Compositions and methods for cleaning comprising polypeptides having thermolysin activity

Abstract

The present disclosure relates to compositions and methods for cleaning. More particularly, the present disclosure relates to compositions and methods for mitigating odor associated with articles such as, but not limited to, textiles or machines, where the machines are washing machines or dishwashers.

Description

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 353,986, filed on June 21, 2022, which is incorporated herein by reference in its entirety.

[0002] The present disclosure relates to compositions and methods for cleaning (eg, hard surface and laundry cleaning) and reducing odors from textiles, hard surfaces, or dishware.

[0003] Reference to a sequence listing submitted electronically

[0004] An official copy of the sequence listing is submitted electronically via the Patent Center as an XML-formatted sequence listing named 20230613_NB42099PCT_SeqListing , created in June 13, 2023 , and the size is 2,128 The sequence list contained in the XML format file is a part of the present specification and is incorporated herein by reference in its entirety. Background Art

[0005] The trend toward cold water washing and synthetic sportswear is driving demand for detergents that eliminate odors, while the industry is moving away from laundry detergent powders that use traditional oxygen bleaches. Therefore, new methods of removing odors associated with laundry and washing machines are needed.

[0006] Despite repeated exposure to surfactants, proteases and amylases from typical laundry detergents, malodorous compounds persist in washing machines and fabrics and textiles and cause hygiene and odor problems. Therefore, there is a need for more effective solutions for reducing odors and improving freshness in cleaning applications such as laundry. Summary of the invention

[0007] One embodiment relates to methods for reducing or treating odor in textiles or machinery, comprising (i) contacting the textile or machinery with a composition comprising: a) 0.2 to 100 parts per million (PPM) of a polypeptide having thermolysin activity; b) 0.00001% to about 2% of a perfume; and c) at least one detergent adjuvant, and (ii) optionally rinsing the textile or machinery.

[0008] Another embodiment is directed to methods for cleaning articles comprising contacting an article in need of cleaning with a composition comprising a) 0.2 to 100 parts per million (PPM) of a polypeptide having thermolysin activity; b) 0.00001% to about 2% of a perfume; and c) at least one detergent adjuvant, and (ii) optionally rinsing the article.

[0009] In another embodiment, methods are provided for improving the freshness of articles after a cleaning process, the methods comprising contacting the articles with a composition comprising a) 0.2 to 100 parts per million (PPM) of a polypeptide having thermolysin activity; b) 0.00001% to about 2% of a perfume; and c) at least one detergent adjuvant, and (ii) optionally rinsing the articles.

[0010] In yet another embodiment, the present disclosure provides the use of a polypeptide having thermolysin activity in a detergent composition for increasing the freshness associated with a perfume of textiles after a laundry washing process, or for enhancing the effect of a perfume in a detergent composition, wherein the detergent comprises at least one perfume compound.

[0011] The present disclosure further provides an enzyme-flavor system comprising about 0.2 to 100 PPM of a polypeptide having thermolysin activity; and about 0.00001% to about 2% of a flavor.

[0012] Further provided herein are detergent compositions comprising about 0.2 to 100 PPM of a polypeptide having thermolysin activity, about 0.00001% to about 2% of a perfume, and optionally at least one detergent adjunct material. DETAILED DESCRIPTION

[0013] The disclosure provides compositions (e.g., enzymes and detergent compositions) and uses such compositions to alleviate odors (e.g., from goods such as hard surfaces or textiles). The disclosure also provides compositions (e.g., enzymes and detergent compositions) and uses such compositions to improve the freshness of articles (e.g., from goods such as hard surfaces or textiles). These compositions are usually used to comprise at least one polypeptide with thermolysin activity and an enzyme-spice combination of spices or to comprise a polypeptide with thermolysin activity and a composition of spices. These compositions also optionally comprise the other components of cleaning detergents, such as one or more surfactants.

[0014] Before describing embodiments of the compositions and methods of the present invention, the following terms are defined.

[0015] Unless otherwise defined herein, all technical and scientific terms used herein have the same meanings as those of ordinary skill in the art to which the present invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, preferred methods and materials are described herein. Therefore, generally by reference to the specification, the terms immediately defined below are more fully described. In addition, unless the context clearly indicates otherwise, as used herein, the singular terms "a / an" and "the" include plural references. It should be understood that the present invention is not limited to the described particular methods, protocols and reagents, as they can vary according to the context used by those skilled in the art.

[0016] Each maximum numerical limit given throughout this specification is intended to include each lower numerical limit, as if such lower numerical limits were expressly written herein. Each minimum numerical limit given throughout this specification will include each higher numerical limit, as if such higher numerical limits were expressly written herein. Each numerical range given throughout this specification will include each narrower numerical range falling within such wider numerical range, as if such narrower numerical ranges were all expressly written herein.

[0017] The term "odor" refers to any undesirable or unexpected smell on an item, for example after cleaning. Examples of odors include volatile compounds with a perceived unpleasant odor, which may be produced by microorganisms. These microorganisms may be gram-positive or gram-negative bacteria (aerobic or anaerobic); algae, protozoa, and / or yeast or filamentous fungi. In some embodiments, the odor may be associated with one or more microorganisms comprising one or more of the following bacterial genera: Acinetobacter sp., Aeromicrobium sp., Brevundimonas sp., Microbacterium sp., Micrococcus luteus, Pseudomonas sp. (e.g., Pseudomonas fluorescens), Staphylococcus sp. (e.g., Staphylococcus epidermidis), and Stenotrophomonas sp., Streptomyces sp., Listeria sp., Streptococcus sp., and Escherichia sp. Another example of bad smell includes unpleasant smells, which may be sweat, or body odor associated with items that have been in contact with people or animals. Another example includes the smell from spices (such as curry or other spices with odor) attached to the item.

[0018] As used herein, term " aromatic effect " refers to people's perception of the fragrance on the articles such as fabric (for example washing clothes) of cleaning.Enzyme-spice combination provided herein or the aromatic effect of the composition comprising enzyme-spice combination can be for example analyzed by using sensory evaluation, for example described in following example 1,2 or 5.Alternately, aromatic effect can be measured by GC-MS analysis and quantitatively expressed as aromatic intensity.Therefore, the aromatic effect of increase can be expressed as the aromatic intensity of increase, make with not having the polypeptide and spices of thermolysin activity or comprising the polypeptide and spices with thermolysin activity similar articles of composition cleaning compared, or before cleaning compared with the same articles, with having the polypeptide and spices of thermolysin activity or comprising the polypeptide and spices with thermolysin activity composition cleaning articles.

[0019] As used herein, "surface" means any surface, including hard surfaces, soft surfaces and porous surfaces. Hard surfaces include, but are not limited to, metals, glass, ceramics, wood, minerals (rocks, stones, marble, granite), aggregate materials such as concrete, plastics, composite materials, hard rubber materials and gypsum. Hard materials can be finished with enamel and paint. Hard surfaces are found in, for example, water treatment and storage equipment and boxes; in dairy and food processing equipment and facilities; in medical equipment and facilities, such as surgical instruments and permanent and temporary implants; in industrial pharmaceutical equipment and factories. Soft surfaces are, for example, hair and all types of textiles. Porous surfaces can also be found in certain ceramics and membranes for filtration. Other surfaces include, but are not limited to, hulls and swimming pools. Other surfaces can be biological surfaces, such as skin, keratin or internal organs.

[0020] The term "fabric" refers to materials such as woven, knitted, and nonwoven materials, as well as staple fibers and filaments that can be converted into, for example, yarns and woven, knitted, and nonwoven fabrics. The term encompasses materials made from natural fibers as well as synthetic (e.g., manufactured) fibers.

[0021] As used herein, term " textile " refers to any textile material, including yarn, yarn intermediate, fiber, non-woven material, natural material, synthetic material and any other textile material, fabric made of these materials and product (for example, clothing and other products) made of fabric. The textile or fabric can be in the form of knitted fabric, woven fabric, denim, non-woven fabric, felt, yarn and towel cloth. The textile can be based on cellulose, such as natural cellulose products, including cotton, flax / linen, jute, ramie, sisal or coconut fiber, or artificial cellulose (for example, derived from wood pulp), including viscose / rayon, cellulose acetate fiber (tricell), lyocell fiber (lyocell) or its blend. The textile or fabric can also not be based on cellulose, such as natural polyamide, including wool, camel hair, cashmere, mohair, rabbit hair and silk, or synthetic polymers such as nylon, aramid, polyester, acrylic acid, polypropylene and spandex / elastic fiber (spandex / elastane) or its blend and based on cellulose and not based on the blend of cellulose fiber. Examples of blends are blends of cotton and / or rayon / viscose with one or more companion 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 (e.g., rayon / viscose fibers, ramie, flax / linen, jute, cellulose acetate fibers, lyocell fibers). Fabrics may be conventional washable clothing, such as household clothing with stains. When the term fabric or garment is used, it is intended to also include the broad term textiles. In the context of the present application, the term "textile" may be used interchangeably with fabric and cloth.

[0022] As used herein, the term "hard surface" refers to any article with a hard surface, including floors, tables, walls, roofs, etc., and the surfaces of hard objects, such as cars (car wash), boat hulls, tableware (dinner dishes), medical equipment, pipes, reservoirs or holding tanks. The term "hard surface" also includes the surfaces of flexible but solid objects, such as the inside of a bendable tube and a supply line or the surface of a deformable tank or container. The term "hard surface" also includes the surfaces inside a cleaning machine, such as the inside of a laundry washing machine or a dishwashing machine, including soap boxes, walls, windows, baskets, racks, nozzles, pumps, sinks, filters, pipes, tubes, joints, seals, gaskets, fittings, impellers, drums, drain pipes, traps, coin trap inlets and outlets. The term hard surface does not cover textiles or fabrics.

[0023] The term "laundry" includes domestic laundry and industrial laundry, and means the process of treating textiles with a solution containing a cleaning or detergent composition as provided herein. The laundry process can be performed, for example, using a domestic or industrial cleaning machine or can be performed by hand.

[0024] The term "wash cycle" refers to a washing operation in which a textile is immersed in a cleaning solution, some mechanical action is applied to the textile to release stains or promote the flow of cleaning solution into and out of the textile, and finally the excess cleaning solution is removed. After one or more wash cycles, the textile is usually rinsed and dried.

[0025] The term "cleaning liquor" is defined herein as a solution or mixture of water and detergent components, optionally including a polypeptide having thermolysin activity and perfume.

[0026] Cleaning Methods

[0027] In one embodiment, methods for preventing, reducing or removing odors are provided, wherein the methods comprise contacting an item with a polypeptide having thermolysin activity and a fragrance or a composition comprising a polypeptide having thermolysin activity and a fragrance.

[0028] In another embodiment, the present disclosure provides a method for preventing, reducing or removing odors associated with textiles or hard surfaces, wherein the method comprises contacting the textile or hard surface with a polypeptide having thermolysin activity and a perfume or a composition comprising a polypeptide having thermolysin activity and a perfume, and optionally rinsing the textile or hard surface.

[0029] In another embodiment, the present disclosure provides methods for reducing odors associated with textiles or hard surfaces, comprising: (i) contacting the textile or hard surface with a polypeptide having thermolysin activity and a perfume or a composition comprising a polypeptide having thermolysin activity and a perfume; and (ii) optionally, rinsing the textile or surface. In some embodiments, the textile or hard surface comprises a protein on the surface of the textile or hard surface. In some embodiments, the odor is reduced by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more compared to the amount of odor present before contacting the textile or hard surface with a polypeptide having thermolysin activity and a perfume or a composition comprising a polypeptide having thermolysin activity and a perfume.

[0030] In some embodiments, the odor is reduced by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more after 1, 2, 3, 4 or 5 or more wash cycles compared to the amount of odor present in textiles or hard surfaces that are not contacted with a polypeptide having thermolysin activity and a perfume or a composition comprising a polypeptide having thermolysin activity and a perfume.

[0031] The textile or surface can be contacted with the polypeptide and spices or the composition comprising the polypeptide and spices with thermolysin activity in a cleaning machine or a manual cleaning bucket (for example, for hand washing). In one embodiment, the textile or surface is contacted with the polypeptide and spices with thermolysin activity or the composition comprising the polypeptide and spices with thermolysin activity in a cleaning solution. In another embodiment, the solution containing the polypeptide and spices with thermolysin activity is hatched together with a hard surface or flows through the hard surface, such as by pumping the solution through a pipe or pipeline or by filling a reservoir with the solution.

