Polypeptides having protease activity for use in detergent compositions

Abstract

In the present invention new protease enzymes are provided. More specifically, genetically engineered protease enzymes, compositions comprising the enzymes, and methods of making and using the enzymes or compositions comprising the enzymes are provided.

Description

[0001] Polypeptides having protease activity for use in detergent compositions

[0002] Field of the invention

[0003] In the present invention new protease enzymes are provided. More specifically, genetically engineered protease enzymes, compositions comprising the enzymes, and methods of making and using the enzymes or compositions comprising the enzymes are provided.

[0004] Background of the invention

[0005] Enzymes are increasingly used in various applications as sustainable alternatives to petrochemistry. Enzymes 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 washing formulations to improve cleaning efficiency and / or reduce energy consumption in a washing step.

[0006] Proteases are enzymes capable of hydrolyzing proteins. Thus, proteases have been employed in the removal of protein stains and have been added to detergent compositions for this purpose. In detergent applications the proteases shall be stable at elevated temperatures and / or within the denaturing conditions of the detergents and the wash liquor.

[0007] WO 2016 / 096711 and WO 2016 / 096714 describe a subtilase variant having improved stability and / or improved wash performance in liquid detergents compared to the parent subtilase and detergents containing the variant. WO 2016 / 001450 and WO 2020 / 002255 discloses subtilase variants with increased stability. WO 2010 / 056640 also describes subtilisin variants. US 6,376,450 discloses multiple-substituted protease variants which provide improved and enhanced cleaning ability. US 2020 / 172890 A1 discloses performance-enhanced and storage-stable protease variants. WO 99 / 20770 A2, WO 03 / 062381 A2, WO 201 1 / 140364 A1 , WO 2011 / 072117 A1 , WO 2011 / 072099 A2, WO 2012 / 151534 A1 , WO 2015 / 144932 A1 , WO 2017 / 207762 A1 , WO 2017 / 192692A1 , WO 2018 / 069158 A1 and WO 2022 / 225696 A2 also disclose protease variants.

[0008] Nevertheless, there is still a need for new protease enzymes, which are stable in harsh detergent compositions and show a good washing performance. Brief summary of the invention

[0009] The present invention is directed to a variant polypeptide having protease activity or a fragment of said polypeptide having protease activity, wherein:

[0010] (i) the polypeptide thereof has an amino acid sequence which is at least 79%, but less than 100%, identical to the amino acid sequence as set forth in SEQ ID NO: NO: 1 and

[0011] (ii) the polypeptide or fragment thereof comprises

[0012] (a) an amino acid substitution at two or more amino acid residues selected from the group consisting of 9, 18, 21 , 38, 59, 228, and 271 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0013] (b) an amino acid substitution at two or more amino acid residues selected from the group consisting of 25, 43, 78, 109, 144, 182, 183, 194, 204, 210, 212, 218, 248, 259, 260 and 261 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, wherein the fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide.

[0014] The present invention further relates to a polynucleotide encoding said variant polypeptide and a composition comprising said variant polypeptide.

[0015] Detailed description of the invention

[0016] The 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.

[0017] Although the present invention will be described with respect to particular embodiments, this description is not to be construed in a limiting sense.

[0018] Definitions

[0019] Unless otherwise noted, the terms used herein are to be understood according to conventional usage by those of ordinary skill in the relevant art.

[0020] 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.

[0021] As 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 %.

[0022] 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.

[0023] Throughout this application, various publications are referenced. The disclosure of all of 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.

[0024] 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.

[0025] “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”. Substitutions can 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”.

[0026] Variants comprising multiple substitutions are separated by “+”, e.g., “Arg170Tyr-t-Gly195Glu”, “R170Y- 9195E” 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., “Arg170[Tyr, Gly]” or “Arg170{Tyr,Gly}” or in short “R170 [Y,G]” or“R170 {Y,G}”.

[0027] The numbering of the amino acid residues of the proteases described herein is as commonly used for proteases in the field (cf. P. N. Bryan, Biochimica et Biophysica Acta 1543 (2000), 203-222, cf. p. 204, left col., 3rdpara.) according to the numbering of the BPN’ subtilisin protease from Bacillus amyloliquefaciens the sequence of which is shown in SEQ ID NO: 2 (i.e. , according to the numbering of SEQ ID NO: 2 or according to “BPN’ numbering”).

[0028] In an alternative way, one can describe the amino acid positions with reference to the numbering of SEQ ID NO: 1 or SEQ ID NO: 3, i.e., according to the numbering of SEQ ID NO: 1 or according to the numbering of SEQ I D NO: 3. Table 1 below shows the amino acid numbering according to SEQ ID NO: 2 and the numbering of the corresponding amino acids in the sequences according to SEQ ID NO: 1 or 3:

[0029] Table 1

[0030] The 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).

[0031] 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 orforeign 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.

[0032] 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 man using gene technology. With regard to a polynucleotide “recombinant” includes all constructs produced by using gene technology / recombinant DNA techniques in which either

[0033] (a) the sequence of the polynucleotide or a part thereof, or

[0034] (b) one or more genetic control sequences, which are operably linked to the polynucleotide, including but not limited to a promoter, or

[0035] (c) both a) and b) are not located in their wild-type genetic environment or have been modified by man.

[0036] A "synthetic" compound is obtained by in vitro chemical and / or enzymatic synthesis.

[0037] 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 a 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.

[0038] 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 Software Suite (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 / length of the alignment region which is showing the respective sequence of the present invention over its complete length) *100.

[0039] A special aspect concerning amino acid substitutions are conservative mutations which often appear to have a minimal effect on protein folding resulting in substantially maintained enzyme properties of the respective enzyme variant compared to the enzyme properties of the parent enzyme. Conservative mutations are those where one amino acid is exchanged with a similar amino acid. Such an exchange most probably does not change enzyme properties.

[0040] Herein the following conservative exchanges are considered:

[0041] Amino acid A is similar to amino acids S

[0042] Amino acid D is similar to amino acids E; N

[0043] Amino acid E is similar to amino acids D; K; Q Amino acid F is similar to amino acids W; Y Amino acid H is similar to amino acids N; Y Amino acid I is similar to amino acids L; M; V Amino acid K is similar to amino acids E; Q; R Amino acid L is similar to amino acids I; M; V Amino acid M is similar to amino acids I; L; V Amino acid N is similar to amino acids D; H; S Amino acid Q is similar to amino acids E; K; R Amino acid R is similar to amino acids K; Q Amino acid S is similar to amino acids A; N; T Amino acid T is similar to amino acids S Amino acid V is similar to amino acids I; L; M Amino acid W is similar to amino acids F; Y Amino acid Y is similar to amino acids F; H; W Conservative amino acid substitutions may occur over the full length of the sequence of a polypeptide sequence of a functional protein such as an enzyme. Preferably, such mutations are not pertaining the functional domains of an enzyme, more preferably conservative mutations are not pertaining the catalytic centers of an enzyme.

[0044] A "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 which essentially retain protease 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 259 consecutive amino acids of the full-length variant 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 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%, 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 82% identical to the amino acid sequence according to SEQ ID NO: 1 and which has the same length as the amino acid sequence according to SEQ ID NO: 1 . In one embodiment, the polypeptide of the present invention does not comprise any internal deletions compared to the amino acid sequence according to SEQ I D NO: 1. 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 “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.

[0045] 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.

[0046] 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 (cf. , for example, Sam- brook, 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.

[0047] 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.

[0048] 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 to be 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”. “Protein formulation” (or “enzyme preparation” or “polypeptide formulation”), e.g., “protease 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 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 detergent 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 detergent 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 detergent composition, wherein in the detergent compositions other compounds are present that serve the cleaning purpose of the detergent composition, e.g., surfactants and / or chelating agents.

[0049] “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, amylases activity involves hydrolysis of glycosidic linkages in polysaccharides, etc. According to the invention, the enzymatic activity is proteolytic activity which can be determined by using Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (Suc-AAPF-pNA; see e.g. DelMar et al. (1979), Analytical Biochem 99, 316-320) or Suc-AAPF-AMC (7-amido-4-methylcoumarin) as substrate. pNA or AMC is cleaved from the substrate molecule by proteolytic cleavage, resulting in release of yellow color of free pNA or change of fluorescence properties which can be quantified either by measuring emission at 460 nm after excitation at 380 nm. Other methods using for example casein as a proteinaceous substrate are known to those skilled in the art. The term “enzyme stability” according to the current invention relates to the retention of enzymatic activity as a function of time during storage or operation. Retention of enzymatic activity as afunction of time during storage is called “storage stability” and is preferred within the context of the invention.

[0050] To determine and quantify changes in catalytic activity of enzymes stored or used under certain conditions over time, the “enzymatic activity” is measured under defined conditions at time zero (100%) and at a certain point in time later (x%). By comparison of the values measured, the residual activity of the enzyme can be determined in its extent. The extent of the residual activity of the enzyme determines an enzyme’s stability or non-stability. “Enzyme inhibitors” as used herein are compounds that slow down or halt enzymatic activity. Enzyme inhibitors frequently also stabilize the enzyme in its three-dimensional structure. Hence, enzyme inhibitors usually also act as “enzyme stabilizers”.

[0051] “pH stability” refers to the ability of an enzyme to exert enzymatic activity after exposure to a certain pH value.

[0052] The term “detergent stability” (also called herein “residual activity in a detergent” or “storage stability in a detergent composition”) refers to the ability of an enzyme to exert catalytic activity or wash performance after storage in a detergent composition, preferably, at a temperature of 37 °C, 45 °C, or 50 °C for up to 14 days in a detergent composition (preferably, in Model B detergent as described herein). Most preferably, “detergent stability” is determined by measuring residual activity of the protease after storage at a temperature of 45 °C for 18 hours and / or 3 days in a detergent composition (preferably, in Model B detergent as described herein).

[0053] 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 detergent composition, i.e. the cleaning performance added to the detergent 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.

[0054] As used herein, the term "specific performance" refers to the cleaning and removal of specific stains or soils per unit of active enzyme. I n some embodiments, the specific performance is determined using stains or soils such as egg, egg yolk, milk, grass, minced meat blood, chocolate sauce, baby food, sebum, etc.

[0055] “Detergent composition” or “detergent” means compositions designated for cleaning soiled material. Detergent compositions are complex formulations as further defined herein. Detergent compositions according to the invention include detergent compositions for different applications such as laundry and hard surface cleaning. Detergent compositions according to the invention also include biodegradable detergent compositions. The term “detergent component” is defined herein to mean a type of chemical, which can be used in detergent compositions. A typical detergent component is a 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. According to its ionic charge, a surfactant is called non-ionic, anionic, cationic, or amphoteric. 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). Detergent compositions typically have a protease concentration of 0.002 to 10 mg / g active enzyme.

[0056] 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 of the present invention. The laundering process 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.

[0057] 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” (a textile made by weaving, knitting or felting fibers) or “garment” (any article of clothing made of textile) as used herein, are intended to include the broader term textile as well.

[0058] 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.

[0059] 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 of the present invention. 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, artho- scopes and related equipment. A particular form of hard surface cleaning is dishwashing, particularly automatic dishwashing (ADW).

[0060] 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. Cleaning performance is evaluated under relevant cleaning conditions. The term "relevant cleaning conditions" herein refers to the conditions, particularly cleaning temperature, time, cleaning mechanics, suds concentration, type of detergent and water hardness, actually used in laundry machines, automatic dish washers or in manual cleaning processes.

[0061] The term “medical device cleaning” refers to the cleaning step in reprocessing reusable medical devices. Medical device cleaning methods can be divided into two categories, manual and mechanical / automated cleaning methods. Manual cleaning is used when mechanical units are not available or medical devices to be cleaned are too fragile or difficult to clean with a mechanical unit. Mechanical / automated cleaning methods remove soiling and microorganisms through an automated cleaning and rinsing process, this includes ultrasonic cleaning and washing.

[0062] 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.

[0063] A “sequestering builder” as used herein is different from a precipitating builder in that no significant amount of precipitate is formed when the builder is used in an amount sufficient to combine with all of the calcium ions in an aqueous solution with 7 °dH hardness (German hardness) initially at neutral pH. A “strong builder” is classified as high efficiency chelators that can bind the divalent cations such as Ca2+ strongly with a logarithmic stability constant (Log Kca ) of the cation / chelator complex of above 4, particular above 5, above 6 or above 7. The stability constants are determined at an ionic strength of 0.1 M and at a temperature of 25°C. A ..strong sequestering builder” combines both of the above- mentioned properties. Strong sequestering builders include, but are not limited to, Ethylenediaminetetraacetic acid (EDTA), Ethylene diamine tetra(methylene phosphonic acid (EDTMP), Nitrilo trimethylene phosphonic acid (NTMP), Diethylenetriamine Penta(Methylene Phosphonic acid) (DTPMP), methylglycinediacetic acid (MGDA), Nitrilotriacetic acid (NTA), 1-Hydroxy Ethylidene-1, 1 -Diphosphonic acid (HEDP), sodium tripolyphosphate (STPP), iminodisuccinic acid (I DS), N,N-diacetic acid tetra sodium salt (GLDA), pyrophosphate and ethylenediaminedisuccinic acid (EDDS), preferably MGDA and / or EDDS.

[0064] An “antimicrobial agent” is a chemical compound that kills microorganisms or inhibits their growth or reproduction. Microorganisms can be bacteria, yeasts or molds. A “preservative” is an antimicrobial agent which may be added to aqueous products and compositions to maintain the original performance, characteristics and integrity of the products and compositions by killing contaminating microorganisms or inhibiting their growth.

[0065] 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 description

[0066] In the present invention new protease enzymes are provided. More specifically, variants of a parent protease, methods of making the variant proteases, compositions comprising the protease variants, and methods of using the variant proteases or compositions comprising the variant proteases are provided.

[0067] Protease Variant

[0068] The present invention is directed to a variant polypeptide having protease activity or a fragment of said polypeptide having protease activity, wherein:

[0069] (i) the polypeptide has an amino acid sequence which is at least 79%, but less than 100%, identical to the amino acid sequence as set forth in SEQ ID NO: NO: 1 and

[0070] (ii) the polypeptide or fragment thereof comprises

[0071] (a) an amino acid substitution at two or more, three or more, four or more, five or more, six or all amino acid residues selected from the group consisting of 9, 18, 21 , 38, 59, 228, and 271 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0072] (b) an amino acid substitution at two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or all amino acid residues selected from the group consisting of 25, 43, 78, 109, 144, 182, 183, 194, 204, 210, 212, 218, 248, 259, 260 and 261 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, wherein the fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide.

[0073] The present invention is directed to a variant polypeptide having protease activity, wherein:

[0074] (I) the polypeptide has an amino acid sequence which is at least 79%, but less than 100%, identical to the amino acid sequence as set forth in SEQ ID NO: NO: 1 and

[0075] (ii) the polypeptide thereof comprises

[0076] (a) an amino acid substitution at two or more, three or more, four or more, five or more, six or all amino acid residues selected from the group consisting of 9, 18, 21 , 38, 59, 228, and 271 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0077] (b) an amino acid substitution at two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or all amino acid residues selected from the group consisting of 25, 43, 78, 109, 144, 182, 183, 194, 204, 210, 212, 218, 248, 259, 260 and 261 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2. The present invention is directed to a variant polypeptide having protease activity or a fragment of said polypeptide having protease activity, wherein:

[0078] (i) the polypeptide has an amino acid sequence which is at least 79%, but less than 100%, identical to the amino acid sequence as set forth in SEQ ID NO: NO: 1 and

[0079] (ii) the polypeptide or fragment thereof comprises

[0080] (a) two or more, three or more, four or more, five or more, six or all amino acid substitutions selected from the group consisting of X9A / Q / E / C / D, X18Q / E / D, X21 l / V / F / W / Y, X38R / K / H / W, X59K / E, X228S and X271 D compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ I D NO: 2 and

[0081] (b) two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or all amino acid substitutions selected from the group consisting of X25D, X43K / R / C / H / D / L / S / W / A / M / Y / Q / F / I, X78N / D / R / W / F / H / K / E / L / Y / M / C / Q, X109K / A, X144N / R, X182K / R / E, X183D / E / C / Q / A / M, X194E / D / C, X204D / E / C / G, X210I / V,

[0082] X212G / M / R / K / P / A / F / H / W / N / Y / D, X218S / T / D, X248Q / R, X259D / E, X260D / E / K and X261 L / M / F / E / W / Y / C compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, wherein the fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide.

[0083] The present invention is directed to a variant polypeptide having protease activity, wherein:

[0084] (I) the polypeptide has an amino acid sequence which is at least 79%, but less than 100%, identical to the amino acid sequence as set forth in SEQ ID NO: NO: 1 and

[0085] (ii) the polypeptide or thereof comprises

[0086] (a) two or more, three or more, four or more, five or more, six or all amino acid substitutions selected from the group consisting of X9A / Q / E / C / D, X18Q / E / D, X21 l / V / F / W / Y, X38R / K / H / W, X59K / E, X228S and X271 D compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ I D NO: 2 and

[0087] (b) two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or all amino acid substitutions selected from the group consisting of X25D, X43K / R / C / H / D / L / S / W / A / M / Y / Q / F / I, X78N / D / R / W / F / H / K / E / L / Y / M / C / Q, X109K / A, X144N / R, X182K / R / E, X183D / E / C / Q / A / M, X194E / D / C, X204D / E / C / G, X210I / V, X212G / M / R / K / P / A / F / H / W / N / Y / D, X218S / T / D, X248Q / R, X259D / E, X260D / E / K and X261 L / M / F / E / W / Y / C compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2.

[0088] The present invention is directed to a variant polypeptide having protease activity or a fragment of said polypeptide having protease activity, wherein:

[0089] (I) the polypeptide has an amino acid sequence which is at least 79%, but less than 100%, identical to the amino acid sequence as set forth in SEQ ID NO: NO: 1 and

[0090] (ii) the polypeptide or fragment thereof comprises (a) two or more, three or more, four or more, five or more, six or all amino acid substitutions selected from the group consisting of X9A / Q, X18Q / E / D, X21 V / l, X38R / K / H / W, X59K / E, X228S and X271 D compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0091] (b) two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or all amino acid substitutions selected from the group consisting of X25D, X43K / R, X78N / D, X109K / A, X144N / R, X182K / R / E, X183D / E, X194E / D, X204D / E, X210I, X212P / A / G / R / K / F / Y / M, X218S / T, X248Q / R, X259D, X260D / E and X261 F / W / Y / L compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ I D NO: 2, wherein the fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide.

[0092] The present invention is directed to a variant polypeptide having protease activity, wherein:

[0093] (I) the polypeptide has an amino acid sequence which is at least 79%, but less than 100%, identical to the amino acid sequence as set forth in SEQ ID NO: NO: 1 and

[0094] (ii) the polypeptide or thereof comprises

[0095] (a) two or more, three or more, four or more, five or more, six or all amino acid substitutions selected from the group consisting of X9A / Q.X18Q / E / D, X21V / I, X38R / K / H / W, X59K / E, X228S and X271 D compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0096] (b) two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or all amino acid substitutions selected from the group consisting of X25D, X43K / R, X78N / D, X109K / A, X144N / R, X182K / R / E, X183D / E, X194E / D, X204D / E, X210I, X212P / A / G / R / K / F / Y / M, X218S / T, X248Q / R, X259D, X260D / E and X261 F / W / Y / L compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ I D NO: 2.

[0097] In a preferred embodiment, the polypeptide of the present invention having protease activity or the fragment thereof comprises amino acid residue D or E at position 101 , preferably E at position 101 , referring to the numbering of SEQ ID NO: 2.

[0098] The present invention is directed to a variant polypeptide having protease activity or a fragment of said polypeptide having protease activity, wherein:

[0099] (I) the polypeptide has an amino acid sequence which is at least 79%, but less than 100%, identical to the amino acid sequence as set forth in SEQ ID NO: NO: 1 and

[0100] (ii) the polypeptide or fragment thereof comprises

[0101] (a) an amino acid substitution at two or more, three or more, four or more, five or more, six or all amino acid residues selected from the group consisting of 9, 18, 21 , 38, 59, 228, and 271 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and (b) an amino acid substitution at two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or all amino acid residues selected from the group consisting of 25, 43, 78, 109, 144, 182, 183, 194, 204, 210, 212, 218, 248, 259, 260 and 261 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, wherein the fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide and wherein the polypeptide comprises amino acid residue D or E at position 101, preferably E at position 101 , referring to the numbering of SEQ ID NO: 2.

[0102] The present invention is directed to a variant polypeptide having protease activity, wherein:

[0103] (I) the polypeptide has an amino acid sequence which is at least 79%, but less than 100%, identical to the amino acid sequence as set forth in SEQ ID NO: NO: 1 and

[0104] (ii) the polypeptide thereof comprises

[0105] (a) an amino acid substitution at two or more, three or more, four or more, five or more, six or all amino acid residues selected from the group consisting of 9, 18, 21 , 38, 59, 228, and 271 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0106] (b) an amino acid substitution at two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or all amino acid residues selected from the group consisting of 25, 43, 78, 109, 144, 182, 183, 194, 204, 210, 212, 218, 248, 259, 260 and 261 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, wherein the polypeptide comprises amino acid residue D or E at position 101 , preferably E at position 101 , referring to the numbering of SEQ ID NO: 2.

[0107] The present invention is directed to a variant polypeptide having protease activity or a fragment of said polypeptide having protease activity, wherein:

[0108] (I) the polypeptide has an amino acid sequence which is at least 79%, but less than 100%, identical to the amino acid sequence as set forth in SEQ ID NO: NO: 1 and

[0109] (ii) the polypeptide or fragment thereof comprises

[0110] (a) two or more, three or more, four or more, five or more, six or all amino acid substitutions selected from the group consisting of X9A / Q / E / C / D, X18Q / E / D, X21 l / V / F / W / Y, X38R / K / H / W, X59K / E, X228S and X271 D compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ I D NO: 2 and

[0111] (b) two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or all amino acid substitutions selected from the group consisting of X25D, X43K / R / C / H / D / L7S / W / A / M / Y / Q / F / I, X78N / D / R / W / F / H / K / E / L / Y / M / C / Q, X109K / A, X144N / R, X182K / R / E, X183D / E / C / Q / A / M, X194E / D / C, X204D / E / C / G, X210I / V, X212G / M / R / K / P / A / F / H / W / N / Y / D, X218S / T / D, X248Q / R, X259D / E, X260D / E / K and X261 L / M / F / E / W / Y / C compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, wherein the fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide and wherein the polypeptide comprises amino acid residue D or E at position 101, preferably E at position 101 , referring to the numbering of SEQ ID NO: 2.

[0112] The present invention is directed to a variant polypeptide having protease activity, wherein:

[0113] (I) the polypeptide has an amino acid sequence which is at least 79%, but less than 100%, identical to the amino acid sequence as set forth in SEQ ID NO: NO: 1 and

[0114] (ii) the polypeptide or thereof comprises

[0115] (a) two or more, three or more, four or more, five or more, six or all amino acid substitutions selected from the group consisting of X9A / Q / E / C / D, X18Q / E / D, X21 l / V / F / W / Y, X38R / K / H / W, X59K / E, X228S and X271 D compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ I D NO: 2 and

[0116] (b) two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or all amino acid substitutions selected from the group consisting of X25D, X43K / R / C / H / D / L7S / W / A / M / Y / Q / F / I, X78N / D / R / W / F / H / K / E / L / Y / M / C / Q, X109K / A, X144N / R, X182K / R / E, X183D / E / C / Q / A / M, X194E / D / C, X204D / E / C / G, X210I / V,

[0117] X212G / M / R / K / P / A / F / H / W / N / Y / D, X218S / T / D, X248Q / R, X259D / E, X260D / E / K and X261 L / M / F / E / W / Y / C compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, wherein the polypeptide comprises amino acid residue D or E at position 101 , preferably E at position 101 , referring to the numbering of SEQ ID NO: 2.

[0118] The present invention is directed to a variant polypeptide having protease activity or a fragment of said polypeptide having protease activity, wherein:

[0119] (I) the polypeptide has an amino acid sequence which is at least 79%, but less than 100%, identical to the amino acid sequence as set forth in SEQ ID NO: NO: 1 and

[0120] (ii) the polypeptide or fragment thereof comprises

[0121] (a) two or more, three or more, four or more, five or more, six or all amino acid substitutions selected from the group consisting of X9A / Q, X18Q / E / D, X21 V / l, X38R / K / H / W, X59K / E, X228S and X271 D compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0122] (b) two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or all amino acid substitutions selected from the group consisting of X25D, X43K / R, X78N / D, X109K / A, X144N / R, X182K / R / E, X183D / E, X194E / D, X204D / E, X210I, X212P / A / G / R / K / F / Y / M, X218S / T, X248Q / R, X259D, X260D / E and X261 F / W / Y / L compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ I D NO: 2, wherein the fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide and wherein the polypeptide comprises amino acid residue D or E at position 101, preferably E at position 101 , referring to the numbering of SEQ ID NO: 2.

[0123] The present invention is directed to a variant polypeptide having protease activity, wherein:

[0124] (I) the polypeptide has an amino acid sequence which is at least 79%, but less than 100%, identical to the amino acid sequence as set forth in SEQ ID NO: NO: 1 and

[0125] (ii) the polypeptide or thereof comprises

[0126] (a) two or more, three or more, four or more, five or more, six or all amino acid substitutions selected from the group consisting of X9A / Q, X18Q / E / D, X21 V / l, X38R / K / H / W, X59K / E, X228S and X271 D compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0127] (b) two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or all amino acid substitutions selected from the group consisting of X25D, X43K / R, X78N / D, X109K / A, X144N / R, X182K / R / E, X183D / E, X194E / D, X204D / E, X210I, X212P / A / G / R / K / F / Y / M, X218S / T, X248Q / R, X259D, X260D / E and X261 F / W / Y / L compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ I D NO: 2, wherein the polypeptide comprises amino acid residue D or E at position 101, preferably E at position 101 , referring to the numbering of SEQ ID NO: 2.

[0128] The present invention is directed to a variant polypeptide having protease activity or a fragment of said polypeptide having protease activity, wherein:

[0129] (I) the polypeptide has an amino acid sequence which is at least 79%, but less than 97.5%, identical to the amino acid sequence as set forth in SEQ ID NO: 1 and

[0130] (ii) the polypeptide or fragment thereof comprises

[0131] (a) an amino acid substitution at two or more, three or more, four or more, five or more, six or all amino acid residues selected from the group consisting of 9, 18, 21 , 38, 59, 228, and 271 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0132] (b) an amino acid substitution at two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or all amino acid residues selected from the group consisting of 25, 43, 78, 109, 144, 182, 183, 194, 204, 210, 212, 218, 248, 259, 260 and 261 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, wherein the fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide.

[0133] The present invention is directed to a variant polypeptide having protease activity, wherein: (iii) the polypeptide has an amino acid sequence which is at least 79%, but less than 97.5%, identical to the amino acid sequence as set forth in SEQ ID NO: 1 and

[0134] (iv) the polypeptide or fragment thereof comprises

[0135] (a) an amino acid substitution at two or more, three or more, four or more, five or more, six or all amino acid residues selected from the group consisting of 9, 18, 21 , 38, 59, 228, and 271 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0136] (b) an amino acid substitution at two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or all amino acid residues selected from the group consisting of 25, 43, 78, 109, 144, 182, 183, 194, 204, 210, 212, 218, 248, 259, 260 and 261 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2.

[0137] The present invention is directed to a variant polypeptide having protease activity or a fragment of said polypeptide having protease activity, wherein:

[0138] (I) the polypeptide has an amino acid sequence which is at least 79%, but less than 97.5%, identical to the amino acid sequence as set forth in SEQ ID NO: 1 and

[0139] (ii) the polypeptide or fragment thereof comprises

[0140] (a) two or more, three or more, four or more, five or more, six or all amino acid substitutions selected from the group consisting of X9A / Q / E / C / D, X18Q / E / D, X21 l / V / F / W / Y, X38R / K / H / W, X59K / E, X228S and X271 D compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ I D NO: 2 and

[0141] (b) two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or all amino acid substitutions selected from the group consisting of X25D, X43K / R / C / H / D / L7S / W / A / M / Y / Q / F / I, X78N / D / R / W / F / H / K / E / L / Y / M / C / Q, X109K / A, X144N / R, X182K / R / E, X183D / E / C / Q / A / M, X194E / D / C, X204D / E / C / G, X210I / V,

[0142] X212G / M / R / K / P / A / F / H / W / N / Y / D, X218S / T / D, X248Q / R, X259D / E, X260D / E / K and X261 L / M / F / E / W / Y / C compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, wherein the fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide.

[0143] The present invention is directed to a variant polypeptide having protease activity, wherein:

[0144] (I) the polypeptide has an amino acid sequence which is at least 79%, but less than 97.5%, identical to the amino acid sequence as set forth in SEQ ID NO: 1 and

[0145] (ii) the polypeptide comprises

[0146] (a) two or more, three or more, four or more, five or more, six or all amino acid substitutions selected from the group consisting of X9A / Q / E / C / D, X18Q / E / D, X21 l / V / F / W / Y, X38R / K / H / W, X59K / E, X228S and X271 D compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ I D NO: 2 and (b) two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or all amino acid substitutions selected from the group consisting of X25D, X43K / R / C / H / D / L / S / W / A / M / Y / Q / F / I, X78N / D / R / W / F / H / K / E / L / Y / M / C / Q, X109K / A, X144N / R, X182K / R / E, X183D / E / C / Q / A / M, X194E / D / C, X204D / E / C / G, X210I / V, X212G / M / R / K / P / A / F / H / W / N / Y / D, X218S / T / D, X248Q / R, X259D / E, X260D / E / K and X261 L / M / F / E / W / Y / C compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2.

[0147] The present invention is directed to a variant polypeptide having protease activity or a fragment of said polypeptide having protease activity, wherein:

[0148] (I) the polypeptide has an amino acid sequence which is at least 79%, but less than 97.5%, identical to the amino acid sequence as set forth in SEQ ID NO: 1 and

[0149] (ii) the polypeptide or fragment thereof comprises

[0150] (a) two or more, three or more, four or more, five or more, six or all amino acid substitutions selected from the group consisting of X9A / Q, X18Q / E / D, X21 V / l, X38R / K / H / W, X59K / E, X228S and X271 D compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0151] (b) two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or all amino acid substitutions selected from the group consisting of X25D, X43K / R, X78N / D, X109K / A, X144N / R, X182K / R / E, X183D / E, X194E / D, X204D / E, X210I, X212P / A / G / R / K / F / Y / M, X218S / T, X248Q / R, X259D, X260D / E and X261 F / W / Y / L compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ I D NO: 2, wherein the fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide.

