EP3385362A1 - Detergent compositions comprising fungal mannanases - Google Patents

Detergent compositions comprising fungal mannanases Download PDF

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Publication number
EP3385362A1
EP3385362A1 EP17164904.9A EP17164904A EP3385362A1 EP 3385362 A1 EP3385362 A1 EP 3385362A1 EP 17164904 A EP17164904 A EP 17164904A EP 3385362 A1 EP3385362 A1 EP 3385362A1
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EP
European Patent Office
Prior art keywords
seq id
amino acid
detergent composition
detergent
enzyme
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Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP17164904.9A
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German (de)
French (fr)
Inventor
Daniela HERBST
Susanne Wieland
Nina Mussmann
Taija LEINONEN
Leena Valtakari
Michael RACHINGER
Kari Juntunen
Jari VEHMAANPERÄ
Pentti Ojapalo
Terhi Puranen
Kristiina JÄRVINEN
Susanna MÄRKINEN
Marja Paloheimo
Jarno Kallio
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Henkel AG and Co KGaA
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Henkel AG and Co KGaA
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Priority to EP17164904.9A priority Critical patent/EP3385362A1/en
Publication of EP3385362A1 publication Critical patent/EP3385362A1/en
Application status is Withdrawn legal-status Critical

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    • CCHEMISTRY; METALLURGY
    • C11ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11DDETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16Organic compounds
    • C11D3/38Products with no well-defined composition, e.g. natural products
    • C11D3/386Preparations containing enzymes, e.g. protease, amylase
    • C11D3/38636Preparations containing enzymes, e.g. protease, amylase containing enzymes other than protease, amylase, lipase, cellulase, oxidase, reductase

Abstract

This invention relates to novel detergent compositions comprising fungal mannanase enzymes. The detergent compositions comprising fungal mannanases are useful in laundry and cleaning applications wherein degradation or modification of mannan is desired. The invention also relates to the use of said detergent compositions in laundry and cleaning applications as well as a method for degrading mannan.

