Polypeptides having pectinase activity for use in cleaning compositions

Abstract

In the present invention pectinase enzymes for cleaning compositions and the cleaning compositions comprising them are provided. Additionally, methods of making and using these enzymes, including recombinant polynucleotides encoding them, are provided.

Description

2200721Polypeptides having pectinase activity for use in cleaning compositionsField of the inventionIn the present invention pectinase enzymes for cleaning compositions and the cleaning compositions comprising them are provided. Additionally, methods of making and using these enzymes, including recombinant polynucleotides encoding them, are provided.Background of the inventionEnzymes are increasingly used in various applications as sustainable alternatives to petrochemistry. They are biodegradable and can be catalytically active already at lower temperatures, which results in reduction of energy consumption. In particular, in the detergent industry, enzymes are implemented in cleaning compositions to improve cleaning efficiency and / or to reduce energy consumption in a washing step.Pectinases are enzymes capable of breaking down pectin polymers and pectate, polysaccharides that are present in the cell walls of plants. Thus, pectinases have been applied e.g. in the food industry or as detergent enzymes. Nevertheless, up to now, pectinases are rarely used in the detergent industry as they are rarely stable at different temperatures and / or within the denaturing conditions of the cleaning compositions and the wash liquor. Thus, the need exists for new pectinases, which meet these requirements.Brief summary of the inventionThus, the present invention is directed to polypeptides having pectinase activity in such conditions, specifically to compositions, preferably cleaning composition, more preferably a laundry cleaning composition or a dish washing composition, comprising these pectinases, wherein the polypeptide having pectinase activity comprises an amino acid sequence that is at least 75%, preferably at least 80% identical to SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5. Additionally, methods for removing stains comprising one or more pectins or pectate using a composition comprising such pectinases are provided. Additionally, methods of making and using these enzymes, including recombinant polynucleotides encoding them, are provided.Detailed description of the inventionThe present invention may be understood more readily by reference to the following detailed description of the embodiments of the invention and the examples included herein.Although the present invention will be described with respect to particular embodiments, this description is not to be construed in a limiting sense.DefinitionsUnless otherwise noted, the terms used herein are to be understood according to conventional usage by those of ordinary skill in the relevant art.Before describing in detail exemplary embodiments of the present invention, definitions important for understanding the present invention are given. Unless stated otherwise or apparent from the nature of the definition, the definitions apply to all compounds, methods and uses described herein.2200722As used in this specification and in the appended claims, the singular forms of "a" and "an" also include the respective plurals unless the context clearly dictates otherwise.In the context of the present invention, the terms "about" and "approximately" denote an interval of accuracy that a person skilled in the art will understand to still ensure the technical effect of the feature in question. The term typically indicates a deviation from the indicated numerical value of ±20 %, preferably ±15 %, more preferably ±10 %, and even more preferably ±5 %.Furthermore, the terms "first", "second", "third" or "(a)", "(b)", "(c)", "(d)" etc. and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein. In case the terms "first", "second", "third" or "(a)", "(b)", "(c)", "(d)", "I", "II" etc. relate to steps of a method or use or assay there is no time or time interval coherence between the steps, i.e. the steps may be carried out simultaneously or there may be time intervals of seconds, minutes, hours, days, weeks, months or even years between such steps, unless otherwise indicated in the application as set forth herein above or below.Throughout this application, various publications are referenced. The disclosure of all these publications and those references cited within those publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.It is to be understood that the term "comprising" is not limiting. For the purposes of the present invention the term "consisting of" is considered to be a preferred embodiment of the term "comprising". If hereinafter a group is defined to comprise at least a certain number of members, this is meant to also encompass a group which consists of these members only."Amino acid substitutions” are described by providing the original amino acid followed by the number of the position within the amino acid sequence, followed by the substituted amino acid. For example, the substitution of histidine at position 120 with alanine is designated as"His120Ala” or"H120A”. Substitutionscan also be described by merely naming the resulting amino acid in the variant without specifying the amino acid of the parent at this position, e.g., by using "X120A” or "120A” or "Xaa120Ala” or “120Ala” .Variants comprising multiple substitutions are separated by "+”, e.g., "Arg170Tyr+Gly 195Glu”, "R170Y+G195E” or "X170Y+X195E” representing a substitution of arginine and glycine at positions 170 and 195 with tyrosine and glutamic acid, respectively. Alternatively, multiple substitutions may be separated by space or a comma, e.g., "R170Y G195E” or "R170Y, G195E” respectively. Where different alternative substitutions can be introduced at a position, the different substitutions are separated by a comma, e.g., “Arg170Tyr,Glu” and "R170T, E”, respectively, represents a substitution of arginine at position 170 with tyrosine or glutamic acid. Alternative substitutions at a particular position can also be indicated as "X120A,G,H”, “120A,G,H", "X120A / G / H”, or “120A / G / H”. Alternatively, different substitutions may be indicated in brackets, e.g., "Arg 170[Tyr, Gly]” or "Arg 170{Tyr, Gly}” or in short "R170 [Y,G]” or “R170 {Y,G}”.2200723The term "native” (or naturally or wild-type or endogenous) cell or organism or polynucleotide or polypeptide refers to the cell or organism or polynucleotide or polypeptide as found in nature (i.e., without there being any human intervention).The term "heterologous” (or exogenous or foreign or recombinant or non-native or non-natural) polypeptide is defined herein as a polypeptide that is not native to the host cell, a polypeptide native to the host cell in which structural modifications, e.g., deletions, substitutions, and / or insertions, have been made by recombinant DNA techniques to alter the native polypeptide, or a polypeptide native to the host cell whose expression is quantitatively altered or whose expression is directed from a genomic location different from the native host cell as a result of manipulation of the DNA of the host cell by recombinant DNA techniques, e.g., a stronger promoter. Similarly, the term "heterologous” (or exogenous or foreign or recombinant or non-native or non-natural) polynucleotide refers to a polynucleotide that is not native to the host cell, a polynucleotide native to the host cell in which structural modifications, e.g., deletions, substitutions, and / or insertions, have been made by recombinant DNA techniques to alter the native polynucleotide, or a polynucleotide native to the host cell whose expression is quantitatively altered as a result of manipulation of the regulatory elements of the polynucleotide by recombinant DNA techniques, e.g., a stronger promoter, or a polynucleotide native to the host cell, but integrated not within its natural genetic environment as a result of genetic manipulation by recombinant DNA techniques. With respect to the relation between two or more polynucleotide sequences or the relation between two or more amino acid sequences, the term "heterologous” is used to characterize that the two or more polynucleotide sequences or two or more amino acid sequences are naturally not occurring in the specific combination with each other.For the purpose of the invention, "recombinant" (or transgenic) with regard to a cell or an organism means that the cell or organism contains a heterologous polynucleotide, which is introduced by people using gene technology. Regarding a polynucleotide "recombinant” includes all constructs produced by using gene technology I recombinant DNA techniques in which either(a) the sequence of the polynucleotide or a part thereof, or(b) one or more genetic control sequences, which are operably linked to the polynucleotide, including but not limited to a promoter, or(c) both a) and b)are not located in their wild-type genetic environment or have been modified by people.A "synthetic" compound is obtained by in vitro chemical and / or enzymatic synthesis.Variant polynucleotide and variant polypeptide sequences may be defined by their sequence identity when compared to a parent sequence.Sequence identity usually is provided as "% sequence identity” or "% identity”. For calculation of sequence identities, in the first step a sequence alignment is produced. According to this invention, a pairwise global alignment is produced, meaning that two sequences are aligned over their complete length, which is usually produced by using a mathematical approach, called alignment algorithm.According to the invention, the alignment is generated by using the algorithm of Needleman and Wunsch (J. Mol. Biol. (1970) 48, p. 443-453). Preferably, the program "NEEDLE” (The European Molecular Biology Open Software2200724Suite (EMBOSS)) is used for the purposes of the current invention, with using the programs default parameter (polynucleotides: gap open=10.0, gap extend=0.5 and matrix=EDNAFULL; polypeptides: gap open=10.0, gap extend=0.5 and matrix=EBLOSUM62). After aligning two sequences, in a second step, an identity value is determined from the alignment produced. For this purpose, the %-identity is calculated by dividing the number of identical residues by the length of the alignment region which is showing the respective sequence of the present invention over its complete length multiplied with 100: %-identity = (identical residues I length of the alignment region which is showing the respective sequence of the present invention over its complete length) *100.A special aspect concerning amino acid substitutions are "conservative mutations”, interchangeably used with "conservative substitutions” herein. Conservative mutations are those where one amino acid is exchanged with a similar amino acid. Herein the following conservative exchanges are considered:Amino acid A is similar to amino acids SAmino acid D is similar to amino acids E; NAmino acid E is similar to amino acids D; K; QAmino acid F is similar to amino acids W; YAmino acid H is similar to amino acids N; YAmino acid I is similar to amino acids L; M; VAmino acid K is similar to amino acids