[0152] The present invention is directed to a variant polypeptide having protease activity, wherein:

[0153] (I) the polypeptide has an amino acid sequence which is at least 79%, but less than 97.5%, identical to the amino acid sequence as set forth in SEQ ID NO: 1 and

[0154] (ii) the polypeptide comprises

[0155] (a) two or more, three or more, four or more, five or more, six or all amino acid substitutions selected from the group consisting of X9A / Q, X18Q / E / D, X21 V / l, X38R / K / H / W, X59K / E, X228S and X271 D compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0156] (b) two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or all amino acid substitutions selected from the group consisting of X25D, X43K / R, X78N / D, X109K / A, X144N / R, X182K / R / E, X183D / E, X194E / D, X204D / E, X210I, X212P / A / G / R / K / F / Y / M, X218S / T, X248Q / R, X259D, X260D / E and X261 F / W / Y / L compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ I D NO: 2. The present invention is directed to a variant polypeptide having protease activity or a fragment of said polypeptide having protease activity, wherein:

[0157] (i) the polypeptide has an amino acid sequence which is at least 79%, but less than 97.5%, identical to the amino acid sequence as set forth in SEQ ID NO: 1 and

[0158] (ii) the polypeptide or fragment thereof comprises

[0159] (a) an amino acid substitution at two or more, three or more, four or more, five or more, six or all amino acid residues selected from the group consisting of 9, 18, 21 , 38, 59, 228, and 271 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0160] (b) an amino acid substitution at two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or all amino acid residues selected from the group consisting of 25, 43, 78, 109, 144, 182, 183, 194, 204, 210, 212, 218, 248, 259, 260 and 261 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, wherein the fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide and wherein the polypeptide comprises amino acid residue D or E at position 101, preferably E at position 101 , referring to the numbering of SEQ ID NO: 2.

[0161] The present invention is directed to a variant polypeptide having protease activity, wherein:

[0162] (I) the polypeptide has an amino acid sequence which is at least 79%, but less than 97.5%, identical to the amino acid sequence as set forth in SEQ ID NO: 1 and

[0163] (ii) the polypeptide comprises

[0164] (a) an amino acid substitution at two or more, three or more, four or more, five or more, six or all amino acid residues selected from the group consisting of 9, 18, 21 , 38, 59, 228, and 271 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0165] (b) an amino acid substitution at two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or all amino acid residues selected from the group consisting of 25, 43, 78, 109, 144, 182, 183, 194, 204, 210, 212, 218, 248, 259, 260 and 261 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, wherein the polypeptide comprises amino acid residue D or E at position 101 , preferably E at position 101 , referring to the numbering of SEQ ID NO: 2.

[0166] The present invention is directed to a variant polypeptide having protease activity or a fragment of said polypeptide having protease activity, wherein:

[0167] (I) the polypeptide has an amino acid sequence which is at least 79%, but less than 97.5%, identical to the amino acid sequence as set forth in SEQ ID NO: 1 and (ii) the polypeptide or fragment thereof comprises

[0168] (a) two or more, three or more, four or more, five or more, six or all amino acid substitutions selected from the group consisting of X9A / Q / E / C / D, X18Q / E / D, X21 l / V / F / W / Y, X38R / K / H / W, X59K / E, X228S and X271 D compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ I D NO: 2 and

[0169] (b) two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or all amino acid substitutions selected from the group consisting of X25D, X43K / R / C / H / D / L / S / W / A / M / Y / Q / F / I, X78N / D / R / W / F / H / K / E / L / Y / M / C / Q, X109K / A, X144N / R, X182K / R / E, X183D / E / C / Q / A / M, X194E / D / C, X204D / E / C / G, X210I / V,

[0170] X212G / M / R / K / P / A / F / H / W / N / Y / D, X218S / T / D, X248Q / R, X259D / E, X260D / E / K and X261 L / M / F / E / W / Y / C compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, wherein the fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide and wherein the polypeptide comprises amino acid residue D or E at position 101, preferably E at position 101 , referring to the numbering of SEQ ID NO: 2.

[0171] The present invention is directed to a variant polypeptide having protease activity, wherein:

[0172] (I) the polypeptide has an amino acid sequence which is at least 79%, but less than 975%, identical to the amino acid sequence as set forth in SEQ ID NO: 1 and

[0173] (ii) the polypeptide comprises

[0174] (a) two or more, three or more, four or more, five or more, six or all amino acid substitutions selected from the group consisting of X9A / Q / E / C / D, X18Q / E / D, X21 l / V / F / W / Y, X38R / K / H / W, X59K / E, X228S and X271 D compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ I D NO: 2 and

[0175] (b) two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or all amino acid substitutions selected from the group consisting of X25D, X43K / R / C / H / D / L / S / W / A / M / Y / Q / F / I, X78N / D / R / W / F / H / K / E / L / Y / M / C / Q, X109K / A, X144N / R, X182K / R / E, X183D / E / C / Q / A / M, X194E / D / C, X204D / E / C / G, X210I / V, X212G / M / R / K / P / A / F / H / W / N / Y / D, X218S / T / D, X248Q / R, X259D / E, X260D / E / K and X261 L / M / F / E / W / Y / C compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, wherein the polypeptide comprises amino acid residue D or E at position 101 , preferably E at position 101 , referring to the numbering of SEQ ID NO: 2.

[0176] The present invention is directed to a variant polypeptide having protease activity or a fragment of said polypeptide having protease activity, wherein:

[0177] (I) the polypeptide has an amino acid sequence which is at least 79%, but less than 975%, identical to the amino acid sequence as set forth in SEQ ID NO: 1 and

[0178] (ii) the polypeptide or fragment thereof comprises (a) two or more, three or more, four or more, five or more, six or all amino acid substitutions selected from the group consisting of X9A / Q, X18Q / E / D, X21 V / l, X38R / K / H / W, X59K / E, X228S and X271 D compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0179] (b) two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or all amino acid substitutions selected from the group consisting of X25D, X43K / R, X78N / D, X109K / A, X144N / R, X182K / R / E, X183D / E, X194E / D, X204D / E, X210I, X212P / A / G / R / K / F / Y / M, X218S / T, X248Q / R, X259D, X260D / E and X261 F / W / Y / L compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ I D NO: 2, wherein the fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide and wherein the polypeptide comprises amino acid residue D or E at position 101, preferably E at position 101 , referring to the numbering of SEQ ID NO: 2.

[0180] The present invention is directed to a variant polypeptide having protease activity, wherein:

[0181] (I) the polypeptide has an amino acid sequence which is at least 79%, but less than 975%, identical to the amino acid sequence as set forth in SEQ ID NO: 1 and

[0182] (ii) the polypeptide comprises

[0183] (a) two or more, three or more, four or more, five or more, six or all amino acid substitutions selected from the group consisting of X9A / Q, X18Q / E / D, X21 V / l, X38R / K / H / W, X59K / E, X228S and X271 D compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0184] (b) two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or all amino acid substitutions selected from the group consisting of X25D, X43K / R, X78N / D, X109K / A, X144N / R, X182K / R / E, X183D / E, X194E / D, X204D / E, X210I, X212P / A / G / R / K / F / Y / M, X218S / T, X248Q / R, X259D, X260D / E and X261 F / W / Y / L compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ I D NO: 2, wherein the polypeptide comprises amino acid residue D or E at position 101 , preferably E at position 101 , referring to the numbering of SEQ ID NO: 2.

[0185] The variant polypeptide having protease activity of the present invention is a non-naturally occurring protease. Preferably, the variant polypeptide having protease activity of the present invention is a purified, isolated, synthetic, and / or recombinant protease. Preferably, the variant polypeptide having protease activity of the present invention is a purified and recombinant protease.

[0186] Proteases according to the invention have “proteolytic activity” or “protease activity”. “Proteolytic activity” or “protease activity” describes the capability for the hydrolysis of peptide bonds in polypeptides. Protease activity may be determined by assays for measurement of protease activity which are known to those skilled in the art. The methods for analyzing proteolytic activity are well-known in the literature (see e.g. Gupta et al. (2002), Appl. Microbiol. Biotechnol. 60: 381-395). For instance, proteolytic activity can be determined by using Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (Suc-AAPF-pNA; see e.g. DelMar et al. (1979), Analytical Biochem 99, 316-320) or Suc-AAPF-AMC (7-amido-4- methylcoumarin) as substrate. pNA or AMC is cleaved from the substrate molecule by proteolytic cleavage, resulting in release of yellow color of free pNA or change of fluorescence properties which can be quantified either by measuring emission at 460 nm after excitation at 380 nm.

[0187] The variant polypeptide of the present invention preferably has a sequence identity of at least 79%, at least 80%, 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 90.5%, at least 91 %, at least 91 .5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.1 %, at least 95.2%, at least 95.3%, at least 95.4%, at least 95.5%, at least 95.6%, at least 95.7%, at least 95.8%, at least 95.9% at least 96%, at least 96. 1%, at least 96.2%, at least 96.3%, at least 96.4%, at least 96.5%, at least 96.6%, at least 96.7%, at least 96.8%, at least 96.9%, at least 97%, at least 97.1 %, at least 97.2%, at least 97.3%, at least 97.4%, but less than 97.5% to the amino acid sequence according to SEQ ID NO: 1 and comprises

[0188] (a) an amino acid substitution at two or more, three or more, four or more, five or more, six or all amino acid residues selected from the group consisting of 9, 18, 21 , 38, 59, 228, and 271 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0189] (b) an amino acid substitution at two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or all amino acid residues selected from the group consisting of 25, 43, 78, 109, 144, 182, 183, 194, 204, 210, 212, 218, 248, 259, 260 and 261 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2.

[0190] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of at least 79%, at least 80%, 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 90.5%, at least 91 %, at least 91 .5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.1 %, at least 95.2%, at least 95.3%, at least 95.4%, at least 95.5%, at least 95.6%, at least 95.7%, at least 95.8%, at least 95.9% at least 96%, at least 96.1 %, at least 96.2%, at least 96.3%, at least 96.4%, at least 96.5%, at least 96.6%, at least 96.7%, at least 96.8%, at least 96.9%, at least 97%, at least 97.1 %, at least 97.2%, at least 97.3%, at least 97.4%, but less than 97.5%, to the amino acid sequence according to SEQ ID NO: 1 and comprises

[0191] (a) two or more, three or more, four or more, five or more, six or all amino acid substitutions selected from the group consisting of X9A / Q / E / C / D, X18Q / E / D, X21 l / V / F / W / Y, X38R / K / H / W, X59K / E, X228S and X271 D compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ I D NO: 2 and

[0192] (b) two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or all amino acid substitutions selected from the group consisting of X25D, X43K / R / C / H / D / L7S / W / A / M / Y / Q / F / I, X78N / D / R / W / F / H / K / E / L / Y / M / C / Q, X109K / A, X144N / R, X182K / R / E, X183D / E / C / Q / A / M, X194E / D / C, X204D / E / C / G, X210I / V,

[0193] X212G / M / R / K / P / A / F / H / W / N / Y / D, X218S / T / D, X248Q / R, X259D / E, X260D / E / K and X261 L / M / F / E / W / Y / C compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2.

[0194] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of at least 79%, at least 80%, 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 90.5%, at least 91 %, at least 91 .5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.1 %, at least 95.2%, at least 95.3%, at least 95.4%, at least 95.5%, at least 95.6%, at least 95.7%, at least 95.8%, at least 95.9% at least 96%, at least 96.1 %, at least 96.2%, at least 96.3%, at least 96.4%, at least 96.5%, at least 96.6%, at least 96.7%, at least 96.8%, at least 96.9%, at least 97%, at least 97.1 %, at least 97.2%, at least 97.3%, at least 97.4%, but less than 97.5% but less than to the amino acid sequence according to SEQ ID NO: 1 and comprises

[0195] (a) two or more, three or more, four or more, five or more, six or all amino acid substitutions selected from the group consisting of X9A / Q, X18Q / E / D, X21 V / l, X38R / K / H / W, X59K / E,X228S and X271 D compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0196] (b) two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or all amino acid substitutions selected from the group consisting of X25D, X43K / R, X78N / D, X109K / A, X144N / R, X182K / R / E, X183D / E, X194E / D, X204D / E, X210I, X212P / A / G / R / K / F / Y / M, X218S / T, X248Q / R, X259D, X260D / E and X261 F / W / Y / L compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ I D NO: 2.

[0197] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of at least 79%, at least 80%, 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 90.5%, at least 91 %, at least 91 .5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.1 %, at least 95.2%, at least 95.3%, at least 95.4%, at least 95.5%, at least 95.6%, at least 95.7%, at least 95.8%, at least 95.9% at least 96%, at least 96.1 %, at least 96.2%, at least 96.3%, at least 96.4%, at least 96.5%, at least 96.6%, at least 96.7%, at least 96.8%, at least 96.9%, at least 97%, at least 97.1 %, at least 97.2%, at least 97.3%, at least 97.4%, but less than 97.5% to the amino acid sequence according to SEQ ID NO: 1 and comprises

[0198] (a) two or more, three or more, four or more, five or more, six or all amino acid substitutions selected from the group consisting of S9A / Q / E / C / D, N18Q / E / D, L21 l / V / F / W / Y, T38R / K / H / W, Q59K / E, A228S and E271 D compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ I D NO: 2 and

[0199] (b) two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or all amino acid substitutions selected from the group consisting of G25D, N43K / R / C / H / D / L / S / W / A / M / Y / Q / F / I, S78N / D / R / W / F / H / K / E / L / Y / M / C / Q, Q109K / A, S144N / R, Q182K / R / E, N183D / E / C / Q / A / M, A194E / D / C, N204D / E / C / G, P210I / V, S212G / M / R / K / P / A / F / H / W / N / Y / D, N218S / T / D, N248Q / R, S259D / E, T260D / E / K and N261 L / M / F / E / W / Y / C compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2.

[0200] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of at least 79%, at least 80%, 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 90.5%, at least 91 %, at least 91 .5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.1 %, at least 95.2%, at least 95.3%, at least 95.4%, at least 95.5%, at least 95.6%, at least 95.7%, at least 95.8%, at least 95.9% at least 96%, at least 96.1 %, at least 96.2%, at least 96.3%, at least 96.4%, at least 96.5%, at least 96.6%, at least 96.7%, at least 96.8%, at least 96.9%, at least 97%, at least 97.1 %, at least 97.2%, at least 97.3%, at least 97.4%, but less than 97.5% to the amino acid sequence according to SEQ ID NO: 1 and comprises

[0201] (a) two or more, three or more, four or more, five or more, six or all amino acid substitutions selected from the group consisting of S9A / Q, N18Q / E / D, L21 V / l, T38R / K / H / W, Q59K / E,A228S and E271 D compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0202] (b) two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or all amino acid substitutions selected from the group consisting of G25D, N43K / R, S78N / D, Q109K / A, S144N / R, Q182K / R / E, N183D / E, A194E / D, N204D / E, P210I, S212P / A / G / R / K / F / Y / M, N218S / T, N248Q / R, S259D, T260D / E and N261 F / W / Y / L compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ I D NO: 2.

[0203] The variant polypeptide of the present invention preferably has a sequence identity of at least 79%, at least 80%, 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 90.5%, at least 91 %, at least 91 .5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.1 %, at least 95.2%, at least 95.3%, at least 95.4%, at least 95.5%, at least 95.6%, at least 95.7%, at least 95.8%, at least 95.9% at least 96%, at least 96. 1%, at least 96.2%, at least 96.3%, at least 96.4%, at least 96.5%, at least 96.6%, at least 96.7%, at least 96.8%, at least 96.9%, at least 97%, at least 97.1 %, at least 97.2%, at least 97.3%, at least 97.4%, but less than 97.5% to the amino acid sequence according to SEQ ID NO: 1 and comprises

[0204] (a) an amino acid substitution at two or more, three or more, four or more, five or more, six or all amino acid residues selected from the group consisting of 9, 18, 21 , 38, 59, 228, and 271 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0205] (b) an amino acid substitution at two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or all amino acid residues selected from the group consisting of 25, 43, 78, 109, 144, 182, 183, 194, 204, 210, 212, 218, 248, 259, 260 and 261 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, wherein said polypeptide comprises amino acid residue D or E at position 101, preferably E at position 101 , referring to the numbering of SEQ ID NO: 2.

[0206] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of at least 79%, at least 80%, 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 90.5%, at least 91 %, at least 91 .5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.1 %, at least 95.2%, at least 95.3%, at least 95.4%, at least 95.5%, at least 95.6%, at least 95.7%, at least 95.8%, at least 95.9% at least 96%, at least 96.1 %, at least 96.2%, at least 96.3%, at least 96.4%, at least 96.5%, at least 96.6%, at least 96.7%, at least 96.8%, at least 96.9%, at least 97%, at least 97.1 %, at least 97.2%, at least 97.3%, at least 97.4%, but less than 97.5%to the amino acid sequence according to SEQ ID NO: 1 and comprises

[0207] (a) two or more, three or more, four or more, five or more, six or all amino acid substitutions selected from the group consisting of X9A / Q / E / C / D, X18Q / E / D, X21 l / V / F / W / Y, X38R / K / H / W, X59K / E, X228S and X271 D compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ I D NO: 2 and

[0208] (b) two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or all amino acid substitutions selected from the group consisting of X25D, X43K / R / C / H / D / L7S / W / A / M / Y / Q / F / I, X78N / D / R / W / F / H / K / E / L / Y / M / C / Q, X109K / A, X144N / R, X182K / R / E, X183D / E / C / Q / A / M, X194E / D / C, X204D / E / C / G, X210I / V,

[0209] X212G / M / R / K / P / A / F / H / W / N / Y / D, X218S / T / D, X248Q / R, X259D / E, X260D / E / K and X261 L / M / F / E / W / Y / C compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, wherein said polypeptide comprises amino acid residue D or E at position 101 , preferably E at position 101 , referring to the numbering of SEQ ID NO: 2.

[0210] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of at least 79%, at least 80%, 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 90.5%, at least 91 %, at least 91 .5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.1 %, at least 95.2%, at least 95.3%, at least 95.4%, at least 95.5%, at least 95.6%, at least 95.7%, at least 95.8%, at least 95.9% at least 96%, at least 96.1 %, at least 96.2%, at least 96.3%, at least 96.4%, at least 96.5%, at least 96.6%, at least 96.7%, at least 96.8%, at least 96.9%, at least 97%, at least 97.1 %, at least 97.2%, at least 97.3%, at least 97.4%, but less than 97.5%to the amino acid sequence according to SEQ ID NO: 1 and comprises

[0211] (a) two or more, three or more, four or more, five or more, six or all amino acid substitutions selected from the group consisting of X9A / Q, X18Q / E / D, X21 V / l, X38R / K / H / W, X59K / E, X228S and X271 D compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0212] (b) two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or all amino acid substitutions selected from the group consisting of X25D, X43K / R, X78N / D, X109K / A, X144N / R, X182K / R / E, X183D / E, X194E / D, X204D / E, X210I, X212P / A / G / R / K / F / Y / M, X218S / T, X248Q / R, X259D, X260D / E and X261 F / W / Y / L compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ I D NO: 2, wherein said polypeptide comprises amino acid residue D or E at position 101, preferably E at position 101 , referring to the numbering of SEQ ID NO: 2.

[0213] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of at least 79%, at least 80%, 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 90.5%, at least 91 %, at least 91 .5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.1 %, at least 95.2%, at least 95.3%, at least 95.4%, at least 95.5%, at least 95.6%, at least 95.7%, at least 95.8%, at least 95.9% at least 96%, at least 96.1 %, at least 96.2%, at least 96.3%, at least 96.4%, at least 96.5%, at least 96.6%, at least 96.7%, at least 96.8%, at least 96.9%, at least 97%, at least 97.1 %, at least 97.2%, at least 97.3%, at least 97.4%, but less than 97.5% to the amino acid sequence according to SEQ ID NO: 1 and comprises

[0214] (a) two or more, three or more, four or more, five or more, six or all amino acid substitutions selected from the group consisting of S9A / Q / E / C / D, N18Q / E / D, L21 l / V / F / W / Y, T38R / K / H / W, Q59K / E, A228S and E271 D compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ I D NO: 2 and

[0215] (b) two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or all amino acid substitutions selected from the group consisting of G25D, N43K / R / C / H / D / L / S / W / A / M / Y / Q / F / I, S78N / D / R / W / F / H / K / E / L / Y / M / C / Q, Q109K / A, S144N / R, Q182K / R / E, N183D / E / C / Q / A / M, A194E / D / C, N204D / E / C / G, P210I / V, S212G / M / R / K / P / A / F / H / W / N / Y / D, N218S / T / D, N248Q / R, S259D / E, T260D / E / K and N261 L / M / F / E / W / Y / C compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, wherein said polypeptide comprises amino acid residue D or E at position 101, preferably E at position 101 , referring to the numbering of SEQ ID NO: 2.

[0216] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of at least 79%, at least 80%, 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 90.5%, at least 91 %, at least 91 .5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.1 %, at least 95.2%, at least 95.3%, at least 95.4%, at least 95.5%, at least 95.6%, at least 95.7%, at least 95.8%, at least 95.9% at least 96%, at least 96.1 %, at least 96.2%, at least 96.3%, at least 96.4%, at least 96.5%, at least 96.6%, at least 96.7%, at least 96.8%, at least 96.9%, at least 97%, at least 97.1 %, at least 97.2%, at least 97.3%, at least 97.4%, but less than 97.5% to the amino acid sequence according to SEQ ID NO: 1 and comprises (a) two or more, three or more, four or more, five or more, six or all amino acid substitutions selected from the group consisting of S9A / Q, N18Q / E / D, L21V / I, T38R / K / H / W, Q59K / E, A228S and E271 D compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0217] (b) two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or all amino acid substitutions selected from the group consisting of G25D, N43K / R, S78N / D, Q109K / A, S144N / R, Q182K / R / E, N183D / E, A194E / D, N204D / E, P210I, S212P / A / G / R / K / F / Y / M, N218S / T, N248Q / R, S259D, T260D / E and N261 F / W / Y / L compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ I D NO: 2.

[0218] The variant polypeptide of the present invention preferably has a sequence identity of 79% to 98%, 80% to 98%,

[0219] 80.5% to 98%, 81 % to 98%, 81 .5% to 98%, 82% to 98%, 82.5% to 98%, 83% to 98%, 83.5% to 98%, 84% to 98%,

[0220] 84.5% to 98%, 85% to 98%, 85.5% to 98%, 86% to 98%, 86.5% to 98%, 87% to 98%, 87.5% to 98%, 88% to 98%,

[0221] 88.5% to 98%, 89% to 98%, 89.5% to 98%, 90% to 98%, 90.3% to 98%, 90.5% to 98%, 90.7% to 98%, 91 % to 98%,

[0222] 91 .3% to 98%, 91 .5% to 98%, 91.7% to 98%, 92% to 98%, 92.3% to 98%, 92.5% to 98%, 92.7% to 98%, 93% to 98%, 93.3% to 98%, 93.5% to 98%, 93.7% to 98%, 94% to 98%, 94.3% to 98%, 94.5% to 98%, 94.7% to 98%, 95% to 98%, 95.1 % to 98%, 95.2% to 98%, 95.3% to 98%, 95.4% to 98%, 95.5% to 98% to the amino acid sequence according to SEQ I D NO: 1 and comprises

[0223] (a) an amino acid substitution at two or more, three or more, four or more, five or more, six or all amino acid residues selected from the group consisting of 9, 18, 21 , 38, 59, 228, and 271 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0224] (b) an amino acid substitution at two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or all amino acid residues selected from the group consisting of 25, 43, 78, 109, 144, 182, 183, 194, 204, 210, 212, 218, 248, 259, 260 and 261 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2.

[0225] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of 79% to 98%, 80% to 98%, 80.5% to 98%, 81 % to 98%, 81 .5% to 98%, 82% to 98%, 82.5% to 98%, 83% to 98%, 83.5% to

[0226] 98%, 84% to 98%, 84.5% to 98%, 85% to 98%, 85.5% to 98%, 86% to 98%, 86.5% to 98%, 87% to 98%, 87.5% to

[0227] 98%, 88% to 98%, 88.5% to 98%, 89% to 98%, 89.5% to 98%, 90% to 98%, 90.3% to 98%, 90.5% to 98%, 90.7% to

[0228] 98%, 91 % to 98%, 91 .3% to 98%, 91 .5% to 98%, 91 .7% to 98%, 92% to 98%, 92.3% to 98%, 92.5% to 98%, 92.7% to 98%, 93% to 98%, 93.3% to 98%, 93.5% to 98%, 93.7% to 98%, 94% to 98%, 94.3% to 98%, 94.5% to 98%, 94.7% to 98%, 95% to 98%, 95.1 % to 98%, 95.2% to 98%, 95.3% to 98%, 95.4% to 98%, 95.5% to 98% to the amino acid sequence according to SEQ ID NO: 1 and comprises (a) two or more, three or more, four or more, five or more, six or all amino acid substitutions selected from the group consisting of X9A / Q / E / C / D, X18Q / E / D, X21 l / V / F / W / Y, X38R / K / H / W, X59K / E, X228S and X271 D compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ I D NO: 2 and

[0229] (b) two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or all amino acid substitutions selected from the group consisting of X25D, X43K / R / C / H / D / L7S / W / A / M / Y / Q / F / I, X78N / D / R / W / F / H / K / E / L / Y / M / C / Q, X109K / A, X144N / R, X182K / R / E, X183D / E / C / Q / A / M, X194E / D / C, X204D / E / C / G, X210I / V,

[0230] X212G / M / R / K / P / A / F / H / W / N / Y / D, X218S / T / D, X248Q / R, X259D / E, X260D / E / K and X261 L / M / F / E / W / Y / C compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2.

[0231] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of 79% to 98%, 80% to 98%, 80.5% to 98%, 81 % to 98%, 81 .5% to 98%, 82% to 98%, 82.5% to 98%, 83% to 98%, 83.5% to

[0232] 98%, 84% to 98%, 84.5% to 98%, 85% to 98%, 85.5% to 98%, 86% to 98%, 86.5% to 98%, 87% to 98%, 87.5% to

[0233] 98%, 88% to 98%, 88.5% to 98%, 89% to 98%, 89.5% to 98%, 90% to 98%, 90.3% to 98%, 90.5% to 98%, 90.7% to

[0234] 98%, 91 % to 98%, 91 .3% to 98%, 91 .5% to 98%, 91 .7% to 98%, 92% to 98%, 92.3% to 98%, 92.5% to 98%, 92.7% to 98%, 93% to 98%, 93.3% to 98%, 93.5% to 98%, 93.7% to 98%, 94% to 98%, 94.3% to 98%, 94.5% to 98%, 94.7% to 98%, 95% to 98%, 95.1 % to 98%, 95.2% to 98%, 95.3% to 98%, 95.4% to 98%, 95.5% to 98% to the amino acid sequence according to SEQ ID NO: 1 and comprises

[0235] (a) two or more, three or more, four or more, five or more, six or all amino acid substitutions selected from the group consisting of X9A / Q, X18Q / E / D, X21 V / l, X38R / K / H / W, X59K / E, X228S and X271 D compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0236] (b) two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or all amino acid substitutions selected from the group consisting of X25D, X43K / R, X78N / D, X109K / A, X144N / R, X182K / R / E, X183D / E, X194E / D, X204D / E, X210I, X212P / A / G / R / K / F / Y / M, X218S / T, X248Q / R, X259D, X260D / E and X261 F / W / Y / L compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ I D NO: 2.

[0237] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of 79% to 98%, 80% to 98%, 80.5% to 98%, 81 % to 98%, 81 .5% to 98%, 82% to 98%, 82.5% to 98%, 83% to 98%, 83.5% to

[0238] 98%, 84% to 98%, 84.5% to 98%, 85% to 98%, 85.5% to 98%, 86% to 98%, 86.5% to 98%, 87% to 98%, 87.5% to

[0239] 98%, 88% to 98%, 88.5% to 98%, 89% to 98%, 89.5% to 98%, 90% to 98%, 90.3% to 98%, 90.5% to 98%, 90.7% to

[0240] 98%, 91 % to 98%, 91 .3% to 98%, 91 .5% to 98%, 91 .7% to 98%, 92% to 98%, 92.3% to 98%, 92.5% to 98%, 92.7% to 98%, 93% to 98%, 93.3% to 98%, 93.5% to 98%, 93.7% to 98%, 94% to 98%, 94.3% to 98%, 94.5% to 98%, 94.7% to 98%, 95% to 98%, 95.1 % to 98%, 95.2% to 98%, 95.3% to 98%, 95.4% to 98%, 95.5% to 98% to the amino acid sequence according to SEQ ID NO: 1 and comprises (a) two or more, three or more, four or more, five or more, six or all amino acid substitutions selected from the group consisting of S9A / Q / E / C / D, N18Q / E / D, L21 l / V / F / W / Y, T38R / K / H / W, Q59K / E, A228S and E271 D compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ I D NO: 2 and

[0241] (b) two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or all amino acid substitutions selected from the group consisting of G25D, N43K / R / C / H / D / L7S / W / A / M / Y / Q / F / I, S78N / D / R / W / F / H / K / E / L / Y / M / C / Q, Q109K / A, S144N / R, Q182K / R / E, N183D / E / C / Q / A / M, A194E / D / C, N204D / E / C / G, P210I / V, S212G / M / R / K / P / A / F / H / W / N / Y / D, N218S / T / D, N248Q / R, S259D / E, T260D / E / K and N261 L / M / F / E / W / Y / C compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2.

[0242] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of 79% to 98%, 80% to 98%, 80.5% to 98%, 81 % to 98%, 81 .5% to 98%, 82% to 98%, 82.5% to 98%, 83% to 98%, 83.5% to 98%, 84% to 98%, 84.5% to 98%, 85% to 98%, 85.5% to 98%, 86% to 98%, 86.5% to 98%, 87% to 98%, 87.5% to 98%, 88% to 98%, 88.5% to 98%, 89% to 98%, 89.5% to 98%, 90% to 98%, 90.3% to 98%, 90.5% to 98%, 90.7% to 98%, 91% to 98%, 91.3% to 98%, 91.5% to 98%, 91.7% to 98%, 92% to 98%, 92.3% to 98%, 92.5% to 98%, 92.7% to 98%, 93% to 98%, 93.3% to 98%, 93.5% to 98%, 93.7% to 98%, 94% to 98%, 94.3% to 98%, 94.5% to 98%, 94.7% to 98%, 95% to 98%, 95.1 % to 98%, 95.2% to 98%, 95.3% to 98%, 95.4% to 98%, 95.5% to 98% to the amino acid sequence according to SEQ ID NO: 1 and comprises

[0243] (a) two or more, three or more, four or more, five or more, six or all amino acid substitutions selected from the group consisting of S9A / Q, N18Q / E / D, L21 V / l, T38R / K / H / W, Q59K / E, A228S and E271 D compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0244] (b) two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or all amino acid substitutions selected from the group consisting of G25D, N43K / R, S78N / D, Q109K / A, S144N / R, Q182K / R / E, N183D / E, A194E / D, N204D / E, P210I, S212P / A / G / R / K / F / Y / M, N218S / T, N248Q / R, S259D, T260D / E and N261 F / W / Y / L compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ I D NO: 2.

[0245] The variant polypeptide of the present invention preferably has a sequence identity of 79% to 98%, 80% to 98%,

[0246] 80.5% to 98%, 81 % to 98%, 81 .5% to 98%, 82% to 98%, 82.5% to 98%, 83% to 98%, 83.5% to 98%, 84% to 98%,

[0247] 84.5% to 98%, 85% to 98%, 85.5% to 98%, 86% to 98%, 86.5% to 98%, 87% to 98%, 87.5% to 98%, 88% to 98%,

[0248] 88.5% to 98%, 89% to 98%, 89.5% to 98%, 90% to 98%, 90.3% to 98%, 90.5% to 98%, 90.7% to 98%, 91 % to 98%,

[0249] 91 .3% to 98%, 91 .5% to 98%, 91.7% to 98%, 92% to 98%, 92.3% to 98%, 92.5% to 98%, 92.7% to 98%, 93% to 98%, 93.3% to 98%, 93.5% to 98%, 93.7% to 98%, 94% to 98%, 94.3% to 98%, 94.5% to 98%, 94.7% to 98%, 95% to 98%, 95.1 % to 98%, 95.2% to 98%, 95.3% to 98%, 95.4% to 98%, 95.5% to 98% to the amino acid sequence according to SEQ I D NO: 1 and comprises (a) an amino acid substitution at two or more, three or more, four or more, five or more, six or all amino acid residues selected from the group consisting of 9, 18, 21 , 38, 59, 228, and 271 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0250] (b) an amino acid substitution at two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or all amino acid residues selected from the group consisting of 25, 43, 78, 109, 144, 182, 183, 194, 204, 210, 212, 218, 248, 259, 260 and 261 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, wherein said polypeptide comprises amino acid residue D or E at position 101, preferably E at position 101 , referring to the numbering of SEQ ID NO: 2.

