CN110381973A - One or more enzymes are preventing, inhibiting or are reducing the purposes in microbial growth on surface - Google Patents

Abstract

The present invention relates to the use of one or more enzymes in preventing, inhibiting or reducing the growth of microorganisms on a surface, wherein the TTC detectable time of said microorganisms on said surface is increased by at least 20% after use of said enzymes, At least 30%, at least 50%, at least 70%, at least 80%, or at least 100%.

Description

The role of one or more enzymes in preventing, inhibiting or reducing the growth of microorganisms on a surface use

References to Sequence Listings

This application contains a Sequence Listing in computer readable form, which is hereby incorporated by reference.

technical field

The present invention relates to the novel use of one or more enzymes to prevent, inhibit or reduce the growth of microorganisms on a surface, wherein the TTC detectable time of said microorganisms on said surface is increased by at least 20% after application of said enzymes. The invention also relates to a method of preventing, inhibiting or reducing the growth of microorganisms on such surfaces by treating the surface with an enzyme to such an extent that after treatment the microorganisms have a TTC detectable time increase of at least 20% on said surface. The invention also relates to a method for demonstrating the inhibition of microbial growth of enzymes on surfaces.

Background technique

In the field of household cleaning, dryers are commonly used to dry clothes and kitchen surfaces such as dishes. However, machine dryers are not very environmentally friendly and are less available in some regions such as Asia. Air drying the surface is often used when machine drying is not possible. But humidity can cause problems during air drying. Specifically, not only does it take longer to dry, but it can also cause hygiene issues.

In humid environments, such as places with frequent precipitation, eg places with regular monsoon seasons or areas that are warm and humid year-round, microorganisms like fungi and bacteria grow rapidly on surfaces like textiles and kitchen utensils. Microbial growth is a problem even after surfaces have been treated through conventional cleaning processes such as laundry. In these areas, the problem of microbial growth on surfaces after washing can be particularly pronounced due to the humidity of the washed surface itself and the prolonged exposure to humid air during air drying. Microbial growth on surfaces can be seen as dark spots on the surface, and microbial growth can further cause unwanted and unpleasant malodor.

A number of enzymes are routinely incorporated into detergent compositions for cleaning benefits. In addition, it has been reported that some enzymes (including but not limited to amylases) can be used to prevent, remove or reduce biofilms on surfaces, see WO 2008112459 A2 (Novozymes, A/S), WO 2006/031554 ( Novozymes). In addition to this, it has been reported that enzymes such as oxidases or peroxidases can be used to inhibit microorganisms in laundry and the effect of polycationic polymers in wash liquors. US 6,287,585 B1, Novozymes.

However, there is no disclosure regarding the use of enzymes in preventing the growth of microorganisms that cause the above-mentioned spotting and malodour problems on household surfaces, especially in humid environments. It is therefore an object of the present invention to provide such use.

Contents of the invention

The present invention relates to the novel use of one or more enzymes to prevent, inhibit or reduce the growth of microorganisms on a surface, wherein the TTC detectable time of said microorganisms on said surface is increased by at least 20% after application of said enzymes.

Another aspect of the present invention relates to a method of preventing, inhibiting or reducing the growth of microorganisms on a surface, as compared to those microorganisms on a surface not treated with said enzyme, by treating the surface with an enzyme for a predetermined period of time until the enzyme The microorganisms have a TTC detectable time increase of at least 20% on said surface after treatment.

Another aspect of the invention relates to a method of demonstrating the microbial growth inhibition or deep cleaning benefit of an enzyme or combination of enzymes on a surface comprising the steps of:

a. Provide two surfaces A and A',

b. Applying a soil comprising an indicator of microbial growth to each surface A and A',

c. washing A with a detergent composition not comprising enzymes and washing A' with said detergent composition comprising one or more enzymes,

d. Prepare to inoculate microorganisms and apply them to surfaces A and A',

e. incubating A and A' under suitable conditions to allow microbial growth for a predetermined period of time,

f. Compare the growth of microorganisms on A and A'.

Description of drawings

Figure 1 relates to the detection of PrestoBlue fluorescence units of Candida Parapsilosis on two differently treated swatches.

Overview of Sequence Listing

SEQ ID NO: 1 is the amino acid sequence of protease

SEQ ID NO: 2 is the amino acid sequence of protease

SEQ ID NO: 3 is the amino acid sequence of protease

SEQ ID NO:4 is the amino acid sequence of protease

SEQ ID NO:5 is the amino acid sequence of protease

SEQ ID NO:6 is the amino acid sequence of protease

SEQ ID NO:7 is the amino acid sequence of protease

SEQ ID NO: 8 is the amino acid sequence of protease

SEQ ID NO:9 is the amino acid sequence of amylase

SEQ ID NO: 10 is the amino acid sequence of amylase

SEQ ID NO: 11 is the amino acid sequence of amylase (SEQ ID NO2 of WO 2000/060060)

SEQ ID NO: 12 is the amino acid sequence of amylase (SEQ ID NO 2 of WO 96/023873)

SEQ ID NO: 13 is the amino acid sequence of amylase (SEQ ID NO3 of WO 2008/112459)

SEQ ID NO: 14 is the amino acid sequence of subtilase (SEQ ID NO: 1 of WO 2004/067737)

SEQ ID NO: 15 is the amino acid sequence of cellulase

SEQ ID NO: 16 is the amino acid sequence of cellulase

SEQ ID NO: 17 is the amino acid sequence of cellulase

SEQ ID NO: 18 is the amino acid sequence of cellulase

definition

TTC Detectable Time : This term identifies the time when microorganisms on a surface become identifiable in situ, as indicated by the color change of triphenyltetrazolium chloride (TTC). TTC is a reduction-oxidation reaction indicator that can be used to distinguish metabolically active from inactive tissues/organisms. The color of TTC is white, and when it is enzymatically reduced by various dehydrogenases in living organisms to water-insoluble TPF (1,3,5-triphenylmethanol ), the color will change to red. Details of the in situ detection of microbial growth are disclosed in later paragraphs herein, in particular the "TTC assay (in situ detection of bacteria)" section in the Assays section.

Sporulation time: This term identifies the time when microorganisms on a surface, especially fungi, become identifiable in situ by microscopic observation. In addition to the TTC detectable time determination described above, this is an additional tool for monitoring microbial growth on surfaces. The time to sporulation method is preferred where the spores have a color that can mask the TTC color change. Details of this procedure are disclosed in later paragraphs herein, particularly the "Fungi SporeFormation Time Assay" section in the Assays section.

Spore Density Score: This term identifies fungal growth as indicated by visible markings on household surfaces caused by spores and/or mycelia of the fungus growing on said surface. On a scale of 0 to 7, 0 indicates no fungal growth on the surface and 7 indicates excessive fungal growth on the surface. The color of the spores can be different for different fungi, but the same 0-7 range can be applied to different fungal growth and spore density measurements. A group of trained panelists can be trained to understand the range and score each surface for spore density according to a preset program.

Detergent composition : The term "detergent composition" refers to a composition for removing undesired compounds from textiles (eg, textiles) to be cleaned. The detergent composition can be used, for example, for cleaning textiles, for both household cleaning and industrial cleaning. These terms encompass any material/compound selected for the particular type of detergent composition and product desired (e.g., liquid, gel, powder, granule, paste, or spray composition) and include, but are not limited to Detergent compositions (eg, liquid and/or solid laundry and delicates detergents; fabric refreshers; fabric softeners; and textile and laundry pre-stainers/pre-treatments). In addition to comprising the DNase of the present invention, the detergent formulation may also contain one or more additional enzymes (such as proteases, amylases, lipases, cutinases, cellulases, endoglucanases, xyloglucanase, pectinase, pectin lyase, xanthanase, peroxidase, haloperoxygenase, catalase and mannanase, or any mixture thereof), ingredients such as Surfactants, builders, chelators or chelating agents, bleach systems or bleach components, polymers, fabric conditioners, suds boosters, suds suppressors, dyes, fragrances, tarnish inhibitors agents, optical brighteners, bactericides, fungicides, soil suspending agents, corrosion inhibitors, enzyme inhibitors or stabilizers, enzyme activators, one or more transferases, hydrolases, oxidoreductases, Blue and fluorescent dyes, antioxidants and solubilizers.

Delta Reflectance (ΔRem): The term "delta reflectance" or "delta reflectance value" is defined herein as the result of a reflectance or reflectance measurement at a wavelength, typically 460 nm. The swatch is measured against a swatch of a similar color, preferably a swatch from repeated laundering, as a background. Swatches representative of each swatch type were measured prior to laundering. Delta reflectance is the reflectance value of the washed swatch minus the reflectance value of the unwashed swatch.

One way of measuring wash performance is the delta enzyme performance value (ΔRem enzyme value): the term "delta enzyme reflectance value" is defined herein as the result of a reflectance or reflectance measurement at 460 nm. The swatch is measured against a swatch of a similar color, preferably a swatch from repeated laundering, as a background. Swatches representative of each swatch type were measured prior to laundering. Delta Enzyme Reflection is the reflectance value of a swatch washed in detergent in the presence of enzyme minus the reflectance value of a similar swatch washed in detergent in the absence of enzyme.

Dishwashing refers to all forms of washing dishes, such as by hand washing or automatic dishwashing. Dishwashing includes but is not limited to cleaning all forms of tableware such as plates, cups, glasses, bowls, all forms of cutlery (e.g. spoons, knives, forks) and serving utensils as well as ceramics, plastics (e.g. melamine), metal , porcelain, glass and acrylic.

Dishwashing composition : The term "dishwashing composition" means a composition intended for cleaning dishes, tableware, pots, pans, eating utensils and all forms of compositions for cleaning hard surface areas in the kitchen. The present invention is not limited to any particular type of dishwashing composition or to any particular detergent.

Enzyme wash benefit : The term "enzyme wash benefit" is defined herein as the beneficial effect of adding an enzyme to a detergent compared to the same detergent without the enzyme. Important wash benefits that can be provided by enzymes are stain removal with no or very little visible soil after washing and/or cleaning, preventing or reducing redeposition of soil released during washing (also known as anti-redeposition effect ), fully or partially restore the whiteness (also known as the effect of bleaching) of textiles that are initially white but acquire a grayish or yellowish appearance after repeated use and washing. Textile care benefits not directly related to catalyzed stain removal or prevention of soil redeposition are also important for enzymatic wash benefits. Examples of such textile care benefits are the prevention or reduction of dye transfer from one fabric to another or another part of the same fabric (also known as dye transfer inhibition or anti-backstaining effects), removal of protruding or broken stains from the surface of fabrics. fibers to reduce the tendency to pill or remove existing pills or fuzz (also known as the anti-pilling effect), improve fabric softness, clarify the color of fabrics and remove particulate dirt trapped in the fibers of fabrics or garments. Enzymatic bleaching is another enzymatic wash benefit where catalytic activity is often used to catalyze the formation of bleaching components such as hydrogen peroxide or other peroxides.

Hard Surface Cleaning : The term "hard surface cleaning" is defined herein as cleaning hard surfaces, where hard surfaces can include floors, tables, walls, roofs, etc., along with surfaces of hard objects such as cars (car wash) and dishes (dishwash) . Dishwashing includes, but is not limited to, cleaning plates, cups, glasses, bowls, eating utensils (such as spoons, knives, forks), serving utensils, ceramics, plastics, metals, china, glass, and acrylics.

Laundry relates to both domestic and industrial laundry and means a process of treating textiles and/or fabrics with a solution containing a detergent composition. The laundry process can eg be performed using eg domestic or industrial washing machines or can be performed manually.

Peroxidase/Oxidase: A peroxidase according to the invention is an enzyme specified by the International Union of Biochemistry and Molecular Biology Nomenclature Committee (IUBMB) encompassed by enzyme classification EC 1.11.1.7, or derived from a presentation therein Any fragment that exhibits peroxidase activity.

Proteases are defined herein as enzymes that hydrolyze peptide bonds. It includes any enzyme belonging to the EC 3.4 enzyme group (including each of its 13 subclasses). EC numbers refer to 1992 Enzyme Nomenclature, NC-IUBMB, Academic Press, San Diego, California, including Supplements 1-5, respectively, published in: Eur. J. Biochem .[European Journal of Biochemistry] 1223:1-5(1994); Eur.J.Biochem.[European Journal of Biochemistry] 232:1-6(1995); Eur.J.Biochem.[European Journal of Biochemistry] 237 :1-5 (1996); Eur.J.Biochem. [European Journal of Biochemistry] 250:1-6 (1997); and Eur.J.Biochem. [European Journal of Biochemistry] 264:610-650 (1999) . The most widely used proteases in the detergent industry such as laundry and dishwashing are the serine proteases or serine peptidases, which are a subgroup of proteases characterized by having a serine in the active site that binds to the base form covalent adducts. In addition, subtilases (as well as serine proteases) are characterized by two active site amino acid residues, a histidine residue and an aspartic acid residue, in addition to serine. Subtilases refer to a subgroup of serine proteases according to Siezen et al., 1991, Protein Engng. 4:719-737 and Siezen et al., 1997, Protein Science 6:501-523. Subtilases can be divided into six subdivisions, namely, the subtilisin family, thermitase family, proteinase K family, lantibiotic peptidase family, Kexin family, and pyrolysin family. The term "protease activity" means proteolytic activity (EC 3.4). Proteases that can be used in detergents are mainly endopeptidases (EC 3.4.21). Several protease activity types exist: the three main activity types are: trypsin-like, where there is cleavage of the amide substrate after Arg or Lys at P1, chymotrypsin-like, where after one hydrophobic amino acid at P1 Cleavage occurs, like elastase, where cleavage occurs after Ala at P1.

Sequence identity: The degree of relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity". For the purposes of the present invention, use as described in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite [EMBOSS: European Molecular Biology Open Software Suite], Rice et al., 2000, Trends Genet. [Genetic Trends] 16:276 -277) (preferably version 5.0.0 or later) the Needleman-Wunsch algorithm implemented in the Needle program (Needleman and Wunsch, 1970, J.Mol.Biol.[ Journal of Molecular Biology] 48:443-453) to determine the sequence identity between two amino acid sequences. The parameters used were a gap opening penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. Use the output of Needle's "longest identity" flag (obtained using the non-simplified (-nobrief) option) as percent identity and calculate as follows: (identical residues x 100)/(length of alignment - length of alignment total number of slots). For the purposes of the present invention, use as in the EMBOSS package (EM-BOSS: The European Molecular Biology Open Software Suite [EM-BOSS: European Molecular Biology Open Software Suite], Rice et al., 2000, supra) (preferably 5.0.0 The Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) implemented in the Needle program (Needleman and Wunsch, 1970 version or later) to determine sequence identity between two deoxyribonucleotide sequences. The parameters used were gap opening penalty of 10, gap extension penalty of 0.5, and EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle's marked "longest identity" (obtained using the non-simplified (-nobrief) option) was used as percent identity and calculated as follows:

(identical deoxyribonucleotides x 100)/(alignment length - total number of gaps in the alignment).

Residual Stains : This term refers to stains that have not been completely removed from household surfaces after cleaning. There can be different levels of residual stains, and they can be ranked from best to worst in terms of surface cleanliness, with 1 being the best. Rating: 1 = complete removal, 2 = very good (acceptable), 3 = very good (borderline), 4 = poor (unacceptable), 5 = no removal (same as original). In some cases, visual symbols can be made to designate the level of residual stain. Here, in the context of the present invention, residual stains include stains ranked from 2 to 5. Stains in the context of the present invention can be from a variety of sources including, but not limited to, starch stains, protein stains, amylose stains, lipid-based stains; food stains; beverage stains, environmental stains, workplace stains, and the like.

Textiles : The term "textile" means any textile material including yarns, yarn intermediates, fibers, nonwoven materials, natural materials, synthetic materials, and any other textile material from which Fabrics and products made from fabrics (eg, clothing and other articles). The textile or fabric may be in the form of knits, wovens, denim, non-wovens, felts, yarns, and terry. The textile may be cellulose based, such as natural cellulose products including cotton, linen/linen, jute, ramie, sisal or coir, or artificial cellulose (e.g. derived from wood pulp) including viscose Fibers/Rayon, cellulose acetate (tricell), lyocell or blends thereof. Textiles or fabrics can also be non-cellulose based, such as natural polyamides, including wool, camel hair, cashmere, mohair, rabbit fur and silk, or synthetic polymers such as nylon, aramid, polyester, acrylic, polypropylene and Spandex/elastane, or blends thereof, and blends of cellulose-based and non-cellulose-based fibers. Examples of blends are blends of cotton and/or rayon/viscose fibers with one or more accompanying materials such as wool, synthetic fibers (e.g. polyamide fibers, acrylic fibers, polyester fibers, Polyvinyl chloride fibers, polyurethane fibers, polyurea fibers, aramid fibers) and/or cellulose-containing fibers (such as rayon/viscose, ramie, linen/linen, jute, cellulose acetate fibers, rayon Cell fiber). The fabric may be conventional washable laundry, such as soiled household laundry. When the term fabric or garment is used, it is intended to also include the broad term textile. In the context of the present invention, the term "textile" is used interchangeably with fabric and cloth.

Variant means a polypeptide having enzymatic activity, but comprising an alteration (ie, substitution, insertion and/or deletion) at one or more (eg, several) positions compared to a parent enzyme or a reference enzyme. Substitution means replacing an amino acid occupying a position with a different amino acid; deletion means removing an amino acid occupying a position; and insertion means adding one or more (e.g., A number of) amino acids (eg, 1, 2, 3, 4 or 5 amino acids).

Wash liquor: The term "wash liquor" is defined herein as a solution or mixture of water and detergent components, optionally including enzymes.

Malodor : With regard to the term "malodor" means an undesired odor. Examples of malodor include compounds with an unpleasant odor, which may be produced by microorganisms. These microorganisms can come from the human or animal body, the interior of the washing machine, or from the rest of the environment. Some examples of such unpleasant smelling compounds are hexanal (grassy), 3-octanone (musty and musty), 2,3-butanedione (skink), benzonitrile (almond-like smell), benzene (gasoline-like smell), or toluene (pungent, benzene-like smell).

Detailed ways

The present invention relates to the use of enzymes for preventing, inhibiting or reducing the growth of microorganisms on surfaces, eg textiles. Air-dried textiles are exposed to a variety of microorganisms that can thrive and colonize textiles. Microbial growth and surfaces can be seen as dark stains and/or foul odors such as mold, damp, or unpleasant odors.

In one aspect, the present invention relates to the use of one or more enzymes in preventing, inhibiting or reducing the growth of microorganisms on a surface, wherein the TTC detectable time of said microorganisms is increased by at least 20% after use of said enzymes on said surface %. The extended TTC detectable time shows that the use of enzymes on such surfaces can prevent, inhibit or reduce the growth of microorganisms.

In one aspect, the TTC detectable time is increased by at least 30%.

In one aspect, the TTC detectable time is increased by at least 40%.

In one aspect, the TTC detectable time is increased by at least 50%.

In one aspect, the TTC detectable time is increased by at least 70%.

In one aspect, the TTC detectable time is increased by at least 80%.

In one aspect, the TTC detectable time is increased by at least 100%.

When the microorganism is a fungus, the effect of using an enzyme on the surface for preventing the growth of the microorganism can be measured as an increase in sporulation time. In one aspect, the microorganism is a fungus, and following application of said enzyme on said surface, the time to sporulation of the fungus is increased by at least 20% compared to the time to sporulation on a surface treated with the enzyme. Prolonged sporulation times following application of the enzymes on these surfaces also indicated that the growth of microorganisms was prevented, inhibited or reduced.

In one aspect, the TTC detectable time is increased by at least 30%.

In one aspect, the sporulation time is increased by at least 40%.

In one aspect, the sporulation time is increased by at least 50%.

In one aspect, the sporulation time is increased by at least 70%.

In one aspect, the sporulation time is increased by at least 80%.

In one aspect, the sporulation time is increased by at least 100%.

When the microorganism is a fungus, the effect of using an enzyme on the surface for preventing the growth of the microorganism can be measured as a reduction in spore density. One aspect relates to the use of one or more enzymes for preventing the growth of microorganisms on a surface, wherein the microorganisms are fungi, and wherein the spore density is on a scale from 0 to 7 when compared to the spore density of a surface not treated with the enzyme Decrease the scale gauge to at least 1.

In another aspect, the reduction in spore density score is at least 2.

In another aspect, the reduction in spore density score is at least 3.

In another aspect, the reduction in spore density score is at least 4.

In another aspect, the reduction in spore density score is at least 5.

In another aspect, the reduction in spore density score is at least 6.

Residual stains often provide nutrients for microorganisms to survive and thrive on the surface. Without being bound by theory, it is believed that the enzymes work in part by removing or reducing residual stains on the surface.

One aspect of the invention relates to the use of one or more enzymes for preventing the growth of microorganisms on a surface, wherein the microorganisms grow on remaining stained areas of the surface.

In one aspect, the invention further relates to the use of one or more enzymes for inhibiting or reducing malodor caused by microorganisms on the surface of textiles.

In one aspect, the invention further relates to the use of one or more enzymes in preventing or reducing the attachment of microorganisms to textile surfaces. Certain cellulases are known to provide color care and whiteness improvement benefits to washed textiles. But it is not known whether cellulase can be used to prevent or reduce microorganisms. Without being bound by theory, it is believed that fluff and damaged fibers are more readily converted to amorphous, that cellulases remove fluff and damaged fibers that form on the surface of fabrics, and that some cellulases are very effective at hydrolyzing amorphous cellulose , and thereby these regions of the fiber are enzymatically removed. Removing these fluffballs and damaged fibers may help prevent or reduce the microbes that can cling to them, among other unknown factors.

Microorganisms targeted by use of enzymes in the present invention may be those that tend to multiply relatively rapidly on household surfaces in hot and humid environments. It can be fungus or bacteria. In one aspect, the microorganism is a fungus or bacterium selected from the group consisting of Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa ), Escherichia coli, Pseudomonas putida, Micrococcus luteus, Staphylococcus epidermidis, Enhydrobacteraerosaccus, Corynebacterium jeikeium , Propionibacterium acnes, Brevundimonas vesicularis, Streptomyces griseus, Streptomyces odorifer, Exophila phaeomuriformis, Fusarium oxysporum, Alternaria alternate, Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Aureobasidium pullulans, Glomus Chaetomium globosum, Cladosporium sphaerospermum, Gliocladium virens, Mucor plumbeus, Penicillium chrysogenum, Penicillium commune, skin Penicillium crustosum, Penicillium rubens, Penicillium variants, Stachybotrys chartarum, Trichoderma viride, Trichophyton rubrum, Candida parapsilosis, Rhodotorulamucilaginosa, Debaryomyces hansenii (Debaryomyces hansenii), Meyerozymaguilliermondii, Candida albicans, and combinations thereof.

In one aspect, the microorganism is a fungus, and preferably Aspergillus niger.

In one aspect, the microorganism is a bacterium, and preferably Escherichia coli or Pseudomonas putida.

Enzymes useful in the present invention may be selected from the group consisting of amylases, proteases, lipases, mannanases, cellulases, pectinases, and combinations thereof.

In one aspect, the enzyme is an amylase or a protease.

In one aspect, the enzyme is not an oxidase or a peroxidase.

In one aspect, the enzyme is an amylase, a protease, a cellulase, or a combination thereof.

In one aspect, the invention further relates to the use of one or more enzymes in preventing or reducing the attachment of microorganisms to textile surfaces.

Enzymes of the invention

The enzymes useful in the present invention may be added to the detergent composition in an amount corresponding to 0.001-200 mg of protein per liter of washing liquor, such as 0.005-100 mg of protein, preferably 0.01-50 mg of protein, more preferably Preferably 0.05-20 mg of protein, even more preferably 0.1-10 mg of protein.

One or more enzymes useful in the invention may be stabilized using conventional stabilizers such as polyols (such as propylene glycol or glycerol), sugars or sugar alcohols, lactic acid, boric acid, or boric acid derivatives (e.g., aromatic boronic acid esters, or phenylboronic acid derivatives (such as 4-formylphenylboronic acid)), and the composition can be formulated as described, for example, in WO 92/19709 and WO 92/19708.

protease

Suitable proteases include those of bacterial, fungal, plant, viral or animal origin, eg plant or microbial origin. Preference is given to microbial origin. Chemically modified mutants or protein engineered mutants are included. It may be an alkaline protease, such as a serine protease or a metalloprotease. A serine protease may eg be of the S1 family (eg trypsin) or the S8 family (eg subtilisin). The metalloprotease may for example be a thermolysin from eg the M4 family or other metalloproteases such as those from the M5, M7 or M8 family.

The term "subtilase" refers to a subgroup of serine proteases according to Siezen et al., Protein Engng. [Protein Engineering] 4 (1991) 719-737 and Siezen et al., Protein Science [Protein Science] 6 (1997) 501-523 . Serine proteases are a subgroup of proteases characterized by having a serine in the active site that forms a covalent adduct with a substrate. Subtilases can be divided into 6 subclasses, namely, subtilisin family, thermophilic protease family, proteinase K family, lanthionine antibiotic peptidase family, Kexin family and Pyrolysin family.

Examples of subtilases are those derived from the genus Bacillus, such as Bacillus lentus, Bacillus alkalophilus, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus pumilus and Gibrin as described in US 7262042 and WO 09/021867. Bacillus; and subtilisin lentus, subtilisin Novo, subtilisin Carlsberg, Bacillus licheniformis, subtilisin BPN', subtilisin 309, subtilisin 147 and subtilisin as described in WO 89/06279 168 and the protease PD138 described in (WO 93/18140). Other useful proteases may be those described in WO 92/175177, WO 01/016285, WO02/026024 and WO02/016547. Examples of trypsin-like proteases are trypsin (e.g. of porcine or bovine origin) and Fusarium protease (described in WO 89/06270, WO 94/25583 and WO 05/040372), and Cellulomonas ( Chymotrypsin of Cellumonas) (described in WO 05/052161 and WO 05/052146).