E; Q; RAmino acid L is similar to amino acids I; M; VAmino acid M is similar to amino acids I; L; VAmino acid N is similar to amino acids D; H; SAmino acid Q is similar to amino acids E; K; RAmino acid R is similar to amino acids K; QAmino acid S is similar to amino acids A; N; TAmino acid T is similar to amino acids SAmino acid V is similar to amino acids I; L; MAmino acid W is similar to amino acids F; YAmino acid Y is similar to amino acids F; H; WA "fragment" or "subsequence” as used herein refers to a portion of an amino acid sequence. Polypeptides comprising a deletion of one or more amino acids at the N terminus and / or the C terminus of the polypeptide and essentially retain pectinase activity are herein designated as "functional fragments". Preferably, the functional fragment has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, 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% of the length of the original full length amino acid sequence. Preferably, the functional fragment comprises 100 to 250 consecutive amino acids of the full-length polypeptide. Also preferably, the functional fragment retains at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80% identical, at least220072581%, 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%, at least 99.5% or at least 100% of the enzyme activity of the original full length amino acid sequence. The functional fragment comprises consecutive amino acids compared to the original full length amino acid sequence, respectively. In the context of the present invention, the "original full length amino acid sequence" is an amino acid sequence which is at least 75%, preferably at least 80%, identical to the amino acid sequence according to SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5 and which has the same length as the amino acid sequence according to SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5, respectively.In one embodiment, the polypeptide of the present invention does not comprise any internal deletions compared to the amino acid sequence according to SEQ ID NO: 1, 3 or 5. However, as discussed above, the polypeptide of the present invention may comprise a deletion of one or more amino acids at the N terminus and / or the C terminus of the polypeptide."Genetic construct” or nucleic acid construct or "expression cassette” as used herein, is a nucleic acid molecule composed of at least one sequence of interest to be expressed, operably linked to one or more control sequences (at least to a promoter) as described herein.The term "vector” as used herein comprises any kind of construct suitable to carry foreign polynucleotide sequences for transfer to another cell, or for stable or transient expression within a given cell. The term "vector” as used herein encompasses any kind of cloning vehicles, such as but not limited to plasmids, phagemids, viral vectors (e.g., phages), bacteriophage, baculoviruses, cosmids, fosmids, artificial chromosomes, and any other vectors specific for specific hosts of interest. Foreign polynucleotide sequences usually comprise a coding sequence which may be referred to herein as "gene of interest”. The gene of interest may comprise introns and exons, depending on the kind of origin or destination of host cell.The term "introduction of a polynucleotide” or "transformation of a polynucleotide” as referred to herein encompasses the transfer of an exogenous polynucleotide into a host cell, irrespective of the method used for transfer. That is, the term "transformation of a polynucleotide” as used herein is independent from vector, shuttle system, or host cell, and it not only relates to the polynucleotide transfer method of transformation as known in the art (of., for example, Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY), but it encompasses any further kind of polynucleotide transfer methods such as, but not limited to, transduction or transfection.A polynucleotide encoding a polypeptide may be "expressed”. The term "expression” or "gene expression” means the transcription of a specific gene or specific genes or specific nucleic acid construct. The term "expression” or "gene expression” means the transcription of a gene or genes or genetic construct into structural RNA (e.g., rRNA, tRNA) or mRNA with or without subsequent translation of the latter into a protein. The process includes transcription of DNA and processing of the resulting mRNA product.The term "purification” or "purifying” refers to a process in which at least one component, e.g., a protein of interest, is separated from at least another component, e.g., a particulate matter of a fermentation broth, and transferred into a different compartment or phase, wherein the different compartments or phases do not necessarily need to2200726be separated by a physical barrier. Examples of such different compartments are two compartments separated by a filtration membrane or cloth, i.e., filtrate and retentate; examples of such different phases are pellet and supernatant or cake and filtrate, respectively. The resulting solution after purifying the enzyme of interest from the fermentation broth is called herein "purified enzyme solution”.A "composition” or formulation is a mixture of components (also called ingredients) prepared according to a specific formula. Compositions include enzyme compositions, e.g. liquid enzyme formulations as described herein, and cleaning compositions, e.g. liquid cleaning compositions as described herein."Protein formulation” (or "enzyme preparation” or "polypeptide formulation” or "enzyme formulation”), e.g., "pectinase formulation”, means any non-complex formulation comprising a small number of ingredients, preferably, 2-8 components, wherein the ingredients serve the purpose of stabilizing the proteins / polypeptides comprised in the protein / polypeptide formulation and / or the stabilization of the protein / polypeptide formulation itself. Preferably, the non-complex protein / polypeptide formulation comprises the protein / polypeptide in higher concentrations than the complex formulation, e.g., than a cleaning composition or detergent formulation. Thus, preferably the non-complex protein formulation is a concentrated protein / polypeptide formulation. Preferably, non-complex protein / polypeptide formulations comprise 20 to 120 mg / g active enzyme, whereas complex formulations, like cleaning compositions, comprise 0.002 to 10 mg / g active enzyme. In contrast to a non-complex formulation, a complex formulation means herein a formulation comprising a higher number of ingredients, preferably, 9-30 components, wherein the ingredients serve the purpose of stabilizing the proteins comprised in the protein formulation and / or the stabilization of the protein formulation itself, but additionally the complex formulation comprises components that serve the purpose of the complex formulation, e.g., a cleaning composition. An example for a non-complex protein formulation is a concentrated enzyme composition that is used as a stock-solution to prepare a complex formulation, e.g., a cleaning composition, wherein in the cleaning compositions other compounds are present that serve the cleaning purpose of the cleaning composition, e.g., surfactants and / or chelating agents.The term "detergent composition” or "cleaning composition" (also called "cleaning formulation”, "detergent formulation” or "detergent” herein), as used herein, refers to compositions and formulations designed for cleaning soiled material. Cleaning compositions are complex formulations as further defined herein. Cleaning compositions also include biodegradable cleaning compositions. The term "detergent component” or "cleaning component” (also called herein as "adjunct cleaning additive”) is defined herein to mean a type of chemical, which can be used in detergent compositions. The term "effective amount of a detergent component” includes amounts of certain components to provide effective stain removal and / or effective cleaning conditions (e.g. pH, temperature, water hardness, quantity of foaming), amounts of certain components to effectively provide optical benefits (e.g. optical brightening, dye transfer inhibition, color care), and amounts of certain components to effectively aid the processing (maintain physical characteristics during processing, storage and use; e.g. rheology modifiers, hydrotropes, desiccants).The term "laundry” or "laundering” relates to both household laundering and industrial laundering and means the process of treating textiles and / or fabrics with a solution containing a detergent composition. The laundering2200727process may be carried out by using technical devices such as a household or an industrial washing machine. Alternatively, the laundering process may be done by hand.The term "textile” means any textile material including yarns (thread made of natural or synthetic fibers used for knitting or weaving), yarn intermediates, fibers, non-woven materials, natural materials, synthetic materials, as well as fabrics made of these materials such as garments, cloths and other articles. The terms "fabric” refers to a textile made by weaving, knitting or felting fibers. The term "garment” refers to any article of clothing made of textile. The term "fibers” includes natural fibers, synthetic fibers, and mixtures thereof. Examples of natural fibers are of plant (such as flax, jute and cotton) or animal origin, comprising proteins like collagen, keratin and fibroin (e.g. silk, sheep's wool, angora, mohair, cashmere). Examples for fibers of synthetic origin are polyurethane fibers such as Spandex® or Lycra®, polyester fibers, polyolefins such as elastofin, or polyamide fibers such as nylon. Fibers may be single fibers or parts of textiles such as knitwear, woven or non-woven fabrics.The term "hard surface cleaning” relates to both household hard surface cleaning and industrial hard surface cleaning and means the process of treating hard surfaces with a solution containing a detergent composition. Hard surfaces may include any hard surfaces in the household or industry, such as floors, furnishing, walls, sanitary ceramics, glass, metallic surfaces including cutlery or dishes and medical devices such as diagnostic instruments, trays, pans, holders, racks, forceps, scissors, shears, saws (e.g. bone saws and their blades), hemostats, knives, chisels, rongeurs, files, nippers, drills, drill bits, rasps, burrs, spreaders, breakers, elevators, clamps, needle holders, carriers, clips, hooks, gouges, curettes, retractors, straightener, punches, extractors, scoops, keratomes, spatulas, expressors, trocars, dilators, cages, glassware, tubing, catheters, cannulas, plugs, stents, endoscopes, arthoscopes and related equipment. A particular form of hard surface cleaning is dishwashing, particularly automatic dishwashing (ADW).The term "dish wash” refers to all forms of washing dishes, e.g. by hand or automatic dish wash. Washing dishes includes, but is not limited to, the cleaning of all forms of crockery 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, china, glass and acrylics.In the field of detergency, usually the term "stains” is used with reference to laundry, e.g., cleaning of textiles, fabric, or fibers, whereas the term "soils” is usually used with