[0032] In certain embodiments, textile or surface is contacted with polypeptide and spices or the composition comprising the polypeptide and spices under the condition of any time period of lasting desired or lasting being enough to prevent, reduce or remove the stink of the textile.In one embodiment, the contacting step is between about 5 minutes and about 10 days.In certain embodiments, contact occurs in cleaning solution, lasts about 5 to about 400 minutes, about 5 minutes to about 300 minutes, about 5 minutes to about 250 minutes, about 5 minutes to about 200 minutes, about 5 minutes to about 150 minutes, about 5 minutes to about 100 minutes, about 5 minutes to about 50 minutes, about 5 minutes to about 30 minutes.

[0033] In some embodiments, the textile or article is contacted with a polypeptide and a perfume or a composition comprising the polypeptide and a perfume under conditions having a temperature that allows the prevention, reduction or removal of the odor of the textile or article. In some embodiments, the temperature in the methods disclosed herein includes those between 10° and 60°C, between 10° and about 45°C, between 15° and about 55°C, between 15° and about 50°C, between 15° and about 45°C, between 20° and about 60°C, between 20° and about 50°C, and between 20° and about 45°C.

[0034] The polypeptides, compositions and methods provided herein can be used in a wide range of applications where it is desired to prevent, reduce or remove odors, such as household cleaning, including washing machines, dishwashers and household surfaces.

[0035] The polypeptides, compositions and methods provided herein are further useful in a wide range of applications where it is desirable to improve the fragrance impact of a given fragrance, such as household cleaning, including cleaning machines, dishwashers, and household surfaces.

[0036] Another embodiment relates to a method for laundering textiles, wherein the method comprises contacting the textiles with a polypeptide having thermolysin activity and a perfume, or a composition comprising a polypeptide having thermolysin activity and a perfume, for a period of time sufficient to prevent, reduce or remove odor from the textiles, and optionally rinsing the textiles.

[0037] Another embodiment is directed to a method for cleaning an article, wherein the method comprises contacting the article with a polypeptide having thermolysin activity and a perfume, or a composition comprising a polypeptide having thermolysin activity and a perfume, under conditions sufficient to reduce or remove the odor of the article, and optionally rinsing the article.

[0038] Composition

[0039] In one embodiment, the present disclosure provides compositions (eg, detergent compositions) for use in the methods provided herein. These compositions typically comprise a polypeptide having thermolysin activity and a perfume and optionally one or more additional detergent components, such as a surfactant.

[0040] The compositions comprising a polypeptide having thermolysin activity and a fragrance that can be used in the methods provided herein can comprise a polypeptide having thermolysin activity at a concentration of 0.001 to 10,000 mg / L, or 0.001 to 2000 mg / L, or 0.01 to 5000 mg / L, or 0.01 to 2000 mg / L, or 0.01 to 1300 mg / L, or 0.1 to 5000 mg / L, or 0.1 to 2000 mg / L, or 0.1 to 1300 mg / L, or 1 to 5000 mg / L, or 1 to 1300 mg / L, or 1 to 5000 mg / L, or 10 to 5000 mg / L, or 10 to 1300 mg / L, or 10 to 500 mg / L. In another embodiment, the composition may contain a polypeptide having thermolysin activity in an amount of 0.002 to 5000 mg protein, such as 0.005 to 1300 mg protein, or 0.01 to 5000 mg protein, or 0.01 to 1300 mg protein, or 0.1 to 5000 mg protein, or 1 to 1300 mg protein, preferably 0.1 to 1300 mg protein, more preferably 1 to 1300 mg protein, even more preferably 10 to 500 mg protein / liter of cleaning liquid, or in an amount of at least 0.002 ppm active thermolysin. In another embodiment, the detergent composition comprises a polypeptide having thermolysin activity in an amount providing between 0.1 and 5000 ppm, about 0.1 and 2500 ppm, about 0.1 and 1500 ppm, about 0.1 and 1300 ppm, about 0.1 and 1000 ppm, about 0.1 and 500 ppm, 1 and 1300 ppm, 10 and 1300 ppm, about 10 and 500 PPM, about 50 and 1300 ppm, about 50 and 500 ppm of thermolysin in the cleaning solution.

[0041] Compositions comprising a polypeptide having thermolysin activity and a fragrance, which are useful for use in the methods provided herein, can comprise fragrance in an amount sufficient to provide a cleaning solution with about 0.001%, 0.0005%, 0.0002%, or 0.0001% fragrance.

[0042] In one embodiment, the composition comprises thermolysin, perfume, and at least one additional detergent component, and optionally one or more additional enzymes.

[0043] Thermolysin polypeptides for use in the methods and compositions herein include any thermolysin polypeptide. As used herein, the term "thermolysin" refers to an enzyme with EC 3.4.24.X or any member of the M4 protease family as described in the MEROPS-peptidase database (see, Rawlings et al., MEROPS: the peptidase database, Nucl Acids Res, Issue 34 database, D270-272

[2006] ), wherein thermolysin (TLN; EC 3.4.24.27) is the prototype. The amino acid sequence of an embodiment of thermolysin is a neutral metalloendopeptidase secreted by Bacillus thermoproteolyticus, and the sequence is shown in UniProtKB / Swiss-Prot accession number P00800 (SEQ ID NO: 1). Thermolysin polypeptides include homologues, variants and active fragments of SEQ ID NO: 1. The terms "thermolysin," "stearolysin," "lysin," "protease-T," "PrT," "thermolysin-like protease," and "TLP" are used interchangeably herein to refer to a neutral metalloprotease having the amino acid sequence of SEQ ID NO: 1 or those having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity to SEQ ID NO: 1.

[0044] As used herein, "homologous genes" refers to pairs of genes from different, but usually related species that correspond to each other and are identical or very similar to each other. The term encompasses genes separated by speciation (i.e., the development of new species) (e.g., orthologous genes) and genes separated by genetic duplication (e.g., paralogous genes).

[0045] As used herein, the term "variant polypeptide" refers to a polypeptide comprising an amino acid sequence that differs from the amino acid sequence of a parent polypeptide or a reference polypeptide (including but not limited to a wild-type polypeptide) by at least one amino acid residue.

[0046] As used herein, "Bacillus" includes all species within the "Bacillus" genus as known to those skilled in the art, including, but not limited to, B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. clausii, B. halodurans, B. megaterium, B. coagulans, B. circulans, B. lautus, and B. thuringiensis. It will be recognized that the genus Bacillus is continually undergoing taxonomic reorganization. Thus, the genus is intended to include species that have been reclassified, including, but not limited to, organisms such as Bacillus stearothermophilus (now called "Geobacillus stearothermophilus"). The production of resistant endospores in the presence of oxygen is considered a defining characteristic of the genus Bacillus, although this characteristic also applies to the more recently named genera Alicyclobacillus, Amphibacillus, Aneurinibacillus, Anoxybacillus, Brevibacillus, Filobacillus, Gracilibacillus, Halobacillus, Paenibacillus, Salibacillus, Thermobacillus, Ureibacillus, and Virgibacillus.

[0047] In some embodiments, thermolysin for use in the compositions and methods provided herein include polypeptides having an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1. In some embodiments, the thermolysin has an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1 and has thermolysin activity. In some embodiments, the thermolysin has an amino acid sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1, and has the ability to prevent, reduce or remove odor-causing residues in laundry washing. In some embodiments, the thermolysin has an amino acid sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1, and has the ability to clean body soil stains. In some embodiments, the thermolysin has an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1 and has the ability to reduce odor by 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% relative to an untreated control.

[0048] As used herein, "thermolysin activity" and "proteolytic activity" refer to proteins or polypeptides that exhibit the ability to hydrolyze peptides or substrates with peptide bonds. Methods for measuring proteolytic activity are known and include comparative assays that analyze the ability of each protease to hydrolyze commercial substrates. Other methods include those provided herein. Exemplary substrates that can be used to analyze proteases or proteolytic activity include, but are not limited to, dimethyl casein (Sigma C-9801), bovine collagen (Sigma C-9879), bovine elastin (Sigma E-1625) and bovine keratin (ICN Biomedical 902111). Colorimetric assays using these substrates are well known in the art (see, for example, WO 99 / 34011 and US 6,376,450). pNA peptide-based assays (see, for example, Del Mar et al., Anal Biochem [Analytical Biochemistry], 99: 316-320, 1979) can also be used to determine the concentration of active enzymes. The assay measures the rate at which p-nitroaniline is released when the enzyme hydrolyzes a soluble synthetic substrate such as succinyl-alanine-alanine-proline-phenylalanine-p-nitroaniline (suc-AAPF-pNA). The rate of generation of the yellow color from the hydrolysis reaction is measured at 405 or 410 nm on a spectrophotometer and is proportional to the active enzyme concentration. In addition, absorbance measurements at 280 nanometers (nm) can be used to determine the total protein concentration in a purified protein sample. The activity of substrate / protein concentration gives the enzyme specific activity.

[0049] As used herein, "% identity or percent identity" refers to sequence similarity. Percent identity can be determined using standard techniques known in the art (see, e.g., Smith and Waterman, Adv. Appl. Math. 2:482

[1981] ; Needleman and Wunsch, J. Mol. Biol. 48:443

[1970] ; Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444

[1988] ; software programs such as GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package (Genetics Computer Group, Madison, WI); and Devereux et al., Nucl. Acid Res. 12:387-395

[1984] ). An example of a useful algorithm is PILEUP. PILEUP uses progressive, pairwise comparisons to create a multiple sequence alignment from a group of related sequences. It can also draw a tree showing the clustering relationship used to create the comparison. PILEUP uses a simplification of the progressive comparison method of Feng and Doolittle (see Feng and Doolittle, J.Mol.Evol. [Journal of Molecular Evolution] 35:351-360

[1987] ). The method is similar to the method described by Higgins and Sharp (see, Higgins and Sharp, CABIOS [Computer Applications in Biological Sciences] 5:151-153

[1989] ). Useful PILEUP parameters include a default gap weight of 3.00, a default gap length weight of 0.10, and a weighted end gap. Other useful algorithms are the BLAST algorithm described by Altschul et al. (See, Altschul et al., J. Mol. Biol. 215:403-410

[1990] ; and Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5787

[1993] ). The BLAST program uses several search parameters, most of which are set to default values.

[0050] As used herein, "homologous proteins" or "homologous proteases" refer to proteins with different similarities in primary, secondary and / or tertiary structures. When comparing proteins, protein homology can refer to the similarity of linear amino acid sequences. Homology can be determined by amino acid sequence alignment, for example using programs such as BLAST, MUSCLE or CLUSTAL. Homology searches for protein sequences can be performed using BLASTP and PSI-BLAST from NCBI BLAST using a threshold (E value cutoff) of 0.001. (Altschul et al., "Gapped BLAST and PSI BLAST a new generation of protein database search programs", Nucleic Acids Res, Group 1; 25(17): 3389-402 (1997)). The BLAST program uses several search parameters, most of which are set to default values. The NCBI BLAST algorithm finds the most related sequences by biological similarity, but is not recommended for query sequences of less than 20 residues (Altschul et al., Nucleic Acids Res, 25:3389-3402, 1997 and Schaffer et al., Nucleic Acids Res, 29:2994-3005, 2001). Exemplary default BLAST parameters for nucleic acid sequence searches include: neighborhood word length threshold = 11; E-value cutoff = 10; Scoring Matrix = NUC.3.1 (match = 1, mismatch = -3); Gap opening = 5; and Gap extension = 2. Exemplary default BLAST parameters for amino acid sequence searches include: word length = 3; E-value cutoff = 10; Scoring Matrix = BLOSUM62; Gap opening = 11; and Gap extension = 1. Using this information, protein sequences can be grouped and / or phylogenetic trees constructed therefrom. Amino acid sequences can be entered in programs such as Vector NTI Advance suite, and guide trees can be created using the Neighbor Joining (NJ) method (Saitou and Nei, Mol Biol Evol [Molecular Biology and Evolution], 4: 406-425, 1987). Tree structures can be calculated using Kimura correction for sequence distances and ignoring positions with gaps. Programs such as AlignX can display calculated distance values ​​in brackets after the molecule name displayed on the phylogenetic tree.