Description

    FIELD OF THE INVENTION
  • This invention relates to novel detergent compositions comprising fungal mannanase enzymes. The detergent compositions comprising fungal mannanases are useful in laundry and cleaning applications wherein degradation or modification of mannan is desired. The invention also relates to the use of said detergent compositions in laundry and cleaning applications as well as a method for degrading mannan.
  • BACKGROUND
  • Mannans are mannose containing polysaccharides found in various plants. Mannans are poorly soluble in an aqueous environment and their physicochemical properties give rise to viscous dispersions. Additionally, mannans have high water- binding capacity. All of these characteristics cause problems in several industries including brewing, baking, animal nutrition, and laundry and cleaning applications.
  • In plant based diets different ß-mannans are present and depending on their amounts and properties they can compromise nutrient digestion, microbial colonisation and growth performance. Enzymatic degradation of mannans reduces digesta viscosity of high water soluble mannans and leads to production of manno-oligosaccharides that may form water-insoluble linear mannans present in leguminoseae. Mannanase increases average daily gain, feed efficiency, weight uniformity and livability in all monogastric animals.
  • For animal feed applications, such as feed for monogastric animals with cereal diets, mannan is a contributing factor to viscosity of gut contents and it thereby adversely affects the feed digestibility and animal growth rate. For ruminants, mannan represents a substantial component of fiber intake and a more complete digestion of mannan would facilitate higher feed conversion efficiencies.
  • For laundry and cleaning applications detergent compositions comprising mannanase can be used to degrade mannan. However, providing mannanases that are stable in varying storage and use conditions while still showing good mannan degrading activity is difficult.
  • It is an object of the present invention to provide detergent compositions comprising novel enzymes exhibiting mannanase activity when applied in these detergent compositions. It is a further object of the present invention to provide detergent compositions having increased stain removal performance on mannan containing stains.
  • SUMMARY
  • According to the first aspect of the invention there is provided a detergent composition comprising at least one enzyme having an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 3 (Man1), and/or at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 9 (Man3), and/or at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 12 (Man 5).
  • In one embodiment of the invention the at least one enzyme comprises an amino acid sequence having 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 the amino acid sequence of SEQ ID NO: 3 (Man1).
  • In one embodiment of the invention the at least one enzyme comprises an amino acid sequence having 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, the amino acid sequence of SEQ ID NO: 9 (Man3).
  • In one embodiment of the invention the at least one enzyme comprises an amino acid sequence having 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 the amino acid sequence of SEQ ID NO: 12 (Man5).
  • In a further embodiment of the invention the at least one enzyme has mannan degrading activity. The mannanases comprised in the detergent composition of the invention are suitable for degrading and modifying mannan containing material in various chemical environments, preferably in detergent compositions.
  • In one embodiment of the present invention the detergent composition further comprises one or more additional enzymes selected from the group consisting of protease, lipase, cutinase, amylase, carbohydrase, cellulase, pectinase, pectatlyase, mannanase, arabinase, galactanase, xylanase, oxidase, xanthanase, laccase, and/or peroxidase, preferably selected from the group consisting of proteases, amylases, cellulases and lipases.
  • In a further embodiment of the present invention the 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 detergent composition can be a laundry detergent composition, preferably a liquid or solid laundry detergent composition.
  • The present invention furthermore relates to the use of the detergent composition as herein disclosed for degrading mannan.
  • In a further embodiment the present invention relates to the use of the detergent composition as herein disclosed in a laundry process.
  • The present invention furthermore relates to a method for removing a stain from a surface, comprising contacting the surface with a detergent composition as herein disclosed.
  • The present invention also relates to a method for degrading mannan comprising applying a detergent composition as herein disclosed to a stain comprising mannan, preferably wherein the stain is on the surface of a textile.
  • BRIEF DESCRIPTION OF THE FIGURES
    • Fig 1 schematically shows the expression cassettes used in the transformation of Trichoderma reesei protoplasts for overproducing the recombinant mannanase proteins. The mannanase genes were under the control of T. reesei cel7A/cbh1 promoter (pcbh1) and the termination of the transcription was ensured by using T. reesei cel7A/cbh1 terminator sequence (tcbh1). The amdS gene was included as a transformation marker.
    • Figure 2 describes the temperature profile of recombinant Man1, Man3, Man5 and Man8 mannanase assayed in 40 mM Britton-Robinson buffer pH 7 using 10 min reaction time, Azurine-crosslinked carob galactomannan was used as a substrate. All measurements were made at least duplicates. The data points are averages of separate measurements.
    • Fig 3 describes the temperature profile of recombinant Man9, Man10, Man11 and Man13 mannanase assayed in 40 mM Britton-Robinson buffer pH 7 using 10 min reaction time, Azurine-crosslinked carob galactomannan was used as a substrate. All measurements were made at least duplicates. The data points are averages of separate measurements.
    • Figure 4a and 4b describes the effect of pH on the activity of recombinant Man1, Man3, Man5 and Man8 mannanase and Man9, Man10, Man11 and Man12 mannanase, respectively, in 40 mM Britton-Robinson buffer at pH 4 to pH 11. Reaction temperature was 50°C and the reaction time was 10 min. Azurine-crosslinked carob galactomannan was used as a substrate. All measurements were made at least duplicates. The data points are averages of separate measurements.
    • Fig 5 shows SDS PAGE analysis of fungal mannanases.
    • Fig 6 describes the stain removal performance of Man1, Man3 and Man5 as an increase of lightness (sum of ΔL*of 3 stains) in the presence of 4.4 g/l of Commercial heavy duty liquid detergent A at 40°C, 16 °dH, 60 min, pH approx. 8.2 and enzymes dosed as activity units
    • Fig 7 describes the stain removal performance of Man8, Man9 and Man11 as an increase of lightness (sum of ΔL*of 3 stains) in the presence of 4.4 g/l of Commercial heavy duty liquid detergent A at 40°C, 16 °dH, 60 min, pH approx. 8.2 and enzymes dosed as activity units
    • Fig 8 describes the stain removal performance of Man10, Man12 and Man13 as an increase of lightness (sum of ΔL*of 3 stains) in the presence of 4.4 g/l of Commercial heavy duty liquid detergent A at 40°C, 16 °dH, 60 min, pH approx. 8.2 and enzymes dosed as enzymes dosed as activity units.
    • Fig 9 describes the stain removal performance of Man29 and Man30 as an increase of lightness (sum of ΔL*of 2 stains) in the presence of 5 g/l of Commercial heavy duty liquid detergent at 40°C, 16 °dH, 60 min, pH approx. 8.2 -8.3 and enzymes dosed as activity units.
    • Fig 10 describes the stain removal performance of Man1, Man3 and Man5 as an increase of lightness (sum of ΔL*of 3 stains) in the presence of 3.8 g/l of Commercial color detergent powder at 40°C, 16 °dH, 60 min, pH approx. 10 and enzymes dosed as activity units.
    • Fig 11 describes the stain removal performance of Man8, Man9, Man11 and Man12 as an increase of lightness (sum of ΔL*of 3 stains) in the presence of 3.8 g/l of Commercial color detergent powder at 40°C, 16 °dH, 60 min, pH approx. 10 and enzymes dosed as activity units.
    • Fig 12 describes the stain removal performance of Man1, Man3 and Man5 as an increase of lightness (sum of ΔL* of 3 stains) in the presence of 4,2 g/l of Commercial bleach detergent powder at 40°C, 16 °dH, 60 min, pH approx. 9.5 and enzymes dosed as activity units.
    • Fig 13 describes the stability of Man1 in liquid detergent (OMO Color) at 37°C. Commercial preparation Mannaway® 4,0 L was used for comparison.
    SEQUENCE LISTINGS
    • SEQ ID NO: 1 nucleotide sequence of man1 gene
    • SEQ ID NO: 2 cDNA sequence of man1
    • SEQ ID NO: 3 deduced amino acid sequence of Man1
    • SEQ ID NO: 4 nucleotide sequence of man2 gene
    • SEQ ID NO: 5 cDNA sequence of man2man2
    • SEQ ID NO: 6 deduced amino acid sequence of Man2
    • SEQ ID NO: 7 nucleotide sequence of man3 gene
    • SEQ ID NO: 8 cDNA sequence of man3
    • SEQ ID NO: 9 deduced amino acid sequence of Man3
    • SEQ ID NO: 10 nucleotide sequence of man5 gene
    • SEQ ID NO: 11 cDNA sequence of man5
    • SEQ ID NO: 12 deduced amino acid sequence of Man5
    • SEQ ID NO: 13 nucleotide sequence of man8 gene
    • SEQ ID NO: 14 codon optimized cDNA sequence of man8
    • SEQ ID NO: 15 deduced amino acid sequence of Man8
    • SEQ ID NO: 16 nucleotide sequence of man9 gene
    • SEQ ID NO: 17 codon optimized cDNA sequence of man9
    • SEQ ID NO: 18 deduced amino acid sequence of Man9
    • SEQ ID NO: 19 nucleotide sequence of man10 gene
    • SEQ ID NO: 20 codon optimized cDNA sequence of man10
    • SEQ ID NO: 21 deduced amino acid sequence of Man10
    • SEQ ID NO: 22 nucleotide sequence of man11 gene
    • SEQ ID NO: 23 codon optimized cDNA sequence of man11
    • SEQ ID NO: 24 deduced amino acid sequence of Man11
    • SEQ ID NO: 25 nucleotide sequence of man12 gene
    • SEQ ID NO: 26 cDNA sequence of man12
    • SEQ ID NO: 27 deduced amino acid sequence of Man12
    • SEQ ID NO: 28 nucleotide sequence of man13 gene
    • SEQ ID NO: 29 codon optimized cDNA sequence of man13
    • SEQ ID NO: 30 deduced amino acid sequence of Man13
    • SEQ ID NO: 31 nucleotide sequence of man17 gene
    • SEQ ID NO: 32 cDNA sequence of man17
    • SEQ ID NO: 33 deduced amino acid sequence of Man17
    • SEQ ID NO: 34 nucleotide sequence of man27 gene
    • SEQ ID NO: 35 cDNA sequence of man27
    • SEQ ID NO: 36 deduced amino acid sequence of Man27
    • SEQ ID NO: 37 nucleotide sequence of man29 gene
    • SEQ ID NO: 38 codon optimized cDNA sequence of man29
    • SEQ ID NO: 39 deduced amino acid sequence of Man29
    • SEQ ID NO: 40 nucleotide sequence of man30 gene
    • SEQ ID NO: 41 codon optimized cDNA sequence of man30
    • SEQ ID NO: 42 deduced amino acid sequence of Man30
    • SEQ ID NO: 43 oligonucleotide Mc_Man for
    • SEQ ID NO: 44 oligonucleotide Mc_Man rev
    • SEQ ID NO: 45 oligonucleotide MANP5
    • SEQ ID NO: 46 oligonucleotide MANP6
    • SEQ ID NO: 47 oligonucleotide Vd_Man3_for
    • SEQ ID NO: 48 oligonucleotide Vd_Man3_rev
    • SEQ ID NO: 49 oligonucleotide Vd_Man2_for
    • SEQ ID NO: 50 oligonucleotide Vd_Man2_rev
    • SEQ ID NO: 51 oligonucleotide Ma_man2_for
    • SEQ ID NO: 52 oligonucleotide Ma_Man2_rev
    • SEQ ID NO: 53 oligonucleotide Ma_Man1for2
    • SEQ ID NO: 54 oligonucleotide Ma_Man1rev
    • SEQ ID NO: 55 oligonucleotide MANP3
    • SEQ ID NO: 56 oligonucleotide MANP4
    DETAILED DESCRIPTION
  • As used herein, "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 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 or decreasing the amount of the substance relative to other components with which it is naturally associated (e.g., recombinant production in a host cell; one or multiple copies of a gene encoding the substance; and use of an alternative promoter to the promoter naturally associated with the gene encoding the substance).In an embodiment a polypeptide, enzyme, polynucleotide, host cell or composition of the invention is isolated.
  • As used herein, the term "comprising" includes the broader meanings of "including", "containing", and "comprehending", as well as the narrower expressions "consisting of" and "consisting only of".
  • As used herein, "fragment" means a protein or a polynucleotide having one or more amino acids or nucleotides deleted. In the context of DNA, a fragment includes both single stranded and double stranded DNA of any length. A fragment may be an active fragment which has the biological function, such as enzyme activity or regulatory activity, of the protein or the polynucleotide. A fragment may also be an inactive fragment, i.e. it does not have one or more biological effects of the native protein or polynucleotide.
  • As used herein, "variant" means a fragment of sequence (nucleotide or amino acid) inserted or deleted by one or more nucleotides/amino acids or which is chemically modified.
  • As used herein, a "peptide" and a "polypeptide" are amino acid sequences including a plurality of consecutive polymerized amino acid residues. For purpose of this invention, peptides are molecules including up to 20 amino acid residues, and polypeptides include more than 20 amino acid residues. The peptide or polypeptide may include modified amino acid residues, naturally occurring amino acid residues not encoded by a codon, and non-naturally occurring amino acid residues. As used herein, a "protein" may refer to a peptide or a polypeptide of any size. A protein may be an enzyme, a protein, an antibody, a membrane protein, a peptide hormone, regulator, or any other protein.
  • The term "polynucleotide" denotes a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end. Polynucleotides include RNA and DNA, and may be isolated from natural sources, synthesized in vitro, or prepared from a combination of natural and synthetic molecules.
  • As used herein, "modification", "modified", and similar terms in the context of polynucleotides refer to modification in a coding or a non-coding region of the polynucleotide, such as a regulatory sequence, 5' untranslated region, 3' untranslated region, up-regulating genetic element, down-regulating genetic element, enhancer, suppressor, promoter, exon, or intron region. The modification may in some embodiments be only structural, having no effect on the biological effect, action or function of the polynucleotide. In other embodiments the modification is a structural modification which provides a change in the biological effect, action or function of the polynucleotide. Such a modification may enhance, suppress or change the biological function of the polynucleotide.
  • As used herein, "identity" means the percentage of exact matches of amino acid residues between two aligned sequences over the number of positions where there are residues present in both sequences. When one sequence has a residue with no corresponding residue in the other sequence, the alignment program allows a gap in the alignment, and that position is not counted in the denominator of the identity calculation. Identity is a value determined with the Pairwise Sequence Alignment tool EMBOSS Needle at the EMBL-EBI website (www.ebi.ac.uk/Tools/psa/emboss_needle/).
  • As used herein, "host cell" means any cell type that is susceptible to transformation, transfection, transduction, mating, crossing or the like with a nucleic acid construct or expression vector comprising a polynucleotide. The term "host cell" encompasses any progeny that is not identical due to mutations that occur during replication. Non-limiting examples of a host cell are fungal cells, filamentous fungal cells from Division Ascomycota, Subdivision Pezizomycotina; preferably from the group consisting of members of the Class Sordariomycetes, Subclass Hypocreomycetidae, Orders Hypocreales and Microascales and Aspergillus, Chrysosporium, Myceliophthora and Humicola; more preferably from the group consisting of Families Hypocreacea, Nectriaceae, Clavicipitaceae, Microascaceae, and Genera Trichoderma (anamorph of Hypocrea), Fusarium, Gibberella, Nectria, Stachybotrys, Claviceps, Metarhizium, Villosiclava, Ophiocordyceps, Cephalosporium, and Scedosporium; more preferably from the group consisting of Trichoderma reesei (Hypocrea jecorina), T. citrinoviridae, T. longibrachiatum, T. virens, T. harzianum, T. asperellum, T. atroviridae, T. parareesei,, Fusarium oxysporum, F. gramineanum, F. pseudograminearum, F. venenatum, Gibberella fujikuroi, G. moniliformis, G. zeaea, Nectria (Haematonectria) haematococca, Stachybotrys chartarum, S. chlorohalonata, Claviceps purpurea, Metarhizium acridum, M. anisopliae, Villosiclava virens, Ophiocordyceps sinensis, Acremonium (Cephalosporium) chrysogenum, and Scedosporium apiospermum, and Aspergillus niger, Aspergillus awamori, Aspergillus oryzae, Chrysosporium lucknowense, Myceliophthora thermophila, Humicola insolens, and Humicola grisea, most preferably Trichoderma reesei. Non-limiting examples of a host cell are bacterial cells, preferably gram positive Bacilli (e.g. Bacillus subtilis, B. licheniformis, B. megaterium, B. amyloliquefaciens, B. pumilus), gram negative bacteria (e.g. Escherichia coli), actinomycetales (e.g. Streptomyces sp.) and yeasts (e.g. Saccharomyces cerevisiae, Pichia pastoris, Yarrowia lipolytica).In an example embodiment of the host cell is a fungal cell, preferably a filamentous fungal cell, such as Trichoderma or Trichoderma reesei. In an example embodiment of the host cell is a fungal cell, preferably a gram positive Bacillus cell, such as B. subtilis, B. licheniformis, B. megaterium, B. amyloliquefaciens, B. pumilus.
  • A "recombinant cell" or "recombinant host cell" refers to a cell or host cell that has been genetically modified or altered to comprise a nucleic acid sequence which is not native to said cell or host cell. In an embodiment the genetical modification comprises integrating the polynucleotide in the genome of the host cell. In another embodiment the polynucleotide is exogenous in the host cell.
  • As used herein, "expression" includes any step involved in the production of a polypeptide in a host cell including, but not limited to, transcription, translation, post-translational modification, and secretion. Expression may be followed by the harvesting, i.e. recovering, the host cells or the expressed product.
  • The term "expression vector" denotes a DNA molecule, linear or circular, that comprises a segment encoding a polypeptide of interest operably linked to additional segments that provide for its transcription. Such additional segments may include promoter and terminator sequences, and may optionally include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, carrier and the like. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both. The expression vector may be any expression vector that is conveniently subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which the vector is to be introduced. Thus, the vector may be an autonomously replicating vector, i.e. a vector, which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.
  • The term "recombinant produced" or "recombinantly produced" used herein in connection with production of a polypeptide or protein is defined according to the standard definition in the art.
  • The term "obtained from" and "obtainable" as used herein in connection with a specific microbial source, means that the polynucleotide is expressed by the specific source (homologous expression), or by a cell in which a gene from the source has been inserted (heterologous expression).
  • The term "enzyme composition" means either a conventional enzymatic fermentation product, possibly isolated and purified, from a single species of a microorganism, such preparation usually comprising a number of different enzymatic activities; or a mixture of monocomponent enzymes, preferably enzymes derived from fungal or fungal species by using conventional recombinant techniques, which enzymes have been fermented and possibly isolated and purified separately and which may originate from different species, preferably fungal or fungal species or the fermentation product of a microorganism which acts as a host cell for production of a recombinant mannanase, but which microorganism simultaneously produces other enzymes.
  • The term "operably linked", when referring to DNA segments, denotes that the segments are arranged so that they function in concert for their intended purposes, e.g. transcription initiates in the promoter and proceeds through the coding segment to the terminator
  • The term "promoter" denotes a portion of a gene containing DNA sequences that provide for the binding of RNA polymerase and initiation of transcription. Promoter sequences are commonly, but not always, found in the 5' non-coding regions of genes.
  • The term "secretory signal sequence" denotes a DNA sequence that encodes a polypeptide (a "secretory peptide") that, as a component of a larger polypeptide, directs the larger polypeptide through a secretory pathway of a host cell in which it is produced. The secretory signal sequence can be native or it can be replaced with secretory signal sequence or carrier sequence from another source. Depending on the host cell, the larger peptide may be cleaved to remove the secretory peptide during transit through the secretory pathway.
  • The term "core region" denotes a domain of an enzyme, which may or may not have been modified or altered, but which has retained its original activity; the catalytic domain as known in the art has remained functional. The core region of a mannanase according to the invention corresponds to the amino acids aligned with the amino acids shown in the Tables 5 and 8.By the term "linker" or "spacer" is meant a polypeptide comprising at least two amino acids which may be present between the domains of a multidomain protein, for example an enzyme comprising an enzyme core and a binding domain such as a carbohydrate binding module (CBM) or any other enzyme hybrid, or between two proteins or polypeptides produced as a fusion polypeptide, for example a fusion protein comprising two core enzymes. For example, the fusion protein of an enzyme core with a CBM is provided by fusing a DNA sequence encoding the enzyme core, a DNA sequence encoding the linker and a DNA sequence encoding the CBM sequentially into one open reading frame and expressing this construct.
