Cleaning methods, enzymes and uses of cleaning compositions

Abstract

Provided is a method of cleaning a medical device comprising contacting the device with a cleaning solution comprising two or more enzymes selected from the group consisting of a protease, an enzyme having DNase activity, and an enzyme having hexosaminidase activity.

Description

[Technical field]

[0001] Sequence Listing Reference This application contains a sequence listing in computer readable form, which is incorporated herein by reference.

[0002] The present invention relates to a method for cleaning a medical device and to the use of an enzyme composition for cleaning a medical device, the enzyme composition comprising two or more enzymes selected from the group consisting of a protease, an enzyme having DNase activity, and an enzyme having hexosaminidase activity.The present invention further relates to a cleaning composition comprising said enzyme composition. [Background technology]

[0003] Medical devices often become heavily contaminated with organic soil as a result of their use, and prior to reuse it is essential that the devices are properly cleaned and sterilized.

[0004] The nature and extent of contamination in healthcare environments is much greater than that typically found in domestic environments, and the need for effective cleaning is high. Of particular concern are microorganisms that are resistant to many types of antibiotics, as they are overrepresented in healthcare environments. Thus, the use of medical equipment that is not adequately reprocessed poses a high risk of cross-infection to other patients whose immune systems may already be compromised by illness, injury, or the trauma of invasive medical procedures.

[0005] Typically, reprocessing procedures for medical devices include cleaning the device to remove organic material, rinsing, sterilizing, and drying. Cleaned medical devices are often still contaminated with organic material that can significantly reduce the effectiveness of subsequent sterilization procedures.

[0006] WO 2017 / 129331 discloses a method for cleaning medical and dental instruments using proteases.

[0007] WO 2019 / 086532 discloses a method for cleaning medical devices using a hexosaminidase having beta-N-acetylglucosaminidase activity. Summary of the Invention [Problem to be solved by the invention]

[0008] There remains a need for improved compositions and methods for cleaning medical devices that can more effectively remove organic soils. [Means for solving the problem]

[0009] The present invention provides a method for cleaning a medical device, comprising: (a) contacting the medical device with a cleaning solution comprising two or more enzymes selected from the group consisting of a protease, an enzyme having DNase activity, and an enzyme having hexosaminidase activity; and optionally (b) rinsing the medical device; The present invention relates to a method comprising the steps of:

[0010] The present invention further relates to the use of an enzyme composition for cleaning a medical device, the enzyme composition comprising two or more enzymes selected from the group consisting of a protease, an enzyme having DNase activity, and an enzyme having hexosaminidase activity.

[0011] Additionally, the present invention relates to a composition for cleaning a medical device, the composition comprising a surfactant and two or more enzymes selected from the group consisting of a protease, an enzyme having DNase activity, and an enzyme having hexosaminidase activity. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] definition As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0013] DNase: The term "DNase" refers to a polypeptide / enzyme with DNase activity that degrades DNA by catalyzing the hydrolytic cleavage of phosphodiester bonds in the DNA backbone. Exodeoxyribonucleases cleave or cleave residues at the ends of the DNA backbone, whereas endodeoxyribonucleases cleave or cleave within the DNA backbone. DNases may cleave only double-stranded DNA or may cleave double-stranded and single-stranded DNA. The terms "DNase" and the phrases "polypeptide with DNase activity" or "enzyme with DNase activity" are used interchangeably throughout this application.

[0014] For the purposes of the present invention, DNase activity is determined according to the procedure described in Assay I. In one embodiment of the invention, the DNase has a DNase activity that is at least 50%, such as at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, or at least 150% of the DNase activity of the mature polypeptide of SEQ ID NO: 1, the enzyme comprising or consisting of the sequence set forth in SEQ ID NO: 2, the enzyme comprising or consisting of the sequence set forth in SEQ ID NO: 3, or the enzyme comprising or consisting of the mature polypeptide of SEQ ID NO: 4.

[0015] Hexosaminidase: The term "hexosaminidase" refers to a polypeptide having hexosaminidase activity (hexosaminidase) and includes enzymes in EC 3.2.1., e.g., that catalyze the hydrolysis of N-acetyl-D-hexosamine or N-acetylglucosamine polymers, e.g., found in biofilms. The term includes dispersins and includes polypeptides having N-acetylglucosaminidase activity and β-N-acetylglucosaminidase activity. The term "polypeptide having hexosaminidase activity" may be used interchangeably with the term hexosaminidase, and similarly, the term "polypeptide having β-N-acetylglucosaminidase activity" may be used interchangeably with the term β-N-acetylglucosaminidase. For purposes of the present invention, hexosaminidase activity is determined according to the procedure described in Assay II. In one aspect, the hexosaminidase of the present invention has a hexosaminidase activity that is at least 40%, such as at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, or at least 120% of the hexosaminidase activity of the mature polypeptide of SEQ ID NO:10.

[0016] Dispersin: The terms "dispersin" and abbreviation "Dsp" refer to a polypeptide having hexosaminidase activity, EC 3.2.1. (catalyzing the hydrolysis of β-1,6-glycosidic bonds in N-acetyl-glucosamine polymers (poly-N-acetylglucosamine), found, for example, in biofilms).

[0017] Bacterial: The term "bacterial" with respect to a polypeptide, such as an enzyme, refers to both a polypeptide that is encoded by the bacterial genome and is thus directly derivable from the bacterial genome, as well as genetically modified variants thereof, e.g., variants of bacterial enzymes that have been modified using protein engineering techniques to result in an enzyme with desired characteristics, such as improved stability and / or increased enzymatic activity. Bacterial polypeptides that are derived directly from a bacterium may be referred to as wild-type enzymes. Variants of wild-type bacterial enzymes may be referred to as being "substantially homologous" to the wild-type sequence, which refers to an enzyme that has at least 80% sequence identity, e.g., at least 85%, at least 90%, at least 95%, or at least 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of a given wild-type enzyme.

[0018] Fungal: The term "fungal" with respect to a polypeptide, such as an enzyme, refers to both a polypeptide that is encoded by the fungal genome and is thus directly derivable from the fungal genome, as well as genetically modified variants thereof, e.g., variants of fungal enzymes that have been modified using protein engineering techniques to result in an enzyme with desired characteristics, such as improved stability and / or increased enzymatic activity. A fungal polypeptide derived directly from a fungus may be referred to as a wild-type enzyme. A variant of a wild-type fungal enzyme may be referred to as being "substantially homologous" to the wild-type sequence, which refers to an enzyme that has at least 80% sequence identity, e.g., at least 85%, at least 90%, at least 95%, or at least 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of a given wild-type enzyme.

[0019] Biofilm: Biofilms can be produced by any group of microorganisms whose cells attach to each other or to a surface, particularly to the surface of a medical device in the context of the present invention. The attached cells are often embedded in a self-generated matrix of extracellular polymeric substances (EPS). Biofilm EPS is generally a polymeric mass composed of extracellular DNA, proteins, and polysaccharides. Bacteria living in biofilms usually have very different properties than planktonic bacteria of the same species, because the dense and protected environment of the film allows these bacteria to cooperate and interact in various ways. One advantage of this environment for microorganisms is increased resistance to detergents and antibiotics, because the dense extracellular matrix and outer layer of cells protect the inside of the community.

[0020] On medical devices, biofilm-producing bacteria of, for example, the following species may be found: Escherichia coli, Klebsiella pneumoniae, Salmonella spp., Mycobacterium spp., Enterococcus faecalis, Enterobacter cloacae, Proteus mirabilis, Serratia marcescens, Staphylococcus aureus, e.g. Staphylococcus aureus ATCC 61164. 6538, Streptococcus spp. (e.g., S. pyogenes, S. agalactiae, or S. pneumoniae), Haemophilus influenzae, Pseudomonas aeruginosa, Clostridium perfringens, Chlamydia trachomatis, Candida albicans, Bacillus anthracis.

[0021] Bacillus subtillis, Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas fluorescens, Yersinia pestis, Aggregatibacter actinomycetemcomitans, Streptococcus pyogenes, Streptococcus dysgalactiae (group C streptococci), Enterococcus faecalis, Listeria monocytogenes, Clostridium difficile, Mycobacterium tuberculosis tuberculosis, Mycobacterium smegmatis, Neisseria meningitides, Neisseria gonorrhea, Haemophilus influenzae, Haemophilus ducreyi, Helicobacter pylori, Campylobacter jejuni, Citrobacter rodentium, Salmonella enterica serovars typhi, Salmonella typhimurium, Candida albicans, Aspergillus flavus, Fusarium solani A wide range of bacterial and fungal microorganisms, including but not limited to, Pseudomonas solani, and Cryptococcus neoformans, have been found to produce poly-N-acetylglucosamine (PNAG) or PNAG-like surface polysaccharides.

[0022] Extracellular DNA (eDNA) is a common matrix component in microbial biofilms and is a common component of the matrix of many strains of bacteria, including but not limited to Acinetobacter baumannii, Actinobacillus actinomycetemcomitans, Bdellovibrio bacterivorous, Bordetella pertussis, Bordetella bronchiseptica, Campylobacter jejuni, Comamonas denitrificans, Escherichia coli, Haemophilus influenza, Klebsiella pneumoniae, and Neisseria meningitidis. meningitides, Pseudomonas aeruginosa, Shewanella oneidensis, Vibrio cholera, Gram-positive bacteria, Bacillus licheniformis, Bacillus subtilis, Enterococcus faecalis, Listeria monocytogenes, Micrococcus luteus, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Streptococcus anginosus anginosus, Streptococcus constellatus, Streptococcus salivarius, Staphylococcus lugdunensishave been identified in species including S. lugdunesis, Streptococcus intermedius, Streptococcus intermedius, Streptococcus mutans, Streptococcus pneumoniae, Streptococcus pyogenes, Aspergillus fumigatus, and Candida albicans.

[0023] Most biofilms are "poly-cultural" because they contain biofilms or EPS from many different species of bacteria.

[0024] Clade: A group of polypeptides clustered together based on homologous features that trace back to a common ancestor. A polypeptide clade can be visualized as a phylogenetic tree, where a clade is a group of polypeptides that consist of a common ancestor and all its direct descendants, for example, the Terribacillus clade or the Terribacillus clade is a group of enzymes that are all related to the same ancestor and share common properties.

[0025] Cleaning ingredients: "Cleaning ingredients" is defined herein to mean a type of chemical that is a component different from the enzyme according to the present invention and can be used in a cleaning composition. Examples of cleaning ingredients are alkali, surfactants, hydrotropes, builders, cobuilders, chelators or chelating agents, bleaching systems or components, polymers, suds suppressors, dispersants, bactericides, fungicides, corrosion inhibitors, soil suspending agents, soil release polymers, anti-redeposition agents, enzyme inhibitors or stabilizers, enzyme activators, antioxidants, preservatives, and solubilizers.

[0026] Cleaning composition: The term "cleaning composition" (which may also be referred to as "detergent composition") refers to a composition used for the removal of undesired compounds from an article to be cleaned, such as a medical device. The cleaning composition of the present invention is particularly suitable for medical cleaning. The term encompasses any material / compound selected for the particular type of cleaning composition desired and product form (e.g., liquid, gel, powder, granule, foam, or spray composition). In addition to containing the enzyme of the present invention, the cleaning composition of the present invention may contain one or more additional enzymes (e.g., amylase, lipase, cellulase, mannanase, hemicellulase, peroxidase, xylanase, phospholipase, esterase, cutinase, keratinase, reductase, oxidase, phenoloxidase, lipoxygenase, ligninase, pullulanase, malanase, β-glucanase, arabinosidase, hyaluronidase, laccase, perhydrolase, and peroxidase, or any mixture thereof) and / or cleaning ingredients described above.

[0027] Deep cleaning: The term "deep cleaning" refers to the reduction or removal of components of a biofilm, such as EPS or parts thereof, polysaccharides, PNAG (poly-N-acetylglucosamine), proteins, DNA, dirt, or other components present in the biofilm.

[0028] Enzyme detergency benefit: The term "enzyme detergency benefit" is defined herein as the beneficial effect that an enzyme can be added to a cleaning composition compared to the same composition without the enzyme. An important detergency benefit that can be provided by an enzyme is stain removal with no or little visible stains after washing and / or cleaning, or prevention or reduction of redeposition of stains released in the cleaning process (effect also called anti-redeposition). In the context of the present invention, this can also include the removal of "invisible" stains that may otherwise remain on a medical device after cleaning with a composition that does not contain the enzyme of the present invention. For the purposes of the present invention, the enzyme detergency benefit can be evaluated by biofilm reduction benefit, as described in Example 1.

[0029] Mature Polypeptide: The term "mature polypeptide" refers to a polypeptide in its final form following translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, and the like.

[0030] It is known in the art that a host cell may produce a mixture of two or more different mature polypeptides (i.e., having different C-terminal and / or N-terminal amino acids) expressed by the same polynucleotide. It is also known in the art that different host cells process polypeptides differently, such that one host cell expressing a polynucleotide may produce a different mature polypeptide (e.g., having different C-terminal and / or N-terminal amino acids) compared to another host cell expressing the same polynucleotide.

[0031] Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity."

[0032] For the purposes of the present invention, sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48; 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 6.6.0 or later, as the "longest identity" output. The parameters used were a gap open penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. In order for the Needle program to report the longest identity, the nobrief option must be specified on the command line. The output of Needle labeled "longest identity" is calculated as follows: (identical residues × 100) / (length of alignment−total number of gaps in the alignment).

[0033] Variant: The term "variant" refers to a polypeptide / enzyme that has the same activity as a parent enzyme and that contains alterations, i.e., substitutions, insertions, and / or deletions, at one or more positions compared to the parent amino acid sequence. A substitution refers to the replacement of an amino acid occupying a position with a different amino acid; a deletion refers to the removal of an amino acid occupying a position; and an insertion refers to the addition of an amino acid adjacent to and immediately after the amino acid occupying a position.

[0034] In the context of the present invention, a variant may be, for example, a variant of a specified DNase that has the enzymatic activity of the parent, i.e. the ability to catalyze the hydrolytic cleavage of phosphodiester bonds in the DNA backbone (deoxyribonuclease activity). In one embodiment, the deoxyribonuclease activity of the variant is increased relative to the parent DNase, e.g. the polypeptide of SEQ ID NO: 2 or 4.

[0035] In the context of the present invention, the variant may also be, for example, a variant of an identified hexosaminidase that has the enzymatic activity of the parent, i.e., the ability to catalyze the hydrolysis of β-1,6-glycosidic bonds of N-acetyl-glucosamine polymers (hexosaminidase activity). In one embodiment, the hexosaminidase activity of the variant is increased relative to the parent hexosaminidase, e.g., the polypeptide of SEQ ID NO: 10.

[0036] Variant nomenclature: In describing enzyme variants herein, the following nomenclature is adopted for ease of reference: IUPAC accepted single-letter or three-letter amino acid abbreviations are generally adopted.

[0037] Substitutions: For amino acid substitutions, the following nomenclature is used: original amino acid, position, substituted amino acid. Thus, a substitution of threonine at position 226 with alanine is designated "T226A." Multiple mutations may be separated by a plus sign ("+") (e.g., "G205R+S411F") or a comma (e.g., "G205R,S411F"), representing the substitution of serine (S) with glycine (G) and phenylalanine (F) with arginine (R) at positions 205 and 411, respectively.

[0038] Deletions: For amino acid deletions, the following nomenclature is used: original amino acid, position, *. Thus, a deletion of glycine at position 195 would be designated "G195*."

[0039] Insertion: For amino acid insertions, the following nomenclature is used: original amino acid, position, original amino acid, inserted amino acid. Thus, an insertion of a lysine after the glycine at position 195 would be designated "G195GK." Reference to an insertion at a particular position is understood to be the insertion after the original amino acid residue. For example, "insertion at position 195" is understood to be the insertion after the original residue at position 195.

[0040] Multiple Changes: Variants containing multiple changes are separated by a plus sign ("+") or a comma, for example, "R170Y+G195E" or "R170Y,G195E" represent the substitution of arginine and glycine at positions 170 and 195 with tyrosine and glutamic acid, respectively.

[0041] Various modifications: Where various modifications can be introduced at a position, the various modifications may be separated by commas, for example, "R170Y,E" represents the substitution of arginine at position 170 with tyrosine or glutamic acid. Thus, "Y167G,A+R170G,A" represents the following variants: "Y167G+R170G", "Y167G+R170A", "Y167A+R170G", and "Y167A+R170A".

[0042] Medical Device: The term "medical device" is intended to refer broadly to any type of medical or dental device, equipment, or equipment that comes into contact with a patient, whether the patient is a human or an animal. Medical devices include devices, instruments, tools, implements, and equipment used in medical or surgical procedures, e.g., for diagnosis or surgery, e.g., in dentistry and veterinary medicine (e.g., those that can be non-heat sterilized, soaked or washed and then heat sterilized, or that can otherwise benefit from cleaning as described herein). Non-limiting examples of medical devices include surgical and diagnostic instruments, such as trays, pans, holders, racks, forceps, scissors, shears, saws (e.g., bone saws and blades), hemostats, knives, chisels, rongeurs, rasps, nippers, drills, bits, rasps, borers, spreaders, breakers, elevators, clamps, needle holders, carriers, clips, hooks, gouges, curettes, retractors, straighteners, punches, ventilators, scoops, keratomes, spatulas, expressors, trocars, dilators, cages, glassware, tubes, catheters, cannulas, plugs, stents, endoscopes, arthroscopes, and related equipment, or combinations thereof. Other examples of medical devices include surgical instruments, such as scalpels, hemostats, Kocher forceps, and tracheotomy instruments.

[0043] The medical device may be an indwelling device, for example a catheter such as a central venous catheter, an intravascular catheter, a urethral catheter, a Hickman catheter, a peritoneal dialysis catheter, or an endotracheal catheter, or a device such as a mechanical heart valve, a cardiac pacemaker, an arteriovenous shunt, a scleral buckle, an artificial joint, a tympanic ventilation tube, a tracheostomy tube, a voice prosthesis, an artificial penis, an artificial urinary sphincter, a synthetic pubovaginal sling, a suture, a bone anchor, a bone screw, an intraocular lens, a contact lens, an intrauterine contraceptive device, an aortofemoral graft, a vascular graft, a needle, a Luer-Lok connector, or a needleless connector.

[0044] The term "at least a part of a medical device" should be understood as the part of the device that will come into contact with the patient, or for example the interior of an endoscope that will come into contact with a fluid or sample that comes into contact with the patient.

[0045] Cleaning Solution: The term "cleaning solution" is intended to mean a solution or mixture of water and / or another solvent and at least one cleaning component, such as a surfactant, used to clean at least some surfaces of a medical device.

[0046] Detailed Description of the Invention The inventors have surprisingly found that cleaning a medical device with two or more enzymes selected from the group consisting of a protease, an enzyme with DNase activity, and an enzyme with hexosaminidase activity confers synergistic cleaning performance, particularly with respect to reducing / removing biofilm, for example from the medical device.

[0047] Thorough cleaning of medical devices, such as endoscopes or surgical instruments, is important to ensure that the medical devices are properly disinfected and sterilized. Residual inorganic and organic materials, such as biofilms, can interfere with the effectiveness of the disinfection and sterilization processes. As a result, infectious pathogens may still live and be present on the medical device after cleaning, disinfection, and sterilization, and may be transmitted to other patients.

[0048] The present invention addresses these challenges by providing a cleaning composition comprising two or more different enzymes that have been found to have synergistic cleaning performance for cleaning medical devices, and thereby can provide deep cleaning benefits to medical devices and greatly reduce the risk of transmitting infectious pathogens from one patient to another.

[0049] Thus, one aspect of the present invention is a method for cleaning an article, e.g., a medical device, comprising: (a) contacting the medical device with a cleaning solution comprising two or more enzymes selected from the group consisting of a protease, an enzyme having DNase activity, and an enzyme having hexosaminidase activity; and optionally (b) rinsing the medical device; The present invention relates to a method comprising the steps of:

[0050] The cleaning performance of a method or composition of the invention can be evaluated by measuring the percentage removal of biofilm present on a medical device compared to cleaning without the enzyme, e.g., as shown in Example 1.

[0051] In one embodiment, the amount of biofilm present on the medical device after cleaning is reduced by at least 1%, e.g., at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, or at least 40%, compared to cleaning with a cleaning solution without the two or more enzymes.

[0052] For example, for the purpose of promoting cleaning performance or sterilization effect, the method may further include a step of pretreating the medical device with a disinfectant before the above-mentioned step a). Suitable disinfectants may include, for example, peracetic acid, hydrogen peroxide, potassium permanganate, chlorine dioxide, and ethanol. Pretreatment may be performed by immersing or contacting the medical device with a solution containing the disinfectant.

[0053] In one embodiment, the medical device is rinsed with water, hi another embodiment, the medical device is rinsed with a solution containing a disinfectant as described above.

[0054] In one embodiment, the medical device is an endoscope, examples of which may include cystoscopes, nephroscopes, bronchoscopes, laryngoscopes, otoscopes, arthroscopes, laparoscopes, gastrointestinal endoscopes, and the like.

[0055] In one embodiment, the medical device is a surgical instrument, such as a scalpel, a hemostat, a forceps, scissors, a retractor, a tracheotomy instrument, and a clamp.