[0251] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of 79% to 98%, 80% to 98%, 80.5% to 98%, 81 % to 98%, 81 .5% to 98%, 82% to 98%, 82.5% to 98%, 83% to 98%, 83.5% to

[0252] 98%, 84% to 98%, 84.5% to 98%, 85% to 98%, 85.5% to 98%, 86% to 98%, 86.5% to 98%, 87% to 98%, 87.5% to

[0253] 98%, 88% to 98%, 88.5% to 98%, 89% to 98%, 89.5% to 98%, 90% to 98%, 90.3% to 98%, 90.5% to 98%, 90.7% to

[0254] 98%, 91 % to 98%, 91 .3% to 98%, 91 .5% to 98%, 91 .7% to 98%, 92% to 98%, 92.3% to 98%, 92.5% to 98%, 92.7% to 98%, 93% to 98%, 93.3% to 98%, 93.5% to 98%, 93.7% to 98%, 94% to 98%, 94.3% to 98%, 94.5% to 98%, 94.7% to 98%, 95% to 98%, 95.1 % to 98%, 95.2% to 98%, 95.3% to 98%, 95.4% to 98%, 95.5% to 98% to the amino acid sequence according to SEQ ID NO: 1 and comprises

[0255] (a) two or more, three or more, four or more, five or more, six or all amino acid substitutions selected from the group consisting of X9A / Q / E / C / D, X18Q / E / D, X21 l / V / F / W / Y, X38R / K / H / W, X59K / E, X228S and X271 D compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ I D NO: 2 and

[0256] (b) two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or all amino acid substitutions selected from the group consisting of X25D, X43K / R / C / H / D / L7S / W / A / M / Y / Q / F / I, X78N / D / R / W / F / H / K / E / L / Y / M / C / Q, X109K / A, X144N / R, X182K / R / E, X183D / E / C / Q / A / M, X194E / D / C, X204D / E / C / G, X210I / V,

[0257] X212G / M / R / K / P / A / F / H / W / N / Y / D, X218S / T / D, X248Q / R, X259D / E, X260D / E / K and X261 L / M / F / E / W / Y / C compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, wherein said polypeptide comprises amino acid residue D or E at position 101 , preferably E at position 101 , referring to the numbering of SEQ ID NO: 2.

[0258] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of 79% to 98%, 80% to 98%, 80.5% to 98%, 81 % to 98%, 81 .5% to 98%, 82% to 98%, 82.5% to 98%, 83% to 98%, 83.5% to

[0259] 98%, 84% to 98%, 84.5% to 98%, 85% to 98%, 85.5% to 98%, 86% to 98%, 86.5% to 98%, 87% to 98%, 87.5% to

[0260] 98%, 88% to 98%, 88.5% to 98%, 89% to 98%, 89.5% to 98%, 90% to 98%, 90.3% to 98%, 90.5% to 98%, 90.7% to

[0261] 98%, 91 % to 98%, 91 .3% to 98%, 91 .5% to 98%, 91 .7% to 98%, 92% to 98%, 92.3% to 98%, 92.5% to 98%, 92.7% to 98%, 93% to 98%, 93.3% to 98%, 93.5% to 98%, 93.7% to 98%, 94% to 98%, 94.3% to 98%, 94.5% to 98%, 94.7% to 98%, 95% to 98%, 95.1 % to 98%, 95.2% to 98%, 95.3% to 98%, 95.4% to 98%, 95.5% to 98% to the amino acid sequence according to SEQ ID NO: 1 and comprises

[0262] (a) two or more, three or more, four or more, five or more, six or all amino acid substitutions selected from the group consisting of X9A / Q, X18Q / E / D, X21 V / l, X38R / K / H / W, X59K / E, X228S and X271 D compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0263] (b) two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or all amino acid substitutions selected from the group consisting of X25D, X43K / R, X78N / D, X109K / A, X144N / R, X182K / R / E, X183D / E, X194E / D, X204D / E, X210I, X212P / A / G / R / K / F / Y / M, X218S / T, X248Q / R, X259D, X260D / E and X261 F / W / Y / L compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ I D NO: 2, wherein said polypeptide comprises amino acid residue D or E at position 101 , preferably E at position 101 , referring to the numbering of SEQ ID NO: 2.

[0264] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of 79% to 98%, 80% to 98%, 80.5% to 98%, 81 % to 98%, 81 .5% to 98%, 82% to 98%, 82.5% to 98%, 83% to 98%, 83.5% to

[0265] 98%, 84% to 98%, 84.5% to 98%, 85% to 98%, 85.5% to 98%, 86% to 98%, 86.5% to 98%, 87% to 98%, 87.5% to

[0266] 98%, 88% to 98%, 88.5% to 98%, 89% to 98%, 89.5% to 98%, 90% to 98%, 90.3% to 98%, 90.5% to 98%, 90.7% to

[0267] 98%, 91 % to 98%, 91 .3% to 98%, 91 .5% to 98%, 91 .7% to 98%, 92% to 98%, 92.3% to 98%, 92.5% to 98%, 92.7% to 98%, 93% to 98%, 93.3% to 98%, 93.5% to 98%, 93.7% to 98%, 94% to 98%, 94.3% to 98%, 94.5% to 98%, 94.7% to 98%, 95% to 98%, 95.1 % to 98%, 95.2% to 98%, 95.3% to 98%, 95.4% to 98%, 95.5% to 98% to the amino acid sequence according to SEQ ID NO: 1 and comprises

[0268] (a) two or more, three or more, four or more, five or more, six or all amino acid substitutions selected from the group consisting of S9A / Q / E / C / D, N18Q / E / D, L21 l / V / F / W / Y, T38R / K / H / W, Q59K / E, A228S and E271 D compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ I D NO: 2 and

[0269] (b) two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or all amino acid substitutions selected from the group consisting of G25D, N43K / R / C / H / D / L / S / W / A / M / Y / Q / F / I, S78N / D / R / W / F / H / K / E / L / Y / M / C / Q, Q109K / A, S144N / R, Q182K / R / E, N183D / E / C / Q / A / M, A194E / D / C, N204D / E / C / G, P210I / V, S212G / M / R / K / P / A / F / H / W / N / Y / D, N218S / T / D, N248Q / R, S259D / E, T260D / E / K and N261 L / M / F / E / W / Y / C compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, wherein said polypeptide comprises amino acid residue D or E at position 101 , preferably E at position 101 , referring to the numbering of SEQ ID NO: 2.

[0270] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of 79% to 98%, 80% to 98%, 80.5% to 98%, 81 % to 98%, 81 .5% to 98%, 82% to 98%, 82.5% to 98%, 83% to 98%, 83.5% to 98%, 84% to 98%, 84.5% to 98%, 85% to 98%, 85.5% to 98%, 86% to 98%, 86.5% to 98%, 87% to 98%, 87.5% to 98%, 88% to 98%, 88.5% to 98%, 89% to 98%, 89.5% to 98%, 90% to 98%, 90.3% to 98%, 90.5% to 98%, 90.7% to 98%, 91% to 98%, 91.3% to 98%, 91.5% to 98%, 91.7% to 98%, 92% to 98%, 92.3% to 98%, 92.5% to 98%, 92.7% to 98%, 93% to 98%, 93.3% to 98%, 93.5% to 98%, 93.7% to 98%, 94% to 98%, 94.3% to 98%, 94.5% to 98%, 94.7% to 98%, 95% to 98%, 95.1 % to 98%, 95.2% to 98%, 95.3% to 98%, 95.4% to 98%, 95.5% to 98% to the amino acid sequence according to SEQ ID NO: 1 and comprises

[0271] (a) two or more, three or more, four or more, five or more, six or all amino acid substitutions selected from the group consisting of S9A / Q, N18Q / E / D, L21V / I, T38R / K / H / W, Q59K / E, A228S and E271 D compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0272] (b) two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or all amino acid substitutions selected from the group consisting of G25D, N43K / R, S78N / D, Q109K / A, S144N / R, Q182K / R / E, N183D / E, A194E / D, N204D / E, P210I, S212P / A / G / R / K / F / Y / M, N218S / T, N248Q / R, S259D, T260D / E and N261 F / W / Y / L compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ I D NO: 2, wherein said polypeptide comprises amino acid residue D or E at position 101, preferably E at position 101 , referring to the numbering of SEQ ID NO: 2.

[0273] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of at least 79%, at least 80%, 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 90.5%, at least 91 %, at least 91 .5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.1 %, at least 95.2%, at least 95.3%, at least 95.4%, at least 95.5%, at least 95.6%, at least 95.7%, at least 95.8%, at least 95.9% at least 96%, at least 96.1 %, at least 96.2%, at least 96.3%, at least 96.4%, at least 96.5%, at least 96.6%, at least 96.7%, at least 96.8%, at least 96.9%, at least 97%, at least 97.1 %, at least 97.2%, at least 97.3%, at least 97.4%, but less than 97.5% to the amino acid sequence according to SEQ ID NO: 1 and comprises

[0274] (a) an amino acid substitution at two or more, three or more, four or more, five or more, six or all amino acid residues selected from the group consisting of 9, 18, 21 , 38, 59, 228, and 271 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0275] (b) an amino acid substitution at two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or all amino acid residues selected from the group consisting of 43, 78, 183, 194, 204, 210, 212, 218, 259, 260 and 261 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2.

[0276] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of at least 79%, at least 80%, 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 90.5%, at least 91 %, at least 91 .5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.1 %, at least 95.2%, at least 95.3%, at least 95.4%, at least 95.5%, at least 95.6%, at least 95.7%, at least 95.8%, at least 95.9% at least 96%, at least

[0277] 96.1 %, at least 96.2%, at least 96.3%, at least 96.4%, at least 96.5%, at least 96.6%, at least 96.7%, at least 96.8%, at least 96.9%, at least 97%, at least 97.1 %, at least 97.2%, at least 97.3%, at least 97.4%, but less than 97.5% to the amino acid sequence according to SEQ ID NO: 1 and comprises

[0278] (a) two or more, three or more, four or more, five or more, six or all amino acid substitutions selected from the group consisting of X9A / Q / E / C / D, X18Q / E / D, X21 l / V / F / W / Y, X38R / K / H / W, X59K / E, X228S and X271 D compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ I D NO: 2 and

[0279] (b) two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or all amino acid substitutions selected from the group consisting of X43K / R / C / H / D / L / S / W / A / M / Y / Q / F / I, X78N / D / R / W / F / H / K / E / L / Y / M / C / Q, X183D / E / C / Q / A / M, X194E / D / C, X204D / E / C / G, X210I / V,

[0280] X212G / M / R / K / P / A / F / H / W / N / Y / D, X218S / T / D, X259D / E, X260D / E / K and X261 L / M / F / E / W / Y / C compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2.

[0281] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of at least 79%, at least 80%, 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 90.5%, at least 91 %, at least 91 .5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.1 %, at least 95.2%, at least 95.3%, at least 95.4%, at least 95.5%, at least 95.6%, at least 95.7%, at least 95.8%, at least 95.9% at least 96%, at least

[0282] 96.1 %, at least 96.2%, at least 96.3%, at least 96.4%, at least 96.5%, at least 96.6%, at least 96.7%, at least 96.8%, at least 96.9%, at least 97%, at least 97.1 %, at least 97.2%, at least 97.3%, at least 97.4%, but less than 97.5% to the amino acid sequence according to SEQ ID NO: 1 and comprises

[0283] (a) two or more, three or more, four or more, five or more, six or all amino acid substitutions selected from the group consisting of X9A / Q, X18Q / E / D, X21 V / l, X38R / K / H / W, X59K / E, X228S and X271 D compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0284] (b) two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or all amino acid substitutions selected from the group consisting of X43K / R, X78N / D, X183D / E, X194E / D, X204D / E, X210I, X212P / A / G / R / K / F / Y / M, X218S / T, X259D, X260D / E and X261 F / W / Y / L compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2.

[0285] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of at least 79%, at least 80%, 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 90.5%, at least 91 %, at least 91 .5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.1 %, at least 95.2%, at least 95.3%, at least 95.4%, at least 95.5%, at least 95.6%, at least 95.7%, at least 95.8%, at least 95.9% at least 96%, at least

[0286] 96.1 %, at least 96.2%, at least 96.3%, at least 96.4%, at least 96.5%, at least 96.6%, at least 96.7%, at least 96.8%, at least 96.9%, at least 97%, at least 97.1 %, at least 97.2%, at least 97.3%, at least 97.4%, but less than 97.5% to the amino acid sequence according to SEQ ID NO: 1 and comprises

[0287] (a) two or more, three or more, four or more, five or more, six or all amino acid substitutions selected from the group consisting of S9A / Q / E / C / D, N18Q / E / D, L21 l / V / F / W / Y, T38R / K / H / W, Q59K / E, A228S and E271 D compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ I D NO: 2 and

[0288] (b) two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or all amino acid substitutions selected from the group consisting of N43K / R / C / H / D / L / S / W / A / M / Y / Q / F / I, S78N / D / R / W / F / H / K / E / L / Y / M / C / Q, N183D / E / C / Q / A / M, A194E / D / C, N204D / E / C / G, P210I / V,

[0289] S212G / M / R / K / P / A / F / H / W / N / Y / D, N218S / T / D, S259D / E, T260D / E / K and N261 L / M / F / E / W / Y / C compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2.

[0290] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of at least 79%, at least 80%, 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 90.5%, at least 91 %, at least 91 .5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.1 %, at least 95.2%, at least 95.3%, at least 95.4%, at least 95.5%, at least 95.6%, at least 95.7%, at least 95.8%, at least 95.9% at least 96%, at least 96.1 %, at least 96.2%, at least 96.3%, at least 96.4%, at least 96.5%, at least 96.6%, at least 96.7%, at least 96.8%, at least 96.9%, at least 97%, at least 97.1 %, at least 97.2%, at least 97.3%, at least 97.4%, but less than 97.5%to the amino acid sequence according to SEQ ID NO: 1 and comprises

[0291] (a) two or more, three or more, four or more, five or more, six or all amino acid substitutions selected from the group consisting of S9A / Q, N18Q / E / D, L21 V / l, T38R / K / H / W, Q59K / E, A228S and E271 D compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0292] (b) two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or all amino acid substitutions selected from the group consisting of N43K / R, S78N / D, N183D / E, A194E / D, N204D / E, P210I, S212P / A / G / R / K / F / Y / M, N218S / T, S259D, T260D / E and N261 F / W / Y / L compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2.

[0293] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of at least 79%, at least 80%, 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 90.5%, at least 91 %, at least 91 .5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.1 %, at least 95.2%, at least 95.3%, at least 95.4%, at least 95.5%, at least 95.6%, at least 95.7%, at least 95.8%, at least 95.9% at least 96%, at least 96.1 %, at least 96.2%, at least 96.3%, at least 96.4%, at least 96.5%, at least 96.6%, at least 96.7%, at least 96.8%, at least 96.9%, at least 97%, at least 97.1 %, at least 97.2%, at least 97.3%, at least 97.4%, but less than 97.5% to the amino acid sequence according to SEQ ID NO: 1 and comprises (a) an amino acid substitution at two or more, three or more, four or more, five or more, six or all amino acid residues selected from the group consisting of 9, 18, 21 , 38, 59, 228, and 271 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0294] (b) an amino acid substitution at two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or all amino acid residues selected from the group consisting of 43, 78, 183, 194, 204, 210, 212, 218, 259, 260 and 261 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, wherein said polypeptide comprises amino acid residue D or E at position 101, preferably E at position 101 , referring to the numbering of SEQ ID NO: 2.

[0295] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of at least 79%, at least 80%, 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 90.5%, at least 91 %, at least 91 .5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.1 %, at least 95.2%, at least 95.3%, at least 95.4%, at least 95.5%, at least 95.6%, at least 95.7%, at least 95.8%, at least 95.9% at least 96%, at least 96.1 %, at least 96.2%, at least 96.3%, at least 96.4%, at least 96.5%, at least 96.6%, at least 96.7%, at least 96.8%, at least 96.9%, at least 97%, at least 97.1 %, at least 97.2%, at least 97.3%, at least 97.4%, but less than 97.5%to the amino acid sequence according to SEQ ID NO: 1 and comprises

[0296] (a) two or more, three or more, four or more, five or more, six or all amino acid substitutions selected from the group consisting of X9A / Q / E / C / D, X18Q / E / D, X21 l / V / F / W / Y, X38R / K / H / W, X59K / E, X228S and X271 D compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ I D NO: 2 and

[0297] (b) two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or all amino acid substitutions selected from the group consisting of X43K / R / C / H / D / L / S / W / A / M / Y / Q / F / I, X78N / D / R / W / F / H / K / E / L / Y / M / C / Q, X183D / E / C / Q / A / M, X194E / D / C, X204D / E / C / G, X210I / V,

[0298] X212G / M / R / K / P / A / F / H / W / N / Y / D, X218S / T / D, X259D / E, X260D / E / K and X261 L / M / F / E / W / Y / C compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, wherein said polypeptide comprises amino acid residue D or E at position 101, preferably E at position 101 , referring to the numbering of SEQ ID NO: 2.

[0299] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of at least 79%, at least 80%, 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 90.5%, at least 91 %, at least 91 .5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.1 %, at least 95.2%, at least 95.3%, at least 95.4%, at least 95.5%, at least 95.6%, at least 95.7%, at least 95.8%, at least 95.9% at least 96%, at least 96.1 %, at least 96.2%, at least 96.3%, at least 96.4%, at least 96.5%, at least 96.6%, at least 96.7%, at least 96.8%, at least 96.9%, at least 97%, at least 97.1 %, at least 97.2%, at least 97.3%, at least 97.4%, but less than 97.5% to the amino acid sequence according to SEQ ID NO: 1 and comprises

[0300] (a) two or more, three or more, four or more, five or more, six or all amino acid substitutions selected from the group consisting of X9A / Q, X18Q / E / D, X21 V / l, X38R / K / H / W, X59K / E, X228S and X271 D compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0301] (b) two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or all amino acid substitutions selected from the group consisting of X43K / R, X78N / D, X183D / E, X194E / D, X204D / E, X210I, X212P / A / G / R / K / F / Y / M, X218S / T, X259D, X260D / E and X261 F / W / Y / L compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, wherein said polypeptide comprises amino acid residue D or E at position 101, preferably E at position 101 , referring to the numbering of SEQ ID NO: 2.

[0302] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of at least 79%, at least 80%, 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 90.5%, at least 91 %, at least 91 .5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.1 %, at least 95.2%, at least 95.3%, at least 95.4%, at least 95.5%, at least 95.6%, at least 95.7%, at least 95.8%, at least 95.9% at least 96%, at least 96.1 %, at least 96.2%, at least 96.3%, at least 96.4%, at least 96.5%, at least 96.6%, at least 96.7%, at least 96.8%, at least 96.9%, at least 97%, at least 97.1 %, at least 97.2%, at least 97.3%, at least 97.4%, but less than 97.5% to the amino acid sequence according to SEQ ID NO: 1 and comprises

[0303] (a) two or more, three or more, four or more, five or more, six or all amino acid substitutions selected from the group consisting of S9A / Q / E / C / D, N18Q / E / D, L21 l / V / F / W / Y, T38R / K / H / W, Q59K / E, A228S and E271 D compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ I D NO: 2 and

[0304] (b) two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or all amino acid substitutions selected from the group consisting of N43K / R / C / H / D / L / S / W / A / M / Y / Q / F / I, S78N / D / R / W / F / H / K / E / L / Y / M / C / Q, N183D / E / C / Q / A / M, A194E / D / C, N204D / E / C / G, P210I / V,

[0305] S212G / M / R / K / P / A / F / H / W / N / Y / D, N218S / T / D, S259D / E, T260D / E / K and N261 L / M / F / E / W / Y / C compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, wherein said polypeptide comprises amino acid residue D or E at position 101, preferably E at position 101 , referring to the numbering of SEQ ID NO: 2.

[0306] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of at least 79%, at least 80%, 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 90.5%, at least 91 %, at least 91 .5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.1 %, at least 95.2%, at least 95.3%, at least 95.4%, at least 95.5%, at least 95.6%, at least 95.7%, at least 95.8%, at least 95.9% at least 96%, at least 96.1 %, at least 96.2%, at least 96.3%, at least 96.4%, at least 96.5%, at least 96.6%, at least 96.7%, at least 96.8%, at least 96.9%, at least 97%, at least 97.1 %, at least 97.2%, at least 97.3%, at least 97.4%, but less than 97.5% to the amino acid sequence according to SEQ ID NO: 1 and comprises

[0307] (a) two or more, three or more, four or more, five or more, six or all amino acid substitutions selected from the group consisting of S9A / Q, N18Q / E / D, L21V / I, T38R / K / H / W, Q59K / E, A228S and E271 D compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0308] (b) two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or all amino acid substitutions from the group consisting of N43K / R, S78N / D, N183D / E, A194E / D, N204D / E, P210I, S212P / A / G / R / K / F / Y / M, N218S / T, S259D, T260D / E and N261 F / W / Y / L compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, wherein said polypeptide comprises amino acid residue D or E at position 101, preferably E at position 101 , referring to the numbering of SEQ ID NO: 2.

[0309] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of 79% to 98%, 80% to 98%, 80.5% to 98%, 81 % to 98%, 81 .5% to 98%, 82% to 98%, 82.5% to 98%, 83% to 98%, 83.5% to

[0310] 98%, 84% to 98%, 84.5% to 98%, 85% to 98%, 85.5% to 98%, 86% to 98%, 86.5% to 98%, 87% to 98%, 87.5% to

[0311] 98%, 88% to 98%, 88.5% to 98%, 89% to 98%, 89.5% to 98%, 90% to 98%, 90.3% to 98%, 90.5% to 98%, 90.7% to

[0312] 98%, 91 % to 98%, 91 .3% to 98%, 91 .5% to 98%, 91 .7% to 98%, 92% to 98%, 92.3% to 98%, 92.5% to 98%, 92.7% to 98%, 93% to 98%, 93.3% to 98%, 93.5% to 98%, 93.7% to 98%, 94% to 98%, 94.3% to 98%, 94.5% to 98%, 94.7% to 98%, 95% to 98%, 95.1 % to 98%, 95.2% to 98%, 95.3% to 98%, 95.4% to 98%, 95.5% to 98% to the amino acid sequence according to SEQ ID NO: 1 and comprises

[0313] (a) an amino acid substitution at two or more, three or more, four or more, five or more, six or all amino acid residues selected from the group consisting of 9, 18, 21 , 38, 59, 228, and 271 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0314] (b) an amino acid substitution at two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or all amino acid residues selected from the group consisting of 43, 78, 183, 194, 204, 210, 212, 218, 259, 260 and 261 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2.

[0315] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of 79% to 98%, 80% to 98%, 80.5% to 98%, 81 % to 98%, 81 .5% to 98%, 82% to 98%, 82.5% to 98%, 83% to 98%, 83.5% to

[0316] 98%, 84% to 98%, 84.5% to 98%, 85% to 98%, 85.5% to 98%, 86% to 98%, 86.5% to 98%, 87% to 98%, 87.5% to

[0317] 98%, 88% to 98%, 88.5% to 98%, 89% to 98%, 89.5% to 98%, 90% to 98%, 90.3% to 98%, 90.5% to 98%, 90.7% to

[0318] 98%, 91 % to 98%, 91 .3% to 98%, 91 .5% to 98%, 91 .7% to 98%, 92% to 98%, 92.3% to 98%, 92.5% to 98%, 92.7% to 98%, 93% to 98%, 93.3% to 98%, 93.5% to 98%, 93.7% to 98%, 94% to 98%, 94.3% to 98%, 94.5% to 98%, 94.7% to 98%, 95% to 98%, 95.1 % to 98%, 95.2% to 98%, 95.3% to 98%, 95.4% to 98%, 95.5% to 98% to the amino acid sequence according to SEQ ID NO: 1 and comprises

[0319] (a) two or more, three or more, four or more, five or more, six or all amino acid substitutions selected from the group consisting of X9A / Q / E / C / D, X18Q / E / D, X21 l / V / F / W / Y, X38R / K / H / W, X59K / E, X228S and X271 D compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ I D NO: 2 and

[0320] (b) two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or all amino acid substitutions selected from the group consisting of X43K / R / C / H / D / L / S / W / A / M / Y / Q / F / I, X78N / D / R / W / F / H / K / E / L / Y / M / C / Q, X183D / E / C / Q / A / M, X194E / D / C, X204D / E / C / G, X210I / V,

[0321] X212G / M / R / K / P / A / F / H / W / N / Y / D, X218S / T / D, X259D / E, X260D / E / K and X261 L / M / F / E / W / Y / C compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2.

[0322] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of 79% to 98%, 80% to 98%, 80.5% to 98%, 81 % to 98%, 81 .5% to 98%, 82% to 98%, 82.5% to 98%, 83% to 98%, 83.5% to 98%, 84% to 98%, 84.5% to 98%, 85% to 98%, 85.5% to 98%, 86% to 98%, 86.5% to 98%, 87% to 98%, 87.5% to 98%, 88% to 98%, 88.5% to 98%, 89% to 98%, 89.5% to 98%, 90% to 98%, 90.3% to 98%, 90.5% to 98%, 90.7% to 98%, 91 % to 98%, 91 .3% to 98%, 91 .5% to 98%, 91 .7% to 98%, 92% to 98%, 92.3% to 98%, 92.5% to 98%, 92.7% to 98%, 93% to 98%, 93.3% to 98%, 93.5% to 98%, 93.7% to 98%, 94% to 98%, 94.3% to 98%, 94.5% to 98%, 94.7% to 98%, 95% to 98%, 95.1 % to 98%, 95.2% to 98%, 95.3% to 98%, 95.4% to 98%, 95.5% to 98% to the amino acid sequence according to SEQ ID NO: 1 and comprises

[0323] (a) two or more, three or more, four or more, five or more, six or all amino acid substitutions selected from the group consisting of X9A / Q, X18Q / E / D, X21 V / l, X38R / K / H / W, X59K / E, X228S and X271 D compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0324] (b) two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or all amino acid substitutions selected from the group consisting of X43K / R, X78N / D, X183D / E, X194E / D, X204D / E, X210I, X212P / A / G / R / K / F / Y / M, X218S / T, X259D, X260D / E and X261 F / W / Y / L compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2.

[0325] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of 79% to 98%, 80% to 98%, 80.5% to 98%, 81 % to 98%, 81 .5% to 98%, 82% to 98%, 82.5% to 98%, 83% to 98%, 83.5% to 98%, 84% to 98%, 84.5% to 98%, 85% to 98%, 85.5% to 98%, 86% to 98%, 86.5% to 98%, 87% to 98%, 87.5% to 98%, 88% to 98%, 88.5% to 98%, 89% to 98%, 89.5% to 98%, 90% to 98%, 90.3% to 98%, 90.5% to 98%, 90.7% to 98%, 91 % to 98%, 91 .3% to 98%, 91 .5% to 98%, 91 .7% to 98%, 92% to 98%, 92.3% to 98%, 92.5% to 98%, 92.7% to 98%, 93% to 98%, 93.3% to 98%, 93.5% to 98%, 93.7% to 98%, 94% to 98%, 94.3% to 98%, 94.5% to 98%, 94.7% to 98%, 95% to 98%, 95.1 % to 98%, 95.2% to 98%, 95.3% to 98%, 95.4% to 98%, 95.5% to 98% to the amino acid sequence according to SEQ ID NO: 1 and comprises (a) two or more, three or more, four or more, five or more, six or all amino acid substitutions selected from the group consisting of S9A / Q / E / C / D, N18Q / E / D, L21 l / V / F / W / Y, T38R / K / H / W, Q59K / E, A228S and E271 D compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ I D NO: 2 and

[0326] (b) two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or all amino acid substitutions selected from the group consisting of N43K / R / C / H / D / L / S / W / A / M / Y / Q / F / I, S78N / D / R / W / F / H / K / E / L / Y / M / C / Q, N183D / E / C / Q / A / M, A194E / D / C, N204D / E / C / G, P210I / V,

[0327] S212G / M / R / K / P / A / F / H / W / N / Y / D, N218S / T / D, S259D / E, T260D / E / K and N261 L / M / F / E / W / Y / C compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2.

[0328] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of 79% to 98%, 80% to 98%, 80.5% to 98%, 81 % to 98%, 81 .5% to 98%, 82% to 98%, 82.5% to 98%, 83% to 98%, 83.5% to

[0329] 98%, 84% to 98%, 84.5% to 98%, 85% to 98%, 85.5% to 98%, 86% to 98%, 86.5% to 98%, 87% to 98%, 87.5% to

[0330] 98%, 88% to 98%, 88.5% to 98%, 89% to 98%, 89.5% to 98%, 90% to 98%, 90.3% to 98%, 90.5% to 98%, 90.7% to

[0331] 98%, 91 % to 98%, 91 .3% to 98%, 91 .5% to 98%, 91 .7% to 98%, 92% to 98%, 92.3% to 98%, 92.5% to 98%, 92.7% to 98%, 93% to 98%, 93.3% to 98%, 93.5% to 98%, 93.7% to 98%, 94% to 98%, 94.3% to 98%, 94.5% to 98%, 94.7% to 98%, 95% to 98%, 95.1 % to 98%, 95.2% to 98%, 95.3% to 98%, 95.4% to 98%, 95.5% to 98% to the amino acid sequence according to SEQ ID NO: 1 and comprises

[0332] (a) two or more, three or more, four or more, five or more, six or all amino acid substitutions selected from the group consisting of S9A / Q, N18Q / E / D, L21 V / l, T38R / K / H / W, Q59K / E, A228S and E271 D compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0333] (b) two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or all amino acid substitutions selected from the group consisting of N43K / R, S78N / D, N183D / E, A194E / D, N204D / E, P210I, S212P / A / G / R / K / F / Y / M, N218S / T, S259D, T260D / E and N261 F / W / Y / L compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2.

