Cutinase

Abstract

A cutinase is provided in the present invention. In addition, compositions comprising cutinase and methods of making and using cutinase are provided.

Description

TECHNICAL FIELD

[0001] The present invention provides cutinases. Additionally, compositions comprising cutinases and methods for preparing and using cutinases are provided. BACKGROUND OF THE INVENTION

[0002] Enzymes are biodegradable and are thus increasingly used as sustainable alternatives to petrochemicals in various applications. Even at lower temperatures, enzymes can have catalytic activity, which results in an overall reduction in energy consumption. In particular, in the detergent industry, enzymes are incorporated into detergent compositions to improve cleaning efficiency and reduce energy consumption in the washing step.

[0003] Cutinases are enzymes that are capable of hydrolyzing, for example, cutin, a waxy polymer that is a major component of the plant cuticle, and other lipid substrates. Thus, cutinases have been proposed as potential detergent enzymes. However, to date, cutinases have rarely been used in the detergent industry because they are rarely stable at different temperatures and / or within the denaturing conditions of detergents and washing liquors. Therefore, there is a need for new cutinases that meet these requirements.

[0004] There is also a need to find new detergent additives that promote the removal of lipid stains, especially sebum stains. SUMMARY OF THE INVENTION

[0005] Accordingly, the present invention relates to new polypeptides having cutinase activity and compositions comprising such cutinases, wherein the polypeptides having cutinase activity comprise an amino acid sequence that is at least 75% identical to SEQ ID NO:2, at least 80% identical to SEQ ID NO:4, or at least 84% identical to SEQ ID NO:6. Additionally, methods for using compositions comprising such cutinases for removing plant-based stains and / or lipid stains, preferably lipid stains, preferably sebum stains, are provided. DETAILED DESCRIPTION

[0006] The present invention can be more readily understood by reference to the following detailed description of the embodiments of the invention and the examples included herein.

[0007] Although the invention will be described with respect to specific embodiments, the description should not be construed in a limiting sense.

[0008] Definition

[0009] Unless otherwise specified, the terms used herein should be understood according to the conventional usage of those of ordinary skill in the relevant art.

[0010] Before describing the exemplary embodiments of the present invention in detail, definitions that are important for understanding the present invention are provided. Unless otherwise specified or obvious from the nature of the definition, the definitions apply to all compounds, methods, and uses described herein.

[0011] As used in this specification and the appended claims, unless the context clearly dictates otherwise, the singular forms "a / an" also include the corresponding plurals and vice versa.

[0012] In the context of the present invention, the terms "about" and "approximately" indicate a range of precision that those skilled in the art will understand still ensures the technical effect of the recited feature. The term typically indicates a deviation of ±20%, preferably ±15%, more preferably ±10%, and even more preferably ±5% from the indicated numerical value.

[0013] Furthermore, the terms "first", "second", "third" or "(a)", "(b)", "(c)", "(d)", etc. in the specification and claims are used to distinguish similar elements and are not necessarily used to describe a sequential or chronological order. It should be understood that the terms so used are interchangeable where appropriate, and the embodiments of the present invention described herein are capable of operating in other orders than those described or shown herein. In the case where the terms "first", "second", "third" or "(a)", "(b)", "(c)", "(d)", "i", "ii", etc. relate to steps of a method or use or experiment, there is no temporal or time interval coherence between these steps, i.e., these steps can be performed simultaneously or there can be a time interval of seconds, minutes, hours, days, weeks, months, or even years between such steps, unless otherwise indicated in the present application as described above or below.

[0014] Throughout this application, various publications are referenced. The disclosures of all these publications and those references cited therein are incorporated herein by reference in their entirety to more fully describe the prior art in the field to which the present invention pertains.

[0015] It should be understood that the term "comprising" is not restrictive. For the purposes of the present invention, the term "consisting of" is considered a preferred embodiment of the term "comprising / including". If a group is defined hereinafter as including at least a certain number of members, this means that a group consisting only of these members is also covered.

[0016] When describing the polypeptides of the present invention, the abbreviations of individual amino acids used are according to the well - recognized IUPAC single - letter or three - letter amino acid abbreviations.

[0017] As used herein, "amino acid change" refers to an amino acid substitution, deletion, or insertion.

[0018] "Substitution" is described by providing the original amino acid, followed by the position number within the amino acid sequence, followed by the amino acid that replaces the original amino acid. For example, substitution of histidine at position 120 by alanine is denoted as "His120Ala" or "H120A". Substitution can also be described, without specifying the initial amino acid at that position, by naming only the resulting amino acid, e.g., "X120A" or "120A" or "Xaa120Ala" or "120Ala".

[0019] "Deletion" is described by providing the original amino acid, followed by the position number within the amino acid sequence, followed by an asterisk. Accordingly, deletion of glycine at position 150 is denoted as "Gly150*" or "G150*". Alternatively, deletion is denoted by, for example, "deletion of D183 and G184".

[0020] "Insertion" is described by providing the original amino acid, followed by the position number within the amino acid sequence, followed by the original amino acid and the newly added amino acid. For example, insertion of lysine at position 180 adjacent to glycine is denoted as "Gly180GlyLys" or "G180GK". When inserting more than one amino acid residue, e.g., inserting Lys and Ala after Gly180, this insertion can be denoted as: "Gly180GlyLysAla" or "G195GKA".

[0021] In the case where substitution and insertion occur at the same position, this can be denoted as "S99A+S99SD" or simply as "S99AD". Sequences containing multiple alterations are separated by the plus sign "+", e.g., "Arg170Tyr+Gly195Glu", "R170Y+G195E" or "X170Y+X195E" represent substitution of arginine and glycine at positions 170 and 195 by tyrosine and glutamate respectively. Alternatively, multiple alterations can be separated by a space or a comma, e.g., "R170Y G195E" or "R170Y,G195E". When different alternative alterations can be introduced at a certain position, the different alterations are separated by a comma, e.g., "Arg170Tyr,Glu" and "R170T,E" represent substitution of arginine at position 170 by tyrosine or glutamate respectively. Alternative substitutions at a specific position can also be denoted as "X120A,G,H", "120A,G,H", "X120A / G / H" or "120A / G / H". Alternatively, different alterations or optional substitutions can be represented in parentheses, e.g., "Arg170[Tyr,Gly]" or "Arg170{Tyr,Gly}" or abbreviated as "R170[Y,G]" or "R170{Y,G}".

[0022] The term "natural" (or naturally or wild-type or endogenous) cell or organism or polynucleotide or polypeptide refers to a cell or organism or polynucleotide or polypeptide found in nature (i.e., without any human intervention).

[0023] The term "isolated" molecule (such as a polypeptide or polynucleotide) is defined herein as a molecule that has been separated from its natural environment.

[0024] The term "heterologous polypeptide" (or exogenous or foreign polypeptide) is defined herein as a polypeptide that is not naturally expressed by a host cell. The term "heterologous nucleotide" (or exogenous or foreign polynucleotide) is defined herein as a polynucleotide that is not naturally contained in a host cell.

[0025] For the purposes of the present invention, "recombinant" (or non-natural or unnaturally) with respect to a cell or organism means that the cell or organism contains a polynucleotide introduced using genetic techniques. Recombinant with respect to a polynucleotide or polypeptide means that the polynucleotide or polypeptide has been newly combined or rearranged in terms of its genetic environment by using recombinant DNA techniques. Thus, recombinant polynucleotides or polypeptides include polypeptides or polynucleotides that are natural to the host cell, the expression of which is quantitatively altered due to manipulation of the host cell DNA by recombinant DNA techniques (such as a stronger promoter), or the expression of which is from a genomic location different from that of the natural host cell. Recombinant polynucleotides or polypeptides can also be heterologous, which means that they can be foreign sequences, but they can also be derived from the same organism into which they are introduced.

[0026] With respect to the relationship between two or more polynucleotides or the relationship between two or more polypeptides, the term "recombinant" is used to characterize that the two or more polynucleotides or two or more polypeptides do not naturally occur in a particular combination with each other.

[0027] A "modified recombinant" polynucleotide or polypeptide means a recombinant polynucleotide or recombinant polypeptide that has been modified by using recombinant DNA techniques to introduce changes (such as deletions, substitutions, and / or insertions) to alter a natural polypeptide or natural polynucleotide.

[0028] A "synthetic" compound is obtained by in vitro chemical and / or enzymatic synthesis.

[0029] Sequence identity is usually provided in the form of "sequence identity %" or "identity %". To calculate sequence identity, the first step is to generate a sequence alignment. According to the present invention, a pairwise global alignment is generated, which means that the two sequences are aligned over their entire lengths. The alignment is usually generated by using a mathematical method called an alignment algorithm.

[0030] According to the present invention, the alignment is generated by using the algorithm of Needleman and Wunsch (J. Mol. Biol. [Journal of Molecular Biology] (1979) 48, pages 443 - 453). Preferably, the program "NEEDLE" (European Molecular Biology Open Software Suite (EMBOSS)) is used for the purposes of the present invention, wherein the program default parameters are used (for polynucleotides: gap open = 10.0, gap extend = 0.5, and matrix = EDNAFULL; for polypeptides: gap open = 10.0, gap extend = 0.5, matrix = EBLOSUM62). After aligning the two sequences, the identity value is determined from the resulting alignment in a second step. For this purpose, the % identity is calculated as follows: dividing the number of identical residues by the length of the alignment region of the corresponding sequence of the present invention shown over its full length and multiplying by 100: Identity % = (identical residues / length of the alignment region of the corresponding sequence of the present invention shown over its full length) * 100.

[0031] For calculating the percentage identity of two polynucleotides, some of the specifications outlined above also apply. For polynucleotides encoding proteins, the pairwise alignment should be performed over the full length of the coding region of the sequences of the present invention (from the start codon to the stop codon, excluding introns). Introns present in other sequences being compared with the sequences of the present invention should also be removed for the pairwise alignment. After aligning the two polynucleotide sequences, the identity value is determined from the resulting alignment in a second step. The percentage identity is calculated as follows: % - identity = (identical residues / length of the alignment region of the sequence of the present invention shown from the start codon to the stop codon and excluding introns over its full length) * 100.

[0032] Here, an exchange of one amino acid for another can be referred to as a "conservative mutation". The definition of similar amino acids according to the present invention is as follows:

[0033] Amino acid A is similar to amino acid S

[0034] Amino acid D is similar to amino acid E; N

[0035] Amino acid E is similar to amino acid D; K; Q

[0036] Amino acid F is similar to amino acid W; Y

[0037] Amino acid H is similar to amino acid N; Y

[0038] Amino acid I is similar to amino acid L; M; V

[0039] Amino acid K is similar to amino acid E; Q; R

[0040] Amino acid L is similar to amino acid I; M; V

[0041] Amino acid M is similar to amino acid I; L; V

[0042] Amino acid N is similar to amino acid D; H; S

[0043] Amino acid Q is similar to amino acid E; K; R

[0044] Amino acid R is similar to amino acid K; Q

[0045] Amino acid S is similar to amino acid A; N; T

[0046] Amino acid T is similar to amino acid S

[0047] Amino acid V is similar to amino acid I; L; M

[0048] Amino acid W is similar to amino acid F; Y

[0049] Amino acid Y is similar to amino acid F; H; W

[0050] As used herein, "fragment" or "subsequence" is a part of a polynucleotide or amino acid sequence. The term "functional fragment" refers to any nucleic acid or amino acid sequence that contains only a part of the full-length amino acid sequence but still has the same or similar activity and / or function. Preferably, the functional fragment is at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80% identical, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99% or at least 99.5% identical to the original full-length amino acid sequence. The functional fragment contains contiguous nucleotides or amino acids that are identical to the corresponding part of the original nucleic acid or original amino acid sequence.

[0051] As used herein, "gene construct" or "expression cassette" is a nucleic acid molecule composed of at least one target sequence to be expressed, which is operably linked to one or more control sequences as described herein (linked to a promoter at least).

[0052] As used herein, the term "vector" encompasses any kind of construct suitable for carrying a polynucleotide sequence for transfer into a cell or for stable or transient expression within a given cell. As used herein, the term "vector" covers any kind of cloning vehicle, such as, but not limited to, plasmids, phagemids, viral vectors (e.g., phages), bacteriophages, baculoviruses, cosmids, fosmids, artificial chromosomes, and any other vector specific to a particular host of interest. The foreign polynucleotide sequence typically contains a coding sequence that may herein be referred to as a "gene of interest". The gene of interest may contain introns and exons, depending on the kind of source or destination of the host cell.

[0053] The term "introduction of polynucleotide" or "transformation of polynucleotide" as referred to herein encompasses the transfer of a polynucleotide from outside the host cell into the host cell, regardless of the method used for transfer. That is, as used herein, the term "transformation of polynucleotide" is independent of the vector, shuttle system, or host cell. In addition, it not only refers to the method of transfer of the transformed polynucleotide (see, for example, Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), it also covers any other kind of polynucleotide transfer method, such as, but not limited to, transduction or transfection.

[0054] A polynucleotide encoding a polypeptide can be "expressed". The term "expression" or "gene expression" means that one or more genes or gene constructs are transcribed into structural RNA (e.g., rRNA, tRNA) or mRNA, and subsequently the latter is either translated into a protein or not. The process includes DNA transcription and the processing of the resulting mRNA product.

[0055] The term "purification" or "purifying" refers to a process in which at least one component (e.g., the protein of interest) is separated from at least another component (e.g., the particulate matter of the fermentation broth) and transferred to a different compartment or phase, where the different compartment or phase does not necessarily need to be separated by a physical barrier. Examples of such different compartments are two compartments separated by a filter membrane or cloth, i.e., the filtrate and the retentate; additional examples of such different phases are the precipitate and the supernatant or the filter cake and the filtrate, respectively. The solution obtained after purifying the enzyme of interest from the fermentation broth is herein referred to as the "purified enzyme solution".

[0056] "Protein formulation" (or "protein preparation") means any non - complex formulation containing a small number of ingredients, where these ingredients are for the purpose of stabilizing the proteins contained in the protein formulation and / or the stability of the protein formulation itself. Preferably, the non - complex protein formulation contains a higher concentration of protein than a complex composition (e.g., than a detergent composition). Thus, preferably, the non - complex protein formulation is a concentrated protein formulation. Preferably, the non - complex protein formulation contains 0.1% to 40% by weight of an enzyme, while a complex formulation (such as a detergent composition) contains 0.0002% to 0.09% by weight of an enzyme, all relative to the total weight of the enzyme formulation.

[0057] "Enzyme properties" include but are not limited to catalytic activity, substrate / cofactor specificity, product specificity, stability over time, thermal stability, pH stability, and chemical stability. "Enzyme activity" or "catalytic activity" means the catalytic effect exerted by an enzyme, expressed as units / mg of enzyme (specific activity) or substrate molecules transformed / minute / enzyme molecule (molecular activity). Enzyme activity can be specified by the actual function of the enzyme. For example, a protease exerts proteolytic activity by catalyzing the hydrolysis cleavage of peptide bonds, a lipase exerts lipolytic activity by the hydrolysis cleavage of ester bonds, amylase activity involves the hydrolysis of glycosidic linkages in polysaccharides, cutinase activity involves the hydrolysis of ester bonds in omega - hydroxy acids and their derivatives, etc.

[0058] The term "enzyme stability" according to the present invention relates to the retention of enzyme activity as a function of time during storage or operation. The retention of enzyme activity as a function of time during storage is called "storage stability" and is the preferred embodiment of enzyme stability within the context of the present invention.

[0059] To determine and quantify the catalytic activity of an enzyme changing over time when stored or used under certain conditions, the "initial enzyme activity" is measured at time zero (100%) and at a later time point (x%) under specified conditions. By comparing these values, the potential loss of enzyme activity can be determined. The degree of enzyme activity loss determines the stability or instability of the enzyme.

[0060] "Enzyme inhibitor" as used herein is a compound that slows down or stops enzyme activity. Enzyme inhibitors can generally also stabilize the three - dimensional structure of the enzyme. Thus, enzyme inhibitors generally also act as "enzyme stabilizers".

[0061] "pH stability" refers to the ability of an enzyme to exert enzyme activity after being exposed to a certain pH value.

[0062] The terms "thermal stability", "thermostability" or "temperature-dependent activity" refer to the ability of an enzyme to exhibit catalytic activity or washing performance after exposure to elevated temperatures, preferably for 28 days at a temperature of 37°C - 45°C, more preferably 56 days, preferably in a detergent composition (preferably, in Detergent Model ES1-C).