  • The term "detergent composition", includes unless otherwise indicated, granular or powder-form all-purpose or heavy-duty washing agents, especially cleaning detergents; liquid, gel or paste-form all-purpose washing agents, especially the so- called heavy-duty liquid (HDL) types; liquid fine-fabric detergents; hand dishwashing agents or light duty dishwashing agents, especially those of the high-foaming type; machine dishwashing agents, including the various tablet, granular, liquid and rinse-aid types for household and institutional use; liquid cleaning and disinfecting agents, car or carpet shampoos, bathroom cleaners; metal cleaners; as well as cleaning auxiliaries such as bleach additives and "stain-stick" or pre-treat types. The terms "detergent composition" and "detergent formulation" are used in reference to mixtures which are intended for use in a wash medium for the cleaning of soiled objects. In some embodiments, the term is used in reference to laundering fabrics and/or garments (e.g., "laundry detergents"). In alternative embodiments, the term refers to other detergents, such as those used to clean dishes, cutlery, etc. (e.g., "dishwashing detergents"). It is not intended that the present invention be limited to any particular detergent formulation or composition. It is intended that in addition to the variants according to the invention, the term encompasses detergents that may contain, e.g., 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, anticorrosion agents, enzyme inhibitors or stabilizers, enzyme activators, transferase(s), hydrolytic enzymes, oxido reductases, bluing agents and fluorescent dyes, antioxidants, and solubilizers.
  • 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 cellulosics (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, rabit and silk or synthetic polymer such as nylon, aramid, polyester, acrylic, polypropylen 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.
  • The term "stability" includes storage stability and stability during use, e.g. during a wash process (in wash stability) and reflects the stability of the protease variant according to the invention as a function of time e.g. how much activity is retained when the protease is kept in solution, in particular in a detergent solution. The stability is influenced by many factors e.g. pH, temperature, detergent composition e.g. amount of builder, surfactants etc. The protease stability may be measured using the 'stability assay' as described in the Materials and Methods section herein. The term "improved stability" or "increased stability" is defined herein as a variant protease displaying an increased stability in solutions, relative to the stability of the parent protease. The terms "improved stability" and "increased stability" includes "improved chemical stability", "detergent stability" or "improved detergent stability.
  • The enzyme comprised in the detergent composition of present invention can be a recombinant fusion protein which, in addition to having the amino acid sequence having mannanase activity, comprises at least one of: an amino acid sequence providing a secretory signal sequence; an amino acid sequence which facilitates purification, such as an affinity tag or His-tag; an amino acid sequence which enhances production, such as an amino acid sequence which is a carrier or CBM; an amino acid sequence having an enzyme activity; and an amino acid sequence providing for the fusion protein with binding affinity, such as a carbohydrate binding moiety.
  • The CBM, carbohydrate binding moiety, as a carrier is advantageous e.g. in Trichoderma production. The CBM may also be a fragment of another protein or enzyme derived from the same or different organism as the mannanase. The amino acid sequence having mannanase activity may be connected to the other functionality of the fusion protein via a linker sequence. Fusion proteins can be engineered to modify properties or production of the mannanase. In an embodiment the mannanase part is connected to the other amino acid sequence with a linker.
  • Detergent composition
  • The present invention relates to novel detergent compositions comprising fungal mannanase enzymes. The detergent compositions comprising fungal mannanases are useful in laundry and cleaning applications wherein degradation or modification of mannan is desired. The invention also relates to the use of said detergent compositions in laundry and cleaning applications as well as a method for degrading mannan.
  • As used herein, the term "mannan" refers to polysaccharides consisting of a mannose backbone linked together by β-1,4-linkages with side-chains of galactose attached to the backbone by α-1,6-linkages. Mannans comprise plant based material such as guar gum and locust bean gum. Glucomannans are polysaccharides having a backbone of more or less regularly alternating β-1,4 linked mannose and glucose, galactomannans and galactoglucomannans are mannans and glucomannans with alpha-1,6 linked galactose sidebranches.
  • As used herein, the term "mannanase" or "galactomannanase" denotes a mannanase enzyme defined according to the art as mannan endo-1,4-beta-mannosidase and having the alternative names beta-mannanase and endo-1,4-mannanase and catalysing hydrolysis of 1,4-beta-D-mannosidic linkages in mannans, galactomannans, glucomannans, and galactoglucomannans. Mannanases are classified according to the Enzyme Nomenclature as EC 3.2.1.78.
  • "Mannanase activity" as used herein refers to the mannan degrading activity of a polypeptide. Degrading or modifying as used herein means that mannose units are hydrolyzed from the mannan polysaccharide by the mannanase. The mannan degrading activity of the polypeptides according to present invention can be tested according to standard test procedures known in the art. Example 6 provides an example of a standard method for determining mannanase activity.
  • According to the first aspect the detergent composition of the present invention comprises at least one enzyme having an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 3 (Man 1), and/or 80% sequence identity to the amino acid sequence of SEQ ID NO: 9 (Man 3), and/or 80% sequence identity to the amino acid sequence of SEQ ID NO: 12 (Man 5).
  • In one embodiment of the invention the at least one enzyme comprises an amino acid sequence having 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: 3 (Man 1).
  • In one embodiment of the invention the at least one enzyme comprises an amino acid sequence having 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: 9 (Man 3).
  • In one embodiment of the invention the at least one enzyme comprises an amino acid sequence having 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: 12 (Man 5).
  • In a further embodiment of the invention the at least one enzyme has mannan degrading activity. The mannanases comprised in the detergent composition of the invention are suitable for degrading and modifying mannan containing material in various chemical environments, preferably in detergent compositions.
  • In one embodiment of the present invention the detergent composition further comprises one or more additional enzymes selected from the group consisting of protease, lipase, cutinase, amylase, carbohydrase, cellulase, pectinase, pectatlyase, mannanase, arabinase, galactanase, xylanase, oxidase, xanthanase, laccase, and/or peroxidase, preferably selected from the group consisting of proteases, amylases, cellulases and lipases.
  • In general the properties of the selected enzyme(s) should be compatible with the selected detergent, (i.e., pH-optimum, compatibility with other enzymatic and non-enzymatic ingredients, etc.), and the enzyme(s) should be present in effective amounts.
  • Cellulases
  • Suitable cellulases include those of fungal or fungal origin. Chemically modified or protein engineered mutants are included. Suitable cellulases include cellulases from the genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, e.g., the fungal cellulases produced from Humicola insolens, 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 described in WO02/099091 ._Other examples of cellulases include the family 45 cellulases described in WO96/29397 , 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, 117, 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, Q119H or Q146 R._Commercially available cellulases include Celluclean™ Celluzyme™, and Carezyme™ (Novozymes A/S), Clazinase™, and Puradax HA™ (Genencor International Inc.), and KAC-500(B)™ (Kao Corporation).
  • Proteases
  • Suitable proteases include those of fungal, fungal, plant, viral or animal origin e.g. vegetable or microbial origin. Microbial origin is preferred. Chemically modified or protein engineered mutants are included. 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 or other metalloprotease such as those from M5, M7 or M8 families.
  • 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. Examples of subtilases are those derived from Bacillus such as Bacillus lentus, B. alkalophilus, B. subtilis, B. amyloliquefaciens, Bacillus pumilus and Bacillus gibsonii described in; US7262042 and WO09/021867 , and subtilisin lentus, subtilisin Novo, subtilisin Carlsberg, Bacillus licheniformis, subtilisin BPN', subtilisin 309, subtilisin 147 and subtilisin 168 described in WO89/06279 and protease PD138 described in ( WO93/18140 ). Other useful proteases may be those described in WO92/175177 , WO01/016285 , WO02/026024 and WO02/016547 . Examples of trypsin-like proteases are trypsin (e.g. of porcine or bovine origin) and the Fusarium protease described in WO89/06270 , WO94/25583 and WO05/040372 , and the chymotrypsin proteases derived from Cellumonas described in WO05/052161 and WO05/052146 . A further preferred protease is the alkaline protease from Bacillus lentus DSM 5483, as described for example in WO95/23221 , and variants thereof, which are described in WO92/21760 , WO95/23221 , EP1921147 and EP1921148 . Examples of metalloproteases are the neutral metalloprotease as described in WO07/044993 (Genencor Int.) such as those derived from Bacillus amyloliquefaciens.
  • Examples of useful proteases are the variants described in: WO92/19729 , WO96/034946 , WO98/20115 , WO98/20116 , WO99/011768 , WO01/44452 , WO03/006602 , WO04/03186 , WO04/041979 , WO07/006305 , WO11/036263 , WO11/036264 , especially the variants with substitutions in one or more of the following positions: 3, 4, 9, 15, 27, 36, 57, 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, 206, 217, 218, 222, 224, 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, S101G,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). Suitable commercially available protease enzymes include those sold under the trade names Alcalase®, DuralaseTm, DurazymTm, Relase®, Relase® Ultra, Savinase®, Savinase® Ultra, Primase®, Polarzyme®, Kannase®, Liquanase®, Liquanase® Ultra, Ovozyme®, Coronase®, Coronase® Ultra, Neutrase®, Everlase® and Esperase® (Novozymes A/S), those sold under the tradename Maxatase®, Maxacal®, Maxapem®, Purafect®, Purafect Prime®, Prefer-enzTm, Purafect MA®, Purafect Ox®, Purafect OxP®, Puramax®, Properase®, EffectenzTm, FN2®, FN3®, FN4®, Excellase®,, Opticlean® and Optimase® (Danisco/DuPont), Axapem™ (Gist-Brocases N.V.), BLAP (sequence shown in Figure 29 of US5352604 ) and variants hereof (Henkel AG) and KAP (Bacillus alkalophilus subtilisin) from Kao.
  • Lipases and Cutinases
  • Suitable lipases and cutinases include those of fungal or fungal origin. Chemically modified or protein engineered mutant enzymes are included. Examples include lipase from Thermomyces, e.g. from T. lanuginosus (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 Burkholderia), 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 Streptomyces lipases ( WO10/065455 ), cutinase from Magnaporthe grisea ( WO10/107560 ), cutinase from Pseudomonas mendocina ( US5,389,536 ), lipase from Thermobifida fusca ( WO11/084412 ), Geobacillus stearothermophilus lipase ( WO11/084417 ), lipase from Bacillus subtilis ( WO11/084599 ), and lipase from Streptomyces griseus ( WO11/150157 ) and S. pristinaespiralis ( WO12/137147 ). Other examples are lipase variants such as those described in EP407225 , WO92/05249 , WO94/01541 , WO94/25578 , WO95/14783 , WO95/30744 , WO95/35381 , WO95/22615 , WO96/00292 , WO97/04079 , WO97/07202 , WO00/34450 , WO00/60063 , WO01/92502 , WO07/87508 and WO09/109500 . Preferred commercial lipase products include include Lipolase™, Lipex™; Lipolex™ and Lipoclean™ (Novozymes A/S), Lumafast (originally from Genencor) and Lipomax (originally from Gist-Brocades). Still other examples are lipases sometimes referred to as acyltransferases or perhydrolases, e.g. acyltransferases with homology to Candida antarctica lipase A ( WO10/111143 ), acyltransferase from Mycobacterium 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 ( WO10/100028 ).
  • Amylases
  • Suitable amylases which can be used together with subtilase variants of the invention may be an alpha-amylase or a glucoamylase and may be of fungal 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 .
  • Suitable amylases include amylases having SEQ ID NO: 3 in WO 95/10603 or variants having 90% sequence 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, 211, 243, 264, 304, 305, 391, 408, and 444.
  • Different suitable amylases include amylases having SEQ ID NO: 6 in WO 02/010355 or variants thereof having 90% sequence identity thereto. Preferred variants are those having a deletion in positions 181 and 182 and a substitution in position 193.
  • Other amylases, which are suitable 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% sequence 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, I201, 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 of WO2006/066594 are those having the substitutions:
    • M197T;
    • H156Y+A181T+N190F+A209V+Q264S; or
    • G48A+T49I+G107A+H156Y+A181T+N190F+I201F+A209V+Q264S.
  • Further amylases which are suitable 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 R181 and G182, or positions H183 and G184.
  • Additional amylases which can be used are those having SEQ ID NO: 1, SEQ ID NO: 3, 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, SEQ ID NO: 3 or SEQ ID NO: 7. Preferred variants of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 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 181 and 182 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 one or more of 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 suitable amylases are amylases having SEQ ID NO: 2 of WO 09/061380 or variants having 90% sequence identity to SEQ ID NO: 2 thereof. Preferred variants of SEQ ID NO: 2 are those having a truncation of the C-terminus and/or 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, T131I, T165I, K178L, T182G, M201L, 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 or of T182 and/or G183. 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+T131I+T165I+K178L+T182G+Y305R+G475K wherein the variants are C-terminally truncated and optionally further comprises a substitution at position 243 and/or a deletion at position 180 and/or position 181.
  • Other suitable amylases are the alpha-amylase having SEQ ID NO: 12 in WO01/66712 or a variant having at least 90% 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 R118K, 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.
  • Other examples are amylase variants such as those described in WO2011/098531 , WO2013/001078 and WO2013/001087 .
  • Commercially available amylases are Ouramyl™, Termamyl™, Fungamyl™, Stainzyme™, Stainzyme Plus™, Natalase™, Liquozyme X and BAN™ (from Novozymes A/S), and Rapidase™, Purastar™/Effectenz™, Powerase and Preferenz S100 (from Genencor International Inc./DuPont).
  • Peroxidases/Oxidases
  • Suitable peroxidases/oxidases include those of plant, fungal 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 .
  • A composition for use in solid laundry detergent, for example, may include 0.000001% - 5%, such as 0.000005%-2%, such as 0.00001%-1%, such as 0.00001%-0,1% of enzyme protein by weight of the composition.
  • A composition for use in laundry liquid, for example, may include 0.000001% - 3%, such as 0.000005 - 1 %, such as 0.00001 %-0,01 % of enzyme protein by weight of the composition.
  • A composition for use in automatic dishwash, for example, may include 0.000001% - 5%, such as 0.000005%-2%, such as 0.00001%-1%, such as 0.00001%-0,1% 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 one embodiment, the invention is directed to detergent compositions comprising an enzyme of the present invention in combination with one or more additional cleaning composition 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 textile care, the consideration of the type of textile 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.
  • 1. 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 nonionic 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 contain 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 sulfate (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 contain 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 ammonium (AQA) compounds, and combinations thereof.
  • When included therein the detergent will usually contain 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 propoxylates, 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), alkylpolyglycosides (APG), alkoxylated amines, fatty acid monoethanolamides (FAM), fatty acid diethanolamides (FADA), ethoxylated fatty acid monoethanolamides (EFAM), propoxylated fatty acid monoethanolamides (PFAM), polyhydroxy alkyl fatty acid amides, or N-acyl N-alkyl derivatives of glucosamine (glucamides, 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 contain 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-hydroxyethyl)amine oxide, fatty acid alkanolamides and ethoxylated fatty acid alkanolamides, and combinations thereof.
    When included therein the detergent will usually contain from about 0% to about 10% by weight of a zwitterionic surfactant. Non-limiting examples of zwitterionic surfactants include betaine, alkyldimethylbetaine, sulfobetaine, and combinations thereof.
  • 2. Hydrotropes
  • 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. Hydrotropes do not display a critical concentration above which self-aggregation occurs as found for surfactants and lipids forming miceller, 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 behavior, 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 contain 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.
  • 3. Builders and Co-Builders
  • The detergent composition may contain 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 typically 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), ethanolamines such as 2-aminoethan-1-ol (MEA), diethanolamine (DEA, also known as iminodiethanol), triethanolamine (TEA, also known as 2,2',2"-nitrilotriethanol), and carboxymethyl inulin (CMI), and combinations thereof.
  • The detergent composition may also contain 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 include a co-builder alone, or in combination with a builder, for example a zeolite builder. Non-limiting examples of co-builders include homopolymers 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 aminocarboxylates, aminopolycarboxylates and phosphonates, and alkyl-or alkenylsuccinic acid. Additional specific examples include 2,2',2"-nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic 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-diphosphonic acid (HEDP), ethylenediaminetetra-(methylenephosphonic acid) (EDTMPA), diethylenetriaminepentakis(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-monopropionic 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), α-alanine-N, N-diacetic acid (α-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), diethanolglycine (DEG), diethylenetriamine penta(methylenephosphonic acid) (DTPMP), aminotris(methylenephosphonic acid) (ATMP), and combinations and salts thereof. Further exemplary builders and/or co-builders are described in, e.g., WO 09/102854 , US 5977053
  • 4. Bleaching Systems
  • The detergent may contain 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 WO98/17767 . A particular family of bleach activators of interest was disclosed in EP624154 and particulary 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:
  1. (i)
    Figure imgb0001
  2. (ii)
    Figure imgb0002
  3. (iii) and mixtures thereof; wherein each R1 is independently a branched alkyl group containing from 9 to 24 carbons or linear alkyl group containing from 11 to 24 carbons, preferably each R1 is independently a branched alkyl group containing from 9 to 18 carbons or linear alkyl group containing from 11 to 18 carbons, more preferably each R1 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
5. Polymers
  • The detergent may contain 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 antiredeposition, 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), poly(vinyl alcohol) (PVA), poly(vinylpyrrolidone) (PVP), poly(ethyleneglycol) 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 polyvinylpyrrolidone-vinylimidazole (PVPVI). Further exemplary polymers include sulfonated polycarboxylates, polyethylene 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.
  • 6. 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 .
  • 7. 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 carboxyl 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-distyryl 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'-disulphonate; 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-triazol-2-yl)stilbene-2,2'-disulphonate; 4,4'-bis-(2-anilino-4(1-methyl-2-hydroxy-ethylamino)-s-triazin-6-ylamino) 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 nonionic or anionic terephthalte 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 alkoxylate 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 2006/113314 (hereby incorporated by reference). Other soil release polymers are substituted polysaccharide structures especially substituted cellulosic structures such as modified cellulose deriviatives 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, zwitterionically 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), polyvinylpyrrolidone (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, hydrotropes, perfumes, pigments, sod suppressors, solvents, and structurants for liquid detergents and/or structure elasticizing agents.
  • In a further embodiment of the present invention the 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 detergent composition can be a laundry detergent composition, preferably a liquid or solid laundry detergent composition. 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 multicompartments. 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 devided into compartments of the pouch. Preferred films are polymeric materials preferably polymers which are formed into a film or sheet. Preferred polymers, copolymers or derivates therof are selected polyacrylates, and water soluble acrylate copolymers, methyl cellulose, carboxy methyl cellulose, sodium dextrin, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, malto dextrin, poly methacrylates, most preferably polyvinyl alcohol copolymers and, hydroxyprpyl 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 detergent 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 dissolvable 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 detergent compositions of present invention may be provided in the form of 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 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.