[0051] The amino acid sequence identity percentage (%) value is determined by the number of matching identical residues divided by the total number of residues in the "reference" sequence (including any gaps created by the program for optimal / maximal alignment). SEQ ID NO: A is a "reference" sequence if the sequence is 90% identical to SEQ ID NO: A. The BLAST algorithm refers to the "reference" sequence as the "query" sequence.

[0052] The CLUSTAL W algorithm is another example of a sequence alignment algorithm (see Thompson et al., Nucleic Acids Res 22:4673-4680, 1994). The default parameters of the CLUSTAL W algorithm include: gap open penalty = 10.0; gap extension penalty = 0.05; protein weight matrix = BLOSUM series; DNA weight matrix = IUB; delayed divergent sequence % = 40; gap separation distance = 8; DNA conversion weight = 0.50; list hydrophilic residues = GPSNDQEKR; use negativity matrix = off; switch special residue penalty = on; switch hydrophilic penalty = on; and switch end gap separation penalty = off. In the CLUSTAL algorithm, deletions occurring at either end are included. For example, a variant having five amino acid deletions at either end of a 500 amino acid polypeptide (or within a polypeptide) has a percent sequence identity of 99% (495 / 500 identical residues x 100) relative to a "reference" polypeptide. Such variants would be encompassed by variants having "at least 99% sequence identity" to the polypeptide.

[0053] In some embodiments, thermolysin enzymes for use herein include those thermolysin enzyme polypeptides described in WO 2015 / 066669.

[0054] In some embodiments, the thermolysin polypeptides for use herein include variants of thermolysin, including those disclosed in WO 2014071410 and US20140099698, US201880073006, EP 3260538, and US20180066244.

[0055] The fragrance ("fragrance" or "perfume") used in the compositions and methods herein includes any available fragrance. The term "fragrance" ("fragrance" or "perfume") includes raw materials and compositions, accords, fragrances and oils, such as essential oils. A wide variety of chemicals are known for fragrance purposes, including compounds such as aldehydes, ketones and esters. Naturally occurring plant and animal oils and secretions that are known to contain complex mixtures of various chemical components are also used as fragrances.

[0056] Fragrances can be blends of volatile compounds of varying volatility that can bind to receptors in the nose and thus have a smell ("smell" or "odor"), usually a pleasant smell. These compounds are also known as fragrances or flavors. Most fragrances have a molar weight of up to about 200 g / mol, in some cases up to about 300 g / mol. Larger molecules are not volatile enough to be perceived by the human nose.

[0057] The volatility of a compound describes how easily it evaporates, either by evaporation or boiling. Fragrance compounds evaporate by evaporation at room temperature and atmospheric pressure (depending on their volatility). Volatility is often described using vapor pressure or boiling point, where a high vapor pressure or low boiling point indicates high volatility. Although the volatility of a compound is related to its molecular weight, other factors such as structure and polarity also play a role, as well as interactions between fragrance compounds.

[0058] The most volatile fragrance compounds are called top notes or head notes, while increasingly less volatile compounds are called heart notes or middle notes, and the least volatile are called base notes or back notes. Top notes are responsible for the first impression of a detergent, and heart notes represent the characteristic smell. Base notes ensure that the effect of the fragrance is more noticeable and lasts longer.

[0059] The top notes, middle notes and base notes can be grouped based on different criteria. One such grouping is Poucher's grouping (Poucher, WA (1993). Poucher's Perfumes, Cosmetics and Soaps, Vol. 2 (9th ed.), Chapman & Hall, p. 55). Poucher classifies fragrance compounds according to the coefficient of evaporation, with top notes having coefficients of 1 to 14, middle notes having coefficients of 15 to 60 and base notes having coefficients of 61 to 100.

[0060] Information about fragrance compounds, such as molecular weight, vapor pressure, and boiling point, can also indicate whether a particular fragrance compound is a top note, a middle note, or a base note. This information is available at iff.com / portfolio / products / fragrance-ingredients / online-compendium and at shop.perfumersapprentice.com.

[0061] The duration of the "freshness" or "cleanliness" effect provided by the fragrance in the detergent composition is affected by how the fragrance and odor are retained on the washed fabrics. Clothing odors can come from various sources, including human body odors as well as odors from the environment, such as kitchen odors, cigarettes, food stains, etc. Another important source of odors is from microorganisms present in textiles, which can metabolize substances transferred from the human body (sweat, dead cells, sebum, etc.) and produce odors during drying, storage or wearing.

[0062] The type of material can also be an important factor in the retention and release of odor compounds. For example, odor compounds can be removed more effectively from cotton than from polyester. This is partly related to the polarity (hydrophilicity) of the odor compounds and the polarity (hydrophilicity) of the textile fibers, where cotton contains mainly cellulose fibers with high polarity, while the fibers of polyester and wool are relatively non-polar compared to cotton fibers. In general, the order of compound polarity of fragrance compounds from high to low is as follows: amide>acid>alcohol>ketone is approximately equal to aldehyde>ester>alkane.

[0063] The fragrance compounds used in laundry detergents can be chemical compounds or essential oils or other natural compounds from any of several different categories. The fragrances that can be used in the context of the present disclosure include all fragrances. Therefore, in particular, the following types of synthetic or natural odorant compounds: esters, ethers, aldehydes (aromatic aldehydes, odorant aldehydes), ketones (aromatic ketones, odorant ketones), alcohols, hydrocarbons, acids, carbonates, aromatic hydrocarbons, aliphatic hydrocarbons, saturated and / or unsaturated hydrocarbons and mixtures of these that can be used as fragrance compounds.

[0064] Individual fragrance compounds, such as synthetic products of esters, ethers, aldehydes, ketones, alcohols and hydrocarbons, and mixtures thereof may be used. However, it is preferred to use mixtures of different fragrance compounds which together generate an attractive fragrance note. Such mixtures may also contain natural fragrance mixtures, such as those obtainable from plant sources, for example pine, citrus, jasmine, patchouli, rose or ylang-ylang oils.

[0065] Fragrances useful for use in the compositions and methods herein include, but are not limited to, those provided below.

[0066] Suitable ester fragrances include, for example, benzyl acetate, phenoxyethyl isobutyrate, p-tert-butylcyclohexyl acetate, linalyl acetate, dimethylbenzyl carbinol acetate (DMBCA), phenethyl acetate, myricylaldehyde, allyl cyclohexylpropionate, styryl propionate, benzyl salicylate, cyclohexyl salicylate, floramate, melusate and jasmacyclate.

[0067] Odor enhancing compounds of the hydrocarbon class include, for example, terpenes, such as limonene and pinene.

[0068] Suitable ether-based fragrances include, for example, benzyl ethyl ether and ambroxol.

[0069] Suitable perfume alcohols include, for example, 10-undecen-1-ol, 2,6-dimethylheptan-2-ol, 2-methylbutanol, 2-methylpentanol, 2-phenoxyethanol, 2-phenylpropanol, 2-tert-butylcyclohexanol, 3,5,5-trimethylcyclohexanol, 3-hexanol, 3-methyl-5-phenylpentanol, 3-octanol, 1-octen-3-ol, 3-phenylpropanol, 4-heptenol, 4-isopropylcyclohexanol, 4-tert-butylcyclohexanol, 6,8-dimethyl-2-nonanol, 6-nonen-1-ol, 9-decen-1-ol, α-methylbenzyl alcohol, terpineol, amyl salicylate, benzyl alcohol, benzyl salicylate, β-terpineol, butyl salicylate, citronellol, water Cyclohexyl salicylate, decanol, dihydromyrcenol, dimethylbenzylcarbinol, dimethylheptanol, dimethyloctanol, ethyl salicylate, ethyl vanillin, anethole, eugenol, geraniol, heptanol, hexyl salicylate, isobornyl alcohol, isoeugenol, isopulegol, linalool, menthol, myrtene alcohol, n-hexanol, nerol, nonanol, octanol, p-menthan-7-ol, phenylethyl alcohol, phenol, phenyl salicylate, tetrahydrogeraniol, tetrahydrolinalool, thymol, trans-2-cis-6-nonadienol, trans-2-nonen-1-ol, trans-2-octenol, undecanol, vanillin and cinnamyl alcohol, wherein when a plurality of perfume alcohols are present, they can be selected independently of one another.

[0070] Suitable perfume ketones can include all ketones that can provide desired fragrance or freshness sensation. Mixtures of different ketones can also be used. For example, the ketone can be selected from the group consisting of: buccoxime, isojasmone, methyl-β-naphthyl ketone, musk indanone, musk tonal / musk+, α-damascone, β-damascone, δ-damascone, isodamascone, damascenone, rose ketone, methyl dihydrojasmonate, menthone, carvone, camphor, anise ketone, α-ionene, β-ionene, dihydro-β-ionene, γ-methyl ionene, jasmine ketone, dihydrojasmone, cis-jasmone, ambergris ketone (iso-E-Super), methyl cedrenyl ketone (methyl cedrylone or methyl cedrylone), acetophenone, methyl acetophenone, p-methoxyacetophenone, benzyl acetone, benzophenone, p-hydroxy-phenyl butanone, celery ketone (celery ket ...hydroxy-phenyl butanone, celery ketone, acetophenone, acetophenone, acetophenone, acetophenone, acetophenone, acetophenone, acetophenone, acetophenone, acetophenone, acetophenone, acetophenone, acetophenone, acetophenone, acetophenone, acetophenone, acetophenone, acetophenone, acetophenone, acetophenone, ketone or livescone), 6-isopropyldecahydro-2-naphthoketone, dimethyloctenone, fresh menthone (frescomenthe), 4-(1-ethoxyvinyl)-3,3,5,5-tetramethylcyclohexanone, methylheptenone, 2-(2-(4-methyl-3-cyclohexen-1-yl)-propyl)cyclopentanone, 1-(p-menthen-6(2)-yl)-1-propanone, 4-(4-hydroxy-3-methoxyphenyl)-2-butanone, 2-acetyl-3,3-dimethylnorbornene, 6,7-dihydro-1,1,2,3,3-pentamethyl-4(5H)-dihydroindanone, 4-damascenaphenone, piperonyl acetone (dulcinyl or cassion), ethyl jasmine (gelsone), hexanone, ambergris ketone (isocyclemone E), methylcyclocitron, methyl lavandinone, irone, p-tert-butylcyclohexanone, fenduoketone, amylcyclopentanone, musk ketone, neobutenone, prikatone, vanolone, 2,4,4,7-tetramethyl-oct-6-ene-3-one, tert-metanone, methyl dihydrojasmonate and mixtures thereof. Preferred ketones may be selected, for example, from α-damascenone, δ-damascenone, isodamascone, carvone, γ-methylionene, ambroxanone, 2,4,4,7-tetramethyl-oct-6-ene-3-one, benzyl acetone, β-damascenone, damascenone, methyl dihydrojasmonate, methyl cedryl ketone, methyl dihydrojasmonate and mixtures thereof.