[0063] The term "detergent stability" or "stability upon storage in a detergent composition" refers to the ability of an enzyme to exhibit catalytic activity or washing performance after storage in a detergent composition (preferably, in Detergent Model ES1-C), preferably for 28 days at a temperature of 37°C or 45°C, more preferably 56 days.

[0064] As used herein, the "washing performance" of an enzyme (also referred to herein as "cleaning performance") refers to the contribution of the enzyme to the cleaning performance of a detergent composition, i.e., the cleaning performance added to the detergent composition by the performance of the enzyme. The term "washing performance" is used herein similarly for laundry and hard surface cleaning. Washing performance is compared under relevant washing conditions. The term "relevant washing conditions" is used herein to indicate the conditions for household use in a detergent market segment, particularly the washing temperature, time, washing machine, concentration of detergent components in the wash liquor (i.e., foam concentration), type of detergent, and water hardness. The term "improved washing performance" is used to indicate better cleaning performance under relevant washing conditions, or, relative to the corresponding control conditions, less enzyme is required on a weight basis to achieve the same final result.

[0065] As used herein, the term "specific performance" refers to the ability of a unit of active enzyme to clean and remove specific stains or soils. In some embodiments, the specific performance is determined using stains or soils such as egg, egg yolk, milk, grass, spinach, minced meat, blood, chocolate sauce, baby food, sebum, etc.

[0066] "Detergent composition" or "detergent" refers to a composition designated for cleaning soiled materials. The detergent compositions according to the present invention include detergent compositions for different applications such as laundry and hard surface cleaning. The term "detergent component" is defined herein to mean a class of chemicals that can be used in a detergent composition. A typical detergent component is a surfactant. "Surfactant" (used synonymously herein with "surface active agent") means an organic chemical that, when added to a liquid, changes the properties of that liquid at the interface. Surfactants are referred to as nonionic surfactants, anionic surfactants, cationic surfactants, or amphoteric surfactants according to their ionic charge. The term "effective amount of a detergent component" includes the amount of certain components that provide effective stain removal and / or effective cleaning conditions (such as pH, temperature, water hardness, amount of foaming), the amount that effectively provides optical benefits (such as optical brightening, dye transfer inhibition, color care), and the amount of certain components that effectively assist in processing (maintaining physical properties during processing, storage, and use; such as rheology modifiers, hydrotropes, desiccants).

[0067] The term "laundry" or "laundering" relates to both household cleaning and industrial cleaning and means the process of treating textiles and / or fabrics with a solution containing the detergent composition of the present invention. The cleaning or washing process can be carried out by using technical equipment such as a household or industrial washing machine. Alternatively, the cleaning or washing process can be done by hand.

[0068] The term "textile" means any textile material, including yarns (threads made of natural or synthetic fibers for knitting or weaving), yarn intermediates, fibers, non-woven materials, natural materials, synthetic materials, and fabrics made from these materials such as clothing, cloth, and other articles. As used herein, the term "fabric" (a textile made by weaving, knitting, or felting fibers) or "garment" (any article of clothing made of a textile) also means to include the broader term textile.

[0069] The term "fiber" includes natural fibers, synthetic fibers, and mixtures thereof. Examples of natural fibers are plant fibers (such as flax, jute, and cotton) or fibers of animal origin, the latter including proteins such as collagen, keratin, and fibroin (such as silk, sheep's wool, angora wool, mohair, cashmere). Examples of fibers of synthetic origin are polyurethane fibers such as or polyester fibers, polyolefins such as elastofin, or polyamide fibers such as nylon. Fibers can be single fibers or part of a textile, such as a knitted fabric, a woven fabric, or a non-woven fabric.

[0070] The term "hard surface cleaning" relates to both domestic and industrial hard surface cleaning and refers to the process of treating hard surfaces with a solution containing the detergent composition of the present invention. Hard surfaces can include any hard surface in the home or industry, such as floors, furniture, walls, sanitary ceramics, glass, metal surfaces (including medical devices), cutlery, and tableware. A special form of hard surface cleaning is dishwashing, including manual dishwashing (MDW) or automatic dishwashing (ADW).

[0071] The term "dishwashing" refers to all forms of dishwashing, such as manual dishwashing or automatic dishwashing. Dishwashing includes, but is not limited to, cleaning all forms of pottery, such as plates, cups, glasses, bowls, all forms of cutlery, such as spoons, knives, forks, and serving utensils, as well as ceramics, plastics such as melamine, metals, porcelain, glass, and acrylic resins.

[0072] Cleaning performance is evaluated under relevant cleaning conditions. In this context, the term "relevant cleaning conditions" refers to the conditions used in washing machines, automatic dishwashers, or during manual cleaning processes, particularly the cleaning temperature, time, cleaning mechanism, foam concentration, detergent type, and water hardness.

[0073] The term "medical device cleaning" refers to the cleaning step of reprocessing reusable medical devices. Medical device cleaning methods can be divided into two categories: manual cleaning methods and mechanical / automatic cleaning methods. Manual cleaning is used when there is no available mechanical device or the medical device to be cleaned is too fragile or difficult to clean with a mechanical device. Mechanical / automatic cleaning methods remove dirt and microorganisms through an automatic cleaning and rinsing process, which includes ultrasonic cleaning and washing.

[0074] In the field of detergency, the term "stain" is usually used to refer to laundry, such as the cleaning of textiles, fabrics, or fibers, while the term "soil" is usually used to refer to hard surface cleaning, such as the cleaning of cutlery and knives. However, in this document, the terms "stain" and "soil" are used interchangeably.

[0075] A "fat stain" is a stain containing fat. Fat means an ester of any fatty acid. Thus, fat stains refer to, but are not limited to, oily stains, solid fat stains, sebum stains, and body soil stains, etc. Sebum stains preferably contain lipids, preferably comprising at least one of triglycerides, wax esters, squalene, and / or cholesterol. More preferably, sebum stains contain a mixture of lipids, preferably one or more of triglycerides, wax esters, squalene, and cholesterol, preferably a mixture of triglycerides, wax esters, squalene, and cholesterol.

[0076] As used herein, a "chelating builder" is different from a precipitating builder in that when a chelating builder is used in an amount sufficient to combine with all calcium ions in an aqueous solution initially having a hardness of 7 °dH (German hardness) at neutral pH, no substantial precipitation is formed. A "strong builder" is classified as a highly efficient chelating agent capable of strongly binding divalent cations (e.g., Ca 2+ ), and the logarithm of the stability constant (Log K Ca ) of the cation / chelating agent complex is greater than 4, particularly greater than 5, greater than 6 or greater than 7. The stability constant is determined at an ionic strength of 0.1 M and a temperature of 25 °C. A "strong chelating builder" combines the above two characteristics.

[0077] A composition "substantially free" of a compound shall herein mean that the corresponding compound is not intentionally added to the composition, and preferably at most a non-effective amount, and most preferably 0% of the compound is included in the composition.

[0078] Detailed description

[0079] Cutinase is provided in the present invention. More particularly, a composition comprising cutinase and methods for preparing and using cutinase are provided.

[0080] Preferably, the cutinase of the present invention is a purified, isolated, synthetic and / or recombinant cutinase. Preferably, the cutinase of the present invention is a purified recombinant cutinase.

[0081] The cutinase according to the present invention has "cutinase activity". "Cutinase activity" describes the ability to hydrolyze ester bonds. Initially, cutinase activity was related to the hydrolysis of ester bonds in the plant polymer cutin. However, cutinase has also been shown to catalyze the hydrolysis of other polymers, triacylglycerols and low molecular weight soluble esters. Cutinase has also been shown to be able to catalyze the hydrolysis of PET (poly(ethylene terephthalate)). Cutinase is a hydrolase classified in the EC class 3.1.1.74.

[0082] Cutinase activity can be determined by assays known to those skilled in the art for measuring cutinase activity. For example, p-nitrophenyl butyrate or 4-nitrophenyl octanoate can be used as substrates to determine cutinase activity. The reaction rate of enzymatic hydrolysis can be determined by spectrophotometrically monitoring the release of p-nitrophenol at 405 nm in a spectrophotometer.

[0083] The cutinase of the present invention is a cutinase comprising an amino acid sequence that is at least 75% identical to SEQ ID NO:2, at least 80% identical to SEQ ID NO:4 or at least 84% identical to SEQ ID NO:6.

[0084] In one embodiment, the cutinase comprises an amino acid sequence that is at least 76%, at least 78%, at least 80%, at least 82%, at least 85%, at least 87%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:2 with increasing preference.

[0085] In another embodiment, the cutinase comprises an amino acid sequence that is at least 82%, at least 85%, at least 87%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:4 with increasing preference.

[0086] In another embodiment, the cutinase comprises an amino acid sequence that is at least 85%, at least 87%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:6 with increasing preference.

[0087] Preferably, the cutinase comprises an amino acid sequence that is 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% identical to SEQ ID NO:2, 4 or 6 with increasing preference.

[0088] More preferably, the cutinase comprises an amino acid sequence that is 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% identical to SEQ ID NO:2 or 6 with increasing preference.

[0089] Most preferably, the cutinase comprises an amino acid sequence that is 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% identical to SEQ ID NO:2 with increasing preference.

[0090] In one embodiment, the cutinase comprises the following or consists of the following: an amino acid sequence that is 100% identical to SEQ ID NO:2, 4 or 6, preferably SEQ ID NO:2 or SEQ ID NO:6, more preferably SEQ ID NO:2.

[0091] In one embodiment, the cutinase comprises or consists of the following: an amino acid sequence that is 100% identical to SEQ ID NO: 2, 4, or 6, preferably SEQ ID NO: 2, but comprising 1 - 20, preferably 1 - 15, more preferably 1 - 10, or even more preferably 1 - 5 amino acid substitutions, preferably conservative amino acid substitutions.

[0092] Methods for introducing amino acid changes (such as substitutions, preferably conservative substitutions) into a protein sequence are well known in the art. Substitutions can be introduced by using any mutagenesis procedure known in the art, which is, for example, site-directed mutagenesis, synthetic gene construction, semi-synthetic gene construction, random mutagenesis, shuffling, etc., followed by relevant screening procedures. See, for example, Scherer and Davis, 1979, Proc. Natl. Acad. Sci. USA 76:4949 - 4955; and Barton et al., 1990, Nucleic Acids Res. 18:7349 - 4966, U.S. Patent Application Publication No. 2004 / 0171154; Storici et al., 2001, Nature Biotechnol. 19:773 - 776; Kren et al., 1998, Nat. Med. 4:285 - 290; and Calissano and Macino, 1996, Fungal Genet. Newslett. 43:15 - 16, Tian et al., 2004, Nature 432:1050 - 1054; Reidhaar-Olson and Sauer, 1988, Science 241:53 - 57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86:2152 - 2156; WO 95 / 17413; or WO 95 / 22625, Lowman et al., 1991, Biochemistry 30:10832 - 10837; U.S. Patent No. 5,223,409; WO 92 / 06204, Derbyshire et al., 1986, Gene 46:145; Ner et al., 1988, DNA 7:127.

[0093] Nucleic acid construct

[0094] The invention also relates to a polynucleotide encoding the cutinase of the invention or its complementary sequence. Preferably, the polynucleotide is a codon-optimized polynucleotide for improving expression in a specific host cell (preferably a Bacillus cell).

[0095] Accordingly, the present invention also relates to polynucleotides, preferably isolated, synthetic and / or recombinant polynucleotides, comprising:

[0096] (a) a polynucleotide having 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% identity, in increasing order of preference, to SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5, wherein the polynucleotide encodes a polypeptide having cutinase activity;

[0097] (b) a polynucleotide encoding a polypeptide having 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% identity, in increasing order of preference, to SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6, wherein the polypeptide has cutinase activity;

[0098] (c) a polynucleotide that hybridizes under high stringency conditions, preferably under very high stringency conditions, to the complementary sequence of:

[0099] (i) the coding sequence of SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6; or

[0100] (ii) the polynucleotide shown in SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5;

[0101] (d) a fragment of (a), (b) or (c), wherein the fragment encodes a polypeptide having cutinase activity; or

[0102] (e) a polynucleotide sequence that is fully complementary to any one of (a) to (d).

[0103] (f) a polynucleotide that differs from any nucleic acid sequence described in (a) to (e) only by the degeneracy of the genetic code.

[0104] In a further embodiment, the present invention also relates to a polypeptide having cutinase activity encoded by a polynucleotide as described herein, preferably an isolated, synthetic or recombinant polypeptide having cutinase activity encoded by a polynucleotide as described herein. Preferably, the polypeptide having cutinase activity is encoded by a polynucleotide that 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% identity with SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5 with increasing preference.

[0105] In another embodiment, the polypeptide having cutinase activity is encoded by a polynucleotide that hybridizes under high stringency conditions (preferably under very high stringency conditions) with increasing preference to the full-length complementary sequence of a polynucleotide having 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% identity with SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5.

[0106] The present invention also relates to a nucleic acid construct (preferably an expression cassette) comprising a polynucleotide as described herein.

[0107] Typically, an expression cassette comprises three elements: a promoter sequence, an open reading frame, and a 3' untranslated region, which in eukaryotes usually contains a polyadenylation site. Additional regulatory elements may include transcriptional as well as translational enhancers. Intron sequences can also be added to the 5' untranslated region (UTR) or the coding sequence to increase the amount of mature messenger accumulated in the cytosol. The expression cassette can be part of a vector or can be integrated into the genome of a host cell and replicated with the genome of its host cell. The expression cassette is generally capable of increasing or decreasing expression.

[0108] The present invention also relates to an expression vector comprising a polynucleotide or a nucleic acid construct as described herein. The expression vector can be a low-copy number vector or a high-copy number vector.

[0109] After transformation into a host cell or host cell organelle, a vector as used herein can provide segments for transcription and translation of polynucleotides. Such additional segments can include regulatory nucleotide sequences, one or more origins of replication required for maintenance and / or replication in a particular cell type, one or more selectable markers, polyadenylation signals, suitable sites for insertion of foreign coding sequences, such as a multiple cloning site. An example is where the vector needs to be maintained as an episomal genetic element (e.g., a plasmid or cosmid molecule) in a bacterial cell. Non-limiting examples of suitable origins of replication include f1-ori and colE1.

[0110] Without integration into the host cell genome, the vector can replicate, e.g., as a plasmid in a bacterial host cell, or it can integrate some or all of its DNA into the host cell genome and thereby cause replication and expression of its DNA.

[0111] A polynucleotide encoding cutinase can be introduced into a vector by standard recombinant DNA techniques. Once introduced into the vector, the polynucleotide containing the coding sequence can be suitably introduced (transformed, transduced, transfected, etc.) into a host cell or host cell organelle. A cloning vector suitable for expression of the polynucleotide sequence in the host cell or host cell organelle can be selected.

[0112] Host cell

[0113] The invention also relates to a host cell comprising a polynucleotide encoding cutinase as described herein, a nucleic acid construct as described herein, or an expression vector as described herein. In one embodiment of the invention, the vector is used for transformation of the host cell.

[0114] The polynucleotide encoding cutinase as described herein can be introduced into a host cell stably or transiently and can remain non-integrated, e.g., as a plasmid. Generally, stable transformation is due to integration of the nucleic acid containing the foreign coding sequence into the chromosome or as an episome (an independent nuclear DNA fragment). Generally, during transient transformation, the nucleic acid containing the foreign nucleic acid sequence is not integrated into the chromosome or as an episome.

[0115] Introduction of nucleic acids into a host cell can be achieved, for example but not limited to, by protoplast transformation (see, e.g., Chang and Cohen, 1979, Molecular General Genetics 168:111-115), by using competent cells (see, e.g., Young and Spizizen, 1961, Journal of Bacteriology 81:823-829, or Dubnau and Davidoff-Abelson, 1971, Journal of Molecular Biology 56:209-221), by electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6:742-751) or by conjugation (see, e.g., Koehler and Thorne, 1987, Journal of Bacteriology 169:5271-5278). Specific transformation protocols for various types of host cells are known in the art (see, e.g., for E. coli protoplast transformation, see Hanahan, 1983, J. Mol. Biol. 166:557-580).

[0116] A variety of host cells can be used to express the nucleic acid constructs described herein. Host cells containing the genetic constructs described herein can be obtained by one of the methods described herein for introducing polynucleotides into such host cells. The host cells of the present invention do not naturally express cutinase. Thus, the host cells are recombinant host cells; the nucleic acid constructs described herein are heterologous to the host cells.