  • The present invention furthermore relates to different uses of the detergent composition as herein disclosed, such as for degrading mannan and for use in a laundry process.
  • The present invention furthermore relates to a method for removing a stain from a surface, comprising contacting the surface with a detergent composition as herein disclosed.
    The present invention also relates to a method for degrading mannan comprising applying a detergent composition as herein disclosed to mannan, preferably wherein the mannan is on the surface of a textile. By degrading mannan attached to the textile or fabric, dirt or soil bound to mannan is released and not capable of binding again to the mannan or mannan stains.
  • Providing mannanases that retain activity in temperatures above ambient temperature and in acidic pH is advantageous for applications wherein mannan degradation is required in such conditions. In an embodiment the mannanase comprised in the detergent compositions of present invention hydrolyses endo-beta-1,4-mannosidic linkages randomly. In an embodiment the mannanase comprised in the detergent compositions of present invention is obtainable or derivable from a fungal source. In an embodiment the mannanase comprised in the detergent compositions of present invention is fused with at least one further polypeptide, thus forming a fusion polypeptide. The mannanase can be connected to the further polypeptide with a linker. The fusion polypeptide or the further polypeptide may have other catalytic or binding activities in addition to those of mannanase. In an embodiment the further polypeptide comprises or consists of carbohydrate binding module, which is optionally a fragment of another protein or enzyme derived from the same or different organism as the mannanase.
  • EXAMPLES
  • The following examples are provided to illustrate various aspects of the present invention. They are not intended to limit the invention, which is defined by the accompanying claims.
  • Example 1: Screening
  • For identification of new beta-1,4-mannanases public databases (NCBI, EBI) and selected proprietary and public genomes were screened. All proprietary and public genomes used in this work are shown in Table 1. All hits were grouped and finally 14 genes of fungal origin were selected for cloning in Trichoderma reesei (Table 2). Table 1: List of proprietary and public genomes used for screening of beta-1,4-mannanases. Identification numbers of proprietary strains, genome sources and identification of strains used for public genomes are shown.
    Species Strain Genome Genome origin
    Melanocarpus albomyces ALKO4237 ROAL
    Acremonium thermophilum ALKO4245 ROAL
    Fusarium equiseti RF6318 ROAL
    Verticillium dahliae RF8261 JGI(a VdLs.17
    Malbranchea cinnamomea ALKO4122 Genozymes(b CBS 343.55
    Aspergillus nishimurae RH3949 AB Enzymes
    Acremonium alcalophilum JGI(a
    Gymnascella citrina JGI(a NRRL 5970
    Plectosphaerella cucumerina JGI(a DS2psM2a2
    (a Joint Genome Institute (United States Department of Energy) at http://genome.jgi.doe.gov/
    (b Genozymes project (Genozymes for Bioproducts and Bioprocesses Development) at http://www.fungalgenomics.ca
    Table 2: List of genes selected for cloning in Trichoderma reesei. Predicted PFAM domains and amino acid lengths of the proteins are shown.
    Sequence ID Species GH family Length
    Man1 Malbranchea cinnamomea 5 410 aa
    Man2 Verticillium dahliae 5 397 aa
    Man3 Verticillium dahliae 5 401 aa
    Man5 Melanocarpus albomyces 5 416 aa
    Man8 Acremonium alcalophilum 5 469 aa
    Man9 Fusarium equisetii 5 446 aa
    Man10 Acremonium thermophilum 5 416 aa
    Man11 Verticillium dahliae 5 492 aa
    Man12 Malbranchea cinnamomea 5 355 aa
    Man13 Aspergillus nishimurae 5 376 aa
    Man17 Melanocarpus albomyces 26 476 aa
    Man27 Acremonium thermophilum 5 379 aa
    Man29 Plectosphaerella cucumerina 5 401 aa
    Man30 Gymnascella citrina 5 411 aa
  • Example 2: PCR-cloning of Malbrachea cinnamomea ALKO4122 man1 and man12, Verticillium dahliae RF8261 man2 and man3, Melanocarpus albomyces ALKO4237 man5 and man17, and Acremonium thermophilum ALKO4245 man27 mannanases.
  • Standard molecular biology methods were used in the isolation and enzyme treatments of DNA (e.g. isolation of plasmid DNA, digestion of DNA to produce DNA fragments), in E. coli transformations, sequencing etc. The basic methods used were either as described by the enzyme, reagent or kit manufacturer or as described in the standard molecular biology handbook, e.g. Sambrook and Russell (2001). Isolation of genomic DNA was performed as described in detail by Raeder and Broda (1985). After analyzing the sequence data from the fungal strains Malbranchea cinnamomea ALKO4122, Verticillium dahliae RF8261, and Melanocarpus albomyces ALKO4237 and Acremonium thermophilum ALKO4245 (see Example 1) 7 mannanase genes from the analyzed genomes were selected for PCR-cloning. The genes encoding mannanases man2 and man3 were PCR-cloned using genomic DNA of RF8261 as a template. Mannanase genes man5 and man17 were cloned using ALKO4237 genomic DNA as template. Man27 was cloned using ALKO4245 genomic DNA and man12 using ALKO4122 genomicDNA as templates. The gene encoding mannanase man1 was PCR-cloned using plasmid pALK4042 as a template. Plasmid pALK4042 consists of pBluescript II KS+ vector and about 4.5 kb DNA fragment from ALKO4122 including the mannanase gene. The genes were amplified by PCR with primers described in Table 3. The PCR mixture contained 1x HF Buffer for Phusion HF Polymerase (NEB/BioNordika, Finland), 0.2 mMdNTP mix (Thermo Fisher Scientific, Finland), 1 µM each primer, 3% DMSO (Thermo Fisher Scientific), 1 unit of Phusion High-Fidelity Polymerase (NEB/BioNordika, Finland) and 1-2 µg genomic DNA or 50 - 200 ng of plasmid DNA as templates. The conditions for the PCR reactions were the following: 30 sec initial denaturation at 98 °C, followed by 28 cycles of 10 sec at 98 °C, 30 sec annealing at one of the following 45/50/55/60 °C, 45 sec extension at 72 °C and the final extension at 72 °C for 7 min.
  • Primer combination described in Table 3 produced specific DNA product having the expected size. The PCR product was isolated from agarose gel with GenJet Gel Extraction Kit (Thermo Fisher Scientific) according to manufacturer's instructions, digested with Sacll and BamHI restriction enzymes (Thermo Fisher Scientific) and cloned into an expression vector cleaved with Sacll and BamHI. Ligation mixtures were transformed into Escherichia coli XL1-Blue (AH Diagnostics) or XL10-Gold (Agilent) cells and plated on LB (Luria-Bertani) plates containing 50-100 µg/ml ampicillin. Several E. coli colonies were collected from the plates and DNA was isolated with GenJet Plasmid Miniprep Kit (Thermo Fisher Scientific). Positive clones were screened using restriction digestions and they were shown to contain inserts of expected sizes. The full-length genes encoding the mannanases man1, man2, man3, man5, man12, man17 and man27 were sequenced and the plasmids were named pALK4073, pALK4070, pALK4071, pALK4271, pALK4080 and pALK4270, respectively (For details see Example 4). The relevant information on the genes and the deduced amino protein sequences (SEQ ID NOs: 1, 3, 4, 6, 7, 9, 10, 12, 25, 27, 31, 33, 34, 36) are summarized in Table 4 and Table 5, respectively. Table 3. The oligonucleotides used as PCR primers to amplify mannanase genes from Malbranchea cinnamomea ALKO4122, Verticillium dahliae RF8261, Melanocarpus albomyces ALKO4237, Acremonium thermophilum ALKO4245 and plasmid pALK4042.
    Template, DNA from Oligonucleotides Length (bp) Sequence(a SEQ ID NO:
    pALK4042 Mc_Man for 53
    Figure imgb0003
    43
    pALK4042 Mc_Man rev 36
    Figure imgb0004
    44
    ALKO4122 Malbranchea cinnamomea MANP5 60
    Figure imgb0005
    45
    ALKO4122 Malbranchea cinnamomea MANP6 52
    Figure imgb0006
    46
    RF8261 Verticillium dahliae Vd_Man3_for 48
    Figure imgb0007
    47
    RF8261 Verticillium dahliae Vd_Man3_rev 37
    Figure imgb0008
    48
    RF8261 Verticillium dahliae Vd_Man2_for 47
    Figure imgb0009
    49
    RF8261 Verticillium dahliae Vd_Man2_rev 38
    Figure imgb0010
    50
    ALKO4237 Melanocarpus albomyces Ma_man2_for 53
    Figure imgb0011
    51
    ALKO4237 Melanocarpus albomyces Ma_Man2_rev 39
    Figure imgb0012
    52
    ALKO4237 Melanocarpus albomyces Ma_Man1for2 47
    Figure imgb0013
    53
    ALKO4237 Melanocarpus albomyces Ma_Man1rev 41
    Figure imgb0014
    54
    ALKO4245 Acremonium thermophilum MANP3 60
    Figure imgb0015
    55
    ALKO4245 Acremonium thermophilum MANP4 54
    Figure imgb0016
    56
    (a "s" in the parenthesis = sense strand, "as" = antisense strand.
    Table 4. The summary on the mannanase encoding genes of Malbranchea cinnamomea ALKO4122, Verticillium dahliae RF8261, Melanocarpus albomyces ALKO4237 and Acremonium thermophilum ALKO4245.
    Gene Origin Length with introns (bp)(a Coding region (bp)(b No of putative introns Lengths of putative introns (bp) SEQ ID NO
    man1 M. cinnamomea 1686 1230 4 119, 124, 121, 89 1
    man2 V. dahliae 1330 1191 2 65, 71 4
    man3 V. dahliae 1388 1203 2 83, 99 7
    man5 M. albomyces 1397 1248 2 81, 65 10
    man 12 M. cinnamomea 1275 1065 3 73, 63, 71 25
    man17 M. albomyces 1761 1428 4 63,69, 103, 95 31
    man27 A. thermophilum 1360 1137 2 107, 113 34
    (a The STOP codon is included
    (b The STOP codon is not included
    Table 5. The summary of the amino acid sequences deduced from the mannanase encoding gene sequences of Malbranchea cinnamomea ALKO4122, Verticillium dahliae RF8261, Melanocarpus albomyces ALKO4237 and Acremonium thermophilum ALKO4245.
    Man protein Origin No of aas Length of ss (a CBM Core (aa-aa) Predicted (Da), ss not included (b Predicted pl, ss not included SEQ ID NO
    Man1 M. cinnamomea 410 18 No 44487 5.37 3
    Man2 V. dahliae 397 22 No 41589 4.56 6
    Man3 V. dahliae 401 17 No 43488 4.71 9
    Man5 M. albomyces 416 20 No 44670 4.51 12
    Man12 M. cinnamomea 355 17 No 37959 4.26 27
    Man17 M. albomyces 476 22 Yes 143-476 50420 4.42 33
    Man27 A. thermophilum 379 18 No 39046 4.86 36
    (a The prediction on the signal sequence was made using the program SignalP v3.0, NN/HMM (Nielsen et al., 1997; Nielsen & Krogh, 1998; Bendtsen et al., 2004).
    (b The predicted signal sequence was not included. The prediction was made using Clone Manager Professional version 9 for Windows, Sci-Ed Software.
  • The comparison of the deduced mannanase sequences Man1 and Man12 from Malbranchea cinnamomea ALKO4122, Man2 and Man3 from Verticillium dahliae RF8261, Man5 and Man17 from Melanocarpus albomyces ALKO4237 and Man27 from Acremonium thermophilum ALKO4245 to the databases is shown in Table 6. Table 6. The highest identity sequences to the deduced Man1, Man2, Man3 Man5, Man12, Man17 and Man27 amino acid sequences from Malbranchea cinnamomea ALKO4122, Verticillium dahliae RF8261, Melanocarpus albomyces ALKO4237 andAcremonium thermophilum ALKO4245. The full-length amino acid sequences including the signal sequences were aligned. The database searches were performed at https:Hwww.ebi.ac.uk/Tools/sss/ncbiblast/https://www.ebi.ac.uk/Tools/psa/emboss_needle/ using NCBI BLAST+ (EMBL-EBI, NCBI BLAST+ - Sequence Similarity Search, UniProt Knowledgebase, BLOSUM62, Gap open 11, Gap extend 1), and EMBOSS Needle (EMBL-EBI, EMBOSS-Needle - Pairwise Sequence Alignment, BLOSUM62, Gap open 10, Gap extend 0.5) for determining the degree of identity. Searches were also performed with https://usgene.sequencebase.com/ using Matrix PAM30, Gap open 9 and Gap extend 1.
    Organism and accession number Identity (%)
    Man1 78.9
    Aspergillus terreus, Q0C8J3 (1 99.5
    Malbranchea cinnamomea, BBN39104 (2
    Man2 100
    Verticillium dahliae, G2XHW3_VERDV (1 63.4
    Myriococcum thermophilum, BBA64714 (2
    Man3 100
    Verticillium dahliae, G2XBE5_VERDV (VDAG_07477) (1 Aspergillus terreus, BBW96507 (2 69.4
    Man5 99.3
    Chaetomium sp. CQ31, G9BZJ2_9PEZI (1 99.3
    Chaetomium sp., BAL41820 (2
    Man12 46.1
    Pseudogymnoascus sp, A0A1B8DBN0_9PEZI (1 100
    Malbranchea cinnamomea, BBN39199 (2
    Man17 81.5
    Madurella mycetomatis, A0A175W047_9PEZI (1 99.8
    Melanocarpus albomyces, BDB24260 (2
    Man27 75.5
    Coniochaeta ligniaria, A0A1J7JVW4_9PEZI (1 100
    Acremonium thermophilum, BDB18590 (2
    (1 Search performed using NCBI BLAST+ (EMBL-EBI, NCBI BLAST+ - Sequence Similarity Search, UniProt Knowledgebase, BLOSUM62, Gap open 11, Gap extend 1), and EMBOSS Needle (EMBL-EBI, EMBOSS-Needle - Pairwise Sequence Alignment, BLOSUM62, Gap open 10, Gap extend 0.5) for determining the degree of identity.
    (2 Search performed with SequenceBase database https://usgene.sequencebase.com/
  • Example 3: Sub cloning of synthetic fungal mannanase genes
  • Standard molecular biology methods were used in the isolation and enzyme treatments of DNA (e.g. isolation of plasmid DNA, digestion of DNA to produce DNA fragments), in E. coli transformations, sequencing etc. The basic methods used were either as described by the enzyme, reagent or kit manufacturer or as described in the standard molecular biology handbook, e.g. Sambrook and Russell (2001). Isolation of genomic DNA was performed as described in detail by Raeder and Broda (1985). Man8, man9, man 10, man11, man13, man29 and man30 from Acremonium alcalophilum, Fusarium equiseti RF6318, Acremonium thermophilum ALKO4245, Verticillium dahliae RF8261,Aspergillus nishimurae RH3949, Plectosphaerella cucmerina and Gymnascella citrina respectively, were ordered from GenScript as synthetic constructs without introns and with codon optimization for Trichoderma reesei.
  • Plasmid DNAs obtained from GenScript including the genes man8, man9, man10, man11, man13, man29 and man30 were re-suspended in sterile water, digested with Sacll and BamHI restriction enzymes (Thermo Fisher Scientific) according to manufacturer's instructions and cloned into an expression vector cleaved with Sacll and BamHI. Ligation mixtures were transformed into Escherichia coli XL1-Blue (AH Diagnostics) and plated on LB (Luria-Bertani) plates containing 50-100 µg/ml ampicillin. Several E. coli colonies were collected from the plates and DNA was isolated with GenJet Plasmid Miniprep Kit (Thermo Fisher Scientific). Positive clones were screened using restriction digestions and they were shown to contain inserts of expected sizes. The full-length genes encoding the Acremonium alcalophilum man8, Fusarium equiseti man9, Acremonium thermophilum man10 and Verticillium dahliae man11 mannanases, and fusion sites to the expression plasmid of Aspergillus nishimurae man13, Plectosphaerella cucumerina man29 and Gymnascella citrina man30 mannanase genes were sequenced and the plasmids were named pALK4363, pALK4091, pALK4084, pALK4090, pALK4413, pALK4411 and pALK4412 respectively (For details see Example 4). The plasmid DNAs including the aforementioned genes delivered by GenScript were also transformed into XL10-Gold E. coli cells (Agilent) and deposited into DSMZ strain collections. The relevant information on the genes and the deduced amino protein sequences (SEQ ID NOs: 14, 17, 20, 23, 29, 38 and 41) are summarized in Table 7 and Table 8, respectively. The E. coli strains RF12388, RF12389, RF12390, RF12391, RF12392 and RF12393 including the plasmids pALK4443, pALK4444, pALK4445, pALK4446, pALK4447 and pALK4448, respectively were deposited to the DSM collection under the accession numbers DSM 32434, DSM 32435, DSM 32436,DSM 32437, DSM 32438 and DSM 32439 respectively. Table 7. The summary on the mannanase encoding synthetic genes designed based on sequences obtained from Acremonium alcalophilum, Fusarium equiseti RF6318, Acremonium thermophilum ALKO4245, Verticillium dahliae RF8261, Aspergillus nishimurae RH3949, Plectosphaerella cucumerina and Gymnascella citrina.
    Gene Length without introns (bp)(a SEQ ID NO
    man8 1410 14
    man9 1341 17
    man10 1251 20
    man11 1479 23
    man13 1131 29
    man29 1206 38
    man30 1236 41
    (a The STOP codon is included
    Table 8. The summary of the amino acid sequences deduced from the mannanase encoding gene sequences from Acremonium alcalophilum, Fusarium equiseti RF6318, Acremonium thermophilum ALKO4245, Verticillium dahliae RF8261, Aspergillus nishimurae RH3949, Plectosphaerella cucumerina and Gymnascella citrina.
    Man protein No of aas Length of ss (a CBM Core (aa-aa) Predicted (Da), ss not included (b Predicted pl, ss not included (b SEQ ID NO
    Man8 469 21 Yes 22-392 49497 4.36 15
    Man9 446 18 Yes 19-387 47728 5.05 18
    Man10 416 19 No 44403 4.97 21
    Man11 492 19 Yes 20-401 51903 5.16 24
    Man13 376 21 No 39351 5.09 30
    Man29 401 18 No 43738 4.55 39
    Man30 411 18 No 44747 5.83 42
    (a The prediction on the signal sequence was made using the program SignalP v3.0, NN/HMM (Nielsen et al., 1997; Nielsen & Krogh, 1998; Bendtsen et al., 2004).
    (b The predicted signal sequence was not included. The prediction was made using Clone Manager Professional version 9 for Windows, Sci-Ed Software.
  • The comparison of the deduced mannanase sequences Man8, Man9, Man10, Man11, Man13, Man29 and Man30 from Acremonium alcalophilum, Fusarium equiseti RF6318, Acremonium thermophilum ALKO4245, Verticillium dahliae RF8261, Aspergillus nishimurae RH3949, Plectosphaerella cucumerina and Gymnascella citrina respectively, to the databases is shown in Table 9. Table 9. The highest identity sequences to the deduced Man8, Man9, Man10, Man11, Man13, Man29 and Man30 amino acid sequences from Acremonium alcalophilum, Fusarium equiseti RF6318, Acremonium thermophilum ALKO4245, Verticillium dahliae RF8261, Aspergillus nishimurae RH3949, Plectosphaerella cucumerina and Gymnascella citrina. The full-length amino acid sequence including the signal sequenceswere aligned. The database searches were performed at https://www.ebi.ac.uk/Tools/sss/ncbiblast/ and https://www.ebi.ac.uk/Tools/psa/emboss_needle/ using NCBI BLAST+ (EMBL-EBI, NCBI BLAST+ - Sequence Similarity Search, UniProt Knowledgebase, BLOSUM62, Gap open 11, Gap extend 1), and EMBOSS Needle (EMBL-EBI, EMBOSS-Needle - Pairwise Sequence Alignment, BLOSUM62, Gap open 10, Gap extend 0.5) for determining the degree of identity. Searches were also performed with https://usgene.sequencebase.com/ using Matrix PAM30, Gap open 9 and Gap extend 1.
    Organism and accession number Identity (%)
    Man8
    Verticillium longisporum, A0A0G4LAG4_9PEZI (1 69.0
    Nectria haematococca, BBX16347 (2 67.7
    Man9
    Fusarium langsethiae, A0A0M9ES31_9HYPO (1 92.2
    Fusarium graminearum, CEF88482.1 (2 91.1
    Man10
    Coniochaeta ligniaria, A0A1J7I5A7_9PEZI (1 69.6
    Thielavia australiensis, BBN39719 (2 67.0
    Man11
    Verticillium longisporum, A0A0G4MTC5_9PEZI (1 99.8
    Nectria haematococca, BBX16347 (2 64.7
    Man13
    Aspergillus udagawae, AOAOK8LCG6_9EURO (1 90.7
    Neosartorya fischeri, BBX16429 (2 87.8
    Man29
    Verticillium dahliae, G2XBE5_VERDV (1 79.4
    Aspergillus terreus, BBW96507 (2 66.9
    Man30
    Aspergillus calidoustus, A0A0U5GVM4_9EURO (1 77.4
    Malbranchea cinnamomea, BBN39104 (2 88.6
    (1 Search performed using NCBI BLAST+ (EMBL-EBI, NCBI BLAST+ - Sequence Similarity Search, UniProt Knowledgebase, BLOSUM62, Gap open 11, Gap extend 1), and EMBOSS Needle (EMBL-EBI, EMBOSS-Needle - Pairwise Sequence Alignment, BLOSUM62, Gap open 10, Gap extend 0.5) for determining the degree of identity.
    (2 Search performed with SequenceBase database https://usgene.sequencebase.com/
  • Example 4: Production of recombinant fungal mannanase proteins in Trichoderma reesei
  • Expression plasmids were constructed for production of recombinant mannanase Man1, Man2, Man3, Man5, Man8, Man9, Man10, Man11, Man12, Man13, Man17, Man27, Man29 and Man30 from Malbranchea cinnamomea ALKO4122, Verticillium dahliae RF8261, Melanocarpus albomyces ALKO4237, Acremonium alcalophilum, Fusarium equiseti RF6318, Acremonium thermophilum ALKO4245, Verticillium dahliae RF8261, Aspergillus nishimurae RH3949, Plectosphaerella cucumerina and Gymnascella citrina (See Examples 2, 3), respectively in Trichoderma reesei. The expression plasmids constructed are listed in Table 10. The recombinant mannanase genes , including their own signal sequences, were fused to the T. reesei cel7A/cbh1 promoter. The transcription termination was ensured by the T. reesei cel7A/cbh1 terminator and the A. nidulans amdS marker gene was used for selection of the transformants as described in Paloheimo et al. (2003). The linear expression cassettes (Fig. 1) were isolated from the vector backbones after NotI or EcoRI digestions and were transformed into T. reesei protoplasts. The host strains used, do not produce any of the four major T. reesei cellulases (CBHI, CBHII, EGI, EGII). The transformations were performed as in Penttilä et al. (1987) with the modifications described in Karhunen et al. (1993), selecting acetamidase as a sole nitrogen source (amdS marker gene). The transformants were purified on selection plates through single conidia prior to sporulating them on PD. Table 10. The expression cassettes constructed to produce Man1, Man2, Man3, Man5, Man8, Man9, Man10, Man11, Man12, Man13, Man17, Man27, Man29 and Man30 recombinant proteins from Malbranchea cinnamomea ALKO4122, Verticillium dahliae RF8261, Melanocarpus albomyces ALKO4237, Acremonium alcalophilum, Fusarium equiseti RF6318, Acremonium thermophilum ALKO4245,, Aspergillus nishimurae RH3949, Plectosphaerella cucumerina and Gymnascella citrina in Trichoderma reesei. The overall structure of the expression cassettes was as described in Fig. 1.
    Mannanase protein Expression plasmid Expression cassette (a
    Man1 pALK4073 7.0 kb NotI
    Man3 pALK4071 6.7 kb NotI
    Man5 pALK4081 6.8 kb NotI
    Man8 pALK4363 6.8 kb NotI
    Man9 pALK4091 6.7 kb NotI
    Man10 pALK4084 6.6 kb NotI
    Man11 pALK4090 6.8 kb NotI
    Man12 pALK4271 6.6 kb NotI
    Man13 pALK4413 6.5 kb NotI
    Man17 pALK4080 7.1 kb EcoRI
    Man27 pALK4270 6.7 kb NotI
    Man29 pALK4411 6.6 NotI
    Man30 pALK4412 6.6 NotI
    (a The expression cassette for T. reesei transformation was isolated from vector backbone by using NotI or EcoRI digestions.
  • The mannanase production of the transformants was analyzed from the culture supernatants of the shake flask cultivations. The transformants were inoculated from the PD slants to shake flasks containing 50 ml of complex lactose-based cellulase inducing medium (Joutsjoki at al. 1993) buffered with 5% KH2PO4. The mannanase protein production of the transformants was analyzed from culture supernatants after growing them for 7 days at 30 °C, 250 rpm. Heterologous production of recombinant proteins was analyzed by SDS-PAGE with subsequent Coomassie staining. The best producing transformants were chosen to be cultivated in laboratory scale bioreactors. The transformants were cultivated in bioreactors either on batch or by additional feeding type of process under protein inducing conditions at a typical mesophilic fungal cultivation temperature and slightly acidic conditions. The cultivation was continued until depletion of the medium sugars or until suitable yield was reached. The supernatants were recovered for application tests by centrifugation or by filtration.
