a half-life improvement factor (HIF) of >1.0; preferably >1 .0, more preferably at least 1.2, such as at least 1 .5, e.g. at least 2.0, relative to a parent xanthan lyase, e.g. a xanthan lyase with SEQ
ID NO: 2.
20. The detergent composition comprising a xanthan lyase variant of paragraph 19, wherein said half-life improvement factor (HIF) is determined after incubation of said xanthan lyase variant in a detergent composition at 25°C for a time period from about 30 min to about 20 hours.
21 . The detergent composition comprising a xanthan lyase variant of any of paragraphs 1 -20, wherein said variant is selected from the group consisting of i) the xanthan lyase variants set forth in Table 1 herein, ii) the xanthan lyase variants set forth in Table 2 herein, iii) the xanthan lyase variants set forth in Table 3 herein, and iv) the xanthan lyase variants set forth in Table 4 herein.
22. The detergent composition of any of paragraph 1-21 , wherein said composition comprises one or more detergent components.
23. The detergent composition of any of paragraph 1-22, further comprising at least one xanthan endoglu- canase.
24. The detergent composition of any of paragraphs 1 -23, further comprising one or more additional en- zymes selected from the group consisting of: endoglucanases, proteases, amylases, lichenases, lipases, cutinases, cellulases, xanthan lyases, xyloglucanases, pectinases, pectin lyases, xanthanases, peroxidases, haloperoxygenases, catalases and mannanases, or any mixture thereof.
25. The detergent composition of any of paragraphs 1 -24, wherein said composition is in form of a bar, a homogenous tablet, a tablet having two or more layers, a pouch having one or more compartments, a regular or compact powder, a granule, a paste, a gel, or a regular, compact or concentrated liquid.
26. Use of a detergent composition of any of paragraphs 1 -25, wherein said use is for degrading xanthan gum, e.g. in a cleaning process, such as laundry or hard surface cleaning such as dish wash.
27. The use of paragraph 26, wherein said xanthan lyase variant has an enzyme detergency benefit.
28. A method for degrading xanthan gum comprising: applying a detergent composition of any of para- graphs 1-25 to a xanthan gum.
29. The method of paragraph 28, wherein said xanthan gum is on a surface or hard surface.
The present invention is further described by the following examples that should not be construed as limiting the scope of the invention.
EXAMPLES
Example 1 : Construction and expression of xanthan lyase variants
Xanthan lyase parent gene (i.e., SEQ ID NO: 1 ) was PCR assembled into a linear cassette containing the promoter system on the upstream and cat selection maker on the downstream. To enable chromosomal integration of the cassette at the Pel locus of B. subtilis host by homologous recombination, >2 kb DNA sequence identical to the site of integration was included on both the sides of the cassette. Genomic DNA prepared from the strain containing xanthan lyase parent gene (SEQ ID NO: 1 ) was used as template for generating the site-directed mutants. Mutagenic forward and reverse primers were used to generate an approximately 6 kb PCR fragment. This fragment was used as a megaprimer along with another forward primer to amplify >8 kb DNA fragment. This fragment contained the complete cassette (promoter system, xanthan lyase and cat gene along with homologous DNA sequence required for recombination at Pel locus) was used for transformation.
The triple promoter system used in the cassette has been described in WO 99/43835 and it consists of promoters from Bacillus licheniformis alpha-amylase gene (amyL), Bacillus amyloliquefaciens alpha-am- ylase gene (amyQ), and the Bacillus thuringiensis crylllA promoter including the stabilizing sequence. Protease signal sequence from Bacillus clausii was included to export the protein out of the cells. Generated variants of the mature parent xanthan lyase of SEQ ID NO: 2 are shown in Table 1 below. The presence of the alteration was confirmed by sequencing.