[0117] In one embodiment, the host cell is a prokaryotic cell or a eukaryotic cell. In another embodiment, the host cell is a bacterial cell, an archaeal cell, a fungal cell, a yeast cell or a eukaryotic cell. In another embodiment, the host cell is a non-human host cell.

[0118] In one embodiment, the host cell is a bacterial cell. The bacterial host cell can be any Gram-positive or Gram-negative bacterium. Gram-positive bacteria include, but are not limited to, Bacillus, Brevibacterium, Corynebacterium, Streptococcus, Streptomyces, Staphylococcus, Enterococcus, Lactobacillus, Lactococcus, Clostridium, Paenibacillus, Geobacillus, and Oceanobacillus. Gram-negative bacteria include, but are not limited to, Escherichia, Pseudomonas, Salmonella, Campylobacter, Basfia, Ensifer, Sinorhizobium, Helicobacter, Acetobacter, Flavobacterium, Fusobacterium, Gluconobacter. In one embodiment, the host cell is a bacterial cell. In a particular embodiment, the host cell is Escherichia or Bacillus. In a particular embodiment, the bacterial host cell is a Bacillus licheniformis cell.

[0119] In the method of the present invention, the bacterial host cell can be any Bacillus cell. Bacillus cells useful in the practice of the present invention include, but are not limited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus methylotrophicus, Bacillus cereus, Bacillus paralicheniformis, Bacillus subtilis, and Bacillus thuringiensis cells. In one embodiment, the bacterial host cell is a Bacillus amyloliquefaciens, Bacillus pumilus, Bacillus lentus, Bacillus licheniformis, Bacillus stearothermophilus, or Bacillus subtilis cell. In a preferred embodiment, the bacterial host cell is a Bacillus licheniformis cell, Bacillus pumilus, or Bacillus subtilis cell. Preferably, the bacterial host cell is a Bacillus licheniformis cell.

[0120] In the method of the present invention, the bacterial host cell can be Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus gasseri, Lactobacillus bulgaricus, Lactobacillus reuteri, Escherichia coli, Staphylococcus aureus, Corynebacterium glutamicum, Corynebacterium acetoglutamicum, Corynebacterium acetoacidophilum, Corynebacterium callunae, Corynebacterium ammoniagenes, Corynebacterium thermoaminogenes, Corynebacterium melassecola, Corynebacterium effiziens, Corynebacterium efficiens, Corynebacterium deserti, Brevibacterium flavum, Brevibacterium lactofermentum, Brevibacterium divarecatum, Pseudomonas putida, Pseudomonas syringae, Streptomyces coelicolor, Streptomyces lividans, Streptomyces albus, Streptomyces avermitilis, Gluconobacter oxydans, Gluconobacter morbifer, Gluconobacter thailandicus, AcetobacterClostridium aceti, Clostridium acetobutylicum, Clostridium saccharobutylicum, Clostridium beijerinckii, Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, Streptococcus equi subsp., Zooepidemicus, or Basfia succiniciproducens.

[0121] In another embodiment, the bacterial host cell may additionally contain modifications (e.g., deletions or disruptions) of other genes that may be harmful to the production, recovery, or application of the polypeptide of interest. In one embodiment, the bacterial host cell is a protease-deficient cell. In another embodiment, the bacterial host cell (e.g., a Bacillus cell) contains disruptions or deletions of extracellular protease genes including but not limited to aprE, mpr, vpr, bpr, and / or epr. In one embodiment, the bacterial host cell does not produce spores. In another embodiment, the bacterial host cell (e.g., a Bacillus cell) contains disruptions or deletions of spoIIAC, sigE, and / or sigG. In one embodiment, the bacterial host cell (e.g., a Bacillus cell) contains a disruption or deletion of one of the genes involved in surfactin biosynthesis (e.g., srfA, srfB, srfC, and / or srfD). See, for example, U.S. Patent No. 5,958,728. In another embodiment, the bacterial host cell contains a disruption or deletion of one of the genes involved in polyglutamic acid biosynthesis. Other genes that are harmful to the production, recovery, or application of the polypeptide of interest (including but not limited to the amyE gene) may also be disrupted or deleted.

[0122] In another embodiment, the bacterial host cell is a standard Escherichia coli cloning host cell, including but not limited to DH5α (Invitrogen), DH10B (Invitrogen), Omnimax (Invitrogen), INV110 (Invitrogen), TOP10 (Invitrogen), HB101 (Promega), SURE (Stratagene), XL1-Blue (Stratagene), TG1 (Lucigen), and JM109 (NEB). In another embodiment, the bacterial host cell is a standard Bacillus subtilis cloning host cell, including but not limited to Bacillus subtilis carrying a defective hsd(RI)R-M- locus, such as Bacillus subtilis IG-20 (BGSC 1A436), or a defective hsdRM1 mutation, such as Bacillus subtilis 1012WT (Mobitec).

[0123] Alternative additional host cells include but are not limited to: Aspergillus niger, Aspergillus oryzae, Hansenula polymorpha, Thermomyces lanuginosus, Fusarium oxysporum, Fusarium heterosporum, Pichia pastoris (also known as Komagataella phaffii), Myceliopthora thermophila C1, Themothelomyces thermophila, Schizosaccharomyces pombe, Trichoderma (preferably Trichoderma reesei), and Saccharomyces (preferably Saccharomyces cerevisiae) or Rhizomucor.

[0124] Preparation method

[0125] The cutinase described herein can be produced on an industrial scale and subsequently purified. Industrial production of enzymes is generally accomplished by culturing a host cell that expresses the enzyme (also referred to as fermentation). Suitable host cells are as described above. The nucleic acid sequence encoding the cutinase described herein can be transformed into a host cell, and the host cell is then cultured under conditions suitable for the production of cutinase by the host cell. In a preferred embodiment, the cutinase is purified from the host cell.

[0126] Accordingly, in yet another embodiment, the present invention relates to a method for producing cutinase, the method comprising the steps of:

[0127] (a) providing a host cell comprising a recombinant nucleic acid construct by introducing a nucleic acid construct comprising a polynucleotide encoding the cutinase described herein into the host cell, the recombinant nucleic acid construct comprising a polynucleotide encoding the cutinase described herein;

[0128] (b) culturing the recombinant host cell of step (a) under conditions that are conducive to the expression of the polynucleotide; and

[0129] (c) optionally, recovering the cutinase encoded by the polynucleotide.

[0130] Culturing of the host cell is generally carried out in a suitable nutrient medium to allow the recombinant cells to grow and express the desired protein. At the end of fermentation, the fermentation broth is collected and can be further processed, wherein the fermentation broth comprises a liquid part and a solid part. The enzyme of interest can be further purified from the fermentation broth.

[0131] The cutinase described herein can be secreted (into the liquid portion of the fermentation broth), or it can remain non-secreted from the microbial cells (and thus be contained in the cells of the fermentation broth). Accordingly, the cutinase can be recovered from the liquid portion of the fermentation broth or from the cell lysate. Preferably, the cutinase is secreted from the cells into the fermentation broth, preferably by a secretion signal peptide added to the amino acid sequence terminus of the cutinase. Recovery of the cutinase can be achieved by methods known to those skilled in the art. Suitable methods for recovering proteins from fermentation broths include, but are not limited to, collection, centrifugation, filtration, extraction, and precipitation. If the product of interest precipitates or crystallizes in the fermentation broth or is at least partially bound to particulate matter in the fermentation broth, additional processing steps may be required to release the product of interest from the biomass or to dissolve the crystals and precipitates of the product of interest. WO 0043502 A1, WO2008110498 A1, and WO 2017097869 A1 describe methods for recovering a product of interest from a fermentation broth that precipitates and / or crystallizes during fermentation. If the desired protein is contained in the cells of the fermentation broth, release of the product of interest from the cells may be required. Release from the cells can be achieved, for example but not limited to, by cell lysis using techniques well known to those skilled in the art, such as lysozyme treatment, sonication, French press, or combinations thereof.

[0132] The cutinase can be purified from the fermentation broth by methods known in the art. For example, the cutinase can be isolated from the fermentation broth by conventional procedures that include, but are not limited to, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation. The isolated polypeptide can then be further purified by a variety of procedures known in the art, including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing (IEF)), differential solubility (e.g., ammonium sulfate precipitation), or extraction (see, for example, Protein Purification, J.-C. Janson and Lars Ryden, eds., VCH Publishers, New York, 1989). The purified polypeptide can then be concentrated by procedures known in the art, including, but not limited to, ultrafiltration and evaporation, particularly thin-film evaporation.

[0133] Composition

[0134] The purified solution of the cutinase described herein can be further processed to form a composition containing the cutinase. Accordingly, also claimed herein is a composition comprising the cutinase described herein and at least one additional component.

[0135] Therefore, the present invention also relates to a method for preparing a composition, the method comprising the steps of: mixing

[0136] a) a cutinase as described herein; and

[0137] b) one or more components as described herein.

[0138] The composition can be a non-compound formulation (such as a cutinase formulation), or a compound formulation (such as a detergent composition).

[0139] Cutinase formulation

[0140] In one embodiment of the present invention, the cutinase is formulated as a cutinase formulation, preferably a concentrated cutinase formulation. The cutinase formulation can be solid or liquid. Such enzyme formulations can be obtained by using techniques known in the art. For example, but not limited to, solid enzyme formulations can be obtained by extrusion or granulation. Suitable extrusion and granulation techniques are known in the art and are described, for example, in WO 94 / 19444A1 and WO 97 / 43482A1.

[0141] The liquid cutinase formulation can contain an enzyme amount in the range of 0.1% to 40%, 0.5% to 30%, 1% to 25%, 1% to 10%, or preferably 1%-6% by weight, all relative to the total weight of the enzyme formulation.

[0142] In one embodiment, the cutinase formulation, especially the liquid enzyme formulation, further contains one or more additional compounds selected from the group consisting of: solvents, salts, pH regulators, preservatives, enzyme stabilizers, and thickeners. Preferably, the cutinase formulation is substantially free of surfactants. The solvent can be water and / or an organic solvent. The cutinase formulation of the present invention can contain water in an amount less than about 60%, less than about 50%, less than about 40%, or less than about 30% by weight, all relative to the total weight of the enzyme formulation. The formulation containing cutinase of the present invention can contain an organic solvent in an amount more than 30%, more than 40%, more than about 50%, more than about 60%, more than about 70%, or more than about 80% but less than 99.9% by weight, all relative to the total weight of the enzyme formulation. The organic solvent can be a water-miscible solvent. The organic solvent can be one or more selected from the group consisting of glycerol, propylene glycol, polypropylene glycol, and polyethylene glycol.

[0143] In one embodiment, the cutinase formulation comprises at least one preservative. Preferably, a preservative means a substance added to a liquid composition for the purpose of preservation, meaning more preferably that compounds known to have preservative characteristics contained in the liquid composition formed during the production process are excluded from the term preservative. In one embodiment, the preservative is selected from the group consisting of: 2-phenoxyethanol, glutaraldehyde, 2-bromo-2-nitropropane-1,3-diol and formic acid (in acid form or its salt form), and 4,4'-dichlor-2-hydroxy diphenyl ether. The liquid composition of the present invention may contain a preservative in an amount less than 10 ppm, for example, in the range of 2 ppm to 5% by weight relative to the total weight of the liquid composition. Preferably, the cutinase formulation is free of preservatives, which means that the content of the preservative is less than 1 ppm, preferably 0 ppm.

[0144] Preferably, the cutinase formulation comprises an enzyme stabilization system. The enzyme stabilization system can be any stabilization system compatible with cutinase.

[0145] Preferably, the enzyme stabilization system comprises at least one compound selected from the group consisting of: polyols (preferably 1,3-propanediol, ethylene glycol, glycerol, 1,2-propanediol, or sorbitol), inorganic salts (preferably CaCl2, MgCl2, or NaCl), short-chain (preferably C1-C3) carboxylic acids or their salts (preferably formic acid, formates (preferably sodium formate), acetic acid, acetates, or lactates).

[0146] Preferably, the liquid cutinase formulation comprises or consists of: cutinase, a solvent, an enzyme stabilization system and optionally a preservative and optionally a second enzyme different from the cutinase. Preferably, the cutinase formulation is substantially free of surfactants.

[0147] Therefore, the present invention also relates to a method for preparing a cutinase formulation (preferably a concentrated cutinase formulation), the method comprising the steps of mixing

[0148] a) cutinase as described herein; and

[0149] b) one or more components selected from the group consisting of: a solvent, an enzyme stabilization system and a preservative

[0150] c) and optionally a second enzyme different from the cutinase as described herein.

[0151] Second enzyme

[0152] In another embodiment, a composition comprising a cutinase as described herein further comprises one or more second enzymes different from the cutinase described herein. Preferably, the second enzyme is selected from the group consisting of: protease, amylase, second lipase, cellulase, mannanase, hemicellulase, phospholipase, esterase, pectinase, lactase, peroxidase, xylanase, second cutinase, pectate lyase, keratinase, reductase, oxidase, phenol oxidase, lipoxygenase, ligninase, pullulanase, tannase, pentosanase, malanase, β-glucanase, arabinosidase, hyaluronidase, chondroitinase, laccase, nuclease, deoxyribonuclease, phosphodiesterase, phytase, carbohydrase, galactanase, xanthanase, xyloglucanase, oxidoreductase, perhydrolase, aminopeptidase, asparaginase, carbohydrase, carboxypeptidase, catalase, chitinase, cyclodextrin glycosyltransferase, α-galactosidase, β-galactosidase, glucoamylase, α-glucosidase, β-glucosidase, glycosidase, invertase, ribonuclease, transglutaminase, and dispersin. More preferably, the one or more second enzymes are selected from the group consisting of: amylase, lipase, cellulase, mannanase, xylanase, DNase, dispersin, pectinase, pectate lyase, glycosidase, and oxidoreductase. Most preferably, the second enzyme is protease or amylase, preferably protease.

[0153] The compositions of the present invention can comprise one type of enzyme or more than one different type of enzyme, such as cutinase and protease, or more than one of the same type of enzyme, such as two or more different proteases, or a mixture thereof, such as cutinase and two different proteases.

[0154] Protease

[0155] Enzymes with proteolytic activity are referred to as "proteases" or "peptidases". Proteases are active proteins that exhibit "protease activity" or "proteolytic activity". Proteolytic activity is related to the rate at which proteins are degraded by proteases or proteolytic enzymes over a defined period of time.

[0156] Methods for analyzing proteolytic activity are well-known in the literature (see, for example, Gupta et al. (2002), Appl. Microbiol. Biotechnol. [Applied Microbiology and Biotechnology] 60: pp. 381-395). Proteolytic activity can be determined by using succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (Suc-AAPF-pNA, abbreviated as AAPF; see, for example, DelMar et al. (1979), Analytical Biochem [Analytical Biochemistry] 99, 316-320) as a substrate. Proteolytic cleavage cleaves pNA from the substrate molecule, resulting in the release of yellow free pNA, which can be quantified by measuring OD 405 for quantification.

[0157] Proteases are members of class EC 3.4. Proteases include aminopeptidases (EC 3.4.11), dipeptidases (EC 3.4.13), dipeptidyl peptidases and tripeptidyl peptidases (EC 3.4.14), peptidyl dipeptidases (EC 3.4.15), serine-type carboxypeptidases (EC 3.4.16), metal carboxypeptidases (EC 3.4.17), cysteine-type carboxypeptidases (EC 3.4.18), ω-peptidases (EC 3.4.19), serine endopeptidases (EC 3.4.21), cysteine endopeptidases (EC 3.4.22), aspartic endopeptidases (EC 3.4.23), metal endopeptidases (EC 3.4.24), threonine endopeptidases (EC 3.4.25) or endopeptidases of unknown catalytic mechanism (EC 3.4.99).

[0158] In one embodiment, the protease may be selected from metalloendopeptidases (EC 3.4.24). The metalloprotease may be, for example, thermolysin from family M4 or another metalloprotease, such as those from families M5, M7 or M8. The metalloprotease may in particular be derived from Bacillus amyloliquefaciens as described in WO 07 / 044993A2, from Bacillus, Brevibacillus, Thermoactinomyces, Geobacillus, Paenibacillus, Lysinibacillus or Streptomyces spp. as described in WO 2014194032, WO 2014194054 and WO 2014194117, from Kribella alluminosa as described in WO2015193488, or from Streptomyces and Lysobacter as described in WO 2016075078.