  • EXAMPLE 5: Purification of Man1 mannanase
  • Cells and solids were removed from the fermentation culture medium by centrifugation for 10 min, 4000 g at 4°C. The supernatant of 10 ml was used for protein purification. The sample was filtered through 0.44 µm PVDF membrane (Millex-HV, Merck Millipore Ltd,Carrigtwohill, IRL). The filtrate was loaded onto a HiPrep 26/10 Desalting column (GE Healthcare, Uppsala, Sweden) equilibrated in 20 mM Tris pH 7. The desalted sample was then loaded onto a 5 ml HiTrap Q HP column (GE Healthcare, Uppsala, Sweden) pre-equilibrated with 20 mM Tris pH 7. After sample loading, the column was washed with the same buffer for 20 ml. Proteins were eluted with linear salt gradient 20 mM Tris, 500 mM NaCl pH 7 in 15 CVs. Fractions of 5 ml were collected and analyzed on SDS-PAGE. The fractions containing target protein were combined and concentrated to 2 ml using Vivaspin 20, 10 kDa MWCO ultrafiltration devices (GE Healthcare). The concentrated sample was further fractionated using Superdex 75 26/60 gel-filtration column equilibrated with 20 mM MES, 200 mM NaCl pH 6,5. Fractions of 2 ml were collected and analyzed by SDS-PAGE. Fractions containing pure mannanase were combined. Enzyme content of the purified sample was determined using UV absorbance 280 nm measurements. Excitation coefficients for each mannanases were calculated on the bases of amino acid sequence of the enzyme by using ExPASy Server http://web.expasy.org/protparam/. (Gasteiger E. et al. 2005). Other mannanases were purified using the same protocol but changing the buffer composition in desalting and ion exchange steps. Buffer compositions are shown in Table 11. Table 11.
    Mannanase Buffers used in ion exchange chromatography
    Man1 20 mM Tris pH 7
    Man3 20 mM MES pH 6
    Man5 20 mM Hepes pH 7
    Man8 20 mM MES pH 6
    Man9 20 mM MES pH 6,5
    Man10 20 mM MES pH 6,5
    Man11 20 mM MES pH 6
    Man12 20 mM MES pH 6
    Man13 20 mM MES pH 6,5
    Purified samples were above 95% pure.
  • pH profiles of mannanases
  • The pH profiles of purified mannanases were determined using the beta-mannazyme tablet assay Azurine-crosslinked carob galactomannan (T-MNZ 11/14) from Megazyme with minor modifications to suggested protocol. The linearity of the assay was checked with each purified enzymes. The assay was performed in 40 mM Britton-Robinson buffer adjusted to pH values between 4 and 11. The enzyme solution was diluted into the assay buffer and 500µl of enzyme solution was equilibrated at 50°C water bath for 5 min before adding one substrate tablet. After 10 minutes, the reaction was stopped by adding 10 ml 2% Tris pH 12. The reaction tubes were left at room temperature for 5 min, stirred and the liquid filtered through a Whatman No.1 paper filter. Release of blue dye from the substrate was quantified by measuring the absorbance at 595 nm. Enzyme activity at each pH was reported as relative activity where the activity at the pH optimum was set to 100%. The pH profiles were shown in Figures 4a and 4b. Relative activity (%) of mannanase is calculated by dividing mannanase activity of a sample by the mannanase activity of a reference sample. In the case of pH profile, the reference sample is a sample at the optimal pH. In the case of temperature profile the reference sample is a sample at the optimal temperature.
  • Temperature profiles of mannanases
  • The temperature optimum of purifed mannanases was determined using the beta-mannazyme tablet assay Azurine-crosslinked carob galactomannan (T-MNZ 11/14) from Megazyme with minor modifications to suggested protocol. The assay was performed at temperatures varying between 30-90°C for 10 minutes in 40 mM Britton-Robinson buffer pH7. Enzyme activity was reported as relative activity where the activity at temperature optimum was set to 100%. The temperature profiles were shown in Figures 2-3. Molecule weight determined in SDS PAGE gel, temperature and pH optimum of enzymes are shown in Table 12. Table 12
    MW SDS PAGE pH optimum Temperature optimum
    Man1 40-60 kDa 5-7 50°C - 60°C
    Man3 40-60 kDa 5-8 40°C - 60°C
    Man5 40-60 kDa 5-8 50°C - 70°C
    Man8 50-70 kDa 6-9 40°C - 60°C
    Man9 50-70 kDa 5-9 40°C - 60°C
    Man10 45-60 kDa 5-8 50°C - 70°C
    Man11 50-90 kDa 5-8 50°C - 70°C
    Man12 30-40 kDa 4-8 50°C - 70°C
  • EXAMPLE 6 Assay of galactomannanase activity by DNS -method
  • Mannanase activity (MNU) was measured as the release of reducing sugars from galactomannan (0.3 w/w-%) at 50°C and pH 7.0 using 5 min reaction time. The amount of released reducing carbohydrates was determined spectrophotometrically using dinitrosalicylic acid. Substrate (0,3 w/w-%) used in the assay was prepared as follows: 0.6 g of locust bean gum (Sigma G-0753) was blended in 50 mM sodium citrate buffer pH 7 (or citrate phosphate buffer pH 7) at about 80 °C using a heating magnetic stirrer and heated up to boiling point. The solution was cooled and let to dissolve overnight in a cold room (2 - 8 °C) with continuous stirring after which insoluble residues were removed by centrifugation. After that solution was filled up to 200 ml volume with the buffer. Substrate was stored as frozen and melted by heating in a boiling water bath to about 80 °C, cooled to room temperature and mixed carefully before use. DNS reagent used in the assay was prepared by dissolving 50 g of 3.5- dinitrosalisylic acid (Sigma D-550) in about 4 liter of water. With continuous magnetic stirring 80.0 g of NaOH was gradually added and let to dissolve. An amount of 1500 g of Rochelle Salt (K-Na-tartrate, Merck 8087) was added in small portions with continuous stirring. The solution that might have been cautiously warmed to a maximum temperature of 45 °C., was cooled to room temperature and filled up to 5000 ml. After that it was filtered through Whatman 1 filter paper and stored in a dark bottle at room temperature. The reaction was started by adding 1.8 ml of substrate solution to two test tubes and let to equilibrate at 50 °C for 5 minutes, after which 200 µl of suitably diluted enzyme solution was added to one of the tubes, mixed well with vortex mixer and incubated exactly for 5 min at 50 °C. Enzyme blanks didn't need to be equilibrated or incubated. The reaction was stopped by adding 3.0 ml of DNS reagent into both tubes and mixed. 200 µl of sample solution was added to the enzyme blank tubes. Both tubes were placed in a boiling water bath. After boiling for exactly 5 minutes, the tubes were placed in a cooling water bath and allowed to cool to room temperature. The absorbance of sample was measured against the enzyme blank at 540 nm and activity was read from the calibration curve and multiplied by the dilution factor. A suitable diluted sample yielded an absorbance difference of 0.15 - 0.4. Standard curve was prepared from 20 mM mannose stock solution by dissolving 360 mg of mannose (SigmaM-6020, stored in a desiccator) to 100 ml of assay buffer and diluted to solutions containing 3, 6, 10 and 14 µmol/ml of mannose. Standards were handled like the samples except for incubation at 50°C. The absorbances were measured against the reagent blank (containing buffer instead of mannose) at 540nm. A calibration curve was prepared for every series of assays. One mannanase unit (MNU) was defined as the amount of enzyme that produces reductive carbohydrates having a reductive power corresponding to one nmol of mannose from galactomannan in one second under the assay conditions (1 MNU = 1 nkat).
  • EXAMPLE 7 Stain removal performance of fungal mannanases with commercial detergents without bleaching agents
  • Cultivation samples of fungal mannanases (Man1, Man3, Man5, Man8, Man9, Man10, Man11, Man12,Man13, Man29 and Man30) produced in Trichoderma (as described in Example 4), were tested for their ability to remove mannanase sensitive standard stains at 40 °C and water hardness of 16°dH with commercial detergents without bleaching agents. The following artificially soiled test cloths from Center for testmaterial B.V. (the Netherlands) were used withMan1, Man3, Man5, Man8, Man9, Man10, Man11, Man12 and Man13 cultivation samples: Chocolate pudding mannanase sensitive on cotton (E-165), Locust bean gum, with pigment on cotton (C-S-73) and Guar gum with carbon black on cotton (C-S-43). With Man29 and Man30 cultivation samples stains E-165 and C-S-73 were used. The fabric was cut in 6 cm x 6 cm swatches and 2 pieces of each were used in test. Commercial heavy duty liquid detergent A containing all other enzymes except mannanase was used at concentration of 4.4 g per liter of wash liquor, commercial heavy duty liquid detergent B was used 5.0 g/l and commercial color detergent powder without enzymes was used at 3.8 g/l. Detergent containing wash liquors we prepared in synthetic tap water with hardness of 16 °dH. Protease Savinase® 16 L (0.5 w/w %) and amylase Stainzyme® 12 L (0.4 w/w %) was added into hard water used with commercial heavy duty liquid detergent B and commercial color detergent powder. The liquid detergent already contained amylase and protease. The pH of the wash liquor of Color detergent powder was approx. 10 and with the liquid detergent approx. 8.2 - 8.3. Mannanase dosages were in range 0 - 0.4 % of detergent weight, the amount depending on test and the strength of enzyme preparation. For the evaluation the dosages were calculated as enzyme activity units (MNU) per ml wash liquor. Activity was measured as described in Example 6. Control sample contained the detergent solution but no mannanase.
  • For synthetic tap water with hardness of 16 °dH the following stock solutions were prepared in deionized water (Milli-Q or equivalent):
    • Stock solution with 1000 °d Calcium-hardness: CaCl2 x 2 H2O (1.02382.1000, Merck KGaA, Germany) 26,22 g/l
    • Stock solution with 200 °d Magnesium-hardness: MgSO4 x 7 H2O (1.05886.1000, Merck KGaA, Germany) 8,79 g/l H2O
    • NaHCO3 stock solution: NaHCO3 (1.06329.1000 Merck KGaA, Germany) 29,6 g/l
    13,3 ml CaCl2 solution, 13,3 ml MgSO4 solution and 10,0 ml of freshly made NaHCO3 solution were added in volumetric flask in the given order, made up to 1 liter with deionized water and mixed. The hardness of water was determined by complexometric titration and found correct. Stain removal treatments were performed in Atlas LP-2 Launder-Ometer as follows. Launder-Ometer was first preheated to 40°C. Then detergent, 250 ml synthetic tap water with hardness of 16 °dH and diluted enzyme (<1.0 ml) were added into 1.2 liter containers. Stains were added and the Launder-Ometer was run at 40°C for 60 min with a rotation speed of 42 rpm. After that the swatches were carefully rinsed under running water and dried overnight at indoor air, on a grid protected against daylight. The stain removal effect was evaluated by measuring the colour as reflectance values with Konica Minolta CM-3610A spectrophotometer using L*a*b* color space coordinates (illuminant D65/10°, 420 nm cut). Fading of the stains, indicating mannanase performance (stain removal efficiency) was calculated as ΔL* (delta L*), which means lightness value L* of enzyme treated fabric minus lightness value L* of fabric treated with washing liquor without mannanase (control). Final results (total stain removal effect) were shown as sum of ΔL* of each stains. Color values of each stain were average of 2 swatches. Mannanase cultivation samples (Man1, Man3, Man5, Man8, Man9, Man10, Man11, Man12, Man13) were tested with 3 different stains in commercial liquid detergent A (Figs. 6-8). Man29 and Man30 cultivation samples were tested with 2 different stains in commercial liquid detergent B (Fig 9). All the mannanases according to the invention increased considerably the stain removal efficiency in liquid detergent. The results obtained with commercial color detergent powder (Figs 10 -11) show that mannanases Man1, Man3, Man5, Man8, Man9, Man11 and Man12 are performing well also in commercial detergent powder. EXAMPLE 8 Stain removal performance of fungal mannanases with bleach containing detergent
  • Fungal mannanases Man1, Man3 and Man5 produced in Trichoderma (as described in Example 4) were tested for their ability to remove mannanase sensitive standard stains at 40 °C and water hardness of 16°dH with commercial bleach detergent powder. Test system was similar to that described in Example 7, except commercial bleach detergent powder was used at concentration of 4,2 g per liter of wash liquor and pH of the wash liquor was approx. 9.5. Protease Savinase® 16 L (0.5 w/w %) and amylase Stainzyme® 12 L (0.4 w/w %) were added into hard water used in test, since the detergent didn't contain any enzymes. The color of the swatches after treatment was measured and results calculated as sum of ΔL* of each 3 stains as described in Example 7. The results obtained with commercial bleach containing detergent (Fig. 12) show that mannanases Man1, Man3 and Man5 increased the stain removal efficiency considerably. In previous tests also cultivations samples of Man8, Man9 and Man11 and Man12 were performing with bleach detergent (data not shown).
  • EXAMPLE 9 Stability of Man1 mannanase in commercial liquid detergent
  • The stability of Man1 preparation produced in Trichoderma (as described in Example 4) was tested in OMO Color liquid obtained from local super market and compared to commercial mannanase preparation Mannaway® 4,0 L. Mannanase preparations were added 0.5 w/w-% in detergents and samples were incubated in plastic tubes with caps at 37°C for 5 or 8 weeks. The activity was measured at certain intervals by activity assay described in Example 6 except using 30 min incubation time. The results in Omo Color (Fig. 13) show that Man1 had considerably better stability compared to Mannaway® 4.0 L. The results of the stability experiments show that the mannanase according to the invention was stable in detergents for several weeks even when stored at high temperature like 37°C. The stability of the mannanase according to the invention (Man1) in liquid detergent is considerably improved compared to a commercial bacterial mannanase.
  • Example 10: Wash performance of liquid detergent compositions according to the invention
  • The wash performance of liquid detergent compositions according to present invention was determined by using standardized stains obtainable from CFT (Center for Testmaterials) B.V., Vlaardingen, Netherlands ("CFT"), Eidgenössische Material- und Prüfanstalt Testmaterialien AG [Federal materials and testing agency, Testmaterials], St. Gallen, Switzerland ("EMPA"), Warwick Equest Ltd Unit 55, Consett Business Park, Consett, County Durham ("Equest").
  • A liquid washing agent with the following composition was used as base formulation (all values in weight percent):
    Chemical name Active substance raw material [%] Active substance detergent formulation [%]
    Water demin. 100 Rest
    Alkyl benzene sulfonic acid 96 2-7
    Anionic surfactants 70 6-10
    C12-C18 Fatty acid sodium salt 30 1-4
    Nonionic surfactants 100 4-7
    Phosphonates 40 0,1-2
    Citric acid 100 1-3
    NaOH 50 1-4
    Boronic acid 100 0,1-2
    Antifoaming agent 100 0,01-1
    Glycerol 100 1-3
    Enzymes 100 0,1-2
    Preserving agent 100 0,05-1
    Ethanol 93 0,5-2
    Optical brightener 90 0,01-1
    Perfume 100 0,1-1
    Dye 100 0,001-0,1
  • The pH of the detergent composition was between 8,2-8,6.
  • Based on this base formulation, liquid detergent compositions 1 - 3 were prepared by adding respective enzymes as indicated below:
    • Composition 1: Enzyme according to SEQ ID NO: 3 (Man 1)
    • Composition 2: Enzyme according to SEQ ID NO: 9 (Man 3)
    • Composition 3: Enzyme according to SEQ ID NO: 12 (Man 5)
  • The wash was performed as follows according to the AISE Method: 3,5 kg Clean ballast cloth, 4 SBL Cloths, Miele washing machine, 20°C and 40°C Short program. All mannanases were added in the same amounts based on total protein content.
  • The dosing ratio of the liquid washing agent was 4.0 grams per liter of washing liquor. The washing procedure was performed for 60 minutes at a temperature of 20°C and 40°C, the water having a water hardness between 15.5 and 16.5° (German degrees of hardness).
  • The results obtained are the difference values between the remission units obtained with the detergents and the remission units obtained with the detergent containing the commercially available reference mannanase (Mannaway 4.0L, obtained from Novozymes). A positive value therefore indicates an improved wash performance of the detergent compositions comprising the mannanases of present invention compared to the same detergent composition comprising the reference mannanase. Within the washing test a large range of stains were tested.
  • The whiteness, i.e. the brightening of the stains, was determined photometrically as an indication of wash performance. A Minolta CM508d spectrometer device was used, which was calibrated beforehand using a white standard provided with the unit.
  • The results obtained are the difference values between the remission units obtained with the detergents and the remission units obtained with the detergent containing the enzyme combinations. A positive value therefore indicates an improved wash performance due to the enzyme combinations present in the detergent. Mannanases of the present invention in detergent compositions show improved performance on a variety of mannan comprising stains. Table 13
    Stain Man1 Man3 Man5
    Chocolate creme (EMPA 160 [CO]) n.d. 1,5 1,5
    Starch/Pigment (WFK 10R [CO]) n.d. 0,6 1,3
    Black currant juice (CFT CS12 [CO]) 1,0 0,9 2,5
    Groundnutoil, pigments, high milk (CFT C10 [CO]) n.d. 0,9 1,0
    Blood (aged) (CFT CS01 [CO]) n.d. 1,5 1,1
    Chocolate Icecream L [CO] (EQUEST) n.d. n.d. 1,2
    Cocoa [CO] (EQUEST) 0,9 1,3 n.d.
  • Example 11: Wash performance of powder detergent compositions according to the invention
  • The wash performance of powder detergent compositions according to present invention was determined by using standardized stains obtainable from CFT (Center for Testmaterials) B.V., Vlaardingen, Netherlands ("CFT"), Eidgenössische Material- und Prüfanstalt Testmaterialien AG [Federal materials and testing agency, Testmaterials], St. Gallen, Switzerland ("EMPA"), Warwick Equest Ltd Unit 55, Consett Business Park, Consett, County Durham ("Equest").
  • A solid washing agent with the following composition was used as base formulation (all values in weight percent):
    Chemical Name Active substance raw material [%] Active substance detergent formulation [%]
    Water demin. 100 1-4
    Alkyl benzene sulfonic acid 97 9-13
    Nonionic surfactants 100 4-7
    Percarbonates 88 9-13
    TAED 92 1-5
    Phosphonates 60 0,1-3
    Polyacrylates 45 1-4
    Soduim silicate 40 5-10
    Sodium carbonate 100 18-22
    Carboxymethylcellulose 69 1-4
    Soil release polymer 100 0,1-1
    Optical brightener 70 0,1-1
    Antifoaming agent t.q. 0,01-1
    Sodium sulfate 100 22-30
    Enzymes 100 0,1-1
    Perfume 100 0,1-1
    NaOH 100 0,1-1
    Rest - 1-4
  • Based on this base formulation, solid detergent compositions 4 and 5 were prepared by adding respective enzymes as indicated below:
    • Composition 4: Enzyme according to SEQ ID NO: 3 (Man1)
    • Composition 5: Enzyme according to SEQ ID NO: 12 (Man5)
  • The wash was performed as follows according to the AISE Method: 3,5 kg Clean ballast cloth, 4 SBL Cloths, Miele washing machine, 20°C and 40°C Short program. All mannanases were added in the same amounts based on total protein content.
  • The dosing ratio of the powder washing agent was 3.8 grams per liter of washing liquor. The washing procedure was performed for 60 minutes at a temperature of 20°C and 40°C, the water having a water hardness between 15.5 and 16.5° (German degrees of hardness). The whiteness, i.e. the brightening of the stains, was determined photometrically as an indication of wash performance. A Minolta CM508d spectrometer device was used, which was calibrated beforehand using a white standard provided with the unit.
  • The results obtained are the difference values between the remission units obtained with the detergents and the remission units obtained with the detergent containing the reference mannanase (Mannaway 4.0L, obtained from Novozymes). A positive value therefore indicates an improved wash performance of the mannanases in the detergent. Mannanases of the invention show improved performance on several stains in table 14. Therefore, it is evident from table 14 that mannanases according to the invention show improved wash performance. Table 14
    Stain Man1 Man5
    Milk/Carbon black (H-MR-B [CO]) n.d. 1,4
    Pudding (EMPA 165 [CO]) 0,6 1,6
    Porridge (EMPA 163 [CO]) n.d. 0,5
    Sebum BEY with carbon black (CFT CS32 [CO]) n.d. 0,7
    Lipstick, diluted, Red (CFT CS216 [CO]) n.d. 0,9
    Chocolate drink, pure (CFT CS44 [CO]) n.d. 1,4
    Groundnutoil, pigments, high milk (CFT C10 [CO]) 0,6 1,6
    Salad dressing, with natural black (CFT CS06 [CO]) n.d. 0,5
  • Without limiting the scope and interpretation of the patent claims, certain technical effects of one or more of the aspects or embodiments disclosed herein are listed in the following: A technical effect is degradation or modification of mannan. Another technical effect is provision of mannanase which has good storage stability.
  • The foregoing description has provided by way of non-limiting examples of particular implementations and embodiments of the invention a full and informative description of the best mode presently contemplated by the inventors for carrying out the invention. It is however clear to a person skilled in the art that the invention is not restricted to details of the embodiments presented above, but that it can be implemented in other embodiments using equivalent means without deviating from the characteristics of the invention.
  • Furthermore, some of the features of the above-disclosed aspects and embodiments of this invention may be used to advantage without the corresponding use of other features. As such, the foregoing description should be considered as merely illustrative of the principles of the present invention, and not in limitation thereof. Hence, the scope of the invention is only restricted by the appended patent claims.
  • In an embodiment at least one component of the compositions of the invention has a different chemical, structural or physical characteristic compared to the corresponding natural component from which the at least one component is derived from. In an embodiment said characteristic is at least one of uniform size, homogeneous dispersion, different isoform, different codon degeneracy, different post-translational modification, different methylation, different tertiary or quaternary structure, different enzyme activity, different affinity, different binding activity, and different immunogenicity
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  • Claims (12)