[0334] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of 79% to 98%, 80% to 98%, 80.5% to 98%, 81 % to 98%, 81 .5% to 98%, 82% to 98%, 82.5% to 98%, 83% to 98%, 83.5% to

[0335] 98%, 84% to 98%, 84.5% to 98%, 85% to 98%, 85.5% to 98%, 86% to 98%, 86.5% to 98%, 87% to 98%, 87.5% to

[0336] 98%, 88% to 98%, 88.5% to 98%, 89% to 98%, 89.5% to 98%, 90% to 98%, 90.3% to 98%, 90.5% to 98%, 90.7% to

[0337] 98%, 91 % to 98%, 91 .3% to 98%, 91 .5% to 98%, 91 .7% to 98%, 92% to 98%, 92.3% to 98%, 92.5% to 98%, 92.7% to 98%, 93% to 98%, 93.3% to 98%, 93.5% to 98%, 93.7% to 98%, 94% to 98%, 94.3% to 98%, 94.5% to 98%, 94.7% to 98%, 95% to 98%, 95.1 % to 98%, 95.2% to 98%, 95.3% to 98%, 95.4% to 98%, 95.5% to 98% to the amino acid sequence according to SEQ ID NO: 1 and comprises

[0338] (a) an amino acid substitution at two or more, three or more, four or more, five or more, six or all amino acid residues selected from the group consisting of 9, 18, 21 , 38, 59, 228, and 271 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and (b) an amino acid substitution at two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or all amino acid residues selected from the group consisting of 43, 78, 183, 194, 204, 210, 212, 218, 259, 260 and 261 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, wherein said polypeptide comprises amino acid residue D or E at position 101, preferably E at position 101 , referring to the numbering of SEQ ID NO: 2.

[0339] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of 79% to 98%, 80% to 98%, 80.5% to 98%, 81 % to 98%, 81 .5% to 98%, 82% to 98%, 82.5% to 98%, 83% to 98%, 83.5% to

[0340] 98%, 84% to 98%, 84.5% to 98%, 85% to 98%, 85.5% to 98%, 86% to 98%, 86.5% to 98%, 87% to 98%, 87.5% to

[0341] 98%, 88% to 98%, 88.5% to 98%, 89% to 98%, 89.5% to 98%, 90% to 98%, 90.3% to 98%, 90.5% to 98%, 90.7% to

[0342] 98%, 91 % to 98%, 91 .3% to 98%, 91 .5% to 98%, 91 .7% to 98%, 92% to 98%, 92.3% to 98%, 92.5% to 98%, 92.7% to 98%, 93% to 98%, 93.3% to 98%, 93.5% to 98%, 93.7% to 98%, 94% to 98%, 94.3% to 98%, 94.5% to 98%, 94.7% to 98%, 95% to 98%, 95.1 % to 98%, 95.2% to 98%, 95.3% to 98%, 95.4% to 98%, 95.5% to 98% to the amino acid sequence according to SEQ ID NO: 1 and comprises

[0343] (a) two or more, three or more, four or more, five or more, six or all amino acid substitutions selected from the group consisting of X9A / Q / E / C / D, X18Q / E / D, X21 l / V / F / W / Y, X38R / K / H / W, X59K / E, X228S and X271 D compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ I D NO: 2 and

[0344] (b) two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or all amino acid substitutions selected from the group consisting of X43K / R / C / H / D / L / S / W / A / M / Y / Q / F / I, X78N / D / R / W / F / H / K / E / L / Y / M / C / Q, X183D / E / C / Q / A / M, X194E / D / C, X204D / E / C / G, X210I / V,

[0345] X212G / M / R / K / P / A / F / H / W / N / Y / D, X218S / T / D, X259D / E, X260D / E / K and X261 L / M / F / E / W / Y / C compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, wherein said polypeptide comprises amino acid residue D or E at position 101, preferably E at position 101 , referring to the numbering of SEQ ID NO: 2.

[0346] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of 79% to 98%, 80% to 98%, 80.5% to 98%, 81 % to 98%, 81 .5% to 98%, 82% to 98%, 82.5% to 98%, 83% to 98%, 83.5% to

[0347] 98%, 84% to 98%, 84.5% to 98%, 85% to 98%, 85.5% to 98%, 86% to 98%, 86.5% to 98%, 87% to 98%, 87.5% to

[0348] 98%, 88% to 98%, 88.5% to 98%, 89% to 98%, 89.5% to 98%, 90% to 98%, 90.3% to 98%, 90.5% to 98%, 90.7% to

[0349] 98%, 91 % to 98%, 91 .3% to 98%, 91 .5% to 98%, 91 .7% to 98%, 92% to 98%, 92.3% to 98%, 92.5% to 98%, 92.7% to 98%, 93% to 98%, 93.3% to 98%, 93.5% to 98%, 93.7% to 98%, 94% to 98%, 94.3% to 98%, 94.5% to 98%, 94.7% to 98%, 95% to 98%, 95.1 % to 98%, 95.2% to 98%, 95.3% to 98%, 95.4% to 98%, 95.5% to 98% to the amino acid sequence according to SEQ ID NO: 1 and comprises (a) two or more, three or more, four or more, five or more, six or all amino acid substitutions selected selected from the group consisting of X9A / Q, X18Q / E / D, X21 V / l, X38R / K / H / W, X59K / E, X228S and X271 D compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0350] (b) two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or all amino acid substitutions selected selected from the group consisting of X43K / R, X78N / D, X183D / E, X194E / D, X204D / E, X210I, X212P / A / G / R / K / F / Y / M, X218S / T, X259D, X260D / E and X261 F / W / Y / L compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ I D NO: 2, wherein said polypeptide comprises amino acid residue D or E at position 101, preferably E at position 101 , referring to the numbering of SEQ ID NO: 2.

[0351] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of 79% to 98%, 80% to 98%, 80.5% to 98%, 81 % to 98%, 81 .5% to 98%, 82% to 98%, 82.5% to 98%, 83% to 98%, 83.5% to

[0352] 98%, 84% to 98%, 84.5% to 98%, 85% to 98%, 85.5% to 98%, 86% to 98%, 86.5% to 98%, 87% to 98%, 87.5% to

[0353] 98%, 88% to 98%, 88.5% to 98%, 89% to 98%, 89.5% to 98%, 90% to 98%, 90.3% to 98%, 90.5% to 98%, 90.7% to

[0354] 98%, 91 % to 98%, 91 .3% to 98%, 91 .5% to 98%, 91 .7% to 98%, 92% to 98%, 92.3% to 98%, 92.5% to 98%, 92.7% to 98%, 93% to 98%, 93.3% to 98%, 93.5% to 98%, 93.7% to 98%, 94% to 98%, 94.3% to 98%, 94.5% to 98%, 94.7% to 98%, 95% to 98%, 95.1 % to 98%, 95.2% to 98%, 95.3% to 98%, 95.4% to 98%, 95.5% to 98% to the amino acid sequence according to SEQ ID NO: 1 and comprises

[0355] (a) two or more, three or more, four or more, five or more, six or all amino acid substitutions selected from the group consisting of S9A / Q / E / C / D, N18Q / E / D, L21 l / V / F / W / Y, T38R / K / H / W, Q59K / E, A228S and E271 D compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ I D NO: 2 and

[0356] (b) two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or all amino acid substitutions selected from the group consisting of N43K / R / C / H / D / L / S / VV / A / M / Y / Q / F / I, S78N / D / R / W / F / H / K / E / L / Y / M / C / Q, N183D / E / C / Q / A / M, A194E / D / C, N204D / E / C / G, P210I / V,

[0357] S212G / M / R / K / P / A / F / H / W / N / Y / D, N218S / T / D, S259D / E, T260D / E / K and N261 L / M / F / E / W / Y / C compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, wherein said polypeptide comprises amino acid residue D or E at position 101, preferably E at position 101 , referring to the numbering of SEQ ID NO: 2.

[0358] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of 79% to 98%, 80% to 98%, 80.5% to 98%, 81 % to 98%, 81 .5% to 98%, 82% to 98%, 82.5% to 98%, 83% to 98%, 83.5% to

[0359] 98%, 84% to 98%, 84.5% to 98%, 85% to 98%, 85.5% to 98%, 86% to 98%, 86.5% to 98%, 87% to 98%, 87.5% to

[0360] 98%, 88% to 98%, 88.5% to 98%, 89% to 98%, 89.5% to 98%, 90% to 98%, 90.3% to 98%, 90.5% to 98%, 90.7% to

[0361] 98%, 91 % to 98%, 91 .3% to 98%, 91 .5% to 98%, 91 .7% to 98%, 92% to 98%, 92.3% to 98%, 92.5% to 98%, 92.7% to 98%, 93% to 98%, 93.3% to 98%, 93.5% to 98%, 93.7% to 98%, 94% to 98%, 94.3% to 98%, 94.5% to 98%, 94.7% to 98%, 95% to 98%, 95.1 % to 98%, 95.2% to 98%, 95.3% to 98%, 95.4% to 98%, 95.5% to 98% to the amino acid sequence according to SEQ ID NO: 1 and comprises

[0362] (a) two or more, three or more, four or more, five or more, six or all amino acid substitutions selected from the group consisting of S9A / Q, N18Q / E / D, L21V / I, T38R / K / H / W, Q59K / E, A228S and E271 D compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0363] (b) two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or all amino acid substitutions selected from the group consisting of N43K / R, S78N / D, N183D / E, A194E / D, N204D / E, P210I, S212P / A / G / R / K / F / Y / M, N218S / T, S259D, T260D / E and N261 F / W / Y / L compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, wherein said polypeptide comprises amino acid residue D or E at position 101, preferably E at position 101 , referring to the numbering of SEQ ID NO: 2.

[0364] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of at least 79%, at least 80%, 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 90.5%, at least 91 %, at least 91 .5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.1 %, at least 95.2%, at least 95.3%, at least 95.4%, at least 95.5%, at least 95.6%, at least 95.7%, at least 95.8%, at least 95.9% at least 96%, at least 96.1 %, at least 96.2%, at least 96.3%, at least 96.4%, at least 96.5%, at least 96.6%, at least 96.7%, at least 96.8%, at least 96.9%, at least 97%, at least 97.1 %, at least 97.2%, at least 97.3%, at least 97.4%, but less than 97.5% to the amino acid sequence according to SEQ ID NO: 1 and comprises

[0365] (a) an amino acid substitution at two or more, three or more, four or all amino acid residues selected from the group consisting of 9, 18, 38, 59, and 271 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0366] (b) an amino acid substitution at two or more, three or more, four or more, five or more, six or more, seven or more, eight or all amino acid residues selected from the group consisting of 43, 78, 183, 194, 204, 212, 218, 259 and 261 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2. In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of at least 79%, at least 80%, 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 90.5%, at least 91 %, at least 91 .5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.1 %, at least 95.2%, at least 95.3%, at least 95.4%, at least 95.5%, at least 95.6%, at least 95.7%, at least 95.8%, at least 95.9% at least 96%, at least 96.1 %, at least 96.2%, at least 96.3%, at least 96.4%, at least 96.5%, at least 96.6%, at least 96.7%, at least 96.8%, at least 96.9%, at least 97%, at least 97.1 %, at least 97.2%, at least 97.3%, at least 97.4%, but less than 97.5% to the amino acid sequence according to SEQ ID NO: 1 and comprises (a) two or more, three or more, four or all amino acid substitutions selected from the group consisting of X9A / Q / E / C / D, X18Q / E / D, X38R / K / H / W, X59K / E and X271 D compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0367] (b) two or more, three or more, four or more, five or more, six or more, seven or more, eight or all amino acid substitutions selected from the group consisting of X43K / R / C / H / D / L / S / W / A / M / Y / Q / F / I, X78N / D / R / W / F / H / K / E / L / Y / M / C / Q, X183D / E / C / Q / A / M, X194E / D / C, X204D / E / C / G, X212G / M / R / K / P / A / F / H / W / N / Y / D, X218S / T / D, X259D / E and

[0368] X261 L / M / F / E / W / Y / C compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2.

[0369] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of at least 79%, at least 80%, 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 90.5%, at least 91 %, at least 91 .5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.1 %, at least 95.2%, at least 95.3%, at least 95.4%, at least 95.5%, at least 95.6%, at least 95.7%, at least 95.8%, at least 95.9% at least 96%, at least 96.1 %, at least 96.2%, at least 96.3%, at least 96.4%, at least 96.5%, at least 96.6%, at least 96.7%, at least 96.8%, at least 96.9%, at least 97%, at least 97.1 %, at least 97.2%, at least 97.3%, at least 97.4%, but less than 97.5% to the amino acid sequence according to SEQ ID NO: 1 and comprises

[0370] (a) two or more, three or more, four or all amino acid substitutions selected from the group consisting of X9A / Q, X18Q / E / D, X21V / I, X38R / K / H / W, X59K and X271 D compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0371] (b) two or more, three or more, four or more, five or more, six or more, seven or more, eight or all amino acid substitutions selected from the group consisting of X43K / R, X78N / D, X183D / E, X194E / D, X204D / E, X212P / A / G / R / K / F / Y / M, X218S / T, X259D and X261 F / W / Y / L compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2.

[0372] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of at least 79%, at least 80%, 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 90.5%, at least 91 %, at least 91 .5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.1 %, at least 95.2%, at least 95.3%, at least 95.4%, at least 95.5%, at least 95.6%, at least 95.7%, at least 95.8%, at least 95.9% at least 96%, at least 96.1 %, at least 96.2%, at least 96.3%, at least 96.4%, at least 96.5%, at least 96.6%, at least 96.7%, at least 96.8%, at least 96.9%, at least 97%, at least 97.1 %, at least 97.2%, at least 97.3%, at least 97.4%, but less than 97.5% to the amino acid sequence according to SEQ ID NO: 1 and comprises

[0373] (a) two or more, three or more, four or all amino acid substitutions selected from the group consisting of S9A / Q / E / C / D, N18Q / E / D, T38R / K / H / W, Q59K / E and E271 D compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and (b) two or more, three or more, four or more, five or more, six or more, seven or more, eight or all amino acid substitutions selected from the group consisting of N43K / R / C / H / D / L / S / W / A / M / Y / Q / F / I, S78N / D / R / W / F / H / K / E / L / Y / M / C / Q, N183D / E / C / Q / A / M, A194E / D / C, N204D / E / C / G, S212G / M / R / K / P / A / F / H / W / N / Y / D, N218S / T / D, S259D / E and

[0374] N261 L / M / F / E / W / Y / C compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2.

[0375] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of at least 79%, at least 80%, 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 90.5%, at least 91 %, at least 91 .5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.1 %, at least 95.2%, at least 95.3%, at least 95.4%, at least 95.5%, at least 95.6%, at least 95.7%, at least 95.8%, at least 95.9% at least 96%, at least 96.1 %, at least 96.2%, at least 96.3%, at least 96.4%, at least 96.5%, at least 96.6%, at least 96.7%, at least 96.8%, at least 96.9%, at least 97%, at least 97.1 %, at least 97.2%, at least 97.3%, at least 97.4%, but less than 97.5% to the amino acid sequence according to SEQ ID NO: 1 and comprises

[0376] (a) two or more, three or more, four or all amino acid substitutions selected from the group consisting of S9A / Q,

[0377] N18Q / E / D, T38R / K / H / W, Q59K and E271 D compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0378] (b) two or more, three or more, four or more, five or more, six or more, seven or more, eight or all amino acid substitutions selected from the group consisting of N43K / R, S78N / D, N183D / E, A194E / D, N204D / E,

[0379] S212P / A / G / R / K / F / Y / M, N218S / T, S259D and N261 F / W / Y / L compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2.

[0380] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of at least 79%, at least 80%, 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 90.5%, at least 91 %, at least 91 .5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.1 %, at least 95.2%, at least 95.3%, at least 95.4%, at least 95.5%, at least 95.6%, at least 95.7%, at least 95.8%, at least 95.9% at least 96%, at least 96.1 %, at least 96.2%, at least 96.3%, at least 96.4%, at least 96.5%, at least 96.6%, at least 96.7%, at least 96.8%, at least 96.9%, at least 97%, at least 97.1 %, at least 97.2%, at least 97.3%, at least 97.4%, but less than 97.5% to the amino acid sequence according to SEQ ID NO: 1 and comprises

[0381] (a) an amino acid substitution at two or more, three or more, four or all amino acid residues selected from the group consisting of 9, 18, 38, 59, and 271 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0382] (b) an amino acid substitution at two or more, three or more, four or more, five or more, six or more, seven or more, eight or all amino acid residues selected from the group consisting of 43, 78, 183, 194, 204, 212, 218, 259 and 261 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, wherein said polypeptide comprises amino acid residue D or E at position 101, preferably E at position 101 , referring to the numbering of SEQ ID NO: 2.

[0383] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of at least 79%, at least 80%, 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 90.5%, at least 91 %, at least 91 .5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.1 %, at least 95.2%, at least 95.3%, at least 95.4%, at least 95.5%, at least 95.6%, at least 95.7%, at least 95.8%, at least 95.9% at least 96%, at least 96.1 %, at least 96.2%, at least 96.3%, at least 96.4%, at least 96.5%, at least 96.6%, at least 96.7%, at least 96.8%, at least 96.9%, at least 97%, at least 97.1 %, at least 97.2%, at least 97.3%, at least 97.4%, but less than 97.5% to the amino acid sequence according to SEQ ID NO: 1 and comprises

[0384] (a) two or more, three or more, four or all amino acid substitutions selected from the group consisting of X9A / Q / E / C / D, X18Q / E / D, X38R / K / H / W, X59K / E and X271 D compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0385] (b) two or more, three or more, four or more, five or more, six or more, seven or more, eight or all amino acid substitutions selected from the group consisting of X43K / R / C / H / D / L / S / W / A / M / Y / Q / F / I, X78N / D / R / W / F / H / K / E / L / Y / M / C / Q, X183D / E / C / Q / A / M, X194E / D / C, X204D / E / C / G, X212G / M / R / K / P / A / F / H / W / N / Y / D, X218S / T / D, X259D / E and

[0386] X261 L / M / F / E / W / Y / C compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, wherein said polypeptide comprises amino acid residue D or E at position 101, preferably E at position 101 , referring to the numbering of SEQ ID NO: 2.

[0387] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of at least 79%, at least 80%, 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 90.5%, at least 91 %, at least 91 .5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.1 %, at least 95.2%, at least 95.3%, at least 95.4%, at least 95.5%, at least 95.6%, at least 95.7%, at least 95.8%, at least 95.9% at least 96%, at least 96.1 %, at least 96.2%, at least 96.3%, at least 96.4%, at least 96.5%, at least 96.6%, at least 96.7%, at least 96.8%, at least 96.9%, at least 97%, at least 97.1 %, at least 97.2%, at least 97.3%, at least 97.4%, but less than 97.5% to the amino acid sequence according to SEQ ID NO: 1 and comprises

[0388] (a) two or more, three or more, four or all amino acid substitutions selected from the group consisting of X9A / Q, X18Q / E / D, X21V / I, X38R / K / H / W, X59K and X271 D compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0389] (b) two or more, three or more, four or more, five or more, six or more, seven or more, eight or all amino acid substitutions selected from the group consisting of X43K / R, X78N / D, X183D / E, X194E / D, X204D / E, X212P / A / G / R / K / F / Y / M, X218S / T, X259D and X261 F / W / Y / L compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, wherein said polypeptide comprises amino acid residue D or E at position 101, preferably E at position 101 , referring to the numbering of SEQ ID NO: 2.

[0390] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of at least 79%, at least 80%, 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 90.5%, at least 91 %, at least 91 .5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.1 %, at least 95.2%, at least 95.3%, at least 95.4%, at least 95.5%, at least 95.6%, at least 95.7%, at least 95.8%, at least 95.9% at least 96%, at least 96.1 %, at least 96.2%, at least 96.3%, at least 96.4%, at least 96.5%, at least 96.6%, at least 96.7%, at least 96.8%, at least 96.9%, at least 97%, at least 97.1 %, at least 97.2%, at least 97.3%, at least 97.4%, but less than 97.5% to the amino acid sequence according to SEQ ID NO: 1 and comprises

[0391] (a) two or more, three or more, four or all amino acid substitutions selected from the group consisting of S9A / Q / E / C / D, N18Q / E / D, T38R / K / H / W, Q59K / E and E271 D compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0392] (b) two or more, three or more, four or more, five or more, six or more, seven or more, eight or all amino acid substitutions selected from the group consisting of N43K / R / C / H / D / L / S / W / A / M / Y / Q / F / I, S78N / D / R / W / F / H / K / E / L / Y / M / C / Q, N183D / E / C / Q / A / M, A194E / D / C, N204D / E / C / G, S212G / M / R / K / P / A / F / H / W / N / Y / D, N218S / T / D, S259D / E and

[0393] N261 L / M / F / E / W / Y / C compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, wherein said polypeptide comprises amino acid residue D or E at position 101, preferably E at position 101 , referring to the numbering of SEQ ID NO: 2.

[0394] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of at least 79%, at least 80%, 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 90.5%, at least 91 %, at least 91 .5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.1 %, at least 95.2%, at least 95.3%, at least 95.4%, at least 95.5%, at least 95.6%, at least 95.7%, at least 95.8%, at least 95.9% at least 96%, at least 96.1 %, at least 96.2%, at least 96.3%, at least 96.4%, at least 96.5%, at least 96.6%, at least 96.7%, at least 96.8%, at least 96.9%, at least 97%, at least 97.1 %, at least 97.2%, at least 97.3%, at least 97.4%, but less than 97.5% to the amino acid sequence according to SEQ ID NO: 1 and comprises

[0395] (a) two or more, three or more, four or all amino acid substitutions selected from the group consisting of S9A / Q, N18Q / E / D, T38R / K / H / W, Q59K and E271 D compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and (b) two or more, three or more, four or more, five or more, six or more, seven or more, eight or all amino acid substitutions selected from the group consisting of N43K / R, S78N / D, N183D / E, A194E / D, N204D / E,

[0396] S212P / A / G / R / K / F / Y / M, N218S / T, S259D and N261 F / W / Y / L compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, wherein said polypeptide comprises amino acid residue D or E at position 101, preferably E at position 101 , referring to the numbering of SEQ ID NO: 2.

[0397] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of 79% to 98%, 80% to 98%, 80.5% to 98%, 81 % to 98%, 81 .5% to 98%, 82% to 98%, 82.5% to 98%, 83% to 98%, 83.5% to

[0398] 98%, 84% to 98%, 84.5% to 98%, 85% to 98%, 85.5% to 98%, 86% to 98%, 86.5% to 98%, 87% to 98%, 87.5% to

[0399] 98%, 88% to 98%, 88.5% to 98%, 89% to 98%, 89.5% to 98%, 90% to 98%, 90.3% to 98%, 90.5% to 98%, 90.7% to

[0400] 98%, 91 % to 98%, 91 .3% to 98%, 91 .5% to 98%, 91 .7% to 98%, 92% to 98%, 92.3% to 98%, 92.5% to 98%, 92.7% to 98%, 93% to 98%, 93.3% to 98%, 93.5% to 98%, 93.7% to 98%, 94% to 98%, 94.3% to 98%, 94.5% to 98%, 94.7% to 98%, 95% to 98%, 95.1 % to 98%, 95.2% to 98%, 95.3% to 98%, 95.4% to 98%, 95.5% to 98% to the amino acid sequence according to SEQ ID NO: 1 and comprises

[0401] (a) an amino acid substitution at two or more, three or more, four or all amino acid residues selected from the group consisting of 9, 18, 38, 59, and 271 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0402] (b) an amino acid substitution at two or more, three or more, four or more, five or more, six or more, seven or more, eight or all amino acid residues selected from the group consisting of 43, 78, 183, 194, 204, 212, 218, 259 and 261 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2. In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of 79% to 98%, 80% to 98%, 80.5% to 98%, 81 % to 98%, 81 .5% to 98%, 82% to 98%, 82.5% to 98%, 83% to 98%, 83.5% to

[0403] 98%, 84% to 98%, 84.5% to 98%, 85% to 98%, 85.5% to 98%, 86% to 98%, 86.5% to 98%, 87% to 98%, 87.5% to

[0404] 98%, 88% to 98%, 88.5% to 98%, 89% to 98%, 89.5% to 98%, 90% to 98%, 90.3% to 98%, 90.5% to 98%, 90.7% to

[0405] 98%, 91 % to 98%, 91 .3% to 98%, 91 .5% to 98%, 91 .7% to 98%, 92% to 98%, 92.3% to 98%, 92.5% to 98%, 92.7% to 98%, 93% to 98%, 93.3% to 98%, 93.5% to 98%, 93.7% to 98%, 94% to 98%, 94.3% to 98%, 94.5% to 98%, 94.7% to 98%, 95% to 98%, 95.1 % to 98%, 95.2% to 98%, 95.3% to 98%, 95.4% to 98%, 95.5% to 98% to the amino acid sequence according to SEQ ID NO: 1 and comprises

[0406] (a) two or more, three or more, four or all amino acid substitutions selected from the group consisting of X9A / Q / E / C / D, X18Q / E / D, X38R / K / H / W, X59K / E and X271 D compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0407] (b) two or more, three or more, four or more, five or more, six or more, seven or more, eight or all amino acid substitutions selected from the group consisting of X43K / R / C / H / D / L / S / W / A / M / Y / Q / F / I, X78N / D / R / W / F / H / K / E / L / Y / M / C / Q, X183D / E / C / Q / A / M, X194E / D / C, X204D / E / C / G, X212G / M / R / K / P / A / F / H / W / N / Y / D, X218S / T / D, X259D / E and X261 L / M / F / E / W / Y / C compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2.

[0408] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of 79% to 98%, 80% to 98%, 80.5% to 98%, 81 % to 98%, 81 .5% to 98%, 82% to 98%, 82.5% to 98%, 83% to 98%, 83.5% to

[0409] 98%, 84% to 98%, 84.5% to 98%, 85% to 98%, 85.5% to 98%, 86% to 98%, 86.5% to 98%, 87% to 98%, 87.5% to

[0410] 98%, 88% to 98%, 88.5% to 98%, 89% to 98%, 89.5% to 98%, 90% to 98%, 90.3% to 98%, 90.5% to 98%, 90.7% to

[0411] 98%, 91 % to 98%, 91 .3% to 98%, 91 .5% to 98%, 91 .7% to 98%, 92% to 98%, 92.3% to 98%, 92.5% to 98%, 92.7% to 98%, 93% to 98%, 93.3% to 98%, 93.5% to 98%, 93.7% to 98%, 94% to 98%, 94.3% to 98%, 94.5% to 98%, 94.7% to 98%, 95% to 98%, 95.1 % to 98%, 95.2% to 98%, 95.3% to 98%, 95.4% to 98%, 95.5% to 98% to the amino acid sequence according to SEQ ID NO: 1 and comprises

[0412] (a) two or more, three or more, four or all amino acid substitutions selected from the group consisting of X9A / Q, X18Q / E / D, X21V / I, X38R / K / H / W, X59K and X271 D compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0413] (b) two or more, three or more, four or more, five or more, six or more, seven or more, eight or all amino acid substitutions selected from the group consisting of X43K / R, X78N / D, X183D / E, X194E / D, X204D / E, X212P / A / G / R / K / F / Y / M, X218S / T, X259D and X261 F / W / Y / L compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2.

[0414] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of 79% to 98%, 80% to 98%, 80.5% to 98%, 81 % to 98%, 81 .5% to 98%, 82% to 98%, 82.5% to 98%, 83% to 98%, 83.5% to

[0415] 98%, 84% to 98%, 84.5% to 98%, 85% to 98%, 85.5% to 98%, 86% to 98%, 86.5% to 98%, 87% to 98%, 87.5% to

[0416] 98%, 88% to 98%, 88.5% to 98%, 89% to 98%, 89.5% to 98%, 90% to 98%, 90.3% to 98%, 90.5% to 98%, 90.7% to

[0417] 98%, 91 % to 98%, 91 .3% to 98%, 91 .5% to 98%, 91 .7% to 98%, 92% to 98%, 92.3% to 98%, 92.5% to 98%, 92.7% to 98%, 93% to 98%, 93.3% to 98%, 93.5% to 98%, 93.7% to 98%, 94% to 98%, 94.3% to 98%, 94.5% to 98%, 94.7% to 98%, 95% to 98%, 95.1 % to 98%, 95.2% to 98%, 95.3% to 98%, 95.4% to 98%, 95.5% to 98% to the amino acid sequence according to SEQ ID NO: 1 and comprises

[0418] (a) two or more, three or more, four or all amino acid substitutions selected from the group consisting of S9A / Q / E / C / D, N18Q / E / D, T38R / K / H / W, Q59K / E and E271 D compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0419] (b) two or more, three or more, four or more, five or more, six or more, seven or more, eight or all amino acid substitutions selected from the group consisting of N43K / R / C / H / D / L / S / W / A / M / Y / Q / F / I, S78N / D / R / W / F / H / K / E / L / Y / M / C / Q, N183D / E / C / Q / A / M, A194E / D / C, N204D / E / C / G, S212G / M / R / K / P / A / F / H / W / N / Y / D, N218S / T / D, S259D / E and

[0420] N261 L / M / F / E / W / Y / C compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2. In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of 79% to 98%, 80% to 98%, 80.5% to 98%, 81 % to 98%, 81 .5% to 98%, 82% to 98%, 82.5% to 98%, 83% to 98%, 83.5% to

[0421] 98%, 84% to 98%, 84.5% to 98%, 85% to 98%, 85.5% to 98%, 86% to 98%, 86.5% to 98%, 87% to 98%, 87.5% to

[0422] 98%, 88% to 98%, 88.5% to 98%, 89% to 98%, 89.5% to 98%, 90% to 98%, 90.3% to 98%, 90.5% to 98%, 90.7% to

[0423] 98%, 91 % to 98%, 91 .3% to 98%, 91 .5% to 98%, 91 .7% to 98%, 92% to 98%, 92.3% to 98%, 92.5% to 98%, 92.7% to 98%, 93% to 98%, 93.3% to 98%, 93.5% to 98%, 93.7% to 98%, 94% to 98%, 94.3% to 98%, 94.5% to 98%, 94.7% to 98%, 95% to 98%, 95.1 % to 98%, 95.2% to 98%, 95.3% to 98%, 95.4% to 98%, 95.5% to 98% to the amino acid sequence according to SEQ ID NO: 1 and comprises

[0424] (a) two or more, three or more, four or all amino acid substitutions selected from the group consisting of S9A / Q,

[0425] N18Q / E / D, T38R / K / H / W, Q59K and E271 D compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0426] (b) two or more, three or more, four or more, five or more, six or more, seven or more, eight or all amino acid substitutions selected from the group consisting of N43K / R, S78N / D, N183D / E, A194E / D, N204D / E,

[0427] S212P / A / G / R / K / F / Y / M, N218S / T, S259D and N261 F / W / Y / L compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2.

[0428] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of 79% to 98%, 80% to 98%, 80.5% to 98%, 81 % to 98%, 81 .5% to 98%, 82% to 98%, 82.5% to 98%, 83% to 98%, 83.5% to

[0429] 98%, 84% to 98%, 84.5% to 98%, 85% to 98%, 85.5% to 98%, 86% to 98%, 86.5% to 98%, 87% to 98%, 87.5% to

[0430] 98%, 88% to 98%, 88.5% to 98%, 89% to 98%, 89.5% to 98%, 90% to 98%, 90.3% to 98%, 90.5% to 98%, 90.7% to

[0431] 98%, 91 % to 98%, 91 .3% to 98%, 91 .5% to 98%, 91 .7% to 98%, 92% to 98%, 92.3% to 98%, 92.5% to 98%, 92.7% to 98%, 93% to 98%, 93.3% to 98%, 93.5% to 98%, 93.7% to 98%, 94% to 98%, 94.3% to 98%, 94.5% to 98%, 94.7% to 98%, 95% to 98%, 95.1 % to 98%, 95.2% to 98%, 95.3% to 98%, 95.4% to 98%, 95.5% to 98% to the amino acid sequence according to SEQ ID NO: 1 and comprises

[0432] (a) an amino acid substitution at two or more, three or more, four or all amino acid residues selected from the group consisting of 9, 18, 38, 59, and 271 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0433] (b) an amino acid substitution at two or more, three or more, four or more, five or more, six or more, seven or more, eight or all amino acid residues selected from the group consisting of 43, 78, 183, 194, 204, 212, 218, 259 and 261 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, wherein said polypeptide comprises amino acid residue D or E at position 101, preferably E at position 101 , referring to the numbering of SEQ ID NO: 2.