[0159] In another embodiment, the protease may be selected from serine proteases (EC 3.4.21). Serine proteases or serine peptidases are characterized by having serine at the catalytic active site, which forms a covalent adduct with the substrate during the catalytic reaction. The serine protease may be selected from the group consisting of: chymotrypsin (e.g., EC 3.4.21.1), elastase (e.g., EC 3.4.21.36, e.g., EC 3.4.21.37 or EC 3.4.21.71), granzyme (e.g., EC3.4.21.78 or EC 3.4.21.79), kallikrein (e.g., EC 3.4.21.34, EC 3.4.21.35, EC3.4.21.118 or EC 3.4.21.119), plasmin (e.g., EC 3.4.21.7), trypsin (e.g., EC3.4.21.4), thrombin (e.g., EC 3.4.21.5) and subtilisin. Subtilisin is also known as subtilopeptidase, e.g., EC 3.4.21.62, which is also referred to hereinafter as "subtilisin".

[0160] Subgroups of serine proteases are trypsin-like or chymotrypsin-like proteases such as trypsin (e.g., of porcine or bovine origin), the Fusarium protease described in WO 89 / 06270, and the chymotrypsin derived from Cellumonas described in WO 05 / 052161 and WO 05 / 052146.

[0161] Another subgroup of serine proteases, tentatively designated as subtilases, has been proposed by Siezen et al. (1991), Protein Eng. 4:719 - 737 and Siezen et al. (1997), Protein Science 6:501 - 523. They are defined by a homology analysis of more than 170 amino acid sequences of serine proteases (previously called subtilisin-like proteases). For a more detailed description of such subtilases and their amino acid sequences, reference is made to Siezen et al. (1997), Protein Science 6:501 - 523.

[0162] For the purposes of the present invention, the protease may be selected from the following: subtilisin from Bacillus amyloliquefaciens BPN' (described by Vasantha et al. (1984) J. Bacteriol. Vol. 159, pp. 811 - 819 and JA Wells et al. (1983) in Nucleic Acids Research, Vol. 11, pp. 7911 - 7925); subtilisin Carlsberg from Bacillus licheniformis (disclosed in EL Smith et al. (1968) J. Biol Chem, Vol. 243, pp. 2184 - 2191 and Jacobs et al. (1985) Nucl. Acids Res, Vol. 13, pp. 8913 - 8926); subtilisin PB92 (the original sequence of alkaline protease PB92 is described in EP 283075A2); subtilisin 147 and / or 309 as disclosed in WO 89 / 06279 (respectively ) proteases from Bacillus lentus as disclosed in WO 91 / 02792, such as subtilisin from Bacillus lentus DSM 5483 or a variant of Bacillus lentus DSM 5483 as described in WO 95 / 23221; subtilisin from Bacillus alkalophilus (DSM 11233) as disclosed in DE 10064983; subtilisin from Bacillus gibsonii (DSM 14391) as disclosed in WO 2003 / 054184; subtilisin from Bacillus sp. (DSM 14390) as disclosed in WO 2003 / 056017; subtilisin from Bacillus sp. (DSM 14392) as disclosed in WO 2003 / 055974; subtilisin from Bacillus gibsonii (DSM 14393) as disclosed in WO 2003 / 054184; subtilisin having SEQ ID NO:4 as described in WO 2005 / 063974; subtilisin having SEQ ID NO:4 as described in WO 2005 / 103244; subtilisin having SEQ ID NO:7 as described in WO 2005 / 103244; and subtilisin having SEQ ID NO:2 as described in application DE 102005028295.4.

[0163] The protease may be subtilisin 309 as disclosed in sequence a) of Table I of WO 89 / 06279 (which may be referred to herein as Savinase) or a variant that is at least 80% identical thereto and has proteolytic activity.

[0164] Further examples of proteases useful according to the invention include variants described in the following: WO 92 / 19729, WO 95 / 23221, WO 96 / 34946, WO 98 / 20115, WO 98 / 20116, WO 99 / 11768, WO 01 / 44452, WO 02 / 088340, WO 03 / 006602, WO 2004 / 03186, WO 2004 / 041979, WO 2007 / 006305, WO 2011 / 036263, WO 2011 / 036264 and WO 2011 / 072099. Suitable examples especially include protease variants of subtilisin having amino acid substitutions at one or more of the following positions derived from SEQ ID NO:22 as described in EP1921147 (which is the sequence of the mature alkaline protease from Bacillus lentus DSM 5483): 3, 4, 9, 15, 24, 27, 33, 36, 57, 68, 76, 77, 87, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 106, 118, 120, 123, 128, 129, 130, 131, 154, 160, 167, 170, 194, 195, 199, 205, 206, 217, 218, 222, 224, 232, 235, 236, 245, 248, 252 and 274 (according to BPN' numbering), which have proteolytic activity. In one embodiment, such subtilisin is not mutated at positions Asp32, His64 and Ser221 (according to BPN' numbering).

[0165] The subtilisin may have SEQ ID NO:22 as described in EP 1921147 (which may be referred to herein as BLAP WT), or a variant thereof that has at least 80% identity with SEQ ID NO:22 as described in EP 1921147 and has proteolytic activity. In one embodiment, the subtilisin has at least 80% identity with SEQ ID NO:22 as described in EP 1921147 and is characterized by having the amino acid glutamate (E), or aspartic acid (D), or asparagine (N), or glutamine (Q), or alanine (A), or glycine (G), or serine (S) at position 101 (according to BPN' numbering) and has proteolytic activity. In one embodiment, the subtilisin is at least 80% identical to SEQ ID NO:22 as described in EP 1921147 and is characterized by having the amino acid glutamate (E) or aspartic acid (D) at position 101 (according to BPN' numbering) and has proteolytic activity. Such subtilisin variants may comprise an amino acid substitution at position 101, such as R101E or R101D alone or in combination with one or more substitutions at positions 3, 4, 9, 15, 24, 27, 33, 36, 57, 68, 76, 77, 87, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 106, 118, 120, 123, 128, 129, 130, 131, 154, 160, 167, 170, 194, 195, 199, 205, 206, 217, 218, 222, 224, 232, 235, 236, 245, 248, 252, and / or 274 (according to BPN' numbering) and has proteolytic activity. In one embodiment, the protease comprises one or more additional substitutions: (a) threonine at position 3 (3T), (b) isoleucine at position 4 (4I), (c) alanine, threonine, or arginine at position 63 (63A, 63T, or 63R), (d) aspartic acid or glutamic acid at position 156 (156D or 156E), (e) proline at position 194 (194P), (f) methionine at position 199 (199M), (g) isoleucine at position 205 (205I), (h) aspartic acid, glutamic acid, or glycine at position 217 (217D, 217E, or 217G), (i) a combination of two or more of the amino acids according to (a) to (h).

[0166] Suitable proteases also include those that are variants of the above proteases and have proteolytic activity. In one embodiment, protease variants include variants having at least 40% to 100% identity to the full-length polypeptide sequence of the parental enzyme disclosed above. In one embodiment, a protease variant having proteolytic activity is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the full-length polypeptide sequence of the parental enzyme disclosed above.

[0167] Suitable subtilisins can be at least 80% identical to SEQ ID NO:22 as described in EP 1921147, and are characterized by containing one amino acid (according to (a)-(h)) or a combination according to (i) with amino acids 101E, 101D, 101N, 101Q, 101A, 101G or 101S (according to BPN' numbering), and having proteolytic activity.

[0168] In one embodiment, the subtilisin is at least 80% identical to SEQ ID NO:22 as described in EP 1921147, and is characterized by containing mutations (according to BPN' numbering) R101E, or S3T+V4I+V205I, or S3T+V4I+R101E+V205I or S3T+V4I+V199M+V205I+L217D or R101E+S156D+L262E, and having proteolytic activity.

[0169] The subtilisin can have an amino acid sequence that is at least 80% identical to SEQ ID NO:22 as described in EP 1921147, and is further characterized by containing R101E, and one or more substitutions selected from the group consisting of: S156D, L262E, Q137H, S3T, R45E,D,Q, P55N, T58W,Y,L, Q59D,M,N,T, G61 D,R, S87E, G97S, A98D,E,R, S106A,W, N117E, H120V,D,K,N, S125M, P129D, E136Q, S144W, S161T, S163A,G, Y171L, A172S, N185Q, V199M, Y209W, M222Q, N238H, V244T, N261T,D and L262N,Q,D (as described in WO 2016 / 096711 and according to BPN' numbering), and having proteolytic activity.

[0170] The protease can be selected from commercially available ones, including but not limited to those sold under the trade name Duralase TM 、Durazym TM 、 Ultra、 Ultra、 Ultra、 Ultra、 and (those sold by Novozymes A / S) under the trade names Prime, Purafect Purafect Purafect and (Danisco / DuPont), Axapem TM (Gist-Brocases N.V.), Bacillus lentus alkaline protease (BLAP; the sequence shown in Figure 29 of US 5,352,604) and its variants, and KAP (Bacillus subtilis protease from Kao Corporation).

[0171] Lipase

[0172] Lipases other than cutinases as described herein can also be included as the second enzyme in the compositions described herein. "Lipase", "lipolytic enzyme" or "lipid esterase" all refer to enzymes of EC class 3.1.1 ("carboxylic ester hydrolases"). Lipase means an active protein having lipase activity (or lipolytic activity; triacylglycerol lipase, EC 3.1.1.3), cutinase activity (EC 3.1.1.74) other than cutinases as described herein, sterol esterase activity (EC 3.1.1.13) and / or wax ester hydrolase activity (EC 3.1.1.50). Lipases include those of bacterial or fungal origin.

[0173] In one aspect of the present invention, suitable lipases as the second enzyme are selected from the following: lipases from Humicola (synonym Thermomyces), such as those described in EP 258068, EP 305216, WO 92 / 05249 and WO 2009 / 109500, for example, lipases from Humicola lanuginosa (Thermomyces lanuginosus), or lipases from Humicola insolens as described in WO 96 / 13580; lipases derived from Rhizomucor miehei as described in WO 92 / 05249; lipases from strains of Pseudomonas (some of which are now renamed Burkholderia), such as lipases from Pseudomonas alcaligenes or Pseudomonas pseudoalcaligenes (EP 218272, WO 94 / 25578, WO 95 / 30744, WO 95 / 35381, WO 96 / 00292), Pseudomonas cepacia (EP 331376), Pseudomonas stutzeri (GB 1372034), Pseudomonas fluorescens, Pseudomonas sp. strain SD705 (WO 95 / 06720 and WO 96 / 27002), Pseudomonas wisconsinensis (WO 96 / 12012), Pseudomonas mendocina (WO 95 / 14783), Pseudomonas glumae (WO 95 / 35381, WO 96 / 00292); lipases from Streptomyces griseus (WO 2011 / 150157) and Streptomyces pristinaespiralis (S.lipases from Pristinaespiralis) (WO 2012 / 137147), GDSL-type Streptomyces lipases (WO 2010 / 065455); lipases from Thermobifida fusca as disclosed in WO 2011 / 084412; lipases from Geobacillus stearothermophilus as disclosed in WO 2011 / 084417; Bacillus lipases, such as those disclosed in WO00 / 60063, as disclosed by Dartois et al. (1992), Biochemica et Biophysica Acta, 1131, 253 - 360 or WO 2011 / 084599, lipases from Bacillus subtilis, Bacillus stearothermophilus (JP S64 - 074992) or Bacillus pumilus (WO 91 / 16422); lipases from Candida antarctica as disclosed in WO 94 / 01541; cutinases from Pseudomonas mendocina (US 5389536, WO 88 / 09367); cutinases from Magnaporthe grisea (WO 2010 / 107560); cutinases from Fusarum solani pisi as disclosed in WO 90 / 09446, WO 00 / 34450 and WO 01 / 92502; and cutinases from Humicola lanuginosa as disclosed in WO 00 / 34450 and WO 01 / 92502.

[0174] Such suitable lipase variants are, for example, those developed by the methods disclosed in WO 95 / 22615, WO 97 / 04079, WO 97 / 07202, WO 00 / 60063, WO 2007 / 087508, EP 407225 and EP 260105.

[0175] Commercially available lipases include, but are not limited to, those sold under the trade names Lipolase TM , Lipex TM , Lipolex TM and Lipoclean TMThose sold by Novozymes A / S, Lumafast (originally from Genencor), Preferenz L (DuPont), and Lipomax (Gist-Brocades / now DSM).

[0176] In one embodiment, the lipase is selected from fungal triacylglycerol lipases (EC class 3.1.1.3). The fungal triacylglycerol lipase can be selected from the lipase of Thermomyces lanuginosus. In one embodiment, the Thermomyces lanuginosus lipase is selected from the triacylglycerol lipase of amino acids 1-269 of SEQ ID NO:2 according to US 5869438 and variants thereof having lipolytic activity.

[0177] The Thermomyces lanuginosus lipase can be selected from variants having lipolytic activity that are 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% identical when compared to the full-length polypeptide sequence of amino acids 1-269 of SEQ ID NO:2 of US 5869438.

[0178] The Thermomyces lanuginosus lipase can be selected from variants having lipolytic activity that contain only conservative mutations and do not involve the functional domains of amino acids 1-269 of SEQ ID NO:2 of US 5869438. The lipase variant of this embodiment having lipolytic activity can be at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similar when compared to the full-length polypeptide sequence of amino acids 1-269 of SEQ ID NO:2 of US 5869438.

[0179] The Thermomyces lanuginosus lipase can be selected from variants having lipolytic activity that contain the following amino acid substitutions when compared to amino acids 1-269 of SEQ ID NO:2 of US 5869438: T231R and N233R. When compared to amino acids 1-269 of SEQ ID NO:2 of US 5869438, the lipase variant can further contain one or more of the following amino acid exchanges: Q4V, V60S, A150G, L227G, P256K.

[0180] The Thermomyces lanuginosus lipase may be a variant with lipolytic activity that includes the amino acid substitutions T231R, N233R, Q4V, V60S, A150G, L227G, P256K within the polypeptide sequence of amino acids 1-269 of SEQ ID NO:2 of US 5869438 and is at least 95%, at least 96%, or at least 97% similar when compared to the full-length polypeptide sequence of amino acids 1-269 of SEQ ID NO:2 of US 5869438.

[0181] The Thermomyces lanuginosus lipase may be a variant with lipolytic activity that includes the amino acid substitutions T231R and N233R within amino acids 1-269 of SEQ ID NO:2 of US5869438 and is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% similar when compared to the full-length polypeptide sequence of amino acids 1-269 of SEQ ID NO:2 of US 5869438.

[0182] The Thermomyces lanuginosus lipase may be a variant with lipolytic activity of amino acids 1-269 of SEQ ID NO:2 of US 5869438, wherein the variant of amino acids 1-269 of SEQ ID NO:2 of US 5869438 is characterized by containing the amino acid substitutions T231R and N233R.

[0183] The Thermomyces lanuginosus lipase may be a variant with lipolytic activity that preferably includes at least one, preferably more than one, more preferably all of the following substitutions within the polypeptide sequence of amino acids 1-269 of SEQ ID NO:1 of WO 2015 / 010009: N11K, A18K, G23K, K24A, V77I, D130A, V154I, V187T, T189Q and is at least 95%, at least 96%, or at least 97% similar when compared to the full-length polypeptide sequence of amino acids 1-269 of SEQ ID NO:1 of WO2015 / 010009.

[0184] Amylase

[0185] "Amylase" (α and / or β) includes amylases of bacterial or fungal origin (EC 3.2.1.1 and 3.2.1.2, respectively). Preferably, the amylase is selected from the group of α-amylases (EC 3.2.1.1).

[0186] The amylase according to the invention has "amylolytic activity" or "amylase activity" which involves the (endo)hydrolysis of glycosidic linkages in polysaccharides.

[0187] The amylase can be from Bacillus licheniformis having SEQ ID NO:2 as described in WO 95 / 10603 and variants that are at least 95% identical thereto. Suitable variants are described in WO 95 / 10603 and include one or more substitutions at 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, which have amylolytic activity. The variants are described in SEQ ID NO:4 of WO 94 / 02597, WO 94 / 018314, WO 97 / 043424, and WO 99 / 019467.

[0188] The amylase can further be from Bacillus stearothermophilus having SEQ ID NO:6 as disclosed in WO 02 / 10355, or an amylase optionally having a C-terminal truncation compared to the wild-type sequence. Suitable variants of SEQ ID NO:6 include variants comprising a deletion at position 179 and / or 181 and / or 182 and / or a substitution at position 193.

[0189] The amylase can further be from Bacillus sp. 707 having SEQ ID NO:6 as disclosed in WO 99 / 19467 and variants that are at least 95% identical thereto. Preferred variants of SEQ NO:6 are those having a substitution, deletion, or insertion at one or more of the following positions: R181, G182, H183, G184, N195, I206, E212, E216, and K269.