    1. Detergent composition comprising at least one enzyme having an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO.3 (Man1) and/or at least 80% sequence identity to the amino acid sequence of SEQ ID NO. 9 (Man 3) and/or at least 80% sequence identity to the amino acid sequence of SEQ ID NO. 12 (Man 5).
    2. Detergent composition of claim 1, wherein the at least one enzyme has an amino acid sequence having 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 the amino acid sequence of SEQ ID NO.3.
    3. Detergent composition of claim 1, wherein the at least one enzyme has an amino acid sequence having 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 the amino acid sequence of SEQ ID NO.9.
    4. Detergent composition of claim 1, wherein the at least one enzyme has an amino acid sequence having 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 the amino acid sequence of SEQ ID NO.12.
    5. Detergent composition of any one of claims 1-4, wherein the at least one enzyme has mannan degrading activity.
    6. The detergent composition of any one of claims 1-5, the composition further comprising one or more additional enzymes selected from the group consisting of protease, lipase, cutinase, amylase, carbohydrase, cellulase, pectinase, pectatlyase, mannanase, arabinase, galactanase, xylanase, oxidase, xanthanase, laccase, and/or peroxidase.
    7. The detergent composition according to any one of claims 1-6, wherein the 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.
    8. The detergent composition of any one of claims 1-7, wherein the detergent composition is a laundry detergent composition, preferably a liquid or solid laundry detergent composition.
    9. Use of a detergent composition of any of claims 1-8 for degrading mannan.
    10. Use of a detergent composition according to any one of claims 1-8 in a laundry process.
    11. A method for removing a stain from a surface, comprising contacting the surface with a detergent composition according to any one of claims 1 - 8.
    12. A method for degrading mannan comprising applying a detergent composition according to any of claims 1 to 8 to a stain comprising mannan, preferably wherein the stain is on the surface of a textile.
    EP17164904.9A 2017-04-05 2017-04-05 Detergent compositions comprising fungal mannanases Withdrawn EP3385362A1 (en)