reference to hard surface cleaning, e.g., cleaning of dishes and cutlery. However, herein the terms "stain” and "soil” shall be used interchangeably.Compositions can be liquid or solid. Liquid compositions of the invention include solutions, emulsions and dispersions, gels etc. as long as the liquid is fluid and pourable. Liquid compositions according to the present invention preferably have a dynamic viscosity in the range of about 50 to about 10000 mPa*s, determined at 25°C according to Brookfield, for example spindle 3 at 20 rpm with a Brookfield viscosimeter LVT-II."Gel", as used herein, means a shear thinning, lamellar gel, with a pouring viscosity in the range of from 100 to 5,000 mPa*s (milli Pascal seconds), more preferably less than 3,000 mPa*s, most preferably less than 1,500 mPa*s.The components of the composition can be separated by formulating them in different compartments, such as different compartments of multi-chamber-pouches or bottles having different chambers, from which the liquids are2200728poured out at the same time in a predefined amount to assure the application of the right amount per individual point of use of each component from each chamber. Such multi-compartment-pouches and bottles etc. are known to a person of skill as well."Enzyme properties” include, but are not limited to, catalytic activity, substrate / cofactor specificity, product specificity, stability in the course of time, thermostability, pH stability, and chemical stability. "Enzymatic activity” or "catalytic activity” means the catalytic effect exerted by an enzyme, expressed as units per milligram of enzyme (specific activity) or molecules of substrate transformed per minute per molecule of enzyme (molecular activity). Enzymatic activity can be specified by the enzyme's actual function, e.g., proteases exerting proteolytic activity by catalyzing hydrolytic cleavage of peptide bonds, lipases exerting lipolytic activity by hydrolytic cleavage of ester bonds, or pectinases exerting pectinolytic activity by degrading pectins or pectate.Pectinolytic activity can be determined by using pectin, pectate, or a complex source that includes pectin or pectate, as substrate. Pectin hydrolysis that results in galacturonic acid can be quantified by measuring absorbance at 235 nm (0.5% Pectin, pH 6, 22 °C). Other methods using, for example following reducing sugars (galacturonic acid) generated during cleavage (DNS-dinitrosalicylic acid reagent) on a pectinolytic substrate are known to those skilled in the art.As used herein, "wash performance" (also called herein "cleaning performance”) of an enzyme refers to the contribution of the enzyme to the cleaning performance of a cleaning composition, i.e. the cleaning performance added to the cleaning composition by the performance of the enzyme. The term "wash performance” is used herein similarly for laundry and hard surface cleaning. Wash performance is compared under relevant washing conditions. The term "relevant washing conditions" is used herein to indicate the conditions, particularly washing temperature, time, washing mechanics, sud concentration, type of detergent and water hardness, actually used in households in a detergent market segment. The term "improved wash performance" is used to indicate that a better end result is obtained in stain removal under relevant washing conditions, or that less enzyme, on weight basis, is needed to obtain the same end result relative to the corresponding control conditions.As used herein, the term "specific performance" refers to the cleaning and removal of specific stains or soils per unit of active enzyme. In some embodiments, the specific performance is determined using stains or soils containing pectins or pectate such as berry jam, fruit, spinach, juices, condiments, baby food, etc.A composition "essentially devoid” of a compound shall mean herein that the respective compound is not added to the composition on purpose, meaning that at most non-effective amounts are present, most preferably 0% of the compound are contained in the composition.Detailed descriptionIn the present invention pectinase enzymes for cleaning compositions and the cleaning compositions comprising them are provided. Additionally, methods of making and using these enzymes, including recombinant polynucleotides encoding them, are provided.2200729PectinasesPectinases according to the invention have "pectinase activity”. "Pectinase activity” describes the capability for degrading pectins and pectate, such as in pectin or pectate containing stains.Pectins are structural polymers of plant cell walls. Pectins are heteropolysaccharides that consist of backbones comprising alternating homogalacturonan, called smooth regions, and rhamnogalacturonan I and II, called hairy regions. The smooth regions are linear polymers composed of 1 ,4-linked alpha-D-galacturonic acid, in which some of the carboxyl groups are esterified with methanol and / or acetyl groups. Rhamnogalacturonan I consists of repeating residues of 1 ,4-linked alpha-D-galacturonic acid and a-1,2-L-rhamnose. Side chains are attached at the rhamnose residues. The side chains are composed of a single sugar residue (galactose and / or arabinose residues) or combined chains of arabinans, galactans, or arabinogalactans. Rhamnogalacturonan II has a backbone that consists of 1 ,4-linked alpha-D-galacturonic acid residues, the backbone being highly branched with side chains at C-2 and C-3. The side chains can include arabinose, apiose, fucose, galactose, rhamnose, aceric acid, glucuronic acid, galacturonic acid, xylose, and fucose (Zdunek A, Pieczywek PM, Cybulska J. The primary, secondary and structures of higher levels of pectin polysaccharides. Compr Rev Food Sci FoodSaf. 2021; 20: 1101-1117. https: / / doi.org / 10.1111 / 1541-4337.12689).Pectate, also known as pectic acid or polygalacturonic acid, is a polysaccharide derived from pectins and an intermediate product of pectin degradation.Pectolytic enzymes, called "pectinases” or "pectinolytic enzymes” herein, are a group of enzymes involved in pectin and pectate degradation. They are often classified based on their preferred substrate, which can be highly methyl-esterified pectin, low methyl-esterified pectin, or polygalacturonic acid (pectate), as well as their reaction mechanism, which can be beta-elimination or hydrolysis.Pectinases are classified in The Enzyme Nomenclature (1992) as pectin esterases (EC 3.1.1.11 and EC 3.1.1.6), pectin depolymerases (endo-polygalacturonases: EC 3.2.1.15, exo-polygalacturonases: EC 3.2.1.67, EC 3.2.1.82, endo-polygalacturonate lyases: EC 4.2.2.2, exo-polygalacturonate lyases: EC 4.2.2.9, endo-polymethylgalacturonate lyases: EC 4.2.2.10) or protopectinases.Specific pectinase activity may be determined by assays for measurement of pectinase activity, which are known to those skilled in the art and which are dependent on the type of pectinase.Pectinolytic activity can, for example, be determined by using pectin, pectate, or a complex source that includes pectin or pectate, as substrate. Pectin hydrolysis that results in galacturonic acid can be quantified by measuring absorbance at 235 nm (0.5% Pectin, pH 6, 22 °C). Other methods using, for example, following reducing sugars (galacturonic acid) generated during cleavage (DNS-dinitrosalicylic acid reagent) on a pectinolytic substrate are known to those skilled in the art.Pectinase esterase activity can, for example, be determined using 0.2% polygalatacturonic acid (Sigma) in 25mM Tris / HCI, 25mM glycine / NaOH, pH 9 buffer as a substrate. Reaction rates of enzymatic hydrolysis can be determined by monitoring the release of unsaturated oligogalaturonides spectrophotometrically as an increase in absorbance at 235 nm in a spectrophotometer.22007210Preferably, pectinases as defined herein are enzymes that have a wash performance in AARGB of at least 0.008, preferably at least 0.009, when tested on pectinase sensitive stains, preferably strawberry banana babyfood (CS77B) or spinach (CS25) stains, preferably when washing for 40 minutes at 30 °C in a LOM using a standard detergent, preferably Persil Non Bio detergent, with an enzyme concentration of 0.1 ppm per wash.AARGB describes the difference in measured RGB values before and after wash and the detergent with and without enzyme.As pectins are naturally found in the cell walls of plants, such as in fruits and vegetables, they are found in plantbased foods and drinks, such as juices. Pectins are also often used in the food industry as gelling agents in jams, yoghurt drinks and smoothies.Pectinases can thus be used in cleaning compositions for the removal of pectins or pectate comprising stains such as fruit and vegetable stains, stains from gelling or thickening agents, grass stains, and wine stains. Further examples include stains such as ketchup, salad dressings, marmalades, juices, yoghurts, milkshakes.The pectinase of the present invention, preferably comprised in a cleaning composition, is a pectinase that comprises an amino acid sequence that is at least 75%, preferably at least 80%, identical to SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5.In one embodiment, the pectinase comprises an amino acid sequence that is with increasing preference 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: 1.In another embodiment, the pectinase comprises an amino acid sequence that is with increasing preference 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: 3.In another embodiment, the pectinase comprises an amino acid sequence that is with increasing preference 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: 5.Preferably, the pectinase comprises an amino acid sequence that is with increasing preference 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: 1, 3 or 5.More preferably, the pectinase comprises an amino acid sequence that is with increasing preference 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: 1 or 3.Most preferably, the pectinase comprises an amino acid sequence that is with increasing preference 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: 1.22007211In one embodiment, the pectinase comprises or consists of an amino acid sequence that is 100% identical to SEQ ID NO: 1 , 3 or 5, preferably SEQ ID NO: 1 or SEQ ID NO: 3, more preferably SEQ ID NO: 1.In one embodiment, the pectinase comprises or consists of an amino acid sequence that is 100% identical to SEQ ID NO: 1, 3 or 5, preferably SEQ ID NO: 1, 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.Ways of introducing amino acid alterations, e.g., substitutions, preferably conservative substitutions, into protein sequence are well known in the art. Substitutions can be introduced by using any mutagenesis procedure known in the art, such as site-directed mutagenesis, synthetic gene construction, semi-synthetic gene construction, random mutagenesis, shuffling, etc., followed by a relevant screening procedure. See, e.g., 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.Nucleic acid constructThe present invention also refers to a polynucleotide encoding the polypeptide having pectinase activity of the present invention. Preferably, the polynucleotide is a codon-optimized polynucleotide for improving expression in a specific host cell, preferably a Bacillus cell, more preferably a B. licheniformis cell.The present invention thus also refers to a polynucleotide, preferably an isolated, a synthetic, and / or a recombinant polynucleotide comprising:(a) a polynucleotide having with increasing preference 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 to SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6, wherein the polynucleotide encodes a polypeptide having pectinase activity;(b) a polynucleotide encoding a polypeptide having with increasing preference 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 to SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5, wherein the polypeptide has pectinase activity; (c) a polynucleotide encoding a polypeptide having pectinase activity comprising an amino acid sequence that is at least 80%, preferably 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% identical to SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5, wherein the polynucleotide preferably has at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6.22007212(d) a polynucleotide that hybridizes under high stringency conditions, preferably under very high stringency conditions, with the complement of(i) a coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5; or(ii) a polynucleotide shown in SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6;(e) a fragment of (a), (b), or (c), wherein the fragment encodes a polypeptide having pectinase activity; or (f) a polynucleotide sequence fully complementary to any of (a) to (d).