[0190] The amylase can further be from Bacillus halmapalus having SEQ ID NO:2 or SEQ ID NO:7 as described in WO 96 / 23872, also described herein as SP-722. Preferred variants are described in WO97 / 3296, WO 99 / 194671, and WO 2013 / 001078.

[0191] The amylase can further be from Bacillus sp. DSM 12649 having SEQ ID NO:4 as disclosed in WO 00 / 22103 and variants that are at least 95% identical thereto.

[0192] The amylase may further be from Bacillus sp. A 7-7 (DSM 12368), which has an amino acid sequence that is at least 95% identical to SEQ ID NO:2, particularly in the region of amino acids 32 to 516 according to SEQ ID NO:2, as disclosed in WO 02 / 10356.

[0193] The amylase may further be from Bacillus sp. strain TS-23 having SEQ ID NO:2 and variants thereof as disclosed in WO 2009 / 061380.

[0194] The amylase may further be from a Cytophaga sp. having SEQ ID NO:1 and variants that are at least 95% identical thereto as disclosed in WO 2013 / 184577.

[0195] The amylase may further be from Bacillus megaterium DSM 90 having SEQ ID NO:1 and variants that are at least 95% identical thereto as disclosed in WO 2010 / 104675.

[0196] The amylase may further be from a Bacillus sp. comprising amino acids 1 to 485 of SEQ ID NO:2 as described in WO 00 / 60060 and variants that are at least 95% identical thereto.

[0197] The amylase may further be from Bacillus amyloliquefaciens or a variant thereof, preferably selected from the amylases according to SEQ ID NO:3 as described in WO 2016 / 092009.

[0198] The amylase may have SEQ ID NO:12 as described in WO 2006 / 002643, or may be an amylase variant thereof, the amylase variants of which comprise the substitutions Y295F and M202LITV within said SEQ ID NO:12.

[0199] The amylase can have SEQ ID NO:6 as described in WO 2011 / 098531, or can be an amylase variant that contains substitutions at one or more positions selected from the group consisting of: 193[G,A,S,T or M], 195[F,W,Y,L,I or V], 197[F,W,Y,L,I or V], 198[Q or N], 200[F,W,Y,L,I or V], 203[F,W,Y,L,I or V], 206[F,W,Y,N,L,I,V,H,Q,D or E], 210[F,W,Y,L,I or V], 212[F,W,Y,L,I or V], 213[G,A,S,T or M] and 243[F,W,Y,L,I or V] within said SEQ ID NO:6.

[0200] The amylase can have SEQ ID NO:1 as described in WO 2013 / 001078, or can be an amylase variant that contains alterations at two or more (several) positions corresponding to positions G304, W140, W189, D134, E260, F262, W284, W347, W439, W469, G476 and G477 within said SEQ ID NO:1.

[0201] The amylase can have SEQ ID NO:2 as described in WO 2013 / 001087, or can be an amylase variant that contains a deletion of positions 181+182, or 182+183, or 183+184 within said SEQ ID NO:2, optionally containing one or two or more modifications at any position corresponding to positions W140, W159, W167, Q169, W189, E194, N260, F262, W284, F289, G304, G305, R320, W347, W439, W469, G476 and G477 within said SEQ ID NO:2.

[0202] The amylase can be a hybrid α-amylase from the above-mentioned amylases, for example, as described in WO 2006 / 066594.

[0203] According to WO 2014 / 183920, a hybrid amylase can have A and B domains and a C domain, where the A and B domains have at least 90% identity with SEQ ID NO:2 of WO 2014 / 183920, and the C domain has at least 90% identity with SEQ ID NO:6 of WO 2014 / 183920, and where the hybrid amylase has amylolytic activity; preferably, the hybrid α-amylase is at least 95% identical to SEQ ID NO:23 of WO 2014 / 183920 and has amylolytic activity.

[0204] According to WO 2014 / 183921, a hybrid amylase can have A and B domains and a C domain, where the A and B domains have at least 75% identity with SEQ ID NO:2, SEQ ID NO:15, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:29, SEQ ID NO:26, SEQ ID NO:32, and SEQ ID NO:39 disclosed in WO 2014 / 183921, and the C domain has at least 90% identity with SEQ ID NO:6 of WO 2014 / 183921, and where the hybrid amylase has amylolytic activity; preferably, the hybrid α-amylase is at least 95% identical to SEQ ID NO:30 disclosed in WO 2014 / 183921 and has amylolytic activity.

[0205] According to WO 2021 / 032881, a hybrid amylase can comprise A and B domains derived from an α-amylase (from Bacillus sp. A7-7 (DSM 12368)), and a C domain derived from an α-amylase (from Bacillus cereus); preferably, the A and B domains are at least 75% identical to the amino acid sequence of SEQ ID NO:42, and the C domain is at least 75% identical to the amino acid sequence of SEQ ID NO:44 - both sequences as disclosed in WO 2021 / 032881; more preferably, the hybrid amylase is at least 80% identical to SEQ ID NO:54 disclosed in WO 2021 / 032881.

[0206] In a preferred embodiment, the amylase is a variant as described in WO 2022 / 175435.

[0207] In one embodiment, at least one amylase is selected from commercially available amylases, which commercially available amylases include but are not limited to those sold under the trade name Duramyl TM 、Termamyl TM 、FungamylTM , Stainzyme TM , Stainzyme Plus TM , Natalase TM , Liquozyme X and BAN TM , Amplify TM , Amplify Prime TM (from Novozymes A / S), and Rapidase TM , Purastar TM , Powerase TM , Effectenz TM (M100 from DuPont), Preferenz TM (S1000, S110 and F1000; from DuPont), PrimaGreen TM (ALL; DuPont), Optisize TM (DuPont).

[0208] Mannanase

[0209] As used herein, "mannanase" is an enzyme selected from the group of mannan-degrading enzymes. Mannan-degrading enzymes may be selected from β-mannosidase (EC 3.2.1.25), endo-1,4-β-mannosidase (EC 3.2.1.78), and 1,4-β-mannobiase (EC 3.2.1.100). Preferably, the mannan-degrading enzyme is selected from the group of endo-1,4-β-mannosidases (EC 3.2.1.78), which enzymes may be referred to herein as endo-β-1,4-D-mannanases, β-mannanases, or mannanases.

[0210] Mannanases may be selected from the alkaline mannanases of family 5 or 26 (i.e., GH5 or GH26). The term "alkaline mannanase" is intended to encompass mannanases having at least 40% of their maximum activity at a given pH of from 7 to 12, preferably from 7.5 to 10.5.

[0211] The mannanase can be selected from mannanases derived from organisms of the genus Bacillus, such as those described below: JP-0304706 [β-mannanase from a Bacillus species], JP-63056289 [alkaline, thermostable β-mannanase], JP-63036774 [Bacillus microorganism FERM P-8856, which produces β-mannanase and β-mannosidase at alkaline pH], JP-08051975 [alkaline β-mannanase from an alkalophilic Bacillus species AM-001], WO97 / 11164 [mannanase from Bacillus amyloliquefaciens], WO 91 / 18974 [mannanase active at extreme pH and temperature], WO 97 / 11164 [mannanase from Bacillus amyloliquefaciens], WO 2014 / 100018 [endo-(3-mannanase (Bleman) cloned from Bacillus circulans or Bacillus lentus strain CMG1240; see US 5,476,775)]. Other suitable mannanases are described in WO 99 / 064619.

[0212] The mannanase can be selected from mannanases derived from organisms of the genus Trichoderma, such as those disclosed in WO 93 / 24622.

[0213] Preferably, the mannanase is a variant as disclosed in any one of WO 2021 / 160816, WO 2021 / 160818 or WO 2021 / 160820.

[0214] Suitable mannanases also include those of variants of the above mannanases having mannanase activity. In one embodiment, the mannanase variant includes a variant having at least 40% to 100% identity when compared to the full-length polypeptide sequence of the parental enzyme disclosed above. In one embodiment, the mannanase variant having mannanase activity is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similar and / or identical to the full-length polypeptide sequence of the parental enzyme disclosed above.

[0215] The mannanase can be selected from commercially available mannanases, such as (Novozymes) or (M100) (DuPont).

[0216] Cellulase

[0217] "Cellulase" refers to an enzyme capable of hydrolyzing cellulose. The cellulase can be selected from cellobiohydrolase (1,4-β-D-glucan cellobiohydrolase, EC 3.2.1.91), endo-β-1,4-glucanase (EC 3.2.1.4), and β-glucosidase (EC 3.2.1.21). The endoglucanase of EC class 3.2.1.4 can be named endoglucanase, endo-1,4-β-D-glucan 4-glucanohydrolase, endo-1,4-β-glucanase, carboxymethyl cellulase, and β-1,4-glucanase.

[0218] The endoglucanase can be selected from family 5 endoglucanases. Also refer to T.-M. Enveri, "Microbial Cellulases", in W.M. Fogarty, Microbial Enzymes and Biotechnology, Applied Science Publishers, pp. 183-224 (1983); Methods in Enzymology, (1988) Vol. 160, pp. 200-391 (edited by Wood, W.A. and Kellogg, S.T.); Béguin, P., "Molecular Biology of Cellulose Degradation", Annu. Rev. Microbiol. (1990), Vol. 44, pp. 219-248; Begun, P. and Aubert, J-P., "The biological degradation of cellulose", FEMS Microbiology Reviews 13 (1994) pp. 25-58; Henrissat, B., "Cellulases and their interaction with cellulose", Cellulose (1994), Vol. 1, pp. 169-196.

[0219] Preferably, at least one cellulase contained in the composition of the present invention is selected from glycoside hydrolase family 7 (GH7, pfam00840), preferably selected from endoglucanase (EC 3.2.1.4).

[0220] Preferably, an alkaline cellulase is used, where "alkaline cellulase" is intended to cover cellulases having enzymatic activity at a pH in a given range of 7 to 12, preferably 7.5 to 10.5.

[0221] In one embodiment, the cellulase is selected from cellulases comprising a cellulose binding domain. In another embodiment, the cellulase comprises a catalytic domain but does not comprise a cellulose binding domain.

[0222] In one embodiment, the composition of the present invention comprises at least one endoglucanase of EC class 3.2.1.4, which is derived from

[0223] · Bacillus, such as Bacillus species CBS 670.93 and CBS 669.93

[0224] · Melanocarpus, such as Melanocarpus albomyces as disclosed in WO 97 / 14804

[0225] · Clostridium, such as Clostridium thermocellum

[0226] · Humicola, such as Humicola insolens (DSM1800) as disclosed in EP 0495257, EP 0531315, EP 0531372, US 4435307, US5648263, US 5776757, WO 89 / 09259, WO 91 / 17244, WO 94 / 07998 (sequence shown in Figure 1, its 43kd human variant), WO 95 / 24471, WO 96 / 11262 and WO 98 / 12307.

[0227] · Fusarium, such as Fusarium oxysporum, for example, strain J79 (DSM2672) as disclosed in EP0495257, EP 0531315, EP 0531372, US 5648263, US 5776757, WO 89 / 09259, WO 91 / 17244, WO 95 / 24471 and WO 96 / 11262

[0228] · Thielavia, such as Thielavia terrestris or Myceliophthora thermophila strain CBS11765 disclosed in EP 0531315, US 5648263, US 5776757, WO 89 / 09259, WO 91 / 17244, WO 95 / 24471, WO 96 / 11262, WO 96 / 29397 (SEQ ID NO:9 and its variants), and WO 98 / 12307.

[0229] · Trichoderma, such as Trichoderma reesei, Trichoderma longibrachiatum, or Trichoderma harzianum disclosed in EP 1305432, EP 1240525, WO 92 / 06165, WO 94 / 21801, WO 94 / 26880, WO 95 / 02043, WO 95 / 24471, and WO 02 / 099091.

[0230] · Aspergillus, such as Aspergillus aculeatus disclosed in WO 93 / 17244

[0231] · Erwinia, such as Erwinia chrysanthermi described by M.H. Boyer et al. in European Journal of Biochemistry, Vol. 162, pp. 311 - 316 (1987).

[0232] · *Acremonium*, such as *Acremonium sp.*, *Acremonium persicinum*, *Acremonium acremonium*, *Acremonium brachypenium*, *Acremonium dichromosporum*, *Acremonium obclavatum*, *Acremonium pinkertoniae*, *Acremonium roseogriseum*, *Acremonium incoloratum*, and *Acremonium furatum*, as disclosed in WO 96 / 11262 and WO 96 / 29397 (SEQ ID NO:5 and its variants).

[0233] · *Cellvibrio*, such as *Cellvibrio mixtus* DSM11683, *Cellvibrio mixtus* DSM 11684, *Cellvibrio mixtus* DSM 11685, *Cellvibrio mixtus* ACM 2601, *Cellvibrio mixtus* DSM 1523, and *Cellvibrio gilvus* DSM 11686, as disclosed in WO 98 / 08940.

[0234] · *Cephalosporium*, such as *Cephalosporium sp.* RYM-202, as disclosed in WO 96 / 11262.

[0235] Suitable cellulases also include those that are variants of the above cellulases with cellulolytic activity. In one embodiment, the cellulase variants include variants having at least 40% to 100% identity when compared to the full-length polypeptide sequence of the parental enzyme disclosed above. In one embodiment, the cellulase variants with cellulolytic activity are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% similar and / or identical to the full-length polypeptide sequence of the parental enzyme disclosed above.

[0236] The cellulase can be the specific Humicola insolens DSM 1800 cellulase complex having endoglucanase, cellobiohydrolase, and β-glucosidase activities.

[0237] The cellulase can be Humicola insolens DSM 1800 endoglucanase (EC 3.2.1.4), preferably having the polypeptide sequence of positions 21 - 435 of SEQ ID NO:2 as disclosed in WO 2018 / 224544 or a variant that is at least 95% identical thereto.

[0238] The cellulase can be Humicola insolens endoglucanase (EC 3.2.1.4) having a molecular weight of 43 kD, preferably having the polypeptide sequence (“43kDhum”) as disclosed in Figure 1a of WO 94 / 07998 or a variant thereof (preferably at least 90% identical thereto), preferably those disclosed in WO 94 / 07998.

[0239] The cellulase can be a Bacillus species cellulase (EC 3.2.1.4) selected from polypeptides that are at least 80% similar and / or identical to the amino acid sequence of positions 1 to 773 of SEQ ID NO:2 of WO 2004 / 053039 or catalytically active fragments thereof. In one embodiment, the cellulase is a mature polypeptide that is at least 95% identical to SEQ ID NO:1 of WO 2018 / 224544.

[0240] The cellulase can be Thielavia terrestris cellulase (EC 3.2.1.4) having a polypeptide that is at least 80% similar and / or identical to the amino acid sequence of positions 1 to 299 of SEQ ID NO:4 of WO 2004 / 053039 or a catalytically active fragment thereof. In one embodiment, the cellulase is a mature polypeptide that is at least 95% identical to SEQ ID NO:4 of WO 2018 / 224544.

[0241] The cellulase can be a mature Sordaria fimicola cellulase, preferably having the polypeptide sequence of SEQ ID NO:5 of WO 2018 / 224544 or a variant that is at least 95% identical thereto.

[0242] At least one cellulase can be selected from and (Novozymes A / S), Clazinase TM and Puradax HA TM (Genencor Int. Inc.) and KAC - 500(B) TM (Kao Corporation).

[0243] Detergent composition

[0244] In one embodiment, the present invention relates to the use of a cutinase as described herein in a detergent composition. Accordingly, the present invention also relates to a detergent composition comprising a cutinase as described herein and one or more detergent components.

[0245] Accordingly, the present invention also relates to a method for preparing a detergent composition, the method comprising the steps of mixing

[0246] a) a cutinase as described herein; and

[0247] b) one or more detergent components as described herein.

[0248] The one or more detergent components may optionally be selected from the group consisting of: additional enzymes different from the cutinase as described herein, enzyme stabilization systems, surfactants, defoamers, builders, polymers, bleaching systems (bleaching agents), rheology modifiers, hydrotropes, softeners, drying agents, optical brighteners, buffers, preservatives, anti-corrosion additives, dyes, and fragrances.

[0249] Preferably, at least one component of the detergent is selected from the group consisting of: surfactants, builders, polymers, preservatives, and a second enzyme different from the cutinase variant.