[0071] Suitable perfume aldehydes can be any aldehydes that produce the desired fragrance or freshness sensation. They can be a single aldehyde or a mixture of aldehydes. Exemplary suitable aldehydes are melon aldehyde, triplal or ligustral, adoc aldehyde, p-methoxybenzaldehyde, cymal, ethyl vanillin, cyanidin, jasminaldehyde, piperonal, hydroxycitronellal, koavone, lauryl aldehyde, lyral, methylnonyl acetaldehyde, para-tert-bucinal, phenylacetaldehyde, undecene aldehyde, vanillin, 2,6,10-trimethyl-9-dodecane, 3-dodecene-1-aldehyde, α-amyl cinnamaldehyde, 4-methoxybenzaldehyde, benzaldehyde, 3-(4-tert-butylphenyl)-propionaldehyde, 2-methyl-3-(p-methyl)-1-ol oxyphenylpropanal), 2-methyl-4-(2,6,6-trimethyl-2(1)-cyclohexen-1-yl)-butyraldehyde, 3-phenyl-2-propenal, cis- / trans-3,7-dimethyl-2,6-octen-1-al, 3,7-dimethyl-6-octen-1-al, [(3,7-dimethyl-6-octenyl)-oxy]-acetaldehyde, 4-isopropylbenzaldehyde, 1,2,3,4,5,6,7,8-octahydro-8,8-dimethyl-2-naphthaldehyde, 2,4-dimethyl-3-cyclohexene-1-carboxaldehyde, 2-methyl-3-(isopropyl-phenyl)-propanal, decanal, 2,6-dimethyl-5-heptenal, 4-(tricyclo-[5.2.10-(2,6)]-decyldiene-8)-butyraldehyde, octahydro-4,7-methylene-1H-indenecarboxaldehyde, 3-ethoxy-4-hydroxybenzaldehyde, p-ethyl-α-α-dimethylhydrocinnamaldehyde, α-methyl-3,4-(methylenedioxy)-hydrocinnamaldehyde, 3,4-methylenedioxybenzaldehyde, α-n-hexylcinnamaldehyde, meta-cymene-7-carboxaldehyde, α-methylphenylacetaldehyde, 7-hydroxy-3,7-dimethyloctanal, undecenal, 2 ,4,6-trimethyl-3-cyclohexene-1-carboxaldehyde, 4-(3)-(4-methyl-3-pentenyl)-3-cyclohexenecarboxaldehyde, 1-dodecane, 2,4-dimethylcyclohexene-3-carboxaldehyde, 4-(4-hydroxy-4-methylpentyl)-3-cyclohexene-1-carboxaldehyde, 7-methoxy-3,7-dimethyloctan-1-carboxaldehyde, 2-methylundecanecarboxaldehyde, 2-methyldecanal, 1-nonanal, 1-octanal, 2,6,10-trimethyl-5, 9-undecadienal, 2-methyl-3-(4-tert-butyl)-propanal, dihydrocinnamaldehyde, 1-methyl-4-(4-methyl-3-pentenyl)-3-cyclohexene-1-carboxaldehyde, 5- or 6-methoxyhexahydro-4,7-methaneindene-1 or 2-carboxaldehyde, 3,7-dimethyloctan-1-aldehyde, 1-undecanal, 10-undecene-1-aldehyde, 4-hydroxy-3-methoxybenzaldehyde, 1-methyl-3-(4-methylpentyl)-3-cyclohexene-1-carboxaldehyde, Hexenecarboxaldehyde, trans-4-decenal, 2,6-nonadienal, p-tolyl-acetaldehyde, 4-methylphenylacetaldehyde, 2-methyl-4-(2,6,6-trimethyl-1-cyclohexen-1-yl)-2-butenal, o-methoxycinnamaldehyde, 3,5,6-trimethyl-3-cyclohexenecarboxaldehyde, 3,7-dimethyl-2-methylene-6-octenal, phenoxyacetaldehyde, 5,9-dimethyl-4,8-decadienal, peony aldehyde aldehyde)(6,1-dimethyl-3-oxa-5,9-undecadien-1-aldehyde), hexahydro-4,7-toluene-1-carboxaldehyde, 2-methyloctanal, α-methyl-4-(1-methylethylphenylacetaldehyde, 6,6-dimethylbicyclo[3.3.1]hept-2-ene-2-propanal, p-methylphenoxyacetaldehyde, 2-methyl-3-phenyl-2-propene-1-aldehyde, 3,5,5-trimethylhexanal, hexahydro-8,8-dimethyl-2-naphthaldehyde, 3-propyl-bicyclo-[2.2.1]-hept-5-ene-2-carboxaldehyde, 9-decenal, 3-methyl-5-phenyl-1-pentanal, 1-p-menthene-q-carboxaldehyde, citral or a mixture thereof, lily of the valley aldehyde, citral, 1-decanal, 2,4-dimethyl-3-cyclohexene-1-carboxaldehyde. Preferred aldehydes may be selected, for example, from cis / trans-3,7-dimethyl-2,6-octadiene-1-aldehyde, piperonal, 2,4,6-trimethyl-3-cyclohexene-1-carboxaldehyde, 2,6-nonadienal, α-n-pentylcinnamaldehyde, α-n-hexylcinnamaldehyde, p-tert-butyraldehyde, lyral, pyralidinaldehyde, methylnonyl acetaldehyde, trans-2-nonenal, lily of the valley aldehyde, trans-2-nonenal and a mixture thereof. .

[0072] The fragrance compound may also be a natural fragrance mixture, such as those obtainable from plant sources, for example pine, citrus, jasmine, patchouli, rose or ylang-ylang oils. Also suitable are musk, sage oil, chamomile oil, clove oil, peppermint oil, cinnamon leaf oil, linden flower oil, juniper berry oil, vetiver oil, olibanum or frankincense oil, galbanum oil and lisdun oil, as well as orange blossom oil or neroli oil, orange peel oil and sandalwood oil. The fragrance compound may also be an essential oil, for example angelica root oil, fennel oil, arnica flower oil, basil oil, osmanthus oil, champaca blossom oil, thyme oil, thyme oil, thyme flower ... oil), silver fir oil, silver fir fruit oil, elemi oil, eucalyptus oil, fennel oil, spruce needle oil, geranium oil, ginger grass oil, guaiac wood oil, gurgling oil, immortelle oil, camphor oil, ginger oil, iris oil, eucalyptus oil, calamus oil, camphor oil, cananga oil, cardamom oil, cinnamon oil, copaiba balsam oil, coriander oil, spearmint oil, artemisia annua oil, fennel oil, lavender oil, lemongrass oil, white lime oil, orange peel oil, lemon balm oil, musk seed oil, myrrh oil, nyori oil, oregano oil, palmarosa oil, Peruvian balsam oil, orange leaf oil, pepper oil, peppermint oil, pimento oil, rosemary oil, celery oil, spike flower oil, stemanis oil (stemanis oil), turpentine, arborvitae, thyme, verbena, absinthe, wintergreen, hyssop, cinnamon, citronella, lemon, and tar.

[0073] Further information on fragrance ingredients can be obtained from the International Fragrance Association (IFRA), which publishes a list of all fragrance ingredients used in consumer products (ifrafragrance.org / initiatives / transparency / ifra-transparency-list).

[0074] In one embodiment, a variety of flavor compounds, such as those listed above or on the list maintained by IFRA, can be included in the compositions provided herein in combination with thermolysin polypeptides.Therefore, the compositions of the present invention can, for example, contain three or more, such as four or more, five or more, six or more or seven or more different flavor components.

[0075] The composition of the present invention will typically contain one or more perfume components in an amount (by weight) of 0.0001% to 2.5%, such as 0.001%-2%, such as 0.01%-1.5%, such as 0.1%-1%, based on the total amount of perfume components and the total weight of the composition. Alternatively, the composition provided herein will contain one or more perfume components in an amount sufficient to provide a concentration between about 0.0000001% and about 2% in the cleaning solution.

[0076] There is no limitation on the type of detergent composition into which the perfumes may be incorporated. For example, they may be included in detergent compositions in the form of liquids, gels, powders, granules, tablets, pods, sachets and soap bars.

[0077] The perfume component can be incorporated into the detergent composition in physical form and using methods known in the art, for example, adding the perfume component as a liquid, solid particles and / or microcapsules.

[0078] Therefore, also provide the detergent composition that can be used in the method provided herein.As used herein, about the composition that is intended to be used in the cleaning medium (for example, cleaning solution) of the object of cleaning pollution or dirty object (comprising specific textile or non-textile object or article), use term " detergent composition " or " detergent preparation ".Such composition of the present invention is not limited to any specific detergent composition or preparation.In fact, in some embodiments, detergent of the present invention comprises at least one thermolysin or metalloproteinase polypeptide (for example, proteinase T), spices, and also comprises one or more surfactants, one or more transferases, hydrolases, oxidoreductases, builders (for example, builders salts), bleaching agents, bleach-activators, bluing agents, fluorescent dyes, caking inhibitors, masking agents, enzyme activators, antioxidants and / or solubilizing agents in addition.In some cases, builders salt is the mixture of silicate and phosphate, preferably has more silicate (for example, sodium metasilicate) than phosphate (for example, sodium tripolyphosphate). Some compositions of the present invention (eg, but not limited to, cleaning compositions or detergent compositions) do not contain any phosphates (eg, phosphates or phosphate builders).

[0079] In some embodiments, the cleaning or detergent compositions of the present invention further comprise adjunct materials including, but not limited to, surfactants, builders, bleaching agents, bleach activators, bleach catalysts, other enzymes, enzyme stabilization systems, chelating agents, optical brighteners, soil release polymers, dye transfer agents, dispersants, foam inhibitors, dyes, colorants, filler salts, hydrotropes, photoactivators, fluorescent agents, fabric conditioners, hydrolyzable surfactants, preservatives, antioxidants, anti-shrinkage agents, Anti-wrinkle agents, bactericides, fungicides, color embellishments, silver care agents, anti-tarnish and / or anti-corrosion agents, alkalinity sources, solubilizers, carriers, processing aids, pigments, and pH control agents (see, e.g., U.S. Pat. Nos. 6,610,642, 6,605,458, 5,705,464, 5,710,115, 5,698,504, 5,695,679, 5,686,014, and 5,646,101, all of which are incorporated herein by reference).

[0080] The detergent or cleaning composition of the present invention is advantageously used in, for example, laundry applications, hard surface cleaning, dishwashing applications, and decorative applications (such as dentures, teeth, hair and skin cleaning). In addition, due to the unique advantages of increased effectiveness in lower temperature solutions, the composition of the present invention is ideally suited for laundry applications. In addition, the composition of the present invention can be used for use in granular and liquid compositions.

[0081] Enzyme component weights are based on total active protein. Unless otherwise indicated, all percentages and ratios are calculated by weight. Unless otherwise indicated, all percentages and ratios are calculated based on the total composition. In laundry detergent compositions, enzyme levels are expressed in ppm, equivalent to mg active protein / kg detergent composition.

[0082] In some embodiments, the laundry detergent compositions described herein further comprise a surfactant. In some embodiments, the surfactant is selected from nonionic surfactants, amphoteric surfactants, semi-polar surfactants, anionic surfactants, cationic surfactants, zwitterionic surfactants, and combinations and mixtures thereof. In yet other embodiments, the surfactant is selected from anionic surfactants, cationic surfactants, zwitterionic surfactants, and combinations thereof. In some embodiments, the laundry detergent compositions described herein comprise from about 0.1% to about 60%, from about 1% to about 50%, or from about 5% to about 40% surfactants by weight of the composition.

[0083] Exemplary surfactants include, but are not limited to, sodium dodecylbenzene sulfonate, C12-14 pareth-7, C12-15 pareth-7, sodium C12-15 pareth sulfate, C14-15 pareth-4, sodium laureth sulfate (e.g., Steol CS-370), sodium hydrogenated coconut oil, C12 ethoxylate (Alfonic 1012-6, Hetoxol LA7, Hetoxol LA4), sodium alkylbenzene sulfonate (e.g., Nacconol 90G), and combinations and mixtures thereof. Anionic surfactants include, but are not limited to, linear alkylbenzene sulfonate (LAS), alpha-olefin sulfonate (AOS), alkyl sulfate (fatty alcohol sulfate) (AS), alcohol ethoxy sulfate (AEOS or AES), secondary alkane sulfonate (SAS), alpha-sulfo fatty acid methyl ester, alkyl- or alkenyl succinic acid, or soap. Nonionic surfactants include, but are not limited to, alcohol ethoxylates (AEO or AE), carboxylated alcohol ethoxylates, nonylphenol ethoxylates, alkyl polyglycosides, alkyl dimethyl amine oxides, ethoxylated fatty acid monoethanolamides, fatty acid monoethanolamides, polyhydroxy alkyl fatty acid amides (e.g., as described in WO 92 / 06154), polyoxyethylene esters of fatty acids, polyoxyethylene sorbitan esters (e.g., TWEEN), polyoxyethylene alcohols, polyoxyethylene isoalcohols, polyoxyethylene ethers (e.g., TRITON and BRIJ), polyoxyethylene esters, polyoxyethylene-p-tert-octylphenol or octylphenyl-ethylene oxide condensates (e.g., NONIDET P40), condensates of ethylene oxide with fatty alcohols (e.g., LUBROL), polyoxyethylene nonylphenols, polyalkylene glycols (SYNPERONIC F108), sugar-based surfactants (e.g., glucopyranosides, thioglucopyranosides), and combinations and mixtures thereof.