Table 1 . Generated variants of the mature parent xanthan lyase of SEQ ID NO: 2
Figure imgf000083_0001
Figure imgf000084_0001
Bacillus organism containing a variant was inoculated in LB broth containing chloramphenicol (6 Mg/ml) and grown overnight at 37°C. For expression of xanthan lyase variants, 2% of overnight culture was added to 300 ml of 10-R medium in 1000 ml baffled flask and grown at 30°C for 96 hrs. at 180 rpm.
10-R medium contained 33 g/L Soluble starch, 6 g/L (NH )2HP0 , 5 g/L Potato peptone, 1.2 g/L (MgS0 x 7H20), 12 g/L KH2PO4, 5 g/L (Na2HP04 x 2H20), 18 mL/L of Trace metal solution, 1.8 g/L K2SO4 and 0.1 g/L (CaCl2 x 2H2O) and 0.5 mL/L SB2121 (anti-foam agent). Trace metal solution was made by mixing 0.49 g/L (MnS04 x H2O), 1.97 g/L (FeS04 x 7H20), 0.1 g/L (CuS04 x 5H20), 0.3 g/L ZnCI2 and 19.6 g/L citric acid. Example 2: Purification of xanthan lyase variants
Prior to purification, Bacillus subtilis broth was clarified by centrifuging at 8000 x g for 30 minutes at 10°C followed by vacuum filtration using a combination of Seitz filter (K250) and WHATMAN glass filter GF/F grade in a Buchner funnel. Finally, the supernatant was filtered through 0.22μ Tangential flow filtration unit.
Xanthan lyase variants were purified using three-step automated tandem column chromatography. Macro-Prep Methyl HIC column was pre-equilibrated with 50 mM Tris, pH 8.0 containing 1 M (NH4)2S04 and 1 mM CaCl2 buffer. During sample loading onto the column the clarified culture supernatant (250 ml_) was diluted 1 :1 in-line with 50 mM Tris, pH 8.0 containing 2 M (NH4)2SO4 andl mM CaCl2 buffer to make the final concentration to 1 M. The unbound or weakly bound protein was washed with the equilibration buffer until the Absorbance at 280 nm comes below 0.1 AU. Elution was carried out using 50 mM Tris pH 8 containing 0.5 M (NH4)2SO4 and 1 mM CaCl2. Eluted protein peak was automatically loaded on MEP-Hy- percel column pre-equilibrated with 50 mM Tris, pH 8 containing 0.5 M (NH4)2SO4 and 1 mM CaCl2. The unbound or weakly bound protein was washed with the equilibration buffer until the Absorbance at 280 nm comes below 0.1 AU. The column was washed again with 50 mM Tris, pH 8 containing 1 mM CaCl2 to remove impurities. The Purified protein was eluted with 50 mM Na-acetate, pH 5 containing 1 mM CaCl2. The eluted purified protein was automatically transferred to Sephadex G-25 column pre-equilibrated with 50 mM MOPS, pH 8 containingl mM CaCl2 for desalting. Example 3: Detergent stability assay
Reagents for the detergent stability assay were prepared as follows:
A stock of 1.0 M MOPS buffer was prepared by dissolving 209.26 g of 3-Morpholinopropanesulfonic acid in Milli Q water. pH was adjusted to 7.5 using NaOH and the final volume of buffer was made up to 1000 ml. This buffer stock was stored at 4°C until use. A 50 mM working solution of MOPS buffer was prepared by adding 50 ml of 1.0 M stock to 950 ml of Milli Q water.
A substrate solution of 0.4% w/v xanthan gum was freshly prepared by dissolving 400 mg of xanthan gum in 100 ml of Milli Q water.
A stock solution mix containing 1.0 M Na2C03, 0.17 M potassium sodium tartrate and 5 mM (Bi(N03)3 x 5H2O) was prepared by dissolving 106.99 g of Na2C03, 47.98 g of potassium sodium tartrate and 2.42 mg of (Bi(N03)3 x 5H2O) in Milli Q water for a final volume of 1000 ml. This stock solution mix was filtered and stored at room temperature.