Further preferred proteases are alkaline proteases from Bacillus lentus DSM 5483 (as described, for example, in WO95/23221), and variants thereof (described in WO 92/21760, WO 95/23221, EP 1921147 and EP 1921148) .

Examples of metalloproteases are neutral metalloproteases such as those derived from Bacillus amyloliquefaciens as described in WO07/044993 (Genencor Int.). Suitable commercially available proteases include those sold under the following trade names: Duralase ^Tm , Durazym ^Tm , Ultra, Ultra, Ultra, Ultra, Blaze 100T, Blaze 125T, Blaze 150T, progress and (Novozymes), those sold under the following trade names: Purafect Purafect Excellenz P1000 ^TM , Excellenz P1250 ^TM , Preferenz P100 ^™ , Purafect Preferenz P110 ^TM , Effectenz P1000 ^TM , Effectenz P1050 ^™ , Purafect Effectenz P2000 ^™ , and (Danisco/DuPont), Axapem ^™ (Gist-Brocases NV), BLAP (sequence shown in Figure 29 of US 5352604) and variants thereof (Henkel AG) and KAP (Bacillus alkalophilus protease) from Kao.

In one aspect, the protease useful in the present invention is selected from the group consisting of:

a) a polypeptide having the amino acid sequence of any one of SEQ ID NO: 1-8, 14;

b) at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, Polypeptides with at least 97%, at least 98%, at least 99% sequence identity;

and combinations thereof.

In one aspect, the protease is a peptide having at least 60% sequence identity to SEQ ID NO: 1 or 2, 3, 4, 5, 6, 7, 8 or 14.

In one aspect, the protease is a peptide having at least 65% sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8 or 14.

In one aspect, the protease is a peptide having at least 70% sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8 or 14.

In one aspect, the protease is a peptide having at least 75% sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8 or 14.

In one aspect, the protease is a peptide having at least 80% sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8 or 14.

In one aspect, the protease is a peptide having at least 85% sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8 or 14.

In one aspect, the protease is a peptide having at least 90% sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8 or 14.

In one aspect, the protease is a peptide having at least 95% sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8 or 14.

In one aspect, the protease is a peptide having at least 96% sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8 or 14.

In one aspect, the protease is a peptide having at least 97% sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8 or 14.

In one aspect, the protease is a peptide having at least 98% sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8 or 14.

In one aspect, the protease is a peptide having at least 99% sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8 or 14.

In one aspect, the protease is a peptide selected from the group consisting of:

i) a protease comprising a substitution at one or more of the following positions compared to the protease shown in SEQ ID NO 1 or SEQ ID NO 2: 3, 4, 9, 15, 24, 27, 42, 55, 59, 60, 66, 74, 85, 96, 97, 98, 99, 100, 101, 102, 104, 116, 118, 121, 126, 127, 128, 154, 156, 157, 158, 161, 164, 176, 179, 182, 185, 188, 189, 193, 198, 199, 200, 203, 206, 211, 212, 216, 218, 226, 229, 230, 239, 246, 255, 256, 268, and 269, wherein the position corresponds to the position of the protease shown in SEQ ID NO 1, or

ii) one or more protease variants of the protease parent, wherein the protease variant comprises one or more mutations selected from the group consisting of: S3T, V4I, S9R, S9E, A15T, S24G, S24R, K27R, N42R, S55P, G59E, G59D, N60D, N60E, V66A, N74D, N85S, N85R, G96S, G96A, S97G, S97D, S97A, S97SD, S99E, S99D, S99G, S99M, S99N, S99R, S99H, S101A、V102I、V102Y、V102N、S104A、G116V、G116R、H118D、H118N、N120S、S126L、P127Q、S128A、S154D、A156E、G157D、G157P、S158E、Y161A、R164S、Q176E、N179E、S182E、Q185N、A188P、 G189E, V193M, N198D, V199I, Y203W, S206G, L211Q, L211D, N212D, N212S, M216S, A226V, K229L, Q230H, Q239R, N246K, N255W, N255D, N255E, L256E, L256A, R2 Position of the protease shown in SEQ ID NO 1, wherein the protease parent is selected from the protease shown in SEQ ID NO 1 and the Bacillus amylolichenifaciens (BPN') shown in SEQ ID NO 2 , and wherein the protease variant has at least 80% sequence identity to SEQ ID NO 1 or 80% sequence identity to SEQ ID NO 2,

iii) a protease comprising a substitution at one or more positions corresponding to positions 171, 173, 175, 179 or 180 of SEQ ID NO: 14 compared to the protease shown in SEQ ID NO 48, wherein the protease becomes The body has at least 75% but less than 100% sequence identity with SEQID NO 14,

iv) a protease comprising the amino acid sequence shown in SEQ ID NO 1 or 2, or a protease with a polypeptide comprising amino acids 1-269 of SEQ ID NO 1 or a polypeptide comprising amino acids 1-275 of SEQ ID NO 2 the polypeptides have at least 80% sequence identity,

v) one or more of the following protease variants selected from the group consisting of:

SEQ ID NO 1, with the changes T22R+S99G+S101A+V102I+A226V+Q239R,

SEQ ID NO 2, with the changes S24G+S53G+S78N+S101N+G128A+Y217Q,

SEQ ID NO 2, with the changes S24G+S53G+S78N+S101N+G128S+Y217Q,

SEQ ID NO 1 with alterations S9E+N42R+N74D+V199I+Q200L+Y203W+S253D+N255W+L256E,

SEQ ID NO 1, having S9E+N42R+N74D+H118V+Q176E+A188P+V199I+Q200L Y203W+S250D+S253D+N255W+L256E,

SEQ ID NO 1, having S9E+N42R+N74D+Q176E+A188P+V199I+Q200L+Y203W S250D+S253D+N255W+L256E,

SEQ ID NO 1 with S3V+N74D+H118V+Q176E+N179E+S182E+V199I+Q200L Y203W+S210V+S250D+S253D+N255W+L256E

SEQ ID NO 1 with the alteration T22A+N60D+S99G+S101A+V102I+N114L+G157D+S182D+T207A+A226V+Q239R+N242D+E265F,

SEQ ID NO 1, with alterations S9E+N42R+N74D+H118V+Q176E+A188P+V199I+Q200L+Y203W+S250D+S253D+N255W+L256E,

SEQ ID NO 1, with alterations S9E+N42R+N74D+Q176E+A188P+V199I+Q200L+Y203W+S250D+S253D+N255W+L256E,

SEQ ID NO 1, with the changes S9E+N42R+N74D+H118V+Q176E+A188P+V199I+Q200L+Y203W+S250D+N255W+L256E+*269aH+*269bH,

SEQ ID NO 1, with the changes S3V+N74D+H118V+Q176E+N179E+S182E+V199I+Q200L+Y203W+S210V+S250D+N255W+L256E,

SEQ ID NO 1 with the alterations S9E+N74D+G113W+G157P+Q176E+V199I+Q200L+Y203W+S250D+T254E+N255W+L256E,

SEQ ID NO 1, with changes S3V+S9R+N74D+H118V+Q176E+N179E+S182E+V199I+Q200L+Y203W+S212V+S250D+N255W+L256E,

SEQ ID NO 1, with alteration S99E, and

SEQ ID NO 2, with alteration L217D,

and combinations thereof.

Amylase:

Suitable amylases that may be used with the enzymes useful in the present invention may be alpha-amylases or glucoamylases and may be of bacterial or fungal origin. Chemically modified mutants or protein engineered mutants are included.

Amylases may include, for example, alpha-amylases obtained from Bacillus, such as certain strains of Bacillus licheniformis (described in more detail in GB 1,296,839).

Suitable amylases include amylases having SEQ ID NO:2 in WO 95/10603 or variants thereof having 90% sequence identity to SEQ ID NO:3. Preferred variants are described in WO 94/02597, WO 94/18314, WO 97/43424 and WO 99/019467 in SEQ ID NO: 4, 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.

Various suitable amylases include amylases having SEQ ID NO:6 in WO 02/010355 or variants thereof having 90% sequence identity to SEQ ID NO:6. Preferred variants of SEQ ID NO: 6 are those with deletions in positions 181 and 182 and substitutions in position 193.

Other suitable amylases are those comprising residues 1-33 of an alpha-amylase derived from Bacillus amyloliquefaciens shown in WO 2006/066594 in SEQ ID NO: 6 and SEQ ID NO in WO 2006/066594: A hybrid alpha-amylase of residues 36-483 of the Bacillus licheniformis alpha-amylase in 4 or a variant thereof having 90% sequence identity. Preferred variants of this hybrid alpha-amylase are those with substitutions, deletions or insertions in one or more of the following positions: G48, T49, G107, H156, A181, N190, M197, I201, A209 and Q264. Hybrid alpha-amylase comprising residues 1-33 of an alpha-amylase derived from Bacillus amyloliquefaciens and residues 36-483 of SEQ ID NO:4 shown in SEQ ID NO:6 of WO 2006/066594 The most preferred variants of enzymes are those with the following substitutions:

M197T;

H156Y+A181T+N190F+A209V+Q264S; or

G48A+T49I+G107A+H156Y+A181T+N190F+I201F+A209V+Q264S.

Further suitable amylases are amylases having SEQ ID NO:6 in WO 99/019467 or a variant having 90% sequence identity to SEQ ID NO:6. Preferred variants of SEQ ID NO: 6 are those having substitutions, deletions or insertions at one or more of the following positions: R181 , G182, H183, G184, N195, I206, E212, E216 and K269. Particularly preferred amylases are those having deletions in positions R181 and G182, or positions H183 and G184.

Additional amylases that may 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 in combination with SEQ ID NO:1, SEQ ID NO: 2. A variant thereof having 90% sequence identity to 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 with substitutions, deletions or insertions in one or more of the following positions: 140, 181, 182, 183, 184, 195, 206, 212, 243, 260, 269, 304, and 476, using SEQ ID 2 of WO 96/023873 for numbering. More preferred variants are those with deletions in two positions selected from 181, 182, 183 and 184 (eg 181 and 182, 182 and 183, or positions 183 and 184). The most preferred amylase variants of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:7 have deletions in positions 183 and 184 and at positions 140, 195, 206, 243, 260, 304 and 476 Those with substitutions in one or more of.

Other amylases that may be used are those having SEQ ID NO: 2 of WO 08/153815, SEQ ID NO: 10 in WO 01/66712 or 90% sequence identity to SEQ ID NO: 2 of WO 08/153815 Or a variant with 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 with substitutions, deletions or insertions at one or more of the following positions: 176, 177, 178, 179, 190, 201, 207, 211 and 264.

Another suitable amylase is the amylase having SEQ ID NO: 2 of WO 09/061380 or a variant having 90% sequence identity to SEQ ID NO: 2. Preferred variants of SEQ ID NO: 2 are those with C-terminal truncations and/or substitutions, deletions or insertions in one or 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 with substitutions at one or 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 those with deletions at positions R180 and/or S181 or T182 and/or G183. The most preferred amylase variants of SEQ ID NO: 2 are those having substitutions at the following positions:

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 these variants are C-terminally truncated and optionally further comprise a substitution at position 243 and/or at position 180 and/or position Deletion at 181.

Further suitable amylases are amylases having SEQ ID NO: 1 in WO13184577 or a variant having 90% sequence identity to SEQ ID NO: 1 . Preferred variants of SEQ ID NO: 1 are those with substitutions, deletions or insertions at one or more of the following positions: K176, R178, G179, T180, G181, E187, N192, M199, I203, S241, R458, T459, D460, G476 and G477. More preferred variants of SEQ ID NO: 1 are those having substitutions at one or more of the following positions: K176L, E187P, N192FYH, M199L, I203YF, S241QADN, R458N, T459S, D460T, G476K, and G477K, and/or those with deletions in positions R178 and/or S179 or T180 and/or G181. The most preferred amylase variants of SEQ ID NO: 1 are those having substitutions at the following positions:

E187P+I203Y+G476K

E187P+I203Y+R458N+T459S+D460T+G476K

Wherein these variants optionally further comprise a substitution at position 241 and/or a deletion at position 178 and/or position 179.

Further suitable amylases are amylases having SEQ ID NO: 1 in WO 10104675 or a variant having 90% sequence identity to SEQ ID NO: 1 . Preferred variants of SEQ ID NO: 1 are those having substitutions, deletions or insertions at one or more of the following positions: N21, D97, V128, K177, R179, S180, I181, G182, M200, L204, E242, G477 and G478. More preferred variants of SEQ ID NO: 1 are those having substitutions at one or more of the following positions: N21D, D97N, V128I, K177L, M200L, L204YF, E242QA, G477K, and G478K, and/or at Those with deletions in positions R179 and/or S180 or I181 and/or G182. The most preferred amylase variants of SEQ ID NO: 1 are those having substitutions at the following positions:

N21D+D97N+V128I

Wherein these variants optionally further comprise a substitution at position 200 and/or a deletion at position 180 and/or position 181.

Other suitable amylases are alpha-amylases having SEQ ID NO: 12 in WO 01/66712 or variants having at least 90% sequence identity to SEQ ID NO: 12. Preferred amylase variants are those having substitutions, deletions or insertions at one or more of the following positions in SEQ ID NO: 12 in WO 01/66712: 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. Particularly preferred amylases include variants having deletions of D183 and G184 and having substitutions R118K, N195F, R320K and R458K, and additionally variants having substitutions at one or more positions selected from the group consisting of: M9, G149, G182, G186, M202, T257, Y295, N299, M323, E345 and A339, most preferred are variants additionally having substitutions at all these positions.

Other examples are amylase variants, such as those described in WO 2011/098531, WO 2013/001078, and WO 2013/001087.

Commercially available amylases are Duramyl ^™ , Termamyl ^™ , Fungamyl ^™ , Stainzyme ^™ , StainzymePlus ^™ , Natalase ^™ , Liquozyme X, Amplify Achieve and BAN ^™ (from Novozymes), and Rapidase ^™ , Purastar ^™ /Effectenz ^™ , Powerase, Preferenz S1000, Preferenz S100 and Preferenz S110 (from Genencor International Ltd/DuPont).

In one aspect, the amylase is selected from the group consisting of:

i) a variant comprising one or more substitutions at the following positions: 9, 26, 30, 33, 82, 37, 106, 118, 128, 133, 149, 150, 160, 178, 182, 186, 193, 195, 202, 203, 214, 231, 256, 257, 258, 269, 270, 272, 283, 295, 296, 298, 299, 303, 304, 305, 311, 314, 315, 318, 319, 320, 323, 339, 345, 361, 378, 383, 419, 421, 437, 441, 444, 445, 446, 447, 450, 458, 461, 471, 482, 484, wherein the position corresponds to SEQ ID NO 11 position;

ii) a variant exhibiting at least 90% identity to SEQ ID NO 12, having deletions at positions 183 and 184,

iii) a variant exhibiting at least 95% identity to SEQ ID NO 13 comprising a mutation at one or more of the following positions: M202, M208, S255, R172 and/or M261,

iv) a polypeptide comprising the amino acid sequence shown in SEQ ID NO:9 or 10,

v) a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96% of SEQ ID NO:9 or 10 , at least 97%, at least 98%, at least 99% sequence identity,

and combinations thereof.

In one aspect, the protease is a peptide having at least 60% sequence identity to SEQ ID NO:9 or 10.

In one aspect, the protease is a peptide having at least 65% sequence identity to SEQ ID NO:9 or 10.

In one aspect, the protease is a peptide having at least 70% sequence identity to SEQ ID NO:9 or 10.

In one aspect, the protease is a peptide having at least 75% sequence identity to SEQ ID NO:9 or 10.

In one aspect, the protease is a peptide having at least 80% sequence identity to SEQ ID NO:9 or 10.

In one aspect, the protease is a peptide having at least 85% sequence identity to SEQ ID NO:9 or 10.

In one aspect, the protease is a peptide having at least 90% sequence identity to SEQ ID NO:9 or 10.

In one aspect, the protease is a peptide having at least 95% sequence identity to SEQ ID NO:9 or 10.

In one aspect, the protease is a peptide having at least 96% sequence identity to SEQ ID NO:9 or 10.

In one aspect, the protease is a peptide having at least 97% sequence identity to SEQ ID NO:9 or 10.

In one aspect, the protease is a peptide having at least 98% sequence identity to SEQ ID NO:9 or 10.

In one aspect, the protease is a peptide having at least 99% sequence identity to SEQ ID NO:9 or 10.

Cellulase - a polypeptide having cellulase activity

The term "cellulase" means an enzyme that hydrolyzes cellulose. In a preferred embodiment of the invention, the cellulase is an endoglucanase. The term "cellulase activity" is defined herein as an enzyme that catalyzes the hydrolysis of 1,4-β-D-glycosidic linkages in β-1,4-glucan (cellulose). For the purposes of the present invention, cellulase activity was determined using AZCL-HE-cellulose (from Megazyme) as the reaction substrate, as shown in Assay IV. Suitable cellulases include those of bacterial or fungal origin. Chemically modified mutants or protein engineered mutants are included. Suitable cellulases include cellulases from Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, for example as disclosed in US 4,435,307, US 5,648,263, Fungal cellulases produced by Humicola insolens, Myceliophthora thermophila and Fusarium oxysporum in US 5,691,178, US 5,776,757 and WO 89/09259.

Particularly suitable cellulases are alkaline or neutral cellulases with color care benefits. Examples of such cellulases are the cellulases described in EP 0 495 257, EP 0 531 372, WO 96/11262, WO 96/29397, WO 98/08940. Further 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 WO 99/001544.

Other cellulases are endo-beta-1,4-glucanases having at least 97% identity to the amino acid sequence from position 1 to position 773 of SEQ ID NO: 2 of WO2002/099091 , or a family 44 xyloglucanase having a sequence at least 60% identical to positions 40-559 of SEQ ID NO: 2 of WO 2001/062903.

Commercially available cellulases include Celluzyme ^™ , and Carezyme ^™ (Novozymes), Carezyme Premium ^™ (Novozymes), Celluclean ^™ (Novozymes), Celluclean Classic ^™ (Novozymes), Cellusoft ^TM (Novozymes), Whitezyme ^TM (Novozymes), Clazinase ^TM , and Puradax HA ^TM (Genencor International Inc.), and KAC-500(B) ^TM (Kao Corporation Kao Corporation), Revitalenz ^™ 1000, Revitalenz ^™ 2000, Revitalenz ^™ 3000 (DuPont).

In one aspect the present invention relates to the use of one or more enzymes in preventing or reducing the attachment of microorganisms to textile surfaces, wherein the enzymes are cellulases.

In one aspect the present invention relates to the use of one or more enzymes, wherein the enzymes are cellulases, for inhibiting or reducing malodor caused by microorganisms on the surface of textiles.

In one aspect of the present invention, the cellulase useful in the present invention comprises a polypeptide having cellulase activity comprising the amino acid sequence of SEQ ID NO:15. In one aspect of the invention, the cleaning composition comprises a polypeptide having cellulase activity comprising at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, even more preferably at least 97%, most preferably at least 98%, or even most preferably at least 99% identical amino acid sequence.

In one aspect of the invention, the amylase useful in the invention comprises a polypeptide having cellulase activity comprising the amino acid sequence of SEQ ID NO:16. In one aspect of the invention, the cleaning composition may comprise a polypeptide having cellulase activity comprising at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably At least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, even more preferably at least 97%, most preferably at least 98%, or even most preferably at least 99% identical amino acid sequence.

In one aspect of the invention, the amylase useful in the invention comprises a polypeptide having cellulase activity comprising the amino acid sequence of SEQ ID NO:17. In one aspect of the invention, the cleaning composition comprises a polypeptide having cellulase activity comprising at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, even more preferably at least 97%, most preferably at least 98%, or even most preferably at least 99% identical amino acid sequence.

In one aspect of the invention, the amylase useful in the invention comprises a polypeptide having cellulase activity comprising the amino acid sequence of SEQ ID NO:18. In one aspect of the present invention, the cleaning composition comprises a polypeptide having cellulase activity comprising at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, even more preferably at least 97%, most preferably at least 98%, or even most preferably at least 99% identical amino acid sequence.

detergent composition

In one aspect, the present invention relates to the use of one or more enzymes contained in a detergent composition in preventing, inhibiting or reducing the growth of microorganisms on a surface, wherein after use of said enzyme, all The TTC detectable time of the microorganism is increased by at least 20%, at least 30%, at least 50%, at least 70%, at least 80%, or at least 100%.

Detergent compositions comprising enzymes useful in the present invention may comprise one or more additional cleaning composition components. Selection of additional components is within the ability of the skilled artisan and includes conventional ingredients, including the exemplary, non-limiting components described below.

For textile care, the selection of components may include considerations of the type of textile to be cleaned, the type and/or degree of soiling, the temperature at which cleaning is performed, and the formulation of the detergent product. Although the below mentioned components are categorized by general headings according to specific functionality, this is not to be construed as limiting, as the components may contain additional functionality as will be understood by those of ordinary skill.

In one aspect, the present invention relates to the use of one or more enzymes comprised in an ADW (automatic dishwashing) composition in combination with one or more additional ADW composition components. Selection of additional components is within the ability of the skilled artisan and includes conventional ingredients, including the exemplary, non-limiting components described below.

In one aspect, the present invention is directed to detergent compositions substantially free of biocides. It is believed that by at least partially replacing the biocide with an enzyme, thereby reducing the need for the biocide, the lesser environmental harm of the biocide can be at least partially prevented.

In one aspect, the invention relates to the use of one or more enzymes for preventing, inhibiting or reducing the growth of microorganisms on a surface, wherein said microorganisms grow on residual stained areas of said surface. Without being bound by theory, it is believed that the residual stain may serve as a source of nutrients, such as carbon and nitrogen, upon which microbial growth depends.

In one aspect, the invention relates to the use of one or more enzymes for preventing, inhibiting or reducing the growth of microorganisms on a surface, where the surface may be a hard surface or a soft surface, such as a fabric/textile surface.

In one aspect, the surface is a textile made of natural fibers, synthetic fibers or mixtures thereof.

In one aspect, the surface is made of cotton, polyester or blends thereof.

In one aspect, the surface is a porous surface, such as a sponge.

In one aspect, the surface is a hard surface, such as a countertop, chopping board, and the like.

Surfactant

The detergent composition may comprise one or more surfactants, which may be anionic and/or cationic and/or nonionic and/or semi-polar and/or zwitterionic, or mixtures thereof . In particular aspects, the detergent composition comprises a mixture of one or more nonionic surfactants and one or more anionic surfactants. The one or more surfactants are typically present in an amount of from about 0.1% to 60% (such as about 1% to about 40%, or about 3% to about 20%, or about 3% to about 10%) by weight. level exists. The one or more surfactants are selected based on the desired cleaning application, and can include any conventional surfactants known in the art.

When included therein, the detergent will generally contain from about 1% to about 40% by weight of anionic surfactants, such as from about 5% to about 30%, including from about 5% to about 15%, or from about 15% to about 20%, or from about 20% to about 25% anionic surfactant. Non-limiting examples of anionic surfactants include sulfates and sulfonates, specifically linear alkylbenzenesulfonates (LAS), isomers of LAS, branched alkylbenzenesulfonates (BABS), Phenyl alkane sulfonate, alpha-olefin sulfonate (AOS), olefin sulfonate, alkene sulfonate, alkane-2,3-diyl bis(sulfate), hydroxy alkane sulfonate As well as disulfonates, alkyl sulfates (AS) (such as sodium dodecyl sulfate (SDS)), fatty alcohol sulfates (FAS), primary alcohol sulfates (PAS), alcohol ether sulfates (AES or AEOS or FES, also known as alcohol ethoxy sulfate or fatty alcohol ether sulfate), secondary alkane sulfonate (SAS), paraffin sulfonate (PS), ester sulfonate, sulfonated fatty acid glycerides , α-sulfo fatty acid methyl ester (α-SFMe or SES) (including methyl ester sulfonate (MES)), alkyl or alkenyl succinic acid, dodecenyl/tetradecenyl succinic acid (DTSA ), fatty acid derivatives of amino acids, diesters and monoesters or fatty acid salts (soaps) of sulfosuccinic acid, and combinations thereof.

When included therein, the detergent will generally comprise from about 1% to about 40% by weight of cationic surfactants, for example from about 0.5% to about 30%, especially from about 1% to about 20% , from about 3% to about 10%, such as from about 3% to about 5%, from about 8% to about 12%, or from about 10% to about 12%. Non-limiting examples of cationic surfactants include alkyl dimethyl ethanol quaternary amines (ADMEAQ), cetyl trimethyl ammonium bromide (CTAB), dimethyl distearoyl ammonium chloride (DSDMAC), and Alkylbenzyldimethylammoniums, alkylquats, alkoxylated quaternary ammonium (AQA) compounds, esterquats, and combinations thereof.

When included therein, the detergent will generally contain from about 0.2% to about 40% by weight of nonionic surfactants, such as from about 0.5% to about 30%, especially from about 1% to about 20% by weight. %, from about 3% to about 10%, such as from about 3% to about 5%, from about 8% to about 12%, or from about 10% to about 12%. Non-limiting examples of nonionic 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), alkyl polyglycosides (APG), alkanes Oxylated amines, fatty acid monoethanolamides (FAM), fatty acid diethanolamides (FADA), ethoxylated fatty acid monoethanolamides (EFAM), propoxylated fatty acid monoethanolamides (PFAM), polyhydroxyalkanes fatty acid amides, or N-acyl N-alkyl derivatives of glucosamine (glucamide (GA), or fatty acid glucamide (FAGA)), and products available under the tradenames SPAN and TWEEN, and combinations thereof .

When included therein, the detergents will generally contain from about 0.5% to about 50%, preferably from about 1% to about 25%, by weight of semi-polar surfactants. Non-limiting examples of semipolar surfactants include amine oxides (AO), such as alkyldimethylamine oxides, N-(cocoalkyl)-N,N-dimethylamine oxides, and N- (tallow-alkyl)-N,N-bis(2-hydroxyethyl)amine oxides, and combinations thereof.