(g) a polynucleotide that differs from any of the nucleic acid sequences described in (a) to (e) merely by the degeneracy of the genetic code.Thus, in one embodiment, the invention relates to polypeptides having pectinase activity which are encoded by a polynucleotide described herein, preferably isolated, synthetic or recombinant polypeptides having pectinase activity, which are encoded by a polynucleotide described herein. Preferably, the polypeptide having pectinase activity is encoded by a polynucleotide having with increasing preference at least 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 to SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6. In a preferred embodiment, the polynucleotide of the present invention encodes a polypeptide having pectinase activity comprising an amino acid sequence that is at least 80%, preferably 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% identical to SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5, wherein the polynucleotide has at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6.In another embodiment, the polypeptide having pectinase activity is encoded by a polynucleotide that hybridizes under high stringency conditions, preferably under very high stringency conditions, with the full-length complement of a polynucleotide having with increasing preference at least 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 to SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6.The present invention also refers to a nucleic acid construct, preferably an expression cassette, comprising the polynucleotide as described herein.Typically, the expression cassette comprises three elements: a promoter sequence, an open reading frame and a 3' untranslated region that, in eukaryotes, usually contains a polyadenylation site. Additional regulatory elements may include transcriptional as well as translational enhancers. An intron sequence may also be added to the 5' untranslated region (UTR) or in the coding sequence to increase the amount of the mature message that accumulates in the cytosol. The expression cassette may be part of a vector or may be integrated into the genome of a host cell and replicated together with the genome of its host cell. The expression cassette usually is capable of increasing or decreasing expression.22007213The present invention also refers to an expression vector comprising the polynucleotide or the nucleic acid construct as described herein. The expression vector can be a low copy number vector or high copy number vector.A vector as used herein may provide segments for transcription and translation of a foreign polynucleotide upon transformation into a host cell or host cell organelles. Such additional segments may include regulatory nucleotide sequences, one or more origins of replication that is required for its maintenance and / or replication in a specific cell type, one or more selectable markers, a polyadenylation signal, a suitable site for the insertion of foreign coding sequences such as a multiple cloning site etc. One example is when a vector is required to be maintained in a bacterial cell as an episomal genetic element (e.g., plasmid or cosmid molecule). Non-limiting examples of suitable origins of replication include the f1-ori and colE1.A vector may replicate without integrating into the genome of a host cell, e.g., as a plasmid in a bacterial host cell, or it may integrate part or all of its DNA into the genome of the host cell and thus lead to replication and expression of its DNA.The polynucleotide encoding the polypeptide having pectinase activity of the present invention may be introduced into a vector by means of standard recombinant DNA techniques. Once introduced into the vector, the polynucleotide comprising a coding sequence may be suitable to be introduced (transformed, transduced, transfected, etc.) into a host cell or host cell organelles. A cloning vector may be chosen suitable for expression of the polynucleotide sequence in the host cell or host cell organelles.Host cellThe present invention also refers to a host cell comprising the polynucleotide encoding the polypeptide having pectinase activity of the present invention, the nucleic acid construct as described herein, or the expression vector as described herein. In one embodiment of the invention, a vector is used for transformation of a host cell. Preferably, the host cell is capable of expressing the polynucleotide encoding the polypeptide having pectinase activity of the present invention.The polynucleotide encoding the polypeptide having pectinase activity of the present invention herein may be transiently or stably introduced into a host cell. The polynucleotide may be maintained non-integrated, for example, as a plasmid. Usually, stable transformation is due to integration of nucleic acid comprising a foreign coding sequence into the host cell chromosomes or as an episome (separate piece of nuclear DNA). Usually, transient transformation is due to nucleic acid comprising a foreign nucleic acid sequence not being integrated into the host cell chromosomes or as an episome. Methods for introducing a nucleic acid into a host cell are well-known in the art.Various host cells can be used for expressing the nucleic acid construct described herein. Preferably, the host cell of the present invention does not naturally express the polypeptide having pectinase activity of the present invention. Thus, the host cell is a recombinant host cell; the nucleic acid construct described herein is heterologous to the host cell.22007214In one embodiment, the host cell is a prokaryote or a eukaryote. In another embodiment, the host cell is a bacterium, an archaeon, a fungal cell, a yeast cell or a eukaryotic cell. In another embodiment, the host cell is a non-human host cell.In a preferred embodiment, the host cell is a bacterial cell. The bacterial host cell may be any gram-positive bacterium or a gram-negative bacterium. Gram-positive bacteria include, but are not limited to, Bacillus, Brevibacterium, Corynebacterium, Streptococcus, Streptomyces, Staphylococcus, Enterococcus, Lactobacillus, Lactococcus, Clostridium, Geobacillus, and Oceanobacillus. Gram-negative bacteria include, but are not limited to, Escherichia, Pseudomonas, Salmonella, Campylobacter, Helicobacter, Acetobacter, Flavobacterium, Fusobacterium, Gluconobacter. In a preferred embodiment the host cell is of the genus Escherichia or Bacillus. In one embodiment, the bacterial host cell is an Escherichia coll cellIn the present invention, the bacterial host cell may 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 preferred embodiment, the bacterial host cell is a Bacillus licheniformis cell, a Bacillus pumilus, or a Bacillus subtilis cell. Preferably, the bacterial host cell is a Bacillus licheniformis cell.Methods of makingThe pectinase described herein can be produced on an industrial scale and subsequently purified. Industrial production of enzymes is usually done by cultivating a host cell, which expresses the enzyme (also called fermentation). Suitable host cells are described above. A nucleic acid sequence encoding the pectinase described herein can be transformed into the host cell, which is subsequently cultivated under conditions suitable for the host cell to produce the pectinase. In a preferred embodiment, the pectinase is purified from the host cell. Hence, in yet another embodiment, the present invention is directed to a method of producing a pectinase comprising the steps of(a) providing a host cell comprising a recombinant nucleic acid construct comprising a polynucleotide encoding the pectinase described herein by introducing the nucleic acid construct comprising the polynucleotide encoding the pectinase as described herein into the host cell;(b) cultivating the recombinant host cell of step (a) under conditions conductive for the expression of the polynucleotide; and(c) optionally, recovering the pectinase encoded by the polynucleotide.Cultivation of the host cell normally takes place in a suitable nutrient medium allowing the recombinant cells to grow and express the desired protein. At the end of fermentation, the fermentation broth is collected and may be22007215further processed, wherein the fermentation broth comprises a liquid fraction and a solid fraction. The enzyme of interest may be further purified from the fermentation broth.The pectinase described herein may be secreted (into the liquid fraction of the fermentation broth) or may not be secreted from the microbial cells (and therefore is comprised in the cells of the fermentation broth). Depending thereon, the pectinase may be recovered from the liquid fraction of the fermentation broth or from cell lysates. Preferably, the pectinase is secreted from the cell into the fermentation broth, preferably by means of a secretion signal peptide also called signal peptide herein added to a terminus, preferably the amino terminus, of the amino acid sequence of the pectinase.Various signal peptides known to those skilled in the art can be used for expressing the pectinase of the present invention. In one embodiment, the signal peptide is the native signal peptide of the pectinase to be expressed. In another embodiment, the signal peptide and the pectinase are recombinant to each other, meaning they are naturally not occurring in the specific combination with each other.In one embodiment, the signal peptide is selected from the group consisting of pelC (e.g. as shown in SEQ ID NO: 7 or 8), wapA (e.g. as shown in SEQ ID NO: 9 or 10), yvcE, or ydjM.In a preferred embodiment, the signal peptide is selected from group consisting of pelC or wapA. Thus, in a preferred embodiment, the signal peptide is selected from a group consisting