[0434] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of 79% to 98%, 80% to 98%, 80.5% to 98%, 81 % to 98%, 81 .5% to 98%, 82% to 98%, 82.5% to 98%, 83% to 98%, 83.5% to 98%, 84% to 98%, 84.5% to 98%, 85% to 98%, 85.5% to 98%, 86% to 98%, 86.5% to 98%, 87% to 98%, 87.5% to 98%, 88% to 98%, 88.5% to 98%, 89% to 98%, 89.5% to 98%, 90% to 98%, 90.3% to 98%, 90.5% to 98%, 90.7% to 98%, 91 % to 98%, 91 .3% to 98%, 91 .5% to 98%, 91 .7% to 98%, 92% to 98%, 92.3% to 98%, 92.5% to 98%, 92.7% to 98%, 93% to 98%, 93.3% to 98%, 93.5% to 98%, 93.7% to 98%, 94% to 98%, 94.3% to 98%, 94.5% to 98%, 94.7% to 98%, 95% to 98%, 95.1 % to 98%, 95.2% to 98%, 95.3% to 98%, 95.4% to 98%, 95.5% to 98% to the amino acid sequence according to SEQ ID NO: 1 and comprises

[0435] (a) two or more, three or more, four or all amino acid substitutions selected from the group consisting of X9A / Q / E / C / D, X18Q / E / D, X38R / K / H / W, X59K / E and X271 D compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0436] (b) two or more, three or more, four or more, five or more, six or more, seven or more, eight or all amino acid substitutions selected from the group consisting of X43K / R / C / H / D / L / S / W / A / M / Y / Q / F / I, X78N / D / R / W / F / H / K / E / L / Y / M / C / Q, X183D / E / C / Q / A / M, X194E / D / C, X204D / E / C / G, X212G / M / R / K / P / A / F / H / W / N / Y / D, X218S / T / D, X259D / E and

[0437] X261 L / M / F / E / W / Y / C compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, wherein said polypeptide comprises amino acid residue D or E at position 101, preferably E at position 101 , referring to the numbering of SEQ ID NO: 2.

[0438] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of 79% to 98%, 80% to 98%, 80.5% to 98%, 81 % to 98%, 81 .5% to 98%, 82% to 98%, 82.5% to 98%, 83% to 98%, 83.5% to

[0439] 98%, 84% to 98%, 84.5% to 98%, 85% to 98%, 85.5% to 98%, 86% to 98%, 86.5% to 98%, 87% to 98%, 87.5% to

[0440] 98%, 88% to 98%, 88.5% to 98%, 89% to 98%, 89.5% to 98%, 90% to 98%, 90.3% to 98%, 90.5% to 98%, 90.7% to

[0441] 98%, 91 % to 98%, 91 .3% to 98%, 91 .5% to 98%, 91 .7% to 98%, 92% to 98%, 92.3% to 98%, 92.5% to 98%, 92.7% to 98%, 93% to 98%, 93.3% to 98%, 93.5% to 98%, 93.7% to 98%, 94% to 98%, 94.3% to 98%, 94.5% to 98%, 94.7% to 98%, 95% to 98%, 95.1 % to 98%, 95.2% to 98%, 95.3% to 98%, 95.4% to 98%, 95.5% to 98% to the amino acid sequence according to SEQ ID NO: 1 and comprises

[0442] (a) two or more, three or more, four or all amino acid substitutions selected from the group consisting of X9A / Q, X18Q / E / D, X21V / I, X38R / K / H / W, X59K and X271 D compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0443] (b) two or more, three or more, four or more, five or more, six or more, seven or more, eight or all amino acid substitutions selected from the group consisting of X43K / R, X78N / D, X183D / E, X194E / D, X204D / E, X212P / A / G / R / K / F / Y / M, X218S / T, X259D and X261 F / W / Y / L compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, wherein said polypeptide comprises amino acid residue D or E at position 101, preferably E at position 101 , referring to the numbering of SEQ ID NO: 2.

[0444] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of 79% to 98%, 80% to 98%, 80.5% to 98%, 81 % to 98%, 81 .5% to 98%, 82% to 98%, 82.5% to 98%, 83% to 98%, 83.5% to 98%, 84% to 98%, 84.5% to 98%, 85% to 98%, 85.5% to 98%, 86% to 98%, 86.5% to 98%, 87% to 98%, 87.5% to 98%, 88% to 98%, 88.5% to 98%, 89% to 98%, 89.5% to 98%, 90% to 98%, 90.3% to 98%, 90.5% to 98%, 90.7% to 98%, 91 % to 98%, 91 .3% to 98%, 91 .5% to 98%, 91 .7% to 98%, 92% to 98%, 92.3% to 98%, 92.5% to 98%, 92.7% to 98%, 93% to 98%, 93.3% to 98%, 93.5% to 98%, 93.7% to 98%, 94% to 98%, 94.3% to 98%, 94.5% to 98%, 94.7% to 98%, 95% to 98%, 95.1 % to 98%, 95.2% to 98%, 95.3% to 98%, 95.4% to 98%, 95.5% to 98% to the amino acid sequence according to SEQ ID NO: 1 and comprises

[0445] (a) two or more, three or more, four or all amino acid substitutions selected from the group consisting of S9A / Q / E / C / D, N18Q / E / D, T38R / K / H / W, Q59K / E and E271 D compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0446] (b) two or more, three or more, four or more, five or more, six or more, seven or more, eight or all amino acid substitutions selected from the group consisting of N43K / R / C / H / D / L / S / W / A / M / Y / Q / F / I, S78N / D / R / W / F / H / K / E / L / Y / M / C / Q, N183D / E / C / Q / A / M, A194E / D / C, N204D / E / C / G, S212G / M / R / K / P / A / F / H / W / N / Y / D, N218S / T / D, S259D / E and

[0447] N261 L / M / F / E / W / Y / C compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, wherein said polypeptide comprises amino acid residue D or E at position 101, preferably E at position 101 , referring to the numbering of SEQ ID NO: 2.

[0448] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of 79% to 98%, 80% to 98%, 80.5% to 98%, 81 % to 98%, 81 .5% to 98%, 82% to 98%, 82.5% to 98%, 83% to 98%, 83.5% to

[0449] 98%, 84% to 98%, 84.5% to 98%, 85% to 98%, 85.5% to 98%, 86% to 98%, 86.5% to 98%, 87% to 98%, 87.5% to

[0450] 98%, 88% to 98%, 88.5% to 98%, 89% to 98%, 89.5% to 98%, 90% to 98%, 90.3% to 98%, 90.5% to 98%, 90.7% to

[0451] 98%, 91 % to 98%, 91 .3% to 98%, 91 .5% to 98%, 91 .7% to 98%, 92% to 98%, 92.3% to 98%, 92.5% to 98%, 92.7% to 98%, 93% to 98%, 93.3% to 98%, 93.5% to 98%, 93.7% to 98%, 94% to 98%, 94.3% to 98%, 94.5% to 98%, 94.7% to 98%, 95% to 98%, 95.1 % to 98%, 95.2% to 98%, 95.3% to 98%, 95.4% to 98%, 95.5% to 98% to the amino acid sequence according to SEQ ID NO: 1 and comprises

[0452] (a) two or more, three or more, four or all amino acid substitutions selected from the group consisting of S9A / Q,

[0453] N18Q / E / D, T38R / K / H / W, Q59K and E271 D compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0454] (b) two or more, three or more, four or more, five or more, six or more, seven or more, eight or all amino acid substitutions selected from the group consisting of N43K / R, S78N / D, N183D / E, A194E / D, N204D / E,

[0455] S212P / A / G / R / K / F / Y / M, N218S / T, S259D and N261 F / W / Y / L compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, wherein said polypeptide comprises amino acid residue D or E at position 101, preferably E at position 101 , referring to the numbering of SEQ ID NO: 2. The variant polypeptide of the present invention preferably has a sequence identity of at least 79%, at least 80%, 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 90.5%, at least 91 %, at least 91 .5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.1 %, at least 95.2%, at least 95.3%, at least 95.4%, at least 95.5%, at least 95.6%, at least 95.7%, at least 95.8%, at least 95.9% at least 96%, at least 96. 1%, at least 96.2%, at least 96.3%, at least 96.4%, at least 96.5%, at least 96.6%, at least 96.7%, at least 96.8%, at least 96.9%, at least 97%, at least 97.1 %, at least 97.2%, at least 97.3%, at least 97.4%, but less than 97.5% to the amino acid sequence according to SEQ ID NO: 1 and comprises

[0456] (a) an amino acid substitution at two or three amino acid residues selected from the group consisting of 18, 21 and 38 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0457] (b) an amino acid substitution at two or more, three or all amino acid residues selected from the group consisting of 78, 183, 194 and 210 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2.

[0458] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of at least 79%, at least 80%, 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 90.5%, at least 91 %, at least 91 .5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.1 %, at least 95.2%, at least 95.3%, at least 95.4%, at least 95.5%, at least 95.6%, at least 95.7%, at least 95.8%, at least 95.9% at least 96%, at least 96.1 %, at least 96.2%, at least 96.3%, at least 96.4%, at least 96.5%, at least 96.6%, at least 96.7%, at least 96.8%, at least 96.9%, at least 97%, at least 97.1 %, at least 97.2%, at least 97.3%, at least 97.4%, but less than 97.5% to the amino acid sequence according to SEQ ID NO: 1 and comprises

[0459] (a) two or three acid substitutions selected from the group consisting of X18Q / E / D, X21I / V / F / W / Y and X38R / K / H / W compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0460] (b) two or more, three or all amino acid substitutions selected from the group consisting of X78N / D / R / W / F / H / K / E / L / Y / M / C / Q, X183D / E / C / Q / A / M, X194E / D / C and X210I / V compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2.

[0461] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of at least 79%, at least 80%, 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 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.1 %, at least 95.2%, at least 95.3%, at least 95.4%, at least 95.5%, at least 95.6%, at least 95.7%, at least 95.8%, at least 95.9% at least 96%, at least 96.1 %, at least 96.2%, at least 96.3%, at least 96.4%, at least 96.5%, at least 96.6%, at least 96.7%, at least 96.8%, at least 96.9%, at least 97%, at least 97.1 %, at least 97.2%, at least 97.3%, at least 97.4%, but less than 97.5% to the amino acid sequence according to SEQ ID NO: 1 and comprises

[0462] (a) two or three amino acid substitutions selected from the group consisting of X18Q / E / D, X21 V and X38R / K / H / W, compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0463] (b) two or more, three or all amino acid substitutions selected from the group consisting of X78N / D, X183D / E, X194E and X210I compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2.

[0464] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of at least 79%, at least 80%, 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 90.5%, at least 91 %, at least 91 .5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.1 %, at least 95.2%, at least 95.3%, at least 95.4%, at least 95.5%, at least 95.6%, at least 95.7%, at least 95.8%, at least 95.9% at least 96%, at least 96.1 %, at least 96.2%, at least 96.3%, at least 96.4%, at least 96.5%, at least 96.6%, at least 96.7%, at least 96.8%, at least 96.9%, at least 97%, at least 97.1 %, at least 97.2%, at least 97.3%, at least 97.4%, but less than 97.5% to the amino acid sequence according to SEQ ID NO: 1 and comprises

[0465] (a) two or three amino acid substitutions selected from the group consisting of N18Q / E / D, L21 l / V / F / W / Y and T38R / K / H / W compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0466] (b) two or more, three or all amino acid substitutions selected from the group consisting of S78N / D / R / W / F / H / K / E / L / Y / M / C / Q, N183D / E / C / Q / A / M, A194E / D / C and P210I / V compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2.

[0467] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of at least 79%, at least 80%, 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 90.5%, at least 91 %, at least 91 .5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.1 %, at least 95.2%, at least 95.3%, at least 95.4%, at least 95.5%, at least 95.6%, at least 95.7%, at least 95.8%, at least 95.9% at least 96%, at least 96.1 %, at least 96.2%, at least 96.3%, at least 96.4%, at least 96.5%, at least 96.6%, at least 96.7%, at least 96.8%, at least 96.9%, at least 97%, at least 97.1 %, at least 97.2%, at least 97.3%, at least 97.4%, but less than 97.5% to the amino acid sequence according to SEQ ID NO: 1 and comprises

[0468] (a) two or three amino acid substitutions selected from the group consisting of N18Q / E / D, L21V and T38R / K / H / W, compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and (b) two or more, three or all amino acid substitutions selected from the group consisting of S78N / D, N183D / E, A194E and P210I compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2.

[0469] The variant polypeptide of the present invention preferably has a sequence identity of at least 79%, at least 80%, 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 90.5%, at least 91 %, at least 91 .5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.1 %, at least 95.2%, at least 95.3%, at least 95.4%, at least 95.5%, at least 95.6%, at least 95.7%, at least 95.8%, at least 95.9% at least 96%, at least 96. 1%, at least 96.2%, at least 96.3%, at least 96.4%, at least 96.5%, at least 96.6%, at least 96.7%, at least 96.8%, at least 96.9%, at least 97%, at least 97.1 %, at least 97.2%, at least 97.3%, at least 97.4%, but less than 97.5% to the amino acid sequence according to SEQ ID NO: 1 and comprises

[0470] (a) an amino acid substitution at two or three amino acid residues selected from the group consisting of 18, 21 and 38 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0471] (b) an amino acid substitution at two or more, three or all amino acid residues selected from the group consisting of 78, 183, 194 and 210 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, wherein said polypeptide comprises amino acid residue D or E at position 101, preferably E at position 101 , referring to the numbering of SEQ ID NO: 2.

[0472] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of at least 79%, at least 80%, 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 90.5%, at least 91 %, at least 91 .5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.1 %, at least 95.2%, at least 95.3%, at least 95.4%, at least 95.5%, at least 95.6%, at least 95.7%, at least 95.8%, at least 95.9% at least 96%, at least 96.1 %, at least 96.2%, at least 96.3%, at least 96.4%, at least 96.5%, at least 96.6%, at least 96.7%, at least 96.8%, at least 96.9%, at least 97%, at least 97.1 %, at least 97.2%, at least 97.3%, at least 97.4%, but less than 97.5% to the amino acid sequence according to SEQ ID NO: 1 and comprises

[0473] (a) two or three amino acid substitutions selected from the group consisting of X18Q / E / D, X21 l / V / F / W / Y and X38R / K / H / W compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0474] (b) two or more, three or all amino acid substitutions selected from the group consisting of X78N / D / R / W / F / H / K / E / L / Y / M / C / Q, X183D / E / C / Q / A / M, X194E / D / C and X210I / V compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, wherein said polypeptide comprises amino acid residue D or E at position 101, preferably E at position 101 , referring to the numbering of SEQ ID NO: 2.

[0475] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of at least 79%, at least 80%, 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 90.5%, at least 91 %, at least 91 .5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.1 %, at least 95.2%, at least 95.3%, at least 95.4%, at least 95.5%, at least 95.6%, at least 95.7%, at least 95.8%, at least 95.9% at least 96%, at least 96.1 %, at least 96.2%, at least 96.3%, at least 96.4%, at least 96.5%, at least 96.6%, at least 96.7%, at least 96.8%, at least 96.9%, at least 97%, at least 97.1 %, at least 97.2%, at least 97.3%, at least 97.4%, but less than 97.5% to the amino acid sequence according to SEQ ID NO: 1 and comprises

[0476] (a) two or three amino acid substitutions selected from the group consisting of X18Q / E / D, X21 V and X38R / K / H / W, compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0477] (b) two or more, three or all amino acid substitutions selected from the group consisting of X78N / D, X183D / E, X194E and X210I compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, wherein said polypeptide comprises amino acid residue D or E at position 101, preferably E at position 101 , referring to the numbering of SEQ ID NO: 2.

[0478] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of at least 79%, at least 80%, 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 90.5%, at least 91 %, at least 91 .5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.1 %, at least 95.2%, at least 95.3%, at least 95.4%, at least 95.5%, at least 95.6%, at least 95.7%, at least 95.8%, at least 95.9% at least 96%, at least 96.1 %, at least 96.2%, at least 96.3%, at least 96.4%, at least 96.5%, at least 96.6%, at least 96.7%, at least 96.8%, at least 96.9%, at least 97%, at least 97.1 %, at least 97.2%, at least 97.3%, at least 97.4%, but less than 97.5% to the amino acid sequence according to SEQ ID NO: 1 and comprises

[0479] (a) two or three amino acid substitutions selected from the group consisting of N18Q / E / D, L21 l / V / F / W / Y and T38R / K / H / W compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0480] (b) two or more, three or all amino acid substitutions selected from the group consisting of S78N / D / R / W / F / H / K / E / L / Y / M / C / Q, N183D / E / C / Q / A / M, A194E / D / C and P210I / V compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, wherein said polypeptide comprises amino acid residue D or E at position 101, preferably E at position 101 , referring to the numbering of SEQ ID NO: 2. In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of at least 79%, at least 80%, 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 90.5%, at least 91 %, at least 91 .5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.1 %, at least 95.2%, at least 95.3%, at least 95.4%, at least 95.5%, at least 95.6%, at least 95.7%, at least 95.8%, at least 95.9% at least 96%, at least 96.1 %, at least 96.2%, at least 96.3%, at least 96.4%, at least 96.5%, at least 96.6%, at least 96.7%, at least 96.8%, at least 96.9%, at least 97%, at least 97.1 %, at least 97.2%, at least 97.3%, at least 97.4%, but less than 97.5% to the amino acid sequence according to SEQ ID NO: 1 and comprises

[0481] (a) two or three amino acid substitutions selected from the group consisting of L21V / I, T38R / K / H / W, P210I and A228S compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0482] (b) two or more, three or all amino acid substitutions selected from the group consisting of N18Q / E / D, N183D and N204D compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 wherein said polypeptide comprises amino acid residue D or E at position 101, preferably E at position 101 , referring to the numbering of SEQ ID NO: 2.

[0483] The variant polypeptide of the present invention preferably has a sequence identity of 79% to 98%, 80% to 98%,

[0484] 80.5% to 98%, 81 % to 98%, 81 .5% to 98%, 82% to 98%, 82.5% to 98%, 83% to 98%, 83.5% to 98%, 84% to 98%,

[0485] 84.5% to 98%, 85% to 98%, 85.5% to 98%, 86% to 98%, 86.5% to 98%, 87% to 98%, 87.5% to 98%, 88% to 98%,

[0486] 88.5% to 98%, 89% to 98%, 89.5% to 98%, 90% to 98%, 90.3% to 98%, 90.5% to 98%, 90.7% to 98%, 91 % to 98%,

[0487] 91 .3% to 98%, 91 .5% to 98%, 91.7% to 98%, 92% to 98%, 92.3% to 98%, 92.5% to 98%, 92.7% to 98%, 93% to 98%, 93.3% to 98%, 93.5% to 98%, 93.7% to 98%, 94% to 98%, 94.3% to 98%, 94.5% to 98%, 94.7% to 98%, 95% to 98%, 95.1 % to 98%, 95.2% to 98%, 95.3% to 98%, 95.4% to 98%, 95.5% to 98% to the amino acid sequence according to SEQ I D NO: 1 and comprises

[0488] (a) two or three amino acid substitutions selected from the group consisting of N18Q / E / D, L21V and T38R / K / H / W, compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0489] (b) two or more, three or all amino acid substitutions selected from the group consisting of S78N / D, N183D / E, A194E and P210I compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, wherein said polypeptide comprises amino acid residue D or E at position 101, preferably E at position 101 , referring to the numbering of SEQ ID NO: 2.

[0490] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of 79% to 98%, 80% to 98%, 80.5% to 98%, 81 % to 98%, 81 .5% to 98%, 82% to 98%, 82.5% to 98%, 83% to 98%, 83.5% to 98%, 84% to 98%, 84.5% to 98%, 85% to 98%, 85.5% to 98%, 86% to 98%, 86.5% to 98%, 87% to 98%, 87.5% to 98%, 88% to 98%, 88.5% to 98%, 89% to 98%, 89.5% to 98%, 90% to 98%, 90.3% to 98%, 90.5% to 98%, 90.7% to 98%, 91 % to 98%, 91 .3% to 98%, 91 .5% to 98%, 91 .7% to 98%, 92% to 98%, 92.3% to 98%, 92.5% to 98%, 92.7% to 98%, 93% to 98%, 93.3% to 98%, 93.5% to 98%, 93.7% to 98%, 94% to 98%, 94.3% to 98%, 94.5% to 98%, 94.7% to 98%, 95% to 98%, 95.1 % to 98%, 95.2% to 98%, 95.3% to 98%, 95.4% to 98%, 95.5% to 98% to the amino acid sequence according to SEQ ID NO: 1 and comprises

[0491] (a) an amino acid substitution at two or three amino acid residues selected from the group consisting of 18, 21 and 38 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0492] (b) an amino acid substitution at two or more, three or all amino acid residues selected from the group consisting of 78, 183, 194 and 210 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2.

[0493] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of 79% to 98%, 80% to 98%, 80.5% to 98%, 81 % to 98%, 81 .5% to 98%, 82% to 98%, 82.5% to 98%, 83% to 98%, 83.5% to 98%, 84% to 98%, 84.5% to 98%, 85% to 98%, 85.5% to 98%, 86% to 98%, 86.5% to 98%, 87% to 98%, 87.5% to 98%, 88% to 98%, 88.5% to 98%, 89% to 98%, 89.5% to 98%, 90% to 98%, 90.3% to 98%, 90.5% to 98%, 90.7% to 98%, 91 % to 98%, 91 .3% to 98%, 91 .5% to 98%, 91 .7% to 98%, 92% to 98%, 92.3% to 98%, 92.5% to 98%, 92.7% to 98%, 93% to 98%, 93.3% to 98%, 93.5% to 98%, 93.7% to 98%, 94% to 98%, 94.3% to 98%, 94.5% to 98%, 94.7% to 98%, 95% to 98%, 95.1 % to 98%, 95.2% to 98%, 95.3% to 98%, 95.4% to 98%, 95.5% to 98% to the amino acid sequence according to SEQ ID NO: 1 and comprises

[0494] (a) two or three amino acid substitutions selected from the group consisting of X21 l / V / F / W / Y, X38R / K / H / W, X210I / V and X228S compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0495] (b) two or more, three or all amino acid substitutions selected from the group consisting of X18Q / E / D, X183D / E / C / Q / A / M and X204D / E / C / G compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2.

[0496] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of 79% to 98%, 80% to 98%, 80.5% to 98%, 81 % to 98%, 81 .5% to 98%, 82% to 98%, 82.5% to 98%, 83% to 98%, 83.5% to 98%, 84% to 98%, 84.5% to 98%, 85% to 98%, 85.5% to 98%, 86% to 98%, 86.5% to 98%, 87% to 98%, 87.5% to 98%, 88% to 98%, 88.5% to 98%, 89% to 98%, 89.5% to 98%, 90% to 98%, 90.3% to 98%, 90.5% to 98%, 90.7% to 98%, 91 % to 98%, 91 .3% to 98%, 91 .5% to 98%, 91 .7% to 98%, 92% to 98%, 92.3% to 98%, 92.5% to 98%, 92.7% to 98%, 93% to 98%, 93.3% to 98%, 93.5% to 98%, 93.7% to 98%, 94% to 98%, 94.3% to 98%, 94.5% to 98%, 94.7% to 98%, 95% to 98%, 95.1 % to 98%, 95.2% to 98%, 95.3% to 98%, 95.4% to 98%, 95.5% to 98% to the amino acid sequence according to SEQ ID NO: 1 and comprises (a) two or three amino acid substitutions selected from the group consisting of X18Q / E / D, X21 V and X38R / K / H / W, compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0497] (b) two or more, three or all amino acid substitutions selected from the group consisting of X78N / D, X183D / E, X194E and X210I compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2.

[0498] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of 79% to 98%, 80% to 98%, 80.5% to 98%, 81 % to 98%, 81 .5% to 98%, 82% to 98%, 82.5% to 98%, 83% to 98%, 83.5% to

[0499] 98%, 84% to 98%, 84.5% to 98%, 85% to 98%, 85.5% to 98%, 86% to 98%, 86.5% to 98%, 87% to 98%, 87.5% to

[0500] 98%, 88% to 98%, 88.5% to 98%, 89% to 98%, 89.5% to 98%, 90% to 98%, 90.3% to 98%, 90.5% to 98%, 90.7% to

[0501] 98%, 91 % to 98%, 91 .3% to 98%, 91 .5% to 98%, 91 .7% to 98%, 92% to 98%, 92.3% to 98%, 92.5% to 98%, 92.7% to 98%, 93% to 98%, 93.3% to 98%, 93.5% to 98%, 93.7% to 98%, 94% to 98%, 94.3% to 98%, 94.5% to 98%, 94.7% to 98%, 95% to 98%, 95.1 % to 98%, 95.2% to 98%, 95.3% to 98%, 95.4% to 98%, 95.5% to 98% to the amino acid sequence according to SEQ ID NO: 1 and comprises

[0502] (a) two or three amino acid substitutions selected from the group consisting of N18Q / E / D, L21 l / V / F / W / Y and T38R / K / H / W compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0503] (b) two or more, three or all amino acid substitutions selected from the group consisting of S78N / D / R / W / F / H / K / E / L / Y / M / C / Q, N183D / E / C / Q / A / M, A194E / D / C and P210I / V compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2.

[0504] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of 79% to 98%, 80% to 98%, 80.5% to 98%, 81 % to 98%, 81 .5% to 98%, 82% to 98%, 82.5% to 98%, 83% to 98%, 83.5% to 98%, 84% to 98%, 84.5% to 98%, 85% to 98%, 85.5% to 98%, 86% to 98%, 86.5% to 98%, 87% to 98%, 87.5% to 98%, 88% to 98%, 88.5% to 98%, 89% to 98%, 89.5% to 98%, 90% to 98%, 90.3% to 98%, 90.5% to 98%, 90.7% to 98%, 91% to 98%, 91.3% to 98%, 91.5% to 98%, 91.7% to 98%, 92% to 98%, 92.3% to 98%, 92.5% to 98%, 92.7% to 98%, 93% to 98%, 93.3% to 98%, 93.5% to 98%, 93.7% to 98%, 94% to 98%, 94.3% to 98%, 94.5% to 98%, 94.7% to 98%, 95% to 98%, 95.1 % to 98%, 95.2% to 98%, 95.3% to 98%, 95.4% to 98%, 95.5% to 98% to the amino acid sequence according to SEQ ID NO: 1 and comprises

[0505] (a) two or three amino acid substitutions selected from the group consisting of N18Q / E / D, L21V and T38R / K / H / W, compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0506] (b) two or more, three or all amino acid substitutions selected from the group consisting of S78N / D, N183D / E, A194E and P210I compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2. The variant polypeptide of the present invention preferably has a sequence identity of 79% to 98%, 80% to 98%,

[0507] 80.5% to 98%, 81 % to 98%, 81 .5% to 98%, 82% to 98%, 82.5% to 98%, 83% to 98%, 83.5% to 98%, 84% to 98%,

[0508] 84.5% to 98%, 85% to 98%, 85.5% to 98%, 86% to 98%, 86.5% to 98%, 87% to 98%, 87.5% to 98%, 88% to 98%,

[0509] 88.5% to 98%, 89% to 98%, 89.5% to 98%, 90% to 98%, 90.3% to 98%, 90.5% to 98%, 90.7% to 98%, 91 % to 98%,

[0510] 91 .3% to 98%, 91 .5% to 98%, 91.7% to 98%, 92% to 98%, 92.3% to 98%, 92.5% to 98%, 92.7% to 98%, 93% to 98%, 93.3% to 98%, 93.5% to 98%, 93.7% to 98%, 94% to 98%, 94.3% to 98%, 94.5% to 98%, 94.7% to 98%, 95% to 98%, 95.1 % to 98%, 95.2% to 98%, 95.3% to 98%, 95.4% to 98%, 95.5% to 98% to the amino acid sequence according to SEQ I D NO: 1 and comprises

[0511] (a) an amino acid substitution at two or three amino acid residues selected from the group consisting of 18, 21 and 38 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0512] (b) an amino acid substitution at two or more, three or all amino acid residues selected from the group consisting of 78, 183, 194 and 210 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, wherein said polypeptide comprises amino acid residue D or E at position 101, preferably E at position 101 , referring to the numbering of SEQ ID NO: 2.

[0513] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of 79% to 98%, 80% to 98%, 80.5% to 98%, 81 % to 98%, 81 .5% to 98%, 82% to 98%, 82.5% to 98%, 83% to 98%, 83.5% to 98%, 84% to 98%, 84.5% to 98%, 85% to 98%, 85.5% to 98%, 86% to 98%, 86.5% to 98%, 87% to 98%, 87.5% to 98%, 88% to 98%, 88.5% to 98%, 89% to 98%, 89.5% to 98%, 90% to 98%, 90.3% to 98%, 90.5% to 98%, 90.7% to 98%, 91 % to 98%, 91 .3% to 98%, 91 .5% to 98%, 91 .7% to 98%, 92% to 98%, 92.3% to 98%, 92.5% to 98%, 92.7% to 98%, 93% to 98%, 93.3% to 98%, 93.5% to 98%, 93.7% to 98%, 94% to 98%, 94.3% to 98%, 94.5% to 98%, 94.7% to 98%, 95% to 98%, 95.1 % to 98%, 95.2% to 98%, 95.3% to 98%, 95.4% to 98%, 95.5% to 98% to the amino acid sequence according to SEQ ID NO: 1 and comprises

[0514] (a) two or three amino acid substitutions selected from the group consisting of X18Q / E / D, X21 l / V / F / W / Y and X38R / K / H / W compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0515] (b) two or more, three or all amino acid substitutions selected from the group consisting of X78N / D / R / W / F / H / K / E / L / Y / M / C / Q, X183D / E / C / Q / A / M, X194E / D / C and X210I / V compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, wherein said polypeptide comprises amino acid residue D or E at position 101, preferably E at position 101 , referring to the numbering of SEQ ID NO: 2.

[0516] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of 79% to 98%, 80% to 98%, 80.5% to 98%, 81 % to 98%, 81 .5% to 98%, 82% to 98%, 82.5% to 98%, 83% to 98%, 83.5% to 98%, 84% to 98%, 84.5% to 98%, 85% to 98%, 85.5% to 98%, 86% to 98%, 86.5% to 98%, 87% to 98%, 87.5% to 98%, 88% to 98%, 88.5% to 98%, 89% to 98%, 89.5% to 98%, 90% to 98%, 90.3% to 98%, 90.5% to 98%, 90.7% to 98%, 91 % to 98%, 91 .3% to 98%, 91 .5% to 98%, 91 .7% to 98%, 92% to 98%, 92.3% to 98%, 92.5% to 98%, 92.7% to 98%, 93% to 98%, 93.3% to 98%, 93.5% to 98%, 93.7% to 98%, 94% to 98%, 94.3% to 98%, 94.5% to 98%, 94.7% to 98%, 95% to 98%, 95.1 % to 98%, 95.2% to 98%, 95.3% to 98%, 95.4% to 98%, 95.5% to 98% to the amino acid sequence according to SEQ ID NO: 1 and comprises

[0517] (a) two or three amino acid substitutions selected from the group consisting of X18Q / E / D, X21 V and X38R / K / H / W, compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0518] (b) two or more, three or all amino acid substitutions selected from the group consisting of X78N / D, X183D / E, X194E and X210I compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, wherein said polypeptide comprises amino acid residue D or E at position 101 , preferably E at position 101 , referring to the numbering of SEQ ID NO: 2.