[0056] In another embodiment, the medical device is a dental instrument used, for example, by a dentist, to remove a tooth or to identify and select a tooth treatment. Examples of such dental instruments may be a scale, a curette, a cotton pliers with serrations, a dental mirror, etc.

[0057] Biofilms present on medical devices can be found among many microorganisms, for example the following species: Enterobacteriacae (e.g. Escherichia coli), Klebsiella pneumoniae, Salmonella spp., Mycobacterium spp., Enterococcus faecalis, Enterobacter cloacae, Proteus mirabilis, Serratia marcescens, Staphylococcus aureus, e.g. Staphylococcus aureus ATCC 6538.In one embodiment, the medical device is selected from the group consisting of Enterobacteriacae (e.g., Escherichia coli), Klebsiella pneumoniae, Salmonella spp., Mycobacterium spp., Enterococcus faecalis, Enterobacter cloacae, Proteus mirabilis, Serratia marcescens, Staphylococcus aureus, e.g., Staphylococcus aureus ATCC 61161 ... 6538, Streptococcus spp. (e.g., S. pyogenes, S. agalactiae, or S. pneumoniae), Haemophilus influenzae, Pseudomonas aeruginosa, Clostridium perfringens, Chlamydia trachomatis, Candida albicans, Bacillus anthracis, or a combination thereof.

[0058] The concentration of each enzyme in the cleaning solution may be in the range of 0.0005 to 100 ppm of enzyme protein, for example, in the range of 0.001 to 50 ppm, in the range of 0.005 to 20 ppm, in the range of 0.01 to 10 ppm, in the range of 0.1 to 15 ppm, or in the range of 0.05 to 5 ppm of enzyme protein.

[0059] Another aspect of the invention provides a composition for cleaning a medical device, the cleaning composition comprising a surfactant and two or more enzymes selected from the group consisting of a protease, an enzyme having DNase activity, and an enzyme having hexosaminidase activity.

[0060] The amount of each enzyme of the present invention to be included in the cleaning composition is at least 0.0001 mg of enzyme protein per gram of composition, e.g., at least 0.001 mg of enzyme protein, at least 0.006 mg of enzyme protein, at least 0.008 mg of enzyme protein, at least 0.01 mg of enzyme protein, at least 0.1 mg of enzyme protein, at least 0.5 mg of enzyme protein, at least 1 mg of enzyme protein, at least 2 mg of enzyme protein, at least 5 mg of enzyme protein, at least 10 mg of enzyme protein, or at least 20 mg of enzyme protein.

[0061] Another aspect of the invention relates to the use of an enzyme composition for cleaning a medical device, such as an endoscope, a surgical instrument, or a dental instrument, the enzyme composition comprising two or more enzymes selected from the group consisting of a protease, an enzyme having DNase activity, and an enzyme having hexosaminidase activity.

[0062] Undesirable microorganisms (e.g., pathogenic bacteria) are not only present in medical equipment, but can also appear in biological manufacturing equipment (e.g., food and beverage production, and biotechnology manufacturing, e.g., enzymatic fermentation processes), such as fermentation and storage tanks, bioreactors, ultrafilters or membranes, pipelines, and other equipment. Such equipment is typically cleaned by cleaning-in-place (CIP) methods, where cleaning is performed without removing or disassembling piping or other equipment.

[0063] Thus, in another aspect, the present invention provides a CIP (clean in place) cleaner comprising two or more enzymes selected from the group consisting of a protease, an enzyme with DNase activity, and an enzyme with hexosaminidase activity.

[0064] The present invention further relates to a CIP cleaning method comprising performing a clean-in-place with two or more enzymes selected from the group consisting of a protease, an enzyme having DNase activity, and an enzyme having hexosaminidase activity; and optionally including a rinsing step.

[0065] Enzymes of the Invention In one embodiment, the DNase according to the invention is an enzyme of the enzyme class EC 3.1, preferably EC 3.1.21, such as EC 3.1.21.X (where X=1, 2, 3, 4, 5, 6, 7, 8, or 9, e.g., DNase I, DNase IV, type I site-specific DNase, type II site-specific DNase, type III site-specific DNase, CC-selective endo-DNase, In one embodiment, the nucleic acid is selected from EC 3.1.22.Y (where Y=1, 2, 4, or 5, e.g., DNase II, Aspergillus DNase K(1), crossover junction endodeoxyribonuclease, DNase X).

[0066] Preferably, the polypeptide having DNase activity is obtained from a microorganism and the DNase is a microbial enzyme. The DNase is preferably of fungal or bacterial origin or a variant of a DNase of fungal or bacterial origin. In one embodiment, a DNase obtained from a fungus, such as Aspergillus oryzae, or a variant thereof is used.

[0067] Suitable bacterial DNases may be obtained, for example, from species of the genus Bacillus and related genera (see Patel and Gupta, Int. J. Syst. Evol. Microbiol. 2020;70:406-438 (which proposed six new genera of the family Bacillaceae from species previously classified as belonging to the genus Bacillus)), such as from the genera Bacillus, Cytobacillus, Metabacillus, Alkalihalobacillus, Rossellomorea, or Mesobacillus. Examples of species from which DNase may be obtained include Bacillus licheniformis, Bacillus subtilis, Sutcliffiella horikoshii, Cytobacillus horneckiae, Metabacillus indicus, Alkalihalobacillus algicola, Rossellomorea vietnamensis, Alkalihalobacillus hwajinpoensis, Mesobacillus campisalis, Bacillus idriensis, Bacillus Preferred bacterial DNases include those obtained from Metabacillus indicus (formerly known as Bacillus cibi) and variants thereof.

[0068] In one embodiment, the DNase of the invention may be a polypeptide having DNase activity and having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO: 1. The enzyme may, for example, differ from the mature polypeptide of SEQ ID NO: 1 by up to 20 amino acids, up to 15 amino acids, or up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0069] In one embodiment, the DNase of the invention may be a polypeptide having DNase activity and having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO: 2. The enzyme may, for example, differ from the mature polypeptide of SEQ ID NO: 2 by up to 20 amino acids, up to 15 amino acids, or up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0070] In one embodiment, the DNase of the invention may be a polypeptide having DNase activity and having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO: 3. The enzyme may, for example, differ from the mature polypeptide of SEQ ID NO: 3 by up to 20 amino acids, up to 15 amino acids, or up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0071] In one embodiment, the DNase of the invention may be a polypeptide having DNase activity and having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO: 4. The enzyme may, for example, differ from the mature polypeptide of SEQ ID NO: 4 by up to 20 amino acids, up to 15 amino acids, or up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0072] In one embodiment, the DNase of the present invention preferably belongs to the NUC1 group of DNases. DNases of the NUC1 group include polypeptides that, in addition to having DNase activity, may contain one or more of the motifs [T / D / S][G / N]PQL (SEQ ID NO: 23) (corresponding to positions 116-120 of SEQ ID NO: 4), [F / L / Y / I]A[N / R]D[L / I / P / V] (SEQ ID NO: 24) (corresponding to positions 111-115 of SEQ ID NO: 4), and C[D / N]T[A / R] (SEQ ID NO: 25) (corresponding to positions 44-47 of SEQ ID NO: 4). DNases of this group preferably further contain the NUC1_A domain [D / Q][I / V]DH (SEQ ID NO: 26) (corresponding to positions 85-88 of SEQ ID NO: 4).

[0073] In one embodiment, the DNase of the present invention preferably belongs to a group of DNases contained in the GYS-clade, which is a group of DNases on the same branch of a phylogenetic tree that have both structural and functional similarities. These NUC1 and / or NUC1_A DNases contain the conserved motifs [D / M / L][S / T]GYSR[D / N] (SEQ ID NO: 27) and / or ASXNRSKG (SEQ ID NO: 28) and share similar structural and functional properties. In the present invention, the motif [D / M / L][S / T]GYSR[D / N] (SEQ ID NO: 27) corresponds to positions 26-32 of SEQ ID NO: 4, and the motif ASXNRSKG (SEQ ID NO: 28) corresponds to positions 125-132 of SEQ ID NO: 4. DNases of the GYS-clade are preferably obtained from species of the genus Bacillus and related genera (see Patel and Gupta, supra).

[0074] In one embodiment, the polypeptide having DNase activity according to the invention comprises one or more motifs (e.g., [T / D / S][G / N]PQL (SEQ ID NO: 23), [F / L / Y / I]A[N / R]D[L / I / P / V] (SEQ ID NO: 24), C[D / N]T[A / R] (SEQ ID NO: 25), [D / Q][I / V]DH (SEQ ID NO: 26), [D / M / L][S / T]GYSR[D / N] (SEQ ID NO: 27), and ASXNRSKG (SEQ ID NO: 28). For example, 2, 3, 4, 5, or 6) and the polypeptide has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO:4.

[0075] In one embodiment, a polypeptide having DNase activity according to the invention comprises one or both motifs [D / M / L][S / T]GYSR[D / N] (SEQ ID NO: 27) and ASXNRSKG (SEQ ID NO: 28), and optionally further comprises a member selected from the group consisting of: [T / D / S][G / N]PQL (SEQ ID NO: 23), [F / L / Y / I]A[N / R]D[L / I / P / V] (SEQ ID NO: 24), C[D / N]T[A / R] (SEQ ID NO: 25), [D / Q][I / V]DH (SEQ ID NO: 26). The polypeptide may comprise one or more (e.g., two, three, four) of the selected motifs, and the polypeptide has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO:4.

[0076] In one embodiment, the enzyme with DNase activity is S26*, D32E,Q, V35I, K36C,H, G37R, F43W, D46G, A55I, N68D, S69V, A76I, K82S,T, P84D,T, K86E,G,L,N,Q,T,V,Y, A91R, L92E, K95I, P97E,N, A101E, Q102E, K105N,G,Q,T,D , A111P, F112Y,W, S115T, V127T, L129K, N133Q, G137R, V138C, N140H, G141Q,R, S144E, N146A, K147N ,E, V148I, A149D,E,F, Q150D,E, P153D,V, S154E, K155E,F,L,S,T, Q157D,E, Q158D, T159Q, K160D, G 161R, T170Q, A172D,E,H,R, G181N, K185*, V187N,Y, N191*, K192A,I, D197K,S, G199Q, Q208V, E211Y,T,P, N213S, N214D, N217A, and Y218D,E, wherein the position numbers correspond to the positions of SEQ ID NO:2, and the variant has at least 80%, e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, and less than 100%, sequence identity to the polypeptide of SEQ ID NO:2 or 3.

[0077] In one embodiment, the enzyme having DNase activity has the amino acid sequence of SEQ ID NO:2, which comprises the substitution D32E and one or more alterations selected from the group consisting of V35I, S69V, K86E, Q102E, K105N, A111P, S115T, V127T, G137R, K147N, Q150E, K155E, T159Q, G161R, A172D, G181N, V187N, K192I, K192A, Q208V, and N217A. wherein the position numbers correspond to positions in SEQ ID NO:2, and the variant has at least 80%, e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, and less than 100%, sequence identity to the polypeptide of SEQ ID NO:2 or 3.

[0078] In one embodiment, the enzyme having DNase activity has the amino acid sequence of SEQ ID NO:2, which comprises the substitution V35I and one or more alterations selected from the group consisting of D32E, S69V, K86E, Q102E, K105N, A111P, S115T, V127T, G137R, K147N, Q150E, K155E, T159Q, G161R, A172D, G181N, V187N, K192I, K192A, Q208V, and N217A. wherein the position numbers correspond to positions in SEQ ID NO:2, and the variant has at least 80%, e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, and less than 100%, sequence identity to the polypeptide of SEQ ID NO:2 or 3.

[0079] In one embodiment, the enzyme having DNase activity has the structure of SEQ ID NO:2, which comprises the substitution K105N and one or more alterations selected from the group consisting of D32E, V35I, S69V, K86E, Q102E, A111P, S115T, V127T, G137R, K147N, Q150E, K155E, T159Q, G161R, A172D, G181N, V187N, K192I, K192A, Q208V, and N217A. wherein the position numbers correspond to positions in SEQ ID NO:2, and the variant has at least 80%, e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, and less than 100%, sequence identity to the polypeptide of SEQ ID NO:2 or 3.

[0080] In one embodiment, the enzyme having DNase activity comprises the substitution A111P and one or more alterations selected from the group consisting of D32E, V35I, S69V, K86E, Q102E, K105N, S115T, V127T, G137R, K147N, Q150E, K155E, T159Q, G161R, A172D, G181N, V187N, K192I, K192A, Q208V, and N217A. wherein the position numbers correspond to positions in SEQ ID NO:2, and the variant has at least 80%, e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, and less than 100%, sequence identity to the polypeptide of SEQ ID NO:2 or 3.

[0081] In one embodiment, the enzyme with DNase activity comprises at least two substitutions selected from D32E, V35I, S69V, Q102E, K105N, A111P, S115T, G161R, and G181N, and further selected from the group consisting of K65E, K67A, K86E, V127T, G137R, K147N, Q150E, K155E, T159Q, A172D, V187N, K192I, K192A, Q208V, and N217A. a variant of SEQ ID NO:2 comprising one or more alterations as set forth above, wherein the position numbers correspond to positions in SEQ ID NO:2, and the variant has at least 80%, e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to the polypeptide of SEQ ID NO:2 or 3.

[0082] In one embodiment the DNase variant comprises or consists of SEQ ID NO:2 with the substitutions S69V+Q102E+K105N+A111P+S115T+Q150E+G161R+G181N+V187N+K192I. In another embodiment the DNase variant comprises or consists of SEQ ID NO:2 with the substitutions D32E+V35I+S69V K86E+Q102E+K105N+A111P+S115T+V127T+G137R+K147N+Q150E+K155E T159Q+G161R+A172D+G181N+V187N+K192A+Q208V+N217A.

[0083] In another embodiment, the enzyme having DNase activity is a variant of SEQ ID NO:4 comprising one or more alterations at positions 1, 13, 22, 25, 27, 33, 39, 42, 56, 57, 59, 65, 76, 77, 109, 116, 127, 144, 147, 149, 167, 175, and 181 of SEQ ID NO:4, wherein the variant has at least 80%, e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, and less than 100%, sequence identity to the polypeptide of SEQ ID NO:4. In a preferred embodiment, the DNase variant comprises one or more of the substitutions T1I, S13Y, T22P, S25P, S27L, L33K, S39P, S42G, D56I, S57W, S59V, T65V, V76L, T77Y, Q109R, S116D, T127V, S144P, A147H, G149N, S167L, G175D, and S181L of SEQ ID NO:4.

[0084] In certain embodiments, the DNase variant comprises or consists of SEQ ID NO:4 with the substitutions T1I, S13Y, T22P, S27L, L33K, S39P, S42G, D56I, S57W, S59V, T65V, V76L, Q109R, S116D, T127V, S144P, A147H, S167L, and G175D. In another embodiment the DNase variant comprises or consists of SEQ ID NO: 4 with the following substitutions: T1I+S13Y+T22P+S25P+S27L+S39P+S42G+S57W+S59V+T65V+V76L+T77Y+Q109R+S116D+S144P+A147H+G149N+S167L+G175D+S181L.

[0085] In one aspect an enzyme having hexosaminidase activity according to the present invention which is useful for the extensive cleaning of medical devices is selected from polypeptides having at least 60%, such as at least 65%, for example at least 70%, such as at least 75%, for example at least 80%, such as at least 85%, for example at least 90%, such as at least 91%, for example at least 92%, such as at least 93%, for example at least 94%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, for example at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO: 10, 11, 19, 20, 21 or 22, preferably wherein the hexosaminidase has N-acetylglucosaminidase activity and / or β-N-acetylglucosaminidase activity.

[0086] In one embodiment the enzyme having hexosaminidase activity according to the invention is obtained from the Terribacillus clade (e.g. from Terribacillus saccharophilus) and has at least 60%, such as at least 65%, for example at least 70%, such as at least 75%, for example at least 80%, such as at least 85%, for example at least 90%, such as at least 91%, for example at least 92%, such as at least 93%, for example at least 94%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, for example at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO:10 or SEQ ID NO:19, preferably the hexosaminidase has N-acetylglucosaminidase activity and / or β-N-acetylglucosaminidase activity.

[0087] In one embodiment the enzyme having hexosaminidase activity according to the present invention is obtained from the genus Lactobacillus, such as Lactobacillus paraplantarum and has at least 60%, such as at least 65%, for example at least 70%, such as at least 75%, for example at least 80%, such as at least 85%, for example at least 90%, such as at least 91%, for example at least 92%, such as at least 93%, for example at least 94%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, such as at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO: 11, preferably the hexosaminidase has N-acetylglucosaminidase activity and / or β-N-acetylglucosaminidase activity.

[0088] In one embodiment the enzyme having hexosaminidase activity according to the invention is obtained from the genus Staphylococcus, such as Staphylococcus cohnii and has at least 60%, such as at least 65%, for example at least 70%, such as at least 75%, for example at least 80%, such as at least 85%, for example at least 90%, such as at least 91%, for example at least 92%, such as at least 93%, for example at least 94%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, for example at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO: 20, preferably the hexosaminidase has N-acetylglucosaminidase activity and / or β-N-acetylglucosaminidase activity.

[0089] In one embodiment the enzyme having hexosaminidase activity according to the invention is obtained from the genus Staphylococcus, such as Staphylococcus fleurettii and has at least 60%, such as at least 65%, for example at least 70%, such as at least 75%, for example at least 80%, such as at least 85%, for example at least 90%, such as at least 91%, for example at least 92%, such as at least 93%, for example at least 94%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, such as at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO: 21, preferably the hexosaminidase has N-acetylglucosaminidase activity and / or β-N-acetylglucosaminidase activity.

[0090] In one embodiment the enzyme having hexosaminidase activity according to the invention is obtained from the genus Aggregatibacter, such as Aggregatibacter actinomycetemcomitans and has at least 60%, such as at least 65%, for example at least 70%, such as at least 75%, for example at least 80%, such as at least 85%, for example at least 90%, such as at least 91%, for example at least 92%, such as at least 93%, for example at least 94%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, such as at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO: 22, preferably the hexosaminidase has N-acetylglucosaminidase activity and / or β-N-acetylglucosaminidase activity.

[0091] In one embodiment, an enzyme with hexosaminidase activity according to the invention may comprise a structural domain of Glyco_hydro_20, such as GH20. A polypeptide comprising a GH20 domain may comprise several motifs. An example is the motif GXDE (SEQ ID NO: 12), located within positions corresponding to positions 158-161 in SEQ ID NO: 10. Residues D and E are the main catalytic residues of GH20 (positions 160 and 161 in SEQ ID NO: 10). GH20 polypeptides may be divided into a number of different subclusters or clades, as shown in WO 2017 / 186943 (hereby incorporated by reference). Examples of specific domains are listed below.

[0092] A further domain is designated IAS, and polypeptides of this domain are characterized by having hexosaminidase activity, e.g. PNAG activity, as well as containing specific motifs, e.g. [EQ][NRSHA][YVFL][AGSTC][IVLF][EAQYN][SN] corresponding to ESYAIAS at positions 44 to 50 of SEQ ID NO: 10 (SEQ ID NO: 13).

[0093] Another domain, preferably shared by the hexosaminidase polypeptides of the present invention, is designated WND. Polypeptides of this domain preferably contain a GH20 domain, are of bacterial origin, and are characterized by containing a specific motif in addition to having PNAG activity. Polypeptides with a WND domain may contain the motif [VIM][LIV]G[GAV]DE[VI][PSA] (SEQ ID NO: 14) corresponding to positions 156-163 of SEQ ID NO: 10, where G (corresponding to position 158 of SEQ ID NO: 10) is completely conserved within the Terribacillus clade, and residues D and E are the key catalytic residues of GH20 (positions 160 and 161 of SEQ ID NO: 10). Another motif that may be comprised by a polypeptide of the invention is WND[SQR][IVL][TLVM] (SEQ ID NO: 15) (positions 193-198 in SEQ ID NO: 10), where W (position 193 in SEQ ID NO: 10) is part of the active site pocket and is predicted to be involved in binding the N-acetyl group of the PNAG substrate.

[0094] Polypeptides of the Terribacillus clade can be further subdivided into a clade designated QSTL, which contains WND domain polypeptides of bacterial origin with PNAG activity. Polypeptides of this clade contain an exemplary motif QSTL (SEQ ID NO:16) (corresponding to positions 216-219 of SEQ ID NO:10), in which all four amino acids are completely conserved in the QSTL clade and are predicted to be involved in substrate binding. Another motif that may be contained by polypeptides of the QSTL clade is NKFFY (SEQ ID NO:17) (positions 273-277 of SEQ ID NO:10). A further motif that may be contained by polypeptides of the QSTL clade is NLD[DR]S (SEQ ID NO:18) (positions 204-208 of SEQ ID NO:10).