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    Citations (127)

    * Cited by examiner, † Cited by third party
    Publication number Priority date Publication date Assignee Title
    GB1296839A (en) 1969-05-29 1972-11-22
    GB1483591A (en) 1973-07-23 1977-08-24 Novo Industri As Process for coating water soluble or water dispersible particles by means of the fluid bed technique
    US4106991A (en) 1976-07-07 1978-08-15 Novo Industri A/S Enzyme granulate composition and process for forming enzyme granulates
    US4435307A (en) 1980-04-30 1984-03-06 Novo Industri A/S Detergent cellulase
    EP0218272A1 (en) 1985-08-09 1987-04-15 Gist-Brocades N.V. Novel lipolytic enzymes and their use in detergent compositions
    US4661452A (en) 1984-05-29 1987-04-28 Novo Industri A/S Enzyme containing granulates useful as detergent additives
    EP0238216A1 (en) 1986-02-20 1987-09-23 Novo Nordisk A/S Protected enzyme systems
    EP0258068A2 (en) 1986-08-29 1988-03-02 Novo Nordisk A/S Enzymatic detergent additive
    EP0305216A1 (en) 1987-08-28 1989-03-01 Novo Nordisk A/S Recombinant Humicola lipase and process for the production of recombinant humicola lipases
    WO1989006279A1 (en) 1988-01-07 1989-07-13 Novo-Nordisk A/S Mutated subtilisin genes
    WO1989006270A1 (en) 1988-01-07 1989-07-13 Novo-Nordisk A/S Enzymatic detergent
    EP0331376A2 (en) 1988-02-28 1989-09-06 Amano Pharmaceutical Co., Ltd. Recombinant DNA, bacterium of the genus pseudomonas containing it, and process for preparing lipase by using it
    WO1989009259A1 (en) 1988-03-24 1989-10-05 Novo-Nordisk A/S A cellulase preparation
    EP0407225A1 (en) 1989-07-07 1991-01-09 Unilever Plc Enzymes and enzymatic detergent compositions
    WO1992005249A1 (en) 1990-09-13 1992-04-02 Novo Nordisk A/S Lipase variants
    EP0495257A1 (en) 1991-01-16 1992-07-22 THE PROCTER &amp; GAMBLE COMPANY Compact detergent compositions with high activity cellulase
    WO1992017577A1 (en) 1991-04-03 1992-10-15 Novo Nordisk A/S Novel proteases
    WO1992019709A1 (en) 1991-04-30 1992-11-12 The Procter & Gamble Company Built liquid detergents with boric-polyol complex to inhibit proteolytic enzyme
    WO1992019708A1 (en) 1991-04-30 1992-11-12 The Procter & Gamble Company Liquid detergents with aromatic borate ester to inhibit proteolytic enzyme
    WO1992019729A1 (en) 1991-05-01 1992-11-12 Novo Nordisk A/S Stabilized enzymes and detergent compositions
    WO1992021760A1 (en) 1991-05-29 1992-12-10 Cognis, Inc. Mutant proteolytic enzymes from bacillus
    EP0531315A1 (en) 1990-05-09 1993-03-17 Novo Nordisk As An enzyme capable of degrading cellulose or hemicellulose.
    EP0531372A1 (en) 1990-05-09 1993-03-17 Novo Nordisk As A cellulase preparation comprising an endoglucanase enzyme.
    WO1993018140A1 (en) 1992-03-04 1993-09-16 Novo Nordisk A/S Novel proteases
    WO1993024618A1 (en) 1992-06-01 1993-12-09 Novo Nordisk A/S Peroxidase variants with improved hydrogen peroxide stability
    WO1994001541A1 (en) 1992-07-06 1994-01-20 Novo Nordisk A/S C. antarctica lipase and lipase variants
    WO1994002597A1 (en) 1992-07-23 1994-02-03 Novo Nordisk A/S MUTANT α-AMYLASE, DETERGENT, DISH WASHING AGENT, AND LIQUEFACTION AGENT
    WO1994007998A1 (en) 1992-10-06 1994-04-14 Novo Nordisk A/S Cellulase variants
    WO1994018314A1 (en) 1993-02-11 1994-08-18 Genencor International, Inc. Oxidatively stable alpha-amylase
    US5352604A (en) 1989-08-25 1994-10-04 Henkel Research Corporation Alkaline proteolytic enzyme and method of production
    WO1994025578A1 (en) 1993-04-27 1994-11-10 Gist-Brocades N.V. New lipase variants for use in detergent applications
    WO1994025583A1 (en) 1993-05-05 1994-11-10 Novo Nordisk A/S A recombinant trypsin-like protease
    EP0624154A1 (en) 1991-12-13 1994-11-17 THE PROCTER &amp; GAMBLE COMPANY Acylated citrate esters as peracid precursors
    US5389536A (en) 1986-11-19 1995-02-14 Genencor, Inc. Lipase from Pseudomonas mendocina having cutinase activity
    WO1995006720A1 (en) 1993-08-30 1995-03-09 Showa Denko K.K. Novel lipase, microorganism producing the lipase, process for producing the lipase, and use of the lipase
    WO1995010602A1 (en) 1993-10-13 1995-04-20 Novo Nordisk A/S H2o2-stable peroxidase variants
    WO1995010603A1 (en) 1993-10-08 1995-04-20 Novo Nordisk A/S Amylase variants
    WO1995014783A1 (en) 1993-11-24 1995-06-01 Showa Denko K.K. Lipase gene and variant lipase
    WO1995022615A1 (en) 1994-02-22 1995-08-24 Novo Nordisk A/S A method of preparing a variant of a lipolytic enzyme
    WO1995023221A1 (en) 1994-02-24 1995-08-31 Cognis, Inc. Improved enzymes and detergents containing them
    WO1995024471A1 (en) 1994-03-08 1995-09-14 Novo Nordisk A/S Novel alkaline cellulases
    WO1995030744A2 (en) 1994-05-04 1995-11-16 Genencor International Inc. Lipases with improved surfactant resistance
    WO1995035381A1 (en) 1994-06-20 1995-12-28 Unilever N.V. Modified pseudomonas lipases and their use
    WO1996000292A1 (en) 1994-06-23 1996-01-04 Unilever N.V. Modified pseudomonas lipases and their use
    WO1996011262A1 (en) 1994-10-06 1996-04-18 Novo Nordisk A/S An enzyme and enzyme preparation with endoglucanase activity
    WO1996012012A1 (en) 1994-10-14 1996-04-25 Solvay S.A. Lipase, microorganism producing same, method for preparing said lipase and uses thereof
    WO1996013580A1 (en) 1994-10-26 1996-05-09 Novo Nordisk A/S An enzyme with lipolytic activity
    WO1996023873A1 (en) 1995-02-03 1996-08-08 Novo Nordisk A/S Amylase variants
    WO1996027002A1 (en) 1995-02-27 1996-09-06 Novo Nordisk A/S Novel lipase gene and process for the production of lipase with the use of the same
    WO1996029397A1 (en) 1995-03-17 1996-09-26 Novo Nordisk A/S Novel endoglucanases
    WO1996034946A1 (en) 1995-05-05 1996-11-07 Novo Nordisk A/S Protease variants and compositions
    WO1997004079A1 (en) 1995-07-14 1997-02-06 Novo Nordisk A/S A modified enzyme with lipolytic activity
    WO1997007202A1 (en) 1995-08-11 1997-02-27 Novo Nordisk A/S Novel lipolytic enzymes
    US5648263A (en) 1988-03-24 1997-07-15 Novo Nordisk A/S Methods for reducing the harshness of a cotton-containing fabric
    WO1997043424A1 (en) 1996-05-14 1997-11-20 Genencor International, Inc. MODIFIED α-AMYLASES HAVING ALTERED CALCIUM BINDING PROPERTIES
    WO1998008940A1 (en) 1996-08-26 1998-03-05 Novo Nordisk A/S A novel endoglucanase
    WO1998012307A1 (en) 1996-09-17 1998-03-26 Novo Nordisk A/S Cellulase variants
    WO1998015257A1 (en) 1996-10-08 1998-04-16 Novo Nordisk A/S Diaminobenzoic acid derivatives as dye precursors
    WO1998017767A1 (en) 1996-10-18 1998-04-30 The Procter & Gamble Company Detergent compositions
    WO1998020115A1 (en) 1996-11-04 1998-05-14 Novo Nordisk A/S Subtilase variants and compositions
    WO1998020116A1 (en) 1996-11-04 1998-05-14 Novo Nordisk A/S Subtilase variants and compositions
    WO1999011768A1 (en) 1997-08-29 1999-03-11 Novo Nordisk A/S Protease variants and compositions
    WO1999019467A1 (en) 1997-10-13 1999-04-22 Novo Nordisk A/S α-AMYLASE MUTANTS
    US5977053A (en) 1995-07-31 1999-11-02 Bayer Ag Detergents and cleaners containing iminodisuccinates
    WO2000034450A1 (en) 1998-12-04 2000-06-15 Novozymes A/S Cutinase variants
    WO2000060063A1 (en) 1999-03-31 2000-10-12 Novozymes A/S Lipase variant
    WO2001016285A2 (en) 1999-08-31 2001-03-08 Novozymes A/S Novel proteases and variants thereof
    WO2001044452A1 (en) 1999-12-15 2001-06-21 Novozymes A/S Subtilase variants having an improved wash performance on egg stains
    WO2001066712A2 (en) 2000-03-08 2001-09-13 Novozymes A/S Variants with altered properties
    WO2001092502A1 (en) 2000-06-02 2001-12-06 Novozymes A/S Cutinase variants
    WO2002010355A2 (en) 2000-08-01 2002-02-07 Novozymes A/S Alpha-amylase mutants with altered stability
    WO2002016547A2 (en) 2000-08-21 2002-02-28 Novozymes A/S Subtilase enzymes
    WO2002026024A1 (en) 2000-08-05 2002-04-04 Haiquan Li An apparatus using recyclable resource
    WO2002099091A2 (en) 2001-06-06 2002-12-12 Novozymes A/S Endo-beta-1,4-glucanase from bacillus
    WO2003006602A2 (en) 2001-07-12 2003-01-23 Novozymes A/S Subtilase variants
    WO2003040279A1 (en) 2001-11-09 2003-05-15 Unilever Plc Polymers for laundry applications
    WO2004003186A2 (en) 2002-06-26 2004-01-08 Novozymes A/S Subtilases and subtilase variants having altered immunogenicity
    WO2004041979A2 (en) 2002-11-06 2004-05-21 Novozymes A/S Subtilase variants
    WO2005003276A1 (en) 2003-06-18 2005-01-13 Unilever Plc Laundry treatment compositions
    WO2005003275A1 (en) 2003-06-18 2005-01-13 Unilever Plc Laundry treatment compositions
    WO2005003274A1 (en) 2003-06-18 2005-01-13 Unilever Plc Laundry treatment compositions
    WO2005040372A1 (en) 2003-10-23 2005-05-06 Novozymes A/S Protease with improved stability in detergents
    WO2005052161A2 (en) 2003-11-19 2005-06-09 Genencor International, Inc. Serine proteases, nucleic acids encoding serine enzymes and vectors and host cells incorporating same
    WO2005056782A2 (en) 2003-12-03 2005-06-23 Genencor International, Inc. Perhydrolase
    WO2006066594A2 (en) 2004-12-23 2006-06-29 Novozymes A/S Alpha-amylase variants
    WO2006108856A2 (en) 2005-04-15 2006-10-19 Basf Aktiengesellschaft Amphiphilic water-soluble alkoxylated polyalkylenimines with an internal polyethylene oxide block and an external polypropylene oxide block
    WO2006113314A1 (en) 2005-04-15 2006-10-26 The Procter & Gamble Company Liquid laundry detergent compositions with modified polyethyleneimine polymers and lipase enzyme
    WO2006130575A2 (en) 2005-05-31 2006-12-07 The Procter & Gamble Company Polymer-containing detergent compositions and their use
    WO2007006305A1 (en) 2005-07-08 2007-01-18 Novozymes A/S Subtilase variants
    WO2007044993A2 (en) 2005-10-12 2007-04-19 Genencor International, Inc. Use and production of storage-stable neutral metalloprotease
    WO2007087508A2 (en) 2006-01-23 2007-08-02 Novozymes A/S Lipase variants
    WO2007087242A2 (en) 2006-01-23 2007-08-02 The Procter & Gamble Company A composition comprising a lipase and a bleach catalyst
    WO2007087244A2 (en) 2006-01-23 2007-08-02 The Procter & Gamble Company Detergent compositions
    WO2007087257A2 (en) 2006-01-23 2007-08-02 The Procter & Gamble Company Enzyme and fabric hueing agent containing compositions
    WO2007087258A2 (en) 2006-01-23 2007-08-02 The Procter & Gamble Company A composition comprising a lipase and a bleach catalyst
    WO2007087243A2 (en) 2006-01-23 2007-08-02 The Procter & Gamble Company Detergent compositions
    WO2007087259A2 (en) 2006-01-23 2007-08-02 The Procter & Gamble Company Enzyme and photobleach containing compositions
    US7262042B2 (en) 2001-12-20 2007-08-28 Henkel Kommanditgesellschaft Auf Aktien (Henkel Kgaa) Alkaline protease from Bacillus gibsonii (DSM 14393) and washing and cleaning products comprising said alkaline protease
    WO2007138054A1 (en) 2006-05-31 2007-12-06 The Procter & Gamble Company Cleaning compositions with amphiphilic graft polymers based on polyalkylene oxides and vinyl esters
    EP1867808A1 (en) 2006-06-06 2007-12-19 Brose Schliesssysteme GmbH &amp; Co. KG Motor vehicle lock
    EP1876226A1 (en) 2006-07-07 2008-01-09 The Procter and Gamble Company Detergent compositions
    WO2008153815A2 (en) 2007-05-30 2008-12-18 Danisco Us, Inc., Genencor Division Variants of an alpha-amylase with improved production levels in fermentation processes
    US20090011970A1 (en) 2007-07-02 2009-01-08 Marc Francois Theophile Evers Laundry multi-compartment pouch composition
    WO2009021867A2 (en) 2007-08-10 2009-02-19 Henkel Ag & Co. Kgaa Agents containing proteases
    WO2009061380A2 (en) 2007-11-05 2009-05-14 Danisco Us Inc., Genencor Division VARIANTS OF BACILLUS sup. TS-23 ALPHA-AMYLASE WITH ALTERED PROPERTIES
    WO2009067279A1 (en) 2007-11-21 2009-05-28 E.I. Du Pont De Nemours And Company Production of peracids using an enzyme having perhydrolysis activity
    WO2009087523A2 (en) 2008-01-04 2009-07-16 The Procter & Gamble Company A laundry detergent composition comprising glycosyl hydrolase
    WO2009102854A1 (en) 2008-02-15 2009-08-20 The Procter & Gamble Company Cleaning compositions
    WO2009109500A1 (en) 2008-02-29 2009-09-11 Novozymes A/S Polypeptides having lipase activity and polynucleotides encoding same
    WO2010065455A2 (en) 2008-12-01 2010-06-10 Danisco Us Inc. Enzymes with lipase activity
    WO2010100028A2 (en) 2009-03-06 2010-09-10 Huntsman Advanced Materials (Switzerland) Gmbh Enzymatic textile bleach-whitening methods
    WO2010107560A2 (en) 2009-03-18 2010-09-23 Danisco Us Inc. Fungal cutinase from magnaporthe grisea
    WO2010111143A2 (en) 2009-03-23 2010-09-30 Danisco Us Inc. Cal a-related acyltransferases and methods of use, thereof
    WO2011036264A1 (en) 2009-09-25 2011-03-31 Novozymes A/S Use of protease variants
    WO2011036263A1 (en) 2009-09-25 2011-03-31 Novozymes A/S Subtilase variants
    WO2011084417A1 (en) 2009-12-21 2011-07-14 Danisco Us Inc. Detergent compositions containing geobacillus stearothermophilus lipase and methods of use thereof
    WO2011084412A1 (en) 2009-12-21 2011-07-14 Danisco Us Inc. Detergent compositions containing thermobifida fusca lipase and methods of use thereof
    WO2011084599A1 (en) 2009-12-21 2011-07-14 Danisco Us Inc. Detergent compositions containing bacillus subtilis lipase and methods of use thereof
    WO2011098531A1 (en) 2010-02-10 2011-08-18 Novozymes A/S Variants and compositions comprising variants with high stability in presence of a chelating agent
    WO2011150157A2 (en) 2010-05-28 2011-12-01 Danisco Us Inc. Detergent compositions containing streptomyces griseus lipase and methods of use thereof
    WO2012137147A1 (en) 2011-04-08 2012-10-11 Danisco Us, Inc. Compositions
    WO2013001078A1 (en) 2011-06-30 2013-01-03 Novozymes A/S Alpha-amylase variants
    WO2013001087A2 (en) 2011-06-30 2013-01-03 Novozymes A/S Method for screening alpha-amylases
    WO2013181760A1 (en) * 2012-06-08 2013-12-12 Concordia University Novel cell wall deconstruction enzymes of scytalidium thermophilum, myriococcum thermophilum, and aureobasidium pullulans, and uses thereof
    WO2014138983A1 (en) * 2013-03-14 2014-09-18 Concordia University Novel cell wall deconstruction enzymes of malbranchea cinnamomea, thielavia australiensis, and paecilomyces byssochlamydoides, and uses thereof
    WO2015109405A1 (en) * 2014-01-22 2015-07-30 Concordia University Novel cell wall deconstruction enzymes of chaetomium thermophilum, thermomyces stellatus, and corynascus sepedonium, and uses thereof
    WO2016090473A1 (en) * 2014-12-09 2016-06-16 Concordia University Novel cell wall deconstruction enzymes of rhizomucor miehei, thermoascus thermophilus (dactylomyces thermophilus), and humicola hyalo thermophila, and uses thereof