of(a) a polynucleotide having with increasing preference 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 to SEQ ID NO: 8 or SEQ ID NO: 10, wherein the polynucleotide encodes a polypeptide having the function of a signal peptide; and (b) a polynucleotide encoding a polypeptide having with increasing preference 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 to SEQ ID NO: 7 or SEQ ID NO: 9, wherein the polypeptide has the function of a signal peptide.In a preferred embodiment, the pectinase that comprises an amino acid sequence that is with increasing preference 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: 1 is expressed using a signal peptide having with increasing preference 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 to SEQ ID NO: 7.In a preferred embodiment, the pectinase that comprises an amino acid sequence that is with increasing preference 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: 3 is expressed using a signal peptide having with increasing preference 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 to SEQ ID NO: 9.Recovery of the pectinase can be achieved by methods known to those skilled in the art. Suitable methods for recovery of proteins from fermentation broth include but are not limited to collection, centrifugation, filtration,22007216extraction, and precipitation. In case the desired protein is comprised in the cells of the fermentation broth, release of the product of interest from the cells might be needed. Release from the cells can be achieved for instance, but not limited thereto, by cell lysis with techniques well known to the skilled person, e.g., lysozyme treatment, ultrasonic treatment, French press or combinations thereof.The pectinase may be purified from the fermentation broth by methods known in the art. For example, the pectinase may be isolated from the fermentation broth by conventional procedures including, but not limited to, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation. The isolated polypeptide may 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 (I EF)), differential solubility (e.g., ammonium sulfate precipitation), or extraction (see, e.g., Protein Purification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989). The purified polypeptide may then be concentrated by procedures known in the art including, but not limited to, ultrafiltration and evaporation, in particular, thin film evaporation.The purified solution of the polypeptide having pectinase activity of the present invention may be further processed to form a pectinase containing composition, preferably a cleaning composition, more preferably a laundry cleaning composition or a dish washing composition.Composition comprising pectinasesThe present invention also refers to a composition, preferably a cleaning composition, more preferably a laundry cleaning composition or a dish washing composition comprising the polypeptide having pectinase activity described herein and at least one additional component.Thus, the present invention therefore also refers to a method for making a composition comprising the steps of mixingI. a polypeptide having pectinase activity as described herein; andii. one or more components described herein.The composition can be a non-complex formulation, e.g., a pectinase formulation, or a complex formulation, e.g., a cleaning composition, as described herein.Preferably, the composition is a cleaning composition, more preferably a laundry cleaning composition or a dish washing composition.Pectinase formulationThe polypeptide having pectinase activity can be formulated as a pectinase formulation, preferably a concentrated pectinase formulation. The pectinase formulation can either be solid or liquid. Protein formulations can be obtained by using techniques known in the art. For instance, without being limited thereto, solid enzyme formulations can be obtained by extrusion or granulation. Suitable extrusion and granulation techniques are known in the art and are described for instance in WO 94 / 19444 A1 and WO 97 / 43482 A1.22007217Liquid pectinase formulations may comprise amounts of pectinase enzyme in the range of 1% to 40%, 1% to 30%, 1% to 25%, 1% to 12%, or preferably 1-10% by weight, all relative to the total weight of the enzyme formulation. In one embodiment, the pectinase formulation, in particular the liquid pectinase formulation, comprises in addition one or more additional components selected from the group consisting of solvent, salt, pH regulator, preservative, enzyme stabilizer, and thickening agent. Preferably, the pectinase formulation is essentially devoid of surfactants, i.e. the pectinase formulation comprises less than 1% surfactant, preferably less than 0.5% surfactant. The solvent may be water and / or an organic solvent. Aqueous pectinase formulations of the invention may comprise water in amounts of more than about 30% by weight, more than about 40% by weight, more than about 50% by weight or more than about 60% by weight, all relative to the total weight of the pectinase formulation. The pectinasecontaining formulations of the invention may comprise an organic solvent in amounts of more than 30%, more than 40%, more than about 50% by weight, more than about 60% by weight, more than about 70% by weight, or more than about 80% by weight, all relative to the total weight of the pectinase formulation. The organic solvent may be a water-miscible solvent. The organic solvent may be one or more selected from the group consisting of glycerol, propanediol, polypropylene glycol, polyethylene glycol, sorbitol or mono propylene glycol.In one embodiment, the pectinase formulation comprises at least one preservative. Preferably, preservative means substances that are added to a liquid composition for the purpose of preservation, meaning more preferably that compounds known to have preserving features comprised in a liquid composition formed in the production process are excluded from the term preservatives. In one embodiment, the preservative is selected from the group consisting of 2-phenoxyethanol, glutaraldehyde, 2-bromo-2-nitropropane-1 ,3-diol, formic acid in acid form or as its salt, and 4,4'-dichloro 2-hydroxydiphenylether. Usually, the liquid compositions of the invention comprise at least one preservative in amounts below 10ppm, such as in amounts ranging from 2 ppm to 5% by weight relative to the total weight of the liquid composition. Preferably, the pectinase formulation is free from preservatives, meaning that preservatives are comprised in amounts less than 1 ppm, preferably 0 ppm.Preferably, the pectinase formulation comprises an enzyme stabilizing system. Preferably, the enzyme stabilizing 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, CaCh, MgCh, or NaCI), short chain (preferably, C1-C3) carboxylic acids or salts thereof (preferably, formic acid, formate (preferably, sodium formate), acetic acid, acetate, or lactate). In one embodiment, the enzyme stabilizing system comprises a combination of at least two of the compounds selected from the group consisting of salts, polyols, and short chain carboxylic acids or salts thereof. Preferably, the pectinase formulation comprises a calcium salt, preferably calcium chloride.Preferably, the liquid pectinase formulation comprises or consists of the polypeptide having pectinase activity of the present invention, a solvent, an enzyme stabilizing system, and optionally a preservative and optionally an additional enzyme different from the pectinase. Preferably, the formulation of the polypeptide having pectinase activity of the present invention is essentially devoid of surfactants, i.e. the pectinase formulation comprises less than 1% surfactant, preferably less than 0.5% surfactant.22007218The present invention therefore also relates to a method for making a pectinase formulation, preferably a concentrated pectinase formulation, comprising the steps of mixinga) a polypeptide having pectinase activity of the present invention; andb) one or more components selected from the group consisting of solvent, enzyme stabilizing system, preservative, and optionally an additional enzyme different from the pectinase.In one embodiment, the formulation is a formulation comprising a microorganism (alive, attenuated or inactivated) containing the polypeptide having pectinase activity of the present invention such as a probiotic or prebiotic formulation.Additional enzymesIn another embodiment, the composition comprising a polypeptide having pectinase activity of the present invention further comprises one or more additional enzymes different from the pectinase. Preferably, the additional enzyme is selected from the group consisting of proteases, amylases, lipases, cellulases, mannanases, hemicellulases, phospholipases, esterases, pectinases other than the polypeptide having pectinase activity of the present invention, lactases, peroxidases, xylanases, cutinases, keratinases, reductases, oxygenases, phenoloxidases, lipoxygenases, ligninases, pullulanases, tannases, pentosanases, malanases, beta-glucanases, arabinosidases, hyaluronidases, chondroitinases, laccases, nucleases, ribonucleases (RNAses), deoxyribonucleases (DNAses), phosphodiesterases, phytases, carbohydrases, galactanases, xanthanases, xyloglucanases, oxidoreductases, perhydrolases, aminopeptidases, asparaginases, carbohydrases, carboxypeptidases, catalases, chitinases, cyclodextrin glycosyltransferases, alpha-galactosidases, beta-galactosidases, glucoamylases, alpha-glucosidases, beta-glucosidases, invertases, transglutaminases and dispersins, and combinations of at least two of the foregoing types. More preferably, the additional enzyme is selected from the group consisting of proteases, amylases, lipases, cellulases, mannanases, esterases, xylanases, DNAses, dispersins, pectinases other than the polypeptide having pectinase activity of the present invention, oxidoreductases, and cutinases, and combinations of at least two of the foregoing types. Further preferred, the additional enzyme is selected from the group consisting of proteases, amylases, lipases, cellulases, mannanases, and combinations of at least two of the foregoing types. Most preferably, the additional enzyme is a protease.The composition of the present invention can comprise more than one enzyme of different types, e.g., a pectinase and a protease, or more than one enzyme of the same type, e.g., two or more different pectinases, or mixtures thereof, e.g., a protease and two different pectinases of which one is the pectinase having pectinase activity of the present invention.Cleaning compositionsIn one embodiment, the present invention is directed to the use of polypeptides having pectinase activity in a cleaning composition. Thus, the present invention is also directed to a cleaning composition, preferably a laundry cleaning composition or a dish washing composition, comprising the polypeptide having pectinase activity and at least one additional component, the additional component preferably being one or more adjunct cleaning additive.22007219The publication IPCOM000274907D published on www.IP.com is regarded as Reference RF1, which is incorporated herein by reference in its entirety. The publication Prior Art Disclosure; Issue 684; paragraphs