[0084] In additional embodiments, the laundry detergent compositions described herein further comprise a surfactant mixture, including but not limited to 5%-15% anionic surfactants, <5% nonionic surfactants, cationic surfactants, phosphonates, soaps, enzymes, perfumes, butylphenyl methylpropionate, geraniol, zeolites, polycarboxylates, hexyl cinnamaldehyde, limonene, cationic surfactants, citronellol, and benzisothiazolinone.

[0085] The laundry detergent compositions described herein may additionally include one or more detergent builders or builder systems, complexing agents, polymers, bleaching systems, stabilizers, foam boosters, foam inhibitors, corrosion inhibitors, soil suspension agents, anti-soil redeposition agents, dyes, bactericides, hydrotropes, optical brighteners, fabric conditioners and perfumes. As provided in more detail herein, the laundry detergent compositions described herein may also include additional enzymes selected from proteases, amylases, cellulases, lipases, mannanases, nucleases, pectinases, xyloglucanases, or perhydrolases.

[0086] In some embodiments, the laundry detergent compositions described herein further comprise from about 1%, from about 3% to about 60%, or even from about 5% to about 40% of a builder by weight of the cleaning composition. Builders may include, but are not limited to, alkali metal, ammonium and alkanolammonium salts of polyphosphates; alkali metal silicates, alkaline earth metal and alkali metal carbonates; aluminosilicates; polycarboxylate compounds; ether hydroxy polycarboxylates; copolymers of maleic anhydride with ethylene or vinyl methyl ether, 1,3,5-trihydroxybenzene-2,4,6-trisulfonic acid, and carboxymethyloxysuccinic acid; various alkali metal, ammonium and substituted ammonium salts of polyacetic acids (such as ethylenediaminetetraacetic acid and nitrilotriacetic acid); and polycarboxylates such as mellitic acid, succinic acid, citric acid, oxydisuccinic acid, polymaleic acid, benzene 1,3,5-tricarboxylic acid, carboxymethyloxysuccinic acid, and soluble salts thereof.

[0087] In some embodiments, builders form water-soluble hardness ion complexes (e.g., chelating builders), such as citrates and polyphosphates (e.g., sodium tripolyphosphate and sodium tripolyphosphate hexahydrate, potassium tripolyphosphate, and mixed sodium and potassium tripolyphosphates, etc.). Any suitable builder can be used in the compositions described herein, including those known in the art.

[0088] In some embodiments, the laundry detergent compositions described herein further comprise adjunct ingredients including, but not limited to, surfactants, builders, bleaching agents, bleach activators, bleach catalysts, additional enzymes, enzyme stabilizers (including, for example, enzyme stabilization systems), chelating agents, optical brighteners, soil release polymers, dye transfer agents, dye transfer inhibitors, catalytic materials, hydrogen peroxide, sources of hydrogen peroxide, preformed peracids, polymeric dispersants, clay soil removers, structural elastic agents, dispersants, foam inhibitors, dyes, perfumes, colorants, filler salts, hydrotropes, photoactivators, fluorescent agents, fabric conditioners, hydrolyzable surfactants, solvents, preservatives, antioxidants, anti-shrinkage agents, anti-wrinkle agents, bactericides, fungicides, color embellishments, anti-corrosion agents, alkalinity sources, solubilizers, carriers, processing aids, pigments, pH control agents, and combinations thereof. (See, e.g., US 6610642, US 6605458, US 5705464, US 5710115, US 5698504, US 5695679, US5686014, and US 5646101). In some embodiments, one or more adjuvants are incorporated, for example, to assist or enhance cleaning performance (for treating substrates to be cleaned), or to modify the aesthetics of the cleaning composition (as in the case of perfumes, colorants, dyes, etc.). Any such adjuvant ingredients are additional to the low-temperature mannanase, low-temperature amylase, and / or low-temperature protease described herein. In some embodiments, the adjuvant ingredients are selected from surfactants, enzyme stabilizers, builder compounds, polymer compounds, bleaching agents, additional enzymes, foam inhibitors, dispersants, lime soap dispersants, soil suspending agents, softeners, anti-redeposition agents, corrosion inhibitors, and combinations thereof.

[0089] In some other embodiments, laundry detergent compositions as described herein include one or more enzyme stabilizers. In certain embodiments, enzyme stabilizers are water-soluble sources of calcium and / or magnesium ions. In certain embodiments, enzyme stabilizers include oligosaccharides, polysaccharides and inorganic divalent metal salts (including alkaline earth metal salts, such as calcium salts). In certain embodiments, enzymes used herein are stabilized by being present in water-soluble sources of zinc (II), calcium (II) and / or magnesium (II) ions in the finished product compositions providing the following such ions for enzymes, and other metal ions (e.g., barium (II), scandium (II), iron (II), manganese (II), aluminum (III), tin (II), cobalt (II), copper (II), nickel (II) and vanadium (IV)). Chlorides and sulfates can also be used in some embodiments. Exemplary oligosaccharides and polysaccharides (e.g., dextrins) are described in, for example, WO 07145964. In some embodiments, the laundry detergent compositions described herein contain reversible protease inhibitors selected from boron-containing compounds (e.g., borates, 4-formylphenylboronic acid, and phenylboronic acid derivatives, such as described in WO 9641859); peptide aldehydes (such as described in WO 2009118375 and WO 2013004636), and combinations thereof.

[0090] The cleaning compositions herein are typically formulated so that during use in aqueous cleaning operations, the wash water has a pH of from about 3.0 to about 11. Liquid product formulations are typically formulated to have a neat pH of from about 5.0 to about 9.0, more preferably from about 7.5 to about 9. Granular laundry detergent products are typically formulated to have a pH of from about 8.0 to about 11.0. Techniques for controlling pH at recommended use levels include the use of buffers, alkalis, acids, etc., and are well known to those skilled in the art.

[0091] Suitable high pH cleaning compositions typically have a neat pH of from about 9.0 to about 11.0, or even a neat pH of from 9.5 to 10.5. Such cleaning compositions typically include a sufficient amount of a pH adjusting agent (such as sodium hydroxide, monoethanolamine or hydrochloric acid) to provide such cleaning compositions with a neat pH of from about 9.0 to about 11.0. Such compositions typically include at least one alkali-stable enzyme. In some embodiments, the compositions are liquids, while in other embodiments, the compositions are solids.

[0092] In one embodiment, the cleaning composition comprises a pH from 7.4 to pH 11.5, or pH 7.4 to pH 11.0, or pH 7.5 to pH 11.5, or pH 7.5 to pH 11.0, or pH 7.5 to pH 10.5, or pH 7.5 to pH 10.0, or pH 7.5 to pH 9.5, or pH 7.5 to pH 9.0, or pH 7.5 to pH 8.5, or pH 7.5 to pH 8.0, or pH 7.6 to pH 11.5, or pH 7.6 to pH 11.0, or pH 7.6 to pH 10.5, or pH 8.7 to pH 10.0, or pH 8.0 to pH 11.5, or pH 8.0 to pH 11.0, or pH 8.0 to pH 10.5, or pH 8.0 to pH 10.5. Those of 10.0.

[0093] The concentration of detergent compositions in typical cleaning solutions throughout the world varies from less than about 800 ppm of detergent composition (a "low detergent concentration geography") (e.g., about 667 ppm in Japan), to between about 800 ppm and about 2000 ppm (a "medium detergent concentration geography") (e.g., about 975 ppm in the United States and about 1500 ppm in Brazil), to greater than about 2000 ppm (a "high detergent concentration geography") (e.g., about 4500 ppm to about 5000 ppm in Europe and about 6000 ppm in high sudsing phosphate builder geography).

[0094] In some embodiments, the detergent compositions described herein can be utilized at temperatures ranging from about 10° C. to about 60° C., or from about 20° C. to about 60° C., or from about 30° C. to about 60° C., from about 40° C. to about 60° C., from about 40° C. to about 55° C., or all ranges within 10° C. to 60° C. In some embodiments, the detergent compositions described herein are used in a “cold water wash” at temperatures ranging from about 10° C. to about 40° C., or from about 20° C. to about 30° C., from about 15° C. to about 25° C., from about 15° C. to about 35° C., or all ranges within 10° C. to 40° C.

[0095] As another example, different geographic locations typically have different water hardnesses. 2+ / Mg 2+ The hardness of water is described by the number of particles in the water. Hardness is the amount of calcium (Ca 2+ ) and magnesium (Mg 2+) in the United States, most water is hard water, but the hardness varies. Moderately hard (60-120 ppm) to hard (121-181 ppm) water has 60 to 181 parts per million (converting parts per million to particles per US gallon is to divide the ppm # by 17.1 to equal particles per gallon) of hardness minerals.

[0096] Table 1. Water hardness levels

[0097]

[0098]

[0099] Typically, European water hardness is greater than about 10.5 (e.g., about 10.5 to about 20.0) granules / gallon of mixed Ca 2+ / Mg 2+ (e.g., about 15 pellets / gallon of mixed Ca 2+ / Mg 2+ ). Typically, North American water hardness is greater than Japanese water hardness, but lower than European water hardness. For example, North American water hardness may be between about 3 and about 10 particles, about 3 to about 8 particles, or about 6 particles. Typically, Japanese water hardness is lower than North American water hardness, usually less than about 4, such as about 3 particles per gallon of mixed Ca 2+ / Mg 2+ .

[0100] In other embodiments, the compositions described herein comprise one or more additional enzymes. The one or more additional enzymes are selected from acyltransferases, α-amylases, β-amylases, α-galactosidases, arabinosidases, arylesterases, β-galactosidases, carrageenase, catalase, cellobiohydrolase, cellulase, chondroitinase, cutinase, DNAse, endo-β-1,4-glucanase, endo-β-mannanase, esterase, exo-mannanase, galactanase, glucoamylase, hexosaminidase, hemicellulase, hyaluronidase, keratinase, laccase, lactase, ligninase, lipase, lipoxygenase , lysozymes, mannanases, additional metalloproteinases, nucleases (e.g., deoxyribonucleases and ribonucleases), oxidases, oxidoreductases, pectate lyases, pectin acetylesterases, pectinases, pentosanases, peroxidases, phenoloxidases, phosphatases, phospholipases, phytases, polygalacturonase, polyesterases, additional proteases, pullulanases, reductases, rhamnogalacturonase, β-glucanase, tannase, transglutaminase, xylan acetylesterase, xylanase, xyloglucanase, xylosidase, and any combination or mixture thereof. Some embodiments relate to a combination (i.e., a "mixture") of enzymes comprising enzymes (e.g., amylases, proteases, lipases, mannanases, and / or nucleases) in combination with one or more thermolysin polypeptides in the compositions provided herein.

[0101] In some embodiments, the compositions provided herein include polypeptides with thermolysin activity in combination with proteases. Proteases used in combination with thermolysin in the compositions disclosed herein include any polypeptides with protease activity. In one embodiment, additional proteases are serine proteases. In another embodiment, additional proteases are additional metalloproteases, fungal subtilisin or alkaline microbial proteases or trypsin-like proteases. Suitable additional proteases include those of animal, plant or microbial origin. In some embodiments, proteases are microbial proteases. In other embodiments, proteases are chemically or genetically modified mutants. In another embodiment, proteases are subtilisin-like proteases or trypsin-like proteases. In other embodiments, additional proteases do not contain epitopes that cross-react with variants, such as measured by antibody binding or other assays available in the art. Exemplary subtilisin include those derived from, for example, Bacillus (e.g., BPN', Carlsberg, subtilisin 309, subtilisin 147, and subtilisin 168) or fungal origin, such as those described in U.S. Patent No. 8,362,222.Exemplary additional proteases include, but are not limited to, WO 92 / 21760, WO 95 / 23221, WO 2008 / 010925, WO 09 / 149200, WO 09 / 149144, WO 09 / 149145, WO 10 / 056640, WO 10 / 056653, WO 2010 / 0566356, WO 11 / 072099, WO 2011 / 13022, WO 11 / 140364, WO 12 / 151534, WO 2015 / 038792, WO 2015 / 089447, WO 2015 / 089441, WO 2017 / 215925 / U.S. Publication No. 2008 / 0090747, US 5,801,039, US 5,340,735, US 5,500,364, US 5,855,625, RE 34,606, US 5,955,340, US 5,700,676, US 6,312,936, US 6,482,628, US 8,530,219, U.S. Provisional Application Nos. 62 / 180673 and 62 / 161077, and PCT Application Nos. PCT / US2015 / 021813, PCT / US2015 / 055900, PCT / US 2015 / 057497, PCT / US2015 / 057492, PCT / US2015 / 057512, PCT / US 2015 / 057526, PCT / US2015 / 057520, PCT / US2015 / 057502, PCT / US 2016 / 022282 and PCT / US16 / 32514, International Publications WO 2016001449, WO 2016087617, WO 2016096714, WO 2016203064, WO 2017089093, and WO2019180111, as well as WO 1999014341, WO 1999033960, WO 1999014342, WO1999034003, WO 2007044993, WO 2009058303, WO 2009058661, WO 2014071410, WO2014194032, WO 2014194034, WO 2014194054 and WO 2014 / 194117. Exemplary additional proteases include, but are not limited to, trypsin (e.g., of porcine or bovine origin) and the Fusarium protease described in WO89 / 06270. Exemplary commercial proteases include, but are not limited to. MAXACAL TM , MAXAPEMTM , OXP、PURAMAX TM 、EXCELLASE TM 、PREFERENZ TM Proteases (e.g. P100, P110, P280), EFFECTENZ TM Proteases (e.g. P1000, P1050, P2000), EXCELLENZ TM Proteases (e.g. P1000), and PURAFAST TM (DuPont); Variant, 16L, ULTRA, DURAZYM TM , LIQUANASE PROGRESS and (Novozymes); BLAP TM and BLAP TM Variant (Henkel AG); LAVERGY TM PRO104L (BASF), KAP (alkaliphilic Bacillus subtilisin (Kao)) and (AB Enzymes).