[0095] In one embodiment, a polypeptide having hexosaminidase activity according to the invention comprises the motif GXDE (SEQ ID NO: 12). In one embodiment, a polypeptide having hexosaminidase activity according to the invention comprises the motif [EQ][NRSHA][YVFL][AGSTC][IVLF][EAQYN][SN] (SEQ ID NO: 13). In one embodiment, a polypeptide having hexosaminidase activity according to the invention comprises the motif [VIM][LIV]G[GAV]DE[VI][PSA] (SEQ ID NO: 14). In one embodiment, a polypeptide having hexosaminidase activity according to the invention comprises the motif WND[SQR][IVL][TLVM] (SEQ ID NO: 15). In one embodiment, a polypeptide having hexosaminidase activity according to the invention comprises the motif QSTL (SEQ ID NO: 16). In one embodiment, a polypeptide having hexosaminidase activity according to the invention comprises the motif NKFFY (SEQ ID NO: 17). In one embodiment, a polypeptide having hexosaminidase activity according to the invention comprises the motif NLD[DR]S (SEQ ID NO: 18).

[0096] In one embodiment, a polypeptide having hexosaminidase activity according to the invention comprises one or more of the motifs GXDE (SEQ ID NO: 12), [EQ][NRSHA][YVFL][AGSTC][IVLF][EAQYN][SN] (SEQ ID NO: 13), [VIM][LIV]G[GAV]DE[VI][PSA] (SEQ ID NO: 14), WND[SQR][IVL][TLVM] (SEQ ID NO: 15), QSTL (SEQ ID NO: 16), NKFFY (SEQ ID NO: 17), NLD[DR]S (SEQ ID NO: 18), and the polypeptide has hexosaminidase activity, preferably N- and having acetylglucosaminidase activity and / or β-N-acetylglucosaminidase activity, wherein the polypeptide has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO:10, 11, 19, 20, 21, or 22.

[0097] In one embodiment, a polypeptide having hexosaminidase activity according to the invention comprises two, three, four, five, six or all seven of the motifs GXDE (SEQ ID NO: 12), [EQ][NRSHA][YVFL][AGSTC][IVLF][EAQYN][SN] (SEQ ID NO: 13), [VIM][LIV]G[GAV]DE[VI][PSA] (SEQ ID NO: 14), WND[SQR][IVL][TLVM] (SEQ ID NO: 15), QSTL (SEQ ID NO: 16), NKFFY (SEQ ID NO: 17), NLD[DR]S (SEQ ID NO: 18), and the polypeptide has hexosaminidase activity, preferably or N-acetylglucosaminidase activity and / or β-N-acetylglucosaminidase activity, and the polypeptide has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO:10, 11, 19, 20, 21, or 22.

[0098] In another embodiment, the hexosaminidase is a variant of the polypeptide of SEQ ID NO: 10 comprising one or more alterations at positions 3, 15, 49, 59, 163, 186, 225, 227, 232, 235, 252, 260, 272, 279, 281, 308, 309, and 312 of SEQ ID NO: 10, wherein the variant has hexosaminidase activity, preferably N-acetylglucosaminidase activity and / or β-N-acetylglucosaminidase activity, and the variant has at least 80%, e.g. at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, and less than 100%, sequence identity to the mature polypeptide of SEQ ID NO: 10. More preferably, the variant has one or more of the motifs GXDE (SEQ ID NO: 12), [EQ][NRSHA][YVFL][AGSTC][IVLF][EAQYN][SN] (SEQ ID NO: 13), [VIM][LIV]G[GAV]DE[VI][PSA] (SEQ ID NO: 14), WND[SQR][IVL][TLVM] (SEQ ID NO: 15), QSTL (SEQ ID NO: 16), NKFFY (SEQ ID NO: 17), NLD[DR]S (SEQ ID NO: 18).

[0099] In one embodiment, the hexosaminidase according to the invention is a variant of the polypeptide of SEQ ID NO: 10 comprising one or more of the substitutions Q3I, H15Y, A49W, N59E, S163P, S186R, S225G, N227T, E232D, G235W, N252P, N260Q, H272V, S279D, Y281P, K308Q, K309E, and K312Q of SEQ ID NO: 10, wherein the variant has hexosaminidase activity, preferably N-acetylglucosidase activity. and / or β-N-acetylglucosaminidase activity, the variant having at least 80%, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to the mature polypeptide of SEQ ID NO: 10. More preferably, the variant has one or more of the motifs GXDE (SEQ ID NO: 12), [EQ][NRSHA][YVFL][AGSTC][IVLF][EAQYN][SN] (SEQ ID NO: 13), [VIM][LIV]G[GAV]DE[VI][PSA] (SEQ ID NO: 14), WND[SQR][IVL][TLVM] (SEQ ID NO: 15), QSTL (SEQ ID NO: 16), NKFFY (SEQ ID NO: 17), NLD[DR]S (SEQ ID NO: 18).

[0100] In one particular embodiment, the hexosaminidase variant comprises or consists of SEQ ID NO: 10 with the substitutions Q3I, H15Y, A49W, N59E, S163P, S186R, S225G, N227T, E232D, G235W, N252P, N260Q, H272V, S279D, Y281P, K308Q, K309E, and K312Q.

[0101] The protease suitable for the present invention may be of any origin, but preferably of bacterial or fungal origin, optionally in the form of a protein engineered or chemically modified variant. The protease may be an alkaline protease, such as a serine protease or a metalloprotease. The serine protease may be, for example, from the S1 family, such as trypsin, or from the S8 family, such as subtilisin. The metalloprotease may be, for example, thermolysin, for example from the M4 family, or another metalloprotease, for example from the M5, M7, or M8 family.

[0102] The term "subtilase" refers to a subgroup of serine proteases according to Siezen et al., Protein Eng. 4 (1991) 719-737 and Siezen et al., Protein Sci. 6 (1997) 501-523. Serine proteases are a subgroup of proteases characterized by having a serine in the active site that forms a covalent adduct with the substrate. Subtilases can be divided into six subdivisions: the subtilisin family, thermitase family, proteinase K family, lantibiotic peptidase family, kexin family, and pyrrolysin family.

[0103] Although proteases suitable for detergent use can be obtained from a variety of organisms, including fungi such as the genus Aspergillus, detergent proteases are generally obtained from bacteria, particularly from the genus Bacillus and related genera (see Patel and Gupta, supra). Examples of Bacillus species from which subtilases are derived include Bacillus lentus, Bacillus alkalophilus, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus pumilus, and Bacillus gibsonii. Particular subtilisins include subtilisin lentus, subtilisin novo, subtilisin Carlsberg, subtilisin BPN', subtilisin 309, subtilisin 147, and subtilisin 168, as well as, for example, protease PD138 (described in WO 93 / 18140). Other useful proteases include, for example, those described in WO 01 / 16285 and WO 02 / 16547.

[0104] Examples of trypsin-like proteases include the Fusarium proteases described in WO 94 / 25583 and WO 2005 / 040372, and the chymotrypsin proteases from the Cellulomonas genus described in WO 2005 / 052161 and WO 2005 / 052146.

[0105] Examples of metalloproteases include the neutral metalloproteases described in WO 2007 / 044993, such as those derived from Bacillus amyloliquefaciens, and the metalloproteases described in WO 2015 / 158723 and WO 2016 / 075078.

[0106] Examples of useful proteases include those described in WO 89 / 06279, WO 92 / 19729, WO 96 / 34946, WO 98 / 20115, WO 98 / 20116, WO 99 / 11768, WO 01 / 44452, WO 03 / 006602, WO These include the protease variants described in WO 2004 / 003186, WO 2004 / 041979, WO 2007 / 006305, WO 2011 / 036263, WO 2014 / 207227, WO 2016 / 087617 and WO 2016 / 174234. Preferred protease variants are, for example: S3T, V4I, S9R, S9E, A15T, S24G, S24R, K27R, N42R, S55P, G59E, G59D, N60D, N60E, V66A, N74D, S85R, A96S, S97G, S97D, S97A, S97SD, S99E, S99D, S99G, S99M, S99N, S99R, S99H, S101A, V102I, V102Y, V102N, S104A, G116V, G116R, H118D, H118N, A120S, S126L, P127Q, S128A, S154D, A156E, G157D, G157P, S158E, Y161A, R164S, Q176E, N179E, S182E, Q185N, A188P, G189E, V193M, N198D, V199I, Q200L , Y203W, S206G, L211Q, L211D, N212D, N212S, M216S, A226V, K229L, Q230H, Q239R, N246K, S253D, N255W, N255D, N255E, L256E, L256D and R269H, wherein the position numbers correspond to the positions of the Bacillus lentus protease shown in SEQ ID NO: 1 of WO 2016 / 001449.A protease variant having one or more of these mutations is preferably a variant of Bacillus lentus protease (Savinase®, also known as subtilisin 309) as set forth in SEQ ID NO: 1 of WO 2016 / 001449, or a variant of Bacillus amyloliquefaciens protease (BPN') as set forth in SEQ ID NO: 2 of WO 2016 / 001449. Such a protease variant preferably has at least 80% sequence identity to SEQ ID NO: 1 or to SEQ ID NO: 2 of WO 2016 / 001449.

[0107] Another protease of interest is the alkaline protease from Bacillus lentus DSM 5483, as described, for example, in WO 91 / 02792, and variants thereof as described, for example, in WO 92 / 21760, WO 95 / 23221, EP 1921147, EP 1921148, and WO 2016 / 096711.

[0108] Alternatively, the protease may be a variant of the TY145 protease having SEQ ID NO:1 of WO 2004 / 067737, for example a variant comprising substitutions at one or more positions corresponding to positions 27, 109, 111, 171, 173, 174, 175, 180, 182, 184, 198, 199 and 297 of SEQ ID NO:1 of WO 2004 / 067737, said protease variant having at least 75% and less than 100% sequence identity to SEQ ID NO:1 of WO 2004 / 067737. TY145 variants of interest are described, for example, in WO 2015 / 014790, WO 2015 / 014803, WO 2015 / 014804, WO 2016 / 097350, WO 2016 / 097352, WO 2016 / 097357, and WO 2016 / 097354.

[0109] In some embodiments, a preferred protease according to the invention may be a protease having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO:5, 6, 7, 8, 9, or 29.

[0110] In another embodiment, a preferred protease according to the invention may be a variant of the polypeptide of SEQ ID NO: 6 comprising an alteration at one or more positions corresponding to positions 3, 4, 9, 15, 43, 68, 76, 99, 101, 103, 104, 160, 167, 170, 194, 199, 205, 206, 209, 217, 218, 222, 245, 261, and 262, where the position numbers correspond to positions in SEQ ID NO: 5 and each alteration is independently a substitution, deletion, or insertion, and the variant has protease activity and has at least 80%, e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, and less than 100%, sequence identity to the polypeptide of SEQ ID NO: 6.

[0111] In one embodiment, the protease of the present invention is selected from the group consisting of: S3T, V4I, S9E, S9R, A15T, V68A, N76D, S99D, S99G, S99A, S99SE, S101E, S101N, S101R, S103A, V104I, G160S, Y167A, R170S, A194P, V199M, V205I, Q206L, Y209W, L217D, L217Q, N218D, M222S, Q245R, N261W, and L262E. A variant of the polypeptide of SEQ ID NO:6 comprising one or more substitutions, wherein the position numbers correspond to the positions of SEQ ID NO:5, wherein the variant has protease activity and has at least 80%, e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and less than 100% sequence identity to SEQ ID NO:6.

[0112] In one embodiment, the protease of the present invention is: S3T, V4I, S9E, S9R, A15T, T22A, N43R, V68A, N76D, S87N, S99D, S99G, S99A, S99SE, S101E, S101N, S101R, S103A, V104I, G118M, S128Q, G160S, Y167A, R170S, N184E, A194P, V199M, V205I, Q206L, Y209W, L217D, L217Q, N218D, M222S, Q245R, S259D, N261W and L262E, wherein the position numbers correspond to positions in SEQ ID NO:5, and the variant has protease activity and has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and less than 100% sequence identity to SEQ ID NO:6.

[0113] In one embodiment, the protease of the invention is a variant of the polypeptide of SEQ ID NO: 6 comprising the substitution S87N, wherein the variant has protease activity, and the position corresponds to the position of SEQ ID NO: 5, wherein the variant has protease activity and has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, and less than 100%, sequence identity to SEQ ID NO: 6.

[0114] In one embodiment, the protease of the invention is a variant of the polypeptide of SEQ ID NO: 6 comprising the substitutions Y167A+R170S+A194P, where the position numbers correspond to the positions of SEQ ID NO: 5, wherein the variant has protease activity and has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, and less than 100%, sequence identity to SEQ ID NO: 6.

[0115] In one embodiment, the protease of the invention is a variant of the polypeptide of SEQ ID NO:6 comprising the substitutions S9E+N43R+N76D+V205I+Q206L+Y209W+S259D+N261W+L262E, where the position numbers correspond to the positions of SEQ ID NO:5, wherein the variant has protease activity and has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, and less than 100%, sequence identity to SEQ ID NO:6.

[0116] In one embodiment, the protease of the invention is a variant of the polypeptide of SEQ ID NO: 6 comprising the substitutions S3T+N43R+N76D+S87N+G118M+S128Q+N184E+V205I+Q206L+Y209W+S259D+N261W+L262E, where the position numbers correspond to the positions of SEQ ID NO: 5, wherein the variant has protease activity and has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, and less than 100%, sequence identity to SEQ ID NO: 6.

[0117] In one embodiment, the protease of the invention is a variant of the polypeptide of SEQ ID NO: 6 comprising the substitutions T22A+N43R+S87N+V205L+Q206L+Y209W+S259D+N261W+L262E, where the position numbers correspond to the positions of SEQ ID NO: 5, wherein the variant has protease activity and has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, and less than 100%, sequence identity to SEQ ID NO: 6.

[0118] In one embodiment, the protease of the invention is a variant of the polypeptide of SEQ ID NO:8 comprising the substitutions A68S+T77N+T78I+G127S+A128P+G165Q+N184Q+A202V+N217S+S258P, where the position numbers correspond to the positions of SEQ ID NO:8, and wherein the variant has protease activity and has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, and less than 100%, sequence identity to SEQ ID NO:8.

[0119] In one embodiment, the protease of the invention is a variant comprising a substitution at one or more positions corresponding to positions 171, 173, 175, 179 or 180 of SEQ ID NO:1 of WO 2004 / 067737, wherein the variant has protease activity and has a sequence identity of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, and less than 100%, to SEQ ID NO:1 of WO 2004 / 067737.

[0120] In one embodiment, the protease of the present invention is selected from the group consisting of X3V, X9[E,R], X22[R,A], X43R, X61[E,D], X62[E,D], X76[D], X87N, X101[E,G,D,N,M], X103A, X104I, X118[V,R,M], X120V, X128[A,L,S,Q], X129Q, X130A, X160D, X184[E,D], X185[E,D], 188[E,D], X191N, X194P, X205I, X206L, X209W, 216V, X217[Q,D,E], X218[D,E,S], X232V, X245R, X248D, X256[E,D], X259[E,D], ,D,W], and X262[E,D], where the position numbers correspond to positions in BPN' (SEQ ID NO:5) and "X" represents any amino acid residue present at the specified position in the parent protease, and the variants have protease activity and have at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to SEQ ID NO:5 or SEQ ID NO:6.

[0121] In one embodiment, the protease of the invention is a variant that comprises any of the following sets of substitutions compared to a parent protease, where the parent protease has the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6, or has at least 80% sequence identity to SEQ ID NO:5 or SEQ ID NO:6, where the position numbers correspond to the positions of BPN' (SEQ ID NO:5), and "X" represents any amino acid residue present at the specified position in the parent protease, and the set of substitutions is: i.X9R+X15T+X68A+X218D+X245R, ii.X9R+X15T+X68A+X245R, iii.X61E+X194P+X205I+X261D, iv.X61D+X205I+X245R, v.X61E+X194P+X205I+X261D, vi.X87N+X118V+X128L+X129Q+X130A, vii.X87N+X101M+X118V+X128L+X129Q+X130A, viii.X76D+X87R+X118R+X128L+X129Q+X130A, ix.X22A+X62D+X101G+X188D+X232V+X245R, x.X103A+X104I, xi.X22R+X101G+X232V+X245R, xii.X103A+X104I+X156D, xiii.X103A+X104I+X261E, xiv.X62D+X245R, xv.X101N+X128A+X217Q, xvi.X101E+X217Q, xvii.X101E+X217D, xviii.X9E+X43R+X262E, xix.X76D+X43R+X209W, xx.X205I+X206L+X209W, xxi.X185E+X188E+X205I, xxii.X256D+X261W+X262E, xxiii.X191N+X209W, xxiv.X261E+X262E, xxv.X261E+X262D, and xxvi.X167A+X170S+X194P is selected from the group consisting of Protease variants have at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to SEQ ID NO:5 or 6.

[0122] In one embodiment, a protease variant of the invention comprises the amino acid sequence of SEQ ID NO:6 with the substitutions Y167A+R170S+A194P, wherein the variant has protease activity and the positions correspond to those of SEQ ID NO:5.

[0123] In one embodiment, a protease variant of the invention comprises the amino acid sequence of SEQ ID NO: 6 with the substitutions S9E+N43R+N76D+V205I+Q206L+Y209W+S259D+N261W+L262E, wherein the variant has protease activity, and the position numbers correspond to the positions in SEQ ID NO: 5.

[0124] In one embodiment, the protease of the present invention is an enzyme having or consisting of the amino acid sequence of SEQ ID NO: 6, 7, 8, 9, or 29.

[0125] In one embodiment, the washing or cleaning composition comprises a protease and an enzyme with DNase activity. In one embodiment, the washing or cleaning composition comprises a protease and an enzyme with hexosaminidase activity. In another embodiment, the washing or cleaning composition comprises an enzyme with DNase activity and an enzyme with hexosaminidase activity. In yet another embodiment, the washing or cleaning composition comprises a protease, an enzyme with DNase activity, and an enzyme with hexosaminidase activity.

[0126] A variety of parent proteases are suitable for variants suitable with DNase and / or hexosaminidase to obtain the beneficial effects described in the present invention, e.g., significantly improved biofilm reduction. It will be apparent to one skilled in the art that the protease may contain additional substitutions.

[0127] Thus, in some embodiments, the washing solution or cleaning composition comprises: a protease having SEQ ID NO:6 with substitutions Y167A+R170S+A194P (position numbers are based on the numbering of SEQ ID NO:5); a DNase having SEQ ID NO:4 with substitutions T1I, S13Y, T22P, S27L, L33K, S39P, S42G, D56I, S57W, S59V, T65V, V76L, Q109R, S116D, T127V, S144P, A147H, S167L, and G175D; and a hexosaminidase having SEQ ID NO:10, 11, 19, 20, 21, or 22.

[0128] In some embodiments, the washing solution or cleaning composition comprises: a protease having SEQ ID NO: 6 with substitutions Y167A, R170S+A194P (position numbers correspond to positions in SEQ ID NO: 5); a DNase having SEQ ID NO: 3 or 4; and a hexosaminidase having SEQ ID NO: 10, 11, 19, 20, 21, or 22.

[0129] In some embodiments, the wash solution or cleaning composition comprises: a protease having SEQ ID NO:8; a DNase having SEQ ID NO:4 with substitutions T1I, S13Y, T22P, S27L, L33K, S39P, S42G, D56I, S57W, S59V, T65V, V76L, Q109R, S116D, T127V, S144P, A147H, S167L, and G175D; and a hexosaminidase having SEQ ID NO:10 with substitutions Q3I, H15Y, A49W, N59E, S163P, S186R, S225G, N227T, E232D, G235W, N252P, N260Q, H272V, S279D, Y281P, K308Q, K309E, and K312Q.

[0130] In some embodiments, the washing solution or cleaning composition comprises: a protease having SEQ ID NO:8 with substitutions A68S+T77N+T78I+G127S+A128P+G165Q+N184Q+A202V+N217S+S258P (position numbers correspond to positions in SEQ ID NO:8); and a hexosaminidase having SEQ ID NO:10 with substitutions Q3I, H15Y, A49W, N59E, S163P, S186R, S225G, N227T, E232D, G235W, N252P, N260Q, H272V, S279D, Y281P, K308Q, K309E, and K312Q.

[0131] In some embodiments, the wash solution or cleaning composition comprises a DNase having SEQ ID NO:4 with substitutions T1I, S13Y, T22P, S27L, L33K, S39P, S42G, D56I, S57W, S59V, T65V, V76L, Q109R, S116D, T127V, S144P, A147H, S167L, and G175D; and a hexosaminidase having SEQ ID NO:10, 11, 19, 20, 21, or 22.

[0132] In some embodiments, the wash solution or cleaning composition comprises a protease having SEQ ID NO:6, 7, or 8 and a DNase having SEQ ID NO:3 or 4.

[0133] Suitable commercially available protease enzymes include Alcalase®, Duralase™, Durazym™, Relase®, Relase® Ultra, Savinase®, Savinase® Ultra, Primase™, Polarzyme®, Kannase®, Liquanase®, Liquanase® Ultra, Ovozyme®, Coronase®, Coronase® Ultra, Blaze®, Blaze Evity® 100T, Blaze Evity® 125T, Blaze Evity® 150T, Blaze Evity® 200T, Neutrase®, Everlase®, Esperase®, Progress® Uno, Progress® In, and Progress® Excel (Novozymes). A / S), including Maxatase™, Maxacal™, Maxapem®, Purafect® Ox, Purafect® OxP, Puramax®, FN2™, FN3™, and FN4. ex(trademark), Excellase®, Excellenz® P1000, Excellenz® P1250, Eraser®, Preferenz® P100, Preferenz® P300, Purafect Prime, Preferenz P110®, Effectenz P1000®, Purafect®, Effectenz P1050®, Purafect® Ox, Effectenz® P2000, Purafast®, Properase®, Opticlean®, and Optimase® (Danisco / DuPont), BLAP (sequence shown in FIG. 29 of U.S. Pat. No. 5,352,604) and variants thereof (Henkel AG), and KAP (Bacillus alkalophilus) from Kao Corporation. Examples of suitable antibacterial agents include those sold under the trade name β-lactamase (β-lactamase), ...