A PAHBAH reagent (1.5% PAHBAH) was freshly prepared by dissolving 1.5 g of p-hydroxybenzoic acid hydrazide (PAHBAH) in the stock solution mix. Detergent stability Assay:
A. Screening of culture supernatants of variants
The in-detergent stability was determined by measuring the enzymatic activity present in culture supernatants of variants or wild-type controls after incubation with detergent (70% Persil Universal Gel deter- gent (PUG), final concentration) at 30°C for one hour.
Detergent stress was carried out by addition of 30μΙ of culture supernatant and 70 μΙ of a Persil Universal Gel detergent (100%) into wells of 96-well microtitre plates which were shaken for 15 min at 1000 rpm. Two identical plates were produced whereof one plate was incubated at 4°C (unstressed plate) and the other plate was incubated at 30°C (stressed plate) for 1 hour. After incubation, samples from unstressed and stressed plates were diluted 50X with dilution buffer (50mM MOPS, 5 mM CaCh, pH 7.5).
To measure the enzyme activity of diluted enzyme-detergent samples, reaction mixtures were prepared in 96-well PCR plates. 50 μΙ of diluted samples were mixed with 50μΙ of freshly prepared substrate solution and incubated at 40°C for 1 hour.
After incubation, 75 μΙ of PAHBAH reagent was added to reaction mixture in the same PCR plate and incubated in a programmable thermal cycler (T-ROBOT) for 10 min at 90°C followed by subsequent cooling at 10°C. Samples (25 μΙ) were transferred to a 384 well microtitre plate and the absorbance was measured at 405 nm using an Infinite M1000 reader (TECAN, Switzerland).
The residual activity (RA) for variants and wild-type controls were calculated as the percentage of enzymatic activity remaining after incubation at 30°C relative to the enzymatic activity remaining after incuba- tion at 4°C, i.e., according to the following formula:
Residual activity (RA) = 100% * Abs405 (sample incubated at 30°C)/Abs405 (sample incubated at 4°C).
The variants with higher detergent stability were picked with respect to the wild-types grown in the plates.
B. Screening of purified variants
The detergent stability of purified variants was determined by measuring the enzyme activity of the purified protein after incubation with detergent (70% Persil Universal Gel detergent (PUG), final concentration for the variants of Table 3, and 90% for Table 4) at 30°C unless otherwise indicated (see Table 4) for the time indicated in Tables 3 and 4 below.
Purified variants were diluted to a concentration of 200 ppm using 50 mM MOPS buffer. For detergent treatment, 10 μΙ of diluted purified samples were mixed with 90 μΙ of Persil Universal Gel detergent (100%) into wells of 96-well microtitre plates which were shaken for 20 min at 1000 rpm. Two identical plates were produced whereof one plate was incubated at 4°C (unstressed plate) and the other plate was incubated at 30°C (stressed plate) for 1 hour. After incubation, samples from unstressed and stressed plates were diluted 50X with dilution buffer (50 mM MOPS, 1 mM CaCI2, pH 7.5). Enzymatic activity analysis of unstressed and stressed samples was done as described in section A. C. Calculating half-lives and half-life improvement factors (HIF)
Half-life (T½ (in hours)) was calculated at a given detergent concentration and storage temperature for the wild-type controls and/or variants, as the degradation follows an exponential decay and the incubation time (hours) is known, i.e., according to the following formulas:
T1/2 (variants) = (Ln (0.5)/Ln (RA-variants/100))*Time
T1/2 (Wild-type) = (Ln (0.5)/Ln (RA-wild-type/100))*Time
Where 'RA' is the residual activity in percent as calculated above and "Time" is the incubation time in hours.
A half-life improvement factor (HIF) under a given set of storage conditions (detergent concentration and temperature) is calculated as HIF = T½ (Variant)/T½ (wild-type), where the wild-type is incubated under the same storage conditions as the variant.