[3000] to

[3061] ; ISSN: 2198-4786; published: February 12, 2024 is regarded as Reference RF2, which is incorporated herein by reference in its entirety.The phrase "cleaning composition" (also called "cleaning formulation”, "detergent composition”, "detergent formulation” or "detergent” herein), as used herein, includes compositions and formulations designed for cleaning soiled material. Such compositions and formulations include those designed for cleaning soiled material or surfaces of any kind, more preferably compositions for Fabric and Home Care. "Cleaning compositions” are defined in more detail in paragraphs

[0001] ,

[0002] ,

[0004] and

[0006] of Reference RF1."Compositions for Fabric and Home Care” include cleaning compositions and formulations including but not limited to laundry cleaning compositions and detergents and hard surface cleaning compositions including dish washing compositions, more preferably liquid laundry formulations, solid laundry compositions, liquid manual dish wash formulations, automatic dish wash (ADW) gels and automatic dish wash (ADW) solid compositions. "Compositions for Fabric and Home Care” are defined in more detail in paragraph

[0003] of Reference RF1.The cleaning compositions of the invention including the inventive enzyme(s) may - and preferably do - contain adjunct cleaning additives (also abbreviated herein as "adjuncts”).Thus, in one embodiment, the additional component comprised in the composition comprising the polypeptide having pectinase activity is an adjunct cleaning additive.Suitable adjunct cleaning additives include polymers, surfactants, builders, cobuilders, further enzymes, enzyme stabilizing systems, structurants or thickeners, clay soil removal / anti-redeposition agents, solubilizing agents, chelating agents, bleaching compounds, bleaching agents, bleach activators, bleach catalysts, brighteners, malodor control agents, pigments, dyes, opacifiers, hueing agents, dye transfer inhibiting agents, chelating agents, suds boosters, suds suppressors (antifoams), color speckles, silver care, anti-tarnish and / or anti-corrosion agents, alkalinity sources, pH adjusters, pH-buffer agents, hydrotropes, scrubbing particles, antibacterial agents, antioxidants, softeners, carriers, processing aids, pro-perfumes, dye fixation agent and perfumes. Preferable adjuncts are polymers, surfactants, or builders.Thus, the present invention also relates to a method for making a cleaning composition comprising the steps of mixinga) a polypeptide having pectinase activity of the present invention; andb) one or more adjunct cleaning additives described herein.The addition of the liquid pectinase formulation to a cleaning composition, preferably liquid cleaning composition, usually occurs in a weight ratio liquid pectinase formulation:cleaning composition of about 1:1000, 1:500, 1:100, 1:50, 1:30, 1:25, 1:20, or 1:10.In preferred embodiments, the cleaning compositions comprise the enzyme having pectinase activity described herein and at least one polymer, preferably cleaning polymers and / or soil release polymers. "Cleaning polymers and soil release polymers” are defined in more detail in paragraphs

[0032] to

[0034] of Reference RF1. These polymers include polycarboxylates, alkoxylated polyalkylenamines, alkoxylated polyalkylenimines, polyether-based polymers, rheology-modifying polymers, dye inhibition polymers and soil release polymers as defined in more detail in paragraphs

[3035] to

[3044] of Reference RF2.Polymers may include, without limitation, "multifunctional alkoxylated polyethylene imines”, "multifunctional alkoxylated diamines” and also terephthalic acid-based polyesters like Clariant's TexCare®, such as TexCare® SRN 170, TexCare® SRN 172, TexCare® SRN 260, TexCare® SRN 260 SG Terra and TexCare® SRA 300 as well as distinct combinations of all of the before mentioned polymers. Also included are graft polymers comprising a polyalkylene oxide-based backbone with grafted side chains of vinyl ester monomer and optionally N-vinylpyrrolidone monomers.In preferred embodiments, the cleaning compositions comprise the enzyme having pectinase activity described herein and at least one surfactant. "Surfactant" (synonymously used herein with "surface active agent”) means an organic chemical that, when added to a liquid, changes the properties of that liquid at an interface. "Surfactants” are anionic, non-ionic, cationic, amphoteric and zwitter-ionic surfactants defined in more detail in paragraphs

[3008] to

[3034] of Reference RF2. In addition, these surfactants are also described in more detail in paragraphs

[0007] to

[0013] of Reference RF1.Anionic surfactants for inventive cleaning compositions include linear alkylbenzenesulfonates (LAS), alkyl sulfates (AS), alkyl alkoxy sulfates (AES), alkyl alkoxy carboxylates, modified alkylbenzene sulfonate (MLAS), methyl ester sulfonate (MES), alkyl sulfosuccinates, alpha-olefin sulfonate (AOS), alkyl polyglycosides (APG) and biosurfactants, such as rhamnolipids and sophorolipids. Non-ionic surfactants for inventive cleaning compositions include alkoxylates, alkoxylated alcohols, alkoxylated fatty acids and alkoxylated (poly-)saccharides. Cationic surfactants for inventive cleaning compositions include surfactants comprising a quaternary ammonium. Amphoteric surfactants for inventive cleaning compositions include amine oxides. Zwitter-ionic surfactants for inventive cleaning compositions include betaines.In preferred embodiments, the cleaning compositions comprise the enzyme having pectinase activity described herein and at least one builder. "Builders” are defined in more detail in paragraphs

[0014] to

[0018] of Reference RF1. These builders include non-phosphate-based builders (NPB) and phosphonates (CoP) described in more detail in paragraphs

[3001] to

[3005] of Reference RF2.Builders may include, without limitation, methylglycinediaceticacid (MGDA), ethylenediaminedisuccinic acid (EDDS), glutamic acid diacetate (GLDA), citric acid and salts thereof.In preferred embodiments, the cleaning compositions comprise the enzyme having pectinase activity described herein and at least one additional enzyme. "(Additional) enzymes” are defined in more detail in paragraphs

[0020] to

[0027] of Reference RF1.These additional enzymes may include hydrolases, such as proteases, amylases, lipases, DNases, cellulases, hemicellulases, phospholipases, esterases, mannanases, xylanases, dispersins, oxidoreductases, cutinases, (additional) pectinases, lactases and peroxidases. In more preferred embodiments, the cleaning composition comprises, in addition to the enzyme having pectinase activity described herein, an enzyme stabilizing system as described herein or in the chapter "Enzyme stabilizing system” of Reference RF1.In preferred embodiments, the cleaning compositions comprise the enzyme having pectinase activity described herein and at least one biocide. "Biocides” are defined in more detail in paragraphs

[0035] and

[0036] of Reference RF1. These biocides also include compounds as defined in more detail in paragraphs

[3006] and

[3007] of Reference RF2.Biocides may include, without limitation, 2-phenoxyethanol and 4,4'-dichoro 2-hydroxydiphenylether.Further adjunct cleaning additives are included and described in more detail in paragraphs

[0005] ,

[0019] ,

[0028] to

[0031] and

[0037] to

[0039] of Reference RF1.Liquid laundry formulations, solid laundry compositions, liquid manual dish wash formulations, automatic dish wash (ADW) gels and automatic dish wash (ADW) solid compositions comprising inventive enzyme(s) are defined in more detail in paragraph