[0102] In certain embodiments, compositions provided herein include polypeptides combined with one or more amylases, with thermolysin activity. In one embodiment, compositions include amylases from about 0.00001% to about 10%, about 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, or about 0.005% to about 0.5% by weight of the composition. Any amylase (e.g., alpha amylase and / or beta amylase) suitable for use in alkaline solutions can be used for use in compositions included in such compositions. Exemplary amylases can be chemical or genetically modified mutants. Exemplary amylases include, but are not limited to, those of bacterial or fungal origin, such as, for example, those described in GB 1,296,839, WO 9100353, WO9402597, WO 94183314, WO 9510603, WO 9526397, WO 9535382, WO 9605295, WO 9623873, WO9623874, WO 9630481, WO 9710342, WO 9741213, WO 9743424, WO 9813481, WO 9826078, WO 9902702, WO 9909183, WO 9919467, WO 9923211, WO 9929876, WO 9942567, WO 9943793, WO9943794, WO 9946399, WO 0029560, WO 0060058, WO 0060059, WO 0060060, WO 0114532, WO0134784, WO 0164852, WO 0166712, WO 0188107, WO 0196537, WO 02092797, WO 0210355, WO0231124, WO 2004055178, WO 2004113551, WO 2005001064, WO 2005003311, WO2005018336, WO 2005019443, WO 2005066338, WO 2006002643, WO 2006012899, WO2006012902, WO 2006031554, WO 2006063594, WO 2006066594, WO 2006066596, WO2006136161, WO 2008000825, WO 2008088493, WO 2008092919, WO 2008101894, WO2008 / 112459, WO 2009061380, WO 2009061381, WO 2009100102, WO 2009140504, WO2009149419, WO 2010 / 059413, WO 2010088447, WO 2010091221, WO 2010104675, WO 2010115021, WO10115028, WO 2010117511, WO 2011076123, WO 2011076897、WO 2011080352、WO2011080353、WO 2011080354、WO 2011082425、WO 2011082429、WO 2011087836、WO2011098531、WO 2013063460、WO 2013184577、WO Amylases described in WO 2014099523, WO 2014164777 and WO2015077126. Exemplary commercial amylases include, but are not limited to STAINZYME STAINZYME STAINZYME and BAN TM (Novozymes); EFFECTENZ TM S1000、POWERASE TM 、PREFERENZ TM S100, PREFERENZ TM S110、EXCELLENZ TM S2000, and P(DuPont).

[0104] In certain embodiments, compositions provided herein comprise polypeptides combined with one or more lipases, having thermolysin activity. In certain embodiments, compositions comprise by weight of the composition from about 0.00001% to about 10%, about 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, or about 0.005% to about 0.5% lipase. Exemplary lipases can be chemical or genetically modified mutants. Exemplary lipases include, but are not limited to, those of, e.g., bacterial or fungal origin, such as, for example, H. lanuginosa lipase (see, e.g., EP 258 068 and EP 305 216), T. lanuginosa lipase (see, e.g., WO 2014 / 059360 and WO 2015 / 010009), Rhizomucor miehei lipase (see, e.g., EP 238 023), Candida lipases such as C. antarctica lipase (e.g., C. antarctica lipase A or B) (see, e.g., EP 238 023). 214761), Pseudomonas lipases such as P. alcaligenes and P. pseudoalcaligenes lipases (see, e.g., EP 218272), Pseudomonas cepacia lipases (see, e.g., EP 331376), Pseudomonas stutzeri lipases (see, e.g., GB 1,372,034), Pseudomonas fluorescens lipases, Bacillus lipases (e.g., Bacillus subtilis lipase (Dartois et al., Biochem. Biophys. Acta [Biochemistry and Biophysics] 1131:253-260 (1993)), Bacillus stearothermophilus lipase (see, e.g., JP 64 / 744992), and Bacillus pumilus lipase (see, e.g., WO 114761), Pseudomonas lipases such as P. alcaligenes and P. pseudoalcaligenes lipases (see, e.g., EP 218272), P. cepacia lipases (see, e.g., EP 331376), P. stutzeri lipases (see, e.g., GB 1,372,034), P. fluorescens lipases, Bacillus lipases (e.g., B. subtilis lipase (Dartois et al., Biochem. Biophys. Acta [Biochemistry and Biophysics] 1131:253-260 (1993)), Bacillus stearothermophilus lipase (see, e.g., JP 64 / 744992), and B. pumilus lipase (see, e.g., 91 / 16422)).Exemplary cloned lipases include, but are not limited to, Penicillium camembertii lipase (see Yamaguchi et al., Gene 103:61-67 (1991)); Geotrichum candidum lipase (see Schimada et al., J. Biochem., 106:383-388 (1989)); and various Rhizopus lipases, such as R. delemar lipase (see Hass et al., Gene 109:117-113 (1991)), R. niveus lipase (Kugimiya et al., Biosci. Biotech. Biochem. 56:716-719 (1992)), and R. oryzae lipase. Other lipolytic enzymes (e.g., cutinases) may also be used in one or more compositions described herein, including, but not limited to, for example, cutinases derived from Pseudomonas mendocina (see WO 88 / 09367) and / or Fusarium solanipisi (see WO 90 / 09446). Exemplary commercial lipases include, but are not limited to, M1 LIPASE. TM 、LUMAFAST TM and LIPOMAX TM (DuPont); and ULTRA (Novozymes); and LIPASE P TM (Amano Pharmaceutical Co. Ltd.).

[0105] In some embodiments, the compositions provided herein include polypeptides having thermolysin activity in combination with one or more mannanases. In one embodiment, the composition comprises from about 0.00001% to about 10%, about 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, or about 0.005% to about 0.5% mannanase by weight of the composition. Exemplary mannanases may be chemically or genetically modified mutants. Exemplary mannanases include, but are not limited to, those of bacterial or fungal origin, such as those described in: WO 2016 / 007929; USPN 6,566,114; 6,602,842; and 6,440,991; and U.S. Provisional Application Nos. 62 / 251516, 62 / 278383, and 62 / 278387. Exemplary commercial mannanases include, but are not limited to (Novozymes) and EFFECTENZ TM M 1000, EFFECTENZ TM M 2000, M 100, and PURABRITE TM (DuPont).

[0106] In some embodiments, the compositions and methods provided herein comprise a polypeptide having thermolysin activity and a fragrance in combination with a nuclease (such as a DNase or an RNase). Exemplary nucleases include, but are not limited to, WO 2015181287, WO2015155350, WO 2016162556, WO 2017162836, WO 2017060475 (e.g., SEQ ID NO: 21), WO2018184816, WO 2018177936, WO 2018177938, WO2018 / 185269, WO 2018185285, WO2018177203, WO 2018184817, WO 2019084349, WO 2019084350, WO 2019081721, WO2018076800, WO 2018185267, WO 2018185280, and WO 2018206553. Other nucleases that can be used in combination with the polypeptides having thermolysin activity and flavoring agents in the compositions and methods provided herein include those described in: Nijland R, Hall MJ, Burgess JG (2010) Dispersal of Biofilms by Secreted, Matrix Degrading, Bacterial DNase. PLoS ONE [Public Library of Science: Comprehensive] 5 (12) and Whitchurch, CB, Tolker-Nielsen, T., Ragas, PC, Mattick, JS (2002) Extracellular DNA required for bacterial biofilm formation [Extracellular DNA required for bacterial biofilm formation]. Science [Science] 295: 1487.

[0107] Still another embodiment relates to a composition comprising one or more thermolysin enzymes described herein, a perfume, and one or more cellulases. In one embodiment, the composition comprises from about 0.00001% to about 10%, 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, or about 0.005% to about 0.5% cellulase by weight of the composition. Any suitable cellulase can be used in the compositions described herein. Exemplary cellulases can be chemically or genetically modified mutants. Exemplary cellulases include, but are not limited to, those of bacterial or fungal origin, such as those described in WO 2005054475, WO 2005056787, US 7,449,318, US 7,833,773, US 4,435,307; EP 0495257; and U.S. Provisional Application No. 62 / 296,678. Exemplary commercial cellulases include, but are not limited to and PREMIUM (Novozymes); REVITALENZ TM 100. REVITALENZ TM 200 / 220, and 2000 (DuPont); and KAC-500 (B) TM (Kao Corporation) In some embodiments, the cellulase is incorporated as a portion or fragment of a mature wild-type or variant cellulase in which a portion of the N-terminus is deleted (see, e.g., US 5,874,276).

[0109] In some embodiments, the laundry detergent compositions described herein comprise at least one chelating agent. Suitable chelating agents may include, but are not limited to, copper, iron and / or manganese chelating agents, and mixtures thereof. In some embodiments, the laundry detergent compositions described herein comprise from about 0.1% to about 15%, or even from about 3.0% to about 10% chelating agents by weight of the composition.

[0110] In some still further embodiments, the laundry detergent compositions described herein comprise at least one deposition aid. Suitable deposition aids include, but are not limited to, polyethylene glycol, polypropylene glycol, polycarboxylates, soil release polymers (such as polyethylene terephthalate), clays such as kaolin, montmorillonite, attapulgite, illite, bentonite, halloysite, and mixtures thereof.

[0111] In some embodiments, the laundry detergent compositions described herein comprise at least one anti-redeposition agent.

[0112] In some embodiments, the laundry detergent compositions described herein contain one or more dye transfer inhibitors. Suitable polymeric dye transfer inhibitors include, but are not limited to, polyvinyl pyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-vinyl pyrrolidone and N-vinylimidazole, polyvinyl oxazolidones and polyvinylimidazoles, or mixtures thereof. In some embodiments, the laundry detergent compositions described herein contain from about 0.0001% to about 10%, from about 0.01% to about 5%, or even from about 0.1% to about 3% dye transfer inhibitors by weight of the composition.

[0113] In some embodiments, the laundry detergent compositions described herein comprise one or more silicates. In some such embodiments, sodium silicates (e.g., sodium disilicate, sodium metasilicate, and crystalline phyllosilicates) may be used. In some embodiments, the laundry detergent compositions described herein comprise from about 1% to about 20%, or from about 5% to about 15% silicates by weight of the composition.

[0114] In yet further embodiments, the laundry detergent compositions described herein comprise one or more dispersants.Suitable water-soluble organic materials include, but are not limited to, homo- or co-polymeric acids or their salts, wherein the polyacid comprises at least two carboxyl groups separated from each other by not more than two carbon atoms.