When included therein, the detergents will generally contain from about 0.5% to about 50%, preferably from about 1% to about 25%, by weight of zwitterionic surfactants. Non-limiting examples of zwitterionic surfactants include betaines, such as alkyldimethylbetaines, sultaines, and combinations thereof.

Hydrotrope

The detergent may comprise 0-10% by weight, such as 0-5% by weight, such as from about 0.5% to about 5%, or from 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 benzenesulfonate, sodium p-toluenesulfonate (STS), sodium xylenesulfonate (SXS), sodium cumenesulfonate (SCS), sodium cymenesulfonate, amine oxide , alcohols and polyethylene glycol ethers, sodium hydroxynaphthoate, sodium hydroxynaphthalene sulfonate, sodium ethylhexyl sulfonate, and combinations thereof.

Builders and co-builders

The detergent composition may comprise from about 0 to 65%, such as from about 5% to about 50%, by weight of a detergent builder or co-builder, or mixtures thereof. In dishwashing detergents, builder levels are typically 40%-65%, especially 50%-65%. Builders and/or co-builders may in particular be chelating agents forming water-soluble complexes with Ca and Mg. Any builder and/or co-builder known in the art for use in laundry/ADW/hard surface cleaning 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, Phyllosilicates (eg, SKS-6 from Hoechst), ethanolamines such as 2-aminoethan-1-ol (MEA), diethanolamine (DEA, also known as 2,2'- aminodiethyl-1-ol), triethanolamine (TEA, also known as 2,2',2"-nitrilotriethan-1-ol), and (carboxymethyl)inulin (CMI), and combination.

The detergent composition may also comprise from 0 to 50%, such as from about 5% to about 30%, by weight of a detergent co-builder. The detergent compositions may include co-builders alone, or in combination with builders such as zeolite builders. Non-limiting examples of co-builders include polyacrylate homopolymers or copolymers thereof, such as poly(acrylic acid) (PAA) or copoly(acrylic acid/maleic acid) (PAA/PMA). Additional non-limiting examples include citrates, chelating agents such as aminocarboxylates, aminopolycarboxylates, and phosphates, and alkyl or alkenyl succinic acids. 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), methylglycine diacetic acid (MGDA), glutamic acid-N,N-diacetic acid (GLDA), 1-hydroxyethane-1, 1-diphosphonic acid (HEDP), ethylenediaminetetra(methylenephosphonic acid) (EDTMPA), diethylenetriaminepenta(methylenephosphonic acid) (DTMPA or DTPMPA), N-(2-hydroxy Ethyl)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), sulfonyl-N,N-diacetic acid (SLDA), taurine-N,N-diacetic acid (TUDA) and sulfonic acid Methyl-N,N-diacetic acid (SMDA), N-(2-hydroxyethyl)-ethylenediamine-N,N',N"-triacetate (HEDTA), diethanolglycine (DEG ), diethylenetriaminepenta(methylenephosphonic acid) (DTPMP), aminotris(methylenephosphonic acid) (ATMP) and combinations and salts thereof. Additional exemplary builders and/or co-builders are described, for example, in WO 09/102854, US 5977053.

bleaching system

The detergent composition may comprise from 0 to 30%, such as from about 1% to about 20%, by weight of a bleaching system. Any bleach system comprising ingredients known in the art for use in cleaning detergents may be utilized. Suitable bleach system components include a source of hydrogen peroxide; a source of peracid; and a bleach catalyst or builder.

· Hydrogen peroxide source

Suitable sources of hydrogen peroxide are inorganic persalts, including alkali metal salts such as sodium percarbonate and sodium perborate (usually monohydrate or tetrahydrate), and hydrogen peroxide-urea (1/1).

· Peracid source

Peracids can be (a) incorporated directly as preformed peracids, or (b) formed in situ in the wash liquor from hydrogen peroxide and bleach activators (perhydrolysis), or (c) from hydrogen peroxide and peroxide Hydrogenase and the latter suitable substrates (eg esters) are formed in situ in the wash solution.

a) Suitable preformed peracids include but are not limited to peroxycarboxylic acids (such as peroxybenzoic acid) and their ring-substituted derivatives, peroxy-alpha-naphthoic acid, peroxyphthalic acid, peroxylauric acid , peroxystearic acid, ε-phthalimidoperoxycaproic acid [phthalimidoperoxycaproic acid (PAP)], and o-carboxybenzamidoperoxycaproic acid; Aliphatic and aromatic diperoxydicarboxylic acids, such as diperoxydodecanedioic acid, diperoxyazelaic acid, diperoxysebacic acid, diperoxybasyl acid, 2-decyl diperoxy Succinic acid, and diperoxyphthalic, -isophthalic, and -terephthalic acids; perimidic acid; peroxymonosulfuric acid; peroxydisulfuric acid; peroxyphosphoric acid; peroxysilicic acid; Mixtures of said compounds. It will be appreciated that in some cases the peracids mentioned may be best added as suitable salts, such as alkali metal salts (e.g. ) or alkaline earth metal salts.

b) Suitable bleach activators include those belonging to the classes of esters, amides, imides, nitriles or anhydrides and, where applicable, their salts. Suitable examples are tetraacetylethylenediamine (TAED), sodium 4-[(3,5,5-trimethylhexanoyl)oxy]benzene-1-sulfonate (ISONOBS), 4-(dodecanoyl Oxy)sodium benzene-1-sulfonate (LOBS), 4-(decanoyloxy)benzene-1-sodium sulfonate, 4-(decanoyloxy)benzoic acid (DOBA), 4-(nonanoyloxy base) sodium benzene-1-sulfonate (NOBS), and/or those disclosed in WO 98/17767. A specific family of bleach activators of interest is disclosed in EP 624154 and particularly preferred within this family is acetyl triethyl citrate (ATC). ATC or short chain triglycerides (like triacetin) have the advantage that they are environmentally friendly. In addition, acetyl triethyl citrate and triacetin have good hydrolytic stability in the product on storage and are effective bleach activators. Finally, ATC is multifunctional because the citrate released in the perhydrolysis reaction can act as a builder.

· Bleach catalyst and booster

The bleach system may also include a bleach catalyst or builder. Some non-limiting examples of bleach catalysts that may be used in the compositions of the present invention include manganese oxalate, manganese acetate, manganese collagen, cobalt-amine catalysts and manganese triazacyclononane (MnTACN) catalysts; manganese in combination with 1, 4,7-Trimethyl-1,4,7-triazacyclononane (Me3-TACN) or 1,2,4,7-tetramethyl-1,4,7-triazacyclononane Complexes of (Me4-TACN), specifically Me3-TACN, such as binuclear manganese complexes [(Me3-TACN)Mn(O)3Mn(Me3-TACN)](PF6)2, and [2,2 ',2"-nitrilotris(ethane-1,2-diylazanylylidene-κN-methylidene)triphenolo-κ3O]manganese(III). These bleach catalysts can also be other Metal compounds such as iron or cobalt complexes.

In some embodiments where a source of peracid is included, an organic bleach catalyst or bleach booster having one of the following formulas may be used:

(i)

(ii)

(iii) and mixtures thereof; wherein each R1 independently is a branched chain alkyl group containing from 9 to 24 carbons or a straight chain alkyl group containing from 11 to 24 carbons, preferably each R1 independently contains from A branched chain alkyl group of 9 to 18 carbons or a straight chain 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, dodecyl, tetradecyl, hexadecyl, octadecyl, isononyl, isodecyl, isodeca Trialkyl and Isopentadecyl.

Other exemplary bleach systems are described, for example, in WO 2007/087258, WO 2007/087244, WO 2007/087259, EP 1867708 (vitamin K) and WO 2007/087242. Suitable photobleaches may for example be sulfonated zinc or aluminum phthalocyanines.

polymer

The detergent may contain 0-10% by weight (eg 0.5%-5%, 2%-5%, 0.5%-2% or 0.2%-1%) of polymer. Any polymer known in the art for use in detergents can be utilized. The polymer may function as a co-builder as mentioned above, or may provide anti-redeposition, fiber protection, soil release, dye transfer inhibition, greasy cleaning and/or suds suppressing properties. Some polymers may have more than one of the properties mentioned above and/or more than one of the motifs mentioned below. Exemplary polymers include (carboxymethyl)cellulose (CMC), poly(vinyl alcohol) (PVA), poly(vinylpyrrolidone) (PVP), poly(ethylene glycol) or poly(ethylene oxide) (PEG ), ethoxylated poly(ethyleneimine), carboxymethylinulin (CMI), and polycarboxylates (such as PAA, PAA/PMA, poly-aspartic acid, and lauryl methacrylate ester/acrylic acid copolymer), hydrophobically modified CMC (HM-CMC) and silicone, copolymers of terephthalic acid and oligoethylene glycol, poly(ethylene terephthalate) and poly(oxyethylene para phthalates) copolymers (PET-POET), PVP, poly(vinylimidazole) (PVI), poly(vinylpyridine-N-oxide) (PVPO or PVPNO), and polyvinylpyrrolidone-vinylimidazole (PVPVI). Additional exemplary polymers include sulfonated polycarboxylates, polyethylene oxide and polypropylene oxide (PEO-PPO), and diquaternary ammonium ethoxysulfate. Other exemplary polymers are disclosed eg in WO2006/130575. Salts of the above-mentioned polymers are also contemplated.

fabric toner

These detergent compositions may also include fabric toners, such as when formulated in a detergent composition, which can deposit on said fabrics when they come into contact with a wash liquor comprising said detergent composition and thus transmit visible light. Dyes or pigments that absorb/reflect to change the color of the fabric. Optical brighteners emit at least some visible light. In contrast, fabric toners change the color of a surface when they absorb at least part of the visible spectrum. Suitable fabric toners 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 those selected from the group consisting of the following dyes falling into the Color Index (C.I.) classification: direct blue, direct red, direct violet, acid blue, acid red, Acid violet, basic blue, basic violet and basic red, or mixtures thereof, such as described in WO 2005/03274, WO 2005/03275, WO 2005/03276 and EP1876226 (which are 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 toner. The composition may comprise from 0.0001 wt% to 0.2 wt% fabric toner, which may be especially preferred when the composition is in unit dose pouch form. Suitable toners are also disclosed in eg WO 2007/087257 and WO 2007/087243.

enzyme

Detergent additives as well as detergent compositions may comprise one or more additional enzymes such as proteases, lipases, cutinases, amylases, carbohydrases, cellulases, pectinases, mannanases, arabinases , galactanase, xylanase, oxidase, such as laccase, and/or peroxidase.

In general, the properties of one or more selected enzymes should be compatible with the selected detergent (i.e., pH optimum, compatibility with other enzymes and non-enzyme ingredients, etc.), and the one or more Enzymes should be present in effective amounts.

cellulase

Suitable cellulases include those of bacterial or fungal origin. Chemically modified mutants or protein engineered mutants are included. Suitable cellulases include cellulases from Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, for example as disclosed in US 4,435,307, US 5,648,263, Fungal cellulases produced by Humicola insolens, Myceliophthora thermophila and Fusarium oxysporum in US 5,691,178, US 5,776,757 and WO 89/09259.

Particularly suitable cellulases are alkaline or neutral cellulases with color care benefits. Examples of such cellulases are the cellulases described in EP 0 495 257, EP 0 531 372, WO 96/11262, WO 96/29397, WO 98/08940. Further 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 WO 99/001544.

Other cellulases are endo-beta-1,4-glucanases having at least 97% identity to the amino acid sequence from position 1 to position 773 of SEQ ID NO: 2 of WO2002/099091 , or a family 44 xyloglucanase having a sequence at least 60% identical to positions 40-559 of SEQ ID NO: 2 of WO 2001/062903.

Commercially available cellulases include Celluzyme ^™ , and Carezyme ^™ (Novozymes), Carezyme Premium ^™ (Novozymes), Celluclean ^™ (Novozymes), Celluclean Classic ^™ (Novozymes), Cellusoft ^™ (Novozymes Corporation), Whitezyme ^™ (Novozymes Corporation), Clazinase ^™ , and Puradax HA ^™ (Genencor International Inc.), and KAC-500(B) ^™ (Kao Corporation).

Suitable cellulases include intact cellulases or monocomponent endoglucanases of bacterial or fungal origin. Chemically or genetically modified mutants are included. The cellulase may, for example, be a single-component or a mixture of single-component endo-1,4-β-glucanases commonly referred to as endoglucanases. Suitable cellulases include fungal cellulases from Humicola insolens (US 4,435,307) or from Trichoderma, eg Trichoderma reesei or Trichoderma viride. Examples of cellulases are described in EP 0 495 257 . Other suitable cellulases are from Thielavia, for example Thielavia terrestris as described in WO 96/29397 or Fusarium oxysporum as described in WO 91/17244, or from Fusarium oxysporum as described in WO 02/ Bacillus species described in 099091 and JP 2000210081. 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. Commercially available cellulases include and (Novozymes), Puradax HA, and Puradax EG (available from Genencor).

Mannanase

Suitable mannanases include those of bacterial or fungal origin. Chemically or genetically modified mutants are included. The mannanase may be a family 5 or 26 alkaline mannanase. It may be wild-type from the genus Bacillus or Humicola, in particular Bacillus agaricus, Bacillus licheniformis, Bacillus alkalophilus, Bacillus clausii or Humicola insolens. Suitable mannanases are described in WO 1999/064619. A commercially available mannanase is Mannaway (Novozymes).

Lipase and cutinase:

Suitable lipases and cutinases include those of bacterial or fungal origin. Chemically modified or protein engineered mutant enzymes are included. Examples include lipases from thermophilic fungi such as from Thermomyces lanuginosa (earlier named Humicola lanuginosa) as described in EP 258068 and EP305216; cutinases from Humicola lanuginosus , such as Humicola insolens (WO 96/13580); lipases from strains of Pseudomonas (some of these are now renamed Burkholderia), such as Pseudomonas alcaligenes or the class Pseudomonas alcaligenes (EP 218272), Pseudomonas cepacia (EP331376), Pseudomonas strain SD705 (WO 95/06720 and WO 96/27002), Pseudomonas wisconsin (P.wisconsinensis) ( WO 96/12012); GDSL-type Streptomyces lipase (WO 10/065455); Cutinase from Magnaporthe grisea (WO 10/107560); Cutinase from Pseudomonas mendoza (US 5,389,536) ; lipase from Thermobifida fusca (WO 11/084412); lipase from Geobacillus stearothermophilus (WO 11/084417); lipase from Bacillus subtilis (WO 11/084599) and lipases from Streptomyces griseus (WO 11/150157) and S. pristinaespiralis (WO 12/137147).

Other examples are lipase variants as described in EP 407225, WO 92/05249, WO 94/01541, WO 94/25578, WO 95/14783, WO 95/30744, WO 95/35381, WO 95/22615, WO 96/ 00292, WO 97/04079, WO 97/07202, WO 00/34450, WO 00/60063, WO 01/92502, WO 07/87508 and WO 09/109500.

Preferred commercial lipase products include Lipolase ^™ , Lipex ^™ ; Lipolex ^™ and Lipoclean ^™ (Novozymes), Lumafast (from Genencor) and Lipomax (from Gistbrocades). (Gist-Brocades)).

Still other examples are lipases sometimes called acyltransferases or perhydrolases, such as the acylase with homology to Candida antarctica lipase A (WO 10/111143), from Clade smegmatis Mycobacterium smegmatis acyltransferase (WO 05/56782), perhydrolase from CE 7 family (WO 09/67279) and variants of Mycobacterium smegmatis perhydrolase (particularly from Huntsman Textiles The S54V variant used in the commercial product Gentle Power Bleach from Huntsman Textile Effects Pte Ltd) (WO 10/100028).

Suitable peroxidases include those of vegetable, bacterial or fungal origin. Chemically modified mutants or protein engineered mutants are included. Examples of useful peroxidases include peroxidases from the genus Pseudomonas, e.g. from C. cinerea (EP 179,486), and variants thereof, as described in WO 93/24618, WO 93/24618, 95/10602 and those described in WO 98/15257.

Peroxidases according to the invention also include haloperoxidases, such as chloroperoxidases, bromoperoxidases and compounds exhibiting chloroperoxidase or bromoperoxidase activity. Haloperoxidases are classified according to their specificity for halide ions. Chloroperoxidase (E.C. 1.11.1.10) catalyzes the formation of hypochlorite from chloride ions.

In one aspect, the haloperoxidase of the invention is a chloroperoxidase. Preferably, the haloperoxidase is a vanadium haloperoxidase, ie a vanadate-containing haloperoxidase. In a preferred method of the invention, a vanadate-containing haloperoxidase is combined with a source of chloride ions.

Haloperoxidases have been isolated from many different fungi, especially from the dematiaceous hyphomycete fungal group, such as Caldariomyces (e.g., C. fumago ), Alternaria, Curvularia (e.g., C. verruculosa and C. inaequalis), Entorhelminthes, Alternaria, and Botrytis.

Haloperoxidases have also been isolated from bacteria such as Pseudomonas (e.g., P. pyrrocinia) and Streptomyces (e.g., S. aureofaciens) .

In a preferred aspect, the haloperoxidase is derived from Curvularia sp., in particular Curvularia verruculosa or Curvularia inaequalis, as described in WO95/27046 Curvularia inequalis CBS 102.42 in or from Curvularia verruculosa CBS 147.63 or Curvularia verruculosa CBS 444.70 as described in WO97/04102; or from Drechslera hartlebii as described in WO 01/79459, as described in WO01/79458 Dendryphiella salina in , Phaeotrichoconis crotalarie as described in WO 01/79461, or Geniculosporium sp. as described in WO 01/79460.

Oxidases according to the invention include in particular any laccases encompassed by enzyme class EC 1.10.3.2 or fragments derived therefrom exhibiting laccase activity, or compounds exhibiting similar activity, such as catechol oxidase (EC 1. 10.3.1), o-aminophenol oxidase (EC 1.10.3.4) or bilirubin oxidase (EC 1.3.3.5).

Preferred laccases are enzymes of microbial origin. The enzyme may be of plant, bacterial or fungal origin (including filamentous fungi and yeast).

Suitable examples from fungi include laccases which may be derived from strains of: Aspergillus, Neurospora (e.g., Neurospora crassa), Podospora, Botrytis, Collybia, Laminaria Fomes, Lentinus, Pleurotus, Trametes (eg Trametes villosa and Trametes versicolor), Rhizoctonia (eg R. solani), Pleurotus Coprinus (for example, C. cinerea, C. comatus, C. friesii and C. plicatilis), Psathyrella (for example, P. condelleana), Pleurotus sp. (e.g., P. papilionaceus), Myceliophthora (e.g., Myceliophthora thermophila), Schytalidium (e.g., S.thermophilum), Polyporus (for example, P. pinsitus), Radix (for example, P. radiata) (WO 92/01046) or Coriolus (for example, P. bacteria (C. hirsutus)) (JP 2238885).

Suitable examples from bacteria include laccases which may be derived from strains of Bacillus.

Preference is given to laccases derived from the genus Pseudocoprinus or Myceliophthora; in particular from Pseudomonas cinerea, as disclosed in WO 97/08325; or from Myceliophthora thermophila, as disclosed in WO 95/33836.

One or more detergent enzymes may be included in the detergent composition by adding a single additive comprising one or more enzymes, or by adding a combined additive comprising all such enzymes. The detergent additives of the present invention, either alone or in combination, can be formulated, for example, as granules, liquids, slurries and the like. Preferred detergent additive formulations are granules, especially non-dusting granules; liquids, especially stabilized liquids; or slurries.

Dust-free granules may for example be produced 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 polyethylene glycol (PEG) with an average molecular weight of 1000 to 20000; ethoxylated nonylphenols with from 16 to 50 ethylene oxide units; alcohols therein containing from 12 Ethoxylated fatty alcohols of up to 20 carbon atoms and in which 15 to 80 ethylene oxide units are present; fatty alcohols; fatty acids; and monoglycerides, and diglycerides, and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application by fluidized bed techniques are given in GB 1483591 . Liquid enzyme preparations can be stabilized, for example, by addition of polyols such as propylene glycol, sugars or sugar alcohols, lactic acid or boric acid according to established methods. Protected enzymes can be prepared according to the method disclosed in EP 238,216.

microorganism

The detergent additives as well as detergent compositions may also comprise one or more microorganisms, such as one or more fungi, yeast, or bacteria.

In one aspect, the one or more microorganisms are dehydrated (eg, by lyophilization) bacteria or yeast, such as Lactobacillus strains.

In another aspect, the microorganism is one or more microbial spores (as opposed to vegetative cells), such as bacterial spores; or fungal spores, conidia, hyphae. Preferably, the one or more spores are Bacillus endospores; even more preferably, the one or more spores are endogenous Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, or Bacillus megaterium spore.

Microorganisms can be included in detergent compositions or additives in the same way as enzymes (see above).

Accessories

Any detergent component known in the art for use in laundry/ADW/hard surface cleaning detergents may also be utilized. Other optional detergent components include anti-corrosion agents, anti-shrinkage agents, anti-soil redeposition agents, anti-wrinkle 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 clay), fillers/processing aids, optical brighteners/optical brighteners, Suds boosters, suds (foam) regulators, fragrances, soil suspending agents, softeners, suds suppressors, tarnish inhibitors and wicking agents, alone or in combination. Any ingredient known in the art for use in laundry/ADW/hard surface cleaning detergents can be utilized. Selection of such ingredients is well within the skill of the skilled artisan.

Dispersant

The detergent compositions useful in the present invention may also contain dispersants. Specifically, powder detergents may contain dispersants. Suitable water-soluble organic materials include homopolymeric or copolymeric acids or salts thereof, wherein the polycarboxylic acid contains at least two carboxyl groups separated from each other by not more than two carbon atoms. Suitable dispersants are described, for example, in Powdered Detergents [Powder Detergents], Surfactant science series [Surfactant Science Series], Volume 71, Marcel Dekker, Inc [Marcel Dekker Company].

dye transfer inhibitor

Detergent compositions useful herein can 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, polyvinyloxazolidone and polyvinyloxazolidone. Vinyl imidazole or mixtures thereof. When present in the subject compositions, dye transfer inhibiting agents may be present in an amount of 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. level exists.

Fluorescent whitening agent

Detergent compositions useful in the present invention will preferably also contain additional ingredients which can tint the item being cleaned, such as optical brighteners or optical brighteners. When present, the brightener is preferably present at a level of from about 0.01% to about 0.5%. Any optical brightener suitable for use in laundry detergent compositions can be used in the compositions of the present invention. The most commonly used optical brighteners are those belonging to the following classes: diaminostilbene-sulfonic acid derivatives, diarylpyrazoline derivatives and diphenyl-distyryl derivatives. Examples of diaminostilbene-sulfonic acid derivatives of optical brighteners include the following sodium salts: 4,4'-bis-(2-diethanolamino-4-anilino-s-triazin-6-ylamino ) stilbene-2,2'-disulfonate, 4,4'-bis-(2,4-dianilino-s-triazin-6-ylamino)stilbene-2.2'-disulfonate, 4 ,4'-bis-(2-anilino-4-(N-methyl-N-2-hydroxy-ethylamino)-s-triazin-6-ylamino)stilbene-2,2'-disulfo salt, 4,4'-bis-(4-phenyl-1,2,3-triazol-2-yl)stilbene-2,2'-disulfonate and 5-(2H-naphtho[1 ,2-d][1,2,3]triazol-2-yl)-2-[(E)-2-phenylethenyl]benzenesulfonate sodium. Preferred optical brighteners are Tinopal DMS and Tinopal CBS available from Ciba-Geigy AG (Basel, Switzerland). Tianlaibao DMS is the disodium salt of 4,4'-bis-(2-morpholino-4-anilino-s-triazin-6-ylamino)stilbene-2,2'-disulfonate. Tianlaibao CBS is the disodium salt of 2,2'-bis-(phenyl-styryl)-disulfonate. It is also preferred that the optical brightener is the commercially available Parawhite KX supplied by Paramount Minerals and Chemicals, Mumbai, India. Other fluorescent agents suitable for use in the present invention include 1-3-diarylpyrazolines and 7-aminoalkylcoumarins.

Suitable fluorescent brightener levels include lower levels of from about 0.01 wt%, from 0.05 wt%, from about 0.1 wt%, or even from about 0.2 wt%, to higher levels of 0.5 wt%, or even 0.75 wt%.

dirt release polymer

The detergent compositions useful in the present invention may also include one or more soil release polymers which aid in the removal of soils from fabrics such as cotton and polyester based fabrics, especially from polyester based fabrics. Hydrophobic dirt. Soil release polymers may for example be nonionic or anionic terephthalic acid based polymers, polyvinylcaprolactam and related copolymers, vinyl graft copolymers, polyester polyamides, see for example Powdered Detergents [powder detergent ], Surfactant science series [Surfactant Science Series], Volume 71, Chapter 7, Marcel Decker Company. Another type of soil release polymer is an amphiphilic alkoxylated oil stain cleaning polymer comprising a core structure and a plurality of alkoxylated groups attached to the core structure. The core structure may comprise polyalkylimine structures or polyalkanolamine structures as described in detail in WO 2009/087523 (incorporated herein by reference). Furthermore, random graft copolymers are suitable soil release polymers. Suitable graft copolymers are described in more detail in WO 2007/138054, WO 2006/108856 and WO 2006/113314 (incorporated herein by reference). Other soil release polymers are substituted polysaccharide structures, especially substituted cellulosic structures, such as modified cellulose derivatives, such as those described in EP 1867808 or WO 2003/040279 (both are incorporated herein by reference ). Suitable cellulosic polymers include cellulose, cellulose ethers, cellulose esters, cellulose amides, and mixtures thereof. Suitable cellulosic polymers include anionically-modified cellulose, non-ionically-modified cellulose, cationic-modified cellulose, zwitterionic-modified cellulose, and mixtures thereof. Suitable cellulosic polymers include methylcellulose, carboxymethylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose, carboxymethylcellulose, and mixtures thereof.

anti redeposition agent

Detergent compositions useful in the present invention may also include one or more anti-redeposition agents such as carboxymethylcellulose (CMC), polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polyoxyethylene and and/or polyethylene glycol (PEG), homopolymers of acrylic acid, copolymers of acrylic acid and maleic acid, and ethoxylated polyethyleneimines. The cellulose-based polymers described above under soil release polymers can also function as anti-redeposition agents.

rheology modifier

Detergent compositions useful herein may also include one or more rheology modifiers, structurants or thickeners other than viscosity reducers. The rheology modifier is selected from the group consisting of non-polymeric crystalline, hydroxyl functional materials, polymeric rheology modifiers which impart shear thinning to the aqueous liquid phase matrix of the liquid detergent composition. characteristics. The rheology and viscosity of detergents can be modified and adjusted by methods known in the art, eg as shown in EP 2169040 .