[0044] of Reference RF1.Method of useThe polypeptide having pectinase activity of the present invention or a composition comprising said polypeptide having pectinase activity can be used in various applications such as in cleaning, preferably laundry cleaning, food processing, in the beverage industry, for juice extraction or juice processing, in the brewing industry, in animal feed, for pulp and paper processing, for baking, mining and oil extraction, in textile or leather processing, in water treatment, in ethanol production, for circular economy, in waste treatment, or recycling. The pectinase of the present invention is preferably used in a cleaning composition.The pectinase of the present invention is thus preferably used in any cleaning process such as laundry or hard surface cleaning, preferably for home care or l&l cleaning.In a preferred embodiment, the polypeptide having pectinase activity comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5 is used for the removal of stains comprising one or more pectins or pectate from an object, preferably a textile or a hard surface.Thus, the present invention also refers to the use of a pectinase as described herein for providing a cleaning composition with improved wash performance, preferably on pectinase sensitive stains, such as plant-based stains, comprising stains such as fruit and vegetable stains, stains from gelling or thickening agents, grass stains, and wine stains. Further examples include stains such as ketchup, salad dressings, marmalades, juices, yoghurts, milkshakes.Thus, the present invention therefore also refers to a method for cleaning, preferably laundry or hard surface cleaning, comprising the step of contacting an object, preferably a textile or a hard surface, with a composition comprising a polypeptide having pectinase activity of the present invention, preferably wherein the composition comprises at least one additional adjunct cleaning additive, preferably a surfactant and / or a builder. Preferably, the hard surfaces are selected from floors, furnishing, walls, sanitary ceramics, glass, metallic surfaces including medical devices, cutlery, and dishes. Preferably, the method for laundry cleaning, comprises the step of contacting a textile with a composition comprising a pectinase as described herein, preferably wherein the composition comprises at least one adjunct cleaning additive, preferably a surfactant and / or a builder. Preferably, the method for hard surface cleaning, comprises the step of contacting a medical device, cutlery, or dish with a composition comprising a pectinase as described herein, preferably wherein the composition comprises at least one adjunct cleaning additive, preferably a surfactant and / or a builder. A particular preferred form of hard surface cleaning is dishwashing, preferably manual dish washing (MDW) or automatic dishwashing (ADW), most preferably automatic dishwashing (ADW).The present invention also refers to the use of a polypeptide having pectinase activity of the present invention for providing a cleaning composition with improved pectinase stability and / or for providing a cleaning composition with improved wash performance, preferably on pectinase sensitive stains.Thus, the present invention is also directed to a method of removing stains comprising one or more pectins or pectate, comprising the step of contacting an object comprising a stain comprising one or more pectins or pectate with a composition comprising a polypeptide having pectinase activity comprising an amino acid sequence that is at least 75%, preferably at least 80%, identical to SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5, as described herein.In one embodiment, the polypeptide having pectinase activity of the present invention is used to improve the sustainability profile of a composition or a method and / or is used in circular economy. With respect to the use in circular economy, the polypeptide having pectinase activity of the present invention can be used in waste treatment or recycling.In one embodiment the present invention is directed to a process for waste treatment, preferably for solubilizing waste, comprising the step of contacting waste with a polypeptide having pectinase activity of the present invention and preferably with one or more enzymes selected from the group consisting of protease, lipase, cellulases, glucanase, amylase, esterase and mannanase, under conditions supporting enzymatic solubilizations of the waste. In one embodiment, the waste is municipal solid waste. In one embodiment, the liquified waste can be used as substrate for microbial fermentation.Preferred embodimentsWhile the invention has been illustrated and described in detail in the foregoing description, the description is to be considered illustrative or exemplary and not restrictive. The invention is not limited to the disclosed embodiments. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing a claimed invention, from a study of the disclosure, and the dependent claims. The detailed description is merely exemplary in nature and is not intended to limit application and uses. The following examples further illustrate the present invention without, however, limiting the scope of the invention thereto. Various changes and modifications can be made by those skilled in the art on the basis of the description of the invention, and such changes and modifications are also included in the present invention.Some preferred embodiments of the present invention include:1. A composition comprising a polypeptide having pectinase activity and at least one additional component, wherein the polypeptide having pectinase activity comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5.2. A composition according to embodiment 1, wherein the pectinase comprises an amino acid sequence that is with increasing preference 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: 1.3. A composition according to embodiment 1, wherein the pectinase comprises an amino acid sequence that is with increasing preference 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: 3.4. A composition according to embodiment 1, wherein the pectinase comprises an amino acid sequence that is with increasing preference 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: 5.5. A composition according to embodiment 1, wherein the pectinase comprises or consists of an amino acid sequence that is 100% identical to SEQ ID NO: 1, 3 or 5, preferably SEQ ID NO: 1 or SEQ ID NO: 3, more preferably SEQ ID NO: 1.246. A composition according to embodiment 1, wherein the pectinase comprises or consists of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5 or wherein the amino acid sequence of the polypeptide having pectinase activity comprises or consists of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5 with 1-20 amino acid substitutions, preferably with 1-10 amino acid substitutions or more preferably with 1-5 amino acid substitutions, preferably wherein the amino acid substitutions are conservative amino acid substitutions.7. The composition according to any of embodiments 1 to 6, wherein the composition is a non-complex pectinase formulation, wherein the additional component is selected from the group consisting of solvent, salt, pH regulator, preservative, enzyme stabilizing system, and thickening agent, preferably wherein the additional component is calcium chloride.8. The composition according to embodiment 7, wherein the composition comprises and enzyme stabilizing system, wherein the enzyme stabilizing 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, CaCI2, MgCI2, or NaCI, short chain carboxylic acids or salts thereof, preferably, formic acid, formate, preferably sodium formate, acetic acid, acetate, or lactate.9. The composition according to embodiment 7 or 8, wherein the composition is liquid and comprises the pectinase in an amount in the range of 1% to 40%, 1% to 30%, 1% to 25%, 1% to 12%, or preferably 1-10% all relative to the total weight of the enzyme formulation.10. The composition according to embodiments 1 to 6, wherein the composition is a cleaning composition, preferably a laundry cleaning composition or a dish washing composition, more preferably a laundry cleaning composition.11. A cleaning composition, preferably a laundry cleaning composition or a dish washing composition, more preferably a laundry cleaning composition, comprising a polypeptide having pectinase activity and at least one additional component, wherein the polypeptide having pectinase activity comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5.12. The cleaning composition according to embodiment 11, wherein the polypeptide having pectinase activity comprises an amino acid sequence that is with increasing preference at least 82%, at least 85%, 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: 1, SEQ ID NO: 3, or SEQ ID NO: 5, more preferably at least 82%, at least 85%, 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: 1 or SEQ ID NO: 3, most preferably at least 82%, at least 85%, 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: 1.13. The composition according to embodiment 10 to 12, wherein the composition comprises the pectinase in an amount in the range of 0.01% to 0.99% by weight, preferably 0.01% to 0.3% by weight, all relative to the total weight of the cleaning composition.2200722514. The composition according to embodiment 10 to 13, wherein the at least one additional component is an adjunct cleaning additive selected from the group consisting of surfactants, builders, polymers, bleaching systems, fluorescent whitening agents, suds suppressors, additional enzymes, stabilizers, hydrotropes, rheology modifiers, preservatives, and corrosion inhibitors, preferably selected from the group consisting of surfactants, builders, and polymers, more preferably selected from the group consisting of surfactants and builders.15. The composition according to embodiment 10 to 13, wherein the composition comprises a surfactant and a builder.16. The composition of embodiments 1 to 15, further wherein the composition comprises at least one additional enzyme selected from the group consisting of protease, oxidoreductase, transferase, hydrolase, lyase, isomerase, ligase, aminopeptidase, amylase, asparaginase, carbohydrase, carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, endo-beta 1,3 glucanase, endo-beta 1,4 glucanase, xanthan endoglucanase, esterase, alpha-galactosidase, betagalactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase, glycosyl hydrolase, hyaluronic acid synthase, invertase, laccase, lipase, mannosidase, mannanase, mutanase, oxygenase, peroxidase, phytase, polyphenoloxidase, pullulanase, ribonuclease, transglutaminase, dispersin, xylanase and other pectinases, preferably, the at least one additional enzyme is selected from the group consisting of protease, amylase, cellulase, mannanase, lipase, dispersin, and DNase, more preferably, the at least one additional enzyme is selected from the group consisting of protease, amylase, cellulase, mannanase, and lipase, preferably a protease.17. A method of removing stains comprising one or more pectins or pectate, comprising the step of contacting an object, preferably a textile or a hard surface, comprising a stain comprising one or more pectins or pectate with a composition according to any of embodiments 1-16.18. Use of a polypeptide having pectinase activity comprising an amino acid sequence that is at least 75% identical to SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5 for the removal of stains comprising one or more pectins or pectate from an object, preferably a textile or a hard surface, for food processing, for juice extraction or juice processing, in the brewing industry, in waste treatment or for recycling waste.19. Use of a polypeptide having pectinase activity comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5 for the removal of stains comprising one or more pectins or pectate from an object, preferably a textile or a hard surface.20. Use according to embodiment 18 or 19, wherein the pectinase comprises an amino acid sequence that is with increasing preference 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: 1, SEQ ID NO: 3, or SEQ ID NO: 5.2200722621. An isolated, synthetic, or recombinant polypeptide having pectinase activity comprising an amino acid sequence that is at least 75% identical to SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5.22. A polypeptide according to embodiment 21, wherein the pectinase comprises an amino acid sequence that is with increasing preference 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: 1, SEQ ID NO: 3, or SEQ ID NO: 5.23. A polypeptide according to embodiment 21, wherein the pectinase comprises or consists of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5 or wherein the amino acid sequence of the polypeptide having pectinase activity comprises or consists of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5 with 1-20 amino acid substitutions, preferably with 1-10 amino acid substitutions or more preferably with 1-5 amino acid substitutions, preferably wherein the amino acid substitutions are conservative amino acid substitutions.24. An isolated, synthetic, or recombinant polynucleotide encoding a polypeptide having pectinase activity comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5, wherein the isolated, synthetic, or recombinant polynucleotide has at least 80% sequence identity to SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6.25. An isolated, synthetic, or recombinant polynucleotide encoding the polypeptide of any of embodiments 21 to 23, wherein the isolated, synthetic, or recombinant polynucleotide preferably has at least 80% sequence identity to SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6.26. A polynucleotide according to embodiment 24 or 25 having with increasing preference 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 to SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6, wherein the polynucleotide encodes a polypeptide having pectinase activity.27. A nucleic acid construct comprising the polynucleotide of embodiments 24 to 26.28. An expression vector comprising the polynucleotide of embodiment 24 to 26, or the nucleic acid construct of embodiment 27.29. A host cell comprising the polynucleotide of embodiment 24 to 26, the nucleic acid construct of embodiment 27, or the expression vector of embodiment 28.30. The host cell according to embodiment 29, wherein the host cell is a Bacillus cell, preferably a B.licheniformis cell.31. A method of making a polypeptide having pectinase activity comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5, comprising providing a polynucleotide according to embodiments 24 to 28, transforming the polynucleotide into a host cell, cultivating the host cell to produce the polypeptide having pectinase activity and optionally purifying the polypeptide having pectinase activity.2200722732. A method of making the polypeptide having pectinase activity of any of embodiments 21 to 23, comprising providing a polynucleotide encoding the polypeptide having pectinase activity, transforming the polynucleotide into a host cell, cultivating the host cell to produce the polypeptide having pectinase activity and optionally purifying the polypeptide having pectinase activity.33. A method according to embodiments 31 or 32, wherein the pectinase is secreted from the cell into the fermentation broth.34. A method according to embodiment 33, wherein the pectinase is secreted by means of a signal peptide added to a terminus, preferably the amino terminus, of the amino acid sequence of the pectinase.35. A method according to embodiment 34, wherein the pectinase and the signal peptide are recombinant to each other.36. A method according to embodiment 35, wherein the signal peptide is selected from the group consisting of pelC, wapA, yvcE, orydjM.37. A method according to embodiment 36, wherein the signal peptide is selected from a group consisting of (a) a polynucleotide having with increasing preference 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 to SEQ ID NO: 8 or SEQ ID NO: 10, wherein the polynucleotide encodes a polypeptide having the function of a signal peptide; and(b) a polynucleotide encoding a polypeptide having with increasing preference 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 to SEQ ID NO: 7 or SEQ ID NO: 9, wherein the polypeptide has the function of a signal peptide.38. A method according to embodiment 37, wherein the pectinase that comprises an amino acid sequence that is with increasing preference 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: 1 is expressed using a signal peptide having with increasing preference 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 to SEQ ID NO: 7.39. A method according to embodiment 37, wherein the pectinase that comprises an amino acid sequence that is with increasing preference 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: 3 is expressed using a signal peptide having with increasing preference 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 to SEQ ID NO: 9.22007228ExamplesExample 1: Generation of B. licheniformis cells expressing potential pectinases and small-scale expression of potential pectinasesPlasmid DNA harboring the gene of potential pectinases (protein sequence of potential pectinases given in SEQ ID NO: 1, 3, 5, 11 or 13, nucleotide sequences used given in SEQ ID NOs: 2, 4, 6, 12 or 14) fused to the pelC signal peptide (nucleotide sequence of signal peptide: SEQ ID NO: 8, protein sequence of signal peptide given in SEQ ID NO: 7) was used to transform chemically competent Bacillus licheniformis by electroporation. The transformed cells were incubated for 2 h at 37 °C with shaking, and then plated on an appropriate selection medium. The plate was incubated for 3 days at 26 °C. Colonies of Bacillus licheniformis harboring the gene of potential pectinases were picked and used to inoculate 0.6 ml of growth medium in a 96 well plate. The samples were incubated 16 h at 30 °C or 37 °C with shaking. This mature seed culture was diluted into an appropriate number of 0.6 ml aliquots in a 96 well plate, and the samples were incubated 48 h at 30 °C or 37 °C with shaking. The samples were clarified by centrifugation and the supernatant was harvested, aliquoted, and stored at -20 °C. Concentrations of the potential pectinases in the supernatants were determined by LabChip (see Table 1).Table 1: Expression levels determined using LabChipAs shown in table 1, the potential pectinases all showed good expression levels leading to a protein concentration of 0.19 to 0.33 mg / ml in the harvested supernatant.Example 2: Wash performance in Launder-o-meter (LOM)Wash experiments, using the enzymes expressed under Example 1, were conducted for 40 minutes at 30 °C in a LOM using Persil Non Bio detergent as described in Table 2.Table 2: Washing conditions LOM essay22007229<>After the LOM run, the swatches were rinsed three times and dried flat overnight. The images were scanned and the ARGB values (before vs after wash) were processed digitally. Wash performance is conveyed as the AARGB between the detergent with enzyme and the detergent alone control swatches (Table 3).Table 3: Wash performance in the LOM (AARGB values)Pectinase sequences having the SEQ ID NOs 1, 3 or 5 showed efficient wash performance on the tested pectinase sensitive stains. Therefore, these sequences were further evaluated.Example 3: Wash performance in terg-o-tometer (TOM)Wash performance of the pectinases having the sequences of SEQ ID NOs 1, 3, or 5 was further evaluated using a TOM wash assay conducted as set out in Table 4.22007230Table 4: Washing conditions TOM assay>After the TOM run, the swatches were rinsed three times and dried flat overnight. The images were scanned and the ARGB values (before vs after wash) were processed digitally. Wash performance is conveyed as the AARGB between the detergent with enzyme and the detergent alone control swatches (Table 5).Table 5: Average delta RGB values in TOM assayAll three sequences showed comparable wash performance in the TOM assay.22007231Example 4: Expression scopingEnzymes having SEQ ID NO: 1 and 3 were expressed using different signal peptides. Combinations listed in Table 6 were expressed in duplicate DASGIP scale fermentation vessels for 66 to 68 hours.Table 6: Expression levels of enzymes having SEQ ID NOs 1 or 3 using different signal peptides.The pectinase having SEQ ID NO: 1 shows improved expression when combined with the signal peptide of SEQ ID NO: 7 in comparison to the expression using the signal peptide of SEQ ID NO: 9. The pectinase having SEQ ID NO: 3 shows improved expression when combined with the signal peptide of SEQ ID NO: 9 in comparison to the expression using the signal peptide of SEQ ID NO: 7.Example 5: Wash performance in launder-o-meter (LOM)Enzymes having SEQ ID NO: 1 and 3 were further tested in a different market laundry cleaning composition (method detergent). A LOM wash assay was conducted in 4 g / l detergent as set out in Table 7.Table 7: Washing conditions LOM essay220072321> Producer: Center for Testmaterials BV, NL-3130 AC VlaardingenHeat treated method laundry detergent (SCJ, including a pectate lyase, amylase, mannanase, and a protease) was used as a control. Enzymes having SEQ ID NO: 1 or 3 were tested for wash performance. After the LOM run, the swatches were rinsed three times and dried flat overnight. The images were scanned and the AE were processed digitally. Wash performance is conveyed as the AAE between the detergent with enzyme having SEQ ID NO: 1 or 3 and the heat-treated detergent control swatches (Tables 7 and 8).Table 8: Results LOM essay using 4 g / l detergentTable 9: Results LOM essay using 2 g / l detergentEnzymes having SEQ ID NO: 1 and 3 showed wash performance on a variety of pectin containing stains.