[0115] In some embodiments, the laundry detergent compositions described herein include one or more bleaching agents, bleach activators, and / or bleach catalysts. In some embodiments, the laundry detergent compositions described herein include one or more inorganic and / or organic bleaching compounds. Inorganic bleaching agents may include, but are not limited to, perhydrate salts (e.g., perborate, percarbonate, perphosphate, persulfate, and persilicate). In some embodiments, the inorganic perhydrate salt is an alkali metal salt. In some embodiments, inorganic perhydrate salts that are crystalline solids but not otherwise protected are included, but in some other embodiments, the salt is coated. Suitable salts include, for example, those described in EP 2100949. Bleach activators are typically organic peracid precursors that enhance bleaching during cleaning at temperatures of 60°C and below. Bleach activators suitable for use herein include compounds that give, under perhydrolysis conditions, preferably aliphatic peroxycarboxylic acids having from about 1 to about 10 carbon atoms, particularly from about 2 to about 4 carbon atoms, and / or optionally substituted peroxybenzoic acid. Bleach catalysts typically include, for example, manganese triazacyclononane and related complexes, and cobalt, copper, manganese and iron complexes, and those described in US4246612, US5227084, US4810410, WO9906521, and EP2100949.

[0116] In certain embodiments, laundry detergent compositions as described herein include one or more catalytic metal complexes. In certain embodiments, metal-containing bleach catalysts can be used. In other embodiments, metal bleach catalysts include a catalytic system containing the following: transition metal cations (e.g., copper, iron, titanium, ruthenium, tungsten, molybdenum or manganese cations) with limited bleach catalytic activity, auxiliary metal cations (e.g., zinc or aluminum cations) with little or no bleach catalytic activity, and chelates with limited stability constants for catalytic and auxiliary metal cations, particularly ethylenediaminetetraacetic acid, ethylenediaminetetrakis (methylenephosphonic acid) and water-soluble salts thereof (see, e.g., US 4430243). In certain embodiments, laundry detergent compositions as described herein are catalyzed by means of manganese compounds. Such compounds and usage levels are well known in the art (see, e.g., US 5576282). In other embodiments, cobalt bleach catalysts can be used in laundry detergent compositions as described herein. Various cobalt bleach catalysts are known in the art (see, for example, US 5597936 and US 5595967) and are readily prepared by known procedures.

[0117] Some embodiments are directed to methods of cleaning comprising contacting an effective amount of a cleaning composition described herein with an item or surface containing a soil, stain, or odor to hydrolyze the soil, stain, or odor.

[0118] Other aspects and embodiments of the compositions and methods of the present invention will be apparent from the foregoing description and the following examples. Various alternative embodiments beyond the embodiments described herein may be employed in practicing the present invention without departing from the spirit and scope of the present invention. Therefore, the claims, rather than the specific embodiments described herein, define the scope of the present invention, and as such, methods and structures within the scope of the claims and their equivalents are thereby covered.

[0119] Examples

[0120] Example 1. Treatment of clothing odor

[0121] Clothing odor is carried out in situ as follows in the Launder-Ometer cleaning machine model system.Tryptic soy broth (TSB) is inoculated with Staphylococcus epidermidis (ATCC 35984), and the culture is grown overnight in an incubator shaker at 30 degrees Celsius and 250RPM.Determine the OD600 of the overnight culture.According to the OD600 value of gained, the solution of tryptic soy broth (TSB) and overnight culture is prepared so that the final cell suspension measures about 0.10-0.15OD600 (minus the culture medium background) with a spectrophotometer.The cell suspension (75mL per dish) is added to a polystyrene culture dish (VWR25384-088), which contains an autoclaved stainless steel plate designed to be inserted into a Launder-Ometer basin (AATCC SDL Atlas).The culture dish with the stainless steel plate is incubated for 48 hours without stirring at 30 degrees Celsius. After incubation, discard the liquid cell culture, and the stainless steel plate is briefly dried (about 25 minutes) in a laminar flow hood.Then the coated stainless steel plate is placed in a fresh sterile culture dish, rinsed with sterile PBS buffer, and then briefly dried again (about 25 minutes).Then the Launder-Ometer plate coated is placed in the Launder-Ometer basin.In each Launder-Ometer basin, a coated plate and two uncoated autoclaved stainless steel plates are placed so that these three plates form an equilateral triangle.In all cases, the microbial residues producing odor are turned away from the center of the triangle outward, to simulate the outside of the cleaning drum and the invisible, other cleaning machine surfaces that may still be coated with the residues causing the odor of the consumer.Plastic covers (Nalgene, Inc. (Nalgene)) of appropriate size are placed inside the equilateral triangle to keep the stainless steel plate fixed inside the Launder-Ometer basin. Three pieces of autoclaved polyester ballast fabric (Testfabrics Inc. Style 730: Textured polyester 56T interlock knit, 8 x 12 cm pieces) were placed in an equilateral triangle within the Launder-Ometer basin.

[0122] Then make the Launder-Ometer basin stand simulated clothing cleaning.Add 200mL of cleaning solution to each basin.Prepare cleaning solution as follows.All basins contain deionized water solution, and the deionized water solution has increased hardness to reach 16 ° dH hardness (3: 1Ca: Mg) and 0.4g / L Tide Original (Tide Original Fragrance) liquid detergent (Procter & Gamble (Procter & Gamble)).The cleaning solution of "detergent only" does not contain other ingredients.The "+spice" and "+enzyme+spice" cleaning solutions contain 0.001% of the spice mixture solution (Skydive Mod, International Flavors & Fragrances (IFF)) in addition.The "+enzyme" and "+enzyme+spice" cleaning solutions contain 80PPM dosage of thermolysin (Protease T, International Flavors & Fragrances) in addition.All cleanings are carried out in the Launder-Ometer set to 25 degrees.In the Launder-Ometer, each basin is cleaned for 25 minutes.After the first cleaning, the cleaning solution is poured out and replaced with the fresh cleaning solution with the same composition used in the first cleaning. Each basin was then subjected to a second 25 minute wash cycle in the Launder-Ometer. After the two washes, all basins were rinsed in the Launder-Ometer for 10 minutes with a rinse solution of 200 mL DI water with 16° dH hardness (3:1 Ca:Mg). The final rinse solution was discarded.

[0123] A synthetic sweat solution (200 mL) that promotes odor production was added to each Launder-Ometer basin (Table 2).

[0124] Table 2. Composition of synthetic sweat solution

[0125] Components Weight % (in water) glucose 1％ Sodium chloride 0.5％ Disodium hydrogen orthophosphate dodecahydrate 0.5％ Leucine 0.2％ Casein amino acids 0.1％ L-Histidine 0.05％ Dipotassium hydrogen phosphate <![CDATA[2.52x 10 -4 %]]> Ammonium Nitrate <![CDATA[3.6x 10 -4 %]]> Ferrous Sulfate <![CDATA[7.2x 10 -7 %]]> Magnesium sulfate <![CDATA[2.52x 10 -4 %]]> Potassium dihydrogen phosphate <![CDATA[2.52x 10 -4 %]]> Zinc sulfate <![CDATA[7.2x 10 -7 %]]>

[0126] The Launder-Ometer pots were sealed, rotated several times to mix, and then incubated at 30 degrees Celsius without stirring for 4 days. After 4 days, the odors in the Launder-Ometer pots were evaluated by an odor perception panel of 11 participants. The odor panelists were asked to lift the lid of the Launder-Ometer pots and evaluate the odor of each pot on a scale from 1 ("bad smell") to 5 ("good smell"). The results are shown in Table 3.

[0127] Table 3. Odor panel evaluation of clothing odor samples.

[0128]

[0129] As shown in Table 3, enzyme-spice mixture shows that odor is significantly improved, far exceeding the error in the experiment, and far exceeding independent enzyme or spice.The benefit of the combination of enzyme and spice is greater than the expected benefit of the sum of the individual effects of enzyme and spice.For example, relative to independent detergent, the benefit (the odor panel score of detergent+spice deducts the odor panel score of independent detergent) provided by spice is 0.8, and the benefit provided by independent detergent+enzyme is 1.4.Therefore, the expected benefit of enzyme and spice combination is expected to be about 2.2 (the sum of the individual benefits of enzyme and spice).However, the benefit of observed enzyme-spice combination (the odor panel score of combination deducts the odor panel score of independent detergent) is 2.9, which is greater than the value predicted by the simple summation of independent effect.Three odor sensory panel participants will score the sample containing spice as good (scoring is greater than 4.0 or higher), and only one participant will score the sample containing enzyme as good (scoring is 4.0 or higher).However, all 11 participants will score the enzyme-spice conformation as good (scoring is 4.0 or higher). Without being limited by theory, cleaning of odor-causing residues by enzymes can reduce the perception of mixed odors (which causes the broad response and high standard deviation in the panel response using fragrance in the absence of enzymes), resulting in a higher and more uniform good response in the combination than expected from the individual effects.

[0130] Example 2. Treatment of clothing odor

[0131] Clothing odor is carried out in situ as follows in the Launder-Ometer cleaning machine model system.Tryptic soy broth (TSB) is inoculated with Staphylococcus epidermidis (ATCC 35984), and the culture is grown overnight in an incubator shaker at 30 degrees Celsius and 250RPM.Determine the OD600 of the overnight culture.According to the OD600 value of gained, the solution of tryptic soy broth (TSB) and overnight culture is prepared so that the final cell suspension measures about 0.10-0.15OD600 (minus the culture medium background) with a spectrophotometer.The cell suspension (75mL per dish) is added to a polystyrene culture dish (VWR25384-088), which contains an autoclaved stainless steel plate designed to be inserted into a Launder-Ometer basin (AATCC SDL Atlas).The culture dish with the stainless steel plate is incubated for 48 hours without stirring at 30 degrees Celsius. After incubation, the liquid cell culture is discarded and the stainless steel plate is briefly dried in a laminar flow hood (about 25 minutes). The coated stainless steel plate is then placed in a fresh sterile culture dish, rinsed with sterile PBS buffer, and then briefly dried again (about 25 minutes). The coated Launder-Ometer plate is then placed in a Launder-Ometer basin. In each Launder-Ometer basin, a coated plate and two uncoated autoclaved stainless steel plates are placed so that the three plates form an equilateral triangle. In all cases, the odor-producing microbial residues are facing outward from the center of the triangle to simulate the outside of the cleaning drum and other cleaning machine surfaces that are invisible to consumers and may still be coated with odor-causing residues. A plastic cover of appropriate size (Le Gene) is placed inside the equilateral triangle to keep the stainless steel plate fixed inside the Launder-Ometer basin ( Figure 1 Three pieces of autoclaved polyester ballast fabric (Testfabrics Ltd. Style 730: Textured polyester 56T interlock knit, 8 x 12 cm pieces) were placed in an equilateral triangle within the Launder-Ometer basin.

[0132] Then make Launder-Ometer basin stand simulated clothing cleaning.Add 200mL of cleaning solution to each basin.Prepare cleaning solution as follows.All basins contain deionized water solution, and this deionized water solution has the hardness of increase to reach 100PPM water hardness (3:1Ca:Mg) and 0.5g / L Tide Original (Tide Original Fragrance) liquid detergent (Procter & Gamble).The cleaning solution of "detergent only" does not contain other ingredients.The "+spice" and "+enzyme+spice" cleaning solutions contain 0.001% spice mixture solution (Skydive Mod, International Flavors & Fragrances Company) in addition.The "+enzyme" and "+enzyme+spice" cleaning solutions contain 80PPM dosage of thermolysin (Protease T, International Flavors & Fragrances Company) in addition.All cleanings are carried out in Launder-Ometer set to 25 degrees.In Launder-Ometer, each basin is carried out to the first cleaning cycle of 35 minutes. After the first cleaning, the cleaning solution in each basin is poured out and replaced with the rinsing solution of 200mL DI water with 100PPM water hardness (3:1Ca:Mg). In the Launder-Ometer, each basin is carried out to the first rinse cycle of 8 minutes. After the rinse cycle, the rinsing solution is poured out and replaced with the fresh cleaning solution with the same composition used in the first cleaning. Then in the Launder-Ometer, each basin is carried out to the second cleaning cycle of 35 minutes. After the second cleaning, the cleaning solution is poured out and replaced with fresh rinsing solution, and then the basin is rinsed in the second 8-minute Launder-Ometer cycle. Final rinsing solution is poured out.

[0133] As described in Example 1, synthetic sweat solution (200 mL) was added to each Launder-Ometer basin to promote odor development.

[0134] The Launder-Ometer pots were sealed, rotated several times to mix, and then incubated at 30 degrees Celsius without stirring for 4 days. After 4 days, the odor in the Launder-Ometer pots was evaluated by an odor perception panel of 9 participants. The odor panel was asked to lift the lid of the Launder-Ometer pot and evaluate the odor of each pot on a scale from 1 ("bad smell") to 5 ("good smell"). The results are shown in Table 4.