[0134] In one aspect, variants of the enzymes (proteases, DNases, and hexosaminidases) according to the invention comprise substitutions, deletions, and / or insertions at one or more positions. In one embodiment, the number of amino acid substitutions, deletions, and / or insertions introduced into the mature polypeptide of SEQ ID NO: 2, 3, 5, 6, 10, or 11 is 20 or less, such as 1 to 15, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The amino acid changes can be minor, i.e., conservative amino acid substitutions or insertions that do not significantly affect protein folding and / or activity; small deletions, typically 1 to 30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; small linker peptides of up to 20 to 25 residues; or small extensions that facilitate purification by altering the net charge or another function, such as polyhistidine tracts, antigenic epitopes, or binding domains.

[0135] Examples of conservative substitutions are in the group consisting of basic amino acids (arginine, lysine, and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamic acid and asparagine), hydrophobic amino acids (leucine, isoleucine, and valine), aromatic amino acids (phenylalanine, tryptophan, and tyrosine), and low molecular weight amino acids (glycine, alanine, serine, threonine, and methionine). Amino acid substitutions that generally do not change the specific activity are known in the art and are described, for example, by H. Neurath and RL Hill, 1979, In, The Proteins, Academic Press, New York. Common substitutions include Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.

[0136] Alternatively, the amino acid changes may be of such a nature that the physicochemical properties of the polypeptide are altered, for example, the amino acid changes may improve the thermostability of the polypeptide, may alter its substrate specificity, may change its pH optimum, etc.

[0137] Essential amino acids in a polypeptide can be identified according to techniques known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resulting mutant molecules are tested for protease, DNase, or hexosaminidase activity to identify amino acid residues important for the activity of the molecule. See also Hilton et al., 1996, J. Biol. Chem. 271:4699-4708. Enzyme active sites or other biological interactions can also be determined by physical analysis of the structure, as measured by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, combined with mutation of putative contact site amino acids. See, e.g., de Vos et al., 1992, Science 255;306-312; Smith et al., 1992, J. Mol. Biol. 224;899-904; Wlodaver et al., 1992, FEBS Lett. 309:59-64. The identities of essential amino acids can also be inferred from alignments with related polypeptides.

[0138] Additional Enzymes One or more additional enzymes, such as amylase, lipase, cellulase, mannanase, hemicellulase, peroxidase, xylanase, phospholipase, esterase, cutinase, keratinase, reductase, oxidase, phenoloxidase, lipoxygenase, ligninase, pullulanase, malanase, β-glucanase, arabinosidase, hyaluronidase, laccase, perhydrolase, and peroxidase.

[0139] In general, the properties of the enzyme selected should be compatible with the selected detergent (i.e., pH optimum, compatibility with other enzymatic and non-enzymatic components, etc.) and the enzyme should be present in an effective amount.

[0140] amylase Suitable amylases that can be used with the protease, DNase, and / or hexosaminidase of the present invention may be alpha-amylases or glucoamylases and may be of bacterial or fungal origin, including chemically modified or protein engineered variants. Amylases include, for example, alpha-amylases obtained from the genus Bacillus, such as special strains of Bacillus licheniformis, which are described in more detail in GB 1,296,839.

[0141] Suitable amylases include amylases having SEQ ID NO: 2 in WO 95 / 10603 or variants thereof having 90% sequence identity to SEQ ID NO: 3. Preferred variants are set out in SEQ ID NO: 4 of WO 94 / 02597, WO 94 / 18314, WO 97 / 43424 and WO 99 / 019467, such as variants having substitutions at one or more of the following positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 178, 179, 181, 188, 190, 197, 201, 202, 207, 208, 209, 211, 243, 264, 304, 305, 391, 408 and 444.

[0142] Different suitable amylases include the amylase having SEQ ID NO: 6 in WO 02 / 010355, or a variant thereof having 90% sequence identity to SEQ ID NO: 6. A preferred variant of SEQ ID NO: 6 is one having deletions at positions 181 and 182 and a substitution at position 193.

[0143] Other suitable amylases include a hybrid alpha-amylase comprising residues 1-33 of the alpha-amylase from B. amyloliquefaciens as set forth in SEQ ID NO: 6 of WO 2006 / 066594 and residues 36-483 of the B. licheniformis alpha-amylase as set forth in SEQ ID NO: 4 of WO 2006 / 066594, or a variant having 90% sequence identity thereto. Preferred variants of this hybrid alpha-amylase are those having substitutions, deletions or insertions at one or more of the following positions: G48, T49, G107, H156, A181, N190, M197, I201, A209, and Q264. The most preferred variant of the hybrid alpha-amylase comprising residues 1-33 of the alpha-amylase from B. amyloliquefaciens as set forth in SEQ ID NO:6 of WO 2006 / 066594 and residues 36-483 of SEQ ID NO:4 comprises the substitution: M197T; H156Y+A181T+N190F+A209V+Q264S; or G48A+T49I+G107A+H156Y+A181T+N190F+I201F+A209V+Q264S It has the following.

[0144] Further suitable amylases are those having SEQ ID NO: 6 in WO 99 / 019467 or variants thereof having 90% sequence identity to SEQ ID NO: 6. Preferred variants of SEQ ID NO: 6 are those having substitutions, deletions or insertions at one or more of the following positions: R181, G182, H183, G184, N195, I206, E212, E216 and K269. Particularly preferred amylases are those having deletions at positions R181 and G182, or H183 and G184.

[0145] Additional amylases that may be used are those having SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:2 or SEQ ID NO:7 of WO 96 / 023873 or variants thereof having 90% sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:7. Preferred variants of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:7 are those having substitutions, deletions or insertions at one or more of the following positions: 140, 181, 182, 183, 184, 195, 206, 212, 243, 260, 269, 304 and 476, using the numbering of SEQ ID NO:2 of WO 96 / 023873. More preferred variants are those having deletions at two positions selected from 181, 182, 183 and 184, for example at positions 181 and 182, 182 and 183 or positions 183 and 184. Most preferred amylase variants of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:7 include those having deletions at positions 183 and 184 and substitutions at one or more of positions 140, 195, 206, 243, 260, 304, and 476.

[0146] Other amylases that may be used include amylases having SEQ ID NO: 2 in WO 08 / 153815, SEQ ID NO: 10 in WO 01 / 66712, or variants thereof having 90% sequence identity to SEQ ID NO: 2 in WO 08 / 153815 or 90% sequence identity to SEQ ID NO: 10 in WO 01 / 66712. Preferred variants of SEQ ID NO: 10 in WO 01 / 66712 include those having substitutions, deletions or insertions at one or more of the following positions: 176, 177, 178, 179, 190, 201, 207, 211, and 264.

[0147] Further suitable amylases include those having SEQ ID NO: 2 of WO 09 / 061380, or variants thereof having 90% sequence identity to SEQ ID NO: 2. Preferred variants of SEQ ID NO: 2 include those having C-terminal truncations and / or substitutions, deletions or insertions at one or more of the following positions: Q87, Q98, S125, N128, T131, T165, K178, R180, S181, T182, G183, M201, F202, N225, S243, N272, N282, Y305, R309, D319, Q320, Q359, K444, and G475. More preferred variants of SEQ ID NO:2 include substitutions at one or more of the following positions: Q87E,R, Q98R, S125A, N128C, T131I, T165I, K178L, T182G, M201L, F202Y, N225E,R, N272E,R, S243Q,A,E,D, Y305R, R309A, Q320R, Q359E, K444E, and G475K, and / or deletions at positions R180 and / or S181 or T182 and / or G183. Most preferred amylase variants of SEQ ID NO:2 include substitutions at one or more of the following positions: Q87E,R, Q98R, S125A, N128C, T131I, T165I, K178L, T182G, M201L, F202Y, N225E,R, N272E,R, S243Q,A,E,D, Y305R, R309A, Q320R, Q359E, K444E, and G475K, and / or deletions at positions R180 and / or S181 or T182 and / or G183. N128C+K178L+T182G+Y305R+G475K; N128C+K178L+T182G+F202Y+Y305R+D319T+G475K; S125A+N128C+K178L+T182G+Y305R+G475K; or S125A+N128C+T131I+T165I+K178L+T182G+Y305R+G475K wherein the variant is C-terminally truncated and optionally further comprises a substitution at position 243 and / or a deletion at positions 180 and / or 181.

[0148] Further suitable amylases include those having SEQ ID NO: 1 of WO13184577, or variants thereof having 90% sequence identity to SEQ ID NO: 1. Preferred variants of SEQ ID NO: 1 include those having substitutions, deletions or insertions at one or more of the following positions: K176, R178, G179, T180, G181, E187, N192, M199, I203, S241, R458, T459, D460, G476, and G477. More preferred variants of SEQ ID NO:1 include substitutions at one or more of the following positions: K176L, E187P, N192FYH, M199L, I203YF, S241QADN, R458N, T459S, D460T, G476K, and G477K, and / or deletions at positions R178 and / or S179 or T180 and / or G181. Most preferred amylase variants of SEQ ID NO:1 include substitutions: E187P+I203Y+G476K E187P+I203Y+R458N+T459S+D460T+G476K Some have The variant optionally further comprises a substitution at position 241, and / or a deletion at positions 178 and / or 179.

[0149] Further suitable amylases include amylases having SEQ ID NO: 1 of WO 10104675 or variants thereof having 90% sequence identity to SEQ ID NO: 1. Preferred variants of SEQ ID NO: 1 include those having substitutions, deletions or insertions at one or more of the following positions: N21, D97, V128 K177, R179, S180, I181, G182, M200, L204, E242, G477 and G478. More preferred variants of SEQ ID NO: 1 include those having substitutions at one or more of the following positions: N21D, D97N, V128I K177L, M200L, L204YF, E242QA, G477K and G478K and / or deletions at positions R179 and / or S180 or I181 and / or G182. The most preferred amylase variant of SEQ ID NO:1 has the substitution: N21D+D97N+V128I Some have The variant optionally further comprises a substitution at position 200 and / or a deletion at positions 180 and / or 181.

[0150] Other suitable amylases are the alpha-amylases having SEQ ID NO:12 in WO 01 / 66712, or variants having at least 90% sequence identity to SEQ ID NO:12. Preferred amylase variants include those that have substitutions, deletions or insertions at one or more of the following positions of SEQ ID NO: 12 in WO 01 / 66712: R28, R118, N174; R181, G182, D183, G184, G186, W189, N195, M202, Y298, N299, K302, S303, N306, R310, N314; R320, H324, E345, Y396, R400, W439, R444, N445, K446, Q449, R458, N471, N484. Particularly preferred amylases include variants having a deletion of D183 and G184 and having the substitutions R118K, N195F, R320K, and R458K, as well as variants additionally having substitutions at one or more positions selected from the group: M9, G149, G182, G186, M202, T257, Y295, N299, M323, E345, and A339, and most preferred are variants additionally having substitutions at all of these positions.

[0151] Other examples include amylase variants such as those described in WO 2011 / 098531, WO 2013 / 001078, and WO 2013 / 001087.

[0152] Commercially available amylases include Duramyl™, Termamyl™, Fungamyl™, Stainzyme™, Stainzyme Plus™, Natalase™, Liquozyme X, and BAN™ (from Novozymes A / S), as well as Rapidase™, Purastar™ / Effectenz™, Powerase, Preferenz S1000, Preferenz S100, and Preferenz S110 (from Genencor International Inc. / DuPont).

[0153] Cellulase Suitable cellulases include single components and mixtures of enzymes of bacterial or fungal origin. Also contemplated are chemically modified or protein engineered variants. The cellulase may be, for example, a single component or a mixture of single component endo-1,4-beta-glucanases, also referred to as endoglucanases.

[0154] Suitable cellulases include those from the genera Bacillus, Pseudomonas, Humicola, Myceliophthora, Fusarium, Thielavia, Trichoderma, and Acremonium. Exemplary cellulases include fungal cellulases from Humicola insolens (U.S. Pat. No. 4,435,307) or from the Trichoderma genus, such as T. reesei or T. viride. Other suitable cellulases include those from the genus Thielavia, e.g., Thielavia terrestris, as described in WO 96 / 29397, or fungal cellulases produced by Myceliophthora thermophila and Fusarium oxysporum, as disclosed in U.S. Pat. Nos. 5,648,263, 5,691,178, 5,776,757, WO 89 / 09259, and WO 91 / 17244. Also relevant are cellulases from the genus Bacillus, as described in WO 02 / 099091 and JP 2000-210081. Suitable cellulases include alkaline or neutral cellulases with care benefits. Examples of cellulases are described in EP 0495257, EP 0531372, WO 96 / 11262, WO 96 / 29397, and WO 98 / 08940.Other examples include cellulase variants such as those described in WO 94 / 07998, EP 0531315, U.S. Pat. No. 5,457,046, U.S. Pat. No. 5,686,593, U.S. Pat. No. 5,763,254, WO 95 / 24471, and WO 98 / 12307.

[0155] Other cellulases include endo-beta-1,4-glucanase enzymes having a sequence that is at least 97% identical to amino acid sequence positions 1 to 773 of SEQ ID NO:2 of WO 2002 / 099091, or Family 44 xyloglucanases, which are xyloglucanase enzymes having a sequence that is at least 60% identical to amino acid sequence positions 40 to 559 of SEQ ID NO:2 of WO 2001 / 062903.

[0156] Commercially available cellulases include Carezyme®, Carezyme® Premium, Celluzyme®, Celluclean®, Celluclast®, Endolase®, Renozyme®; Whitezyme®, Celluclean® Classic, Cellusoft® (Novozymes A / S), Puradax®, Puradax HA, and Puradax EG (available from Genencor International Inc.), and KAC-500(B)™ (Kao Corporation).

[0157] Mannanase Suitable mannanases include those of bacterial or fungal origin. Chemically or genetically modified variants are included. The mannanase may be an alkaline mannanase of family 5 or 26. It may be a wild type from the genus Bacillus or Humicola, in particular B. agaradhaerens, B. licheniformis, B. halodurans, B. clausii, or H. insolens. Suitable mannanases are described in WO 1999 / 064619. Commercially available mannanases include Mannaway (Novozymes A / S).

[0158] Lipase and cutinase Suitable lipases and cutinases include those of bacterial or fungal origin. Chemically modified or protein engineered mutant enzymes are included. Examples include those from the genus Thermomyces, such as T. lanuginosus (formerly Humicola lanuginosa), as described in EP 258068 and EP 305216. lanuginosa), cutinases from the genus Humicola, e.g. H. insolens (WO 96 / 13580), the genus Pseudomonas (some of which have now been renamed Burkholderia), e.g. P. alcaligenes or P. pseudoalcaligenes (EP 218272), P. cepacia (EP 331376), P. sp. strain SD705 (WO 95 / 06720 and WO 96 / 27002), P. wisconinensis (P.wisconsinensis (WO 96 / 12012), GDSL-type Streptomyces lipases (WO 10 / 065455), Magnaporthe grisea cutinase (WO 10 / 107560), Pseudomonas mendocina cutinase (U.S. Pat. No. 5,389,536), Thermobifida fusca lipase (WO 11 / 084412), Geobacillus stearothermophilus lipase (WO 11 / 084417), Bacillus Examples include lipases from S. subtilis (WO 11 / 084599), as well as lipases from Streptomyces griseus (WO 11 / 150157) and S. pristinaespiralis (WO 12 / 137147).

[0159] Other examples include lipase variants such as those described in EP 407225, WO 92 / 05249, WO 94 / 01541, WO 94 / 25578, WO 95 / 14783, WO 95 / 30744, WO 95 / 35381, WO 95 / 22615, WO 96 / 00292, WO 97 / 04079, WO 97 / 07202, WO 00 / 34450, WO 00 / 60063, WO 01 / 92502, WO 07 / 87508 and WO 09 / 109500.

[0160] Preferred commercially available lipase products include Lipolase™, Lipex™; Lipolex™, and Lipoclean™ (Novozymes A / S), Lumafast (originally manufactured by Genencor), and Lipomax (originally manufactured by Gist-Brocades).

[0161] Further examples include lipases sometimes referred to as acyltransferases or perhydrolases, such as the acyltransferase with homology to Candida antarctica lipase A (WO 10 / 111143), the acyltransferase from Mycobacterium smegmatis (WO 05 / 56782), the perhydrolases from the CE 7 family (WO 09 / 67279), as well as variants of M. smegmatis perhydrolase, in particular the S54V variant used in the product Gentle Power Bleach from Huntsman Textile Effects Pte Ltd (WO 10 / 100028).

[0162] Peroxidase / Oxidase Suitable peroxidases / oxidases include those of plant, bacterial or fungal origin. Chemically modified or protein engineered variants are included. Examples of useful peroxidases include peroxidases from the genus Coprinus, such as C. cinereus, and variants thereof, as described in WO 93 / 24618, WO 95 / 10602, and WO 98 / 15257. Commercially available peroxidases include Guardzyme™ (Novozymes A / S).

[0163] Suitable peroxidases preferably include the peroxidase enzymes encompassed by the enzyme classification EC 1.11.1.7 as set forth by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMB), or any fragment derived therefrom which exhibits peroxidase activity.

[0164] Suitable peroxidases also include haloperoxidase enzymes, such as chloroperoxidase, bromoperoxidase, and compounds that exhibit chloroperoxidase or bromoperoxidase activity. Haloperoxidases are classified according to their specificity for halide ions. Chloroperoxidase (EC 1.11.1.10) catalyzes the formation of hypochlorite from chloride ions. The haloperoxidase may be a chloroperoxidase. Preferably, the haloperoxidase is a vanadium peroxidase, i.e., a vanadate-containing haloperoxidase. In a preferred method of the present invention, a vanadate-containing haloperoxidase is combined with a source of chloride ions.

[0165] Haloperoxidases have been isolated from many species of fungi, particularly from the fungal group of the dematiaceous hyphomycetes, such as the genera Caldariomyces, e.g., C. fumago, Alternaria, Curvularia, e.g., C. verruculosa and C. inaequalis, Drechslera, Ulocladium, and Botrytis.

[0166] Haloperoxidases can also be derived from bacteria such as those of the genus Pseudomonas, e.g., P. pyrrocinia, and Streptomyces, e.g., S. aureofaciens.

[0167] Haloperoxidases can be produced from Curvularia species, in particular Curvularia verruculosa or Curvularia inaequalis, e.g. C. inaequalis CBS 102.42 as described in WO 95 / 27046; C. verruculosa CBS 147.63 or C. verruculosa CBS 444.70 as described in WO 97 / 04102; Drechslera hartlebii as described in WO 01 / 79459, Dendrophiella salina as described in WO 01 / 79458, salina, Phaeotrichoconis crotalarie as described in WO 01 / 79461, or Geniculosporium species as described in WO 01 / 79460.

[0168] Suitable oxidases include in particular any laccase enzyme encompassed by the enzyme classification EC 1.10.3.2, or any fragment derived therefrom exhibiting laccase activity, or a compound exhibiting similar activity, such as catechol oxidase (EC 1.10.3.1), o-aminophenol oxidase (EC 1.10.3.4), or bilirubin oxidase (EC 1.3.3.5).

[0169] Preferred laccase enzymes are those of bacterial origin. The enzymes may be derived from plants, bacteria, or fungi (including filamentous fungi and yeasts).

[0170] Suitable examples of fungal origin include those from the genera Aspergillus, Neurospora, e.g. N. crassa, Podospora, Botrytis, Collybia, Fomes, Lentinus, Pleurotus, Trametes versicolor, and the like. s), such as T. villosa and T. versicolor, Rhizoctonia, such as R. solani, Coprinopsis, such as C. cinerea, C. comatus, C. friesii, and C. p licatilis, Psathyrella such as P. condoelleana, Panaeolus such as P. papilionaceus, Myceliophthora such as M. thermophila, Schytalidium such as S. thermophilum, Polyporus such as P. pinsitus, Phlebia such as P. radiata (WO 92 / 01046), or Coriolus such as C. hirsutus (JP 2238885 A).

[0171] Suitable examples of bacterial origin include laccases derived from strains of the genus Bacillus.

[0172] Laccases derived from the genus Coprinopsis or Myceliophthora are preferred; in particular, laccases derived from Coprinopsis cinerea as disclosed in WO 97 / 08325; or laccases derived from Myceliophthora thermophila as disclosed in WO 95 / 33836.

[0173] Protease Stabilizers / Inhibitors The proteases of the invention can be stabilized with compounds that act by temporarily reducing proteolytic activity (reversible inhibitors).

[0174] Thus, cleaning compositions of the present invention may also include protease inhibitors / stabilizers that are reversible inhibitors of protease activity, such as serine protease activity.