Other suitable excipients include, but are not limited to, anti-shrink agents, anti-wrinkle agents, bactericides, binders, carriers, dyes, enzyme stabilizers, fabric softeners, fillers, foam regulators, hydrotropes, fragrances, pigments, Suds suppressors, solvents and structurants and/or structural elastic agents for liquid detergents.

Preparations for detergent products

The detergent compositions useful herein may be in any conventional form, such as bars, uniform tablets, tablets with two or more layers, sachets with one or more chambers, regular or compressed powders, Granules, pastes, gels, or regular, compressed or concentrated liquids.

Bags can be configured as single or multi-chambered. It may be of any form, shape and material suitable for holding the composition, for example not allowing the composition to be released from the pouch prior to contact with water. The bag is made of a water-soluble film, which contains an inner volume. The internal volume can be divided into chambers of bags. Preferred films are polymeric materials, preferably polymers forming films or sheets. Preferred polymers, copolymers or derivatives thereof are selected from polyacrylates, and water-soluble acrylate copolymers, methylcellulose, carboxymethylcellulose, sodium dextrin, ethylcellulose, hydroxyethylcellulose , hydroxypropylmethylcellulose, maltodextrin, polymethacrylates, most preferably polyvinyl alcohol copolymers, and hydroxypropylmethylcellulose (HPMC). Preferably, the level of polymer in the film, such as PVA, is at least about 60%. The preferred average molecular weight will typically be from about 20,000 to about 150,000. The film may also be a blend composition comprising a hydrolytically degradable and water soluble polymer blend such as polylactic acid and polyvinyl alcohol (known under trade reference M8630 as manufactured by MonoSol Ltd. of Indiana, USA). Responsibility company sales) plus plasticizers like glycerin, ethylene glycol, propylene glycol, sorbitol and mixtures thereof. These bags may contain a solid laundry cleaning composition or fractions and/or a liquid cleaning composition or fractions separated by a water soluble film. Chambers that can be used for liquid components can be constructed differently than those containing solids: US 2009/0011970A1.

The detergent ingredients may be physically separated from each other by compartments in the water soluble pouch or in different layers of the tablet. Thus, undesirable storage interactions between components can be avoided. Different dissolution profiles for each compartment can also cause delayed dissolution of selected components in wash solutions.

Liquid or gel detergents for non-unit administration may be aqueous, typically containing at least 20% and up to 95% water by weight, such as up to about 70% water, up to about 65% water, up to about 55% water % water, up to about 45% water, up to about 35% water. Other types of liquids including, but not limited to, alkanols, amines, glycols, ethers, and polyols may be included in the aqueous liquid or gel. Aqueous liquid or gel detergents may contain from 0-30% organic solvents.

Liquid or gel detergents can be non-aqueous.

Enzyme formulations in co-granules

Enzymes useful in the present invention for preventing microbial growth on household surfaces can be formulated as granules, eg, as co-granules that incorporate one or more enzymes. Each enzyme will then be present in multiple granules which ensure a more even distribution of the enzymes in the detergent. This also reduces the physical segregation of different enzymes due to different particle sizes. A method for producing multi-enzyme co-granules for the detergent industry is disclosed in IP.com disclosure IPCOM000200739D.

Another example of the formulation of enzymes by using co-granules is disclosed in WO 2013/188331, which relates to detergent compositions comprising: (a) multi-enzyme co-granules; (b) less than 10 wt zeolite (anhydrous and (c) less than 10wt phosphate (anhydrous basis), wherein the enzyme co-granules comprise from 10wt% to 98wt% water sink component (sink component), and the composition additionally Also comprising from 20% to 80% by weight of a detergent water sink component. WO2013/188331 also relates to a method of treating and/or cleaning a surface, preferably a textile surface, comprising the steps of: (i) subjecting said surface in an aqueous wash liquor with a detergent as claimed and described herein The composition is contacted, (ii) rinsing and/or drying the surface.

The multi-enzyme co-granules may comprise the enzyme of the present invention and (a) one or more enzymes selected from the group consisting of first wash lipase, cleaning cellulase, xyloglucanase, Hydrolases, peroxidases, lipoxygenases, laccases, and mixtures thereof; and (b) one or more enzymes selected from the group consisting of hemicellulases, proteases, care fiber Sulfase, cellobiose dehydrogenase, xylanase, phospholipase, esterase, cutinase, pectinase, mannanase, pectin lyase, keratinase, reductase, oxidase, phenol oxidase , ligninase, pullulanase, tannase, pentosanase, lichenase, dextranase, arabinosidase, hyaluronidase, chondroitinase, amylase, and mixtures thereof.

method and use

In another aspect of the present invention relates to a method of preventing, inhibiting or reducing the growth of microorganisms on a surface by treating the surface with an enzyme as defined in the preceding paragraphs and aspects of the enzyme part of the present invention to a microbial TTC on said surface after treatment The detectable time is increased to an extent of at least 20%.

In one aspect, it relates to the above method, wherein the microorganism is a fungus, and the fungus on said surface has at least a 20% increase in sporulation time after treatment of the surface with an enzyme useful in the invention.

In one aspect, it relates to the above method, wherein the microorganism is a fungus, and the spore density score of the fungus on said surface is reduced by at least 1 on a scale from 0 to 7 following treatment of the surface with an enzyme useful in the invention.

In another aspect of the present invention relates to a method of demonstrating the microbial growth inhibition or deep cleaning benefits of an enzyme as defined in the preceding paragraph in the partial name of the enzyme of the present invention on a surface, the method comprising the steps of:

a. Provide two surfaces A and A',

b. Applying a soil comprising an indicator of microbial growth to each surface A and A',

c. washing A with a detergent composition not comprising enzymes and washing A' with said detergent composition comprising one or more enzymes,

d. Prepare to inoculate microorganisms and apply them to surfaces A and A',

e. incubating A and A' under suitable conditions to allow microbial growth for a predetermined period of time,

f. Compare the growth of microorganisms on A and A'.

The surface mentioned here can be any household surface, hard or soft like a fabric/textile surface. In one aspect, the surface is a textile made of natural fibers, synthetic fibers or mixtures thereof. In one aspect, the surface is made of cotton, polyester or blends thereof.

In one aspect, it relates to the method mentioned in the preceding aspect, wherein the microbial growth indicator is selected from the group consisting of triphenyltetrazolium chloride (TTC), INT (2-(4-iodo Substituted phenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazole), MTT(3-(4,5-dimethylthiazol-2-yl)-2,5- Diphenyltetrazolium bromide), XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazole-5-carboxanilide), MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazole), WST (water soluble tetrazolium salts) and CCK-8 (Cell Counting Kit-8), carotenoids, fluoresceins, fluorescent proteins/fluorescein-producing microorganisms, ATP, X-Gal, MUG, and combinations thereof. In one aspect, the microbial growth indicator is triphenyltetrazolium chloride (TTC).

In one aspect, it relates to the method mentioned in the previous aspect, wherein the surfaces A and A' provided in step a. described in the previous aspect are made of substantially the same material.

Commercial detergent compositions and textile pretreatment compositions

The detergent compositions mentioned below may incorporate enzymes suitable for the uses and methods of the invention.

Liquid detergent: Standard A

Liquid Detergent: Standard N

Liquid detergent: Standard O

Powder Detergent: Standard X

Liquid Detergent: Standard SEA

Biotex Black (Liquid)

5%-15% anionic surfactants, <5% nonionic surfactants, fragrances, enzymes, DMDM and hydantoins.

Ariel Sensitive White & Color Composition, Liquid Detergent Composition

Water, Alcohol Ethoxysulfate, Alcohol Ethoxylate, Amoxide, Citric Acid, C12-18 Topped Palm Kernel Fatty Acid, Protease, Glycosidase, Amylase, Ethanol, 1,2 Propylene Glycol, Sodium Formate, Chloride Calcium, Sodium Hydroxide, Silicone Emulsion, Trans Sulfate EHDQ (the ingredients are listed in descending order).

Standard Detergent A Composition (Liquid)

Ingredients: 12% LAS, 11% AEO Biosoft N25-7 (NI), 7% AEOS (SLES), 6% MPG (Propylene Glycol), 3% Ethanol, 3% TEA, 2.75% Cocoa Soap, 2.75% Soybean Soap, 2 % Glycerin, 2% Sodium Hydroxide, 2% Sodium Citrate, 1% Sodium Formate, 0.2% DTMPA and 0.2% PCA (all percentages are w/w).

Persil Bio Tablets

Sodium Carbonate, Sodium Carbonate Peroxide, Sodium Bicarbonate, Zeolite, Water, Sodium Silicate, Sodium Lauryl Sulfate, Cellulose, TAED, Sodium Dodecylbenzene Sulfonate, Hemicellulose, Lignin, Lauryl Glucose Glycoside, Sodium Acrylate/MA Copolymer, Bentonite, Sodium Chloride, Fragrance, Tetrasodium Etidronate, Sodium Sulfate, Sodium Polyacrylate, Simethicone, Anilinomorpholinotriazinylaminostilbene Sulfonate Disodium Salt, Dodecylbenzenesulfonic Acid, Trimethylsiloxysilicate, Calcium Carbonate, Cellulose, PEG-75, Titanium Dioxide, Dextrin, Protease, Modified Corn Starch, Sucrose, CI 12490, Polyarylsulfone Sodium Disulfide, Sodium Thiosulfate, Amylase, Kaolin.

Composition of Ariel Actilif (powder)

INGREDIENTS: 5%-15% Anionic Surfactants, Oxygen Based Bleach, <5% Nonionic Surfactants, Phosphates, Polycarboxylates, Zeolites, Optical Brighteners, Enzymes, Fragrances, Styrene Styrene Methyl propionaldehyde, coumarin, hexyl cinnamaldehyde.

The invention can also be described by the following paragraphs:

1. Use of one or more enzymes in preventing, inhibiting or reducing the growth of microorganisms on a surface, wherein after using said enzyme, the TTC detectable time of said microorganisms on said surface increases by at least 20%, at least 30%, at least 50%, at least 70%, at least 80%, or at least 100%.

2. The use according to paragraph 1, wherein the microorganism is a fungus, and the fungus has a sporulation time increased by at least 20% compared to a fungus on a surface not treated with an enzyme, and/or a spore density score from Decreases the spore density scale meter from 0 to 7 by at least 1.

3. Use according to paragraph 1 or 2, for inhibiting or reducing malodor caused by microorganisms on the surface of textiles.

4. Use according to paragraph 1 or 2, for preventing or reducing the attachment of microorganisms to textile surfaces.

5. Use according to any one of paragraphs 1-4, wherein the microorganism is a fungus or bacterium selected from the group consisting of Staphylococcus aureus, Klebsiella pneumoniae Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli, Pseudomonas putida, Micrococcusluteus, Staphylococcus epidermidis, Enhydrobactera erosaccus, Corynebacterium jeikeium, Propionibacterium acnes, Brevundimonas vesicularis, Streptomyces griseus, Streptomyces odorifer), Exophila phaeomuriformis, Fusarium oxysporum, Alternaria alternate, Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger niger), Aureobasidium pullulans, Chaetomium globosum, Cladosporium sphaerospermum, Gliocladium virens, Mucor plumbeus, yellow-green Penicillium chrysogenum, Penicillium commune, Penicillium crustosum, Penicillium rubens, Penicillium variants, Penicillium citrinum, Staphylococcus papyrus (Stachybotrys chartarum), Trichoderma viride, Trichophyton rubrum, Candida parapsilosis sis), Rhodotorula mucilaginosa, Debaryomyces hansenii, Meyerozyma guilliermondii, Candida albicans, and combinations thereof.

6. The use according to any one of paragraphs 1-5, wherein the microorganisms grow on remaining stained areas of the surface.

7. The use according to any one of paragraphs 1-6, wherein the one or more enzymes are selected from the group consisting of amylase, protease, lipase, mannanase, fiber Sulfase, pectinase, and combinations thereof.

8. The use according to any one of paragraphs 1-7, wherein the enzyme is an amylase, a protease, a cellulase, or a combination thereof.

9. The use according to paragraph 3 or 4, wherein the enzyme is a cellulase.

10. Use according to any one of the preceding paragraphs, wherein the protease is selected from the group consisting of:

i) a protease comprising a substitution at one or more of the following positions compared to the protease shown in SEQ ID NO 1 or SEQ ID NO 2: 3, 4, 9, 15, 24, 27, 42, 55 ,59,60,66,74,85,96,97,98,99,100,101,102,104,116,118,121,126,127,128,154,156,157,158,161,164 ,176,179,182,185,188,189,193,198,199,200,203,206,211,212,216,218,226,229,230,239,246,255,256,268,269 , wherein said position corresponds to the position of the protease shown in SEQ ID NO 1, or

ii) One or more protease variants of a protease parent, wherein said protease variant comprises one or more mutations selected from the group consisting of: S3T, V4I, S9R, S9E, A15T, S24G . 、S101A、V102I、V102Y、V102N、S104A、G116V、G116R、H118D、H118N、N120S、S126L、P127Q、S128A、S154D、A156E、G157D、G157P、S158E、Y161A、R164S、Q176E、N179E、S182E、Q185N、A188P , G189E, V193M, N198D, V199I, Y203W, S206G, L211Q, L211D, N212D, N212S, M216S, A226V, K229L, Q230H, Q239R, N246K, N255W, N255D, N255E, L256E, T2686D, among which the R2696D The position corresponds to the position of the protease shown in SEQ ID NO 1, wherein the protease parent is selected from the protease shown in SEQ ID NO 1 and the Bacillus amyloliquefaciens (Bacillus amylolichenifaciens) protease shown in SEQ ID NO 2 ( BPN'), and wherein said protease variant has at least 80% sequence identity to SEQ ID NO 1 or has 80% sequence identity to SEQ ID NO 2,

iii) a protease comprising a substitution at one or more positions corresponding to positions 171, 173, 175, 179 or 180 of SEQ ID NO: 14 compared to the protease shown in SEQ ID NO 14, wherein the the protease variant has at least 75% but less than 100% sequence identity to SEQ ID NO 14,

iv) a protease comprising the amino acid sequence shown in SEQ ID NO 1 or 2, or a protease with a polypeptide comprising amino acids 1-269 of SEQ ID NO 1 or comprising amino acids 1-269 of SEQ ID NO 2 275 polypeptides having at least 80% sequence identity,

v) one or more of the following protease variants selected from the group consisting of:

SEQ ID NO 1, with the changes T22R+S99G+S101A+V102I+A226V+Q239R,

SEQ ID NO 2, with the changes S24G+S53G+S78N+S101N+G128A+Y217Q,

SEQ ID NO 2, with the changes S24G+S53G+S78N+S101N+G128S+Y217Q,

SEQ ID NO 1 with alterations S9E+N42R+N74D+V199I+Q200L+Y203W+S253D+N255W+L256E,

SEQ ID NO 1, having S9E+N42R+N74D+H118V+Q176E+A188P+V199I+Q200L Y203W+S250D+S253D+N255W+L256E,

SEQ ID NO 1, having S9E+N42R+N74D+Q176E+A188P+V199I+Q200L+Y203W S250D+S253D+N255W+L256E,

SEQ ID NO 1, having S3V+N74D+H118V+Q176E+N179E+S182E+V199I+Q200L Y203W+S210V+S250D+S253D+N255W+L256E,

SEQ ID NO 1 with the alteration T22A+N60D+S99G+S101A+V102I+N114L+G157D+S182D+T207A+A226V+Q239R+N242D+E265F,

SEQ ID NO 1, with alterations S9E+N42R+N74D+H118V+Q176E+A188P+V199I+Q200L+Y203W+S250D+S253D+N255W+L256E,

SEQ ID NO 1, with alterations S9E+N42R+N74D+Q176E+A188P+V199I+Q200L+Y203W+S250D+S253D+N255W+L256E,

SEQ ID NO 1, with the changes S9E+N42R+N74D+H118V+Q176E+A188P+V199I+Q200L+Y203W+S250D+N255W+L256E+*269aH+*269bH,

SEQ ID NO 1, with the changes S3V+N74D+H118V+Q176E+N179E+S182E+V199I+Q200L+Y203W+S210V+S250D+N255W+L256E,

SEQ ID NO 1 with the alterations S9E+N74D+G113W+G157P+Q176E+V199I+Q200L+Y203W+S250D+T254E+N255W+L256E,

SEQ ID NO 1, with changes S3V+S9R+N74D+H118V+Q176E+N179E+S182E+V199I+Q200L+Y203W+S212V+S250D+N255W+L256E,

SEQ ID NO 1, with alteration S99E, and

SEQ ID NO 2, with alteration L217D,

and combinations thereof.

11. Use according to any one of the preceding paragraphs, wherein said amylase is selected from the group consisting of:

i) variants comprising one or more substitutions at the following positions: 9, 26, 30, 33, 82, 37, 106, 118, 128, 133, 149, 150, 160, 178, 182, 186 ,193,195,202,203,214,231,256,257,258,269,270,272,283,295,296,298,299,303,304,305,311,314,315,318,319 , 320, 323, 339, 345, 361, 378, 383, 419, 421, 437, 441, 444, 445, 446, 447, 450, 458, 461, 471, 482, 484, wherein the positions correspond to SEQ The location of ID NO 11;

ii) a variant exhibiting at least 90% identity to SEQ ID NO 12, having deletions at positions 183 and 184,

iii) a variant exhibiting at least 95% identity to SEQ ID NO 13 comprising a mutation at one or more of the following positions: M202, M208, S255, R172 and/or M261,

iv) a polypeptide comprising the amino acid sequence shown in SEQ ID NO:9 or 10,

v) a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96% of SEQ ID NO:9 or 10 %, at least 97%, at least 98%, at least 99% sequence identity,

and combinations thereof.

12. Use according to any one of the preceding paragraphs, wherein said cellulase is selected from the group consisting of:

a. a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity.

13. Use according to any one of the preceding paragraphs, wherein the surface is a textile made of natural fibers such as cotton, synthetic fibers such as polyester, or mixtures thereof.

14. Use according to any of the preceding paragraphs, wherein the one or more enzymes are incorporated into a detergent composition substantially free of biocides.

15. Use according to any of the preceding paragraphs, wherein the one or more enzymes are incorporated into a powder detergent or a liquid detergent.

16. A method of preventing, inhibiting or reducing microbial growth on a surface, said method comprising the steps of:

a. Treating said surface with an enzyme as defined in any one of paragraphs 10-12 to such an extent that the TTC detectable time of at least one microorganism on said surface increases by at least 20% after said treatment.

17. A method of demonstrating the microbial growth inhibition or deep cleaning benefit of an enzyme or combination of enzymes as defined in paragraphs 10-12 on a surface, said method comprising the steps of:

a. Provide two surfaces A and A',

b. Applying a soil comprising an indicator of microbial growth to each surface A and A',

c. washing A with a detergent composition not comprising enzymes and washing A' with said detergent composition comprising one or more enzymes,

d. Prepare to inoculate microorganisms and apply them to surfaces A and A',

e. incubating A and A' under suitable conditions to allow microbial growth for a predetermined period of time,

f. Compare the growth of microorganisms on A and A'.

18. The method of paragraph 17, wherein the microbial growth indicator is triphenyltetrazolium chloride (TTC).

19. The method according to paragraph 16 or 17, wherein said surfaces A and A' provided in step a. according to paragraph 14 are made of substantially the same material.

determination

washing assay

Terg-O-to-meter (TOM) washing assay

Tergo-To-Meter (TOM) is a medium-scale standard washing system, which can be applied to test 16 different washing conditions simultaneously. The TOM is basically a large temperature-controlled water bath with up to 16 open metal beakers submerged into it. Each beaker constitutes a small top loading washing machine and during the experiment each of them will contain a solution of a particular detergent/enzyme system and test its performance on soiled and unsoiled fabrics. The mechanical stress was obtained by rotating the stirring arm, which stirred the liquid in each beaker. Because the TOM cup does not contain a lid, it is possible to retrieve the sample during the TOM experiment and analyze the information online during the wash.

The TOM Standard Wash System is primarily used for mid-scale testing of detergents and enzymes under US or LA/AP wash conditions. Factors such as ballast to soil ratio and fabric to wash liquor ratio can be varied in a TOM experiment. Thus, TOM provides a link between small-scale experiments such as AMSA and microwashing, and more time-consuming full-scale experiments in top-loading washers.

Equipment: The water bath has 16 steel beakers with 1 swivel arm per beaker, each beaker has a capacity of 500 mL to 1200 mL of detergent solution. The temperature range is from 3.5°C to 60°C. The water bath must be filled with deionized water. The speed can be set up to 40 to 200rpm/min.

Set the temperature in the Terg-O-Tometer and start the rotation in the water bath. Wait for temperature adjustment (tolerance is +/-0.5°C). All beakers, stirring arms, and filters should be dishwasher clean and free from traces of previously tested material.

Prepare a wash solution with the desired amount of detergent and water hardness in a bucket. The detergent was allowed to dissolve for 10 min during magnetic stirring, and the pH of the detergent solution was measured after stirring for 10 min. Washing solutions should be used within 30 to 60 minutes of preparation.

Add 1000ml of wash solution to the TOM beaker. The wash solution was stirred at 120 rpm and allowed to spin until the temperature was correct. A small swatch is sprinkled into the beaker and ballast load, and one or more enzymes are then optionally added to the beaker. Time measurement begins when the swatch and ballast are added to the beaker. The swatches were washed for 20 minutes, after which time the agitation was stopped. Subsequently, the wash load was transferred from the TOM beaker to a container and rinsed with cold tap water. Separate the solid swatches from the ballast load. Under running water, transfer the soil swatch to a 5L beaker containing cold tap water for 5 minutes. Store ballast loads separately for upcoming inactivation. Gently press out the water from the small swatches by hand and place on a paper lined tray. Place another sheet of paper on top of the small swatch. The swatches were allowed to dry overnight before being subjected to analysis, eg, measuring color strength using Color Eye as described herein.

Full Scale Wash (FSW) Assay

FSW, Testing Product Performance in a Washing Machine under Scientifically Designed Conditions, is a home care application assay that is at its largest scale and most relevant to consumers at the end of the screening process. FSW system can be used in US or LA, AP and EU washing conditions according to different regional machines. Test swatches and ballast are added to each wash along with detergent and enzymes.

Equipment: AP washing machine - Panasonic (Panasonic) XQB65-Q680U automatic washing machine. The water used in these machines is deionized water. The normal wash program is the default program for mixing clothes and is used herein. Other suitable washing machines can also be used.

Enzymes: Enzymes can be added on a molar concentration, protein amount, or activity-based basis, for example.

Ballast: Clean white cloth (without optical brightener) consisting of cotton, polyester or cotton/polyester. The composition of the ballast was a mixture of different items in a cotton/polyester ratio of 65/35 by weight. Ballast weight, dryness and item composition must be the same in each wash. The ballast was inactivated after each wash in an industrial scrubber at 85°C/15min or in a wash without detergent (EU machine) at 90°C. Example of standard AP ballast composition, co/pe ratio at 65/35 (1.5kg±50g): 1 T-shirt (100% cotton) + 5 short sleeve shirts (55% cotton/45% polyester )+2 pillowcases (35% cotton/65% polyester, 110x 75cm)+3 tea towels (100% cotton). Ballast weight adjusted to correct ballast with shirt, short sleeves.

Test small swatches: Test small swatches, ie technical and natural stains, commercial or NZ produced. All washes in the test must use the same batch. Different swatch sizes are available. The swatches were attached to the tea towels with a stapler; the same swatch type (stain) was placed on different towels or in different locations on the towels. Each swatch is individually marked for identification and to indicate the front. It's important to keep small swatches in the dark and always limit exposure, as many stains are light sensitive. The total weight of the textile includes the weight of the ballast and the test swatches.

Water Hardness: Natural waters contain varying levels of metal ions (mainly Ca ²⁺ and Mg ²⁺ ), depending on the geographic region. The metal ions thought to constitute water hardness are those of the precipitated fatty acid soaps (mainly Ca ²⁺ and Mg ²⁺ , but not eg Na ⁺ ). Natural water also contains varying levels of bicarbonate, HCO ₃ ⁻ , with an average molar number 1.5 times that of the sum of Ca ²⁺ and Mg ²⁺ on a molar basis, and may contain many other anions, including Cl ⁻ and SO ₄ ²⁻ . The presence of HCO ₃ ^- is important for the buffering capacity of water and affects the pH of the wash solution prepared by adding detergent. Artificial hard water is made by adding CaCl ₂ , MgCl ₂ and NaHCO ₃ to Milli-Q water or deionized water.

Washing conditions: Standard AP washing conditions are as follows

Other wash conditions may apply. Washing conditions for each zone of normal heavy duty washing are exemplified in the table below.

*°dH: adjusted by adding CaCl ₂ *2H ₂ O, MgCl ₂ *6H ₂ O and NaHCO ₃ to milli-Q water.