[0135] Table 4. Odor panel evaluation of clothing odor samples.

[0136]

[0137] As shown in Table 4, enzyme-spice mixture shows that odor is significantly improved, far exceeding the error in the experiment, and far exceeding independent enzyme or spice.As in Example 1, the benefit of the combination of enzyme and spice is greater than the expected benefit of the sum of the individual effects of enzyme and spice.For example, relative to independent detergent, the benefit (the odor panel score of detergent+spice deducts the odor panel score of independent detergent) provided by spice is 1.1, and the benefit provided by independent detergent+enzyme is 0.7.Therefore, the expected benefit of enzyme and spice combination is expected to be about 1.8 (the sum of the individual benefits of enzyme and spice).However, the benefit of observed enzyme-spice combination (the odor panel score of combination deducts the odor panel score of independent detergent) is 2.6, greater than the value predicted by the simple addition of independent effect.In this experiment, three participants scored the sample containing spice as good (scoring greater than 4.0 or higher), and two participants scored the sample containing enzyme as good (scoring 4.0 or higher).However, all 9 participants scored the enzyme-spice conformation as good (scoring 4.0 or higher).

[0138] Example 3. GC-MS analysis of odor compounds

[0139] The volatile odor compounds present in the Launder-Ometer odor system were analyzed as follows. Following the same procedure as in Examples 1 and 2, a culture of Staphylococcus epidermidis was grown for 48 hours in an autoclaved 20 mL GC-MS vial (Agilent Technologies part number 5188-2753) containing 4 mL of cell suspension in the GC-MS vial. After 48 hours, the residue coated on the bottom of the vial was rinsed with PBS and then the PBS was removed. 4 mL of synthetic sweat solution (as in Example 1) was then added to the vial and the vial was placed at 30°C for 4 days with the cap screwed on (the cap was Agilent Technologies part number 5188-2759). Samples were analyzed in SPME mode using an Agilent 7890 / 5975 GC-MS system with a CTC GC PAL autosampler, starting at 40 degrees Celsius and ramping up to 240 degrees Celsius using splitless mode. The column was Zebron ZB-FFAP (30m x 0.32mm x 0.5um). The SPME fiber used was Supelco SPME fiber (blue tip-carboxen / PDMS 23 specification). SPME GCMS results identified some odor compounds present in the clothing odor model system, such as ethanol, isopentanol, acetoin, acetic acid, and isovaleric acid.

[0140] Example 4. Cleaning of odor-causing residues

[0141] In order to further evaluate the enzymatic cleaning of the residues producing odor, the enzymatic removal of body dirt stains was tested. The contaminated fabric, i.e. Consumertec Collar and Cuff dingy monitor DINGY_TN / CP / USA (Consumer Technology Company (Consumertec)) is cut into small cubes, about 0.5-0.6cm on each side. The contaminated fabric square is placed in the hole of 96-well plate for cleaning test. In each well, 250 microliters of 1:1200 diluted Tide Original (Tide Original fragrance) liquid laundry detergent in water is added, and the thermolysin (Protease T, International Flavors & Fragrances Company) of enzyme concentration is 0,2 or 10PPM. In order to simulate the cleaning cycle, the microplate is sealed and then placed in the iEMS incubator oscillator set to 25 degrees and 1150rpm for 30 minutes. After the simulation cleaning cycle, 200 microliters of liquid are removed to fresh 96-well plates, and the absorbance at 500nm is read.

[0142] Table 5. Absorbance at 500 nm of wash solutions treated with 0, 2, and 10 PPM enzyme. The body dirt is released into the solution.

[0143]

[0144] As shown in Table 5, more stain was released in the solutions with 2 PPM and 10 PPM enzyme compared to the no enzyme control.

[0145] Example 5. Scent Benefits of Fragrance

[0146] Table 6. Composition of standard unscented laundry detergent

[0147]

[0148] Wash solutions consisting of 2 g / L of standard detergent (Table 6) in deionized water and the following concentrations of fragrance (SkydiveMod, International Flavors & Fragrances) were prepared: 0.001%, 0.0005%, 0.0002%, 0.0001%, and no fragrance. The samples were prepared by adding fragrance to an aliquot of the standard detergent and then dissolving the standard detergent in water.

[0149] The solutions were placed in scintillation vials (10 ml each). A volunteer odor sensory panel was asked to smell the solutions and rate them on a scale of 1 (moderate or non-fresh) to 5 (very fresh) based on their freshness perception ("How fresh did the sample smell?"). The results are shown in Table 7.

[0150] Table 7. Odor sensory panel evaluation of cleaning solutions containing various fragrance concentrations .

[0151] Spice Dosage Odor panel rating (n=7) 0 1±0 0.1x 3.1±1.0 0.2x 3.6±1.3 0.5x 4.4±0.8 1x 4.5±0.8

[0152] As shown in Table 7, panelists reported increased freshness relative to the unperfumed control at all concentrations tested.

[0153] Example 6. Combination of Metalloproteases and Perfume Ingredients

[0154] Combinations of metalloproteases and specific flavor ingredient classes are also contemplated herein. Metalloproteases such as thermolysin or lysozyme with one or more flavor ingredients such as tertiary alcohols (e.g., 2,6-dimethyl-7-octen-2-ol), secondary alcohols (e.g., 2-hydroxycamphane), primary alcohols (e.g., 3-methyl-5-phenyl-1-pentanol), ketones (e.g., damascenone delta), nitriles (e.g., 3,7-dimethyl-6-octenonitrile), lactones (e.g., gamma undecanoic acid lactone), aldehydes (e.g., undecanal), ethers (e.g., diphenyl ether), or esters (e.g., isoborneol acetate).

[0155] Although the present disclosure has been described in conjunction with the specific embodiments of the present disclosure, it is apparent that many substitutions, modifications and variations will be apparent to those skilled in the art. Therefore, the present disclosure is intended to cover all such substitutions, modifications and variations that fall within the spirit and broad scope of the appended claims.

[0156] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference in their entirety, to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application should not be construed as an admission that such reference is available as prior art to the present disclosure. To the extent that section headings are used, they should not be construed as necessarily limiting.

Claims

1. A method for treating odor in textiles or machinery, the method comprising: (i) contacting a textile or a machine with a composition comprising: a) 0.2 to 100 parts per million (PPM) of a polypeptide having thermolysin activity; b) 0.00001% to about 2% fragrance; and c) at least one detergent adjuvant, and (ii) optionally rinsing the textile or machine.

2. The method of claim 1, wherein the machine is a washing machine or a dishwasher.

3. A method as claimed in any one of the preceding claims, wherein the composition comprises a polypeptide having thermolysin activity in an amount selected from 0.001 to 10,000 mg / L, or 0.001 to 2000 mg / L, or 0.01 to 5000 mg / L, or 0.01 to 2000 mg / L, or 0.01 to 1300 mg / L, or 0.1 to 5000 mg / L, or 0.1 to 2000 mg / L, or 0.1 to 1300 mg / L, or 1 to 5000 mg / L, or 1 to 1300 mg / L, or 1 to 5000 mg / L, or 10 to 5000 mg / L, or 10 to 1300 mg / L, or 10 to 500 mg / L.

4. The method of any one of the preceding claims, wherein the fragrance is selected from the group consisting of alcohols, aldehydes, ketones, nitriles, lactones, ethers, esters, essential oils, and mixtures thereof.

5. A method as claimed in any preceding claim wherein the composition is a laundry detergent composition.

6. A method for cleaning an article, the method comprising contacting an article in need of cleaning with a composition comprising a) 0.2 to 100 parts per million (PPM) of a polypeptide having thermolysin activity; b) 0.00001% to about 2% of a perfume; and c) at least one detergent adjuvant, and (ii) optionally rinsing the article.

7. The method of claim 6, wherein the article is a textile or a hard surface.

8. The method of claim 7, wherein the hard surface is a washing machine, dishware or dishwasher.

9. The method of any one of claims 6-8, wherein the composition comprises a polypeptide having thermolysin activity in an amount selected from 0.001 to 10,000 mg / L, or 0.001 to 2000 mg / L, or 0.01 to 5000 mg / L, or 0.01 to 2000 mg / L, or 0.01 to 1300 mg / L, or 0.1 to 5000 mg / L, or 0.1 to 2000 mg / L, or 0.1 to 1300 mg / L, or 1 to 5000 mg / L, or 1 to 1300 mg / L, or 1 to 5000 mg / L, or 10 to 5000 mg / L, or 10 to 1300 mg / L, or 10 to 500 mg / L.

10. The method of any one of claims 6-9, wherein the fragrance is selected from the group consisting of alcohols, aldehydes, ketones, nitriles, lactones, ethers, esters, essential oils, and mixtures thereof.

11. The method of any one of claims 6-10, wherein the composition is a laundry detergent composition.

12. A method for improving the freshness of items after a cleaning process, the method comprising contacting the items with a composition comprising a) 0.2 to 100 parts per million (PPM) of a polypeptide having thermolysin activity; b) 0.00001% to about 2% perfume; and c) at least one detergent adjuvant, and (ii) optionally rinsing the items.

13. The method of claim 12, wherein the freshness of the items after the cleaning process is improved compared to the freshness of the items before the cleaning process, or compared to the freshness of similar items after a cleaning process using a composition lacking (i) the polypeptide having thermolysin activity, (ii) the fragrance; or (iii) both the polypeptide having thermolysin activity and the fragrance.

14. The method of any of the preceding claims, wherein the composition further comprises one or more additional enzymes selected from the group consisting of: acyltransferases, alpha-amylases, beta-amylases, alpha-galactosidases, arabinosidases, arylesterases, beta-galactosidases, carrageenase, catalase, cellobiohydrolase, cellulase, chondroitinase, cutinase, endo-beta-1,4-glucanase, endo-beta-mannanase, esterase, exo-mannanase, galactanase, glucoamylase, hemicellulase, hexosaminidase, hyaluronidase, keratinase, lacquer enzymes, lactases, ligninases, lipases, lipoxygenases, lysozymes, mannanases, other metalloproteinases, nucleases (e.g., deoxyribonucleases and ribonucleases), oxidases, oxidoreductases, pectate lyases, pectin acetylesterases, pectinases, pentosanases, peroxidases, phenoloxidases, phosphatases, phospholipases, phytases, polygalacturonases, polyesterases, additional proteases, pullulanases, reductases, rhamnogalacturonases, β-glucanases, tannases, transglutaminases, xylan acetylesterases, xylanases, xyloglucanases, xylosidases, and any combination or mixture thereof.

15. Use of a polypeptide having thermolysin activity in a detergent composition for increasing the freshness associated with perfumes on textiles after a laundry washing process, or for enhancing the effect of perfumes in a detergent composition, wherein the detergent comprises at least one perfume compound.

16. The use according to claim 15, wherein the polypeptide having thermolysin activity is a polypeptide having at least about 60% sequence identity with SEQ ID NO:

1.

17. The use according to any one of claims 15-16, wherein the detergent composition is a laundry detergent or a fabric softener.

18. The use according to any one of claims 15 to 17, wherein the detergent composition further comprises a nuclease.

19. An enzyme-flavor system, comprising: About 0.2 to 100 PPM of a polypeptide having thermolysin activity; and From about 0.00001% to about 2% fragrance.

20. A detergent composition comprising about 0.2 to 100 PPM of a polypeptide having thermolysin activity, about 0.00001% to about 2% of a perfume, and optionally at least one detergent adjunct material.

21. The detergent composition of claim 20, wherein the composition further comprises one or more adjunct materials selected from the group consisting of builders, bleaches, bleach activators, bleach catalysts, other enzymes, enzyme stabilization systems, chelating agents, optical brighteners, soil release polymers, dye transfer agents, dispersants, foam inhibitors, dyes, perfumes, colorants, filler salts, hydrotropes, photoactivators, fluorescent agents, fabric conditioners, hydrolyzable surfactants, preservatives, antioxidants, anti-shrinkage agents, anti-wrinkle agents, bactericides, fungicides, color embellishments, silver care agents, anti-tarnish and / or anti-corrosion agents, alkalinity sources, solubilizers, carriers, processing aids, pigments, and pH control agents.

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