[0175] Preferably, the protease inhibitor is a (reversible) subtilisin protease inhibitor. In particular, the protease inhibitor may be a peptide aldehyde, boric acid, or boronic acid; or a derivative of any of these.

[0176] Boronic Acids The protease inhibitor may be a boronic acid or a derivative thereof; preferably, a phenylboronic acid or a derivative thereof. In one embodiment of the present invention, the phenylboronic acid derivative is of the following formula: [ka] (wherein R is selected from the group consisting of hydrogen, hydroxy, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkenyl, and substituted C1-C6 alkenyl.) Preferably, R is hydrogen, CH3, CH3CH2, or CH3CH2CH2.

[0177] In a preferred embodiment, the protease inhibitor (phenylboronic acid derivative) is 4-formyl-phenyl-boronic acid (4-FPBA).

[0178] In another particular embodiment, the protease inhibitor is selected from the group consisting of thiophene-2-boronic acid, thiophene-3-boronic acid, acetamidophenylboronic acid, benzofuran-2-boronic acid, naphthalene-1-boronic acid, naphthalene-2-boronic acid, 2-FPBA, 3-FBPA, 4-FPBA, 1-thianthreneboronic acid, 4-dibenzofuranboronic acid, 5-methylthiophene-2-boronic acid, thionaphthreneboronic acid, furan-2-boronic acid, furan-3-boronic acid, 4,4-biphenyl-diboronic acid, 6-hydroxy-2-naphthalene, 4-(methylthio)phenylboronic acid, 4-(trimethyl-silyl)phenylboronic acid, 3-bromothiopheneboronic acid, 4-methylthiopheneboronic acid, 2-naphthylboronic acid, 5-bromothipheneboronic acid. acid), 5-chlorothiopheneboronic acid, dimethylthiopheneboronic acid, 2-bromophenylboronic acid, 3-chlorophenylboronic acid, 3-methoxy-2-thiophene, p-methyl-phenylethylboronic acid, 2-thianthreneboronic acid, dibenzothiopheneboronic acid, 4-carboxyphenylboronic acid, 9-anthrylboronic acid, 3,5-dichlorophenylboronic acid, diphenylboronic anhydride, o-chlorophenylboronic acid, p-chlorophenylboronic acid, m-bromophenylboronic acid, p-bromophenylboronic acid, p-fluorophenylboronic acid, p-tolylboronic acid, o-tolylboronic acid, octylboronic acid, 1,3,5-trimethylphenylboronic acid, 3-chloro-4-fluorophenylboronic acid, 3-aminophenylboronic acid, 3,5-bis-(trifluoromethyl)phenylboronic acid, 2,4-dichlorophenylboronic acid, and 4-methoxyphenylboronic acid.

[0179] Further boronic acid derivatives suitable as protease inhibitors in cleaning compositions are described in U.S. Pat. No. 4,963,655, U.S. Pat. No. 5,159,060, WO 95 / 12655, WO 95 / 29223, WO 92 / 19707, WO 94 / 04653, WO 94 / 04654, U.S. Pat. No. 5,442,100, U.S. Pat. No. 5,488,157, and U.S. Pat. No. 5,472,628.

[0180] Peptide aldehyde or ketone The protease stabilizer may have the formula: PALB-B0-R*: R* is H (hydrogen), CH3, CX3, CHX2, or CH2X, where X is a halogen atom, in particular F (fluorine); preferably, R*=H (the stabilizer is thus a peptide aldehyde having the formula PALB-B0-H); L is absent or is a linker group of the formula -C(=O)-, -C(=O)-C(=O)-, -C(=S)-, -C(=S)-C(=S)-, or -C(=S)-C(=O)-; A is absent if L is absent, or is one or two amino acid residues linked to L via the N-terminus; thus A can represent A1 or A2-A1, where A2 and A1 each represent one amino acid residue; B may be one, two, or three amino acid residues; thus B may represent B1, B2-B1, or B3-B2-B1 linked via the C-terminus to B0, where B3, B2, and B1 each represent one amino acid residue; B0 is a single amino acid residue having the L- or D-configuration of the formula -NH-CH(R)-C(=O)-; R is independently optionally substituted with one or more identical or different substituents R′; 1~6 Alkyl, C 6~10 Aryl or C 7~10 arylalkyl; R' is independently selected from the group consisting of halogen, -OH, -OR'', -SH, -SR'', -NH2, -NHR'', -NR''2, -CO2H, -CONH2, -CONHR'', -CONR''2, -NHC(=N)NH2; R'' is C 1~6 is an alkyl group; P is selected from the group consisting of hydrogen, or, if L is absent, an N-terminal protecting group; B0 may be a single amino acid residue having an L- or D-configuration linked to H through the C-terminus of the amino acid. B0 has the formula -NH-CH(R)-C(=O)-, where R is C 1~6 Alkyl, C 6~10 Aryl or C 7~10 Arylalkyl side chains, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, phenyl, or benzyl, where R may be optionally substituted with one or more identical or different substituents R'. Particular examples of B0 are the D- or L-forms of arginine (Arg), 3,4-dihydroxyphenylalanine, isoleucine (Ile), leucine (Leu), methionine (Met), norleucine (Nle), norvaline (Nva), phenylalanine (Phe), m-tyrosine, p-tyrosine (Tyr), and valine (Val). Particular embodiments are where B0 is leucine, methionine, phenylalanine, p-tyrosine, or valine. Particularly preferred is p-tyrosine.

[0181] B1, which is linked to B0 via the C-terminus of the amino acid, can be an aliphatic amino acid, a hydrophobic amino acid, and / or a neutral amino acid. Examples of B1 include alanine (Ala), cysteine ​​(Cys), glycine (Gly), isoleucine (Ile), leucine (Leu), norleucine (Nle), norvaline (Nva), proline (Pro), serine (Ser), threonine (Thr), and valine (Val). Particular examples of B1 include alanine, glycine, isoleucine, leucine, and valine. Particular embodiments are where B1 is alanine, glycine, or valine.

[0182] B2, if present, is linked to B1 via the C-terminus of the amino acid, and may be an aliphatic amino acid, a hydrophobic amino acid, a neutral amino acid, and / or a polar amino acid. Examples of B2 include alanine (Ala), arginine (Arg), capreomycin (Cpd), cysteine ​​(Cys), glycine (Gly), isoleucine (Ile), leucine (Leu), norleucine (Nle), norvaline (Nva), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), and valine (Val). Particular examples of B2 include alanine, arginine, capreomycin, glycine, isoleucine, leucine, phenylalanine, and valine. Particular embodiments are where B2 is arginine, glycine, leucine, phenylalanine, or valine.

[0183] B3, if present, is linked to B2 via the C-terminus of the amino acid and may be a large aliphatic amino acid, an aromatic amino acid, a hydrophobic amino acid, and / or a neutral amino acid. Examples of B3 are isoleucine (Ile), leucine (Leu), norleucine (Nle), norvaline (Nva), phenylalanine (Phe), phenylglycine, tyrosine (Tyr), tryptophan (Trp), and valine (Val). Specific examples of B3 include leucine, phenylalanine, tyrosine, and tryptophan.

[0184] The linker group L may be absent or selected from the group consisting of -C(=O)-, -C(=O)-C(=O)-, -C(=S)-, -C(=S)-C(=S)-, or -C(=S)-C(=O)-. A particular embodiment of the invention is where L is absent or where L is a carbonyl group -C(=O)-.

[0185] A1, if present, is linked to L via the N-terminus of the amino acid and may be an aliphatic amino acid, an aromatic amino acid, a hydrophobic amino acid, a neutral amino acid, and / or a polar amino acid. Examples of A1 include alanine (Ala), arginine (Arg), capreomycin (Cpd), glycine (Gly), isoleucine (Ile), leucine (Leu), norleucine (Nle), norvaline (Nva), phenylalanine (Phe), threonine (Thr), tyrosine (Tyr), tryptophan (Trp), and valine (Val). Particular examples of A1 include alanine, arginine, glycine, leucine, phenylalanine, tyrosine, tryptophan, and valine. Particular embodiments are where B2 is leucine, phenylalanine, tyrosine, or tryptophan.

[0186] A2, if present, is linked to A1 via the N-terminus of the amino acid and may be a large aliphatic amino acid, an aromatic amino acid, a hydrophobic amino acid, and / or a neutral amino acid. Examples of A2 include arginine (Arg), isoleucine (Ile), leucine (Leu), norleucine (Nle), norvaline (Nva), phenylalanine (Phe), phenylglycine, tyrosine (Tyr), tryptophan (Trp), and valine (Val). Particular examples of A2 include phenylalanine and tyrosine.

[0187] The N-terminal protecting group P (if present) may be selected from formyl, acetyl (Ac), benzoyl (Bz), trifluoroacetyl, methoxysuccinyl, aromatic and aliphatic urethane protecting groups such as fluorenylmethyloxycarbonyl (Fmoc), methoxycarbonyl (Moc), (fluoromethoxy)carbonyl, benzyloxycarbonyl (Cbz), t-butyloxycarbonyl (Boc), and adamantyloxycarbonyl; p-methoxybenzylcarbonyl, benzyl (Bn), p-methoxybenzyl (PMB), p-methoxyphenyl (PMP), methoxyacetyl, methylaminocarbonyl, methylsulfonyl, ethylsulfonyl, benzylsulfonyl, methylphosphoramidyl (MeOP(OH)(=O)), and benzylphosphoramidyl (PhCHOP(OH)(=O)).

[0188] Suitable peptide aldehydes are described in WO 94 / 04651, WO 95 / 25791, WO 98 / 13458, WO 98 / 13459, WO 98 / 13460, WO 98 / 13461, WO 98 / 13462, WO 07 / 141736, WO 07 / 145963, WO 09 / 118375, WO 10 / 055052, and WO 11 / 036153.More specifically, the peptide aldehydes include Cbz-Arg-Ala-Tyr-H, Ac-Gly-Ala-Tyr-H, Cbz-Gly-Ala-Tyr-H, Cbz-Gly-Ala-Tyr-CF3, Cbz-Gly-Ala-Leu-H, Cbz-Val-Ala-Leu-H, Cbz-Val-Ala-Leu-CF3, Moc-Val-Ala-Leu-CF3, Cbz-Gly-Ala-Phe-H, Cbz-Gl y-Ala-Phe-CF3, Cbz-Gly-Ala-Val-H, Cbz-Gly-Gly-Tyr-H, Cbz-Gly-Gly-Phe-H, Cbz-Arg-Val-Tyr-H, Cbz-Leu-Val -Tyr-H, Ac-Leu-Gly-Ala-Tyr-H, Ac-Phe-Gly-Ala-Tyr-H, Ac-Tyr-Gly-Ala-Tyr-H, Ac-Phe-Gly-Ala-Leu-H, Ac-Phe -Gly-Ala-Phe-H, Ac-Phe-Gly-Val-Tyr-H, Ac-Phe-Gly-Ala-Met-H, Ac-Trp-Leu-Val-Tyr-H, MeO-CO-Val-Ala-Leu- H, MeNCO-Val-Ala-Leu-H, MeO-CO-Phe-Gly-Ala-Leu-H, MeO-CO-Phe-Gly-Ala-Phe-H, MeSO2-Phe-Gly-Ala-Leu-H, M The stabilizer may be eSO2-Val-Ala-Leu-H, PhCHO-P(OH)(O)-Val-Ala-Leu-H, EtSO2-Phe-Gly-Ala-Leu-H, PhCH2SO2-Val-Ala-Leu-H, PhCHO-P(OH)(O)-Leu-Ala-Leu-H, PhCHO-P(OH)(O)-Phe-Ala-Leu-H, or MeO-P(OH)(O)-Leu-Gly-Ala-Leu-H. A preferred stabilizer for use in the liquid composition of the present invention is Cbz-Gly-Ala-Tyr-H, or a hydrosulfite adduct thereof, where Cbz is benzyloxycarbonyl.

[0189] Further examples of such peptide aldehydes include α-MAPI, β-MAPI, Phe-C(=O)-Arg-Val-Tyr-H, Phe-C(=O)-Gly-Gly-Tyr-H, Phe-C(=O)-Gly-Ala-Phe-H, Phe-C(=O)-Gly-Ala-Tyr-H, Phe-C(=O)-Gly-Ala-LH, Phe-C(=O)-Gly-Ala-Nva- ... Ly-Ala-Nle-H, Tyr-C(=O)-Arg-Val-Tyr-H, Tyr-C(=O)-Gly-Ala-Tyr-H, Phe-C(=S)-Arg-Val-Phe-H, Phe-C(=S)-Arg-Val-Tyr-H, Phe-C(=S)-Gly-Ala-Tyr-H, antipain, GE20372A, GE20372B, chymostatin A, chymostatin B, and chymostatin C.

[0190] The protease stabilizer may be a hydrosulfite adduct of the above-mentioned peptide aldehyde or ketone, e.g., as described in WO 2013 / 004636. The adduct may have the formula PAL-BN(H)-CHR-CH(OH)-SO3M, where P, A, L, B, and R are defined above and M is H or an alkali metal, preferably Na or K.

[0191] Aqueous solutions of hydrosulfite adducts may be prepared by reacting the corresponding peptide aldehyde with an aqueous solution of sodium bisulfite (sodium hydrogen sulfite, NaHSO3); potassium bisulfite (KHSO3), by known methods, e.g., as described in WO 98 / 47523; U.S. Pat. No. 6,500,802; U.S. Pat. No. 5,436,229; J. Am. Chem. Soc. (1978) 100, 1228; Org. Synth., Coll. vol. 7:361.

[0192] Particularly preferred peptide aldehyde protease stabilizers have the formula P-B3-B2-B1-B0-H or a hydrosulfite adduct having the formula P-B3-B2-B1-N(H)-CHR-CHOH-SO3M, i) H is hydrogen; ii) B0 is a single amino acid residue of the formula -NH-CH(R)-C(=O)- having the L- or D-configuration; iii) B1 and B2 are, independently, a single amino acid residue; iv) B3 is a single amino acid residue or is absent; v) R is independently a C optionally substituted with one or more identical or different substituents R′. 1~6 Alkyl, C 6~10 Aryl or C 7~10 arylalkyl; vi) R' is independently selected from the group consisting of halogen, -OH, -OR'', -SH, -SR'', -NH2, -NHR'', -NR''2, -CO2H, -CONH2, -CONHR'', -CONR''2, -NHC(=N)NH2; vii) R'' is C 1~6 is an alkyl group; viii) P is an N-terminal protecting group, preferably methoxycarbonyl (Moc) or benzyloxycarbonyl (Cbz); ix) M is H or an alkali metal, preferably Na or K.

[0193] In an even more preferred embodiment, the peptide aldehyde protease stabilizer has the formula P-B2-B1-B0-H, or an adduct having the formula P-B2-B1-N(H)-CHR-CHOH-SO3M, i) H is hydrogen; ii) B0 is a single amino acid residue of the formula -NH-CH(R)-C(=O)- having the L- or D-configuration; iii) B1 and B2 are, independently, a single amino acid residue; iv) R is independently a C optionally substituted with one or more identical or different substituents R′. 1~6 Alkyl, C 6~10 Aryl or C 7~10 arylalkyl; v) R' is independently selected from the group consisting of halogen, -OH, -OR'', -SH, -SR'', -NH2, -NHR'', -NR''2, -CO2H, -CONH2, -CONHR'', -CONR''2, -NHC(=N)NH2; vi) R'' is C 1~6 is an alkyl group; vii) P is an N-terminal protecting group, preferably methoxycarbonyl (Moc) or benzyloxycarbonyl (Cbz); viii) M is H or an alkali metal, preferably Na or K.

[0194] Preferred embodiments of B0, B1, B2, B3, and P are as described above.

[0195] When the peptide aldehyde has the formula P-B3-B2-B1-B0-H or its hydrosulfite adduct, P is preferably acetyl, methoxycarbonyl, benzyloxycarbonyl, methylaminocarbonyl, methylsulfonyl, benzylsulfonyl, and benzylphosphoramidyl.

[0196] When the peptide aldehyde has the formula P-B2-B1-B0-H or its hydrosulfite adduct, P is preferably acetyl, methoxycarbonyl, methylsulfonyl, ethylsulfonyl, and methylphosphoramidyl.

[0197] The molar ratio of said peptide aldehyde (or hydrosulfite adduct) to the protease may be at least 1:1 or 1.5:1, or may be less than 1000:1, more preferably less than 500:1, even more preferably 100:1 to 2:1 or 20:1 to 2:1, or most preferably the molar ratio is 10:1 to 2:1.

[0198] Also, formate salts (e.g., sodium formate) and formic acid have shown good effects as inhibitors of protease activity. Formate salts can be used synergistically with the above-mentioned protease inhibitors, as shown in WO 2013 / 004635. Formate salts may be present in the composition in an amount of at least 0.1 w / w% or 0.5 w / w%, for example at least 1.0%, at least 1.2%, or at least 1.5%. The amount is typically less than 5 w / w%, less than 4%, or less than 3%.

[0199] In one embodiment, the protease is a metalloprotease and the inhibitor is a metalloprotease inhibitor, such as a protein hydrolysate-based inhibitor (such as those described in WO 2008 / 134343).

[0200] Surfactants The cleaning composition of the present invention may include one or more surfactants, which may be anionic and / or cationic and / or nonionic and / or semi-polar and / or zwitterionic, or a mixture thereof. In certain embodiments, the cleaning composition includes a surfactant system (comprising two or more surfactants), for example, a mixture of one or more nonionic surfactants and one or more anionic surfactants. In one embodiment, the detergent includes at least one anionic surfactant and at least one nonionic surfactant, and the weight ratio of the anionic surfactant to the nonionic surfactant may be from 10:1 to 1:10. In one embodiment, the amount of nonionic surfactant is greater than the amount of anionic surfactant, for example, the weight ratio of the nonionic surfactant to the anionic surfactant may be from 10:1 to 1.1:1 or from 5:1 to 1.5:1. Also, the amount of anionic surfactant to the nonionic surfactant may be equal or may be a weight ratio of 1:1. Medical cleaning detergents (e.g., cleaning compositions for cleaning medical devices) typically include more nonionic surfactant than anionic surfactant. The total weight of surfactant present in the composition is typically about 0.1% by weight to about 60% by weight, for example, about 1% by weight to about 40% by weight, or about 3% by weight to about 20% by weight, or about 3% by weight to about 10% by weight. The surfactant is selected based on the desired cleaning application and may include any conventional surfactant known in the art. If included, the cleaning composition will usually contain about 1% by weight to about 30% by weight of anionic surfactant, for example, about 5% by weight to about 20% by weight, or about 5% by weight to about 10% by weight of anionic surfactant.Non-limiting examples of anionic surfactants include, typically, sodium or potassium salts or salts of monoethanolamine (MEA, 2-aminoethan-1-ol) or triethanolamine (TEA, 2,2′2″-nitrilotriethane-1-ol); in particular linear alkylbenzenesulfonates (LAS), isomers of LAS, such as branched alkylbenzenesulfonates (BABS) and phenylalkanesulfonates; olefinsulfonates, in particular alpha-olefinsulfonates (AOS); alkyl sulfates (AS), in particular fatty alcohol sulfates (FAS), i.e. primary alcohol sulfates (PAS), such as dodecyl sulfate; alcohols. These include ether sulfates (AES or AEOS or FES, also known as alcohol ethoxy sulfates or fatty alcohol ether sulfates); paraffin sulfonates (PS), including alkane-1-sulfonates and secondary alkane sulfonates (SAS); sulfonic acid esters, including sulfonated fatty acid glycerol esters and alpha-sulfo fatty acid methyl esters (alpha-SFMe or SES or MES); alkyl- or alkenyl succinic acids, for example dodecenyl / tetradecenyl succinic acid (DTSA); diesters and monoesters of sulfosuccinic acid; sulfates and sulfonates available as fatty acid derivatives of amino acids. Furthermore, the salts of fatty acids (soaps) may be mentioned.

[0201] When included, the cleaning composition will typically contain from about 1% to about 30% by weight, such as from about 0.5% to about 20% by weight, particularly from about 1% to about 15% by weight, from about 3% to about 10% by weight, such as from about 3% to about 5% by weight of cationic surfactant. Non-limiting examples of cationic surfactants include alkyl dimethylethanolamine quats (ADMEAQ), cetyltrimethylammonium bromide (CTAB), dimethyldistearylammonium chloride (DSDMAC), and alkylbenzyldimethylammonium, alkyl quaternary ammonium compounds, alkoxylated quaternary ammonium (AQA) compounds, ester quats, and combinations thereof.

[0202] When included, the cleaning composition will typically contain about 1 wt % to about 40 wt %, for example, about 5 wt % to about 30 wt %, particularly about 2 wt % to about 20 wt %, about 3 wt % to about 10 wt %, for example, about 5 wt % to about 25 wt %, or about 8 wt % to about 15 wt % of a nonionic surfactant. Non-limiting examples of nonionic surfactants include alcohol ethoxylates (AE or AEO), such as the AEO series, e.g., AEO-7, alcohol propoxylates, in particular propoxylated fatty alcohols (PFA), ethoxylated and propoxylated alcohols, alkoxylated fatty acid alkyl esters, such as ethoxylated and / or propoxylated fatty acid alkyl esters (in particular methyl ester ethoxylates, MEE), alkyl polyglycosides (APG), alkoxylated amines, fatty acid monoethanolamides (FAM), fatty acid diethanolamides (FADA), ethoxylated fatty acid monoethanolamides (EFAM), propoxylated fatty acid monoethanolamides (PFAM), polyhydroxyalkyl fatty acid amides, or N-acyl N-alkyl derivatives of glucosamine (glucamides, GA, or fatty acid glucamides, FAGA), as well as products available under the trade names SPAN and TWEEN®, and combinations thereof.