[0250] A detergent component may have more than one function in the final application of the detergent composition, and thus any detergent component mentioned herein in the context of a specific function may also have another function in the final application of the detergent composition. The function of a particular detergent component in the final application of the detergent composition generally depends on its amount in the detergent composition, i.e., the effective amount of the detergent component. The type and / or amount of the detergent component in the detergent composition depends on the desired application, such as washing white textiles, colored textiles, and wool. The further selection of one or more components depends on the physical form of the detergent composition (liquid, solid, gel, provided in sachets or as tablets, etc.). For example, the further selection of one or more components for the detergent composition depends on regional practices, which in turn are related to aspects such as the washing temperature used, the mechanics of the washing machine (vertical axis compared to horizontal axis machines), the water consumption per wash cycle, etc., and geographical features such as the average hardness of the water.

[0251] In one embodiment, the detergent composition comprises more than two detergent components, wherein at least one component is effective in removing stains, at least one component is effective in providing optimal cleaning conditions, and at least one component is effective in maintaining the physical properties of the detergent.

[0252] The detergent composition can be a liquid or solid detergent composition or a combination of liquid and solid detergent compositions. The liquid detergent composition is preferably a gel detergent composition. The solid detergent composition can be a soap bar or a powder detergent composition, preferably a powder detergent composition, where the powder detergent composition can be pressed into tablets.

[0253] The detergent composition can be a unit dose or multi-dose composition. The detergent composition can be in the form of a sachet (including multi-compartment sachets). The detergent composition can be a laundry or dishwashing detergent composition, suitable for home care and / or industrial and institutional (I&I) cleaning. Both the laundry and dishwashing compositions can be in the form of hand-wash or automatic wash compositions. Preferably, the dishwashing composition is an automatic dishwashing (ADW).

[0254] The detergent sachet can be of any form, shape, and material suitable for containing the composition, for example, not allowing the composition to be released from the sachet before contact with water. The sachet is made of a water-soluble film that encloses an internal volume. The internal volume can be divided into compartments of the sachet. The preferred film is a polymeric material, preferably a polymer forming a film or sheet. Preferred polymers, copolymers, or their derivatives are selected from polyacrylates and water-soluble acrylate copolymers, methylcellulose, carboxymethylcellulose, sodium dextrin, ethylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose, maltodextrin, polymethacrylate, most preferably polyvinyl alcohol copolymers and hydroxypropylmethylcellulose (HPMC). Preferably, the level of polymer in the film, such as PVA, is at least about 60%. The preferred average molecular weight will typically be from about 20,000 to about 150,000. The film can also be a blend composition containing a hydrolyzable and water-soluble polymer blend, such as polylactic acid and polyvinyl alcohol (known, for example, as sold by Chris Craft In. Prod., Gary, Indiana, USA, under the commercial reference number M8630) plus plasticizers such as glycerol, ethylene glycol, propylene glycol, sorbitol, and mixtures thereof. The sachet can contain a solid laundry detergent composition and / or a liquid detergent composition. The compartment for the liquid component can be compositionally different from the compartment containing the solid (see, for example, US2009 / 0011970).

[0255] The detergent composition can contain an amount of enzyme in the range of 0.0002% to 0.09% by weight, preferably in the range of 0.0002% to 0.01%, all relative to the total weight of the detergent composition. In one embodiment, the pH of the detergent composition is in the range of 5 - 12, preferably in the range of 6 - 11, more preferably in the range selected from 6 - 10, 7 - 9, and 7.5 - 8.5. In one embodiment, the composition is a detergent composition, preferably a liquid detergent composition.

[0256] In one embodiment, the detergent composition according to the present invention comprises one or more surfactants. Depending on their ionic charge, surfactants are referred to as nonionic surfactants, anionic surfactants, cationic surfactants or amphoteric surfactants.

[0257] The detergent composition of the present invention may comprise one or more surfactants, which may be anionic and / or cationic and / or nonionic and / or semi-polar and / or zwitterionic, or mixtures thereof. In a preferred embodiment, the detergent composition of the present invention comprises at least one surfactant. In a particular embodiment, the detergent composition of the present invention comprises a mixture of one or more nonionic surfactants and one or more anionic surfactants. One or more surfactants are typically present at a level of about 0.1 wt.-% to 60 wt.-% (such as 1 wt.-% to 40 wt.-%, 3 wt.-% to 20 wt.-% or 3 wt.-% to 10 wt.-%). One or more surfactants are selected based on the desired cleaning application and include any one or more conventional surfactants known in the art.

[0258] In one embodiment, the detergent composition of the present invention comprises a reduced amount of surfactant compared to a conventional detergent without cutinase as described herein.

[0259] Any surfactant known in the art for use in detergents can be used. Non-limiting examples of surfactants are disclosed in McCutcheon's 2016 Detergents and Emulsifiers, and McCutcheon's 2016 Functional Materials, both in North American and International editions, MC Publishing Co, 2016 edition. Other useful examples are disclosed in earlier editions of the same publications known to those skilled in the art.

[0260] The detergent composition according to the present invention may comprise one or more compounds selected from complexing agents (chelating agents (chelants), sequestrating agents), precipitating agents and ion exchange compounds, which can form water-soluble complexes with calcium and magnesium. Such compounds may be referred to herein as "builders" or "building agents", but are not meant to limit such compounds to this function in the final application of the detergent composition.

[0261] In one embodiment, the detergent composition of the present invention comprises at least one builder selected from non-phosphate-based builders, which non-phosphate-based builders are, for example, sodium gluconate, one or more sodium citrates, one or more sodium silicates, one or more sodium carbonates, one or more sodium phosphonates, one or more sodium aminocarboxylates, one or more polycarboxylates, one or more polysulfonates and one or more sodium polyphosphonates. In one embodiment, the detergent composition of the present invention comprises a strong chelating builder. Preferably, the detergent composition of the present invention is phosphate-free, meaning substantially free of phosphate-based builders. As used herein, "substantially free of phosphate" is understood to mean that, as determined by gravimetry and with reference to the corresponding detergent composition of the present invention, the total content of phosphates and polyphosphates is in the range of 10 ppm to 1% by weight. In another preferred embodiment, the detergent composition comprises a phosphonate, wherein the phosphonate is preferably DTPMP and / or HEDP.

[0262] In one embodiment, the detergent composition of the present invention comprises at least one "citrate", which is selected from the mono-alkali metal salts and di-alkali metal salts of citric acid, in particular the monosodium salt of citric acid and preferably the trisodium salt, the ammonium salts or substituted ammonium salts of citric acid, and citric acid itself.

[0263] The detergent composition of the present invention may comprise one or more silicates. "One or more silicates" in the context of the present invention particularly includes disodium silicate and sodium metasilicate, aluminosilicates such as sodium aluminosilicate such as zeolite A (i.e., Na 12 (AlO2) 12 (SiO2) 12 *27H2O) and sheet silicates, in particular those having the formulas α-Na2Si2O5, β-Na2Si2O5 and δ-Na2Si2O5.

[0264] The detergent composition of the present invention may comprise one or more carbonates. The term "one or more carbonates" includes alkali metal carbonates and alkali metal bicarbonates, preferably sodium salts. Particularly suitable is sodium carbonate (Na2CO3).

[0265] The detergent composition of the present invention may comprise one or more phosphonates. "Phosphonates" include but are not limited to 2-phosphonobutane-1,2,4-tricarboxylic acid (PBTC); ethylenediaminetetra(methylenephosphonic acid) (EDTMPA); 1-hydroxyethane-1,1-diphosphonic acid (HEDP), CH2C(OH)[PO(OH)2]2; aminotri(methylenephosphonic acid) (ATMP), N[CH2PO(OH)2]3; sodium aminotri(methylenephosphonate) (ATMP), N[CH2PO(ONa)2]3; 2-hydroxyethyliminobis(methylenephosphonic acid), HOCH2CH2N[CH2PO(OH)2]2; diethylenetriaminepenta(methylenephosphonic acid) (DTPMP), (HO)2POCH2N[CH2CH2N[CH2PO(OH)2]2]2; sodium diethylenetriaminepenta(methylenephosphonate), C9H (28-x) N3Na x O 15 P5(x = 7); potassium hexamethylenediamine(tetramethylenephosphonic acid), C 10 H (28-x) N2K x O 12 P4(x = 6); and bis(hexamethylene)triamine(pentamethylenephosphonic acid), (HO2)POCH2N[(CH2)2N[CH2PO(OH)2]2]2. Their salts may also be suitable.

[0266] The detergent composition of the present invention may comprise one or more aminocarboxylates. Non-limiting examples of suitable "aminocarboxylates" include, but are not limited to: diethanol glycine (DEG), dimethyl glycine (DMG), nitrilotriacetic acid (NTA), N-hydroxyethyliminodiacetic acid, ethylenediaminetetraacetic acid (EDTA), N-(2-hydroxyethyl)iminodiacetic acid (HEIDA), hydroxyethylenediaminetriacetic acid, N-hydroxyethyl-ethylenediaminetriacetic acid (HEDTA), hydroxyethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid (DTPA), and methylglycine diacetic acid (MGDA), glutamic acid-diacetic acid (GLDA), iminodisuccinic acid (IDS), hydroxyminodisuccinic acid, ethylenediaminedisuccinic acid (EDDS), aspartic acid-diacetic acid, and their alkali metal salts or ammonium salts. Also suitable are aspartic acid-N-monoacetic acid (ASMA), aspartic acid-N,N-diacetic acid (ASDA), aspartic acid-N-monopropionic acid (ASMP), N-(2-sulfomethyl)aspartic acid (SMAS), N-(2-sulfoethyl)aspartic acid (SEAS), N-(2-sulfomethyl)glutamic acid (SMGL), N-(2-sulfoethyl)glutamic acid (SEGL), N-methyliminodiacetic acid (MIDA), α-alanine-N,N-diacetic acid (α-ALDA), serine-N,N-diacetic acid (SEDA), isoserine-N,N-diacetic acid (ISDA), phenylalanine-N,N-diacetic acid (PHDA), anthranilic acid-N,N-diacetic acid (ANDA), sulfanilic acid-N,N-diacetic acid (SLDA), taurine-N,N-diacetic acid (TUDA), and sulfomethyl-N,N-diacetic acid (SMDA) and their alkali metal salts or ammonium salts. As used in this context, the term "ammonium salt" refers to a salt having a cation with at least one nitrogen atom carrying a permanently or temporarily quaternized nitrogen atom. Examples of cations carrying at least one permanently quaternized nitrogen atom include tetramethylammonium, tetraethylammonium, dimethyldiethylammonium, and n-C 10 -C 20 -alkyltrimethylammonium. Examples of cations carrying at least one temporarily quaternized nitrogen atom include protonated amines and ammonia, such as monomethylammonium, dimethylammonium, trimethylammonium, monoethylammonium, diethylammonium, triethylammonium, n-C 10 -C 20 -alkyldimethylammonium 2-hydroxyethylammonium, bis(2-hydroxyethyl)ammonium, tris(2-hydroxyethyl)ammonium, N-methyl-2-hydroxyethylammonium, N,N-dimethyl-2-hydroxyethylammonium, and especially NH4 + .

[0267] In one embodiment, the detergent composition of the present invention comprises more than one builder. Preferably, the detergent composition of the present invention contains less than 0.2% by weight of nitrilotriacetic acid (NTA), or 0.01% to 0.1% by weight of NTA, relative to the total weight of the detergent composition.

[0268] In one embodiment, the detergent composition of the present invention comprises at least one aminocarboxylate selected from the group consisting of methylglycine diacetate (MGDA), glutamate diacetate (GLDA), and their corresponding salts, such as their alkali metal (such as sodium) salts, in an amount in the range of 0.1% to 25.0% by weight, in the range of 1.0% to 18.0% by weight, in the range of 3.0% to 15.0% by weight, in the range of 3.0% to 10.0% by weight, or in the range of 5.0% to 8.0% by weight, relative to the total weight of the detergent composition.

[0269] The detergent composition of the present invention may comprise one or more hydrotropes. The one or more hydrotropes may be selected from organic solvents such as ethanol, isopropanol, ethylene glycol, 1,2 - propylene glycol, and additional organic solvents known in the art that are miscible with water under normal conditions, but are not limited thereto. In one embodiment, the detergent composition of the present invention comprises 1,2 - propylene glycol in an amount in the range of 5% - 10% by weight, preferably about 6% by weight, all relative to the total weight of the detergent composition. Other non - limiting examples of hydrotropes include sodium benzenesulfonate, sodium p - toluenesulfonate (STS), sodium xylenesulfonate (SXS), sodium cumenesulfonate (SCS), sodium cymenesulfonate, amine oxides, alcohols and polyethylene glycol ethers, sodium hydroxynaphthoate, sodium hydroxynaphthalenesulfonate, sodium 2 - ethylhexyl sulfate, and combinations thereof.

[0270] In one embodiment, the detergent composition comprises at least one preservative. Preferably, a preservative means a substance added to a liquid composition for the purpose of preservation, meaning more preferably that compounds known to have preservative characteristics contained in the liquid composition formed during the production process are excluded from the term preservative. In one embodiment, the preservative is selected from the group consisting of 2 - phenoxyethanol, glutaraldehyde, 2 - bromo - 2 - nitropropane - 1,3 - diol and formic acid (in acid form or its salt form), and 4,4'-dichloro - 2 - hydroxy diphenyl ether. The liquid composition of the present invention may comprise at least one preservative in an amount less than 10 ppm, for example, in the range of 2 ppm to 5% by weight relative to the total weight of the liquid composition. Preferably, the liquid composition is free of preservatives, which means the content of the preservative is less than 1 ppm.

[0271] In one embodiment, a detergent composition comprising a cutinase as described herein further comprises one or more second enzymes different from the cutinase. Preferably, the second enzyme is selected from the group consisting of: protease, amylase, second lipase, cellulase, mannanase, hemicellulase, phospholipase, esterase, pectinase, lactase, peroxidase, xylanase, pectate lyase, keratinase, reductase, oxidase, phenol oxidase, lipoxygenase, ligninase, pullulanase, tannase, pentosanase, malanases, β-glucanase, arabinosidase, hyaluronidase, chondroitinase, laccase, nuclease, DNase, phosphodiesterase, phytase, carbohydrase, galactanase, xanthanase, xyloglucanase, oxidoreductase, perhydrolase, aminopeptidase, asparaginase, carbohydrase, carboxypeptidase, catalase, chitinase, cyclodextrin glycosyltransferase, α-galactosidase, β-galactosidase, glucoamylase, α-glucosidase, β-glucosidase, invertase, ribonuclease, transglutaminase, and dispersins, and combinations of at least two of the foregoing types. More preferably, the second enzyme is selected from the group consisting of: protease, amylase, lipase, cellulase, mannanase, xylanase, DNase, dispersin, pectinase, pectate lyase, glycosidase, and oxidoreductase, and combinations of at least two of the foregoing types. Most preferably, the second enzyme is amylase or protease, preferably protease.

[0272] Particularly preferred additional enzymes are disclosed elsewhere herein, and that description is also incorporated by reference into this part of the specification.

[0273] The compositions of the present invention can contain one type of enzyme or more than one different type of enzyme, such as amylase and protease, or more than one of the same type of enzyme, such as two or more different proteases, or a mixture thereof, such as amylase and two different proteases.

[0274] The detergent composition can contain a water-soluble calcium and / or magnesium ion source. In one embodiment, the detergent composition comprises an enzyme stabilization system as described herein.

[0275] In one embodiment, the present invention relates to a method of providing a detergent composition, preferably a liquid detergent composition, more preferably a liquid laundry detergent composition, the method comprising the step of mixing in one or more steps:

[0276] (a) at least one cutinase according to the present invention, preferably wherein the cutinase is provided in a cutinase formulation as described herein; and

[0277] (b) at least one detergent component, preferably selected from surfactants, builders, polymers, preservatives, and

[0278] (c) Optionally, a second enzyme different from the cutinase as described herein,

[0279] (b) and (c) are present in an amount effective for cleaning performance and / or effective for maintaining the physical properties of the detergent.

[0280] In one embodiment, the present invention relates to a detergent composition comprising

[0281] a) a cutinase as described herein;

[0282] b) one or more surfactants, preferably at a concentration of 0.2% - 65%, preferably 0.2% - 40%,

[0283] c) one or more builders, preferably at a concentration of 0.01% - 25%, and

[0284] d) Optionally, one or more additional compounds selected from the group consisting of: additional enzymes different from the cutinase in a), defoamers, polymers, bleaching systems (bleaching agents), rheology modifiers, hydrotropes, softeners, desiccants, optical brighteners, buffers, preservatives, anti-corrosion additives, dyes, and fragrances;

[0285] Preferably, the detergent composition is a liquid, powder, sachet or capsule detergent composition.