    Patent Citations (135)

    * Cited by examiner, † Cited by third party
    Publication number Priority date Publication date Assignee Title
    GB1296839A (en) 1969-05-29 1972-11-22
    GB1483591A (en) 1973-07-23 1977-08-24 Novo Industri As Process for coating water soluble or water dispersible particles by means of the fluid bed technique
    US4106991A (en) 1976-07-07 1978-08-15 Novo Industri A/S Enzyme granulate composition and process for forming enzyme granulates
    US4435307A (en) 1980-04-30 1984-03-06 Novo Industri A/S Detergent cellulase
    US4661452A (en) 1984-05-29 1987-04-28 Novo Industri A/S Enzyme containing granulates useful as detergent additives
    EP0218272A1 (en) 1985-08-09 1987-04-15 Gist-Brocades N.V. Novel lipolytic enzymes and their use in detergent compositions
    EP0238216A1 (en) 1986-02-20 1987-09-23 Novo Nordisk A/S Protected enzyme systems
    EP0258068A2 (en) 1986-08-29 1988-03-02 Novo Nordisk A/S Enzymatic detergent additive
    US5389536A (en) 1986-11-19 1995-02-14 Genencor, Inc. Lipase from Pseudomonas mendocina having cutinase activity
    EP0305216A1 (en) 1987-08-28 1989-03-01 Novo Nordisk A/S Recombinant Humicola lipase and process for the production of recombinant humicola lipases
    WO1989006279A1 (en) 1988-01-07 1989-07-13 Novo-Nordisk A/S Mutated subtilisin genes
    WO1989006270A1 (en) 1988-01-07 1989-07-13 Novo-Nordisk A/S Enzymatic detergent
    EP0331376A2 (en) 1988-02-28 1989-09-06 Amano Pharmaceutical Co., Ltd. Recombinant DNA, bacterium of the genus pseudomonas containing it, and process for preparing lipase by using it
    US5691178A (en) 1988-03-22 1997-11-25 Novo Nordisk A/S Fungal cellulase composition containing alkaline CMC-endoglucanase and essentially no cellobiohydrolase
    WO1989009259A1 (en) 1988-03-24 1989-10-05 Novo-Nordisk A/S A cellulase preparation
    US5648263A (en) 1988-03-24 1997-07-15 Novo Nordisk A/S Methods for reducing the harshness of a cotton-containing fabric
    US5776757A (en) 1988-03-24 1998-07-07 Novo Nordisk A/S Fungal cellulase composition containing alkaline CMC-endoglucanase and essentially no cellobiohydrolase and method of making thereof
    EP0407225A1 (en) 1989-07-07 1991-01-09 Unilever Plc Enzymes and enzymatic detergent compositions
    US5352604A (en) 1989-08-25 1994-10-04 Henkel Research Corporation Alkaline proteolytic enzyme and method of production
    US5686593A (en) 1990-05-09 1997-11-11 Novo Nordisk A/S Enzyme capable of degrading cellulose or hemicellulose
    EP0531315A1 (en) 1990-05-09 1993-03-17 Novo Nordisk As An enzyme capable of degrading cellulose or hemicellulose.
    EP0531372A1 (en) 1990-05-09 1993-03-17 Novo Nordisk As A cellulase preparation comprising an endoglucanase enzyme.
    US5457046A (en) 1990-05-09 1995-10-10 Novo Nordisk A/S Enzyme capable of degrading cellullose or hemicellulose
    US5763254A (en) 1990-05-09 1998-06-09 Novo Nordisk A/S Enzyme capable of degrading cellulose or hemicellulose
    WO1992005249A1 (en) 1990-09-13 1992-04-02 Novo Nordisk A/S Lipase variants
    EP0495257A1 (en) 1991-01-16 1992-07-22 THE PROCTER &amp; GAMBLE COMPANY Compact detergent compositions with high activity cellulase
    WO1992017577A1 (en) 1991-04-03 1992-10-15 Novo Nordisk A/S Novel proteases
    WO1992019708A1 (en) 1991-04-30 1992-11-12 The Procter & Gamble Company Liquid detergents with aromatic borate ester to inhibit proteolytic enzyme
    WO1992019709A1 (en) 1991-04-30 1992-11-12 The Procter & Gamble Company Built liquid detergents with boric-polyol complex to inhibit proteolytic enzyme
    WO1992019729A1 (en) 1991-05-01 1992-11-12 Novo Nordisk A/S Stabilized enzymes and detergent compositions
    WO1992021760A1 (en) 1991-05-29 1992-12-10 Cognis, Inc. Mutant proteolytic enzymes from bacillus
    EP0624154A1 (en) 1991-12-13 1994-11-17 THE PROCTER &amp; GAMBLE COMPANY Acylated citrate esters as peracid precursors
    WO1993018140A1 (en) 1992-03-04 1993-09-16 Novo Nordisk A/S Novel proteases
    WO1993024618A1 (en) 1992-06-01 1993-12-09 Novo Nordisk A/S Peroxidase variants with improved hydrogen peroxide stability
    WO1994001541A1 (en) 1992-07-06 1994-01-20 Novo Nordisk A/S C. antarctica lipase and lipase variants
    WO1994002597A1 (en) 1992-07-23 1994-02-03 Novo Nordisk A/S MUTANT α-AMYLASE, DETERGENT, DISH WASHING AGENT, AND LIQUEFACTION AGENT
    WO1994007998A1 (en) 1992-10-06 1994-04-14 Novo Nordisk A/S Cellulase variants
    WO1994018314A1 (en) 1993-02-11 1994-08-18 Genencor International, Inc. Oxidatively stable alpha-amylase
    WO1994025578A1 (en) 1993-04-27 1994-11-10 Gist-Brocades N.V. New lipase variants for use in detergent applications
    WO1994025583A1 (en) 1993-05-05 1994-11-10 Novo Nordisk A/S A recombinant trypsin-like protease
    WO1995006720A1 (en) 1993-08-30 1995-03-09 Showa Denko K.K. Novel lipase, microorganism producing the lipase, process for producing the lipase, and use of the lipase
    WO1995010603A1 (en) 1993-10-08 1995-04-20 Novo Nordisk A/S Amylase variants
    WO1995010602A1 (en) 1993-10-13 1995-04-20 Novo Nordisk A/S H2o2-stable peroxidase variants
    WO1995014783A1 (en) 1993-11-24 1995-06-01 Showa Denko K.K. Lipase gene and variant lipase
    WO1995022615A1 (en) 1994-02-22 1995-08-24 Novo Nordisk A/S A method of preparing a variant of a lipolytic enzyme
    EP1921147A2 (en) 1994-02-24 2008-05-14 Henkel Kommanditgesellschaft auf Aktien Improved enzymes and detergents containing them
    WO1995023221A1 (en) 1994-02-24 1995-08-31 Cognis, Inc. Improved enzymes and detergents containing them
    EP1921148A2 (en) 1994-02-24 2008-05-14 Henkel Kommanditgesellschaft auf Aktien Improved enzymes and detergents containing them
    WO1995024471A1 (en) 1994-03-08 1995-09-14 Novo Nordisk A/S Novel alkaline cellulases
    WO1995030744A2 (en) 1994-05-04 1995-11-16 Genencor International Inc. Lipases with improved surfactant resistance
    WO1995035381A1 (en) 1994-06-20 1995-12-28 Unilever N.V. Modified pseudomonas lipases and their use
    WO1996000292A1 (en) 1994-06-23 1996-01-04 Unilever N.V. Modified pseudomonas lipases and their use
    WO1996011262A1 (en) 1994-10-06 1996-04-18 Novo Nordisk A/S An enzyme and enzyme preparation with endoglucanase activity
    WO1996012012A1 (en) 1994-10-14 1996-04-25 Solvay S.A. Lipase, microorganism producing same, method for preparing said lipase and uses thereof
    WO1996013580A1 (en) 1994-10-26 1996-05-09 Novo Nordisk A/S An enzyme with lipolytic activity
    WO1996023873A1 (en) 1995-02-03 1996-08-08 Novo Nordisk A/S Amylase variants
    WO1996027002A1 (en) 1995-02-27 1996-09-06 Novo Nordisk A/S Novel lipase gene and process for the production of lipase with the use of the same
    WO1996029397A1 (en) 1995-03-17 1996-09-26 Novo Nordisk A/S Novel endoglucanases
    WO1996034946A1 (en) 1995-05-05 1996-11-07 Novo Nordisk A/S Protease variants and compositions
    WO1997004079A1 (en) 1995-07-14 1997-02-06 Novo Nordisk A/S A modified enzyme with lipolytic activity
    US5977053A (en) 1995-07-31 1999-11-02 Bayer Ag Detergents and cleaners containing iminodisuccinates
    WO1997007202A1 (en) 1995-08-11 1997-02-27 Novo Nordisk A/S Novel lipolytic enzymes
    WO1997043424A1 (en) 1996-05-14 1997-11-20 Genencor International, Inc. MODIFIED α-AMYLASES HAVING ALTERED CALCIUM BINDING PROPERTIES
    WO1998008940A1 (en) 1996-08-26 1998-03-05 Novo Nordisk A/S A novel endoglucanase
    WO1998012307A1 (en) 1996-09-17 1998-03-26 Novo Nordisk A/S Cellulase variants
    WO1998015257A1 (en) 1996-10-08 1998-04-16 Novo Nordisk A/S Diaminobenzoic acid derivatives as dye precursors
    WO1998017767A1 (en) 1996-10-18 1998-04-30 The Procter & Gamble Company Detergent compositions
    WO1998020115A1 (en) 1996-11-04 1998-05-14 Novo Nordisk A/S Subtilase variants and compositions
    WO1998020116A1 (en) 1996-11-04 1998-05-14 Novo Nordisk A/S Subtilase variants and compositions
    WO1999011768A1 (en) 1997-08-29 1999-03-11 Novo Nordisk A/S Protease variants and compositions
    WO1999019467A1 (en) 1997-10-13 1999-04-22 Novo Nordisk A/S α-AMYLASE MUTANTS
    WO2000034450A1 (en) 1998-12-04 2000-06-15 Novozymes A/S Cutinase variants
    WO2000060063A1 (en) 1999-03-31 2000-10-12 Novozymes A/S Lipase variant
    WO2001016285A2 (en) 1999-08-31 2001-03-08 Novozymes A/S Novel proteases and variants thereof
    WO2001044452A1 (en) 1999-12-15 2001-06-21 Novozymes A/S Subtilase variants having an improved wash performance on egg stains
    WO2001066712A2 (en) 2000-03-08 2001-09-13 Novozymes A/S Variants with altered properties
    WO2001092502A1 (en) 2000-06-02 2001-12-06 Novozymes A/S Cutinase variants
    WO2002010355A2 (en) 2000-08-01 2002-02-07 Novozymes A/S Alpha-amylase mutants with altered stability
    WO2002026024A1 (en) 2000-08-05 2002-04-04 Haiquan Li An apparatus using recyclable resource
    WO2002016547A2 (en) 2000-08-21 2002-02-28 Novozymes A/S Subtilase enzymes
    WO2002099091A2 (en) 2001-06-06 2002-12-12 Novozymes A/S Endo-beta-1,4-glucanase from bacillus
    WO2003006602A2 (en) 2001-07-12 2003-01-23 Novozymes A/S Subtilase variants
    WO2003040279A1 (en) 2001-11-09 2003-05-15 Unilever Plc Polymers for laundry applications
    US7262042B2 (en) 2001-12-20 2007-08-28 Henkel Kommanditgesellschaft Auf Aktien (Henkel Kgaa) Alkaline protease from Bacillus gibsonii (DSM 14393) and washing and cleaning products comprising said alkaline protease
    WO2004003186A2 (en) 2002-06-26 2004-01-08 Novozymes A/S Subtilases and subtilase variants having altered immunogenicity
    WO2004041979A2 (en) 2002-11-06 2004-05-21 Novozymes A/S Subtilase variants
    WO2005003276A1 (en) 2003-06-18 2005-01-13 Unilever Plc Laundry treatment compositions
    WO2005003274A1 (en) 2003-06-18 2005-01-13 Unilever Plc Laundry treatment compositions
    WO2005003275A1 (en) 2003-06-18 2005-01-13 Unilever Plc Laundry treatment compositions
    WO2005040372A1 (en) 2003-10-23 2005-05-06 Novozymes A/S Protease with improved stability in detergents
    WO2005052161A2 (en) 2003-11-19 2005-06-09 Genencor International, Inc. Serine proteases, nucleic acids encoding serine enzymes and vectors and host cells incorporating same
    WO2005052146A2 (en) 2003-11-19 2005-06-09 Genencor International, Inc. Serine proteases, nucleic acids encoding serine enzymes and vectors and host cells incorporating same
    WO2005056782A2 (en) 2003-12-03 2005-06-23 Genencor International, Inc. Perhydrolase
    WO2006066594A2 (en) 2004-12-23 2006-06-29 Novozymes A/S Alpha-amylase variants
    WO2006113314A1 (en) 2005-04-15 2006-10-26 The Procter & Gamble Company Liquid laundry detergent compositions with modified polyethyleneimine polymers and lipase enzyme
    WO2006108856A2 (en) 2005-04-15 2006-10-19 Basf Aktiengesellschaft Amphiphilic water-soluble alkoxylated polyalkylenimines with an internal polyethylene oxide block and an external polypropylene oxide block
    WO2006130575A2 (en) 2005-05-31 2006-12-07 The Procter & Gamble Company Polymer-containing detergent compositions and their use
    WO2007006305A1 (en) 2005-07-08 2007-01-18 Novozymes A/S Subtilase variants
    WO2007044993A2 (en) 2005-10-12 2007-04-19 Genencor International, Inc. Use and production of storage-stable neutral metalloprotease
    WO2007087242A2 (en) 2006-01-23 2007-08-02 The Procter & Gamble Company A composition comprising a lipase and a bleach catalyst
    WO2007087258A2 (en) 2006-01-23 2007-08-02 The Procter & Gamble Company A composition comprising a lipase and a bleach catalyst
    WO2007087243A2 (en) 2006-01-23 2007-08-02 The Procter & Gamble Company Detergent compositions
    WO2007087259A2 (en) 2006-01-23 2007-08-02 The Procter & Gamble Company Enzyme and photobleach containing compositions
    WO2007087244A2 (en) 2006-01-23 2007-08-02 The Procter & Gamble Company Detergent compositions
    WO2007087508A2 (en) 2006-01-23 2007-08-02 Novozymes A/S Lipase variants
    WO2007087257A2 (en) 2006-01-23 2007-08-02 The Procter & Gamble Company Enzyme and fabric hueing agent containing compositions
    WO2007138054A1 (en) 2006-05-31 2007-12-06 The Procter & Gamble Company Cleaning compositions with amphiphilic graft polymers based on polyalkylene oxides and vinyl esters
    EP1867808A1 (en) 2006-06-06 2007-12-19 Brose Schliesssysteme GmbH &amp; Co. KG Motor vehicle lock
    EP1876226A1 (en) 2006-07-07 2008-01-09 The Procter and Gamble Company Detergent compositions
    WO2008153815A2 (en) 2007-05-30 2008-12-18 Danisco Us, Inc., Genencor Division Variants of an alpha-amylase with improved production levels in fermentation processes
    US20090011970A1 (en) 2007-07-02 2009-01-08 Marc Francois Theophile Evers Laundry multi-compartment pouch composition
    WO2009021867A2 (en) 2007-08-10 2009-02-19 Henkel Ag & Co. Kgaa Agents containing proteases
    WO2009061380A2 (en) 2007-11-05 2009-05-14 Danisco Us Inc., Genencor Division VARIANTS OF BACILLUS sup. TS-23 ALPHA-AMYLASE WITH ALTERED PROPERTIES
    WO2009067279A1 (en) 2007-11-21 2009-05-28 E.I. Du Pont De Nemours And Company Production of peracids using an enzyme having perhydrolysis activity
    WO2009087523A2 (en) 2008-01-04 2009-07-16 The Procter & Gamble Company A laundry detergent composition comprising glycosyl hydrolase
    WO2009102854A1 (en) 2008-02-15 2009-08-20 The Procter & Gamble Company Cleaning compositions
    WO2009109500A1 (en) 2008-02-29 2009-09-11 Novozymes A/S Polypeptides having lipase activity and polynucleotides encoding same
    WO2010065455A2 (en) 2008-12-01 2010-06-10 Danisco Us Inc. Enzymes with lipase activity
    WO2010100028A2 (en) 2009-03-06 2010-09-10 Huntsman Advanced Materials (Switzerland) Gmbh Enzymatic textile bleach-whitening methods
    WO2010107560A2 (en) 2009-03-18 2010-09-23 Danisco Us Inc. Fungal cutinase from magnaporthe grisea
    WO2010111143A2 (en) 2009-03-23 2010-09-30 Danisco Us Inc. Cal a-related acyltransferases and methods of use, thereof
    WO2011036264A1 (en) 2009-09-25 2011-03-31 Novozymes A/S Use of protease variants
    WO2011036263A1 (en) 2009-09-25 2011-03-31 Novozymes A/S Subtilase variants
    WO2011084417A1 (en) 2009-12-21 2011-07-14 Danisco Us Inc. Detergent compositions containing geobacillus stearothermophilus lipase and methods of use thereof
    WO2011084412A1 (en) 2009-12-21 2011-07-14 Danisco Us Inc. Detergent compositions containing thermobifida fusca lipase and methods of use thereof
    WO2011084599A1 (en) 2009-12-21 2011-07-14 Danisco Us Inc. Detergent compositions containing bacillus subtilis lipase and methods of use thereof
    WO2011098531A1 (en) 2010-02-10 2011-08-18 Novozymes A/S Variants and compositions comprising variants with high stability in presence of a chelating agent
    WO2011150157A2 (en) 2010-05-28 2011-12-01 Danisco Us Inc. Detergent compositions containing streptomyces griseus lipase and methods of use thereof
    WO2012137147A1 (en) 2011-04-08 2012-10-11 Danisco Us, Inc. Compositions
    WO2013001078A1 (en) 2011-06-30 2013-01-03 Novozymes A/S Alpha-amylase variants
    WO2013001087A2 (en) 2011-06-30 2013-01-03 Novozymes A/S Method for screening alpha-amylases
    WO2013181760A1 (en) * 2012-06-08 2013-12-12 Concordia University Novel cell wall deconstruction enzymes of scytalidium thermophilum, myriococcum thermophilum, and aureobasidium pullulans, and uses thereof
    WO2014138983A1 (en) * 2013-03-14 2014-09-18 Concordia University Novel cell wall deconstruction enzymes of malbranchea cinnamomea, thielavia australiensis, and paecilomyces byssochlamydoides, and uses thereof
    WO2015109405A1 (en) * 2014-01-22 2015-07-30 Concordia University Novel cell wall deconstruction enzymes of chaetomium thermophilum, thermomyces stellatus, and corynascus sepedonium, and uses thereof
    WO2016090473A1 (en) * 2014-12-09 2016-06-16 Concordia University Novel cell wall deconstruction enzymes of rhizomucor miehei, thermoascus thermophilus (dactylomyces thermophilus), and humicola hyalo thermophila, and uses thereof

    Non-Patent Citations (5)

    * Cited by examiner, † Cited by third party
    Title
    "Powdered Detergents, Surfactant science series", vol. 71, MARCEL DEKKER, INC, article "chapter 7"
    "Powdered Detergents, Surfactant science", vol. 71, MARCEL DEKKER, INC.
    DHAWAN SAMRITIL ET AL: "Microbial mannanases: An overview of production and applications", CRC CRITICAL REVIEWS IN BIOTECHNO, CRC PRESS, BOCA RATON, FL, US, vol. 27, no. 4, 1 January 2007 (2007-01-01), pages 197 - 216, XP009161462, ISSN: 0738-8551, DOI: 10.1080/07388550701775919 *
    SIEZEN ET AL., PROTEIN ENGNG., vol. 4, 1991, pages 719 - 737
    SIEZEN ET AL., PROTEIN SCIENCE, vol. 6, 1997, pages 501 - 523

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