[0203] When included, cleaning compositions will typically contain from about 0.01 to about 10% by weight of a semi-polar surfactant. Non-limiting examples of semi-polar surfactants include amine oxides (AOs), such as alkyl dimethyl amine oxides, particularly N-(cocoalkyl)-N,N-dimethyl amine oxide and N-(tallow-alkyl)-N,N-bis(2-hydroxyethyl) amine oxide, and combinations thereof.

[0204] If included, the cleaning composition will typically contain from about 0.01% to about 10% by weight of a zwitterionic surfactant. Non-limiting examples of zwitterionic surfactants include betaines, such as alkyldimethylbetaines, sulfobetaines, and combinations thereof.

[0205] Additional bio-based surfactants may be used, for example, the surfactant is a sugar-based non-ionic surfactant which may be hexyl-β-D-maltopyranoside, thiomaltopyranoside, or cyclic maltopyranoside, as described, for example, in EP 2516606 B1.

[0206] Hydrotropes A hydrotrope is a compound that solubilizes hydrophobic compounds in aqueous solutions (or substances of opposite polarity in a non-polar environment). Typically, hydrotropes have both hydrophilic and hydrophobic properties (so-called amphiphilicity, as known from surfactants); however, the molecular structure of hydrotropes generally does not favor spontaneous self-aggregation (see, for example, the review by Hodgdon and Kaler (2007), Current Opinion in Colloid & Interface Science 12:121-128). Hydrotropes do not exhibit a limiting concentration above which self-aggregation occurs, as found for surfactants and lipids that form micellar, lamellar, or other well-defined mesophases. Instead, many hydrotropes exhibit a sustained aggregation process in which the size of the aggregates increases with increasing concentration. However, many hydrotropes modify the phase behavior, stability, and colloidal properties of systems containing polar and non-polar substances, including mixtures of water, oil, surfactants, and polymers. Hydrotropes are classically used throughout industries ranging from pharmaceutical, personal care, and food to technical applications. The use of hydrotropes in cleaning compositions allows for more concentrated formulations of surfactants (such as processes that concentrate liquid detergents by removing water) without inducing undesirable phenomena such as phase separation or high viscosity.

[0207] The cleaning composition may contain 0-10% by weight, such as 0.2-8% by weight, such as about 0.5% to about 5% or about 3% to about 5% of a hydrotrope. Any hydrotrope known in the art for use in cleaning compositions may be utilized. Non-limiting examples of hydrotropes include sodium benzenesulfonate, sodium p-toluenesulfonate (STS), sodium xylenesulfonate (SXS), sodium cumenesulfonate (SCS), sodium cymenesulfonate, amine oxides, alcohols and polyglycol ethers, sodium hydroxynaphthoate, sodium hydroxynaphthalenesulfonate, sodium ethylhexyl sulfate, and combinations thereof.

[0208] Builders and co-builders The cleaning composition may contain about 0 to 50% by weight, for example, about 0.5% to about 30% by weight, 1% to 10% by weight of detergent builder or co-builder, or mixtures thereof. The builder and / or co-builder may specifically be a chelating agent that forms a water-soluble complex with Ca and Mg. Any builder and / or co-builder known in the art for use in cleaning detergents may be utilized.

[0209] Non-limiting examples of builders include zeolites, diphosphates (pyrophosphates), triphosphates such as sodium triphosphate (STP or STPP), carbonates such as sodium carbonate, soluble silicates such as sodium metasilicate, layered silicates (e.g., SKS-6 from Clariant), ethanolamines such as 2-aminoethan-1-ol (MEA), diethanolamine (DEA, also known as 2,2'-iminodiethane-1-ol), triethanolamine (TEA, also known as 2,2',2''-nitrilotriethane-1-ol), and (carboxymethyl)inulin (CMI), and combinations thereof.

[0210] The cleaning composition may also contain about 0% to 50% by weight, for example about 5% to about 30% by weight, of a detergent co-builder. The cleaning composition may include the co-builder alone or in combination with a builder, for example a zeolite builder. Non-limiting examples of co-builders include homopolymers of polyacrylates or copolymers thereof, for example poly(acrylic acid) (PAA) or copoly(acrylic acid / maleic acid) (PAA / PMA). Further non-limiting examples include citrates, chelating agents, for example aminocarboxylates, aminopolycarboxylates, and phosphonates, and alkyl or alkenyl succinic acids.Additional examples include 2,2',2"-nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), iminodisuccinic acid (IDS), ethylenediamine-N,N'-disuccinic acid (EDDS), methylglycine diacetate (MGDA), glutamic acid-N,N-diacetate (GLDA), 1-hydroxyethane-1,1-diylbis(phosphonic acid (HEDP)), ethylenediaminetetramethylenetetrakis(phosphonic acid) ( EDTMPA), diethylenetriaminepentamethylenepentakis(phosphonic acid) (DTMPA or DTPMPA), N-(2-hydroxyethyl)iminodiacetic acid (EDG), aspartic acid-N-monoacetic acid (ASMA), aspartic acid-N,N-diacetic acid (ASDA), aspartic acid-N-monopropionic acid (ASMP), iminodisuccinic acid (IDA), N-(2-sulfomethyl)aspartic acid (SMAS), N-(2-sulfoethyl)aspartic acid (SEAS ), N-(2-sulfomethyl)glutamic acid (SMGL), N-(2-sulfoethyl)glutamic acid (SEGL), N-methyliminodiacetic acid (MIDA), α-alanine-N,N-diacetic acid (α-ALDA), serine-N,N-diacetic acid (SEDA), isoserine-N,N-diacetic acid (ISDA), phenylalanine-N,N-diacetic acid (PHDA), anthranilic acid-N,N-diacetic acid (ANDA), sulfanilic acid-N,N-diacetic acid (SLDA), taurine-N,N-diacetic acid Acid (TUDA), sulfomethyl-N,N-diacetic acid (SMDA), N-(2-hydroxyethyl)ethylenediamine-N,N',N''-triacetic acid (HEDTA), diethanolglycine (DEG), and aminotrimethylenetris(phosphonic acid) (ATMP), and combinations and salts thereof. Further exemplary builders and / or cobuilders are described, for example, in WO 09 / 102854 and U.S. Pat. No. 5,977,053.

[0211] polymer The cleaning composition may contain 0.005 to 10% by weight of the polymer, for example, 0.5 to 5% by weight, 2 to 5% by weight, 0.5 to 2% by weight, or 0.2 to 1% by weight. Any polymer known in the art for use in detergents may be utilized. The polymer may function as a co-builder as described above, or may provide anti-redeposition, fiber protection, soil release, dye transfer inhibition, grease cleaning, and / or defoaming properties. Some polymers may have two or more of the above properties. Exemplary polymers include (carboxymethyl)cellulose (CMC), poly(vinyl alcohol) (PVA), poly(ethylene glycol) or poly(ethylene oxide) (PEG or PEO), ethoxylated poly(ethyleneimine), (carboxymethyl)inulin (CMI), carboxylate polymers and d, as well as lauryl methacrylate / acrylic acid copolymers, hydrophobically modified CMC (HM-CMC), silicones, copolycarboxylates such as polyacrylates, maleic acid / acrylic acid copolymers, acrylic acid copolymers, and the like. Examples of suitable copolymers include acrylate / styrene copolymers, poly(aspartic)acipolymers of terephthalic acid and oligomeric glycols, copolymers of poly(ethylene terephthalate) and poly(oxyethene terephthalate) (PET-POET), poly(vinylpyrrolidone) (PVP), poly(vinylimidazole) (PVI), poly(vinylpyridine-N-oxide) (PVPO or PVPNO), and copoly(vinylimidazole / vinylpyrrolidone) (PVPVI). Suitable examples include PVP-K15, PVP-K30, ChromaBond S-400, ChromaBond S-403E, and ChromaBond S-100 manufactured by Ashland Aqualon, and Sokalan® HP 165, Sokalan® HP 50 (dispersant), Sokalan® HP 53 (dispersant), Sokalan® HP 59 (dispersant), Sokalan® HP 56 (stain transfer inhibitor), Sokalan® HP 66 K (stain transfer inhibitor) manufactured by BASF.Further exemplary polymers include sulfonated polycarboxylates, polyethylene oxide and polypropylene oxide (PEO-PPO), and diquaternium ethoxysulfate. A particularly preferred polymer is the ethoxylated homopolymer Sokalan® HP 20 from BASF, which helps prevent redeposition of soils in the cleaning solution. Further exemplary polymers include sulfonated polycarboxylates, ethylene oxide-propylene oxide copolymers (PEO-PPO), copolymers of PEG with vinyl acetate, and diquaternium ethoxysulfate or quaternized sulfated ethoxylated hexamethylenediamine. Other exemplary polymers are disclosed, for example, in WO 2006 / 130575. Also contemplated are salts of the above-mentioned polymers.

[0212] Auxiliary substances Also, any detergent auxiliary ingredient known in the art may be utilized.Exemplary auxiliary substances may include anti-corrosion agents, anti-shrinkage agents, anti-soil redeposition agents, germicides, corrosion inhibitors (or "rust inhibitors"), disintegrants, dyes, enzyme stabilizers (including boric acid, borates, CMC, and / or polyols, such as propylene glycol), suds suppressors, alone or in combination.The selection of such ingredients is well within the skill of the art.In some embodiments, the cleaning composition of the present invention may include a corrosion inhibitor to prevent rust or corrosion of medical devices after washing or cleaning.

[0213] Cleaning composition product formulation The cleaning compositions of the present invention may be formulated in any convenient form, such as a liquid, a tablet, a pouch having one or more compartments, a foam, or a spray. In one embodiment, the cleaning composition is formulated as a liquid detergent.

[0214] The pouch can be configured as a single compartment or multiple compartments. It can be of any form, shape, and material suitable for holding the composition, such as by preventing release of the composition from the pouch prior to contact with water. The pouch is made of a water-soluble film that encompasses the content. The content can be divided into the pouch compartments. The preferred film is a polymeric material, preferably a polymer that is formed into a film or sheet. Preferred polymers, copolymers, or derivatives thereof are selected polyacrylates and water-soluble acrylate copolymers, methylcellulose, carboxymethylcellulose, sodium dextrin, ethylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose, maltodextrin, polymethacrylates, and most preferably polyvinyl alcohol copolymers and hydroxypropylmethylcellulose (HPMC). Preferably, the level of polymer, such as PVA, in the film is at least about 60%. The preferred average molecular weight will typically be about 20,000 to about 150,000. The film can also be a blended composition comprising a hydrolytically degradable water-soluble polymer blend, such as polylactide and polyvinyl alcohol (known under the trade name M8630 sold by MonoSol LLC, Indiana, USA), and a plasticizer, such as glycerol, ethylene glycerol, propylene glycol, sorbitol, and mixtures thereof. The pouch can contain solid cleaning compositions or components and / or liquid cleaning compositions or components separated by a water-soluble film.

[0215] The detergent or cleaning ingredients may be physically separated from one another by compartments in a water-soluble pouch or in different layers of a tablet, thereby avoiding undesirable storage interactions between the ingredients.

[0216] The non-unit dose liquid or gel detergent may be water-soluble and typically contains at least 20% by weight and up to 95% water, for example up to about 70%, up to about 65%, up to about 55%, up to about 45%, up to about 35% water. Other types of liquids may be included in the aqueous liquid or gel, including but not limited to alkanols, amines, diols, ethers, and polyols. The water-soluble liquid or gel detergent may contain 0-30% organic solvent. The liquid or gel detergent may be non-water-soluble.

[0217] Formulation of Enzymes in Cogranules The enzymes of the invention may be formulated, for example, as co-granules combining one or more enzymes, where each enzyme will be present in more granules to ensure uniform distribution of the enzymes in the detergent. This also reduces physical separation of the various enzymes due to different particle sizes. A method for making multi-enzyme co-granules for the detergent industry is disclosed in IP.com disclosure IPCOM000200739D.

[0218] Another example of formulating enzymes using co-granules is disclosed in WO 2013 / 188331, which relates to a cleaning composition comprising: (a) a multi-enzyme co-granule; (b) less than 10 wt. of a zeolite (on anhydrous basis); and (c) less than 10 wt. of a phosphate (on anhydrous basis), wherein the enzyme co-granule comprises 10-98 wt. % of a moisture sink component, and the composition additionally comprises 20-80 wt. % of a detergent moisture sink component.

[0219] The multi-enzyme co-granules may comprise an enzyme of the invention and one or more additional enzymes selected from the group consisting of amylase, lipase, cellulase, mannanase, hemicellulase, peroxidase, xylanase, phospholipase, esterase, cutinase, keratinase, reductase, oxidase, phenoloxidase, lipoxygenase, ligninase, pullulanase, malanase, β-glucanase, arabinosidase, hyaluronidase, laccase, perhydrolase, peroxidase, and mixtures thereof.