Note (this is an estimate rather than absolute, different regions and different machines have special conditions)

washing program

a. Prepare ballast and test swatch (stitched to tea towel).

b. Select parameters for washing: program and water level. Note that when using autofill, the known water level is not the true water level. For the 32L program, the actual water intake is 33L. Enzyme and detergent additions should be adjusted to reflect this water level.

c. Program and start the automatic water meter system.

d. Press the start/pause button to start water filling and adjust the water temperature during dispensing. Water consumption is automatically registered during this period.

e. Add Ca/Mg according to water consumption

a. Press the button to stir the water.

b. Stop and check the water hardness.

f. Add detergent and powdered NaHCO ₃ to machine - short mix (10 seconds).

g. Add enzyme solution or pellets - short mix (10 seconds).

h. Add ballast and swatches to washer.

i. Quickly restart the washing machine, select the washing program and water level, and start washing by pressing the start/pause button. If soaking, stir for 30s, then maintain soaking time. If a low water level is detected, the machine will automatically fill with water (0.2-1L) during the main wash.

j. Measure the pH after washing for 8 or 10 min.

k. The washing machine rinses 2 times by default. Rinse with water of the same hardness. If excessive foaming is detected, the machine will automatically add 1 rinse. Water consumption is automatically registered during this period.

l. After the wash is complete, remove the test swatch from the tea towel and place on a tray to dry - make sure the swatch is dried in complete darkness as many stains are light sensitive. The small swatch is allowed to dry overnight and must be completely dry before measuring.

Mini Launder-O-Meter (Mini LOM) Standard Washing System

Mini-LOM is a modified mini-wash system of Launder-O-Meter (LOM), which is a mid-scale standard wash system, which can be applied to test up to 32 different wash conditions simultaneously. The mini LOM model washing system is mainly used for washing sanitation experiments. The Mini LOM is a spinner that includes free 50ml x 16 (usually 6-32) tube spinner fittings, each tube holds 10 to 20mL of detergent solution. The water bath must be filled with sterile deionized water. Each tube will contain a solution of the specific detergent/enzyme system and soiled and unsoiled fabrics whose performance has been tested. Mix them horizontally or vertically by rotating the axis to achieve mechanical stress. It can be used at ambient temperatures from 4°C to 55°C, and the rotation range is from 10 to 70rpm. In mini-LOM experiments, factors such as microbial load and fabric pieces can be varied. Tubes should be discarded after washing.

Prepare a wash solution with the desired amount of detergent and water hardness in a 1 L beaker. The detergent was allowed to dissolve for 10 min during magnetic stirring, and the pH of the detergent solution was measured after stirring for 10 min. Washing solutions should be used within 30 to 60 minutes of preparation.

Add 20ml of wash solution to sterile mini-LOM tubes. The wash solution was agitated at 20 rpm. Scatter small swatches into the beaker and load of microbial broth. All of the above operations should be performed on a clean bench. Time measurement begins when the swatch and microbial culture are added to the beaker and the mini-LOM machine is moved to the chamber with the target temperature. The swatches were washed for 60 minutes, after which time the agitation was stopped. After washing, the swatches were transferred from the tubes to a beaker with 400ml sterile water and rinsed for 10 min. The small swatches were then processed according to the reduced attachment assay.

Stain Recipe

Preparation of soiled swatches:

A clean swatch (6 x 6 cm) was placed in the cylindrical bottom of a 25 mL round bottom flask (bottom diameter was about 3.8 mm) and the swatch was held in place with one hand. The prepared soil was loaded into the center of the swatch with a pipette, and the stain solution was then spread evenly over the surface of the swatch with fingers. Three different types of swatches from the Center For Testmaterials BV in the Netherlands mentioned below were used.

dirt assessment

Wash performance is expressed as delta reflectance value (ΔRem). After washing and rinsing, the swatches were spread out and allowed to air dry overnight at room temperature. All washes were evaluated the day after washing. Light reflectance evaluation of small swatches was performed using a MacbethColor Eye 7000 reflectance spectrophotometer with a very small aperture. The measurement was performed without ultraviolet (UV) light in the incident light and the reflectance at 460 nm was extracted. Measurements were made on unwashed and washed swatches. Place the test swatch to be measured on top of another swatch of the same type and color (pair swatches). There was only one swatch of each in each beaker, and swatches from duplicate washes were used in this way. The reflectance values for individual swatches were calculated by subtracting the reflectance values for the unwashed swatches from the reflectance values for the washed swatches. The total wash performance for each soiled swatch set was calculated as the sum of the individual ΔRems.

The enzyme effect for each stain was calculated by taking the measurement from the swatch washed with enzyme and subtracting the measurement from the swatch not washed with enzyme. Overall enzyme performance was calculated as the sum of the individual ΔRem _enzymes .

TTC assay (in situ detection of microbial growth)

Triphenyltetrazolium chloride (TTC) is a reduction-oxidation reaction indicator and can be used to distinguish metabolically active from inactive tissues/organisms. It is white in color, and when TTC is enzymatically reduced in living cells to water-insoluble TPF (1,3,5-triphenylmethanol ), the color turns red due to the activity of various dehydrogenases (enzymes that play an important role in the oxidation of organic compounds and thus in cellular metabolism). Scheme I below shows the reaction from TTC to TPF. For this reason, TTC can be used to detect microbial growth.

Option I

Other microbial growth indicators with TTC-like microbial growth indicator function can be used in this study. They can include redox indicators, live cell dyes, colored metabolites, and metabolite detection assays. Redox indicators include all redox assays such as INT (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazole), MTT (3- (4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), XTT (2,3-bis-(2-methoxy-4-nitro-5 -sulfophenyl)-2H-tetrazole-5-carboxanilide), MTS(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)- 2-(4-sulfophenyl)-2H-tetrazole), WST (water soluble tetrazolium salt) and CCK-8 (Cell Counting Kit-8). Viability dyes may include Acridine Orange, Calcein-AM and Hoechst. All colored metabolite assays/microorganisms should be included, such as all carotenoids, fluoresceins, fluorescent proteins/microorganisms that produce fluorescence. Metabolite detection assays include all metabolite and enzyme detection assays such as ATP detection assays using X-Gal or MUG to detect β-galactosidase.

Material:

a. TTC solution.

b. Dissolve 0.1 g of 2,3,5-triphenyltetrazolium chloride (TTC) (Sigma, cas: 298-96-4) in 2 mL of sterilized Mili-Q water as 1000 × stock solution (50 mg/mL).

c. Medium

Nutrient agar medium: in 1 liter of distilled water, 5g peptone, 30g beef extract, 5g NaCl, 15g agar, adjust the pH to 7.0-7.2. This medium is used to grow Pseudomonas putida

• Potato Dextrose Agar (PDA): 200 g of sliced unpeeled potatoes were boiled in 1 liter of distilled water for 30 min. Strain through cheesecloth, reserving the outflow, which is the potato extract (or use a commercially dehydrated form). Mix with 20g dextrose and add water to dilute to 1L. This was distributed to flasks, and 15 g/L agar was added to each flask, followed by autoclaving at 121 °C for 15 min. 20-25 ml portions were then dispensed into sterile 15 x 100 mm Petri dishes. This medium is used to grow fungi.

d. PBS buffer (0.01M, pH 7.4), sterilized at 121°C for 15min, and stored at 4°C.

program:

1. Preparation of Microbe/TTC Solution

a) Incubate the bacteria or fungi on each respective solid medium until colonies form or spores are seen.

b) Inoculate a single colony into a liquid medium and incubate overnight at the optimum growth temperature for each microorganism according to the specification of the microorganism until the OD600 reaches about 0.6-0.8), and then culture a part at a volume ratio of 0.1%-0.5% solution was added to fresh liquid medium, incubated at 28°C-37°C for about 8-12 hours, then the culture pellet was collected and resuspended in sterile PBS buffer twice, and a solution with an OD600 of about 0.10-0.13 was prepared.

c) Add TTC solution (50mg/ml) to the microbial solution from step 2 to bring the TTC concentration to 50mg/L, mix well and use immediately.

2. Preparation of Stained Small Cloth Swatches

a) After the washing procedure described in the Washing Determination section the swatch having completed the washing step is rinsed with water for 7-10 min and the swatch is placed on the plate. Plates were sterilized by immersion in 75% ethanol and irradiated with UV light for 0.5-1 hr before use.

b) Inject 300 μL of bacterial or fungal spore solution evenly on each swatch by using a pipette

c) Incubate the thus prepared swatches in a sterile culture chamber at 34°C, 90%-98% RH, regardless of whether the microorganisms applied to the swatches are bacteria or fungi.

3. TTC in situ detection of microbial growth on small cloth samples.

Small swatches are removed from the incubator at 6-hour intervals and can be evaluated on the scanner. First, scan a small piece of cloth with a scanner (e.g., Epson Expression 10000XL) at predetermined settings (for example, Silverfast's software can be used and set to scan at 200dpi and 48→24 bit color) and then analyze the scanned small piece Image of cloth swatch. A software with a color vector program for RGB readout can be used and a circled area with a diameter 1.2 times the stain diameter is selected for color analysis purposes. Color intensity on swatches is calculated according to the following equation. The strength of the tested swatch was then compared to that of a similarly soiled and washed (but no enzyme used in the present invention) swatch, and if the change in strength was a reduction of 4, the time was recorded as TTC detects time.

When the surface is of a type other than a swatch, essentially the same procedure as above will be applied for TTC determination purposes, except that the washing conditions need to be adjusted to suit the specific surface washing purpose.

Determination of fungal spore/mycelium formation time (mainly used for in situ detection of fungal growth)

program:

The above steps described above in the TTC determination section apply here, with the only difference that no TTC is added to the culture solution (as in step 1c) and the microorganisms inoculated are fungi instead of bacteria.

After step 2c), small swatches were taken at 12 hr intervals for microscopic examination to check for sporulation. An Olympus SZX16 microscope can be used and any conidia visible in 3 fields of view can be randomly selected under the microscope using 10X magnification. Depending on the species of fungus, their spores/mycelia may appear black, green or red in color. Colors can be ordered by studying the specifications of the fungus.

Once the spores (conidia, sporangia, or other types of spores) or mycelia are screened for inspection under a 10X stereomicroscope, then at a higher magnification of 50X to confirm the size of the conidia heads, if conidia The diameter of the spore head is not less than 20 μm (for Aspergillus niger) or the diameter of the mycelium is not less than 2 μm, and then the time of fungal sporulation time is recorded.

Spore Density Scoring Determination

The spore density score was used to determine the growth of the fungus as indicated by the visible markings on the household surface caused by the spores/conidia of the fungus growing on the surface.

Small swatches from the TTC assay section above were taken to measure spore density scores after 7 days of incubation.

A range of 0 to 7 was set for measuring spore density values, where 0 indicates no fungal growth on the surface and 7 indicates excessive fungal growth on the surface. The color of the spores can be different for different fungi, but the same 0-7 range can be applied to different fungal growth and spore density measurements. A group of panelists was first trained to know the range and corresponding spore density values, and then scored for spore density by comparing each surface (swatch) tested to the range.

Screening of Aspergillus niger used in examples

The filamentous fungus Aspergillus niger (Novozymes internal strain number 57825) was used. The strain was isolated in 2014 from moldy corn in Liaoning Province, China. It was isolated and cultured in a potato dextrose agar (PDA) medium at 25°C for 3 days. Alternatively, other commercially available Aspergillus nigers may be used interchangeably with this strain in the examples.

example

The following examples further describe and demonstrate aspects within the scope of the present invention. The examples given are for the purpose of illustration only and are not to be construed as limitations of the invention, since many variations thereof are possible without departing from the spirit and scope of the invention.

Example 1 Prevention of Fungal Growth on Dirt on Chopping Boards Treated with Enzymatic Hand Dishwashing (HDW) Detergents

Material

1. HDW Dirt Formula:

35% potato starch (food grade, Huaou Company, China) + 2% semi-skimmed milk (Oldenburger, Germany) + 2% egg yolk (from fresh eggs) + 1.3% soy sauce (Lee Kum Kee Company, China)

2. Fungus - Aspergillus niger

The filamentous fungus Aspergillus niger (Novozymes internal strain number 57825) was used. The strain was isolated in 2014 from moldy corn in Liaoning Province, China. It was isolated and cultured in a potato dextrose agar (PDA) medium at 25°C for 3 days. It is believed that, in examples, other commercially available Aspergillus nigers may be used interchangeably with this strain.

3. Cutting board

Wooden chopping boards from supermarkets (Taixing, ginkgo wood)

4. Detergent: SEA standard detergent, washing liquid 0.75g/L

5. Enzyme: protease (SEQ ID NO:3), amylase (SEQ ID NO:9)

6. Grouping:

program:

1. Procedure for Preparation of Fouling Solution

5% potato starch was gelatinized in tap water at 85°C for 5 min to form a hot paste, and then 2% semi-skimmed milk and 2% egg yolk were added, and further 1.3% soy sauce was added to make a homogeneous solution.

2. Preparation of Fungal Spores

Aspergillus niger was incubated on petri dishes (PDA or czapek's medium) in an incubator at 30°C for 3 days. The spores were collected by washing the plate with sterile PBS buffer (0.01 nM, pH 7.4); the spore solution thus prepared was vortexed vigorously for 5 min; the spore solution was then diluted so that the final _OD600 reached between 0.10-0.13.

3. Enzyme treatment of chopping boards and comparison of fungal growth

0.5 g of the freshly made soil solution according to step 1 above in this example was applied to each panel separately, and the panels were then dried at 25°C for 2 hours.

Each group then added its corresponding wash solution as listed in the grouping table shown above. The plates were then left to stand for 10 min. Afterwards, sponge scrub the board with 5 scrubs under running tap water.

At a concentration of OD = 0.124, 1 ml of the Aspergillus niger spore solution prepared according to the procedure described in the TTC measurement section of the above measurement section was inoculated onto each plate by using a pipette, respectively. The chopping plate was then incubated at 34°C, 90%-98% RH, where the incubator was sterilized with UV before incubating the plate.

Fungal growth was observed every 12 hours. By following the procedure described in the fungal sporulation assay section in the assay section, record the time when visible black sporangia/spores are identified by the naked eye.

result:

1. Sporulation time

Table 1. Time to visible conidia formation of Aspergillus niger on chopping board swatches

It can be seen from Table 1 that all the chopping boards from the enzyme-treated test groups 1-3 had longer sporulation times than the blank group and the contaminated negative control group, and the blank group and the negative control group were only Wash with water or with a liquid detergent that does not itself contain this protease and amylase. Specifically, the sporulation time of the test group was at least 1.5 fold, ie at least 50% increased compared to the negative control group.

The results clearly demonstrate the effectiveness of protease and amylase used in water or liquid detergent in inhibiting the growth of Aspergillus niger on chopping boards.

2. Spore Density Score Measurement

After 7 days of culture by following the procedure described in the Spore Density Scoring Determination Section of the Determination Section in the preceding paragraph, the spore density score of each cutting board was assessed by panel observation.

Table 2. Spore Density Scores - Panel Scores and Means

As shown in Table 2, all test groups 1-3 chopping boards from the enzyme treatment had much lower spore density scores than the blank group (score 7), and at least one score was lower than the negative control group (score 3), The blank and negative controls were soiled and washed with water only or with a liquid detergent which itself did not contain the protease and amylase.

This clearly shows the effect of the protease and amylase used in the water or liquid detergent to help inhibit the growth of the fungus, Aspergillus niger, on the chopping board.

Example 2. Inhibition of Bacterial Growth by Washing with Enzymatic Liquid Detergents Containing Different Doses of Enzymes

Material

1. Bacteria: Pseudomonas putida (China General Microorganism Culture Collection Center (CGMCC), strain number 1.3096)

2. Detergent: SEA standard detergent

3. Enzyme: protease (SEQ ID NO:3), amylase (SEQ ID NO:9)

4. Surface: Plastic chopping board (Jie neng brand purchased from a supermarket, polypropylene), slightly sliced with a saw, and a few lines were drawn on its surface to simulate the board used in real life.

5. Stain Recipe: Baby Food No. 2

6. Grouping:

program:

1. Procedure for Preparation of Fouling Solution

Baby food soil No. 2 was prepared as described in the Stain Recipe section in the Assays section. Apply 1 mL of each soil solution to each corresponding chopping board. The plates were then air dried at room temperature for 3 hrs.

2. Enzyme treatment of chopping boards and comparison of bacterial growth

Then, 1 mL of its corresponding wash solution was added to the plates from each group, as listed in the grouping table shown above. The plates were then left to stand for 10 min. Afterwards, sponge scrub the board with 5 scrubs under running tap water.

At a concentration of OD = 0.124, 700 uL of bacterial solutions prepared according to the procedure described in the TTC measurement section of the above measurement section were inoculated onto each plate by using a pipette, respectively. The chopping plate was then incubated at 34°C, 90%-98% RH, where the incubator was sterilized with UV before incubating the plate. Observe the TTC detectable time of the plate by following the procedure described in the TTC Assay section in the Assay section.

result

1. TTC detectable time

The TTC detectable time was measured following the procedure in the TTC Determination section in the Assay section.

table 3

blank test 1 test 2 negative control test 3 Test 3/Negative 24 hours >48hr >48hr 24 hours >48hr >2

The results in Table 3 show that for plates from all test groups 1-3, washed with water or a detergent containing a blend of protease and amylase, respectively, the TTC detectable time was significantly prolonged compared to the negative control group (ratio >2). This suggests that bacterial growth was inhibited by using these enzymes in water or a liquid detergent applied to plastic chopping board surfaces.

Example 3. Inhibition of fungal growth by washing with an enzymatic liquid detergent comprising a combination of amylase and protease

Material

1. Microorganism-Aspergillus niger (same as in example 2)

2. Stain: Baby Food No. 3

3. Detergent: BlueMoon Deepclean liquid detergent

4. Small swatch types: PCN01, W30A and CN42

5. Enzyme: protease (SEQ ID NO:3), amylase (SEQ ID NO:9)

6. Grouping:

program:

1. Prepare soiled swatches according to the procedure described in the Washing Assay section above.

2. Use the TOM washing program. Washing conditions: TOM, 30°C, 120rpm, 14°dH (Ca ²⁺ :Mg ²⁺ =3:2), 10/20min (when the textile of the test small piece of cloth is CN42, wash for 20min, when the test Small pieces of textiles are PCN01 or W30A, washed for 10 minutes).

3. Preparation of fungal spore inoculum and application to washed swatches and observation of fungal growth on swatches from different groups as described in the Assay section Sporulation Assay section.

result

1. Sporulation time

Table 4. Time to visible conidia formation of Aspergillus niger on swatches

As can be seen from the results in Table 4, the test group swatches were washed with detergents containing protease and amylase, at least PCN01 and W30A showed the same results as the unsoiled and unwashed swatches. The results from the test group and the blank control group were significantly longer than the two negative control groups, which were soiled but not washed, or soiled and washed with liquid detergent itself without this protease and amylase . This clearly shows that protease and amylase can be used to inhibit the growth of the fungus, Aspergillus niger.

2. Spore Density Score Measurement

Each swatch was observed by a panel and scored for size and density of spore-conidia. Measurements refer to the "Spore Density Scoring" section under the Assays section.

Table 5. Spore Density Scores - Panel Scores and Means

As indicated by the results shown in Table 5, the test group swatches were washed with detergents containing protease and amylase, at least the PCN01 and W30A swatches showed a similarity with the unsoiled and unwashed swatches. same result.

In addition to this, the results from both the test group and the blank control group were significantly smaller than the two negative control groups, which were either soiled but not washed, or soiled and treated with water without this protease and amylase. The detergent itself washes.

These two aspects clearly show that proteases and amylases can be used to prevent, inhibit or reduce the growth of the fungus, Aspergillus niger.

Example 4. Inhibition of fungal growth by washing with an enzyme powder detergent comprising a combination of amylase and protease

Material

1. Microorganism-Aspergillus niger (same as in example 2)

2. Stain: Baby Food No. 3

3. Detergent: Standard X

4. Small swatch type: W30A and CN42

5. Enzyme: a blend of protease (SEQ ID NO: 1 ) and amylase (SEQ ID NO: 10).

6. Grouping:

program:

1. Prepare soiled swatches according to the procedure described in the Washing Assay section above.

2. Use the TOM washing program. Washing conditions: TOM, 30°C, 120rpm, 14°dH (Ca ²⁺ :Mg ²⁺ =3:2), 20min.

3. Preparation of fungal spore inoculum and application to washed swatches and observation of fungal growth on swatches from different groups as described in the Assay section Sporulation Assay section.

result

1. Sporulation time

Aspergillus niger is a filamentous fungus with black spores that form asexual spores called conidospores (conidia) during growth. Aspergillus niger conidia heads can be observed with the naked eye. Record when visible black conidia can be seen.

Table 6

As can be seen from the results in Table 6, the test group swatches were washed with a detergent containing protease and amylase, at least the W30A swatches showed the same results as the unsoiled and unwashed swatches . In addition, the above results show that the test group and the blank control group have significantly longer sporulation time than the two negative control groups, which are soiled but not washed, or soiled and treated with a method without this protease. And amylase powder detergent itself for washing. Two aspects of the results clearly show that protease and amylase can be used to inhibit the growth of the fungus, Aspergillus niger.

2. Spore Density Score Measurement

Each swatch was observed by a panel and scored for size and density of spore-conidia. Measurements refer to the "Spore Density Scoring" section under the Assays section.

Table 7. Spore Density Scoring

The results in Table 7 above show that the test group swatches washed with a detergent comprising protease and amylase showed essentially the same results as the unsoiled and unwashed swatches from the blank control group. The results from both the test group and the blank control group were significantly smaller than the two negative control groups which were either soiled but not washed, or soiled and washed themselves with a detergent that did not contain this protease and amylase of. This clearly shows the effect of protease and amylase for preventing, inhibiting or reducing the fungus, Aspergillus niger.

Example 5. Inhibition of fungal growth by washing with enzyme liquid detergents containing different doses of enzymes

Material

1. Microorganism - Aspergillus niger. (same as in instance 2)

2. Stain: Baby Food No. 1

3. Detergent: Standard O

4. Small swatch type: PCN01 and CN42

5. Different enzyme groups:

A: protease alone (SEQ ID NO:3),

B: amylase alone (SEQ ID NO:9),

C: A blend of protease (SEQ ID NO:3), amylase (SEQ ID NO:9) in a weight ratio of 4:1.

6. Grouping:

program:

1. Prepare soiled swatches according to the procedure described in the Washing Assay section above.

2. Use the TOM washing program. Washing conditions: TOM, 30°C, 120rpm, 14°dH (Ca ²⁺ :Mg ²⁺ =3:2), 20min.

3. Preparation of fungal spore inoculum and application to washed swatches and observation of fungal growth on swatches from different groups as described in the Assay section Sporulation Assay section.

result

1. Sporulation time

A. niger conidia can be observed by naked eye following the same procedure as in the previous examples. Record when visible black conidia can be seen.

Table 8. Visible conidia formation time

As can be seen from the results in Table 8, for test groups C and B, they were washed with detergents comprising a blend of protease and amylase and amylase itself, respectively. For both PCN01 and W30A swatches, the above results show that at the lower enzyme dose, the fungal sporulation time was significantly prolonged compared with the negative control group, and at the higher dose, the fungal sporulation time was even compared with Unstained and unwashed blanks were the same.

This result demonstrates the fungal growth inhibition benefit of using enzymes in powder detergents. It can also be seen that W30A is better than PCN01 in delaying fungal sporulation.

2. Spore Density Score Measurement

Each swatch was observed by a panel and scored for size and density of spore-conidia. Measurements refer to the "Spore Density Scoring" section under the Assays section.

Table 9

The results in Table 9 show the swatches from all three test groups A-C, and for the PCN01 and W30A swatches, showed a lower fungal density score at lower enzyme doses compared to the negative control group. Much more, and at higher doses, fungal sporulation times were even the same as unstained and unwashed controls.

This clearly shows that proteases and amylases can be used to prevent, inhibit or reduce the growth of the fungus, Aspergillus niger.

3. CFU count

In addition, CFU counts were performed to quantify fungi on small swatches. A PCN01 swatch from test group C was used for this purpose. The number of spores was calculated from CFU by the following equation: ((mean CFU at dose of 10 ^-n ) x 10 ⁿ /0.2 mL) x 10 Ml.

Table 10

The results in Table 10 clearly show that the test group had a significantly lower amount of fungal residue on the swatches treated with the enzymatic detergent comprising protease and amylase.

Example 6. Inhibition of Bacterial Growth by Washing with Enzyme Liquid Detergents Containing Different Doses of Enzymes

Material

1. Bacteria: Pseudomonas putida (China General Microorganism Culture Collection Center (CGMCC), strain number 1.3096)

2. Detergent: Standard O, washing solution concentration 2g/L

3. Enzyme: protease (SEQ ID NO:3), amylase (SEQ ID NO:9)

4. Small swatches: CN42 and W30A

5. Stain Recipe: Baby Food No. 2

6. Grouping:

program:

1. Prepare soiled swatches according to the procedure described in the Washing Assay section above.

2. Use the TOM washing program. Washing conditions: TOM, 30°C, 120rpm, 14°dH (Ca ²⁺ :Mg ²⁺ =3:2), for 10/20min (when the textile of the test small piece of cloth is CN42, wash for 20min, when the test The textiles of the small cloth samples are PCN01 or W30A, washed for 10 minutes).

3. Preparation of bacterial inoculum and its application to washed swatches, and growth of TTC color changes observed on swatches from different groups as described in the TTC Assay section in the Assay section.

result

1. TTC detectable time

The TTC detectable time was measured following the procedure in the TTC Determination section in the Assay section.

Table 11

As can be seen from the results in Table 11, both W30A and CN42 showed a significantly prolonged TTC compared to the negative control 1 group for the test group swatches washed with a detergent comprising a blend of protease and amylase Detectable times, and test/negative 1 ratios were 2 and 1.5 for the W30A swatches and CN42 swatches, respectively.

This suggests a bacterial growth inhibition benefit for cleaning fabric surfaces by using these enzymes in liquid detergents.