[0519] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of 79% to 98%, 80% to 98%, 80.5% to 98%, 81 % to 98%, 81 .5% to 98%, 82% to 98%, 82.5% to 98%, 83% to 98%, 83.5% to

[0520] 98%, 84% to 98%, 84.5% to 98%, 85% to 98%, 85.5% to 98%, 86% to 98%, 86.5% to 98%, 87% to 98%, 87.5% to

[0521] 98%, 88% to 98%, 88.5% to 98%, 89% to 98%, 89.5% to 98%, 90% to 98%, 90.3% to 98%, 90.5% to 98%, 90.7% to

[0522] 98%, 91 % to 98%, 91 .3% to 98%, 91 .5% to 98%, 91 .7% to 98%, 92% to 98%, 92.3% to 98%, 92.5% to 98%, 92.7% to 98%, 93% to 98%, 93.3% to 98%, 93.5% to 98%, 93.7% to 98%, 94% to 98%, 94.3% to 98%, 94.5% to 98%, 94.7% to 98%, 95% to 98%, 95.1 % to 98%, 95.2% to 98%, 95.3% to 98%, 95.4% to 98%, 95.5% to 98% to the amino acid sequence according to SEQ ID NO: 1 and comprises

[0523] (a) two or three amino acid substitutions selected from the group consisting of N18Q / E / D, L21 l / V / F / W / Y and T38R / K / H / W compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0524] (b) two or more, three or all amino acid substitutions selected from the group consisting of

[0525] S78N / D / R / W / F / H / K / E / L / Y / M / C / Q, N183D / E / C / Q / A / M, A194E / D / C and P210I / V compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, wherein said polypeptide comprises amino acid residue D or E at position 101, preferably E at position 101 , referring to the numbering of SEQ ID NO: 2.

[0526] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of 79% to 98%, 80% to 98%, 80.5% to 98%, 81 % to 98%, 81 .5% to 98%, 82% to 98%, 82.5% to 98%, 83% to 98%, 83.5% to 98%, 84% to 98%, 84.5% to 98%, 85% to 98%, 85.5% to 98%, 86% to 98%, 86.5% to 98%, 87% to 98%, 87.5% to 98%, 88% to 98%, 88.5% to 98%, 89% to 98%, 89.5% to 98%, 90% to 98%, 90.3% to 98%, 90.5% to 98%, 90.7% to 98%, 91% to 98%, 91.3% to 98%, 91.5% to 98%, 91.7% to 98%, 92% to 98%, 92.3% to 98%, 92.5% to 98%, 92.7% to 98%, 93% to 98%, 93.3% to 98%, 93.5% to 98%, 93.7% to 98%, 94% to 98%, 94.3% to 98%, 94.5% to 98%, 94.7% to 98%, 95% to 98%, 95.1 % to 98%, 95.2% to 98%, 95.3% to 98%, 95.4% to 98%, 95.5% to 98% to the amino acid sequence according to SEQ ID NO: 1 and comprises

[0527] (a) two or three amino acid substitutions selected from the group consisting of N18Q / E / D, L21V and T38R / K / H / W, compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0528] (b) two or more, three or all amino acid substitutions selected from the group consisting of S78N / D, N183D / E, A194E and P210I compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, wherein said polypeptide comprises amino acid residue D or E at position 101, preferably E at position 101 , referring to the numbering of SEQ ID NO: 2.

[0529] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of at least 79%, at least 80%, 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 90.5%, at least 91 %, at least 91 .5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.1 %, at least 95.2%, at least 95.3%, at least 95.4%, at least 95.5%, at least 95.6%, at least 95.7%, at least 95.8%, at least 95.9% at least 96%, at least 96.1 %, at least 96.2%, at least 96.3%, at least 96.4%, at least 96.5%, at least 96.6%, at least 96.7%, at least 96.8%, at least 96.9%, at least 97%, at least 97.1 %, at least 97.2%, at least 97.3%, at least 97.4%, but less than 97.5% to the amino acid sequence according to SEQ ID NO: 1 and comprises

[0530] (a) a combination of amino acid substitutions selected from the group consisting of: compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0531] (b) a combination of amino acid substitutions selected from the group consisting of: compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2. In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of at least 79%, at least 80%, 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 90.5%, at least 91 %, at least 91 .5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.1 %, at least 95.2%, at least 95.3%, at least 95.4%, at least 95.5%, at least 95.6%, at least 95.7%, at least 95.8%, at least 95.9% at least 96%, at least 96.1 %, at least 96.2%, at least 96.3%, at least 96.4%, at least 96.5%, at least 96.6%, at least 96.7%, at least 96.8%, at least 96.9%, at least 97%, at least 97.1 %, at least 97.2%, at least 97.3%, at least 97.4%, but less than 97.5% to the amino acid sequence according to SEQ ID NO: 1 and comprises

[0532] (a) a combination of amino acid substitutions selected from the group consisting of: compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0533] (b) a combination of amino acid substitutions selected from the group consisting of: compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, wherein said polypeptide comprises amino acid residue D or E at position 101, preferably E at position 101 , referring to the numbering of SEQ ID NO: 2.

[0534] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of 79% to 98%, 80% to 98%, 80.5% to 98%, 81 % to 98%, 81 .5% to 98%, 82% to 98%, 82.5% to 98%, 83% to 98%, 83.5% to

[0535] 98%, 84% to 98%, 84.5% to 98%, 85% to 98%, 85.5% to 98%, 86% to 98%, 86.5% to 98%, 87% to 98%, 87.5% to

[0536] 98%, 88% to 98%, 88.5% to 98%, 89% to 98%, 89.5% to 98%, 90% to 98%, 90.3% to 98%, 90.5% to 98%, 90.7% to

[0537] 98%, 91 % to 98%, 91 .3% to 98%, 91 .5% to 98%, 91 .7% to 98%, 92% to 98%, 92.3% to 98%, 92.5% to 98%, 92.7% to 98%, 93% to 98%, 93.3% to 98%, 93.5% to 98%, 93.7% to 98%, 94% to 98%, 94.3% to 98%, 94.5% to 98%, 94.7% to 98%, 95% to 98%, 95.1 % to 98%, 95.2% to 98%, 95.3% to 98%, 95.4% to 98%, 95.5% to 98% to the amino acid sequence according to SEQ ID NO: 1 and comprises

[0538] (a) a combination of amino acid substitutions selected from the group consisting of: compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0539] (b) a combination of amino acid substitutions selected from the group consisting of: compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2. In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of 79% to 98%, 80% to 98%, 80.5% to 98%, 81 % to 98%, 81 .5% to 98%, 82% to 98%, 82.5% to 98%, 83% to 98%, 83.5% to

[0540] 98%, 84% to 98%, 84.5% to 98%, 85% to 98%, 85.5% to 98%, 86% to 98%, 86.5% to 98%, 87% to 98%, 87.5% to

[0541] 98%, 88% to 98%, 88.5% to 98%, 89% to 98%, 89.5% to 98%, 90% to 98%, 90.3% to 98%, 90.5% to 98%, 90.7% to

[0542] 98%, 91 % to 98%, 91 .3% to 98%, 91 .5% to 98%, 91 .7% to 98%, 92% to 98%, 92.3% to 98%, 92.5% to 98%, 92.7% to 98%, 93% to 98%, 93.3% to 98%, 93.5% to 98%, 93.7% to 98%, 94% to 98%, 94.3% to 98%, 94.5% to 98%, 94.7% to 98%, 95% to 98%, 95.1 % to 98%, 95.2% to 98%, 95.3% to 98%, 95.4% to 98%, 95.5% to 98% to the amino acid sequence according to SEQ ID NO: 1 and comprises

[0543] (a) a combination of amino acid substitutions selected from the group consisting of: compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0544] (b) a combination of amino acid substitutions selected from the group consisting of: compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, wherein said polypeptide comprises amino acid residue D or E at position 101, preferably E at position 101 , referring to the numbering of SEQ ID NO: 2.

[0545] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of at least 79%, at least 80%, 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 90.5%, at least 91 %, at least 91 .5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.1 %, at least 95.2%, at least 95.3%, at least 95.4%, at least 95.5%, at least 95.6%, at least 95.7%, at least 95.8%, at least 95.9% at least 96%, at least 96.1 %, at least 96.2%, at least 96.3%, at least 96.4%, at least 96.5%, at least 96.6%, at least 96.7%, at least 96.8%, at least 96.9%, at least 97%, at least 97.1 %, at least 97.2%, at least 97.3%, at least 97.4%, but less than 97.5% to the amino acid sequence according to SEQ ID NO: 1 and comprises one of the combinations of amino acid substitutions of Table 2 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2. Table 2

[0546]

[0547]

[0548]

[0549] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of at least 79%, at least 80%, 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 90.5%, at least 91 %, at least 91 .5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.1 %, at least 95.2%, at least 95.3%, at least 95.4%, at least 95.5%, at least 95.6%, at least 95.7%, at least 95.8%, at least 95.9% at least 96%, at least 96.1 %, at least 96.2%, at least 96.3%, at least 96.4%, at least 96.5%, at least 96.6%, at least 96.7%, at least 96.8%, at least 96.9%, at least 97%, at least 97.1 %, at least 97.2%, at least 97.3%, at least 97.4%, but less than 97.5% to the amino acid sequence according to SEQ ID NO: 1 , comprises amino acid residue D or E at position 101 , preferably E at position 101 , referring to the numbering of SEQ ID NO: 2 and comprises one of the combinations of amino acid substitutions of Table 2 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2

[0550] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of 79% to 98%, 80% to 98%, 80.5% to 98%, 81 % to 98%, 81 .5% to 98%, 82% to 98%, 82.5% to 98%, 83% to 98%, 83.5% to

[0551] 98%, 84% to 98%, 84.5% to 98%, 85% to 98%, 85.5% to 98%, 86% to 98%, 86.5% to 98%, 87% to 98%, 87.5% to

[0552] 98%, 88% to 98%, 88.5% to 98%, 89% to 98%, 89.5% to 98%, 90% to 98%, 90.3% to 98%, 90.5% to 98%, 90.7% to

[0553] 98%, 91 % to 98%, 91 .3% to 98%, 91 .5% to 98%, 91 .7% to 98%, 92% to 98%, 92.3% to 98%, 92.5% to 98%, 92.7% to 98%, 93% to 98%, 93.3% to 98%, 93.5% to 98%, 93.7% to 98%, 94% to 98%, 94.3% to 98%, 94.5% to 98%, 94.7% to 98%, 95% to 98%, 95.1 % to 98%, 95.2% to 98%, 95.3% to 98%, 95.4% to 98% or 95.5% to 98% to the amino acid sequence according to SEQ ID NO: 1 and comprises one of the combinations of amino acid substitutions of Table 2 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2.

[0554] In one embodiment the variant polypeptide of the present invention preferably has a sequence identity of 79% to 98%, 79.5% to 98%, 80% to 98%, 80.5% to 98%, 81 % to 98%, 81 .5% to 98%, 82% to 98%, 82.5% to 98%, 83% to

[0555] 98%, 83.5% to 98%, 84% to 98%, 84.5% to 98%, 85% to 98%, 85.5% to 98%, 86% to 98%, 86.5% to 98%, 87% to

[0556] 98%, 87.5% to 98%, 88% to 98%, 88.5% to 98%, 89% to 98%, 89.5% to 98%, 90% to 98%, 90.3% to 98%, 90.5% to

[0557] 98%, 90.7% to 98%, 91 % to 98%, 91 .3% to 98%, 91 .5% to 98%, 91 .7% to 98%, 92% to 98%, 92.3% to 98%, 92.5% to 98%, 92.7% to 98%, 93% to 98%, 93.3% to 98%, 93.5% to 98%, 93.7% to 98%, 94% to 98%, 94.3% to 98%, 94.5% to 98%, 94.7% to 98%, 95% to 98%, 95.1 % to 98%, 95.2% to 98%, 95.3% to 98%, 95.4% to 98% or 95.5% to 98% to the amino acid sequence according to SEQ I D NO: 1 , comprises amino acid residue D or E at position 101 , preferably E at position 101 , referring to the numbering of SEQ ID NO: 2 and comprises one of the combinations of amino acid substitutions of Table 2 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2.

[0558] In one embodiment the variant polypeptide of the present invention or a fragment of said polypeptide having protease activity additionally comprises an amino acid substitution at one or more amino acid residues selected from the group consisting of 24, 103 and 160 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2.

[0559] In one embodiment the variant polypeptide of the present invention or a fragment of said polypeptide having protease activity additionally comprises at least one amino acid substitution selected from the group consisting of X24K, X103S and X160H compared to the amino acid sequence as set forth in SEQ I D NO: 3 and referring to the numbering of SEQ ID NO: 2.

[0560] In one embodiment the variant polypeptide of the present invention or a fragment of said polypeptide having protease activity additionally comprises at least one amino acid substitution selected from the group consisting of S24K, A103S and S160H compared to the amino acid sequence as set forth in SEQ I D NO: 3 and referring to the numbering of SEQ ID NO: 2.

[0561] The present invention is directed to a variant polypeptide having protease activity or a fragment of said polypeptide having protease activity, wherein:

[0562] (I) the polypeptide has an amino acid sequence which is at least 79%, but less than 100%, identical to the amino acid sequence as set forth in SEQ ID NO: 1 and

[0563] (ii) the polypeptide or fragment thereof comprises

[0564] (a) an amino acid substitution at two or more, three or more, four or more, five or more, six or all amino acid residues selected from the group consisting of 9, 18, 21 , 38, 59, 228, and 271 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2,

[0565] (b) an amino acid substitution at two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or all amino acid residues selected from the group consisting of 25, 43, 78, 109, 144, 182, 183, 194, 204, 210, 212, 218, 248, 259, 260 and 261 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, and

[0566] (c) an amino acid substitution at one or more, two or all amino acid residues selected from the group consisting of 24, 103 and 160 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, wherein the fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide. The present invention is directed to a variant polypeptide having protease activity, wherein:

[0567] (i) the polypeptide has an amino acid sequence which is at least 79%, but less than 100%, identical to the amino acid sequence as set forth in SEQ ID NO: 1 and

[0568] (ii) the polypeptide comprises

[0569] (a) an amino acid substitution at two or more, three or more, four or more, five or more, six or all amino acid residues selected from the group consisting of 9, 18, 21 , 38, 59, 228, and 271 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2,

[0570] (b) an amino acid substitution at two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or all amino acid residues selected from the group consisting of 25, 43, 78, 109, 144, 182, 183, 194, 204, 210, 212, 218, 248, 259, 260 and 261 compared to the amino acid sequence as set forth in SEQ I D NO: 3 and referring to the numbering of SEQ ID NO: 2, and c) an amino acid substitution at one or more, two or all amino acid residues selected from the group consisting of 24, 103 and 160 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2.

[0571] The present invention is directed to a variant polypeptide having protease activity or a fragment of said polypeptide having protease activity, wherein:

[0572] (I) the polypeptide has an amino acid sequence which has a sequence identity of 95.5% to 98% to the amino acid sequence as set forth in SEQ ID NO: 1 and

[0573] (ii) the polypeptide or fragment thereof comprises

[0574] (a) an amino acid substitution at two or more, three or more, four or more, five or more, six or all amino acid residues selected from the group consisting of 9, 18, 21 , 38, 59, 228, and 271 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2,

[0575] (b) an amino acid substitution at two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or all amino acid residues selected from the group consisting of 25, 43, 78, 109, 144, 182, 183, 194, 204, 210, 212, 218, 248, 259, 260 and 261 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, and

[0576] (c) an amino acid substitution at one or more, two or all amino acid residues selected from the group consisting of 24, 103 and 160 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, wherein the fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide.

[0577] The present invention is directed to a variant polypeptide having protease activity, wherein: (i) the polypeptide has an amino acid sequence which has a sequence identity of 95.5% to 98% to the amino acid sequence as set forth in SEQ ID NO: 1 and

[0578] (ii) the polypeptide comprises

[0579] (a) an amino acid substitution at two or more, three or more, four or more, five or more, six or all amino acid residues selected from the group consisting of 9, 18, 21 , 38, 59, 228, and 271 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2,

[0580] (b) an amino acid substitution at two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or all amino acid residues selected from the group consisting of 25, 43, 78, 109, 144, 182, 183, 194, 204, 210, 212, 218, 248, 259, 260 and 261 compared to the amino acid sequence as set forth in SEQ I D NO: 3 and referring to the numbering of SEQ ID NO: 2, and c) an amino acid substitution at one or more, two or three amino acid residues selected from the group consisting of 24, 103 and 160 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2.

[0581] The present invention is directed to a variant polypeptide having protease activity or a fragment of said polypeptide having protease activity, wherein:

[0582] (I) the polypeptide has an amino acid sequence which is at least 79%, but less than 100%, identical to the amino acid sequence as set forth in SEQ ID NO: 1 and

[0583] (ii) the polypeptide or fragment thereof comprises

[0584] (a) an amino acid substitution at two or more, three or more, four or more, five or more, six or all amino acid residues selected from the group consisting of 9, 18, 21 , 38, 59, 228, and 271 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2,

[0585] (b) an amino acid substitution at two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or all amino acid residues selected from the group consisting of 25, 43, 78, 109, 144, 182, 183, 194, 204, 210, 212, 218, 248, 259, 260 and 261 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, and

[0586] (c) an amino acid substitution at one or more, two or all amino acid residues selected from the group consisting of 24, 103 and 160 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, wherein the fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide and wherein said polypeptide comprises amino acid residue D or E at position 101 , preferably E at position 101 , referring to the numbering of SEQ ID NO: 2.

[0587] The present invention is directed to a variant polypeptide having protease activity, wherein: (i) the polypeptide has an amino acid sequence which is at least 79%, but less than 100%, identical to the amino acid sequence as set forth in SEQ ID NO: 1 and

[0588] (ii) the polypeptide comprises

[0589] (a) an amino acid substitution at two or more, three or more, four or more, five or more, six or all amino acid residues selected from the group consisting of 9, 18, 21 , 38, 59, 228, and 271 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2,

[0590] (b) an amino acid substitution at two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or all amino acid residues selected from the group consisting of 25, 43, 78, 109, 144, 182, 183, 194, 204, 210, 212, 218, 248, 259, 260 and 261 compared to the amino acid sequence as set forth in SEQ I D NO: 3 and referring to the numbering of SEQ ID NO: 2, and c) an amino acid substitution at one or more, two or all amino acid residues selected from the group consisting of 24, 103 and 160 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, wherein said polypeptide comprises amino acid residue D or E at position 101 , preferably E at position 101 , referring to the numbering of SEQ ID NO: 2.

[0591] The present invention is directed to a variant polypeptide having protease activity or a fragment of said polypeptide having protease activity, wherein:

[0592] (I) the polypeptide has an amino acid sequence which has a sequence identity of 95.5% to 98% to the amino acid sequence as set forth in SEQ ID NO: 1 and

[0593] (ii) the polypeptide or fragment thereof comprises

[0594] (a) an amino acid substitution at two or more, three or more, four or more, five or more, six or all amino acid residues selected from the group consisting of 9, 18, 21 , 38, 59, 228, and 271 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2,

[0595] (b) an amino acid substitution at two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or all amino acid residues selected from the group consisting of 25, 43, 78, 109, 144, 182, 183, 194, 204, 210, 212, 218, 248, 259, 260 and 261 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, and c) an amino acid substitution at one or more, two or three amino acid residues selected from the group consisting of 24, 103 and 160 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, wherein the fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide and wherein said polypeptide comprises amino acid residue D or E at position 101 , preferably E at position 101 , referring to the numbering of SEQ ID NO: 2. The present invention is directed to a variant polypeptide having protease activity, wherein:

[0596] (i) the polypeptide has an amino acid sequence w which has a sequence identity of 95.5% to 98% to the amino acid sequence as set forth in SEQ ID NO: 1 and

[0597] (ii) the polypeptide comprises

[0598] (a) an amino acid substitution at two or more, three or more, four or more, five or more, six or all amino acid residues selected from the group consisting of 9, 18, 21 , 38, 59, 228, and 271 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2,

[0599] (b) an amino acid substitution at two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or all amino acid residues selected from the group consisting of 25, 43, 78, 109, 144, 182, 183, 194, 204, 210, 212, 218, 248, 259, 260 and 261 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, and c) an amino acid substitution at one or more amino acid residues selected from the group consisting of 24, 103 and 160 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ I D NO: 2, wherein said polypeptide comprises amino acid residue D or E at position 101 , preferably E at position 101, referring to the numbering of SEQ ID NO: 2.

[0600] In one embodiment the variant polypeptide of the present invention or a fragment of said polypeptide having protease activity additionally comprises at least one amino acid substitution at an amino acid residue selected from the group consisting of 3, 76 and 256 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2.

[0601] Hence, in one embodiment the present invention relates to a variant polypeptide having protease activity or a fragment of said polypeptide having protease activity, wherein:

[0602] (I) the polypeptide has an amino acid sequence which is at least 79%, but less than 100%, identical to the amino acid sequence as set forth in SEQ ID NO: NO: 1 and

[0603] (ii) the polypeptide or fragment thereof comprises

[0604] (a) an amino acid substitution at two or more, three or more, four or more, five or more, six or all amino acid residues selected from the group consisting of 9, 18, 21 , 38, 59, 228, and 271 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0605] (b) an amino acid substitution at two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or all amino acid residues selected from the group consisting of 25, 43, 78, 109, 144, 182, 183, 194, 204, 210, 212, 218, 248, 259, 260 and 261 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, (c) at least one, two or three amino acid substitutions at an amino acid residue selected from the group consisting of 3, 76 and 256 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, wherein the fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide.

[0606] Hence, in one embodiment the present invention relates to a variant polypeptide having protease activity or a fragment of said polypeptide having protease activity, wherein:

[0607] (i) the polypeptide has an amino acid sequence which is at least 79%, but less than 100%, identical to the amino acid sequence as set forth in SEQ ID NO: NO: 1 and

[0608] (ii) the polypeptide or fragment thereof comprises

[0609] (a) an amino acid substitution at two or more, three or more, four or more, five or more, six or all amino acid residues selected from the group consisting of 9, 18, 21 , 38, 59, 228, and 271 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0610] (b) an amino acid substitution at two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or all amino acid residues selected from the group consisting of 25, 43, 78, 109, 144, 182, 183, 194, 204, 210, 212, 218, 248, 259, 260 and 261 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2,

[0611] (c) at least one, two or three amino acid substitutions at an amino acid residue selected from the group consisting of 3, 76 and 256 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, wherein the fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide and wherein said variant polypeptide comprises amino acid residue D or E at position 101 , preferably E at position 101 , referring to the numbering of SEQ ID NO: 2.

[0612] Hence, in one embodiment the present invention relates to a variant polypeptide having protease activity or a fragment of said polypeptide having protease activity, wherein:

[0613] (i) the polypeptide has an amino acid sequence which is 95.5% to 98% identical to the amino acid sequence as set forth in SEQ ID NO: 1 and

[0614] (ii) the polypeptide or fragment thereof comprises

[0615] (a) an amino acid substitution at two or more, three or more, four or more, five or more, six or all amino acid residues selected from the group consisting of 9, 18, 21 , 38, 59, 228, and 271 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0616] (b) an amino acid substitution at two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or all amino acid residues selected from the group consisting of 25, 43, 78, 109, 144, 182, 183, 194, 204, 210, 212, 218, 248, 259, 260 and 261 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2,

[0617] (c) at least one, two or three amino acid substitutions at an amino acid residue selected from the group consisting of 3, 76 and 256 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, wherein the fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide.

[0618] Hence, in one embodiment the present invention relates to a variant polypeptide having protease activity or a fragment of said polypeptide having protease activity, wherein:

[0619] (i) the polypeptide has an amino acid sequence which is 95.5% to 98% identical to the amino acid sequence as set forth in SEQ ID NO: 1 and

[0620] (ii) the polypeptide comprises

[0621] (a) an amino acid substitution at two or more, three or more, four or more, five or more, six or all amino acid residues selected from the group consisting of 9, 18, 21 , 38, 59, 228, and 271 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0622] (b) an amino acid substitution at two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or all amino acid residues selected from the group consisting of 25, 43, 78, 109, 144, 182, 183, 194, 204, 210, 212, 218, 248, 259, 260 and 261 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2,

[0623] (c) at least one, two or three amino acid substitutions at an amino acid residue selected from the group consisting of 3, 76 and 256 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, wherein the fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide and wherein said variant polypeptide comprises amino acid residue D or E at position 101 , preferably E at position 101 , referring to the numbering of SEQ ID NO: 2.

[0624] In one embodiment the variant polypeptide of the present invention or a fragment of said polypeptide having protease activity additionally comprises at least one amino acid substitution selected from the group consisting of X3T / Q / V, X76D and X256E / T / D / R / P compared to the amino acid sequence as set forth in SEQ I D NO: 3 and referring to the numbering of SEQ ID NO: 2.

[0625] In one embodiment the variant polypeptide of the present invention or a fragment of said polypeptide having protease activity additionally comprises at least one amino acid substitution selected from the group consisting of S3T / Q / V, N76D and S256E / T / D / R / P compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2.

[0626] In one embodiment the variant polypeptide of the present invention or a fragment of said polypeptide having protease activity comprises amino acid substitutions at positions 18, 21 , 25, 38, 78, 109, 144, 182, 183, 194, 204, 210, 212, 218, 228, 248, 260 and 271 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and comprises amino acid residue D or E at position 101 , preferably E at position 101 , referring to the numbering of SEQ ID NO: 2.

[0627] In one embodiment the variant polypeptide of the present invention or a fragment of said polypeptide having protease activity comprises the amino acid substitutions X18Q / E / D, X21 V / l, X25D, X38W / K, X56D, X78N / D, X109K / A, X144N / R, X182K / R / E, X183D / E, X194E / D, X204D / E, X210I, X212P / A / G / R / K / F / Y / M, X228S, X248Q / R, X260D / E and X271 D compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and comprises amino acid residue D or E at position 101 , preferably E at position 101 , referring to the numbering of SEQ ID NO: 2.

[0628] In one embodiment the variant polypeptide of the present invention or a fragment of said polypeptide having protease activity comprises the amino acid substitutions N18Q / E / D, L21V / I, G25D, T38W / K, S56D, S78N / D, Q109K / A, S144N / R, Q182K / R / E, N183D / E, A194E / D, N204D / E, P210I, S212P / A / G / R / K / F / Y / M, N218S / T, A228S, N248Q / R, T260D / E and E271 D compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and comprises amino acid residue D or E at position 101 , preferably E at position 101 , referring to the numbering of SEQ ID NO: 2.

[0629] Hence, in one embodiment the present invention relates to a variant polypeptide having protease activity or a fragment of said polypeptide having protease activity, wherein:

[0630] (I) the polypeptide has an amino acid sequence which is at least 79%, but less than 100%, identical to the amino acid sequence as set forth in SEQ I D NO: 1 and

[0631] (ii) the polypeptide or fragment thereof comprises amino acid substitutions at positions 18, 21, 25, 38, 78, 109, 144, 182, 183, 194, 204, 210, 212, 218, 228, 248, 260 and 271 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and comprises amino acid residue D or E at position 101 , preferably E at position 101 , referring to the numbering of SEQ ID NO: 2.

[0632] Hence, in one embodiment the present invention relates to a variant polypeptide having protease activity or a fragment of said polypeptide having protease activity, wherein:

[0633] (I) the polypeptide has an amino acid sequence which is at least 79%, but less than 100%, identical to the amino acid sequence as set forth in SEQ I D NO: 1 and

[0634] (ii) the polypeptide or fragment thereof comprises the amino acid substitutions X18Q / E / D, X21V / I, X25D, X38W / K, X78N / D, X109K / A, X144N / R, X182K / R / E, X183D / E, X194E / D, X204D / E, X210I, X212P / A / G / R / K / F / Y / M, X228S, X248Q / R, X260D / E and X271 D compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ I D NO: 2 and comprises amino acid residue D or E at position 101 , preferably E at position 101 , referring to the numbering of SEQ ID NO: 2.

[0635] Hence, in one embodiment the present invention relates to a variant polypeptide having protease activity or a fragment of said polypeptide having protease activity, wherein:

[0636] (I) the polypeptide has an amino acid sequence which is at least 79%, but less than 100%, identical to the amino acid sequence as set forth in SEQ I D NO: 1 and

[0637] (ii) the polypeptide or fragment thereof comprises the amino acid substitutions N18Q / E / D, L21V / I, G25D, T38W / K, S78N / D, Q109K / A, S144N / R, Q182K / R / E, N183D / E, A194E / D, N204D / E, P210I, S212P / A / G / R / K / F / Y / M, N218S / T, A228S, N248Q / R, T260D / E and E271 D compared to the amino acid sequence as set forth in SEQ I D NO: 3 and referring to the numbering of SEQ ID NO: 2 and comprises amino acid residue D or E at position 101 , preferably E at position 101 , referring to the numbering of SEQ ID NO: 2.

[0638] Hence, in one embodiment the present invention relates to a variant polypeptide having protease activity or a fragment of said polypeptide having protease activity, wherein:

[0639] (I) the polypeptide has an amino acid sequence which is 95.5% to 98% identical to the amino acid sequence as set forth in SEQ ID NO: 1 and

[0640] (ii) the polypeptide or fragment thereof comprises amino acid substitutions at positions 18, 21, 25, 38, 78, 109, 144, 182, 183, 194, 204, 210, 212, 218, 228, 248, 260 and 271 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and comprises amino acid residue D or E at position 101 , preferably E at position 101 , referring to the numbering of SEQ ID NO: 2.

[0641] Hence, in one embodiment the present invention relates to a variant polypeptide having protease activity or a fragment of said polypeptide having protease activity, wherein:

[0642] (I) the polypeptide has an amino acid sequence which is 95.5% to 98% identical to the amino acid sequence as set forth in SEQ ID NO: 1 and

[0643] (ii) the polypeptide or fragment thereof comprises the amino acid substitutions X18Q / E / D, X21V / I, X25D, X38W / K, X78N / D, X109K / A, X144N / R, X182K / R / E, X183D / E, X194E / D, X204D / E, X210I, X212P / A / G / R / K / F / Y / M, X228S, X248Q / R, X260D / E and X271 D compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ I D NO: 2 and comprises amino acid residue D or E at position 101 , preferably E at position 101 , referring to the numbering of SEQ ID NO: 2.

[0644] Hence, in one embodiment the present invention relates to a variant polypeptide having protease activity or a fragment of said polypeptide having protease activity, wherein:

[0645] (I) the polypeptide has an amino acid sequence which is 95.5% to 98% identical to the amino acid sequence as set forth in SEQ ID NO: 1 and

[0646] (ii) the polypeptide or fragment thereof comprises the amino acid substitutions N18Q / E / D, L21V / I, G25D, T38W / K, S78N / D, Q109K / A, S144N / R, Q182K / R / E, N183D / E, A194E / D, N204D / E, P210I, S212P / A / G / R / K / F / Y / M, N218S / T, A228S, N248Q / R, T260D / E and E271 D compared to the amino acid sequence as set forth in SEQ I D NO: 3 and referring to the numbering of SEQ ID NO: 2 and comprises amino acid residue D or E at position 101 , preferably E at position 101 , referring to the numbering of SEQ ID NO: 2.