[0220] The invention is further described in the following paragraphs. 1. A method for cleaning a medical device, comprising: (a) contacting the medical device with a cleaning solution comprising two or more enzymes selected from the group consisting of a protease, an enzyme having DNase activity, and an enzyme having hexosaminidase activity; and optionally (b) rinsing the medical device; The method includes: 2. The method according to paragraph 1, wherein the medical device is or includes an endoscope, e.g., a cystoscope, nephroscope, bronchoscope, laryngoscope, otoscope, arthroscope, laparoscope, and gastrointestinal endoscope; a surgical instrument, e.g., a scalpel, a hemostat, a forceps, a scissors, a retractor, a tracheotomy instrument, and a clamp; and a dental instrument, e.g., a scale, a curette, a serrated cotton pliers, a dental mirror. 3. The method according to paragraph 1 or 2, wherein the method further comprises the step of pre-treating the medical device with a disinfectant prior to step (a), preferably the disinfectant is selected from the group consisting of peracetic acid, hydrogen peroxide, potassium permanganate, chlorine dioxide, and ethanol. 4. A method according to any of the preceding paragraphs, wherein the medical device is rinsed with water or with a solution containing a disinfectant, such as peracetic acid, hydrogen peroxide, potassium permanganate, chlorine dioxide, or ethanol. 5. Medical devices are contaminated with biofilms, which are composed of bacteria such as Escherichia coli, Klebsiella pneumoniae, Salmonella spp., Mycobacterium spp., Enterococcus faecalis, Enterobacter cloacae, Proteus mirabilis, Serratia marcescens, Staphylococcus aureus, e.g. Staphylococcus aureus ATCC 61161, and / or Staphylococcus aureus ATCC 61161. 6538, the method according to any of the preceding paragraphs, wherein the antibody is produced or partially produced by a Streptococcus spp. (e.g., S. pyogenes, S. agalactiae, or S. pneumoniae), Haemophilus influenzae, Pseudomonas aeruginosa, Clostridium perfringens, Chlamydia trachomatis, Candida albicans, and / or Bacillus anthracis. 6. The method according to any of the preceding paragraphs, wherein the enzyme with DNase activity is of microbial origin, such as of fungal or bacterial origin. 7. The method according to any of the preceding paragraphs, wherein the enzyme having DNase activity is a polypeptide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO:1, 2, 3, or 4. 8. The enzyme having DNase activity has one or more (e.g., 2, 3, 4, 5, or 6) motifs selected from the group consisting of [T / D / S][G / N]PQL (SEQ ID NO: 23), [F / L / Y / I]A[N / R]D[L / I / P / V] (SEQ ID NO: 24), C[D / N]T[A / R] (SEQ ID NO: 25), [D / Q][I / V]DH (SEQ ID NO: 26), [D / M / L][S / T]GYSR[D / N] (SEQ ID NO: 27), and ASXNRSKG (SEQ ID NO: 28). 4、38%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 100% sequence identity to the mature polypeptide of SEQ ID NO:4. 9. The enzyme having DNase activity comprises one or both of the motifs [D / M / L][S / T]GYSR[D / N] (SEQ ID NO: 27) and ASXNRSKG (SEQ ID NO: 28), and optionally further comprises one or more (e.g., 2, 3, 4) of the motifs selected from the group consisting of [T / D / S][G / N]PQL (SEQ ID NO: 23), [F / L / Y / I]A[N / R]D[L / I / P / V] (SEQ ID NO: 24), C[D / N]T[A / R] (SEQ ID NO: 25), [D / Q][I / V]DH (SEQ ID NO: 26). The method of any of the preceding paragraphs, wherein the enzyme having DNase activity comprises at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO:4. 10. Enzymes with DNase activity include S26*, D32E,Q, V35I, K36C,H, G37R, F43W, D46G, A55I, N68D, S69V, A76I, K82S,T, P84D,T, K86E,G,L,N,Q,T,V,Y, A91R, L92E, K95I, P97E,N, A101E, Q102E, K105N,G,Q,T,D, A111P, F112Y,W, S115T, V127T, L129K, N133Q, G137R, V138C, N140H, G141Q,R, S144E, N146A, K147N,E, V148I, A149D,E, F, Q150D,E, P153D,V, S154E, K155E,F,L,S,T, Q157D,E, Q158D, T159Q, K160D, G161R, T170Q, A172D,E,H ,R, G181N, K185*, V187N,Y, N191*, K192A,I, D197K,S, G199Q, Q208V, E211Y,T,P, N213S, N214D, N217A and Y218D,E, wherein the position numbers correspond to the positions of SEQ ID NO:2, and the variant has DNase activity and has at least 80%, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, and less than 100%, sequence identity to the polypeptide of SEQ ID NO:2 or 3. 11. The method according to any of the preceding paragraphs, wherein the enzyme having DNase activity is a variant comprising one or more alterations at positions 1, 13, 22, 25, 27, 33, 39, 42, 56, 57, 59, 65, 76, 77, 109, 116, 127, 144, 147, 149, 167, 175, and 181 of SEQ ID NO:4, wherein the variant has DNase activity and has at least 80%, e.g. at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to the mature polypeptide of SEQ ID NO:4. 12. The enzyme having DNase activity is a variant of SEQ ID NO: 4 and comprises one or more substitutions of T1I, S13Y, T22P, S25P, S27L, L33K, S39P, S42G, D56I, S57W, S59V, T65V, V76L, T77Y, Q109R, S116D, T127V, S144P, A147H, G149N, S167L, G175D, and S181L, e.g., T22P+S167L and / or G175D, where the position numbers correspond to the positions in SEQ ID NO: 4, and the variant has DNase activity and is at least 80%, e.g., at least 10. The method according to paragraph 9, having 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% and less than 100% sequence identity, preferably wherein the variant comprises or consists of SEQ ID NO: 4 with the substitutions T1I, S13Y, T22P, S27L, L33K, S39P, S42G, D56I, S57W, S59V, T65V, V76L, Q109R, S116D, T127V, S144P, A147H, S167L, and G175D. 13. The method according to any of the preceding paragraphs, wherein the enzyme having DNase activity is a variant of SEQ ID NO: 4 with the following substitutions: T1I+S13Y+T22P+S27L+L33K+S39P+S42G+D56I+S57W+S59V+T65V+V76L+Q109R+S116D+T127V+S144P+A147H+S167L+G175D. 14. The method according to any of the preceding paragraphs, wherein the enzyme having DNase activity is a variant of SEQ ID NO: 4 with the following substitutions: T1I+S13Y+T22P+S25P+S27L+S39P+S42G+S57W+S59V+T65V+V76L+T77Y+Q109R+S116D+S144P+A147H+G149N+S167L+G175D+S181L. 15. The method according to any of the preceding paragraphs, wherein the enzyme with DNase activity is a variant of SEQ ID NO:2 with the following substitutions: S69V+Q102E+K105N+ A111P+S115T+Q150E+G161R+G181N+V187N+K192I. 16. The method according to any of the preceding paragraphs, wherein the enzyme having DNase activity is a variant of SEQ ID NO:2 with the following substitutions: D32E+V35I+S69V+K86E+Q102E+K105N+A111P+S115T+V127T+G137R+K147N+Q150E+K155E+T159Q+G161R+A172D+G181N+V187N+K192A+Q208V+N217A. 17. The method according to any of the preceding paragraphs, wherein the enzyme having hexosaminidase activity is of microbial origin, such as of fungal or bacterial origin. 18. The method according to any of the preceding paragraphs, wherein the enzyme having hexosaminidase activity has N-acetylglucosaminidase activity and / or β-N-acetylglucosaminidase activity. 19. The method according to any of the preceding paragraphs, wherein the enzyme having hexosaminidase activity is a polypeptide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO: 10, 11, 19, 20, 21, or 22. 20. The method according to any of the preceding paragraphs, wherein the enzyme having hexosaminidase activity is a variant of the polypeptide of SEQ ID NO: 10 comprising one or more alterations at positions 3, 15, 49, 59, 163, 186, 225, 227, 232, 235, 252, 260, 272, 279, 281, 308, 309, and 312 of SEQ ID NO: 10, wherein the variant has hexosaminidase activity and has at least 80%, e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, and less than 100%, sequence identity to the mature polypeptide of SEQ ID NO: 10. 21. The enzyme having hexosaminidase activity is a variant of the polypeptide of SEQ ID NO: 10, comprising one or more substitutions of Q3I, H15Y, A49W, N59E, S163P, S186R, S225G, N227T, E232D, G235W, N252P, N260Q, H272V, S279D, Y281P, K308Q, K309E, and K312Q of SEQ ID NO: 10, where the position numbers correspond to the positions of SEQ ID NO: 10, and the variant has hexosaminidase activity and is at least 80%, e.g., at least 85%, relative to the mature polypeptide of SEQ ID NO: 10. 19. The method according to paragraph 18, wherein the variant has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% and less than 100% sequence identity with the variant, preferably wherein the variant comprises or consists of SEQ ID NO: 10 with the substitutions Q3I, H15Y, A49W, N59E, S163P, S186R, S225G, N227T, E232D, G235W, N252P, N260Q, H272V, S279D, Y281P, K308Q, K309E, and K312Q. 22. The method according to any of the preceding paragraphs, wherein the polypeptide having hexosaminidase activity comprises one or more motifs selected from the group consisting of GXDE (SEQ ID NO: 12), [EQ][NRSHA][YVFL][AGSTC][IVLF][EAQYN][SN] (SEQ ID NO: 13), [VIM][LIV]G[GAV]DE[VI][PSA] (SEQ ID NO: 14), WND[SQR][IVL][TLVM] (SEQ ID NO: 15), QSTL (SEQ ID NO: 16), NKFFY (SEQ ID NO: 17), and NLD[DR]S (SEQ ID NO: 18). 23. The method according to any of the preceding paragraphs, wherein the protease has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO:5, 6, 7, 8, 9, or 29. 24. The protease is a variant of the polypeptide of SEQ ID NO:6 comprising an alteration at one or more positions corresponding to positions 3, 4, 9, 15, 22, 43, 68, 76, 87, 99, 101, 103, 104, 118, 128, 160, 167, 170, 184, 194, 199, 205, 206, 209, 217, 218, 222, 245, 259, 261, and 262, where the position numbers correspond to positions in SEQ ID NO:5, and each alteration is independently selected from the group consisting of: 21. The method according to paragraph 20, wherein the variant has protease activity and has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, and less than 100%, sequence identity to the polypeptide of SEQ ID NO:6. 25. Proteases: (a) a variant of the polypeptide of SEQ ID NO: 6, selected from the group consisting of: S3T, V4I, S9E, S9R, A15T, T22A, N43R, V68A, N76D, S87N, S99D, S99G, S99A, S99SE, S101E, S101N, S101R, S103A, V104I, G118M, S128Q, G160S, Y167A, R170S, N184E, A194P, V199M, V205I, Q206L, Y209W, L217D, L217Q, N218D, M222S, Q245R, S259D, N261W, and L262E. a variant having one or more substitutions selected from the group consisting of N76D+Q206L+Y209W or Y209W+N261W+L262E substitutions, wherein the position numbers correspond to the positions of SEQ ID NO:5, the variant having protease activity and having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to SEQ ID NO:6; (b) a variant of the polypeptide of SEQ ID NO:6 comprising the substitution S87N, wherein the variant has protease activity, and the position corresponds to the position of SEQ ID NO:5, wherein the variant has protease activity and has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to SEQ ID NO:6; (c) a variant of the polypeptide of SEQ ID NO: 6 comprising the substitutions Y167A+R170S+A194P, wherein the position numbers correspond to the positions of SEQ ID NO: 5, wherein the variant has protease activity and has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to SEQ ID NO: 6; (d) a variant of the polypeptide of SEQ ID NO:6 comprising the substitutions S9E+N43R+N76D+V205I+Q206L+Y209W+S259D+N261W+L262E, where the position numbers correspond to the positions of SEQ ID NO:5, wherein the variant has protease activity and has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to SEQ ID NO:6; (e) a variant of the polypeptide of SEQ ID NO:6 comprising the substitutions S3T+N43R+N76D+S87N+G118M+S128Q+N184E+V205I+Q206L+Y209W+S259D+N261W+L262E, wherein the position numbers correspond to the positions of SEQ ID NO:5, wherein the variant has protease activity and has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, and less than 100%, sequence identity to SEQ ID NO:6; (f) a variant of the polypeptide of SEQ ID NO: 6 comprising the substitutions T22A+N43R+S87N+V205L+Q206L+Y209W+S259D+N261W+L262E, where the position numbers correspond to the positions of SEQ ID NO:5, wherein the variant has protease activity and has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, and less than 100%, sequence identity to SEQ ID NO: 6; (g) a variant of the polypeptide of SEQ ID NO:8 comprising the substitutions A68S+T77N+T78I+G127S+A128P+G165Q+N184Q+A202V+N217S+S258P, where the position numbers correspond to positions in SEQ ID NO:8, wherein the variant has protease activity and has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, and less than 100%, sequence identity to SEQ ID NO:8; (h) a variant comprising a substitution at one or more positions corresponding to positions 171, 173, 175, 179 or 180 of SEQ ID NO: 1 of WO 2004 / 067737, wherein the variant has protease activity and has a sequence identity of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, to SEQ ID NO: 1 of WO 2004 / 067737; (i)X3V, X9[E,R], X22[R,A], X43R, X61[E,D], X62[E,D], X76[D], X87N, X101[E,G,D,N, M], X103A, X104I, X118[V,R,M], X120V, X128[A,L,S,Q], X129Q, X130A, X160D, X184[E,D ], X185[E,D], 188[E,D], X191N, X194P, X205I, X206L, X209W, X216V, X217[Q,D,E], X218 Consists of [D,E,S], X232V, X245R, X248D, X256[E,D], X259[E,D], X261[E,D,W], and X262[E,D] a protease variant comprising one or more substitutions compared to a parent protease selected from the group, wherein the position number corresponds to a position in BPN' (SEQ ID NO:5) and "X" represents any amino acid residue present at the designated position in the parent protease, wherein the variant has protease activity and has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, and less than 100%, sequence identity to SEQ ID NO:5 or SEQ ID NO:6; and (j) A protease variant comprising any of the following sets of substitutions compared to a parent protease, wherein the parent protease has an amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6, or has at least 80% sequence identity to SEQ ID NO:5 or SEQ ID NO:6, where the position numbers correspond to positions in BPN' (SEQ ID NO:5), and "X" represents any amino acid residue present at the specified position in the parent protease, and the set of substitutions is: i.X9R+X15T+X68A+X218D+X245R, ii.X9R+X15T+X68A+X245R, iii.X61E+X194P+X205I+X261D, iv.X61D+X205I+X245R, v.X61E+X194P+X205I+X261D, vi.X87N+X118V+X128L+X129Q+X130A, vii.X87N+X101M+X118V+X128L+X129Q+X130A, viii.X76D+X87R+X118R+X128L+X129Q+X130A, ix.X22A+X62D+X101G+X188D+X232V+X245R, x.X103A+X104I, xi.X22R+X101G+X232V+X245R, xii.X103A+X104I+X156D, xiii.X103A+X104I+X261E, xiv.X62D+X245R, xv.X101N+X128A+X217Q, xvi.X101E+X217Q, xvii.X101E+X217D, xviii.X9E+X43R+X262E, xix.X76D+X43R+X209W, xx.X205I+X206L+X209W, xxi.X185E+X188E+X205I, xxii.X256D+X261W+X262E, xxiii.X191N+X209W, xxiv.X261E+X262E, xxv.X261E+X262D, and xxvi.X167A+X170S+X194P is selected from the group consisting of The protease variant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to SEQ ID NO: 5 or 6. 2. A method according to any of the preceding paragraphs, 26. The method according to any of the preceding paragraphs, wherein the protease variant comprises the amino acid sequence of SEQ ID NO: 6 with the substitutions Y167A+R170S+A194P, wherein the variant has protease activity, and the position numbers correspond to the positions of SEQ ID NO: 5. 27. The method according to any of the preceding paragraphs, wherein the protease variant comprises the amino acid sequence of SEQ ID NO: 6 with the substitutions S9E+N43R+N76D+V205I+Q206L+Y209W+S259D+N261W+L262E, wherein the variant has protease activity, and the position numbers correspond to the positions of SEQ ID NO: 5. 28. The method according to any of the preceding paragraphs, wherein the protease is an enzyme having or consisting of the amino acid sequence of SEQ ID NO: 6, 7, 8, or 9. 29. A method according to any of the preceding paragraphs, wherein the amount of biofilm present on the medical device after cleaning is reduced compared to cleaning with a cleaning solution without the two or more enzymes. 30. The method according to any of the preceding paragraphs, wherein the wash solution further comprises one or more additional enzymes selected from the group consisting of amylase, lipase, cellulase, mannanase, hemicellulase, peroxidase, xylanase, phospholipase, esterase, cutinase, keratinase, reductase, oxidase, phenoloxidase, lipoxygenase, ligninase, pullulanase, malanase, β-glucanase, arabinosidase, hyaluronidase, laccase, perhydrolase, and peroxidase. 31. The method according to any of the preceding paragraphs, wherein the concentration of each enzyme in the cleaning solution is in the range of 0.0005 to 100 ppm enzyme protein, e.g., in the range of 0.001 to 50 ppm, in the range of 0.005 to 20 ppm, in the range of 0.01 to 10 ppm, or in the range of 0.05 to 5 ppm enzyme protein. 32. A method according to any of the preceding paragraphs, wherein the cleaning solution comprises a cleaning composition according to any of paragraphs 37-50. 33. Use of an enzyme composition for cleaning a medical device, the enzyme composition comprising two or more enzymes selected from the group consisting of a protease, an enzyme having DNase activity, and an enzyme having hexosaminidase activity. 34. Medical devices include endoscopes, such as cystoscopes, nephroscopes, bronchoscopes, laryngoscopes, otoscopes, arthroscopes, laparoscopes, and gastrointestinal endoscopes; surgical instruments, such as scalpels, hemostats, forceps, scissors, retractors, tracheotomy instruments, and clamps; and dental instruments, such as scales, curettes, serrated cotton pliers, dental mirrors, in accordance with the preceding use paragraph. 35. Medical devices are contaminated with biofilms, which are composed of Enterobacteriacae (e.g. Escherichia coli), Klebsiella pneumoniae, Salmonella spp., Mycobacterium spp., Enterococcus faecalis, Enterobacter cloacae, Proteus mirabilis, Serratia marcescens, Staphylococcus aureus, e.g. Staphylococcus aureus ATCC 61161, and / or other bacteria. 6538, the use according to any of the preceding use paragraphs, which is produced or partly produced by Streptococcus spp. (e.g. S. pyogenes, S. agalactiae, or S. pneumoniae), Haemophilus influenzae, Pseudomonas aeruginosa, Clostridium perfringens, Chlamydia trachomatis, Candida albicans, and / or Bacillus anthracis. 36. Use according to any of the preceding use paragraphs, wherein the enzyme having DNase activity is as defined in any of the preceding paragraphs 6 to 16. 37. Use according to any of the preceding use paragraphs, wherein the enzyme having hexosaminidase activity is as defined in any of the preceding paragraphs 17 to 22. 38. Use according to any of the preceding use paragraphs, wherein the protease is as defined in any of the preceding paragraphs 23 to 28. 39. Use according to any of the previous use paragraphs, wherein the amount of biofilm present on the medical device after cleaning is reduced compared to cleaning with a composition without said two or more enzymes. 40. Use according to any of the preceding use paragraphs, in which a composition according to paragraphs 41 to 54 is used. 41. A composition for cleaning a medical device, comprising a surfactant and two or more enzymes selected from the group consisting of a protease, an enzyme having DNase activity, and an enzyme having hexosaminidase activity. 42. Medical devices are contaminated with biofilms, which are composed of Enterobacteriacae (e.g. Escherichia coli), Klebsiella pneumoniae, Salmonella spp., Mycobacterium spp., Enterococcus faecalis, Enterobacter cloacae, Proteus mirabilis, Serratia marcescens, Staphylococcus aureus, e.g. Staphylococcus aureus ATCC 61161, and / or other bacteria. 6538, the composition according to paragraph 39 being produced or partly produced by Streptococcus (e.g. S. pyogenes, S. agalactiae, or S. pneumoniae), Haemophilus influenzae, Pseudomonas aeruginosa, Clostridium perfringens, Chlamydia trachomatis, Candida albicans, Bacillus anthracis. 43. A composition according to any of the preceding composition paragraphs, wherein the enzyme having DNase activity is as defined in any of the preceding paragraphs 6 to 16. 44. A composition according to any of the preceding composition paragraphs, wherein the enzyme having hexosaminidase activity is as defined in any of the preceding paragraphs 17 to 22. 45. A composition according to any of the preceding composition paragraphs, wherein the protease is as defined in any of the preceding paragraphs 23 to 28. 46. ​​A composition according to any of the preceding composition paragraphs, wherein the amount of biofilm present on a medical device after cleaning with the composition is reduced compared to cleaning with a composition lacking the two or more enzymes. 47. A composition according to any of the preceding composition paragraphs, wherein the surfactant comprises at least a nonionic surfactant, and optionally further comprises a cationic surfactant and / or an anionic surfactant. 48. The composition according to paragraph 45, wherein the non-ionic surfactant is selected from the group consisting of alcohol ethoxylates (AE or AEO), alcohol propoxylates, propoxylated fatty alcohols (PFA), alkoxylated fatty acid alkyl esters such as ethoxylated and / or propoxylated fatty acid alkyl esters, alkylphenol ethoxylates (APEs), nonylphenol ethoxylates (NPEs), alkyl polyglycosides (APGs), alkoxylated amines, fatty acid monoethanolamides (FAMs), fatty acid diethanolamides (FADAs), ethoxylated fatty acid monoethanolamides (EFAMs), propoxylated fatty acid monoethanolamides (PFAMs), polyhydroxyalkyl fatty acid amides, and N-acyl N-alkyl derivatives of glucosamine (glucamides, GAs, or fatty acid glucamides, FAGAs). 49. Anionic surfactants include linear alkylbenzene sulfonates (LAS), isomers of LAS, branched alkylbenzene sulfonates (BABS), phenylalkanesulfonates, alpha-olefinsulfonates (AOS), olefinsulfonates, alkenesulfonates, alkane-2,3-diylbis(sulfates), hydroxyalkanesulfonates and disulfonates, alkyl sulfates (AS) such as sodium dodecyl sulfate (SDS), fatty alcohol sulfates (FAS), primary alcohol sulfates (PAS), alcohol ethers. 46. ​​The composition according to paragraph 45, selected from the group consisting of sulfates (AES or AEOS or FES, secondary alkane sulfonates (SAS), paraffin sulfonates (PS), sulfonate esters, sulfonated fatty acid glycerol esters, alpha-sulfo fatty acid methyl esters (alpha-SFMe or SES) sulfonic acid methyl esters (MES), alkyl- or alkenyl succinic acids, dodecenyl / tetradecenyl succinic acid (DTSA), fatty acid derivatives of amino acids, di- and monoesters of sulfosuccinic acid, or salts of fatty acids (soaps). 50. The composition according to paragraph 45, wherein the cationic surfactant is selected from the group consisting of bis(acyloxyethyl)hydroxyethylmethylammonium methosulfate, dipermoylethylhydroxyethylmonium methosulfate, dihydrogenated tallow hydroxyethylmonium methosulfate, distearoylethylhydroxyethylmonium methosulfate, dioleoylethylhydroxyethylmonium methosulfate alkyl quaternary ammonium compounds, alkoxylated quaternary ammonium (AQA) compounds, other ester quats, and combinations thereof. 51. A composition according to any of the preceding composition paragraphs, wherein the composition comprises at least 1 wt.%, such as at least 2 wt.%, at least 5 wt.% of a non-ionic surfactant, and optionally further comprises 0.2 to 10 wt.% of a builder. 52. The composition according to any of the preceding composition paragraphs, wherein the composition further comprises one or more additional enzymes selected from the group consisting of amylase, lipase, cellulase, mannanase, hemicellulase, peroxidase, xylanase, phospholipase, esterase, cutinase, keratinase, reductase, oxidase, phenoloxidase, lipoxygenase, ligninase, pullulanase, malanase, β-glucanase, arabinosidase, hyaluronidase, laccase, perhydrolase, and peroxidase. 53. A composition according to any of the preceding composition paragraphs, wherein the composition is a liquid detergent, a foam detergent, or a spray detergent. 54. A composition according to any of the preceding composition paragraphs, wherein the composition comprises at least 0.0001 mg of enzyme protein, at least 0.001 mg of enzyme protein, at least 0.006 mg of enzyme protein, at least 0.008 mg of enzyme protein, at least 0.01 mg of enzyme protein, at least 0.1 mg of enzyme protein, at least 0.5 mg of enzyme protein, at least 1 mg of enzyme protein, at least 2 mg of enzyme protein, at least 5 mg of enzyme protein, at least 10 mg of enzyme protein, or at least 20 mg of enzyme protein per gram of composition. 55. A CIP (clean in place) cleaning method comprising carrying out a clean in place process with two or more enzymes selected from the group consisting of a protease, an enzyme having DNase activity, and an enzyme having hexosaminidase activity; and optionally including a rinsing step.

[0221] The present invention is further illustrated in the following non-limiting examples. EXAMPLES

[0222] Materials and Methods Model detergent MC (liquid) This is an example of a cleaning composition that can be used in combination with the enzymes of the present invention.

[0223] Ingredients: 5% MPG (propylene glycol), 5% Pluronic® PE 4300 (PO / EO block polymer; 70% / 30%, approximately 1750 g / mol), 2% Plurafac LF 305 (fatty alcohol alkoxylate; C6-10+EO / PO), 1% MGDA (methylglycine diacetic acid, 1% TEA (triethanolamine) (all percentages are w / w). The pH was adjusted to 8.7 with phosphoric acid.

[0224] Enzyme assays Assay I Testing for DNase activity DNase activity can be determined with DNase Test Agar with Methyl Green (BD, Franklin Lakes, NJ, USA), which is prepared according to the supplier's manual. Briefly, 21 g of agar is dissolved in 500 ml of water and then autoclaved at 121°C for 15 min. The autoclaved agar is brought to 48°C in a water bath, and 20 ml of the agar is poured into a Petri dish and incubated overnight at room temperature to solidify. 5 μl of enzyme solution is added onto the solidified agar plate, and DNase activity is observed as a colorless zone around the spotted enzyme solution.

[0225] Assay II Hexosaminidase activity test Hexosaminidase activity can be determined using 4-nitrophenyl N-acetyl-β-D-glucosaminide (Sigma-Aldrich) as substrate. Enzyme reactions are carried out in triplicate in 96-well flat-bottom polystyrene microtiter plates (Thermo Scientific) with the following conditions: 50 mM 2-(N-morpholino)ethanesulfonic acid pH 6 buffer, 1.5 mg / ml 4-nitrophenyl N-acetyl-β-D-glucosaminide, and 10, 20, or 50 μg / ml purified enzyme sample in a total reaction volume of 100 μl. A blank sample without polypeptide is run simultaneously. Reactions are carried out at 37° C. in a Thermomixer comfort (Eppendorf). After 10 min incubation, 5 μl of 1 M NaOH is added to each reaction mixture to stop the enzymatic reaction. The absorbance is read at 405 nm using a POLAR star Omega plate reader (BMG LABTECH) to assess the formation of 4-nitrophenolate ions released due to enzymatic hydrolysis of the 4-nitrophenyl N-acetyl-β-D-glucosaminide substrate. The measured absorbance of the reaction carried out with the hexosaminidase polypeptide is higher than that of the blank without the polypeptide, indicating that the tested polypeptide exhibits hexosaminidase activity.

[0226] Assay III Protease activity test Proteolytic activity can be determined by a method using Suc-AAPF-pNA substrate. Suc-AAPF-pNA is the abbreviation of N-succinyl-alanine-alanine-proline-phenylalanine-p-nitroanilide, which is a blocked peptide that can be cleaved by endoproteases. After proteolytic cleavage, yellow free pNA molecules are released and can be measured by visible spectrophotometry at a wavelength of 405 nm. Suc-AAPF-PNA substrate is produced by Bachem (catalog number L1400, dissolved in DMSO).

[0227] The protease samples to be analyzed are diluted in the remaining activity buffer (100 mM Tris, pH 8.6). The assay is performed by transferring 30 μl of the diluted enzyme sample to a 96-well microtiter plate and adding 70 μl of substrate working solution (0.72 mg / ml in 100 mM Tris, pH 9). The solutions are mixed at room temperature and absorbance is measured at OD 405 nm every 20 seconds for 5 minutes.

[0228] The slope of the time-dependent absorbance curve (absorbance per minute) is directly proportional to the activity of the protease of interest under a given set of conditions. The protease sample should be diluted to a level where the slope is linear.