Example 7. Use of cellulase in reducing bacterial attachment to textiles

Material:

1. Detergent: liquid detergent: standard A; powder detergent: standard X

2. Microorganism: Pseudomonas putida (China General Microorganism Culture Collection Center (CGMCC), strain number 1.3096)

3. Pigment soiling: WFK09V from Center For Testmaterials BV (CFT). It is the standard pigment mix in all soiled/stained test fabrics containing "pigment". The addition of pigment soils simulates real washing situations where relatively dirty laundry is present releasing soil into the wash liquor.

4. Washing machine:

a. Mini LOM, 25℃, 60min, 20rpm, 20ml washing solution, rinse in 400ml washing beaker for 10min, when using liquid detergent standard N

b.TOM, 25°C, 30 or 20min, 120rpm, 500ml washing solution, rinse in a 500ml washing beaker for 10min, when using powder detergent standard X

5. Small piece of fabric sample: Emp252 (knitted striped cotton textile)

program:

1. Pre-washing of small swatches: the small swatches are first aged in the Wascator machine, program 151, aging time: 10h. The test swatches were then washed with a standard detergent, either Liquid Detergent Standard A containing 0.075 ppm cellulase (SEQ ID NO: 15); or 0.05 ppm cellulase (SEQ ID NO: 15) powder detergent X; and the control swatches were washed in FSW with the respective standard detergent only. After 20 cycles of washing, the swatches were cut into circles with a diameter of 1.9 cm and then sterilized in an autoclave at 121 °C for 15 min and dried in an oven at 60 °C.

Table 12. FSW Wash Conditions for Standard A:

EU washing machine washing conditions washing program standard volume 15L Washing time (main washing) 51min temperature 40℃ water hardness 15°dH(2:1:7.5) rinse 2 Ballast 3Kg ballast (mixed 60% cotton + 40% polyester)

Table 13. FSW Wash Conditions for Standard X:

AP washing machine washing conditions washing program standard volume 33L Washing time (main washing) 15min temperature 25°C water hardness 14°dH(2:1:4.5) rinse 2 ballast 1.5Kg ballast (mixed 60% cotton + 40% polyester)

2. Second wash above pre-washed swatches plus pigment soil and bacterial inoculation

1) Prepare fresh Pseudomonas putida culture solution up to OD ₆₀₀ =0.3 according to routine procedures.

2) Load the detergent and pigment soil into the mini LOM beaker (or 500mL wash solution of the TOM beaker) by adding water (water hardness 14°dH, Ca ²⁺ : Mg ²⁺ :Na ⁺ = 2:1:4.5) In, the concentration of pigment soil was 0.7g/L, and the concentration of detergent was 2g/L, to prepare 20ml of washing liquid. Then 100 μL of fresh bacterial culture (OD ₆₀₀ =0.3) for mini-LOM wash or 1900 ul for TOM wash was added to the wash.

3) Put 3 pre-washed swatches into each of the mini-LOM beaker or TOM beaker, for mini-LOM, wash at 25°C, 20rpm for 60min, for TOM, wash at 30°C, 120rpm for 30min .

4) After washing, remove the swatch from the beaker and place in 400 mL sterile water after washing in TOM, in mini-LOM or 500 mL sterile water. Rinse for 10 minutes.

3. Measure the amount of bacteria remaining on the swatches: Transfer the different swatches to the nutrient agar plate and let it sit for 20 seconds. Incubate the agar plate in an incubator at 28°C for about 12-24 hours until colonies form and determine the CFU.

result:

Table 14. CFU of bacteria attached to Emp252 swatches after prewashing with liquid detergent

Group Average CFU Small swatches prewashed without cellulase 142 A swatch pre-washed with cellulase (SEQ ID NO: 15), 108 Percentage reduction 23.9%

As can be seen from the results in the table above, for those small pieces of cloth swatches prewashed with a liquid detergent containing cellulase (SEQ ID NO: 15), when it is further co-washed with relatively dirty clothes ( By adding pigment soil and bacteria to mimic those released into the wash solution from this relatively dirty laundry), the prewashed swatches were compared with those swatches that were only prewashed with a detergent that did not contain cellulase. 23.9% fewer bacterial CFU remained on the surface of the small swatch. This result shows that cellulases can be effectively used to reduce bacterial attachment to textiles.

Table 15. Bacterial CFU residues on Emp252 swatches after prewashing with powder detergent

As can be seen from the results in the table above, for those small pieces of cloth swatches prewashed with a liquid detergent containing cellulase (SEQ ID NO: 15), when it is further co-washed with relatively dirty clothes ( By adding pigment soil and bacteria to mimic those released into the wash solution from this relatively dirty laundry), the prewashed swatches were compared with those swatches that were only prewashed with a detergent that did not contain cellulase. 33.0% fewer bacterial CFUs remained on the surface of the swatch. This result shows that cellulases can be effectively used to reduce bacterial attachment to textiles.

Example 8 Cellulase is used in reducing the adhesion of fungi to textiles

The carrying out of example 8 is basically the same as the previous example, and the difference is that the fungus Candida parapsilosis (China Common Microorganism Culture Collection and Management Center, (strain number: 2.1846)) replaced the bacteria Pseudomonas putida, Pseudomonas parapsilosis The amount and culture conditions of Trichozyme were correspondingly different from the foregoing examples.

1) The amount of yeast loading is: load 100 μL of fresh yeast culture solution (OD ₆₀₀ =0.3) into the washing solution for mini-LOM, and load 1000 μL of fresh yeast culture solution (OD ₆₀₀ =0.3) into the washing solution for TOM middle.

2) The washing time of TOM is 20min.

3) The medium used for Candida parapsilosis is yeast extract-peptone-dextrose (YPD) medium: 10g/L yeast extract, 20g/L peptone, 20g/L dextrose (the dextrose Sugar is sterilized separately from other components and mixed together after sterilization), 20g/L agar is used for solid medium.

Independent of the CFU determination, the swatches were subjected to fluorescence detection from the microorganisms remaining on the swatches using the PrestoBlue ^™ Cell Viability Reagent to compare the viability of the microorganisms remaining on them after the second wash.

PrestoBlue ^™ cell viability reagent (Thermo Fisher Scientific, catalog number: A13261 ) was used. PrestoBlue is a resazurin-based cell permeable solution that acts as an indicator of cell viability by quantitatively measuring the proliferation of cells using the reducing power of living cells. When added to cells, PrestoBlue reagent is modified by the reducing environment of living cells and turns red in color, becoming highly fluorescent. Fluorescence or absorbance measurements can be used to detect this color change. PrestoBlue solution was prepared at a ratio of PrestoBlue ^™ cell viability reagent:YPD medium=1:9.

After washing in the TOM or mini-LOM, the swatches in different beakers were rinsed with 500 mL of sterile water for 10 min, and excess water was carefully removed from the swatches. Transfer small swatches to a 12-well plate. 2ml of PrestoBlue solution was added to each well, then the plate was covered and the fluorescence kinetics were measured (excitation: 544nm, emission: 590nm; interval: 30min, duration: 22h).

result:

Table 16 Fungal CFU residues on Emp252 swatches after prewashing with liquid detergent.

As can be seen from the results in the table above, for those small pieces of cloth swatches prewashed with a liquid detergent containing cellulase (SEQ ID NO: 15), when it is further co-washed with relatively dirty clothes ( By adding pigment soil and bacteria to mimic those released into the wash solution from this relatively dirty laundry), the prewashed swatches were compared with those swatches that were only prewashed with a detergent that did not contain cellulase. 67.3% fewer fungal CFU remained on the surface of the swatch. This result shows that cellulases can be effectively used to reduce fungal attachment to textiles.

Table 17. Fungal CFU residues on Emp252 swatches after prewashing with powder detergent

Group average value Small swatches prewashed without cellulase 43.5 A swatch pre-washed with cellulase (SEQ ID NO: 15), 6.5 reduce 85%

As can be seen from the results in the table above, for those small pieces of cloth swatches prewashed with a liquid detergent containing cellulase (SEQ ID NO: 15), when it is further co-washed with relatively dirty clothes ( By adding pigment soil and bacteria to mimic those released into the wash solution from this relatively dirty laundry), the prewashed swatches were compared with those swatches that were only prewashed with a detergent that did not contain cellulase. 85% fewer fungal CFUs remained on the surface of the swatch. This result shows that cellulases can be effectively used to reduce fungal attachment to textiles.

Table 18 PrestoBlue fluorescence emission results

The same results corresponding to Table 18 are also shown in FIG. 1 .

As can be seen from the above table and the PestoBlue fluorescence measurement results of Figure 1, for those small pieces of cloth swatches prewashed with a liquid detergent containing cellulase (SEQ ID NO: 15), when it is further mixed with relatively dirty clothes When co-washing (by adding pigment soil and bacteria to mimic those released into the wash solution from such relatively soiled laundry), the fluorescence kinetics plots showed that compared to prewashing with only a detergent that did not contain cellulase, The fungal viability remaining on the pre-washed swatches was much lower compared to those swatches. Specifically, the enzymatically prewashed swatches showed a slower and flatter rise in fluorescence unit values, and this was significant when the swatches had been incubated for a period of more than 900 min. This result shows that cellulases can be effectively used to reduce fungal attachment to textiles.

Example 9. Use of enzymes to inhibit bacteria produced by enzyme liquid detergents

Material

1. Bacteria: Pseudomonas putida (China General Microorganism Culture Collection Center (CGMCC), strain number 1.3096)

2. Detergent: Lanyue deep cleaning liquid detergent, the concentration of washing liquid is 2g/L

3. Enzymes:

a. Protease, SEQ ID NO: 3

b. Enzyme Mix 1: 0.41 ppm Protease (SEQ ID NO:3), 0.052 ppm Amylase (SEQ ID NO:9)

c. Enzyme Mix 2: 0.62ppm Protease (SEQ ID NO:3), 0.052ppm Amylase (SEQ ID NO:9) and 0.16ppm Cellulase (SEQ ID No:17)

4. Small swatch: pre-aged PCN01: The small swatch was aged in the Wascator machine, program 151, aging time: 10h.

5. Stains: No. 2 baby food, braised beef stains

program:

1. Prepare baby food soiled swatches or braised beef soiled swatches according to the procedure described in the assay section: Preparation of soiled swatches.

2. Use the FSW wash program to wash the above soiled swatches. Washing conditions: AP top-loading machine, 25°C, 14°dH (Ca ²⁺ :Mg ²⁺ =3:2), main wash time 15min, rinse twice. For the different groups, the swatches were washed with detergent only (Blue Moon Deep Clean) or detergents containing various enzymes or combinations of enzymes.

3. Place the swatches from step 2 in a sterile petri dish (diameter = 9 cm) and one swatch in a petri dish for inoculation with Pseudomonas putida. The preparation of the bacterial inoculum and its application to the washed swatches, and the growth of the TTC color change on the swatches was observed following the relevant description in the TTC Assay section in the Assays section.

4. After incubating the small cloth swatches for 30 ± 4 hours, remove all Petri dishes from the incubator and perform odor panel assessment using the instructions above.

5. Odor panel assessment

Small swatches removed from the incubator were allowed to equilibrate for 30 min at room temperature and humidity before being moved into the panel room for evaluation. Panel members are selected from a pool of experienced and qualified candidates. Panelists evaluate without seeing the swatches. Given two samples in two Petri dishes to be evaluated, the panelist smelled each sample for 2 seconds and then sucked in fresh air for more than 10 seconds before smelling the other sample. After breathing fresh air for more than 10 s, they can follow the same procedure to evaluate the next pair of samples.

6. Simultaneously with the panel evaluation, the swatches from step 4 were also subjected to GS-MS measurements for malodor:

Remove the microbiologically soiled swatch from the incubator. Use sterilized scissors to excise a small piece of stain 1.5 cm x 1.5 cm from the stained area. Each cut cloth swatch was then transferred to a 20 mL GC headspace vial using clean sterile forceps, and the headspace from the capped vial was then analyzed.

Headspace from capped vials was analyzed by GC-MS:

GCMS Agilent 7890GC with split/splitless injector and 5977MS with extractor ion source coupled to Gerstel MPS2 sampler with HS/SPME, SPME needle heater.

Gerstel MPS SPME incubator: agitator. Incubation temperature: 60°C. Incubation time: 10.00min. Stirring speed: 250rpm. Sample parameters: extraction time: 20min; injection and desorption time: 120s.

SPME (Solid Phase Micro Extraction) Fiber Type: Carbon Molecular Sieve (Carboxen)/Polydimethylsiloxane (CAR/PDMS)

The method used was: GC oven temperature: initial 40 °C; hold 2 min; rate 5 °C/min until 150 °C; rate 35 °C/min until 240 °C; hold 3 min. Front SS inlet He: mode split flow; T ^a 230°C, split ratio 10:1; split flow 15mL/min. Column: Agilent 19091F-433: FFAP-01HP-FFAP 30m x 250μm x 0.25μm

MS Information: Acquisition Mode: Scan. Solvent delay (minutes): 1. Scan parameters: Start time: 1. Low mass: 35. High mass: 350. Threshold: 100. A/D sample: 4. MS zone: MS source: 230°C. MS Quad: 150°C

result

1. Smell panel

Table 19. Odor evaluation of baby food and Pseudomonas putida soiled swatches by 5 panelists:

The results in Table 19 above show that for those swatches soiled with baby food washed with detergents containing different enzymes or enzyme mixtures and subsequently exposed to bacteria (P. They all showed significantly better malodor suppression results as indicated by the panel evaluation compared to the swatch parallel group.

Table 20. Odor evaluation by 11 panelists of baby food and Pseudomonas putida soiled swatches:

The results in Table 20 above show that for those swatches soiled with baby food washed with detergents containing different enzymes or enzyme mixtures and subsequently exposed to bacteria (P. They all showed significantly better malodor suppression results as indicated by the panel evaluation compared to the swatch parallel group.

2. GCMS results

Table 21. GCMS malodor test results on baby food and Pseudomonas putida soiled swatches:

The four VOC molecules are representative malodorous molecules.

The results in Table 21 above show that for those swatches soiled with baby food washed with detergents containing different enzymes or enzyme mixtures and subsequently exposed to bacteria (Pseudomonas VOC levels were significantly lower compared to those parallel groups of swatches washed with detergent.

Table 22. GCMS odor detection results of braised beef stains and small cloth samples stained with Pseudomonas putida:

Seven kinds of VOC molecules can be representative malodorous molecules, especially 3-octanone has a taste and a musty smell, and 2,3-butanedione has a bad smell.

The results in Table 22 above show that for those swatches soiled with braised beef that had been washed with detergents containing different enzymes or enzyme mixtures and subsequently exposed to bacteria (Pseudomonas VOC levels were significantly lower compared to those parallel groups of swatches washed with detergent.

Example 10 Inhibition of fungal malodour by washing with a multi-enzyme liquid detergent containing enzymes

Example 9 was carried out essentially the same as the previous example except that the fungus Aspergillus niger (same strain information as in Example 2) was substituted for the bacterium Pseudomonas putida. The preparation of the fungal inoculum and its application to the washed swatches, and the observation of fungal growth on the swatches corresponding to The preceding examples are different. The swatches were evaluated using only GS-MS to measure malodor.

result

Table 23. GCMS malodor results on baby food and Aspergillus niger soiled swatches:

The results in Table 23 above show that for those swatches soiled with baby food that were washed with detergents containing different enzymes or enzyme mixtures and subsequently exposed to A. VOC levels were significantly lower compared to the small swatches.

Table 24. GCMS odor detection results of braised beef stains and Aspergillus niger stained swatches:

The results in Table 24 above show that for those swatches soiled with braised beef that had been washed with detergents containing different enzymes or enzyme mixtures and subsequently exposed to A. VOC levels were significantly lower compared to the small swatches.

sequence listing

<110> Novozymes A/S

<120> Use of one or more enzymes to prevent, inhibit or reduce microbial growth on surfaces

<130> 14376-WO-PCT

<160> 18

<170> PatentIn Version 3.5

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Ala Gln Ser Val Pro Trp Gly Ile Ser Arg Val Gln Ala Pro Ala Ala

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His Asn Arg Gly Leu Thr Gly Ser Gly Val Lys Val Ala Val Leu Asp

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Thr Gly Ile Ser Thr His Pro Asp Leu Asn Ile Arg Gly Gly Ala Ser

35 40 45

Phe Val Pro Gly Glu Pro Ser Thr Gln Asp Gly Asn Gly His Gly Thr

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His Val Ala Gly Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly Val Leu

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Gly Val Ala Pro Ser Ala Glu Leu Tyr Ala Val Lys Val Leu Gly Ala

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Ser Gly Ser Gly Ser Val Ser Ser Ile Ala Gln Gly Leu Glu Trp Ala

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Gly Asn Asn Gly Met His Val Ala Asn Leu Ser Leu Gly Ser Pro Ser

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Pro Ser Ala Thr Leu Glu Gln Ala Val Asn Ser Ala Thr Ser Arg Gly

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Val Leu Val Val Ala Ala Ser Gly Asn Ser Gly Ala Gly Ser Ile Ser

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Tyr Pro Ala Arg Tyr Ala Asn Ala Met Ala Val Gly Ala Thr Asp Gln

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Asn Asn Asn Arg Ala Ser Phe Ser Gln Tyr Gly Ala Gly Leu Asp Ile

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Val Ala Pro Gly Val Asn Val Gln Ser Thr Tyr Pro Gly Ser Thr Tyr

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Ala Ser Leu Asn Gly Thr Ser Met Ala Thr Pro His Val Ala Gly Ala

210 215 220

Ala Ala Leu Val Lys Gln Lys Asn Pro Ser Trp Ser Asn Val Gln Ile

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Arg Asn His Leu Lys Asn Thr Ala Thr Ser Leu Gly Ser Thr Asn Leu

245 250 255

Tyr Gly Ser Gly Leu Val Asn Ala Glu Ala Ala Thr Arg

260 265

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<211> 275

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<213> Bacillus amyloliquefaciens

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Ala Gln Ser Val Pro Tyr Gly Val Ser Gln Ile Lys Ala Pro Ala Leu

1 5 10 15

His Ser Gln Gly Tyr Thr Gly Ser Asn Val Lys Val Ala Val Ile Asp

20 25 30

Ser Gly Ile Asp Ser Ser His Pro Asp Leu Lys Val Ala Gly Gly Ala

35 40 45

Ser Met Val Pro Ser Glu Thr Asn Pro Phe Gln Asp Asn Asn Ser His

50 55 60

Gly Thr His Val Ala Gly Thr Val Ala Ala Leu Asn Asn Ser Ile Gly

65 70 75 80

Val Leu Gly Val Ala Pro Ser Ala Ser Leu Tyr Ala Val Lys Val Leu

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Gly Ala Asp Gly Ser Gly Gln Tyr Ser Trp Ile Ile Asn Gly Ile Glu

100 105 110

Trp Ala Ile Ala Asn Asn Met Asp Val Ile Asn Met Ser Leu Gly Gly

115 120 125

Pro Ser Gly Ser Ala Ala Leu Lys Ala Ala Val Asp Lys Ala Val Ala

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Ser Gly Val Val Val Val Ala Ala Ala Gly Asn Glu Gly Thr Ser Gly

145 150 155 160

Ser Ser Ser Thr Val Gly Tyr Pro Gly Lys Tyr Pro Ser Val Ile Ala

165 170 175

Val Gly Ala Val Asp Ser Ser Asn Gln Arg Ala Ser Phe Ser Ser Val

180 185 190

Gly Pro Glu Leu Asp Val Met Ala Pro Gly Val Ser Ile Gln Ser Thr

195 200 205

Leu Pro Gly Asn Lys Tyr Gly Ala Tyr Asn Gly Thr Ser Met Ala Ser

210 215 220

Pro His Val Ala Gly Ala Ala Ala Leu Ile Leu Ser Lys His Pro Asn

225 230 235 240

Trp Thr Asn Thr Gln Val Arg Ser Ser Leu Glu Asn Thr Thr Thr Lys

245 250 255

Leu Gly Asp Ser Phe Tyr Tyr Gly Lys Gly Leu Ile Asn Val Gln Ala

260 265 270

Ala Ala Gln

275

<210> 3

<211> 269

<212> PRT

<213> Bacillus lentus

<400> 3

Ala Gln Ser Val Pro Trp Gly Ile Glu Arg Val Gln Ala Pro Ala Ala

1 5 10 15

His Asn Arg Gly Leu Thr Gly Ser Gly Val Lys Val Ala Val Leu Asp

20 25 30

Thr Gly Ile Ser Thr His Pro Asp Leu Arg Ile Arg Gly Gly Ala Ser

35 40 45

Phe Val Pro Gly Glu Pro Ser Thr Gln Asp Gly Asn Gly His Gly Thr

50 55 60

His Val Ala Gly Thr Ile Ala Ala Leu Asp Asn Ser Ile Gly Val Leu

65 70 75 80

Gly Val Ala Pro Ser Ala Glu Leu Tyr Ala Val Lys Val Leu Gly Ala

85 90 95

Ser Gly Ser Gly Ser Val Ser Ser Ile Ala Gln Gly Leu Glu Trp Ala

100 105 110

Gly Asn Asn Gly Met His Val Ala Asn Leu Ser Leu Gly Ser Pro Ser

115 120 125

Pro Ser Ala Thr Leu Glu Gln Ala Val Asn Ser Ala Thr Ser Arg Gly

130 135 140

Val Leu Val Val Ala Ala Ser Gly Asn Ser Gly Ala Gly Ser Ile Ser

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Tyr Pro Ala Arg Tyr Ala Asn Ala Met Ala Val Gly Ala Thr Asp Gln

165 170 175

Asn Asn Asn Arg Ala Ser Phe Ser Gln Tyr Gly Ala Gly Leu Asp Ile

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Val Ala Pro Gly Val Asn Ile Leu Ser Thr Trp Pro Gly Ser Thr Tyr

195 200 205

Ala Ser Leu Asn Gly Thr Ser Met Ala Thr Pro His Val Ala Gly Ala

210 215 220

Ala Ala Leu Val Lys Gln Lys Asn Pro Ser Trp Ser Asn Val Gln Ile

225 230 235 240

Arg Asn His Leu Lys Asn Thr Ala Thr Ser Leu Gly Asp Thr Trp Glu

245 250 255

Tyr Gly Ser Gly Leu Val Asn Ala Glu Ala Ala Thr Arg

260 265

<210> 4

<211> 269

<212> PRT

<213> Bacillus lentus

<400> 4

Ala Gln Ser Val Pro Trp Gly Ile Glu Arg Val Gln Ala Pro Ala Ala

1 5 10 15

His Asn Arg Gly Leu Thr Gly Ser Gly Val Lys Val Ala Val Leu Asp

20 25 30

Thr Gly Ile Ser Thr His Pro Asp Leu Arg Ile Arg Gly Gly Ala Ser

35 40 45

Phe Val Pro Gly Glu Pro Ser Thr Gln Asp Gly Asn Gly His Gly Thr

50 55 60

His Val Ala Gly Thr Ile Ala Ala Leu Asp Asn Ser Ile Gly Val Leu

65 70 75 80

Gly Val Ala Pro Ser Ala Glu Leu Tyr Ala Val Lys Val Leu Gly Ala

85 90 95

Ser Gly Ser Gly Ser Val Ser Ser Ile Ala Gln Gly Leu Glu Trp Ala

100 105 110

Gly Asn Asn Gly Met Val Val Ala Asn Leu Ser Leu Gly Ser Pro Ser

115 120 125

Pro Ser Ala Thr Leu Glu Gln Ala Val Asn Ser Ala Thr Ser Arg Gly

130 135 140

Val Leu Val Val Ala Ala Ser Gly Asn Ser Gly Ala Gly Ser Ile Ser

145 150 155 160

Tyr Pro Ala Arg Tyr Ala Asn Ala Met Ala Val Gly Ala Thr Asp Glu

165 170 175

Asn Asn Asn Arg Ala Ser Phe Ser Gln Tyr Gly Pro Gly Leu Asp Ile

180 185 190

Val Ala Pro Gly Val Asn Ile Leu Ser Thr Trp Pro Gly Ser Thr Tyr

195 200 205

Ala Ser Leu Asn Gly Thr Ser Met Ala Thr Pro His Val Ala Gly Ala

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Ala Ala Leu Val Lys Gln Lys Asn Pro Ser Trp Ser Asn Val Gln Ile