Claims

22007233CLAIMS1. A cleaning composition, preferably a laundry cleaning composition or a dish washing composition, comprising a polypeptide having pectinase activity and at least one additional component, wherein the polypeptide having pectinase activity comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5.

2. The cleaning composition according to claim 1, wherein the polypeptide having pectinase activity comprises an amino acid sequence that is with increasing preference at least 82%, at least 85%, 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: 1, SEQ ID NO: 3, or SEQ ID NO: 5, more preferably at least 82%, at least 85%, 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: 1 or SEQ ID NO: 3, most preferably at least 82%, at least 85%, 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: 1.

3. The cleaning composition according to claim 1 or 2, wherein the at least one additional component is selected from the group consisting of surfactants, builders, polymers, bleaching systems, fluorescent whitening agents, suds suppressors, additional enzymes, stabilizers, hydrotropes, rheology modifiers, preservatives, and corrosion inhibitors, preferably selected from the group consisting of surfactants, builders, and polymers, more preferably selected from the group consisting of surfactants and builders.

4. The cleaning composition according to any preceding claim, further wherein the composition comprises at least one additional enzyme selected from the group consisting of protease, oxidoreductase, transferase, hydrolase, lyase, isomerase, ligase, aminopeptidase, amylase, asparaginase, carbohydrase, carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, endo-beta 1,3 glucanase, endo-beta 1,4 glucanase, xanthan endoglucanase, esterase, alpha-galactosidase, betagalactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase, glycosyl hydrolase, hyaluronic acid synthase, invertase, laccase, lipase, mannosidase, mannanase, mutanase, oxygenase, peroxidase, phytase, polyphenoloxidase, pullulanase, ribonuclease, transglutaminase, dispersin, xylanase and other pectinases, preferably, the at least one additional enzyme is selected from the group consisting of protease, amylase, cellulase, mannanase, lipase, dispersin, and DNase, more preferably, the at least one additional enzyme is selected from the group consisting of protease, amylase, cellulase, mannanase, and lipase, most preferably, a protease.

5. A method of removing stains comprising one or more pectins or pectate, comprising the step of contacting an object comprising a stain comprising one or more pectins or pectate with a composition according to any of claims 1-4.220072346. Use of a polypeptide having pectinase activity comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 1 , SEQ ID NO: 3, or SEQ ID NO: 5 for the removal of stains comprising one or more pectins or pectate from an object, preferably a textile or a hard surface.

7. An isolated, synthetic, or recombinant polynucleotide encoding a polypeptide having pectinase activity comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5, wherein the isolated, synthetic, or recombinant polynucleotide has at least 80% sequence identity to SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6.

8. A nucleic acid construct comprising the polynucleotide of claim 7.

9. An expression vector comprising the polynucleotide of claim 7 or the nucleic acid construct of claim 8.

10. A host cell comprising the polynucleotide of claim 7, the nucleic acid construct of claim 8, or the expression vector of claim 9.

11. The host cell according to claim 10, wherein the host cell is a Bacillus cell.

12. A method of making a polypeptide having pectinase activity comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5, comprising providing a polynucleotide according to claim 7, transforming the polynucleotide into a host cell, cultivating the host cell to produce the polypeptide having pectinase activity and optionally purifying the polypeptide having pectinase activity.