[0229] enzyme DNase 1: DNase corresponding to the polypeptide of SEQ ID NO:3. DNase 2: DNase corresponding to the polypeptide of SEQ ID NO:4. DNase 3: A polypeptide of SEQ ID NO: 4 having the following substitutions: T1I+S13Y+T22P+S27L+L33K+S39P+S42G+D56I+S57W+S59V+T65V+V76L+Q109R+S116D+T127V+S144P+A147H+S167L+G175D. DNase 4: A polypeptide of SEQ ID NO:2 having the following substitutions: S69V+Q102E+K105N+ A111P+S115T+Q150E+G161R+G181N+V187N+K192I. DNase 5: Polypeptide of SEQ ID NO:2 with the following substitutions: D32E+V35I+S69V+K86E+Q102E+K105N+A111P+S115T+V127T+G137R+K147N+Q150E+K155E+T159Q+G161R+A172D+G181N+V187N+K192A+Q208V+N217A. DNase 6: A polypeptide of SEQ ID NO: 4 having the following substitutions: T1I+S13Y+T22P+S25P+S27L+S39P+S42G+S57W+S59V+T65V+V76L+T77Y+Q109R+S116D+S144P+A147H+G149N+S167L+G175D+S181L. Protease 1: Polypeptide of SEQ ID NO: 6 with the following mutations: Y167A+R170S+A194P (position numbers are based on the numbering of SEQ ID NO: 5). Protease 2: Protease corresponding to the polypeptide of SEQ ID NO:8. Protease 3: Protease corresponding to the polypeptide of SEQ ID NO:7. Protease 4: Polypeptide of SEQ ID NO: 6 having the following mutations: S9E+N43R+N76D+V205I+Q206L+Y209W+S259D+N261W+L262E (position numbers are based on the numbering of SEQ ID NO: 5). Hexosaminidase 1: A hexosaminidase corresponding to the polypeptide of SEQ ID NO:10. Hexosaminidase 2: Polypeptide of SEQ ID NO: 10 having the following mutations: Q3I+H15Y+A49W+N59E+S163P+S186R+S225G+N227T+E232D+G235W+N252P+N260Q+H272V+S279D+Y281P+K308Q+K309E+K312Q. Hexosaminidase 3: A hexosaminidase corresponding to the polypeptide of SEQ ID NO:11. Hexosaminidase 4: A hexosaminidase corresponding to the polypeptide of SEQ ID NO:19. Hexosaminidase 5: a hexosaminidase corresponding to the polypeptide of SEQ ID NO:20. Hexosaminidase 6: a hexosaminidase corresponding to the polypeptide of SEQ ID NO:21. Hexosaminidase 7: A hexosaminidase corresponding to the polypeptide of SEQ ID NO:22.

[0230] Example 1 Biofilm removal test using a medical cleaning model detergent MC Staphylococcus aureus ATCC 6538 was used as a model microorganism in this example. The strains were restreaked onto TSA plates (15 g / L tryptone, 5 g / L soy peptone, 5 g / L NaCl, and 15 g / L agar, pH 7.3±0.2) and incubated overnight at 30° C. The strains were then inoculated into 5 mL TSB medium (15 g / L tryptone, 5 g / L soy peptone, 5 g / L NaCl, pH 7.3±0.2) and incubated at 30° C. for 8 hours with shaking at 200 rpm. Cultures were then diluted (1:100 v / v) in TSB + 0.25 wt% glucose (G7528, Sigma-Aldrich) and 100 μL aliquots were added to wells of a 96-well polystyrene microplate (167008, Nunc™ MicroWell™ 96-well, Nunclon Delta-Treated, Flat-Bottom Microplate). Sterile medium was added to control wells. The 96-well microtiter plates were then incubated for 20 hours at 30° C. under static conditions. After incubation, the cultures were removed and rinsed once with deionized water. The bacteria were then killed by 0.1% peracetic acid before a washing step.

[0231] Liquid medical cleaning model detergent MC with the composition given above was used in this example. Washing solutions (5 g / L model detergent MC in deionized water) containing no enzyme (i.e. control) or 5 ppm of one or more enzymes (protease, DNase, and / or hexosaminidase) were added to a 96-well microtiter plate and the plate was incubated at 40° C. for 20 min. Afterwards, the wells were rinsed with deionized water and stained with 0.1% crystal violet (C6158, Sigma-Aldrich) for 5 min at room temperature. Subsequently, the wells were rinsed once with deionized water. The remaining dye was dissolved with 30% acetic acid and the resulting mixture was read at 595 nm (A) with a microplate reader (SpectraMax® M2, Molecular Devices). 595The biofilm removal benefit of the enzymes alone or in combination was expressed as a percentage (%) of biofilm reduction, calculated using the following formula: Biofilm reduction % = (A1-A2) / A1 x 100% A1: A of biofilm treated with detergent without enzyme 595 (i.e. control) A2: A of biofilm treated with detergent plus enzyme 595

[0232] The results are summarized below in Table 1. A higher % biofilm reduction indicates better biofilm removal benefit.

[0233] [Table 1]

[0234] [Table 2]

[0235] [Table 3]

[0236] As can be seen from the above, Tables 1, 2, and 3 show that adding enzymes to medical cleaning detergents provides biofilm removal benefits. The combination of two or three enzymes (protease, DNase, and / or hexosaminidase) showed a synergistic effect on biofilm removal, significantly reducing the biofilm present on the object to be cleaned. This is seen by the biofilm reduction percentage of the enzyme combination being higher than the sum of the biofilm reduction percentages of the individual enzymes. Tables 1, 2, and 3 show that a particularly good effect is obtained by combining dispersin with DNase, especially when dispersin is combined with both DNase and protease.

[0237] Example 2 Cleaning test using model detergent for medical cleaning Staphylococcus aureus 15981 (kindly provided by Inyigo Lasa (Valle et al., Mol Microbiol. 2003 May;48(4):1075-87)) and Staphylococcus epidermidis DSM3270 were used as model microorganisms in this example. Strains were restreaked onto Tryptone Soya Agar (TSA) (pH 7.3) (CM0131; Oxoid Ltd, Basingstoke, UK) and incubated overnight at 37°C. A single colony was then inoculated into 10 mL of TSB and incubated at 37°C, 200 rpm for 16 hours. After growth, cultures were diluted in fresh TSB + 1 wt% glucose (24563; Roquette Freres) to a final ratio of 1:25 (S. aureus:S. epidermidis) and 100 μL aliquots were added to wells of a 96-well polystyrene microplate (167008; Nunc™ MicroWell™ 96-Well, Nunclon Delta-Treated, Flat-Bottom Microplate). Sterile medium was added to control wells. After 18 h at 37°C (static incubation), the microplates were aspirated and treated with a model medical cleaning solution (5 g / L model detergent MC in 5° dH hardness water) with or without enzymes for 1 h at 30°C under static conditions. The microplates were subsequently rinsed with 0.9% NaCl solution and stained with 0.095% crystal violet (SIGMA V5265) for 15 min. After an additional rinsing step, the remaining dye was dissolved using 30% acetic acid solution. The absorbance was measured at 595 nm. The biofilm removal benefit of the enzymes alone or in combination was expressed as a biofilm reduction percentage (%), calculated as described in Example 1. The results are shown in Tables 4-9.

[0238] [Table 4]

[0239] [Table 5]

[0240] [Table 6]

[0241] [Table 7]

[0242] [Table 8]

[0243] [Table 9]

[0244] The results clearly show that the polypeptides of the present invention have cleaning properties in medical cleaning-related detergents, i.e., they destroy and / or remove the tested biofilms or biofilm components, when compared to samples treated with cleaning solutions that do not contain enzymes.The results also show that the enzyme combinations containing protease and hexosaminidase provide superior cleaning properties in model detergents compared to the individual enzymes, given that the cleaning performance (% biofilm reduction) of the enzyme combination compared to the control detergent without enzymes clearly exceeds the sum of the cleaning performances of the individual enzymes.This clearly suggests that a synergistic effect exists between the two enzymes.

[0245] Example 3 Cleaning test using model detergent for medical cleaning Staphylococcus epidermidis DSM3270 was used as a model microorganism in this example. The strain was restreaked on Tryptone Soya Agar (TSA) (pH 7.3) (CM0131; Oxoid Ltd, Basingstoke, UK) and incubated overnight at 37°C. A single colony was then inoculated into 10 mL of TSB and incubated at 37°C, 200 rpm for 16 hours. After growth, the culture was diluted (1:100) into fresh TSB + 1 wt% glucose (24563; Roquette Freres) and 100 μL aliquots were added to wells of a 96-well polystyrene microplate (167008; Nunc™ MicroWell™ 96-Well, Nunclon Delta-Treated, Flat-Bottom Microplate). Sterile medium was added to the control wells. After 18 hours at 37°C (static incubation), the microplates were aspirated and treated with a model medical cleaning solution (5g / L model detergent MC in 5° dH hardness water) with or without enzymes for 1 hour at 30°C under static conditions. The microplates were subsequently rinsed with 0.9% NaCl solution and stained with 0.095% crystal violet (SIGMA V5265) for 15 minutes. After an additional rinsing step, the remaining dye was dissolved using 30% acetic acid solution. The absorbance was measured at 595 nm. The biofilm removal benefit of the enzymes alone or in combination was expressed as a biofilm reduction percentage (%), calculated as described in Example 1. The results are shown in Tables 10 and 11.

[0246] [Table 10]

[0247] [Table 11]

[0248] The results clearly show that the polypeptides of the present invention have cleaning properties in medical cleaning related detergents, i.e., they destroy and / or remove the tested biofilms or biofilm components, when compared to samples treated with cleaning solutions that do not contain enzymes.The results also show that the enzyme combinations containing protease and DNase provide superior cleaning properties in model detergent MC compared to the individual enzymes, given that the cleaning performance (% biofilm reduction) of the enzyme combination clearly exceeds the sum of the performance of the individual enzymes.This clearly suggests that a synergistic effect exists between the two enzymes.

[0249] Example 4 Removal of Pseudomonas aeruginosa (PSA) and PSA biofilms from ultrafiltration membranes Undesirable microorganisms (e.g., pathogenic bacteria) may not only be present in medical devices, but also accumulate in biological manufacturing equipment (e.g., food and beverage production, and biotechnological manufacturing, e.g., enzymatic fermentation processes), such as fermentation and storage tanks, bioreactors, ultrafilters or membranes, pipelines, and other equipment. Such equipment is typically cleaned by cleaning-in-place (CIP) methods, and cleaning is performed without removing or dismantling piping or other equipment. An example of such equipment is the ultrafiltration membrane assembly in a separation module that is often used in enzymatic fermentation processes. Between two batches of fermentation, the ultrafiltration membrane assembly is cleaned using the CIP (clean-in-place) method. Insufficient cleaning may cause pathogenic bacteria, such as PSA, to grow and accumulate on the ultrafiltration membrane assembly. On the other hand, bacteria are difficult to remove, since they form biofilms to protect themselves from detergents and antibiotics. This in turn promotes the growth of PSA on the ultrafiltration membrane, causing contamination of the fermentation broth. In this Example 4, the effect of the enzyme combination of the present invention on removing PSA and / or PSA biofilm was evaluated.

[0250] CIP cleaning process / procedure 1. Add enzymes and water to the tank to begin the cleaning procedure where the tank and ultrafiltration membrane assembly are washed with the enzyme mixture for 2 hours at 45° C. The enzymes and dosages used in this process are as follows: 0.1 g / L hexosaminidase 2; 0.1 g / L of Protease 1; and 0.1 g / L DNase 3. 2. Rinse the tank and ultrafiltration membrane assembly with water for 10 minutes to remove released biofilm and PSA. 3. Add Ultrasil 115 and water to the tank (final concentration of Ultrasil 115 is 0.5 wt% in water) to begin the cleaning procedure. Clean the tank and ultrafiltration membrane assembly at 52°C for 40 minutes. Ultrasil 115 is a strong alkaline liquid detergent available from Ecolab Inc. In this step, residual PSA can be further removed or killed by Ultrasil 115. 4. Rinse the tank and ultrafiltration membrane assembly with water for 10 minutes. 5. Add Ultrasil 78 and water to the tank (final concentration of Ultrasil 78 is 0.7 wt% in water). Wash the tank and ultrafiltration membrane assembly for 40 minutes at 52° C. Ultrasil 78 is a phosphorus-free, acidic liquid detergent available from Ecolab Inc. 6. Rinse the tank and ultrafiltration membrane assembly with water for 10 minutes. 7. Measure the amount of PSA in the 250 ml rinse water from step 6. The results are recorded as "Enzyme 1 Wash" in Table 12 below. The fermentation process and CIP wash described above in steps 1-7 are repeated for two more cycles. The amount of PSA in the final 250 ml rinse water is then measured and recorded as "Enzyme Wash 3" in Table 12 below.

[0251] [Table 12]

[0252] It is clear from Table 12 that the use of the enzymes of the present invention in a CIP cleaning process can significantly improve the removal of PSA. By using the enzymes in a CIP process after each batch of fermentation, the PAS removal can be further improved and PSA can be maintained at very low levels (e.g., <10 cfu / 250 ml after 3 washes), a level that is particularly desirable in food related manufacturing processes.

[0253] Example 5 Biofilm removal test using a medical cleaning model detergent MC Various enzymes / dosages were evaluated in Example 5. The experimental procedures were the same as those described in Example 1. The results are summarized in Tables 13-15 below.

[0254] [Table 13]

[0255] [Table 14]

[0256] [Table 15]

Claims

1. 1. A method for cleaning a medical device, comprising: (a) contacting the medical device with a cleaning solution comprising a protease, an enzyme having DNase activity, and an enzyme having hexosaminidase activity; and optionally (b) rinsing the medical device; Including, the protease has 60% or greater sequence identity with the polypeptide of SEQ ID NO:6; the enzyme having DNase activity has a sequence identity of 60% or more with the polypeptide of SEQ ID NO: 4; the enzyme having hexosaminidase activity has a sequence identity of 60% or more with the polypeptide of SEQ ID NO: 10; method.

2. 10. The method of claim 1, wherein the medical device is or includes an endoscope, such as a cystoscope, nephroscope, bronchoscope, laryngoscope, otoscope, arthroscope, laparoscope, and gastrointestinal endoscope; a surgical instrument, such as a scalpel, hemostat, forceps, scissors, retractors, tracheotomy instruments, and clamps; and a dental instrument, such as a scale, curette, serrated cotton pliers, or dental mirror.

3. The medical device is contaminated with a biofilm, the biofilm being selected from the group consisting of Escherichia coli, Klebsiella pneumoniae, Salmonella spp., Mycobacterium spp., Enterococcus faecalis, Enterobacter cloacae, Proteus mirabilis, Serratia marcescens, Staphylococcus aureus, e.g., Staphylococcus aureus. aureus ATCC 6538, Streptococcus spp. (e.g., S. pyogenes, S. agalacticae, or S. pneumoniae), Haemophilus influenzae, Pseudomonas aeruginosa, Clostridium perfringens, Chlamydia trachomatis, Candida albicans, and / or Bacillus anthracis 2. The method of claim 1, wherein the soluble ...

4. 2. The method of claim 1, wherein the enzyme having DNase activity is a polypeptide having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO:

4.

5. 5. The method of claim 4, wherein the enzyme having DNase activity is a variant of the polypeptide of SEQ ID NO: 4 comprising a mutation at one or more positions selected from the group consisting of positions 1, 13, 22, 25, 27, 33, 39, 42, 56, 57, 59, 65, 76, 77, 109, 116, 127, 144, 147, 149, 167, 175 and 181 of SEQ ID NO: 4, and the variant has a sequence identity of 80% or more to SEQ ID NO:

4.

6. The method of claim 5, wherein the enzyme having DNase activity is a variant of the polypeptide of SEQ ID NO: 4 comprising one or more substitution mutations selected from the group consisting of T1I, S13Y, T22P, S25P, S27L, L33K, S39P, S42G, D56I, S57W, S59V, T65V, V76L, T77Y, Q109R, S116D, T127V, S144P, A147H, G149N, S167L, G175D and S181L.

7. 6. The method of claim 5, wherein the enzyme having DNase activity is a variant of the polypeptide of SEQ ID NO: 4, said variant comprising or consisting of SEQ ID NO: 4 with the following substitution mutations: T1I, S13Y, T22P, S27L, L33K, S39P, S42G, D56I, S57W, S59V, T65V, V76L, Q109R, S116D, T127V, S144P, A147H, S167L and G175D.

8. 2. The method of claim 1, wherein the enzyme having hexosaminidase activity is a polypeptide having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO:

10.

9. 9. The method of claim 8, wherein the enzyme having hexosaminidase activity is a variant of the polypeptide of SEQ ID NO: 10 comprising one or more alterations at positions 3, 15, 49, 59, 163, 186, 225, 227, 232, 235, 252, 260, 272, 279, 281, 308, 309, and 312 of SEQ ID NO: 10, wherein the variant has at least 80% sequence identity to the mature polypeptide of SEQ ID NO:

10.

10. 9. The method of claim 8, wherein the variant comprises one or more substitutions selected from the group consisting of Q3I, H15Y, A49W, N59E, S163P, S186R, S225G, N227T, E232D, G235W, N252P, N260Q, H272V, S279D, Y281P, K308Q, K309E and K312Q, and wherein the variant has 80% or greater sequence identity to the mature polypeptide of SEQ ID NO:

10.

11. 9. The method of claim 8, wherein the enzyme having hexosaminidase activity is a variant of the polypeptide of SEQ ID NO: 10, wherein the variant consists of or comprises the polypeptide of SEQ ID NO: 10 with the following substitutions: Q3I, H15Y, A49W, N59E, S163P, S186R, S225G, N227T, E232D, G235W, N252P, N260Q, H272V, S279D, Y281P, K308Q, K309E and K312Q.

12. 2. The method of claim 1, wherein the protease has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO:

6.

13. 13. The method of claim 12, wherein the protease is a variant of the polypeptide of SEQ ID NO: 6 comprising an alteration at one or more positions corresponding to positions 3, 4, 9, 15, 22, 43, 68, 76, 87, 99, 101, 103, 104, 118, 128, 160, 167, 170, 184, 194, 199, 205, 206, 209, 217, 218, 222, 245, 259, 261, and 262, where the position numbers correspond to positions in SEQ ID NO: 5 and each alteration is independently a substitution, deletion, or insertion, and the variant has protease activity and at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95% and less than 100% sequence identity to the polypeptide of SEQ ID NO:

6.

14. The protease may be: (a) a variant of the polypeptide of SEQ ID NO: 6, comprising: S3T, V4I, S9E, S9R, A15T, T22A, N43R, V68A, N76D, S87N, S99D, S99G, S99A, S99SE, S101E, S101N, S101R, S103A, V104I, G118M, S128Q, G160S, Y167A, R170S, N184E, A194P, V199M, V205I, Q206L, Y209W, L217D, L217Q, N218D, M222S, Q245R, S259D, N a variant comprising one or more substitutions selected from the group consisting of N76D+Q206L+Y209W or Y209W+N261W+L262E, for example N76D+Q206L+Y209W or Y209W+N261W+L262E substitutions, wherein the position numbers correspond to positions in SEQ ID NO: 5, said variant having protease activity and having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% or at least 95%, but less than 100%, sequence identity to SEQ ID NO: 6; (b) a variant of the polypeptide of SEQ ID NO: 6 comprising the substitution S87N, said variant having protease activity, wherein the position corresponds to the position in SEQ ID NO: 5, said variant having protease activity and having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to SEQ ID NO: 6; (c) a variant of the polypeptide of SEQ ID NO: 6 comprising the substitutions Y167A+R170S+A194P, wherein the position numbers correspond to the positions of SEQ ID NO: 5, said variant having protease activity and having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95%, but less than 100%, sequence identity to SEQ ID NO: 6; (d) a variant of the polypeptide of SEQ ID NO: 6 comprising the substitutions S9E+N43R+N76D+V205I+Q206L+Y209W+S259D+N261W+L262E, wherein the position numbers correspond to the positions in SEQ ID NO: 5, said variant having protease activity and having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95%, but less than 100%, sequence identity to SEQ ID NO: 6; (e) a variant of the polypeptide of SEQ ID NO: 6 comprising the substitutions S3T+N43R+N76D+S87N+G118M+S128Q+N184E+V205I+Q206L+Y209W+S259D+N261W+L262E, wherein the position numbers correspond to the positions in SEQ ID NO: 5, and wherein the variant has protease activity and has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95%, but less than 100%, sequence identity to SEQ ID NO: 6; (f) a variant of the polypeptide of SEQ ID NO: 6 comprising the substitutions T22A+N43R+S87N+V205L+Q206L+Y209W+S259D+N261W+L262E, wherein the position numbers correspond to the positions in SEQ ID NO: 5, and wherein the variant has protease activity and has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95%, but less than 100%, sequence identity to SEQ ID NO: 6; The method of claim 13, wherein

15. 14. Use of an enzyme composition for cleaning a medical device, the enzyme composition comprising an enzyme having DNase activity, an enzyme having hexosaminidase activity, and a protease; the enzyme having DNase activity is as defined in any one of claims 4 to 7, the enzyme having hexosaminidase activity is as defined in any one of claims 8 to 11, and the protease is as defined in any one of claims 12 to 14.

16. 1. A composition for cleaning a medical device, comprising a surfactant, an enzyme having DNase activity, an enzyme having hexosaminidase activity, and a protease; wherein the enzyme having DNase activity is as defined in any one of claims 4 to 7, the enzyme having hexosaminidase activity is as defined in any one of claims 8 to 11, and the protease is as defined in any one of claims 12 to 14.