225 230 235 240

Arg Asn His Leu Lys Asn Thr Ala Thr Asp Leu Gly Asp Thr Trp Glu

245 250 255

Tyr Gly Ser Gly Leu Val Asn Ala Glu Ala Ala Thr Arg

260 265

<210> 5

<211> 269

<212> PRT

<213> Bacillus lentus

<400> 5

Ala Gln Ser Val Pro Trp Gly Ile Glu Arg Val Gln Ala Pro Ala Ala

1 5 10 15

His Asn Arg Gly Leu Thr Gly Ser Gly Val Lys Val Ala Val Leu Asp

20 25 30

Thr Gly Ile Ser Thr His Pro Asp Leu Arg Ile Arg Gly Gly Ala Ser

35 40 45

Phe Val Pro Gly Glu Pro Ser Thr Gln Asp Gly Asn Gly His Gly Thr

50 55 60

His Val Ala Gly Thr Ile Ala Ala Leu Asp Asn Ser Ile Gly Val Leu

65 70 75 80

Gly Val Ala Pro Ser Ala Glu Leu Tyr Ala Val Lys Val Leu Gly Ala

85 90 95

Ser Gly Ser Gly Ser Val Ser Ser Ile Ala Gln Gly Leu Glu Trp Ala

100 105 110

Gly Asn Asn Gly Met His Val Ala Asn Leu Ser Leu Gly Ser Pro Ser

115 120 125

Pro Ser Ala Thr Leu Glu Gln Ala Val Asn Ser Ala Thr Ser Arg Gly

130 135 140

Val Leu Val Val Ala Ala Ser Gly Asn Ser Gly Ala Gly Ser Ile Ser

145 150 155 160

Tyr Pro Ala Arg Tyr Ala Asn Ala Met Ala Val Gly Ala Thr Asp Gln

165 170 175

Asn Asn Asn Arg Ala Ser Phe Ser Gln Tyr Gly Pro Gly Leu Asp Ile

180 185 190

Val Ala Pro Gly Val Asn Ile Leu Ser Thr Trp Pro Gly Ser Thr Tyr

195 200 205

Ala Ser Leu Asn Gly Thr Ser Met Ala Thr Pro His Val Ala Gly Ala

210 215 220

Ala Ala Leu Val Lys Gln Lys Asn Pro Ser Trp Ser Asn Val Gln Ile

225 230 235 240

Arg Asn His Leu Lys Asn Thr Ala Thr Ser Leu Gly Asp Thr Trp Glu

245 250 255

Tyr Gly Ser Gly Leu Val Asn Ala Glu Ala Ala Thr Arg

260 265

<210> 6

<211> 269

<212> PRT

<213> Bacillus lentus

<400> 6

Ala Gln Ser Val Pro Trp Gly Ile Glu Arg Val Gln Ala Pro Ala Ala

1 5 10 15

His Asn Arg Gly Leu Thr Gly Ser Gly Val Lys Val Ala Val Leu Asp

20 25 30

Thr Gly Ile Ser Thr His Pro Asp Leu Arg Ile Arg Gly Gly Ala Ser

35 40 45

Phe Val Pro Gly Glu Pro Ser Thr Gln Asp Gly Asn Gly His Gly Thr

50 55 60

His Val Ala Gly Thr Ile Ala Ala Leu Asp Asn Ser Ile Gly Val Leu

65 70 75 80

Gly Val Ala Pro Ser Ala Glu Leu Tyr Ala Val Lys Val Leu Gly Ala

85 90 95

Ser Gly Ser Gly Ser Val Ser Ser Ile Ala Gln Gly Leu Glu Trp Ala

100 105 110

Gly Asn Asn Gly Met His Val Ala Asn Leu Ser Leu Gly Ser Pro Ser

115 120 125

Pro Ser Ala Thr Leu Glu Gln Ala Val Asn Ser Ala Thr Ser Arg Gly

130 135 140

Val Leu Val Val Ala Ala Ser Gly Asn Ser Gly Ala Gly Ser Ile Ser

145 150 155 160

Tyr Pro Ala Arg Tyr Ala Asn Ala Met Ala Val Gly Ala Thr Asp Glu

165 170 175

Asn Asn Asn Arg Ala Ser Phe Ser Gln Tyr Gly Pro Gly Leu Asp Ile

180 185 190

Val Ala Pro Gly Val Asn Ile Leu Ser Thr Trp Pro Gly Ser Thr Tyr

195 200 205

Ala Ser Leu Asn Gly Thr Ser Met Ala Thr Pro His Val Ala Gly Ala

210 215 220

Ala Ala Leu Val Lys Gln Lys Asn Pro Ser Trp Ser Asn Val Gln Ile

225 230 235 240

Arg Asn His Leu Lys Asn Thr Ala Thr Asp Leu Gly Asp Thr Trp Glu

245 250 255

Tyr Gly Ser Gly Leu Val Asn Ala Glu Ala Ala Thr Arg

260 265

<210> 7

<211> 269

<212> PRT

<213> Bacillus lentus

<400> 7

Ala Gln Ser Val Pro Trp Gly Ile Glu Arg Val Gln Ala Pro Ala Ala

1 5 10 15

His Asn Arg Gly Leu Thr Gly Ser Gly Val Lys Val Ala Val Leu Asp

20 25 30

Thr Gly Ile Ser Thr His Pro Asp Leu Arg Ile Arg Gly Gly Ala Ser

35 40 45

Phe Val Pro Gly Glu Pro Ser Thr Gln Asp Gly Asn Gly His Gly Thr

50 55 60

His Val Ala Gly Thr Ile Ala Ala Leu Asp Asn Ser Ile Gly Val Leu

65 70 75 80

Gly Val Ala Pro Ser Ala Glu Leu Tyr Ala Val Lys Val Leu Gly Ala

85 90 95

Ser Gly Ser Gly Ser Val Ser Ser Ile Ala Gln Gly Leu Glu Trp Ala

100 105 110

Gly Asn Asn Gly Met Val Val Ala Asn Leu Ser Leu Gly Ser Pro Ser

115 120 125

Pro Ser Ala Thr Leu Glu Gln Ala Val Asn Ser Ala Thr Ser Arg Gly

130 135 140

Val Leu Val Val Ala Ala Ser Gly Asn Ser Gly Ala Gly Ser Ile Ser

145 150 155 160

Tyr Pro Ala Arg Tyr Ala Asn Ala Met Ala Val Gly Ala Thr Asp Glu

165 170 175

Asn Asn Glu Arg Ala Ser Phe Ser Gln Tyr Gly Pro Gly Leu Asp Ile

180 185 190

Val Ala Pro Gly Val Asn Ile Leu Ser Thr Trp Pro Gly Ser Thr Tyr

195 200 205

Ala Ser Leu Asn Gly Thr Ser Met Ala Thr Pro His Val Ala Gly Ala

210 215 220

Ala Ala Leu Val Lys Gln Lys Asn Pro Ser Trp Ser Asn Val Gln Ile

225 230 235 240

Arg Asn His Leu Lys Asn Thr Ala Thr Asp Leu Gly Ser Thr Trp Glu

245 250 255

Tyr Gly Ser Gly Leu Val Asn Ala Glu Ala Ala Thr Arg

260 265

<210> 8

<211> 269

<212> PRT

<213> Bacillus lentus

<400> 8

Ala Gln Val Val Pro Trp Gly Ile Ser Arg Val Gln Ala Pro Ala Ala

1 5 10 15

His Asn Arg Gly Leu Thr Gly Ser Gly Val Lys Val Ala Val Leu Asp

20 25 30

Thr Gly Ile Ser Thr His Pro Asp Leu Asn Ile Arg Gly Gly Ala Ser

35 40 45

Phe Val Pro Gly Glu Pro Ser Thr Gln Asp Gly Asn Gly His Gly Thr

50 55 60

His Val Ala Gly Thr Ile Ala Ala Leu Asp Asn Ser Ile Gly Val Leu

65 70 75 80

Gly Val Ala Pro Ser Ala Glu Leu Tyr Ala Val Lys Val Leu Gly Ala

85 90 95

Ser Gly Ser Gly Ser Val Ser Ser Ile Ala Gln Gly Leu Glu Trp Ala

100 105 110

Gly Asn Asn Gly Met Val Val Ala Asn Leu Ser Leu Gly Ser Pro Ser

115 120 125

Pro Ser Ala Thr Leu Glu Gln Ala Val Asn Ser Ala Thr Ser Arg Gly

130 135 140

Val Leu Val Val Ala Ala Ser Gly Asn Ser Gly Ala Gly Ser Ile Ser

145 150 155 160

Tyr Pro Ala Arg Tyr Ala Asn Ala Met Ala Val Gly Ala Thr Asp Glu

165 170 175

Asn Asn Glu Arg Ala Glu Phe Ser Gln Tyr Gly Ala Gly Leu Asp Ile

180 185 190

Val Ala Pro Gly Val Asn Ile Leu Ser Thr Trp Pro Gly Ser Thr Tyr

195 200 205

Ala Val Leu Asn Gly Thr Ser Met Ala Thr Pro His Val Ala Gly Ala

210 215 220

Ala Ala Leu Val Lys Gln Lys Asn Pro Ser Trp Ser Asn Val Gln Ile

225 230 235 240

Arg Asn His Leu Lys Asn Thr Ala Thr Asp Leu Gly Ser Thr Trp Glu

245 250 255

Tyr Gly Ser Gly Leu Val Asn Ala Glu Ala Ala Thr Arg

260 265

<210> 9

<211> 482

<212> PRT

<213> Artificial

<220>

<223> fusion protein

<400> 9

His Asp Gly Thr Asn Gly Thr Ile Met Gln Tyr Phe Glu Trp Asn Val

1 5 10 15

Pro Asn Asp Gly Gln His Trp Asn Arg Leu His Asn Asn Ala Gln Asn

20 25 30

Leu Lys Asn Ala Gly Ile Thr Ala Ile Trp Ile Pro Pro Ala Trp Lys

35 40 45

Gly Thr Ser Gln Ser Asp Thr Gly Tyr Gly Ala Tyr Asp Leu Tyr Asp

50 55 60

Leu Gly Glu Phe Asn Gln Arg Gly Thr Val Arg Thr Lys Tyr Gly Thr

65 70 75 80

Lys Ala Glu Leu Glu Arg Ala Ile Arg Ser Leu Lys Ala Asn Gly Ile

85 90 95

Gln Val Tyr Gly Asp Val Val Met Asn His Lys Ala Gly Ala Asp Gln

100 105 110

Thr Glu Gln Val Gln Ala Val Glu Val Asn Pro Gln Asn Arg Asn Gln

115 120 125

Glu Val Ser Gly Thr Tyr Gln Ile Glu Ala Trp Thr Gly Phe Asn Phe

130 135 140

Pro Gly Arg Gly Asn Gln His Ser Ser Phe Lys Trp Arg Trp Tyr His

145 150 155 160

Phe Asp Gly Thr Asp Phe Asp Gln Ser Arg Gly Leu Ser Asn Arg Ile

165 170 175

Tyr Lys Phe Arg Thr Lys Ala Trp Asp Trp Glu Val Asp Thr Glu Phe

180 185 190

Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Leu Asp Met Asp His Pro

195 200 205

Glu Val Ile Asn Glu Leu Asn Arg Trp Gly Val Trp Tyr Ala Asn Thr

210 215 220

Leu Asn Leu Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys Phe

225 230 235 240

Ser Phe Met Arg Asp Trp Leu Gly His Val Arg Gly Gln Thr Gly Lys

245 250 255

Asn Leu Phe Ala Val Ala Glu Tyr Trp Lys Asn Asp Leu Gly Ala Leu

260 265 270

Glu Asn Tyr Leu Ser Lys Thr Asn Trp Thr Met Ser Ala Phe Asp Val

275 280 285

Pro Leu His Tyr Asn Leu Tyr Gln Ala Ser Asn Ser Ser Gly Asn Tyr

290 295 300

Asp Met Arg Asn Leu Leu Asn Gly Thr Leu Val Gln Arg His Pro Ser

305 310 315 320

His Ala Val Thr Phe Val Asp Asn His Asp Thr Gln Pro Gly Glu Ala

325 330 335

Leu Glu Ser Phe Val Gln Gly Trp Phe Lys Pro Leu Ala Tyr Ala Thr

340 345 350

Ile Leu Thr Arg Glu Gln Gly Tyr Pro Gln Val Phe Tyr Gly Asp Tyr

355 360 365

Tyr Gly Ile Pro Ser Asp Gly Val Pro Ser Tyr Arg Gln Gln Ile Asp

370 375 380

Pro Leu Leu Ala Ala Arg Gln Gln Tyr Ala Tyr Gly Thr Gln His Asp

385 390 395 400

Tyr Leu Asp Asn Gln Asp Val Ile Gly Trp Thr Arg Glu Gly Asp Ser

405 410 415

Ala His Ala Gly Ser Gly Leu Ala Thr Val Met Ser Asp Gly Pro Gly

420 425 430

Gly Ser Lys Thr Met Tyr Val Gly Thr Ala His Ala Gly Gln Val Phe

435 440 445

Lys Asp Ile Thr Gly Asn Arg Thr Asp Thr Val Thr Ile Asn Ser Ala

450 455 460

Gly Asn Gly Thr Phe Arg Cys Asn Lys Gly Ser Val Ser Ile Trp Val

465 470 475 480

Lys Gln

<210> 10

<211> 483

<212> PRT

<213> Bacillus species

<400> 10

His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr

1 5 10 15

Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Ser Asp Ala Ser

20 25 30

Asn Leu Lys Asp Lys Gly Ile Ser Ala Val Trp Ile Pro Pro Ala Trp

35 40 45

Lys Gly Ala Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr

50 55 60

Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Ile Arg Thr Lys Tyr Gly

65 70 75 80

Thr Arg Asn Gln Leu Gln Ala Ala Val Asn Ala Leu Lys Ser Asn Gly

85 90 95

Ile Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp

100 105 110

Ala Thr Glu Met Val Lys Ala Val Glu Val Asn Pro Asn Asn Arg Asn

115 120 125

Gln Glu Val Ser Gly Glu Tyr Thr Ile Glu Ala Trp Thr Lys Phe Asp

130 135 140

Phe Pro Gly Arg Gly Asn Thr His Ser Asn Phe Lys Trp Arg Trp Tyr

145 150 155 160

His Phe Asp Gly Val Asp Trp Asp Gln Ser Arg Lys Leu Asn Asn Arg

165 170 175

Ile Tyr Lys Phe Arg Gly Lys Gly Trp Asp Trp Glu Val Asp Thr Glu

180 185 190

Phe Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Met Asp His

195 200 205

Pro Glu Val Val Asn Glu Leu Arg Asn Trp Gly Val Trp Tyr Thr Asn

210 215 220

Thr Leu Gly Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His Ile Lys

225 230 235 240

Tyr Ser Phe Thr Arg Asp Trp Ile Asn His Val Arg Ser Ala Thr Gly

245 250 255

Lys Asn Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu Gly Ala

260 265 270

Ile Glu Asn Tyr Leu Asn Lys Thr Asn Trp Asn His Ser Val Phe Asp

275 280 285

Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Lys Ser Gly Gly Asn

290 295 300

Tyr Asp Met Arg Gln Ile Phe Asn Gly Thr Val Val Gln Lys His Pro

305 310 315 320

Met His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro Glu Glu

325 330 335

Ala Leu Glu Ser Phe Val Glu Glu Trp Phe Lys Pro Leu Ala Tyr Ala

340 345 350

Leu Thr Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr Gly Asp

355 360 365

Tyr Tyr Gly Ile Pro Thr His Gly Val Pro Ala Met Lys Ser Lys Ile

370 375 380

Asp Pro Ile Leu Glu Ala Arg Gln Lys Tyr Ala Tyr Gly Arg Gln Asn

385 390 395 400

Asp Tyr Leu Asp His His Asn Ile Ile Gly Trp Thr Arg Glu Gly Asn

405 410 415

Thr Ala His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp Gly Ala

420 425 430

Gly Gly Asn Lys Trp Met Phe Val Gly Arg Asn Lys Ala Gly Gln Val

435 440 445

Trp Thr Asp Ile Thr Gly Asn Lys Ala Gly Thr Val Thr Ile Asn Ala

450 455 460

Asp Gly Trp Gly Asn Phe Ser Val Asn Gly Gly Ser Val Ser Ile Trp

465 470 475 480

Val Asn Lys

<210> 11

<211> 485

<212> PRT

<213> Bacillus species

<400> 11

His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr

1 5 10 15

Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Ser Asp Ala Ser

20 25 30

Asn Leu Lys Asp Lys Gly Ile Ser Ala Val Trp Ile Pro Pro Ala Trp

35 40 45

Lys Gly Ala Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr

50 55 60

Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Ile Arg Thr Lys Tyr Gly

65 70 75 80

Thr Arg Asn Gln Leu Gln Ala Ala Val Asn Ala Leu Lys Ser Asn Gly

85 90 95

Ile Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp

100 105 110

Ala Thr Glu Met Val Arg Ala Val Glu Val Asn Pro Asn Asn Arg Asn

115 120 125

Gln Glu Val Ser Gly Glu Tyr Thr Ile Glu Ala Trp Thr Lys Phe Asp

130 135 140

Phe Pro Gly Arg Gly Asn Thr His Ser Asn Phe Lys Trp Arg Trp Tyr

145 150 155 160

His Phe Asp Gly Val Asp Trp Asp Gln Ser Arg Lys Leu Asn Asn Arg

165 170 175

Ile Tyr Lys Phe Arg Gly Asp Gly Lys Gly Trp Asp Trp Glu Val Asp

180 185 190

Thr Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Met

195 200 205

Asp His Pro Glu Val Val Asn Glu Leu Arg Asn Trp Gly Val Trp Tyr

210 215 220

Thr Asn Thr Leu Gly Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His

225 230 235 240

Ile Lys Tyr Ser Phe Thr Arg Asp Trp Ile Asn His Val Arg Ser Ala

245 250 255

Thr Gly Lys Asn Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu

260 265 270

Gly Ala Ile Glu Asn Tyr Leu Asn Lys Thr Asn Trp Asn His Ser Val

275 280 285

Phe Asp Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Lys Ser Gly

290 295 300

Gly Asn Tyr Asp Met Arg Gln Ile Phe Asn Gly Thr Val Val Gln Arg

305 310 315 320

His Pro Met His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro

325 330 335

Glu Glu Ala Leu Glu Ser Phe Val Glu Glu Trp Phe Lys Pro Leu Ala

340 345 350

Tyr Ala Leu Thr Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr

355 360 365

Gly Asp Tyr Tyr Gly Ile Pro Thr His Gly Val Pro Ala Met Lys Ser

370 375 380

Lys Ile Asp Pro Ile Leu Glu Ala Arg Gln Lys Tyr Ala Tyr Gly Arg

385 390 395 400

Gln Asn Asp Tyr Leu Asp His His Asn Ile Ile Gly Trp Thr Arg Glu

405 410 415

Gly Asn Thr Ala His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp

420 425 430

Gly Ala Gly Gly Asn Lys Trp Met Phe Val Gly Arg Asn Lys Ala Gly

435 440 445

Gln Val Trp Thr Asp Ile Thr Gly Asn Arg Ala Gly Thr Val Thr Ile

450 455 460

Asn Ala Asp Gly Trp Gly Asn Phe Ser Val Asn Gly Gly Ser Val Ser

465 470 475 480

Ile Trp Val Asn Lys

485

<210> 12

<211> 485

<212> PRT

<213> Bacillus sp. NCIB 12513

<400> 12

His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp His

1 5 10 15

Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Asp Asp Ala Ser

20 25 30

Asn Leu Arg Asn Arg Gly Ile Thr Ala Ile Trp Ile Pro Pro Ala Trp

35 40 45

Lys Gly Thr Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr

50 55 60

Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly

65 70 75 80

Thr Arg Ser Gln Leu Glu Ser Ala Ile His Ala Leu Lys Asn Asn Gly

85 90 95

Val Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp

100 105 110

Ala Thr Glu Asn Val Leu Ala Val Glu Val Asn Pro Asn Asn Arg Asn

115 120 125

Gln Glu Ile Ser Gly Asp Tyr Thr Ile Glu Ala Trp Thr Lys Phe Asp

130 135 140

Phe Pro Gly Arg Gly Asn Thr Tyr Ser Asp Phe Lys Trp Arg Trp Tyr

145 150 155 160

His Phe Asp Gly Val Asp Trp Asp Gln Ser Arg Gln Phe Gln Asn Arg

165 170 175

Ile Tyr Lys Phe Arg Gly Asp Gly Lys Ala Trp Asp Trp Glu Val Asp

180 185 190

Ser Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp Met

195 200 205

Asp His Pro Glu Val Val Asn Glu Leu Arg Arg Trp Gly Glu Trp Tyr

210 215 220

Thr Asn Thr Leu Asn Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His

225 230 235 240

Ile Lys Tyr Ser Phe Thr Arg Asp Trp Leu Thr His Val Arg Asn Ala

245 250 255

Thr Gly Lys Glu Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu

260 265 270

Gly Ala Leu Glu Asn Tyr Leu Asn Lys Thr Asn Trp Asn His Ser Val

275 280 285

Phe Asp Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Asn Ser Gly

290 295 300

Gly Asn Tyr Asp Met Ala Lys Leu Leu Asn Gly Thr Val Val Gln Lys

305 310 315 320

His Pro Met His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro

325 330 335

Gly Glu Ser Leu Glu Ser Phe Val Gln Glu Trp Phe Lys Pro Leu Ala

340 345 350

Tyr Ala Leu Ile Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr

355 360 365

Gly Asp Tyr Tyr Gly Ile Pro Thr His Ser Val Pro Ala Met Lys Ala

370 375 380

Lys Ile Asp Pro Ile Leu Glu Ala Arg Gln Asn Phe Ala Tyr Gly Thr

385 390 395 400

Gln His Asp Tyr Phe Asp His His Asn Ile Ile Gly Trp Thr Arg Glu

405 410 415

Gly Asn Thr Thr His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp

420 425 430

Gly Pro Gly Gly Glu Lys Trp Met Tyr Val Gly Gln Asn Lys Ala Gly

435 440 445

Gln Val Trp His Asp Ile Thr Gly Asn Lys Pro Gly Thr Val Thr Ile

450 455 460

Asn Ala Asp Gly Trp Ala Asn Phe Ser Val Asn Gly Gly Ser Val Ser

465 470 475 480

Ile Trp Val Lys Arg

485

<210> 13

<211> 485

<212> PRT

<213> Bacillus sp. No. 707

<400> 13

His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr

1 5 10 15

Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Asn Ser Asp Ala Ser

20 25 30

Asn Leu Lys Ser Lys Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Trp

35 40 45

Lys Gly Ala Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr

50 55 60

Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly

65 70 75 80

Thr Arg Ser Gln Leu Gln Ala Ala Val Thr Ser Leu Lys Asn Asn Gly

85 90 95

Ile Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp

100 105 110

Ala Thr Glu Met Val Arg Ala Val Glu Val Asn Pro Asn Asn Arg Asn

115 120 125

Gln Glu Val Thr Gly Glu Tyr Thr Ile Glu Ala Trp Thr Arg Phe Asp

130 135 140

Phe Pro Gly Arg Gly Asn Thr His Ser Ser Phe Lys Trp Arg Trp Tyr

145 150 155 160

His Phe Asp Gly Val Asp Trp Asp Gln Ser Arg Arg Leu Asn Asn Arg

165 170 175

Ile Tyr Lys Phe Arg Gly His Gly Lys Ala Trp Asp Trp Glu Val Asp

180 185 190

Thr Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Met

195 200 205

Asp His Pro Glu Val Val Asn Glu Leu Arg Asn Trp Gly Val Trp Tyr

210 215 220

Thr Asn Thr Leu Gly Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His

225 230 235 240

Ile Lys Tyr Ser Phe Thr Arg Asp Trp Ile Asn His Val Arg Ser Ala

245 250 255

Thr Gly Lys Asn Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu

260 265 270

Gly Ala Ile Glu Asn Tyr Leu Gln Lys Thr Asn Trp Asn His Ser Val

275 280 285

Phe Asp Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Lys Ser Gly

290 295 300

Gly Asn Tyr Asp Met Arg Asn Ile Phe Asn Gly Thr Val Val Gln Arg

305 310 315 320

His Pro Ser His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro

325 330 335

Glu Glu Ala Leu Glu Ser Phe Val Glu Glu Trp Phe Lys Pro Leu Ala

340 345 350

Tyr Ala Leu Thr Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr

355 360 365

Gly Asp Tyr Tyr Gly Ile Pro Thr His Gly Val Pro Ala Met Arg Ser

370 375 380

Lys Ile Asp Pro Ile Leu Glu Ala Arg Gln Lys Tyr Ala Tyr Gly Lys

385 390 395 400

Gln Asn Asp Tyr Leu Asp His His Asn Ile Leu Gly Trp Thr Arg Glu

405 410 415

Gly Asn Thr Ala His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp

420 425 430

Gly Ala Gly Gly Ser Lys Trp Met Phe Val Gly Arg Asn Lys Ala Gly

435 440 445

Gln Val Trp Ser Asp Ile Thr Gly Asn Arg Thr Gly Thr Val Thr Ile

450 455 460

Asn Ala Asp Gly Trp Gly Asn Phe Ser Val Asn Gly Gly Ser Val Ser

465 470 475 480

Ile Trp Val Asn Lys

485

<210> 14

<211> 311

<212> PRT

<213> Bacillus sp. TY145

<400> 14

Ala Val Pro Ser Thr Gln Thr Pro Trp Gly Ile Lys Ser Ile Tyr Asn

1 5 10 15

Asp Gln Ser Ile Thr Lys Thr Thr Gly Gly Ser Gly Ile Lys Val Ala

20 25 30

Val Leu Asp Thr Gly Val Tyr Thr Ser His Leu Asp Leu Ala Gly Ser

35 40 45

Ala Glu Gln Cys Lys Asp Phe Thr Gln Ser Asn Pro Leu Val Asp Gly

50 55 60

Ser Cys Thr Asp Arg Gln Gly His Gly Thr His Val Ala Gly Thr Val

65 70 75 80

Leu Ala His Gly Gly Ser Asn Gly Gln Gly Val Tyr Gly Val Ala Pro

85 90 95

Gln Ala Lys Leu Trp Ala Tyr Lys Val Leu Gly Asp Asn Gly Ser Gly

100 105 110

Tyr Ser Asp Asp Ile Ala Ala Ala Ile Arg His Val Ala Asp Glu Ala

115 120 125

Ser Arg Thr Gly Ser Lys Val Val Ile Asn Met Ser Leu Gly Ser Ser

130 135 140

Ala Lys Asp Ser Leu Ile Ala Ser Ala Val Asp Tyr Ala Tyr Gly Lys

145 150 155 160

Gly Val Leu Ile Val Ala Ala Ala Gly Asn Ser Gly Ser Gly Ser Asn

165 170 175

Thr Ile Gly Phe Pro Gly Gly Leu Val Asn Ala Val Ala Val Ala Ala

180 185 190

Leu Glu Asn Val Gln Gln Asn Gly Thr Tyr Arg Val Ala Asp Phe Ser

195 200 205

Ser Arg Gly Asn Pro Ala Thr Ala Gly Asp Tyr Ile Ile Gln Glu Arg