[0647] In one embodiment, the amino acid substitution at amino acid residue 9 is X9A and / or the amino acid substitution at amino acid residue 18 is X18Q; and / or the amino acid substitution at amino acid residue 21 is X21 V; and or the amino acid substitution at amino acid residue 38 is X38K / W; and / or the amino acid substitution at amino acid residue 43 is X43K / R; and / or the amino acid substitution at amino acid residue 59 is X59K; and / or the amino acid substitution at amino acid residue 78 is X78N; and / or the amino acid substitution at amino acid residue 109 is X109K; and / or the amino acid substitution at amino acid residue 144 is X144N; and / or the amino acid substitution at amino acid residue 182 is X182K; and / or the amino acid substitution at amino acid residue 183 is X183D; and / or the amino acid substitution at amino acid residue 194 is X194E; and / or the amino acid substitution at amino acid residue 204 is X204D; and / or the amino acid substitution at amino acid residue 210 is X210I; and / or the amino acid substitution at amino acid residue 212 is X212P; and / or the amino acid substitution at amino acid residue 218 is X218S; and / or the amino acid substitution at amino acid residue 248 is X248Q; and / orthe amino acid substitution at amino acid residue 259 is X259D; and / or the amino acid substitution at amino acid residue 260 is X260D; and / or the amino acid substitution at amino acid residue 261 is X261 F.

[0648] In one embodiment the variant polypeptide of the present invention or a fragment of said polypeptide having protease activity additionally comprises at least one amino acid substitution at an amino acid residue selected from the group consisting of 8, 108, 133, 137 and 215 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2.

[0649] Hence, in one embodiment the present invention relates to a variant polypeptide having protease activity or a fragment of said polypeptide having protease activity, wherein:

[0650] (i) the polypeptide has an amino acid sequence which is at least 79%, but less than 100%, identical to the amino acid sequence as set forth in SEQ ID NO: 1 and

[0651] (ii) the polypeptide comprises

[0652] (a) an amino acid substitution at two or more, three or more, four or more, five or more, six or all amino acid residues selected from the group consisting of 9, 18, 21 , 38, 59, 228, and 271 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0653] (b) an amino acid substitution at two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or all amino acid residues selected from the group consisting of 25, 43, 78, 109, 144, 182, 183, 194, 204, 210, 212, 218, 248, 259, 260 and 261 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, (c) at least one, at least two, at least three, four or five amino acid substitutions at an amino acid residue selected from the group consisting of 8, 108, 133, 137 and 215 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, wherein the fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide.

[0654] Hence, in one embodiment the present invention relates to a variant polypeptide having protease activity or a fragment of said polypeptide having protease activity, wherein:

[0655] (i) the variant polypeptide has a sequence identity of 95.5% to 98% to the amino acid sequence according to SEQ ID NO: 1 and

[0656] (ii) the polypeptide comprises

[0657] (a) an amino acid substitution at two or more, three or more, four or more, five or more, six or all amino acid residues selected from the group consisting of 9, 18, 21 , 38, 59, 228, and 271 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0658] (b) an amino acid substitution at two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or all amino acid residues selected from the group consisting of 25, 43, 78, 109, 144, 182, 183, 194, 204, 210, 212, 218, 248, 259, 260 and 261 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2,

[0659] (c) at least one, at least two, at least three, four or five amino acid substitution at an amino acid residue selected from the group consisting of 8, 108, 133, 137 and 215 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2.

[0660] In one embodiment the variant polypeptide of the present invention or a fragment of said polypeptide having protease activity additionally comprises at least one amino acid substitution selected from the group consisting of X8V, X108V, X133L, X137L / H and X215K / R / Q / N / S / T / W compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2.

[0661] In one embodiment, the variant polypeptide comprises at least one amino acid substitution at an amino acid residue selected from the group consisting of 4, 15, 35, 40, 45, 55, 58, 61 , 68, 69, 72, 87, 97, 98, 104, 106, 114, 115, 117, 118, 120, 124, 128, 129, 130, 136, 138, 143, 145, 147, 149, 156, 158, 161 , 163, 166, 167, 169, 170, 171, 172, 177, 185, 187, 188, 189, 191 , 195, 199, 205, 206, 209, 21 1 , 216, 217, 222, 225, 235, 238, 244, 245, 255, and / or 262 compared to the amino acid sequence as set forth in SEQ I D NO: 3 and referring to the numbering of SEQ ID NO: 2.

[0662] In one embodiment, the variant polypeptide comprises at least one amino acid substitution selected from the group consisting of X4I, X15T, X35XD, X40E, X45E / D / Q, X55N, X58W / L / N, X61 E / D / R, X68A, X69S, X72V / A, X87E / D / N / R, X97AD / XE / D / F, X98D / E / R, X104F / V, X106A / W, X114V, X115T / W, X1 17E, X118R / V, X120D / V / K / N / I / T, X124M, X128A / L / R, X129D / Q / E, X130A / G, X136Q, X138Q, X143W, X145L / W / Y, X147W, X149C / N, X156D / G / Q, X158E, X161 E / T / C, X163A, X166Q, X167A / T, X169G, X170S, X171 L, X172S, X177L, X185E / Q / C, X187D, X188A / C / E / D, X189R, X191 R / N, X195E, X199M, X205I, X206L / A / S / T / C / E / I / K / V, X209WA / / K, X211 P, X216T / I / V, X217D / Q / E, X222A / S / Q / C / R, X225A, X235L / N, X238H, X244T / R, X245R / K, X255C / E and / or X262E / D / Q / N / C compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ I D NO: 2.

[0663] I n one embodiment the variant polypeptide or fragment thereof comprises a combination of substitutions selected from the group consisting of:

[0664] (a) X9A;X18Q;X21 V;X24K;X25D;X38W;X43K;X56D;X78N;X103S;X109K;X144N;X160H;

[0665] X182K;X183D;X194E;X204D;X21 Ol ;X212P;X218S;X228S;X248Q;X260D;X261 F;X271 D;

[0666] (b) X9A;X18Q;X21V;X24K;X25D;X38K;X43K;X56D;X78N;X103S;X109K;X144N;X160H;

[0667] X182K;X183D;X194E;X204D;X21 Ol ;X212P;X218S;X228S;X248Q;X260D;X271 D;

[0668] (c) X9A;X18Q;X21 V;X24K;X25D;X38K;X43K;X56D;X78N;X103S;X109K;X144N;X160H;

[0669] X182K;X183D;X194E;X204D;X21 Ol ;X212P;X218S;X228S;X248Q;X260D;X261 F;X271 D;

[0670] (d) X9A;X18Q;X21V;X24K;X25D;X38K;X43K;X56D;X78N;X103S;X109K;X144N;X160H;

[0671] X182K;X183D;X194E;X204D;X21 Ol ;X212P;X218S;X228S;X248Q;X259D;X260D;X261 F;

[0672] X271 D;

[0673] (e) X18Q;X21V;X24K;X25D;X38K;X56D;X78N;X103S;X109K;X144N;X160H;X182K;

[0674] X183D;X194E;X204D;X210l;X212P;X218S;X228S;X248Q;X260D; X261 F;X271 D;

[0675] (f) X18Q;X21 V;X24K;X38W;X56D;X78N;X103S;X109K;X144N;X160H;X182K;X183D;

[0676] X194E;X204D;X210l;X212P;X218S;X228S;X248Q;X260D;X261 F;X271 D;

[0677] (g) X9A;X18Q;X21V;X24K;X25D;X38K;X56D;X78N;X103S;X109K;X144N;X160H;X182K;

[0678] X183D;X204D;X210l;X212P;X218S;X228S;X248Q;X259D;X260D;X261 F;X271 D;

[0679] (h) X9A;X18Q;X21 V;X24K;X38K;X56D;X78N;X103S;X109K;X144N;X160H;X182K;X183D;

[0680] X194E;X204D;X210l;X212P;X218S;X228S;X248Q;X260D;X261 F;X271 D;

[0681] (I) X9A;X18Q;X21 V;X24K;X38K;X56D;X78N;X103S;X109K;X144N;X160H;X182K;X183D;

[0682] X194E;X204D;X210l;X212P;X218S;X228S;X248Q;X259D;X260D;X261 F;X271 D;

[0683] Q) X18Q;X21 V;X24K;X38K;X56D;X78N;X103S;X109K;X144N;X160H;X182K;X183D;

[0684] X204D;X210l;X212P;X218S;X228S;X248Q;X259D;X260D;X261 F;X271 D;

[0685] (k) X18Q;X21 V;X24K;X25D;X38K;X43K;X56D;X78N;X103S;X109K;X144N;X160H;X182K;

[0686] X183D;X194E;X204D;X210l;X218S;X228S;X248Q;X260D;X261 F;X271 D;

[0687] (l) X9A;X18Q;X21V;X24K;X25D;X38K;X43K;X56D;X78N;X103S;X109K;X144N;X160H;

[0688] X182K;X183D;X194E;X204D;X210l;X218S;X228S;X248Q;X260D;X261 F;X271 D;

[0689] (m) X9A;X18Q;X21 V;X24K;X25D;X38K;X43K;X56D;X78N;X103S;X109K;X144N;X160H;

[0690] X182K;X183D;X204D;X210l;X218S;X228S;X248Q;X260D;X261 F;X271 D;

[0691] (n) X9A;X18Q;X21V;X24K; X38K;X43K;X56D;X78N;X103S;X109K;X144N;X160H;X182K; X183D;X194E;X204D;X210l;X218S;X228S;X248Q;X260D;X261 F;X271 D;

[0692] (o) X9A;X18Q;X21V;X24K;X38K;X56D;X78N; X109K;X144N;X160H;X182K;X183D;X204D;

[0693] X210l ; X218S; X228S; X248Q; X260D;X271 D;

[0694] (p) X18Q;X21 V;X24K;X25D;X38K;X43R;X56D;X78N;X103S;X109K;X144N;X160H;X182K;

[0695] X183D;X194E;X204D;X210l;X212P;X218S;X228S;X248Q;X261 F;

[0696] (q) X18Q;X21 V;X24K;X25D;X38K;X43R;X56D;X78N;X109K;X144N;X160H;X182K;X183D;

[0697] X194E;X204D;X210l;X212P;X218S;X228S;X248Q;

[0698] (r) X9A;X18Q;X21 V;X24K;X25D;X38K;X43R;X56D;X78N;X103S;X109K;X144N;X160H;

[0699] X182K;X183D;X194E;X204D;X210l;X212P;X218S;X228S;X248Q;X261 F;X271 D;

[0700] (s) X18Q;X21 V;X24K;X25D;X38K;X43K;X56D;X78N;X109K;X144N;X160H;X182K;X183D;

[0701] X194E;X204D;X210l;X212P;X218S;X228S;X248Q;X259D;X261 F;

[0702] (t) X9A;X18Q;X21 V;X24K;X25D;X38W;X43K;X56D;X78N;X103S;X109K;X144N;X160H;

[0703] X182K;X183D;X204D;X210l;X212P;X218S;X228S;X248Q;X259D;X261 F;X271 D;

[0704] (u) X9A;X18Q;X21V;X24K;X25D;X38K;X43K;X56D;X78N;X103S;X109K;X144N;X160H;

[0705] X182K;X183D;X194E;X204D;X210l;X212P;X218S;X228S;X248Q;X259D;X271 D;

[0706] (v) X9A;X18Q;X21 V;X24K;X25D;X38K;X43K;X56D;X78N;X103S;X109K;X144N;X160H;

[0707] X182K;X183D;X194E;X204D;X210l;X212P;X218S;X228S;X248Q;X259D;X261 F;

[0708] (w) X9A;X18Q;X21 V;X24K;X25D;X38K;X43K;X56D;X78N;X103S;X109K;X144N;X160H;

[0709] X182K;X183D;X204D;X210l;X212P;X218S;X228S;X248Q;X259D;X271 D;

[0710] (x) X9A;X18Q;X21 V;X24K;X25D;X38K;X43K;X56D;X78N;X103S;X109K;X144N;X160H;

[0711] X182K;X183D;X204D;X210l;X212P;X218S;X228S;X248Q;X259D;X261 F;X271 D;

[0712] (y) X9A;X18Q;X21 V;X24K;X38K;X43K;X56D;X78N;X103S;X109K;X144N;X160H;X182K;

[0713] X183D;X194E;X204D;X210l;X212P;X218S;X228S;X248Q;X259D;X261 F;

[0714] (z) X9A;X18Q;X21 V;X24K;X38K;X43K;X56D;X78N;X103S;X109K;X144N;X160H;X182K;

[0715] X183D;X194E;X204D;X210l;X212P;X218S;X228S;X248Q;X259D;X261 F;X271 D;

[0716] (aa) X9A;X18Q;X21 V;X24K;X38K;X43K;X56D;X78N;X103S;X109K;X144N;X160H;X182K;

[0717] X183D;X204D;X210l;X212P;X218S;X228S;X248Q;X259D;X261 F;X271 D;

[0718] (bb) X18Q;X21 V;X24K;X38W;X56D;X59K;X78N;X103S;X109K;X144N;X160H;X182K;X183D;

[0719] X194E;X204D;X210l;X212P;X218S;X228S;X248Q;X260D;X261 F;X271 D;

[0720] (cc) X9A;X18Q;X21 V;X24K;X25D;X38W;X56D;X59K;X78N;X103S;X109K;X144N;X160H;

[0721] X182K;X183D;X194E;X204D;X21 Ol ;X212P;X218S;X228S;X248Q;X260D;X261 F;X271 D;

[0722] (dd) X9A;X18Q;X21 V;X24K;X25D;X38W;X56D;X59K;X78N;X103S;X109K;X144N;X160H;

[0723] X182K;X183D;X204D;X21 Ol ;X212P;X218S;X228S;X248Q;X259D;X260D;X261 F;X271 D;

[0724] (ee) X9A;X18Q;X21 V;X24K;X25D;X38W;X56D;X59K;X78N;X109K;X144N;X160H;X182K; X183D;X204D;X210l;X212P;X218S;X228S;X248Q;X260D;X271 D;

[0725] (ff) X9A;X18Q;X21V;X24K;X25D;X38K;X56D;X59K;X78N;X103S;X109K;X144N;X160H;

[0726] X182K;X183D;X194E;X204D;X210l;X212P;X218S;X228S;X248Q;X260D;X271 D;

[0727] (gg) X9A;X18Q;X21 V;X24K;X25D;X38K;X56D;X59K;X78N;X103S;X109K;X144N;X160H;

[0728] X182K;X183D;X194E;X204D;X21 Ol ;X212P;X218S;X228S;X248Q;X260D;X261 F;X271 D;

[0729] (hh) X9A;X18Q;X21 V;X24K;X25D;X38K;X56D;X59K;X78N;X103S;X109K;X144N;X160H;

[0730] X182K;X183D;X194E;X204D;X21 Ol ;X212P;X218S;X228S;X248Q;X259D;X260D;X271 D;

[0731] (ii) X9A;X18Q;X21 V;X24K;X25D;X38K;X56D;X59K;X78N;X103S;X109K;X144N;X160H;

[0732] X182K;X183D;X194E;X204D;X21 Ol ;X212P;X218S;X228S;X248Q;X259D;X260D;X261 F;

[0733] X271 D;

[0734] Gj) X9A;X18Q;X21 V;X24K;X25D;X38K;X56D;X59K;X78N;X103S;X109K;X144N;X160H;

[0735] X182K;X183D;X204D;X21 Ol ;X212P;X218S;X228S;X248Q;X259D;X260D;X261 F;X271 D;

[0736] (kk) X9A;X18Q;X21 V;X24K;X25D;X38K;X56D;X59K;X78N;X109K;X144N;X160H;X182K;

[0737] X183D;X204D;X210l;X212P;X218S;X228S;X248Q;X259D;X260D;X271 D;

[0738] (II) X9A;X18Q;X21 V;X24K;X38W;X56D;X59K;X78N;X103S;X109K;X144N;X160H;X182K;

[0739] X183D;X204D;X210l;X212P;X218S;X228S;X248Q;X259D;X260D;X261 F;X271 D;

[0740] (mm) X18Q;X21V;X24K;X38K;X56D;X59K;X78N;X103S;X109K;X144N;X160H;X182K;

[0741] X183D;X194E; X204D;X210l;X212P;X218S;X228S;X248Q;X260D;X271 D; and

[0742] (nn) X18Q;X21 V;X24K;X38K;X56D;X59K;X78N;X103S;X109K;X144N;X160H;X182K;

[0743] X183D;X194E; X204D;X210l;X212P;X218S;X228S;X248Q;X260D;X261 F;X271 D, compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2. In one embodiment the variant polypeptide comprises a combination of substitutions selected from the group consisting of:

[0744] (b) S9A;N18Q; L21V;S24K;G25D;T38W;N43K;S56D;S78N;A103S;Q109K;S144N;S160H;

[0745] Q182K; N 183D; A194E; N204D; P2101 ; S212P; N218S;A228S; N248Q;T260D; N261 F; E271 D;

[0746] (b) S9A;N18Q;L21 V;S24K;G25D;T38K;N43K;S56D;S78N;A103S;Q109K;S144N;S160H;

[0747] Q182K;N183D;A194E;N204D;P210l;S212P;N218S;A228S;N248Q;T260D;E271 D;

[0748] (c) S9A;N18Q;L21 V;S24K;G25D;T38K;N43K;S56D;S78N;A103S;Q109K;S144N;S160H;

[0749] Q182K; N 183D; A194E; N204D; P2101 ; S212P; N218S;A228S; N248Q;T260D; N261 F; E271 D;

[0750] (d) S9A;N18Q;L21 V;S24K;G25D;T38K;N43K;S56D;S78N;A103S;Q109K;S144N;S160H;

[0751] Q182K;N183D;A194E;N204D;P210l;S212P;N218S;A228S;N248Q;S259D;T260D;N261 F;

[0752] E271 D;

[0753] (e) N18Q;L21V;S24K;G25D;T38K;S56D;S78N;A103S;Q109K;S144N;S160H;Q182K;

[0754] N183D;A194E;N204D;P210l;S212P;N218S;A228S;N248Q;T260D;N261 F;E271 D; (f) N18Q;L21 V;S24K;T38W;S56D;S78N;A103S;Q109K;S144N;S160H;Q182K;N183D;

[0755] A194E;N204D;P210l;S212P;N218S;A228S;N248Q;T260D;N261 F;E271 D;

[0756] (g) S9A;N18Q;L21 ;S24K;G25D;T38K;S56D;S78N;A103S;Q109K;S144N;S160H;Q182K;

[0757] N183D;N204D;P210l;S212P;N218S;A228S;N248Q;S259D;T260D;N261 F;E271 D;

[0758] (h) S9A;N18Q;L21 V;S24K;T38K;S56D;S78N;A103S;Q109K;S144N;S160H;Q182K;N183D;

[0759] A194E;N204D;P210l;S212P;N218S;A228S;N248Q;T260D;N261 F;E271 D;

[0760] (i) S9A;N18Q;L21 V;S24K;T38K;S56D;S78N;A103S;Q109K;S144N;S160H;Q182K;N183D;

[0761] A194E;N204D;P210l;S212P;N218S;A228S;N248Q;S259D;T260D;N261 F;E271 D;

[0762] Q) N18Q;L21V;S24K;T38K;S56D;S78N;A103S;Q109K;S144N;S160H;Q182K;N183D;

[0763] N204D;P210l;S212P;N218S;A228S;N248Q;S259D;T260D;N261 F;E271 D;

[0764] (k) N18Q;L21 V;S24K;G25D;T38K;N43K;S56D;S78N;A103S;Q109K;S144N;S160H;Q182K;

[0765] N183D;A194E; N204D; P210I; N218S;A228S; N248Q;T260D; N261 F; E271 D;

[0766] (l) S9A;N18Q;L21 V;S24K;G25D;T38K;N43K;S56D;S78N;A103S;Q109K;S144N;S160H;

[0767] Q182K;N183D;A194E;N204D;P210l;N218S;A228S;N248Q;T260D;N261 F;E271 D;

[0768] (m) S9A;N18Q;L21 V;S24K;G25D;T38K;N43K;S56D;S78N;A103S;Q109K;S144N;S160H;

[0769] Q 182K; N 183D; N204D; P210l ; N218S; A228S; N248Q;T260D; N261 F; E271 D;

[0770] (n) S9A;N18Q;L21 V;S24K;T38K;N43K;S56D;S78N;A103S;Q109K;S144N;S160H;Q182K;

[0771] N183D;A194E; N204D; P21 Ol; N218S;A228S; N248Q;T260D; N261 F; E271 D;

[0772] (o) S9A;N18Q;L21 V;S24K;T38K;S56D;S78N;Q109K;S144N;S160H;Q182K;N183D;N204D;

[0773] P210l;N218S;A228S;N248Q;T260D;E271 D;

[0774] (p) N18Q;L21V;S24K;G25D;T38K; N43R;S56D;S78N;A103S;Q109K;S144N;S160H;Q182K;

[0775] N183D;A194E; N204D; P21 Ol; S212P; N218S;A228S; N248Q; N261 F;

[0776] (q) N18Q;L21 V;S24K;G25D;T38K; N43R;S56D;S78N;Q109 K; S 144N ; S 160H;Q182K; N183D; A 194E; N204D; P210l ; S212P; N218S; A228S; N248Q;

[0777] (r) S9A;N18Q;L21 V;S24K;G25D;T38K;N43R;S56D;S78N;A103S;Q109K;S144N;S160H;

[0778] Q182K;N183D;A194E;N204D;P210l;S212P;N218S;A228S;N248Q;N261 F;E271 D;

[0779] (s) N18Q;L21 V;S24K;G25D;T38K;N43K;S56D;S78N;Q109K;S144N;S160H;Q182K;N183D;

[0780] A194E;N204D;P210l;S212P;N218S;A228S;N248Q;S259D;N261 F;

[0781] (t) S9A;N18Q;L21 V;S24K;G25D;T38W;N43K;S56D;S78N;A103S;Q109K;S144N;S160H;

[0782] Q182K;N183D;N204D;P210l;S212P;N218S;A228S;N248Q;S259D;N261 F;E271 D;

[0783] (u) S9A;N18Q;L21 V;S24K;G25D;T38K;N43K;S56D;S78N;A103S;Q109K;S144N;S160H;

[0784] Q182K;N183D;A194E;N204D;P210l;S212P;N218S;A228S;N248Q;S259D;E271 D;

[0785] (v) S9A;N18Q;L21 V;S24K;G25D;T38K;N43K;S56D;S78N;A103S;Q109K;S144N;S160H;

[0786] Q182K;N183D;A194E;N204D;P210l;S212P;N218S;A228S;N248Q;S259D;N261 F; (w) S9A;N18Q;L21 V;S24K;G25D;T38K;N43K;S56D;S78N;A103S;Q109K;S144N;S160H;

[0787] Q 182K; N 183D; N204D; P210l ; S212P; N218S; A228S; N248Q; S259D; E271 D;

[0788] (x) S9A;N18Q;L21 V;S24K;G25D;T38K;N43K;S56D;S78N;A103S;Q109K;S144N;S160H;

[0789] Q182K;N183D;N204D;P210l;S212P;N218S;A228S;N248Q;S259D;N261 F;E271 D;

[0790] (y) S9A; N18Q; L21 ; S24K;T38K; N43K; S56D;S78N;A103S; Q109K; S144N ; S 160H; Q182K;

[0791] N183D;A194E;N204D;P210l;S212P;N218S;A228S;N248Q;S259D;N261 F;

[0792] (z) S9A; N18Q; L21 ; S24K;T38K; N43K; S56D;S78N;A103S; Q109K; S144N ; S 160H; Q182K;

[0793] N183D;A194E;N204D;P210l;S212P;N218S;A228S;N248Q;S259D;N261 F;E271 D;

[0794] (aa) S9A;N18Q;L21V;S24K;T38K;N43K;S56D;S78N;A103S;Q109K;S144N;S160H;Q182K;

[0795] N183D; N204D; P21 Ol ; S212P; N218S;A228S; N248Q;S259D; N261 F; E271 D;

[0796] (bb) N18Q;L21 V;S24K;T38W;S56D;Q59K;S78N;A103S;Q109K;S144N;S160H;Q182K;N183D;

[0797] A194E;N204D;P210l;S212P;N218S;A228S;N248Q;T260D;N261 F;E271 D;

[0798] (cc) S9A;N18Q;L21 V;S24K;G25D;T38W;S56D;Q59K;S78N;A103S;Q109K;S144N;S160H;

[0799] Q182K;N183D;A194E;N204D;P210l;S212P; ;N218S;A228S;N248Q;T260D;N261 F;E271 D;

[0800] (dd) S9A;N18Q;L21 V;S24K;G25D;T38W;S56D;Q59K;S78N;A103S;Q109K;S144N;S160H;

[0801] Q182K;N183D;N204D;P210l;S212P;N218S;A228S;N248Q;S259D;T260D;N261 F;E271 D;

[0802] (ee) S9A;N18Q;L21 V;S24K;G25D;T38W;S56D;Q59K;S78N;Q109K;S144N;S160H;Q182K;

[0803] N183D;N204D; P21 Ol ; S212P; N218S;A228S;N248Q;T260D; E271 D;

[0804] (ff) S9A;N18Q;L21 V;S24K;G25D;T38K;S56D;Q59K;S78N;A103S;Q109K;S144N;S160H;

[0805] Q182K;N183D;A194E;N204D;P210l;S212P;N218S;A228S;N248Q;T260D;E271 D;

[0806] (gg) S9A;N18Q;L21V;S24K;G25D;T38K;S56D;Q59K;S78N;A103S;Q109K;S144N;S160H;

[0807] Q182K; N 183D; A194E; N204D; P2101 ; S212P; N218S;A228S; N248Q;T260D; N261 F; E271 D;

[0808] (hh) S9A;N18Q;L21V;S24K;G25D;T38K;S56D;Q59K;S78N;A103S;Q109K;S144N;S160H;

[0809] Q182K;N183D;A194E;N204D;P210l;S212P;N218S;A228S;N248Q;S259D;T260D;E271 D;

[0810] (ii) S9A;N18Q;L21 V;S24K;G25D;T38K;S56D;Q59K;S78N;A103S;Q109K;S144N;S160H;

[0811] Q182K;N183D;A194E;N204D;P210l;S212P;N218S;A228S;N248Q;S259D;T260D;N261 F;

[0812] E271 D;

[0813] Gj) S9A;N18Q;L21 V;S24K;G25D;T38K;S56D;Q59K;S78N;A103S;Q109K;S144N;S160H;

[0814] Q182K;N183D;N204D;P210l;S212P;N218S;A228S;N248Q;S259D;T260D;N261 F;E271 D;

[0815] (kk) S9A;N18Q;L21V;S24K;G25D;T38K;S56D;Q59K;S78N;Q109K;S144N;S160H;Q182K;

[0816] N183D;N204D;P210l;S212P;N218S;A228S;N248Q;S259D;T260D;E271 D;

[0817] (II) S9A;N18Q;L21 V;S24K;T38W;S56D;Q59K;S78N;A103S;Q109K; S144N; S160H;Q182K;

[0818] N183D;N204D;P210l;S212P;N218S;A228S;N248Q;S259D;T260D;N261 F;E271 D;

[0819] (mm) N18Q;L21V;S24K;T38K;S56D;Q59K;S78N;A103S;Q109K;S144N;S160H;Q182K; N183D;A194E;N204D;P210l;S212P;N218S;A228S;N248Q;T260D;E271 D; and

[0820] (nn) N18Q;L21V;S24K;T38K;S56D;Q59K;S78N;A103S;Q109K;S144N;S160H;Q182K;

[0821] N183D;A194E;N204D;P210l;S212P;N218S;A228S;N248Q;T260D;N261 F;E271 D, compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2. In one embodiment, the variant protease has an amino acid sequence with a sequence identity of at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99% or of 100% to the amino acid sequence according to any one of SEQ ID NO:5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 1 1 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ I D NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22,

[0822] SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ I D NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29,

[0823] SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32, SEQ I D NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36,

[0824] SEQ ID NO: 37, SEQ I D NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43 and SEQ ID NO: 44.

[0825] In one embodiment, the variant protease has an amino acid sequence with a sequence identity of at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99% or of 100% to the amino acid sequence according to any one of SEQ ID NO: 120, SEQ ID NO: 121 , SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ I D NO: 125, SEQ ID NO: 126, SEQ I D NO: 127, SEQ ID NO: 128, SEQ I D NO: 129, SEQ ID NO: 130, SEQ I D NO: 131 , SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ I D NO: 139 and SEQ ID NO: 140.

[0826] In one embodiment, the protease variant of the present invention exhibits one or more improved properties compared to the parent protease, preferably compared to the protease according to SEQ ID NO: 3 or 4, preferably compared to the protease according to SEQ ID NO: 4.

[0827] Preferably, the variant polypeptide having protease activity of the present invention shows an increased stability, in particular storage stability, compared to the parent protease according to SEQ ID NO: 3 or 4, preferably compared to the protease according to SEQ ID NO: 4. The increased stability is preferably indicated as residual activity after stability challenge. Preferably, storage stability is indicated as residual activity after storage under the respective storage conditions, preferably, after storage in a detergent composition (preferably in a laundry or dishwash detergent, preferably laundry detergent). Preferably, the residual activity of the protease variant is increased compared to the residual activity of the parent protease SEQ ID NO: 3 or 4, preferably the protease according to SEQ I D NO: 4, after storage. Preferably, the residual activity of the protease variant after storage is at least 0.5%, at least 1 %, at least 2%, at least 3%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, or at least 200% increased compared to the residual activity of the parent protease, preferably compared to the protease SEQ ID NO: 3 or 4, preferably compared to the protease according to SEQ ID NO: 4.

[0828] Preferably, the improved property is one or more property selected from the group consisting of an increase in stability, thermostability, performance in a detergent, performance in a laundry detergent, performance in an ADW detergent. Preferably, the improved activity is improved wash performance, wash performance of a laundry detergent, and / or wash performance of an ADW detergent.

[0829] Preferably, the improved property is improved stability in a detergent composition, preferably a laundry detergent composition, preferably in a detergent composition comprising 1-20%, more preferably 5-10% anionic surfactants, preferably LAS and / or greater than 50% water, preferably in a Model B type detergent as described herein. In one embodiment, the improved property is improved stability in a detergent composition, preferably a laundry detergent composition, preferably in a detergent composition comprising 0. 1-5%, preferably 0.3-3%, of a strong sequestering builder. In this embodiment, the detergent composition preferably comprises 1 -20%, more preferably 5-10% anionic surfactants, preferably LAS, and / or greater than 50% water.

[0830] Nucleic acid construct

[0831] The present invention also refers to a polynucleotide encoding the variant polypeptide having protease 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.

[0832] The present invention also refers to a nucleic acid construct, preferably an expression cassette, comprising the polynucleotide as described herein.

[0833] 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.

[0834] The 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.

[0835] 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 col E1 .

[0836] 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.

[0837] The polynucleotide encoding the variant polypeptide having protease 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.

[0838] Host cell

[0839] The present invention also refers to a host cell comprising the polynucleotide encoding the variant polypeptide having protease 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 variant polypeptide having protease activity of the present invention.