[0286] Preferably, the detergent composition of the present invention, preferably the powder detergent composition, in addition to containing the cutinase described herein, further comprises

[0287] a) one or more surfactants selected from the group consisting of: alcohol ethoxylate 7EO, coconut fatty acids C12 - 18, C12 - C14 fatty alcohol ether sulfate (2EO), linear alkylbenzene sulfonic acid, preferably at a concentration of 0.2% - 65%,

[0288] b) one or more builders selected from the group consisting of: HEDP, MGDA, GLDA, and DTPMP, preferably at a concentration of 0.01% - 25%, and

[0289] c) one or more compounds selected from the group consisting of: sodium acetate, sodium citrate, sodium silicate, sodium carbonate, sodium phosphate, sodium bicarbonate, zeolite 4A, sodium sulfate, sodium chloride, optical brightener, and polymer, and optionally a bleach activator and percarbonate.

[0290] Preferably, the detergent composition of the present invention, preferably the liquid detergent composition, in addition to containing the cutinase described herein, further comprises

[0291] a) One or more surfactants selected from the group consisting of: alcohol ethoxylate 7EO, coconut fatty acids C12-18, C12C14-fatty alcohol ether sulfate (2EO), linear alkylbenzene sulfonic acid, preferably at a concentration of 0.2% - 65%,

[0292] b) One or more builders selected from the group consisting of: HEDP, MGDA, GLDA, and DTPMP, preferably at a concentration of 0.01% - 25%, and

[0293] c) One or more compounds selected from the group consisting of: sulfonic acid, 1,2-propanediol, triethanolamine, monoethanolamine, NaOH, glycerol, ethanol, sodium citrate, and polymers.

[0294] Preferred composition

[0295] In one embodiment, the cutinase described herein is included in a composition comprising one or more, preferably all, compounds selected from the group consisting of (all percentages are w / w):

[0296] · A formulation comprising the cutinase as described herein, 0.05% to 0.2%;

[0297] · Anionic detergent surfactants (such as alkylbenzene sulfonates, alkyl ethoxylated sulfates, and mixtures thereof), 8% to 15%;

[0298] · Nonionic detergent surfactants (such as alkyl ethoxylated alcohols), 0.5% to 4%;

[0299] · Cationic detergent surfactants (such as quaternary ammonium compounds), 0% - 4%;

[0300] · Other detergent surfactants (such as zwitterionic detergent surfactants, amphoteric surfactants, and mixtures thereof), 0% to 4%;

[0301] · Carboxylic acid polymers (such as copolymers of maleic acid and acrylic acid), 1% to 4%;

[0302] · Polyethylene glycol polymers (such as polyethylene glycol polymers containing polyvinyl acetate side chains), 0.5% to 4%;

[0303] · Polyester soil release polymers (such as Repel-o-tex and / or Texcare polymers), 0.1% to 2%;

[0304] · Cellulose polymers (such as carboxymethyl cellulose, methyl cellulose, and combinations thereof), 0.5% to 2%;

[0305] · Other polymers (such as amine polymers, dye transfer inhibitor polymers, hexamethylenediamine derivative polymers, and mixtures thereof), 0% to 4%;

[0306] · Zeolite builders and phosphate builders (such as zeolite 4A and / or sodium tripolyphosphate), 0% to 4 wt%;

[0307] · Other builders (such as sodium citrate and / or citric acid), 0% to 3%;

[0308] · Carbonates (such as sodium carbonate and / or sodium bicarbonate), 15% to 30%;

[0309] · Silicates (such as sodium silicate), 0% to 10%;

[0310] · Fillers (such as sodium sulfate and / or biological fillers), 10% to 40%;

[0311] · Available oxygen sources (such as sodium percarbonate), 10% to 20%;

[0312] · Bleach activators (such as tetraacetylethylenediamine (TAED) and / or nonanoyloxybenzenesulfonate (NOBS)), 2% to 8%;

[0313] · Bleach catalysts (such as quaternary oxaziridinium-based bleach catalysts and / or transition metal bleach catalysts), 0% to 0.1%;

[0314] · Other bleaching agents (such as reducing bleaching agents and / or preformed peracids), 0% to 10%;

[0315] · Chelating agents (such as ethylenediamine-N'N'-disuccinic acid (EDDS) and / or hydroxyethane diphosphonic acid (HEDP)), 0.2% to 1%;

[0316] · Optical bleaching agents (such as zinc sulfonated phthalocyanine and / or aluminum sulfonated phthalocyanine), 0% to 0.1%;

[0317] · Colorants (such as direct violet 99, acid red 52, acid blue 80, direct violet 9, solvent violet 13, and any combination thereof), 0% to 1%;

[0318] · Brighteners (such as brightener 15 and / or brightener 49), 0.1% to 0.4%;

[0319] · Fabric softeners (such as montmorillonite clay and / or polydimethylsiloxane (PDMS)), 0% to 4%;

[0320] · Flocculants (such as polyethylene oxide), 0% to 1%;

[0321] · Foam inhibitors (such as silicone and / or fatty acids), 0% to 0.1%;

[0322] · Perfumes (such as perfume microcapsules, spray perfumes, starch-encapsulated perfume notes, zeolites loaded with perfume, and any combination thereof), 0.1% to 1%; and

[0323] · Cosmetic agents (such as colored soap rings and / or colored spots / strips), 0% to 1%; and

[0324] · Optionally one or more second enzymes as described herein, the balance.

[0325] In another embodiment, the cutinase described herein is included in a composition comprising one or more, preferably all, compounds selected from the group consisting of (all percentages are w / w):

[0326] · A formulation containing the cutinase as described herein, 0.05% to 0.2%;

[0327] · Carboxyl-containing polymers (comprising about 60% to about 70% by mass of acrylic-based monomers (A); and about 30% to about 40% by mass of monomers containing sulfonic acid groups (B); and wherein the average molecular weight is about 23,000 to about 50,000, preferably in the range of about 25,000 to about 38,000 (as described in WO 2014032269)), about 0.5 wt% to about 1.5 wt%;

[0328] · Anionic detergent surfactants (such as alkylbenzene sulfonates, alkyl ethoxylated sulfates, and mixtures thereof), about 8 wt% to about 15 wt%;

[0329] · Nonionic detergent surfactants (such as alkyl ethoxylated alcohols) about 0.5 wt% to 4 wt%;

[0330] · Cationic detergent surfactants (such as quaternary ammonium compounds), about 0 wt% to about 4 wt%;

[0331] · Other detergent surfactants (such as zwitterionic detergent surfactants, amphoteric surfactants, and mixtures thereof), about 0 wt% to 4 wt%;

[0332] · Carboxylate polymers (such as copolymers of maleic acid and acrylic acid), about 1 wt% to about 4 wt%;

[0333] · Polyethylene glycol polymers (such as polyethylene glycol polymers containing polyvinyl acetate side chains), about 0 wt% to about 4 wt%;

[0334] · Polyester soil removal polymers (such as Repel-O-Tex(R) and / or Texcare(R) polymers), about 0.1 wt% to about 2 wt%;

[0335] · Cellulose polymers (such as carboxymethyl cellulose, methyl cellulose, and combinations thereof), from about 0.5 wt% to about 2 wt%;

[0336] · Other polymers (such as amine polymers, dye transfer inhibitor polymers, hexamethylenediamine derivative polymers, and mixtures thereof), from about 0 wt% to about 4 wt%;

[0337] · Zeolite builders and phosphate builders (such as zeolite 4A and / or sodium tripolyphosphate), from about 0 wt% to about 4 wt%;

[0338] · Other auxiliaries (such as sodium citrate and / or citric acid), from about 0 wt% to about 3 wt%;

[0339] · Carbonates (such as sodium carbonate and / or sodium bicarbonate), from about 15 wt% to about 30 wt%;

[0340] · Silicates (such as sodium silicate), from about 0 wt% to about 10 wt%;

[0341] · Fillers (such as sodium sulfate and / or biological fillers), from about 10 wt% to about 40 wt%;

[0342] · Available oxygen source (such as sodium percarbonate), from about 10 wt% to about 20 wt%;

[0343] · Bleaching activators (such as tetraacetylethylenediamine (TAED) and / or nonanoyloxybenzenesulfonate (NOBS), from about 2 wt% to about 8 wt%;

[0344] · Bleaching catalysts (such as quaternary oxaziridinium-based bleaching catalysts and / or transition metal bleaching catalysts), from about 0 wt% to about 0.1 wt%;

[0345] · Other bleaching agents (such as reducing bleaching agents and / or preformed peracids), from about 0 wt% to about 10 wt%;

[0346] · Chelating agents (such as ethylenediamine-N,N'-disuccinic acid (EDDS) and / or hydroxyethane diphosphonic acid (HEDP), from about 0.2 wt% to about 1 wt%;

[0347] · Optical brighteners (such as optical brightener 15 and / or optical brightener 49), from about 0.1 wt% to about 0.4 wt%;

[0348] · Colorants (such as direct violet 99, acid red 52, acid blue 80, direct violet 9, solvent violet 13, and any combination thereof), from about 0 wt% to about 0.5 wt%;

[0349] · Brighteners (such as brightener 15 and / or brightener 49), from about 0.1 wt% to about 0.4 wt%;

[0350] · Fabric softener (such as montmorillonite clay and / or polydimethylsiloxane (PDMS)), 0 wt% to 15 wt%;

[0351] · Flocculant (such as polyethylene oxide), 0 wt% to 1 wt%;

[0352] · Foam inhibitor (such as silicone and / or fatty acid), 0 wt% to 0.1 wt%;

[0353] · Perfume (such as perfume microcapsules, spray perfume, starch-encapsulated perfume notes, zeolite loaded with perfume, and any combination thereof), 0.1 wt% to 1 wt%; and

[0354] · Cosmetic agent (such as colored soap rings and / or colored spots / strips), 0 wt% to 1 wt%; and optionally one or more second enzymes as described herein.

[0355] · The balance.

[0356] The cutinase as described herein can be incorporated into one of the following detergent compositions.

[0357]

[0358]

[0359]

[0360]

[0361]

[0362]

[0363]

[0364]

[0365] Method of use

[0366] The present invention also relates to the use of the cutinase as described herein in a cleaning process (such as for laundry or hard surface cleaning), preferably for household care or I&I cleaning. Accordingly, the present invention also relates to the use of the cutinase as described herein for providing a detergent composition having improved washing performance, preferably against cutinase-sensitive stains, such as plant-based stains and / or fatty stains, preferably fatty stains, preferably sebum stains.

[0367] Accordingly, the present invention also relates to a method for cleaning, preferably for cleaning laundry or hard surfaces, which comprises the step of bringing a test substance, preferably a textile or a hard surface, into contact with a composition comprising a cutinase as described herein, preferably wherein the composition comprises at least one further detergent component, preferably a surfactant and / or a builder. Preferably, the hard surfaces are selected from floors, furniture, walls, sanitary ceramics, glass, metal surfaces including medical devices, knives and tableware. Preferably, the method for laundry cleaning comprises the step of bringing a textile into contact with a composition comprising a cutinase as described herein, preferably wherein the composition comprises at least one further detergent component, preferably a surfactant and / or a builder. Preferably, the method for hard surface cleaning comprises the step of bringing a medical device, a knife or tableware into contact with a composition comprising a cutinase as described herein, preferably wherein the composition comprises at least one further detergent component, preferably a surfactant and / or a builder. A particularly preferred form of hard surface cleaning is dishwashing, preferably manual dishwashing (MDW) or automatic dishwashing (ADW), most preferably automatic dishwashing (ADW).

[0368] Furthermore, the present invention also relates to a method for improving the washing performance of a detergent composition, preferably for cutinase-sensitive stains, such as plant-based stains and / or fatty stains, preferably fatty stains, preferably sebum stains, the method comprising the step of formulating a cutinase as described herein in the detergent composition.

[0369] The present invention also relates to a method for removing plant-based stains and / or fatty stains, preferably fatty stains, more preferably sebum stains, the method comprising the step of bringing an object comprising a plant-based stain and / or a fatty stain (preferably sebum stain) into contact with a composition comprising a polypeptide having cutinase activity, the polypeptide comprising an amino acid sequence that is at least 75% identical to SEQ ID NO:2, at least 80% identical to SEQ ID NO:4 or at least 84% identical to SEQ ID NO:6, as described herein.

[0370] Accordingly, the present invention relates to a method for removing plant-based stains, the method comprising the step of bringing an object comprising a plant-based stain into contact with a composition comprising a polypeptide having cutinase activity, the polypeptide comprising an amino acid sequence that is at least 75% identical to SEQ ID NO:2, at least 80% identical to SEQ ID NO:4 or at least 84% identical to SEQ ID NO:6, as described herein.

[0371] The present invention also relates to a method for removing fatty stains (preferably sebum stains), which method comprises the step of contacting an object comprising a fatty stain (preferably a sebum stain) with a composition comprising a polypeptide having cutinase activity, said polypeptide comprising an amino acid sequence that is at least 75% identical to SEQ ID NO:2, at least 80% identical to SEQ ID NO:4 or at least 84% identical to SEQ ID NO:6, as described herein.

[0372] The present invention also relates to the use of a polypeptide having cutinase activity for removing plant-based stains and / or fatty stains, preferably fatty stains, more preferably sebum stains, from an object (preferably a textile or a hard surface), said polypeptide comprising an amino acid sequence that is at least 75% identical to SEQ ID NO:2, at least 80% identical to SEQ ID NO:4 or at least 84% identical to SEQ ID NO:6, as described herein.

[0373] Furthermore, enzymes play an increasingly important role in animal nutrition and they are used in feed formulations to improve efficiency and at the same time reduce costs by helping the animal, for example, to digest its feed more efficiently or by detoxifying. Accordingly, the present invention also relates to the use of a cutinase as described herein in animal feed. Accordingly, the present invention also relates to an animal feed composition comprising a cutinase as described herein.

[0374] The present invention also relates to the use of a cutinase as described herein for increasing the digestibility of animal feed.

[0375] Preferred embodiment

[0376] Specifically, preferred herein is:

[0377] 1. A composition comprising a polypeptide having cutinase activity and at least one additional component, wherein the polypeptide having cutinase activity comprises an amino acid sequence that is at least 75% identical to SEQ ID NO:2, at least 80% identical to SEQ ID NO:4 or at least 84% identical to SEQ ID NO:6.

[0378] 2. A composition comprising a polypeptide having cutinase activity and at least one additional component, wherein the polypeptide having cutinase activity comprises an amino acid sequence that is at least 75%, at least 78%, at least 80%, at least 82%, at least 85%, at least 88%, 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% identical to SEQ ID NO:2 with increasing preference.

[0379] 3. A composition comprising a polypeptide having cutinase activity and at least one additional component, wherein the polypeptide having cutinase activity comprises an amino acid sequence that is at least 80%, at least 82%, at least 85%, at least 88%, 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% identical to SEQ ID NO:4 with increasing preference.

[0380] 4. A composition comprising a polypeptide having cutinase activity and at least one additional component, wherein the polypeptide having cutinase activity comprises an amino acid sequence that is at least 84%, at least 85%, at least 88%, 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% identical to SEQ ID NO:6 with increasing preference.

[0381] 5. A composition comprising a polypeptide having cutinase activity and at least one additional component, wherein the polypeptide having cutinase activity comprises an amino acid sequence that is at least 84%, at least 85%, at least 88%, 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% identical to SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6, more preferably SEQ ID NO:2 or SEQ ID NO:6, and most preferably SEQ ID NO:2 with increasing preference.

[0382] 6. The composition according to any one of Examples 1 to 5, wherein the composition is a non-compound formulation, preferably a cutinase formulation, preferably a concentrated cutinase formulation, and wherein the additional component is selected from the group consisting of: solvents, salts, pH regulators, preservatives, enzyme stabilizers and thickeners.

[0383] 7. The composition according to Example 6, wherein the formulation comprises an enzyme stabilization system, and wherein the enzyme stabilization system preferably comprises at least one compound selected from the group consisting of: polyols (preferably 1,3-propanediol, ethylene glycol, glycerol, 1,2-propanediol, or sorbitol), inorganic salts (preferably CaCl2, MgCl2, or NaCl), short-chain (preferably C1-C3) carboxylic acids or their salts (preferably formic acid, formates (preferably sodium formate), acetic acid, acetates, or lactates).