210 215 220

Asp Ile Glu Val Ser Ala Pro Gly Ala Ser Val Glu Ser Thr Trp Tyr

225 230 235 240

Thr Gly Gly Tyr Asn Thr Ile Ser Gly Thr Ser Met Ala Thr Pro His

245 250 255

Val Ala Gly Leu Ala Ala Lys Ile Trp Ser Ala Asn Thr Ser Leu Ser

260 265 270

His Ser Gln Leu Arg Thr Glu Leu Gln Asn Arg Ala Lys Val Tyr Asp

275 280 285

Ile Lys Gly Gly Ile Gly Ala Gly Thr Gly Asp Asp Asp Tyr Ala Ser Gly

290 295 300

Phe Gly Tyr Pro Arg Val Lys

305 310

<210> 15

<211> 278

<212> PRT

<213> Thielavia terrestris

<400> 15

Ala Ser Gly Ser Gly Gln Ser Thr Arg Tyr Trp Asp Cys Cys Lys Pro

1 5 10 15

Ser Cys Ala Trp Pro Gly Lys Ala Ala Val Ser Gln Pro Val Tyr Ala

20 25 30

Cys Asp Ala Asn Phe Gln Arg Leu Ser Asp Phe Asn Val Gln Ser Gly

35 40 45

Cys Asn Gly Gly Ser Ala Tyr Ser Cys Ala Asp Gln Thr Pro Trp Ala

50 55 60

Val Asn Asp Asn Leu Ala Tyr Gly Phe Ala Ala Thr Ser Ile Ala Gly

65 70 75 80

Gly Ser Glu Ser Ser Trp Cys Cys Ala Cys Tyr Ala Leu Thr Phe Thr

85 90 95

Ser Gly Pro Val Ala Gly Lys Thr Met Val Val Gln Ser Thr Ser Thr

100 105 110

Gly Gly Asp Leu Gly Ser Asn Gln Phe Asp Ile Ala Met Pro Gly Gly

115 120 125

Gly Val Gly Ile Phe Asn Gly Cys Ser Ser Gln Phe Gly Gly Leu Pro

130 135 140

Gly Ala Gln Tyr Gly Gly Ile Ser Ser Arg Asp Gln Cys Asp Ser Phe

145 150 155 160

Pro Ala Pro Leu Lys Pro Gly Cys Gln Trp Arg Phe Asp Trp Phe Gln

165 170 175

Asn Ala Asp Asn Pro Thr Phe Thr Phe Gln Gln Val Gln Cys Pro Ala

180 185 190

Glu Ile Val Ala Arg Ser Gly Cys Lys Arg Asn Asp Asp Ser Ser Phe

195 200 205

Pro Val Phe Thr Pro Pro Ser Gly Gly Asn Gly Gly Thr Gly Thr Pro

210 215 220

Thr Ser Thr Ala Pro Gly Ser Gly Gln Thr Ser Pro Gly Gly Gly Ser

225 230 235 240

Gly Cys Thr Ser Gln Lys Trp Ala Gln Cys Gly Gly Ile Gly Phe Ser

245 250 255

Gly Cys Thr Thr Cys Val Ser Gly Thr Thr Cys Gln Lys Leu Asn Asp

260 265 270

Tyr Phe Ser Gln Cys Leu

275

<210> 16

<211> 470

<212> PRT

<213> Humicola insolens

<400> 16

Met Arg Ser Ser Pro Leu Leu Arg Ser Ala Val Val Ala Ala Leu Pro

1 5 10 15

Val Leu Ala Leu Ala Ala Asp Gly Arg Ser Thr Arg Tyr Trp Asp Cys

20 25 30

Cys Lys Pro Ser Cys Gly Trp Ala Lys Lys Ala Pro Val Asn Gln Pro

35 40 45

Val Phe Ser Cys Asn Ala Asn Phe Gln Arg Ile Thr Asp Phe Asp Ala

50 55 60

Lys Ser Gly Cys Glu Pro Gly Gly Val Ala Tyr Ser Cys Ala Asp Gln

65 70 75 80

Thr Pro Trp Ala Val Asn Asp Asp Phe Ala Leu Gly Phe Ala Ala Thr

85 90 95

Ser Ile Ala Gly Ser Asn Glu Ala Gly Trp Cys Cys Ala Cys Tyr Glu

100 105 110

Leu Thr Phe Thr Ser Gly Pro Val Ala Gly Lys Lys Met Val Val Gln

115 120 125

Ser Thr Ser Thr Gly Gly Asp Leu Gly Ser Asn His Phe Asp Leu Asn

130 135 140

Ile Pro Gly Gly Gly Val Gly Ile Phe Asp Gly Cys Thr Pro Gln Phe

145 150 155 160

Gly Gly Leu Pro Gly Gln Arg Tyr Gly Gly Ile Ser Ser Arg Asn Glu

165 170 175

Cys Asp Arg Phe Pro Asp Ala Leu Lys Pro Gly Cys Tyr Trp Arg Phe

180 185 190

Asp Trp Phe Lys Asn Ala Asp Asn Pro Ser Phe Ser Phe Arg Gln Val

195 200 205

Gln Cys Pro Ala Glu Leu Val Ala Arg Thr Gly Cys Arg Arg Asn Asp

210 215 220

Asp Gly Asn Phe Pro Ala Val Gln Ile Pro Ser Ser Ser Thr Ser Ser

225 230 235 240

Pro Val Asn Gln Pro Thr Ser Thr Ser Thr Thr Thr Ser Thr Ser Thr Thr Thr

245 250 255

Ser Ser Pro Pro Val Gln Pro Thr Thr Pro Ser Gly Cys Ala Asp Gly

260 265 270

Arg Ser Thr Arg Tyr Trp Asp Cys Cys Lys Pro Ser Cys Gly Trp Ala

275 280 285

Lys Lys Ala Pro Val Asn Gln Pro Val Phe Ser Cys Asn Ala Asn Phe

290 295 300

Gln Arg Ile Thr Asp Phe Asp Ala Lys Ser Gly Cys Glu Pro Gly Gly

305 310 315 320

Val Ala Tyr Ser Cys Ala Asp Gln Thr Pro Trp Ala Val Asn Asp Asp

325 330 335

Phe Ala Leu Gly Phe Ala Ala Thr Ser Ile Ala Gly Ser Asn Glu Ala

340 345 350

Gly Trp Cys Cys Ala Cys Tyr Glu Leu Thr Phe Thr Ser Gly Pro Val

355 360 365

Ala Gly Lys Lys Met Val Val Gln Ser Thr Ser Thr Gly Gly Asp Leu

370 375 380

Gly Ser Asn His Phe Asp Leu Asn Ile Pro Gly Gly Gly Val Gly Ile

385 390 395 400

Phe Asp Gly Cys Thr Pro Gln Phe Gly Gly Leu Pro Gly Gln Arg Tyr

405 410 415

Gly Gly Ile Ser Ser Arg Asn Glu Cys Asp Arg Phe Pro Asp Ala Leu

420 425 430

Lys Pro Gly Cys Tyr Trp Arg Phe Asp Trp Phe Lys Asn Ala Asp Asn

435 440 445

Pro Ser Phe Ser Phe Arg Gln Val Gln Cys Pro Ala Glu Leu Val Ala

450 455 460

Arg Thr Gly Cys Arg Arg

465 470

<210> 17

<211> 425

<212> PRT

<213> Escherichia coli

<400> 17

Thr Ala Leu Leu Leu Gly Leu Val Asn Gly Gln Lys Pro Gly Glu Thr

1 5 10 15

Lys Glu Val His Pro Gln Leu Thr Thr Phe Arg Cys Thr Lys Arg Gly

20 25 30

Gly Cys Lys Pro Ala Thr Asn Phe Ile Val Leu Asp Ser Leu Ser His

35 40 45

Pro Ile His Arg Ala Glu Gly Leu Gly Pro Gly Gly Cys Gly Asp Trp

50 55 60

Gly Asn Pro Pro Pro Lys Asp Val Cys Pro Asp Val Glu Ser Cys Ala

65 70 75 80

Lys Asn Cys Ile Met Glu Gly Ile Pro Asp Tyr Ser Gln Tyr Gly Val

85 90 95

Thr Thr Asn Gly Thr Ser Leu Arg Leu Gln His Ile Leu Pro Asp Gly

100 105 110

Arg Val Pro Ser Pro Arg Val Tyr Leu Leu Asp Lys Thr Lys Arg Arg

115 120 125

Tyr Glu Met Leu His Leu Thr Gly Phe Glu Phe Thr Phe Asp Val Asp

130 135 140

Ala Thr Lys Leu Pro Cys Gly Met Asn Ser Ala Leu Tyr Leu Ser Glu

145 150 155 160

Met His Pro Thr Gly Ala Lys Ser Lys Tyr Asn Pro Gly Gly Ala Tyr

165 170 175

Tyr Gly Thr Gly Tyr Cys Asp Ala Gln Cys Phe Val Thr Pro Phe Ile

180 185 190

Asn Gly Leu Gly Asn Ile Glu Gly Lys Gly Ser Cys Cys Asn Glu Met

195 200 205

Asp Ile Trp Glu Ala Asn Ser Arg Ala Ser His Val Ala Pro His Thr

210 215 220

Cys Asn Lys Lys Gly Leu Tyr Leu Cys Glu Gly Glu Glu Cys Ala Phe

225 230 235 240

Glu Gly Val Cys Asp Lys Asn Gly Cys Gly Trp Asn Asn Tyr Arg Val

245 250 255

Asn Val Thr Asp Tyr Tyr Gly Arg Gly Glu Glu Phe Lys Val Asn Thr

260 265 270

Leu Lys Pro Phe Thr Val Val Thr Gln Phe Leu Ala Asn Arg Arg Gly

275 280 285

Lys Leu Glu Lys Ile His Arg Phe Tyr Val Gln Asp Gly Lys Val Ile

290 295 300

Glu Ser Phe Tyr Thr Asn Lys Glu Gly Val Pro Tyr Thr Asn Met Ile

305 310 315 320

Asp Asp Glu Phe Cys Glu Ala Thr Gly Ser Arg Lys Tyr Met Glu Leu

325 330 335

Gly Ala Thr Gln Gly Met Gly Glu Ala Leu Thr Arg Gly Met Val Leu

340 345 350

Ala Met Ser Ile Trp Trp Asp Gln Gly Gly Asn Met Glu Trp Leu Asp

355 360 365

His Gly Glu Ala Gly Pro Cys Ala Lys Gly Glu Gly Ala Pro Ser Asn

370 375 380

Ile Val Gln Val Glu Pro Phe Pro Glu Val Thr Tyr Thr Asn Leu Arg

385 390 395 400

Trp Gly Glu Ile Gly Ser Thr Tyr Gln Glu Val Gln Lys Pro Lys Pro

405 410 415

Lys Pro Gly His Gly Pro Arg Ser Asp

420 425

<210> 18

<211> 773

<212> PRT

<213> Bacillus subtilis

<400> 18

Ala Glu Gly Asn Thr Arg Glu Asp Asn Phe Lys His Leu Leu Gly Asn

1 5 10 15

Asp Asn Val Lys Arg Pro Ser Glu Ala Gly Ala Leu Gln Leu Gln Glu

20 25 30

Val Asp Gly Gln Met Thr Leu Val Asp Gln His Gly Glu Lys Ile Gln

35 40 45

Leu Arg Gly Met Ser Thr His Gly Leu Gln Trp Phe Pro Glu Ile Leu

50 55 60

Asn Asp Asn Ala Tyr Lys Ala Leu Ala Asn Asp Trp Glu Ser Asn Met

65 70 75 80

Ile Arg Leu Ala Met Tyr Val Gly Glu Asn Gly Tyr Ala Ser Asn Pro

85 90 95

Glu Leu Ile Lys Ser Arg Val Ile Lys Gly Ile Asp Leu Ala Ile Glu

100 105 110

Asn Asp Met Tyr Val Ile Val Asp Trp His Val His Ala Pro Gly Asp

115 120 125

Pro Arg Asp Pro Val Tyr Ala Gly Ala Glu Asp Phe Phe Arg Asp Ile

130 135 140

Ala Ala Leu Tyr Pro Asn Asn Pro His Ile Ile Tyr Glu Leu Ala Asn

145 150 155 160

Glu Pro Ser Ser Asn Asn Asn Asn Gly Gly Ala Gly Ile Pro Asn Asn Glu

165 170 175

Glu Gly Trp Asn Ala Val Lys Glu Tyr Ala Asp Pro Ile Val Glu Met

180 185 190

Leu Arg Asp Ser Gly Asn Ala Asp Asp Asn Ile Ile Ile Val Gly Ser

195 200 205

Pro Asn Trp Ser Gln Arg Pro Asp Leu Ala Ala Asp Asn Pro Ile Asn

210 215 220

Asp His His Thr Met Tyr Thr Val His Phe Tyr Thr Gly Ser His Ala

225 230 235 240

Ala Ser Thr Glu Ser Tyr Pro Pro Glu Thr Pro Asn Ser Glu Arg Gly

245 250 255

Asn Val Met Ser Asn Thr Arg Tyr Ala Leu Glu Asn Gly Val Ala Val

260 265 270

Phe Ala Thr Glu Trp Gly Thr Ser Gln Ala Asn Gly Asp Gly Gly Pro

275 280 285

Tyr Phe Asp Glu Ala Asp Val Trp Ile Glu Phe Leu Asn Glu Asn Asn

290 295 300

Ile Ser Trp Ala Asn Trp Ser Leu Thr Asn Lys Asn Glu Val Ser Gly

305 310 315 320

Ala Phe Thr Pro Phe Glu Leu Gly Lys Ser Asn Ala Thr Asn Leu Asp

325 330 335

Pro Gly Pro Asp His Val Trp Ala Pro Glu Glu Leu Ser Leu Ser Gly

340 345 350

Glu Tyr Val Arg Ala Arg Ile Lys Gly Val Asn Tyr Glu Pro Ile Asp

355 360 365

Arg Thr Lys Tyr Thr Lys Val Leu Trp Asp Phe Asn Asp Gly Thr Lys

370 375 380

Gln Gly Phe Gly Val Asn Ser Asp Ser Pro Asn Lys Glu Leu Ile Ala

385 390 395 400

Val Asp Asn Glu Asn Asn Thr Leu Lys Val Ser Gly Leu Asp Val Ser

405 410 415

Asn Asp Val Ser Asp Gly Asn Phe Trp Ala Asn Ala Arg Leu Ser Ala

420 425 430

Asp Gly Trp Gly Lys Ser Val Asp Ile Leu Gly Ala Glu Lys Leu Thr

435 440 445

Met Asp Val Ile Val Asp Glu Pro Thr Thr Val Ala Ile Ala Ala Ile

450 455 460

Pro Gln Ser Ser Lys Ser Gly Trp Ala Asn Pro Glu Arg Ala Val Arg

465 470 475 480

Val Asn Ala Glu Asp Phe Val Gln Gln Thr Asp Gly Lys Tyr Lys Ala

485 490 495

Gly Leu Thr Ile Thr Gly Glu Asp Ala Pro Asn Leu Lys Asn Ile Ala

500 505 510

Phe His Glu Glu Asp Asn Asn Met Asn Asn Ile Ile Leu Phe Val Gly

515 520 525

Thr Asp Ala Ala Asp Val Ile Tyr Leu Asp Asn Ile Lys Val Ile Gly

530 535 540

Thr Glu Val Glu Ile Pro Val Val His Asp Pro Lys Gly Glu Ala Val

545 550 555 560

Leu Pro Ser Val Phe Glu Asp Gly Thr Arg Gln Gly Trp Asp Trp Ala

565 570 575

Gly Glu Ser Gly Val Lys Thr Ala Leu Thr Ile Glu Glu Ala Asn Gly

580 585 590

Ser Asn Ala Leu Ser Trp Glu Phe Gly Tyr Pro Glu Val Lys Pro Ser

595 600 605

Asp Asn Trp Ala Thr Ala Pro Arg Leu Asp Phe Trp Lys Ser Asp Leu

610 615 620

Val Arg Gly Glu Asn Asp Tyr Val Ala Phe Asp Phe Tyr Leu Asp Pro

625 630 635 640

Val Arg Ala Thr Glu Gly Ala Met Asn Ile Asn Leu Val Phe Gln Pro

645 650 655

Pro Thr Asn Gly Tyr Trp Val Gln Ala Pro Lys Thr Tyr Thr Ile Asn

660 665 670

Phe Asp Glu Leu Glu Glu Ala Asn Gln Val Asn Gly Leu Tyr His Tyr

675 680 685

Glu Val Lys Ile Asn Val Arg Asp Ile Thr Asn Ile Gln Asp Asp Thr

690 695 700

Leu Leu Arg Asn Met Met Ile Ile Phe Ala Asp Val Glu Ser Asp Phe

705 710 715 720

Ala Gly Arg Val Phe Val Asp Asn Val Arg Phe Glu Gly Ala Ala Thr

725 730 735

Thr Glu Pro Val Glu Pro Glu Pro Val Asp Pro Gly Glu Glu Thr Pro

740 745 750

Pro Val Asp Glu Lys Glu Ala Lys Lys Glu Gln Lys Glu Ala Glu Lys

755 760 765

Glu Glu Lys Glu Glu

770

Claims (19)

1. Use of one or more enzymes in preventing, inhibiting or reducing the growth of microorganisms on a surface, wherein after using said enzyme, the TTC detectable time of said microorganisms on said surface increases by at least 20%, at least 30%, at least 50%, at least 70%, at least 80%, or at least 100%. 2. The use according to claim 1, wherein the microorganism is a fungus, and the sporulation time of the fungus is increased by at least 20% compared to fungi on surfaces not treated with enzymes, and/or the spore density is scored by Decreases the spore density scale from 0 to 7 by at least 1. 3. The use according to claim 1 or 2, for suppressing or reducing malodor caused by microorganisms on the surface of textiles. 4. Use according to claim 1 or 2 for preventing or reducing the attachment of microorganisms to the surface of textiles. 5. The use according to any one of claims 1-4, wherein the microorganism is a fungus or bacterium selected from the group consisting of Staphylococcus aureus, Klebsiella pneumoniae, aeruginosa Pseudomonas, Escherichia coli, Pseudomonas putida, Micrococcus luteus, Staphylococcus epidermidis, Corynebacterium japonica, Propionibacterium acnes, Brevundimonas vesicularis, Streptomyces griseus, Soil Streptomyces odoriferous, Exophyllum obscura, Fusarium oxysporum, Alternaria alternaria, Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Aureobasidium pullulans, Chaetomium globosa, Cladosporium coccidioides, Viridans viridans Mold, Mucoralum, Penicillium chrysogenum, Penicillium vulgaris, Penicillium dermatodes, Penicillium erythrogenes, Penicillium mutatis, Penicillium citrinum, Grape ear mold, Trichoderma viride, Trichophyton rubrum, Candida parasmoothum Saccharomyces, Rhodotorula marcescens, Debaria hansenii, Pichia mongoliana, Candida albicans, and combinations thereof. 6. Use according to claims 1-5, wherein the micro-organisms grow on remaining soiled areas of the surface. 7. Use according to any one of claims 1-6, wherein the one or more enzymes are selected from the group consisting of amylases, proteases, lipases, mannanases, Cellulases, pectinases, and combinations thereof. 8. The use of any one of claims 1-7, wherein the enzyme is an amylase, a protease, a cellulase, or a combination thereof. 9. Use according to claim 3 or 4, wherein the enzyme is a cellulase. 10. Use according to any one of the preceding claims, wherein the protease is selected from the group consisting of: i) a protease comprising a substitution at one or more of the following positions compared to the protease shown in SEQ ID NO 1 or SEQ ID NO 2: 3, 4, 9, 15, 24, 27, 42, 55 ,59,60,66,74,85,96,97,98,99,100,101,102,104,116,118,121,126,127,128,154,156,157,158,161,164 ,176,179,182,185,188,189,193,198,199,200,203,206,211,212,216,218,226,229,230,239,246,255,256,268,269 , wherein said position corresponds to the position of the protease shown in SEQ ID NO 1, or ii) One or more protease variants of a protease parent, wherein said protease variant comprises one or more mutations selected from the group consisting of: S3T, V4I, S9R, S9E, A15T, S24G . 、S101A、V102I、V102Y、V102N、S104A、G116V、G116R、H118D、H118N、N120S、S126L、P127Q、S128A、S154D、A156E、G157D、G157P、S158E、Y161A、R164S、Q176E、N179E、S182E、Q185N、A188P , G189E, V193M, N198D, V199I, Y203W, S206G, L211Q, L211D, N212D, N212S, M216S, A226V, K229L, Q230H, Q239R, N246K, N255W, N255D, N255E, L256E, T2686D, among which the R2696D a position corresponding to the position of the protease shown in SEQ ID NO 1, wherein the protease parent is selected from the protease shown in SEQ ID NO 1 and the Bacillus amyloliquefaciens protease (BPN') shown in SEQ ID NO 2, and wherein said protease variant has at least 80% sequence identity to SEQ ID NO 1 or 80% sequence identity to SEQ ID NO 2, iii) a protease comprising a substitution at one or more positions corresponding to positions 171, 173, 175, 179 or 180 of SEQ ID NO: 14 compared to the protease shown in SEQ ID NO 14, wherein the the protease variant has at least 75% but less than 100% sequence identity to SEQ ID NO 14, iv) a protease comprising the amino acid sequence shown in SEQ ID NO 1 or 2, or a protease with a polypeptide comprising amino acids 1-269 of SEQ ID NO 1 or comprising amino acids 1-269 of SEQ ID NO 2 275 polypeptides having at least 80% sequence identity, v) one or more of the following protease variants selected from the group consisting of: SEQ ID NO 1, with the changes T22R+S99G+S101A+V102I+A226V+Q239R, SEQ ID NO 2, with the changes S24G+S53G+S78N+S101N+G128A+Y217Q, SEQ ID NO 2, with the changes S24G+S53G+S78N+S101N+G128S+Y217Q, SEQ ID NO 1 with alterations S9E+N42R+N74D+V199I+Q200L+Y203W+S253D+N255W+L256E, SEQ ID NO 1, having S9E+N42R+N74D+H118V+Q176E+A188P+V199I+Q200L Y203W+S250D+S253D+N255W+L256E, SEQ ID NO 1, having S9E+N42R+N74D+Q176E+A188P+V199I+Q200L+Y203W S250D+S253D+N255W+L256E, SEQ ID NO 1, having S3V+N74D+H118V+Q176E+N179E+S182E+V199I+Q200L Y203W+S210V+S250D+S253D+N255W+L256E, SEQ ID NO 1 with the alteration T22A+N60D+S99G+S101A+V102I+N114L+G157D+S182D+T207A+A226V+Q239R+N242D+E265F, SEQ ID NO 1, with alterations S9E+N42R+N74D+H118V+Q176E+A188P+V199I+Q200L+Y203W+S250D+S253D+N255W+L256E, SEQ ID NO 1, with alterations S9E+N42R+N74D+Q176E+A188P+V199I+Q200L+Y203W+S250D+S253D+N255W+L256E, SEQ ID NO 1, with the changes S9E+N42R+N74D+H118V+Q176E+A188P+V199I+Q200L+Y203W+S250D+N255W+L256E+*269aH+*269bH, SEQ ID NO 1, with the changes S3V+N74D+H118V+Q176E+N179E+S182E+V199I+Q200L+Y203W+S210V+S250D+N255W+L256E, SEQ ID NO 1 with the alterations S9E+N74D+G113W+G157P+Q176E+V199I+Q200L+Y203W+S250D+T254E+N255W+L256E, SEQ ID NO 1, with changes S3V+S9R+N74D+H118V+Q176E+N179E+S182E+V199I+Q200L+Y203W+S212V+S250D+N255W+L256E, SEQ ID NO 1, with alteration S99E, and SEQ ID NO 2, with alteration L217D, and combinations thereof. 11. Use according to any one of the preceding claims, wherein the amylase is selected from the group consisting of: i) variants comprising one or more substitutions at the following positions: 9, 26, 30, 33, 82, 37, 106, 118, 128, 133, 149, 150, 160, 178, 182, 186 ,193,195,202,203,214,231,256,257,258,269,270,272,283,295,296,298,299,303,304,305,311,314,315,318,319 , 320, 323, 339, 345, 361, 378, 383, 419, 421, 437, 441, 444, 445, 446, 447, 450, 458, 461, 471, 482, 484, wherein the positions correspond to SEQ The location of ID NO 11; ii) a variant exhibiting at least 90% identity to SEQ ID NO 12, having deletions at positions 183 and 184, iii) a variant exhibiting at least 95% identity to SEQ ID NO 13 comprising a mutation at one or more of the following positions: M202, M208, S255, R172 and/or M261, iv) a polypeptide comprising the amino acid sequence shown in SEQ ID NO:9 or 10, v) a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96% of SEQ ID NO:9 or 10 %, at least 97%, at least 98%, at least 99% sequence identity, and combinations thereof. 12. Use according to any one of the preceding claims, wherein the cellulase is selected from the group consisting of: a. a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity. 13. The use according to any one of the preceding claims, wherein the surface is a textile made of natural fibers such as cotton, synthetic fibers such as polyester, or mixtures thereof. 14. Use according to any one of the preceding claims, wherein the one or more enzymes are incorporated into a detergent composition substantially free of biocides. 15. Use according to any one of the preceding claims, wherein the one or more enzymes are incorporated into a powder detergent or a liquid detergent. 16. A method of preventing, inhibiting or reducing microbial growth on a surface, said method comprising the steps of: a. Treating said surface with an enzyme as defined in any one of claims 10-12 to such an extent that the TTC detectable time of at least one microorganism on said surface increases by at least 20% after said treatment. 17. A method of demonstrating microbial growth inhibition or deep cleaning benefits on a surface according to an enzyme or combination of enzymes as defined in claims 10-12, said method comprising the steps of: a. Provide two surfaces A and A', b. Applying a soil comprising an indicator of microbial growth to each surface A and A', c. washing A with a detergent composition not comprising enzymes and washing A' with said detergent composition comprising one or more enzymes, d. Prepare to inoculate microorganisms and apply them to surfaces A and A', e. incubating A and A' under suitable conditions to allow microbial growth for a predetermined period of time, f. Compare the growth of microorganisms on A and A'. 18. The method of claim 17, wherein the microbial growth indicator is triphenyltetrazolium chloride (TTC). 19. The method according to claim 16 or 17, wherein said surfaces A and A' provided in step a. according to claim 14 are made of substantially the same material.