[0840] The polynucleotide encoding the variant polypeptide having protease activity of the present invention herein may be transiently or stably introduced into a host cell. The polynucleotidemay be maintained non- 1 nt eg rated, 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.

[0841] Various host cells can be used for expressing the nucleic acid construct described herein. The host cell of the present invention does not naturally express the variant polypeptide having protease activity of the present invention. Thus, the host cell is a recombinant host cell; the nucleic acid construct described herein is heterologous for the host cell.

[0842] In one embodiment, the host cell is a prokaryote or a eukaryote. In another embodiment, the host cell is a bacteria, an archaea, afungal cell, a yeast cell or a eukaryotic cell. In another embodiment, the host cell is a non-human host cell. In one 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, Corynebac- terium, Streptococcus, Streptomyces, Staphylococcus, Enterococcus, Lactobacillus, Lactococcus, Clostridium, Geobacillus, and Oceanobacillus. Gram-negative bacteria include, but are not limited to, Escherichia, Pseudomonas, Sal- monella, Campylobacter, Helicobacter, Acetobacter, Flavobacterium, Fusobacterium, Gluconobacter. In a specific embodiment, the bacterial host cell is a Echerichiacoli cell. In one embodiment, the host cell is a bacterial cell. In aspecific embodiment the host cell is of the genus Escherichia or Bacillus.

[0843] In the methods of 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.

[0844] Methods of making

[0845] Another embodiment of the present invention is a method of obtaining a protease variant of a parent protease comprising the steps of introducing into a parent protease, preferably into the protease according to SEQ ID NO: 1,

[0846] (a) an amino acid substitution at two or more amino acid residues selected from the group consisting of 9, 18, 21 , 38, 59, 228, and 271 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and

[0847] (b) an amino acid substitution at two or more amino acid residues selected from the group consisting of 25, 43, 78, 109, 144, 182, 183, 194, 204, 210, 212, 218, 248, 259, 260 and 261 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, thereby providing a variant polypeptide of said parent protease, wherein said variant has at least 79%, but less than 100%, sequence identity to the amino acid sequence to the polypeptide of SEQ I D NO: 1, and wherein said variant polypeptide has protease activity and preferably wherein the variant polypeptide has an improved property relative to said parent protease.

[0848] Ways of introducing amino acid substitutions into a protein sequence are well known in the art. The variants may be prepared using any mutagenesis procedure known in the art, such as site-directed mutagenesis, synthetic gene construction, semi-synthetic gene construction, random mutagenesis, shuffling, etc.

[0849] The variant polypeptide having protease activity of the present invention can be produced in an industrial scale and subsequently purified. Industrial production of enzymes usually is done by cultivating a host cell (also called fermentation) which expresses the enzyme. Suitable host cells are described herein. A nucleic acid sequence encoding the variant polypeptide having protease activity of the present invention can be transformed into the host cell, which is subsequently cultivated under conditions suitable for the host cell to produce the variant polypeptide having protease activity of the present invention. I n a preferred embodiment, the variant polypeptide having protease activity of the present invention is purified from the host cell.

[0850] Hence, in yet another embodiment, the present invention is directed to a method of producing a variant polypeptide having protease activity, comprising the steps of

[0851] (a) providing a host cell comprising a heterologous nucleic acid construct comprising a polynucleotide encoding the variant polypeptide having protease activity;

[0852] (b) cultivating the recombinant host cell of step (a) under conditions conductive for the expression of the polynucleotide; and

[0853] (c) optionally, recovering the variant polypeptide having protease activity encoded by the polynucleotide.

[0854] 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 comprising a liquid fraction and a solid fraction is collected and may be further processed. The variant polypeptide having protease activity may be further purified from the fermentation broth.

[0855] The variant polypeptide having protease activity 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 on this, the variant polypeptide having protease activity may be recovered from the liquid fraction of the fermentation broth or from cell lysates. Preferably, the variant polypeptide having protease activity is secreted from the cell into the fermentation broth, preferably by means of a secretion signal peptide added to the N- terminus of the amino acid sequence, forming a propeptide of the polypeptide. The polypeptide is folded upon cleavage of the propeptide and the mature protease is released as active variant. Within the cultivation process measures might be taken to actively protect the protein from autocatalysis such as described in WO 2024 / 028340 or WO 2024 / 028338. Recovery of the variant polypeptide having protease activity 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, extraction, and precipitation. If the protein of interest precipitates or crystallizes in the fermentation broth or binds at least in part to the particulate matter of the fermentation broth additional treatment steps might be needed to release the protein of interest from the biomass or to solubilize protein of interest crystals and precipitates. WO 00 / 43502 A1 , WO 2008 / 110498 A1 , and WO 2017 / 097869 A1 describe a method for recovering a protein of interest, which precipitates and / or crystallizes during fermentation, from the fermentation broth. In case the desired protein is comprised in the cells of the fermentation broth release of the protein of interest from the cells might be needed. Release from the cells can be achieved for instance, but not being limited thereto, by cell lysis using techniques well known to the skilled person, e.g., lysozyme treatment, ultrasonic treatment, French press or combinations thereof. The variant polypeptide having protease activity may be purified from the fermentation broth by methods known in the art. For example, the variant polypeptide having protease activity may be isolated from the fermentation broth by con- ventional 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, chromatofocussing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing (IEF), 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.

[0856] Protease Compositions

[0857] The purified solution of the variant polypeptide having protease activity of the present invention may be further processed to form a protease containing composition. Hence, also claimed herein is a composition comprising the variant polypeptide having protease activity of the present invention and at least one additional component.

[0858] Thus, the present invention therefore also refers to a method for making a composition comprising the steps of mixing I. a variant polypeptide having protease activity as described herein; and ii. one or more components described herein.

[0859] Further, the present invention also refers to a method for improving protease stability in a composition comprising the steps of mixing

[0860] (a) a variant polypeptide having protease activity as described herein; and

[0861] (b) one or more components described herein.

[0862] The composition can be a non-complex formulation, e.g., a protease formulation, or a complex formulation, e.g., a detergent composition, as described herein.

[0863] I n one embodiment of the present invention, the variant polypeptide having protease activity is formulated as a protease formulation, preferably a concentrated protease formulation. The protease formulation can be either 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 .

[0864] Liquid protease formulations may comprise amounts of protease enzyme in the range of 2% to 40%, 2% to 30%, 2% to 25%, 2% to 12%, or preferably 2-7.5% by weight, all relative to the total weight of the enzyme formulation.

[0865] In one embodiment, the protease formulation, in particular the liquid protease formulation, comprises in addition one or more additional compounds selected from the group consisting of solvent, salt, pH regulator, preservative, enzyme stabilizer, and thickening agent. Preferably, the protease formulation is essentially devoid of surfactants, i.e. the protease formulation comprises less than 1 % surfactant, preferably less than 0.5% surfactant. The solvent may be water and / or an organic solvent. Aqueous protease 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 protease formulation. The protease-containing 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 protease 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, and polyethylene glycol.

[0866] In one embodiment, the present invention relates to a composition comprising the variant polypeptide having protease activity of the present invention and at least one solubilizing agent selected from the group consisting of neopentyl glycol, gamma-valerolactone, 1,6-hexanediol, and diethylene glycol. In one embodiment, the composition comprising the variant polypeptide having protease activity of the present invention and at least one solubilizing agent selected from the group consisting of neopentyl glycol, gamma-valerolactone, 1 ,6-hexanediol, and diethylene glycol additionally comprises glycerol and / or monopropylene glycol (MPG).

[0867] Thus, the present invention also provides a composition comprising the variant polypeptide having protease activity of the present invention, neopentyl glycol and glycerol. The present invention also provides a composition comprising the variant polypeptide having protease activity of the present invention, neopentyl glycol and MPG. The present invention also provides a composition comprising the variant polypeptide having protease activity of the present invention, neopentyl glycol, glycerol and MPG. The present invention also provides a composition comprising the variant polypeptide having protease activity of the present invention, 1,6-hexanediol and glycerol. The present invention also provides a composition comprising the variant polypeptide having protease activity of the present invention, 1,6-hexanediol and MPG. The present invention also provides a composition comprising the variant polypeptide having protease activity of the present invention, 1 ,6-hexanediol, glycerol and MPG. The present invention also provides a composition comprising the variant polypeptide having protease activity of the present invention, 1 ,6-hexanediol, neo-pentyl glycol, glycerol and MPG. The present invention also provides a composition comprising the variant polypeptide having protease activity of the present invention, glycerol and MPG.

[0868] In one embodiment, the protease 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’-di- chloro 2-hydroxydipheny lether. 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 protease formulation is free from preservatives, meaning that preservatives are comprised in amounts less than 1 ppm, preferably 0 ppm. Preferably, the protease 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, CaCfe, 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), borate, boric acid, boronic acids (preferably, 4-formyl phenylboronic acid (4-FPBA)), peptide aldehydes (preferably, Benzyloxycarbony-VAL-H (Z-VAL-H or Cbz-VAL-H) or Benzyloxycarbony-GAY-H (Z-GAY-H or Cbz-GAY-H), peptide acetals, and peptide aldehyde hydrosulfite adducts. Preferably, 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 and preferably one or more of the compounds selected from the group consisting of borate, boric acid, boronic acids (preferably, 4-formyl phenylboronic acid (4-FPBA)), peptide aldehydes, peptide acetals, and peptide aldehyde hydrosulfite adducts. In a particularly preferred embodiment, the stabilizing system comprises a protease inhibitor, preferably selected from borate, boric acid, boronic acids (preferably, 4-FPBA), peptide aldehydes (preferably, peptide aldehydes like Z-VAL-H or Z-GAY-H), peptide acetals, and peptide aldehyde hydrosulfite adducts, preferably the protease inhibitor is a peptide aldehyde, preferably Z-VAL-H or Z-GAY-H. In one embodiment, the stabilizing system does not comprise a protease inhibitor. Preferably, the composition is boron-free. Preferably, the protease formulation comprises a calcium salt, preferably calcium chloride.

[0869] In one embodiment, the protease formulation comprises a protease inhibitor, preferably a peptide aldehyde, in amounts in the range of about 0.04% to 0.8%, 0.04% to 0.7%, 0.04% to 0.6%, 0.04% to 0.5%, 0.04% to 0.4% or 0.04% to 0.3% by weight relative to the total weight of the liquid protease variant formulation. Preferably, the peptide aldehyde is comprised in amounts in the range of about 0.1 % to 0.6% by weight, of about 0.1 % to 0.5% by weight, of about 0.1 % to 0.4%, or of about 0. 1 % to 0.35% by weight, all relative to the total weight of the liquid protease formulation.

[0870] Preferably, the liquid protease formulation comprises or consists of the variant polypeptide having protease activity of the present invention, a solvent, an enzyme stabilizing system, and optionally a preservative and optionally an additional enzyme different from the protease variant. Preferably, the formulation of the variant polypeptide having protease activity of the present invention is essentially devoid of surfactants, i.e. the protease formulation comprises less than 1 % surfactant, preferably less than 0.5% surfactant.

[0871] The present invention therefore also relates to a method for making a protease formulation, preferably a concentrated protease formulation, comprising the steps of mixing a) a variant polypeptide having protease activity of the present invention; and b) one or more components selected from the group consisting of solvent, enzyme stabilizing system, preservative, and an additional enzyme different from the protease.

[0872] Further, the present invention relates to a method for improving protease stability in a composition with comprising the steps of mixing a) a variant polypeptide having protease activity of the present invention; and b) one or more components selected from the group consisting of solvent, enzyme stabilizing system, preservative, and an additional enzyme different from the protease.

[0873] In one embodiment, the variant polypeptide having protease activity of the present invention is part of a microorganism (alive, attenuated or inactivated), probiotic, or prebiotic.

[0874] Additional enzymes

[0875] In another embodiment, the composition comprising a variant polypeptide having protease activity of the present invention further comprises one or more additional enzymes different from the protease. Preferably, the additional enzyme is selected from the group consisting of amylases, lipases, proteases other than the polypeptide having protease activity of the present invention, cellulases, mannanases, hemicellulases, phospholipases, esterases, pectinases, lactases, peroxidases, xylanases, cutinases, pectate lyases, keratinases, reductases, oxidases, 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 amylases, lipases, proteases other than the polypeptide having protease activity of the present invention, cellulases, mannanases, xylanases, DNAses, dispersins, pectinases, 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 amylases, lipases, proteases other than the polypeptide having protease activity of the present invention, cellulases, mannanases, and combinations of at least two of the foregoing types. Most preferably, the additional enzyme is an amylase, preferably, an alpha-amylase.

[0876] The composition of the present invention can comprise more than one enzyme of different types, e.g., an amylase and a protease, or more than one enzyme of the same type, e.g., two or more different proteases, or mixtures thereof, e.g., an amylase and two different proteases of which one is the variant polypeptide having protease activity of the present invention.

[0877] Proteases

[0878] Proteases other than the variant polypeptide having protease activity of the present invention can comprise an amino acid sequence having with increasing preference at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but be- low 100% sequence identity with SEQ I D NO: 45 and comprising amino acid substitutions in one or more of the following positions 3, 4, 9, 15, 27, 33, 36, 57, 68, 77, 87, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104, 106, 118, 120, 123, 128, 129, 130, 131 , 154, 160, 167, 170, 194, 199, 205, 206, 217, 218, 222, 224, 232, 235, 236, 245, 252 and 274 (according to the BPN' numbering), which has proteolytic activity. In one embodiment, such a protease is not mutated at positions Asp32, His64 and Ser221 (according to the BPN' numbering). Preferably, the protease used in combination with the variant polypeptide having protease activity of the present invention comprises an amino acid sequence having with increasing preference at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but below 100% sequence identity with SEQ ID NO: 45 and is further characterized by having amino acid glutamic acid (E), or aspartic acid (D), or asparagine (N), or glutamine (Q), or alanine (A), or glycine (G), or serine (S), preferably glutamic acid (E), at position 101 (according to the BPN' numbering) and has proteolytic activity. Mostly preferred is a protease which has at least 80%, but below 100% sequence identity with SEQ I D NO: 45 and is characterized by having amino acid glutamic acid (E) at position 101 (according to the BPN' numbering) and has proteolytic activity. The protease may comprise an amino acid substitution at position 101 , such as R101 E, alone or in combination with one or more substitutions at positions 3, 4, 9, 15, 27, 33, 36, 57, 68, 77, 87, 95, 96, 97, 98, 99, 100, 102, 103, 104, 106, 118, 120, 123, 128, 129, 130, 131 , 154, 160, 167, 170, 194, 199, 205, 206, 217, 218, 222, 224, 232, 235, 236, 245, 252 and / or 274 (according to the BPN' numbering) and has proteolytic activity. In one embodiment, said protease comprises one or more further substitutions: (a) threonine at position 3 (3T), (b) isoleucine at position 4 (4I), (c) alanine, threonine or arginine at position 63 (63A, 63T, or63R), (d) aspartic acid or glutamic acid at position 156 (156D or 156E), (e) proline at position 194 (194P), (f) methionine at position 199 (199M), (g) isoleucine at position 205 (205I), (h) aspartic acid, glutamic acid or glycine at position 217 (217D, 217E or 217G), (i) combinations of two or more amino acids according to (a) to (h), according to the BPN' numbering. A suitable protease may be at least 80% identical to SEQ ID NO: 45 and is characterized by comprising one amino acid (according to (a)-(h)) or combinations according to (i) together with the amino acid 101 E, 101 D, 101 N, 101 Q, 101A, 101 G, or 101 S (according to the BPN' numbering) and has proteolytic activity. In one embodiment, the protease is at least 80% identical to SEQ ID NO:45 and is characterized by comprising the mutation (according to the BPN' numbering) R101 E, or S3T + V4I + V205I, or S3T + V4I + R101 E + V205I or S3T + V4I + V199M + V205I + L217D, and has proteolytic activity. In another embodiment, the protease comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 45 and being further characterized by comprising S3T + V4I + S9R + A15T + V68A + D99S + R101 S + A103S + 1104V + N218D (according to the BPN' numbering) and has proteolytic activity. I n another embodiment, the protease may have an amino acid sequence being at least 80% identical to SEQ ID NO:45 and being further characterized by comprising R101 E, and one or more substitutions selected from the group consisting of S156D, L262E, Q137H, S3T, R45E, D,Q, P55N, T58W,Y, L, Q59D, M,N,T, G61 D.R, S87E, G97S, A98D,E,R, S106A,W, N117E, H120V,D, K,N, S125M, P129D, E136Q, S144W, S161T, S163A.G, Y171 L, A172S, N185Q, V199M, Y209W, M222Q, N238H, V244T, N261T, D and L262N,Q,D (according to the BPN' numbering), and has proteolytic activity.

[0879] In this embodiment, preferably, the protease used in combination with the variant polypeptide having protease activity according to the present invention comprises an amino acid sequence which is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% identical to SEQ ID NO: 45 and the protease comprises compared to SEQ ID NO: 45 the amino acid substitutions R101 E and S156D and / or L262E, and optionally at least one further mutation selected from I104T, H120D, Q137H, S141 H, R145H and S163G according to the BPN' numbering.

[0880] In another embodiment, the protease used in combination with the variant polypeptide having protease activity according to the present invention has at least 60%, preferably at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to SEQ ID NO: 45 and comprises compared to SEQ ID NO: 45 the amino acid substitution R101 D or R101 E, preferably R101 E, and one or more of the amino acid substitutions selected from the group consisting of S3T, V4I, and V205I, preferably all of the amino acid substitutions S3T, V4I, and V205I, according to the BPN' numbering, and one or more substitutions at positions according to the BPN' numbering selected from the group consisting of 76, 138, 145, 156, 166, 167, 169, 177, 187, 189, 191 , 206, 209, 215, 218, and 262, preferably selected from the group consisting of N76D / E / Q, A138Q, R145L / W / Y, S156D / Q, S166G, Y167T, A169G, V177 L, A187 D, F189R, Q191 R, Q206A / L / S / T, Y209K / V / W, A215K / W, N218S / T, and L262D / E / Q

[0881] In this embodiment, preferably, the protease used in combination with the variant polypeptide having protease activity according to the present invention has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to SEQ ID NO: 45 and comprises compared to SEQ ID NO: 45 the amino acid substitution R101 D or R101 E, preferably R101 E, and one or more of the amino acid substitutions selected from the group consisting of S3T, V4I, and V205I, preferably all of the amino acid substitutions S3T, V4I, and V205I, according to the BPN' numbering, and one or more substitutions at positions according to the BPN' numbering selected from the group consisting of 156, 166, 187, 189, 191 , 206, 209, 215 and 262, preferably selected from the group consisting of S156D, S166G, A187 D, F189R, Q191 R, Q206L, Y209W, A215K und L262E.

[0882] In this embodiment, particularly preferred, the protease used in combination with the variant polypeptide having protease activity according to the present invention has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to SEQ ID NO: 45 and comprises compared to SEQ ID NO: 45 according to the BPN' numbering the amino acid substitutions

[0883] (I) S3T-V4I-R101 E-V199I-Q206L-Y209W;

[0884] (II) S3T-V4I-R101 E-V199I-N218S;

[0885] (ill) S3T-V4I-R101 E-V199I-N76D; (iv) S3T-V4I-R101 E-V199I-S156D-L262E;

[0886] (v) S3T-V4I-R101 E-V199I-Q206L-Y209W-S156D-L262E;

[0887] (vi) S3T-V4I-R101 E-V199I-N76D-Q206L-Y209W;

[0888] (vii) S3T-V4I-R101 E-V199I-N76D-S156D-Q206L-Y209W-L262E;

[0889] (viii) S3T-V4I-R101 E-V199I-N76D-N218S;

[0890] (ix) S3T-V4I-R101 E-V199I-N76D-S156D-Y209W-L262E;

[0891] (x) S3T-V4I-R101 E-V199I-N76D-Y209W;

[0892] (xi) S3T-V4I-R101 E-V199I-N76D-S156D-Q206L-L262E;

[0893] (xii) S3T-V4I-R101 E-V199I-N76D-Q206L;

[0894] (xi i i) S3T-V4I-R101 E-V199I-S156D-Q206L-Y209W;

[0895] (xiv) S3T-V4I-R101 E-V199I-Q206L-Y209W-L262E;

[0896] (xv) S3T-V4I-R101 E-V199I-A138Q-R145W-Y167 T-Q206L;

[0897] (xvi) S3T-V4I-R101 E-V199I-N76D-R145Y-A215W-N218S-L262E;

[0898] (xvii)S3T-V4l-R101 E-V199I-A138Q-S156D-V177 L-Q206L;

[0899] (xviii) S3T-V4I-R101 E-V199I-Q206L-Y209W-A215K-S156D-L262E;

[0900] (xix) S3T-V4I-R101 E-V199I-S156D-S166G-Q191 R-Q206L-Y209W-L262E; or

[0901] (xx) S3T-V4I-R101 E-V199I-S156D-A187 D-F189R-Q206L-Y209W-L262E.

[0902] Commercially available protease enzymes include but are not limited to those sold under the trade names Progress® Uno, Alcalase®, Blaze®, Duralase™, Durazym™, Relase®, Relase® Ultra, Savinase®, Savinase® Ultra, Primase®, Polarzyme®, Kannase®, Liquanase®, Liquanase® Ultra, Ovozyme®, Coronase®, Coronase® Ultra, Neutrase®, Ev- erlase® and Esperase® (Novozymes A / S) and those sold under the tradename Maxatase®, Maxacai®, Maxapem®, Purafect®, Purafect® Prime, Purafect MA®, Purafect Ox®, Purafect OxP®, Puramax®, Properase®, FN2®, FN3®, FN4®, Excellase®, Eraser®, Ultimase®, Opticlean®, Effectenz®, Preferenz® (P100, P200, P300 and P400 from IFF) and Optimase® (Danisco / DuPont), Axapem™ (Gist-Brocases N.V.), KAP (Bacillus alkalophilus subtilisin) from Kao Corporation and Lavergy Pro (BASF).

[0903] Lipases

[0904] “Lipases”, “lipolytic enzyme”, “lipid esterase”, all refer to an enzyme of EC class 3.1.1 (“carboxylic ester hydrolase”). Lipase means active protein having lipase activity (or lipolytic activity; triacylglycerol lipase, EC 3.1.1.3), cutinase activity (EC 3.1 .1 .74; enzymes having cutinase activity may be called cutinase herein), sterol esterase activity (EC 3.1.1.13) and / or wax-ester hydrolase activity (EC 3.1.1.50). Lipases include those of bacterial or fungal origin.

[0905] The methods for determining lipolytic activity are well-known in the literature (see e.g. Gupta et al. (2003), Biotechnol. Appl. Biochem. 37, p. 63-71). For instance, the lipase activity may be measured by ester bond hydrolysis in the substrate para-nitrophenyl palmitate (pNP-Palmitate, C: 16) and releases pNP which is yellow and can be detected at 405 nm. In one aspect of the invention, a suitable lipase (component (b)) is selected from the following: lipases from Humicola (synonym Thermomyces), e.g. from H. lanuginosa (T. lanuginosus) as described in EP 258068, EP 305216, WO 92 / 05249 and WO 2009 / 109500 or from H. insolens as described in WO 96 / 13580; lipases derived from Rhizomucor miehei as described in WO 92 / 05249; lipase from strains of Pseudomonas (some of these now renamed to Burkhold- eria), e.g. from P. alcaligenes or P. pseudoalcaligenes (EP 218272, WO 94 / 25578, WO 95 / 30744, WO 95 / 35381 , WO 96 / 00292), P. cepacia (EP 331376), P. stutzeri (GB 1372034), P. fluorescens, Pseudomonas sp. strain SD705 (WO 95 / 06720 and WO 96 / 27002), P. wisconsinensis (WO 96 / 12012), Pseudomonas mendocina (WO 95 / 14783), P. glumae (WO 95 / 35381 , WO 96 / 00292); lipase from Streptomyces griseus (WO 2011 / 150157) and S. pristinaespiralis (WO 2012 / 137147), GDSL-type Streptomyces lipases (WO 2010 / 065455); lipase from Thermobifida fusca as disclosed in WO 2011 / 084412; lipase from Geobacillus stearothermophilus as disclosed in WO 201 1 / 084417; Bacillus lipases, e.g. as disclosed in WO 00 / 60063, lipases from B. subtilis as disclosed in Dartois et al. (1992), Biochemica et Biophys...

Claims

CLAIMS1. A variant polypeptide having protease activity or a fragment of said polypeptide having protease activity, wherein:(I) the polypeptide has an amino acid sequence which is at least 79%, but less than 100%, identical to the amino acid sequence as set forth in SEQ ID NO: NO: 1 and(ii) the polypeptide or fragment thereof comprises(a) an amino acid substitution at two or more amino acid residues selected from the group consisting of 9, 18, 21, 38, 59, 228, and 271 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and(b) an amino acid substitution at two or more amino acid residues selected from the group consisting of 25, 43, 78, 109, 144, 182, 183, 194, 204, 210, 212, 218, 248, 259, 260 and 261 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, wherein the fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide.

2. The variant polypeptide of claim 1 , wherein the variant polypeptide comprises:(a) an amino acid substitution at two or more amino acid residues selected from the group consisting of 9, 18, 21, 38, 59, 228, and 271 compared to the amino acid sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and(b) an amino acid substitution at two or more amino acid residues selected from the group consisting of 43, 78, 183, 194, 204, 212, 210, 218, 259, 260 and 261 compared to the amino acid sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2.

3. The variant polypeptide of claim 1 or 2, wherein the variant polypeptide comprises:(a) an amino acid substitution at two or more amino acid residues selected from the group consisting of 9, 18, 38, 59, and 271 compared to the amino acid sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and(b) an amino acid substitution at two or more amino acid residues selected from the group consisting of 43, 78, 183, 194, 204, 212, 218, 259 and 261 compared to the amino acid sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2.

4. The variant polypeptide or fragment thereof of claim 1 or 2, wherein the polypeptide or fragment thereof comprises(a) an amino acid substitution at two or more amino acid residues selected from the group consisting of 18, 21 and 38 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and(b) an amino acid substitution at one or more amino acid residues selected from the group consisting of 78, 183, 194 and 210 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2.

5. The variant polypeptide or fragment thereof of claim 1 or 2, wherein the polypeptide or fragment thereof comprises(a) an amino acid substitution at two or more amino acid residues selected from the group consisting of 21, 38, 59, 228, and 271 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2 and(b) an amino acid substitution at two or more amino acid residues selected from the group consisting of 25, 210, 212, 248, 259 and 261 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2.

6. The variant polypeptide of any one of the preceding claims, wherein:(a) the amino acid substitution at amino acid residue 9 is X9A / Q / E / C / D, preferably is X9A / Q; and / or(b) the amino acid substitution at amino acid residue 18 is X18Q / E / D; and / or(c) the amino acid substitution at amino acid residue 21 is X21 l / V / F / W / Y, preferably is X21 V / l; and / or(d) the amino acid substitution at amino acid residue 25 is X25D; and / or(e) the amino acid substitution at amino acid residue 38 is X38R / K / H / W; and / or(f) the amino acid substitution at amino acid residue 43 is X43K / R / C / H / D / L / S / W / A / M / Y / Q / F / I, preferably isX43K / R; and / or(g) the amino acid substitution at amino acid residue 59 is X59K / E; and / or(h) the amino acid substitution at amino acid residue 78 is X78N / D / R / W / F / H / K / E / L / Y / M / C / Q, preferably isX78N / D; and / or(i) the amino acid substitution at amino acid residue 109 is X109K / A; and / or(k) the amino acid substitution at amino acid residue 144 is X144N / R; and / or(l) the amino acid substitution at amino acid residue 182 is X182K / R / E; and / or(m) the amino acid substitution at amino acid residue 183 is X183D / E / C / Q / A / M, preferably is X183D / E; and / or(n) the amino acid substitution at amino acid residue 194 is X194E / C / D, preferably is X194E / D; and / or(o) the amino acid substitution at amino acid residue 204 is X204D / E / C / G, preferably is X204D / E; and / or(p) the amino acid substitution at amino acid residue 210 is X210IA / , preferably is X210I; and / or(q) the amino acid substitution at amino acid residue 212 is X212G / M / R / K / P / A / F / H / W / N / Y / D, preferably isX212P / A / G / RK / F / Y / M; and / or(r) the amino acid substitution at amino acid residue 218 is X218S / T; and / or(s) the amino acid substitution at amino acid residue 228 is X228S; and / or(t) the amino acid substitution at amino acid residue 248 is X248Q / R; and / or(u) the amino acid substitution at amino acid residue 259 is X259D / E, preferably is X259D; and / or(v) the amino acid substitution at amino acid residue 260 is X260D / E / K, preferably is X260D / E; and / or(w) the amino acid substitution at amino acid residue 261 is X261 L / M / F / E / W / Y / C, preferably is X261 I\NIYI\_ and / or(x) the amino acid substitution at amino acid residue 271 is X271 D.

7. The variant polypeptide or fragment thereof of any of the preceding claims, wherein the variant polypeptide or fragment thereof additionally comprises an amino acid substitution at one or more amino acid residues selected from the group consisting of 24, 103 and 160 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2.

8. The variant polypeptide or fragment thereof of any of the preceding claims, wherein the variant polypeptide or fragment thereof additionally comprises an amino acid substitution at one or more amino acid residues selected from the group consisting of 3, 76 and 256 compared to the amino acid sequence as set forth in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2.

9. The variant polypeptide of claim 8, wherein:(a) the amino acid substitution at amino acid residue 24 is X24K; and / or(b) the amino acid substitution at amino acid residue 103 is X103S; and / or(c) the amino acid substitution at amino acid residue 160 is X160H / S / D / E / P, preferably is X160H.

10. The variant polypeptide of any one of the preceding claims, wherein said polypeptide comprises amino acid residue D or E at position 101, preferably E at position 101, referring to the numbering of SEQ ID NO: 2.11 . The variant polypeptide of any one of the preceding claims, wherein the polypeptide exhibits one or more improved properties compared to the protease according to SEQ ID NO: 3, preferably wherein the improved properties are selected from:(I) increase in stability,(ii) increase in storage stability, and(ill) increase in storage stability in a detergent composition.

12. A polynucleotide encoding the variant polypeptide of any one of the preceding claims.

13. A composition comprising the variant polypeptide of any one of claims 1 to 11 and at least one additional component, preferably wherein the composition comprises an 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 (preferably, C1-C3) carboxylic acids or salts thereof (preferably, formic acid, formate (preferably, sodium formate), acetic acid, acetate, or lactate), borate, boric acid, boronic acids (preferably, 4-formyl phenylboronicacid (4-FPBA)), peptide aldehydes (preferably, Z-VAL-H or Z-GAY-H), peptide acetals, and peptide aldehyde hy- drosulfite adducts, preferably peptide aldehydes (preferably, Z-VAL-H or Z-GAY-H).

14. The composition of claim 13, wherein the composition comprises one or more second enzymes different from the variant polypeptide referred to in any of the preceding claims, preferably one or more second enzymes selected from the group consisting of amylases, second proteases, lipases, cellulases, hemicellulases, mannanases, xylanases, DNases, dispersins, pectinases, oxidoreductases, and cutinases, preferably selected from amylases, mannanases, and lipases, most preferably amylases.

15. The composition of any one of claims 13 or 14, wherein the composition is a detergent composition, preferably a laundry detergent composition or a hard surface cleaning detergent composition, preferably a biodegradable detergent composition, preferably wherein the composition comprises one or more surfactants and / or one or more builder, preferably strong sequestering builder.

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