[0384] 8. A composition as described in Example 6 or 7, wherein the composition is a liquid and contains cutinase, and the amount of the cutinase is in the range of 0.1% to 40%, 0.5% to 30%, 1% to 25%, 1% to 10%, or preferably 1 - 6%, all relative to the total weight of the enzyme formulation.

[0385] 9. A composition as described in any one of Examples 1 to 5, wherein the composition is a detergent composition, preferably a laundry detergent composition or a dishwashing detergent composition, and wherein the additional component is at least one detergent component, preferably wherein the detergent composition contains cutinase, and the amount of the cutinase is in the range of 0.0002% to 0.09% by weight, preferably 0.0002% to 0.01%, all relative to the total weight of the detergent composition.

[0386] 10. A composition as described in Example 9, wherein the detergent component is selected from the group consisting of: surfactants, builders, polymers, bleaching systems, optical brighteners, foam inhibitors, stabilizers, hydrotropes, rheology modifiers, preservatives, and corrosion inhibitors.

[0387] 11. A composition as described in any one of Examples 7 to 10, which further comprises at least one second enzyme selected from the group consisting of: proteases, amylases, lipases, cellulases, mannanases, xylanases, DNases, dispersins, pectinases, pectate lyases, oxidoreductases, glycosidases, glucoamylases, xanthan lyases, esterases, and additional cutinases, preferably the at least one second enzyme is a protease and / or an amylase, most preferably a protease.

[0388] 12. A composition as described in any one of Examples 9 to 11, wherein the composition contains one or more surfactants and / or one or more builders, preferably strong chelating builders.

[0389] 13. A composition as described in Example 12, wherein the composition contains a builder, and the builder is selected from MDGA, GLDA, DTPMP, HEDP, and EDDS, preferably MDGA or EDDS.

[0390] 14. A composition as described in any one of Examples 9 to 13, wherein the composition contains a surfactant, and the surfactant is selected from nonionic surfactants, anionic surfactants, cationic surfactants, amphoteric surfactants, and combinations thereof.

[0391] 15. A composition as described in any one of Examples 9 to 14, wherein no anionic surfactant is added to the detergent composition.

[0392] 16. The composition according to any one of embodiments 9 to 15, wherein the surfactant and / or the builder is biodegradable and / or bio-based.

[0393] 17. The composition according to any one of embodiments 9 to 16, wherein the detergent composition is liquid or solid.

[0394] 18. The composition according to any one of embodiments 9 to 17, wherein the detergent composition is in the form of a sachet.

[0395] 19. A method for removing plant-based stains and / or fatty stains, preferably fatty stains, more preferably sebum stains, the method comprising the step of contacting an object comprising plant-based stains and / or fatty stains, preferably fatty stains, preferably sebum stains with the composition according to any one of embodiments 1 - 18, preferably using the detergent composition according to any one of embodiments 9 - 18, preferably the method is a method for removing fatty stains, more preferably sebum stains, the method comprising the step of contacting an object comprising fatty stains, preferably sebum stains with the composition according to any one of embodiments 1 - 18, preferably using the detergent composition according to any one of embodiments 9 - 18.

[0396] 20. Use of a polypeptide having cutinase activity for removing plant-based stains and / or fatty stains, preferably fatty stains, more preferably sebum stains, the polypeptide comprising an amino acid sequence that is at least 75% identical to SEQ ID NO:2, at least 80% identical to SEQ ID NO:4 or at least 84% identical to SEQ ID NO:6.

[0397] 21. Use of a polypeptide having cutinase activity, the polypeptide comprising an amino acid sequence that is at least 75%, at least 78%, at least 80%, at least 82%, at least 85%, at least 88%, 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% identical to SEQ ID NO:2 with increasing preference.

[0398] 22. Use of a polypeptide having cutinase activity, the polypeptide comprising an amino acid sequence that is at least 80%, at least 82%, at least 85%, at least 88%, 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% identical to SEQ ID NO:4 with increasing preference.

[0399] 23. Use of a polypeptide having cutinase activity, said polypeptide comprising an amino acid sequence that is at least 84%, at least 85%, at least 88%, 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% identical to SEQ ID NO:6 with increasing preference.

[0400] 24. Use of a polypeptide having cutinase activity, said polypeptide comprising an amino acid sequence that is at least 84%, at least 85%, at least 88%, 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% identical to SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6, more preferably SEQ ID NO:2 or SEQ ID NO:6, most preferably SEQ ID NO:2 with increasing preference.

[0401] 25. Use of a polypeptide having cutinase activity as described in any one of Examples 20 to 24, said use being for removing plant-based stains and / or fatty stains, preferably fatty stains, more preferably sebum stains.

[0402] 26. Use of a polypeptide having cutinase activity as described in any one of Examples 20 to 24, said use being in animal feed.

[0403] 27. An isolated, synthetic or recombinant polypeptide having cutinase activity, said polypeptide comprising an amino acid sequence that is at least 75% identical to SEQ ID NO:2, at least 80% identical to SEQ ID NO:4 or at least 84% identical to SEQ ID NO:6.

[0404] 28. An isolated, synthetic or recombinant polypeptide having cutinase activity, wherein said recombinant polypeptide having cutinase activity comprises an identity of at least 75%, at least 77%, at least 80%, at least 82%, at least 85%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% to SEQ ID NO:2 with increasing preference.

[0405] 29. An isolated, synthetic or recombinant polypeptide having cutinase activity, wherein the recombinant polypeptide having cutinase activity comprises at least 80%, at least 82%, at least 85%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with SEQ ID NO:4 in increasing order of preference.

[0406] 30. An isolated, synthetic or recombinant polypeptide having cutinase activity, wherein the recombinant polypeptide having cutinase activity comprises at least 84%, at least 85%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with SEQ ID NO:6 in increasing order of preference.

[0407] 31. An isolated, synthetic or recombinant polypeptide having cutinase activity, wherein the recombinant polypeptide having cutinase activity comprises at least 84%, at least 85%, at least 88%, 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% identity with SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6, more preferably SEQ ID NO:2 or SEQ ID NO:6, most preferably SEQ ID NO:2 in increasing order of preference.

[0408] 32. The polypeptide according to any one of Examples 27 - 31, wherein the amino acid sequence of the polypeptide having cutinase activity comprises or consists of: SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6, more preferably SEQ ID NO:2 or SEQ ID NO:6, most preferably SEQ ID NO:2, or wherein the amino acid sequence of the polypeptide having cutinase activity comprises or consists of: SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6, more preferably SEQ ID NO:2 or SEQ ID NO:6, most preferably SEQ ID NO:2, with 1 - 20 amino acid substitutions, preferably with 1 - 10 amino acid substitutions or more preferably with 1 - 5 amino acid substitutions, preferably wherein these amino acid substitutions are conservative amino acid substitutions.

[0409] 33. An isolated, synthetic or recombinant polynucleotide encoding an isolated, synthetic or recombinant polypeptide as described in any one of Examples 27 to 32, wherein the isolated, synthetic or recombinant polynucleotide preferably has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5 in increasing order of preference, preferably wherein when the polypeptide having cutinase activity has at least 90% sequence identity with SEQ ID NO:2, the polynucleotide encoding the polypeptide having cutinase activity has at least 70% sequence identity with SEQ ID NO:1, or wherein when the polypeptide having cutinase activity has at least 90% sequence identity with SEQ ID NO:4, the polynucleotide encoding the polypeptide having cutinase activity has at least 70% sequence identity with SEQ ID NO:3, or wherein when the polypeptide having cutinase activity has at least 90% sequence identity with SEQ ID NO:6, the polynucleotide encoding the polypeptide having cutinase activity has at least 70% sequence identity with SEQ ID NO:5.

[0410] 34. A nucleic acid construct comprising the polynucleotide as described in Example 33.

[0411] 35. An expression vector comprising the polynucleotide as described in Example 33 or the nucleic acid construct as described in Example 34.

[0412] 36. A host cell comprising the polynucleotide as described in Example 33, the nucleic acid construct as described in Example 34, or the expression vector as described in Example 35.

[0413] 37. The host cell as described in Example 36, wherein the host cell is a Bacillus cell, preferably a Bacillus licheniformis cell.

[0414] 38. A method for preparing a polypeptide having cutinase activity as described in Examples 27 - 32, the method comprising providing a polynucleotide encoding the polypeptide having cutinase activity, preferably the polynucleotide as described in Example 33, transforming the polynucleotide into a host cell, preferably a Bacillus cell, more preferably a Bacillus licheniformis cell, culturing the host cell to produce the polypeptide having cutinase activity and optionally purifying the polypeptide having cutinase activity.

[0415] Examples

[0416] Example 1: Cutinase Expression

[0417] One hundred and four sequences extracted from public databases were transformed into Bacillus and expressed in Bacillus.

[0418] Therefore, plasmid DNA containing the target gene and prepared from Bacillus subtilis was used to transform chemically competent Bacillus licheniformis by electroporation. The transformed cells were incubated with shaking at 37 °C for 2 hours and then plated on an appropriate selection medium. The plates were incubated at 26 °C for 3 days. Bacillus licheniformis colonies containing the target gene were used to inoculate 0.6 ml of growth medium in a 96-well plate. The samples were incubated with shaking at 30 °C or 37 °C for 16 hours. The mature seed cultures were diluted into appropriate amounts of 0.6 ml aliquots in a 96-well plate, and the samples were incubated with shaking at 30 °C or 37 °C for 48 hours. The samples were clarified by centrifugation and the supernatants were harvested, aliquoted and stored at -20 °C. The presence of the target gene product was confirmed by LabChip.

[0419] Eighteen cultures expressed active cutinase. When tested at a medium expression scale, five of them (SEQ ID NO: 2, 4, 6, 8 and 10) expressed at levels greater than 0.1 mg / ml of supernatant. Medium expression followed the same method as described above, using multiple wells for each growth harvested by centrifugation. The supernatants were harvested, pooled, the cutinase concentration was determined by Lab Chip and the aliquots were stored at -20 °C. SEQ ID NO: 2 showed a particularly high expression of >1 mg / ml of supernatant.

[0420] Example 2: Washing performance in a Terg-O-Tometer (TOM)

[0421] The washing performance of five well-expressed cutinases (SEQ ID NO: 2, 4, 6, 8 and 10) was tested on different stains in a TOM at 200 rpm and 22 °C for 60 min. The enzymes were quantitatively added to the detergent composition as shown in Table 1.

[0422] Table 1: Washing conditions, TOM assay

[0423]

[0424]

[0425] 1) CFT-CS 06, manufacturer: Center for Testmaterials BV, NL-3130AC Vlaardingen

[0426] TOM was run followed by three water rinses, drying overnight, scanning the dried small cloth samples and performing image analysis via Digimizer to determine the RGB values.

[0427] The δRGB values were calculated using the RGB values measured when washing with detergents (ES1C or Tide Hygienic Clean) with and without added enzyme.

[0428] Table 2: Average δRGB values determined by TOM

[0429]

[0430] As shown in Table 2, the cutinases of SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6 showed washing performance against sebum stain WFK20D (standard sebum stain) and other stains such as CS25 spinach or PCS132 sebum shell. SEQ ID NO:8 and SEQ ID NO:10 did not show any detectable washing performance under the test conditions.

[0431] Example 3: Beaker assay - WFK20D

[0432] Using stain WFK20D and quantitatively adding cutinases SEQ ID NO:2 and SEQ ID NO:6 in different detergent compositions (ES1C, Tide Hygienic Clean, see Table 3), small-scale beaker assays were performed with 50 ml or 25 ml of washing solution (in a 50 ml beaker).

[0433] Table 3: Washing conditions for beaker assay

[0434]

[0435]

[0436] 1) CFT-CS 06, manufacturer: Center for Testmaterials BV, NL-3130AC Vlaardingen

[0437] The washing solution was stirred at 22 °C between 200 rpm and 250 rpm for 45 min. This was followed by three water rinses, drying overnight, scanning the dried small cloth samples and performing image analysis via Digimizer to determine the RGB values.

[0438] Table 4: δRGB values for beaker assay in the case of stain WFK 20D in 25 ml of ES1C detergent

[0439] WFK 20D in ES1C detergent SEQ ID NO:2 0,053 SEQ ID NO:6 0,023

[0440] Table 5: δRGB values measured in a beaker for stain WFK 20D in 50 ml of Tide Hygienic Clean

[0441] WFK 20D in Tide Hygienic Clean SEQ ID NO:2 0,035 SEQ ID NO:6 0,012

[0442] As shown in Tables 4 and 5, both cutinases SEQ ID NO:2 and SEQ ID NO:6 exhibited beneficial cleaning effects. The benefits of SEQ ID NO:2 and SEQ ID NO:6 in sebum cleaning were clearly demonstrated on the WFK 20D stain (pigment / sebum on polyester / cotton stain). SEQ ID NO:2 and SEQ ID NO:6 both showed good improvements in washing performance in a commercial detergent (Tide Hygienic Clean) as well as in ES1C (3 g / L in hard water). SEQ ID NO:2 showed better cleaning performance than SEQ ID NO:6.

Claims

1. A composition comprising a polypeptide having cutinase activity and at least one additional component, wherein the polypeptide having cutinase activity comprises an amino acid sequence that is at least 75% identical to SEQ ID NO:2, at least 80% identical to SEQ ID NO:4, or at least 84% identical to SEQ ID NO:

6.

2. The composition according to claim 1, wherein the composition is a detergent composition, preferably a laundry detergent composition or a dishwashing detergent composition, and wherein the additional component is at least one detergent component.

3. The composition according to claim 2, wherein the detergent component is selected from the group consisting of: surfactants, builders, polymers, bleaching systems, optical brighteners, foam inhibitors, stabilizers, hydrotropes, rheology modifiers, preservatives, and corrosion inhibitors.

4. The composition according to claim 2 or 3, which further comprises at least one second enzyme selected from the group consisting of: proteases, amylases, lipases, cellulases, mannanases, xylanases, DNases, dispersins, pectinases, pectate lyases, oxidoreductases, glycosidases, glucoamylases, xanthan lyases, esterases, and additional cutinases.

5. A method for removing plant-based stains and / or fatty stains, preferably fatty stains, more preferably sebum stains, the method comprising the step of contacting an object comprising plant-based stains and / or fatty stains, preferably sebum stains, with the composition according to any one of claims 1-4.

6. Use of a polypeptide having cutinase activity comprising an amino acid sequence that is at least 75% identical to SEQ ID NO:2, at least 80% identical to SEQ ID NO:4, or at least 84% identical to SEQ ID NO:6 for removing plant-based stains and / or fatty stains, preferably fatty stains, more preferably sebum stains from an object, preferably a textile or a hard surface.

7. An isolated, synthetic or recombinant polypeptide having cutinase activity, the polypeptide comprising an amino acid sequence that is at least 75% identical to SEQ ID NO:2, at least 80% identical to SEQ ID NO:4, or at least 84% identical to SEQ ID NO:

6.

8. The isolated, synthetic or recombinant polypeptide having cutinase activity according to claim 7, the polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90% or at least 95% identical to SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6, more preferably SEQ ID NO:2 or SEQ ID NO:6, most preferably SEQ ID NO:

2.

9. The polypeptide according to claim 7 or claim 8, wherein the amino acid sequence of the polypeptide having cutinase activity comprises or consists of the following: SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6, or wherein the amino acid sequence of the polypeptide having cutinase activity comprises or consists of the following: SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6, with 1 - 20 amino acid substitutions, preferably with 1 - 10 amino acid substitutions, or more preferably with 1 - 5 amino acid substitutions, preferably wherein these amino acid substitutions are conservative amino acid substitutions.

10. An isolated, synthetic, or recombinant polynucleotide encoding a polypeptide according to any one of claims 7 - 9, wherein the isolated, synthetic, or recombinant polynucleotide preferably has at least 80% sequence identity with SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:

5.

11. A nucleic acid construct comprising the polynucleotide according to claim 10.

12. An expression vector comprising the polynucleotide according to claim 10 or the nucleic acid construct according to claim 11.

13. A host cell comprising the polynucleotide according to claim 10, the nucleic acid construct according to claim 11, or the expression vector according to claim 12.

14. The host cell according to claim 13, wherein the host cell is a Bacillus cell.

15. A method for producing a polypeptide having cutinase activity according to any one of claims 7 - 9, the method comprising providing a polynucleotide encoding the polypeptide having cutinase activity, transforming the polynucleotide into a host cell, culturing the host cell to produce the polypeptide having cutinase activity, and optionally purifying the polypeptide having cutinase activity.

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