In cases where the difference in stability between wild-type and variants is too large to accurately assess half-life for both wild-type and variant using the same incubation time (see Table 4), the incubation time for wild-type and variant is different, e.g. 1 h for wild-type and up to 168 h for the most stable variants. Further, in order to determine the stability (half-life) within a shorter duration of incubation time for the more stable variants, e.g. <168 h, the incubation temperature for some variants in Table 4 was increased by 2-5 degrees Celsius.
The half-lives and calculated half-life improvement factor (HIF) values for culture supernatants of single mutation variants are provided in Table 2 below. Tables 3 and 4 show the half-life for purified variants having single, double or multiple mutations, as well as half-life improvement factor (HIF) values for the variants of Table 3. HIFs of all variants were calculated based on the wild-type xanthan lyase (SEQ ID NO: 2) incubated at the same detergent concentration and temperature (HIF of wild-type = 1.0 in all tables, per definition).
Figure imgf000087_0001
Figure imgf000088_0001
The obtained half-life and HIF values for purified variants tested at a 70% detergent concentration (30°C, incubation time one hour) are shown in Table 3 below.
Figure imgf000088_0002
Figure imgf000089_0001
Figure imgf000090_0001
The obtained half-life values for purified variants tested at a 90% detergent concentration (temperature and incubation time as indicated) are shown in Table 4 below.
Figure imgf000090_0002
Figure imgf000091_0001
Figure imgf000092_0001
Figure imgf000093_0001
Figure imgf000094_0001
Figure imgf000095_0001
Figure imgf000096_0001
Example 4
Calculating Half-lives and Half-life improvement factors (HIF) for Xanthan lyase variants
Half-life (T½ (in hours)) was calculated at a given detergent concentration and storage temperature (Persil Universal Gel (PUG) 95%, 30°C, 4 wk or more) for the wild-type controls and/or variants, as the residual activity follows an exponential decay and the incubation time (hours) is known, i.e., according to the following formulas:
T1/2 (variants) = (Ln (0.5)/Ln (RA-variants/100))*Time
T1/2 (Wild-type) = (Ln (0.5)/Ln (RA-wild-type/100))*Time
A half-life improvement factor (HIF) is calculated as HIF = T½ (Variant)/T½ (Wild-type), where the wild- type is incubated under the same storage condition as the variant. In the cases where the difference in stability between Wild-type and variants is too big to accurately assess half-life for both wild-type and variant using the same incubation time, the incubation time for wild-type and variant is different e.g. 1 h for wild- type and 840 h for the most stable variants, HIF values could not be calculated as the half-life of the wild- type could not be determined accurately. Stability of these variants are reported in terms of half-life (in hours).
Figure imgf000096_0002
Figure imgf000097_0001
Figure imgf000098_0001

Claims

A detergent composition comprising a xanthan lyase variant, comprising an alteration at one or more 1 .
positions selected from the group consisting of:
i) region 7 corresponding to amino acids 1 to 153 of SEQ ID NO: 2,
ii) region 8 corresponding to amino acids 177 to 613 of SEQ ID NO: 2,
iii) region 9 corresponding to amino acids 659 to 730 of SEQ ID NO: 2,
iv) region 10 corresponding to amino acids 804 to 806 of SEQ ID NO: 2,
v) region 11 corresponding to amino acids 847 to 871 of SEQ ID NO: 2,
vi) region 12 corresponding to amino acids 886 to 902 of SEQ ID NO: 2, and
vii) region 13 corresponding to amino acids 1005 to 1037 of SEQ ID NO: 2,
wherein said variant has at least 60% and less than 100% sequence identity to SEQ ID NO: 2.
2. The detergent composition comprising a xanthan lyase variant of claim 1 , wherein said variant has at least 61 %, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 2.
3. The detergent composition comprising a xanthan lyase variant of claim 1 or 2, wherein said alteration at one or more positions is selected from the group consisting of alterations in positions: 9, 15, 46, 58, 66, 89, 95, 100, 106, 109, 183, 188, 190, 203, 204, 221 , 229, 234, 238, 240, 242, 243, 257, 258, 291 , 293, 316, 320, 324, 329, 333, 339, 341 , 352, 354, 360, 377, 399, 400, 419, 440, 450, 451 , 454, 458, 481 , 492, 567, 568, 578, 579, 582, 664, 672, 703, 728, 843, 855, 887, 892, 1008 and 1016 of SEQ ID NO: 2, wherein numbering is according to SEQ ID NO: 2.
4. The detergent composition comprising a xanthan lyase variant of any of claims 1-3, further comprising one or more alterations at one or more positions selected from the group consisting of positions 624, 631 , 635, 649, 656, 752, 752, 754, 757, 769, 775, 777, 800, 801 , 875, 91 1 , and 915 wherein numbering is according to SEQ ID NO: 2.
5. The detergent composition comprising a xanthan lyase variant of any of claims 1 -3, having one or more substitutions selected from the group consisting of: K9R, N15T, L46D, A58L, S66H, Q89Y, K95E, S100D, N106Y, Q109R, Q109D, Q109F, Q109K, Q109A, K183Q, K183R, V188I, A190Q, A203P, K204R, A221 P, E229N, E229S, I234V, I238W, I238L, I238M, I240W, N242S, G243V, Y257W, R258E, K291 R, A293G, A293P, K316R, K320R, L324Q, K329R, K333R, L339M, 1341 P, V352I, S354P, K360G, K360R, F377Y, N399K, K400R, F419Y, N440K, D450P, K451 E, K451 R, A454V, D458S, K481 R, A492L, A492H, K567R, G568A, S578K, S578R, S579R, S579K, S582K, A624E, T631 N, S635E, T649K, I656V, T664K, N672D, I703L, M728V, G738L, P752K, P752R, G753E, S754E, S754R, S757D, A769D, L775A, D777R, V800P, D801 G, A843P, K855R, K875T, K887R, N892Y, N892W, N892F, A911V, T915A, N1008D and K1016T wherein numbering is according to SEQ ID NO: 2.
6. The detergent composition comprising a xanthan lyase variant of any of claims 1-5, wherein the variant has one of the following set of substitutions:
Figure imgf000100_0001
Figure imgf000101_0001
7. The detergent composition comprising a xanthan lyase variant of any of claims 1 -6, wherein said variant has an improved stability in a detergent composition compared to a parent xanthan lyase, e.g. a xanthan lyase with SEQ ID NO: 2.
8. The detergent composition comprising a xanthan lyase variant of any of claims 1 -7, wherein said variant has a half-life improvement factor (HIF) of >1.0, preferably at least 1.2, such as at least 1.5, e.g. at least 2.0, relative to a parent xanthan lyase, e.g. a xanthan lyase with SEQ ID NO: 2.
9. The detergent composition comprising a xanthan lyase variant of any of claims 1 -8, wherein said variant is selected from the group consisting of i) the xanthan lyase variants set forth in Table 3 herein, and ii) the xanthan lyase variants set forth in Table 4 herein.
10. The detergent composition comprising a xanthan lyase variant of any of claims 1 -9, wherein said variant does not comprise any alteration (substitution, deletion or insertion) at a position in a region selected from the group consisting of: region 1 corresponding to amino acids 154 to 176 of SEQ ID NO: 2, region 2 corresponding to amino acids 614 to 658 of SEQ ID NO: 2, region 3 corresponding to amino acids 731 to 803 of SEQ ID NO: 2, region 4 corresponding to amino acids 807 to 846 of SEQ ID NO: 2, region 5 corresponding to amino acids 872 to 885 of SEQ ID NO: 2, and region 6 corresponding to amino acids 903 to 1004 of SEQ ID NO: 2.
11 . The detergent composition of any of claims 1 -10, wherein said composition comprises one or more detergent components.
12. Use of a detergent composition of any of claims 1-11 , wherein said use is for degrading xanthan gum, e.g. in a cleaning process, such as laundry or hard surface cleaning such as dish wash.
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